Biomedical subjects

F A Robey

Publications and source records attributed to F A Robey.

58 records · Page 4Linked to original sources

PubMedDate not supplied

Synthesis of N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine: its use in peptide synthesis for placing a bromoacetyl cross-linking function at any desired sequence position.

A new amino acid derivative, N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a side-chain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates, and polymers. BBAL is synthesized by condensation of N-bromoacetyl-beta-alanine with N alpha-Boc-L-lysine and is a white powder which is readily stored, weighed, and used with a peptide synthesizer, programmed for N alpha-Boc amino acid derivatives. BBAL residues are stable to final HF deprotection/cleavage. BBAL peptides can be directly coupled to other molecules or surfaces which possess free sulfhydryl groups by forming stable thioether linkages. Peptides containing both BBAL and cysteine residues can be self-coupled to produce either cyclic molecules or linear peptide polymers, also linked through thioether bonds. Products made with BBAL peptides may be characterized by amino acid analysis of acid hydrolyzates by quantification of beta-alanine, which separates from natural amino acids in suitable analytical systems. Where sulfhydryl groups on coupling partners arise from cysteine residues, S-(carboxymethyl)cysteine in acid hydrolyzates may also be assayed for this purpose. Examples are given of the use of BBAL in preparing peptide polymers and a peptide conjugate with bovine albumin to serve as immunogens or model vaccine components.

Amino Acid Sequence↗

PubMedDate not supplied

Synthesis and use of a new bromoacetyl-derivatized heterotrifunctional amino acid for conjugation of cyclic RGD-containing peptides derived from human bone sialoprotein.

A new amino acid derivative, N alpha-(tert-butyloxycarbonyl)-N beta-(bromoacetyl)diaminopropionic acid (BBDap), has been synthesized as a reagent for introducing side-chain bromaocetyl groups into any position of a peptide sequence during solid-phase peptide synthesis. By using minor modifications to the protocol of the automated peptide synthesizer and a two-step in situ neutralization procedure, the syntheses of (bromoacetyl)diaminopropionic acid (BDap) in Arg-Gly-Asp-containing peptides from human bone sialoprotein were optimized and completed. Following HPLC purification, the BDap-derivatized peptides were cyclized or/and conjugated to carrier protein or to glass cover slips. In addition, a new procedure for site-specific conjugation of cyclic peptides to protein carriers or to glass was developed. The cell attachment activity of the peptide derivatives and conjugates was tested in cell adhesion assays with human osteoblasts, and the specificity of the binding was confirmed by competition with linear and/or cyclic forms of GRGDS. The results show that conjugates containing the linear and cyclic derivatives of the peptide EPRGDNYR supported cell attachment and spreading in a dose-dependent manner when the peptides were immobilized as described. Cell attachment to the intact bone sialoprotein and to conjugates containing the linear peptides was abolished by competition with linear and cyclic RGD-containing peptides, whereas the attachment to conjugates containing the cyclic peptide was inhibited only partially, and the cell spreading was preserved even in the presence of RGD-peptides.

Amino Acid Sequence↗

PubMedDate not supplied

Peptomer aluminum oxide nanoparticle conjugates as systemic and mucosal vaccine candidates: synthesis and characterization of a conjugate derived from the C4 domain of HIV-1MN gp120.

Peptomers are polymers composed of peptides that are specifically cross-linked in a head-to-tail fashion. Recently, a peptomer composed of an amphipathic peptide from the C4 domain of HIV-1MN gp120 was shown to display a prominent alpha-helical conformation that, as an immunogen, elicited rabbit antibodies recognizing native and recombinant gp120 [Robey et al. (1995) J. Biol. Chem. 270, 23918-23921]. For the present study, we synthesized a conjugate composed of the C4 peptomer covalently linked to calcinated aluminum oxide nanoparticles. The nanoparticles were first reacted with (3-aminopropy])-triethoxysilane to provide an amine load of 15.9 mmol of R-NH2/g of solid. The amine-modified aluminum oxide nanoparticles then were reacted with N-acetylhomocysteine thiolactone at pH 10 to place a reactive thiol on the nanoparticles. A bromoacetylated C4 peptomer, modified at the epsilon-amines of lysine residues, then was reacted with the thiolated nanoparticles to give the peptomer covalently linked to aluminum oxide via a thioether bond. The peptomer load was determined to be 16 mg of peptomer/g of particles, a 55% theoretical yield. Particle shape and size of the peptomer-conjugated alumina were analyzed by electron microscopy and displayed a mean maximum diameter of 355 nm and a mean minimum diameter of 113 nm, well within the desired size range of 300 nm believed to be optimal for mucosal immunization purposes. Experimentally determined values of mean particle diameters, specific surface area, and specific peptomer load provided the information necessary to calculate the mean antigen load, which was determined to be 53000 +/- 42000 peptomer epitopes per particle. Peptomer-alumina conjugates, such as that described here, could form the basis of a new class of biomaterial that combines a chemically defined organic immunogen with a nontoxic chemically defined inorganic adjuvant.

AIDS Vaccines↗

PubMedDate not supplied

Effective use of free thiols as scavengers for HF cocktails to deprotect bromo- and chloroacetylated synthetic peptides.

A variety of thiol-containing compounds, in combination with m-cresol, were tested as scavengers in hydrogen fluoride (HF) cocktails that are used to deprotect haloacetylated peptidyl resins. Our results indicate that brome and chloroacetyl moieties on a synthetic peptide remain intact following HF treatment when the HF cocktail contains m-cresol along with either thiophenol, m-thiocresol or 1,2-ethanedithiol. The free thiols prevent the formation of a number of impurities in the preparation of bromo- and chloroacetylated peptides that contain amino acids that could be oxidized in a nonreducing HF environment. Ethvimethylsulfide, however, could not be used with bromoacetylated peptides, but it could be used with chloroacetylated peptides.

Acetylation↗