CENTRAL EUROPEAN NMR DISCUSSION GROUPS
th
17 NMR Valtice
8.-10.4.2002
Valtice, Czech Republic
THIS CONFERENCE IS SPONSORED BY
Bruker Analytik, GmbH
ChemStar, Plzeń
Humosoft, Praha
Spectra Stable Isotope Group
Merck, s.r.o. Praha
Scientific Instruments Brno, s.r.o.
SciTech®, s.r.o. Praha
Sigma-Aldrich, Praha
RototecSpintec, Ramshalten
Varian NMR Instruments
2
PROGRAMME
Monday April 8, 2002
Afternoon 2 p.m.
14:00
Opening of the Conference
14:10
Markéta Munzarová, Vladimír Sklenář:
Parameterization of the Karplus Equation for the Glycosidic Torsion in Nucleic
Acids: Ab initio Study.
14:25
Lukáš Trantírek, Erik Caha, Michael Akke, Radovan Fiala:
Base and Sugar Dynamics in GCAA RNA Hairpin Tetraloop.
14:40
Petr Padrta, Lukáš Žídek, Vladimír Sklenář:
Refinement of d(GCGAAGC) Hairpin Structure Using One- and Two-Bonds
Residual Dipolar Couplings.
14:55
Josef Chmelík, Petr Padrta:
Evaluation of Protein Torsion Angles Using Program MULDER.
15:10
Monika Nálezková, Josef Chmelík, Lukáš Žídek:
Structural Studies of Major Urinary Protein I
15:25
Karin Hohenthanner, Guido Pintacuda, Norbert Müller:
Progress in Application of Cross Correlated Curie Spin Relaxation for Structure
Determination in Paramagnetic Proteins.
15:40
Break
16:15
Richard Hrabal, Václav Veverka, Jan Lang, Helena Bauerová, Iva Pichová:
Progress in Structural Study of Protease from Mason-Pfizer Monkey Virus.
16:30
Václav Veverka, Jan Lipov, Michaela Rumlová, Tomáš Ruml, Richard Hrabal:
NMR Study of Phenotypically Relevant Mutants of Matrix Protein from MasonPfizer Monkey Virus.
16:45
Dana Kurková, Jaroslav Kříž, Pavel Schmidt, Jiří Dybal, José Carlos Rodríguez-
3
Cabello, Matilde Alonso:
Inverse temperature transition in poly(Gly-Val-Gly-Val-Pro) and poly(Ala-ValGly-Val-Pro) studied by NMR spectroscopy.
17:00
Jiří Brus, Jiří Dybal:
1
H-1H Interatomic Distance Measured by Multidimensional CRAMPS
Experiments.
17:15
Gábor Szalontai:
Residual Dipolar Couplings in 31P and 29Si MAS Spectra of Co(I) Complexes.
18:00
Dinner
20:00
Get-together party in wine cellar sponsored by Varian
Tuesday
April 9, 2002
Morning 8.30 a.m.
8:30
Jiří Vlach, Veronika Michlová, Jan Budka, Jan Lang:
Determination of Calix[4]arene Complex Geometry Using Full Relaxation
Matrix.
8:45
Hana Dvořáková, Jan Lang, Pavel Lhoták:
NMR Study of Conformational and Dynamic Properties of Thiacalix[4]arenes: the
Pinched Cone/Pinched Cone Conversion.
9:00
Tamás Gáti, Gábor Tóth, Helmut Duddeck, Shahid Malik:
Complexation of Selenium to (R)-Rh2(MTPA)4: Thermodynamics and
Stoichiometry by 1H, 13C and 77Se NMR.
9:15
József Kovács, Gábor Tóth, András Simon, Albert Lévai, Erich Kleinpeter:
Stereochemistry of 3-Arylidene-1-thioflavan-4-on sulfoxides and sulfones by 1H,
13
C, 17O NMR and Quantum-Chemical Calculations.
9:30
9:45
Antonín Lyčka, Jaroslav Holeček:
O NMR Spectra of Some Organotin(IV) Acetates.
17
Tomáš Lébl, Jaroslav Holeček
The Investigation of Intramolecular Donor-Acceptor Interaction in
3-Methoxypropylstannanes by Means of Long-Range nJ(1H-119Sn) Coupling
Constants.
4
10:00
Break
10:30
Ján Imrich, Karel D. Klika, Naďa Prónayová, Juraj Alföldi, Tibor Liptaj, Juraj
Bernát, Pavol Kristian, Kalevi Pihlaja, Mária Vilková, Eva Balentová:
NMR Properties of New Spirocyclic and Heterocyclic Acridine Derivatives.
10:45
Radek Marek, Marcela Lukášková, Jaromír Toušek, Jiří Brus, Zdeněk Trávníček:
Protonation and Tautomerism of 6-Benzylaminopurine Derivatives.
11:00
Franc Perdih, Alojz Demšar, Janez Košmrlj, Andrej Pevec:
NMR Studies of the Solvation of Lithium Cation in [{Li(Cp*2Ti2F7)(L)}2].
11:15
Miroslav Holík, Miloslav Nechvátal:
Computations with Three-dimensional Matrix of Chemical Shift Data.
12:00
Lunch
14:00
Excursion to the 'Cross' wine cellar
16:30
Information about new products of our sponsors
18:30
Dinner
20:00
Meeting of the NMR Discussion Groups - refreshment sponsored by Bruker
and Scientific Instruments Brno
Wednesday April 10, 2002
Morning 8.30 a.m.
8:30
V. Mlynárik, Z. Starčuk, Z. Starčuk, Jr., S. Gruber, E. Moser:
Proton NMR Spectroscopy of Human Brain at 3 Tesla.
8:45
Zoltán Berente, Erzsébet Ősz, Balázs Sümegi:
In vivo and in situ Metabolic Studies Using Magnetic Resonance Spectroscopy
(MRS) and Imaging (MRI).
9:00
Milan Mazur:
5
„Lens Effect“ in Magnetic Resonance Spectroscopy.
9:15
Lothar Brecker, Ulrich Zähringer:
High Resolution NMR Analysis of Lipid A – A Methodological Approach
Towards the NMR Analysis of a Charged Amphiphile.
9:30
Christian Steindl, Christina Schäffer, Paul Messner, Norbert Müller:
NMR – Structure Elucidation of the Carbohydrate Portion of the Secondary Cell Wall
Polymer From Aneurinibacillus Thermoaerophilus Dsm 10155.
9:45
Vilko Smrečki, Norbert Müller :
A Density Functional Theory Study of N-15 Chemical Shielding Anisotropy Tensors in
Peptides.
10:00
Gy. Batta, F. Sztaricskai, S.S. Printsevskaya, M.N. Preobrazehnskaya:
Cell-Wall Analogue Induced Effects in Glycopeptides.
10:15
M. Kuzma, P. Sedmera, V. Havlíček, A. Jegorov:
Some New Cyclic Peptides and Depsipeptides.
10:30
Jiřina Bohdálková, Ervín Kozubek, Miroslav Kaloč:
NMR Spectroscopy of Bituminous Coal Tars.
10:45
Martin Albert, Petra Feiertag, Gertraud Hayn, Helmut Hönig and Robert Saf:
Polyguanidine Biocides: A Comparative 1H-, 13C- And 15N-NMR Study
Complemented by MALDI-TPF-MS Spectra.
11:00
Closing of the Conference
6
ABSTRACTS
7
PARAMETERIZATION OF THE KARPLUS EQUATION FOR THE
GLYCOSIDIC TORSION IN NUCLEIC ACIDS:
AB INITIO STUDY.
Markéta Munzarová and Vladimír Sklenář
National Center for Biomolecular Research, Faculty of Science,
Masaryk University, Kotlářská 2, CZ-611 37 Brno, Czech Republic
NMR vicinal spin-spin coupling constants 3J(C2/4-H1') and 3J(C6/8-H1') have been studied
theoretically for deoxyribonucleosides as well as several modified bicyclo-nucleosides as a
function of the torsion angle χ. Geometry of purine and pyrimidine nucleosides have been
optimized at the DFT (density functional theory) level keeping the exocyclic backbone torsion
angles frozen to their average experimental values and varying the glycosidic torsion angle χ. On
the optimized structures, the DFT calculations of the spin-spin coupling constants have been
performed using the combination of the FPT method (for the FC term) and the SOS_DFPT
approach (for the PSO, DSO, and the SO terms) as implemented in the program deMon.[1]
Our calculations provide separate parameterizations of the Karplus equation for the vicinal C-H
couplings for all of the deoxyribonucleosides studied. The results demonstrate that, contrary to
approaches adopted earlier, different parameterizations are to be used for different nucleosides.
For a given χ, the differences in the spin-spin coupling between different nucleosides reach up to
2 Hz. Vicinal spin-spin coupling across the glycosidic bond significantly differs for pyrimidines
as compared to purines (in the syn region, the maximum of 3J(C6/8-H1') is 4.0 Hz for G but 5.8
Hz for C). At the same time, also the particular purine/pyrimidine base has a significant influence
(in the syn region, the maximum of 3J(C2/4-H1') is 5.9 Hz for C but 7.2 Hz for T).
Another interesting result of our study is the observation of an "anti-Karplus" behavior of the
vicinal coupling for some of the bases. The spin-spin coupling is thus more effective for the synperiplanar than for the anti-periplanar arrangement of the coupled nuclei. This and other
interesting features of the vicinal coupling mentioned above have been interpreted using the
qualitative-MO approach together with the quantitative analysis of contributions to the coupling
obtained by the FPT-DFT approach. An interesting through-space contribution to the spin-spin
coupling has been identified as the source of the anti-Karplus behavior for cytidine, as opposed to
guanosine.
References
[1] deMon-NMR program: Salahub, D. R.; Fournier, R.; Mlynarski, P.; Papai, I.; St-Amant, A.;
Ushio, J. In Density Functional Methods in Chemistry; Labanovski, J.; Andzelm, J., Eds.;
Springer, New York, 1991.
8
BASE AND SUGAR DYNAMICS IN GCAA RNA HAIRPIN TETRALOOP
Lukáš Trantírek1, Erik Caha1, Mikael Akke2 and Radovan Fiala1
1
National Centre for Biomolecular Research, Kotlářska 2, CZ-611 37 Brno,
Czech Republic
2
Department of Biophysical Chemistry, Lund University, Box 124, SE-221 00,
Lund, Sweden
A full understanding of the structure-function relationship of RNAs requires characterization of
both static structure and conformational dynamics of the molecule. Hairpins with tetraloops of
GNRA family (where N is any nucleotide and R is purine) are the most frequently occurring
hairpins in biologically active RNAs. These hairpins are particularly stable and often play
important roles in tertiary contact, in RNA folding and as protein binding sites.
In order to bring further insight into the internal dynamics of RNAs we have performed a 13C
NMR relaxation study of a 14-mer hairpin including GCAA tetraloop. We have measured R1 and
R1ρ relaxation rates for C8 of purines, C2 of adenines, C6 and C5 of pyrimidines as well as for
C1’ of the ribose sugars at several magnetic field strengths.
The data has been interpreted in the framework of modelfree analysis [1, 2] characterizing the
internal dynamics of the molecule by order parameters and correlation times for fast motions on
picosecond to nanosecond time scale and by contributions of chemical exchange. The fast
dynamics reveals a rather rigid stem and a significantly more flexible loop. The cytosine and the
last adenine bases in the loop as well as all the loop sugars exhibit a significant contribution of
conformational equilibrium on microsecond to millisecond time scale. The high R1ρ values
detected on both base and sugar moieties of the loop indicate coordinated motions in this region.
A semiquantitative analysis of the conformational equilibrium suggests the exchange rates on the
order of 104 s−1. The results are in general agreement with dynamics studies of GAAA loops by
NMR relaxation [3] and fluorescence spectroscopy [4].
References
1
G. Lipari and A. Szabo, J. Am. Chem. Soc. 1982, 104, 4546-4559.
2
G. Lipari and A. Szabo, J. Am. Chem. Soc. 1982, 104, 4559-4570.
3
C. G. Hoogstraten, J. R. Wank and A. Pardi, Biochemistry 2000, 39, 9951-9958.
4
M. Menger, F. Eckstein and D. Porschke, Biochemistry 2000, 39, 4500-4507.
9
REFINEMENT OF D(GCGAAGC) HAIRPIN STRUCTURE USING ONEAND TWO-BONDS RESIDUAL DIPOLAR COUPLINGS
Petr Padrta, Lukáš Žídek, Vladimír Sklenář
National Centre for Biomolecular Research. Faculty of Science, Masaryk University,
Kotlářská 2, CZ-611 37, Brno, Czech Republic
e-mail:[email protected], [email protected]
The structure of the 13C,15N-labeled d(GCGAAGC) hairpin, as determined by NMR spectroscopy
and refined using molecular dynamics with NOE-derived distances, torsion angles, and residual
dipolar couplings (RDCs), will be presented. Although the studied molecule is of small size, it
will be demonstrated that the incorporation of diminutive RDCs [1] can significantly improve
local structure determination of regions undefined by the conventional restraints. The effect of
individual types of NMR restraints on the structure will be demonstrated by presenting several
preliminary MD calculations. The quality of the calculated structures will be discussed while
comparing the values of atomic RMSD, of the alignment tensor, and violations of experimental
restraints,. The final structure will be discussed in context of the extraordinary stability [2-4] of
the d(GCGAAGC) hairpin.
References
1.
2.
3.
4.
Žídek, L., et al., Journal of Biomolecular NMR, 2001. 21: p. 153 - 160.
Hirao, I., et al., Nucleic Acids Research, 1994. 22(4): p. 576-582.
Yoshizawa, S., et al., Nucleic Acids Research, 1994. 22(12): p. 2217-2221.
Yoshizawa, S., et al., Biochemistry, 1997. 36(16): p. 4761-4767.
10
EVALUATION OF PROTEIN TORSION ANGLES USING PROGRAM
MULDER
Josef Chmelík, Petr Padrta
National Centre for Biomolecular Research. Faculty of Science, Masaryk University,
Kotlářská 2, CZ-611 37, Brno, Czech Republic
e-mail: [email protected], [email protected]
3
J–couplings carry one of the most important structural informations – torsion angles. As first
described by Karplus [1], the magnitude of a 3J–coupling constant is a function of the torsion
angle θ:
3
J(θ) = A cos2 (θ + phase) + B cos (θ + phase) + C.
The Karplus equation can have up to four torsion angle solutions. The degeneracy may be
removed or at least partially reduced if some additional information about the torsion angle is
known. Thus 3J-couplings may be used directly as restraints in molecular dynamics
calculation(s), together with other types of NMR restraints, letting the MD care about the
degeneracy internally. This approach, however convenient, because it saves additional data
manipulation, tends to be rather non-transparent as the rejection of false solution(s) in the course
of numerical optimization is difficult to trace. Nevertheless, the degeneracy may be also dealed
with independently of MD by exloiting several 3J-couplings, solving appropriate Karplus
equations and looking for those solutions common to all solved equations. This alternative
approach has been implemented in program MULDER (P. Padrta, manuscript in preparation)
and presented in last year Valtice (16th Valtice). At that time, the program has been tested mostly
on nucleic acids but in the mean time, the program proved to be general enough to work also on
other biologically interesting molecules. This presentation will show application of program
MULDER to proteins where it can be used for determination of backbone torsion angles φ and ψ
(Table 1) and, in favorable cases, even for torsion angles in side chains, e.g. torsion angle χ1 can
be determined from 3J(NHβ) and 3J(NCβ).
Table 1: Determination of φ and ψ backbone angles from 3J–couplings
Angle
φ
φ
φ
φ
φ
φ
ψ
ψ
Coupling Phase
3
J(HNC’) -120°
3
J(HNCβ)
60°
3
α
J(H C’)
-60°
3
J(HNHα)
-60°
3
J(C’Cβ) -120°
3
J(C’iCβi-1)
0°
3
α
J(NiH i-1) -120°
3
J(NiNi+1)
0°
References
1. M. Karplus, J. Phys. Chem. 30, 11–15 (1959)
11
STRUCTURAL STUDIES OF MAJOR URINARY PROTEIN I
Monika Nálezková, Josef Chmelík, Lukáš Žídek
National Centre for Biomolecular Research, Masaryk University, Kotlářská 2, 611 37 Brno
MUP-I is a member of the lipocalin superfamily of proteins, it is abundant in urine of the Mus
musculus and its main biological function is to bind feromones, like 2-sec.-butyl-4,5dihydrothiazole.
High resolution NMR spectroscopy has become one of the most important methods for the
determination of 3D structure of biological macromolecules and for the description of the
dynamic properties of molecules of biological interests. Binding site of the complex of major
urinary protein I (MUP-I) with pheromone have been studied and its full structure determination
is in progress.
Assignment of backbone and side chain resonances has been completed. A set of spectra
providing structure information has been recorded. This set includes 13C/15N edited-NOESY,
HNCA[CB]-E.COSY, HNCA[HA]-E.COSY, HNCA[CO]-E.COSY, HNCACB[CO]-E.COSY,
Cα-coupled HNCO and CO-coupled IPAP 15N HSQC (the last two spectra recorded with
isotropic and partially aligned samples). Currently, structural parameters including NOEs,
3
J(HNCβ), 3J(HNHα), 3J(HNC‘), 3J(CβC’i-1), 1D(NHN), 1D(CαC‘), 1D(NC‘), 2D(C’HN) are being
extracted from the spectra.
References
1
L. Žídek et. al., Biochemistry, 1999, 38, 9850-61.
12
PROGRESS IN APPLICATION OF CROSS CORRELATED CURIE SPIN
RELAXATION FOR STRUCTURE DETERMINATION IN
PARAMAGNETIC PROTEINS
Karin Hohenthanner1, Guido Pintacuda2, and Norbert Müller1
1
Institut für Chemie, Johannes Kepler Universität, A-4040 Linz, Austria
2
MBB, Karolinska Institutet, S-17177 Stockholm, Sweden
Cross correlation effects in NMR relaxation can be exploited for sensitivity improvement (e.g.
TROSY experiments) and also to derive molecular geometry information. Paramagnetic
interactions, in particular Curie spin relaxation (CSR), the susceptibility contributions of
paramagnetic centres to nuclear relaxation, is source of substantial cross-correlation effects.
We have recently introduced a new approach to exploit cross-correlated CSR for structure
determination in proteins[1]. Interference effects of CSR and dipolar (DD) relaxation in
paramagnetic systems are relatively large and depend on orientation of bonds relative to the
electron center, which is external to the nuclear spin system. This angular dependence gives
information on the orientations of internuclear vectors relative to this center. The effects decrease
only with the third power of the distance from the paramagnetic center. However due to
experimental error and uncertainty about the superimposed CSAxDD cross correlation effects a
practical distance limit to the application exists.
We have investigated means to push that limit to longer distances from the electronic centre. In
this we have addressed both systematic and statistical errors of the data evaluation and made use
of the anisotropic paramagnetic shifts to improve the interpretation of the data.
Acknowledgement
This research has been supported by the Austrian Science Funds (FWF) projects P12696-CHE
and P15380.
[1] P. K. Madhu, Rita Grandori, Karin Hohenthanner, Pravat K. Mandal, and Norbert Müller;
Geometry dependent two-dimensional heteronuclear multiplet effects in paramagnetic
proteins; J. Biomol. NMR 20, 31-37 (2001)
[2] P. K. Madhu, Pravat K. Mandal and Norbert MüllerCross-correlation Effects Involving Curie
Spin Relaxation in Methyl Groups, J. Magn. Reson., in print (2002)
13
PROGRESS IN STRUCTURAL STUDY OF PROTEASE FROM MASONPFIZER MONKEY VIRUS.
Richard Hrabala, Václav Veverkaa, Jan Langa, Helena Bauerováb, and Iva Pichováb,
a
Laboratory of NMR Spectroscopy, Institute of Chemical Technology, Technická 5,
166 28 Prague 6, Czech Republic, e-mail: [email protected]
b
Department of Biochemistry, Institute of Organic Chemistry and Biochemistry, Academy of
Sciences of the Czech Republic, 166 10 Prague 6, Czech Republic
As a continuation of our effort to obtain a structure of the shortest (12 kDa) form of
protease from Mason-Pfizer monkey virus we will present our most recent results. Based on the
calculation of chemical shift index (CSI) of assigned backbone resonances (Hα, Cα, and CO) we
managed to find out secondary structure elements along the whole amino acid sequence (Veverka
et al. 2001). Their positions were further refined by the identification of typical sequential and
long-range NOE contacts. In the next step, a set of X-edited and doubly edited (X=
15
N) NOESY spectra were acquired to assign more side chain 1H and
13
13
C and/or
C resonances and to
identify more NOE interactions to provide a computational system ARIA (Nilges et al., 1997)
with decent input data. The current progress of computing the structure of the protease will be
presented. Relaxation rates of the backbone 15N nuclei have been measured, namely R1, R2 and
heteronuclear 1H-15N NOE. The processing of this data is in progress and the some preliminary
results will also be presented.
The work has been supported by the Grant Agency of the Czech Republic (Grant 203/00/1241)
References
1. M. Nilges, J. Macias, S.I. O’Donoghue, H. Oschkinat, J.Mol.Biol., 1997, 269, 408-422.
2. V. Veverka, H. Bauerová, A. Zábranský, I. Pichová, and R. Hrabal, J. Biomolecular NMR,
291-292 (2001).
14
NMR STUDY OF PHENOTYPICALLY RELEVANT MUTANTS OF
MATRIX PROTEIN FROM MASON-PFIZER MONKEY VIRUS
Václav Veverkaa, Jan Lipovb, Hana Dvořákováa, Michaela Rumlovác,
Tomáš Rumlb, c and Richard Hrabala
a
Laboratory of NMR Spectroscopy, bDepartment of Biochemistry and Microbiology,
Institute of Chemical Technology, Technická 5, 166 28 Prague 6, Czech Republic,
c
Department of Biochemistry, Institute of Organic Chemistry and Biochemistry, Academy of
Sciences of the Czech Republic, 166 10 Prague 6, Czech Republic
Matrix protein (MA) is important not only for correct folding of Gag poly-proteins but
also for their targeting and interactions during self-assembly of immature capsids in retroviruses.
Several functionally characterized MA mutants affect different steps of intracellular transport,
membrane interaction and budding [1].
We have focused on three mutants of MA from Mason-Pfizer monkey virus. R55F
mutation results in reversion of the type of assembly from D-type to a C-type, i.e. capsids are
assembled at the plasma membrane instead of the intracytoplasmic assembly typical for MasonPfizer monkey virus [2]. Mutation A18V results in formation of transport deficient immature
capsids that are accumulated in cytoplasm. The last is a double mutant T41I/T78I that has a
unique phenotype: It assembles capsids and trans-ports them to the plasma membrane with wild
type kinetics. However, it is defective in initiating the process of budding through the membrane.
The project that we have recently launched should provide us with an answer to the
following question: Are the changes of morphogenesis of Mason-Pfizer monkey virus caused
only by local structural changes of the matrix protein or by larger changes of the global fold of
this protein? Within the frame of the project three mutants of the matrix protein will be subjected
to comparative structural studies.
We will present complete resonace assignment of R55F mutant. From chemical shift
index we estimated approximate positions of secondary structure elements. We found out the
prevalence of helical motifs (5 α-helices) and no signs for the presence of regular β-structure.
References
1
S. S. Rhee, E. Hunter, EMBO J., 1991, 10(3), 535-546
2
S. S. Rhee, E. Hunter, Cell, 1990, 5, 63(1), 77-86
15
INVERSE TEMPERATURE TRANSITION IN POLY(GLY-VAL-GLY-VALPRO) AND POLY(ALA-VAL-GLY-VAL-PRO) STUDIED BY NMR
SPECTROSCOPY.
Dana Kurková1, Jaroslav Kříž1, Pavel Schmidt1, Jiří Dybal1, José Carlos Rodríguez-Cabello2,
Matilde Alonso3
1
Institute of Macromolecular Chemistry, Academy of Sciences of Czech Republic, 162 06 Prague 6, CR 2Department
of Condensed Matter/E.T.S. I.I, University of Valladolid, 47011 Valladolid, 3SpainDepartment of Analytical
Chemistry, E. U. P., University of Valladolid, 47014 Valladolid, Spain
Two types of polymers, poly(Gly-Val-Gly-Val-Pro) and poly(Ala-Val-Gly-Val-Pro),
[Gly, Val, Pro, Ala means glycine, valine, proline, alanine aminoacids], water-soluble below the
critical temperature but undergoing a reversible inverse temperature transition leading to
conformational change and aggregation at about 28 oC, were studied by NMR.
We performed 1H, 13C, 15N NMR spectral and T1 and T2 relaxation studies of both
polymers in D2O and H2O. Relaxation of HOD in the system in D2O was also studied. Pulsedgradient spin-echo (PGSE) was used to measure self-diffusion coefficients of polymers and HOD
and thus the size of polymer molecules. Temperature-induced conformation changes were probed
using chemical shifts (1H, 13C, 15N), 3JH-H coupling constants and NOE factors.
Both polymers form rigid parts above the transition temperature stabilized by inner
CO…NH hydrogen bonds. These are more frequent in the case of poly(Ala-Val-Gly-Val-Pro),
where they prevent chemical exchange of NH protons with water. A part of water molecules is
confined in the loops formed by the polymer and thus hindered in motion. In poly(Gly-Val-GlyVal-Pro), the inner CO…NH bridges are less frequent compared with poly(Ala-Val-Gly-ValPro); consequently, chemical exchange between water molecules and NH protons is less
restricted. However, confinement and hindered motion of a part of water molecules are also
observable.
T1 and T2 relaxation studies show that CH3 groups of valine residues are in both polymers
in two forms: (a) the first has a similar temperature dependence of correlation time of the
rotation/translation movement (τc) as water molecules (HOD) in the system; the proportion of
this type decreases with increasing temperature; (b) the other does not correlate with the motions
of water and its τc is shorter than in (a). With increasing temperature, the proportion of this form
increases. From (a) it follows that water molecules are released from the vicinity of CH3 groups
with increasing temperature in both types of polymers, which is in accord with the previous
results – vibrational studies and model calculations. From comparison of the correlation times τc
of motion of the form (a) in both polymers it follows that at higher temperatures, τc is longer in
poly(Gly-Val-Gly-Val-Pro) compared with poly(Ala-Val-Gly-Val-Pro). This means that the
motion of measured valine CH3 groups of poly(Gly-Val-Gly-Val-Pro) at higher temperatures is
slower compared with poly(Ala-Val-Gly-Val-Pro), which is in accord with lower rigidity of the
globules in the former case.
Acknowledgement: The authors wish to thank the Grant Agency of the Czech Republic (grant No. 203/00/1320), the Grant
Agency of the Academy of Sciences of Czech Republic (grant No. A4050208), to the “Junta de Castilla y León“ (Programmes
VA30/00B) and to the “Comission Interministerial de Ciencia y Tecnologia/CICYT” (Programme MAT 98-0731) for financial
support.
16
1
H-1H INTERATOMIC DISTANCE MEASURED BY
MULTIDIMENSIONAL CRAMPS EXPERIMENTS
Jiří Brus, Jiří Dybal
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic,
162 06 Prague 6, Czech Republic, Tel.: +420 2 20403380; fax: +420-2-3535981;
e-mail: [email protected] .
In general, 1H spin diffusion is highly useful and efficient NMR technique to investigate structure
properties of polymer systems. Experiments probing geometry on the nm length scale are well
established. Crucial limitation of theses techniques follows from the low resolution of 1H NMR
spectra and that is why predominantly large length scale characteristics such as morphology,
degree of mixing various components or size of domains were investigated up to now. The
increase in spectral resolution making possible to determine 1H-1H intramolecular distances has
been recently achieved by Lee-Goldburg homodecoupling sequences leading to a design of
highly resolved 3D 1H-1H-13C experiments [1] or by indirect detection of the 1H-1H spin
exchange process via 13C resonances leading to 2D 13C-13C correlation experiments [2,3].
In this contribution we show limitations of these techniques and suggest the ways to overcame
them.
1. Serious complication resulting from local molecular motions even in highly rigid crystalline
organic solids strongly affect accurate determination of interatomic distances. It will be
demonstrated that without the exact knowledge of molecular dynamics the determination of 1H1
H distances is highly uncertain.
2. Fast spin diffusion during cross-polarization steps used for coherence transfer to 13C spins in
the 2D 13C-13C correlation experiment lead to intermolecular correlation and relayed transfer.
This causes undesired coherence pathways affecting quantitative evaluation of spin exchange in
the case of short mixing times. That is why we performed some modification of the 2D 13C-13C
correlation experiment to avoid 1H-1H spin diffusion during cross-polarization steps making
possible to measure short interatomic distances.
References:
1. D. Sakellariou, A. Lesage, L. Emsley, J. Am. Chem. Soc. 123, 5604 (2001).
2. F. M. Mulder, W. Heinen, M. van Duin, J. Lugtenburg, H. J. M. de Groot, J. Am. Chem. Soc.
120, 12891 (1998).
3. M. Wilhelm, H. Feng, U. Tracht, H. W. Spiess, J. Magn. Reson., 134, 255 (1998).
17
RESIDUAL DIPOLAR COUPLINGS IN 31P AND 29SI MAS SPECTRA OF
COBALT COMPLEXES
Gábor Szalontai
University of Veszprém, NMR laboratory, H-8200 Veszprém, Pf.158. Hungary
Spin-spin interaction between spin-1/2 and quadrupolar nuclei has been the subject of several
recent solid-state NMR studies1,2. The MAS experiment cannot average the dipolar interaction
between such spin-pair to zero because the quadrupolar nucleus is not solely quantized by the
applied external magnetic field, but also by the anisotropic quadrupolar interaction. Therefore the
easiest way to look at the effect is to record the MAS spectrum of the spin-1/2 nucleus. Spin pairs
such as 13C-14N, 13C-35,37Cl, 31P-63,65Cu, 119Sn-35,37Cl, 31P-35,37Cl, 13C-2H, etc. have been studied in
detail.
Under favourable conditions (first-order perturbation theory applies, J and q tensors have axial
symmetry, the dipolar and scalar coupling main axises coincide) scalar and dipolar couplings or
the sum of them (note that normally Jiso is not available from solution spectra) can be obtained
for the spin-pair involved.
We have studied in particular the characteristic of 31P-59Co (I=7/2) spin-pair in mono-, bi- and
trinuclear Co complexes and clusters. An example for the 29Si-59Co pair, not reported so far, will
also be given. At the same time no fine structure or second-order shift but line broadening was
observed for the 31P-105Pd (I=5/2) spin-pairs of A-frame dimeric palladium complexes.
31
P MAS (121.4 MHz) spinning rate: 5700 Hz
7
S
8
CO
CO
1 2 3 4 5 6
PPh 3
1
160
150
140
29
120
C
Co
Co
S
S
CO
CO
PPh 3
C
SC(CH 3)3
31
J( Co- P) ≅ 440 Hz
130
SC(CH 3)3
110
100
90
ppm
Several examples be shown for small and larger second-order quadrupolar shifts to illustrate the
effect and to help the interpretation of such spectra.
References
(1)
R.K.Harris, A.Olivieri, Progress in NMR Spectrocopy, 24, 435 (1992)
(2)
D.Freude, J.Haase, NMR Basic Principles and Progress, Vol.29. Spinger, Berlin, 1999.
18
DETERMINATION OF CALIX[4]ARENE COMPLEX GEOMETRY USING
FULL RELAXATION MATRIX.
Jiří Vlach,a Veronika Michlová,b Jan Budka,b Jan Langa
a
Laboratory of NMR Spectroscopy, bDepartment of Organic Chemistry
Institute of Chemical Technology, Technická 5, Prague 6, Czech Republic
Calixarenes are popular building blocks in supramolecular chemistry. They possess useful
features, such as facile synthesis, broad derivatization potential, well defined geometry, ability to
interact with ions and neutral guests, etc. [1]. However, most of the calixarene-based ligands
have been prepared so far to interact with cations rather than with anions.
The recently synthesized calixarene host-molecule 1 {5,17-bis(N'-fenylureido)25,26,27,28-tetrapropoxycalix[4]arene} [2] is designed to form complexes with anions
specifically. We focus on the structure determination of the complex of 1 with o-toluate anion.
Series of 2D-NOESY spectra are analyzed using the full relaxation matrix approach.
Bu4N+
HN
NH
HN
O
O
O
O
CH3
NH
PrO
-
PrO OPr
OPr
compound 1
References
[1] Calixarenes: A Versatile Class of Macrocyclic Compounds, Vinsen J., Böhmer V., Eds.;
Kluwer, Dordrecht 1991
[2] Budka J., Lhoták P., Michlová V., Stibor I.: Tetrahedron Lett., 2000, 42, 1583
19
NMR STUDY OF CONFORMATIONAL AND DYNAMIC PROPERTIES
OF THIACALIX[4]ARENES: THE PINCHED CONE/PINCHED CONE
CONVERSION
Hana Dvořákováa, Jan Langa, Pavel Lhotákb
a
Laboratory of NMR Spectroscopy, bDepartment of Organic Chemistry, Institute of Chemical
technology, Technická 5, 166 28 Praha 6, Czech Republic
Thiacalix[4]arenes [1], new members of supramolecular “family”, posses many interesting
properties when compared with the “clasical” calixarenes. The presence of sulphur atoms instead
of methylene groups has changed dramatically the conformational and complexation behaviour
of the thia analogs.
Thiacalix[4]arenes as well as their parent compounds can adopt four extreme conformations
in the solution: cone, partial cone, 1,3-alternate and 1,2-alternate. The symmetrical cone
conformation, usually observed in 1H NMR spectra, is in fact time averaged structure of two
pinched cone conformations possesing lower C2ν symmetry. In contrast to methylene bridged
calix[4]arenes the transition between the two pinched cones of some thiacalix[4]arenes (R = Me,
Et, Pr) is readily ovservable by NMR techniques [2].
S
S
O
R
S
O O
R
R
C2v
pinched cone
S
O
R
S
S
O
R
S
O O
R
R
S
O
R
S
S
S
O
O O O
R
R
R
R
S
C2v'
C4
cone
pinched cone
Dynamic NMR measurments were used to obtain thermodynamic parameters of the pinched
cone/pinched cone interconversion. The rate constants were determined using complete line
shape analysis and exchange spectroscopy (2D EXSY).
The activation parameters are strongly dependent on the solvent. The effect of interaction of
both calix[4]arene types (thia and methylene bridged) with solvent molecules has been
qualitatively investigated by the use of temperature dependent 1H NMR measurments in
chloroform, 1,1,2,2-tetrachloroethane, chlorobenzene and dimethylformamide.
References
1
H. Kumagai, M. Hasegawa, S. Miyanari, Y. Sugawa, Y. Sato, T. Hori, S. Ueda, H.
Kamiyama, S. Miyano, Tetrahedron Letters, 1997, 38, 3971- 3972.
2
J. Lang, H. Dvořáková, I. Bartošová, P. Lhoták, I. Stibor, R. Hrabal, Tetrahedron
Letters, 1999, 40, 373- 376.
20
COMPLEXATION OF SELENIUM TO RH2[(R)-MTPA]4:
THERMODYNAMICS AND STOICHIOMETRY BY 1H, 13C AND
77
SE NMR.
Tamás Gáti1, Gábor Tóth1, Helmut Duddeck2 and Shahid Malik2
1
Technical Analytical Research Group of the Hungarian Academy of Sciences, Institute for General and Analytical Chemistry of the Technical University, Szt. Gellért tér 4, H-1111 Budapest, Hungary
2
Universität Hannover, Institut für Organische Chemie, Schneiderberg 1B, D-30167 Hannover, Germany
R
R
O
O
R
Rh
O
O
O
R
O
Rh
R
O
O
+
L
O
O
R
Rh*
Rh
O
O
O
O
Rh
O
R
O
OMe
L
R= C
Ph
CF3
R
1
3
2
Se
1
Organoselenium compounds play an increasing role in modern stereo- and regioselective
reactions. It is often necessary to monitor the composition of mixtures of enantiomers. The
dirhodium complex Rh2[(R)-MTPA]4 (Rh*, MTPA-H ≡ methoxytrifluoromethylphenylacetic
acid, Mosher's acid) is a potent solvating agent for the determination of enantiomeric ratios of
various chiral ligands (L). This “dirhodium method“ is particularly suitable for soft-base
functionalities such as epoxides, olefines, nitriles, selenides and iodides where the classical
method using chiral lanthanide shift reagents (CLSR) fails.
A study of the adducts of 3-phenylselenenyl-1-phenyl-1-propene (1) with Rh* will be presented. An investigation of that system at different complex-ligand ratios and different temperatures showed that the equilibra (see formula scheme) are strongly shifted towards the adduct, and
both 1:1- and 1:2-adducts are formed. In the case of selenide excess the system favours the 1:2adduct, and intermolecular selenide exchange can be observed. This exchange process can be
monitored by variable-temperature 1H and 77Se NMR spectoscopy and the energy barrier
estimated. The results of some other selenide ligands will be discussed, too.
In addition, a powerful indirect NMR method – {77Se} 1H HMBC spectroscopy – will be
presented for observing 77Se resonances.
This work was supported by the Deutsche Forschungsgemeinschaft and the Hungarian Academy of Sciences (436
UNG 113/148).
21
STEREOCHEMISTRY OF 3-ARYLIDENE-1-THIOFLAVAN-4-ON
SULFOXIDES AND SULFONES BY 1H, 13C, 17O NMR AND QUANTUMCHEMICAL CALCULATIONS
József Kovács1, Gábor Tóth1,András Simon1, Albert Lévai2 and Erich Kleinpeter3
1
Institute for General and Analytical Chemistry of the Technical University, Szt. Gellért tér
4, H-1111 Budapest, Hungary
2
Department of Organic Chemistry, University of Debrecen, H-4010 Debrecen, P.O.Box 20,
Hungary
3
Universität Potsdam, Institut für Organische Chemie und Strukturanalytik, P.O.Box 691553,
D 14415 Potsdam, Germany
We have recently reported on stereoselective epoxidation of E-3-arylidene-1-thioflavanones with
nucleophilic oxidants, viz. alkaline hydrogen peroxide and sodium hypochloride that resulted
trans,cis (1) and trans,trans (2) epoxides.1,2
Oxidation of the separated trans,cis and trans,trans epoxide isomers with a slight excess
dimethyldioxirane (DMD) produces sulfoxide derivates (3 and 4), whereas application of five
equivalents DMD yielded the sulfon compounds (5 and 6).
O
O
O
S
Ph
S
Ar
O
O
Ph
DMD
Ar
H
O
1
S
O
DMD
Ph
O
3
O
O
2
O
S
Ar
Ph
H
O
4
O
O
Ph
Ar
DMD
H
O
O
6
Now we report on the determination of the isomers, the preferred conformations and the
configuration of the sulfoxide group by NMR methods and by ab initio MO calculations (HF/631G*). The research was supported by Hungarian National Science Foundation (OTKA D032830
and T029171).
References
[1] G. Tóth, J. Kovács, A. Lévai E. Kleinpeter, A. Koch, Magn. Reson. Chem. 2001, 39, 251-258.
[2] 15th NMR Valtice, J. Kovács, G. Tóth, A. Lévai and B. Balázs, pp. 28, 2000.
22
H
5
S
Ar
DMD
O
Ar
H
O
S
Ph
H
17
O NMR SPECTRA OF SOME ORGANOTIN(IV) ACETATES
a
Antonin Lyčkaa and Jaroslav Holečekb
Research Institute for Organic Syntheses, CZ-532 18 Pardubice-Rybitvi, Czech Republic
[email protected]
b
Department of General and Inorganic Chemistry, University of Pardubice,
CZ-532 10 Pardubice, Czech Republic
Purely organic esters provide two signals in their
17
O NMR spectra as shown in Fig. 1. On the
contrary, the only a single resonance was observed for both oxygen atoms of the –COOSn groups
in 17O NMR spectra of
17
O enriched dibutyltin(IV) acetate (Fig. 2) and dibutyltin(IV) diacetate
in coordinating and non-coordinating solvents in wide temperature range1.
Figure 1:
17
O NMR spectra of tert-butyl acetate in CD2Cl2
Figure 2:
17
O NMR spectra of tributyltin(IV) acetate in CD2Cl2
The explanation of the existence of only single 17O signal in 17O NMR spectra of
enriched tributyltin(IV) acetate and dibutyltin(IV) diacetate will be presented.
17
O isotope
Acknowledgement: The authors thank the Grant Agency of the Czech Republic (Grant No.
203/00/0920) for financial support.
References
Lyčka A., Holeček J.: Magn. Reson. Chem. 2002, 40, 289.
23
THE INVESTIGATION OF INTRAMOLECULAR DONOR-ACCEPTOR
INTERACTION IN 3-METHOXYPROPYLSTANNANES BY MEANS OF
LONG-RANGE nJ(1H-119Sn) COUPLING CONSTANTS
Tomáš Lébl, Jaroslav Holeček
Department of General and Inorganic Chemistry, University of Pardubice, Čs Legií, 565, 532 10
Pardubice, Czech Republic, e-mail: [email protected]
For 3-methoxypropylstannanes of general formula (CH3OCH2CH2CH2)xSnCl4-x where x = 1-4
the structures in the solution of non-coordinating (CDCl3) and coordinating (dmso-d6) solvent
were determined by means of multinuclear NMR spectroscopy. In the case of
3methoxypropyltin dichloride (x = 2) and trichloride (x = 3), X-ray diffraction was carried out,
too.1
The structural study revealed that the strength of O→Sn intramolecular donor-acceptor
interaction increases with Lewis acidity of central tin atom, i.e. with number of coordinated
chlorine atoms.
This paper deal with long-range nJ(1H-119Sn) coupling constants acquired for
3-methoxypropylstannanes by means of 1H-119Sn J-HMBC spectroscopy.2 The values of longrange nJ(1H-119Sn) coupling constants will be discussed as novel tool for evaluation of the
strength of O→Sn donor-acceptor interaction.
References
1 T. Lébl, A. Smička, C. Bruhn, Eur. J. Inorg. Chem., submitted.
2 J. C. Martins, M. Biesemans, R. Willem, Prog. NMR Spectrosc., 2000, 36, 271- 322.
24
NMR PROPERTIES OF NEW SPIROCYCLIC AND HETEROCYCLIC
ACRIDINE DERIVATIVES
Ján Imricha, Karel D. Klikab, Naďa Prónayovác, Juraj Alföldid, Tibor Liptajc, Juraj Bernáta, Pavol
Kristiana, Kalevi Pihlajab, Mária Vilkováa, and Eva Balentováa
a
Department of Organic Chemistry, Faculty of Science, P. J. Šafárik University, Moyzesova 11, SK-041
67 Košice, Slovak republic, E-mail: [email protected]
b
Department of Chemistry, University of Turku, SF-20014 Turku, Finland
c
Faculty of Chemical and Food Technology, Central Laboratories, Slovak Technical University,
Radlinského 9, SK-81237 Bratislava, Slovak Republic
d
Chemical Institute, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84238 Bratislava, Slovak
Republic
9-Isothiocyanatoacridines 1 known as fluorescent, intercalating and biologically active
compounds afford with nucleophilic agents new classes of spirocyclic hetero- cycles of a
dihydroacridine-9(10H),4´-thiazoli(di)ne type 3, 5, 6, 8. Depending on conditions used,
alternative products 4, 7 and 9 were also obtained. Analogous reaction of 9-aminomethylacridine
with isothiocyanates and bromoacetonitrile afforded a new type of heterocycle,
spiro[dihydroacridine-9(10H),2′-(2′,7′-dihydro-3′H-imidazo[1.2-c]thiazol-5′-ylidene-amine] 10,
and imidazolidine 11 as a side-product.
R
COOMe
MeOOC
S
CH3 X
O
N
R1
2
N
H N
R1
R2
4
X = O, S
R1 = H, Cl
R1
N
H
R2
3
N
5
R2 = H, Me
R2 = H, Me, Br
R3 = H, Me, Meo
R2
S
S
R1
R = Bn,i -Pr, 2-furylmethyl
cyclohexyl, t-Bu
R1 = COOMe, CN
S
N
R
R3
N
N
93
S
H
N
N
N
N
8
R1 = COOMe, CN
R = Bn,i -Pr, 2-furylmethyl
S
N
R1
N
H
7
6
R1 = COOMe, CN
R2 = H, Cl, Me
N
S
N
N
H
R3
R1 = COOMe, CN
R3 N
N
R1
R2
N
N
H
S
S
O
O
N
R-HN
S
N
H
R2
10
R1 = H, Br R2 = H, Me, MeO
R = p-NO2Ph, p-BrPh
R
N
H
11
R = subst. Ph
NMR
studies allowed assorting of acridine structures into several classes which were extensively
studied. The data obtained can be applied for prediction of the structure, ionic state and
stereochemistry of new 9-acridinyl compounds.
R3 = Me, Bn
References
1. P. Kristian, J. Bernát, J. Imrich, et al.: Molecules, 1996, 1, 181.
2. K. D. Klika, J. Bernát, J. Imrich, et al.: J. Org. Chem., 2001, 66, 4416.
The authors are grateful to the Ministry of Education of the Slovak republic and the Grant Agency for
Science for the financial support (VEGA grant No. 1/9245/02).
25
PROTONATION AND TAUTOMERISM OF
6-BENZYLAMINOPURINE DERIVATIVES
Radek Mareka, Marcela Lukáškováa, Jaromír Toušekb,
Jiří Brusc, Zdeněk TRÁVNÍČEKd
a
National Center for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská
2, CZ - 61137 BRNO, Czech Republic; Email [email protected]
b
Department of Theoretical and Physical Chemistry, Faculty of Science, Masaryk University,
Kotlářská 2, CZ - 61137 BRNO, Czech Republic
c
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic,
Heyrovského nám. 2, CZ - 16206 PRAGUE, Czech Republic
d
Laboratory of Growth Regulators, Institute of Experimental Botany AS, Palacký University,
Šlechtitelů 11, CZ - 78371 OLOMOUC, Czech Republic
Analogues of 6-benzylaminopurine have been extensively investigated for their biological
activity (inhibition of cyclin-dependent kinases) [1]. The nitrogen atoms of the purine derivatives
are the centers of primary interactions with biomolecules. Due to the presence of several nitrogen
atoms in these compounds, the knowledge of tautomeric equilibria and protonation sites is of
great importance for determining their reactivity and biological activity. 15N NMR spectroscopy
is a very sensitive probe for studying the tautomeric equilibria and protonation processes [2].
The 15N NMR parameters were measured by inverse and direct
methods in solution at various temperatures. 15N CP/MAS data of
selected compounds were recorded in order to study the principal
values of 15N chemical shifts [3]. Ab initio calculations of nitrogen
chemical shifts were used for assigning the nitrogen resonances
observed in the solid-state spectra and for determining the
orientation of principal components of the chemical shift tensors
[3]. The partial results of this project including solution-state
temperature measurements will be discussed [4].
X
HN
N
N
1
N
N
3
7
9
H
This work was supported by the Ministry of Education of the Czech Republic (LN00A016) and the Grant Agency of
the Czech Republic (203/00/0152).
References
1
L. Havlíček, J. Hanuš, J. Veselý, S. Leclers, L. Meijer, G. Shaw, M. Strnad, J. Med. Chem.,
1997, 40, 408.
2
R. Marek, A. Lyčka, Curr. Org. Chem., 2002, 6, 35.
3
R. Marek, J. Brus, J. Toušek, L. Kovács, D. Hocková, Magn. Reson. Chem., 2002, 40, 353.
4
R. Marek, M. Lukášková, J. Brus, J. Toušek, J. Marek, Z. Trávníček, K. Doležal,
unpublished results.
26
NMR STUDIES OF THE SOLVATION OF LITHIUM CATION IN
[{Li(Cp*2Ti2F7)(L)}2]
Franc Perdih, Alojz Demšar, Janez Košmrlj, Andrej Pevec
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000
Ljubljana, Slovenia
In the last decade important advance was achieved in fluoro chemistry. New suitable
fluorinating reagents allow preparations of cyclopentadienyl substituted fluorides of group 4
metals with high yields [1]. This improved synthesis made possible further to investigate the
interactions with other inorganic compounds. The most common ligand in our research group is
[Cp'2Ti2F7]- anion (Cp' – substituted cyclopentadienyl), which forms complexes with several
cations. The presence of fluorine atoms in the first coordination shell of the metal cation enables
us to study the dinamic behaviour of the metal centre by 19F NMR spectroscopy. This approach
was found useful in the lithium complex [(Cp*TiF3)4(LiF)] [2] and we applied it also to another
type of lithium complex [{Li(Cp*2Ti2F7)(L)}2] (1), where L is THF, H2O, HMPA, DMSO,
dioxane and 12-crown-4. In the solid state the title compounds consist of dimeric units, while two
temperature dependant sets are found in variable temperature (VT) 19F NMR studies, which
reveal a solution equilibria between monomeric (Cp*2Ti2F7)Li (2) complex and its adduct with
the ligand 2·L. We also employed quantum mechanical calculations (HF, B3LYP) in order to
study the interactions between Li[Cp2Ti2F7] and selected ligands.
Cp*
F
F
Ti
F
F
Cp*
Li
F
Ti
F
L
F
F
Ti
F
F
Li
L
1
F
F
F
Cp*
dissociation
F
F
F
Ti
F
F
Cp*
F
Ti
Cp*
F
Ti
F
Cp*
Li
F
Ti
Cp*
2·L
F
L
F
F
F
Li
+ L
F
Ti
Cp*
F
2
References
1 Reviews: (a) H. W. Roesky, Inorg. Chem., 1999, 38, 5934-5943; (b) H. W. Roesky, I. Haiduc,
J. Chem. Soc., Dalton Trans., 1999, 2249-2264.
2 A. Demsar, A. Pevec, L. Golič, S. Petriček, A. Petrič, H. W. Roesky, Chem. Commun., 1998,
1029-1030.
27
COMPUTATIONS WITH THREE-DIMENSIONAL MATRIX
OF CHEMICAL SHIFT DATA.
Miroslav Holíka and Miloslav Nechvátalb
a
Department of Theoretical and Physical Chemistry, Faculty of Science, Masaryk University,
Kotlářská 2, CZ 61137 Brno, [email protected],
b
Pliva-Lachema, Karásek 1, CZ 62133 Brno,[email protected]
H-1 NMR chemical shifts of five methyl groups in four substituted benzamides I (R = H, Cl, Br,
I ) were measured in six solvents ( C2Cl3F3, C2Cl4, C4Cl6, CCl4, C2D2Cl4, and CDCl3).
R
CH3
N(CH3 ) 2
CH3
C
I
O
CH3
The data forming 3-dimensional matrix DM(5,4,6) were worked up by different methods to get
insight into the substituent and solvent effects in the molecular system.
In addition to averaging and Principal Component analysis also the Fourier Transform of the data
and calculations of confidence ellipses were used [1,2]. The resulting vectors were correlated to
some substituent and solvent constants like σI, σR, π*, etc.
References
[1]
[2]
M.Holík, J.Halámek: Confidence Ellipses for the Quantitative Analysis from the
Whole NMR Spectrum, 16th NMR Valtice, 23.-25.4.2001, The Abstract Book, p.44.
M.Holík, J.Halámek, M.Chmelařová: J.Near Infrared Spectrosc., in print.
28
PROTON NMR SPECTROSCOPY OF HUMAN BRAIN
AT 3 TESLA
V. Mlynárik,a Z. Starčuk,b Z. Starčuk, Jr.,b S. Gruber,a E. Mosera
a
Institute of Medical Physics, University of Vienna, Währingerstr. 13, A-1090 Vienna, Austria;
Institute of Scientific Instruments, Academy of Sciences of Czech Republic, Brno, Czech
Republic
b
Until now, proton MR spectra of human brain have been almost exclusively measured on 1.5
Tesla scanners. Due to the development of 3 Tesla clinical scanners, it was desirable to test
specific features of proton spectroscopy at this field and to optimise measurement protocols.
Experiments on animals at 11 T indicated1 that the high field proton spectra of human brain
should have had much better resolution at higher fields. However, it has been observed that the
expected improvement of spectral dispersion in human measurements on 4 T and 7 T research
scanners2,3 was considerably compromised by an increase of a spectral line width. To explain this
finding, T1 and T2 relaxation times of spectral lines of major brain metabolites were measured on
a 3 Tesla Bruker MEDSPEC system.4 It was found that the T1 relaxation times of the metabolites
did not change substantially with increasing magnetic field while the T2 relaxation times
decreased in comparison with the 1.5 T data. However, the decrease in T2 did not justify the
observed increase in the line width.
In spite of broader spectral lines, the spectra measured at a higher field offer more accurate and
reliable information on metabolite concentrations. Higher signal-to-noise ratio enables to reduce
the volume of the tissue necessary for obtaining high quality spectra in a reasonable period of
time. This is especially useful for multivoxel acquisition techniques such as spectroscopic
imaging. By proper phase and/or Hadamard encoding of the excited volume the nominal voxel
size can be decreased down to 0.1 cm3 or less. The signal-to-noise ratio at such extremely high
resolution is enhanced by a relative decrease of the line width.5 High resolution spectroscopic
imaging allows to study small and irregular anatomic or pathological structures in human brain. It
has been demonstrated that the addition of the spectra corresponding to a specific part of a
tumour can provide a spectrum with a sufficiently high signal-to-noise ratio and free from partial
volume effects.
References
1. Tkáč I., Starčuk Z., Choi I.Y., Gruetter R. In vivo 1H NMR spectroscopy of rat brain at 1 ms echo time. Magn.
Reson. Med. 41, 649-656-(1999).
2. Gruetter R., Weisdorf S.A., Rajanayagan V., Terpstra M., Merkle H., Truwit C.L., Garwood M., Nyberg S.L.,
Ugurbil K. Resolution improvements in in vivo 1H NMR spectra with increased magnetic field strength. J. Magn.
Reson. 135, 260-264 (1998).
3. Tkáč I., Andersen P., Adriany G., Merkle H., Ugurbil K., Gruetter R. In vivo 1H NMR spectroscopy of the human
brain at 7 T. Magn. Reson. Med. 46, 451-456 (2001).
4. Mlynárik V., Gruber S., Moser E. Proton T1 and T2 relaxation times of human brain metabolites at 3 Tesla. NMR
Biomed. 14, 325-331 (2001).
5. Moser E., Gruber S., Mlynárik V. High resolution 3D 1H MRS of the human brain at 3 Tesla. Part I: SNR and
linewidth vs. spatial resolution. Magn. Reson. Med., submitted.
29
IN VIVO AND IN SITU METABOLIC STUDIES USING MAGNETIC
RESONANCE SPECTROSCOPY (MRS) AND IMAGING (MRI)
Zoltán Berente, Erzsébet Ősz, Balázs Sümegi
30
"LENS EFFECT" IN MAGNETIC RESONANCE SPECTROSCOPY
Milan Mazúr
Department of Physical Chemistry, Faculty of Chemical and Food Technology,
Slovak Technical University, Radlinského 9, SK-812 37 Bratislava, Slovakia.
The response of the cavity to the insertion of the samples with variable wall thickness of
the sample tubes and a quartz Dewar inside the Bruker single TE102 rectangular cavity has been
analyzed. The experimental dependencies of the EPR signal intensity on the wall thickness of the
quartz sample tube, δ, for the line-like samples with identical length of the sample material
columns, L = 30 mm, recorded in the empty microwave cavity showed the following: A directly
proportional increase of the relative "lens effect" to the increase of the wall thickness of the
sample tubes in the interval, δ ∈ <0.1, 0.5 mm>, which can be approximated by the linear
function (correlation, r ≈ 0.98) with identical intercept, p ≈ 1.0, for both diameters of the sample
material columns of 1 and 1.3 mm, but different slope, q, which increased from 0.03 to 0.07 mm1 as the diameter of the sample material column increased from 1 to 1.3 mm. It is obvious that:
(i) In the case of the identical sample material columns, the relative "lens effect" increased
linearly with the increase of the wall thickness of the quartz sample tubes from 0.1 to 0.5 mm. (ii)
In the case of the identical wall thickness of the sample tubes, the relative "lens effect" increased
with the increase of the diameter of the sample material column from 1 to 1.3 mm. The insertion
of the variable-temperature double-walled quartz Dewar (home-built, resonant frequency shift, ca
- 300 MHz) inside the single TE102 rectangular cavity showed the following: An identical
relative "lens effect" of the quartz Dewar, ca 1.5, within experimental error of 1.4 % or less, for a
point-like sample and all line-like samples with material column diameter of 1 and 1.3 mm,
length of 30 mm, and wall thickness of the sample tubes, δ ∈ <0.1, 0.5 mm>. Each of the above
phenomena may be a serious source of significant errors in quantitative EPR spectroscopy. Linelike samples to be compared in quantitative EPR should have identical wall thickness of the
sample tubes, and quantitative EPR spectra must be recorded in the identical quartz Dewar when
there is one inserted inside the microwave cavity.
31
HIGH RESOLUTION NMR ANALYSIS OF LIPID A –
A METHODOLOGICAL APPROACH TOWARDS THE NMR
ANALYSIS OF A CHARGED AMPHIPHILE
Lothar Brecker and Ulrich Zähringer
Research Center Borstel, Division of Immunochemistry,
Parkallee 22, D-23845 Borstel, Germany
Lipid A constitutes the lipid component of bacterial lipopolysaccharide (LPS, endotoxin). It is
covalently bound to the polysaccharide and known as the endotoxic principle of LPS. Some
isolated lipid A show endotoxic activities when tested in human mononuclear cells, whereas
others have no or even LPS antagonistic activities. For understanding these different lipid A
receptor interactions a systematic knowledge about dissimilar primary and secondary structures
of lipid A is required.
However, the tendency of the amphiphilic lipid A to form micelles in several solvents restricts
the NMR spectroscopic efficiency. Therefore we tested hexa-acyl lipid A from Escherichia coli
in several solvents with the intention to determine conditions that lead to the best possible
disaggregation and consequently to the optimal line resolution, signal form and signal separation.
CDCl3, dioxan-d8 or pyridin-d5, each mixed with 25% (vol.) methanol-d4 and DMSO-d6 are the
organic solvents, that lead to the best line resolution. In D2O a 0.1 M addition of N(C2D5)3 cause
an intensive improvement of the spectral resolution and makes NMR spectra accessible. Spectra
taken in all these solvents allow an univocal determination of the lipid A configuration.
In all these solvents we investigate line width at half high, transversal relaxation time and
longitudinal relaxation time of proton signals from different parts of the molecule. Differences
between the spin-lattice and spin-spin relaxation time indicate that aggregates are formed in all
solvents. However T2 time variation of selected protons in different solvents indicate dissimilar
tumbling of some molecular parts and give hints to the lipid A aggregation form. Additionally
nuclear Overhauser effects and coupling constants between several nuclei allow conclusions
about the conformation, that is obviously different in all solvents.
Furthermore the influences of short T2 times on spectra with long homo- or heteronuclear
transversal magnetization transfer have been examined. For ROESY spectra we demonstrate that
the initial linear rate approximation of the growing ROE at short mixing times is blotted out by
cross relaxation at mixing times of ~200 ms. In HMBC spectra we correlate strong and weak
cross peaks of 2J- and 3J-couplings with long and short transverse relaxation times of the
affiliated protons, respectively. These findings indicate the limitation of lipid A NMR analysis.
The variety of suitable solvents enables more systematic determination of lipid A configurations
by NMR. Conformational aspects are actually coming into the focus of interest and will make
molecular modeling obvious to verify structural details. Most interesting solvent in future will be
D2O, as water is the natural solvent and lipid A receptor interaction can only be clarified in the
natural environment.
32
NMR – STRUCTURE ELUCIDATION OF THE CARBOHYDRATE
PORTION OF THE SECONDARY CELL WALL POLYMER FROM
ANEURINIBACILLUS THERMOAEROPHILUS DSM 10155
Steindl Christian1, Schäffer Christina2, Messner Paul2, Müller Norbert1
1
Institut für Chemie, Johannes-Kepler-Universität Linz, A-4040 Linz,
Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für
Molekulare Nanotechnologie, Universität für Bodenkultur Wien, A-1180 Wien
2
The surface-layer (S-layer) glycoprotein, the peptidoglycan and the secondary cell wall polymer
(SCWP) are the major glycosylated cell wall components of Aneurinibacillus thermoaerophilus
DSM 10155 [1].
By means of one- and two-dimensional nuclear magnetic resonance spectroscopy, we have
achieved the first structure description of a branched SCWP.
It contains similar repeating trisaccharides in each branch. The non-reducing end of each branch
is built by a single 2-acetamido-2-deoxy-α-D-glucopyranoside unit (α-D-GlcpNAc).
In addition, there is another repeating disaccharide unit, containing the branching point, which is
linked to carbon 6 of muramic acid of the peptidoglycan sacculus via an additional α-D-GlcpNAc
unit and, probably, a pyrophosphate bridge [2].
The anomeric configurations of the glycoses could be shown by a non-decoupled HSQC
experiment, receiving 13C-1H coupling constants of ~165 Hz for β-configuration and ~172 Hz
for α-configuration.
After assignment of 1H and 13C shift values the glycosidic linkages were determined by NOE,
ROESY and, in some cases, HMBC (long-range HSQC) spectra.
The complete structure of the carbohydrate portion of the SCWP, determined in this work, is
proposed to be:
α-D-GlcpNAc
1
↓
3
[β-D-ManpNAc-(1→4)-β-D-Glcp-(1→3)-α-D-GlcpNAc]2
1
↓
4
[β-D-ManpNAc-(1→3)- α-D-GlcpNAc-(1→3)]2- α-D-GlcpNAc-(1-O)-R
3
↑
1
[β-D-ManpNAc-(1→4)-β-D-Glcp-(1→3)-α-D-GlcpNAc]2
3
↑
1
α-D-GlcpNAc
References
1
P. Messner & C. Schäffer, in (R.Doyle, ed.) Glycomicrobiology, Kluwer Academic /
Plenum Press, New York, 2000, p. 93 - 125
2
C. Schäffer, N. Müller, P. K. Mandal, R. Christian, S. Zayni & P. Messner,
Glycoconjugate J., 2000, 17, 681 - 690.
33
A DENSITY FUNCTIONAL THEORY STUDY OF N-15 CHEMICAL
SHIELDING ANISOTROPY TENSORS IN PEPTIDES
Vilko Smrecki1,21 and Norbert Mueller1
1
Institute of Chemistry, Johannes Kepler University,
Altenbergerstr. 69, A-4040, Linz, Austria
2
Rudjer Boskovic Institute, Dept. Org. Chem. & Biochem.,
Bijenicka 54, POB 180, HR-10002 Zagreb, Croatia
Chemical shielding (or shift) anisotropy (CSA) is attracting increasing scientific interest in
particular in the context of structure determination of biomolecules. Current NMR techniques
enable more accurate determination of larger numbers of CSA's not only in solid-state NMR of
powder protein samples [1], but also in liquid state NMR both in isotropic solution [2] and in
fluid partially oriented media [3].
Renewed interest in cross-correlation phenomena involving CSA has arisen since these can be
exploited to derive structural constraints [4-6], using their dependence on the angle Θ spanned by
the principal axes of the respective interaction tensors as (3cos2Θ-1)/2.
As a prerequisite to structural interpretation of experimental CSA values we used Density
Functional Theory (DFT) calculations in order to quantitatively predict the influence of
secondary structure (phi and psi angles) on CSA tensors. The DFT calculations were conducted
using the Gaussian98 program package employing implemented methods, basis sets and
approaches (e.g., the 6-311G(d,p) basis set for geometry optimization, as well as frequency
calculation and 6-311++G(3df,3pd) basis set for NMR parameters calculation). The Becke's three
parameter hybrid functional using the Lee, Yang and Parr correlation functional (B3LYP) was
employed in DFT calculation., while the gauge-independence requirement for NMR parameter
calculation was treated with the Gauge-Invariant Atomic Orbital (GIAO) approach.
The first molecular model used in the simulation of secondary structure elements was a capped
dipeptide (Ac-Ala-Ala-NH2).
The results of DFT simulations of 15N CSA showing dependence on secondary protein structure
will be presented.
Acknowledgement
This work was supported by the Austrian Science Fond (FWF) project P15380. One of us (V.S.) would like to thank
the Austrian Exchange Service (OEAD) for the fellowship.
References
[1] Y. Wei, D.-K. Lee, and A. Ramamoorthy, J. Am. Chem. Soc., 2001, 123, 6118-26.
[2] N. Tjandra and A. Bax, J. Am. Chem. Soc., 1997, 119, 8076-82.
[3] G. Cornilescu and A. Bax, J. Am. Chem. Soc., 2000, 122, 10143-54.
[4] R. Brueschweiler, C. Griesinger, and R. R. Ernst, J. Am. Chem. Soc., 1989, 111, 8034-5.
[5] R. Brueschweiler and R. R. Ernst, J. Chem. Phys., 1992, 96, 1758-66.
[6] B. Reif, M. Hennig, and C. Griesinger, Science, 1997, 276, 1230-3.
1
Present address: #1
34
CELL-WALL ANALOGUE INDUCED EFFECTS IN GLYCOPEPTIDES
Gy. Batta, F. Sztaricskai, S.S. Printsevskaya, M.N. Preobrazhenskaya
Research Group for Antibiotics of the Hungarian Academy of Sciences, University of Debrecen,
Debrecen Egyetem tér 1, H-4010 HUNGARY
Gause institute of New Antibiotics, Moscow, B. Pirogovskaya 11, 119867 RUSSIA
For some time, glycopeptide antibiotics have been considered the last line of defense against
Methicillin Resistant Staphylococcus Aaureus (MRSA). However, vancomycin resistance of
Gram-positive bacteria is an increasingly emerging worldwide health problem. The mode of
action of glycopeptide antibiotics involves binding of peptidoglycan cell-wall fragments
terminating in D-Ala-D-Ala sequence to the carboxylate anion binding pocket of the antibiotic.
Simultaneously, dimerization (oligomerization) of the antibiotic enhances the antibacterial effect.
Oligomers associated with contiguous back to back and face to face dimers were observed in the
crystal structure of vancomycin bound to Nac-D-Ala; the question arises if they persist in
aqueous solution which more closely mimics in-vivo conditions.
References
1. P. J. Loll, R.Miller, C. M. Weeks, P. H. Axelsen, Chemistry & Biology 1998, 5, 293-298.
2. Gy. Batta, F. Sztaricskai, M. O. Makarova, E. G. Gladkikh, V. V. Pogozheva, T. F.
Berdnikova, Chemical Communications , 2001, 501-502.
35
SOME NEW CYCLIC PEPTIDES AND DEPSIPEPTIDES
Marek Kuzma, Petr Sedmera, Vladimír Havlíček,
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20
Prague 4, Czech Republic
Alexandr Jegorov
Galena Co., Branišovská 31, 370 05 České Budějovice,Czech Republic
Our continuing interest in cyclosporins fuelled by their alternative uses (treatment of rheumatic
arthritis, psoriasis, multidrug resistance) besides the reputed immunosuppressing power of
cyclosporin A lead us to discovery of several new representatives of this compound class.
Recently, we have elucidated the structures of two natural cyclosporins: cyclo[-MeBmt1-Abu2Sar3-MeLeu4-Abu5-MeLeu6-Ala7-D-Ala8-MeLeu9-MeLeu10-MeVal11-] and cyclo[-MeBmt1Abu2-Sar3-MeLeu4-Val5-MeLeu6-Ala7-D-Ala8-MeLeu9-Nva10-MeVal11-], where Bmt is (4R)-4((E)-2-butenyl)-4-methyl-L-threonin, Sar is sarcosin (methylalanin), and Nva is norvalin. The
amino acid composition was deduced from COSY and TOCSY. Sequence information was
determined in three independent ways: i) ROESY, ii) HMQC + HMBC, iii) from MS
(confirmation)1.
Investigation of depsipeptides from microbial strains known to produce biopesticides afforded
two new beauverolides: cyclo[-3-hydroxy-4-methyloctanoyl-Val-Ala-Leu-] and cyclo[-3hydroxy-4-methyloctanoyl-Tyr-Ala-Leu-] whose structures were solved by an analogous
manner2. “New” roseotoxin turned to be destruxin B, cyclo[-2-hydroxy-4-methylpentanoyl-ProIle-MeVaal-MeAla- β-Ala-], not known from this source before.
This work was supported by grants 203//00/1255 issued by Grant Agency of the Czech Republic
and AV0Z5020903 (Institutional Research Concept, Institute of Microbiology).
References
1. M. Kuzma, P. Sedmera, V. Havlíček, A. Jegorov, J. Nat. Prod., submitted.
2. M. Kuzma, A. Jegorov, P. Kačer, V. Havlíček, J. Mass Spectrom., 36, 1108 (2001).
36
NMR SPECTROSCOPY OF BITUMINOUS COAL TARS
Jiřina Bohdálková*, Ervín Kozubek*, Miroslav Kaloč**
*
Department of Analytical Chemistry and Material Testing, **Department of Chemistry, FMME,
VŠB – TU Ostrava, Czech Republic
Raw coking tar is defined as dense black liquid of characteristic odour that is complex
blend of various compounds, namely aromatic and heterocyclic ones. It is assumed that their total
number is about 10 000. Coal tar is a very important product of bituminous-coal coking
processing [1]. Basic technological operation of processing bituminous-coal tar is a distillation.
Particular fractions of the tar distillation (according to their increasing boiling points) are: light
oil, tar-acid oil, naphthalene oil, wash oil I, wash oil II, anthracene oil I, anthracene oil II,
anthracene oil III, pitch [2]. In this paper are shown the NMR spectra of the first two oils
obtained by distillation of so-called mixture of raw tars (MRT) from Deza, Valašské Meziříčí
(see Fig. 1,2). MRT consists of several raw materials. The main constituent of MRT is raw tar
from coking plants (80-90 vol. %), the others are mixture oil, recycled tar-acid oil and
impregnational oil.
The spectra were obtained by means of 80 MHz FT HR NMR Spectrometer Tesla situated
in the Department of Analytical Chemistry and Material Testing, FMME, VŠB – Technical
University Ostrava.
Fig. 1: 13C NMR – light oil
Fig. 2: 13C NMR – tar-acid oil
The first absorption maximum on the right side (see Fig. 1,2) corresponds to the
standard (HMDSA). The wedges occurring within the interval from 20 to 50 ppm show the
presence of aliphatic groups and other ones from 110 to 160 ppm represent aromatic and
heteroaromatic compounds. Absorption maxima about 80 ppm characteristic the deuterium
chloroform.
The authors wish to express their thanks to the Grant Agency of the Czech Republic (project no.105/00/1698) for its
financial support while dealing with this topic.
References
1 M. Kaloč, Průmyslový uhlík, VŠB-TU Ostrava, 1993, 38-46.
2 J. Vymětal, M. Plesník, Zpracování černouhelného dehtu a smoly, DEZA a. s., Valašské
Meziříčí, 1994, 6-142.
37
POLYGUANIDINE BIOCIDES: A COMPARATIVE 1H-, 13C- AND 15N-NMR
STUDY COMPLEMENTED BY MALDI-TOF-MS SPECTRA
Martin Alberta,§, Petra Feiertaga, Gertraud Haynb, Helmut Höniga* and Robert Safb
a
Institute of Organic Chemistry and bInstitute of Chemical Technology of Organic Materials,
Graz University of Technology, Stremayrgasse 16, A-8010 Graz, Austria.
There is a wealth of guanidine biocides on the market, the most widely used and best known
being chlorhexidine digluconate [1]. Poor water solubility, relatively high toxicity and some
other unfavorable properties thereof have prompted the search for alternatives in this powerful
class of disinfectants showing biocidal activities in the ppm range [2].
In this respect, several polymeric analogs have been synthesized and tested in the past decades
like PHMG (Korcid, PMG, Polisept) or PHMB (Arlagard E, BG-IR, Cosmocil CQ, Lonzabac BG
1, Polyhexanide, PP 073, Proxel IB, Reputex 20, Vantocil IB, Revacil) [3].
To our knowledge, the exact structures and molecular weight distributions of these substances
have never been studied in detail. This prompted the work reported in this contribution.
At first glance, the proton nmr spectra seemed to corroborate structures and degree of
polymerization estimated by intrinsic viscosity measurements [4]. More detailed 13C- and 15Nnmr investigations however gave indications of a more diverse and complex nature of these
substances. So, MALDI-TOF-MS was used to gain more insight into the molecular structures
involved. The MS spectra suggested four different main classes of structures of acyclic as well as
of cyclic nature. Up to our knowledge, cyclic structures were not thought of in the first instance.
However, the usual ambiguities of MS-spectra did not allow exact distinctions between
postulated isomeric structures.
Therefore, 13C-(guanidine) enriched species were synthesized and at least 6 different
environments of guanidine carbon atoms were detected, which could be assigned to a high extent.
Still not all minor components are identified, but the present study clearly indicated the necessity
to use multiple spectroscopic approaches to gain deeper insight into complex polymeric mixtures.
Financial support by P.O.C. Oil Industry Technology, Vienna is gratefully acknowledged.
§
Present address: Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1,
D-45470 Mühlheim an der Ruhr
References
[1] J. M. Ascenzi, Handbook of Disinfectants and Antiseptics, Marcel Dekker, New York, 1996,
p. 235 ff
[2] see e.g.: P. Werle, M. Trageser, R. Stober, DE 4002403 A1, 8.1.1991
[3] Chemical Abstracts Search, Dec. 2001.
[4] O. Schmidt, WO9954291 A1, 18. 10.1999
38
LIST OF PARTICIPANTS
39
BATTA Gyula
H
36
BERENTE Zoltán
H
31
BRECKER Lothar
D
33
BROŽKOVÁ Marie
CZ
—
BRUS Jiří
CZ
18, 27
DVOŘÁKOVÁ Hana
CZ
16, 21
FIALA Radovan
CZ
10
FISCHER Roland
A
—
GÁTI Tamás
H
22
HANYKOVÁ Lenka
CZ
—
HOENIG Helmut
A
39
HOLÍK Miroslav
CZ
29
HOLUB Josef
CZ
—
HRABAL Richard
CZ
15, 16
HULOVÁ Dagmar
CZ
—
HUMPA Otakar
CZ
—
CHMELÍK Josef
CZ
12, 13
ILLASZEWICZ Carina
A
—
IMRICH Ján
SK
26
KESSLER Pavel
D
—
KLEINPETER Erich
D
23
KLIŠOVÁ Marie
CZ
—
KOLONIČNÝ Alois
CZ
—
KOVÁCS József
H
23
KOZUBEK Ervín
CZ
38
KUBÍČKOVÁ Božena
CZ
—
KURKOVÁ Dana
CZ
17
KUZMA Marek
CZ
37
LÉBL Tomáš
CZ
25
LYČKA Antonín
CZ
24
MANNSCHRECK Albrecht
D
—
40
MAREK Radek
CZ
27
MAZUR Milan
SK
32
MAZÁČ Jiří
CZ
—
MLYNÁRIK Vladimír
SK
30
MÜLLER Norbert
A
14, 34, 35
NÁLEZKOVÁ Monika
CZ
13
NECHVÁTAL Miloslav
CZ
29
ŐSZ Erzsébet
H
31
PADRTA Petr
CZ
11, 12
PEJCHAL Vladimír
CZ
—
PELNÁŘ Jan
CZ
—
PERDIH Franc
SL
28
POTÁČEK Milan
CZ
—
PŮČEK Ladislav
CZ
—
RIEDL František
CZ
—
SCHOEFBERGER Wolfgang
A
—
SEČKÁŘOVÁ Pavlína
CZ
—
SEDMERA Petr
CZ
37
SIMON András
H
23
SKLENÁŘ Vladimír
CZ
9, 11
SMREČKI Vilko
HR
35
STEINDL Christian
A
34
SÜMEGI Balázs
H
31
SZALONTAI Gábor
H
19
TÓTH Gábor
H
22, 23
TUREČKOVÁ Milena
CZ
—
VEVERKA Václav
CZ
15, 16
VLACH Jiří
CZ
20
VONTOROVÁ Jiřina
CZ
38
VRÁTNÍČKOVÁ Zlata
CZ
—
WEBER Hansjoerg
A
—
41
ZELLHOFER Thomas
CH
—
ŽÍDEK Lukáš
CZ
11, 13
42