ORIGEN DE LA CARICATURA

C 0

CH

CH

-(-N H C O C H

C 0

CH

CH

98 R = CH3, X = Cl

99 R = N 0 2, X = Br

^

100 R = CH3

101 R = NO

H+

102 R = CH3

103 R = N 0 2

Scheme 28. Preparation of the substituted phenylalanines 102 and 103.

59 R' = CH3, R" = H

102 R' = H, R" = CH3

61 R' = N 0 2, R" = H

103 R' = H, R" = N 0 2

104 R' = CH3, R" = H

105 R' = H, R" = CH3

106 R' = N 0 2, R" = H

107 R' = H, R" = N 0 2

Scheme 29.

Preparation of the phenylalanine derivatives 104-107 from the

phenylalanines 59, 61, 102 and 103.

Results and Discussion, Chapter VI

followed by acid catalysed hydrolysis and decarboxylation.133,171 Each of the adducts 100 and 101 and each of the phenylalanines 102 and 103 was identified by comparison o f its observed physical and spectroscopic data with those reported.133,171

Treatm ent of the phenylalanines 59, 61, 102 and 103 with thionyl chloride pretreated methanol and then with trifluoroacetic anhydride afforded the phenylalanine derivatives 104-107 (Scheme 29), which were fully characterised. The NMR spectrum of each of the products 104-107 includes a resonance corresponding to the hydrogen nuclei of a methyl ester at ca. 83.8. The presence of a trifluoroacetyl group in the products 104-107 is confirmed by the presence o f characteristic peaks in their 13C NMR spectra. Signals corresponding to the quaternary carbon of a trifluoroacetyl group (q, 7p = 288 Hz) at ca. 5115 and to the carbonyl carbon of a trifluoroacetyl group (q, 7p = 38 Hz) at ca. 5155 are observed in each case.

The phenylalanine derivatives 104-107 are insoluble in water. For the hydrolysis of each of the nitrophenylalanines 94 and 95 by protease VIII reported in the literature,104 the limited aqueous solubility of the substrates 94 and 95 was overcome through carrying out the reactions in a biphasic system consisting of dichloromethane and pH 7.5 phosphate buffer. Such a system is incompatible with the use of cyclodextrins, as cyclodextrins have been shown to form insoluble com plexes with halogenated hydrocarbons.172 The phenylalanine derivatives 104-107 are soluble in 10% acetonitrile in water. Trial experiments established that protease VIII retains activity in this solvent mixture. Thus, subsequent reactions monitoring the hydrolysis of the phenylalanine derivatives 104-107 by protease VIII were carried out in 10% acetonitrile in pH 7.5 phosphate buffer at 291 K. The buffer was used to ensure the pH of the mixtures did not change as the reactions proceeded. The pH value was chosen as that where the enzyme shows m axim um a ctiv ity .103,104 The tem perature used corresponded to room temperature in a controlled laboratory.

The cy clo d ex trin s used in th is study w ere ß - c y c lo d e x tr in ( 8 ) , 6A-am ino-6A-deoxy-ß-cyclodextrin (40), prepared as discussed in Chapter II of the Results and D iscussion, and hydroxypropyl-ß-cyclodextrin (108). The modified cyclodextrin 108 is commercially available and is the reaction product of ß-cyclodextrin (8) and propylene oxide. Reaction occurs on both the primary and secondary hydroxyl groups of the cyclodextrin 8 and the average number of hydroxypropyl groups added per cyclodextrin moiety is characteristic of the modified cyclodextrin 108 batch. This degree o f substitution has previously been calculated using soft-ionisation mass and NMR spectroscopic techniques.109,173 In this case, ]H NM R spectroscopy was used to determine the degree of substitution of the batch of hydroxypropyl-ß-cyclodextrin (108)

used. This involved comparison of the ratio of the integration of the resonance due to the methyl protons (81.12, d, J = 8 Hz) to those of the signals corresponding to the acetal protons (85-5.3, m) and the remaining protons (83.4-4.1, m). This comparison gave an average degree of substitution of 3.65 hydroxypropyl side chains per cyclodextrin moiety for the modified cyclodextrin 108 batch used.

The modified cyclodextrin 108 has an increased aqueous solubility when compared to ß-cyclodextrin (8).174,175 The protonated form of the amine 40 has a pA"a of 8.7 ,111 so the amine 40 is present in solution predominantly in the protonated form at pH 7.5. Tliis form of the amine 40 has an increased aqueous solubility when compared to the parent cyclodextrin 8 .111 Thus, the modified cyclodextrins 40 and 108 can be present in solution at greater concentrations than ß-cyclodextrin (

8

of a guest by a host is a bimolecular reaction, a higher concentration of the host results in a greater proportion of the guest being complexed. That is, it was envisaged that the cyclodextrins 40 and 108 might have greater effects than the naturally occurring cyclodextrin 8 on the rates of hydrolysis of the phenylalanine derivatives 104-107 by protease VIII, through removing a greater proportion of the phenylalanines 104-107 from solution.

A series of experiments was carried out observing the hydrolysis of each of the phenylalanines 104-107 by protease VIII in the presence of either no cyclodextrin, ß-cyclodextrin (8) (9.42 x lO 3 mol dm*3), 6A-am ino-6A-deoxy-ß-cyclodextrin (40) (93.5 x 10'3 mol dm 3) or hydroxypropyl-ß-cyclodextrin (108) (86.0 x 10'3 mol dm-3). The extent of reaction in each case was monitored through analysis of aliquots of the reaction mixture using HPLC. This involved comparison of the integrations of the signals due to the acid produced and the residual starting material. Hydrolysis of each of the phenylalanines 104-107 by protease VIII ceased at 50% reaction. This is consistent with only the (S)-isomer of each o f the phenylalanine derivatives 104-107 being hydrolysed, in agreement with literature reports,102' 104 though this was not further

Results and Discussion, Chapter VI

verified. Through trial experiments, the amount of enzyme required in each assay for the