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. 2012 May 30;134(21):8958-67.
doi: 10.1021/ja301994d. Epub 2012 May 16.

Exploring Post-Translational Arginine Modification Using Chemically Synthesized Methylglyoxal Hydroimidazolones

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Free PMC article

Exploring Post-Translational Arginine Modification Using Chemically Synthesized Methylglyoxal Hydroimidazolones

Tina Wang et al. J Am Chem Soc. .
Free PMC article

Abstract

The methylglyoxal-derived hydroimidazolones (MG-Hs) 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, multigram-scale syntheses of all MG-H-amino acid building blocks, suitably protected for solid-phase peptide synthesis, 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. Access to synthetic MG-H-peptide adducts has enabled detailed 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 provide insight into 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.

Figures

Figure 1
Figure 1
Stuctures and retrosynthesis of the methylglyoxal-derived hydorimidazolone (MG-H) class of AGEs. (A) The MG-Hs consist of three isomeric structures as shown, each formed through condensation of methylglyoxal with arginine. (B) Proposed “divergent” retrosynthesis of all three isomers derived from a single, readily-available starting material.
Figure 2
Figure 2
MG-H3-containing peptides possess antioxidant activity comparable with ascorbic acid. (A) Peptides 26–29, 30–33, and ascorbic acid (abbreviated “AA”) were evaluated for their ability to reduce yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to purple formazan at room temperature. Data represents the mean of triplicate experiments ± standard deviation. Data in the indicated columns (***, P < 0.0001) were found to differ from all other data sets in the Figure using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparison posthoc test. (B) Radical quenching activity of peptides 26–29 was determined by treatment with the stable radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH). Values represent the mean of triplicate experiments ± standard deviation. (C) Effect of pH on the observed rate of DPPH quenching by both MG-H3–peptide and MG-H3-amino acid conjugates. Note that determination of DPPH quenching rate of free MG-Hs at pH 7 was impeded by high assay background. Values represent the mean of triplicate experiments ± standard deviation. (D) Antioxidant activity of MG-H–peptide conjugates in cellular assays. RAW 264.7 macrophages were pre-incubated with dihydrorhodamine 123, a cell-permeable fluorogenic probe converted to a highly fluorescent product on exposure to various cellular oxidants, then treated with oxidant (H2O2) and additive (peptide conjugates or AA). Two treatment regimes were explored; in the first, oxidant concentration was varied at a fixed level of additive (left-hand panel), while in the second, additive was varied at a fixed amount of oxidant (right-hand panel). Reported “Antioxidant Activity” values reflect the extent to which each additive is capable of suppressing H2O2-induced fluorescence. These values (± standard error) were calculated from linear least-squares fits of fluorescence intensity data plotted versus additive concentration as detailed in the supporting information. P-values (two-tail) represent the probabilities that pair-wise differences in slope (plus versus minus additive) could have arisen from randomly chosen data points, and are reported as follows: ***, P < 0.0001; *, P < 0.05. All reported trends were observed on at least two separate occasions.
Figure 3
Figure 3
LC/MS and LC/MS-MS analyses of hydroimidazolone-peptide conjugates in MTT antioxidant assays. (A) LC traces of MTT assay reaction mixtures. Two new peaks are observed in assays involving peptide 29, while experiments employing peptides 26, 27, and 28, indicate only starting materials. (B) Mass spectra corresponding to material contained in the LC peaks labeled 1 and 2 in panel A. These experiments indicate a loss of two mass units in newly formed material versus the parent peptide 29. (C) LC/MS-MS fragmentation analysis of material in peak 2. This data suggests that the decrease in reaction product mass originates at the MG-H3-modified residue. (D) Schematic reaction mechanism illustrating the overall transformation from MG-H3-modified peptide 29 to the corresponding Arg-modified sequence (26) and pyruvate (35) by way of imidazolone 34. For simplicity, one representitve MG-H3 tautomer is shown; the rest are provided in Scheme S4.
Figure 4
Figure 4
Proposed model for the regulation of arginine glycation. Accessible Arg sidechains react with MGO to form a mixture of isomeric MG-H adducts, of which MG-H3 is believed to be the kinetic product. MG-H3 is sensitive toward local perturbations in pH or redox balance. Path A. At neutral pH, MG-H3 undergoes rapid oxidative conversion to MG-I3, which then spontaneously hydrolyzes to regenerate Arg plus one equivalent of pyruvate. Path B. In the presence of low local pH or nearby protic residues, MG-H3-H+ is formed, and this species is likely stable and relatively insensitive to changes in redox balance. Path C. At higher local pH or in the presence of basic residues, however, ring-opening of MG-H3 affords carboxyethylarginine (CEA) and MG-H1, which both appear to be relatively environmentally insensitive. *For simplicity, only one of two likely MG-H3 tautomers is shown. Please see Supporting Information (Scheme S4) for more detail.
Scheme 1
Scheme 1
Scheme 2
Scheme 2
Scheme 3
Scheme 3

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