Carbohydrate purification

In few areas of natural product chemistry does the injunction first, catch your hare so succinctly summarise the main difficulties. Whereas a battery of well-established techniques can be used for the purification of a protein from biological materials, each sub-field, and even each laboratory, in the carbohydrate purification and sequencing area tends to adopt its own extraction procedures. Polysaccharides in vivo are often heterogeneous with respect to molecular weight, which restricts the usefulness of size-exclusion chromatography. Some polysaccharides are covalently attached to other macromolecular... 

When ionic liquids are used as replacements for organic solvents in processes with nonvolatile products, downstream processing may become complicated. This may apply to many biotransformations in which the better selectivity of the biocatalyst is used to transform more complex molecules. In such cases, product isolation can be achieved by, for example, extraction with supercritical CO2 [50]. Recently, membrane processes such as pervaporation and nanofiltration have been used. The use of pervaporation for less volatile compounds such as phenylethanol has been reported by Crespo and co-workers [51]. We have developed a separation process based on nanofiltration [52, 53] which is especially well suited for isolation of nonvolatile compounds such as carbohydrates or charged compounds. It may also be used for easy recovery and/or purification of ionic liquids. 

Mond process The purification of nickel by the formation and decomposition of nickel carbonyl, monomer A small molecule from which a polymer is formed. Examples CH2=CH2 for polyethylene NH2(CH2)6NH2 for nylon, monoprotic acid A Bronsted acid with one acidic hydrogen atom. Example CH COOI I. monosaccharide An individual unit from which carbohydrates are considered to be composed. Example C6H(206, glucose, multiple bond A double or triple bond between two atoms. 

This class of polysaccharide was well known in sugar refineries as the causative agent of ropiness it was formed from cane or beet sugar by bacteria of the Leuconostoc genus. Over many years, numerous papers were published, mainly with E. J. Bourne [Adv. Carbohydr. Chem. Biochem., 34 (1977) 1-22] and S. A. Barker as co-authors, describing the isolation, purification, properties, and structural features of dextrans. 

The type of data produced in a f.a.b. experiment is affected by the pH and ionic strength of the matrix. The former may be controlled either by the addition of acids or bases, although, in practice, it is usually preferable to keep the matrix acidic. The ionic strength is partly dictated by the purity of the sample (many biological compounds are still contaminated with salts, even after extensive purification) and partly by exogenous additives. Three additives are especially useful for carbohydrate work. They are as follows. 

Crude chloroform-methanol-water (30 60 8, v/v) extracts of immunostainedTLC bands were analyzed without further purification by nanoelectrospray low-energy mass spectrometry. The authors showed that this effective PLC/MS-joined procedure offers a wide range of applications for any carbohydrate-binding agents such as bacterial toxins, plant lectins, and others. Phenyl-boronic acid (PBA) immobilized on stationary support phases can be put to similar applications. This technology, named boronate affinity chromatography (BAC), consists of a chemical reaction of 1,2- and 1,3-diols with the bonded-phase PBA to form a stable... 

It should be possible to use the special properties of chiral structures for particular separation problems. According to Belinski and Tencer, one possible way in which nature solved the ribose problem could have involved an enantioselective and diastereoselective purification process acting on a mixture of biomolecules, which left ribose as the only molecule available for further reactions. The authors propose a theoretical mechanism in which a type of chromatographic process occurs at chiral mineral surfaces. This paper is likely to stimulate new experiments as well as the quest for as yet unknown surfaces which can separate racemic carbohydrate mixtures. The question arises, however, as to whether there were minerals present on the young Earth which are now unknown, as they no longer exist on the Earth of today (Belinski and Tencer, 2007). 

Silica-supported reagents have been exploited as nontoxic, inexpensive, reusable, and environmentally acceptable catalysts for developing stoichiometric reaction methods in organic chemistry and specifically in carbohydrate chemistry. Apart from being easy to handle and to store, these systems allow facile workup, the catalyst being removed by simple filtration, and the reaction products isolated by chromatographic purification, if necessary. 

J. F. Robyt and T. F. Walseth, Production, purification, and properties of dextransucrase from Leuconostoc mesenteroides NRRL B-512F, Carbohydr. Res., 68 (1979) 95-111. 

Purification of the conjugates may be done by reverse phase HPLC separation. Dry the reaction solution under a nitrogen stream and reconstitute in a minimum volume of acetonitrile/water (1 1, v/v). Apply the sample to a 5 pm Cig-silica HPLC column (250 X 4.6 mm, Nucleosil). Elute with a gradient of water to acetonitrile at a flow rate of 1 ml/minute over a time course of 30 minutes. Free BNAH and BNAH-glycan derivatives can be monitored by absorbance at 275 nm. The conjugate peak also will be positive for carbohydrate by reaction with orcinol, which can be detected by spray after spotting a small eluted sample on a TLC plate. 

By means of gel electrophoresis on cross-linked, hydrolyzed starch,99 with simultaneous checking for proteins, lipids, and pectinesterase activity, it was found, however, that the product isolated after the separation on CM-Sephadex C-50 constitutes but one of five multiple forms of tomato pectinesterase, and is the one present in preponderant proportion98 (see Fig. 4). The accompanying lipid and sugar components were separated from this pectinesterase form in the course of the purification procedure. After analysis of the hydro-lyzate of the final product for fatty acids, as well as for carbohydrate components, it was possible to exclude the possibility of a lipoprotein,30 as well as glycoprotein,100 character of this form of tomato pectinesterase. 

J2. Jagannathan, V., Rangachari, P. N., and Damodaran, M., Carbohydrate metabolism in citric acid fermentation. 5. Purification and properties of Zwischen-ferment from Aspergillus niger. Biochem. ]. 64, 477-481 (1956). 

An attractive feature of this dehydrative coupling approach is that it avoids the need for isolation of intermediate glycosyl donors. This can be desirable if a glycosyl donor is not stable to isolation or purification. Moreover, the use of a hemiacetal donor reduces the number of synthetic manipulations of the carbohydrate donor by avoiding hemiacetal derivatization to alternative donor types. In this way, the approach has the potential to streamline time and labor-intensive multiglycosylation sequences. Although there increasingly have been reports of these direct dehydrative... 

The synthesis of complex carbohydrate structures can preferably be accomplished using several enzymes in one-pot or sequential mode. Crude intermediate products may be either directly processed by the follow-up enzyme, or a fast and simple purification step may be included like desalting, concentrating, etc. This approach saves both time and costs, however, it requires a relatively high specificity, regioselectivity and yields in all the steps, which somehow limits the choice of glycosidases available. 

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