Donor Promiscuity of a Thermostable Transketolase by Directed Evolution: Efficient Complementation of 1‐Deoxy‐d‐xylulose‐5‐phosphate Synthase Activity

Enzymes catalyzing asymmetric carboligation reactions typically show very high substrate specificity for their nucleophilic donor substrate components. Structure‐guided engineering of the thermostable transketolase from Geobacillus stearothermophilus by directed in vitro evolution yielded new enzyme variants that are able to utilize pyruvate and higher aliphatic homologues as nucleophilic components for acyl transfer instead of the natural polyhydroxylated ketose phosphates or hydroxypyruvate. The single mutant H102T proved the best hit toward 3‐methyl‐2‐oxobutyrate as donor, while the double variant H102L/H474S showed highest catalytic efficiency toward pyruvate as donor. The latter variant was able to complement the auxotrophic deficiency of Escherichia coli cells arising from a deletion of the dxs gene, which encodes for activity of the first committed step into the terpenoid biosynthesis, offering the chance to employ a growth selection test for further enzyme optimization.     

A Water‐Bridged H‐Bonding Network Contributes to the Catalysis of the SAM‐Dependent C‐Methyltransferase HcgC

[Fe]‐hydrogenase hosts an iron‐guanylylpyridinol (FeGP) cofactor. The FeGP cofactor contains a pyridinol ring substituted with GMP, two methyl groups, and an acylmethyl group. HcgC, an enzyme involved in FeGP biosynthesis, catalyzes methyl transfer from S ‐adenosylmethionine (SAM) to C3 of 6‐carboxymethyl‐5‐methyl‐4‐hydroxy‐2‐pyridinol ( 2 ). We report on the ternary structure of HcgC/S ‐adenosylhomocysteine (SAH, the demethylated product of SAM) and 2 at 1.7 Å resolution. The proximity of C3 of substrate 2 and the S atom of SAH indicates a catalytically productive geometry. The hydroxy and carboxy groups of substrate 2 are hydrogen‐bonded with I115 and T179, as well as through a series of water molecules linked with polar and a few protonatable groups. These interactions stabilize the deprotonated state of the hydroxy groups and a keto form of substrate 2 , through which the nucleophilicity of C3 is increased by resonance effects. Complemented by mutational analysis, a structure‐based catalytic mechanism was proposed.     

Improved Synthesis and Reaction of 11‐Chloroneocryptolepines,Strategic Scaffold for Antimalaria Agent,and Their 6‐Methyl Congener from Indole‐3‐carboxylate

Various 11‐chloro‐5‐methyl‐5H‐indolo[2,3‐b]quinolines (neocryptolepines) with different substituents on the quinoline ring, key intermediates for antimalaria agents, are prepared from the substituted N‐methylanilines, easily accessible by the N‐methylation of anilines, and indole‐3‐carboxylate as a counterpart. This protocol is benign in terms of the reduced number of steps to reach the target, compared with the known method using anilines, and easy product purification. Alternatively, their 6‐methyl congener is prepared by N‐methylation of the indole moiety of 2‐arylaminoindole‐3‐carboxylate followed by successive cyclization and chlorination. 11‐Chloroneocryptolepines are found more reactive than their 6‐methyl congener in the nucleophilic substitution at the C11 position.     

Synthesis and NMR characteristics of N‐acetyl‐4‐nitro,N‐acetyl‐5‐nitro,N‐acetyl‐6‐nitro and N‐acetyl‐7‐nitrotryptophan methyl esters

N‐acetyl‐4‐nitrotryptophan methyl ester (2), N‐acetyl‐5‐nitrotryptophan methyl ester (3), N‐acetyl‐6‐nitrotryptophan methyl ester (4) and N‐acetyl‐7‐nitrotryptophan methyl ester (5) were synthesized through a modified malonic ester reaction of the appropriate nitrogramine analogs followed by methylation with BF₃‐methanol. Assignments of the ¹H and ¹³C NMR chemical shifts were made using a combination of ¹H–¹H COSY, ¹H–¹³C HETCOR and ¹H–¹³C selective INEPT experiments. Copyright © 2008 Crown in the right of Canada. Published by John Wiley & Sons, Ltd     

The Catalytic Mechanism of the Class C Radical S‐Adenosylmethionine Methyltransferase NosN

S ‐Adenosylmethionine (SAM) is one of the most common co‐substrates in enzyme‐catalyzed methylation reactions. Most SAM‐dependent reactions proceed through an S_N2 mechanism, whereas a subset of them involves radical intermediates for methylating non‐nucleophilic substrates. Herein, we report the characterization and mechanistic investigation of NosN, a class C radical SAM methyltransferase involved in the biosynthesis of the thiopeptide antibiotic nosiheptide. We show that, in contrast to all known SAM‐dependent methyltransferases, NosN does not produce S ‐adenosylhomocysteine (SAH) as a co‐product. Instead, NosN converts SAM into 5′‐methylthioadenosine as a direct methyl donor, employing a radical‐based mechanism for methylation and releasing 5′‐thioadenosine as a co‐product. A series of biochemical and computational studies allowed us to propose a comprehensive mechanism for NosN catalysis, which represents a new paradigm for enzyme‐catalyzed methylation reactions.     

Homology Modeling and Molecular Docking Studies of (S)‐Scoulerine 9‐O‐Methyltransferase from Coptis chinensis

(S)‐Scoulerine 9‐O‐methyltransferase (SMT), belonging to the S‐adenosyl‐L‐methionine (SAM)‐dependent O‐methyltransferase family, is an essential enzyme in the berberine biosynthetic pathways. In order to study the interactions of SMT with its substrate and further to understand the catalytic mechanism and substrate specificity, a three dimensional model of SMT from Coptis chinensis was constructed by homology modeling using the crystal structure of caffeic acid/5‐hydroxyferulic acid 3/5‐O‐methyltransferase (COMT) as a template. The three dimensional structure of SMT, which was mainly composed of α‐helices and some β‐sheets, was similar to that of COMT. In contrast with COMT, the non‐conserved residues in the substrate binding pocket of SMT might be responsible for their differences in the substrate specificity. Val119 and Asp254 in SMT were the key residues for orienting substrate for methylation as both residues had H‐bonds with (S)‐scoulerine. The methylation of (S)‐scoulerine involved deprotonation of the 9‐hydroxyl group by His253 and Asp254 in SMT followed by a nucleophilic attack on the SAM‐methyl resulting in the product, (S)‐tetrahydrocolumbamine.     

Structure analysis of some α‐(n‐methyl‐n‐formylaminomethyl)‐quinolines

The ¹H and ¹³C nmr spectra of the rotational isomers 3a and 3b of 6‐N‐methyl‐N‐formylaminomefhyl)‐thioquinanthrene were completely assigned with a combination of 1D and 2D nmr techniques. The key‐parts of this methodology were long‐range proton‐carbon correlations and NOE experiments with N‐methyl‐N‐formylaminomethyl substituent. The X‐ray study of 4‐methyl‐2‐N‐methyl‐N‐formylaminomethyl)quinoline 4a as well as ¹H and ¹³C nmr spectra show that N‐methyl‐N‐formylaminomethyl substituent in 4a and 4b has a different steric arrangement than the same substituent in 3a and 3b .     

Synthesis and Selected Transformations of 1H‐Imidazole 3‐Oxides Derived from Amino Acid Esters

A series of new optically active 1H‐imidazole 3‐oxides 5 with a substituted acetate group at N(1) as the chiral unit were prepared by the reaction of α‐(hydroxyimino) ketones, α‐amino acid methyl esters, and formaldehyde. In an analogous reaction, ethyl 2‐(hydroxyimino)‐3‐oxobutyrate and 1,3,5‐trialkylhexahydro‐1,3,5‐triazines gave 3‐oxido‐1H‐imidazole‐4‐carboxylates 14 , which easily rearranged into the 2‐oxo derivatives 15 . Selected examples of N‐oxides 5 could be transformed into the corresponding 2,3‐dihydro‐1H‐imidazole‐2‐thione derivatives 10 via a ‘sulfur‐transfer reaction’, and the reduction of the histidine derivative 5i with Raney‐Ni yielded the optically active 2,3‐bis(imidazolyl)propanoate 12 . Furthermore, reaction of the (1H‐imidazol‐1‐yl)acetates with primary amines yielded the corresponding acetamides.     

Stereochemistry of the Methyl Group in (R)‐3‐Methylitaconate Derived by Rearrangement of 2‐Methylideneglutarate Catalysed by a Coenzyme B12‐Dependent Mutase

2‐Methylideneglutarate mutase is an adenosylcobalamin (coenzyme B₁₂)‐dependent enzyme that catalyses the equilibration of 2‐methylideneglutarate with (R)‐3‐methylitaconate. This reaction is believed to occur via protein‐bound free radicals derived from substrate and product. The stereochemistry of the formation of the methyl group of 3‐methylitaconate has been probed using a `chiral methyl group'. The methyl group in 3‐([²H₁,³H]methyl)itaconate derived from either (R)‐ or (S)‐2‐methylidene[3‐²H₁,3‐³H₁]glutarate was a 50 : 50 mixture of (R)‐ and (S)‐forms. It is concluded that the barrier to rotation about the C−C bond between the methylene radical centre and adjacent C‐atom in the product‐related radical [^.CH₂CH(⁻O₂CC=CH₂)CO₂⁻] is relatively low, and that the interaction of the radical with cob(II)alamin is minimal. Hence, cob(II)alamin is a spectator of the molecular rearrangement of the substrate radical to product radical.     

Synthesis and Pharmacological Evaluation of a Novel Series of 2‐((2‐Aryl thiazol‐4‐yl)methyl)‐5‐(alkyl/alkylnitrile thio)‐1,3,4‐oxadiazole Derivatives as Possible Antifungal Agents

In the present investigation, a novel series of 2‐[(2‐arylthiazol‐4‐yl)methyl]‐5‐(alkyl/alkylnitrile thio)‐1,3,4‐oxadiazole derivatives were synthesized by cyclo‐condensation of 2‐(2‐substituted thiazol‐4‐yl)aceto hydrazide with carbon disulfide followed by S‐alkylation with alkyl halide in dry acetone. All the newly synthesized compounds were characterized by spectral (IR, ¹H NMR, ¹³C NMR, mass, and elemental analysis) methods. The title compounds were screened for in vitro antifungal activity and most of the synthesized compounds show moderate to good antifungal activity.     

Methylation of Arenols through Ni‐catalyzed C—O Activation with Methyl Magnesium Bromide

Direct alkylation of arenols with alkyl organometallic reagents has never been approached. Herein we reported the first successful example of nickel‐catalyzed methylation of arenols with methyl Grignard reagents to construct C(sp²)‐C(sp³) bond under mild conditions. The transformation was compatible with broad substrate scope of 2‐naphthol derivatives. Benzyl alcohol and biphenols were also suitable substrates for this methylation.     

Synthesis and properties of some 2,3‐disubstituted 6‐fluoro‐7‐(4‐methyl‐1‐piperazinyl)quinoxalines

The 2,3‐disubstituted 6‐fluoro‐7‐(4‐methyl‐1‐piperazinyl)‐quinoxalines ( 3–11 ) were synthesized for bioassay via reaction of 1.2‐diamino‐4‐fluoro‐5‐(4‐methyl‐1‐piperazinyl)benzene (2) with the appropriate 1,2‐dicarbonyl compounds. However, none of the tested compounds 3–11 showed significant in vitro activ ity against E. coli ATCC11229, S. aureus ATCC6538 and C.albicans SATCC10231.     

5‐Methyl‐2‐thiouracil

The molecular structure of the title compound, also known as 2‐thio­thymine [systematic name: 2,3‐di­hydro‐5‐methyl‐2‐thioxopyrimidin‐4(1H)‐one], C₅H₆N₂OS, is similar to that of thymine, with only small changes in the ring structure, apart from a significant difference at the substitution site [S=C = 1.674 (1) Å]. The mol­ecules are connected by hydrogen bonds, with N—H?O = 2.755 (2) Å and N—H?S = 3.352 (1) Å. The hydrogen‐bond network is different from that in thymine, since it involves all the donor and acceptor atoms.     

2,3‐Dihydro‐3‐methyl‐2‐nitrimino‐1,3‐thiazole

The title compound {alternatively, 3‐methyl‐2‐[oxido(oxo)hydrazono]‐2,3‐dihydro‐1,3‐thiazole}, C₄H₅N₃O₂S, was obtained by methyl­ation of N‐(2‐thia­zolyl)­nitr­amine. The molecule lies on a mirror plane and the thia­zole ring is planar, regular in shape and aromatic. The S atom participates in the aromatic sextet via an electron pair on the 3p_z orbital. In the crystal, the mol­ecules are arranged in parallel layers, bound to each other by weak C—H?O and C—H?N hydrogen bonds and by S?O dipolar interactions, with an interlayer separation of 3.23 Å.     

Novel Synthesis and Antimicrobial Activity of 3‐Substituted 5‐bromo‐7‐methyl‐1,2,4‐triazolo‐[3,4‐b]‐benzothiazoles

4‐Methyl acetanilide ( 1 ) on treatment with bromine in acetic acid, followed by hydrolysis with dilute HCl/NaOH solution, yielded 2‐bromo‐4‐methyl aniline ( 2 ), which on treatment with sodium thiocyanate in acetic acid afforded 2‐amino‐4‐bromo‐6‐methyl benzothiazole ( 3 ). Compound 3 in ethylene glycol was heated at 150°C with 80% hydrazine hydrate to get 4‐bromo‐2‐hydrazino‐6‐methyl benzothiazole ( 4 ). This hydrazino compound 4 on heating with formic acid for 3 h yielded 4‐bromo‐2‐hydrazinoformyl‐6‐methyl benzothiazole ( 5 ). Same compound 4 when heated independently with formic acid for 6 h/urea for 3 h/carbon disulfide in alkali afforded 5‐bromo‐7‐methyl ( 6 )/5‐bromo‐3‐hydroxy‐7‐methyl ( 7 )/5‐bromo‐3‐mercapto‐7‐methyl ( 8 )‐1,2,4‐triazolo‐[3,4‐b]‐benzothiazoles, respectively. Compound 4 on heating with acetic acid/acetic anhydride gave acetyl benzothiazolyl derivative 9 , which on cyclization with orthophosphoric acid yielded 5‐bromo‐3,7‐dimethyl‐1,2,4‐triazolo‐[3,4‐b]‐benzothiazole ( 10 ). All these newly synthesized compounds were screened for antimicrobial activity against Escherichia coli (Gram ?ve), Bacillus subtilis (Gram +ve), Erwinia carotovora, and Xanthomonas citri using ampicillin, streptomycin, and penicillin as a standard for comparison.     

New approach to l'C‐modified riboside scaffold via stereoselective functionalization of D‐(+)‐ribonic‐y‐lactone

We describe the preparation and spectroscopic properties of a novel class of nucleoside analogues in which a phenyl sulfonyl methylene group is attached to the 1′‐carbon atom of P‐D‐ribofuranose. The glyco‐sylation of 5‐O‐(tert‐butyldiphenylsilyl)‐2,3‐O‐isopropylidene‐D‐ribofuranolactone lb with phenyl methyl‐lithium sulfone in THF at ?60° C afforded 5‐O‐(tert‐butyldiphenylsilyl)‐1′‐(benzenesulfonylmethylene)‐2′,3′‐O‐isopropylidene‐α‐D‐ribofuranose 2b . When subjected to deoxydative reaction conditions with boron trifluoride etherate in the presence of triethylsilane at ?45° C, lactol 2b was converted into 2′,3′‐O‐isopro‐pylidene‐1′‐deoxy‐1′‐(benzenesulfonylmethylene)‐β‐D‐ribofuranose 4b with excellent stereocontrol over the anomeric carbon in moderate yield. This method has the potential for the development of a wider array of useful probes derived from 1′‐deoxy‐β‐D‐ribofuranose for nucleic acid research and for antisense therapeutic agents through further functionalization of the coupled sulfonyl group.     

Reactions of (1E)‐Buta‐1,3‐dien‐1‐yl Acetate with Diazocarbonyl Compounds

Carbene transfer to appropriate substrates is a highly versatile tool for the construction of carbon frameworks with increased functional and structural complexity. In this study, some novel cyclopropane derivatives were synthesized via carbenoid reactions and their further reactivities were investigated. (1E)‐Buta‐1,3‐dien‐1‐yl acetate was reacted with four different diazocarbonyl compounds, ethyl diazoacetate, dimethyl diazomalonate, 1‐diazo‐1‐phenylpropan‐2‐one, and methyl (3E)‐2‐diazo‐4‐phenylbut‐3‐enoate, in the presence of two catalysts. All synthesized substituted cyclopropanes were obtained chemoselectively with respect to less‐hindered C?C bonds. Under the applied conditions, while cyclopropanes 7a and 7d underwent further reactions, cyclopropanes 7b and 7c were stable enough. Cyclopropanes 7a and an additional equivalent of ethyl diazoacetate yielded polyfunctionalized cyclohexenes. Cyclopropanes from methyl (3E)‐2‐diazo‐4‐phenylbut‐3‐enoate yielded polyfunctionalyzed cycloheptadiene isomers by Cope rearrangement.     

Decomposition of model alkoxyamines in simple and polymerizing systems. II. Diastereomeric N‐(2‐methylpropyl)‐N‐(1‐diethyl‐phosphono‐2,2‐dimethyl‐propyl)‐aminoxyl‐based compounds

Thermal reactions of the alkoxyamine diastereomers DEPN‐R′ [DEPN: N‐(2‐methylpropyl)‐N‐(1‐diethylphosphophono‐2,2‐dimethyl‐propyl)‐aminoxyl; R′: methoxy‐carbonylethyl and phenylethyl] with (R,R) + (S,S) and (R,S) + (S,R) configurations have been investigated by ¹H NMR at 100 °C. During the overall decay the diastereomers interconvert, and an analytical treatment of the combined processes is presented. Rate constants are obtained for the cleavage and reformation of DEPN‐R′ from NMR, electron spin resonance, and chemically induced dynamic nuclear polarization experiments also using 2,2,6,6‐tetramethylpiperidinyl‐1‐oxyl (TEMPO) as a radical scavenger. The rate constants depend on the diastereomer configuration and the residues R′. Simulations of the kinetics observed with styrene and methyl methacrylate containing solutions yielded rate constants for unimeric and polymeric alkoxyamines DEPN‐(M)_n‐R′. The results were compatible with the known DEPN mediation of living styrene and acrylate polymerizations. For methyl methacrylate the equilibrium constant of the reversible cleavage of the dormant chains DEPN‐(M)_n‐R′ is very large and renders successful living polymerizations unlikely. Mechanistic and kinetic differences of DEPN‐ and TEMPO‐mediated polymerizations are discussed. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 3264–3283, 2002     

Rhodium‐Catalyzed 1,4‐Addition of Lithium 2‐Furyltriolborates to Unsaturated Ketones and Esters for Enantioselective Synthesis of γ‐Oxo‐Carboxylic Acids By Oxidation of the Furyl Ring with Ozone

Rhodium‐catalyzed 1,4‐addition of lithium 5‐methyl‐2‐furyltriolborate ([ArB(OCH₂)₃CCH₃]Li, Ar=5‐methyl‐2‐furyl) to unsaturated ketones to give β‐furyl ketones was followed by ozonolysis of the furyl ring for enantioselective synthesis of γ‐oxo‐carboxylic acids. [Rh(nbd)₂]BF₄ (nbd=2,5‐norbornadiene) chelated with 2,2′‐bis(diphenylphosphino)‐1,1′‐binaphthyl (binap) or 2,3‐bis(diphenylphosphino)butane (chiraphos) gave high yields and high selectivities in a range of 91–99 % ee at 30 °C in a basic dioxane/water solution. The corresponding reaction of unsaturated esters, such as methyl crotonate, had strong resistance under analogous conditions, but the 1,4‐adduct was obtained in 70 % yield and with 94 % ee when more electron‐deficient phenyl crotonate was used as the substrate.     

Synthesis and Characterization of Novel 1‐Methyl‐3‐(4‐phenyl‐4H‐1,2,4‐triazol‐3‐yl)‐1H‐indazole Derivatives

A series of novel 1‐methyl‐3‐(4‐phenyl‐4H‐1,2,4‐triazol‐3‐yl)‐1H‐indazoles was synthesized in three steps from 5‐(1‐methyl‐1H‐indazol‐3‐yl)‐4‐phenyl‐2H‐1,2,4‐triazole‐3(4H)‐thiones. 5‐(1‐Methyl‐1H‐indazol‐3‐yl)‐4‐phenyl‐2H‐1,2,4‐triazole‐3(4H)‐thiones were converted into 1‐methyl‐3‐(5‐(methylsulfonyl)‐4‐phenyl‐4H‐1,2,4‐triazol‐3‐yl)‐1H‐indazoles upon methylation followed by treatment with aq. KMnO₄. The reaction of 1‐methyl‐3‐(5‐(methylsulfonyl)‐4‐phenyl‐4H‐1,2,4‐triazol‐3‐yl)‐1H‐indazoles with Raney nickel resulted in desulphonylation to afford corresponding 1‐methyl‐3‐(4‐phenyl‐4H‐1,2,4‐triazol‐3‐yl)‐1H‐indazoles. All the new synthesized compounds were characterized by spectral techniques.