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Interaction between singlet oxygen and biologically active compounds in aqueous solution III. Physical and chemical 1O2-quenching rate constants of 6,6′-diapocarotenoids
J. Photo&em.
Photobiol. B: Biol., 8 (1990) 51-61
51
Interaction between singlet oxygen and biologically active compounds in aqueous solution III. Physical and chemical 102-quenching rate constants of 6,6 ‘-diapocarotenoids+ Giovanna
Speranza++, Paolo Man&to
and Diego Monti
Dipartimento di Chimica Organica e Industriale, Universitd di Milan0 and Centro di Studio suL?e Sostanze 0rganiche Naturali, Consigli~ Nazimmle &lb Ricer&e, via Vent&an 21, I-20133 Milan (Italy)
(Received March 12, 1990; accepted May 16, 1990)
Keywords. Singlet oxygen, 6,6’-diapocarotenoids, constants, energy transfer, electron transfer, bixin.
‘02-quenching rate
AbStnrct
The chemical ‘O,-quenching rate constant ka and the physical ‘02-quenching rate constant k, of a number of 6,6’-diapocarotenoids (bixinoids) were measured using a thermal source of ‘02, lAP Values of k, were found to be close to diffusion-controlled rate constants both in water and in polar organic solvents, whereas ka showed a strong dependence on the medium. These results suggest the co-occurrence of energy and election transfer mechanisms in the quenching of IO2 by blxinoids. Biological implications are discussed.
1. Introduction
The study of the interaction between singlet oxygen 02, lAg and polyolefhrs is of great relevance to the understanding of the protective role played by carotenoids in photosynthesis [l-3] as well as against photodynamic effects L4-61. In continuation of our work [7, 81 in this field, we report here the determination of the physical ‘O,-quenching rate constant k, and the chemical ‘Oa-quenching rate constant ka for a number of 6,6’-diapocarotenoids (l-6). These compounds can be collectively named bixinoids owing to their structural relationship with the most representative member of the group, i.e. bixin (1) [9]. Bixin is the main constituent of annatto, a commercial product obtained from the seeds of Bixu orellunu [lo] and largely used as a food colouring agent. +For details of part II of this paper, see ref. 8. ++Authorto whom correspondence should be addressed.
loll-1344/90/$3.50
Q Elsevier Sequoia/Printedin The Netherlands
52
1
R1 = H
R* = Me
.2
R1 = R2 = Me
METHYLBIXIN
3
R1 = R2 = H
NORBIXIN
BIXIN
C02R2
4
RI = R2 = fle
taanr-METHYLBIXIN
5
RI = R2 = H
trwwNORBIXIN
kinetic method used for the simultaneous measurement of k, and kR has previously been developed by us and shown to be characterized by a satisfactory accuracy [ 7, 81. It is based on the following experimental facts and theoretical assumptions. The water-soluble 1,4_endoperoxide of 3-(4-methyl-l-naphthyl)propionic acid (6) [ 111 was used to produce singlet oxygen non-photochemically (thus avoiding photochemical side reactions, e.g. cis-trans isomerization of the polyene chain [12] and dye-sensitized 02- production [13]). The following sequence of reactions was taken into account: The
Ckt,Cki,C0,~ 6
AOz LA+
‘02
‘02 - kS 302 ‘02--
02
kQ3 +c
‘o,+c
2
product
where AOp is the endoperoxide (6), A the parent naphthalenic acid, C the carotenoid examined for its physical quenching rate constant k, and chemical quenching rate constant ka; kD and ks are the rate constants for the decomposition of the endoperoxide and for the natural decay of ‘02 respectively. When analysed under a steady state approximation for ‘02 concentration, the above system affords the following kinetic equation [7, 81: 2, --a1 tb v, [Cl,
(1)
53
where a= -ks kR
-G=kJAOJo
(2)
exp(-h&
(4)
[AO&being the initial concentration of the endoperoxide corrected for the yield in ‘Oz, and [Cl, and V, the actual concentration of the carotenoid and its rate of disappearance respectively at the time t. Equation (1) is linear (I/& US. l/[C],) if Z,= constant, i.e. for a set of experiments carried out with different initial concentration of the carotenoid, provided that the same [AO,], is used and the two experimental variables are measured at the same time t. This time must be chosen within the interval where the steady state equation
v= -y
=k,[C]
holds, as shown by monitoring [C] by means of its visible absorption at a given wavelength and plotting ln[C] VS. t. Thus the quenching rate constants kR and k, can be calculated from the slope a and the intercept b of eqn. (1). 2. Materials
and methods
UV spectra were recorded on a Perkin-Elmer model 554 spectrophotometer and fluorescence spectra on a Perkin-Elmer model 650-10s spectrofluorometer both equipped with thermostated cells. ‘H (300 MHz) and 13C(75.47 MHz) nuclear magnetic resonance (NMR) spectra were determined on a Bruker CXP 300 spectrometer in dimethylsulphoxide (DMSO) ds using thesignalsolventasinternalstandard(6=2.50and39.5fromtetramethylsilane for ‘H and 13C respectively). Cyclic voltammograms were recorded with an Amel model 466 polarographic analyser. Thin layer chromatography (TLC) was performed with silica gel 60 Fzs4 precoated plates (Merck, 0.25 mm layer) and flash chromatography with commercial Merck silica gel 60 (230-400 mesh ASTM) using in both cases CHC13-MeOH (9:1, v/v) as eluent, unless otherwise stated. All chemicals for kinetic measurements were analytical grade and used without further purification. 2.1. Pmparaticm of biximids 2.1.1. Bixin (1) Commercial bixin (1.2 g) (Roth) was purified by flash chromatography. Fractions showing one orange spot in TLC (l&=0.56) were collected and
54
evaporated under vacuum. The residue (0.9 g) was found to be pure bixin (1) by comparison of its spectral properties (UV [14], and ‘H and 13C NMR [15]) with those previously reported. The UV A,, (log e) values are as follows: in HaO, pH 7.8, 432 nm (4.99), 457 nm (5.13), 486 nm (5.08); in DMSO, 443 nm (4.95), 469 nm (5.08), 499 nm (5.03); in dimethylformamide (DMF), 436 nm (4.95), 464 nm (5.07), 494 nm (5.02). 2.1.2. Methylbixin (2) A slow stream of CHzNz (prepared from DiazaId, Aldrich, 3.2 g) [ 161 was bubbled (N, as carrier) for 2 h into a solution of 1 (380 mg) in CHCl,-MeOH (2:1, v/v) (15 ml). After evaporation of the solvent, the residue was fIash chromatographed giving a compound which was shown to be pure methylbixin (2) (290 mg) by TLC (l&=0.77) and spectral data (UV [ 17, 181, and ‘H [ 181 and 13C NMR [ 191). The UV A,, (log l) values are as follows: in DMSO, 445 nm (4.97), 473 run (5.09) 503 nm (5.03); in DMF, 440 nm (4.97), 466 run (5.09), 496 run (5.02). 2.1.3. Norbixin (3) A solution of bixin (1)(500 mg) in MeOH (35 ml) containing KOH (2 g) was stirred at room temperature under N2 overnight. After evaporation of the solvent under reduced pressure, the residue was dissolved in Ha0 (20 ml). Addition of concentrated HCl solution under stirring gave a red precipitate which was filtered, repeatedly washed with Ha0 and dried under vacuum (60 “C). This product was found to be pure norbixin 3 (330 mg) by TLC (j&=0.33), UV [ 141 and NMR data [ 151. The UV A,, (Iog l) values are as follows: in HaO, pH 7.8, 428 nm (5.07), 450 nm (5.15), 479 nm (5.07); in DMSO, 439 nm (5.01) 466 run (5.08), 495 run (5.03); in DMF, 437 nm (5.00), 462 nm (5.07), 492 nm (5.02). 2.1.4. Tram+methylbixin (4) Iodine (1.5 mg) was added to methylbixin (2) (25 mg) dissolved in CHCla (10 ml> and the solution refluxed for 4 h under Na. After cooling, the reaction mixture was washed with 0.1 N Na$Oa (2 X 10 ml) and then with Ha0 (15 ml) and dried on N&SO+ Removal of the solvent under vacuum and purikation by flash chromatography gave a compound shown to be pure trans-methylbixin (4) (20 mg) by TLC (&= 0.77) and spectral data (UV and ‘H and 13CNMR) [ 17-191. The UV &,,, (log l) values are as follows: in DMSO, 450 run (4.97), 479 nm (5.11), 509 nm (5.07); in DMF, 446 nm (5.00), 472 nm (5.10) 502 run (5.06). 2.1S. Trans-norbixin (5) This compound was prepared from trans-methylbixin (4) as reported for 3. It was shown to be trans-norbixin (6) by TLC (l&=0.33) and ‘H and 13C NMR data [15]. The UV A,, (log e) values are as follows: in DMSO, 443 nm (4.99), 471 nm (5.09), 500 nm (5.03); in DMF, 440 nm (4.99), 465 nm (5.09), 495 run (5.02).
55
2.2. Pmparatim of 3-(1,4-epidioxy-4-methyL1,4-dihydro-l-nuphthylJrwqpionic acid (6) Compound 6 was prepared according to Saito et al. [ 111. After chromatographic purification (silica gel column at 0 “C, elution with hexane-ethyl acetate-acetone-acetic acid 10:3:2:1, v/v) [8] it was shown to be 98% pure by KI titration and TLC (&=0.35 using the above eluent). 2.3. Determination of the rate constant for the decomposition of the en&peroxide (6_) The first-order rate constants k,, for the decomposition of 6 both in water and in organic solvents were determined by monitoring the increase in the UV absorption of the resulting naphthylpropionic acid. The UV &,, values are as follows: in HzO, pH 7.8, 287 run; in DMSO, 289 nm; in DMF, 290 nm. 2.4. Determination of the ‘02-quenching rate constants 2.4.1. Bleaching method In a typical’experiment in 0.1 M phosphate buffer (pH 7.8), two separated solutions of the bixinoid (8.0X 10V6 M) and of 6 (8.0X 10T3 M) were mixed at 0 “C. At this temperature the decomposition of the endoperoxide is negligible [ 111. After deoxygenation by Nz bubbling, the resulting solution was rapidly heated to 35 + 1 “C and allowed to stand at 35 f 0.1 “C. Electronic absorption spectra taken automatically at 3 ruin intervals were recorded in the range 350-600 nm. The disappearance of the bixinoid was monitored by the decrease in the absorbance of the central maximum (Fig. 1). For
OL
I
I
I
I
I
I
350
400
450
500
550
600
Fig. 1. Bleaching of norbixin (3) (5.4X lo-’
of the endoperoxide (6) (7.7
X
A hm)
M) in phosphate buffer @H 7.8) in the presence lo-” M) at 35 “C. Spectra are recorded at 3 n-dn intervals.
56
all experiments performed, a linear relationship of the type ln [C] = -k,t + constant was obtained in the interval l-30 ruin (Fig. 2), thus allowing the time of 6 min after reaching the reaction temperature to be taken as standard. To calculate kR and k, (eqns. (l)-(4)) in aqueous solution and in organic solvents the following constants were used: ka, 2.4 X lo5 s-’ in Hz0 [20], 1.4X lo5 s-’ in DMF [21], 5.2~ lo4 s-’ in DMSO [22]; lcn (measured as indicated above), 2.75~ 10v4 s-l in 0.1 M phosphate buffer at pH 7.8 (in ref. 11, 4.9 X 10e4 s-l in 0.26 M phosphate buffer at pH 7.0), 1.47~ 10m4 s-’ in DMF, 1.70~ 10m4 s-’ in DMSO. In addition, the values of 0.45 in Hz0 [23] and 0.82 in organic solvents [ll] were assumed for the yield in ‘02 formed by decomposition of the endoperoxide (6). 2.4.2. Youn& method [24] Solutions containing compound 6 (2 X 10B4 M), 1,3diphenylisobenzofuran (DPBF) (about 10 -6 M) and varying amount of bixinoids (l-5) (O-5 X 10e5 M) at 0 “C were carefully deoxygenated by Nz bubbling, rapidly heated to 35 f 1 “C and allowed to stand at 35 f 0.1 “C. The disappearance of DPBF was followed by monitoring the decrease in fluorescence intensity of the DPBF (excitation, A= 405 run; emission, A= 458 run) at 3 min intervals. When measurements were carried out in aqueous solution, sodium dodecyl sulfate (SDS) (0.1 M) was used to solubilize DPBF [25]. 2.5. Measurmt of oxidation potentials Oxidation potentials (peak potentials) were measured by cyclic voltammetry using a three-electrode cyclic voltammetric celI (glassy carbon electrode, platinum auxiliary electrode and reference saturated calomel electrode (SCE)) at 25 “C. Sample solutions were deaerated by bubbling Na for 15 min. KC1 (0.1 M) and 0.1 M tetraethylammonium perchlorate (TEAP) were used as supporting electrolytes in HZ0 and organic solvents respectively. The voltammograms were scanned at 100 mV s-‘.
0
I
I
I
9
10
27
min.
Fig. 2. Plot of the decline in the absorbance of norbixin the time, under the conditions reported in Fig. 1.
(3) at A=450
nm as a function
of
57
3. Results and discussion The structures of bixin (l), methylbixin (2), norbixin (3), transmethylbixin (4) and trans-norbixin (6) were confirmed by ‘H and 13C NMR spectra [ 15, 18, 191. The purity of each carotenoid was checked by TLC before use. When a cold aqueous solution of norbixin (3) and the endoperoxide (6) was rapidly heated to 35 “C and kept at this temperature, a bleaching of the reaction mixture was observed as shown in Fig. 1. Using the decrease in visible absorbance to monitor norbixin disappearance (Fig. 2), the kinetic analysis reported above allowed the physical and chemical ‘02-quenching rate constants to be calculated. Analogously, the rate constants for bixin (1) were determined. The values of these constants are reported in Table 1 together with the oxidation potentials (peak potentials) of the two bixinoids. The ‘02-quenching rate constants and the oxidation potentials for 1 and 3, as well as for the other compounds 2, 4 and 6, were also determined in aprotic solvents as shown in Table 2. In this case, a parallel measurement of the total ‘Oa-quenching rate constant (A+= k, + kR) was performed using Young’s method [24, 251 in order to estimate the reliability of the bleaching procedure in those non-aqueous media. In fact, in DMF and DMSO, the bleaching of bixinoids by ‘02 appears to be slow and the ratio k,Jk, higher than 103. These circumstances strongly limit the accuracy of kR and k, values obtainable by the kinetic analysis developed by us [7, 81, as can be seen by considering eqns. (5) or (3). However, good agreement was found between the values of i& resulting from the two methods, thus reinforcing the following conclusions drawn from these experimental data. The marked decrease in the kR value (about two orders of magnitude) observed on going from water (Table 1) to solvents characterized by lower dielectric constants, more positive E, for bixinoids, and more negative redox TABLE
1
‘Oa-quenching 35kO.l “C
Bixin (1) Bixin (l)d Norbixin (3) Norbixin (3)d
rate con&ants
of bixin
(1)and norbixin (3) in aqueous solution (pH 7.8) at
IO-’ kRp (M-i s-i)
lo-" k
0.9(*0.2)
1.8 1.1 2.3 0.9
0.7 (jzO.2) 1.5 (*0.4) 0.8 (&0.3)
(M-’
a
soi) (*0.4) (*0.4) (*OS) (*0.3)
“Mean values of four independent measurements on five bleaching experiments. bDetermined by Young’s technique [24, 251. ‘Measured by cyclic voltammetry. din the presence of 0.1 M SDS.
10-l'k T b (M-l
s-r)
&Xc w
(SW)
0.57 0.7 0.50 0.6
(standard deviation in parentheses),
each based
58
TABLE 2 ‘02-quenching rate constants and oxidation and dimethylsulphoxide at 35 f 0.1 “CL
potentials
of bixinoids
(l-6)
IO-"kR
IO-lok
IO-lo
(M-’
(M-r
so’)
(M-l
s-r)
in dimethylformamide
k b s’r)
&Xc (V (SW)
DMF
Bixin (1) Methylbixin (2) Norbixin (3) Truns-methylbixin (4) Runs-norbixin (5)
1.6 2.1 1.7 2.0 0.8
(51.1) (kO.7) (*Lo) (k0.8) (kO.5)
2.3 3.0 1.2 0.6 0.3
( f 1.0) (f 1.2) (k0.8) (kO.4) (*0.2)
2.0 1.6 1.7 0.9 0.8
0.87 0.92 0.92 0.92
1.4 1.0 1.9 0.8 0.9
(fO.7) (kO.6) (k0.8) (f0.6) (rtO.5)
2.1 1.3 1.7 0.4 0.3
(kO.6) (kO.8) (k0.6) (*0.3) (fO.2)
1.5 2.2 0.9 0.4 0.4
0.82 0.83 0.88 0.86
DMSO
Bixin (1) Methylbixin (2) Norbixin (3) Trans-methylbixin (4) Truns-norbixin (5)
‘Standard deviations are given in parentheses. bDetermined by Young’s technique [24, 251. cMeasured by cyclic voltammetry.
potentials for O.JOa- (E” (vs. SCE) = -0.40 V in HsO, -0.71 V in DMF and -0.58 V in DMSO, using unit concentration as the standard state reference [26]) (Table 2) strongly suggests that an electron transfer [27] process is responsible for the polyene bleaching in aqueous solution. In this medium, AGO< 0 can be calculated by the well-known Rehm-Weller [28] equation using Eo,o= 22.53 kcal mol-’ for O2 excitation energy [29]. The same trend was observed for 8,8’-diapocarotenoids (crocinoids) whose reaction with O2 lAg was explained in terms of an electron transfer mechanism [7]. The close resemblance of the two polyene families (nine and seven noncarboxy double bonds) in their chemical behaviour towards singlet oxygen is a probable reflection of their very similar oxidation potentials [7]. Considering that a reversible exciplex mechanism is generally involved in singlet-oxygen reactions [30, 311, the formation of an intermediate (or a transition state) stabilized by some degree of charge transfer from substrate to O2 ‘Ag is also plausible for bixinoids as an alternative to a full electron transfer [8]. In addition to the electron transfer mechanism, singlet-oxygen addition to the polyene chain to give a zwltterion (C+-O-O-) [31, 321 cannot be ruled out as a reaction contributing to the carotenoid bleaching. Solvent addition to a similar zwitterion has been demonstrated [33]. In organic solvents as well as in Hz0 the k, values approximate to the diffusion-controlled rate constant, which can reasonably be estimated to be (2-3)x 10” M-l s-’ (kdiff of crocetin and molecular oxygen in water was calculated to be about 2 X 10” M-’ s-’ by the Smoluchowsky equation) [ 71. In this respect, bixinoids differ from crocinoids in showing a higher total
59
102-quenching capacity (about one order of magnitude). This fact is best explained assuming that an energy transfer mechanism of the type [34] ‘c + lo,* -ac*
+ao,
where C=carotenoid is responsible for the physical quenching. Such an ‘0,~scavenging mechanism has been proposed for crocetin [7, 351 and p-carotene [Z, 361 on the basis of spectroscopic evidence for the formation of triplet carotenoid. The fact that the energy transfer appears to be slightly endothermic (k,
We are indebted to Dr. T. F. Conway (Moffet Technical Center, SummitArgo, IL, U.S.A.) who kindly made available the ‘H and 13C NMR spectra of bixinoids. Thanks are due to the Minister0 dell’ Universit& e della Ricerca Scientiilca e Tecnologica and Consiglio Nazionale delle Ricerche of Italy for financial support. References 1 C. S. Foote and R. W. Denny, Chemistry of singlet oxygen. VII. Quenching by p-carotene, J. Am. Chem. Sot., 90 (1968) 6233-6235. 2 C. S. Foote, Y. C. Chang and R. W. Denny, Chemistry of singlet oxygen. X. Carotenoid quenching parallels biological protection, J. Am. Chem. Sot., 92 (1970) 5216-5218. C. S. Foote, Y. C. Chang and R. W. Denny, Chemistry of singlet oxygen. XI. Cis-trsns isomerization of carotenoids by singlet oxygen and a probable quenching mechanism, J. Am. Chem. Sot., 92 (1970) 5218-5219. 3 R. J. Cogdell, Carotenoids in photosynthesis, Pure Appl. Chem., 57 (1985) 723-728. P. Mathis and C. C. Schenck, The functions of carotenoids in photosynthesis, in G. Britton
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9 10 11 12
13
14
15
16 17 18 19 20
21 22 23
and T. W. Goodwin (eds.), Carotenoid Chemistry and Biochemistry, Pergamon, Oxford, 1982, pp. 339351. N. I. Krinsky, Function, in 0. Isler (ed.), Carotenoids, Birkhauser, Berlin, 1971, pp. 669-716. M. M. Mathews-Roth, Carotenoids quench evolution of excited species in epidermis exposed to UV-B (290-320 nm) light, Photo&em. Photobiol., 43 (1986) 91-93. M. M. Mathews-Roth, Beta-carotene therapy for erythropoietic protoporphyria and other photosensitivity diseases, Biochimie, 68 (1986) 875-384. E. L. Schrott, Carotenoids in plant photoprotection, Pure Apple Chem., 57 (1985) 729-734. N. I. Krinsky, Carotenoid protection against oxidation, Pure Appl. Chem., 51 (1979) 649-660. P. Man&to, G. Speranza, D. Monti and P. Gramatica, Singlet oxygen reactions in aqueous solution. Physical and chemical quenching rate constants of crocin and related carotenoids, Tetrahedron L&t., 28 (1987) 4221-4224. P. Mar&to, G. Speranxa and V. Malatesta, The quenching of ‘Oe (‘43 by anthracyclines in aqueous solution, II. Evidence for a charge-transfer mechanism, Chem. Phps. L&t., 159 (1989) 310-314. 0. Straub, Key to Carotenoids, Birkhauser, Basel, 2nd edn., 1987, pp. 207-208. J. C. Bauernfeind, G. B. Brubacher, H. M. Klaui and W. L. Marusich, Use of carotenoids, in 0. Isler (ed.), Caroterwids, Birkhauser, Berlin, 1971, pp. 743-770. I. Saito, T. Matsuura and K. Inoue, Formation of superoxide ion via one-electron transfer from electron donors to singlet oxygen, J. Am. Chem. Sot., 10.5 (1983) 3200-3206. L. Zechmeister, Cis-Trans Is0m.eri.c Carotenoids, Vitamins A andArylpo&mes, Springer, Berlin, 1962. G. Speranza, G. Dada, P. Ma&to, D. Monti and P. Gramatica, 13-cti-crocim a new crocmoid of saffron, Gazz. Chim. Ital., I14 (1984) 189-192. G. S. Cox, D. G. Whitten and C. Giannotti, Interaction of porphyrin and metailoporphyrin excited states with molecular oxygen. Energy-transfer versus electron-transfer quenching mechanisms in photo oxidations, Chem. Phus. L&t., 67 (1979) 511615. P. Karrer and T. Takahashi, Pilanzenfarbstoffe XLVII. Uber die Isomerieverhaltnisse beim Bixin. Bemerkungen zu den Theorien uber die Bildung von Carotinoidpigmenten in der Pfianze, nelv. Chim. Acta, 16 (1933) 287-292. T. F. Conway, C. K. Finger, W. A. Jacaway and B. Urbas, Application of two-dimensional NMR techniques for complete proton and carbon-13 assignments of the cis and trans isomers of bixin and norbixin, in K. A. Mueller, R. Kind and J. Ross (eds.), Proc. 22nd Ampere Congr. on Magnetic Resonance and Related Phewmena, Zurich Ampere Commission, Zurich, 1984, pp. 331332; C.A., IO3 (1985) 543192. M. A. Walker, D. R. Roberts and E. B. Dumbroff, Convenient apparatus for methylating small samples with diazomethane, J. Chromatogr., 241 (1982) 390-391. L. Zechmeister and R. B. Escue, A stereochemical study of methylbixin, J. Am C&m. Sot., 66 (1944) 322-330. G. Pattenden, J. E. Way and B. C. L. Weedon, Carotenoids and related compounds. Part XXVI. Synthesis of methyl natural bixin, J. Chem. Sot. C, (1970) 235-241. G. P. Moss, Carbon-13 NMR spectra of carotenoids, Pure Appl. Chem., 47 (1976) 97-102. M. A. J. Rodgers, Solvent-induced deactivation of singlet oxygen: additivity relationships in nonaromatic solvents, J. Am. Chem. Sot., 105 (1983) 6201-6205. R. Schmidt and H. D. Brauer, Radiationless deactivation of singlet oxygen (‘Ad by solvent molecules, J. Am. Chem. Sot., 109 (1987) 6976-6981. D. R. Adams and F. Wilkinson, Lifetime of singlet oxygen in liquid solution, J. Chem. Sot., Faraday Trans. II, 68 (1978) 586-593. H. J. Guiraud and C. S. Foote, Chemistry of superoxide ion. III. Quenching of singlet oxygen, J. Am. Chem. Sot., 98 (1976) 1984-1986. M. V. M. Laileuer, A. W. M. Nieuwint, J. M. Aubry, H. Kortbeek, F. Arwert and H. Joeme, DNA damage by chemically generated singlet oxygen, Free Radic. Res. Commun., 2 (1987) 343350. J. M. Aubry, !l’h.&e de Doctorat d%tat,
Universite de Paris VI, 1982, pp. 126-129.
61 24 R. H. Young, K. Wehrly and R. L. Martin, Solvent effects in dye-sensitized photooxidation reactions, J. Am. Chem. Sot., 93 (1971) 5774-5779. 25 A. A. Gorman, G. Lovering and M. A. J. Rodgers, The photosensitized formation and reaction of singlet oxygen, 02’ (‘A), in aqueous micellar systems, Photo&em. PhotobioL, 23 (1976) 399-403. 26 D. T. Sawyer and J. S. Valentine, How super is superoxide?, Act. Chem. Res., 14 (1981) 393-400. 27 L. Eberson, Electron-transfer reactions in organic chemistry, Adv. Phys. Org. Chem., 18 (1982) 79-185. 28 D. Rehm and A. Weller, Kinetics of fluorescence quenching by electron and H-atom transfer, In-. J. Chem., 8 (1970) 259-271. 29 H. H. Wasserman and R. W. Murray (eds.), Singlet Oxygen, Academic Press, New York, 1979. 30 M. J. Thomas and C. S. Foote, Chemistry of singlet oxygen - XXVI. Photooxygenation of phenols, Photo&em. Photobiol., 27 (1978) 683-693. A. A. Gorman, I. R. Gould and I. Hamblett, Time-resolved study of the solvent and temperature dependence of singlet oxygen (‘Ad reactivity toward enol ethers: reactivity parameters typical of rapid reversible exciplex formation, J. Am. Chem. Sot., 204 (1982) 7098-7104. B. Cazin, J. M. Aubry and J. Rigaudy, Is water the best or the worst solvent for [2+4]cycloadditions of singlet oxygen to aromatic compounds?, J. Chem. Sot., Chem. Commun., (1986) 952-953. 31 K. Goiinick and A. Griesbeck, [4+ 2lCycloaddition of singlet oxygen to conjugated acyclic hexadienes: evidence of singlet oxygen induced cis+ trans isomerization, Tetrahedron I&t., 24 (1983) 3303-3306. 32 Z. Jiancheng and C. S. Foote, Photooxidation of substituted indenes at low temperature, Tetrahedron L&t., 27 (1986) 6153-6156. L. E. Manring, R. C. Kanner and C. S. Foote, Chemistry of singlet oxygen. 43. Quenching by conjugated oleiins, J. Am. Chem. Sot., 105 (1983) 4707-4710. L. E. Manring and C. S. Foote, Chemistry of singlet oxygen. 44. Mechanism of photooxidation of 2,5-dimethylhexa-2,4diene and 2-methyl-2-pentene, J. Am. Chem. Sot., 10.5 (1983) 4710-4717. 33 K. E. O’Shea and C. S. Foote, Chemistry of singlet oxygen. 51. Zwitterionic intermediates from 2,4-hexadienes, J. Am. Chem. Sot., I10 (1988) 7167-7170. 34 N. Turro, Moakrn Molecular Photochemistry, Benjamin, Menlo Park, 1978, pp. 296-361. 35 I. B. C. Matheson and M. A. J. Rodgers, Crocetin, a water soluble carotenoid monitor for singlet molecular oxygen, Photo&em. Photobiol., 36 (1982) l-4. 36 A. Farmiio and F. W-on, On the mechanism of quenching of singlet oxygen in solution, Photo&em. Photobid., 18 (1973) 447-450. A. Gamer and F. Wilkinson, The use of p-carotene as a probe for singlet oxygen yields and lifetime, in B. Ranby and J. F. Rabek (eds.), Singlet Oxygen. Reactions with Organic Ccmpwunds and Polymers, Wiley, New York, 1978, pp. 48-53. M. A. J. Rodgers and A. L. Bates, Kinetic and spectroscopic features of some carotenoid triplet states: sensitization by singlet oxygen, Photo&em. Photobiol., 31 (1980) 533-537. 37 P. Murasecco-Suardi, E. Oliveros, A. M. Braun and H.-J. Hansen, Singlet-oxygen quenching by carotenoids: steady-state luminescence experiments, Helv. Chim. Acta, 71 (1988) 1005-1010. 38 D. Beilus, Physical quenchers of singlet molecular oxygen, Adv. Photo&em., 11 (1979) 105-205. 39 U. Takahama, Suppression of carotenoid photobleaching by kaempferol in isolated chloroplasts, Plant Cell Physiol., 23 (1982) 859-864, and references cited therein. J. P. Knox and A. D. Dodge, Singlet oxygen and plants, Phytochemistry, 24 (1985) 889-896. 40 M. M. Mathews-Roth, T. Wilson, E. Fujimori and N. I. Krinsky, Carotenoid chromophore length and protection against photosensitization, Photochem. Photobid., 19 (1974) 217-222.
Photobiol. B: Biol., 8 (1990) 51-61
51
Interaction between singlet oxygen and biologically active compounds in aqueous solution III. Physical and chemical 102-quenching rate constants of 6,6 ‘-diapocarotenoids+ Giovanna
Speranza++, Paolo Man&to
and Diego Monti
Dipartimento di Chimica Organica e Industriale, Universitd di Milan0 and Centro di Studio suL?e Sostanze 0rganiche Naturali, Consigli~ Nazimmle &lb Ricer&e, via Vent&an 21, I-20133 Milan (Italy)
(Received March 12, 1990; accepted May 16, 1990)
Keywords. Singlet oxygen, 6,6’-diapocarotenoids, constants, energy transfer, electron transfer, bixin.
‘02-quenching rate
AbStnrct
The chemical ‘O,-quenching rate constant ka and the physical ‘02-quenching rate constant k, of a number of 6,6’-diapocarotenoids (bixinoids) were measured using a thermal source of ‘02, lAP Values of k, were found to be close to diffusion-controlled rate constants both in water and in polar organic solvents, whereas ka showed a strong dependence on the medium. These results suggest the co-occurrence of energy and election transfer mechanisms in the quenching of IO2 by blxinoids. Biological implications are discussed.
1. Introduction
The study of the interaction between singlet oxygen 02, lAg and polyolefhrs is of great relevance to the understanding of the protective role played by carotenoids in photosynthesis [l-3] as well as against photodynamic effects L4-61. In continuation of our work [7, 81 in this field, we report here the determination of the physical ‘O,-quenching rate constant k, and the chemical ‘Oa-quenching rate constant ka for a number of 6,6’-diapocarotenoids (l-6). These compounds can be collectively named bixinoids owing to their structural relationship with the most representative member of the group, i.e. bixin (1) [9]. Bixin is the main constituent of annatto, a commercial product obtained from the seeds of Bixu orellunu [lo] and largely used as a food colouring agent. +For details of part II of this paper, see ref. 8. ++Authorto whom correspondence should be addressed.
loll-1344/90/$3.50
Q Elsevier Sequoia/Printedin The Netherlands
52
1
R1 = H
R* = Me
.2
R1 = R2 = Me
METHYLBIXIN
3
R1 = R2 = H
NORBIXIN
BIXIN
C02R2
4
RI = R2 = fle
taanr-METHYLBIXIN
5
RI = R2 = H
trwwNORBIXIN
kinetic method used for the simultaneous measurement of k, and kR has previously been developed by us and shown to be characterized by a satisfactory accuracy [ 7, 81. It is based on the following experimental facts and theoretical assumptions. The water-soluble 1,4_endoperoxide of 3-(4-methyl-l-naphthyl)propionic acid (6) [ 111 was used to produce singlet oxygen non-photochemically (thus avoiding photochemical side reactions, e.g. cis-trans isomerization of the polyene chain [12] and dye-sensitized 02- production [13]). The following sequence of reactions was taken into account: The
Ckt,Cki,C0,~ 6
AOz LA+
‘02
‘02 - kS 302 ‘02--
02
kQ3 +c
‘o,+c
2
product
where AOp is the endoperoxide (6), A the parent naphthalenic acid, C the carotenoid examined for its physical quenching rate constant k, and chemical quenching rate constant ka; kD and ks are the rate constants for the decomposition of the endoperoxide and for the natural decay of ‘02 respectively. When analysed under a steady state approximation for ‘02 concentration, the above system affords the following kinetic equation [7, 81: 2, --a1 tb v, [Cl,
(1)
53
where a= -ks kR
-G=kJAOJo
(2)
exp(-h&
(4)
[AO&being the initial concentration of the endoperoxide corrected for the yield in ‘Oz, and [Cl, and V, the actual concentration of the carotenoid and its rate of disappearance respectively at the time t. Equation (1) is linear (I/& US. l/[C],) if Z,= constant, i.e. for a set of experiments carried out with different initial concentration of the carotenoid, provided that the same [AO,], is used and the two experimental variables are measured at the same time t. This time must be chosen within the interval where the steady state equation
v= -y
=k,[C]
holds, as shown by monitoring [C] by means of its visible absorption at a given wavelength and plotting ln[C] VS. t. Thus the quenching rate constants kR and k, can be calculated from the slope a and the intercept b of eqn. (1). 2. Materials
and methods
UV spectra were recorded on a Perkin-Elmer model 554 spectrophotometer and fluorescence spectra on a Perkin-Elmer model 650-10s spectrofluorometer both equipped with thermostated cells. ‘H (300 MHz) and 13C(75.47 MHz) nuclear magnetic resonance (NMR) spectra were determined on a Bruker CXP 300 spectrometer in dimethylsulphoxide (DMSO) ds using thesignalsolventasinternalstandard(6=2.50and39.5fromtetramethylsilane for ‘H and 13C respectively). Cyclic voltammograms were recorded with an Amel model 466 polarographic analyser. Thin layer chromatography (TLC) was performed with silica gel 60 Fzs4 precoated plates (Merck, 0.25 mm layer) and flash chromatography with commercial Merck silica gel 60 (230-400 mesh ASTM) using in both cases CHC13-MeOH (9:1, v/v) as eluent, unless otherwise stated. All chemicals for kinetic measurements were analytical grade and used without further purification. 2.1. Pmparaticm of biximids 2.1.1. Bixin (1) Commercial bixin (1.2 g) (Roth) was purified by flash chromatography. Fractions showing one orange spot in TLC (l&=0.56) were collected and
54
evaporated under vacuum. The residue (0.9 g) was found to be pure bixin (1) by comparison of its spectral properties (UV [14], and ‘H and 13C NMR [15]) with those previously reported. The UV A,, (log e) values are as follows: in HaO, pH 7.8, 432 nm (4.99), 457 nm (5.13), 486 nm (5.08); in DMSO, 443 nm (4.95), 469 nm (5.08), 499 nm (5.03); in dimethylformamide (DMF), 436 nm (4.95), 464 nm (5.07), 494 nm (5.02). 2.1.2. Methylbixin (2) A slow stream of CHzNz (prepared from DiazaId, Aldrich, 3.2 g) [ 161 was bubbled (N, as carrier) for 2 h into a solution of 1 (380 mg) in CHCl,-MeOH (2:1, v/v) (15 ml). After evaporation of the solvent, the residue was fIash chromatographed giving a compound which was shown to be pure methylbixin (2) (290 mg) by TLC (l&=0.77) and spectral data (UV [ 17, 181, and ‘H [ 181 and 13C NMR [ 191). The UV A,, (log l) values are as follows: in DMSO, 445 nm (4.97), 473 run (5.09) 503 nm (5.03); in DMF, 440 nm (4.97), 466 run (5.09), 496 run (5.02). 2.1.3. Norbixin (3) A solution of bixin (1)(500 mg) in MeOH (35 ml) containing KOH (2 g) was stirred at room temperature under N2 overnight. After evaporation of the solvent under reduced pressure, the residue was dissolved in Ha0 (20 ml). Addition of concentrated HCl solution under stirring gave a red precipitate which was filtered, repeatedly washed with Ha0 and dried under vacuum (60 “C). This product was found to be pure norbixin 3 (330 mg) by TLC (j&=0.33), UV [ 141 and NMR data [ 151. The UV A,, (Iog l) values are as follows: in HaO, pH 7.8, 428 nm (5.07), 450 nm (5.15), 479 nm (5.07); in DMSO, 439 nm (5.01) 466 run (5.08), 495 run (5.03); in DMF, 437 nm (5.00), 462 nm (5.07), 492 nm (5.02). 2.1.4. Tram+methylbixin (4) Iodine (1.5 mg) was added to methylbixin (2) (25 mg) dissolved in CHCla (10 ml> and the solution refluxed for 4 h under Na. After cooling, the reaction mixture was washed with 0.1 N Na$Oa (2 X 10 ml) and then with Ha0 (15 ml) and dried on N&SO+ Removal of the solvent under vacuum and purikation by flash chromatography gave a compound shown to be pure trans-methylbixin (4) (20 mg) by TLC (&= 0.77) and spectral data (UV and ‘H and 13CNMR) [ 17-191. The UV &,,, (log l) values are as follows: in DMSO, 450 run (4.97), 479 nm (5.11), 509 nm (5.07); in DMF, 446 nm (5.00), 472 nm (5.10) 502 run (5.06). 2.1S. Trans-norbixin (5) This compound was prepared from trans-methylbixin (4) as reported for 3. It was shown to be trans-norbixin (6) by TLC (l&=0.33) and ‘H and 13C NMR data [15]. The UV A,, (log e) values are as follows: in DMSO, 443 nm (4.99), 471 nm (5.09), 500 nm (5.03); in DMF, 440 nm (4.99), 465 nm (5.09), 495 run (5.02).
55
2.2. Pmparatim of 3-(1,4-epidioxy-4-methyL1,4-dihydro-l-nuphthylJrwqpionic acid (6) Compound 6 was prepared according to Saito et al. [ 111. After chromatographic purification (silica gel column at 0 “C, elution with hexane-ethyl acetate-acetone-acetic acid 10:3:2:1, v/v) [8] it was shown to be 98% pure by KI titration and TLC (&=0.35 using the above eluent). 2.3. Determination of the rate constant for the decomposition of the en&peroxide (6_) The first-order rate constants k,, for the decomposition of 6 both in water and in organic solvents were determined by monitoring the increase in the UV absorption of the resulting naphthylpropionic acid. The UV &,, values are as follows: in HzO, pH 7.8, 287 run; in DMSO, 289 nm; in DMF, 290 nm. 2.4. Determination of the ‘02-quenching rate constants 2.4.1. Bleaching method In a typical’experiment in 0.1 M phosphate buffer (pH 7.8), two separated solutions of the bixinoid (8.0X 10V6 M) and of 6 (8.0X 10T3 M) were mixed at 0 “C. At this temperature the decomposition of the endoperoxide is negligible [ 111. After deoxygenation by Nz bubbling, the resulting solution was rapidly heated to 35 + 1 “C and allowed to stand at 35 f 0.1 “C. Electronic absorption spectra taken automatically at 3 ruin intervals were recorded in the range 350-600 nm. The disappearance of the bixinoid was monitored by the decrease in the absorbance of the central maximum (Fig. 1). For
OL
I
I
I
I
I
I
350
400
450
500
550
600
Fig. 1. Bleaching of norbixin (3) (5.4X lo-’
of the endoperoxide (6) (7.7
X
A hm)
M) in phosphate buffer @H 7.8) in the presence lo-” M) at 35 “C. Spectra are recorded at 3 n-dn intervals.
56
all experiments performed, a linear relationship of the type ln [C] = -k,t + constant was obtained in the interval l-30 ruin (Fig. 2), thus allowing the time of 6 min after reaching the reaction temperature to be taken as standard. To calculate kR and k, (eqns. (l)-(4)) in aqueous solution and in organic solvents the following constants were used: ka, 2.4 X lo5 s-’ in Hz0 [20], 1.4X lo5 s-’ in DMF [21], 5.2~ lo4 s-’ in DMSO [22]; lcn (measured as indicated above), 2.75~ 10v4 s-l in 0.1 M phosphate buffer at pH 7.8 (in ref. 11, 4.9 X 10e4 s-l in 0.26 M phosphate buffer at pH 7.0), 1.47~ 10m4 s-’ in DMF, 1.70~ 10m4 s-’ in DMSO. In addition, the values of 0.45 in Hz0 [23] and 0.82 in organic solvents [ll] were assumed for the yield in ‘02 formed by decomposition of the endoperoxide (6). 2.4.2. Youn& method [24] Solutions containing compound 6 (2 X 10B4 M), 1,3diphenylisobenzofuran (DPBF) (about 10 -6 M) and varying amount of bixinoids (l-5) (O-5 X 10e5 M) at 0 “C were carefully deoxygenated by Nz bubbling, rapidly heated to 35 f 1 “C and allowed to stand at 35 f 0.1 “C. The disappearance of DPBF was followed by monitoring the decrease in fluorescence intensity of the DPBF (excitation, A= 405 run; emission, A= 458 run) at 3 min intervals. When measurements were carried out in aqueous solution, sodium dodecyl sulfate (SDS) (0.1 M) was used to solubilize DPBF [25]. 2.5. Measurmt of oxidation potentials Oxidation potentials (peak potentials) were measured by cyclic voltammetry using a three-electrode cyclic voltammetric celI (glassy carbon electrode, platinum auxiliary electrode and reference saturated calomel electrode (SCE)) at 25 “C. Sample solutions were deaerated by bubbling Na for 15 min. KC1 (0.1 M) and 0.1 M tetraethylammonium perchlorate (TEAP) were used as supporting electrolytes in HZ0 and organic solvents respectively. The voltammograms were scanned at 100 mV s-‘.
0
I
I
I
9
10
27
min.
Fig. 2. Plot of the decline in the absorbance of norbixin the time, under the conditions reported in Fig. 1.
(3) at A=450
nm as a function
of
57
3. Results and discussion The structures of bixin (l), methylbixin (2), norbixin (3), transmethylbixin (4) and trans-norbixin (6) were confirmed by ‘H and 13C NMR spectra [ 15, 18, 191. The purity of each carotenoid was checked by TLC before use. When a cold aqueous solution of norbixin (3) and the endoperoxide (6) was rapidly heated to 35 “C and kept at this temperature, a bleaching of the reaction mixture was observed as shown in Fig. 1. Using the decrease in visible absorbance to monitor norbixin disappearance (Fig. 2), the kinetic analysis reported above allowed the physical and chemical ‘02-quenching rate constants to be calculated. Analogously, the rate constants for bixin (1) were determined. The values of these constants are reported in Table 1 together with the oxidation potentials (peak potentials) of the two bixinoids. The ‘02-quenching rate constants and the oxidation potentials for 1 and 3, as well as for the other compounds 2, 4 and 6, were also determined in aprotic solvents as shown in Table 2. In this case, a parallel measurement of the total ‘Oa-quenching rate constant (A+= k, + kR) was performed using Young’s method [24, 251 in order to estimate the reliability of the bleaching procedure in those non-aqueous media. In fact, in DMF and DMSO, the bleaching of bixinoids by ‘02 appears to be slow and the ratio k,Jk, higher than 103. These circumstances strongly limit the accuracy of kR and k, values obtainable by the kinetic analysis developed by us [7, 81, as can be seen by considering eqns. (5) or (3). However, good agreement was found between the values of i& resulting from the two methods, thus reinforcing the following conclusions drawn from these experimental data. The marked decrease in the kR value (about two orders of magnitude) observed on going from water (Table 1) to solvents characterized by lower dielectric constants, more positive E, for bixinoids, and more negative redox TABLE
1
‘Oa-quenching 35kO.l “C
Bixin (1) Bixin (l)d Norbixin (3) Norbixin (3)d
rate con&ants
of bixin
(1)and norbixin (3) in aqueous solution (pH 7.8) at
IO-’ kRp (M-i s-i)
lo-" k
0.9(*0.2)
1.8 1.1 2.3 0.9
0.7 (jzO.2) 1.5 (*0.4) 0.8 (&0.3)
(M-’
a
soi) (*0.4) (*0.4) (*OS) (*0.3)
“Mean values of four independent measurements on five bleaching experiments. bDetermined by Young’s technique [24, 251. ‘Measured by cyclic voltammetry. din the presence of 0.1 M SDS.
10-l'k T b (M-l
s-r)
&Xc w
(SW)
0.57 0.7 0.50 0.6
(standard deviation in parentheses),
each based
58
TABLE 2 ‘02-quenching rate constants and oxidation and dimethylsulphoxide at 35 f 0.1 “CL
potentials
of bixinoids
(l-6)
IO-"kR
IO-lok
IO-lo
(M-’
(M-r
so’)
(M-l
s-r)
in dimethylformamide
k b s’r)
&Xc (V (SW)
DMF
Bixin (1) Methylbixin (2) Norbixin (3) Truns-methylbixin (4) Runs-norbixin (5)
1.6 2.1 1.7 2.0 0.8
(51.1) (kO.7) (*Lo) (k0.8) (kO.5)
2.3 3.0 1.2 0.6 0.3
( f 1.0) (f 1.2) (k0.8) (kO.4) (*0.2)
2.0 1.6 1.7 0.9 0.8
0.87 0.92 0.92 0.92
1.4 1.0 1.9 0.8 0.9
(fO.7) (kO.6) (k0.8) (f0.6) (rtO.5)
2.1 1.3 1.7 0.4 0.3
(kO.6) (kO.8) (k0.6) (*0.3) (fO.2)
1.5 2.2 0.9 0.4 0.4
0.82 0.83 0.88 0.86
DMSO
Bixin (1) Methylbixin (2) Norbixin (3) Trans-methylbixin (4) Truns-norbixin (5)
‘Standard deviations are given in parentheses. bDetermined by Young’s technique [24, 251. cMeasured by cyclic voltammetry.
potentials for O.JOa- (E” (vs. SCE) = -0.40 V in HsO, -0.71 V in DMF and -0.58 V in DMSO, using unit concentration as the standard state reference [26]) (Table 2) strongly suggests that an electron transfer [27] process is responsible for the polyene bleaching in aqueous solution. In this medium, AGO< 0 can be calculated by the well-known Rehm-Weller [28] equation using Eo,o= 22.53 kcal mol-’ for O2 excitation energy [29]. The same trend was observed for 8,8’-diapocarotenoids (crocinoids) whose reaction with O2 lAg was explained in terms of an electron transfer mechanism [7]. The close resemblance of the two polyene families (nine and seven noncarboxy double bonds) in their chemical behaviour towards singlet oxygen is a probable reflection of their very similar oxidation potentials [7]. Considering that a reversible exciplex mechanism is generally involved in singlet-oxygen reactions [30, 311, the formation of an intermediate (or a transition state) stabilized by some degree of charge transfer from substrate to O2 ‘Ag is also plausible for bixinoids as an alternative to a full electron transfer [8]. In addition to the electron transfer mechanism, singlet-oxygen addition to the polyene chain to give a zwltterion (C+-O-O-) [31, 321 cannot be ruled out as a reaction contributing to the carotenoid bleaching. Solvent addition to a similar zwitterion has been demonstrated [33]. In organic solvents as well as in Hz0 the k, values approximate to the diffusion-controlled rate constant, which can reasonably be estimated to be (2-3)x 10” M-l s-’ (kdiff of crocetin and molecular oxygen in water was calculated to be about 2 X 10” M-’ s-’ by the Smoluchowsky equation) [ 71. In this respect, bixinoids differ from crocinoids in showing a higher total
59
102-quenching capacity (about one order of magnitude). This fact is best explained assuming that an energy transfer mechanism of the type [34] ‘c + lo,* -ac*
+ao,
where C=carotenoid is responsible for the physical quenching. Such an ‘0,~scavenging mechanism has been proposed for crocetin [7, 351 and p-carotene [Z, 361 on the basis of spectroscopic evidence for the formation of triplet carotenoid. The fact that the energy transfer appears to be slightly endothermic (k,
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