Our recent advances in enzymatic polymerization, defined as chemical polymer syntheses in vitro (in test tubes) via non-biosynthetic pathways catalyzed by an isolated enzyme, have been mainly reviewed. The major target macromolecules formed via the enzymatic polymerizations described in this article are polysaccharides, polyesters, and polyaromatics. For synthesis of polysaccharides and polyesters, hydrolases are used as catalysts; hydrolases catalyzing a bond-cleavage reaction by water induce the reverse reaction of hydrolysis, leading to polymer production by a bond-forming reaction in vitro. Specific enzyme catalysis provides a novel synthetic route with precise structure control not only for natural biopolymers such as cellulose, xylan, and chitin, but also for unnatural polysaccharides and polyesters, many of which are difficult to be synthesized by conventional methodologies. Oxidoreductases act as catalysts for oxidative polymerization of phenol, aniline, and their derivatives. A new class of phenolic polymers are synthesized without use of toxic formaldehyde under mild reaction conditions. Enzyme-model complexes also catalyze polymerizations for the synthesis of new high-performance polymeric materials.
Hyaluronan (hyaluronic acid, HA) is one of natural polysaccharides classified into glycosaminoglycans (GAGs) that is widely found in living cells and extracellular matrix (ECM), in particular, smooth muscle cells, 1 fibroblasts, 1 vitreous of the human eye, 2 synovial joint fluid, 2 and rooster comb. 2 As a member of ECM, HA plays important functions in vivo, for example, tissue proliferation, regeneration, wound healing, etc. 3 HA is a typical heteropolysaccharide consisting of two kinds of sugar units, glucuronic acid (GlcA) and 2-acetamido-2-deoxy-D-glucose (GlcNAc), and has a structure of a GlcAβ(1f3)GlcNAc disac-
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.
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.
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.
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