Biomedical subjects

A M Bray

Publications and source records attributed to A M Bray.

8 recordsLinked to original sources

PubMed1994

Multiple peptide synthesis on acid-labile handle derivatized polyethylene supports.

Using the multipin peptide synthesis approach, a range of peptides with native amide and carboxylate C-termini were generated using an acid-labile approach. Polyethylene crowns grafted with hydroxyethylmethacrylate (HEMA) polymer were functionalized with either 4-hydroxymethylphenoxyacetic acid for the generation of peptide-carboxylate or p-[(R,S)-alpha-[1-(9H-fluoren-9-yl)methoxyformamido]-2,4-dim ethoxy- benzyl]phenoxyacetic acid for peptide-amide. A range of known peptide hormone sequences and other peptides with native C-termini were assembled by sequential incorporation of N alpha-Fmoc protected amino acids. Peptides were sidechain deprotected and cleaved from crowns with TFA/scavengers within 2 mL centrifuge tubes, and isolated by a series of ether/petrol wash and centrifugation steps. In this way it was possible to avoid a cleavage and isolation botteneck, allowing rapid processing of large numbers of peptides.

Amino Acid Sequence↗

PubMed1993

Systematic study of substance P analogs. I. Evaluation of peptides synthesized by the multipin method for quantitative receptor binding assay.

The multipin peptide synthesis technique, a method for simultaneous multiple peptide synthesis, was developed for large-scale screening of oligopeptides [Geysen et al. (1984) Proc. Natl. Acad. Sci. USA, 81, 3998-4002]. A modification of the technique allows the peptides assembled on polyethylene pins to be cleaved in their native amide form and reconstituted into physiologically compatible solutions. In this study, the suitability of these peptides for quantitative receptor binding assay was evaluated. Substance P and 18 analogs, including a set of N-terminal truncated substance P and a set of naturally occurring substance P analogs, were synthesized by the multipin methods. An average yield of 20 +/- 3 nmol of peptide per pin was obtained. The purity of the peptides was estimated to be ca. 90%. The binding activities of these peptides were determined in a competition assay against 125I-BHSP binding to a rat brain synaptosome preparation. The rank order of the affinities of these peptides depicted a typical pharmacological profile of central NK1 receptor. The IC50 values obtained were also in good agreement with data reported by other groups using similar experimental conditions, except that bulk synthesized peptides were used. This study demonstrates that the peptides synthesized with the multipin technique are suitable for quantitative receptor studies, particularly for a high-volume screening of bioactive peptides.

Amino Acid Sequence↗

PubMed1993

Systematic study of substance P analogs. II. Rapid screening of 512 substance P stereoisomers for binding to NK1 receptor.

Recent developments in multiple peptide synthesis have made it feasible to synthesize and screen large numbers of peptide analogs simultaneously. We report here a model study of large scale screening of stereoisomers of substance P with systematic D-amino acid replacements. Such studies are useful in exploring conformational requirements of peptide-receptor interaction and to provide empirical information for peptide drug design. 512 stereoisomers of SP were prepared by the multipin peptide synthesis method. Receptor binding affinities of these analogs were estimated by an iterative competition assay. Results obtained form a comprehensive database on the stereochemical requirement of SP binding to central NK1 receptor. Data from analogs with single D-amino acid replacement are consistent with those previously reported showing that SP binding is highly sensitive to the chirality change of the C-terminal residues (Gln6-Leu10), but less sensitive to the chirality change of the N-terminal residues (Arg1-Gln5). A qualitative analysis of the database by comparison of series of peptide pairs revealed a repeated pattern of affinity change with D-amino acid replacement, suggesting a largely additive binding activity of SP from each residue. On the other hand, possible intramolecular interactions between some N-terminal and C-terminal residues to form an optimal binding conformation were also found. A set of 189 peptides with IC50 values less than 5 microM was subjected to an empirical QSAR analysis using a linear additive model. The relative contribution coefficients obtained agreed with the observation that the predominant contribution comes from the C-terminal residues, suggesting considerable independency of each residue on binding to NK1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Simultaneous multiple synthesis of peptide-carrier conjugates.

Recently, the multipin approach for simultaneous multiple peptide synthesis was applied to the analysis of T cell determinants by using a novel cleavage method (Maeji et al., 1990). A diketopiperazine forming linker allowed cleavage of peptides into aqueous buffer which, without further purification, could be used immediately in cell culture assays. Another potential application of the technique is the simultaneous cleavage and coupling of peptides to immunogenic carriers. Without further purification the resulting conjugates can be used for the production of antipeptide antisera. The choice of carrier and conjugation chemistry is not restricted as peptide/pin cleavage occurs in aqueous solution over a range of pH and ionic strength. The method was assessed using the 2,4-dinitrophenyl group as a model hapten, diphtheria toxoid as the carrier, and N-(epsilon-maleimidocaproyloxy)succinimide as the cross-linking reagent. The resulting DNP-DT conjugate was used to prepare high titered specific anti-DNP antisera in mice.

2,4-Dinitrophenol↗

PubMed1991

Synthesis of peptide analogues using the multipin peptide synthesis method.

Modification of the multipin peptide synthesis method which allows the simultaneous synthesis of large numbers of different peptide analogues is described. Peptides were assembled on polyethylene pins derivatized with a 4-(beta-alanyloxymethyl)benzoate (beta-Ala-HMB) handle. For comparative purposes, peptides were also assembled on the diketopiperazine-forming handle N epsilon-(beta-alanyl)lysylprolyloxylactate. In model studies it was demonstrated that beta-Ala-HMB-linked peptides were cleaved from polyethylene pins with dilute sodium hydroxide or 4% methylamine/water to yield analogues with beta-Ala-free acid (beta-Ala-CO2H) and beta-Ala-methylamide (beta-Ala-CONHCH3), respectively. To assess the suitability of this approach for T-cell determinant analysis, analogues of a known T-cell determinant were synthesized with the various C-terminal endings. Peptides were characterized by amino acid analysis and fast atom bombardment-mass spectrometry. HPLC of the crude cleaved peptides indicated that 22 of the 24 peptides were greater than 95% pure. These crude peptide solutions were nontoxic in sensitive cell culture assays without further purification. All three cleavage procedures gave comparable activities in T-cell proliferation assays. These results demonstrate the potential of the multipin peptide synthesis method for the production of large numbers of different peptide analogues.

Amino Acid Sequence↗

PubMed1990

Multi-pin peptide synthesis strategy for T cell determinant analysis.

Techniques to synthesize many peptides simultaneously exist, however their individual cleavage and subsequent purification constitutes a bottleneck to total throughput. Biological screening of peptides is generally carried out at physiological pH in aqueous solutions. However, peptides, unless individually purified are usually contaminated by residual compounds used in their preparation such as trifluoroacetic acid, organic solvents, scavengers etc. In testing with cellular systems, such as T cell determinant analysis, such contaminations must be rigorously excluded. We have extended the pin synthesis technique of synthesizing and screening large number of peptides (Geysen et al., 1984) to the analysis of T cell determinants. Peptides can be synthesized on polyethylene pins, the side chain protective groups removed and the peptides washed free of contaminants. A linker system stable under these conditions can then be triggered to cleave the peptides from the pins in an aqueous solution at neutral pH. This strategy enables the rapid mapping of T cell determinants. It is also applicable to other systems where large numbers of solution phase peptides are required, for example, in the study of hormone analogues.

Amino Acid Sequence↗

PubMedDate not supplied

Systematic screening for bioactive peptides.

Using simultaneous multiple peptide synthesis by the multipin approach, the feasibility of systematic large-scale pharmacological screening of peptide ligands was investigated. The method involves the assembly of small quantities of peptides (ca. 50 nmol) on plastic pins derivatized with an ester linker based on glycolate and 4-(hydroxymethyl)benzoate. These esters are stable under peptide synthesis and side-chain deprotection conditions but cleave under relatively mild basic conditions to generate peptides having C-terminal acid, amide and methylamide. A two-step approach to side-chain deprotection and cleavage from the solid support allows potentially toxic reagents to be removed (washed) from the peptide prior to cleavage. Consequently, the resulting peptide solutions can be used in bioassays with minimal processing. A series of angiotensin II and substance P analogs were synthesized and evaluated in an in vivo rat model and in vitro radioreceptor assay, respectively. Structure-activity studies on analogs of these bioactive peptides are well documented. The data obtained were consistent with that reported in the literature.

Amino Acid Sequence↗

PubMedDate not supplied

Larger scale multipin peptide synthesis.

The multipin peptide synthesis approach originated as an immunological tool for epitope mapping. However, continuing evolution of the basic technology has allowed synthesis at scales up to 10 mumol per pin. At this loading, the methodology can no longer be considered just a screening tool. The overall synthesis efficiency of this approach was assessed by the synthesis of 2913 different peptides having little or no sequence homology and ranging up to a 46-mer in length. High performance liquid chromatography analysis of the crude peptides indicates overall quality of synthesis was high. The method is suitable for multi-milligram synthesis of peptides without sacrificing any of the inherent advantages of the 96-well format.

Amino Acid Sequence↗