At the ABRF Satellite Meeting in San Diego, Hank Wolfe moderated a Peptide Synthesis Workshop entitled "Combining BOC and FMOC Chemistries for Protein Synthesis". Hank Wolfe presented his work on "Synthesis of a Biologically Active Kunitz Protease Inhibitor" and Gregg Fields spoke on "Synthesis of Triple-Helical Collagen Peptides". Both speakers followed a self-imposed ground rule of showing bad along with good results. In addition to the interesting biochemical and biophysical aspects that were discussed, a number of practical tips were covered which are briefly summarized:
1. Solubility is a major factor in obtaining good coupling yields with protected peptides and this should be built into the synthetic strategy. Analysis of such protected peptides should include analysis of the deprotected peptide as well, since these are much more amenable to high resolution chromatographic methods for detecting deletions or insertions.
2. Recovery and analysis of the deprotected peptides is also severely restricted in the case of peptides designed to self associate (i.e., spontaneously fold). Gregg found that hydrophobic interaction chromatography provided reproducible analysis without peak broadening due to on-column aggregation .
3. Both speakers addressed the issue of tryptophan modification in FMOC chemistry but from different directions. Hank Wolfe was able to eliminate modification of a tryptophan from the Wang resin "handle" through the use of a mixture of scavengers. This mix, which he dubbed "Gesellchen solvent" (after Paul Gesellchen who developed it several years ago) virtually eliminated side reactions. Briefly, the peptide resin is stirred at room temperature in the solvent (50 ml/g resin; TFA/DMS/EDT/m-cresol, 65:25:5:5) for four hours before being quenched by dripping into cold diethyl ether and collecting the precipitate for purification. Gregg Fields utilized the recently available Trp(Boc) group to avoid the problem altogether. The N-carboxyindole intermediate formed during deprotection of the peptides with standard reagents (i.e., Reagent K) serve to protect the tryptophan ring system from the short-lived carbonium ions generated during cleavage. Gregg's parallel studies with several peptides provided convincing evidence of the utility of this derivative.
4. Gregg Fields described a nice addition to FMOC removal which allows the use of the non-controlled DBU to remove FMOC groups with only a minor amount of piperidine required to scavenge the dibenzofulvene liberated during deprotection. He utilized a mixture in DMF or NMP (2% DBU, 2% Pip in DMF, v/v) to remove the FMOC group over a 20 minute period.
Finally, Hank Wolfe detailed some extreme efforts at attempting to incorporate protected peptide segments with poor solubility. One particular segment (a decapeptide being attached to a low-substitution tripeptide resin) required eight different attempts using reagents such as DIC, BOP, OPfp esters and HOBT esters in a variety of solvents ranging from neat chloroform to 20% hexamethylphosphoramide, 20% DMSO in NMP, to obtain a final coupling yield of only 76%. Work with analogous segments led him to the rule of checking all his segments for solubility and coupling competence on a microscale before committing to a more reasonable scale. Based on the results of these initial studies the protection scheme or peptide is then altered to improve the coupling yields.
Overall, the response to this workshop was good (~35-40 attendees), with the candor in data presentation giving rise to a lively discussion both during and after the workshop. This is certainly a workshop worth continuing in the future and the ABRF welcomes persons interested in participating in or running such a session.
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