November 29
PROGRAM

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Glycoscience : diversity and integration

Heparanomics :Synthetic approaches forcharacterization ofspecific interactions with proteins
Shang-Cheng Hung, Ph.D.
Distinguished Research Fellow
Genomics Research Center, Academia Sinica, TAIWAN

Shang-Cheng Hung
ProfileShang-Cheng Hung studied at the National
Tsing Hua University (Hsinchu, Taiwan)
and obtained his Ph.D. in organic chemistry under the supervision of Prof. BiingJiun Uang and Prof. Chun-Chen Liao in1992. After two years of military service,
he spent a year with Prof. Andrew Streitwieser at the University of California, Berkeley as a postdoc. In 1995, he moved to theScripps Research Institute for his second
post-doctoral research with Prof. ChiHuey Wong for three years. He then wasrecruited back to Taiwan as an AssistantResearch Fellow at the Institute of Chemistry, Academia Sinica and was promoted
to the rank of Associate Research Fellow
in 2002. He moved to the Department ofChemistry, National Tsing Hua University as an Associate Professor (2005) andwas promoted as a full professor (2006)
and Distinguished Professor (2007). In
2009, he joined the Genomics ResearchCenter, Academia Sinica as a ResearchFellow and, in 2012, advanced to the rankof Distinguished Research Fellow. His research interests focus on the synthesis of
biologically important sugars, particularly,
glycosaminoglycans and mycobacterialcell envelope components, together with
their bio-evaluations. He received several
awards including the Distinguished Research Award from the National Science
Council of Taiwan, the 17th Teco Award
from the Teco Technology Foundation,and the Outstanding Scholar Chair fromthe Foundation for the Advancement ofOutstanding Scholarship (FAOS). He is
also a member of the Editorial Board of
Carbohydrate Research.

Heparan sulfate (HS) is a linear polysaccharide that is widely distributed in metazoan cell surfaces, extracellular matrices,and basement membranes, where it exists
as proteoglycan component. The HS
chain is initially assembled as a simple
1→4-linked copolymer of N-acetyl-α-Dglucosamine and β-D-glucuronic acid
(GlcA), but the seemingly regulated but
non-template driven modifications cause
extensive microheterogeneity along thesugar backbone comprised of around 50
to 200 disaccharide units. These modifications, which include N-deacetylation,
N-sulfonation, GlcA 5-C epimerization
forming α-L-iduronic acid, and multiple
O-sulfonations, are implemented by several enzyme isoforms of varying specificities and are always incomplete, accounting
to theoretically 48 disaccharide variations.
Organisms, tissues, or particular cells,
thus, express a range of HS structures that,in its entirety, comprise what is collectively
called the heparanome. With such ubiquity and structural complexity, numerous
proteins acquired the ability to utilize HS
as an accessory in eliciting their bioactivity. Consequently, HS play crucial roles inmany physiological and pathophysiological processes such as signaling, development, tumor progression and metastasis,
viral and bacterial infections, and inflammatory response.
The myriad of functional group patterns
decorating the sugar backbone allowed
HS to encode a high density of structural information that rivals proteins andnucleic acids. Such array of modifications
is responsible for mediating or modulating
protein activity. While some proteins bind
non-specifically through ionic interactions
with the negatively charged sugar chain, alarge number of proteins are believed torequire particular modification patterns
for optimum activity. It should be notedthat the actual binding site represents only44

the 20th Anniversary of the Mizutani Foundation for Glycosciencea small segment of the HS chain, usuallyaround 2-10 disaccharides. These biologically active domains within the heparanome, the character of their interaction
with protein ligands, and the influences
they exert in physiology as a whole are thesubjects of the emergent heparanomics
field. Keen interests are focused in deciphering the molecular level details of theHS-protein interactions because they may
present therapeutic opportunities. However, unlike the fully developed methodsof protein acquisition, the preparationof structurally defined oligosaccharides
based on potential HS binding sites is considered a bottleneck in conducting structure–activity relationship studies.
Chemical synthesis has so far proved reliable in the procurement of well-defined
HS oligosaccharides at a considerable
scale suitable for biological assays. Synthetic approaches aimed at constructing
HS-based compounds are met with challenges concerning the preparation of sugarbuilding blocks particularly the rare L-idoconfigured derivatives, the regio- and stereoselective formation of glycosidic linkages, and the regioselective functionalization of similarly reactive hydroxyl groups
to emulate the complex modification patterns of the naturally occurring materials.
To access the L-idose building block, ourgroup developed methodologies startingfrom the commercially abundant diacetone α-D-glucose with 5-C-epimerization,
1,6-anhydro-ring formation, and metal
triflate-mediated acetolysis as key steps.
We also established protocols for the regioselective one-pot protection and stereoselective one-pot protection-glycosylationthat effectively reduced the amount of timeand effort in constructing building blocks
and sugar backbones. The preparations ofD-glucose and D-glucosamine acceptors
and donors using this strategy are being
routinely carried out in our laboratory. AFigure 1. Synthetic HS-based oligosaccharides are important tools in understanding the character of HS-protein interactions.

completely stereoselective glycosylationthat generates the α-D-glucosamine unit
is usually difficult to accomplish. Aftera systematic evaluation of protecting
groups for the 2-azido-2-deoxy-D-glucosyl donors, we found that the installation of tert-butyldiphenylsilyl group at6-O, 2-naphthylmethyl group at 4-O, andp-bromobenzyl group at the 3-O position
confer exclusive α-stereoselectivity regardless of leaving group, activator, and sugar
acceptor. Even more noteworthy is thatthese protecting groups are orthogonal
and flexible enough to allow functionalgroup manipulations, enabling us to generate several disaccharide building blocks
from a single precursor. Complementing
the potential disaccharides present in HS,
our laboratory prepared 48 disaccharidebuilding blocks that may be assembled
and eventually transformed to target any
structure that could be found in the natural compound. We have accomplished thetotal synthesis of several lengths of HSbased oligosaccharides containing identical repeating disaccharide components.
The length-dependent interactions of thesesugars with eosinophil cationic protein,
eosinophil-derived neurotoxin, fibroblast
growth factors, and mycobacterial heparin-binding hemagglutinin were examinedusing either cell-based inhibition or directbinding assays. In one groundbreaking
achievement, the chemical preparation of
two HS-based 3-O sulfonated octasaccharides possessing non-regular disaccharide
repeats were completed by us. This effortwas also remarkable because one of thetarget compounds carry free amino, acetamido, and sulfonatamido functionalities,
a difficult feat to realize in HS-based oligosaccharide synthesis. Both oligosaccharides inhibited the herpes simplex virus
1 infection of Vero cells. Identifying theoptimal sugar sequence sought by binding proteins require a series of compoundswith variable functional group patterns.
However, for making such a compound
library to be practical, efficient strategies
that minimize the number of synthetic
steps must be adopted. By this manner,
we undertook the procurement of all 48
HS-based disaccharides from only twoorthogonally protected disaccharide precursors using different combinations andsequence of functional group transformation steps. Binding assays conducted
between the synthesized disaccharides
and fibroblast growth factor-1 using isothermal titration calorimetry showed that
only four compounds significantly boundthe growth factor. The molecular details of
the interactions were revealed by the X-ray
co-crystal analysis of the identified sugars
and the protein. Altogether, our findings
illustrate the value of synthetic chemistryin advancing the current understanding ofthe functions embedded within the heparanome.

References1) 	Bishop JR, Schuksz M, Esko, JD. Heparan sulphate proteoglycans fine-tune 7) 	Wang CC, Lee JC, Luo SY, Kulkarni SS, Huang YW, Lee CC, Chang KL, Hungmammalian physiology, Nature (446): 1030-1037, 2007.
SC. Regioselective one-pot protection of carbohydrates, Nature (446): 8962) 	Turnbull JE. Heparan sulfate glycomics: towards systems biology strategies,899, 2007.
Biochem. Soc. Trans. (38): 1356-1360, 2010.
8) 	Hu YP, Lin SY, Huang CY, Zulueta MML, Liu JY, Chang W, Hung SC. Synthe3) 	Hsu CH, Hung SC, Wu CY, Wong CH. Toward automated oligosaccharide
sis of 3-O-sulfonated heparan sulfate octasaccharides that inhibit the herpes
synthesis, Angew. Chem., Int. Ed. (50): 11872-11923, 2011.
simplex virus type 1 host–cell interaction, Nat. Chem. (3): 557-563, 2011.
4) 	Lee JC, Lu XA, Kulkarni SS, Wen YS, Hung SC. Synthesis of heparin oligosac- 9) 	Fan TC, Fang SL, Hwang CS, Hsu CY, Lu XA, Hung SC, Lin SC, Chang MDT.
charides, J. Am. Chem. Soc. (126): 476-477, 2004.
Characterization of molecular interactions between eosinophil cationic pro5) 	Lu LD, Shie CR, Kulkarni SS, Pan GR, Lu XA, Hung SC. Synthesis of 48 ditein and heparin, J. Biol. Chem. (283): 25468-25474, 2008.
saccharide building blocks for the assembly of a heparin and heparan sulfate 10) 	Zulueta MML, Lin SY, Lin YT, Huang CJ, Wang CC, Ku CC, Shi Z, Chyoligosaccharide library, Org. Lett. (8): 5995-5998, 2006.
an CL, Irene D, Lim LH, Tsai TI, Hu YP, Arco SD, Wong CH, Hung SC.
6) 	Hung SC, Lu XA, Lee JC, Chang MDT, Fang SL, Fan TC, Zulueta MML,
α-Glycosylation by D-glucosamine-derived donors: synthesis of heparosan
Zhong YQ. Synthesis of heparin oligosaccharides and their interaction withand heparin analogs that interact with mycobacterial heparin-binding hemeosinophil-derived neurotoxin, Org. Biomol. Chem. (10): 760-772, 2012.
agglutinin, J. Am. Chem. Soc. (134): 8988-8995, 2012.

Commemorative Symposium

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