Methylation-GC-MS analysis of arabinofuranose-and galactofuranose-containing structures : rapid synthesis of partially O-methylated alditol acetate standards

Arabinose and galactose were treated with MeOH containing traces of H2SO4 or HCl at 25◦C to give mixtures of their methyl alphaand beta-furanosides, as shown by 1D and 2D nuclear magnetic resonance (NMR). Oxidation of the Me alpha,beta-Araf mixture with NaIO4 preferentially oxidised the beta-isomer, to give pure Me alpha-Araf . Each product was progressively O-methylated using the Purdie reagent (MeI/Ag2O) at 25◦C and resulting mixtures of partially methylated glycosides (PMGs) were rapidly assayed by thin layer chromatography (TLC) first to favour higher yields of mono-O-methyl derivatives and later for products with higher degrees of methylation. The products were converted to complex mixtures of partially O-methylated alditol acetate derivatives (PMAAs) by successive hydrolysis, reduction with NaBD4, and acetylation. These can be used as gas chromatography-mass spectrometry (GC-MS) standards in methylation analysis of complex carbohydrates containing arabinofuranosyl and galactofuranosyl units. Of particular interest were the retention times and electron impact MS of the difficult to prepare alditol acetates of 5,6-Me2Gal, 2,5-Me2Gal, 2,5,6-Me3Gal, 3,5,6-Me3Gal, 5-MeAra, 2,5-Me2Ara, and 3,5-Me2Ara. The relative reactivities of hydroxyl groups for mixtures of Me alphaand Me beta-Galf were HO-2 > HO-3 > HO-6 > HO-5, that of Me alphaand Me beta-Araf HO-2 > HO-3 > HO-5, and that of Me alpha-Araf HO-2 > HO-3 ≥ HO-5.


INTRODUCTION
Arabinose and galactose are widely found in complex carbohydrates and are present in pyranosyl and furanosyl rings in molecules encompassing a great variety of glycosidic linkages with αand βanomeric configurations.Units of Araf are found in polysaccharides of plants, as in gums and free or protein-linked arabinogalactans: to the latter are highly immunogenic to them.The Galfcontaining molecules of bacteria, protozoans, and fungi appear to play different roles, but their influence in the recognition and/or invasion parasitehost have been observed (Suzuki et al. 1997, Levery et al. 1998), probably by their β-Galf -containing epitopes.
Methylation analysis of complex sugars provides information on the substitution position of their glycosidic linkages.In this procedure the molecule can be first completely O-methylated by the methods of Haworth 1915, Kuhn et al. 1955, and Hakomori 1964, but nowadays the most used frequently in our hands is that of Ciucanu and Kerek 1984.The products can be converted by successive hydrolysis to give partially O-methylated aldoses, which are reduced with NaBH 4 , followed by acetylation to provide partially O-methylated alditol acetates (PMAAs), which on GC-MS, have typical retention times (R t ) and electron impact (e.i.) spectra (Jansson et al. 1970).Sometimes, the product suffers from a symmetry problem, for example 2,3di-O-= 3,4-di-O-methylxylitol acetate, but this can be overcome by reduction with NaBD 4 , which introduces deuterium at C-1 (Carpita and Shea 1989).
For the preparation of PMAA standards, there is an option of preparing each one individually or all at once by partial O-methylation of methyl aldosides.For the latter experiment, Haworth (Me 2 SO 4 / NaOH), Purdie (MeI/Ag 2 O), Kuhn (MeI/DMF/ Ag 2 O), and Hakomori (MeI/methylsulfinylmethanide/Me 2 SO) methylations have been carried out.The methylation of methyl α-mannopyranoside was performed out by Handa and Montgomery (1969), using the Haworth, Kuhn, and Hakomori procedures, and by Fournet and Montreuil (1973) with the Kuhn methylation, which formed all possible O-methyl derivatives, except for the mono-Omethyl ones.This was rectified by Fournet et al. (1974) who prepared and converted them to their mono-O-methyl alditol acetates, which were examined by GC-MS, sodium borodeuteride being used in the intermediate reduction step.A similar procedure was used for preparation of 15 O-methyl alditol acetates starting from methyl α-galactopyranoside (Fournet et al. 1978) and the MS data were presented in a tabular form.
Another of the early investigations directed to the preparation of methylation standards for carbohydrate analysis was by Elkin et al. (1975), who partially and fully O-methylated the methyl pyranosides of Rha, Fuc, Ara, Xyl, Man, Gal, and Glc, among others with the Purdie reagent (Purdie and Irvine 1903), but the resulting O-methyl derivatives were only poorly resolved by GLC and MS and not employed.
Although they did not contain the synthesis of PMAAs, the classic publications of Jansson et al. (1970) and Carpita and Shea (1989) contained the retention times (R t 's) and e.i.profiles on GC-MS, with few exceptions, of those that can arise from pyranosyl and furanosyl structures.Lomax and Conchie (1982) used the Purdie reagent to partially and fully methylate the same methyl pyranosides and converted them to PMAAs, which were subjected to GC and although MS was mentioned, no details were included.Methyl arabinofuranoside but not methyl galactofuranoside were investigated.Doares et al. (1991) carried out partial to complete methylation of methyl pyranosides of Rha, Fuc, Ara, Xyl, Man, Gal, and Glc with potassium methylsulphinylmethanide in Me 2 SO/MeI to form PMGs. Their R t s of resulting PMAAs, formed using an intermediate sodium borodeuteride reduction, were recorded and identification was accomplished by referring to the e.i.profiles of Carpita and Shea (1989).A study incorporating synthesis of partially to fully O-methylated PMG derivatives from the same methyl pyranosides, followed by their conversion to all possible PMAAs, with the exception of the 6-O-methyl derivative, and determination of their R t s and e.i.breakdown profiles, was carried out by Sassaki et al. (2005) using the relatively easy to handle Purdie reagent.
The same approach has now been extended to the furanoside series, starting from synthesised methyl arabinofuranosides and methyl galactofura- RAPID SYNTHESIS OF PMMs ARAf AND GALf FOR GC-MS 225 nosides, to finally form all the PMAAs necessary for GC-MS analysis of arabinofuranose-and galactofuranose-containing structures.

Preparation of Methyl Glycofuranosides
Galactose and arabinose were obtained from Sigma-Aldrich, MO, U.S.A and each (500 mg) was stirred in 0.5% w/w MeOH-HCl (100 ml) or 0.5% w/w MeOH-H 2 SO 4 (100 ml) at 25 • C until complete dissolution (for Ara: 6 h, for Gal: 16 h).The solution was neutralised with excess pyridine, evaporated to a small volume, and acetylated with Ac 2 O-pyridine (2 ml; 1:1, v/v) overnight at room temperature.The mixture was added to excess icewater and after 1 h, it was extracted with CHCl 3 , which was evaporated to dryness.The residue was dissolved in MeOH containing NaOMe (5 ml, 200 mM) and after 2 h, the solution was evaporated to dryness, and then treated with an aq.suspension of Dowex 50W-X8 H + , providing after evaporation Me αβ-Araf or Me αβ-Galf .

NMR Analysis of Methyl Arabinoside and Methyl Galactofuranoside Preparations
Each methyl glycofuranoside preparation was deuterium exchanged by repeated D 2 O dissolution, followed by evaporation.1D and 2D 1 H and coupled and decoupled 13 C NMR spectra were obtained using a Bruker Avance DRX-400 spectrometer with a 5 mm inverse probe. 13C-NMR acquisitions were performed using the following parameters: FIDRES-0.8466Hz, AQ-0.5906 s, DW 15.75 s, DE-5.5 µs D1-110 msec, D2-3.4 msec, PL12-17dB (decoupler 1 H), waltz 16 pulse program.Coupled 13 C NMR spectrum was obtained under similar con-ditions using PL12 of 60db.The spectra were obtained in D 2 O either at 30 • C or 70 • C, and chemical shifts measured in relation to Me 4 Si (δ = 0).

Purdie Methylation of Methyl Glycofuranosides
Each glycoside (10 mg) was submitted to vigorous stirring in MeI (1.5 ml), containing Ag 2 O (250 mg) at room temperature over a period of 2 h.Each one gradually dissolved in the suspension and the degree of methylation was monitored at 30 min intervals using aliquots, which were removed and spotted on to TLC plates (solvent: CHCl 3 -EtOH, 9:1 v/v), developed with orcinol-H 2 SO 4 spray -100 • C for 5 min (Skipiski 1975), and the spot intensities measured by Scion Imaging.

Preparation of PMAAs
PMG mixtures were evaporated to dryness and the residue was hydrolysed with M H 2 SO 4 for 8 h at 100 • C. The solution was neutralised (BaCO 3 ), and the mixture containing partially O-methylated aldoses reduced with NaBD 4 (5 mg) for 4 h at room temperature.The solution was neutralised with 50 µl glacial HOAc, dried under reduced pressure, and co-distilled with 100 µl of MeOH at 50 • C, this step being repeated thrice.The product was acetylated with Ac 2 O-pyridine (500 µl; 1:1, v/v) overnight at room temperature.The PMAAs were extracted with CHCl 3 and washed with 2% aq.CuSO 4 and the organic layer containing PMAAs dried at room temperature, and the residue dissolved in acetone before GC-MS analysis.

GC-MS
Each PMAA mixture was dissolved in acetone and examined by GC-MS at a range from m/z 80 to 220, using a Varian GC, Model 3300 coupled to a Finnigan MS with ion trap detector (model ITD 800).The PMAA was applied to an OV-225 fused silica capillary column (Quadrex -30 m × 0.25 mm i.d.), with He as carrier gas.β-Galp (Fig. 2A) and J = 170.2Hz (Fig. 2B) for Me α-Galp.The J values are consistent with those of Chambat et al. (1978), who found C-1 of Me β-Galf to have 172.5 Hz and C-1 of Me α-Galf to have ∼ 175 Hz and Perlin and Casu (1969), who reported 169 Hz for α-Glcp and 160 Hz for β-Glcp.
Methylation of the methyl arabinofuranosides and galactofuranosides with the Purdie reagent over a period of time gave rise to PMGs with progressive increase in their degree of O-methylation.It The Purdie methylation has the advantage of being easier to handle than those of Kuhn et al. (1955) and Doares et al. (1991), since TLC examination could be carried out by directly spotting the reaction mixture at intervals of 30 minutes on to TLC plates, without prior removal of non-volatile solvents.
Each product obtained after 1 and 2 hours reaction time was converted to mixtures of PMAAs via successive hydrolysis, reduction with NaBD 4 , and acetylation.The resulting PMAA mixtures contained the necessary components for analysis of all, with one exception, arabinofuranose-and galacto- furanose-containing structures by GC-MS, by virtue of their typical R t s and e.i.breakdown patterns (Table I and Fig. 4).The e.i.profile of each PMAA was recorded over a range of m/z 80 to 220 and its key ions are represented in Figure 4.Although some of the yields were rather low, the required derivatives of 3,5,6-Me 3 Gal (1.4%), 2,5,6-Me 3 Gal (5.6%), 2,5-Me 2 Gal (1.3%), 3,5-Me 2 Gal (2.1%), and 5,6-Me 2 Gal (5.3%) were obtained, being were detectable on GC-MS.One exception in the rapid synthe-sis was the acetate of 5-O-methylgalactitol.However, its presence in any methylation analysis mixture could be detected since it has an R t identical to that of the 2-O-methyl derivative and would give a similar EI-MS, but with a key ion at m/z 117 instead of 118 (Figure 4, B12).For determination of the relative reactivities of hydroxyl groups in the Me αβ-Galf mixture, attention was paid to the formation of mono-Omethyl derivatives, since further methylation of - CHOH groups vicinal to those of -CHOCH 3 would be more rapid.Consequently we can interpret (Table I) and that in the case of the αβ-mixture of Me-Galf the order is HO-2 > HO-3 > HO-6 > HO-5, which differs from those of Me αand β-Galp, which is HO-3 > HO-2 > HO-4 > HO-6 (Sassaki et al. 2005).

alditol acetates obtained following Purdie methy- lation synthesis after 2 h reaction with methyl furanosides of Ara and Gal.
a Mixture of αβ-anomers obtained on methyl glycosidation; b 5,6-Me 2 Gal eluted before 2,3,5-Me 3 Galf ; c Indentifiable in methylation analysis mixtures with identical R t as that of 2-MeGal, but with a key ion at m/z 117 instead of 118.