phytoalexin accumulation in arabidopsis during the … · an arabidopsis phytoalexin purification...

Plant Physiol. (1992) 98, 1304-13090032-0889/92/98/1 304/06/$01 .00/0

Received for publication September 3, 1991Accepted November 15, 1991

Phytoalexin Accumulation in Arabidopsis thalianaduring the Hypersensitive Reaction toPseudomonas syringae pv syringae1

Jun Tsuji, Evelyn P. Jackson, Douglas A. Gage, Raymond-Hammerschmidt, and Shauna C. Somerville*Michigan State University-Department of Energy Plant Research Laboratory (J. T., S.C.S.), Max T. Rogers NuclearMagnetic Resonance Facility (E.P.J.), Michigan State University-National Institute of Health Mass Spectrometry

Facility, Department of Biochemistry (D.A.G.), and Department of Botany and Plant Pathology (R.H., S.C.S.),Michigan State University, East Lansing, Michigan 48824

ABSTRACT

Inoculation of leaves of Arabidopsis thaliana (L.) Heynh. withthe wheat pathogen, Pseudomonas syringae pv syringae, resultedin the expression of the hypersensitive reaction and in phyto-alexin accumulation. No phytoalexin accumulation was detectedafter infiltration of leaves with a mutant of P. s. syringae deficientin the ability to elicit a hypersensitive reaction; with the cruciferpathogen, Xanthomonas campestris pv campestris; or with 10milimolar potassium phosphate buffer (pH 6.9). Phytoalexin ac-cumulation was correlated with the restricted in vivo growth of P.s. syringae. A phytoalexin was purified by a combination ofreverse phase flash chromatography, thin layer chromatography,followed by reverse phase high performance liquid chromatog-raphy. The Arabidopsis phytoalexin was identified as 3-thiazol-2'-yl-indole on the basis of ultraviolet, infrared, mass spectral,1H-nuclear magnetic resonance, and '3C-nuclear magnetic reso-nance data.

Phytoalexins are low mol wt, antimicrobial compounds ofplant origin that accumulate after inoculation with a plantpathogen (14). A number of observations support the hypoth-esis that phytoalexins play a role in the defense response ofplants to pathogens. Phytoalexins are absent in healthy tissuesand accumulate after infection by fungal (18, 24, 30) orbacterial (9, 1 1) pathogens in monocotyledonous plants (24)as well as in dicotyledonous plants (9, 18, 30). Phytoalexinshave been demonstrated to accumulate rapidly at the site ofattempted infection in sufficient quantities to inhibit the invitro growth of fungi (18, 30) and bacteria (9). Virulence ofthe fungus Nectria haematococca on pea is correlated withthe ability to detoxify the phytoalexin pisatin (28). Further-more, transformation of Cochliobolus heterostrophus with thegene encoding pisatin demethylase allowed this maize patho-

Supported by Michigan Agricultural Experiment Station (No.1648), National Institutes for Health-National Centers for ResearchResources (PH l-RR00480 to J. Watson), U.S. Department of Energy(DE-FG02-90ER2002 1), U.S. Department ofAgriculture (No. 8080),and the Michigan State University Foundation. J.T. was supportedin part by a fellowship from the College of Natural Science.

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gen to cause limited infections on pea (20). These observationssuggest that phytoalexins contribute to disease resistance.Although a large body of evidence supports the defensive

role for phytoalexins, phytoalexin accumulation is not theonly disease resistance-associated response that has been ob-served in plants. Plants often respond to infection with a HR,2a rapid localized necrosis that is a common response of plantsto bacteria, fungi, and viruses (10). Plants have also beenobserved to deposit lignin and hydroxyproline-rich glycopro-teins in their cell walls and to synthesize the fungal cell wall-degrading enzymes chitinase and ,3-1,3-glucanase after infec-tion (5). Because a number of host responses have beencorrelated with disease resistance, the relative contribution ofphytoalexins to disease resistance remains controversial.We have initiated an investigation of phytoalexin accu-

mulation in Arabidopsis thaliana (L.) Heynh. to address thecontribution ofphytoalexins to disease resistance. Arabidopsisoffers many advantages for physiological and molecular stud-ies of disease resistance in plants (12). Pseudomonas syringaepv syringae van Hall, a bacterial wheat pathogen, is nonpath-ogenic on Arabidopsis and elicits a HR. In this paper, wereport on the purification and structural determination of aphytoalexin from Arabidopsis and examine the accumulationof this phytoalexin during the HR to P. s. syringae.

MATERIALS AND METHODS

Biological Materials

Arabidopsis thaliana race Columbia was grown as described(29). The following bacterial strains were used in this study:Pseudomonas syringae pv syringae Nal', PSSD20, a wheatpathogen (23); P. s. syringae Nalr, Kanr, Hrp-, PSSD20::Tn5,PSSD22, a nonpathogenic mutant (23); and Xanthomonascampestris pv campestris (Pammel) Dowson Rifr, 2D520, acauliflower pathogen (21). Bacteria were grown, suspended in10 mM potassium phosphate buffer (pH 6.9) to 108 cfu/mL,and infiltrated into leaves of 3-week-old plants as describedpreviously (29).

2 Abbreviations: HR hypersensitive reaction; cfu, colony formingunit; EI, electron impact.

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AN ARABIDOPSIS PHYTOALEXIN

Purification and Quantification of the ArabidopsisPhytoalexin

Phytoalexin accumulation was elicited by spraying leavesof 3-week-old Arabidopsis with 10 mm silver nitrate in 0.1%Tween 20. After 1 d, leaves were harvested, quickly weighed,and placed in boiling 80% methanol for 15 min. The extractwas cooled to room temperature and then filtered throughWhatman filter paper No. 1. The methanol was then removedby rotary evaporation at 35°C, and the resulting aqueoussolution was extracted three times each with 2 volumes ofchloroform. The chloroform-soluble extracts were pooled,evaporated to dryness, and stored at -20°C.The chloroform-soluble residue was dissolved in 50%

ethanol and fractionated by flash chromatography on C,8(25). The C,8 column (2 x 7.5 cm) was developed with a stepgradient of 0, 25, 50, 75, and 95% ethanol, and I0-mLfractions were collected and assayed for antifungal activity asdescribed below. Fungitoxic fractions were combined, and thesolvent was removed by evaporation under nitrogen. Thesample was dissolved in chloroform and fractionated by TLCon silica gel. TLC plates were developed in chloro-form:methanol (9:1, v/v), air-dried, and assayed for inhibitionof fungal growth by the Cladosporium bioassay as describedbelow. The Arabidopsis phytoalexin was eluted from the silicagel with methanol, concentrated under nitrogen, and purifiedby reverse phase HPLC. HPLC was performed on a Varian500 instrument with a Waters ,uBondapac C,8 column (0.78x 30 cm) as previously described (16) except elution was witha linear gradient of 1 to 100% acetonitrile at a flow rate of2.0 mL/min.

Purified Arabidopsis phytoalexin was quantified in metha-nol at 318 nm using a molar extinction coefficient of 14,800M-'cm_'. This molar extinction coefficient was calculatedusing the Beer-Lambert law by measuring the UV absorbancesofweighed samples. A linear relationship between phytoalexinconcentration and absorbance at 318 nm was observed withphytoalexin concentrations between 300 ng/mL and 30 ,ug/mL methanol.

Spectral Analysis

UV absorption spectra were obtained on a Beckman DU-70 spectrophotometer; IR spectra were obtained on a NicoletFTIR/42; EI and high resolution (R = 5000) mass spectrawere obtained on a JEOL AX505 double focusing massspectrometer; and the fast atom bombardment mass spectrumwas acquired on a JEOL HX 110 double focusing mass spec-trometer with nitrobenzyl alcohol as the liquid matrix. 'H-and '3C-NMR spectra were obtained on a Varian VXR 500spectrometer at 500 and 125 MHz, respectively. Chemicalshifts were referenced to the solvent, CDC13.

Fungal and Bacterial Growth Inhibition Assays

Antifungal activity was detected by the Cladosporium-TLCbioassay (1). Extracts were spotted onto silica gel 60A K6FTLC plates (Whatman) and the plates were developed inchloroform:methanol (9:1, v/v). The plates were then air-dried and sprayed with a dense conidial suspension of Cla-dosporium cucumerinum Ellis & Arth. suspended in double-

strength potato dextrose broth warmed to 40°C. The plateswere then incubated under high humidity in the dark at roomtemperature for 3 d.

Antibacterial activity was detected as described (I 1) exceptthat an agar overlay was used. Purified phytoalexin prepara-tions were spotted onto silica gel TLC plates, and the plateswere overlayed with a mixture of P. s. syringae suspended in50°C King's B agar (19) amended with 0.05% 2,3,5-triphenyltetrazolium chloride. The plates were then incubated in amoist chamber in the dark at room temperature for 3 d.

Bacterial Growth Study

Leaves ofArabidopsis were infiltrated with a suspension ofP. s. syringae PSSD20 at a concentration of 1 x 108 cfu/mL.At 0, 1, 2, 3, and 5 d post-inoculation, an oval leaf disc (0.44cm2) was removed from the inoculation site, rinsed threetimes in sterile distilled water, and then macerated in 1 mLof 10 mm potassium phosphate buffer (pH 6.9). Viable countswere determined by plating serial dilutions of the leaf extracton King's B agar (19) containing 200 ,ug nalidixic acid/mL.

RESULTS

Purification of the Arabidopsis Phytoalexin

The Arabidopsis phytoalexin was eluted from the C,8 flashchromatography column with 95% ethanol. This fraction wasfurther purified by TLC on silica gel. The phytoalexin mi-grated on the TLC plates with an RF value of about 0.56.Final purification of the Arabidopsis phytoalexin wasachieved by HPLC on a C,8 column. Purified phytoalexineluted from the column as a single peak with 61% acetonitrile(retention time, 37 min) (Fig. 1). By this purification method,2.31 mg of the Arabidopsis phytoalexin was recovered from1.28 kg fresh weight of elicited leaf tissue. The phytoalexinwas also purified from P. s. syringae-inoculated leaves andwas determined to be identical to that purified from silvernitrate-treated leaves based on the UV and mass spectra.

E

(N

LiJ

z

Kuz0L()aD

1100

0 1 0 20 30 40 50 60

LiiJ

75 zF-z0

50 L,

25 Lw(-9ci:LLiici-

0

RETENT ION TIME (min)

Figure 1. Reverse phase HPLC of fungitoxic fractions obtained fromsilica gel TLC plates. Elution was with a linear gradient of 1 to 100%acetonitrile.

I

I

.1I

.1

I

I

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Plant Physiol. Vol. 98, 1992

Antifungal activity was also detected in a more polar frac-tion that eluted from the C18 HPLC column with 42% ace-

tonitrile (retention time, 25 min). This fraction was presentin minor quantities and was unstable when rechromato-graphed on the reverse phase column. This fraction was notpurified to homogeneity and was not investigated further.

Structural Determinafion of the Arabidopsis Phytoalexin

The Arabidopsis phytoalexin was obtained as a stable,colorless solid that fluoresced bright blue-purple on a TLCplate under long wavelength UV (302 nm) illumination. Thefluorescence was visible when as little as 3.5 ng of the Arabi-dopsis phytoalexin was spotted on a TLC plate.

In the El mass spectrum, the purified Arabidopsis phyto-alexin displayed a prominent molecular ion as the base peakat m/z 200. This mol wt determination was confirmed by fastatom bombardment MS, which displayed an intense peak forthe protonated molecule (MH+) at m/z 201. High resolutionEI MS indicated that the formula for the Arabidopsis phyto-alexin was C1iH8N2S (measured, 200.0401; calculated,200.0408), which is consistent with the isotope distributionpattern observed in the low resolution mass spectrum. Thisformula suggested that the compound contained a combinedtotal of nine rings and double bonds (rings + double bonds =x - y/2 + z/2 + 1, for CxHyNz).The UV spectrum of a methanolic solution of the Arabi-

dopsis phytoalexin contained absorbance maxima (log E) at318 nm (4.17), 275 nm (3.92), and 215 nm (4.36), in accordwith the high degree ofconjugation indicated by the molecularformula.The 'H-NMR spectrum (500 MHz, CDC13) displayed six

one-proton and one two-proton downfield signals between7.2 and 8.5 ppm, which could be grouped into three spincoupling systems based on selective decoupling experiments(Fig. 2). The first spin system consisted of the broad singlet at8.45 ppm, which was exchangeable with D20, and coupled toa narrow doublet at 7.87 ppm (J = 2.2 Hz). The second spin

system involved four protons: a one-proton multiplet at 8.25ppm, a one-proton multiplet at 7.43 ppm, and a two-proton,ABXX' resonance at 7.28 ppm. Taken together, these twospin systems were suggestive of a monosubstituted indolesubstituted at the 3 position, with the broad singlet at 8.45ppm representing the N-H proton and the narrow doublet at7.87 ppm representing H-2. The two one-proton signals at8.25 and 7.43 ppm were assigned to H-4 and H-7, respectively,and the two-proton signal at 7.28 ppm was assigned to H-5and H-6. The assignments were confirmed by comparison ofthe 500 MHz 'H-NMR spectrum of the Arabidopsis phyto-alexin with those of 3-cyanoindole, 3-methyl indole, andbrassinin at the same field strength. The '3C-NMR data ofthe Arabidopsis phytoalexin were also consistent with theseinterpretations.The two additional coupled doublets at 7.82 and 7.23 ppm

(J = 3.3 Hz) remained to be accounted for in the spectrum.Excluding these latter two protons, the indole structure ac-

counted for eight carbons, six hydrogens, and one of thenitrogen atoms in the molecular formula. In addition, six ofthe nine degrees of unsaturation were fulfilled. Only a fewpossible structures can be proposed to fit the last three car-

bons, one sulfur, one nitrogen, two hydrogens, and the re-

maining three degrees of unsaturation into the molecule. Onepossibility, the a,f-unsaturated isothiocyanate, can be ex-cluded on the basis of the stability of the Arabidopsis phyto-alexin relative to the expected instability of the isothiocyanateand the absence of a prominent IR absorption between 2000and 2300 cm-'.Three alternate structures seemed more likely. The peaks

at 163.1 and 142.6 ppm in the '3C-NMR spectrum, the twocoupled protons at 7.82 and 7.23 ppm in the 'H-NMRspectrum that could be assigned to the two carbon peaks at142.6 and 115.9 ppm by heteronuclear multiquantum coher-ence data, and the fragment ion m/z 58 (C2H2S) in the mass

spectrum indicated that the substituent attached to the indolewas a thiazole or an isothiazole (15). These possibilities werediscriminated by 'H-NMR. The coupling constant (J = 3.3

H2 H4'

H7H4

NH

IH5'

8.5- 8.4 8.3 8.2 8.1 8.0 7. 9 7.t8 7.7 7.6 7.5 7.4 ppm

Figure 2. 'H-NMR spectrum (500 MHz, CDC13) of the Arabidopsis phytoalexin.

1 306 TSWI ET AL.




1

Figure 3.indole.

Structure of the Arabidopsis phytoalexin, 3-thiazol-2'-yl-

Hz) between the doublets at 7.82 and 7.23 ppm is character-istic of a monosubstituted thiazole substituted at the 2' posi-tion (3). Hence, the structure of the Arabidopsis phytoalexinis 3-thiazol-2'-yl-indole as shown in Figure 3.

Phytoalexin Accumulation in Arabidopsis andRelationship with the HR to P. s. syringae

When leaves of Arabidopsis were inoculated with the non-host pathogen, P. s. syringae PSSD20 (approximately 1 x 108cfu/mL), a confluent, grayish, sunken necrosis characteristicof the HR (10) was observed within 24 h. During the sameneriod leaves infiltrated with P. s. svrin-aae PSSD22. the Hr,-mutant of.potassiumThe vial

Arabidopsiinoculated

6

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0)

0

-j

5

4

Figure 4. Tiinto leaves cStandard eradditional ex0.5% sodiun

--- 150N I

E

>/

~ oo

0 1 0 20 30 40 50

Hours post-inoculationFigure 5. Time course of the accumulation of phytoalexin activity.Leaves of Arabidopsis race Columbia were infiltrated with (0) P. s.syringae PSSD20, (O) P. s. syringae PSSD22, (x) X. c. campestris2D520, or (P) 10 mm potassium phosphate buffer (pH 6.9). Chloro-form-soluble fractions of methanol leaf extracts (50 mg fresh weight)were assayed for fungitoxic activity, and the area of fungal growthinhibition in MM2 was recorded. Each point is the mean of threeseparate experiments. Standard error bars are shown.

P. s. syringae PSSD20; X. c. campestris; or 10 mM teria by dilution plating. In Arabidopsis, populations of P. s.phosphate buffer (pH 6.9) were symptomless. syringae increased within 24 h after inoculation but thenbility of P. s. syringae inoculated into leaves of exhibited a decline in the number of viable bacteria after 24ts was investigated by reisolating P. s. syringae from h (Fig. 4).leaves and determining the number of living bac- To detect the presence of a phytoalexin, inoculated leaves

were harvested and chloroform-soluble fractions of methanolextracts were prepared. From the Cladosporium-TLC bioas-say, antifungal activity was detected as a single zone of fungalgrowth inhibition with an RF value of approximately 0.56.Phytoalexin activity increased rapidly between 12 and 24 hafter inoculation with P. s. syringae and reached maximumactivity between 24 and 48 h postinoculation (Fig. 5). Duringthe same time period, little or no antifungal activity was

-i\ T | detected by the same method in extracts prepared from leavesinfiltrated with 10 mm potassium phosphate buffer (pH 6.9);

I I\T | the crucifer pathogen, X. c. campestris; or a mutant of P. s.I syringae deficient in the ability to elicit a hypersensitiveI reaction in tobacco or Arabidopsis (Fig. 5).I Once a method for purifying the Arabidopsis phytoalexin

had been developed, the accumulation of this compound inleaves of Arabidopsis was assessed. After inoculation with P.s. syringae, the amount of the phytoalexin increased in par-allel with the level of phytoalexin activity to a maximum level

0 1 2 3 4 5 6 of about 8 ,ug/g fresh weight (Fig. 6). No fluorescence under

Days post-inoculation long UV illumination was observed with extracts preparedfrom uninoculated leaves. The antimicrobial activity of the

ime course of growth of P. s. syringae PSSD20 infiltrated purified phytoalexin was quantified by determining the min-)f Arabidopsis. Each point is the mean of six replicates. imum amount of the phytoalexin able to reproducibly inhibitror bars are shown. Similar results were obtained in two the growth of either the fungus C. cucumerinum or the bac-Kperiments in which leaf discs were surface sterilized in terium P. s. syringae. An aliquot of 250 ng completely inhib-n hypochlorite for 1 to 2 min before maceration. ited the growth of C. cucumerinum. A higher amount, 1 ,ug,

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Plant Physiol. Vol. 98, 1992

10

8

6

4

n

- T

I

T

0 10 20 30

Hours post-inoculat

Figure 6. Time course of the accumulation of 3-thiazfollowing inoculation with P. s. syringae PSSD20. Thwas purified from inoculated leaves of Arabidopsis e

using a molar extinction coefficient of 14,800 M-1 cm

is the mean of two separate experiments. Standard i

shown.

was required to inhibit the growth of P. s. syringshown).

DISCUSSION

Leaves of Arabidopsis exhibited a classic HRlated with the wheat pathogen P. s. syringae.'were restricted within the necrotic tissue anddecline in the number of viable cells. To deternthe decline in P. s. syringae population levels an

sion of nonhost resistance may be due in partmulation of antimicrobial compounds by Araused the Cladosporium-TLC bioassay to detectof phytoalexins. Using this relatively sensitivewere able to detect the accumulation of a low molthat met the criteria of a phytoalexin as define(14). This phytoalexin accumulated rapidly in I

12 h postinoculation with P. s. syringae and w

with the cell collapse that occurred during the ethe HR. The Arabidopsis phytoalexin was fouithe in vitro growth of P. s. syringae, and the acci

the Arabidopsis phytoalexin was negatively co]the in vivo growth of P. s. syringae. Although fuare needed, these initial observations suggest th;alexin plays a role in the general defense mArabidopsis against pathogens.The Arabidopsis phytoalexin was found to acc

maximum level of about 8 ltg/g fresh weight baccumulation was greater than the maximum 1lmulation of brassilexin (240 ng/g fresh weight)Brassica juncea 6 d after inoculation with Lmaculans (17), but less than the maximum levelation of cyclobrassinin and methoxybrassinin (-

_ 100 ,ug/g fresh weight, respectively) in leaves of B. napus 10d after inoculation with L. maculans (4). Thus, although thelevels of accumulation of the Arabidopsis phytoalexin werenot comparable to those of the isoflavanoid phytoalexins (9,30), the levels were within the range exhibited by the othercruciferous phytoalexins.While this paper was in preparation, Browne et al. (2)

reported the isolation of two phytoalexins, camalexin andmethoxycamalexin, from leaves of Camelina sativa. Thechemical structure reported for camalexin is identical to thestructure we elucidated for the Arabidopsis phytoalexin. Com-parison of the spectral data of the Arabidopsis phytoalexinwith those reported for camalexin suggests that the two phy-toalexins are the same compound, thus providing independ-ent confirmation for the structure of 3-thiazol-2'-yl-indole. In

40 50D addition, Slusarenko and Mauch-Mani (22) reported the pres-ence of an antibacterial compound in extracts prepared from

ion inoculated Arabidopsis. Further spectral data are required todetermine whether this compound is identical or related to 3-

zol-2'-yl-indole thiazol-2'-yl-indole.ie phytoalexin Eight other phytoalexins have been isolated from five otherand quantified species of the Cruciferae (6, 7, 13, 26, 27). Camalexin is-1. Each point structurally similar to these phytoalexins in that it is a sulfur-error bars are containing indole derivative. The structural similarity be-

tween the cruciferous phytoalexins suggests that they mayshare common biosynthetic intermediates. All of the phyto-alexins are structurally similar to indolyl 3-methyl isothiocy-

wae (data not anate, which is the product of the hydrolysis of glucobrassicinby myrosinase. However, a direct link between the indoleglucosinolates and the cruciferous phytoalexins has not beenestablished. Devys et al. (6) speculated that indole-3-carbox-aldehyde may be a common precursor of the indole phyto-

when inocu- alexins and have recently isolated a significant amount of thisThe bacteria compound from leaves of Brassica oleracea (8). Indole-3-exhibited a carboxaldehyde may also be an intermediate to camalexin

nine whether biosynthesis. The Arabidopsis phytoalexin may be biosynthe-d the expres- sized through the cyclization of indole-3-carboxaldehyde withto the accu- cysteine with the subsequent loss of CO2. We are currentlyibidopsis, we investigating this possible biosynthetic pathway.the presence With the description of an Arabidopsis phytoalexin, thebioassay, we molecular and genetic tools available in Arabidopsis can beI wt molecule employed to determine the relative contribution of this phy-d by Paxton toalexin in host defenses against both bacterial (29) and fungalleaves within pathogens (22). In addition, the collections of auxotrophicas associated mutants of Arabidopsis (12) will aid in the elucidation of theexpression of biosynthetic pathway of 3-thiazol-2'-yl-indole and its meta-nd to inhibit bolic regulation in Arabidopsis.umulation ofrrelated with ACKNOWLEDGMENTSirther studiesat the phyto- The authors thank Dr. Soledad Pedras (Plant Biotechnology Insti-iechanism of tute, National Research Council, Saskatoon, Saskatchewan, Canada)

for the samples of brassinin and 3-cyanoindole, Philip Jensen (Mich-umulate to a igan State University) for the sample of 3-methyl indole, and Dr.)y 36 h. This William Reusch (Michigan State University) for the Fourier trans-yve36 hccu form IR spectra.[evel of accu- APPENDIXin leaves of

,eptosphaeria 3-Thiazol-2'-yl-indole. Isolated as a colorless solid. UV max1 of accumu- (MeOH, log e): 318 nm (4.17), 275 nm (3.92), and 215 nm (4.36);about 50 and Fourier transform IR (CHC13, cm-'): 3692 (med), 3610 (small), 3480

a)

c .m*- a1)

~cI)- 3:

r_

(N (1I L

0N E

L..

I L

0) V.

3F

TSWI ET AL.1 308




(large), 1600 (med), 1585 (med), and 1580 (med); HRMS (EI) m/zmeasured 200.0401; calculated 200.0408 for CI,HgN2S; EIMS m/z(relative intensity): 200 (C1 H8N2S, 100), 142 (CqH6N2, 21), 115(CxH5N, 7), 100 (6), 86 (4), 58 (C2H2S, 17); 'H-NMR (500 MHz,CDCI3): 8.45 (br s, 1H, D20 exchangeable, N-H), 8.25 (m, 1H, H-4),7.87 (d, J = 2.2 Hz, 1H, H-2), 7.82 (d, J = 3.4 Hz, 1H, H-4'), 7.43(m, 1H, H-7), 7.28 (m, 2H, H-5, H-6), 7.23 (d, J = 3.2 Hz, lH, H-5'). I3C-NMR (125 MHz, CDC13): 163.1 (s, C-2'), 142.6 (d, C-4'),136.4 (s, C-7a), 124.7 (d, C-2), 124.4 (s, C-3a), 123.2 (d, C-5),121.5 (d, C-6), 120.7 (d, C-4), 115.9 (d, C-5'), 112.7 (s, C-3), 111.9(d, C-7).

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