Photo-induced generation of the riboflavin-tryptophan adduct and a vibrational interpretation of its structure

173

Vibrational Spectroscopy, 6 (19941173-183 Elsevier Science Publishers B.V., Amsterdam

Photo-induced generation of the riboflavin-tryptophan adduct and a vibrational interpretation of its structure M.M. Campos Vallette Universidad de Chile, Facultad de Ciencias, Departamento de Quimica, Casilla 653, Santiago (Chile)

R.G. Diaz F. Universidad de Playa Ancha, Departamento de Quimica, Casilla 34-V Valparaiso (Chile)

A.M. Edwards, S. Kennedy and E. Silva Pontificia Universidad Catolica de Chile, Facultad de Q&mica, Departamento de Quimica Biologica, Casilla 306, Correo 22, 536 Santiago (Chile)

J. Derouault and M. Rey-Lafon Laboratoire de Spectroscopic Moleculaire et Crzktalline, URA 124 CNRS, Universite’de Bordeaux, 351, Cours de la Liberation, 33405 Talence Cedex (France) (Received 20th October 1992)

Abstract

A pure fraction of a riboflavin-tryptophan adduct was obtained for the first time from a mixture of products resulting from the anaerobic photoirradiation of riboflavin and tryptophan in solution. The procedure used to isolate the adduct is described. Characterization of the compound was performed by using absorption spectra and fluorescence measurements. A vibrational interpretation of the structure of this adduct was performed on the basis of its infrared spectrum. The vibrational assignment suggests an important electronic redistribution in the structure of riboflavin and ttyptophan when the adduct is formed. From this fact can be inferred at least two ways of binding between the indole and isoalloxaxine rings. Stability of the adduct is also discussed. Keywords: Infrared spectrometry; Riboflavin; Tryptophan

Fluorimetry;

Raman spectrometry;

The nutritionally essential amino acid tryptophan (Trp) and the vitamin riboflavin (Rb) are common components of our diet. Notwithstanding this fact, the simultaneous presence of these Correspondence to: M.M. Campos Vallette, Universidad de Chile, Facultad de Ciencias, Departamento de Quimica, Casilla 653, Santiago (Chile). 0924-2031/94/$07.00

Band assignment;

Photo-induced

coupling;

two compounds in biological systems exposed to the action of visible light has been related to hepatic disfunction observed during parenteral nutrition [1,2] and to cytotoxic effects in culture media [3,4]. During the last past years, we have described [5-8,111 the generation of an adduct between Rb and free or protein-bound Trp following irradia-

0 1994 - Elsevier Science Publishers B.V. All rights reserved

174

tion under anaerobic conditions, and this compound has been proposed to be one of the photoproducts which may be responsible for hepatic disfunction [7] and cytotoxicity in irradiated culture media [8]. However, it has not yet been possible to elucidate the precise chemical nature of the binding between these compounds, because of the simultaneous modifications of Rb by the action of light [9] and the complexity of the indole photochemistry [10]. On the basis that photoadduct formation by intermolecular photoreduction of Rb, promoted by substrates of low oxidation potential, is a common process involving addition to the sensitizer at the Caa , C 8 or N 5 positions [9], a binding between the C 3 atom of the indole and the N 5 atom of the flavin has been postulated [11] (see Figs. 1 and 2). Infrared and Raman spectroscopy are very useful for the structural study of molecules. A band is the result of the existence of a bond or a simple interaction between molecules or atoms. However, interactions such as hydrogen bonding or intermolecular binding are normally weak; in such a case the spectral study can be made through the spectral modifications of molecular modes sensitive to these interactions. An infrared band assignment of Rb in the region 1750-250 cm -1 was proposed by Abe et al. [12]; a normal coordinate treatment for the lumiflavin compound allowed a better understanding of the vibrational spectrum of the isoalloxazine skeleton [13]. Infrared and Raman spectra of N-acetyl-L-amino acid methylamides were registered in the regions 4000-100 cm -1 and 20-4000 cm -1, respectively [14]; on this basis a tentative assignment for the indolyl group was proposed. An assignment of the low-energy infrared bands (650-30 cm -1) was performed for Trp [15]. Harada et al. [16] reported a vibrational study on the influence of the surroundings on Trp in proteins, following spectral modifications of the infrared Raman doublet at about 1350 cm-1. Some band assignments resulted from a theoretical analysis of the resonance Raman spectrum of Trp [17]. A vibrational interpretation of the structure of the Rb-Trp adduct has not been published yet.

M.M. Campos Vallette et aL / Vib. Spectrosc. 6 (1994) 173-183

HC

H C/ '

[lo

~9

c

c~

1

~c

N

~C/S,NNf~,N~/3~ I°

'

'ii

u "

Rb: R~-Cl-I2 - CH(OH ) - CH (OH) - CH(OH) - CH~OH

H

f

?

I

\

H

H

Trp: R"=CH2-CHICOOH }- NH2 Fig. 1. Structure and numbering of the isoalloxazine (top) and tryptophan (bottom) rings.

Interaction between flavin and indole rings was studied by means of the x-ray data of the model complex 7,8-dimethylisoalloxazine-10-acetic acid-

RI

Ra

Fig. 2. Structure of the riboflavin-tryptophan adduct (Rb-Trp) after Ref. 11.

175

M.M. Campos Vallette et al. / VT. Spectrosc. 6 (1994) 173-183

L-tryptophan methyl ester [181; the adduct formation is stabilized by a ~~~,,,,~-rr~_~~~~charge transfer between the indole ring and the isoalloxazine skeleton. The position of the rings is nearly parallel and most of the inter-ring distances are smaller than the normal Van der Waals distance (3.4 A). This work aims to obtain, by means of vibrational spectroscopy, an insight into the way of binding between Trp and Rb.

EXPERIMENTAL

Riboflavin and tryptophan were obtained from Sigma and were used as received. 14C-Riboflavin and 14C-tryptophan were purchased from Amersham International. Solutions of Rb (3.5 x 10m5 M) and Trp (1.05 x 10v4 M) in 0.05 M phosphate buffer of pH 7.0 were irradiated for 15 min in a l-cm light path cell thermostated at 37°C. Anaerobic condition was reached by nitrogen bubbling. Light from a 150 W slide projector lamp, equipped with an interference filter, was employed for irradiation at 452 nm. The energy flux was 3.0 J rnw2 s-l. Gel filtration was carried out on a 42 x 1.8 nm I.D. Sephadex G-15 column equilibrated with distilled water. Liquid chromatography (LC) was performed with a reversed-phase CL-Bondapack C,, column (30 x 8 cm I.D., Waters). The mobile phase was 5 mM sodium acetate-citric acid buffer, pH 3.6 and 25% methanol (v/v). Results were registered and evaluated with Waters 746 Data Module. Absorption spectra were recorded on a Varian Super Scan III spectrophotometer and the fluorescence measurements were performed with a Perkin-Elmer 650-10 S fluorescence spectrometer. Infrared spectra of solids Rb, Trp and Rb-Trp were recorded from 4000 to 190 cm-’ in CsI mulls by using the Bruker 113-V and PerkinElmer 621 spectrometers. The Raman spectra displayed a strong fluorescence which prevented the identification of the vibrational bands. Highly diluted samples were used for the spectral measurements in the region 4000-2000 cm-‘.

RESULTS AND DISCUSSION

Riboflavin-tryptophan adduct characterization The elution profiles in a Sephadex G-15 column of a solution of Trp (10.5 X 10m5 M) and Rb (3.5 x lo-’ M) in 0.05 M phosphate buffer of pH 10) without irradiation and irradiated anaerobically with monochromatic light (452 nm) during 15 min are shown in Fig. 3. A significant decrease in the signals of the reactants is observed (peaks III and V, corresponding to unreacted Rb and Trp) along with the appearance of products with higher molecular weights than those of Rb and Trp (peaks I and II>. Peak IV has spectral characteristics similar to those of lumichrom, a known product of Rb photodegradation [191. Peak I is the main and major fraction and gives a positive ninhydrin test indicating the amino acidic character of this fraction. The absorption (Fig. 4a) and emission (Fig. 4b) spectra confirm the flavinic origin of peak I. When this fraction was excited at 260 nm, a typical flavinic emission at 520 nm was found along with two other emission bands, at

0.2

a

t

b

w :

m Iv v

II

I

I

I

I

0

30

60

0

30

60

Tube number

Fig. 3. Elution pattern on a Sephadex G-15 column (42~ 1.8 cm I.D.) of solutions of tryptophan and riboflavin (a) both unirradiated and (b) irradiated with monochromatic light (A = 450 nm) during 15 min under anaerobic atmosphere at pH 7.0 in 0.05 M phosphate buffer.

MM. Campos Valktte et al. / Vii. Spectrosc. 6 (1994) 173-183

a

b

,

10

M

6aJ

I

500

I

LOO

20

30 Time (mif]

Fig. 5. LC chromatogram in a reversed-phase C1s column of peak I shown in Fig. 3. LC conditions are given in Experimental.

\i L

34lo

A (nm)

Fig. 4. (a) Absorption and (b) emission (Aex = 280 nm) spectra of peak I shown in Fig. 3.

355 nm and at 460 nm, indicating the presence of at least two other components in this fraction. Peak I was re-chromatographed through the same G-15 column and subsequently applied to a reversed-phase C,, column; the chromatogram shown in Fig. 5 was obtained. A great number of fractions are observed, with peak X being the main and major. Peak X was collected, concentrated and applied to the Sephadex G-15 column for desalting, and the fraction obtained (fraction X) gave the absorption spectra shown in Fig. 6. A noticeable zone is observed fn the visible region, with flavinic-like characterist~arallel experiments using r4C-tryptophan and r4C-riboflavin allow us to distinguish the flavinic and the indolic origin of fraction X; the molecular size of this fraction is larger than that of the reactants. Fraction X is therefore attributed to the riboflavintryptophan adduct.

Viirational assignment and structural hterpretation Compared with those of Rb and Trp, the spectrum of the adduct displays broad absorptions and considerable band shifts in particular in the spectral range 1700-1400 cm-’ (see Figs. 7 and 8). The infrared band assignment for the Rb-Trp adduct and its precursors Rb and Trp was carried

t!i

6

n ‘0 QOS tn

9

300

LOO

500

1

io

Akim)

Fig. 6. Absorption spectrum of peak X shown in Fig. 5.

M.M. Campos Vallette et al. /Vii.

Spectrosc. 6 (1994) 173-183

out on the basis of partial data reported for Rb and Trp [12-171, vibrational studies of indole [20] and pyrrole [21] in solution, and general data from spectral tables [22] and related molecules [23-331. Self-association of Rb and Trp. The qualitative and comparative analysis of the Rb and Trp spectra suggests the existence of associated species in the solid state (see Figs. 7 and 8). In the case of Rb this fact is manifested mainly by the presence of the absorption at 3217 cm-’ corresponding to an NH stretching MNH)] [24], and of the broad bands localized at 3500 and 3374 cm-’ [22], which correspond to the v(OH) modes. The absorptions at 1733 and 1650 cm-’

4ow

3200

2400 WAVENUMBER

1600

177

were attributed to the v(C4=O) and v(+O) modes, respectively, according to the assignment proposed for lumiflavin and riboflavin. derivatives [12,13]; the C,=O bond was found weaker than the C,=O bond, suggesting strong intra- and intermolecular hydrogen bonds between the oxygen atom of the C,=O group and the ribitylic OH functions and the hydrogen atom bound to the nitrogen in position 3. The band at 1035 cm-‘, only observed in the spectrum of Rb, is attributed to v(C-OH) ribitylic vibrations [22]; this result confirms the participation of the alcoholic function in the self-association. The self-association of Trp is verified mainly by the v(N,H) and the deformation S(N,H) modes which appear at 3404

800

CM-1

Fig. 7. Infrared spectra of riboflavin (Rb), tryptophan (Trp) and riboflavin-tryptophan 4000-600 cm-‘.

adduct (Rb-Trp)

in the spectral range

M.M. Campos Valkvte et al. / Vii. Spectrosc. 6 (1994) 173-183

178

600

500

400

WAVENUMBER

300

I

30

CM-1

Fig. 8. Infrared spectra of riboflavin (Rb), tryptophan (Trp) and riboflavin-tryptophan adduct (Rb-Trp) in the spectral range 700-200 cm-‘.

and 1116 cm-‘, respectively; in indol, these bands are shifted to 3500 and 1080 cm-’ by dilution t201. Zwitterionic structure of Tip. The presence of the Trp bands at about 3079, 2568, 2081, 1666, 1591,1487, 1415 and 589 cm-’ is compatible with a zwitterionic structure for the amino acidic group [15,22,32,331. The bands at 3079 and 1487 cm-’ are assigned to the stretching and bending vibrations of the NI-If group; the absorptions at 1666 and 1591 cm-l, and 1415 cm-’ are due mainly to the asymmetric and symmetric v(COO-1 modes, respectively [22]. The weak band at 589 cm-’ is assigned to a wagging of the COO- anion, following Husain et al. [153; this mode is probably coupled to a pyrrolic out-of-plane deformation [161. The other two absorptions at 2568 and 2081 cm-’ were ascribed to v(NH *- - 0) [25].

Ring vibrations of Rb and Trp. The weak absorption at 1622 cm-’ of Rb could be assigned to a Y(W) mode of ring I (see Fig. 1); the corresponding band in the spectrum of Trp is not directly observed because of the strong absorptions at 1666 and 1591 cm-‘. The well-defined bands at 1582, 1549 and 1505 cm-’ in the spectrum of Rb and the broad one in that of Trp at 1591 cm-‘, which is composed by at least two absorptions, have been assigned to v(CC) and v(CN) [22]; these modes are currently coupled. Following the spectral assignment proposed by Takeuchi and Harada [26] for indole and the results of a normal coordinate analysis performed by Abe and Kyogoku [13] for lumiflavin, we propose these bands to be attributed to the v(C,C,) and v(C,C,) modes in Trp and v(C,,N,) and v(C,,,N,) in Rb. The bands at 1357 and 1347 cm-i were assigned to the coupled vibrations v(C,C,) and &C,N,) in Trp and u(C,,,N,~) and v(C,,C,) in Rb, respectively, following Takeuchi and Harada [26] and Abe and Kyogoku [13]. Three bands around 510 and 450 cm-’ in the spectra of Rb and Trp are attributable to out-ofplane deformations of the pyrrolic, pyrimidinic and pyrazinic rings. Adduct spectrum. The broad bands at 3495 and 3460 cm-’ correspond to the free v(OH) vibrations of both the amino acidic COOH function and the ribitylic CHOH and CH,OH groups [22] coupled to the v(NH) pyrrolic mode; this mode appears as a strong band around 3500 cm-’ in the spectrum of indole solutions 1201.The medium broad band at 3315 cm-’ has been proposed as due to a pyrimidinic or amino acidic v(NH) rather than to a pyrrolic one. The weak broad band at about 2940 cm-’ is assigned to the CH bonds of the CH, groups. The CO bands at 1733 cm-’ [Y(C~O)] and 1650 cm-’ [v(C,O)] for Rb are shifted to higher energies by adduct formation. One of the components of the band at 1591 cm-’ in the spectrum of Trp could be associated to the CO function of the COO- group; this band is also shifted to higher frequencies by adduct formation. On this basis the strong broad bands at 1754 and 1711 cm-i of the adduct are undoubtly assigned to the u(C0) modes; the first one should correspond to

179

M.M. Campos Vallette et al. / Vi&.Spectrosc. 6 (1994) 173-183 TABLE 1 Fundamental infrared band frequencies (cm-‘) for riboflavin (Rb), tryptophan (Trp) and riboflavin-tryptophan and their corresponding assignment ‘. Rb

Trp

Rb-Trp

Most probable assignment

3500 w 3374 bw 3217 w 3115 vw 3037 bvw 2939 w

3404 s

3495 m 3460 w > 3315 bm 3200 w

v(OHrib.+ aminoacid) + v(NHpyTA VW-l amino acid 1 v@JH,) + v(CHknzene)

2940 vw

v(CH*)

3079 sh 3050 bms

_I.

2568 vw 2081 m > 1733 s 1650 s 1622 m 1582 ms 1549 s 1505 m 1458 dwm 1435 bw 1399 m 1369 w 1347 m

1666 s 1591 bs b

1211 sh 1180 m 1155 w

1079 dms 1061 sh 1035 w 1014 m 988 vw 928

VW

1754 s 1711 s 1623 vw

V(Copyrimidine)

v(CC

’

1357 s 1337 sh 1316 wm 1294 vw 1279 w 1251 w 1231 m 1207 vm 1190 vwm 1158 m 1144m 1132 VW 1116 vw 11OOm 1077 wm 1052 bw 1022 sh 1008 m 988 m > 964 vw 926 m

+ v(coamino

+ v(CNiscmlloxazine)

v(Coandno

acid)

6(NH;)

S(OH) + AC01

1430 >

1360 1339 1321 ms b 1307 1278 1244

V(CNmmidine) 6(CH, + %N,H) v(CC) + v(CN) 60-I,) o(CH,) v(CC benzene) OKH,) v(CN) v(CC WTKk

1218 1199 s b 1170 1143

+pyrimid&) + ACN)

? v(CC pyrimidine) NCH

+ 6(mbenzene)

benzene )

p(NH:) 6m-o ~(~,rok)

1082 m 1054 m >

Izoalloxazine ring def. +6(CH t+znzene)+ V(CNpyrimidine) ? ~(C-OHebityl) “(CNtimidine + indole)

967 sh

?

946 dms 906m >

Ring def. + ACH,) + P(CH indole )

896 sh

884m 867 vw 850 m 821 w 808 m

883ms

S(CCCbenzene

865 mult. ms

+ indolyl )

I 820 m 803 wm

acid)

benzene )

v(CCindole)

1392

1306 w 1276 w 1239 bm

&NH:)

1591 bs 1537 sh 1487 w 1458 ms 1415 bs

adduct (Rb-Trp),

S(mCpyrimidine+pymzizinc)

>

+P(~,,,“J

M.M. Camps Vallette et al. / VT%Spectrosc. 6 (1994) 173-183

180 TABLE 1 (continued) Rbs 784 sh 775 m

Trp 778 vw 764 sh 760 s

Most probable assignment

Rb-Trp

v(CC pyde)+ v(C-cHJ

?

~(CHpyrro,,)

782 s

v(CC benzene

742 w 718 bw 681 w 674 sh 656 vw 626 VW 611 sh 596 wm

575 m 533 m 519 m 501 m 474 486 wm > 450 s 410 m 384 ww

295 bm 275 w 258 w 248 VW 233 w 218vw

)

6(OH)

707 VW 683 m

?

697 dm

N’JH

2) + P(a

benzene)

?

656 w 627 m

642 bm >

S(CCC)

+ S(CO)

? 596 sh 589 wm 581 m 558 m 549 m 528 ms 510 s 498 m

S(CNC

pyrrole + pyrimidine

1

o(coo-)+X(py&)

599 bm 551bm

S(CNC ryrrote)+ GtNCCisoattoxazine)

> 503dbms

Abenzene) + G(C(J’J,,i”,acid) x( pyrmle

+ pyrimidine

+ pynzine

)

+ pyrimidine

+ pynzine

1

S(C0) 456 m 426 s

425 bw >

x( pyrrole 6CCO)

397 vw 348 dm 325m 269 m 252wrn

?

387bs > 282bm

p(NH) + amino acid group def. 7(interring

Trp)

T(CC)

252w

X(benzene) ?

237 m 217 m 210 sh

g(CCCribityl)

210bw >

+ a(CCNamino

acid)

alcohol sensitive

a Abbreviations: s, strong; m, medium; w, weak, sh, shoulder (irrespective of strength); v, stretching; S, in-plane deformation; p, out-of-plane deformation; r, torsion; w, wagging; ,y, out-of-plane ring deformation; mult., multiple; b, broad; d, double. b Massif.

the CO groups of the isoalloxazine skeleton while the second one may be due to the carboxyhc function of Trp (Table 1). The bands of Trp assigned to different vibrations of the COO- and NH: ions are not observed at similar frequencies in the spectrum of the adduct; this fact suggests structural modifications of NH: and COO-. Therefore the zwitterionic structure disappears by adduct formation and the band at about 3315 cm-’ should then correspond to a v(NH) of the NH, group.

Bands ascribed to the v(C,Cs) and v(C,C,) modes in Trp and Y(C~~N~)and ~(Cr,,~Nrj in Rb were not observed in the region 1650-1450 cm-’ of the adduct spectrum, suggesting an important electronic density redistribution in the corresponding bonds involved in the adduct formation. Weak bands of Rb at 1458 and 1435 cm-’ are assigned to aromatic Y(CC) and ribitylic S(OH) modes, respectively [12]. The strong bands of Trp at 1458 and 1415 cm-’ are attributed to the %CH,) and V&COO-) modes, following the as-

M.M. Campos Vallette et al. / vib. Spectrosc. 6 (1994) 173-183

sigmnent given for glycine and alanine [32,331. All these bands are in correspondence with the one observed at about 1430 cm-’ in the spectrum of the adduct. This band belongs to a group of strong bands constituted by at least five absorptions displayed between 1450 and 1280 cm-‘. The spectral modifications in this region are associated with both the disappearance of the ribitylC,O interaction and the change of COO- into COOH. The band at 1399 cm-‘, which is observed only in the spectrum of Rb, is shifted to 1392 cm-’ in the adduct. It has been assigned to a mixture of Y(C~NJ and v(C,N,) modes in flavin compounds [13]; the corresponding bonds are slightly sensitive to the adduct formation. The weak absorption at 1369 cm- ’ is observed only in the spectrum of Rb; it is assigned to a coupled vibration of the 6cCH.J and 6(N,H) modes [12]. The fact that this mode is not observed at a similar frequency in the spectrum of the adduct could be related to both the dissapearence of self-associated species of Rb during the adduct formation and the eventually high sensitivity of the N,H bond to the Rb-Trp interaction. On the basis of that proposed for Trp and Rb in the region 1370-1340 cm-‘, we assign the band of the adduct at 1360 cm-’ either to v(CC) or v(CN) vibrations of the isoalloxazine and indole rings. No evident spectral modifications of the precursor bands at 1307 and 1278 cm-’ upon adduct formation were observed. The first absorption is a benzenic v(CC) while the second one contains an important 6@JH) + Y(CN) character [26]. Five maxima were observed between 1244 and 1143 cm-‘. Two of these bands at 1218 and 1244 cm- ’ correspond probably to the v(CC) and v(CN) coupled modes. Bands at 1231 and 1251 cm-’ of Trp were assigned to the Y(C&,) and 6(CH) [S(C,H) and 6(&H)] modes, respectively, according to the results of a normal coordinate analysis for indol [26]; the broad band at 1239 cm-’ in Rb is a v(CC) vibration with a v(CN) contribution: Abe and Kyogoku [13] suggested that this band in flavin derivatives is due mainly to the v(C,N,) and v(C,C4,) modes. The fre-

181

quency shifts are associated with an electronic density redistribution, mainly in the C,C, indolic isoalloxazinic bonds of the adduct. and C&, The frequency shift of the bands at 1158 cm-‘, associated mainly with 6(C,H) + 6(C,H) 1261 in Trp and at 1155 cm-‘, assigned to v(C&,,,) and v(C,C,,) [13] in Rb, allows one to infer that the absorption in the adduct at 1170 cm- ’ expresses an important dependence of the corresponding bonds on the Rb-Trp interaction. The band observed at 1143 cm-’ in the spectra of the adduct and Trp with similar relative intensity and frequency was assigned to a 6(CH) benzenic mode; such a result suggests that this mode is not involved in the Rb-Trp interaction. The weak band at 1132 cm-‘, only active in the spectrum of Trp, was attributed to a p(NHl) vibration following the assignment proposed by several authors [25,27,30,32,331; its disappearance upon adduct formation confirms the change of NH; into NH,. A medium absorption at 1100 cm- ’ of Trp is assigned to an indolic 6(CH) mode in agreement with that proposed for indole and indolizine rings [26,31]; this band was not observed in the spectra of the adduct and Rb. This could be due to an important participation of that mode in the RbTrp interaction. Adduct absorptions at 1082 and 1054 cm-’ are observed at about the same frequencies in the spectra of the precursors; these bands were assigned to vibrations of rings I and II coupled to 6(CH) benzenic and v(CN) pyrimidinic modes. The absorptions at 1008 and 988 cm-’ of Trp and at 1014 and 988 cm-’ in the Rb spectrum, are shifted in the spectrum of the adduct. Matsuura et al. 1141suggest that these bands involve indolic vibrational modes; Abe and Kyogoku [13] propose, from normal coordinate calculation for isoalloxazine rings, that these absorptions are mainly due to v(CN) vibrations of the pyrimidinic ring: v(N,C,) and Y(N&). From this result it can be inferred that these modes in the adduct are involved in the Rb-Trp interaction. It is usual to expect the ribitylic vibrations WCC), v(COH), 6(CH), S(COH) and S(CCC)] below 1000 cm-‘; these vibrations are normally coupled, which makes their spectral assignment

182

difficult. In terms of the results of Takeuchi and Harada [26] for indole and the assignment of bands in N-acetyl+amino acid derivatives of Matsuura et al. [14], bands at 946 and 906 cm-’ in the spectrum of the adduct can be attributed to ring deformations coupled to CH, rocking and p(C,H) indolic modes. The band of the adduct at 883 cm-r is due to benzene deformations [20] coupled to indolic modes [14,29,31]; this absorption appears in the region 884-850 cm-’ in the spectrum of Rb and around 865 cm-’ in that of Trp. The Rb and Trp bands at 821 and 760 cm-’ are observed at 820 and 782 cm-‘, respectively, in the spectrum of the adduct. Ring II and III deformation modes S(CNC) [12,22], indolic v(E) vibrations and benzenic S(CCC) and pD(CH) modes [26], are expected in the region 850-720 cm-‘. On the basis of calculations for indole [26], the Trp band at 778 cm-’ is assigned to a v(C,C,) mode; the double band of Rb at 775 cm-’ probably contains a v(C-CH,) mode. The spectral shift of the adduct band at 782 cm-’ is attributed to the indolic p(C,H) vibration [26]. The band of the adduct at 697 cm-r is observed at 683 and 681 cm-’ in the spectra of Trp and Rb, respectively; we propose this absorption to be assigned to p(CH), probably strongly coupled to S(NH,) [22]. This spectral shift seems to be associated with the change of NH: into NH,. Bands between 660 and 620 cm-’ of Rb and Trp are observed around 642 cm-r in the spectrum of the adduct; this band assigned to a benzene ring deformation could be coupled to a 6(CO) deformation as proposed by Husain et al. 1153. The spectral shift probably arises from this vibrational coupling. The spectral shift of the Trp band at 581 cm-r is associated with the adduct formation; this absorption is attributed to a coupled vibration of the iXCCN) and G(CNC) modes. The broad band of the adduct at 551 cm-r is mainly due to a coupled vibration of the out-of-plane benzene ring deformations and the amino acidic S(CCN) modes. The spectral shift upon adduct formation of the out-of-plane deformation of the pyrrolic, pyrimidinic and pyrazinic rings near 510 and 450

M.M. Campos Vallette et al. / Vi&.Spectrosc. 6 (1994) 173483

cm-’ of Rb and Trp, suggests that the adduct broad bands at 503 and 425 cm-r are highly sensitive to the interaction between the isoalloxazine and indole systems. The 6(CO) and amino acidic deformations at about 480 and 340 cm- ’ of Rb and Trp resulted to be particularly sensitive to adduct formation. The strong and well-isolated band of the adduct at 387 cm-’ is assigned to a p(NH) vibration. Aliphatic C-C, R-CH, and ring torsions are active below 300 cm-’ The band at 282 cm-’ is assigned to an indolic CsC, torsion mode [26], and the band at 252 cm-’ is attributable to a benzenic deformation; in both cases the corresponding modes are observed in the spectra of precursors at about the same frequency. Conchions

Products of higher molecular weight than the reactants were obtained when a solution containing Trp and Rb (pH = 10) was anaerobically irradiated with monochromatic visible light (450 nm). Using a reversed-phase C,, LC column we have isolated the main and major of these products, which had flavinic and indolic characteristics, and corresponded to a photo-induced Rb-Trp adduct. A vibrational interpretation of the structure of this adduct was performed on the basis of its infrared spectrum. Particular spectral characteristics of bands assigned to NH and CO vibrations of Rb and Trp suggest associated species in the solid state. Concerning the Trp moiety, the formation of the adduct mainly involves the pyrrolic NH bond; a zwitterion structure for the amino acidic group is not ruled out. The second moiety, Rb, displays intra- and/or intermolecular interactions between the CO group and the NH and OH functions of the pyrimidinic ring and the ribitylic chain, respectively. The band assignment of the adduct spectrum suggests an important electronic energy redistribution in the isoalloxazine and indole systems; the self-association of precursors Rb and Trp disappears upon formation of the adduct. These facts are well evidenced by the spectral modifications of the vibrational transitions concerning surroundings of the nitrogen atoms in position 1 and

Spectrosc. 6 (1994) 173-183

183

5 of the isoalloxazine ring and the indolic C,, C, and C, atoms. It has been also observed that the C,-0 and N-H bonds of the pyrimidinic ring and vibrations of the COO- and NH: groups are energetically reinforced by the formation of the adduct, suggesting a modification of the zwitterion structure of the amino acidic chain. The spectral interpretation is compatible with the radical structure of precursors proposed by Silva et al. [ll]. The adduct formation involves a strong interaction between the indole and isoalloxazine systems; the nature of this association could be attributed to a r-rr charge transfer as proposed by Inoue et al. [18] or to a photoinduced binding C, . . . N, [111. The great chemical stability of the adduct could only be explained by a cooperative effect of both types of interactions. A formal C,-N, bond cannot be ruled out; however, because of the presence of strong groups of bands around 1380 and 1200 cm-‘, it was not possible in this paper to confirm this possibility.

7 M.N. Donoso, A. Valenzuela and E. Silva, Nutr. Rep. Int., 37 (1988) 599. 8 E. Silva, M. Salim-Hanna, MI. Becker and A. De Ioannes, Int. J. Vitam. Nutr. Res., 58 (1988) 394. 9 P.F. Heelis, Chem. Sot. Rev., 11 (1983) 15. 10 D. Creed, Photochem. Photobiol., 39 (1984) 537. 11 E. Silva, M. Salim-Hanna, A.M. Edwards, M.I. Becker and A.E. De Ioannes, in M. Friedman (e.d.), Nutritional and Toxicological Consequences of Food Processing, Plenum Press, New York, 1991, pp. 33-48. 12 M. Abe, Y. Kyogoku, T. Kitagawa, K. Kawano, N. Ohishi, A. Takai-Suzuki and K. Yagi, Spectrochim. Acta, 42A (1986) 1059. 13 M. Abe and Y. Kyogoku, Spectrochim. Acta, 43A (1987) 1027. 14 H. Matsuura, K. Kasegawa and T. Miyazawa, Spectrochim. Acta, 42A (1986) 1181. 15 S.K. Husain, J.B. Hasted, D. Rosen, E. Nicol and J.R. Birch, Infrared Phys., 24 (1984) 201. 16 I. Harada, T. Miura and H. Takeuchi, Spectrochim. Acta, 42A (1986) 307. 17 G. Marconi, J. Raman Spectrosc., 22 (1991) 361. 18 M. Inoue, Y. Okuda, T. Ishida and M. Nakagaki, Arch. Biochem. Biophys., 227 (1983) 52. 19 M. Sun, T.A. Moore and P.-S. Song, J. Am. Chem. Sot., 94 (1972) 1730. 20 R. Barraza, M. Campos-Vallette, K.A. Figueroa, V. Manriquez and V. Vargas C., Spectrochim. Acta, 46A (1990) 1375. 21 M. Campos-Vallette, K.A. Figueroa and V. Vargas C., Spectrochim. Acta, 44A (1988) 601. 22 G. Socrates, in Infrared Characteristic Group Frequencies, Wiley, New York, 1980. 23 C.A. Acevedo-Gonzalez, M.M. Campos-Vallette and R.E. Clavijo C., Spectrochim. Acta, 42A (1986) 919. 24 T. Uno and K. Machida, Bull. Chem. Sot. Jpn., 34 (1961) 545; 34 (1961) 551. 25 E. Steger, A. Turcu und V. Macovei, Spectrochim. Acta, 19 (1963) 293. 26 H. Takeuchi and I. Harada, Spectrochim. Acta, 42A (1986) 1069. 27 K. Machida, M. Izumi and A. Kagayama, Spectrochim. Acta, 35A (1979) 1333. 28 J. Bandekar, L. Genzel, F. Kremer and L. Santo, Spectrochim. Acta, 39A (1983) 357. 29 L. Colombo, P. Bleckmann, B. Schrader, R. Schneider and Th. Plesser, J. Chem. Phys., 61 (1974) 3270. 30 M. Tsuboi, T. Takenishi and A. Nakamura, Spectrochim. Acta, 19 (1963) 271. 31 A. Lautie, M.F. Lautie, A. Gruger and S.A. Fakhri, Spectrochim. Acta, 36A (1980) 85. 32 M. Tsuboi, K. Onishi, I. Nakagawa, T. Shimanouchi and S. Mizushima, Spectrochim. Acta, 12 (1958) 253. 33 K. Fukushima, T. Onishi, T. Shimanouchi and S. Mizushima, Spectrochim. Acta, 15 (1959) 236.

MM Campos Vaflette et al. /I&.

The authors M.C.-V. and G.D. thank FONDECYT, Grant 0935-92, DIG1 Univ. de Playa Ancha, Grant 319192 and DTI Univ. de Chile, Grant Q-3075-9012. M.C.-V. also thanks the Association France-Chilienne pour le D&61oppement de 1’Enseignement et de la Recherche Universitaire en Chimie, AFCUC, for supporting a research state at the Universite de Bordeaux I. E.S. and A.M.E. thank FONDECYT Grants 0389-89 and 19305171.

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