Enzymatic Synthesis Of Homogeneous Hyaluronan And Related Biological Functions

Glycosaminoglycans(GAGs)are naturally heteropolysaccharides in mammals that are consisting of repeating disaccharide repeats and are present in proteoglycans or free forms.The important physiological functions of GAGs and their close relationships with diseases have been well-documented by scientists in recent years.Hyaluronan(HA)is the most simple GAQ which is linked by disaccharide repeating units of[-D-GlcA-?1,3-GlcNAc-?1,4-].Unlike other GAGs,HA is not sulfated and does not covalently bind to the protein and is present in free form.HA is widely present in animal tissues and certain microorganisms,and has been widely used in food,cosmetics and medicine fields because of its excellent water retention,viscoelasticity and biocompatibility.In recent years,HA has been involved in the important physiological processes such as cell recognition and signal transduction,and has played a multiple biological effects in the development of inflammatory reaction and tumor.Metabolic abnormality is an important feature of tumor.In tumor microenvironment,the dynamic equilibrium of HA synthesis and degradation is abnormal,and the change of sugar chain length is an important manifestation and one of the evaluation indexes of tumor malignancy.There are numerous HA-specific receptors and binding proteins on the surface of tumor cells.HA interacts with receptors or binding proteins to activate subsequent multiple signal pathways in the cells and participates in the biological effects of tumor development and inflammatory response.It is noteworthy that,unlike the synthetic process of other biological effect molecules,the biological activity of HA is regulated by it sugar chain length,and HA exhibits a chain length dependent biological effects.The commercial HA has a high degree of heterogeneity,which seriously hindering the illustration of HA chain length-effect relationship.Currently,commercial HA mainly produce in two methods.First,animal tissue extraction,the yield is low,and has many problems like restricted animal materials and remains of animal virus.Second,microbial fermentation,this method has low cost,large scale and unlimited by raw materialsin,but risk of contaminations with bacterial endotoxin,nucleic acid,protein and heavy metal still existed.The above method has been developed in the preparation of commercial HA,however,homogeneous HA with any desired chain length is still unacessibile.In this dissertation,we studied the catalytic mechanism of HA synthase from Pasteurella multiflora(PmHAS),and developed a general strategy for the synthesis of homogeneous HA conjugates which combined stepwise PmHAS-catalyzed HA oligosaccharide synthesis and one-pot homogeneous HA polymerization.In the second chapter,we studied the catalytic reaction mechanism of PmHAS,and found that PmHAS was able to use an HA trisaccharide as the necessary primer to catalyze the formation of homogeneous HA polymers.The acceptor/donor molar ratio was the key factor for final HA chain length.Homogeneous HMW-and LMW-HAs with a range of desired HA chain lengths could be efficiently synthesized by controlling the acceptor/donor molar ratio involved in the reaction system.More importantly,owing to flexible substrate specificity of PmHAS,the general synthetic strategy was fiirther exploited to produce homogeneous HA conjugates.A library of homogeneous HA-biotin conjugates with definite HA chain lengths were efficiently prepared and used as fluorescently labeled probes to investigate HA biological functions in tumor cells.A series of homogeneous HA-drug conjugates with both definite chain lengths and specific modification sites were generated to illustrate the crucial relationships between HA chain length and biological functions.In the third chapter,we developed a strategy for the synthesis of hyaluronan-chondroitin(HA-CS)hybrid GAG Due to the structural similarity of HA and CS,and the adaptability of PmHAS,four kinds of HA-CS hybrid GAG(CS2HA2-N3,HA2CS2HA2-N3,CS2HA4-N3 and CS4HA4-N3 HA-CS)were synthesized in a step-wise enzymatic synthesis manner,which could facilitate the studies on structure-function relationship of HA.The strategy can also be applied to the synthesis of other hybrid GAGs.In the fourth chapter,based on our previous studes on sugar nucleotides biosynthesis,a sequential one-pot multienzyme(OPME)strategy for in vitro synthesis of HA and its derivatives was developed.The strategy that combined sugar nucleotides in situ generation and HA synthase catalyzed chain polymerization could convert cheaper starting monosaccharides into HA polymers without consuming exogenous sugar nucleotides donors.More importantly,azide-modified HA derivative was well-controlled synthesized and applied for crosslinking hydrogen studies.This work highlights the potential application of sequential OPME strategy in glycoconjugates biosynthesis.