Photochemistry and Phofobiology. 1976. Vol. 23, pp. 7-1 I
Pergamon Press
Printed in Great Britain
THE PHOTOSENSITIZED OXIDATION OF TYROSINE DERIVATIVES IN THE PRESENCE OF ALGINATE-11: REACTION UNDER HETEROGENEOUS CONDITIONS G . R. SEELY and R. L. HART Charles F. Kettering Research Laboratory*, Yellow Springs, Ohio 45387, U.S.A (Received 9 June 1975; uccepted 23 September 1975)
Abstract-The thionine sensitized photooxidations of several tyrosine derivatives have been compared in a system in which the dye, bound to the anionic pOlySdCchdridC alginate, is separated by a dialysis membrane from a much larger external phase containing most of the substrate. Brown oxidation products appear both inside the bag and outside, but at considerably greater concentration inside. Oxidation products inside the bag retard the oxidation of the original substrate by competing with it for photooxidant. Substrates were divided into two groups, related to tyrosine and tyramine respectively, according to their rates of oxidation. The groups differ as to whether the prevailing phenolate form of the substrate is a zwitterion or an anion. In case of the latter, the Donnan equilibrium set up by the polyelectrolyte reduces its concentration in the polymer phase, and thereby the rate of oxidation. Titration and dialysis data support this interpretation. INTRODUCTION
MATERIALS AND METHODS
In the preceding paper (Seely and Hart, 1976), (hereafter Part I), we compared the photosensitized oxidations of tyrosine (tyr) and tyramine in solutions which did or did not contain the anionic polysaccharide, alginate. The purpose of the experiments was to determine whether interaction of the ionic charge of the substrate with that of the polyelectrolyte would affect the rate of oxidation sensitized by a dye bound to the latter. Although some differences in the rate and course of the reactions could be interpreted as consequences of associations of some of the oxidation products with the sensitizing dye and with the polymer, it was not possible to establish a direct effect of the ionic charge on the polymer per se. We have also performed experiments under heterogeneous conditions, in which the alginate and the dye bound to it were separated from a much larger volume of weakly buffered substrate solution by a dialysis membrane. In these experiments there was a large difference between the rates of oxidation of tyramine and tyrosine and derivatives of each, which we explain mainly as an effect of the Donnan equilibrium set up by the charge on the polyelectrolyte. One of the purposes of this series of experiments was to effect a crude separation of the brown products of the reaction into those that pass through a dialysis membrane in a reasonable time and those that do not. Reference has been made in Part I to the chromatography of brown products accumulated this way. For reasons discussed in the preceding paper it will be assumed that tyr and its derivatives are oxidized by a singlet oxygen mehanism.
The following substrates were obtained commercially and used as received: tyr, tyramine hydrochloride, tyr amide hydrochloride, N-acetyltyrosine amide, tyr ethyl ester hydrochloride, hordenine (N-dimethyltyramine) sulfate and p-chlorophenol. Candicine (N-trimethyltyraminium) iodide was prepared by methylation of hordenine with CH,I in methanol. Tetraethylammonium (TEA) alginatc solution, 7.5 x lo-, N, was prepared by neutralization of a solution of alginic acid, prepared in turn by acidification and dialysis of Na alginate solution. Use of TEA as the chief counterion minimizes site binding to the polymer chain. The reactions were conducted in a 1 / flask suspended in a water bath for cooling to cu. 20". The flask contained a solution of 0.1 mmol of substrate and 0.2 mmol of TEA carbonate buffer in 750m( of deionized water. The pH was adjusted to about 9.4 with TEA hydroxide (0.1 mmol or less required). In the center of the flask was suspended a bag of 27 mm dialysis tubing, containing 0.225 meq of TEA alginate and 0.0030 mmol of thioninc in 33 m/ water After a dark equilibration period of 3 W 5 min the flask was irradiated by light from a 750 W projector lamp, 22 cm from the bag. Oxygen was bubbled through the outer phase, and the solution was stirred magnetically. At intervals during the first 12 h the spectrum of the outer phase was recorded on a Cary 14 spectrophotometer. A t 22 h the reaction was stopped and both phases were analyzed spectrophotometrically. The dye survived the reaction and remained in the polymer phase throughout; its conccntration in the outer phase did not exceed M before or during the reaction. A control was also run in which the polymer was replaced by 0.225 mmol of TEA glucuronate in the dialysis bag, and irradiation begun immediately. Dye escaped rapidly from the bag, and the oxidation of the substrate, tyramine, also proceeded rapidly. This reaction stopped after 11 h because of exhaustion of the dye, although about 30% of the original tyramine remained. The progress of the reaction was followed by the fall of substrate bands in the uv and by the increase of oxidation product absorption. The oxidation products absorb through the visible region and increasingly into the uv, but were measured at 320 nm, where there is no absorption by substrates.
*Contribution No, 545. 7
G. R. SEELYand R. L. HART
8
Since the pH decreases as the oxidation proceeds, thereby shifting the state of substrate ionization from phenolate to phenol, and since oxidation product absorptivity exceeds that of the substrate in much of the uv, use of isosbestic points is preferred in the estimation of substrate consumption. Candicine, hordenine, tyramine and tyr were estimated from the difference in absorbance at their phenol-phenolate isosbestic points at 265 and 278 nm. The estimation was calibrated with candicine on the assumption that its oxidation was 95% complete. Chlorophenol was similarly estimated from its isosbestic points at 268 and 283 nm. The corresponding isosbestic points for tyr amide. N-acetyltyrosine amide, and tyr ethyl ester were inconveniently close together for this method to be used. Instead. the difference between the 242 nm phenolate band and a substrate-product isosbestic point at 259-260 nm was followed; the intensity of the corresponding 225 nm phenol band changed very little during the oxidation of these compounds, so that the loss of phenolate was a fairly good estimate of the total loss of substrate. Equilibrium dialyses of tyramine, tyr amide, and N-acetyltyrosine amide against sodium alginate were conducted in a 25°C bath for 2 or 3 days. Potentiometric titration of tyramine and hordenine was performed with a Corning Model 12 pH Meter.
refer to the outer phase, where the progress of the reaction was followed. The meaning of the parameters B and @(r) will be discussed. The increase in product absorption with time, measured at 320 nm in the outer phase, is shown in Fig. 1. It is clear that by this criterion, tyramine, hordenine and candicine were oxidized rather more rapidly than the other tyrosine derivatives. On the other hand, it is evident in Table 1 that a larger fraction of products absorbing at 320 nm is retained in the polymer phase with tyr and its amides than with the tyramine derivatives. However, as comparison of Fig. 1 and Table 1 shows, the total amount of brown products retained within the membrane is not greater. Furthermore, absorbance at 320 nm reflects the extent of oxidation of products as well as the amount. Products from chlorophenol, not a tyr derivative, are retained least by the membrane. The control without alginate in the dialysis bag showed that permeation of dye, and presumably other small molecules, through the membrane was fast comRESULTS pared to rates of photoreaction in the presence of Data pertaining to the runs are summarized in alginate. It also demonstrated again the stabilizing Table L. The pH values and initial phenolate fractions effect of binding to polymer on thionine, reported in Table 1. Data pertaining to the thionine-sensitized photooxidation of tyrosine derivatives under heterogeneous conditions
No.
Name
1.
Tyrosine
CH2CHNH,,+
0.49
9.38
9.07
8.55
0.95
1.6
0.51
9.21
-
8.34
0.95
1.6
0.59
9.38
-
7.62
0.95
1.7
0.46
9.26
8.38T
6.12
0.95
2.7
3.6
1 coo 2.
3.
4.
Tyrosine amide
CH~CHNH
N-Acetyl tyrosine amide
CH2CHNHCOCH3
Tyrosine ethyl ester
CH2CHNH:
+
l
3
corn2
I
CONH2
1
COOCZH5
5.
p-Chlorophenol
C1
0.97
9.38
8.69
3.03
0.95
6.
Tyramine
CH2CH2NH3+
0.73
9.38
9.05
6.30
0.67
30
7.
Hordenine
CH CH N(CH3)2
0.68
9.45
9.06
4.17
0.67
18
0.82
9.31
8.85
3.66
0.67
66
0.75
-
8.65
2.16
(control)
+ '8
+ 8. 9.
Candicine
ryramine
CH2CH2;
Pergamon Press
Printed in Great Britain
THE PHOTOSENSITIZED OXIDATION OF TYROSINE DERIVATIVES IN THE PRESENCE OF ALGINATE-11: REACTION UNDER HETEROGENEOUS CONDITIONS G . R. SEELY and R. L. HART Charles F. Kettering Research Laboratory*, Yellow Springs, Ohio 45387, U.S.A (Received 9 June 1975; uccepted 23 September 1975)
Abstract-The thionine sensitized photooxidations of several tyrosine derivatives have been compared in a system in which the dye, bound to the anionic pOlySdCchdridC alginate, is separated by a dialysis membrane from a much larger external phase containing most of the substrate. Brown oxidation products appear both inside the bag and outside, but at considerably greater concentration inside. Oxidation products inside the bag retard the oxidation of the original substrate by competing with it for photooxidant. Substrates were divided into two groups, related to tyrosine and tyramine respectively, according to their rates of oxidation. The groups differ as to whether the prevailing phenolate form of the substrate is a zwitterion or an anion. In case of the latter, the Donnan equilibrium set up by the polyelectrolyte reduces its concentration in the polymer phase, and thereby the rate of oxidation. Titration and dialysis data support this interpretation. INTRODUCTION
MATERIALS AND METHODS
In the preceding paper (Seely and Hart, 1976), (hereafter Part I), we compared the photosensitized oxidations of tyrosine (tyr) and tyramine in solutions which did or did not contain the anionic polysaccharide, alginate. The purpose of the experiments was to determine whether interaction of the ionic charge of the substrate with that of the polyelectrolyte would affect the rate of oxidation sensitized by a dye bound to the latter. Although some differences in the rate and course of the reactions could be interpreted as consequences of associations of some of the oxidation products with the sensitizing dye and with the polymer, it was not possible to establish a direct effect of the ionic charge on the polymer per se. We have also performed experiments under heterogeneous conditions, in which the alginate and the dye bound to it were separated from a much larger volume of weakly buffered substrate solution by a dialysis membrane. In these experiments there was a large difference between the rates of oxidation of tyramine and tyrosine and derivatives of each, which we explain mainly as an effect of the Donnan equilibrium set up by the charge on the polyelectrolyte. One of the purposes of this series of experiments was to effect a crude separation of the brown products of the reaction into those that pass through a dialysis membrane in a reasonable time and those that do not. Reference has been made in Part I to the chromatography of brown products accumulated this way. For reasons discussed in the preceding paper it will be assumed that tyr and its derivatives are oxidized by a singlet oxygen mehanism.
The following substrates were obtained commercially and used as received: tyr, tyramine hydrochloride, tyr amide hydrochloride, N-acetyltyrosine amide, tyr ethyl ester hydrochloride, hordenine (N-dimethyltyramine) sulfate and p-chlorophenol. Candicine (N-trimethyltyraminium) iodide was prepared by methylation of hordenine with CH,I in methanol. Tetraethylammonium (TEA) alginatc solution, 7.5 x lo-, N, was prepared by neutralization of a solution of alginic acid, prepared in turn by acidification and dialysis of Na alginate solution. Use of TEA as the chief counterion minimizes site binding to the polymer chain. The reactions were conducted in a 1 / flask suspended in a water bath for cooling to cu. 20". The flask contained a solution of 0.1 mmol of substrate and 0.2 mmol of TEA carbonate buffer in 750m( of deionized water. The pH was adjusted to about 9.4 with TEA hydroxide (0.1 mmol or less required). In the center of the flask was suspended a bag of 27 mm dialysis tubing, containing 0.225 meq of TEA alginate and 0.0030 mmol of thioninc in 33 m/ water After a dark equilibration period of 3 W 5 min the flask was irradiated by light from a 750 W projector lamp, 22 cm from the bag. Oxygen was bubbled through the outer phase, and the solution was stirred magnetically. At intervals during the first 12 h the spectrum of the outer phase was recorded on a Cary 14 spectrophotometer. A t 22 h the reaction was stopped and both phases were analyzed spectrophotometrically. The dye survived the reaction and remained in the polymer phase throughout; its conccntration in the outer phase did not exceed M before or during the reaction. A control was also run in which the polymer was replaced by 0.225 mmol of TEA glucuronate in the dialysis bag, and irradiation begun immediately. Dye escaped rapidly from the bag, and the oxidation of the substrate, tyramine, also proceeded rapidly. This reaction stopped after 11 h because of exhaustion of the dye, although about 30% of the original tyramine remained. The progress of the reaction was followed by the fall of substrate bands in the uv and by the increase of oxidation product absorption. The oxidation products absorb through the visible region and increasingly into the uv, but were measured at 320 nm, where there is no absorption by substrates.
*Contribution No, 545. 7
G. R. SEELYand R. L. HART
8
Since the pH decreases as the oxidation proceeds, thereby shifting the state of substrate ionization from phenolate to phenol, and since oxidation product absorptivity exceeds that of the substrate in much of the uv, use of isosbestic points is preferred in the estimation of substrate consumption. Candicine, hordenine, tyramine and tyr were estimated from the difference in absorbance at their phenol-phenolate isosbestic points at 265 and 278 nm. The estimation was calibrated with candicine on the assumption that its oxidation was 95% complete. Chlorophenol was similarly estimated from its isosbestic points at 268 and 283 nm. The corresponding isosbestic points for tyr amide. N-acetyltyrosine amide, and tyr ethyl ester were inconveniently close together for this method to be used. Instead. the difference between the 242 nm phenolate band and a substrate-product isosbestic point at 259-260 nm was followed; the intensity of the corresponding 225 nm phenol band changed very little during the oxidation of these compounds, so that the loss of phenolate was a fairly good estimate of the total loss of substrate. Equilibrium dialyses of tyramine, tyr amide, and N-acetyltyrosine amide against sodium alginate were conducted in a 25°C bath for 2 or 3 days. Potentiometric titration of tyramine and hordenine was performed with a Corning Model 12 pH Meter.
refer to the outer phase, where the progress of the reaction was followed. The meaning of the parameters B and @(r) will be discussed. The increase in product absorption with time, measured at 320 nm in the outer phase, is shown in Fig. 1. It is clear that by this criterion, tyramine, hordenine and candicine were oxidized rather more rapidly than the other tyrosine derivatives. On the other hand, it is evident in Table 1 that a larger fraction of products absorbing at 320 nm is retained in the polymer phase with tyr and its amides than with the tyramine derivatives. However, as comparison of Fig. 1 and Table 1 shows, the total amount of brown products retained within the membrane is not greater. Furthermore, absorbance at 320 nm reflects the extent of oxidation of products as well as the amount. Products from chlorophenol, not a tyr derivative, are retained least by the membrane. The control without alginate in the dialysis bag showed that permeation of dye, and presumably other small molecules, through the membrane was fast comRESULTS pared to rates of photoreaction in the presence of Data pertaining to the runs are summarized in alginate. It also demonstrated again the stabilizing Table L. The pH values and initial phenolate fractions effect of binding to polymer on thionine, reported in Table 1. Data pertaining to the thionine-sensitized photooxidation of tyrosine derivatives under heterogeneous conditions
No.
Name
1.
Tyrosine
CH2CHNH,,+
0.49
9.38
9.07
8.55
0.95
1.6
0.51
9.21
-
8.34
0.95
1.6
0.59
9.38
-
7.62
0.95
1.7
0.46
9.26
8.38T
6.12
0.95
2.7
3.6
1 coo 2.
3.
4.
Tyrosine amide
CH~CHNH
N-Acetyl tyrosine amide
CH2CHNHCOCH3
Tyrosine ethyl ester
CH2CHNH:
+
l
3
corn2
I
CONH2
1
COOCZH5
5.
p-Chlorophenol
C1
0.97
9.38
8.69
3.03
0.95
6.
Tyramine
CH2CH2NH3+
0.73
9.38
9.05
6.30
0.67
30
7.
Hordenine
CH CH N(CH3)2
0.68
9.45
9.06
4.17
0.67
18
0.82
9.31
8.85
3.66
0.67
66
0.75
-
8.65
2.16
(control)
+ '8
+ 8. 9.
Candicine
ryramine
CH2CH2;
