Isotactic polyethylenimines induce formation of L-amino acids in transamination

Article abstract of DOI:10.1021/ol0630604

(Chemical Equation Presented) Isotactic polyethylenimines with (S)-benzyl side chains were synthesized from 4-(S)-4-benzyl-2-oxazolines. When α-keto acids were subjected to transamination in the presence of this polymer, and a pyridoxamine coenzyme modified with hydrophobic chains, enantioselectivity toward the natural isomer (L > D) was observed, followed by racemization of the amino acid products. However, the racemization did not occur when the coenzyme was covalently attached to the polymer.

Full text of DOI:10.1021/ol0630604

ORGANIC
LETTERS
2007
Vol. 9, No. 6
1009-1012
Isotactic Polyethylenimines Induce
Formation of -Amino Acids in
Transamination
L
Subhajit Bandyopadhyay, Wenjun Zhou, and Ronald Breslow*
Department of Chemistry, Columbia UniVersity, 3000 Broadway,
New York, New York 10027
rb33@columbia.edu
Received December 18, 2006
ABSTRACT
Isotactic polyethylenimines with (S)-benzyl side chains were synthesized from 4-(S)-4-benzyl-2-oxazolines. When
r
-keto acids were subjected
to transamination in the presence of this polymer, and a pyridoxamine coenzyme modified with hydrophobic chains, enantioselectivity toward
the natural isomer ( ) was observed, followed by racemization of the amino acid products. However, the racemization did not occur when
L
> D
the coenzyme was covalently attached to the polymer.
Natural enzymes catalyze reactions with regio- and ste-
reospecificity. Chemists, inspired by the natural enzymes,
try to mimic them in order to develop synthetic systems that
can achieve high selectivity in chemical transformations.1
Achievement of high chiral induction in the amino acid
products from transaminase mimics has attracted a number
of research groups worldwide.
used a molecularly imprinted polymer6 as transaminase
mimics. Our group has achieved a high degree of chiral
induction in transamination reactions with pyridoxamine
systems having rigidly attached chiral side chains,7 and more
recently, enzyme mimics consisting of pyridoxamines co-
valently linked to reduced peptide have induced moderate
to high chirality.8
Besides our work in this field, Schultz used catalytic
antibodies,2 Imperiali used polypeptide-pyridoxamine chi-
meras,3 Destefano used an adipocyte lipid binding protein,4
Jorgensen used metal-chiral ligand systems,5 and Nicholls
Transaminase enzymes perform a series of general acid
and general base catalyzed reactions in chiral environments
to afford chiral amino acids.9 Polyethylenimines (PEIs) titrate
over a range of pH 3-13, having both effective amino group
(1) Breslow, R. Artificial Enzymes; Breslow, R., Ed.; Wiley-VCH:
Weinheim, Germany, 2005.
(2) Pham, T. R.; Schultz, P. G.; Sugasawara, R.; Schultz, P. G. J. Am.
Chem. Soc. 1991, 113, 6670-6672.
(3) Imperiali, B.; Roy, R. S. J. Am. Chem. Soc. 1994, 116, 12083-12084.
(4) Kuang, H.; Brown, M. L.; Davies, R. R.; Young, E. C.; Distefano,
M. D. J. Am. Chem. Soc. 1996, 118, 10702-10706.
(5) Bachman, S.; Knudsen, K.; Jorgensen, K. A. Org. Biomol. Chem.
2004, 2, 2044-2049.
(6) Svenson, J.; Zheng, N.; Nicholls, I. A. J. Am. Chem. Soc. 2004, 126,
2004, 8554-8560.
(7) Zimmerman, S. C.; Breslow, R. J. Am. Chem. Soc. 1984, 106, 1490-
1491.
(8) Zhou, W. J.; Yerkes, N.; Chruma, J. J.; Liu, L.; Breslow, R. Bioorg.
Med. Chem. Lett. 2005, 15, 1351-1355.
(9) (a) Bruice, T. C.; Benkovic, S. Bioorganic Mechanisms; W. A.
Benjamin: New York, 1966; Vol. 2, Chapter 8. (b) Walsh, C. Enzymatic
Reaction Mechanisms; W. H. Freeman: San Francisco, 1979; Chapter 24.
10.1021/ol0630604 CCC: $37.00
© 2007 American Chemical Society
Published on Web 02/23/2007

general bases and ammonium group general acids near
neutrality, and thus can act as efficient transaminase mimics.
Recently, we have described transaminase mimics consisting
of pyridoxamines covalently linked to branched PEIs,
carrying long-chain alkyl chains. They converted R-keto
acids to amino acids with up to a 240000-fold rate enhance-
ment relative to the reaction with simple pyridoxamine at
the same total pyridoxamine concentration.10-12 We have also
used noncovalent polymer-pyridoxamine systems as enzyme
mimics, in which pyridoxamines carrying hydrophobic side
chains boundsalong with the substratessinto hydrophobic
regions of the PEIs.13 The latter showed even better rate
enhancement, as much as 725000-fold with the hydrophobic
indolepyruvic acid that forms tryptophan.14 In all of these
cases, the polymer, being achiral, formed racemic mixtures
of the product amino acids.
Scheme 2. Synthesis of 1
ylenimine) 2 with a base (Scheme 2), following Saegusa’s
method.16,17 The methyl group of the butoxy group served
as the NMR probe for the determination of degree of
polymerization. Complete absence of the formyl groups in
the hydrolyzed product 1 was confirmed by IR spectroscopy,
where the strong formyl CdO stretching at 1668 cm-1 of 2
1
Incorporating the features of hydrophobicity in a chiral
environment, and general acid-general base catalyst, we
have briefly described a transaminase mimic with non-
covalently bound pyridoxamine-chiral polyethylenimine
systems (Scheme 1).15
disappeared, and also by H NMR spectroscopy, where the
broad formyl proton peak around δ 7.7 of the precursor was
absent.
The average degree of polymerization (DP) with 2 mol
% of initiator (compared to the oxazoline) was ca. 50, with
3 mol % was ca. 30, and with 6 mol % was ca. 13. The
polydispersity of the chiral PEI’s determined by GPC were
1.01, 1.07, and 1.20 for the 50-mer, 30-mer, and the 13-
mer, respectively. The low polydispersity values are in
accordance with the highly living nature of the polymeriza-
tion, where the DP values are very close to the oxazoline/
initiator ratio.18 Prior to our work, Goodman et al. had
synthesized optically active polyoxazolines (including the
50 mer 2) following Saegusa’s method, and from circular
dichroism studies had shown that polyoxazolines obtained
via this kind of cationic ring opening polymerization are
isotactic in nature.19
Scheme 1. Transamination with 1a
The (S)-benzyl side chains of the polymers 1 can form a
chiral hydrophobic pocket in which the nonpolar alkyl chains
of the pyridoxamine coenzyme 413 can bind, and thus, the
transamination reaction can take place in a chiral environ-
ment, as in the natural system, to give chiral induction in
the amino acid products. Indeed, high enantioselectivity was
achieved when the 50-mer in ca. 40% aqueous methanol (v/
v%) at pH 7.3-7.8 produced L/D valine with a ratio of 83/
17 (ca. 5-7% conversion), but unfortunately the amino acid
racemized at higher conversion. The ratio of L/D valine with
the 30- and 13-mers were slightly less (78/22 and 71/29,
respectively). Enantioselectivity at the initial stage of tran-
samination, followed by racemization of the amino acid
products, was also reported by Bernauer with Cu(II) com-
plexes of C2-symmetric ligands and Schiff bases of keto acids
and pyridoxamine.20
a Reaction conditions: 5.0 × 10-4 mol/L of pyridoxamine 4, 10
g/L of PEI, 5 × 10-4 EDTA, 5 × 10-3 mol/L of 3-methyl-2-
oxopyruvic acid, t ) 20-25 °C. pH values reported here are as
read with a glass-electrode calibrated against aqueous buffers.
The chiral PEI 1 with (S)-benzyl side chains was synthe-
sized from 4-(S)-4-benzyl-2-oxazoline 3 via a cationic ring-
opening polymerization using methyl tosylate as the initiator
and 1-butanol as the terminator and subsequent deformylation
of the N-formamide groups of poly(N-formyl-1-benzyleth-
Interestingly, racemization was never observed with py-
ridoxamines that were covalently attached to other catalytic
units, including amines.7,8 We believe that the Schiff base
(10) Liu, L.; Rozenman, M.; Breslow, R. J. Am. Chem. Soc. 2002, 124,
12660-12661.
(11) Zhou, W. J.; Liu, L.; Breslow, R. HelV. Chim. Acta 2003, 86, 3560-
3567.
(12) Liu, L.; Breslow, R. J. Am. Chem. Soc. 2002, 124, 4978-4979.
(13) Liu, L.; Zhou, W. J.; Chruma, J.; Breslow, R. J. Am. Chem. Soc.
2004, 126, 8136-8137.
(16) Saegusa, T.; Fujii, H.; Ikeda, H. Polym. J. 1972, 3, 35-39.
(17) Saegusa, T.; Fujii, H.; Ikeda, H. Macromolecules 1972, 5, 108.
(18) Kobayashi, S.; Iijima, S.; Igarashi, T.; Saegusa, T. Macromolecules
1987, 20, 1729-1734.
(14) Chruma, J. J.; Liu, L.; Zhou, W.; Breslow, R. Bioorg. Med. Chem.
2005, 13, 5873-5883.
(19) Oh, Y. S.; Yamazaki, T.; Goodman, M. Macromolecules 1992, 25,
6322-6331.
(15) Breslow, R.; Bandyopadhyay, S.; Levine, M.; Zhou, W. Chembio-
Chem 2006, 7, 1491-1496.
(20) Chuard, T.; Gretillat, F.; Bernauer, K. Chimia 1993, 47, 215-217.
1010
Org. Lett., Vol. 9, No. 6, 2007

product between a pyridoxal and the amino acid can move
into water and racemize before hydrolysis. Thus in order to
overcome the problem of racemization, we have now
explored pyridoxamine systems that are covalently attached
to a chiral isotactic linear polyethylenimine carrying (S)-
benzyl side chains. When the cofactor is covalently attached
to the polymer, it is in a chiral environment where racem-
ization is slow.
reaction time or the conversion did not affect the relative
amounts of the two enantiomers to any significant extent,
which indicated that the problem of racemization that was
observed in the noncovalent system was eliminated with this
covalently attached system. Under the same conditions, when
phenylpyruvic acid was subjected to transamination, the ratio
of L/D phenylalanine was found to be 69/31 ( 2 %, an
increase that can be attributed to better hydrophobic binding
of this keto acid to the polymer-pyridoxamine system.
4-Hydroxyphenylpyruvic acid, which is less hydrophobic
than phenylpyruvic acid, gave a ratio of 63/37 ( 2% of the
L/D tyrosine. Thus, with this system the highest enantio-
selectivity so far was achieved with phenylalanine, reflecting
its stronger interaction with the hydrophobic polymer.
We examined whether a shorter spacer, instead of a
thiopropionyl spacer, could bring the pyridoxamine reaction
center closer to the chiral units and increase the stereo-
selectivity. Thus, we synthesized the chiral PEI-pyridox-
amine system 6 via a substitution reaction of the Boc-
protected pyridoxamine dimesyl ester 7 with the chiral PEI,
and subsequent deprotection of the mesyl group with sodium
ethoxide in ethanol, followed by the deprotection of the Boc
group with trifluoroacetic acid (Scheme 4). The resulting
PEI 1 was acylated with the N-hydroxysuccinimide ester
of 3,3′-dithiopropionic acid (Scheme 3). The disulfide bonds
Scheme 3. Synthesis of 5
Scheme 4. Synthesis of 6
were reduced with sodium borohydride to produce the
thiolate groups in situ, which were then alkylated using the
5-bromomethyl analogue of pyridoxamine to yield the chiral
PEI-pyridoxamine system 5. The polymer was precipitated
from a solution of sodium carbonate to remove any unbound
3-thiopropionic acid and pyridoxamine impurities, centri-
1
fuged, washed with water, and lyophilized. From the H
pyridoxamine-PEI 6 was purified by the method descibed
previously. The H NMR spectrum of 6 indicated that ca.
8% of the polymer nitrogens were attached to the pyridox-
amine (UV-vis measurement indicated 9%).
When phenylpyruvic acid was transaminated in the pres-
ence of this system under conditions identical to those
described above, the ratio of L/D phenylalanine was 71/29
( 3%, essentially the same as obtained with the PEI-
pyridoxamine system 5. Changing the length of the tether
did not enhance the chiral induction significantly, and both
of these systems are less effective in inducing chirality than
the noncovalently bound pyridoxamine (4)-chiral PEI (1)
NMR integration, it was found that ca. 9% of the polymer
nitrogens carried the pyridoxamine units. From the intensity
of the UV-vis spectrum of 5 at 318 nm, comparing it with
the absorption of a standard solution of pyridoxamine, the
amount of pyridoxamine attached to the polymer was
estimated to be ca. 10% of the maximum possible value, in
reasonable agreement with the NMR results.
When the sodium salt of 3-methyl-2-oxobutanoic acid was
subjected to transamination with the chiral PEI-pyridoxamine
system 5 in aqueous methanol (35-40% v/v) at pH 5.5 in
the presence of EDTA, and the product, after derivatization
with o-phthalaldehyde and N-acetylcysteine, was analyzed
by reversed-phase HPLC,21 an enantiomeric ratio of 58/42
( 3% of valine (L > D) was observed. In this case, the
1
(21) Liu, L.; Rozenman, M.; Breslow, R. Bioorg. Med. Chem. 2002, 10,
3973-3979.
Org. Lett., Vol. 9, No. 6, 2007
1011

PEI 1 was partially N-acylated using 0.2 equiv of 4-tert-
butylbenzoyl chloride to give polymer 8 (Scheme 5). The
NMR spectrum indicated that about 15% of the polymer
nitrogens were acylated. Polymer 8 was then alkylated with
5-deoxymethyl-5-bromomethylpyridoxamine in dichloro-
methane-DMF in the presence of sodium carbonate to give
9. When 0.25 equiv (with respect to polymer 8) of 5-deoxy-
methyl-5-bromomethylpyridoxamine was used, it was found
that only ca. 8% of the polymer nitrogens of 9 were attached
to the pyridoxamines. Removal of the 4-tert-butylbenzoyl
groups of the polymer 9 with potassium hydroxide in ethanol
gave 10.
Scheme 5. Synthesis of 10
When phenylpyruvic acid was subjected to transamination
with polymer 10, the ratio of L/D phenylalanine was found
to increase up to 76/24 ( 3% (highest 79/21).
The efficacy of PEI chains in transamination with 5, 6,
and 10 is established by the rate accelaration relative to
pyridoxamine without any PEI under identical conditions.
Polymer 5, 6, and 10 showed rate enhancement of the
pseudo-first-order reactions with factors of 1369, 1178, and
1021, respectively, at pH 5.5. The agreement between
different runs was within 15%.
Thus, in this work, we have demonstrated enantioselec-
tivity in transamination reaction with a new isotactic PEI in
a less than fully aqueous environment. The reversibly bound
systems were somewhat more effective than the covalently
linked version, but the reversible systems showed product
racemization that was not seen in the covalent cases.
Regeneration experiments to afford turnover, as we have
described with our nonchiral systems,13,14 are currently in
progress.
system. However, the benefit of using the covalent systems
5 and 6 was that the problem of racemization encountered
in the noncovalent system was solved here.
We envisaged that one reason for the lower values of chiral
induction in these cases (with 5 and 6) could be that the
pyridoxamine coenzyme 4 was able to bind to the most
hydrophobic part of the polymer, whereas with the covalently
bound pyridoxamine-polymer systems 5 and 6, the mobility
of the pyridoxamine moiety is lost and it may link to the
most accessible part of the polymer, not necessarily the most
catalytically effective part. We thus decided to block the more
accessible part of the chiral PEI by acylating it, so that the
pyridoxamines could be attached to the interior part of the
polymer.
Acknowledgment. We thank Ms. Meghann White for her
help with the measurement of the polydispersity. We thank
the NSF and NIH for financial support of this work.
Supporting Information Available: Experimental pro-
cedures and characterization data for the polymers. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL0630604
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Org. Lett., Vol. 9, No. 6, 2007