OOE+OO 3.00E-02 4.00E-

[6A-amino-6A-deoxy-ß-cyclodextrin (40)] (mol dm '3)

Figure 12. !H NMR chem ical shift difference plotted against the concentration of 6A-amino-6A-deoxy-ß-cyclodextrin (40) for the difference in chemical shifts between the resonances corresponding to the oc-proton and the 4'-proton of trans-cinnamate (4) in 0.05 mol dm '3 phosphate buffered deuterium oxide at pH 7.0 and 298 K.

cyclodextrin.139' 141 However, the conditions used for the chromatography are limited to those for the system in which the association constant is required, in this case, 0.05 mol dm '3 phosphate buffer at pH 7.0. Under these conditions, the signals corresponding to the host 40 and the guest (S)-3 could not be resolved, due to the similar chromatographic properties of these species, so this method could not be used.

Since direct methods of measurement proved unsuitable, the use of an indirect method o f association constant calculation was considered. The displacem ent of a fluorophore from a cyclodextrin cavity by another guest has been used previously to calculate the association constant of the complex form ed.142' 147 Initial studies using 2-(4-toluidino)naphthalene-6-sulfonate (82) as the fluorophore, indicated that complexes consisting o f one guest 82 and two host 40 molecules are formed, as well as 1 : 1 complexes. This complicates any analysis of changes in fluorescence, so this method was not pursued.

H

It was anticipated that the displacement of the cinnamate 4from the cavity of the cyclodextrin 40 by (^)-phenylalanine ((5)-3) could be used to calculate the association constant of the complex of the guest (S)-3 with the host 40. Any displacement of the

cinnamate 4by (^-phenylalanine ((S)-3) would result in an increase in the proportion of the cinnam ate 4 free in solution and hence a change in the chem ical shifts of the resonances due to it in the NMR spectrum of the solution. It was envisaged that any change in chem ical shift of the signals due to the cinnam ate 4 on addition of the phenylalanine (S)-3 could be used to calculate the association constant of the complex of the guest ( S ) ^ with the host 40. A complete summary of the calculations involved is

described in Appendix 2.

To evaluate this method, a trial experiment involving the addition of the adamantane derivative 10to a solution of the cinnamate 4 and the host 40 was carried out. The adam antane derivative 10 is reported to include in ß-cyclodextrins with association constants of >104 mol-1 dm3,46 so it was expected to displace the guest 4 from the cavity of the cyclodextrin 40. On addition of the carboxylate 10, the chemical

shifts of the signals corresponding to the cinnamate 4protons in the ]H NMR spectrum of the solution were observed to change, indicating that the method was appropriate to monitor displacement of the cinnamate 4from the cavity of the cyclodextrin 40.

The association constant for the complex of the phenylalanine (S)-3 with the amine 40was calculated by observing the change in chemical shift difference between the signals due to the a-p ro to n and the 4 ’-proton of the cinnam ate 4 in the ]H NMR spectrum of the solution at various concentrations of the phenylalanine (S)-3 and fixed concentrations of the host 40 and the cinnamate 4. Only a very small change in the

chemical shift of the signals corresponding to the cinnamate 4protons was observed on addition of the phenylalanine (S)-3, with the association constant of the complex between (S)-phenylalanine ((S')-3) and the host 40thus being calculated as 9.6 ± 0.3 m ob1 dm3.

This value is much lower than had been expected. The effect of the amine 40 on the ratio of the total amount of the cinnamate 4to the total amount of the phenylalanine (S)-3 present at equilibrium in a solution containing ammonium ions and PAL suggests that the amine 40 complexes the phenylalanine (S)-3 in preference to the cinnamate 4.

That is, it was expected that the association constant for the com plex o f the phenylalanine (S)-3 with the amine 40 would be greater than that o f the cinnamate 4with the amine 40. However, this is not the case, as the association constants were calculated

as 9.6 ± 0.3 mol-1 dm 3 and 270 ± 20 mol*1 dm3, respectively. Further, given that the cyclodextrins 7and 40were used at similar concentrations and have similar association constants for their com plexes with the guest (S)-3, their effects to remove the phenylalanine (5)-3 from solution and hence limit its digestion by PAL would be

Results and Discussion, Chapter IV

expected to be similar. However, while no effect of the cyclodextrin 7to decrease PAL activity by removing the substrate (5)-3 from solution was observed (Figure 7), the amine 40 significantly decreased PAL activity (Figure 11) suggesting that more of the substrate (S)-3 was removed from solution in that case.

The effect of the adamantane derivative 10 to disrupt the com plex of the cinnamate 4and the amine 40indicates that the cinnamate 4is complexed in the cavity of the cyclodextrin 40. The experiments which showed that the phenylalanine (S)-3 does

not effectively displace the cinnamate 4from the cyclodextrin annulus, and the low value of the association constant for the complex of the host 40 and the guest (S)-3 reflecting this do not take into account binding to the exterior surface of the host 4 0. The

inconsistency of the calculated association constant with the observed effects of the host 40 on systems containing (^-phenylalanine ((S)-3) and the cinnamate 4 suggests that the interaction of (S)-phenylalanine ((S)-3) with the host 40 may be non-inclusive in nature. The interaction of molecules with the surface of cyclodextrins has been reported and is particularly significant when cyclodextrins are used as chiral stationary phases in separation technology.148

In any event, the work described in this Chapter has illustrated that in a solution containing two compounds which are interconverted by an enzyme which is also present, the ratio of the amounts of these compounds present at equilibrium may be altered on addition of a cyclodextrin. Further, different cyclodextrins were shown to be capable of altering the ratio in different directions and to different extents. A limitation of the use of enzymes in synthesis is that the thermodynamics of an enzyme catalysed reaction might be such that, at equilibrium, little of the desired product is present. It now appears that there is the potential to overcome this limitation through the addition of a cyclodextrin which preferentially complexes the desired product, resulting in an increase in the total amount of the desired product present at equilibrium.

Chapter V

Acylase I Catalysed Hydrolysis of para-Substituted