Chem. Res. Toxicol. 1993,6, 82-90
82
Studies on the Nitroso-Glyoxylate Reaction. Relative Hydroxamic Acid Production by Glyoxylate, Pyruvate, and Formaldehyde in Reactions with 4-Nitrosobiphenyl Michael D. Corbett*vtJ and Bernadette R. Corbettt Pesticide Research Laboratory and Department of Pharmacology & Therapeutics, University of Florida, Gainesville, Florida 3261 1 Received July 20, 1992
The pH rate profiles for the reactions of 4-nitrosobiphenyl with three carbonyl substrates in aqueous buffers were determined by use of chromatographic and spectrophotometric methods. Glyoxylate and formaldehyde caused the conversion of 4-nitrosobiphenyl to N(4-biphenyl)formohydroxamic acid, while pyruvate resulted in the production of N-U-biphenyl)acetohydroxamic acid. The dramatic effect of pH on the kinetics of these reactions provided considerable information concerning the nature of these reactions. The reactions with pyruvate and formaldehyde displayed similar pH rate profiles and were significant only a t acidic pH. Glyoxylate displayed a pH rate profile that differed markedly from those of pyruvate and formaldehyde as the pH was increased beyond 2.0. The ability of glyoxylate to convert 4-nitrosobiphenyl to the hydroxamic acid increased rapidly in the pH range 2.0-4.0, above which the pH dependency was constant. This biphasic appearance of the pH rate profile was unique to glyoxylate, since the reactions of pyruvate and formaldehyde became extremely slow as solution neutrality was approached. A second substrate, 4-chloronitrosobenzene, displayed similar pH rate profiles in its reactions with these carbonyl substrates. For 4-nitrosobiphenyl, hydroxamic acid formation by glyoxylate was lo4 times faster than that by pyruvate a t neutral pH, but only about 3-fold faster a t pH 1.0. The appearance of the pH rate profile for glyoxylate suggested that this a-oxo acid reacts with nitrosoarenes a t neutrality via a pathway that is insignificant for pyruvate or formaldehyde. Thus, the nitroso-glyoxylate reaction is unique to this a-oxo acid under physiological pH conditions. Two mechanisms were proposed to distinguish the H+-catalyzed production of hydroxamic acids from the nitrose-glyoxylate reaction. Potential mechanistic variations for the nitroso-glyoxylate reaction were considered in terms of carbonyl hydration, Hammett analyses on existing rate data, and the dichotomy of pyruvate and glyoxylatereactivities.
Introduction In 1980we reported on anew chemical reaction between C-nitrosoaromatic compounds (Ar-NO)' and glyoxylic acid in aqueous solutions, which resulted in the direct production of N-formyl-derived hydroxamic acids (1). This unusual reaction was first detected during our continuing studies of the interaction of thiamine-dependent enzymes with Ar-NO. The conversions of Ar-NO to various hydroxamic acids had been reported earlier as a general reaction of thiamine enzymes with most Ar-NO that had been investigated (2-11). An interesting aspect of the thiamine enzyme production of hydroxamic acids is that novel hydroxamic acid structures are produced by enzymes such as transketolase (4, 11) and a-ketoglutarate dehydrogenase (8-1 01,while pyruvate decarboxylatingenzymes produce N-acetyl-derived hydroxamic acids (3, 6, 7,201. The nature of the N-acyl group in the product hydroxamic acid depends on the basic biochemical function of the enzyme under study (SchemeI). It was during an attempt * Address correspondence to this author at The Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha NE 68198-6805. + Pesticide Research Laboratory.
*
Department of Pharmacology & Therapeutics. Abbreviations: Ar-NO,aromatic nitroso compound; 4-NOB, 4-nitrosobiphenyl; Bi-AHA, N-(4-biphenyl)acetohydroxamicacid; Bi-FHA, N-(4-biphenyl)formohydroxamic acid; 4-CNB, 4-chloronitrosobenzene; Cl-FHA, N-(4-chlorophenyl)formohydroxamicacid; AHA, acetylhydroxamic acid; FHA, formylhydroxamic acid.
Scheme I. Thiamine-Mediated conversion of Ar-NO to Hydroxamic Acids
CH;
- Thlamine no
o
I II N-C-CH2R
@
R
:
H
R : OH
a c e t y l (AHA1 glycolyl
R = CH2CO2H succinyl
R'
to force pyruvate decarboxylase to convert Ar-NO to the .N-formylhydroxamicacids (FHA)that we discoveredwhat is now referred to as the 'nitroso-glyoxylate reaction" ( 2 , 22,13). Since pyruvate decarboxylasewas knowntoeffect the decarboxylationof glyoxylic acid (141,it was reasonable to expect this enzyme to catalyze the production of the FHA accordingto expected thiamine chemistry at enzyme (activesites (i.e., Scheme I). Surprisingly,the conversion of Ar-NO to FHA was found not to require the presence of any enzyme. The reaction appears to be a general property of C-nitrosoaromatic compounds since it occurs in cases where the aromatic ring possesses either a strong electron withdrawing group (13) or strong electron do@ :I993 American Chemical Society
Chem. Res. Toxicol., Vol. 6, No. 1, 1993 83
Nitroso-Clyoxylate Reaction
Scheme 111. Chemical Conversions Investigated in the Present Study
Scheme 11. The Nitroso-Glyoxylate Reaction u+
HO 0
6
on ,o-
0
R
+N-C-C02H
+
OHC-C02H
e
I
HO 0 I
+N=C
@v\H
R
9
'H
COP
glyoxylote
Y
R
Experimental Section Caution: The following chemicals are hazardous (potential carcinogens) and should be handled according to NIH Guidelines: 4-NOB, Bi-AHA, and Bi-FHA. Sodium glyoxylate,sodium pyruvate, HCHO, and NaC104were obtained from Sigma Chemical Co. (St. Louis, MO). 4-CNB and 4-NOB were synthesized according to a previously described method (II),andCl-FHAandBi-FHAwere synthesizedaccording to the title reaction method (I). Determination of Products and Rate Constants by UV. The reactions were carried out in both citrate and phosphate
4
or HCHO
FHA
nating group (15). On the basis of kinetic and isotope labeling studies, we proposed a mechanism (1)to explain this novel reaction (Scheme 11). The mechanism is quite different from the potential mechanisms by which thiamine enzymes effect the conversion of Ar-NO to hydroxamic acids (10). The unique aspect of this mechanism is that the nitroso group acts as a nucleophile (1,16,17). In addition to being a useful synthetic conversion,the nitromglyoxylate reaction provides a convenient method for the detection and trapping of Ar-NO metabolites and for a sensitive assay for glyoxylic acid production in enzymatic reactions (18,19). The potential toxicological significance of the nitrosoglyoxylate reaction should be considered since the reaction occurs rapidly under physiologicalconditions. It is possible that this reaction could compete with the well-known reaction of Ar-NO with organic -SH chemicals (20). The resulting FHA products are much more stable than the precursor nitroso metabolites, yet are reasonably expected to be activated by some of the pathways responsible for the activation of the more common N-acetylhydroxamic acids. On the other hand, some research suggests that N-formyl-derived hydroxamic acids might be activated by unique metabolic pathways or behave in a considerably different manner toward the classical activation pathways known for N-acetylhydroxamic acids (21-23). Such factors could account for differences in target organ by genotoxic arylamine chemicals and cause other differences in the actions of an arylamine entity toward living cells. Much work remains to be done concerning different latent forms of the same chemical toxicant, and how such differences are manifested by a change in the nature of the ultimate toxic lesions. Another laboratory proposed that the nitrom-glyoxylate reaction was a more specific case of the general reaction of Ar-NO with a-oxo acids (24). The reaction of HCHO with Ar-NO was also reported to produce FHA (16). We had previously reported that pyruvic acid and HCHO did not react with Ar-NO in a manner similar to that of glyoxylic acid (I). To clarify this situation, we reinvestigated the reaction of Ar-NO with glyoxylic acid, pyruvic acid, and HCHO. We now report that, under conditions consistent with living cells, Ar-NO compounds react nonenzymatically only with glyoxylic acid a t sufficient rates to produce hydroxamic acids.
II
N-C-H
II
Y=CI
6
Y'C6H5
CI-FHA SI-FHA
Y
Y=CI
4-CNS
Y'CgHg
4-NOS
HO 0 I II N-C-CH3 pyruvate
*
@ YzCI Y'C6H5
Y CI-AHA SI-AHA
buffers for the pH range 1.0-8.0. The citrate buffer (50 mM) consisted of citric acid and disodium citrate for pH values 22.5 rmd citric acid and HCl for pH value below 2.5. Phosphate buffer (10mM), containing 0.5 mM MgClz and 0.137 M NaCl, was used for the pH value 22.5. For the pH values below 2.5, all or part of the NaCl was replaced with HCl to achieve the desired pH. In certain pH studies, 50 mM NaClO, was present. A 2.9-mL aliquot of the appropriate buffer was placed in a cuvette and warmed to 37 OC. The nitroso substrates were dissolved in ethanol at appropriate concentrations so that the addition of 10-pL aliquota to the buffer solution gave concentrations of 13,18, or 5!5 pM 4-NOB and 50,85, or 120 pM 4-CNB. The actual initial concentrations in the buffered solutions were determined using the extinction coefficienta for 4-NOB (c = 13 380 at 360 nm)and 4-CNB (a = 6330 at 340 nm) as determined in the reaction buffer. The solution was then allowed to stand for 10 min at 37 "C to ensure solubilization of the nitroso substrate. The reaction was initiated by the addition of 0.10 mL of an aqueous solution of siodium glyoxylate (to make 0.5,0.75, and 1.0 mM for 4-NOB or 11,2, and 4 mM for 4-CNB), sodium pyruvate (1mM for 4-NOB), or HCHO (100,430, and 890 mM for 4-NOB). The decrease in absorbance was monitored at 360 nm for 4-NOB and 340 nm for 4-CNB using a Beckman Model 35 spectrophotometer equipped with a heated cuvette holder. The reactions were carried out until at least 10% of the nitroso substrate had reacted. This period of time ranged from 10 to 60 min for the glyoxylate and MCHO reactions, and from 1to 72 h for the pyruvate reactions, depending upon concentration and pH. Incubations were also ricanned periodically in the UV region to follow the conversion of the Ar-NO to the hydroxamic acid. In one pH-dependency ntudy, simultaneous analyses by HPLC methods were conducted by removing 20-pL aliquote from the UV cuvette, followed by direct injection of the aliquot into the HPLC system described below. Determination of Products and Rate Constants by HPLC. Solutions of the a-oxo acids were prepared by dissolving 5.7 mg (50 pmol) of sodium glyoxylate monohydrate or 5.5 mg (50 pmol) of sodium pyruvate in 10.0 mL of the appropriate buffer. The pH of the solution was adjusted as necessary using 0.1 M HCl or 0.1 M NaOH. Further dilutions with media were made as needed. The solutions of the a-OXO acid were equilibrated to 37 OC and 1.0-mL portions transferred to 1.0-mL Wheaton septum vials with Teflon-lined septum closures. The nitroso substrate was then added as a solution in ethanol so aa not to exceed 1% of the total reaction volume. The reactions were monitored by HPLC on a Waters Associates CIS-pBondapak column (10 pm, 3.9 mm X 300 mm)at t = 0,15, and 30 min using the solvent 60% methanol buffered at pH 3.5 with 0.05 M KHzPOJHsPO4 and containing 0.01 % desferal mesylate for 4-NOB. Aliquota of 50 pL each were directly injected into the HPLC system at the appropriate reaction time. The reactions were monitored for up
84
Chem. Res. Toxicol., Vol. 6, No. 1, 1993
Corbett and Corbett
Table I. Second-OrderRate Constants (ksi SD for n = 3) As Determined by HPLC for the Reaction of 4-Nitrosobiphenyl with Carbonyl Compounds at Selected pH at 37 O C substrate
DH7.4
DH1.0 ~~
glyoxylate pyruvate
0.19 i 0.02 O.ooOo20 i 0.000005
07r
~
~
0.091 i 0.003 0.0226 f 0.0012
to 24 h in certain cases. Producta were identified by cochromatography with authentic standards, coupled with the observation of identical UV spectra as determined by use of a diode array spectrophotometric detector.
Results Products from the nitroso-glyoxylate reaction had been previously characterized as N-formyl-derived hydroxamic acids (FHA) with C02 originating from the glyoxylate carboxyl group (I). Bi-FHA produced from 4-NOB was characterized by similar chromatographic methods, colorimetric analysis, and mass spectral analyses. Bi-FHA and Bi-AHA have been previously reported in the chemical literature (26). The conversions of the two Ar-NO substrates to hydroxamic acids by the three carbonyl substrates are illustrated in Scheme 111. In this study, our initial kinetic investigations employed HPLC analyses and first demonstrated a large difference in the rates of reactions of two Ar-NO substrates with glyoxylate and pyruvate. The reactions with pyruvate a t near-neutral pH were too slow to monitor, and only at a more acidic pH did the pyruvate reaction proceed at a discernible rate (Table I). Because a more detailed pH study was desirable, our analytical method for determining the reaction kinetics was modified to employ spectrophotometry. HPLC analyses were done concurrently with certain of these studies in order to determine product composition. The n ?r* absorption bands of the nitroso functional group are centered at 319 and 350 nm for 4-CNB and 4-NOB, respectively, in aqueous solutions and provide a method to follow the reaction kinetics. Our computations of rate constants by spectral methods involved the measurement of the nitroso concentration as a function of time. The values for c at the Amax of the Ar-NO were 10520 and 13 750 for 4-CNB and 4-NOB, respectively. The quantitative conversion of Ar-NO to the appropriate hydroxamic acid was assumed in employing the second-order rate equation, k2 = W(Ao - Bo) X In (ABo/AoB)l/s, where k2 is the second-order rate constant, A0 is the initial concentration of the carbonyl substrate, and Bo is the initial concentration of the nitroso substrate determined for each experiment from e. An excess of glyoxylate, pyruvate, and especially HCHO was employed since the solubility in aqueous buffer of the Ar-NO was a limiting factor (1mM and 50 r M for 4-CNB and 4-NOB, respectively). Even though the experimental conditions were under pseudofirst-order conditions, our data are expressed as the calculated second-order rate constants, k2, because these are more useful for comparison of the reactivities of the carbonyl substrates toward 4-NOB. HPLC analysis was employed to detect any deviation in the quantitative conversion of Ar-NO to hydroxamic acids during the time span over which a spectral measurement was made. The deviations from quantitative conversions of Ar-NO to hydroxamic acids occurred primarily with pyruvate and HCHO, and this effect probably gave slightly higher computed rate constants for the pyruvate and HCHO
-
250
300
350
250
400
X
300
350
400
(nm)
Figure 1. UV spectral analyses of the reaction of 4-NOB with glyoxylate. The reactions were conducted at 37 O C with 40 pM 4-NOB and 1 mM glyoxylate and were kept out of the UV light path during times between scans. Panel a shows the UV scans of the reaction at pH 1.0, and panel b shows the W scans of the reaction at pH 7.4, where t = 0 (curve l ) , 15 min (curve 2), 30 min (curve3), 60min (curve4), 120 min (curve51,180 min (curve 6), 240 min (curve 7), and 360 min (curve 8). T h e dashed lime (- - -) in panel a is the UV spectrum of authentic Bi-FHA.
reactions than might be expected at certain pH values. HPLC studies were consistent with this point. Nitro and azoxy aromatics were detected by HPLC as significant products after several days under neutral conditions in which hydroxamic acid production was very slow for pyruvate and HCHO. The UV spectral scans of the reaction of 4-NOB with glyoxylate a t pH 1.0 and 7.4 are presented in Figure 1.At pH 7.4, the UV patterns changed with time from that of 4-NOB to one superimposable with authentic Bi-FHA, with an isosbestic point at 308 nm. At pH 1.0, the 4-NOB was consumed over the course of several hours, but the UV spectra became distorted due to the acid instability of Bi-FHA. However, the reaction rates were computed on the basis of measurements when only about 10% of the nitroso substrate had been consumed, and HPLC showed no significant product decomposition for this short reaction time. The acid decomposition of Bi-FHA was shown by HPLC to produce several products (Figure 21, with a single major product as the terminal product (retention time of 12 min). Under the same conditions, 4-nitrobiphenyl gave a retention time of -14 min. The same general pattern of products was observed as was seen for the glyoxylate reaction with 4-NOB at pH 1.0. Although the kinetics of the acid decomposition of Bi-FHA were not quantitatively examined, the acid instability was negligible (at 4 h) above pH 3.0. Analogous UV scans, but over necessarily longer time periods, were obtained for pyruvate and HCHO; however, it was not possible to detect the near-quantitative conversion of 4-NOB to the hydroxamic acid by either pyruvate or HCHO at any pH. Figure 3 illustrates the W scans for the pyruvate reaction at pH 1.0, superimposed with a scan of Bi-AHA. Even at this favorable pH for the pyruvate reaction, the quantitative conversion of the nitroso substrate to Bi-AHA was not observed due to subsequent acid decomposition of the product. Over shorter time periods (10% substrate consumption), thia instability of Bi-AHA did not prevent accurate memure-
-
Chem. Res. Toricol., Vol. 6, No. 1, 1993 85
Nitroso-Glyorylate Reaction 6.0- I
0.25r
v)
67
0.20
It I1
-r
//
I
0
4.0-
X v
W 0
c
O
n L 0
:2 0 v)
PH
0
IO
20
Time ( m i n ) Figure 2. HPLC analysis of the reaction between 4-NOB and glyoxylate at pH 1.0. A 50-pL aliquot of the reaction of 40 pM 4-NOB and 1mM glyoxylate at pH 1.0 and 37 OC was removed at t = 5 hand injected into the HPLC system, which is described in the Experimental Section. The detector wavelength was 254 nm with a solvent flow of 1.5 mL/min. In this chromatogram, 4-NOBelutes at -16 min and Bi-FHA as the large peak at -6 min. 0.4
' \
I
0.3
al 0
E 0.2
2 v)
n
a
0.1
I
0 250
300
X
,
,
350
,
!
400
(nm)
Figure 3. UV spectral analyses of the reaction of 4-NOB with pyruvate. The reactions were conducted at 37 O C with 28 p M 4-NOBand 1 mM pyruvate at pH 1.0 and were kept out of the UV light path during times between scans. This figure shows the UV scans of the reaction at t = 0 (curve I), 1 h (curve 21, 2 h (curve 3),3 h (curve 4), 4 h (curve 5 ) , 5 h (curve 6), 24 h (curve 7), and 48 h (curve 8). The dashed line (- - -) is the UV curve of authentic Bi-AHA.
menta of the reaction kinetics by spectrophotometry. Although the rate of acid decomposition of Bi-AHA was slower than that for Bi-FHA, it appears from HPLC studies
Figure 4. pH rate profiles for the reaction of 4-NOB with glyoxylate. Plots of kz vs pH for ( 0 )the reaction in phosphate buffer (pH 0.7-7.4) and (X) the reaction in citrate buffer (pH 1.0-7.4). Bars indicate ASD for n = 5 and are shown only for phosphate buffer. Similar variability was observed for citrate buffer (n = 3).
that the same terminal product was formed from both hydroxamic acids. The chemical structure of this decomposition product is being elucidated. In earlier experiments we employed KH2P04 buffer (0.05 M) over the pH range of 3.0-9.0,for which we reported a weak pH dependency with a maximum at about 6.0 (I). In the present study, we reexamined this pH effect, particularly at more acidic values. As shown in Figure 4, the nitroso-glyoxylate reaction with 4-NOB displayed a similar pH effect as reported previously, but at pH values more acidic than 4.0, the kinetics of the reaction decreased markedly to a minimum in the 1.5-2.0 region, followed by an abrupt increase at lower pH. A nearly identical pH profile, but with rates approximately one-fourth those for 4-NOB, was observed in the case of 4-CNB in ita reaction with glyoxylate (data not shown). The reactions with glyoxylate were observed to be first order in both this carbonyl substrate and 4-NOB over the pH range of 1.08.0. All second-order rate constants (k2 for -d(Ar-NO)/dt = k2[glyoxylatel [Ar-NO]) are expressed under defined [H+l; thus, the rate equation does not include a term for acidity. Similarly, the rate constants, k2, for the analogous reactions with pyruvate and HCHO are expressed under defined [H+l in this paper. This approach readily allowed for a direct comparison of the second-order rate constants for the reactions of Ar-NOwith the three different carbonyl substrates. That ionic effects on the reaction rates were not significant was established by the inclusion of 50 mM NaClO4 in certain studies over the pH range (data not shown). Our interest in the physiological role of the nitrosoglyoxylate reaction prompted our particular emphasis on the absolute rate constants at pH 7.4, which are identical within experimental error to those observed at pH 6.0. In the physiological phosphate buffer (designed after Dulbecco's phosphate-buffered saline media) at pH 7.4, the rate constants at 37 OC for the glyoxylate reaction with 4-NOB and 4-CNB were 0.25 f 0.03 M-' s-l (n = 58) and 0.070 f 0.006M-ls-l (n= 19),respectively. The Hammett reaction constant, p o = -1.7, was3 calculated (27) from the data in our original study of eight nitroso substrates (1).
Corbett and Corbett
86 Chem. Res. Toxicol., Vol. 6, No. 1, 1993 0.25l-
I
A
0.20
N Y
0.0 O " O5:
01
v
u '
10
20
30
40
X Y
-
-* I
-5
1.0-
I
N
x
50
60
7.0
:
1
1.0
PH
Figure 5. pH rate profiles for the reaction of 4-NOBwith glyoxylate in citrate buffer as determined by UV and HPLC. The plots of kp vs pH are from a single experiment with simultaneous analysis by UV ( 0 )and by HPLC (X).
-
2.0
3.0
4.0
5.0
6.0
7.0
PH
Figure 6. pH rate profiles for the reactions of 4-NOBwith pyruvate and HCHO. The reactions were conducted in phosphate buffer at 37 O C . Plots of kz vs pH for (x) pyruvate and ( 0 ) HCHO.
In that study, the kinetics for 4-CNB were determined by HPLC methods, and the kz (pH 6.0 at 30 "C) for 4-CNB was reported to be 0.034 M-' 5-l. On the basis of the Hammett relationship in our first report ( l )calculations , for the expected rate of reaction of 4-NOB (employinguop = 0.05) predict kz = 0.22 M-l s-l under the conditions where T = 37 OC. Thus, the current results, which are based largely on spectrophotometric methods to determine the reactions kinetics, are consistent with the results of our initial report. The pH profile for the glyoxylate reaction with 4-NOB in citrate buffer (Figure 4) was the same as seen in phosphate buffer. HPLC analyses were conducted simultaneously with spectrophotometric measurements to determine if the nature of the initial reaction products changed over the pH range. No such product changes were detected, and the reaction kinetics as determined by simultaneous HPLC analyses were nearly identical to values obtained by spectral methods (Figure 5). The rate constants from HPLC data were calculated from measurements of Bi-FHA production. Under the short incubation times in this study ( 2.0. The pK:s of hydrated and dehydrated pyruvic acid are 3.7 and 2.2, respectively (30).The presence of the pyruvate methyl group must place severe constraints upon this pathway since glyoxylate reacts lo4times faster (pH 7.4) than pyruvate. In contrast, the H+-catalyzedreaction at pH 1.0is only 3 times faster for glyoxylate than pyruvate. An effect greater than lo3 on the relative rates of these closely related a-oxo acids in the two parallel pathways was surprising. With such large differences in rate constants between glyoxylate and pyruvate, the nitroso-glyoxylate reaction, as originally reported in a broad physiological pH range (11, is essentially unique to glyoxylate. Hydroxamicacids from the reactions of the other carbonyls probably arise only as products of the H+-catalyzedpathway. The pH rate profiles for glyoxylate (Figures 4 and 5) can be viewed as the composite of these two pathways since glyoxylate produces hydroxamic acids via both pathways. In keeping with the original intent (1, lo), we believe the term ‘nitroso-glyoxylate” reaction should be applied only to describe the pathway for hydroxamic acid formation that occurs significantly at neutral pH. A generalization of the mechanism in Scheme I1 to include other a-oxo acids provides a reaction mechanism for the H+-catalyzedpathway to hydroxamic acids. The obvious driving force for the reaction is decarboxylation. Kronja and co-workers proposed a similar mechanism to explain H+-catalyzed hydroxamic acid formation in the case of HCHO (16). In this case, loss of H+ rather than COz was invoked to explain FHA production. Our results are consistent with their proposal, which also provides an explanation as to the relatively low rate of reaction of HCHO with Ar-NO even under H+ conditions (Figure 6). The report that pyruvate and other a-oxo acids converted Ar-NO to various hydroxamic acids (24) seemed to contradict our earlier observations, until the necessity for H+ catalysis was revealed. In that study, a mechanism was proposed which included a cyclic reaction intermediate (Scheme IV). Neither of these closely related mechanism (Schemes I1 and IV) provides an obvious explanation for the unique behavior of glyoxylate at neutral pH, and the cyclic mechanism (Scheme IV) is especially untenable since the acid moiety will be ionized. An explanation for the existerxe of two pathways for hydroxamic acid production, one of which is unique to glyoxylate, must reside in a physicochemical difference between pyruvate and glyoxylate. The methyl group of pyruvate eliminates the ability of this a-oxo acid to react
Corbett and Corbett
88 Chem. Res. Toxicol., Vol. 6, No. 1, 1993
Scheme V. Proposed Mechanism for the Nitroso-Glyoxylate Reaction: Nucleophilic Attack of Hydrated a-Oxo Acids on Ar-NO
Scheme VI. Proposed Alternate Mechanism for the Nitroso-Glyoxylate Reaction: Nucleophilic Attack of the Nitroso Group on the Carbonyl Group of a-Oxo Acids
I
Ar
R
CO;
Ar
Ar
I Ar
R'
'CO,
I Ar
I R
--' -
/
Ar
-\
co,
0, - o; \ +N=C
a/, significantly via the nitroso-glyoxylate pathway. A possible explanation of this phenomenon resides with the marked difference between carbonyl hydration of these substrates. The rates of hydration and dehydration of these substrates are fast and are highly dependent upon general acid/base catalysis (29, 30). The &(hydrate/ carbonyl) are higher for the free acid forms of glyoxylic acid [k, = 300 (2913 and pyruvic acid [k,, = 1.6 (3011than for the ionized forms of glyoxylate [k, = 15 (29)l and pyruvate [k, = 0.064 (30)l. The k, for hydration of HCHO under mild acid conditions is 2000 (29). No general correlation is apparent between the degree of hydration and the rates of reactions of glyoxylate, pyruvate, and HCHO with 4-NOB. The glyoxylicacid/glyoxylatesystem displays both the greatest hydration and reactivity toward 4-NOB throughout the pH range 1.0-8.0, but in this system, the rate of reaction with 4-NOB actually increases in going from a high degree of hydration (k, = 300) to a 20-fold lower degree of hydration (k, = 15). The opposite effect is seen for the case of the pyruvic acid/pyruvate system, for which virtually no reactivity toward 4-NOB occurs under conditions that favor maximal free carbonyl group. At pH 1 4.0, the hydration of glyoxylate is more than 200 times greater than that of pyruvate. It is this difference which suggests an important role of the hydrate in the carboxylate-dependent nitroso-glyoxylate reaction and, thus, an explanation for the unique ability of glyoxylate to react with Ar-NO at neutral pH. We originally proposed a mechanistic variation by which hydrated glyoxylate might react with Ar-NO to produce FHA (1). An attractive feature of a mechanism requiring hydrated glyoxylate is that it would explain our earlier observation that the reaction appears to be unique to aqueous solvents in addition to being restricted to glyoxylate (I). In contrast, the H+-catalyzedpathway appears not to be restricted to aqueous solvents (24),which further suggests a major difference in reaction mechanism. Our present data on glyoxylate are consistent with the mechanism presented in Scheme V, while the negative pyruvate results can also be explained on the basis of this mechanism. Ionized glyoxylate is still largely hydrated but pyruvate is not; thus, the mechanism in Scheme V might be very slow for pyruvate because the intermediate l b would be formed only in trace amounts relative to the analogousintermediate la from glyoxylate. The difference in equilibria for the formation of la and -b is probably even greater between the two a-oxo acids than a difference in relative hydration of 200 would predict, because steric effects due to the methyl group of pyruvate might further
R'
destabilize such an intermediate. The pyruvate methyl might slow or prevent the subsequent intramolecular displacement which gives rise to the intermediate 2. Formation of the hydroxamic acid resonant, 3, via decarboxylation of 2 (Scheme V) would be proportionately slower in the case of pyruvate because the equilibrium concentration of 2b is likely to be much lower than that of 2a formed from glyoxylate. The intermediate, 2, is an oxaziridineN-oxide which has been proposed as a reactive intermediate in related reactions that approximate the reverse of the mechanism in question. These reactions include the peracid oxidation of imines and oxaziranes to yield nitroso and carbonyl products (31,32). That the nitroso-glyoxylate reaction might proceed via an initial nucleophilic attack of the glyoxylate hydrate on the N-position of the nitroso ?r-bond (Scheme V) is consistent with the known electronic effects of aromatic ring substituents on the kinetics of these reactions (1). Our initial preference for a mechanism (Scheme VI) involving nucleophilic attack of the nonbonding electron pair localized at the nitroso N-position on the carbonyl carbon to produce intermediate 4 is also consistent with this electronic effect (1). It led us to propose a nucleophilic role for the nitroso functional group, which is unusual since most reactions of the nitroso group are viewed as it serving the role of an electrophile to various nucleophiles (33). An interesting aspect of the hydrate pathway (Scheme V) is that the reaction would be expected to display the "apparent nucleophilicity" of the nitroso N in the form of real nucleophilicity by the hydroxylaminelike intermediate 1. Hydroxylamines are enhanced nucleophiles due to the a-effect of the N-0 bond (11,34). Thus, the transition from 1 to 2 might be a favorable process, especially when coupled with the irreversible decarboxylation of 2 to produce 3, which has the kinetic effect of shifting the equilibrium of 1t;2 to the right. It is not obvious as to why this pathway is not favorable at lower pH, because even a higher degree of hydration occurs for un-ionized glyoxylic acid, and this could be expected to facilitate the formation of la (protonated form). Possible explanations includethe ability of the carboxylate anion to stabilize a mechanistic intermediate (vide infra) or that decarboxylation occurs more readily through the ionized form rather than free acid or H-bonded free acid. The formation of an intermediate such as 2 or 4 is a probable necessity in the nitroso-glyoxylatereaction, since
Chem. Res. Toxicol., Vol. 6, No. 1, 1993 89
Nitroso-Glyoxylate Reaction
Scheme VII. Potential Role of the Carboxylate Anion in the Alternate Mechanism for the Nitroso-GlyoxyIate Reaction
Ar R
4
' 0 /C\o /
c
.-H..
0'
\
-
+N=C I
"0
/
\
R
Ar
3
decarboxylation of either 2 or 4 results in 3, which is an acknowledged resonant structure for hydroxamic acids (35). Intermediates 2 and 4 are tautomeric structures and, thus, illustrate a high degree of similarity between the two mechanistic variants that we have proposed for this reaction (Schemes V and VI). Nevertheless, these mechanisms differ radically with respect to the nature of the reactive form of glyoxylate that interacts with the nitroso functional group. The hydrate pathway 1 2 3 is a more likely explanation for the unique role of glyoxylate as a substrate in this reaction than is the more direct 4 3 pathway, which we originally proposed. The hydrate pathway (Scheme V) is attractive since it can account for at least a 200-fold difference in rates in the first step alone of a possible mechanism (i.e., equilibrium formation of 1). It is then only necessary to postulate an approximate 10-fold difference for two subsequent steps in the reaction (i.e., 1 2 and 2 3) to account for the overall difference in reactivities between glyoxylate and pyruvate. On the other hand, we cannot exclude the mechanism depicted in Scheme VI. It is possible that the steric crowding introduced by the CH3 group in pyruvate could account fully for such a large difference in reaction rates and that the correlation with hydration is coincidental. Steric hindrance in an intermediate such as 4 is not expected to be severe, which could explain the fact that, in the H+-catalyzed reaction, glyoxylate is only about 3 times more reactive than pyruvate. While the difference of 104between glyoxylate and pyruvate seems difficult to reconcile by the simple sequence of AI-NO 4 3, it is possible that certain required transitional intermediates between 4 and 3 might be extremely susceptible to steric crowding. Some possibilities for such intermediates are presented in Scheme VII. The charge stabilization suggested by 5 is consistent with the required ionized form of the acid in the reaction. It is difficult to estimate the effects of the replacement of H with CH3 for the R group in this intermediate; however, when extrapolated to the fused bicyclic intermediate, 6, the potential for severe steric crowding becomes more evident since the Ar and R groups must be in a cis relationship. If a transition state similar to 6 is required, then a reaction mechanism independent of the hydrated acid might yet be reasonable for the nitroso-glyoxylate reaction. An experimental approach to distinguish between these mechanistic variants has yet to be designed. Further evidence for the distinct mechanistic differences between the H+-catalyzed reaction and the nitroso-
--
-
--
-
-
glyoxylate reaction is suggested by the results of Hammett analyses for these pathways. Uematsu's group reported an excellent correlation between Brown's u+ conatants and the rates of reaction of four Ar-NO substrates with pyruvic and glyoxylic acids under acidic conditions (24). We had earlier reported a good correlation between Hammett's u constants and the rates of reaction of eight Ar-NO with glyoxylic acid at pH 6.0 (I). An even better correlation was subsequently found when we employed uo constante (27) in this analysis. Most noteworthy ie our current observation that the Hammett analysis of the data from our initial report does not yield a good correlation with u+ constants. The resonance stabilization (Scheme IV) proposed by Uematsu's group (24) would not be expected to provide stabilization of the intermediates 1, 2, or 6 proposed in Schemes V-VII, yet such resonance stabilization is reasonable for 4 and especially for the cyclic 6-memberedintermediate (SchemeIV). Thus, it appears that resonance stabilization by the aromatic substituent of a positive charge in a transitional intermediate is of no importance in the nitroso-glyoxylate reaction. Nevertheless, increased electron density in the aromatic ring and at the nitroso N does favor the reaction as evidenced by po = -1.7 for the nitroso-glyoxylate reaction at pH 6.0. These observations are consistent with both of the mechanistic variants that we have proposed for the nitrose glyoxylate reaction (Schemes V and VI-VII). Under conditions of physiological pH, the reactions of Ar-NO are kinetically significant only for glyoxylate. We feel confident in concluding that the chemical reactions between Ar-NO and pyruvic acid or higher a-oxo acids are vanishingly slow and that they are irrelevant to hydroxamic acid production in living organisms. Any direct metabolic conversion of such a-oxo acids to hydroxamic acids requirea enzymatic action according to the thiamine cofactor chemistry (Scheme I). The exception is glyoxylate which may be sufficiently reactive with many Ar-NO substrates to effect their conversion to FHA within cellular systems via a purely chemical reaction. The chemical reaction of Ar-NO with sulfhydryl groups, particularly reduced glutathione, is even more facile on the basis of a comparison of rate constants under similar conditions. The rapid, reversible reaction of glutathione with nitrosobenzene produces an unstable semimercaptal with a second-order rate constant reported to be 5.5 X lo3 M-' s-l(20). This general reaction displays a dependency on electronic factors that is opposite from that of the nitroeo-glyoxylate reaction, which suggests that the nitroso group behaves as an electrophile in this reaction. The Hammett reaction constant has been reported to be +2.1(20) and +1.9 (36) for semimercaptal formation, and the value of p = +3.2 was reported for the equilibrium formation of this initial adduct (36). Subsequent reactions of these semimercaptala are affected differentially by ring substituents (20, 36). Reduced glutathione probably plays a major role in maintaining very low concentrations of free Ar-NO under many biochemical conditions, although certain of the products that can arise from the semimercaptal are likely to be electrophilic reactants and possible mediators of toxicity (36). Under situations of depleted glutathione sulfhydryls, it would be expected that the nitroso-glyoxylate reaction may become metabolically important to the overall fate of Ar-NO. The availability of glyoxylate in various cellular and extracellular environments would then become the
90
limiting factor for FHA production. The most obvious of such environmenta are the peroxisomes, which are thought to be present as microbodies in nearly all eucaryotic cells, although their functions and enzyme contents are highly variable among species and tissue types (37,381. Common features of peroxisomes include the production and utilization of both HzOz and glyoxylate with catalase as a marker enzyme. Glyoxylate concentrations in various peroxisomes are not known, but likely range from 0.1 to 1.0 mM, which suggests that glyoxylate in cells tends to be concentrated in these subcellular components (37,381. Because of their highly oxidized nature, free sulfhydryl groups are probably minimal in these subcellular organelles and glutathione is not known to be present. This same prooxidant situation would also favor the nitroso oxidation state in the arylhydroxylamine =F Ar-NO equilibrium (39). Research on the Occurrence of the nitroso-glyoxylate reaction in peroxisomal systems is being conducted.
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