Aspartic acid, aspartimide formation

Another competing cyclisation during peptide synthesis is the formation of aspartimides from aspartic acid residues [15]. This problem is common with the aspartic acid-glycine sequence in the peptide backbone and can take place under both acidic and basic conditions (Fig. 9). In the acid-catalysed aspartimide formation, subsequent hydrolysis of the imide-containing peptide leads to a mixture of the desired peptide and a (3-peptide. The side-chain carboxyl group of this (3-peptide will become a part of the new peptide backbone. In the base-catalysed aspartimide formation, the presence of piperidine used during Fmoc group deprotection results in the formation of peptide piperidines. 

A Karsltrom, A Unden. Design of protecting groups for the P-carboxylic group of aspartic acid that minimize base-catalyzed aspartimide formation, (dimethylpentyl) Int J Pept Prot Res 48, 305, 1996. 

A Karsltrom, A Unden. A new protecting group for aspartic acid that minimizes piperidine-catalyzed aspartimide formation in Fmoc solid phase peptide synthesis. (3-methylpent-3-yl) Tetrahedron Lett 37, 4234, 1996. 

The cyclization of aspartic acid residues to form aspartimide is the most likely side-reaction observed in routine SPPS (Fig. 10). This is a sequence-dependent side-reaction that occurs either during chain elongation or during final TFA cleavage when Asp(OtBu)-AA sequence (AA = Ala, Gly, Ser, Asn(Trt)) is present in the peptide. Hydrolysis of the aspartimide ring leads to a mixture of both a- and P-peptides. Its reaction with piperidine used for Fmoc removal also leads to the formation of a- and p-piperidides. Normally, in Fmoc-based SPPS, Asp (OtBu) provides sufficient protection. However, for particular sequences such as Asp(OtBu)-Asn(Trt) particularly sensitive to aspartimide formation, addition of HOBt to the piperidine solution or protection of the aspartyl amide bond with the 2-hydroxy-4-methoxybenzyl (Hmb) group should be considered (36). 

Aspartimide formation is sometimes a significant side-reaction in sequences containing Asp-Gly, Asp-Ser, and Asp-Ala residues. This side-product can be easily hydrolyzed to give a mixture of a- and P-peptides or, if this is deemed undesirable, then moving the aspartic acid to the C-terminal position can easily prevent it, as aspartimide formation is not possible with Asp or Glu at the C-terminal posi-... 

Cyclization of aspartic acid and asparagine to form aspartimides, and to a lesser extent of glutamic acid and glutamine to form glutarimide is an acid- and base-catalyzed common side reaction in peptide synthesis (see also Section 2.2.2). In SPPS it is particularly troublesome when Asp-Gly, Asp-Ala, and Asp-Ser sequences are present,but also with Asp-Asn.P P Piperidine-catalyzed aspartimide formation can be very rapid,and in this context DBU is even worse than piperidine.P The formation of aspartimide is reduced by the addition of HOBt or 2,4-dinitrophenol, but more efficiently it is reduced by protecting the aspartyl amide bond with the 2-hydroxy-4-methoxybenzyl (Hmb) group (see Section 2.3.2).P 1... 

Aspartic acid P-esters are incompatible with this group since cyclization to aspartimide occurs during the base-mediated deprotection. Even aspartic acid P-tert-butyl ester residues undergo this reaction with the related a P transpeptidation (see Section 2.2.2). Similarly, with C-terminal asparagine tert-butyl ester rapid succinimide formation was ob-... 

In order to prevent base-catalyzed aspartimide formation, a protecting group which provides steric hindrance in the form of both bulkiness and conformational flexibility (compared to, for example, the fert-butyl group) is required. For this purpose, the 2,4-dimethylpent-3-yl (Dmpn) ester of aspartic acid was proposed (Scheme which was shown to be very... 

Cycloalkyl esters for the side-chain protection of aspartic acid in SPPS have been developed to increase resistance to aspartimide formation. Based on mechanistic studies of this side reaction, these protection groups should fulfill the following criteria provide steric hindrance to intramolecular aminolytic attack of the ester by the amide nitrogen in acidic and basic media, provide increased stability toward repetitive TFA treatments but quantitative cleavage by HE, as well as stabilization of the carbenium ion produced by cleavage of the protecting group to prevent recapture by the peptide. The secondary cycloalkyl esters are more acid stable and more sterically hindered if compared to the primary benzyl esters. In Scheme 7, different cycloalkyl esters are shown. 

A comparative study of the (3-menthyl (Men) ester of aspartic acid with other cycloalkyl esters showed that this ester is less susceptible to base-catalyzed aspartimide formation than the cPe, Cy, cHp, or cOc derivatives. Boc-Asp(OMen)-OH is prepared by esterification of Boc-Asp-OBzl with menth-l-ol by the DCC/DMAP procedure (see Section 2.2.2.5), followed by hydrogenolysis. The menthyl ester is stable to TFA treatment and is cleaved by HF or 1M TfOH/thioanisole in TFA within 60 min in an ice bath. For some syntheses, dependent on the peptide sequence, an excess (1.4 equiv) of Boc-Asp(OMen)-OSu has to be used, presumably owing to the bulkiness of the protected side chain. For the cleavage of the Men group, in some cases diphenyl sulfide (20 equiv) has to be added as a scavenger to the 1M TfOH/thioanisole/TFA mixture to facilitate its removal. However, acid-catalyzed aspartimide formation cannot be fully prevented. 

FIGURE 15.6 Aspartic acid residues in peptides often undergo aspartimide formation during aetivation, prior to attempts to couple to glycosyl amines. This side-reaction gives rise to formation of peptides that are linked either through the a- or the P-carboxy group as by-products. 

Triphenylmethyl esters are not always stable in aqueous solution, but are stable to oxymercuration. The related 4-pyridyldiphenylmethyl and the 9-phenylfluoren-9-yl esters have been prepared of aspartic acid but these were found unsuitable for the prevention of aspartimide formation during peptide synthesis. ... 

Base-catalyzed aspartimide formation, which has been described in detail in the literature, can be a serious problem during chain assembly in peptide synthesis [29]. This side reaction involves attack by the nitrogen atom attached to the a-carboxy group of either aspartic acid or asparagine on the side chain ester or amide group. 

One major factor influencing aspartimide formation upon activation of the aspartic acid s P-carboxyl group is the identity of the neighboring amino acid located C-teiminal to the aspartic acid. Bodanszky et al. have demonstrated that peptides with Gly, Ala, Ser, Thr, Asn and Glu in this position are especially susceptible to aspartimide formation (44). Fields et al. have shown that peptides... 

Removal of the Fmoc group from the N-terminus of the resin-bound peptide chain is normally achieved by treating the peptidyl resin with 20-50% piperidine in DMF. The reaction is typically complete within 4-10 min, depending on the nature of the peptide being synthesized. With peptides containing aspartic acid and asparagine, inclusion of 0.1 M HOBt in the deprotection mixture has been found to be partially effective in suppressing aspartimide formation (18). 

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