Model Studies on the Modification of Proteins by Lipoxidation-Derived

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Chem. Res. Toxicol. 2003, 16, 232-241

Model Studies on the Modification of Proteins by Lipoxidation-Derived 2-Hydroxyaldehydes† Zhongfa Liu and Lawrence M. Sayre* Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106 Received October 17, 2002

2-Hydroxyaldehydes have been previously identified as products of lipid peroxidation, and although they represent the simplest reducing sugars, their potential for modification of proteins under physiological conditions has not been investigated. Here, 2-hydroxyaldehydes were found to condense with amines in two ways, implicating potential pathways for modification of lysine residues. A fluorescent 4,5-dialkyl-3-hydroxypyridinium with ex/em 327/390 nm and a nonfluorescent 4-alkylimidazolium cross-linking product were isolated and characterized by 1H NMR, 13C NMR, high-resolution mass spectrometry, and, in the former case, through independent synthesis. Both reactions appear to proceed through Amadori rearrangement of the initial Schiff base. On the basis of the UV absorbance of the 3-hydroxypyridinium, the latter was estimated to represent modification of 1.5% of the lysines of RNase incubated with 0.5 mM 2-hydroxyheptanal for 10 days at 25 °C. The 4-alkylimidazolium is proposed to contribute to the protein cross-linking observed by gel electrophoresis in the incubation of RNase with higher concentrations of 2-hydroxyheptanal.

Introduction A large variety of aldehydic products of lipid peroxidation have been shown in recent years to be capable of modifying nucleophilic protein side chains, most prominently the -amino group of lysines. Such modifications are believed to play key roles in aging and pathophysiological processes, such as atherogenesis (1). The early emphasis on the easily measurable malondialdehyde was soon augmented by an increasing interest in R,βunsaturated aldehydes (2, 3), most notably trans-4hydroxy-2-nonenal (HNE) (4-11). In addition, short chain and long chain aliphatic 2-hydroxyaldehydes were more recently identified by trapping methods in the oxidation of unsaturated fatty acids, low-density lipoprotein (LDL), and some tissues in vitro by Spiteller (1215). Short and long chain 2-hydroxyaldehydes were proposed to arise from two different sources: the former from decomposition of bis-dihydroperoxides of unsaturated fatty acids (14) and the latter from hydrolysis of the epoxides of the enol ether plasmalogens formed upon enzyme-mediated epoxidation at the expense of monohydroperoxides of unsaturated fatty acids such as linoleic acid (15). The most abundant 2-hydroxyaldehyde found in tissue is 2-hydroxyheptanal, which has been shown to arise from the ω-6 polyunsaturated arachidonic and linoleic acids (16). 2-Hydroxybutanal has also been found to arise from ω-3 polyunsaturated fatty acids (17). The reaction of 2-hydroxyheptanal with amines was reported to be † Presented in preliminary form: Liu, Z., Xu, G., and Sayre, L. M. (2001) Abstracts of Papers, 222nd National Meeting of the American Chemical Society, Chicago, IL, August 26-30, 2001, American Chemical Society, Washington, DC (ORGN 25). Sayre, L. M. (2001) Abstracts of Papers, 222nd National Meeting of the American Chemical Society, Chicago, IL, August 26-30, 2001, American Chemical Society, Washington, DC (TOXI 17). * To whom correspondence should be addressed. Tel: (216)368-3704. Fax: (216)368-3006. E-mail: [email protected].

limited to Schiff base formation under basic conditions, as supported by characterization of the secondary amine product generated upon quenching with NaBH4 (18). However, 2-hydroxyaldehydes are the simplest forms of reducing sugars, which are known to condense with lysine groups to give, following Amadori rearrangement, R-ketoamines (eq 1) (19, 20). We suspected that the latter chemistry would ensue under neutral conditions at relatively long incubation times that could be relevant in vivo.

The 2-hydroxyaldehyde function also occurs naturally in the maturation of the connective tissue protein collagen. Lysyl oxidase-mediated deamination of D-hydroxylysine side chains formed by the action of lysine hydroxylase results in “hydroxylysine aldehyde” moieties 1 that condense with other δ-hydroxylysine or lysine residues, ultimately forming the trifunctional pyridinium crosslink structures pyridinoline (5a) or deoxypyridinoline (5b), respectively (Scheme 1) (21, 22). These stable and fluorescent moieties (λmax 325 nm at neutral pH) are widely used urinary markers to assess turnover of connective tissue in diseases such as osteoporosis and bone cancer (23, 24). The biosynthesis of these pyridinium species is described to be initiated by condensation/ Amadori rearrangement of hydroxylysine aldehyde (1) with the -amino groups of δ-hydroxylysine or lysine. Studies on the total syntheses of pyridinoline or deoxypyridinoline (25, 26), which can be generated in high yield from bis-R-ketoamines 2 independently prepared from the corresponding R-bromoketones, support the biosynthetic route shown in Scheme 1, involving an initial

10.1021/tx020095d CCC: $25.00 © 2003 American Chemical Society Published on Web 01/18/2003

Lipoxidation-Derived 2-Hydroxyaldehydes

Chem. Res. Toxicol., Vol. 16, No. 2, 2003 233 Scheme 1

double condensation/Amadori process. In the synthetic studies, base-catalyzed cyclization of 2 typically results in pyridiniums 5 without detection of dihydropyridone intermediate 3, apparently because of the ease of autoxidation of the latter under the basic conditions. However, in a model reaction wherein 1-phenethylamine was alkylated by 2 equiv of 1-bromopentan-2-one, Allevi and co-workers showed that base treatment of the resulting bis-R-ketoamine in the absence of oxygen permitted the corresponding 3-like dihydropyridone to be isolated and completely characterized (25). The latter result indicated that conversion of 3 to 5 represented strictly an O2dependent autoxidation (presumably of the enol-enamine tautomer 4) rather than a redox disproportionation process. The present paper deals with an exploration of the reaction of simple 2-hydroxyaldehydes with amines as lysine surrogates. The finding in preliminary studies that the incubation of amines with 2-hydroxyaldehydes gave rise to a fluorescent product with ex/em 327/390 nm led to its characterization as a 4,5-dialkyl-3-hydroxypyridinium, apparently duplicating the chemistry of pyridinoline biosynthesis. In addition, as it was observed that treatment of proteins with 2-hydroxyheptanal led to protein cross-linking in addition to fluorophore generation, model studies were directed at determining a crosslink structure, leading to identification of a 4-alkylimidazolium.

Experimental Procedures General Methods. 1H NMR (300 or 200 MHz) and 13C NMR (75.1 or 50.1 MHz) spectra were recorded on Varian Gemini 300 or 200 instruments. In all cases, the solvent peak served as the internal standard for reporting chemical shifts, which are expressed as parts per million downfield from TMS (δ scale). In the 13C NMR line listings, attached proton test (APT) designations are given as (+) or (-) following the chemical shift. High-resolution mass spectra (HRMS) were obtained at 20 eV on a Kratos MS-25A instrument. TLC was performed on glass plates precoated with silica gel 60F254. Compounds on the developed plate were visualized by short wavelength UV light (λ ) 254 nm) by placing the plate in a chamber filled with iodine vapor or by spraying with ammonium phosphomolybdic acid solution or ninhydrin solution. Preparative column chromatography was performed using 32-63 µm silica gel. Electrophoresis studies utilized an SE 200 series minigel caster (Hoefer Scientific Instruments, San Francisco, CA) and a Fisher Biotech FB

570 power supply (Fisher Scientific, Pittsburgh, PA). The water that was used for all studies was purified with a Millipore system. UV-vis spectra were obtained using a Perkin-Elmer Lambda 20 spectrophotometer. The fluorescent spectra were obtained using a Varian Cary Eclipse fluorescence spectrometer. Sodium phosphate buffers in D2O were prepared from the corresponding pH-adjusted sodium phosphate buffer in H2O by complete removal of H2O and reconstitution of the residue to the same volume with D2O, repeated two more times. Diethyl acrolein acetal, 2-methoxyethylamine, 70% ethylamine solution, 6-aminocaproic acid, and NR-Cbz-Lys were purchased from Aldrich Chemical Co. Ribonuclease A (RNase A) was purchased from Sigma Chemical Company (St. Louis, MO). All other reagents and chemicals were AR or ACS grade. Unless stated otherwise, all operations were conducted at room temperature and all concentrations were conducted at reduced pressure. 2-Hydroxybutanal Diethyl Acetal (26). Acrolein diethyl acetal (11.44 g, 88 mmol), benzonitrile (9.2 mL), and 30% aqueous H2O2 (10.56 g) were added sequentially to a suspension of KHCO3 (1.5 g, 15 mmol) in methanol (50 mL). The mixture was stirred for 2 h and then heated to 40-45 °C for 4 h. Methanol was removed, and the residue was dissolved in water (50 mL). The mixture was filtered, and the filtrate was extracted with chloroform (3 × 50 mL). The organic layers were combined and evaporated, and hexane (100 mL) was added to the residue to precipitate benzamide. Benzamide was filtered off, and hexane was evaporated. The residue was subjected to silica gel chromatography eluted with ethyl acetate-hexane (1:4, v/v) to yield 2-(diethoxymethyl)oxirane (27) in 60% yield. A solution of the latter (1.46 g, 10 mmol) and cuprous bromide (40 mg) in THF (20 mL) was cooled with ice and charged with argon, to which a solution of methylmagnesium bromide in THF (4 mL, 2.8 M) was added in 30 min. The reaction mixture was then heated to 60-70 °C for 17 h and then cooled, and water (100 mL) was added. The water layer was separated and extracted with ether (3 × 50 mL). The organic layers were combined and evaporated, and the crude product was subjected to silica gel chromatography (eluant hexanes-ethyl acetate 1:1, v/v) to afford 2-hydroxybutanal diethyl acetal in a yield of 92.6%. 2-Hydroxyheptanal Diethyl Acetal. A solution of 2-(diethoxymethyl)oxirane (1.46 g, 10 mmol) and cuprous bromide (40 mg) in THF (20 mL) was cooled with ice and charged with argon, to which a solution of n-butylmagnesium chloride in THF (5.6 mL, 2.0 M) was added dropwise in 30 min. The reaction mixture was then heated to 60-70 °C for 17 h and worked up as above to yield the crude product, which was subjected to chromatography (eluant hexanes-ethyl acetate 3:1, v/v) to afford 2-hydroxyheptanal diethyl acetal in a yield of 85.3%. 1H NMR (CDCl3): δ 0.89 (t, 3H, J ) 5.86 Hz), 1.22 (t, 3H, J ) 7.0 Hz), 1.25 (t, 3H, J ) 8.1 Hz), 1.30-1.42 (6H), 1.52-1.56 (2H),

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2.16 (br, 1H), 3.54-3.62 (3H), 3.69-3.79 (2H), 4.26 (dd, 1H, J ) 6.00, 0.80 Hz). 13C NMR (CDCl3): δ 14.12 (-), 15.48 (-), 15.54 (-), 22.69 (+), 25.29 (+), 31.80 (+), 31.99 (+), 63.46 (+), 63.55 (+), 71.84 (-), 105.25 (-). EI HRMS m/z calcd for C9H19O2 (MOEt)+, 159.1385; found, 159.1380. General Procedure for Isolation of 3-Hydroxypyridinium from Long-Term Incubation of 2-Hydroxybutanal with Simple Amines. Dowex 50W-X8 H+ (400 mg) was added to a solution of 2-hydroxybutanal diethyl acetal (1.20 g, 7.4 mmol) in phosphate buffer (200 mL, 1.5 M, pH 7.4). The mixture was stirred at room temperature for 6 h, and then, 30 mmol of either 2-methoxyethylamine or ethylamine was added, and the mixture was stirred at room temperature for 2 weeks. The reaction mixture was extracted with ethyl acetate, and the aqueous layer was evaporated. The residue was subjected to silica gel chromatography eluted with methanol to give the corresponding 3-oxidopyridinium zwitterionic “inner salts”. 1,5-Diethyl-4-methyl-3-oxidopyridinium (6). 1H NMR (methanol-d4): δ 1.23 (t, 3H, J ) 7.36 Hz), 1.56 (t, 3H, J ) 7.32 Hz), 2.27 (s, 3H), 2.70 (q, 2H, J ) 7.52 Hz), 4.30 (q, 2H, J ) 7.34 Hz), 7.53 (s, 1H), 7.62 (s, 1H). 13C NMR (methanol-d4): δ 12.48 (-), 14.52 (-), 17.09 (-), 25.24 (+), 56.72 (+), 127.23 (-), 129.76 (-), 143.00 (+), 145.23 (+), 167.54 (+). FAB HRMS m/z calcd for C10H16NO (M + H)+, 166.1232; found, 166.1232. 5-Ethyl-1-(2-methoxyethyl)-4-methyl-3-oxidopyridinium (20). 1H NMR (D2O): δ 1.13 (t, 3H, J ) 7.44 Hz), 2.19 (s, 3H), 2.62 (q, 2H, J ) 7.44 Hz), 3.31 (s, 3H), 3.87 (t, 2H, J ) 4.26 Hz), 4.39 (t, 2H, J ) 4.26 Hz), 7.49 (s, 1H), 7.55 (s, 1H). 13C NMR (D O): δ 11.90 (-), 13.14 (-), 23.81 (+), 58.40 (-), 2 59.47 (+), 70.69 (+), 128.87 (-), 129.43 (-), 142.40 (+), 145.18 (+), 162.78 (+). EI HRMS m/z calcd for C11H17NO2 M+, 195.1259; found, 195.1259. N,N-Bis(2-hydroxybutyl)benzylamine (9) (29). Benzylamine (2.72 mL, 25.0 mmol) was added to 1,2-epoxybutane (3.6 g, 50 mmol) under nitrogen, and the reaction mixture was heated to 100 °C in a sealed tube for 24 h. The mixture was cooled and purified by silica gel column chromatography (eluant hexanes-ethyl acetate 3:2, v/v) to afford 9 in 85% yield. N,N-Bis(2-hydroxybutyl)amine (10). To a solution of 9 (259 mg, 1 mmol) in 4.4% methanolic formic acid solution (26 mL) was added 10% Pd/C (26 mg) (30). The mixture was stirred at room temperature for 5 h and filtered through a pad of Celite powder, and the pad was washed with methanol. The solvent was evaporated, and the residue was used for the next step directly. 1H NMR (CDCl3): δ 0.97 (t, 6H, J ) 7.49 Hz), 1.471.57 (4H), 2.89-3.16 (4H), 3.92(m, 2H), 8.48 (br, 3H). tert-Butyl N,N-Bis(2-hydroxybutyl)carbamate (11). The crude amine 10 (159 mg, 1 mmol) was dissolved in THF (2 mL) and di-tert-butyl dicarbonate (545 mg, 2.5 mmol) was added at room temperature under nitrogen. The mixture was stirred at room temperature for 4 h, the solvent was evaporated, and the crude product was dissolved in ethyl acetate (5 mL) and water (2 mL). The aqueous layer was separated and extracted with ethyl acetate (2 × 3 mL). The combined organic layer was washed with brine (2 mL), dried (anhydrous Na2SO4), and concentrated. The crude product was subjected to silica gel column chromatography (eluant hexanes-ethyl acetate 3:2, v/v) to afford 240 mg of 11 in 90% yield as a mixture of RR/SS and RS/SR diastereomers. Fast moving fraction (11a): 1H NMR (CDCl3): δ 0.97 (t, 6H, J ) 7.56 Hz), 1.37-1.51 (4H), 1.46 (s, 9H), 2.83 (br, 2H), 3.38 (br, 2H, exchangeable), 3.62 (app dd, 2H, J ) 2.1, 14.4 Hz), 3.86 (br, 2H). 13C NMR (CDCl3): δ 9.82 (-), 28.03 (+), 28.49 (-), 57.14 (+), 72.9 (br, -), 80.27 (+), 164.5 (-). Slow moving fraction (11b): 1H NMR (CDCl3): δ 0.97 (t, 6H, J ) 7.46 Hz), 1.37-1.51 (m, 4H), 1.46 (s, 9H), 2.92 (s, 2H, exchangeable), 3.28 (br, 2H), 3.79 (br m, 2H). 13C NMR (CDCl3): δ 9.99 (-), 27.97 (+), 28.47 (-), 55.36 (br, +), 72.13 (-), 80.57 (+), 167.91 (-). EI HRMS m/z calcd for C13H28NO4 M+, 262.2018; found, 262.2032. tert-Butyl N,N-Bis(2-oxobutyl)carbamate (12). Dimethyl sulfoxide (0.35 mL, 5 mmol) in methylene chloride (2.5 mL) was added slowly via syringe to a cooled solution of oxalyl chloride

Liu and Sayre (318 mg, 2.5 mmol) in methylene chloride (4 mL) at -78 °C under nitrogen (31). After the mixture was stirred for 30 min, a solution of 11 (262 mg, 1 mmol) in methylene chloride (3 mL) was added via a syringe at -78 °C. The mixture was stirred for 3.5 h and allowed to warm to -60 °C slowly. Triethylamine (1.2 mL, 10 mmol) was added at -60 °C, and the mixture was stirred for 15 min. The reaction was quenched with water (2 mL), and the aqueous layer was separated and extracted with methylene chloride (2 × 3 mL). The combined organic layer was washed with brine (2 mL), dried (anhydrous Na2SO4), and evaporated, and the residue was subjected to silica gel chromatography (eluant hexanes-ethyl acetate 7:3, v/v) to afford 12 (233 mg) in 83.3% yield. 1H NMR (CDCl3): δ 1.04 (t, 3H, J ) 7.32 Hz), 1.06 (t, 3H, J ) 7.38 Hz), 1.39 (s, 9H), 2.34-2.48 (4H), 3.98 (s, 2H), 4.10 (s, 2H). 13C NMR (CDCl3): δ 7.40 (+), 7.57 (+), 28.19 (+), 32.85 (-), 33.05 (-), 56.63 (-), 57.10 (-), 80.94 (-), 155.29 (-), 206.92 (-), 207.22 (-). EI HRMS m/z calcd for C13H23NO4 M+, 257.16270; found, 257.16341. 5-Ethyl-4-methyl-3-hydroxypyridine (13). According to a published method (26), a suspension of potassium t-butoxide (96 mg, 0.8 mmol) in THF (1.5 mL) was cooled in an ice bath under nitrogen, and a solution of 12 (51.4 mg, 0.2 mmol) in THF (0.6 mL) was added. After 30 min, the bath was removed, and the reaction mixture was stirred at room temperature for 24 h and then recooled (ice bath) and diluted with methanol (3 mL). The solvent was evaporated, and the residue was dissolved in ethyl acetate (5 mL) and water (2 mL). The aqueous layer was separated and extracted with ethyl acetate (2 × 3 mL). The combined organic layer was washed with brine (2 mL), dried (anhydrous Na2SO4), and evaporated, and the residue was subjected to silica gel column chromatography (eluant methanolethyl acetate 5:95, v/v) to afford 13 in 73.0% yield. 1H NMR (CDCl3): δ 1.22 (t, 3H, J ) 7.7 Hz), 2.27 (s, 3H), 2.66 (q, 2H, J ) 7.7 Hz), 7.90 (s, 1H), 8.13 (s, 1H). 13C NMR (CDCl3): δ 11.08 (-), 14.51 (-), 23.93 (-), 132.89 (-), 134.07 (+), 138.18 (+), 138.57 (-), 152.84 (+). EI HRMS m/z calcd for C8H11NO M+, 137.0841; found, 137.0847. Preparation of 1,5-Diethyl-4-methyl-3-oxidopyridinium (6) from 13. Compound 13 (68.5 mg, 0.5 mmol) was added to a solution of ethyl iodide (41.5 mg, 0.25 mmol) dissolved in anhydrous 1,4-dioxane (2.5 mL) under nitrogen. The mixture was heated to reflux for 10 h, the solvent was evaporated, and the crude product was subjected to silica gel column chromatography eluted with methanol to afford 6, in 85.6% yield, as the inner salt as verified by the lack of reaction with AgNO3 expected if the product was the 3-hydroxypyridinium iodide. General Procedure for Isolation of 3-Hydroxypyridiniums from Long-Term Incubation of 2-Hydroxybutanal with Nr-Cbz-Lys and 6-Aminocaproic Acid. Dowex 50WX8 H+ (400 mg) was added to a solution of 2-hydroxybutanal diethyl acetal (1200 mg) in phosphate buffer (200 mL, 0.1 M, pH 7.4). The mixture was stirred at room temperature for 6 h and then decanted into a solution of either 6-aminocaproic acid or N-Cbz-Lys (4 equiv) in 1.5 M pH 7.4 sodium phosphate buffer (600 mL), and the mixture was stirred at room temperature for 2 weeks. The reaction mixture was extracted with ethyl acetate, and the aqueous layer was evaporated. The residue was subjected to silica gel chromatography eluted with methanol to give the corresponding 3-hydroxypyridinium carboxylates in 5-10% yield. 1-(5-Benzyloxycarbonylamino-5-carboxypentyl)-5-ethyl4-methyl-3-hydroxypyridinium (14a). 1H NMR (methanold4): δ 1.20 (t, 3H, J ) 7.65 Hz), 1.39 (m, 2H), 1.71 (m, 1H), 1.83-1.92 (3H), 2.24 (s, 3H), 2.66 (q, 2H, J ) 7.62 Hz), 4.01 (t, 1H, J ) 3.78 Hz), 4.18 (t, 2H, J ) 6.45 Hz), 5.05 (s, 2H), 7.287.33 (5H), 7.45 (s, 1H), 7.53 (s, 1H). 13C NMR (methanol-d4): δ 13.35 (-), 15.41 (-), 24.32 (+), 26.07 (+), 32.90 (+), 34.36 (+), 58.12 (-), 62.13 (+), 68.26 (+), 128.223 (-), 129.70 (-), 129.83 (-), 130.34 (-), 130.93 (+), 139.27 (+), 143.58 (+), 146.07 (+), 159.01 (+), 168.61 (+), 170.06 (+). FAB HRMS m/z calcd for C22H29N2O5 (M + H)+, 401.2076; found, 401.2059.

Lipoxidation-Derived 2-Hydroxyaldehydes 1-(5-Carboxypentyl)-5-ethyl-4-methyl-3-hydroxypyridinium (14b). 1H NMR (D2O): δ 1.10 (t, 3H, J ) 7.65 Hz), 1.24 (m, 2H), 1.50 (m, 2H), 1.85 (m, 2H), 2.11 (t, 2H, J ) 7.29 Hz), 2.17 (s, 3H), 2.61 (q, 2H, J ) 7.65 Hz), 4.23 (t, 2H, J ) 7.18 Hz), 7.67 (s, 1H), 7.73 (s, 1H). 13C NMR (D2O): δ 11.50 (-), 13.27 (-), 23.77 (+) 25.18 (+), 25.30 (+), 30.35 (+), 37.12 (+), 60.43 (+), 128.47 (-), 130.22 (-), 142.66 (+), 144.22 (+), 161.01 (+), 183.07 (+). FAB HRMS m/z calcd for C14H22NO3 (M + H)+, 252.1600; found, 252.1602. 1H NMR Spectroscopic Monitoring of the Reaction of 2-Hydroxybutanal with Excess 2-Methoxyethylamine. Dowex 50W-X8 H+ (10 mg) was added to a solution of 2-hydroxybutanal diethyl acetal (32.4 mg, 0.2 mmol) in phosphate buffer in D2O (0.5 mL, 0.1 M, pH 7.4). 1H NMR spectroscopy monitoring indicated that the reaction was complete in 6 h. Then, 2-methoxyethylamine (0.8 mmol) was added, and the 1H NMR spectrum of the mixture was taken after 2 h. 1H NMR Spectroscopic Monitoring of the Reaction of 2-Hydroxybutanal with 2-Methoxyethylamine under Buffer Conditions. Dowex 50W-X8 H+ (2.7 mg) was added to a solution of 2-hydroxybutanal diethyl acetal (0.05 mmol, 8.1 mg) in phosphate buffer in D2O (100 µL), and the mixture was kept at room temperature for 6 h. The resulting solution (100 µL) was decanted into a solution of 2-methoxyethylamine (0.2 mmol) in 1.5 M pH 7.4 sodium phosphate in D2O (400 µL) in an NMR tube. The progress of the reaction was monitored by 1H NMR spectroscopy at 0, 2, 5, 10, 24, and 48 h. Reductive Quenching (NaBH4) of the Reaction of 2-Hydroxybutanal with 2-Methoxyethylamine. Dowex 50W-X8 H+ (40 mg) was added to a solution of 2-hydroxybutanal diethyl acetal (120 mg) in phosphate buffer (20 mL, 0.1 M, pH 7.4). The mixture was stirred at room temperature for 6 h and then decanted into a solution of 2-methoxyethylamine (4 equiv) in 1.5 M pH 7.4 phosphate buffer (60 mL), and the mixture was stirred at room temperature for 10 h. Then, NaBH4 (40 mg) was added to the mixture, and the reaction mixture was stirred at room temperature for 24 h and adjusted to pH 12 with 1 N NaOH. The solution was extracted with ethyl acetate (6 × 50 mL), and the combined organic layer was evaporated to give a residue that was subjected to silica gel chromatography (eluant hexanes-ethyl acetate 1:3, v/v) to give 21 and 22. N-(2-Methoxyethyl)-2-hydroxybutylamine (21). 1H NMR (CDCl3): δ 0.93 (t, 3H, J ) 7.53 Hz), 1.42 (m, 2H), 2.43 (dd, 1H, J ) 12.03, 9.33 Hz), 2.69 (dd, 1H, J ) 12.03, 3.03 Hz), 2.77 (m, 2H), 3.33 (s, 3H), 3.44-3.52 (3H). 13C NMR (CDCl3): 10.01 (-), 27.94 (+), 49.00 (+), 54.93 (+), 58.81 (-), 70.85 (-), 71.92 (+). EI HRMS m/z calcd for C7H17NO2 M+, 147.1259; found, 147.1255. N-(2-Methoxyethyl)-N-(2-hydroxybutyl)-2-hydroxybutylamine (22). 1H NMR (CDCl3): δ 0.93 (t, 6H, J ) 7.30 Hz), 1.38 and 1.39 (2q, 2H each), 2.40 (dd, 2H, J ) 13.32, 9.84 Hz), 2.62 (dd, 2H, J ) 13.32, 3.30 Hz), 2.79 (t, 2H, J ) 5.12 Hz), 3.35 (s, 3H), 3.41 (t, 2H, J ) 5.30 Hz), 3.32-3.52 (4H). 13C NMR (CDCl3): δ 10.03 (-), 27.47 (+), 56.40 (+), 58.87 (-), 63.48 (+), 71.30 (-), 71.42 (+). EI HRMS m/z calcd for C11H25NO3 M+, 219.1834; found, 219.1815. Reaction of 2-Hydroxyheptanal with 6-Aminocaproic Acid. Dowex 50W-X8 H+ (330 mg) was added to a solution of 2-hydroxyheptanal diethyl acetal (5 mmol, 1.002 g) in 95% aqueous acetonitrile (30 mL), and the mixture was sonicated for 3.5 h. The solution was diluted with acetonitrile (50 mL) and decanted to a solution of 6-aminocaproic acid (10 mmol, 1.31 g) in 0.1 M pH 7.4 phosphate buffer (300 mL). The mixture was stirred at room temperature for 2 weeks. Acetonitrile was evaporated, and the aqueous solution was extracted with ethyl acetate (3 × 50 mL). The aqueous layer was evaporated under vacuum, and the residue was dissolved in methanol (100 mL). The mixture was filtered, the filtrate was evaporated, and the residue was subjected to silica gel chromatography (eluant methanol-ethyl acetate 2:1, v/v) to give 15 and 23 as their carboxylate inner salts. 4-Butyl-1-(6-carboxylpentyl)-5-pentyl-3-hydroxypyridinium (15). Yield: 39 mg, 4.7%. 1H NMR (methanol-d4): δ

Chem. Res. Toxicol., Vol. 16, No. 2, 2003 235 0.87-0.98 (6H), 1.28-1.67 (14H), 1.91 (m, 2H), 2.16 (t, 2H, J ) 7.47 Hz), 2.63-2.73 (4H), 4.21 (t, 2H, J ) 7.35 Hz), 7.46 (s, 1H), 7.56 (s, 1H). 13C NMR (methanol-d4, two aliphatic carbon signals overlapping): δ 14.38 (-), 23.53 (+), 24.35 (+), 26.93 (+), 27.27 (+), 27.40 (+), 31.46 (+), 31.71 (+), 32.22 (+), 32.84 (+), 38.66 (+), 61.34 (+), 128.20 (-), 130.53 (-), 141.12 (+), 149.67 (+), 167.42 (+), 182.30 (+). FAB HRMS m/z calcd for C20H34NO3 (M + H)+, 336.2539; found, 336.2529. 1,3-Bis-(6-carboxypentyl)-4-pentylimidazolium (23). Yield: 90 mg, 10.6%. 1H NMR (methanol-d4): δ 0.94 (t, 3H, J ) 7.08 Hz), 1.32-1.42 (8H), 1.44-1.67 (6H), 1.70-1.91 (4H), 2.17 (t, 4H, J ) 7.20 Hz), 2.67 (t, 2H, J ) 7.35 Hz), 4.13 and 4.15 (2t, 2H each, J ) 7.5 Hz), 7.42 (s, 1H), 8.93 (s, 1H). 13C NMR (methanol-d4): δ 14.33 (-), 23.42 (+), 24.37 (+), 26.67 (+), 26.75 (+), 27.10 (+), 27.20 (+), 28.18 (+), 30.53 (+), 30.77 (+), 32.41 (+), 120.13 (-), 137.47 (+), 175.23 (+). 13C NMR (D2O, one aliphatic carbon signal overlapping): δ 13.23 (-), 21.63 (+), 22.71 (+), 24.84 (+), 24.95 (+), 25.16 (+), 25.30 (+), 26.31 (+), 28.76 (+), 28.89 (+), 30.35 (+), 36.65 (+), 46.42 (+), 49.16 (+), 118.60 (+), 136.16 (-), 182.58 (2C, +). FAB HRMS m/z calcd for C20H35N2O4 M+, 367.2597; found, 367.2589. Incubation of RNase with 2-Hydroxyheptanal. To 2.50 mL aliquots of RNase A (7.5 mM based on Lys) in 0.1 M pH 7.4 phosphate buffer were added 2.50 mL aliquots of freshly prepared 2-hydroxybutanal (15, 7.5, 3.75, 1.88, 0.94, 0.47, 0.24, and 0 mM) in a 3:1 mixture of 0.1 M pH 7.4 phosphate buffer and acetonitrile, and the UV-vis spectrum of each incubation was taken from 200 to 600 nm at 1, 2, 3, 4, 8, 10, and 14 days. Aliquots of the reaction mixtures (20 µL) at 2 and 4 days were mixed with 20 µL of denaturing buffer for electrophoresis (0.3 M pH 6.8 Tris, 5% SDS, 5% 2-mercaptoethanol, 50% glycerol, and 0.02% bromophenol blue, pH 6.8) and heated in boiling water for 5 min. Then, 3 µL samples were analyzed by SDSPAGE using 5% stacking and 14% resolving gels. Protein bands were stained by Gelcode Blue stain reagent (Pierce, Rockford, IL). Trinitrobenzenesulfonic Acid (TNBS) Assay for Protein Lys Modification (32). Solutions of TNBS (30 mM) and 0.1 M Na2B2O7‚10H2O in 0.1 M NaOH were prepared in doubly distilled water. The protein solution (100 µL) was added to 2.90 mL of the borate buffer, and then, 75 µL of the TNBS solution was added. The mixture was allowed to stand at room temperature for 30 min. The absorbance was measured at 420 nm. The values were used to estimate the percent of reactive lysyl groups modified relative to the native protein.

Results and Discussion Synthesis of 2-Hydroxyaldehydes. 2-Hydroxybutanal and 2-hydroxyheptanal were prepared from acrolein diethyl acetal according to a modification of literature methods (26, 27). In brief, acrolein diethyl acetal was converted to its epoxide with hydrogen peroxide under basic conditions in the presence of benzonitrile. Opening of the epoxide with methylmagnesium bromide or nbutylmagnesium chloride in the presence of a catalytic amount of cuprous bromide yielded the corresponding 2-hydroxyalkanal diethyl acetals. The free 2-hydroxyalkanals were generated in situ by hydrolysis of the diethylacetals with Dowex 50W-X8 H+ in pH 7.4 phosphate buffer (for 2-hydroxybutanal) or in 95% aqueous acetonitrile (for 2-hydroxyheptanal). Fluorescent Advanced End Product, 3-Oxidopyridinium, from the Incubation of 2-Hydroxyalkanals with Primary Amines. Exposure of 2-hydroxybutanal to primary amines in aqueous buffer over the course of several days led to production of a fluorescent product with a λmax near 325 nm. Using the UV absorbance as an indicator of fluorophore generation, it was found that the use of higher concentrations and an excess

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Figure 2. Observed NOEs in compound 6.

Figure 1. Fluorescence spectrum of compound A in H2O (647 mM).

of amine over aldehyde resulted in optimal apparent yields of the fluorophore. To simplify the structural characterization of what was presumed to be a single major chromophoric product, the incubation of 2-hydroxybutanal (40 mM) with 2-methoxyethylamine (160 mM) was carried out in pH 7.4 phosphate buffer for 2 weeks. The choice of amine was based on the advantage of the methoxy singlet in the 1H NMR spectrum in assessing the stoichiometry of the reaction by integration. Workup of the reaction yielded a fluorescent species A with ex/ em 327/390 nm (Figure 1) as the main isolable product (silica gel column chromatography and preparative TLC) that was formed in a 50% yield based on starting aldehyde, as revealed by integration of the crude 1H NMR spectrum. The 1H NMR spectrum (D2O) of the purified product showed that it had one methoxy group from 2-methoxyethylamine, an ethyl, and a methyl group implicating two molecules of 2-hydroxybutanal and two aryl singlets at 7.6 and 7.7 ppm. The 13C NMR APT spectrum showed that five of the total of 11 carbons are in an aromatic ring; two bonded to hydrogen resonate at relatively high field and the other three resonate at relatively low field. Electron ionization (EI) HRMS indicated a M+ peak at m/z 195.1259, consistent with one molecule of 2-methoxyethylamine and two molecules of 2-hydroxybutanal with the molecular formula C11H17NO2. Because the sum formula for two 2-hydroxybutanals and one 2-methoxyethylamine would predict C11H25NO5, the stoichiometry requires three dehydrations and one two electron oxidation. A similar product was isolated from the incubation of 2-hydroxybutanal with ethylamine.

Taking the ethylamine-derived product as an example, two possible hydroxypyridine-like structures 6 and 7 were considered presuming starting material-related connectivity. Structure 6 is shown as the 3-oxidopyridinium inner salt (betaine) (33, 34) that would be the expected neutral form eluting from a chromatography column. The structure was assigned as 6 rather than 7 based on the two-dimensional nuclear Overhauser effect (NOE) spectrum. All NOEs observed for the aryl singlets were as indicated in Figure 2: the distinguishing findings were that (i) no aryl singlet showed an NOE to the C4 methyl singlet and (ii) both aryl singlets exhibited NOEs

to the methylene signal at 4.3 ppm. The betaine structure was also supported by comparing the aryl hydrogen and aryl carbon NMR chemical shifts in DMSO-d6 and D2O with those reported for 1-methyl-3-oxidopyridinium (33, 34). The core 3-oxidopyridinium structure was unambiguously confirmed by independent synthesis of 6, conducted according to a strategy used for the total synthesis of pyridinoline and model compounds (26), as shown in Scheme 2. Thus, 1,2-epoxybutane (8) was reacted with Scheme 2

benzylamine to afford N,N-bis(2-hydroxybutyl)benzylamine (9) (29). Hydrogenolytic N-debenzylation (30) to give 10 and treatment with di-tert-butyl dicarbonate afforded two diastereomers (meso and d.l) of the Boc derivative 11, which were individually characterized but recombined and converted to azadiketone 12 by Swern oxidation (31). Treatment of azadiketone 12 with potassium tert-butoxide gave 5-ethyl-4-methyl-3-hydroxypyridine (13), which was alkylated with ethyl iodide to afford target molecule 6, found to be identical to that isolated from the incubation of 2-hydroxybutanal and ethylamine. To confirm that the 3-hydroxypyridinium would also form from the reaction of the -amino group of Lys with 2-hydroxybutanal, the incubation of NR-Cbz-Lys (50 mM) with 2-hydroxybutanal (12.5 mM) was carried out in 0.1 M pH 7.4 phosphate buffer for 2 weeks. The 1H NMR spectrum of the reaction product in D2O after several column chromatographic and preparative TLC separations exhibited two characteristic peaks at about 7.4 and 7.5 ppm. Also, the 13C NMR APT spectrum supports the 3-hydroxypyridinium core structure. FAB HRMS showed the exact mass is 401.2059, which is consistent with the quasi-ion mass of 1-(5-benzyloxycarbonylamino-5-carboxypentyl)-5-ethyl-4-methyl-3-hydroxypyridinium (14a). This model study suggests that the 3-hydroxypyridinium motif could represent a stable oxidative stress protein modification generated by the action of 2-hydroxyaldehydes on lysyl residues. Thus, to prepare 3-hydroxypyridinium haptens, from the most predominant and next predominant lipoxidation-derived 2-hydroxyaldehydes, that could be conveniently conjugated to a carrier protein for production of antibodies, the reaction was repeated using 2-hydroxyheptanal or 2-hydroxybutanal with 6-aminocaproic acid as a tether, leading to isolation of 14b and

Lipoxidation-Derived 2-Hydroxyaldehydes

15. In the cases of 14 and 15, the presence of the carboxyl moiety led to the neutral compounds being isolated as 3-hydroxypyrinidinium carboxylates rather than as the 3-oxido species.

Mechanism of the Reaction of 2-Hydroxyalkanals with Amines. To investigate the mechanism of pyridinium formation, which contrasts the simple Schiff base formation observed by Spiteller (18), the reaction of 2-hydroxybutanal with 2-methoxyethylamine was monitored by 1H NMR spectroscopy. First, generation of 2-hydroxybutanal from the diethyl acetal precursor was monitored in buffered D2O by observing the release of ethanol. The aldehydic group did not appear in the 1H NMR spectrum, indicating that 2-hydroxybutanal exists as its hydrate form in this medium. With addition of 2-methoxyethylamine (4 equiv) to the solution, which exceeded the buffer and made the reaction basic, the 1H NMR spectrum after 2 h showed a doublet at 7.65 ppm and no resonance from 1.8 to 2.6 ppm, suggesting the formation of the Schiff base 16 (CH3CH2CH(OH)CHd N-) (eq 2). In contrast, when the pH 7.4 buffer concentration was increased to exceed that of the added amine, the 1H NMR spectrum showed slow disappearance of the

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starting aldehyde diastereotopic C3 multiplets at 1.39 and 1.62 ppm (CH3CH2CH(OH)CH(OH)2) (Figure 3A) and growth of a quartet at 2.4 ppm (Figure 3B), a transformation that was nearly complete after 10 h (Figure 3C). The quartet at 2.4 ppm is consistent with R-aminoketone 17 (CH3CH2C(dO)CH2N) formed via the Amadori reaction (eq 2). These results suggested that whereas the simple Schiff base is stable under basic conditions, tautomerization (Amadori rearrangement) proceeds under pH 7.4 buffer conditions. This explains the difference between our results and those obtained by Spiteller (18).

Further spectral monitoring over the period of 10-24 h revealed the appearance of a singlet at 1.7 ppm and a quartet at 2.2 ppm, the intensities of which were seen to grow with a corresponding decrease in intensity of the presumed R-aminoketone peak at 2.4 ppm (Figure 3D). The two newly formed peaks, which are distinct from the singlet at 2.0 ppm and quartet at 2.6 ppm seen in the final pyridinium product 20 (previously isolated compound A), are consistent with structure 19 proposed as

Figure 3. Progress of the reaction of 2-hydroxybutanal (SM) (0.1 mmol) with 2-methoxyethylamine (0.4 mmol) as monitored by 1H NMR in D2O (pH 7.4 sodium phosphate buffer): (A) 5 min progress, (B) 2 h progress, (C) 10 h progress, and (D) 24 h progress.

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the 3-oxidopyridinium precursor (25). Although the possible intermediacy of the azadiketone 18 could not be discerned distinctly from 17 by 1H NMR, an identical reaction mixture to that monitored by NMR was quenched with NaBH4 after 10 h to permit isolation of the more stable alcohols potentially reflective of the coexistence of 17 and 18. After work up, two major products were obtained and characterized as the N-(2-hydroxybutyl) 21 and N,N-bis(2-hydroxybutyl) 22 derivatives of 2-methoxyethylamine (eq 3). Once isolated, compounds 21 and 22 could be determined to form in a ratio of 4:3 from the crude 1H NMR spectrum. The finding of 21 and 22 is consistent with the sequence of reaction 17 f 18 f 19 f 20 (eq 2). Figure 4. Fluorescence spectrum of RNase A (10 mg/mL) treated with 7.5 mM 2-hydroxyheptanal in 0.1 M pH 7.4 phosphate buffer for 10 days.

On the basis of all of the above information, we propose that generation of 3-oxidopyridiniums in the reaction of primary amines with 2-hydroxyaldehydes follows the same general pathway described in Scheme 1 for the biosynthesis of pyridinoline. The initial stages of reaction follow well-precedented Amadori chemistry of reducing sugars, and following the known cyclization to dihydropyridone (25), its enol-enamine tautomer is readily autoxidized to the pyridinium. On the basis of incorporation of two molecules of aldehyde per molecule of amine, it is not readily apparent why the yield of pyridinium was found to be optimal using excess amine over aldehyde. One possibility is that use of excess amine completely and efficiently ties up the aldehyde in the form of the Schiff base-Amadori equilibrium mixture 16/17 and that the protonated Schiff base 16H+ is a more reactive aldehyde donor in reacting with 17 (to give 18) than is the free aldehyde. Detection of a Fluorescent Adduct in the Incubation of RNase with 2-Hydroxyaldehydes. Whereas a fluorescent product (ex/em 327/390 nm) was observed in the model reaction of 2-hydroxyaldehydes with primary amines, fluorescence (ex/em 325/409 nm) was also seen when the 13.7 kDa protein RNase A was incubated with 7.5 mM 2-hydroxyheptanal as shown in Figure 4. The similarity of the absorbance-emission spectrum is consistent with the 3-hydroxypyridinium adduct identified above in the model studies. We found no evidence for an alternative amine-derived fluorescent product in the above model reactions, and other studies (results not shown) failed to provide evidence for a fluorophore (or any other stable adduct) when other potentially reactive protein side chain groups (4-alkylimidazole for His and N-alkylguanidine for Arg) were incubated with 2-hydroxyheptanal. Assuming a protein-bound 3-hydroxypyridinium that has a similar extinction coefficient at 325 nm as does the model pyridinium compound 15 at 327 nm, the level of

3-hydroxypyridinium in the sample of RNase (10 mg/mL, 7.5 mM of Lys) treated with 7.5 mM 2-hydroxyheptanal for 10 days could be estimated to involve 3.8% of the lysines in RNase A. Because the TNBS assay (32) on the same sample indicated that 75% of the lysines of the protein was modified, it is clear that only a small fraction of the lysines modified by 2-hydroxyheptanal were converted to the 3-hydroxypyridinium. This is not surprising in view of the fact that formation of the pyridinium requires the initial 1:1 R-aminoketone Amadori product to react with a second molecule of 2-hydroxyheptanal before it is converted (e.g., oxidized) to another species. Although it constitutes a minor fraction of Lys modification, the pyridinium adduct represents a convenient and stable marker for protein modification by 2-hydroxyheptanal. As far as the nature of the adducts responsible for the main loss of TNBS reactivity of lysines, it is probable that the initial R-aminoketone mono-Amadori product (eq 4, P-NH2 ) protein Lys -amine) is the major contributor, although this would have to be confirmed by further studies, e.g., by analysis of protein hydrolysate following reductive stabilization.

Figure 5 shows that the 3-hydroxypyridinium began to form after only 2 days of incubation and reached a plateau on the eighth day. Incubations of various concentrations of 2-hydroxyheptanal with RNase A for 10 days demonstrated that the formation of the fluorophore was dependent on the concentration of 2-hydroxyheptanal (Figure 6). Even a concentration of 2-hydroxyheptanal as low as 0.5 mM resulted in pyridinylation of 1.5% of RNase lysines. At even lower concentrations of aldehyde likely to be present under physiological conditions, the 3-hydroxypyridinium adduct would be expected to form only after a longer period of time. Isolation and Characterization of a Potential 2-Hydroxyaldehyde-Induced Protein Cross-Linking Moiety. In the incubation of RNase with 2-hydroxyheptanal, in addition to fluorophore generation, gel electrophoretic analysis revealed that 2-hydroxyheptanal also induces protein cross-linking (Figure 7), although the pyridinium adduct would not explain this. Notwith-

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gested that the compound had an imidazolium structure, where it is known that C2 undergoes equilibrium deprotonation to give a ylide (35, 36). Furthermore, because of the H(D) exchange at C2, the carbon resonance was not observed in the 13C NMR spectrum. Nonetheless, the FAB HRMS indicated the exact mass was 367.2589, consistent with the molecular formula C20H35N2O4 (367.2597) for the 4-pentylimidazolium compound 23.

Figure 5. Time course of formation of 3-hydroxypyridinium in the incubation of RNase (10 mg/mL) with 2-hydroxyheptanal (7.5 mM) in a 3:1 (v/v) mixture of 0.1 M pH 7.4 phosphate buffer and acetonitrile.

The generation of 23 from 2-hydroxyheptanal requires one intact aldehyde and a second aldehyde molecule that serves as the donor of the lone C2 carbon. A possible mechanism starting from the previously described enolenamine intermediate is shown in Scheme 3. The enolScheme 3

Figure 6. Concentration dependence of 3-hydroxypyridinium formation in 2-hydroxyheptanal-treated RNase for 10 days.

Figure 7. Modification of RNase with 2-hydroxyheptanal. Solutions of RNase A (10 mg/mL, control RNase A in lane 1) containing 0.117, 0.235, 0.469, 0.938, 1.875, 3.75, 7.5, 15, and 30 mM 2-hydroxyheptanal (lanes 2-10), respectively, were incubated at room temperature for 4 days.

standing, in addition to the pyridinium products isolated in the incubations with 2-hydroxybutanal, the crude 1H NMR spectra indicated the presence of a minor nonfluorescent product. Under the conditions of the incubation of 6-aminocaproic acid with 2-hydroxyheptanal (mixed phosphate buffer and CH3CN), this alternate product was actually the major product accompanying the generation of pyridinium 15. The 1H NMR spectrum of the purified compound in CD3OD exhibited two singlets at 7.42 and 8.93 ppm in a ratio of 1:1. The hydrogen resonating at 8.93 underwent exchange over time. This result sug-

enamine species 24 can suffer autoxidation in the reaction mixture to produce the 2-oxoaldehyde Schiff base 25, which can react with another molecule of amine to form the same R-diimine intermediate 26 that appears to be involved in the formation of imidazolium structures in the incubation of 2-oxoaldehydes with amines (36). The species 26 may then react with another molecule of 2-hydroxyaldehyde to give an adduct 27 that decomposes with loss of chain-shortened aldehyde to the species 28 that readily dehydrates to 23. According to this mechanism, the second molecule of 2-hydroxyheptanal that donates its C1 would end up as hexanal, a well-known aldehyde generated in lipid peroxidation. Although generation of the imidazolium in our reactions here requires 2 mol of Lys side chain and 2 mol of 2-hydroxyaldehyde, the same 2:2 stoichiometry is required to generate the corresponding imidazoliums from glyoxal and methylglyoxal, termed GOLD and MOLD, respectively, which have been observed in physiological samples (37). Thus, imidazolium formation from 2-hydroxyaldehydes under physiological situations should not be deterred on the basis of a seemingly unlikely stoichiometry.

Conclusion 2-Hydroxyaldehydes are important products of lipid oxidation, but their potential for modification of nucleo-

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philic side chains in proteins under aerobic physiomimetic conditions has not been explored. 2-Hydroxyaldehydes were found to be capable of condensing with amines in two ways, implicating potential pathways for modification of Lys residues. A fluorescent 4,5-dialkyl3-hydroxypyridinium with ex/em 327/390 nm and a nonfluorescent 4-alkylimidazolium cross-linking product were isolated and characterized by NMR and HRMS and, in the former case, through independent synthesis. Mechanisms have been proposed for formation of these adducts, both of which depend on a two electron autoxidation step. The 4-alkylimidazolium is proposed to contribute to the protein cross-linking observed by gel electrophoresis in the incubation of RNase with 2-hydroxyheptanal. In this same incubation, the extent of generation of the fluorescent 3-hydroxypyridinium was estimated based on the UV absorbance. The results of this study suggest that the 3-hydroxypyridinium is a convenient marker for the modification of proteins by 2-hydroxyaldehydes. It is of interest to consider the possible biological significance of this pyridinium relative to the several pyridines that form enzymatically and nonenzymatically in the connective tissue proteins collagen and elastin (21-26, 38, 39). The preparative conditions chosen here to conveniently access end products involved nonphysiological, high concentrations of aldehyde; nonetheless, the identified structures would be expected to form physiologically in small amounts over time. Because 2-hydroxyheptanal is the predominant 2-hydroxyalkanal found in lipid oxidation, a 6-aminocaproic acid-based hapten for the 4-butyl5-pentyl-3-hydroxypyridinium was prepared for the purposes of conjugating to keyhole limpet hemocyanin, to permit the production of antibodies recognizing this epitope. Studies on the use of these antibodies to confirm the generation of the 3-hydroxypyridinium in proteins exposed to oxidized lipid preparations and in in vitro oxidized LDL will be reported separately.

Acknowledgment. We thank the National Institutes of Health for support of this work through Grant HL 53315. Supporting Information Available: 1H and 13C NMR spectra of new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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