Optimization of the synthesis of peptide combinatorial libraries using a one-pot method

Abstract

Conditions for the synthesis of synthetic peptide combinatorial libraries (SPCLs) from mixtures of amino acids were explored. In a one-pot synthesis, the effect of the starting concentrations of amino acids on the resulting library composition was studied, and the optimum balance of amino acids was determined. Protein sequencing, MALDI-TOF, and amino acid analysis were used for the evaluation of the libraries, and their relative merits-are discussed. The effects of continuous-flow automated synthesis instrumentation in conjunction with polyethylene glycol-polystyrene (PEG-PS) graft supports and various cleavage cocktails on the successful synthesis of SPCLs were examined.

A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

“…The resin was introduced in the ABI-431A peptide synthesizer and all Fmoc-amino acids were coupled using the DCC/HOBt protocol from the company (4-fold excess of the Fmoc-amino acid, 90 min per coupling). The peptide was cleaved from the resin with a mixture of TFA: TIS: H 2 O (95%: 2.5%: 2.5%) (5 ml) yielding the crude DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala peptide which was precipitated and washed with cold ether (5 ml X 5). The crude DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala (385 mg) was purified by preparative HPLC (gradient b) and lyophilized to give 170 mg of the peptide product as a white solid.…”

“…Cells were placed in 1.5ml tubes. In parallel, unlabeled DUP-1, DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala, 15-mer and pegylated Rak-2 [1] were dissolved in fresh RPMI containing 1% BSA at different concentrations (from 50 nM to 0.6 mM). 250 µl of each concentration were transferred to the 1.5ml tubes containing the cells and pre-incubated for 40 min at 37 0 C in the orbital shaker.…”

“…Therefore, two convoluted sub-libraries were synthesized on labeled microspheres as mixtures of 12 biased peptides in each sub-library. The biased libraries I and II were synthesized by the systematic introduction of a kinetic mixture of Fmoc-amino acids [12] at each of two arginine positions in the backbone of the peptide (see Fig. 4).…”

“…Compound Rak-2, previously validated as having good affinity for PC-3 cells, was synthesized in a pegylated form to improve solubilization and analyzed together with the new peptide candidate. (FITC-13 Lys)DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) standard peptide was incubated with PC-3 cells at a concentration of 1µM in the presence of different concentrations of non-labeled DUP-1, negative control peptide 15-mer or ligand hits as previously shown [1]. The cells were analyzed by flowcytometry and the affinity was determined using the geometric means obtained from FACS plots at the different concentrations of ligands [13].…”

“…In the experiments we compared the displacement of (FITC-13 Lys)DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) fluorescence by non-labeled DUP-1 () and by a new peptide DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala () found to have high affinity to PC-3 cells in our screening or by a small organic Ugi molecule Rak-2 () found to have good to PC-3 cells using the same screening technique in a previous work. We can clearly acknowledge that the affinity range of DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala () is slightly higher than that of the native DUP-1 () and the affinity range of small molecule Rak-2 () is slightly lower but similar to that of the native DUP-1.…”