Transition-state Analogue Substrate Research Articles

Keratanase II‐Catalyzed Synthesis of Keratan Sulfate Oligomers by Using Sugar Oxazolines as Transition‐State Analogue Substrate Monomers: A Novel Insight into the Enzymatic Catalysis Mechanism

Keratan sulfate (KS) oligomers with well-defined structures were synthesized by keratanase II (KSase II)-catalyzed transglycosylation. N-Acetyllactosamine [Galbeta(1-->4)GlcNAc; LacNAc] oxazoline derivatives with sulfate groups at the C-6 (1 a) and both the C-6 and the C-6' (1 b) were prepared as transition-state analogue substrate monomers for KSase II. Monomer 1 a was effectively oligomerized by the enzyme under weak alkaline conditions, to give alternating 6-sulfated KS oligomers (2 a) in good yields, and with total control of regioselectivity and stereochemistry. KSase II also recognized 1 b, which provided fully 6-sulfated KS oligomers (2 b) in good yields under similar conditions. Nonsulfated LacNAc oxazoline was difficult to oligomerize enzymatically. These results imply that the catalysis mechanism of KSase II involves a sugar oxazolinium ion that requires the 6-sulfate group in the GlcNAc residue not only in hydrolysis of KS chains, but also in oligomerization of oxazoline monomers. This is the first report of KSase II-catalyzed transglycosylation to form beta(1-->3)-glycosidic bond through a substrate-assisted mechanism.

Enzymatic Polymerization to Unnatural Hybrid Polysaccharides

AbstractUnnatural hybrid polysaccharides with an alternating structure of different kinds of monosaccharide units have been synthesized by enzymatic polymerization catalyzed by a glycoside hydrolase. For the synthesis, various activated monomers with a disaccharide structure were designed on the basis of the concept of transition‐state analogue substrate (TSAS). The monomers include two types of TSAS monomer structures: a sugar fluoride type and a sugar oxazoline type. Hydrolase catalysis successfully induces polycondensation or ring‐opening polyaddition of these monomers to produce unnatural hybrid polysaccharides in good yield. Characterization of the products, mechanistic features of the polymerization, and specific properties of the product polysaccharides are discussed.magnified image

New developments of polysaccharide synthesis via enzymatic polymerization

This review focuses on the in vitro synthesis of polysaccharides, the method of which is “enzymatic polymerization” mainly developed by our group. Polysaccharides are formed by repeated glycosylation reactions between a glycosyl donor and a glycosyl acceptor. A hydrolysis enzyme was found very efficient as catalyst, where the monomer is designed based on the new concept of a “transition-state analogue substrate” (TSAS); sugar fluoride monomers for polycondensation and sugar oxazoline monomers for ring-opening polyaddition. Enzymatic polymerization enabled the first in vitro synthesis of natural polysaccharides such as cellulose, xylan, chitin, hyaluronan and chondroitin, and also of unnatural polysaccharides such as a cellulose–chitin hybrid, a hyaluronan–chondroitin hybrid, and others. Supercatalysis of hyaluronidase was disclosed as unusual enzymatic multi-catalyst functions. Mutant enzymes were very useful for synthetic and mechanistic studies. In situ observations of enzymatic polymerization by SEM, TEM, and combined SAS methods revealed mechanisms of the polymerization and of the self-assembling of high-order molecular structure formed by elongating polysaccharide molecules.(Communicated by Hitosi NOZAKI, M.J.A.)

Stepwise synthesis of chitooligosaccharides through a transition-state analogue substrate catalyzed by mutants of chitinase A1 from Bacillus circulans WL-12

AbstractSeveral mutants of chitinase A1 fromBacillus circulansWL-12 have been prepared by site-directed mutagenesis of tryptophan 433. The hydrolytic activity of these mutants towardp-nitrophenyl chitobioside was lower than that of wild-type chitinase A1. The mutants were found to catalyze the transglycosylation reaction of the 1,2-oxazoline derivative ofN-acetyllactosamine (Gal-GlcNAc-oxa) toN,N′′-diacetylchitobiose (GlcNAc-GlcNAc), giving rise to a transglycosylated tetrasaccharide product (Gal-GlcNAc-GlcNAc-GlcNAc) in high yield. Based on these results, a one-pot synthesis of chitooligosaccharides by combined use of mutant W433Y and β-galactosidase has been demonstrated. By controlling the reaction conditions, chitotriose and chitotetraose were obtained in 74% and 40% yield (determined by HPLC), respectively, without isolating any intermediate products.

A Hyaluronidase Supercatalyst for the Enzymatic Polymerization to Synthesize Glycosaminoglycans

Hyaluronidase (HAase) catalyzes multiple enzymatic polymerizations with controlling regio- and stereoselectivity perfectly. This behavior, that is, the single enzyme being effective for multireactions and retaining the enzyme catalytic specificity, is not usual, and hence, HAase is a supercatalyst. Various sugar oxazoline monomers prepared based on the concept "transition-state analogue substrate" were successfully polymerized and copolymerized with HAase catalysis, yielding natural and unnatural glycosaminoglycans.

Chemo-enzymatic Synthesis of Novel Oligo-N-acetyllactosamine Derivatives having a β(1-4)–β(1-6) Repeating Unit by Using Transition State Analogue Substrate

Chitinase-catalyzed hydrolytic and transglycosylating behavior of 1,2-oxazoline derivative of N-acetyllactosamine (LacNAc-oxa) 1 has been investigated. An extremely rapid hydrolysis (ring-opening of the oxazoline moiety) could be observed, suggesting that 1 behaves as a transition state analogue substrate for chitinase A1 (Bacillus circulans WL-12). This disaccharide monomer 1 was found to polymerize under basic conditions, giving rise to novel oligosaccharides having a β(1-4)–β(1-6) repeating unit in the main chain. The degree of polymerization of the resulting oligosaccharides was up to 5. This is the first example of enzymatic glycosylation reaction forming a β(1-6) bond catalyzed by chitinase.

Enzymatic Polymerization to Novel Polysaccharides Having a Glucose-N-acetylglucosamine Repeating Unit, a Cellulose−Chitin Hybrid Polysaccharide

A cellulose-chitin hybrid polysaccharide having alternatingly beta(1-->4)-linked D-glucose (Glc) and N-acetyl-d-glucosamine (GlcNAc) was synthesized via two modes of enzymatic polymerization. First, a sugar oxazoline monomer of Glcbeta(1-->4)GlcNAc (1) was designed as a transition-state analogue substrate (TSAS) monomer for chitinase catalysis. Monomer 1 was recognized by chitinase from Bacillus sp., giving rise to a cellulose-chitin hybrid polysaccharide (2) via ring-opening polyaddition with perfect regioselectivity and stereochemistry. Molecular weight (M(n)) of 2 reached 4030, which corresponds to 22 saccharide units. Second, a sugar fluoride monomer of GlcNAcbeta(1-->4)Glc (3) was synthesized for the catalysis of cellulase from Trichoderma viride. The enzyme catalyzed polycondensation of 3, providing a cellulose-chitin hybrid polysaccharide (4) in regio- and stereoselective manner. M(n) of 4 reached 2840, which corresponds to 16 saccharide units. X-ray diffraction measurements revealed that these hybrid polysaccharides did not form any characteristic crystalline structures. Furthermore, these unnatural hybrids of 2 and 4 were successfully digested by lysozyme from human neutrophils.

Chitinase-Catalyzed Synthesis of Alternatingly N-Deacetylated Chitin: A Chitin−Chitosan Hybrid Polysaccharide

A chitin-chitosan hybrid polysaccharide (2) having a beta(1-->4)-linked alternating structure of an N-acetyl-D-glucosamine (GlcNAc) unit and a D-glucosamine (GlcN) unit was synthesized via chitinase-catalyzed polymerization of an oxazoline derivative of a GlcNbeta(1-->4)GlcNAc monomer (1). Monomer 1 was designed as a transition-state analogue substrate (TSAS) monomer for chitinase catalysis, which belongs to the glycoside hydrolase family 18. Monomer 1 was effectively polymerized by the catalysis of enzymes from Bacillus sp., Serratia marcescens and Streptomyces griseus, under weak alkaline conditions, giving rise to a water-soluble hybrid polysaccharide (2) in good yields. Molecular weights of 2 reached 2,020 with using chitinase from Serratia marcescens, which corresponds to 10-12 saccharide units.

Enzymatic Synthesis of Chondroitin 4-Sulfate with Well-Defined Structure

Synthesis of chondroitin sulfate (ChS) with well-defined structure was achieved for the first time by hyaluronidase-catalyzed polymerization. N-Acetylchondrosine (GlcAbeta(1-->3)GalNAc) oxazoline derivatives sulfated at C4 (1a), C6 (1b), and both C4 and C6 (1c) in the GalNAc unit were synthesized as transition state analogue substrate monomers for hyaluronidase (HAase) catalysis. Compound 1a was effectively polymerized by the enzyme, giving rise to synthetic ChS sulfated perfectly at the C4 position in all N-acetylgalactosamine units (Ch4S, 2a) in good yields. Molecular weights (Mn) of 2a ranged from 4000 to 18,400, which were controlled by varying reaction conditions. Compounds 1b and 1c were not catalyzed by the enzyme, affording the corresponding disaccharides through the oxazoline ring-opening without formation of polysaccharides.

Enzymatic Synthesis of Chondroitin and Its Derivatives Catalyzed by Hyaluronidase.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Enzymatic synthesis of chondroitin and its derivatives catalyzed by hyaluronidase.

The enzymatic polymerization to provide synthetic chondroitin and its derivatives is reported here, the first example of such in vitro synthesis to date. N-Acetylchondrosine (GlcAbeta(1-->3)GalNAc) oxazoline (1a) and its derivatives (1b-1f) were designed and synthesized as novel transition state analogue substrate monomers for catalysis by hyaluronidase. Hyaluronidase is a hydrolysis enzyme of chondroitin that also catalyzes the formation of repeated glycosidic bonds in in vitro synthesis, rather than in the catabolic direction. Monomers of 2-methyl (1a), 2-ethyl (1b), and 2-vinyl (1f) oxazoline derivatives were polymerized using this enzyme, via ring-opening polyaddition with total control of regioselectivity and stereochemistry. These reactions provided the corresponding synthetic chondroitin (natural type; N-acetyl, 2a) and the derivatives (unnatural type) with N-propionyl (2b) and N-acryloyl (2f) functional groups at the C2 position of all the galactosamine units, in good yields. Monomers of 2-n-propyl (1c) and 2-isopropyl (1d) oxazoline derivatives were polymerized to produce 2c and 2d in low yield. The 2-phenyl oxazoline derivative (1e) did not afford any enzyme-catalyzed products. M(n) values of 2a and 2b reached 4800 and 4000, respectively. The M(n) value of 2a corresponds to that of the naturally occurring chondroitin. Thus, hyaluronidase catalysis allows the in vitro production of not only natural type but also the formation of unnatural type chondroitins.

Synthesis of Artificial Chitin: Irreversible Catalytic Behavior of a Glycosyl Hydrolase through a Transition State Analogue Substrate

Synthesis of Artificial Chitin: Irreversible Catalytic Behavior of a Glycosyl Hydrolase through a Transition State Analogue Substrate