Dissertation/Thesis Abstract

Konvergente Synthesemethoden für N- und O-glycosylierte Proteine
by Rädisch, Marisa, Dr.Nat., Universitaet Bayreuth (Germany), 2016, 201; 27600555
Abstract (Summary)

Using the convergent glycopeptide synthesis it is possible to synthesize libraries of homogeneously glycosylated peptides. By means of these homogeneous glycoforms the structure-activity relationship can be investigated. The HIV-1 envelop protein gp120 is highly glycosylated. This represents a target for selective recognition through broadly neutralizing, monoclonal antibodies (PG9). In the context of HIV-1 vaccine design glycopeptide mimetics of gp120 could be synthesized, which are recognized by antibodies like PG9. In cooperation with the research group of J. A. Robinson the synthesis of a glycosylated HIV-1 B/C loop mimetic of CAP45 from Phe-159 to Ala-172 was pursued. In order to stabilize the β-turn of 12, the templat D-Pro/LPro was incorporated. A pseudoproline dipeptide was incorporated adjacent to the glycosylation site Asn-160 in order to reduce aspartimde formation. The peptide 7 (1.8 mM) was cyclised with HATU/HOAt and subsequently desallylated. Afterwards GlcNAc-NH2 11 and Man5GlcNAc2- NH2 14 respectively was coupled. Both mimetics 12 and 15 were not recognized by the antibody PG9, since the peptide backbone did not form a stable β-hairpin structure. Consequently, alanine-172 was replaced with valine and arginine-166 and Leucin-165 were removed. This led to the optimised glycopeptide 19, which was recognized by the antibody PG9. In the context of the synthesis of bioconjugates via click chemistry a glycopeptide was obtained. The conjugate 25 was obtained by a copper(I) catalysed cycloaddition of the alkyne 23 to the azide 22. The V1/V2 mimetic was then immobilised on an appropriate carrier. In cooperation with the R. G. Lichtenstein research group the synthesis of a library of homogeneously glycosylated IgG1 Fc-fragments was pursued. The assumption is that death of neuronal cells in ALS patients is related to the structure of the N-glycans conjugated to the Fc of ALS-specific antibodies. The interaction of glycosylated Fc-fragments and its Fc-receptors should be investigated. The peptide thioesters IgG1 Fc 223-260 36 and IgG1 Fc 261-286 43 were synthesized by solid phase peptide synthesis. The glycopeptide hydrazide 50 was obtained by convergent glycopeptide synthesis. Besides IgG1 Fc 287-320 53 with bisected N-glycan was obtained. The glycopeptide IgG1 Fc 261-286 59 was accessible by the deprotection of the glycopeptide 50 and subsequent native chemical ligation with the thioester 43. By metal free desulfurization the Cys-287, which was incorporated in order to enable a NCL, was converted to a native Alanine. During desulfuration the native Cys-261 was protected by a PhAcm group. Subsequent deprotection of the PhAcm group was performed enzymatically by Penicillin G Acylase in 2 M GdmCl. Thiolysis of the hydrazide 73 and the last NCL with the Cys-fragment 75 was not successfull, because of the ε-caprolactam formation of the C-terminal lysine of the thioester 74. The investigation of the glycosaminoglycans of proteoglycans is important, since deficient GAG’s causes human diseases. Herein the semisynthesis of the core proteoglycan bikunin was described. Bikunin was split into three fragments. The O-glycopeptide bikunin 1-25 88 and the N-glycopeptide bikunin 26-50 108 was synthesized as a thioester or hydrazide. The coupling condition of the O-glycosyl aminoacid 82 was optimized in order to prevent racemization. The N-glycopeptide was obtained in a convergent approach by use of the glycosylamine 48. The Cys-fragment bikunin 51-147 120 was recombinantly expressed in E. coli and liberated by enzymatic removal of the His6-SUMO tag. The native chemical ligation of the thioester 88 and the Cys-fragment 108 led to bikunin 1-50 111. Finally, the N- and O-glycosylated bikunin 1-147 121 was achieved after the conversion of the hydrazide 111 to the thioester 116 and the subsequnt native chemical ligation with bikunin 51-147 120. After oxidative refolding bikunin 121a was obtained, which showed activity against trypsin.

Indexing (document details)
Advisor:
Commitee:
School: Universitaet Bayreuth (Germany)
School Location: Germany
Source: DAI-C 81/4(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Bioengineering, Biochemistry, Bioinformatics
Keywords: Glycopeptides
Publication Number: 27600555
ISBN: 9781687909657
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