DOI: 10.1002/cbic.200600120
Water Exclusion and Enantioselectivity in
Catalysis**
Ronald Breslow,* Subhajit Bandyopadhyay, Mindy Levine, and Wenjun Zhou[a]
Water Exclusion in Enzymes
Enzymes are large molecules. A number of arguments can be
offered as to why this should be so, including the need for a
large protein, consisting of a linear sequence of amino acids,
to have a preferential folding into the biologically effective
three-dimensional structure. There are also arguments that the
dynamics of segment motion in a large protein can contribute
to catalytic effectiveness. However, there is another argument
for the large molecules characteristic of enzymes that is quite
convincing—their size and structure let reactions occur inside
a hydrophobic, nonaqueous, and nonpolar region of the protein while the outside of the protein has polar groups and is
compatible with the aqueous solvents.
Two interesting effects result from this. On the one hand,
substrates that have hydrophobic groups will tend to bind in
the nonpolar interior of the protein, where the catalytic groups
can be located. On the other hand, water is in one respect an
enemy of rate, even though the hydrophobic effect depends
on water for its positive contribution to reaction rates. In a
ACHTUNGREreaction in which acid and base groups play a catalytic role,
water that is hydrogen bonded to these groups must normally
be removed before they can effectively act on the substrate.
The energy cost of desolvation of these catalytic groups—and
perhaps also desolvation of the substrates—can slow the reaction considerably. For this reason it has often been proposed
that the interior of the protein, without any water molecules
or highly polar groups in it, is a better place to perform a catalyzed reaction and that such a medium will increase the rates
of the processes.
As we have described, we synthesized our transaminase
mimics by using a variety of polyethylenimines. In the first
system, we used a rather large polyethylenimine with a
number-average molecular weight of about 60 000 and with
high polydispersity; the weight-average molecular weight was
750 000.[1] This commercial polymer has about 1400 nitrogens;
about 25 % of them are primary amino groups, 50 % are secondary amines, and 25 % are tertiary amines. We alkylated about
10 % of the nitrogens with alkyl halides ranging from methyl
iodide up to dodecyl iodide, and then acylated about 5 % of
the nitrogens with a dipropionyldisulfide that we used to
attach the pyridoxamine. We reductively methylated the remaining NH groups, then reduced the disulfide linkages and
attached pyridoxamines to the resulting thiols, which were covalently linked to the polymers. The schematic of this polymer
(1), and the structure of pyridoxamine (2), are shown in
Scheme 1.
We then compared the ability of a pyridoxamine unit in this
polymer to the ability of a simple pyridoxamine molecule in
solution as a transaminase mimic, converting keto acids to aamino acids. The process of transamination is quite well understood. As Scheme 2 shows, it involves a sequence of acid- and
base-catalytic steps in which protons are successively added
Water Exclusion in Polymeric Enzyme Mimics
In order to explore this, we have synthesized a number of
enzyme mimics based on polymers.[1–7] Our most extensive
studies have been directed to branched polyethylenimines, a
group of commercial polymers whose catalytic properties have
been previously studied by Klotz,[8, 9] by Suh,[9, 10] and by
Kirby.[11] They explored hydrolysis and cleavage reactions, while
we have been interested in synthetic processes. In our first
studies,[1–5] we examined the amination of keto acids by pyridoxamine units attached to the polymers. Later, we examined
the even more effective system in which the pyridoxamine
units were reversibly bound to the hydrophobic core of such a
polymer, in imitation of the binding of coenzymes by the natural enzymes.[6]
ChemBioChem 2006, 7, 1491 – 1496
Scheme 1. A pyridoxamine unit attached to a polyethylenimine carrying
ACHTUNGREdodecyl chains (1) and pyridoxamine (2).
[a] Prof. Dr. R. Breslow, Dr. S. Bandyopadhyay, M. Levine, W. Zhou
Department of Chemistry, Columbia University
New York, NY 10027 (USA)
Fax: (+ 1)
E-mail: [email protected]
[**] Based on a lecture delivered by Ronald Breslow at the Symposium on The
Interface between Chemistry and Biology, PacifiChem 2005, Honolulu
Hawaii
@ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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R. Breslow et al.
Scheme 2. Transamination of a keto acid by pyridoxamine derivatives.
and subtracted from reaction intermediates. Under most conditions, the first few steps are reversible, and the rate-limiting
step is the one in which a proton is removed from the methylene group of the fourth structure in the sequence. This sequence does not even show the full range of steps in which
proton transfers are involved, since at the end of the sequence,
the imine group in the penultimate product must be hydrolyzed by acid and base catalysis to generate a pyridoxal structure and the product a-amino acid.
Polyethylenimines are very interesting species. The nitrogens
are so close together that the polymer titrates with acids over
a very broad pH range, starting from almost pH 13 on the
basic side for the first added proton and going all the way
down to pH 3 before the last proton can be added. This is the
result of electrostatic repulsions by the positively charged protonated amino groups. There is about 50 % protonation of the
polymer at pH 8, and at this pH it contains the strongest bases
and acids that can exist in equilibrium. Any stronger acids
would lose a proton to the medium, and any stronger base
would already have picked up a proton from the medium. In
natural enzymes, this situation is normally achieved by using
catalytic groups such as the imidazole rings of the a-amino
acid histidine, which has a pKa close to the normal operating
pH of the enzyme. In polyethylenimines, this desirable situation is achieved because of electrostatic repulsion. As a result,
we find that our catalysts show essentially no buffer catalysis
in transaminations, whereas simple pyridoxamine does show
strong catalysis by buffer species. The polyethylenimine catalyst species have their own internal buffer acid and base
groups.
When we examined the transamination of pyruvic acid to
form alanine, we found that our catalyst carrying long dodecyl
hydrocarbon chains gave a 2300-fold acceleration relative to
simple pyridoxamine in buffer solution at the same pH, but
the external buffer was catalyzing the simple reaction. When
the buffer concentration was extrapolated to zero, the actual
advantage of the enzyme mimic was 10 000-fold for each pyridoxamine unit compared with a simple pyridoxamine species
itself in aqueous solution. We achieved some amine catalysis
by simply taking a pyridoxamine and attaching monoamines,
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diamines, or even tetramines to
it, but the maximum acceleration was of the order of 68-fold
in water, not the 10 000-fold of
our polymer. The polymer also
imposes an important medium
effect.
We saw that the acceleration
by our polymer/pyridoxamine
species in transaminating pyruvate depended strongly on the
size of the chains that were attached in the polymers. With
simple methyl groups, the reaction was not nearly as rapid as it
was with the dodecyl chains,
and with C-15 or C-18 chains the
rate enhancement was slightly higher than with the C-12
chains. Thus a big proportion of the acceleration we see with
our material has to do with the presence of these hydrophobic
chains. When we plotted the rate of transamination of pyruvate as a function of the concentration of the pyruvate, kinetic
saturation was observed, with the rate leveling off at high pyruvate concentrations.
This is exactly what one sees in normal enzyme reactions, in
which the rate levels off as the enzyme becomes saturated
with substrate; our artificial enzyme is also being saturated.
We observed what is generally called Michaelis–Menten kinetics, which can be analyzed in terms of the rate constant for
the reaction of the catalyst/substrate complex and the equilibrium constant for dissociation of the catalyst/substrate complex. (This Michaelis constant, abbreviated KM, is not literally a
dissociation constant, but involves not only the return of the
catalyst/substrate complex to the two separate species but
also its forward reaction to the products as in typical enzyme
Michaelis–Menten kinetics. If the forward rate constant is small
compared with the rate constant for dissociation of the complex, which is likely in our case, then the KM is essentially a dissociation constant of the complex.)
With this analysis we were able to show what the effects of
changes in our catalyst were on the rate constants for converting complexes to products, and also what the effects were on
the equilibrium constants for complexing (listed as dissociation
constants). These are listed in Table 1. As that table shows, the
rate constant for converting pyruvate to alanine increased by
about 40-fold when C-12 chains were attached in the polymer
rather than simple methyl groups. At the same time, the disso-
Table 1. Michaelis constants for transamination by the polymeric pyridoxamine reagents based on polyethylenimine (PEI) with Mn = 60 000 with
and without added C-12 units.
reagent
pyruvic acid
Km
kcat I 1000
ACHTUNGRE[min 1]
[mm]
phenylpyruvic acid
kcat I 1000
Km
ACHTUNGRE[min 1]
[mm]
1 without C-12
1 with C-12
7.2 0.7
290 60
13 1
370 30
@ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
12 2
42 6
27 3
4.0 0.6
ChemBioChem 2006, 7, 1491 – 1496
Water Exclusion and Enantioselectivity in Catalysis
ciation constant of the pyruvate to the catalyst is larger with
the C-12 hybrid than with the C-1 hybrid; this indicates that
the hydrophobic groups are making the binding less favorable
for pyruvate. This is not surprising—pyruvate is really not a hydrophobic molecule, and it is apparently undesirable to bury
its charge into a hydrophobic region. By contrast, with phenylpyruvate, which is converted to phenylalanine, both the rate
constant and the binding constant were improved by the hydrocarbon side chains.
It should be mentioned that what we call binding of the
substrate to the catalyst can involve covalent links, since the
first few steps of the transamination process are reversible,
and the rate-determining steps come later. Every reversible
process before the rate-determining step will play a role in the
dissociation constant. In the case of phenylpyruvate, the hydrophobic binding of the phenyl group of the substrate to the
hydrophobic core of the catalyst overcomes the dissociation
effect of the negative charge that was seen with simple pyruvate anion.
We also saw that the dissociation constants were sensibly affected by the amount of hydrophobic surface in the substrates.
As Table 2 shows, the dissociation constants became smaller as
Table 2. Michaelis constants for the PEI-laurylated transaminase mimic 1
with various substrates
substrate
kcat I 1000 [min 1]
Km [mm]
glyoxylic acid
pyruvic acid
4-methyl-2-oxopentanoic acid
a-ketoglutaric acid
phenylpyruvic acid
indole-3-pyruvic acid
92 6
100 0
55 4
30 5
28 1
13 3
120 0
200 1
110 2
7.4 0.9
5.5 0.6
1.3 0.2
kcat/Km
3.1
3.6
4.2
16
36
85
the hydrocarbon regions of the substrates became larger up to
indolepyruvate, whose transamination produces the a-amino
acid tryptophan. As a result of this, the 10 000-fold acceleration
in the transamination of pyruvate, in comparison with a pyridoxamine that has no added buffer catalysis, became 24 times
larger with indolepyruvate. We have seen previously that pyruvate and indolepyruvate have about the same rate of transamination with simple pyridoxamine,[12] so the reaction of indolepyruvate is 240 000 times faster with the C-12 polyethylenimine hybrid than it is with simple pyridoxamine in solution.
We also examined the system in which the pyridoxamine
was not covalently linked to the polymer but was simply reversibly bound to it in water through hydrophobic association.[6]
We attached hydrocarbon side chains to the pyridoxamine,
such as the two C-10 side chain groups of compound 3, and
saw that this strongly bound into the polymer that had its
own attached C-12 hydrophobic chains. This was an even
better catalytic system, converting indolepyruvate to tryptophan with a 725 000-fold rate increase per pyridoxamine coenzyme compared with the rate with simple pyridoxamine itself
in aqueous solution without the polymer. This is a very substantial rate increase.
Of course, the conversion of pyridoxamine and a substrate
keto acid into pyridoxal and a product a-amino acid is not a
true catalytic sequence. For catalytic transamination, the enzymes then take the pyridoxal species and react it with a sacrificial a-amino acid to reverse the transamination process and
convert the sacrificial a-amino acid to a keto acid while converting the pyridoxal species back to pyridoxamine. We and
many others have looked at this reverse process in enzyme
model systems and have concluded that it is very slow, since
with all the systems that have been looked at so far, it is actually thermodynamically uphill to reverse the process of transamination and convert pyridoxal back to pyridoxamine. However, we were able to show that a somewhat different process
would indeed easily carry out this reversal.[6]
If the a-amino acid is a glycine species disubstituted on the
a-carbon, then the product of the reverse transamination is a
ketone, not an aldehyde, and furthermore there is irreversible
decarboxylation associated with the process shown in
Scheme 3. This is actually a mimic of an enzyme known as diACHTUNGREalkylglycine decarboxylase, and it is a process that we have
studied extensively.[13] For our current purposes it is enough to
point out that the combination of the forward transamination
of a keto acid and the reverse oxidative deamination and decarboxylation of this disubstituted glycine unit constitute an
overall catalytic cycle, as Scheme 4 shows, and one that we
have been able to carry out with up to 100 turnovers.
We have made a number of studies of other polymers,
ACHTUNGREincluding shorter versions of the polyethylenimines and some
other catalytic polymer units.[4] Furthermore, we have described transaminases in which we have constructed dendrimers instead of linear or cross-linked polymers.[3, 5] The systems
we have looked at to date have a pyridoxamine at the core of
the dendrimer. We saw that we achieved catalytic transamination with these dendrimers provided there were amino groups
nominally on the outside face of the dendrimer. This is striking
evidence that dendrimers do not in fact have the tree-like
structures usually invoked, but instead have structures in
which many of the branches of the tree are curled back into
otherwise empty space, and can reach to the center of the
dendrimer and catalyze processes there. We are currently investigating such dendrimers further.
Scheme 3. Decarboxylative transamination that converts a pyridoxal derivative to the
corresponding pyridoxamine.
ChemBioChem 2006, 7, 1491 – 1496
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they induce the transamination of the keto acid 7 to
form the a-amino acid l-valine (8) with chiral induction in the product.[14] With the 50-mer, the initial
transamination process has enantioselectivity leading
to 83 % of l-valine and 17 % of d-valine. The 30-mer
is almost as effective, the 13-mer somewhat less.
Thus indeed the chirality of the polymer side chains
is being expressed in the selectivity of the process.
Unfortunately, the a-amino acids are produced initially as the Schiff bases with pyridoxal, a species that
can undergo relatively rapid racemization. Thus, in
this particular system, the product becomes less and
less enantiopure as the process is carried further in
Scheme 4. Turnover catalytic transaminations of a keto acid with sacrificial a-methyltime, so this is not a practical system for producing
ACHTUNGREphenylglycine.
a-amino acids with high enantiopurity. However, it is
clear that the problem simply is that the product
itself can be rapidly racemized because of its special
Chiral Polyamine Catalysts
activation in the pyridoxal Schiff base. With other chemical
steps and processes—leading to products that are not rapidly
Our polymers are not chiral, and the a-amino acids products
racemized—this class of polymers could be very effective
we produced were racemic. In order to achieve enantioselecchiral catalysts, am idea that we are currently pursuing.
tivity in the formation of a-amino acids from keto acids, we
The second approach to this set of homochiral isotactic
needed to produce polyethylenimines that were derived from
polyamines has been achieved by a very interesting novel prochiral units; we have done this in two different ways so far.
cess, the selective deoxygenation of polypeptides. We have
In our first series, we produced homochiral polyamines by
seen that, with borane, such polypeptides can be converted to
the cationic polymerization of oxazolines (Scheme 5).[14] These
polyamines with no scrambling of the asymmetric centers and
species can be prepared from amino alcohols derived from ano cleavage of the chains.[17, 18] This has been successful with
quite long polypeptides. In some earlier work, we also showed
that with even relatively small polypeptides we could convert
a covalently linked peptide/pyridoxamine structure such as 9
into the corresponding amine derivative 10 and achieve interesting chiral selectivity in transaminations (Scheme 6).[17] The
product amines were quite good catalysts for transaminations,
with enantioselectivity of up to 85 % ee, while the corresponding polypeptides from which these were derived gave less
than 5 % enantioselectivity.
Scheme 5. Synthesis of a polyformamide (5) and the corresponding polyamine (6) from cationic polymerization of oxazoline 4, and the conversion of
keto acid 7 into valine (8).
amino acids. The system we examined first is polymer 6, which
is derived from l-phenylalanol via the oxazoline 4. As Saegusa[15] and later Goodman[16] showed, oxazolines like this have
the interesting feature that they can be rapidly methylated
with a reagent such as methyl tosylate, and they will then
slowly undergo nucleophilic attack by the neutral oxazolines
to grow significant linear polymer chains with low polydispersity, in which each unit carries a benzyl group from phenylalanine in a homochiral (isotactic) polymeric amide, and the nitrogens carry formyl groups. This polyamide 5 can be hydrolyzed
with base to generate the linear polyamine 6 with two carbons
separating every nitrogen, as in the polyethylenimines discussed above, but with chirally attached benzyl groups on
each unit in a position to influence the transamination process.
We have examined several of these polymers, including a
13-mer, a 30-mer, and 50-mer, and in all cases have seen that
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Scheme 6. Borane reduction converts peptide 9 to polyamine 10, which
shows enantioselectivity in transaminations.
@ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2006, 7, 1491 – 1496
Water Exclusion and Enantioselectivity in Catalysis
In this series, the various segments of the polyamines do
not have to carry the same side chain units since they can be
derived from synthetic or natural polypeptides with well-defined and different side chains in various positions. Thus, this
class of chiral polyamines is particularly interesting. We are actively pursuing both synthetic studies of this process and evaluation of the properties of the unusual polyamines we can
derive from this deoxygenation.
It should be mentioned that a third approach to homochiral
polyamines has been reported very recently.[19] Under strong
base anionic polymerization conditions, a substituted aziridine
was polymerized to a system related to the ones we have described above, but in which a long alkyl chain is appended to
each unit. No studies on catalysis by this polymer have yet
been described.
Meteorites Bring Chirality to our Planet
Our studies on the transamination process—in which we were
able to recycle pyridoxal units back to pyridoxamines with amethylated a-amino acids—raised our interest in a fascinating
set of findings that have been reported over the years in
chemical and nonchemical journals. Meteorites deposit large
amounts of organic compounds on earth, and these include
ordinary a-amino acids, reflecting Strecker reactions in interstellar space between carbonyl compounds and ammonia and
HCN. The normal a-amino acids found in these meteoritics are
racemic—hardly surprising since they can relatively easily racemize by ionizing the a-hydrogen, as happened in our catalytic
system described above. However, some a-amino acids are
also found in these meteoritic deposits that are C,C-disubstituted glycines and incapable of racemization. These species are
found with some enantiomeric excesses (ees). The chiralities
observed so far have (S)-a-amino acids with ees ranging from
3 % up to 15 %.[20–25]
The findings are intrinsically very interesting, and could hold
the clue to the often considered question[26] of the origin of
homochirality in biomolecules on earth. Such a-alkylated aamino acids may be formed by Strecker reactions involving ketones instead of aldehydes; the ketones could be formed in interstellar hydrocarbon clouds by the hydration of acetylenes,
for instance. The enantioselectivity is normally explained by
the proposal that high-energy circularly polarized light is generated from neutron stars and can selectively destroy one of
the two enantiomers of the original racemic products,[27] although this particular explanation is not universally accepted.
However, the really important point is that these materials
bring to earth chiral materials with ees that could serve as the
initiators for the observed ees in our natural biomolecules.
Several things are required for this to be a believable idea.
First of all, it is necessary to see whether such ees in these unusual a-amino acids can be transferred to normal biomolecules
under credible prebiotic conditions. Secondly, it is important to
see how the partial ees observed can be amplified to the full
homochirality that we see in biological molecules today. Pizzarello has reported that one of these meteoritic a-amino acids,
(S)-a-ethylalanine, can catalyze the aldol condensation of two
ChemBioChem 2006, 7, 1491 – 1496
glycolaldehyde molecules to form erythrose and threose.[28]
The erythrose was formed with some enantiomeric excess of
the l product 11, which is not the natural material in biomole-
cules today, but the threose did have a small enantiomeric
excess of d-threose 12. We are examining this system further,
and its extension into other sugar chemistry in processes related to the formose reaction.[29]
We have found and reported that one of the meteoritic aamino acids that is found with an excess of the S enantiomer,
a-methylvaline, can perform a transamination with phenylpyruvic acid 13 to produce phenylalanine (15) with a small enantiomeric excess.[30] In this process, shown in Scheme 7, there is
Scheme 7. Conversion of keto acid 13 into phenylalanine (15) by direct
treatment of 13 with a-methylvaline (14). With (R)-14 (shown), the phenylACHTUNGREalanine is formed with an excess of the S enantiomer, l-phenylalanine
(shown).
a direct Schiff-base formation between the two reactants, and
then a decarboxylation with subsequent enantioselective
proton transfer to produce the observed product. In the particular example we examined, the commercially available R meteoritic a-amino acid 14 produced an excess of l-phenylalanine.
Thus the meteoritic S enantiomer of 14 would produce d-phenylalanine, not the l-phenylalanine of our normal proteins.
However, d-a-amino acids are known biomolecules, being
found in a number of organisms.[31, 32] In any case, using these
meteoritic a-amino acids to induce chirality in normal biomolecules is in its infancy, and one can expect much more from it
in the future.
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The other interesting question has to do with amplification.
There are some striking examples of amplification of chirality
in chemical systems, but they are generally not under credible
prebiotic conditions, and, for that matter, do not involve credible prebiotic compounds.[33] We have now demonstrated a believable process by which such enantiomeric amplification can
indeed occur under credible prebiotic conditions. This work
will be reported elsewhere.
To summarize, polyamines can be very potent catalysts for a
simple process such as transamination, and such polyamines
with appropriate side chains can be synthesized with homochirality that can be expressed in the enantioselectivity of the
products of catalysis. Some of the transamination processes
observed in these model systems can also be used with products of meteoritic processes to perform chirality transfers into
normal biomolecules. All these findings open attractive avenues for future exploration that could have a major impact
both on our ability to imitate enzymes even more closely and
on our ability to understand how chirality in our biomolecules
could have arisen from “chemical seeds” brought to the earth
in meteoritic deposits.
Acknowledgements
This work was supported by grants from the U.S. National Science Foundation and National Institutes of Health.
Keywords: amino acids · biomimetic synthesis · meteorites ·
polyethylenimines · transamination
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Received: March 28, 2006
Published online on September 15, 2006
@ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2006, 7, 1491 – 1496