Effect of lysine residues on the deamidation reaction of asparagine side chains

Capasso, Sante; Balboni, Gianfranco; Cerbo, Paola Di

doi:10.1002/(sici)1097-0282(200002)53:2<213::aid-bip11>3.0.co;2-c

Cited by 16 publications

(19 citation statements)

References 18 publications

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“…Comparison with authentic samples of the deamidate forms of I , III and RNase A showed that the deamidation site is the Asn 67 in the enzyme and the corresponding Asn 3 in the peptides. In agreement with a previous report (23) the relative yield of β‐Asp and Asp derivatives changed markedly with the pH. For all three substrates, the ratio β‐Asp/Asp derivatives ranged from about three at basic pH to less than one at acidic pH.…”

Section: Resultssupporting

confidence: 92%

“…The peptides Ac‐Cys‐Lys‐Asn‐Gly‐Gln‐Thr‐Asn‐Cys‐NH 2 ( I ) and Ac‐Cys‐Lys‐Asp‐Gly‐Gln‐Thr‐Asn‐Cys‐NH 2 ( II ) were synthesized by solid phase procedure with a Milligen 9050 synthesizer as previously reported (23). Ac‐Cys(Me)‐Lys‐Asn‐Gly‐Gln‐Thr‐Asn‐Cys(Me)‐NH 2 ( III ) and Ac‐Cys(Me)‐Lys‐Asp‐Gly‐Gln‐Thr‐Asn‐Cys(Me)‐NH 2 ( IV ) were synthesized as I and II using Fmoc‐Cys(Me)OH instead of Fmoc‐Cys(tBu)OH.…”

Section: Methodsmentioning

confidence: 99%

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Effect of the three‐dimensional structure on the deamidation reaction of ribonuclease A

Capasso

Salvadori

1999

The Journal of Peptide Research

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Kinetic data on the deamidation reaction of Asn67 in RNase A and of Asn3 in the two peptides Ac-Cys-Lys-Asn-Gly-Gln-Thr-Asn-Cys-NH2 and Ac-Cys(Me)-Lys-Asn-Gly-Gln-Thr-Asn-Cys(Me)-NH2, whose sequences are similar to that of the deamidation site in the enzyme, have been determined in a wide range of pH and buffer concentrations. The values of the observed rate constant (k) for the enzyme are markedly lower than those for the peptides. However, the k dependence on pH and buffers is similar for all three substrates, indicating a similar reaction mechanism. The lower k-values for the enzyme have been quantitatively related to the thermal stability and the three-dimensional structure of the enzyme.

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Section: Resultssupporting

confidence: 92%

Section: Methodsmentioning

confidence: 99%

Effect of the three‐dimensional structure on the deamidation reaction of ribonuclease A

Capasso

Salvadori

1999

The Journal of Peptide Research

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“…Succinimide formation involves proton removal from the backbone amide to form the highly nucleophilic amide anion, and thus would be expected to exhibit specific base catalysis,49 but this is often not observed 16, 50, 51. Tyler‐Cross and Schirch52 reported a slope of only 0.2 for a plot of log k obs versus pH in their studies of the deamidation of the peptide VANSV from pH 6 to 10 in 20 mM sulfonate buffers, and on this basis concluded that the reaction was not subject to specific base catalysis.…”

Section: Discussionsupporting

confidence: 81%

The Role of the Cyclic Imide in Alternate Degradation Pathways for Asparagine‐Containing Peptides and Proteins

DeHart

Anderson

2007

Journal of Pharmaceutical Sciences

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“…It is well‐known that side reactions such as Asi formation are highly dependent on the peptide sequence and on the Asp β ‐carboxy protecting group . The peptide sequence Ac‐ Xaa ‐Asp‐ Yaa ‐Ala‐Lys‐Phe‐NH 2 has been defined to further investigate the influence of different parameters such as the flanking amino acid residues Xaa and Yaa, the Fmoc cleavage conditions, and the Asp carboxy protecting group on the level of side reaction.…”

Section: Resultsmentioning

confidence: 99%

The aspartimide problem persists: Fluorenylmethyloxycarbonyl‐solid‐phase peptide synthesis (Fmoc‐SPPS) chain termination due to formation of N‐terminal piperazine‐2,5‐diones

Samson

Rentsch

Minuth

et al. 2019

Journal of Peptide Science

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Aspartimide (Asi) formation is a notorious side reaction in peptide synthesis that is well characterized and described in literature. In this context, we observed significant amounts of chain termination in Fmoc‐SPPS while synthesizing the N‐terminal Xaa‐Asp‐Yaa motif. This termination was caused by the formation of piperazine‐2,5‐diones. We investigated this side reaction using a linear model peptide and independently synthesizing its piperazine‐2,5‐dione derivative. Nuclear magnetic resonance (NMR) data of the side product present in the crude linear peptide proves that exclusively the six‐membered ring is formed whereas the theoretically conceivable seven‐membered 1,4‐diazepine‐2,5‐dione is not found. We propose a mechanism where nucleophilic attack of the N‐terminal amino function takes place at the α‐carbon of the carbonyl group of the corresponding Asi intermediate. In addition, we systematically investigated the impact of (a) different adjacent amino acid residues, (b) backbone protection, and (c) side chain protection of flanking amino acids. The side reaction is directly related to the Asi intermediate. Hence, hindering or avoiding Asi formation reduces or completely suppresses this side reaction.

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Effect of lysine residues on the deamidation reaction of asparagine side chains

Cited by 16 publications

References 18 publications

Effect of the three‐dimensional structure on the deamidation reaction of ribonuclease A

Effect of the three‐dimensional structure on the deamidation reaction of ribonuclease A

The Role of the Cyclic Imide in Alternate Degradation Pathways for Asparagine‐Containing Peptides and Proteins

The aspartimide problem persists: Fluorenylmethyloxycarbonyl‐solid‐phase peptide synthesis (Fmoc‐SPPS) chain termination due to formation of N‐terminal piperazine‐2,5‐diones

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