Pergamon

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Biochemical Systematics and Ecology, Vol. 23, No. 7/8, pp. 717-721,1995 Copyright © 1995 Bsevier Science Ltd

Printed in Great Britain. All rights reserved 0305-1878/95 $9,50+0,00

Large-scale Production and Chemical Characterization of the Protective Higher Plant Allelochemicals: L-Canavanine and L-Canaline

MICHAEL BASS,* LEVI HARPER,* GERALD A. ROSENTHAL,* SUPINAN NA PHUKETt and PETER A. CROOKSt

*Laboratory of Biochemical Ecology and tCollege of Pharmacy, University of Kentucky, Lexington, KY 40506, U.S.A.

Key Word Index--L-Canavanine; L-canaline; Canavalia ensiformis.

Abstract--Procedures are presented for the large-scale preparation of L-canavanine, and its principal catabolic product L-canaline, compounds of demonstrated importance in biochemical ecology studies, and as higher plant chemotherapeutic agents. Mass spectroscopic and NMR data are provided for these experimentally-valuable natural products.

Introduct ion Canavanine (1), a non-protein amino acid of leguminous plants, is a potent arginine antagonist (Rosenthal, 1982) able to manifest antimetabolic effects in viruses, bacteria, fungi, as well as in lower and higher plants and animals (Rosenthal, 1977a). Canavanine has proven invaluable in understanding the biochemical ecology of toxic secondary metabolites that function as protective allelochemicals in higher plant- insect interactions (Bleiler et al., 1988; Rosenthal, 1983a, 1988). Investigations focusing on the canavanine-sensitive tobacco hornworm, Manduca sexta have provided an insightful and fundamental understanding of how this pernicious non- protein amino acid functions in protecting the plant (Rosenthal, 1991). The biochemical response of insects naturally resistant to canavanine, such the tobacco budworm, Heliothis virescens, and the canavanine-adapted bruchid beetle, Caryedes brasiliensis, provides a meaningful picture of the biochemical basis for the response of insects to this toxic allelochemical (Berge and Rosenthal, 1990, 1991; Rosenthal, 1983b).

Our studies of canavanine's efficacy against a solid rat colon tumor confirmed the marked potential of this higher plant natural product as an anticancer agent (Thomas et al., 1986). Companion toxicological and metabolic investigations in the rat established that L-canaline (2), the product of arginase-mediated (EC 3.5.3.1) hydro- lysis of canavanine, was the quantitatively significant degradation product (Thomas and Rosenthal, 1987). Canaline has also proven a valuable tool for studying the biochemical ecology of higher plant detoxification of xenobiotics (Rosenthal et al., 1989). The need to continue animal studies on the antitumor effects of canavanine and canaline as well as their utility in a host of basic biochemical ecology investiga- tions prompted our development of methods for large-scale production of these non- protein amino acids.

Abbreviations used: TSP, the sodium salt of 3- (trimethylsilyl) propionic acid; TMS, tetramethyl silane; and BSTFA, bis(trimethylsilyl)trifluomacetamide.

(Received 12 April 1995; accepted 6 July 1995)

717

718 M. BASS ETAL.

o o

H O ~ , . v . ~ . . ~ O ~ N ~ N H 2 H O ~ , - ' / " - . . ~ / O - N H 2

[1]

SCHEME 1. STRUCTURES OF CANAVINE (1) and CANALINE (2).

[2]

We report on the isolation of L-canavanine from canavanine-containing seeds and a method for producing large quantities of L-canaline from L-canavanine. Additional chemical characterization of these non-protein amino acids is also presented.

Materials and Methods Jack bean seeds (Canavalia ensiformis), can be obtained from Sigma Chemical Co., St. Louis or purchased in bulk from Mr T. Stewart, Waldron, Arkansas. Dioclea megacarpa seeds were collected in Parque Nacional de Guanacaste Province, Costa Rica. Trisodium pentacyanoammonioferate was obtained from Fisher Scientific Co. and picric acid from Baker Chemical Co.; D20, the sodium salt of 3-(trimethylsilyl)- propionic acid (TSp3), tetramethyl silane (TMS) and bis(trimethylsilyl)trifluoroacetamide (BSTFA) were obtained from the Aldrich Chemical Co.

Melting points (uncorrected) were determined on a Fisher-Johns apparatus. Nuclear magnetic reso- nance spectra were performed on a Varian Associates VXR-300 spectrometer using TMS or the sodium salt of TSP asn an internal standard. Mass spectra were obtained on a Finigan-Mat Incos 50 spectrometer utilizing a 15 m DB5-MS column. The mass range was 40-550 AMU, spectra were recorded at 20 EV, and GC conditions were: a 50°C hold for 1 min followed by a 10°C per min increase to 280°C.

Results and Discussion The procedures presented provide a convenient method for the large scale preparation of the biologically active amino acids L-canavanine and L-canaline from the readily available canavanine-containing seeds of jack bean, Canavalia ensiformis (Leguminosae). Initially, seed canavanine is extracted with aqueous methanolic HCI and the amino acid isolated on Dowex 50 resin. Elution of the resin with aqueous ammonia, followed by concentration in vacuo afforded a crude produce that could be crystallized from ethanol. Following this procedure, 21 g of analytically pure cana- vanine can be prepared from 2 kg of hydrated seed, representing a yield of 75% of the total canavanine content of the seed.

L-Canaline can be prepared from L-canavanine by enzymic cleavage utilizing arginase which can be isolated conveniently from the leaves of C, ensiformis. Canaline generated during this enzymic reaction can be isolated via the stable dipicrate salt and subsequently converted to the free base by treatment with either 2N H2SO 4 followed by Ba(OH)2, or 2N HCI followed by triethylamine. In these procedures, care must be taken to keep the pH of the media between 7.0 and 7.2, since canaline is unstable at higher pHs. The latter procedure gave the best results, affording a yield of 65% L-canaline from L-canaline dipicrate.

L-Canavanine and L-canaline isolated via the above procedures were fully characterized and their structures and purity confirmed by NMR spectroscopy, specific rotation, and elemental analysis. In addition, GLC-MS studies were carried out on both products after conversion to their respective trimethylsilyl derivatives. L- Canavanine afforded two silylation products with rRS of 11.6(minor) and 12.3(major) min, corresponding to the tri- and tetrasilylated derivatives, respectively. Similar deri- vitization of L-canaline also afforded tri- and tetra-silylated products (tR 9.1 and 9.6. respectively). In this case, the tri-silyl derivative was a significant product; however, only weak molecular ions for both of these silylated products were obtained. Attempts were made to utilize the glyoxylate oxime of canaline in GLC-MS studies;

PRODUCTION OF L-CANAVANINE AND L-CANALINE 719

this effort was unsuccessful as the silylated glyoxylate oxime decomposed during analysis.

L-Canavanine production Plant extract. We report on canavanine isolation conducted from the jack bean,

Canavalia ensiformis, as well as the neotropical legume, Dioclea megacarpa; however, its isolation in principle should be achievable from any canavanine-containing seed. The canavanine-containing seeds were allowed to hydrate overnight in slowly flowing tap water prior to removal of the seed coat, which adversely affects the purification process. Hydrated seeds (2.0 kg) were ground mechanically with 6.01 of 60% aqueous methanol containing 60 ml of conc. HCI. This procedure extracts greater than 95% of the seed canavanine. The resulting slurry was expressed through cheesecloth prior to filtering over Whatman 451 paper. After titrating the supernatant solution to pH 3.2-3.3 with concentrated HCI, the turbid solution was clarified by a

final filtration over Whatman 1 paper. Canavanine isolation. The filtrate was stirred mechanically with Dowex-50 (NH;)

(41 of settled resin volume) overnight at 6-8°C. Twenty-five litres of deionized water was used to wash fully the ionic-exchange resin. The resin was stirred mechanically with 11 of cold deionized water at 6-8°C and canavanine elution initiated by the addition of 35 ml of concentrated ammonium hydroxide. After 5 h, the resin was fil- tered in vacuo; filtration was continued until all of the filtrate was collected. The resin bed was washed with 0.51 of deionized water and filtered as above. The washing was repeated thrice and the combined ammoniacal filtrate was stored at 6-8°C. Under these alkaline conditions, canavanine slowly cyclizes irreversibly to deamino- canavanine (Rosenthal, 1972); thus, ammonia was removed as rapidly as possible by rotary evaporation in vacuo.

Canavanine crystallization. The filtrate was concentrated by rotary evaporation in vacuo to about 700 ml and the resulting solution stirred mechanically with 12 g of decolorizing charcoal at least 1 h. After filtering in vacuo, the filtrate was concentrated by rotary evaporation in vacuo to about 30 ml. Residual charcoal and other debris were removed by vacuum filtration employing a Millipore filter (GS membrane, 0.221~m). Further rotary evaporation in vacuo was employed to create a highly viscous solution which was treated with an excess of absolute ethanol chilled to -20°C. The isolated canavanine was dissolved in a minimum of deionized water and recrystallized from the mother liquor with excess absolute ethanol chilled to -20°C. Recrystallization resulted in a 5% product loss.

The above procedures can be completed in 3 days, scaled down without difficulty, and applied to canavanine isolation from other canavanine-containing seeds without modification. A typical yield was 20 or 27 g from Canavalia ensiformis or Dioclea megacarpa, respectively. L-Canavanine free base had a mp of 172 + 0.5°C (dec.); [a] 22 (1.4%, H20 = + 4.9 -t-_ 0.1°, Found: C, 34.12, H, 6.87; N, 31.98~/o; C5H1203N 2 requires C, 34.09; H, 6.87; N, 31.80; 1H-NMR, d 2.20 (2H, m, 3.92 (2H, rn, C.~_2H -CH 2- O-), 3.82 (1H, dd, I~-proton), 3.92 (2H, rn, CH2-3.92 (2H, m, CH2-0) ppm; ' C - ° " NMR, d 32.67 (b-C), 55.85 (~C), 72.30 (g-C), 161.34 (C = N-), 177.75 (COOH) ppm. GLC-MS (BSTFA-pyridine) analysis afforded tri-silylated (minor, tR = 11.6 min) and tetra-sily- lated (major, tR = 12.3 min) products. The tri-silyl derivative afforded rn/z 392 (M +, 20), 377(10), 320(6), 276(6), 248(12), 220(6), 204(100), 188(60), and 171 (80); the tetra-silyl derivative afforded m/z 464(M+,8), 449(3), 320(2), 276(5), 262(5), 245(6), 220(6), 204(100), 188(25) and 171 (20).

L-Canaline production Crude arginase. Freshly hydrated jack bean seeds were grown in vermiculite for 10-

12 days under ambient greenhouse conditions in June or July. The primary, cordate

720 M. BASS ETAL.

leaves and petioles were stored at -75°C. Unless otherwise indicated, the buffer consisted of sodium tricine (pH 7.6) containing 1.0 mM MnCI2 and 0.1% (v/v) 2- mercaptoethanol. Frozen leaf material (350 g) was ground with a Sorvall Omni-mixer at full speed for 30 s with 25 mM buffer. The resulting slurry was expressed through cheesecloth and clarified by centrifugation at 12,000 g for 25 min. The supernatant solution was taken to 55% saturation (v/v) with saturated liquid ammonium sulfate (taken to pH 7.2 with concentrated ammonium hydroxide) and allowed to stand for 90 rain. After collection of the precipitated protein by centrifugation as above, the pellet was dissolved in 50 mM buffer and dialysed overnight against 500 volumes of buffer; the dialysis buffer was changed once. The crude arginase solution was taken to 150ml with 50mM buffer and stored at -75°C. The stored enzyme solution is a rich source of both arginase and urease. The latter enzyme is required to degrade the urea, formed in canaline formation, to ammonia and carbon dioxide.

L-Canaline synthesis. The crude arginase solution was reacted with 25g of canavanine (taken to pH 7.6 with 2N HCI) and 1.0 mM MnCI2 in a final vol of 300 ml. The reaction mixture was shaken mechanically at 37°C. At 30min intervals, the reaction mixture was evaluated for canavanine hydrolysis by monitoring the loss of the canavanine chromogen via the PCAF-colorimetric assay for canavanine (Rosenthal, 1977b). Canavanine degradation is complete within 6 -8 h. At the appro- priate time, the reaction was transferred to a 11 flask, immersed in a salt-ice bath, and swirled to cool the mixture. Forty millilitres of 50% (w/v) trichloroacetic acid was added slowly to the chilled reaction mixture. This process resulted in the evolution of gaseous reaction products which could eject violently the reaction mixture. After standing for 30 rain, the turbid solution was clarified by centrifugation as above. Tri- chloroacetic acid was removed by repetitive extraction with anhydrous ether prior to concentrating the aqueous phase by rotary evaporation in vacuo. The concentrated reaction mixture was evaluated for canaline by a colorimetric assay predicated upon chemical carbamylation of canaline to O-ureidohomoserine and colorimetric analysis of the latter compound (Rosenthal, 1973).

L-Canaline dipicrate. The concentrated reaction mixture was added to a 2.1 molar excess of picric acid heated to 80°C in a minimum volume of deionized water. After standing overnight at 6°C, the crystallized canaline dipicrate was collected by filtration. The canaline dipicrate crystals were washed sparingly with cold absolute ethanol, anhydrous ether, dried overnight at 60°C, and stored at -75°C. Production of the dipicrate salt of canaline has the advantages of allowing successful isolation of canaline from a complex reaction mixture while providing a stable form that can be stored for years without detectable chemical decomposition. L-Canaline dipicrate has mp 193-194°C (dec.). Found: C, 32.5; H, 2.6; N18.8% ; C1sH16N8017 requires C, 32.4; H, 2.7; N, 18.9.

L-Canaline via the sulfate salt. Canaline dipicrate (23.8 g, 4.0 mmole) was treated with 250 ml of 4N sulfuric acid at 60°C for 1.5 h with vigorous mechanical stirring. The reaction was terminated by filtration in vacuo, and the filtrate extracted twice with anhydrous ether to remove free picric acid. Canaline sulfate, reacted with suffi- cient saturated Ba(OH)2 to bring the pH to 7.2, was allowed to stand in an ice-water bath for 20min prior to centrifugation at 14,000g for 20min. The supernatant solution was concentrated by rotary evaporation in vacuo, treated with decolorizing charcoal for 30 min, filtered, and concentrated as above. The concentrated canaline solution was filtered over a Millipore filter (GS membrane), concentrated to a viscous solution and treated with -20°C absolute ethanol. Canaline was collected by fil- tration, washed sparingly with cold absolute ethanol, anhydrous ether, and dried at no more than 50°C overnight in vacuo. The final product was dissolved in a minimum amount of deionized water and recrystallized as above. Canaline free base is hygro- scopic and must be stored with a desiccant at -20°C or colder. Final yield typically is

PRODUCTION OF L-CANAVANINE AND L-CANALINE 721

2.5g. L-Canaline free base has a mp of 213°C (dec.). [a] 22 (1.4%, H20) = + 7 . 7 ___ 0.2 °, Found: C, 34.88; H, 7.40; N, 20.10%; C4H1oN203 .1/4 H20 requires (3, 34.66; H, 7.58; N, 20.22. tH-NMR, d 2.17 (2H, m, - - 3 . 9 2 (2H, m, - CH2CH2-O), 3.86 (3H, m superimposed on dd, -CH23.92 (2H, m, *CH2-O and I~-CH) ppm; t3C-NMR, d 31.74 (b-C), 55.83 (~C), 74.87 g-C), 176.78 (COOH)) ppm. GLC-MS (BSTFA/pyridine) analysis afforded tri-silylated (tR=9.1min) and tetrasilylated (tR=9.6min) products in approximately equal amounts. The tri-silyl derivative afforded m/z 351 (M ÷, 0.5), 276(0.5), 262(0.3), 246(2.0), 230(0.8), 216(2), 202(2.1), 190(1.8), 174(40), 159(20), 146(100); the tetra-silyl derivative afforded m/z 423(M +, 0.1), 407(0.3), 279(0.2), 317(0.3), 302(0.25), 274(2), 262(5), 246(30), 231(6), 218(45), 144(80), and 128(100).

L-Canaline via the hydrochloride salt. L-Canaline dipicrate (5.94g, 10mmol) was treated with 50 ml of 2N HCI at 60°C for 90-129 min and the reaction mixture was stirred vigorously. After cooling the reaction mixture in an ice-water bath, it was filtered in vacuo. The filtrate was chilled for 5 rain and filtered a second time as above. The combined filtrates and cold deionized water washes were extracted three times with anhydrous ether to remove picric acid prior to concentrating by rotary evaporation in vacuo. Rotary evaporation was repeated exhaustively to remove HCI. At that time, the residue was taken up in a minimum amount of deionized water, and the solution adjusted to pH 7.0 with triethylamine. Free canaline was deposited from the mother liquor by the addition of absolute ethanol and anhydrous ether (1:1, v/v) at -20°C and standing at this temperature overnight. Canaline free base was collected by filtration in vacuo and dried overnight in vacuo at no more than 50°C. The typical yield from this procedure is 65%. The product was identical in physical and spectroscopic characteristics to L-canaline free base obtained via the sulfate salt as described above.

Acknowledgements--The authors gratefully acknowledge support from National Science Foundation Grant (IBN-9302875), NSF support under the REU program, and the Howard Hughes Medical Institute Undergraduate Initiative Program (71192-56501 ).

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