Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2007.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2007
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| Online Access: | http://hdl.handle.net/1721.1/38661 |
| _version_ | 1826204505632407552 |
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| author | Lohman, Gregory J. S |
| author2 | JoAnne Stubbe. |
| author_facet | JoAnne Stubbe. Lohman, Gregory J. S |
| author_sort | Lohman, Gregory J. S |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2007. |
| first_indexed | 2024-09-23T12:56:41Z |
| format | Thesis |
| id | mit-1721.1/38661 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T12:56:41Z |
| publishDate | 2007 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/386612019-04-10T12:12:52Z Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate Lohman, Gregory J. S JoAnne Stubbe. Massachusetts Institute of Technology. Dept. of Chemistry. Massachusetts Institute of Technology. Dept. of Chemistry. Chemistry. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2007. Vita. Includes bibliographical references. Efforts towards the Synthesis of Fully N-Differentiated Heparin-like Glycosaminoglycans. Heparin-like glycosaminoglycans (HLGAGs) are complex information-carrying biopolymers and are an important component of the coagulation cascade. They have also been implicated in interactions with growth factors, cytokines, virus entry, and other functions. Currently, no general synthesis of arbitrary HLGAG sequences has been demonstrated. The modular synthesis of glycosaminoglycans requires straightforward methods for the production of large quantities of protected uronic acid building blocks. An efficient route to methyl 3-0- benzyl-1,2-O-isopropylidene-a-L-idopyranosiduronate from diacetone glucose in nine steps and 36% overall yield is described. Additionally, a general method for the conversion of glycals to the corresponding 1,2-cis-isopropylidene-a-glycosides is reported. Epoxidation of glycals with dimethyldioxirane followed by ZnC12-catalyzed addition of acetone converted a variety of protected glycals into 1,2-cis-isopropylidene-a-glycosides in good yield. The reaction is compatible with a range of protecting groups, as well as free hydroxyl groups. This method has been applied to develop a synthesis of 3-O-benzyl-1,2-O-isopropylidene-P-D-glucopyranosiduronate in seven steps and 32% overall yield. (cont.) These compounds are useful as glycosyl acceptors and as intermediates that may be further elaborated into uronic acid trichloroacetimidate glycosyl donors for the assembly of glycosaminoglycan structures. The glucosamine residues in HLGAGs have been found to exist as amines, acetamides, and N-sulfonates. In order to develop a completely general, modular synthesis of heparin, three degrees of orthogonal nitrogen protection are required. Reported is a strategy for the synthesis of fully N-differentiated heparin oligosaccharides in the context of target octasaccharide 3-1, which contains an N-acetate, N-sulfonates, and a free amine. The protecting group scheme used in the synthesis blocked the N-acetate as a N-diacetate, the N-sulfonates as azido groups, and the amine as a N-CBz; free hydroxyls were masked as benzyl ethers and O-sulfonates as acetate esters. Disaccharide and tetrasaccharide modules were synthesized using this strategy; however, the union of tetrasaccharide trichloroacetimidate 3-4 with disaccharide acceptor 3-5 unexpectedly formed the undesired P-linked glycoside in addition to the a-linkage anticipated for iduronic acid nucleophiles, resulting in an inseparable 6:1 a: p mixture of products. Detailed studies into the basis for this unexpected result were conducted and are also reported. (cont.) Investigations into the Mechanism of Inactivation of RTPR by Gemcitabine Triphosphate. Ribonucleoside triphosphate reductase (RTPR) is an adenosylcobalamin (AdoCbl) dependant enzyme that catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates via controlled radical chemistry. The antitumor agent 2',2'-difluoro-2'- deoxycytidine (gemcitabine, F2C) has been shown to owe some of its in vivo activity to inhibition of human RNR by the 5'-diphosphate (F2CDP). Previous studies have shown that RTPR is rapidly inactivated by one equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP). This inactivation is associated with the release of two equivalents of fluoride and modification of RTPR by a Co-S bond between C419 and the cobalamin cofactor. In order to further characterize this inactivation, isotopically labeled derivatives of F2CTP were synthesized: radiolabeled 1'-[3H]-F2C and mass labeled 1'-[2H]-F2C and 3'-[2H]-F2C. These compounds were converted to F2CTP through a set of enzymatic phosphorylation steps which overcome difficulties found using traditional, chemical methods. Biochemical investigations were performed using these labeled derivatives to track the fate of the base and sugar during RTPR inactivation by F2CTP. (cont.) The release of cytosine base, previously overlooked in this system, was detected utilizing 5-[3H]-F2CTP: 0.7 equiv. of cytosine were released, with 0.15-0.2 equiv. of unreacted F2CTP remaining. Size exclusion chromatography (SEC) was used to quantify covalent labeling of RTPR by F2CTP: 0.15 equiv. were detected using 5-[3H]-F2CTP, 0.45 equiv. were detected using 1'-[3H]-F2CTP. A small molecule nucleotide product was identified in inactivation mixtures quenched with NaBH4 and identified as an isomer of cytidine, indicating the loss of both fluorides and the addition of an oxygen at the 2' carbon. RTPR inactivated with 1'-[3H]-F2CTP was digested with trypsin and peptides containing radioactivity purified. Identical peptides were prepared using partially deuterated F2CTP, allowing identification by MALDI-MS. Post source decay (PSD) MS/MS methods were used to further characterize these peptides, identifying the site of label as the C-terminal tryptic peptide of RTPR at C731 and C736. The cysteines were labeled through conjugate addition with a furanone-like precursor that had lost cytosine, triphosphate, and both fluorines. The results of these studies have allowed for the first time the proposal of a mechanistic hypothesis for RTPR inactivation by F2CTP. by Gregory J.S. Lohman. Ph.D. 2007-08-29T20:41:08Z 2007-08-29T20:41:08Z 2006 2007 Thesis http://hdl.handle.net/1721.1/38661 163249285 eng M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 2 v. (591 p.) application/pdf Massachusetts Institute of Technology |
| spellingShingle | Chemistry. Lohman, Gregory J. S Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate |
| title | Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate |
| title_full | Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate |
| title_fullStr | Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate |
| title_full_unstemmed | Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate |
| title_short | Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate |
| title_sort | efforts towards the synthesis of fully n differentiated heparin like glycosaminoglycans and investigations into the mechanism of inactivation of rtpr by gemcitabine triphosphate |
| topic | Chemistry. |
| url | http://hdl.handle.net/1721.1/38661 |
| work_keys_str_mv | AT lohmangregoryjs effortstowardsthesynthesisoffullyndifferentiatedheparinlikeglycosaminoglycansandinvestigationsintothemechanismofinactivationofrtprbygemcitabinetriphosphate |