Pyridoxamine: An Extremely Potent Scavenger of 1,4-Dicarbonyls

Irene Zagol-Ikapitte , Venkataraman Amarnath , Olivier Boutaud , and John A. ..... Isabella Dalle-Donne , Roberto Maffei Facino , Aldo Milzani , M...
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Chem. Res. Toxicol. 2004, 17, 410-415

Pyridoxamine: An Extremely Potent Scavenger of 1,4-Dicarbonyls Venkataraman Amarnath,* Kapil Amarnath,† Kalyani Amarnath, Sean Davies, and L. Jackson Roberts II Departments of Pathology and Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232 Received October 28, 2003

1,4-Dicarbonyl compounds, which include 2,5-hexanedione and recently discovered endogenous 4-ketoaldehydes (levuglandins, isoketals, and neuroketals), exhibit severe toxicity. The key step in the toxicity of these compounds is their reaction with the lysyl residues of proteins to form pyrrole adducts. To screen for effective scavengers of these toxic compounds, we determined the reaction rates of pyrrole formation for a series of primary amines with a model 4-ketoaldehyde, 4-oxopentanal (OPA). We found pyridoxamine (PM) to react extremely rapidly, with a second-order rate constant at physiological pH being ∼2300 times faster than that of NR-acetyllysine. The extreme reactivity of PM was unique to 1,4-dicarbonyls, as its reactions with methylglyoxal and 4-hydroxy-2(E)-nonenal were much slower and only slightly faster than with NR-acetyllysine. The phenolic group of PM was found to be essential to its high reactivity, and the rate constant for pyrrole formation with OPA exhibited a maximum at pH 7.5, close to the second pKa of PM. We therefore propose a mechanism involving transfer of the phenolic proton to the carbonyl of the initially formed hemiacetal, which facilitates subsequent nucleophilic attack and ring closure. Only 1,4-dicarbonyls are likely to participate in the proposed mechanism, thereby conferring unique sensitivity of this class of compounds to scavenging by PM.

Introduction The toxicity of many carbonyl compounds is substantially augmented by the presence of another carbonyl function. The reactivity and the mechanism of toxicity of dicarbonyls are highly dependent upon the relative positions of these groups (Scheme 1). Vicinal dicarbonyls, such as MGO1 (1) and glyoxal, are formed during the uncatalyzed oxidation of carbohydrates. The initial step for a series of their reactions with proteins is the addition of two amino groups to the adjacent carbonyl groups (1). 1,3-Dicarbonyls, including MDA (2), can be formed during the oxidation of polyunsaturated fatty acids. One of the targets of MDA is the guanine residue of DNA, which is converted to a pyrimdo[1,2-a]purin-10(3H)-one ring (2). The most important reaction of dicarbonyls separated by two carbons is cyclization to form furans andswith primary aminesspyrroles. These cyclizations, known as Paal-Knorr reactions (3, 4), are not only very useful synthetically but are also relevant in the toxicity of the 1,4-dicarbonyl derivatives. Because the formation of furans is catalyzed by acid, at physiological pH, the pyrrole formation with amino groups of proteins appears to be biologically significant (5, 6). The exact mechanism for the toxicity of 1,4-dicarbonyls remains to be fully elucidated and is likely to include a number of different * To whom correspondence should be addressed. E-mail: [email protected]. † Voluntary worker from Massachusetts Institute of Technology, Cambridge, MA. 1 Abbreviations: AcHD, 3-acetyl-2,5-hexanedione; AcLys, NR-acetyllysine; DEHD, 3,4-diethyl-2,5-hexanedione; IsoK, 15-E2-isoketal; MDA, malondialdehyde; MGO, methylglyoxal; OPA, 4-oxopentanal; PM, pyridoxamine.

Scheme 1. Reactive Dicarbonyls and HNE

pathways. The pyrrole modification on a protein by itself may be detrimental as it alters both the charge and the bulk of adducted lysyl residues. Because lysyl residues often are critical for both protein-protein recognition and enzymatic functions, these alterations could significantly hamper cellular viability. In addition, the electron-rich pyrrole ring, through oxidation, could lead to crosslinking and cause further damage. One source of γ-diketones is exposure to the common solvent hexane that is metabolically activated to 2,5hexanedione (3) (7). The toxicity of various 2,5-hexanedione analogues directly correlates to their rate of pyrrole formation and subsequent oxidation of the resulting

10.1021/tx0300535 CCC: $27.50 © 2004 American Chemical Society Published on Web 02/13/2004

Pyridoxamine: Scavenger of 1,4-Dicarbonyls

pyrrole adducts. Recently, oxidation of arachidonic acid, either by prostaglandin H2 synthases or by free radicalmediated mechanisms, has been shown to form 4-ketoaldehydes termed levuglandins and isoketals (such as 4), respectively (8, 9). Isoketals and levuglandins are highly reactive γ-ketoaldehydes that show a marked proclivity to form pyrroles on proteins. The pyrrole adducts rapidly oxidize to form cross-links as well as lactam and hydroxylactam adducts, (8) which are highly toxic to neuronal cell cultures (10). Finally, another product of fatty acid peroxidation, 4-hydroxy-2(E)-nonenal (5, HNE), is also known to form pyrroles with amino groups on proteins (11). An obvious first approach to protect against the assault of dicarbonyls is to reduce their generation; however, in many situations, this approach is ineffective. Therefore, an adjuvant remedy is to scavenge the dicarbonyls using competing primary amines. The toxicity of most amines has limited the success of this approach. However, during the past few years, PM (6f), which is essentially nontoxic (12), has shown promise. PM is effective in trapping the 1,2-dicarbonyl intermediates of carbohydrate degradation including MGO and glyoxal in models of diabetes (13). We explored the potential of PM in preventing the 1,4dicarbonyls from attacking sensitive amino functions of proteins. We determined the reactivity of PM with model 1,4-dicarbonyls and found that they react with PM at rates vastly exceeding that with lysine, thereby making PM an effective scavenger of those reactive metabolites. We also explored the mechanism for this uniquely rapid reactivity of PM for the possibility of designing more effective analogues.

Experimental Section Chemicals. OPA (7), 3-acetylhexane-2,5-dione (9, AcHD), d,l and meso isomers of 3,4-diethylhexane-2,5-dione (11E, d,lDEHD; and 11M, meso-DEHD), and 2-hydroxybenzylamine (6h) were synthesized using published procedures (14-17). OPA was freshly distilled before use in kinetic experiments. A mixture of diastereomers of IsoK was freshly prepared by the hydrolysis of the dimethoxy acetal.2 Other amines and MGO were obtained from commercial sources. Pyrroles. PM dihydrochloride (120 mg, 0.5 mmol) in 0.2 M phosphate buffer (20 mL, pH 7.5) and OPA (60 mg, 0.6 mmol) were stirred at room temperature for 1 h and evaporated. The residue was purified on a column of silica that was prepared and eluted with 10% methanol in ethyl acetate to get 1-(2hydroxy-6-hydroxymethyl-3-methylpyridin-4-ylmethyl)-2-methylpyrrole (8f); 100 mg (86%); mp 206-208 °C. UV (pH 7.4) λmax nm (): 326.5 (6930), 250.2 (4430), and 218.6 (19 300). MS (Thermo-Finnigan TSQ 700 mass spectrometer) m/z 233 (M + H)+ and 152 (2-hydroxy-6-hydroxymethyl-3-methylpyridinylmethyl). 1H NMR (Bruker DPX-300) (CD3OD): δ 2.23 (s, 3H, pyrrole-CH3), 2.46 (s, 3H, pyridine-CH3), 4.39 (s, 2 H, CH2O), 5.16 (CH2N), 5.66 (d, 1 H, J ) 2.4 Hz, C3-H), 5.77 (d, 1 H, J ) 2.4 Hz, C4-H), 6.7 (m, 1 H, C5-H), 7.93 (s, 1 H, H on pyridine). 13C NMR: δ 12.1 (pyrrole-CH ), 18.8 (pyridine-CH ), 41.3 3 3 (CH2N), 60.2 (CH2O), 107.9 and 108.6 (C-3 and C-4), 120.9 (C5), 130.2 (C-2), 121.0, 130.4, 134.0, 137.1, and 147.6 (pyridine). 3-Acetyl-1-(2-hydroxy-6-hydroxymethyl-3-methylpyridin-4-ylmethyl)-2,5-dimethylpyrrole (10f) was similarly prepared using AcHD in place of OPA; 120 mg (83%); mp 196-198 °C. MS m/z 289 (M + H)+ and 247 (loss of acetyl). 1H NMR (CD3OD): δ 2.18 (CH3CO), 2.23 and 2.24 (s, 3H each, pyrrole-CH3), 3.05 (s, 3H, pyridine-CH3), 4.26 (s, 2 H, CH2O), 5.25 (CH2N), 6.26 (s, 1 H, C4-H), 7.91 (s, 1 H, H on pyridine). 13C NMR: δ 11.8 and 2

Amarnath, V., and Amarnath, K.

Chem. Res. Toxicol., Vol. 17, No. 3, 2004 411 11.9 (pyrrole-CH3), 18.2 (pyridine-CH3), 27.8 (CH3CO), 41.2 (CH2N), 58.5 (CH2O), 108.1 (C-4), 119.2 (C-3), 128.9 and 129 (C-2 and C-5), 131.5, 134.4, 135.7, 136.5, and 144.9 (pyridine), 204.0 (CdO). 3,4-Diethyl-1-(2-hydroxy-6-hydroxymethyl-3-methylpyridin4-ylmethyl)-2,5-dimethylpyrrole (12) had a single spot when checked by TLC (silica-ethyl acetate) with an Rf of 0.45 and gave a MS with m/z of 303 (M + 1)+ and 152 (2-hydroxy-6hydroxymethyl-3-methylpyridin-4-ylmethyl); it could not be further purified. 2-Aminomethanol (0.25 g, 4 mmol) and OPA (0.5 g, 5 mmol) were stirred in dichloromethane (20 mL) for 2 h and evaporated. The residue was purified by column chromatography (silica, 4:1 hexanes-ethyl acetate) followed by distillation to give 0.3 g (75%) of 1-(2-hydroxyethyl)-2-methylpyrrole (8c); 43-45 °C/0.8 Torr. MS m/z 126 (M + 1)+. 1H NMR (CDCl3): δ 2.19 (s, 3 H, CH3), 3.65 (t, 2 H, J ) 6.6 Hz, CH2O), 3.84 (t, 2 H, J ) 6.6 Hz, CH2N), 5.83 (s, 1 H, C3-H), 6.01 (t, 1 H, J ) 2.3 Hz, C4-H), 6.56 (t, 1 H, J ) 2.3 Hz, C5-H). 13C NMR: δ 12.0 (CH3), 48.7 (CH2N), 62.4 (CH2O), 106.0 and 106.9 (C-3 and C-4), 120.4 (C-5), 128.7 (C-2). The following pyrroles were similarly prepared with the solvent used for purification by column chromatography, and the yield is given in the parenthesis. 1-(3-Hydroxypropyl)-2methylpyrrole (8d) (5:1 hexanes-ethyl acetate, 65%). MS m/z 140 (M + 1)+. 1H NMR (CDCl3): δ 1.83 (m, 2 H, CCH2C), 2.15 (s, 3H, CH3), 3.52 (t, 2 H, J ) 6.0 Hz, CH2O), 3.84 (t, 2 H, J ) 6.9 Hz, CH2N), 5.79 (s, 1 H, C3-H), 5.96 (t, 1 H, J ) 3.0 Hz, C4-H), 6.51 (t, 1 H, J ) 3.0 Hz, C5-H). 13C NMR: δ 11.9 (CH3), 33.6 (CCH2C), 43.1 (CH2N), 59.5 (CH2O), 106.6 and 106.8 (C-3 and C-4), 119.9 (C-5), 128.4 (C-2). 1-(Pyridin-4-ylmethyl)-2methylpyrrole (8e) (ethyl acetate, 85%); mp 59-60 °C. MS m/z 173 (M + H)+ and 92 (pyridinylmethyl). 1H NMR (CDCl3): δ 2.09 (s, 3H, CH3), 5.00 (CH2), 5.97 (s, 1 H, C3-H), 6.13 (s, 1 H, C4-H), 6.61 (s, 1 H, C5-H), 6.85 (s, 2H, C3-H on pyridine), 8.50 (s, 2 H, C2-H on pyridine). 13C NMR: δ 11.7 (CH3), 49.2 (CH2N), 107.7 and 107.8 (C-3 and C-4), 120.8 (C-5), 128.5 (C-2), 121.0, 147.7, and 150.1 (pyridine). 1-(2-Hydroxybenzyl)-2-methylpyrrole (8h) (3:1 hexanes-ethyl acetate, 80%). MS m/z 188 (M + H)+ and 107 (hydroxybenzyl). 1H NMR (CDCl ): δ 2.15 (s, 3H, CH ), 5.00 (CH ), 5.95 (s, 1 H, 3 3 2 C3-H), 6.12 (s, 1 H, C4-H), 6.63 (s, 1 H, C5-H), 6.65 (m, 2 H), 6.78 (t, 1 H, J ) 7.5 Hz), and 7.08 (t, 1 H, J ) 7.5 Hz, phenyl). 13C NMR: δ 11.8 (CH ), 45.6 (CH N), 106.8 and 107.0 (C-3 and 3 2 C-4), 120.8 (C-5), 125.0 (C-2), 115.1, 128.0, 128.1, 128.4, 129.1, and 153 (phenyl ring). 1-(2-Methoxybenzyl)-2-methylpyrrole (8i) (5:1 hexanes-ethyl acetate, 85%); mp 53-54 °C. MS m/z 202 (M + H)+ and 121 (methoxybenzyl). 1H NMR (CDCl3): δ 2.09 (s, 3H, CH3), 3.74 (CH3O), 4.95 (CH2), 5.91 (s, 1 H, C3-H), 6.08 (s, 1 H, C4-H), 6.56 (s, 1 H, C5-H), 6.45 (d, 1 H, J ) 7.2 Hz), 6.78 (m, 2 H,), and 7.12 (t, 1 H, J ) 7.2 Hz, phenyl). 13C NMR: δ 11.5 (pyrrole-CH3), 45.2 (CH2N), 54.9 (CH3O), 106.6 and 106.7 (C-3 and C-4), 120.6 (C-5), 126.7 (C-2), 109.5, 120.5, 126.8, 128.1, 128.5, and 155.9 (phenyl ring). Pyrroles 8g, 10a, and 10c have been described previously (15, 18), and the formation of 8a on mixing OPA with AcLys has been shown by NMR (19). Kinetic Experiments. A reaction mixture (1 mL) containing 2 mM each of PM and OPA in 100 mM phosphate buffer (pH 7.4) was taken in a 1.5 mL eppendorf tube that was mixed at 25 °C in an Eppendorf ThermoMix. At each time point, an aliquot (40 µL, smaller volumes at later time points) was diluted with 460 µL of water and heated with 0.5 mL of Ehrlich’s reagent (80 mM 4-dimethylaminobenzaldehyde in 1:1 methanol0.6 M HCl) for 2 min at 67-69 °C. After the mixture was cooled, the absorbances at 528 and 568 nm were measured (Shimadzu UV-1601 spectrophotometer) and used for calculating the concentration of 2-methylpyrrole (8). The sum of extinction coefficients for Ehrlich adducts of pyrroles in this study was 68 000 ( 500. Kinetic studies using other amines and OPA were similarly performed but with higher concentrations of reactants. The pH of the reaction mixture in the range 6.5-9.0 was

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controlled by phosphate and adjusted to the reported value before starting the reaction. The rate of pyrrole formation between PM and AcHD (5 mM each) was followed at pH 7.4 (100 mM phosphate buffer) at 25 °C. A 20 µL aliquot was diluted to 500 µL with 15:85 acetonitrile-1% formic acid that was also the solvent system used to analyze 40 µL (delivered by an autoinjector) of the diluted solution by HPLC to determine the concentration of the pyrrole. A Zorbax SB-Aq column (4.7 mm × 250 mm; 5 µm) in a Waters 2960 connected to a 996 photodiode array detector was used at the flow rate of 1.0 mL/min. The rate constant for the reaction between PM and MGO was similarly determined by measuring the concentration of the unreacted PM. The concentrations of pyrrole in the reaction between AcHD and AE or AcLys were measured spectrophotometrically after appropriate dilution of aliquots from the reaction mixture. The molar absorptivity of 3-acetylpyrrole at 258 nm (15) was used in the calculations. A reaction mixture containing 200 µL each of 100 mM PM, 100 mM 1-octanol, and 100 mM d,l (or meso) DEHD, 400 µL of 0.2 M phosphate buffer (pH 7.4), and 1.0 mL of water was agitated at 45 °C. At each time point, a 200 µL aliquot was extracted with 100 µL of ethyl acetate and ∼1 µL of the extract was analyzed by a Hewlett-Packard gas chromatograph 5890A with a flame ionization detector. The column (Phenomenex Zebron ZB-wax, 15 m × 0.53 mm × 1 mm) was heated at 80 °C for 2 min and then at the rate of 20 °C/min up to 220 °C. The ratio of the peak areas of 11E or 11M to 1-octanol was used for determining its concentration. Competition Experiments with IsoK and Lysine. IsoK (100 µM final concentration) was added to PBS solutions (250 µL final volume) containing 1 mM lysine with tracer quantities of 3H-lysine and either vehicle (PBS), 1 mM PM, or 10 mM PM. A reaction with lysine but not IsoK was also performed as a negative control. After incubation at 37 °C for 4 h, aliquots were removed for analysis of IsoK-lysine adducts. Aliquots were applied to a Waters tC18 Sep-Pak cartridge preequilibrated with methanol and water. Unreacted lysine was eluted from the cartridge using water and then 15% MeOH in water. IsoKlysine adducts were eluted with 100% MeOH. The 3H label in both eluants was determined by liquid scintillation counting with the total amount of IsoK-lysine adducts formed calculated as the percent of 3H label in the 100% MeOH eluant. Additionally, levels of the stable oxidized pyrrole product, IsoK-lysyllactam, were determined by LC/MS/MS as previously described (8) with 13C6-IsoK-lysyl-lactam added to each aliquot as an internal standard.

Results Reaction with OPA. Our desire to obtain an effective scavenger of isoketals and levuglandins (9) prompted us to determine the reaction rates of γ-ketoaldehydes with AcLys (6a) and a series of primary amines including PM. Because the tetra-substituted pyrroles resulting from the reaction of isoketals or levuglandins with amines rapidly oxidize, OPA (7) was chosen for our studies to determine the rate of pyrrole formation. The 1,2-dialkyl substituted pyrrole (8) from OPA was relatively stable to oxidation and was determined as its adduct with 4-dimethylaminobenzaldehyde (Ehrlich’s reagent). The absorption spectra of the reaction between the 2-methylpyrroles and the reagent exhibited two maxima at 528 and 568 nm, which were attributed to 3(4)- and 5-substituted products, respectively (20). Because the composition of the mixture was difficult to control and changed with time, the sum of the absorbances at the two wavelengths was used as a measure of the pyrrole concentration. The second-order rate constants for AcLys and other amines are compared in Table 1. The reactivity slightly

Amarnath et al.

increased for 2-aminoethanol (and 3-aminopropanol) and increased further for benzylamine and 4-picolinylamine. Strikingly, PM showed extremely high reactivity (∼2300 times greater) as compared to AcLys. Such a drastic difference suggested the potential of PM to protect lysyl residues against γ-ketoaldehydes by effectively competing as substrates. To test this scavenging potential, 1 mM lysine containing tracer amounts of 3H-lysine was reacted with 100 µM isoketal in the presence of vehicle (PBS), 1 mM PM, or 10 mM PM. The resulting lysyl adducts were quantified by separation of the highly polar free lysine from more nonpolar adduction products by solid phase extraction. One millimolar PM reduced the amount of lysine adducts by 80% and 10 mM PM reduced adducts by 97% (Figure 1A). The levels of lysyl-lactam adduct, measured by LC/ MS/MS, were reduced in these same reactions by 97 and 99.8%, respectively, for 1 and 10 mM PM (Figure 1B). To further understand the enhanced reactivity of PM, the influence of pH on the rate of pyrrole formation with OPA was studied. The log of rate constant for AE increased linearly with pH (Figure 2). In contrast, the reactivity of PM and 2-hydroxybenzylamine (6h) exhibited maxima at pH 7.5 and 8.5, respectively. Reaction with Other Dicarbonyls and HNE. We next examined the relative reactivity of PM with other biologically relevant carbonyls. The second-order rate constant for reaction between PM and OPA was ∼190 Table 1. Second-Order Rate Constants for Pyrrole Formation with OPA: pH 7.4, T ) 25 °C amine

k × 106 M-1 s-1

relative rate

AcLys (6a) N-acetyllysine (6b) 2-aminoethanol (6c) 3-aminopropanol (6d) 4-picolinylamine (6e) PM (6f) benzylamine (6g) 2-hydroxybenzylamine (6h) 2-methoxybenzylamine (6i)

180.5 ( 2.5 234.5 ( 3.5 793 ( 30 830.7 ( 20.7 4785 ( 112 416 800 ( 2770 1955 ( 82 177 467 ( 4658 651 ( 12.3

1 1.3 4.4 4.6 26.5 2309 10.8 983 9.1

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Chem. Res. Toxicol., Vol. 17, No. 3, 2004 413

Figure 2. Effect of pH on the second-order rate constant (log k) of pyrrole formation with OPA. While the rate constant for AE (b) increases with pH, that for PM ([) or 6h (2) exhibits a maximum suggesting the importance of the phenolic proton. Table 2. Second-Order Rate Constants for Reaction with MGO, AcHD, and DEHD at pH 7.4a amine

dione

k × 106 M-1 s-1

PM (6f) AcLys (6a) 2-aminoethanol (6c) PM (6f) PM (6f) PM (6f)

MGO (1) AcHD (9) AcHD (9) AcHD (9) d,l-DEHD (11E) meso-DEHD (11M)

2230 ( 85 329 ( 2.2 1533 ( 22 38 925 ( 2290 3303 ( 200 930 ( 65

a The reaction with DEHD was performed at 45 °C while the rest were carried out at 25 °C.

Figure 1. Effect of PM on the adduction of lysine by IsoK. Lysine (1 mM) containing tracer levels of 3H-lysine was incubated for 4 h with 100 µM IsoK in the presence of vehicle (PBS), 1 mM PM, or 10 mM PM. (A) Total levels of IsoK-lysine adducts were determined by scintillation counting after separation of unreacted lysine from adducted lysine using solid phase extraction. (B) Levels of the specific oxidized pyrrole product, IsoKlysyl-lactam, were determined by LC/MS/MS using an internal standard.

(∼120 times) over AcLys in the rate (Table 2), although the advantage was less than that observed for cyclization with OPA. The bulkiness of PM may have reduced its reactivity toward a diketone. The rate constant for the reaction between the PM and the enantiomeric mixture of DEHD was higher (3.6 times) than that for reaction with the meso isomer of DEHD. Such differences have been observed for other amines during cyclization with the diastereomers of 3,4-disubstituted hexanediones (16).

times greater than that of PM and MGO. In the reaction mixture containing HNE and PM, only traces of pyrrole were detected. For studying the reaction between PM and a γ-diketone, AcHD (9) was chosen as a model, again due

Discussion

to the stability and the convenient UV absorbance of the resulting pyrrole. PM exhibited a considerable edge

Our results indicate a unique sensitivity of 1,4-dicarbonyls to scavenging by PM and suggest a plausible mechanism for this selectivity (Scheme 2). The first step in the reaction between a primary amine and a 1,4dicarbonyl compound (13) is the formation of the hemi-

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Chem. Res. Toxicol., Vol. 17, No. 3, 2004 Scheme 2. Possible Mechanism of Pyrrole Formation between PM and 1,4-Dicarbonyls

aminal 14, which in the case of a ketoaldehyde, may likely involve attack at the aldehyde group (21). The initially formed hemiaminal 14 could lose a molecule of water leading to the imine 15. This reversible reaction is probably involved in the reaction between PM and other carbonyl compounds such as MGO (13) and HNE (19). The reaction rate of PM does not exhibit great advantage over AcLys during this step with only an increase of 2-4 times faster reported for condensation with glycolaldehdye and glyoxal (13). For 1,4-dicarbonyls, however, the hemiaminal 14 can also undertake electrophilic attack on the other carbonyl, which appears to be the rate-limiting step (3, 21). The aromatization of the resulting cyclic diol 17 to pyrrole (16) is irreversible and is the driving force for the Paal-Knorr reaction. Therefore, any proposed mechanism for the enhanced reactivity of PM should rationalize increased cyclization. The cyclization of 14 requires the deprotonated amine, and therefore, the reaction rate is dependent on the pKa of the amine. The small increase in reactivity (up to 30fold) from 6a to 6e results from substitution on the amine that lowers its pKa. In the range 6.5 to 8.5, the rate for 6c increased with pH, which can be expected from increased ionization of the hemiaminal with raising pH. The second ionization of PM has a pKa of 7.9, and spectrophotometric data suggest the existence of 6f-I and 6f-II in equilibrium in the pH range 5-8, with 6f-I predominating (22). In structure 6f-I, the phenolic anion could attract a proton from the amine, thereby making it more basic. Such a structure explains the increased reactivity of PM toward carbonyl compounds in general but fails to account for the 3 orders of magnitude increase in the rate of pyrrole formation. Any contribution of the alcoholic group of PM toward its reactivity is unlikely since the rate constants for 6c,d were nearly the same. The remaining possibility is that the acidic phenol protonates the carbonyl and holds it in position thereby facilitating the attack of the hemiaminal amine, as in structure 18. Because such an intermediate does not exist for the reaction with MGO, the second-order rate constant for its reaction with PM was considerably slower than that between PM and OPA. In the reaction between HNE and a primary amine, the pathway from the imine 15 to pyrrole does not involve 18 (11, 19) and it explains why only negligible amounts of pyrrole were formed when PM and HNE were incubated at 25 °C at pH 7.4.

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The proposed participation of the phenolic group in the rate-limiting step is supported by the observation that the rate constant for PM does not increase with pH as in the case of 6c but exhibits a maximum around pH 7.5. An intermediate, such as 18, requires the presence of 6fII so that the proton can be transferred to the carbonyl group. As the pH increases above 7.6, the loss of proton from the phenol increases so that the rate constant decreases sharply. The above hypothesis is also consistent with the rate constants for benzylamines 6g-i given in Table 1. Salicylamine (6h) with a 2-phenolic function is approximately 100 times more reactive than benzylamine, probably due to the participation of the phenolic proton as in 18. Such an advantage is lost with 2-methoxybenzylamine (6i) that does not possess an acidic proton and with 3,4-dihydroxybenzylamine whose phenolic groups cannot reach the carbonyl group (data not shown). The rate constant for 6h also reaches a maximum at 8.5 as it approaches the pKa of the phenolic group (23) before decreasing in more basic solutions. Further evidence for this mechanism comes from the influence of stereochemistry on the γ-diketone reaction rates. The cyclization of 14 in the rate-limiting step requires that the meso isomer of 3,4-disubstituted hexanedione reacts slower than the chiral isomer because of the increased crowding in the transition state of the former as compared to the latter. In addition, the stereoisomeric purity of the unreacted isomer should be preserved since hydrogen at the 3(4)-position is lost only after the slow step. Both of these criteria were met in the reaction between the PM and the stereoisomers of DEHD (11). The rate of reaction for PM with the enantiomeric mixture of DEHD (11E) was indeed higher than that for the meso isomer 11M (Table 2), and in both reaction mixtures, the unreacted diastereomer was found not to undergo isomerization.

Conclusion PM was found to react with 1,4-dicarbonyls at rates many orders of magnitude greater than AcLys, and the high reactivity appeared to be selective for this class of dicarbonyls. PM significantly reduced adduction of an endogenous 4-ketoaldehyde, IsoK, to lysine when added at equimolar concentrations in vitro. Therefore, PM and related phenolic amines have the potential to scavenge endogenous 4-ketoaldehydes postulated to be overproduced in biological tissues during many degenerative diseases and thereby sharply reduce their adverse effects.

Acknowledgment. The work was supported by the National Institutes of Health Grant GM 42056.

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