Chemoenzymatic synthesis with the hyaluronan synthase; production of a multitude of defined authentic, derivatized, and analog polymers.

Hyaluronan (HA; [-3-GlcNAc-1-beta-4-GlcA-1-beta] ), an essential matrix polysaccharide of vertebrates and the molecular camouflage coating in certain pathogens, is polymerized by "HA synthase" (HAS) enzymes. Three HAS classes have been identified with biotechnological utility, but only the Class II PmHAS from Type A has been useful for preparation of very defined HA polymers in vitro. Two general chemoenzymatic strategies with different size products are possible: (1) repetitive step-wise extension reactions by sequential addition of a single monosaccharide from a donor UDP-sugar onto an acceptor (or "primer") comprised of a short glycosaminoglycan chain (e.g., HA di-, tri- or tetrasaccharide) or an artificial glucuronide yielding homogeneous oligosaccharides in the range of 2 to ~20 monosaccharide units ( = 1 to ~10), or (2) "one-pot" polymerization reactions employing acceptor-mediated synchronization with stoichiometric size control yielding quasi-monodisperse (i.e., polydispersity approaching 1; very narrow size distributions) polysaccharides in the range of ~7 kDa to ~2 MDa ( = ~17 to 5000). In either strategy, acceptors containing non-carbohydrate functionalities (e.g., biotin, fluorophores, amines) can add useful moieties to the reducing termini of HA chains at 100% efficiency. As a further structural diversification, PmHAS can utilize a variety of unnatural UDP-sugar analogs thus adding novel groups (e.g., trifluoroacetyl, alkyne, azide, sulfhydryl) along the HA backbone and/or at its nonreducing terminus. This review discusses the current understanding and recent advances in HA chemoenzymatic synthesis methods using PmHAS. This powerful toolbox has potential for creation of a multitude of HA-based probes, therapeutics, drug conjugates, coatings, and biomaterials.