Bacteriocin small of Rhizobium leguminosarum belongs to the class of N-acyl-L-homoserine lactone molecules, known as autoinducers and as quorum sensing co-transcription factors.

Small bacteriocin was isolated from the culture broth of the gram-negative bacterium Rhizobium leguminosarum, which forms symbiotic nitrogen-fixing root nodules on a number of leguminous plants. The structure of the molecule was elucidated by spectroscopic methods and identified as N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone. The absolute configuration of both asymmetric carbon atoms in the molecule was determined by the use of the chiral solvating agents S-(+)- and R-(-)-2,2,2-trifluoro-1-(9-anthryl)-ethanol. small bacteriocin is structurally related to the quorum sensing co-transcription factors for genes from other bacteria such as Vibrio fischeri, Pseudomonas aeruginosa, Erwinia carotovora, and Agrobacterium tumefaciens which are involved in animal-microbe or plant-microbe interactions. The mechanism of regulation of such interactions by this kind of co-transcription factors is still unknown in R. leguminosarum.

Conformational analysis of the dinucleotides 5'-methylphospho-N6-dimethyladenylyl-uridine (mpm62A-U) and 5'methylphospho-uridylyl-N6-dimethyladenosine (mpU-m62A) and of the trinucleotide U-m62A-U. A nuclear magnetic resonance and circular dichroic study.

NMR and CD studies were carried out on the dinucleotides 5'-methylphospho-N6-dimethyladenylyl-uridine (mpm62-U) and 5'-methylphospho-uridylyl-N6-dimethyladenosine (mpU-m62A) and on the trinucleotide U-m62A-U. A detailed comparison is given of the conformational features of mpm62A-U and mpU-m62A with the corresponding 5'-nonphosphorylated dinucleotides m62A-U and U-m62A, respectively. The behaviour of the trinucleotide U-m62A-U is compared with the properties of the constituent dinucleotides U-m62A and mpm62A-U. Chemical-shift and CD data were used to determine the amount of stacking interactions. For each compound NMR spectra were recorded at two or three sample concentrations in order to separate intermolecular and intramolecular base-base interactions. The coupling constants of the ribose ring are interpreted in terms of the N/S equilibrium, and population distributions along the backbone angles beta, gamma and epsilon are presented. The combined data indicate a strong similarity between mpm62A-U and m62A-U both in degree and in mode of stacking. In contrast, the existence of different types of stacking interactions in mpU-m62A and U-m62A is suggested in order to explain the NMR and CD data. It is concluded that dinucleoside bisphosphates serve better as a model for the behaviour of trinucleotides than dinucleoside monophosphates. The trinucleotide U-m62A-U adopts a regular single-stranded stacked RNA structure with preference for N-type ribose and gamma+ and beta t backbone torsion angles. The difference in behaviour between the U-m62A- part of U-m62A-U and the dimer U-m62A is seen as a typical example of conformational transmission.

Conformational characteristics of the hexanucleoside pentaphosphate AUAUAU: a 2D NMR study at 500 MHz.

All 36 ribose proton resonances and most of the base proton resonances of the hexanucleoside pentaphosphate AUAUAU have been assigned unequivocally using 2D J-resolved spectroscopy, spin echo correlated spectroscopy (SECSY) and 2D NOE spectroscopy (NOESY). The NMR parameters of AUAUAU are compared with those of smaller fragments that contain methylated adenine bases: m62AU, m62AUm62A, m62AUm62AU and m62AUm62AUm62A. Previous studies on this series of compounds have shown that in all these cases purine-pyrimidine-purine sequences prefer to adopt a mixture of states which have as common feature that the interior pyrimidine residues are bulged out, whereas the purine residues stack upon each other. Chemical shift data, proton-proton coupling constants, as well as the observation of imino-proton resonances for AUAUAU show unambiguously that upon lowering the temperature the high-temperature "bulged out" situation reverts to a normal A-RNA-like double helix.

Carbon-13 NMR in conformational analysis of nucleic acid fragments. 4. The torsion angle distribution about the C3'-O3' bond in DNA constituents.

Carbon-13 and proton NMR spectra of a series of oligodeoxynucleotides (d(CT), d(CC), d(TA), d(AT), d(CG), d(GC), d(AG), d(AAA), d(TATA) and d(GGTAAT] were measured at various temperatures. The three coupling constants that are related to the magnitude of backbone angle epsilon (J(C4'-P), J(C2'-P) and J(H3'-P] are analyzed in terms of a three-state equilibrium about this bond. Two epsilon (trans) angles occur, which differ in magnitude depending on the conformation (N or S) of the adjoining deoxyribose ring. The S-type deoxyribose ring is associated with a smaller epsilon (trans) angle: epsilon (t,S) = 192 degrees. The N-type deoxyribose ring is associated with a larger epsilon (trans) angle epsilon (t,N) = 212 degrees. The third rotamer participating in the conformational equilibrium, is a gauche(-) (epsilon (-] conformer and occurs exclusively in combination with the S-type sugar ring (epsilon (-,S) = 266 degrees). Within the limits of experimental error, the magnitude of these three angles appears to be independent of the particular base sequence, except in the case of d(CG) where a slightly larger epsilon (t,S) angle (197 degrees) is indicated. A simple equation is proposed which may be used to calculate the population of epsilon (t,S) conformer in cases where only J(H3'-P) is known.

Carbon-13 NMR in conformational analysis of nucleic acid fragments. 3. The magnitude of torsional angle epsilon in d(TpA) from CCOP and HCOP NMR coupling constants.

Carbon-13 NMR spectra of the deoxyribonucleotide d(TpA), 3',5'-cyclic AMP and 3',5'-cyclic dAMP were measured. It is shown that the different substitution of C2' in deoxyribonucleotides versus ribonucleotides does not affect the vicinal C2'-C3'-O3'-P coupling to a measurable extent. Therefore, the same set of Karplus parameters may be used for the C2'-C3-O3'-P couplings in ribonucleotides and in deoxyribonucleotides. Vicinal carbon-phosphorus and proton-phosphorus coupling constants are used to calculate the magnitude of the torsion angle epsilon (C4'-C3'-O3'-P), which amounts to 195(0) in the trans conformer and to 261(0) in the gauche(-) conformer.

Carbon-13 NMR in conformational analysis of nucleic acid fragments. 2. A reparametrization of the Karplus equation for vicinal NMR coupling constants in CCOP and HCOP fragments.

13C-31P coupling constants of 10 oligoribonucleoside phosphates, measured at a number of temperatures, are presented. The combination of these data with 1H-31P couplings of the same compounds leads to the derivation of two new and mutually consistent sets of Karplus parameters: J(CCOP) = 6.9cos2 phi--3.4cos phi + 0.7 J(HCOP) = 15.3cos2 phi--6.1cos phi + 1.6 At the same time new values for the base sequence dependent magnitude of the trans conformer of the backbone angle epsilon (C4'-C3'-O3'-P) are calculated. The present results show that the magnitude of epsilon(t) in right-handed ribo helices is confined to the range 214 degrees-226 degrees (average 219 degrees), which is in much better agreement with single crystal X-ray studies (average 218 degrees) than were previous deductions from NMR spectroscopic results (average 208 degrees).

Carbon-13 NMR in conformational analysis of nucleic acid fragments. Heteronuclear chemical shift correlation spectroscopy of RNA constituents.

The assignment of the non-quaternary 13C resonances by means of two-dimensional heteronuclear chemical shift correlation spectroscopy is presented for several oligoribonucleotides: The dimers m6(2)AU, m6(2)Am6(2)A and mpUm6(2)A and the trimers m6(2)AUm6(2)A and m4(2)Cm4(2)Cm6(2)A. The temperature and concentration dependency of the 13C chemical shifts are studied with emphasis on the behaviour of the dimer m6(2)AU. The present study shows that in the 5-50 mM range the concentration-dependent chemical shift changes of the ribose carbons are negligible compared to chemical shift changes due to intramolecular events. All compounds studied show a surprising correlation between the chemical shifts of the carbon atoms of the ribose ring and the sugar conformational equilibrium as expressed by the percentage N or S conformer. Thus the chemical shift data can be used to obtain the thermodynamical parameters of the two-state N/S equilibrium. Parameters deduced for m6(2)AU are Tm = 306 K and delta S = -25 cal mol-1 K-1, which values are in satisfactory agreement with results obtained earlier from 1H NMR and from Circular Dichroism.

Conformational analysis of a ribopentanucleoside tetraphosphate in aqueous solution. A two-dimensional NMR study at 500 MHz.

The 30 ribose proton resonances of the pentaribonucleoside tetraphosphate m6(2)AUm6(2)AUm6(2)A have been assigned unequivocally by means of spin-echo-correlated spectroscopy, 2D J-resolved spectroscopy and Nuclear Overhauser difference spectroscopy, carried out at 500 MHz. A detailed comparison of the conformational properties of the title compound with its constituent fragments m6(2)AUm6(2)AU, m6(2)AUm6(2)A, m6(2)AU and the relevant monomers is given. Chemical shift data indicate the existence of a doubly "bulged out" conformer, in which the two interior U-fragments are not involved in regular nearest neighbour stacking interactions. The coupling constants of the ribose-ring are interpreted in terms of the N/S equilibrium, and population distributions along the backbone angles beta and gamma are presented. The combined data suggest a strong similarity between the 5'-terminal triplets in m6(2)AUm6(2)AUm6(2)A, m6(2)AUm6(2)AU and m6(2)AUm6(2)A2.

Thermodynamics of stacking and of self-association of the dinucleoside monophosphate m2(6)A-U from proton NMR chemical shifts: differential concentration temperature profile method.

Chemical shifts of base and sugar protons of the modified ribodinucleoside monophosphate N6-dimethyladenylyl(3'-5')uridine (m2(6)A-U) were measured at 100, 360 and 400 MHz in aqueous solution. Seven different samples were used with concentrations ranging from 0.28 mM to 32.7 mM. The temperature was varied from -5 degrees C to 105 degrees C. An internal temperature calibration was used. The effects of intermolecular self-association and of intramolecular stacking on the chemical shifts were quantitatively separated by means of a new approach: differential concentration/temperature profiles (DCTP). Several computational models were tested and the analysis allowed deeper insight into the behaviour of m2(6)A-U at the molecular level. The simple two-state approach for both self-association and stacking already afforded a significant improvement over models in which the association is entirely neglected. A computer least-squares analysis of the chemical shift behaviour of each individual proton yielded thermodynamic parameters for self-association and stacking. However, the two-state model did not suffice to reproduce accurately all of the observations. A satisfactory fit required two additional assumptions: (a) the aromatic protons experience different association shifts in stacked and in unstacked molecules: (b) a temperature-dependent conformational equilibrium exists between sets of unstacked microstates. The stacked state is taken to represent a single conformational species. The implementation of this extended model in the least-squares optimization allowed the reproduction of over one thousand chemical shift observations within experimental error. Thermodynamic equilibrium parameters deduced for intramolecular stacking are: delta H degrees x = -28.8 kJ mol-1, delta S degrees x = -93 J mol-1 K-1. These numbers agree well with those obtained earlier by us from circular dichroism spectra. The equilibrium enthalpy and entropy values deduced for the association process are: delta H degrees A = -35 kJ mol-1 and delta S degrees A = -95 J mol-1 K-1.

Cyanogenesis in Passifloraceae.

A novel cyclopentenoid cyanogenic bis-glycoside has been isolated from Passiflora capsularis L. Its structure has been tentatively determined as 4-boivinosyl tetraphyllin B. The name passicapsin is proposed for this compound. Linamarin has been isolated from Passiflora warmingii Mast. Extracts of Passiflora perfoliata L. yielded a (probably glycosidic) cyclopentenone dervative, which possibly originated from an unstable glycoside having a free alpha-hydroxynitrile group.

Isolation of the secoiridoid-glucoside sweroside from Tabernaemontana psorocarpa.

The known secoiridoid glucoside sweroside (structure I) has been isolated from the leaves of Tabernaemontana psorocarpa and identified by chemical, chromatographic and spectroscopical data. Its (1)H-NMR at 360 MHz is discussed.