Peptides stepwise monitoring

B. Stepwise Monitoring of the Peptide Synthesis with HPLC and MS... 

Figure 3 The collapse of the peptide Ace-Nle30-Nme under deeply quenched poor solvent conditions monitored by both radius of gyration (Panel A) and energy relaxation (Panel B). MC simulations were performed in dihedral space 81% of moves attempted to change angles, 9% sampled the w angles, and 10% the side chains. For the randomized case (solid line), all angles were uniformly sampled from the interval —180° to 180° each time. For the stepwise case (dashed line), dihedral angles were perturbed uniformly by a maximum of 10° for 4>/ / moves, 2° for w moves, and 30° for side-chain moves. In the mixed case (dash-dotted line), the stepwise protocol was modified to include nonlocal moves with fractions of 20% for 4>/ J/ moves, 10% for to moves, and 30% for side-chain moves. For each of the three cases, data from 20 independent runs were combined to yield the traces shown. CPU times are approximate, since stochastic variations in runtime were observed for the independent runs. Each run comprised of 3 x 107 steps. Error estimates are not shown in the interest of clarity, but indicated the results to be robust.

The linker 36 was prepared on pMeBHA resin using Fmoc chemistry. After the tetrapeptide was synthesized, the Fmoc group was removed by piperidine/NMP (1 4) for 20 min and the Boc group was removed by TFA/CH2C12 (1 1) for 20 min followed by neutralization with DIPEA/NMP (1 19). Fmoc-p-Ala-OH and bromoacetic acid were then condensed stepwise to the peptide-resin. Completion of each reaction was monitored by the ninhydrin test. The peptide-resin was treated with TFA/3-cresol/TfOH (9 1 1 v/v) for 1 h at rt. Subsequently, H20 was added to the mixture with cooling and the mixture was washed with pentane (2 x ) followed by Et20 (2 x ). The aqueous soln was applied to RP-HPLC, and the purified peptide analyzed by FAB-MS m/z 1067.0 Da [M + H]+ (calcd 1067.2 Da). 

The solubilizing effect of PEG on the attached peptide and the absence of any direct influence of the polyoxyethylene chain on the physicochemical properties of the peptides provide a wider range of possibilities for analytical control during the liquid phase peptide synthesis than those in the solid phase method. The reactions employed in the stepwise liquid phase synthesis can thus be quantitatively monitored by several analytical methods. 

Stepwise analysis is an efficient and direct method to monitor the progress of side reactions in Fmoc chemistry. The analysis involves stepwise micro-scale TFA cleavage in conjunction with HPLC and MS. Micro-scale TFA cleavage can be conveniently carried out on a small amount of sample. MS analysis of peptide ladders provides a rapid method for monitoring and identification of side reactions in peptide synthesis. 

Peptide/flavonoids supramolecular complexes are sufficiently stable to accept internal energy shifts without early disruption, and sufficiently fragile to undergo dissociation by a controlled ERMS procedure. Trapped ionized complexes were submitted to stepwise activation by CID, and the intensities of the precursor and product ions were monitored while increasing %NCE. 

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