2026-09-19T17:57:20Zhttps://dspace.mit.edu/server/oai/requestoai:dspace.mit.edu:1721.1/96562022-01-13T07:54:20Zcom_1721.1_7582com_1721.1_7581col_1721.1_131022
Satoru Masamune.
Fleck, Roman (Roman W.), 1966-
Massachusetts Institute of Technology. Department of Chemistry
2005-08-19T19:14:33Z
2005-08-19T19:14:33Z
1999
1999
http://hdl.handle.net/1721.1/9656
42423662
Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1999.
Vita.
Includes bibliographical references (leaves 101-107).
The first part of the thesis describes a novel approach to generate amide cleaving antibodies. Two haptens, O1 and O2, were designed and synthesized to create a hydrophobic pocket for an auxiliary nucleophile as well as elicit complementary basic/acidic side chain residues in the antibody combining binding site. The recently established heterologous immunization protocol was utilized to induce multiple catalytic side chain residues in antibodies. Three antibodies were isolated capable of catalyzing the hydrolysis of propionyl-p-nitroanilide in the presence of phenol, as the nucleophilic cofactor. Further studies with substrate and nucleophile analogs clearly showed that the nucleophilic phenolic hydroxyl group is essential for catalysis, much like amide hydrolysis catalyzed by serine proteases. The second part of the thesis describes the generation of catalytic antibodies capable of removing a protection group for the simultaneous activation of multiple prodrugs. To this end, a protective group was designed which could be attached to any kind of cancer drug, in order to convert a cancer drug into a prodrug. The protective group relies on a [beta]-sulfone elimination process to release the active drug. The corresponding hapten design includes a non-specific element to allow broad substrate tolerance with regard to the drug portion. Two haptens, RI and R2, both structurally similar, but with different functionalities were designed and synthesized. Both haptens include a perfectly placed ammonium residue for a-proton abstraction. The second hapten design, RI, further sought to induce an acidic residue for carbamate leaving group stabilization. A comparison of both haptens showed that leaving group stabilization did not improve antibody performance. Another subject crucial to the prodrug activation project concerns the detection of catalytic activity in hybridoma supematants. A new method called "Capture"-CatELISA was developed, which allowed the efficient screening for catalysis of hundreds of hybridoma supematants in a very short period of time. To this end, a substrate Cl was developed, which upon activation covalently traps catalytic antibodies, and therefore immobilizes them on a solid support. A conventional ELISA-assay was then applied to identify the catalytic clones.
by Roman Fleck.
Ph.D.
138 leaves
9178708 bytes
9178465 bytes
application/pdf
application/pdf
eng
Massachusetts Institute of Technology
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http://dspace.mit.edu/handle/1721.1/7582
Chemistry
Catalytic antibodies for amide cleavage and prodrug activation
Catalytic antibodies from amide hydrolysis to prodrug activation
Thesis
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<Abstract>The first part of the thesis describes a novel approach to generate amide cleaving antibodies. Two haptens, O1 and O2, were designed and synthesized to create a hydrophobic pocket for an auxiliary nucleophile as well as elicit complementary basic/acidic side chain residues in the antibody combining binding site. The recently established heterologous immunization protocol was utilized to induce multiple catalytic side chain residues in antibodies. Three antibodies were isolated capable of catalyzing the hydrolysis of propionyl-p-nitroanilide in the presence of phenol, as the nucleophilic cofactor. Further studies with substrate and nucleophile analogs clearly showed that the nucleophilic phenolic hydroxyl group is essential for catalysis, much like amide hydrolysis catalyzed by serine proteases. The second part of the thesis describes the generation of catalytic antibodies capable of removing a protection group for the simultaneous activation of multiple prodrugs. To this end, a protective group was designed which could be attached to any kind of cancer drug, in order to convert a cancer drug into a prodrug. The protective group relies on a [beta]-sulfone elimination process to release the active drug. The corresponding hapten design includes a non-specific element to allow broad substrate tolerance with regard to the drug portion. Two haptens, RI and R2, both structurally similar, but with different functionalities were designed and synthesized. Both haptens include a perfectly placed ammonium residue for a-proton abstraction. The second hapten design, RI, further sought to induce an acidic residue for carbamate leaving group stabilization. A comparison of both haptens showed that leaving group stabilization did not improve antibody performance. Another subject crucial to the prodrug activation project concerns the detection of catalytic activity in hybridoma supematants. A new method called "Capture"-CatELISA was developed, which allowed the efficient screening for catalysis of hundreds of hybridoma supematants in a very short period of time. To this end, a substrate Cl was developed, which upon activation covalently traps catalytic antibodies, and therefore immobilizes them on a solid support. A conventional ELISA-assay was then applied to identify the catalytic clones.</Abstract>
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