655
THEREVERSIBLE HYDRATION O F 2- AND 4-PYRIDINECARBOXALDEHYDES
The Reversible Hydration of 2- and 4-Pyridinecarboxaldehydes. 11. General Acid-Base and Metal Ion Catalysis’ by Y. Pocker2and J. E. Meany Department of Chemistry, University of Washington, Seattle, Washington 08106
(Received August 8, 1967)
The reversible hydrations of both 2- and 4-pyridinecarboxaldehyde have been found to exhibit general acid and general base catalysis. The catalytic rate coefficients associated with the acid-base components of acetic acid, phosphate, and veronal buffers have been deduced a t 0.0” a t an ionic strength of 0.1, employing a spectrophotometric method. The use of these buffers also allowed the determination of the respective catalytic rate coefficients for water, hydroxide ions, and hydronium ions. The catalysis of the hydration of 4-pyridinecarboxaldehyde exceeds that of the corresponding 2-pyridinecarboxaldehyde reaction by a factor of about 2 for the bases studied, and by a factor of about 8 for the acids. Investigations have also been carried out which pertain to the metal ion catalyzed hydrations. In contrast to catalysis by general acids and bases, metal ion catalysis was found to be distinctly selective, promoting almost exclusively the reversible hydration of 2-pyridinecarboxaldehyde. For the reaction, the magnitude of metal ion catalysis follows the order Cu2+> Go2+ > Ni2+ > Zn2+> Cd2+> Mn2+> Mg2+‘v Ca2+, an order which may be dictated, a t least in part, by the ability of the metal ion to coordinate with 2-pyridinecarboxaldehyde and form a transient complex in which attack by water on the carbonyl carbon is facilitated in the hydration process. The formation of a chelate between metal ion and conjugate hydrate of 2-pyridinecarboxaldehyde similarly facilitates the reverse process. The corresponding metal ion catalysis of the reversible hydration of 4-pyridinecarboxaldehyde was found to be small in comparison.
Introduction I n earlier publications from this laboratory, we have presented evidence showing that the hydrations of both 2- and 4-pyridinecarboxaldehyde are strongly catalyzed by the zinc metalloenzyme, erythrocyte carbonic a n h y d r a ~ e . ~ We ~ ~have further shown that, in contrast to the metalloenzyme, divalent zinc and cobalt3 ions are powerful catalysts only in the 2-pyridinecarboxaldehyde hydration. Our earlier measurements were carried out in dilute buffered solutions in order to minimize the contribution of the buffer components to the over-all rate. It is the purpose of the present paper to examine in some detail the actual magnitude of the catalysis afforded to the hydrations of both 2- and 4-pyridinecarboxaldehyde by several general acids and bases as well as by water, hydroxide, and hydronium ions. Since no previous kinetic work on the hydration of either of these substrates has been described, these measurements provide a body of information which allows comparisons to be made between 2- and 4-pyridinecarboxaldehyde with respect to the magnitude of their “spontaneous” rates of hydration, catalysis by general acids and bases, as well as to their susceptibility to enzymatic and metal ion catalysis.
We also report in this paper the results from an extended study on the catalysis of the hydration of 2pyridinecarboxaldehyde by several metal ions. As will be shown later in this paper, the catalytic efficiency of these various ions appears to parallel their ability to promote certain hydrolytic r e a ~ t i o n s ~and ~ ~ to b be very similar to their capacity to coordinate with certain bidentate ligand~.~CWe further show that, in contrast to the efficiency of the metal ion catalysis of 2-pyridinecarboxaldehyde hydration, where the metal ion can bind to the nitrogen and at the same time remain close to the aldehydic group, the corresponding catalysis of the 4-pyridinecarboxaldehyde reaction is very small.
(1) Support of this work by the National Institutes of Health of the Public Health Service is gratefully acknowledged. (2) Author to whom correspondence should be addressed. (3) Y . Pocker and J. E. Meany, J. Am. Chem. SOC.,89, 631 (1967). (4) Y. Pocker and J. E. Meany, Biochemistry, 6 , 239 (1967). (6) (a) H. Kroll, J . Am. Chem. Soc., 74, 2036 (1962); M. L. Bender and B . H. Turnquest, ibid., 79, 1809 (1967); W. L. Koltun, M. Fried, and F. R. N. Gurd, ibid., 82, 233 (1960); (b) L. Meriwether and F . H. Westheimer, ibid., 85, 3039 (1963); M. D. Alexander and D. H. Busch, ibid., 88, 1130 (1966); (c) F. L. Eplattenier, I. Murase, and A. E. Martell, ibid., 89, 837 (1967).
Volume 79,Number 8 February 1068
656
Y. POCKERAND J. E. MEANY
Experimental Section The purification of 2- and 4-pyridinecarboxaldehyde, of diethylmalonic acid, and of acetonitrile has been described earlier.3 With the exception of diethylmalonate,G the buffer solutions employed in this investigation were prepared from commercially available compounds, analytical or reagent grade or of comparable purity. The various metal salts were obtained, in reagent grade, from Baker and Adamson either as nitrates or chlorides. In the study of general-acid general base catalysis, absorbancies were monitored at 305 mp ([Zpyridinecarboxaldehyde] = 4.91 X 10-3M) and at 320 mp ([4-pyridinecarboxaldehyde] = 4.96 X lo-* M ) . For the determination of metal ion catalysis, the diminution in absorbancy was monitored at 278 mp for both aldehydes, since much lower substrate concentrations ( [2-pyridinecarboxyaldehyde] = 1.13 X M ; [4-pyridinecarboxaldehyde] = 6.94 X lop6 M ) were employed. I n these latter studies, diethylmalonate buffers were used to control pH, because the respective acid-base components, HA- and A2-, contribute negligibly to the over-all rates of hydration. The apparatus, procedures, and the method for deducing pseudo-first-order constants were described in an earlier study.a Results Since the hydrations of 2- and 4-pyridinecarboxaldehyde are reversible, the experimental rate coefficient for equilibration, hob& is actually the sum of forwardand reverseratecoefficients, kobsd = kf k,. Recent quantitative measurements by Pocker, Meany, and Nist7 show the variation of the fractions of hydration, x, of 2- and 4-pyridinecarboxaldehyde with pH. The fraction of hydration of 2-pyridinecarboxaldehyde is 0.49 in the region 6-10, and that of 4-pyridinecarboxyaldehyde is 0.67 in the region 7-10. I n the above regions of pH, the products of hydration are believed t o be zwitterionic (I).S
+
HI
111 I1
I
For both aldehydes, a decrease in pH below 6 (2-pyridinecarboxaldehyde) or below 7 (4-pyridinecarboxaldehyde) causes an increase in the fraction of hydration7-g in which the hydrate is now distributed between forms I and 11, form I1 becoming more dominant with decreasing pH. It should be noted that even in acetate buffers, the concentrations of the conjugate acids of both 2- and 4-pyridinecarboxaldehydes are negligibly small. This arises because the pK,'s of The Journal of Physical Chemistry
the pyridine aldehydes are significantly lower than those of their respective hydrates. As the pH increases beyond 10, the fractions of hydration again increase for 2- and 4-pyridinecarboxThe hydrates are now partitioned between forms I and 111, form 111 becoming more dominant with increasing pH. The forward rate coefficients, kt = Xkobsd, for each kinetic run were evaluated using the fraction of hydration corresponding t o the actual pH of the solution being studied (Table I). The hydrations of 2- and 4pyridinecarboxaldehyde are general acid-general base catalyzed and the over-all pseudo-first-order rate constants, kf, contain catalytic contributions from the various acids and bases present in the reaction solution, eq 1
kf = ko
+ JGH~o+
[H30+]
+
ko~-[OH-l f kA[Al
+ kB[Bl
(1)
Table I: Fractions of Hydration, x , of 2- and 4Pyridinecerboxaldehyde at Various Values of pH Buffer
Acetate
Phosphate
Verona1
P H Z - P A ~ ~ ~ X2-PAa
4.28 4.68 5.26 5.61 5.65 6.94 7.44 7.68 7.80 8.25 8.59 8.86
0.68 0.59 0.52 0.50 0.50 0.49 0.49 0.49 0.49 0.49 0.49 0.49
PH4-PAaPb
%&PAa
4.38 4.73 5.32 5.58 5.76 6.95 7.42 7.67 7.82 8.25 8.58 8.87
0.92 0.88 0.78 0.73 0.72 0.67 0.67 0.67 0.67 0.67 0.67 0.67
a Refers to 0.0'. The fractions of hydration of 2- and 4pyridinecarboxaldehyde at much higher and much lower values of pH have been reported earlier.?
I n earlier publication^,^^^ we did not convert the experimentally determined values of the activities of hydronium and hydroxide ions into their respective concentration and have reported values of ~ ' H ~ o += k ~ ~ o + / f and & OH- = k O H - / f f . In the present paper, we followed Bell and Darwente and have deduced the constants, k H a 0 + and OH-, from eq 2 ksolv =
ko
kHaO+(aH80+/ff)
+ k O H -(Kw/aIIaO
+ff) (2)
(6) Y. Pocker and J. E. Meany, Biochemistry, 4, 2535 (1965). (7) Y. Pocker, J. E. Meany, and B. J. Nist, J. Phys. Chem., in press. (8) (a) K. Nakamoto and H. E. Martell, J . Am. Chem. Soc., 81, 5857 (1959); (b) 8. Cabani and P . Cecchi, Ann. Chim. (Rome), 44, 205 (1959). (9) R. P. Bell and P. G . Evans, Proc. Roy. SOC.(London), A291, 297 (1966).
THEREVERSIBLE HYDRATION OB 2- AND
657
4-PYRIDINECARBOXALDEHYDES
Table 11: Catalytic Rate Coefficients for Acids and Bases in the Hydration of 2- and 4-Pyridinecarboxaldehydes a t 0.0"" krZ-PA,
4
3
[ H30C]
2
I
O
4
2
X I O 5 ( m o l e 1:)'
[OH-]
6
8
10
12
X IO7 ( m o l e I - ' ]
Figure 1. Catalysis of the hydrations of 2- and 4-pyridinecarboxaldehydeby HzO, HaO+, and OH- at 0.0": for 2-pyridinecarboxaldehyde hydration, ko = 0.25 min. -1; for 4-pyridinecarboxaldehyde hydration, ko = 0.50 min. -1; upper curves in the acidic and basic regions refer to 4-pyridinecarboxaldehydehydration; lower curves refer to 2-pyridinecarboxyaldehyde hydration; AJ acetate buffers; 0, phosphate buffers; and 0, veronal buffers.
where the mean ion activity coefficient, f f, was calculated from expression 3, where I and z stand for the ionic strength and charge, respectively logf, = -0.49z21"'/(1
4- 1.51"')
= ko
Speoies
PKS
min-1
min-1
HzO OHVeronal H + Veronal sodium HzPOdHP04'HOAc" OAcHaO+
16.69 8.27
0.25"55.5 1 800,000 d
7.12
4 d
0.50b/55.5 4,700,000 d 8
d 52 11 3.8 84,000
24 1.4 1.7 11,000
4.78 -1.74
a Sodium chloride was used in this work to maintain a constant ionic strength of 0.1. The numerator refers to ko min-l. As ~ k0/55.5 O 1. mole-' min-1. ' It should be noted indicates, ~ H = that the fraction of hydration, x, increases with decreasing p H in the region of p H in which these constants were determined. The rate coefficients associated with these catalytic species for the forward process were evaluated using the fraction of hydration corresponding to the actual p H of the solution being studied Too small to detect. (see Table I).
[ M2*]
1
6-
kcoz+
:33,000
I. rnole-'rnin~'
k N i z + = 31,000 i. mola-'rnin:l kZnz+
0'
4
S
[ M2+]
+
These hydrations, if carried out with high substrate concentrations, give decreasing first-order rate coefficients, because the product of hydration, 2-pyridinecarboxaldehyde hydrate, appears to be a better chelating agent for the metal ion than the reactant or the buffer. This effect is particularly prominent with Cu2+ where a 44-fold increase in the concentration of 2-pyridinecarboxaldehyde (from 1.13 X M to M ) gave a ca. 8-fold decrease in the Cu2+ 4.91 X catalysis, as measured around 30% reaction. Consequently, in the present work, we purposely chose very low substrate concentrations (1.13 X M ) such
X IO6 ( mole I.-'
7
k~a~l+[HaO+] -I- ~oH-IOH-]-I~ A [ A ] ~ B [ B ] k~a+[M'"+](4)
+
1. mole-1
(3)
For a given buffer ratio, r = [A]/[B], plots of kf vs. [A] were found to be linear with a slope, S, = k~ -Ik g / r , and an intercept equal to ksolv. The catalytic coefficients, kp, and k g , were evaluated by deducing values of S, corresponding t o several reciprocal buffer ratios. It was found that S, is a linear function of l/r for each buffer pair studied in the hydrations of 2The constants k ~ , and 4-pyridinecarboxaldehyde. kHsO+, and koII- were obtained from values of ksolv a t various hydronium or hydroxide ion concentrations, as shown in Figure 1. The catalytic rate coefficients for HzO, H30+, and OH-, as well as those associated with the various buffer components studied, are given in Table 11. I n the presence of metal ions, the over-all rate coefficients for the forward process, ki, consist of a sum of catalytic terms given by eq 4
h
kS"PA,
1. mole-1
kCdi+
0
8
12
28,000 1. moli'rnin.' 16
X 10' ( m o l e ' : 1
I
20
24
28
40
48
58
1
= 4,000 I. mole-' mi",'
16
[M2+]
24
X IO4
32 ( m o l e ' :1
)
Figure 2. Metal ion catalysis of 2-pyridinecarboxaldehyde hydration. Upper box: 0, Cuz+ catalysis; middle box: 0, Goz* catalysis; A, Niz+ catalysis; A, ZnZCcatalysis; lower box: A, Cdz+ catalysis; A, Mnz+ catalysis. Volume 78, Number 8 February 1868
Y.POCKER AND J. E. MEANY
658 Table 111: Metal Ion Catalysis of ZPyridinecarboxaldehyde Hydration at pH 7.2,O.Ooo kM"+,
Ion
[M"+lrnax, mole 1.-1
cu2+ CoZ + Ni2 + ZnZ+ Cda+
8 x 10-6 1.7 x 10-4 11 x 10-6 2.0 x 10-4 7 . 3 x 10-4
1. mole-1 min-1
1 ,100,000 33,000 31,000 28,000 5 ,000
kY"+,
1. mole-1 min-1
Ion
5.6 X 10-8 0.007 0.012 0.016 0.016
Mnz+ Fe8+ Cr*+ Cat+ Mg2+
460 b, c b, d b b
'
[2-Pyridinecarboxaldehyde] = 1.13 X lo-' M in diethylmalonate buffer (0.01M ) . Acceleration in rate by the [Mn+lm, given in the table was too small to be detected. ' The spectral absorbancy of Fe8+a t 278 mfi precluded the use of higher concentrations of this ion, The addition of Cra+ caused radical decreases in pH, SO that for this ion the undetected acceleration corresponds to [CrS+] 0.012 M at pH 3 4 . (I
that product inhibition was largely avoided. The catalytic coefficients, k ~ n + , were evaluated for 2pyridinecarboxyaldehyde from series of runs in which only the metal ion concentrations were varied. Within these series of runs, plots of kr vs. metal ion concentration (Figure 2) were used to evaluate the respective catalytic rate coefficients associated with the various metal ions under consideration. The specific rates of hydrations of 2-pyridinecarboxaldehyde were linear in metal ion concentration and the values of k M n + are given in Table 111. In contrast to the very efficient catalysis of 2-pyridinecarboxaldehyde hydration by several metal ions, the promotion of the 4pyridinecarboxaldehyde reaction is exceedingly mild, kyn+ 100 1. mole-' min-l, for all the metal ions studied thus far (kzni+ N 100 1. mole-' min-l; kco~+ N 25 1. mole-l min-l; the spectral absorbancies of Cu2f and Fe*+ at 278 mp precluded the accurate determination of the respective catalytic coefficients of these ions, however both kcu2+ and ~ F G +are less than 100 1. mole-l min-'; for the remainder of the ions, such high concentrations were required that it was difficult to quantify their catalytic efficiencies).
koa-PA,implying that neither transition state IV nor V plays any significant role in The Journal of Physical Chemistry
the spontaneous hydration of 2-pyridinecarboxaldehyde, since the similar transition states in the hydration of 4-pyridinecarboxaldehyde are prohibited, owing to the distance separating the ring nitrogen and the carbonyl carbon.
Iv
H'
V It is apparent from Table I1 that these conclusions also apply to cyclic transition states analogous to IV and V in which the general acid or general base is explicitly considered. It is also apparent from Table I1 that catalytic rate coefficients associated with HP042for the hydrations of both 2- and 4-pyridinecarboxaldehyde are considerably higher than those associated with veronal sodium, even though the pK, of its conjugate acid is significantly higher. Preliminary experiments also show that catalysis by HP042- is much stronger than that by either imidazole or diethylmalonate dianion, in spite of the similarity in values of pK, for the conjugate acids of these bases. Thus, in agreement with recent studies involving the hydration of acetaldehyde,'(' catalysis of the hydrations of 2and 4-pyridinecarboxaldehyde by HP0d2- is greater than one might have expected. The metal ion catalysis of the hydration of 2pyridinecarboxaldehyde provides additional evidence that certain metal ions in particular environments can be extremely efficient hydration catalysts. We have previously reported the potent catalysis of the zinc metalloenzyme, erythrocyte carbonic anhydrase, in respect to several carbonyl ~ y s t e m s . ~ll-u . ~ ~ 6 With ~ (10) Y.Pocker and J. E. Meany, J . Phy8. Chem., in press. (11) Y. Pocker and J. E. Meany, Abstracts of the Sixth International Congress of Biochemistry, Vol. 11,New York, N. Y . , 1964,p 327. (12) Y. Pocker, J. E. Meany, D. G. Dickerson, and J. T. Stone, Science, 150,382 (1966).
THEREVERSIBLE HYDRATION OF 2- AND
4-PYRIDINECARBOXALDEHYDES
this catalysis, convincing evidence leads to the conclusion that the one zinc ion per enzyme molecule constitutes an obligatory component for its hydrase activity.lB We have also shown that zinc ions in the presence of imidazole buffers markedly accelerate the hydration of acetaldehyde,6i11-13 and that divalent zinc and cobadt ions are powerful catalysts for the hydration of 2-pyridinecarboxaldehyde. Because of the magnitude of metal ion catalysis associated with the hydration of 2-pyridinecarboxaldehyde (Table 111), one must assert that it is not merely an example of general acid catalysis. Thus the copper ion promoted hydration of 2-pyridinecarboxaldehyde is -2 X 108 more efficiient than that by water alone, 100-fold more efficient than that by H30,+ and -60% as powerful as that due to hydroxide ions. We postulate that the powerful acceleration of this reaction by certain metal ions involves either their ability to act as conveniently located Lewis acids held by the ring nitrogen and interacting with the carbonyl oxygen, or it may involve their capacity to function as a bridge between substrate and water and thereby participate in the direct transfer of metal bound water to the aldehydic group.3 While only the former explanation requires 2-pyridinecarboxaldehyde to act as a bidentate ligand, both of these postulated mechanisms necessitate the near proximity of the ring nitrogen and the carbonyl group. Complexes between the oximes of 2-pyridinecarboxaldehyde and divalent cations are well documented.” Although there is not as much evidence regarding complexes between these dications and 2-pyridinecarboxaldehyde, their existence, at least in equilibrium concentrations, has often been postulated.ls Indeed, a green complex composed of 1 mole each of CuC12, 2-pyridinecarboxaldehyde, and ethanol was isolated from these coimponents in absolute ethanol.lD The powerful inhibition of the cupric ion assisted hydration by the product, appears to be due to the capacity of 2-pyridinecarboxaldehyde hydrate to act as an efficient bidentate ligand which forms a stable five-membered ring with cupric ions. It is also interesting to note that the stability constants of metal complexes of N,N’-di(2-hydroxybenzy1)ethylenediamine-N,N-diaceticacid60 follow the order Cu2+ > Ni2+ > Go2+ > Zn2+ > Cd2+ > Mn2+ > Mg2+ > Ca2+, an order which is indeed very similar to that followed by the ability of these same divalent ions to catalyze the hydration of 2-pyridinecarboxaldehyde (Cu2+ > Go2+ > Ni2+ > Zn2+ > Cd2+ > Mn2+ > Mg2+ or Ca2+) or to promote the hydrolysis of amino acid esters5* or amides.5b I n comparison to the very powerful catalysis observed in the hydration of 2-pyridinecarboxaldehyde by Cu2+,Co2+,Ni2+, Zn2+, Cd2+, and R!tn2+,the mag-
659
nitude of the acceleration afforded by these same ions in the corresponding 4-pyridinecarboxaldehyde re6 100 1. mole-’ min-’). action is negligible (/can.+ It is again implied that the vast difference in catalytic behavior of these metal ions for the two reactions is due to the special arrangement of the ring nitrogen and aldehydic group in 2-pyridinecarboxaldehyde which is absent in 4-pyridinecarboxaldehyde. In order to test the generality of this phenomenon, we are presently considering other substrates which may possess structural characteristics conducive to enhanced metal ion catalysis, i.e., compounds to which metal ions may bind close to the site of hydration or dehydration. I n this connection, we have established that the dehydration of pyruvic acid hydrate is powerfully catalyzed by certain transition metal ions.20 We have further shown catalysis of this dehydration by carbonic anhydrase. Enzymatic and metal ion catalysis involved in the pyruvic acid-water systems will be reported in a later paper. I n contrast t o metal ion catalysis of 2-pyridinecarboxaldehyde hydration, where the metal ion can bind with the pyridine nitrogen and a t the same time remain close to or actually interact with the aldehydic group, the enzyme appears to overcome these restrictions on substrate binding and is an efficient catalyst for both 2- and 4-pyridinecarboxaldehyde. Additionally, it is interesting to note that although both Go2+ and Zn2+ act as efficient coenzymes to apocarbonic anhydrase,16 the use of divalent copper to replace zinc in the native enzyme produces a completely inactive enzyme.21 It would thus appear that divalent copper, although it is actually a more powerful catalyst than native carbonic anhydrase with respect to the hydration of 2-pyridinecarboxaldehyde, does not possess the very special properties necessary to activate the apoenzyme. The particularly strong affinity of Cu2+ for amino acid residues in the enzyme coupled with its strong preference for regular geometry may preclude it from holding the water molecule which is generally regarded as necessary for the hydrase activity of both Zn(I1) and Co(I1) carbonic anhydrase. (13) Y. Pocker and J. E. Meany, J. Am. Chem. Soc., 87, 1809 (1965). (14) Y. Pocker and J. T. Stone, ibid., 87, 5497 (1965). (15) Y.Pocker and J. T . Stone, Biochemistry, 6, 608 (1967). (16) S. Lindskog and B. G. Malmstrom, J . BioZ. Chem., 237, 4129 (1962). (17) S. Bolton and R. I. Ellin, J . Pharm. Sci., 51, 533 (1962). (18) B. Kirson and 5. Yariv, Bull. SOC.Chim. France, 2969 (1964). (19) D.Kutscher and E. Hayer, 2. Chem., 3, 68 (1913). (20) Y. Pocker and J. E. Meany, unpublished data. (21) B. G. Malmstrom, P. 0. Nyman, B. Strandberg, and B. Tilander in “Structure and Activity of Enzymes,” T . W. Goodwin, J. I. Harris, and B. S. Hartley, Ed., Academic Press, New York, N. Y., 1964, p 121; T. A. Duff and J. E. Coleman, Biochemistry, 5, 2009 (1966).
Volume 79,Number 9 February 1068
