J . Am. Chem. SOC.1982, 104, 1016-1024
1016
can be rationalized by low IPS for all but higher potential energies due to heat of vaporization differences.21 Since low IP does seem to be important, a polar reaction mechanism would seem reasonable, perhaps as follows for Al:
-
E+
-
the reaction must be more complex and Mg clusters may be involved. In the case of transition metals, high IPS,low M-Br bond strengths, and unfilled d orbitals lead to the preference of CH,Br---M complexation rather than oxidative addition.
Complete electron transfer may occur during the reaction, and only one AI (or Ga or In) atom is involved. In the case of Mg
Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for the support of this work. Numerous discussions with Dr. R. Hauge of Rice University and G. A. Ozin of the University of Toronto have been very helpful.
(21) Professor P. S. Skell has found a similar reactivity dependency in M-H20 reactions (28th IUPAC Congress, Aug 16-21, 1981, Vancouver, B.C., Canada, paper IN 002) and has used the term "atom potential" to describe this inherent reactivity.
Registry No. CH3Br, 74-83-9; AI, 7429-90-5; Ga, 7440-55-3; In, 7440-74-6; Mg, 7439-95-4; Fe, 7439-89-6; Cu, 7440-50-8; Pb, 7439-92-1; Co, 7440-48-4; Ni, 7440-02-0; Pd, 7440-05-3; Ag, 7440-22-4; Au, 7440-57-5; TI, 7440-28-0; Ge, 7440-56-4; Sn, 7440-31-5.
AI t CH3-Br
,AI.
H3C Z z - - - b -
st
H3C-AI
s-
Br
Mechanism of the Cyanide-Induced Formation of Methyl Acetate from [ ( Methoxycarbonyl)methyl]cobalaminl William W. Reenstra, Robert H. Abeles,* and William P. Jencks* Contribution No. 1389 from the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254. Received July 6, 1981
Abstract: The addition of cyanide to [(methoxycarbonyl)methyl]cobalamin (Me02CCH2Cbl,MCMB,,) results in the rapid, reversible formation of a cyanide addition compound (KO= 0.044 M), followed by cleavage of the carbon-cobalt bond and protonation to give methyl acetate. An analogous, slower reaction occurs with (carboxymethy1)cobalamin (KO= 0.77 M). At pH 9, and the absence of general-acid catalysis show that protonation of the leaving carbon atom does not occur in the ratedetermining step. There is discrimination against the incorporation of tritium from the solvent into the product, with ['H]/['H] = 4.3-4.9 at pH >9 and [1H]/[3H]= 8.6 at pH 1. These isotope effects establish a stepwise reaction mechanism, in which protonation of the intermediate occurs after the rate-determining step. a-Deuterium isotope effects of k 2 ~ / k 2= ~1.19 f 0.06 at pH 11.4 and k2H/k2D= 1.11 f 0.06 at pH 0.9 demonstrate a significant change toward sp2 hybridization of the a-carbon atom in the rate-determining transition state. Partial inversion of configuration at the a-carbon atom in the methyl acetate produced from (R)-[2H]MCMB12in tritiated water shows that protonation must occur, at least in part, before separation of the enolate from cobalamin and provides evidence that the intermediate is a K complex with a lifetime of >lo" s. The small equilibrium isotope effect of KHcN/KDcN= 2.3 for the dissociation of hydrogen cyanide is attributed to the low bending frequency of the C-H bond.
The ability of adenosylcobalamin and other alkylcobalamins to undergo both cleavage and formation of carbon-cobalt bonds with remarkable ease is probably responsible for the biological activity of these compounds in mediating reactions that cannot be brought about by the simpler chemistry utilized by other coenzymes. This facile carbon-cobalt bond cleavage occurs to give electron-deficient, radical, or carbanionic products, depending on the ability of substituents on carbon to stabilize one or another of these species. The ease with which these different reactions occur reflects the easy accessibility of the three oxidation states of the products, Cob(I)alamin, Cob(II)alamin, and Cob(II1)alamin, that are formed in the three modes of cleavage. The expulsion of leaving groups with a pair of electrons, leaving behind a Cob(III)alamin, is greatly facilitated by the addition to the trans position of cyanide anion or other groups that increase the electron density on cobalt.2 The experiment's described here were undertaken in order to learn more about the chemistry of heterolytic carbon-obalt bond (1) Supported by grants from the National Institutes of Health (Grant GM-21633) to R.H.A. and the National Science Foundation (Grant PCM77-08369) and National Institutes of Health (Grant GM-20888) to W.P.J. W.W.R. was supported by a training grant from the National Institutes of Health (Grant 5 R01 GM-00212). (2) Pratt, J. M. "Inorganic Chemistry of Vitamin BI2";Academic Press: New York, 1972.
0002-7863/82/l504-1016$01.25/0
cleavage. Adenosylcobalamin and [ (methoxycarbonyl)methyl]cobalamin (MCMBI2)undergo cyanide-induced fragmentations that give Cob(II1)alamin products and appear to proceed through the electron-rich, carbanionic class of rnechani~m.~-~ It has been proposed that the cleavage of MCMB,,, to give methyl acetate as the product, proceeds through a direct, front-side displacement to expel the enolate anion of methyl acetate4 or through an intermediate adduct in which cyanide ion adds to the a (trans) position, replacing the dimethylbenzimidazole ligand5 (eq 1, in 0
I1
H,CCOCH,
@
0
p II
HzCCOCH3
-
-
H0
pot"'
H,C.-C
HD.
CN
CN
N
N 0
(3) Barker, H. A.; Weissbach, H.; Smyth, R. D. Proc. Natl. Acad. Sei. U.S.A. 1958,44, 1093-1097. Johnson, A. W.; Shaw, N. J . Chem. Soc. 1962, 4608-46 14. (4) Hogenkamp, H. P. C.; Rush, J. E.; Swenson, C. A. J . B i d . Chem. 1965, 240, 3641-3644. ( 5 ) Fenton, W. A. Thesis, Brandeis University, 1974.
0 1982 American Chemical Society
Addition of CN to [(MethoxycarbonyI)methyl]cobalamin this and subsequent equations the corrin ring of cobalamin is represented by a square and the dimethylbenzimidazole ligand by N). Our results show that the reaction proceeds in at least three steps, with rapid formation of the cyanide adduct at equilibrium (eq 1, first cyanide adduct), followed by cleavage of the C-Co bond to give the enolate or enol of methyl acetate in the rate-determining step, and then protonation to give methyl acetate. The remarkable result is that protonation occurs preferentially on one face of the enolate. This requires that protonation of the enolate occurs before separation from the cobalamin and provides evidence for cleavage of the carbon-cobalt u bond to give complex with a lifetime of >lo" s. A prean intermediate i~ liminary communication of this work has appeareda6
Experimental Section Materials. Hydroxocobalamin (OHB,,) and cyanocobalamin (CNBI2) were kindly given by Glaxo Laboratories and were used without further purification. (R)-[3H,2H]Aceticacid was a gift from Dr. J. W. Cornforth. [(R)-(MethoxycarbonyI)deuteriomethyl]cobalamin [(R)[2H]MCMB12]was prepared by F e n t ~ n .'H ~ N M R analysis showed that (R)-[2H]MCMB,2contained 7), 361 nm (pH 2-7), or 359 nm (pH 85% at pH 4.6 and >95% at pH 9.0) by extraction into ether of products from the cleavage of 14C-labeled MCMBI2. The dependence on pH of the observed second-order rate constants, based on total cyanide concentration, shows three pHindependent regions (Figure 1). This behavior is described by the three terms of the rate law of eq 4. Above pH 8 an unca-
+ ke[MCMB,2] [CN-] [H+] + kc[MCMBI2H+][CN-] [H']
t
5.0
-
(4)
talyzed reaction of MCMB12and cyanide anion occurs with the rate constant kA. Near neutral pH the predominant pathway is an acid-catalyzed reaction of MCMBI2and cyanide anion, which is described by the rate constant kB. Below pH 5 the reaction involves acid catalysis of the reaction of cyanide anion with the protonated, base-off species of MCMB12and can be described by the rate constant kc or by the kinetically equivalent rate constant kc' (eq 5), which is related to kc by the dissociation
0
/
0
Y
50
25
050
I00
?5
125Y-'
I/[CN-!
Figure 2. Double-reciprocal plot of the observed rate constants for the reaction of cyanide anion with MCMBI2at pH 11.27 at varying cyanide concentrations at 30 OC and ionic strength 1.8 M (KCI). Inset: Dependence on cyanide concentration of the ratio k o M / k l (0)and the ratio U/U,, (01, where AA = &bsd - A M C M Band , ~ Urn,, = A M C M B , ~ C N - AUCMBI2; AoM is the initial absorbance after the addition of cyanide. The solid line is calculated from the equation Y = [CN-]/([CN-] + KO) with KO = 0.044 M.
is inhibited by the amines piperidine (50% at 0.76 M) and N,Ndimethylglycine. This inhibition may be caused by complexation with the amine, but no change in the spectrum of MCMB,, was observed in the presence of 1.0 M piperidine. At concentrations of hydrochloric acid greater than 1 M the rate of cleavage was found to decrease. The values of log k in the presence of 0.1 M hydrogen cyanide follow H024with a slope of 0.9 and there is a rate decrease by a factor of 50 at Ho = -2.7. The absorption spectrum of MCMB,, in 4 M hydrochloric acid does not show significant changes, such as are observed upon ionization of CMB,, and (aminoethyl)cobalamin, and binding of chloride ion is expected to give only a small inhibition under these conditions (the association constant for aquocobalamin and chloride ion is 1.3 M-l and binding to an alkylcobalamin is expected to be weaker;,, furthermore, an increase in the concentration of chloride ion from 1.8 to 4 M could not cause a rate decrease by a factor of 50). At high pH values there is a leveling of the observed rate constants and a change in the spectrum of the reaction mixture, extrapolated to zero time, with increasing concentration of cyanide anion. This is caused by accumulation of the a-cyano addition compound (eq 6). A plot of l/kobsdagainst l/[CN-] (Figure
@
&
CH,COOMe
CH2COOMe
kc[MCMBl2H+] [H'] [CN-] = kc'[MCMBl2H+] [HCN]
(5) constant of hydrogen cyanide. The lines a, b, and c in Figure 1 show the contributions of the different terms in eq 4 to the observed rate constants and are based on the rate and equilibrium constants in Tables I and 11. No general-acid or -base catalysis was detected in buffers of trifluoroethanol (pH 11.24, up to 0.9 M), cacodylic acid (pH 6.05, up to 0.8 M), or acetic acid (pH 4.50, up to 1.3 M). The reaction
/
$
-25
a 30 "C and ionic strength 1.8 M (KC1). The rate constants are defined in eq 4 and 6. Total cyanide concentration. 97% deuterium oxide, 3% water. From Figure 2. Observed rate constant at 1.0 M [ KCN]. kc' = ~ C K H C=N0.24 M-' s-' in H,O and 0.28 M-' s-' in D,O.
u = k~[McMB12][CN-]
1c.c
: I
IID
+
CN-
e KO
Xi
products
(6)
N
2) gives a limiting first-order rate constant of k l = 4.65 X s-l
at high cyanide concentrations, a second-order rate constant (24) Paul, M. A,; Long, F.A. Chem. Reu. 1957, 57, 1-45.
Reenstra, Abeles, and Jencks
4m.Chem. SOC.,Vol. 104, No. 4, 1982
Table IV. Secondary Deuterium Isotope Effects for the Reaction of MCMB,, with Cyanide' -
-
-
k(MCMB,,)/
term k,/s-l
kc/M-' s - ~
pH
11.39 00.91 4 M HC1
[cyanidel/Mb runs k([*H]MCMB,,)" 0.98 0.05 0.10
8 12 6
1.19 1.11 1.21
i
t t
0.065 0.06 0.03d
' At 30 "C and ionic strength 1.8 M (KCI). Total cyanide concentration. " Observed rate constant ratio for the di-H and d k 2 H compounds. Ionic strength = 4.1 M. Table V. Solvent [ 3 H ] H , 0 Selection Isotope Effect for Discrimination against 3H Incorporation into Methyl Acetate' spec act
spec act
of H,O/ @Ci of
of MAC/ @Ci of 3H/fimol of MA)
selection isotope
0.36 0.40 0.49 0.22
4.9 4.3 4.4 8.7
3H/l.rmol pH 11.3 11.3 9 .O 1.0 nrn
Figure 3. Spectra of 0.038 mM MCMBll in the absence (dashed line, pH 8) and the presence (solid lines) of 0.5 M potassium cyanide at pH 11.3, 30 OC, and ionic strength 1.8 M (KCI). The spectra were taken 180, 600, 1500, 3600, and 11 340 s after the addition of cyanide to MCMBIZ. Table 111. Solvent Deuterium Isotope Effects for the Reaction of MCMB,, with Cyanide' k~ ,o l k ~o, or
I."
term kA1M-l k,/s-' Ko/M
S-'
k,/M-' s-' kc/M-' E'
(cyanide]/Mb runs K H ~ / K ' 0 D
10.4, 11.3d 11.4
0.002-0.010 0.98
6.2-6.7 0.9-1.35 8-10
0.10 0.05
11 12 17 20 10 28
1.4 i 0.16 1.44 f. 0.08 1.02 f 0.20 0.37 i 0.04e 0.37 i 0.01 2.29 0.1
KHCN/M * ' 30 "C, ionic strength 1.8 M (KCl); data from Tables I and 11.
Total concentration of cyanide. In 97% deuterium oxide. Runs in deuterium oxide at pD = 11.3. The observed rate constants have not been corrected for the contributions from kA and kC; see text. @
of k A = 1.06 X M-I s-l a t low cyanide concentration, and a dissociation constant of KO= 0.044 M. The same dissociation constant describes the change in absorbance extrapolated to zero time (inset, Figure 2); this change was found to be complete by the time of the first measurement a few seconds after the addition of cyanide. Figure 3 shows the spectra of a reaction mixture containing 0.5 M cyanide at pH 11.3 at different times. The same behavior was observed with the carboxylate compound, CMB12, at pH 11.5, with values of k , = 1.8 X s-l and K,' = 0.77 M. The second-order rate constant of 2.3 X M-' s-l at low cyanide concentration agrees with an extrapolation of data obtained by Fentons The products of this reaction are acetic acidlo and an equilibrium mixture of cyanocobalamin and dicyanocobalamin, as determined spectrophotometrically. Solvent deuterium isotope effects were determined in the three plateau regions of the pH-rate profile and are reported in Table 111. The isotope effect on kB was determined at pH (pD) = 6.2-6.7, where the k B term accounts for >90% of the observed reaction. The isotope effects are small at high pH but are inverse at intermediate and low pH, with kD20/kH20= 2.7. The isotope effects for rate constants at pH
