High rates and substrate selectivities in water by polyvinylimidazoles as transaminase enzyme mimics with hydrophobically bound pyridoxamine derivatives as coenzyme mimics

Article abstract of DOI:10.1021/ja9072589

(Chemical Equation Presented) Free-radical polymers of 4-vinylimidazole and copolymers with 1-dodecyl-4-vinylimidazole were used as enzyme mimics to transaminate pyruvic acid to alanine, phenylpyruvic acid to phenylalanine, and indole-3-pyruvic acid to tryptophan in water at pH 7.5 and 20 °C using pyridoxamines carrying hydrophobic side chains as coenzyme mimics. The best enzyme mimic accelerated the transamination of indole-3-pyruvic acid by a factor of 4 million relative to the rate without the polymer, a higher rate ratio than we had previously achieved with a polyaziridine-based enzyme mimic. The properties of various polyvinylimidazoles were compared, including those prepared with the RAFT modification of the polymerization process.

Full text of DOI:10.1021/ja9072589

Published on Web 10/13/2009
High Rates and Substrate Selectivities in Water by Polyvinylimidazoles as
Transaminase Enzyme Mimics with Hydrophobically Bound Pyridoxamine
Derivatives as Coenzyme Mimics
Rachid Skouta, Sujun Wei, and Ronald Breslow*
Department of Chemistry, Columbia UniVersity, New York, New York 10027
Received August 29, 2009; E-mail: rb33@columbia.edu
We have studied enzyme mimics for many years,1 including
those that performed transamination of ketoacids to amino acids.
Our systems normally focused on the first step, the reaction of a
pyridoxamine with the ketoacid to form a pyridoxal and the amino
acid, although we have also shown how this can be part of a
complete catalytic cycle by using decarboxylative transamination
of 2,2-disubstituted glycines to convert the pyridoxal back to the
pyridoxamine form.2 In our recent work, we used polyaziridines
to mimic the transaminase enzyme itself.3 Klotz,4 Suh,5 and Kirby6
had used those polymers to mimic hydrolytic enzymes.
Using 1 mol % azobis(isobutyronitrile) (AIBN) for the polymers
listed in Table 1 (except for 5 mol % AIBN for the polymers in
entries 9 and 10 of the table, as noted), we obtained a mixture of
20- to 40-mer polymers along with some methanol-insoluble larger
polymers (for the mass spectrum, see the Supporting Information)
and used the entire polymer mixture to catalyze the reaction of
pyridoxamine A (Scheme 2) with pyruvic acid to form alanine and
pyridoxal in water. The rate of the reaction at pH 7.5 and 20 °C
was followed by the appearance of the UV spectrum of the product
pyridoxal (see Table 1 for the data and the reaction conditions), as
we have described previously.2 We saw that the reaction was 100-
fold faster with a 37.5 mg/L P4VIm mixture (entry 2) than without
the polymer (entry 1).
We used commercially available polyaziridines, as did Klotz,
Suh, and Kirby, and compared the results for different sizes of the
polymers, different N-alkylations of the nitrogens using alkyl groups
from methyl to dodecyl, and different amounts of such alkylations.
We used either covalently attached pyridoxamine derivatives or,
even better, reversibly bound pyridoxamines with hydrophobic side
chains as coenzyme mimics.7 We saw that the most effective
polymers had a limited amount of dodecylation; with enough we
achieved a hydrophobic core in the polymer, while with too much
the materials were insoluble in water.
Scheme 1. Synthesis of P4VIm and Partially Dodecylated P4VIm
We used water as the solvent to take advantage of hydrophobic
binding of substrates and cofactors into the polymer as well as the
higher rates achieved when the reaction is performed in an interior
nonaqueous core of the polymer, as with natural enzymes. We
achieved an increase by a factor of as much as 350 000 in the rate
of transamination of hydrophobic phenylpyruvic acid to form
phenylalanine relative to the rate of the same process and same
pyridoxamine in water without the polymer. An even higher
acceleration was observed with indolepyruvic acid to form tryptophan.
We then turned to the use of hydrophobic derivatives of
pyridoxamine and copolymers of 4-vinylimidazole with 1-dode-
cylated-4-vinylimidazole (obtained from 4-vinylimidazole and
dodecyl iodide; the NMR spectrum indicated that it was 83%
4-vinyl-1-dodecylimidazole and 17% 5-vinylimidazole). We pre-
pared the pyridoxamine derivatives B-E (Scheme 2) as described
previously2 and examined them with simple P4VIm (entries 3-6
in Table 1); we also used cofactor E with copolymers of 4-vi-
nylimidazole and its dodecylated monomer (entries 11-15). We
also examined the use of the reversible addition-fragmentation
chain transfer (RAFT)-modified radical polymerization of 4-vi-
nylimidazole, which normally affords a more monodisperse poly-
mer, and saw by MALDI-TOF that this changed somewhat the
distribution of polymer but did not lead to significantly less
polydispersity. The modified (entry 10) and unmodified polymers
We also used polyaziridines with thiazolium and imidazolium
coenzyme mimics carrying hydrophobic side chains to catalyze the
benzoin condensation of two benzaldehyde molecules in water.8
In the transaminase mimics, the polymer furnished general acid
and general base catalysis of the reaction using the partially
protonated main-chain amino groups. However, the benzoin
condensation was not subject to general acid or base catalysis but
simply to electrostatic catalysis by positive charges on the amino
groups of the polyaziridines. In this report, we will describe the
contrasting results and even better catalyses of the transamination
reactions using a polymer series different from the polyaziridines.
Overberger9 studied polyvinylimidazoles (PVIm’s) formed by
radical polymerization of either 4-vinylimidazole or 1-vinylimida-
zole. We were attracted to these compounds because they also
incorporate base (imidazole) and acid (imidazolium) catalytic
species and have side chains large enough to have some hydro-
phobic binding character of their own. We synthesized the polymer
of 4-vinylimidazole (P4VIm, Scheme 1) according to the Over-
berger procedure (see the Supporting Information) and characterized
the methanol soluble fraction by MALDI-TOF mass spectrometry.
Scheme 2. Structures of P1VIm (Table 1, Entry 7) and
Pyridoxamine Derivatives A-E
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10.1021/ja9072589 CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Kinetic Study of the Transamination Reaction under Various Conditionsa
entry
pyridoxamine
polymer
ktransamination (min-1
)
krelative
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
A
A
B
C
D
E
E
E
E
E
E
E
E
E
E
no polymer
P4VIm
P4VIm
P4VIm
(1.4 ( 0.3) × 10-6
1.00b
100
500
(1.4 ( 0.2) × 10-4
(7.0 ( 0.2) × 10-4
(1.3 ( 0.1) × 10-2
(3.3 ( 0.2) × 10-2
(1.7 ( 0.1) × 10-1
(31.2 ( 0.1) × 10-3
(1.2 ( 0.1) × 10-2
(1.4 ( 0.1) × 10-1
(1.5 ( 0.1) × 10-1
(1.2 ( 0.1) × 10-1
(1.3( 0.1) × 10-1
(1.1 ( 0.1) × 10-1
(6.9 ( 0.1) × 10-2
-
9300
P4VIm
23600
121400
22300
8600b
100000
107200
85700
92900
78600
49300
-
P4VIm 0% dodecylated
P1VIm 0% dodecylated
PEI 0% dodecylated
P4 VIm 0% dodecylatedc
P4 VIm 0% dodecylatedd
P4VIm 2.3% dodecylated
P4VIm 4.5% dodecylated
P4VIm 10.1% dodecylated
P4VIm 14.8% dodecylated
P4VIm 30% dodecylated
a Reaction conditions: 1.5 × 10-4 mol/L pyridoxamine, 37.5 mg/L polymer, 5.0 × 10-3 mol/L pyruvic acid, 2.0 × 10-3 mol/L EDTA, 20 °C, pH 7.5.
b Data from ref 2. c Polymerization was done with 5 mol % AIBN instead of the normal 1 mol %. d Polymerization was done with 5 mol % AIBN
instead of the normal 1 mol % and 8 mol % chain-transfer agent (CTA) reagent.
(entry 9) had similar rates with cofactor E. We found that simple
P4VIm with cofactor E (entry 6) was 14-fold more effective as a
catalyst than our polyaziridine without dodecyl groups under the
same conditions (entry 8). Poly(1-vinylimidazole) (P1VIm) was also
prepared (see the Supporting Information), and the transamination
rate was determined to be enhanced 22 300-fold (entry 7), meaning
that P1VIm is only 2.6-fold more effective than the polyaziridine
(entry 8).
of oxazoline ionic polymerization, as we employed previously with
phenylalanine derivatives.11 Since those ionic polymers are not only
almost monodisperse but also isotactic, retaining the chirality of
the parent amino acids, analogues derived from histidine could well
be polyimidazoles with useful chiral selectivities for products and
starting materials.
Acknowledgment. We thank the NIH and NSF for financial
support of this work. R.S. has an FQRNT (Fonds Que´be´cois de la
Recherche sur la Nature et les Technologies) postdoctoral fellow-
ship. We thank Dr. Mary Ann Gawinowicz from the Protein Core
Facility at Columbia University College of Physicians & Surgeons
for the MALDI-TOF experiments.
Our copolymers with dodecyl groups are more effective with
phenylpyruvic acid than with simple pyruvic acid. This reflects the
better binding of a hydrophobic substrate into the more hydrophobic
polymer. The reaction of phenylpyruvic acid was too rapid to follow
by the UV method under our conditions, so we determined the
selectivity of a competition reaction using 30:30:1 phenylpyruvic
acid/pyruvic acid/pyridoxamine derivative mixture to form a
phenylalanine/alanine ratio that was determined by HPLC, as
previously.2 We observed a ratio of 3:1 for the reaction using simple
P4VIm and (19 ( 1):1 for the reaction with the copolymer having
4.5% dodecylated monomer. The latter is slightly higher than the
14:1 ratio we had seen previously with the dodecylated polyaziri-
dine.2 Since amination by this imidazole copolymer (entry 12) was
92 900-fold more rapid than the reaction without polymer, and since
we had shown previously that simple pyridoxamines aminate
pyruvic acid and phenylpyruvic acid at the same rate,10 this means
that the acceleration of the amination of phenylpyruvic acid by the
polymer with cofactor E is (1.77 ( 0.1) × 106 (i.e., almost
2-million-fold). The amination of indolepyruvic acid, forming
tryptophan, was accelerated (3.9 ( 0.1) × 106-fold (i.e., 4-million-
fold) under the same conditions.
Supporting Information Available: Synthesis procedures and
pertinent spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Artificial Enzymes; Breslow, R., Ed.; Wiley-VCH: Weinheim, Germany,
2005.
(2) Liu, L.; Zhou, W. J.; Chruma, J.; Breslow, R. J. Am. Chem. Soc. 2004,
126, 8136.
(3) (a) Zhou, W. J.; Liu, L.; Breslow, R. HelV. Chim. Acta 2003, 86, 3560. (b)
Liu, L.; Breslow, R. Bioorg. Med. Chem. 2004, 12, 3277.
(4) Delaney, E. J.; Wood, L. E.; Klotz, I. M. J. Am. Chem. Soc. 1982, 104,
799.
(5) Suh, J. Synlett 2001, 1343, and references therein.
(6) Hollfelder, F.; Kirby, A. J.; Tawfik, D. S. J. Org. Chem. 2001, 66, 5866.
(7) (a) Liu, L.; Breslow, R. J. Am. Chem. Soc. 2002, 124, 4978. (b) Liu, L.;
Rozenman, M.; Breslow, R. J. Am. Chem. Soc. 2002, 124, 12660.
(8) Zhao, H.; Foss, F. W.; Breslow, R. J. Am. Chem. Soc. 2008, 130, 12590.
(9) (a) Overberger, C. G.; Vorcheheimer, N. J. Am. Chem. Soc. 1963, 85, 951.
(b) Overberger, C. G.; Corett, R.; Salamone, J. C.; Yaroslavsky, S.
Macromolecules 1968, 1, 331. (c) Overberger, C. G.; Shen, C.-M. Bioorg.
Chem. 1971, 1, 1. (d) Overberger, C. G.; Glowaky, R. C.; Vandewyer,
P. H. J. Am. Chem. Soc. 1973, 95, 6008.
The use of these polyvinylimidazoles in the benzoin condensation
with thiazolium and imidazolium coenzyme mimics is under
investigation. We are also pressing forward with an attempt to
produce polymers with smaller polydispersity, including the use
(10) Breslow, R.; Hammond, M.; Lauer, M. J. Am. Chem. Soc. 1980, 102, 421.
(11) Bandyopadhyay, S.; Zhou, W. J.; Breslow, R. Org. Lett. 2007, 9, 1009.
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