abstracts poster presentations (l)

Journal of Inorganic Biochemistry 86 (2001) 231

A MRI contrast agent responsive of esterase enzymatic activity

S. Geninatti Crich a, S. Aime a, C. Cabella a, L. Lattuada b, S. Pavesio ~, G. Tarone c Department o f Chemistry IFM, University of Torino, via P. Giuria 7, 10125, Torino, Italy. Bracco Imaging SpA, via E. Folli 50, 20134, Milano, Italy.

CDepartment of Genetics, Biology and Biochemistry, via Santena 5bis, 10126, Torino, Italy. (e-mail: [email protected])

Nowadays it is well established that several magnetic resonance imaging (MR/) procedures take advantage from the use of Gd(III) based contrast agents. However it is also a commonly accepted view that the next generation of contrast agents will be represented by systems either endowed with improved targeting capabilities either able to act as reporter of a specific parameter of the microenvironment "i • ° whereby they distribute. In the field of systems whose relaxivity is sensitive to the ii "

/

activity of a given enzyme we propose an insoluble Gd-complex that becomes soluble ~ o only upon a selective enzymatic cleavage of the functionality responsible for its o insolubilization. The Gd(III) chelate used in this work is a DTPA derivative containing •

sO ~S ~0 SS ~0 two C 18-aliphatic chains bound to the ligand surface through ester functionalities. Upon ...., esterase activity there is the release of a soluble Gd(III) chelate whose amount can be directly assessed by the increase of water protons relaxation rate. An assay for evaluating the esterase activity in phagocytic cells of reticuloendothelial system has been set up. The results obtained upon cellular uptake have been compared with those ones obtained "in vitro" in the presence of different esterases.

1. Aime S., Botta M., Gianolio E., Terreno E., Angew. Chem. Int. Ed., 39, 747, (2000) 2. Caravan P., Ellison J.J., McMurry T.J., Lauffer R.B., Chem. Rev., 99, 2293-2352, (1999)

Chemical studies of diiron(I) organometallic complexes

Irene P. Georgakaki, Xuan Zhao, Joseph H. Reibenspies and Marcetta Y. Darensbourg Department o f Chemistry, Texas A&M University, College Station, TX 77843, USA

(e-mail. 'georgakaki@mail. chem. tamu. edu)

The structural similarity of the active site of Fe-only hydrogenase I to low valent di-Fe(I) organometallic complexes (~t-SRS)(Fe2(CO)6 2, 3 suggests that these type of complexes can be used to define the chemistry related to this active site. 4 It is known that the mechanism of hydrogenase , ~ _ activity involves the reversible heterolytic splitting of molecular H2 to H ÷ and H. \S',#S~.. /S--4Fe4S However the way that this function is facilitated by the active site has not been . . . . Fe,,, F~.. established. Towards this direction, studies of model complexes with different sources of O,C~ Z C"" -S~,CN hydrogen (H ÷, H or H2) are designed to give insight into the enzymatic mechanism in N O ~'O terms of plausible intermediates formed and the role of the two Fe centers in the active site.

Theoretical (DFT) calculations identify the HOMO of (~-SRS)Fe2(CO)6 as the Fe--Fe bond density. 3 Protonation of (bt-SCHzCH2CH2S)Fe2(CO)4L2 n- (L = PMe3, n = 0 or L = CN, n = 2) is observed to give products with a high field resonance typical for /.t-H species. Similar Fe--Fe distances in the FeIFe I neutral (p-SCH2CH2CHzS)Fez(CO)4(PMe3)2 (2.556 A) and the protonated FenFe H (p-H)(~t-SCHzCH2CH2S)Fe2(CO)4(PMe3)2 + (2.575 A) species were determined by x-ray analysis. Characterization attempts designed to establish the pKa value show that both strong anionic bases like CN and weak bases like CI rapidly deprotonate the HFe2 + giving back the parent compound; whereas, the neutral Et3N abstracted the [.t-H very slowly and incompletely. Studies of the reactivity of HFe2 + species with D2 are underway.

1. Peters J. W., et al., Science, 282, 1853-1858 (1998), Nicolet Y., et al., Structure, 7, 13-23 (1999) 2. Lyon E. J., Georgakaki I. P., Reibenspies J. H., Darensbourg M. Y., Angew. Chem. Int. Ed., 38, 3178-3180 (1999) 3. Lyon E. J., Georgakaki I. P., Reibenspies J. H., Darensbourg M. Y., J. Am. Chem. Soc., 123, 3268-3278 (2001) 4. Cao Z. and Hall M. B., J. Am. Chem. Soc., 123, 3734-3742 (2001)

232 Journal of Inorganic Biochemistry 86 (2001)

Stopped- f low in frared spec troscopy reveals a 6 -coordinate in termedia te in the format ion of p r o x i m a l l y b o u n d 5-coordinate N O adduct o f c y t o c h r o m e c"

Simon J. George, a Colin R. Andrew, b David M. Lawson, a Roger N.F. Thomeley, a Robert R. Eady. a

Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, UK (e-mail: [email protected], uk)

b Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 NW Walker Road, Beaverton, Oregon 97006-8921, USA.

Stopped-flow Fourier transform infrared (SF-FTIR) spectroscopy has been used to monitor the novel binding chemistry of NO to cytochrome c ' from Alcaligenes xylosoxidans. ~ A recent crystallographic analysis of this protein showed that NO binds as a 5-coordinate complex (5c-NO) with the novel binding of NO at the proximal side of the heme. 2 Using time-dependent isotope difference (~4NO - 'sNO) spectroscopy, we show that NO first binds to form a 6- coordinate NO-heme complex (6c-NO) with v(N=O) at 1625 cm l and presumably distal NO coordination. This is then converted to 5-coordinate NO-heme (5c-NO) whose infrared spectrum comprises at least three conformers at 1678, 1666 and 1654 cm 1. The SF-FTIR data also show isotope insensitive time-dependent changes which may be interpreted in terms of distortion of the polypeptide and heme as well as the dissociation of the proximal histidine. The rates of both steps increase with increasing NO concentration. A mechanism is proposed in which 5c-NO is formed from 6c-NO through attack by a second NO from the proximal side of the heme. This novel chemistry may provide the mechanistic key for ligand-discrimination and signal transduction in heme-based gas sensor proteins such as soluble guanylate cyclase (sGC).

1. George, S.J.; Andrew, C.R.; Lawson, D.M.; Thorneley, R.N.F. and Eady, R.R.J. Amer. Chem. Soc. in thepress. 2. Lawson, D.M.; Stevenson, C.E.M.; Andrew, C.R. and Eady, R.R. EMBO J., 19, 5661-5671 (2000)

We thank the BBSRC (UK) and the NIH (USA) for f'mancial support.

X-ray s tudies of artif icial meta l loprote ins with 4-hel ix bund le mot i f

S. Geremia a, L. Di Costanzo a, W. F. DeGrado b, A. Lombardi c, V. Pavone c , L. Randaccio a

" CERB, Centro di Eccellenza per la Ricerca inBiocristallografia, Department of Chemical Sciences, University of Trieste, Via L. Giorgieri 1, 1-34127 Trieste, Italy

b The Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104-6059, USA

c Department of Chemistry, University of Napoli, Via Cintia, 1-80126 Napoli, Italy

Artificial metalloproteins mimicking natural systems are important for development of novel materials, catalysts, and biosensors. A very simple model protein, DF1, has been recently designed, l DF1 is a homodimer of helix-loop-helix hairpins assembled to form a 4-helix bundle (Figure) with a site similar to the binuclear iron site of bacterioferritin. Recently, we solved the crystal structure of the DF1 zinc derivative at 2.5 A of resolution.l The core of DF 1 is extremely well packed, and Leu 13 and Leu 13' occupy the space proximal to the di-metal site, preventing access of substrate molecules. Substitution of these residue with smaller side chain amino acids, like Ala and Gly, form a "hole" near the di-metallic site, which should allow the access of substrates. The di-Mn(II) form of DF l-L13A and DF1-L 13G variants were crystallized and structurally characterized by X- ray diffraction experiments at the Elettra Synchrotron. An important electron density near to the two metal ions has been found in the "hole" formed by the substitution of the Leul 3. The X-ray structures of these artificial metalloproteins will be described.

1. Lombardi, A., Summa, C., Geremia, S., Randaccio, L., Pavone, V. & DeGrado, W.F. Proc. Natl. Acad. Sci. USA, 97, 6298-6305 (1999).

The Ministero della Ricerca Scientifica e Tecnologica (MURST) of Italy is acknowledged for Financial support.


S u p r a l i g a n d fine tuning of Zn-based enzymes: the case of Horse Liver alcohol d e h y d r o g e n a s e

Francesco Luigi Gervasio ~, Vincenzo Schettino a, Stefano Mangani b, Paolo Carloni ', Michele Parrinello a "Department o f Chemistry, University of Firenze, via Gino Capponi 9,I-5012, Firenze, Italy (e-mail: gervasio@chim, unifi, it) b Dipartimento di Chimica, University o f Siena, v iaA. Moro, 1-53100 Siena, Italy

International School for Advanced Studies (SISSA/ISAS), via Beirut 4, 1-34014, Trieste, Italy J Max-Planck-Ins t i tu t fur FestKorperforschung, Heisenbergstrasse 1, D-70569, Stuttgart, Germany

The existence of specific and conserved structural motifs ~ involving metal ligands H- bonded to surrounding aminoacids suggests that second shell ligands (and perhaps beyond) play a role for the chemistry of these enzymes, In this respect, zinc enzymes are /~'~ a typical example. Histidinate ligands, present in the majority of Zn proteins 2 most often ~,O) ~,67 H-bonds to Glu and Asp forming a Zn-His-carboxyl(ate) triad. Here we investigate the c,~74 [ c,~ importance this structural motif in modulating the catalytic function of many zinc ~'-~,'~""it enzymes by studying the case of horse liver alcohol dehydrogenase, one of the most " .... ~--_ studied zinc enzymes. His geometry, electronic structure and reactivity were studied by ~ stateoftheartab-initiodensityfunctionalmethods. Themodelsystem(inFig.)was . ~ . ~ @ ) ~ studied in presence or absence of aspartate hydrogen bonded to the zinc ligand histidine o N and of an highly conserved water molecule bonded to the aspartate. Both the aspartate

NAD* and the water molecule were found to be essential to correctly reproduce the chemistry of the active site.

1. Christianson, D. W. Adv.Prot.Chem. 1991, 42, 281-355. 2. Coleman, J. E. Curr.Opin.Chem Biol. 1998, 2, 222-234.

R e l a x o m e t r i c agents respons ive to se l f -assembl ing processes

E. Gianolio a, S. Aimea,, G.C. Morelli b, C. Pedone b, D. Tesauro b Dipartimento di Chimica I.F.M., Universith di Torino, Via P. Giuria 7, 10125 Torino, Italy

(e-mail: gianolio@ch, unito, i 0 b Centro di Biocristallografia del CNR, Via Mezzocannone 6/8, 80134 Napoli, Italy

In solutions containing Gd(III) chelates, the measure of water proton relaxation time assess the occurrence of self-assembling processes. In fact the relaxation enhancement promoted by the exchange of coordinated water is highly sensitive to the molecular reorientational time of the paramagnetic complex. This effect has been largely exploited in MRI applications 1. Porphyrins are particularly suited building blocks for investigating self-assembling processes. The "stacking" of porphyrins is the result of the Van Der Waals n-n interactions in the hydrophobic environment between the porphyrin n-clouds which result in close contacts z. Herein the relaxometric properties of a porphyrin derivative containing four DTPA moieties conjugated to its external surface [Por(DTPAh] are reported. It has been found that the aggregation of metal complexes of [Por(DTPA)4] can be conveniently controlled by modulating the charge repulsion between the individual molecular components. Thus, whereas [Por(GdDTPA)(DTPA)3] displays a relaxivity of 11.3 raM" s l , [Por(GdDTPA)4] reaches a value of 39 mM t s t

~rovides an excellent probe to

A .

=" ~ ~ ? " ' 7 " , -

[Por(GdDTPAhl

per Gd unit. Analogously several [Por(GdDTPA)(M-DTPA)3] systems (where M is a diamagnetic metal ion either divalent or trivalent) have been investigated and the obtained results discussed in terms of self-assembly processes modulated by the charge of the chelated M ions.

1. Caravan P., Ellison J.J., McMurry T.J. Lauffer R.B., Chem. Rev.,99,2293-2352,1999 2. White W.I., The Porphyrins, Dolphin D.Ed.; Academic: New York, Vol. 5 Chapter 7, 1978


Inner- and outer-sphere co-ordination of the minor groove site of adenine by alkali metal ions

Ashleigh E Gibson, Clayton Price, William Clegg and Andrew Houlton. Department of Chemistry, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU UNITED KINGDOM (e-mail: a.e.gibson@ncl, ae. uk)

Group I metal ions, particularly Na + and K*, are ubiquitous in their association with nucleic acid structures. The condensation of these cations is essential for the assembly of DNA duplexes to offset the repulsive interactions of the anionic phosphodiester backbone. Reliably detecting these ions by X-ray crystallography of synthetic oligonucleotides has proved challenging.I A feature of these studies is the so-called 'spine of hydration' in the minor groove of duplex DNA. Subsequently this has been reinterpreted as also containing metal ions. Here we have considered the nature of the interaction between Group I cations and the minor groove sites on adenine. 2'3 We achieved such interactions by complexation of cations into an aza-18-crown-6 macrocycle which was tethered at the N-9 position (1). Using crystallographic methods we have demonstrated two distinct binding modes from cation to N-3: Co-ordinate bond formation and outer sphere binding via a co-ordinated water molecule. Synthesis and structural data will be presented

1. Tereshko, et al. Nuc. Acids Res., 29, 1208-1215 (2001). 2. De Wall, ,Z Chem. Cryst., 30, 227-231 (2000). 3. Meadows etal. J. Am. Chem. Soc., 123, 3092-3107 (2001)

NH2

o¢

The BBSRC and Leverhulme Trust is acknowledged for financial support.

Protein-based radicals in M. tuberculosis catalase-peroxidase (KatG)

Stefania Girotto a, Salem Chouchane a, Vladimir Krymov b, Gary Gerfen u and Richard S. Magliozzo a Brooklyn College and CUNY Graduate School, Dept. of Chemistry, 2900 Bedford Ave., Brooklyn, NY 11210, USA

b Albert Einstein College of Medicine of Yeshiva University, Dept. of Physiology and Biophysics, 1300 Morris Park Ave., Bronx, NY 10461, USA. (stefania girotto@hotmail, com)

Catalase- peroxidase (KatG) is a heme enzyme in M. tuberculosis responsible for activation of the first-line antibiotic pro-drug, isoniazid (isonicotinic acid hydrazide), used to treat tuberculosis infection. Optical stopped-flow experiments demonstrate the formation of a short-lived Compound I (pi-cation radical) species upon reaction of the resting (ferric) enzyme with alkyl hydroperoxidesJ Rapid freeze-quench electron paramagnetic resonance (EPR) experiments .were also performed to follow this reaction. At early time points (10-500 ms) after mixing enzyme with peroxyacetic acid, a transient doublet signal was found (major splitting 16 Gauss, overall linewidth 32 Gauss) in X-band EPR spectra. As the doublet signal decreased in intensity, a singlet with poorly resolved hyperf'me features appeared (linewidth 27 Gauss) and persisted beyond 10 sec. Examination of the singlet species using high field EPR (microwave frequency 130 GHz) revealed a spectrum dominated by anisotropy in the electron Zeeman interaction, with g-values consistent with an assignment to tyrosyl radical. The g-values and proton nuclear hyperfme interaction parameters observed for the singlet were similar to those evaluated for tyrosyl radicals in other systems. The high-field EPR spectrum of the initial doublet species does not exhibit the g-anisotropy found for the singlet. Simulation of the doublet epr spectrum at X-band, along with a preliminary analysis of its high-field epr spectrum suggest that it may arise from a neutral tryptophan radical though other radical species cannot be ruled out at present. The doublet signal is rapidly quenched upon addition of isoniazid, suggesting that this radical may have a catalytic function in KatG separate from the known function of Compound I in drug activation.

1. Chouchane S., Lippai I., and Magliozzo R. S., Biochemistry, 39, 9975-9983 (2000).


Metal ion interaction with duplex and triplex forms of polyA and polyU

G.O. G ladchenko , V.A. Sorokin , V.A. Va leev , E.A. Onyshchenko , M.V. Degtyar , Yu.P. Blagoi

B.I. Verkin Institute for Low Temperature Physics and Engineering, National Academy of Sciences of Ukraine, Lenin ave 47, 61164 Kharkov, Ukraine, (e-mail: gladchenko@kharkov, ua )

Ni 2+, Cu 2+, Cd 2÷ and Zn 2+ interactions with single-stranded (1S) polyA and polyU, double (2H) - and triple-helical (3H) structures formed by these homopolynucleotides in buffer solutions (pH 6-7) with different Na + ion contents (10 2 - 10 -t) M were studied by the method of difference UV spectroscopy.. Diagrams of the phase equilibrium between IS, 2H and 3H structures were built for the concentration range 10 .5 - 10 -3 M of divalent ions.

State diagrams for system of (polyA+polyU)+ Ni 2+ are qualitatively similar to those of polyA+polyU+Na* ,Mg 2+ [1,2].

Like Mg 2÷ , Ni 2÷ stabilizes 2- and 3-helical structures and preferentially multy-H of the higher hierarchy degree, tm values for 2H---~lS transitions in the presence of Mg 2+ and Ni z+ coincide. At [Mt]>3. 10-SM (a triple point at the phase diagram) the phase 3H appears the existence region of which broadens with the rise of [Ni z÷ ] due to the increase of tm 3H--~l S. Temperatures of transitions 2H-~3H exceed those for Mg 2+. The effect of Ni 2÷ ions upon the thermal stability of 2H(polyA-polyU) and 3H (polyA.2polyU) is mainly connected with their different binding to multy-stranded helices and polyU. The constants of the Ni 2+ binding are found.

Zn z+ stabilizes both 2H and 3H. Cd z+ weakly affects 3H--*2H transition while Cu 2÷ destabilizes triplexes. The Me 2+ effect on tm 3H---~2H obeys to the Ni_>Mg>Zn>Cd>Cu raw. High concentrations of transition metal ions ([Cu2+]> 10 a, [Cd2+]> 2.10 4, [Zn2+]> 10 "3) induce aggregation of I S.

1. Krakauer H., Biochem., 13,2579- 2589(1974) 2. Gladchenko G.O., Sorokin V.A., Valeev V.A., Degtyar M.V., Blagoi Yu.P, in: "Spectroscopy of Biological

Molecules: New Directions", Kluwer Acad. Publ., 259- 260, (1999)

Triazacorrole macrocycles from porphyrazine and phthalocyanine precursors: new porphyrinoid compounds for the stabilization of high oxidation states

David P. Goldberg , B o b b y Ramdhan ie , and Joseph P. Fox

Department of Chemistry, The Johns Hopkins University, 3400 N. Charles Street, Baltimore MD, 21218, USA (email: [email protected])

Corroles are a class of porphyrinoid macrocycles that have a remarkable ability to stabilize high oxidation states (e.g. Mn v, Fe TM, and CoV). ~ Although there is great interest in studying these compounds in terms of their biological relevance and the promise they hold for catalysis, difficulties in their synthesis have prevented their widespread use. Recently, more facile methods of corrole preparation have been described. 2 Here we describe our results concerning the facile synthesis of novel triazacorrole compounds from readily accessible porphyrazine precursors, 3 following ring-contraction procedures first observed in phthalocyanine chemistry. 4 The metal-free (free base) form of the triazacorrole system has been prepared by a novel reductive demetallation reaction. With the free base form in hand, a large class of new transition metal corrole-type compounds is now accessible. The synthesis and characterization of the cobalt, iron and copper triazacorroles will be discussed, along with a phosphorus(V) triazacorrole that has been characterized by X-ray crystallography, absorption spectroscopy, cyclic voltammetry and multinuclear NMR (tH, 31p, 13C) methods.

Erben, C., Will, S. and Kadish, K. M. In The Porphyrin Handbook; Kadish, K. M., Smith, K. M. and Guilard, R., Eds.; Academic Press: New York, 2000; Vol. 2 pp 233-300. Gross, Z., Galili, N., Simkhovich, L., Saltsman, I., Botoshansky, M., Blaser, D., Boese, R., and Goldberg, I. Org. Lea., 1, 599-602 1999. Ramdhanie, B., Stern, C. L., and Goldberg, D. P., submitted for publication 2001. Li, J., Subramanian, L. R., and Hanack, M. Eur. J. Org. Chem., 2759-2767 1998.


Models of sulfur reactivity in [NiFe] and [FeFe] hydrogenases

Melissa L. Golden, Matthew L. Miller, and Marcetta Y. Darensbourg Department of Chemistry, Texas A&M University, 77843, College Station, USA (e-mail: snodgrass@mail, chem. tam u. edu)

Sulfur rich metalloenzyme active sites in [NiFe] and [FeFe] hydrogenases present opportunity for sulfur-based transformation of organic moieties. Terminal cysteine thiolate sulfurs bound to Ni are in proximity to the terminus of a substrate delivery channel in [NiFe] hydrogenase I, and the 2Fe2S unit in the H-cluster of [FeFe] hydrogenase strongly resembles derivatives of (p.-Sz)Fe2(CO)6, well known to template C-S bond forming reactions. Model compounds for the Ni site in [NiFe]H2ase also demonstrate sulfur-carbon bond formation in reaction with alkylating agents.

The active site of [NiFe] and [FeFe] hydrogenases are unusual in that the diatomic ligands CO and CN- are found bound to iron in each case; a Fe(CO)(CN)2 moiety is present in the former and two Fe(CO)(CN) units are in the latter. In pursuit of reaction model

Me 0% S

/ X/C--Me 0

chemistry that would provide evidence for the likelihood that these toxic ligands are delivered in a benign form, [O3P(=O)C(=O)NI-I2];-, 2 we explore the possibility that the carbamoyl phosphate serves as an alkylating agent to nickel dithiolate or iron sulfides and results in degradation to C N and CO diatomic ligands. The cis-dithiolate model complex (bme-dach)Ni demonstrates reactivity with MeC(=O)C1 and with MezNC(=O)C1. The crystal structure of the product of acetyl chloride and (bme-dach)Ni demonstrates acetylation of both thiolates, with one thioester remaining bound to Ni and the other displaced by C1- illustrating the weak binding ability of the resulting thioester. A thorough examination of the degradation products from carbamoylation is underway.

1. Montet Y., Amara P., Volbeda A., Vernede X., Hatchikian E. C., Field M.J., Frey M., and Fontecilla-Camps J. C., Nat. Struct. Biol., 4, 523-526 (1997)

2. Paschos A., Glass R. S., and B6ch A., FEBS Letters, 488, 9-12 (2001)

Functionalization of manganese corroles and the first third-generation corroles- based catlyst

Galina Golubkov a, Israel Goldberg b, Jesper Bendix a and Zeev Gross a'* aDepartment of Chemistry, Technion - Israel Institute of Technology, Haifa 32000, Israel. bSchool o f C h e m i s t r y , T e l - A v i v U n i v e r s i t y , R a m a t A v i v , I s rae l .

We have recently shown that the iron and rhodium complexes of 5,10,15- tris(pentafluorophenyl)corrole are potent catalysts for oxygen- and carbene-transfer to olefins and alkanes. 1 In contrast to the fully characterized c~V(o) and FeW(C0 corroles, only manganese(III) complexes were reported in the literature. 2 We have now found that Mnm(tpfc) is a mild epoxidation catalyst and were also able to characterize the relatively stable oxomanganese(V) corrole. 3

In addition, we report here the isolation and the X-ray structures of two manganese(IV) corroles, characterization of a nitrido- manganese(V)corrole, as well as preliminary results for the first per- halogeneted manganese(III) corrole and its activity as oxidation catalyst. 4

1. Gross, Z., Simkhovich, L., Galili, N., Chem. Commun, 7, 599-600 (1999).

2. Bendix, J., Gray, H. B., Golubkov, G., Gross, Z., Chem. Commun., 19, 1957-1958 (2000) and references therein.

3. Gross, Z., Golubkov, G., Simkhovich, L., Angew. Chem. Int. Ed., 39, 4045-4047 (2000).

4.

X-ray structure of (tpfc)MnlVBr

Golubkov, G., Bendix, J., Gray, H.B., Mahammed, A., Goldberg, I., DiBilio, A. J., Gross Z., Angew. Chem. Int. Ed., 40, 2132-2134 (2001).


1,3,4-Oxadiazolecopper(II) complexes: crystal structures of [Cu(CaHsN40)2(H20)2](NO3)2 and [Cu(CsHsN40)(oxalate)(H20)].H20.

Javier Garc ia -To ja l a' Pa t r ic ia G 6 m e z - S a i z a, Migue l A. Maes t ro b, Jos6 M a h i a b

"Dpto de Quimica, Univ. de Burgos, Misael Ba~uelos, s/n, 09001 Burgos, SPAIN (e-mail: [email protected]) bEdificio Anexo Facultade de Ciencias, Univ. da Coru~a, E-15071, A Coru~a, SPAIN

1,3,4-Oxadiazoles have a wide variety of applications and exhibit relevant biological properties) Complexes of formula [Cu(CsHsN40)2(H20)z](NO3)2 (1) and [Cu(CsHsN40)(oxalate)(H20)].H20 (2), where (CsHsN40) = 2- methylamino-5-pyridin-2-yl-1,3,4-0xadiazole), have been characterized. Both compounds show a monomeric character. Their structural features and a comparative study with analogous compounds-' will be discussed. Biological studies are in progress.

"r"

q

0 4

(1) (2)

1. Sharma B. L., Tandon S. K., Pharmazie, 39 H-12, 858-859 (1984) 2. Lagrenee M., Sueur S., Wignacourt J. P., Memari B., Boukhari A., J. Chim. Phys., 88, 2075-2082 (1991)

Cellular effects by substituted transpyridine platinum complexes

Adoraci6n Gomez-Quiroga a, C. Navarro-Ranninger, J.M. Perez, N. Farrell Departamento de Quimica Inorgdnica, Facultad de Ciencias, Universidad Aut6noma de Madrid,

Cantoblanco, 28049-Madrid, Spain (e-mail: [email protected]) Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284-2006, USA.

Transplatinum compounds represent an interesting challenge for anticancer drug development. The activation of the transplatinum geometry was first demonstrated in vitro by substituting heterocyclic ligands (L') such as pyridine ligands for NH3 in the general structure trans-PtL(L')X2 where L=L ' or L = NH3 ~L'. ~ More recently. It was found that similar activation may be obtained using simple aliphatic amines (L = isopropylamine, L' = dimethylamine). 2 This contribution compares the patterns of cytotoxicity and apoptotic effects using transplatinum compounds containing either heterocyclic pyridine ligands or aliphatic amines in tumor cells transformed by ras oncogenes (Pam 212-ras). Further the effect of substituents in the pyridine ring is examined.

1. Farrell, N., Kelland, L.R., Roberts, J.D. and Van Beusichem, M.: Cancer Res. 52:5065 (1992). 2. Montero EI, Diaz S, Gonzfilez-Vadillo AM, P6rez JM, Alonso C, Navarro-Ranninger C.: J Med Chem. 42:4264

(1999).

238 Journal of lnorganic Biochemistry 86 (2001)

Deve lopment of novel S OD mimics and D N A cleavage systems based on copper( lI ) complexes of Benzothiazole-N-Sulfonamides.

Marta Gonz~lez-/klvarez ~, Gloria Alzuet ~, Joaquin Borrfis ~ , Lucas del Castillo Agudo b, Jose Manuel Montejo-Bernardo c, Santiago Garcia-Granda c, Virtudes Moreno o, Maria J. Prieto e. a Departamento de Quimica Inorgdnica. b Departamento de Microbiologia y Ecologia.

Universidad de Valencia. ,4vda. Vicent AndrOs Estellds s/n. 46100 - Burjassot, Spain c Departamento de Quimica-Fisica y Analitica. Universidad de Oviedo. Avda. Julidn Claveria 8,

33006-Oviedo, Spain d Departamento de Quimica Inorgdnica. e Departamento de Microbiologia. Universidad de

Barcelona. Diagonal 647, 08028-Barcelona, Spain

Copper(II) ternary complexes based on the novel benzothiazole N-sulfonamides and pyridine have been synthesized and characterized. The crystal structure of the complexes [Cu(L1)2(py)2] (1) and [Cu(L2)2(py)2] (2) were determined by single crystal X-ray analysis. In both compounds the metal ion adopts a square planar geometry. A new in vivo method has been developed for determining the SOD-like activity of the complexes. It consists on the protection by the complexes of the Asodl mutant Saccharomyces cerevisiae against free radicals generated by hydrogen peroxide and menadione. Figure shows the effect of complex 1 on the growth inhibition generated by oxidative stress in S. cerevisiae Asodl mutant. Complex I shows high SOD- mimetic activity. The nuclease activity of complex 1 has been tested from electrophoretic and Atomic Force Microscopy (AFM) methods. The results suggest that this compound is a potent chemical nuclease.

Cycit and Ministerio de Ciencia y Tecnologia are acknowleged for financial support (PM-97-105-C02-01) and for the doctoral grant of M. Gonz,qlez-~i, lvarez.

A bioinorganic insight into the Zn requirement for the in vivo folding of copper metallothioneins

Pilar Gonz,-ilez-Duarte b, Sflvia Atrian a, Roger Bofill b, Merc~ Capdevila b, Neus Cols a, Roser GonzAlez-Duarte a, Marc Vails ~. O Departament de Gen~tica, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645,

E-08028, Barcelona, Spain b Departament de Quimica, Universitat Autdnoma de Barcelona, E-08193, Bellaterra, Barcelona,

Spain, (pilar. gonzalez, duarte@uab, es)

Metallothioneins (MT) are small proteins with a high Cys content that are characterised by their extreme ability to bind essential heavy metal ions, such as Zn(lI) and Cu(I), and toxic heavy metal ions, such as Cd(ll) or Hg(II). In order to elucidate the structural significance of Zn(II) in Cu-MT species in physiological conditions, we have undertaken the analysis of recombinant mouse MTI and its ct and 13 fragments, i Homartt~ americanus MT (MTIT) and its two 1313MTH and 13aMTH domains, 2 and Drosophila MTN 3, synthesised by E.coli cells grown in Zn- and Cu-supplemented media. In addition, the Cd and Cu aggregates obtained in vitro by Zn replacement in Zn-MT proteins have also been characterized by using different spectroscopic techniques (UV-Vis, circular dichroism, emission and optical ICP) and through the use of electrospray ionization mass spectrometry (ESI-MS). The results obtained have enabled us to conclude that Zn(II) is essential for the in vivo-folding of recombinant mouse MT and recombinant lobster MT in the presence of Cu(I), while it is not the case for recombinant Drosophila MTN. By using a protein-sequence similarity approach, the binding ability of MT with respect to physiologically active metals (Zn, Cu) can be related to the primary structure of MT.

1. Bofill R., Capdevila M., Cols N., Atrian S. and Gonzfilez-Duarte P., J. Biol. lnorg. Chem., 6, 405-417 (2001) 2. Vails M., Bofill R., Gonzhlez-Duarte R., Gonzhlez-Duarte P., Capdevila, M. and Atrian, S., Submitted 3. Cols N., Romero-lsart N., Bofill R., Capdevila M., Gonz/dez-Duarte P., Gonzidez-Duarte R. and Atrian S., Prot.

Eng., 12, 265-269 (1999)

Journal of Inorganic Biochemistry 86 (2000 239

Immobilization of pinch type ligands onto polystyrene-divinylbenzene copolymers and their complexes

with iron (III)-protoporphyrin ix

Enrique GonzMez-Vergara, Alejandro Alonso-Calder6n, Vladimir Carranza-Tdllez, Brenda Sfinchez- Gayt/m, y Laboratorio Regional de Investigaciones Biol6gicas y Centro de Quimica. lnstituto de Ciencias de la Benem&ita Universidad Autdnoma de Puebla.

l~ Sur 6301. C. U. San Manuel. C.P. 72570.( e-mail. engonza l@s iu .buap .mx)_

Immobilization of two pinch type ligands I and their complexes with iron(III)- protoporphyrin IX are here reported. Ligands with two terminal imidazole groups bridged by Glutamic acid moieties were synthesized in a 1:3 water-dioxane mixture using Histidine and Carnosine as building blocks. The Glutamic acid was previously activated in acetone with

1,1-carbonildiimidazole. The characterization was carried out by UV, IR, and I~C y ~H spectroscopies. Solubilization of heroin in water was performed in the presence of the ligands and analysis of the UV spectra of the complexes showed a mixture of high and low spin species, being the latest the more abundant even at low concentrations of the ligands. Reduction with dithionite affords low spin iron (II) complexes with similar spectra to cytochrome bs. Immobilization was achieved at pH 8 by reaction of a polystyrene-divinylbenzene copolymer previously functionalized with triglycine and activated with CDI

Proposed structure for the immobilized

pmch-porphyrin

1. Reyes Ortega Y., ,/dvarez Toledano C., Ramirez Rosales C., Sfinchez Sandoval A., Gonzglez Vergara E. y Zamorano Ulloa R.; Pinch-Porphyrins, new spectroscopic and kinetic models of peroxidases; Journal of the Chemical Society Dalton Transactions, (004): 667-674, 1998.

Funds were provided by FOMES 2000, CECYTand CONACYT M6xico. and CONACYT M6xico

Synthesis of Pt(II) complexes with diazenecarboxamides

Sabina Grabner, Janez Kosmrlj, Nata~a Bukovec Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia

GSH (glutathion) reduces the cellular toxicity of chemotherapy agents such as platinum compounds] It has been postulated that one form of drug resistance results from the elevated levels of GSH in tumors. Recent study has shown that diazenecarboxamides as selective oxidants lowered GSH concentraction and with combination with anticancer drug may be useful in the treatment of certain tumor types. 2

We have been studying the interaction of [PtCl(dmso)(en)]Cl, K2PtC14 and [PtCl(acv-N7)(en)]C1 (acv is [9-(2- hydroxyethoxymethyl)guanine]) with R-NHCONN-R1 (R = phenyl, pyridine; R1 = phenyl, trifluoromethylphenyl ) and with their reduced form semicarbazides. Reactions were carried out in DMF, water and acetone at different molar ratio and temperatures. Multinuclear NMR spectroscopy (IH,195pI, 2D [IH-13C] HMQC, 2D [tH-13C] HMBC, 1D NOE) was the main method for the determination of the coordination site of the ligands. The chemical shifts of the ~95pt NMR spectra suggested that complexes with PtN4 coordination sphere were formed.

1. l.Akiyama S.I., Chen Z.S., Sumizawa Z.S. and Furukawa T., Anti-Cancer Drug Design, 14, 1143 - 151 (1999), 2. Nielsen D., Maare C. and Skovsgaard T., Gen.Pharmacol., 27,251-255(1996) 3. 2.Osmak M., Bordukalo T., Ambriovid R., Jernej B., Kosmrlj J. and Polanc S., Neoplasma, 47, 390-395 (2000)


Synthetic models of nitrile hydratase

Craig A. Grapperhaus, Apurba K. Patra, Selma Poturovic Department of Chemistry, University of Louisville, Louisville, Kentucky, 40292, USA

Nitrile hydratase (NHase) incorporates either a non-heme iron(III) or non-corrin cobalt(III) for the catalytic hydration of nitriles to amides.~ NHase possesses some intriguing properties from an inorganic standpoint including an unusual role for a common metal, a novel NO-regulation mechanism, and an unprecedented coordination sphere. The metal sits in a N2S3 donor environment comprised of two amido nitrogen donors from the protein backbone and three cysteine sulfurs. X-ray structural characterization of the NO-inactivated form of NHase from Rhodococcus sp. N-771 at 1.7 A resolution indicated two of the cysteines had been post-translationally modified resulting in a unique, mixed thiolato (RS-), sulfenato (RS(O)-), sulfinato (RSO2-) donor set. 2 Sulfur oxygenation was subsequently shown to be required for catalytic activity although the role it serves remains undetermined)

Currently, we are developing synthetic model complexes of nitrile hydratase based on the penta-coordinate, dithiolato ligand 4,7-Bis(2-methyl-2-mercaptopropyl)-l-thia-4,7- diazacyclononane (brnmp-TASN, H2L ~) to unravel the role of the oxygenated cysteine residues. To date a series of iron(III) complexes, L~FeX, have been prepared and thoroughly characterized. [LIFeNO] + displays a single NO band in the infrared at 1856 cm -~ comparable to the NO-inactivated form of NHase; 1852 cm ~. The photostability of [L~FeNO] + will be reported. Reactivity of each LIFeX complex with dioxygen and hydrogen peroxide has also been investigated. Related cobalt derivatives are currently being studied.

f o rX = CI, CN, O-FeL~; n = 0 f o r X = N O ; n = 1

n+

1. Nelson, M. J.; Jin, H.; Turner, I. M., Jr.; Grove, G.; Scarrow, R. C.; Brennan, B. A.; Que, L., Jr. J. Am. Chem. Soc. 1991, 113, 7072-7073. 2. Nagashima, S.; Nakasako, M.; Dohmae, N.; Tsujimura, M.; Tokoi, K.; Odaka, M.; Yohda, M.; Kamiya, N.; Endo, I. Nat. Struct. Biol. 1998, 5, 347-351. 3. Murakami, T.; Nojiri, M.; Nakayama, H.; Odaka, M.; Yohda, M.; Dohmae, N.; Takio, K.; Nagamune, T.; Endo, I. Protein Sci. 2000, 9, 1024-1030.

Mutual trans effect in thiolate-ligated ferryl species

Michael T. Green a, John H. Dawson a, and Harry B. Gray" "Beckman Institute, California Institute of Technology, Pasadena, CA 91125 (e-mai!." mgreen@caltech, edu)

To examine the role of thiolate ligation in oxidative heme chemistry, we applied extended x-ray absorption spectroscopy and density functional theory to intermediates in the catalytic cycles of eytochrome P450, chloroperoxidase, and horseradish peroxidase. Our results indicate a strong trans influence acts between the thiolate and oxo ligands of thiolate-ligated ferryl intermediates. This mutual trans effect, which is not seen in systems with non-thiolate ligands, may be responsible for the unique oxygen transfer chemistry of thiolate-heme proteins.

MTG receives support from the National Institutes of Health and the Burroughs-Wellcome Fund. MTG thanks Michele McGuirl, Graham George, and Matthew Latimer for helpful discussions.

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