Andrew M. Bray

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.

The simultaneous multiple production of solution phase peptides ; assessment of the geysen method of simultaneous peptide synthesis

Abstract Peptides produced by the Geysen technique, a method allowing very large numbers of peptides to be prepared simultaneously, have been fully characterized and shown to be of high quality. A technique allowing peptides to be cleaved directly into pH 7 buffer now enables this powerful screening technique to be applied to biological/immunological problems requiring solution phase assays.

Gas phase cleavage of Peptides from a solid support with ammonia vapour. Application in simultaneous multiple Peptide Synthesis.

Abstract Support-bound peptide esters are cleaved with ammonia vapour and then eluted from the support with a solvent of choice. The method is demonstrated in conjunction with the multipin method of peptide synthesis.

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.

Grafted supports used with the multipin method of peptide synthesis

Abstract Radiation grafting of a range of monomers allows a diversity of surface characteristics to be generated on a common plastic support. Taken together with the modular 8-column, 12-row format used with the multipin method of peptide synthesis, many thousands of peptides can be generated not only for epitope mapping and other solid-phase applications but also to produce larger quantities of cleaved peptides.

Rapid optimization of organic reactions on solid phase using the multipin approach: Synthesis of 4-aminoproline analogues by reductive amination

Abstract The Multipin method of multiple solid phase synthesis was used in conjunction with high throughput characterization methods (ion spray mass spectrometry and HPLC) to optimize reactions rapidly for solid phase synthesis. The approach is demonstrated in the synthesis of 4-aminoproline analogues by reductive amination under a wide range of conditions, using a diverse set of amines.

A simple linker for the attachment of aldehydes to the solid phase. Application to solid phase synthesis by the Multipin™ method

Abstract A simple linker has been developed for the attachment of aldehyde functionality. The aldehyde substrate is condensed with a support-bound serine or threonine to give an oxazolidine. The oxazolidine linker is stable to the conditions of Fmoc peptide synthesis, including TFA treatment, but is cleaved by mild aqueous acid at 60°C. The method is used, in conjunction with the Multipin method, to prepare a set of peptide aldehydes.

Multipin solid phase synthesis of ethers using modified Mitsunobu chemistry

Abstract Formation of ether derivatives of phenolic containing structures using solid phase Mitsunob functionalized polyethylene pins was investigated. Using the Multipin approach, a range of reaction parameters were systematically varied in parallel experiments including solvent, temperature, time, reactant concentrations, base, phosphine and alcohol to determine optimum reaction conditions. Solid phase reaction of three phenols with a range of alcohols to form the ethers was found to proceed smoothly using 0.15M PPh3/DEAD/alcohol in THF at 37°C for 4 days in the presence of 0.45M triethylamine.

Synthesis of phosphopeptides by the Multipin method: Evaluation of coupling methods for the incorporation of Fmoc-Tyr(PO3Bzl,H)-OH, Fmoc-Ser(PO3Bzl,H)-OH and Fmoc-Thr(PO3Bzl,H)-OH

The efficiency of various coupling methods for the incorporation of the three monobenzyl phosphorodiesterprotected derivatives, Fmoc-Tyr(PO3Bzl,H)-OH, Fmoc-Ser(PO3Bzl,H)-OH and Fmoc-Thr(PO3Bzl,H)-OH, was examined through the test synthesis of Ala-Ser-Gln-Gly-Xxx(PO3H2)-Leu-Glu-Asp-Pro-Ala-NH2 (Xxx=Tyr, Ser, Thr) using the Multipin method of multiple peptide synthesis. The coupling methods examined were (1) PyBrop/DIEA; (2) BOP/HOBt/NMM; (3) BOP/HOBt/DIEA; (4) HBTU/HOBt/DIEA; (5) HATU/HOAt/DIEA; (6) HATU/DIEA; (7) DIC/HOBt; (8) DIC/HOBt/DIEA; (9)DIC/HOAt; (10) DIC/HOAt/DIEA. While all four DIC-based coupling procedures resulted in incomplete incorporation, both the HBTU/HOBt/DIEA and HATU/HOAt/DIEA coupling procedures provided most efficient incorporation of the three Fmoc-Xxx (PO3Bzl,H)-OH derivatives. In the subsequent synthesis of the α-helical Tyr(P)-peptide, Glu-Thr-Gly-The-Lys-Ala-Glu-Leu-Leu-Ala-Lys-Tyr(PO3H2)-Glu-Ala-Thr-His-Lys-NH2, analysis of the crude peptide by electrospray MS confirmed that several residue deletions had occurred but that complete incorporation of the Tyr(P)-residue had been accomplished using HBTU/HOBt/DIEA coupling of Fmoc-Tyr(PO3Bzl,H)-OH.

Efficient synthesis of thioether-based cyclic peptide libraries

Abstract A new method for the synthesis of cyclic peptide libraries has been developed where the key cyclisation step involves reaction between a C-terminal cysteine side chain and an N-terminal bromoacyl group. We report conditions whereby liberation of peptides from the solid support and cyclisation occur concurrently to form thioether-linked cyclic peptides in generally >95% yield.