Two-dimensional NOE Spectroscopy Research Articles

Enhancing adsorption capacity and herbicidal efficacy of 2,4-D through supramolecular self-assembly: Insights from cocrystal engineering to solution chemistry

Highly water-soluble pesticides have garnered significant attention due to their poor durability and severe environmental damage caused by easy leaching. Cocrystallization based on a supramolecular self-assembly mechanism has been demonstrated to be an effective method for tuning the dissolution of pharmaceuticals and agrochemicals. However, as a strategy to tune the properties of solid materials, cocrystal engineering appears to be challenging to apply to many liquid formulations of pesticides. Herein, cocrystal of the 2,4-dichlorophenoxyacetic acid (2,4-D) and phenazine (PHE) were developed to explore whether supramolecular assembly in the solid state could be extended to the solution. In the crystalline state, the cocrystal reduced the solubility and dissolution rate of 2,4-D by 71.51 % and 50.55 %, respectively. Notably, when applied in solution, 2,4-D - PHE cocrystal exhibits greater soil adsorption capacity and lower leaching characteristics than pure 2, 4-D. Specifically, only 75% of the cocrystal was leached when 2,4-D was fully leached. Under simulated rainfall, the biological activity experiments in the greenhouse demonstrated that the control efficacy of cocrystal was almost twice as that of the pure 2,4-D. Additionally, two-dimensional NOE Spectroscopy (2D NOESY) and molecular simulations were employed to reveal the mechanism of supramolecular assembly in crystal lattice and aqueous solution. This work reveals that cocrystal engineering is a promising strategy for bridging solid-state and solution chemistry through supramolecular self-assembly to improve the persistence and decrease the off-target threat of highly water-soluble pesticides.

Exploring the intermolecular interactions acting in solvent-modified MEKC by Molecular Dynamics and NMR: The effect of n-butanol on the separation of diclofenac and its impurities

An integrated approach involving CE experiments, Molecular Dynamics (MD) simulations and two-dimensional NOE spectroscopy (2D-NOESY) experiments was employed to elucidate the intermolecular interactions and the separation mechanisms involved in a solvent-modified MEKC method for the simultaneous determination of diclofenac sodium and its impurities. The CE findings indicated that the addition of n-butanol (nBuOH) to the SDS micellar solution played a primary role for controlling the partitioning into the mixed micelles and the migration of the analytes and that the presence of nBuOH as cosurfactant was compulsory for achieving the complete separation of the compounds. The different capacity factors of the analytes were calculated and a change in solute association with the mixed micelle when changing the SDS/nBuOH molar ratio was highlighted. The optimal SDS/nBuOH molar ratio for the electrophoretic separation was 1:8. On the other hand, both MD simulations and NMR experiments indicated that the most favorable molar ratio for the formation of mixed SDS/nBuOH micelles was 1:2. These results suggested that probably there is an excess of nBuOH in the background electrolyte, both as free molecules and in form of aggregates, which is able to interact with the analytes, and thus may compete with mixed micelles for the considered compounds. The calculated values of gain in potential energy of the analytes when included in mixed micelles were in agreement with the observed migration order of the compounds. The role of methyl-β-cyclodextrin (MβCyD) in the background electrolyte was also investigated, since the addition of this CyD to the solvent-modified MEKC system was found to be useful to reduce the analysis time. MD simulations and 2D-NOESY spectra highlighted the formation of inclusion complexes with MβCyD not only with the analytes, but also with SDS. MβCyD may lower the availability of both SDS and nBuOH for forming micelles and mostly may compete with the mixed micelle as a second pseudostationary phase.

Combined approach using capillary electrophoresis, NMR and molecular modeling for ambrisentan related substances analysis: Investigation of intermolecular affinities, complexation and separation mechanism

A comprehensive investigation on the CE separation mechanisms and on the inclusion complexation with CyDs of the chiral drug S-ambrisentan (S-AMB), its R-enantiomer and other impurities was performed by different techniques. A CE method was previously set up allowing the simultaneous determination of the enantiomeric purity and of impurities of S-AMB, based on the addition of SDS micelles and γ-cyclodextrin (γCyD) to borate buffer. In this study, the electrophoretic behavior of the analytes in terms of selectivity and mobility with respect to the addition of different CyDs was first investigated, evidencing the presence of interactions for all the CyDs, but the unique ability of γCyD for obtaining the separation of all the compounds. By molecular modeling, aggregates between SDS micelles and analytes, and inclusion complexes between CyDs, SDS and/or analytes of different stoichiometries were simulated. The potential and the gain energy of complexes were calculated on the minimized conformations, showing the great tendency of γCyD of forming mixed complexes with one or two SDS molecules and with the analyte, even if with different affinities among the analytes. For 1:1:1 mixed complexes with different CyDs, the highest difference of potential energy between the enantiomers’ complexes was observed for γCyD. Two-dimensional NOE spectroscopy experiments were performed for S-AMB and I1 and pointed out the interactions of the aromatic moiety of the analytes and of SDS aliphatic chain with γCyD protons, confirming the existence of γCyD mixed complexes. The high affinity of SDS for the γCyD cavity was suggested to justify the fundamental role of SDS in modulating and achieving the CE separation, due to its influence both on the stability and on the type of complexes between γCyD and the analytes.

1D and 2D NMR investigations of the interaction between oppositely charged polymers and surfactants

Proton chemical shifts and two-dimensional COSY and NOE spectroscopy (NOESY) experiments have been used to examine the interaction of various oppositely charged surfactant and polyelectrolyte systems, namely, the cationic surfactant dodecyltrimethyammonium bromide (DTAB) and a series of alkanediyl-α,ω-bis(alkyldimethylammonium bromide) surfactants (Gem 12-s-12, where s is the length of the methylene spacer group) with the anionic polyelectrolyte poly(styrene sulfonate) or PSS. In all cases, we observe substantial aromatic-solute-induced chemical shifts (ASIS) in the surfactant peaks of the polymer/surfactant complexes versus that of the pure surfactant spectra. In the case of the DTAB/PSS system, the chemical-shift changes as a function of changing ratio of surfactant to polymer are interpreted in terms of structural changes that occur in the complex with increasing polymer concentration. For the Gem 12-s-12/PSS systems, the interaction of these gemini surfactants with the anionic polyelectrolyte, as deduced from the interpretation of the1H ASIS shifts and the NOESY cross peaks, is dependent on the length of the methylene spacer. From the NOESY experiments, we observed significant NOESY cross peaks that correlated well with the expected mechanism of interaction as observed in the literature for the single-tailed surfactant/polymer system. NMR techniques are shown to provide information on the molecular arrangement of these molecules in aqueous solution.

Minimum FGF2 Binding Structural Requirements of Heparin and Heparan Sulfate Oligosaccharides As Determined by NMR Spectroscopy

Heparin and heparan sulfate (HS) glycosaminoglycans (HSGAGs) are sulfated polysaccharidesthat play important roles in fundamental biological processes by binding to proteins. The prototypic exampleof HSGAG-protein interactions is that with the fibroblast growth factors (FGFs), specifically FGF1 andFGF2. Structural and biochemical studies have shown that the chain length, sulfation pattern, andconformation of HSGAGs play a critical role in FGF binding and activity. Previously, we showed that atetrasaccharide of the form ANS,6X-I2S-ANS,6X-I2S-OPr (where X is OH or O-sulfate and Pr is propyl) withat least one of the ANS,6X residues having a 6-O sulfate group was the minimum binding motif for FGF1[Guerrini, M., Agulles, T., Bisio, A., Hricovini, M., Lay, L., Naggi, A., Poletti, L., Sturiale, L., Torri, G.,and Casu, B. (2002) Biochem. Biophys. Res. Commun. 292, 222-230]. We report NMR structural analysisusing two-dimensional NOE spectroscopy (2D-NOESY) and transferred NOESY (trNOESY) on a non-6-O-sulfated synthetic tetrasaccharide TETRA (ANS-I2S-ANS-I2S-OPr) both in its free state and bound toFGF2. This tetrasaccharide comprises both the structural trisaccharide motif ANS-I2S-ANS that forms "kinks"in longer heparin chains induced by FGF binding [Raman, R., Venkataraman, G., Ernst, S., Sasisekharan,V., and Sasisekharan, R. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 2357-2362] and the common bindingmotif I2S-ANS-I2S present in octasaccharides that exhibited strong FGF2 binding [Kreuger, J., Salmivirta,M., Sturiale, L., Gimenez-Gallego, G., and Lindahl, U. (2001) J. Biol. Chem. 276, 30744-30752]. Thesedata suggest that TETRA could be the shortest HSGAG oligosaccharide that binds to FGF2. Furthermore,our study confirms that both the IdoA residues in TETRA adopt the chair 1C4 conformation upon FGF2binding to provide the best molecular fit in contrast to an analogous 6-O-sulfated tetrasaccharide motifobserved in the FGF2-HSGAG cocrystal structure where one of the IdoAs adopts skew-boat 2SOconformation. Thus, our study highlights the fact that the conformational plurality of IdoA is able toaccommodate the changes in the sulfation pattern to provide the necessary specificity for protein binding.

Consequences of N-Acylation on Structure and Membrane Binding Properties of Dermaseptin Derivative K4-S4-(1-13)

Acyl conjugation to antimicrobial peptides is known to enhance antimicrobial properties. Here, we investigated the consequences of aminolauryl (NC(12)) conjugation to the dermaseptin derivative K(4)-S4-(1-13) (P) on binding properties to bilayer models mimicking bacterial plasma membrane, which is often cited as the ultimate site of action. Isothermal titration calorimetry revealed that acylation was responsible for enhancing the binding affinity of NC(12)-P compared with P (K = 13 x 10(5) and 1.5 x 10(5) m(-1), respectively). Surface plasmon resonance measurements confirmed the isothermal titration calorimetry results (K(app) = 12.6 x 10(5) and 1.53 x 10(5) m(-1), respectively) and further indicated that enhanced adhesion affinity (K(adhesion) = 3 x 10(5) and 1 x 10(5) m(-1), respectively) was coupled to enhanced tendency to insert within the bilayer (K(insertion) = 4.5 and 1.5, respectively). To gain insight into the molecular basis for these observations, we investigated the three-dimensional structures in the presence of dodecylphosphocholine using NMR. The ensemble of NMR-calculated structures (backbone root mean square deviation <0.6 A) showed that the acyl moiety was responsible for a significant molecular reorganization, possibly affecting the electrostatic potential distribution in NC(12)-P relative to that of P. The combined data present compelling evidence in support of the hypothesis that N-acylation affects antimicrobial properties by modifying the secondary structure of the peptide in a manner that facilitates contact with the membrane and consequently increases its disruption.

Solution Structure of the C-terminal Domain of TFIIH P44 Subunit Reveals a Novel Type of C4C4 Ring Domain Involved in Protein-Protein Interactions

The human general transcription factor TFIIH is involved in both transcription and DNA nucleotide excision repair. Among the 10 subunits of the complex, p44 subunit plays a crucial role in both mechanisms. Its N-terminal domain interacts with the XPD helicase, whereas its C-terminal domain is involved specifically in the promoter escape activity. By mutating an exposed and non-conserved cysteine residue into a serine, we produced a soluble mutant of p44-(321-395) suitable for solution structure determination. The domain adopts a C4C4 RING domain structure with sequential organization of beta-strands that is related to canonical RING domains by a circular permutation of the beta-sheet elements. Analysis of the molecular surface and mutagenesis experiments suggests that the binding of p44-(321-395) to TFIIH p34 subunit is not mediated by electrostatic interactions and, thus, differs from previously reported interaction mechanisms involving RING domains.

A 2-D NMR investigation of the micellar solubilization site in ionic micellar solutions

Two-dimensional NOE spectroscopy (NOESY) experiments have been used to examine the dissolution of two typical solubilizates (1-butanol and benzene) in anionic sodium dodecyl sulfate (SDS) micelles and of benzene in cationic dodecyltrimethyammonium (DTAB) micelles. In all cases, significant, positive NOESY cross peaks were obtained that appeared to correlate well with the expected locus of solubilization of these solubilizates in these surfactant systems. The NOESY technique, which is a noninvasive experiment, is found to provide significant insight in terms of the solubilization of these molecules in the interior of ionic micelles.Key words: solubilization, surfactants, NMR spectroscopy, 2-D NOESY experiments.

Structure of the O-specific polysaccharide of a serologically separate strain Proteus penneri 2 from a new proposed serogroup O66.

O-specific polysaccharide chain of Proteus penneri strain 2 lipopolysaccharide was studied by full and partial acid hydrolysis, Smith degradation, methylation analysis, and NMR spectroscopy, including two-dimensional rotating-frame NOE spectroscopy (ROESY) and 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. Together with D-glucose and 2-acetamido-2-deoxy-D-glucose, the polysaccharide was found to contain two rarely occurring sugars, 6-deoxy-L-talose (L-6dTal) and 2,3-diacetamido-2,3,6-trideoxy-L-mannose (L-RhaNAc3NAc), and the following structure of a non-stoichiometrically O-acetylated tetrasaccharide repeating unit was established: [equation: see text] The O-specific polysaccharide studied has a unique composition and structure and, accordingly, P. penneri 2 is serologically separate among Proteus strains. Therefore, we propose for P. penneri 2 a new Proteus O-serogroup O66 where this strain is at present the single representative.

A 2-D NMR investigation of the micellar solubilization site in ionic micellar solutions

Two-dimensional NOE spectroscopy (NOESY) experiments have been used to examine the dissolution of two typical solubilizates (1-butanol and benzene) in anionic sodium dodecyl sulfate (SDS) micelles and of benzene in cationic dodecyltrimethyammonium (DTAB) micelles. In all cases, significant, positive NOESY cross peaks were obtained that appeared to correlate well with the expected locus of solubilization of these solubilizates in these surfactant systems. The NOESY technique, which is a noninvasive experiment, is found to provide significant insight in terms of the solubilization of these molecules in the interior of ionic micelles.

Structure of the O-specific polysaccharide of Proteus penneri strain 41 from a new proposed serogroup O62.

O-specific polysaccharide of Proteus penneri strain 41 was studied using 1H- and 13C-NMR spectroscopy, including two-dimensional COSY, heteronuclear 13C,1H-correlation (HETCOR) and one-dimensional NOE spectroscopy, and the following structure of a non-stoichiometrically O-acetylated hexasaccharide repeating unit was established:[structure: see text] where RGlcNAc is 2-acetamido-4-O-[(S)-1-carboxyethyl]-2-deoxyglucose. Cross-reactivity of anti-P. penneri 41 O-serum with other P. penneri strains is discussed, and a new, separate O62 serogroup is proposed which is the next Proteus O-serogroup containing P. penneri strains only.

Preparation oftrans- andcis-2-allyl-6-alkyl(aryl)-1,2,3,6-tetrahydropyridines based on the reductivetrans-2,6-dialkylation of pyridine. Synthesis of (±)-epidihydropinidine and (±)-dihydropinidine

A general method for the preparation of unsymmetricaltrans-2-allyl-6-alkyl(aryl)-1,2,3,6-tetrahydropyridines6 based on a combination of 1,2-addition of RLi to pyridine andtrans-6-allylation with triallylborane in the presence of methanol was elaborated. It was shown thattrans-piperideines (6 (R=Alk, Ph) isomerize into the correspondingcis-2-allyl-6-alkyl(phenyl)-3-piperideines14 on heating with triallylborane followed by deboronation of aminoborane (16) with methanol and an alkali. The stereochemistry of compounds6 and14 was determined by two-dimensional NOE spectroscopy. A possible mechanism of the formation oftrans-amines6 and their transformation intocis-isomers14 is discussed. Alkaloids (±)-epidihydropinidine (trans-2-methyl-6-propylpiperidine2a, 70%) and (±)-dihydropinidine (cis-2-methyl-6-propylpiperidine1d, 71%) were synthesized by hydrogenation of compound6a (R=Me) and14a (R=Me), respectively, over Raney nickel.

Use of cross-links to study the conformational dynamics of triplex DNA.

The conformational dynamics of a 34-base-long pyrimidine purine-pyrimidine motif intramolecular DNA triple helix possessing three cytosine residues in the Hoogsteen strand (1) and a disulfide cross-linked analog (2) were studied by two-dimensional exchange and NOE spectroscopy and by measuring base-catalyzed imino proton exchange rates. Under acidic conditions that stabilize triplexes containing Hoogsteen strand cytosines (pH 6.0 and 1 degree C), sequences 1 and 2 exhibit a small and identical degree of conformational heterogeneity. However, at a higher temperature (pH 6.0 and 37 degrees C), 1 exhibits much more extensive conformational heterogeneity than 2. The exchange times for Watson-Crick imino protons are approximately 1 h for both triplexes. However, the Hoogsteen base-pair lifetimes of 1 could not be measured because this sequence is conformationally labile under the alkaline conditions necessary to conduct these experiments. Because of the extraordinary pH stability conferred by the cross-link, it is possible to estimate the base-pair lifetimes for 2. The lifetimes of the Hoogsteen base pairs range from about 3 to 370 ms, and in all cases are shorter than that of the Watson-Crick base pair contained in the same triplet. These experiments represent the first measurement of base-pair lifetimes within Hoogsteen triplets. The ability to measure individual base-pair lifetimes may prove useful in studies that attempt to modulate triplex properties through rational design.

Structural studies on the lipopolysaccharide from a rough strain of Ochrobactrum anthropi containing a 2,3-diamino-2,3-dideoxy- d-glucose disaccharide lipid A backbone

A degradation protocol using de- O-acylation and subsequent alkaline de- N-acylation was applied to the lipopolysaccharide of Ochrobactrum anthropi rough strain LMG 3301. Three main oligosaccharide bisphosphates containing core–lipid A backbone structures were obtained after fractionation by anion-exchange HPLC. Using 1 H and 13 C NMR spectroscopy, including two-dimensional COSY, TOCSY, and NOE spectroscopy (ROESY and NOESY), the following structures were established: where Kdo is 3-deoxy- d- manno-octulosonic acid, d-GlcN3N is 2,3-diamino-2,3-dideoxy- d-glucose and R is H or α- d-Gal pA or 4-deoxy- β- l- threo-hex-4-enopyranuronic acid, the latter sugar being derived from α- d-Gal pA by β-elimination of a substituent attached to O-4. This is the first report on the isolation from a lipopolysaccharide of an oligosaccharide containing GlcN3N in the lipid A backbone [ β- d-Glc pN3N4 P-(1→6)- α- d-Glc pN3N1 P]. Sugar and methylation analysis confirmed the presence of the GalA→Kdo disaccharide and non-stoichiometric substitution of GalA. It is suggested that Glc is the substituent at O-4 in GalA and that in the non-degraded lipopolysaccharide the amino group of GlcN is not acylated.

Structures of the O‐specific Polysaccharide Chains of Pectinatus cerevisiiphilus and Pectinatus frisingensis Lipopolysaccharides

Mild acid hydrolysis of the smooth-type lipopolysaccharide (LPS) of Pectinatus frisingensis afforded no polysaccharide but monomeric 6-deoxy-L-altrose (L-6dAlt) which was identified by anion-exchange chromatography in borate buffer, GLC/MS, 1H-NMR spectroscopy, and optical rotation. LPS was degraded with alkali under reductive conditions to give a completely O-deacylated polysaccharide, which was studied by methylation analysis, 1H-NMR and 13C-NMR spectroscopy, including sequential, selective spin-decoupling, two-dimensional correlation spectroscopy (COSY), COSY with relayed coherence transfer, two-dimensional heteronuclear 13C, 1H-COSY, one-dimensional NOE and two-dimensional rotating-frame NOE spectroscopy. It was found that the O-specific polysaccharide chain of P. frisingensis LPS is a homopolymer of 6-deoxy-L-altrofuranose built up of tetrasaccharide-repeating units having the following structure: [sequence: see text] Similarly, mild acid degradation of smooth-type LPS of Pectinatus cerevisiiphilus resulted in depolymerisation of the polysaccharide chain to give a disaccharide consisting of D-glucose and D-fucose. Study of the disaccharide by methylation analysis and alkali-degraded LPS by one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectroscopy showed that the O-specific polysaccharide of P. cerevisiiphilus has the following structure: -->2)-beta-D-Fucf-(1-->2)-alpha-D-Glcp-(1-->.

Structure and Epitope Characterisation of the O-specific Polysaccharide of Proteus mirabilis O28 Containing Amides of d-galacturonic Acid with l-serine and l-lysine

The O-specific polysaccharide of Proteus mirabilis O28 was found to contain D-galactose, D-galacturonic acid (GalA), 2-acetamido-2-deoxy-D-glucose, L-serine, L-lysine, and O-acetyl groups in molar ratios 1:2:1:1:1:1, the amino acids being linked via their alpha-amino group to the carboxyl group of GalA. The polysaccharide was studied using 1H- and 13C-NMR spectroscopy, including selective spin-decoupling, one-dimensional total correlation spectroscopy, two-dimensional homonuclear correlation spectroscopy (COSY), heteronuclear 13C,1H COSY, one-dimensional NOE, and two-dimensional rotating-frame NOE spectroscopy and partial acid hydrolysis followed by borohydride reduction, methylation, and GLC/MS analysis of the derived glycosyl alditols. The following structure of the repeating unit was established: [formula: see text] Epitope specificity of the P. mirabilis O28 polysaccharide was analysed using a homologous rabbit polyclonal antiserum in quantitative precipitation, passive immunohemolysis, and inhibition of passive immunohemolysis. Study with related synthetic glycopolymers (2-acrylamidoethyl glycosides of amides of alpha-D-GalA with amino acids copolymerised with acrylamide) showed the importance of D-GalA(L-Lys) for manifesting serological specificity of the O-antigen. Serological cross-reactions between P. mirabilis O28, S1959, and R14/S1959 (a transient-like form) are discussed.

NMR conformational study of a model tetradecapeptide mimicking the RXVRG consensus cleavage site of a Xenopus luevis skin endoprotease

A model tetradecapeptide, used for the purification of the RXVRG-endoprotease from Xenopus laevis skin exudate, has been studied by two-dimensional NMR, correlation (COSY) and NOE (NOESY) spectroscopy. This peptide has the 5-9 consensus sequence (RXVRG), along with an acidic moiety (1-4) and a hydrophobic domain (10-14). Variations with temperature of NH chemical shifts in a dimethyl sulfoxide solution (low thermal coefficients at residues 6, 7 and 8) and quantified NOE values from four spectra at different mixing times clearly showed a structural organization in the consensus domain with psi-angles around [-40, -10 degrees] for residues 7 and 8, and two NOE correlations of alpha HiNHi + 2 type (5-7 and 6-8). Moreover, a privileged rotamer in the side chain is established for three residues (Val2, Asp3 and Val7) and limited possibilities are discussed for seven others. Most of the folding trends were not observed in the [Ser7] derivative, underlying the relationship between the conformations and a full consensus sequence. In the model tetradecapeptide an equilibrium between two beta-turns of type I, fragments 4-7 and 5-8, seems the most probable. Comparison between this tetradecapeptide and its 4-14 fragment, also a substrate for RXVRG-endoprotease, shows that the 1-3 moiety (DVD) influences the consensus domain structure(s) and clearly stabilizes the folded one(s). Finally, two analytical methods are developed in order to determine: (1) the trifluoroacetic acid content of the peptide samples, on the basis of 19F NMR spectroscopy; (2) the mean phi- and psi-angles of each residue, from the whole set of NH/alpha H coupling constants (3JN alpha) and NOE data at a local level.

Structure and epitope characterisation of the O-specific polysaccharide of Proteus mirabilis O28 containing amides of D-galacturonic acid with L-serine and L-lysine.

The O-specific polysaccharide of Proteus mirabilis O28 was found to contain D-galactose, D-galacturonic acid (GalA), 2-acetamido-2-deoxy-D-glucose, L-serine, L-lysine, and O-acetyl groups in molar ratios 1:2:1:1:1:1, the amino acids being linked via their alpha-amino group to the carboxyl group of GalA. The polysaccharide was studied using 1H- and 13C-NMR spectroscopy, including selective spin-decoupling, one-dimensional total correlation spectroscopy, two-dimensional homonuclear correlation spectroscopy (COSY), heteronuclear 13C,1H COSY, one-dimensional NOE, and two-dimensional rotating-frame NOE spectroscopy and partial acid hydrolysis followed by borohydride reduction, methylation, and GLC/MS analysis of the derived glycosyl alditols. The following structure of the repeating unit was established: [formula: see text] Epitope specificity of the P. mirabilis O28 polysaccharide was analysed using a homologous rabbit polyclonal antiserum in quantitative precipitation, passive immunohemolysis, and inhibition of passive immunohemolysis. Study with related synthetic glycopolymers (2-acrylamidoethyl glycosides of amides of alpha-D-GalA with amino acids copolymerised with acrylamide) showed the importance of D-GalA(L-Lys) for manifesting serological specificity of the O-antigen. Serological cross-reactions between P. mirabilis O28, S1959, and R14/S1959 (a transient-like form) are discussed.

Determination of the absolute configurations of the epimers of the P-chiral phosphine Ph 2PCH 2CH 2P ☆Ph( L-(−)-methyl) by use of two-dimensional NMR spectroscopy in combination with a palladium(II) “reporter complex”

The two epimers of the new P-chiral phosphine Ph 2 PCH 2 CH 2 P ☆ Ph(L-(−)- menthyl) ( 1-Rp and 1-Sp) were synthesized by treating Ph 2PH with the diastereomerically pure forms of Ph( L-(−)-methyl)CHCH 2)P). The palladium(II) complexes [ Pd(( S)-o- C 6 H 4 CHMeNMe 2)( 1-Rp)][ PF 6], 2-Rp and [ Pd(( S)-o- C 6 H 4 CHMeNMe 2) ( 1-Sp)[ PF 6] , 2-Sp, were obtained by treating the corresponding bisphosphines with [Pd 2Cl 2((S)- o-CH 6 H 4CHeNMe 2) 2]. Each of these complex exists in the form of two regioisomers differing in the orientation of the chiral phosphine relative to the C-donor. Constraints from close interligand, intramolecular hydrogen-hydrogen contacts within three of these palladium complexes were detected by two-dimensional NOE spectroscopy, and were used to determine the absolute configurations at the chiral phosphorus centers in the Ph 2PCH 2CH 2PPh( L-(−)-menthyl) isomers. The conformations of the two chelate rings in the more abundant of these isomers were also determined.

Physico-chemical and spectroscopic properties of the monohemic cytochrome C552 from Pseudomonas nautica 617.

A c-type monohemic ferricytochrome C552 (11 kDa) was isolated from the soluble extract of a marine denitrifier, Pseudomonas nautica strain 617, grown under anaerobic conditions with nitrate as final electron acceptor. The NH2-terminal sequence and the amino acid composition of the cytochrome were determined. The heme iron of the cytochrome C552 has histidine-methionine as axial ligands, and a pH-dependent mid-point redox potential, equal to 250 mV at pH 7.6. The presence of methionine was demonstrated by visible, EPR and NMR spectroscopies. The assignment of most of the hemic protons was performed applying two-dimensional NOE spectroscopy (NOESY), and the aromatic region was assigned through two-dimensional correlated spectroscopy (COSY) experiments. The EPR spectrum of the oxidised form of the cytochrome C552 is typical of a low-spin ferric heme.