Design And Synthesis Heparan Sulfate(HS)-Derived Oligosaccharide Thioglycoside Heparanase(HPSE) Inhibitors

Multiple myeloma(MM),the second most common hematologic malignancy,is a Bcell malign ancy that is difficult to cure.Chemotherapy is the most widely used strategy for the treatment of multiple myeloma.In most myeloma patients,after several rounds of intensive th erapy,tumor cells still survive and proliferate,leading to relapse and drug resistance.Many studies have found that this resistance is closely related to high expression of hep aranase(HPSE).HPSE is the only β-D-glucuronidase in mammals,which can degrade heparan sulfate(HS)of heparan sulfate proteoglycans(HSPGs)in the extracellular matrix(ECM),release growth factors,and reconstruct network of ECM,leading to tumor growth and metastasis.Lysosomes HPSE causes drug resistance through enhanced autophagy.Clinically,HPSE that is closely related to tumor growth,m etastasis and drug resistance is significantly up-regulated in all the tested tumors.Therefore,the development of HPSE inhibitors has become a new potential strategy.In this study,the repeating disaccharide unit contained in natural substrate HS was used as the lead structure,and the crystal model of HPSE and its the minimum substrate trisaccharide complex was applied as a design guide,and then an oxygen(O)atom at the HPSE cleavage site was replaced with a sulfur(S)atom using isostere principle.Based on those strategies,we design and synthesize structurally defined HS-based HPSE inhibitors with the maintained specificity and enhaced half-lives.The work included the following three aspects:i)Synthesis of sugar receptors and donors of HS thioglycoside;ii)Syntheses of HS disaccharide thioglycoside and HS trisaccharide thioglycoside;iii)Establishment of an evaluation model for HPSE inhibitors.(1)Synthesis of the monosaccharide receptors and donors for HS thioglycosidesLike HS structure,the designed target compounds HS sulfonates were composed of a negatively charged glucuronic acid(GlcA)and an N-acetylglucosamine(GlcNAc)or Nglucosamine sulfonate(GlcNS)in the formation of a thioside linkage.Firstly,1-OH glucoglucuronic acid donor acitivated by-SAc and 4-OH-N-acetyl/trifluoroacetyl glucosanine receptor activated by-OTf were synthesized,and then,they are,under the action of catalysts,coupled to disaccharide thioglycosides,an d furth er elongated to trisaccharide thioglycosides with enzym e-catalyzed assembly.The synthesis of 1-SAc-substituted glucuronic acid as a donor started from glucuronic acid.After peracetylation,hydrolysis and methylation of position 6,activation and substitution with 1-SAc at position 1,the activated glucuronic acid donor(G6’)was obtained in a five-step reaction with a total yield of 31.7%.The reaction route was short and well repeated.N-acetylglucosamine was a starting point for the synthesis of 4-OTf-Nacetylglucosamine(GlcNHAc-C4-OTf)(N8),which was obtained through a seven-step reaction including 4,6-OH protected with benzaldehyde,acetylation,two-time configuration inversion at position 4,and selective benzoylation of position 6 in a total 11.9%yield.N-acetylglucosamine hydrochloride severed as a starting material for the synthesis of 4site-OTf-activated trifluoroacetaminosamine(GlcNHTFA-C4-OTf)receptor(T9),which was achieved by an 8-step reaction such as 2-protec ted with TFA,selective benzoylation of position 6,and two-time configuration inversion at position 4 in a total 6.73%yield.(2)Synthesis of HS disaccharide thioglycoside and HS trisaccharide thioglycosideGlucuronic acid donor(G6’)and activated 4-OTf-N-acetylglucosamine(GlcNHAc-C4OTf)receptor(N8)were coupled to form HS disaccharide thioglycosides,followed by deacetylation,activation and coupling to offor p-nitrobenzyl substituted-N-acetyl thiodisaccharide(E4).The glucuronic acid donor(G6’)and activated 4-OTf-N-trifluoroacetylglucosamine(GlcNHTFA-C4-OTf)receptor(T9)underwent the same reaction as compound E4 to obtain p-nitrobenzyl-N-trifluoroacetyl thiodisaccharide(T13).The starting materials for synthesizing trisaccharides were N-acetylglucosamine(GlcNHAc)or trifluoroacetaminosamine(GlcNHTFA)and the fully deprotected compound EHS-1 from the disaccharide thioglycoside(E4).In the Tris-HCl(pH=7.0)buffer solution,N-acetylglucosamine(GlcNHAc)or N-trifluoroacetaminosamine(GlcNHTFA)was activated with enzyme Nahk to transfer a phosphate group from ATP to the 1-OH of a sugar residue,and in situ react with UTP to obtain the UDP-1-GlcNHAc or UDP-1-GlcNHTFA under the action of two enzymes,GlmU and PPA.Finally,in Tris-HCl(pH=7.5)solution,under the action of PmHS2 disaccharide thioglycoside(EHS-1)was elongated,respectively,to N-acetyltrisaccharide thioglycoside(EHS-2)and trifluoroacetyltrisaccharide thioglycoside(EHS-3).(3)Establishment of an evaluation model for HPSE inhbitorsIt is important to evaluate the inhibitory activity of heparan sulphate thioglycosides towards HPSE,so we have constructed a high-throughput screening model for HPSE inhibitors.To establish a simple,convenient and accurate screeing model with easy operation and wide practicability,we compared our previous evaluation models for determination of heparanase activity.In this thesis,we adopted an improved sulfonate heparin(FPX)-based fluorescence m ethod,where sulfonate heparin(FPX)as the substrate was degraded by HPSE to form a trisaccharide and a reducing disaccharide,and the fluorescence colorimetric formation of a reducing disaccharide and 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5tetrazolyl]-1,3-benzodisulfonate(WST-1)reflected quantitatively the HPSE degradation.capacity.Adding inhibitors tested to competitively interfer with the abilities of HPSE degradation affect the fluorescence intensity,and the concentration and fluorescence intensity were proportional.We plotted the standard curves for different reaction time,enzyme concentration and substrate concentration.and then obtained the optimal reaction conditions as follows:the reaction time was 2 hours,the enzymatic concentration was 2 μg/mL and the FPX constration was 50 pg/ml,wh ich laid a solid foundation for discovering HPSE inhibitors using high-throughput screening technology.Collectively,the thesis decribed the synthesis of three kinds of sugar resides required for heparin sulfate thioglycoside,including activated 4-OTf-N-acetyl glucosamine,and activated 4-OTf-N-acetyl glucosamine,and 1-thioacetyl glucuronic acid.Based on these.residues,two disaccharides thiogylcosides were syntesized by chemical methods,and onetrisaccharides thiogylcosides were assembled using a chemo-enzymatic synthesis stategy,and finally,a high-throughput screening model for HPSE inhibitors was established.These works provided a good experimental basis for the synthesis of heparan sulphate thioglycosides and screening the anti-HPSE activities.