South Dakota State University Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange Electronic Theses and Dissertations 1972 Biosynthesis of Canavanine Shu-Chiung W. Chen Follow this and additional works at: https://openprairie.sdstate.edu/etd Recommended Citation Chen, Shu-Chiung W., "Biosynthesis of Canavanine" (1972). Electronic Theses and Dissertations. 4639. https://openprairie.sdstate.edu/etd/4639 This Thesis - Open Access is brought to you for free and open access by Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contact [email protected]. BIOSYNTHESIS OF CANAVANINE BY SHU-CHIUNG W • CHEN A thesis submitted in partial fulfillment of the requirements for the degree Moster of Science, Maior in Chemistry, South Dakota · State University 1972 SOUTH DAKOTA STATE UN·IVERSITY l ARY BIOSYNTHESIS OF CANAVANINE This thesis is approved as a creditable and independent investigation · by a candidate for the degree, Master of Science, and is acceptable as · meeting the thesis requirements for this degree. Acceptance of this thesis does not imply that the conclusions reached_ by the candidate are neces­ sarily the conclusions of the maior department.· ryisff'Adviser Date Head, Chemistry Department Date ACKNOWLEDGEMENTS The author wishes to express her sincere appredation to · Dr. Terry J. Gilbertson under whose direction and advice the research was carried out. A word of thanks also goes to the Chemistry Department .and Pharmacy College for supplying the materials and equipment to make thfa work possible. The duthor also wishes to extend her gratitude to Mr. Jeong-Shwu Liu for drawing the figures. TABLE. OF CONTENTS INTRODUCTION Page Discovery of Canavanine . Physical Properties and Structure Proof • • • • • I• • • 1 Methods of Isolation . 5 • • • • . Biosynthe sis . 9 PROPOSAL ON BIOSYNTHESIS OF FOUR CARBON CHA IN OF CANAVANINE Methionine Hypothesis • • • • • • • • • • • 10 Glutamic Acid Hypothesis • 91 • . 12 RESULTS . 16 • . CONCLUSION . 19 EXPERIMENTAL . Description of Instrumentation Used • � • I • • • • • • • • • 21 Description of Pentacyanoammonioferrate and Ni nhydrin Tes ts . • • • • • • . • • • 22 Paper and Thin-layer Chromatography Used • • . • 25 Isolation of Canavanine • • • . 28 Degradation of Canavanine • . • . 32 Method of Feeding Plants and Growth of Plants . 34 • • • • • • • . Literature Cited • • • . • 35 TABLE OF FIGURES Figure Page 1 • Reactions of canal ine . • • • . • • 3 2. Van Slyke's reaction for canavanine and Enyzmati c degradation of canavanine . • . 4 3. Synthesis of Canavanine from canaline- • • • • • • • • • • • • • • 6 4. Reaction of 0-ethers of hydroxylamine with ha lide acid 0 • . • • 7 5. Reaction of canavanine with hydrobromide • • • • . • • 7 6. Synthesis of canavanine from Y-butyrolactone • . 11 7. lntramolecular nucleophilic displacement of . S-adenosyl methionine • • • • • • • • . .. 12 8. Proposed biosynthetic route from methionine • • . 13 9. Proposed biosynthetic route from glutamic acid • • . • • 15 10. Activity of effluent from AG-50W-8X column • . 18 11. A standard curve of PCAF test for canavanine sulfate • . 24 12. A ninhydrin test and a PCAF test for effluent from AG-50W-8X column • • • • • • • • • • • • • • • • • • • · • 30 13 0 A ninhydrin test and a PCAF test for effluent from IR- 4B co I umn • • • • • • • • • • • • • • • . • 31 14. Degradation of canavanine • • • • • • • • • • • • • • • • • • • 33 1 INTRODUCTION DISCOVERY OF CANAVANINE Canavanine, o{ -amino- Y-guanidoxy-butyric acid, was first discovered in Jack bean {Canavalia ensiformis) by Kitagawa and Tomiyama (1-3). The com- pound is basic and is found free in the non-protein fraction of Jack bean. It is soluble in 50% alcohol, is precipitated as a viscous mass in absolute alcohol, and can be hydrolyzed by the enzyme, arginase, to urea and a new diamino acid, canaline, C H o N • Subsequently in 1939 Damodaran and Narayanan 4 10 3 2 found it in the seed of C.Obtusifolia. (4) So far it has been found mainly in the seeds of Leguminosae, subfamily Papilionoideae. The concentration is occasionally as high as _3.5% of the dry weight. (5-8) PHYSICAL PROPERTIES AND STRUCTUR E PROOF Canavanine H2NC(NH)NHOCH 2CH 2CH(N�)C02H (9) _ ° 2 mol.wt. 176.18 mp 184 (o<) g = +7.9 (w •. c.=2) It crystallizes from alcohol but crystalline form is not reported. It dissolves in water but not in alcohol, ether or benzene. It has some derivatives as listed below: Sulfate: crystallized from alcohol 0 2 mp 17 deco mp. (o< ) I� = + 19. 4 1 0 Copper salt: mp 205-8 decomp. 0 Tribenzoyl: mp 86 decomp. 2 ° 163-4 Picrate: mp ° 212 Flavianate: yellow needles from water mp o The constitution of canavanine C 5H12 3N4 has chiefly been determined H o N , by studying the simpler amino acid, canaline C 4 10 3 2 which is formed together with urea by the degradation of canavanine with the enzyme, arginase. to Kitagawa assigned the structure, H2N-O-C� 2CH 2CH(NH2)C02H, canaline based on the fact that it contains N which is not detected either by Van Slyke's 1). H reaction nor by the formol reaction (Fig. On reduction with 2 and Pt 1 1 black in AcOH or MeOH, it absorbed mole of H2 and set free mole of N H3 • A substance which was regarded as identical with synthetic o( -amino­ 1). Y-hydroxybutyric acid was isolated from the reaction (Fig. No free hy­ droxyl group could be found in canaline. When canaline was warmed with mineral acid, it did not give a lactone. The hydroxyl group appeared in the N Y-position after catalytic reduction. Therefore, this non-amino group on -ON� (10-1.3). canaline was considered to be combined with 0 as 2 Canaline also was synthesized from ethyl o(-benzoylamino-l"-iodobutyrate. (13, 14) The natural and synthetic products were shown to be the same, namely, o<-amino-Y-0-hydroxylaminobutyric acid. 7. 93), From the properties of canavanine, its basic character (pl its fission into urea and an amino compound, and the reaction of only two of its four nitrogen atoms with nitrous acid in the Van Slyke determination of amino .. nitrogen, (Fig .2) Kitagawa et aL suggested that the guanidine group 3 Van Slyke's Reaction Forrnol Reaction HC-HII > 0 HN-CH20H 9. N-(CH2 OH)2 HC-H H2N-0-(CH2kCHtr-OH > H2N -0-(CH2)2-CH�-OH 0 0 Reduction with H2 1. ·Fig. Reactions of canaline. 4 + HONO · O OH 2. Fig. Van Slyke's reaction for canavanine. arginase 2. Fig. Enzymatic degradation of canavanine. 5 H2N·C( :NH}•NH was probably present in canavanirie. They prepared dibenzoyl canaline from canaline with BzCl and NaOH. When it was 10% o( treated with Ac20 first, then H2S041 -benzoyl canaline was ob­ tained. o<-benzoyl canaline reacted with methylisourea and MeOH in the 10% cold. The precipitate was decomposed with HC1 then �lavianic acid 3) was added. Canavanine flavianate was obtaJned. (Fig. This confirmed (15) that canavanine is a guanido derivative of canaline. Then 0-ethers of hydroxylamine reacted with hot halide acids, they yielded the alkyl halide and hydroxylamine. (Fig. 4 ) Gulland and Morris (16) treated canavanine with concentrated hydrobromic acid in a sealed ° 5 160 . tube for hours at Canavanine was converted into ammonia, guanidine, and a substance which must be regarded as o(-amino-¥-butyro­ 5) lactone hydrobromide in view of its properties. (Fig. When this sub­ stance was heated with concentrated hydrobromic acid, it yielded opti• cal ly inactive Y-bromo-o<-amino butyric acid hydrobromide, i den tical · o< with a specimen prepared by the same �ethod from synthetic -amino- Y-butyro-lactone. These results proved canavanine has the structure, METHODS OF ISOLATION (2) The Jack bean plant is still the main source of canavanine. Kitagawa 50% extracted canavanine from Jack bean with alcohol and treated it with flavianic acid but the %N was lower t�an the ·theoretical value··for this 6 . CsH5COCI Ac20 cool > 3. from Fig. Synthesis of canavanine canaline 7 - R-O NH2 + HCI > RCI + NH20H 4. Fig . Reaction of 0-ethers of hydroxylamine with halide acid . �H2·HBr �H2·HBr CH2CH-C-OH CH2CH2CHC=O + HBr ----.) Br-CH2 I O_J. 0 Fig. 5. Reaction of canavanine with hydrobromide. 8 flavianate. In 1937 Kitagawa and Tsukamoto {17) recommended the destruction of the impurity by digesting the first crude canavanine precipitate with 10% HC1. They reported that this gave a high purity canavanine flavian­ ote1 but they did not give a detailed method nor the yield. In 1935 GuHand and Morris (16), who also had the same difficulty, suggested the purification of the base liberated from the flavianic acid �a lt by conversion into the rufianate. In 1939 Damodaran {18) treated the crude canavanine with a solu- tion of basic lead acetate, and then with flavianic acid. He got a. high purity flavianate successfully, but had trouble removing the excess lead. In 1962 a method was devised using ion-exchange resin to prevent the formation of desaminocanavanine with cold 99% alcohol from the concentrated canavanine extract, then treated it with flavianic acid, and decomposed the flavianate with hot saturated Ba(OH) • The filtrate was passed through IR-48 2 - (OH form) ion-exchange resin to remove the impurity. The effluent was con- centrated under reduced pressure and canavanine crystallized upon the addi- tion of absolute alcohol. The merits of this method are that it gives the free canavanine in high purity, easily and in a good yield, directly from its flavianate. (19) In the experiment of the author, the filtrate from decomposition with Ba(OH) was passed through AG-50W-8X resin and ca navanine•2HC1 was 2 eluted with 4N HCl. The effluent contain_ing canavanfoe.2HC1 was passed through IR-4B (OH-form) resin to get free canavanine.