A Novel Synthetic Approach to C-Glycosyl-D- and L-Alanines

Abstract

C-Glycosyl-(S)- and (R)-alanines 12a and 12b were synthesized from the known β-C-glycoside 1. The nitrogen function was introduced by aza-Claisen rearrangement of the allylic thiocyanate 7, derived from the corresponding alcohol 6. The absolute configuration of the newly created chiral carbon center (C-3) was assigned by X-ray diffraction analysis of the intermediate 3(S)-isothiocyanato-d-glycero-d-galacto-decose 8a.

Introduction

Glycoconjugates [1] have a significant pharmaceutical potential and intensive research on understanding the functions of these structures in biological events has become a major target for many scientific groups in the recent years.

This increasing interest has been recently turned to modified glycosyl amino acids such as C-glycosyl α-amino acids or fused sugar amino acids [2,3,4,5], in which carbohydrate and amino acid are linked directly to the anomeric centre of the sugar either via a carbon-carbon bond or an entire α-amino acid (glycinyl moiety) [2]. They represent a significant class of building blocks for the construction of C-glycosylated peptides [3,6]. The incorporation of unnatural C-glycosyl amino acids in glycopeptide mimetics may serve for preparing analogues with enhanced resistance to enzymatic hydrolysis but also in the development of glycopeptide-based drugs with interesting pharmacological properties [3,4,5]. For the construction of the C-glycosyl amino acids, several synthetic approaches have been developed [2,5,6,7,8,9,10].

Results and Discussion

We report here a synthetic strategy for the preparation of diastereomerically pure C-glycosyl-alanines 12a, 12b, starting from the know β-C-glycoside 1 [11] and based on the aza-Claisen rearrangement of allylic thiocyanates previously developed in our laboratory [12,13]. The starting β-C-glycoside 1 was synthesized together with its α-anomer 2 via a Wittig-intramolecular Michael-type ring closure sequence from the known 2,3:5,6-di-O-isopropylidene-α-d-mannofuranose and a stabilized ylide (Ph₃P=CHCO₂CH_3, acetonitrile, reflux). Subsequent reduction of 1 with lithium aluminum hydride in dry diethyl ether gave alcohol 3 (88%, Scheme 1). The 2,5-anhydroalditol 3 was subsequently oxidized with o-iodoxybenzoic acid [14] (IBX) in acetonitrile to yield the corresponding aldehyde 4 in 93% yield. The aldehyde 4 was then treated with the stabilized ylide Ph₃P=CHCO₂CH₃ to afford (E)-α,β-unsaturated ester 5 in 87% yield (Scheme 1).

Scheme 1. Synthesis of protected 1-thiocyanato-d-glycero-d-galacto-dec-2(E)-enitol 7.

Scheme 1. Synthesis of protected 1-thiocyanato-d-glycero-d-galacto-dec-2(E)-enitol 7.

Reagents and conditions: (i) LiAlH₄, Et₂O; (ii) IBX, CH₃CN; (iii) Ph₃P=CHCO₂CH₃, CH₂Cl₂; (iv) DIBAH, CH₂Cl₂; (v) MsCl, Et₃N/KSCN, CH₃CN.

Its structure was determined by ¹H- and ¹³C-NMR spectroscopy (for data see Experimental part). The observed coupling constant in 5 (J_3,2 = 15.7 Hz) accounted for a trans-configuration of the double bond. The ester 5 was subjected to reduction with diisobutylaluminum hydride in CH₂Cl₂ to give the allylic alcohol 6 (75%). The required thiocyanate 7 was easily prepared in 76% overall yield by a two-step process of mesylation of alcohol 6 followed by displacement using KSCN in acetonitrile (Scheme 1). The thermal aza-Claisen rearrangement of thiocyanate 7, which was carried out at 90 ^oC in dry n-heptane under a nitrogen atmosphere for 6 h, afforded a mixture of diastereomeric isothiocyanates 8a and 8b (Scheme 2), with high yield (83%) but without selectivity (8a:8b ≈ 1:1, as determined by ¹H- NMR). The microwave (MW) induced rearrangement of thiocyanate 7 realized under the same conditions (90 ^oC, n-heptane, Scheme 2) gave a 1:1 mixture of 8a and 8b in 86% yield, within 2 h. The reaction was performed in closed vessel in a focused microwave reactor (CEM Discover, see Experimental part).

Figure 1. A molecular structure of 8a, showing crystallographic numbering.

Figure 1. A molecular structure of 8a, showing crystallographic numbering.

We have observed that the use of microwave irradiation remarkably accelerated rearrangement of 7→8a, 8b with reduction to one-third of the reaction time, in comparison with the conventional thermal conditions, but it had practically no influence on the selectivity of the rearrangement.

Fortunately, these diastereoisomers were easily separated by chromatography and compound 8a was isolated in crystalline state. In order to determine the absolute configuration of compound 8a, we tried to recrystallize 8a to obtain single crystals for X-ray diffraction analysis. The isothiocyanate 8a crystallized well from a mixture of ether and hexane, forming colorless prisms suitable for X-ray measurements. The crystallographic structure of compound 8a, shown in Figure 1, confirmed that the newly introduced stereocentre at C-3 in 8a possesses S configuration. Consequently, the isothiocyanate 8b must be the 3R-epimer.

Scheme 2. Synthesis of C-glycosyl-(S)- and (R)-alanines.

Scheme 2. Synthesis of C-glycosyl-(S)- and (R)-alanines.

Reagents and conditions: (i) a) n-heptane, 90 °C b) n-heptane, 90 °C, microwaves; (ii) CH₃ONa, CH₃OH; (iii) MNO, CH₃CN; (iv) O₃, -78 °C, Ph₃P; (v) NaClO₂, CH₃CN/t-BuOH/2-methylbut-2-ene = 4:4:1.

Our approach to the build-up of C-glycosyl-(S)- and (R)-alanines 12a and 12b was based on four subsequent steps which were conducted with pure diastereoisomers 8a and 8b. In the first step, the reaction of 8a and 8b with CH₃ONa in dry methanol at room temperature gave a nearly quantitative yield of thiourethanes 9a and 9b, which were used immediately in the next step without purification to avoid problems connected with their possible instability. The treatment of 9a and 9b with mesitonitrile oxide (MNO) [15] in acetonitrile afforded in 85% and 92% yields, respectively, carbamates 10a and 10b (Scheme 2), whose structure was confirmed by ¹H- and ¹³C-NMR spectroscopy (for data see experimental part). Ozonolysis of 10a and 10b at -78 ^oC in methanol afforded the corresponding aldehydes 11a and 11b. After a short pad filtration on silica gel (to remove arising triphenylphosphine oxide), these products were used immediately in the next step due to instability of α-amino aldehydes. The structure of 11a and 11b was determined by ¹H-NMR; the observed chemical shift of aldehyde proton in 11a δ = 9.64 ppm and in 11b δ = 9.52 ppm. The aldehydes 11a and 11b were selectively oxidized to protected C-glycosyl-(S)- and (R)-alanines 12a and 12b (Scheme 2) by treatment with sodium chlorite (NaClO₂) in CH₃CN/tert-butyl alcohol/2-methyl-2-butene at 0 ^oC in 74% and 73% yields, respectively after flash chromatography.

Conclusions

In summary, the novel synthetic approach to the chiral non-racemic C-glycosylated alanines 12a and 12b has been developed. The obtained compounds 12a and 12b differ in the stereochemistry of the newly formed chiral carbon atom (C-2), one having the l-configuration (12a) and the other the d-configuration (12b). These novel amino acids 12a and 12b can be useful in modifying the properties of some glycopeptides by virtue of the presence of a stable anomeric C-C bond instead of the C-O or C-N bond and an additional amino group at C-2.

Experimental

General

All commercially available reagents were used without purification and solvents were dried according to standard procedures. Product purification was carried out using flash chromatography on silica gel (Merck silica gel 60 (0.040-0.063 mm). TLC was run on Merck silica gel 60 F₂₅₄ analytical plates; detection was carried out with either UV, iodine and spraying with a solution of KMnO₄, with subsequent heating. The melting points were determined on the Kofler block, and are uncorrected. Optical rotations were measured in chloroform, using a P3002 Krüss polarimeter and reported as follows: [α]_d²⁵ (c in g/100 mL, solvent). NMR spectra were recorded at room temperature on a FT NMR spectrometer Varian Mercury Plus 400 (¹H at 400.13 MHz and ¹³C at 100.6 MHz) using CDCl₃ as the solvent and TMS as internal reference. For ¹H δ are given in parts per million relative to TMS (0 ppm), for ¹³C relative to CDCl₃ (77 ppm). ¹³C-NMR multiplicities were determined by a DEPT pulse sequence. IR spectra were recorded on a Perkin-Elmer 599 IR spectrometer in CHCl_3. All reactions were performed under nitrogen atmosphere when anhydrous solvents were used. Microwave experiments were conducted using a focused microwave system (CEM Discover). All experiments were performed in glass vessels (10 mL) sealed with a septum. At the end of reaction, the vessels and contents were cooled rapidly using a stream of compressed air.

Methyl 3,6-anhydro-2-deoxy-4,5:7,8-di-O-isopropylidene-d-glycero-d-galacto-octanoate (1) and Methyl 3,6-anhydro-2-deoxy-4,5:7,8-di-O-isopropylidene-d-glycero-d-talo-octanoate (2). 1: ¹H-NMR: δ 1.33 (3H, s, CH₃), 1.37 (3H, s, CH₃), 1.44 (3H, s, CH₃), 1.46 (3H, s, CH₃), 2.73 (1H, dd, J_2,2=16.7 Hz, J_3,2=6.4 Hz, H₂), 2.81 (1H, dd, J_2,2=16.7 Hz, J_3,2=7.3 Hz, H₂), 3.52 (1H, m, H₆), 3.70 (3H, s, OCH₃), 3.94 (1H, m, H₃), 4.04 (1H, dd, J_8,8=8.7 Hz, J_8,7=4.7 Hz, H₈), 4.07 (1H, dd, J_8,8=8.7 Hz, J_8,7=6.1 Hz, H₈), 4.38 (1H, ddd, J_7,6=7.5 Hz, J_8,7=6.1 Hz, J_8,7=4.7 Hz, H₇), 4.76 (2H, m, H₄, H₅); ¹³C- NMR: δ 24.6, 25.2, 25.7, 26.9, 33.3, 51.8, 66.9, 73.1, 77.7, 80.7, 81.0, 81.6, 109.1, 112.6, 171.4. The procedure and [α]_D were consistent with those reported [11]. 2: ¹H-NMR: δ 1.34 (3H, s, CH₃), 1.37 (3H, s, CH₃), 1.45 (3H, s, CH₃), 1.51 (3H, s, CH₃), 2.47 (1H, dd, J_2,2=15.2 Hz, J_3,2=7.1 Hz, H₂), 2.54 (1H, dd, J_2,2=15.2 Hz, J_3,2=7.7 Hz, H₂), 3.71 (3H, s, OCH₃), 3.79 (1H, dd, J_7,6=7.7 Hz, J_6,5=3.7 Hz, H₆), 4.00 (1H, dd, J_8,8=8.7 Hz, J_8,7=4.4 Hz, H₈), 4.08 (1H, dd, J_8,8=8.7 Hz, J_8,7=6.3 Hz, H₈), 4.39 (1H, ddd, J_7,6=7.7 Hz, J_8,7=6.3 Hz, J_8,7=4.4, H₇), 4.49 (1H, dd, J_3,2=7.7 Hz, J_3,2=7.1 Hz, H₃), 4.64 (1H, d, J_5,4=6.0 Hz, H₄), 4.81 (1H, dd, J_5,4=6.0 Hz, J_6,5=3.7 Hz, H₅); ¹³C-NMR: δ 24.7, 25.2, 26.1, 27.0, 36.2, 51.9, 66.9, 73.3, 80.8, 80.9, 80.9, 84.9, 109.2, 112.9, 170.6. The procedure, m.p. and [α]_d were consistent with those reported [11].

3,6-Anhydro-2-deoxy-4,5:7,8-di-O-isopropylidene-d-glycero-d-galacto-octitol (3): LiAlH₄ (0.87 g, 23.0 mmol) was added at 0 ^oC to a solution of ester 1 (3.83 g, 12.1 mmol) in dry Et₂O (70 mL). The reaction mixture was stirred at 0 ^oC for 15 min and then for 45 min at room temperature. The reaction was quenched by careful addition of water (3 mL) and the precipitate was removed by filtration. The filtrate was dried (Na₂SO₄) and concentrated under reduced pressure. The chromatography of the residue on silica gel (hexane-ethyl acetate, 2:1) afforded 3.03 g (88%) of alcohol 3 as a colorless oil; [α]_d²⁵ = -23 (c 0.49, CHCl₃); ¹H-NMR: δ 1.34 (3H, s, CH₃), 1.38 (3H, s, CH₃), 1.44 (3H, s, CH₃), 1.48 (3H, s, CH₃), 1.93 (1H, m, H₂), 2.05 (1H, m, H₂), 3.54 (1H, dd, J_7,6=7.2 Hz, J_6,5=3.7 Hz, H₆), 3.70 (1H, ddd, J_3,2=8.4 Hz, J_3,2=5.1 Hz, J_4,3=3.7 Hz, H₃), 3.80 (2H, m, H₁), 4.05 (1H, dd, J_8,8=8.7 Hz, J_8,7=4.8 Hz, H₈), 4.09 (1H, dd, J_8,8=8.7 Hz, J_8,7=6.1 Hz, H₈), 4.40 (1H, m, H₇), 4.66 (1H, dd, J_5,4=6.1 Hz, J_4,3=3.7 Hz, H₄), 4.76 (1H, dd, J_5,4=6.1 Hz, J_6,5=3.7 Hz, H₅); ¹³C-NMR: δ 24.6, 25.3, 25.7, 26.9, 31.1, 60.4, 66.8, 73.1, 80.5, 80.6, 81.7, 81.7, 109.0, 112.4; Anal. Calcd for C₁₄H₂₄O₆ (288.34): C 58.32, H 8.39; found C 58.54, H 8.66.

3,6-Anhydro-2-deoxy-4,5:7,8-di-O-isopropylidene-d-glycero-d-galacto-octose (4): To a solution of alcohol 3 (3.0 g, 10.5 mmol) in CH₃CN (55 mL) was added IBX (4.41 g, 15.7 mmol). The resulting suspension was heated under reflux for 40 min. Then the reaction was cooled to room temperature and filtered through a medium glass frit. The filter cake was washed with further portions of acetonitrile (2 x 15 mL). The combined filtrates were concentrated under reduced pressure. The chromatography of the residue on silica gel (hexane-ethyl acetate, 3:1) afforded 2.73 g (93%) of aldehyde 4 as a colorless oil; [α]_d²⁵ = -12 (c 0.68, CHCl₃); ¹H-NMR: δ 1.32 (3H, s, CH₃), 1.38 (3H, s, CH₃), 1.45 (3H, s, CH₃), 1.46 (3H, s, CH₃), 2.87-2.89 (2H, m, H₂), 3.55 (1H, dd, J_7,6=7.4 Hz, J_6,5=3.3 Hz, H₆), 3.99 (1H, ddd, J_3,2=6.4 Hz, J_3,2=6.4 Hz, J_4,3=3.3 Hz, H₃), 4.03 (1H, dd, J_8,8=8.7 Hz, J_8,7=4.7 Hz, H₈), 4.08 (1H, dd, J_8,8=8.7 Hz, J_8,7=6.2 Hz, H₈), 4.39 (1H, ddd, J_7,6=7.4 Hz, J_8,7=6.2 Hz, J_8,7=4.7 Hz, H₇), 4.76 (1H, dd, J_5,4=6.1 Hz, J_4,3=3.3 Hz, H₄), 4.79 (1H, dd, J_5,4=6.1 Hz, J_6,5=3.3 Hz, H₅), 9.81 (1H, t, J=1.3 Hz, CHO); ¹³C-NMR: δ 24.5, 25.2, 25.6, 26.9, 42.8, 66.8, 73.0, 76.6, 80.6, 81.0, 81.6, 109.1, 112.6, 199.9; Anal. Calcd for C₁₄H₂₂O₆ (286.33): C 58.73, H 7.74; found C 58.49, H 7.51.

Methyl 5,8-anhydro-2,3,4-trideoxy-6,7:9,10-di-O-isopropylidene-d-glycero-d-galacto-dec-2(E)-enoate (5): [(Methoxycarbonyl)methylidene]triphenylphosphorane (3.82 g, 11.4 mmol) was added to a solution of aldehyde 4 (2.73 g, 9.5 mmol) in dry CH₂Cl₂ (25 mL). The reaction mixture was stirred for 1.5 h at room temperature. The solvent was removed under reduced pressure and the residue was purified by chromatography on silica gel (hexane-ethyl acetate, 5:1) to afford 2.79 g (87%) of (E)-5 as a colorless oil; [α]_d²⁵ = -12 (c 0.34, CHCl₃); ¹H-NMR: δ 1.33 (3H, s, CH₃), 1.38 (3H, s, CH₃), 1.44 (3H, s, CH₃), 1.48 (3H, s, CH₃), 2.61 (2H, m, H₄), 3.50 (1H, dd, J_9,8=7.5 Hz, J_8,7=3.6 Hz, H₈), 3.59 (1H, ddd, J_5,4=6.8 Hz, J_5,4=6.8 Hz, J_6,5=3.6 Hz, H₅), 3.73 (3H, s, OCH₃), 4.05 (1H, dd, J_10,10=8.7 Hz, J_10,9=4.8 Hz, H₁₀), 4.09 (1H, dd, J_10,10=8.7 Hz, J_10,9=6.1 Hz, H₁₀), 4.39 (1H, ddd, J_9,8=7.5 Hz, J_10,9=6.1 Hz, J_10,9=4.8 Hz, H₉), 4.63 (1H, dd, J_7,6=6.1 Hz, J_6,5=3.6 Hz, H₆), 4.76 (1H, dd, J_7,6=6.1 Hz, J_8,7=3.6 Hz, H₇), 5.94 (1H, ddd, J_3,2=15.7 Hz, J_4,2=1.5 Hz, J_4,2=1.5 Hz, H₂), 6.99 (1H, ddd, J_3,2=15.7 Hz, J_4,3=7.1 Hz, J_4,3=7.1 Hz, H₃); ¹³C-NMR: δ 24.6, 25.3, 25.7, 26.9, 31.4, 51.5, 66.9, 73.1, 80.3, 80.7, 81.1, 81.7, 109.1, 112.6, 123.0, 144.9, 166.8; Anal. Calcd for C₁₇H₂₆O₇ (342.39): C 59.64, H 7.65; found C 59.73, H 7.79.

5,8-Anhydro-2,3,4-trideoxy-6,7:9,10-di-O-isopropylidene-d-glycero-d-galacto-dec-2(E)-enitol (6): To a solution of ester 5 (2.79 g, 8.15 mmol) in dry CH₂Cl₂ (37 mL) diisobutylaluminum hydride (24.6 mL of 1.2 M toluene solution) was added dropwise at -15 °C. The resulting mixture was stirred for 45 min at the same temperature and then quenched with methanol (6.2 mL). The mixture was allowed to warm to room temperature and poured into 30% aqueous K/Na tartrate (123 mL). After stirring for 30 min, the product was extracted with CH₂Cl₂ (3 x 37 mL). The combined organic layers were dried (Na₂SO₄) and the solvent evaporated under reduced pressure. Chromatography of the residue on silica gel (hexane-ethyl acetate, 1:1) afforded 2.34 g (91%) of allylic alcohol 6 as a colorless oil; [α]_d²⁵ = +22 (c 0.28, CHCl₃); ¹H-NMR: δ 1.34 (3H, s, CH₃), 1.38 (3H, s, CH₃), 1.45 (3H, s, CH₃), 1.48 (3H, s, CH₃), 2.47 (2H, m, H₄), 3.48 (1H, m, H₈), 3.52 (1H, m, H₅), 4.05 (1H, dd, J_10,10=8.7 Hz, J_10,9=4.8 Hz, H₁₀), 4.08 (1H, dd, J_10,10=8.7 Hz, J_10,9=6.0 Hz, H₁₀), 4.11 (2H, m, H₁), 4.40 (1H, ddd, J_9,8=7.4 Hz, J_10,9=6.0 Hz, J_10,9=4.8 Hz, H₉), 4.62 (1H, dd, J_7,6=6.1 Hz, J_6,5=3.6 Hz, H₆), 4.74 (1H, dd, J_7,6=6.1 Hz, J_8,7=3.7 Hz, H₇), 5.76 (2H, m, H₂, H₃); ¹³C-NMR: δ 24.7, 25.3, 25.8, 26.9, 31.2, 63.6, 66.9, 73.2, 80.7, 81.1, 81.5, 81.6, 109.0, 112.4, 128.2, 131.5; Anal. Calcd for C₁₆H₂₆O₆ (314.38): C 61.13, H 8.34; found C 61.32, H 8.50.

5,8-Anhydro-1,2,3,4-tetradeoxy-6,7:9,10-di-O-isopropylidene-1-thiocyanato-d-glycero-d-galacto-dec-2(E)-enitol (7): To a solution of alcohol 6 (2.34 g, 7.44 mmol) in dry dichloromethane (26 mL) were added triethylamine (1.55 mL, 11.17 mmol) and CH₃SO₂Cl (0.69 mL, 8.93 mmol) at 0 ^oC. The mixture was stirred at 0 ^oC for 15 min and then further 45 min at room temperature. The solvent was evaporated under reduced pressure. The residue was diluted with diethyl ether (40 mL) and the solid was removed by filtration. The solvent was evaporated to afford the crude mesylate which was used in the subsequent reaction directly without further purification. To the crude mesylate dissolved in CH₃CN (26 mL), KSCN (1.09 g, 11.17 mmol) was added. After stirring at room temperature for 1 h, the solvent was evaporated. The residue was diluted with diethyl ether (40 mL) and the solid was removed by filtration. Evaporation of the solvent and chromatography of the residue (hexane-ethyl acetate, 5:1) afforded 2.0 g (76%) of thiocyanate 7 as white crystals; m.p. 81–82 °C; [α]_d²⁵ = +23 (c 0.28, CHCl₃); ¹H-NMR: δ 1.34 (3H, s, CH₃), 1.38 (3H, s, CH₃), 1.44 (3H, s, CH₃), 1.48 (3H, s, CH₃), 2.50-2.54 (2H, m, H₄), 3.49 (1H, dd, J_9,8=7.5 Hz, J_8,7=3.7 Hz, H₈), 3.52-3.55 (3H, m, 2 x H₁, H₅), 4.05 (1H, dd, J_10,10=8.7 Hz, J_10,9=4.8 Hz, H₁₀), 4.09 (1H, dd, J_10,10=8.7 Hz, J_10,9=6.1 Hz, H₁₀), 4.39 (1H, ddd, J_9,8=7.5 Hz, J_10,9= 6.1 Hz, J_10,9=4.8 Hz, H₉), 4.67 (1H, dd, J_7,6=6.1 Hz, J_6,5=3.6 Hz, H₆), 4.74 (1H, dd, J_7,6=6.1 Hz, J_8,7=3.7 Hz, H₇), 5.70 (1H, m, H₂), 5.87 (1H, m, H₃); ¹³C-NMR: δ 24.6, 25.3, 25.8, 26.9, 31.4, 36.3, 66.9, 73.1, 80.7 81.0, 81.2, 81.6, 109.0, 111.9, 112.4, 124.9, 134.0; Anal. Calcd for C₁₇H₂₅NO₅S (355.46): C 57.44, H 7.09, N 3.94; found C 57.61, H 7.28, N 4.04.

5,8-Anhydro-1,2,3,4-tetradeoxy-6,7:9,10-di-O-isopropylidene-3(S)-isothiocyanato-d-glycero-d-galacto-dec-1-enitol (8a) and 5,8-Anhydro-1,2,3,4-tetradeoxy-6,7:9,10-di-O-isopropylidene-3(R)-isothiocyanato-d-glycero-d-galacto-dec-1-enitol (8b): Conventional method for the preparation of 8a, 8b: A solution of thiocyanate 7 (1.80 g, 5.06 mmol) in dry n-heptane (30 mL) was heated at 90 ^oC for 6 h under nitrogen atmosphere. The solvent was evaporated under reduced pressure. The chromatography of the residue on silica gel (hexane-ethyl acetate, 9:1) afforded isothiocyanates 8a (0.75 g, 42%) and 8b (0.73 g, 41%).

Microwave-assisted synthesis of 8a, 8b: The (E)-thiocyanate 7 (20 mg, 0.056 mmol) was weighed in a 10 ml glass pressure microwave tube equipped with a magnetic stirrer bar. Dry n-heptane (0.4 mL) was added, the tube was closed with a silicon septum and the reaction mixture was subjected to microwave irradiation for 2 h (power: 150 W, temperature: 90 ^oC, pressure: 12 bar). The reaction mixture was allowed to cool to room temperature and transferred into a round bottom flask. The solvent was evaporated under reduced pressure. The chromatography of the residue on silica gel (hexane-ethyl acetate, 9:1) gave 0.16 mg (86%) of isothiocyanates 8a, 8b. Compound 8a: white crystals; m.p. 54-56 ^oC; [α]_d²⁵ = -19 (c 0.27, CHCl₃); υ_max (liquid film) 2033 (NCS) cm^-1; ¹H-NMR: δ 1.33 (3H, s, CH₃), 1.38 (3H, s, CH₃), 1.45 (3H, s, CH₃), 1.46 (3H, s, CH₃), 1.93 (1H, m, H₄), 2.07 (1H, ddd, J_4,4=14.3 Hz, J_5,4=9.3 Hz, J_4,3=3.7 Hz, H₄), 3.54 (1H, dd, J_9,8=7.6 Hz, J_8,7=3.7 Hz, H₈), 3.71 (1H, ddd, J_5,4=9.3 Hz, J_6,5=3.7 Hz, J_5,4=3.5 Hz, H₅), 4.06 (1H, dd, J_10,10=8.7 Hz, J_10,9=4.8 Hz, H₁₀), 4.09 (1H, dd, J_10,10=8.7 Hz, J_10,9=6.0 Hz, H₁₀), 4.39 (1H, ddd, J_9,8=7.6 Hz, J_10,9=6.0 Hz, J_10,9=4.8 Hz, H₉), 4.45 (1H, m, H₃), 4.64 (1H, dd, J_7,6=6.1 Hz, J_6,5=3.7 Hz, H₆), 4.78 (1H, dd, J_7,6=6.1 Hz, J_8,7=3.7 Hz, H₇), 5.24 (1H, dd, J_2,1cis=10.2 Hz, J_3,1cis=1.4 Hz, H_1cis), 5.39 (1H, dd, J_2,1trans=16.9 Hz, J_3,1trans=1.6 Hz, H_1trans), 5.83 (1H, ddd, J_2,1trans=16.9 Hz, J_2,1cis=10.2 Hz, J_3,2=5.4 Hz, H₂); ¹³C-NMR: δ 24.5, 25.3, 25.7, 26.9, 35.3, 57.5, 66.9, 73.0, 78.1, 80.8, 81.5, 81.7, 109.2, 112.5, 116.5, 132.9, 135.2; Anal. Calcd for C₁₇H₂₅NO₅S (355.46): C 57.44, H 7.09, N 3.94; found C 57.25, H 7.20, N 4.11. Compound 8b: a colorless oil; [α]_d²⁵ = -10 (c 0.41, CHCl₃); υ_max (liquid film) 2020 (NCS) cm^-1; ¹H-NMR: δ 1.33 (3H, s, CH₃), 1.38 (3H, s, CH₃), 1.44 (3H, s, CH₃), 1.47 (3H, s, CH₃), 2.03 (1H, m, H₄), 2.16 (1H, m, H₄), 3.49 (1H, dd, J_9,8=7.6 Hz, J_8,7=3.6 Hz, H₈), 3.61 (1H, ddd, J_5,4=7.4 Hz, J_5,4=6.1 Hz, J_6,5=3.7 Hz, H₅), 4.05 (1H, dd, J_10,10=8.8 Hz, J_10,9=4.6 Hz, H₁₀), 4.09 (1H, dd, J_10,10=8.8 Hz, J_10,9=6.2 Hz, H₁₀), 4.36–4.41 (2H, m, H₃, H₉), 4.64 (1H, dd, J_7,6=6.1 Hz, J_6,5=3.7 Hz, H₆), 4.77 (1H, dd, J_7,6=6.1 Hz, J_8,7=3.6 Hz, H₇), 5.25 (1H, ddd, J_2,1´cis=10.2 Hz, J_3,1cis=1.1 Hz, J_1cis,1trans=0.5 Hz, H_1cis), 5.36 (1H, ddd, J_2,1trans=16.8 Hz, J_3,1trans=1.3 Hz, J_1cis,1trans=0.5 Hz, H_1trans), 5.81 (1H, ddd, J_2,1trans=16.8 Hz, J_2,1cis=10.2 Hz, J_3,2=6.0 Hz, H₂); ¹³C-NMR: δ 24.6, 25.2, 25.7, 27.0, 34.8, 57.3, 66.9, 73.0, 78.4, 80.7, 81.0, 81.7, 109.1, 112.6, 117.2, 132.7, 134.8; Anal. Calcd for C₁₇H₂₅NO₅S (355.46): C 57.44, H 7.09, N 3.94; found C 57.62, H 6.94, N 3.88.

5,8-Anhydro-1,2,3,4-tetradeoxy-6,7:9,10-di-O-isopropylidene-3(S)-(methoxycarbonylamino)-d-glycero-d-galacto-dec-1-enitol (10a): To a solution of isothiocyanate 8a (0.54 g, 1.52 mmol) in dry methanol (15 mL) was added sodium methoxide (90 mg, 1.67 mmol). The reaction mixture was stirred for 3 h at room temperature under nitrogen atmosphere. The solvent was evaporated and the residue was partitioned between CH₂Cl₂ (25 mL) and water (7 mL). The organic layer was dried (Na₂SO₄) and the solvent was evaporated under reduced pressure to provide the crude thiourethane 9a which was used in the subsequent reaction directly without further purification. To a solution of 9a (436 mg, 1.12 mmol) in dry acetonitrile (10.8 mL) was added mesitonitrile oxide (218 mg, 1.35 mmol). The mixture was stirred at room temperature for 2 h under nitrogen atmosphere. The solvent was evaporated under reduced pressure. The chromatography of the residue (hexane-ethyl acetate, 3:1) gave 0.35 g (85%) of 10a as a colorless oil; ¹H-NMR: δ 1.33 (3H, s, CH₃), 1.38 (3H, s, CH₃), 1.45 (3H, s, CH₃), 1.47 (3H, s, CH₃), 1.88 (1H, m, H₄), 2.04 (1H, m, H₄), 3.51 (1H, dd, J_9,8=6.7 Hz, J_8,7=3.7 Hz, H₈), 3.63 (1H, m, H₅), 3.66 (3H, s, CH₃O), 4.05 (1H, dd, J_10,10=8.7 Hz, J_10,9=4.8 Hz, H₁₀), 4.10 (1H, dd, J_10,10=8.7 Hz, J_10,9=6.2 Hz, H₁₀), 4.36-4.41 (2H, m, H₃, H₉), 4.59 (1H, dd, J_7,6=6.1 Hz, J_6,5=3.7 Hz, H₆), 4.72 (1H, dd, J_7,6=6.1 Hz, J_8,7=3.7 Hz, H₇), 5.13 (1H, d, J_2,1cis=10.4 Hz, H_1cis), 5.20 (1H, d, J_2,1trans=17.1 Hz, H_1trans), 5.38 (1H, d, J_3,NH=6.4 Hz, NH), 5.80 (1H, ddd, J_2,1trans=17.1 Hz, J_2,1cis=10.4 Hz, J_3,2=5.1 Hz, H₂); ¹³C- NMR: δ 24.5, 25.3, 25.7, 26.9, 32.6, 51.0, 52.0, 66.8, 73.1, 79.1, 80.5, 81.7, 81.8, 109.1, 112.5, 114.7, 138.2, 156.4; Anal. Calcd for C₁₈H₂₉NO₇ (371.43): C 58.21, H 7.87, N 3.77; found C 58.46, H 7.61, N 3.92.

5,8-Anhydro-1,2,3,4-tetradeoxy-6,7:9,10-di-O-isopropylidene-3(R)-(methoxycarbonylamino)-d-glycero-d-galacto-dec-1-enitol (10b): To a solution of isothiocyanate 8b (416 mg, 1.17 mmol) in dry methanol (11.6 mL) was added sodium methoxide (69.5 mg, 1.29 mmol). The reaction mixture was stirred for 4 h at room temperature under nitrogen atmosphere. The solvent was evaporated and the residue was partitioned between CH₂Cl₂ (20 mL) and water (6 mL). The organic layer was dried (Na₂SO₄). The solvent was evaporated under reduced pressure to give the crude thiourethane 9b which was used in the subsequent reaction directly without further purification. To a solution of 9b (288 mg, 0.74 mmol) in dry acetonitrile (7 mL) was added mesitonitrile oxide (144 mg, 0.89 mmol). The mixture was stirred at room temperature for 2 h under nitrogen atmosphere. The solvent was evaporated under reduced pressure and the chromatography of the residue (hexane-ethyl acetate, 3:1) afforded 0.25 g (92%) of 10b as a colorless oil; ¹H-NMR: δ 1.33 (3H, s, CH₃), 1.37 (3H, s, CH₃), 1.44 (3H, s, CH₃), 1.47 (3H, s, CH₃), 1.84 (1H, ddd, J_4,4=14.2 Hz, J_4,3=9.5 Hz, J_5,4=6.5 Hz, H₄), 2.00 (1H, m, H₄), 3.47 (1H, dd, J_9,8=7.5 Hz, J_8,7=3.6 Hz, H₈), 3.59 (1H, ddd, J_5,4=6.5 Hz, J_5,4=6.5 Hz, J_6,5=3.6, H₅), 3.67 (3H, s, CH₃O), 4.03 (1H, dd, J_10,10=8.7 Hz, J_10,9=4.6 Hz, H₁₀), 4.08 (1H, dd, J_10,10=8.7 Hz, J_10,9=6.2 Hz, H₁₀), 4.29 (1H, m, H₃), 4.38 (1H, ddd, J_9,8=7.5 Hz, J_10,9=6.2 Hz, J_10,9=4.6 Hz, H₉), 4.67 (1H, dd, J_7,6=6.1 Hz, J_6,5=3.6 Hz, H₆), 4.73 (1H, dd, J_7,6=6.1 Hz, J_8,7=3.6 Hz, H₇), 4.93 (1H, m, NH), 5.12 (1H, dd, J_2,1cis=10.4 Hz, J_3,1cis=1.3 Hz, H_1cis), 5.21 (1H, dd, J_2,1trans=17.0 Hz, J_3,1trans=1.2 Hz, H_1trans), 5.80 (1H, ddd, J_2,1trans=17.0 Hz, J_2,1cis=10.4 Hz, J_3,2=5.6 Hz, H₂); ¹³C-NMR: δ 24.6, 25.2, 25.8, 26.9, 33.7, 51.4, 52.1, 66.9, 73.1, 79.5, 80.6, 81.4, 81.7, 109.1, 112.4, 114.8, 138.5, 156.6; Anal. Calcd for C₁₈H₂₉NO₇ (371.43): C 58.21, H 7.87, N 3.77; found C 58.05, H 7.54, N 3.53.

4,7-Anhydro-2,3-dideoxy-5,6:8,9-di-O-isopropylidene-2(S)-(methoxycarbonylamino)-d-glycero-d-galacto-nononic acid (12a): A solution of 10a (0.28 g, 0.76 mmol) in methanol (28 mL) was cooled to -78 ^oC. Ozone was then passed through the solution under vigorous stirring. The maximum time for the ozone treatment was 30 min. This resulted in the formation of a bluish solution. Dry nitrogen was passed through the cold solution in order to remove excess ozone. Ph₃P (0.20 g, 0.76 mmol) and CH₂Cl₂ (11 mL) were added and the solution was allowed to warm up to room temperature while stirring was continued for 1.5 h. The solvent was removed under reduced pressure and the chromatography of the residue (hexane-ethyl acetate, 2:1) afforded 0.25 g (87%) of 11a as a colorless oil which was used immediately in the next step. ¹H-NMR: δ 1.32 (3H, s, CH₃), 1.38 (3H, s, CH₃), 1.44 (3H, s, CH₃), 1.47 (3H, s, CH₃), 2.07-2.17 (1H, m, H₃), 2.24 (1H, m, H₃), 3.54 (1H, dd, J_8,7=6.8 Hz, J_7,6=3.7 Hz, H₇), 3.62-3.68 (1H, m, H₄), 3.70 (3H, s, CH₃O), 4.03 (1H, dd, J_9,9=8.7 Hz, J_9,8=4.8 Hz, H₉), 4.08 (1H, dd, J_9,9=8.7 Hz, J_9,8=6.3 Hz, H₉), 4.32-4.40 (2H, m, H₂, H₈), 4.60 (1H, dd, J_6,5=6.1 Hz, J_5,4=3.7 Hz, H₅), 4.74 (1H, dd, J_6,5=6.1 Hz, J_7,6=3.7 Hz, H₆), 5.77 (1H, m, NH), 9.64 (1H, bs, CH=O). A solution of NaClO₂ (80%, 0.57 g, 6.3 mmol) and NaH₂PO₄ (0.71 g, 4.5 mmol) in 3.8 mL of water was added dropwise to a solution of aldehyde 11a (0.25 g, 0.68 mmol) in acetonitrile/tert-butyl alcohol/2-methyl-2-butene (15 mL, 4:4:1) at 0 ^oC over 5 min and stirred at the same temperature for 35 min. The reaction mixture was poured into brine (12 mL) and extracted with ethyl acetate (2 x 25 mL). The combined organic layers were dried (Na₂SO₄). The solvent was removed under reduced pressure and chromatography of the residue on silica gel (hexane-ethyl acetate, 1:2) afforded 0.20 g (74%) of carboxylic acid 12a as a colorless oil; [α]_d²⁵ = -33 (c 0.12, CHCl₃); ¹H-NMR: δ 1.31 (3H, s, CH₃), 1.37 (3H, s, CH₃), 1.44 (3H, s, CH₃), 1.45 (1H, s, CH₃), 2.18-2.26 (2H, m, H₃), 3.51 (1H, dd, J_8,7=6.8 Hz, J_7,6=3.6 Hz, H₇), 3.62-3.66 (4H, m, H₄, CH₃O), 4.02-4.08 (2H, m, H₉), 4.37 (1H, ddd, J_8,7=6.8 Hz, J_9,8=6.1 Hz, J_9,8=4.8 Hz, H₈), 4.48 (1H, m, H₂), 4.59 (1H, dd, J_6,5=6.1 Hz, J_5,4=3.7 Hz, H₅), 4.70 (1H, dd, J_6,5=6.1 Hz, J_7,6=3.6 Hz, H₆), 5.76 (1H, d, J_2,NH=7.9 Hz, NH); ¹³C-NMR: δ 24.6, 25.3, 25.7, 26.9, 30.9, 52.1, 52.3, 66.7, 73.1, 80.0, 2 x 81.0, 81.6, 109.2, 112.5, 156.7, 171.2; Anal. Calcd for C₁₇H₂₇NO₉ (389.40): C 52.44, H 6.99, N 3.60; found C 52.10, H 7.00, N 3.41.

4,7-Anhydro-2,3-dideoxy-5,6:8,9-di-O-isopropylidene-2(R)-(methoxycarbonylamino)-d-glycero-d-galacto-nononic acid (12b): A solution of 10b (185 mg, 0.498 mmol) in methanol (18 mL) was cooled to -78 ^oC. Ozone was then passed through the solution under vigorous stirring. The maximum time for the ozone treatment was 30 min. This resulted in the formation of a bluish solution. Dry nitrogen was passed through the cold solution in order to remove excess ozone. Ph₃P (0.13 g, 0.498 mmol) and CH₂Cl₂ (7 mL) were added and the solution was allowed to warm up to room temperature while stirring was continued for 1.5 h. The solvent was removed under reduced pressure and the chromatography of the residue (hexane-ethyl acetate, 2:1) gave 0.16 g (88%) of aldehyde 11b as a colorless oil which was used immediately in the next step. ¹H-NMR: δ 1.33 (3H, s, CH₃), 1.36 (3H, s, CH₃), 1.43 (3H, s, CH₃), 1.48 (3H, s, CH₃), 2.11-2.21 (1H, m, H₃), 2.26-2.36 (1H, m, H₃), 3.48 (1H, dd, J_8,7=7.0 Hz, J_7,6=3.6 Hz, H₇), 3.62-3.67 (1H, m, H₄), 3.70 (3H, s, CH₃O), 3.94 (1H, dd, J_9,9=8.7 Hz, J_9,8=4.8 Hz, H₉), 4.04 (1H, dd, J_9,9=8.7 Hz, J_9,8=6.2 Hz, H₉), 4.32-4.36 (2H, m, H₂, H₈), 4.68 (1H, dd, J_6,5=6.1 Hz, J_5,4= 3.5 Hz, H₅), 4.74 (1H, dd, J_6,5=6.1 Hz, J_7,6=3.6 Hz, H₆), 5.63 (1H, m, NH), 9.52 (1H, bs, CH=O). A solution of NaClO₂ (80%, 0.37 g, 4.1 mmol) and NaH₂PO₄ (0.46 g, 2.9 mmol) in 2.5 mL of water was added dropwise to a solution of aldehyde 11b (0.16 g, 0.43 mmol) in acetonitrile/tert-butyl alcohol/2-methyl-2-butene (10 mL, 4:4:1) at 0 ^oC over 5 min and stirred at the same temperature for 45 min. The reaction mixture was poured into brine (8 mL) and extracted with ethyl acetate (2 x 16 mL). The combined organic layers were dried (Na₂SO₄). The solvent was removed under reduced pressure and the chromatography of the residue on silica gel (hexane-ethyl acetate, 1:2) gave 0.12 g (73%) of carboxylic acid 12b as a white viscous oil; [α]_d²⁵ = -7 (c 0.49, CHCl₃); ¹H-NMR: δ 1.32 (3H, s, CH₃), 1.35 (3H, s, CH₃), 1.42 (3H, s, CH₃), 1.45 (3H, s, CH₃), 2.05-2.13 (1H, m, H₃), 2.29-2.36 (1H, m, H₃), 3.44 (1H, m, H₇), 3.60-3.63 (1H, m, H₄), 3.65 (3H, s, CH₃O), 3.98-4.04 (2H, m, H₉), 4.34 (1H, ddd, J_8,7=7.3 Hz, J_9,8=6.2 Hz, J_9,8=5.4 Hz, H₈), 4.39-4.40 (1H, m, H₂), 4.70 (2H, m, H₅, H₆), 5.61 (1H, d, J_2,NH=7.1 Hz, NH); ¹³C-NMR: δ 24.7, 25.3, 25.8, 26.9, 31.4, 51.9, 52.1, 66.8, 73.1, 80.7, 2 x 81.4, 81.6, 109.0, 112.4, 156.7, 173.4; Anal. Calcd for C₁₇H₂₇NO₉ (389.40): C 52.44, H 6.99, N 3.60; found C 52.68, H 7.03, N 3.82.

Crystal structure determination of 8a

A single crystal of 8a suitable for X-ray structure analysis was prepared by growth under slow evaporation of a mixture of diethyl ether and hexane at room temperature in a form of the colorless prisms. The intensities were collected at 295 K on a diffractometer Oxford Diffraction Gemini R CCD using Mo-Kα radiation (0.71073 Å). Details of crystal data, data collection and refinement parameters are given in Table 1.

Table 1. Crystal and experimental data for compound 8a

Empirical formula	C17 H25 N1O5 S1
Formula weight	355.46
Temperature, T (K)	100 K
Wavelength, λ (Å)	0.71093
Crystal system	Trigonal
Space group	P3121
Unit cell dimensions(Å)	a = 10.4026(2) γ = 120°
	b = 10.4026(2)
	c = 30.8227(5)
Unit-cell volume, V (Å3)	2889(1)
Formula units per unit cell, Z	6
Calculated density, Dx (g cm-3)	1.233
Absorption coefficient, μ (mm-1)	0.192
F(000)	744
Crystal size (mm)	0.630 x 0.085 x 0.050
Theta range for data collection, (°)	3.00 - 29.47
Index ranges	-12 ≤ h ≤ 12, -12 ≤ k ≤ 12, -38 ≤ l ≤ 38
Independent reflections [I>2σ(I)]	3483 (Rint = 0.048)
Absorption correction	Empiric Psi-scan
Max. and min. transmission	0.927 and 0.966
Refinement method	Full-matrix least-squares on F2
Data / parameters	3922 / 263
Goodness-of-fit (all)	1.09
Final R indices [I>2σ(I)]	R1 = 0.0480, wR2 = 0.0119
R indices (all data)	R1 = 0.0527, wR2 = 0.1212
Largest diff. peak and hole	0.33 and -0.37 (e Å-3)

Table 1. Crystal and experimental data for compound 8a

Empirical formula	C17 H25 N1O5 S1
Formula weight	355.46
Temperature, T (K)	100 K
Wavelength, λ (Å)	0.71093
Crystal system	Trigonal
Space group	P3121
Unit cell dimensions(Å)	a = 10.4026(2) γ = 120°
	b = 10.4026(2)
	c = 30.8227(5)
Unit-cell volume, V (Å3)	2889(1)
Formula units per unit cell, Z	6
Calculated density, Dx (g cm-3)	1.233
Absorption coefficient, μ (mm-1)	0.192
F(000)	744
Crystal size (mm)	0.630 x 0.085 x 0.050
Theta range for data collection, (°)	3.00 - 29.47
Index ranges	-12 ≤ h ≤ 12, -12 ≤ k ≤ 12, -38 ≤ l ≤ 38
Independent reflections [I>2σ(I)]	3483 (Rint = 0.048)
Absorption correction	Empiric Psi-scan
Max. and min. transmission	0.927 and 0.966
Refinement method	Full-matrix least-squares on F2
Data / parameters	3922 / 263
Goodness-of-fit (all)	1.09
Final R indices [I>2σ(I)]	R1 = 0.0480, wR2 = 0.0119
R indices (all data)	R1 = 0.0527, wR2 = 0.1212
Largest diff. peak and hole	0.33 and -0.37 (e Å-3)

The structure was solved by direct methods [16]. All non-hydrogen atoms were refined anisotropically by full-matrix least-squares calculations based on F2 [16]. The hydrogen atoms bonded to nitrogen atoms were found in a difference Fourier map and their coordinates and isotropic thermal parameters have been refined freely. All other hydrogen atoms were included in calculated positions as riding atoms, with SHELXL97 [16] defaults. PLATON [17] program was used for structure analysis and molecular and crystal structure drawings preparation. The following crystal structure has been deposited at the Cambridge Crystallographic Data Centre and allocated the deposition number CCDC 697340. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.