Predictive Analysis of the Side Chain Conformation of the Higher Carbon Sugars: Application to the Preorganization of the Aminoglycoside Ring 1 Side Chain for Binding to the Bacterial Ribosomal Decoding A Site

Abstract

With a view to facilitating prediction of the exocyclic bond to the pyranoside ring in higher carbon sugars a model is advanced that relates the relative configuration of the three stereogenic centers comprised of the branchpoint and of the two flanking centers (C4–C5–C6 in aldoheptoses and higher, and C5–C6–C7 in sialic and ulosonic acids) to that of the simple ring-opened pentoses. Assignment of a given stereotriad as arabino, lyxo, ribo, or xylo by inspection of the Fischer projection formulae permits prediction of conformation of the exocyclic bond by comparison with the known solution (= crystal in all cases) conformations of the simple pentitols. More remote stereogenic centers in the side chain, as in the 8-position of N-acetylneuraminic acid, have little impact on the conformation of the exocyclic bond. On the basis of this model the conformation of the exocyclic bond in ring I of 6’-homologated 4,5-disubstituted 2-deoxystreptamine class aminoglycoside antibiotics was predicted and was borne out by NMR analysis of newly synthesized derivatives in D₂O at pD5. The antiribosomal and antibacterial activity of these derivatives is briefly presented and discussed in terms of preorganization of the side chain for binding to the ribosomal decoding A site. It is anticipated that this predictive analysis will also find use in the prediction of the conformation of the exocyclic bonds in other 2-(1-hydroxyalkyl)-3-hydroxytetrahydropyrans and tetrahydrofurans.

Graphical Abstract

Introduction

It is well-appreciated that the conformation of the side chain (exocyclic C5–C6 bond) in the hexopyranosides makes an important contribution to reactions at the anomeric center. Three staggered conformers resulting from rotation about the exocyclic bond are possible and known as gauche,gauche, gauche,trans, and trans,gauche (gg, gt, and tg, respectively) where the first and second descriptors refer to the position of the C6–O6 bond relative to C5–O5 and C5–C4 bonds, respectively, as illustrated for D-glucopyranose and D-galactopyranose in Figure 1.^1–3 In an alternative nomenclature the gg, gt, and tg conformers are called the -g, +g, and t-conformers, respectively. In this Article we use the gg, gt, and tg nomenclature consistent with our recent practice and to avoid confusion when drawing on (pseudo)enantiomeric series of sugars (D- and L-series).

Figure 1.

The staggered conformations of the D-galacto- and D-glucopyranose side chains and their relative populations in free solution.

The relative proportions of the three staggered conformers in free, as opposed to protein-bound, sugars can be determined by NMR spectroscopy,^1–3 or by statistical analysis of X-ray crystal structure databases,⁴ and generally fall into two broad groups according to whether the C-O bond at the 4-position of the pyranose ring is axially or equatorially disposed, as exemplified by D-galacto- and D-glucopyranose, respectively (Figure 1). Absent a C-O bond at the 4-position (4-deoxy-D-xylo-hexopyranose) the side chain population closely mirrors that in D-glucopyranose itself.⁵ Much is also known about the manner in which the anomeric configuration and protecting groups influence the side chain conformation,^6–12 although these effects tend to be relatively small with the obvious exception of those that include the side chain in a fused ring such as the 4,6-acetals. The distribution of conformations among the gg, gt, and tg conformers in the hexopyranoses (Figure 1) is widely understood to be the result of the gauche effect, which stabilizes gauche conformations of vicinal diols,¹³ and of repulsion between syn-coplanar C-O dipoles in 1,3-diols, known as the Hassel-Ottar effect,¹⁴ and evident in the gg conformation of D-galactopyranose and the tg conformation of D-glucopyranose (Figure 1). This latter effect, hereinafter simply called a 1,5-syn interaction and estimated¹⁵ at 1.9 kcal.mol⁻¹, is the heteroatomic equivalent of the syn-pentane interaction.

In the so-called higher carbon sugars the situation is complicated by the presence of the additional C6–C7 bond and a consequent further stereogenic center at the 6-position such that, taking into account the stereogenic centers at the 4- and 6-positions only, a total of four diastereomers are possible, each with three staggered conformers, as illustrated in Figure 2 for the simplest case of the heptopyranosides.

Figure 2.

The Different Configurations and Conformations of the Heptopyranoses in the D-Galacto and D-Gluco Series

Our interest in the chemistry and biology of the bacterial oligosaccharides led us to appreciate that the conformation of the exocyclic bond to the side chain in the sialic and ulosonic acids is governed by the relative configuration at the 5- and 7-positions, which are equivalent to the 4- and 6-positions in the simple hexo- and heptopyranoses. Thus, N-acetylneuraminic acid 1 and its derivatives very predominantly adopt the gg conformation, whereas both the 5- and the 7-epi-series take up the gt conformation, as revealed for example by 2 and 3, respectively.^{16, 17} Pseudaminic acid 4, which is epimeric at the 5- and 7-positions relative to 1,¹⁸ and its pseudo-enantiomer KDO 5 both adopt the tg conformation (Figure 3).¹⁹ Raines, Kiessling, and coworkers have reached analogous conclusions on the basis of a computational analysis for KDO and other octoses and applied it to their recognition by lectins.²⁰ The importance of the 1,5-syn interaction between two C-O bonds is illustrated by 8-epi-N-acetylneuraminic acid 6, which adopts a bent conformation about the 7,8-bond.²¹ Importantly however, 6 retains the gg conformation of the exocyclic bond highlighting the dominant influence of the relative configuration of the C5–C7 centers on the conformation of the exocyclic bond. The importance of the 1,5-syn interaction is further emphasized by the two stereoisomeric acyclic alditols 7 and 8 obtained on reduction of 1 with sodium borohydride. Thus, the D-erythro-L-gluco isomer 7 predominantly adopts the zig zag conformation of its entire nine carbon backbone, which is free of 1,5-syn interactions. The D-erythro-L-manno isomer 8 on the other hand is considered to be an approximately equimolar mixture of three conformers: the zig zag conformation depicted with the offending 1,5-syn interaction, and two others arising from rotation about the 2,3- and 3,4-bonds respectively which lack the said interaction.²²

Figure 3.

Epimeric sialic and ulosonic acids 1-6, and their predominant side chain conformations, and alditols 7 and 8 in their zig zag conformations

Looking to generalize the lessons from our work on the conformational analysis of the sialic and ulosonic acids, namely the realization that the conformation about the exocyclic bond (henceforth termed exocyclic bond conformation for the sake of simplicity) is controlled by the relative configuration in a simple stereotriad consisting of the point of attachment of the side chain to the pyranose ring and its immediate neighbors, we sought to correlate the exocyclic bond conformation with that of the simple acyclic pentitols: arabinitol, lyxitol, ribitol, and xylitol. We show that the carbon chains comprised of C4–C7 of the pyranose forms of the higher carbon sugars, or C5–C8 of the sialic and ulosonic acids, follow the same conformational patterns as the acyclic pentitols, making the prediction of side chain conformation possible by simple inspection of the Fischer projection formulae. We apply this insight to the analysis of the side chain conformation of aminoglycoside derivatives substituted at the 6’-position of ring I and show how the introduction of a simple alkyl group at that position with the correct configuration can be used to pre-organize the exocyclic bond and its substituent for binding to the decoding A site in helix 44 of the bacterial ribosome.

Results and Discussion

Examination of the studies outlined in Figure 3 suggests that exocyclic bond conformation in the higher carbon sugars is largely determined by the relative configurations at the 4-, 5-, and 6-positions (which correspond to the 5-, 6-, and 7-positions in the neuraminic and ulosonic acid series). In the hexopyranose series, provided a standard chair conformation is adopted with the side chain in an equatorial position, the four carbon chain from C3–C6 is held in the favored zig zag conformation (Figure 4A, bold red line). When the side chain is extended by one carbon as in a heptopyranose, three staggered conformations about the C5–C6 bond are possible (Figure 4). One of these conformations retains the full zig zag conformation for the C3–C7 carbon backbone and, following the usual rules of conformational analysis of saturated carbon chains and in the absence of further substitution,²³ is considered to be lower in energy than the remaining two which represent bent or sickle conformations of the C3–C7 carbon backbone (Figure 4B–D, bold red lines). Hydroxylation of the red chain at positions 3–6 (Figure 4A) gives rise to the tetritols erythritol and threitol, depending on the configuration of substitution at C4. Similarly, hydroxylation at positions 3–7 in any of Figs 4B–D affords the pentitols arabinitol, lyxitol, ribitol, and xylitol depending on relative configuration. It follows that consideration of the conformations of the simple pentitols, as determined by NMR spectroscopy or X-ray crystallography, should facilitate prediction of side chain conformation in the higher carbon sugars.

Figure 4.

Zig zag and bent or sickle conformations of the carbon chain from C3–C6 (A) and C3–C7 (B-D) in hexo- and heptopyranoses (hydroxyl groups are omitted for clarity)

The corresponding four D-pentitols are represented as both their Fischer projection formulae, and with the zig zag conformation of the carbon-carbon backbone, preferred by some²⁴ and more familiar to organic chemists, in Figure 5. The zig zag conformation of D-arabinitol (= D-lyxitol) has two sets of vicinal diols with the favored gauche relationship, and no 1,5-syn-interactions, and is therefore the lowest energy conformation. Consistent with this logic arabinitol crystallizes in the zig zag conformation.²⁵

Figure 5.

The D-Pentitols Represented as Fischer Projection Formulae, with Extended Zig Zag Conformations of their Carbon Backbones, Actual Conformations in the Crystal and Aqueous Solution, with Stablizing gauche Interactions in Blue and Destabilizing 1,3-syn Interactions in Red.

In the extended zig zag conformation ribitol does not benefit from any stabilizing gauche interactions between pairs of vicinal C-O bonds, and it is destabilized by the presence of a 1,5-syn interaction. It can therefore be expected that it undergoes a change of conformation to gain gauche interactions and to escape from the 1,5-syn interaction. Indeed, the single crystal X-ray structure of ribitol exhibits a sickle conformation achieved by rotation about the C3–C4 bond by 120°; in doing so it gains a gauche interaction and removes the 1,5-syn interaction (Figure 5).²⁶ Xylitol represents an intermediate case between arabinitol and ribitol in that in the extended zig zag conformation it benefits from two stabilizing gauche interactions, but suffers from a destabilizing syn-1,3-diol motif. Xylitol crystallizes as a sickle conformation that retains two gauche interactions, but which avoids the 1,5-syn-interaction (Figure 5).²⁷ Overall, as summarized by Jeffrey and Kim,²⁸ a combination of stabilizing gauche interactions and the avoidance of destabilizing 1,5-syn interactions is sufficient to overcome the usual steric interactions, and any crystal packing forces, that favor the zig zag conformation of straight chain alkanes. Turning to the more relevant solution phase structures, Horton and Wander studied the conformations of a wide variety of open-chain D-aldopentose derivatives by ¹H NMR spectroscopy in CD₃OD at 100 MHz using a lanthanide shift reagent to increase the dispersion of chemical shifts.²⁹ They found that open-chain derivatives of D-arabinose and of D-lyxose predominantly adopted the zig zag conformation of the carbon backbone. Open-chain derivatives of D-ribose and D-xylose on the other hand were found to be either conformationally unstable or to adopt gauche conformations about either the C2–C3 or the C3–C4 bonds; pertinently none of the ribose and xylose derivatives studied retained the zig zag conformation of the carbon backbone equivalent to those seen in the corresponding pentitols. Subsequently, Hawkes and Lewis studied the conformations of the tetritols, pentitols, and hexitols themselves in D₂O solution by ¹H NMR spectroscopy at 400 MHz.³⁰ It was found that D-arabinitol predominantly adopts the extended zig zag conformation of its carbon backbone akin to that found in the crystal (Figure 5). Ribitol and xylitol on the other hand were found to exhibit twist conformers, alternating between out of plane rotation of the C2–C3 and C3–C4 bonds (which are mirror images because of the meso-configuration), again consistent with the conformations found in the X-ray crystal structures (Figure 5).³⁰ In the hexitols, D-galactitol and D-mannitol were found to adopt very predominantly the zig zag conformation of their carbon backbones, which lack any syn-1,3-diol motifs, whereas the other stereoisomers were all found to contain gauche conformations about one or more bonds so as to avoid the presence of destabilizing 1,5-syn-interactions. These studies were later extended by Lewis and Angyal to include the heptitols, where broadly similar conclusions were reached.^{31, 32} The 1-deoxyalditols were also found to follow the same configuration dependent pattern of conformations indicating that the terminal hydroxyl groups of the alditols themselves do not play a major role in the determination of conformation.³³ Subsequently, Leino and coworkers studied the solution-phase and single crystal conformations of a series of extended D-mannitol and D-glucitol derivatives by modern NMR and computational methods, and consistent with the earlier work found that crystallization and gel formation is facilitated by the presence of a zig zag conformation of the carbon backbone, which in turn is stabilized by the presence of gauche interactions between vicinal C-O bonds and destabilized by 1,5-syn interactions: mannitol derivatives crystallize more readily than glucitol ones.^34–36 Overall, it is clear that the factors which determine the conformation of polyols are the maximization of gauche effects between vicinal diols, the minimization of 1,5-syn-interactions, and the steric preference for the zig zag conformation of the carbon backbone (Figure 5), with the combination of the former two overriding the latter in conflicting cases.

Returning to the issue of prediction of side chain conformation in higher carbon pyranoses, Figure 6 displays the Fischer projections of the neuraminic and sialic acids 1–5, with the relative configurations of the three stereogenic centers C5, C6, and C7 indicated, namely the point of attachment of the side chain and a single stereogenic center either side of it. The pyranose forms of these sugars are depicted below the Fischer projections with the experimentally determined conformations of their side chains. The five carbon chains of the pentitols spanning the point of attachment of the side chain are highlighted in bold red.

Figure 6.

Fischer Projections and Solution Phase Side Chain Conformations of Neuraminic and Sialic Acids

In all of the five cases illustrated, the red five carbon chain adopts the conformation predicted by the above analysis of the corresponding pentitol, thereby setting the conformation of the exocyclic C6–C7 bond. Thus, NeuAc 1 has the D-lyxo configuration of C5–C7 and the C6–C7 bond takes up the gg conformation, corresponding to the zig-zag conformation of the carbon backbone of lyxitol. Both 5- and 7-epi-NeuAc derivatives 2 and 3 take up a gt conformation of the substituents about their exocyclic bonds corresponding to the sickle conformation of the carbon backbones with the D-xylo and L-ribo configurations, respectively. Pseudaminic acid 4 has the L-arabino configuration and KDO 5 has the D-arabino configuration, both of which enforce the tg conformation of the exocyclic bond and the zig zag conformation of the red carbon backbone.

Applying the same analysis to the 6-methyl D-galacto- and glucopyranoses (Figure 7), and consistent with an earlier observation by Hindsgaul and coworkers,³⁷ it is predicted that the D-glycero-D-gluco and L-glycero-D-galacto configurations should favor the gt conformation. The gg conformation should be preferred in the L-glycero-D-gluco configuration, and the tg conformation is likely adopted in the D-glycero-D-galacto configuration. Based on this analysis it was hypothesized that substitution of the 6-position of an aminoglycoside antibiotic could be used to tune its activity.

Figure 7.

D- and L-Glycero-D-gluco- and D- and L-Glycero-D-galacto Heptopyranoses and the Predicted Conformations of their Exocyclic Bonds

Aminoglycoside antibiotics (AGAs) bind to the aminoacyl site (A site) in the ribosomal RNA of bacteria where they inhibit protein synthesis.³⁸ A critical interaction between ring I of the 2-deoxystreptamine AGAs and the ribosome is the pseudobase-pair interaction with residue A1408 involving hydrogen bonds from both the side chain heteroatom and the ring oxygen of the AGA ring I to the adenine residue (Figure 8). Crystal structures of AGAs bound to the ribosome show that participation in this interaction enforces the gt conformation of the side chain³⁹ despite the approximately equal population of the gg and gt conformations in the unbound drug.⁴⁰ Preorganization of the exocyclic bonds into the gt conformation can therefore be expected to increase affinity for the ribosomal decoding A site, as was borne out with paromomycin 9 and its bicyclic analog 11. In contrast to paromomycin, neomycin 10 saw no improvement of activity upon cyclization to 12 (Figure 9). Application of the above analysis of exocyclic bond conformation in higher carbon sugars (Figure 7) suggests that the (R)-6’-methylparomomycin and neomycin derivatives should preferentially adopt the gt conformation, and so be preorganized for binding to the bacterial ribosome. The (S)-6’-methyl derivatives should have the opposite effect as they are predicted to enforce the gg conformation, increasing the entropic penalty for binding. Jorgensen and coworkers have discussed how the addition of a single methyl group to a ligand can influence its affinity for its protein target and concluded that “the greatest improvements in activity arise from coupling the conformational gain with the burial of the methyl group in a hydrophobic region of the protein”.⁴¹

Figure 8.

Pseudobase-pair Interaction Between the Paromomycin and Neomycin Ring I and A1408

Figure 9.

Paromomycin, Neomycin, and their Bicyclic Analogs

Chemical Synthesis

Synthesis began with known diol 13 (Scheme 1),⁴² of which selective oxidation of the 6’-hydroxy group with BAIB and TEMPO gave the carboxylic acid 14 in 99% yield,⁴³ whose coupling to N,O-dimethylhydroxylamine using DCC and DMAP gave Weinreb amide⁴⁴ 15 in 67% yield. After protection of the 4’-hydroxy group of 15 as a trimethylsilyl ether with hexamethyldisilazane in acetonitrile,⁴⁵ alkylation with the methyl Grignard reagent in THF gave ketone 16 in 39% yield. Reduction of 16 with sodium borohydride then resulted in a 1:1 mixture of diastereomers 17(R) and 17(S) in 75% yield. For comparison purposes the 6’,6’-dimethyl paromomycin was accessed by a further Grignard reaction on methyl ketone 16, affording the 6’,6’-dimethyl alcohol 18 in 78% yield. The 6’-methylneomycin derivatives were accessed through conversion of 16 to the oxime followed by reduction using sodium cyanoborohydride in acidic methanol, resulting in a mixture of hydroxylamines 19(R) and 19(S) in a 2:1 ratio favoring the (R)-configuration (Scheme 1). Compound 19(R) was isolated in 39% yield and compound 19(S) was isolated in 23% yield.

Scheme 1.

Synthesis of 6’-Methylparomomycin and Neomycin Intermediates

In an alternative approach to the 6-methyl neomycin derivatives, triflation of 17(R) with triflic anhydride and pyridine followed by displacement with lithium azide gave the inverted 6’-azido derivative 20 in 40% yield as a single diastereomer (Scheme 2). However, attempted application of the same method to the epimeric 17(S) alcohol afforded a considerably less stable triflate, which failed to give the desired product on exposure to sodium azide. These observations are consistent with the L-glycero-D-gluco configured 17(S)-alcohol and the derived triflate predominantly adopting the predicted gg conformation of the ring I exocyclic bond in which the leaving group is antiperiplanar to the C5’-H5’ bond and so primed for elimination. On the other hand, the D-glycero-D-gluco configuration configured (R)-isomer should have the gt conformation about the exocyclic bond to ring I, which is less prone to elimination and so better suited to substitution.

Scheme 2.

Alternate synthesis of a 6’-(S)-methylneomycin Derivative

An alternative route was explored for the 6’-ethylparomomycin derivatives starting from diol 13, whose 6’-hydroxy group was protected as the triisopropylsilyl ether using TIPSOTf and 2,6-lutidine to give 21 in 82% yield (Scheme 3). The 4’-hydroxy group of 21 was then converted to the 4-methoxybenzyl ether with 4-methoxybenzyl chloride and sodium hydride to give 22 in 89% yield. Removal of the silyl ether from 22 with TBAF gave the 6’-hydroxy compound 23 in 88% yield. Oxidation of 23 to the corresponding aldehyde under Swern conditions⁴⁶ was followed by treatment with the ethyl Grignard reagent, affording an inseparable 3:1 mixture of diastereomers, which was determined retroactively to favor the (S)-isomer. Acidic hydrolysis of the PMB ether from this mixture was achieved with trifluoroacetic acid and gave a mixture of 24(R) and 24(S) in 79% yield. Further purification using preparative HPLC gave 24(R) and 24(S) in 9% and 35% isolated yield, respectively. The corresponding 6’(R)- and 6’(S)-allyl paromomycin derivatives 25(R) and 25(S) were obtained as previously described.⁴⁷ In each of the 6’-methyl and ethyl paromomycin series, the configuration at the 6’-position was established by conversion to rigid 4’,6’-O-benzylidene derivatives and NMR analysis (see Experimental Section). Final global deprotection of all compounds, including reduction of alkenes, azides and hydroxylamines was achieved by hydrogenolysis (Scheme 4).

Scheme 3.

Synthesis of 6’-Ethylparomomycin Intermediates

Scheme 4. Final Deprotections^a.

a) Hydrogenolysis of 17(R)-, 17(S)-, and 18 was preceded by cleavage of the silyl ether from the 4’-position with Bu₄NF in THF.

NMR Analysis of Side Chain Conformation

NMR spectroscopic studies were conducted on compounds 26(R) and 26(S) to determine the conformation of the ring I side chain in aqueous solution at pD5. On the basis of the ³J_H5’,H6’ coupling constant alone (2.5 Hz) it is not possible to distinguish between the gg and gt conformers for 26(R) as the ideal coupling constants for the two conformers are both 2.5–3.0 Hz as determined from extensive studies of model compounds.⁴⁸ However, in the ROESY spectrum, the methyl group of 26(R) exhibits a strong correlation to H4’ and a much weaker one to H5’ (Figure 10) very strongly suggesting that the gt conformation of 26(R) predominates in solution.⁴⁸ On the other hand, 26(S) has a ³J_H5’,H6’ coupling constant of 1.6 Hz, which is consistent with that expected (~1.1 Hz) from model compounds for the gg conformer, and not with the expected values (~ 9.5 and ~4.5 Hz) for the gt and tg conformers, respectively.⁴⁸ The 6’-methyl group of 26(S) to displays an ROE correlation to H-5’ that is double that to H-4’, consistent with a predominant gg conformation mixed with a relatively minor amount of the gt and/or tg conformations (Figure 10).⁴⁸ This NMR analysis bears out the prediction that 26(R) with the D-glycero-D-gluco configuration of ring I will adopt the gt conformation of its side chain, and so be preorganized for formation of a pseudobase pair with A1408 in the ribosomal decoding A site, while 26(S) with the L-glycero-D-gluco configuration occupies mainly the predicted gg conformation. The 6’-ethyl and propyl derivatives are considered to adopt comparable conformations of the side chain as the comparably configured 6’-methyl derivatives on the basis of their homologous structures and the comparable magnitudes of the diagnostic ³J_H5’,H6’ coupling constants. For the 6’-methylneomycin derivatives, with a ³J_H5’,H6’ coupling constants of 3.0 Hz and a strong ROESY correlation between the methyl group and H4’ but not H5’, the 28(R) isomer predominantly populates the gt conformation like the corresponding alcohol. In the 28(S) isomer the strong ROE correlation from the methyl group to H-5’ and weaker one to H-4’ point strongly toward the gg conformation. However, the ³J_H5’,H6’ coupling constants of 2.9 Hz, which is somewhat larger than in the corresponding alcohol, suggest that this is mixed with a larger amount of the gt or tg conformer than is the case with the corresponding alcohol (Figure 10). Overall, the experimentally determined side chain conformations of this set of compounds are consistent with the predictions (Figure 7).

Figure 10.

Diagnostic Coupling Constants, Major ROE Interactions, and Predominant Side Chain Conformations of 26(R), 26(S), 28(R), and 28(S) in D₂O at pD5.

Antiribosomal and Antibacterial Activity

All compounds were screened for the ability to disrupt ribosomal protein synthesis by cell free translational assays with engineered M. smegmatis ribosomes as previously described (Table 1).^{49, 50} These assays employed with wild type M. smegmatis ribosomes (bacterial) and to assess toxicity with hybrid M. smegmatis ribosomes engineered to carry the complete decoding A site of the human mitochondrial (Mit13), mutant mitochondrial (1490G), and cytoplasmic (Cyt14) ribosomes. Inhibition of the human mitochondrial and especially mutant mitochondrial ribosomes is known to be a significant factor in aminoglycoside induced ototoxicity, where inhibition of the cytoplasmic ribosome is considered to cause broader systemic toxicity.⁵¹ Selectivity factors (Table 1) are calculated by dividing the IC₅₀ of given mutant ribosome by that of the bacterial ribosome and show the preferential inhibition of the bacterial over the hybrid ribosomes.

Table 1.

Cell Free Translation Assays with Wild Type Bacterial and Hybrid Ribosomes

Compound	Alkyl Substituent	IC50, μM (Bacterial/Hybrid Selectivity)
		Bacterial	Mit13	1490G	Cyt14
Paromomycin	-	0.04	142 (3703)	12 (323)	31 (801)
26(R)	methyl	0.02	185 (7693)	8.4 (350)	2.6 (109)
26(S)	methyl	0.06	188 (3128)	88 (1471)	70 (1171)
27	dimethyl	0.06	328 (5460)	87 (1452)	47 (782)
29(R)	ethyl	0.03	170 (5667)	14 (467)	10 (333)
29(S)	ethyl	0.19	841 (4446)	220 (1163)	482 (2548)
30(R)	propyl	0.06	162 (2706)	30 (505)	51 (858)
30(S)	propyl	0.24	499 (2079)	142 (592)	394 (1642)
Neomycin	-	0.04	4.3 (119)	0.4 (11)	36 (1011)
28(R)	methyl	0.01	7.9 (603)	0.7 (55)	64 (4908)
28(S)	methyl	0.01	8.9 (650)	0.8 (61)	26 (1905)

In each of the 6’-alkyl paromomycin derivatives, the (R) isomer displays between two- and four-fold greater activity against the bacterial ribosome than the corresponding (S) isomer, with the greatest activity residing in the 6’(R)-methyl derivative 26(R), and the least activity in the 6’(S)-propyl derivative 30(S). Antibacterioribosomal activity therefore correlates with the configuration and so conformation of the side chain, and inversely with the size of the appended alkyl group. Further, the 6’-methyl derivative 26(R) has antibacterioribosomal activity two-fold greater than that of the parent indicating that preorganization of the side chain into the gt conformation is beneficial provided that the added steric bulk is minimized. The activity of the gem-dimethyl compound 27 is less than that of the parent and comparable to that of the 26(S)-methyl derivative. In the neomycin series both diastereomers of 28 showed a comparable increase in activity over the parent.

With regard to inhibition of the hybrid ribosomes, the R-isomers in the 6’-alkyl paromomycin series are consistently more active than the S-isomers. The R-isomers show low levels of activity toward the hybrid mitochondrial (Mit 13) ribosome with little influence of chain length, reflecting the already low activity of the parent. For the mutant mitochondrial (1490G) ribosomes, with their more bacteria-like decoding A sites, strong activity is again observed in the R-series, with a reverse dependence on alkyl chain length. The 6’(R)-methyl paromomycin derivative 26(R) and even its immediate homolog 29(R) show enhanced activity toward inhibition of the hybrid cytoplasmic ribosome (Cyt14) paralleling the known high read-through activity of geneticin 31.^52–54

In the 6’-methyl neomycin series the two diastereomers have the same IC₅₀ for the bacterial ribosome, which is four-fold greater than that of the parent and presumably related to the increased basicity and more complete protonation conveyed by methylation. We suggest that the lack of discrimination between 28(R) and 28(S) isomers is related to the 10:90:0 gg:gt:tg population of side chain conformers in methyl 2,6-diamino-2,6-dideoxy-α-D-glucopyranoside,⁴⁰ and by extension in neomycin and its derivatives, which obscures any smaller advantage conveyed on 25(R) by preorganization of the side chain conformation. Any small advantage due to side chain preorganization is further hidden by the increased strength of the RNH₃⁺-A1408 hydrogen bond in the neomycin series over the ROH-A1408 H bond in the paromomycin series (estimated increase >3 kcal.mol⁻¹)⁵⁵ (Figure 8). The reduced activity of neomycin and its alkyl congeners 28(R) and 28(S) toward the humanized cytoplasmic ribosome Cyt14 is expected and is a function of the A1408G replacement in the cytoplasmic ribosome,⁵⁶ resulting in a much reduced pseudobase pair interaction compared to the bacterial and humanized mitochondrial ribosomes (Figure 8). The inverted activity ratio of 28(R) and 28(S) toward Cyt14 compared to the corresponding alcohols 26(R) and 26(S) is presumably also a function of the A1408G replacement and the resultant different steric environment.

Antibacterial Activity.

All compounds were screened for activity against wild-type Escherichia coli and engineered strains carrying aminoglycoside modifying enzymes^57–60 acting on the 4’- and 6’-positions to check for the influence of the 6’-alkylation on activity and its ability to suppress modification by AME’s in its immediate vicinity (Table 2). Consistent with the antiribosomal activity pattern, the 6’(R)-alkyl paromomycin derivatives are all more active than their (S)-isomers and activity is inversely proportional to the length of the alkyl chain. The most active 6’(R)-methyl derivative 26(R) has comparable activity to the parent. The gem-dimethyl derivative 27 has comparable activity to the 6’(S)-methylparomomycin derivative 26(S). Again consistent with the antiribosomal data little or no difference was found between the two diastereomers of 6’-methyl neomycin. 6’-Alklyation does not lead to increased activity in the presence of the ANT(4’)-type aminoglycoside modifying enzyme as is apparent from the lack of activity against E. coli carrying this resistance determinant. On the other hand, both diastereomers of 6’-methylneomycin do display significantly increased activity over the parent in the presence of the 6’-aminoacyltransferase (AAC6’) type of AME, clearly pointing to the ability of alkylation at the 6-position to suppress 6’-acetamide formation. All compounds were also screened for activity against wild type isolates of the Gram-negative ESKAPE pathogens Acinetobacter baumannii, Klebsiella pneumonia, and Enterobacter cloacae, as well as against the Gram-positive methicillin-resistant Staphylococcus aureus (Table 3), with the same overall pattern of activity.

Table 2.

Antibacterial Activity Against Wild Type and Resistant E. coli Strains

Compd	Alkyl Substituent	E. coli MIC (mg/L) (pathogen, feature, strain)
		ATCC Ref WT	WT	WT	AAC(6’)-Ib	ANT(4’) ANT(4”)
		AG212	AG001	AG055	AG009	AG036
Paromomycin	-	2–4	2–4	2	2–4	64
26(R)	methyl	2	4–8	4	2–4	32–64
26(S)	methyl	4	8–16	8–16	4	64
27	dimethyl	4	8	8	4	16–32
29(R)	ethyl	-	-	-	-	-
29(S)	ethyl	8	8–16	8	8	-
30(R)	propyl	2	2	2	4	32–64
30(S)	propyl	8	32	32	16	64
Neomycin	-	0.5–1	1	1	4	4–8
28(R)	methyl	1	2	2	2	16
28(S)	methyl	1	2	2	0.5	16

Table 3.

Antibacterial Activity Against Selected ESKAPE Pathogens

Compd	Alkyl Subs	MIC (mg/L) (pathogen, feature, strain)
		A. baum	K. pneu	E. Cloac	MRSA
		WT	WT	WT	WT
		AG225	AG215	AG290	AG044
Paromomycin	-	2	1	2	4–8
26(R)	methyl	2	1	1	2–4
26(S)	methyl	4	2	2–4	4
27	dimethyl	4	1–2	2	4–8
29(R)	ethyl	2	0.5–1	2	-
29(S)	ethyl	4–8	2	2–4	-
30(R)	propyl	2	1	1	2
30(S)	propyl	8–16	4	4–8	16
Neomycin	-	1–2	0.25–0.5	1	0.5–1
28(R)	methyl	2	1	1	2
28(S)	methyl	1	0.5	0.5	1

Conclusion.

The conformation about the exocyclic bond in higher carbon sugars can be predicted with confidence based on the configuration of its point of attachment to the pyranose ring relative to the two flanking adjacent centers by simple inspection of Fischer projection formula. This predictive model derives from consideration of the solid and solution state conformations of the acyclic pentitols arabinitol (lyxitol), ribitol, and xylitol, which in turn are compensatory functions of the maximization of the gauche effect of 1,2-diols, the minimization of 1,5-syn interactions in 1,3-diols and of steric effects in the carbon backbone. Additional more remote stereogenic centers in the side chain, as in the C8-epimer of N-acetylneuraminic acid, have less influence on the conformation of the exocyclic bond itself. While this analysis primarily targets higher carbon sugars, it should also be directly applicable to any 2-(1-hydroxyalkyl)-3-hydroxytetrahydropyran or related tetrahydrofuran nucleus.

The model was applied to the prediction of side chain conformation of 6’-alkyl substituted 4,5-disubstituted 2-deoxystreptamine type aminoglycoside derivatives, enabling the successful design, synthesis and evaluation of aminoglycosides with ring I side chains preorganized for binding to the bacterial ribosome.

Experimental Section.

General Experimental

All reagents were purchased from commercial sources and used without further purification unless otherwise specified. Thin-layer chromatography was performed on glass backed silica gel plates with UV 254 dye. Chromatographic purifications were carried out over silica gel 60 230–400 mesh unless otherwise specified. High resolution mass spectra were collected on a Waters LCT Premier XE ESI-TOF mass spectrometer. Optical rotations were measured using an automatic polarimeter in a 1 dm cell. NMR spectra were collected with 400, 500, 600, or 900 MHz spectrometers as indicated. NMR spectra were assigned with the aid of advanced 1D and 2D techniques including COSY, HSQC, HMBC, and ROESY.

1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-1,3,2’,2”’,6”’-pentadeaminoparomomycin-6’-carboxylic acid (14).

TEMPO (87.5 mg, 0.56 mmol) and BAIB (1.98 g, 6.16 mmol) were added to a stirred solution of 13⁴² (3.60 g, 2.80 mmol) in 33 mL of 1:1 MeCN/H₂O. After 3.5 h the MeCN was removed under vacuum and the aqueous solution was extracted with ethyl acetate. The organic layer was washed with 20% aqueous Na₂S₂O₃ solution, 1 N HCl, and brine followed by drying over sodium sulfate and silica gel column chromatography in 70% EtOAc in hexanes with 1% AcOH to give 3.60 g (2.77 mmol, 99%) of the orange foam 14. [α]_D²³ = 75.0 (c = 1.0, CHCl₃), ¹H NMR (600 MHz, CD₃OD/CDCl₃ 4:1) δ 7.40 – 7.13 (m, 30H), 6.12 (d, J = 3.6 Hz, 1H, H-1’), 5.56 (d, J = 4.8 Hz, 1H, H-1”), 4.90 (d, J = 11.0 Hz, 1H, PhCH₂O), 4.87 (d, J = 10.8 Hz, 1H, PhCH₂O), 4.79 (d, J = 1.9 Hz, 1H, H-1”’), 4.71 – 4.67 (m, 2H, PhCH₂O), 4.57 (d, J = 11.7 Hz, 1H, PhCH₂O), 4.53 (d, J = 9.4 Hz, 1H, H-5’), 4.51 – 4.40 (m, 6H, PhCH₂O), 4.35 (d, J = 12.2 Hz, 1H, PhCH₂O), 4.33 (d, J = 12.2 Hz, 1H, PhCH₂O), 4.19 – 4.14 (m, 2H, H-3”, H-5”), 3.92 (t, J = 4.6 Hz, 1H, H-2”), 3.85 (dd, J = 9.9, 8.4 Hz, 1H, H-3’), 3.80 (t, J = 2.9 Hz, 1H, H-3”’), 3.79 – 3.71 (m, 4H, H-5, H-4’, H-5”, H-5”’), 3.68 (t, J = 9.4 Hz, 1H, H-4), 3.55 – 3.50 (m, 2H, H-5”, H-6”’), 3.50 – 3.42 (m, 2H, H-1, H-3), 3.31 (t, J = 2.3 Hz, 1H, H-2”’), 3.29 – 3.23 (m, 2H, H-6, H-4”’), 3.11 (dd, J = 9.8, 3.6 Hz, 1H, H-2’), 3.03 (dd, J = 12.9, 4.1 Hz, 1H, H-6”’), 2.20 (dt, J = 12.8, 4.6 Hz, 1H, H-2eq), 1.39 (q, J = 12.6 Hz, 1H, H-2ax). ¹³C{¹H} NMR (151 MHz, CD₃OD/CDCl₃ 4:1) δ 171.9 (C-6’), 138.2, 138.1, 138.0, 137.6, 137.4, 137.3, 128.3, 128.2, 128.13, 128.10, 128.0, 127.94, 127.91, 127.9, 127.84, 127.82, 127.7, 127.58, 127.55, 127.4, 127.3, 127.2 (Ar), 106.7 (C-1”), 98.5 (C-1”’), 96.2 (C-1’), 83.9 (C-6), 82.0 (C-3”), 81.8 (C-5), 81.5 (C-2”), 78.7 (C-3’), 75.8 (C-4), 75.7 (C-4”), 74.8 (PhCH₂O), 74.6 (C-5”), 74.2 (PhCH₂O), 73.2 (C-3”), 73.1 (PhCH₂O), 72.9 (PhCH₂O), 72.3 (PhCH₂O), 71.9 (C-4”’), 71.8 (C-4’), 71.7 (PhCH₂O), 70.1 (C-5”), 62.1 (C-2’), 60.4 (C-1), 59.7 (C-3), 57.2 (C-2”’), 50.9 (C-6”’), 31.8 (C-2). HRMS (ESI) m/z: Calcd for C₆₉H₆₉N₁₅O₁₅Na [M+Na]⁺ 1322.4995; Found 1322.5044.

N-Methoxy-N-methyl-1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-1,3,2’,2”’,6”’-pentadeaminoparomomycin-6’-carboxamide (15).

Compound 14 (4.64 g, 3.57 mmol), DMAP (0.091 g, 0.72 mmol), and N,O-dimethylhydroxylamine hydrochloride (0.524 g, 5.37 mmol) were stirred under argon in 30 mL DCM followed by addition of DCC (1.105 g, 5.36 mmol) in 5.7 mL of DCM. After two hours DCC (0.368 g, 1.79 mmol) in 1 mL of DCM was added to the reaction mixture. After another hour, no starting material was detected by TLC and the reaction mixture was concentrated under vacuum. The crude residue was dissolved in EtOAc and washed with 1N HCl and brine, dried with Na₂SO₄, filtered, and concentrated. The crude residue was then subjected to flash column chromatography over silica gel with 40% EtOAc in hexanes. Following chromatography, the product still contained some dicyclohexyl urea, which was removed by dissolving the residue in a minimal amount of toluene and filtering while cold. Concentration of the filtrate gave 3.22 g (2.40 mmol 67%) of 15 as a white foam. [α]_D²³ = 89.10 (c = 1.0, CHCl₃) ¹H NMR (600 MHz, CDCl₃) δ 7.45 – 7.10 (m, 30H, Ar-H), 6.28 (d, J = 3.5 Hz, 1H, H-1’), 5.66 (d, J = 6.2 Hz, 1H, H-1”), 4.97 (d, J = 10.5 Hz, 1H, PhCH₂O), 4.88 – 4.82 (m, 3H, H-1”’, PhCH₂O), 4.79 (br d, J = 9.1 Hz, 1H, H-5’), 4.67 (d, J = 10.5 Hz, 1H, PhCH₂O), 4.61 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.53 (d, J = 11.7 Hz, 1H, PhCH₂O), 4.50 (d, J = 11.6 Hz, 1H, PhCH₂O), 4.44 – 4.38 (m, 3H, PhCH₂O), 4.30 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.27 – 4.23 (m, 2H, H-4”’, PhCH₂O), 4.22 (dd, J = 5.0, 2.0 Hz, 1H, H-3”), 4.04 (t, J = 9.4 Hz, 1H, H-3’), 4.01 – 3.95 (m, 2H, H-5, H-4’), 3.89 (dd, J = 6.2, 5.0 Hz, 1H, H-2”), 3.78 – 3.73 (m, 4H, H-4, H-5”, H-3”’, H-5”’), 3.72 (s, 3H, OCH₃), 3.60 (dd, J = 12.9, 8.4 Hz, 1H, H-6”’), 3.54 (dd, J = 10.4, 3.0 Hz, 1H, H-5”), 3.52 – 3.42 (m, 2H, H-1, H-3), 3.32 (t, J = 2.5 Hz, 1H, H-2”’), 3.28 (s, 3H, NCH₃), 3.24 (t, J = 9.3 Hz, 1H, H-6), 3.11 (d, J = 2.4 Hz, 1H, H-4”’), 3.03 (dd, J = 10.1, 3.5 Hz, 1H, H-2’), 2.89 (dd, J = 13.0, 4.1 Hz, 1H, H-6”’), 2.24 (dt, J = 13.1, 4.6 Hz, 1H, H-2eq), 1.33 (q, J = 12.8 Hz, 1H, H-2ax). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 169.2 (C-6’), 138.2, 138.0, 137.8, 137.5, 137.0, 136.9, 128.7, 128.49, 128.47, 128.43, 128.40, 128.36, 128.3, 128.24, 128.17, 128.1, 127.8, 127.62, 127.61, 127.5 (Ar), 105.8 (C-1”), 98.8 (C-1”’), 96.3 (C-6), 84.5 (C-2”), 82.5 (C-4”), 82.2 (C-5), 81.8 (C-3’), 78.5 (C-3”), 75.6 (PhCH₂O), 75.1 (PhCH₂O), 74.6 (C-4), 74.3 (C-5”’), 73.4 (PhCH₂O), 73.3 (PhCH₂O), 72.9 (C-3”’), 72.4 (PhCH₂O), 71.9 (C-4’), 71.7 (PhCH₂O), 71.4 (C-4”’), 69.9 (C-5”), 68.5 (C-5’), 62.2 (C-2’), 62.1 (OCH₃), 60.4 (C-1), 60.3 (C-3), 57.2 (C-2”’), 51.0 (C-6”’), 32.8 (C-2), 32.4 (NCH₃). HRMS (ESI) m/z: Calcd for C₆₇H₇₄N₁₆O₁₅Na [M+Na]⁺ 1365.5417; Found 1365.5453.

1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-6’-methyl-6’-oxo-4’-O-trimethylsilyl-1,3,2’,2”’,6”’-pentadeaminoparomomycin (16).

Hexamethyldisilazane (0.55 mL, 2.6 mmol) was added to a stirred solution of compound 15 (1.17 g, 0.87 mmol) in 8.7 mL MeCN under argon. After 3 h no starting material was detected by TLC. The reaction mixture was concentrated under vacuum and the white foam was used without further purification. HRMS (ESI) m/z: Calcd for C₇₀H₈₂N₁₆O₁₅SiNa [M+Na]⁺ 1437.5813; Found 1437.5868. 0.6 mL of 3M MeMgCl in THF were added to a stirred solution of the above silylated amide in 8.8 mL of THF at −78 °C . The reaction mixture was stirred for 5 min then transferred to an ice bath and stirred for another 10 min before quenching with 1 mL of aqueous NH₄Cl. The THF was then removed under vacuum, diluted with Et₂O, and washed with aqueous NH₄Cl and brine. The organic layer was dried with Na₂SO₄, filtered, and concentrated. The crude residue was purified over silica gel with gradient elution of 0% ethyl acetate in hexanes to 80% to give 0.4673 g (0.341 mmol, 39%) of ketone 16 as a white foam. [α]_D²³ = 104.2 (c = 1.0, CHCl₃) ¹H NMR (600 MHz, CDCl₃) δ 7.39 – 7.15 (m, 30H, Ar-H), 6.09 (d, J = 3.7 Hz, 1H, H-1’), 5.61 (d, J = 5.4 Hz, 1H, H-1”), 4.93 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.88 (d, J = 1.9 Hz, 1H, H-1”’), 4.78 (s, 2H, PhCH₂O), 4.72 (d, J = 10.7 Hz, 1H, PhCH₂O), 4.62 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.54 (d, J = 11.7 Hz, 1H, PhCH₂O), 4.51 (d, J = 11.8 Hz, 1H, PhCH₂O), 4.47 – 4.41 (m, 3H, PhCH₂O), 4.40 (d, J = 4.5 Hz, 1H, H-5’), 4.34 – 4.23 (m, 4H, H-3”, H-4”, PhCH₂O), 3.94 – 3.88 (m, 2H, H-5, H-2”), 3.82 – 3.76 (m, 3H, H-3’, H-5”, H-5”’), 3.75 (t, J = 2.9 Hz, 1H, H-3”’), 3.63 (dd, J = 13.0, 8.5 Hz, 1H, H-6”’), 3.61 – 3.58 (m, 2H, H-4, H-4’), 3.56 (dd, J = 10.5, 3.4 Hz, 1H, H-5”), 3.48 – 3.39 (m, 2H, H-1, H-3), 3.35 (t, J = 2.7 Hz, 1H, H-2”’), 3.27 (t, J = 9.3 Hz, 1H, H-6), 3.13 (d, J = 2.6 Hz, 1H, H-4”’), 2.94 – 2.88 (m, 2H, H-2’, H-6”’), 2.27 (dt, J = 13.2, 4.6 Hz, 1H, H-2eq), 2.21 (s, 3H, CH₃), 1.42 (q, J = 12.7 Hz, 1H, H-2ax), 0.04 (s, 9H, SiCH₃). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 204.6 (C-6’), 138.3, 138.1, 137.8, 137.6, 137.0, 136.9, 128.7, 128.5, 128.4, 128.34, 128.32, 128.28, 128.23, 128.18, 127.82, 127.77, 127.74, 127.71, 127.54, 127.53, 127.49, 127.47 (Ar), 106.3 (C-1”), 98.5 (C-1”’), 96.3 (C-1’), 84.0 (C-6), 82.2 (C-2”), 82.0 (C-4”), 81.9 (C-5), 80.0 (C-3’), 76.1 (C-5’), 75.5 (C-4), 75.4 (C-3”), 75.2 (PhCH₂O), 75.0 (PhCH₂O), 74.3 (C-5”’), 73.3 (PhCH₂O), 73.1 (PhCH₂O), 72.9 (C-3”’), 72.5 (C-4’), 72.4 (PhCH₂O), 71.7 (PhCH₂O), 71.5 (C-4”’), 70.2 (C-5”), 62.8 (C-2’), 60.3 (C-1), 59.9 (C-3), 57.3 (C-2”’), 51.1 (C-6”’), 32.4 (C-2), 28.6 (CH₃), 0.5 (SiCH₃). HRMS (ESI) m/z: Calcd for C₆₉H₇₉N₁₅O₁₄SiNa [M+Na]⁺ 1392.5598; Found 1392.5637.

1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-6’-C-methyl-4’-O-trimethylsilyl-1,3,2’,2”’,6”’-pentadeaminoparomomycin (17(R) and 17(S)).

NaBH₄ (0.018 g, .47 mmol) was added to a stirred solution of compound 16 (0.323 g, 0.236 mmol) in 2.4 mL of 1:1 THF/MeOH. The reaction mixture was stirred for 20 mins then concentrated and the crude residue was dissolved in EtOAc, washed with water and brine, dried over Na₂SO₄, and concentrated to afford a 1:1 mixture of isomers of 17. Silica gel chromatography eluting with 16% EtOAc in hexanes followed by 18% then 20% afforded the compounds 17(S) (119 mg, 0.086 mmol, 37%) and 17(R) (123 mg, 0.090 mmol, 38%) both as white foams. 17(R) [α]_D²³ = 97.0 (c = 1.0, CHCl₃), ¹H NMR (600 MHz, CDCl₃) δ 7.39 – 7.15 (m, 30H, Ar-H), 6.09 (d, J = 3.8 Hz, 1H, H-1’), 5.63 (d, J = 5.4 Hz, 1H, H-1”), 4.95 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.91 (d, J = 1.9 Hz, 1H, H-1”’), 4.82 (d, J = 11.2 Hz, 1H, PhCH₂O), 4.75 – 4.70 (m, 2H, PhCH₂O), 4.62 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.56 (d, J = 11.8 Hz, 1H, PhCH₂O), 4.52 – 4.39 (m, 4H, PhCH₂O), 4.33 – 4.30 (m, 2H, H-3”, PhCH₂O), 4.29 (q, J = 2.8 Hz, 1H, H-4”), 4.25 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.02 – 3.97 (m, 1H, H-6’), 3.96 – 3.90 (m, 3H, H-5, H-5’, H-2”), 3.83 – 3.75 (m, 4H, H-3’, H-5”, H-3”’, H-5”’), 3.70 – 3.63 (m, 2H, H-4, H-6”’), 3.57 (dd, J = 10.5, 3.4 Hz, 1H, H-5”), 3.50 – 3.41 (m, 2H, H-1, H-3), 3.37 (t, J = 2.6 Hz, 1H, H-2”’), 3.29 (t, J = 9.3 Hz, 1H, H-6), 3.21 (dd, J = 9.9, 8.5 Hz, 1H, H-4’), 3.13 (t, J = 2.6 Hz, 1H, H-4”’), 2.88 (dd, J = 13.0, 3.8 Hz, 1H, H-6”’), 2.82 (dd, J = 10.2, 3.8 Hz, 1H, H-2’), 2.24 (dt, J = 13.2, 4.6 Hz, 1H, H-2eq), 1.42 (q, J = 12.7 Hz, 1H, H-2ax), 1.14 (d, J = 6.5 Hz, 3H, H-7’), 0.07 (s, 9H, SiCH₃). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 138.4, 138.2, 137.9, 137.6, 137.0, 136.9, 128.7, 128.5, 128.4, 128.35, 128.33, 128.28, 128.2, 127.82, 127.79, 127.75, 127.71, 127.5, 127.45, 127.38, 127.37, 127.2 (Ar), 106.3 (C-1”), 98.5 (C-1”’), 95.7 (C-1’), 84.1 (C-6), 82.2 (C-2”), 81.99 (C-5), 81.96 (C-4”), 80.3 (C-3’), 75.4 (C-3”), 75.02 (PhCH₂O), 74.98 (C-4), 74.8 (PhCH₂O), 74.4 (C-5”’), 74.3 (C-5’), 73.23 (C-4’), 73.21 (PhCH₂O), 73.1 (PhCH₂O), 72.9 (C-3”’), 72.4 (PhCH₂O), 71.7 (PhCH₂O), 71.5 (C-4”’), 70.4 (C-5”), 67.0 (C-6’), 63.3 (C-2’), 60.4 (C-1), 60.1 (C-3), 57.3 (C-2”’), 51.2 (C-6”’), 32.5 (C-2), 16.4 (C-7’), 0.7 (SiCH₃). HRMS (ESI) m/z: Calcd for C₆₉H₈₁N₁₅O₁₄SiNa [M+Na]⁺ 1394.5754; Found 1394.5784. 17(S) [α]_D²³ = 97.20 (c = 1.0, DCM), ¹H NMR (600 MHz, CDCl₃) δ 7.39 – 7.16 (m, 30H, Ar-H), 6.11 (d, J = 3.8 Hz, 1H, H-1’), 5.68 (d, J = 5.7 Hz, 1H, H-1”), 4.99 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.95 (d, J = 1.9 Hz, 1H, H-1”’), 4.78 (s, 2H, PhCH₂O), 4.73 (d, J = 10.5 Hz, 1H, PhCH₂O), 4.63 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.58 (d, J = 11.9 Hz, 1H, PhCH₂O), 4.52 – 4.44 (m, 3H, PhCH₂O), 4.42 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.36 (dd, J = 4.8, 2.5 Hz, 1H, H-3”), 4.32 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.30 (q, J = 2.5 Hz, 1H, H-4”), 4.25 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.00 (dd, J = 5.7, 4.8 Hz, 1H, H-2”), 3.94 (t, J = 8.9 Hz, 1H, H-5), 3.93 – 3.88 (m, 1H, H-6’), 3.84 – 3.80 (m, 2H, H-5”, H-5”’), 3.79 – 3.75 (m, 2H, H-3’, H-3”’), 3.68 (dd, J = 13.0, 8.7 Hz, 1H, H-6”’), 3.61 (d, J = 9.7 Hz, 1H, H-5’), 3.58 (dd, J = 10.4, 2.9 Hz, 1H, H-5”), 3.54 (dd, J = 9.8, 8.7 Hz, 1H, H-4), 3.47 – 3.41 (m, 3H, H-1, H-3, H-4’), 3.39 – 3.36 (m, 1H, H-2”’), 3.28 (t, J = 9.3 Hz, 1H, H-6), 3.14 – 3.12 (m, 1H, H-4”’), 2.88 (dd, J = 13.1, 3.7 Hz, 1H, H-6”’), 2.76 (dd, J = 10.4, 3.8 Hz, 1H, H-2’), 2.24 (dt, J = 13.1, 4.5 Hz, 1H, H-2eq), 1.35 (q, J = 12.5 Hz, 1H, H-2ax), 1.27 (d, J = 6.5 Hz, 3H, CH₃), 0.10 (s, 9H, SiCH₃). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 138.4, 138.3, 137.9, 137.6, 137.0, 136.9, 128.7, 128.5, 128.41, 128.36, 128.32, 128.27, 128.19, 127.81, 127.79, 127.65, 127.58, 127.53, 127.51, 127.4, 127.1 (Ar), 106.2 (C-1”), 98.6 (C-1”’), 95.7 (C-1’), 84.4 (C-6), 82.4 (C-2”), 82.1 (C-4”), 82.0 (C-5), 80.1 (C-3’), 75.4 (C-3”), 75.1 (PhCH₂O), 75.0 (PhCH₂O), 74.8 (C-4), 74.6 (C-5’), 74.5 (C-5”’), 73.2 (PhCH₂O), 73.0 (PhCH₂O), 72.9 (C-3”’), 72.4 (PhCH₂O), 71.7 (PhCH₂O), 71.5 (C-4”’), 71.3 (C-4’), 70.3 (C-5”), 64.3 (C-6’), 63.1 (C-2’), 60.4 (C-1), 60.1 (C-3), 57.3 (C-2”’), 51.2 (C-6”’), 32.7 (C-2), 20.7 (C-7’), 0.6 (SiCH₃). HRMS (ESI) m/z: Calcd for C₆₉H₈₁N₁₅O₁₄SiNa [M+Na]⁺ 1394.5754; Found 1394.5760.

6’-(R)-C-Methylparomomycin pentaacetate salt (26(R)).

1M TBAF in THF (0.051 mL) was added dropwise to a stirred solution of compound 17(R) (26.7 mg, 0.017 mmol) in 1.7 mL of THF under argon. When the starting material was no longer visible by TLC, the reaction mixture was diluted with Et₂O and washed with of NaHCO₃ solution and brine. The organic layer was then dried with Na₂SO₄, filtered, and concentrated to give the intermediate alcohol which was used without further purification. The previous alcohol was stirred in 0.4 mL of 1:1 dioxane/10% AcOH in water with 58.0 mg of Pd/C under 50 psi of H₂ for 18 h. Once the reaction was determined to be complete by LCMS the reaction mixture was diluted with water and filtered through Celite. The resulting crude product was purified over a CM Sephadex C-25 column. The column was washed with 100 mL of DI water and eluted with NH₄OH in water starting at 0.1% and increasing stepwise by 0.1% every 20 mL to 0.8%. Lyophilization of the pure fractions with AcOH gave 2.6 mg (0.003 mmol, 18%) of the pentaacetate salt 26(R) as a white solid. [α]_D²³ = 41.3 (c = 0.6, H₂O), ¹H NMR (600 MHz, D₂O) δ 5.55 (d, J = 3.9 Hz, 1H, H-1’), 5.31 (d, J = 2.8 Hz, 1H, H-1”), 5.19 (d, J = 1.8 Hz, 1H, H-1”’), 4.44 (dd, J = 6.5, 5.0 Hz, 1H, H-3”), 4.28 (dd, J = 5.0, 2.8 Hz, 1H, H-2”), 4.25 – 4.21 (m, 1H, H-5”’), 4.16 – 4.11 (m, 3H, H-6’, H-4”, H-3”’), 3.86 – 3.79 (m, 2H, H-5’ [1DTOCSY 3.82 (dd, J = 10.1, 2.5 Hz)], H-5”), 3.77 – 3.72 (m, 3H, H-5, H-3’, H-4”’), 3.70 (dd, J = 12.4, 4.9 Hz, 1H, H-5”), 3.61 (t, J = 9.4 Hz, 1H, H-4), 3.52 (dd, J = 10.4, 9.2 Hz, 1H, H-6), 3.48 – 3.43 (m, 1H, H-2”’), 3.38 – 3.32 (m, 2H, H-4’, H-6”’), 3.29 (dd, J = 13.6, 3.9 Hz, 1H, H-6”’), 3.20 (dd, J = 10.7, 3.9 Hz, 1H, H-2’), 3.17 – 3.06 (m, 2H, H-1, H-3), 2.20 (dt, J = 12.8, 4.3 Hz, 1H, H-2eq), 1.84 (s, 15H, AcOH), 1.51 (q, J = 12.6 Hz, 1H, H-2ax), 1.15 (d, J = 6.6 Hz, 3H, H-7’). ¹³C{¹H} NMR (151 MHz, D₂O) δ 181.4 (AcOH), 109.7 (C-1”), 96.9 (C-1’), 95.9 (C-1”’), 84.5 (C-6), 81.3 (C-4”), 81.0 (C-4), 75.3 (C-3”), 75.1 (C-5’), 73.5 (C-5), 73.4 (C-2”), 70.37 (C-4’), 70.35 (C-3’), 70.26 (C-5”’), 68.0 (C-3”’), 67.5 (C-4”’), 65.7 (C-6’), 60.3 (C-5”), 54.3 (C-2’), 51.0 (C-2”’), 50.2 (C-1), 49.3 (C-3), 40.4 (C-6”’), 31.1 (C-2), 23.3 (AcOH), 14.8 (C-7’). HRMS (ESI) m/z: Calcd for C₂₄H₄₇N₅O₁₄ [M+H]⁺ 630.3198; Found 630.3212.

6’-(S)-C-Methylparomomycin pentaacetate salt (26(S)).

1M TBAF in THF (0.075 mL) was added dropwise to a stirred solution of compound 17(S) (32.8 mg, 0.024 mmol) in 2.3 mL of THF under argon. When the starting material was no longer visible by TLC, the reaction mixture was diluted with Et₂O and washed with of aqueous NaHCO₃ and brine. The organic layer was then dried with Na₂SO₄, filtered, and concentrated to give the intermediate alcohol which was used without further purification. The crude alcohol was stirred in 0.4 mL of 1:1 dioxane/10% AcOH in water with 58.0 mg of Pd/C under 50 psi of H₂ for 18 h. Once the reaction was determined to be complete by LCMS the reaction mixture was diluted with water and filtered through Celite. The resulting crude product was purified over a CM Sephadex C-25 column. The column was washed with 100 mL of DI water and eluted with NH₄OH in water starting at 0.1% and increasing stepwise by 0.1% every 20 mL to 0.8%. Lyophilization of the pure fractions with AcOH gave 5.2 mg (0.006 mmol, 25%) of the pentaacetate salt 26(S) as a white solid. [α]_D²³ = 50.9 (c = 0.6, H₂O), ¹H NMR (600 MHz, D₂O) δ 5.61 (d, J = 3.8 Hz, 1H, H-1’), 5.31 (d, J = 2.5 Hz, 1H, H-1”), 5.20 (d, J = 1.8 Hz, 1H, H-1”’), 4.46 (dd, J = 6.7, 4.9 Hz, 1H, H-3”), 4.29 (dd, J = 5.0, 2.5 Hz, 1H, H-2”), 4.26 – 4.21 (m, 1H, H-5”’), 4.17 – 4.10 (m, 3H, H-6’ [1DTOCSY 4.13 (qd, J = 6.6, 1.6 Hz)], H-4”, H-3”’), 3.85 (dd, J = 12.4, 3.1 Hz, 1H, H-5”), 3.79 (dd, J = 10.8, 8.6 Hz, 1H, H-3’), 3.76 – 3.68 (m, 3H, H-5, H-5”, H-4”’), 3.59 – 3.50 (m, 4H, H-4, H-6, H-4’, H-5’), 3.49 – 3.45 (m, 1H, H-2”’), 3.35 (dd, J = 13.7, 6.9 Hz, 1H, H-6”’), 3.29 (dd, J = 13.6, 3.9 Hz, 1H, H-6”’), 3.21 (dd, J = 10.8, 3.8 Hz, 1H, H-2’), 3.16 (ddd, J = 12.4, 10.4, 4.2 Hz, 1H, H-1), 2.96 (ddd, J = 12.1, 9.6, 4.3 Hz, 1H, H-3), 2.15 (dt, J = 12.9, 4.3 Hz, 1H, H-2eq), 1.84 (s, 15H, AcOH), 1.47 (q, J = 12.6 Hz, 1H, H-2ax), 1.21 (d, J = 6.6 Hz, 3H, H-7’). ¹³C{¹H} NMR (151 MHz, D₂O) δ 181.4 (AcOH), 109.8 (C-1”), 96.4 (C-1’), 95.8 (C-1”’), 84.8 (C-5), 81.8 (C-4), 81.2 (C-4”), 75.4 (C-5’), 75.2 (C-3”), 73.4 (C-2”), 73.2 (C-6), 70.3 (C-5”’), 69.7 (C-3’), 69.5 (C-4’), 68.0 (C-3”’), 67.4 (C-4”’), 64.1 (C-6’), 60.1 (C-5”), 54.3 (C-2’), 51.0 (C-2”’), 50.4 (C-1), 49.4 (C-3), 40.4 (C-6”’), 31.5 (C-2), 23.3 (AcOH), 18.9 (C-7’). HRMS (ESI) m/z: Calcd for C₂₄H₄₇N₅O₁₄ [M+H]⁺ 630.3198; Found 630.3209.

1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-6’,6’-di-C-methyl-4’-O-trimethylsilyl-1,3,2’,2”’,6”’-pentadeaminoparomomycin (18).

0.1 mL of 3M MeMgCl in THF were added to a stirred solution of compound 16 (0.152 g, 0.111 mmol) in 1.1 mL of THF at −30 °C. The reaction mixture was stirred for 30 mins before quenching with 0.5 mL of aqueous NH₄Cl. The reaction mixture was then diluted with Et₂O then washed with NH₄Cl solution and brine. The organic layer was dried with Na₂SO₄, filtered, and concentrated. The crude residue was purified over silica gel in 20% EtOAc in hexanes to give 0.120 g (0.087 mmol, 78%) of compound 18 as a white foam. [α]_D²³ = 102.3 (c = 1.0, CHCl₃), ¹H NMR (600 MHz, CDCl₃) δ 7.40 – 7.15 (m, 30H, Ar-H), 6.10 (d, J = 3.9 Hz, 1H, H-1’), 5.67 (d, J = 5.8 Hz, 1H, H-1”), 4.97 (d, J = 1.9 Hz, 1H, H-1”’), 4.96 (d, J = 11.2 Hz, 1H, PhCH₂O), 4.94 (d, J = 12.2 Hz, 1H, PhCH₂O), 4.75 (d, J = 10.5 Hz, 1H, PhCH₂O), 4.64 (d, J = 11.5 Hz, 1H, PhCH₂O), 4.63 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.58 (d, J = 11.9 Hz, 1H, PhCH₂O), 4.49 – 4.41 (m, 4H, PhCH₂O), 4.37 – 4.31 (m, 2H, H-3”, PhCH₂O), 4.28 (q, J = 2.7 Hz, 1H, H-4”), 4.25 (d, J = 12.1 Hz, 1H, PhCH₂O), 3.97 – 3.91 (m, 2H, H-5, H-2”), 3.85 – 3.76 (m, 4H, H-3’, H-3”, H-5”, H-5”’), 3.74 (d, J = 9.6 Hz, 1H, H-5’), 3.70 (dd, J = 13.0, 8.7 Hz, 1H, H-6”’), 3.64 (dd, J = 9.8, 8.9 Hz, 1H, H-4), 3.58 – 3.54 (m, 2H, H-5”, OH), 3.50 – 3.42 (m, 2H, H-1, H-3), 3.41 – 3.37 (m, 2H, H-4’, H-2”’), 3.28 (t, J = 9.4 Hz, 1H, H-6), 3.14 – 3.11 (m, 1H, H-4”’), 2.86 (dd, J = 13.1, 3.6 Hz, 1H, H-6”’), 2.70 (dd, J = 10.0, 3.9 Hz, 1H, H-2’), 2.24 (dt, J = 13.3, 4.6 Hz, 1H, H-2eq), 1.38 (q, J = 12.7 Hz, 1H, H-2ax), 1.26 (s, 3H, −CH₃), 1.19 (s, 3H, −CH₃), 0.08 (s, 9H, −OTMS). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 138.4, 138.3, 137.9, 137.6, 137.0, 136.9, 128.7, 128.5, 128.42, 128.38, 128.34, 128.31, 128.2, 128.14, 127.8, 127.68, 127.56, 127.45, 127.2, 127.1, 126.9 (Ar), 106.1 (C-1”), 98.6 (C-1”’), 95.6 (C-1’), 84.4 (C-6), 82.2 (C-2”), 82.0 (C-4”), 81.8 (C-5), 79.7 (C-3’), 75.3 (C-3”), 75.2 (PhCH₂O), 74.9 (C-4), 74.8 (C-5’), 74.6 (C-5”’), 73.6 (PhCH₂O), 73.4 (C-4’), 73.1 (PhCH₂O), 73.0 (PhCH₂O), 72.8 (C-3”’), 72.4 (PhCH₂O), 72.0 (C-6’), 71.7 (PhCH₂O), 71.5 (C-4”’), 70.4 (C-5”), 63.5 (C-2’), 60.5 (C-1), 60.2 (C-3), 57.2 (C-2”’), 51.2 (C-6”’), 32.7 (C-2), 27.0 (−CH₃), 24.7 (−CH₃), 0.9 (-OTMS). HRMS (ESI) m/z: Calcd for C₇₀H₈₃N₁₅O₁₄SiNa [M+Na]⁺ 1408.5911; Found 1408.5900.

6’,6’-Di-C-methylparomomycin pentaacetate salt (27).

A 1M TBAF solution in THF (0.13 mL) was added dropwise to a stirred solution of compound 18 (0.056 g, 0.041 mmol) in 1.6 mL of THF under argon. When the starting material was no longer visible by TLC, the reaction mixture was diluted with Et₂O and washed with of aqueous NaHCO₃ and brine. The organic layer was then dried with Na₂SO₄, filtered, and concentrated to give the intermediate alcohol which was used without further purification. The crude alcohol was stirred in 0.6 mL of 1:1 dioxane/10% AcOH in water with 107 mg of Pd/C under 50 psi of H₂ for 21 h. Once the reaction was determined to be complete by LCMS the reaction mixture was diluted with water and filtered through Celite. The resulting crude product was purified over a CM Sephadex C-25 column. The column was washed with 100 mL of DI water and eluted with NH₄OH in water starting at 0.1% and increasing stepwise by 0.1% every 20 mL to 0.8%. Lyophilization of the pure fractions with AcOH gave 20.4 mg (0.022 mmol, 54%) of the penta acetate salt 27 as a white solid. [α]_D²³ = 35.0 (c = 1.0, H₂O), ¹H NMR (600 MHz, D₂O) δ 5.59 (d, J = 3.9 Hz, 1H, H-1’), 5.23 (d, J = 2.9 Hz, 1H, H-1”), 5.15 (d, J = 1.8 Hz, 1H, H-1”’), 4.36 (t, J = 5.7 Hz, 1H, H-3”), 4.20 (dd, J = 5.1, 3.0 Hz, 1H, H-2”), 4.17 (td, J = 4.8, 3.9, 2.3 Hz, 1H, H-5”’), 4.09 (t, J = 3.2 Hz, 1H, H-3”’), 4.08 – 4.06 (m, 1H, H-4”), 3.77 (dd, J = 12.4, 3.1 Hz, 1H, H-5”), 3.74 – 3.65 (m, 4H, H-4, H-5, H-3’, H-4”’), 3.62 (dd, J = 12.4, 4.9 Hz, 1H, H-5”), 3.54 – 3.49 (m, 2H, H-6, H-5’), 3.44 (br. s, 1H, H-2”’), 3.40 (t, J = 9.3 Hz, 1H, H-4’), 3.29 (dd, J = 13.7, 6.6 Hz, 1H, H-6”’), 3.26 – 3.21 (m, 2H, H-2’, H-6”’), 3.21 – 3.12 (m, 2H, H-1, H-3), 2.21 (dt, J = 12.8, 4.3 Hz, 1H, H-2eq), 1.77 (s, 15H, AcOH), 1.56 (q, J = 12.6 Hz, 1H, H-2ax), 1.20 (s, 3H, −CH₃), 1.14 (s, 3H, −CH₃). ¹³C{¹H} NMR (151 MHz, D₂O) δ 181.2 (AcOH), 109.8 (C-1”), 95.9 (C-1’), 95.5 (C-1”’), 84.4 (C-5), 81.4 (C-4”), 79.7 (C-4), 77.2 (C-5’), 75.4 (C-3”), 73.3 (C-2”), 72.6 (C-6), 72.3 (C-6’), 70.7 (C-4’), 70.2 (C-5”’), 69.7 (C-3’), 67.6 (C-3”’), 67.2 (C-4”’), 60.2 (C-5”), 53.9 (C-2’), 50.8 (C-2”’), 49.9 (C-1), 49.2 (C-3), 40.3 (C-6”’), 29.8 (C-2), 26.3 (−CH₃), 23.3 (−CH₃), 23.1 (AcOH). HRMS (ESI) m/z: Calcd for C₂₅H₅₀N₅O₁₄ [M+H]⁺ 644.3354; Found 644.3358.

1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-6’-O-triisopropylsilyl-1,3,2’,2”’,6”’-pentadeaminoparomomycin (21).

TIPSOTf (1.25 mL, 4.7 mmol) was added to a stirred solution of 13 (5.06 g, 3.93 mmol) and lutidine (2.3 mL, 19.7 mmol) in DCM (79 mL) under argon. The reaction mixture was stirred for 1 h with monitoring by TLC and LCMS then was quenched with methanol and concentrated under vacuum. The crude residue was dissolved in EtOAc and washed with saturated aqueous NaHCO₃ and brine. The organic layer was dried with Na₂SO₄, filtered, and concentrated. The crude product was purified over silica gel eluting with 18–20% EtOAc in hexanes to give 21 (4.64 g, 3.22 mmol) as a white foam in 82% yield. [α]_D²³ = 68.1 (c = 1.0, DCM), ¹H NMR (600 MHz, CDCl₃) δ 7.44 – 7.11 (m, 30H, Ar-H), 6.14 (d, J = 3.7 Hz, 1H, H-1’), 5.67 (d, J = 5.8 Hz, 1H, H-1”), 4.97 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.87 (d, J = 1.9 Hz, 1H, H-1”’), 4.85 (s, 2H, PhCH₂O), 4.69 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.62 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.59 (d, J = 11.7 Hz, 1H, PhCH₂O), 4.52 (d, J = 11.9 Hz, 1H, PhCH₂O), 4.45 (d, J = 11.8 Hz, 1H, PhCH₂O), 4.43 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.40 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.30 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.29 – 4.27 (m, 1H, H-4”), 4.26 – 4.22 (m, 2H, H-3”, PhCH₂O), 4.01 (dt, J = 9.6, 5.7 Hz, 1H, H-5’), 3.98 – 3.91 (m, 4H, H-5, H-3’, H-6’, H-2”), 3.86 (dd, J = 10.1, 5.9 Hz, 1H, H-6’), 3.80 (dd, J = 10.4, 2.3 Hz, 1H, H-5”), 3.77 – 3.73 (m, 2H, H-3”’, H-5”’), 3.70 (dd, J = 9.8, 8.9 Hz, 1H, H-4), 3.63 (dd, J = 13.0, 8.6 Hz, 1H, H-6”’), 3.56 (dd, J = 10.4, 3.2 Hz, 1H, H-5”), 3.50 – 3.40 (m, 3H, H-1, H-3, H-4’), 3.34 (t, J = 2.6 Hz, 1H, H-2”’), 3.26 (t, J = 9.4 Hz, 1H, H-6), 3.11 (t, J = 2.5 Hz, 1H, H-4”’), 3.03 (s, 1H, 4’-OH), 2.96 (dd, J = 10.3, 3.7 Hz, 1H, H-2’), 2.87 (dd, J = 13.0, 3.9 Hz, 1H, H-6”’), 2.23 (dt, J = 13.2, 4.6 Hz, 1H, H-2eq), 1.36 (q, J = 12.8 Hz, 1H, H-2ax), 1.19 – 1.12 (m, 3H, TIPS-CH), 1.12 – 1.08 (m, 18H, TIPS−CH₃). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 138.34, 138.29, 137.9, 137.6, 137.0, 136.9, 128.7, 128.50, 128.47, 128.42, 128.34, 128.32, 128.29, 128.24, 128.18, 127.83, 127.80, 127.77, 127.75, 127.5, 127.4, 127.3 (Ar), 106.0 (C-1”), 98.6 (C-1”’), 95.7 (C-1’), 84.3 (C-6), 82.5 (C-2”), 82.1 (C-4”), 81.9 (C-5), 79.4 (C-3’), 75.5 (C-3”), 75.0 (PhCH₂O), 74.6 (C-4), 74.3 (C-5”’), 74.2 (C-4’), 73.3 (PhCH₂O), 73.2 (PhCH₂O), 72.8 (C-3”’), 72.4 (PhCH₂O), 71.7 (PhCH₂O), 71.5 (C-4”’), 70.8 (C-5’), 70.2 (C-5”), 65.6 (C-6’), 62.5 (C-2’), 60.3 (C-1), 60.1 (C-3), 57.3 (C-2”’), 51.1 (C-6”’), 32.6 (C-2), 18.0 (OTIPS−CH₃), 18.0 (OTIPS−CH₃), 11.8 (OTIPS-CH). HRMS (ESI) m/z: Calcd for C₇₄H₉₅N₁₆O₁₄Si [M + NH₄]⁺ 1459.6983; Found 1459.7007.

1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-4’-O-(4-methoxybenzyl)-6’-O-triisopropylsilyl-1,3,2’,2”’,6”’-pentadeaminoparomomycin (22).

NaH (0.237 g, 9.88 mmol) and TBAI (0.147 g, 0.40 mmol) were added to a stirred solution of compound 21 (5.61 g, 3.89 mmol) in DMF (33 mL) at 0 °C. After 20 minutes PMBCl (1.6 mL, 11.7 mmol) was added and the reaction mixture was warmed to rt. After 1.5 h the reaction was quenched with 2 mL of saturated aqueous NH₄Cl, diluted with EtOAc and washed with saturated NH₄Cl solution, water, and brine. The organic layer was dried with Na₂SO₄, filtered, and concentrated. The crude residue was purified over silica gel to give compound 22 (5.39g, 89%) as a white foam. [α]_D²³ = 64.8 (c = 1.0, CHCl₃), ¹H NMR (600 MHz, CDCl₃) δ 7.39 – 7.12 (m, 32H, Ar-H), 6.87 – 6.84 (m, 2H, Ar-H), 6.12 (d, J = 3.7 Hz, 1H, H-1’), 5.65 (d, J = 5.9 Hz, 1H, H-1”), 4.94 (d, J = 10.7 Hz, 1H, PhCH₂O), 4.88 (d, J = 1.9 Hz, 1H, H-1”’), 4.86 – 4.82 (m, 2H, PhCH₂O), 4.77 (d, J = 11.1 Hz, 1H, PhCH₂O), 4.66 (d, J = 10.7 Hz, 1H, PhCH₂O), 4.63 – 4.60 (m, 2H, PhCH₂O), 4.58 – 4.54 (m, 2H, PhCH₂O), 4.46 (d, J = 11.7 Hz, 1H, PhCH₂O), 4.44 – 4.39 (m, 2H, PhCH₂O), 4.31 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.26 (q, J = 2.8 Hz, 1H, H-4”), 4.25 – 4.21 (m, 2H, H-3”, PhCH₂O), 4.08 (dd, J = 10.4, 9.0 Hz, 1H, H-3’), 3.99 – 3.90 (m, 3H, H-5, H-5’, H-2”), 3.86 (dd, J = 11.0, 1.7 Hz, 1H, H-6’), 3.80 (s, 3H, −OCH₃), 3.79 – 3.72 (m, 4H, H-4, H-5”, H-3”’, H-5”’), 3.69 (dd, J = 10.9, 5.7 Hz, 1H, H-6’), 3.60 (dd, J = 12.9, 8.4 Hz, 1H, H-6”’), 3.55 (dd, J = 10.5, 3.3 Hz, 1H, H-5”), 3.48 – 3.37 (m, 2H, H-1, H-3), 3.37 – 3.32 (m, 2H, H-4’, H-2”’), 3.24 (t, J = 9.3 Hz, 1H, H-6), 3.11 (t, J = 2.5 Hz, 1H, H-4”’), 3.05 (dd, J = 10.4, 3.7 Hz, 1H, H-2’), 2.88 (dd, J = 12.9, 4.2 Hz, 1H, H-6”’), 2.20 (dt, J = 13.1, 4.6 Hz, 1H, H-2eq), 1.35 (q, J = 12.7 Hz, 1H, H-2ax), 1.08 (d, J = 4.5 Hz, 21H, OTIPS). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 159.2, 138.3, 138.1, 137.9, 137.7, 137.03, 136.97, 130.5, 129.5, 128.7, 128.5, 128.41, 128.39, 128.35, 128.33, 128.31, 128.22, 128.20, 128.16, 127.82, 127.76, 127.73, 127.5, 127.4, 113.8 (Ar), 105.9 (C-1”), 98.6 (C-1”’), 95.5 (C-1’), 84.2 (C-6), 82.6 (C-2”), 82.0 (C-4”), 81.7 (C-5), 80.3 (C-3’), 77.9 (C-4’), 75.6 (C-3”), 75.4 (PhCH₂O), 75.0 (PhCH₂O), 74.4 (C-5”’), 74.3 (C-4), 74.2 (PhCH₂O), 73.3 (PhCH₂O), 73.2 (PhCH₂O), 72.9 (C-3”’), 72.7 (C-5’), 72.3 (PhCH₂O), 71.7 (PhCH₂O), 71.4 (C-4”’), 70.0 (C-5”), 63.5 (C-2’), 63.0 (C-6’), 60.3 (C-1), 60.0 (C-3), 57.3 (C-2”’), 55.2 (OCH₃), 51.0 (C-6”’), 32.5 (C-2), 18.12 (OTIPS−CH₃), 18.10 (OTIPS−CH₃), 12.0 (OTIPS-CH). HRMS (ESI) m/z: Calcd for C₈₂H₉₉N₁₅O₁₅SiNa [M + Na]⁺ 1584.7112; Found 1584.7095.

1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-4’-O-(4-methoxybenzyl)-1,3,2’,2”’,6”’-pentadeaminoparomomycin (23).

A 1M solution of TBAF in THF (7.5 mL) was added to a stirred solution of 22 (3.92 g, 2.51 mmol) in THF (43 mL) and the reaction mixture was stirred under argon for 3 h with monitoring by TLC. After completion, the reaction mixture was concentrated under vacuum and the residue was dissolved in ethyl acetate and washed with saturated aqueous NaHCO₃ followed by brine. The organic layer was dried with Na₂SO₄, filtered, and concentrated under vacuum. Purification over silica gel eluting with 20–30% EtOAc in hexanes gave the product 23 (3.1 g, 2.20 mmol) in 88% yield as a white foam. [α]_D²³ = 79.1 (c = 1.0, CHCl₃), ¹H NMR (600 MHz, CDCl₃) δ 7.39 – 7.13 (m, 32H, Ar-H), 6.86 – 6.83 (m, 2H, Ar-H), 6.13 (d, J = 3.7 Hz, 1H, H-1’), 5.67 (d, J = 5.6 Hz, 1H, H-1”), 4.97 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.89 (d, J = 1.9 Hz, 1H, H-1”’), 4.83 (s, 2H, PhCH₂O), 4.75 (d, J = 10.9 Hz, 1H, PhCH₂O), 4.71 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.62 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.57 (d, J = 11.8 Hz, 1H, PhCH₂O), 4.54 (d, J = 11.0 Hz, 1H, PhCH₂O), 4.51 (d, J = 11.9 Hz, 1H, PhCH₂O), 4.49 – 4.43 (m, 2H, PhCH₂O), 4.40 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.34 – 4.29 (m, 3H, H-3”, H-4”, PhCH₂O), 4.24 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.02 (dd, J = 10.3, 9.0 Hz, 1H, H-3’), 3.99 (dd, J = 5.7, 4.5 Hz, 1H, H-2”), 3.94 (t, J = 8.8 Hz, 1H, H-5), 3.90 (dt, J = 10.2, 3.1 Hz, 1H, H-5’), 3.82 (dd, J = 10.5, 2.1 Hz, 1H, H-5”), 3.80 – 3.77 (m, 4H, H-5”’, −OCH₃), 3.76 (t, J = 2.8 Hz, 1H, H-3”’), 3.75 – 3.72 (m, 1H, H-6’), 3.66 (dd, J = 13.0, 8.6 Hz, 1H, H-6”’), 3.63 – 3.56 (m, 3H, H-4, H-6’, H-5”), 3.46 – 3.39 (m, 2H, H-1, H-3), 3.37 – 3.33 (m, 2H, H-4’, H-2”’), 3.29 (t, J = 9.3 Hz, 1H, H-6), 3.11 (t, J = 2.5 Hz, 1H, H-4”’), 2.94 (dd, J = 10.3, 3.7 Hz, 1H, H-2’), 2.86 (dd, J = 13.0, 3.7 Hz, 1H, H-6”’), 2.22 (dt, J = 13.2, 4.6 Hz, 1H, H-2eq), 1.39 (q, J = 12.7 Hz, 1H, H-2ax). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 159.2, 138.3, 138.0, 137.9, 137.5, 137.0, 136.9, 130.3, 129.4, 128.7, 128.5, 128.42, 128.40, 128.34, 128.29, 128.26, 128.20, 128.1, 127.83, 127.79, 127.75, 127.70, 127.6, 127.5, 127.1, 113.8 (Ar), 106.2 (C-1”), 98.6 (C-1”’), 95.7 (C-1’), 84.2 (C-6), 82.5 (C-2”), 82.1 (C-4”), 82.0 (C-5), 79.8 (C-3’), 77.4 (C-4’), 75.5 (C-3”), 75.3 (PhCH₂O), 75.0 (PhCH₂O), 74.9 (C-4), 74.4 (C-5”’), 73.2 (PhCH₂O), 72.8 (C-3”’), 72.4 (PhCH₂O), 71.7 (PhCH₂O), 71.6 (C-5’), 71.4 (C-4”’), 70.3 (C-5”), 63.2 (C-2’), 61.6 (C-6’), 60.3 (C-1), 60.0 (C-3), 57.3 (C-2”’), 55.3 (−OCH₃), 51.1 (C-6”’), 32.4 (C-2). HRMS (ESI) m/z: Calcd for C₇₃H₇₉N₁₅O₁₅Na [M + Na]⁺ 1428.5778; Found 1428.5724.

1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-6’-C-ethyl-1,3,2’,2”’,6”’-pentadeaminoparomomycin (24(R) and 24(S)).

Oxalyl chloride (0.125 mL, 1.42 mmol) was added to a stirred solution of DMSO (0.21 mL, 2.96 mmol) in DCM (7.1 mL) at −78 °C under argon. After 15 minutes compound 23 (1.001 g, 0.71 mmol) was dissolved in DCM (3 mL) and added to the cold reaction mixture dropwise. After 1 h triethylamine (0.44 mL, 3.16 mmol) was added and the reaction mixture was allowed to slowly warm to room temperature before dilution with ether and washing with saturated NH₄Cl solution, DI water, and brine. The organic layer was dried with Na₂SO₄, filtered, and concentrated to give the intermediate aldehyde as a white foam (0.982 g, 0.70 mmol) in 98% yield which was used in the next step without purification. Freshly prepared EtMgBr 1M solution (1.4 mL) was added to a stirred solution of aldehyde (0.470 g, 0.34 mmol) in THF (6.7 mL) at −78 °C. After 1 h the reaction was quenched with 1 mL aqueous saturated NH₄Cl solution, diluted with Et₂O, washed with aqueous NH₄Cl and brine, dried with Na₂SO₄, and concentrated. The crude residue was then purified using silica gel column chromatography eluting with 25% EtOAc in hexanes to give the intermediate alcohols (0.303 g, 0.211 mmol, 63%) as an inseparable mixture of diastereomers which were used without further purification. ESI-HRMS: m/z calcd for C₇₅H₈₃N₁₅O₁₄Na [M + Na]⁺ 1456.6091, found 1456.6062. TFA (0.33 mL) was added to a stirred solution of these alcohols (0.283 g, 0.20 mmol) in DCM (3 mL) at 0 °C. After 1 h the reaction mixture was diluted with Et₂O and washed with aqueous saturated NaHCO₃ and brine. The organic layer was dried with Na₂SO₄ and concentrated followed by purification using silica gel column chromatography eluting with 28% EtOAc in hexanes to give 24(R) (23.9 mg, 0.018 mmol) in 9% isolated yield, 24(S) (90.3 mg, 0.069) in 35% isolated yield, as well as a mixture of 24(R) and 24(S) (91.5 mg, 0.070 mmol) in 35% yield. 24(R) [α]_D²³ = 88.0 (c = 0.5, DCM), ¹H NMR (600 MHz, CDCl₃) δ 7.42 – 7.12 (m, 30H, Ar-H), 6.14 (d, J = 3.7 Hz, 1H, H-1’), 5.67 (d, J = 5.7 Hz, 1H, H-1”), 4.97 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.91 – 4.87 (m, 2H, H-1”’, PhCH₂O), 4.72 – 4.69 (m, 2H, PhCH₂O), 4.62 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.56 (d, J = 11.8 Hz, 1H, PhCH₂O), 4.48 (d, J = 11.7 Hz, 1H, PhCH₂O), 4.46 – 4.42 (m, 2H, PhCH₂O), 4.40 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.32 – 4.27 (m, 3H, H-3”, H-4”, PhCH₂O), 4.24 (d, J = 12.1 Hz, 1H, PhCH₂O), 3.97 – 3.93 (m, 2H, H-5, H-2”), 3.88 (dd, J = 10.3, 8.6 Hz, 1H, H-3’), 3.81 (dd, J = 10.3, 1.9 Hz, 1H, H-5”), 3.79 – 3.74 (m, 3H, H-5’, H-3”’, H-5”’), 3.68 – 3.62 (m, 2H, H-6’, H-6”’), 3.62 – 3.55 (m, 2H, H-4, H-5”), 3.48 – 3.41 (m, 3H, H-1, H-3, H-4’), 3.35 (t, J = 2.6 Hz, 1H, H-2”’), 3.28 (t, J = 9.3 Hz, 1H, H-6), 3.12 (t, J = 2.6 Hz, 1H, H-4”’), 2.89 – 2.85 (m, 2H, H-2’, H-6”’), 2.24 (dt, J = 13.3, 4.6 Hz, 1H, H-2eq), 1.76 (dqd, J = 14.4, 7.5, 3.5 Hz, 1H, H-7’), 1.49 (ddd, J = 14.4, 8.6, 7.1 Hz, 1H, H-7’), 1.38 (q, J = 12.8 Hz, 1H, H-2ax), 1.02 (t, J = 7.4 Hz, 3H, H-8’). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 138.2, 138.1, 137.9, 137.5, 137.0, 136.9, 128.7, 128.6, 128.5, 128.4, 128.33, 128.32, 128.28, 128.26, 128.18, 128.15, 128.0, 127.80, 127.78, 127.76, 127.72, 127.5, 127.4, 127.2 (Ar), 106.1 (C-1”), 98.6 (C-1”’), 95.6 (C-1’), 84.3 (C-6), 82.4 (C-2”), 82.1 (C-4”), 81.9 (C-5), 79.6 (C-3’), 75.5 (C-3”), 75.4 (C-6’), 75.1 (PhCH₂O), 75.04 (PhCH₂O), 75.01 (C-4), 74.4 (C-5”’), 73.7 (C-4’), 73.2 (PhCH₂O), 73.1 (PhCH₂O), 72.9 (C-3”’), 72.4 (PhCH₂O), 72.1 (C-5’), 71.7 (PhCH₂O), 71.5 (C-4”’), 70.3 (C-5”), 62.5 (C-2’), 60.4 (C-1), 60.3 (C-3), 57.2 (C-2”’), 51.1 (C-6”’), 32.6 (C-2), 25.6 (C-7’), 9.8 (C-8’). HRMS (ESI) m/z: Calcd for C₆₇H₇₅N₁₅O₁₄Na [M + Na]⁺ 1336.5516; Found 1336.5537. 24(S) [α]_D²³ = 74.3 (c = 1.0, DCM), ¹H NMR (600 MHz, CDCl₃) δ 7.40 – 7.15 (m, 30H, Ar-H), 6.17 (d, J = 3.6 Hz, 1H, H-1’), 5.70 (d, J = 5.9 Hz, 1H, H-1”), 5.01 (d, J = 10.5 Hz, 1H, PhCH₂O), 4.94 – 4.90 (m, 2H, PhCH₂O), 4.72 (d, J = 10.5 Hz, 1H, PhCH₂O), 4.68 (d, J = 11.4 Hz, 1H, PhCH₂O), 4.63 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.59 (d, J = 11.9 Hz, 1H, PhCH₂O), 4.49 (d, J = 11.9 Hz, 1H, PhCH₂O), 4.47 (s, 2H, PhCH₂O), 4.41 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.34 – 4.30 (m, 3H, H-3”, H-4”, PhCH₂O), 4.25 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.00 (dd, J = 6.0, 4.6 Hz, 1H, H-2”), 3.96 (t, J = 8.9 Hz, 1H, H-5), 3.88 (dd, J = 10.3, 8.9 Hz, 1H, H-3’), 3.84 (dd, J = 10.4, 2.2 Hz, 1H, H-5”), 3.80 (ddd, J = 8.7, 3.8, 1.9 Hz, 1H, H-5”’), 3.77 (t, J = 2.9 Hz, 1H, H-3”’), 3.73 (dd, J = 9.8, 1.4 Hz, 1H, H-5’), 3.70 – 3.64 (m, 2H, H-6’, H-6”’), 3.59 (dd, J = 10.4, 2.7 Hz, 1H, H-5”), 3.54 (t, J = 9.3 Hz, 1H, H-4), 3.52 – 3.49 (m, 1H, H-4’), 3.49 – 3.41 (m, 2H, H-1, H-3), 3.37 (t, J = 2.6 Hz, 1H, H-2”’), 3.29 (t, J = 9.4 Hz, 1H, H-6), 3.14 – 3.11 (m, 1H, H-4”’), 2.87 (dd, J = 13.0, 3.8 Hz, 1H, H-6”’), 2.82 (dd, J = 10.3, 3.7 Hz, 1H, H-2’), 2.25 – 2.20 (m, 2H, H-2eq, −OH), 1.65 – 1.56 (m, 2H, H-7’, −OH), 1.52 (dqd, J = 14.7, 7.5, 4.7 Hz, 1H, H-7’), 1.34 (q, J = 12.8 Hz, 1H, H-2ax), 1.02 (t, J = 7.4 Hz, 3H, H-8’). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 138.3, 138.1, 137.9, 137.5, 137.0, 136.9, 128.7, 128.6, 128.5, 128.41, 128.36, 128.33, 128.26, 128.2, 128.1, 128.0, 127.8, 127.7, 127.6, 127.5, 127.0 (Ar), 106.1 (C-1”), 98.7 (C-1”’), 95.9 (C-1’), 84.4 (C-6), 82.5 (C-2”), 82.2 (C-4”), 82.0 (C-5), 79.8 (C-3’), 75.5 (C-3”), 75.1 (PhCH₂O), 74.9 (PhCH₂O), 74.8 (C-4), 74.5 (C-5”’), 73.1 (PhCH₂O), 72.9 (C-3”’), 72.7 (C-5’), 72.4 (PhCH₂O), 71.7 (PhCH₂O), 71.5 (C-4”’), 70.7 (C-6’), 70.2 (C-5”), 70.0 (C-4’), 62.5 (C-2’), 60.4 (C-1), 60.3 (C-3), 57.2 (C-2”’), 51.2 (C-6”’), 32.7 (C-2), 27.0 (C-7’), 10.5 (C-8’). HRMS (ESI) m/z: Calcd for C₆₇H₇₅N₁₅O₁₄Na [M + Na]⁺ 1336.5516; Found 1336.5549.

6’-(R)-C-Ethylparomomycin pentaacetate salt (29(R)).

Compound 24(R) (18.8 mg, 0.014 mmol) was dissolved in 0.2 mL of dioxane and 0.2 mL of 10% AcOH in water. Pd/C (38 mg) was added and the mixture subjected to 48 psi H₂ for 48 h. Once the reaction was determined to be complete by LCMS the reaction mixture was diluted with water and filtered through Celite. The resulting crude product was purified using a CM Sephadex C-25 column. The column was washed with 50 mL of DI water and eluted with NH₄OH in water starting at 0.1% and increasing stepwise by 0.1% every 20 mL to 0.8%. Lyophilization with AcOH gave the acetate salt 29(R) (2.3 mg, 0.0024 mmol) in 34% yield as a white powder. [α]_D²³ = 57.6 (c = 0.1, H₂O), ¹H NMR (600 MHz, D₂O) δ 5.53 (d, J = 4.0 Hz, 1H, H-1’), 5.24 (d, J = 2.8 Hz, 1H, H-1”), 5.16 (d, J = 1.8 Hz, 1H, H-1”’), 4.38 (t, 1H, H-3”), 4.22 (dd, J = 5.1, 2.9 Hz, 1H, H-2”), 4.20 – 4.16 (m, 1H, H-5”’), 4.10 (t, J = 3.1 Hz, 1H, H-3”’), 4.09 – 4.06 (m, 1H, H-4”), 3.81 – 3.68 (m, 7H, H-4, H-5, H-3’, H-5’ [J_5’,6’ = 2.5 Hz extracted from HSQC], H-6’, H-5”, H-4”’), 3.64 (dd, J = 12.4, 4.6 Hz, 1H, H-5”), 3.54 (t, J = 9.5 Hz, 1H, H-6), 3.46 (t, J = 2.3 Hz, 1H, H-2”’), 3.39 (t, J = 9.5 Hz, 1H, H-4’), 3.36 – 3.15 (m, 5H, H-1, H-3, H-2’, H-6”’, H-6”’), 2.29 (dt, J = 13.2, 4.1 Hz, 1H, H-2eq), 1.80 (s, 15H, AcOH), 1.65 (q, J = 12.5 Hz, 1H, H-2ax), 1.54 – 1.44 (m, 1H, H-7’), 1.43 – 1.33 (m, 1H, H-7’), 0.84 (t, J = 7.4 Hz, 3H, H-8’). ¹³C{¹H} NMR (151 MHz, D₂O) δ 180.5 (AcOH), 109.7 (C-1”), 96.5 (C-1’), 95.4 (C-1”’), 84.1 (C-5), 81.3 (C-4”), 79.2 (C-4), 75.6 (C-5’), 75.2 (C-3”), 73.4 (C-2”), 72.5 (C-6), 71.1 (C-6’), 70.1 (C-5”’), 69.7 (C-4’), 69.5 (C-3’), 67.6 (C-3”’), 67.2 (C-4”’), 60.0 (C-5”), 53.8 (C-2’), 50.8 (C-2”’), 49.7 (C-1), 49.0 (C-3), 40.4 (C-6”’), 28.9 (C-2), 22.7 (AcOH), 22.2 (C-7’), 9.8 (C-8’). HRMS (ESI) m/z: Calcd for C₂₅H₄₉N₅O₁₄ [M + H]⁺ 644.3354; Found 644.3358.

6’-(S)-C-Ethylparomomycin pentaacetate salt (29(S)).

Compound 24(S) (38.1 mg, 0.029 mmol) was dissolved in 0.4 mL of dioxane and 0.4 mL of 10% AcOH in water. Pd/C (77.5 mg) was added and the reaction mixture was subjected to 50 psi H₂ for 48 h. Once the reaction was determined to be complete by LCMS the reaction mixture was diluted with water and filtered through Celite. The resulting crude product was purified using a CM Sephadex C-25 column. The column was washed with 250 mL of DI water and eluted with NH₄OH in water starting at 0.1% and increasing stepwise by 0.1% every 20 mL to 0.8%. Lyophilization with AcOH gave the acetate salt 29(S) (9.5 mg, 0.010 mmol) in 35% yield as a white powder. [α]_D²³ = 41.3 (c = 0.4, H₂O), ¹H NMR (600 MHz, D₂O) δ 5.65 (d, J = 3.8 Hz, 1H, H-1’), 5.26 (d, J = 2.6 Hz, 1H, H-1”), 5.16 (d, J = 1.8 Hz, 1H, H-1”’), 4.39 (dd, J = 6.6, 5.0 Hz, 1H, H-3”), 4.23 (dd, J = 5.0, 2.6 Hz, 1H, H-2”), 4.20 – 4.16 (m, 1H, H-5”’), 4.10 (t, J = 3.1 Hz, 1H, H-3”’), 4.08 (ddd, J = 7.1, 4.5, 3.2 Hz, 1H, H-4”), 3.80 – 3.70 (m, 5H, H-4, H-5, H-3’, H-6’ [1dTOCSY 3.73, dd, J = 9.9, 3.9 Hz], H-5”), 3.69 – 3.68 (m, 1H, H-4”’), 3.64 (dd, J = 12.4, 4.7 Hz, 1H, H-5”), 3.57 – 3.50 (m, 3H, H-6, H-4’, H-5’), 3.46 – 3.45 (m, 1H, H-2”’), 3.29 (dd, J = 13.7, 6.7 Hz, 1H, H-6”’), 3.26 – 3.15 (m, 4H, H-1, H-3, H-2’, H-6”’), 2.25 (dt, J = 12.1, 3.5 Hz, 1H, H-2eq), 1.81 (s, 15H, AcOH), 1.62 (q, J = 12.7 Hz, 1H, H-2ax), 1.52 (ddq, J = 14.6, 9.3, 7.3 Hz, 1H, H-7’), 1.38 (dqd, J = 14.6, 7.3, 3.7 Hz, 1H, H-7’), 0.83 (t, J = 7.5 Hz, 3H, H-8’). ¹³C{¹H} NMR (151 MHz, D₂O) δ 180.3 (AcOH), 109.7 (C-1”), 95.8 (C-1’), 95.3 (C-1”’), 84.2 (C-5), 81.3 (C-4”), 78.8 (C-4), 75.23 (C-5’), 75.19 (C-3”), 73.4 (C-2”), 72.4 (C-6), 70.1 (C-5”’), 69.7 (C-6’), 69.2 (C-4’), 68.9 (C-3’), 67.6 (C-3”’), 67.2 (C-4”’), 60.0 (C-5”), 53.8 (C-2’), 50.8 (C-2”’), 49.9 (C-1), 49.2 (C-3), 40.3 (C-6”’), 29.1 (C-2), 26.1 (C-7’), 22.6 (AcOH), 9.9 (C-8’). HRMS (ESI) m/z: Calcd for C₂₅H₄₉N₅O₁₄ [M + H]⁺ 644.3354; Found 644.3369.

6’-(R)-C-Propylparomomycin pentaacetate salt (30(R)).

Compound 25(R) (33.2 mg, 0.0251 mmol) was dissolved in 0.4 mL of dioxane and 0.4 mL of 10% AcOH in water, treated with 67.4 mg of Pd/C and subjected to 48 psi H₂ for 22 h. Once the reaction was determined to be complete by LCMS the reaction mixture was diluted with water and filtered through Celite. The resulting crude product was purified using a CM Sephadex C-25 column. The column was washed with 50 mL of DI water and eluted with NH₄OH in water starting at 0.1% and increasing stepwise by 0.1% every 20 mL to 0.8%. Lyophilization with AcOH gave the acetate salt 30(R) (10.0 mg, 0.010 mmol) in 42% yield as a white powder. [α]_D²³ = 48.8 (c = 0.3, H₂O), ¹H NMR (600 MHz, D₂O) δ 5.52 (d, J = 4.0 Hz, 1H, H-1’), 5.24 (d, J = 2.9 Hz, 1H, H-1”), 5.16 (d, J = 1.8 Hz, 1H, H-1”’), 4.38 (t, J = 5.7 Hz, 1H, H-3”), 4.21 (dd, J = 5.2, 2.9 Hz, 1H, H-2”), 4.20 – 4.15 (m, 1H, H-5”’), 4.10 (t, J = 3.2 Hz, 1H, H-3”’), 4.09 – 4.06 (m, 1H, H-4”), 3.92 – 3.88 (m, 1H, H-6’), 3.82 (t, J = 9.5 Hz, 1H, H-4), 3.79 – 3.68 (m, 5H, H-5, H-3’, H-5’ [J_5’,6’ = 2.7 Hz extracted from HSQC trace], H-5”, H-4”’), 3.64 (dd, J = 12.4, 4.6 Hz, 1H, H-5”), 3.56 (t, J = 9.8 Hz, 1H, H-6), 3.46 (s, 1H, H-2”’), 3.42 – 3.36 (m, 2H, H-3, H-4’), 3.32 – 3.17 (m, 4H, H-1, H-2’, H-6”’), 2.32 (dt, J = 11.4, 4.0 Hz, 1H, H-2eq), 1.82 (s, 15H, AcOH), 1.69 (q, J = 12.8 Hz, 1H, H-2ax), 1.44 – 1.32 (m, 3H, H-7’, H-8’), 1.27 – 1.15 (m, 1H, H-8’), 0.78 (t, J = 7.0 Hz, 3H, H-9’). ¹³C{¹H} NMR (151 MHz, D₂O) δ 179.9 (AcOH), 109.7 (C-1”), 96.6 (C-1’), 95.5 (C-1”’), 84.0 (C-5), 81.4 (C-4”), 78.9 (C-4), 75.9 (C-5’), 75.2 (C-3”), 73.4 (C-2”), 72.3 (C-6), 70.1 (C-5”’), 69.7 (C-4’), 69.4 (C-3’), 69.0 (C-6’), 67.6 (C-3”’), 67.2 (C-4”’), 59.9 (C-5”), 53.8 (C-2’), 50.8 (C-2”’), 49.6 (C-1), 49.0 (C-3), 40.3 (C-6”’), 31.0 (C-7’), 28.4 (C-2), 22.5 (AcOH), 18.5 (C-8’), 13.0 (C-9’). HRMS (ESI) m/z: Calcd for C₂₆H₅₁N₅O₁₄ [M + H]⁺ 658.3511; Found 658.3529.

6’-(S)-C-Propylparomomycin pentaacetate salt (30(S)).

Compound 25(S) (33.3 mg, 0.025 mmol) was dissolved in 0.4 mL of dioxane and 0.4 mL of 10% AcOH in water, treated with 64.8 mg of Pd/C and subjected to 50 psi H₂ for 22 h. Once the reaction was determined to be complete by LCMS the reaction mixture was diluted with water and filtered through Celite. The resulting crude product was purified using a CM Sephadex C-25 column. The column was washed with 250 mL of DI water and eluted with NH₄OH in water starting at 0.1% and increasing stepwise by 0.1% every 20 mL to 0.8%. Lyophilization with AcOH gave the acetate salt 30(S) (11.9 mg, 0.012 mmol) in 49% yield as a white powder. [α]_D²³ = 40.6 (c = 0.4, H₂O), ¹H NMR (600 MHz, D₂O) δ 5.65 (d, J = 3.9 Hz, 1H, H-1’), 5.26 (d, J = 2.5 Hz, 1H, H-1”), 5.16 (d, J = 1.9 Hz, 1H, H-1”’), 4.39 (dd, J = 6.6, 4.9 Hz, 1H, H-3”), 4.23 (dd, J = 5.0, 2.5 Hz, 1H, H-2”), 4.17 (ddd, J = 6.4, 4.1, 1.5 Hz, 1H, H-5”’), 4.09 (t, J = 3.1 Hz, 1H, H-3”’), 4.07 (ddd, J = 7.0, 4.4, 3.0 Hz, 1H, H-4”), 3.85 (ddd, J = 9.8, 3.4, 1.7 Hz, 1H, H-6’), 3.79 – 3.74 (m, 2H, H-3’, H-5”), 3.74 – 3.70 (m, 2H, H-4, H-5), 3.70 – 3.68 (m, 1H, H-4”’), 3.64 (dd, J = 12.4, 4.6 Hz, 1H, H-5”), 3.56 – 3.47 (m, 3H, H-6, H-4’, H-5’), 3.45 (dt, J = 3.0, 1.4 Hz, 1H, H-2”’), 3.29 (dd, J = 13.7, 6.6 Hz, 1H, H-6”’), 3.25 – 3.15 (m, 4H, H-1, H-3, H-2’, H-6”’), 2.25 (dt, J = 12.8, 4.3 Hz, 1H, H-2eq), 1.81 (s, 15H, AcOH), 1.63 (q, J = 12.7 Hz, 1H, H-2ax), 1.53 (dtd, J = 13.7, 9.7, 9.1, 4.3 Hz, 1H, H-7’), 1.40 – 1.26 (m, 2H, H-7’, H-8’), 1.26 – 1.19 (m, 1H, H-8’), 0.78 (t, J = 7.2 Hz, 3H, H-9’). ¹³C{¹H} NMR (151 MHz, D₂O) δ 180.3 (AcOH), 109.7 (C-1”), 95.8 (C-1’), 95.4 (C-1”’), 84.2 (C-5), 81.3 (C-4”), 78.9 (C-4), 75.6 (C-5’), 75.2 (C-3”), 73.4 (C-2”), 72.4 (C-6), 70.1 (C-5”’), 69.2 (C-4’), 68.9 (C-3’), 67.62 (C-3”’), 67.58 (C-6’), 67.2 (C-4”’), 60.0 (C-5”), 53.7 (C-2’), 50.8 (C-2”’), 49.9 (C-1), 49.1 (C-3), 40.3 (C-6”’), 34.9 (C-7’), 29.1 (C-2), 22.6 (AcOH), 18.6 (C-8’), 12.9 (C-9’). HRMS (ESI) m/z: Calcd for C₂₆H₅₁N₅O₁₄ [M + H]⁺ 658.3511; Found 658.3528.

1,3,2’,6’,2”’,6”’-Hexaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-6’-(S)-C-methyl-1,3,2’,6’,2”’,6”’-hexaadeaminoneomycin (20).

Trifluoromethanesulfonic anhydride (35 μL, 0.24 mmol) was added to a stirred solution of 17(R) (0.151 g, 0.110 mmol) and pyridine (0.09 mL, 1.1 mmol) in DCM (2.2 mL) at 0 °C. After 20 min the reaction was quenched with MeOH (0.02 mL, 0.49 mmol) and concentrated under vacuum. The resulting triflate was dissolved in DMF (1.1 mL) and LiN₃ (56.0 mg, 1.14 mmol) was added. After 1 h the reaction mixture was diluted with Et₂O, washed with 1N HCl and brine, and concentrated. The crude residue was purified using silica gel column chromatography in 22% EtOAc in hexanes to give 20 (58.6 mg, 0.044 mmol) in 40% yield. [α]_D²³ = 90.6 (c = 0.7, CHCl₃), ¹H NMR (600 MHz, CDCl₃) δ 7.39 – 7.15 (m, 30H, Ar-H), 6.16 (d, J = 3.7 Hz, 1H, H-1’), 5.64 (d, J = 5.7 Hz, 1H, H-1”), 4.96 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.93 (d, J = 11.4 Hz, 1H, PhCH₂O), 4.87 (d, J = 2.0 Hz, 1H, H-1”’), 4.71 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.63 – 4.60 (m, 2H, PhCH₂O), 4.58 (d, J = 11.7 Hz, 1H, PhCH₂O), 4.54 (d, J = 11.9 Hz, 1H, PhCH₂O), 4.47 – 4.43 (m, 2H, PhCH₂O), 4.40 (d, J = 11.9 Hz, 1H, PhCH₂O), 4.31 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.27 (q, J = 2.9 Hz, 1H, H-4”), 4.26 – 4.23 (m, 2H, H-3”, PhCH₂O), 3.97 – 3.92 (m, 2H, H-5, H-2”), 3.83 – 3.72 (m, 6H, H-3’, H-5’, H-6’, H-5”, H-3”’, H-5”’), 3.61 (dd, J = 13.0, 8.4 Hz, 1H, H-6”’), 3.59 – 3.54 (m, 2H, H-4, H-5”), 3.51 (td, J = 9.3, 2.8 Hz, 1H, H-4’), 3.49 – 3.40 (m, 2H, H-1, H-3), 3.34 (t, J = 2.6 Hz, 1H, H-2”’), 3.27 (t, J = 9.3 Hz, 1H, H-6), 3.13 (d, J = 2.6 Hz, 1H, H-4”’), 2.95 (dd, J = 10.3, 3.8 Hz, 1H, H-2’), 2.91 (dd, J = 12.9, 4.1 Hz, 1H, H-6”’), 2.24 (dt, J = 13.2, 4.6 Hz, 1H, H-2eq), 2.07 (d, J = 3.1 Hz, 1H, 4’-OH), 1.44 (d, J = 6.9 Hz, 3H, H-7’), 1.35 (q, J = 12.7 Hz, 1H, H-2ax). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 138.2, 138.0, 137.8, 137.6, 137.0, 136.9, 128.70, 128.66, 128.5, 128.41, 128.39, 128.34, 128.31, 128.28, 128.16, 128.12, 128.08, 127.81, 127.80, 127.79, 127.77, 127.6, 127.5 (Ar), 106.1 (C-1”), 98.7 (C-1”’), 95.9 (C-1’), 84.3 (C-6), 82.3 (C-2”), 82.0 (C-4”), 81.7 (C-5), 80.0 (C-3’), 75.5 (C-3”), 75.1 (C-4), 75.1 (PhCH₂O), 74.8 (PhCH₂O), 74.3 (C-5”’), 73.6 (C-5’), 73.3 (PhCH₂O), 73.2 (PhCH₂O), 72.9 (C-3”’), 72.4 (PhCH₂O), 71.7 (PhCH₂O), 71.5 (C-4”’), 70.4 (C-4’), 70.0 (C-5”), 62.4 (C-2’), 60.4 (C-1), 60.2 (C-3), 57.3 (C-2”’), 55.0 (C-6’), 51.0 (C-6”’), 32.7 (C-2), 15.3 (C-7’). HRMS (ESI) m/z: Calcd for C₆₆H₇₂N₁₈O₁₃ [M + Na]⁺ 1347.5424; Found 1347.5458.

1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-6’-hydroxylamino-6’-C-methyl-1,3,2’,6’,2”’,6”’-hexaadeaminoneomycin (19(R) and 19(S)).

Hydroxylamine hydrochloride (0.125 g, 1.80 mmol) was added to a stirred solution of 16 (0.507 g, 0.370 mmol) in 1:1 DCM/MeOH (7.4 mL). After 3 h the reaction mixture was diluted with Et₂O, washed with 1 N HCl and brine, dried with Na₂SO₄, and concentrated. The resulting oxime was used in the next step without further purification. 10% HCl MeOH solution (0.5 mL) was added to a stirred solution of this oxime and NaBH₃CN (0.114 g, 1.81 mmol) in MeOH (7.4 mL) at 60 °C. HCl in MeOH was added at 20 minutes (1 mL), 50 mins (0.5 mL), and 1 h (0.4 mL) to ensure reaction mixture was acidic. NaBH₃CN (0.113 g, 1.80 mmol) was added at 30 minutes. After 1.5 h the reaction mixture was diluted with Et₂O, washed with aqueous saturated NaHCO₃ and brine, dried with Na₂SO₄, and concentrated. The crude residue was purified using silica gel column chromatography in 40–60 % EtOAc in hexanes to give 19(R) (0.192 g, 0.15 mmol) in 39% yield and 19(S) (0.114 g, 0.087 mmol) in 23% yield. 19(R) [α]_D²³ = 83.2 (c = 1.0, CHCl₃), ¹H NMR (600 MHz, CDCl₃) δ 7.42 – 7.13 (m, 30H, Ar-H), 6.15 (d, J = 3.7 Hz, 1H, H-1’), 5.68 (d, J = 5.9 Hz, 1H, H-1”), 4.99 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.900 (d, J = 11.1 Hz, 1H, PhCH₂O), 4.897 (d, 1.8 Hz, 1H, H-1”’), 4.75 – 4.69 (m, 2H, PhCH₂O), 4.63 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.58 (d, J = 11.8 Hz, 1H, PhCH₂O), 4.50 (d, J = 11.8 Hz, 1H, PhCH₂O), 4.48 – 4.39 (m, 3H, PhCH₂O), 4.33 – 4.28 (m, 3H, H-3”, H-4”, PhCH₂O), 4.25 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.08 (dd, J = 10.0, 4.4 Hz, 1H, H-5’), 3.97 – 3.93 (m, 2H, H-5, H-2”), 3.90 (dd, J = 10.3, 8.7 Hz, 1H, H-3’), 3.83 (dd, J = 10.4, 2.2 Hz, 1H, H-5”), 3.80 – 3.75 (m, 2H, H-3”’, H-5”’), 3.73 (t, J = 9.3 Hz, 1H, H-4), 3.67 (dd, J = 13.0, 8.6 Hz, 1H, H-6”’), 3.58 (dd, J = 10.4, 3.1 Hz, 1H, H-5”), 3.49 – 3.38 (m, 2H, H-1, H-3), 3.37 (t, J = 2.4 Hz, 1H, H-2”’), 3.35 – 3.28 (m, 3H, H-6, H-4’, H-6’), 3.12 (t, J = 2.4 Hz, 1H, H-4”’), 2.91 – 2.84 (m, 2H, H-2’, H-6”’), 2.20 (dt, J = 13.1, 4.6 Hz, 1H, H-2eq), 1.40 (q, J = 12.8 Hz, 1H, H-2ax), 1.09 (d, J = 6.7 Hz, 3H, H-7’). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 138.3, 138.05, 137.96, 137.5, 137.0, 136.9, 128.7, 128.6, 128.5, 128.43, 128.36, 128.34, 128.27, 128.22, 128.0, 127.83, 127.82, 127.79, 127.78, 127.5, 127.2 (Ar), 106.1 (C-1”), 98.6 (C-1”’), 95.7 (C-1’), 84.3 (C-6), 82.4 (C-2”), 82.1 (C-4”), 82.0 (C-5), 80.0 (C-3’), 75.5 (C-3”), 75.1 (PhCH₂O), 74.6 (PhCH₂O), 74.4 (C-5”’), 73.24 (PhCH₂O), 73.18 (PhCH₂O), 72.8 (C-3”’), 72.6 (PhCH₂O), 72.4 (C-4’), 71.7 (PhCH₂O), 71.5 (C-4”’), 70.3 (C-5”), 69.1 (C-5’), 62.7 (C-2’), 60.4 (C-1), 60.3 (C-3), 58.3 (C-6’), 57.2 (C-2”’), 51.1 (C-6”’), 32.6 (C-2), 12.0 (C-7’). HRMS (ESI) m/z: Calcd for C₆₆H₇₅N₁₆O₁₄ [M + H]⁺ 1315.5649; Found 1315.5668. 19(S) [α]_D²³ = 82.7 (c = 1.0, CHCl₃), ¹H NMR (600 MHz, CDCl₃) δ 7.46 – 7.12 (m, 30H, Ar-H), 6.09 (d, J = 3.8 Hz, 1H, H-1’), 5.67 (d, J = 5.6 Hz, 1H, H-1”), 4.97 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.88 (d, J = 1.9 Hz, 1H, H-1”’), 4.86 (d, J = 11.1 Hz, 1H, PhCH₂O), 4.82 (d, J = 11.2 Hz, 1H, PhCH₂O), 4.71 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.62 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.56 (d, J = 11.7 Hz, 1H, PhCH₂O), 4.50 (d, J = 11.8 Hz, 1H, PhCH₂O), 4.47 – 4.42 (m, 2H, PhCH₂O), 4.41 (d, J = 11.9 Hz, 1H, PhCH₂O), 4.34 – 4.28 (m, 3H, H-3”, H-4”, PhCH₂O), 4.25 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.10 (dd, J = 10.1, 3.2 Hz, 1H, H-5’), 3.97 (dd, J = 5.7, 4.4 Hz, 1H, H-2”), 3.93 (t, J = 8.9 Hz, 1H, H-5), 3.88 (dd, J = 10.3, 8.7 Hz, 1H, H-3’), 3.82 (dd, J = 10.4, 2.0 Hz, 1H, H-5”), 3.79 – 3.74 (m, 2H, H-3”’, H-5”’), 3.69 – 3.59 (m, 3H, H-4, H-4’, H-6”’), 3.57 (dd, J = 10.7, 2.9 Hz, 1H, H-5”), 3.47 – 3.38 (m, 3H, H-1, H-3, H-6’), 3.35 (t, J = 2.4 Hz, 1H, H-2”’), 3.27 (t, J = 9.3 Hz, 1H, H-6), 3.12 (t, J = 2.3 Hz, 1H, H-4”’), 2.92 (dd, J = 10.3, 3.8 Hz, 1H, H-2’), 2.87 (dd, J = 13.0, 3.8 Hz, 1H, H-6”’), 2.21 (dt, J = 13.1, 4.6 Hz, 1H, H-2eq), 1.39 (q, J = 12.8 Hz, 1H, H-2ax), 1.13 (d, J = 6.8 Hz, 3H, H-7’). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 138.3, 138.2, 137.9, 137.6, 137.0, 136.9, 128.7, 128.51, 128.48, 128.4, 128.34, 128.27, 128.19, 128.17, 127.83, 127.79, 127.75, 127.51, 127.50, 127.2 (Ar), 106.2 (C-1”), 98.6 (C-1”’), 96.0 (C-1’), 84.2 (C-6), 82.4 (C-2”), 82.1 (C-4”), 81.9 (C-5), 79.7 (C-3’), 75.5 (C-3”), 75.12 (PhCH₂O), 75.06 (C-4), 74.4 (C-5”’), 73.2 (PhCH₂O), 73.1 (PhCH₂O), 72.8 (C-3”’), 72.4 (PhCH₂O), 71.8 (C-4’), 71.7 (PhCH₂O), 71.5 (C-4”’), 70.3 (C-5”), 69.9 (C-5’), 62.6 (C-2’), 60.4 (C-1), 60.1 (C-3), 58.0 (C-6’), 57.3 (C-2”’), 51.1 (C-6”’), 32.5 (C-2), 13.0 (C-7’). HRMS (ESI) m/z: Calcd for C₆₆H₇₅N₁₆O₁₄ [M + H]⁺ 1315.5649; Found 1315.5677.

6’-(R)-C-Methylneomycin hexaacetate salt (28(R)).

Compound 19(R) (43.7 mg, 0.033 mmol) was stirred in 0.4 mL of 1:1 dioxane/10% AcOH in water with 79.0 mg of Pd/C under 50 psi of H₂ for 12 h. Once the reaction was determined to be complete by LCMS the reaction mixture was diluted with water and filtered through Celite. The resulting crude product was purified over a CM Sephadex C-25 column. The column was washed with 250 mL of DI water and eluted with NH₄OH in water starting at 0.1% and increasing stepwise by 0.1% every 20 mL to 0.8%. Lyophilization with AcOH gave the acetate salt 28(R) (11.8 mg, 0.012 mmol) as a white solid in 36% yield. [α]_D²³ = 44.9 (c = 0.4, H₂O), ¹H NMR (600 MHz, D₂O) δ 5.92 (d, J = 3.9 Hz, 1H, H-1’), 5.29 (d, J = 2.8 Hz, 1H, H-1”), 5.16 (d, J = 1.8 Hz, 1H, H-1”’), 4.34 (dd, J = 6.2, 5.0 Hz, 1H, H-3”), 4.24 (dd, J = 5.1, 2.8 Hz, 1H, H-2”), 4.18 (ddd, J = 6.0, 4.1, 1.5 Hz, 1H, H-5”’), 4.12 – 4.08 (m, 2H, H-4”, H-3”’), 3.88 – 3.81 (m, 2H, H-3’, H-5’), 3.80 – 3.73 (m, 2H, H-5, H-5”), 3.71 – 3.66 (m, 3H, H-4, H-6’, H-4”’), 3.60 (dd, J = 12.3, 5.4 Hz, 1H, H-5”), 3.51 (dd, J = 10.6, 9.0 Hz, 1H, H-6), 3.45 (t, J = 1.2 Hz, 1H, H-2”’), 3.38 (t, J = 9.5 Hz, 1H, H-4’), 3.32 – 3.21 (m, 3H, H-2’, H-6”’), 3.20 – 3.12 (m, 2H, H-1, H-3), 2.20 (dt, J = 12.6, 4.3 Hz, 1H, H-2eq), 1.79 (s, 18H, AcOH), 1.58 (q, J = 12.6 Hz, 1H, H-2ax), 1.19 (d, J = 6.9 Hz, 3H, H-7’). ¹³C{¹H} NMR (151 MHz, D₂O) δ 180.6 (AcOH), 110.1 (C-1”), 95.5 (C-1’), 95.1 (C-1”’), 85.2 (C-5), 81.5 (C-4”), 77.1 (C-4), 75.5 (C-3”), 73.6 (C-2”), 72.8 (C-6), 71.4 (C-5’), 70.1 (C-5”’), 69.8 (C-4’), 68.5 (C-3’), 67.6 (C-3”’), 67.3 (C-4”’), 60.3 (C-5”), 53.6 (C-2’), 50.8 (C-2”’), 50.1 (C-1), 48.6 (C-3), 47.1 (C-6’), 40.4 (C-6”’), 29.9 (C-2), 22.8 (AcOH), 11.2 (C-7’). HRMS (ESI) m/z: Calcd for C₂₄H₄₈N₆O₁₃ [M+H]⁺ 629.3358; Found 629.3362.

6’-(S)-C-Methylneomycin hexaacetate salt (28(S)) from Hydroxylamine 19(S).

Compound 19(S) (32.6 mg, 0.0248 mmol) was stirred in 0.4 mL of 1:1 dioxane/10% AcOH in water with 65.0 mg of Pd/C under 50 psi of H₂ for 12 h. Once the reaction was determined to be complete by LCMS the reaction mixture was diluted with water and filtered through Celite. The resulting crude product was purified over a CM Sephadex C-25 column. The column was washed with 250 mL of DI water and eluted with NH₄OH in water starting at 0.1% and increasing stepwise by 0.1% every 20 mL to 0.8%. Lyophilization with AcOH gave the acetate salt 28(S) (8.3 mg, 0.008 mmol) as a white solid in 34% yield. [α]_D²³ = 40.4 (c = 0.3, H₂O), ¹H NMR (600 MHz, D₂O) δ 5.78 (d, J = 3.9 Hz, 1H, H-1’), 5.27 (d, J = 2.8 Hz, 1H, H-1”), 5.16 (d, J = 1.8 Hz, 1H, H-1”’), 4.36 (dd, J = 6.3, 5.0 Hz, 1H, H-3”), 4.24 (dd, J = 5.0, 2.8 Hz, 1H, H-2”), 4.18 (td, J = 5.1, 4.7, 2.3 Hz, 1H, H-5”’), 4.12 – 4.07 (m, 2H, H-4”, H-3”’), 3.83 – 3.71 (m, 4H, H-5, H-3’, H-5’, H-5”), 3.70 – 3.69 (m, 1H, H-4”’), 3.69 – 3.64 (m, 2H, H-4, H-6’), 3.62 (dd, J = 12.4, 5.0 Hz, 1H, H-5”), 3.52 (dd, J = 10.5, 9.1 Hz, 1H, H-6), 3.47 – 3.42 (m, 2H, H-4’, H-2”’), 3.30 (dd, J = 13.7, 6.4 Hz, 1H, H-6”’), 3.27 – 3.22 (m, 2H, H-2’, H-6”’), 3.19 – 3.14 (m, 1H, H-1), 3.13 – 3.08 (m, 1H, H-3), 2.19 (dt, J = 12.9, 4.3 Hz, 1H, H-2eq), 1.80 (s, 18H, AcOH), 1.54 (q, J = 12.6 Hz, 1H, H-2ax), 1.29 (d, J = 6.9 Hz, 3H, H-7’). ¹³C{¹H} NMR (151 MHz, D₂O) δ 180.5 (AcOH), 109.9 (C-1”), 95.6 (C-1’), 95.5 (C-1”’), 84.9 (C-5), 81.5 (C-4”), 79.0 (C-4), 75.4 (C-3”), 73.5 (C-2”), 72.8 (C-6), 72.0 (C-5’), 70.1 (C-5”’), 69.9 (C-4’), 68.8 (C-3’), 67.6 (C-3”’), 67.3 (C-4”’), 60.2 (C-5”), 53.5 (C-2’), 50.8 (C-2”’), 50.1 (C-1), 48.8 (C-3), 47.3 (C-6’), 40.4 (C-6”’), 30.1 (C-2), 22.8 (AcOH), 14.9 (C-7’). HRMS (ESI) m/z: Calcd for C₂₄H₄₈N₆O₁₃ [M+H]⁺ 629.3358; Found 629.3354.

Alternative Preparation of 6’-(S)-C-Methylneomycin hexaacetate salt (28(S)) from Azide 20.

Compound 20 (0.039 g, 0.030 mmol) was stirred in 0.8 mL of 1:1 dioxane/10% AcOH in water with 77.4 mg of Pd/C under 50 psi of H₂ for 72 h. Once the reaction was determined to be complete by LCMS the reaction mixture was diluted with water and filtered through Celite. The resulting crude product was purified over a CM Sephadex C-25 column. The column was washed with 250 mL of DI water and eluted with NH₄OH in water starting at 0.1% and increasing stepwise by 0.1% every 20 mL to 0.8%. Lyophilization with AcOH gave the acetate salt 28(S) (10.5 mg, 0.0167 mmol) as a white solid in 56% yield, with spectral data identical to the above isolated sample.

1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-4’,6’-O-benzylidene-6’-(S)-C-methyl-1,3,2’,2”’,6”’-pentadeaminoparomomycin (31).

A 1 M TBAF solution in THF (0.38 mL) was added to a stirred solution of compound 17(S) (0.172 g, 0.13 mmol) in THF (4.6 mL) under Ar. After 2 h the reaction mixture was diluted with Et₂O, washed with aqueous saturated NaHCO₃ and brine, dried with Na₂SO₄, and concentrated. The resulting diol (0.163 g, 0.13 mmol, 99%) was used in the next step without purification. Benzaldehyde dimethyl acetal (23 μL, 0.15 mmol) was added to a stirred solution of the above diol (0.163 g, 0.13 mmol) and CSA (3.2 mg, 14 μmol) in MeCN (3.3 mL). After 30 min CSA (2.2 mg, 9.5 μmol) and benzaldehyde dimethyl acetal (22 μL, 0.15 mmol) were added and the reaction mixture was stirred for an additional 30 min monitoring by LCMS and TLC until all starting material was consumed. The reaction was quenched with Et₃N, diluted with Et₂O, and washed with aqueous saturated NaHCO₃ and brine. The organic layer was concentrated and the resulting residue was purified using silica gel column chromatography in 20 % EtOAc in hexanes to give the acetal 31 (0.100 g, 0.072 mmol) in 60 % yield as a white foam. [α]_D²³ = 66.1 (c = 1.0, CHCl₃), ¹H NMR (600 MHz, C₆D₆) δ 7.64 – 7.59 (m, 2H, Ar-H), 7.52 – 7.49 (m, 2H, Ar-H), 7.46 – 7.42 (m, 2H, Ar-H), 7.32 – 7.28 (m, 4H, Ar-H), 7.20 (t, J = 7.8 Hz, 2H, Ar-H), 7.18 – 6.96 (m, 23H, Ar-H), 6.41 (d, J = 3.8 Hz, 1H, H-1’), 5.98 (d, J = 5.5 Hz, 1H, H-1”), 5.71 (s, 1H, PhCH(O)₂), 5.06 (d, J = 11.5 Hz, 1H, PhCH₂O), 5.00 (d, J = 2.1 Hz, 1H, H-1”’), 4.96 (d, J = 10.5 Hz, 1H, PhCH₂O), 4.90 (d, J = 11.5 Hz, 1H, PhCH₂O), 4.67 (dd, J = 10.3, 5.9 Hz, 1H, H-5’), 4.63 (q, J = 6.5 Hz, 1H, H-6’), 4.60 – 4.58 (m, 2H, H-4”, PhCH₂O), 4.50 (dd, J = 4.8, 2.7 Hz, 1H, H-3”), 4.45 – 4.37 (m, 4H, H-3’, PhCH₂O), 4.31 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.29 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.14 (t, J = 5.2 Hz, 1H, H-2”), 4.08 (d, J = 11.8 Hz, 1H, PhCH₂O), 4.00 – 3.97 (m, 2H, PhCH₂O), 3.95 (dd, J = 10.5, 2.3 Hz, 1H, H-5”), 3.83 – 3.77 (m, 2H, H-5, H-4’), 3.75 (ddd, J = 8.4, 4.1, 2.0 Hz, 1H, H-5”’), 3.67 (t, J = 3.1 Hz, 1H, H-3”’), 3.59 (dd, J = 10.5, 3.1 Hz, 1H, H-5”), 3.56 (t, J = 9.3 Hz, 1H, H-4), 3.43 (dd, J = 12.8, 8.4 Hz, 1H, H-6”’), 3.35 (t, J = 2.8 Hz, 1H, H-2”’), 3.13 (dd, J = 10.3, 3.8 Hz, 1H, H-2’), 2.96 (t, J = 2.6 Hz, 1H, H-4”’), 2.85 (t, J = 9.5 Hz, 1H, H-6), 2.78 – 2.68 (m, 2H, H-3, H-6”’), 2.54 (ddd, J = 12.3, 9.7, 4.5 Hz, 1H, H-1), 1.36 (dt, J = 12.9, 4.6 Hz, 1H, H-2eq), 1.26 (d, J = 6.8 Hz, 3H, H-7’), 0.82 (q, J = 12.6 Hz, 1H, H-2ax). ¹³C{¹H} NMR (151 MHz, C₆D₆) δ 138.7, 138.6, 138.44, 138.35, 137.9, 137.4, 137.3, 128.44, 128.35, 128.33, 128.24, 128.22, 128.18, 128.17, 128.15, 128.01, 128.00, 127.98, 127.90, 127.88, 127.6, 127.3, 127.2, 126.4 (Ar), 106.3 (C-1”), 98.8 (C-1”’), 97.3 (C-1’), 94.0 (PhCH(O)₂), 83.9 (C-6), 82.6 (C-2”), 82.5 (C-4”), 81.8 (C-5), 76.3 (C-4’), 76.3 (C-3’), 75.9 (C-3”), 75.5 (C-4), 74.94 (PhCH₂O), 74.85 (PhCH₂O), 74.2 (C-5”’), 73.5 (C-3”’), 73.1 (PhCH₂O), 72.9 (PhCH₂O), 72.4 (C-4”’), 72.2 (PhCH₂O), 71.6 (PhCH₂O), 70.4 (C-5”), 70.3 (C-6’), 65.3 (C-5’), 62.8 (C-2’), 60.0 (C-1), 59.9 (C-3), 56.7 (C-2”’), 51.0 (C-6”’), 31.8 (C-2), 11.1 (C-7’). HRMS (ESI) m/z: Calcd for C₇₃H₇₇N₁₅O₁₄Na [M+Na]⁺ 1410.5672; Found 1410.5674.

1,3,2’,2”’,6”’-Pentaazido-6,3’,2”,5”,3”’,4”’-hexa-O-benzyl-4’,6’-O-benzylidene-(R)-6’-C-ethyl-1,3,2’,2”’,6”’-pentadeaminoparomomycin (32).

Benzaldehyde dimethyl acetal (10 μL, 67 μmol) was added to a stirred solution of 24(R) (24.3 mg, 18.5 μmol) and CSA (3.0 mg, 13 μmol) in MeCN (0.5 mL) under argon. The reaction mixture was stirred for 1 h monitoring by LCMS and TLC then quenched with Et₃N. The reaction mixture was diluted with Et₂O, washed with aqueous saturated NaHCO₃ and brine, dried with Na₂SO₄, and concentrated. The crude residue was purified using silica gel column chromatography with 18 % EtOAc in hexanes to give acetal 32 (12.3 mg, 8.8 μmol) in 47 % yield as a white foam. [α]_D²³ = 92.8 (c = 0.2, CHCl₃), ¹H NMR (600 MHz, CDCl₃) δ 7.49 (d, J = 7.3 Hz, 2H, Ar-H), 7.40 – 7.13 (m, 33H, Ar-H), 6.19 (d, J = 3.9 Hz, 1H, H-1’), 5.66 (d, J = 5.6 Hz, 1H, H-1”), 5.56 (s, 1H, PhCH(O)₂), 4.97 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.92 (d, J = 11.2 Hz, 1H, PhCH₂O), 4.88 (d, J = 1.9 Hz, 1H, H-1”’), 4.76 (d, J = 11.2 Hz, 1H, PhCH₂O), 4.72 (d, J = 10.6 Hz, 1H, PhCH₂O), 4.62 (d, J = 12.0 Hz, 1H, PhCH₂O), 4.56 (d, J = 11.8 Hz, 1H, PhCH₂O), 4.52 (d, J = 11.9 Hz, 1H, PhCH₂O), 4.47 – 4.42 (m, 2H, PhCH₂O), 4.40 (d, J = 11.9 Hz, 1H, PhCH₂O), 4.34 – 4.27 (m, 3H, H-3”, H-4”, PhCH₂O), 4.25 (d, J = 12.1 Hz, 1H, PhCH₂O), 4.09 (t, J = 9.6 Hz, 1H, H-3’), 4.00 – 3.92 (m, 2H, H-5, H-2”), 3.82 – 3.74 (m, 3H, H-5”, H-3”’, H-5”’), 3.69 (t, J = 9.3 Hz, 1H, H-5’), 3.64 (dd, J = 12.9, 8.6 Hz, 1H, H-6”’), 3.60 (t, J = 9.2 Hz, 1H, H-4), 3.58 – 3.51 (m, 2H, H-6’, H-5”), 3.49 – 3.40 (m, 3H, H-1, H-3, H-4’), 3.35 (t, J = 2.4 Hz, 1H, H-2”’), 3.29 (t, J = 9.3 Hz, 1H, H-6), 3.12 (br s, 1H, H-4”’), 3.06 (dd, J = 10.1, 3.9 Hz, 1H, H-2’), 2.88 (dd, J = 13.0, 3.9 Hz, 1H, H-6”’), 2.23 (dt, J = 13.4, 4.7 Hz, 1H, H-2eq), 1.96 (dtt, J = 15.3, 7.9, 4.6 Hz, 1H, H-7’), 1.62 (dp, J = 15.5, 7.7 Hz, 1H, H-7’), 1.37 (q, J = 12.7 Hz, 1H, H-2ax), 1.10 (t, J = 7.4 Hz, 3H, H-8’). ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 138.3, 138.1, 137.9, 137.8, 137.6, 137.0, 136.9, 128.72, 128.66, 128.5, 128.4, 128.33, 128.31, 128.27, 128.21, 128.18, 128.14, 127.81, 127.77, 127.74, 127.73, 127.67, 127.5, 127.4, 127.3, 126.1 (Ar), 106.1 (C-1”), 100.8 (PhCH(O)₂), 98.6 (C-1”’), 96.1 (C-1’), 84.3 (C-6), 82.4 (C-2”), 82.1 (C-4”), 81.8 (C-5), 81.7 (C-4’), 80.5 (C-6’), 76.1 (C-3’), 75.5 (C-3”), 75.2 (C-4), 75.0 (PhCH₂O), 74.9 (PhCH₂O), 74.4 (C-5”’), 73.2 (PhCH₂O), 73.1 (PhCH₂O), 72.9 (C-3”’), 72.4 (PhCH₂O), 71.7 (PhCH₂O), 71.5 (C-4”’), 70.3 (C-5”), 67.2 (C-5’), 62.8 (C-2’), 60.4 (C-1), 60.0 (C-3), 57.3 (C-2”’), 51.1 (C-6”’), 32.6 (C-2), 24.7 (C-7’), 9.6 (C-8’). HRMS (ESI) m/z: Calcd for C₇₄H₇₉N₁₅O₁₄Na [M + Na]⁺ 1424.5829; Found 1424.5809.

Ribosome inhibition assays.

IC₅₀ values were determined by cell‐free translation inhibition assays with bacterial S30 extracts (University of Zurich) as described previously.⁶¹ Firefly luciferase mRNA was used as reporter to monitor translation activity..

Antibacterial inhibition assays.

The minimal inhibitory concentrations (MIC) of synthesized compounds were determined by broth microdilution assays according to CLSI reference methodology M0720 as described previously.⁶² Clinical bacterial isolates were obtained from the diagnostic laboratories of the Institute of Medical Microbiology, University of Zurich. Whole genome sequencing of the bacterial isolates and bioinformatic annotation of resistance genes was done as described previously.⁶²