1,2,4-Triazoliums: Applications in Biocatalysis, Organocatalysis and Stable Radical Synthesis

Abstract

Herein we report the exploration of carbenes derived from 1,2,4-triazoliums in the novel rearrangement reaction to stable Blatter radicals, biocatalysis and organocatalysis.
Nitron is an intriguing 1,2,4-triazolium, with a C(5)-N-anilino substituent which permits establishment of a tautomeric equilibrium between the C(3) and C(5)-N positions. This work continues from our initial report of the unusual rearrangement of Nitron to Blatter radicals. The synthesis of Nitron was revisited and contemporary synthetic strategies were employed to enhance isolated yields and scope. Evaluating the series of Nitron derivatives for propensity to form Blatter radicals enabled the isolation of seven new radicals and implicated the role of the carbenic tautomer in the initial hydrolytic ring opening step. Evaluation of the tautomeric equilibrium was performed by spectrophotometric determination of C(5)-NH pKa values and NMR spectroscopic kinetic monitoring of C(3)-H/D exchange. A 10-fold drop in carbene proportion at equilibrium when Nitron is substituted with a 4-F3CC6H4 group at the C(5)-N-anilino position was attributed to stabilisation of the formal N- charge in the zwitterionic form. The experimental evidence supports a faster ring opening from the carbene tautomer compared to the zwitterion.
Owing to the growing interest in stable organic diradicals, a range of synthetic strategies was explored to couple Blatter radicals. The intolerance of radical character to synthetic conditions and cross-reactivity of the ring and exocyclic nitrogen positions complicated this coupling. A synthetic strategy to a coupled di-Nitron was developed and evaluation for di-radical formation provided circumstantial evidence. The lack of a C(3)-H for carbene formation appears to limit hydrolytic ring opening. Future work could explore conditions to promote hydrolytic ring opening at the 1,2,4-triazolium C(3)-position.
The use of heterocyclic azolium-derived carbenes for organocatalysis originated from the mechanistic evaluation of thiamine (pyrophosphate, TPP) (a thiazolium) as a cofactor in TPP-dependent enzymes. Since this discovery, the field of NHC organocatalysis has grown exponentially, with chemists developing catalysts centred about the more acidic 1,2,4-triazolium scaffold. We report the first synthesis of a 1,2,4-triazolium replica of TPP in attempt to improve the yield, selectivity and scope of TPP-dependent biocatalytic transformations. Kinetic evaluation of H/D exchange of TPP and the isolated 1,2,4- triazolium mimic using NMR spectroscopy permitted estimation of C(2/3)-H pKa values and showed a ~1.5 unit decrease for 1,2,4-triazolium mimics in comparison to thiamine.
Initial evaluation of 1,2,4-triazolium mimics focussed on model benzoin condensation reactions, in the absence of enzyme. An approximate 10-fold enhancement in rates of initial hydroxyaryl adduct were determined through kinetic monitoring. Assessment of the 1,2,4-triazolium mimic as a cofactor was performed with TPP-dependent enzymes pyruvate decarboxylase from Saccharomyces cerevisiae and pyruvate oxidase from Aerococcus sp. via spectrophotometric coupled assays and direct NMR spectroscopic analysis. Loss of enzyme activity in the presence of the cofactor mimic was observed implying that binding to the active site occurred but the mimic was less active. This decreased activity was thought to be due to a conformational discrepancy within the active site. Future work will screen a greater range of more tolerant TPP-dependent enzymes to fully explore compatibility of the 1,2,4-triazolium mimic.
Complementary studies evaluated the hydrolytic stability of TPP by 31P NMR spectroscopy in a series of deuterated buffers from pD 0-13. Under physiological conditions (pD 4-7) TPP is very stable with minimal hydrolysis of the pyrophosphate over several months (t1/2 = 300-500 days). Under acidic conditions (pD < 3), hydrolysis of the pyrophosphate becomes more prevalent (t1/2 down to 2 days). Above pD 8, hydrolysis of the pyrophosphate group is negligible, but cleavage of the methylene linker occurs.
Finally, 1,2,4-triazoliums isolated within these projects were evaluated as catalysts to the benzoin condensation, a key NHC organocatalysed transformation. Intriguingly, Nitron derivatives showed no propensity for catalysis, despite showing similar rates constants for H/D-exchange as other commonly employed 1,2,4-triazolium catalysts. This result was unexpected and attributed to the unusual chemical nature of carbenes derived from Nitron derivatives. The evaluation of more sustainable reaction conditions for NHC organocatalysis led to the observation that rate enhancements were observed when the benzoin condensation was performed under aqueous conditions with a more traditional 1,2,4-triazolium catalyst. This observation paves the way for significant future work to develop an understanding of NHC-organocatalysed transformations in water.