Exploring Post-translational Arginine Modification Using Chemically Synthesized Methylglyoxal Hydroimidazolones

Abstract

The methylglyoxal-derived hydroimidazolones (MG-Hs, Figure 1A) comprise the most prevalent class of non-enzymatic, post-translational modifications of protein arginine residues found in nature. These adducts form spontaneously in the human body, and are also present at high levels in the human diet. Despite numerous lines of evidence suggesting that MG-H–arginine adducts play critical roles in both healthy and disease physiology in humans, detailed studies of these molecules have been hindered by a lack of general synthetic strategies for their preparation in chemically homogeneous form, and on scales sufficient to enable detailed biochemical and cellular investigations. To address this limitation, we have developed efficient, multi-gram-scale syntheses of all MG-H–amino acid monomers in 2–3 steps starting from inexpensive, readily available starting materials. Thus, MG-H derivatives were readily incorporated into oligopeptides site-specifically using standard solid-phase peptide synthesis (SPPS). Access to synthetic MG-H-peptide adducts has enabled detailed biochemical investigations, which have revealed a series of novel and unexpected findings. First, one of the three MG-H isomers – MG-H3 – was found to possess potent, pH-dependent antioxidant properties in biochemical and cellular assays intended to replicate redox processes that occur in vivo. Computational and mechanistic studies suggest that MG-H3-containing constructs are capable of participating in mechanistically distinct H-atom-transfer and single-electron-transfer oxidation processes. Notably, the product of MG-H3 oxidation was unexpectedly observed to disassemble into the fully unmodified arginine residue and pyruvate in aqueous solution. We believe these observations to reflect meaningfully on the role(s) of MG-H–protein adducts in human physiology, and expect the synthetic reagents reported herein to enable investigations into non-enzymatic protein regulation at an unprecedented level of detail.

Site specific modification of the human plasma proteome by methylglyoxal

“…One of the most quantitatively and functionally important MG adducts is the methylglyoxal-derived hydroimidazolone (MG-HI) adduct on arginine, producing a loss of positive charge via formation of a neutral hydroimidazolone (Scheme 1). The pKa of MG-HI is 4.58, a drastic change from the arginine side chain pKa of 12.48 (Wang et al , 2012). MG will also adduct to the side chains on lysine and cysteine residues, but adducts formed on these residues are transient and exhibit faster off-rate kinetics (Lo et al , 1994).…”

Functional Lysine Modification by an Intrinsically Reactive Primary Glycolytic Metabolite

“…Many intermediates in primary metabolic pathways reversibly bind to proteins as a form of feedback or feedforward regulation (2). Covalent PTMs are, on the other hand, typically introduced onto proteins by enzyme-catalyzed processes, but can also result from enzyme-independent interactions between reactive metabolites and nucleophilic residues in proteins (4–7). The scope and broad functional significance of non-enzymatic modifications of proteins, however, remain poorly understood.…”

Copper‐Catalyzed Domino Synthesis of 2‐Imino‐1H‐imidazol‐5(2H)‐ones and Quinoxalines Involving CC Bond Cleavage with a 1,3‐Dicarbonyl Unit as a Leaving Group

“…When 1,4‐enedione 6 a was used as substrate (R 2 =R 3 =Ph), only a low yield of the desired product was obtained (7 %; entry 19), possibly because the two bulky phenyl substituents on the 1,3‐dicarbonyl units were not beneficial for this reaction. Unfortunately, only an inseparable mixture was obtained for other alkyl substituted guanidine derivatives, such as guanidine hydrochloride ( 2 b ), 1,1‐dimethylguanidine ( 2 c ), and L ‐Arginine ( 2 d ), possibly because the corresponding products could easily undergo hydrolysis reactions 11…”

“…The synthesis of novel heterocyclic skeletons is always a hot topic in chemistry because it can provide enormous opportunities for the discovery of new pharmaceuticals and materials 10. 2‐Imino‐1 H ‐imidazol‐5(2 H )‐one derivatives are important intermediates in metabolism,11 but methods for their synthesis have rarely been investigated. Considering their potential applications in the pharmaceutical industry,12 it is highly desirable to develop an efficient method for their synthesis.…”