Enzymatic Synthesis of Heparin - Sustainable Biotechnology: Sources of Renewable Energy - page 270

heparin, suggesting that our enzyme-based modification system is indeed capable of
converting HS backbones to anticoagulant polysaccharides. We also measured the
binding affinity of AT to HS1 by surface plasmon resonance (SPR), which showed
that the anticoagulant activities of HS1 correlated to its binding affinity to AT. We
also generated other types of HS polysaccharides which bind to FGF2 or HSV-1
gD. Taken together, this demonstrates the feasibility of large-scale chemoenzymatic
synthesis of heparin/HS with desired biological activities and could be used as a
unique tool to explore the biosynthesis of heparin/HS.

4.4 Use of an Enzymatic Combinatorial Approach to Identify
Novel Anticoagulant HS Structures

We applied this approach to the synthesis of a small HS library with different
sulfation patterns [41]. In this study, N -sulfo heparosan had been chemoenzymat-
ically prepared from heparosan starting material. A combination of recombinant
HS biosynthetic enzymes was used to modify N -sulfo heparosan. Our lab dis-
covered one polysaccharide, known as Recomparin (Fig. 7). Recomparin showed
strong AT-mediated anticoagulant activity. Disaccharide analysis suggested that
the Recomparin consists of a repeating tetrasaccharide (-GlcUA-GlcNS3S

6S-
GlcUA-GlcNS6S-). It was somewhat surprising to discover Recomparin has strong
anticoagulant activity despite the fact that Recomparin contains no IdoUA2S unit.
Previous studies showed that the IdoUA2S unit was critical for a pentasaccharide
bind to AT [7]. Furthermore, IdoUA adopts a skew boat ( 2 S 0 ) or chair ( 1 C 4 ) con-
formation, while GlcUA is mainly found in the chair conformation ( 4 C 1 ) (Fig. 1)
[13]. The 2 S 0 conformation was generally believed to be necessary for binding to
AT [55]. Our results indicated that the structural flexibility of IdoUA unit is less
important in polysaccharide-AT interaction. Indeed, further experimental data sug-
gest that IdoUA unit is essential for binding to AT if the oligosaccharide is smaller
than a hexasaccharide, while IdoUA unit is not essential when the oligosaccha-
ride is larger than an octasaccharide [41]. Since IdoUA2S units are responsible
for heparin binding to PF4 [28] and FGF [27], our results can help design novel
heparin-based anticoagulant drugs with reduced chance of inducing HIT or stimulat-
ing cell growth. Indeed, we found that Recomparin, unlike heparin, had no activity
in stimulating FGF/FGFR mediated cell proliferation, demonstrating that the anti-
coagulant activity and the activity in stimulating cell proliferation can be separated
at the polysaccharide levels.

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4.5 Preparation of 3-O-Sulfated Octasaccharide that Inhibits
the Entry of HSV-1

We also utilized the enzyme-based approach to prepare a structure-defined octasac-
charide with the purpose of developing novel anti-herpes drugs by targeting the viral