Aspartimide formation

Cycloalkyl esters have.been used to protect the /3-CO2H group in aspartyl peptides to minimize aspartimide formation during acidic or basic reactions. Aspartimide foimation is limited to 2-3% in TFA (20 h, 25°), 5-7% with HF at 0°, and 1.5-4% TfOH (thioanisole in TFA). Cycloalkyl esters are also stable to Et3N, whereas use of the benzyl ester leads to 25 % aspartimide formation during Et3N treatment. Cycloalkyl esters are stable to CF3COOH, but are readily cleaved with HF or TfOH. - ... 

Cleavage is effected with acid. The following table compares the acidolysis rates with Bn and cyclohexyl esters in TFA/phenol at 43°. The Dmp group reduces aspartimide formation during Fmoc-based peptide synthesis. 

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. 

JP Tam, TW Wong, MW Reimen, FS Tjoeng, RB Merrifield. Cyclohexyl ester as a new protecting group for aspartyl peptides to minimize aspartimide formation in acidic and basic treatments. Tetrahedron Lett 4033, 1979. 

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. 

JP Tam, MW Rieman, RB Merrifield. Mechanisms of aspartimide formation the effects of protecting groups, acid, base, temperature and time. Pept Res 1, 6, 1988. 

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. 

DBU = l,8-diazabicyclo[5.4.0]undec-7-ene (15) is a nonnucleophilic base employed in conjunction with piperidine in dimethylformamide (1 1 48) for removal of fluorenylmethyl-based protectors. The piperidine is necessary as a nucleophile to trap the expelled moiety that does not react with DBU. DBU has no effect on phthalimido [Pth-NH of -Lys(Pht)-], dialky-lphosphoryl [-Tyr(P03R2)-], or Dde-NH [-Lys(Dde)- see Section 6.4], but it promotes aspartimide formation at the pertinent residues of susceptible sequences (see Section 6.13). In dichloromethane, it promotes a reaction between two molecules of urethane-protected amino acid /V-carboxyanhy-dride with release of carbon dioxide (see Section 7.14). 

During Fmoc-based syntheses the aspartyl side chain can undergo intramolecular cyclization during repetitive base treatment, especially Asp-Asn, Asp-Gly, Asp-Gin, and Asp-Ser. Backbone protection prevents aspartimide formation and is introduced wherever prudent. 20 ... 

Scheme 1 Potential Side Reactions of Glycopeptides (A) with Acid, (B) with Base, and (C) by Aspartimide Formation 47-49 ...

Scheme 37 Aspartimide Formation during Convergent /V-Glycopeptide Synthesis 3951...

Offer, J., Quibell, M., and Johnson, T. (1996) On-resin solid-phase synthesis of asparagine-AMinked glycopeptides use of /V-(2-accloxy-4-mc(hoxybenzyl) (AcHmb) aspartyl amide-bond protection to prevent unwanted aspartimide formation./. Am. Chem. Soc. Perkin TransI., 175-182. 

The convergent approach to N-glycopeptides was first demonstrated by Peter Lansbury et al. (Scheme 11.4) [52, 53], Condensation of an oligosaccharyl amine with the Asp side chain of a suitably protected peptide was examined in solution phase but can also be carried out in solid phase. However, the drawback of this method is that the Asp unit meant to be coupled to an oligosaccharyl amine is prone to cyclic aspartimide formation with the amide nitrogen atom of the peptide backbone. Samuel Danishefsky and coworkers examined several conditions to overcome... 

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). 

Fig. 10. Aspartimide formation and succinimide ring reopening in basic medium.

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-... 

Figure 1. Manually synthesized sequence derived from an epidermal growth factor-like domain in blood coagulation factor X. The numbers in the parentheses show the sequence position relative to the C-terminal. The arrow indicates the location of aspartimide formation and subsequent ring opening by piperidine to form an adduct.

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... 

It must be remembered that hydrazine treatment of peptides containing allyl-based protecting groups also results in reduction of the aUyl moiety 0 4 this is successfully prevented by adding a 200-fold excess of allyl alcohol as a scavenger.O Moreover, hydrazine treatment of peptides may lead to aspartimide formation in the case of cyclization-prone Asn-Xaa and Asp-Xaa sequences (see Section 2.2.2). 

Scheme 1 Reaction Mechanism of Base-Catalyzed Aspartimide Formation...

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. 

In general the /er/-butyl esters, as used in the Z/tBu strategy in solution, are stable towards hydrazinolysis. Therefore, peptide intermediates containing Glu(OtBu) residues are readily converted into the corresponding hydrazides, although in the case of Asp(OtBu) residues controversial results were reported depending upon the reaction conditions and apparently even upon the peptide sequence.t In the case of the base-sensitive Asp-Gly and Asp-Ser sequences the tert-butyl ester protection does not prevent aspartimide formation similarly, in cases of Asn-Gly sequences a P transpeptidation on treatment with hydrazine was found to occur although to a lower extent. 

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