Cofactor maturase NifEN: A prototype ancient nitrogenase?

Nitrogenase plays a key role in the global nitrogen cycle; yet, the evolutionary history of nitrogenase and, particularly, the sequence of appearance between the homologous, yet distinct NifDK (the catalytic component) and NifEN (the cofactor maturase) of the extant molybdenum nitrogenase, remains elusive. Here, we report the ability of NifEN to reduce N2 at its surface-exposed L-cluster ([Fe8S9C]), a structural/functional homolog of the M-cluster (or cofactor; [(R-homocitrate)MoFe7S9C]) of NifDK. Furthermore, we demonstrate the ability of the L-cluster-bound NifDK to mimic its NifEN counterpart and enable N2 reduction. These observations, coupled with phylogenetic, ecological, and mechanistic considerations, lead to the proposal of a NifEN-like, L-cluster-carrying protein as an ancient nitrogenase, the exploration of which could shed crucial light on the evolutionary origin of nitrogenase and related enzymes.

ATP-Independent Turnover of Dinitrogen Intermediates Captured on the Nitrogenase Cofactor.

Nitrogenase reduces N2 to NH3 at its active-site cofactor. Previous studies of an N2-bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N2 species via asymmetric belt-sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N2 reduction involving all cofactor belt-sulfur sites; yet, the evidence for the existence of multiple N2 species on Av1* remains elusive. Here we report a study of ATP-independent, EuII/SO3 2--driven turnover of Av1* using GC-MS and frequency-selective pulse NMR techniques. Our data demonstrate incorporation of D2-derived D by Av1* into the products of C2H2- and H+-reduction, and decreased formation of NH3 by Av1* concomitant with the release of N2 under H2; moreover, they reveal a strict dependence of these activities on SO3 2-. These observations point to the presence of distinct N2 species on Av1*, thereby providing strong support for our proposed mechanism of stepwise reduction of N2 via belt-sulfur mobilization.

Mechanistic Insight from Lewis-Acid-Dependent Selectivity and Reversible Haloboration, as Harnessed for Boron-Based Electrophilic Cyclization Reactions.

Different reaction selectivity occurs with the Lewis acids B-chlorocatecholborane (ClBcat), B-bromocatecholborane (BrBcat), and BBr3, favoring either alkyne haloboration, electrophilic cyclization of a tethered nucleophilic sulfur onto the alkyne, or group transfer of the nucleophile. This reaction selectivity also depends on the chain length of the tethered nucleophile, revealing a subtle interplay of relative kinetics and thermodynamics. In all cases, BBr3 reacts readily with alkynes to form haloborated products; however, this process is reversible, and this reversibility can be harnessed to ultimately access regio- and stereodefined cyclic sulfonium zwitterions via the slower but thermodynamically favored electrophilic cyclization pathway. Reversibility was noted by following the reaction by NMR spectroscopy, and by characterizing the kinetic and thermodynamic products by a combination of 2D NMR spectroscopy and single-crystal X-ray diffraction. The "mixed" reagent bromocatechol borane (BrBcat) displayed reactivity between ClBcat and BBr3, producing bromoboration in some cases and electrophilic cyclization in others. With this enhanced understanding of the reaction dynamics, it becomes possible to use boron Lewis acids in a predictable manner in cases where haloboration is the kinetic product but in which the reversibility of this reaction maintains access to eventual alternative reactivity leading to desired building blocks in organic synthesis.

NHC induced radical formation via homolytic cleavage of B-B bonds and its role in organic reactions.

New borylation methodologies have been reported recently, wherein diboron(4) compounds apparently participate in free radical couplings via the homolytic cleavage of the B-B bond. We report herein that bis-NHC adducts of the type (NHC)2·B2(OR)4, which are thermally unstable and undergo intramolecular ring expansion reactions (RER), are sources of boryl radicals of the type NHC-BR2˙, exemplified by Me2ImMe·Bneop˙ 1a (Me2ImMe = 1,3,4,5-tetramethyl-imidazolin-2-ylidene, neop = neopentylglycolato), which are formed by homolytic B-B bond cleavage. Attempts to apply the boryl moiety 1a in a metal-free borylation reaction by suppressing the RER failed. However, based on these findings, a protocol was developed using Me2ImMe·B2pin23 for the transition metal- and additive-free boryl transfer to substituted aryl iodides and bromides giving aryl boronate esters in good yields. Analysis of the side products and further studies concerning the reaction mechanism revealed that radicals are likely involved. An aryl radical was trapped by TEMPO, an EPR resonance, which was suggestive of a boron-based radical, was detected in situ, and running the reaction in styrene led to the formation of polystyrene. The isolation of a boronium cation side product, [(Me2ImMe)2·Bpin]+I-7, demonstrated the fate of the second boryl moiety of B2pin2. Interestingly, Me2ImMe NHC reacts with aryl iodides and bromides generating radicals. A mechanism for the boryl radical transfer from Me2ImMe·B2pin23 to aryl iodides and bromides is proposed based on these experimental observations.

Repurposing π Electrophilic Cyclization/Dealkylation for Group Transfer.

A metal-free regio- and stereocontrolled group-transfer route toward the synthesis of trisubstituted alkenes is described. In this route, an electrophilic heterocyclization is followed by ring-opening group transfer. Specifically, a thioboration reaction transforms readily available alkynyl sulfide precursors into alkenyl boronates and alkenyl sulfides with defined regio- and stereochemistry in one synthetic step using commercially available B-chlorocatecholborane (ClBcat). Mechanistic studies identified the likely pathway as proceeding through zwitterionic rather than haloborated intermediates. The regio- and stereochemistry set in the initial cyclization step is preserved in the final acyclic alkene product, producing alkenes with up to four modifiable substituents with predictable regio- and stereochemistry. Downstream functionalization reactions showcase the versatility of the substitutions of the resulting alkenes. The mechanistic concept maps onto future reaction designs, given the abundance of known electrophiles and nucleophiles for electrophilic heterocyclization/dealkylation sequences.

FBpin and its adducts and their role in catalytic borylations.

We present herein investigations concerning the reactivity of B2pin2 and FBpin (pin = pinacolato) with respect to fluoride ions and NHCs (NHC = N-Heterocyclic Carbene) in order to understand better the role of added fluoride in our recently reported Ni-catalyzed C-F borylation process, to confirm the nature of the NHC-adducts of FBpin, and to explain the formation of the species detected as side products of the defluoroborylation reaction. We report the calculated gas phase fluoride ion affinities (FIA) of relevant boron species and demonstrate that the presence of one equivalent of added F- in solution will bind and activate B2pin2 (FIA = +247 kJ mol-1), but it will also bind even more effectively to FBpin (FIA = +287 kJ mol-1) formed in the reaction. We prepared the NHC-adducts of FBpin by two different methods and isolated and characterized several examples of the type FBpin·NHC (NHC = iPr2Im, 1; Me2Im, 2; MeiPrIm, 3; nPr2Im, 4; Mes2Im 5). The salts [pinBF2][NMe4] 6, as well as the significantly more soluble analogue [pinBF2][NnBu4] 7, were also isolated and characterized, providing confirmation of the presence of the pinBF2 anion in various stoichiometric and catalytic borylation reactions.

Cytochrome P450 2C19 inhibitory activity of common berry constituents.

The cytochrome P450 enzyme CYP2C19 is involved in the metabolism of many commonly prescribed drugs, including proton pump inhibitors, antiepileptics and antidepressants. CYP2C19 inhibitors from food and food supplements may augment the toxicity of these agents and lead to noncompliance with treatment. The present investigation addresses CYP2C19 inhibition by 18 berry constituents using a chemiluminescent assay. Test compounds displayed inhibitory properties in a concentration-dependent fashion, with IC(50) values ranging from 20.2 microM up to >316 microM. In the order of decreasing effect size, anthocyanidins were followed by anthocyanidin-monoglycosides and procyanidins. Anthocyanidin-diglucosides exhibited weak and biphasic effects. When compared with the CYP2C19 inhibitor fluvoxamine, the flavonoids under study were 50- to 750-fold less potent. It is concluded that the above natural substances are moderate to poor inhibitors of CYP2C19 in vitro.