Succinimides, formation

Fig. 1.3 The currently accepted chemical mechanism of protein splicing. 1 N-S(O) acyl shift, 2 transesterification, 3 cleavage by succinimide formation, 4 S(0)-N acyl shift.

Before doing so, we briefly examine the influence of conformation and flexibility. Indeed, formation of succinimide is limited in proteins due to conformational constraints, such that the optimal value of the and ip angles (Sect. 6.1.2) around the aspartic acid and asparagine residues should be +120° and -120°, respectively [99], These constraints often interfere with the reactivity of aspartic acid residues in proteins, but they can be alleviated to some extent by local backbone flexibility when it allows the reacting groups to approach each other and, so, favors the intramolecular reactions depicted in Fig. 6.27. When compared to the same sequence in more-flexible random coils, elements of well-formed secondary structure, especially a-helices and 13-turns, markedly reduce the rate of succinimide formation and other intramolecular reactions [90][100],... 

Histidine flanking an aspartic acid residue has been found to increase the rate of succinimide formation up to tenfold (Fig. 6.27, Pathway a) [96][100]. This effect may be due to the ability of the histidine residue to facilitate succinimide formation by protonating the OH leaving group of the aspartic acid side. 

S. Clarke, Propensity for Spontaneous Succinimide Formation from Aspartyl and Asparaginyl Residues in Cellular Proteins , Int. J. Pept. Protein Res. 1987, 30, 808-821. 

The mechanism of splicing is related to the chemistry of pyruvoyl enzyme activation (Eq. 14-41), succinimide formation from asparagine residues (Eq. 2-24), and protein carboxymethylation (Box 12-A). The intein always contains serine or cysteine in its N-terminal (l)-position and asparagine in its C-terminal position. The latter is always followed by cysteine, serine, or threonine in the N-terminal... 

However, FTIR spectroscopy is not accurate enough to differentiate between the succinimide formation either by a nucleophilic addition of an aromatic diamine on the maleimide or by radical crosslinking. NMR spectroscopy offers a possibility to analyze the two different mechanisms and Fig. 8 shows the 13C chemical shifts of different carbon atoms as a function of the product structures [17]. 

Fig. 2. Continued) chain of Cys+i (N-terminal amino acid of the C-extein) on the thioester results in the formation of a branched intermediate. Excision of the intein occurs by peptide bond cleavage coupled to succinimide formation at the C-terminal asparagine of the intein. The ligated exteins undergo a spontaneous S-N acyl rearrangement to create a stable amide bond.

Figure 4 Mechanism of trans-protein splicing, (a) Initial association of the intein halves to form a functional intein. (b) Activation of the N-terminal splice-junction via an N-S acyl shift, (c) Formation of a branched intermediate upon transthioesterification. (d) Branch resolution and intein release by succinimide formation. Spontaneous S-N acyl rearrangement yields the processed product with a native peptide backbone.

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-... 

The cyclopentyl (cPe) ester group was introduced by Blake for the protection of carboxy groups. The ester bond is stable against TFA and readily cleaved with HF at 0 °C. This ester is 14 times more stable than the corresponding benzyl ester when exposed to 55% TFA in dichloromethane. However, there is little difference in the extent of HF-catalyzed succini-mide formation between peptidyl-resins containing benzyl- or cyclopentyl-protected aspartyl residues, although the latter protection reduces base-catalyzed succinimide formation. 

Stephenson, R. C. Clarke, S. (1989). Succinimide formation from aspartyl and asparginyl peptides as a model for the spontaneous degradation of proteins. J. Biol. Chem. 264,6164-6170. 

The mechanism of splicing is related to the chemistry of pyruvoyl enzyme activation (Eq. 14-41), succinimide formation from asparagine residues (Eq. 2-24), and protein carboxymethylation (Box... 

Treatment with DBU (1 to 2.5% solution in DMF or DMA) can cause succinimide formation from cyclization of aspartyl residues [77] (Fig. 8) and decomposition when silyl-based groups are used for the protection of carbohydrate hydroxy groups [78]. 

Figure 8 DBU-mediated succinimide formation from cyclization of aspartyl residues.

After the incorporation of the preceding building block and for the rest of the synthesis, the removal of the Fmoc group is performed with a 2.5% solution of DBU in DMA for 10 min. Caution DBU can cause succinimide formation from cyclization of aspartyl residues. 

Irrespective of whether solution phase or SPPS is practiced, if Boc amino acids are used with monoamino dicarboxylic acids, then the side-chain carboxyl usually is protected as its Bzl ester. Because of the potential for intramolecular succinimide formation with Asp(Bzl) peptides, however, the replacement of Bzl with a cyclohexyl (cHex) ester reduces this undesirable side reaction (14). On the other hand, if Fmoc amino acids are used, then the side-chain carboxyl usually is protected as the Bu ester. Similarly, when using Boc-Ser and Boc-Thr, the hydroxyl function is protected as the Bzl ether. In the case of Tyr, however, the Bzl ether is not stable enough during repeated TFA treatments needed to cleave Boc groups. Therefore, in the case of... 

An ice-cooled soln. of formic acid in dry ether satd. with ketene, the excess of which is then removed by a stream of dry air, ether and N-hydroxysuccinimide added to the soln. of the resulting acetic formic anhydride, stirred 3 hrs. at room temp., and allowed to stand overnight -> succinimide formate. Y 85.6%. Also formylation of phenols and f. procedures s. S. Sofuku, I. Muramatsu, and A. Hagi-tani. Bull. Chem. Soc. Japan 40, 2942 (1967). 

While with y -benzyl aspartyl residue succinimide formation can be quite extensive, the corresponding tert.butyl ester are fairly inert toward intramolecular attack. 

Fig. 7. Succinimide formation at the Asn-Gly sequence. The ultimate ratio of alfi peptides is determined by the thermodynamic stability of each sequence.

A comprehensive survey of the degradation products of 73 protein pharmaceuticals indicates that the primary chemical pathway of degradation is succinimide formation at Asn and Asp residues to yield Asp and isoAsp at both residues. Deamidation at Gin in Gln-Gly sequences, hydrolysis at Asp-Pro bonds and Met oxidation were also observed at a lesser extent. These reactions are briefly discussed below. 

FIGURE 3 Deamidation of asparagine by succinimide formation. L-aspartic acid and L-isoaspartic acid residues are the products of L-asparagine degradation via a succinimide intermediate. Aspartic acid residues can also form the succinimide intermediate by a similar mechanism with a loss of water. Figure based on that of Clarice et al. ... 

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