Stereospecific Synthesis of Methyl 2-Amino-2,4-dideoxy-6S-deuterio-α-D-xylo-hexopyranoside and Methyl 2-Amino-2,4-dideoxy-6S-deuterio-4-propyl-α-D-glucopyranoside: Side Chain Conformation of the Novel Aminoglycoside Antibiotic Propylamycin.

Abstract

The stereospecific syntheses of methyl 2-amino-2,4-dideoxy-4-C-propyl-α-D-glucopyranoside and of methyl 2-amino-2,4-dideoxy-α-D-xylo-hexopyranoside and of their 6S-deuterioisotopomers are described as models for ring I of the aminoglycoside antibiotics propylamycin and 4’-deoxyparomomycin, respectively. Analysis of the ¹H NMR spectra of these compounds and of methyl 2-amino-2-deoxy-α-D-glucopyranoside, a model for paromomycin itself, reveals that neither deoxygenation at the 4-position, nor substitution of the C-O bond at the 4-postion by a C-C bond significantly changes the distribution of the side chain population between the three possible staggered conformations. From this it is concluded that the beneficial effect on antiribosomal and antibacterial activity of the propyl group in propylamycin does not derive from a change in side chain conformation. Rather, enhanced basicity of the ring oxygen and increased hydrophobicity and/or solvation effects are implicated.

Graphical abstract

1. Introduction

Propylamycin, 4’-deoxy-4’-C-propyl paromomycin, 1 is a novel synthetic 4,5-disubstituted 2-deoxystreptamine class aminoglycoside antibiotic that shows increased antibacterial and antibacterioribosomal activity compared to the parent paromomycin 2, as well as reduced ototoxicity in the guinea pig model.¹ 4’-Deoxyparomomycin 3 on the other hand is somewhat less active than the parent 2, with which it shows no significant difference in selectivity for prokaryotic over eukaryotic ribosomes.^{1, 2}

In this paper we explore the hypothesis that substitution at the 4’-position in ring I of paromomycin affects the distribution of the ring I side chain between the three staggered conformations, gauche,gauche (gg), gauche,trans (gt) and trans,gauche (tg) (Figure 1)^3–5 and so influences a critical hydrogen bonding interaction between the aminoglycoside and the drug binding pocket in the decoding A site of helix 44 on the small ribosomal subunit.

Figure 1.

Staggered Conformation of the Hexopyranose Side Chain

It is known that binding of deoxystreptamine type aminoglycosides to the decoding A site on the bacterial ribosome locks the side chain of ring I into the gt conformation by virtue of its participation in a pseudo-base pair interaction with A1408.^{6, 7} Indeed, we have demonstrated that a model of 2 with the ring I side locked into the gt conformation shows activity and selectivity approaching that of propylamycin.⁸ On the basis of these observations we were driven to study the influence of the 4’-deoxy and 4’-deoxy-4’-C-propyl modifications on the side chain conformation of ring I in the paromomycin series. In view of the complexity of the NMR spectra of the aminoglycosides, and the need for unambiguous assignment of the diastereotopic side chain protons H6R and H6S, we elected to study simplified monosaccharide models of ring I. To this end we describe the synthesis of methyl 2-amino-2,4-dideoxy-α-D-xylo-hexopyranoside 4α and methyl 2-amino-2,4-dideoxy-4-C-propyl-α-D-glucopyranoside 5, and for purposes of spectral elucidation their 6S-deuterio isotopomers, and the assignment of their side chain conformations by analysis of the 5,6R and 5,6S ¹H,¹H ³J coupling constants. Methyl 2-amino-2-deoxy-α-D-glucopyranoside 6⁹ and its 6S-deuterioisotopomer is employed as model for ring I of paromomycin itself.

2. Results and Discussion

2.1. Synthesis

Methyl 2-amino-2,4-dideoxy-α-D-xylo-hexopyranoside 4α was obtained by hydrogenolysis of the corresponding benzylcarbamate 7, which was prepared according to Mayer (Scheme 1).¹⁰ The synthesis of its 6S-deuterio isotopomer began with stereospecific introduction of deuterium into the 6-exo-position of 1,6-anhydro-D-glucose giving 8 under radical conditions following the literature protocol,^{11, 12} as modified in our laboratory⁹ by replacement of tetrachloromethane by the more environmentally friendly α,α,α-trifluorotoluene.¹³ Following the established protocol, 8 was converted to the epoxide 10 in two straightforward steps.^{9, 14–16} Ring opening of 10 with BF₃·OEt₂ and NaBH₄ in dimethoxyethane gave 11 in 99% yield (Scheme 1), which, on treatment with sodium methoxide formed the 2,3-epoxide 12 in 99% yield. Microwave heating of 12 to 110 °C in DMF with lithium azide and benzoic acid afforded 43% of a 1.3:1 mixture of regioisomeric azidoalcohols 13 and 14 from which 5% of the desired 14 was isolated by HPLC purification. Ring opening of 14 with TFA and acetic anhydride was followed by Fischer glycosylation in methanolic HCl and hydrogenolysis, resulting in an 85% yield of the target 6S-²H-4 in a mixture with the corresponding β-anomer, from which it could not be easily separated. Nevertheless, the NMR spectra of 6S-²H-4α in the mixture of anomers were well resolved, and permitted unambiguous assignment of the resonances from the diastereotopic 6-H_R and 6-H_S in isotopomers 4α.

Scheme 1.

Synthesis of Methyl 2-amino-2,4-dideoxy–α-D-xylo-hexopyranoside 4 and its 6S-Deuterio Isotopomer

The synthesis of methyl 2-amino-2,4-dideoxy-4-propyl-α-D-glucopyranoside (Scheme 2) began with the 3,4-epoxy tosylate 10, which was converted to the alcohol 15 by treatment with allylmagnesium chloride in the presence of copper(I) iodide and then to the 2,3-epoxide 16 with sodium methoxide as described in the literature.^{17, 18} Opening of epoxide 16 with sodium azide under a variety of conditions was unsuccessful, but heating in benzylamine to 155 °C gave the Fürst-Plattner¹⁹ product 17 in 77% yield (Scheme 2). Hydrogenolysis of the benzyl amine and saturation of the double bond gave the amine 18 in 79% yield, which was converted with Stick’s reagent^20–23 to the corresponding azido derivative 19 in 91% yield. Ring opening of 19 with TFA and acetic anhydride followed by Fischer glycosylation in methanolic HCl resulted in a 1.5:1 anomeric mixture of 20 in 69% yield, from which the individual anomers 20α and 20β were isolated in 22% and 12% yield, respectively. Hydrogenolysis of 20α gave the acetate salt 5 in 96% yield (Scheme 2). The isotopomer 6S-²H-5 was synthesized analogously and with comparable yield from 6-exo-deuterio 10. Comparison of the ¹H NMR spectra of 5 and its 6Sdeuterio isotopomer enabled assignment of the diastereotopic protons at the 6-position.

Scheme 2.

Synthesis of Methyl 2-amino-2,4-dideoxy-4-propyl–α-D-glucopyranoside 5 and its 6S-Deuterio Isotopomer

The differing regioselectivity in the ring openings of epoxides 12 and 16 with lithium azide and benzylamine, respectively, is of interest. The opening of 16 with benzylamine (Scheme 2) is consistent with the Fürst-Plattner rule and stereoelectronic control, with 1,3-diaxial interactions between the incoming nucleophile and the propyl substituent minimized due to the flattening of the pyranose ring imposed by the 1,6-anhydro bridge^{24, 25} and consistent with the anti-reflex effect.^{26, 27} On the other hand, the opening of 12 with azide affords a mixture of regioisomers (Scheme 1) in which the Fürst-Plattner product 14, formed directly in a chair conformation with trans-diaxial substituents, is minor. In contrast the major isomer 13 is necessarily formed in a twist boat conformation before relaxation to the observed chair with its two equatorial substituents. Presumably this diversion from the usual Fürst-Plattner^{19, 28} is due to the build up of positive charge on carbon during the ring opening of the protonated epoxide by azide, which is better accommodated on C3 adjacent to the methylene group than on C2 adjacent to the electron-withdrawing anomeric center (Scheme 3).

Scheme 3.

Differing Mechanisms and Regioselectivities in the Openings of Epoxides 12 and 16

A related example was recently observed in a synthesis of bradyrhizose,²⁹ wherein a cyclohexane-based trisubstituted epoxide underwent ring opening under acidic conditions in an anti-Fürst Plattner manner; an observation that was rationalized by preferential localization of partial positive charge on the tertiary carbon at the transition state (Scheme 4).

Scheme 4.

Anti-Fürst-Plattner Regioselectivity in the Opening of an Epoxide en route to Bradyrhizose

2.2. Analysis of Side Chain Populations

The ³J_H5,H6R and ³J_H5,H6S coupling constants for 4α and 5, taken from ¹H NMR spectra recorded at pH 5 in D₂O at room temperature, are reported in Table 1. The corresponding coupling constants for methyl 2-amino-2-deoxy-α-D-glucopyranoside 6 (Table 1) are literature values from our laboratory recorded under the same conditions.⁹

Table 1.

Experimental Chemical Shifts and ³J_H5,H6R and ³J_H5,H6S Coupling Constants

Ring I Model	δ H5 (ppm)	δ H6R (ppm)	δ H6S (ppm)	3JH5,H6R (Hz)	3JH5,H6S (Hz)
6	3.53	3.59	3.77	4.9	2.2
4α	3.79	3.43	3.51	5.8	2.5
5	3.62	3.54	3.66	5.3	2.2

Using limiting values of ³J_R,gg-³J_S,tg for each of the pure gg, gt, and tg conformers (Table 2) taken from a recent study employing a series of conformationally rigid bicyclic models,³⁰ the side chain populations of all compounds (f_gg – f_tg, (Table 3)) were determined in the standard manner^{3, 31–33} with the aid of eqs 1–3. The population of −2% calculated for the tg conformers of 6 and 5 is an artifact and arises from the errors in the coupling constants, estimated from the digital resolution of the specstra to be 0.4 Hz, and is considered to be indistinguishable from a 0% population.

Table 2.

Limiting Coupling Constants for the Ideal Staggered Conformers

	gg	gt	tg
3JR (Hz)	1.0	11.0	4.8
3JS (Hz)	2.2	2.5	10.2

Table 3.

Side Chain Populations of the Model Compounds

	fgg	fgt	ftg
6s	62	40	−2
4	51	47	2
5	58	44	−2

$${}^3{J}_{\text{H}5,\text{H}6\text{R}}={}^3{J}_{R,gg}{f}_{gg}+{}^3{J}_{R,gt}{f}_{gt}+{}^3{J}_{R,tg}{f}_{tg}$$ eq 1

$${}^3{J}_{\text{H}5,\text{H}6\text{S}}={}^3{J}_{S,gg}{f}_{gg}+{}^3{J}_{S,gt}{f}_{gt}+{}^3{J}_{S,tg}{f}_{tg}$$ eq 2

$$1=f_{gg}+f_{gt}+f_{tg}$$ eq 3

A modest increase in the population of the gt conformation, at the expense of a corresponding decrease in the gg conformation, is observed on replacement of the equatorial hydroxyl group in methyl 2-amino-2-deoxy-α-D-glucopyranoside 6 by a proton in methyl 2amino-2,4-dideoxy-α-D-glucopyranoside 4α (Table 3, entries 1 and 2). This pattern of changes mirrors that observed earlier by Bock and Duus³ on going from methyl α-D-glucopyranoside to methyl 4-deoxy-α-D-glucopyranoside, supporting our earlier conclusions that the presence of protonated amino group at the 2-position has little influence on the side chain conformation.⁹ Replacement of the hydroxyl group in 6 by the propyl group in methyl 2-amino-2,4-dideoxy-4propyl-α-D-glucopyranoside 5 (Table 3, entries 1 and 3) results in a side chain population that is intermediate between that of 6 and 4α. Clearly, the overall influence of the 4-OH group on the side chain conformation of 6, whether derived from dipolar interactions, hydrogen bonding, and/or solvation effects, is qualitatively reproduced by the propyl group in 5.

3. Conclusion

Analysis of the model compounds 6, 4α, and 5 indicates that neither deoxygenation nor substitution of the C-O bond at the 4-position by a C-C bond causes a significant change in the relative distribution of the side chain conformation between the three staggered conformations. Consequently, the increased activity of propylamycin in the inhibition of bacterial ribosomes and the inhibition of bacterial growth does not derive from a reduction in the entropic penalty paid on binding to the ribosome with a fixed gt side chain conformation. As suggested previously¹ any reduction in binding energy to the ribosome on deoxygenation at the 4’-position of paromomycin, due to the loss of a hydrogen bond to a backbone phosphate, is offset by the increased basicity of the ring oxygen (O5’) and the concomitant increase in the critical hydrogen bond with A1408 in the binding pocket. The difference in activity between the 4’-deoxy derivative 2 and propylamycin 1 with the 4’-deoxy-4’-propyl substitution pattern is therefore presumably related to hydrophobicity and/or solvation.

4. Experimental

GENERAL EXPERIMENTAL

All reagents were purchased from commercial sources and used without further purification unless otherwise specified. Chromatographic purifications were carried out in Fisher 230–400 mesh silica gel unless otherwise specified. Microwave reactions were conducted with a Biotage Initiator microwave reactor. High resolution mass spectra were collected on a Waters LCT Premier XE ESI-TOF mass spectrometer. Optical rotations were measured using an automated polarimeter in a 1 dm cell. NMR spectra were collected at 400, 500, or 600 MHz as indicated. ¹H NMR spectra were assigned with the aid of 1D and 2D techniques including COSY, HSQC, HMBC, and TOCSY.

Methyl 2-amino-2,4-dideoxy-α-D-xylo-hexopyranoside acetate salt (4α).

Compound 7 (93.8 mg, 0.30 mmol) was dissolved in a 1:1 mixture of 1,4-dioxane and 10% aqueous AcOH (0.8 mL) followed by addition of Pd/C (20.8 mg). The reaction mixture was stirred under 50 psi H₂ for 5 h followed by filtration over Celite® and lyophilization to obtain 4 as an off white solid (69.1 mg, 97%). [α]_D²³ = 110.6 (c = 0.14, water), ¹H NMR (600 MHz, D₂O) δ 4.86 (d, J = 3.7 Hz, 1H, H1), 3.91 (td, J = 11.0, 5.1 Hz, 1H, H3), 3.79 (ddt, J = 11.8, 5.8, 2.5 Hz, 1H, H5), 3.51 (dd, J = 12.1, 3.1 Hz, 1H, H6), 3.43 (dd, J = 12.1, 6.2 Hz, 1H, H6’), 3.24 (s, 3H, OMe), 3.02 (dd, J = 10.5, 3.6 Hz, 1H, H2), 1.86 (ddd, J = 12.8, 5.1, 2.1 Hz, 1H, H4eq), 1.76 (s, 3H, AcOH), 1.33 (q, J = 12.0 Hz, 1H, H4ax). ¹³C NMR (151 MHz, D₂O) δ 180.4 (AcOH), 96.6 (C1), 68.8 (C5), 64.3 (C3), 63.3 (C6), 55.2 (C2), 55.0 (OMe), 34.2 (C4), 22.6 (AcOH). ESI-HRMS: m/z calcd for C₇H₁₅NO₄Na [M + Na]⁺ 200.0899, found 200.0891.

1,6-Anhydro-4-deoxy-2-O-p-toluenesulfonyl-6S-deuterio-D-glucopyranose (11).

NaBH₄ (0.43 g, 11.4 mmol) and BF₃·OEt₂ (0.85 mL, 6.9 mmol) were added to an ice cold solution of 10 (0.84 g, 2.8 mmol) in 1,2-dimethoxyethane (9.4 mL). After 5 h the reaction mixture was diluted with Et₂O and washed with saturated NaHCO₃ solution and brine then dried with Na₂SO₄ and concentrated under vacuum to give 11 as a clear gum (0.85 g, 2.8 mmol, 99%) which was used in the next step without purification. [α]_D²³ = −27.0 (c = 1.0, CHCl₃), ¹H NMR (600 MHz, CDCl₃) δ 7.81 (d, J = 8.3 Hz, 2H, Ar-H), 7.36 – 7.33 (m, 2H, Ar-H), 5.27 (d, J = 1.9 Hz, 1H, H-1), 4.52 (d, J = 4.1 Hz, 1H, H-5), 4.20 (q, J = 1.7 Hz, 1H, H-2), 4.09 (s, 1H, H-6), 3.92 (t, J = 6.3 Hz, 1H, H-3), 2.46 (d, J = 6.3 Hz, 1H, -OH), 2.44 (s, 3H, Ar-CH₃), 2.31 (ddd, J = 15.0, 5.7, 4.5 Hz, 1H, H-4ax), 1.70 (ddt, J = 15.0, 1.9, 1.0 Hz, 1H, H-4eq). ¹³C NMR (151 MHz, CDCl₃) δ 145.3, 133.2, 130.1, 127.9 (Ar), 99.3 (C-1), 76.8 (C-2), 71.3 (C-5), 67.5 (t, J = 23.3 Hz, C-6), 66.5 (C-3), 32.5 (C-4), 21.7 (Ar-CH₃). ESI-HRMS: m/z calcd for C₁₃H₁₅DO₆SNa [M + Na]⁺ 324.0628, found 324.0627.

1,6;2,3-Bisanhydro-4-deoxy-6S-deuterio-D-lyxopyranose (12).

NaOMe (0.34 g, 6.3 mmol) was added to an ice cold stirred solution of 11 (0.824 g, 2.73 mmol) in 1:1 CHCl₃/MeOH (13.6 mL). After 4 h the reaction mixture was diluted with CH₂Cl₂ and washed with water. The aqueous phase was back extracted with CH₂Cl₂ and the combined organic layers were washed with brine then dried with Na₂SO₄ and concentrated under vacuum to give 12 as a colorless oil (0.351 g, 99%). [α]_D²³ = −28.6 (c = 1.0, CH2CL2), ¹H NMR (400 MHz, CDCl₃) δ 5.64 (dd, J = 3.2, 1.0 Hz, 1H, H-1), 4.38 (br d, J = 6.0 Hz, 1H, H-5), 3.63 (d, J = 1.7 Hz, 1H, H-6), 3.33 (t, J = 3.6 Hz, 1H, H-2), 3.10 (dd, J = 3.3 Hz, 1H, H-3), 2.19 (ddd, J = 15.3, 5.9, 3.2 Hz, 1H, H-4ax), 1.94 (d, J = 15.3 Hz, 1H, H-4eq). ¹³C NMR (101 MHz, CDCl₃) δ 98.0 (C-1), 68.0 (t, J = 23.1 Hz, C-6), 67.2 (C-5), 53.7 (C-2), 46.3 (C-3), 30.0 (C-4). ESI-HRMS: m/z calcd for C₆H₇DO₃Na [M + Na]⁺ 152.0434, found 152.0438.

1,6-Anhydro-2-azido-2,4-dideoxy-D-xylo-hexopyranose (14).

Compound 12 (75.0 mg, 0.58 mmol), LiN₃ (145 mg, 2.96 mmol), benzoic acid (109 mg, 0.89 mmol), and DMF (2.0 mL) were added to a microwave vial and heated to 110 °C for 75 mins in a microwave reactor. The reaction mixture was then diluted with CH₂Cl₂ and washed with aqueous saturated NaHCO₃ solution and brine. The organic layer was dried with Na₂SO₄ and filtered followed by concentration. The residue was subjected to silica gel preparative HPLC eluting with a gradient from 20 % to 60 % EtOAc in hexanes to give 14 (4.7 mg, 0.027 mmol, 5%). [α]_D²³ = 19.6 (c = 0.2, CHCl₃), ¹H NMR (600 MHz, CDCl₃) δ 5.53 (t, J = 2.0 Hz, 1H, H-1), 4.59 (d, J = 4.4 Hz, 1H, H-5), 4.19 (s, 1H, H-6), 4.00 – 3.96 (m, 1H, H-3), 3.30 (d, J = 2.2 Hz, 1H, H-2), 2.55 (d, J = 7.7 Hz, 1H, -OH), 2.31 (dt, J = 15.2, 4.9 Hz, 1H, H-4ax), 1.84 (ddt, J = 15.3, 2.9, 1.6 Hz, 1H, H-4eq). ¹³C NMR (151 MHz, CDCl₃) δ 100.8 (C-1), 72.0 (C-5), 67.6 (t, J = 23.3 Hz, C-6), 66.9 (C-3), 61.7 (C-2), 33.6 (C-4).

Methyl 2-amino-2,4-dideoxy-6S-deuterio-D-xylo-hexopyranoside acetate salt (6S-²H-4).

TFA (0.05 mL) was added to a stirred solution of 14 (4.6 mg, 0.27 mmol) in Ac₂O (0.5 mL) at 0 ° C and the reaction mixture was stirred for 3 h with monitoring by TLC. The reaction mixture was then diluted with Et₂O and washed with aqueous saturated NaHCO₃ solution and brine, dried with Na₂SO₄, and concentrated to give an inseparable mixture of anomers (8.3 mg, 0.026 mmol, 97 %), which were used in the next step without purification. ESI-HRMS: m/z calcd for C₁₂H₁₆DN₃O₇Na [M + Na]⁺ 339.1014, found 339.1027. The mixture of anomers from the previous step (8.3 mg, 0.026 mmol) were stirred in a 10 % HCl methanol solution (0.6 mL) under reflux for 3 h. After the reaction was complete by TLC and LCMS, the reaction mixture was concentrated under vacuum and the resulting inseparable mixture of anomers of methyl glycosides was used in the next step without purification. ESI-HRMS: m/z calcd for C₇H₁₂DN₃O₄Na [M + Na]⁺ 227.0867, found 227.0864. 10 wt% Pd/C (5.0 mg) was added to a solution of the methyl glycosides (6.1 mg, 0.30 mmol) in 1:1 dioxane/10% aqueous acetic acid (0.4 mL) and the reaction mixture was stirred under 50 psi H₂ for 6 h. The reaction mixture was then filtered through Celite® and concentrated under vacuum to give 6S-²H-4 (5.4 mg, 0.023 mmol, 76%) as a mixture of anomers in a ratio of 2:1 α/β as the acetate salts. ESI-HRMS: m/z calcd for C₇H₁₄DNO₄Na [M + Na]⁺ 201.1962, found 201.1956. 6S-²H-4α: ¹H NMR (600 MHz, D₂O) δ 4.87 (d, J = 3.6 Hz, 1H, H-1), 3.92 (td, J = 11.0, 5.0 Hz, 1H, H-3), 3.80 (ddd, J = 12.1, 6.3, 2.2 Hz, 1H, H-5), 3.42 (d, J = 6.0 Hz, 1H, H-6), 3.26 (s, 3H, -OCH₃), 3.03 (dd, J = 10.4, 3.7 Hz, 1H, H-2), 1.87 (ddd, J = 12.1, 5.0, 2.1 Hz, 1H, H-4eq), 1.78 (s, 3H, AcOH), 1.34 (q, J = 12.1 Hz, 1H, H-4ax). ¹³ C NMR (151 MHz, D₂O) δ 180.2 (AcOH), 96.6 (C-1), 68.7 (C-5), 64.3 (C-3), 63.2 – 62.8 (m, C-6), 55.2 (C-2), 55.0 (-OCH₃), 34.2 (C-4), 22.5 (AcOH). 6S-²H-4β: ¹H NMR (600 MHz, D₂O) δ 4.37 (d, J = 8.4 Hz, 1H, H-1), 3.76 (dt, J = 10.9, 5.4 Hz, 1H, H-3), 3.57 (ddd, J = 11.8, 6.7, 2.1 Hz, 1H, H-5), 3.45 (d, J = 6.6 Hz, 1H, H-6), 3.41 (s, 3H, -OCH₃), 2.69 (dd, J = 10.3, 8.4 Hz, 1H, H-2), 1.91 (ddd, J = 12.9, 5.2, 2.0 Hz, 1H, H-4eq), 1.78 (s, 3H, AcOH), 1.31 (dt, J = 12.9, 11.5 Hz, 1H, H-4ax). ¹³C NMR (151 MHz, D₂O) δ 180.2 (AcOH), 100.1 (C-1), 72.7 (C-5), 66.8 (C-3), 63.1 – 62.6 (m, C-6), 57.4 (C-2), 57.2 (−OCH₃), 34.4 (C-4), 22.5 (AcOH).

4-C-Allyl-1,6-anhydro-2-N-benzyl-2,4-dideoxy-D-glucopyranose (17).

A solution of 16 (0.319 g, 1.90 mmol) in benzylamine (5.0 mL) was stirred for 3 days at 155 °C. After concentration under reduced pressure the residue was purified by flash column chromatography on silica gel eluting with 40% ethyl acetate and 1% triethylamine in hexanes to afford 17 (0.402 g, 77%). [α]²³D = −49.4 (c = 1.00, CHCl₃). ¹H NMR (500 MHz, CDCl₃) δ 7.38 – 7.23 (m, 5H: aromatic), 5.83 (ddq, J = 16.4, 14.4, 6.2, 5.3 Hz, 1H: CH₂CHCH₂-C4), 5.47 (s, 1H: H1), 5.21 – 5.10 (m, 2H: CH₂CHCH₂-C4), 4.40 (d, J = 5.0 Hz, 1H: H5), 4.06 (d, J = 7.0 Hz, 1H: H6a), 3.93 – 3.85 (m, 5H: PhCH₂), 3.80 – 3.72 (m, 1H, H6b), 3.65 (td, J = 2.9, 1.6 Hz, 1H: H3), 2.68 – 2.63 (m, 1H: H2), 2.49 – 2.33 (m, 2H: CH₂CHCH₂-C4), 1.77 (t, J = 7.2 Hz, 1H: H4). ¹³C NMR (125 MHz, CDCl₃) δ 139.9, 136.1 (CH₂CHCH₂-C4), 128.5, 128.1, 127.2, 117.5 (CH₂CHCH₂-C4), 102.6 (C1), 74.6 (C5), 70.3 (C3), 68.5 (C6), 62.2 (C2), 51.7 (PhCH₂), 44.6 (C4), 36.5 (CH₂CHCH₂-C4). ESI-HRMS: m/z calcd for C₁₆H₂₂NO₃Na [M+Na]⁺ 276.1600, found 276.1600.

2-Amino-1,6-anhydro-2,4-dideoxy-4-C-propyl-D-glucopyranose acetate salt (18).

Compound 17 (0.4020 g, 1.46 mmol) and Pd/C (0.1316 g) were stirred in a 1:2 mixture of 10% aqueous acetic acid and 1,4-dioxane (6 mL) under 40 psi H₂ for 7 h. Pd/C (0.23 g) was added after 7 h and the reaction mixture was stirred for an additional 14 h before a final addition of Pd/C (0.39 g). After an additional 28 h the reaction mixture was filtered through Celite® and concentrated to give 18 (0.286 g, 79%). [α]_D²³ = −45.9 (c = 1.0, MeOH), ¹H NMR (600 MHz, CD₃OD) δ 5.32 (s, 1H, H-1), 4.41 (d, J = 5.2 Hz, 1H, H-5), 3.98 (d, J = 6.8 Hz, 1H, H-6a), 3.68 (dd, J = 6.9, 5.3 Hz, 1H, H-6b), 3.43 (t, J = 4.0 Hz, 1H, H-3), 2.82 (d, J = 4.0 Hz, 1H, H-2), 1.92 (s, 3H, AcOH), 1.64 – 1.47 (m, 4H, H-4, -CH₂CH₂-), 1.45 – 1.34 (m, 1H, -CH₂CH₂-), 0.97 (t, J = 7.3 Hz, 3H,-CH₃). ¹³C NMR (151 MHz, CD₃OD) δ 104.9 (C-1), 79.1 (C-5), 74.6 (C-6), 72.6 (C-3), 60.7 (C-2), 48.8 (C-4), 37.9 (-CH₂CH₂CH₃), 25.4 (AcOH), 23.9 (-CH₂CH₂CH₃), 16.9 (-CH₂CH₂CH₃). ESI-HRMS: m/z calcd for C₉H₁₈NO₃ [M + H]⁺ 188.1287, found 188.1286.

1,6-Anhydro-2-azido-2,4-dideoxy-4-C-propyl-D-glucopyranose (19).

Stick’s reagent (0.466 g, 2.23 mmol) was added to an ice cold stirred solution of CuSO₄ (0.024 g, 0.15 mmol), triethylamine (0.62 mL, 4.5 mmol), and 18 (0.278 g, 1.12 mmol) in 4:1 MeCN/water (14.8 mL). The reaction mixture was stirred for 1 h before MeCN was removed under vacuum and the residue was diluted with EtOAc, washed with 1N HCl, saturated NaHCO₃ solution, and brine. The organic layer was dried with Na₂SO₄ and concentrated to give 19 as a colorless gum (0.219 g, 91%). [α]_D²³ = −113.2 (c = 1.0, CHCl₃) ¹H NMR (500 MHz, CDCl₃) δ 5.46 (t, J = 1.4 Hz, 1H, H-1), 4.43 (d, J = 5.1 Hz, 1H, H-5), 4.10 (dd, J = 7.0, 0.8 Hz, 1H, H-6a), 3.78 (dd, J = 7.1, 5.1 Hz, 1H, H-6b), 3.65 (tt, J = 2.4, 1.2 Hz, 1H, H-3), 3.47 (t, J = 2.0 Hz, 1H, H-2), 1.72 – 1.56 (m, 3H, H-4, CH₂CH₂CH₃), 1.55 – 1.45 (m, 1H, -CH₂CH₂CH₃), 1.44 – 1.34 (m, 1H, -CH₂CH₂CH₃), 0.97 (t, J = 7.2 Hz, 3H, -CH₂CH₂CH₃). ¹³C NMR (126 MHz, CDCl₃) δ 100.6 (C-1), 75.0 (C-5), 71.2 (C-3), 68.6 (C-6), 63.6 (C-2), 44.3 (C-4), 33.3 (CH₃CH₂CH₂-), 20.5 (CH₃CH₂CH_2-), 14.0 (CH₃CH₂CH_2-).

Methyl 2-azido-2,4-dideoxy-4-C-propyl-D-glucopyranoside (20α and 20β).

Compound 19 (0.219 g, 1.00 mmol) was dissolved in Ac₂O (8.6 mL) followed by addition of TFA (0.86 mL) and stirred under argon for 45 mins. The reaction mixture was then diluted with Et₂O and washed with saturated NaHCO₃ solution and brine, dried with Na₂SO₄, filtered, and concentrated under vacuum. The residue was then dissolved in 10% HCl MeOH solution (7 mL) and heated to reflux for 4.5 h followed by concentration under vacuum to give a 1.5:1 mixture of 20α and 20β (0.174 g, 69%). The resulting residue was subjected to flash column chromatography over silica gel in 45% to 50% ethyl acetate in hexanes, which afforded 20α (55 mg, 22%) and 20β (31 mg, 12%). 20α: [α]_D²³ = 125.4 (c = 1.0, MeOH), ¹H NMR (600 MHz, CD₃OD) δ 4.76 (d, J = 3.5 Hz, 1H, H1), 3.78 (t, J = 10.2 Hz, 1H, H3), 3.74 – 3.68 (m, 1H, H6), 3.62 – 3.54 (m, 2H, H5, H6), 3.37 (s, 3H, OMe), 3.10 (dd, J = 10.0, 3.5 Hz, 1H, H2), 1.62 – 1.50 (m, 2H, H4, CH₂CH₂-), 1.49 – 1.28 (m, 3H, -CH₂CH₂-), 0.91 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (151 MHz, CD₃OD) δ 99.2 (C1), 71.8 (C5), 68.5 (C3), 65.2 (C2), 61.8 (C6), 53.9 (OMe), 43.1 (C4), 28.8 (-CH₂CH₂-), 18.7 (-CH₂CH₂-), 13.7 (-CH₃). ESI-HRMS: m/z calcd for C₁₀H₁₉N₃O₄Na [M + Na]⁺ 268.1273, found 268.1273. 20β: [α]_D²³ = −31.8 (c = 1.0, MeOH), ¹H NMR (600 MHz, CD₃OD) δ 4.13 (d, J = 8.1 Hz, 1H, H1), 3.77 (dd, J = 12.1, 2.1 Hz, 1H, H6), 3.59 (dd, J = 12.1, 5.5 Hz, 1H, H6), 3.52 (s, 3H, OMe), 3.32 – 3.24 (m, 2H, H3, H5), 3.03 (dd, J = 9.5, 8.1 Hz, 1H, H2), 1.58 – 1.25 (m, 5H, H4, -CH₂CH₂-), 0.90 (t, J = 7.2 Hz, 3H, -CH₃). ¹³C NMR (151 MHz, CD₃OD) δ 102.7 (C1), 76.2 (C5), 72.6 (C3), 68.6 (C2), 61.8 (C6), 55.6 (OMe), 42.6 (C4), 28.7 (-CH₂CH₂-), 18.6 (-CH₂CH₂-), 13.7 (-CH₃). ESI-HRMS: m/z calcd for C₁₀H₁₉N₃O₄Na [M + Na]⁺ 268.1273, found 268.1261.

Methyl 2-amino-2,4-dideoxy-4-C-propyl-α-D-glucopyranoside (5).

Compound 20α (13.6 mg, 0.30 mmol) was dissolved in a 1:1 mixture of 1,4-dioxane and 10% aqueous AcOH (0.6 mL) followed by addition of Pd/C (2.9 mg). The reaction mixture was stirred under 50 psi H₂ for 1.5 h followed by filtration over Celite® and lyophilization to obtain 5 as an off white solid (15.4 mg, 99%). [α]_D²³ = 80.5 (c = 0.7, water), ¹H NMR (600 MHz, D₂O) δ 4.84 (d, J = 3.6 Hz, 1H, H1), 3.70 (t, J = 10.6 Hz, 1H, H3), 3.66 (dd, J = 12.3, 2.2 Hz, 1H, H6), 3.62 (ddd, J = 10.9, 5.3, 2.2 Hz, 1H, H5), 3.54 (dd, J = 12.3, 5.3 Hz, 1H, H6), 3.24 (s, 3H, OMe), 3.07 (dd, J = 10.3, 3.6 Hz, 1H, H2), 1.79 (s, 3H, AcOH), 1.49 (tt, J = 10.8, 4.0 Hz, 1H, H4), 1.41 – 1.32 (m, 1H, -CH₂CH₂-), 1.32 – 1.24 (m, 1H, CH₂CH₂-), 1.23 – 1.06 (m, 2H, -CH₂CH₂-), 0.71 (t, J = 7.2 Hz, 3H, -CH₃). ¹³C NMR (151 MHz, D₂O) δ 96.3 (C1), 71.6 (H5), 67.0 (C3), 61.1 (6), 55.3 (C2), 54.9 (OMe), 42.0 (C4), 27.7 (-CH₂CH₂-), 17.8 (CH₂CH₂-), 13.8 (-CH₃). ESI-HRMS: m/z calcd for C₁₀H₂₂NO₄ [M + H]⁺ 220.1549, found 220.1539.

4-C-Allyl-1,6-Anhydro-6S-deuterio-2,4-dideoxy-2-O-p-toluenesulfonyl-D-glucopyranose (6S-²H-15).

Freshly prepared 0.5 M allylMgCl THF solution (16 mL) was added to an ice-cold stirred solution of epoxide 6S-²H-10 (0.590 g 1.97 mmol) and CuI (0.38 g, 0.20 mmol) under argon in THF (20 mL). The reaction mixture was stirred for 11 h followed by addition of further allylMgCl solution (8 mL). After another 17 h the reaction mixture was concentrated under vacuum then diluted with Et₂O and washed with aqueous saturated NH₄Cl solution and brine. The organic layer was dried with Na₂SO₄ and concentrated. The residue was then purified using silica gel flash column chromatography in 35 to 40 % EtOAc in hexanes to give 6S-²H-15 (0.24 g, 36 %). [α]_D²³ = −51.3 (c = 1.0, CH₂Cl₂), ¹H NMR (600 MHz, CDCl₃) δ 7.83 – 7.78 (m, 2H, Ar-H), 7.37 – 7.33 (m, 2H, Ar-H), 5.74 (ddt, J = 16.3, 10.5, 7.1 Hz, 1H, CH₂CHCH₂-), 5.27 (d, J = 1.5 Hz, 1H, H1), 5.13 (dq, J = 6.1, 1.2 Hz, 1H, CH₂CHCH₂-), 5.10 (t, J = 1.3 Hz, 1H, CH₂CHCH₂-), 4.39 (s, 1H, H-5), 4.18 (dt, J = 2.5, 1.2 Hz, 1H, H-2), 3.99 (s, 1H, H-6), 3.69 (tt, J = 2.7, 1.2 Hz, 1H, H-3), 2.45 (s, 3H, ArCH₃), 2.37 – 2.33 (m, 2H, CH₂CHCH₂-), 1.68 (t, J = 7.7 Hz, 1H, H-4). ¹³C NMR (151 MHz, CDCl₃) δ 135.3 (CH₂CHCH₂-), 130.0 (Ar), 127.9 (Ar), 118.0 (CH₂CHCH₂-), 99.7 (C-1), 78.9 (C-2), 74.2 (C-5), 70.0 (C-3), 68.0 (t, J = 23.4 Hz, C-6), 43.0 (C-4), 35.4 (CH₂CHCH₂-), 21.7 (ArCH₃). ESI-HRMS: m/z calcd for C₁₆H₁₉DO₆Na [M + Na]⁺ 364.0941, found 364.0936.

4-C-Allyl-1,6;2,3-bisanhydro-6S-deuterio-4-deoxy-β-D-mannopyranose (6S-²H-16).

NaOMe (0.088 g, 1.63 mmol) was added to an ice-cold stirred solution of compound 6S-²H-15 (0.240 g, 0.70 mmol) in 1:1 mixture of methanol and chloroform (3.5 mL) and the solution was allowed to warm to room temperature. After 2 h the reaction mixture was diluted with Et₂O and washed with aqueous saturated NH₄Cl solution and brine. The organic layer was dried with Na₂SO₄ and concentrated to give 6S-²H-16 (0.118 g, 99 %) as a white waxy solid which was used without further purification. [α]_D²³ = −15.2 (c = 1.0, CH₂Cl₂), ¹H NMR (600 MHz, CDCl₃) δ 5.82 (ddt, J = 17.2, 10.2, 7.0 Hz, 1H, CH₂CHCH₂-), 5.65 (d, J = 3.2, 1H, H-1), 5.16 – 5.10 (m, 2H, CH₂CHCH₂-), 4.23 (s, 1H, H-5), 3.70 (d, J = 1.6 Hz, 1H, H-6), 3.34 (ddd, J = 3.8, 3.1, 0.7 Hz, 1H, H2), 2.93 (dd, J = 4.0, 1.3 Hz, 1H, H-3), 2.32 (tt, J = 7.2, 1.3 Hz, 2H, CH₂CHCH₂-), 2.01 (t, J = 7.6 Hz, 1H, H-4). ¹³C NMR (151 MHz, CDCl₃) δ 135.2 (CH₂CHCH₂-), 117.8 (CH₂CHCH₂-), 98.0 (C-1), 70.9 (C-5), 68.2 (t, J = 23.0 Hz, C-6), 53.8 (C-2), 50.4 (C-3), 39.0 (C-4), 35.1 (CH₂CHCH₂-). ESI-HRMS: m/z calcd for C₉H₁₁DO₃Na [M + Na]⁺ 192.0747, found 192.0746.

4-C-Allyl-1,6-anhydro-6S-deuterio-2-N-benzyl-2,4-dideoxy-D-glucopyranose (6S-²H-17).

A stirred solution of epoxide 6S-²H-17 (0.118 g, 0.427 mmol) in benzylamine (5.0 mL) was heated to 155 ^°C for 1.5 days before concentration under vacuum. The residue was then purified using silica gel column chromatography in 40 % EtOAc and 1% triethylamine in hexanes to give amine 6S-²H-17 (0.166 g, 86 %). [α]_D²³ = −32.9 (c = 1.0, CH2CL2), ¹H NMR (400 MHz, CDCl₃) δ 7.36 – 7.30 (m, 4H, Ar-H), 7.29 – 7.23 (m, 1H, Ar-H), 5.88 – 5.76 (m, 1H, CH₂CHCH₂-), 5.46 (s, 1H, H-1), 5.16 – 5.10 (m, 2H, CH₂CHCH₂-), 4.38 (s, 1H, H-5), 4.03 (s, 1H, H-6), 3.90 (d, J = 13.2 Hz, 1H, PhCH₂N-), 3.86 (d, J = 13.2 Hz, 1H, PhCH₂N-), 3.64 (dq, J = 3.0, 1.4 Hz, 1H, H-3), 2.64 (p, J = 1.2 Hz, 1H, H-2), 2.50 – 2.30 (m, 2H, CH₂CHCH₂-), 1.76 (t, J = 7.7 Hz, 1H, H-4). ¹³C NMR (101 MHz, CDCl₃) δ 139.9 (Ar), 136.1 (CH₂CHCH₂-), 128.5 (Ar), 128.1 (Ar), 127.2 (Ar), 117.5 (CH₂CHCH₂-), 102.6 (C-1), 74.6 (C-5), 70.3 (C-3), 68.2 (t, J = 23.4 Hz, C-6), 62.2 (C-2), 51.7 (PhCH₂N-), 44.6 (C-4), 36.5 (CH₂CHCH₂-). ESI-HRMS: m/z calcd for C₁₆H₂₁DNO₃ [M + H]⁺ 277.1657, found 277.1664.

2-Amino-1,6-anhydro-6S-deuterio-2,4-dideoxy-4-C-propyl-D-glucopyranose acetate salt (6S-²H-18).

Pd/C (10 % w/w, 27 mg) was added to a solution of 6S-²H-17 (0.137 g, 0.50 mmol) in a 1:1 mixture of 10 % aqueous acetic acid and 1,4-dioxane (0.6 mL) followed by pressurization to 40 psi of H₂. The reaction mixture was stirred vigorously for 9 h before filtration through Celite® and concentration under vacuum to give amine 6S-²H-18 (0.122 g, 99%) as the acetate salt, which was used without further purification. [α]_D²³ = −48.6 (c = 4.0, MeOH), ¹H NMR (600 MHz, D₂O) δ 5.41 (s, 1H, H-1), 4.45 (s, 1H, H-5), 3.87 (s, 1H, H-6), 3.47 (t, J = 4.6 Hz, 1H, H-3), 3.01 (dd, J = 4.4, 1.0 Hz, 1H, H-2), 1.75 (s, 3H, AcOH), 1.53 (tdd, J = 6.8, 4.6, 1.5 Hz, 1H, H-4), 1.43 – 1.37 (m, 2H, CH₃CH₂CH₂-), 1.32 (dp, J = 13.3, 7.2 Hz, 1H, CH₃CH₂CH₂-), 1.21 (tdd, J = 15.1, 13.4, 7.1 Hz, 1H, CH₃CH₂CH₂-), 0.76 (t, J = 7.3 Hz, 3H, CH₃CH₂CH₂-). ¹³C NMR (151 MHz, D₂O) δ 98.5 (C-1), 75.3 (C-5), 68.7 (t, J = 23.5 Hz, C-6), 68.3 (C-3), 55.6 (C-2), 43.6 (C4), 33.0 (CH₃CH₂CH₂-), 23.0 (AcOH), 19.3 (CH₃CH₂CH₂-), 13.0 (CH₃CH₂CH₂-). ESI-HRMS: m/z calcd for C₉H₁₇DNO₃ [M + H]⁺ 189.1349, found 189.1357.

1,6-Anhydro-2-azido-6S-deuterio-2,4-dideoxy-4-C-propyl-D-glucopyranose (6S-²H-19).

Stick’s reagent (0.209 g, 1.00 mmol) was added to an ice cold stirred solution of CuSO₄ (11 mg, 0.07 mmol), triethylamine (0.28 mL, 2.0 mmol), and compound 6S-²H-18 (0.123 g, 0.495 mmol) in 4:1 MeCN/water (6.6 mL). The reaction mixture was stirred for 9 h before MeCN was removed under vacuum and the residue was diluted with EtOAc, washed with 1N HCl, saturated NaHCO₃ solution, and brine. The organic layer was dried with Na₂SO₄ and concentrated to give 6S-²H-19 as a colorless gum (0.101 g, 96%). [α]_D²³ = −40.5 (c = 1.0, CH₂Cl₂), ¹H NMR (600 MHz, CDCl₃) δ 5.41 (s, 1H, H-1), 4.38 (s, 1H, H-5), 4.04 (s, 1H, H-6), 3.60 (s, 1H, H-3), 3.42 (s, 1H, H-2), 2.71 (br s, 1H, -OH), 1.66 – 1.51 (m, 3H, H-4, CH₃CH₂CH₂-), 1.50 – 1.41 (m, 1H, CH₃CH₂CH₂-), 1.40 – 1.30 (m, 1H, CH₃CH₂CH₂-), 0.93 (t, J = 7.3 Hz, 3H, CH₃CH₂CH₂-). ¹³C NMR (151 MHz, CDCl₃) δ 100.5 (C-1), 74.9 (C-5), 71.1 (C-3), 68.2 (t, J = 23.2 Hz, C-6), 63.7 (C-2), 44.2 (C-4), 33.3 (CH₃CH₂CH₂-), 20.5 (CH₃CH₂CH₂-), 14.0 (CH₃CH₂CH₂-).

Methyl 2-azido-6S-deuterio-2,4-dideoxy-4-C-propyl-D-glucopyranoside (6S-²H-20α and 6S-²H-20β).

Compound 6S-²H-19 (0.091 g, 0.42 mmol) was dissolved in Ac₂O (4 mL) followed by addition of TFA (0.40 mL) and stirred under argon for 2 h. The reaction mixture was then diluted with Et₂O and washed with saturated NaHCO₃ solution and brine, dried with Na₂SO₄, filtered, and concentrated. The residue was then dissolved in 10 % HCl MeOH solution (3.4 mL) and heated to reflux for 10 h followed by concentration under vacuum to give a 2:1 mixture of 6S-²H-20α and 6S-²H-20β (80.8 mg, 77%). The resulting residue was subjected flash column chromatography over silica gel in 45 % to 50 % ethyl acetate in hexanes which afforded 6S-²H-20α (11.7 mg, 11 %), 6S-²H-20β (9.5 mg, 9 %), and (11.8 mg, 11 %) of a mixture of anomers. ESIHRMS: m/z calcd for C₁₀H₁₈DN₃O₄Na [M + Na]⁺ 269.1336, found 269.1326. 6S-²H-20α: [α]_D²³ = 98.8 (c = 1.0, MeOH), ¹H NMR (600 MHz, CD₃OD) δ 4.76 (d, J = 3.5 Hz, 1H, H-1), 3.78 (t, J = 10.2 Hz, 1H, H-3), 3.58 (dd, J = 10.6, 5.4 Hz, 1H, H-5), 3.55 (d, J = 5.5 Hz, 1H, H-6), 3.37 (s, 3H, -OMe), 3.09 (dd, J = 10.1, 3.5 Hz, 1H, H-2), 1.61 – 1.49 (m, 2H, -CH₂CH₂-, H-4), 1.49 – 1.27 (m, 3H, CH₂CH₂-), 0.91 (t, J = 7.2 Hz, 3H, -CH₃). ¹³C NMR (151 MHz, CD₃OD) δ 99.2 (C-1), 71.8 (C-5), 68.5 (C-3), 65.2 (C-2), 61.5 (t, J = 21.3 Hz, C-6), 53.9 (-OMe), 43.1 (C-4), 28.8 (-CH₂CH₂-), 18.7 (CH₂CH₂-), 13.6 (-CH₃). ESI-HRMS: m/z calcd for C₁₀H₁₈DN₃O₄Na [M + Na]⁺ 269.1336, found 269.1334. 6S-²H-20β: [α]_D²³ = −35.4 (c = 0.003, MeOH), ¹H NMR (600 MHz, CD₃OD) δ 4.13 (d, J = 8.1 Hz, 1H, H-1), 3.57 (d, J = 5.5 Hz, 1H, H-6), 3.52 (s, 3H, -OMe), 3.30 – 3.24 (m, 2H, H-3, H-5), 3.02 (dd, J = 9.5, 8.1 Hz, 1H, H-2), 1.58 – 1.24 (m, 5H, H4, -CH₂CH₂-), 0.90 (t, J = 7.1 Hz, 3H, -CH₃). ¹³ C NMR (151 MHz, CD₃OD) δ 102.7 (C-1), 76.1 (C-5), 72.6 (C-3), 68.6 (C-2), 61.5 (t, J = 21.9 Hz, C-6), 55.6 (-OMe), 42.6 (C-4), 28.6 (-CH₂CH₂-), 18.6 (-CH₂CH₂-), 13.6 (-CH₃).

Methyl 2-amino-6-(S)-deuterio-2,4-dideoxy-4-C-propyl-α-D-glucopyranoside (6S-²H-5).

Compound 6S-²H-20α (9.0 mg, 0.037 mmol) was dissolved in a 1:1 mixture of 1,4-dioxane and 10 % aqueous AcOH (0.6 mL) followed by addition of Pd/C (1.8 mg). The reaction mixture was stirred under 50 psi H₂ for 1 h followed by filtration over Celite® and lyophilization to obtain 6S-²H-5 as an off white solid (10.2 mg, 99 %). [α]_D = 56.4 (c = 0.5, H₂O), H NMR (600 MHz, D₂O) δ 4.84 (d, J = 3.6 Hz, 1H, H1), 3.71 (t, J = 10.5 Hz, 1H, H3), 3.62 (dd, J = 10.9, 5.4 Hz, 1H, H5), 3.53 (d, J = 5.5 Hz, 1H, H6), 3.25 (s, 3H, OMe), 3.08 (dd, J = 10.4, 3.6 Hz, 1H, H2), 1.80 (s, 3H, AcOH), 1.49 (tt, J = 10.8, 4.0 Hz, 1H, H4), 1.40 – 1.33 (m, 1H, -CH₂CH₂-), 1.32 – 1.24 (m, 1H, -CH₂CH₂-), 1.24 – 1.06 (m, 2H, -CH₂CH₂-), 0.72 (t, J = 7.2 Hz, 3H, -CH₃). ¹³C NMR (151 MHz, D₂O) δ 179.9 (AcOH), 96.3 (C1), 71.6 (C5), 67.0 (C3), 60.99 (t, J = 21.0 Hz, C6), 55.3 (C2), 54.9 (OMe), 42.0 (C4), 27.7 (−CH₂CH₂-), 22.3 (AcOH), 17.8 (-CH₂CH₂-), 13.8 (-CH₃). ESI-HRMS: m/z calcd for C₁₀H₂₁DNO₄ [M + H]⁺ 221.1612, found 221.1605.

Highlights:

• Stereospecific Deuterium Labeling

• Assignment of pro-R and pro-S Hydrogens in Side Chain

• Side Chain Conformation