8057 K4
[Rh(diphos)]++ >C=C
C=CC=CC=CC=CC=CC=C
Jack Halpern,* Dennis P. Riley Albert S. C. Chan, Joseph J. Pluth Department of Chemistry, The University of Chicago Chicago, Illinois 60637 Received August 17, 1977
Models for NADH Coenzymes. Isotope Effects in the N-Benzyldihydronicotinamide/N-Benzylnicotinamide Salt Transhydrogenation Reaction Sir: The study of oxidation-reduction reactions of models for nicotinamide coenzymes has provided a great deal of information about the mechanism of such pr0cesses.I Perhaps the most fundamental redox reaction involving the dihydropyridine/pyridinium salt redox couple is the transhydrogenation reaction. An in-depth study of the transhydrogenation reaction has special appeal because of the symmetry between reactants
Communications t o the Editor
8058
R-
gOl \
14
ao
12
10
-*
, I 50
n
I
c
-
(P
8
c
40
-
30
-
20
-
0
L
5
!! a
2t/v 0.04
M E
Concentrrtlon l o t /
Y
1
50
I
100
I
I
150
200
*
,
"400
Time (min)
OC.
Figure 1. Time course of the N-benzyldihydronicotinamide(O)/N-benzylnicotinamide salt (m)transhydrogenase reaction in an acetonitrile-0.1 M borate (pH 8.8) buffer mixture (1:3 v:v) at 40.0 i 0.1 OC. Initial conand 0.03 I M ditions: 0.029 M N-[benzyl-3H]-l,4-dihydronicotinamide N-benzylnicotinamide chloride.
Scheme I
of the transhydrogenation reaction is evaluated by observing the percent of the total 3H activity of each component as a function of time.2bA typical run is shown in Figure 1. In principle, the transhydrogenation reaction is an example of a simple isotope exchange reaction. A*
1
+ B + A + B*
(1)
Although such reactions represent second-order processes, their kinetic behavior are first order as a result of a constant concentration term.3 Furthermore, such reactions can be shown to fit the following mathematical scheme
2
and products which greatly simplifies the mechanistic picture. In this communication, we wish to report on our studies of the N-benzyl- 1,4-dihydronicotinamide/N-benzylnicotinamide chloride transhydrogenation reaction. We wish to report two major findings: (a) the measured secondary isotope effects during transhydrogenation are exactly opposite to those predicted by considering changes in hybridization at the 4 position in going to the transition state, and (b), although the evidence supports a multistep process, the 1,2- or 1,6-dihydropyridine may be excluded as an obligatory intermediate. The course of the transhydrogenation reaction was monitored by using a radiochemically labeled dihydronicotinamide or nicotinamide salt and observing the change in the specific activity of the reaction components (Scheme I) as a function of time. In a typical experiment, pre-equilibrated buffered solutions of N- [ben~yl-~H]dihydronicotinamide~~ and N benzylnicotinamide chloride are mixed and maintained at 40.0 f 0.1 "C. Aliquots are removed at various times and the components separated by a high pressure liquid chromatograph coupled to a UV detector. All peaks are individually collected and assayed for radioactivity by liquid scintillation counting. Observed counts per minute are corrected for quenching by the use of an external standard. We observe that the sum of the 3H activity over all peaks remains constant during the course of the transhydrogenation reaction and is equal to the total 3H activity injected into the chromatograph. Furthermore, the peaks corresponding to N-benzyldihydronicotinamide and N-benzylnicotinamide salt account for >97% of the total 3H activity during 3 half-lives of transhydrogenation. The progress Journal of the American Chemical Society / 99:24
IM)
Figure 2. Variation of observed first-order rate constant of transhydrogenation (kobsd) as a function of the sum of the dihydronicotinamide and nicotinamide salt concentrations. Reactions were carried out in acetonitrile-0.1 M phosphate (pH 8 . 6 ) buffer mixture (1:3 v:v) at 40.0 f 0.1
In ( B * , - B*!) = -kz(A*o
+ Bo)t + In (B*, - Bo)
(2)
where k2 is the second-order rate constant and the subscripts t , 0, and 03 correspond to the conditions at a given time, at the beginning of the reaction, and at infinity, respectively. The relevance of the above scheme to the transhydrogenation reaction is demonstrated in the following experiment. Using N-benzyl- [7-I4C]nicotinamide chloride, the appearance of I4C activity in the N-benzyldihydronicotinamidecomponent follows first-order kinetics for more than 4 half-lives. Moreover, the variation of the observed first-order rate constant, kobsd, as a function of the sum of the concentration of N-benzyldihydronicotinamide and N-benzylnicotinamide chloride is linear (Figure 2). The slope of 0.1 19 f 0.003 L/mol-' min-' represents the second-order rate constant of transhydrogenation at the indicated conditions. One possible mechanistic pathway for the transhydrogenation reaction requires that the isomeric 1,2- or 1,6-dihydronicotinamide be an obligatory intermediate in the reaction. The interconversion of 1,4-dihydropyridines to their isomeric species has been the subject of several report^.^ We have tested this hypothesis by synthesizing N-benzyl-4,4-dideuteriodihydronicotinamide and N-benzyl-4-deuterionicotinamide chloride and examining the deuterium content at the 4 position before and after transhydrogenation. If the 1,2- or 1,6-dihydronicotinamide was an obligatory intermediate (Scheme 11), the deuterium label would be scrambled after transhydrogenation. NMR examination of the reaction components after >4 half-lives of transhydrogenation indicated that 97 f 2% of the deuterium is retained in the original 4 position. This
/ November 23, 1977
8059 Table I. Isotope Effects for the N-Benzyl- 1,4-dihydronicotinamide/N-Benzylnicotinamide Salt Transhydrogenation Reaction Reaction A B C D E
1,4-Dihydronicotinamide (1)
Nicotinamide chloride salt (2) N-Benzyl- (2c)
N-Benzyl-[7-I4C]- (la) N - [ b e n ~ y l - ~ H(1 ] -b) N-Benzyl- (IC)
N - Benzyl- [7-I4C]- (2a) N - [ b e n ~ y l - ~ H(2b) ]N-Benzyl- (2c)
N - B e n ~ y l - [ 7 - ~ ~ (Cl ]a-) N - Benzyl-[4-3H]- (la) N-Benzyl- (IC)
N-Benzyl- [7-I4C]- (2a) N - B e n ~ y l - [ 4 - ~ H(2d) ]-
N - B e n ~ y l - [ 7 - ~ ~ (la) C]N-[ben~yl-~H]-[4-~H (le) ]-
N-Benzyl- (2c)
Kinetic ratiob
*
1.oo 0.01 0.95 f 0.02 1.05 f 0.03 0.93 f 0.07 1.58 f 0.02 1.67 f 0.14 1.39 f 0.05 1.16 f 0.07 1.24 f 0.07 1.51 f 0.07 1.46 f 0.10
IsotoDe effectC 0.99 f 0.02 1.03 f 0.07 0.79 f 0.02f 1.30 f 0.12 6.2 f 2.0g
Notesd
-
Controle (SP3 SP2) Controle (SP2 SP3) Secondary tritium (SP3 SP2) Secondary tritium (SP2 SP3)
-
-
Primary deuterium (SP3 SP2) At 40.0 f 0.1 OC in acetonitrile-0.1 M aqueous sodium borate buffer (pH 8.8) mixture (1.3 v:v); all kinetic runs were carried out at a concentration of -0.02 M in each reactant. The ratios of the specific rates of the two isotopically labeled nicotinamides [k(14C)/k(3H)] were obtained from the fractional conversion of the two isotopic nicotinamides to their conjugate species as described in text (kinetic ratio = [3H/14C]~[14C/3H],); the errors represent the standard deviation of the intercept of the least-squares best-fit line of the observed kinetic ratio as a function of time for each experiment. Isotope effects calculated from kinetic ratios corrected for multiple pathways by eq 5 and 6; represents the weighted mean of two or more kinetic runs.14 Hybridization change at the 4 position incurred by the labeled species is given in parenthesis. e Control reactions involve transhydrogenation of nicotinamide species labeled with tritium at nontransferable positions. SO = fcalculated from the kinetic ratio using eq 4. g Calculated from the kinetic ratio using eq 6 and the assumption that ST = 0.85 f 0.02.
Scheme 111
Scheme I1 D
O
D
O
kinetic ratio (reaction C) = 2 k ~ / h ~ ' 2c + Id "identity transfer" T o assure that the procedure for the simultaneous assay of
3H and I4C activity was unbiased, the kinetic ratios of two control reactions (reactions A and B in Table I) were measured. Since the radiochemical labels were located some distance from the transferable hydrogen, no isotope effect was expected and a kinetic ratio of 1 may be predicted. The exdemonstrates that the transhydrogenation reaction results from cellent agreement between the observed and predicted kinetic the specific transfer of a hydrogen from the 4 position of the ratios provides strong support for the accuracy of the procedihydronicotinamide to the 4 position of the nicotinamide salt. dure. Moreover, this result assures that nicotinamides, isotopically Substitution of tritium or deuterium on a carbon which labeled a t the 4 position, will not undergo label scrambling changes from sp3 to sp2 hybridization in the rate-determining during the kinetic isotope effect studies discussed below. step of a reaction produces a secondary kinetic isotope effect6 The measurement of the secondary isotope effects during of 20 and 15%, respectively; i.e., kH/kT N 1.2. Substitution the transhydrogenation reaction (Table I) provides anomalous of tritium or deuterium on a carbon undergoing a hybridization secondary isotope effects. Isotope effects were calculated from change in the reverse direction, from sp2 to sp3, produces a the measured competition reaction between two isotopically secondary kinetic isotope effect of about the same magnitude labeled nicotinamides and their conjugate redox specie^.^ For but in the inverse direction; i.e., kH/kT 0.83. If the mechexample, a solution of N-ben~yI-[4-~H]dihydronicotinamide anism of transhydrogenation involves a simple hydride transfer and N-benzy1-[7-l4C]dihydronicotinamide was reacted with a solution of unlabeled N-benzylnicotinamide chloride in a Scheme IV buffered solvent mixture (reaction C on Table I). During the 2kH course of the transhydrogenation reaction (always
