Bioconjugate Chem. lQ02, 3, 514-518
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Acidic Derivatives of Homocysteine Thiolactone: Utility as Anionic Linkers W. J. Leanza, L. S. Chupak, R. L. Tolman, and S. Marburg' Department of Medicinal Chemistry, Merck Research Laboratories, Box 2000, Rahway, New Jersey 07065. Received June 15,1992 Homocysteine thiolactone (2) derivatives in which the nitrogen is acylated with groups containing acidic functionalities have been synthesized. These include the succinyl (31, the carboxymethylglutaryl (4), the 3-phosphonopropionyl(7),and the 3-sulfopropionyl(8) derivatives. These thiolactones can be used to introduce a thiol functionality into proteins such as the outer membrane protein complex of Neisseria meningitidis (OMPC) allowing conjugation with electrophilic ligands. This chemistry is the same as with N-acetylhomocysteine thiolactone (11, but their pK, values are such that at pH 7 concomitant negative charge is introduced into the conjugate. Such negative charge should neutralize some excess positive charge introduced when arginine- and lysine-rich peptides are bonded as ligands. In the case of OMPC, introduction of such positive charge appears to effect irreversible precipitation. The system has been studied using the maleimidopropionyl and bromoacetyltriarginine (9 and 10) derivatives as models. In select instances anionic spacers reduce the degree of precipitation relative to N-acetylhomocysteine thiolactone derivatives.
INTRODUCTION
The outer membrane protein complex of Neisseria meningitidis112(OMPC) is a very effective immunogenic carrier of polysaccharide antigens (1, 2) and has been approved by regulatory agencies worldwide as a vaccine component (PedvaxHIB). It follows that OMPC is a potential immunogenic carrier for peptide antigens, especially those derived from the V3 domain of gp120, an envelope protein of the human immunodeficiency virus. The principal neutralization determinant of this V3 domain has been extensively mapped ( 4 ) and has been shown to reside in the V3 disulfide loop. Indeed, substantial antipeptide responses are observed for such OMPC-peptide conjugates (unpublished data). These peptide conjugates were prepared by reacting an electrophilic derivative of the peptide with a thiol derivative of OMPC which was obtained by reacting the protein with N-acetylhomocysteine thiolactone, 1 (R = methyl), as outlined in Scheme I. This thiolactone has been used to introduce a thiol functionality into small molecules and macromoleculesfor a variety of purposes outlined in Table I and has been particularly useful because it unambiguously defines covalency by a "bigeneric spacer" concept (1).
Frequently, however, the OMPC-peptide preparations were characterized by very poor protein recoveries (
