Enzymes, Abzymes, ChemzymesâTheozymes? - ACS Symposium

Chapter 5

Enzymes, Abzymes, Chemzymes—Theozymes? Claudia Müller, Li-Hsing Wang, and Hendrik Zipse

Downloaded by UNIV OF ARIZONA on August 7, 2012 | http://pubs.acs.org Publication Date: April 8, 1999 | doi: 10.1021/bk-1999-0721.ch005

Institute of Organic Chemistry, TU Berlin, Str. d. 17. Juni 135, D-10623 Berlin, Germany

The reaction of amines with esters has been investigated theoretically as well as experimentally. The uncatalyzed reaction proceeds through a direct displacement pathway for esters with good leaving groups. The reaction barrier is mainly caused by unfavorable proton transfer geometries. The reaction can be accelerated substantially through bifunctional catalysts such as 2pyridones to such an extent, that proton transfer is not rate limiting anymore. Formation of a tetrahedral zwitterionic structure, which becomes rate limiting in the pyridone-catalyzed case, can be facilitated through the presence of a specifically oriented second pyridone molecule. 1. Introduction. In recent years, various new approaches for the development of catalysts that rival enzymes in terms of their reactivity and selectivity have been devised. The concept of catalytically active antibodies (1,2) ("antibody enzymes", "abzymes") takes advantage of the ability of the immune system to produce antibodies which specifically bind a selected antigen. Provided a hapten can be found that resembles the transition state of a reaction, one must expect that antibodies raised against this hapten are catalytically active for the respective reaction. Following a more traditional approach combining mechanistic insight and empirical optimization, the development of man-made catalysts ("chemzymes") (3) has probably been the most successful route to develop catalysts for many organic transformations. The development of catalysts can also be based on theoretical studies of the influence of various functional groups on the mechanism of a given reaction. This "theozyme" approach (4) has been pioneered by Houk et al. and applied to

© 1999 American Chemical Society

In Transition State Modeling for Catalysis; Truhlar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

61

reactions as diverse as the Diels-Alder reaction, the intramolecular ring opening of epoxides, and the ring opening of isoxazoles. In all these examples, the theoretical studies provide a mechanistic basis for the rationalization of the catalytic activities of catalytic antibodies in these reactions. We are now trying here to use the theozyme approach to develop catalysts for the reaction of amines with esters, in which a peptide bond is formed. In order to keep the experimental work as close to the theoretical studies as possible all experiments discussed in the following have been conducted in a medium of low polarity (chlorobenzene). The reaction of primary amines with active esters such as /?ara-nitrophenyl actetate has been studied in apolar media already in quite some detail before.


OH (1) N0

2

Polyethers as well as various basic compounds have been found to accelerate the rate of the reaction (5,6). In the following we will first take a look at theoretical results from model studies for the uncatalyzed reaction. As a second step, catalysts for the ester aminolysis will be derived from the theoretical results following various strategies. 2. Theoretical study of the uncatalyzed reaction The smallest possible model system for the reaction of esters with amines consists of ammonia (1) and formic acid (2). This system has been studied at several different levels of theory in the gas phase (7,8). In all cases the reaction has been found to proceed through two distincly different reaction pathways, which can best be described as "addition/elimination" and "direct substitution" (Figure 1). The addition/elimination pathway involves formation of an uncharged tetrahedral intermediate 6 through addition of ammonia to the formic acid C - 0 double bond. Elimination of water (3) through transition state 7 yields formamide (4). The same products are formed in a single kinetic step from the reactants through transition state 8 for the direct displacement pathway. Somewhat surprisingly, the reaction barriers are comparable for both pathways. In order to verify that both pathways are viable options in more realistic model systems, the reaction between phenyl acetate (10) and methyl amine (9) has been studied at the same level of theory (Figure 2). In this larger model system the barriers for the addition/elimination and the direct displacement pathways are reduced by 6.9 and by 15.6 kcal/mol, respectively, relative to the parent system shown in Figure 1. This leaves the direct substitution process as the preferred mode of action in this system. The structure as well as the charge distribution of transition state 12 was consequently used in the development of an A M B E R force field representation of this transition state that can be used to investigate the interaction of 12 with potential catalysts. The complex between 12 and tetraglyme depicted in Figure 3 was obtained in this way (8).


63

+42.1

HN Downloaded by UNIV OF ARIZONA on August 7, 2012 | http://pubs.acs.org Publication Date: April 8, 1999 | doi: 10.1021/bk-1999-0721.ch005

3

H

O A HO H

•

1

2

HO

1 HN

3

H

2

4

OH

H--0

Q-H H NH 6

HN

+39.0

•

H0

2

OH

+41.5

2

NH

2

7 Figure 1. Addition/elimination and direct substitution pathways in the reaction of ammonia with formic acid. Energy differences between reactants and transition states are given in kcal/mol at the MP2/6-31G(d,p)//HF/6-31G(d,p) + AZPE level of theory.

O

x H N-CH 2

9

H-0

H-N-W11

+32.1

0

3

+

hbCÔ 10

HaC,

V

C H

3 /=\

7

12

+26.6

Figure 2. Transition states for the addition/elimination and direct substitution pathways in the reaction of methyl amine (9) with phenyl acetate (10). Energy differences between reactants and transition states are given in kcal/mol at the MP2/6-31G(d,p)//HF/6-31G(d,p) + AZPE level of theory.



64

Figure 3. Complex between tetraglyme and transition state 12 as optimized with the A M B E R force field. Using this force field approach, the interaction energy between various catalysts and the transition state can be estimated. The difference between transition state and ground state complexation energy should form a sufficient basis for the evaluation of the catalytic potential of various catalysts. The catalytic potential of a number of polyethers and polyalcohols has been evaluated using this force field approach. The estimated barrier lowering has then been compared to the experimentally determined catalytic rate constant for reaction of butyl amine with para-nitrophenyl acetate (Figure 4). Unfortunately, no good correlation between theoretically predicted and experimentally determined catalytic activity could be found. The limited predictive accuracy of this approach apparent from Figure 4 might be due to several factors. First of all, the differences in catalytic activity found experimentally are rather small and the intrinsic inaccuracy of the force field used might simply not allow for a prediction of relative barriers to within 0.5 kcal/mol. Inspection of the ground state complexes formed by polyalcohols also points to the fact that reactions involving catalysts with hydroxyl groups might proceed through a different pathway, eliminating 12 as a good representation of the rate limiting transition state. This suggests that an accurate prediction of the catalytic activity of a wide range of structurally diverse catalysts is only possible under the condition that the transition structure is reoptimized for each new catalyst (8). 2. Pyridone Catalysis A second approach for the de novo design of catalysts based on the results of quantum chemical calculations involves an analysis of the barrier formation of the uncatalyzed reaction. For the reaction of methyl amine with para-nitrophenyl acetate shown in Figure 5, this turns out to be remarkably easy. While formation of the C-N bond and cleavage of the C - 0 bond starts quite early along the reaction coordinate, no proton transfer can be observed before the transition state. It is only



65

Figure 4. Correlation between the theoretically predicted and experimentally found catalytic efficiency of various polyethers and polyalcohols in the reaction of butyl amine with /?ara-nitrophenyl acetate in chlorobenzene at room temperature.


66


after this point that N to O proton transfer commences, indicating the difficulty of this part of the overall process.

R(C-N) [A]

2.43

1.80

1.57

1.45

1.37

R(C-O) [A]

1.41

1.51

1.98

2.30

2.35

R(N-H) [A]

1.00

1.01

1.03

1.32

2.10

Figure 5. Selected structural data along the direct substitution pathway for the reaction of methyl amine with para-nitrophenyl acetate as calculated at the HF/321G level of theory.

Inspection of the transition state structure involved in this reaction reveals a rather acute proton transfer angle of 115°, far from the optimum value of 180°. This suggests that inclusion of any structural motive that leads to a more favorable proton transfer angle will lower the reaction barrier and thus function as a catalyst. This goal can be achieved in several ways as shown schematically in Figure 6. Inclusion of a structure X - H , in which X may be any electronegative atom, will lead to a six-membered ring transition structure, in which proton transfer occurs through a more favorable geometry. The ideal situation of collinear proton transfer can, however, only be achieved with a bridging unit C(=X)YH as shown in Figure 6. In this structure the center X acts as a proton donor, while the center Y delivers a proton to the ester leaving group. One additional requirement for these Afunctional catalysts is that the two possible tautomers should not be too different energetically and that the catalyst be neither strongly acidic nor basic in order to avoid unwanted side reactions. One compound that fits all these requirements is 2pyridone (13) that can exist as either tautomer 13a or 13b, with 13a slightly preferred in the gas phase (9,10). The catalytic potential of 13 was evaluated theoretically in the reaction of methyl acetate with methyl amine as model substrates.


67

Bifunctional Catalysis

H

>c;-> ci-a R

H

0--H--Y Ar'

> R


Ar Catalysis by X-H

v

N

O

H

H

13a

H

13b

O-H'

Ar

Figure 6. Two different strategies for the improvement of the proton transfer step in the reaction of amines with esters.

a H

H :

: H

,Oi| ,

Me^'N^

lr

u

O

14

M Me e

_

/

^ +12.8

+12.4

|

H

Ô.//

H

o

\

15

Figure 7. Most favorable reaction pathways in the pyridone-catalyzed reaction of methyl acetate with methyl amine as calculated at the Becke3LYP/6-31G(d)// HF/3-21G + AZPE level of theory. Energies are given in kcal/mol.



68 Several different reaction pathways have been investigated (9), and the most favorable pathway shown in Figure 7 corresponds to a fully conceited mechanism, in which the products are formed in a single kinetic step starting from the reactant complex. In this case there is little difference between the catalytic ability of tautomer 13a, which leads to transition state 15, and tautomer 13b, which leads to transition state 14. Despite the fact that the reaction is concerted in both cases, the double proton transfer involved in these reactions is decidedly asynchronous. While proton transfer from pyridone to the methoxide leaving group is halfway complete in 14 as well as 15, the second proton transfer from methyl amine back to the pyridone molecule has not yet begun. This indicates that the energetically most demanding step in the pyridone catalyzed reaction of methyl amine with methyl acetate corresponds to proton transfer from pyridones to the methoxy leaving group. Somewhat surprisingly then, pyridones function as acids in this reaction.

Table 1. Theoretically predicted and experimentally found catalytic activity of pyridones in the reaction of n-butyl amine with /?ara-nitrophenyl acetate in chlorobenzene at room temperature.

N

H-O

H CN

01

ex xx 6. ^N 13^ O H

^ N 16< ^ O H

d

At A

=0.0

SÔH

^NÔH

-1.24

-0.6

-2.4 (R=H)

15.1 ±7%

170 ±32%

1.4 ± 5 % (R=C6H-| )

[kcal/mol] kcat^M-V ]

5.0 ±13%

1

K[M' ] 1

Solubility [mM] a

3

71 (±30%) 246

3.0

0.3

>4.0 (R=C Hi3) 6

b

Exerimental results from Ref. 9. Experimental results from Ref. 11

Based on this observation catalysts with improved activity can be generated by simply enhancing the acidity through introduction of electron withdrawing substituents into the pyridone ring. Reinvestigation of the reaction shown in Figure 7 using several cyano-substituted pyridones supports this hypothesis. The relative barriers given in Table 1 correspond to the reaction pathway through the



69 pyridone N-H-tautomers as in transition structure 15. Relative activation barriers have been calculated at the Becke3LYP/6-31G(d)//HF/3-21G + AZPE level of theory and the barrier for the unsubstituted pyridone 13 has been taken as the point of reference. Introduction of a cyano group at the C4-position of the pyridone ring system as in 17 is predicted to lead to a small barrier lowering of 0.6 kcal/mol, while introduction of a cyano group in the 3-position as in 18 leads to a much larger effect. Introduction of an additional methyl group in the 6-position as in 16 exerts steric effects, which reduces the catalytic efficiency somewhat relative to that for pyridone 18. In order to test these theoretical predictions the reaction of butyl amine with /?ara-nitrophenyl acetate has again been investigated experimentally in chlorobenzene and the pyridones shown in Table 1 (as well as some other pyridones not shown here) have been used as catalysts. How do the theoretical predictions compare to the experimental results? Introduction of a cyano group does indeed lead to improved catalytic activities, as is most evident for pyridone 17. The catalytic rate constant for this catalyst is more than 30 times larger than that for the parent pyridone 13. Unfortunately, the high catalytic potential of pyridone 18 did not materialize in the experiments, indicating a lack of correlation between the theoretically predicted and the experimentally found catalytic efficiencies for substituted pyridones in this study. Moreover, kinetic measurements are complicated through two properties of pyridones that have not been apparent in the modeling studies. The first complication arises from the formation of dimers that bind substantial amounts of the catalyst in a catalytically unreactive dimeric form. That the equilibrium constant for dimer formation could only be determined in some of the cases is related to the low solubility of some of the substituted pyridones in chlorobenzene, which represents the second problem encountered here. This problem is most severe for catalyst 17 that can hardly be dissolved in chlorobenzene at all.

3. Catalysis by Bispyridones The lack of correlation between the theoretically predicted and the experimentally determined catalytic activity described in Table 1 suggests that the reaction between methyl amine and methyl acetate might not be representative enough for the experiments performed with much more reactive esters. The catalytic potential of pyridone 13 was therefore also studied theoretically for the reaction of methyl amine with /?

Enzymes, Abzymes, ChemzymesâTheozymes? - ACS Symposium

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Enzymes, Abzymes, ChemzymesâTheozymes? - ACS Symposium