6-Diazo-5-oxo-L-norleucine

6-Diazo-5-oxo-L-norleucine

Legal status
Legal status	not approved[ where? ]
Identifiers
IUPAC name (5S)-5-Amino-1-diazonio-6-hydroxy-6-oxohex-1-en-2-olate [1]
CAS Number	157-03-9  Y
PubChem CID	5359375
ChemSpider	16735775  N
UNII	03J0H273KZ
ChEBI	CHEBI:138889
ChEMBL	ChEMBL97485  N
CompTox Dashboard (EPA)	DTXSID501028846
ECHA InfoCard	100.150.017
Chemical and physical data
Formula	C6H9N3O3
Molar mass	171.156 g·mol−1
3D model (JSmol)	Interactive image
SMILES O=C(CC[C@H](N)C(O)=O)\C=[N+]=[N-]
InChI InChI=1S/C6H9N3O3/c7-5(6(11)12)2-1-4(10)3-9-8/h3,5H,1-2,7H2,(H,11,12)/t5-/m0/s1 N Key:YCWQAMGASJSUIP-YFKPBYRVSA-N N
NY  (what is this?)    (verify)

6-Diazo-5-oxo-L-norleucine (DON) is a glutamine antagonist, which was isolated originally from Streptomyces in a sample of Peruvian soil. This diazo compound is biosynthesized from lysine by three enzymes in bacteria. ^[2] It is one of the most famous non-proteinogenic amino acid and was characterized in 1956 by Henry W Dion et al., ^[3] who suggested a possible use in cancer therapy. This antitumoral efficacy was confirmed in different animal models. ^[4] DON was tested as chemotherapeutic agent in different clinical studies, but was never approved. In 2019, DON was shown to kill tumor cells while reversing disease symptoms and improve overall survival in late-stage experimental glioblastoma in mice, when combined with calorie-restricted ketogenic diet. ^[5]

Chemistry

DON is a water-soluble yellowish powder, which can be dissolved also in aqueous solutions of methanol, acetone or ethanol, but dissolution in absolute alcohols is difficult. Solutions of at least 50 μM DON in 0.9% NaCl are lightly yellowish. The crystalline form appears as yellowish greenish needles. The specific rotation is [α]₂₆^D +21° (c = 5.4% in H₂O). In phosphate buffer, pH 7 are the ultraviolet absorption maxima at 274 nm (E1%1 cm. 683) and 244 nm (E1%1 cm 376). ^{[3] [6]}

Biochemistry

DON is used as inhibitor of different glutamine utilizing enzymes. Due to its similarity to glutamine, it can enter catalytic centres of these enzymes and inhibits them by covalent binding, or more precisely, by alkylation. ^{[7] [8]} The following table gives a survey of DON targets.

Selection of enzymes inhibited by DON

Enzyme	Metabolic pathway	References
Carbamoyl phosphate synthase (CAD)	Pyrimidine-De-Novo-Synthesis	[7] [9]
CTP synthase (CTPS)	Pyrimidine-De-Novo-Synthesis	[7] [9]
FGAR amidotransferase	Purine-De-Novo-Synthesis	[7] [10]
Guanosine monophosphate synthetase (GMPS)	Purine-De-Novo-Synthesis	[7] [11]
PRPP amidotransferase	Purine-De-Novo-Synthesis	[7] [11]
Mitochondrial glutaminase	First step of glutaminolysis	[7] [11]
NAD synthase	Coenzyme of the electron transport chain	[7] [12]
Asparagine synthetase	Amino acid synthesis	[7] [13]

Pharmacology

DON is a cytotoxic inhibitor of many enzymes of nucleotide synthesis. It could be shown in vitro that DON treatment led to apoptosis, or programmed cell death. Different pathways were investigated; it could be shown that the inner mitochondrial membrane was damaged, ^[14] and that single strand DNA breaks occurred. ^[15] The exact mode of action remains unclear and needs further research.

DON has not been approved as a pharmaceutical agent; however, it has been tested in combination with a recombinant glutaminase in clinical trials for the treatment of different solid tumors. ^[16]

See also

• JHU-083, a prodrug of 6-diazo-5-oxo-L-norleucine

References

1. "CID 5359375". PubChem. United States National Library of Medicine.
2. Kawai S, Sugaya Y, Hagihara R, Tomita H, Katsuyama Y, Ohnishi Y (April 2021). "Complete Biosynthetic Pathway of Alazopeptin, a Tripeptide Consisting of Two Molecules of 6-Diazo-5-oxo-l-norleucine and One Molecule of Alanine". Angewandte Chemie. 60 (18): 10319–10325. doi:10.1002/anie.202100462. PMID   33624374. S2CID   232039107.
3. Dion HW, Fusari SA, Jakubowski ZL, Zora JG, Bartz QR, et al. (1954). 6-diazo-5-oxo-L-norleucine, A new tumor inhibitory substance. II: Isolation and Characterization. Antibiotics and Chemotherapy. Vol. 78. pp. 3075–7.
4. Yoshioka K, Takehara H, Okada A, Komi N (June 1992). "Glutamine antagonist with diet deficient in glutamine and aspartate reduce tumor growth". The Tokushima Journal of Experimental Medicine. 39 (1–2): 69–76. PMID   1412455.
5. Mukherjee P, Augur ZM, Li M, Hill C, Greenwood B, Domin MA, et al. (29 May 2019). "Therapeutic benefit of combining calorie-restricted ketogenic diet and glutamine targeting in late-stage experimental glioblastoma". Communications Biology. 2 (1): 200. doi:10.1038/s42003-019-0455-x. PMC   6541653 . PMID   31149644.
6. DeWald HA, Moore AM (August 1958). "6-Diazo-5-oxo-L-norleucine, a New Tumor-inhibitory Substance.^1a Preparation of L-, D- and DL-Forms^1b". Journal of the American Chemical Society. 80 (15): 3941–3945. doi:10.1021/ja01548a036.
7. Pinkus LM (1977). "Glutamine binding sites". Affinity labeling. Methods in Enzymology. Vol. 46. pp. 414–27. doi:10.1016/S0076-6879(77)46049-X. ISBN   978-0-12-181946-0. PMID   909432.
8. Ortlund E, Lacount MW, Lewinski K, Lebioda L (February 2000). "Reactions of Pseudomonas 7A glutaminase-asparaginase with diazo analogues of glutamine and asparagine result in unexpected covalent inhibitions and suggests an unusual catalytic triad Thr-Tyr-Glu". Biochemistry. 39 (6): 1199–1204. doi:10.1021/bi991797d. PMID   10684596.
9. Eidinoff ML, Knoll JE, Marano B, Cheong L (1 January 1958). "Pyrimidine Studies: I. Effect of DON (6-Diazo-5-oxo-l-norleucine) on Incorporation of Precursors into Nucleic Acid Pyrimidines". Cancer Research. 18 (1): 105–109.
10. Levenberg B, Melnick I, Buchanan JM (March 1957). "Biosynthesis of the purines. XV. The effect of aza-L-serine and 6-diazo-5-oxo-L-norleucine on inosinic acid biosynthesis de novo". The Journal of Biological Chemistry. 225 (1): 163–176. doi: 10.1016/S0021-9258(18)64919-1 . PMID   13416227.
11. Ahluwalia GS, Grem JL, Hao Z, Cooney DA (1990). "Metabolism and action of amino acid analog anti-cancer agents". Pharmacology & Therapeutics. 46 (2): 243–271. doi:10.1016/0163-7258(90)90094-I. PMID   2108451.
12. Barclay RK, Phillipps MA (February 1966). "Effects of 6-diazo-5-oxol-norleucine and other tumor inhibitors on the biosynthesis of nicotinamide adenine dinucleotide in mice". Cancer Research. 26 (2): 282–286. PMID   4285554.
13. Rosenbluth RJ, Cooney DA, Jayaram HN, Milman HA, Homan ER (August 1976). "DON, CONV and DONV-II. Inhibition of L-'asparagine synthetase in vivo". Biochemical Pharmacology. 25 (16): 1851–1858. doi:10.1016/0006-2952(76)90189-1. PMID   9091.
14. Wu F, Lukinius A, Bergström M, Eriksson B, Watanabe Y, Långström B (July 1999). "A mechanism behind the antitumour effect of 6-diazo-5-oxo-L-norleucine (DON): disruption of mitochondria". European Journal of Cancer. 35 (7): 1155–1161. doi:10.1016/S0959-8049(99)00099-4. PMID   10533463.
15. Hiramoto K, Fujino T, Kikugawa K (June 1996). "DNA strand cleavage by tumor-inhibiting antibiotic 6-diazo-5-oxo-L-norleucine". Mutation Research. 360 (2): 95–100. doi:10.1016/0165-1161(95)00073-9. PMID   8649470.
16. Mueller C, Al-Batran S, Jaeger E, Schmidt B, Bausch M, Unger C, Sethuraman N (2008). "A phase IIa study of PEGylated glutaminase (PEG-PGA) plus 6-diazo-5-oxo-L-norleucine (DON) in patients with advanced refractory solid tumors". J Clin Oncol. 26 (May 20 Suppl): abstr 2533. doi:10.1200/jco.2008.26.15_suppl.2533.