Chem. Res. Toricol. 1993, 6,19-22
19
Communications Pyrrole Formation from 4-Hydroxynonenal and Primary Amines Lawrence M. Sayre,* Pramod K. Arora, Rajkumar S. Iyer, and Robert G. Salomon Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106 Received September IO, 1992
The reaction of trans-4-hydroxy-2-nonenal (4-HNE) with primary amines was investigated to elucidate chemistry that may clarify the nature of its physiological covalent binding with protein-based primary amino groups. Such binding of 4-HNE, generated endogenously from lipid peroxidation, appears to be a pathophysiologic factor in the modification of low-density lipoprotein and perhaps other instances. We now show that 4-HNE reacts with primary amines in aqueous acetonitrile at p H 7.8 t o afford after workup, in 14-23% yield, the corresponding pyrroles, which were characterized by independent synthesis from 4-oxononanal. Additional, mostly unstable adducts are also formed, some of which eventually "age" to the pyrrole. Hydride reduction after initial adduct formation permits the isolation of more stable materials, one of which has been identified as the reduced amine Michael addition product. Pyrrole formation may constitute a physiologically important reaction of 4-hydroxyalkenals.
Introduction There is substantial current interest in clarifying the cytotoxic consequences of physiologic covalent binding by trans-4-hydroxy-2-nonenal(4-HNE)l and trans-4hydroxy-2-hexenal, generated endogenously from peroxidation of n-6 (1) and n-3 (2)fatty acids, respectively. trans-4-Hydroxy-2-hexenal has also been identified as a metabolite of cytotoxic macrocyclic pyrrolizidine alkaloids (3-5). 4-Hydroxy-2-alkenals readily undergo Michael addition of biological thiols (1,6-8), giving stable adducts which exist predominantly as cyclic hemiacetals (eq 1)(9, 10). Although thiol adduction is generally considered to R
Lo +
R+Y=-
R'SH
SR'
R = CHsCHz, CH3(CH2)4
organelles, which could be modeled by reacting 4-HNE with amino-containing but not trimethylammoniumcontaining phosphatides (15). In an effort to clarify the chemical consequence of 4-hydroxyenal modification of amino groups, a pyridinium adduct was obtained from a model reaction of glycinewith 4-hydroxy-2-pentenal at alkaline pH (161,but this particular structure contains two molecules of aldehyde and is thus of unlikely physiologic significance. On the basis of our own studies on y-diketones (17,181and y-ketoaldehydes (191,which undergo the Pad-Knorr condensation with primary amines to give pyrroles, we were intrigued by the isomeric relationship between y-dicarbonyls and 4-hydroxyenals. In fact, y-hydroxyenones are known to isomerize to y-diketones with acid (20).In this communication we describe the reaction of 4-HNE with primary amines and report the formation of pyrroles (along with other adducts) under physiomimetic pH 7.8 conditions.
Rq OH
SR'
,,t
I' I
constitute the chemically preferred transformation (7,8), the reaction of 4-HNE with protein-based lysine e-amino groups has been documented in several instances (8).In particular, covalent modification of low-densitylipoprotein (LDL) by 4-HNE (11, 12) may be of pathophysiologic importance and may involve cross-linking (13). Multisite binding of 4-HNE to LDL, which increases the overall negative charge of the protein (141,is associated with the generation of epitopes which cross-react with antigenic loci created in the reaction of 4-HNE with polylysines (12).However, the enhancement of antigenicity resulting from "reductive alkylation" conditions (12) makes it difficult to interpret the chemical nature of the epitope and is suggestiveof a fair degree of heterogeneity. 4-HNE was also found to generate a fluorophore with membranous 'Abbreviations: 4-HNE, trans-4-hydroxy-2-nonenal; LDL, low-density lipoprotein; APT, attached proton test; HRMS,high-resolution mass spectrometry.
Experimental Section General. 'H and 13C NMR spectra were recorded in CDCl3 on either Varian XL-200or Gemini 300 instruments, and chemical shifts are reported as ppm downfield from tetramethylsilane. Attached proton test (APT) designations for 13C NMR signals are given as (+) for quaternary and methylene carbons and (-) for methine and methyl carbons. High-resolution electron impact mass spectra (HRMS) were obtained on a Kratos MS-25A instrument at 20 eV. Analytical and preparative TLC was performed on Merck silica gel 60 F-254 precoated plates (EM Science, Gibbstown, NJ). All reagents and solvents used were ACS grade; benzylamine and phenethylamine were distilled prior to use. 4-Oxononanal was prepared as described previously (21) and purified by flash chromatography using 15:85 EtOAchexane: 'H NMR d 9.81 (s, 1 H), 2.75 (s, 4 H, C&-H), 2.47 (t, 2 H,J = 7.4 Hz, Cs-H), 1.67-1.22 (m, 6 H),0.89 (t, 3 H,J = 6.6 Hz,C9-H);HRMScalcdforC~H1~02m/z 156.1150,found 156.1160 (M+,1.7). 4-HNE was prepared by a modification of the reported method (22)by carrying out the reduction of 4-hydroxy-2-nonynal
1993 American Chemical Society
20 Chem. Res. Taxicol., Val. 6, No. 1, 1993 diethylacetalwith NainNH3at-78 OCunder N2,yieldingstrictly the desired tram-alkene (23).Deprotection with 1% aqueous citric acid (3 h), followed by extraction into CHCl3, gave 4-HNE in an overall yield of 74% following purification by flash chromatography (CHCl3, 2% EtOH): lH NMR 6 0.94 (t, 3 H, CH3),1.3-1.5 (m, 6 H), 1.66 (app t, 2 H, J = 6.8 Hz), 2.69 (br 8, 1 H, OH), 4.34 (ddt, 1 H, J = 1.4,4.5, and 6.8 Hz), CHOH), 6.29 (ddd, 1 H, J = 1.4, 7.8, and 15.7 Hz, CZvinyl), 6.91 (dd, 1 H, J = 4.5 and 15.7 Hz, C3 vinyl), 9.62 (d, 1 H,J = 7.8 Hz, C H 4 ) . Preparation of 1-Alkyl-2-pentylpyrroles.PhCHzNHz or PhCH2CHzNHZ (0.21 mmol) was added slowly to a solution of 4-oxononanal (0.19 mmol) in 2 mL EtOH, under argon, at 25 OC. Evaporation of solvent after 2 h and preparative thin-layer or flash chromatography of the residue (EtOAc-hexane, 15) afforded the pyrrole in 30-50 % yield. 1-Benzyl-2-pentylpyrrole: lH NMR 6 7.33-6.96 (5 H, m), 6.61 (1H, m), 6.13 (1H, t,
Communicatione H, ArH), 3.50-3.74 (m, -1.5 H), 3.2S3.45 (m, -0.5 H), 3.0-3.3 (br s, 3 H, OH and NH), 2.76-3.05 (m, 6 H), 1.52-1.69 (m, 2 H),
1.26-1.50(m,8H),0.88(t,3H,~~6.4Hz);1*CNMR6139.26(+), 128.64(-), 128.56(-), 126.35(-), 75.74(-), 74.85(-), 74.12(-), 73.86 (-), 50.37(+), 47.62(+), 47.17(+), 35.90(+), 33.61(+), 32.30(+), 31.92(+), 31.78(+), 27.97(+), 25.76(+), 25.54(+), 22.58(+), 14.04 (-); HRMS calcd for C1,HmN02 mlz 279.2199, found 279.1960 (M+,0.2),calcd for CIOHZZNOZ ([M - PhCH21+) mlz 188.1651, found 188.1631 (79).
Results and Discussion In considering suitable conditions for conducting model reactions of 4-HNE with primary amines, we chose 50% aqueous acetonitrile as the solvent in order to guarantee the solubility of the starting 4-HNE as well as any possible J=3.1Hz),5.96(1H,m),5.02(2H,s),2.44(2H,t,J=7.7Hz), intermediates and products. We propose this solvent 1.60-1.22(6H,m),0.85(3H,t,J=7Hz);13CNMR6139.2,134.3,mixture to be a, reasonable compromise for modeling 129.3(2ArC),127.9,126.9(2ArC),121.3,107.7,106.4,50.8,32.2, biochemical reactions which, as far as we know, could be 29.1,26.8,23.1,14.6 (CH,); HRMS calcd for C d h N m l z 227.1673, occurring in relatively low dielectric microenvironments found 227.1672 (M+, 38). 1-(2-Phenethyl)-2-pentylpyrrole: rather than the aqueous milieu. We chose to use a slightly 1H NMR 6 7.35-7.09 (5 H, m), 6.56 (1H, m), 6.08 (1 H, t, J = basic medium, to promote reaction of the amine, but 3.15 Hz), 5.87 (1 H, m), 4.01 (2 H, t, J = 7.65 Hz), 2.98 (2 H, t, without deviating too far from physiologic pH. In initial J = 7.62 Hz), 2.41 (2 H, t, J = 7.7 Hz), 1.63-1.26 (6 H, m), 0.89 studies, we reacted 4-HNE with equimolar quantities of (3 H, t, J = 6.9 Hz); '3C NMR 6 138.5,133.3,128.7 (2 Ar C), 128.6 (2ArC),126.6,119.4,106.8,105.1,47.9,38.3,31.8,28.5,26.1,22.5, amines, but 'H NMR analysis of the adducts indicated 14.1 (CH,); HRMS calcd for C17H23N m/z 241.1830, found incorporation of two (or more) equivalents of aldehyde 241.1826 (M+, 41.3). per amine molecule, even under high-dilution conditions. 2-Acetamido-6-(2-pentylpyrrol-l-yl)hexanoic Acid. A soThe key factor permitting generation of 1:l adducts was lution of Nu-acetyl-L-lysine(140mg, 0.74 mmol) and 4-oxononanal found to be the use of a 5-fold excess of amine. (0.33 mmol) in 5 mL of EtOH was kept under argon at 25 OC for Upon reacting 4-HNE with 5 equiv of either benzyl2 days. Concentration of the solvent, partitioning of the residue amine, phenethylamine, or N*-acetyl-L-lysinein CH3CNbetween dilute HCl and EtOAc, drying and then evaporation of (1:l) at pH 7.8, TLC after 48 h indicated complete H2O the organic extract, and flash chromatography of the residue disappearance of aldehyde and the presence of several (EtOAc-2-propanol,3:1)gave the pyrrole in 30% yield: 'H NMR products, the fastest moving of which in each case was 6 6.55 (1H, m), 6.08 (1H, m), 4.88 (1H, m), 4.57 (1H, m), 3.77 identified as the correspondingpyrrole by the appearance (2 H, t, J = 7.26 Hz), 2.68 (2 H, m), 2.47 (2 H, m), 2.05 (3 H, s), 1.78-1.22 (10 H, m), 0.89 (3 H, m); 13C NMR 6 180.04, 170.70, of an instant blue-violet color on spraying with Ehrlich's 133.22, 119.61, 106.75, 105.36, 52.18, 45.99, 42.72, 36.82, 31.63, reagent [acidified 4-(dimethylamino)benzaldehydel.Af30.73,28.64,27.48,26.23,22.52,13.99; HRMScalcd for C17Hd203 ter workup (see Experimental Section), yields of pyrroles m/z 308.2100, found 308.2096 (M+, 13.1). were determined from 'H NMR integration of the unique Reaction of 4-HNE with Primary Amines. 4-HNE (47 mg, downfield pyrrole signals: 177% in the case of benzylamine, 0.3 mmol) was added to 10 mL of CH~CN-HZO(l:l), pH 7.8 (0.2 23 5% in the case of phenethylamine, and 14% in the case M phosphate buffer), containing 1.5mmol of either benzylamine, of N"-acetyl-L-lysine, based on starting 4-HNE. Verifiphenethylamine, or N'-acetyl-L-lysine, and the resulting solution cation of the identity of pyrrole in each case was through was kept in the dark under Nz for 48 h at 25 "C. The reaction comparison, by chromatography and 'H NMR, to the mixture was concentrated in vacuo, the residue was triturated authentic pyrroles,synthesized independently from Padwith CHC13, and the organic extract was dried (anhydrous NapKnorr condensation of the above three amines with SO,) and evaporated. A weighed quantity of hexamethylbenzene was added as an internal integration standard prior to 'H NMR 4-oxononanal. analysis. The reaction of 4-HNE with phenethylamine was also Preparative Reaction of 4-HNE with Phenethylamine. carried out on a larger scale (still 5-fold excess amine) in Phenethylamine (1.8 g, 15 mmol) was added to 400 mL of HzOorder to permit isolation of the pyrrole and other adducts. CH3CN (l:l), the pH was adjusted to 7.8 with dilute HCl, 4-HNE Concentration of the reaction mixture when TLC and UV (0.47 g, 3 mmol) dissolved in 5 mL of CHaCN was added, and the monitoring indicated disappearance of 4-HNE (3 days) reaction was kept under N2 at room temperature and monitored resulted in a residue which showed changes in composition by UV at 222 nm in order to assess disappearance of the starting (TLC analysis) for 2-3 weeks (under Nz), including an 4-HNE. After various times, aliquota of the reaction solution increase in pyrrole content. After 1month TLC (EtOAcwere evaporated in vacuo for analysis by NMR and product CHC13,l:l) indicated fluorescentmaterials at Rf0.00,0.55, separation by preparative TLC. and 0.70; 12-active and (2,4-dinitrophenyl)hydrazine3-(2-Phenethylamino)-l,4-nonanediol.In one of the preceding preparations, 150 mL of the reaction solution (containing positive materials at Rf 0.19 and 0.27; the pyrrole at Rf 1.13 mmol of aldehyde) was treated after 6 days with 142 mg of 0.77; and a material at Rf 0.82 which gave a positive NaBH3CN (2.25 mmol) under Nz. After 2 h, the mixture was Ehrlich's color upon strong heating. All bands were concentrated to half ita volume, and excess NaBHd (150 mg, 4 isolated by preparative TLC. The top band contained a mmol) was added prior to dilution with water and extraction mixture of the pyrrole and the Rf 0.82 material, which with CHC13. The residue was subjected to preparative TLC were separated by rechromatographing with hexane(MeOH-EtOAc, 60:40). Two major bands were isolated: the CHCl3 (1:l). The material which did not run in EtOAcjust above lower, weakly ninhydrin reactive band at R3,0.25-0.50, CHCla (1:l)was shown to contain two fluorescent materiale the unreacted phenethylamine band at R, 0 . 2 3 , was rechroby rechromatography using MeOH (1%aqueous NH4matographed (MeOH-EtOAc, 2:l) to afford a diastereomeric OH). All materials other than the pyrrole and the Rf0.82 mixture of the aminodiol as an oil: lH NMR 6 7.14-7.33 (m, 5
Communications
compound displayed uncharacterizable lH NMR absorptions, and some appearedto be derived either from 4-HNE alone or from two (or more) 4-HNE molecules per phenethylamine unit. The Rf 0.82 material is a stable product which forms only at long reaction times, and which so far has eluded structural identification. When we attempted to isolate adducts after shorter reaction times, intermediate R, materials isolated by preparative TLC were found to be unstable, in part undergoing conversion to the pyrrole. Pyrrole formation was found to be rather sensitive to the reaction conditions. For example, heating at reflux a 95 % aqueous ethanol solution of 4-HNE and the HCl salt of PhCH2NH2 (both 0.15 M) gave only a trace of pyrrole and mainly the 4-HNE-derived 2-pentylfuran: 'H NMR (CDCl3) 6 0.91 (t,3 H, CH3), 1.34 (m, 4 H), 1.67 (m, 2 H), 2.61 (t, 2 H, J = 7.5 Hz, ArCH2), 6.00 and 6.31 (2 m, 1H each, C3/Cd-H), 7.33 (m, 1 H, Cs-H). In contrast, the addition of 5 mol 7% CuCl2 (based on amine) was found to increase the efficiency of pyrrole formation. The latter preliminary observation could be important in view of the alleged role of transition metal ions in physiologic oxidative modifications of LDL and the frequent use of Cu(I1) in the experimental preparation of "oxidized LDL" (24). Pyrrole formation did not occur in the presence of thiols; e.g., the reaction of 4-HNE (30 mM), PhCH2NH2 (45mM), and PhCH2SH (45 mM) in CH&N-H20 (1:l)(pH 7.65) produced only the 4-HNE-PhCHzSH adduct. Also, the reaction of 4-HNE (6.4 mM) and cysteine (12.8 mM) in CH3CN-H20 (1:l) (pH 7.9) gave no Ehrlich's-positive material. It thus appears, as expected ( 1 , 7 ) ,that amines cannot compete with thiols in reacting with 4-hydroxy2-alkenals and, therefore, that 4-HNE-amine adducts should not form in physiologic compartments containing high glutathione concentration. In another preparative-scale reaction between 4-HNE and phenethylamine, the solution obtained after 6 days was divided in two portions, one of which was treated with NaBH3CN (and then quenched withNaBH4)and the other exposed to H2 in the presence of Pd/C. TLC and lH NMR of the residues obtained upon evaporation of solvent appeared similar. Preparative chromatography of the hydride-reduced material yielded two major bands which ran in between the pyrrole and the recovered phenethylamine. Spectral characterization of the slower-moving of these bands implicated the reduced product of Michael addition of PhCHzCHzNHz to 4-HNE, 342-phenethylamino)-l,4-nonanediol, formed as a mixture of diastereomers (four 13C NMR signals are seen for the chiral C-3 and C-4 methine carbons, and four of the remaining nine aliphatic carbons also come as a pair of resonances). The faster-moving band is itself a mixture which appears to contain the reduced Schiff base, but the latter has not yet been obtained in pure form. A mechanism consistent with our results is shown in Scheme I. Although there is no chemical literature which delineates the amine reactivity of 4-hydroxyenals, the most frequent outcome of reacting primary amines with simple a,@-unsaturatedaldehydes is Schiff base formation (2528). The Schiff base would be in, albeit unfavorable, equilibrium with an enamine, which, in this case, is the enol tautomer of an intermediate on the pathway of the Paal-Knorr condensation (29) between amine and the corresponding 7-ketoaldehyde, thereby rationalizing pyrrole formation. Nonetheless, there is also precedent for
Chem. Res. Toxicol., Vol. 6, No. 1, 1993 21
R = PhCH2, PhCH2CH2, H O O C C H ( H W C ) C H ~ C H ~ C H ~ C H ~
Michael addition of amines to a,@-unsaturatedaldehydes and/or competing 1,2- and 1,Caddition (30, 31). Some Michael adducts may be only reversibly formed, in view of the well-known ability of primary amines to catalyze cis-trans isomerization of unsaturated carbonyl compounds (32)(the generation of 2-pentylfuran in the reaction of 4-HNE with PhCH2NHzeHCl n a y represent a formal trans-to-cis isomerization). Our ability to isolate the hydride-reduced product of 1,4-addition of phenethylamine to 4-HNE verifies the generation of 4-HNE-amine Michael adducts, a suspicion raised recently by other investigators (33). The substoichiometric production of pyrrole even at long reaction time, coupled with the fact that the overall pyrrole yield increases with attempted isolation of (unstable) 4-HNE-amine adducts, suggests an effectively nonequilibrating endurance in solution of one or more 4-HNE-amine adducts which are not on the pathway to pyrrole. Likely candidates are the Michael adduct, probably stabilized as the cyclic hemiacetal, and possibly (at least in our experimental regime using excess amine) the Schiff base of the Michael adduct (a 2:l amine-4HNE adduct). Preliminary "NMR tube" studies (D2OCD3CN), conducted under the same conditions as our preparative reactions, indicate that at the time of disappearance of starting 4-HNE (2-3 days), the levels of pyrrole in solution are significantly lower than the isolated yields obtained upon workup (evaporation and extraction). In DMSO&, however, pyrrole content after 3 days appears to be comparable to the isolated yields. Clearly, additional work is needed to clarify mechanistic details and the factors which govern pyrrole formation, as well as the nature of other 4-HNE-amine adducts generated in these reactions. Sensitive assays, such as an immunoassay for pyrrolated protein or GC-MS and HPLC detection of pyrrolated lysine from protein digests, must now be developed to confirm the physiologic occurrence and biological significance of 4-HNE-derived pyrroles. Acknowledgment. This research was supported by Grants NS 22688 (L.M.S.) and GM 21249 (R.G.S.) from the National Institutes of Health. L.M.S. also acknowledges a Research Career Development Award (1987-1992) from NIH. References (1) Esterbauer, H.(1982) Aldehydic producta of lipid peroxidation. In Free Radicals, Lipid Peroxidation and Cancer (McBrien, D. C . H., and Slater, T. F., Eds.) pp 101-122, Academic Press, London.
22 Chem. Res. Toxicol., Vol. 6, No. 1, 1993 (2) Van Kuijk, F. J. G. M., Holte, L. L., and Dratz, E. A. (1990) 4-Hydroxyhexenal: a lipid peroxidation product derived from oxidized docosahexaenoic acid. Biochim. Biophys. Acta 1043,116118. (3) Segall, H. J., Wilson, D. W., Dallas, J. L., and Haddon, W. F. (1985) Trans-4-hydroxy-2-hexenal:A reactive metabolite from the macrocyclic pyrrolizidine alkaloid senecionine. Science 229, 472-475. (4) Grasse, L. D., Lame, M. W., and Segall, H. J. (1985) In vivo covalent binding of trans-4-hydroxy-2-hexenal to rat liver macromolecules. Toxicol. Lett. 29, 43-49. (5) Griffin, D. S., and Segall, H. J. (1986)Genotoxicity and cytotoxicity of selected pyrrolizidine alkaloids. A possible alkenal metabolite of the alkaloids, and related alkenals. Toxicol. Appl. Pharmacol. 86, 227-234. (6) Esterbauer, H., Zollner, H., and Scholz, N. (1975) Reaction of glutathione with conjugated carbonyls. 2. Naturforsch. 30c, 466473. (7) Witz, G. (1989) Biological interactionsof a,p-unsaturated aldehydes. Free Radical Biol. Med. 7,333-349. (8) Esterbauer, H., Schaur, R. J., and Zollner, H. (1991)Chemistry and biochemistry of 4-hydroxynonenal, malondialdehyde and related aldehydes. Free Radical Biol. Med. 11, 81-128. (9) Schauenstein, E., Dorner, F., and Sonnenbichler,J. (1968) Binding of 4-hydroxy-2,3-en& to protein sulfhydryl groups. 2.Naturforsch. 23b, 316319. (10) Esbrbauer, H., Ertl, A., and Scholz,N. (1976)Thereaction of cysteine with a&unsaturated aldehydes. Tetrahedron 32, 285-289. (11) Palinski, W., Rosenfeld, M. E., Yla-Herttuala, S., Gurtner, G. C., Socher, S. S.,Butler,S. W., Parthasarathy,S.,Carew,T. E.,Steinberg, D., and Witztum, J. L. (1989) Low density lipoprotein undergoes oxidative modification in vivo. Proc. Acad. Sci. U.S.A. 86, 13721376. (12) Palinski, W., Yla-Herttuala, S., Rosenfeld, M. E., Butler, S. W., Socher, S. A., Parthasarathy, S., Curtiss, L. K., and Witztum, J. L. (1940) Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Arteriosclerosis 10, 325-335. (13) Hoff, H. F., O’Neil, J., Chisolm, G. M., 111,Cole,T. B., Quehenberger, O.,Esterbauer, H., and J q e n s , G. (1989) Modificationof low density lipoprotein with 4-hydroxynonenal induces uptake by macrophages. Arteriosclerosis 9, 538-549. (14) Jurgens, G., Lang, J., and Esterbauer, H. (1986) Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal. Biochim. Biophys. Acta 875, 103-114. (15) Esterbauer,H.,Koller,E.,Slee,R. G., andKoster,J. F. (1986)Possible involvement of the lipid-peroxidationproduct 4-hydroxynonenal in the formation of fluorescent chromolipids. Biochem. J. 239,405409. (16) Napetschnig, S., Schauenstein, E., and Esterbauer, H. (1988) Formation of a pyridinium derivative by reaction of 4-hydroxypentenal with glycine. Chem.-Biol. Interact. 68, 165-177. (17) Sayre, L. M., Autilio-Gambetti, L., and Gambetti, P. (1985) Pathogenesis of experimental giant neurofilamentous axonopa-
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