SYNTHESIS & APPLICATIONS OF LABELLED FORMYLATING REAGENTS: AMINO ACIDS & PEPTIDE

合成

• 批准号: 6220428
• 负责人: MANOUCHEHR SALJOUGHIAN
• 金额: $ 4.24万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6220428
• 关键词: biological products; biomedical resource; proteins; technology /technique

Efficient reagents for formylation have a wide utility in organic synthesis. In addition to simple and direct formylation of reactive functional groups, they are used for one carbon extension reactions, and cyclization of aliphatic and aromatic compounds, through methine insertion reactions. Several formylating reagents such as formaldehyde, formic acid, formyl fluoride, formic anhydride or formic acetic anhydride have been available for non-radioactive synthesis of a formyl group in organic synthesis for many years. In contrast, only tritiated formaldehyde at a specific activity of 25-100 mCi/mmole in 1:99 mixture with water is commercially available for tritioformylation reactions. Several biologically important compounds, such as folinic acid, an antidote to drugs that act as folic acid antagonists, Chlorophyll b, an essential compound in photosynthesis, and a group of bioactive peptides known as chemotactic peptides (leukocyte chemoattractants which direct the migration of cells) bear a formyl group necessary for their action. In this last group, the chemotactic response of the rabbit and human neutrophils to the synthetic tripeptide N-formyl-methionyl-leucyl-phenylalanine is well established to be mediated via interaction of the peptide with a specific receptor. I n addition to stimulating chemotaxis, this peptide/receptor interaction initiates a number of events in neutrophils such as lysosomal enzyme release, superoxide formation and release of calcium. The N-formyl group appears to be essential for good activity since N-acetylation, removal of the _-amino group, or replacement by an ethyl group results in loss of biological activity by a factor of 1,000-10,000. The development of an efficient and high specific activity tritiated formylation reagent would be extremely useful for the elucidation of many biological processes. We propose three possible tritioformylating reagents: p-Nitrophenyl tritioformate, N-tritioformyloxyphthalimide, and acetic formic anhydride (AFA). All candidates were synthesized and evaluated during our exploratory chemistry, but AFA was the most selective and efficient reagent. The deuterated form of AFA was synthesized in three steps: 1. Synthesis of superdeuteride from LiD; 2. Reduction of CO2 to lithium formate (DCOOLi) by superdeuteride; and 3. Reaction of acetyl chloride with DCOOLi to form acetic formic anhydride (CH3COOCOD) The deuteriated AFA was used for formylation of L-phenylalanine and L-methionine. 3H AFA formed from supertritide at 20% tritium abundance was used for N-tritioformylation of methionyl-leucylphenylalanine (MLP) and the formylated peptide was isolated in high yield at 100% radiochemical purity. The analogous synthesis using supertritide at 100% tritium content yielded the radiochemically pure N-tritioformyl-MLP at 95% chemical yield. To our knowledge, this is the first demonstration of N-formylation at 100% tritium incorporation. The following general procedure describes the synthesis of the reagent at the maximum specific activity and its highly efficient application in the N-tritioformylation of a tripeptide. Supertritide (0.2 mmole) at the theoretical specific activity was prepared prior to the synthesis of tritiated lithium formate. Carbon dioxide (8 mL) was injected in the reaction vessel and reduced for 1 hour to furnish high specific activity T-COOLi. Acetyl chloride (0.18 mmole, 7 _L) in dry ether (100 _L) was then injected into the reaction vessel and the suspension was stirred at room temperature for two hours to form acetic [3H]formic anhydride. The reagent in ether solution was vapor transferred to a second reaction vessel. A solution of methionylleucylphenylalanine (MLP, 10 mg, 0.0025 mmole) in glacial acetic acid (l mL) was injected, and the reaction was stirred for 1 hour at room temperature to form N-[3H]-formylmethionyl-leucylphenylalanine (F-MLP). At the end of the reaction, excess reagent was destroyed by the addition of methanol (0.5 mL), and the solvents were removed under vacuum. The dried residue was dissolved in methanol (1 mL) for analysis. Radio-HPLC analysis gave a specific activity of 28.6 Ci/mmole, and 95% chemical yield. Extensive 1H/3H NMR analysis showed the desired product with the formyl tritium at _ 8.1 ppm in the tritium spectrum and close to 100% radiochemical purity.

甲酰化的高效试剂在有机化学中有着广泛的应用