Planta (BEE.) 102, 85-90 (1972) 9 by Springer-Verlag 1972

Short Communication

Monomethyl-4-Chloroindolyl-3-Acetyl-L-Aspartate and Absence of Indolyl-3-Acetic Acid in Immature Seeds of Pisum sativum HIROYUKI ~tATTO~I and SH~NOO MARUMO Department of Agricultural Chemistry, Nagoya University, Nagoya, Japan Received August 9, 1971 Summary. A new auxin-related metabolite has been isolated from immature seeds of Pisum sativum and its structure has been determined as monomethyl4-ehloroindolyl-3-acetyl-L-aspartate. Neither indolyl-3-acetic acid nor its methyl ester have been detected in immature seeds by gas-liquid chromatographic analysis. We have previously shown t h a t the main auxin in i m m a t u r e seeds of P i s u m sativum is methyl-4-chloroindolyl-3-aeetate (Me-4-Cl-IA) and the minor one is its free acid (4-CI-IAA) (Marumo et al., 1968a, b). T h e y are chlorinated auxins found in nature for the first time since the discovery of indolyl-3-aeetie acid (IAA) as " h e t e r o a u x i n " (KSgl et al., 1934). W h e n assayed b y the hypoeotyl swelling test with Phaseolus mungo methanol extracts of i m m a t u r e pea seeds showed an auxinlike activity other t h a n those of the above compounds. The activity was detected on paper ehromatograms at the Rf region between 0.1 and 0.3 (solvent, isopropanol: a m m o n i a :water, 8 : 1 : 1, v/v). We have isolated three active compounds from this Rf zone, and two of t h e m have been identified b y synthesis as ~-N-carbomethoxyaeetyl- and ~-N-carboethoxyacetyl-D-4-chlorotryptophane (Marumo and H a t t o r i , 1970). This is the first evidence t h a t l)-4-chlorotryptophane occurs in nature. I n this communication, the identification of the third compound as monomethyl-4-chloroindolyl-3-aeetyl-L-aspartate [1 ] is reported. I n addition, we show t h a t neither I A A nor methylindolyl-3-acetate (Me IA) is present in i m m a t u r e seeds of P i s u m sativum. CH2CONHCHCOOR CH2COOR' H R,R'= H and CH3 or CH3 and H

(I]

86

H. Hattori and S. ~arumo:

9

It

,.-,',,,.,./""

,

V I

I

3500 3000

,

I

2000

,

.

1700

,

I

1500

,

,

,

,

I

,

,

1000

Fig. 1. Infrared spectra (CHC13). Solid line, methyl ester of the natural compound isolated from immature pea seeds; broken line, synthetic dimethyl-4-chloroindolyl3-acetyl-L-aspartate

The isolation procedure of the active compound was described in detail in Marumo and Hattori (1970). Immature pea seeds (Pisum 8ativum L. cv. Usui), 241 kg, were purchased from a vegetable market and extracted with ethanol. The extract was processed to yield an acidic fraction which was soluble in acetonitrile and insoluble in hexane. The acidic substance was purified by (1) 11 transfers of counter-current-distribution (solvent, ethyl acetate and tartrate buffer, pH 4.1), (2) passing an ethanolic solution of the active substances through Sephadex LH-20, and (3) partition chromatography on silicie acid (stationary phase, 1VI/15tartarate buffer, pH 4.0; mobile phase, ethyl acetat~-hexane). The active compound was eluted from the column with 70 % ethyl acetate in hexane. Final purification was achieved by preparative thin-layer chromatography (TLC) (Kiesel-gel I-I) after converting the active compound into the methyl ester with ethereal diazomethane. In this manner, 2.2 mg of the active compound was obtained as methyl ester. The s t r u c t u r e of the active c o m p o u n d was deduced as ~ollows. The ultraviolet s p e c t r u m showed absorptions at ~ t o H 223, 2 7 7 , 2 8 2 and ~m0,x 291 mff (log s 4.66, 3.82, 3.85 a n d 3.78), indicating the presence of an indole nucleus. The infrared s p e c t r u m (Fig. 1) showed b a n d s of N t I (indole) (vc~cl. em -1 3490), secondary amide (3370, 1685 a n d 1530) a n d ester (1735)i I n the mass spectrum, the molecular ion peak was observed at m/e 352 along with 30 % i n t e n s i t y of p-[-2, clearly indicating t h a t t h e c o m p o u n d contains one chlorine a t o m a n d should h a v e at least two (or an even n u m b e r of) nitrogens in the molecule, according to the " n i t r o g e n rule" (Sflverstein a n d Bassler, 1963). The

Monomethyl-4-Chloroindolyl-3-Aeetyl-s-Aspar~atein Piaum

87

base peak, m/e 164 (100 %) and its fragmented peaks at m/e 129 (15%), 128 (i8%), and 101 (10%) found also in the spectrum of 2Vie-4-CI-IA, were assigned to the chloroquinolium ion and a series of its decomposed ions produced by successive elimination of C1. -~ tIC1, C1. ~-HCN and HC1 -~ HCN. This indicates that the active compound contains a chloroindolyl-3-methylene moiety which cleaves predominantly at the/3-linkage of the side chain, producing the chloroquinolium ion. The position of chlorine on the indole nucleus was not determined from the mass spectrum. A peak at m/e 191 (35%) should be assigned to the chloroindolyl-3-ketene ion (III) which was produced through four-centered transition (Budzikiewicz et al., 1964, p. 67) from the amide (II) as shown in the scheme. Thus, the methyl ester of Cl

"~-I-

CI

~~H (1I) role352

CH:O:OI+

CIII) role t9'i

the active compound was shown to consist of two components connected by an amide bond. One is the chloroindolyl-3-acetyl group and the other the amino-acid moiety; the mass number of the latter should be 161 by calculation. This amino acid was most probably aspartie acid. To confirm this, the active compound (70/,g) was hydrolyzed with 1 N HC1 at 120 ~ C for 3 h and the hydrolyzate separated into the ethyl-acetate- and the water-soluble fractions. The former fraction gave with the Ehrlich reagent only one spot on a thin-layer ehromatogr~m (Kiesel-gel H; isopropyl ether: acetic acid, 95:5, v/v); its Rf value coincided with that of authentic 4-Cl-IAA and differed from those of the 5-, 6- and 7-CI-IAAs according to the method previously reported (Marumo et al., 1971). When the water-soluble fraction was applied to TLC on cellulose powder (Aviccl SF; solvent, butanol:acetic acid: water, 4:1:2, v/v), a ninhydrin-positivc substance appeared at an Rf value corresponding to that of aspartic acid. The optical configuration was determined by optical rotatory dispersion ; the amino acid (30 fig) isolated from the hydrolyzate showed a positive plain curve in 1 N HC1, indicating that it is the L-form. On the basis of these experiments, the structure, dimethyl-4-chloroindolyl-3-acetyl-L-aspartate was assigned to the active compound isolated as the methyl ester. Final structural confirmation was done by

88

H. Hattori and S. Marumo:

synthesis. 4-Chloroindolyl-3-acetie acid was converted into a carboxylie carbonic a n h y d r i d e with e t h y l chlorocarbonate (Greenstein and Wintz, 1961); this was t h e n condensed with dimethyl-L-aspartate in tetrah y d r o f u r a n . The desired p r o d u c t was o b t a i n e d as an oil, yield 22 %. The n a t u r a l c o m p o u n d isolated f r o m i m m a t u r e pea seeds was completely identical with the synthetic c o m p o u n d with respect to infrared spectra (Fig. 1), mass spectra a n d R f value (0.44) on TLC (Kiesel-gel H ; solvent, ethyl acetate). As the isolation was carried out after converting the active comp o u n d into t h e methylester, it h a d to be clarified whether t w o e a r b o x y l groups of the aspartic-acid m o i e t y of the original c o m p o u n d in the seeds were b o t h free or w h e t h e r either one was m e t h y l a t e d . W e conclude t h a t the original f o r m is a m o n o m e t h y l ester, based on the following observation. On c o u n t e r - c u r r e n t - d i s t r i b u t i o n the comp o u n d was transferred to the same t u b e n u m b e r s as ~:N-earbomethoxyaeetyl- and e-N-carboethoxyacetyl-D-4-chlorotryptophane. This suggests t h a t while being p a r t i t i o n e d between e t h y l - a c e t a t e a n d t a r t r a t e buffer, p H 4.1, the c o m p o u n d behaves as a substance carrying a m o n o c a r b o x y l i c acid like the two c o m p o u n d s just mentioned. W h e n i m m a t u r e pea seeds were e x t r a c t e d with acetone instead of m e t h a n o l a n d t h e acidic f r a c t i o n was subjected to c o u n t e r - c u r r e n t - d i s t r i b u t i o n (pH 4.1 ; 7 transfers) in parallel with t h a t f r o m the m e t h a n o l extract, t h e auxin activities of b o t h fractions a p p e a r e d in the same tubes (No. 3-5). These facts show t h a t the m o n o m e t h y l ester is n o t a n artifact derived f r o m a reaction between a carboxylie acid and m e t h a n o l during isolation, b u t is a genuine constituent of the seeds. However, it is a t present n o t clear which c a r b o x y l group, ~ or fl, of the L-aspartie acid is m e t h y l a t e d . In this and previous work we have thus shown the presence in immature seeds of Pisum sativum of five new auxins and related metabolites, all of which are derivatives of either 4-C1-L4A or 4-Cl-tryptophane. An important problem to be clarified was whether the immature pea seeds contain only 4-CI-IAA and its derivatives as native auxins. We have examined the possible presence of IAA and MeIA by means of gas-liquid chromatography (GLC). The acidic substances obtained from the methanol extract of 4 kg immature pea seeds were subjected to 11 transfers of counter-current-distribution (solvent, ethyl acetate and phosphate buffer, pI-I 6.50). The pH of the buffer was carefully adjusted so that IAA came to the middle tubes of the whole transfer distribution. In this ease, theoretically 25 % of the total amount of IAA applied is transferred to tube No. 6. This fraction was concentrated to dryness and the residue was iraetionated by preparative TLC (Kiesel-gel I-I; isopropyl ether:acetic acid, 95:5, v/v). Substances in the same Rf zone as IAA were extracted and methylated with etherial diazomethane. The sample thus prepared was analyzed by GLC at the three different conditions: a) 3 % SE-30, 210 ~ C, retention time (r.t.) of Me IA, 9.Stain; b) 1.5% OV-17, 180~ r.t. 7.5min; c) 1.5% PEG-20M, 150-200~

Monomethyl-4-Chloroindolyl-3-Acetyl-L.Aspartate in Pisum

89

(3 ~ C/rain), r.t. 23.5 min. The neutral substance was purified by alumina (Merck, activation grade I I - I I I ) column chromatography. The column was eluted by a stepwise increase of ethyl acetate in hexane, 5, 8, 10 and 15 %. Me-IA was found to come from the column with 8 % ethyl acetate in hexane, and ~e-4-Cl-IA with 15 %. Both eluates were evaporated and the resultant residues analyzed by GLC under the three different conditions described above. I n every case, neither the neutral nor the methylated acidic fractions did show any appreciable peak at the same retention time as Me-IA, but both fractions exhibited a strong peak corresponding to Me-4-CI-IA. In those chromatographic conditions, 0.02 ~g of authentic Ne-IA can be detected. From these result, it was calculated that 4 kg of pea seeds do not contain even as little as 4 ~zg of ~e-IA in both the neutral and the methylated acidic fractions. On the other hand, the immature seeds contain about 400 ~g of Me-4-CI-IA in the neutral and 60 ~g of 4-CI-IAA in the acidic fractions; these amounts are deduced by calculation from the actual amounts obtained as crystals in our previous work. We thus conclude that immature pea seeds contain only 4-CI-IAA and its derivatives, and neither IAA nor Me-IA, or at least that they contain neither IAA nor its methyl ester above the minimum analytical limit of GLC (4 lzg per kg). I n our screening work to find n e w a u x i n s or a u x i n - r e l a t e d comp o u n d s i n p l a n t s , we o b s e r v e d t h a t p l a n t e x t r a c t s often e x h i b i t e d a u x i n a c t i v i t y a t a low R f zone (0-0.3) i n p a p e r c h r o m a t o g r a p h y (solvent, i s o p r o p y l alcohol: a m m o n i a : w a t e r , 8 : 1 : 1 , v/v). I n 1953, B e n n e t - C l a r k a n d K e f f o r d (1953) d e s i g n a t e d this a c t i v i t y as acceler a t o r ~. T h e m o n o m e t h y l - 4 - c h l o r o i n d o l y l - 3 - a c e t y l - L - a s p a r t a t ewe h a v e i d e n t i f i e d i n this r e p o r t has been isolated from this low l~f zone; hence, it m a y be considered as one of accelerators ~ i n i m m a t u r e pea seeds, i n a d d i t i o n to t h e a l r e a d y i d e n t i f i e d two 4 - c h l o r o t r y p t o p h a n e derivatives which also a p p e a r e d i n this Rf zone. T h e two t y p e s of m e t a bolites, i.e., t h e L - a s p a r t a t e of 4-CI-IAA a n d t h e 4 - c h l o r o t r y p t o p h a n e d e r i v a t i v e s , differ i n t h e i r biological a c t i v i t y i n a s f a r as t h e f o r m e r i n d u c e s h y p o c o t y l swelling i n Phaseolus mungo a b o u t I d a y a f t e r applic a t i o n ; whereas t h e l a t t e r n e e d a n o t h e r 1.5 d a y s for i n d u c i n g a s i m i l a r response.

References BenneVClark, T. A., Kefford, N. P. : Chromatography of the growth substances in plant extracts. Nature (Lond.) 171, 645-647 (1953). Budzikiewiez, H., Djerassi, C., Williams, D. 1~I.: Luterpretation of mass spectra of organic compounds. San Francisco: Holden-Day 1964. Greestein, J.P., Winitz, M.: Chemistry of the amino acids, vol. 2. NewYork: Wiley & Sons 1961. KSgl, F., tIaagen- Stair, A. J., Erxleben, H. : t3ber ein neues Auxin (" Heteroauxin") aus ttarn. XII. Hoppe-Seylers Z. physiol. Chem. 228, 90-103 (1934). Marumo, S., Abe, H., Hattori, H., Munakata, K. : Isolation of a novel auxin, methyl 4-chloroindolacetate from immature seeds of Pisum sativum. Agric. Biol. Chem. (Japan) 32, 117-118 (1968a).

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H . H a t t o r i and S.Marumo: lVlonomethyl-4-Chloroindolyl-3-Acetyl-L-Aspartat~

Marumo, S., ttattori, It.: Isolation of D-4-chlorotryptophane derivatives as auxin-related metabolites from immature seeds of P i s u m sativum. Planta (BEE.) 99, 208-211 (1970). - - - - Abe, H. : Chromatography of a new natural auxin, 4-chloroindolyl-3-aeetie acid, and related chloroderivatives. Anal. Biochem. 49, 488-490 (1971). - - - - - - Munakata, K. : Isolation of 4-chloroindolyl-3-acetic acid from immature seeds of P i s u m sativum. Nature (Load.) 219, 959-960 (1968b). Silverstein, R. M., Bassler, G. C. : Spectrometric identification of organic compounds. New York-London: Wiley & Sons 1963. Dr. Hiroyuki Hattori Department of Agricultural Chemistry Nagoya University Furo-cho, Chikusa-ku, Nagoya, Japan