α-Azido bisphosphonates: synthesis and nucleotide analogues

Abstract

The first examples of α-azido bisphosphonates [(RO)₂P(O)]₂CX(N₃) (1, R = i-Pr, X = Me; 2, R = i-Pr, X = H; 3, R = H, X = Me; 4, R = H, X = H) and corresponding β,γ-CX(N₃) dGTP (5–6) and α,β-CX(N₃) dATP (7–8) analogues are described. The individual diastereomers of 7 (7a, 7b) were obtained by HPLC separation of the dADP synthetic precursor (14a/b).

Triphosphate analogues in which a bridging oxygen is replaced by a functionalized carbon atom have been of interest as enzymatic probes since the early 1980s.^1,2 Recently, we described the preparation of a series of highly purified and well characterized β,γ-CXY^3,4 and α,β-CXY⁵ dNTP derivatives and applied them as probes of structure, function, and fidelity in DNA polymerase β (pol β),^3–5 a base excision repair (BER) enzyme that is overexpressed in some cancers. By appropriate substitution on the CXY carbon (X, Y= H, Me, F, Cl, Br) a range of stereoelectronic properties is made available to probe binding and catalytic interactions with the enzyme. Linear free-energy relationship (LFER) plots of DNA pol β-catalyzed dGMP incorporation rates into nascent DNA from β,γ-CXY dGTPs have revealed a base match- dependent leaving group effect,⁴ and X-ray crystallographic data have provided evidence for stereospecific binding of analogues containing a CXF group.^3,6

To extend these studies, we wished to construct β,γ (5, 6) and α,β (7, 8) dNTPs that incorporate a bridging CX(N₃) group. As a pseudohalide⁷ the azido functionality could usefully extend the tunability of the analogue series, providing more information on molecular interactions at the active site arising from the stereochemistry at the bridging carbon. Introduction of the azido group might also facilitate separation of individual CXY diastereomers, which has not been accomplished with the various X,Y = methyl and halide derivatives prepared thus far.^3,5 These considerations, combined with the prospect of developing a new and potentially useful bisphosphonate synthon, motivated an investigation of α-azido bisphosphonic esters and acids, a previously unknown class of compounds (Figure 1).

FIGURE 1.

Structures of α-azido bisphosphonate esters and acids.

Electrophilic azido transfer was explored as a methodology offering a direct route to 1 and 2 from readily available starting materials. In this approach, a sulfonyl azide reacts with a carbanion species to generate a triazene intermediate that can fragment into either diazo or azido products.⁸ Various factors influence the fragmentation pathway, but the nature of the sulfonyl azide is particularly important. Sulfonyl azides with sterically bulky and electron-donating substituents, such as 2,4,6-triisopropylbenzenesulfonyl azide (trisyl azide; TrN₃), favor azido transfer^8,9 while those containing strongly electron-withdrawing moieties, e.g. trifluoromethylsulfonyl azide (triflyl azide; TfN₃), favor diazo transfer.^9,10

Although the methylene group of a bisphosphonate ester such as tetraisopropyl methylenebis(phosphonate) 9 is exposed in principle to either pathway, replacement of one α-H by a methyl group, as in tetraisopropyl ethylidenebis(phosphonate) 10, will enforce azido transfer. In the synthesis of 1 from 10, methylation of 9 with potassium tert-butoxide (t-BuOK) followed by methyl iodide gave a mixture of 78% 10 and 20% α,α-dimethyl side product. Fortunately, this crude 10 preparation could be directly treated with t-BuOK and then p-toluenesulfonyl azide (tosyl azide; TsN₃) in DMSO at room temperature to give the azido ester 1 in 70% yield after purification by column chromatography (Scheme 1).

SCHEME 1.

Synthesis of 1.

Attempts to synthesize 2 from 9 using tosyl azide led to the unwanted diazo transfer product. Reaction of a carbanion derived from an activated methylene group with a sulfonyl azide is a known route to tetraalkyl diazomethylenebis(phosphonates) and trialkyl α-diazo phosphonoacetates.¹¹ However, use of trisyl azide with careful optimization of conditions (THF, −78° C, potassium counterion, fivefold azide reagent excess, immediate quenching by AcOH) generated 2 from the carbanion of 9 in greater than 95% yield by ³¹P NMR (Scheme 2). Despite the efficient conversion, purification of 2 proved to be problematic initially due to difficulty in removing the large excess of trisyl azide and a minor side product, tetraisopropyl diazomethylenebis(phosphonate) (11), by preparative chromatography on silica gel. Pure 2 (50% yield) was eventually obtained by crystallization of remaining trisyl azide, filtration, and selective oxidation of 11 with t-butyl hypochlorite¹² in wet ethyl acetate¹³ prior to chromatography.

SCHEME 2.

Synthesis of 2.

Care must be taken during work-up of 2, which retains a relatively acidic α-H, because it decomposes slowly on silica gel, more quickly in aqueous solution at pH 6, and rapidly at pH 8. This contrasts with the reported stability of the malonate derivative¹⁴ and presumably reflects the resonance stabilization available in the enolate form of the latter compound.

Exposure of 2 to bases such as BuLi, NaH, KHMDS, and t-BuOK results in the release of N₂ with formation of diisopropyl cyanophosphate (12)¹⁵ and diispropyl phosphite (13) (Scheme 3). The base-induced elimination of nitrogen from α-azido esters,¹⁶ malonates,¹⁴ and sulfones¹⁷ has been previously reported and transition metal-catalyzed versions of this reaction have found synthetic utility.^18,19 In contrast to diethyl azidomalonate, in which the carbanion was further derivatized at −15 °C without the loss of N₂,¹⁴ 2 loses N₂ rapidly upon exposure to a variety of bases in organic solvents at −78 °C and does not give appreciable amounts of adducts in the presence of a large excess of N-chlorosuccinimide.

SCHEME 3.

Base-induced decomposition of 2.

The structures of 1–4 are supported by their suite of IR, NMR, and MS data. Esters 1 and 2 are both colorless oils with strong IR absorbances in the azido region (1: 2123, 2086 cm⁻¹; 2: 2104, sh. at 2134 cm⁻¹). The ¹H NMR spectrum of 1 includes a CH₃ triplet at δ 1.60 ppm (t, J_HP 15 Hz) and that of 2 has an α-H triplet at δ 3.53 ppm (J_HP 21 Hz). The ¹³C NMR spectrum of 1 has resonances at δ 17.6 (CH₃) and δ 59.8 ppm (C_α t, J_CP 150 Hz). The C_α of 2 resonates at δ 55.0 ppm (t, J_CP 145 Hz). The ³¹P chemical shifts of 1 (δ 17.0 ppm) and 2 (δ 13.3 ppm) are consistent with a previously observed 3–4 ppm downfield effect of α-methylation in other bisphosphonate esters.^20,21

Reaction of 1 and 2 with bromotrimethylsilane (BTMS)²² in acetonitrile at room temperature quantitatively gave the tetrakis(trimethylsilyl) esters, which display the expected upfield ³¹P shifts, Δδ +16–17 ppm. Desilylation with aqueous ethanol produced the acids 3 and 4 (> 98% overall) as hygroscopic white solids. These acids are stable in dilute aqueous solution pH 2–12 for at least several days at room temperature. They have IR bands at 2126, 2085 (sh) cm⁻¹ (3) and 2113 cm⁻¹ (4) attributed to the azido group. The ¹H NMR spectra include resonances at δ 1.43 ppm (C(CH₃)N₃, t, J_HP 13 Hz) for 3 and δ 3.26 ppm (CHN₃, t, J_HP 17 Hz) for 4 (CHN₃). The ¹³C NMR displays resonances at δ 23.5 (CH₃) and δ 68.4 ppm (C_α, t, J_CP 129 Hz) for 3, and at δ 65.1 ppm (t, J_CP 124 Hz) (C_α) for 4. The ³¹P chemical shifts are in the expected regions, δ 18.3 ppm (3) and δ 13.5 ppm (4). The HRMS m/z values for the [M−H]⁻ anion of 3 and 4 match calculated values within error, confirming the assigned structures.

The present study and our previous interest in diazo transfer chemistry^23,24 prompted us to reinvestigate a reported azido transfer to triethyl phosphonoacetate using triflyl azide in the presence of triethyl amine.²⁵ Triflyl azide is known to be a powerful diazo transfer reagent⁹ and attempts by other authors to extend the procedure to benzocyclic β-keto esters were unsuccessful;⁸ nevertheless, the original reaction has been cited as a viable azido transfer reaction in recent reviews.^26,27 In our hands, the procedure of Hakimelahi and Just²⁵ produced triethyl diazophosphonoacetate, characterized by ¹H, ¹³C, ³¹P NMR and MS (ESI and APCI) and generated no detectable amounts of azido transfer product. In light of these results and analysis of the literature,²⁸ we propose that the reaction of triflyl azide with triethylphosphonoacetate is consistent with known diazo transfer chemistry and is not an anomalous case of azido transfer.

With acids 3 and 4 in hand, we then proceeded to the synthesis of β,γ (5, 6) and α,β (7, 8) azido methylene nucleotide triphosphate analogues. The β,γ-CXN₃ dGTP analogues 5 and 6 were prepared by coupling^3,4 the Bu₃NH⁺ salts of 3 and 4 with the dGMP-morpholidate.²⁹ α,β-CXN₃ analogues of dATP (7, 8) were synthesized by reaction of the Bu₄N⁺ salt of 3 or 4 with dA-5’-tosylate followed by enzymatic phosphorylation of the resulting α,β-CX(N₃) dADP intermediates (14, 15) by ATP-phosphoenolpyruvate (PEP) catalyzed by pyruvate kinase (PK) and nucleoside diphosphate kinase (NDPK) in HEPES buffer, pH 7.5⁵ (Scheme 4). The final compounds were purified by dual-pass (strong-anion exchange ‘SAX’ followed by reverse phase ‘RP’) HPLC to ≥ 97% as determined by ³¹P NMR and analytical HPLC, and were characterized by NMR (¹H, ³¹P) and HRMS.

SCHEME 4.

Synthesis of nucleotide azidomethylene analogues.

Nucleotide triphosphate α,β-CXY or β,γ-bisphosphonate analogues with an asymmetrically substituted bridging carbon (X ≠ Y) are obtained as mixtures of diastereomers by existing synthetic procedures starting from a prochiral bisphosphonate precursors, and thus far none have been separated chromatographically.^{3, 5, 6} Diastereomeric β,γ-fluoromethylene dNTPs can be individually observed in the synthetic mixture by ¹⁹F NMR.^3,6 In addition to the expected nucleoside resonances for dGuo, the ¹H NMR of 5a/b shows a pair of doublets centered at δ 1.58–1.59 ppm (C(CH₃)N₃) and that of 6a/b an apparent triplet at δ 3.40 ppm (CHN₃). However, although the proton-decoupled P_α and P_γ ³¹P NMR signals of 5a/b at 202 MHz are doublets at δ −9.45 and 14.5 ppm respectively, two resolvable (Δδ 0.03 ppm) diastereomeric pairs of doublets (J = 34, 19 Hz) are observed for P_β. The δ vs. J assignments were confirmed by comparing ³¹P spectral data obtained at 162 and 202 MHz.

A similar pattern is seen with 6a/6b except that resolvable diastereomeric multiplets are observed for both P_α (δ −9.78, −9.81 ppm) and P_β (δ 9.04, 9.06 ppm), but not for P_γ (10.4 ppm). This difference between 5a,b and 6a,b with respect to the sensitivity of the P_α chemical shift to the P_βγ CXN₃ chiral center invites further investigation (we speculate that it might implicate an H-bonding effect involving the relatively acidic CHN₃ hydrogen, or alternatively reflects a more asymmetric shielding effect of the CHN₃ group on the P_α nucleus).

In the preparation of the α,β-C(CH₃)N₃ ATP analogues 7a and 7b, we found that the corresponding diphosphate intermediates (14a/b) could be isolated individually by isocratic RP HPLC (Figure 2). Subsequent enzymatic phosphorylation as described above then generated the individual diastereomers 7a and 7b. CD spectra measured for 7a and 7b showed prominent features of opposite polarity centered near 260 nm (7a, (−); 7b (+)). Curiously, the α,β-CHN₃ dADP intermediate (15a/b) did not separate under similar conditions and its phosphorylation yielded the triphosphates 8a/b as a mixture of diastereomers which could not be distinguished by ³¹P NMR.

FIGURE 2.

Separation of 14 diastereomers by RP preparative HPLC. Left: ³¹P NMR spectrum of 14a/b as synthesized displays two pairs of doublets for both P_α and P_β. Right: ³¹P NMR (14a and 14b) spectra of separated diastereomers show single pairs of doublets for P_α and P_β.

In conclusion, we have described the first examples of α-azido bisphosphonate esters and acids. The latter compounds can be used to synthesize novel nucleotide analogues containing a CHN₃ or CMeN₃ at either the α,β or β,γ bridging position. The ability to obtain for the first time the individual diastereomers of the α,β-CH₃N₃ dADP (14a, 14b) and corresponding dATP analogues (5a, 5b) provides more refined probes for stereochemical interactions of these compounds with appropriate enzymes. A further use of these new bisphosphonates may be to prepare derivatives made more conveniently or uniquely accessible by the presence of the synthetically versatile azido function.

Experimental

General Experimental Methods

The nucleotide triphosphate analogues were prepared by adapting our previously published methods for β,γ-CXY^3,4,6 and α,β-CXY⁵ dNTP analogues. Analytical HPLC analysis was conducted on a Varian PureGel SAX 10 mm × 100 mm 7 µL column eluted with A: H₂O B: 0.5 M (0–50%) LiCl gradient over 30 min at a 4 mL/min flow rate. Products were detected at 259 nm for adenosine derivatives and at 253 nm for guanosine derivatives. All dNTP derivatives were prepared as triethylammonium salts.^3–6 CD spectra were obtained on a JASCO J-815 spectropolarimeter.

Tetraisopropyl (1-azidoethane-1,1-diyl)bis(phosphonate), 1

Tetraisopropyl methylenebis(phosphonate) (1.75 g, 5 mmol) was treated with 1.2 equiv. t-BuOK and 1.2 equiv. MeI in anhydrous acetonitrile overnight at rt. Volatiles were removed at reduced pressure and the residue was dissolved in water, extracted into DCM and dried over MgSO₄. Evaporation of the solvent left an oil (1.84 g) that contained ~20% dimethylated product, ~2% unmethylated material, and 78% monomethylated product. This mixture (~4 mmol monomethylated product) was dissolved in 20 mL DMSO and stirred with 0.689 g (6 mmol) t-BuOK. TsN₃ 1.05 g (5 mmol) was added dropwise. The reaction mixture was stirred for 30 s, quenched with 1.5 mL of AcOH, and then allowed to stir for several hours at rt. The reaction mixture was extracted with hexanes and the organic phase was dried over MgSO₄. Following concentration by evaporation, the product was purified on silica gel using ACE:DCM (1:3), R_f = 0.69, giving 1 as a colorless oil. Yield: 1.45 g (70% overall). IR: ν (film) 2123 cm⁻¹, 2086 (N₃). δ_H (399.8 MHz; CDCl₃): 1.33-1.37 (24 H, m, 4 × OCH(CH₃)₂), 1.60 (3H, t, J_HP 15.2 Hz, PCCH₃N₃P), 4.83 (4 H, m, 4 × OCH(CH₃)₂); δ_C (100.5 MHz; CDCl₃): 17.6 (t, J_CP 3.9 Hz, PCCH₃N₃P), 23.7-23.8, 24.3-24.4 (OCH(CH₃)₃, 59.8 (t, J_CP 150.3 Hz, PCP), 72.6-72.8 ((OCH(CH₃)₃; δ_P (161.9 MHz; CDCl₃; 85% H₃PO₄): 17.0 ({¹H} s; coupled J_PH 15.2 Hz (qm), J_PC 150 Hz (satellites)).

Tetraisopropyl (azidomethanediyl)bis(phosphonate), 2

In a 500 mL RB flask under N₂, ~6 g (19 mmol) of TrN₃ were dissolved in 30 mL of anhydrous THF and stirred vigorously at −78 °C. A solution of 1.517 g (4.41 mmol) of tetraisopropyl methylenebis(phosphonate) and 4.41 mL 1M KHMDS in 10 mL THF was mixed at rt, cooled to −78 °C and added at once to the TrN₃ solution by syringe. The solution was stirred for 10 s and then quenched with 2.5 mL AcOH. The flask was moved to a −20 °C freezer and allowed to stand for 48 h, or until the triazene had decomposed as determined by ³¹P NMR. The precipitate was filtered and volatiles removed at reduced pressure. The reaction mixture was then seeded with solid TrN₃ and allowed to sit overnight at 0 °C. The resulting precipitate was washed with H₂O and filtered off. After removing water with rotovap, the residue was dissolved in 20 mL wet EtOAc. Ten drops of t-BuOCl were added and stirred at rt until evolution of N₂ ceased. EtOAc was washed with a saturated aqueous NH₄Cl solution, dried and concentrated. The residue (95% by ³¹P NMR) was purified on silica gel eluted with ACE:DCM (1:9), R_f = 0.36. Contaminating phosphite was removed by rotary evaporation (oil pump) with gentle warming to afford 0.83 g of 2 as a colorless oil (yield: 50% overall). IR: ν (film) 2134 (m), 2104 cm⁻¹ (N₃). NMR: δ_H (399.8 MHz; CDCl₃): 1.31-1.34 (24 H, m, 4 × OCH(CH₃)₂), 3.53 (1H, t, J_HP 20.8 Hz, PCHN₃P), 4.79 (4 H, m, 4 × OCH(CH₃)₂); δ_C (100.5 MHz; CDCl₃): 23.7-23.8, 24.1-24.2 **(OCH(CH₃)₃, 55.0 (t, J_CP 144.8 Hz, PCP), 72.7, 72.9 (2t, J_CP 3.5 Hz OCH(CH₃)₃); δ_P (161.9 MHz; CDCl₃; 85% H₃PO₄): 13.3 ({¹H} coupled, s; J_PH 20.5 Hz (dm), J_PC 145 Hz).

Base-induced decomposition of 2

5 mg (13 µmol) of 2 was dissolved in 1.5 mL anhydrous THF and cooled to −78 °C. 1.5 equiv. t-BuOK was added at once, causing rapid evolution of gas. Identical results were obtained with BuLi and KHMDS. NMR: δ_P (202.5 MHz; CDCl₃; 85% H₃PO₄): 22.29 (diisopropyl cyanophosphate),¹⁵ 4.8 (diispropyl phosphite) with integration 1:1.

(1-Azidoethane-1,1-diyl)bis(phosphonic acid), 3 and (azidomethanediyl)bis(phosphonic acid), 4

The appropriate bisphosphonate ester was dissolved in 2 mL anhydrous acetonitrile and 6 equiv. of BTMS were added by syringe. Stirring overnight gave the tetrakis(trimethylsilyl) esters (100% by ³¹P NMR). From 1: δ_P (202.5 MHz; CHCN, ext. 85% H₃PO₄): 0.9; from 2: δ_P −3.2 (s). Hydrolysis with ~1 mL of EtOH:H₂O (1:1) and removal of volatiles by prolonged evaporation at low pressure provided the acids as hygroscopic white solids (>98% overall yield). Acid is further dried under vacuum in desiccator containing P₂O₅.

3: white solid, IR: ν (KBr): 2085 (m), 2126 cm⁻¹ (N₃). NMR: δ_H (399.8 MHz; D₂O; pH 10.88): 1.43 (t, J_HP 13.2 Hz, PCCH₃N₃P); δ_C (100.5 MHz; D₂O; ext. pH 10.88): 23.5 (s, PCCH₃P), 68.4 (t, J_CP 129, PCP); δ_P (161.9 MHz; D₂O; 85% ext. H₃PO₄; pH 10.88): 18.3 ({¹H} s; coupled, J_PH 13.2 Hz (q); J_PC 129 Hz (satellites)). HRMS (ESI/APCi) [M−H]⁻: calcd for C₂H₆N₃O₆P₂: 229.9737. Found: 229.9734.

4: IR: ν (KBr): 2113 cm⁻¹ (N₃). δ_H (399.8 MHz; D₂O; pH 10.88): 3.26 (t, J_HP 16.8 Hz, PCHN₃P); δ_C (100.5 MHz; D₂O; pH 10.88): 65.1 (t, J_CP 124 Hz, PCP); δ_P (161.9 MHz; D₂O; 85% ext. H₃PO₄; pH 10.88): 13.5 ({¹H} s; coupled, J_PH 16.5 Hz (d); J_PC 122 Hz (satellites)); HRMS (ESI/APCi) [M−H]⁻: calcd for CH₄N₃O₆P₂, 215.9581. Found: 215.9584.

Ethyl diazo(diethoxyphosphoryl)acetate

from trifyl azide and ethyl(diethylphosphoryl)acetate. According to the procedure of Hakimelahi and Just,²⁵ 72 mg NaN₃ (1.1 mmol) was suspended in 20 mL anhydrous DMF and 106 µL (1.0 mmol) CF₃SO₂Cl was added under N₂. After several min, a solution of 222 mg (1.0 mmol) of ethyl(diethylphosphoryl)acetate and 101 mg (1.0 mmol) Et₃N in 5 mL anhydrous DMF was added dropwise. After 40 min, a mixture of compound was detected by ³¹P NMR with δp (DMF) 20.8, 18.3, 10.4 in a ratio of 5:4:1. Gentle heating for an additional 30 min gave a binary mixture of the compounds at 10.4 (40%) and 20.8 (60%). Ether was added and the reaction mixture was washed 5 × with water. The organic layer was dried over MgSO₄ and evaporated. A portion of the residue was purified by preparative TLC on silica gel eluted with ACE:DCM (1:9) giving a single mobile band (UV), R_f = 0.65 which was identified as ethyl diazo(diethoxyphosphoryl)acetate. NMR: δ_H (400.2 MHz; CDCl₃): 1.32 (3H, t, J_HH 7 Hz), 1.38 (6H, dt, J_HH 7 Hz, J_HP 0.8 Hz), 4.22 (4H, m), 4.29 (2H, q, J_HH 7 Hz); δ_P (202.5 MHz, CDCl₃; 85% H₃PO₄) 10.3 (s). MS (APCi, m/z), 251 (M+1)⁺; an MS/MS analysis of the m/z 251 species, gave a major fragment at m/z 223.¹¹

2’-Deoxyguanosine 5’-triphosphate β,γ CCH₃N₃, 5a/b

Compound 3 (2 equiv. as the 1.5 tributylammonium salt) was coupled to 100 mg (0.242 mmol) dGuo-morpholidate in anhydrous DMSO to yield a white solid, 32.5 mg (23.8%) after dual pass (SAX then RP) HPLC purification.⁴ Analytical HPLC: retention time = 9.6 min; purity = 98%. NMR: δ_H (399.8 MHz; D₂O; pH 10.88): 1.58 (dd, PCCH₃N₃P) 2.48-2.54, 2.72-2.79, 4.11-4.21, 4.12, 6.32 8.05. δ_P (161.9 MHz; D₂O; 85% H₃PO₄; pH 10.88): −9.5 (d, J_αβ 33.7 Hz, P_α), 13.28 (dd, J_αβ 33.7 Hz, J_βγ 19.4 Hz, P_β), 13.31 (dd, J_αβ 33.7 Hz, J_βγ 19.4 Hz, P_β) 14.5 (d, J_βγ 19.4 Hz, P_γ). HRMS (ESI/APCi) [M−H]⁻: calcd for C₁₂H₁₈N₈O₁₂P₃, 559.0263. Found: 559.0266.

2’-Deoxyguanosine 5’-triphosphate β,γ CHN₃, 6a/b

Compound 4 (2 equiv. as the 1.5 tributylammonium salt) was coupled to 100 mg (0.242 mmol) dGuo-morpholidate in anhydrous DMSO to yield a white solid, 31.1 mg (23.5%) 6a/b after dual pass (SAX then RP) HPLC purification.⁴ Analytical HPLC: retention time = 10.3 min, purity = 98%. NMR: δ_H (499.8 MHz; D₂O; pH 10.88): 2.48-2.53, 2.71-2.76, 3.53 (ddd, PCHN₃P), 4.10-4.20, 4.25, 6.31 (t, J 6.0 Hz) 8.03, 8.03; δ_P (161.9 MHz; D₂O; 85% H₃PO₄; pH 10.88): −9.78 (d, J_αβ 27.8 Hz, P_α), −9.81 (d, J 27.8 hz P_α), 9.04 (dd, J_αβ 27.8 Hz, J_βγ 12.9 Hz, P_β), 9.06 (dd, J_αβ 27.8 Hz, J_βγ 12.9 Hz, P_β) 10.414 (d, J_βγ 12.9 Hz, P_γ). HRMS (ESI/APCi) [M−H]⁻: calcd for C₁₁H₁₆N₈O₁₂P₃, 545.0106. Found: 545.0121.

2’-Deoxyguanosine 5’-diphosphate, α,β C(CH₃)N₃, 14a/b

The tris(tetrabutylammonium) salt from 60 mg (0.260 mmol) of 3 was allowed to react with 115 mg (~1.1 equiv.) dA-5’-Ts in anhydrous acetonitrile⁵ to yield 65.1 mg of the nucleoside diphosphonate diasteromers in a ratio of 2:3; (54% crude conversion), which could be separated by RP HPLC. 14a: NMR: δ_H (399.8 MHz; D₂O; pH 10.88): 1.50 (dd, J_HP 12.4, J_HP 15.2 Hz, PCCH3N3P), 2.57-2.63, 2.82-2.91, 4.10-4.16, 4.22-4.27, 6.48 (t, 6.4 Hz), 8.25, 8.52; δ_P (161.9 MHz; D₂O; 85% H₃PO₄; pH 10.88) 14.5 (d, J 15 Hz, P_β); 21.7 (d, J 15 Hz, P_α). 14b: NMR: δ_H (399.8 MHz; D₂O; pH 10.88): 1.50 (dd, J_HP J_HP 12.8, 15.2 Hz, PCCH3N3P), 2.57-2.63, 2.82-2.88, 4.10-4.16, 4.22-4.27, 6.47 (t, 6.4 Hz), 8.22, 8.50; δ_P (161.9 MHz; D₂O; 85% H₃PO₄; pH 10.88) 14.4 (d, J 15 Hz, P_β); 21.6 (d, J 15 Hz, P_α).

2’-Deoxyadenosine 5’-triphosphate α,β C(CH₃)N₃, 7a/b

Enzymatic phosphorylation⁵ of 14a and 14b followed by HPLC purification of each product gave 17.3 mg (yield: 12.2% overall from starting acid) 7a and 14.9 mg (10.5%) 7b. 7a: Analytical HPLC: retention time = 9.5 min; purity = 98%. NMR: δ_H (499.8 MHz; D₂O; pH 10.88): 1.58 (t, J_HP 15.0 Hz, PCCH₃N₃P), 2.58-2.63, 2.83-2.89, 4.21-4.29, 6.50 (t, 6.5 Hz), 8.25, 8.54; δ_P (202.5 MHz; D₂O; 85% H₃PO₄; pH 10.88) −4.5 (d, J_βγ 31.9 Hz, P_γ); 7.0 (dd, P_β); 18.51 (d, J_αβ 19.0 Hz, P_α). HRMS (ESI/APCi) [M−H]⁻: calcd for C₁₂H₁₈N₈O₁₁P₃, 543.0313. Found: 543.0333. 7b: Analytical HPLC: retention time = 9.5 min; purity = 96%. NMR: δ_H (499.8 MHz; D₂O; pH 10.88): 1.59 (t, J_HP 15 Hz, PCCH₃N₃P), 2.58-2.63, 2.85-2.90, 4.17-4.22, 6.51 (t, 6.5 Hz), 8.26, 8.56; δ_P (202.3 MHz; D₂O; 85% H₃PO₄; pH 10.88) −4.4 (d, J_βγ 30.1 Hz, P_γ); 8.0 (dd, P_β); 18.47 (d, J_αβ 18.2 Hz, P_α). HRMS (ESI/APCi) [M−H]⁻: calcd for C₁₂H₁₈N₈O₁₁P₃, 543.0315. Found: 543.0315.

2’-Deoxyguanosine 5’-diphosphate, α,β CHN₃, 15a/b

The tris(tetrabutylammonium) salt was prepared from 60 mg (0.276 mmol) of 4 and allowed to react with 120 mg (~1.1 equiv.) dAdo-5’-Ts in anhydrous acetonitrile⁵ to yield a white solid, 32 mg (26%). NMR: δ_p (242.8; D₂O; pH 10.88): 9.9 (s, P_β); 17.2 (s, P_α).

2’-deoxyadenosine 5’-triphosphate α,β CHN₃, 8a/b

Enzymatic phosphorylation⁵ followed by HPLC purification gave 33.2 mg (22.7%) 8a/b. Analytical HPLC: retention time = 10.4 min; purity = 98%. NMR: δ_H (400.2 MHz; D₂O; pH 10.88): 2.57-2.63, 2.82-2.90, 3.86 (dt, PCHN₃P), 4.11-4.25, 4.29, 6.50 (t, J 6.5 Hz), 8.24, 8.528, 8.532; δ_P (202.3 MHz; D₂O; 85% H₃PO₄; pH 10.88) −5.1 (d, J_βγ 25.1 Hz, P_γ); 2.8 (dd, P_β); 14.3 (d, J_αβ 13.8 Hz, P_α). HRMS (ESI/APCi) [M−H]⁻: calcd for C₁₁H₁₆N₈O₁₁P₃, 529.0157. Found: 529.0147.