Methylation technique

III. Labeling Studies of Glycophorin A by Way OF THE Reductive, [ C]Methylation Technique... 

In order further to determine whether the reductive, [ C]methylation technique possibly perturbs the structure of these glycoproteins and glycopeptides, natural-abundance C-n.m.r.-spectral data were obtained for unmodified, and for reductively [ C]methylated, compound 10 (for struc-... 

Table I shows the results of periodate oxidation and methylation applied to amylopectins from various sources. If the percentage of amylose in a whole starch is known, the length of unit chain of the amylopectin component can be calculated from the results of periodate oxidation of the whole starch (see Table II). The length of unit chain appears to depend on the botanical species, but not the variety, from which the starch wras isolated. In the case of tapioca and corn amylopectins, sub-fractionation of these by precipitation with methanol, followed by periodate oxidation, showed the sub-fractions had the same degree of branching as the original amylopectins.71 The action of periodate on whole starches and amylopectins is now so well established and accurate, that it may well completely supersede the methylation technique for pure starches, in view of its many advantages.

METHYLATION TECHNIQUES IN THE STRUCTURAL ANALYSIS OF GLYCOPROTEINS AND GLYCOLIPIDS... 

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. 

It has been observed that the neuraminic acid linkages can also be conveniently analyzed by the methylation technique.33,34 Some important fragments, the origins of which were confirmed by deuteriumlabelling experiments,33 are shown for two common, N-acetylneura-minic acid derivatives, namely, the terminal sugar derivative 10 and the 8-O-substituted N-acetylneuraminic acid (11). 

Tipson devoted most of his years in Levene s laboratory accomplishing seminal work on the components of nucleic acids. To determine the ring forms of the ribose component of the ribonucleosides he applied Haworth s methylation technique and established the furanoid structure for the sugar in adenosine, guanosine, uridine, and thymidine. He showed that formation of a monotrityl ether is not a reliable proof for the presence of a primary alcohol group in a nucleoside, whereas a tosyl ester that is readily displaced by iodide affords clear evidence that the ester is at the 5-position of the pentofuranose. Acetonation of ribonucleosides was shown to give the 2, 3 -C -isopropyl-idene derivatives, which were to become extensively used in nucleoside and nucleotide chemistry, and were utilized by Tipson in the first chemical preparation of a ribonucleotide, inosinic acid. 

The methylation technique consists of total methylation of the oligosaccharide, followed by hydrolysis to the partially methylated monosaccharide units, and determination of the unmethylated positions in each unit, these being the positions of linkage between units. 

The analysis of monosaccharide mixtures as the permethylated derivatives was proposed early in the application of gas-liquid chromatography to carbohydrates, but the method has now been superseded by more convenient procedures.230,231 There are, however, situations in which this method is useful, such as during a structural study of a polysaccharide by the methylation technique. The mixture of partially methylated monosaccharides obtained by methanolysis may then be fully methylated, and the proportions of the various monosaccharides determined. This approach has been used, for example, in studies on a galactomannan392 and on tamarind-kernel polysaccharide.393 Such an analysis also constitutes a useful check to ensure that no significant change in the composition of the polysaccharide occurred during methylation. 

As regards the l,l-bis(acylamido)-l-deoxyalditols, the first structural study was that of Fletcher and coworkers,69 who conducted oxidations with lead tetraacetate. These authors postulated an empirical relationship between the position of the oxidation curve in a graph and the number of hydroxyl groups present. They compared the curves afforded by a series of l,l-bis(acetamido)-l-deoxyalditols with those of alditols having the same number of free hydroxyl groups, and observed a close correspondence. At present, periodate oxidation is widely applied to these mono- or di-saccharide derivatives. Under controlled conditions, the methylation technique can also be applied.42... 

The uniqueness of the aldofuranosides, as compared to the aldopyrano-sides, was recognized initially through the difference in rates of hydrolysis by acid. This difference was confirmed by the use of methylation techniques the latter have, to a great extent, been supplanted by the periodate oxidation of Jackson and Hudson120 and by oxidation with lead tetraacetate. Hudson s rules of isorotation were also employed to a great extent. 

If the extracted lipid is to be stored, flush the tube with nitrogen gas and tightly close the cap, and leave at -20°C for future applications. Otherwise, proceed to the methylation technique of choice (see Basic Protocol 1 and 2 also see Alternate Protocol). 

Sequential Methylation Technique. A sequential methylation-extraction technique has been devised primarily to separate phenolics from aromatic and aliphatic acids. The procedure, outlined below, allows quantitative conversion of phenolic hydroxyls to methyl ethers in one step and esterification of carboxyl groups in another. 

In general the direct GLC methods without derivative formation tend to produce asymmetric peaks which increase the difficulty of quantitation by measurement of peak heights. The on-colunn methylation techniques appear to be convenient to run and sharper peaks are obtained at lower temperatures. 

However, it must be pointed out that to date, the application of the phosphorylation and on-column methylation techniques have had only limited application. 

No partly methylated ribose derivatives have been isolated from natural sources up to the present, for although such nucleic acids as yeast nucleic acid contain D-ribose phosphate residues, the methylation technique fails in structural studies in this group because of the hydrolytic action of alkaline reagents. Trimethyl-D-ribofuranose has, how-... 

Branched-chain mannans from Candida species were analyzed by the methylation technique, and the presence of 2,6-di-O-substituted mannofuranosyl units was suggested on the basis of supposed 3,5-di-O-methyl-D-mannose isolated.100 However, this compound proved to be identical to synthetic 2,4-di-O-methyl-D-mannose,101 and it differed from the authentic 3,5-di-O-methyl isomer,102 which is consistent with 3,6-di-O-substituted D-mannopyranosyl units. 

The methylation technique is of outstanding importance in structural polysaccharide chemistry. The procedure involves the preparation of the exhaustively methylated polysaccharide, hydrolysis to a mixture of monomers, and the separation, identification, and quantitative estimation of the components of this mixture the original points of substitution wiU correspond to the unsubstituted hydroxyl groups in these monomeric methyl ethers. Although laborious and time-consuming, the method gives valuable... 

Codeine occurs in opium in relatively small amounts (—0.3%) and yet it is in great demand as an analgesic. Much of the morphine derived from opium is therefore converted by methylation to codeine. Conventional methylation techniques have been employed, including dimethyl sulfate, diazomethane, and phenyltrimethylammonium chloride. 

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