Document Detail


Conjugation of unprotected trisuccin, N-[tris[2-[(N-hydroxyamino)carbonyl]ethyl]methyl]succinamic acid, to monoclonal antibody CC49 by an improved active ester protocol.
MedLine Citation:
PMID:  9327144     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
For the conjugation of the trihydroxamate bifunctional chelating agent N-[tris[2-[[N-(benzyloxy)amino]-carbonyl]ethyl]methyl]succinamic acid (trisuccin, 1) to antibodies, we originally used the corresponding 2,3,5,6-tetrafluorophenyl active ester followed by the postconjugation removal of the benzyl protecting groups by catalytic hydrogenation. It was of interest to us to design a conjugation protocol capable of incorporating deblocked hydroxamates into peptides and proteins. Reported procedures that were expected to be compatible with the functionalities present in trisuccin were used with no success, as judged by the lack of ability of the products to radiolabel with 188Re. A simple conjugation method was then developed utilizing the o-nitrophenol (ONP) activated ester of the unprotected trisuccin, N-[tris[2-[(N-hydroxyamino)carbonyl]ethyl]methyl]succinamic acid, 3, which eliminates the need for the postconjugation deblocking. An assay for indirect estimation of the active ester content, based on the concentration of its decomposition byproduct, ONP-OH, was developed. Comparison of the indirectly estimated concentrations with those obtained directly from purified products showed > 90% accuracy for this assay. This procedure has the advantage of rapidly using the unpurified active ester, eliminating the possibilities of its decomposition through solvolysis or self-condensation by the unprotected hydroxamate functions. A colorimetric assay was developed for estimation of the number of ligands per molecule of protein. This assay and the fact that all conjugates consistently radiolabeled with 188Re show that this procedure conjugated the unprotected hydroxamate ligands to the CC49 monoclonal antibody. These results indicate the potential applicability of this technique to conjugation of unprotected hydroxamate derivatives with other proteins and peptides.
Authors:
A Safavy; A Sanders; H Qin; D J Buchsbaum
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Bioconjugate chemistry     Volume:  8     ISSN:  1043-1802     ISO Abbreviation:  Bioconjug. Chem.     Publication Date:    1997 Sep-Oct
Date Detail:
Created Date:  1997-11-28     Completed Date:  1997-11-28     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  9010319     Medline TA:  Bioconjug Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  766-71     Citation Subset:  IM    
Affiliation:
Department of Radiation Oncology, University of Alabama at Birmingham 35294, USA. radt004@uabdpo.dpo.uab.edu
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MeSH Terms
Descriptor/Qualifier:
Antibodies, Monoclonal / chemistry*
Chromatography, High Pressure Liquid
Cross-Linking Reagents / chemistry*
Esters / chemical synthesis,  chemistry
Hydroxamic Acids / chemistry*
Isotope Labeling
Ligands
Proteins / chemistry
Rhenium
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Spectrophotometry, Ultraviolet
Succinates / chemistry*
Grant Support
ID/Acronym/Agency:
CA44173/CA/NCI NIH HHS; CA62550/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Antibodies, Monoclonal; 0/Cross-Linking Reagents; 0/Esters; 0/Hydroxamic Acids; 0/Ligands; 0/Proteins; 0/Succinates; 147219-26-9/trisuccin; 7440-15-5/Rhenium

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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