Methylation permethylation

An important general method in characterization of carbohydrates is the classical exhaustive methylation (permethylation). Repeated treatment with a methylating agent such as methyl iodide converts all free OH groups to OCH3 groups. Then, complete acid... 

MeSSiMe3 or PhSSiMe3, Znl2, Bu4N I. In this case the 6-O-methyl ether was cleaved selectively from permethylated glucose. 

Three types of carbohydrate sample have been reported to give data above mass 4000, namely, permethylated polysaccharides, permethylated glycosphingolipids, and naturally acylated forms of a mycobacterial O-methyl-D-glucose polysaccharide. All are hydrophobic, and desorption is probably facilitated by their inability to form strongly hydrogen-bonded aggregates, either with themselves or with the matrix. 

A detailed study of the 0-linked oligosaccharides present on the surface of normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells has been completed. Structures were elucidated by f.a.b.-m.s. after permethylation, and methylation analysis before and after specific exo-glycosidase treatments. Some of the components were shown by f.a.b.-m.s. to be poly(N-acetyllactosaminyl) oligosaccharides, for example, 29. 

Data from f.a.b.-m.s., and also f.d.-m.s., revealed the existence of naturally occurring, large cyclic polysaccharides. The first indication that a molecule may be cyclic comes from its precise molecular-weight determination. Cyclic molecules are 18 mass units less than their linear counterparts. Loss of water may, of course, occur in a number of ways, for example, by dehydration or lactonization, and conclusive evidence for the presence of a cyclic molecule can only be obtained from f.a.b.-m.s. of suitable derivatives, such as the permethyl derivative. Cyclic and dehydrated linear polymers are distinguishable after permethylation, as the cyclic polymer will incorporate one methyl group less than the linear molecule. 

That this difference in (4+2)-cycloaddition behavior most likely has steric origins—the methyl groups in 150 or the derived monoadduct preventing an ortho -addition of two equivalents of the dienophile—is supported by the observation that permethyl[6]radialene 95 is inert even towards the extremely reactive dienophile 4-phenyl-1,2,4-triazolinedione68. 

In this method, Furstner converts N-BOC protected pyrrole to the 2,5-dibromo compound (122) with NBS and this is followed by metalation and carbomethoxylation with t-butyl lithium in THF and subsequent trapping of the metalated species with methyl chloroformate to yield a pyrrole diester (123). Bromination of this diester at positions 3 and 4 with bromine in water followed by Suzuki cross-coupling with 3,4,5-trimethoxyphenyl boronic acid yields the symmetrical tetrasubstituted pyrrole (125). Base-mediated N-alkylation of this pyrrole with 4-methoxyphenethyl bromide produces the key Boger diester (126) and thereby constitutes a relay synthesis of permethyl storniamide A (120). 

A pullulan reported to contain 6% of a-D-(l—>3)-linkages112 was reinvestigated by 13C-n.m.r. spectroscopy of its per-O-methylated derivative,55 which was shown to contain < 3% of this type of unit. Similarly, the permethyl ether of a dextran containing < -D-(l—>4)-, a-D-(l—>6)-, and a-D-(l— 3)-linkages was used for quantitation purposes, but the H-l resonances in the proton-n.m.r. spectrum were better resolved than those of the C-l atom in the 13C-n.m.r. spectrum and, furthermore, the p.m.r. method was superior, as T, and n.O.e. effects were not involved. 

IV. Degradation of the Permethylated Sample, and Analysis of the Partially Methylated Sugars... 

Depolymerization of the permethylated carbohydrate is achieved by hydrolysis with acid. Under these conditions, the amino sugar residues are N-deacetylated, and the aminohexosidic linkages become resistant to hydrolysis. Stellner and coworkers29 showed that, when the acid degradation is conducted in 95% acetic acid, the amino sugar residues are also liberated, and can be analyzed by the methylation technique.29 Therefore, acetolysis followed by acid hydrolysis is now commonly used, as it allows the analysis both of hexose and hexosamine residues. 

The partially methylated monosaccharides obtained on depolymerization of the permethylated sample are preferably analyzed as acetates by g.l.c.-m.s., as shown by Bjomdal and coworkers.41,42 The neutral sugars and the amino sugars obtained in acetolysis-acid hydrolysis are reduced, and acetylated for the analysis, and the amino-hexitol and the neuraminic acid residues are acetylated after methanolysis. Identification with the aid of g.l.c.-m.s. has been described for all of the common components of protein- and lipid-linked glycans and oligosaccharides from animal cells, namely, the neutral sugars,41-43 hexitols,44 hexosamines,29,43,45,46 aminohexitols,31,32 and neuraminic acids.33,34,47... 

Fig. 3.—Mass Fragmentography of Methylated Neuraminic Acids. [Detection at m/e 274 A, disialosyl-lactosylceramide B, the disialosyl ganglioside isolated C, B after treatment with neuraminidase D, the fraction containing the trisialosyl ganglioside. Detection at m/e 330 E, the disialosyl ganglioside F, the trisialosyl fraction. The peaks identified are (1) the permethylated (terminal) N-acetylneuraminic acid, and (2) the 8-O-acetyl (8-O-substituted) derivative of the methylated N-acetylneuraminic acid. Conditions 1% of SE-30, at 240°. Reproduced, by permission, from Ref. 80.]...

For the analysis of fatty acids amide-linked to GlcN(I), several chemical degradation procedures are available. One comprises periodate oxidation of 0-deacylated and 2H-reducedlipid A (lipid A-OH ). Following permethyl-ation, g.l.c.-m.s. analysis revealed a 2-deoxy-1 -deuterio-1,3-di-O-methyl-2-(iV-methyl-3-methoxyacylamido)glycerol derivative in which the amide-finked fatty acid at GlcN(I) was identified as 14 0(3-OH) in B. pertussis (93), and as 16 0(3-OH) and 18 0(3-OH) in R. trifolii 81). 

Methylation analysis Permethylation of intact oligosaccharides Hydrolysis of glycosidic linkages Reduction to monosaccharide alditols Acetylation of free hydroxyl groups previously involved in link-... 

Recently, it was found that the commercially available heptakis(2,3,6-tri-0-methyl)-/i-cyclodextrin (permethylated /1-cyclodextrin, TRIMEB), induced nonequivalence in the NMR spectra, in CD3OD, of enantiomeric mixtures of trisubstituted allenes devoid of polar functional groups, thus affording a simple and general way to determinations of their enantiomeric purity63. 

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