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 copolymerization to hybrid glycosaminoglycans: a novel strategy for intramolecular hybridization of polysaccharides.

Hybrid glycosaminoglycans (GAGs) having an intramolecularly hybridized structure of hyaluronan-chondroitin (3a) and hyaluronan-chondroitin 4-sulfate (3b) have been synthesized via enzymatic copolymerization catalyzed by hyaluronidase (HAase). N-Acetylhyalobiuronate (GlcAbeta(1-->3)GlcNAc)-derived oxazoline (1) was copolymerized with N-acetylchondrosine (GlcAbeta(1-->3)GalNAc)-derived oxazoline (2a) by HAase catalysis at pH 7.5 and 30 degrees C, giving rise to copolymer 3a with Mn 7.4 x 103 in a 50% yield. Also, HAase-catalyzed copolymerization of monomer 1 with N-acetylchondrosine oxazoline having a sulfate group at C4 on GalNAc (2b) was carried out to produce copolymer 3b with Mn 1.4 x 104 in a 60% yield. The copolymer compositions were controllable by varying the comonomer feed ratio. These hybrid GAGs were successfully digested by the catalysis of hyaluronan lyase, clearly exhibiting that the products are not a blend of different homopolymers but an intramolecularly hybridized GAG.

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.

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.

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.