A Novel Synthetic Approach to C-Glycosyl-d- and l-Alanines

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.

OPEN ACCESS
This increasing interest has been recently turned to modified glycosyl amino acids such as Cglycosyl 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 αamino 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].
Its structure was determined by 1 H-and 13 C-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 CH 2 Cl 2 to give the allylic alcohol 6 (75%). The required thiocyanate 7 was easily prepared in 76% overall yield by a twostep 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 o C in dry nheptane 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 1 H-NMR). The microwave (MW) induced rearrangement of thiocyanate 7 realized under the same conditions (90 o C, 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). 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. 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 CH 3 ONa 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 1 H-and 13 C-NMR spectroscopy (for data see experimental part). Ozonolysis of 10a and 10b at -78 o C 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 1 H-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 (NaClO 2 ) in CH 3 CN/tert-butyl alcohol/2-methyl-2-butene at 0 o C 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 Dconfiguration (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.

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 F 254 analytical plates; detection was carried out with either UV, iodine and spraying with a solution of KMnO 4 , 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] D 25 (c in g/100 mL, solvent). NMR spectra were recorded at room temperature on a FT NMR spectrometer Varian Mercury Plus 400 ( 1 H at 400.13 MHz and 13 C at 100.6 MHz) using CDCl 3 as the solvent and TMS as internal reference. For 1 H δ are given in parts per million relative to TMS (0 ppm), for 13 C relative to CDCl 3 (77 ppm). 13 C-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.   (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 CH 3

5,8-Anhydro-1,2,3,4-tetradeoxy-6,7:9,10-di-O-isopropylidene-3(S)-(methoxycarbonylamino)-Dglycero-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 CH 2 Cl 2 (25 mL) and water (7 mL). The organic layer was dried (Na 2 SO 4 ) 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 ( 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. 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 data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.