New Evidence for the Diversity of Mechanisms and Protonated Schiff Bases Formed in the Non-Enzymatic Covalent Protein Modification (NECPM) of HbA by the Hydrate and Aldehydic Forms of Acetaldehyde and Glyceraldehyde.

Acetaldehyde is a physiological species existing in blood. Glyceraldehyde is a commonly-used surrogate for glucose in studies of nonenzymatic glycation. Both species exist in dynamic equilibrium between two forms, an aldehyde and a hydrate. Nonenzymatic covalent protein modification (NECPM) is a process whereby a protein is covalently modified by a non-glucose species. The purpose here was to elucidate the NECPM mechanism(s) for acetaldehyde and glyceraldehyde with human hemoglobin (HbA). For the first time, both aldehydic and hydrate forms of acetaldehyde and glyceraldehyde were considered. Computations and model reactions followed by 1H NMR were employed. Results demonstrated that the aldehyde and hydrate forms of acetaldehyde bind and covalently-modify Val1 of HbA via different chemical mechanisms, yet generated an identical protonated Schiff base (PSB). The aldehyde and hydrate of glyceraldehyde also covalently-modified Val1 via mechanisms distinct from one another, yet generated an identical PSB. It is noteworthy that the PSB from acetaldehyde and glyceraldehyde were different structures. The PSB from acetaldehyde is proposed to proceed to covalent adducts that have been implicated in alcohol toxicity. Conversely, the PSB generated from glyceraldehyde can form an Amadori which has been implicated in diabetic complications. Thus, the PSB structure generated from acetaldehyde versus glyceraldehyde may be central to pathophysiological outcomes because it determines the structure of the stable covalent adduct formed.

Insights into the Progression of Labile Hb A1c to Stable Hb A1c via a Mechanistic Assessment of 2,3-Bisphosphoglycerate Facilitation of the Slow Nonenzymatic Glycation Process.

Nonenzymatic glycation (NEG) of human hemoglobin (Hb A) consists of initial non covalent, reversible steps involving glucose and amino acid residues, which may also involve effector reagent(s) in the formation of labile Hb A1c (the conjugate acid of the Schiff base). Labile Hb A1c can then undergo slow, largely irreversible, formation of stable Hb A1c (the Amadori product). Stable Hb A1c is measured to assess diabetic progression after labile Hb A1c removal. This study aimed to increase the understanding of the distinctions between labile and stable Hb A1c from a mechanistic perspective in the presence of 2,3-bisphosphoglycerate (2,3-BPG). 2,3-Bisphosphoglycerate is an effector reagent that reversibly binds in the Hb A1c pocket and modestly enhances overall NEG rate. The deprotonation of C2 on labile Hb A1c in the formation of the Amadori product was previously proposed to be rate-limiting. Computational chemistry was used here to identify the mechanism(s) by which 2,3-BPG facilitates the deprotonation of C2 on labile Hb A1c. 2,3-Bisphosphoglycerate is capable of abstracting protons on C2 and the α-nitrogen of labile Hb A1c and can also deprotonate water and/or amino acid residues, therefore preparing these secondary reagents to deprotonate labile Hb A1c. Parallel reactions not leading to an Amadori product were found that include formation of the neutral Schiff base, dissociation of glucose from the protein, and cyclic glycosylamine formation. These heretofore under appreciated parallel reactions may help explain both the selective removal of labile from stable Hb A1c and the slow rate of NEG.

Potential roles of inorganic phosphate on the progression of initially bound glucopyranose toward the nonenzymatic glycation of human hemoglobin: mechanistic diversity and impacts on site selectivity.

Nonenzymatic glycation (NEG) begins with the non-covalent binding of a glucopyranose to a protein. The bound glucopyranose must then undergo structural modification to generate a bound electrophile that can reversibly form a Schiff base, which can then lead to Amadori intermediates, and ultimately to glycated proteins. Inorganic phosphate (Pi) is known to accelerate the glycation of human hemoglobin (HbA), although the specific mechanism(s) of Pi as an effector reagent have not been determined. The aim of this study was to determine whether Pi and a glucopyranose can concomitantly bind to HbA and react while bound within the early, noncovalent stages to generate electrophilic species capable of progress in NEG. 31P and 1HNMR of model reactions confirm that bimolecular reactions between Pi and glucopyranose occur generating modified glucose electrophiles. Computations of protein/substrate interactions predict that Pi can concomitantly bind with a glucopyranose in HbA pockets with geometries suitable for multiple acid/base mechanisms that can generate any of four transient electrophiles. Pi-facilitated mechanisms in the noncovalent stages predict that the glycation of ß-Val1 of HbA to HbA1c is a "hot spot" because the ß-Val1 pocket facilitates many more mechanisms than any other site. The mechanistic diversity of the Pi effect within the early noncovalent stages of NEG predicts well the overall site selectivity observed from the in vivo glycation of HbA in the presence of Pi. These insights extend our basic understanding of the NEG process and may have clinical implications for diabetes mellitus and even normal aging.

A Perspective on Reagent Diversity and Non-covalent Binding of Reactive Carbonyl Species (RCS) and Effector Reagents in Non-enzymatic Glycation (NEG): Mechanistic Considerations and Implications for Future Research.

This perspective focuses on illustrating the underappreciated connections between reactive carbonyl species (RCS), initial binding in the nonenzymatic glycation (NEG) process, and nonenzymatic covalent protein modification (here termed NECPM). While glucose is the central species involved in NEG, recent studies indicate that the initially-bound glucose species in the NEG of human hemoglobin (HbA) and human serum albumin (HSA) are non-RCS ring-closed isomers. The ring-opened glucose, an RCS structure that reacts in the NEG process, is most likely generated from previously-bound ring-closed isomers undergoing concerted acid/base reactions while bound to protein. The generation of the glucose RCS can involve concomitantly-bound physiological species (e.g., inorganic phosphate, water, etc.); here termed effector reagents. Extant NEG schemes do not account for these recent findings. In addition, effector reagent reactions with glucose in the serum and erythrocyte cytosol can generate RCS (e.g., glyoxal, glyceraldehyde, etc.). Recent research has shown that these RCS covalently modify proteins in vivo via NECPM mechanisms. A general scheme that reflects both the reagent and mechanistic diversity that can lead to NEG and NECPM is presented here. A perspective that accounts for the relationships between RCS, NEG, and NECPM can facilitate the understanding of site selectivity, may help explain overall glycation rates, and may have implications for the clinical assessment/control of diabetes mellitus. In view of this perspective, concentrations of ribose, fructose, Pi, bicarbonate, counter ions, and the resulting RCS generated within intracellular and extracellular compartments may be of importance and of clinical relevance. Future research is also proposed.

Structures of gas-phase C2F 7 (+) ions.

In an ion cyclotron resonance spectrometer, less than 96% of the C7F 7 (+) cation formed on electron ionization of perfluorotoluene reacts with hexamethyldisilazane. In contrast, the C7F 7 (+) from perfluoronorbornadiene or perfluorobicyclo[3.2.O]hepta-2,6-diene is nonreactive with hexamethyldisilazane. Collision-induced dissociation results support this dichotomy, although the evidence is not as clear-cut. The reactive ion is assigned the benzyl structure and the nonreactive ion the tropyl structure, on the basis of analogy with the protio cases. By AM1 calculations, the perfluorobenzyl ion is 25 kcal/mol more stable than the perfluorotropyl ion, the opposite of the situation for the protio analogs (- 12 kcal/mol). Ab initio calculations at the 3-21G level agree with the semiempirical energy difference to within 0.4 kcal/mol; at the more appropriate 6-31G*/MP2 level, the perfluorobenzyl cation is 9.7 kcal/mol more stable than the perfluorotropyl cation.