Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins

Nonenzymatic intramolecular reactions can result in the deamidation, isomerization, and racemization of protein and peptide asparaginyl and aspartyl residues via succinimide intermediates. To understand the sequence dependence of these reactions, we measured the rate of succinimide formation in a series of synthetic peptides at pH 7.4. These peptides (Val-Tyr-Pro-X-Y-Ala) contained an internal aspartyl, asparaginyl, aspartyl beta-methyl ester, or aspartyl alpha-methyl ester residue (X) followed by a glycyl, seryl, or alanyl residue (Y). The rates of succinimide formation of the asparaginyl peptides were found to be 13.1-35.6 times faster than those of the aspartyl peptides. The rates of succinimide formation for the glycyl peptides were 6.5-17.6 times faster than those of the alanyl peptides, while the rates for the seryl peptides were 1.6-4.5 times faster than those of the alanyl peptides. The overall 232-fold range in these reaction rates for aspartyl and asparaginyl residues suggests that sequence can be an important determinant in their stability in flexible peptides. In proteins, there may be a much larger range in the rates of succinimide formation because specific conformations may greatly enhance or inhibit this reaction.     

Cyclopropane amino acid ester dipeptide sweeteners

A series of esters of L-aspartyl-1-aminocyclopropane carboxylic acid has been prepared and their sweet tastes determined. The sweetest ester prepared was about 300 times sweeter than sucrose. An attempt to use basic conditions during preparation of the dipeptide allyl ester led to succinimide formation of the aspartyl peptide even though the beta-carboxyl group was protected by a t-butyl ester function. The X-ray structure of the propyl ester (1c) was determined and its conformation is discussed.     

Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation

Aspartyl and asparaginyl deamidation, isomerization, and racemization reactions have been studied in synthetic peptides to model these spontaneous processes that alter protein structure and function. We show here that the peptide L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala undergoes a rapid deamidation reaction with a half-life of only 1.4 days at 37 degrees C, pH 7.4, to give an aspartyl succinimide product. Under these conditions, the succinimide product can further react by hydrolysis (half-time, 2.3h) and by racemization (half-time, 19.5 h). The net product of the deamidation reaction is a mixture of L- and D-normal aspartyl and beta-transpeptidation (isoaspartyl) hexapeptides. Replacement of the asparagine residue by an aspartic acid residue results in a 34-fold decrease in the rate of succinimide formation. Significant racemization was found to accompany the deamidation and isomerization reactions, and most of this could be accounted for by the rapid racemization of the succinimide intermediate. Replacement of the glycyl residue in the asparagine-containing peptide with a bulky leucyl or prolyl residue results in a 33-50-fold decrease in the rate of degradation. Peptide cleavage products are observed when these Asn-Leu and Asn-Pro-containing peptides are incubated. Our studies indicate that both aspartic acid and asparagine residues may be hot spots for the nonenzymatic degradation of proteins, especially in cells such as erythrocytes and eye lens, where these macromolecules must function for periods of about 120 days and 80 years, respectively.     

Chemical conversion of aspartyl peptides to isoaspartyl peptides. A method for generating new methyl-accepting substrates for the erythrocyte D-aspartyl/L-isoaspartyl protein methyltransferase

Mammalian protein carboxyl methyltransferases have recently been proposed to recognize atypical configurations of aspartic acid and may possibly function in the metabolism of covalently altered cellular proteins. Consistent with this proposal, the tetrapeptide tetragastrin, containing a single "normal" L-aspartyl residue (L-Trp-L-Met-L-Asp-L-Phe-NH2) was found here not to be an in vitro substrate for erythrocyte carboxyl methyltransferase activity. However, chemical treatment of tetragastrin by methyl esterification and then de-esterification of the aspartic acid residue yielded a mixture of peptide products, the major one of which could now be enzymatically methylated. We show here that this new peptide species is the isomeric beta-aspartyl form of tetragastrin (L-iso-tetragastrin; L-Trp-L-Met-L-Asp-L-Phe-NH2), and it appears that isomerization proceeds via an intramolecular succinimide intermediate during the de-esterification procedure. L-iso-Tetragastrin is stoichiometrically methylated (up to 90% in these experiments) with a Km for the enzyme of 5.0 microM. Similar chemical treatment of several other L-aspartyl peptides also resulted in the formation of new methyltransferase substrates. This general method for converting normal aspartyl peptides to isoaspartyl peptides may have application in the reverse process as well.     

Induction of beta-sheet structure in amyloidogenic peptides by neutralization of aspartate: a model for amyloid nucleation.

Amyloid fibril formation is widely accepted as a critical step in all types of amyloidosis. Amyloid fibrils derived from different amyloidogenic proteins share structural elements including beta-sheet secondary structure and similar tertiary structure. While some amyloidogenic proteins are rich in beta-sheet in their soluble form, others, like Alzheimer beta-amyloid peptide (Abeta) or serum amyloid A, must undergo significant structural transition to acquire a high beta-sheet content. We postulate that Abeta and other amyloidogenic proteins undergo a transition to beta-sheet as a result of aging-related chemical modifications of aspartyl residues to the form of succinimide or isoaspartyl methyl ester. We hypothesize that spontaneous cyclization of aspartate residues in amyloidogenic proteins can serve as a nucleation event in amyloidogenesis. To test this hypothesis, we synthesized a series of designed peptides having the sequence VTVKVXAVKVTV, where X represents aspartic acid or its derivatives. Studies using circular dichroism showed that neutralization of the aspartate residue through the formation of a methyl ester or an amide, or replacement of aspartate with glutamate led to an increased beta-sheet content at neutral and basic pH. A higher content of beta-sheet structure correlated with increased propensity for fibril formation and decreased solubility at neutral pH.     

Metabolism of a synthetic L-isoaspartyl-containing hexapeptide in erythrocyte extracts. Enzymatic methyl esterification is followed by nonenzymatic succinimide formation

The synthetic peptide, L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala, is a substrate for the erythrocyte and brain protein carboxyl methyltransferases. These enzymes catalyze the methyl esterification of the free alpha-carboxyl group of the isoaspartyl residue, to which the glycyl residue is linked through the side chain beta-carboxyl group. In this work, we show that the alpha-methyl ester of this peptide was rapidly demethylated (t1/2 = 4 min at 37 degrees C, pH 7.4) in erythrocyte cytosolic extracts and that the product of this reaction appears to be the succinimide ring derivative of the peptide. The rate of demethylation, measured at either pH 6.0 or 7.4, was the same in buffer and erythrocyte extracts, suggesting that succinimide formation was a nonenzymatic reaction. The L-succinimide is more stable than the ester, but can be hydrolyzed in buffer at pH 7.4 (t1/2 = 180 min at 37 degrees C) to give a mixture of about 75% isoaspartyl peptide and 25% normal aspartyl peptide. The metabolism of the succinimide hexapeptide in erythrocyte extracts appears to be more complex, however. The implications of this work for the methylation and demethylation of cellular proteins containing structurally altered aspartyl residues are discussed.     

Problem of aspartimide formation in Fmoc-based solid-phase peptide synthesis using Dmab group to protect side chain of aspartic acid.

The sequence-dependent, acid- or base-catalysed aspartimide formation is one of the most serious side reactions in solid-phase synthesis of peptides containing aspartic acid. In the present work, we investigated the susceptibility of 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino)benzyl (Dmab), an aspartic acid beta-carboxy side-chain protecting group, for aspartimide formation. As a model, 15-amino acid-residue galanin fragment analogue containing the Asp-Ala motif was used during Fmoc-based solid-phase synthesis. Our study showed a strong tendency of Dmab-protected peptide to form aspartimide with unusual high efficiency. Furthermore, to investigate the susceptibility of Asp-Ala motif for aspartimide formation during the synthesis using Asp(ODmab), a 5-amino acid-residue galanin fragment LGPDA, different types of resin linkers, variety of Fmoc-deprotection conditions and coupling methods were applied.     

Rapid degradation of D- and L-succinimide-containing peptides by a post-proline endopeptidase from human erythrocytes

We have been interested in the metabolic fate of proteins containing aspartyl succinimide (Asu) residues. These residues can be derived from the spontaneous rearrangement of Asp and Asn residues and from the spontaneous demethylation of enzymatically methylated L-isoAsp and D-Asp residues. Incubation of the synthetic hexapeptide N-Ac-Val-Tyr-Pro-Asu-Gly-Ala with the cytosolic fraction of human erythrocytes resulted in rapid cleavage of the prolyl-aspartyl succinimide bond producing the tripeptide N-Ac-Val-Tyr-Pro. The rate of this reaction is equal for both L- and D-Asu-containing peptides and is 10-fold greater than the rate of cleavage of a corresponding peptide containing a normal Pro-Asp linkage. When the aspartyl succinimide ring was replaced with an isoaspartyl residue, the cleavage rate was about 5 times that of the normal Pro-Asp peptide. The tripeptide-producing activity copurified on DEAE-cellulose chromatography with an activity that cleaves N-carbobenzoxy-Gly-Pro-4-methylcoumarin-7-amide, a post-proline endopeptidase substrate. These two activities were both inhibited by an antiserum to rat brain post-proline endopeptidase, and it appears that they are catalyzed by the same enzyme. This enzyme has a molecular weight of approximately 80,000 and is covalently labeled and inhibited by [3H]diisopropyl fluorophosphate. The facile cleavage of the succinimide- and isoaspartyl-containing peptides by this post-proline endopeptidase suggests that it may play a role in the metabolism of peptides containing altered aspartyl residues.     

Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins

One mechanism for the spontaneous degradation of polypeptides is the intramolecular attack of the peptide bond nitrogen on the side chain carbonyl carbon atom of aspartic acid and asparagine residues. This reaction results in the formation of succinimide derivatives and has been shown to be largely responsible for the racemization, isomerization, and deamidation of these residues in several peptides under physiological conditions (Geiger, T. & Clarke, S. J. Biol. Chem. 262, 785-794 (1987]. To determine if similar reactions might occur in proteins, I examined the sequence and conformation about aspartic acid and asparagine residues in a sample of stable, well-characterized proteins. There did not appear to be any large bias against dipeptide sequences that readily form succinimides in small peptides. However, it was found that aspartyl and asparaginyl residues generally exist in native proteins in conformations where the peptide bond nitrogen atom cannot approach the side chain carbonyl carbon to form a succinimide ring. These orientations also represent energy minimum states, and it appears that this factor may account for a low rate of spontaneous damage to proteins by succinimide-linked reactions. The presence of aspartic acid and asparagine residues in other conformations, such as those in partially denatured, conformationally flexible regions, may lead to more rapid succinimide formation and contribute to the degradation of the molecule. The possible role of isoimide intermediates, formed by the attack of the peptide oxygen atom on the side chain carboxyl group, in protein racemization, isomerization, and deamidation is also considered.     

Spontaneous peptide bond cleavage in aging alpha-crystallin through a succinimide intermediate

Cleavage of specific peptide bonds occurs with aging in the alpha A subunit of bovine alpha-crystallin. One of the breaks occurs at residue Asn-101. This same residue undergoes in vivo deamidation, isomerization, and racemization. Deamidation and isomerization are known to occur via succinimide ring formation of labile asparagine residues. Model studies on peptides have shown that imide formation can also lead to peptide bond cleavage (Geiger, T., and Clarke, S. (1987) J. Biol. Chem. 262, 785-794). In that case, both asparagine and aspartic acid amide would be expected as C termini of the truncated polypeptide, and this is indeed the case in the alpha A-(1-101)-chain. This thus represents a first example of nonenzymatic in vivo peptide bond cleavage in an aging protein through the formation of a succinimide intermediate. In addition, we found that in bovine lens no detectable conversion (through the action of protein-carboxyl methyltransferase) of isoaspartyl to normal aspartyl residues occurs in vivo after deamidation of Asn-101.     

Oligonucleotides conjugated to short lysine chains

A new method for synthesizing oligonucleotide peptide conjugates by an in-line approach is presented. A phosphorothioate oligonucleotide with the sequence of bcl-2 targeted Oblimersen by employing a modified 2'-amino-2'-desoxy-uridine nucleotide bearing a succinyl linker at the 2' position was prepared. The carboxyl group was protected for solid-phase synthesis as the benzyl ester. Ester cleavage was afforded by a phase transfer reaction using palladium nanoparticles as catalyst and cyclohexadiene as hydrogen donor. Short tails of up to three lysyl residues were conjugated to the oligonucleotide by an inverse stepwise peptide synthesis. The conjugates were characterized by HPLC, mass spectrometry, and circular dichroism. Influence of lysyl tails on CD spectra were minimal. Melting profiles revealed only minimal destabilizing effects on duplexes by conjugation of peptides.     

The peptides of α-aminosuberic acid I New intermediates for synthetic studies

Summary. The paper describes the synthesis of α-aminosuberic acid derivatives suitable for the synthesis of peptides. These include Z-, Boc- and Fmoc-protection on the α-amino group, benzyl ester, Boc-hydrazide and Z-hydrazide as well as the free carboxylic function in the side chain, and methyl ester, benzyl ester or free α-carboxylic group. Their use is demonstrated on the synthesis of the respective derivatives of Asu-Val-Leu. The enzyme catalyzed reaction was succesfully used both as a route to L-Asu from the D,L-compound as well as for the direct synthesis of the optically active tripeptide derivative from the Z-D,L-Asu-OH. Received February 5, 1999     

Benzylprotected aromatic phosphonic acids for anchoring peptides on titanium

The development of biocompatible coatings is an ongoing issue. Mimicking the physiological adhesion process of osteoblasts to the extracellular matrix improves cell adhesion of osteoblasts in vitro and results in improved and earlier osseous integration of implants in vivo. Titanium, an often used material in implant surgery, can be easily coated by peptides bearing phosphonic acid groups. We report here, the synthesis of benzyl protected phosphonic acids suitable for solid-phase peptide synthesis (SPPS), which can be easily deprotected with TFA.     

Solid-phase synthesis of bombesin by continuous flow procedure using Fmoc-amino acids

Bombesin has been synthesized by the continuous flow solid-phase procedure on the derivatized Kieselguhr-supported polydimethylacrylamide resin. Preformed Fmoc-amino acid symmetrical anhydrides (Met, Leu, and Arg) and Fmoc-amino acid active esters were used for amine acylation. The Mtr and the Pmc groups have been alternatively used for masking the side chain function of Arg-3. The progress of the synthesis was monitored by different analytical methods including quantitative solid-phase Edman degradation. Cleavage from the resin and simultaneous formation of the C-terminal amide function were achieved with a methanolic ammonia solution yielding indistinguishable crude peptides which have been purified by HPLC and fully characterized. Preliminary pharmacological experiments indicated that the activity of the synthetic peptides is similar to that previously measured for other synthetic bombesins. For comparison bombesin has also been prepared by solid-phase synthesis on 4-methyl benhydrylamine resin using the Boc chemistry. The results of the two strategies are discussed and compared.     

Solid-phase synthesis of phosphopeptides

We report the solid-phase synthesis of peptides containing O-phosphoserine. Coupling was with commercially available Fmoc-amino acid pentafluorophenyl esters, with base used at each cycle to cleave Fmoc. Phosphorylation of those serine residues left unprotected on the peptide-resin was achieved with dibenzylphosphochloridate, and finally trifluoroacetic acid was used to remove side-chain protecting groups (including the benzyl groups used for the phosphate), and to cleave the peptide from the resin in the same step. This synthetic strategy enables the preparation of peptides with individual, selectively phosphorylated residues. Alternative approaches to introduce protected phosphate and continue with coupling of further amino acids were less advantageous due to the lability of the phosphate group to base and to steric hindrance.     

The 4‐pyridylmethyl ester as a protecting group for glutamic and aspartic acids: ‘flipping’ peptide charge states for characterization by positive ion mode ESI‐MS

Use of the 4‐pyridylmethyl ester group for side‐chain protection of glutamic acid residues in solid‐phase peptide synthesis enables switching of the charge state of a peptide from negative to positive, thus making detection by positive ion mode ESI‐MS possible. The pyridylmethyl ester moiety is readily removed from peptides in high yield by hydrogenation. Combining the 4‐pyridylmethyl ester protecting group with benzyl ester protection reduces the number of the former needed to produce a net positive charge and allows for purification by RP HPLC. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI‐MS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A simple protocol for combinatorial cyclic depsipeptide libraries sequencing by matrix‐assisted laser desorption/ionisation mass spectrometry

Short cyclic peptides have a great interest in therapeutic, diagnostic and affinity chromatography applications. The screening of ‘one‐bead‐one‐peptide’ combinatorial libraries combined with mass spectrometry (MS) is an excellent tool to find peptides with affinity for any target protein. The fragmentation patterns of cyclic peptides are quite more complex than those of their linear counterparts, and the elucidation of the resulting tandem mass spectra is rather more difficult. Here, we propose a simple protocol for combinatorial cyclic libraries synthesis and ring opening before MS analysis. In this strategy, 4‐hydroxymethylbenzoic acid, which forms a benzyl ester with the first amino acid, was used as the linker. A glycolamidic ester group was incorporated after the combinatorial positions by adding glycolic acid. The library synthesis protocol consisted in the following: (i) incorporation of Fmoc‐Asp[2‐phenylisopropyl (OPp)]‐OH to Ala‐Gly‐oxymethylbenzamide‐ChemMatrix, (ii) synthesis of the combinatorial library, (iii) assembly of a glycolic acid, (iv) couple of an Ala residue in the N‐terminal, (v) removal of OPp, (vi) peptide cyclisation through side chain Asp and N‐Ala amino terminus and (vii) removal of side chain protecting groups. In order to simultaneously open the ring and release each peptide, benzyl and glycolamidic esters were cleaved with ammonia. Peptide sequences could be deduced from the tandem mass spectra of each single bead evaluated. The strategy herein proposed is suitable for the preparation of one‐bead‐one‐cyclic depsipeptide libraries that can be easily open for its sequencing by matrix‐assisted laser desorption/ionisation MS. It employs techniques and reagents frequently used in a broad range of laboratories without special expertise in organic synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Site-specific racemization in aging alpha A-crystallin

Of all aspartyl residues in bovine alpha A-crystallin, only Asp-151 exhibits pronounced racemization. Asp-151 is also one of the sites where peptide bond cleavage occurs in in vivo aging alpha A-crystallin. This aspartyl residue is followed by an alanyl residue and resides in a flexible carboxyl terminal extension of alpha-crystallin. Both in vivo and in vitro racemization studies indicate that the pronounced and site-specific racemization of Asp-151 proceeds via formation of a succinimide intermediate. The in vivo racemization of aspartyl residues in alpha A-crystallin is discussed with regard to the proposed tertiary structure of alpha-crystallin.     

Ritter-based glycoconjugation of amino acids and peptides--access to novel glycoconjugates displaying a beta-amide linkage between amino acid and sugar moiety

Beta-peptidic-D-gluco-, D-galacto-, and L-fuco-configured glycosyl amino acids can be prepared from the corresponding 2-deoxy-oct-3-ulopyranosonic acids via a one-pot intramolecular Ritter reaction. Initially, a ketopyranoside-based acid condenses under Lewis acid promoted conditions with nitriles (PhCN, MeCN) and a partially protected diamino ester (Boc-DAB-O-t-Bu, Boc-Orn-O-t-Bu) to form a beta-peptidic glycosyl amino t-butylesters. The glycosyl amino t-butylesters can be converted into Fmoc-protected glycosyl amino acids that are suitably protected for solid-phase glycopeptide synthesis. Furthermore, replacement of the protected diamino ester by immobilized peptide amines permits post-synthetic N-terminal- and N(epsilon)-glycoconjugation of peptides on the solid phase.