Coronatine, a phytotoxin produced by plant pathogenic bacteria

Pergamon Press Ltd .

Toxi.con, Supp1.3, pp .187-190, 1983 . Printed in Great Britain.

CORONATINE, A PHYTOTOBIN PRODUCED BY PLANT PATHOGENIC BACTERIA Akitami Ichihara and Sadao Sakamura Department of Agricultural Chemistry, Hokkaido IIniveraity,

Sapporo 060, Japan

INTRODUCTION The chemical structures of a number of fungal phytotoxins have been determined to date because of widespread availability of modern instruments of analysis . However, structural assignments of bacterial phytotoxins have been done only on several phytopsthogenic bacteria . The classical example of a bacterial toxin is tabtoxin active in the wildfire disease of tobacco. Recently the structure of phaeeolotoxin produced by the bean haloblight bacterium was elucidated (MITCHELL et al ., 1981) . Pseudomonas eyringae pv . atropurpurea causes chocolate spot disease on Italian ryegrase (NISHIYAMA et al ., 1976) . During the study of bacterial physiology, it was found isolates of the bacterium caused hypertrophy on potato tuber tissues . that the virulent The same activity was also found in the filtrates of the culture fluids . The fact suggests that the activity may be one of the essential requirements for the pathogenicity of the bacterium and may thus used as a bioassay in isolation . In 1975 the substance responsible for the activity was isolated and named 'Coronatine' . Coronatine is a vivotoxin, since it was present in the leaves of Italian ryegraes infected by the bacterium. Coronatine is also produced by two other pathovars, glycinea and moraprunorum . ISOLATION AND PHYTOTO%IC EFFECTS OF CORONATINE raoiation Coronatine was isolated from the culture fluid of a virulent iaglates of Ps . 9yringae pv . atropurpurea . This bacterium was incubated at 23 ° C for 3 days in glucose-potassium under aeration . The cultured celle were removed by centrifugation . The nitrate~um supernatant and 2x active carbon-celite (1 : 1 w/w) was mined well and the adsorbent was packed in a column, which was washed with water and eluted with acetone . The slants was concentrated to small volume in vacuo , adjusted to pH 2 .5, and extracted with ethyl ace~ate . The extracts were concentrated, and the concentrate was chromatographed on a column containing eilicic acid and hyflo supercel (10 : 1 w/w) . The column was first eluted with benzene, then stepwise with benzene-ethyl acetate and finally with methanol . Fractions from the column were tested for hypertrophy-inducing activity on potato tuber discs . The fractions showing physiological activity were combined and further chromatographed on silicic acid column which was eluted successively with isopropyl ether-acetic acid (95 : 5 v/v), acetone, and methyl alcohol. Coronafacic acids were obtained from the isopropyl ether-acetic acid slants . The acetone eluates were separated twice on eilicic acid columns using ether and benzene-acetone (1 : 1 v/v) as eluent . Finally active fraction was purified by preparative TLC developing with benzene-acetone (1 : 1 v/v) . Recrystallization from ethyl acetate yielded pure coronatine, as needles, m.p . 151-153°C. phvtotoaic effects of coronatine Coronatine produced lesions on leaves of Italian ryegrass on injection (1 ~) of an aqueous acetone solution at 3.2 to 3 .200 ag/ 1 . The necrosis with chlorotic halo produced by coronatine was similar to the symptom on the leaves inoculated with the pathogenic bacterium. In addition, a substance isolated from infected plants had some properties identical with those of coronatine, this substance was isolated neither from healthy plants nor from the bacterial cells (NISHIYAMA et al ., 1977) . From the results of the phytotoaicity tests, coronatiae was suggested to be achloroeis-inducing toxin in the pathogenesis . In order to determine host-specificity, tests were carried out on many plant species, and coron~atine was tozi.c to 21 species of plant in 17 genera in 4 families out of 25 species in 20 genera in 6 families tested . 8vidently, coronatine ie not a host-specific toxin . STRUCTURE AND SYNTH);SIS OF CORONATINE PLe structure of coronatine Coronatine ~ needles, mp 151-153°C, was+formulated as C HO,N by elemental analysis and high resolution mass spectrometry (m/z M 319 .1753, calcd, 319.1731) (ICHIHARA at al ., 187

A . ICHIHARA and S . SARAMURA

18 8

1977a) . The W~H E IR and NMR spectra of coronatine ~dicate the presence of following 208 moiety, UV)~x , (t 8378) (O=C~=C-) IR~ 1740 (five membered ring C=0), 1620 e 0 .94 (6H, t, J=7Hz, CH,CH, a 2), (C=C), 3270, 1645, 1525 cm ' (-CONH-), Nl~t~~ce 3 .15 (1H, br .q -CHCO-), 6 .50 (1H, s, =CH) . The high resolution mesa spectrum of ,lr indicates that coronatine ie bonded to each other by an amide linkage . Other ion peaks below m/z 191 are quite similar to those of coronafacic acids 2a and ~, which were isolated directly from the culture broth . In fact, hydrolysis of coronatine gave an acid, whose Rf value on TLC is identical with that of 2,~, and a-amino acid which is later identified as coronamic acid (4~a) (FIG . 1) .

,N Cv

0-

3

1 N

H COX

FIG . 1

?,

~ ~

X = OH

X = OCH3 X = C1

STRUCTURE OF CORONATINE AND ITS DERIVATIVES

Treatment of coronatine with acetic anhydride-pyridine afforded anhydrocoronatine 3~ m/z 301 (M'~), whose IR spectrum exhibited characteristic peaks at 1800, 1605 cm' aesignable to an azlactone moiety . Since the NMR spectrum of ,~ (and N also) shows the presence of two ethyl groups, one of which is known to be from coronafacic acid, the amino acid must be depicted as 4, considering the degree of unsaturation . The structure including relative configuration ~NHs/CHzCH, try) was also confirmed by the synthesis of (_+)-coronamic acid (ICHIHARA et al ., 1977a , ICHIHARA et al ., 1977b) . Coronafacic acids exist as two stereoieomers, 2~ and ~ depending on the conditions of recrystallization . The isomer ~ is easily convertible through enolisation to ~ . Esterification of e~hO gf ~ and ~ by methanolic HC1 afforded the same methyl ester 6f The UV spectrum Qmaâ 21 nm, ~ 558) of ~ ie compatible with a,ß-unsaturated acid, whose double bond is located in a six-membered ring . The IR spectra (1740 cm 1 in CHC1,, five2a and 2b, D,0 treatment o£ 2b, (~/z l~f~ 211), the NMR spectrum of membered ring ketone) of r (315, quintet, J=lOHz, 7Hz,6Hz, CO-C_H-(CH)-CH,), and the mass spectrum of 2+a (m/z 55, aCeO ) suggest a 1-hydrindaaone structure (~) for coronafacic acid . Dlcisive structure of coronafacic acid has been established 2a and 2}3 by x-ray analysis, and the plane structure of coronatine was confirmed . Stereochemietry of coronatine The relative configurations of coronafacic acid (2a) and coronamic acid (~ were already confirmed by x-ray analysis and synthesis respectively . The absolute configuration of coronatine (~ has been established by partial synthesis of coronatine (~ from natural coronafacic aci (2a) and optically active, synthetic coronamic acid (~ whose absolute configurations weré determined separately (ICHIHARA et al ., 1977b, ICHIHARA et al ., 1979) . The absolute configuration of Za1 has been confirmed by ORD and CD measurements, which exhibited positive Cotton effect, ((a)  o + 2912°, ~a)a  + 2641) and ((9)s,s + 333,

Coronatine, a Bacterial Phytotoxin

189

, + 1333) . Since the octant projection of ~a indicates that all the carbon atome, i~ -5, C-6, C-7, C-9 and C-10 lie in positive octant in the moat plausible conformation, the absolute configuration must be as depicted in ~. Possible four atereoisomers(~, ~, ,~, ,~) of coronamic acid were synthesized (FIG . 2) . Synthetic (_+)-coronamic acid (~ was identical with the natural sample in the behavior on paper chromatography and retention time on the amino acid analyzer . Optical resolution of ~)-coronamic acid (4) was carried out using the quinine salts of N-formylcoronamic acid dained and after several fractional recryatallizations, the two c ~talline materials o~t were separately hydrolysed to yield optically active 4a,~a]~ + 14 .7 and xb, [a] D - 14 .2 respectively . Enzymatic resolution of (_+)-N-acetylcoronamic acid using L-acylase also gave directly ,~, and the recovered ~ acetate was hydrolysed to ~. Ia the same way, (+)allocoronamic acid was etereoselectively ynthesized and resolved through the quinin salt to give (+)-allocoronamic acid (~), ~a~ ~~ + 65 .0 and (-)-allocoronamic acid(5b~ (aj~ l 68 .4 (SHIRAISHI et al ., 1977) . It was expected that if (+)-coronamic acid has the stereochemistry ~a and ie cleaved at _a by hydrogenolysis, D-ieoleucine and L-alloisoleucine would be produced (ICHIHABA et _al ., 1979) . In fact, hydrogenolysis of ~a over lOb Pd-C uader pressure of 14 Rg/cm at 80°C occurred at all three bonds (a, _b and _c) of the cyclopropane ring and afforded four amino acids, ieoleucine, alloisoleucine, norleucine, and 2-amino-2methyl valeric acid, whose structures were identified by the N[~fft spectra and amino acid analyzer . When the reaction products were treated with L-amino acid oxidase, only the peak ascribed to alloisoleucine was decreased in the amino acid analyzer . On the other hand, treatment of the reaction products with D-amino acid oxidase resulted in decreased ieoleucine. These observations indicate that the alloisoleucine and ieoleucine have L- and Dconfiguration respectively, and the stereochemietry of their precursor, (+)-coronamic acid, should be represented as (+)-(1S,2S)-1-amino-2-ethylcyclopropane-l-carboxylic acid (4~) . The same treatment of (-)-coronamic acid (4b) and (+)-allocoronamic acid (5,~a,) confirmed that these have (1R, 2R) and (lR, 2S) configuration respectively . The absolute configuration of 4~1. was further confirmed by %-ray analysis of-ita N-acetate -Condensation of the acid chloride ,Z, with ~a afforded synthetic coronatine (~ which is identical with a natural sample in spectral data and biological activity . From these results, absolute configuration of coronatine is as depicted in 1.

4a

4b

FIG. 2

FOIIR STEREOISOMLRS OF CORONAMIC ACID

,cY Vnthesi9 ôf coronatine : synthesis oL (~-corbudfAcic acid (~) Since partial synthesis of coronatine (~ from natural coronafacic acid and optically active coronamic acid has been completed, the synthesis of (+)-coronafacic acid means the total synthesis of 1~ in a formal sense . Synthesis of ~y has been carried out by two routes ICHIHARA et a1 .,1977c) . Sere only the former route ie described {ICHIHARA et al ., 1980, (FIG . 3) . Condensation of the enamine from butanal and dimethylamine with diethyl maleate, quaternization with p-TsOMe, and subsequent elimination yielded the known diaster,~ The atereochemistry of the diester 8 was confirmed to be traps which is a necessary requirement ¬or the formation of E,E-thane through conrotatory ring opening at a later stage . Reduction of 8~ oxidation and ketalization afforded an acetal, which was further oxidized to an ,~ A methyl ketone ~( was prepared from dimethylfulvene and methyl vinyl ketone . a],dehyde .9 Aldol condensation of the methyl ketone ~ with the aldehyde yielded a ketol 11, which was treated with mesyl chloride and then diazabicycloundecene to give an a,ß-unsaturated ketone . Selective reduction of the conjugated double bond was accomplished using sodium

r

A . ICHIRARA and S . SARAMURA

19 0

dihydrobis(2-methoxyethoxy)aluminate to give a saturated ketone ~. Thermal reaction of ß7y involved three successive reactions, 1) conrotatory opening of t e cyclobutane ring, 2) retro-Diels-Alder reaction, and 3) intramolecular Diels-Alder reaction, affording 92% yield of a single products ~ . Jones oxidation of ~3~, accompanied by deacetalization, ieomerization, and oxidation, produced (+)-coronafacic acid (~a), whose spectral data are identical with those of a natural sample (ICHIHARA et al ., 1980) .

COyCyHS

1)Lj~4

2)Mn02

CH20H ~,~~

~,,

Cr03'PY

ÇHO

C O2C2 H5 3)HOCH2CH20H 1)MsCI'Py

LDA 11 N

0

Cr03 acetone 13 N

FIG . 3

3)SMEAH

0 12 ..r

H}-~ H COZH 2a N SYNTHESIS OF CORONAFACIC ACID

Structure-activity relationships The etereoisomers and analogs of coronatine were sgnthesized by replacing the coronamic acid moiety with other amino compounds . The hypertrophic response of potato tubers was used for the bioassay of the analogs (SHIRAISHI et al ., 1979) . A carboxyl group in coronamic acid was essential for induction of the activity . Moreover, the configuration at the acarbon atom in the amino acid was closely related to increase in activity . Bulkiness of the alkyl groups in the amino acid moiety was associated with the activity . AF~~CES ICHIHARA,A ., SHIRAISHI,R ., SATO,H ., SARAMQRA,S ., NISHIYAMA,K ., SARAI,R ., FIIRIISARI,A . and MATSIIMDTO,T . (1977x) . J . Am . Chew . Soc . 99, 636 . ICHIHARA,A ., SHIRAISHI,R ., SARAMDRA,S . NISHIYAMA,R . and SARAI,R . (1977b) . Tetrahedron Lett . 269--272 . ICHIHA&A,A ., RIMURA,R ., I~RIYASD,R . and SARAMURA,S . (1977c) . Tetrahedron Lett . , 4331-4334 . ICHIHARA,A ., SHIRAISHI,R ., SARAMITßA,S ., FIIHUSARI,A ., HASHIBA,N, and MATSIIMOTO,T . (1979) . Tetrahedron Lett . , 365-368 . ICHIHARA,A ., RIMURA,R ., YAMADA,S . and SARAMURA,S . (1980) . J . Am . Chem . Soc . , 102, 63536355 . MITCHELL,R .E . (1981), Tozins in Plant Disease, p .259-293 (DIIRBIN,R .D ., Eda .) . New York : Academic Press . NISHIYAMA,R ., SARAI,R ., EZIIRA,A ., ICHIHAEA,A ., SHIRAISHI,R ., OGASAFiARA,M ., SATO,H . and SARAMQRA,S . (1976) . Ann . Phytopathol . Soc . Japan , 42, 61314 . NISHIYAMA,R ., SARAI,R ., EZURA,A ., ICHIHARA,A ., SHIRAISHI,R . and SARAMURA,S . (1977) . Ann . Phytopathol . Soc . Japan , _43, 219220 . SHIRAISHI,R ., ICHIHARA,A . and SARAMDRA,S . (1977) . Agric . Biol . Chem . 41, 24972498 . SHIEAISHI,R ., RONOMA,R ., SATO,H ., ICHIHARA,A . and SARAMDßA,S . (1979) .Ägric . Biol . Chin . 1753-1755 .