N,N,N',N'-Tetramethylazodicarboxamide, TMAD, was found to be more versatile in the Mitsunobu reaction than traditional diethyl azodicarboxylate or recently developed 1,1′-(azodicarbonyl)dipiperidine, when used in combination with tributylphosphine in benzene. The usefulness of the reagent was demonstrated by the highly efficient two-step synthesis of benzylcrotylamine from N-benzyltrifluoroacetamide.

N,N,N',N'-Tetramethylazodicarboxamide-Tributylphosphine Reagents

Four secondary alcohols of different steric environment reacted with five carboxylic acids of different acidity in the presence of N,N,N',N'-Tetramethylazodicarboxamide and tributylphosphine to give the corresponding epi-esters in better yield than with diethyl azodicarboxylate-triphenylphosphine in most cases. Of special merit is the combination of the new reagent system with p-methoxybenzoic acid to achieve complete inversion of sterically congested secondary alcohols. The reaction, however, is very sensitive to the steric situation around the carbinyl carbon;the yield of the epi-ester decreases sharply with sterically congested alcohols. Although this difficulty has partly been overcome by changing the solvent or applying stronger carboxylic acids, further improvement is still needed for the reaction of sterically congested secondary alcohols. Since the combination of N,N,N',N'-Tetramethylazodicarboxamide (TMAD)and tributylphosphine (TBP)was recently found by us to be the most versatile Mitsunobu reagent system applicable to nucleophiles of up to 13.5,we investigated the characteristics of this combination for the Mitsunobu inversion, Presented herein are the results of these studies which reveal this combination to be more satisfactorily applicable to the inversion of sterically congested secondary alcohols than that of diethyl azodicarboxylate (DEAD)and triphenylphosphine (TPP).[1]

Typical experimental procedure: Under dry Atmosphere, solid N,N,N',N'-Tetramethylazodicarboxamide (1.5 mmol)was added in one portion to the dry benzene solution (3 mL)of an alcohol (1 mmol),TBP and a carboxylic acid (1.5 mmol each)at 0°C with stirring. After 10 min, the reaction mixture was heated at 60°C for 24 h with stirring, during which time dihydro-TMAD crystallized out. The epimeric mixture of esters obtained was analyzed as follows. The inversion ratios were determined by capillary GLC or H-NMR on the crude products obtained by the evaporation of the solvent in vacuo. The yields were obtained after the product isolation using SiO, column chromatography. Thus, the TMAD-TBP system was shown to have larger tolerance to the steric congestion of the carbinyl carbon but to be more sensitive to the acidity of nucleophiles than the DEAD-TPP system. For the Mitsunobu inversion of sterically congested alcohols, the combination of N,N,N',N'-Tetramethylazodicarboxamide -TBP system and p- methoxybenzoic acid is the reaction of choice.

N,N,N',N'-Tetramethylazodicarboxamide insecticides targeting insect ryanodine

N,N,N',N'-Tetramethylazodicarboxamide insecticide is a kind of pesticide that specifically targets the ryanodine receptor of Lepidopteran pests, which makes it safe, effective, targeted, and low toxicity to mammals. So, it is one of the most concerned and fastest-growing pesticide products after neonicotinoid pesticides. Intracellular Ca2+ concentration can be regulated by ryanodine receptors, and the continuous release of Ca2+ eventually leads to the death of pests and achieve the insecticidal effect. as well as its specific target-ryanodine receptor, and analyzes how the diamide insecticide acts on the ryanodine receptor and how its mechanism of action can provide a theoretical basis for the rational use of highly effective insecticides and solve the resistance problem.[2]

N,N,N',N'-Tetramethylazodicarboxamide insecticides are the fastest-growing pesticide products in recent years, and chlorantraniliprole is one of the best-selling insecticides. With the widespread use of diamide insecticides, the problem of insecticide resistance has become increasingly prominent. The amino acid I4790M mutation of RyRs in Plutella xylostella resulted in a 10-fold reduction in the efficacy of chlorantraniliprole. The multipoint mutation in RyRs of Chilo suppressalis confers resistance to N,N,N',N'-Tetramethylazodicarboxamide insecticides. It was found that the simultaneous mutation of M4758I and G4915E had higher resistance to chlorantraniliprole (153.1-fold) and cyantraniliprole (323.5-fold). We should always pay attention to the problem of pesticide resistance. Long-term use of pesticides, especially single pesticides, will cause many hazards, such as a sharp rise in resistance and increased environmental risks. The harm caused by the long-term use of pesticides is one of the urgent problems to be solved, which deserves attention.

N,N,N',N'-Tetramethylazodicarboxamide Triggers Mainly S Thiolations

Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile N,N,N',N'-Tetramethylazodicarboxamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. The toxicity of diamide is based on disulfide bond formation, which was recently visualized in B. subtilis and S. aureus by the fluorescence alkylation of oxidized thiols (FALKO) assay. It was thought that the formation of nonnative inter- and intramolecular disulfide bonds results in damage of proteins. Depletion of the free cysteine pool in B. subtilis after exposure to N,N,N',N'-Tetramethylazodicarboxamide supports this finding. To assess if GSH may have a bearing on the thiol redox buffer of B. subtilis, the gshF gene of Listeria monocytogenes (gshFLm) was expressed in B. subtilis, enabling GSH biosynthesis.[3]

In general, the diagonal gel electrophoresis approach of N,N,N',N'-Tetramethylazodicarboxamide -treated B. subtilis and S. aureus cells supports the idea that the majority of disulfide bonds are mixed disulfides of protein thiols with LMW thiols rather than inter- and intramolecular disulfide bonds. However, we have to consider that some cysteine-containing proteins may be below the detection level of this assay or could have cysteine residues that are not solvent exposed and not accessible for thiol modifications. In summary, different approaches were used to analyze the nature of reversible thiol oxidations in B. subtilis and S. aureus in response to N,N,N',N'-Tetramethylazodicarboxamide stress. S thiolation is the major mechanism used to protect cellular protein thiols in both bacteria after exposure to diamide.

References

[1]Tsunoda, T., Yamamiya, Y., Kawamura, Y., & It?, S. (1995, April 3). Mitsunobu acylation of sterically congested secondary alcohols by N,N,N',N'-tetramethylazodicarboxamide-tributylphosphine reagents. Tetrahedron Letters, 36(14), 2529 - 2530. 

[2]Du, Juan, and Yuejun Fu. “Diamide insecticides targeting insect ryanodine receptors: Mechanism and application prospect.” Biochemical and biophysical research communications vol. 670 (2023): 19-26. doi:10.1016/j.bbrc.2023.05.107

[3]P?ther DC, Liebeke M, Hochgr?fe F, Antelmann H, Becher D, Lalk M, Lindequist U, Borovok I, Cohen G, Aharonowitz Y, Hecker M. Diamide triggers mainly S Thiolations in the cytoplasmic proteomes of Bacillus subtilis and Staphylococcus aureus. J Bacteriol. 2009 Dec;191(24):7520-30. doi: 10.1128/JB.00937-09. Epub 2009 Oct 16. PMID: 19837798; PMCID: PMC2786588.