Microwave Heating for Solid-Phase Peptide Synthesis: General Evaluation and Application to 15-mer Phosphopeptides Malene Brandt, 1,2 Steen Gammeltoft, 2 and Knud J. Jensen 1,3 (Accepted June 28, 2006; Online publication July 27, 2006) Here we present a comprehensive study of microwave heating in manual solid-phase peptide synthesis. Three different solid supports and three different handles (linkers) were evaluated for the synthesis of one short and two medium length peptides, including a phosphopeptide. Microwave heating to 60°C was applied to different kinds of amide bond formation, reductive amination, removal of the Fmoc protecting group, and to the acidolytic release of peptides from different handles. Using microwave heating, reaction times were significantly reduced, while maintaining the high purity of the crude products. However, control experiments showed that reaction times as short as 3–4 min at rt, at least for some applications, are sufficient for acylations (couplings). While microwave heating can be used in all steps in solid- phase peptide synthesis, particularly relatively slow steps benefit from this method. KEY WORDS: microwave heating; peptide synthesis; phosphopeptide. INTRODUCTION Current methods for solid-phase peptide synthesis (SPPS) can reliably generate a very wide range of peptides. However, peptide synthesis can be a rather time-consuming process and accelerating reactions using elevated temperatures to enhance coupling rates has been explored (Tam, 1985, 1987; Lloyd et al., 1989). Furthermore, despite the many advances in the chemistry of peptide synthesis, assembly of Ôdifficult sequences’ often remains a problem, to which precise heating could provide a solution. Pre- viously, Varanda and coworkers (Varanda and Miranda, 1997) synthesized the peptide acyl carrier protein (ACP(65–74)) and unsulfated cholecystoki- nin-8 as model peptides at different temperatures, and found 60°C to be the most appropriate for the cou- pling steps. Kaplan and coworkers (1998) synthesized long peptides (84–107 residues) using elevated tem- peratures in both the deprotection (40°C) and cou- pling (55°C) steps. The interest in accelerating reactions by micro- wave heating grew in the early 1990’s, however, as domestic microwave ovens were used, the condi- tions and results proved difficult to reproduce (Kappe, 2004). The synthesis of ACP(65–74) was improved with respect to reaction time (from 2 to 3 h at room temperature to 6 min with microwave heating at 60°C) and the coupling efficiency of side- chain hindered amino acids using a domestic microwave oven was increased (Yu et al., 1992). Dedicated microwave instruments for heating in the chemical laboratory offer the prospect of very precise heating (fast, direct heating of the reaction mixture rather than the reaction vessel, precise 1 Department of Natural Sciences, Section for Bioorganic Chem- istry, Royal Veterinary and Agricultural University, DK-1871, Frederiksberg, Denmark. 2 Department of Clinical Biochemistry, Glostrup Hospital, DK- 2600, Glostrup, Denmark. 3 Correspondence should be addressed to: Knud J. Jensen, Department of Natural Sciences, Section for Bioorganic Chem- istry, Royal Veterinary and Agricultural University, DK-1871, Frederiksberg, Denmark. Tel.: +45-3528-2430; Fax: +45-3528- 2398; e-mail: kjj@kvl.dk. International Journal of Peptide Research and Therapeutics, Vol. 12, No. 4, December 2006 (Ó 2006) pp. 349–357 DOI: 10.1007/s10989-006-9038-z 349 1573-3149/06/1200–0349/0 Ó 2006 Springer Science+Business Media, Inc.