High-Affinity Alkynyl Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT)

Abstract

Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that methylates nicotinamide (NAM) using cofactor S-adenosylmethionine (SAM). NNMT overexpression has been linked to diabetes, obesity, and various cancers. In this work, structure-based rational design led to the development of potent and selective alkynyl bisubstrate inhibitors of NNMT. The reported nicotinamide-SAM conjugate (named NS1) features an alkyne as a key design element that closely mimics the linear, 180° transition state geometry found in the NNMT-catalyzed SAM → NAM methyl transfer reaction. NS1 was synthesized in 14 steps and found to be a high-affinity, subnanomolar NNMT inhibitor. An X-ray co-crystal structure and SAR study revealed the ability of an alkynyl linker to span the methyl transfer tunnel of NNMT with ideal shape complementarity. The compounds reported in this work represent the most potent and selective NNMT inhibitors reported to date. The rational design principle described herein could potentially be extended to other methyltransferase enzymes.

Graphical Abstract

INTRODUCTION

Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme responsible for the methylation of nicotinamide (NAM) using the cofactor S-adenosylmethionine (SAM), resulting in the production of 1-methylnicotinamide (MNAM) and S-adenosylhomocysteine (SAH).^1–2 While initially established as a vitamin B3 clearance enzyme, recent work has shown NNMT to be an important regulator of several intracellular pathways in fat, liver, and cancer cells.² NNMT can regulate methyl donor metabolism by direct interaction with proteins of the methionine cycle³ and can regulate hepatic nutrient metabolism through Sirt1 stabilization⁴. By decreasing cellular SAM levels, NNMT can modulate the epigenetic landscape of cancer cells⁵ and embryonic-stem cells⁶.

Accordingly, NNMT has emerged as a disease-relevant enzyme and a possible point of therapeutic intervention. Abnormal NNMT expression is implicated in several diseases, including diabetes^7–8, obesity^{7, 9}, and a variety of cancers^{2, 5, 10}. Increased NNMT protein expression was observed in adipose and liver tissue of obese and diabetic mice⁷, while increased NNMT mRNA expression was observed in humans¹¹. NNMT is overexpressed in glioblastoma, leading to cellular SAM depletion, DNA hypomethylation, and accelerated tumor growth.¹² Recently, NNMT was shown to be a master metabolic regulator of cancer-associated fibroblasts, with NNMT expression supporting ovarian cancer migration, proliferation, and metastasis.¹³ The successful development of potent and selective NNMT inhibitors would aid efforts to elucidate the role of NNMT in disease, potentially enabling new strategies to treat a variety of metabolic disorders, cancers, and other pathologies.

Several NNMT inhibitors have been reported, including methylated quinolines¹⁴, nicotinamide analogues^15–16, covalent inhibitors^17–18, and amino-adenosine derived bisubstrate inhibitors^19–21. Bisubstrate inhibitors are compounds that bind both the substrate and cofactor binding pockets within an enzyme active site. The design of bisubstrate inhibitors relates closely to two well-established ligand design strategies: transition-state mimicry and fragment-based design.²² Importantly, if two weak inhibitors that bind a protein at distinct sites are linked in an optimal manner, the resulting bisubstrate inhibitors can obtain several orders of magnitude increase in binding affinity.^23–24 As a result, linker identity is crucial to properly position the key interacting groups of the two component molecules and minimize unfavorable interactions between the linker and the protein.

In this work, we used a combination of structure-based design and molecular docking to develop high-affinity alkynyl bisubstrate inhibitors of NNMT. To begin, we examined the substrate-bound NNMT co-crystal structure (PDB ID 3ROD) and noted that the NAM nitrogen and SAH sulfur atoms are positioned ~ 4 Å apart and reside in a small tunnel that facilitates the S_N2-type methyl transfer reaction (Figure 1a,b).²⁵ We envisioned an alkyne as the optimal linker between a NAM-like and a SAM-like fragment, as an alkyne linker resembles the linear, 180° transition state geometry found in the SAM → NAM methyl transfer reaction (Figure 1c,d).

Figure 1.

Overview of the alkynyl bisubstrate strategy. (a) Binding pose of ligands SAH and NAM (teal) rendered from previously published co-crystal structure (PDB ID 3ROD). (b) Graphical representation of the SAM → NAM methyl transfer reaction catalyzed by NNMT. (c) Graphical representation of alkynyl bisubstrate inhibitor NS1 posed to mimic the binding geometry of SAM/NAM. (d) Molecular docking output pose of alkynyl bisubstrate inhibitor NS1 (black) overlaid with SAH and NAM from structure 3ROD (teal).

Thus, we rationally designed alkynyl nicotinamide-SAM conjugate 1 (named NS1, 10). Molecular docking with Glide²⁶ predicted NS1 to be a better binder than SAM by > 3 kcal/mol (see Supporting Information Part 1). The docked pose of NS1 (Figure 1d) overlays well with NAM and SAH in the previously published substrate-bound crystal structure; the alkyne linker fits well into the methyl transfer tunnel. With molecular docking suggesting NS1 as a potential NNMT inhibitor, we aimed to synthesize and test NS1 in an NNMT inhibition assay.

6’-alkynyl nucleosides were first proposed and synthesized in 1990 as mixtures of alkynyl and amino acid diastereomers.²⁷ Unfortunately, the designed alkynyl nucleosides were poor inhibitors when tested against catechol O-methyltransferase (COMT).²⁸ In this work, we demonstrate that the alkynyl bisubstrate strategy toward SAM-dependent methyltransferase inhibition is a valuable approach. We synthesized the rationally designed alkynyl bisubstrate compound NS1 as a single enantiomer and diastereomer in 14 steps and showed it to be a high-affinity, selective NNMT inhibitor with a K_i of 500 pM.

RESULTS AND DISCUSSION

NS1 Synthesis.

The synthesis of NS1 (10, Scheme 1) began with commercially available acetonide 1. Alkylation of the corresponding triflate with lithium TMS-acetylide followed by silyl deprotection generated alkyne 2. Copper-catalyzed semi-reduction²⁹ of 2 rendered terminal alkene 3, which was converted to enal 4 by cross-metathesis with crotonaldehyde. Solvent and catalyst screening (Table S1, Figure S1) revealed methyl tert-butyl ether (MTBE) and the commercially available Nitro-Grela olefin metathesis catalyst³⁰ as an optimal solvent/catalyst system to effect this transformation. The key C6’ stereocenter was installed by a rhodium-catalyzed asymmetric alkynylation³¹ to afford aldehyde 5 as a single diastereomer (see Supporting Information Part 1, Section 3 for numbering scheme). A Horner-Wadsworth-Emmons olefination with a phosphonoglycine derivative yielded enamide 6. The C17’ stereocenter was set via rhodium-catalyzed asymmetric hydrogenation with a Burk-type 1,2-bis(phospholano)ethane (BPE) ligand,³² providing (S)-trifluoroacetamide 7 as a single diastereomer. Subsequent acylation and Vorbrüggen glycosylation promoted by a 2,6-di-tert-butyl-4-methylpyridinium triflate salt³³ delivered nucleoside 8. Introduction of the benzamide moiety by a Sonogashira cross-coupling and a global deprotection sequence completed the synthesis of NS1 (10) in a total of 14 steps with an overall yield of 9.1%. The small-molecule X-ray crystal structure of NS1•TFA was obtained, confirming the regiochemistry of the adenine nucleobase (N9-linkage) as well as the configuration of the C6’ and C17’ stereocenters.

Scheme 1.

Synthesis and X-Ray Crystal Structure of NS1 (10)

Biochemical Evaluation of NS1 and Analogues.

A fluorescence-based NNMT inhibition assay³⁴ revealed NS1 to be a highly potent, subnanomolar (500 pM) NNMT inhibitor. In the assay, NNMT catalyzes the SAM-dependent methylation of quinoline, forming 1-methyquinolinium (1MQ) and SAH. Quinoline as opposed to NAM as methyl-group acceptor has been previously employed by Loring and Thompson³⁵ who determined the detailed kinetic mechanism of NNMT (Rapid Equilibrium Random Bi Bi in Cleland’s nomenclature^36–38) and thus indirectly established that NAM and quinoline are mechanistically equivalent as mutually interchangeable methyl group acceptors.

More importantly for the purposes of our inhibition study, quinoline enables continuous real-time monitoring of the reaction progress, using fluorescence detection to monitor the appearance of 1MQ. In contrast, previously described NNMT assays using NAM as native substrate were forced to rely on discontinuous (end-point) detection³⁹ and are thus unsuitable for real-time recording of continuous reaction progress, as was done in our study. Utilization of a quinoline-based continuous real-time assay was especially important because many enzymatic progress curves were prominently nonlinear, both in the presence and in the absence of inhibitors (Figure 2). In the specific case of NS1, the reaction progress curves were strongly nonlinear even within the first two minutes of the inhibition assay and therefore no “initial linear region” could be identified. Thus, as a matter of principle, the inhibitors reported in this study could not be studied by conventional initial rate kinetic methods.^40–41

Figure 2.

Kinetic studies of NNMT. A time course of the NNMT kinetic assay in the absence (red circles) or in the presence (remaining symbols) of NS1. Inhibitor concentrations are shown at top right. ΔF are scaled fluorescence changes in relative fluorescence units (“rfu”). The smooth curves represent the best least-squares fit to a system of first-order differential equations automatically generated by the DynaFit software package^42–43 according to the reaction mechanism shown in the Supporting Information (Part 3: Enzyme Kinetics).

Therefore we analyzed the full reaction time-course using the software package DynaFit.^42–43 The mathematical models for each of several postulated kinetic mechanisms were systems of first-order Ordinary Differential Equations (ODEs) automatically derived by the DynaFit software. Inhibition constants were computed as the ratio K_i = k_off/k_on, where k_off is the microscopic rate constant for the dissociation of the enzyme-inhibitor complex and k_on is the corresponding microscopic association rate constant. (for details, see Supporting Information Part 3, Section 2, pp. 13–15). Because the K_i values reported in this work span six orders of magnitude, they are presented and discussed after logarithmic scaling, as pK_i (Figures 4, 5).

Figure 4.

Structure-activity relationship (SAR) studies of bisubstrate NNMT inhibitors. pKi values (−log₁₀Ki) are shown in red and are used to facilitate comparison between inhibitors of widely varying potency. (a) Exploration of the alkynyl bisubstrate approach to NNMT inhibition. (b) Amino acid modifications.

Figure 5.

SAR studies of bisubstrate inhibitors. pKi values (−log₁₀K_i) are shown in red and are used to facilitate comparison between inhibitors of widely varying potency. (a) Modification of the benzamide moiety of NS1. (b) Previously published amino-bisubstrate NNMT inhibitors were synthesized and tested alongside NS1 and analogues.

Note that the inhibition constants, K_i, reported in this study are true dissociation constants of the enzyme—inhibitor complex, as opposed to apparent inhibition constants, K_i^app, and therefore are entirely independent of the nature of the particular substrate (quinoline vs. NAM).⁴⁴ This interpretation is based on the assumption, supported by the X-Ray structural studies reported herein, that bisubstrate inhibitors in principle bind to the target enzyme in such a way that the binding of a bisubstrate inhibitor precludes any simultaneous binding of either of the two co-substrates.

X-Ray Co-Crystal Structure of NS1 Bound to hNNMT.

We determined the co-crystal structure of NS1-bound NNMT (Figure 3, PDB ID 6ORR, Table S2) and observed an NS1-shaped density in the NAM/SAM binding pocket in the electron density map (Figure 3a). Fitting NS1 into the density decreased the R_free value and improved the map quality, revealing that NS1 occupies both the NAM and SAM binding sites in the same orientation as the native substrates, with the alkynyl linker occupying the methyl transfer tunnel. The NS1 alkyne is an optimal surrogate for the SAM-dependent methyl transfer reaction of NNMT and effectively positions both structural components of NS1 properly in space, allowing NS1 to capture similar binding interactions as the native substrates (Figure 3b).

Figure 3.

Co-crystal structure of NS1-bound NNMT (PDB ID 6ORR). (a) A 2F_o-F_c omit map contoured at 1σ reveals NS1-shaped electron density in the binding pocket of NNMT. (b) NS1 (black) binds NNMT in an orientation that closely resembles previously published co-crystal structure containing ligands SAH and NAM (cyan; PDB ID 3ROD). (c–e) NS1 also exploits the same protein interactions that NNMT uses to bind its native substrates. Ligand-interacting residues from NNMT are shown in yellow; black dotted lines represent hydrogen bonds. (f) MS2756-bound NNMT (brown; PDB ID 6B1A) superimposed on NS1-bound NNMT.

NS1 exploits the same amino acids that contact the native substrates to bind NNMT. To stabilize the adenosine portion of NS1, D142 forms a hydrogen bond with the primary amine on the adenine nucleobase, while the hydroxyl groups of the ribose hydrogen-bond extensively with the backbone nitrogen atom of S87 and the side chains of D85 and N90 (Figure 3c). The carboxylic acid of the NS1 amino acid accepts four hydrogen bonds from Y20, Y25, Y69, and T163, while the amino group hydrogen-bonds with the backbone carbonyl of G63 (Figure 3d). A hydrophobic clamp positions the aromatic ring of the benzamide moiety between Y204 and L164, with S201 and S213 stabilizing the amide group through hydrogen bonds (Figure 3e).

Stucture-Activity Relationships (SAR).

The synthesis of NS1 was designed to be highly modular to facilitate a systematic structure-activity analysis of our bisubstrate approach (Figures 4,5). SAR analysis began with the minimal motif desthia-SAH (11) and built up to the full NS1 structure incrementally (Figure 4a). Desthia-SAH, completely lacking an alkynyl-benzamide side chain, was a poor NNMT inhibitor. Addition of an acetylene (12) or a phenyl-acetylene (13) side chain yielded equally poor NNMT inhibitors. The potency of the parent compound NS1 (10) highlights the importance of the benzamide functionality in driving compound potency.

A fundamental aspect of our design strategy relied on the C6’ stereocenter to properly direct the NS1 alkyne toward the NAM binding pocket. Analysis of the NNMT crystal structure (3ROD) suggested that the S stereochemistry would best capture the transition state geometry modeled in Figure 1. Accordingly, NS1 was a better inhibitor than the NS1-C6’ epimer (14) by > 200-fold in terms of the inhibition constant K_i. The linear vector maintained by the alkynyl linker of NS1 proved optimal; NS1-alkane (15) was almost 10-fold less potent than NS1 itself (in terms of K_i). The NS1-alkane 6’ epimer (16) and alkynyl epimer 14 had similar pK_i values (6.97 vs. 6.91, respectively). These results may be explained by the shape of the NNMT binding pocket; the narrow width of the methyl transfer tunnel may accommodate linear alkynyl geometry better than a staggered aliphatic chain. Furthermore, the rigidity of the alkynyl linker could lower the entropic cost of binding relative to alkane containing analogues.

More drastic structural modifications to the NS1 scaffold included excision of the entire amino-acid side chain (17) or the entire adenine nucleobase (19). Compound 17, a simplified bisubstrate inhibitor designed to target the adenosine and NAM binding pockets of NNMT, was a weaker inhibitor than NS1 by ~ 500-fold in terms of K_i. Attempts to improve the inhibitory activity of 17 by conformational restriction (via the installation of a cyclopropyl group; 18) proved detrimental. Removal of the adenine nucleobase (19) completely abrogated NNMT inhibition.

SAR: Amino Acid Modification.

Alteration of the NS1 amino acid (Figure 4b) revealed the C17’ amino group of NS1 to be critical for inhibition, as compound 20 (lacking the C17’ amine) was a poor NNMT inhibitor. Removal of the carboxylic acid was better tolerated, as compound 21 (which retains the C17’ amine) had a pK_i of 7.24 ± 0.02. Amide- (22) and methyl ester-containing (23) analogues were poor NNMT inhibitors (5.98 ± 0.01 and 6.36 ± 0.08, respectively), while amino-amide- (24) or urea-containing (25) analogues were slightly better (7.77 ± 0.02 and 7.12 ± 0.05, respectively). These results are consistent with the structural features of 24 and 25, as both contain the same number of potential-hydrogen bonding groups as NS1 and native substrate SAH (although differing slightly in their donor/acceptor capacities and positions). Compound 22 has one fewer hydrogen-bonding atom than NS1. In 23, the methyl ester likely adds too much bulk to fit in the amino acid pocket while also reducing the hydrogen bonding ability of the amino acid carboxylate.

Only one side chain modification yielded a more potent compound than NS1: the addition of a single methylene unit in the NS1 backbone (26, homo-NS1). Homo-NS1 had a pK_i of 9.62 ± 0.05 (compared to NS1 with pK_i = 9.30 ± 0.07), indicating a ~ 2-fold increase in inhibitory activity in terms of K_i. This finding is consistent with prior reports showing that the addition of a methylene unit in amino-bisubstrate NNMT inhibitors can improve potency.²⁰ We also synthesized and tested previously reported compounds MS2734 and MS2756 (Figure 5b). In our hands, MS2734—a one-carbon chain-extended homologue of MS2756—was a 10-fold better inhibitor than MS2756 in terms of K_i (pK_i of 7.05 ± 0.01 vs. 6.04 ± 0.06). The benefit of an extra methylene group in bisubstrate inhibitors could arise because an added methylene better mimics the longer C—S bonds of the native substrates. The trimethylene linker of homo-NS1 would also be more flexible than the dimethylene linker of NS1, which may allow the molecule more room to optimally satisfy hydrogen bonds between the amino acid, benzamide, ribose and adenine moieties to the NNMT binding pocket.

SAR: Aryl Modification.

After examining the impact of side chain modifications on inhibitor potency, we tested the effects of aryl group modification. We purposely designed the NS1 synthesis to accommodate a late-stage Sonogashira cross-coupling reaction to allow for easy introduction of a desired aryl moiety late in our synthesis. Leveraging this capability, we prepared gram quantities of intermediate 8 (Scheme 1) and synthesized a variety of aryl substituted NS1 analogues (Figure 5a).

These studies revealed that proper positioning of an amide group on NS1 is essential for NNMT binding, as para-benzamide (27), ortho-benzamide (28), and sulfonamide (29) substituted NS1 analogues were much less potent than NS1. We designed fluorine (30), methyl (31), trifluoromethyl (32), and chlorine (33) containing analogues to 1) bind a small hydrophobic pocket in the nicotinamide binding site, 2) modulate the pK_a of the NS1 benzamide group through inductive effects, and 3) potentially alter the rotation of the NS1 amide relative to the NS1 benzamide phenyl ring. While 30, 31, and 32 were potent NNMT inhibitors (pK_i > 8.0), only chloro-substitution (33) yielded a more potent NNMT inhibitor than NS1 (pK_i = 9.72 ± 0.15). As shown in Figure 6a, there is a small hydrophobic region in the NAM binding pocket para to the alkyne of the NS1 scaffold. The small, hydrophobic chlorine atom of 33 likely makes Van der Waals contacts with this region, providing additional binding affinity.

Figure 6.

The chemical properties and geometry of NS1 closely match the hydrophobic profile and shape of the NNMT binding pocket. (a) The binding pocket is buried in the hydrophobic core of the protein (gray). (Inset) A solvent-accessible surface representation of the binding pocket colored by the Eisenberg hydrophobicity scale⁴⁶ reveals a hydrophobic patch (dark red) that contacts the C6’ carbon atom of NS1. (b) A side-on view shows that the binding pocket has shape complementarity specific to the S diastereomer of NS1 at the C6’ carbon.

After interrogating ring substitution at the para-position, we attempted to restrict rotation of the NS1 amide by converting the benzamide moiety to a benzolactam (Figure 5a, 34 and 35). Benzolactams 34 and 35 were moderate NNMT inhibitors, with a six-membered ring (34) better tolerated than a five-membered ring (35). We also examined C13’/C14’ substitution with methylenedioxy analogue 36 (pK_i = 7.49 ± 0.05) or introduced a heterocyclic nitrogen atom (to better mimic NAM, 37, 38, 39, 40). Compounds 36–40 all had pK_i values below that of NS1, demonstrating that C13’/C14’ substitution or inclusion of a nitrogen atom are not beneficial in these cases. Lastly, we designed and synthesized amino naphthalene derivative 41 (a bisubstrate analogue of the previously reported NNMT inhibitor 5-amino-1-methylquinolinium⁴⁵), but 41 was also a poor inhibitor (pK_i = 5.43 ± 0.07).

NS1-Alkane (15) vs. Amino-Bisubstrate Inhibitor MS2756.

In the final stage of our SAR analysis we compared our inhibitors with previously reported amino bisubstrate NNMT inhibitors (Figure 5b). Previously reported compounds VH45¹⁹ , MS2756²⁰, and MS2734²⁰ use a tertiary amine core and an alkyl tether to link a SAM-like fragment to a NAM-like fragment. An alignment of NS1-bound NNMT with MS2756-bound NNMT²⁰ shows the similar binding modes of the two compounds (Figure 3f). We synthesized and tested all three of these compounds alongside NS1-alkane (15, Figure 4a) using the same assay protocols and kinetic analyses to determine K_i values. We compared MS2756 and NS1-alkane directly because they are close structural analogues in which the tertiary amine core of MS2756 has been replaced with an sp³ hybridized carbon stereocenter. These studies revealed that NS1-alkane was approximately 200-fold more potent than MS2756 in terms of K_i.

The contrast between the observed activity of MS2756 and NS1-alkane is noteworthy; changing a single non-hydrogen atom (tertiary nitrogen to sp³ carbon stereocenter) substantially alters compound potency. We hypothesize that a variety of factors could be responsible for this observation. It is evident that the stereochemical definition at the C6’ stereocenter of NS1-alkane is beneficial as evidenced by the ~ 20-fold difference in K_i between NS1-alkane and its C6’ epimer (16). A much larger difference (~ 250-fold) in K_i between NS1 and its C6’ epimer (14) was also observed, demonstrating that as linker rigidity increases, C6’ stereodefinition becomes more important.

While NS1-alkane and NS1 both show large differences in K_i values between their C6’ stereoisomers, the tertiary nitrogen atom in MS2756 maintains sp³ tetrahedral geometry but is free to invert rapidly at room temperature. In solution, we imagine that NS1-alkane and NS1 assume the optimal tetrahedral geometry at C6’ necessary for binding, whereas MS2756 is free to occupy conformations that are unproductive for binding because of nitrogen inversion. It is also likely that the hydrophobic, carbon-based scaffolds of NS1-alkane and NS1 are preferred over the polar, tertiary amine core of MS2756. NS1-alkane and NS1 likely form favorable Van der Waals contacts with the NNMT active site, as the closest residue to the NS1-alkane/NS1 C6’ carbon stereocenter is F15 (~ 4 Å away). This phenylalanine residue presents a large hydrophobic⁴⁶ surface in the NNMT ligand binding pocket (Figure 6a), near the NS1-alkane/NS1 C6’ stereocenter.

Finally, the transition state structure of PNMT (phenylethanolamine N-methyltransferase⁴⁷), the second closest homologue of NNMT, involves even distribution of positive charge along the entire axis of methyl transfer.⁴⁸ If NNMT has a transition state structure similar to that of PNMT, analogues like MS2756, which localize positive charge on a single nitrogen atom, do not accurately capture the transient electrostatics of the methyl transfer transition state. While not positively charged, carbon-based linkers avoid the localized positive charge of MS2756 and other amino-bisubstrate inhibitors.

Selectivity Evaluation.

While several previously reported NNMT inhibitors were described as selective for NNMT, none of them have been tested against the closest homologues²⁵ of NNMT or other small-molecule methyltransferases^49–50. We aimed to critically evaluate the selectivity profile of NS1 by testing it against the methyltransferases most like NNMT (Table 1). Sequence similarity analysis and generation of a sequence similarity network^51–52 (Figure S2, Table S3) showed that NNMT, INMT (indolethylamine N-methyltransferase⁵³), and PNMT cluster tightly, with INMT and PNMT having 53% and 39% sequence identity to NNMT, respectively. A complementary approach based on structural homology analysis via the DALI⁵⁴ server demonstrated that INMT and PNMT are also the closest structural homologues of NNMT (Figure S3, S4, Table S4).²⁵ In addition to the selectivity of NS1 amongst small-molecule methyltransferases, we tested NS1 against a few representative DNA, histone, and protein-arginine methyltransferases (Table 1, Table S5). These studies revealed thiopurine S-methyltransferase (TPMT) to be the closest off-target of NS1, with an IC₅₀ of ~ 1 μM. This result might be explained by the flexibility of the TPMT active site⁵⁵ and the ability of TPMT to bind a variety of small molecule substrates and inhibitors⁵⁶. INMT was the second closest off-target of NS1, with an IC₅₀ of 3.4 μM (Figure S5).

Table 1.

Methyltransferase Selectivity Profile of NS1

small-molecule methyltransferase	inhibition at 10 μM (%)	inhibition at 1 μM (%)
TPMTa	76	51
INMTb,*	53	14
COMTc	43	0
PNMTd	26	0
GAMTe	26	0
GNMTf	23	4
HNMTg	2	6
protein or DNA methyltransferase	IC50 (μM)
hDNMT3ah	7.6
PRMT1i	>10
ASH1Lj	>10
DOT1Lk	>10
EHMT1l	>10
G9am	>10
SETDB1n	>10

^athiopurine S-methyltransferase.

^bindolethylamine N-methyltransferase;

^*values interpolated from the full IC₅₀ curve shown in Figure S5.

^ccatechol O-methyltransferase.

^dphenylethanolamine N-methyltransferase.

^eguanidinoacetate N-methyltransferase.

^fglycine N-methyltransferase.

^ghistamine N-methyltransferase.

^hDNA (cytosine-5)-methyltransferase 3A.

ⁱprotein arginine N-methyltransferase 1.

^jhistone-lysine N-methyltransferase ASH1L.

^khistone-lysine N-methyltransferase, H3 lysine-79 specific.

^lhistone-lysine N-methyltransferase EHMT1.

^mhistone-lysine N-methyltransferase EHMT2.

ⁿhistone-lysine N-methyltransferase SETDB1.

While in this case NS1 proved selective for NNMT, the introduction of different aryl groups on the NS1 scaffold could likely be used to target other methyltransferases selectively. In this work, cross coupling of compound 8 (Scheme 1) with 3-iodobenzamide generated NS1; cross coupling of 8 with indole or thiopurine fragments could provide inhibitors of INMT and TPMT, respectively. In cases where off-target binding or inhibition is observed, aryl groups could be modified until a desired selectivity profile is achieved.

Thermal Stabilization and Cell-Based Evaluation.

Along with biochemical profiling of NS1 and analogues, we found that NS1 binds and stabilizes NNMT in lysate of K562 cells (a chronic myelogenous leukemia line known to express NNMT¹⁷). The cellular thermal shift assay⁵⁷ revealed that NS1 and 25 shift the melting curve of NNMT by 8 and 5 °C, respectively (Figures S6, S8) and in a dose-dependent manner (Figures S7, S9). We also evaluated several compounds for cytotoxicity via the CellTiter-Glo luminescent cell viability assay in U2OS cells (a human bone osteosarcoma line known to express NNMT¹⁵). None of the compounds tested (NS1, 21, 23, 24, 25) were toxic to U2OS cells after 48 h at a 100 μM dose (Table S7).

We tested the ability of NS1, 21, 23, 24, and 25 to decrease MNAM levels in U2OS cells, but the effects were modest (Table S8). NS1-methyl ester (23), designed to be more cell permeable than NS1, performed best in this assay, decreasing MNAM levels by 30% at a 31.6 μM dose. A-B permeability testing (Caco-2, pH 6.5/7.4) showed that NS1 and analogues have limited permeability (most permeable out of those tested was NS1-amine 21, 1.3 × 10⁻⁶ cm/s, Table S9). Ongoing work is aimed at developing more cell-penetrant alkynyl NNMT inhibitors that would be useful probes in cell-based studies.

CONLUSIONS

In this work, we designed and synthesized the most potent and selective NNMT inhibitors reported to date. A modular, scalable synthesis of alkynyl nucleosides enabled us to make and test a variety of alkynyl bisubstrate inhibitors, leading to the discovery of NS1 as a selective, picomolar NNMT inhibitor. Alkynyl bisubstrate inhibition could be a generalizable strategy; covalent tethering of alkynyl nucleosides to substrate mimics of other small molecule methyltransferases could provide a method for selective inhibition of these enzymes. The stereodefined alkyne present in the NS1 scaffold could be used for click chemistry or other functionalization, enabling the development of bisubstrate probes or inhibitors of protein arginine/lysine methyltransferases.^58–59

Finally, this work demonstrates the value of alkynes in small-molecule ligand design in the context of methyltransferase inhibition. Alkynes provide unique rigidity, geometry, and spatial occupancy not captured by common fragment linkers. Current state-of-the-art bisubstrate inhibitor designs often rely upon reductive amination, alkylation, and amide-bond forming reactions to link fragments together. These reactions are generally fast and easy to implement, but often produce highly flexible structures characterized by relatively weak overall binding affinity. This report demonstrates that C6’ alkynyl nucleosides—while more synthetically complex—can be used to generate potent bisubstrate methyltransferase inhibitors.

EXPERIMENTAL SECTION

General Chemistry Procedures

All reactions were performed under an atmosphere of nitrogen in flame- or oven-dried glassware unless otherwise noted. Reactions were monitored by thin layer chromatography (TLC) using Merck silica gel 60 covered glass backed plates (0.25 mm, F₂₅₄). TLC plates were visualized under UV light and/or exposure to an acidic solution of p-anisaldehyde, an aqueous solution of ceric ammonium molybdate (CAM), or an aqueous solution of potassium permanganate, followed by heating. Reactions were also monitored by analytical LCMS using an Agilent 6120 Quadrupole MS and an Agilent 1260 Infinity II LC system equipped with (1) a Phenomenex Kinetex F5 column (2.6 μm, 100 Å, 100 × 3.0 mm), (2) a Phenomenex Kinetex Biphenyl column (2.6 μm, 100 Å, 100 × 3.0 mm) or (3) a Thermo Scientific Accucore aQ C18 column (2.6 μm, 100 × 2.1 mm). For analytical LCMS, solvent A consisted of 0.1% (v/v) FA/water and solvent B consisted of 0.1% (v/v) FA/CH₃CN. Two methods were used: method A for columns 1, 2, and 3: 5% B for 1 min, 5% → 95% B over 8 min, 95% B for 2 min, and reequilibration for 2 min; flow rate: 0.55 mL/min (1), 0.95 mL/min (2), 0.70 mL/min (3), column temperature: 50 °C; UV₂₅₄, and method B for columns 2 and 3: 0% B for 1 min, 0% → 40% B over 8 min, 40% → 95% B over 2 min, 95% B for 2 min, and reequilibration for 2 min; flow rate: 0.95 mL/min (2), 0.70 mL/min (3), column temperature: 50 °C; UV₂₅₄. Column 2 (method B) was used for the final analysis of very polar compounds, including NS1 (10) and analogues. Flash column chromatography was performed on an Interchim PuriFlash 215 system with prepacked silica gel cartridges (Büchi FlashPure (35 to 45 μm), Büchi Reveleris HP (16 to 24 μm), Teledyne Isco RediSep Rf (35 to 70 μm) and Teledyne Isco RediSep Rf Gold (20 to 40 μm). Preparative HPLC was performed on an Agilent 1260 Infinity II Preparative LC system equipped with a Kromasil C18 column (10 μm, 100 Å, 21.2 × 250 mm) using solvents A (0.1% (v/v) FA/water) and B (0.1% (v/v) FA/CH₃CN) or A’ (0.1% (v/v) TFA/water) and B’ (0.1% (v/v) TFA/CH₃CN) at flow rate 10 mL/min delivering gradients specified in individual experimental protocols. All compounds reported in this manuscript and Supporting Information have a purity of ≥ 95% as determined by analytical LCMS and NMR (¹H and ¹³C) unless otherwise noted.

Instrumentation

¹H NMR spectra were recorded on Varian INOVA-600 or Varian INOVA-500 spectrometers at ambient temperature. Chemical shifts are reported in ppm (δ scale) relative to residual undeuterated solvent (CDCl₃: 7.26 (CHCl₃), CD₃OD: 3.31 (CD₂HOD), CD₃CN: 1.94 (CD₂HCN), D₂O: 4.79 (HOD)). Data are reported as follows: chemical shift (δ ppm) (integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, app = apparent, or a combination of these), coupling constant(s) J (Hz)). When a mixture of deuterated solvents is employed, the residual undeuterated solvent used for reference is indicated in bold. ¹³C NMR spectra were recorded on Varian INOVA-500, Varian MERCURY-400 and JEOL J400 spectrometers at ambient temperature. Data are reported as follows: chemical shifts (δ scale, ppm) relative to the carbon resonances of the solvent (CDCl₃: 77.16, CD₃OD: 49.00, CD₃CN: 1.32). When a mixture of deuterated solvents is employed, the solvent used for reference is indicated in bold. ¹⁹F NMR spectra were recorded on Varian INOVA-500 and Varian MERCURY-400 spectrometers at ambient temperature. Data are reported as follows: chemical shifts (δ scale, ppm) relative to CFCl₃ and referenced to benzotrifluoride (BTF), hexafluorobenzene (HFB), or trifluoroacetic acid (TFA) as internal standards (BTF IStd: δ −63.7, HFB IStd: δ −164.9, TFA IStd: δ −76.6). ³¹P NMR spectra were recorded on a Varian MERCURY-400 spectrometer at ambient temperature. Data are reported as follows: chemical shifts (δ scale, ppm), calibrated to an external standard of triphenyl phosphate in CDCl₃ (0.0485 M, δ −17.6 relative to H₃PO₄ at δ 0). Infrared (FTIR) spectra were recorded on a Bruker Alpha FT-IR spectrophotometer. Data is reported in frequency of absorption (cm⁻¹). The IR spectrum of each compound (solid or liquid) was acquired directly neat or on a thin layer at ambient temperature. High resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF-Q II or an Agilent 6220 LC-TOF equipped with an electrospray ionization source (ESI+).

Materials

Commercial reagents and solvents were used as received unless otherwise noted. All solvents were of commercially available anhydrous grade (DriSolv® or equivalent) unless otherwise noted. The molarity of n-butyllithium solutions was determined by titration using N-benzylbenzamide⁶⁰ or N-pivaloyl-o-toluidine⁶¹ with similar results (average of three determinations). [Rh(OAc)(C₂H₄)₂]₂ was prepared according to a known procedure⁶² using [RhCl(C₂H₄)₂]₂ (CAS: 12081-16-2) obtained from Strem Chemicals, Inc. (catalog #: 45–0270). The following catalysts were all obtained from Strem Chemicals, Inc.: nitro-Grela catalyst ([1,3-bis(2,4,6-trimethylphenylimidazolidin-2-ylidene)]-(2-i-propoxy-5-nitrobenzylidene)ruthenium(II) dichloride), CAS: 502964-52-5, catalog #: 44–0758; (S,S)-MeBPERh catalyst⁶³ ((−)-1,2-bis((2S,5S)-2,5-dimethylphospholano)ethane(1,5-cyclooctadiene)rhodium(I) tetrafluoroborate), CAS: 213343-65-8, catalog #: 45–0169; (S)-DTBMSegPhos ((S)-(+)-5,5’-bis[di(3,5-di-t-butyl-4-methoxyphenyl)phosphino]-4,4’-bi-1,3-benzodioxole), CAS: 210169-40-7, catalog #: 15–0067; [Rh(nbd)₂]BF₄, CAS: 36620-11-8, catalog #: 45–0230; (S,S)-MeDUPHOS ((+)-1,2-Bis((2S,5S)-2,5-dimethylphospholano)benzene), CAS: 136735-95-0, catalog #: 15–0092. (±)-Z-α-phosphonoglycine trimethyl ester, CAS: 88568-95-0, was obtained from Chem-Impex International, (Catalog #: 14125).

Synthesis of NS1 (10)

(3aR,4R,6R,6aR)-4-methoxy-2,2-dimethyl-6-(prop-2-yn-1-yl)tetrahydrofuro[3,4-d][1,3]dioxole (2).

The following is an adaption of a published procedure.⁶⁴ To a stirred solution of 2,6-lutidine (9.85 mL, 84.6 mmol, 1.1 equiv.) in CH₂Cl₂ (130 mL) at −78 °C was added trifluoromethanesulfonic anhydride (13.7 mL, 81.5 mmol, 1.06 equiv.) over 5 min (flask A, 1 L rbf), followed by the addition of a solution of alcohol 1 (15.7 g, 76.9 mmol) in CH₂Cl₂ (33 mL) over 10 min. The mixture was stirred at −78 °C for 2 h and warmed to 0 °C. Meanwhile, to a separate flask (flask B, 500 mL rbf) containing a solution of TMS-acetylene (32.0 mL, 231 mmol, 3.00 equiv.) in dry THF (130 mL) at −78 °C was added n-butyllithium (2.56 M in hexanes, 93.7 mL, 240 mmol, 3.12 equiv.) at a rate of 5 mL/min via syringe. The resulting mixture was stirred at −78 °C for 1.5 h and warmed to 0 °C. While the contents of flask B remained at 0 °C, flask A was removed from the ice/water bath, volatiles were removed in vacuo (bath temperature at 20 °C) and a brief subjection to high vacuum yielded a red-orange paste. The crude triflate in flask A was resuspended in dry THF (65 mL) and DMPU (35 mL) was added. The contents were stirred vigorously for 10 min until complete solubilization was noted and cooled to −78 °C. The contents of flask B were cooled to −78 °C and transferred to flask A via cannula. The resulting mixture was stirred at −78 °C for 1 h and at −12 °C for 3 h (NaCl/ice bath), before the slow addition of TBAF (caution, gas evolution!, 1.0 M in THF, 250 mL, 250 mmol, 3.25 equiv.) via cannula. The mixture was allowed to warm to RT and was stirred for another 30 min before volatiles were removed in vacuo. The reddish-brown residue was resuspended in MTBE (350 mL) and a sat. aq. NH₄Cl solution (300 mL) was added. The layers were roughly separated and the organic phase, largely emulsified, was filtered through a pad of CaCO₃/celite to give two clear layers in the filtrate. The layers were separated and the combined aqueous phases were extracted with MTBE (2 × 200 mL) and EtOAc (200 mL). The combined organic extracts were washed with a 5% aq. LiCl solution (800 mL) and then with brine (800 mL), dried over MgSO₄, filtered, and concentrated. The crude residue was purified by dry column vacuum chromatography⁶⁵ (cyclohexane/EtOAc, 0 → 30%) to yield pure 2 as well as semi-pure 2 which was repurified by silica gel chromatography (hexanes/EtOAc, 0 → 30%). Compound 2 (11.1 g, 52.3 mmol, 68%) was obtained as a light-yellow oil. R_f = 0.60 (hexanes/EtOAc, 67:33); FTIR (neat), ν_max(cm⁻¹): 3286, 2989, 2938, 2835, 1438, 1209, 1090, 1053, 1041; ¹H NMR (600 MHz, CDCl₃): δ 4.95 (s, 1H), 4.69 (d, J = 5.9 Hz, 1H), 4.60 (d, J = 5.9 Hz, 1H), 4.30 (dd, J = 9.3, 6.6 Hz, 1H), 3.32 (s, 3H), 2.52 (ddd, J = 16.7, 6.6, 2.7 Hz, 1H), 2.42 (ddd, J = 16.6, 9.4, 2.7 Hz, 1H), 2.04 (t, J = 2.7 Hz, 1H), 1.46 (s, 3H), 1.31 (s, 3H); ¹³C NMR (151 MHz, CDCl₃): δ 112.3, 109.6, 85.2, 85.1, 83.3, 80.2, 70.4, 54.7, 26.4, 24.9, 24.6; HRMS (ESI+): [C₁₁H₁₆O₄Na]⁺ calc. 235.0941, meas. 235.0938, Δ 1.0 ppm.

(3aR,4R,6R,6aR)-4-allyl-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole (3).

Alkyne 2 (10.6 g, 49.9 mmol) was reduced according to protocols adapted from Cox et al.⁶⁶ Crude material was purified by silica gel chromatography (hexanes/EtOAc, 0 ➔ 30%) to afford alkene 3 (9.97 g, 46.5 mmol, 93%) as a clear, viscous oil. The flash column chromatography removed ca. 98% of PHMS, yielding material that was suitable for use in subsequent reactions. An analytically pure sample was prepared by subjecting 2.0 g of the post-column material to short path distillation (Kugelrohr) at 130 °C and 200 mTorr. R_f = 0.50 (hexanes/EtOAc, 80:20); FTIR (neat), ν_max (cm⁻¹): 3078, 2987, 2938, 2833, 1643, 1381, 1372, 1209, 1193, 1105, 1090, 1059, 1031; ¹H NMR (600 MHz, CDCl₃): δ 5.82 (dddd, J = 16.7, 10.3, 7.4, 6.2 Hz, 1H), 5.15–5.09 (m, 2H), 4.96 (s, 1H), 4.61 (d, J = 5.9 Hz, 1H), 4.57 (dd, J = 6.0, 1.0 Hz, 1H), 4.23 (td, J = 7.8, 1.0 Hz, 1H), 3.34 (s, 3H), 2.46–2.38 (m, 1H), 2.32–2.24 (m, 1H), 1.48 (s, 3H), 1.31 (s, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 134.5, 117.7, 112.4, 109.6, 86.6, 85.7, 83.6, 55.0, 39.6, 26.6, 25.2; HRMS (ESI+): [C₁₁H₁₈O₄Na]⁺ calc. 237.1095, meas. 237.1097, Δ 1.0 ppm.

(E)-4-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)but-2-enal (4).

To a solution of alkene 3 (2.0 g, 9.3 mmol) in MTBE (degassed prior to addition by sparging with nitrogen for 15 min, 28 mL) at RT, were added E-crotonaldehyde (3.9 mL, 47 mmol, 5.0 equiv.) and nitro-Grela metathesis catalyst (63 mg, 93 μmol, 1 mol %) sequentially. The flask headspace was purged with nitrogen for 10 min and the mixture was stirred at 40 °C for 48 h, at which point TLC analysis indicated ca. 50% conversion. More nitro-Grela catalyst (63 mg, 93 μmol, 1 mol %) and E-crotonaldehyde (3.9 mL, 47 mmol, 5.0 equiv.) were added and the reaction mixture was stirred at 40 °C for another 24 h, at which point complete conversion was indicated by TLC analysis. Volatiles were removed in vacuo and the crude material was purified by silica gel chromatography (hexanes/EtOAc, 2 → 50%) to afford enal 4 (1.60 g, 6.60 mmol, 71%) as a yellow oil. R_f = 0.41 (hexanes/EtOAc, 50:50); FTIR (neat), ν_max (cm⁻¹): 2990, 2936, 2832, 2735, 1688, 1641, 1453, 1382, 1377, 1298, 1269, 1263, 1242, 1210, 1194, 1161, 1140, 1104, 1089, 1058, 1026; ¹H NMR (600 MHz, CDCl₃): δ 9.54 (d, J = 7.8 Hz, 1H), 6.84 (dt, J = 15.7, 6.8 Hz, 1H), 6.21 (ddt, J =15.7, 7.8, 1.5 Hz, 1H), 4.98 (s, 1H), 4.64 (d, J = 5.9 Hz, 1H), 4.56 (dd, J = 6.0, 1.1 Hz, 1H), 4.35 (ddd, J= 8.8, 6.4, 1.0 Hz, 1H), 3.34 (s, 3H), 2.68 (dddd, J = 15.4, 8.5, 6.6, 1.6 Hz, 1H), 2.58 (dtd, J = 15.2, 6.7,1.5 Hz, 1H), 1.48 (s, 3H), 1.32 (s, 3H); ¹³C NMR (151 MHz, CDCl₃): δ 193.6, 153.5, 134.8, 112.6, 109.8, 85.4, 85.3, 83.7, 54.7, 38.4, 26.5, 25.0; HRMS (ESI+): calcd. for [C₁₂H₁₈O₅Na]⁺ 265.1046, meas. 265.1062, Δ 6.0 ppm.

(R)-3-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-5-(triisopropylsilyl)pent-4-ynal (5).

Aldehyde 5 was prepared using protocols adapted from Nishimura et al.³¹ A 250 mL flask was charged with [Rh(OAc)(C₂H₄)₂]₂ catalyst (126 mg, 0.289 mmol, 2.5 mol %), (S)-DTBM-SegPhos (818 mg, 0.693 mmol, 6.0 mol%), and a large stir bar. The flask headspace was evacuated and backfilled with nitrogen (3 ×) and methanol was added (22 mL). The resulting orange suspension was stirred vigorously at RT until a red-orange solution was obtained (30 min). At this time, a solution of enal 4 (2.80 g, 11.6 mmol) in MeOH (10 mL) and TIPS-acetylene (5.19 mL, 23.1 mmol, 2.0 equiv.) were added sequentially to the reaction mixture. The resulting red mixture was stirred at 40 °C for 15 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 0 → 50%) to afford aldehyde 5 (4.60 g, 10.8 mmol, 94%) as a single diastereomer, as a light yellow oil. R_f = 0.42 (cyclohexane/EtOAc, 75:25); FTIR (neat), ν_max (cm⁻¹): 2941, 2865, 2722, 2167, 1728, 1463, 1381, 1371, 1271, 1241, 1210, 1193, 1160, 1093, 1058, 1018; ¹H NMR (600 MHz, CDCl₃): δ 9.82 (t, J = 2.1 Hz, 1H), 4.95 (s, 1H), 4.61 (dd, J = 5.9, 1.2 Hz, 1H), 4.58 (d, J = 5.9 Hz, 1H), 4.49 (ddd, J = 10.6, 4.2, 1.2 Hz, 1H), 3.35 (s, 3H), 3.16 (dtd, J = 11.2, 6.9, 3.9 Hz, 1H), 2.60 (ddd, J = 16.5, 7.5, 2.2 Hz, 1H), 2.55 (ddd, J = 16.5, 6.4, 2.1 Hz, 1H), 1.74 (ddd, J = 13.3, 10.6, 4.0 Hz, 1H), 1.64 (ddd, J = 13.2, 11.1, 4.2 Hz, 1H), 1.47 (s, 3H), 1.31 (s, 3H), 1.11–0.97 (m, 21H); ¹³C NMR (151 MHz, CDCl₃): δ 200.7, 112.6, 109.9, 108.6, 85.5, 85.3, 84.5, 84.2, 55.4, 49.0, 40.7, 26.7, 25.4, 25.1, 18.7, 11.3; HRMS (ESI+): calcd. for [C₂₃H₄₀O₅Si₁Na]⁺ 447.2537, meas. 447.2527, Δ 2.3 ppm.

Methyl (S,Z)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-7-(triisopropylsilyl)hept-2-en-6-ynoate (6).

A solution of methyl 2-(dimethoxyphosphoryl)-2-(2,2,2-trifluoroacetamido)acetate (S2, 3.11 g, 10.6 mmol, 1.5 equiv.) in THF (21 mL) was added dropwise over 20 min to a stirred suspension of NaH (95%, 254 mg, 10.6 mmol, 1.5 equiv.) in THF (10 mL) at 0 °C. The resulting cloudy mixture was stirred at 0 °C for 30 min and a solution of aldehyde 5 (3.00 g, 7.06 mmol) in THF (7.1 mL) was added over 5 min. The resulting mixture was allowed to warm gradually to RT and was stirred for 2 h, at which point the reaction media was heated to 40 °C and stirred for another 6 h. The mixture was allowed to cool to RT and volatiles were removed in vacuo. The residue was partitioned between MTBE (25 mL) and 1× PBS (15 mL). The mixture was stirred vigorously for 10 min and the layers were separated. The aqueous phase was extracted with MTBE (3 × 25 mL). The combined organic extracts were washed with brine (80 mL), dried over MgSO₄, filtered, and concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 0 → 30%) to yield enamide 6 (3.74 g, 6.32 mmol, 89%) as a 6:1 mixture of Z and E isomers as a light yellow oil. *Note: Z and E isomers are not separated for the following step, as they both react to form the desired product. R_f = 0.48 (hexanes/EtOAc, 67:33); FTIR (neat), ν_max (cm⁻¹): 3297, 2943, 2866, 1720, 1664, 1528, 1463, 1439, 1382, 1373, 1335, 1283, 1210, 1160, 1106, 1092, 1062, 1043, 1016; ¹H NMR (600 MHz, CDCl₃): δ (Z-isomer) 7.73 (s, 1H), 7.07 (t, J = 7.2 Hz, 1H), 4.94 (s, 1H), 4.61 (dd, J = 6.0, 1.1 Hz, 1H), 4.58 (d, J = 5.9 Hz, 1H), 4.44 (ddd, J = 10.4, 4.4, 1.1 Hz, 1H), 3.81 (s, 3H), 3.32 (s, 3H), 2.87 (ddd, J = 8.3, 5.5, 3.7 Hz, 1H), 2.40 (ddd, J = 15.8, 7.2, 5.1 Hz, 1H), 2.26 (ddd, J = 15.8, 8.7, 7.2 Hz, 1H), 1.71 (ddd, J = 13.3, 10.4, 4.2 Hz, 1H), 1.65 (ddd, J = 13.4, 10.8, 4.5 Hz, 1H), 1.46 (s, 3H), 1.30 (s, 3H), 1.06–1.02 (m, 21H); ¹³C NMR (126 MHz, CDCl₃): δ (Z-isomer) 163.3, 155.1 (q, ²J_C-F = 38 Hz), 138.3, 133.3, 123.8, 115.6 (q, ¹J_C-F = 288 Hz), 112.0, 109.4, 109.1, 85.2, 84.2, 83.7, 54.5, 52.2, 40.6, 34.4, 29.2, 26.2, 24.8, 18.3, 11.0; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ (Z-isomer) −76.4; HRMS (ESI+): calcd. for [C₂₈H₄₄F₃N₁O₇Si₁K]⁺ 630.2471, meas. 630.2462, Δ 1.4 ppm.

Methyl (2S,5S)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-7-(triisopropylsilyl)hept-6-ynoate (7).

To a flask charged with olefin 6 (300 mg, 0.507 mmol), as a 6:1 mixture of Z and E isomers, was added MeOH (20 mL) at RT under a nitrogen atmosphere. The contents were stirred until full dissolution was noted. Separately, (S,S)-MeBPE-Rh (28 mg, 0.051 mmol, 10 mol %) was weighed out into a vial in a glove box. The vial was removed from the glove box, placed under a nitrogen atmosphere, and MeOH (10 mL) was added. The methanolic solution of (S,S)-MeBPE-Rh was transferred via syringe to the methanolic solution of olefin 6. The flask headspace was purged with hydrogen (balloon), the outlet needle was removed, and the balloon was refilled with hydrogen. The contents were stirred vigorously at RT under a hydrogen atmosphere (balloon) for 4 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 0 → 50%) to afford protected amino acid 7 (276 mg, 0.465 mmol, 92%) as a single diastereomer, as a clear viscous oil. R_f = 0.45 (hexanes/EtOAc, 67:33); FTIR (neat), ν_max (cm⁻¹): 3321, 2942, 2865, 2165, 1750, 1719, 1549, 1462, 1440, 1382, 1372, 1208, 1160, 1095, 1060, 1017; ¹H NMR (600 MHz, CDCl₃): δ 6.84 (d, J = 7.8 Hz, 1H), 4.93 (s, 1H), 4.66 (td, J = 7.7, 4.6 Hz, 1H), 4.61 (dd, J = 5.9, 1.2 Hz, 1H), 4.57 (d, J = 5.9 Hz, 1H), 4.43 (ddd, J = 10.1, 4.8, 1.2 Hz, 1H), 3.79 (s, 3H), 3.30 (s, 3H), 2.70–2.62 (m, 1H), 2.14 (dddd, J = 14.0, 10.8, 5.8, 4.6 Hz, 1H), 2.08–1.99 (m, 1H), 1.68–1.57 (m, 2H), 1.54 (dddd, J = 13.1, 10.7, 5.9, 4.5 Hz, 1H), 1.46 (s, 3H), 1.36 (dddd, J = 13.1, 11.2, 9.7, 4.4 Hz, 1H), (s, 3H), 1.08–1.03 (m, 21H); ¹³C NMR (126 MHz, CDCl₃): δ 171.4, 156.9 (q, ²J_C-F = 38 Hz), 115.7 (q, ¹J_C-F = 288 Hz), 112.5, 109.8, 109.5, 85.6, 85.5, 84.5, 83.7, 55.1, 53.0, 52.3, 41.1, 31.1, 30.1, 29.8, 26.7, 25.4, 18.7, 11.3; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.8; HRMS (ESI+): calcd. for [C₂₈H₄₆F₃N₁O₇Si₁Na]⁺ 616.2888, meas. 616.2890, Δ 0.4 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-ethynyl-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (8).

To a suspension of N⁶-benzoyladenine (742 mg, 3.10 mmol, 2.15 equiv.) in propionitrile (dried over 4 Å molecular sieves, 10 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (0.97 mL, 4.0 mmol, 2.75 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which complete solubilization of N⁶-benzoyladenine was observed. To a separate flask charged with triacetate S4 (dried by azeotropic distillation with benzene (4 ×), 735 mg, 1.44 mmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (51 mg, 0.14 mmol, 10 mol %) was added propionitrile (10 mL). The resulting mixture was stirred at RT for 5 min until a clear solution was obtained and the solution of silylated N⁶-benzoyladenine was added. The reaction mixture was stirred at 95 °C for 3 h, cooled to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (PhMe/MeCN, 0 → 50%) to afford nucleoside 8 (663 mg, 0.963 mmol, 67%) as a white amorphous solid. R_f = 0.48 (PhMe/MeCN, 50:50); FTIR (thin-film), ν_max (cm⁻¹): 3273, 3086, 2928, 1748, 1720, 1611, 1582, 1511, 1487, 1456, 1374, 1329, 1243, 1217, 1182, 1160, 1099, 1074, 1047, 1029; ¹H NMR (500 MHz, CDCl₃): δ 9.09 (s, 1H), 8.77 (s, 1H), 8.09 (s, 1H), 8.03–7.99 (m, 2H), 7.62–7.57 (m, 1H), 7.53–7.48 (m, 2H), 7.06 (d, J = 7.9 Hz, 1H), 6.12 (d, J = 5.4 Hz, 1H), 6.07 (t, J = 5.4 Hz, 1H), 5.55 (dd, J = 5.5, 4.5 Hz, 1H), 4.61 (td, J = 7.8, 5.0 Hz, 1H), 4.48 (ddd, J = 10.9, 4.5, 2.9 Hz, 1H), 3.77 (s, 3H), 2.59 (ddtd, J = 11.4, 9.1, 4.4, 2.5 Hz, 1H), 2.16 (d, J = 2.4 Hz, 1H), 2.15 (s, 3H), 2.11–2.00 (m, 2H), 2.06 (s, 3H), 1.95 (dddd, J = 13.9, 10.9, 7.7, 4.6 Hz, 1H), 1.86 (ddd, J = 13.8, 11.2, 2.9 Hz, 1H), 1.61–1.54 (m, 1H), 1.45 (dddd, J = 13.6, 10.9, 9.3, 4.6 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.2, 169.8, 169.6, 165.2, 157.0 (q, ²J_C-F = 37.6 Hz), 152.6, 151.6, 149.9, 142.4, 133.5, 132.8, 128.8, 128.1, 123.9, 115.7 (q, ¹J_C-F = 288 Hz), 87.0, 84.7, 80.7, 73.5, 72.9, 71.7, 53.0, 52.3, 38.2, 30.8, 29.6, 27.9, 20.6, 20.4; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.7; HRMS (ESI+): calcd. for [C₃₁H₃₁F₃N₆O₉Na]⁺ 711.1988, meas. 711.1997, Δ 1.2 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((3-carbamoylphenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (9).

To a vial charged with alkyne 8 (79 mg, 0.11 mmol) were added 3-iodobenzamide (57 mg, 0.23 mmol, 2.0 equiv.), CuI (4 mg, 0.02 mmol, 20 mol %), and Pd(PPh₃)₄ (7 mg, 6 μmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 3.5 mL) was added at RT. The resulting mixture was stirred at 70 °C for 3 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (PhMe/MeCN, 0 → 100%) to afford protected NS1 9 (83 mg, 0.10 mmol, 90%) as a white amorphous solid. R_f = 0.17 (EtOAc/i-PrOH, 50:50); FTIR (neat), ν_max (cm⁻¹): 3255, 3064, 2929, 1744, 1717, 1666, 1610, 1578, 1511, 1486, 1455, 1374, 1329, 1240, 1212, 1180, 1158, 1096, 1073, 1046, 1029; ¹H NMR (600 MHz, CD₂Cl₂): δ 9.03 (br s, 1H), 8.75 (s, 1H), 8.14 (s, 1H), 8.00 (t, J = 1.3 Hz, 1H), 7.99 (d, J = 1.5 Hz, 1H), 7.87 (td, J = 1.8, 0.6 Hz, 1H), 7.77 (ddd, J = 7.8, 1.9, 1.1 Hz, 1H), 7.64 (ddt, J = 8.0, 7.0, 1.3 Hz, 1H), 7.57–7.54 (m, 2H), 7.53 (dd, J = 1.6, 1.0 Hz, 1H), 7.40 (td, J = 7.8, 0.6 Hz, 1H), 7.15 (d, J = 7.7 Hz, 1H), 6.53 (br s, 1H), 6.18 (d, J = 5.8 Hz, 1H), 6.15 (t, J = 5.5 Hz, 1H), 5.70 (br s, 1H), 5.65 (dd, J = 5.3, 4.0 Hz, 1H), 4.65 (td, J = 7.5, 5.5 Hz, 1H), 4.54 (dt, J = 10.1, 3.9 Hz, 1H), 3.77 (s, 3H), 2.83 (ddt, J = 11.0, 9.0, 4.4 Hz, 1H), 2.19 (ddd, J = 13.8, 10.1, 4.0 Hz, 1H), 2.15 (s, 3H), 2.11 (dt, J = 10.5, 5.1 Hz, 1H), 2.11–1.99 (m, 2H), 2.04 (s, 3H), 1.68 (ddt, J = 13.0, 10.7, 5.6 Hz, 1H), 1.57 (dddd, J = 13.5, 10.8, 9.3, 4.8 Hz, 1H); ¹³C NMR (101 MHz, CD₂Cl₂): δ 171.6, 170.4, 170.1, 168.8, 165.4, 157.4 (q, ²J_C-F = 37 Hz), 152.8, 152.1, 150.3, 142.9, 134.8, 134.1, 134.0, 133.1, 131.1, 129.1, 128.9, 128.3, 127.4, 124.6, 123.9, 116.1 (q, ¹J_C-F = 287 Hz), 91.9, 87.3, 83.1, 81.5, 74.1, 73.0, 53.3, 53.0, 38.8, 31.4, 29.8, 29.1, 20.8, 20.6; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.7; HRMS (ESI+): calcd. for [C₃₈H₃₇F₃N₇O₁₀]⁺ 808.2549, meas. 808.2538, Δ 1.3 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-carbamoylphenyl)hept-6-ynoic acid (NS1, 10).

To a solution of alkyne 9 (158 mg, 196 μmol) in THF (2.0 mL) at RT, was added a 0.5 M aq. LiOH solution (2.0 mL). The resulting mixture was stirred for 2.5 h, cooled to 0 °C, and a 10% aq. AcOH solution was added dropwise until pH 7 was reached. Volatiles were removed in vacuo and the residue was dissolved in a solution of ammonia in methanol (7 N, 8 mL) at RT. The resulting mixture was stirred for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (gradient elution: 5% → 55% B over 45 min) to afford NS1 10 (107 mg, 172 μmol, 88% over 2 steps) as the TFA salt. Single crystals suitable for X-ray diffraction studies were grown from a 4:1:1 volumetric mixture of 10 mM aq. NS1-TFA:n-BuOH:i-PrOH via slow evaporation at 4 °C over two weeks. Full small molecule X-ray crystallographic data for NS1 (10) are reported in the Supporting Information. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.37 (s, 1H), 8.34 (s, 1H), 7.78 (td, J = 1.8, 0.6 Hz, 1H), 7.72 (ddd, J = 7.8, 1.9, 1.2 Hz, 1H), 7.52 (dt, J = 7.8, 1.4 Hz, 1H), 7.40 (td, J = 7.8, 0.6 Hz, 1H), 6.01 (d, J = 5.1 Hz, 1H), 4.74 (t, J = 5.1 Hz, 1H), 4.38 (ddd, J = 10.3, 4.4, 3.2 Hz, 1H), 4.23 (t, J = 4.8 Hz, 1H), 4.02 (dd, J = 6.6, 5.8 Hz, 1H), 2.81 (ddt, J = 10.6, 9.1, 4.5 Hz, 1H), 2.17 (dddd, J = 14.4, 11.5, 6.7, 4.5 Hz, 1H), 2.10–1.97 (m, 2H), 1.95–1.89 (m, 1H), 1.81–1.72 (m, 1H), 1.61–1.52 (m, 1H); ¹³C NMR (101 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.1, 171.3, 151.0, 149.4, 145.2, 144.1, 135.8, 134.4, 131.6, 129.9, 128.1, 124.5, 120.1, 92.8, 90.0, 83.9, 83.4, 74.8, 74.5, 53.5, 38.9, 30.9, 29.6, 28.7; HRMS (ESI+): calcd. for [C₂₄H₂₈N₇O₆]⁺ 510.2096, meas. 510.2082, Δ 2.6 ppm.

Synthesis of Compounds 11–41

(S)-2-amino-6-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)hexanoic acid (11).

To a solution of nucleoside S11 (73 mg, 0.10 mmol) in a 4:2:1 mixture of MeOH, THF and water (35 mL) at RT was added CsOH•xH₂O (100 mg). The resulting mixture was stirred for 16 h and a 10% AcOH aq. solution was added dropwise until neutral pH was reached. Volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 95% B’ over 80 min) to afford the partially deprotected carbamate. To a solution of the carbamate intermediate in a 10:1 mixture of i-PrOAc and t-BuOH (25 mL) at RT, was added Pd/C (10%, 65 mg, 61 μmol). The reaction mixture was purged with hydrogen (3 ×) and stirred at RT under a hydrogen atmosphere (balloon) for 2 h. The reaction rate appeared sluggish so Pd(OH)₂/C (20%, 105 mg, 150 μmol) was added as a suspension in a 1:1 mixture of i-PrOAc and AcOH (10 mL) and the resulting mixture was stirred at 50 °C for 2 h. The reaction remained slow, so EtOH (20 mL) was added. The temperature was lowered to 40 °C and the reaction media was stirred for 3 h, upon which complete cleavage of the Cbz protecting group was observed.⁷⁰ The flask headspace was purged with nitrogen under vigorous stirring. The contents were filtered over a pad of celite and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (2.5% → 27.5% B’ over 80 min) to afford 11 (33 mg, 69 μmol, 66% over 2 steps) as the TFA salt, as a fluffy white powder. ¹H NMR (600 MHz, D₂O): δ 8.48 (s, 1H), 8.45 (s, 1H), 6.12 (d, J = 5.1 Hz, 1H), 4.82 (t, J = 5.2 Hz, 1H), 4.29 (t, J = 5.0 Hz, 1H), 4.21–4.16 (m, 1H), 4.00 (t, J = 6.2 Hz, 1H), 1.98 (app. ddt, J = 14.2, 10.9, 5.5 Hz, 1H), 1.94–1.86 (m, 1H), 1.88–1.77 (m, 2H), 1.59–1.39 (m, 4H); ¹³C NMR (101 MHz, D₂O): δ 173.2, 150.3, 148.6, 144.9, 143.0, 119.2, 88.4, 85.1, 74.1, 73.4, 53.6, 32.5, 30.0, 24.7, 24.2; HRMS (ESI+): calcd. for [C₁₅H₂₃N₆O₅]⁺ 367.1724, meas. 367.1728, Δ 1.1 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)hept-6-ynoic acid (12).

To a solution of 8 (42 mg, 61 μmol) in THF (1.0 mL) at RT, was added a 0.5 M LiOH aq. solution (1.0 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 10% AcOH aq. solution was added dropwise until pH 7 was reached. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 3 mL) at RT. The resulting mixture was stirred for 16 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (2% → 42% B over 30 min followed by 42% → 95% B over 5 min) to afford 12 (22 mg, 44 μmol, 72% over 2 steps) as the TFA salt upon treatment with d-TFA as part of sample preparation for NMR analysis. ¹H NMR (500 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.87 (s, 2H), 6.50 (d, J = 5.0 Hz, 1H), 5.19 (t, J = 5.1 Hz, 1H), 4.68 (t, J = 4.8 Hz, 1H), 4.49 (dd, J = 6.7, 5.8 Hz, 1H), 3.08 (dddd, J = 13.7, 7.2, 5.8, 4.3 Hz, 1H), 3.02 (d, J = 2.4 Hz, 1H), 2.59 (dddd, J = 14.2, 11.6, 6.8, 4.7 Hz, 1H), 2.53–2.45 (m, 1H), 2.41 (td, J = 10.0, 5.2 Hz, 1H), 2.33 (ddd, J = 14.0, 10.9, 3.4 Hz, 1H), 2.19 (tt, J = 12.3, 5.0 Hz, 1H), 1.99 (dddd, J = 13.4, 11.9, 9.1, 4.6 Hz, 1H); ¹³C NMR (101 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.0, 150.9, 149.3, 145.2, 144.0, 120.0, 89.8, 86.1, 83.7, 74.8, 74.4, 73.1, 53.4, 38.8, 30.7, 28.8, 28.4; HRMS (ESI+): calcd. for [C₁₇H₂₃N₆O₅]⁺ 391.1724, meas. 391.1722, Δ 0.7 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-phenylhept-6-ynoic acid (13).

To a solution of S12 (74 mg, 97 μmol) in THF (7.0 mL) at RT, was added a 0.5 M LiOH aq. solution (1.7 mL). The resulting mixture was stirred for 3 h, cooled to 0 °C, and a 10% AcOH aq. solution was added dropwise until pH 7 was reached. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 10 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 45 min followed by 45% → 95% B over 5 min) to afford 13 (35 mg, 60 μmol, 62% over 2 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.84 (s, 1H), 8.82 (s, 1H), 7.84–7.80 (m, 2H), 7.78–7.74 (m, 3H), 6.47 (d, J = 5.1 Hz, 1H), 5.19 (t, J = 5.1 Hz, 1H), 4.83 (ddd, J = 10.5, 4.5, 3.2 Hz, 1H), 4.68 (t, J = 4.8 Hz, 1H), 4.48 (dd, J = 6.7, 5.7 Hz, 1H), 3.27 (ddt, J = 10.7, 9.2, 4.5 Hz, 1H), 2.63 (dddd, J = 14.3, 11.5, 6.8, 4.5 Hz, 1H), 2.56–2.44 (m, 2H), 2.38 (ddd, J = 13.9, 10.8, 3.3 Hz, 1H), 2.23 (tt, J = 12.6, 5.0 Hz, 1H), 2.03 (dddd, J = 13.4, 11.9, 9.2, 4.5 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.0, 151.0, 149.4, 145.2, 144.0, 132.4, 129.5, 129.2, 123.9, 120.1, 91.7, 89.9, 84.2, 83.9, 74.8, 74.5, 53.5, 39.0, 30.9, 29.5, 28.7; HRMS (ESI+): calcd. for [C₂₃H₂₆N₆O₅Na]⁺ 489.1857, meas. 489.1841, Δ 3.2 ppm.

(2S,5R)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-carbamoylphenyl)hept-6-ynoic acid (14).

To a solution of S19 (50 mg, 62 μmol) in THF (1.2 mL) at RT, was added a 0.5 M LiOH aq. solution (1.2 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.22 mL) was added until pH 7 was reached. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 5 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 14 (38 mg, 62 μmol, quant. over 2 steps) as the TFA salt upon treatment with d-TFA (50 μL), as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (50 μL)): δ 8.37 (s, 1H), 8.31 (s, 1H), 7.70 (ddd, J = 7.7, 1.7, 1.3 Hz, 1H), 7.67 (ddd, J = 1.7, 1.5, 0.4 Hz, 1H), 7.41 (ddd, J = 7.7, 1.5, 1.3 Hz, 1H), 7.36 (app td, J = 7.7, 0.4 Hz, 1H), 6.00 (d, J = 5.2 Hz, 1H), 4.79 (app t, J = 5.2 Hz, 1H), 4.28 (ddd, J = 8.6, 5.1, 4.0 Hz, 1H), 4.24 (dd, J = 5.2, 4.0 Hz, 1H), 4.01 (app t, J = 6.4 Hz, 1H), 2.81 (app tt, J = 8.6, 5.3 Hz, 1H), 2.21 (dddd, J = 14.2, 11.5, 6.4, 5.4 Hz, 1H), 2.08 (app dt, J = 13.9, 8.6 Hz, 1H), 2.03–1.94 (m, 2H), 1.76–1.63 (m, 2H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (50 μL)): δ 172.2, 171.6, 150.9, 149.4, 145.2, 144.1, 135.7, 134.2, 131.3, 129.9, 128.1, 124.5, 120.0, 93.8, 90.0, 84.6, 82.9, 74.7, 74.7, 53.6, 38.9, 30.7, 29.8, 28.6; HRMS (ESI+): calcd. for [C₂₄H₂₈N₇O₆]⁺ 510.2096, meas. 510.2112, Δ 3.2 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-carbamoylphenyl)heptanoic acid (15).

To a solution of 9 (20 mg, 25 μmol) in MeOH (1.3 mL) at RT was added Pd/C (10%, 8 mg, 7 μmol, 30 mol %). The vial headspace was evacuated and refilled with hydrogen (3 ×) and the resulting mixture was stirred vigorously at 50 °C under a hydrogen atmosphere (balloon) for 17 h and filtered through a nylon syringe filter (13 mm, 0.22 μm). The solids were washed with MeOH (5 × 1 mL) and the filtrates were concentrated. Crude alkane was used directly in the next step without further purification. To a solution of crude alkane in THF (0.5 mL) at RT, was added a 0.5 M LiOH aq. solution (0.46 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C and a 1 M HCl aq. solution (0.11 mL) was added to reach pH 7. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford NS1-alkane (15) (16 mg, 25 μmol, quant. over 3 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.32 (s, 1H), 8.31 (s, 1H), 7.57– 7.56 (m, 1H), 7.54 (app dt, J = 7.3, 1.7 Hz, 1H), 7.32 (app dt, J = 7.7, 1.7 Hz, 1H), 7.30 (ddd, J = 7.7, 7.3, 0.5 Hz, 1H), 5.93 (d, J = 4.7 Hz, 1H), 4.64 (app t, J = 4.7 Hz, 1H), 4.13–4.10 (m, 1H), 4.10 (dd, J = 5.3, 4.7 Hz, 1H), 3.94 (app t, J = 6.1 Hz, 1H), 2.65–2.53 (m, 2H), 1.93–1.87 (m, 1H), 1.86–1.78 (m, 1H), 1.76–1.70 (m, 2H), 1.68–1.60 (m, 1H), 1.60–1.53 (m, 2H), 1.52–1.45 (m, 1H), 1.45–1.38 (m, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.5, 172.3, 151.0, 149.4, 145.3, 144.2, 144.0, 133.8, 133.4, 129.7, 128.4, 125.9, 120.0, 89.9, 83.4, 74.8, 74.7, 53.9, 37.4, 34.8, 34.3, 32.8, 29.0, 27.9; HRMS (ESI+): calcd. for [C₂₄H₃₂N₇O₆]⁺ 514.2414, meas. 514.2437, Δ 4.5 ppm.

(2S,5R)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-carbamoylphenyl)heptanoic acid (16).

To a solution of 14 (10 mg, 16 μmol) in a 9:1 mixture of EtOH and water (2.0 mL) at RT was added Pd/C (10%, 5 mg, 5 μmol, 30 mol %). The vial headspace was evacuated and refilled with nitrogen (3 ×) followed by hydrogen (3 ×), and the resulting mixture was stirred vigorously at 50 °C under a hydrogen atmosphere (balloon) for 2.5 h and filtered through a nylon syringe filter (13 mm, 0.22 μm). The solids were washed with EtOH (3 × 1 mL) and the filtrates were concentrated. Crude material was purified by preparative HPLC (5% → 45% B’ over 30 min followed by 45% → 95% B’ over 5 min) to afford 16 (9 mg, 14 μmol, 89%) as the TFA salt. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)): δ 8.26 (s, 1H), 8.24 (s, 1H), 7.56–7.52 (m, 2H), 7.29–7.25 (m, 2H), 5.92 (d, J = 4.6 Hz, 1H), 4.64 (dd, J = 5.0, 4.6 Hz, 1H), 4.09 (app t, J = 5.0 Hz, 1H), 4.09–4.05 (m, 1H), 3.80 (app t, J = 6.1 Hz, 1H), 2.59–2.52 (m, 2H), 1.90–1.83 (m, 1H), 1.80–1.70 (m, 2H), 1.67 (ddd, J = 14.3, 7.6, 3.5 Hz, 1H), 1.64–1.51 (m, 3H), 1.48–1.37 (m, 2H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)): δ 173.1, 172.3, 152.4, 149.5, 147.5, 144.2, 143.0, 133.8, 133.2, 129.6, 128.3, 125.8, 119.9, 89.5, 83.6, 74.8, 74.6, 54.6, 37.7, 35.6, 34.3, 32.7, 28.1, 27.5; HRMS (ESI+): calcd. for [C₂₄H₃₂N₇O₆]⁺ 514.2409, meas. 514.2423, Δ 2.9 ppm.

3-(4-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)but-1-yn-1-yl)benzamide (17).

To a vial charged with S26 (58 mg, 97 μmol) was added a solution of ammonia in methanol (7 N, 8.0 mL) at RT. The resulting mixture was stirred for 17 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (10% → 45% B’ over 35 min followed by 45% → 95% B’ over 5 min) to afford 17 (35 mg, 86 μmol, 88%) as a white fluffy solid. ¹H NMR (600 MHz, CD₃CN (400 μL)/D₂O (400 μL)): δ 8.36 (s, 1H), 8.31 (s, 1H), 7.70–7.69 (m, 1H), 7.71–7.67 (m, 1H), 7.46–7.43 (m, 1H), 7.38–7.35 (m, 1H), 5.98 (d, J = 5.1 Hz, 1H), 4.70 (dd, J = 5.1, 4.7 Hz, 1H), 4.22–4.18 (m, 2H), 2.53 (app dt, J = 17.3, 6.9 Hz, 1H), 2.49 (app dt, J = 17.3, 7.2 Hz, 1H), 2.03–1.96 (m, 2H); ¹³C NMR (126 MHz, CD₃CN (400 μL)/D₂O (400 μL)): δ 171.3, 150.9, 149.4, 145.3, 143.8, 135.6, 134.2, 131.2, 129.9, 127.9, 124.6, 119.9, 91.6, 89.6, 84.8, 80.9, 74.8, 74.1, 32.6, 16.3; HRMS (ESI+): calcd. for [C₂₀H₂₀N₆O₄Na]⁺ 431.1438, meas. 431.1453, Δ 3.4 ppm.

3-(((1R,2R)-2-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)cyclopropyl)ethynyl)benzamide (18).

To a vial charged with S33 (50 mg, 82 μmol) was added a solution of ammonia in methanol (7 N, 6.0 mL) at RT. The resulting mixture was stirred for 17 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (10% → 45% B’ over 30 min followed by 45% → 95% B’ over 5 min) to afford 18 (32 mg, 76 μmol, 93%) as a white fluffy solid. ¹H NMR (500 MHz, CD3CN (350 μL)/D2O (350 μL)): δ 8.39 (s, 1H), 8.32 (s, 1H), 7.71 (td, J = 1.8, 0.6 Hz, 1H), 7.67 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.44 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.35 (td, J = 7.8, 0.6 Hz, 1H), 5.95 (d, J = 4.7 Hz, 1H), 4.73 (dd, J = 5.2, 4.7 Hz, 1H), 4.36 (dd, J = 5.2, 4.8 Hz, 1H), 3.45 (dd, J = 9.1, 4.8 Hz, 1H), 1.62 (dddd, J = 9.1, 8.8, 5.9, 4.4 Hz, 1H), 1.53 (ddd, J = 8.6, 5.4, 4.4 Hz, 1H), 1.05 (ddd, J = 8.8, 5.4, 4.8 Hz, 1H), 1.02 (ddd, J = 8.6, 5.9, 4.8 Hz, 1H); ¹³C NMR (126 MHz, CD3CN (350 μL)/D2O (350 μL)): δ 171.2, 151.1, 149.3, 145.4, 143.8, 135.6, 134.3, 131.3, 129.8, 127.8, 124.5, 120.0, 93.6, 89.7, 88.5, 76.7, 74.9, 74.4, 25.2, 12.9, 6.4; HRMS (ESI+): calcd. for [C₂₁H₂₁N₆O₄]⁺ 421.1619, meas. 421.1627, Δ 1.9 ppm.

(2S,5S)-2-amino-7-(3-carbamoylphenyl)-5-(((2R,3S,4R,5R)-3,4-dihydroxy-5-methoxytetrahydrofuran-2-yl)methyl)hept-6-ynoic acid (19).

To a solution of S35 (23 mg, 44 μmol) in THF (2.0 mL) at RT was added a 0.5 M LiOH aq. solution (2.0 mL). The reaction mixture was stirred at RT for 3 h and a 10% AcOH aq. solution was added slowly until pH 5 was obtained. Volatiles were removed in vacuo and the residue was purified by preparative HPLC (5% → 55% B’ over 60 min followed by 55% → 95% B’ over 5 min) to afford 19 (11 mg, 28 μmol, 63%) as the TFA salt. ¹H NMR (600 MHz, D₂O): δ 7.84 (t, J = 1.8 Hz, 1H), 7.74 (ddd, J = 7.9, 1.9, 1.0 Hz, 1H), 7.65 (ddt, J = 7.7, 1.6, 0.8 Hz, 1H), 7.47 (tt, J = 7.9, 0.6 Hz, 1H), 4.90 (d, J = 1.4 Hz, 1H), 4.31 (ddd, J = 10.2, 6.3, 3.0 Hz, 1H), 4.13 (dd, J = 6.3, 4.7 Hz, 1H), 4.11–4.06 (m, 2H), 3.40 (s, 3H), 2.97–2.89 (m, 1H), 2.25 (dddd, J = 14.2, 11.5, 6.7, 4.6 Hz, 1H), 2.16 (ddt, J = 14.4, 11.2, 5.5 Hz, 1H), 1.93 (ddd, J = 13.8, 10.9, 3.1 Hz, 1H), 1.85–1.80 (m, 1H), 1.77 (ddd, J = 13.7, 10.6, 4.5 Hz, 1H), 1.70–1.59 (m, 1H); ¹³C NMR (101 MHz, D₂O): δ 172.6, 172.3, 135.3, 133.1, 130.5, 129.0, 127.2, 123.2, 108.2, 92.1, 82.7, 81.0, 74.6, 74.4, 55.2, 53.2, 39.5, 29.9, 29.1, 28.0; HRMS (ESI+): calcd. for [C₂₀H₂₇N₂O₇]⁺ 407.1813, meas. 407.1809, Δ 1.0 ppm.

(S)-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-carbamoylphenyl)hept-6-ynoic acid (20).

To a solution of S41 (105 mg, 0.151 mmol) in a 2:1 mixture of MeOH and THF (24 mL) at 0 °C was added a 0.2 M LiOH aq. solution (7.5 mL, 3.01 mmol, 20 equiv.). The resulting mixture was stirred at RT for 24 h and organics were removed in vacuo. A 1 M HCl aq. solution (1.7 mL) was added and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (30% → 95% B over 35 min followed by 95% B over 5 min) to afford 20 (45 mg, 91 μmol, 60%) as a fluffy white solid. ¹H NMR (600 MHz, CD₃CN (400 μL)/D₂O (400 μL)/d-TFA (100 μL)): δ 8.36 (s, 1H), 8.34 (s, 1H), 7.76 (dd, J = 1.8, 1.2 Hz, 1H), 7.70 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.49 (app dt, J = 7.8, 1.2 Hz, 1H), 7.37 (app t, J = 7.8 Hz, 1H), 6.01 (d, J = 5.3 Hz, 1H), 4.81–4.77 (dd, J = 5.3, 5.0 Hz, 1H), 4.41 (ddd, J = 10.6, 4.0, 3.1 Hz, 1H), 4.25–4.21 (dd, J = 5.0, 4.0 Hz, 1H), 2.78–2.71 (m, 1H), 2.36–2.26 (m, 2H), 2.01 (ddd, J = 13.9, 10.6, 4.1 Hz, 1H), 1.87 (ddd, J = 13.9, 11.0, 3.1 Hz, 1H), 1.83–1.75 (m, 1H), 1.73–1.64 (m, 1H), 1.61–1.49 (m, 2H); ¹³C NMR (126 MHz, CD₃CN (400 μL)/D₂O (400 μL)/d-TFA (100 μL)): δ 178.3, 171.9, 151.1, 149.5, 145.3, 144.4, 136.0, 134.2, 131.5, 130.0, 128.1, 124.9, 120.2, 94.0, 90.3, 84.5, 82.9, 74.9, 74.8, 39.2, 35.2, 34.5, 29.8, 23.5; HRMS (ESI+): calcd. for [C₂₄H₂₇N₆O₆]⁺ 495.1987, meas. 495.1984, Δ 0.4 ppm.

3-((S)-6-amino-3-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)hex-1-yn-1-yl)benzamide (21).

To a vial charged with S47 (79 mg, 0.11 mmol) was added a solution of ammonia in methanol (7 N, 15 mL) at RT. The resulting mixture was stirred for 24 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 21 (33 mg, 57 μmol, 54%) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.36 (s, 1H), 8.33 (s, 1H), 7.76 (ddd, J = 1.8, 1.5, 0.4 Hz, 1H), 7.71 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.50 (ddd, J = 7.8, 1.5, 1.2 Hz, 1H), 7.39 (app td, J = 7.8, 0.4 Hz, 1H), 6.00 (d, J = 5.0 Hz, 1H), 4.71 (dd, J = 5.2, 5.0 Hz, 1H), 4.36 (ddd, J = 10.3, 4.5, 3.4 Hz, 1H), 4.21 (dd, J = 5.2, 4.5 Hz, 1H), 2.97–2.89 (m, 2H), 2.80–2.74 (m, 1H), 1.98 (ddd, J = 14.1, 10.3, 4.3 Hz, 1H), 1.90 (ddd, J = 14.1, 10.7, 3.4 Hz, 1H), 1.89–1.82 (m, 1H), 1.78–1.70 (m, 1H), 1.61 (app ddt, J = 13.2, 10.7, 5.4 Hz, 1H), 1.54 (dddd, J = 13.2, 10.6, 9.1, 5.1 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 171.4, 150.9, 149.4, 145.2, 144.0, 135.7, 134.2, 131.4, 129.9, 128.0, 124.5, 120.0, 93.3, 89.8, 83.9, 83.1, 74.8, 74.4, 40.2, 38.9, 32.3, 29.5, 25.8; HRMS (ESI+): calcd. for [C₂₃H₂₈N₇O₄]⁺ 466.2197, meas. 466.2219, Δ 4.7 ppm.

3-((S)-7-amino-3-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-oxohept-1-yn-1-yl)benzamide (22).

To a vial charged with S41 (105 mg, 0.151 mmol) was added a solution of ammonia in methanol (7 N, 18 mL) at RT. The resulting mixture was stirred at 40 °C for 5 days and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 22 (57 mg, 0.12 mmol, 77%) as a white fluffy solid. ¹H NMR (600 MHz, CD₃CN (400 μL)/D₂O (400 μL)/d-TFA (50 μL)): δ 8.35 (s, 1H), 8.32 (s, 1H), 7.75 (app t, J = 1.5 Hz, 1H), 7.69 (app dt, J = 7.8, 1.5 Hz, 1H), 7.48 (app dt, J = 7.8, 1.5 Hz, 1H), 7.36 (app t, J = 7.8 Hz, 1H), 5.99 (d, J = 5.2 Hz, 1H), 4.75 (app t, J = 5.2 Hz, 1H), 4.38 (ddd, J = 10.6, 4.1, 3.1 Hz, 1H), 4.21 (dd, J = 5.2, 4.1 Hz, 1H), 2.73 (dddd, J = 10.9, 9.2, 5.4, 4.0 Hz, 1H), 2.23 (ddd, J = 14.6, 8.0, 7.2 Hz, 1H), 2.20 (ddd, J = 14.6, 7.8, 7.1 Hz, 1H), 1.99 (ddd, J = 13.8, 10.6, 4.1 Hz, 1H), 1.85 (ddd, J = 13.8, 11.0, 3.1 Hz, 1H), 1.82–1.73 (m, 1H), 1.71–1.62 (m, 1H), 1.59–1.47 (m, 2H); ¹³C NMR (126 MHz, CD₃CN (400 μL)/D₂O (400 μL)/d-TFA (50 μL)): δ 179.5, 171.4, 150.9, 149.4, 145.2, 144.2, 135.8, 134.2, 131.4, 129.9, 128.0, 124.7, 120.1, 93.9, 90.1, 84.3, 82.8, 74.8, 74.6, 39.1, 35.6, 35.1, 29.7, 24.2; HRMS (ESI+): calcd. for [C₂₄H₂₇N₇O₅Na]⁺ 516.1966, meas. 516.1986, Δ 3.9 ppm.

Methyl (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-carbamoylphenyl)hept-6-ynoate (23).

To a solution of S51 (32 mg, 34 μmol) in a 1:1 mixture of MeCN and CH₂Cl₂ (2.0 mL) at RT was added piperidine (0.50 mL). The resulting mixture was stirred for 20 min and volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B’ over 35 min followed by 45% → 95% B’ over 5 min) to afford 23 (12 mg, 19 μmol, 27% over 2 steps) as the TFA salt. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)): δ 8.33 (s, 1H), 8.30 (s, 1H), 7.77 (ddd, J = 1.9, 1.5, 0.5 Hz, 1H), 7.72 (ddd, J = 7.8, 1.9, 1.2 Hz, 1H), 7.51 (ddd, J = 7.8, 1.5, 1.2 Hz, 1H), 7.40 (app td, J = 7.8, 0.5 Hz, 1H), 5.98 (d, J = 5.0 Hz, 1H), 4.68 (dd, J = 5.2, 5.0 Hz, 1H), 4.34 (ddd, J = 10.0, 4.7, 3.4 Hz, 1H), 4.19 (dd, J = 5.2, 4.7 Hz, 1H), 4.04 (dd, J = 6.6, 6.1 Hz, 1H), 3.73 (s, 3H), 2.82–2.75 (m, 1H), 2.13 (dddd, J = 14.3, 11.6, 6.6, 4.6 Hz, 1H), 2.06–1.95 (m, 2H), 1.91 (ddd, J = 13.9, 10.5, 3.4 Hz, 1H), 1.71 (app ddt, J = 12.8, 11.6, 5.1 Hz, 1H), 1.52 (dddd, J = 12.8, 11.7, 9.7, 4.6 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)): δ 171.2, 170.9, 151.5, 149.4, 146.2, 143.6, 135.7, 134.3, 131.4, 129.9, 128.1, 124.3, 119.9, 92.7, 89.7, 83.6, 83.4, 74.7, 74.3, 54.3, 53.5, 38.7, 30.7, 29.5, 28.7; HRMS (ESI+): calcd. for [C₂₅H₃₀N₇O₆]⁺ 524.2252, meas. 524.2271, Δ 3.6 ppm.

3-((3S,6S)-6,7-diamino-3-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-oxohept-1-yn-1-yl)benzamide (24).

To a vial charged with 9 (44 mg, 54 μmol) was added a solution of ammonia in methanol (7 N, 15 mL) at RT. The resulting mixture was stirred at 40 °C for 36 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 24 (31 mg, 50 μmol, 91%) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (50 μL)): δ 8.36 (s, 1H), 8.33 (s, 1H), 7.78–7.76 (m, 1H), 7.71 (ddd, J = 7.8, 1.9, 1.2 Hz, 1H), 7.51 (ddd, J = 7.8, 1.5, 1.2 Hz, 1H), 7.39 (app td, J = 7.8, 0.6 Hz, 1H), 6.00 (d, J = 5.1 Hz, 1H), 4.73 (app t, J = 5.1 Hz, 1H), 4.37 (ddd, J = 10.3, 4.5, 3.4 Hz, 1H), 4.22 (dd, J = 5.1, 4.5 Hz, 1H), 3.96 (app t, J = 6.4 Hz, 1H), 2.79 (dddd, J = 10.6, 9.2, 4.5, 4.3 Hz, 1H), 2.13 (dddd, J = 14.2, 12.4, 6.4, 4.4 Hz, 1H), 2.00 (ddd, J = 14.3, 10.3, 4.3 Hz, 1H), 1.98–1.89 (m, 2H), 1.67 (dddd, J = 13.1, 12.4, 4.8, 4.5 Hz, 1H), 1.56 (dddd, J = 13.1, 12.7, 9.2, 4.4 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (50 μL)): δ 172.4, 171.6, 151.0, 149.4, 145.2, 144.1, 135.9, 134.2, 131.6, 130.0, 128.1, 124.5, 120.1, 92.9, 90.0, 84.0, 83.4, 74.9, 74.5, 53.8, 38.8, 30.7, 29.8, 29.7; HRMS (ESI+): calcd. for [C₂₄H₂₈N₈O₅Na]⁺ 531.2075, meas. 531.2089, Δ 2.7 ppm.

3-((S)-3-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-5-ureidopent-1-yn-1-yl)benzamide (25).

To a vial charged with S58 (31 mg, 45 μmol) was added a solution of ammonia in methanol (7 N, 5.0 mL) at RT. The resulting mixture was stirred for 17 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 35 min followed by 45% → 95% B over 5 min) to afford 25 (18 mg, 36 μmol, 80%) as a fluffy white solid. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.54 (s, 1H), 8.51 (s, 1H), 7.96 (t, J = 1.7 Hz, 1H), 7.90 (ddd, J = 7.8, 1.9, 1.1 Hz, 1H), 7.70 (dt, J = 7.7, 1.3 Hz, 1H), 7.58 (dd, J = 7.8, 0.6 Hz, 1H), 6.18 (d, J = 5.2 Hz, 1H), 4.93 (t, J = 5.2 Hz, 1H), 4.56 (dt, J = 10.6, 3.5 Hz, 1H), 4.40 (t, J = 4.7 Hz, 1H), 3.50 (ddd, J = 13.3, 7.7, 5.4 Hz, 1H), 3.43 (dt, J = 13.7, 7.5 Hz, 1H), 2.98 (tt, J = 9.7, 4.5 Hz, 1H), 2.19 (ddd, J = 14.4, 10.5, 4.2 Hz, 1H), 2.08 (ddd, J = 13.9, 10.9, 3.2 Hz, 1H), 2.00–1.91 (m, 1H), 1.86 (dddd, J = 13.2, 9.7, 7.4, 5.4 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 171.6, 161.7, 151.0, 149.5, 145.2, 144.3, 135.9, 134.3, 131.5, 130.0, 128.2, 124.6, 120.1, 93.0, 90.1, 84.1, 83.2, 74.8, 74.6, 39.5, 39.0, 35.3, 27.6; HRMS (ESI+): calcd. for [C₂₃H₂₆N₈O₅Na]⁺ 517.1918, meas. 517.1912, Δ 1.2 ppm.

(2S,6S)-2-amino-6-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-8-(3-carbamoylphenyl)oct-7-ynoic acid (26).

To a solution of S66 (69 mg, 84 μmol) in THF (1.5 mL) at RT was added a 0.5 M LiOH aq. solution (1.5 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.20 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 25% B’ over 30 min followed by 25% → 95% B’ over 5 min) to afford 26 (9 mg, 14 μmol, 17% over 2 steps) as the TFA salt. ¹H NMR (600 MHz, D₂O (700 μL)): δ 8.42 (s, 1H), 8.30 (s, 1H), 7.70–7.65 (m, 1H), 7.51–7.48 (m, 1H), 7.37–7.33 (m, 2H), 6.08 (d, J = 5.0 Hz, 1H), 4.95 (dd, J = 5.1, 5.0 Hz, 1H), 4.47 (ddd, J = 9.2, 4.6, 3.2 Hz, 1H), 4.40 (dd, J = 5.1, 4.6 Hz, 1H), 3.95 (app t, J = 6.1 Hz, 1H), 2.89–2.84 (m, 1H), 2.08 (ddd, J = 14.4, 9.2, 3.7 Hz, 1H), 2.00 (ddd, J = 14.4, 9.8, 3.2 Hz, 1H), 1.96–1.91 (m, 2H), 1.72–1.60 (m, 3H), 1.56–1.49 (m, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)): δ 172.5, 171.3, 151.2, 149.4, 145.7, 143.8, 135.7, 134.2, 131.4, 129.9, 127.9, 124.5, 120.0, 93.7, 89.7, 83.9, 82.9, 74.7, 74.4, 53.7, 38.9, 34.9, 30.5, 29.5, 23.2; HRMS (ESI+): calcd. for [C₂₅H₃₀N₇O₆]⁺ 524.2252, meas. 524.2277, Δ 4.7 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(4-carbamoylphenyl)hept-6-ynoic acid (27).

To a solution of S67 (18 mg, 22 μmol) in THF (0.41 mL) at RT was added a 0.5 M LiOH aq. solution (0.41 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.10 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B’ over 30 min followed by 45% → 95% B’ over 5 min) to afford 27 (7 mg, 11 μmol, 50% over 2 steps) as the TFA salt. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)): δ 8.32 (s, 1H), 8.30 (s, 1H), 7.73–7.70 (m, 2H), 7.43–7.39 (m, 2H), 5.97 (d, J = 5.0 Hz, 1H), 4.71 (dd, J = 5.3, 5.0 Hz, 1H), 4.33 (ddd, J = 10.3, 4.5, 3.3 Hz, 1H), 4.19 (dd, J = 5.3, 4.5 Hz, 1H), 3.90 (dd, J = 6.8, 5.7 Hz, 1H), 2.83–2.76 (m, 1H), 2.10 (dddd, J = 14.2, 11.5, 6.8, 4.6 Hz, 1H), 2.03–1.96 (m, 2H), 1.94–1.88 (m, 1H), 1.73 (dddd, J = 12.8, 12.0, 5.4, 4.6 Hz, 1H), 1.55 (m, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)): δ 172.6, 171.4, 151.7, 149.4, 146.5, 143.6, 133.2, 132.5, 128.6, 127.7, 120.0, 94.7, 89.7, 83.7, 83.4, 74.7, 74.4, 53.9, 38.6, 30.9, 29.6, 28.8; HRMS (ESI+): calcd. for [C₂₄H₂₈N₇O₆]⁺ 510.2096, meas. 510.2097, Δ 0.2 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(2-carbamoylphenyl)hept-6-ynoic acid (28).

To a solution of alkyne S68 (18 mg, 22 μmol) in THF (0.41 mL) at RT was added a 0.5 M LiOH aq. solution (0.41 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.10 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B’ over 30 min followed by 45% → 95% B’ over 5 min) to afford 28 (8 mg, 13 μmol, 58% over 2 steps) as the TFA salt. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)): δ 8.32 (s, 1H), 8.29 (s, 1H), 7.56–7.52 (m, 1H), 7.44–7.41 (m, 1H), 7.40 (app td, J = 7.6, 1.5 Hz, 1H), 7.37 (ddd, J = 7.6, 7.1, 2.0 Hz, 1H), 5.97 (d, J = 5.1 Hz, 1H), 4.71 (app t, J = 5.1 Hz, 1H), 4.34 (ddd, J = 10.1, 4.5, 3.4 Hz, 1H), 4.20–4.18 (m, 1H), 3.88 (dd, J = 6.7, 5.9 Hz, 1H), 2.84–2.76 (m, 1H), 2.09 (dddd, J = 14.2, 11.7, 6.7, 4.6 Hz, 1H), 2.01–1.95 (m, 2H), 1.95–1.89 (m, 1H), 1.72 (app ddt, J = 13.4, 11.7, 4.9 Hz, 1H), 1.60–1.52 (m, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)): δ 172.8, 151.7, 149.4, 146.5, 143.6, 137.3, 134.1, 131.7, 129.4, 128.8, 121.3, 120.0, 97.1, 89.7, 83.7, 81.8, 74.6, 74.4, 54.1, 38.5, 30.9, 29.7, 28.9; HRMS (ESI+): calcd. for [C₂₄H₂₈N₇O₆]⁺ 510.2096, meas. 510.2095, Δ 0.2 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-sulfamoylphenyl)hept-6-ynoic acid (29).

To a solution of S69 (41 mg, 49 μmol) in THF (1.3 mL) at RT was added a 0.5 M LiOH aq. solution (1.1 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.20 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 5 mL) at RT. The resulting mixture was stirred for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 29 (26 mg, 39 μmol, 81% over 2 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.36 (s, 1H), 8.34 (s, 1H), 7.82 (dd, J = 1.9, 1.5 Hz, 1H), 7.77 (ddd, J = 7.8, 1.9, 1.2 Hz, 1H), 7.57 (ddd, J = 7.8, 1.5, 1.2 Hz, 1H), 7.48 (app t, J = 7.8 Hz, 1H), 6.00 (d, J = 5.1 Hz, 1H), 4.73 (dd, J = 5.2, 5.1 Hz, 1H), 4.36 (ddd, J = 10.3, 4.5, 3.3 Hz, 1H), 4.22 (dd, J = 5.2, 4.5 Hz, 1H), 4.01 (dd, J = 6.7, 5.8 Hz, 1H), 2.86–2.78 (m, 1H), 2.16 (dddd, J = 14.4, 11.7, 6.7, 4.5 Hz, 1H), 2.10–2.02 (m, 1H), 2.01 (ddd, J = 14.3, 10.3, 4.1 Hz, 1H), 1.96–1.90 (m, 1H), 1.77 (app ddt, J = 13.0, 11.7, 5.1 Hz, 1H), 1.58 (dddd, J = 13.0, 11.7, 9.6, 4.5 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.1, 151.0, 149.4, 145.3, 144.1, 143.7, 136.4, 130.6, 129.6, 126.4, 125.2, 120.1, 93.8, 90.1, 83.9, 82.8, 74.9, 74.5, 53.6, 38.8, 30.9, 29.6, 28.7; HRMS (ESI+): calcd. for [C₂₃H₂₈N₇O₇S₁]⁺ 546.1765, meas. 546.1764, Δ 0.3 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-carbamoyl-4-fluorophenyl)hept-6-ynoic acid (30).

To a solution of S71 (44 mg, 53 μmol) in THF (1 mL) at RT, was added a 0.5 M LiOH aq. solution (1 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.20 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 5 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 30 (17 mg, 26 μmol, 50% over 2 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.36 (s, 1H), 8.34 (s, 1H), 7.76 (dd, J = 7.1, 2.3 Hz, 1H), 7.51 (ddd, J = 8.6, 4.8, 2.3 Hz, 1H), 7.15 (dd, J = 11.1, 8.6 Hz, 1H), 6.00 (d, J = 5.1 Hz, 1H), 4.72 (app t, J = 5.1 Hz, 1H), 4.35 (ddd, J = 10.3, 4.5, 3.3 Hz, 1H), 4.21 (dd, J = 5.1, 4.5 Hz, 1H), 4.01 (dd, J = 6.7, 5.8 Hz, 1H), 2.82–2.76 (m, 1H), 2.15 (dddd, J = 14.4, 11.7, 6.7, 4.5 Hz, 1H), 2.07–2.02 (m, 1H), 1.99 (ddd, J = 13.9, 10.3, 4.5 Hz, 1H), 1.91 (ddd, J = 13.9, 10.8, 3.3 Hz, 1H), 1.75 (app ddt, J = 12.9, 11.7, 5.0 Hz, 1H), 1.56 (dddd, J = 12.9, 11.9, 9.5, 4.5 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.2, 167.5 (d, J_C-F = 2.0 Hz), 160.7 (d, J_C-F = 253 Hz), 151.0, 149.4, 145.3, 144.1, 137.7 (d, J_C-F = 9.5 Hz), 134.8 (d, J_C-F = 2.7 Hz), 122.4 (d, J_C-F = 14 Hz), 120.9 (d, J_C-F = 3.7 Hz), 120.1, 117.9 (d, J_C-F = 25 Hz), 92.5, 90.0, 84.0, 82.4, 74.9, 74.5, 53.6, 38.9, 30.9, 29.6, 28.8; ¹⁹F NMR (471 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL), HFB IStd): δ −113.6; HRMS (ESI+): calcd. for [C₂₄H₂₆F₁N₇O₆Na]⁺ 550.1821, meas. 550.1821, Δ 0.0 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-carbamoyl-4-methylphenyl)hept-6-ynoic acid (31).

To a solution of S73 (26 mg, 32 μmol) in THF (1.5 mL) at RT was added a 0.5 M LiOH aq. solution (0.9 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.17 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 5 mL) at RT. The resulting mixture was stirred for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 31 (11 mg, 17 μmol, 55% over 2 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.36 (s, 1H), 8.33 (s, 1H), 7.37 (br d, J = 1.8 Hz, 1H), 7.29 (dd, J = 7.9, 1.8 Hz, 1H), 7.17 (br d, J = 7.9 Hz, 1H), 5.99 (d, J = 5.1 Hz, 1H), 4.73 (app t, J = 5.1 Hz, 1H), 4.35 (ddd, J = 10.3, 4.5, 3.2 Hz, 1H), 4.21 (dd, J = 5.1, 4.5 Hz, 1H), 4.00 (dd, J = 6.7, 5.8 Hz, 1H), 2.82–2.75 (m, 1H), 2.32 (s, 3H), 2.14 (dddd, J = 14.3, 11.7, 6.7, 4.5 Hz, 1H), 2.07–2.00 (m, 1H), 1.99 (ddd, J = 13.9, 10.3, 4.3 Hz, 1H), 1.90 (ddd, J = 13.9, 10.7, 3.2 Hz, 1H), 1.78–1.70 (app ddt, J = 13.0, 11.7, 5.1 Hz, 1H), 1.55 (dddd, J = 13.0, 11.8, 9.3, 4.5 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 174.5, 172.2, 151.0, 149.4, 145.2, 144.1, 136.9, 136.5, 133.9, 132.1, 130.9, 121.4, 120.1, 92.1, 90.0, 83.9, 83.5, 74.8, 74.5, 53.6, 38.9, 30.9, 29.6, 28.8, 19.8; HRMS (ESI+): calcd. for [C₂₅H₂₉N₇O₆Na]⁺ 546.2072, meas. 546.2074, Δ 0.5 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-carbamoyl-4-(trifluoromethyl)phenyl)hept-6-ynoic acid (32).

To a solution of S75 (51 mg, 58 μmol) in THF (5.0 mL) at RT was added a 0.5 M LiOH aq. solution (1.5 mL). The resulting mixture was stirred for 3 h, cooled to 0 °C, and a 10% AcOH aq. solution was added dropwise until pH 7 was reached. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 8 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 35% B over 30 min followed by 35% → 95% B over 5 min) to afford 32 (22 mg, 32 μmol, 55% over 2 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.82 (s, 1H), 8.80 (s, 1H), 8.12 (d, J = 8.2 Hz, 1H), 8.01 (ddt, J = 8.2, 1.8, 1.0 Hz, 1H), 8.00–7.97 (m, 1H), 6.45 (d, J = 5.0 Hz, 1H), 5.16 (t, J = 5.1 Hz, 1H), 4.82–4.79 (m, 1H), 4.65 (t, J = 4.8 Hz, 1H), 4.46 (dd, J = 6.7, 5.8 Hz, 1H), 3.32–3.25 (m, 1H), 2.60 (dddd, J = 14.3, 11.5, 6.7, 4.5 Hz, 1H), 2.54–2.43 (m, 2H), 2.41–2.35 (m, 1H), 2.27–2.18 (m, 1H), 2.03 (dddd, J = 13.4, 11.8, 9.3, 4.5 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.0, 171.9, 151.0, 149.4, 145.2, 144.0, 136.0, 133.8, 132.0, 128.3, 127.8, 127.8, 127.7, 126.6, 126.3, 125.5, 123.4, 120.0, 117.9, 115.6, 113.3, 95.7, 89.9, 83.8, 82.3, 74.8, 74.4, 53.5, 38.7, 30.7, 29.6, 28.7; HRMS (ESI+): calcd. for [C₂₅H₂₆F₃N₇O₆Na]⁺ 600.1789, meas. 600.1776, Δ 2.2 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-carbamoyl-4-chlorophenyl)hept-6-ynoic acid (33).

To a solution of S77 (45 mg, 53 μmol) in THF (1 mL) at RT was added a 0.5 M LiOH aq. solution (1 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.20 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 5 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 33 (35 mg, 53 μmol, quant. over 2 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.36 (s, 1H), 8.34 (s, 1H), 7.48–7.47 (m, 1H), 7.38–7.36 (m, 2H), 5.99 (d, J = 5.1 Hz, 1H), 4.72 (app t, J = 5.1 Hz, 1H), 4.34 (ddd, J = 10.4, 4.5, 3.2 Hz, 1H), 4.20 (dd, J = 5.1, 4.5 Hz, 1H), 4.00 (dd, J = 6.7, 5.8 Hz, 1H), 2.79 (app ddt, J = 10.7, 9.2, 4.5 Hz, 1H), 2.14 (dddd, J = 14.3, 11.6, 6.7, 4.5 Hz, 1H), 2.07–2.01 (m, 1H), 2.00 (ddd, J = 14.1, 10.4, 4.5 Hz, 1H), 1.91 (ddd, J = 14.1, 10.7, 3.2 Hz, 1H), 1.75 (dddd, J = 13.1, 11.6, 5.0, 4.5 Hz, 1H), 1.55 (dddd, J = 13.1, 11.8, 9.2, 4.5 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.1, 171.1, 151.0, 149.4, 145.2, 144.1, 136.0, 135.1, 132.7, 131.3, 131.0, 123.1, 120.1, 93.9, 90.0, 83.9, 82.4, 74.8, 74.5, 53.5, 38.8, 30.8, 29.6, 28.7; HRMS (ESI+): calcd. for [C₂₄H₂₆Cl₁N₇O₆Na]⁺ 566.1525, meas. 566.1528, Δ 0.4 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(1-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)hept-6-ynoic acid (34).

To a solution of S78 (29 mg, 35 μmol) in THF (0.65 mL) at RT was added a 0.5 M LiOH aq. solution (0.65 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.14 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 5 mL) at RT. The resulting mixture was stirred for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 34 (23 mg, 35 μmol, quant. over 2 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.36 (s, 1H), 8.33 (s, 1H), 7.08 (br d, J = 7.7 Hz, 1H), 6.96 (dd, J = 7.7, 1.6 Hz, 1H), 6.82 (d, J = 1.6 Hz, 1H), 5.99 (d, J = 5.1 Hz, 1H), 4.73 (app t, J = 5.1 Hz, 1H), 4.35 (ddd, J = 10.4, 4.5, 3.2 Hz, 1H), 4.21 (dd, J = 5.1, 4.5 Hz, 1H), 4.00 (dd, J = 6.7, 5.6 Hz, 1H), 2.89–2.85 (m, 2H), 2.78 (dddd, J = 10.8, 9.3, 4.7, 4.3 Hz, 1H), 2.53–2.48 (m, 2H), 2.13 (dddd, J = 14.3, 11.8, 6.7, 4.6 Hz, 1H), 2.03 (dddd, J = 14.3, 11.7, 5.6, 5.2 Hz, 1H), 1.99 (ddd, J = 14.0, 10.4, 4.3 Hz, 1H), 1.89 (ddd, J = 14.0, 10.8, 3.2 Hz, 1H), 1.74 (dddd, J = 13.0, 11.8, 5.2, 4.7 Hz, 1H), 1.54 (dddd, J = 13.0, 11.7, 9.3, 4.6 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 174.6, 172.2, 151.0, 149.4, 145.2, 144.1, 138.1, 129.2, 127.5, 125.6, 122.8, 120.1, 119.1, 91.7, 90.0, 83.9, 83.8, 74.8, 74.5, 53.5, 38.9, 30.9, 30.7, 29.5, 28.7, 25.4; HRMS (ESI+): calcd. for [C₂₆H₃₀N₇O₆]⁺ 536.2252, meas. 536.2268, Δ 3.0 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(3-oxoisoindolin-5-yl)hept-6-ynoic acid (35).

To a solution of 8 (107 mg, 0.155 mmol) and 6-iodoisoindolin-1-one (101 mg, 0.388 mmol, 2.5 equiv.) in a 5:3:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 4.7 mL) at RT were added CuI (7 mg, 0.04 mmol, 25 mol %) and Pd(PPh₃)₄ (9 mg, 8 μmol, 5 mol %) rapidly. The resulting mixture was stirred at 70 °C for 3 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 85:15) to afford the desired crude reaction product which was used directly in the next step without further purification. To a solution of the crude reaction product in THF (3 mL) at RT was added a 0.5 M LiOH aq. solution (3 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.60 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 18 mL) at RT. The resulting mixture was stirred for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 35 (35 mg, 55 μmol, 36% over 3 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.36 (s, 1H), 8.33 (s, 1H), 7.65 (br s, 1H), 7.54 (dd, J = 7.9, 1.5 Hz, 1H), 7.48–7.45 (m, 1H), 6.01 (d, J = 5.1 Hz, 1H), 4.74 (app t, J = 5.1 Hz, 1H), 4.40 (br s, 2H), 4.38 (ddd, J = 10.2, 4.4, 3.3 Hz, 1H), 4.23 (app t, J = 4.8 Hz, 1H), 4.03 (app t, J = 6.2 Hz, 1H), 2.82 (app ddt, J = 10.7, 9.2, 4.5 Hz, 1H), 2.18 (dddd, J = 14.4, 11.4, 6.7, 4.5 Hz, 1H), 2.06 (app ddt, J = 14.5, 11.5, 5.6 Hz, 1H), 2.01 (ddd, J = 14.1, 10.2, 4.2 Hz, 1H), 1.96–1.90 (m, 1H), 1.77 (app ddt, J = 12.9, 11.5, 5.0 Hz, 1H), 1.57 (dddd, J = 13.4, 11.7, 9.4, 4.4 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.8, 172.2, 150.9, 149.4, 145.2, 145.1, 144.1, 136.1, 132.7, 127.0, 124.9, 123.8, 120.0, 92.7, 90.0, 84.0, 83.4, 74.8, 74.5, 53.6, 47.0, 38.8, 30.9, 29.6, 28.8; HRMS (ESI+): calcd. for [C₂₅H₂₈N₇O₆]⁺ 522.2096, meas. 522.2103, Δ 1.4 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(6-carbamoylbenzo[d][1,3]dioxol-4-yl)hept-6-ynoic acid (36).

To a solution of S81 (27 mg, 32 μmol) in THF (0.60 mL) at RT was added a 0.5 M LiOH aq. solution (0.60 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. Solution (90 μL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 40% B’ over 40 min followed by 40% → 95% B’ over 5 min) to afford 36 (16 mg, 24 μmol, 76% over 2 steps) as the TFA salt. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)): δ 8.31 (s, 1H), 8.29 (s, 1H), 7.32 (d, J = 1.8 Hz, 1H), 7.19 (d, J = 1.8 Hz, 1H), 6.05 (d, J = 1.0 Hz, 1H), 6.04 (d, J = 1.0 Hz, 1H), 5.96 (d, J = 5.1 Hz, 1H), 4.71 (app t, J = 5.1 Hz, 1H), 4.32 (ddd, J = 10.4, 4.4, 3.2 Hz, 1H), 4.19 (dd, J = 5.1, 4.4 Hz, 1H), 3.88 (dd, J = 6.7, 5.8 Hz, 1H), 2.84–2.78 (m, 1H), 2.08 (dddd, J = 14.2, 11.7, 6.7, 4.7 Hz, 1H), 2.03–1.97 (m, 2H), 1.90 (ddd, J = 13.9, 10.7, 3.2 Hz, 1H), 1.72 (app ddt, J = 13.1, 11.7, 5.0 Hz, 1H), 1.55 (dddd, J = 13.1, 11.7, 9.2, 4.7 Hz, 1H); ¹³C NMR (101 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.4, 171.4, 152.8, 151.1, 149.5, 149.1, 145.4, 144.4, 127.8, 126.9, 120.1, 108.6, 105.4, 104.0, 97.2, 90.3, 84.1, 77.5, 74.9, 74.6, 53.8, 38.7, 30.9, 29.9, 28.9; HRMS (ESI+): calcd. for [C₂₅H₂₈N₇O₈]⁺ 554.1994, meas. 554.2004, Δ 1.8 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(6-carbamoylpyridin-2-yl)hept-6-ynoic acid (37).

To a solution of S82 (28 mg, 35 μmol) in THF (0.66 mL) at RT was added a 0.5 M LiOH aq. solution (0.66 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.13 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 4 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative (5% → 20% B over 30 min followed by 20% → 95% B over 5 min) to afford 37 (19 mg, 30 μmol, 88% over 2 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.37 (s, 1H), 8.34 (s, 1H), 7.99 (dd, J = 7.9, 1.4 Hz, 1H), 7.96 (dd, J = 7.9, 7.5 Hz, 1H), 7.63 (dd, J = 7.5, 1.4 Hz, 1H), 6.00 (d, J = 5.0 Hz, 1H), 4.74 (dd, J = 5.2, 5.0 Hz, 1H), 4.37 (ddd, J = 10.1, 4.5, 3.4 Hz, 1H), 4.23 (dd, J = 5.2, 4.5 Hz, 1H), 4.02 (dd, J = 6.7, 5.8 Hz, 1H), 2.91–2.85 (m, 1H), 2.15 (dddd, J = 14.3, 11.6, 6.7, 4.6 Hz, 1H), 2.10–2.02 (m, 2H), 1.98 (ddd, J = 13.9, 10.6, 3.4 Hz, 1H), 1.80 (app ddt, J = 13.1, 11.6, 5.0 Hz, 1H), 1.64 (dddd, J = 13.1, 11.8, 9.2, 4.6 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.1, 167.9, 151.0, 149.6, 149.4, 145.2, 144.2, 142.3, 140.6, 131.6, 123.1, 120.1, 95.1, 90.0, 83.8, 82.2, 74.8, 74.4, 53.5, 38.3, 30.6, 29.6, 28.7; HRMS (ESI+): calcd. for [C₂₃H₂₇N₈O₆]⁺ 511.2048, meas. 511.2059, Δ 2.1 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(2-carbamoylpyridin-4-yl)hept-6-ynoic acid (38).

To a solution of S83 (35 mg, 43 μmol) in THF (0.8 mL) at RT was added a 0.5 M LiOH aq. solution (0.8 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.16 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 4 mL) at RT. The resulting mixture was stirred for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 20% B over 30 min followed by 20% → 95% B over 5 min) to afford 38 (19 mg, 30 μmol, 70% over 2 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.68 (dd, J = 5.8, 0.6 Hz, 1H), 8.37 (s, 1H), 8.35 (s, 1H), 8.27 (dd, J = 1.6, 0.6 Hz, 1H), 7.90 (dd, J = 5.8, 1.6 Hz, 1H), 6.01 (d, J = 5.0 Hz, 1H), 4.73 (dd, J = 5.2, 5.0 Hz, 1H), 4.36 (ddd, J = 10.5, 4.4, 3.3 Hz, 1H), 4.22 (dd, J = 5.2, 4.4 Hz, 1H), 4.01 (dd, J = 6.6, 5.9 Hz, 1H), 2.95 (app ddt, J = 10.9, 9.3, 4.8 Hz, 1H), 2.14 (dddd, J = 14.3, 11.6, 6.6, 4.6 Hz, 1H), 2.09–2.02 (m, 2H), 1.99 (ddd, J = 14.0, 10.9, 3.3 Hz, 1H), 1.82 (app ddt, J = 13.3, 11.6, 4.8 Hz, 1H), 1.64 (dddd, J = 13.3, 11.9, 9.3, 4.6 Hz, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.1, 165.6, 151.0, 149.4, 145.5, 145.3, 145.2, 144.2, 141.1, 131.6, 127.6, 120.1, 105.9, 90.1, 83.6, 81.0, 74.8, 74.5, 53.5, 38.3, 30.5, 30.2, 28.7; HRMS (ESI+): calcd. for [C₂₃H₂₇N₈O₆]⁺ 511.2048, meas. 511.2050, Δ 0.4 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(5-carbamoylpyridin-3-yl)hept-6-ynoic acid (39).

To a solution of S84 (26 mg, 32 μmol) in THF (0.6 mL) at RT was added a 0.5 M LiOH aq. solution (0.6 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.13 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 20% B over 30 min followed by 20% → 95% B over 5 min) to afford 39 (16 mg, 26 μmol, 80% over 2 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (500 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 9.04 (d, J = 1.8 Hz, 1H), 8.89 (d, J = 1.8 Hz, 1H), 8.81 (app t, J = 1.8 Hz, 1H), 8.37 (s, 1H), 8.36 (s, 1H), 6.01 (d, J = 4.9 Hz, 1H), 4.72 (app t, J = 4.9 Hz, 1H), 4.37 (ddd, J = 10.3, 4.9, 3.1 Hz, 1H), 4.22 (app t, J = 4.9 Hz, 1H), 4.01 (app t, J = 6.3 Hz, 1H), 2.95–2.86 (m, 1H), 2.15 (dddd, J = 14.3, 11.6, 6.3, 4.8 Hz, 1H), 2.10–2.01 (m, 2H), 1.98 (ddd, J = 13.7, 10.7, 3.1 Hz, 1H), 1.85–1.77 (m, 1H), 1.68–1.59 (m, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.1, 165.8, 151.1, 149.4, 147.7, 147.0, 145.3, 144.1, 141.1, 133.9, 125.1, 120.1, 101.4, 90.1, 83.7, 77.4, 74.9, 74.5, 53.5, 38.4, 30.6, 30.0, 28.7; HRMS (ESI+): calcd. for [C₂₃H₂₇N₈O₆]⁺ 511.2048, meas. 511.2069, Δ 4.0 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(4-carbamoylpyridin-2-yl)hept-6-ynoic acid (40).

To a solution of S85 (22 mg, 27 μmol) in THF (1.0 mL) at RT was added a 0.5 M LiOH aq. solution (1.0 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.20 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 4 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 20% B over 30 min followed by 20% → 95% B over 5 min) to afford 40 (14 mg, 22 μmol, 82% over 2 steps) as the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analysis. ¹H NMR (500 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.73 (dd, J = 6.1, 0.7 Hz, 1H), 8.37 (s, 1H), 8.35 (s, 1H), 8.22 (dd, J = 1.9, 0.7 Hz, 1H), 8.14 (dd, J = 6.1, 1.9 Hz, 1H), 6.01 (d, J = 4.9 Hz, 1H), 4.73 (dd, J = 5.1, 4.9 Hz, 1H), 4.35 (ddd, J = 10.2, 4.8, 3.4 Hz, 1H), 4.23 (dd, J = 5.1, 4.8 Hz, 1H), 4.01 (app t, J = 6.3 Hz, 1H), 3.03–2.96 (m, 1H), 2.18–1.99 (m, 4H), 1.89–1.80 (m, 1H), 1.74–1.64 (m, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.1, 166.8, 151.0, 150.0, 149.4, 145.3, 144.8, 144.2, 137.5, 129.7, 124.8, 120.1, 106.3, 90.1, 83.4, 76.1, 74.8, 74.4, 53.5, 38.0, 30.3, 30.2, 28.6; HRMS (ESI+): calcd. for [C₂₃H₂₇N₈O₆]⁺ 511.2048, meas. 511.2055, Δ 1.3 ppm.

(2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-7-(5-aminonaphthalen-1-yl)hept-6-ynoic acid (41).

To a solution of S86 (34 mg, 41 μmol) in THF (0.76 mL) at RT was added a 0.5 M LiOH aq. solution (0.76 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution (0.17 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 41 (20 mg, 26 μmol, 64% over 2 steps) as the TFA salt upon treatment with d-TFA as part of sample preparation for NMR analysis. ¹H NMR (600 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 8.33 (s, 1H), 8.29 (app dt, J = 8.4, 1.0 Hz, 1H), 8.29 (s, 1H), 7.89 (app dt, J = 8.5, 1.0 Hz, 1H), 7.70 (dd, J = 7.2, 1.0 Hz, 1H), 7.63 (dd, J = 7.5, 1.0 Hz, 1H), 7.61 (dd, J = 8.5, 7.2 Hz, 1H), 7.57 (dd, J = 8.4, 7.5 Hz, 1H), 6.00 (d, J = 5.0 Hz, 1H), 4.77 (dd, J = 5.2, 5.0 Hz, 1H), 4.44 (ddd, J = 10.0, 4.6, 3.3 Hz, 1H), 4.28 (dd, J = 5.2, 4.6 Hz, 1H), 4.06 (dd, J = 6.7, 5.6 Hz, 1H), 3.01–2.95 (m, 1H), 2.23 (dddd, J = 14.4, 11.6, 6.7, 4.6 Hz, 1H), 2.17–2.10 (m, 1H), 2.11 (ddd, J = 14.0, 10.0, 4.5 Hz, 1H), 2.05 (ddd, J = 14.0, 10.3, 3.3 Hz, 1H), 1.84 (app ddt, J = 13.1, 11.6, 5.1 Hz, 1H), 1.72–1.64 (m, 1H); ¹³C NMR (126 MHz, CD₃CN (350 μL)/D₂O (350 μL)/d-TFA (40 μL)): δ 172.1, 150.8, 149.4, 145.1, 144.2, 134.6, 132.5, 128.5, 128.4, 127.5, 127.5, 127.4, 123.0, 122.7, 122.0, 119.9, 98.5, 90.1, 83.9, 81.3, 74.7, 74.4, 53.5, 38.6, 30.8, 29.7, 28.8; HRMS (ESI+): calcd. for [C₂₇H₃₀N₇O₅]⁺ 532.2303, meas. 532.2325, Δ 4.2 ppm.

Synthesis of Compounds S2–S86

Methyl 2-(dimethoxyphosphoryl)-2-(2,2,2-trifluoroacetamido)acetate (S2).⁶⁷

To a solution of (±)-Z-α-phosphonoglycine trimethyl ester (S1) (3.31 g, 9.99 mmol) in CH₂Cl₂ (50 mL) at RT, was added Pd/C (10%, 0.53 g, 0.50 mmol, 5 mol %). The flask headspace was evacuated and refilled with hydrogen (3 ×) and the reaction mixture was stirred vigorously under a hydrogen atmosphere (balloon) for 24 h. The flask headspace was evacuated and refilled with nitrogen, and the contents were filtered through a pad of celite. The filtrate was cooled to 0 °C and TFAA (3.47 mL, 25.0 mmol, 2.5 equiv.) was added dropwise. The reaction mixture was stirred at RT for 12 h and the solution was concentrated to yield an orange residue. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 0 → 100%) to afford TFA protected phosphonate S2 (2.05 g, 6.99 mmol, 70% over 2 steps). R_f = 0.41 (EtOAc); FTIR (thin film), cm−1: ν_max (cm⁻¹): 3197, 3046, 2964, 2861, 1757, 1722, 1559, 1433, 1360, 1307, 1257, 1212, 1180, 1150, 1035; ¹H NMR (600 MHz, CDCl₃): δ 7.23 (br s, 1H), 5.14 (dd, ²J _H-P = 21.3, J = 8.7 Hz, 1H), 3.89 (s, 3H), 3.87 (d, ³J _H-P , J = 11.2 Hz, 3H), 3.83 (d, ³J _H-P , J = 11.1 Hz, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 165.5, 157.2 (qd, ²J _C-F = 38 Hz, ³J _C-P = 5.9 Hz), 115.7 (q, ¹J _C-F = 287 Hz), 54.5 (d, J _C-P = 6.6 Hz), 54.1 (d, ²J _C-P = 6.9 Hz), 53.3, 50.3 (d, ¹J _C-P = 152 Hz); ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.5; ³¹P NMR (162 MHz, CDCl₃): δ 16.1; HRMS (ESI+): calcd. for [C₇H₁₂F₃N₁O₆P₁]⁺ 294.0360, meas. 294.0349, Δ 3.7 ppm.

Methyl (2S,5S)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)hept-6-ynoate (S3).

To a vial charged with 7 (182 mg, 0.307 mmol) was added DMF (3 mL) at RT under a nitrogen atmosphere. A separate vial was charged with TASF (253 mg, 0.920 mmol, 3.0 equiv.) in a glovebox, put under a nitrogen atmosphere, and DMF (2 mL) was added at RT. The resulting solution was added to the vial containing alkyne 7 and the reaction mixture was stirred at 60 °C for 2 h. Volatiles were removed in vacuo. The residue was partitioned between Et₂O (40 mL) and a 1:1 mixture of brine and 5% LiCl aq. solution (40 mL). The layers were separated and the aqueous phase was extracted with Et₂O (3 × 30 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 5 → 50%) to yield S3 (128 mg, 0.293 mmol, 95%) as a colorless oil. R_f = 0.35 (hexanes/EtOAc, 60:40); FTIR (neat), ν_max (cm⁻¹): 3292, 3089, 2990, 2954, 2938, 2836, 1747, 1719, 1553, 1455, 1440, 1383, 1275, 1209, 1160, 1104, 1060; ¹H NMR (600 MHz, CDCl₃): δ 6.94 (d, J = 7.9 Hz, 1H), 4.93 (s, 1H), 4.64 (td, J = 7.8, 4.9 Hz, 1H), 4.59 (d, J = 6.0 Hz, 1H), 4.55 (dd, J = 6.0, 1.0 Hz, 1H), 4.45 (ddd, J = 11.2, 3.9, 0.9 Hz, 1H), 3.80 (s, 3H), 3.31 (s, 3H), 2.66–2.58 (m, 1H), 2.14 (d, J = 2.4 Hz, 1H), 2.10 (ddq, J = 14.0, 10.8, 5.2 Hz, 1H), 2.05–1.94 (m, 1H), 1.67 (ddd, J = 13.3, 11.2, 4.0 Hz, 1H), 1.55 (dddd, J = 21.7, 13.2, 10.9, 4.6 Hz, 2H), 1.47 (s, 3H), 1.40 (dddd, J = 16.5, 14.4, 8.7, 3.9 Hz, 1H), 1.30 (s, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 171.3, 157.0 (q, ²J _C-F = 38 Hz), 115.1 (q, ¹J _C-F = 287 Hz), 112.5, 110.1, 85.6, 85.1, 85.0, 84.5, 71.6, 55.3, 53.1, 52.6, 52.5, 40.4, 30.7, 29.8, 28.6, 26.6, 25.1; HRMS (ESI+): calcd. for [C₁₉H₂₆F₃N₁O₇Na]⁺ 460.1554, meas. 460.1569, Δ 3.3 ppm.

(3R,4R,5R)-5-((2S,5S)-2-ethynyl-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-2,3,4-triyl triacetate (S4).

To a solution of acetonide S3 (1.21 g, 2.77 mmol) in CH₂Cl₂ (16.5 mL) at RT, was added a 2:1 mixture of TFA and water (51 mL). The reaction mixture was stirred at RT for 21 h and volatiles were removed in vacuo. The residue was resuspended in Ac₂O (6.0 mL, 64 mmol, 23 equiv.) and pyridine (12.0 mL, 149 mmol, 54 equiv.). The resulting mixture was stirred at RT for 22 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 0 → 40%) to afford triacetate S4 (735 mg, 1.44 mmol, 52% over 2 steps) as a 1:1.7 mixture of α and β anomers, as a light yellow oil. R_f = 0.37 (α-anomer) / 0.46 (β-anomer) (hexanes/EtOAc, 50:50); FTIR (thin-film), ν_max (cm⁻¹): 3290, 2956, 1748, 1724, 1553, 1438, 1373, 1219, 1178, 1112, 1049, 1013; ¹H NMR (600 MHz, CDCl₃): δ α-anomer: 6.92 (d, J = 7.8 Hz, 1H), 6.36 (d, J = 4.6 Hz, 1H), 5.22 (dd, J = 6.8, 4.6 Hz, 1H), 5.05 (dd, J = 6.8, 3.7 Hz, 1H), 4.63 (td, J = 7.6, 5.1 Hz, 1H), 4.43 (dt, J = 10.2, 3.6 Hz, 1H), 3.80 (s, 3H), 2.62 (dqd, J = 13.8, 4.6, 2.4 Hz, 1H), 2.13 (d, J = 2.4 Hz, 1H), 2.12 (s, 3H), 2.08 (s, 3H), 2.08–2.04 (m, 1H), 2.07 (s, 3H), 2.01 (dddd, J = 18.5, 9.1, 5.3, 2.9 Hz, 1H), 1.84 (ddd, J = 14.1, 10.8, 3.5 Hz, 1H), 1.65–1.59 (m, 1H), 1.59–1.53 (m, 1H), 1.40 (dddd, J = 13.1, 11.0, 9.5, 4.8 Hz, 1H); β-anomer: 7.02 (d, J = 8.1 Hz, 1H), 6.10 (s, 1H), 5.31 (dd, J = 4.9, 0.9 Hz, 1H), 5.16 (dd, J = 7.2, 4.7 Hz, 1H), 4.64 (td, J = 8.2, 4.6 Hz, 1H), 4.33 (ddd, J = 10.1, 7.1, 2.7 Hz, 1H), 3.81 (s, 3H), 2.66 (ttd, J = 9.6, 4.9, 2.4 Hz, 1H), 2.13 (d, J = 2.4 Hz, 1H), 2.12 (s, 3H), 2.10 (s, 3H), 2.08–2.04 (m, 1H), 2.06 (s, 3H), 2.05–1.97 (m, 1H), 1.84 (ddd, J = 13.1, 9.9, 2.8 Hz, 1H), 1.69–1.61 (m, 1H), 1.60–1.55 (m, 1H), 1.47 (dddd, J = 13.2, 11.3, 8.9, 4.5 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ α-anomer: 171.1, 169.9, 169.5, 169.3, 156.9 (q, ²J _C-F = 38 Hz), 115.7 (q, ¹J _C-F = 288 Hz), 93.7, 84.6, 81.2, 72.5, 71.5, 69.7, 52.9, 52.3, 38.9, 30.4, 29.1, 28.4, 21.0, 20.5, 20.2; β-anomer: 171.1, 170.1, 169.7, 169.4, 156.9 (q, ²J _C-F = 38 Hz), 115.6 (q, ¹J _C-F = 288 Hz), 98.3, 84.9, 79.8, 74.3, 73.8, 71.3, 52.9, 52.3, 39.6, 30.6, 29.5, 28.0, 21.0, 20.6, 20.2; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −75.8; HRMS (ESI+): calcd. for [C₂₁H₂₆F₃N₁O₁₀Na]⁺ 532.1401, meas. 532.1407, Δ 1.2 ppm.

Methyl 6-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)hexanoate (S6).⁶⁸

To a 3-necked flask charged with pyridine (10 mL) were added NaI (1.49 g, 10.0 mmol, 1.0 equiv.) and FeCl₃•6H₂O (1.35 g, 5.00 mmol, 50 mol %) sequentially, both in one portion. Zinc powder (3.92 g, 60.0 mmol, 6.0 equiv.) was added portionwise to the reaction mixture (Caution, exothermic), monitoring the temperature so that it remains below 40 °C. Upon complete addition of the zinc powder, the resulting mixture was allowed to cool gradually to RT and a solution of riboside S5⁶⁹ (3.14 g, 10.0 mmol) in methyl acrylate (6.0 mL) was added over 10 min via syringe. The reaction mixture was stirred at RT for 2 h, before the addition of toluene (100 mL) and filtration over celite. Volatiles were removed in vacuo to give methyl ester S6 (2.52 g, 9.25 mmol, 93%) which was of suitable purity to be used in the next step without further purification. R_f = 0.42 (cyclohexane/EtOAc, 67:33); FTIR (thin film), ν_max (cm⁻¹): 2989, 2940, 1735, 1437, 1372, 1270, 1239, 1208, 1194, 1159, 1090, 1057, 1011; ¹H NMR (600 MHz, CDCl₃): δ 4.94 (s, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.52 (dd, J = 5.9, 1.1 Hz, 1H), 4.13 (ddd, J = 8.9, 6.4, 1.1 Hz, 1H), 3.67 (s, 3H), 3.34 (s, 2H), 2.35 (td, J = 7.4, 1.4 Hz, 2H), 1.86 – 1.76 (m, 1H), 1.71 (ddtd, J = 13.2, 10.1, 7.3, 5.7 Hz, 1H), 1.62 (dddd, J = 14.1, 10.1, 8.9, 5.1 Hz, 1H), 1.53 (ddt, J = 13.4, 10.4, 6.0 Hz, 1H), 1.47 (s, 3H), 1.31 (s, 3H); ¹³C NMR (151 MHz, CDCl₃): δ 173.5, 112.1, 109.5, 86.7, 85.5, 84.1, 54.9, 51.4, 34.4, 33.5, 26.4, 24.9, 21.7; HRMS (ESI+): calcd. for [C₁₃H₂₂O₆Na]⁺ 297.1309, meas. 297.1307, Δ 0.7 ppm.

4-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)butanal (S7).

To an oversized flask (250 mL) containing a solution of methyl ester S6 (2.15 g, 7.83 mmol) in toluene (20 mL) at −85 °C (i-PrOH/liquid nitrogen bath) was added i-Bu₂AlH (1.0 M in hexanes, 8.0 mL, 8.0 mmol, 1.02 equiv.) over 40 min via syringe pump, down the side-wall of the flask. Upon complete addition of i-Bu₂AlH, the reaction mixture was stirred at −85 °C for 1 h, before the slow addition of MeOH (4 mL) over 20 min down the side-wall of the flask, followed by a potassium sodium tartrate sat. aq. solution (40 mL) and EtOAc (50 mL). The reaction mixture was removed from the cooling bath and allowed to warm gradually to RT. Water (10 mL) and brine (10 mL) were added, and the biphasic mixture was stirred for another 10 min. The layers were separated and the aqueous phase was extracted with EtOAc (3 × 50 mL). The combined organic extracts were washed with brine (200 mL), dried over MgSO₄, filtered and concentrated to give aldehyde S7 (1.80 g, 7.37 mmol, 94%) as a light-yellow oil which was of suitable purity to be used in the next step without further purification. R_f = 0.31 (cyclohexane/EtOAc, 67:33); FTIR (thin film), ν_max (cm⁻¹): 2988, 2938, 2832, 2723, 1724, 1457, 1412, 1381, 1372, 1273, 1240, 1209, 1193, 1160, 1087, 1058, 1018; ¹H NMR (600 MHz, CDCl₃): δ 9.73 (t, J = 1.6 Hz, 1H), 4.89 (s, 1H), 4.55 (d, J = 6.0 Hz, 1H), 4.48 (dd, J = 6.0, 1.1 Hz, 1H), 4.09 (ddd, J = 9.0, 6.3, 1.1 Hz, 1H), 3.30 (s, 3H), 2.45 (dddd, J = 8.7, 7.1, 3.4, 1.6 Hz, 2H), 1.81–1.71 (m, 1H), 1.72–1.63 (m, 1H), 1.59 (dtd, J = 13.6, 9.2, 5.2 Hz, 1H), 1.49 (ddt, J = 13.5, 10.1, 6.1 Hz, 1H), 1.43 (s, 3H), 1.27 (s, 3H); ¹³C NMR (151 MHz, CDCl₃): δ 202.0, 112.3, 109.6, 86.8, 85.5, 84.1, 55.1, 43.4, 34.4, 26.6, 25.0, 18.9.

Methyl (Z)-2-(((benzyloxy)carbonyl)amino)-6-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)hex-2-enoate (S8).

To a stirred suspension of (±)-Cbz-α-phosphonoglycine trimethyl ester (S1) (343 mg, 1.04 mmol, 1.2 equiv.) in THF (3.0 mL) at −78 °C was added 1,1,3,3-tetramethylguanidine (0.12 mL, 0.95 mmol, 1.1 equiv.). The resulting mixture was stirred at −78 °C for 15 min, before the addition of a solution of aldehyde S7 (211 mg, 0.864 mmol) in THF (2.0 mL). The reaction mixture was stirred at −78 °C for 30 min and then placed in an ice/water bath and stirred for 1 h. The reaction was quenched by the addition of a 10% AcOH aq. solution (2 mL) and water (10 mL) was added. The layers were separated and the aqueous phase was extracted with CH₂Cl₂ (3 × 15 mL). The combined organic extracts were washed with water (10 mL) and brine (10 mL), dried over MgSO₄, filtered and concentrated. ¹H NMR analysis of the crude revealed a 15:1 mixture of Z and E isomers. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 50%). Z and E isomers separated and only fractions containing Z isomers were collected to afford enamide S8 (353 mg, 0.785 mmol, 91 %) as a clear viscous oil. R_f = 0.44 (cyclohexane/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3316, 2988, 2942, 1716, 1657, 1500, 1455, 1437, 1381, 1373, 1270, 1213, 1160, 1105, 1089, 1047; ¹H NMR (600 MHz, CDCl₃): δ 7.41–7.30 (m, 5H), 6.61 (t, J = 7.2 Hz, 1H), 5.14 (s, 2H), 4.93 (s, 1H), 4.58 (d, J = 5.9 Hz, 1H), 4.49 (d, J = 5.9 Hz, 1H), 4.12 (dd, J = 8.3, 6.3 Hz, 1H), 3.75 (s, 3H), 3.33 (s, 3H), 2.31–2.18 (m, 2H), 1.68–1.48 (m, 4H), 1.47 (s, 3H), 1.30 (s, 3H); ¹³C NMR (151 MHz, CDCl₃): δ 165.1, 137.5, 136.1, 128.6, 128.3, 128.2, 112.3, 109.6, 86.9, 85.6, 84.2, 67.4, 55.0, 52.5, 34.7, 28.1, 26.6, 25.1; HRMS (ESI+): calcd. for [C₂₃H₃₂NO₈]⁺ 450.2122, meas. 450.2124, Δ 0.3 ppm.

Methyl (S)-2-(((benzyloxy)carbonyl)amino)-6-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)hexanoate (S9).

To a flask charged with enamide S8 (179 mg, 0.398 mmol, exclusively Z isomer) was added MeOH (5 mL) at RT under a nitrogen atmosphere. The contents were stirred until full dissolution was noted. Separately, [Rh(nbd)₂]BF₄ (49 mg, 0.12 mmol, 30 mol %) and (S,S)-Me-DUPHOS (43 mg, 0.14 mmol, 35 mol %) were weighed out into the same vial in a glove box. The vial was removed from the glove box, placed under a nitrogen atmosphere and MeOH (10 mL) was added. The flask headspace was evacuated and backfilled with hydrogen (3 ×). The methanolic solution of enamide S8 was transferred to the vial containing the catalyst (2 mL of MeOH used for vial rinse) and the contents were stirred vigorously at RT under a hydrogen atmosphere (balloon) for 1 h. Volatiles were removed in vacuo and crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 50%) to afford protected amino acid S9 (172 mg, 0.381 mmol, 96%) as a clear viscous oil and as a single diastereomer. R_f = 0.32 (cyclohexane/EtOAc, 67:33); FTIR (thin film), ν_max (cm⁻¹): 3338, 2988, 2939, 2862, 1720, 1523, 1455, 1439, 1381, 1373, 1348, 1259, 1239, 1209, 1161, 1106, 1087, 1056, 1026; ¹H NMR (600 MHz, CDCl₃): δ 7.38–7.28 (m, 5H), 5.31 (d, J = 8.4 Hz, 1H), 5.09 (s, 2H), 4.92 (s, 1H), 4.57 (d, J = 5.9 Hz, 1H), 4.48 (d, J = 6.0 Hz, 1H), 4.37 (td, J = 8.0, 5.1 Hz, 1H), 4.10 (dd, J = 8.9, 5.8 Hz, 1H), 3.73 (s, 3H), 3.31 (s, 3H), 1.88–1.79 (m, 1H), 1.64 (app. h, J = 8.1 Hz, 1H), 1.58 (app. q, J = 8.8 Hz, 1H), 1.49–1.43 (m, 2H), 1.46 (s, 3H), 1.40–1.32 (m, 3H), 1.30 (s, 3H); ¹³C NMR (151 MHz, CDCl₃): δ 173.0, 155.9, 136.3, 128.5, 128.2, 128.1, 112.2, 109.4, 87.0, 85.6, 84.1, 67.0, 54.9, 53.8, 52.3, 34.8, 32.6, 26.5, 25.8, 25.0; HRMS (ESI+): calcd. for [C₂₃H₃₄NO₈]⁺ 452.2280, meas. 452.2285, Δ 1.1 ppm.

(3R,4R,5R)-5-((S)-5-(((benzyloxy)carbonyl)amino)-6-methoxy-6-oxohexyl)tetrahydrofuran-2,3,4-triyl triacetate (S10).

To a vial charged with acetonide S9 (171 mg, 0.378 mmol) was added a 4:1 mixture of acetic acid and water (7.5 mL) at RT. The resulting mixture was stirred at 85 °C for 8 h. Volatiles were removed in vacuo and the residue was dried by azeotropic distillation with benzene (1 × 10 mL). Crude material was used directly in the next step without further purification. To a solution of the crude triol in CH₂Cl₂ (4.0 mL) at RT, were added pyridine (0.50 mL, 6.2 mmol, 16 equiv.), Ac₂O (0.50 mL, 5.3 mmol, 14 equiv.) and DMAP (5 mg, 0.04 mmol, 0.11 equiv.) sequentially. The resulting mixture was stirred at RT for 2 h and the volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 100%) to afford triacetate S10 (152 mg, 0.290 mmol, 77% over 2 steps) as a ca. 1:1 mixture of α and β anomers as a colorless oil. R_f = 0.28 (α-anomer)/0.37 (β-anomer) (EtOAc); FTIR (thin film), ν_max (cm⁻¹): 3359, 2950, 1743, 1721, 1523, 1455, 1437, 1371, 1216, 1110, 1047, 1010; ¹H NMR (600 MHz, CDCl₃): δ α-anomer: 7.38–7.27 (m, 5H), 6.34 (d, J = 4.6 Hz, 1H), 5.29 (d, J = 8.2 Hz, 1H), 5.18 (dd, J = 6.8, 4.6 Hz, 1H), 5.09 (s, 2H), 5.01 (dd, J = 6.8, 3.6 Hz, 1H), 4.35 (td, J = 7.8, 5.1 Hz, 1H), 4.20–4.15 (m, 1H), 3.73 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 1.83–1.77 (m, 1H), 1.69–1.54 (m, 3H), 1.47–1.42 (m, 1H), 1.39–1.28 (m, 3H); β-anomer: 7.38–7.28 (m, 5H), 6.10 (d, J = 1.1 Hz, 1H), 5.31 (d, J = 8.1 Hz, 1H), 5.29 (dd, J = 4.8, 1.2 Hz, 1H), 5.14 (dd, J = 6.9, 4.8 Hz, 1H), 5.10 (s, 2H), 4.36 (td, J = 7.8, 5.0 Hz, 1H), 4.12 (td, J = 7.4, 5.2 Hz, 1H), 3.73 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.85–1.78 (m, 1H), 1.68–1.55 (m, 2H), 1.51–1.42 (m, 1H), 1.40–1.29 (m, 4H); ¹³C NMR (126 MHz, CDCl₃): δ mixture of α and β-anomers: 172.9, 170.2, 169.8, 169.4, 155.9, 136.3, 128.6, 128.2, 128.2, 98.3, 94.0, 83.3, 81.4, 74.6, 73.6, 72.3, 69.9, 67.0, 53.8, 52.4, 33.8, 33.1, 32.5, 25.0, 25.0, 24.9, 24.8, 21.1, 21.1, 20.7, 20.6, 20.6, 20.4; HRMS (ESI+): calcd. for [C₂₅H₃₄NO₁₁]⁺ 524.2126, meas. 524.2134, Δ 1.5 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-5-(((benzyloxy)carbonyl)amino)-6-methoxy-6-oxohexyl)tetrahydrofuran-3,4-diyl diacetate (S11).

To a suspension of N⁶-benzoyladenine (149 mg, 0.624 mmol, 2.15 equiv.) in propionitrile (dried over 4 Å M.S., 6.0 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (0.21 mL, 0.87 mmol, 3.00 equiv.). The resulting mixture was stirred at 95 °C for 10 min, upon which complete solubilization of N⁶-benzoyladenine was observed. To a separate flask charged with triacetate S10 (152 mg, 0.290 mmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (21 mg, 58 μmol, 20 mol %) was added propionitrile (6.0 mL). The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained. This clear solution was added to the solution of silylated N⁶-benzoyladenine. The reaction mixture was stirred at 95 °C for 2.5 h, cooled to RT and the volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 100% then EtOAc/i-PrOH, 0 → 15%) to afford nucleoside S11 (148 mg, 0.211 mmol, 73%) as a white amorphous solid. R_f = 0.28 (EtOAc); FTIR (thin film), ν_max (cm⁻¹): 3330, 2950, 1744, 1703, 1609, 1581, 1510, 1487, 1454, 1408, 1373, 1330, 1239, 1214, 1177, 1095, 1047, 1028; ¹H NMR (600 MHz, CDCl₃): δ 9.28 (br s, 1H), 8.74 (s, 1H), 8.11 (s, 1H), 8.01–7.97 (m, 2H), 7.59–7.53 (m, 1H), 7.49–7.44 (m, 2H), 7.33–7.24 (m, 5H), 6.12 (d, J = 5.3 Hz, 1H), 5.94 (t, J = 5.5 Hz, 1H), 5.45 (d, J = 3.5 Hz, 1H), 5.44 (t, J = 5.3 Hz, 1H), 5.07 (s, 2H), 4.34 (td, J = 8.0, 5.1 Hz, 1H), 4.15 (dt, J = 8.8, 4.9 Hz, 1H), 3.69 (s, 3H), 2.11 (s, 3H), 2.04 (s, 3H), 1.85–1.70 (m, 3H), 1.65–1.57 (m, 1H), 1.51–1.42 (m, 1H), 1.41–1.30 (m, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 172.9, 169.7, 169.5, 165.0, 155.9, 152.7, 151.8, 149.9, 141.9, 136.3, 133.6, 132.8, 128.8, 128.5, 128.2, 128.1, 128.0, 123.9, 86.5, 82.4, 73.3, 73.1, 67.0, 53.7, 52.4, 32.9, 32.5, 24.9, 24.9, 20.6, 20.4; HRMS (ESI+): calcd. for [C₃₅H₃₈N₆O₁₀Na]⁺ 725.2542, meas. 725.2573, Δ 4.3 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-6-methoxy-6-oxo-2-(phenylethynyl)-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S12).

To a vial charged with nucleoside 8 (82 mg, 0.12 mmol) were added iodobenzamide (49 mg, 0.24 mmol, 2.0 equiv.), CuI (5 mg, 0.02 mmol, 20 mol %) and Pd(PPh₃)₄ (7 mg, 6 μmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 3.5 mL) was added at RT. The resulting mixture was stirred at 70 °C for 3 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 5%) to afford alkyne S12 (76 mg, 99 μmol, 83%) as a white amorphous solid. R_f = 0.48 (EtOAc); FTIR (thin-film), ν_max (cm⁻¹): 3297, 3082, 2954, 1745, 1717, 1610, 1582, 1509, 1489, 1455, 1373, 1328, 1239, 1212, 1176, 1159, 1096, 1072, 1047, 1029; ¹H NMR (500 MHz, CDCl₃): δ 8.95 (s, 1H), 8.81 (s, 1H), 8.13 (s, 1H), 8.01 (d, J = 7.6 Hz, 2H), 7.61 (t, J = 7.2 Hz, 1H), 7.52 (t, J = 7.5 Hz, 2H), 7.40 (ddt, J = 5.3, 2.7, 1.4 Hz, 2H), 7.31 (tt, J = 3.8, 2.2 Hz, 3H), 6.92 (d, J = 5.8 Hz, 1H), 6.14 (d, J = 5.4 Hz, 1H), 6.09 (t, J = 5.4 Hz, 1H), 5.59 (t, J = 4.9 Hz, 1H) 4.66 (td, J = 7.7, 5.0 Hz, 1H), 4.53 (ddd, J = 10.8, 4.5, 2.9 Hz, 1H), 3.78 (s, 3H), 2.82 (ddt, J = 10.9, 9.1, 4.5 Hz, 1H), 2.16 (s, 3H), 2.19–2.09 (m, 1H), 2.07 (s, 3H), 2.06–1.92 (m, 2H), 1.70–1.60 (m, 2H), 1.58–1.46 (m, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.2, 169.8, 169.6, 164.8, 157.0 (q, ²J_C-F = 38 Hz), 152.9, 151.7, 149.8, 142.4, 133.5, 132.9, 131.8, 128.4, 128.2, 128.0, 124.0, 123.2, 115.7 (q, ¹J_C-F = 288 Hz), 90.0, 87.1, 83.8, 81.0, 77.3, 73.7, 73.0, 53.1, 52.4, 38.8, 31.1, 30.0, 28.8, 20.7, 20.5; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.7; HRMS (ESI+): calcd. for [C₃₇H₃₆F₃N₆O₉]⁺ 765.2490, meas. 765.2504, Δ 1.8 ppm.

(S)-3-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-5-(triisopropylsilyl)pent-4-ynal (S13).

A 50 mL flask was charged with [Rh(OAc)(C₂H₄)₂]₂ (60 mg, 0.14 mmol, 2.5 mol %) and (R)-DTBMSegPhos (389 mg, 0.329 mmol, 6.0 mol %). The flask headspace was purged with nitrogen for 10 min followed by the addition of MeOH (14 mL). The resulting orange suspension was stirred vigorously at RT, until a clear solution was obtained (15 min), followed by the sequential addition of enal 4 (1.33 g, 5.49 mmol) in MeOH (4 mL) and TIPS-acetylene (2.46 mL, 11.0 mmol, 2.0 equiv.). The resulting red mixture was stirred at 40 °C for 15 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/acetone, 0 → 30%) to afford aldehyde S13 (2.00 g, 4.71 mmol, 86 %) as a single diastereomer, as a light yellow oil. R_f = 0.31 (hexanes/acetone, 85:15); FTIR (thin film), ν_max (cm⁻¹): 2942, 2866, 2169, 1729, 1464, 1382, 1211, 1160, 1108; ¹H NMR (600 MHz, CDCl₃): δ 9.83 (dd, J = 2.1, 1.7 Hz, 1H), 4.95 (s, 1H), 4.60 (s, 2H), 4.39 (app t, J = 7.7 Hz, 1H), 3.34 (s, 3H), 3.09 (app tdd, J = 7.7, 6.5, 6.0 Hz, 1H), 2.65 (ddd, J = 16.6, 6.0, 1.7 Hz, 1H), 2.60 (ddd, J = 16.6, 7.7, 2.1 Hz, 1H), 1.89 (app dt, J = 13.6, 7.7 Hz, 1H), 1.73 (ddd, J = 13.6, 7.7, 6.5 Hz, 1H), 1.47 (s, 3H), 1.31 (s, 3H), 1.06–1.03 (m, 21H); ¹³C NMR (126 MHz, CDCl₃): δ 200.8, 112.6, 109.6, 108.8, 85.6, 84.8, 83.9, 83.8, 55.2, 47.8, 39.9, 26.7, 25.3, 24.2, 18.7, 11.3; HRMS (ESI+): calcd. for [C₂₃H₄₀O₅Si₁Na]⁺ 447.2537, meas. 447.2523, Δ 3.2 ppm.

Methyl (R,Z)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-7-(triisopropylsilyl)hept-2-en-6-ynoate (S14).

To a suspension of NaH (95%, 45 mg, 1.8 mmol, 1.5 equiv.) in THF (2.5 mL) at 0 °C, was added a solution of phosphonate S2 (517 mg, 1.76 mmol, 1.5 equiv.) in THF (2.5 mL) dropwise. The resulting cloudy mixture was stirred at 0 °C for 30 min and a solution of aldehyde S13 (499 mg, 1.18 mmol) in THF (2.5 mL) was added in one portion. The yellow reaction mixture was stirred at 40 °C for 1.5 h and cooled to 0 °C, before the addition of an aqueous pH 7 buffer (6 mL) and Et₂O (6 mL). The layers were separated and the aqueous phase was extracted with Et₂O (3 × 6 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 1 → 35%) to afford olefin S14 (651 mg, 1.10 mmol, 94%) as a 6:1 mixture of Z and E isomers as a colorless oil. R_f = 0.28 (hexanes/EtOAc, 80:20); FTIR (thin film), ν_max (cm⁻¹): 3305, 2943, 2865, 2168, 1721, 1665, 1536, 1463, 1439, 1382, 1274, 1210, 1160, 1107, 1061; ¹H NMR (600 MHz, CDCl₃): δ 7.78 (s, 1H), 7.09 (dd, J = 7.5, 7.2 Hz, 1H), 4.95 (s, 1H), 4.60 (d, J = 6.0 Hz, 1H), 4.55 (dd, J = 6.0, 0.9 Hz, 1H), 4.37 (app td, J = 7.5, 0.9 Hz, 1H), 3.81 (s, 3H), 3.34 (s, 3H), 2.81 (dddd, J = 9.0, 7.5, 7.0, 4.6 Hz, 1H), 2.45 (ddd, J = 15.7, 7.2, 4.6 Hz, 1H), 2.29 (ddd, J = 15.7, 9.0, 7.5 Hz, 1H), 1.89 (app dt, J = 13.7, 7.5 Hz, 1H), 1.70 (ddd, J = 13.7, 7.5, 7.0 Hz, 1H), 1.46 (s, 3H), 1.30 (s, 3H), 1.06–1.04 (m, 21H); ¹³C NMR (101 MHz, CDCl₃): δ 163.8, 155.2 (q, ²J _C-F = 38 Hz), 137.7, 123.7, 115.8 (q, ¹J_C-F = 288 Hz), 112.6, 109.5, 109.3, 85.6, 84.8, 83.9, 55.1, 52.9, 40.0, 33.8, 28.8, 26.6, 25.2, 18.7, 11.3; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.3, −77.2; HRMS (ESI+): calcd. for [C₂₈H₄₄F₃N₁O₇Si₁Na]⁺ 614.2731, meas. 614.2735, Δ 0.7 ppm.

Methyl (2S,5R)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-7-(triisopropylsilyl)hept-6-ynoate (S15).

To a flask charged with enamide S14 (6:1 mixture of Z and E isomers, 651 mg, 1.10 mmol) was added MeOH (32 mL) under a nitrogen atmosphere. The contents were stirred until full solubilization was observed. In a glove box, a separate vial was charged with (S,S)-MeBPE-Rh (31 mg, 55 μmol, 5 mol %). The vial was removed from the glove box, placed under a nitrogen atmosphere and MeOH (4 mL) was added. The resulting orange solution was transferred to the flask containing the methanolic solution of enamide S14 via syringe. The flask headspace was evacuated and refilled with hydrogen (3 ×), and the reaction mixture was stirred vigorously under a hydrogen atmosphere (balloon) for 1 h. Volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 1 → 50%) to afford protected amino acid S15 (627 mg, 1.06 mmol, 96%) as a single diastereomer, as a colorless oil. R_f = 0.30 (hexanes/EtOAc, 85:15); FTIR (thin film), ν_max (cm⁻¹): 3319, 2942, 2865, 2166, 1749, 1720, 1549, 1461, 1382, 1208, 1159, 1106, 1092, 1061; ¹H NMR (500 MHz, CDCl₃): δ 6.90 (d, J = 7.5 Hz, 1H), 4.93 (s, 1H), 4.64 (app td, J = 7.5, 5.2 Hz, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.53 (dd, J = 5.9, 0.8 Hz, 1H), 4.36 (app td, J = 7.8, 0.8 Hz, 1H), 3.79 (s, 3H), 3.33 (s, 3H), 2.58–2.51 (m, 1H), 2.29 (dddd, J = 13.9, 11.4, 5.2, 4.7 Hz, 1H), 1.90–1.81 (m, 2H), 1.63 (ddd, J = 13.6, 7.8, 6.7 Hz, 1H), 1.59–1.44 (m, 2H), 1.46 (s, 3H), 1.30 (s, 3H), 1.06–1.02 (m, 21H); ¹³C NMR (126 MHz, CDCl₃): δ 171.4, 156.9 (q, ²J_C-F = 38 Hz), 115.7 (q, ¹J_C-F = 288 Hz), 112.6, 109.6, 109.5, 85.6, 84.9, 83.9, 83.5, 55.1, 53.1, 52.7, 40.3, 30.4, 30.1, 29.3, 26.6, 25.2, 18.7, 11.3; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.8; HRMS (ESI+): calcd. for [C₂₈H₄₆F₃N₁O₇Si₁Na]⁺ 616.2888, meas. 616.2890, Δ 0.3 ppm.

Methyl (2S,5R)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)hept-6-ynoate (S16).

To a solution of TIPS alkyne S15 (108 mg, 0.182 mmol) in DMF (4 mL) at RT, was added a solution of TASF (0.15 g, 0.55 mmol, 3.0 equiv.) in DMF (3.3 mL). The resulting mixture was stirred at 60 °C for 2.5 h and volatiles were removed in vacuo. The residue was partitioned between Et₂O (7 mL) and a 1:1 mixture of brine and 5% LiCl aq. solution (7 mL). The layers were separated and the aqueous phase was extracted with Et₂O (3 × 7 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 5 → 50%) to afford alkyne S16 (80 mg, 0.18 mmol, quant.) as a colorless oil. R_f = 0.26 (hexanes/EtOAc, 70:30); FTIR (thin film), ν_max (cm⁻¹): 3288, 2936, 1747, 1719, 1552, 1456, 1440, 1383, 1374, 1209, 1160, 1106, 1091, 1059; ¹H NMR (500 MHz, CDCl₃): δ 6.96 (d, J = 7.5 Hz, 1H), 4.93 (s, 1H), 4.63 (ddd, J = 7.5, 7.0, 5.8 Hz, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.51 (dd, J = 5.9, 1.0 Hz, 1H), 4.33 (app td, J = 7.5, 1.0 Hz, 1H), 3.80 (s, 3H), 3.33 (s, 3H), 2.50 (ddddd, J = 9.5, 7.3, 6.8, 4.6, 2.4 Hz, 1H), 2.22 (dddd, J = 14.0, 11.2, 5.8, 4.8 Hz, 1H), 2.13 (d, J = 2.4 Hz, 1H), 1.90–1.82 (m, 2H), 1.64 (ddd, J = 13.7, 7.5, 6.8 Hz, 1H), 1.60–1.44 (m, 2H), 1.47 (s, 3H), 1.30 (s, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 171.3, 156.9 (q, ²J_C-F = 38 Hz), 115.7 (q, ¹J_C-F = 288 Hz), 112.6, 109.8, 85.6, 85.4, 84.6, 84.0, 71.2, 55.2, 53.1, 52.6, 39.8, 29.8, 29.6, 28.2, 26.6, 25.1; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.8; HRMS (ESI+): calcd. for [C₁₉H₂₆F₃N₁O₇Na]⁺ 460.1554, meas. 460.1562, Δ 1.9 ppm.

(3R,4R,5R)-5-((2R,5S)-2-ethynyl-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-2,3,4-triyl triacetate (S17).

To a solution of acetonide S16 (80 mg, 0.18 mmol) in CH₂Cl₂ (0.8 mL) at 0 °C, was added a 4:1 mixture of TFA and water (3.6 mL). The resulting mixture was stirred at RT for 5.5 h. Volatiles were removed in vacuo and the residue was dried by azeotropic distillation with benzene (2 × 0.8 mL). Crude material was used directly in the next step without further purification. To a solution of the crude triol in CH₂Cl₂ (3.6 mL) at RT, were added pyridine (0.15 mL, 1.8 mmol, 10 equiv.), Ac₂O (0.17 mL, 1.8 mmol, 10 equiv.) and DMAP (16 mg, 0.13 mmol, 0.70 equiv.) sequentially. The resulting mixture was stirred at RT for 15 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 5 → 70%) to afford triacetate S17 (60 mg, 0.12 mmol, 64% over 2 steps) as a 2:1 mixture of diastereomers, as a colorless oil. R_f = 0.35/0.43 (hexanes/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3290, 2930, 1746, 1722, 1554, 1438, 1372, 1214, 1111, 1011; ¹H NMR (500 MHz, CDCl₃): δ 7.12 (d, J = 8.1 Hz, 2H), 6.98 (d, J = 8.0 Hz, 1H), 6.35 (d, J = 4.5 Hz, 1H), 6.06 (d, J = 1.0 Hz, 2H), 5.30 (dd, J = 4.8, 1.0 Hz, 2H), 5.25–5.20 (m, 3H), 5.17 (dd, J = 6.9, 3.5 Hz, 1H), 4.66–4.59 (m, 3H), 4.38–4.32 (m, 3H), 3.80 (s, 3H), 3.79 (s, 6H), 2.60–2.50 (m, 3H), 2.25–2.15 (m, 2H), 2.14 (d, J = 2.5 Hz, 1H), 2.13 (d, J = 2.5 Hz, 2H), 2.12 (s, 6H), 2.11 (s, 3H), 2.11 (s, 3H), 2.09 (s, 6H), 2.07 (s, 6H), 2.06 (s, 3H), 1.93–1.83 (m, 4H), 1.83–1.74 (m, 6H), 1.61–1.47 (m, 6H); ¹³C NMR (126 MHz, CDCl₃): δ 171.2, 170.2, 170.0, 169.9, 169.7, 169.6, 169.5, 157.1 (q, ²J_C-F = 38 Hz), 156.9 (q, ²J_C-F = 37 Hz), 115.8 (q, ¹J_C-F = 287 Hz), 115.7 (q, ¹J_C-F = 288 Hz), 98.5, 93.9, 85.2, 85.0, 81.3, 79.2, 74.4, 73.7, 72.0, 71.6, 71.2, 69.9, 53.1, 53.1, 52.6, 52.5, 38.4, 37.9, 29.7, 29.6, 29.5, 27.8, 27.3, 21.2, 21.1, 20.8, 20.6, 20.6, 20.4; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.75, −76.76; HRMS (ESI+): calcd. for [C₂₁H₂₆F₃N₁O₁₀Na]⁺ 532.1401, meas. 532.1416, Δ 2.8 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2R,5S)-2-ethynyl-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S18).

To a suspension of N⁶-benzoyladenine (113 mg, 0.471 mmol, 4.0 equiv.) in propionitrile (dried over 4 Å M.S., 2.4 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (0.14 mL, 0.59 mmol, 5.0 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which complete solubilization of N⁶-benzoyladenine was observed. To a separate flask charged with triacetate S17 (dried by azeotropic distillation with benzene (4 ×), 60 mg, 0.12 mmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (21 mg, 59 μmol, 50 mol %) was added propionitrile (2.4 mL). The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained, and the solution of silylated N⁶-benzoyladenine was added. The reaction mixture was stirred at 95 °C for 5.5 h, cooled to RT and the volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 30 → 100%) to afford nucleoside S18 (53 mg, 77 μmol, 65%) as a colorless oil. R_f = 0.33 (hexanes/EtOAc, 20:80); FTIR (thin film), ν_max (cm⁻¹): 3283, 3084, 2929, 1745, 1720, 1612, 1583, 1511, 1487, 1456, 1374, 1244, 1218, 1075, 1029; ¹H NMR (500 MHz, CDCl₃): δ 9.07 (s, 1H), 8.79 (s, 1H), 8.11 (s, 1H), 8.04–7.98 (m, 2H), 7.63–7.57 (m, 1H), 7.54–7.49 (m, 2H), 7.04 (br d, J = 7.6 Hz, 1H), 6.12 (d, J = 5.5 Hz, 1H), 6.04 (app t, J = 5.5 Hz, 1H), 5.58 (dd, J = 5.5, 4.5 Hz, 1H), 4.60 (ddd, J = 7.6, 7.0, 5.4 Hz, 1H), 4.38 (ddd, J = 7.5, 5.8, 4.5 Hz, 1H), 3.76 (s, 3H), 2.57 (dddd, J = 14.0, 8.2, 6.4, 2.4 Hz, 1H), 2.23–2.12 (m, 2H), 2.15 (s, 3H), 2.15 (d, J = 2.4 Hz, 1H), 2.06 (s, 3H), 2.00–1.93 (m, 1H), 1.86–1.77 (m, 1H), 1.54–1.48 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 171.2, 169.8, 169.6, 164.8, 156.9 (q, ²J_C-F = 38 Hz), 153.0, 151.8, 149.9, 142.0, 133.5, 133.0, 129.0, 128.0, 124.0, 115.7 (q, ¹J_C-F = 288 Hz), 86.7, 85.1, 80.6, 73.3, 72.8, 71.5, 53.2, 52.5, 37.7, 29.6, 29.6, 27.5, 20.7, 20.5; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.7; HRMS (ESI+): calcd. for [C₃₁H₃₂F₃N₆O₉]⁺ 689.2177, meas. 689.2178, Δ 0.1 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2R,5S)-2-((3-carbamoylphenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S19).

To a solution of nucleoside S18 (53 mg, 77 μmol) and 3-iodobenzamide (48 mg, 0.19 mmol, 2.5 equiv.) in a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 3.9 mL) at RT, were introduced CuI (4 mg, 0.02 mmol, 25 mol %) and Pd(PPh₃)₄ (4 mg, 4 μmol, 5 mol %) rapidly. The resulting mixture was stirred at 60 °C for 2 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 5%) to afford alkyne S19 (50 mg, 62 μmol, 80%) as a colorless oil. R_f = 0.43 (EtOAc/i-PrOH, 95:5); FTIR (thin film), ν_max (cm⁻¹): 3284, 2932, 1746, 1716, 1668, 1611, 1580, 1455, 1375, 1242, 1215, 1183, 1160, 1075; ¹H NMR (600 MHz, CDCl₃): δ 9.23 (s, 1H), 8.75 (s, 1H), 8.18 (s, 1H), 8.04–7.98 (m, 2H), 7.84–7.82 (m, 1H), 7.80 (ddd, J = 7.7, 1.9, 1.3 Hz, 1H), 7.61–7.56 (m, 1H), 7.52–7.47 (m, 2H), 7.46 (app dt, J = 7.7, 1.3 Hz, 1H), 7.36 (br d, J = 7.3 Hz, 1H), 7.34 (app t, J = 7.7 Hz, 1H), 6.91 (br s, 1H), 6.16 (d, J = 5.7 Hz, 1H), 6.13 (dd, J = 5.7, 5.4 Hz, 1H), 5.99 (br s, 1H), 5.76 (dd, J = 5.4, 4.3 Hz, 1H), 4.68–4.63 (m, 1H), 4.43 (ddd, J = 7.0, 5.5, 4.3 Hz, 1H), 3.76 (s, 3H), 2.87–2.81 (m, 1H), 2.33–2.26 (m, 1H), 2.26 (ddd, J = 14.2, 8.4, 7.0 Hz, 1H), 2.14 (s, 3H), 2.06 (s, 3H), 2.05–2.01 (m, 1H), 1.97–1.89 (m, 1H), 1.61–1.55 (m, 2H); ¹³C NMR (126 MHz, CDCl₃): δ 171.3, 170.0, 169.6, 168.8, 165.0, 157.1 (q, ²J_C-F = 38 Hz), 152.9, 151.9, 149.9, 142.2, 134.5, 133.6, 133.5, 133.0, 130.6, 129.0, 128.8, 128.1, 127.7, 124.1, 123.3, 115.7 (q, ¹J_C-F = 288 Hz), 91.7, 86.7, 82.9, 81.1, 73.2, 72.7, 532, 52.7, 37.6, 30.2, 29.6, 28.0, 20.8, 20.5; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.7; HRMS (ESI+): calcd. for [C₃₈H₃₇F₃N₇O₁₀]⁺ 808.2549, meas. 808.2541, Δ 0.9 ppm.

3-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)propan-1-ol (S21).⁷¹

To a solution of known α,β-unsaturated methyl ester S20⁷² (1.22 g, 4.72 mmol) in a 10:1 mixture of Et₂O and MeOH (22 mL) at 0 °C, was added a solution of LiBH₄ (2.0 M in THF, 9.45 mL, 18.9 mmol, 4.0 equiv.) dropwise. The resulting mixture was allowed to warm gradually to RT and stirred for 17 h, before the addition of a 1:1 mixture of a NH₄Cl sat. aq. solution and water (35 mL). The layers were separated and the aqueous phase was extracted with Et₂O (3 × 35 mL). The combined organic extracts were washed with brine (100 mL), dried over MgSO₄, filtered and concentrated to give alcohol S21 (1.10 g, 4.72 mmol, quant.) as a colorless oil. R_f = 0.24 (hexanes/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3422, 2989, 2937, 1449, 1373, 1210, 1160, 1088, 1054, 1015; ¹H NMR (600 MHz, CDCl₃): δ 4.95 (s, 1H), 4.61 (d, J = 5.9 Hz, 1H), 4.54 (dd, J = 5.9, 1.0 Hz, 1H), 4.20–4.16 (m, 1H), 3.70–3.67 (m, 2H), 3.36 (s, 3H), 1.76–1.59 (m, 4H), 1.48 (s, 3H), 1.32 (s, 3H); ¹³C NMR (151 MHz, CDCl₃): δ 112.4, 109.7, 87.2, 85.5, 84.3, 62.3, 55.1, 31.6, 29.6, 26.6, 25.1; HRMS (ESI+): calcd. for [C₁₁H₂₀O₅Na]⁺ 255.1203, meas. 255.1196, Δ 2.6 ppm.

3-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)propanal (S22).⁷³

To a solution of alcohol S21 (400 mg, 1.72 mmol) in CH₂Cl₂ (11.5 mL) at RT, was added DMP (876 mg, 2.07 mmol, 1.2 equiv.) in one portion. The resulting mixture was stirred at RT for 1 h, before the addition of a 1:1 mixture of a Na₂S₂O₃ sat. aq. solution and a NaHCO₃ sat. aq. solution (10 mL). The biphasic mixture was stirred vigorously at RT for 10 min and the layers were separated. The aqueous phase was extracted with Et₂O (3 × 10 mL) and the combined organic extracts were washed with brine (40 mL), dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (pentane/EtOAc, 2 → 50%) to afford aldehyde S22 (324 mg, 1.41 mmol, 82%) as a colorless oil. R_f = 0.32 (hexanes/EtOAc, 75:25); FTIR (thin film), ν_max (cm⁻¹): 2988, 2936, 2834, 2724, 1721, 1373, 1209, 1160, 1090, 1058; ¹H NMR (500 MHz, CDCl₃): δ 9.79 (t, J = 1.3 Hz, 1H), 4.93 (s, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.52 (dd, J = 5.9, 1.1 Hz, 1H), 4.14 (ddd, J = 9.2, 6.5, 1.1 Hz, 1H), 3.32 (s, 3H), 2.62 (dddd, J = 18.1, 7.9, 6.6, 1.3 Hz, 1H), 2.57 (dddd, J = 18.1, 8.2, 6.9, 1.3 Hz, 1H), 1.89–1.82 (m, 2H), 1.45 (s, 3H), 1.30 (s, 3H); ¹³C NMR (151 MHz, CDCl₃): δ 201.4, 112.5, 109.9, 86.3, 85.6, 84.1, 55.3, 40.8, 27.5, 26.6, 25.1; HRMS (ESI+): calcd. for [C₁₁H₁₈O₅Na]⁺ 253.1046, meas. 253.1036, Δ 4.0 ppm.

(3aR,4R,6R,6aR)-4-(but-3-yn-1-yl)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole (S23).⁷⁴

To a solution of aldehyde S22 (324 mg, 1.41 mmol) in MeOH (6.0 mL) at RT, was added K₂CO₃ (583 mg, 4.22 mmol, 3.0 equiv.), followed by a solution of dimethyl-1-diazo-2-oxopropyl phosphonate (338 mg, 1.76 mmol, 1.25 equiv.) in MeOH (3.0 mL) under vigorous stirring. The resulting yellow suspension was stirred at RT for 3 h. The reaction was quenched by the addition of a NH₄Cl sat. aq. solution (15 mL) and Et₂O (25 mL) was added. The layers were separated and the aqueous phase was extracted with Et₂O (3 × 25 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (hexane/Et₂O, 2 → 50%) to afford alkyne S23 (254 mg, 1.12 mmol, 80%) as a colorless oil. R_f = 0.50 (hexanes/EtOAc, 75:25); ¹H NMR (600 MHz, CDCl₃): δ 4.95 (s, 1H), 4.60 (d, J = 5.9 Hz, 1H), 4.56 (dd, J = 5.9, 1.0 Hz, 1H), 4.30 (ddd, J = 9.8, 5.8, 1.0 Hz, 1H), 3.34 (s, 3H), 2.36 (dddd, J = 16.8, 7.8, 5.9, 2.6 Hz, 1H), 2.31 (dddd, J = 16.8, 7.8, 7.3, 2.6 Hz, 1H), 1.98 (t, J = 2.6 Hz, 1H), 1.80 (dddd, J = 13.4, 9.8, 7.3, 5.9 Hz, 1H), 1.74 (app dtd, J = 13.4, 7.8, 5.8 Hz, 1H), 1.48 (s, 3H), 1.31 (s, 3H); ¹³C NMR (151 MHz, CDCl₃): δ 112.5, 109.8, 85.9, 85.7, 84.1, 83.3, 69.1, 55.3, 33.9, 26.6, 25.1, 15.6; HRMS (ESI+): calcd. for [C₁₂H₁₈O₄Na]⁺ 249.1097, meas. 249.1118, Δ 8.4 ppm.

(3R,4R,5R)-5-(but-3-yn-1-yl)tetrahydrofuran-2,3,4-triyl triacetate (S24).

To a vial charged with acetonide S23 (50 mg, 0.22 mmol) was added a 4:1 mixture of acetic acid and water (2.5 mL) at RT. The resulting mixture was stirred at 80 °C for 5 h. Volatiles were removed in vacuo and the residue was dried by azeotropic distillation with benzene (2 × 1.0 mL). Crude material was used directly in the next step without further purification. To a solution of the crude triol in CH₂Cl₂ (2.5 mL) at RT, were added pyridine (0.18 mL, 2.2 mmol, 10 equiv.), Ac₂O (0.21 mL, 2.2 mmol, 10 equiv.) and DMAP (19 mg, 0.15 mmol, 0.70 equiv.) sequentially. The resulting mixture was stirred at RT for 2 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 2 → 60%) to afford triacetate S24 (58 mg, 0.19 mmol, 88% over 2 steps) as a 1:1 mixture of diastereomers, as a colorless oil; R_f = 0.42/0.50 (hexanes/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3285, 2958, 2920, 2850, 2117, 1742, 1433, 1371, 1214, 1107, 1010; ¹H NMR (600 MHz, CDCl₃): δ 6.36 (d, J = 4.6 Hz, 1H), 6.12 (d, J = 1.0 Hz, 1H), 5.30 (dd, J = 4.8, 1.0 Hz, 1H), 5.21 (dd, J = 6.8, 4.6 Hz, 1H), 5.21 (dd, J = 7.0, 4.8 Hz, 1H), 5.10 (dd, J = 6.8, 3.6 Hz, 1H), 4.33 (ddd, J = 8.5, 4.9, 3.6 Hz, 1H), 4.27 (ddd, J = 8.5, 7.0, 4.6 Hz, 1H), 2.39–2.25 (m, 4H), 2.11 (s, 6H), 2.10 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 1.97 (t, J = 2.8 Hz, 1H), 1.96 (t, J = 2.6 Hz, 1H), 1.95–1.86 (m, 2H), 1.86–1.78 (m, 2H); ¹³C NMR (126 MHz, CDCl₃): δ 170.2, 170.0, 169.8, 169.6, 169.5, 169.2, 98.3, 93.9, 83.0, 82.8, 82.1, 80.3, 74.7, 73.5, 72.1, 70.0, 69.4, 69.2, 33.2, 32.3, 21.2, 21.2, 20.8, 20.7, 20.6, 20.4, 14.9, 14.8; HRMS (ESI+): calcd. for [C₁₄H₁₈O₇Na]⁺ 321.0945, meas. 321.0952, Δ 2.3 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-(but-3-yn-1-yl)tetrahydrofuran-3,4-diyl diacetate (S25).

To a suspension of N⁶-benzoyladenine (160 mg, 0.670 mmol, 4.0 equiv.) in propionitrile (dried over 4 Å M.S., 5.0 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (0.20 mL, 0.84 mmol, 5.0 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which complete solubilization of N⁶-benzoyladenine was observed. To a separate flask charged with triacetate S24 (dried by azeotropic distillation with benzene (4 ×), 50 mg, 0.17 mmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (30 mg, 84 μmol, 50 mol %) was added propionitrile (5.0 mL). The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained, and the solution of silylated N⁶-benzoyladenine was added. The reaction mixture was stirred at 95 °C for 17 h, cooled to RT and the volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 100%) to afford nucleoside S25 (73 mg, 0.15 mmol, 91%) as a colorless oil. R_f = 0.31 (EtOAc); FTIR (thin film), ν_max (cm⁻¹): 3291, 3091, 2934, 1747, 1699, 1609, 1582, 1510, 1486, 1455, 1373, 1239, 1217, 1099, 1073, 1053; ¹H NMR (600 MHz, CDCl₃): δ 9.12 (s, 1H), 8.78 (s, 1H), 8.09 (s, 1H), 8.04–7.98 (m, 2H), 7.62–7.57 (m, 1H), 7.54–7.48 (m, 2H), 6.13 (d, J = 5.4 Hz, 1H), 6.04 (dd, J = 5.6, 5.4 Hz, 1H), 5.57 (dd, J = 5.6, 4.7 Hz, 1H), 4.37 (ddd, J = 9.2, 4.7, 4.3 Hz, 1H), 2.38 (dddd, J = 17.0, 7.0, 5.6, 2.6 Hz, 1H), 2.31 (dddd, J = 17.0, 8.5, 6.8, 2.6 Hz, 1H), 2.15 (s, 3H), 2.16–2.09 (m, 1H), 2.06 (s, 3H), 2.06–2.00 (m, 1H), 1.99 (t, J = 2.6 Hz, 1H); ¹³C NMR (151 MHz, CDCl₃): δ 169.8, 169.6, 164.8, 153.0, 151.7, 149.9, 142.1, 133.6, 133.0, 129.0, 128.0, 124.0, 86.9, 82.8, 81.3, 73.3, 73.1, 69.6, 31.8, 20.7, 20.5, 14.8; HRMS (ESI+): calcd. for [C₂₄H₂₄N₅O₆]⁺ 478.1721, meas. 478.1728, Δ 1.5 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-(4-(3-carbamoylphenyl)but-3-yn-1-yl)tetrahydrofuran-3,4-diyl diacetate (S26).

To a vial charged with alkyne S25 (72 mg, 0.15 mmol) were added 3-iodobenzamide (75 mg, 0.30 mmol, 2.0 equiv.), CuI (7 mg, 38 μmol, 25 mol %) and Pd(PPh₃)₄ (9 mg, 8 μmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 6.0 mL) was added at RT. The resulting mixture was stirred at 80 °C for 1 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 20%) to afford alkyne S26 (58 mg, 97 μmol, 64%) as a colorless oil. R_f = 0.24 (EtOAc/i-PrOH, 90:10); FTIR (thin film), ν_max (cm⁻¹): 3187, 2931, 1747, 1664, 1609, 1578, 1511, 1486, 1454, 1373, 1239, 1215, 1097, 1049; ¹H NMR (600 MHz, CDCl₃): δ 9.30 (s, 1H), 8.76 (s, 1H), 8.17 (s, 1H), 8.04–7.97 (m, 2H), 7.83 (app t, J = 1.5 Hz, 1H), 7.74 (app dt, J = 7.8, 1.5 Hz, 1H), 7.59–7.55 (m, 1H), 7.50–7.45 (m, 3H), 7.33 (app t, J = 7.8 Hz, 1H), 6.64 (br s, 1H), 6.17 (d, J = 5.6 Hz, 1H), 6.13 (br s, 1H), 6.11 (app t, J = 5.6 Hz, 1H), 5.71 (dd, J = 5.6, 4.3 Hz, 1H), 4.40 (ddd, J = 8.1, 4.9, 4.3 Hz, 1H), 2.61 (app dt, J = 17.2, 6.3 Hz, 1H), 2.53 (ddd, J = 17.2, 8.4, 6.1 Hz, 1H), 2.23–2.17 (m, 1H), 2.14 (s, 3H), 2.16–2.09 (m, 1H), 2.05 (s, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 170.0, 169.7, 168.9, 165.0, 152.9, 151.9, 149.9, 142.2, 134.7, 133.7, 133.5, 133.0, 130.6, 128.9, 128.7, 128.1, 127.1, 124.1, 124.0, 89.7, 86.7, 81.8, 80.8, 73.3, 73.0, 31.7, 20.8, 20.5, 15.6; HRMS (ESI+): calcd. for [C₃₁H₂₉N₆O₇]⁺ 597.2092, meas. 597.2097, Δ 0.8 ppm.

((1R,2R)-2-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)cyclopropyl)methanol (S28).

The following protocol was adapted from an early publication reporting the asymmetric cyclopropanation of allylic alcohols with dioxaborolane ligands.⁷⁵ To a stirred solution of diethylzinc in hexanes (1.0 M in hexanes, 4.46 mL, 4.46 mmol, 2.23 equiv.) was added CH₂Cl₂ (8.0 mL). The mixture was cooled to 0 °C and diiodomethane (0.72 mL, 8.9 mmol, 4.45 equiv.) was added dropwise over 5 min. The reaction mixture was stirred at 0 °C for 15 min (white precipitate was formed), and a pre-cooled (0 °C) solution of butylboronic acid N,N,N’,N’-tetramethyl-D-tartaric acid diamide ester (360 mg, 2.28 mmol, 1.14 equiv.) and allylic alcohol S27⁷² (457 mg, 2.00 mmol) in CH₂Cl₂ (12 mL) was rapidly added via syringe. The resulting mixture was stirred at RT for 1 h, cooled to 0 °C and a NH₄Cl sat. aq. solution (15 mL) was added slowly. The layers were separated and the aqueous phase was extracted with CH₂Cl₂ (3 × 30 mL). The combined organic extracts were passed through a phase separator (Biotage Isolute), dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 100%) to afford cyclopropyl alcohol S28 (382 mg, 1.56 mmol, 78%) as a 12:1 mixture of cyclopropyl diastereomers (major=desired), as a colorless oil. R_f = 0.24 (cyclohexane/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3446, 2991, 2935, 2251, 2041, 1458, 1382, 1373, 1274, 1210, 1194, 1159, 1105, 1089, 1053, 1030; ¹H NMR (600 MHz, CDCl₃): δ 4.95 (s, 1H), 4.74 (d, J = 5.9 Hz, 1H), 4.66 (d, J = 5.9 Hz, 1H), 3.51–3.43 (m, 3H), 3.38 (s, 3H), 1.45 (s, 3H), 1.31 (s, 3H), 1.21 (dddd, J = 12.0, 8.4, 6.9, 5.1 Hz, 1H), 0.94 (ddt, J = 10.4, 9.0, 4.7 Hz, 1H), 0.53 (ddt, J = 17.2, 8.4, 5.1 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 112.1, 109.1, 91.1, 85.3, 84.0, 65.5, 54.4, 26.3, 24.8, 20.8, 20.4, 8.1; HRMS (ESI+): calcd. for [C₁₂H₂₀O₅Na]⁺ 267.1203, meas. 267.1202, Δ 0.2 ppm.

(1R,2R)-2-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)cyclopropane-1-carbaldehyde (S29).

To a solution of cyclopropyl alcohol S28 (245 mg, 1.00 mmol) in CH₂Cl₂ (20 mL) open to atmosphere at RT was added DMP (510 mg, 1.20 mmol, 1.2 equiv.) portionwise over 5 min. The reaction mixture was stirred for 2 h and was diluted with Et₂O (10 mL). The suspension was filtered and the volatiles were removed in vacuo. The crude residue was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 50%) to yield aldehyde S29 (182 mg, 0.751 mmol, 75%) as a clear oil. In our hands, aldehyde S29 was best prepared, purified and used immediately in the next reaction. R_f = 0.39 (cyclohexane/EtOAc, 67:33); ¹H NMR (600 MHz, CDCl₃): δ 9.16 (d, J = 4.9 Hz, 1H), 4.96 (s, 1H), 4.75 (d, J = 5.9 Hz, 1H), 4.66 (d, J = 5.8 Hz, 1H), 3.55 (d, J = 10.0 Hz, 1H), 3.28 (s, 3H), 2.02 (app dtd, J = 7.8, 4.9, 3.9 Hz, 1H), 1.79 (app tdd, J = 10.1, 8.9, 6.4, 4.0 Hz, 1H), 1.45 (s, 3H), 1.33 (dd, J = 8.9, 4.9 Hz, 1H), 1.31 (s, 3H), 1.07 (ddd, J = 8.3, 6.4, 4.9 Hz, 1H); ¹³C NMR (151 MHz, CDCl₃): δ 199.9, 112.5, 109.2, 89.3, 85.4, 84.0, 54.6, 28.9, 26.4, 26.1, 25.0, 12.7.

(3aR,4R,6R,6aR)-4-((1R,2R)-2-ethynylcyclopropyl)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole (S30).

To a solution of aldehyde S29 (182 mg, 0.751 mmol) in MeOH (4.0 mL) at RT was added K₂CO₃ (312 mg, 2.26 mmol, 3.0 equiv.) followed by a solution of dimethyl-1-diazo-2-oxopropyl phosphonate (180 mg, 0.939 mmol, 1.25 equiv.) in MeOH (3.0 mL) under vigorous stirring. The resulting yellow suspension was stirred at RT for 3 h. The reaction was quenched by the addition of a NH₄Cl sat. aq. solution (10 mL) and CH₂Cl₂ (15 mL) was added. The layers were separated and the aqueous phase was extracted with CH₂Cl₂ (3 × 15 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (cyclohexane/EtOAc, 0 → 50%) to afford alkyne S30 (98 mg, 0.41 mmol, 55 %) as a colorless oil. Alkyne S30 crystallized when stored as a neat oil at −20 °C for 1 week. A small molecule X-ray structure of S30 was obtained, confirming its absolute configuration as shown in the Supporting Information. R_f = 0.68 (cyclohexane/EtOAc, 67:33); FTIR (thin film), ν_max (cm⁻¹): 3283, 2989, 2935, 2834, 2116, 1458, 1407, 1382, 1373, 1307, 1273, 1233, 1209, 1194, 1159, 1104, 1090, 1056, 1034, 1023; ¹H NMR (600 MHz, CDCl₃): δ 4.99 (s, 1H), 4.73 (d, J = 5.9 Hz, 1H), 4.66 (d, J = 5.9 Hz, 1H), 3.42 (s, 3H), 3.39–3.34 (m, 1H), 1.80 (d, J = 2.0 Hz, 1H), 1.45 (s, 3H), 1.44–1.38 (m, 1H), 1.38–1.32 (m, 1H), 1.3 (s, 3H), 0.94 (app dt, J = 8.5, 5.0 Hz, 1H), 0.79–0.74 (m, 1H); ¹³C NMR (151 MHz, CDCl₃): δ 112.4, 109.1, 90.5, 85.9, 85.6, 84.1, 64.8, 54.6, 26.5, 26.1, 25.0, 13.2, 6.1; HRMS (ESI+): calcd. for [C₁₃H₁₉O₄]⁺ 239.1278, meas. 239.1288, Δ 4.4 ppm.

(3R,4R,5R)-5-((1R,2R)-2-ethynylcyclopropyl)tetrahydrofuran-2,3,4-triyl triacetate (S31).

To a vial charged with acetonide S30 (97 mg, 0.41 mmol) was added a 4:1 mixture of acetic acid and water (5.0 mL) at RT. The resulting mixture was stirred at 80 °C for 6 h. Volatiles were removed in vacuo and the residue was dried by azeotropic distillation with benzene (2 × 1.0 mL). Crude material was used directly in the next step without further purification. To a solution of the crude triol in CH₂Cl₂ (5.0 mL) at RT, were added pyridine (0.99 mL, 12 mmol, 30 equiv.), Ac₂O (0.50 mL, 5.3 mmol, 13 equiv.) and DMAP (10 mg, 0.082 mmol, 0.20 equiv.) sequentially. The resulting mixture was stirred at RT for 2 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 100%) to afford triacetate S31 (63 mg, 0.20 mmol, 50% over 2 steps) as a 4:5 mixture of α and β anomers, as a colorless oil. R_f = 0.60 (α-anomer) / 0.74 (β-anomer) (cyclohexane/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3284, 2580, 1742, 1431, 1370, 1210, 1103, 1044, 1009; ¹H NMR (400 MHz, CDCl₃): δ α-anomer: 6.38 (dd, J = 3.9, 0.8 Hz, 1H), 5.26–5.19 (m, 2H), 3.85 (dd, J = 6.8, 2.9 Hz, 1H), 2.12 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 1.82 (d, J = 2.1 Hz, 1H), 1.42 (dddd, J = 8.8, 6.7, 6.0, 4.5 Hz, 1H), 1.31 (app. dtd, J = 9.5, 5.0, 2.1 Hz, 1H), 0.97 (app. dt, J = 8.7, 5.1 Hz, 1H), 0.91 (ddd, J = 8.9, 6.1, 4.8 Hz, 1H); β-anomer: 6.12 (d, J = 1.1 Hz, 1H), 5.33 (dd, J = 4.8, 1.1 Hz, 1H), 5.30 (dd, J = 6.6, 4.8 Hz, 1H), 3.75 (t, J = 6.8 Hz, 1H), 2.12 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 1.82 (d, J = 2.1 Hz, 1H), 1.39 (dddd, J = 8.8, 7.0, 6.0, 4.4 Hz, 1H), 1.37–1.26 (m, 1H), 0.95 (app. dt, J = 8.7, 5.0 Hz, 1H), 0.85 (ddd, J = 8.8, 6.0, 4.8 Hz, 1H); ¹³C NMR (151 MHz, CDCl₃): δ α-anomer: 170.1, 169.7, 169.4, 93.9, 85.2, 84.5, 72.6, 70.0, 65.3, 23.7, 21.1, 20.8, 20.4, 11.7, 4.6; β-anomer: 169.8, 169.5, 169.3, 98.1, 82.8, 74.6, 73.8, 65.1, 55.5, 24.0, 21.2, 20.7, 20.6, 11.7, 4.4; HRMS (ESI+): calcd. for [C₁₅H₁₈O₇Na]⁺ 333.0945, meas. 333.0970, Δ 7.5 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((1R,2R)-2-ethynylcyclopropyl)tetrahydrofuran-3,4-diyl diacetate (S32).

To a suspension of N⁶-benzoyladenine (114 mg, 0.478 mmol, 2.15 equiv.) in propionitrile (dried over 4 Å M.S., 6.0 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (0.16 mL, 0.67 mmol, 3.00 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which complete solubilization of N⁶-benzoyladenine was observed. To a separate flask charged with triacetate S31 (dried by azeotropic distillation with benzene (4 ×), 69 mg, 0.22 mmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (16 mg, 44 μmol, 20 mol %) was added propionitrile (6.0 mL). The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained, and the solution of silylated N⁶-benzoyladenine was added. The reaction mixture was stirred at 95 °C for 2.5 h, cooled to RT and the volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 90:10) to afford alkyne S32, which was further purified by preparative HPLC (5% → 95% B’ over 40 min) to afford alkyne S32 (63 mg, 0.13 mmol, 58%) as a white amorphous solid. R_f = 0.19 (EtOAc); FTIR (thin film), ν_max (cm⁻¹): 3295, 3020, 2931, 1748, 1696, 1650, 1612, 1583, 1512, 1488, 1458, 1375, 1240, 1215, 1072, 1050; ¹H NMR (500 MHz, CDCl₃): δ 8.80 (s, 1H), 8.24 (s, 1H), 8.07–8.02 (m, 2H), 7.65–7.59 (m, 1H), 7.56–7.50 (m, 2H), 6.13 (d, J = 5.4 Hz, 1H), 6.07 (app t, J = 5.4 Hz, 1H), 5.66 (dd, J = 5.4, 4.3 Hz, 1H), 3.68 (dd, J = 8.3, 4.3 Hz, 1H), 2.15 (s, 3H), 2.08 (s, 3H), 1.83 (d, J = 2.1 Hz, 1H), 1.78–1.71 (m, 1H), 1.45–1.39 (m, 1H), 1.06 (app dt, J = 8.8, 5.2 Hz, 1H), 0.92 (ddd, J = 8.8, 5.8, 5.0 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 169.7, 169.5, 165.1, 152.0, 151.9, 149.7, 142.5, 133.2, 133.1, 129.0, 128.3, 123.7, 87.1, 85.2, 84.9, 73.6, 73.2, 65.5, 23.6, 20.7, 20.5, 12.1, 5.3; HRMS (ESI+): calcd. for [C₂₅H₂₄N₅O₆]⁺ 490.1721, meas. 490.1725, Δ 0.8 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((1R,2R)-2-((3-carbamoylphenyl)ethynyl)cyclopropyl)tetrahydrofuran-3,4-diyl diacetate (S33).

To a vial charged with alkyne S32 (48 mg, 98 μmol) were added 3-iodobenzamide (61 mg, 0.25 mmol, 2.5 equiv.), CuI (5 mg, 0.03 mmol, 25 mol %) and Pd(PPh₃)₄ (6 mg, 5 μmol, 5 mol %). The vial headspace was purged with nitrogen for 10 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 5.0 mL) was added at RT. The resulting mixture was stirred at 60 °C for 1.5 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 75:25) to afford benzamide S33, which was further purified by preparative HPLC (15% → 95% B’ over 30 min) to afford benzamide S33 (50 mg, 82 μmol, 84%) as a white amorphous solid. R_f = 0.35 (EtOAc/i-PrOH, 95:5); FTIR (thin film), ν_max (cm⁻¹): 3351, 3199, 3067, 2934, 1748, 1664, 1611, 1583, 1514, 1457, 1375, 1241, 1215, 1072; ¹H NMR (500 MHz, CDCl₃): δ 8.80 (s, 1H), 8.35 (s, 1H), 8.03–7.99 (m, 2H), 7.76–7.74 (m, 1H), 7.74–7.71 (m, 1H), 7.62–7.57 (m, 1H), 7.52–7.46 (m, 3H), 7.33 (t, J = 7.7 Hz, 1H), 6.88 (br s, 1H), 6.80 (br s, 1H), 6.28 (dd, J = 5.8, 5.3 Hz, 1H), 6.17 (d, J = 5.8 Hz, 1H), 5.70 (dd, J = 5.3, 3.5 Hz, 1H), 3.69 (dd, J = 9.0, 3.5 Hz, 1H), 2.17 (s, 3H), 2.08 (s, 3H), 2.00–1.94 (m, 1H), 1.61–1.56 (m, 1H), 1.17 (app dt, J = 8.7, 5.2 Hz, 1H), 1.02 (app dt, J = 8.7, 5.4 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 170.2, 169.8, 169.6, 165.4, 151.9, 151.8, 149.6, 143.2, 135.4, 133.4, 132.8, 132.7, 130.7, 129.0, 128.9, 128.3, 127.2, 124.1, 123.6, 92.1, 87.5, 86.3, 76.3, 74.1, 72.9, 24.3, 20.8, 20.5, 13.0, 6.4; HRMS (ESI+): calcd. for [C₃₂H₂₉N₆O₇]⁺ 609.2092, meas. 609.2114, Δ 3.6 ppm.

Methyl (2S,5S)-7-(3-carbamoylphenyl)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)hept-6-ynoate (S34).

To a vial charged with alkyne S3 (140 mg, 0.320 mmol) were added 3-iodobenzamide (197 mg, 0.799 mmol, 2.5 equiv.), CuI (15 mg, 80 μmol, 25 mol %) and Pd(PPh₃)₄ (19 mg, 16 μmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 7.1 mL) was added at RT. The resulting mixture was stirred at 60 °C for 1 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 100%) to afford alkyne S34 (72 mg, 0.13 mmol, 40%) as a colorless solid. R_f = 0.43 (EtOAc); FTIR (thin-film), ν_max (cm⁻¹): 3307, 3066, 2935, 2253, 1719, 1665, 1612, 1575, 1438, 1382, 1274, 1209, 1158; ¹H NMR (500 MHz, CDCl₃): δ 7.83 (td, J = 1.8, 0.6 Hz, 1H), 7.75 (ddd, J = 7.8, 1.9, 1.2 Hz, 1H), 7.51 (dt, J = 7.7, 1.4 Hz, 1H), 7.41–7.30 (m, 2H), 6.47 (br. s, 1H), 5.97 (br. s, 1H), 4.95 (s, 1H), 4.69 (td, J = 7.6, 5.2 Hz, 1H), 4.61 (s, 2H), 4.50 (dd, J = 10.8, 4.2 Hz, 1H), 3.80 (s, 3H), 3.34 (s, 3H), 2.87 (ddt, J = 10.6, 9.0, 4.4 Hz, 1H), 2.26–2.04 (m, 2H), 1.81–1.50 (m, 4H), 1.48 (s, 3H), 1.31 (s, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 171.5, 168.8, 157.2 (q, ²J_C-F = 38 Hz), 134.9, 133.5, 130.8, 128.7, 127.2, 123.9, 115.8 (q, ¹J_C-F = 288 Hz), 112.5, 110.2, 91.7, 85.6, 85.3, 84.5, 83.0, 55.4, 53.2, 52.7, 40.5, 30.9, 29.7, 29.4, 26.6, 25.0; ¹⁹F NMR (376 MHz, CDCl₃, BTF IStd): δ −76.8; HRMS (ESI+): calcd. for [C₂₆H₃₁F₃N₂O₈Na]⁺ 579.1925, meas. 579.1928, Δ 0.5 ppm.

Methyl (2S,5S)-7-(3-carbamoylphenyl)-5-(((2R,3S,4R,5R)-3,4-dihydroxy-5-methoxytetrahydrofuran-2-yl)methyl)-2-(2,2,2-trifluoroacetamido)hept-6-ynoate (S35).

To a vial charged with acetonide S34 (38 mg, 68 μmol) was added a 2:1 mixture of MeOH and TFA (6.0 mL) at RT. The vial was sealed tightly and the reaction mixture was stirred at 85 °C for 6 h. Volatiles were removed in vacuo and the residue was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 15%) to afford, in order of elution, β-anomer S35 (15 mg, 29 μmol, 42%) and the α-anomer counterpart (14 mg, 27 μmol, 40%) as off-white solids. Only β-anomer S35 was used in the subsequent step. R_f = 0.37 (β-anomer) (EtOAc/MeOH, 91:9); FTIR (thin-film), ν_max (cm⁻¹): 3349, 3076, 2921, 1719, 1665, 1613, 1598, 1573, 1438, 1383, 1250, 1211, 1180, 1161, 1127, 1103, 1063, 1029; ¹H NMR (600 MHz, (CD₃)₂CO): δ β-anomer: 8.82 (d, J = 8.1 Hz, 1H), 7.96 (t, J = 1.8 Hz, 1H), 7.88 (dt, J = 7.9, 1.4 Hz, 1H), 7.56 (dt, J = 7.7, 1.4 Hz, 1H), 7.54 (br. s, 1H), 7.44 (t, J = 7.7 Hz, 1H), 6.70 (br. s, 1H), 4.74 (s, 1H), 4.62 (ddd, J = 9.6, 8.0, 4.9 Hz, 1H), 4.23 (d, J = 4.1 Hz, 1H), 4.18 (ddd, J = 10.5, 6.5, 2.8 Hz, 1H), 4.12 (d, J = 7.2 Hz, 1H), 3.98 (td, J = 6.7, 4.8 Hz, 1H), 3.91 (t, J = 4.4 Hz, 1H), 3.74 (s, 3H), 3.29 (s, 3H), 2.98 (ddd, J = 15.3, 9.5, 4.7 Hz, 1H), 2.23 (dddd, J = 13.7, 10.9, 6.2, 4.8 Hz, 1H), 2.15 (ddt, J = 13.8, 9.6, 5.0 Hz, 1H), 1.91 (ddd, J = 13.4, 10.6, 2.9 Hz, 1H), 1.80 (dddd, J = 13.1, 10.0, 6.2, 4.9 Hz, 1H), 1.75–1.67 (m, 1H), 1.65 (ddd, J = 13.3, 10.6, 4.4 Hz, 1H); ¹³C NMR (101 MHz, (CD₃)₂CO): δ β-anomer: 171.9, 168.3, 135.7, 135.1, 131.5, 129.4, 127.9, 124.9, 109.6, 93.3, 83.1, 82.0, 76.4, 76.2, 54.9, 53.6, 52.9, 42.1, 32.3; ¹⁹F NMR (471 MHz, CDCl₃ (500 μL)/(CD₃)₂CO (50 μL), BTF IStd): δ β-anomer: −76.6; HRMS (ESI+): calcd. for [C₂₃H₂₈F₃N₂O₈]⁺ 517.1792, meas. 517.1788, Δ 0.8 ppm.

Methyl (S,E)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-7-(triisopropylsilyl)hept-2-en-6-ynoate (S36).

To a solution of 5 (728 mg, 1.71 mmol) in PhMe (16 mL) at RT, was added methyl (triphenylphosphoranylidene)acetate (1.06 g, 3.18 mmol, 1.85 equiv.) in one portion. The resulting mixture was stirred at 50 °C for 2 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 2 → 40%) to afford S36 (747 mg, 1.55 mmol, 91%) as a 13:1 mixture of E and Z isomers, as a colorless oil. R_f = 0.35 (hexanes/Et₂O, 70:30); FTIR (thin film), ν_max (cm⁻¹): 2942, 2865, 2169, 1726, 1660, 1463, 1436, 1381, 1371, 1270, 1210, 1159, 1093, 1060; ¹H NMR (600 MHz, CDCl₃): δ 7.00 (app dt, J = 15.6, 7.3 Hz, 1H), 5.88 (app dt, J = 15.6, 1.4 Hz, 1H), 4.93 (s, 1H), 4.61 (dd, J = 5.9, 1.1 Hz, 1H), 4.57 (d, J = 5.9 Hz, 1H), 4.45 (ddd, J = 10.6, 4.3, 1.1 Hz, 1H), 3.71 (s, 3H), 3.31 (s, 3H), 2.80 (dddd, J = 11.2, 7.5, 6.0, 3.7 Hz, 1H), 2.43–2.34 (m, 2H), 1.69 (ddd, J = 13.3, 10.6, 3.7 Hz, 1H), 1.60 (ddd, J = 13.3, 11.2, 4.3 Hz, 1H), 1.46 (s, 3H), 1.30 (s, 3H), 1.06–1.02 (m, 21H); ¹³C NMR (126 MHz, CDCl₃): δ 166.7, 145.9, 123.3, 112.5, 109.8, 109.3, 85.6, 85.5, 84.5, 83.7, 55.2, 51.5, 40.6, 38.4, 29.7, 26.7, 25.4, 18.8, 11.3; HRMS (ESI+): calcd. for [C₂₆H₄₄O₆Si₁Na] 503.2799, meas. 503.2787, Δ 2.5 ppm.

Methyl (S)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-7-(triisopropylsilyl)hept-6-ynoate (S37).

To a vial charged with S36 (747 mg, 1.55 mmol) were added PhMe (degassed by sparging with nitrogen for 15 min, 16 mL) and t-BuOH (degassed by sparging with nitrogen for 15 min, 0.45 mL, 4.7 mmol, 3.0 equiv.) at RT, followed by the addition of (BDP)CuH (1.0 M in PhMe, 3.1 mL, 3.1 mmol, 2.0 equiv.). The resulting mixture was stirred for 4.5 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 1 → 25%) to afford S37 (750 mg, 1.55 mmol, quant.) as a colorless oil. R_f = 0.37 (hexanes/EtOAc, 80:20); FTIR (thin film), ν_max (cm⁻¹): 2942, 2865, 2165, 1741, 1462, 1381, 1371, 1209, 1160, 1092, 1059; ¹H NMR (600 MHz, CDCl₃): δ 4.93 (s, 1H), 4.63 (dd, J = 5.9, 1.1 Hz, 1H), 4.57 (d, J = 5.9 Hz, 1H), 4.46 (ddd, J = 10.4, 4.6, 1.1 Hz, 1H), 3.65 (s, 3H), 3.31 (s, 3H), 2.63 (app dtd, J = 11.0, 7.1, 3.7 Hz, 1H), 2.38–2.29 (m, 2H), 1.90–1.82 (m, 1H), 1.82–1.73 (m, 1H), 1.67 (ddd, J = 13.3, 10.4, 3.7 Hz, 1H), 1.59 (ddd, J = 13.3, 11.0, 4.6 Hz, 1H), 1.51–1.46 (m, 2H), 1.46 (s, 3H), 1.30 (s, 3H), 1.07–1.03 (m, 21H); ¹³C NMR (126 MHz, CDCl₃): δ 173.9, 112.4, 110.5, 109.8, 85.8, 85.6, 84.6, 82.8, 55.2, 51.6, 41.1, 35.2, 33.9, 30.0, 26.7, 25.4, 22.8, 18.8, 11.4; HRMS (ESI+): calcd. for [C₂₆H₄₆O₆Si₁Na] 505.2956, meas. 505.2961, Δ 0.9 ppm.

Methyl (S)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)hept-6-ynoate (S38).

To a solution of TIPS alkyne S37 (750 mg, 1.55 mmol) in THF (16 mL) at 0 °C, was added TBAF (1.0 M in THF, 1.94 mL, 1.94 mmol, 1.25 equiv.) slowly. The resulting mixture was stirred at RT for 1 h, followed by the addition of water (15 mL). The layers were separated and the aqueous phase was extracted with Et₂O (3 × 15 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 1 → 30%) to afford alkyne S38 (468 mg, 1.43 mmol, 92%) as a colorless oil. R_f = 0.35 (hexanes/EtOAc, 75:25); FTIR (thin film), ν_max (cm⁻¹): 3273, 2959, 2926, 1738, 1438, 1373, 1258, 1089, 1014; ¹H NMR (600 MHz, CDCl₃): δ 4.93 (s, 1H), 4.59 (d, J = 6.0 Hz, 1H), 4.57 (dd, J = 6.0, 0.8 Hz, 1H), 4.48 (ddd, J = 11.1, 3.9, 0.8 Hz, 1H), 3.66 (s, 3H), 3.32 (s, 3H), 2.64–2.57 (m, 1H), 2.37–2.29 (m, 2H), 2.11 (d, J = 2.4 Hz, 1H), 1.88–1.80 (m, 1H), 1.79–1.72 (m, 1H), 1.69 (ddd, J = 13.3, 11.2, 3.9 Hz, 1H), 1.56 (ddd, J = 13.3, 11.1, 4.0 Hz, 1H), 1.52–1.48 (m, 2H), 1.48 (s, 3H), 1.30 (s, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 173.9, 112.4, 110.1, 85.9, 85.7, 85.2, 84.6, 70.9, 55.3, 51.6, 40.4, 34.8, 33.8, 28.7, 26.6, 25.2, 22.6; HRMS (ESI+): calcd. for [C₁₇H₂₆O₆Na]⁺ 349.1622, meas. 349.1621, Δ 0.3 ppm.

(3R,4R,5R)-5-((S)-2-ethynyl-6-methoxy-6-oxohexyl)tetrahydrofuran-2,3,4-triyl triacetate (S39).

To a vial charged with acetonide S38 (468 mg, 1.43 mmol) at 0 °C, was added a 4:1 mixture of TFA and water (10 mL). The resulting mixture was stirred at RT for 5.5 h. Volatiles were removed in vacuo and the residue was dried by azeotropic distillation with benzene (2 × 2 mL). Crude material was used directly in the next step without further purification. To a solution of the crude triol in CH₂Cl₂ (14 mL) at RT, were added pyridine (1.16 mL, 14.3 mmol, 10 equiv.), Ac₂O (1.36 mL, 14.3 mmol, 10 equiv.) and DMAP (123 mg, 1.00 mmol, 0.70 equiv.) sequentially. The resulting mixture was stirred at RT for 1 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 2 → 60%) to afford triacetate S39 (200 mg, 0.500 mmol, 35% over 2 steps) as a 2:1 mixture of diastereomers, as a colorless oil; R_f = 0.27/0.32 (hexanes/EtOAc, 60:40); FTIR (thin film), ν_max (cm⁻¹): 3280, 2952, 2923, 1739, 1436, 1371, 1216, 1110, 1011; ¹H NMR (600 MHz, CDCl₃): δ 6.35 (d, J = 4.6 Hz, 1H), 6.13 (d, J = 1.1 Hz, 2H), 5.31 (dd, J = 4.8, 1.1 Hz, 2H), 5.22 (dd, J = 6.8, 4.6 Hz, 1H), 5.18 (dd, J = 6.9, 4.8 Hz, 2H), 5.06 (dd, J = 6.8, 3.7 Hz, 1H), 4.46 (app dt, J = 10.3, 3.7 Hz, 1H), 4.40 (ddd, J = 10.3, 6.9, 3.1 Hz, 2H), 3.66 (s, 9H), 2.63–2.57 (m, 3H), 2.37–2.28 (m, 6H), 2.11 (s, 6H), 2.11 (s, 3H), 2.10 (s, 3H), 2.07 (s, 6H), 2.06 (s, 6H), 2.05 (s, 3H), 1.89–1.80 (m, 3H), 1.80– 1.69 (m, 6H), 1.65 (ddd, J = 13.9, 10.3, 4.2 Hz, 2H), 1.62 (ddd, J = 13.4, 10.3, 4.0 Hz, 1H), 1.55–1.45 (m, 6H); ¹³C NMR (126 MHz, CDCl₃): δ 173.9, 173.9, 170.2, 170.0, 169.8, 169.6, 169.5, 169.2, 98.5, 93.9, 85.7, 85.4, 81.5, 79.9, 74.7, 74.0, 72.7, 71.1, 70.9, 69.9, 51.7, 40.2, 39.2, 34.7, 34.7, 33.8, 33.8, 28.5, 28.2, 22.5, 22.5, 21.2, 21.2, 20.8, 20.7, 20.7, 20.4; HRMS (ESI+): calcd. for [C₁₉H₂₆O₉Na]⁺ 421.1469, meas. 421.1490, Δ 4.9 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-2-ethynyl-6-methoxy-6-oxohexyl)tetrahydrofuran-3,4-diyl diacetate (S40).

To a suspension of N⁶-benzoyladenine (240 mg, 1.00 mmol, 4.0 equiv.) in propionitrile (dried over 4 Å M.S., 4.2 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (0.31 mL, 1.3 mmol, 5.0 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which complete solubilization of N⁶-benzoyladenine was observed. To a separate flask charged with triacetate S39 (dried by azeotropic distillation with benzene (4 ×), 100 mg, 0.251 mmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (45 mg, 0.13 mmol, 50 mol %) was added propionitrile (4.2 mL). The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained, and the solution of silylated N⁶-benzoyladenine was added. The reaction mixture was stirred at 95 °C for 2.5 h, cooled to RT and the volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (PhMe/MeCN, 2 → 60%) to afford nucleoside S40 (102 mg, 0.177 mmol, 70%) as a colorless oil. R_f = 0.44 (EtOAc); FTIR (thin film), ν_max (cm⁻¹): 3285, 2952, 2925, 2855, 1744, 1703, 1610, 1582, 1510, 1486, 1455, 1241, 1219, 1096, 1074, 1047; ¹H NMR (600 MHz, CDCl₃): δ 8.98 (br s, 1H), 8.80 (s, 1H), 8.07 (s, 1H), 8.04–8.00 (m, 2H), 7.63–7.59 (m, 1H), 7.55–7.50 (m, 2H), 6.14–6.11 (m, 2H), 5.59–5.54 (m, 1H), 4.53 (ddd, J = 11.0, 4.4, 2.8 Hz, 1H), 3.66 (s, 3H), 2.59–2.51 (m, 1H), 2.35–2.25 (m, 2H), 2.16 (s, 3H), 2.16–2.10 (m, 1H), 2.13 (d, J = 2.4 Hz, 1H), 2.07 (s, 3H), 1.88–1.80 (m, 2H), 1.75–1.67 (m, 1H), 1.55–1.48 (m, 2H); ¹³C NMR (126 MHz, CDCl₃): δ 173.8, 169.8, 169.5, 164.7, 152.9, 151.7, 149.9, 142.4, 133.6, 133.0, 129.0, 128.0, 124.1, 87.2, 85.5, 81.0, 73.8, 72.9, 71.2, 51.7, 38.4, 34.7, 33.8, 28.1, 22.5, 20.8, 20.5; HRMS (ESI+): calcd. for [C₂₉H₃₂N₅O₈]⁺ 578.2245, meas. 578.2242, Δ 0.5 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-2-((3-carbamoylphenyl)ethynyl)-6-methoxy-6-oxohexyl)tetrahydrofuran-3,4-diyl diacetate (S41).

To a solution of alkyne S40 (102 mg, 0.177 mmol) and 3-iodobenzamide (131 mg, 0.530 mmol, 3.0 equiv.) in a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 9.0 mL) at RT, were introduced CuI (8 mg, 0.04 mmol, 25 mol %) and Pd(PPh₃)₄ (10 mg, 8.8 μmol, 5 mol %) rapidly. The resulting mixture was stirred at 60 °C for 2.5 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 10%) to afford benzamide S41 (105 mg, 0.151 mmol, 85%) as a white amorphous solid. R_f = 0.39 (EtOAc/i-PrOH, 95:5); FTIR (thin film), ν_max (cm⁻¹): 3340, 3198, 2925, 2855, 1747, 1669, 1611, 1581, 1513, 1487, 1456, 1375, 1244, 1221, 1097, 1047; ¹H NMR (600 MHz, CDCl₃): δ 9.12 (s, 1H), 8.79 (s, 1H), 8.11 (s, 1H), 8.03–8.00 (m, 2H), 7.87–7.86 (m, 1H), 7.75 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.62–7.57 (m, 1H), 7.54–7.47 (m, 3H), 7.36 (app t, J = 7.8 Hz, 1H), 6.51 (br s, 1H), 6.18 (dd, J = 5.9, 5.2 Hz, 1H), 6.15 (d, J = 5.9 Hz, 1H), 5.98 (br s, 1H), 5.66 (dd, J = 5.2, 3.8 Hz, 1H), 4.53 (app dt, J = 10.0, 3.8 Hz, 1H), 3.65 (s, 3H), 2.76 (app dtd, J = 11.1, 7.0, 3.7 Hz, 1H), 2.39–2.29 (m, 2H), 2.20 (ddd, J = 13.6, 10.0, 3.7 Hz, 1H), 2.15 (s, 3H), 2.05 (s, 3H), 1.98 (ddd, J = 13.6, 11.1, 3.8 Hz, 1H), 1.92–1.84 (m, 1H), 1.81–1.73 (m, 1H), 1.63–1.57 (m, 2H); ¹³C NMR (126 MHz, CDCl₃): δ 173.9, 170.0, 169.7, 168.8, 164.8, 152.9, 151.8, 149.9, 142.4, 134.9, 133.6, 133.6, 133.0, 130.8, 129.0, 128.7, 128.0, 127.0, 124.3, 124.0, 92.3, 86.9, 82.5, 81.5, 74.0, 72.8, 51.7, 38.7, 35.0, 33.8, 29.0, 22.7, 20.8, 20.6; HRMS (ESI+): calcd. for [C₃₆H₃₆N₆O₉Na]⁺ 719.2436, meas. 719.2452, Δ 2.3 ppm.

Triisopropyl((S,E)-3-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-6-nitrohex-5-en-1-yn-1-yl)silane (S42).

To a solution of aldehyde 5 (385 mg, 0.907 mmol) in MeNO₂ (9 mL) at 0 °C, was added Et₃N (0.13 mL, 0.91 mmol, 1.00 equiv.). The resulting mixture was stirred at RT for 17 h and volatiles were removed in vacuo. To a solution of the residue in CH₂Cl₂ (9 mL) at −78 °C were added i-Pr₂NEt (0.40 mL, 2.3 mmol, 2.50 equiv.) and MsCl (90 μL, 1.1 mmol, 1.25 equiv.) sequentially. The resulting mixture was stirred at −78 °C for 2 h, before the addition of a NH₄Cl sat. aq. solution (8 mL). The biphasic mixture was allowed to warm to RT and the layers were separated. The aqueous phase was extracted with CH₂Cl₂ (3 × 8 mL). The combined organic extracts were dried over Na₂SO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 2 → 25%) to afford S42 (319 mg, 0.682 mmol, 75% over 2 steps) as a colorless oil. R_f = 0.46 (hexanes/EtOAc, 75:25); FTIR (thin film), ν_max (cm⁻¹): 2941, 2865, 2168, 1655, 1528, 1463, 1351, 1210, 1092; ¹H NMR (600 MHz, CDCl₃): δ 7.33 (ddd, J = 13.4, 7.9, 7.5 Hz, 1H), 7.06 (app dt, J = 13.4, 1.4 Hz, 1H), 4.95 (s, 1H), 4.62 (dd, J = 5.9, 1.1 Hz, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.46 (ddd, J = 10.6, 4.3, 1.1 Hz, 1H), 3.34 (s, 3H), 2.89 (dddd, J = 10.9, 7.9, 5.5, 4.0 Hz, 1H), 2.49 (dddd, J = 14.6, 7.5, 5.5, 1.4 Hz, 1H), 2.41 (app dtd, J = 14.6, 7.9, 1.4 Hz, 1H), 1.72 (ddd, J = 13.3, 10.6, 4.0 Hz, 1H), 1.66 (ddd, J = 13.3, 10.9, 4.3 Hz, 1H), 1.47 (s, 3H), 1.31 (s, 3H), 1.07–1.03 (m, 21H); ¹³C NMR (126 MHz, CDCl₃): δ 141.0, 139.2, 112.7, 110.0, 108.0, 85.5, 85.3, 85.1, 84.4, 55.4, 40.8, 34.4, 29.6, 26.7, 25.4, 18.7, 11.3; HRMS (ESI+): calcd. for [C₂₄H₄₁N₁O₆Si₁Na]⁺ 490.2595, meas. 490.2580, Δ 3.2 ppm.

2,2,2-trifluoro-N-((S)-4-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-6-(triisopropylsilyl)hex-5-yn-1-yl)acetamide (S43).

To a solution of S42 (319 mg, 0.682 mmol) in THF (6.8 mL) at 0 °C, was added LiAlH₄ (1.0 M in Et₂O, 4.8 mL, 4.8 mmol, 7.0 equiv.) dropwise. The resulting mixture was stirred at RT for 4 h. The reaction was quenched by the dropwise addition of water (0.18 mL), a 15% NaOH aq. solution (0.18 mL) and water (0.54 mL), sequentially, at 0 °C and Na₂SO₄ was added. The mixture was stirred vigorously for 20 min and filtered through a cotton plug to remove the white solids. The filtrate was concentrated and the crude material was used directly in the next step without further purification. To a solution of the crude amine in THF (6.8 mL) at 0 °C, were added Et₃N (5.7 mL, 41 mmol, 60 equiv.) and ethyl trifluoroacetate (2.4 mL, 21 mmol, 30 equiv.) sequentially. The resulting mixture was stirred at RT for 17 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 2 → 50%) to afford trifluoroacetamide S43 (252 mg, 0.470 mmol, 69% over 2 steps) as a colorless oil. R_f = 0.47 (hexanes/EtOAc, 65:35); FTIR (thin film), ν_max (cm⁻¹): 3306, 2941, 2865, 2166, 1705, 1559, 1463, 1381, 1209, 1161, 1106, 1061; ¹H NMR (600 MHz, CDCl₃): δ 6.27 (br s, 1H), 4.94 (s, 1H), 4.62 (dd, J = 5.9, 0.7 Hz, 1H), 4.58 (d, J = 5.9 Hz, 1H), 4.45 (ddd, J = 10.3, 4.7, 0.7 Hz, 1H), 3.44–3.39 (m, 2H), 3.32 (s, 3H), 2.69–2.63 (m, 1H), 1.88–1.80 (m, 1H), 1.76 (app ddq, J = 13.4, 9.9, 6.7 Hz, 1H), 1.68 (ddd, J = 13.4, 10.3, 4.1 Hz, 1H), 1.62 (ddd, J = 13.4, 10.7, 4.7 Hz, 1H), 1.56–1.48 (m, 2H), 1.47 (s, 3H), 1.31 (s, 3H), 1.08–1.03 (m, 21H); ¹³C NMR (126 MHz, CDCl₃): δ 157.3 (q, ²J_C-F = 37 Hz), 116.0 (q, ¹J_C-F = 288 Hz), 112.5, 110.1, 109.8, 85.7, 85.6, 84.5, 83.4, 55.2, 41.2, 39.8, 32.8, 30.0, 26.9, 26.7, 25.4, 18.8, 11.3; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.9; HRMS (ESI+): calcd. for [C₂₆H₄₄F₃N₁O₅Si₁Na]⁺ 558.2833, meas. 558.2858, Δ 4.4 ppm.

2,2,2-trifluoro-N-((S)-4-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)hex-5-yn-1-yl)acetamide (S44).

To a solution of TIPS alkyne S43 (196 mg, 0.366 mmol) in DMF (3 mL) at RT, was added a solution of TASF (0.30 g, 1.2 mmol, 3.0 equiv.) in DMF (4 mL). The resulting mixture was stirred at 60 °C for 2.5 h and volatiles were removed in vacuo. The residue was partitioned between Et₂O (7 mL) and a 1:1 mixture of brine and 5% LiCl aq. solution (7 mL). The layers were separated and the aqueous phase was extracted with Et₂O (3 × 7 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 5 → 50%) to afford alkyne S44 (117 g, 0.308 mmol, 84%) as an amorphous white solid. R_f = 0.32 (hexanes/EtOAc, 65:35); FTIR (thin film), ν_max (cm⁻¹): 3298, 2937, 1706, 1558, 1454, 1382, 1209, 1159, 1104, 1059; ¹H NMR (600 MHz, CDCl₃): δ 6.43 (br s, 1H), 4.94 (s, 1H), 4.59 (d, J = 6.0 Hz, 1H), 4.57 (dd, J = 6.0, 0.9 Hz, 1H), 4.47 (ddd, J = 11.1, 4.0, 0.9 Hz, 1H), 3.42–3.37 (m, 2H), 3.32 (s, 3H), 2.66–2.60 (m, 1H), 2.14 (d, J = 2.4 Hz, 1H), 1.86–1.78 (m, 1H), 1.74 (app ddt, J = 14.0, 10.3, 7.0 Hz, 1H), 1.69 (ddd, J = 13.3, 11.1, 4.0 Hz, 1H), 1.59 (ddd, J = 13.3, 10.9, 4.0 Hz, 1H), 1.53–1.49 (m, 2H), 1.48 (s, 3H), 1.32–1.30 (s, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 157.4 (q, ²J_C-F = 37 Hz), 116.0 (q, ¹J_C-F = 288 Hz), 112.5, 110.1, 85.6, 85.6, 85.1, 84.5, 71.3, 55.3, 40.5, 39.8, 32.3, 28.6, 26.8, 26.6, 25.1; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.9; HRMS (ESI+): calcd. for [C₁₇H₂₄F₃N₁O₅Na]⁺ 402.1499, meas. 402.1510, Δ 2.7 ppm.

(3R,4R,5R)-5-((S)-2-ethynyl-5-(2,2,2-trifluoroacetamido)pentyl)tetrahydrofuran-2,3,4-triyl triacetate (S45).

To a solution of S44 (117 mg, 0.308 mmol) in CH₂Cl₂ (1.5 mL) at 0 °C, was added a 4:1 mixture of TFA and water (6.0 mL). The resulting mixture was stirred at RT for 15 h. Volatiles were removed in vacuo and the residue was dried by azeotropic distillation with benzene (2 × 2 mL). Crude material was used directly in the next step without further purification. To a solution of the crude triol in CH₂Cl₂ (6.0 mL) at RT, were added pyridine (0.37 mL, 4.6 mmol, 15.0 equiv.), Ac₂O (0.44 mL, 4.6 mmol, 15.0 equiv.) and DMAP (39 mg, 0.32 mmol, 1.05 equiv.) sequentially. The resulting mixture was stirred at RT for 2.5 h, before the addition of water (6 mL). The layers were separated and the aqueous phase was extracted with CH₂Cl₂ (3 × 6 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 5 → 70%) to afford S45 (74 mg, 0.16 mmol, 53% over 2 steps) as a 1:1 mixture of diastereomers, as a colorless oil. R_f = 0.30/0.41 (hexanes/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3288, 2933, 1749, 1720, 1558, 1435, 1373, 1218, 1180, 1158, 1012; ¹H NMR (600 MHz, CDCl₃): δ 6.70–6.62 (m, 2H), 6.34 (d, J = 4.5 Hz, 1H), 6.10 (d, J = 0.9 Hz, 1H), 5.30 (dd, J = 4.8, 0.9 Hz, 1H), 5.21 (dd, J = 6.7, 4.5 Hz, 1H), 5.16 (dd, J = 7.0, 4.8 Hz, 1H), 5.04 (dd, J = 6.7, 3.7 Hz, 1H), 4.44 (app dt, J = 10.1, 3.7 Hz, 1H), 4.36 (ddd, J = 10.1, 7.0, 3.0 Hz, 1H), 3.42– 3.33 (m, 4H), 2.66–2.59 (m, 2H), 2.13 (d, J = 2.4 Hz, 2H), 2.11 (s, 3H), 2.11 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 1.87–1.76 (m, 4H), 1.75–1.68 (m, 2H), 1.68–1.60 (m, 2H), 1.57–1.43 (m, 4H); ¹³C NMR (126 MHz, CDCl₃): δ 170.3, 170.1, 169.9, 169.7, 169.5, 169.4, 157.4 (q, ²J_C-F = 37 Hz), 157.4 (q, ²J_C-F = 37 Hz), 116.0 (q, ¹J_C-F = 288 Hz), 116.0 (q, ¹J_C-F = 288 Hz), 98.5, 93.8, 85.5, 85.2, 81.5, 79.9, 74.5, 73.9, 72.7, 71.4, 71.2, 69.9, 40.1, 39.7, 39.6, 39.3, 32.1, 32.0, 28.5, 28.1, 26.6, 26.3, 21.2, 21.2, 20.8, 20.7, 20.6, 20.4; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.88, −76.90; HRMS (ESI+): calcd. for [C₁₉H₂₄F₃N₁O₈Na]⁺ 474.1346, meas. 474.1358, Δ 2.5 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-2-ethynyl-5-(2,2,2-trifluoroacetamido)pentyl)tetrahydrofuran-3,4-diyl diacetate (S46).

To a suspension of N⁶-benzoyladenine (72 mg, 0.30 mmol, 4.0 equiv.) in propionitrile (dried over 4 Å M.S., 1.5 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (92 μL, 0.38 mmol, 5.0 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which complete solubilization of N⁶-benzoyladenine was observed. To a separate flask charged with triacetate S45 (dried by azeotropic distillation with benzene (4 ×), 34 mg, 75 μmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (13 mg, 38 μmol, 50 mol %) was added propionitrile (1.5 mL). The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained, and the solution of silylated N⁶-benzoyladenine was added. The reaction mixture was stirred at 95 °C for 4 h, cooled to RT and the volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (PhMe/MeCN, 0 → 50%) to afford nucleoside S46 (35 mg, 56 μmol, 74%) as a colorless oil. R_f = 0.31 (hexanes/EtOAc, 25:75); FTIR (thin film), ν_max (cm⁻¹): 3289, 2928, 1749, 1708, 1612, 1583, 1457, 1245, 1218, 1159, 1076, 1049; ¹H NMR (600 MHz, CDCl₃): δ 9.15 (s, 1H), 8.75 (s, 1H), 8.07 (s, 1H), 8.04–7.98 (m, 2H), 7.62–7.57 (m, 1H), 7.53–7.48 (m, 2H), 6.71–6.62 (m, 1H), 6.13–6.09 (m, 2H), 5.55 (app t, J = 4.3 Hz, 1H), 4.49 (ddd, J = 10.8, 4.3, 3.1 Hz, 1H), 3.34 (m, 2H), 2.59–2.52 (m, 1H), 2.16 (s, 3H), 2.15 (d, J = 2.4 Hz, 1H), 2.12–2.06 (ddd, J = 13.5, 10.8, 4.2 Hz, 1H), 2.06 (s, 3H), 1.87 (ddd, J = 13.5, 11.2, 3.1 Hz, 1H), 1.82–1.74 (m, 1H), 1.67 (app tdd, J = 13.4, 8.5, 6.5 Hz, 1H), 1.56–1.45 (m, 2H); ¹³C NMR (126 MHz, CDCl₃): δ 169.9, 169.6, 164.9, 157.4 (q, ²J_C-F = 37 Hz), 152.8, 151.7, 149.9, 142.4, 133.5, 133.0, 129.0, 128.0, 124.1, 116.0 (q, ¹J_C-F = 288 Hz), 87.2, 85.2, 80.9, 73.7, 72.9, 71.5, 39.6, 38.5, 32.2, 28.0, 26.7, 20.8, 20.5; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.9; HRMS (ESI+): calcd. for [C₂₉H₃₀F₃N₆O₇]⁺ 631.2123, meas. 631.2142, Δ 3.1 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-2-((3-carbamoylphenyl)ethynyl)-5-(2,2,2-trifluoroacetamido)pentyl)tetrahydrofuran-3,4-diyl diacetate (S47).

To a solution of S46 (33 mg, 52 μmol) and 3-iodobenzamide (39 mg, 0.16 mmol, 3.0 equiv.) in a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 2.6 mL) at RT, were introduced CuI (3 mg, 0.01 mmol, 25 mol %) and Pd(PPh₃)₄ (3 mg, 3 μmol, 5 mol %) rapidly. The resulting mixture was stirred at 60 °C for 2 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 10%) to afford alkyne S47 (32 mg, 43 μmol, 82%) as a white amorphous solid. R_f = 0.28 (EtOAc); FTIR (thin film), ν_max (cm⁻¹): 3306, 2928, 1749, 1709, 1667, 1611, 1581, 1456, 1376, 1245, 1217, 1157, 1098, 1049; ¹H NMR (500 MHz, CDCl₃): δ 9.32 (s, 1H), 8.75 (s, 1H), 8.12 (s, 1H), 8.04–7.99 (m, 2H), 7.89–7.87 (m, 1H), 7.76 (ddd, J = 7.7, 1.9, 1.2 Hz, 1H), 7.61–7.56 (m, 1H), 7.52–7.47 (m, 3H), 7.34 (app td, J = 7.7, 0.5 Hz, 1H), 7.19–7.16 (m, 1H), 6.80 (br s, 1H), 6.18 (dd, J = 5.8, 5.2 Hz, 1H), 6.14 (d, J = 5.8 Hz, 1H), 6.08 (br s, 1H), 5.63 (dd, J = 5.2, 3.8 Hz, 1H), 4.51 (ddd, J = 10.2, 3.8, 3.6 Hz, 1H), 3.43–3.31 (m, 2H), 2.76 (m, 1H), 2.15 (s, 3H), 2.19–2.12 (m, 1H), 2.06 (s, 3H), 1.96 (ddd, J = 13.3, 11.0, 3.6 Hz, 1H), 1.88–1.79 (m, 1H), 1.79–1.69 (m, 1H), 1.64–1.52 (m, 2H); ¹³C NMR (126 MHz, CDCl₃): δ 170.1, 169.7, 168.9, 165.1, 157.6 (q, ²J_C-F = 37 Hz), 152.7, 151.8, 150.0, 142.6, 134.7, 133.6, 133.5, 133.0, 131.0, 129.0, 128.7, 128.1, 127.3, 124.3, 123.7, 116.0 (q, ¹J_C-F = 288 Hz), 91.8, 87.0, 82.9, 81.3, 73.9, 72.7, 39.9, 38.7, 32.5, 28.8, 26.7, 20.8, 20.6; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.8; HRMS (ESI+): calcd. for [C₃₆H₃₄F₃N₇O₈Na]⁺ 772.2313, meas. 772.2306, Δ 0.9 ppm.

Methyl (2S,5S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)hept-6-ynoate (S48).

To a solution of acetonide S3 (136 mg, 311 μmol) in THF (3.0 mL) was added a 0.5 M LiOH aq. solution (1.3 mL) at 0 °C. After 75 min, more 0.5 M LiOH aq. solution (1.5 mL) was added. The reaction mixture was allowed to warm gradually to RT and was stirred for 8 h. More 0.5 M LiOH aq. solution (1.5 mL) was added and the resulting mixture was stirred for 1 h. The reaction mixture was cooled to 0 °C and a 1 M HCl aq. solution was added dropwise until pH 6 was obtained. A NaHCO₃ sat. aq. solution (5.0 mL) was added slowly followed by 1,4-dioxane (3.0 mL). A solution of Fmoc-Cl in 1,4-dioxane (0.39 M, 1.0 mL, 0.39 mmol, 1.25 equiv.) was added dropwise over 5 min under vigorous stirring. After 1 h, more Fmoc-Cl in 1,4-dioxane (0.39 M, 2.0 mL, 0.78 mmol, 2.50 equiv.) was added and the reaction mixture was stirred for 2 h. The reaction was quenched by the slow addition of a 1 M HCl aq. solution (8 mL) at 0 °C followed by CH₂Cl₂ (8 mL). The layers were separated using a phase separator (Biotage Isolute) and the aqueous phase was extracted with CH₂Cl₂ (2 × 8 mL). The combined organic extracts were concentrated and the residue was dissolved in MeOH (20 mL). (Trimethylsilyl)diazomethane (2.0 M in hexanes, 5.0 mL) was added to the methanolic solution over 2 min and the reaction mixture was stirred for 1 h. More (trimethylsilyl)diazomethane (2.0 M in hexanes, 3.0 mL) was added and the resulting mixture was stirred for 1 h. The reaction was quenched by the dropwise addition of glacial AcOH (4 mL) over 10 min and the volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 100%) to afford carbamate S48 (83 mg, 0.15 mmol, 47% over 3 steps) as an amorphous white solid. R_f = 0.56 (cyclohexane/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3300, 2987, 2947, 1723, 1526, 1447, 1377, 1341, 1212, 1163, 1099, 1061; ¹H NMR (500 MHz, CDCl₃): δ 7.77 (dq, J = 7.5, 1.0 Hz, 2H), 7.60 (t, J = 6.8 Hz, 2H), 7.40 (tq, J = 7.4, 1.0 Hz, 2H), 7.32 (tt, J = 7.5, 1.4 Hz, 2H), 5.31 (d, J = 8.5 Hz, 1H), 4.94 (s, 1H), 4.58 (q, J = 6.0 Hz, 2H), 4.48 (dd, J = 11.1, 4.0 Hz, 1H), 4.46–4.33 (m, 3H), 4.23 (t, J = 7.1 Hz, 1H), 3.77 (s, 3H), 3.31 (s, 3H), 2.66–2.60 (m, 1H), 2.14 (d, J = 2.3 Hz, 1H), 2.06–1.95 (m, 1H), 1.95–1.85 (m, 1H), 1.69 (ddd, J = 14.8, 11.2, 4.0 Hz, 1H), 1.58 (ddd, J = 16.3, 12.3, 4.7 Hz, 2H), 1.52fz (m, 1H), 1.49 (s, 3H), 1.31 (s, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 172.9, 156.1, 144.0, 143.8, 141.4, 127.8, 127.2, 125.2, 125.1, 120.1, 112.4, 110.1, 110.1, 85.6, 85.1, 84.5, 71.4, 67.2, 55.3, 53.8, 52.6, 47.3, 40.4, 31.1, 30.5, 28.7, 26.6, 25.1; HRMS (ESI+): calcd. for [C₃₂H₃₇N₁O₈Na]⁺ 586.2411, meas. 586.2407, Δ 0.8 ppm.

(3R,4R,5R)-5-((2S,5S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-ethynyl-6-methoxy-6-oxohexyl)tetrahydrofuran-2,3,4-triyl triacetate (S49).

To a vial charged with acetonide S48 (43 mg, 76 μmol) was added a 4:1 mixture of acetic acid and water (3.5 mL) at RT. The resulting mixture was stirred at 80 °C for 10 h. Volatiles were removed in vacuo and the residue was dried by azeotropic distillation with benzene (2 × 1.0 mL). Crude material was used directly in the next step without further purification. To a solution of the crude triol in CH₂Cl₂ (3.5 mL) at RT, were added pyridine (60 μL, 0.76 mmol, 10 equiv.), Ac₂O (70 μL, 0.76 mmol, 10 equiv.) and DMAP (7 mg, 53 μmol, 0.70 equiv.) sequentially. The resulting mixture was stirred at RT for 2 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 5 → 60%) to afford S49 (42 mg, 66 μmol, 87% over 2 steps) as a 1:1 mixture of diastereomers, as a colorless oil. R_f = 0.30/0.38 (hexanes/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3355, 3294, 2954, 2924, 2853, 1744, 1722, 1526, 1451, 1372, 1218, 1107, 1050, 1012; ¹H NMR (500 MHz, CDCl₃): δ 7.78–7.75 (m, 4H), 7.63–7.58 (m, 4H), 7.42–7.38 (m, 4H), 7.34–7.29 (m, 4H), 6.36 (d, J = 4.6 Hz, 1H), 6.14 (d, J = 1.0 Hz, 1H), 5.38 (br d, J = 8.5 Hz, 1H), 5.34 (br d, J = 8.4 Hz, 1H), 5.32 (dd, J = 4.8, 1.0 Hz, 1H), 5.23 (dd, J = 6.8, 4.6 Hz, 1H), 5.18 (dd, J = 7.1, 4.8 Hz, 1H), 5.07 (dd, J = 6.8, 3.6 Hz, 1H), 4.46 (app dt, J = 10.2, 3.6 Hz, 1H), 4.42–4.36 (m, 7H), 4.23 (app t, J = 7.0 Hz, 2H), 3.76 (s, 6H), 2.69–2.57 (m, 2H), 2.13 (d, J = 2.4 Hz, 1H), 2.12 (d, J = 2.4 Hz, 1H), 2.12 (s, 3H), 2.11 (s, 3H), 2.10 (s, 3H), 2.07 (s, 6H), 2.06 (s, 3H), 2.00–1.75 (m, 6H), 1.70–1.43 (m, 6H); ¹³C NMR (126 MHz, CDCl₃): δ 172.9, 172.8, 170.2, 170.0, 169.8, 169.6, 169.5, 169.3, 156.1, 156.0, 144.0, 144.0, 143.9, 143.8, 141.4, 141.4, 127.8, 127.2, 125.2, 120.1, 120.1, 98.5, 93.9, 85.3, 85.1, 81.4, 79.9, 74.6, 73.9, 72.6, 71.5, 71.3, 69.9, 67.1, 53.8, 53.7, 52.6, 47.3, 40.1, 39.2, 31.0, 30.4, 30.3, 28.6, 28.2, 21.2, 21.2, 20.8, 20.7, 20.7, 20.4; HRMS (ESI+): calcd. for [C₃₄H₃₇N₁O₁₁Na]⁺ 658.2259, meas. 658.2248, Δ 1.7 ppm.

(2R,3R,4R,5R)-2-((2S,5S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-ethynyl-6-methoxy-6-oxohexyl)-5-(6-benzamido-9H-purin-9-yl)tetrahydrofuran-3,4-diyl diacetate (S50).

To a suspension of N⁶-benzoyladenine (63 mg, 0.26 mmol, 4.0 equiv.) in propionitrile (dried over 4 Å M.S., 1.5 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (80 μL, 0.33 mmol, 5.0 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which complete solubilization of N⁶-benzoyladenine was observed. To a separate flask charged with triacetate S49 (dried by azeotropic distillation with benzene (4 ×), 42 mg, 66 μmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (12 mg, 33 μmol, 50 mol %) was added propionitrile (1.5 mL). The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained, and the solution of silylated N⁶-benzoyladenine was added. The reaction mixture was stirred at 90 °C for 4 h, cooled to RT and the volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 30 → 100%) to afford nucleoside S50 (44 mg, 54 μmol, 82%) as a colorless oil; R_f = 0.49 (EtOAc); FTIR (thin film), ν_max (cm⁻¹): 3300, 2953, 2926, 2854, 1745, 1704, 1610, 1582, 1510, 1452, 1241, 1217, 1098, 1073, 1047; ¹H NMR (500 MHz, CDCl₃): δ 8.79 (s, 1H), 8.13 (s, 1H), 8.02–7.97 (m, 2H), 7.76–7.72 (m, 2H), 7.62–7.57 (m, 3H), 7.53–7.49 (m, 2H), 7.40–7.35 (m, 2H), 7.31–7.27 (m, 2H), 6.13 (br d, J = 5.5 Hz, 1H), 6.05 (br t, J = 5.5 Hz, 1H), 5.55 (br t, J = 5.0 Hz, 1H), 5.35 (br d, J = 8.4 Hz, 1H), 4.54–4.48 (m, 1H), 4.46–4.35 (m, 3H), 4.23 (br t, J = 7.1 Hz, 1H), 3.74 (s, 3H), 2.67–2.58 (m, 1H), 2.16 (s, 3H), 2.15 (d, J = 2.4 Hz, 1H), 2.12–2.05 (m, 1H), 2.07 (s, 3H), 2.00–1.92 (m, 1H), 1.92–1.80 (m, 2H), 1.64–1.45 (m, 2H); ¹³C NMR (126 MHz, CDCl₃): δ 172.8, 169.8, 169.6, 164.8, 156.1, 152.8, 151.7, 149.8, 144.0, 143.8, 142.3, 141.4, 133.5, 133.0, 129.0, 128.0, 127.8, 127.2, 125.2, 123.9, 120.1, 87.1, 85.1, 80.9, 73.7, 73.0, 71.6, 67.1, 53.6, 52.6, 47.3, 38.5, 31.0, 30.5, 28.0, 20.8, 20.5; HRMS (ESI+): calcd. for [C₄₄H₄₃N₆O₁₀]⁺ 815.3035, meas. 815.3028, Δ 0.9 ppm.

(2R,3R,4R,5R)-2-((2S,5S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-((3-carbamoylphenyl)ethynyl)-6-methoxy-6-oxohexyl)-5-(6-benzamido-9H-purin-9-yl)tetrahydrofuran-3,4-diyl diacetate (S51).

To a vial charged with S50 (61 mg, 75 μmol) were added 2-iodobenzamide (46 mg, 0.19 mmol, 2.5 equiv.), CuI (4 mg, 0.02 mmol, 25 mol %) and Pd(PPh₃)₄ (4 mg, 4 μmol, 5 mol %). The vial headspace was purged with nitrogen for 10 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 2.5 mL) was added at RT. The resulting mixture was stirred at 60 °C for 2 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 25%) to afford S51 (68 mg, 73 μmol, 97%) as a light yellow oil. FTIR (thin film), ν_max (cm⁻¹): 3357, 3070, 2951, 2928, 1746, 1703, 1669, 1613, 1582, 1520, 1452, 1376, 1245, 1219, 1103, 1080, 1050; ¹H NMR (500 MHz, CDCl₃): δ 8.78 (s, 1H), 8.36 (s, 1H), 8.01 (br d, J = 7.7 Hz, 2H), 7.90–7.86 (m, 1H), 7.77 (dt, J = 7.9, 1.5 Hz, 1H), 7.74–7.69 (m, 2H), 7.63–7.58 (m, 1H), 7.57–7.52 (m, 3H), 7.52–7.48 (m, 2H), 7.39–7.33 (m, 3H), 7.28–7.24 (m, 2H), 6.92 (br s, 1H), 6.82 (br s, 1H), 6.17 (br d, J = 5.8 Hz, 1H), 6.10 (br t, J = 5.8 Hz, 1H), 5.71–5.65 (m, 1H), 5.51 (br d, J = 8.3 Hz, 1H), 4.56–4.50 (m, 1H), 4.49–4.41 (m, 1H), 4.41–4.32 (m, 2H), 4.18 (br t, J = 7.0 Hz, 1H), 3.75 (s, 3H), 2.89–2.80 (m, 1H), 2.16 (s, 3H), 2.19–2.12 (m, 1H), 2.06 (s, 3H), 2.12–2.02 (m, 2H), 1.99–1.89 (m, 1H), 1.74–1.64 (m, 1H), 1.64–1.54 (m, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 172.8, 170.3, 170.2, 169.8, 165.6, 156.2, 152.0, 151.8, 149.4, 143.9, 143.7, 143.3, 141.4, 135.2, 133.5, 132.6, 131.1, 129.0, 128.9, 128.4, 127.9, 127.9, 127.4, 127.2, 127.2, 125.1, 123.9, 123.3, 120.1, 92.0, 87.1, 82.9, 81.7, 73.8, 72.9, 67.2, 53.6, 52.8, 47.2, 38.6, 31.1, 30.5, 28.8, 20.8, 20.5; HRMS (ESI+): calcd. for [C₅₁H₄₇N₇O₁₁Na]⁺ 956.3226, meas. 956.3214, Δ 1.2 ppm.

(S)-3-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-5-(triisopropylsilyl)pent-4-yn-1-ol (S52).

To a solution of 5 (5.50 g, 12.9 mmol) in MeOH (100 mL) at −78 °C was added NaBH₄ (588 mg, 15.5 mmol, 1.2 equiv.) portionwise. The reaction mixture was allowed to warm to 0 °C over 1 h, then was warmed to RT and stirred for 1 h. Volatiles were removed in vacuo. The residue was partitioned between CH₂Cl₂ (30 mL) and a NH₄Cl sat. aq. solution (25 mL). The layers were separated and the aqueous phase was extracted with CH₂Cl₂ (3 × 25 mL). The combined organic extracts were dried over MgSO₄ and filtered over a silica plug to yield S52 as a yellow oil (5.29 g, 12.4 mmol, 95%) which was of suitable purity to be used in the next step without further purification. While the protocol reported here was performed on purified starting material, it was found that direct treatment of the rhodium-catalyzed conjugate alkynylation reaction mixture (5) with NaBH₄ at 0 °C reliably generated alcohol S52 in > 90% yield over two steps (alkynylation/reduction). R_f = 0.28 (hexanes/EtOAc, 67:33); FTIR (thin-film), ν_max (cm⁻¹): 3424, 2936, 2891, 2864, 2163, 1463, 1377, 1372, 1347, 1272, 1242, 1211, 1193, 1161, 1104, 1092, 1057, 1019; ¹H NMR (600 MHz, CDCl₃): δ 4.95 (s, 1H), 4.63 (dd, J = 6.0, 1.2 Hz, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.48 (ddd, J = 10.3, 4.6, 1.2 Hz, 1H), 3.88–3.80 (m, 2H), 3.33 (s, 3H), 2.83 (dddd, J = 10.9, 9.3, 5.2, 4.0 Hz, 1H), 1.80–1.62 (m, 5H), 1.47 (s, 3H), 1.31 (s, 3H), 1.10–1.00 (m, 21H); ¹³C NMR (151 MHz, CDCl₃): δ 112.5, 110.6, 109.7, 85.6, 84.5, 83.3, 61.2, 55.2, 41.2, 38.5, 27.4, 26.7, 25.4, 18.8, 11.3; HRMS (ESI+): calcd. for [C₂₃H₄₃O₅Si₁]⁺ 427.2874, meas. 427.2870, Δ 0.9 ppm.

(S)-3-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)pent-4-yn-1-ol (S53).

To a solution of TIPS-alkyne S52 (3.81 g, 8.92 mmol) in THF (15 mL) was added TBAF (1.0 M in THF, 15.0 mL, 15.0 mmol, 1.7 equiv.) at RT. The reaction mixture was stirred for 3 h and the volatiles were removed in vacuo. The residue was partitioned between Et₂O (40 mL) and a NH₄Cl sat. aq. solution (40 mL). The layers were separated and the aqueous phase was extracted with Et₂O (4 × 40 mL). The combined organic extracts were washed with brine (120 mL), dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 0 → 100%) to afford alkyne S53 (2.22 g, 8.21 mmol, 92%) as a colorless oil. The pure material crystallized upon storage at −20 °C over three weeks. The crystals were of suitable quality to obtain X-ray crystallographic data which is reported in the Supporting Information. R_f = 0.31 (cyclohexane/EtOAc, 50:50); FTIR (thin-film), ν_max (cm⁻¹): 3446, 3281, 2988, 2937, 2836, 1142, 1382, 1374, 1274, 1241, 1210, 1161, 1090, 1056; ¹H NMR (500 MHz, CDCl₃): δ 4.94 (s, 1H), 4.60 (d, J = 6.0 Hz, 1H), 4.58 (dd, J = 6.0, 0.8 Hz, 1H), 4.50 (ddd, J = 11.2, 4.0, 0.8 Hz, 1H), 3.87–3.76 (m, 2H), 3.33 (s, 3H), 2.86–2.76 (m, 1H), 2.15 (d, J = 2.4 Hz, 1H), 1.79–1.66 (m, 3H), 1.62 (ddd, J = 13.3, 11.1, 4.0 Hz, 1H), 1.48 (s, 3H), 1.31 (s, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 112.4, 110.1, 85.9, 85.7, 85.1, 84.5, 71.2, 60.8, 55.4, 40.5, 38.0, 26.6, 25.8, 25.1; HRMS (ESI+): calcd. for [C₁₄H₂₃O₅]⁺ 271.1540, meas. 271.1547, Δ 2.6 ppm.

N-((S)-3-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)pent-4-yn-1-yl)-2-nitrobenzenesulfonamide (S54).

To a solution of alcohol S53 (550 mg, 2.04 mmol), DMEAD⁷⁶ (1.19 g, 5.09 mmol, 2.5 equiv.) and PPh₃ (1.39 g, 5.29 mmol, 2.6 equiv.) in a 4:1 mixture of toluene and THF (110 mL) stirred at 0 °C over activated 4 Å M.S. (1.65 g), was added a solution of 2-NsNH₂ (1.44 g, 7.12 mmol, 3.5 equiv.) in THF (22 mL, prepared over 1.65 g of activated 4 Å M.S.) over 5 min. The reaction mixture was allowed to warm gradually to RT and was stirred for 72 h. The media was filtered and the volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 0 → 100%) to afford S54 (496 mg, 1.09 mmol, 54%) as a white amorphous solid and unreacted S53 (81 mg, 0.30 mmol, 15%). R_f = 0.27 (hexanes/EtOAc, 50:50); FTIR (thin-film), ν_max (cm⁻¹): 3289, 2989, 2936, 2836, 1540, 1442, 1415, 1363, 1343, 1300, 1274, 1241, 1210, 1192, 1162, 1087, 1056; ¹H NMR (600 MHz, CDCl₃): δ 8.16–8.11 (m, 1H), 7.89–7.83 (m, 1H), 7.78–7.70 (m, 2H), 5.50 (t, J = 6.1 Hz, 1H), 4.93 (s, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.54 (dd, J = 6.0, 0.9 Hz, 1H), 4.43 (ddd, J = 11.1, 3.9, 0.9 Hz, 1H), 3.32 (s, 3H), 3.30–3.26 (m, 2H), 2.71 (dddd, J = 14.6, 8.9, 4.5, 2.9 Hz, 1H), 2.13 (d, J = 2.4 Hz, 1H), 1.77 (dtd, J = 13.2, 7.5, 4.8 Hz, 1H), 1.71–1.63 (m, 2H), 1.58 (ddd, J = 13.3, 11.0, 4.0 Hz, 1H), 1.47 (s, 3H), 1.30 (s, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 148.1, 133.7, 133.6, 132.9, 131.1, 125.5, 112.4, 110.1, 85.5, 84.8, 84.7, 84.4, 71.9, 55.4, 41.9, 40.3, 35.2, 26.5, 26.5, 25.0; HRMS (ESI+): calcd. for [C₂₀H₂₆N₂O₈S₁Na]⁺ 477.1302, meas. 477.1287, Δ 3.2 ppm.

(3R,4R,5R)-5-((S)-2-(2-((4-nitrophenyl)sulfonamido)ethyl)but-3-yn-1-yl)tetrahydrofuran-2,3,4-triyl triacetate (S55).

To a solution of acetonide S54 (210 mg, 0.462 mmol) in CH₂Cl₂ (0.80 mL) at 0 °C, was added a 4:1 mixture of TFA and water (4.0 mL). The resulting mixture was stirred at RT for 15 h. Volatiles were removed in vacuo and the residue was dried by azeotropic distillation with benzene (2 × 1.0 mL). Crude material was used directly in the next step without further purification. To a solution of the crude triol in CH₂Cl₂ (4.8 mL) at RT, were added pyridine (0.37 mL, 4.6 mmol, 10 equiv.), Ac₂O (0.44 mL, 4.6 mmol, 10 equiv.) and DMAP (11 mg, 92 μmol, 20 mol %) sequentially. The resulting mixture was stirred at RT for 1 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 15 → 75%) to afford S55 (70 mg, 0.13 mmol, 29% over 2 steps) as a 1.8:1 mixture of diastereomers, as a colorless oil. R_f = 0.31/0.40 (hexanes/EtOAc, 40:60); FTIR (thin film), ν_max (cm⁻¹): 3288, 2925, 2853, 1747, 1541, 1424, 1368, 1222, 1167, 1124, 1082, 1058, 1012; ¹H NMR (600 MHz, CDCl₃): δ 8.16–8.11 (m, 2.8H), 7.89–7.84 (m, 2.8H), 7.79–7.71 (m, 5.6H), 6.32 (d, J = 4.6 Hz, 1.0H), 6.11 (d, J = 1.0 Hz, 1.8H), 5.55 (app t, J = 6.1 Hz, 1.0H), 5.52 (app t, J = 6.1 Hz, 1.8H), 5.29 (dd, J = 4.9, 1.0 Hz, 1.8H), 5.19 (dd, J = 6.7, 4.6 Hz, 1.0H), 5.16 (dd, J = 6.9, 4.9 Hz, 1.8H), 5.02 (dd, J = 6.7, 3.7 Hz, 1.0H), 4.40 (ddd, J = 10.3, 3.7, 3.3 Hz, 1.0H), 4.34 (ddd, J = 10.2, 6.9, 3.1 Hz, 1.8H), 3.33–3.22 (m, 5.6H), 2.70–2.61 (m, 2.8H), 2.14 (d, J = 2.4 Hz, 2.8H), 2.12 (s, 5.4H), 2.11 (s, 3.0H), 2.10 (s, 3.0H), 2.08 (s, 5.4H), 2.06 (s, 5.4H), 2.06 (s, 3.0H), 1.86–1.73 (m, 5.6H), 1.71–1.60 (m, 4.6H), 1.56 (ddd, J = 14.0, 10.3, 4.2 Hz, 1.0H); ¹³C NMR (126 MHz, CDCl₃): δ 170.2, 170.0, 169.8, 169.6, 169.5, 169.2, 148.2, 133.8, 133.7, 133.7, 132.9, 132.9, 131.2, 131.2, 125.6, 125.6, 98.4, 93.8, 84.5, 84.3, 81.2, 79.6, 74.6, 73.8, 72.5, 72.1, 72.0, 69.9, 41.9, 40.1, 39.0, 35.2, 35.0, 26.6, 26.2, 21.2, 21.2, 20.8, 20.7, 20.7, 20.4; HRMS (ESI+): calcd. for [C₂₂H₂₆N₂O₁₁S₁Na]⁺ 549.1150, meas. 549.1138, Δ 2.1 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-2-(2-((4-nitrophenyl)sulfonamido)ethyl)but-3-yn-1-yl)tetrahydrofuran-3,4-diyl diacetate (S56).

To a suspension of N⁶-benzoyladenine (93 mg, 0.39 mmol, 2.15 equiv.) in propionitrile (dried over 4 Å M.S., 4.5 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (121 μL, 0.497 mmol, 2.75 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which complete solubilization of N⁶-benzoyladenine was observed. To a separate flask charged with triacetate S55 (dried by azeotropic distillation with benzene (4 ×), 95 mg, 0.18 mmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (13 mg, 36 μmol, 20 mol %) was added propionitrile (4.5 mL). The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained, and the solution of silylated N⁶-benzoyladenine was added. The reaction mixture was stirred at 95 °C for 30 h, cooled to RT and the volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (PhMe/MeCN, 0 → 50%) and lyophilized to afford nucleoside S56 (120 mg, 0.170 mmol, 94%) as a white fluffy solid. R_f = 0.36 (EtOAc); FTIR (thin-film), ν_max (cm⁻¹): 3295, 3097, 2945, 1746, 1699, 1610, 1582, 1540, 1509, 1485, 1455, 1423, 1365, 1338, 1298, 1240, 1218, 1164, 1074, 1047; ¹H NMR (600 MHz, CDCl₃): δ 9.09 (s, 1H), 8.76 (s, 1H), 8.10 (dd, J = 7.7, 1.6 Hz, 1H), 8.07 (s, 1H), 8.02 (dt, J = 7.1, 1.4 Hz, 2H), 7.80 (dd, J = 7.6, 1.6 Hz, 1H), 7.72 (td, J = 7.6, 1.6 Hz, 1H), 7.68 (td, J = 7.6, 1.6 Hz, 1H), 7.63–7.59 (m, 1H), 7.55–7.49 (m, 2H), 6.10 (d, J = 4.7 Hz, 2H), 5.55 (q, J = 6.2, 5.4 Hz, 2H), 4.46 (ddd, J = 11.0, 4.5, 2.8 Hz, 1H), 3.25 (dt, J = 7.5, 6.2 Hz, 2H), 2.62 (dddq, J = 11.2, 9.0, 4.6, 2.5 Hz, 1H), 2.17 (d, J = 2.4 Hz, 1H), 2.16 (s, 3H), 2.07–2.12 (m, 1H), 2.07 (s, 3H), 1.85 (ddd, J = 13.8, 11.2, 2.9 Hz, 1H), 1.82–1.73 (m, 2H), 1.69 (ddt, J = 13.5, 9.9, 6.3 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 169.9, 169.6, 164.8, 152.9, 151.6, 150.0, 148.1, 142.5, 133.8, 133.7, 133.6, 133.0, 133.0, 131.2, 129.0, 128.1, 125.5, 124.1, 87.2, 84.4, 80.8, 77.4, 73.7, 72.9, 72.2, 41.9, 38.3, 35.1, 26.1, 20.8, 20.6; HRMS (ESI+): calcd. for [C₃₂H₃₂N₇O₁₀S₁]⁺ 706.1926, meas. 706.1922, Δ 0.5 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-4-(3-carbamoylphenyl)-2-(2-((4-nitrophenyl)sulfonamido)ethyl)but-3-yn-1-yl)tetrahydrofuran-3,4-diyl diacetate (S57).

To a vial charged with S56 (54 mg, 77 μmol) were added 3-iodobenzamide (29 mg, 0.12 mmol, 1.5 equiv.), CuI (3 mg, 0.02 mmol, 20 mol %) and Pd(PPh₃)₄ (4 mg, 4 μmol, 5 mol %). The vial headspace was purged with nitrogen for 10 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 3.0 mL) was added at RT. The resulting mixture was stirred at 70 °C for 4 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 15%) to afford benzamide S57 (50 mg, 60 μmol, 78%) as a white amorphous solid. R_f = 0.16 (EtOAc/i-PrOH, 91:9); FTIR (thin-film), ν_max (cm⁻¹): 2926, 1746, 1667, 1610, 1579, 1540, 1511, 1484, 1455, 1366, 1339, 1298, 1241, 1215, 1163, 1073, 1047; ¹H NMR (500 MHz, CDCl₃): δ 9.13 (s, 1H), 8.76 (s, 1H), 8.11 (s, 1H), 8.10–8.07 (m, 1H), 8.05–8.00 (m, 2H), 7.87–7.86 (m, 1H), 7.80 (ddd, J = 7.8, 1.9, 1.2 Hz, 1H), 7.79–7.76 (m, 1H), 7.71–7.64 (m, 2H), 7.63–7.58 (m, 1H), 7.55–7.47 (m, 3H), 7.38 (td, J = 7.7, 0.6 Hz, 1H), 6.56 (br s, 1H), 6.17 (t, J = 5.5 Hz, 1H), 6.13 (d, J = 5.8 Hz, 1H), 5.84 (br s, 1H), 5.79 (t, J = 6.1 Hz, 1H), 5.64 (dd, J = 5.3, 4.0 Hz, 1H), 4.50 (dt, J = 10.3, 3.7 Hz, 1H), 3.38–3.29 (m, 1H), 3.32–3.23 (m, 1H), 2.87 (tt, J = 10.2, 4.2 Hz, 1H), 2.23–2.10 (m, 1H), 2.16 (s, 3H), 2.06 (s, 3H), 1.98 (ddd, J = 13.5, 11.1, 3.6 Hz, 1H), 1.92–1.73 (m, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 170.1, 169.7, 168.6, 164.9, 152.8, 151.7, 150.0, 148.1, 142.6, 134.7, 133.8, 133.6, 133.6, 133.5, 133.0, 133.0, 131.2, 130.9, 129.0, 128.8, 128.1, 127.5, 125.5, 124.3, 123.4, 91.1, 87.1, 83.5, 81.2, 77.4, 77.2, 76.9, 73.8, 72.7, 42.1, 38.5, 35.3, 27.0, 20.8, 20.6; HRMS (ESI+): calcd. for [C₃₉H₃₇N₈O₁₁S₁]⁺ 825.2297, meas. 825.2300, Δ 0.4 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-4-(3-carbamoylphenyl)-2-(2-ureidoethyl)but-3-yn-1-yl)tetrahydrofuran-3,4-diyl diacetate (S58).

To a solution of S57 (112 mg, 0.136 mmol) in a 2:1 mixture of MeCN and DMF (10.5 mL) at 0 °C, were added thiophenol (56 μL, 0.54 mmol, 4.0 equiv.) and Cs₂CO₃ (354 mg, 1.09 mmol, 8.0 equiv.) sequentially. The resulting mixture was stirred at RT for 1 h and was filtered twice through a nylon syringe filter (13 mm, 0.22 μm). Volatiles were removed in vacuo. The crude amine was used directly in the next step without further purification. To a solution of the crude amine in a 1:1 mixture of CH₂Cl₂ and i-PrOH (13.5 mL) at RT, was added (trimethylsilyl)isocyanate (1.29 mL, 9.51 mmol, 70 equiv.). The resulting mixture was stirred at RT for 17 h and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC (5% → 95% B over 40 min) to afford S58 (24 mg, 35 μmol, 26% over two steps) as a white fluffy solid. ¹H NMR (600 MHz, CD₃OD): δ 8.78 (s, 1H), 8.58 (s, 1H), 8.13–8.08 (m, 2H), 7.94 (t, J = 1.8 Hz, 1H), 7.83 (dt, J = 7.9, 1.6 Hz, 1H), 7.71–7.64 (m, 1H), 7.61–7.54 (m, 3H), 7.43 (t, J = 7.8 Hz, 1H), 6.35 (d, J = 5.3 Hz, 1H), 6.20 (t, J = 5.5 Hz, 1H), 5.68 (t, J = 5.2 Hz, 1H), 4.60 (ddd, J = 10.5, 4.8, 3.1 Hz, 1H), 3.40 (ddd, J = 13.1, 7.4, 5.3 Hz, 1H), 3.28 (dt, J = 13.5, 7.4 Hz, 1H), 2.91 (ddt, J = 11.0, 9.3, 4.5 Hz, 1H), 2.28 (ddd, J = 14.1, 10.5, 4.0 Hz, 1H), 2.18 (s, 3H), 2.07 (s, 3H), 2.03 (ddd, J = 14.0, 11.2, 3.2 Hz, 1H), 1.82 (dtd, J = 12.7, 7.6, 5.1 Hz, 1H), 1.74 (dddd, J = 13.0, 9.5, 7.3, 5.4 Hz, 1H); ¹³C NMR (126 MHz, CD₃OD): δ 171.6, 171.4, 171.3, 168.2, 162.1, 153.5, 153.1, 151.4, 145.2, 135.7, 135.3, 134.9, 133.9, 131.8, 129.8, 129.7, 129.5, 128.1, 125.7, 125.2, 92.8, 88.5, 83.5, 82.1, 75.2, 74.2, 39.6, 36.9, 28.0, 20.5, 20.3; HRMS (ESI+): calcd. for [C₃₄H₃₅N₈O₈]⁺ 683.2572, meas. 683.2598, Δ 3.8 ppm.

(S)-4-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-6-(triisopropylsilyl)hex-5-ynenitrile (S59).

To a solution of S52 (0.54 g, 1.3 mmol) in benzene (44 mL) at RT, were added acetone cyanohydrin (0.17 mL, 1.9 mmol, 1.5 equiv.), Pn-Bu₃ (0.47 mL, 1.9 mmol, 1.5 equiv.) and a solution of TMAD (0.33 g, 1.9 mmol, 1.5 equiv.) in benzene (20 mL). The resulting mixture, which quickly became thick and cloudy, was stirred at RT for 2 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 2 → 40%) to afford S59 (0.49 g, 1.1 mmol, 89%) as a colorless oil. R_f = 0.44 (hexanes/EtOAc, 70:30); FTIR (thin film), ν_max (cm⁻¹): 2941, 2865, 2247, 2166, 1463, 1381, 1371, 1241, 1210, 1192, 1160, 1092, 1057, 1018; ¹H NMR (500 MHz, CDCl₃): δ 4.94 (s, 1H), 4.61 (dd, J = 6.0, 1.0 Hz, 1H), 4.58 (d, J = 6.0 Hz, 1H), 4.45 (ddd, J = 10.6, 4.5, 1.0 Hz, 1H), 3.34 (s, 3H), 2.81 (dddd, J = 10.6, 10.2, 4.6, 4.2 Hz, 1H), 2.59 (ddd, J = 16.9, 8.0, 5.6 Hz, 1H), 2.54 (ddd, J = 16.9, 8.0, 7.8 Hz, 1H), 1.86 (app dtd, J = 13.2, 8.0, 4.6 Hz, 1H), 1.74 (dddd, J = 13.2, 10.2, 7.8, 5.6 Hz, 1H), 1.69 (ddd, J = 13.2, 10.6, 4.2 Hz, 1H), 1.64 (ddd, J = 13.2, 10.6, 4.5 Hz, 1H), 1.46 (s, 3H), 1.30 (s, 3H), 1.07–1.03 (m, 21H); ¹³C NMR (100 MHz, CDCl₃): δ 119.4, 112.6, 109.9, 108.0, 85.5, 85.3, 84.8, 84.4, 55.4, 40.8, 31.6, 29.8, 26.7, 25.4, 18.8, 15.4, 11.3; HRMS (ESI+): calcd. for [C₂₄H₄₁N₁O₄Si₁Na]⁺ 458.2697, meas. 458.2691, Δ 1.2 ppm.

Methyl (S)-6-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-8-(triisopropylsilyl)oct-2-en-7-ynoate (S61).

To a solution of nitrile S59 (0.49 g, 1.1 mmol) in PhMe (22 mL) at −78 °C, was added DIBALH (1.0 M in hexanes, 1.68 mL, 1.68 mmol, 1.5 equiv.) dropwise. The resulting mixture was stirred at −78 °C for 1.3 h. The reaction was quenched by the addition of a potassium sodium tartrate sat. aq. solution (22 mL), followed by the addition of Et₂O (15 mL) and brine (15 mL). The biphasic mixture was stirred vigorously at RT for 45 min. The layers were separated and the aqueous phase was extracted with Et₂O (3 × 25 mL). The combined organic extracts were dried over Na₂SO₄, filtered and concentrated. Crude material was filtered through a short pad of silica gel (hexanes/EtOAc, 75:25) to afford crude aldehyde S60 which was used directly in the next step without further purification. To a suspension of NaH (95%, 33 mg, 1.3 mmol, 1.5 equiv.) in THF (1.7 mL) at 0 °C, was added a solution of phosphonate S2 (0.39 g, 1.3 mmol, 1.5 equiv.) in THF (2.0 mL) dropwise. The resulting cloudy mixture was stirred at 0 °C for 30 min and a solution of aldehyde S60 (0.38 g, 0.88 mmol) in THF (3.7 mL) was added in one portion. The yellow reaction mixture was stirred at 45 °C for 20 h and cooled to 0 °C, before the addition of an aqueous pH 7 buffer (5 mL) and Et₂O (5 mL). The layers were separated and the aqueous phase was extracted with Et₂O (3 × 10 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 1 → 40%) to afford olefin S61 (0.44 g, 0.72 mmol, 82%) as a 1:1 mixture of Z and E isomers, as a colorless oil. R_f = 0.17/0.31 (hexanes/EtOAc, 80:20); FTIR (thin film), ν_max (cm⁻¹): 3305, 2942, 2865, 2164, 1729, 1661, 1541, 1463, 1439, 1382, 1372, 1209, 1159, 1104, 1092, 1060; ¹H NMR (500 MHz, CDCl₃): δ 8.30 (s, 1H), 7.60 (s, 1H), 7.37 (app t, J = 7.6 Hz, 1H), 6.86 (app t, J = 7.3 Hz, 1H), 4.94 (s, 1H), 4.94 (s, 1H), 4.63 (dd, J = 6.0, 1.2 Hz, 1H), 4.62 (dd, J = 6.0, 1.2 Hz, 1H), 4.58 (d, J = 6.0 Hz, 1H), 4.58 (d, J = 6.0 Hz, 1H), 4.49–4.43 (m, 2H), 3.87 (s, 3H), 3.80 (s, 3H), 3.32 (s, 3H), 3.32 (s, 3H), 2.89–2.75 (m, 2H), 2.74–2.61 (m, 2H), 2.45–2.36 (m, 1H), 2.36–2.26 (m, 1H), 1.74–1.57 (m, 8H), 1.46 (s, 6H), 1.31 (s, 6H), 1.08–1.00 (m, 42H); ¹³C NMR (126 MHz, CDCl₃): δ 163.9, 163.8, 155.1 (q, ²J_C-F = 38 Hz), 155.0 (q, ²J_C-F = 38 Hz), 140.7, 135.5, 123.6, 122.9, 115.8 (q, ¹J_C-F = 288 Hz), 115.6 (q, ¹J_C-F = 288 Hz), 112.5, 112.5, 110.1, 109.8, 109.8, 109.8, 85.7, 85.6, 85.6, 84.5, 84.5, 83.4, 83.2, 55.2, 55.2, 53.1, 53.0, 41.2, 41.1, 35.6, 34.1, 30.3, 30.2, 27.2, 26.7, 25.4, 25.4, 18.8, 18.8, 11.4, 11.3; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.4, −77.2; HRMS (ESI+): calcd. for [C₂₉H₄₆F₃N₁O₇Si₁Na]⁺ 628.2888, meas. 628.2894, Δ 1.0 ppm.

Methyl (2S,6S)-6-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-8-(triisopropylsilyl)oct-7-ynoate (S62).

In a glove box, [Rh(nbd)₂]BF₄ (47 mg, 0.13 mmol, 20 mol %) and (S,S)-MeBPE (36 mg, 0.14 mmol, 22 mol %) were placed in two separate vials, capped with a septum and placed under an atmosphere of nitrogen out of the glovebox. MeOH (3 mL) was added to the vial containing (S,S)-MeBPE and the resulting colorless solution was transferred to the vial charged with [Rh(nbd)₂]BF₄. More MeOH (2 mL) was used for rinsing. The red solution of (S,S)-MeBPE-Rh was added to a solution of olefin S61 (0.38 g, 0.63 mmol) in MeOH (16 mL). The flask headspace was evacuated and refilled with hydrogen (3 ×), and the reaction mixture was stirred vigorously at RT under an atmosphere of hydrogen (balloon) for 2 h. Volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 1 → 60%) to afford protected amino acid S62 (0.35 g, 0.57 mmol, 90%) as a colorless oil. R_f = 0.40 (hexanes/EtOAc, 70:30); FTIR (thin film), ν_max (cm⁻¹): 3317, 2941, 2865, 2165, 1749, 1724, 1549, 1462, 1382, 1208, 1160, 1105, 1094, 1060; ¹H NMR (500 MHz, CDCl₃): δ 6.84 (d, J = 7.8 Hz, 1H), 4.94 (s, 1H), 4.65–4.60 (m, 2H), 4.58 (d, J = 5.9 Hz, 1H), 4.44 (ddd, J = 10.1, 4.7, 1.1 Hz, 1H), 3.79 (s, 3H), 3.31 (s, 3H), 2.65–2.58 (m, 1H), 1.98–1.89 (m, 1H), 1.88–1.78 (m, 1H), 1.69–1.54 (m, 3H), 1.51–1.42 (m, 3H), 1.46 (s, 3H), 1.31 (s, 3H), 1.07–1.01 (m, 21H); ¹³C NMR (126 MHz, CDCl₃): δ 171.4, 156.9 (q, ²J_C-F = 38 Hz), 115.7 (q, ¹J_C-F = 288 Hz), 112.5, 110.3, 109.8, 85.7, 85.6, 84.5, 82.9, 55.2, 53.1, 52.7, 41.2, 35.3, 31.9, 30.1, 26.7, 25.4, 22.7, 18.8, 11.4; ¹⁹F NMR (376 MHz, CDCl₃, BTF IStd): δ −76.8; HRMS (ESI+): calcd. for [C₂₉H₄₈F₃N₁O₇Si₁Na]⁺ 630.3044, meas. 630.3046, Δ 0.3 ppm.

Methyl (2S,6S)-6-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)oct-7-ynoate (S63).

To a solution of TIPS alkyne S62 (381 mg, 0.627 mmol) in DMF (3.2 mL) at RT, was added a solution of TASF (518 mg, 1.88 mmol, 3.0 equiv.) in DMF (3.2 mL). The resulting mixture was stirred at 60 °C for 2.5 h and volatiles were removed in vacuo. The residue was partitioned between Et₂O (15 mL) and a 1:1 mixture of brine and 5% LiCl aq. solution (15 mL). The layers were separated and the aqueous phase was extracted with Et₂O (3 × 15 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 2 → 50%) to afford alkyne S63 (250 mg, 0.554 mmol, 88%) as a colorless oil. R_f = 0.48 (hexanes/EtOAc, 60:40); FTIR (thin film), ν_max (cm⁻¹): 3309, 2937, 2867, 1747, 1717, 1550, 1440, 1374, 1208, 1158, 1089, 1058; ¹H NMR (500 MHz, CDCl₃): δ 6.90 (d, J = 7.5 Hz, 1H), 4.93 (s, 1H), 4.63 (ddd, J = 7.5, 7.1, 5.6 Hz, 1H), 4.59 (d, J = 6.0 Hz, 1H), 4.56 (dd, J = 6.0, 0.9 Hz, 1H), 4.46 (ddd, J = 11.0, 4.1, 0.9 Hz, 1H), 3.79 (s, 3H), 3.31 (s, 3H), 2.63–2.55 (m, 1H), 2.10 (d, J = 2.4 Hz, 1H), 1.97–1.89 (m, 1H), 1.80 (dddd, J = 13.8, 10.4, 7.1, 4.6 Hz, 1H), 1.66 (ddd, J = 13.4, 11.0, 4.1 Hz, 1H), 1.60–1.52 (m, 2H), 1.47 (s, 3H), 1.51–1.39 (m, 3H), 1.30 (s, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 171.4, 156.9 (q, ²J_C-F = 38 Hz), 115.7 (q, ¹J_C-F = 288 Hz), 112.4, 110.1, 85.6, 85.6, 85.2, 84.5, 71.1, 55.3, 53.1, 52.6, 40.5, 34.4, 31.6, 28.5, 26.6, 25.1, 22.5; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.8; HRMS (ESI+): calcd. for [C₂₀H₂₈F₃N₁O₇Na] 474.1710, meas. 474.1721, Δ 2.3 ppm.

(3R,4R,5R)-5-((2S,6S)-2-ethynyl-7-methoxy-7-oxo-6-(2,2,2-trifluoroacetamido)heptyl)tetrahydrofuran-2,3,4-triyl triacetate (S64).

To a solution of acetonide S63 (125 mg, 0.277 mmol) in CH₂Cl₂ (1.1 mL) at 0 °C, was added a 4:1 mixture of TFA and water (5.5 mL). The resulting mixture was stirred at RT for 5.5 h. Volatiles were removed in vacuo and the residue was dried by azeotropic distillation with benzene (2 × 1.5 mL). Crude material was used directly in the next step without further purification. To a solution of the crude triol in CH₂Cl₂ (5.5 mL) at RT, were added pyridine (0.22 mL, 2.8 mmol, 10 equiv.), Ac₂O (0.26 mL, 2.8 mmol, 10 equiv.) and DMAP (24 mg, 0.19 mmol, 0.70 equiv.) sequentially. The resulting mixture was stirred at RT for 18 h and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 5 → 70%) to afford triacetate S64 (93 mg, 0.18 mmol, 64% over 2 steps) as a 2:1 mixture of diastereomers, as a colorless oil. R_f = 0.23/0.29 (hexanes/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3293, 2954, 2867, 1747, 1723, 1553, 1438, 1372, 1216, 1180, 1162, 1113, 1051, 1012; ¹H NMR (500 MHz, CDCl₃): δ 6.96–6.89 (m, 3H), 6.35 (d, J = 4.6 Hz, 1H), 6.12 (d, J = 1.0 Hz, 2H), 5.30 (dd, J = 4.8, 1.0 Hz, 2H), 5.22 (dd, J = 6.8, 4.6 Hz, 1H), 5.17 (dd, J = 7.1, 4.8 Hz, 2H), 5.05 (dd, J = 6.8, 3.8 Hz, 1H), 4.65–4.60 (m, 3H), 4.45 (ddd, J = 10.3, 3.8, 3.3 Hz, 1H), 4.38 (ddd, J = 10.2, 7.1, 3.0 Hz, 2H), 3.79 (s, 6H), 3.79 (s, 3H), 2.63–2.55 (m, 3H), 2.12 (s, 6H), 2.11 (s, 3H), 2.10 (s, 3H), 2.09 (d, J = 2.4 Hz, 2H), 2.09 (d, J = 2.4 Hz, 1H), 2.07 (s, 6H), 2.07 (s, 6H), 2.05 (s, 3H), 1.99–1.89 (m, 3H), 1.85–1.73 (m, 6H), 1.66–1.53 (m, 6H), 1.53–1.37 (m, 9H); ¹³C NMR (126 MHz, CDCl₃): δ 171.4, 170.2, 170.1, 169.9, 169.7, 169.5, 169.3, 156.9 (q, ²J_C-F = 38 Hz), 156.9 (q, ²J_C-F = 38 Hz), 115.7 (q, ¹J_C-F = 288 Hz), 98.5, 93.8, 85.3, 85.1, 81.4, 79.8, 74.6, 73.9, 72.6, 71.2, 71.1, 69.9, 53.1, 52.6, 40.3, 39.3, 34.4, 34.4, 31.6, 31.6, 28.5, 28.1, 22.5, 22.4, 21.2, 21.2, 20.8, 20.7, 20.7, 20.4; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.8; HRMS (ESI+): calcd. for [C₂₂H₂₈F₃N₁O₁₀Na]⁺ 546.1558, meas. 546.1565, Δ 1.3 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,6S)-2-ethynyl-7-methoxy-7-oxo-6-(2,2,2-trifluoroacetamido)heptyl)tetrahydrofuran-3,4-diyl diacetate (S65).

To a suspension of N⁶-benzoyladenine (302 mg, 1.26 mmol, 4.0 equiv.) in propionitrile (dried over 4 Å M.S., 6.0 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (0.39 mL, 1.6 mmol, 5.0 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which complete solubilization of N⁶-benzoyladenine was observed. To a separate flask charged with triacetate S64 (dried by azeotropic distillation with benzene (4 ×), 165 mg, 0.315 mmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (56 mg, 0.16 mmol, 50 mol %) was added propionitrile (6.0 mL). The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained, and the solution of silylated N⁶-benzoyladenine was added. The reaction mixture was stirred at 95 °C for 5.5 h, cooled to RT and the volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 30 → 100%) to afford nucleoside S65 (209 mg, 0.297 mmol, 94%) as a colorless oil. R_f = 0.43 (hexanes/EtOAc, 20:80); FTIR (thin film), ν_max(cm⁻¹): 3287, 2955, 2925, 2868, 1748, 1721, 1612, 1583, 1511, 1488, 1457, 1245, 1219, 1187, 1161, 1049; ¹H NMR (600 MHz, CDCl₃): δ 9.13 (br s, 1H), 8.76 (s, 1H), 8.07 (s, 1H), 8.04–7.98 (m, 2H), 7.61–7.57 (m, 1H), 7.53–7.48 (m, 2H), 7.05 (br d, J = 7.9 Hz, 1H), 6.12–6.10 (m, 2H), 5.57–5.54 (m, 1H), 4.60 (ddd, J = 7.9, 7.4, 5.4 Hz, 1H), 4.50 (ddd, J = 11.0, 4.5, 2.8 Hz, 1H), 3.75 (s, 3H), 2.55–2.48 (m, 1H), 2.15 (s, 3H), 2.12 (d, J = 2.4 Hz, 1H), 2.10–2.03 (m, 1H), 2.06 (s, 3H), 1.89 (app ddt, J = 13.9, 10.8, 5.4 Hz, 1H), 1.83 (ddd, J = 13.8, 11.4, 2.8 Hz, 1H), 1.74 (dddd, J = 13.9, 10.7, 7.4, 4.8 Hz, 1H), 1.61–1.52 (m, 1H), 1.52–1.42 (m, 2H), 1.42–1.33 (m, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.4, 169.8, 169.6, 164.8, 156.9 (q, ²J_C-F = 38 Hz), 152.8, 151.7, 149.9, 142.4, 133.6, 133.0, 129.0, 128.0, 124.2, 115.7 (q, ¹J_C-F = 288 Hz), 87.2, 85.1, 80.9, 73.7, 72.9, 71.4, 53.0, 52.5, 38.6, 34.4, 31.6, 28.1, 22.6, 20.7, 20.5; ¹⁹F NMR (376 MHz, CDCl₃, BTF IStd): δ −76.8; HRMS (ESI+): calcd. for [C₃₂H₃₄F₃N₆O₉]⁺ 703.2334, meas. 703.2326, Δ 1.1 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,6S)-2-((3-carbamoylphenyl)ethynyl)-7-methoxy-7-oxo-6-(2,2,2-trifluoroacetamido)heptyl)tetrahydrofuran-3,4-diyl diacetate (S66).

To a vial charged with nucleoside S65 (89 mg, 0.13 mmol) were added 3-iodobenzamide (78 mg, 0.32 mmol, 2.5 equiv.), CuI (6 mg, 0.03 mmol, 25 mol %) and Pd(PPh₃)₄ (7 mg, 6 μmol, 5 mol %). The vial headspace was purged with nitrogen for 10 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 4.2 mL) was added at RT. The resulting mixture was stirred at 60 ◦C for 2 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 95:5) to afford benzamide S66, which was further purified by preparative HPLC (15% → 95% B’ over 30 min) to afford benzamide S66 (69 mg, 84 μmol, 66%) as a white amorphous solid. R_f = 0.51 (EtOAc/i-PrOH, 95:5); FTIR (thin film), ν_max (cm⁻¹): 3279, 3071, 2928, 1748, 1719, 1667, 1612, 1580, 1512, 1487, 1457, 1438, 1377, 1245, 1219, 1159, 1097; ¹H NMR (500 MHz, CDCl₃): δ 8.79 (s, 1H), 8.46 (s, 1H), 8.07–8.01 (m, 2H), 7.87 (app t, J = 1.8 Hz, 1H), 7.80 (ddd, J = 7.8, 1.8, 1.3 Hz, 1H), 7.64–7.60 (m, 1H), 7.55–7.49 (m, 3H), 7.39 (app t, J = 7.8 Hz, 1H), 7.18 (br d, J = 7.8 Hz, 1H), 7.12 (br s, 2H), 6.20 (d, J = 5.8 Hz, 1H), 6.12 (dd, J = 5.8, 5.4 Hz, 1H), 5.65 (dd, J = 5.4, 4.0 Hz, 1H), 4.63 (app td, J = 7.8, 5.1 Hz, 1H), 4.54 (app dt, J = 9.6, 4.0 Hz, 1H), 3.74 (s, 3H), 2.80–2.72 (m, 1H), 2.17 (s, 3H), 2.13 (ddd, J = 13.8, 9.6, 3.6 Hz, 1H), 2.06 (s, 3H), 2.05–1.92 (m, 2H), 1.85–1.76 (m, 1H), 1.71–1.45 (m, 4H); ¹³C NMR (126 MHz, CDCl₃): δ 171.5, 170.7, 170.2, 169.9, 165.9, 157.2 (q, ²J_C-F = 38 Hz), 152.0, 151.2, 149.2, 143.5, 135.3, 133.7, 132.3, 132.2, 130.9, 129.1, 128.9, 128.5, 127.5, 123.9, 122.8, 115.7 (q, ¹J_C-F = 288 Hz), 92.0, 87.4, 82.7, 81.8, 73.8, 72.9, 53.1, 52.7, 38.8, 34.6, 31.8, 29.0, 23.1, 20.8, 20.5; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.8; HRMS (ESI+): calcd. for [C₃₉H₃₈F₃N₇O₁₀Na]⁺ 844.2524, meas. 844.2532, Δ 0.8 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((4-carbamoylphenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S67).

To a vial charged with alkyne 8 (20 mg, 29 μmol) were added 4-iodobenzamide (18 mg, 73 μmol, 2.5 equiv.), CuI (1 mg, 7 μmol, 25 mol %) and Pd(PPh₃)₄ (2 mg, 2 μmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 1.5 mL) was added at RT. The resulting mixture was stirred at 60 °C for 3 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 95:5) to afford alkyne S67, which was further purified by preparative HPLC (15% → 95% B’ over 30 min) to afford alkyne S67 (19 mg, 24 μmol, 81%) as a white amorphous solid. ¹H NMR (500 MHz, CD₃OD): δ 8.79 (s, 1H), 8.63 (s, 1H), 8.11–8.07 (m, 2H), 7.86–7.80 (m, 2H), 7.70–7.65 (m, 1H), 7.60–7.55 (m, 2H), 7.48–7.44 (m, 2H), 6.34 (d, J = 5.0 Hz, 1H), 6.16 (dd, J = 5.7, 5.0 Hz, 1H), 5.66 (dd, J = 5.7, 5.0 Hz, 1H), 4.56 (ddd, J = 10.3, 5.0, 3.6 Hz, 1H), 4.53 (dd, J = 9.6, 4.9 Hz, 1H), 3.73 (s, 3H), 2.92–2.84 (m, 1H), 2.23 (ddd, J = 14.0, 10.3, 3.9 Hz, 1H), 2.15 (s, 3H), 2.18–2.10 (m, 1H), 2.06 (s, 3H), 2.05–2.00 (m, 2H), 1.74–1.60 (m, 2H); ¹³C NMR (126 MHz, CD₃OD): δ 172.5, 171.6, 171.5, 171.3, 168.7, 159.1 (q, ²J_C-F = 37 Hz), 153.1, 153.0, 151.0, 145.5, 134.6, 134.2, 134.2, 132.6, 129.8, 129.6, 128.7, 128.2, 125.0, 117.4 (q, ¹J_C-F = 287 Hz), 94.3, 88.8, 83.9, 82.2, 75.0, 74.2, 53.8, 53.1, 39.4, 32.6, 29.9, 29.7, 20.5, 20.3; ¹⁹F NMR (376 MHz, CD₃OD, BTF IStd): δ −76.5; HRMS (ESI+): calcd. for [C₃₈H₃₇F₃N₇O₁₀]⁺ 808.2549, meas. 808.2556, Δ 0.9 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((2-carbamoylphenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S68).

To a vial charged with alkyne 8 (20 mg, 29 μmol) were added 2-iodobenzamide (18 mg, 73 μmol, 2.5 equiv.), CuI (1 mg, 7 μmol, 25 mol %) and Pd(PPh₃)₄ (2 mg, 2 μmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 1.5 mL) was added at RT. The resulting mixture was stirred at 60 °C for 3 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 75:25) to afford alkyne S68, which was further purified by preparative HPLC (15% → 95% B’ over 30 min) to afford alkyne S68 (18 mg, 22 μmol, 77%) as a white amorphous solid. ¹H NMR (600 MHz, CD₃OD): δ 8.80 (s, 1H), 8.68 (s, 1H), 8.13–8.07 (m, 2H), 7.70–7.67 (m, 1H), 7.61–7.57 (m, 3H), 7.48–7.45 (m, 1H), 7.42 (app td, J = 7.5, 1.6 Hz, 1H), 7.39 (app td, J = 7.5, 1.6 Hz, 1H), 6.36 (d, J = 5.2 Hz, 1H), 6.15 (dd, J = 5.7, 5.2 Hz, 1H), 5.62 (dd, J = 5.7, 5.0 Hz, 1H), 4.62 (ddd, J = 10.4, 5.0, 3.0 Hz, 1H), 4.52 (dd, J = 9.8, 4.9 Hz, 1H), 3.73 (s, 3H), 2.92–2.86 (m, 1H), 2.22 (ddd, J = 13.7, 10.4, 4.2 Hz, 1H), 2.16 (s, 3H), 2.16–2.09 (m, 1H), 2.07 (s, 3H), 2.08–2.01 (m, 2H), 1.72–1.61 (m, 2H); ¹³C NMR (126 MHz, CD₃OD): δ 173.2, 172.6, 171.7, 171.3, 168.6, 159.1 (q, ²J_C-F = 38 Hz), 153.1, 152.4, 150.6, 145.5, 139.2, 134.6, 134.2, 134.1, 131.2, 129.9, 129.8, 129.6, 129.2, 128.8, 122.1, 117.4 (q, ¹J_C-F = 287 Hz), 96.7, 88.8, 82.3, 82.2, 74.9, 74.2, 53.8, 53.1, 39.2, 32.5, 30.0, 29.7, 20.6, 20.3; ¹⁹F NMR (471 MHz, CD₃OD, BTF IStd): δ −76.5; HRMS (ESI+): calcd. for [C₃₈H₃₇F₃N₇O₁₀]⁺ 808.2549, meas. 808.2547, Δ 0.1 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-6-methoxy-6-oxo-2-((3-sulfamoylphenyl)ethynyl)-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S69).

To a solution of alkyne 8 (41 mg, 59 μmol) and 3-iodobenzenesulfonamide (25 mg, 89 μmol, 1.5 equiv.) in a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 2.0 mL) at RT, were introduced CuI (3 mg, 0.01 mmol, 25 mol %) and Pd(PPh₃)₄ (3 mg, 3 μmol, 5 mol %) rapidly. The resulting mixture was stirred at 50 °C for 17 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 10%) to afford alkyne S69 (41 mg, 49 μmol, 82%) as a colorless oil. R_f = 0.29 (EtOAc/i-PrOH, 95:5); FTIR (thin film), ν_max (cm⁻¹): 3301, 3074, 2924, 1748, 1721, 1612, 1583, 1456, 1336, 1245, 1219, 1162, 1097; ¹H NMR (600 MHz, CDCl₃): δ 9.11 (s, 1H), 8.75 (s, 1H), 8.16 (s, 1H), 8.02–7.96 (m, 2H), 7.85 (app t, J = 1.2 Hz, 1H), 7.77 (app dt, J = 7.8, 1.2 Hz, 1H), 7.62–7.57 (m, 1H), 7.53–7.48 (m, 2H), 7.47 (app dt, J = 7.8, 1.2 Hz, 1H), 7.37 (app t, J = 7.8 Hz, 1H), 7.27 (br d, J = 7.7 Hz, 1H), 6.15 (d, J = 5.7 Hz, 1H), 6.13 (dd, J = 5.7, 5.3 Hz, 1H), 5.63 (br s, 2H), 5.62 (dd, J = 5.3, 4.2 Hz, 1H), 4.65 (app td, J = 7.7, 5.5 Hz, 1H), 4.47 (ddd, J = 9.7, 4.2, 3.5 Hz, 1H), 3.77 (s, 3H), 2.86–2.81 (m, 1H), 2.21–2.14 (m, 1H), 2.16 (s, 3H), 2.10 (app ddt, J = 14.0, 11.1, 5.5 Hz, 1H), 2.06 (s, 3H), 2.05–1.98 (m, 2H), 1.69–1.60 (m, 1H), 1.60–1.52 (m, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.3, 170.1, 169.8, 165.0, 157.2 (q, ²J_C-F = 38 Hz), 152.8, 151.8, 149.8, 142.7, 142.6, 135.2, 133.4, 133.1, 129.8, 129.1, 129.0, 128.1, 125.5, 124.4, 124.1, 115.7 (q, ¹J_C-F = 288 Hz), 92.7, 87.0, 82.2, 81.1, 73.6, 72.8, 53.3, 52.5, 38.1, 30.8, 29.8, 28.6, 20.8, 20.6; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.6; HRMS (ESI+): calcd. for [C₃₇H₃₇F₃N₇O₁₁S₁]⁺ 844.2218, meas. 844.2198, Δ 2.4 ppm.

2-fluoro-5-iodobenzamide (S70).

To a vial charged with 2-fluoro-5-iodobenzonitrile (0.99 g, 4.0 mmol) was added H₂SO₄ (98%, 4.0 mL). The resulting mixture was stirred at 70 °C for 2 h, cooled to 0 °C, before the addition of a 1 N NaOH aq. solution (15 mL) and a NaHCO₃ sat. aq. solution (5 mL). The resulting white precipitate was filtered to yield 2-fluoro-5-iodobenzamide (S70) as a pale orange, off-white powder (1.1 g, 4.0 mmol, quant.). R_f = 0.43 (hexanes/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3361, 3305, 3165, 1710, 1656, 1625, 1598, 1564, 1475, 1427, 1363, 1254, 1228; ¹H NMR (500 MHz, CD₃OD): δ 8.09 (1H, dd, J = 6.8, 2.4 Hz), 7.85 (1H, ddd, J = 8.7, 4.7, 2.4 Hz), 7.03 (1H, dd, J = 10.7, 8.7 Hz); ¹³C NMR (101 MHz, CD₃OD): δ 166.9, 161.5 (d, J_C-F = 251 Hz), 143.3 (d, J_C-F = 8.7 Hz), 140.5 (d, J_C-F = 2.6 Hz), 125.7 (d, J_C-F = 15 Hz), 119.6 (d, J_C-F = 25 Hz), 88.0 (d, J_C-F = 3.7 Hz); ¹⁹F NMR (471 MHz, CD₃OD, BTF IStd): δ −116.3; HRMS (ESI+): calcd. for [C₇H₅F₁I₁N₁O₁Na]⁺ 287.9292, meas. 287.9283, Δ 3.1 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((3-carbamoyl-4-fluorophenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S71).

To a vial charged with nucleoside 8 (31 mg, 46 μmol) were added 2-fluoro-5-iodobenzamide (S70) (20 mg, 75 μmol, 1.65 equiv.), CuI (2 mg, 9 μmol, 20 mol %) and Pd(PPh₃)₄ (3 mg, 2 μmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 3.5 mL) was added at RT. The resulting mixture was stirred at 60 °C for 1 h. More 2-fluoro-5-iodobenzamide (10 mg, 38 μmol, 0.83 equiv.) in a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (2.0 mL) was added and the reaction mixture was stirred at 60 °C for another 3 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 10%) to afford alkyne S71 (34 mg, 41 μmol, 91%) as a white amorphous solid. FTIR (thin-film), ν_max (cm⁻¹): 3256, 3082, 3060, 3026, 2924, 2853, 1748, 1719, 1672, 1611, 1583, 1513, 1491, 1453, 1366, 1245, 1216, 1183, 1159, 1110, 1074, 1048, 1029; ¹H NMR (600 MHz, CDCl₃): δ 9.02 (s, 1H), 8.80 (s, 1H), 8.14 (dd, J = 7.5, 2.3 Hz, 1H), 8.12 (s, 1H), 8.03 (d, J = 7.4 Hz, 2H), 7.69–7.59 (m, 1H), 7.55–7.44 (m, 3H), 7.09 (dd, J = 11.5, 8.5 Hz, 1H), 7.05 (br d, J = 7.8 Hz, 1H), 6.68 (br d, J = 10.9 Hz, 1H), 6.14 (d, J = 5.4 Hz, 1H), 6.11 (t, J = 5.4 Hz, 1H), 5.95 (br s, 1H), 5.60 (t, J = 5.0 Hz, 1H), 4.65 (td, J = 7.7, 5.0 Hz, 1H), 4.50 (ddd, J = 10.7, 4.6, 3.0 Hz, 1H), 3.79 (s, 3H), 2.82 (ddt, J = 10.9, 9.1, 4.5 Hz, 1H), 2.17 (s, 3H), 2.18–2.08 (m, 2H), 2.08 (s, 3H), 2.04–1.92 (m, 2H), 1.64 (ddd, J = 18.6, 9.3, 5.4 Hz, 1H), 1.52 (dddd, J = 13.5, 11.0, 9.4, 4.6 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 171.3, 169.9, 169.7, 164.7, 164.1, 164.1, 161.6, 159.1, 157.3, 156.9, 156.5, 152.9, 151.7, 149.9, 142.4, 137.1, 137.0, 135.8, 135.8, 133.6, 133.0, 132.3, 132.2, 132.1, 132.1, 129.0, 128.7, 128.6, 128.0, 124.1, 120.5, 120.5, 120.4, 120.3, 117.2, 116.7, 116.4, 114.3, 91.1, 91.0, 87.2, 81.9, 80.9, 77.4, 73.7, 73.0, 53.3, 52.5, 38.6, 31.0, 30.0, 28.7, 23.6, 20.8, 20.6; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.7, −113.0; HRMS (ESI+): calcd. for [C₃₈H₃₅F₄N₇O₁₀Na]⁺ 848.2272, meas. 848.2274, Δ 0.2 ppm.

5-iodo-2-methylbenzamide (S72).⁷⁷

To a vial charged with 5-iodo-2-methylbenzonitrile (0.12 g, 0.50 mmol) and t-BuOK (0.17 g, 1.5 mmol, 3.0 equiv.) was added t-BuOH (4.5 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 60 °C for 24 h, before the addition of water (10 mL) and EtOAc (20 mL). The layers were separated and the aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic extracts were washed with brine (80 mL), dried over MgSO₄, filtered and concentrated. Crude material was dissolved in hot toluene, recrystallized and washed with hexanes to yield 5-iodo-2-methylbenzamide (S72) (85 mg, 0.33 mmol, 65% yield) as pale yellow crystals. R_f = 0.20 (hexanes/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3371, 3187, 1649, 1582, 1559, 1401, 1373; ¹H NMR (600 MHz, CDCl₃): δ 7.77 (d, J = 1.8 Hz, 1H), 7.65 (dd, J = 8.2, 1.8 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H), 5.68 (br s, 1H), 5.62 (br s, 1H), 2.43 (s, 3H); ¹³C NMR (126 MHz, CDCl₃): δ 170.2, 139.3, 137.3, 136.2, 135.7, 133.2, 90.2, 19.7; HRMS (ESI+): calcd. for [C₈H₉I₁N₁O₁]⁺ 261.9723, meas. 261.9721, Δ 0.9 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((3-carbamoyl-4-methylphenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S73).

To a solution of alkyne 8 (50 mg, 73 μmol) and 5-iodo-2-methylbenzamide (S72) (47 mg, 0.18 mmol, 2.5 equiv.) in a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 3.6 mL) at RT, were introduced CuI (3 mg, 0.02 mmol, 25 mol %) and Pd(PPh₃)₄ (4 mg, 4 μmol, 5 mol %) rapidly. The resulting mixture was stirred at 60 °C for 2 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 95:5) to afford alkyne S73, which was further purified by preparative HPLC (15% → 95% B over 30 min) to afford alkyne S73 (40 mg, 49 μmol, 67%) as a white amorphous solid. R_f = 0.34 (EtOAc); FTIR (thin film), ν_max (cm⁻¹): 3341, 1747, 1721, 1668, 1612, 1584, 1457, 1376, 1246, 1220, 1099, 1075, 1052; ¹H NMR (500 MHz, CDCl₃): δ 9.28 (s, 1H), 8.74 (s, 1H), 8.13 (s, 1H), 8.04–7.98 (m, 2H), 7.61–7.56 (m, 1H), 7.52–7.47 (m, 2H), 7.46 (d, J = 1.8 Hz, 1H), 7.42 (br d, J = 7.7 Hz, 1H), 7.28 (dd, J = 7.8, 1.8 Hz, 1H), 7.11 (br d, J = 7.8 Hz, 1H), 6.32 (br s, 1H), 6.16 (br s, 1H), 6.13 (d, J = 5.6 Hz, 1H), 6.11 (dd, J = 5.6, 5.1 Hz, 1H), 5.61 (dd, J = 5.1, 4.2 Hz, 1H), 4.64 (app td, J = 7.7, 5.2 Hz, 1H), 4.48 (ddd, J = 10.0, 4.2, 3.6 Hz, 1H), 3.75 (s, 3H), 2.79 (dddd, J = 10.9, 9.3, 4.8, 4.0 Hz, 1H), 2.42 (s, 3H), 2.13 (s, 3H), 2.17–2.07 (m, 2H), 2.05 (s, 3H), 2.07–1.99 (m, 1H), 1.96 (ddd, J = 13.9, 10.9, 3.6 Hz, 1H), 1.63 (dddd, J = 13.4, 10.8, 5.8, 4.8 Hz, 1H), 1.53 (dddd, J = 13.4, 10.6, 9.3, 4.8 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.4, 171.4, 170.0, 169.6, 165.0, 157.1 (q, ²J_C-F = 38 Hz), 152.8, 151.8, 149.9, 142.4, 136.8, 135.2, 133.5, 133.1, 133.0, 131.3, 130.6, 128.9, 128.1, 124.2, 120.6, 115.7 (q, ¹J_C-F = 288 Hz), 90.5, 86.9, 83.0, 81.2, 73.7, 72.8, 53.2, 52.5, 38.5, 31.0, 29.8, 28.7, 20.8, 20.5, 20.1; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.6; HRMS (ESI+): calcd. for [C₃₉H₃₉F₃N₇O₁₀]⁺ 822.2705, meas. 822.2718, Δ 1.6 ppm.

5-iodo-2-(trifluoromethyl)benzamide (S74).

To a vial charged with 5-iodo-2-(trifluoromethyl)benzonitrile (0.30 g, 1.0 mmol) and t-BuOK (0.67 g, 6.0 mmol, 6.0 equiv.) was added t-BuOH (5.0 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 60 °C for 5 min, cooled to RT and stirred for 48 h. The reaction was quenched by the addition of water (10 mL). The resulting suspension was filtered to produce an orange solid. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 0 → 100%) to yield 5-iodo-2-(trifluoromethyl)benzamide (S74) as a white solid (0.13 g, 0.41 mmol, 41%). R_f = 0.46 (hexanes/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3369, 3183, 1649, 1588, 1569, 1400, 1378, 1311, 1287; ¹H NMR (500 MHz, CD₃OD): δ 8.01 (1H, ddq, J = 8.3, 1.7, 0.9 Hz), 7.95–7.92 (1H, m), 7.50 (1H, br d, J = 8.3 Hz); ¹³C NMR (126 MHz, CD₃OD): δ 171.3, 140.3, 138.6 (q, J_C-F = 2.1 Hz), 138.3, 129.0 (q, J_C-F = 4.8 Hz), 127.7 (q, J_C-F = 32.6 Hz), 125.1 (q, J_C-F = 273 Hz), 99.4 (q, J_C-F = 1.4 Hz); ¹⁹F NMR (471 MHz, CD₃OD, BTF IStd): δ −60.3; HRMS (ESI+): calcd. for [C₈H₅F₃I₁N₁O₁Na]⁺ 337.9260, meas. 337.9260, Δ 0.0 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((3-carbamoyl-4-(trifluoromethyl)phenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S75).

To a vial charged with alkyne 8 (82 mg, 0.12 mmol) were added 5-iodo-2-(trifluoromethyl)benzamide (S74) (62 mg, 0.20 mmol, 1.65 equiv.), CuI (5 mg, 24 μmol, 20 mol %) and Pd(PPh₃)₄ (7 mg, 6 μmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 3.5 mL) was added at RT. The resulting mixture was stirred at 70 °C for 3 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 5%) to afford alkyne S75 (90 mg, 0.10 mmol, 86%) as a white amorphous solid. R_f = 0.48 (PhMe/MeCN, 50:50); FTIR (thin-film), ν_max (cm⁻¹): 3306, 2926, 2165, 1747, 1719, 1672, 1609, 1582, 1511, 1487, 1456, 1410, 1373, 1313, 1289, 1242, 1216, 1175, 1133, 1114, 1074, 1039; ¹H NMR (500 MHz, CDCl₃): δ 8.98 (s, 1H), 8.78 (s, 1H), 8.11 (s, 1H), 8.04–7.98 (m, 2H), 7.63–7.59 (m, 2H), 7.58 (dd, J = 1.6, 0.8 Hz, 1H), 7.57–7.49 (m, 2H), 7.48 (ddt, J = 8.2, 1.8, 0.9 Hz, 1H), 7.13 (d, J = 7.7 Hz, 1H), 6.20 (br s, 1H), 6.16–6.12 (m, 2H), 6.05 (br s, J = 2.7 Hz, 1H), 5.62 (td, J = 4.2, 0.8 Hz, 1H), 4.65 (td, J = 7.6, 5.2 Hz, 1H), 4.46 (dt, J = 10.3, 3.5 Hz, 1H), 2.86 (ddt, J = 10.6, 9.0, 4.5 Hz, 1H), 2.23–2.17 (m, 1H), 2.16 (s, 3H), 2.12 (ddd, J = 10.7, 8.4, 5.4 Hz, 1H), 2.07 (s, 3H), 2.04–1.96 (m, 2H), 1.66 (ddt, J = 13.1, 10.7, 5.4 Hz, 1H), 1.56 (ddd, J = 8.9, 6.3, 4.6 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 171.3, 170.0, 169.6, 169.3, 165.0, 157.2 (q, ²J_C-F = 38 Hz), 152.7, 151.7, 149.9, 142.5, 135.2, 133.4, 133.0, 132.8, 132.2, 132.1, 131.7, 128.9, 128.6, 128.5, 128.1, 127.4, 126.6, 126.5, 126.3 (q, ²J_C-F = 32 Hz), 124.7, 123.4 (q, ¹J_C-F = 274 Hz), 115.7 (q, ¹J_C-F = 287 Hz), 94.4, 87.0, 81.7, 81.0, 77.3, 73.6, 72.7, 53.1, 52.4, 38.1, 30.8, 29.7, 28.6, 20.7, 20.5; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −60.0, −76.7; HRMS (ESI+): calcd. for [C₃₉H₃₆F₆N₇O₁₀]⁺ 876.2422, meas. 876.2426, Δ 0.4 ppm.

2-chloro-5-iodobenzamide (S76).

To a vial charged with 2-chloro-5-iodobenzonitrile (1.32 g, 5.01 mmol) and t-BuOK (1.69 g, 15.0 mmol, 3.0 equiv.) was added t-BuOH (10.0 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 60 °C for 5 min, cooled to RT and stirred for 1 h. The reaction was quenched by the addition of water (10 mL). The solid amide was filtered and recrystallized from absolute ethanol to give 2-chloro-5-iodobenzamide (S76) as a white solid (896 mg, 3.18 mmol, 64%). R_f = 0.35 (hexanes/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3373, 3180, 1655, 1618, 1462, 1397, 1362; ¹H NMR (500 MHz, CD₃OD): δ 7.82 (1H, d, J = 2.2 Hz), 7.76 (1H, dd, J = 8.5, 2.2 Hz), 7.24 (1H, d, J = 8.5 Hz); ¹³C NMR (151 MHz, CD₃OD): δ 170.5, 141.1, 139.2, 138.6, 132.9, 131.8, 92.0; HRMS (ESI+): calcd. for [C₇H₆Cl₁I₁N₁O₁]⁺ 281.9177, meas. 281.9188, Δ 3.9 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((3-carbamoyl-4-chlorophenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S77).

To a solution of alkyne 8 (50 mg, 73 μmol) and 2-chloro-5-iodobenzamide (S76) (51 mg, 0.18 mmol, 2.5 equiv.) in a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 3.6 mL) at RT, were introduced CuI (3 mg, 0.02 mmol, 25 mol %) and Pd(PPh₃)₄ (4 mg, 4 μmol, 5 mol %) rapidly. The resulting mixture was stirred at 70 °C for 3 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 90:10) to afford alkyne S77, which was further purified by preparative HPLC (15% → 95% B over 30 min) to afford alkyne S77 (45 mg, 53 μmol, 74%) as a white amorphous solid. R_f = 0.48 (EtOAc/i-PrOH, 95:5); FTIR (thin film), ν_max (cm⁻¹): 3320, 3085, 1748, 1719, 1673, 1613, 1583, 1456, 1374, 1246, 1219, 1047; ¹H NMR (600 MHz, CDCl₃): δ 9.29 (s, 1H), 8.74 (s, 1H), 8.12 (s, 1H), 8.04–7.97 (m, 2H), 7.69 (d, J = 2.0 Hz, 1H), 7.60–7.56 (m, 1H), 7.50–7.46 (m, 3H), 7.31 (dd, J = 8.3, 2.0 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 6.65 (s, 1H), 6.48 (s, 1H), 6.16–6.08 (m, 2H), 5.58 (app t, J = 4.5 Hz, 1H), 4.63 (app td, J = 7.8, 5.1 Hz, 1H), 4.46 (ddd, J = 10.5, 4.5, 3.1 Hz, 1H), 3.75 (s, 3H), 2.80 (dddd, J = 11.1, 9.1, 5.3, 4.6 Hz, 1H), 2.14 (s, 3H), 2.19–2.11 (m, 1H), 2.12–2.06 (m, 1H), 2.05 (s, 3H), 2.03–1.96 (m, 1H), 1.94 (ddd, J = 13.8, 11.1, 3.1 Hz, 1H), 1.63 (app ddt, J = 13.0, 10.8, 5.3 Hz, 1H), 1.58–1.49 (m, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.4, 170.0, 169.7, 167.7, 165.0, 157.2 (q, ²J_C-F = 38 Hz), 152.8, 151.7, 149.9, 142.5, 134.4, 134.1, 133.6, 133.5, 133.0, 130.5, 130.5, 128.9, 128.1, 124.1, 122.5, 115.7 (q, ¹J_C-F = 288 Hz), 92.5, 87.1, 81.9, 80.9, 73.6, 72.8, 53.2, 52.5, 38.3, 30.9, 29.8, 28.7, 20.8, 20.5; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.6; HRMS (ESI+): calcd. for [C₃₈H₃₆Cl₁F₃N₇O₁₀]⁺ 842.2159, meas. 842.2168, Δ 1.0 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-6-methoxy-6-oxo-2-((1-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)ethynyl)-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S78).

A vial was charged with alkyne 8 (40 mg, 58 μmol), 7-bromo-3,4-dihydroisoquinolin-1(2H)-one (33 mg, 0.15 mmol, 2.5 equiv.) and CuI (3 mg, 0.02 mmol, 25 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (degassed by sparging with nitrogen for 15 min, 1.5 mL) was added at RT. The resulting mixture was further degassed by sparging with nitrogen for 5 min and a solution of Pd(Pt-Bu₃)₂ (3 mg, 6 μmol, 10 mol %) in a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (1.5 mL) was added. The resulting mixture was stirred at 55 °C for 6 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 70:30) to afford alkyne S78, which was further purified by preparative HPLC (15% → 95% B over 30 min) to afford alkyne S78 (29 mg, 35 μmol, 60%) as a white amorphous solid. FTIR (thin film), ν_max (cm⁻¹): 3262, 3082, 2938, 1749, 1720, 1681, 1612, 1579, 1513, 1482, 1456, 1373, 1243, 1218, 1097, 1048; ¹H NMR (600 MHz, CDCl₃): δ 9.16 (s, 1H), 8.78 (s, 1H), 8.78 (s, 1H), 8.13 (s, 1H), 8.05–8.00 (m, 2H), 7.62–7.58 (m, 1H), 7.54–7.49 (m, 2H), 7.24 (br d, J = 7.7 Hz, 1H), 7.08 (d, J = 7.8 Hz, 1H), 7.01 (dd, J = 7.8, 1.2 Hz, 1H), 6.83 (d, J = 1.2 Hz, 1H), 6.14 (d, J = 5.6 Hz, 1H), 6.11 (dd, J = 5.6, 5.3 Hz, 1H), 5.59 (dd, J = 5.3, 4.3 Hz, 1H), 4.64 (app td, J = 7.7, 5.6 Hz, 1H), 4.49 (ddd, J = 10.4, 4.3, 3.1 Hz, 1H), 3.76 (s, 3H), 2.93 (app dd, J = 8.5, 6.6 Hz, 2H), 2.82–2.76 (m, 1H), 2.61 (app dd, J = 8.5, 6.6 Hz, 2H), 2.17–2.10 (m, 1H), 2.15 (s, 3H), 2.10–2.05 (m, 1H), 2.06 (s, 3H), 2.04–1.97 (m, 1H), 1.96 (ddd, J = 13.8, 11.0, 3.1 Hz, 1H), 1.64 (app ddt, J = 12.9, 10.6, 5.3 Hz, 1H), 1.57–1.49 (m, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.8, 171.4, 169.9, 169.6, 164.9, 157.1 (q, ²J_C-F = 38 Hz), 152.8, 151.8, 150.0, 142.4, 137.4, 133.5, 133.0, 129.0, 128.1, 128.0, 126.7, 124.2, 124.0, 122.5, 118.4, 115.7 (q, ¹J_C-F = 288 Hz), 90.4, 87.0, 83.1, 81.1, 73.8, 72.9, 53.2, 52.5, 38.7, 31.1, 30.6, 30.0, 28.8, 25.4, 20.8, 20.6; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.6; HRMS (ESI+): calcd. for [C₄₀H₃₉F₃N₇O₁₀]⁺ 834.2705, meas. 834.2702, Δ 0.3 ppm.

7-iodobenzo[d][1,3]dioxole-5-carboxamide (S80).

To a solution of known aldehyde 7-iodobenzo[d][1,3]dioxole-5-carbaldehyde (S79)^78–79 (500 mg, 1.81 mmol) in t-BuOH (20 mL) at RT, was added a solution of NaClO₂ (328 mg, 3.62 mmol, 2.0 equiv.) and NaH₂PO₄ (0.11 g, 0.91 mmol, 0.50 equiv.) in water (4 mL). The resulting pale yellow solution was cooled to 0 °C and 2-methyl-2-butene (4.0 mL, 38 mmol, 21 equiv.) was added dropwise. The reaction mixture was stirred at RT for 20 h, upon which more NaClO₂ (164 mg, 1.81 mmol, 1.0 equiv.) and NaH₂PO₄ (54 mg, 0.45 mmol, 0.25 equiv.) were added as a solution in water (2 mL). The resulting mixture was stirred at RT for 72 h and diluted with a 10% H₃PO₄ aq. solution (10 mL). Water (20 mL) and EtOAc (40 mL) were added. The layers were separated and the aqueous phase was extracted with EtOAc (2 × 40 mL). The combined organic extracts were washed with brine (80 mL), dried over Na₂SO₄, filtered and concentrated to give a white solid. Crude material was used directly in the next step without further purification. To a flask charged with the crude carboxylic acid and HATU (758 mg, 1.99 mmol, 1.1 equiv.) were added DMF (20 mL) and i-Pr₂NEt (0.63 mL, 3.6 mmol, 2.0 equiv.) at RT. The resulting brown mixture was stirred for 30 min and NH₄Cl (116 mg, 2.18 mmol, 1.2 equiv.) was added. The flask headspace was flushed with nitrogen and the reaction mixture was stirred for 24 h, before the addition of EtOAc (30 mL) and water (20 mL). The layers were separated and the aqueous phase was extracted with EtOAc (3 × 30 mL). The combined organic extracts were washed with a 5% LiCl aq. solution (100 mL) and brine (2 × 100 mL), dried over Na₂SO₄, filtered and concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 5 → 100%) to afford iodobenzamide S80 (348 mg, 1.20 mmol, 66% over 2 steps) as a white solid. R_f = 0.15 (hexanes/EtOAc, 50:50); FTIR (thin film), ν_max (cm⁻¹): 3355, 3187, 2924, 2854, 1658, 1592, 1479, 1424, 1254, 1044; ¹H NMR (500 MHz, CD₃OD): δ 7.77 (d, J = 1.6 Hz, 1H), 7.31 (d, J = 1.6 Hz, 1H), 6.11 (s, 2H); ¹³C NMR (101 MHz, CD₃OD): δ 170.1, 153.9, 148.1, 132.3, 130.7, 108.7, 103.0, 70.6; HRMS (ESI+): calcd. for [C₈H₇I₁N₁O₃]⁺ 291.9465, meas. 291.9461, Δ 1.4 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((6-carbamoylbenzo[d][1,3]dioxol-4-yl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S81).

To a vial charged with alkyne 8 (30 mg, 44 μmol) were added iodobenzamide S80 (32 mg, 0.11 mmol, 2.5 equiv.), CuI (2 mg, 0.01 mmol, 25 mol %) and Pd(PPh₃)₄ (3 mg, 2 μmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr₂NEt (degassed by sparging with nitrogen for 15 min, 2.3 mL) was added at RT. The resulting mixture was stirred at 60 °C for 3 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 80:20) to afford alkyne S81, which was further purified by preparative HPLC (15% → 95% B’ over 30 min) to afford alkyne S81 (27 mg, 32 μmol, 73%) as a white amorphous solid. FTIR (thin film), ν_max (cm⁻¹): 3351, 3222, 3077, 2937, 1746, 1717, 1659, 1593, 1424, 1243, 1203, 1045; ¹H NMR (500 MHz, CD₃OD): δ 8.80 (s, 1H), 8.70 (s, 1H), 8.12–8.06 (m, 2H), 7.70–7.65 (m, 1H), 7.60–7.55 (m, 2H), 7.45 (d, J = 1.7 Hz, 1H), 7.28 (d, J = 1.7 Hz, 1H), 6.34 (d, J = 5.0 Hz, 1H), 6.14 (dd, J = 5.7, 5.0 Hz, 1H), 6.10 (d, J = 1.1 Hz, 1H), 6.10 (d, J = 1.1 Hz, 1H), 5.65 (dd, J = 5.7, 5.1 Hz, 1H), 4.58 (ddd, J = 10.2, 5.1, 3.2 Hz, 1H), 4.52 (dd, J = 9.6, 5.0 Hz, 1H), 3.73 (s, 3H), 2.93–2.86 (m, 1H), 2.22 (ddd, J = 14.1, 10.2, 4.1 Hz, 1H), 2.15 (s, 3H), 2.17–2.10 (m, 1H), 2.06 (s, 3H), 2.07–1.98 (m, 2H), 1.72–1.58 (m, 2H); ¹³C NMR (126 MHz, CD₃OD): δ 172.5, 171.6, 171.3, 170.7, 168.7, 159.1 (q, ²J_C-F = 38 Hz), 153.0, 152.9, 152.2, 150.4, 149.4, 145.6, 134.4, 134.3, 129.8, 129.6, 128.9, 126.7, 124.3, 117.0 (q, ¹J_C-F = 288 Hz), 108.4, 105.6, 103.9, 96.7, 88.9, 82.2, 77.8, 74.9, 74.3, 53.7, 53.1, 39.3, 32.5, 29.9, 29.6, 20.5, 20.3; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.7; HRMS (ESI+): calcd. for [C₃₉H₃₇F₃N₇O₁₂]⁺ 852.2447, meas. 852.2483, Δ 4.3 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((6-carbamoylpyridin-2-yl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S82).

A vial was charged with alkyne 8 (35 mg, 51 μmol), 6-bromopyridine-2-carboxamide (26 mg, 0.13 mmol, 2.5 equiv.) and CuI (2 mg, 0.01 mmol, 25 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (degassed by sparging with nitrogen for 15 min, 1.3 mL) was added at RT. The resulting mixture was further degassed by sparging with nitrogen for 5 min and a solution of Pd(Pt-Bu₃)₂ (3 mg, 5 μmol, 10 mol %) in a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (1.3 mL) was added. The resulting mixture was stirred at 50 °C for 5 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 95:5) to afford alkyne S82, which was further purified by preparative HPLC (15% → 95% B over 30 min) to afford alkyne S82 (28 mg, 35 μmol, 68%) as a white amorphous solid. R_f = 0.43 (EtOAc/i-PrOH, 95:5); FTIR (thin film), ν_max (cm⁻¹): 3292, 2928, 1748, 1718, 1690, 1611, 1584, 1511, 1487, 1457, 1377, 1245, 1220, 1161, 1075; ¹H NMR (600 MHz, CDCl₃): δ 9.16 (s, 1H), 8.78 (s, 1H), 8.14 (s, 1H), 8.10 (dd, J = 7.8, 1.1 Hz, 1H), 8.05–7.99 (m, 2H), 7.95 (d, J = 4.0 Hz, 1H), 7.78 (app t, J = 7.8 Hz, 1H), 7.62–7.57 (m, 1H), 7.54–7.48 (m, 3H), 7.33 (br d, J = 7.7 Hz, 1H), 6.15 (d, J = 5.9 Hz, 1H), 6.14 (dd, J = 5.9, 4.8 Hz, 1H), 5.92 (d, J = 4.0 Hz, 1H), 5.60 (dd, J = 4.8, 3.9 Hz, 1H), 4.66 (app td, J = 7.7, 5.1 Hz, 1H), 4.51 (ddd, J = 10.2, 3.9, 3.4 Hz, 1H), 3.77 (s, 3H), 2.90–2.83 (m, 1H), 2.22 (ddd, J = 14.0, 10.2, 4.1 Hz, 1H), 2.15 (s, 3H), 2.18–2.11 (m, 1H), 2.09–2.02 (m, 2H), 2.06 (s, 3H), 1.71 (app ddt, J = 13.1, 10.7, 5.4 Hz, 1H), 1.65–1.57 (m, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.3, 169.9, 169.6, 166.3, 164.9, 157.1 (q, ²J_C-F = 38 Hz), 152.8, 151.8, 150.0, 149.9, 142.4, 141.9, 137.6, 133.5, 133.0, 129.8, 129.0, 128.1, 124.2, 121.7, 115.7 (q, ¹J_C-F = 288 Hz), 91.3, 87.0, 82.9, 81.2, 73.8, 72.7, 53.2, 52.5, 38.3, 30.8, 29.8, 28.7, 20.8, 20.5; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.6; HRMS (ESI+): calcd. for [C₃₇H₃₆F₃N₈O₁₀]⁺ 809.2501, meas. 809.2504, Δ 0.4 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((2-carbamoylpyridin-4-yl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S83).

A vial was charged with alkyne 8 (40 mg, 58 μmol), 4-bromopicolinamide (29 mg, 0.15 mmol, 2.5 equiv.) and CuI (3 mg, 0.02 mmol, 25 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (degassed by sparging with nitrogen for 15 min, 1.5 mL) was added at RT. The resulting mixture was further degassed by sparging with nitrogen for 5 min and a solution of Pd(Pt-Bu₃)₂ (3 mg, 6 μmol, 10 mol %) in a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (1.5 mL) was added. The resulting mixture was stirred at 50 °C for 5 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 70:30) to afford alkyne S83, which was further purified by preparative HPLC (15% → 95% B over 30 min) to afford alkyne S83 (35 mg, 43 μmol, 75%) as a white amorphous solid. FTIR (thin film), ν_max (cm⁻¹): 3283, 3079, 2931, 1748, 1721, 1690, 1611, 1599, 1583, 1456, 1356, 1243, 1218, 1074; ¹H NMR (600 MHz, CDCl₃): δ 9.07 (s, 1H), 8.78 (s, 1H), 8.48 (dd, J = 5.0, 0.8 Hz, 1H), 8.12 (dd, J = 1.6, 0.8 Hz, 1H), 8.11 (s, 1H), 8.04–8.00 (m, 2H), 7.82 (d, J = 4.2 Hz, 1H), 7.62–7.58 (m, 1H), 7.54–7.49 (m, 2H), 7.38 (dd, J = 5.0, 1.6 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 6.15–6.11 (m, 2H), 5.93 (d, J = 4.2 Hz, 1H), 5.62–5.58 (m, 1H), 4.65 (app td, J = 7.8, 5.4 Hz, 1H), 4.47 (ddd, J = 10.8, 4.3, 3.0 Hz, 1H), 3.78 (s, 3H), 2.88 (app ddt, J = 10.8, 9.2, 4.6 Hz, 1H), 2.22–2.15 (m, 1H), 2.17 (s, 3H), 2.11 (ddt, J = 14.2, 11.0, 5.4 Hz, 1H), 2.08 (s, 3H), 2.03–1.95 (m, 2H), 1.67 (app ddt, J = 13.4, 10.8, 5.4 Hz, 1H), 1.57 (dddd, J = 13.4, 11.0, 9.2, 4.6 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.3, 169.9, 169.7, 166.4, 164.8, 157.1 (q, ²J_C-F = 38 Hz), 152.9, 151.7, 149.9, 149.6, 148.5, 142.5, 133.6, 133.0, 133.0, 129.0, 128.4, 128.1, 124.8, 124.2, 115.7 (q, ¹J_C-F = 288 Hz), 96.7, 87.3, 81.1, 80.8, 73.7, 72.9, 53.3, 52.4, 38.3, 30.8, 29.9, 28.9, 20.8, 20.6; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.6; HRMS (ESI+): calcd. for [C₃₇H₃₆F₃N₈O₁₀]⁺ 809.2501, meas. 809.2491, Δ 1.2 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((5-carbamoylpyridin-3-yl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S84).

A vial was charged with alkyne 8 (40 mg, 58 μmol), 5-bromonicotinamide (29 mg, 0.15 mmol, 2.5 equiv.) and CuI (3 mg, 0.02 mmol, 25 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (degassed by sparging with nitrogen for 15 min, 1.5 mL) was added at RT. The resulting mixture was further degassed by sparging with nitrogen for 5 min and a solution of Pd(Pt-Bu₃)₂ (3 mg, 6 μmol, 10 mol %) in a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (1.5 mL) was added. The resulting mixture was stirred at 50 °C for 5 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 70:30) to afford alkyne S84, which was further purified by preparative HPLC (15% → 95% B over 30 min) to afford alkyne S84 (26 mg, 32 μmol, 55%) as a white amorphous solid. FTIR (thin film), ν_max (cm⁻¹): 3263, 3085, 2954, 2928, 1748, 1720, 1677, 1612, 1583, 1456, 1376, 1245, 1219, 1075; ¹H NMR (600 MHz, CDCl₃): δ 9.11 (s, 1H), 8.94 (d, J = 1.4 Hz, 1H), 8.77 (s, 1H), 8.67 (d, J = 2.0 Hz, 1H), 8.16–8.11 (m, 2H), 8.01 (m, 2H), 7.63–7.58 (m, 1H), 7.55–7.48 (m, 2H), 7.46 (br d, J = 7.5 Hz, 1H), 6.83 (br s, 1H), 6.17 (dd, J = 5.7, 5.1 Hz, 1H), 6.15 (d, J = 5.7 Hz, 1H), 6.05 (br s, 1H), 5.66 (dd, J = 5.1, 4.0 Hz, 1H), 4.65 (app td, J = 7.5, 5.4 Hz, 1H), 4.48 (ddd, J = 9.8, 4.0, 3.8 Hz, 1H), 3.79 (s, 3H), 2.88–2.82 (m, 1H), 2.21–2.15 (m, 1H), 2.16 (s, 3H), 2.15–2.08 (m, 1H), 2.07 (s, 3H), 2.08–1.99 (m, 2H), 1.72–1.64 (m, 1H), 1.63–1.54 (m, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.3, 170.1, 169.8, 166.9, 165.0, 157.3 (q, ²J_C-F = 38 Hz), 154.5, 152.8, 151.8, 149.9, 147.6, 142.6, 138.1, 133.5, 133.1, 129.0, 128.5, 128.1, 124.3, 120.2, 115.7 (q, ¹J_C-F = 288 Hz), 95.3, 87.1, 81.2, 79.9, 73.7, 72.7, 53.3, 52.6, 38.3, 30.9, 29.7, 28.9, 20.8, 20.6; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.6; HRMS (ESI+): calcd. for [C₃₇H₃₆F₃N₈O₁₀]⁺ 809.2501, meas. 809.2511, Δ 1.2 ppm.

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((4-carbamoylpyridin-2-yl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S85).

A vial was charged with alkyne 8 (35 mg, 51 μmol), 2-bromoisonicotinamide (26 mg, 0.13 mmol, 2.5 equiv.) and CuI (2 mg, 0.01 mmol, 25 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (degassed by sparging with nitrogen for 15 min, 1.3 mL) was added at RT. The resulting mixture was further degassed by sparging with nitrogen for 5 min and a solution of Pd(Pt-Bu₃)₂ (3 mg, 5 μmol, 10 mol %) in a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (1.3 mL) was added. The resulting mixture was stirred at 50 °C for 5 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 95:5) to afford alkyne S85, which was further purified by preparative HPLC (15% → 95% B over 30 min) to afford alkyne S85 (22 mg, 27 μmol, 54%) as a light yellow amorphous solid. R_f = 0.31 (EtOAc/i-PrOH, 95:5); FTIR (thin film), ν_max (cm⁻¹): 3289, 2925, 1747, 1719, 1681, 1612, 1583, 1549, 1457, 1414, 1376, 1245, 1220, 1162, 1047; ¹H NMR (600 MHz, CDCl₃): δ 9.18 (s, 1H), 8.77 (s, 1H), 8.64 (dd, J = 5.2, 0.9 Hz, 1H), 8.13 (s, 1H), 8.05–7.98 (m, 2H), 7.79 (dd, J = 1.7, 0.9 Hz, 1H), 7.65 (dd, J = 5.2, 1.7 Hz, 1H), 7.62–7.58 (m, 1H), 7.59–7.56 (m, 1H), 7.55–7.48 (m, 2H), 7.11 (br s, 1H), 6.14 (br s, 1H), 6.14 (dd, J = 5.4, 5.0 Hz, 1H), 6.12 (d, J = 5.4 Hz, 1H), 5.66 (dd, J = 5.0, 4.4 Hz, 1H), 4.62 (app q, J = 6.9 Hz, 1H), 4.53 (ddd, J = 10.1, 4.4, 3.6 Hz, 1H), 3.77 (s, 3H), 2.85 (app ddt, J = 10.9, 9.0, 4.3 Hz, 1H), 2.15 (s, 3H), 2.19–2.09 (m, 3H), 2.06 (s, 3H), 2.07–2.01 (m, 1H), 1.73–1.66 (m, 1H), 1.65–1.57 (m, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.2, 170.1, 169.8, 166.8, 165.0, 157.4 (q, ²J_C-F = 38 Hz), 152.8, 151.7, 150.7, 149.9, 143.9, 142.6, 141.0, 133.5, 133.1, 129.0, 128.1, 125.2, 124.2, 120.9, 115.7 (q, ¹J_C-F = 288 Hz), 92.3, 87.2, 83.1, 80.9, 73.7, 72.9, 53.3, 52.8, 38.2, 31.0, 29.6, 28.8, 20.8, 20.6; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.6; HRMS (ESI+): calcd. for [C₃₇H₃₆F₃N₈O₁₀]⁺ 809.2501, meas. 809.2502, Δ 0.1 ppm.

(2R,3R,4R,5R)-2-((2S,5S)-2-((5-aminonaphthalen-1-yl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)-5-(6-benzamido-9H-purin-9-yl)tetrahydrofuran-3,4-diyl diacetate (S86).

A vial was charged with alkyne 8 (42 mg, 61 μmol), 5-bromonaphthalen-1-ylamine (34 mg, 0.15 mmol, 2.5 equiv.) and CuI (3 mg, 0.02 mmol, 25 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (degassed by sparging with nitrogen for 15 min, 1.5 mL) was added at RT. The resulting mixture was further degassed by sparging with nitrogen for 5 min and a solution of Pd(Pt-Bu₃)₂ (3 mg, 6 μmol, 10 mol %) in a 5:1:1.5 mixture of toluene, DMF and i-Pr₂NH (1.5 mL) was added. The resulting mixture was stirred at 50 °C for 5 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/iPrOH, 70:30) to afford alkyne S86, which was further purified by preparative HPLC (20% → 95% B over 30 min) to afford alkyne S86 (34 mg, 41 μmol, 67%) as a light yellow amorphous solid. R_f = 0.42 (EtOAc); FTIR (thin film), ν_max (cm⁻¹): 3315, 3066, 2924, 1748, 1721, 1611, 1582, 1514, 1456, 1369, 1243, 1218, 1179, 1074; ¹H NMR (600 MHz, CDCl₃): δ 9.02 (s, 1H), 8.80 (s, 1H), 8.12 (s, 1H), 8.03–7.99 (m, 2H), 7.80 (app dt, J = 8.5, 1.0 Hz, 1H), 7.70 (app dt, J = 8.3, 1.0 Hz, 1H), 7.62–7.58 (m, 2H), 7.53–7.50 (m, 2H), 7.37 (dd, J = 8.5, 7.1 Hz, 1H), 7.34 (dd, J = 8.3, 7.4 Hz, 1H), 7.04 (br d, J = 7.7 Hz, 1H), 6.80 (dd, J = 7.4, 1.0 Hz, 1H), 6.15 (d, J = 5.3 Hz, 1H), 6.09 (dd, J = 5.5, 5.3 Hz, 1H), 5.61 (dd, J = 5.5, 4.8 Hz, 1H), 4.69 (app td, J = 7.7, 4.9 Hz, 1H), 4.65 (ddd, J = 10.8, 4.8, 2.8 Hz, 1H), 4.32 (br s, 2H), 3.74 (s, 3H), 2.97 (app ddt, J = 10.8, 9.1, 4.5 Hz, 1H), 2.24–2.16 (m, 2H), 2.13 (s, 3H), 2.14–2.04 (m, 2H), 2.06 (s, 3H), 1.72 (dddd, J = 13.3, 10.8, 6.0, 4.8 Hz, 1H), 1.61 (dddd, J = 13.3, 10.9, 9.4, 4.5 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 171.3, 169.9, 169.6, 164.7, 157.0 (q, ²J_C-F = 38 Hz), 152.9, 151.7, 149.9, 142.7, 142.3, 134.5, 133.6, 133.0, 130.7, 129.0, 128.0, 127.3, 124.1, 124.1, 123.5, 121.5, 121.3, 117.0, 115.7 (q, ¹J_C-F = 288 Hz), 110.4, 94.9, 87.2, 82.4, 81.0, 73.7, 73.1, 53.2, 52.5, 38.9, 31.2, 30.3, 29.3, 20.8, 20.6; ¹⁹F NMR (471 MHz, CDCl₃, BTF IStd): δ −76.7; HRMS (ESI+): calcd. for [C₄₁H₃₉F₃N₇O₉]⁺ 830.2756, meas. 830.2772, Δ 1.9 ppm.

Cellular Thermal Shift Assay (CETSA)

K562 Cell Culture. The human chronic myeloid leukemia cell line K562 (ATCC CCL-243) was cultured in RPMI 1640 with Glutamax (Gibco) supplemented with 10% fetal bovine serum (Atlanta Biologicals), and 100 IU penicillin and 100 mg/mL streptomycin (Corning). Cells were cultured in T175 flasks with 50 mL of media per flask (Corning) in a 37 °C incubator with 5% CO₂ until the cells reached a density of 2 × 106 cells/mL.

Preparation of K562 Cell Lysate. K562 cells were cultured as described above. Cells were washed with PBS and then resuspended in an appropriate volume of PBS supplemented with cOmplete protease inhibitor cocktail (Roche) to obtain a final cell count of 3.0 × 10⁷ to 3.6 × 10⁷ cells/mL. The suspension was transferred to microcentrifuge tubes and subjected to three rounds of freeze-thaw using liquid nitrogen and a room temperature water bath. The cell lysate was clarified by centrifuging at 17,000g for 20 min at 4 °C, and the supernatant was transferred to a new microcentrifuge tube. Lysate protein concentration was determined by BCA assay and the lysate was diluted to a protein concentration of 3 mg/mL with PBS supplemented with protease inhibitor cocktail. Aliquots were snap frozen in liquid nitrogen and stored at −80 °C.

CETSA with K562 Cell Lysate. The CETSA assay was adapted from previously published protocols.⁸⁰⁻⁸¹ K562 lysate prepared as described above was incubated at room temperature with DMSO, 100 μM of NS1 (10), or 100 μM of compound 25 for 30 min. A Bio-Rad C1000 Touch Thermal Cycler was preheated to the following temperatures using the gradient setting: 37 °C, 38.8 °C, 41.6 °C, 45.2 °C, 50.1 °C, 53.7 °C, 56 °C, 57.9 °C, 60.8 °C, 64.3 °C, 69.1 °C, 72.9 °C. DMSO-treated and compound-treated lysate were aliquotted into twelve PCR tubes, and each tube was heated at one of the temperatures listed above for 3 min. The samples were cooled at room temperature for 3 min, then snap frozen in liquid nitrogen. When thawed, the samples were centrifuged at 17,000g for 20 min at 4 °C. The supernatant was transferred to a new tube, mixed with NuPAGE LDS sample buffer (Novex) and β-mercaptoethanol (2.5%). The samples were heated at 70 °C for 10 min and analyzed by SDS-PAGE and western blot using an anti-NNMT mouse antibody (Abcam, ab119758, 1:1000 dilution), an anti-actin HRP-conjugated mouse antibody (Abcam, ab49900, 1:250000 dilution), and an anti-mouse HRP-conjugated IgG (Promega, W402B, 1:5000 dilution).

Isothermal Dose Response (IDTR) CETSA with K562 Cell Lysate. K562 lysate prepared as described above was incubated at room temperature with DMSO or various concentrations of NS1 (10) or compound 25 for 30 min. A Bio-Rad C1000 Touch thermal cycler was preheated to 57 °C. Samples were aliquotted into PCR tubes, and each tube was heated at 57 °C in the thermal cycler for 3 min. The samples were cooled at room temperature for 3 min and then snap frozen in liquid nitrogen. When thawed, the samples were centrifuged at 17,000g for 20 min at 4 °C. The supernatant was prepared for western blot analysis as described in the CETSA procedure above.

Cytotoxicity Assay in U2OS Cells.

Cytotoxicity evaluation was performed with the Promega CellTiter-Glo Luminescent Cell Viability Assay. U2OS cells were maintained in DMEM-F12 glutamax complete growth media supplemented with 10% heat-inactivated fetal bovine serum (HI FBS). CellTiter-Glo Buffer & CellTiter-Glo Substrate were thawed at room temperature prior to use. An appropriate volume (10 mL) of CellTiter-Glo Buffer was transferred into the amber bottle containing CellTiter-Glo Substrate to reconstitute the lyophilized enzyme/substrate mixture. The contents were gently vortexed to obtain a homogeneous solution. U2OS cells were seeded at 10000 cells/150 μL/well in 96 well cell culture plate and incubated overnight at 37 °C and 5% CO₂. The next day, media was changed (150 μL), various concentrations of compounds (50 μL from 4× stock) were tested in duplicates, at eight concentrations, in semi log dilution pattern, with starting concentration of 100 μM. Final concentration of DMSO in the assay was 1 %. 10 μM, 5 μM and 3 μM of doxorubicin hydrochloride was used as positive control in the assay. The plates were incubated for 24 h and 48 h at 37 °C with 5% CO₂. After 24 h and 48 h of incubation, CellTiter-Glo Reagent (Promega) preparation was added to the cell culture medium present in each well in a 1:1 ratio by volume (e.g., add 50 μL of reagent to 50 μL of medium containing cells). The plate was gently vortexed for 10 min on an orbital shaker to induce cell lysis. The contents of the plate were transferred to an opaque half area 96 well plate. The plate was incubated at RT for 10 min to stabilize the luminescence signal. Luminescence output was read on a Tecan Spark plate reader. The % cell viability was calculated relative to DMSO control.

MNAM Measurement in U2OS Cells by LC-MS/MS

Materials.

Acetonitrile (Millipore Sigma 1000292500), D4-MNAM (BioOrganics SRK(I)-257–3226), MNAM (BioOrganics BST(I)-256–3271), DPBS (Invitrogen 14190–136), Top Seal-A Plus (Perkin Elmer 6050185), 96-well plate sterile TC treated (Eppendorf 0030730119), 96 well plate transfer plate (Costar 3364), reference compound 6-methoxy nicotinamide/(alternate name: JBSNF-0088) (Arbor chemical corporation Limited, P180810). U2OS cells were obtained from ATCC and maintained in DMEM-F12 growth media containing 10% HI FBS and 1% Pen-Strep (filter sterilized) in 37 °C incubator with 5% CO₂. Extraction buffer was prepared by adding internal standard (D4 MNAM, 20 ng/mL) to 100% acetonitrile.

Protocol.

Cells were maintained in cell culture flasks until seeding into microwell plates. Media was removed, cells were washed with DPBS (−/−) and then detached with TrypLE Express (4mL) into a 150 cm² flask (incubate at 37 °C for 5 min). The reaction was stopped by the addition of 5 mL media and cells were counted with a Vi-CELL cell counter. Cells were seeded into a 96-well microplate (100 μL/well = 10000 cells/well) and incubated for 24 h at 37 °C, 5% CO₂, 95% humidity with the lid on. Media was replaced by addition of 100 μL of media/compound mixture and cells were incubated for 24 h at 37 °C, 5% CO₂, 95% humidity with the lid on. The media/compound mixture was removed and cells were washed two times with 150 μL of DPBS(−/−). The DPBS was removed and 100 μL of extraction buffer was added. The plate was incubated for 20 min at RT with mild shaking. 100 μL autoclaved water was added and the plate was mixed gently. The plate was centrifuged at 5000g for 10 min and then 150 μL of the extraction buffer/water mixture (final D4 MNAM concentration of 10 ng/mL) was transferred into a 96-well microplate. MNAM levels were quantified by LC-MS/MS according to methods previously reported by Kannt et al.¹⁵

ABBREVIATIONS

NNMT

nicotinamide N-methyltransferase

SAM

S-adenosylmethionine

NAM

nicotinamide

MNAM

1-methylnicotinamide

SAH

S-adenosylhomocysteine

COMT

catechol O-methyltransferase

BPE

1,2-bis(phospholano)ethane

INMT

indolethylamine N-methyltransferase

PNMT

phenylethanolamine N-methyltransferase

TPMT

thiopurine S-methyltransferase

IStd

internal standard; rbf, round-bottom flask

PMHS

polymethylhydrosiloxane