Alberto Cassetta

Synthesis and spectroscopic investigations (IR, NMR and Mössbauer) of tin(IV) and organotin(IV) derivatives of bis(pyrazol-1-yl) alkanes: X-ray crystal structures of bis(4-methylpyrazol-1-yl) methane and its dimethyltin(IV) dichloride adduct

A series of 1:1 adducts of the type [(L)RnSnX4−n]·zH2O (L ≡ bis(4-methylpyraazol-1-yl)ethane (L4), bis(3,4,5-timethylpyrazol-1-yl)methane (LT), 1,2-bis(pyrazol-1-yl)ethane (LA) or 1,2-bis(3,5-dimethylpyrazol-1-yl)ethane (LB); R ≡ Me, Et, Bu or Ph; X ≡ I, Br or Cl; n = 0, 1 or 2; z = 1, 1.5 or 2), and the likely polynuclear [(LA)5(SnCl4)4]·(H2O)5 and [(LB)2(SnCl4)3]·12[Et2O] have been characterized in the solid state and in solution by analyses, spectral (IR, Mossbauer, and 1H, 13C and 119Sn NMR) data and conductivity measurements. When LT reacts with SnCl4, cleavage of a carbon (sp3)-nitrogen bond was observed and the adduct [(3,4,5-trimethyl-pyrazole)2SnCl4] was obtained. The diorganotin(IV) complexes generally dissociate in chloroform or in acetone solution, while the trichlorotin(IV) and tetrachlorotin(IV) adducts probably retain the hexacoordinate configuration. The crystal structures of L4 and of the adduct [(L4)(CH3)2SnCl2] have been determined by X-ray analysis. In the adduct the tin atom is coordinated to two halide atoms, two methyl groups and two N atoms, with trans-(CH3)2Sn in a slightly distorted octahedral configuration. The long SnN bond distance (2.436(6)A) indicated weak donor ability of the N2 donor L4. Comparison is made with the X-ray crystal structures of several R2SnX2N2-type compounds.

Dissecting NGF Interactions with TrkA and p75 Receptors by Structural and Functional Studies of an Anti-NGF Neutralizing Antibody

The anti-nerve growth factor (NGF) monoclonal antibody alphaD11 is a potent antagonist that neutralizes the biological functions of its antigen in vivo. NGF antagonism is expected to be a highly effective and safe therapeutic approach in many pain states. A comprehensive functional and structural analysis of alphaD11 monoclonal antibody was carried out, showing its ability to neutralize NGF binding to either tropomyosine receptor kinase A (TrkA) or p75 receptors. The 3-D structure of the alphaD11 Fab fragment was solved at 1.7 A resolution. A computational docking model of the alphaD11 Fab-NGF complex, based on epitope mapping using a pool of 44 NGF mutants and experimentally validated by small-angle X-ray scattering, provided the structural basis for identifying the residues involved in alphaD11 Fab binding. The present study pinpoints loop II of NGF to be an important structural determinant for NGF biological activity mediated by TrkA receptor.

Tin(IV) and organotin(IV) complexes containing mono or bidentate N-donor ligands: III. 1-methylimidazole derivatives: synthesis, spectroscopic and structural characterization

Abstract A series of adducts of the type [(L # ) y R n SnX 4− n ]· z H 2 O (L # =1-methylimidazole, y =1 or 2, R=Me, Et, Bu n or Ph, n =1, 2 or 3, X=Cl, Br or I, z =0, 1/2 or 1) has been characterized in the solid state and in solution by analyses, spectral (IR, 119 Sn Mossbauer, and 1 H, 13 C and 119 Sn NMR) data and conductivity measurements. The molecular weight determinations and the NMR data indicate that these organotin(IV) complexes partly dissociate in chloroform and acetone solution. The donor L # interacts with [(CH 3 ) 3 SnNO 3 ], yielding the 2:1 ionic complex [(L # ) 2 (CH 3 ) 3 Sn]NO 3 . The derivative [(L # ) 2 (CH 3 ) 2 SnCl 2 ] reacts with NaClO 4 , AgNO 3 , NaBPh 4 and KSCN in ethanol and diethyl ether giving the complexes [(L # ) 2 (CH 3 ) 2 Sn(ClO 4 ) 2 ], [(L # )(CH 3 ) 2 Sn(NO 3 ) 2 (H 2 O)], [(L # )(CH 3 ) 2 SnCl(H 2 O) 2 ]BPh 4 and [(L # ) 2 (CH 3 ) 2 Sn(NCS) 2 ], respectively, whereas when [(L # )(CH 3 ) 3 SnCl] interacts with an equimolar quantity of NaClO 4 , [(L # ) 2 (CH 3 ) 3 Sn]ClO 4 ·1/2H 2 O and (CH 3 ) 3 Sn(ClO 4 ) in 1:1 ratio are obtained. The stability towards self-decomposition of the complexes obtained decreases with increasing number of the Sn-bonded aryl or alkyl groups. The derivative [(L # ) 2 (CH 3 ) 2 SnBr 2 ] reacts with 1,10-phenanthroline (Phen), yielding immediately the complex [(Phen)(CH 3 ) 2 SnBr 2 ], whereas from the reaction between [(L # ) 2 (CH 3 ) 2 Sn(ClO 4 ) 2 ] and Phen, the mixed ligand complex [(L # )(Phen)(CH 3 ) 2 Sn](ClO 4 ) 2 is obtained. A different behaviour has been shown from the diiodide complex [(L # ) 2 (C 2 H 5 ) 2 SnI 2 ] which reacts with Phen, yielding the compound with the 3:2 stoichiometry [(Phen) 3 {(C 2 H 5 ) 2 SnI 2 } 2 ]. Both the crystal structures of [(L # ) 2 (CH 3 ) 2 SnBr 2 ] and [(L # ) 2 (C 2 H 5 ) 2 SnI 2 ] show the tin atom in an all- trans octahedral regular configuration, whereas in [(L # )(C 6 H 5 ) 3 SnCl] the tin atom exhibits a distorted trigonal bipyramidal geometry, with the phenyl groups in the equatorial positions. A comparison was made with structural data of other R 3 SnXN-type derivatives. The molecular parameters of 1-methylimidazole in the tin(IV) complexes were used, together with other structural data in literature, to derive empirical rules concerning the imidazole donor.

Triorganotin(IV) derivatives of several 4-acyl-5-pyrazolonato ligands: synthesis, spectroscopic characterization and behavior in solution. Crystal structure of aquotrimethyl(4-p-methoxybenzoyl-1-phenyl-3-methyl-pyrazolon-5-ato)tin(IV).

New triorganotin(IV) derivatives [(Q)SnR3 · x(H2O)] (x = 0, R  Ph; x = 1, R  Me and nBu) (in general QH  1-R′-3-methyl-4-R″ (CC)-pyrazol-5-one; in detail Q′H: R′ = C6H5, R″ = C6H5; QAH:R′ = C6H5, R″ = p-CH3OC6H4; QNH: R′ C6H5, R″  p-NO2C6H4; QBrH:R′ = C6H5, R″ = p -BrC6H4; Q″H: R′ = C6H5,R″ = CH3; QClH: R′ = C6H5, R″ = CCl3; QFH: R′ = C6H5, R″ = CF3; QMH: R′ = CH3 R″ = C6H5; QDH: R′ = CH3, R″ = CH3) have been synthesized and characterized by analysis and spectral (IR and 1H, 13C and 119Sn NMR) data. The (Q)SnPh3 derivatives are five-coordinated in the solid state, with a likely skewed cis-trigonal bipyramidal (cis-TBP) geometry around the tin center and the ligand (Q)− acting in the bidentate form. In [(Q)SnR3 · (H2O)] derivatives (R = nBu or Me) a coordination site is occupied by water, with the ligand (Q)− coordinating in a monodentate fashion. The crystal structure of [(QASnMe3 · (H2O)] has been determined: the tin atom is found in a distorted TBP environment, with the methyl in the equatorial positions. Two of the SnC bond lengths are normal (2.11(1) and 2.08(2) A) whereas the third is longer (2.18(2) A); the ligand binds the metal atom through one carbonyl oxygen in the apical position (SnO = 2.10(1) A). The bond lenght between H2O and Sn is longer (2.41(2) A), and the OSnO angle is 174.9(5)°. H atoms of water are involved in an intermolecular H-bond network with uncoordinated carbonyl and the pyridinic N atom of the ligand. In chloroform solution the[(Q)Snr3 · (H2O)] derivatives (R = Me or nBu) lost the molecule of water and adopt a tetrahedral arrangement. They also give rise to a slow disproportionation, yielding SnR4 and [(Q)2SnR2] derivatives.

Tin(IV) and organotin(IV) complexes containing mono or bidentate N-donor ligands II. 14-Phenylimidazole derivatives. Crystal and molecular structure of [bis(4-phenylimidazole) trimethyltin(IV) ] chloride

Abstract A series of 2:1 adducts of the type [(L′) 2 R n SnX 4− n ]· z H 2 O (L′ = 4-phenylimidazole, R = Me, Et, n Bu, Ph or Cy, X = I, Br or Cl, n = 0, I , 2 or 3, z = 0 or 1) has been characterized in the solid state and in solution by analyses, spectral (IR, far-IR, 1 H and 13 C) data and conductivity measurements. The derivatives [(L′) 2 (Me) 3 Sn]Cl ( 1 ) and [(L′) 2 (Me) 2 SnCl 2 ] ( 3 ) react with NaClO 4 in THF giving the ionic complexes [(L′) 2 (Me) 3 Sn]ClO 4 and (L′) 3 [(Me) 2 Sn(ClO 4 ) 2 ]·(H 2 O) 4 respectively. Whereas the triorganotin(IV) derivatives are completely dissociated in acetone solution, the diorganotin(IV) derivatives dissociate only partly and the tri- and tetrahalidetin(IV) complexes probably retain the hexacoordinate configuration. The crystal structure of [(L′)2(Me) 3 Sn]Cl ( 1 ) has been determined by X-ray analysis. The tin atom is coordinated to three methyl groups and two 4-phenylimidazole donors in a substantially regular trigonal bipyramidal geometry. The ionic chloride group and the two N-H moieties are involved in a hydrogen bond network.

(1-Phenyl-3-methyl-4-acetylpyrazolon-5-ato)rhodium(I) complexes, synthesis, structural and spectroscopical characterization: Reactivity of diolefin- and dicarbonyl-rhodium complexes toward N-, P- and O-donors

Novel complexes of rhodium(I) [Rh(diolefin)(Q″)] (where HQ″=1-phenyl-3-methyl-4-acetylpyrazol-5-one and diolefin=cycloocta-1,5-diene (COD), bicyclo[2.2.1]hepta-2,5-diene (NBD) or 1,5-hexadiene (HEX)) were synthesized and characterized by analytical and spectral data. [Rh(COD)(Q″)] interacts with 4,5-dimethyl-1,10-phenanthroline (Me 2 Phen) and 2,2′-bipyridil (Bipy) yielding the cationic derivatives [Rh(COD)(Me 2 Phen)](Q″)(H 2 O), [Rh(COD)(Bipy)](Q″)(H 2 O) upon displacement of the (Q″) − donor from the coordination sphere of the metal center. Whereas [Rh(COD)(Q″)] interacts with 2-benzoylpyridine (Bzpy) yielding the 1:1 adduct [Rh(COD)(Bzpy)(Q″)] in which Bzpy acts as N-monodentate donor. On the other hand the monodentate P-donors triphenylphosphine, triphenylphosphite, tricyclohexylphosphine and the bidentate bis(diphenylphosphino)ethane (DPPE) displace the COD ligand from [Rh(COD)(Q″)] giving the neutral derivatives [Rh(PR 3 ) 2 (Q″)] (PR 3 =PPh 3 , or P(OPh) 3 ) and [Rh(DPPE)(Q″)](H 2 O). HQ″ reacts with the dinuclear [Rh(CO) 2 Cl] 2 . The tetradentate cycloocto-tetraene (COT) reacts with [Rh(CO) 2 (Q″)] yielding the derivative [Rh(CO) 2 (HQ″)Cl] in which HQ″ acts as neutral monodentate O-donor ligand. Whereas in presence of NEt 3 HQ″ reacts with [Rh(CO) 2 Cl] 2 yielding [Rh(CO) 2 (Q″)]. In this complex, one molecule of CO can be replaced by one mole of Phen and Bipy or by two moles of PPh 3 and AsPh 3 yielding the derivatives [Rh(CO)(L) n (Q″)]·x(H 2 O) (L=Me 2 Phen or Bipy, n =1; L=PPh 3 or AsPh 3 , n =2) whereas one mole of DPPE displaces both the molecules of CO, yielding [Rh(DPPE)(Q″)] yielding the derivative [Rh(COT)(Q″)]. The X-ray crystal structure determination of [Rh(COD)(Q″)] establishes that the rhodium atom is in a square planar configuration with two adjacent sites occupied by the (Q″) − ligand in the O 2 -bidentate form (Rh–O distances=2.054(2) and 2.061(2) A). The COD ring has a twisted boat conformation with Rh–C distances in the range 2.101(3)–2.110(3) A. Comparison was made with structural data reported for several related tetracoordinated (COD)Rh(I) adducts.

Crystallization, X-ray diffraction analysis and phasing of 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus

17beta-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) is an NADP(H)-dependent enzyme that preferentially catalyses the oxidoreduction of oestrogens and androgens. The enzyme belongs to the short-chain dehydrogenase/reductase superfamily and is the only fungal hydroxysteroid dehydrogenase known to date. 17beta-HSDcl has recently been characterized and cloned and has been the subject of several functional studies. Although several hypotheses on the physiological role of 17beta-HSDcl in fungal metabolism have been formulated, its function is still unclear. An X-ray crystallographic study has been undertaken and the optimal conditions for crystallization of 17beta-HSDcl (apo form) were established, resulting in well shaped crystals that diffracted to 1.7 A resolution. The space group was identified as I4(1)22, with unit-cell parameters a = b = 67.14, c = 266.77 A. Phasing was successfully performed by Patterson search techniques. A catalytic inactive mutant Tyr167Phe was also engineered, expressed, purified and crystallized for functional and structural studies.

Purification, crystallization, X-ray diffraction analysis and phasing of a Fab fragment of monoclonal neuroantibody αD11 against nerve growth factor

The rat monoclonal neuroantibody alphaD11 is a potent antagonist that prevents the binding of nerve growth factor (NGF) to its tyrosine kinase A receptor (TrkA) in a variety of systems, most notably in two in vivo systems linked to crucial pathological states, such as Alzheimers disease and HIV infection. To provide further insights into the mechanism of action of this potentially therapeutic monoclonal antibody, structural studies of the antigen-binding fragment (Fab) of alphaD11 were performed. alphaD11 IgG2a immunoglobulin was obtained from hybridomas by in vitro tissue culture. The alphaD11 Fab crystallizes in two crystal forms. Form I belongs to space group P1, with unit-cell parameters a = 42.7, b = 50.6, c = 102.7 A, alpha = 82.0, beta = 89.1, gamma = 86.0 degrees. With two molecules in the asymmetric unit, V(M) is 2.3 A(3) Da(-1) and the solvent content is 46%. A complete data set has been collected at 2.7 A resolution on beamline XRD-1 (ELETTRA, Trieste, Italy). Form II belongs to space group C2, with unit-cell parameters a = 114.8, b = 69.4, c = 64.10 A, beta = 117.0 degrees. With one molecule in the asymmetric unit, V(M) is 2.4 A(3) Da(-1) and the solvent content is 48%. A complete data set has been collected at 1.7 A resolution on beamline ID14-1 (ESRF, Grenoble, France). Phasing was successfully performed by Patterson search techniques and refinement of the structures is currently under way. Crystal forms I and II display a close-packing pattern.

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159–180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1–B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.

The Conundrum of the High-Affinity NGF Binding Site Formation Unveiled?

The homodimer NGF (nerve growth factor) exerts its neuronal activity upon binding to either or both distinct transmembrane receptors TrkA and p75(NTR). Functionally relevant interactions between NGF and these receptors have been proposed, on the basis of binding and signaling experiments. Namely, a ternary TrkA/NGF/p75(NTR) complex is assumed to be crucial for the formation of the so-called high-affinity NGF binding sites. However, the existence, on the cell surface, of direct extracellular interactions is still a matter of controversy. Here, supported by a small-angle x-ray scattering solution study of human NGF, we propose that it is the oligomerization state of the secreted NGF that may drive the formation of the ternary heterocomplex. Our data demonstrate the occurrence in solution of a concentration-dependent distribution of dimers and dimer of dimers. A head-to-head molecular assembly configuration of the NGF dimer of dimers has been validated. Overall, these findings prompted us to suggest a new, to our knowledge, model for the transient ternary heterocomplex, i.e., a TrkA/NGF/p75(NTR) ligand/receptors molecular assembly with a (2:4:2) stoichiometry. This model would neatly solve the problem posed by the unconventional orientation of p75(NTR) with respect to TrkA, as being found in the crystal structures of the TrkA/NGF and p75(NTR)/NGF complexes.