Determination of an Optimal Solid Support for Use with Microwave-Assisted Solid-Phase Peptide Synthesis

  • Vigil-Cruz S
  • Peck A
  • Aldrich J
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Abstract

Introduction The success or failure of peptide synthesis is affected by many factors including the sequence of the peptide, complete removal of N-terminal amine protection, efficiency of coupling reactions and the solid support. The use of microwave energy to facilitate peptide synthesis, by accelerating coupling and deprotection reactions, was first reported over a decade ago using a conventional kitchen microwave oven [1]. Solvents play a key role in swelling the support during solid phase synthesis and also affect the transfer of microwave energy [2]. In order to explore how resins and solvents affect microwave-assisted peptide synthesis, three different types of resins, the polystyrene Wang resin, the polyethylene glycol-polystyrene PEG-PS resin and the non-polystyrene CLEAR resin were evaluated in microwave-assisted solid phase peptide synthesis (SPPS). These resins were examined under three different solvent conditions (N,N-dimethylformamide (DMF), dichloromethane (DCM)/DMF and N-methylpyrrolidone (NMP)) for coupling and washings. The acyl carrier protein (ACP) decapeptide was used as the model peptide to evaluate the resins and different solvent combinations (Fig. 1). ACP-(65-74) Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-GlyOH Fig. 1. Structure of the acyl carrier protein (ACP) decapeptide. Results and Discussion The peptides were synthesized on the CEM Liberty™ Peptide Synthesizer using a 5-fold excess of Fmoc-amino acids relative to the resin. Couplings were achieved using PyBOP (amino acid/PyBOP/HOBt/NiPr 2 Et = 1/0.9/0.9/1.8). The microwave conditions consisted of 5 cycles for 5 min total, with each cycle using 20 sec microwave power at 25W and 40 sec with the microwave power off, reaching a maximum temperature of 65°C. The deprotection reactions used 20% piperdine in DMF for a 1 min, and then a 2 min cycle; the microwave cycle involved 30 sec microwave power at 25W and them 30 sec off, reaching 75°C maximum. The Fmoc-protected peptides were removed from the instrument, the final Fmoc deprotection was performed manually and the peptides were cleaved using Reagent B [3] for 2 hrs at RT. The crude peptides were analyzed by HPLC and the quantity of the desired peptide and various side products were estimated based on the area under the curve (AUC) of absorbance at 214 nm. The crude peptides were also analyzed by LC-MS for the desired product and impurity identification. The N-terminal Fmoc on the assembled resin-bound peptides was quantified by UV absorption after piperidine treatment. The results in Table 1 indicate that all three resins could be used for microwave-assisted solid phase peptide synthesis. The peptide loading at the end of the synthesis as determined by quantitative Fmoc analysis were >80% of the expected values, except for the syntheses performed in NMP (data not shown), indicating that the peptidyl-resins were stable under the microwave conditions. Since NMP has a Understanding Biology Using Peptides Sylvie E. Blondelle (Editor) American Peptide Society, 2005 163 high dielectric loss (8.86) that would allow for efficient transfer of microwave energy [2], this solvent was expected to be an optimal solvent for microwave-assisted SPPS. However, except for the synthesis on the Wang resin (75% AUC for the ACP decapeptide), the yield of the ACP decapeptide synthesized in NMP was low (20-45% AUC). Unexpectedly syntheses in the mixed solvent system resulted in products of the highest purity despite the low dielectric loss (0.38) of DCM [2]. The side products identified were similar for the syntheses performed on the Wang and PEG-PS resins and under different solvent conditions, with the quantities varying depending on the synthesis conditions (Table 1). Three deletion sequences lacking Asn and the β-branched residues Ile and Val were detected by LC-MS in varying amounts for all three resins. A unique side product with a molecular weight higher (Δ m/z + 72) than the ACP decapeptide was detected following syntheses on the CLEAR resin; MS/MS analysis indicated that the modification was on the C-terminus of the peptide, suggesting that the side product arose from the Fmoc-Gly-OH CLEAR resin. In conclusion, all three resins were compatible with microwave-assisted solid phase peptide synthesis. However, the purity of the ACP decapeptide varied substantially depending upon the solvent utilized. The mixed DCM/DMF solvent system resulted in the ACP decapeptide with the highest purity for all three resins.

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Vigil-Cruz, S. C., Peck, A. M., & Aldrich, J. V. (2010). Determination of an Optimal Solid Support for Use with Microwave-Assisted Solid-Phase Peptide Synthesis. In Understanding Biology Using Peptides (pp. 162–163). Springer New York. https://doi.org/10.1007/978-0-387-26575-9_65

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