Tools and ingredients for the biocatalytic synthesis of carbohydrates and glycoconjugates

Abstract

The presence of multiple functional groups and stereocentres in carbohydrates and glycoconjugates make them challenging targets for synthesis. Although progress in chemical synthesis and engineering is impressive, there is still a need to selectively introduce and remove protecting groups in the total synthesis of target molecules of increasing complexity. Multiple hydroxyl-groups with similar reactivities have to be differentiated in order to form the desired glycosidic bonds in a regioand stereospecific way. To complement the existing chemical tools and ingredients, biocatalysts for selective carbonÁcarbon bond formation and glycosylation reactions have been developed. The availability of auxiliary ingredients like transfer reagents is a prerequisite for the development of viable biocatalytic process steps. In the case of dihydroxyacetonephosphate-dependent aldolases, e.g. fructose-1,6-bisphosphate aldolase (EC 4.1.2.13), the large-scale availability of dihydroxyacetone-phosphate (DHAP) eliminates the need to synthesize the donor DHAP. For the pyruvate-dependent aldolases, e.g. the N-acetylneuraminic acid aldolase (EC 4.1.3.3) and acetaldehyde-dependent aldolases like the 2-deoxy-Dribose-5-phosphate aldolase (4.2.1.4), the donors pyruvate and acetaldehyde are also available on a large scale. A broad range of natural and recombinant aldolases have been produced in stable lyophilized form. Recombinant transketolase together with a new synthesis of hydroxypyruvates has provided a platform technology for the preparation of monosaccharides, whereby the carbon backbone is extended by a two-carbon unit (C2-elongation). Natural and recombinant glycosyltransferases have been prepared on a large-scale to establish biocatalytic glycosylations in water as highly regio-and stereospecific reaction methodologies without the need for laborious protecting group manipulations, solubility adaptations and complex synthetic schemes. In order to simplify the synthetic manipulations for specific glycosylations, toolkits for b-1,4-galactosylations, a-1,3-galactosylations and a-1,3-fucosylations have been developed for rapid quantitative conversions. The introduction of matched pairs of biocatalysts and transfer reagents as ingredients together with the optimized reaction methodology as tool provide an important starting point for biocatalytic glycomics.

Substrate and Process Engineering for Biocatalytic Synthesis and Facile Purification of Human Milk Oligosaccharides

“…Biocatalysis is environmentally friendly, sustainable, highly efficient, and selective for the desired molecular transformation [2] . It has an especially high potential in industrial production of carbohydrates and glycoconjugates that are soluble in aqueous solution, a preferred medium for enzyme‐catalyzed reaction [1,3] . The enzyme stabilization power of high concentration carbohydrate substrates has also been shown, [1] adding another advantage for the production of carbohydrates by biocatalysis.…”

“…[2] It has an especially high potential in industrial production of carbohydrates and glycoconjugates that are soluble in aqueous solution, a preferred medium for enzyme-catalyzed reaction. [1,3] The enzyme stabilization power of high concentration carbohydrate substrates has also been shown, [1] adding another advantage for the production of carbohydrates by biocatalysis. With the growing demands for carbohydrates as pharmaceuticals, nutraceuticals, and ingredients for foods and health beneficial products, [1,4] developing biocatalytic strategies for their synthesis is becoming increasingly important especially if their natural sources are limited and their chemical synthesis is challenging.…”

Rational substrate and enzyme engineering of transketolase for aromatics

“…While many carbon-carbon bond forming chemical reactions can be achieved in a single step, several challenges still exist, 1 and enzymes such as transketolase (TK) offer an attractive alternative due to their high stereospecificity and selectivity. [2][3][4][5][6][7][8] Transketolase (E.C. 2.2.1.1) is a thiamine diphosphate (ThDP) dependent enzyme which plays a crucial role in the non-oxidative phase of the pentose phosphate pathway and Calvin cycle, 9 where it catalyses a reversible transfer of a two-carbon ketol group from a ketose sugar to aldose.…”

Biocatalytic Methods for CC Bond Formation

“…The donor can be produced in situ from serine using a serine hydroxygenase. A robust and scalable synthetic procedure for the production of hydroxypyruvate 99 has been developed recently 117. The use of transketolases has been facilitated by recombinant protein expression and the large‐scale synthesis of ketol donors.…”