The biosynthesis of the thiazole phosphate moiety of thiamin: the sulfur transfer mediated by the sulfur carrier protein ThiS

Thiamin-pyrophosphate is an essential cofactor in all living systems. The biosynthesis of both the thiazole and the pyrimidine moieties of this cofactor involves new biosynthetic chemistry. Thiazole-phosphate synthase (ThiG) catalyses the formation of the thiazole moiety of thiamin-pyrophosphate from 1-deoxy-D-xylulose-5-phosphate (DXP), dehydroglycine and the sulfur carrier protein (ThiS), modified on its carboxy terminus as a thiocarboxylate (ThiS-thiocarboxylate). Thiazole biosynthesis is initiated by the formation of a ThiG/DXP imine, which then tautomerizes to an amino-ketone. In this paper we study the sulfur transfer from ThiS-thiocarboxylate to this amino-ketone and trap a new thioenolate intermediate. Surprisingly, thiazole formation results in the replacement of the ThiS-thiocarboxylate sulfur with an oxygen from DXP and not from the buffer, as shown by electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) using (18)O labeling of the 13C-, 15N-depleted protein. These observations further clarify the mechanism of the complex thiazole biosynthesis in bacteria.     

Magnesium Catalysis for the Hydroboration of Carbodiimides

A β‐diketiminato magnesium alkyl complex, [CH{C(Me)NDipp}₂}MgnBu] (Dipp=2,6‐iPr₂C₆H₃), was shown to be an effective pre‐catalyst for the first reported catalytic hydroboration of alkyl‐ and aryl‐substituted carbodiimides with pinacol borane (HBpin). The catalytic reactions proceed under mild conditions to afford the corresponding N‐borylated formamidine compounds in good yields. The reactions were observed to proceed through the intermediacy of magnesium amidinate and formamidinatoborate intermediates and an example of one of these latter species has been structurally characterised by an X‐ray diffraction analysis. Crucially, no formation of the N‐boryl formamidine products was observed in the absence of additional equivalents of the carbodiimide and HBpin substrates. This observation, supported by the evolution of a sigmoidal kinetic profile for the hydroboration of dicyclohexylcarbodiimide, has been rationalised as the consequence of an allosteric effect of the pinacol borane and carbodiimide on the magnesium formamidinatoborate intermediates.     

Silver phosphanes partnered with carborane monoanions: synthesis, structures and use as highly active Lewis acid catalysts in a hetero-Diels-Alder reaction

Four Lewis acidic silver phosphane complexes partnered with [1-closo-CB(11)H(12)](-) and [1-closo-CB(11)H(6)Br(6)](-) have been synthesised and studied by solution NMR and solid-state X-ray diffraction techniques. In the complex [Ag(PPh(3))(CB(11)H(12))] (1), the silver is coordinated with the carborane by two stronger 3c-2e B-H-Ag bonds, one weaker B-H-Ag interaction and a very weak Ag.C(arene) contact in the solid state. In solution, the carborane remains closely connected with the [Ag(PPh(3))](+) fragment, as evidenced by (11)B chemical shifts. Complex 2 [Ag(PPh(3))(2)(CB(11)H(12))](2) adopts a dimeric motif in the solid state, each carborane bridging two Ag centres. In solution at low temperature, two distinct complexes are observed that are suggested to be monomeric [Ag(PPh(3))(2)][CB(11)H(12)] and dimeric [Ag(PPh(3))(2)(CB(11)H(12))](2). With the more weakly coordinating anion [CB(11)H(6)Br(6)](-) and one phosphane, complex 3 [Ag(PPh(3))(CB(11)H(6)Br(6))] is isolated. Complex 4, [Ag(PPh(3))(2)(CB(11)H(6)Br(6))], has been characterised spectroscopically. All of the complexes have been assessed as Lewis acids in the hetero-Diels-Alder reaction of N-benzylideneaniline with Danishefsky's diene. Exceptionally low catalyst loadings for this Lewis acid catalysed reaction are required (0.1 mol %) coupled with turnover frequencies of 4000 h(-1) (quantitative conversion to product after 15 minutes using 3 at room temperature). Moreover, the reaction does not occur in rigorously dry solvent as addition of a substoichiometric amount of water (50 mol %) is necessary for turnover of the catalyst. It is suggested that a Lewis assisted Br?nsted acid is formed between the water and the silver. The effect of changing the counterion to [BF(4)](-), [OTf](-) and [ClO(4)](-) has also been studied. Significant decreases in reaction rate and final product yield are observed on changing the anion from [CB(11)H(6)Br(6)](-), thus demonstrating the utility of weakly coordinating carborane anions in organic synthesis.     

Mechanistic studies on pyridoxal phosphate synthase: the reaction pathway leading to a chromophoric intermediate

Two routes for the de novo biosynthesis of pyridoxal-5'-phosphate (PLP) have been discovered and reconstituted in vitro. The most common pathway that organisms use is dependent upon the activity of just two enzymes, known as Pdx1 (YaaD) and Pdx2 (YaaE) in bacteria. Pdx2 has been shown to have glutaminase activity and most likely channels ammonia to the active site of the PLP synthase subunit, Pdx1, where ribose-5-phosphate (R5P), glyceraldehyde-3-phosphate (G3P), and ammonia are condensed in a complex series of reactions. In this report we investigated the early steps in the mechanism of PLP formation. Under pre-steady-state conditions, a chromophoric intermediate (I320) is observed that accumulates upon addition of only two of the substrates, R5P and glutamine. The intermediate is covalently bound to the protein. We synthesized C5 monodeuterio (pro-R, pro-S) and dideuterio R5P and showed that there is a primary kinetic isotope effect on the formation of this intermediate using the pro-R but not the pro-S labeled isomer. Furthermore, it was shown that the phosphate unit of R5P is eliminated rather than hydrolyzed in route to intermediate formation and also that there is an observed C5-deuterium kinetic isotope effect on this elimination step. Interestingly, it was observed that the formation of the intermediate could be triggered in the absence of Pdx2 by the addition of high concentrations of NH4Cl to a preincubated solution of Pdx1 and R5P. The formation of I320 was also monitored using high-resolution electrospray ionization Fourier transform mass spectrometry and revealed a species of mass 34,304 Da (Pdx1 + 95 Da). These results allow us to narrow the mechanistic possibilities for the complex series of reactions involved in PLP formation.     

Calix[4]arene Schiff bases—potential ligands for fluorescent studies

A series of novel double-armed calix[4]arene derivatives incorporating imine substituents were synthesised from the Schiff-base reaction of 25,27-bis[2-[(1-formyl-2-phenyl)oxy]ethyl]-p-tert-butylcalix[4]arene (1) with the appropriate amine or hydrazone. All compounds were characterised by various spectroscopic and analytical techniques, and in three cases, by X-ray crystallographic studies. In the case of compound 2, inclusion compounds were synthesised with both m-xylene and dimethylformamide and their X-ray structures revealed these inclusion sites—between the pendant arms and in the upper cavity, respectively. In all cases, the pendant arms are bent away from each rather than adopting a face-to-face conformation.     

Stoichiometric and catalytic reactivity of the N-heterocyclic carbene ruthenium hydride complexes [Ru(NHC)(L)(CO)HCl] and [Ru(NHC)(L)(CO)H(eta2-BH4)] (L=NHC, PPh3)

Thermolysis of [Ru(AsPh3)3(CO)H2] with the N-aryl heterocyclic carbenes (NHCs) IMes (1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene), IPr (1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene) or the adduct SIPr.(C6F5)H (SIPr=1,3-bis(2,6-diisopropylphenyl)-4,5-dihydroimidazol-2-ylidene), followed by addition of CH2Cl2, affords the coordinatively unsaturated ruthenium hydride chloride complexes [Ru(NHC)2(CO)HCl] (NHC=IMes , IPr , SIPr ). These react with CO at room temperature to yield the corresponding 18-electron dicarbonyl complexes . Reduction of and [Ru(IMes)(PPh3)(CO)HCl] () with NaBH4 yields the isolable borohydride complexes [Ru(NHC)(L)(CO)H(eta2-BH4)] (, L=NHC, PPh3). Both the bis-IMes complex and the IMes-PPh3 species react with CO at low temperature to give the eta1-borohydride species [Ru(IMes)(L)(CO)2H(eta1-BH4)] (L=IMes , PPh3), which can be spectroscopically characterised. Upon warming to room temperature, further reaction with CO takes place to afford initially [Ru(IMes)(L)(CO)2H2] (L=IMes, L=PPh3) and, ultimately, [Ru(IMes)(L)(CO)3] (L=IMes , L=PPh3). Both and lose BH3 on addition of PMe2Ph to give [Ru(IMes)(L)(L')(CO)H2](L=L'=PMe2Ph; L=PPh3, L'=PMe2Ph). Compounds and have been tested as catalysts for the hydrogenation of aromatic ketones in the presence of (i)PrOH and H2. For the reduction of acetophenone, catalytic activity varies with the NHC present, decreasing in the order IPr>IMes>SIMes.     

Modelling of solid state voltammetry of immobilized microcrystals assuming an initiation of the electrochemical reaction at a three-phase junction

The electrochemical reduction of a solid compound characterized by mixed ionic/electronic conductivity, immobilized on an electrode surface and in contact with an electrolyte solution, has been studied theoretically. The uptake or expulsion of electrons and electrolyte cation is coupled to maintain electroneutrality and is assumed to obey Fick's law of diffusion. Starting with the fully oxidized species, the simultaneous uptake of cations and electrons will be possible at the three-phase junction only, where electrode, solid and electrolyte solution meet. From this point, electrons and cations diffuse perpendicularly into the crystal lattice. The reaction zone grows owing to the formation of the electronically and ionically conducting reduced product. Two- and three-dimensional models have been utilized to simulate the diffusion and the current flow in response to an applied potential step. The resulting chronoamperometric curves have been analyzed with the help of fitting procedures. Under certain conditions, a transition of the three-phase reaction to a pure two-phase reaction occurs. This transition to a two-phase condition is the reason that a number of equations for the exhaustive conversion are similar to those known for planar diffusion, for example. To illustrate this, and for a better understanding of the phenomena, concentration profiles are presented for different degrees of the reaction and for varied simulation conditions. It is demonstrated how geometrical properties like crystal shape (cuboid with x ≠ y ≠ z) and crystal size as well as physical properties, e.g. the diffusion coefficients, govern the electrochemical behavior of mixed ionic/electronic conductors and form the basis of the current-time functions. The numerical simulation of a two-dimensional semi-infinite model of the reaction at the three-phase junction gives results comparable to an algebraical approach. The finite-difference method turned out to be suitable to solve the problems arising from the three-dimensional and finite diffusion conditions and from different crystal shapes. Received: 24 November 1999 / Accepted: 22 February 2000     

Unexpected Migratory Insertion Reactions of M(alkyl)2 (M=Zn,Cd) and Diamidocarbenes

The electrophilic character of free diamidocarbenes (DACs) allows them to activate inert bonds in small molecules, such as NH₃ and P₄. Herein, we report that metal coordinated DACs also exhibit electrophilic reactivity, undergoing attack by Zn and Cd dialkyl precursors to afford the migratory insertion products [(6‐MesDAC‐R)MR] (M=Zn, Cd; R=Et, Me; Mes=mesityl). These species were formed via the spectroscopically characterised intermediates [(6‐MesDAC)MR₂], exhibiting barriers to migratory insertion which increase in the order MR₂ = ZnEt₂ < ZnMe₂ < CdMe₂. Compound [(6‐MesDAC‐Me)CdMe] showed limited stability, undergoing deposition of Cd metal, by an apparent β‐H elimination pathway. These results raise doubts about the suitability of diamidocarbenes as ligands in catalytic reactions involving metal species bearing nucleophilic ligands (M‐R, M‐H).