284
Chem. Res. Toxicol. 1996,8,284-291
Structural Definition of Early Lysine and Histidine Adduction Chemistry of 4-Hydroxynonenal Durgesh V. Nadkarni and Lawrence M. Sayre* Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106 Received August 9,1994@
The lipid peroxidation product trarzs-4-hydroxy-2-nonenal (HNE) has been implicated in the covalent modification of low-density lipoproteins (LDL) thought to contribute to the overaccumulation of LDL in the arterial wall in the initial stages of atherosclerosis. Proposals for the exact structures of “early” protein side-chain modifications until now have been based on indirect evidence. I n this paper, the structures of first-formed His- and Lys-based adducts were elucidated by correlating NMR spectral properties with those obtained on models with reduced chiral center content, in some cases following hydride reduction. In this manner, we could confirm unambiguously the structure of a HNE-His imidazole(“) Michael adduct, stabilized as a cyclic hemiacetal and isolated from a neutral aqueous 1:lstoichiometry reaction mixture. I n the case of Lydamine reactivity, where an excess of amine is needed to avert HNE aldol condensation, the predominance of a 1 : l Michael adduct in homogeneous aqueous solution and a 1:2 Michael-Schiff base adduct under two-phase aqueous-organic conditions could be verified by isolation of the respective borohydride-reduced forms. The 1:2 adduct, shown to exist as the cyclic hemiaminal, could represent a stable lysine-based cross-link in certain protein microenvironments.
Introduction Substantial evidence suggests that the overaccumulation of oxidatively modified low-density lipoprotein (oxLDLY by macrophages in the arterial wall contributes to the pathogenesis of atherosclerotic plaques (1-4). Oxidative modification of LDL involves the derivatization of its constituent apolipoprotein B (apo B) by breakdown products of lipid peroxidation, to a large degree by trans4-hydroxy-2-nonenal (HNE) (5,6). The chemistry of HNE-protein adduction is highly complex, being heterogeneous, in part subject to reversible equilibria, and associated with time-dependent and autoxidationdependent adduct “aging”. Higher concentration of HNE, which may, however, occur physiologically (7), induce the aggregation (and increased macrophage uptake) of LDL in a manner seen in highly oxidized LDL (8)and appears to involve covalent cross-linking of apo B (8,9).Overall, it is important to clarify the HNE covalent adduction chemistry that occurs as a function of time and concentration, including a n elucidation of the apparent predisposition of HNE toward covalent cross-linking, with the ultimate goal of establishing which modifications are of the greatest atherogenic significance. The formation of thiol-derived Michael adducts (1012),stabilized as cyclic hemiacetals (13,14), a t one time was considered to constitute the main biochemical reactivity of HNE. However, it has become increasingly clear that, for LDL, Lys c-amino groups are the major targets of HNE binding (6,7, 15),and neutralization of Lys residues through adduct formation would explain the observed increase in the overall negative charge of LDL particles accompanying HNE modification (3). Other Abstract published in Advance ACS Abstracts, February 1, 1995. Abbreviations: ox-LDL, oxidatively modified LDL; LDL, lowdensity lipoprotein; apo B, apolipoprotein B; HNE, 4-hydroxy-2nonenal; GAF’DH, glyceraldehyde-3-phosphate dehydrogenase; 2,4DNP, 2,4-dinitrophenylhydrazine;HBE, 4-hydroxy-2-butenal; DIBAL, diisobutylaluminum hydride; HRMS, high-resolution mass spectrometry. @
immunochemical studies suggest the additional involvement of Tyr, Arg, and His in HNE-modified apo B (16). Interestingly, borohydride reduction following HNE incubation enhances the antigenicity of HNE-LDL (3)and is thus usually employed in the generation of monoclonal antibodies. Enhanced immunogenicity implies either that a portion of HNE covalent binding represents a semireversible type of modification that is cemented by hydride reduction and/or that reduction of the HNE adducts liberates more antigenic functional groups. This finding points to the importance of elucidating what chemical changes accompany hydride reduction of HNE amino acid adducts. Indirect structural information on the nature of HNE modification of protein amino acid side chains has recently been described by Stadtman and co-workers (17-21 1. Using a combination of amino acid analysis, 2,4-dinitrophenylhydrazine(2,4-DNP) reactivity, and radiolabel incorporation using tritiated NaB&, these workers concluded that Michael addition of nucleophilic amino acid side chain groups to the C=C of HNE represents the dominant reaction pathway for Cys sulfhydryl, Lys c-amino, and His imidazole, in each case existing as cyclic hemiacetals (1-3).Also, observed intra- and inter-
R
1
FI
R
NH-c-
MI-CA .-,
MI-c-
2
3
molecular cross-linking in HNE-treated glyceraldehyde3-phosphate dehydrogenase (GAPDH)was postulated to arise from protein-based Lys Schiff base formation a t HNE C-1, subsequent to Michael addition of proteinbased Lys, Cys, or His nucleophiles a t HNE C-3 (19).
0893-228x/95/2708-0284$09.00/0 0 1995 American Chemical Society
Chem. Res. Toxicol., Vol. 8, No. 2, 1995 286
Early Adduction Chemistry of 4-Hydroxynonenal
Scheme 1 L
o
n G h 1
*
L
N
R
L
&NHR OH
nG.Hl1
n.C5H1 1
RNH2
n-CsH1l
1
N
H
R
1.
6
n-CsH11 &OH
[ . C 5 H 1 ~ - n - c s H f & ~ ~
NaBH4
-
n-Cs
11
NHR
7
R I AcNHCH(COOH)CH~CH~CH~CH~, PhCH2CH2
However, a suspicious lack of 2,4-DNP reactivity of some of the proposed Michael adduct hemiacetals (171, as well as a curious selectivity for Lys and Cys over His as the Michael added group in the putative C-1 Lys Schiff base cross-link (191, gives reason to pursue a more direct assessment of adduct structure. In a previous publication (22), we described model studies implicating the formation of a Lys-derived pyrrole 4 as one of the “late” HNE adducts consistent with the increase in negative charge of LDL observed upon modification with HNE (Scheme 1). Additional, in part unstable, adducts were also seen, some of which eventually “age” to the pyrrole in concentrated solution. Hydride treatment after initial HNE reaction allowed the isolation of reductively stabilized adducts, one of which we identified as the reduced diastereomeric mixture 7 of Michael adduct 5 (R = PhCHZCHz), the persistence of which may reflect its tendency to exist as cyclic hemiacetal 6. Such reductive stabilization is consistent with the observed increased antigenicity of HNE-LDL upon hydride reduction (3). At this time, we describe studies aimed a t the unambiguous assignment of the structure of the first-formed HNE adducts with His imidazole and Lys amino, on the basis of detailed NMR and mass spectral analysis using (i) simplified models for the protein nucleophiles and (ii) a simplified model of HNE, trans-4-hydroxy-2-butenal (HBE), which eliminates the otherwise chiral C-4 center. Subsequently, the more complex NMR spectral data for the actual HNE-amino acid adducts could be correlated by comparing chemical shift and peak integration data.
Experimental Section General Methods. ‘H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded on a Varian Gemini 300 instrument. In all cases, tetramethylsilane or the solvent peak served as an internal standard for reporting chemical shifts. NMR assignments were made on the basis of extensive ‘H NMR homonuclear decoupling experiments, 13C NMR AFT experiments (assignments listed as or -1, andor chemical shift comparisons to model compounds. lH NMR assignments t o major and minor diastereomer signals (when well separated) are indicated as major and minor. For NMR listings, chemical shifts of the minor diastereomeric signals are given in brackets when present. High-resolution mass spectra (HRMS) were obtained at 20 eV on a Kratos MS-25A instrument. Analytical and preparative thin-layer chromatography was performed using Merck silica gel 60 plates with 254 nm indicator. HNE was prepared according to a recent literature procedure (23,241.All other materials were reagent grade.
+
Preparation of trans-4-Hydroxy-2-butenal (€€BE). According to a modification of the published procedure (251, fumaraldehyde bis(dimethy1acetal) (8 g, 0.045 mol) was dissolved in a mixture of 80 mL of acetone and 4 mL of water. Strongly acidic Amberlyst-15 ion exchange resin (Aldrich) (0.8 g) was added to the solution. The reaction mixture was stirred a t 0 “C for 1 h. The reaction mixture was filtered and the solvent was evaporated. Methylene chloride was added to the oily residue and the solution was dried over NazS04. Filtration followed by evaporation of the filtrate yielded a yellowish oil (-5 g), which contained monoaldehyde and dialdehyde in a ratio of 86:14. This mixture (4.08 g, 0.031 mol) was dissolved in 60 mL of dry benzene. DIBAL (1.5 M solution in toluene, Aldrich) (0.031 mol, 20.92 mL) was added t o the preceding solution at -78 “C under argon. The reaction mixture was warmed to 0 “C and stirred a t that temperature for 5 h. Excess water was added to the reaction mixture at 0 “C to precipitate out the aluminum salts. The precipitated aluminum salts were filtered and washed with CHzClz, and the filtrate was evaporated to yield crude 4,4-dimethoxy-2-butenol. The latter material (-3.9 g) was dissolved in 60 mL of acetone and 4 mL of water, and Amberlyst-15 ion exchange resin (0.7 g) was added to it. The reaction mixture was stirred for 30 min a t room temperature. the solution was filtered, and the solvent was evaporated to furnish an oily residue that was dissolved in CHzClz and dried over NazS04. Filtration followed by evaporation of the solvent yielded crude HBE, which was purified by silica gel chromatography using 20% EtOAc in CHzClz as eluent t o yield 1.3 g of HBE (48.3%). This compound was previously prepared by a more lengthy route without spectral characterization (26).IH NMR (CDC13): 6 4.47 (br, 2H), 6.38 (dd, l H , J = 7.94 and 15.69 Hz), 6.92 (dt, l H , J = 3.69 and 15.69 Hz), 9.58 (d, l H , J=7.94Hz). l3CNMR(CDC13): 661.81,130.62,156.17, 193.58. Preparation of NU-Acetyl-Nr-(2-hydroxytetrahydrofuran-4-y1)histamine(10). To a solution of HBE (0.200 g, 2.325 mmol) in 4 mL of aqueous 1 M phosphate buffer (pH 7) was added Na-acetylhistamine (0.356 g, 2.325 mmol). The reaction mixture was stirred a t 37 “C for 16 h under argon. The solvent water was evaporated, and the residue was dried at high vacuum and then extracted with methanol. The combined washings were evaporated to yield an oily residue. NMR analysis of the crude product revealed the presence of 10 as a 2:l mixture of diastereomers (0.520 g, 93.5%). IH NMR (CD3CN): 6 1.82 ( 8 , 3H), 2.29 (m, 2H, H-31, 2.60 (t, J = 7.02 Hz, major) and 2.61 (t, J = 6.87 Hz, minor) (total 2H, Hp), 3.30 (m, 2H, Ha), 3.81 (dd, J = 2.63 and 9.60 Hz, major) and 3.90 (dd, J = 5.71 and 9.31 Hz, minor) (total l H , H-51, 4.20 (dd, J = 6.04 and 9.41 Hz, major) and 4.19 (dd, J = 5.62 and 9.83 Hz, minor) (total l H , H-51, 4.76 (m, minor) and 4.88 (m, major) (total lH, H-4), 5.53 (dd, J = 2.02 and 5.62 Hz, minor) and 5.67 (t, J = 4.02 Hz, major) (total l H , H-21, 6.81 (br, major) and 7.03 (br, minor) (total lH, H-5’1, 7.43 (d, J = 1.27 Hz, major) and 7.53
286 Chem. Res. Toxicol., Vol. 8, No. 2, 1995 (d, J = 1.27 Hz, minor) (total lH, H-2'). I3C NMR (DMSO-&): 6 22.60 (-), 28.35 (+I, 40.36 L40.271 (+I, 41.14 (+I, 55.90 L54.721 (-1, 70.84 [70.77] (+), 97.29 (-1, 113.84 [114.291(-), 135.58 (-1, 139.46 [139.33] (+I, 168.95 (+). HRMS: calcd for CllH1703N3 239.1271, found 239.1269. Preparation of Na-Acetyl-Nr-2-(1,4-dihydroxybutyl)hst a m i n e (11). The adduct 10 (0.05 g, 0.210 mmol) was treated with a 2 mL solution of NaBH4 (0.0103 g, 0.273 mmol) in 0.1 N NaOH. The reaction mixture was stirred a t 37 "C for 1h and then quenched with 1 M HC1 (5 mL) and neutralized (pH 7) with aqueous NaOH. The solvent was evaporated, and the residue was dried a t high vacuum. The residue was then triturated with a minimum amount of methanol, and the washings were collected and evaporated to yield a single product as an oil, which was found to be of satisfactory purity by NMR analysis (0.048 g, 96%). 'H NMR (CD30D): 6 1.91 (s, 3H, CH3), 1.93-2.02 (m, 2H, H-3), 2.70 (t, 2H, J = 7.48 Hz, Hp), 3.263.33 (m, lH), 3.40 (t, 2H, J = 7.48 Hz, Ha), 3.45-3.56 (m, lH), 3.76 (d, 2H, J = 5.13 Hz), 4.30 (m, lH), 6.98 (s, lH), 7.60 (9, 1H). '3C NMR (CD30D): 6 22.60, 29.04, 35.21, 40.48, 58.15, 58.79, 65.68, 115.74, 137.99, 140.19, 173.22. HRMS: calcd for CllH1903N3 241.1428, found 241.1409. Preparation of Na-Acetyl-~-(2-hydroxy-5pentyltetrahydrofuran-4-y1)histamine (13). To a solution of HNE (0.961 mmol, 0.150 g) in 4 mL of pH 7 phosphate buffer (1 M) and 1 mL of CH3CN was addedNa-acetylhistamine (0.961 mmol, 0.147 g). The reaction mixture was stirred under argon a t 37 "C for 24 h. The reaction mixture was cooled to room temperature and extracted with CHZC12. The CHzClz extracts were combined, dried (NazSO4),and evaporated to furnish a yellow oil. NMR analysis revealed the presence of pure 13 (0.256 g, 86%) and a mixture of diastereomers. 'H NMR (CDC13): 6 0.80 (overlapping triplets, 3H, H-11, 1.13-1.55 (m, 8H, CHz), 1.92 (5, 3H, CH3), 1.99-2.66 (m, 2H, H-31, 2.70 (t, 2H, J = 6.4 Hz, Hp), 3.45 (t, 2H, J = 6.4 Hz, Ha), 4.12-4.27 (m, l H , H-5),4.504.58 (m, l H , H-4), 5.64 (d, J = 5.19 Hz) and 5.80 (t, J = 4.54 Hz) (total l H , H-2), 6.63-7.55 (several singlets, 2H, H-2' and H-5'). NMR (CDC13): 6 13.98 (-1, 22.49 L22.421 (+I, 23.22 (-), 25.40 [25.60,26.06] (+I, 27.53 [28.581 (+I, 31.67 L33.731 (+I, 35.45 (+), 39.57 [39.45,39.35] (+I, 41.25 [41.42,42.551(+I, 61.22 [60.98, 59.871 (-1, 82.66 L83.97, 79.431 (-1, 96.96 L97.33, 96.551 (-), 114.96 [115.46, 113.941, (-1, 135.75 L135.871 (-), 140.54 [140.79, 140.041 (+), 170.47 [170.391 (+I. HRMS: calcd for C16H~7N303309.2054, found 309.2058. Preparation of Na-Acetyl-Nr-3-(1,4-dihydroxynonyl)hst a m i n e (14). In the same manner by which 11was prepared from 10, adduct 13 (0.076 g, 0.246 mmol) was treated with a 3 mL solution of NaBH4 (0.030 g, 0.738 mmol) in 0.1 N NaOH. Workup as before yielded a diastereomeric mixture of a material that was >95% pure as revealed by TLC and 'H NMR. 'H NMR (CD3CN): 6 0.86 (t, 3H, J = 6.38 Hz), 1.15-1.40 (m, 8H), 1.82 (s, 3H, CH&=O), 1.90-2.15 (m, 2H, H-2), 2.61 (t,2H, J = 6.92 Hz, Hp), 3.11-3.19 (m, lH), 3.34 (app q, 2H, Ha), 3.43 (m, W, 3.61-3.72 (m, lH), 4.06 (m, lH), 6.69 (br t, l H , NH), 6.82 (major) and 6.85 (minor) (29, total lH), 7.36 (s, 1H). NMR (CD&N, aliphatic region only): 6 14.29 (-1, 23.13 (-1, 23.26 (+I, 26.09 (+), 28.96 (+), 32.39 (+I, 33.76 L34.211 (+), 34.68 L35.861 (+I, 40.07 (+), 58.56 (+I, 60.16 [59.561 (-1, 74.21 L73.101 (-1. HRMS: calcd for C16H29N303 311.2211, found 311.2208. Preparation of Na-Acetyl-N7-(2-hydroxy-5-pentyltetrahydrofuran-4-y1)histidine(16). To a solution of HNE (0.088 g, 0.564 mmol) in 3.4 mL of pH 7.3 phosphate buffer (1 M) and 0.6 mL of CH3CN was added Na-acetylhistidine (0.121 g, 0.564 mmol). The reaction mixture was stirred under argon a t 37 "C for 16 h and then cooled to room temperature and extracted with CHZC12. The aqueous phase was separated and evaporated to yield a solid residue, which was extracted with MeOH. The combined MeOH washings were evaporated to furnish a white solid (mp 132-140 "C). 'H NMR (CDsOD, characteristic peaks): 6 0.85 (overlapping triplets, 3H), 1.21.3 (m, 8H), 1.90 (overlapping singlets, 3H), 5.56 and 5.75 (2m, total 1H).
Nadkarni and Sayre Preparation of the 2,4-Dinitrophenylhydrazoneof NuAcetyl-N7-3-(l-oxo-4-hydroxybuty1)histamine(17). To a solution of 2,4-DNP (0.420 mmol, 0.083 g) in 1N HC1 [prepared by stirring and heating the suspension of 2,4-DNP (0.083 g) in 60 mL of aqueous 1 N HC1 until all the solid dissolved1 was added a solution of adduct 10 (0.100 g, 0.420 mmol) in 5 mL of 1N HC1 a t room temperature. The yellowish reaction mixture was then neutralized (pH 7) with aqueous 10%NaOH (it should be noted that higher pH causes the formation of a dark brown solution, resulting in the partial decomposition of the product), and the solvent water was evaporated to dryness. The yellowish solid was dried a t high vacuum for 2 h and then thoroughly triturated with methanol. All methanol washings were collected and partially evaporated to reduce the volume of the methanol. The inorganic salts that precipitated out of the methanol a t this stage were removed by filtration. The methanolic filtrate was evaporated to afford the crude product, which was purified by column chromatography on silica gel using 1:l MeOH-EtOAc as eluent, yielding 0.105 g (94.6%)(mp 116-118 "C). 'H NMR (CD30D): 6 1.90 (s, 3H, CH3), 2.69 (t, 2H, J = 7.14 Hz, H-2), 2.95 (t, 2H, J = 6.04 Hz, Hp), 3.34 (br m, 2H, Ha), 3.86 (m, 2H, H-4), 4.62 (m, l H , H-3), 7.12 (s, l H , Im H-5'1, 7.68 (t, l H , J = 4.91 Hz, H-l), 7.73 (d, l H , J = 9.6 Hz), 7.78 (s, l H , Im H-2'1, 8.21 (dd, l H , J = 2.63 and 9.46 Hz), 8.86 (d, l H , J = 2.5 Hz). I3C NMR (CD30D): 6 22.70 (-1, 28.93 (+I, 35.57 (+I, 40.50 (+I, 58.76 (-), 65.40 (+I, 116.17 (-), 117.50 (-1, 123.95 (-1, 130.31 (+), 130.63 (-), 137.90 (-1, 138.86 (+I, 139.96 (+I, 146.12 (+I, 150.07 (-), 173.26 (-1. HRMS: calcd for C10H1005N4 (elimination of Nu-acetylhistamine) 266.0651, found 266.0654 (Mf was not observed). Preparation of 3-(Butylamino)-1,4-nonanediol(2Ob). A solution of HNE (0.075 g, 0.48 mmol) and butylamine (0.236 mL, 2.4 mmol) in a mixture of 3 mL of CH3CN and 13 mL of 1.2 M pH 7 potassium phosphate buffer was kept under argon for 18 h a t 37 "C. After this time an oily layer had separated, and the solution was made homogeneous by the addition of water (10 mL) and CH&N (6 mL). To this solution was then added NaBH4 (0.1 g , 2.64 mmol), and after i t was stirred for 1 h a t room temperature, the solution was acidified with 1 M aqueous HCl and extracted with CHzC12. Drying and evaporation of the organic layer, followed by silica gel chromatography (3:l CH2Clz-MeOH eluant), yielded 20b as an oil (0.40 g, 36%). 1H NMR (CDC13): 6 0.90 (2t, 3H each), 1.30-1.67 (m, 14 H), 2.48-2.76 (m, 3H), 3.72-3.81 (m, 3H). I3C NMR (CDC13): 6 13.99 (-), 14.05 (-1, 20.48 (+), 22.63 (+I, 26.10 (+I, 29.05 (+I, 31.91 (+), 32.64 (+I, 34.01 (+I, 32.64 (+I, 46.71 (+I, 62.37 (+I, 62.69 (-), 70.21 (-). HRMS: calcd for C13HzgOzN 231.2198, found 231.2194. Preparation of 2,4-Bis(butylamino)tetrahydrofuran (22a). To a solution of HBE (0.08 g, 0.93 mmol) in 10 mL of 1 M pH 7 phosphate buffer was added n-butylamine (0.34 g, 0.46 mL, 4.65 mmol). The reaction mixture was stirred under argon a t 37 "C for 24 h and then cooled to room temperature and extracted with CHzClz. The combined CHzClz extract was dried (Na2S04) and evaporated to give a crude yellowish product. NMR spectral analysis revealed the presence of 22a (0.190 g, 95%) as a diastereomeric mixture (57:43). The product was found to be unstable to silica gel chromatography. Also, even simple storage of the adduct a t -9 "C for 2-3 weeks resulted in partial decomposition to a polymeric material. 'H NMR (CDC13): 6 0.89 (t,6H, J = 7.141, 1.23-1.57 (m, 8H), 1.68-1.77 (m, l H , H-3), 2.20-2.29 (m, l H , H-31, 2.54 (m, 4H, NHCHz), 3.32 (9, l H , NH), 3.55-3.64 (m, l H , H-4), 3.84-3.99 (m, 2H, H-5),4.73(t,J=4.76Hz,major)and4.87(t, J = 7.5Hz,minor) (total l H , H-2). I3C NMR (CDC13): 6 13.98,20.49,32.62 132.671, 32.43, 39.43 [39.561, 45.55 [45.681, 48.19 [47.961, 57.96 L58.041, 71.86 [71.35], 91.20 [90.731. HRMS: calcd for ClzH26N20 214.2046, found 214.2052. Preparation of 2,4-Bis(butyl~o~-5-pentyltetrahydrofuran (22b). To a solution of HNE (0.134 g, 0.859 mmol) in 15 mL of 1.5 M pH 7 phosphate buffer and 5 mL of acetonitrile was added n-butylamine (0.69 mL, 6.87 mmol). The reaction mixture was stirred a t 37 "C for 24 h under argon. The reaction
Chem. Res. Toxicol., Vol. 8, No. 2, 1995 287
Early Adduction Chemistry of 4-Hydroxynonenal
Scheme 2
I
i) DIBAL ii) H+, HzO
H
O
1
H+,H20
T
H
0 4-hydmxybutenal@E)
0
4-hydmxynonenal (I-INE)
mol) under argon. The reaction mixture was warmed to 0 "C with stirring for 3 h. Methanol was added slowly to the reaction mixture a t 0 "C to precipitate out the aluminum salts, which were removed by filtration through Celite. The filtrate was evaporated, and the residue was taken up in brine and extracted with CHzC12. The CHZC12 extracts were combined, dried (NazSOd), and evaporated to furnish the crude product as an oil. A part of the crude product was purified by preparative TLC using 4:l CH&Iz-EtOAc as eluent to give an analytical sample of the desired lactol (diastereomeric mixture) as a colorless oil. IH NMR (CDC13) 6 1.17 (d, J = 6.17 Hz, major) and 1.29 (d, J = 6.10 Hz, minor) (total 3H), 1.62-2.17 (m, 4H), 4.06 (major) and 4.27 (minor) (2m, total lH, H-5), 5.33 (d, J = 3.30 Hz, minor) and 5.42 (dd, J = 2.38 and 4.82 Hz, major) (total l H , H-2). 13C NMR (CD30D): 6 21.37 [22.93](-), 32.33 L31.981 (+), 34.48 L34.271 (+I, 75.22 177.651 (-), 99.33 r99.391 (-1. The reaction of 3 drops of y-valerolactol with 1 mL of 2,4DNP reagent (prepared by dissolving 0.250 g of 2,4-DNP in 200 mL of 1N HCl) resulted in the immediate precipitation of the 2,4-DNP derivative (mp 122-124 "C). lH NMR (CD30D): 6 1.21 (d, 3H, J = 6.10 Hz), 1.73 (m, 2H), 2.48 (m, 2H), 3.82 (m, lH), 7.76 (t, l H , J = 5.16 Hz),7.92 (d, l H , J = 9.71 Hz), 8.29 (dd, l H , J = 2.63 and 9.71 Hz), 9.01 (d, l H , J = 2.63 Hz). 13C NMR (CD30D): 6 23.55, 30.22, 36.30, 67.89, 117.41, 124.09, 130.62, 130.87, 138.65, 146.44, 154.64. Reaction of NQ-Acetylhistamine-HNEAdduct 13 with Butylamine. To a solution of 13 (0.074 g, 0.24 mmol) in 2 mL of 1 M pH 7 phosphate buffer and 1 mL of CH3CN was added Preparation of NQ-Acetyl-N'-[l-(2-hydroxyethyl)-2-hy- butylamine (0.070 g, 0.96 mmol). The reaction mixture was stirred a t 37 "C for 16 h under argon and, after cooling, extracted droxyheptyll-L-lysineN-Methylamide (26). To a solution with CHzClz. NMR spectral analysis of the residue obtained of HNE (0.116 g, 0.746 mmol) in a mixture of 10 mL of 1M pH upon evaporation of the dried (Na~S04)organic layer indicated 7.8 phosphate buffer and 5 mL of CH3CN was added W-AC-Lthe presence of largely unreacted 13 accompanied by other Lys-NHCH3 (0.750 g, 3.73 mmol). The reaction mixture was products. However, only weak signals characteristic of a heated a t 37 "C under argon for 30 h and was then cooled and hemiaminal cross-link (e.g., IH a t 6 4.7) were observed. treated with 0.15 g (4 mmol) of NaBH4 with stirring. After 30 min a t room temperature, 20 mL of 1M HC1 was added to break Results and Discussion up the borate salts. The mixture was then made basic with 10% aqueous NaOH to pH 11 and extracted with CHzC12. The "Early"Histidine Adduction Chemistry. Synthesis combined organic extracts were dried (Na2S04) and evaporated. of HNE ( 2 3 , 2 4 )and its analog HBE used in this study The resulting residue was applied to a preparative silica gel TLC was accomplished from the biddimethyl acetal) of fuplate (2 mm), which was eluted with EtOAc-MeOH (2:3). The maric dialdehyde (25),as outlined in Scheme 2. Using band at Rf = 0.2 was extracted with MeOH, affording, after Na-acetylhistamine (8) as a n achiral model of N"-Ac-Lfiltration and evaporation, 0.026 g (10%) of 26 as a hygroscopic His, we observed that Michael addition to HBE occurred white solid. No other identifiable product containing HNE could cleanly using a 1:lstoichiometry in aqueous CH&N at be isolated from either the organic or water layers obtained from pH 7. The Michael adduct 9 exists completely in the ringworkup of the NaBH4 reduction. lH NMR (CDC13): 6 0.88 (t, 3H, J = 6.53 Hz), 1.29-1.78 (m, 16 HI, 2.00 (s, 3H), 2.50 (m, closed hemiacetal form 10,in both CD3CN and DMSOlH), 2.62 (m, lH), 2.76 and 2.78 (28, total 4H), 3.80 (m, 3H), d6 solvents, as a mixture of two diastereomers. The 4.36 (app p, l H , J = 7.08 Hz). 13C NMR (CDCl3): 6 14.06 (-), structure could be assigned unambiguously from the 22.62 L22.901 (+I, 23.06 L23.111 (-1, 26.14 (+I, 26.20 (-1, 28.28 combined lH and 13C NMR spectra and the appropriate L28.451 (+), 29.14 129.341 (+I, 31.21 (+I, 31.91 (+I, 34.01 (+I, selective decoupling information with verification by a 45.51 L45.801 (+I, 53.21 L53.271 (+I, 62.75 L62.821 (+), 63.13 HETCOR experiment (supplementary material). Also, [63.441(-), 69.80 [69.961(-), 170.93 L170.121 (+I, 172.66 (172.601 NaBH4 reduction of 9 gave a single diol isomer 11. (+I. HRMS: calcd for C18H37N304 359.2786, found 359.2779. Although alkylation of His imidazole has been observed Preparation of 4-HydroxypentanalHemiacetal (y-Valto occur a t both Nn and sterically more accessible N7 erolactol). To a solution of y-valerolactone (2.0 g, 0.02 mol) in positions, the NMR spectra indicated that Michael alkylbenzene (30 mL) at -78 "C was added DIBAL (13.3 mL, 0.02
mixture was cooled to room temperature and extracted with CHzCl2. The CH2Clz extracts were combined, dried (NazS04), and evaporated to yield a yellowish oil. NMR analysis revealed the presence of a diastereomeric mixture (4258) of 22b (0.234 g, 96%). lH NMR (CDC13): 6 0.78-0.84 (overlapping triplets, 9H, CH3), 1.22-1.51 (m, 16H, CHZ), 1.64-2.21 (m, 2H, H-31, 2.42-2.57 (m, 4H, NHCHz), 2.79-2.85 (m, l H , H-4), 3.47-3.49 (minor) and 3.58-3.64 (major) (2m, total l H , H-51, 4.69-4.74 (m, l H , H-2). 13C NMR (CDCl3): 6 13.89 (-1, 13.92 (-1, 13.97 (-), 20.43 (+), 22.56 (+), 25.86 (+I, 31.92 (+I, 32.35 (+I, 32.71 (+), 34.86 [35.861 (+I, 39.12 L39.371 (+I, 45.15 L45.531 (+I, 48.03 (+), 62.86 162.441(-), 82.82 [83.451(-), 90.31 (-1. HRMS: calcd for C17H36N20 284.2829, found 284.2826. Preparation of 1,3-Bis(butylamino)-4-nonanol (23b). The adduct 22b (0.05 g, 0.176 mmol) was treated with a solution of N&& (0,009 g, 0.234 mmol) in 2 mL of 0.1 N aqueous NaOH for 1 h at 37 "C. The reaction mixture was cooled to room temperature, quenched with 1M aqueous HCl(5 mL), adjusted to pH 10 with aqueous NaOH, and extracted with CHzClZ. The extracts were combined, dried (NazSOd), and evaporated to yield an oil. The crude product was found to be a single diastereomer of 295% purity (0.046 g, 92%) by NMR analysis. IH NMR (CDC13): 6 0.90 (overlapping triplets, 9H), 1.29-1.70 (m, 18H), 2.49-2.72 (m, 7H, CHzN and CHN), 3.55 (m, l H , H-4). 13C NMR (CDC13): 6 14.04 (-),20.47 (+I, 20.55 (+), 22.69 (+I, 26.17 (+), 28.65 (+), 32.01 (+), 32.12 (+I, 32.66 (+I, 34.15 (+I, 45.77 (+), 47.21 (+), 49.43 (+I, 60.94 (-1, 70.83 (-). HRMS: calcd for C17H3~N20286.3031, found 286.2970.
288 Chem. Res. Toxicol., Vol. 8, No. 2, 1995
Nadkarni and Sayre
R
A H
8
+
HBE
OH
11
OH
Na.Ac-L-His t
OH
HNE
ation was occurring exclusively a t one position. Our assignment of structures 9-11 as involving N‘ alkylation was on the basis that the imidazole vinyl singlets in these cases could, upon amplification in aprotic solvents, be seen to be doublets with a coupling of -1.30 Hz, arising from (transannular) 4J coupling between the two imidazole vinyls. By studying a very large series of substituted imidazoles, Matthews and Rapoport observed that the 4J coupling was always in the ranges 1.20-1.40 Hz for N‘ substitution (J2,5) and 0.9-1.1 Hz for N” substitution ( J z , (27). ~ ) Our observation of exclusive N‘ alkylation is not without precedent (28)and must reflect a combination of the high chemoselectivity and large steric requirements of this particular Michael addition reaction, as opposed to, for example, simple alkylation by primary alkyl halides. Relative to the case of HBE, reaction of HNE with Naacetylhistamine in a 1:l molar ratio gave a stoichiometric adduct according to mass spectrometry. The NMR spectra, although more complicated, did not exhibit the complexity expected of a mixture of imidazole positional isomers, and the borohydride-reduced material 14 appeared as a simple mixture of two diastereomers. On the basis of this information and a comparison of the chemical shift positions (IH and 13C NMR) and peak integrations (IH NMR) for focal ranges of the spectra for the HNE-8 adduct and 10,we assign the structure of the HNE-8 adduct as a mixture of four diastereomers of the hemiacetal 13 of the Michael adduct 12 (itself a mixture of two diastereomers). Similar regional chemical shift comparisons could be made in the 13CNMR spectra.
H
8
+ ...nnr
OH
12
13
14
With these model results in hand, we could then confirm that the same chemistry occurs between HNE and Na-Ac-L-His, except that cyclic hemiacetal form 16 of the Michael adduct 16 in this case is a mixture of four diastereomers, with complicating ‘H NMR diastereotopic splittings by the amino acid C, chiral center. Again, although the NMR spectra of 16 were too complex to analyze directly, we could assign structure 16 by comparing the lH NMR chemical shifts and peak integrations for focal ranges of the spectra for 16 and 13. Of particular note are the characteristic signals (CD3OD) of the hemiacetal C-H a t 6 -5.6 (major diastereomer) and 6 -5.8 (minor diastereomer). Our establishment of the structure of the L-His Michael adduct hemiacetal 16 confirms the recent proposal of such by Uchida and Stadtman made on the basis of indirect biochemical experiments, although they assumed a mixture of imidazole positional alkylation (17). These workers noted that the adduct should retain 2,4-DNP
OH
16
15
reactivity a t C-1, even though the aldehyde group is present in hemiacetal form with the C-4 hydroxyl. Their observation of a lack of 2,4-DNP reactivity thus was unsettling (17), but it could be a consequence of their particular reaction conditions. We thus felt it was important to establish that Michael adduct hemiacetals of the type 10113116 did retain 2,4-DNP reactivity. DIBAL reduction of the lactone of 4-hydroxypentanoic acid afforded the hemiacetal form of 4-hydroxypentanal, which immediately formed the 2,4-DNP derivative in 1 N HC1. On the basis of this finding, we exposed the Naacetylhistamine adduct 10 to excess 2,4-DNP reagent made up in 1N HC1 for 1h a t room temperature, followed by adjustment of the pH to 7 and MeOH extraction of the solid obtained upon evaporation of the solvent. The expected 2,4-dinitrophenylhydrazone 17 was thereby isolated as the only HNE-containing material. It remains unclear why Uchida and Stadtman were unable to observe 2,4-DNP reactivity of what they believed was adduct 16.
R
8
NH-CCH,
i) 1N HCI
with MeOH
17
“Early” Lysine Adduction Chemistry. In our previous studies on HNE-amine chemistry, we found that a large excess of amine over HNE was needed to ensure the formation of 1:ladducts (22). The use of equimolar quantities afforded mainly adducts containing two (or more) molecules of HNE per amine molecule. Although this suggested a t the time that HNE was inherently biased toward condensation (e.g., aldol) chemistry, our ability to obtain 1:l Michael adducts from equimolar HNE-histamine reactions earlier indicates that the amine is responsible for stimulating HNE condensations, presumably via Schiff base formation a t the HNE C-1 carbonyl. Amine catalysis of aldol condensation via Schiff base formation is well-known (29). Lysine-based HNE aldol adducts on LDL may well contribute to aggregatory andlor fluorescent properties, and thus elucidation of these structures would be a worthwhile pursuit. At the current time, in order to focus on adducts involving single HNE molecules in the absence of confounding condensation chemistry, we limited model reactions to those using excess n-butylamine as an achiral model for the side chain of lysine. Our plan was to take advantage of the volatility of n-butylamine to facilitate the isolation of the desired product. Reaction of HBE with n-butylamine in aqueous buffer, followed by extraction with CH2C12, led to the isolation of the 1:2 HNEamine Schiff base Michael adduct 21a, which existed in CDC13 solution in the form of the hemiaminal 22a (Scheme 3). The structure of 22a, representing a mixture
Chem. Res. Toxicol., Vol. 8, No.2, 1995 289
Early Adduction Chemistry of 4-Hydroxynonenal Scheme 3 f
a: R = H , H B E b: R = n-C$I1; HNE
I :
+
~
i
Amine ?
j + Amine
i t
1
"v
21
of two diastereomers, could be unambiguously determined by 'H and 13C NMR spectroscopy. Similar reaction of n-butylamine with HNE led to the same chemistry, although the NMR spectra of the diastereomeric mixture of 22b could be assigned only by comparison to the spectra for 22a through chemical shift and peak integration correlations. Of note is the characteristic lH NMR signal of the hemiaminal H-1 a t 6 -4.7, compared to 6 -5.6 for the Michael adduct hemiacetals. Interestingly, borohydride reduction of isolated 22b afforded hydroxy diamine 23b as a single diastereomer. This finding demonstrates a singular n-butylamine Michael addition stereochemistry in the adduct 21b. In contrast to the n-butylamine case, we could not utilize an organic solvent extraction and evaporation sequence to isolate the HNE adduct ofN"-Ac-L-Lys. Even upon resorting to the neutral nucleophile N"-Ac-L-LYsNHCH3, the attempted isolation of the HNE adduct resulted in an impure product mixture. However, the predominance of 1 : l HNE-amine Michael adduct 24 in solution, presumably stabilized in hemiacetal form 26, was demonstrated by in situ reduction with NaB&. The amino diol adduct 26 was obtained cleanly, with no evidence for the presence of the 1,3-diamino-4-hydroxy reduction product expected from any 1:2 adduct (e.g., 21/ 22) also present in solution. Although 26 was a mixture of two diastereomers, consistent with what we observed previously in the reaction of HNE with PhCHzCHZNHz (221,its structure could be confidently assigned through a n analysis of the NMR chemical shifts ('H and 13C)and peak integrations ('H) for the focal ranges of the spectra compared to those for Na-Ac-~-Lys-NHCH3 and the other reduced HNE Michael adducts. HNE+
H3N-Y
excess
= ? s;o :c/
-4'
NHAc
"\ /C0"CH3
24
?WAC
Our finding that NaBH4 reduction traps a 1 : l adduct for HNE in the case ofNa-Ac-L-Lys-NHCH3,whereas 1:2
.
adducts 22 were isolated in the case of n-butylamine, suggested that the latter result reflected an equilibration driven by the excess n-butylamine present during the CH& extraction stage. Although 22b was shown to be stable in both protic and aprotic organic solvents, its dissolution in aqueous pH 7 buffer and reextraction with CHZC12 resulted in the recovery of HNE, verifying the reversible nature of HNE-amine adduction chemistry. Reinvestigation of the HNE n-butylamine reaction revealed that, in the absence of a n organic cosolvent, the buffered (pH 7) aqueous reaction mixture is not totally homogeneous and contains a highly dispersed organic oil, which separates upon cessation of stirring. Addition of NaB& with stirring under these conditions affords mainly the 1,3-diamino-4-hydroxycompound 23b, identical to that obtained from the reduction of isolated 22b. However, if instead the reaction mixture is brought to homogeneity by dilution with CH3CN prior to NaBH4 reduction, one obtains exclusively the amino diol 20b, arising from Michael adduct 18b/19b. These results, combined with those reported earlier for N"-Ac-L-LYsNHCH3, demonstrate that the main HNE-amine adduct present in aqueous solution is the 1:l Michael adduct (e.g., 18/19), even in the presence of excess amine. On the other hand, the principal HNE-amine adduct present in organic solution, when the amine is in excess, is the 1:2 Schiff base Michael adduct (e.g., 21/22). In this regard, although Uchida and Stadtman previously suggested the possibility of a 1:2 HNE-amine cross-link of type 21, their own efforts to isolate N"-AC-L-LYS adducts of HNE resulted in HPLC isolation of a 1 : l adduct (as deduced by mass spectrometry) (19). All of these results suggest to us that although 1:2 HNE-lysine cross-links 27 would have only limited stability in bulk water, they could represent a legitimate physiological cross-link in a nonaqueous microenvironment. Cross-linking by HNE of LDL-based lysines is believed to contribute to HNEinduced LDL aggregatory behavior (30))and it is crucial to establish the cross-link structures. The Nature of Lys Schiff Base Michael Adduct Cross-Links. As postulated by Uchida and Stadtman (19), one would theoretically predict the occurrence not only of a Lys-Lys cross-link 27 but of two additional cross-links involving the Michael addition of Cys sulfhydryl (see 28) or His imidazole (see 291, rather than Lys amino at C-4 of a n HNE-Lys Schiff base. These workers presumed that Michael addition was always the preferred initial reaction, in which case cross-links would form from
Nadkarni and Sayre
290 Chem. Res. Toxicol., Vol. 8, No. 2, 1995 Schiff base condensation of the C-1 carbonyl of the respective HNE Michael adducts with lysine +amino groups. I
I
NL
NH-k mo
(.
Hisimidamle
\
a
w
- Lys E - amino 1
29
In this regard, we attempted to obtain the mixed crosslink corresponding to 29 by exposure of the Nu-acetylhistamine adduct 13 to excess n-butylamine. However, under both homogeneous (aqueous CH3CN, pH 7) and heterogeneous (aqueous buffer with CHzClz extraction workup) reaction conditions, the starting 13 was mostly recovered unchanged (NMR indicated the formation of only minor amounts of the possible cross-linked structures). Our inability to obtain the mixed 1:2 adduct is not a consequence of a nonequilibrating persistence of Michael adduct 12 in unreactive cyclic hemiacetal form 13,because NMR spectra of the adduct in protic media (e.g., CD30D) indicate its presence as a mixture of cyclic and open (acyclic) forms. Our result thus appears to indicate that the formation of cross-links 27-29 may first require C-1 HNE-lysine Schiff base formation and then C-3 Michael addition of side-chain nucleophiles, stepwise order opposite that discussed by Uchida and Stadtman. In the case of our HNE reactions with excess amine, although again the most obvious route to 1:2 adducts 211 22 is the reaction of 1:l adducts 18/19with amine, we have no evidence to support this pathway (broken arrow with ? in Scheme 3). I n fact, our inability to isolate the mixed cross-link above suggests that 1:lMichael and 1:2 adducts represent separate parallel pathways, the latter forming via Schiff base intermediates. We see no direct evidence for such Schiff base adducts themselves, indicating that they are efficiently trapped by Michael addition of the amine present in excess. The equilibration of 1:2 adducts 21/22 with 1:l adducts 18/19upon the dilution of buffered aqueous reaction medium with CH3CN may not reflect Schiff base hydrolysis of 21/22, occurring instead by the reversion of 21/22to starting HBE/HNE (via retro-Michael and then Schiff base hydrolysis) followed by re-formation of Michael adduct 181 19. The latter route is compatible with the finding that HNE is regenerated upon the exposure of isolated 22b to pH 7 buffer. If our preceding analysis of cross-link formation is correct, the preparation of mixed cross-links 28 and 29 under model reaction conditions will require the generation of a t least some of the requisite HNE C - l Lys Schiff bases in solution. The latter cannot be guaranteed by reacting 1:l mixtures of HNE and amine on account of the great tendency toward (aldol) condensation mentioned earlier, and the use of excess amine to avoid this
problem will result mainly in isolation of the diamine adduct (e.g., 20121). No such limitation would exist for cross-link formation with proteins, because single HNE molecules could form Schiff base adducts with isolated Lys
