Planta (Berl.) 90, 208--211 (1970)
Short Communication
Isolation of D-4-Chlorotryptophane Derivatives as Auxin-Related Metabolites from Immature Seeds of Pisum sativum SHINGO MAttUMO a n d 1-IIRoYUKI tIATTORI Department of Agricultural Chemistry, Nagoya University,
Nagoya, Japan Received July 31/September 29, 1969 Summary. Three active compounds which induce hypocotyl swelling in Phaseolus mungo were isolated from immature seeds of Pisum sativum. For purification of the compounds, our three-step procedure (counter-current distribution, Sephadex LIt 20, and partition chromatography on silicic acid) were used. The chemical structures of two of the compounds were determined by synthesis to be ~-N-carboethoxyacetyl- and e-N-earbomethoxyacetyl-D-4-chlorotryptophane. D-4-Chlorotryptophane is the first naturally occurring chlorinated amino acid found.
W e h a v e p r e v i o u s l y shown (Marumo, A b e et al., 1968; Marumo, t i a t t o r i et aI., 1968) t h a t auxins in i m m a t u r e seeds of P i s u m sativum are 4-ehloroindolyl-3-acetic acid (4-CI-IAA) a n d its m e t h y l ester. A n auxin-like a c t i v i t y was also detected, b y means of t h e h y p o c o t y l swelling t e s t with Phaseolu8 mungo, in a zone between Rf 0.1 a n d 0.3 on p a p e r e h r o m a t o g r a m s (solvent, i s o p r o p a n o l : a m m o n i a : water, 8 : 1 : 1). A u x i n a c t i v i t y in this a r e a is f o u n d in m a n y plants, a n d B e n n e t Clark a n d K e f f o r d (1953) d e s i g n a t e d it as accelerator c~. However, t h e active principle has n o t been chemically identified. W e now r e p o r t on the isolation of three active principles which a p p e a r in this Rf zone a n d which induce h y p o c o t y l swelling in Ph. mungo, a n d describe briefly the identification of two of t h e m as ~ - N - c a r b o e t h o x y a e e t y l - a n d ~ - N - e a r b o m e t h o x y a e e t y l - D - 4 - e h l o r o t r y p t o p h a n e , respectively. This is first t i m e t h a t D - 4 - e h l o r o t r y p t o p h a n e has been f o u n d to occur n a t u r a l l y . For isolation of the compounds, methanol extracts of immature pea seeds (Pisum sativum L. cv. Usui) (241 kg) were subjected to preliminary purification as follows. (1) Methanol extracts were dissolved in acetonitrile. (2) This solution was washed with hexane, then evaporated to dryness. (3) The residue was dissolved in ethyl acetate. (4) The soluble material, after evaporation of the solvent, was dissolved in water by adjusting the pit to 10 with 1 N Na0I-I. (5) The aqueous solution was shaken with ethyl acetate to remove basic and neutral substances, acidified to pit 3 with 1 N It~SO4, and the acidic substances extracted from it with ethyl acetate.
D-4-Chlorotryptophane Derivatives in Pisum Seeds
209
The acidic substances (14.8 g) thus obtained were subjected to 11 transfers of counter-current distribution (solvent, ethyl acetate and phosphate buffer, pH 4.1). The biological activity was located in tubes No. 5--8. The contents of these tubes were combined and evaporated to dryness. The material was further purified by passing an ethanol solution of the active substances through a column of Sephadex LIt 20, and by subsequent partition chromatography on silicie acid (stationary phase, M/15 phosphate buffer, pH 4.0; mobile phase, ethyl acetate-hexane). The substances containing the activity were eluted from the column with 60% ethyl acetate in hexane. The eluate, after evaporation and recrystallization from ethyl acetate and hexane, gave a crystalline mixture of two active compounds (I and II, 12 rag). Further elution of the column with 70% ok the same solvent mixture yielded the third active compound (III). Final purification was achieved by preparative thin-layer chromatography (Kieselgel H; solvent, ethyl acetate) after converting the compounds into the methyl ester with ethereal diazomethane. The three active compounds thus obtained in pure form as the methyl esters were n~med, in order of decreasing Rf values, I-Me (yield, 3.9 rag), II-1Ke (1.5 rag) und III-Me (2.2 rag), respectively. We should emphasize that our three-step isolation procedure, n~mely, (1) countercurrent distribution, (2) Sephadex LH 20, ~nd (3) partition chromatography on silieic acid, is quite effective for erystMlization of n~tural auxins and related metabolites. Especially the use of the proper pH of the buffer makes selective isolation of different auxins possible (e.g., indolyl-3-aeetic acid, pK 6.3; 4-CI-IAA, pit 7.1). I-Me is a colourless off having the molecular formula, C~vH19N205CI (measured m/e 366.096 and calculated m/e 366.098; the P~-2 has 30% intensity of the molecular peak). The ultraviolet spectrum, ~Emt~O~m~z (s) 226 (39700), 276sh (6190), 282 (6750) ~nd292 (5730), shows the presence of an indole nucleus. The infrared spectrum, as shown in Fig. 1, indicates bands of N H (indo]e) (rcnc' 3 cm -1, 3500), secondary amide (3375, 1675 and 1530) and ester (1735). W h e n I-Me was hydrolyzed with 1 N tIC1, 4-chlorotryptophane was liberated. I-Me was thus established to be the m e t h y l ester of 4-chlorotryptophane having the remaining portion of the molecule, CsHvQ. This was assumed to be the ~-Ncarboethoxyaeetyl group, on the basis of the nuclear-magnetic-resonance spectrum and the fragmentation p a t t e r n of the mass spectrum. The structure was finally confirmed b y synthesis. 4-Chlorotryptophane was synthesized from 4-chloroindole (Uhle, 1949) and ethyl acetoamidomalohate (Snyder et al., 1944) t h r o u g h a four-step procedures, l~eaction of the m e t h y l ester of 4-chlorotryptoph~ne with malonylchloride in ethanol and triethylamine yielded the desired product; the latter was completely identical to I-Me with respect to infrared (Fig. 1) and ultraviolet spectra and the Rf value on thin-layer c h r o m a t o g r a p h y (Kieselgel I-I; solvent, ethyl acetate). II-Me has quite similar spectroscopic properties as those of I-Me, except t h a t the molecular peak in the mass spectrum is 14 mass units less t h a n t h a t of I-Me, showing t h a t II-Me might have a m e t h o x y l group instead of ethoxy]. Structural identification was completed b y 14
P l a n t ~ (Berl.), :Bd. 90
210
S. Marumo
and H. Ilattori:
~9
"~? ~Z
*c
dT~
~
9
N
,
m
N
showing full coincidence of the infrared spectra (Fig. 2) of the natural and the synthetic compound; the latter was prepared by an analogous method as I-Me. D-Configuration of the amino acid was indicated by the optical rotatory dispersion of I-Me, [~]400 m z : --90~ Details on the chemistry of the compounds are to be published in a separate paper. Biological activity in the Ph. mungo test was measured by using the crystalline mixture of I and I I which had been obtained in the course of isolation. The mixture induced hypocotyl swelling and root shortening (66% inhibition) at 1.0 rag/1. This activity is slightly less than the
D-4-Chlorotryptoph~ne Derivatives in Pisum Seeds
211
74% inhibition b y (~=)4-chlorotryptophane (1 rag/l), and comparable to the 70% inhibition caused b y 0.05 rag/1 of 4-CI-IAA. I t is interesting t h a t the response appears 2.5 days after application, t h a t it about one d a y later t h a n in the ease of 4-CI-IAA and 4-ehlorotryptophane, suggesting t h a t the compounds m u s t be metabolized to 4-CI-IAA through 4-chlorotryptophane before becoming active.
References Bennet-Clark, T.A., Kefford, N.P.: Chromatography of the growth substances in plant extracts. Nature (Lend.) 171, 645--647 (1953). Marumo, S., Abe, H., Hattori, I t , Munak~ta, K.: Isolation of a novel auxin, methyl 4-chloroindoleacetate from immature seeds of Pisum sativum. Agric. Biol. Chem. (J~pan) 82, 117--118 (1968). - - Hattori, H., Abe, It., ~unakata, K. : Isolation of 4-chloroidolyl-3-acetic acid from immature seeds of Pisum sativu~. ;Nature (Lend.) 219, 959--960 (1968). Snyder, It. R., Smith, C. W. : A convenient synthesis of dI-tryptophane. J. Amer. chem. Soc. 66, 350--351 (1944). Uhle, F. C.: The synthesis of 5-keto-l,3,4,5-tetrahydrobenz(cd)indole. A synthesis of 4-substituted indote. J. Amer. chem. Soc. 71, 761--766 (1949). Dr. Shingo Marumo, t-Iiroyuki Hattori Department of Agricultural Chemistry ;Nagoya University Nagoya, Japan
14"
Short Communication
Isolation of D-4-Chlorotryptophane Derivatives as Auxin-Related Metabolites from Immature Seeds of Pisum sativum SHINGO MAttUMO a n d 1-IIRoYUKI tIATTORI Department of Agricultural Chemistry, Nagoya University,
Nagoya, Japan Received July 31/September 29, 1969 Summary. Three active compounds which induce hypocotyl swelling in Phaseolus mungo were isolated from immature seeds of Pisum sativum. For purification of the compounds, our three-step procedure (counter-current distribution, Sephadex LIt 20, and partition chromatography on silicic acid) were used. The chemical structures of two of the compounds were determined by synthesis to be ~-N-carboethoxyacetyl- and e-N-earbomethoxyacetyl-D-4-chlorotryptophane. D-4-Chlorotryptophane is the first naturally occurring chlorinated amino acid found.
W e h a v e p r e v i o u s l y shown (Marumo, A b e et al., 1968; Marumo, t i a t t o r i et aI., 1968) t h a t auxins in i m m a t u r e seeds of P i s u m sativum are 4-ehloroindolyl-3-acetic acid (4-CI-IAA) a n d its m e t h y l ester. A n auxin-like a c t i v i t y was also detected, b y means of t h e h y p o c o t y l swelling t e s t with Phaseolu8 mungo, in a zone between Rf 0.1 a n d 0.3 on p a p e r e h r o m a t o g r a m s (solvent, i s o p r o p a n o l : a m m o n i a : water, 8 : 1 : 1). A u x i n a c t i v i t y in this a r e a is f o u n d in m a n y plants, a n d B e n n e t Clark a n d K e f f o r d (1953) d e s i g n a t e d it as accelerator c~. However, t h e active principle has n o t been chemically identified. W e now r e p o r t on the isolation of three active principles which a p p e a r in this Rf zone a n d which induce h y p o c o t y l swelling in Ph. mungo, a n d describe briefly the identification of two of t h e m as ~ - N - c a r b o e t h o x y a e e t y l - a n d ~ - N - e a r b o m e t h o x y a e e t y l - D - 4 - e h l o r o t r y p t o p h a n e , respectively. This is first t i m e t h a t D - 4 - e h l o r o t r y p t o p h a n e has been f o u n d to occur n a t u r a l l y . For isolation of the compounds, methanol extracts of immature pea seeds (Pisum sativum L. cv. Usui) (241 kg) were subjected to preliminary purification as follows. (1) Methanol extracts were dissolved in acetonitrile. (2) This solution was washed with hexane, then evaporated to dryness. (3) The residue was dissolved in ethyl acetate. (4) The soluble material, after evaporation of the solvent, was dissolved in water by adjusting the pit to 10 with 1 N Na0I-I. (5) The aqueous solution was shaken with ethyl acetate to remove basic and neutral substances, acidified to pit 3 with 1 N It~SO4, and the acidic substances extracted from it with ethyl acetate.
D-4-Chlorotryptophane Derivatives in Pisum Seeds
209
The acidic substances (14.8 g) thus obtained were subjected to 11 transfers of counter-current distribution (solvent, ethyl acetate and phosphate buffer, pH 4.1). The biological activity was located in tubes No. 5--8. The contents of these tubes were combined and evaporated to dryness. The material was further purified by passing an ethanol solution of the active substances through a column of Sephadex LIt 20, and by subsequent partition chromatography on silicie acid (stationary phase, M/15 phosphate buffer, pH 4.0; mobile phase, ethyl acetate-hexane). The substances containing the activity were eluted from the column with 60% ethyl acetate in hexane. The eluate, after evaporation and recrystallization from ethyl acetate and hexane, gave a crystalline mixture of two active compounds (I and II, 12 rag). Further elution of the column with 70% ok the same solvent mixture yielded the third active compound (III). Final purification was achieved by preparative thin-layer chromatography (Kieselgel H; solvent, ethyl acetate) after converting the compounds into the methyl ester with ethereal diazomethane. The three active compounds thus obtained in pure form as the methyl esters were n~med, in order of decreasing Rf values, I-Me (yield, 3.9 rag), II-1Ke (1.5 rag) und III-Me (2.2 rag), respectively. We should emphasize that our three-step isolation procedure, n~mely, (1) countercurrent distribution, (2) Sephadex LH 20, ~nd (3) partition chromatography on silieic acid, is quite effective for erystMlization of n~tural auxins and related metabolites. Especially the use of the proper pH of the buffer makes selective isolation of different auxins possible (e.g., indolyl-3-aeetic acid, pK 6.3; 4-CI-IAA, pit 7.1). I-Me is a colourless off having the molecular formula, C~vH19N205CI (measured m/e 366.096 and calculated m/e 366.098; the P~-2 has 30% intensity of the molecular peak). The ultraviolet spectrum, ~Emt~O~m~z (s) 226 (39700), 276sh (6190), 282 (6750) ~nd292 (5730), shows the presence of an indole nucleus. The infrared spectrum, as shown in Fig. 1, indicates bands of N H (indo]e) (rcnc' 3 cm -1, 3500), secondary amide (3375, 1675 and 1530) and ester (1735). W h e n I-Me was hydrolyzed with 1 N tIC1, 4-chlorotryptophane was liberated. I-Me was thus established to be the m e t h y l ester of 4-chlorotryptophane having the remaining portion of the molecule, CsHvQ. This was assumed to be the ~-Ncarboethoxyaeetyl group, on the basis of the nuclear-magnetic-resonance spectrum and the fragmentation p a t t e r n of the mass spectrum. The structure was finally confirmed b y synthesis. 4-Chlorotryptophane was synthesized from 4-chloroindole (Uhle, 1949) and ethyl acetoamidomalohate (Snyder et al., 1944) t h r o u g h a four-step procedures, l~eaction of the m e t h y l ester of 4-chlorotryptoph~ne with malonylchloride in ethanol and triethylamine yielded the desired product; the latter was completely identical to I-Me with respect to infrared (Fig. 1) and ultraviolet spectra and the Rf value on thin-layer c h r o m a t o g r a p h y (Kieselgel I-I; solvent, ethyl acetate). II-Me has quite similar spectroscopic properties as those of I-Me, except t h a t the molecular peak in the mass spectrum is 14 mass units less t h a n t h a t of I-Me, showing t h a t II-Me might have a m e t h o x y l group instead of ethoxy]. Structural identification was completed b y 14
P l a n t ~ (Berl.), :Bd. 90
210
S. Marumo
and H. Ilattori:
~9
"~? ~Z
*c
dT~
~
9
N
,
m
N
showing full coincidence of the infrared spectra (Fig. 2) of the natural and the synthetic compound; the latter was prepared by an analogous method as I-Me. D-Configuration of the amino acid was indicated by the optical rotatory dispersion of I-Me, [~]400 m z : --90~ Details on the chemistry of the compounds are to be published in a separate paper. Biological activity in the Ph. mungo test was measured by using the crystalline mixture of I and I I which had been obtained in the course of isolation. The mixture induced hypocotyl swelling and root shortening (66% inhibition) at 1.0 rag/1. This activity is slightly less than the
D-4-Chlorotryptoph~ne Derivatives in Pisum Seeds
211
74% inhibition b y (~=)4-chlorotryptophane (1 rag/l), and comparable to the 70% inhibition caused b y 0.05 rag/1 of 4-CI-IAA. I t is interesting t h a t the response appears 2.5 days after application, t h a t it about one d a y later t h a n in the ease of 4-CI-IAA and 4-ehlorotryptophane, suggesting t h a t the compounds m u s t be metabolized to 4-CI-IAA through 4-chlorotryptophane before becoming active.
References Bennet-Clark, T.A., Kefford, N.P.: Chromatography of the growth substances in plant extracts. Nature (Lend.) 171, 645--647 (1953). Marumo, S., Abe, H., Hattori, I t , Munak~ta, K.: Isolation of a novel auxin, methyl 4-chloroindoleacetate from immature seeds of Pisum sativum. Agric. Biol. Chem. (J~pan) 82, 117--118 (1968). - - Hattori, H., Abe, It., ~unakata, K. : Isolation of 4-chloroidolyl-3-acetic acid from immature seeds of Pisum sativu~. ;Nature (Lend.) 219, 959--960 (1968). Snyder, It. R., Smith, C. W. : A convenient synthesis of dI-tryptophane. J. Amer. chem. Soc. 66, 350--351 (1944). Uhle, F. C.: The synthesis of 5-keto-l,3,4,5-tetrahydrobenz(cd)indole. A synthesis of 4-substituted indote. J. Amer. chem. Soc. 71, 761--766 (1949). Dr. Shingo Marumo, t-Iiroyuki Hattori Department of Agricultural Chemistry ;Nagoya University Nagoya, Japan
14"
