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

CHEMICAL SCIENCES

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

Guilherme L. Sassaki; Marcello Iacomini; Philip A.J. Gorin^*

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. Postal 19046, 81531-990 Curitiba, PR, Brasil

^{Correspondence}

ABSTRACT

Arabinose and galactose were treated with MeOH containing traces of H₂SO₄ or HCl at 25ºC to give mixtures of their methyl alpha- and 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 25ºC 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 alpha- and Me beta-Galf were HO-2 > HO-3 > HO-6 > HO-5, that of Me alpha and Me beta-Araf HO-2 > HO-3 > HO-5, and that of Me alpha-Araf HO-2 > HO-3 > HO-5.

Key words: partially O-methylated alditol acetates, GC-MS standards, Purdie methylation, OH reactivity, NMR.

RESUMO

Arabinose e galactose foram tratadas com metanol anidro contendo quantidades catalíticas de H₂SO₄ e HCl a 25ºC visando a formação de suas respectivas misturas de alpha- e beta-metil- glicofuranose, as quais foram confirmadas por ressonância magnética nuclear (RMN) unidimensional e bidimensional. A mistura de alpha, beta-Me-Araf foi oxidada com NaIO₄, dando origem somente à Me-alpha-Araf, mostrando que o isômero beta é oxidado preferencialmente. Cada derivado furanosídico foi metilado pelo reagente de Purdie (Ag₂O/MeI) a 25ºC, dando origem a misturas de metil glicofuranosídeos parcialmente metilados (Me-GFP), os quais tiveram seus respectivos graus de metilação monitorados por cromatografia em camada delgada (CCD), que variaram inicialmente em altos rendimentos de derivados mono metilados de Me-GFP a derivados com alto grau de metilação. Os produtos da reação foram convertidos a derivados alditol acetato parcialmente metilados (AAPM), após hidrólise, seguida de redução com NaBD₄ e posterior acetilação. Os derivados obtidos podem ser utilizados como padrões de AAPM em CG-EM para auxiliar na análise de metilação de carboidratos complexos que possam conter unidades de arabinofuranose e galactofuranose. Os derivados AAPM de 6-MeGal, 5,6-Me₂Gal, 2,5-Me₂Gal, 2,5,6-Me₃Gal, 3,5,6-Me₃Gal, 5-MeAra, 2,5-Me₂Ara e 3,5-Me₂Ara mostraram espectros de eletroimpacto e tempos de retenção de maior interesse pois são de difícil síntese. A avaliação da reatividades dos grupos hidroxílicos nesta metilação mostrou que a mistura de alpha, beta-Me-Galf reage preferencialmente em HO-2 > HO-3 > HO-6 > HO-5, a mistura de alpha, beta-Me-Araf em HO-2 > HO-3 > HO-5, alpha-Me-Araf em HO-2 > HO-3 > HO-5.

Palavras-chave: alditol acetatos parcialmente metilados, padrões p/ CG-EM, metilação de Purdie, reatividade grupos hidroxílicos, RMN.

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 a- and b-anomeric configurations. Units of Araf are found in polysaccharides of plants, as in gums and freeor protein-linked arabinogalactans: to the lattermany functions have been assigned, including plant growth and development, as well as cell proliferation, expansion and death (Svetek et al. 1999). On the other hand, galactofuranosyl-containing molecules do not occur in green plants, but are widespread in glycolipids, proteoglycans, cell-wall polysaccharides, and exopolysaccharides (EPS) of fungi and yeasts (Barreto-Bergter and Gorin 1983, Levery et al. 1998, Sassaki et al. 2002, Jones et al. 2004). Galf units are also present in macromolecules of bacteria and protozoans, but not in mammals, which are highly immunogenic to them. The Galf-containing molecules of bacteria, protozoans, and fungi appear to play different roles, but their influence in the recognition and/or invasion parasite-host have been observed (Suzuki et al. 1997, Levery et al. 1998), probably by their b-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 NaBH₄, 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 2,3-di-O- = 3,4-di-O-methylxylitol acetate, but this can be overcome by reduction with NaBD₄, 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 (Me₂SO₄/ NaOH), Purdie (MeI/Ag₂O), Kuhn (MeI/DMF/Ag₂O), and Hakomori (MeI/methylsulfinylmethanide/Me₂SO) methylations have been carried out. The methylation of methyl a-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-O-methyl 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 a-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 Me₂SO/MeI to form PMGs. Their R_ts 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_ts 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 galactofuranosides, to finally form all the PMAAs necessary for GC-MS analysis of arabinofuranose- and galactofuranose-containing structures.

MATERIALS AND METHODS

PREPATAION 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-H₂SO₄ (100 ml) at 25ºC 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 Ac₂O-pyridine (2 ml; 1:1, v/v) overnight at room temperature. The mixture was added to excess ice-water and after 1 h, it was extracted with CHCl₃, 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 ab-Araf or Me ab-Galf.

Me ab-Araf (113 mg) with treated with 2 molar equivalents of NaIO₄ (295 mg) in H₂O (10 ml) for 30 min at 25ºC. The solution was treated with a mixture of Amberlite IR-120 (H⁺) and IR-400 (OH^–), the latter to remove dialdehyde, the suspension filtered, and the filtrate freeze dried to give Me a-Araf (19 mg).

NMR ANALYSIS OF METHYL ARABINOSE AND METHYL GALACTOFURANOSIDE PREPARATIONS

Each methyl glycofuranoside preparation was deuterium exchanged by repeated D₂O dissolution,followed by evaporation. 1D and 2D ¹H and coupled and decoupled ¹³C 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- 0.8466 Hz, AQ- 0.5906 s, DW 15.75 s, DE- 5.5 µs D1- 110 msec, D2- 3.4 msec, PL12-17dB (decoupler ¹H), waltz 16 pulse program. Coupled ¹³C NMR spectrum was obtained under similar conditions using PL12 of 60db. The spectra were obtained in D₂O either at 30ºC or 70ºC, and chemical shifts measured in relation to Me₄Si (d = 0).

PURDIE METHYLATION OF METHYL GLYCOFURANOSIDES

Each glycoside (10 mg) was submitted to vigorous stirring in MeI (1.5 ml), containing Ag₂O (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: CHCl₃-EtOH, 9:1 v/v), developed with orcinol-H₂SO₄ spray - 100ºC for 5 min (Skipiski 1975), and the spot intensities measured by Scion Imaging.

PREPARATION OF PMAAS

PMG mixtures were evaporated to dryness andthe residue was hydrolysed with M H₂SO₄ for 8 h at 100ºC. The solution was neutralised (BaCO₃), and the mixture containing partially O-methylated aldoses reduced with NaBD₄ (5 mg) for 4 h atroom temperature. The solution was neutralised with 50 µl glacial HOAc, dried under reduced pressure, and co-distilled with 100 µl of MeOH at 50ºC, this step being repeated thrice. The product was acetylated with Ac₂O-pyridine (500 µl; 1:1, v/v) overnight at room temperature. The PMAAs were extracted with CHCl₃ and washed with 2% aq.CuSO₄ 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. Conditions: electron impact at 70 eV; injector temp.: 250ºC; initial temp.: 50ºC (1 min); temp. program: 40ºC min^–1 to 220ºC, maintained for 25 min.

RESULTS AND DISCUSSION

Arabinose and galactose were each converted tomixtures of methyl glycofuranosides using MeOH containing traces of H₂SO₄ 25ºC. The acid was removed by neutralisation with pryridine, followed by acetylation and deacetylation, ¹³C-NMR examination of the Me ab-Gal product at 30ºC in D₂O showed that they had furanosyl rings, as two typical low field signals at d 108.1 and 102.0 (Fig. 1A) were present (Gorin and Mazurek 1975) in a ratio of ~ 1.95 : 1. Its ¹³C, ¹H-coupled spectrum contained C-1 doublets centred at d 108.1 with J = 172.4 Hz) from Me b-Galf, whereas that centred at d 102.0 had J = 176.8 Hz (Fig. 1B). These values differed from those of a standard mixture of Me b-Galp of Me a-Galp, which gave respective signals at d 103.5 (Fig. 2A), J = 162.1 Hz (Fig. 2B) and d 99.1 for Me b-Galp (Fig. 2A) and J = 170.2 Hz (Fig. 2B) for Me a-Galp. The J values are consistent with those of Chambat et al. (1978), who found C-1 of Me b-Galf to have 172.5 Hz and C-1 of Me a-Galf to have ~ 175 Hz and Perlin and Casu (1969), who reported 169 Hz for a-Glcp and 160 Hz for b-Glcp.

Using a similar procedure, the decoupled and coupled ¹³C-NMR spectra of the methyl glycosidic mixture formed from arabinose contained C-1 signals for Me a-Araf and Me b-Araf respectively at d 108.7 (Fig. 3A) and J = 173.1 Hz (Fig. 3B) and 102.6 (Fig. 3A) and J = 173.1 Hz (Fig. 3B) in a ratio of ~ 1.5 : 1 (Fig. 3).

When the Me ab-Araf mixture was treated with an excess of sodium periodate over a short period of time, the product after removal of dialdehyde with resin only gave the signals of Me a-Araf at d 108.7 (C-1), 84.2 (C-4), 81.1 (C-2), 76.9 (C-3), 61.7 (C-5), and 55.6 OCH₃, showing that the Me b-Araf was preferentially oxidised.

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 was found using TLC that after 1 hour Me ab-Araf gave rise mainly to mono-O- (R_f 0.31-0.50) and di-O-methyl derivatives (R_f 0.52-0.62) and after two hours the tri-O-methyl derivative (R_f 0.75) was formed. The derivatives of Me ab-Galf were mainly mono-O- (R_f 0.31-0.50) and di-O-methyl (R_f 0.52-0.62) after 1 hour and tri-O-methyl (R_f 0.64) and tetra-O-methyl (R_f 0.75) after two hours. 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 NaBD₄, and acetylation. The resulting PMAA mixtures contained the necessary components for analysis of all, with one exception, arabinofuranose- and galactofuranose-containing structures by GC-MS, by virtue of their typical R_ts 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-Me₃Gal (1.4%), 2,5,6-Me₃Gal (5.6%), 2,5-Me₂Gal (1.3%), 3,5-Me₂Gal (2.1%), and 5,6-Me₂Gal (5.3%) were obtained, being were detectable on GC-MS. One exception in the rapid synthesis 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 ab-Galf mixture, attention was paid to the formation of mono-O-methyl derivatives, since further methylation of -CHOH groups vicinal to those of -CHOCH₃ would be more rapid. Consequently we can interpret (Table I) and that in the case of the ab-mixture of Me-Galf the order is HO-2 > HO-3 > HO-6 > HO-5, which differs from those of Me a- and b-Galp, which is HO-3 > HO-2 > HO-4 > HO-6 (Sassaki et al. 2005).

The relative reactivities for the Me ab-Araf mixture was HO-2 > HO-3 > HO-5, and for Me a-Araf, they were not markedly different, with HO-2 > HO-3 > HO-5 (Table I).

ACKNOWLEDGMENTS

The authors wish to thank the Brazilian agencies Financiadora de Estudos e Projetos (FINEP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação Araucária, State of Paraná for financial support.

Manuscript received on December 12, 2004; accepted for publication on February 24, 2005; contributed by PHILIP A.J. GORIN*

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*

Member Academia Brasileira de Ciências

Publication Dates

• Publication in this collection
  09 May 2005
• Date of issue
  June 2005

History

• Received
  12 Dec 2004
• Accepted
  24 Feb 2005