Pergamon Tetrahedron Letters 39 (1998) 8529-8532 TETRAHEDRON LETTERS Racemization Studies of Fmoc-Ser(tBu)-OH During Stepwise Continuous-Flow Solid-Phase Peptide Synthesis Armida Di Fenza, Mariella Tancredi, Claudia Galoppini and Paolo Rovero* Istituto di Mutagenesi e Differenziamento, CNR, Laboratorio Sintesi Peptidica, Via Svezia 2A, 56124 Pisa, Italy. Received 30 July 1998; accepted 7 September 1998 Abstract: We observed unexpectedly high levels of racemization of Fmoc-Ser(tBu)-OH during automated stepwise solid-phase peptide synthesis under standard continuous-flow conditions. We set up a model assay, based on the solid-phase assembly of the tripeptide H-GIy-Ser-Phe-NH2, in order to test the effect of the coupling conditions on serine racemization. Protocols based on collidine as the tertiary base added to the coupling reagent enabled incorporation of serine with less than 1% racemization. © 1998Elsevier ScienceLtd. All rights reserved. Keywords:Amino acids and derivatives; Racemization;Solid-phasesynthesis. The key step in the preparation of synthetic peptides is the controlled formation of a peptide bond between two amino acids, the so-called coupling reaction. It is well known that this reaction, which requires the activation of the carboxyl group of one amino acid, is particularly prone to racemization. Accordingly, coupling methods which preserve the configurational integrity of the carboxylic component have been widely studied. 1 In stepwise solid-phase peptide synthesis (SPPS) the problem of racemization is generally assumed to be less dramatic than for other strategies. In fact, amino acids NCt-protected by a urethane type blocking group, such as 9-fluorenyl- methoxycarbonyl (Fmoc) or tert-butoxycarbonyl (Boc), are resistant to racemization during activation and coupling. 2 Moreover, the large excess of reagents generally used in SPPS makes the coupling reaction faster than in solution, thus minimising the loss of chirality. Consequently, it is widely accepted that in SPPS the risk of racemization is reduced to a negligible level, if not completely absent. However, recent reports indicate that racemization of cysteine residues can be a serious concern in Fmoc SPPS.3 We now report a similar observation regarding high levels of racemization of Fmoc-Ser(tBu)-OH 4 during stepwise SPPS under standard continuous- flow conditions. In the course of our structure-activity relationship studies of bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser- Pro-Phe-Arg-OH), which imply the synthesis of several Ser-containing analogues using standard automated continuous-flow SPPS methods, 5 we consistently observed in the crude a side-product with the same mass as the desired peptide, as shown by EI-MS. The racemization analysis of the side-product, isolated by semi- preparative RP-HPLC, indicated that racemization occurred at the level of the Ser residue. We decided to set up a 0040-4039/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved. PII: S0040-4039(98)01891-7