Search

An NMR method is described which should provide a rapid means for determining and assigning antiparallel sheets and helices in small proteins. It begins by locating apparent NOESY crosspeaks which suggest the presence of the secondary structure; this is followed by searches for MCD patterns (Englander & Wand (1987) Biochemistry 22, 5953) which are characteristic of these structures. As a result, only spin-systems of the amino acids within the secondary structure need to be defined. A triple-stranded, antiparallel sheet and a helix have been found and assigned for both alpha-cobratoxin and the scorpion toxin AaH III.

RESUME La mucoviscidose est la maladie genetique letale la plus repandue dans la population caucasienne. Les efforts considerables accomplis dans le domaine de la therapie geniquen’ont pas pour l’instant apporte de solution satisfaisante. A partir de l’observation d’un cas clinique surprenant, nous avons ebauche une approche consistant, par l’utilisation d’un traitement de type anti-cancereux, a activer l’expression de proteines de la famille des transporteurs ABC, tres proches de la proteine deficiente CFTR, et pouvant restaurer des fonctions communes.

The first iridium-catalyzed isomerization of an exocyclic into an endocyclic double bond is described. A mechanism is proposed for this reaction. Crabtree's catalyst thus allows the migration of a double bond that does not occur under classical conditions.

In a recent study, the dynamics of large magnetization subject to a RF feedback field from the detection circuit of the probe was investigated both theoretically and experimentally. A simple model including non-linear modification of the Bloch equations allowed us to predict the occurrence of repeated bursts of NMR transverse magnetization during evolution (named self-sustained maser pulses). However, the decay of the amplitude of these repeated maser pulses with a characteristic time 4T1 predicted by this simple model could not be observed and the experiments showed a regime of non-attenuated self-sustained maser pulses. This was interpreted as a direct consequence of B0 inhomogeneities. In this paper we present new experimental evidence that support this assumption and that further demonstrate the role of B0 inhomogeneity. To cite this article : A. Louis-Joseph et al., C. R. Chimie 7 (2004).

Ras1p and Ras2p, from Saccharomyces cerevisiae, are GTP-binding proteins that are essential elements in the signaling cascade leading to the activation of adenylyl cyclase. To overcome proteolytic activities that have hampered biochemical studies of Ras1p so far, its gene was genetically modified after which full-length Ras1p could be obtained. The interaction of famesylated and unprenylated Ras1p with guanine nucleotides, guanine nucleotide exchange factors, GTPase activating proteins, and adenylyl cyclase was compared to Ras2p and human Ha-Ras interactions. Farnesylation of Ras proteins was demonstrated to be a prerequisite for membrane-bound guanine nucleotide exchange factor dependent formation of Ras-GTP complexes, and for efficient Ras-mediated adenylyl cyclase activation. To relate observed functional deviations with sequence differences between Ras1p and Ras2p, which reside almost exclusively within the hypervariable region, truncated versions and chimaeras of the Ras proteins were made. The characteristics of these constructs point to the presence of the hypervariable region of yeast Ras proteins for an efficient activation of adenylyl cyclase. The importance of the latter was confirmed as inhibition of the activation of adenylyl cyclase by an isolated farnesylated hypervariable region of Ras2p could be shown. This strongly suggests that the hypervariable region of Ras proteins can interact directly with adenylyl cyclase.

By coupling the toxic restriction endoribonuclease RegB, from the bacteriophage T4, to the prokaryotic T7 and the eukaryotic GAL1 promoters, we constructed a two-function plasmid called pTOXR-1. This plasmid is a zero-background cloning vector. It allows an efficient positive selection of recombinant plasmids without the need to completely digest, dephosphorylate, or purify the vector prior to the ligation step. The pTOXR-1 positive selection system requires no special Escherichia coli strains, no special culture media, and no addition of inducer to the selective plates. In addition, since this vector carries all signals required for both prokaryotic and eukaryotic expression, it allows the overproduction of heterologous proteins, fused to a polyhistidine tag, in the bacterium E. coli and in the yeast Saccharomyces cerevisiae from a single plasmid. Hence, this vector may be a useful time-saving tool for one-step cloning and versatile protein expression in bacteria and yeast.

A highly functionalised precursor of the antifeedant natural product Clerodin has been synthesised with good diastereocontrol. Key steps include a three-component version of the Vaultier tandem sequence, and an oxidative decarboxylation with a simple experimental procedure.

International audience; Hydrazone 30, precursor of a taxoid C-ring, was synthesized in 7 steps from commercial 2-methyl-1,3-cyclohexanedione. An enantioselective approach to 30 was also investigated, using a reduction of diketone 11 with bakers' yeast to introduce enantioselectivity. During the alkyne isomerization step, anionic cyclizations of alkoxides and enolates onto the triple bond were observed, and a thorough study of these reactions is reported here. © Wiley-VCH Verlag GmbH, 2000.

The asymmetric isomerisation of 5,7-dibenzyloxy-6-(trialkylsilyloxy)cyclohepta-1,3-diene 1 to the corresponding 1,6-dibenzyloxy-7-(trialkylsilyloxy)cyclohepta-1,3-diene 2 can be accomplished at 90°C in a 75/25 mixture of toluene and DME in the presence of a [Rh(BIPNOR)(cod)] + catalyst with 92% ee. A much lower ee is observed with BINAP. In both cases, the observed ee's increase with temperature. A mechanism involving an η 5 -pentadienyl–rhodium complex is proposed.

The aldol reaction between methyl (methyl-3-O-benzyl-2-O-methoxymethyl-α-D-xylofurano-side)uronate and D-(R)-glyceraldehyde acetonide, promoted by cerium(III) chloride, led to three diastereoisomers in good yield and high level of stereoselectivity at the newly created quaternary center of the furanoside. Under acidic conditions, the aldol derivatives undergo transketalization to afford different bicyclic ketal isomers. This reaction was used as the key step in the synthesis of a functionalized bicyclic core of the zaragozic acids.

Cycloaddition of chiral nitroso derivatives with cyclohepta-1,3-diene gave one single stereoisomer with an excellent selectivity. The structures including absolute configurations have been assigned by spectroscopy and X-ray crystallography. These studies have been applied to the total synthesis of the naturally occurring calystegine B 2 .

Applied to the new iron(II) compound Fe(t-msbpy)2(NCS)2 (t-msbpy=4-methyl-4′-trans-styryl-2,2′-bipyridine), a novel strategy based on the so-called '‘ligand-driven light-induced spin change’' (or LD-LISC) effect, which consists in triggering the metal ion electronic spin-state crossover in a complex by a photochemical modification of the ligand, allowed this photomagnetic effect to be observed for the first time at room temperature. The compound, studied in CH3CN or CD3CN, was found to be initially nearly diamagnetic (S=0) by means of NMR experiments. Then, irradiation of a solution to the photostationary state with λexc=334 nm was shown, by using UV-VIS spectrometry, to result in the trans→cis isomerization of the styryl substituent of msbpy and in the subsequent S=0 (low spin)→S=2 (high spin) conversion of a significant fraction of the iron(II) ions.

In this Letter, the lagged cross-moments and cross-spectra of the Bloch equations driven by a radiofrequency field whose amplitude is fluctuating randomly with Markovian statistics are calculated. Exact expressions for the lineshape are derived in the special case of a Markovian binary noise. The limiting case of white noise is investigated and compared with results found in the literature.

The osmylation of dienic sulfone 4, a potential key intermediate in forskolin 1 and polyhydroxylated drimanes or labdanes derivatives, occurred exclusively on the Δ6–7 double bound and preferentially from the α-face of the molecule (40–100% de). The influence of the substituent at C-1 on the stereoselectivity of the reaction is studied.

A synthesis of the highly functionalized bicyclic core of the zaragozic acids is described, in which the key step involves the construction of the C4 quaternary carbon center by a CeCl3-promoted aldol reaction between the α-D-xylofuranuronic acid derivative 2 and D-(R)-glyceraldehyde acetonide.

The direct microbial 3β-hydroxylation of drimanic terpenes by filamentous fungi, followed by a transfer of the functionalization from the 3β- to the 1α-position has been used for preparing derivatives of antifeedant terpenic compounds bearing an additional 1α-hydroxyl group. The semisynthesis of 1α-hydroxypolygodial is described as an example.

Two different reactions were experimented for the construction of the eight-membered B-ring of taxane skeleton through the formation of the C9C10 bond. A SmI2 promoted radical reaction on seco-alkynyl aldehydes 11cis or 11trans failed. A Nozaki/Kishi reaction carried out on the seco-iodovinyl aldehyde 12trans led, through an unprecedented intramolecular SN2′ reaction, to the “abnormally” cyclised product 14, the structure of which has been confirmed by X-ray crystallography.

Human angiogenin is a member of the pancreatic ribonuclease superfamily that induces blood vessel formation. Its three-dimensional solution structure has been determined to high resolution by heteronuclear NMR spectroscopy. 30 structures were calculated, based on a total of 1441 assigned NOE correlations, 64 coupling constants and 50 hydrogen bonds. The backbone atomic rms difference from the mean coordinates is 0.067 +/- 0.012 nm and 0.13 nm from the previously determined crystal structure. The side-chain of Gln117 was found to obstruct the active site as observed in the crystal state. There was no evidence of an alternative open form of angiogenin, although two sets of chemical shifts were observed for some residues, mainly around the active site and in the C-terminal segment. The topology of the ribonucleolytic active site is described with a particular emphasis on the conformation and protonation of active-site His residues. The side-chain of His114 adopts two main conformations in solution. In contrast to pancreatic ribonuclease A, His13 was shown to be more basic than His114, with pKa values of 6.65 and 6.05 respectively. The His47 residue is located in an environment very resistant to protonation with a pKa lower than 4.

Intramolecular Diels-Alder reactions have been used for the synthesis of cis-hydroisobenzofuranones 23 or 21, which represents a potential precursor of taxol C-ring. Easy preparation of β-ketoesters 7a and 7b is also described.

The first synthesis of a functionalized tricyclic skeleton of vinigrol is described. The key step involved an anionic oxy-Cope rearrangement of bicyclic allylic alcohol 18, readily prepared by highly stereoselective addition of vinyl magnesium chloride to the hydroxy enone 15b. Introduction of the tertiary hydroxy group at carbon 8a was achieved by an unexpected hydration of 30 with aqueous trifluoroacetic acid.

Stannylcupration of enyne 5 was performed with the homostannylcuprate (Bu 3 Sn) 2 CuCNLi 2 to deliver in a stereoselective way the distal dienylstannane 7 in 77% yield. This reaction was reproducible only when methanol was added to the cuprate solution. In comparison, hydroboration or hydrozirconation reactions failed in this case to produce the corresponding boron or zirconium derivative.

A new N-channel (or multichannel) deconvolution method of 1D and 2D NMR spectra is presented based on the Maximum Entropy method. In the general case of spectra exhibiting several lines of various fine structures, the method allows simultaneous deconvolution of all lines. While deconvolution is performed, determination of the values of the coupling constants is possible. Reconstruction of “decoupled” 1D and 2D spectra is also performed in a fully automated approach. The efficiency of the method is illustrated on experimental 1D and 2D spectra. The method has been developed in the GIFA software, a complete package for processing of NMR data available from the authors.

Initiation factor IF3 from Escherichia coli plays a critical role in the selection of the correct initiation codon. This protein is composed of two domains, connected by a lysine-rich hydrophilic linker. The conformation of native IF3 was investigated by heteronuclear NMR spectroscopy. The two domains are independent and show little or no interaction. Heteronuclear relaxation studies of a sample selectively labelled on lysine residues demonstrates that the inter-domain linker is highly flexible, exhibiting increased 15N T2 values and negative 1H{15N} nuclear Overhause effects over a length of at least eight residues. Analysis of the rotational correlation times further shows that the motions of the two domains are most likely uncorrelated. The inter-domain linker thus displays almost totally unrestricted motions. Accordingly, the amide protons in the central region are shown to be in fast exchange with water. Such a high degree of flexibility of the inter-domain linker might be required for IF3 domains to interact with distant regions of the ribosome.

An approach of taxol synthesis based on the use of (E)-bis-1,2-(tributylstannyl)ethylene 6 as a readily available two-carbon 1,2-link between A and C-ring is described. The conjugate addition of a mixed higher-order stannylvinyl cyanocuprate derived from 6 on chiral cyclohexenone 5 is completely stereoselective. A model study on 3-methylcyclohex-2-enone showed that the afforded stannylvinyl cyclohexenone 12 can be engaged without further transformation in a Stille cross-coupling reaction with the vinyl triflate 4 corresponding to the A-ring of taxol to give seco-taxane 14.

Calystegine B 2 was prepared by the intramolecular cyclisation of a polyhydroxylated 4-amino-cycloheptanone. This intermediate results from a heteroycloaddtion between an acylnitroso compound and a cyclohepta-1,3-diene. The diene is in turn obtained from iron-complexed tropone.

The new NMR concept of selective excitation generated by pulses whose envelope is slaved to that of a signal originating from the sample is described. It is shown that these kinds of pulses exhibit interesting features such as a sort of auto-calibration of the pulse length and phase. These particular features are discussed and experimental evidence is given. Illustration of the use of slaved pulses is demonstrated in the case of 1D transient NOE by selective inversion.

The allenyl titanium 11a, prepared using Yamamoto's conditions reacted with different aldehydes 12a-c to produce the anti acetylenic diols 13a-c. In a synthetic approach to erigerol 1 this reaction was applied to the aldehyde 4 and thr expected key intermediate 13d was obtained in 40% yield after removal of the THP group. During this study the α-alkoxypropargylstannane 14 and the α-alkoxyallenylstannane 15 were prepared in good yields after transmetallation of the corresponding lithio derivatives 5b and 7b by Ti(OiPr)4 or Et2ZnCl.

Preparation of the “Ziegler key intermediate” 2 from lactone 3,readily obtained from hydroxy-β-ionone, was studied. In a first exploratory approach, lactones 11 and 13 were obtained but futher transformations aimed at setting the ring junction were unsuccessful due to unexpected rearrangements. Through a straightforward synthetic sequence involving two new rearrangements, lactone 3 was converted to diosphenol 21 with the desired trans A/B ring junction. 21 was in turn transformed into the well-known acetonide 2 in a good yield. This constitutes a new formal total synthesis of forskolin.

A highly diastereoselective synthesis of the taxane potential precursor 16 is achieved. An unexpected diastereoselectivity was observed upon condensation of the α-trimethylsilyloxyaldehydes 9a or 9b with the cyclohexenyllithium 10 . The single diol isomers 13A and 14A exhibiting the required 1β,2α relative configuration are obtained in good yields.

Structure of beticolin O has been established through examination of its spectroscopic data including X-Ray diffraction and through its chemical transformation into the previously identified beticolin 2.

The three-dimensional structure of bovine angiogenin has been determined using two- and three-dimensional proton NMR spectroscopy. The solution structure is very close to that recently determined by X-ray diffraction analysis. This structure appears well defined, even if five loops and one helix exhibit greater flexibility. Analysis of the active site geometry confirms the position of the Glu-118 residue which obstructs the pyrimidine binding site. There is no experimental evidence of an unobstructed conformation of angiogenin in solution. In addition, it appears that the Glu-118 and Ser-119 residues and the cell receptor binding loop may play an important role in the differences of C-terminal fragment organization and ribonucleolytic activity observed between angiogenins and ribonuclease A.

Title compounds were prepared by the intramolecular cyclisation of 4-aminocycloheptanones as the key step. The functionalisation of the iron-complexed tropone gave cyclohepta-1,3-dienes. The 4-aminocycloheptanones result from a heterocycloaddition between an acylnitroso compound and a eyclohepta-1,3-diene.

An unprecedented remote effect of the hydroxyl group on π-facial selectivity was observed in the addition of Grignard reagents to bicyclo[2.2.2]octan-2-ones, where nucleophilic attack occured from the slerically more hindered face.

OUr goeal was to create a magnetization-dependent field in the sample, which exactly compensates the existing radiation-damping field.

Starting from thiopyridyl compounds bearing a radical stabilizing group in α-position to the thiopyridyl moiety, the thiopyridyl group can be easily removed upon reduction by zinc metal in acetic acid.

In a synthetic approach to forskolin 1 , a 6-endo-trig radical cyclisation was performed on propargylic derivative 12 to furnish in 60% yield compound 17 which contains the A-B ring system of forskolin. In the same way yn-one 14 submitted to Bu 3 SnH gave the expected cyclised product 19 in 55% yield. A particular 5-exo-dig cyclisation took place, leading to compound 18 in 10% yield, when silylated propargylic alcohol 13 was treated under the same conditions.

The electrocyclisation of allenyldiene easily obtained from hydroxy-β-ionone has been studied with the aim to set up an efficient access to polyhydroxylated decalinic systems. Some aspects of the stereoselectivity of this reaction are discussed and the transformation into valuable highly functionalized labdane precursors is described.

A general method for the synthesis of chiral cis epoxides, which we have previously described, is used to synthetize the active enantiomer 1 of the sex pheromone of Boarmia selenaria.

The synthesis of polyhydroxylated 6-N-benzylpiperidin-2-ones is described from the corresponding N-benzylaminoglycoside. These compounds are easily reduced to the differently protected 1, 5-dideoxy-1, 5-imino-D-xylitol.

Initiation factor IF3 from Escherichia coli is composed of two domains connected by a hydrophilic peptide. In this study, the N-terminal domain (residues 7–83) has been overexpiessed, 15N labelled and purified. NMR assignments for this domain have been obtained by two-dimensional and three-dimensional heteronuclear and homonuclear spectroscopy. Using distance geometry and simulated annealing, a three-dimensional solution structure was calculated using 506 NOE and 56 dihedral angle restraints. The resulting structure is composed of a five-stranded antiparallel β sheet surrounded by two α helices. Since the heteronuclear 1H-15N correlation spectrum of the N-terminal domain of IF3 is an almost exact subset of that of the native protein, the assignments obtained and the structure calculated should be directly transposable to the full-length protein.

From the dihydrofuran derivative 3, an mCPBA oxidation followed by an LAH reduction led to the 9β-hydroxy lactol 11, the Jones' oxidation of which gave the 9β-hydroxy lactone 13 together with the formyl ketone compound 7. Treatment of 13 with SOCl2-Py finally produced the Ziegler-Corey-Ikegami intermediate lactone 2 leading to a formal synthesis of (±)-forskoline.

A general method for the synthesis of chiral cis-epoxides substituted bearing a saturated chain and an unsaturated chain is described. This method is used to synthesize both enantiomers of compound 1 .

a general chemical transformation of the “linear” beticolin skeleton (beticolin 2 and 4) into a “bent” beticolin skeleton (cebetin A and beticolin 3 respectively) is described. Beticolins 6 and 8, minor components of mycelial extract are also characterized as minor compounds resulting from these transformations.

From available hydroxy-aldehyde 6 , propargylic derivative 8 was prepared and treated with Bu 3 SnH to give in 95% yield the bicyclic vinyl stannae 10 . After 10 was converted into the diol 15 , this latter was oxidated with the Dess-Martin reagent into the dialdehyde 4 . Compound 4 was then submitted to a pinacolic coupling reaction leading to the bicyclic cis-diol 3 in a stereospecific manner.

1,2-Metallate rearrangements were performed on α-(carbamoyl)alkenyl stannnane 6 with high yield in alkyl transfer to give 10 after quenching with MeI; disappointing results were obtained for preparation of 11 and 2b . Using dihydrofuran 13 , optimized conditions were found to realize 1,2-stannyl and 1,2-(stannyl)vinyl transfers, and methylation, leading to stannyl derivative 15 and stannyl diene 16 in good yields.

Application of the Mitsunobu reaction to various 3β-(equatorial)-hydroxy-4,4-dimethyl terpenic compounds provides the 2-ene-derivatives in good yields. A discussion about the mechanisms involved, and full details on the modified experimental protocol are given.

Various fragments of the N-terminal, DNA-binding domain of the yeast Saccharomyces cerevisiae transcriptional activator CYP1(HAP1) have been cloned and expressed in Escherichia coli. The corresponding polypeptides have been analysed biochemically and we have undertaken a more extensive physical study of a fragment consisting of amino acids 49-126 [CYP1(49-126)]. We show that this CYP1(49-126) peptide requires zinc or cadmium in the growth medium in order to maintain a stable structure. A method to purify CYP1(49-126) is presented. We demonstrate that the purified CYP1(49-126) fragment contains two zinc ions/fragment or two cadmium ions/fragment, which are necessary for DNA binding. 113Cd one-dimensional NMR data suggest that CYP1(HAP1) has a tetrahedral coordination, and that it forms a zinc-cluster complex like GAL4.

The synthesis of the tricyclic system of forskolin by an intramolecular Diels-Alder reaction is described, starting from tri-O-acetyl-D-galactal.