Solid-phase peptide synthesis (SPPS) in the N-to-C direction (inverse SPPS), opposite to the classical C-to-N direction of peptide synthesis,2) provides the synthetically versatile peptide C-terminal carboxyl group for further modification into C-terminally modified peptide mimetics. These are of general interest as potential bioactive agents, and of particular interest in the search for potent and specific protease inhibitors. In addition, the ability to elaborate the C-terminus on the solid-phase would facilitate the synthesis of combinatorial libraries of peptide mimetics.3-5) Several strategies for inverse SPPS have been described.6-10) Recently described strategies for inverse SPPS include using amino acid 9-fluorenylmethyl esters, 8) amino acid tri-tert-butyloxysilyl esters, 9) and amino acid allyl esters. 10) These strategies appear less than ideal, since suitable amino acid derivatives are not generally available commercially and can be difficult to prepare, and due to instability of reactants and/or intermediates. Amino acid tert-butyl esters are easily prepared, stable, and a large selection are commercially available. Another advantage is that the well established chemistry, instrumentation, and side chain protecting groups developed for the analogous Boc strategy for normal phase SPPS syntheses can be used as guides in the development and application of amino acid tert-butyl ester based inverse SPPS. This report describes an amino acid tert-butyl ester based strategy for inverse SPPS of peptides and peptide trifluoromethylketones.Our initial strategy for inverse SPPS used succinylated Pam resin, as illustrated by an example synthesis of the model peptide Suc-Ala-Leu-Pro-Phe (1) (Fig. 1). To compare different coupling procedures this model peptide was synthesized with dicyclohexylcarbodiimide (DCC)/N-hydroxybenztriazol (HOBT), O-benzotriazol-1-yl-1,1,3,3- and O-7-azabenzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/2,4,6-trimethylpyridine (TMP) based coupling methods. With HBTU/DIEA both in situ and preactivation protocols were tested. After completion of chain assembly succinylated peptide was released from the solid support by treatment with trifluoromethanesulfonic acid (TFMSA)/trifluoroacetic acid (TFA), and products were analyzed by liquid chromatography (LC)/MS. Coupling with DCC/HOBT gave the target peptide (60%) and two deletion peptides-Suc-Ala-Leu-Pro (20%) and Suc-AlaPro-Phe (20%)-resulting from incomplete incorporation of Phe and Leu residues respectively. HBTU/DIEA and HATU/TMP coupling methods yielded high purity (Ͼ80%) target peptide.To compare the effect of preactivation (resin attached carboxyl group activated prior to addition of the amino acid tert-butyl ester) with in situ activation (all reagents mixed together nearly at once) on racemization, peptide samples were synthesized with HBTU/DIEA both with in situ and preactivation, and analyzed for racemization using Marfey's reagent 11,12) (1 a) and 1 b) in Table 1). The first and second residues underwent subst...