Journal of Peptide Science J. Peptide Sci. 11: 658–664 (2005) Published online 9 May 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/psc.675 α - and β - Aspartyl peptide ester formation via aspartimide ring opening PANAGIOTIS STATHOPOULOS, SERAFIM PAPAS, SARANTOS KOSTIDIS and VASSILIOS TSIKARIS* Department of Chemistry, University of Ioannina, GR-45110 Ioannina, Greece Received 11 January 2005; Accepted 28 February 2005 Abstract: The undesirable reaction of aspartimide formation has been proved to occur under both acid and base conditions in solid-phase peptide synthesis and is dependent on the β-carboxyl protecting group, the acid or base used during the synthesis, as well as the peptide sequence. The hydrolysis of aspartimide-containing peptides, especially during HPLC purification, yields a mixture of α- and β-aspartyl peptides that can not be purified easily. A previous study demonstrated that treatment of aspartimide-containing peptides with methanol in the presence of 2% diisopropylethylamine in solution leads to α- and β-aspartyl peptide methyl esters. Taking advantage of these results and aiming at elucidating the optimal conditions for aspartimide ring opening, the effect of different types and concentrations of alcohols (primary and secondary) and bases (diisopropylethylamine, collidine, 4-pyrrolidinopyridine, 1-methyl-2-pyrrolidone, piperidine and KCN) was tested at various temperatures and reaction times. The best results were obtained with a combination of a primary alcohol and diisopropylethylamine, while aspartimide ring opening by secondary alcohols occurred only at high temperatures. The optimal conditions were also applied to solid-phase peptide synthesis. Copyright  2005 European Peptide Society and John Wiley & Sons, Ltd. Keywords: aspartimide; aspartimide chemistry; α- and β-aspartyl peptide esters INTRODUCTION The main side reaction in the synthesis of Asp- containing peptides is aspartimide formation (Asi: aspartimide; the abbreviation Asu was used in some publications to denote aspartimide [1,2] while the same abbreviation is proposed for α-aminosuberic acid [3]). In order to avoid future confusion we propose the use of Asi as abbreviation, derived from As parti mide. Asi formation occurs under both acid and base conditions [1,4] (Figure 1). In Boc (tert- butyloxycarbonyl)-based solid-phase peptide synthesis (SPPS), acid-catalysed aspartimide formation has been reported to occur in the presence of both strong acids, such as HF and trifluoromethanesulfonic acid- trifluoroacetic acid (TFMSA-TFA), and milder acids, such as TFA [5]. In Fmoc (9-fluorenylmethoxycarbonyl)- based peptide chemistry aspartimide formation occurs via base catalysis, particularly with the prolonged use of piperidine or stronger bases [6,7] for the Fmoc group cleavage during the synthetic cycles. It has been established that this intramolecular cyclization occurs at higher rates under base conditions [4]. Several detailed studies have related the extent of Asi formation in Asp-containing peptides to: (i) the protocols used in peptide synthesis, i.e. the choice of Fmoc-deprotecting or coupling reagents [8,9], (ii) the type of Asp β -carboxyl protecting group * Correspondence to: V. Tsikaris, Department of Chemistry, University of Ioannina, GR-45110 Ioannina, Greece; e-mail: btsikari@cc.uoi.gr [5,8], (iii) the amino acid which follows Asp in the sequence, -Asp-Xaa- (imide formation have been reported to occur when Xaa is Gly, Asn, Asp, Ser, Ala, Arg, Cys, His and Gln, while the extent of the reaction depends on whether the Boc/Bzl (benzyl) or the Fmoc/Bu t (tert-butyl) synthetic approach is followed [7,10] and (iv) the peptide conformation [6,11]. Various methods have been proposed aimed at minimizing Asi formation. The use of HOBt (1- hydroxybenzotriazole) or 2,4-dinitrophenol (Dnp) with the base during Fmoc deprotection can partly, but not fully, suppress this side reaction [12]. In addition, these two reagents are not convenient when the peptide is attached to the resin via a base-labile linker. Another approach includes the protection of the amide group of the Xaa residue in the sequence -Asp-Xaa- with the Hmb (2-hydroxy-4-methoxybenzyl) group [13]. However, this method is not compatible with the Boc-based chemistry. In addition, difficulties have been reported in the Fmoc-based chemistry, including incomplete couplings due to the bulkiness of Hmb, as well as incomplete backbone deprotection in cases of amino acids other than glycine [10]. In general, electron donating and/or sterically hindered β -carboxyl protecting groups, such as cyclohexyl ester in the Boc-based chemistry [5] and tert-butyl ester in the Fmoc/Bu t approach [8], can also minimize Asi formation. In some cases, and especially in the synthesis of long peptides, considerable amounts of Asi-containing peptides and related by-products have been obtained. Copyright  2005 European Peptide Society and John Wiley & Sons, Ltd.