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CENTRAL EUROPEAN NMR DISCUSSION GROUPS

16th NMR Valtice

23.-25.4.2001

Valtice, Czech Republic

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THIS CONFERENCE IS SPONSORED BY

Bruker Analytik, GmbH

ChemStar, Plzen

Martek Biosciences Corp.

Merck, s.r.o. Praha

Scientific Instruments Brno, s.r.o.

SciTech®, s.r.o. Praha

RototecSpintec, Ramshalten

Varian NMR Instruments

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PROGRAMME Monday April 23, 2001 Afternoon 2 p.m. 14:00 Opening of the Conference 14:10 Hana Dvořáková, Marcela Tkadlecová, Tomáš Tobrman, Dalimil Dvořák. Conformational and D-NMR study of chelated (η2-diallylamino)carbene complexes of Cr, W and Fe. 14:25 Zdeněk Friedl and Svatopluk Zeman, Theoretical and 15N-NMR Study of Some High Energetic Nitramines. 14:40 Roman Jambor, Aleš Růžička, Jiří Brus, Jaroslav Holeček, Solution and CP/MAS NMR Investigation of Intramolecular Coordination Sn- N in Some Organotin(IV) C,N-Chelates. 14:55 V. Macháček, A. Lyčka, J. Kaválek, 17O NMR Spectra of Some Meisenheimer Adducts. 15:10 Radek Marek, Jiří Brus, Jaromír Toušek, N7- and N9-substituted purine analogues – 15N NMR study. 15:25 Tomáš Lébl, Jaroslav Holeček, Marek Dymák, Dirk Steinborn, Reactions of 2-functionalised vinylstannanes with acetyl bromide. 15:40 J. Pavlovský, E. Kozubek, P.Jelínek, L.Mokoš, J.Bohdálková, 29Si NMR Spectroscopy Studies of Selected Water Glass Samples for Foundry Technology. 15:55 Break 16:30 A. Perjéssy, W.-D. Rudorf, P. Meyer, E. Kolehmainen, D. Loos, K. Laihia, M. Nissinen, J. Koivisto, R. Kauppinen, Conformation and Transmission of Substituent Effects in Disubstituted Tetrahydropyridazine and Diazabicycloheptene Derivatives. 16:45 Andrej Petrič, Spectroscopic Properties vs. Molecular Geometry in a Series of Fluorescent Compounds. 17:00 Aleš Růžička, Libor Dostál, R.Jambor, J.Brus, J.Holeček, Structure of (2,6-Bis(dimethylamino)methyl)-diphenyltin(IV) derivatives. 17:15 Pavlína Sečkářová, Jiří Dostál, Roger Dommisse, Radek Marek, Structure elucidation of benzophenanthridine and protoberberine alkaloids. 17:30 J. Bohdalková, E. Kozubek, M. Peňáz, J. Pavlovský, NMR and GC-MS Analysis of Selected Coal Extracts 17:45 Alojz Demšar, NMR Study of the Solution Structures of C5Me5TiF3 18:30 Dinner 20:00 Get-together party in wine cellar sponsored by Varian

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Tuesday April 24, 2001 Morning 8 a.m. 8:00 Robert Glaser, Dror Shiftan, and Artem Novoselsky, Solid-State NMR Studies of Conformationally Polymorphic Crystalline Solids for Stereochemical Interpretation of Fast Exchange Limit Solution-State NMR Spectra. 8:30 Jaroslav Kříž, NMR studies of supramolecular self-assembly: pre-micellar aggregation of sodium p-dodecylbenzenesulfonate. 8:45 Jiří Spěváček, Jiří Brus, Solid state 1H CRAMPS NMR study of molecular complexes of poly(ethylene oxide) and benzene derivatives. 9:00 Radovan Fiala, Lukáš Trantírek, Erik Caha and Mikael Akke 13C Relaxation Study of RNA Loop Dynamics. 9:15 Richard Hrabal , Václav Veverka, Simplification of NMR spectra of Mason-Pfizer monkey virus protease. 10:00 Break 10:30 M.Hricovini, G.Torri, M.Guerrini and B.Casu, NMR analysis of protein-carbohydrate interactions through evaluation of chemical shifts, coupling constants and transferred NOEs. 10:45 Hana Křížová, Lukáš Žídek, Martin Stone & Vladimír Sklenář, NMR & Proteins Dynamics. 11:00 Dana Kurková, Jaroslav Kříž, Pavel Schmidt, José Carlos Rodríguez-Cabello+, NMR spectroscopy of an elastin-like polypeptide. 11:15 Václav Veverka, Helena Bauerová, Aleš Zábranský, Iva Pichová., Richard Hrabal, 1H, 13C, 15N Assignment and Secondary Structure Identification of the Protease from Mason-Pfizer Monkey Virus. 11:30 Petr Sedmera, Jindřich Volc, Ronald Mathä, Dietmar Haltrich, Biotransformations in NMR sample tube. 11:45 Daniela Suciu, Pulse NMR used in study of bound water in biological systems. 12:30 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

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Wednesday April 25, 2001 Morning 8 a.m. 8:00 Christoph Seger, Wolfgang Robien, CSEARCH online - a free spectrum estimation tool for structure elucidation utilizing stereochemical information. 8:15 Vladimír Sychrovský, Jaroslav Vacek, Pavel Hobza, and Vladimír Sklenář, DFT Calculation of NMR Spin-Spin Coupling Constants in Adenine, Thymine, Cytosine, and Guanine. 8:30 Petr Padrta, Vladimír Sklenář, MULDER – program for extracting of torsion angles from NMR data. 8:45 Erich Kleinpeter and Sabrina Klod, Ab-Initio MO Calculation of the Anisotropy Effect of Multiple Bonds and the Ring Current Effect of Arenes - Application in Conformational and Configurational Analysis. 9:00 Marek Kuzma, Petr Sedmera, 4J(H,C) - What are they good for? 9:15 Milan Mazúr, Quantity in Magnetic Resonance Spectroscopy. - Part 4. 9:30 Karin Hohenthanner, P. K. Madhu, Rita Grandori and Norbert Müller, Cross-correlation Effects in Paramagnetic Systems. 9:45 Zenon Starčuk, Zenon Starčuk, Jr., Jaroslav Horký, Water suppression – recent developments. 10:00 Jan Lang, Jiří Vlach, Hana Dvořáková, Pavel Lhoták and Richard Hrabal, Kinetic study of thermal isomerisation of 25,26,27,28-tetrapropoxy-2,8,14,20- tetrathiacalix[4]arene. 10:15 Herwig Häusler, Arnold. E. Stütz, Peter Greimel and Hansjörg Weber, Mechanistic Studies on d-Xylose (d-Glucose) Isomerase (EC 5.3.1.5): NMR Spectrometric Investigations. 10:30 Hana Navrátilová, Enantiomeric Analysis of (3S, 4R)-4-(4-Fluorophenyl)-3-hydroxymethyl-1- methylpiperidine by 19F NMR Spectroscopy. 10:45 Miroslav Holík and Josef Halámek, Confidence Ellipses for the Quantitative Analysis from the whole NMR Spectrum. 11:00 Closing of the Conference

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A B S T R A C T S

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CONFORMATIONAL AND D-NMR STUDY OF CHELATED (η2-DIALLYLAMINO) CARBENE COMPLEXES OF Cr, W AND Fe

Hana DVOŘÁKOVÁa, Marcela TKADLECOVÁb, Tomáš TOBRMANc, Dalimil

DVOŘÁKc

aLaboratory of NMR Spectroscopy, bDepartment of Analytical Chemistry, cDepartment of Organic Chemistry, Institute of Chemical Technology, Technická 5, 166 28 Prague

6, Czech Republic

Fischer carbene complexes of transition metals have found wide synthetic applications. Processes like the cyclopropanation1 and the metathesis or polymerisation of olefins2 are of equal industrial and theoretical importance. Significant intermediates in these reactions are carbene-(η2-alkene) complexes that are usually not stable and could be isolated only in exceptional cases. However, chelated (η2-allylamino)carbene complexes of Cr, W and Fe are stable and can therefore serve as a model for study of the properties of carbene-(η2-alkene) complexes of transition metals.

Among other interesting features of these systems restricted rotation around

aromate – carbene carbon bond has been observed. The aim of this study was to find the origin of the restricted rotation and to examine factors that influence activation parameters. Full line shape analyses of signals of diastereotopic ortho aromatic protons in carbene complexes of Cr, W and Fe was performed by the use of program gNMR 4.1. The values of activation parameters were calculated using exchange rate constants, which were determined in the temperature range of 233 - 313 K in CDCl3.

It was found that the energy barrier is caused by the steric hindrance of the N-substituent and ortho aromatic protons. The transition metal does not influence substantially the value of activation energy, however, the electronic nature of p- aromatic substituent is of a great significance. A special attention was also paid to determination of spatial orientation of complexed double bond that was found to be perpendicular to carbon – transition metal double bond. This work was supported by the Grant Agency of the Czech Republic (grant No. 203/00/0316). References [1] Doyle, M.P., Forbes, D. C. Chem. Rev. 1998, 98, 911. [2] Buchmeiser, M.R. Chem. Rev. 2000, 100, 1565.

M = Fe, W; R = HM = Cr; R = H, CH3, CF3, OCH3, CO2CH3, Br

(CO)nMN

R

11

THEORETICAL AND 15N-NMR STUDY OF SOME HIGH ENERGETIC NITRAMINES

Zdeněk FRIEDL and Svatopluk ZEMAN

Faculty of Chemistry, Brno University of Technology, Purkyňova 118, CZ-61200 Brno, Czech Republic (friedl@fch.vutbr.cz) and Department of Theory and Technology of

Explosives, University of Pardubice, CZ-53210 Pardubice, Czech Republic (svatopluk.zeman@upce.cz)

Electronic charges q at nitrogen atoms of twenty nitramines of general formula R1,R2-N-NO2 in the range from the simplest monomethylnitramine (MNA) to the sophisticated cage structure of 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-hexaazawurtzitane (HNIW, CL-20) were calculated at the ab initio HF/6-31G**//B3LYP/6-31G** level [1]. The relationships between δN chemical shifts of nitrogen atom in the nitro group of the nitramine and squares of detonation velocities or, as the case may be, the heats of explosion were confirmed [2-5]:

X = a δN + b where X can be the heat of explosion, Q [kJ kg-1 ], or square of detonation velocity, D2

[km2 s-2 ], and δΝ the 15N NMR chemical shifts corresponding to the primarily leaving nitro groups in the process of initiation of detonation. These relationships can be considered as an analogue of modified Evans-Polanyi-Semenov equation and such in the given manner directly specify the most reactive nitro group of nitramine molecule in the detonation and consequently the N-NO2 bond primarily split in this process. References 1 R. Huczala, S. Zeman, Z. Friedl, Proc. 4th Seminar “New Trends in Research of

Energetic Materials”, P07. Pardubice, April 2001. 2 S. Zeman, Propellants, Explos. Pyrotech., 2000, 25, 66. 3 S. Zeman, Thermochim. Acta, 1999, 333, 121-129. 4 S. Zeman, J. Energetic. Mat., 1999, 17, 305-329. 5 S. Zeman, Thermochim. Acta, 1992, 202, 191.

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SOLUTION AND CP/MAS NMR INVESTIGATION OF INTRAMOLECULAR COORDINATION Sn-N IN SOME

ORGANOTIN(IV) C, N- CHELATES

Roman JAMBOR, a Aleš RŮŽIČKA,a Jiří BRUS,b and Jaroslav HOLEČEKa

aDepartment of General and Inorganic Chemistry and, Faculty of Chemical Technology, University of Pardubice, Studentská 95, CZ-532 10, Pardubice, Czech

Republic, Fax: + 420 40 603 7068, E-mail: ales.ruzicka@upce.cz bInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic,

Heyrovsky sq.2, 162 06 Praha 6, Czech Republic

An intramolecular donor-acceptor Sn-N bonding connection in a set of triphenyl- and diphenyl-(halogeno)tin(IV) C,N-chelates, Ph2XSnL, where Ph = C6H5, X = Ph, Cl or Br and L1 = 2-(dimethylaminomethyl)phenyl-, C6H4(CH2NMe2)-2, and L2 = 2,6-bis[(dimethylaminomethyl)-phenyl]-, C6H3(CH2NMe2)2-2,6, respectively, was studied by 119Sn, 15N, 13C and 1H NMR spectroscopy in solution of non-coordinating solvent (CDCl3) and by 119Sn CP/MAS NMR techniques1 in the solid state. The existence of Sn-N co-ordination bonds was confirmed in studied compounds and their strengths were evaluated through the values of NMR spectra parameters of nuclei directly involved in Sn-N connection, namely by characteristic changes of chemical shifts δ(119Sn) and δ(15N) and values of J(119Sn,13C), J(119Sn,15N) and J(119Sn,14N) coupling constants.2

The set was extended by compound [2,6-C6H3(CH2NMe2)2]PhSnCl2 , that is the decomposition product of compound [2,6-C6H3(CH2NMe2)2]Ph2SnCl . This decomposition product was characterised by NMR spectroscopy and its structure was estimated by X-ray diffraction techniques. References: 1. R.K. Harris, A. Sebald, Organometallics 7 (1988) 388 2. A. Růžička, V. Pejchal, J. Holeček, A. Lyčka, K. Jacob, Collect. Czech. Chem.

Commun. 63 (1998) 977.

N

SnR1R2R3

N

SnR1R2R3

N

R1,R2,R3 = Ph, Cl, Br

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17O NMR SPECTRA OF SOME MEISENHEIMER ADDUCTS

Vladimír MACHÁČEK1, Antonín LYČKA2 and Jaromír KAVÁLEK1

1Department of Organic Chemistry, University of Pardubice, CZ-53210 Pardubice, Czech Republic

2Research Institute for Organic Syntheses, CZ-53218 Pardubice-Rybitví, Czech Republic

17O NMR spectra of the so-called Meisenheimer adducts (Scheme 1, Nu– stands for a nucleophile) were measured.

O2N NO2

NO2

+ Nu_

NuHNO2O2N

NO2 Scheme 1 The 17O chemical shifts (referred to external water) are collected in Tab. 1. Table 1: 17O Chemical shifts and half-widths of signals in 17O NMR spectra of 1,3,5-trinitrobenzene (TNB) and adducts 1, 2, and 3 Adduct, Nu δ(17O) 2,6-NO2 half-width (Hz) δ(17O) 4-NO2 half-width (Hz) Solvent TNB 581 550 581 550 a TNB 580 900 580 900 b 1, H 538 900 491 1400 a 2, CH2COCH3 534 1100 496 1500 a 3, OCH3 539 1700 493 2000 b a Acetonitrile, b DMSO-d6 - methanol. A higher upfield shift is exhibited by oxygen atoms of 4-nitro group (by about 90 ppm), lower ones by those of 2- and 6-nitro groups (about 40 ppm). The value of shift is independent of structure or bulkiness of the nucleophile bound at the tetrahedral centre C1 in the adduct, being also independent of the counter ion and solvent (acetonitrile or mixture of DMSO-d6 and methanol). The measured δ(17O) values indicate that in the Meisenheimer adducts the negative charge at oxygen atoms is larger in 4-NO2 than in 2- and 6-NO2 groups. All details are given in ref. [1]. A.L. thanks the Grant Agency of the Czech Republic for the financial support (Grant No.104/99/0835). References [1] V. Macháček, A. Lyčka, J. Kaválek, Magn. Reson. Chem., 2000, 38, 1001.

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N7- AND N9-SUBSTITUTED PURINE ANALOGUES – 15N NMR STUDY

Radek MAREKa, Jiří BRUSb, Jaromír TOUŠEKc

a National Center for Biomolecular Research, Faculty of Science, Masaryk University,

Kotlářská 2, CZ-61137 BRNO, Czech Republic; Email rmarek@chemi.muni.cz b Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic,

Heyrovského nám. 2, CZ - 16206 PRAGUE, Czech Republic c Department of Theoretical and Physical Chemistry, Faculty of Science, Masaryk

University, Kotlářská 2, CZ - 611 37, BRNO, Czech Republic

15N NMR chemical shifts of N7- and N9-substituted purine (guanine and adenine) analogues were systematically studied at natural abundance 15N level. NMR chemical shifts determined in liquid state were assigned using GHSQC, GHMQC, GHMBC and GSQMBC experiments [1]. 15N CP/MAS data of selected compounds were recorded in order to study the principal values of 15N chemical shifts. The calculations of chemical shifts (IGLO and GIAO) with the geometrical parameters obtained by X-ray analysis and/or RHF/6-31G** were used for assigning the nitrogen resonances observed in solid state spectra and for determining the orientation of the principal components of the chemical shift tensor. The results obtained [2] for N-3 and N-1 nitrogen atoms of compounds 1 and 2 are summarized in Table 1. Table 1a CP/MAS Compound Atom Liquid δ Calcul. δ δiso δ11 δ22 δ33

N-1 249.1 241.8 248.5 448.0 317.6 -19.9 1 N-3 256.5 265.8 255.5 462.5 321.7 -16.0 N-1 252.2 242.8 261.0 457.4 347.5 -13.1 2 N-3 234.3 228.7 233.0 449.0 273.0 -20.1

1: R1 = -N=CH-NMe2 , R2 = -H, R3 = -CH2CF3; 2: R1 = -N=CH-NMe2 , R2 = -H, R3 = -CH2CN a 15N NMR chemical shifts are reported relatively to the liquid ammonia This work was supported by the grant of Ministry of Education (LN00A016). References

1 R. Marek, A. Lyčka, Curr. Org. Chem., submitted. 2 R. Marek, J. Brus, J. Toušek, L. Kovács, D. Hocková, Magn. Reson. Chem., in

preparation.

N

N

N

N

R1

2RR3

N

N

N

N

R1

2R

R3

1 2

1

3

7

9

15

REACTIONS OF 2-FUNCTIONALISED VINYLSTANNANES

WITH ACETYL BROMIDE

Tomáš LÉBLa, Jaroslav HOLEČEKa, Marek DYMÁKa, Dirk STEINBORNb

a Department of General and Inorganic Chemistry, University of Pardubice, Cs. Legii 565, 532 10 Pardubice, Czech Republic

b Department of Inorganic Chemistry, Martin Luther Universitaet Halle-Wittenberg, Kurt-Mothes-Strasse 2, 06120 Halle (Saale), Germany.

In 1975 KAZANKOVA et al. reported on reactions of 2-functionalised vinylstannanes R”3SnCR’=CHYRn (R, R’, R” is alkyl or H, Y is O or N, n is 1 or 2) with acetyl bromide [1]. It is solitary report on heterolytic fragmentation of this type of compounds (Scheme 1a). However, for reactions of Ph3SnC(R’)=CHYRn (YRn = NMe2, OEt, SMe, SEt; R’ = Ph, n-Bu, n-pentyl, H) with acetic acid it was shown that electrophilic attack preferably takes place at α-carbon atom resulting in the cleavage of Sn-C= bonds (Scheme 1b) [2].

C CH

E

R' YRnR''3SnX +

R''3SnX C C H+ R' + EYRn

+ E XC CHR''3Sn

R' YRn

(a)

(b)E X = CH3CO Br or H OOCCH3

Scheme 1

In order to explain this disaccord the set of four analogous 2-functionalised vinylstannanes (E)/(Z)-Bu3SnCR’=CHOEt (R’ = n-Bu or H) was prepared and fully characterised using 1H, 13C, 119Sn, 1H-13C gs HMQC, 1H-13C gs HMBC and 1H-119Sn gs HMBC NMR spectroscopy. Investigations of reactions with acetyl bromide and acetic acid by means of NMR spectroscopy revealed that even for reactions of 2-functionalised vinylstannanes with acetyl bromide the cleavage of Sn-C= bond (Scheme 1b) is the most preferable pathway. The financial support from the Ministry of Education, Youth and Sports of Czech Republic (project LN 00 A028) is gratefully acknowledged. References 1 a) M.A. Kazankova, T.I. Zverkova, M.Z. Levin and I.F. Lutsenko, Zh. Obshch.

Khim., 1975, 45, 73. b) M.A. Kazankova, T.I. Zverkova and I.F. Lutsenko, Zh. Obshch. Khim. 1975, 45, 2044.

2 T. Lebl, J. Holecek, M. Dymak, D. Steinborn, J. Organomet. Chem., in press.

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29Si NMR SPECTROSCOPY STUDIES OF SELECTED WATER GLASS SAMPLES FOR FOUNDRY TECHNOLOGY

J. PAVLOVSKÝ *), E. KOZUBEK **), P. JELÍNEK *), L. MOKOŠ **), J. BOHDÁLKOVÁ **)

*) Department of Foundry Industry, **) Department of Analytical Chemistry and Material Testing, FMME, VŠB-TUO, 17. listopadu 15, 708 33

Ostrava-Poruba, Czech Republic

Water glass is a „liquid“ solution of silicates sodium or potasium. These solutions of samples can be prepared from silicate sodium, sodium hydroxide and water [1,2].

Measurements were performed also by means of FT HR NMR spectrometer BRUKER at ČVUT, Prague (Fig. 1). In the frame of measurement establishement of 29Si NMR spectra in the VŠB-TU Ostrava the measurements of water glasses solutions were tested. Measurements were performed by means of FT HR NMR spectrometer TESLA BS 587 A. Then the spectra were evaluated and compared with the spectrum given in Fig. 2.

Fig.1:

2299SSii NNMMRR ssppeeccttrruumm ooff wwaatteerr ggllaassss bbyy mmeeaannss ooff FFTT Fig. 2:

2299SSii NNMMRR ssppeeccttrruumm ooff wwaatteerr ggllaassss bbyy mmeeaannss ooff FFTT

NNMMRR ssppeeccttrroommeetteerr BBrruukkeerr HHRR NNMMRR ssppeeccttrroommeetteerr TTeessllaa 8800 MMHHzz BBSS 558877 AA

The spectrum (see Fig. 1) contains: from the left monomer (Q0) in weight per cents (1,19), dimer (Q1) (9,68), substitued cyclic trimer {Q2(X)} (2,61), tricyclic octamer {Q2(A)}, cyclic tetramer (Q2Z/Q3), substitued cyclic trimer {Q3(M)}, pentacyclic heptamer {Q3(A)} altogether (42,68), bridged cyclic tetramer {Q3(X)}- two, two tricyclic octamers {Q3(X)} (41,69) and colloid phase (Q4) (2,14). The last spectrum (see Fig. 2) contains the same structures as see Fig. 1, in addition close to chemical shift along –84,5 ppm – tricyclic hexamer (transoid) {Q2(A)}, close to –96,3 ppm – bicyclic hexamer {Q3(X)}. From the quantitative point of evaluating has been perform in weight percentages: dimer (Q1) is represented by (7,00), tricyclic octamer {Q2(A)}, cyclic tetramer (Q2Z/Q3), substitued cyclic trimer {Q3(M)}, pentacyclic heptamer {Q3(A)} (45,83), bridged cyclic tetramer {Q3(X)}- two, two tricyclic octamers {Q3(X)} (47,17). The authors thank the Grant Agency of the Czech Republic for its financial support given under Grant 60/1013.

References 1 S. D. Tepjakov, Struktura a vlastnosti vodního skla, Litějnoje proizvidstvo,

1984, 5, 18-20. 2 P. Jelínek, Struktura alkalických silikátů a možnosti ovlivňování jejich pojivo-

vých vlastností, Slévárenství XLIV, 1996, 4, 287-291.

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CONFORMATIONS AND TRANSMISSION OF SUBSTITUENT EFFECTS IN DISUBSTITUTED TETRAHYDROPYRIDAZINE

AND DIAZABICYCLOHEPTENE DERIVATIVES

Alexander PERJÉSSYa, Wolf-Dieter RUDORFb, Peter MEYERb, Erkki KOLEHMAINENc, Dušan LOOSa, Katri LAIHIAc, Maija NISSINENc,

Jari KOIVISTOc, Reijo KAUPINENc

aDepartment of Organic Chemistry and Institute of Chemistry, Faculty of Natural Sciences, Comenius University, SK - 842 15 Bratislava, Slovak Republik,

E-mail: perjessy@fns.uniba.sk bInstitute of Organic Chemistry, Martin-Luther University, D-06099 Halle (Saale),

Germany cDepartment of Chemistry, University of Jyväskylä, FIN-40351, Jyväskylä, Finland

The 1H, 13C, 15N NMR and FTIR spectra of 22 substituted ethyl 2-arylcarbamoyl-4, 5-dimethyl-1, 2, 3, 6-tetrahydropyridazine-1-carboxylates (I) and substituted ethyl 3-arylcarbamoyl-2,3-diazabicyclo[2.2.1]hept-5-ene-2-carboxylates (II) were measured and assigned. The structure of some compounds were confirmed by X-ray structure analysis. The electronic structure and conformations were calculated by semiempirical AM1 and PM3 methods.

O

NN

N H

X

O

O-CH2-CH3

CH3

CH3

O

NH

O

O-CH2-CH3

X

N

N

I II

I X : 4-CH3, 2-CH3, 4-C2H5, 3-CH3, H, 4-Cl, 4-Br, 4-Br, 3-Cl, 4-OCH3, 4-OCH3, 4-NO2, 2-Cl

II X : 4-OCH3, 4-CH3, 4-C2H5, 3-CH3, H, 4-Cl, 4-Br, 3,4-Cl2, - 4-NO2 NMR spectral assignments were based on DQF 1H,1H COSY, 1H,1H EXSY, PFG 1H,13C HMQC and PFG 1H,13C HMBC measurements. In FTIR spectral analysis the data fitting by Lorenz - Gaussian functions were employed. Using correlation analysis between the spectral, empirical and theoretical structural data, the transmission of substituent effects as well as the preferential conformations of compounds I and II connected with synchronous double nitrogen inversion and the mutual orientation of N-H and C=O bond was assessed.

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SPECTROSCOPIC PROPERTIES vs. MOLECULAR GEOMETRY

IN A SERIES OF FLUORESCENT COMPOUNDS

Andrej PETRIČ

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia

In search for novel fluorescent probes for medical research we found that 2-{1-[6-(dimethylamino)-2-naphthyl]ethylidene}malononitrile (1, DDNP)1 exhibits rather remarkable sensitivity of spectroscopic properties to solvent polarity/viscosity. We also found that DDNP can be used for labeling of cell membranes in confocal fluorescence microscopy. In our attempts to prepare novel fluorescent probes by modification of the structure of DDNP, we realized that optical properties change significantly on formal substitution of the N,N-dimethylamino group with various secondary amines.2 This prompted us to investigate these changes and to try correlating them with the respective molecular geometry.

Analogues of DDNP (1) have been prepared by formal substitution of the dimethylamino group by an azetidine, aziridine, pyrrolidine, or piperidine ring (2, n = 0−3).

Fluorescence excitation and emission maxima and 1H NMR chemical shifts variation for derivatives 2 in solvents with different polarity/viscosity were correlated with the geometry features of the molecules, obtained from the single crystal X-Ray analyses.

References: 1 A. F. Jacobson, A. Petrič, A. Sinur, J. R. Barrio, J. Am. Chem. Soc. 1996, 118, 5572-5579. 2 G. Ambrožič, S. Čeh, A. Petrič, Magn. Reson. Chem. 1998, 36, 873-877; A. Petrič, T.

Špes, J. R. Barrio, Monatsh. Chem. 1998, 129, 777-786; A. Petrič, A. F. Jacobson, J. R. Barrio, Bioorg. Med. Chem.Lett. 1998, 8, 1455-1460; A. Petrič, J. R. Barrio, Acta Chim. Slov. 1998, 45, 475-486.

N

CNNC

1

N(CH2)n

CNNC

2

19

STRUCTURE OF [2,6-BIS(DIMETHYLAMINO)METHYL] DIPHENYLTIN(IV) DERIVATIVES

Aleš RŮŽIČKA,a Libor DOSTÁL,a Roman JAMBOR, a Jiří BRUS,b

and Jaroslav HOLEČEKa

aDepartment of General and Inorganic Chemistry and, Faculty of Chemical Technology, University of Pardubice, Studentská 95, CZ-532 10, Pardubice, Czech

Republic, Fax: + 420 40 603 7068, E-mail: ales.ruzicka@upce.cz bInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic,

Heyrovsky sq.2, 162 06 Praha 6, Czech Republic.

The organotin(IV) compounds have been extensively studied and screened in vitro and in vivo for antitumor activity, usually against P388 lymphocytic leukaemia and especially triorganotin(IV) derivatives have been recently received considerable attention due to their high antifungal activities against some medically important fungi The low aqueous solubility of organotin compounds is limiting matter of their further research in the medicinal area.

Figure 1: The crystal structure of [2,6-bis(dimethylamino)methyl]diphenyltin(IV) bromide hydrate. We have prepared and characterized a set of seven organotin(IV) compounds, which have the ionic matter, and are well soluble in water. The structure in the solid state (CP MAS, X-ray) and in solution will be discussed.

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STRUCTURE ELUCIDATION OF BENZOPHENANTHRIDINE AND PROTOBERBERINE ALKALOIDS

Pavlína SEČKÁŘOVÁa, Jiří DOSTÁLb, Roger DOMMISSEc, Radek MAREKa

a National Center for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, CZ - 611 37 Brno, Czech Republic, e-mail: pavlina@chemi.muni.cz,

rmarek@chemi.muni.cz b Department of Biochemistry, Faculty of Medicine, Masaryk University, Komenského

nám. 2, CZ - 662 43 Brno, Czech Republic, e-mail: jrdostal@med.muni.cz c Department of Chemistry, University of Antwerp RUCA, Groenenborgerlaan 171, B -

2020 Antwerpen, Belgium, e-mail: dommisse@ruca.ua.ac.be

Quaternary benzo[c]phenanthridine alkaloids (QBA) and quaternary protoberberine alkaloids (QPA) show considerable biological activity, for example antimicrobial, antitumour and anti-inflammatory.1-3

NCH3

R

R

RR

RR H OH

N

R

R

R

R

R HOH

1 2 In the presence of OH- anion QBA (resp. QPA) are converted into 6-

hydroxyderivatives4 (resp. 8-hydroxyderivatives) called pseudobases 1 (resp. 2). Free bases of QBA alkaloids chelerythrine, sanguilutine, sanguirubine, chelirubine, chelilutine, sanguinarine, nitidine and fagaronine and QPA alkaloids berberine, palmatine, coptisine and corysamine were studied by 1H- and 13C-NMR spectroscopy (1D and 2D techniques). Structures of free bases were also confirmed by mass spectrometry. QBA pseudobases may change to bimolecular aminoacetals by elimination of H2O molecule.5,6 QPA pseudobases are less stable and undergo an oxidation process during a time. The differences between the chemical behavior of QBA and QPA with respect to their pK values will be discussed.

This work was supported by the grant of Ministry of Education (LN00A016).

References:

1. Colombo M. L., Bosisio E., Pharmacol. Res. 33, 128-132 (1996) 2. Preininger V., The Alkaloids 15, 207-261 (1975) 3. Iwasa K., Kim H. S., Wataya Y., Lee D. U., Eur. J. Med. Chem. 33, 65-69 (1998) 4. Sečkářová P., Marek R., Dostál J., Dommisse R., manuscript in preparation 5. Marek R, Sklenář V, Dostál J, Slavík J. Tetrahedron Lett. 36, 1655 (1996) 6. Marek R, Toušek J, Králík L, Dostál J, Sklenář V. Chem. Lett., 369 (1997)

21

NMR AND GC-MS ANALYSIS OF SELECTED COAL EXTRACTS

J. BOHDÁLKOVÁ*, E. KOZUBEK*, M. PEŇÁZ*, J. PAVLOVSKÝ**,

*Department of Analytical Chemistry and Material Testing, **Department of Foundry Industry, FMME, VŠB – TU Ostrava, Czech Republic

Coal originated for the most part from vegetable sediments accumulated in the areas of the mild climatic zone in rivers, streams, lakes, sea gulfs and lagoons. The Earth geological reserves of the black coal are assessed to be 15. 1012 tons. The reserves of solid caustobioliths contain approximately 90 % of fossil energy [1,2]. Mechanical treatment samples of naturally altered (from the "9. květen" mine) and coal altered in a laboratory (in the temperature of 250°C in an air stream for the period of 4 days) were allowed to macerate in a deuterium solvent. Altered coal is a bituminous coal destroyed either by heat, oxidation or by heat and oxidation. The measurements were performed by means of a NMR spectrometer of the type 80 MHz FT HR NMR Tesla BS 587 A (13C method of Inverse Gate Decoupling Hetero) and by a GC/MS HP 5890. This article shows an example the 13C spectrum of altered coal (for naturally altered coal see Fig. 1) and GC of deuterium benzene extract of naturally altered coal (see Fig. 2).

Fig. 1: 13C NMR-Nat. alt. coal in deuteroacetone Fig. 2: GC-Nat. alt. coal in deuterobenzene The first absorption maximum on the right (see Fig. 1) corresponds to the standard (HMDSA). The wedges occurring within the interval from 20 to 50 ppm show the presence of aliphatic groups. The most marked absorption maximum is the characteristic of deuterium acetone. The wedges occurring within the interval from 120 to 170 ppm belong to aromatic compounds which are partially overlapped by the solvent. Groups of carboxylic acids lie above 180 ppm. For the comparison Fig. 2 demonstrates GC of deuterium benzene macerate prepared from the naturally altered bituminous coal. The comparison of the NMR spectra shows that there is only a slight difference between naturally and laboratory altered coal. The authors wish to express thanks to the grant agency of the Czech Republic (project No. 105/99/0225) for its financial support for the dealing with this topic.

References 1 E. Kozubek, J. Bohdálková, J. Pavlovský, NMR of Humic Acids. In: Proc. of 15th

NMR Valtice. Central European NMR Discussion Groups, 2000, 33. 2 V. Roubíček, J. Buchtele, Chemie uhlí a jeho využití, VŠB-TU Ostrava, 1996, 13-17.

22

NMR STUDY OF THE SOLUTION STRUCTURES OF C5ME5TIF3

Alojz DEMŠAR

Faculty of Chemistry and Chemical Technology, University of Ljubljana.

Aškerčeva 5, SLO - 1000 Ljubljana, Slovenia

The variable temperature 1H and 19F NMR spectroscopy revealed an temperature and

concentration dependent equilibria of monomer, dimer, two tetramers (in different

concentrations) and two minor additional low-temperature species, likely trimers of

pentamethylcyclopentadienyl-titanium trifluoride, [(C5Me5)TiF3] 1, in solution. The

higher temperature and lower concentration favor smaller species. The dimer-monomer

dissociation was studied in a fast-exchange 19F NMR regime and the thermodynamic

parameters were evaluated. We followed the geometry of nonmethylated analogue of 1,

[(C5H5)TiF3]2 along the dissociation path by ab initio methods. The equilibrium of

tetramers and dimer, slow on a 1H NMR time scale, was studied . The structure of a

major tetramer species in the solid state was determinated by X-ray crystallography and

is equal to the solution structure.

23

SOLID-STATE NMR STUDIES OF CONFORMATIONALLY POLYMORPHIC CRYSTALLINE SOLIDS FOR

STEREOCHEMICAL INTERPRETATION OF FAST EXCHANGE LIMIT SOLUTION-STATE NMR SPECTRA

Robert GLASER, Dror SHIFTAN, and Artem NOVOSELSKY

Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

A change of configuration at a medium-size ring atom can result in a conformational change with concomitant change in the NMR spectral parameters. For example, in many cases, a methylene proton in medium-size rings typically points towards the center of the ring and thus suffers transannular interactions, while the other one has a sterically free exo orientation. Methyl substitution at this exo-position usually leaves the preferred conformation invariant (compared to the unsubstituted parent compound), while substitution at the sterically hindered endo-position affords a conformational change. Cpmas 13C NMR spectra of the two methylated diastereomers provide chemical shift values for a particular nucleus that are diagnostic for a conformational type. In the solution-state, many molecules exist as a mixture of conformations. Their NMR spectral parameters thus represent weighted time averaged values from each participant at the fast exchange limit for conformational equilibrium. The solution-state energy barriers for conformational interconversion are usually low, and thus attainment of slow exchange limit spectra represents a difficult undertaking. Packing considerations due to residence within a crystal lattice usually result in freezing out one of the low energy conformational possibilities. Changing the crystallization conditions can result in new crystals containing a different conformation (so called conformational polymorphism or pseudopolymorphism). The cpmas 13C NMR spectra of conformational polymorphs can also provide chemical shift values for a particular nucleus that are diagnostic of the conformational type [1,2]. These solid-state chemical shift values can be used to interpret the conformational equilibrium from the solution-state weighted time-average values. While the crystal lattice generally provides constraints against conformational interchange, the appropriate crystal design can sometimes engineer intermolecular voids which enable conformational change also within the solid [2]. References 1 R. Glaser, D. Shiftan, and M. Drouin, J. Org. Chem, 1999, 64, 9217-9224. 2 R. Glaser, A. Novoselsky, D. Shiftan, and M. Drouin, J. Org. Chem., 2000, 65, 6345- 6353.

24

NMR STUDIES OF SUPRAMOLECULAR SELF-ASSEMBLY: PREMICELLAR AGGREGATION OF SODIUM

DODECYLBENZENESULFONATE

Jaroslav KŘÍŽ

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic

kriz@imc.cas.cz Ionic surfactants start binding cooperatively to complementary polyions at concentrations lower than their critical micellar concentration. To understand this behavior, pre-micellar aggregation has to be studied. First such observation was accomplished by NMR on sodium 4-dodecylbenzenesulfonate (SDDBS). Gradual aggregation can be observed in the development of the aromatic part of 1H NMR spectrum with increasing SDDBS concentration, the shift of m-protons being due to increasing long-range shielding:

The concentration dependence of the population of individual aggregates can be described by a cooperative self-assembly model:

∏=−

=

1

11][A][A

j

ii

jj K , ∑ =−∏ +

=

−

=

n

j

j

ii

j Kj2

01

1

111 0][A][A)][A(

The increasing size of the aggregates can be established by PGSE. Swift exchange between the aggregates follows from the concentration dependence of the self-diffusion coefficients and transverse relaxation times of individual resonances: The dynamics of this exchange obtained from T1ρ(ω1) dependences reveals lower stability of the medium-sized species. Combining 23Na NMR PGSE and T1 measurements, a progressively tighter binding of the Na+ counterions in larger aggregates can be established. NMR is the only method able to reveal all these features.

0.0 0.5 1.0 1.5 2.0 2.5

100

120

140

160

180

200

220

240

260

280

300

320

340

T 1 [

ms]

670

700

730

760

790

820

850

880

910

940

970

h

gf

d

a+b+c

T 2 [

ms]

log(c)+3.5

1H NMR spectra (aromatic region) of SDDBS at increasing concentration

T2 (full) and T1 (dashed) values of individual species of DDBS.

25

SOLID STATE 1H CRAMPS NMR STUDY OF MOLECULAR COMPLEXES OF POLY(ETHYLENE OXIDE) AND

BENZENE DERIVATIVES

Jiří SPĚVÁČEK and Jiří BRUS

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, 162 06 Prague 6, Czech Republic

Using methods of solid state 1H CRAMPS (combined rotation and multiple pulse spectroscopy) NMR spectroscopy, we studied crystalline intercalate complexes of poly(ethylene oxide) (PEO) with hydroxybenzenes (resorcinol, 2-methylresorcinol (2MR), hydroquinone, 4-nitrophenol (4NP)), stabilized by hydrogen bonds, or with 1,4-dichlorobenzene. The 1H chemical shifts were correlated with the hydrogen bond strength as characterized by infrared spectra (OH stretching band) [1]. The increasing hydrogen bond strength can lead both to larger values (PEO protons, OH protons) and to smaller values (aromatic protons) of chemical shifts. Measurements of two-dimensional 1H CRAMPS exchange spectra have been used to characterize proton distances in complexes. For all the complexes studied, a close contact between PEO and the hydroxybenzene (proton distance 0.45 nm) was found. For the PEO/4NP and PEO/2MR-β complexes, even a closer contact (0.32 nm) between aromatic and PEO protons was detected. This result together with 13C NMR spectra (hydroxybenzene resonances) [2] indicates that interactions of other functional groups of 4NP or 2MR with PEO can be important in these complexes, in addition to hydrogen bonds. Two-dimensional 1H CRAMPS exchange spectra also evidenced a close contact (proton distance 0.32 nm) between both components in the PEO/1,4-dichlorobenzene intercalate complex stabilized by weak van der Waals interactions. References: 1 J. Spěváček, L. Paternostre, P. Damman, A.C. Draye, M. Dosiere,

Macromolecules, 1998, 31, 3612-3616. 2 J. Spěváček, M. Suchopárek, Macromol. Symp., 1997, 114, 23-34.

26

13C RELAXATION STUDY OF RNA LOOP DYNAMICS

Radovan FIALAa, Lukáš TRANTÍREKa, Erik CAHAa and Mikael AKKEb

aNational Centre for Biomolecular Research, Masaryk University, Kotlářská 2, CZ-611 37 Brno, Czech Republic

bDepartment of Physical Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, Sweden

The RNA sequence CUNCGG (where N is any nucleotide, the nucleotides forming loops are underlined) is an abundant structural element of extraordinary high stability that has been proposed as nucleation site for RNA folding and as protein nucleation site. We have performed an NMR relaxation study to characterize the equilibrium conformational fluctuations occurring on a time scale of picoseconds and nanoseconds. The investigation of intramolecular motions can provide further insight into the relationship between the structure, thermodynamic stability and biological function of the tetraloop. Previously, we have characterized intramolecular dynamics of guanine and uracil bases in a tetradecamer RNA hairpin containing the UUCG loop by 15N spin relaxation1. Since the 15N relaxation studies rely on imino nitrogen, which is not represented in adenine and cytosine bases, a nitrogen relaxation study is bound to be incomplete. The present study probes the relaxation properties of the RNA oligonucleotide through purine C8 carbons and C2 carbon atoms of adenine, C6 carbons of pyrimidine nucleotides and C1’ atoms of ribose. We have measured T1 and T2 relaxation times and steady-state heteronuclear NOE at 400, 500 and 600 MHz in a uniformly 13C- and 15N-labeled 14-mer RNA hairpin containing a UUCG nucleotide sequence. The relaxation data were interpreted in the framework of reduced spectral density mapping and model-free formalism. The results of experimental study are compared with the characteristics based on molecular dynamics calculations in explicit solvent. The talk will concentrate on methodological issues of using 13C relaxation as a probe for the study of dynamics in fully 13C- and 15N-labeled oligonucleotides. References: 1. Akke M, Fiala R, Jiang F, Patel D, Palmer A G. 1997. RNA 3, 702-709.

27

SIMPLIFICATION OF NMR SPECTRA OF MASON-PFIZER MONKEY VIRUS PROTEASE.

Richard HRABAL, and Václav VEVERKA

NMR Laboratory, Institute of Chemical Technology in Prague, Technická 5, CZ-16628 Prague, Czech Republic, e-mail hrabalr@vscht.cz

NMR spectra of large proteins are frequently so complicated that even using of

triple resonance, multidimensional experiments does not help. In the process of resonance assignment it is inevitable to find responses of amino acid residues with typical cross peak patterns, which then serve as starting points. In case of heavily overlapped resonances, amino acid selective 1H-15N spectra might serve this purpose [1,2]. The selectivity of these experiments is based on a topology of side chains typical for each amino acid residue. The experiments utilize either multiplicity selective in-phase coherence transfer MUSIC (for selection of Gly, Ala, Thr, Ile, Val, Asn, Gln), selective excitation (Ser) or other typical features like e.g. an absence of amino proton (Pro).

We have used this strategy to speed up the assignment of 1H, 13C, and 15N resonances of Mason-Pfizer monkey virus protease. A combination of intra- and inter-residual coherence transfer experiments, e.g. Gly(i)-Gly(i+1) or Ala(i)-Ala(i+1) and others was utilized. In this way unique cross peak patterns of some amino acid residues were found making it possible to complete the sequence specific assignment by classical triple resonance 3D NMR experiments [3]. Usefulness of this approach will be discussed. The work was supported by the Grant Agency of the Czech Republic (Grant 203/00/1241). References

1. M. Schubert, M. Smalla, P. Schmieder, and H. Oschkinat, J. Magn. Reson., 1999, 141, 34-43.

2. M. Schubert, L.J. Ball, H. Oschkinat, and P. Schmieder, J. Biomolecular NMR, 2000, 17, 331-335.

3. V. Veverka, H. Bauerová, A. Zábranský, I. Pichová, and R. Hrabal, J. Biomolecular NMR, submitted.

28

NMR ANALYSIS OF PROTEIN-CARBOHYDRATE INTERACTIONS THROUGH EVALUATION OF CHEMICAL

SHIFTS, COUPLING CONSTANTS AND TRANSFERRED NOES.

M.HRICOVÍNI,1 G.TORRI,2 M.GUERRINI2 and B.CASU2

1Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia 2Institute for Chemical and Biochemical Research “G.Ronzoni”, Milano, Italy

Some carbohydrates are known for their ability to induce various important biochemical processes through their interactions with proteins. The knowledge of three-dimensional (3D) structure of complexes of these carbohydrates with proteins has the potential to assist in the design of new carbohydrate-based therapeutic agents. For systems in fast chemical exchange, NMR-based analysis of protein-carbohydrate complexes utilizes mainly the evaluation of transferred NOEs; changes of chemical shifts and scalar couplings constants can be used as well. In the present case, the example will be given of the 3D structure analysis of biologically important glycosaminoglycans (e.g. heparin, heparin-derived oligosaccharides, dermatan sulfate, etc.) in the complex with plasma protein antithrombin. Both 1H and 13C chemical shifts, proton-proton and proton-carbon coupling constants as well as transferred NOEs have been monitored in tetrasaccharide (GlcN,6-SO3-α(1-4)-GlcA-β(1-4)-GlcN,3,6-SO3-α(1-4)IdoA-αOMe) in aqueous solution and in the complex with protein antithrombin (molar ratio 20:1 and 10:1, carbohydrate:protein). Considerable changes in 1H and 13C chemical shifts were observed for both 20:1 and 10:1 ligand/protein ratios. The analysis of transferred NOEs indicated that the conformations at the glycosidic linkages changed during the binding process. Furthermore, the data indicated that the conformational equilibrium in the IdoA residue is shifted towards the 1C4 chair form. The study of a structurally similar pentasaccharide showed the variations at the glycosidic bonds conformations in the complex with respect to the free state. The evidence is also provided that the protein drives the conformationally flexible 2-O-sulfated iduronic acid residue towards the skewed 2S0 form.

29

NMR & PROTEIN DYNAMICS

Hana KŘÍŽOVÁ1, Lukáš ŽÍDEK1, Martin STONE2, Vladimír SKLENÁŘ1 1Národní centrum pro výzkum biomolekul, Masarykova univerzita, Kotlářská 2, 611 37

Brno 2Indiana University, Department of Chemistry, Bloomington, IN 47405, USA

High resolution NMR spectroscopy has become one of the most important methods for the determination of 3D structures of biological macromolecules and for the description of the dynamic properties of molecules of biological interests. The biological function is connected with the dynamical properties of protein structure in solution. Backbone dynamics of mouse Major Urinary Protein I (MUP-I) was studied. MUP-I is an abundant protein excreted into mouse urine. The protein belongs to the family of lipocalines and is known to bind several pheromones, including 2-sec.butyl-4,5-dihydrothiazole. Effects of ligand binding on dynamics of MUP-I have been studied. Longitudinal and transverse relaxation rates as well as NOEs were measured with N-15 labeled sample. These data were used for calculation of parametres describing the molecular motion using the model-free approach. The results were compared to the previously reported data. Finanční podporu poskytly NIH (granty DC02418 a GM55055), NSF (grant MCB-9600968) a GAČR (grant 203/00/0511). References: L.Žídek et.al., Biochemistry, 38 (1999), 9850 - 61 Lipari, G., Szabo, A., J. Am. Chem. Soc., 104 (1982), 4546 - 59

30

NMR SPECTROSCOPY OF AN ELASTIN-LIKE poly(Gly1-Val1-Gly2-Val2-Pro) POLYMER

Dana KURKOVÁ1, Jaroslav KŘÍŽ1, Pavel SCHMIDT1, and José Carlos RODRÍGUEZ-

CABELLO2

1Institute 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, Spain

Poly(G1V1G2V2P) is a polymer undergoing reversible inverse temperature transition at about 28 oC, which is water-soluble below the critical temperature but tends to aggregate above it. According to previous studies, it forms a β-turn between proline and glycine1. Our aim is to clarify conformation changes and the function of hydration water before and after thermal transition of the polymer. We performed 1H, 13C, 15N NMR spectral and relaxation studies. Signal assignments were done using COSY, NOESY, HXCORR, HSQC, HMBC and SSLR INEPT techniques. Temperature-induced conformation changes were studied using chemical shifts (1H, 13C, 15N) 3JH-H coupling constants and NOE factors. Selective hydration (hydrophilic or hydrophobic) was explored using NOESY, ROESY, and both selective and non-selective longitudinal relaxation of water (HOD). In 1H NMR spectra, the signals of Gly1 and Gly2 as well as Val1 and Val2 are mostly superimposed except for that of Val1-αCH. NH proton signals are well-resolved. In 13C NMR, there are two different Val1-αCH and Val2-αCH signals. In 15N NMR, there are four NH signals corresponding to Val1-NH, Val2-NH, Gly1-NH and Gly2-NH. As expected, detectable temperature changes in chemical shifts were found in NH 1H signals in H2O, the largest difference being that of Val2-NH (∆δ = -0.34 ppm). In 15N NMR, the largest temperature changes are shown by Val2- NH and Gly2- NH (∆δ = -0.5 ppm), whereas those in 13C are shown by Val2-αCH, Pro-βCH2, and Val1-C=O (the respective ∆δ being -0.2, -0.2, and -0.3 ppm). These changes agree with the idea of a β-turn between Pro and Gly1. The values of 3JH-H change in the interval ∼ 6.8-7.96 Hz in temperature area ∼7-37 oC. The calculated change of dihedral angles φ of the NH–CH torsion were from 201 to 208o (or –81 to -88o) for Val1 and from 206 to 210o (or –86 to -90o) for Val2. These values of 3JH-H indicate, that the chains of the polymer are relatively extended in this temperature area and undergo a conformation change in the value of the angle φ. NOESY measurements at temperatures ∼7-37 oC gave distances in the range from 2.0 to 3.0 Å between the groups of Val1,2-βCH and Val1,2-γCH3, Pro-δCH2 and Val1,2-γCH3, Gly-αCH2 and Val1,2-γCH3, Pro-δCH2 and Gly-αCH2, Val1-αCH and Val1,2-γCH3, Pro-γCH2 and Pro-βCH2. All atempts to prove selective hydration of the polymer and its thermal changes, both on the grounds of NOE and relaxation, have failed so far. The reason could be a too low residence time of water molecules in the hydration sphere of the polymer. The authors wish to thank to the Grant Agency of the Czech Republic (Grant No. 203/00/1320), to the “Junta de Castilla y León“ (Programmes VA30/97 and VA30/00B) and to the “Comission Interministerial de Ciencia y Tecnologia/CICYT” (Programme MAT 98-0731) for financial support.

31

1H, 13C, 15N ASSIGNMENT AND SECONDARY STRUCTURE IDENTIFICATION OF THE PROTEASE FROM

MASON-PFIZER MONKEY VIRUS

Václav VEVERKAa, Helena BAUEROVÁb, Aleš ZÁBRANSKÝb, Iva PICHOVÁb, Richard HRABALa

aLaboratory of NMR Spectroscopy, Institute of Chemical Technology, Technická 5,

166 28 Prague 6, Czech Republic, e-mail: Vaclav.Veverka@vscht.cz bDepartment of Biochemistry, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague 6, Czech Republic

Mason-Pfizer monkey virus encodes an aspartic protease (M-PMV PR), which is

essential for the maturation of the virion particles by processing viral protein precursors

yielding fully functional structural proteins and enzymes. The overall fold of all known

retroviral proteases is quite conservative despite rather large varieties of their amino

acid sequences. However, Mason-Pfizer Monkey Virus protease exists in three active

forms, which is a feature making this enzyme unique within the this family. In vivo

Mason-Pfizer Monkey Virus protease occurs as a 17 kDa (per monomer) molecule. In

vitro it undergoes a rapid self-processing at the C-terminus yielding 13 kDa and finally

12 kDa forms [1]. We will present backbone resonance assignment and secondary

structure identification of shortest 12 kDa form of protease from Mason-Pfizer Monkey

Virus [2].

The work was supported by the Grant Agency of the Czech Republic (Grant 203/00/1241).

References:

1 Zábranský A., Andreanský M., Hrušková-Heidingsfeldová O., Havlíček V., Hunter E., Ruml T., Pichová I., Virology, 1998, 245(2), 250-256.

2 V. Veverka, H. Bauerová, A. Zábranský, I. Pichová, and R. Hrabal, J. Biomolecular NMR, submitted.

32

BIOTRANSFORMATIONS IN NMR SAMPLE TUBE

Petr SEDMERA1, Jindřich VOLC1, Ronald MATHÄ2, Dietmar HALTRICH2

1Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic

2Division of Biochemical Engineering, Institute of Food Technology, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria

Growing interest in NMR monitoring of biotransformations [1-3] lead us to use this approach for the detection of reaction intermediates and structure elucidation of the products. The biotransformations were performed with highly purified and concentrated enzymes, substrates and benzoquinone in D2O directly in NMR sample tube. The structural information was extracted with the help of gCOSY, 1D-TOCSY, HMQC, and HMBC. The reaction of selected glycosides with pyranose dehydrogenase from Agaricus meleagris proceeds quantitatively within several hours to two days. αGlcp(OMe) was converted into 3-keto derivative; with βGlcp(OMe) and αGalp(OMe) the reaction was slower and hydrated ketone was also formed. 3-Keto-trehalose was spectroscopically identified as an intermediate leading to the final bis-3-ketoglucose. Oxidation of sucrose at C-3 was also observed. Melezitose and erlose were oxidized in the terminal glucose unit only. The transformation of ribose by the same enzyme gave γ-ribonolactone and its δ-isomer. Pyranose dehydrogenase from Macrolepiota rhacodes oxidizes glucose at the position 3 only. The composition of resulting tautomeric mixture [4] was revised on the basis of our results. The use of [1-13C] glucose showed that the α-anomer reacts 20 times faster than the β one. Partial information (oxidation at C-1) was obtained from the reaction of lactose with enzyme from Agaricus xanthoderma. This deduction was confirmed by comparison of 13C NMR spectra with those of lactobionic acid. References 1 P. Spangenberg, V. Chiffoleau-Giraud, C. André, M. Dion, C. Rabiller, Tetrahedron

Asymm. 10, 2905 (1999). 2 L. Brecker, D. W. Ribbons, Trends Biotechnol. 18, 197 (2000). 3 H. Weber, L. Brecker, Curr. Opin. Biotech. 11, 572 (2000). 4 P. E. Morris, K. D. Hope, D. E. Kiely, J. Carbohydr. Chem. 8, 515 (1989).

33

PULSE NMR USED IN STUDY OF BOUND WATER IN BIOLOGICAL SYSTEMS

Daniela SUCIU

Dept. of Biophysics, Faculty of Stomatology, University of Medicine and Pharmacy

“Gr. T. Popa” Iasi, Romania

Nuclear Magnetic Resonance is a powerful technique that provides information on biochemical status and physiological processes both in vitro and in vivo. The metabolism of intact cells and tissues can be studied in a continuous manner, and thus, NMR is a non-invasive research tool enabling detection of the metabolic changes as they occur [2]. The proton has the highest NMR sensitivity, and is the most abundant nucleus in biological molecules. This offers the possibility to investigate the changes that appear to the bound intracellular water in different metabolic and physiological status.

Relaxation times, experimentally determined, demonstrate the presence of many phases of structured water. There are in minimum three ways in which water is bound with protein in the tissues. This structured water depends on the type of tissue, external factors, pathological or physiological changes, ultrasounds, x-rays, etc [1, 3].

Comparative studies of high resolution NMR and pulse NMR for the water protons have shown important differences in structured water in cerebral tumors comparing with normal tissue, obtaining for T2 relaxation times the higher values than in normal tissue. In the same time there were obtained for T2 relaxation time the different values depending on the type of the cerebral tumor which was under study [4].

These results can be explained as the evolution of the malignity determining essentially changes to intracellular water which is less structured in early stages of the evolution of the malignity than that in the last stages. This play, less structured water – more structured water, determines changes in the dynamics of the macromolecules and malfunction of the cells. These changes can be used to determine the earliest stages of the malignity. References

1. Baciu Daniela, Baciu I., The Water Structuralisation in Biological Tissues Studied by Pulse NMR, International School of Structural Biology and Magnetic Resonance – Protein Dynamics, Function and Design, Erice, Italy, April 1997, pag. 5.

2. Berényi E; Repa I; Bogner P; Dóczi T; Sulyok E, Pediatr Res., 1998 Mar, 43:3, 421-5

3. Hazlewood C.F., Baylor College of Medicine, Departments of Pediatrics and Physiology, Houston, Texas, 1982.Poliquen, Rivet, Gallier, Le Jeune, Anticancer Research, 1993, Jan. - Feb. 13, 49-55.

5. Yamada T, Adv. Exp. Med. Biol., 1998, 453:, 145-54; discussion 154-5

34

CSEARCH ONLINE A FREE SPECTRUM ESTIMATION TOOL FOR STRUCTURE

ELUCIDATION UTILIZING STEREOCHEMICAL INFORMATION

Christoph Seger1, Wolfgang Robien2

1Institute of Pharmaceutical Chemistry and Pharmaceutical Technology,

University of Graz, Universitätsplatz 1, A - 8010 Graz, Austria 2Institute of Organic Chemistry, University of Vienna, Währinger Str. 38, A-1090

Vienna, Austria

The usage of the free CSEARCH server, accessible through the internet and based on the well known CSEARCH - database technology [1] will be demonstrated on examples from our current research. Beside giving an outline of the server access procedure the advantages and limits of the system will be discussed.

Database access: http://mailbox.univie.ac.at/Wolfgang.Robien/csearch_main.html

References: [1] V. Schütz, V. Purtuc, S. Felsinger, W. Robien, Fresenius J. Anal. Chem., 359, 33 (1997) [2] C. Seger, B. Jandl, G. Brader, W. Robien, O. Hofer, H. Greger, J. Anal. Chem., 359, 42 (1997)

35

DFT CALCULATION OF NMR SPIN-SPIN COUPLING CONSTANTS IN ADENINE, THYMINE, CYTOSINE, AND

GUANINE

aVladimír SYCHROVSKÝ, aJaroslav VACEK, aPavel HOBZA, and bVladimír SKLENÁŘ

aJ.Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejškova 3, 182 23, Praha 8, Czech Republic, e-mail sych@jh-inst.cas.cz

bLaboratory of Biomolecular Structure and Dynamics, Faculty of Science, Masaryk University, Kotlářská 2, CZ-611 37, Brno, Czech Republic,

e-mail sklenar@chemi.muni.cz Coupled Perturbed DFT (CP DFT) calculation of all four contributions to the total indirect reduced NMR spin – spin coupling constants ( DSO – diamagnetic spin orbit, PSO – paramagnetic spin orbit, FC – Fermi contact, SD – spin dipolar) was performed using the hybrid DFT exchange – correlation functional B3LYP in DNA basis of Adenine, Thymine, Cytosine, and Guanine. The functional of B3LYP was found to perform well previously [1]. In the present study we used the basis set of (9s5p1d/5s1p) [6s4p1d/3s1p] usually called Iglo II and the molecules were optimized at the level B3LYP/6-31G**. The calculated 1J and 2J coupling constants of Carbon - Hydrogen, Carbon - Carbon, Nitrogen - Hydrogen, and Carbon - Nitrogen coupled pairs are in a good agreement with the experimental data. The deviation of calculated from experimental values can be demonstrated by the mean absolute error μ=4.2 Hz. References 1. V. Sychrovský, J. Gräfenstein, and D. Cremer J. Chem. Phys., 2000, 113, 3530- 3547.

36

MULDER - PROGRAM FOR EXTRACTING OF TORSION ANGLES FROM NMR DATA

Petr PADRTA, Vladimír SKLENÁŘ

National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, CZ-611 37, Brno, Czech Republic

E-mail: padrta@chemi.muni.cz http://www.chemi.muni.cz/ncbr/mulder.html

For NMR structure refinement, it is very important to have as many NMR restraints as possible. In order to evaluate the torsion angles unambiguously, we must reduce the degeneracy in solutions of Karplus equation:

3Jxy(ϕ) = P1 cos2(ϕ + phase) + P2 cos(ϕ + phase) + P3 To solve this problem we could 1) feed 3J-coupling constants directly into r-MD calculation together with appropriate Karplus parameterization(s) or 2) reduce the number of solutions to Karplus equation before r-MD calculation by exploiting more coupling constants for each torsion angle. While the first approach is definitely faster, the user does not have full control over what is going on inside r-MD, i.e., it behaves as a sort of black box. Moreover, r-MD programs supporting this feature (e.g., XPLOR, CNS, AMBER) know by default just the simple Karplus equation, represented by the above equation, and do not allow for using of more sophisticated forms referred to as generalized Karplus equations like those by Haasnoot, Donders, etc…

The second way offers more control over the geometry of resulting structure(s) but the amount of work is a bit larger. Program MULDER has been written to make the process of extracting one torsion angle from more 3J-coupling constants easier and as transparent as possible. In addition to coupling constants, MULDER can also use NOE-derived distances as functions of torsion angles by introducing the concept of pseudoKarplus equation.

Another problem during extraction of NMR restraints is caused by fast (compared to NMR time scale) interconversion of sugar conformations thus resulting in averaging of measurable 3J-coupling constants. Most often, program PSEUROT is used to assess the dynamic equilibrium of 5-membered rings but this program lacks any means for evaluation of calculated results. Therefore, MULDER has been extended to postprocess the PSEUROT output, filtering out “bad” records and calculating exo-/endo-cyclic torsion angles from pseudorotation parameters in remaining records. The presentation will also contain examples of graphical output of the program which is its key feature allowing to display all pertinent information in concise and publication-quality form.

37

AB-INITIO MO CALCULATION OF THE ANISOTROPY EFFECT OF MULTIPLE BONDS AND THE RING CURRENT

EFFECT OF ARENES - APPLICATION IN CONFORMATIONAL AND CONFIGURATIONAL ANALYSIS

Erich KLEINPETER and Sabrina KLOD

Universität Potsdam, Institut für Organische Chemie und Strukturanalytik, P. O. Box

601553, D-14415 Potsdam, F. R. Germany

Substituents containing magnetically anisotropic chemical bonds, e.g. double

bonds, triple bonds, or the aromatic phenyl ring, influence the shielding of any nucleus

in the molecule by their anisotropy effect dependent on its geometrical position. This

effect of the magnetic anisotropy of neighbouring groups on the chemical shift of nuclei

is usually specified qualitatively by the anisotropy cone. Along a theoretical MO study,

the magnetic anisotropy effect of unsaturated chemical bonds and the ring current effect

in arenes has been calculated quantitatively as nuclear independent chemical shieldings

(NICSs) in a three dimensional grid of “ghost atoms“ around the molecule using the

GIAO method implemented into the Gaussian 94 calculation program. Plotting the

shielding/deshielding data thus obtained as iso-chemical-shielding surfaces (ICSS)

around the magnetically anisotropic moieties allows to quantify both direction and scale

of the anisotropy effect.

The calculation of the anisotropy effect of double and triple bonds, and the ring current

effect of the phenyl ring, has been applied to a number of stereochemical problems; esp.

in conforma-tional analysis this proceeding proved very useful in quantitatively

assigning 1H chemical shifts and hereby the stereochemistry of the molecules studied. In

addition, contributions to 1H chemical shifts based on the anisotropy effect of

neighbouring groups and based on other substituent effects could be differentiated

quantitatively.

Considerable deviations from the qualitative sketches of the anisotropy effects of double

and triple bonds published in text books were found.

38

4J(H,C) - WHAT ARE THEY GOOD FOR?

Marek KUZMA and Petr SEDMERA

Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic

The textbook statement [1] |3JCH| > |2JCH| ≈ |4JCH| means that heteronuclear couplings over four bonds should be observable. The early source of data were measurements on C-13 specifically enriched compounds, analysis of proton-coupled 13C NMR spectra, and long-range heteronuclear correlations optimized for small J's. We have routinely observed 4JCH couplings in gradient HMBC experiments. According to our experience (>2 years), several categories could be recognized: i) across a benzene ring ii) through a double bond from one aromatic/heteroaromatic ring to another iii) from a N-methyl or C-methyl to a carbon atom in an adjacent ring iv) from OAc or NAc protons to the carbon bearing these groups Such couplings are mostly observed with protons resonating as singlets or narrow multiplets, especially with methyls. "Trans" coupling pathway favours the observation of a crosspeak, the involvement of heteroatoms is beneficial. The possible uses are verification of assignments, linking the spin systems (partial structures) together, determination of sites of acetylation (e.g., in sugars), and acetyl group assignments. Advantages and disadvantages of this approach will be discussed. References 1 J. L. Marshall, Carbon-Carbon and Carbon-Proton NMR Couplings: Application

to Organic Stereochemistry and Conformational Analysis, p. 46. Verlag Chemie International, Deerfield Beach, Florida, U.S.A., 1983.

39

QUANTITY IN MAGNETIC RESONANCE SPECTROSCOPY PART 4.

Milan MAZÚR

Department of Physical Chemistry, Faculty of Chemical Technology,

Slovak Technical University, Radlinského 9, SK-812 37 Bratislava, Slovakia.

The response of the cavity to the movement of the "over full-length cavity" cylindrical samples with lengths from 50 to 100 mm along the x-axis of the Bruker double TE104 and single TE102 rectangular cavity has been analyzed. The

experimentally observed dependencies of the EPR signal intensity, Ipp, versus sample position in the cavity showed the following: (i) a "sloping plateau", which can be approximated by the linear function (correlation, r=0.85-0.96); (ii) an additional oscillating signal superimposed on the "sloping plateau" region; (iii) an identical absolute value of the maximal signal intensity on the "sloping plateau" region for all sample lengths, within experimental error of 1.27 % . The trends of the above dependencies with the "sloping plateau" were independent of the length and internal diameter of the "over full-length cavity" samples, but, the width of the "sloping plateau" regions progressively increased with the increase of the sample lengths, from ca 30 mm for the 50-mm sample to ca 80 mm for the 100-mm sample. Theoretical predictions of the experimentally observed dependencies of the signal intensity, Ipp, versus sample position in the cavity were calculated using the "modified" and "revised" sine-squared functions, and the correlation between observed and theoretically computed dependencies is very good. The experimental dependence of the "over full-length cavity" sample position in the cavity, (x-coordinate), at which the signal intensity was a maximum, as a function of sample length, L, is non-linear. The experimentally determined x-coordinate of these sample positions in the cavity oscillated between the upper, L - a/2, and lower, L - a, theoretically computed limiting values, where, a (23.5 mm), is the length of the active part of the Bruker microwave rectangular cavity. Each of the above phenomena may be a serious source of significant errors in quantitative EPR spectroscopy. "Over full-length cavity" cylindrical samples to be compared in quantitative EPR should be of identical length and must be identically positioned in the microwave cavity.

40

CROSS CORRELATION EFFECTS IN PARAMAGNETIC SYSTEMS

Karin HOHENTHANNER, P.K. MADHU, Rita GRANDORI and Norbert MÜLLER

Institut für Chemie, Johannes Kepler Universität , A-4040 Linz, Austria The existence of cross correlation effects cause different relaxation rates and therefore

different linewidths within a spin multiplet. This phenomena (cross correlation between

chemical shift anisotropy CSA and internuclear dipolar interaction DD) is used for

instance in transverse relaxation optimised spectroscopy (TROSY), where only the

slowest relaxing component is selected.

In paramagnetic proteins the electron spin provides an additional relaxation mechanism

termed Curie Spin Relaxation (CSR) and cross correlation between CSR and

internuclear dipolar interaction (DD) can occur.

This cross correlation effect is shown to contain information regarding both distance

and angular orientation of a 15N- 1H vector with respect to the electron centre . The

relative intensities of the multiplet components observed in an αβ-HSQC-αβ type

experiment are directly correlated with the orientation of the 15N- 1H vector relative to

the electron centre.

This research has been supported by the Austrian Science Funds (FWF) projects P12696-CHE and M531-CHE and by the province government of Upper Austria.

41

WATER SUPPRESSION – RECENT DEVELOPMENTS

Zenon.STARČUK, Zenon.STARČUK, Jr., Jaroslav.HORKÝ

Institute of Scientific Instruments, Academy of Sciences of the CR, Královopolská 147, 61264 Brno, ČR

In the last decade, especially the use of magnetic field gradients has provided the basis for new and/or more efficient means of water suppression both in in vivo and in vitro (high-resolution) NMR experiments.

Most commonly used gradient-enhanced WS methods can generally be classified into two basic categories: (1) frequency-selective refocusing flanked by gradient pulses which dephase unwanted transverse magnetization and (2) frequency-selective excitation followed by dephasing the created transverse magnetization with gradient pulses. Basic representatives of the former category of spin-echo based WS techniques are WATERGATE techniques [1,2]. Since their introduction, numerous related WS techniques, such as MEGA, BASING, excitation sculpting, and some others have been developed to improve the performance of the original WATERGATE techniques. WS techniques of the latter category, commonly referred to by an acronym CHESS (chemical-shift selective), have been developed for attaining the performance robustness needed in in vivo applications. Increased attention has especially been paid to the development of various WET (water suppression enhanced through T1 effects) techniques [3]. In general it can be noticed that there is reduced interest in WS techniques that are tricky to set-up, that involve tedious optimization whenever a sample is changed or the probe is replaced, that introduce phase roll or baseline distortions, that are sensitive to B1 field inhomogeneities, that require too much RF power (especially for in vivo experiments), that are sensitive to T1 or T2 relaxation times, etc. Despite the progress achieved in the development of WS techniques, no scheme exists nowadays that meets all requirements imposed on WS under all experimental circumstances. Therefore, the development of WS techniques still remains one of the active areas of development in NMR. This work was supported by the Academy of Sciences of the Czech Republic (Grant A4065901) and by a project of Czech-Austrian cooperation KONTAKT (Grant 2000/9). References 1 V.Sklenář et al., J. Biomol.NMR, 1992, 2, 661. 2 V.Sklenář et al., J. Magn. Reson., 1993, A 102, 241. 3 Z.Starčuk,jr.et al., J. Magn. Reson. – submitted for publication.

42

KINETIC STUDY OF THERMAL ISOMERISATION OF 25,26,27,28-TETRAPROPOXY-2,8,14,20-

TETRATHIACALIX[4]ARENE

Jan LANGa, Jiří VLACHa, Hana DVOŘÁKOVáa, Pavel LHOTÁKb, and Richard HRABALa

aLaboratory of NMR Spectroscopy, bDepartment of Organic Chemistry Institute of Chemical Technology, Technická 5, Prague 6, Czech Republic

Thiacalix[4]arenes, analogues of the popular building blocks in supramolecular

chemistry - calix[4]arenes [1], possess new chemical features with respect to the parent compounds, for instance due to possible oxidation of sulphur atoms to sulfone or sulfoxide groups or due to their high affinity to transition-metal cations. [2,3]

These compounds can, in principle, adopt four basic conformations – cone, partial cone, 1,2-alternate and 1,3-alternate (Fig. 1) – that can in some cases interconvert. The interconversion barriers depend on size of substituents at lower rim. 25,26,27,28-Tetrapropoxy-2,8,14,20-tetrathiacalix[4]arene 1 undergoes conformational transitions only at elevated temperature yielding equilibrium mixture of all four conformers. [4] This process was continuously monitored by 1H NMR spectro-scopy. Reaction rate and equilib-rium constants were computed using an iterative procedure that involves a numerical solution of the set of kinetic differential equations. The calculation proce-dure utilized all available spectral information, including overlapping resonances. Reference [1] P. Lhoták, S. Shinkai, J. Synth. Org. Chem., Jpn., 1995, 53, 963 [2] G. Mislin, M. W. Hosseini, A. DeCian and J. Fischer, Tetrahedron Lett., 1999, 40,

1129 [3] N. Iki, N. Morohashi, F. Narumi, S. Miyano, Bull. Chem. Soc. Jpn., 1998, 71, 1597 [4] J. Lang, J. Vlach, H. Dvořáková, P. Lhoták, M. Himl, R. Hrabal, I. Stibor, J. Chem.

Soc., Perkin Trans. 2, 2001, in press

Fig. 1 Conformations of 1

43

MECHANISTIC STUDIES ON D-XYLOSE (D-GLUCOSE) ISOMERASE (EC 5.3.1.5): NMR SPECTROMETRIC

INVESTIGATIONS

Herwig HÄUSLER, Arnold. E. STÜTZ, Peter GREIMEL and Hansjörg WEBER

Institut für Organische Chemie, Technische Universität Graz, Stremayrgasse 16, A-8010 Graz Austria

D-Xylose (D-Glucose) isomerase has been used industrially for more than twenty years on a multi-ton scale for the production of High Fructose Corn Syrup from glucose1. The reaction catalyzed by the enzyme is shown in the following scheme:

OH

OHO

OHOH

OH

OH

OH

OHOH

OOHD-xylose isomerase

This enzyme has high synthetic value due to its ability to convert various pentoses and hexoses as well as a range of modified non-natural carbohydrates. We became interested in the mechanism of this reaction which had also been investigated by Bock2 and coworkers with NMR methods in 1983. It could be shown employing deuterated substrates that the pro-R hydrogen on C-1 of fructose or the corresponding deuterium is shuttled betwen C-1 and C-2 in D-glucose with a very high degree of stereoselectivity. NMR spectra of extracts of the conversion of deuterated glucose with the enzyme confirmed this result in our laboratory:

Figure 1. A) Isomerisation of (1-2H)-D-Glucose. B) Isomerisation of (2-2H)-D-glucose. C) Isomerisation of D-glucose/D-fructose. Arrows indicate the signals of proton(s) of A) the 1S, B) the 1R, C) the unsubstituted ß-D-fructopyranose tautomer.

Results obtained with a range of other aldopentoses as substrates for xylose isomerase will be presented3.

References: 1. Bosale S.H., Rao M.B., Deshande V.V., Microbiol. Rev. 60 (1996) 280-300 and references cited therein. 2. Bock K., Meldal B., Meyer B., Wiebe L., Acta Chem. Scand. B 37 (1983) 101-108. 3. Haeusler H., Weber H., Stuetz A.E., J. Carbohydrate Res. (2001) in press.

A B

C

44

ENANTIOMERIC ANALYSIS OF (3S, 4R)-4-(4-FLUOROPHENYL)-3-HYDROXYMETHYL-1-METHYLPIPERIDINE BY 19F NMR

SPECTROSCOPY

Hana NAVRÁTILOVÁ

Department of Organic Chemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic

A sensitive method of determining enantiomeric purity of 1a using S-Mosher acid 2 as a chiral solvating agent [3] is reported. The compoud 1a is an important synthetic precursor of an antidepressive drug paroxetine [1,2]. Formation of a diastereomeric salt complex with S-Mosher acid 2 resulted in chemical-shift anisochrony of some enantiotopic nuclei including fluorine in 4-fluorophenyl substituent of 1.

The magnitude of chemical-shift non-equivalence in fluorine spectra was strongly dependent on solvent polarity, concentration and enantiomeric composition. With help of 13C satellite signals detected in fluorine spectra it was possible to evaluate [4] very low amounts (0.2-1%) of an enantiomeric impurity 1b in the resolved product 1a. Figure A part of 282.37 MHz 19F {1H} NMR spectrum of enantiomerically pure 1a (trace a) and of a sample containing 1 % of 1b (trace b); recorded in the presence of S-Mosher acid 2 at 303K and calibrated to CFCl3.

References 1 Christensen, J. A., Squires, R. F. US patent 4,007,196, 1977. 2 Engelstoft, M., Hansen, J.B. Acta Chem. Scan., 1996, 50:164-169. 3 R. Fullwood and D. Parker, J. Chem. Soc. Perkin Trans. 2, 1994, 57-64. 4 K. McLeod and M. B. Comisarow, J. Magn. Reson., 1989, 84, 490-500.

F

N

CH3

OH**3

4

COOHF3C

H3CO

1: racemic 1a: (3S,4R) 1b: (3R,4S)

2

-116.2-116.0-115.8-115.6-115.4(ppm)

a

b

1a

1b

45

CONFIDENCE ELLIPSES FOR THE QUANTITATIVE ANALYSIS FROM THE WHOLE NMR SPECTRUM

Miroslav Holík1 and Josef Halámek2

1Department of Theoretical and Physical Chemistry, Faculty of Science, Masaryk University, Kotlářská 2, CZ 611 37 Brno, Czech Republic,

2Institute of Scientific Instruments, Academy of Sciences of the Czech Republic, Královopolská 147, CZ 612 64 Brno, Czech Republic

If the NMR spectrum does not contain any signal suitable for integration, a quantivative determination can be made from the whole spectrum which was properly broadened by an exponential multiplication of the FID. Then the mathematical procedure comprises either a multivariate calibration or a nonparametric estimation [1]. For both methods an accuracy of the results is not easy to find out. However, it can be estimated from a confidence ellipse. For this graphical presentation a calibration set of, e.g., 7 NMR spectra from repeated measurement of the same sample with the concentration of analyte corresponding to that which will be determined is obtained and each spectral curve is transformed into two coefficients x and y. These are in fact real and imaginary parts of the first complex number after the Fourier transform of the NMR spectrum. Around their average the confidence ellipses are drawn and thean the point from sample with different concentration is added. If the point,e.g., 2 is pretty out of the ellipses, the corresponding concentration will be easy to determine. The point 1 shows probable limit of the quantitative determination. References: 1 M.Holík, 22th Discussion Meeting of FMR, September 27-30, 2000, Regenburg, Germany, Book of Abstracts, p.72

∑∑==

−π=

−π=

n

1hh

n

1hh n

)1h(2sinAn1y

n)1h(2cosA

n1x

46

L I S T O F P A R T I C I P A N T S

47

48

BOHDÁLKOVÁ Jiřina CZ 15, 20 BOROS Sándor HU CAHA Erik CZ 25 CHMELÍK Josef CZ CROSS David DE DEMŠAR Alojz SI 21 DVOŘÁKOVÁ Hana CZ 9, 41 FIALA Radovan CZ 25 FRIEDL Zdeněk CZ 10 GLASER Robert IL 22 HOENIG Helmut AT HOHENTHANNER Karin AT 39 HOLÍK Miroslav CZ 44 HOLUB Josef CZ HORKÝ Jaroslav CZ 40 HRABAL Richard CZ 26, 30, 41 HRICOVINI Miloš SK 27 HUMPA Otakar CZ ILLASZEWICZ Carina AT JAMBOR Roman CZ 11, 18 KESSLER Pavel DE KLÍČOVÁ Zlata CZ KOZUBEK Ervin CZ 15, 20 KLEINPETER Erich DE 36 KŘÍŽOVÁ Hana CZ 28 KŘÍŽ Jaroslav CZ 23 KUBÍČEK Karel CZ KURKOVA Dana CZ 29 KUZMA Marek CZ 37 LÉBL Tomáš CZ 14 LUKÁŠKOVÁ Marcela CZ LYČKA Antonín CZ 12 MAREK Radek CZ 13, 19

49

MASOJÍDKOVÁ Milena CZ MAZÁČ Jiří CZ MAZÚR Milan SK 38 MUELLER Norbert AT 39 NÁLEZKOVÁ Monika CZ NAVRÁTILOVÁ Hana CZ 43 NECHVÁTAL Miloslav CZ PADRTA Petr CZ 35 PAVLOVSKÝ Jiří CZ 15, 20 PERJÉSSY Alexander SK 16 PETRIČ Andrej SI 17 PŮČEK Ladislav CZ RIEDL František CZ RŮŽIČKA Aleš CZ 11, 18 SCHRAML Jan CZ SEČKÁŘOVÁ Pavlína CZ 19 SEDMERA Petr CZ 31, 37 SEGER Christoph AT 33 SEJBAL Jan CZ SIMON András HU SKLENÁŘ Vladimír CZ 28, 34, 35 SPĚVÁČEK Jiří CZ 24 STARČUK Zenon CZ 40 SUCIU Daniela RO 32 SYCHROVSKÝ Vladimír CZ 34 SZÕLLÕSY Áron HU TOTH Gábor HU TRANTÍREK Lukáš CZ 25 TUREČKOVÁ Milena CZ VACEK Jaroslav CZ 34 VEVERKA Václav CZ 26, 30 VLACH Jiří CZ 41 WEBER Hansjoerg AT 42 ŽÍDEK Lukáš CZ 28

50