Synthesis of the methyl ethers of methyl 6-deoxy-3-C -methyl- α - L - talopyranoside and - α - L -mannopyranoside. Examination of the conformation and chromatographic properties of the compounds

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Introduction
Two of the mycobacteria (M.tuberculosis and M. leprae) are specifically responsible for human pathogenic infections.On the contrary, the members of the serocomplex of Mycobacterium avium belong to the opportunistic human mycobacteria [1][2][3] .Of the known cell-surface glycopeptide (GLP)-type antigens, the serovariant 19 of M. avium possesses the most complicated structure 4 with respect both to the sugar component, and the immunodeterminant pentasaccharide composition (Fig. 1.).The chemists 4 who isolated and studied the structure searched for the configuration of carbon C-4 of the branched-monosaccharide unit (i.e.6-deoxy-3-C-methyl-2,4-di-O-methyl-L-manno-or talopyranose), which is the penultimate monosaccharide moiety.However, they could not determine this configuration unequivocally.In order to synthesize the pentasaccharide, we prepared methyl 6-deoxy-3-C-methyl-4-Omethyl-α-L-manno-(22) and -talopyranoside (4), as well as methyl 6-deoxy-3-C-methyl-2,4-di-O-methyl-α-L-manno-(23) and -talopyranoside (5) 5 .In this way it was convincingly established that the sugar next to the last in the pentasaccharide has a mannose-configuration.Upon glycosylation of compounds 5 and 23 with 2,6-di-O-acetyl-3,4-di-O-methyl-D-glucopyranosyl trichloroacetimidate the conformation of the aglycone 5 changed from 1 C 4 (L) to 4 C 1 (L), no such change occurred in the case of 23, and the disaccharide also possessed the 1 C 4 conformation.We observed that the chromatographic behaviour (TLC, HPLC) of the similarly substituted mannoand talopyranose derivatives are rather different.For the discovery and explanation of these anomalies we decided to synthesize each of the methyl ethers of the two sugars, and also to study and compare their conformational and chromatographic properties.

Synthesis
The synthesis of the representatives of the two series was carried out in a similar way.The only exceptions were the selective alkylation reactions (methylation, benzylation).The required selective alkylations of the talopyranosides was achieved by sodium hydride-mediated reactions, whereas in the case of the mannopyranosides phase transfer-type catalysts were found most suitable.Methyl 6-deoxy-2,3-O-isopropylidene-3-C-methyl-α-L-talopyranoside (1) 5 , and -α-Lmannopyranoside (19) 5 were methylated by means of the Brimacombe method 6 to obtain the

Scheme 1
Hydrolysis of the isopropylidene group of 3 and 21 with 60% acetic acid and a small quantity of trifluoroacetic acid in dichloromethane at room temperature furnished methyl 4-O-benzyl-6deoxy-3-C-methyl-α-L-talopyranoside (6) and 4-O-benzyl-6-deoxy-3-C-methyl-α-Lmannopyranoside (24), respectively.Since both of these two sugars (i.e. 6 and 24) contain a less reactive tertiary hydroxyl group (OH-3) besides the secondary OH-2, the selective methylation of the secondary hydroxyl group in both sugars was considered.However, the two sugars possessed rather different reactivities.Methylation of 6 gave 60% of 12.At the same time, the regioselectivity in the case of 24 was very low under similar conditions, and a complex productmixture was obtained.Finally, under phase-transfer-conditions 7 methylation of 24 led to the isolation of 30 in 59% yield.Removal of the benzyl ether functions was accomplished by catalytic hydrogenation to afford methyl 6-deoxy-3-C-methyl-2-O-methyl-α-L-talopyranoside (13) and methyl 6-deoxy-3-C-methyl-4-O-methyl-α-L-mannopyranoside (31).The previous sugar (13) is the methyl glycoside of the naturally occurring saccharide vinelose 8,9 .Regioselective benzylation of the sugars 6 and 24 at OH-2 resulted in a similar result as observed for the methylation: in the case of 6 benzylation was carried out in the presence of sodium hydride to give 9, but this reaction for 24 proceeded only under phase transfer catalysis to yield 27. The free OH-3 group of 9 and 27 was methylated in the presence of sodium hydride, and the corresponding methyl ethers 10 and 28, respectively, were obtained with 80% yield.Following hydrogenolysis of the benzyl groups, methyl 6-deoxy-3-C-methyl-3-O-methyl-α-Ltalopyranoside (11) and methyl 6-deoxy-3-C-methyl-3-O-methyl-α-L-mannopyranoside (29) were isolated.
The synthesis of methyl 6-deoxy-3-C-methyl-3,4-di-O-methyl-α-L-talopyranoside (16), and of the -α-L-mannopyranoside (34) was done by means of the regioselective benzylation of 4 and 22; compounds 4 and 22 were benzylated in the presence of NaH, and under phase transfer condition, respectively, to obtain the saccharides 14 and 32.Methylation of the OH-3 groups led to 15 and 33, which were debenzylated to obtain the final products 16 and 34.

Conformational studies
Each representative of both of the synthesized carbohydrate series possesses two separated spin systems, and by the determination of the 3 J 1,2 and 3 J 4,5 coupling costants the conformation of all of the prepared compounds can be studied.All of the 6-deoxy-3-C-methyl-α-L-mannopyranoside derivatives adopt the 1 C 4 conformation: 3 J 1,2 ≤ 2 Hz and 3 J 4,5 ≥ 9 Hz.The situation in the case of the 6-deoxy-3-C-methyl-α-L-talopyranosides is completely different: for the fully substituted glycosides (7, 10, 15, 18 and 38) the 4 C 1 conformation is predominant, as proved by the coupling constants ( 3 J 1,2 ≈ 5-6 Hz and 3 J 4,5 ≈ 4-4.5 Hz).However, it has to be noted that all of the mono-and disubstituted talopyranoside derivatives exist exclusively in the 1 C 4 conformation.We suppose that the steric hindrance is responsible for the change of the conformation and it appears only when each of the three OH groups is substituted.
The fact that in the anomeric proton signals in the 1 H NMR spectra of the tetraglycosyl alditol 4 , isolated by the degradation of the antigen of the serovariant 19, three ca. 1 Hz and one 7.75 Hz coupling constants could be determined, and that this last coupling constant (7.75 Hz) was assigned to the 3,4-di-O-methyl-β-D-glucuronic acid moiety, proves that the building block next to the last one possesses L-manno (thus, not L-talo) configuration.

Chromatographic studies
In the case of carbohydrates carrying the same substituents, the chromatographic mobility is determined by the following structural features: -the substitution degree/pattern, -the steric position of the hydroxyl groups, -the primary, secondary, or tertiary order/character of the OH groups, -the shape of the molecule, and -the role of the hydrogen-bonds.In our studies the thin layer chromatographic and HPLC examination of the methyl ethers in the 6-deoxy-3-C-methyl-α-L-mannopyranosyl (22, 23, 26, 29, 31, 34 and 36) and -α-Ltalopyranosyl series (4, 5, 8, 11, 13, 16 and 18), as well as of the disaccharides 38 and 39 was accomplished.In the case of the mannopyranoside derivatives the first three of the above molecular/structural features play a decisive role in the chromatographic behaviour.Considering the substitution degree/pattern , the mono-, di-, and trimethyl ethers can be readily and securely separated (compounds 22 and 26 possess identical R f values).The equatorial OH groups are more polar (OH-4 > OH-3 > OH-2) than the axial ones, and the order: primary, secondary and tertiary leads to a decreasing polarity.A same behaviour, very similar to the TLC properties, was observed with the HPLC method (LiChros-pherSi-60).The chromatographic mobilities of the methyl ethers of 6-deoxy-3-C-methyl-α-Ltalopyranosides are completely different as compared to those of the L-mannopyranosides.The most polar is the 2,4-di-O-methyl ether (R f : 0.14), the following is the 2,3,4-tri-O-methyl glycoside (R f : 0.16), and the most apolar is the 3,4-di-O-methyl derivative (R f : 0.47).The R f and R t values are summarized in Table 3.In the talopyranoside series the substitution degree does not influence the chromatographic properties.The steric position of the hydroxyl groups cannot be correlated either with the R f or the R t values, and no observable role of the order of the OH functions was experienced.

Table 3. Chromatographic properties (TLC, HPLC) of the synthesized methyl ethers
The low R f and R t values found for the tri-O-substituted talopyranosides is presumably in connection with the shape of the molecule.This assumption is substantiated, not only by the totally different mobilities of the two tri-O-methyl ethers (talo: R f : 0.16; manno: R f : 0.52), but also by the fact that despite the presence of the bulky O-3 substituent (2,6-di-O-acetyl-3,4-di-Omethyl-β-D-glucopyranosyl), the mobilities of the two disaccharides are rather different (R f : 0.23 and 0.37).Another example is that the chromatographic mobilities of the 4-O-benzyl-2,3-di-O-methyl ether 7, possessing 4 C 1 conformation, [R f : 0.40 (dichloromethane-acetone 95:5) and R f : 0.30 (hexane-ethyl acetate 7:3)] obtained by the methylation of the 4-O-benzyl ether 6 [R f : 0.37 (dichloromethane-acetone 95:5) and R f : 0.30 (hexane ethyl acetate 7:3)] are practically the same, and independent of the methylation of the two free OH groups.
Upon chromatography there is a competition between the intramolecular H-bonds existing in the molecule, and those of the intermolecular H-bonds ensuring binding to the adsorbent and elution by the solvent.The role of the H-bonds influencing the chemical reactions of saccharides has been extensively studied 11 .By examining the above models, in this work we can only qualitatively analyse and consider that in the case of the talopyranoside derivatives, (i) the hydrogen bondings could be stronger, and (ii) there is a possibility for adopting various resonance-like boundary states.In such a process the presence of OH-3 in free or substituted form may be a determining factor.
In the case of the tri-O-substituted saccharide ethers no H-bondings are in operation, but due to the change of the conformation, the equatorial steric orientation of the OCH 3 groups may ensure a favourable fit to the surface of silicagel.

Conclusions
By the synthesis and the studies of the conformational properties of the title compounds, it was established that the penultimate monosaccharide unit in the pentasaccharide-type antigen of the serovariant 19 of M. avium is 6-deoxy-3-C-methyl-2,4-di-O-methyl-α-L-mannopyranose.For the explanation of the chromatographic mobilities of the prepared sugars, the number, the steric position, and order of the OH groups, as well as the role of the conformation of the pyranosyl skeleton, and of the hydrogen bondings were evaluated.

Experimental Section
General Procedures.Optical rotations were measured at room temperature with a Perkin-Elmer 241 automatic polarimeter in CHCl 3 .TLC was performed on Kieselgel 60 F254 (Merck) with detection by charring with 50 % aqueous sulfuric acid.Column chromatography was performed on Silica gel 60 (Merck 63-200 mesh).For HPLC a Merck Hitachi liquid chromatograph equipped with a refractive index detector L-7490, programmable autosampler L-7250, pump L-7100, interface L-7000 was used.The samples were separated on a LiChrospher Si-60 (250*4 mm, 5 µm) by an isocratic system with n-hexane/ethyl acetate 50:50 or 40:60 eluent.The flowrate was 1 mL/min at 25 ˚C.Detection time was 20 minutes.The quality of the n-hexane and ethyl acetate were HPLC grade.Quantitative results are given on the basis of retention time.The 1 H (200, 360 and 500 MHz) and 13 C NMR (50.3, 90.54, 125.76 MHz) spectra were recorded with Bruker WP-200 SY, Bruker AM-360 and Bruker DRX-500 spectrometers.Internal references: TMS (0.00 ppm for 1H), CDCl 3 (77.00ppm for 13 C for organic solutions).Elemental analyses were performed at the analytical laboratories in Debrecen.Abbreviations: Ac = acetyl, Bn = benzyl, Me = methyl.
. Sodium hydride (91 mg) was added to a solution of 24 (214 mg, 0.77 mmol) in dry DMF (5 ml) at 0 °C, the mixture was stirred for 1 h, then methyl iodide (282 µl) was added dropwise.After 1 h stirring at 0 °C, methanol (0.5 ml) was added, the mixture was concentrated.The residue was diluted with CH 2 Cl 2 , extracted with water, dried and concentrated in vacuo.The resulting syrup was purified on a column of silica gel (hexane/ethyl acetate 7:3) to yield 25 (152 mg, 64 %) as a colourless syrup.