Nikolai Ignatiev

Applying metagenomics for the identification of bacterial cellulases that are stable in ionic liquids

Ionic liquids (ILs) are novel and chemically inert solvents for a wide range of reactions in organic synthesis and biocatalysis, and at least one of them is known to dissolve cellulose. ILs would provide novel options for cellulose degradation in homogenous catalysis if cellulases were sufficiently stable and active. By screening metagenomic libraries 24 novel cellulase clones were identified and tested for their performance in the presence of ILs. Most enzyme clones showed only very poor or no activities. Three enzyme clones (i.e. pCosJP10, pCosJP20 and pCosJP24) were moderately active and stable in the presence of 1-butyl-1-methyl-pyrrolidinium trifluoromethanesulfonate. The corresponding genes of these environment-derived cosmids were similar to known cellulases from Cellvibrio japonicus and a salt-tolerant cellulase from an uncultured microorganism, S. Voget, H. L. Steele and W. R. Streit, J. Biotechnol., 2006, 126, 26-36.1 The most active protein (CelA10) belonged to GH5 family cellulases and was active at IL concentrations of up to 30% (v/v). Recombinant CelA10 was extremely tolerant to 4 M NaCl and KCl. Furthermore improved cellulase variants of CelA10 were isolated in a directed evolution experiment employing SeSaM-technology. Analysis of these variants revealed that the N-terminal cellulose binding domain plays a pivotal role for IL resistance.

“Umpolung” at Boron by Reduction of [B(CN)4]− and Formation of the Dianion [B(CN)3]2−

Species containing nucleophilic boron, which, for example, convert protons into hydridic hydrogen atoms in B H bonds, are well-investigated. For instance, nucleophilic boron in metal borides (e.g., MgB2 ) and clusters (e.g., [B11H13] 2 ) are common, but in the remaining boron chemistry they are more scarce. 4] In 1924, Krause reported the first compound containing a nucleophilic boron atom. 6] In the reaction of sodium with triphenylboron in diethyl ether, formation of “NaBPh3” was postulated. Later this reaction was elucidated, and “NaBPh3” was identified as a mixture of dimers (e.g. Na2[Ph3B C6H5=BPh2], Na2[Ph3B C6H4 BHPh2], Scheme S1). In the reaction of sodium with triphenylboron [BPh3] [8] was indeed detected as intermediate and it was found that the radical anions [BAr3] are generally unstable. 9] Only the radical anion with mesityl ligands at boron can be isolated, because its dimerization is kinetically hindered. 11] The blue [Li([12]crown-4)2][BMes3] is stable up to 230 8C, and its structure was determined by single crystal X-ray diffraction. The dianions [B(C10H7)3] 2 [12, 13] and [BMes3] 2 [14] are scarcely characterized, and their existence is doubtful. Moreover, the claimed syntheses of [Ph2B] , [Bu2B] , and [Bu2BNEt3] [17, 18] turned out to be questionable. 19–22] Nevertheless during the last 20 years, besides “(C6H11)3PBH2Li” as intermediate, [23,24] some examples for nucleophilic boron species have been described (Scheme 1), which are fully characterized and have found use in organic synthesis. Herein we describe an unprecedented dianion in the salts M2[B(CN)3] (M = Li, Na, K), which contains nucleophilic boron in the formal oxidation state + 1. This tricyanoborate anion was obtained by reductive B C bond fission in [B(CN)4] by alkaline metals in liquid ammonia according to Equation (1).

The Bis(pentafluoroethyl)phosphinous Acid (C2F5)2POH

The industrial product (C(2)F(5))(3)PF(2) is transformed into the phosphinic acid chloride (C(2)F(5))(2)P(O)Cl, which reacts with an excess of Bu(3)SnH in a clean, multistep reaction to give the stannyl derivative (C(2)F(5))(2)POSnBu(3). Subsequent treatment with gaseous HBr leads to the formation of (C(2)F(5))(2)POH, which is isolated in 70 % yield. Besides (CF(3))(2)POH, bis(pentafluoroethyl)phosphinous acid, (C(2)F(5))(2)POH, represents the second known example of a phosphinous acid that is predicted by using density functional theory calculations at the B3PW91/6-311G(3d,p) level to be more stable than the phosphane oxide tautomer, the energy difference being 11.7 kJ mol(-1). Only the phosphinous acid isomer is detectable in the gas phase and in solution. However, investigations of the neat liquid reveal a temperature-dependent tautomeric equilibrium with the phosphane oxide isomer (C(2)F(5))(2)P(O)H, which is characterized by vibrational and multinuclear NMR spectroscopic methods in combination with quantum-chemical calculations.

Development of low viscous ionic liquids: the dependence of the viscosity on the mass of the ions

Straightforward syntheses of selected non-deuterated, partially deuterated, and fully deuterated room temperature ionic liquids (ILs) with the anions tris(pentafluoroethyl)trifluorophosphate, tetracyanoborate, and methyl sulfate were developed. The viscosity and the density of these ionic liquids were measured at various temperatures. The dissociation rate of the ionic liquid, the sizes of the cation and the anion, and the nature of the inter-ionic interaction are not influenced by deuteration, but viscosity is. All deuterated ionic liquids possess a higher viscosity and density in comparison to non-deuterated ones. The explanation of this phenomenon is given and discussed based on the modified Stokes-Einstein equation with a parameter which reflects the mass of the diffused particles. This new knowledge supports the recent development of novel low viscous inert ionic liquids which are of particular interest for the application in energy conversion devices. Deuterated ionic liquids are valuable media for the investigation of chemical reactions, natural products, and bio-materials by means of the NMR spectroscopy.

Synthesis and Characterization of Bis[bis(pentafluoroethyl)phosphinyl]imides, M+N[(C2F5)2P(O)]2-, M = H, Na, K, Cs, Ag, Me4N†

A convenient synthesis and a full characterization of the strong acid bis[bis(pentafluoroethyl)phosphinyl]imide and some of its salts M (+)N[(C 2F 5) 2P(O)] 2 (-), M = Na, K, Cs, Ag, Me 4N, are presented. Their thermal (mp, T dec.) and spectroscopic (IR, Raman, NMR) properties are discussed. A single crystal structure of [Me 4N][N{P(O)(C 2F 5) 2} 2] has been obtained, and the structural parameters of the anion are compared with the results of quantum-chemical calculations. The observed properties are comparable to those of bis((trifluoromethyl)sulfonyl)imide and their derivatives.

ω-(Bis(trifluoromethyl)amino)alkane-1-sulfonates: synthesis and mass spectrometric study of the biotransformation products

In search of fluorinated functional groups which could undergo defluorination, and therefore be included in novel non-polluting fluorinated surfactants, omega-(bis(trifluoromethyl)amino)alkane-1-sulfonates (BTFMA-AS) with a homologue distribution from seven to thirteen methylene groups were synthesized and investigated for aerobic biodegradation applying both a standardized test and a fixed-bed bioreactor (FBBR). These compounds were prepared as part of a screening study for potentially mineralizable fluorinated endgroups.Application of hybrid triple quadrupole-linear ion trap mass spectrometry (QqQ(LIT)-MS) coupled to high-performance liquid chromatography (HPLC) allowed the tracking of primary degradation as well as the detection and structural elucidation of biotransformation intermediates. An understanding of the fragmentation pathway of the test compounds allowed selective precursor ion scans to reveal the presence of stable fluorinated metabolites. Structures were confirmed by enhanced product ion scans and MS(3) scans in the linear ion trap mode.The primary biodegradation rate and the extent of biodegradation were found to be chain-length dependent, with higher homologues being completely primarily degraded within 10 days. For the first time, two simultaneous metabolic pathways for substituted linear alkane-1-sulfonates were discovered: Desulfonation, oxidation to a carboxylic acid and subsequent chain-length shortening by beta-oxidation dominated the metabolism. This pathway resulted in the formation of 3-(bis(trifluoromethyl)amino)propionic acid and bis(trifluoromethyl)aminoacetic acid, which showed recalcitrance in this experiment. Oxidation of the alkyl chain to the respective carbonyl derivative represents the minor pathway. Only the long-chain homologues of these oxidized species were partially degraded; the short-chain homologues were not attacked.