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

DeHart, Michael P.; Anderson, Bradley D.

doi:10.1002/jps.20905

Cited by 34 publications

(25 citation statements)

References 64 publications

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“…This explains the fact that the diketopiperazines were only minor degradation products in this study at 808C while they were the dominant products in the pH range 7.5-9.5 at 258C. 32 At pH 10 and 258C the linear peptides also exceeded the diketopiperazines. 32 Extrapolating the plots of k À1 and k À2 to 258C results in values of 0.58 and 1.89 h À1 , respectively.…”

Section: Kinetics Of Isomerization and Enantiomerizationmentioning

confidence: 56%

“…The diketopiperazines are formed from the succinimides via intramolecular attack of the amino terminus. 32 It can be speculated that higher temperatures favor succinimide hydrolysis over diketopiperazine formation. This assumption is supported by the fact that higher concentrations of the diketopiperazines were observed in incubations of the succinimide peptide L-Phe-L-Asu-GlyOH at lower temperatures (see the discussion below).…”

Section: Peptide Degradationmentioning

confidence: 99%

See 1 more Smart Citation

Kinetics of Aspartic Acid Isomerization and Enantiomerization in Model Aspartyl Tripeptides under Forced Conditions

Conrad

Fahr

Scriba

2010

Journal of Pharmaceutical Sciences

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Section: Kinetics Of Isomerization and Enantiomerizationmentioning

confidence: 56%

Section: Peptide Degradationmentioning

confidence: 99%

Kinetics of Aspartic Acid Isomerization and Enantiomerization in Model Aspartyl Tripeptides under Forced Conditions

Conrad

Fahr

Scriba

2010

Journal of Pharmaceutical Sciences

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“…Because peptides containing Asn in second position may undergo additional intramolecular reactions involving the N-terminal ␣-amino group, with loss of ammonia (17 Da) and formation of a sevenmembered ring (41), we hypothesize that the Ϫ17 Da molecular species correspond to this product. Alternatively, the ␣-amino group of the succinimide intermediate can react with the succinimide ring forming a six-membered ring (diketopiperazine, DKP) (40). These reactions are not mutually exclusive and given that both DKP and seven-ring membered compound are characterized by the same molecular mass, it is likely that the Ϫ17 Da product is a mixture of both, although in an unknown proportion.…”

Section: Discussionmentioning

confidence: 99%

“…It is also known that peptides containing Asn in the second position may undergo additional intramolecular reactions involving the N-terminal ␣-amino group, loss of ammonia (Ϫ17 Da), and formation of six/seven-membered rings (40,41). To characterize the structural changes occurring in linear and cyclic peptides after degradation, we monitored peptide degradation by MALDI-TOF MS.…”

Section: Linear and Disulfide-constrained Ngr Peptides Generate Diffementioning

confidence: 99%

Critical Role of Flanking Residues in NGR-to-isoDGR Transition and CD13/Integrin Receptor Switching

Curnis

Cattaneo

Longhi

et al. 2010

Journal of Biological Chemistry

110

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Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycine-arginine (isoDGR), a ligand of ␣v␤3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the ␣-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind ␣v␤3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind ␣v␤3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (␣v␤5, ␣v␤6, ␣v␤8, and ␣5␤1), although with 10 -100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas ␣-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.Various peptides containing the Asn-Gly-Arg (NGR) motif have been discovered by peptide-phage library panning in tumor-bearing mice (1). The tumor-homing properties of these peptides rely on the interaction with aminopeptidase N (CD13), a membrane protease expressed by the tumor neovasculature (2, 3). Because of this property, these peptides have been exploited for ligand-directed delivery of various drugs and particles to tumor vessels, in the attempt to increase their antitumor activity (4). For instance, we have shown that peptides containing cyclic CNGRC and linear GNGRG motives can be used for delivering tumor necrosis factor ␣ (TNF) 3 (5-7), interferon ␥ (8 -10), and liposomal doxorubicin (11, 12) to tumor neovasculature, improving their therapeutic properties. The CNGRC-TNF conjugate, called NGR-TNF, is currently tested in phase II clinical studies (13-15). Other investigators have used the NGR motif embedded in similar or different molecular scaffolds for delivering chemotherapeutic drugs, antiangiogenic drugs, tissue factor, viruses, and other compounds to tumor vessels (1, 16 -32). Recently, a CNGRC peptide with an acetylated N-terminal ␣-amino group has been successfully exploited also for quantitative molecular magnetic resonance imaging of ...

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“…The nucleophilic functional group with free amine in the chemical molecule like hydrazines and hydroxylamines can potentially react with the cyclic succinimide intermediate formed as part of degradation pathway in the asparagine residue with all the possible factors leading to a covalent adduct [35,[38][39][40][41][42][43][44][45][46]. Tris with its free amine, a potential nucleophilic functional group, reacts with the cyclic asparagine-succinimide intermediate leading to the formation of in-solution covalent adduct that gives the mass of 104 Da addition at the asparagine site adjacent to the glycine residue (Scheme 1).…”

Section: Specific Polypeptide Sequence Motif Governs Tris Conjugate Fmentioning

confidence: 99%

Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics

Kabadi

Sankaran

Palanivelu

et al. 2016

J. Am. Soc. Mass Spectrom.

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Abstract. We present here extensive mass spectrometric studies on the formation of a Tris conjugate with a therapeutic monoclonal antibody. The results not only demonstrate the reactive nature of the Tris molecule but also the sequence and reaction conditions that trigger this reactivity. The results corroborate the fact that proteins are, in general, prone to conjugation and/or adduct formation reactions and any modification due to this essentially leads to formation of impurities in a protein sample.Further, the results demonstrate that the conjugation reaction happens via a succinimide intermediate and has sequence specificity. Additionally, the data presented in this study also shows that the Tris formation is produced in-solution and is not an in-source phenomenon. We believe that the facts given here will open further avenues on exploration of Tris as a conjugating agent as well as ensure that the use of Tris or any ionic buffer in the process of producing a biopharmaceutical drug is monitored closely for the presence of such conjugate formation.

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

Cited by 34 publications

References 64 publications

Kinetics of Aspartic Acid Isomerization and Enantiomerization in Model Aspartyl Tripeptides under Forced Conditions

Kinetics of Aspartic Acid Isomerization and Enantiomerization in Model Aspartyl Tripeptides under Forced Conditions

Critical Role of Flanking Residues in NGR-to-isoDGR Transition and CD13/Integrin Receptor Switching

Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics

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