Photochemistry ond Phorobralogy. Vol. 29. pp, 893 10 897 0 Perg,imun Press Lld. 1979. Printcd in Great Brilmn

W31-8655 7910501-0893802 oO/O

ISOLATION AND IDENTIFICATION OF TRYPTOPHAN PHOTOPRODUCTS FROM AQUEOUS SOLUTIONS OF

TRYPTOPHAN EXPOSED TO NEAR-UV LIGHT*

MING SUN? and SEYMOUR ZIGMAN Departments of Ophthalmology and Biochemistry,

University of Rochester School of Medicine and Dentistry, Box 314, 601 Elmwood Avenue, Rochester, NY 14642, U.S.A.

(Received 18 July 1978; accepted 7 November 1978)

Abstract-One of the previously unidentified photoproducts isolated from the photolysate of aqueous tryptophan solutions was identified as 2-carboxy-3a-hydroxy-1,2.3,3a,8,8a-hexahydropyrrolo(2,3b)- indole by direct comparison with the authentic reference compound synthesized using the established procedure. This pyrroloindole alcohol has been shown to be the reduction product of the 3a-hydro- peroxy intermediate (structure 4 in Fig. 1) by Nakagawa er al. (1977). The isolation and identification of this derivative and the detection of the peroxy intermediate 3a-hydroperoxypyrrolidinoindole (4). from irradiated aqueous tryptophan solutions suggests that the direct photooxidation of L-tryptophan to fromylkynurenine may follow a pathway via a tricyclic intermediate instead of the energetically unfavorable dioxotane intermediate. This scheme is similar to the mechanistic model proposed by Nakagawa et a/. (1977) for the rose bengal sensitized photooxidation of tryptophan.

I N T R O D U ~ I O N

Photochemistry of tryptophan has been a popular topic of research for many chemists and bioscientists, not only because it is one of the major aromatic amino acids responsible for protein absorptivity in the UV and near-UV regions, but also because some of its photoproduct (s) and derivatives demonstrated a wide range of biological activities. For example, there are reports indicating that near-UV exposed trpf photoproduct(s) are able to bind to DNA (Glatzer et al., 1976), and to kill bacteria and cells in cultures (Yoakum and Eisenstark, 1972; McCor- mick rf al. 1976). N-Formylkynurenine, one of the identified trp photoproducts, served as a photodyna- mic sensitizer in the inactivation of bovine carbonic anhydrase (Walrant and Santus, 1974). We have also demonstrated that certain trp photoproduct (s) are capable of binding to and inhibiting macromolecule synthesis and mitosis (Zigman et al., 1973, 1976, 1977, 1978), and to inhibit the activities of bovine catalase (Zigman et a/., 1976) and prostaglandin 15-hydroxy- dehydrogenase (Sun et al., 1979). Many of these trp

*Portions of the results were presented at the 6th annual meeting of the American Society for Photobiology in Bur- lington. VT. June 11-15, 1978.

tPresent address: Abbott Laboratories, Abbott Park, North Chicago, IL 60064, U.S.A.

Supported by: N.I.H. Grant. No. EY 00459-09 (National Eye Institute).

SAbhretiations: trp, tryptophan ; HPI, trp photoproduct suspected to be hexahydropyrroloindole compound (5); PPI. try photoproduct suspected to be 3a-hydroperoxy- hexahydropyrroloindole compound (4); NFK, N-formyl- kynurenine; Ky, kynurenine; TLC, thin layer chroma- tography; NMR, nuclear magnetic resonance.

p *. p 29,5--l 893

photoproduct (s) referred to in these works have yet to be identified and isolated.

Recent work by Nakagawa et al. (1977) reported the successful isolation of a tricyclic compound, 2-carboxy- 3a-hydroperoxy- 1,2,3,3a,8,8a- hexah ydrop yrrolo (2.3 b)- indole (structure 4, Fig. l), from rose bengal sensitized photooxygenation of trp solution. Reduction of compound 4 yielded the tricyclic pyrroloindole com- pound 5, N-formylkynurenine (NFK, compound 6) and kynurenine (Ky, structure 7). Thus, they proposed a new model for the dye-sensitized photooxidation of trp to NFK via the tricyclic 3a-hydroperoxy inter- mediate (4).

' m t C O O H

4 0 - A COOH -t &coon H H NC=O

6

'4 HH i

COOH H H

Figure 1. Photooxidative conversion of trp to NFK and Ky via the tricyclic intermediate PPI (4). Compounds 4. 5 , 6 and 7 have been isolated or detected. Compounds 2 and 3 in parenthesis are the proposed short-lived inter- mediates and derivatives to fit this trp photooxidative

pathway.

894 MING S U N and SEYMOUR ZICMAN

In the present communication, we will report the isolation and identification of both compounds 4 and 5 from the direct photolysis of aqueous trp solutions with a near-UV light source and in the absence of dye-sensitizer. We therefore, propose that the direct photooxidative pathway of near-UV exposed trp follows a scheme similar to that for the dye-sensitized photooxygenation of trp.

MATERIALS AND METHODS

L-Tryptophan, butanol. acetic acid, 2.2-thiodiethano1, rose bengal and acetic anhydride were purchased from Eastman Organic Chemical Company. Hydrogen peroxide (50% solution), 2-6-lutidine and KI/starch strip were from Fischer Scientific Company; Ninhydrin was from Pierce Chemical Company ; 2-mercaptoethanol and kynurenine were from Sigma Chemical Company; pyridine was from J. T. Baker Chemical Company; and N-formylkynurenine was from Calbiochemical Company. Pre-scored TLC plates were from Analtech, Inc., the dimensions of TLC plates were 10 x 20cm and 0.25 mm thickness. Irradiation was carried out with a UV lamp (PCQ 008L; U V Products, Inc.) emitting principally at 365 nm at an inten- sity of 30 W/mZ. The temperature of the solutions was kept at either 37°C or 4°C with a Neslab PBC-4 water bath. cooler, and heater in tandem. The procedure for photolysis was described previously (Zigman et a/., 1976b).

Peroxyacetic acid was prepared according to Savige (1975) and Findley et a/. (1945). To synthesize (5), 5.1 g of trp was dissolved in 500mP of distilled water (concen- tration 50 mM), 9.3 m/ of the peroxyacetic acid solution (2.75 M ) was slowly added to the trp solution at 0-5"C which was kept in a refrigerator at this temperature for 24 h. A small amount of mercaptoethanol or 2,2-thiodieth- anol was then added to the mixture until the Kl/starch reaction became negative. The pH of the solution was adjusted to about 3-4 with pyridine. The deep yellow- colored solution was lyophilized to dryness. The residue was either redissolved in 25 m/ of water, and then submit- ted to recrystallization, or dissolved in about 80mC of water, filtering off the insoluble matter, and then applied to a Sephadex G-10 column (1 10 x 5.5 cm). After the void volume (800 mt), 150 fractions at 15 m/ each were col- lected. This column was also used for the preliminary sep- aration of rose bengal from trp photoproducts in the dye- sensitized reactions. The fractionation profile was con- structed by measuring the absorption at 290 nm.

Absorption spectra were measured with a Cary 14 recording spectrophotometer. Fluorescence emission and excitation spectra were measured with a Perkin-Elmer model 512 double beam spectrophotometer at 5nm or lOnm resolution. Mass spectra were obtained using a Dupont Mass Spectrometer (21-490B) at 75 and 20 eV with the chamber temperature at 280°C. The 3a-hydroperoxy compound (4) was synthesized photochemically in an air saturated tryptophan solution in the presence of 0.4 mmol of rose bengal, irradiated with a near-UV light source (Sun er d., 1979) at 4°C for 12h. followed by alumina and Sephadex G column separation. KIistarch positive frac- tions were pooled.

Further purification was carried out on alumina oxide or silica gel precoated TLC plates at 0.25 mm thickness. After developing the TLC plates for 2 h in methanol: H,O (9:l) they were dried in a hood and sprayed with 0.2% ninhydrin and with 1% 2,6-lutidin in acetone. they were then heated in an oven at 70°C for 5min to develop the color. Melting point, NMR and mass spectra were measured at the Chemistry Department, University of Rochester, with the assistance of department personnel.

RESULTS AND DISCUSSION

'Although at least nine trp photoproducts have been detected after exposure of trp to flash photolysis in the presence of oxygen, most of these photoproducts. except NFK and Ky, have not been identified (Savige. 1971 ; Pailthorpe et af. (1973). This report rcpresents new progress in a long search for the structures of these unknown compounds (Zigman et ul., 1973, 1976; Sun et al., 1978). Figure 1 describes schemati- cally the transformation of trp to NFK and KY via the tricyclic peroxy-pyrroloindole compound (4). The intermediate (4) is relatively unstable. HPI (5) and NFK (6) are produced at room temperature from this intermediate from the TLC isolation procedure.

Figure 2a shows the chromatographic profile of near-UV light induced photoproducts from a Sephadex G-10 column. Four peaks labelled A. B. C and D, respectively, were separated from the un- reacted trp (peak T) after the void volume. Figure Zb shows the elution profile of rose bengal sensitized trp photooxidative products (after a preliminary run on a larger G-10 column to eliminate rose bengal). The experimental conditions were different from that de- scribed by Nakagawa et a / . (1977) in that we have not continuously bubbled oxygen into the solution during the irradiation. Also, we used a Xe irradiation source with maximal output a t about 365 nm instead of the halogen lamp source employed by Nakagawa et al. (1975). Nevertheless, a large number of fractions of the eluents in the last portion of peak G. gave a positive KI/starch test. as marked by '+' signs.

Figure 2c indicates the separation of the chemically synthesized 3a-hydroxypyrroloindole as peak H. Peak H was easily identified as compound (5) from its known physical properties (Savige. 1975) and from its fingerprint absorption spectrum, and was located in the same fractionation region as that of peak C and F. Further analysis indicated that the samples in fractions C and F behaved similarly to those of H in their movements on the TLC plates and in their reaction with ninhydrin and ninhydrin plus lutidin. Also, their absorption spectra resemble each other closely in that they exhibit absorption maxima at about 235 and 290nm with a minimum at 260-270 nm. The ratio between the two absorption maxima of the compounds isolated from peaks C and F were slightly different from that of the synthesized compound. The KI/starch test indicated that the per- oxy derivative (PPI) is located between fraction 34 and 42, separate from compound (5). Thus, Fig. 2. provided a close comparison of Sephadex G-10 fractionations of the trp products derived from three different procedures: a, direct near-UV irradiation ; b, dye-sensitized photooxidation : and c, peroxyacetic acid chemical synthesis, respectively. Unreacted trp (peak T) was clearly separated from all of its deriva- tives, and was unmistakably identified from its absorption spectrum. Peak H was found to consist of compound (5). Peaks C and F are located in the

Identification of trp photoproducts 895

\ i! ‘. r\ ROSE BENGAL I+ SENSITIZED

i F G + x L * - - - - d f T

CHEMICALLY SYNTHESIZED

20

0 10 20 30 40 50 60 70 80 90 FRACTION NUMBER

Figure 2. Sephadex G-10 filtration. The column (480 x 22 mm) was equilibrated at 5°C with deionized water; the flow rate was 0.5m//min. A lorn/ portion of the concentrated reaction mixtures were first applied to the column and Sm//tube fractions were collected. Figure 2a is the fractionation profile of a 1% aqueous trp solution after exposure to near-UV radiation for 24 h (2592 J). Figure 2b is the fractionation of 1% trp irradiated for 12 h (1296J) in the presence of 0.4mM rose bengal as photosensitizer. Figure 2c is the elution profile of aqueous trp solutions treated with peroxyacetic

acid (see method section).

same fractionation region as that of H, indicating that C and F have the same constituents as found in peak H.

Peaks A, B and D of Fig. 2 have not been identified yet. When these fractions were concentrated and applied to TLC, several bands appeared in the devel- oped plates. NFK and Ky could be detected in the mixture, particularly under peak D. In future reports of this series we will reveal the identity of the other trp photoproducts.

Further purification by TLC of the material from peak C yielded essentially pure HPI. judging from its absorption spectrum, mass spectrum and melting point. Solvent selection and TLC coating material are important in this isolation procedure, particularly in the isolation of the PPI. In butanol: acetic acid: H 2 0 (4: 1 : 1) at room temperature, PPI shows three nin-

hydrin-lutidin positive spots on TLC (Table 1). The R,’s of these spots as well as the color appearance from the ninhydrin-lutidin reaction (Table 1) indi- cated that in addition to PPI (R, of 0.385. purple), HPI ( R , of 0.32, purple) and NFK (R, of 0.23, bright yellow) were formed. When methanol/H20 (9: 1) was used as the developing solvent, only one spot could be observed (Rf, 0.59 purple, KI/starch positive). These results indicated that HPI and NFK could form from PPI during TLC purification using the butanol-acetic acid-water solvent system.

Figure 3 is the mass spectrum of HPI isolated from the photolysate of 1% trp aqueous solution after exposure to the near-UV light source for 14 h and then isolated by Sephadex G-10 and TLC (methanol: H20, 9: 1). It shows a primary peak at m/e 220, corre-

Table 1. TLC properties of different trp derivatives in butano1:acetic acid:H,O (4 : l : 1) and in methanol:H20 (9: 1). Compounds were detected using a UV fluorescent lamp and the color reaction with ninhydrinflutidin

BUOH :ACETIC AC1D:HZO (4: I : 1) ME0H:HIO (9:l) Compound Rf Ninh ydrinllutidin Rf Ninhydrin/lufidin

TRY 0.40 KY 0.33 N FK 0.33, 0.22 5-OH TRY 0.34 3-OH KY 0.36 H PI 0.33 PPI 0.385

0.32 0.23

Brown 0.57 Brown Violet 0.49 Bright yellow 0.49. 0.44 Yellow Brown - 0.54 Mauve 0.52 Purple Purple 0.59 Purple Mauve Bright yellow

-

0.57 Dark brown -

896

I

MING SUN and SEYMOUR ZIGMAN

133 158

175

I I I, 1111 121

I I I 105 94 202

1111

Figure 3. Mass spectrum of HP. at 20 eV; ni/e of the major peaks are indicated.

sponding to the molecular weight of compound (5). The other major peaks are at 202 (-HzO), 175 (-carboxyl)? 158 (-carboxy, -H20), 146 (-glycyl) and 133 (-alanyl). Considerable difficulties were encountered in obtaining the NMR of the isolated PPI and HPI during the experiments. In D20, CDJl and deuterated acetone solvents, the NMR of the lyo- philized compounds was dominated by the absorp- tion of H 2 0 . Extensive drying procedures at an ele- vated temperature (70°C) in the presence of air led to the decomposition of the photoproducts. Vacuum dried HPI demonstrated multiplet a t 6.75-7.50, and doublet and singlet at 5.40. 5.80, 4.73. 4.40, which are comparable with those reported by Savige (1975) and Nakagawa, (1977). The melting point of HPI is 219-221°C. slightly lower than those measured by the above authors for their synthesized compound (5), probably reflects the existence of a mixture of both trum and cis isomers.

The mass spectrum (Fig. 3) of the isolated HPI, with its primary peak at m/e 220 and all other major peaks, as indicated, is matched closely with those from the synthesized reference compound. Melting point and NMR are also in general agreement with the published results (Nakagawa et al., 1977; Savige. 1975).

Figure 4 shows the absorption spectrum of isolated HPI. The absorption maxima are at 237 and 293 nm with a minimum at 262 nm. The extinction coefficient

I

1.0 {

0 220 260 300 340

WAVELENGTH (nm) Figure 4. Absorption spectrum of HPI isolated from the photolysate of trp after irradiation for 24 h with a near-UV

light source (2592 J).

.is 2000 at 293 nm. These values are very close to those of the synthesized compound (5) and entirely different from the known trp derivatives, NFK and Ky.

WAVELENGTH (nm) Figure 5. Fluorescence excitation and emission spectra of HPI in H,O at room temperature (3 nm resolution). Emission was scanned with the excitation set at 287nm. and excitation was scanned with

the emission set at 334nm.

Identification of trp photoproducts 897

Figure 5 presents the excitation and emission spec- tra of HPI in water at room temperature. The sample appeared to be spectroscopically pure after the TLC isolation step. The uncorrected emission maximum is at 320nm with a broad shoulder a t 340nm. There were n o other detectable emissions from the sample. Flourescent characteristics of HPI provided us with another fingerprint to identify the compound as well as a criterion to judge the purity of the isolated HPI. Since fluorescent emission measurement is about one thousand-fold more sensitive than absorption measurement, frequently a single well-separated frac- tion from the chromatographic column could be suffi- cient to detect the presence of a minute amount of this compound. This detection method is of great convenience and is advantageous, since pooling of the fractions may reduce the resolution. Concentration or other handling procedures may enhance the possi- bility of decomposition or other types of alteration. I t is noted that the fluorescent spectrum is substan- tially different from those of many other indoles (Sun

and Song, 1977). Predictably, the quantum yield and the emission spectra at low temperature will be differ- ent from all other indoles also, since the tricyclic pyrroloindole molecular framework of (5) should be more rigid than that of the other indoles.

Thus, comparisons of physical properties, including Sephadex G-10 column chromatographic behavior. TLC. melting points, absorption, emission, NMR and mass spectra, between the isolated photoproduct HPI and the synthesized tricyclic compound (5). have indi- cated that they are the same. These results prove the presence of HPI (5) in the near-UV photolysate of trp. Although the direct near-UV light irradiation procedure is entirely different from the procedure used by Nakagawa, we have been able to isolate HPI and to detect PPI (4). We also observed that NFK (6) and Ky (7) could be readily generated from PPI. Thus, the Fig. 1 scheme could be applied to near-UV photolysis of aqueous trp solutions similar to that presented by Nakagawa et a!. (1977) for the dye- sensitized photo-oxygenation of trp.

REFERENCES

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Proc. Narl. Acad. Sci. US. 74, 4730-4733. Nakagawa. M.. K. Yoshikawa and T. Hino (1975) J. Am. Chem. SOC. 97. 649fj-6501. Pailthorpe, M. T.. J. P. Bonjour and C. H. Nicholls (1973) Photocheni. Photobiol. 17. 209-223. Savige, W. E. (1971) Aust. J. Chem. 24, 128551293, Savige. W. E. (1975) Aust J. Chem. 28. 2275-2287. Sun, M. and P. S. Song (1977) Photochem. Photobiol. 25. 3-9. Sun. M.. S. Zigman and H. H. Tai (1979) Photochem. Photobiol. 29. 63-66. Yoakum. G. and A. Eisenstark (1972) J. Bacteriol. 112, 653-655. Walrant, P. and R. Santus (1974) Phorochem. Photobiol. 20, 455460. Zigman. S., J. Schultz, T. Yulo and G. Griess (1973) Expl Eye Res. 17, 209-217. Zigman, S., J. D. Hare, T. Yulo and D. Ennist (1978) Photochem. Photobiof. 27. 281-284. Zigman, S. and J. D. Hare (1976a) Mol. Cell. Biochem. 10. 131-135. Zigman. S.. T. Yulo and G . A. Griess (1976b) Mol. Cell Biochem. 11, 149-154.

(1945) J. Am. Chem. SOC. 67, 412. Biochim. Bioplrys. Acta 418, 137-145.