Enzymatic Synthesis of Chondroitin and Its Derivatives Catalyzed by Hyaluronidase

Abstract

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.

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

“…[16][17][18][19][20][21] Notably, we have achieved the successful synthesis of hyaluronan (HA), chondroitin (Ch) and their derivatives, which belong to GAG family, by hyaluronidase-catalyzed polymerization. [22,23] Very recently, hybrid GAGs that are composed of HA, Ch and Ch-4-sulfate have been prepared via hyaluronidase-catalyzed copolymeriza-tions. [24] In these polymerizations, synthetic carbohydrate monomers that are activated at the anomeric carbons by fluorine atoms [17,20] or a dihydrooxazole (oxazoline) structure [18][19][20][21][22][23][24] were enzymatically polymerized in a perfectly regioselective and stereocontrolled fashion.…”

New developments of polysaccharide synthesis via enzymatic polymerization

“…Therefore, on the basis of TSAS concept an oxazoline monomer (Ch-oxa(R)) was designed again for the synthesis of Ch via HAase-catalyzed ring-opening polyaddition (Scheme 13). 24) In addition to the 2-methyl-oxazoline monomer, polymerization of various oxazoline monomers with 2-ethyl, 2-n-propyl, 2-isopropyl, 2-vinyl, and 2-phenyl substituents was investigated. Monomers with methyl, ethyl and vinyl substituents were efficiently catalyzed by HAase at pH 7.5, providing synthetic Ch and its derivatives bearing the corresponding N-acyl groups under perfect structure control.…”

Design and Utilization of Chitinases with Low Hydrolytic Activities

“…The enzymatic polyaddition of 7 and 8 took place smoothly to give artificial hyaluronic acid 9 and artificial chondroitin 10, respectively ( Fig. 3(b)) (16,17). A disaccharide substrate of Man β(1-4) GlcNAcoxazoline 11 was synthesized as a probe for detection o f t h e t r a n s g l y c o s y l a t i n g a c t i v i t y o f e n d o -β -Nacetylglucosaminidase.…”