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Bifunctional acid—base catalysis by imidazole groups in enzyme mimics
JOURNAL OF
MOLECULAR CATALYSIS Journal of Molecular Catalysis 91 ( 1994) 161-174
ELSEVIER
Review
Bifunctional acid-base catalysis by irnidazole groups in enzyme mimics Ronald Breslow Department
of Chemistry, Columbia University, New York, NY 10027, USA
(Received January 17, 1994; accepted February 17, 1994)
Abstract The cleavage of RNA can be catalyzed by imidazole buffer, and shows sequential bifunctional catalysis. When the two imidazole groups are attached to the same catalyst, as in enzymes or in cyclodextrin bis-imidazole derivatives, simultaneous bifunctional catalysis is seen. The geometric preference for such bifnnctional catalysis is revealed by the use of three isomeric cyclodextrin bis-
imidazoles. Different isomers are preferred for phosphate ester hydrolysis and for ketone enolization, and the preferences reveal details of the catalytic mechanisms. Keywords: acid-base catalysis; cyclodextrin; enolization; hydrolysis; imidazole groups; phosphate esters; ribonuclease A mimics
1. Introduction
Imidazole groups of the amino acid histidine are common in enzymatic catalysis. With a pKnear neutrality, imidazole (Im) groups are the strongest bases that can exist unprotonated at neutral pH, while their protonated forms - imidazolium cations (ImH+) - are the strongest acids that can exist under neutral conditions. Thus there has been considerable interest over the years in the catalytic properties of imidazole and its derivatives [ l-91. We were interested in building mimics of the enzyme ribonuclease A, which uses two imidazole rings of His- 12 and His- 119 as its principal catalytic groups. Our earliest work [ 10,111 involved construction of mimics based on the widely accepted mechanism for the enzyme. However, we then did mechanistic studies on the cleavage of RNA by simple imidazole buffer [ 12-151, and based on that work we concluded that an alternative mechanism was preferred in solution, and probably as well for the enzyme. In this review we will first *Corresponding author; fax. ( + l-212)9321289. 0304-5102/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDIO304-5102(94)00046-X
R. Breslow /Journal of Molecular Catalysis 91 (1994) 161-174
162
describe the mechanistic studies on RNA cleavage by imidazole buffers, then describe the bifunctional catalysts whose geometric preferences reflect these mechanistic results.
2. Bifunctional cleavage of RNA by imidazole buffers The cleavage of RNA by the enzyme ribonuclease A involves bifunctional catalysis by the imidazole groups of His- 12 and His- 119, one acting as a base while the other, protonated, acts as an acid. The result is a bell-shaped pH vs. rate profile, in which the rate reaches a maximum at pH 6.3 or so, where one imidazole is protonated and the other not. The mechanism of cleavage involves an ester exchange, converting the linear 3’S phosphate diester 1 to a cyclic 2’,3’ phosphate diester 2 by attack of the 2’ hydroxyl group (Scheme 1) . In a subsequent step this cyclic phosphate ester is hydrolyzed to a 3’ phosphate 3, with regeneration of the 2’ hydroxyl group. The same two imidazoles also catalyze this process. In the textbook mechanism for action of this enzyme, the Im removes a proton from the attacking 2’ OH group while the ImH + protonates the leaving 5” oxygen (Scheme 1) . This is only one possible role of the two catalytic groups; our mechanistic studies addressed the question of whether this was the best mechanism for Im/ImH+ bifunctional catalysis of phosphate diester cleavage. In our first study [ 131, polyuridylic acid was used as substrate. We saw that its cleavage was catalyzed by imidazole buffer, and with interesting kinetics. The rate of cleavage was simply first order in total buffer concentration (Fig. 1) , but the pH vs. rate profile was bell shaped (Fig. 2)) with a rate maximum at ca. 40% Im, 60% ImH+. The latter result showed that both Im and ImH+ were required for the catalysis, but the first order kinetics in total buffer indicated that the two species were not operating at the same time. Thus we were observing sequential bifunctional catalysis; one buffer component converted the substrate to an intermediate, and the other buffer component converted this intermediate to the cleaved product. However, simple kinetic methods could not establish which was the first catalyst, Im or ImH+, since the two alternative mechanisms are kinetically equivalent. \
CHz
\ Base
k 0
0 .o--‘P=O
0
H'lm
Im:
Im:
OH
0, I
O\
\ LH-“,H
Base
k 0
0,
Base
k
OH
bH
+
CH,
o
o-\p= 0
HO,
-o-y=0
0
o&‘P’ \ 20
_
W
OH
-o-+=0
O\ Scheme 1.Textbook mechanism
for RNA cleavage by ribonuclease
A.
R. Breslow / Journal of Molecular
I 0
.I
1
.
.
.
1000
500
163
Catalysis 91 (1994) 161-l 74
Total concentration
.
I
.
.
.
.
I. 2000
1500
of buffer (mM)
Fig. 1. Rate of cleavage of polyuridylic acid by imidazole buffer vs. the total buffer concentration at l/4 Im/ ImH+ and 3/4 Im/ImH+. The lines are theoretical curves using the same parameters as those for the line in Fig. 2.
0.0
0.2
0.4
0.6
0.8
1.0
State of protonation Fig. 2. Rate of cleavage of polyuridylic acid by imidazole buffer vs. mole fraction of the buffer as imidazolium ion. The corrected data are by subtraction of the rate at the same pH but zero buffer concentration. The curve is the theoretical line by fitting the data to an equation similar to that of EIq. 2. See [ 171 for details.
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164
Later studies [ 14,151 established the sequence in detail. In this work we studied the cleavage of simple nucleotide dimers, 3’,5”-UpU (uridyluridine) or 3’,5”-ApA (adenosyladenosine) (4)) and saw that imidazole buffer catalyzed two simultaneous processes. One was the conversion of 4 to the cyclic phosphate 5 and uridine 6, while the other was the conversion of 4 to the 2’S’ isomer 7. We saw that the cleavage reaction showed a bellshaped pH vs. rate profile, just as it had with polyU substrate, but the isomerization was catalyzed only by ImH+. Of course one possible interpretation is that the two processes have completely independent pathways, but we found evidence that this is not the case. The other possibility is that the two paths branch not from the starting material but instead from the phosphorane intermediate that had been required to explain our cleavage kinetics. A reaction with two steps must have an intermediate, in this case a phosphorane 8 that is the only sensible intermediate between a linear phosphate diester and a cyclic phosphate ester. Kinetic arguments [ 131 indicated that the phosphorane cleavage intermediate had to be the monoanion, 8, as shown. Other evidence [ 161 indicates that a phosphate migration from the 3’ to the 2’ oxygen must also proceed through a phosphorane intermediate that has enough lifetime to undergo a process known as pseudorotation. If the two pathways branch from this common phosphorane intermediate, as shown in Scheme 2, we could solve the kinetic ambiguity in the cleavage reaction. Since the isomerization of 4 to 7 was catalyzed only by ImH+, that must be the catalyst for the common first step in which 4 is converted to 8. Thus the question was simply whether indeed these reactions branch from a common phosphorane intermediate, as in Scheme 2. Other kinetic evidence indicated that this was the case. When we examined reactions with varying buffer ratios, we found that the isomerization reaction was not simply uncatalyzed by Im, it showed negative catalysis. That is, increasing
Ho\
ImH’ caralys~s
CHz Base 0
k 0
OH H’
-o-‘PC0
i==
!
O\H,
Base
k 0
HO 4
OH
3'.5"-upu or ApA
Ho\
CH,
0
Z.5”.upu or ApA
-HI
0
Base
Base
(a) 0
P OS-P-OH
0
‘p’o (a 5
Scheme 2.
0
‘P’ o” lo.
HO?&0
6R
Base
k 0
ImH’
HO
I
CHz
k- k
CHz
,
Ho\
Ho\
7.3’ cyclic phosphate
+
R-OH nucleoside 6
R. Bnsbw/Journal
of Molecular Catalysis 91 (1994) 161-174
165
the concentration of Im while keeping ImH+ constant led to a slowing of the isomerization reaction (but not of the cleavage reaction, which went faster). Of course changing the buffer ratio causes a change in pH, but we corrected for this effect by the normal method of extrapolating the rate to zero buffer concentration with a given buffer ratio. This negative catalysis by Im is exactly as expected for a reaction pathway in which ImH+ converts the substrate to the phosphorane 8, and then two paths branch from it. The isomerization does not show further buffer catalysis, but the cleavage does. The mechanism described is shown in Scheme 2, and the kinetic equations that derive from this mechanism (with added terms for other catalytic species) are Eqs. 1 and 2. As those equations make clear, increasing the concentration of Im will slow the isomerization reaction because [ Im] appears only in the denominator of its equation (but also in the numerator of the equation for cleavage).
ksomerization =
klk3[BH+]
k_,[BH+]
+k,[B]
+k;k,
(1)
+k3 +k,
of UpU, B = imidazole or morpholine k cleavage=
hkz[BH+l[Bl k-l [BH+]
+k,[B]
+G +k3 +k,
+
k’[B] +k”[BH+] k_, [BH+]
+k,[B]
+k, +k,
(2)
of upu. Physically, increasing the [ Im] decreases the concentration of the intermediate 8, because it speeds its conversion to the cleavage products. The negative rate effect is clear evidence that the two paths branch from a common intermediate, and that it is Im that is the second catalyst in the cleavage sequence since that is the species that made a negative contribution to the isomerization rate. There was really no doubt about these conclusions, but they were confirmed in another recent study [ 17,181. In this case we used morpholine buffer, not imidazole buffer. Morpholine is more basic than imidazole, so we expected that the negative catalytic effect of the basic buffer component - which is the key piece of evidence about the mechanism would be even clearer in this case. By using a high B/BH+ ratio we were able to see a negative catalytic effect without having to make any corrections for changing pH effects. That is, we increased the concentration of the buffer but kept a constant B/BH+ ratio and constant ionic strength so the pH was unchanged. We saw that the rate of isomerization decreased (Fig. 3) as the buffer concentration increased, but the rate of cleavage (Fig. 4) increased as expected. Thus there was a negative catalytic effect by the buffer itself, not just by its base component. However, the negative effect was much smaller with a lower B/BH+ ratio, making it clear that it was the basic component that caused it. The data fit very well to theoretical lines generated from the appropriate equations. What detailed mechanism do these results indicate? They show that the conversion of 4 to the intermediate 8 is catalyzed by BHC, but for such general acid catalysis there are always two mechanistic possibilities. One is that the BH+ acts directly, and the other is that there is an indirect mechanism - reversible protonation followed by catalysis by the basic form B. This is called the specific acid-general base sequence, and it is completely kinetically equivalent to the general acid one-step mechanism. It is the sequence we invoke. As
R. Breslow / Journal
166
of Molecular Catalysis 91 (1994) 161-174
K ._ Y
0.4
1
0.2
I
0.4
I
0.6
Buffer Concentration
I
0.8
1
1.0
(M)
Fig. 3. Rate of isomerization of 3’5”-uridyluridine (UpU) to 2’S”-UpU by morpholine buffer at a 9/l B/BH+ ratio. Negative catalysis is seen, the rate decreasing as the buffer concentration increases. The solid curve is from a plot of Eq. 1 to fit these data.
20
0.2
0.4
0.6
Buffer Concentration
0.8
1.0
(M)
Fig. 4. Rate of cleavage of 3’,5”-UpU to form &dine-2’,3’-cyclic phosphate and uridine by morpholine buffer at a 9/ 1 B/BH+ ratio. The solid curve is a fit of Eq. 2 to the data; the dashed lines are the contributions from the first term (b) and the second term (c) of Eq. 2.
we show in Scheme 2, reversible protonation converts the phosphate anion to the phosphoric acid, a more electrophilic group, and then the buffer base B assists attack by the 2’ OH group to form the phosphorane anion intermediate 8. This then undergoes rate-determining pseudorotation followed by ring opening along the isomerization path, but the cleavage requires catalysis by the buffer base B in the second stage of the process.
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of Molecular Catalysis 91 (1994) 161-174
167
The base B - Im or morpholine - must be removing a proton from the phosphorane 8 to labilize it toward cleavage, but then it is likely that the resulting BH+ uses its proton to assist the departure of the leaving oxygen, as shown. We have explained elsewhere [ 13,14,19,20] that in such sequential processes the BH+ will be kinetically invisible, so the sequence seems to involve only base catalysis. There is thus no direct evidence for this BHf protonation step, but it is likely on the basis of microscopic reversibility. Addition of an alcohol to the cyclic phosphate 5, to form the phosphorane 8, should show catalysis by B just as in the attack of the 2’ OH group in Scheme 2. Thus the reverse reaction would have to show BHC catalysis.
3. What does this imply for the enzyme mechanism? In solution simple acid and base catalytic groups would normally have to act sequentially, not simultaneously, but of course an enzyme has no such restriction. Thus we have proposed a mechanism for the action of the enzyme ribonuclease A (Scheme 3) that is a simultaneous version of the one we outlined above. In it the protonation of the phosphate anion is simultaneous with the deprotonation and attack by the 2’ OH group, so the ImH+ does act as a simple general acid here while the other imidazole acts as a base. Further proton transfers lead to the cyclization product, with RNA chain cleavage. Then in a hydrolysis sequence the entire process is run backward, but substituting water for the leaving ROH group.
‘CH2 Base 0 His-12
His-12 k
I
.Jm
.-Am
-
: Z-proton shift
H:Lp&.-. >’ :, i ‘R
b
16
‘13
I
NH3+
I His-l 19
\ Lys‘ll
His-l 19
Z== NH; \ Lys-41
\ His-12
I
_
0\
/
0
.;P; 2.proton shlfr
+Hlm *I’ *I’
0. Im-...__
I
His-l 19
Scheme 3. Our new proposed
- HO,
R
mechanismfor
ribonuclease A catalysis
168
R. Breslow/Journal
of Molecular Catalysis 91 (1994) 161-174
As we have described [ 14,17,21], there Some data on the enzyme are better suited Karplus [ 221 finds this a better mechanism Thus this mechanistic study may well have is a model.
is some evidence supporting this mechanism. to this proposal, and a theoretical treatment by than the classical process previously invoked. furnished insight into the enzyme for which it
4. Catalysis by synthetic enzyme mimics Cyclodextrins (CDs) are useful building blocks for enzyme mimics. They have reasonable affinity for hydrophobic groups in water solution, so mimics of enzyme-substrate complexes can be formed. They are also easily derivatized to attach catalytic groups. We have used them to make a number of enzyme mimics over the years. A single functional group can be attached to the primary face of a CD by using the CD tosylate, selectively formed on a primary hydroxyl group. Reaction with imidazole affords /3-CD6imidazole 9 (P-cyclodextrin is the highly available cycloheptaamylose, with a cavity diameter of 7 A or so). With bridging reagents, it is also possible to selectively functionalize two different primary hydroxyl groups having a well defined geometric relationship [ 23,24 1. In this way we have prepared [24] (Scheme 4) P-CD-bisimidazole isomers with the two imidazole groups located on neighboring glucose units (the AB isomer lo), one glucose one unit apart (the AC isomer 11) or on glucoses separated by two units on one side, by three units on the other side of the seven glucose ring (the AD isomer 12). Some years ago we initiated a study of these compounds as catalysts for the hydrolytic cleavage of a cyclic phosphate ester. The substrate chosen was 13 (Scheme 5)) with a hydrophobic t-butylphenyl group that binds well into the P-CD cavity and a catechol cyclic phosphate that mimics the cyclic phosphate group in 7, but is somewhat more reactive. We found that all our cyclodextrin imidazoles were catalysts for this hydrolysis.
OH
10 substitutedon neighboring(A,B) glucoses 11 substitutedon A.C &xoses 12 substitutedon A,D glucoses
Scheme 4.
R. Breslow/Journal
of Molecular Catalysis 91 (1994) 161-174
169
phosphorme
?/OH
=OH (P 0-P-0'
I
Minor
product
Major
product
Scheme 5.
The first studies [ 10,111 were based on the idea that the classical mechanism for the enzyme was correct. If the base Im delivers water to the phosphate group while the acid ImH+ protonates the leaving oxygen, the preferred geometry for a bifunctional catalyst should have the two imidazoles mounted on opposite sides of the cyclodextrin cavity. The attacking water oxygen and leaving oxygen atom should be 180” apart, so the catalysts should also be far apart. We saw [ lo] that indeed the AD isomer 12 of CD-bisimidazole in which the two imidazoles are mounted 154” apart - was a bifunctional catalyst for the hydrolysis of 13. There was a bell-shaped pH vs. rate profile, with an optimum at pH 6.3 (cf., Fig. 5). In this early work we saw that the AC isomer 11 was also effective, but we did not examine the AB isomer 10.By the classical mechanism, two imidazoles mounted only 5 1” apart would not be suitable to catalyze the attack and departure of two oxygens that are 180” apart. However, with the new mechanistic work it was clear that this omission was a mistake. We expected that the enzyme mimic would use simultaneous bifunctional catalysis, just as the enzyme does, and the new mechanism requires that the Im deliver an attacking water molecule while the ImH+ puts a proton on the phosphate oxyanion (Scheme 5)) not on the leaving oxygen. The projection angle between an attacking water oxygen and the phosphate oxyanion is only 90”, not 180”. Because of the angles made by O-H bonds or hydrogen bonds, the catalytic groups can be much closer than 90” and still fit the transition state with its O-P-O angle of 90”. Molecular models and computer modelling indicated that the AB
R. Breslow / Journal of Molecular Catalysis 91 (1994) 161-l 74
170
4.0
5.0
6.0
7.0
6.0
PH
Fig. 5. The rate of cleavage of substrate 13 according to Scheme 4 by three different P-cyclodextrin-bisimidazoles, with imidazoles mounted on neighboring glucose units (AB), on glucose units one apart (AC), or on glucose units two units apart (AD). The rate optimum as a function of pH, a bell-shaped curve, indicates catalysis by one Im and one ImH+ group.
isomer 10 should be able to catalyze hydrolysis of 13 by the new mechanism, but not by the classical one. This prediction was correct. We saw (Fig. 5) that the AB isomer was not only a competent catalyst, it was the best of the three [ 241. This clearly indicates that the mechanism used is that of Scheme 5, as our mechanistic work had suggested.
5. Proton
inventory
as a proof of simultaneous
bifunctional
catalysis
There is a technique - called “proton inventory” [ 251 - for determining whether one proton or more are moving in the transition state of a reaction. The reaction is performed in H,O/D,O mixtures, in which all exchangeable protons will be deuterated to various extents. If one proton is “in flight” in the rate-determining step then it is expected that there will be a straight line plot of the rate versus the mole fraction composition of the solvent (cf., Fig. 7). If two protons are moving, the plot will be curved (cf., Fig. 6). In a sense, this plot is a function of the kinetic order in isotope effect, and is linear or curved just as a plot of rate versus catalyst concentration would be linear for one catalyst molecule in the transition state and curved for two (or more). This technique was applied to the enzyme ribonuclease A [ 261, and it was found that a curved plot was seen which could be fit with a theoretical line for two protons moving, with isotope effects of 1.75 each. This is consistent with the mechanism we have written in
R. Breslow / Journal of Molecular Catalysis 91 (1994) 161-I 74
171
‘.’ I
i
l.O-
O.Q-
0.8 -
2
3
0.7 -
0.6 -
0.5 -
0.4 -
0.3 -
0.2 I
. , . I , 1 . I . I . I. 0.0 0.1 0.2 0.3 0.4 0.5
I . I * 1 0.6 0.7 0.6
1 r I . 0.9 1 0
n; Mole Fraction Deuterium Oxide Fig. 6. The rate of cleavage of substrate 13 at kinetic saturation with catalyst 10 as a function of the H20/D20 composition of the solvent. The lower curved line fits the observed data if two protons are “in flight” in the ratedetermining step. The upper curve supports this; the plot of the square root of the relative rates fits a straight line.
3, in which two protons are moving in the simultaneous bifunctional acid-base catalysis. We applied the same technique to our AB isomer 10of CD-bisimidazole [27], and it also showed a curved plot (Fig. 6) that could be fit by the theoretical equation for two protons in flight. The isotope effects needed for this fit were 2.12 and 1.90, a little higher than those for the enzyme. Thus this is also consistent with the proposal that the CDbisimidazole enzyme mimic uses simultaneous bifunctional catalysis, just as in the enzyme. We were able to test the method in our case, although a similar thing is not possible with the enzyme. We examined [ 271 cleavage by CD-monoimidazole 9, which acts only as a base catalyst. We saw that in this case the plot (Fig. 7) was linear, confirming the idea that the monofunctional catalyst can perform only a monofunctional process in which one proton is being removed by the base group. This helps to confirm the validity of the method, and of the conclusions in our bifunctional case. Scheme
172
R. Breslow / Journal of Molecular Catalysis 91(1994) 161-174
ro
3
0.6 -
0.2 I .
1 . , . , . , . , . , . , . , . , * 1 . 1 . 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.6 0.9 1.0
n; Mole Fraction Deuterium Oxide Fig. 7. The rate of cleavage of substrate 13 at kinetic saturation with catalyst 9 as a function of the H,0/D20 composition of the solvent. The lower straight line fits the observed data if one proton is “in flight” in the ratedetermining step. The upper carve supports this; the plot of the square root of the relative rates does not fit a straight line.
6. Other reactions catalyzed by these enzyme mimics The availability of a set of three CD bisimidazoles 10-12 - and of the monoimidazole 9 as well - makes it possible to investigate other reactions that could be susceptible to bifunctional acid-base catalysis. We have just started on this program, but already have seen striking results. The first systems examined are ketones, whose conversion to the enol could show such bifunctional catalysis. It seems likely that enzymes that perform carbonyl chemistry in which enols are formed use both a base to remove the proton from carbon and an acid to protonate the oxygen, forming the enol rather than the enolate ion. We have found a similar process with our catalysts. The first example (Scheme 6) was a simple deuterium exchange into a ketone, p-tbutylacetophenone 14. Again the t-butylphenyl group binds well into the P-CD cavity in water solution, and then the imidazoles are in a position to catalyze enolization. If they remove a proton but then exchange rapidly with solvent before they catalyze the reverse reaction - ketonization - the result will be exchange of deuterium into the methyl group of
R. Breslow / Journal of Molecular Catalysis 91 (1994) 161-174
173
Scheme 6.
the ketone. We observed such deuteration at pH 6.2 and 35°C with all our catalysts, the CD monoimidazole and the three CD bisimidazoles [ 281. However, the geometric preference was quite different from that seen for phosphate cleavage. The AB 10 and AC 11 isomers of CD bisimidazole were not much more effective than was CD monoimidazole 9, although all three catalyzed the reaction under conditions in which essentially no deuteration was seen in the absence of catalyst. By contrast, the AD isomer 12 of the CD bisimidazole catalyst was clearly more effective than the others. It showed a bell-shaped pH vs. rate profile indicating that it indeed had its optimum rate at pH 6.3 or so, where the Im is expected to be ca. 50% protonated. It seems that the AB and AC isomers are fundamentally monofunctional base catalysts, but the AD isomer is a bifunctional catalyst. The preferred geometry is interesting, since molecular models suggested that the AC isomer 11 should be able to align the ImH+ directly with the unshared electron pair on the carbonyl oxygen that gets protonated in the enol, while the Im lines up with the C-H bond that has to break. The preference for the AD isomer means that the geometry of C-H cleavage is not linear; we explained the likely stereoelectronic reason for this in our publication [ 281. The finding that in this case the AD isomer is the best, while for phosphate cleavage the AB isomer was the most effective, adds support to our claim that these preferences reflect the geometric demands of the mechanisms for these processes. Very recently we have seen [29] that the same AD isomer 12 can catalyze an aldol condensation under these neutral conditions, conditions in which no reaction occurs in the absence of the catalyst. It seems likely that these CD bisimidazoles, and related compounds, will be able to perform many other reactions with selectivity under very mild conditions, acting as true enzyme mimics.
7. Conclusions Mechanistic studies demonstrate the pathways by which imidazole catalysts perform the cleavage of RNA and its accompanying isomerization. The path involves sequential acid-
R. Breslow/Journal
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of Molecular Catalysis 91 (1994) 161-174
base bifunctional catalysis; an analogous simultaneous pathway seems most likely as the mechanism for the enzyme ribonuclease A. This mechanistic work guided the synthesis of a set of enzyme mimics whose mechanisms of catalysis closely parallel that of the enzyme. These enzyme mimics can also catalyze other processes with geometric preferences; the geometric requirement for simultaneous bifunctional acid-base catalysis of enolization is completely different from that for phosphate ester hydrolysis, for reasons that are understood.
References [ 11 [2] [3] ]4] [5] [6] [7] [8] [9]
[ 101 [ 111 [ 121 [ 131 [ 141 [ 151 [ 161 [ 171 [ 181 [ 191 [20] [21] [22] [23] [24] [25] [26] [27] [28] [29]
D. van Aken, L.M. Paulissen and H.M. Buck, J. Am. Chem. Sot., 103 (1981) 3189. K.W. Abel1 and A.J. Kirby, J. Chem. Sot., Perkin Trans., 2 (1983) 1171. A.J. Kirby and M. Younas, J. Chem. Sot. B, (1970) 510. K. Taira, T. Fanni and D.G. Gorenstein, J. Am. Chem. Sot., 106 ( 1984) 1521. M.R. Eftink and R.L. Biltonen, Biochemistry, 22 (1983) 5134. D.G. Oakenfull, D.I. Richardson and D.A. Usher, J. Am. Chem. Sot., 89 ( 1967) 5491. K. Satoh and Y. moue, Chem Lett., ( 1972) 1097. C.H. Tung, Z. Weis, M.J. Labowitz and M.J. Stein, Proc. Natl. Acad. Sci. USA, 89 (1992) 7114. (a) K.N. Dalby, A.J. Kirby and F. Hollfelder, J. Chem. Sot. Perkin Trans., 2 (1993) 1269; (b) D.S. Sigman, A. Mazumder, and D.M. Perrin, Chem. Rev., 93 (1993) 2295. R. Breslow, J. Doherty, G. Guillot and C. Lipsey, J. Am. Chem. Sot., 100 (1978) 3227. R. Breslow, P. Bovy and C. Lipsey Hersh, J. Am. Chem. Sot., 102 (1980) 2115. R. Corcoran, M. Labelle, A.W. Czarnik and R. Breslow, Anal. Biochem., 144 (1985) 563. R. Breslow and M. Labelle, J. Am. Chem. Sot., 108 (1986) 2655. E. Anslyn and R. Breslow, J. Am. Chem. Sot., 111 (1989) 4473. (a) R. Breslow and D.-L. Huang, J. Am. Chem. Sot., 112 (1990) 9621; (b) R. Breslow, Accts. Chem. Res., 24 (1991) 317. F.H. Westheimer, Act. Chem. Res., 1 (1968) 70. R. Breslow and R. Xu, Proc. Natl. Acad. Sci. USA, 90 (1993) 1201. R. Breslow and R. Xu, J. Am. Chem. Sot., 115 (1993) 10705. R. Breslow, J. Chem. E&c., 67 (1990) 228. R. Breslow, Proc. Natl. Acad. Sci. USA, 90 (1993) 1208. R. Breslow, Deeng-Lih Huang and E. Anslyn, Proc. Natl. Acad. Sci. USA, 86 (1989) 1746. K. Haydock, C. Lim, A.T. Brllnger and M. Karplus, J. Am. Chem. Sot., 112 ( 1990) 3826. I. Tabushi, K. Shimokawa, H. Shirakata, and K. Fujita, J. Am. Chem. Sot., 98 (1976) 7855. E. Anslyn and R. Breslow, J. Am. Chem. Sot., 111 (1989) 5972. K.B. Schowen and R.L. Schowen, Methods Enzymol., 87 (1982) 551. M.S. Matta and D.T. Vo, J. Am. Chem. Sot., 108 (1986) 5316. E. Anslyn and R. Breslow, J. Am. Chem. Sot., 111 (1989) 8931. R. Breslow and A. Graff, J. Am. Chem. Sot., 115, (1993) 10988. R. Breslow and J. Desper, unpublished work.
MOLECULAR CATALYSIS Journal of Molecular Catalysis 91 ( 1994) 161-174
ELSEVIER
Review
Bifunctional acid-base catalysis by irnidazole groups in enzyme mimics Ronald Breslow Department
of Chemistry, Columbia University, New York, NY 10027, USA
(Received January 17, 1994; accepted February 17, 1994)
Abstract The cleavage of RNA can be catalyzed by imidazole buffer, and shows sequential bifunctional catalysis. When the two imidazole groups are attached to the same catalyst, as in enzymes or in cyclodextrin bis-imidazole derivatives, simultaneous bifunctional catalysis is seen. The geometric preference for such bifnnctional catalysis is revealed by the use of three isomeric cyclodextrin bis-
imidazoles. Different isomers are preferred for phosphate ester hydrolysis and for ketone enolization, and the preferences reveal details of the catalytic mechanisms. Keywords: acid-base catalysis; cyclodextrin; enolization; hydrolysis; imidazole groups; phosphate esters; ribonuclease A mimics
1. Introduction
Imidazole groups of the amino acid histidine are common in enzymatic catalysis. With a pKnear neutrality, imidazole (Im) groups are the strongest bases that can exist unprotonated at neutral pH, while their protonated forms - imidazolium cations (ImH+) - are the strongest acids that can exist under neutral conditions. Thus there has been considerable interest over the years in the catalytic properties of imidazole and its derivatives [ l-91. We were interested in building mimics of the enzyme ribonuclease A, which uses two imidazole rings of His- 12 and His- 119 as its principal catalytic groups. Our earliest work [ 10,111 involved construction of mimics based on the widely accepted mechanism for the enzyme. However, we then did mechanistic studies on the cleavage of RNA by simple imidazole buffer [ 12-151, and based on that work we concluded that an alternative mechanism was preferred in solution, and probably as well for the enzyme. In this review we will first *Corresponding author; fax. ( + l-212)9321289. 0304-5102/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDIO304-5102(94)00046-X
R. Breslow /Journal of Molecular Catalysis 91 (1994) 161-174
162
describe the mechanistic studies on RNA cleavage by imidazole buffers, then describe the bifunctional catalysts whose geometric preferences reflect these mechanistic results.
2. Bifunctional cleavage of RNA by imidazole buffers The cleavage of RNA by the enzyme ribonuclease A involves bifunctional catalysis by the imidazole groups of His- 12 and His- 119, one acting as a base while the other, protonated, acts as an acid. The result is a bell-shaped pH vs. rate profile, in which the rate reaches a maximum at pH 6.3 or so, where one imidazole is protonated and the other not. The mechanism of cleavage involves an ester exchange, converting the linear 3’S phosphate diester 1 to a cyclic 2’,3’ phosphate diester 2 by attack of the 2’ hydroxyl group (Scheme 1) . In a subsequent step this cyclic phosphate ester is hydrolyzed to a 3’ phosphate 3, with regeneration of the 2’ hydroxyl group. The same two imidazoles also catalyze this process. In the textbook mechanism for action of this enzyme, the Im removes a proton from the attacking 2’ OH group while the ImH + protonates the leaving 5” oxygen (Scheme 1) . This is only one possible role of the two catalytic groups; our mechanistic studies addressed the question of whether this was the best mechanism for Im/ImH+ bifunctional catalysis of phosphate diester cleavage. In our first study [ 131, polyuridylic acid was used as substrate. We saw that its cleavage was catalyzed by imidazole buffer, and with interesting kinetics. The rate of cleavage was simply first order in total buffer concentration (Fig. 1) , but the pH vs. rate profile was bell shaped (Fig. 2)) with a rate maximum at ca. 40% Im, 60% ImH+. The latter result showed that both Im and ImH+ were required for the catalysis, but the first order kinetics in total buffer indicated that the two species were not operating at the same time. Thus we were observing sequential bifunctional catalysis; one buffer component converted the substrate to an intermediate, and the other buffer component converted this intermediate to the cleaved product. However, simple kinetic methods could not establish which was the first catalyst, Im or ImH+, since the two alternative mechanisms are kinetically equivalent. \
CHz
\ Base
k 0
0 .o--‘P=O
0
H'lm
Im:
Im:
OH
0, I
O\
\ LH-“,H
Base
k 0
0,
Base
k
OH
bH
+
CH,
o
o-\p= 0
HO,
-o-y=0
0
o&‘P’ \ 20
_
W
OH
-o-+=0
O\ Scheme 1.Textbook mechanism
for RNA cleavage by ribonuclease
A.
R. Breslow / Journal of Molecular
I 0
.I
1
.
.
.
1000
500
163
Catalysis 91 (1994) 161-l 74
Total concentration
.
I
.
.
.
.
I. 2000
1500
of buffer (mM)
Fig. 1. Rate of cleavage of polyuridylic acid by imidazole buffer vs. the total buffer concentration at l/4 Im/ ImH+ and 3/4 Im/ImH+. The lines are theoretical curves using the same parameters as those for the line in Fig. 2.
0.0
0.2
0.4
0.6
0.8
1.0
State of protonation Fig. 2. Rate of cleavage of polyuridylic acid by imidazole buffer vs. mole fraction of the buffer as imidazolium ion. The corrected data are by subtraction of the rate at the same pH but zero buffer concentration. The curve is the theoretical line by fitting the data to an equation similar to that of EIq. 2. See [ 171 for details.
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164
Later studies [ 14,151 established the sequence in detail. In this work we studied the cleavage of simple nucleotide dimers, 3’,5”-UpU (uridyluridine) or 3’,5”-ApA (adenosyladenosine) (4)) and saw that imidazole buffer catalyzed two simultaneous processes. One was the conversion of 4 to the cyclic phosphate 5 and uridine 6, while the other was the conversion of 4 to the 2’S’ isomer 7. We saw that the cleavage reaction showed a bellshaped pH vs. rate profile, just as it had with polyU substrate, but the isomerization was catalyzed only by ImH+. Of course one possible interpretation is that the two processes have completely independent pathways, but we found evidence that this is not the case. The other possibility is that the two paths branch not from the starting material but instead from the phosphorane intermediate that had been required to explain our cleavage kinetics. A reaction with two steps must have an intermediate, in this case a phosphorane 8 that is the only sensible intermediate between a linear phosphate diester and a cyclic phosphate ester. Kinetic arguments [ 131 indicated that the phosphorane cleavage intermediate had to be the monoanion, 8, as shown. Other evidence [ 161 indicates that a phosphate migration from the 3’ to the 2’ oxygen must also proceed through a phosphorane intermediate that has enough lifetime to undergo a process known as pseudorotation. If the two pathways branch from this common phosphorane intermediate, as shown in Scheme 2, we could solve the kinetic ambiguity in the cleavage reaction. Since the isomerization of 4 to 7 was catalyzed only by ImH+, that must be the catalyst for the common first step in which 4 is converted to 8. Thus the question was simply whether indeed these reactions branch from a common phosphorane intermediate, as in Scheme 2. Other kinetic evidence indicated that this was the case. When we examined reactions with varying buffer ratios, we found that the isomerization reaction was not simply uncatalyzed by Im, it showed negative catalysis. That is, increasing
Ho\
ImH’ caralys~s
CHz Base 0
k 0
OH H’
-o-‘PC0
i==
!
O\H,
Base
k 0
HO 4
OH
3'.5"-upu or ApA
Ho\
CH,
0
Z.5”.upu or ApA
-HI
0
Base
Base
(a) 0
P OS-P-OH
0
‘p’o (a 5
Scheme 2.
0
‘P’ o” lo.
HO?&0
6R
Base
k 0
ImH’
HO
I
CHz
k- k
CHz
,
Ho\
Ho\
7.3’ cyclic phosphate
+
R-OH nucleoside 6
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of Molecular Catalysis 91 (1994) 161-174
165
the concentration of Im while keeping ImH+ constant led to a slowing of the isomerization reaction (but not of the cleavage reaction, which went faster). Of course changing the buffer ratio causes a change in pH, but we corrected for this effect by the normal method of extrapolating the rate to zero buffer concentration with a given buffer ratio. This negative catalysis by Im is exactly as expected for a reaction pathway in which ImH+ converts the substrate to the phosphorane 8, and then two paths branch from it. The isomerization does not show further buffer catalysis, but the cleavage does. The mechanism described is shown in Scheme 2, and the kinetic equations that derive from this mechanism (with added terms for other catalytic species) are Eqs. 1 and 2. As those equations make clear, increasing the concentration of Im will slow the isomerization reaction because [ Im] appears only in the denominator of its equation (but also in the numerator of the equation for cleavage).
ksomerization =
klk3[BH+]
k_,[BH+]
+k,[B]
+k;k,
(1)
+k3 +k,
of UpU, B = imidazole or morpholine k cleavage=
hkz[BH+l[Bl k-l [BH+]
+k,[B]
+G +k3 +k,
+
k’[B] +k”[BH+] k_, [BH+]
+k,[B]
+k, +k,
(2)
of upu. Physically, increasing the [ Im] decreases the concentration of the intermediate 8, because it speeds its conversion to the cleavage products. The negative rate effect is clear evidence that the two paths branch from a common intermediate, and that it is Im that is the second catalyst in the cleavage sequence since that is the species that made a negative contribution to the isomerization rate. There was really no doubt about these conclusions, but they were confirmed in another recent study [ 17,181. In this case we used morpholine buffer, not imidazole buffer. Morpholine is more basic than imidazole, so we expected that the negative catalytic effect of the basic buffer component - which is the key piece of evidence about the mechanism would be even clearer in this case. By using a high B/BH+ ratio we were able to see a negative catalytic effect without having to make any corrections for changing pH effects. That is, we increased the concentration of the buffer but kept a constant B/BH+ ratio and constant ionic strength so the pH was unchanged. We saw that the rate of isomerization decreased (Fig. 3) as the buffer concentration increased, but the rate of cleavage (Fig. 4) increased as expected. Thus there was a negative catalytic effect by the buffer itself, not just by its base component. However, the negative effect was much smaller with a lower B/BH+ ratio, making it clear that it was the basic component that caused it. The data fit very well to theoretical lines generated from the appropriate equations. What detailed mechanism do these results indicate? They show that the conversion of 4 to the intermediate 8 is catalyzed by BHC, but for such general acid catalysis there are always two mechanistic possibilities. One is that the BH+ acts directly, and the other is that there is an indirect mechanism - reversible protonation followed by catalysis by the basic form B. This is called the specific acid-general base sequence, and it is completely kinetically equivalent to the general acid one-step mechanism. It is the sequence we invoke. As
R. Breslow / Journal
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of Molecular Catalysis 91 (1994) 161-174
K ._ Y
0.4
1
0.2
I
0.4
I
0.6
Buffer Concentration
I
0.8
1
1.0
(M)
Fig. 3. Rate of isomerization of 3’5”-uridyluridine (UpU) to 2’S”-UpU by morpholine buffer at a 9/l B/BH+ ratio. Negative catalysis is seen, the rate decreasing as the buffer concentration increases. The solid curve is from a plot of Eq. 1 to fit these data.
20
0.2
0.4
0.6
Buffer Concentration
0.8
1.0
(M)
Fig. 4. Rate of cleavage of 3’,5”-UpU to form &dine-2’,3’-cyclic phosphate and uridine by morpholine buffer at a 9/ 1 B/BH+ ratio. The solid curve is a fit of Eq. 2 to the data; the dashed lines are the contributions from the first term (b) and the second term (c) of Eq. 2.
we show in Scheme 2, reversible protonation converts the phosphate anion to the phosphoric acid, a more electrophilic group, and then the buffer base B assists attack by the 2’ OH group to form the phosphorane anion intermediate 8. This then undergoes rate-determining pseudorotation followed by ring opening along the isomerization path, but the cleavage requires catalysis by the buffer base B in the second stage of the process.
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167
The base B - Im or morpholine - must be removing a proton from the phosphorane 8 to labilize it toward cleavage, but then it is likely that the resulting BH+ uses its proton to assist the departure of the leaving oxygen, as shown. We have explained elsewhere [ 13,14,19,20] that in such sequential processes the BH+ will be kinetically invisible, so the sequence seems to involve only base catalysis. There is thus no direct evidence for this BHf protonation step, but it is likely on the basis of microscopic reversibility. Addition of an alcohol to the cyclic phosphate 5, to form the phosphorane 8, should show catalysis by B just as in the attack of the 2’ OH group in Scheme 2. Thus the reverse reaction would have to show BHC catalysis.
3. What does this imply for the enzyme mechanism? In solution simple acid and base catalytic groups would normally have to act sequentially, not simultaneously, but of course an enzyme has no such restriction. Thus we have proposed a mechanism for the action of the enzyme ribonuclease A (Scheme 3) that is a simultaneous version of the one we outlined above. In it the protonation of the phosphate anion is simultaneous with the deprotonation and attack by the 2’ OH group, so the ImH+ does act as a simple general acid here while the other imidazole acts as a base. Further proton transfers lead to the cyclization product, with RNA chain cleavage. Then in a hydrolysis sequence the entire process is run backward, but substituting water for the leaving ROH group.
‘CH2 Base 0 His-12
His-12 k
I
.Jm
.-Am
-
: Z-proton shift
H:Lp&.-. >’ :, i ‘R
b
16
‘13
I
NH3+
I His-l 19
\ Lys‘ll
His-l 19
Z== NH; \ Lys-41
\ His-12
I
_
0\
/
0
.;P; 2.proton shlfr
+Hlm *I’ *I’
0. Im-...__
I
His-l 19
Scheme 3. Our new proposed
- HO,
R
mechanismfor
ribonuclease A catalysis
168
R. Breslow/Journal
of Molecular Catalysis 91 (1994) 161-174
As we have described [ 14,17,21], there Some data on the enzyme are better suited Karplus [ 221 finds this a better mechanism Thus this mechanistic study may well have is a model.
is some evidence supporting this mechanism. to this proposal, and a theoretical treatment by than the classical process previously invoked. furnished insight into the enzyme for which it
4. Catalysis by synthetic enzyme mimics Cyclodextrins (CDs) are useful building blocks for enzyme mimics. They have reasonable affinity for hydrophobic groups in water solution, so mimics of enzyme-substrate complexes can be formed. They are also easily derivatized to attach catalytic groups. We have used them to make a number of enzyme mimics over the years. A single functional group can be attached to the primary face of a CD by using the CD tosylate, selectively formed on a primary hydroxyl group. Reaction with imidazole affords /3-CD6imidazole 9 (P-cyclodextrin is the highly available cycloheptaamylose, with a cavity diameter of 7 A or so). With bridging reagents, it is also possible to selectively functionalize two different primary hydroxyl groups having a well defined geometric relationship [ 23,24 1. In this way we have prepared [24] (Scheme 4) P-CD-bisimidazole isomers with the two imidazole groups located on neighboring glucose units (the AB isomer lo), one glucose one unit apart (the AC isomer 11) or on glucoses separated by two units on one side, by three units on the other side of the seven glucose ring (the AD isomer 12). Some years ago we initiated a study of these compounds as catalysts for the hydrolytic cleavage of a cyclic phosphate ester. The substrate chosen was 13 (Scheme 5)) with a hydrophobic t-butylphenyl group that binds well into the P-CD cavity and a catechol cyclic phosphate that mimics the cyclic phosphate group in 7, but is somewhat more reactive. We found that all our cyclodextrin imidazoles were catalysts for this hydrolysis.
OH
10 substitutedon neighboring(A,B) glucoses 11 substitutedon A.C &xoses 12 substitutedon A,D glucoses
Scheme 4.
R. Breslow/Journal
of Molecular Catalysis 91 (1994) 161-174
169
phosphorme
?/OH
=OH (P 0-P-0'
I
Minor
product
Major
product
Scheme 5.
The first studies [ 10,111 were based on the idea that the classical mechanism for the enzyme was correct. If the base Im delivers water to the phosphate group while the acid ImH+ protonates the leaving oxygen, the preferred geometry for a bifunctional catalyst should have the two imidazoles mounted on opposite sides of the cyclodextrin cavity. The attacking water oxygen and leaving oxygen atom should be 180” apart, so the catalysts should also be far apart. We saw [ lo] that indeed the AD isomer 12 of CD-bisimidazole in which the two imidazoles are mounted 154” apart - was a bifunctional catalyst for the hydrolysis of 13. There was a bell-shaped pH vs. rate profile, with an optimum at pH 6.3 (cf., Fig. 5). In this early work we saw that the AC isomer 11 was also effective, but we did not examine the AB isomer 10.By the classical mechanism, two imidazoles mounted only 5 1” apart would not be suitable to catalyze the attack and departure of two oxygens that are 180” apart. However, with the new mechanistic work it was clear that this omission was a mistake. We expected that the enzyme mimic would use simultaneous bifunctional catalysis, just as the enzyme does, and the new mechanism requires that the Im deliver an attacking water molecule while the ImH+ puts a proton on the phosphate oxyanion (Scheme 5)) not on the leaving oxygen. The projection angle between an attacking water oxygen and the phosphate oxyanion is only 90”, not 180”. Because of the angles made by O-H bonds or hydrogen bonds, the catalytic groups can be much closer than 90” and still fit the transition state with its O-P-O angle of 90”. Molecular models and computer modelling indicated that the AB
R. Breslow / Journal of Molecular Catalysis 91 (1994) 161-l 74
170
4.0
5.0
6.0
7.0
6.0
PH
Fig. 5. The rate of cleavage of substrate 13 according to Scheme 4 by three different P-cyclodextrin-bisimidazoles, with imidazoles mounted on neighboring glucose units (AB), on glucose units one apart (AC), or on glucose units two units apart (AD). The rate optimum as a function of pH, a bell-shaped curve, indicates catalysis by one Im and one ImH+ group.
isomer 10 should be able to catalyze hydrolysis of 13 by the new mechanism, but not by the classical one. This prediction was correct. We saw (Fig. 5) that the AB isomer was not only a competent catalyst, it was the best of the three [ 241. This clearly indicates that the mechanism used is that of Scheme 5, as our mechanistic work had suggested.
5. Proton
inventory
as a proof of simultaneous
bifunctional
catalysis
There is a technique - called “proton inventory” [ 251 - for determining whether one proton or more are moving in the transition state of a reaction. The reaction is performed in H,O/D,O mixtures, in which all exchangeable protons will be deuterated to various extents. If one proton is “in flight” in the rate-determining step then it is expected that there will be a straight line plot of the rate versus the mole fraction composition of the solvent (cf., Fig. 7). If two protons are moving, the plot will be curved (cf., Fig. 6). In a sense, this plot is a function of the kinetic order in isotope effect, and is linear or curved just as a plot of rate versus catalyst concentration would be linear for one catalyst molecule in the transition state and curved for two (or more). This technique was applied to the enzyme ribonuclease A [ 261, and it was found that a curved plot was seen which could be fit with a theoretical line for two protons moving, with isotope effects of 1.75 each. This is consistent with the mechanism we have written in
R. Breslow / Journal of Molecular Catalysis 91 (1994) 161-I 74
171
‘.’ I
i
l.O-
O.Q-
0.8 -
2
3
0.7 -
0.6 -
0.5 -
0.4 -
0.3 -
0.2 I
. , . I , 1 . I . I . I. 0.0 0.1 0.2 0.3 0.4 0.5
I . I * 1 0.6 0.7 0.6
1 r I . 0.9 1 0
n; Mole Fraction Deuterium Oxide Fig. 6. The rate of cleavage of substrate 13 at kinetic saturation with catalyst 10 as a function of the H20/D20 composition of the solvent. The lower curved line fits the observed data if two protons are “in flight” in the ratedetermining step. The upper curve supports this; the plot of the square root of the relative rates fits a straight line.
3, in which two protons are moving in the simultaneous bifunctional acid-base catalysis. We applied the same technique to our AB isomer 10of CD-bisimidazole [27], and it also showed a curved plot (Fig. 6) that could be fit by the theoretical equation for two protons in flight. The isotope effects needed for this fit were 2.12 and 1.90, a little higher than those for the enzyme. Thus this is also consistent with the proposal that the CDbisimidazole enzyme mimic uses simultaneous bifunctional catalysis, just as in the enzyme. We were able to test the method in our case, although a similar thing is not possible with the enzyme. We examined [ 271 cleavage by CD-monoimidazole 9, which acts only as a base catalyst. We saw that in this case the plot (Fig. 7) was linear, confirming the idea that the monofunctional catalyst can perform only a monofunctional process in which one proton is being removed by the base group. This helps to confirm the validity of the method, and of the conclusions in our bifunctional case. Scheme
172
R. Breslow / Journal of Molecular Catalysis 91(1994) 161-174
ro
3
0.6 -
0.2 I .
1 . , . , . , . , . , . , . , . , * 1 . 1 . 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.6 0.9 1.0
n; Mole Fraction Deuterium Oxide Fig. 7. The rate of cleavage of substrate 13 at kinetic saturation with catalyst 9 as a function of the H,0/D20 composition of the solvent. The lower straight line fits the observed data if one proton is “in flight” in the ratedetermining step. The upper carve supports this; the plot of the square root of the relative rates does not fit a straight line.
6. Other reactions catalyzed by these enzyme mimics The availability of a set of three CD bisimidazoles 10-12 - and of the monoimidazole 9 as well - makes it possible to investigate other reactions that could be susceptible to bifunctional acid-base catalysis. We have just started on this program, but already have seen striking results. The first systems examined are ketones, whose conversion to the enol could show such bifunctional catalysis. It seems likely that enzymes that perform carbonyl chemistry in which enols are formed use both a base to remove the proton from carbon and an acid to protonate the oxygen, forming the enol rather than the enolate ion. We have found a similar process with our catalysts. The first example (Scheme 6) was a simple deuterium exchange into a ketone, p-tbutylacetophenone 14. Again the t-butylphenyl group binds well into the P-CD cavity in water solution, and then the imidazoles are in a position to catalyze enolization. If they remove a proton but then exchange rapidly with solvent before they catalyze the reverse reaction - ketonization - the result will be exchange of deuterium into the methyl group of
R. Breslow / Journal of Molecular Catalysis 91 (1994) 161-174
173
Scheme 6.
the ketone. We observed such deuteration at pH 6.2 and 35°C with all our catalysts, the CD monoimidazole and the three CD bisimidazoles [ 281. However, the geometric preference was quite different from that seen for phosphate cleavage. The AB 10 and AC 11 isomers of CD bisimidazole were not much more effective than was CD monoimidazole 9, although all three catalyzed the reaction under conditions in which essentially no deuteration was seen in the absence of catalyst. By contrast, the AD isomer 12 of the CD bisimidazole catalyst was clearly more effective than the others. It showed a bell-shaped pH vs. rate profile indicating that it indeed had its optimum rate at pH 6.3 or so, where the Im is expected to be ca. 50% protonated. It seems that the AB and AC isomers are fundamentally monofunctional base catalysts, but the AD isomer is a bifunctional catalyst. The preferred geometry is interesting, since molecular models suggested that the AC isomer 11 should be able to align the ImH+ directly with the unshared electron pair on the carbonyl oxygen that gets protonated in the enol, while the Im lines up with the C-H bond that has to break. The preference for the AD isomer means that the geometry of C-H cleavage is not linear; we explained the likely stereoelectronic reason for this in our publication [ 281. The finding that in this case the AD isomer is the best, while for phosphate cleavage the AB isomer was the most effective, adds support to our claim that these preferences reflect the geometric demands of the mechanisms for these processes. Very recently we have seen [29] that the same AD isomer 12 can catalyze an aldol condensation under these neutral conditions, conditions in which no reaction occurs in the absence of the catalyst. It seems likely that these CD bisimidazoles, and related compounds, will be able to perform many other reactions with selectivity under very mild conditions, acting as true enzyme mimics.
7. Conclusions Mechanistic studies demonstrate the pathways by which imidazole catalysts perform the cleavage of RNA and its accompanying isomerization. The path involves sequential acid-
R. Breslow/Journal
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of Molecular Catalysis 91 (1994) 161-174
base bifunctional catalysis; an analogous simultaneous pathway seems most likely as the mechanism for the enzyme ribonuclease A. This mechanistic work guided the synthesis of a set of enzyme mimics whose mechanisms of catalysis closely parallel that of the enzyme. These enzyme mimics can also catalyze other processes with geometric preferences; the geometric requirement for simultaneous bifunctional acid-base catalysis of enolization is completely different from that for phosphate ester hydrolysis, for reasons that are understood.
References [ 11 [2] [3] ]4] [5] [6] [7] [8] [9]
[ 101 [ 111 [ 121 [ 131 [ 141 [ 151 [ 161 [ 171 [ 181 [ 191 [20] [21] [22] [23] [24] [25] [26] [27] [28] [29]
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