Peptide combinatorial library cleavage

It is important to further develop the concept of structure-activity relationships to precisely define the structural requirements of glutathione action. Thus, this section introduces the design, synthesis, and screening of a peptide combinatorial library to obtain multiple glutathione analogs. Combinatorial libraries will be composed of mixtures of peptides (consisting of natural or noncoded amino acids) on solid support. After cleavage from the resin, the mixtures of the peptides will be screened directly in different specific assays. 

Automated synthesis of peptide and oligonucleotide libraries was initiated about 10 years ago [4], Within the last three years, there has been much attention focused on the generation of combinatorial libraries of small molecules. As with biopolymers, the use of solid resin support was central to the advance of this field. In solid-phase synthesis, one of the reactants is covalently bound to the solid support and an excess of the other reactants may be used in each step to drive reactions to completion. Purification of the intermediates and final product is easily achieved through extensive washing of the resin after each chemical step. For the purpose of high throughput synthesis, cleavage of the final... 

This chapter provides a manual for a laboratory-hased short course to introduce the common techniques of solid-phase peptide synthesis (SPPS). The course provides students the opportunity to design and manually synthesize analogs of glutathione using relatively simple equipment available in any unsophisticated laboratory. The manual provides compact protocols for both the different steps of SPPS and the final cleavage of peptides from resin supports. We also introduce a simple method for the synthesis of combinatorial libraries of glutathione analogs that is suitable for those relatively unfamiliar with the field of peptide chemistry. 

Future routine uses of MALDI mass spectrometry for the detection of combinatorial library components could include techniques that enable the analytical chemist to directly analyze reaction products from beads without using prior cleavage reactions, as is shown in Fig. 7. This means that standard linker molecules would have to be designed in such a way that cleavage from the bead is obtained by the laser irradiation used for the ionization process as has been employed by Oda et al. and others (71,78) for the identification of peptides bound to a resin (72). 

For some applications it might be desirable to cleave the product from a support in two or more portions. This can be realized by derivatizing a functionalized support with a mixture of different linkers that enable a sequential cleavage [9]. The resulting support can, for instance, be used to prepare and screen combinatorial peptide libraries by the mix-and-split method ([10-12] one different peptide on each bead). The first portion of peptide released would be tested for biological activity, and, once an active peptide had been identified, the remaining peptide on the support could be used for structure elucidation. 

The combinatorial approach is attractive for the synthesis of libraries of hydroxamic acids for screening as metalloenzyme inhibitors. A variety of hydroxylamine-linked resins (shown in Schemes 9-12) have been developed that allow peptide condensation reactions followed by cleavage of the hydroxamic acid from the resin. 

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