Search PubMed⌕ Search

Biomedical subjects

S B Kent

Publications and source records attributed to S B Kent.

119 records · Page 7Linked to original sources
PubMed1982

Weak acid-catalyzed pyrrolidone carboxylic acid formation from glutamine during solid phase peptide synthesis. Minimization by rapid coupling.

Formation of pyrrolidone carboxylic acid (pyroglutamic acid) residues from amino-terminal glutaminyl residues in peptides was shown to be catalyzed by weak acids, but not by strong acids. During dicyclohexylcarbodiimide-mediated coupling reactions the N alpha-protected amino acid reagent accelerated this cyclization and resulted in a significant amount of chain termination. The side reaction could be minimized by accelerating the coupling reaction and simultaneously reducing the time of exposure to weak acids. The most effective procedure was to couple in dimethylformamide with the preformed symmetric anhydride of the amino acid.

Chemical Phenomena↗
PubMed1979

Mechanisms and prevention of trifluoroacetylation in solid-phase peptide synthesis.

A novel mechanism for trifluoroacetylation in solid-phase peptide synthesis, independent of the coupling step, has been elucidated. It involves the presence of trifluoroacetoxymethyl groups on the resin support, which react with resin-bound amines by an intersite nucleophilic reaction. The trifluoroacetoxymethyl groups are generated from preexisting hydroxymethyl sites during treatment with trifluoroacetic acid in dichloromethane or by acidolysis of the benzyl ester bond between the peptide and the resin. The transfer of trifluoroacetyl from hydroxyl to amine occurs during the subsequent neutralization with tertiary amine. The mechanism was first elucidated by model studies with aminomethyl-resins. Then the expected transfer of trifluoroacetyl groups from trifluoroacetoxymethyl-resin to the alpha-amino group of N(epsilon)-benzyloxycarbonyllysine benzyl ester in solution was demonstrated; k(2), 6 x 10(-4) M(-1). Lysine-resins were used to examine the extent of trifluoroacetylation under the conditions of solid-phase peptide synthesis. After a series of acid/base cycles simulating synthetic conditions but without coupling, the poorly nucleophilic alpha-amino group was approximately 1-2% trifluoroacetylated per cycle when attached to resins already containing hydroxymethyl groups. Standard benzyl ester resins without preexisting hydroxymethyl groups gave comparable levels of trifluoroacetylation after the first few synthetic cycles because of gradual acid cleavage of the ester and accumulation of trifluoroacetoxymethyl sites. Peptide chain termination resulting from trifluoroacetylation by this mechanism could be prevented (<0.02% per cycle) by the use of the aminoacyl-4-(oxymethyl)-phenylacetamidomethyl-resin support, which can be synthesized free from extraneous functionalities and which is stable to trifluoroacetic acid under the conditions of solid-phase peptide synthesis.

Acetylation↗
PubMedDate not supplied

Delineation of contiguous determinants essential for biological functions of the pre-S sequence of the hepatitis B virus envelope protein: its antigenicity, immunogenicity and cell-receptor recognition.

The preS region of the hepatitis B virus (HBV) envelope protein has the following properties: (1) exposure on the surface of the virus; (2) high immunogenicity; (3) involvement in the reaction of the virus with cell receptors and (4) elicitation of antibodies protective against infection. Attempts to mimic B- and T-cell epitopes on the native protein by synthetic peptides were highly successful. This success depended on identification of those regions within the preS sequences which are the most important for biological function of the virus and for immunity, and on the synthesis of long peptides (20-40 residues) containing both B- and T-cell epitopes. Results presented here highlight those subregions of the preS sequence which are the most essential for the antigenicity and immunogenicity of HBV.

Amino Acid Sequence↗
PubMedDate not supplied

Hepatitis B virus contains pre-S gene-encoded domains.

One of the open reading frames on hepatitis B virus (HBV) DNA comprises the coding region (designated the env gene) for the virus envelope proteins. Studies on messenger RNA transcription suggest that this gene has the potential to code for three related proteins: (1) a protein of 226 amino acids identified as a major protein constituent of the HBV envelope, termed S-protein; (2) a protein with 55 additional amino acids at the N-terminal coded for by a portion of the env gene upstream of the S-gene (pre-S); (3) a protein corresponding to the entire env gene (pre-S + S). Synthetic peptides from the N-terminals of proteins (2) and (3), and antisera to them have been used to study the occurrence and properties of pre-S sequences. The results presented here provide unambiguous evidence that all three env encoded proteins are present in HBV particles; synthetic peptides corresponding to the gene encoding pre-S are highly immunogenic and can be used in diagnostic tests for detection in human sera of antibodies preferentially recognizing HBV; such antibodies, specific for pre-S determinants, are elicited during hepatitis B infection and by immunization with HBV proteins (2) and (3); the hepatitis B vaccine licensed in the United States does not contain pre-S proteins; and the pre-S proteins of the HBV envelope contain domains specifically recognized by liver cells. These findings suggest that pre-S determinants are important in virus-neutralizing responses and should be present in HBV vaccines.

Antibody Specificity↗
PubMedDate not supplied

Fluorescence detection in automated DNA sequence analysis.

We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific for the bases A, C, G and T. The reaction mixtures are combined and co-electrophoresed down a single polyacrylamide gel tube, the separated fluorescent bands of DNA are detected near the bottom of the tube, and the sequence information is acquired directly by computer.

Automation↗
PubMedDate not supplied

In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences.

Simple, effective protocols have been developed for manual and machine-assisted Boc-chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (> 0.2 M) of Boc-amino acid-OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine-assisted synthesis (75 residues per day). No racemization was detected in the base-catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA-DMF; chain-termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc-amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for "difficult" sequences arising from sequence-dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc-chemistry SPPS protocols or in Fmoc-chemistry SPPS. Reported syntheses include HIV-1 protease(1-50,Cys.amide), HIV-1 protease(53-99), and the full length HIV-1 protease(1-99).

Acetylation↗