Buchan, Alexander
Description
This thesis presents an investigation into the development of enzyme catalysis of
cycloaddition reactions.
Tyrosyl-tRNA synthetase was initially investigated as a catalyst for the
cycloaddition between azides and alkynes and nitrile oxides and alkenes. From
modeling studies, substrates were proposed based on the natural substrates of tyrosyltRNA
synthetase. Each substrate was appended with either an azide, alkene, alkyne or
nitrile oxide moiety. Tyrosyl-tRNA synthetase was expressed in E....[Show more] coli and purified by
FPLC. A variety of commercial and literature based assays were attempted to determine
the activity of tyrosyl-tRNA synthetase but each assay failed to give a satisfactory
result. An HPLC based assay was developed that could determine the amount of AMP
produced by tyrosyl-tRNA synthetase and therefore the activity of the enzyme. The
assay was found to be broadly applicable with the activities of pyruvate kinase and
potato apyrase also being determined. The binding affinities of the azide and alkyne
substrates and their corresponding cycloadducts were determined using the HPLC assay
and these data were used to plan experiments to investigate catalysis of the cycloaddtion
between the azide and alkyne. Under the experimental conditions, it was found that the
rate of non-enzyme catalysed cycloaddition between the azide and alkyne was too slow
to be measured. The rate of reaction in the presence of tyrosyl-tRNA synthetase was
also too slow to be measured, therefore, it could not be determined if catalysis was
occurring in this system. Copper catalysis of the cycloaddtion reaction was attempted to
increase the non-enzyme catalysed rate, however, in the presence of the enzyme the reaction was found to be inhibited. Due to the instability of the nitrile oxide and the
alkene, catalysis was not investigated in this system. Also, as tyrosyl-tRNA synthetase
was only accessible in small quantities, it was decided to continue investigating
catalysis of cycloaddition reactions with Chymotrypsin as this enzyme could be bought
in bulk and this meant the concentration of the enzyme could be increased and would
allow a greater proportion of the substrates to be enzyme bound.
Chymotrypsin was investigated as a catalyst for the cycloaddition between
benzonitrile oxide and alkenes. Initial modeling studies provided aliphatic alkenes as the
complementary substrates to benzonitrile oxide. The binding affinities of the alkenes
and the benzonitrile oxide precursor benzaldoxime as well as the corresponding
cycloadducts were directly measured using isothermal titration calorimetry. The binding
data were used to plan the experiments to investigate catalysis of the cycloaddition
between the aliphatic alkene and benzonitrile oxide by Chymotrypsin. The results
showed that the reaction between the alkene and benzonitrile oxide was too slow.
Although the formation of the cycloadduct was detectable, impurities present in the
mixture containing Chymotrypsin made it difficult to isolate the product by HPLC. The
amount of cycloadduct produced in the presence of Chymotrypsin could not be
accurately determined but at most was similar to that produced in the non-enzyme
catalysed reaction. The alkene substrate was then redesigned to be more reactive
towards cycloaddition with benzonitrile oxide to increase the rate of reaction. The
reaction rate was found to be greater than that of the previous system by a factor of
approximately 105. The reaction between the activated alkene and benzonitrile oxide produced a mixture of regioisomers with the 5-substituted
regioisomer being the major product. Under the chosen reaction conditions, the reaction
between the alkene and benzonitrile oxide was found to be too fast with the reaction
being complete after an hour. On lowering the concentrations of both reactants it was
found that the reaction still did not proceed beyond half an hour and also that the yields
of the cycloadducts were lower. It was determined from these results that the
benzonitrile oxide was taking part in a competing reaction which was likely to be first
order rather than the second order dimerisation. As the benzonitrile oxide was reacting
too quickly, the reaction rate between the alkene and benzonitrile oxide could not be
determined. However, an increase in the ratio of the 5- to 4-substituted cycloadducts in
the presence of Chymotrypsin indicated that approximately 10% of the reaction may be
occurring on the enzyme if the reaction on the enzyme is completely regioselective.
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