cysteine (HHPC)

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Identification of [6-Hydroxy-2-(hydroxymethyl)-5-oxo-5,6dihydro-2H-pyran-3-yl]-cysteine (HHPC) as a Cysteine-specific Modification Formed from 3,4-Dideoxyglucosone-3-ene (3,4-DGE) Sabrina Gensberger-Reigl, Lisa Atzenbeck, Alexander Göttler, and Monika Pischetsrieder Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00320 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 19, 2019

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Chemical Research in Toxicology

Identification of [6-Hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2H-pyran-3-yl]cysteine (HHPC) as a Cysteine-specific Modification Formed from 3,4Dideoxyglucosone-3-ene (3,4-DGE) Sabrina Gensberger-Reigl†, Lisa Atzenbeck†, Alexander Göttler, Monika Pischetsrieder*

Friedrich-Alexander University Erlangen-Nürnberg (FAU), Department of Chemistry and Pharmacy, Food Chemistry, Nikolaus-Fiebiger-Str. 10, 91058 Erlangen, Germany

†

These authors contributed equally to the work.

* Correspondence to MP, phone: +49-9131-8565592; E-mail: [email protected]

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Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Glucose degradation products (GDPs) are formed from glucose and other reducing sugars during heat treatment, for example in heat-sterilized peritoneal dialysis fluids or foods. Due to their reactive mono- and dicarbonyl structure, they react readily with proteins resulting in the formation of advanced glycation end products (AGEs), loss of protein functionality and cytotoxicity. Among the GDPs, 3,4-dideoxyglucosone-3-ene (3,4-DGE) exerts the strongest effects despite its relatively low concentration levels. The goal of the present study was therefore to identify the structure of specific protein modifications deriving from 3,4-DGE. A nonapeptide containing the reactive amino acids lysine, arginine, and cysteine was incubated with 3,4-DGE and the dominant GDPs 3-deoxyglucosone (3-DG) and 3-deoxygalactosone (3-DGal) in concentrations as present in peritoneal dialysis fluids (235 µM 3-DG, 100 µM 3Gal and 11 µM 3,4-DGE). Glycation rate and product formation was determined by ultraHPLC–MS/MS (UHPLC–MS/MS). 3,4-DGE showed the strongest glycation activity. After 2 h of incubation, 3,4-DGE had modified 57% of the nonapeptide, whereas 3-DG had modified only 2% and 3-DGal 29% of the peptide. A stable 3,4-DGE-derived cysteine modification was isolated. Its structure was determined by comprehensive NMR- and MS experiments as [6-hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2H-pyran-3-yl]-cysteine (HHPC), which represents a novel cysteine-AGE derived from 3,4-DGE. The results indicate that 3,4-DGE might contribute to a severe loss of protein functionality by forming cysteine-specific AGEs, such as HHPC.



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INTRODUCTION Glucose degradation products (GDPs) are formed by the nonenzymatic degradation of glucose and other reducing carbohydrates.1, 2 The tendency of GDPs to form advanced glycation end products (AGEs) considerably exceeds the glycation activity of the carbohydrates themselves. Exposure to GDPs in vivo has health-damaging effects,3, 4 as AGE formation leads to the loss of protein function and, consequently, tissue function.5, 6 Additionally, GDPs are strongly cytotoxic 7, 8 and form DNA-adducts 9-11 leading to a loss of genetic integrity.12 For humans, major GDP sources are the treatment with heat-sterilized drugs such as peritoneal dialysis fluids and infusion solutions,1 as well as food intake.2, 13, 14 In addition, GDPs are formed endogenously, especially under pathological conditions such as diabetes.15, 16 The term GDP summarizes different structures, mainly mono- and α-dicarbonyl compounds,2 which show different reactivity and toxicity. In particular 3,4-dideoxyglucosone-3-ene (3,4DGE), which is formed by the dehydration of the primary GDP 3-deoxyglucosone (3-DG), has strong adverse impact, even if it is present in considerably lower concentration than other GDPs.17, 18 According to a previous study, 3,4-DGE induced a similar decrease of RNase activity as the other highly reactive GDPs 3-deoxygalactosone (3-DGal) and glucosone, even though 3,4-DGE was present at only 13, respectively 46% of their concentration levels. The moderately reactive GDPs 3-DG, glyoxal, and methylglyoxal led to considerably less enzyme inactivation.19 Likewise, the strong loss of cell viability after two days of incubation with a heat-sterilized peritoneal dialysis solution could be completely explained by the presence of low concentrations of 3,4-DGE.19 Pronounced effects of 3,4-DGE on cell viability have been observed in various cell lines under varying incubation conditions.20-26 Additionally, it was reported that 3,4-DGE in infusion fluids suppressed the secretion of inflammatory cytokines after bacterial infection of neutrophils.27 4 ACS Paragon Plus Environment

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The molecular mechanisms of 3,4-DGE’s high reactivity against various cellular targets are not clear. In contrast to other GDPs, 3,4-DGE contains a β,γ-unsaturated α-dicarbonyl structure, which is prone to Michael addition of nucleophils. Consequently, it was shown that 3,4-DGE reacts preferentially with cysteine residues, whereas the other GDPs with αdicarbonyl structure mainly address lysine and arginine residues.28 Furthermore, it was observed that 3,4-DGE leads to cellular glutathione depletion by forming a covalently linked adduct.24 The aim of the present study was to investigate the molecular mechanisms leading to the high reactivity of 3,4-DGE against proteins as the main cellular target. Thus, we investigated its glycating activity and binding-site specificity in comparison to 3-DG and 3-DGal in concentrations that are common in drugs or food. Furthermore, the structure of the main cysteine adduct was elucidated.

EXPERIMENTAL PROCEDURES Reagents and Chemicals The nonapeptide Ac-GWGCGRGKG-NH2 (N-terminus acetylated, C-terminus amidated) was synthesized with a purity ≥ 95% by JPT Peptide Technologies (Berlin, Germany). Jutta Eichler (FAU Erlangen, Germany) generously provided the dipeptide Ac-FC-NH2 (N-terminus acetylated, C-Terminus amidated). 3-DG (purity > 95%) was obtained from Chemos (Regenstauf, Germany). Acetonitrile (LC-MS CHROMASOLV®), DMSO‑d6 (99.9%), D-glucose (≥ 99.5%), and o-phenylenediamine (≥ 98%) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Ammonium formate (99%) and TMS (99.9%) were obtained from Acros (Geel, Belgium) and DTT (≥ 99%) from Roth (Karlsruhe, Germany). Formic acid (for MS), disodium phosphate dihydrate (analytical grade), and sodium 5 ACS Paragon Plus Environment


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dihydrogen phosphate dihydrate (analytical grade) were purchased from Fluka (SigmaAldrich, Taufkirchen, Germany) and methanol for LC/MS from Fisher Scientific (Schwerte, Germany). For sample and eluent preparation, purified water from a Synergy-185 system (Merck Millipore, Darmstadt, Germany) was used.

Synthesis of α-Dicarbonyls 3-DGal was prepared as previously described by Madson and Feather29 with modifications by Hellwig et al.30 and Gensberger et al.31 3,4-DGE was synthesized analogously to Mittelmaier et al.18

Incubation of 3-DG, 3-DGal, and 3,4-DGE with the Nonapeptide to Evaluate their Glycating Activity Incubation experiments were performed following the method of Mittelmaier et al.28 with minor changes. Fifteen microliters of target peptide (20 µM) in water was incubated with 120 µL of aqueous GDP solutions and 15 µL of phosphate buffer (1 M, pH 7.2) in a dry block shaker (Eppendorf, Hamburg, Germany) for 2, 4, 8, 24, and 32 h at 37 °C and 500 rpm. The final GDP concentrations were 235 µM (3-DG), 100 µM (3-DGal), and 11 µM (3,4-DGE). Control samples were incubated in parallel containing 120 µL of water instead of GDPs to analyze the thermal degradation of the peptide. After incubation, the samples were stored at −20 °C until analysis. Additional aliquots were frozen directly after mixing and served as unheated controls. All incubations were performed in independent triplicates and were used to quantify the loss of α-dicarbonyls and peptide as indicators for the glycation activity as described below.


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Quantification of the α-Dicarbonyl Loss by UHPLC–DAD After thawing, the samples were directly derivatized with o-phenylenediamine and quantified according to Mittelmaier et al. by UHPLC and hyphenated diode array detection (DAD).18 To determine the α-dicarbonyl loss caused by heating and glycation, the results obtained from the test samples were related to the concentration in unheated controls, which was set to 100%.

Analysis of the Glycating Activity by the Quantification of Target Peptide Loss by UHPLC–MS/MS-MRM Product-independent analysis of the glycating activity was achieved by quantifying the remaining unmodified target peptide by UHPLC–MS/MS in multiple reaction monitoring (MRM) mode following the method of Mittelmaier et al. with minor modifications.28 First, 50 µL of the incubated peptide mixture was diluted with the same volume of DTT solution (5 mM in 0.1 M phosphate buffer, pH 7.2) to reduce the disulfide bonds formed during incubation. For UHPLC–MS/MS analysis, an Ultimate 3000 RS UHPLC system was used consisting of degasser, binary pump, autosampler, and column oven (Thermo Scientific, Germering, Germany). The system was connected to an API 4000 QTRAP mass spectrometer (AB Sciex, Darmstadt, Germany) equipped with electrospray ionization source controlled by Analyst 1.6 software. The analytes were separated on an ACQUITY UPLC® BEH300 C18ec column (2.1 x 100 mm, 1.7 µm particle size, Waters, Eschborn, Germany) equipped with a precolumn of the same material (2.1 x 5 mm, Waters) at 30 °C. The mass spectrometer was operated in positive ionization mode with ion spray voltage of 5000 V at a temperature of 500 °C. Declustering potential and collision energy were set to +30 V each. Three specific 7 ACS Paragon Plus Environment


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fragments of the target peptide were used for qualification and quantification: m/z 633.4 (y7, quantifier), m/z 576.4 (y6, qualifier), and m/z 473.4 (y5, qualifier). For the relative quantification of peptide loss caused exclusively by glycation, control samples containing water instead of GDP solution were treated analogously. The peak areas obtained from the incubated GDP samples were related to those of the concurrently analyzed controls, which were set to 100%. The experiments were carried out in independent triplicates. The level of residual unmodified peptide is indirectly proportional to the glycating activity.

Incubation of the Nonapeptide with 3,4-DGE for the Untargeted Analysis of Modification Products For the analysis of modification products formed from 3,4-DGE (final concentration 10 µM) after different incubation times, mixtures of 10 µL of the nonapeptide (5 mM), 10 µL of phosphate buffer (1 M, pH 7.2), and 80 µL of 3,4-DGE (12.5 µM) were incubated in a dry block shaker (37 °C, 500 rpm) for 4 or 12 h, and 1, 2, 3, 5, or 7 days. Controls containing water instead of peptide and controls containing water instead of 3,4-DGE were treated accordingly. All incubations were performed in independent triplicates. Untargeted analysis of modification products was carried out analogously to Mittelmaier et al.28 with minor modifications: Enhanced-resolution scans and enhanced product ion (EPI) scans were recorded independently of precursor ion scans. The declustering potential of +50 V was used and collision energies were optimized for each modification product.

Incubation of Cysteine-Containing Dipeptide and Preparation of the Novel AGE For structure elucidation, the novel cysteine-AGE was prepared from a cysteine-containing dipeptide. The formation of the corresponding adduct was monitored during dipeptide 8 ACS Paragon Plus Environment

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incubation by LC–MS analysis. For this purpose, mixtures of 10 µL of Ac-FC-NH2 (5 mM in methanol/water 1:1, v/v), 80 µL of 3,4-DGE (0.625 mM), and 10 µL of phosphate buffer (100 mM, pH 7.2) were incubated in a dry block shaker (37 °C, 500 rpm) for 12 h and 1, 3, 5, 7, 9, 11, 14, 18, or 21 days. Additional controls were prepared containing 10 µL of water instead of peptide or 80 µL of water instead of 3,4-DGE. Before analysis, all samples were diluted 1:20 with water. Using the UHPLC–MS/MS setup described above, measurements were carried out with the gradient: A, formic acid (0.1% in water) and B, acetonitrile; (time (min)/% B): −4.0/5, 0.0/5, 15.0/30, 15.5/95, 20.0/95. The flow rate was set to 0.3 mL/min. Full-scan spectra were recorded in enhanced mass spectrum mode with declustering potential of +50 V and mass traces of putative modified peptides were extracted. The third quadrupole Q3 was used as linear ion trap to enhance the mass resolution and to determine the charge state. To obtain the dipeptide-bound AGE in sufficient quantity for isolation, several aliquots consisting of 40 µL of Ac-FC-NH2 (5 mM in methanol/water 1:1, v/v), 320 µL of 3,4-DGE (0.625 mM), and 40 µL of phosphate buffer (100 mM, pH 7.2) were incubated in a dry block shaker (50 °C, 300 rpm) for 36 h. The solutions were combined and freeze-dried. The dried product was solved in a mixture of methanol and water (1:2, v/v) to a concentration of 5 mg/mL. The dipeptide-bound AGE was isolated by semipreparative HPLC (Jasco Plus HPLC system consisting of 980-50 degasser, two 2087 pumps, 3 mL mixing chamber, 2057 autosampler and 2077 UV/Vis-detector; Gross-Umstadt, Germany). The analytes were separated on a Nucleodur C18ec column (250 x 10 mm, Macherey-Nagel, Dueren, Germany) equipped with a precolumn of the same material (8 x 10 mm, Macherey-Nagel) at room temperature using the following gradient: A, formic acid (0.1% in water) and B, acetonitrile; (time (min)/% B) 0/8, 20/30, 25/80, 30/80, 33/8, 45/8. The flow rate was set to 3 mL/min and aliquots of 400 µL were injected. Chromatograms were recorded at 200 nm and 284 nm by 9 ACS Paragon Plus Environment


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Chrompass 1.7 software. The modified dipeptide eluted at 15.8 min and was collected by a CHF 122 SB fraction collector (Advantec MFS, Dublin, CA, USA) from 15.2 to 16.5 min. The organic solvent was evaporated under reduced pressure at 40 °C and the remaining solution was lyophilized.

Structure Elucidation of the Dipeptide Modification with a Mass Shift of +142 Da For UHPLC–MS experiments, aliquots of the isolated compound were analyzed using the above-mentioned chromatographic conditions. Full-scan spectra were acquired in enhanced mass spectrum mode. For fragmentation spectra, the collision energy was set to 25 V with a collision energy spread of ±5 V. The UV/Vis-absorption of the novel AGE was measured by UHPLC–DAD. Data were acquired with Chromeleon 6.80 software at 195 nm and 284 nm. Furthermore, a full DAD spectrum (190–500 nm) was recorded to obtain the UV/Vis-absorption spectra. High-resolution (HR) mass spectra of the dipeptide modification were obtained using a TripleTOF 6600 mass spectrometer (AB Sciex, Darmstadt, Germany). An aliquot of the dried product was solved in 0.1% formic acid in aqueous methanol (40/60, v/v) in a concentration of 1 µg/mL and was directly infused with a flow of 5 µL/min. Full-scan spectra were acquired in positive ionization mode using an ion spray voltage of 5000 V at a temperature of 200 °C with a declustering potential of 80 V. Data acquisition and analysis was carried out with Analyst 1.7 TF and PeakView 2.2 software. The accurate mass of the modified product obtained from HR-MS experiments enabled the calculation of elemental composition. NMR spectra were recorded for structure elucidation. The isolated product (3 mg) was solved in DMSO-d6, placed in a NMR tube and 1D- as well as 2D spectra were recorded by an Avance 600 spectrometer (600 MHz, Bruker, Rheinstetten, Germany). The measurements 10 ACS Paragon Plus Environment

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included 1H, 13C, distortion less enhancement by polarization transfer (DEPT), correlation spectroscopy (COSY), heteronuclear multiple bond correlation (HMBC), heteronuclear single quantum coherence (HSQC), rotating frame Overhauser enhancement (ROE), total correlation spectroscopy (TOCSY), selective TOCSY and selective ROE. TMS was used as internal standard. Data acquisition and processing were performed with Bruker TopSpin 3.5 software. Chemical shifts are expressed in ppm in relation to the signal of TMS.

RESULTS The goal of the present study was to assess the glycating activity of 3,4-DGE in comparison to its hydration products 3-DG and 3-DGal and to identify the structure of the major AGE formed by the reaction of 3,4-DGE. The structures of 3-DG, 3-DGal, and 3,4-DGE are depicted in the Supporting Information, Scheme S-1.

Glycating Activity of 3,4-DGE, 3-DG, and 3-DGal Against the Nonapeptide AcGWGCGRGKG-NH2 A previous study demonstrated that 3,4-DGE has much higher glycating activity than its hydration products 3-DG and 3-DGal indicating its high relevance for toxic effects of heated carbohydrate solutions. However, 3,4-DGE is usually formed in considerably lower concentration than 3-DG and 3-DGal.18, 31 Therefore, the glycating activity of the three GDPs 3,4-DGE, 3-DG, and 3-DGal was measured in the respective concentrations present in peritoneal dialysis fluids or food. For this purpose, a nonapeptide was synthesized that contained the amino acids lysine, arginine, and cysteine as possible glycation targets. The model peptide was incubated with the three different GDPs for 0, 2, 4, 8, 24, and 32 h at 37



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°C. Subsequently, UHPLC–MS/MS-MRM and UHPLC–DAD analyses determined the residual contents of α-dicarbonyls and the nonapeptide. The latter is directly correlated to the glycating activity.18, 28 After 32 h of incubation, 96% of the initial 3-DG content was found indicating that 3-DG was quite stable under the reaction conditions. 3-DGal showed a similar behavior as 3-DG: almost 80% of the initial 3-DGal concentration was detected after 32 h of incubation. In contrast to 3-DG and 3-DGal, 3,4-DGE was almost fully degraded and only 8% of the initial 3,4-DGE amount was left (Figure 1). Because 3,4-DGE was degraded to a similar extent in controls without peptide (data not shown), the decline could not result from the reaction between the peptide and 3,4-DGE alone. Thus, it can be concluded that 3,4-DGE was mainly degraded to other GDPs, such as 5-hydroxymethyl-2-furaldehyde.32 Because the 3,4-DGE concentration decreased to almost nil after 32 h of incubation, the maximum incubation time to test for glycating activity was set to 32 h. The incubation of 2 µM nonapeptide with the GDPs led to a time-dependent decrease of the peptide indicating that glycation took place at the amino acid side chains. Immediately after adding 3-DGal or 3,4-DGE, respectively, a significant peptide loss of 20% was observed, whereas 3-DG did not decrease the peptide content (Figure 2). In the course of prolonged incubation, 3,4-DGE caused a significantly higher peptide loss than 3-DG or 3-DGal. After 2 h, 98% (3-DG), 71% (3-DGal), and 43% (3,4-DGE) of the initial peptide contents were still detectable. Prolonged incubation for up to 32 h with 3-DG resulted in 77% of unmodified peptide. Peptide modification by 3,4-DGE, however, increased only marginally during the extended time, which can be explained by the degradation of 3,4-DGE during incubation. Despite its low concentration (11 µM), 3,4-DGE led to a more pronounced peptide loss than 3-DG (235 µM) and 3-DGal (100 µM) that was significantly higher at all measuring points (Figure 2). These results confirm the high relevance of 3,4-DGE for GDP-induced protein modification. 12 ACS Paragon Plus Environment

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AGE-Profiling after Incubation of the Nonapeptide with 3,4-DGE Although 3,4-DGE has high glycating activity, the structures of the resulting AGEs are mostly unknown. Therefore, untargeted UHPLC–MS/MS experiments were conducted to analyze which structures were formed. For this purpose, 500 µM nonapeptide was incubated with 10 µM 3,4-DGE for various times (4 or 12 h, and 1, 2, 3, 5, or 7 days) before analysis by UHPLC–MS/MS. This experiment included time frames of more than 32 h to determine whether the AGEs were stable or if any stable AGEs were formed from intermediates during prolonged incubation. Four major different mass shifts could be observed, namely +162, +144, +142, and +126 Da (Table 1). Whereas the mass shifts of +162, +144, and +126 Da could already be detected after 4 h of incubation, the modification characterized by the mass shift of +142 Da appeared after more than two days. These results indicate that the modification of +142 Da depicts a stable AGE, which may be of particular relevance for the loss of protein function. A modification of +142 Da had also been detected as a major product, when 3,4-DGE was incubated with a peptide under different conditions 28 indicating a broader relevance. The mass difference of +142 Da could not be related to any known AGE structure. Therefore, further experiments were carried out to identify the structure of this AGE, which seems to be selectively formed from 3,4-DGE. The other detected mass shifts could be explained by 3-DG/3-DGal-derived hemiaminal or hemithioacetal (+162 Da),28 Michael adduct (+144 Da),24 and 3,4-DGE-derived imidazolinone (+126 Da). EPI scans supported these assignments and revealed the unmodified peptide in the fragment spectra of the adducts +162 Da and +144 Da indicating a relatively loosely bound modification in accordance with a hemiaminal/hemithioacetal or Michael adduct (see Supporting Information, Figure S-1).



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Structure Elucidation of the 3,4-DGE-Derived Cysteine-AGE Fragmentation patterns of the 3,4-DGE-derived peptide adduct +142 Da were recorded by EPI scans to determine the modification site. The presence of several modified fragments y6 and y7, but only unmodified fragments y4 and y5 indicates that the modification is bound to the cysteine residue (see Supporting Information, Figure S-1). To identify the cysteine modification with a mass shift of +142 Da, a dipeptide consisting of Ac-FC-NH2 was incubated with 3,4-DGE. We used this dipeptide consisting of cysteine and phenylalanine because phenylalanine enhances retention in reversed-phase chromatography and allows UV detection. Targeted MS-screening showed that an adduct with a mass shift of +142 was also formed during the incubation of 3,4-DGE with Ac-FC-NH2 confirming the presence of a cysteine modification. To increase the yield for further structure elucidation, 3,4-DGE and Ac-FCNH2 were then incubated in equimolar concentrations. UHPLC–DAD experiments revealed a bathochromic shift of the UV spectra with an additional maximum at 284 nm (Figure 3B) of the modified dipeptide. Therefore, the adduct had to contain an additional chromophore. Chromatographic separation revealed a double peak at the retention time of 5.85 min (Figure 3A). Both signals showed the same UV spectra and collision-induced fragmentation pattern indicating isomeric structures. The product-ion spectra revealed a characteristic neutral loss of one ammonia molecule (−17 Da, −NH3, m/z 435), and one (m/z 417) or two (m/z 399) water molecules (−18 Da each) proceeding from the parent ion m/z 452. The most intensive signal (m/z 263) could be assigned to the modified cysteine. Starting from this fragment, the characteristic neutral loss of one or two water molecules can be observed indicating at least two hydroxyl groups within the fragment (Figures 3C/D). HR-MS experiments revealed a


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singly charged ion with m/z 452.14851 [M+H+] which is in accordance with the elemental formula C20H25N3O7S (calculated mass 452.14915 [M+H+]). NMR experiments were performed for unequivocal structure elucidation. To this end, the modified dipeptide was isolated by semipreparative HPLC–UV, freeze-dried, and subsequently analyzed by 1D and 2D NMR. The NMR signals referring to the modification are displayed in Table 2 and discussed below. Based on these experiments, the cysteine modification could be assigned to [6-hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2H-pyran-3-yl]-cysteine (HHPC, Scheme 1).

DISCUSSION GDPs are powerful glycating agents so that even small amounts may have adverse health effects. Recent studies revealed that the α-dicarbonyl 3,4-DGE is much more active, for example in respect to enzyme inactivation, than the short-chain GDPs glyoxal and methylglyoxal.19 Furthermore, cytotoxic effects impairing the biocompatibility of drugs have been described for 3,4-DGE.19, 23, 24, 33 The first goal of the present study was to analyze the glycating activity and specificity of 3,4-DGE applied in concentrations as present, for instance, in peritoneal dialysis fluids or foods and to compare it to the activity of the more prevalent GDPs 3-DG and 3-DGal.18, 31, 34 All three α-dicarbonyls (3,4-DGE, 3-DG, and 3DGal) are in equilibrium in aqueous solution (see Supporting Information, Scheme S-1).35 The peptide Ac-GWGCGRGKG-NH2 with acetylated N-terminus and amidated C-terminus was used as glycation target. Lysine, arginine, and cysteine are the main targets in proteins affected by AGE formation. In contrast to free amino acids, the model peptide allowed for the simultaneous analysis of all putative glycation sites combined in one peptide. Additionally, the use of the model nonapeptide had the advantage over proteins that no enzymatic hydrolysis was necessary before analysis that could be negatively influenced by AGEs.36, 37 15 ACS Paragon Plus Environment


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Glycine served as a spacer between the reactive amino acid side chains in the model to avoid steric hindrance. The loss of 3,4-DGE during incubation most likely resulted from thermal treatment and glycation reactions with the target peptide (see also below). Significant hydration of 3,4-DGE yielding 3-DG or 3-DGal, respectively, was not observed, because the content of these two compounds did not increase distinctly (data not shown). However, interconversion of 3,4DGE to 3-DGal took place, but only in very small quantities. In contrast, no 3-DG was detected in the incubated solutions of 3,4-DGE. In parallel, the peptide loss during incubation was monitored by targeted UHPLC–MS/MS-MRM analysis. When incubated with 3-DG and 3-DGal, the concentrations of unmodified peptide decreased to 77, resp. 65% after 32 h. Even though 3-DGal differs from 3-DG only by the stereochemistry of the hydroxyl group at C-4, 3-DGal led to a significantly higher peptide loss than 3-DG for up to eight hours of incubation despite being present in lower concentration. This finding is in line with results by Distler et al.,19 who observed RNase inhibition after incubation with 3-DG or 3-DGal. In the reported model, 3-DGal seemed to be more active than its diastereomer 3-DG.19 The reason for the higher reactivity of 3-DGal compared to 3-DG is not known yet, but it had been suggested that the configuration of the hydroxyl group at C-4 may shift the equilibrium of the cyclic hemiacetal forms towards more reactive isomers.19 The incubation with 3,4-DGE resulted in even more pronounced peptide modification. The present observation confirms previous reports, which demonstrated that 3,4-DGE strongly inhibited RNAse activity.19 Remarkably, 3-DG and 3-DGal were used in much higher concentration levels (approximately factor 21 and factor 10, respectively) than 3,4-DGE, but the latter had the most pronounced effect in both models. These findings substantiate the critical role of 3,4DGE impairing the biocompatibility of drugs.


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It seems probable that nucleophilic amino acid side chains can react with the Michael system of 3,4-DGE and, thus, form AGEs. A previous study showed that 3,4-DGE mainly reacted with cysteine residues.28 Due to their ability to form disulfide bridges, cysteine residues are highly important for protein stability and functionality. Additionally, cysteine residues are often located at the active site of enzymes. Thus, it is of high interest how 3,4-DGE can modify cysteine and consequently impair protein- or enzyme activity. To address this issue, a dipeptide was incubated with 3,4-DGE and the reaction product showing a mass shift of +142 Da was isolated for further experiments. The modification with a mass shift of +142 Da seems to be specific for 3,4-DGE.28 The structure could be unequivocally assigned to [6hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2H-pyran-3-yl]-cysteine (HHPC; Scheme 1). An isolated C-H proton without any direct coupling partners on C-4 is characteristic for this structure, which could be confirmed by the COSY spectra, where no coupling partner was detected. However, signal splitting at 6.24 ppm was observed in 1H NMR spectra. Hence, ROE, TOCSY and selective TOCSY experiments were performed. The experiments demonstrated the interaction between the C-4 proton and one proton located on the CH2 group of the cysteine residue confirming the signal splitting. The proton on C-6 can undergo coupling with the proton located on the C-6 hydroxy group, which was confirmed by COSY experiments. It is probable that the proton of C-2 undergoes vinylogous keto-enoltautomerism with the carbonyl group on C-5. This hypothesis could be confirmed by hydrogen-deuterium (H/D) exchange experiments because no signal for H-2 at 4.37 ppm could be observed after D2O addition indicating an acidic proton on C-2. This result was also confirmed by the COSY experiments, which showed only a very weak signal for the coupling between C-2 and C-7 protons. The NMR data, however, demonstrated that the major amount of the AGE structure exists in the carbonyl form because a strong signal could be detected at 198 ppm for the carbon atom, but no signal for an additional hydroxyl group or a hydroxyl-



Page 18 of 34

substituted unsaturated carbon atom, which was expected at about 180 ppm. Both signals for the hydroxyl groups located on C-6 and C-7 (5.39 ppm and 5.85 ppm) were confirmed by H/D exchange experiments, which clearly indicated the complete signal loss in the presence of D2O. The aliphatic protons at carbon atom C-7 were also confirmed by the chemical shift of 3.57 ppm and 3.31 ppm and coupling signals. The substitution pattern of the carbon atoms could be confirmed by DEPT, HSQC, HMBC, and TOCSY experiments, which all obtained the same structural properties. Thus, a new cysteine-AGE derived from 3,4-DGE could be unequivocally identified to be HHPC. This structure is also in good accordance with the presence of two signals in the chromatograms, because C-2 is a CH-acidic position resulting in stereoisomers (Figure 3). Further, α- and β-isomers can be formed at C-6. The proposed structure is also in accordance with the UV-absorption spectrum, because an α,β-unsaturated carbonyl moiety leads to an additional UV-absorption maximum of the dipeptide. A proposed formation mechanism leading to this new AGE is displayed in Scheme 2. In the first step, the Michael system is attacked by the thiol group of cysteine, which is a stronger nucleophile than the amine group of lysine. The resulting thioether is easily oxidized leading to the formation of a sulfoxide. A chromatographic signal corresponding to this sulfoxide (+158 Da) was observed in extracted EMS scans. Subsequently, vinylogous tautomerization steps can follow eventually promoting the elimination of water. In the last step, a hemiacetal is formed to yield HHPC. In conclusion, we could demonstrate that 3,4-DGE forms a novel AGE modifying specifically cysteine residues, which might have negative impact on the structure and function of proteins and, thus, contribute to adverse health effects of this GDP in drugs. Additionally, 3,4-DGE showed the highest glycating activity against a model peptide despite its very low concentration compared to 3-DGal and 3-DG. Thus, 3,4-DGE should serve as a


Page 19 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


highly relevant marker for product quality during drug development to achieve maximal biocompatibility.

Supporting Information Figure S1. Enhanced product ion spectra of the modified nonapeptide Ac-GWGCGRGKGNH2 after incubation with 3,4-dideoxyglucosone-3-ene Scheme S1. Structures of 3,4-dideoxyglucosone-3-ene (3,4-DGE) and its hydration products 3-deoxyglucosone (3-DG) and 3-deoxygalactosone (3-DGal)



Page 20 of 34

Funding Information This study was supported by the Deutsche Forschungsgemeinschaft (DFG), grant PI 276/101. Additionally, the contribution of the Deutsche Forschungsgemeinschaft (DFG) to the applied UHPLC–MS/MS unit is gratefully acknowledged.

Acknowledgments We thank Rebecca Hoffmann and Jannis Beutel for the synthesis of the dipeptide, Anke Seitz for performing the NMR measurements, and Christine Meissner for proofreading the manuscript.

Abbreviation list GDP, glucose degradation product; AGE, advanced glycation end product; 3,4-DGE, 3,4dideoxyglucosone-3-ene; 3-DG, 3-deoxyglucosone; 3-DGal, 3-deoxygalactosone; DAD, diode array detection; MRM, multiple reaction monitoring; EPI, enhanced product ion; HR, high resolution; DEPT, distortion less enhancement by polarization transfer; COSY, correlation spectroscopy; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum coherence; ROE, rotating frame Overhauser enhancement; TOCSY, total correlation spectroscopy; HHPC, [6-hydroxy-2-(hydroxymethyl)-5-oxo-5,6dihydro-2H-pyran-3-yl]-cysteine ; H/D, hydrogen-deuterium.


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REFERENCES (1) Pischetsrieder, M., Gensberger-Reigl, S., Atzenbeck, L., and Weigel, I. (2016) Chemistry and clinical relevance of carbohydrate degradation in drugs. Drug Discov. Today 21, 1620-1631. (2) Hellwig, M., Gensberger-Reigl, S., Henle, T., and Pischetsrieder, M. (2018) Foodderived 1,2-dicarbonyl compounds and their role in diseases. Semin. Cancer Biol. 49, 1-8. (3) Linden, T., Forsback, G., Deppisch, R., Henle, T., and Wieslander, A. (1998) 3deoxyglucosone, a promoter of advanced glycation end products in fluids for peritoneal dialysis. Perit. Dial. Int. 18, 290-293. (4) Tauer, A., Knerr, T., Niwa, T., Schaub, T. P., Lage, C., Passlick-Deetjen, J., and Pischetsrieder, M. (2001) In vitro formation of n[epsilon]-(carboxymethyl)lysine and imidazolones under conditions similar to continuous ambulatory peritoneal dialysis. Biochem. Biophys. Res. Commun. 280, 1408-1414. (5) Augner, K., Eichler, J., Utz, W., and Pischetsrieder, M. (2014) Influence of nonenzymatic posttranslational modifications on constitution, oligomerization and receptor binding of s100a12. PloS one 9, e113418. (6) Rojas, A., and Morales, M. A. (2004) Advanced glycation and endothelial functions: A link towards vascular complications in diabetes. Life Sci. 76, 715-730. (7) Wieslander, A., and Linden, T. (1996) Glucose degradation and cytotoxicity in pd fluids. Perit. Dial. Int. 16 Suppl. 1, S114-118. (8) Wieslander, A. P., Nordin, M. K., Kjellstrand, P. T., and Boberg, U. C. (1991) Toxicity of peritoneal dialysis fluids on cultured fibroblasts, l-929. Kidney Int. 40, 77-79. (9) Frischmann, M., Bidmon, C., Angerer, J., and Pischetsrieder, M. (2005) Identification of DNA adducts of methylglyoxal. Chem. Res. Toxicol. 18, 1586-1592.



Page 22 of 34

(10) Wang, H., Cao, H., and Wang, Y. (2010) Quantification of n2-carboxymethyl-2'deoxyguanosine in calf thymus DNA and cultured human kidney epithelial cells by capillary high-performance liquid chromatography-tandem mass spectrometry coupled with stable isotope dilution method. Chem. Res. Toxicol. 23, 74-81. (11) Li, H., Nakamura, S., Miyazaki, S., Morita, T., Suzuki, M., Pischetsrieder, M., and Niwa, T. (2006) N2-carboxyethyl-2'-deoxyguanosine, a DNA glycation marker, in kidneys and aortas of diabetic and uremic patients. Kidney Int. 69, 388-392. (12) Tamae, D., Lim, P., Wuenschell, G. E., and Termini, J. (2011) Mutagenesis and repair induced by the DNA advanced glycation end product n2-1-(carboxyethyl)-2'-deoxyguanosine in human cells. Biochemistry 50, 2321-2329. (13) Marceau, E., and Yaylayan, V. A. (2009) Profiling of alpha-dicarbonyl content of commercial honeys from different botanical origins: Identification of 3,4-dideoxyglucoson-3ene (3,4-dge) and related compounds. J. Agric. Food Chem. 57, 10837-10844. (14) Gensberger, S., Glomb, M. A., and Pischetsrieder, M. (2013) Analysis of sugar degradation products with alpha-dicarbonyl structure in carbonated soft drinks by uhplc-dadms/ms. J. Agric. Food Chem. 61, 10238-10245. (15) Niwa, T. (1999) 3-deoxyglucosone: Metabolism, analysis, biological activity, and clinical implication. J. Chromatogr. B Biomed. Sci. Appl. 731, 23-36. (16) Thornalley, P. J. (1996) Pharmacology of methylglyoxal: Formation, modification of proteins and nucleic acids, and enzymatic detoxification--a role in pathogenesis and antiproliferative chemotherapy. Gen. Pharmacol. 27, 565-573. (17) Frischmann, M., Spitzer, J., Funfrocken, M., Mittelmaier, S., Deckert, M., Fichert, T., and Pischetsrieder, M. (2009) Development and validation of an hplc method to quantify 3,4dideoxyglucosone-3-ene in peritoneal dialysis fluids. Biomed. Chromatogr. 23, 843-851.


Page 23 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18) Mittelmaier, S., Funfrocken, M., Fenn, D., Berlich, R., and Pischetsrieder, M. (2011) Quantification of the six major alpha-dicarbonyl contaminants in peritoneal dialysis fluids by uhplc/dad/msms. Anal. Bioanal. Chem. 401, 1183-1193. (19) Distler, L., Georgieva, A., Kenkel, I., Huppert, J., and Pischetsrieder, M. (2014) Structure- and concentration-specific assessment of the physiological reactivity of alphadicarbonyl glucose degradation products in peritoneal dialysis fluids. Chem. Res. Toxicol. 27, 1421-1430. (20) Catalan, M. P., Santamaria, B., Reyero, A., Ortiz, A., Egido, J., and Ortiz, A. (2005) 3,4-di-deoxyglucosone-3-ene promotes leukocyte apoptosis. Kidney Int. 68, 1303-1311. (21) Lee, D.-H., Choi, S.-Y., Ryu, H.-M., Kim, C.-D., Park, S.-H., Chung, H.-Y., Kim, I.S., and Kim, Y.-L. (2009) 3,4-dideoxyglucosone-3-ene induces apoptosis in human peritoneal mesothelial cells. Perit. Dial. Int. 29, 44-51. (22) Erixon, M., Linden, T., Kjellstrand, P., Carlsson, O., Ernebrant, M., Forsback, G., Wieslander, A., and Jonsson, J. A. (2004) Pd fluids contain high concentrations of cytotoxic gdps directly after sterilization. Perit. Dial. Int. 24, 392-398. (23) Linden, T., Cohen, A., Deppisch, R., Kjellstrand, P., and Wieslander, A. (2002) 3,4dideoxyglucosone-3-ene (3,4-dge): A cytotoxic glucose degradation product in fluids for peritoneal dialysis. Kidney Int. 62, 697-703. (24) Yamamoto, T., Tomo, T., Okabe, E., Namoto, S., Suzuki, K., and Hirao, Y. (2009) Glutathione depletion as a mechanism of 3,4-dideoxyglucosone-3-ene-induced cytotoxicity in human peritoneal mesothelial cells: Role in biocompatibility of peritoneal dialysis fluids. Nephrol. Dial. Transplant. 24, 1436-1442. (25) Sanchez-Nino, M. D., Poveda, J., Sanz, A. B., Carrasco, S., Ruiz-Ortega, M., Selgas, R., Egido, J., and Ortiz, A. (2014) 3,4-dge is cytotoxic and decreases hsp27/hspb1 in podocytes. Arch. Toxicol. 88, 597-608.



Page 24 of 34

(26) Justo, P., Sanz, A. B., Egido, J., and Ortiz, A. (2005) 3,4-dideoxyglucosone-3-ene induces apoptosis in renal tubular epithelial cells. Diabetes 54, 2424-2429. (27) Bryland, A., Broman, M., Erixon, M., Klarin, B., Linden, T., Friberg, H., Wieslander, A., Kjellstrand, P., Ronco, C., Carlsson, O., and Godaly, G. (2010) Infusion fluids contain harmful glucose degradation products. Intensive Care Med. 36, 1213-1220. (28) Mittelmaier, S., and Pischetsrieder, M. (2011) Multistep ultrahigh performance liquid chromatography/tandem mass spectrometry analysis for untargeted quantification of glycating activity and identification of most relevant glycation products. Anal. Chem. 83, 9660-9668. (29) Madson, M. A., and Feather, M. S. (1981) An improved preparation of 3-deoxy-erythro-hexos-2-ulose via the bis(benzoylhydrazone) and some related constitutional studies. Carbohydr. Res. 94, 183-191. (30) Hellwig, M., Degen, J., and Henle, T. (2010) 3-deoxygalactosone, a "new" 1,2dicarbonyl compound in milk products. J. Agric. Food Chem. 58, 10752-10760. (31) Gensberger, S., Mittelmaier, S., Glomb, M. A., and Pischetsrieder, M. (2012) Identification and quantification of six major alpha-dicarbonyl process contaminants in highfructose corn syrup. Anal. Bioanal. Chem. 403, 2923-2931. (32) Erixon, M., Wieslander, A., Linden, T., Carlsson, O., Forsback, G., Svensson, E., Jonsson, J. A., and Kjellstrand, P. (2005) Take care in how you store your pd fluids: Actual temperature determines the balance between reactive and non-reactive gdps. Perit. Dial. Int. 25, 583-590. (33) Witowski, J., Korybalska, K., Wisniewska, J., Breborowicz, A., Gahl, G. M., Frei, U., Passlick-Deetjen, J., and Jorres, A. (2000) Effect of glucose degradation products on human peritoneal mesothelial cell function. J. Am. Soc. Nephrol. 11, 729-739.


Page 25 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(34) Hellwig, M., Nobis, A., Witte, S., and Henle, T. (2016) Occurrence of (z)-3,4dideoxyglucoson-3-ene in different types of bbeer and malt beer as a result of 3deoxyhexosone interconversion. J. Agric. Food Chem. 64, 2746-2753. (35) Mittelmaier, S., Funfrocken, M., Fenn, D., and Pischetsrieder, M. (2011) 3deoxygalactosone, a new glucose degradation product in peritoneal dialysis fluids: Identification, quantification by hplc/dad/msms and its pathway of formation. Anal. Bioanal. Chem. 399, 1689-1697. (36) Deng, Y., Wierenga, P. A., Schols, H. A., Sforza, S., and Gruppen, H. (2017) Effect of maillard induced glycation on protein hydrolysis by lysine/arginine and non-lysine/arginine specific proteases. Food Hydrocoll. 69, 210-219. (37) Sheng, B., Larsen, L. B., Le, T. T., and Zhao, D. (2018) Digestibility of bovine serum albumin and peptidomics of the digests: Effect of glycation derived from alpha-dicarbonyl compounds. Molecules 23, E712.



Page 26 of 34

Table 1 Detected modifications after incubation of the peptide Ac-GWGCGRGKG-NH2 with 3,4dideoxyglucosone-3-ene (3,4-DGE;10 µM) at 37 °C for different incubation times (n=3). Incubation time m/z

z

MWa [Da]

Δ MW [Da]

4h

12 h

1d

2d

3d

5d

7d

540.6

2

1079.2

162

xb

x

x

x

x

x

x

531.6

2

1061.2

144

x

x

x

x

x

x

x

530.6

2

1059.2

142

n.d.c

n.d.

n.d.

n.d.

x

x

x

522.7

2

1043.4

126

x

x

x

x

x

x

x

a

MW, molecular weight; b “x” indicates detectable modification; c n.d., not detected


Page 27 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2 1H and 13C NMR spectral data observed for the newly identified cysteine modification [6hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2H-pyran-3-yl]-cysteine (HHPC). The modification resulted in a mass shift of +142 Da. Measurements were performed before and after H/D exchange. Shift [ppm] Multiplicity Coupling constant [Hz] Assigned atom 3.04

dd

13.52; 4.72

cysteine alkyl –CH2

3.16

dd

13.51; 4.72

cysteine alkyl –CH2

3.31

t

10.29

H-7

3.57

t

11.43

H-7

4.02

d

5.94

H-6

4.37 a

s

-

H-2

5.39 a

s

-

OH-6

5.85 a

d

13.98

OH-7

6.24

d

5.82

H-4

63.5

C-7

81.5

C-6

83.1

C-2

122.3

C-4

175.4

C-3

197.7

C-5

a

signals were not detectable in H/D exchange experiments.



Page 28 of 34

Figure Legends

Figure 1. Loss of 3-deoxyglucosone (3-DG, ■, initial concentration 235 µM), 3deoxygalactosone (3-DGal, ▲, initial concentration 100 µM), and 3,4-dideoxyglucosone-3ene (3,4-DGE, ●, initial concentration 11 µM) during the incubation (37 °C, pH 7.2) with the nonapeptide Ac-GWGCGRGKG-NH2 (2 µM). The peak areas of the quinoxaline derivatives of the controls at time point 0 h were set as 100% (n=3, means ± SD).

Figure 2. Contents of unmodified peptide Ac-GWGCGRGKG-NH2 after incubation with 3deoxyglucosone (3-DG, 235 µM), 3-deoxygalactosone (3-DGal, 100 µM), or 3,4dideoxyglucosone-3-ene (3,4-DGE, 11 µM) at 37 °C, pH 7.2. The amounts measured in heated control samples were set as 100% (n=3, means ± SD). Asterisks indicate significant differences between different glucose degradation products (GDPs) at certain time points (unpaired Student’s t-test, p < 0.001 ***; n.s., not significant).

Figure 3. (A) Extracted ion chromatogram (m/z 452.2) of a reaction mixture of the Ac-FCNH2 dipeptide (25 µM) and 3,4-dideoxyglucosone-3-ene (3,4-DGE, 25 µM, after 11 days of incubation). This m/z represents a peptide modification with a mass shift of +142 Da and gives a double peak at retention time 5.8–5.9 min. (B) Both signals showed the same UV/Vis absorption with an additional maximum at 284 nm (black) in comparison to the unmodified dipeptide (grey). Enhanced product ion scans of the signals (m/z 452.2) at retention time (C) 5.80 min and (D) 5.90 min yielded the same product ion spectra indicating that the signals belong to isomeric structures.


Page 29 of 34

Figure 1

100%

remaining dicarbonyl

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80% 60% 40% 20% 0% 0

5

10

15

20

25

30

35

incubation time [h]



*** 120%

***

***

***

***

*** n.s.

***

***

***

*** n.s.

n.s.

100%

remaining peptide

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 34

***

***

***

***

***

80% 60% 40% 20% 0% 0

2

4

8

24

32

incubation time [h] 3-DG 235 µM

3-DGal 100 µM

3,4-DGE 11 µM

Figure 2


Page 31 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3



1

Page 32 of 34

SCHEME LEGENDS

2 3

Scheme 1. Structure of the novel cysteine-derived advanced glycation end product [6-

4

hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2H-pyran-3-yl]-cysteine (HHPC).

5 6

Scheme 2. Proposed reaction mechanism for the formation of the 3,4-dideoxyglucosone-3-

7

ene (3,4-DGE)-derived cysteine modification [6-hydroxy-2-(hydroxymethyl)-5-oxo-5,6-

8

dihydro-2H-pyran-3-yl]-cysteine (HHPC) via an intermediate sulfoxide.


Page 33 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O 5

H O

C

H

HC

C

NH H

4

3

S

O 2 7

H

H 6

H

OH

1

H OH

Scheme 1



C HC

H C

S

NH H

+

H

OH

OH

O

H

-H+/+H+

O

H OH

C

H

HC

C

H S

oxidation

O

H OH

NH H

C

H

O

H

HC

C

S

H

vinylogous keto-enol tautomerism

OH O

OH

NH H

C

H

O

HC

C

S

H OH

NH H

CH2OH

CH2OH

CH2OH

OH

O

O

O O

Page 34 of 34

CH2OH

sulfoxide

sulfoxide vinylogous keto-enol tautomerism

O H

H O

C

H

HC

C

NH H

O OH

O O

O

S

H H H

C

H

HC

C

NH H

H S OH CH2OH

OH

HHPC - hemiacetal

O OH -H2O

O

C

H

OH

HC

C

S OH

NH H

CH2OH

HHPC - open chain form

Scheme 2


H

cysteine (HHPC)

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