Dissertation/Thesis Abstract

Synthese von Glycoformen des humanen Erythropoietins
by Seeleithner, Simone Tamara, Dr.Nat., Universitaet Bayreuth (Germany), 2019, 175; 27600399
Abstract (Summary)

Glycoproteins play an important role at many biological processes. Since naturally occuring glycoproteins always exhibit microheterogeneity, but for structure-activity relationship studies homogeneous glycoforms are necessary, they need to be assembled by chemical and enzymatic synthesis. Immunoglobulin G1 possesses one N-glycan in the Fc region. The Fc 287-320 glycopeptide hydrazide 1 was assembled by Fmoc-SPPS and pseudoproline-assisted Lansbury aspartylation. A TFAc protective group was used to prevent cyclization of the C-terminal lysine and its applicability for ligations was confirmed. Erythropoietin regulates the oxygen homoiostasis in the human body by stimulation of erythropoiesis. Human erythropoietin possesses three N-glycans whose structure influences the in vivo activity significantly. To obtain monoglycosylated EPO A through semisynthesis the EPO 1 28 (nona) hydrazide 23 was assembled by convergent glycopeptide synthesis and the sugar was sialylated enzymatically. After thioesterification and following ligation with the recombinant EPO 29-166 peptide fragment C oxidative refolding provided EPO (undeca) A with an α-2,6-sialylated complex N-glycan at Asn-24 in 33% yield. The total synthesis strategy for triglycosylated EPO D envisaged the sequential NCL of the five fragments B2-H2 with desulfurization of three cysteines. Different protective group strategies were investigated. Within the Trt/Nvoc strategy protection of the native cysteines-29, 33 and 161 was maintained by an acid labile trityl group, which was introduced afterwards at the deprotected peptides. The C terminal Lys-97 was protected by a Nvoc group in the side chain. EPO 29-67 (2xSTrt, nona) hydrazide 29 gave low yields at retritylation due to the hydrophobic trityl protective groups. The synthesis of the peptide fragment EPO 98-166 (STrt) 33 was carried out by NCL. The poorly soluble biglycosylated EPO 29-97 (2xSTrt, Nvoc) hydrazide 34 was connected to 33 after thioesterification, however, the trityl groups hampered the isolation of EPO 29-166 (3xSTrt, 2xNona, Nvoc) 36. Due to the difficulties the Acm/TFAc strategy was applied for assembling EPO with three complex N-glycans D. The glycopeptide hydrazide EPO 29-67 40 was synthesized with an Acm protective group at Cys 29 and 33. The lability of the Acm group at palladium catalyzed deprotection of the glycosylation site was circumvented by use of a PhiPr protective group. EPO 68-97 (nona) F2 was assembled with a TFAc protected Lys-97. The α 2,6-sialylation of the biantennary N-glycans at the glycopeptide hydrazides 40 and F2 was successful. After conversion of 40 into the thioester E2 ligation with F2 to biglycosylated EPO 29-97 (2xSAcm, TFAc) 47 and its thioesterification was carried out. At the peptide EPO 98-166 (Thz, SPhacm) 51 removal of both Cys protective groups with PdCl2 was tested. Here a higher lability of Phacm compared to Thz was observed. At the ligation product EPO 29 166 (3xSAcm, 2xnona, TFAc) 55 the non-native cysteines-68, 98 and 128 were desulfurized to native alanines. Removal of the Acm groups of the three remaining cysteines by PdCl2 resulted in EPO 29-166 (3xSH, 2xnona, TFAc) 58. Basic removal of the TFAc group tended to side reactions, but finally succeeded at 58. The EPO 1-28 (nona) thioester B2 and EPO 29 166 (3xSH, 2xnona) 59 were ligated to the full length glycoprotein Dred and oxidatively refolded to EPO (3xnona) D. Because of numerous problems removing the TFAc protective group the synthesis of triglyco-sylated EPO D was pursued according to the Acm strategy. First of all the TFAc group at Lys-97 of the EPO 68-97 (TFAc) glycopeptide hydrazide F2 was removed, which proved to be non-problematic at this step. By thioesterification and sequential NCL EPO 29 166 (3xSAcm, 2xnona) 62 was obtained. An ε-caprolactam formation at Lys-97 did not occur. After desulfurization and removal of the Acm groups NCL with the EPO 1 28 (nona) thioester B2 and following oxidative refolding lead to the desired tri-N-glycosylated EPO D. The yield accounted for 11%. Thus, with the Acm strategy a viable procedure for the total synthesis of homogeneous tri-N-glycosylated EPO was established.

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: Biochemistry, Organic chemistry
Keywords: Erythropoietin
Publication Number: 27600399
ISBN: 9781687902702
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