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The purpose of this work was to probe the rate and mechanism of rapid decarboxylation of pyruvic acid in the presence of hydrogen peroxide (H2O2) to acetic acid and carbon dioxide over the pH range 2-9 at 25 °C, utilizing UV spectrophotometry, high performance liquid chromatography (HPLC), and proton and carbon nuclear magnetic resonance spectrometry ((1)H, (13)C-NMR). Changes in UV absorbance at 220 nm were used to determine the kinetics as the reaction was too fast to follow by HPLC or NMR in much of the pH range. The rate constants for the reaction were determined in the presence of molar excess of H2O2 resulting in pseudo first-order kinetics. No buffer catalysis was observed. The calculated second-order rate constants for the reaction followed a sigmoidal shape with pH-independent regions below pH 3 and above pH 7 but increased between pH 4 and 6. Between pH 4 and 9, the results were in agreement with a change from rate-determining nucleophilic attack of the deprotonated peroxide species, HOO(-), on the α-carbonyl group followed by rapid decarboxylation at pH values below 6 to rate-determining decarboxylation above pH 7. The addition of H2O2 to ethyl pyruvate was also characterized., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Elucidating protein structure in amorphous solids is central to the rational design of stable lyophilized protein drugs. Hydrogen/deuterium (H/D) exchange with electrospray ionization mass spectrometry was applied to lyophilized powders containing calmodulin (17 kDa) and exposed to D(2)O vapor at controlled relative humidity (RH) and temperature. H/D exchange was influenced by RH and by the inclusion of calcium chloride and/or trehalose in the solid. The effects were not exhibited uniformly along the protein backbone but occurred in a site-specific manner, with calcium primarily influencing the calcium-binding loops and trehalose primarily influencing the alpha-helices. The results demonstrate that the method can provide quantitative and site-specific structural information on proteins in amorphous solids and on changes in structure induced by protein cofactors and formulation excipients. Such information is not readily available with other techniques used to characterize proteins in the solid state, such as Fourier transform infrared, Raman, and near-infrared spectroscopy.

Asparagine (Asn) degradation kinetics in two model peptides, Gly-Gln-Asn-Gly-Gly (GQNGG) and Val-Tyr-Pro-Asn-Gly-Ala (VYPNGA), were studied at 50 degrees C in pH 7 buffer solutions in the presence and absence of 5% (w/v) sucrose or mannitol and at 50 degrees C and 30% relative humidity in solid samples lyophilized from these solutions. Solid formulations were characterized using Karl Fischer coulometric titration, thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), Fourier-transform infrared spectrometry (FTIR), and solid-state nuclear magnetic resonance (NMR) spectroscopy. GQNGG and VYPNGA showed similar pseudo first-order deamidation rates in solution in the absence of sucrose and mannitol. Adding 5% sucrose or mannitol decreased the rates by no more than 17%. The model peptides degraded 2- to 80-fold more slowly in the solid formulations of sucrose and mannitol than in 5% solutions of these carbohydrates. Ratios of deamidation rates of the model peptides depended upon the solid matrix. In the mannitol solid, the ratio of deamidation rates of GQNGG and VYPNGA (GQNGG:VYPNGA) was approximately 8, while in the sucrose solid, the model peptides deamidated at similar rates (GQNGG:VYPNGA congruent with 1). DSC showed the mannitol formulations to be largely amorphous immediately after lyophilization with some ordered, crystalline-like structure; the extent of ordered structure increased during storage as shown by FTIR and ssNMR. In contrast, the sucrose formulation was largely amorphous after lyophilization and remained so during storage. Together, the results showed that 5% sucrose or mannitol in solution does not significantly change the rates of Asn deamidation of the model peptides, while sucrose stabilizes the model peptides against deamidation more than mannitol in the solid state., ((c) 2005 Wiley-Liss, Inc. and the American Pharmacists Association)

The deamidation kinetics of four model peptides (AcGQNGG, AcGQNDG, AcGQNEG, and AcGQNQG) were studied in solution (70 degrees C, pH 5-10) and in lyophilized solids [70 degrees C, 50% relative humidity, "effective pH" ('pH') 5-10] containing polyvinyl pyrrolidone. AcGQNGG, AcGQNEG, and AcGQNQG degraded exclusively through Asn deamidation, whereas AcGQNDG also displayed Asp isomerization, and Asp-Gly peptide bond cleavage. The pH/'pH'-rate profiles were consistent with a shift in the rate-determining step of Asn deamidation from carbonyl addition to expulsion of ammonia with increasing pH. In solution, AcGQNGG deamidated up to 38-fold faster than the other peptides, indicating the importance of steric effects of the N + 1 residue. AcGQNGG and AcGQNQG had up to 60 times slower rates of deamidation in the solid state than in solution. In contrast, the deamidation rates of AcGQNEG and AcGQNDG in the solid state were similar to those in solution. N + 1 Glu or Asp residue may enhance local hydration, so that the deamidation of Asn in the solid formulations actually proceeds in a solution-like environment., (Copyright 2005 Wiley-Liss, Inc. and the American Pharmacists Association.)

The chemical stability of recombinant human lymphotoxin (rhLT) was evaluated at pH 7, 9, and 11 and 40 degrees C using quantitative tryptic map and urea-IEF methods. Degradation products were characterized by mass spectrometry. The stability of denatured rhLT protein was also evaluated to elucidate the effects of three-dimensional structures on Asn deamidation in rhLT. Two sites that underwent Asn deamidation were identified in rhLT, Asn(19) and Asn(40)-Asn(41). At pH 11 and 40 degrees C, deamidation at Asn(19) and Asn(40)-Asn(41) had half-lives of 14 +/- 4 and 80 +/- 24 days, respectively. Upon denaturation, 31- and ninefold acceleration in the degradation rates was observed at the Asn(19) and Asn(40)-Asn(41) sites, respectively. The rate of Asn(19) degradation in denatured rhLT was comparable to that of the model peptide possessing the same primary sequence as the Asn(19)-containing region in rhLT. Analysis of the rhLT crystal structure revealed that both Asn deamidation sites were located in beta-turn structures with extensive hydrogen-bonding networks created with nearby residues in the tertiary structures. The results suggested that these tertiary and secondary structures, if held true in solution, were probably responsible for the stabilization of Asn in the native rhLT protein by reducing flexibility, thus preventing adoption of the favorable conformation required for cyclic-imide formation., (Copyright 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:869-880, 2003)

During stability studies at high temperature (70 degrees C) and low relative humidity ( approximately 0%), the recovery of an asparagine containing hexapeptide (VYPNGA) and its known deamidation products from solid polyvinylpyrrolidone (PVP) matrices was incomplete. To determine the causes of this mass loss, formulations were prepared by lyophilizing solutions containing PVP, glycerol, and the Asn-hexapeptide in pH 7.5 phosphate buffer, followed by storage at 70 degrees C and 0% relative humidity. Asn-hexapeptide loss was mono-exponential and reached a plateau at about 30% remaining. Total recovery of the peptide and its known deamidation products was approximately 30% of peptide load. Size exclusion chromatography with fluorescence detection indicated the formation of a PVP-peptide adduct that was stable in the presence of 6 M guanidine hydrochloride. Similar stability studies using N-acetyl phenylalanine, phenylalanine ethyl ester, and N-acetyl tyrosine ethyl ester demonstrated that the reaction involves the peptide N-terminus. The adduct was disrupted in the presence of carboxypeptidase-A, suggesting the formation of an amide bond between the peptide and PVP. (15)N solid-state nuclear magnetic resonance spectroscopy using (15)N-labeled valine as a model of the peptide N-terminus showed different populations of (15)N, suggesting that noncovalent peptide-polymer interactions precede amide bond formation., (Copyright 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association)

For the creation of powerfully catalytic antibodies, the technique of reactive immunization solves the problem inherent in immunization with transition-state analogs (TSAs), namely, that many interesting target reactions are multistep reactions, with multiple transition states, and thus in general, no single analog can adequately simulate all the transition states along the reaction path. In contrast, immunization with chemically reactive antigens such as phosphonylating agents, which phosphonylate B-cells during the immune response, produces antibodies that have been "trained" to recognize, bind, and stabilize all the actual transition states involved in the phosphonylation reaction. Therefore, catalytic antibodies have been selected by the immune system on the basis of their capacity to stabilize any number of transition states that occur during the target reaction. Somewhat surprisingly, phosphonolysis catalysts generated in this way commonly also catalyze esterolysis reactions. Esterolysis reactions should pass through transition states with a roughly tetrahedral disposition of ligands about a central carbon atom, while phosphonolysis reactions should pass through transition states with a roughly trigonal-bipyramidal disposition of ligands about a central phosphorus atom. These two divergent transition-state geometries suggest that the same active site should have difficulty recognizing and stabilizing both kinds of transition state. The observations thus indicate a puzzling form of "cross-reactivity" toward transition states. A possible explanation arises from evidence that at least some nucleophilic displacements at phosphorus do not pass through a trigonal-bipyramidal adduct, with a bond-formation transition state preceding it and a bond-fission transition state succeeding it. Instead a single transition state is traversed in which both bond-formation and bond-fission occur simultaneously. Such a concerted-reaction transition state should have two weak, partial bonds to phosphorus, one for formation of the nucleophile-P bond and one for fission of the P-leaving group bond. In a stepwise reaction through an intermediate, only one bond is partial and weak in each of the two transition states. The concerted-reaction transition state, with two weak bonds to phosphorus, may be more easily compressed, expanded, and otherwise distorted because of the lower force constants associated with partial bonds; particularly distortions of angles involving the two partial bonds should require relatively low energies. This may lend a high level of flexibility to phosphonolysis transition states, allowing them to be accommodated within an active site (or a range of active sites) with strong catalytic stabilization. Included among these active sites may be a majority that can also stabilize esterolysis transition states. Indeed many of the target esterolysis reactions studied to date may occur through a single concerted-reaction transition state rather than through separate transition states before and after a tetrahedral intermediate. Thus, the esterolysis transition states may also be highly flexible. Finally, flexibility present in germline antibodies may be specifically preserved in reactive immunization. The high flexibility of both kinds of ligands and of the antibody combining site may then account for the catalytic "cross-reactivity" of these antibodies.

This technical note provides evidence for the degradation of Tris buffer in a peptide formulation stored at elevated temperature (70 degrees C). The buffer degrades to liberate formaldehyde, which is shown to react with the peptide tyrosine residue. Those involved in peptide/protein formulation should be aware of the possible instability in this common biological buffer., (Copyright 2001 Wiley-Liss, Inc.)

The rate of Asn deamidation of a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala) was measured as a function of effective pH ('pH') in glassy and rubbery polymeric solids containing poly(vinyl pyrrolidone) (PVP) and in solution controls at 70 degrees C. The reaction exhibited pseudo-first-order kinetics in all samples over a wide 'pH' range (0.5 < 'pH' < 12); the formation of similar products suggests that the reaction mechanism is unaffected by matrix type. Rates of deamidation were comparable for the polymeric and solution samples in the acidic range ('pH' < 4). Solution-state rates were faster than those in polymeric solids at neutral 'pH' (6 < 'pH' < 8), increasing to a > 10,000-fold difference in the basic range ('pH' > 8). Specific base catalysis was observed in solution and in the polymeric solids under neutral conditions (6 < 'pH' < 8). In solution, the reaction exhibited general base catalysis for 'pH' > 8, whereas the reaction was 'pH'-independent in the polymeric solids in this range. The 'pH'-rate profile and supporting buffer catalysis data are consistent with a change in the rate-determining step in the basic range from 'pH'-dependent attack of the deprotonated backbone amide nitrogen on the Asn side chain in solution to 'pH'-independent ammonia expulsion in the polymeric solids. The results suggest that polymer matrix incorporation not only affects the magnitude of the deamidation rate constant but also the 'pH' dependency of the reaction and the rate-determining step in the basic 'pH' range.

The mechanistic role of water in the deamidation of a model asparagine-containing hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) in lyophilized formulations containing poly(vinylpyrrolidone) (PVP) and glycerol was investigated. Glycerol was used as a plasticizer to vary formulation glass transition temperature (T(g)) without significantly changing water content or activity. Increases in moisture and glycerol contents increased the rate of peptide deamidation. This increase was strongly correlated with T(g) at constant water content and activity, suggesting that increased matrix mobility facilitates deamidation. In rubbery systems (T > T(g)), deamidation rates appeared to be independent of water content and activity in formulations with similar T(g)s. However, in glassy formulations with similar T(g)s, deamidation increased with water content, suggesting a solvent/medium effect of water on reactivity in this regime. An increase in water content also affected the degradation product distribution; less of the cyclic imide intermediate and more of the hydrolytic products, isoAsp- and Asp-hexapeptides, were observed as water content increased. Thus, residual water appears to facilitate deamidation in these solid PVP formulations both by enhancing molecular mobility and by solvent/medium effects, and also participates as a chemical reactant in the subsequent breakdown of the cyclic imide.

This paper examines the effect of water content, water activity, and glass transition temperature (T(g)) on the deamidation of an asparagine-containing hexapeptide (VYPNGA; Asn-hexapeptide) in lyophilized poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) at 50 degrees C. The rate of Asn-hexapeptide deamidation increases with increasing water content or water activity and, hence, decreasing T(g). The rate of deamidation is more sensitive to changes in these parameters in PVA than in PVP. Deamidation is clearly evident in the glassy state in both formulations. In the glassy state, the peptide is more stable in PVA than in PVP formulations but is less stable in the rubbery state. No single variable (water content, water activity, or T(g)) could account for the variation in deamidation rates in PVA and PVP formulations. Deamidation rates were correlated with the degree of plasticization by water (distance of T(g) from the dry intrinsic glass transition temperature); coincident curves for the two polymers were obtained with this correlation. Deamidation in PVA and PVP was closely correlated with the extent of water-induced plasticization experienced by the formulation relative to its glass transition at 50 degrees C, suggesting that the physical state of formulations could be used to predict chemical stability.

The deamidation reactions of asparagine residues in alpha-helical and beta-turn secondary structural environments of peptides and proteins are reviewed. Both kinds of secondary structure tend to stabilize asparagine residues against deamidation, although the effects are not large. The effect of beta-sheet structures on asparagine stability is unclear, although simple considerations suggest a stabilization in this environment also.

The dehydration of the beta-hydroxycyclopentanone, 11,16-dihydroxy-16-methyl-9-oxo-13-trans-protenoic acid methyl ester, an analogue of prostaglandin E1, proceeds with acid catalysis (pH less than 3), by uncatalyzed routes (pH congruent to 4 and congruent to 7), and with base catalysis (pH congruent to 5-6 and greater than 8). Deuterium from the solvent is not introduced alpha to the reactant keto function at 60% reaction at pH congruent to 1, but approximately 30% exchange has occurred at pH congruent to 5, 50% at pH congruent to 7, and 80% at pH congruent to 9. The data are consistent with a mechanism in which the substrate is initially enolized with catalysis by acid, base, and water to a 1,3-enediol, which looses water with catalysis by acid, base, and water. The first stage is rate determining in very acidic solution, while the second stage assumes the limitation of rate to an ever greater degree as the solution becomes more basic.