Trifluoroacetamide is a good quencher of tryptophan fluorescence. Trifluoroacetamide was used as probe for determination of membrane potential and used in a convenient alternative to the Gabriel synthesis of primary amines from halides by N-alkylation followed by cleavage of the readily-hydrolyzed trifluoroacetyl group.

Trifluoroacetamide applied in the synthesis of a reactive oxygen species

A new heterobifunctional reactive oxygen species (ROS) responsive thioketal linker and its synthesis are described. This linker allows for developing new ROS-responsive agents with two distinct functionalities using universal bioconjugation methods. Three different ester products, O,S-thioketal, enol thioether, and S,S-thioketal, were separated from the reaction mixture (structure determined with 1H NMR, data not shown) with the desired O,S-thioketal as the major product in 90% yield. However, the following transthioketalization reaction with trifluoroacetamide protected β-mercaptoethylamine was unsuccessful despite several attempts using various acids (p-toluenesulfonic acid, hydrochloric acid, and aluminum chloride). In an effort to synthesize the heterobifunctional S,S-thioketal using an alternative but facile method, we chose to prepare the linker in a one-pot fashion. Stoichiometric amount of methyl 3-mercaptopropionate, trifluoroacetamide protected β-mercaptoethylamine and acetone were mixed with boron trifluoride etherate at 0 °C.To prepare the linker for conjugation, it is necessary to selectively remove either the methyl ester or the trifluoroacetamide protecting group. Both methyl ester and trifluoroacetamide are popular small molecule protecting groups in bioconjugation that are relatively stable in many conditions but may be readily hydrolyzed in alkaline condition.[1]

Although reports of selective deprotection of methyl ester in the presence of trifluoroacetamide are known, the reaction conditions are not universally transferable to other structures due to poor efficiency or selectivity. Unfortunately, attempts using typical literature conditions (lithium hydroxide or triethylamine in aqueous methanol) were unsuccessful. To resolve this chemoselectivity problem, we decided to use porcine liver esterase (PLE) to remove the methyl ester. PLE earned most of its fame in enzymatic organic synthesis for its remarkable performance in asymmetric hydrolysis of methyl esters. The reaction yield was later improved to 73% by carefully maintaining the pH of the reaction mixture around 7 – 8 with the addition of sodium hydroxide solution as the reaction proceeded. The resulting carboxylic acid can then be coupled with a desired moiety using classical procedure before the trifluoroacetamide protection is removed for the second conjugation.In summary, we have developed a new heterobifunctional ROS-responsive thioketal linker with a carboxylic acid and a primary amine group as the conjugation sites. Such a heterobifunctional linker allows for developing ROS-responsive agents with two distinct functionalities, therefore, it has great promise in biomedical applications where on - demand photo activated bond cleavage is desirable. In addition, we found that PLE could be a useful tool to selectively remove methyl ester in the presence of trifluoroacetamide protecting group. Lastly, kinetic study showed that ROS effectively cleaved this thioketal linker and the cleavage reaction followed pseudo-first order kinetics in the presence of sufficient amount of ROS.

Argentation chromatography of molecular trifluoroacetamides

An effective resolution of intact phosphatidylserines on the basis of unsaturation has been achieved by conventional argentation thin layer chromatography (TLC) following trifluoracetylaction. The trifluoroacetamides are prepared by treatment with trifluoroacetic anhydride or N-methyl-bis-trifluoroacetamide. The acetamides are resolved with chloroform-methanol-water (65:25:4, v/v/v) on Silica Gel G containing 20% silver nitrate. Subfractions with 0-6 double bonds per molecule were obtained for the phosphatidylserines of pig and ox brain, pig erythrocytes, rat liver, and rabbit skeletal muscle. The preparation of trifluoroacetamides is also advantageous for the silver ion fractionation of phosphatidylethanolamines. The method is applicable to metabolic studies of molecular species using radioactive precursors of neutral lipids, phosphorus, and nitrogenous bases.[2]

Inhibitory activity of Trifluoroacetamide analogues

Many naturally occurring and synthetic azasugars are potent and specific inhibitors for enzymes associated with carbohydrate metabolism, and they have the potential to produce a number of kinds of beneficial therapeutic effects such as antihyperglycemic, antimetastatic, antifungal, and antiviral activities. Recent studies have provided considerable evidence of increased levels of B-glucuronidase activity in human tumors and suggested that B-glucuronidase may play a role in the metastasis of tumor cells. A trifluoroacetamide analogue of siastatin B, (3S,4S,5R,6R)-6-(trifluoroacetamido)-4,5-dihydroxy-3-piperidine carboxylic acid has been chemically synthesized. This compound, as well as the previously synthesized analogue, (3R,4R,5R,6R)-6-(trifluoroacetamido)-3,4,5-trihydroxy-3-piperid inecarboxylic acid, showed marked inhibitory activity against beta-glucuronidase and significant inhibition of experimental pulmonary metastasis of the highly metastatic melanoma B16.[3]

References

[1]Ling X, Zhang S, Shao P, Wang P, Ma X, Bai M. Synthesis of a reactive oxygen species responsive heterobifunctional thioketal linker. Tetrahedron Lett. 2015 Sep 9;56(37):5242-5244. doi: 10.1016/j.tetlet.2015.07.059. PMID: 26309336; PMCID: PMC4545510.

[2]Yeung SK, Kuksis A, Marai L, Myher JJ. Resolution of molecular species of intact serine and ethanolamine phosphatides by argentation chromatography of their trifluoroacetamides. Lipids. 1977 Jul;12(7):529-37. doi: 10.1007/BF02533377. PMID: 895400.

[3]Nishimura Y, Kudo T, Kondo S, Takeuchi T, Tsuruoka T, Fukuyasu H, Shibahara S. Totally synthetic analogues of siastatin B. III. Trifluoroacetamide analogues having inhibitory activity for tumor metastasis. J Antibiot (Tokyo). 1994 Jan;47(1):101-7. doi: 10.7164/antibiotics.47.101. PMID: 8119851.