CAW. 3. BIBCHEM. VOL. 51, 1973

An Improved Method for the PreparaGon of Isomeric a,t-Diaminopimelic Acid from Glutaraldehyde'

]W. B. ROY AND M. KAREL Department of Nutaidion and Food Science, AIassachus~tts Insbidute of Techno8ogy,

Cambridge, Mass. 02639

Received November 1 1, 1972

ROY, W. B., and KAWEE, M. An improved method for the preparation of isomeric ape-diarnino- pimelic acid from glutaraldehyde. Cam. J. Biochem. 51,942-943 ( 1973).

An aqueous solution of glutaraldehyde was reacted with a mixture of sodium cyanide and ammonium chloride giving a high yield of 2,6-dicyanopiperidim. Refluxing of this compound with a mixture sf ammonium hydroxide and ammonium bicarbonate produced 5,s'-trimethylene dihydantoin, which on acid hydrolysis gave a, c -diaminopimelic acid.

ROY, R. B., et KAREE, M. An improved method for the preparation of isomeric a,e-diamino- pimelic acid from glutaraldehyde. Can. J. Biochem. 51,942-943 ( I973 ) .

La rkaction d'une solution aqueuse de glutaraldkhyde avec un mClamge de cyanrrre de sodiaarn et de chlorure &ammonium donne la 2,6-dicyanopipCridimae avec un rendement klevC. Par Cbulli- tion A reflux de ce compost5 avec pl~a mClange d'hydroxyde d'ammonium et de bisr dr b owafe d'ammonium om sbtient la 5,s'-trimCthyl5ne dihydantolne qui, par hydrolyse aside, donne l'acide a, c -diamimopimClique. [Traduit par le journal]

a,~-Diaminopimelic acid is an imps termediate for the preparation of lysine on a commercial scale by following a biological pro- cedure (1 ). It serves as a starting material in the synthesis of many interesting peptides (2). It can be synthesized by a number of methods de- scribed in the literature (3-51, yielding, in almost all cases, three isomers: DD, LL, and meso.

During the course of our studies on the reac- tion of lysine with hydroproxides, we needed compounds with the general formula HPO2CH- QNW2) -(CH2) ,-CH (NH,) C02H (a t = 3.8) for comparison with isolated reaction products. Roger ( 6 ) and Hause ('7) have described the preparation of a,€-diaminopimelic acid by a modified Strecker procedure, but the handling of the highly toxic hydrogen cyanide gas makes their method inconvenient. We report a modified method of Roger (6) and Hause ('7) which can be used to prepare large quantities of isomeric a,~-diarninopimelic acid with an inexpensive and commercially available aqueous solution of glu- taraldehyde (available in 25 % aqueous solu- tion). Rhuland et a&. (10) have reported a preparation of this compound via another method. In most of the solvent systems, the synthetic

'This work was supported by grant 5PO1-ES-00597- 02 from the National Institutes of Health.

diaminopimelic acid was found to give a single ninhydrin-reactive spot (Rf 0.27, phenol-am- monia), which was identical with that given by the authentic meso- or DL-diamiaopimelic acid. The synthetic product was treated with carbo- benzoxy chloride in the presence of' a dilute sodium hydroxide solution and was worked up to give two isomeric dicarbobenzoxy derivatives, which may consist of a mixture of DL- and meso- foms of diaminopimelic acid.

2,6-Bicyanopiperi$iate A mixture of sodium cyanide (18.0 g) and

ammonium chloride (20.0 g) was dissolved in water (150 ml). To this, concentrated ammo- nium h y d r o ~ d e solution (35 mI, 28-30% aqueous solution) was added and the reaction mixture was cooled to 0-3" in ice. An aqueous solution of glutaraldehyde (58 ml, 25% practi- cal grade available from Eastman Kodak Com- pany) was added dropwise. The reaction flask was kept below 5" for 4-5 h and the separated crystals were filtered off. These crystals were dissolved in benzene and washed with water. The organic layer was dried over anhydrous mag- nesium sulfate and filtered. On concentration, the filtrate gave white crystals: m.p. 112-1 14" (lit. (6) mop. 11 1-1 f 4"), yield 85% (calcu- lated on glutaraldehyde). This compound had expected peaks in infrared and nuclear magnetic resonance spectra.

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NOTES 943

5,5'-Tri~~zetktylenedi/aydan&oin A mixture of 2,6-dicyanopiperidine ( 10.0 g ) ,

ammonium bicarbonate (20.0 g) , and concen- trated ammonium hydroxide solution (40.0 ml) was refluxed for 3 h using a long air condenser. Additional ammonium bicarbonate (10.0 g) was added and the reaction mixture was refluxed for another 3 h (total time, 6 h) . The reaction mix- ture was concentrated to dryness under reduced pressure on a water bath. The residue obtained was crystallized from hot water to give white crystals: m.p. 236-238", yield 9.5 g (53%). The dihydantion was characterized by infrared spectra and by the comparison of melting p in ts reported for this compound (7 ,8 ) . Psoarmeric a,~-Biaminopimek Acid

Bihydantion (5.0 g), obtained as described above, was refluxed with 8 N hydrochloric acid

with carbobenzoxy chloride (1 ml) in 2 N NaOH solution 45 ml) and worked up to get white crystals: m.p. 165-1 68" (from ethyl acetate), yield 300 mg. On further concentration of the mother liquor, more white crystals were ob- tained: m.p. 128-1 30°, yield 1 20 mg. The melt- ing point reported in literature (9) for the di- carbobenzoxy derivatives of the DL- and meso- form of diaminopimelic acid were 164-1 65 " and 123-1 25 ", respectively. The mixed melting points with authentic dicarbobenzoxy derivatives of DL- and naeso-form of diaminopimelic acid, respectively, were not depressed.

1. CASIDA, L. E., JR.: U.S. Patent 2, 771, 396 (1956). 2. NIGOT, C., VAN HEIGENOORT, J., LEFMNCPER, P.,

and BRIGAS, E. : J. Org. Chem. 30, 3746 ( f 965). 3. SORENSON, S. P. L., and ANDERSON, A. C.: %. Phy-

siol. Chem. 56,250 ( 1908). solution for 24 h. The reaction iixture was con- 4- S H E E ~ N , J. C., and BQLHQFER, W. A.: J. Am.

centrated to dryness under reduced pressure on Sot. 72, 2786 (1950)- 5. AKABQRI, S., IZUMI, Y., and FUJIWARA, To: J. a water bath. The residue obtained was dissolved Chem. Sot. 75, 993 ( 1954).

in a small volume of water and neutralized with 6. ROGER, A. O.: Chem. Abstr. 61, 13287 (1964). ~ ~ r i d i n e . The white crystals which separated 7. HAUSE, N- L.: Chem. Abstr. 61,4363 ( 1964). (3.2 g, yield 81 % ) were filtered ofl%. o n a Paper 8. EMMIGK, B., and LONBERGAN, T. E.: Chem. Abstr. chromatogram (Whatman No. 1) using meth- 57,5805 (1962).

Y w

mol-water-pyridine, 80:20:4, as a solvent sys- 9- W ~ E , R., B ~ B A U M , S. M., WINITZ, M., KOEGEL,

tern, a single ninhydrin-reactive spot (Rf 0.21 ; R. J., and GREENSTEIN, J. P.: J. Am. Chem. Soc. 79,648 (1957).

lit. (10) 0.26) was obtained. 10. RHULAND, E., WORK, E., BENMAN, R. P., and The above compound (1.0 g) was treated HOAX, D. S.: J. Am. Chem. SW. 77,4844 (1955).

Effects of Elevated IrntracelIular ATP and GTP Concentrations on Purine Ribonucleotide Synthesis and Interconversion'

FLOYD F. SNYDER AND J. FRANK HENDERSON Cancer Research Unit (McEachern Laboratory) and Department of Biochemistry,

University of Alberta, Edmonton, Alberta TBG 2EP

Received February 5, 1973

SNYDER, F. F., and HENDERSON, J. F. Effects of elevated intracellular ATP and CTP concentra- tions on purine ribonucleotide synthesis and interconversion. Can. J. Biochem. 51, 943-948 (1973).

The effects of elevated intracellular ATP and CTP concentrations on enzymes 0% purine ribonuclestide synthesis and interconversion were examined in intact Ehrlich ascites tumor cells. Elevated ATP and CTP concentrations inhibit nueleotide synthesis from purine bases up to 56% and elevated concentrations of GTP severely inhibit IMP dehydrogenase (60%). In contrast, adenylate deminase activity was inhibited 16% at the highest concentration of GTP studied and elevated ATP concentrations had only a slight inhibitory effect on adenylosuccinate synthetase plus lyase (9% ) .

'This work was supported by the Medical Research Council and National Cancer Institute of Canada.

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