Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Am Chem Soc. Author manuscript; available in PMC 2013 May 30.
Published in final edited form as:
PMCID: PMC3397831

Exploring Post-Translational Arginine Modification Using Chemically Synthesized Methylglyoxal Hydroimidazolones (MG-Hs)


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).

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” ...

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.


Advanced glycation end-products (AGEs) comprise a structurally diverse class of post-translational protein modifications that form through non-enzymatic chemical processes in all living organisms, primarily on lysine and arginine side-chains.1,2 Out of more than eight arginine-derived AGEs reported to date, the methylglyoxal-derived hydroimidazolones (MG-Hs, Figure 1A) are believed to be the most prevalent in humans.3 Estimates suggest that at least one MG-H adduct is present on 3–13% of the proteins found in the human body,4,5 and that MG-H modifications comprise 1.3% by weight of total protein in heat-treated foods (e.g., baked goods, cereals, and dairy products).6

MG-H-protein adducts form both intra-and extracellularly as a mixture of three regioisomers (Figure 1A), via rapid reactions between arginine and methylglyoxal (MGO), a dicarbonyl metabolite produced by all living cells.7 Notably, elevated levels of MGO and MG-H-adducts have been shown to induce a range of cellular and subcellular effects including perturbations in cell signaling, inhibition of protein synthesis and cell growth,8 induction of oxidative stress and pro-inflammatory cytokine release,914 decreases in the adhesive properties of vascular basement membrane,15 alterations in chaperone function,16 and numerous other processes.17,18 MGO concentrations have also been observed to increase in various disease states, including diabetes,1922 cancer,23 cardiovascular disease,24 and renal failure.25 Taken together, these observations suggest that MG-H modifications may play a role in the pathophysiology of these illnesses.

Despite their putative biological properties and their ubiquity in humans, synthetic studies of the MG-Hs, and AGEs in general, are in their infancy. Available synthetic procedures for MG-H adducts are limited to providing products on analytical scales,6 in very low purities,26 or as a subset of the natural isomers.27 The vast majority of published data regarding the biological functions of MG-Hs has been obtained using highly heterogeneous mixtures prepared via prolonged incubation of model proteins (such as albumins) with MGO.28 Although these mixtures have provided useful information regarding certain properties and functions of MGO-protein adducts, their heterogeneous nature complicates efforts to identify and characterize the specific structure(s) responsible for bioactivity.29 Moreover, these samples are prone to contamination with varying levels of bioactive impurities (e.g., bacterial lipopolysaccharide).30

Here we report preparative-scale synthetic routes that for the first time afford access to the entire class of methylglyoxal-derived hydroimidazolones (MG-Hs), both as amino acids and peptide conjugates. These routes are concise (2–3 steps starting from readily accessible materials) and high yielding (44–52% overall yield), and MG-H monomers can readily be incorporated site-specifically into synthetic oligopeptides using automated Fmoc solid phase peptide synthesis (SPPS). Critically, access to chemically characterized MG-H-containing peptide constructs has enabled us to perform biochemical evaluations, which have revealed a series of notable findings. First, we have discovered that MG-H3-containing oligopeptides are redox-active, both in vitro and in a cellular system. Notably, this redox activity is pH-dependent and has a molar potency comparable to that of ascorbic acid. Computational and mechanistic studies suggest that the MG-H3 core heterocycle possesses a versatile reactivity profile and is capable of participating in distinct one- and two-electron oxidation processes. Also, we unexpectedly found that the product of MG-H3 oxidation disassembles spontaneously in aqueous solution to provide the fully unmodified arginine side-chain and pyruvate. Taken together, these data suggests that MG-H3 adducts may serve as pH-sensitive reducing agents under physiological conditions, leading to spontaneous de-glycation of the hydroimidazolone core to regenerate the native arginine side-chain. These results underscore the significant potential of chemically homogeneous MG-H adducts to facilitate chemical and biological investigations into these poorly understood species at an unprecedented level of detail.


Synthetic Studies

The arginine-derived hydroimidazoles MG-H1 through MG-H3 (Figure 1A) present substantial synthetic challenges. These compounds have been reported to decompose rapidly, and undergo spontaneous ring-opening, H/D exchange, isomerization and rearrangement reactions.31 Also, because MG-Hs likely exert their biological actions as peptide- or protein-conjugates in vivo, we required a synthesis that provided AGE-amino acid monomers suitably protected for Fmoc SPPS (Scheme 1). We therefore designed the retrosynthetic analysis shown in Figure 1B. We envisioned accessing protected isomeric MG-H monomers 13 via the regioselective cyclization of intermediates 46. Regiocontrol in these ring-forming processes was anticipated to derive from the strategic placement of functional groups R1 through R3. We also expected that the appropriate protecting groups could be used to stabilize the acid-and base-labile hydroimidazolone ring during SPPS. Due to published evidence indicating that the ring methine proton in all three MG-H isomers rapidly undergoes epimerization or exchange in water,6,32 we decided to target these compounds first as diastereomeric mixtures. Ultimately, 46 could be derived from the commercial reagent Nα-Fmoc-Nδ-Boc-L-ornithine (7) via simple alkylation and/or guanidylation processes.33

Initial efforts focused on a suitably protected MG-H1-arginine adduct. Based on our preliminary studies, and prior observations suggesting that the MG-H1 isomer is kinetically disfavored,42 we hypothesized that a blocking group on the ornithine side-chain (e.g., R1 in 4) would be necessary to direct cyclization through the desired pathway. Furthermore, we discovered that guanidylation required the side-chain N-atom to be electron rich,34 thus ruling out the majority of common N-protecting groups. After initial experiments, which revealed that sterically unhindered substituents (e.g., trimethoxybenzyl) led to spontaneous internal removal of the Fmoc group, we settled on the bis-(4-methoxyphenyl)-methyl (Dod) group to block the ornithine side-chain (Scheme 1).35,36 Application of this derivative to Fmoc-ornithine-OBn•HCl (8), followed by HgCl2-promoted coupling with Cbz-thiourea 9, smoothly provided the corresponding guanidine (10) in good yield (50% over two steps).34 Notably, this two-step sequence could be executed on preparative scales (11.0 g) without difficulty. Subjection of 10 to catalytic hydrogenolysis cleaved the Cbz and benzyl ester groups in a single operation, and the resulting intermediate (11), underwent spontaneous intramolecular cyclization to provide MG-H1 building block 12 as the exclusive product in 88% yield. Overall, this sequence provides 12 in 43% overall yield in only three synthetic steps, and can be conducted starting with >12 g of ornithine-derivative 8. Two-step removal of the remaining protecting groups afforded the free MG-H1–arginine adduct as a TFA salt (12free•TFA), whose spectral properties (1H-NMR, 13C-NMR, and HRMS) agreed well with literature values reported for a sample isolated from bakery products.6 Assignment of the hydroimidazolone ring in 12 and 12free as the indicated tautomer was based on calculations (Table S2) and comparisons with previously reported data in homologous systems.37,38

Having identified a successful route to the MG-H1 monomer (12), we next turned our attention to MG-H2 (18, Scheme 2). Toward this end, we developed a simple sequence consisting of reductive amination (814), guanidylation (1416) and cyclization (1617), which afforded an intermediate iminohydantoin (17) in 55% overall yield. Chemoselective cleavage of the benzyl ester in 17 provided side-chain- and backbone-protected MG-H2 monomer 18 in only three synthetic steps and in 48% overall yield. Global protecting group removal proceeded smoothly to yield MG-H2-arginine adduct 18free•TFA, which matched previously reported spectroscopic data.32,39 Connectivity of 18free•TFA was also independently confirmed by heteronuclear HMBC coupling between the ring methine proton and the side-chain carbon (Figure S5). Tautomeric forms of 18 and 18free were assigned based upon calculations (Table S3) and comparison to previously reported simplified systems.37,38

Finally, MG-H3 precursor 22 was obtained simply by rearranging chemical transformations that had proved successful in the routes above. This sequence (Scheme 3) began with mercury-mediated guanidylation of 8 with 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf)-derived thiourea 19,46 which afforded an intermediate N,N′-bis-alkyl-guanidine (20). Although, in theory, pathways for forming either MG-H1 or MG-H3 ring systems were both accessible via 20, in situ cyclization provided MG-H3 precursor 21 as a single regioisomer. With 21 in hand, benzyl ester removal provided compound 22 in 64% yield. Overall, this sequence allowed us to access monomer 22 in only two steps and 52% overall yield starting from 8. Protecting group removal provided 22free•TFA whose spectral properties matched previously reported values.32,39 Notably, as for the MG-H1 series (Scheme 1), synthetic sequences for accessing both MG-H2 and MG-H3 precursors could be carried out effectively on large scales (6.7–10.7 g). Connectivity assignments in 22free•TFA were inferred from heteronuclear HMBC couplings between the side-chain protons and the ring carbonyl carbon (Figure S6). However, the tautomeric preference of 22free could not be unequivocally established (see Supporting Information).37

With protected monomeric hydroimidazolone derivatives 12, 18, and 22 in hand, we set out to construct several AGE-containing oligopeptide sequences. Toward this end, we synthesized oligopeptides derived from human serum albumin (HSA), site-specifically substituting the arginine residues with the corresponding hydroimidazolone derivatives (Table 1). These included HSA fragments (residues 111–120 and 407–415), chosen because of their established propensities toward glycation in vitro,40,41 and were prepared in good to excellent yields, with purities universally greater than 95%.37 Spectroscopic data for peptides 2325 was found to match perfectly with data previously reported for these compounds.32,39 Overall, this strategy represents the first feasible route for making AGE-peptide conjugates on large scale, and also enables straightforward access to multiply AGE-derivatized constructs (Table 1, entries 8–11).32,39

Table 1
Synthesis of Hydroimidazolone-containing peptides

Although the ring stereocenters in MG-H1 and MG-H3 have been reported previously to undergo epimerization,6 the rate of epimerization has not yet been quantified. Thus, we investigated rates of H/D exchange using constructs 2325. Dissolution of these peptides as a formate salt in neat D2O revealed a gradual disappearance of the hydroimidazolone ring α-proton for all three MG-H isomers as monitored by 1H NMR spectroscopy. The half-lives of H/D exchange in these peptides were calculated to equal 40.5, 28.5, and 15.4 hours (Figures S1–3), respectively.37 Other workers have observed an increase in the rate of epimerization at pH 5.0 versus 7.4, suggesting that this process may occur rapidly under physiological conditions.42 Taken together with the mechanism for formation of MG-Hs from an achiral molecule (MGO), this data suggests that MG-Hs likely exist as thermodynamic mixtures of diastereomers in the body.

Biochemical Investigations

With various MG-H–peptide constructs in hand, we were intrigued by strong positive correlations between oxidative stress and AGEs formation, reported in both in vitro and in vivo assays.4347 In particular, MGO-protein adducts have been suggested to induce the formation of reactive oxygen species (ROS) in tissue culture,914 and treatment of bovine-serum albumin (BSA) with MGO has been shown to cause free radical generation by EPR spectroscopy.48 Interestingly, glycated proteins believed to contain MG-H adducts have also been shown to possess anti-oxidant properties in vitro.49,50 Taken together, these observations led us to hypothesize that hydroimidazolone adducts might be capable of participating in redox processes.

To test this hypothesis, we evaluated the redox activity of MG-H-peptide conjugates 2629 in vitro, in the MTT and DPPH assay systems (Figure 2). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a tetrazolium salt that can be reduced to a purple formazan by mitochondrial enzymes,51 superoxide,52 or direct electron transfer mechanisms.53 This indicator has been used to measure cell viability,54 superoxide production,52 and the antioxidant activity of natural products.55 DPPH (1,1-diphenyl-2-picryl-hydrazyl), on the other hand, is a stable radical that can be quenched by either hydrogen atom or proton-coupled electron transfer, and is commonly used to estimate the ability of substrates to serve as H-atom donors in aqueous media.56 Peptide conjugates containing the MG-H3 modification (29 and 33) exhibited anti-oxidant activities in both MTT (Figure 2A) and DPPH (Figure 2B) assays at comparable molar potencies to ascorbic acid (Vitamin C), an essential nutrient that serves as both an antioxidant57 and enzyme cofactor.58 Surprisingly, despite their high structural similarities to MG-H3, neither MG-H1 nor MG-H2 exhibited any redox activities under these conditions.

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 ...

We also evaluated the effect of pH on the rate of DPPH quenching in the presence of various MG-H constructs (Figure 2C). Once again, MG-H3 adducts demonstrated significant activity in this assay, both as peptide conjugate 29, and as the free amino acid monomer (22free), while MG-H1 and MG-H2 constructs were inactive. Furthermore, the activity of MG-H3 conjugates was observed to increase at pH values approaching 7, which is consistent with the results of pKa titrations and computational studies (see Table 2, below). Interestingly, pH changes were found to exert a far greater effect on DPPH reduction rate for MG-H3–peptide conjugate 29 than the corresponding free amino acid (22free). Taken together with observations in MTT assays (Figure 2A), this context-dependent pH sensitivity indicates the potential for neighboring amino acid residues to influence the chemical properties of AGEs, and may be an important factor in dictating the processes in which theses AGEs participate in vivo.

Table 2
pKa Values and Calculated Energies for MG-H Deriva-tives. a,b

To determine the cellular relevance of this redox behavior, we explored the effects of MG-H–peptide conjugates using a well-established assay protocol.59 Thus, RAW 264.7 macrophages were treated with dihydrorhodamine 123, a cell-permeable fluorogenic probe converted to a highly fluorescent product on exposure to cellular oxidants in the presence of varying concentrations of oxidant (H2O2) and MG-H–peptide conjugates.60 As shown in Figure 2D, MG-H3 conjugate 29 was found to possess a significant level of cellular antioxidant activity, which was both concentration-dependent (right panel), and robust over a range of physiologically relevant H2O2 concentrations (left panel, see Supporting Information for more detail). Interestingly, these effects were slightly smaller in magnitude than those of ascorbic acid, perhaps due to the relatively poor membrane permeability of this specific peptide sequence (Supplemental Figure S17). Because MGO and corresponding MG-H adducts are present physiologically both inside and outside of the cytosol, these data may underestimate the effects of MG-H3 adducts in vivo. Interestingly, MG-H2 conjugate 28 was found to possess detectable antioxidant effects, which were only revealed under conditions of variable H2O2 concentration. This observation suggests that MG-H2 adducts may also possess cellular redox activities that emerge under conditions of high oxidative stress. As before, MG-H1 conjugate 27 was found to be inactive in this assay. Taken together, these studies demonstrate the capacity of MG-H3–peptide conjugates to serve as antioxidants in a cellular milieu in the context of physiologically relevant reactive oxygen species.

To characterize the products of redox reactions involving MG-H species, we next performed LC/MS analyses of MTT assay mixtures using peptides 26–29. These experiments indicated the formation of a new species (Figure 3A). Mass spectrometric analyses revealed this newly formed material to be two mass units less than parent peptide 29 (Figure 3B), suggesting oxidation to the corresponding imidazolone (MG-I3, 34).61 Tandem mass spectrometry (MS/MS) fragmentation analysis (Figure 3C) further confirmed this decrease in mass to originate at the MG-H3 residue. Detailed NMR experiments, performed on the oxidized intermediate 34, further support our proposed structural assignment (Supplemental Figure S8). Interestingly, the analogous MG-H1-derived imidazolone was postulated in prior work to form under aerobic conditions in model reactions between MGO and BSA or model peptides;61,62 however, subsequent studies revealed this structural assignment to be incorrect, and the adduct initially believed to be the imidazolone was actually the argpyrimidine modification.62

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 ...

Based on the electron-deficient nature of the imidazolone oxidation product, we speculated that perhaps this material could react with arginine and/or lysine residues to form inter- or intramolecular crosslinks. Formation of such crosslinks is common in protein glycation processes, and has been cited as a critical contributing factor in the impact of AGE formation on protein structure and function.63 Unexpectedly, dissolution of oxidized peptide 34 in physiological buffer led to spontaneous hydrolysis to provide the parent arginine adduct 26 and pyruvate (35, Figure 3D, Supplemental Figures S9-S11). This hydrolysis process was found to proceed with first-order kinetics (kobs = 1.84 × 10−5 s−1; t1/2 = 10.5 h) at a rate compatible with the lifetime of many intracellular proteins (Supplemental Figure S11). Hydrolysis of imidazolone adducts to provide unmodified arginine residues is therefore kinetically competent to occur under physiological conditions.64 Notably, these results provide a novel mechanistic framework through which MG-H3-modified proteins can directly participate in cellular redox processes and recycle back to unmodified species. Furthermore, the observation of the pyruvate byproduct in these studies supports the intermediacy of MG-I3 (34), and also provides evidence that MG-H adducts can non-enzymatically cleave to give rise to a metabolically useful intermediate.

We next conducted mechanistic studies aimed at obtaining insight into the origin of MG-H3’s antioxidant activity. We first prepared derivatives of 29 in which the methine C–H bond in the MG-H3 core heterocycle was replaced with deuterium. Indeed, the effect of isotopic substitution on reduction rate differed substantially between the MTT and DPPH assays; a kinetic isotope effect (KIE) of 1.38 was observed using the former assay system, and 2.27 using the latter (Supplemental Figures S15–16). The relatively large KIE observed in DPPH assays supports a mechanism involving C–H/D bond homolysis as the rate-limiting step,65 while the relatively small KIE for the MTT assay is consistent with speculations that electron transfer pathways are rate-limiting under these conditions.53 Taken together, these data suggest that MG-H3 is capable of functioning as an antioxidant by way of two distinct mechanistic pathways.

To shed light on the observed reactivity differences between MG-H isomers, we performed a series of pKa titrations and Density Functional Theory (DFT) calculations (B3LYP/6–311++G(2df, 2p), Table 2). These experiments revealed a number of intriguing features, which may provide explanations for many of MG-H3’s unique properties. For example, indications that the MG-H3 core heterocycle (38) possesses C–H bond-dissociation energy (BDE) and ionization energy (IE) values that are both significantly lower than isomeric compounds 36 and 37 may explain the multi-modal activity of MG-H3 derivatives in both MTT and DPPH assays. Furthermore, the observation that 38 possesses a pKa value near neutrality, taken together with calculations suggesting that protonation of the core heterocycle (39) increases both BDE and IE, may explain the deleterious effect of decreasing pH on antioxidant activity (Figure 2C). Interestingly, the observed pKa trends for various MG-H isomers are in agreement with experimental observations of structurally homologous isomers of creatinine.38,66 Because the observed reactivity differences between MG-H isomers do not correlate with differences between free energy of oxidation (ΔGox) values (Table 2), we speculate that they reflect kinetic rather than thermodynamic factors.


Here we disclose robust synthetic routes that enable straightforward incorporation of all known variations of hydroimidazolone-modified arginine residues into peptides via automated Fmoc SPPS. These constructs have allowed us to undertake among the first detailed biochemical investigations into chemically homogeneous MG-Hs. Previously, such studies have been hampered due to limited availability of preparative-scale synthetic strategies for peptide-MG-H constructs. Our investigations have revealed a series of unexpected findings. First, using well-characterized assays to measure redox behavior, we have determined that adducts containing MG-H3 can serve as potent antioxidants in vitro and in cellular systems, while isomeric MG-H1 and MG-H2 conjugates are completely inactive under identical conditions. Based on kinetic isotope effect data and computational results, we believe that MG-H3 conjugates participate in redox reactions through two distinct mechanisms. In DPPH assays, we postulate this mechanism to involve rate-limiting H-atom transfer,65 while we speculate the MTT reduction to proceed by way of rate-limiting single-electron transfer, as previously described.53 Furthermore, results from both experimental and computational studies support that protonation of MG-H3’s core heterocycle takes place within the physiological range and diminishes its reactivity, suggesting a possible role for local pH contexts in regulating MG-H oxidation in vivo. Finally, we have observed that MG-H3 oxidation leads to spontaneous decomposition to pyruvate and fully unmodified arginine in aqueous solution. This result provides strong support for a novel “deglycation” mechanism for MG-H-modified arginine residues, which may also be relevant in vivo.

In light of our observations, as well as previous reports, we hypothesize that non-enzymatic post-translational modification of protein arginine residues may occur in vivo through the pathway shown in Figure 4. First, unmodified arginine residues react to form MG-H isomers at a rate related to MGO concentration, pH, and other protein- and context-specific variables (e.g., subcellular localization, sequence-dependent rate accelerations, etc.), based on previous studies. Our data suggests that, once formed, MG-H3 adducts are greatly influenced by their local environment. At physiological pH, MG-H3 adducts are expected to exist as a nearly equimolar mixture of unprotonated and protonated (MG-H3-H+) forms on the basis of pKa determinations (Table 2). Thus, upon encountering appropriate oxidizing agents (e.g., reactive oxygen species or free radicals), un-protonated MG-H3 can undergo a net two-electron oxidation to yield MG-I3 (Figure 4, Path A), which spontaneously hydrolyzes to afford arginine and pyruvate. Because oxidation is most likely a faster process than hydrolysis at neutral pH – for example, DPPH scavenging by peptide 29 has an observed half-life of approximately 6 minutes (Figure S11), while hydrolysis has a half-life of 10.5 h (Figures S5–S7) – the possibility exists that significant concentrations of MG-I3 can accumulate physiologically, perhaps leading to protein-protein crosslinking or increased oxidative stress.67

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 ...

In situations where protonation is favored (Figure 4, Path B), MG-H3 adducts are protected from oxidation (Figure 2C, Table 2) and ring-opening.31 They are therefore believed to be quite stable – the t1/2 of 22free at pH 5.4 and 37 °C is approximately 14.8 days.31 One might also speculate that the presence of a positive charge in MG-H3-H+ serves to mitigate destabilizing effects of side-chain modification on protein structure,68 as opposed to other MG-H modifications, which would be uncharged at neutral pH. Further studies will be necessary to investigate these possibilities in detail. Finally, in more basic environments (Figure 4, Path C), we anticipate that MG-H3 will rapidly convert to carboxyethyl arginine (CEA) and MG-H1; previous studies have shown these two species to be the predominant products of MG-H3 hydrolytic cleavage,42 and at pH 9.4, MG-H3 decomposes with a half-life of approximately 20 minutes.31 MG-H1 is also expected to be the most thermodynamically stable MG-H isomer; our calculations suggest it to be approximately 2.39 kcal/mol lower in energy than MG-H3 (Supporting Information, Table S5), a trend which is supported by previous experimental studies.42 MG-H1 is therefore expected to represent a “long-lived” modification of the arginine side-chain that is susceptible neither to hydrolytic nor oxidative removal. Indeed, MG-H1 is often identified as the major MG-H formed in vivo,32 perhaps because of its stability relative to MG-H3, and the scarcity of MG-H2.

Also worth noting are the apparent functional similarities between MG-H3 and ascorbic acid in redox assays (Figure 2). Intriguingly, like MG-H3, ascorbic acid has been documented to serve as a reducing agent through both single-electron-and H-atom-transfer mechanisms, leaving open the possibility that these species perform similar functions in vivo.69 Similarly, ascorbate’s paradoxical oxidative activity, observed in vitro in the presence of transition metals,70 may be mechanistically linked to pro-oxidant effects reported for MGO-modified proteins.71 Based on reported literature values,17,72 we estimate that concentrations of MG-H–arginine adducts can range from 100 nM to 3 μM in plasma or cytosol (higher levels are observed among diabetic patients), while ascorbate is found in the plasma at approximately 6–60 μM and in cytosol at approximately 35–500 μM.70,73 These rough estimates suggest that MG-H3 may function as a bulk antioxidant physiologically. Also, the high cellular permeability of MGO suggests the existence of MG-H–protein adducts both inside and outside of cells, although these modified proteins themselves may be impermeable.

Taken together, we hypothesize that both formation and removal of MG-H adducts can occur in a pH- and/or redox-regulated manner, and that these processes could be involved in normal physiology and perturbed in various disease processes. Validation of this theory will require follow-up studies of significant scope, which will be enabled by chemically homogeneous AGE derivatives, and have the potential to implicate MG-Hs and other AGE family members in a much broader range of life processes than previously suspected.

Supplementary Material


updated SI


We thank Prof. K. Wiberg for invaluable discussions and assistance regarding mechanistic studies and computational chemistry. We also thank Prof. Seth Herzon and Christopher Parker for helpful discussions. This work was supported by the Ellison Medical Foundation through the New Scholar Award in Aging Research and by the National Institutes of Health through the NIH Director’s New Innovator Award Program (DP22OD002913).


Supporting Information Available: Detailed experimental procedures and compound characterizations are provided.


1. Henle T. Protein and Peptide Letters. 2010;35:S32–S37.
2. Maillard LC. Comptes Rendus del’ Académie des Sciences. 1912;154:66–68.
3. Ahmed N, Thornalley PJ, Dawczynski J, Franke S, Strobel J, Stein G, Haik GM. Invest Ophthalmol Vis Sci. 2003;44:5287–92. [PubMed]
4. Thornalley P, Battah S, Ahmed N, Karachalias N, Agalou S, Babaei-Jadidi R, Dawnay A. Biochem J. 2003;375:581–592. [PubMed]
5. Ahmed N, Dobler D, Dean M, Thornalley P. J Biol Chem. 2005;280:5724–5732. [PubMed]
6. Henle T, Walter AW, Haessner R, Klostermeyer H. Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung. 1994;199:55–58. [PubMed]
7. Gomes RA, Vicente Miranda H, Silva MS, Graca G, Coelho AV, Ferreira AE, Cordeiro C, Freire AP. FEBS J. 2006;273:5273–87. [PubMed]
8. Egyud LG, Szent-Gyorgyi A. Proc Natl Acad Sci U S A. 1966;56:203–7. [PubMed]
9. Godbout JP, Pesavento J, Hartman ME, Manson SR, Freund GG. J Biol Chem. 2002;277:2554–61. [PubMed]
10. Westwood ME, Thornalley P. J Immunol Lett. 1996;50:17–21. [PubMed]
11. Abordo EA, Westwood ME, Thornalley P. J Immunol Lett. 1996;53:7–13. [PubMed]
12. Fan X, Subramaniam R, Weiss M, Monnier V. Arch Biochem Biophys. 2003;409:274–286. [PubMed]
13. Glorieux G, Helling R, Henle T, Brunet P, Deppisch R, Lameire N, Vanholder R. Kidney Int. 2004;66:1873–1880. [PubMed]
14. Van Herreweghe F, Mao JQ, Chaplen FWR, Grooten J, Gevaert K, Vandekerckhove J, Vancompernolle K. Proc Natl Acad Sci. 2002;99:949–954. [PubMed]
15. Thornalley PJ, Rabbani N. Seminars in Dialysis. 2009;22:400–404. [PubMed]
16. Gangadhariah MH, Wang B, Linetsky M, Henning C, Spanneberg R, Glomb MA, Nagaraj RH. Bba-Mol Basis Dis. 2010;1802:432–441. [PMC free article] [PubMed]
17. Thornalley PJ. Drug Metabol Drug Interact. 2008;23:125–50. [PMC free article] [PubMed]
18. Rabbani N, Thornalley P. J Ann N Y Acad Sci. 2008;1126:124–127. [PubMed]
19. Fosmark DS, Bragadottir R, Stene-Johansen I, Berg JP, Berg TJ, Lund T, Sandvik L, Hanssen KF. Acta Ophthalmol Scand. 2007;85:618–22. [PubMed]
20. Fosmark DS, Torjesen PA, Kilhovd BK, Berg TJ, Sandvik L, Hanssen KF, Agardh CD, Agardh E. Metabolism. 2006;55:232–6. [PubMed]
21. Kilhovd BK, Giardino I, Torjesen PA, Birkeland KI, Berg TJ, Thornalley PJ, Brownlee M, Hanssen KF. Metabolism. 2003;52:163–7. [PubMed]
22. Han Y, Randell E, Vasdev S, Gill V, Curran M, Newhook LA, Grant M, Hagerty D, Schneider C. Clin Biochem. 2009;42:562–9. [PubMed]
23. Thornalley PJ. Gen Pharmacol. 1996;27:565–73. [PubMed]
24. Kilhovd BK, Juutilainen A, Lehto S, Ronnemaa T, Torjesen PA, Hanssen KF, Laakso M. Atherosclerosis. 2009;205:590–4. [PubMed]
25. Agalou S, Ahmed N, Babaei-Jadidi R, Dawnay A, Thornalley PJ. J Am Soc Nephrol. 2005;16:1471–85. [PubMed]
26. Gruber P, Hofmann TJ. Pept Res. 2005;66:111–124. [PubMed]
27. Hellwig M, Geissler S, Matthes R, Peto A, Silow C, Brandsch M, Henle T. Chembiochem. 2011;12:1270–9. [PubMed]
28. Lieuw-a-Fa MLM, Schalkwijk CG, Engelse M, van Hinsbergh VWM. Thromb Haemostasis. 2006;95:320–328. [PubMed]
29. Waanders F, van den Berg E, Schalkwijk C, van Goor H, Navis G. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association-European Renal Association. 2007;22:3093–4. [PubMed]
30. Valencia JV, Mone M, Koehne C, Rediske J, Hughes TE. Diabetologia. 2004;47:844–852. [PubMed]
31. Ahmed N, Argirov OK, Minhas HS, Cordeiro CAA, Thornalley PJ. Biochem J. 2002;364:1–14. [PubMed]
32. Ahmed N, Thornalley PJ. Biochemical Journal. 2002;364:15–24. [PubMed]
33. Linton BR, Carr AJ, Orner BP, Hamilton AD. The Journal of organic chemistry. 2000;65:1566–8. [PubMed]
34. Levallet C, Lerpiniere J, Ko SY. Tetrahedron. 1997;53:5291–5304.
35. Jonsson D. Tetrahedron Letters. 2002;43:4793–4796.
36. Jonsson D, Unden A. Tetrahedron Letters. 2002;43:3125–3128.
37. Details of these experiments can be found in the Supporting Information.
38. Kenyon GL, Rowley GL. Journal of the American Chemical Society. 1971;93:5552.
39. Gruber P, Hofmann T. Journal of Peptide Research. 2005;66:111–124. [PubMed]
40. Ahmed N, Thornalley PJ. Maillard Reaction: Chemistry at the Interface of Nutrition, Aging, and Disease. 2005;1043:260–266.
41. Ahmed N, Dobler D, Dean M, Thornalley PJ. Journal of Biological Chemistry. 2005;280:5724–5732. [PubMed]
42. Klöpfer A, Spanneberg R, Glomb MAJ. Agric Food Chem. 2011;59:394–401. [PubMed]
43. Wolff SP, Jiang ZY, Hunt JV. Free Radic Biol Med. 1991;10:339–52. [PubMed]
44. Schleicher E, Friess U. Kidney Int Suppl. 2007:S17–26. [PubMed]
45. Fan X, Subramaniam R, Weiss MF, Monnier VM. Arch Biochem Biophys. 2003;409:274–86. [PubMed]
46. Schmitt A, Bigl K, Meiners I, Schmitt J. Biochim Biophys Acta. 2006;1763:927–36. [PubMed]
47. Deuther-Conrad W, Loske C, Schinzel R, Dringen R, Riederer P, Munch G. Neurosci Lett. 2001;312:29–32. [PubMed]
48. Lee C, Yim MB, Chock PB, Yim HS, Kang SOJ. Biol Chem. 1998;273:25272–8. [PubMed]
49. Dittrich R, El-Massry F, Kunz K, Rinaldi F, Peich CC, Beckmann MW, Pischetsrieder MJ. Agric Food Chem. 2003;51:3900–4. [PubMed]
50. Murakami M, Shigeeda A, Danjo K, Yamaguchi T, Takamura H, Matoba T. Journal of Food Science. 2002;67:93–96.
51. Slater TF, Sawyer B, Straeuli U. Biochim Biophys Acta. 1963;77:383–93. [PubMed]
52. Burdon RH, Gill V, Rice-Evans C. Free Radic Res Commun. 1993;18:369–80. [PubMed]
53. Marques EP, Zhang JJ, Tse YH, Metcalfe RA, Pietro WJ, Lever ABP. Journal of Electroanalytical Chemistry. 1995;395:133–142.
54. Mosmann T. J Immunol Methods. 1983;65:55–63. [PubMed]
55. Liu Y, Nair MG. J Nat Prod. 2010;73:1193–5. [PubMed]
56. Brandwilliams W, Cuvelier ME, Berset C. Food Science and Technology-Lebensmittel-Wissenschaft & Technologie. 1995;28:25–30.
57. Padayatty SJ, Katz A, Wang YH, Eck P, Kwon O, Lee JH, Chen SL, Corpe C, Dutta A, Dutta SK, Levine M. Journal of the American College of Nutrition. 2003;22:18–35. [PubMed]
58. Arrigoni O, De Tullio MC. Biochimica Et Biophysica Acta-General Subjects. 2002;1569:1–9. [PubMed]
59. Kim CY, Lee C, Park GH, Jang JH. Arch Pharm Res. 2009;32:869–81. [PubMed]
60. Emmendorffer A, Hecht M, Lohmann-Matthes ML, Roesler J. J Immunol Methods. 1990;131:269–75. [PubMed]
61. Lo TW, Westwood ME, McLellan AC, Selwood T, Thornalley PJ. J Biol Chem. 1994;269:32299–305. [PubMed]
62. Uchida K, Khor OT, Oya T, Osawa T, Yasuda Y, Miyata T. FEBS Lett. 1997;410:313–8. [PubMed]
63. Goh SY, Cooper ME. J Clin Endocrinol Metab. 2008;93:1143–52. [PubMed]
64. Dice JF. FASEB J. 1987;1:349–57. [PubMed]
65. Baciocchi E, Calcagni A, Lanzalunga O. J Org Chem. 2008;73:4110–5. [PubMed]
66. Matsumoto K, Rapoport H. J Org Chem. 1968;33:552–558.
67. Westwood ME, Thornalley PJ. J Protein Chem. 1995;14:359–72. [PubMed]
68. Biswas S, Chida AS, Rahman I. Biochem Pharmacol. 2006;71:551–64. [PubMed]
69. Halliwell B. Free Radic Res. 1999;31:261–72. [PubMed]
70. Duarte TL, Lunec J. Free Radic Res. 2005;39:671–86. [PubMed]
71. Halliwell B. Free Radical Res. 1999;31:261–272. [PubMed]
72. Yatscoff RW, Tevaarwerk GJ, MacDonald JC. Clin Chem. 1984;30:446–9. [PubMed]
73. Korcok J, Yan R, Siushansian R, Dixon SJ, Wilson JX. Brain Res. 2000;881:144–51. [PubMed]