C O M M U N I C A T I O N S
An HPLC analysis of the reaction products under the same
conditions (as a function of time) detected alanine whose rate of
formation had a rate constant of (1.5 ( 0.3) × 10-2 min-1 with
the polymer, and (1.8 ( 0.6) × 10-6 min-1 with pyridoxamine, a
rate constant ratio of 8300. Furthermore, the final yield of alanine
corresponded to 80% of the original pyridoxamine in the polymer,
indicating that essentially all the pyridoxamine units in the polymer
are of comparable reactivity, with no special “hot spots.” We believe
that the unreactive 20% of the pyridoxamine units in the polymer
are trapped by the pyridoxals that are formed. At pH 7.0 the rate
enhancement by the polymer is still 2300 times, while at pH 8.0,
the optimum for pyridoxamine itself, it is 1900 times (full data in
Supporting Information).
Transamination by simple pyridoxamine shows strong metal ion
catalysissadding 1 equiv of CuCl2 per pyridoxamine unit to the
pH 5.0 solution (without added EDTA) increases the free pyridox-
amine rate constant to 1.1 × 10-2 min-1, a 6000-fold rate increase;
however, the rate for the polymer increases by only 30-fold. The
transaminase enzymes do not use metal ions.
We believe that this sequence of general acid and general base
catalyses can be particularly well accomplished by the polyethyl-
eneimine species. The proximity of the many nitrogens means that
even at low pH there will be some unprotonated nitrogens able to
act as bases, and indeed bases strong enough that they are just too
weak to be protonated. These are the strongest bases that can exist
in the system at a given pH. Similarly, there are protonated amine
cations almost strong enough as acids to lose their protons, the
strongest general acids that can exist at equilibrium. Combined with
the somewhat nonpolar environment that the long alkyl chains
provide, the general acids and bases with pK’s close to the operating
pH are features that are common to many enzymes.
With phenylglycine as the sacrificial reagent, we see catalytic
formation of alanine with our polymeric catalyst, but with only
2.5 turnovers. It remains to be seen whether such polymers carrying
coenzyme groups can perform other enzymelike processes, includ-
ing stereoselective reactions. However, our early results reported
here do indicate that such artificial polymer-based enzyme/
coenzyme systems have interesting potential.
The rate enhancement of the polymer over that of simple
pyridoxamine was a steep function of the length of the alkyl chains
added, in polymers with roughly the same percentage of alkylation
and of pyridoxamine attachment. At pH 7.0 and 30 °C, the
acceleration over the rate with pyridoxamine, from kinetics by UV,
was 160 for C-1 chains, 180 for C-3, 500 for C-6, 1000 for C-9,
2300 for C-12, and 2500 for the C-15 and C-18 normal alkyl chains.
This chain effect seems unlikely to involve hydrophobic binding
of a substrate as hydrophilic as pyruvic acid. Instead we believe
that the hydrophobic chains modify the pK’s of the amino groups
in the polymer, as seen previously,12 and also create a cavity in
which the transamination can take place in a less than fully aqueous
environment.13
The transamination sequence involves several steps in which
general acid and general base catalyses are involved. This includes
addition of the amino group of a pyridoxamine unit to the carbonyl
group of the ketoacid, in which a proton must be removed from
zwitterionic intermediate 1 and a proton must be added to the
oxyanion of that intermediate to form the neutral carbinolamine 2.
Then a general acid must protonate the hydroxyl group of 2 and
later a proton must be removed from the nitrogen to form the imine
3. At some point the pyridine nitrogen is protonated or at least
hydrogen-bonded by a general acid, and then the proton of the
methylene group must be removed by a general base to produce
intermediate 4. A general acid must protonate the R carbon of the
ketoacid unit, and the resulting Schiff base 5 must then be
hydrolyzed with the assistance of general acids and bases, reversing
the sequence by which imine 3 was originally formed.
Acknowledgment. We thank Mary Rozenman and Wenjun
Zhou for some experimental studies, and the NIH and NSF for
financial support of this work
Supporting Information Available: Details of the polymer catalyst
syntheses and characterization, and the kinetic studies and data (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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