extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Chemistry Biology

text:
Theoretical study on β-cyclodextrin inclusion complexes with propiconazole and protonated propiconazole

The synthesis of the β-cyclodextrin/propiconazole nitrate inclusion complex and the advantages of the encapsulation of this drug were recently reported, but the experimental data only partially revealed the structure of the supramolecular complex due to the limitations in understanding the intermolecular association mechanism. The present work describes the equilibrium molecular geometries of β-cyclodextrin/propiconazole and β-cyclodextrin/protonated propiconazole, established by the AM1 and PM3 semi-empirical methods. The affinity between different parts of the guest molecule and the cyclodextrin cavity was studied considering that propiconazole possesses three residues able to be included into the host cavity through primary or secondary hydroxyl rims. The results have revealed that the most stable complex is formed when the azole residue of the propiconazole enters the cavity of the cyclodextrin through the narrow hydroxyl’s rim.

Introduction
The occurrence of fungal diseases has dramatically increased during the past 20 years. Extremely rare ten years ago, nowadays, antifungal drug resistance has become an important problem in treatment of fungal diseases for various categories of patients, especially those infected with HIV. Excessive and prolonged treatment with azole-containing medicines has led to fungal resistance to this class of compounds, especially in the case of HIV patients with repeated recurrent episodes [1,2]. Today, the number of reported cases of clinic resistance to antifungal drugs is growing and mycologists have warned about an increasingly large-scale expansion of this phenomenon [3]. Consequently, the development of new therapeutic conjugates able to combine new antifungal properties with water solubility of the drug has become a major direction of research in the field of antifungal therapy. In this respect, one of the expected methods consists of the complexation of antifungals with cyclodextrins and/or with soluble polymers.
Propiconazole (PP) is a triazole derivative effective as a fungicide, with a broad spectrum, designed and launched by Janssen Pharmaceutics (Belgium). It is widely used in agriculture as a systemic foliar fungicide and, lately, for its fungistatic action. Propiconazole nitrate was tested in order to reduce the toxicity of the unmodified PP, but little information is available on this topic. Recently synthesized positively charged protonated propiconazole (PPH + ) showed an increased antifungal activity compared to unmodified PP. The inclusion compound based on β-cyclodextrin (β-CD) and PPH + (further abbreviated as β-CD/ PPH + ) was preliminarily investigated in vitro, and its antifungal activity was reported [4].
Cyclodextrins (CDs) are macrocyclic oligosaccharides consisting of six to twelve glucopyranose units joined in a truncated cone-shaped structure [5]. They exhibit a hydrophobic cavity delimited by two rims, a wide and a narrow one, composed of secondary and primary hydroxy groups. By virtue of this structure, CDs are able to generate inclusion complexes with a wide variety of hydrophobic organic compounds in aqueous solution. The driving forces leading to complexation are numerous, varying from van der Waals to hydrophobic and to dipole-dipole interactions [6,7]. Since CDs and their complexes are widely used in pharmaceutical sciences and synthesis, there is currently a great interest in the theoretical study of their supramolecular associates.
Accurately representing the size of CD cavities and their chain flexibility represents a challenge for molecular simulation when quantum methods are employed. Since the ab initio approach is time consuming for this kind of molecule, quantum semi-empirical methods, such as CNDO, AM1 and PM3 were widely used in the theoretical investigation of CDs. The PM3 method has proved to be a powerful tool in the conformational study of supramolecular systems, such as CD inclusion compounds and provides better performance compared to the AM1 method for molecular geometry optimization, due to its improved description of hydrogen bonds and steric effects [8][9][10]. Advanced methods, such as Hartree-Fock (HF) and density functional theory (DFT), were also applied in cyclodextrin chemistry to explain experimental data [11,12]. Very often, ab initio methods are used in tandem with the semi-empirical PM3 method [13][14][15][16][17]. Because of the experimental limitations, the geometric details and the interactions that stabilize the molecular architecture of β-CD/PPH + inclusion compounds are still poorly understood. For this reason the present study theoretically investigates the interaction between PP, PPH + and β-CD molecules by means of AM1 and PM3 semi-empirical quantum-mechanical calculations, to examine in detail the insertion pathways and to determine the intimate configurations of the β-CD/propiconazole (β-CD/PP and β-CD/PPH + ) inclusion complexes. Bearing in mind that the CD cavity cannot fully incorporate the guest molecule, the aim of this work is to identify the part of the molecular cavity that is more suitable for complexation with the PPH + . This information can be useful to predict which of the hydroxy groups of cyclodextrin can be chemically modified (by pegylation for example) in order to avoid the shielding of the cavity during the inclusion process, and, as a consequence, to improve the systemic bioavailability and pharmacokinetics of the inclusion complexes.

Results and Discussions
The most stable conformations of the β-CD/PP and β-CD/PPH + inclusion compounds were selected by considering the binding energy as being the difference between the heat of formation of the complex and the heat of formation of the involved free molecules: (1) where E CD/PP , E PP and E CD represent the heat of formation of the complex, of the free β-CD, and of the free guest molecule, respectively. The higher the negative value of the stabilization energy, the more thermodynamically favorable is the pathway of inclusion-complex formation. The particular shape of the PP molecule allows its inclusion into the β-CD cavity following three different ways. Additionally, each residue of PP can be well accommodated either by the secondary or the primary face of the β-CD cavity ( Figure 1a). Therefore, six configurations must be considered in pursuing the most stable molecular struc-ture of the inclusion complex. For simplicity, let us note each PP residue as it is shown in Figure 1b. The orientation of PP toward β-CD will be named according to the PP residue that is first included through the wider (A) or narrower (B) cavity rim (e.g., aliphatic A, aliphatic B, if the aliphatic residue is included through the wider or narrower hydroxy rim of the cyclodextrin). A suitable methodology for finding the equilibrium molecular geometry of cyclodextrin inclusion complexes is to place the guest on the cavity axis and to move it through the cavity in steps, simultaneously optimizing the conformations. Since there are six possible configurations, it is crucial to find which propiconazole residue has maximum compatibility with the cyclodextrin cavity. To save computational resources, this can be achieved by the methodology described in the computational method section of the paper. The first step in attaining this goal involves the setting of some conformational constraints, followed by the energetic minimization of the resulted conformation. Hence, the optimized molecular geometries will contain the guest molecule inside or outside of the β-CD, depending on the molecular hindrance and on the affinity between the guest residues and CD cavities. Finally, the structures are to be subjected to PM3 calculations, without any constraints, to obtain the heats of formation and to compare the stability of the conformers.
Looking at Figure 2 it is obvious that all structures are stable since, in all cases, negative binding energies were obtained. It can also be observed that the PP deeply entered into the cavity during the calculation, demonstrating a high probability of complex formation. The binding energy is not very high, which could be explained by the absence of hydrogen bonds that strongly stabilize the molecular association. Analyzing the numerical values summarized in Figure 2, one can see that the stability of the complexes is inversely related to their global electric dipoles. There is an obvious correlation between the global electric dipole moments (p) of the complexes and their binding energies, for each paired situation including the PP residues. Such a fact suggests that a dipole-dipole coupling mechanism could be involved in the complex formation. The inclusion of the triazole ring is energetically favored, since the resulting binding energy is the lowest. The obtained theoretical results confirm the experimental data published on complexation of β-CD and PP, which highlights the inclusion of the triazole ring in the cyclodextrin cavity [4]. Hence, the developed interaction model is accurate.

Inclusion compounds with non-protonated propiconazole (β-CD/PP)
In order to determine the exact structure of the obtained complex, further calculations were carried out. Taking into account previous theoretical and experimental results, we assumed that the β-CD/PP complex is formed by triazole ring inclusion into the cyclodextrin cavity. The further study was performed considering two orientations of the triazole ring in relation to the CD cavity: one with the triazole ring pointing toward the negative sense of the z axis, denoted by A (Figure 3a), and the other with the triazole ring pointing toward the positive sense of the cavity axis, denoted by B (Figure 3b). The intermolecular distance was measured between the cavity center and the "dummy" atom of the triazole ring (marked with an asterisk). Initially, the triazole ring was placed in the center of the cavity and a complete rotation was performed to establish the preferred angular orientation of PP during the inclusion process. Keeping constant the resulting angular orientation, PP was then moved along the z axis simultaneously with geometry optimization, in the absence of any symmetry constraints ( Figure 3a and Figure 3b) as discussed above.
Scanning the binding energy during the movement along the z axis, by using the PM3 method, always provided negative values for both A and B configurations (Figure 4a and Figure 4b).
The most stable equilibrium molecular geometry is obtained when PP is deeply included in the β-CD cavity, suggesting an enthalpically driven process (Figure 5a and Figure 5b). The obtained results confirm that PP penetration through the rim of the primary hydroxy is thermodynamically favored, and that the complex is stabilized in the B orientation, which has a stabilization energy about ~20 kJ smaller as compared to the A configuration (Table 1). These facts are in good agreement with those exposed in Figure 2, confirming a higher probability of azole ring inclusion into the β-CD cavity through the narrow rim, according to the B configuration. The results are also consistent with the DFT single-point computations applied on the PM3 equilibrium geometries.
The results are similar to those reported by Fatiha [18], where the imidazole ring of sulconazole enters through the narrow rim into the β-cyclodextrin cavity. The β-CD/sulconazole is stabilized by van der Waals interactions and hydrogen bonds. In fact, the author explained the difference between the A and B orientations in the case of sulconazole by the occurrence of hydrogen bonds. In the present case, no hydrogen bonds were evidenced and, since there are no steric constraints to explain the difference between the A and B orientations in terms of stability, other forces must be considered. It is already known that cyclodextrin molecules have a rather high electric dipole moment; the correlation between this parameter and the complex stability by means of quantum-mechanical calculations has been previously reported [19,20]. The PP molecule has a permanent electric dipole, and the dipole-dipole interaction can     make the difference between the A and B configurations. The decrease of the global dipole moment together with the increase of the binding energy (see Table 1), points to the dipole-dipole interactions as a major contributor to the stabilization of the B structure.
As depicted in Figure 6a and Figure 6b, the molecular geometry of the AM1 optimized inclusion compounds shows that the PP molecule is deeply inserted in the cavity of β-CD for both A and B configurations.
In terms of binding energy, the difference between the A and B orientations was found to be 9.62 kJ. Similarly to the preceding PM3 calculation, the AM1 optimized B configuration of the β-CD/PP complex has a higher stability and no hydrogen bonds where identified. After AM1 optimization too, the same correlation between the stabilization energies and dipole moments of the inclusion compounds was revealed (Table 1).

Inclusion compounds with protonated propiconazole (β-CD/PPH + )
In order to study the complex formation between β-CD and the positively charged guest molecule, a model of PPH + was built. The proton affinity (PA) was calculated for each of the five protonation sites of the azole ring (according to the notation scheme shown in Figure 1b). According to reference [21,22], PA was defined as the energy variation in the proton-addition equilibrium: (2) In this model the heat of formation of the proton was taken to be zero because of the lack of electrons, and the reaction system was considered in vacuo, at 0 K. The method does not provide the exact value of the PA parameter, but the calculation is useful to compare the stability of different protonation forms of the PP molecule. Table 2 shows that the PA values are considerably higher when PP is protonated at the iminic nitrogen atom (in the c position), this position being therefore the best protonation site.   To find the energy-minimized molecular geometry of the β-CD/ PPH + complex, only the inclusion of the protonated azole ring into the β-CD cavity was considered. The procedure was similar to that used in the case of the β-CD/PP inclusion complex, the azole ring being progressively introduced into the β-CD cavity. Also, similar to the β-CD/PP complex, two orientations of the guest molecule relative to the β-CD cavity were taken into account and the same notations are kept.
Scanning the binding energy by using the PM3 method revealed that, for all step intervals along the z axis, the energy of the complex is substantially lower compared with the sum of the energies of the isolated host and guest molecules (Figure 7a and Figure 7b). According to the values of the energy of the two configurations, it results that the B orientation of the PPH + molecule relative to the β-CD cavity provides the most stable inclusion complex, the difference being about 13.8 kJ/mol when compared to the A configuration (Table 3). Results are also consistent with the DFT single point computations applied on the PM3 equilibrium geometries.
As depicted in Figure 8a and Figure 8b, the equilibrium molecular geometries of the A and B PM3 optimized inclusion compounds are quite different. In the A orientation, the stable complex is formed with the protonated azole ring located outside of the β-CD cavity (Figure 8a), while in the B orientation, the azole ring is completely included in the cavity (Figure 8b).
The AM1 method shows the same trend as the PM3 regarding the inclusion pathway of PPH + in the β-CD cavity, where the complex has the highest stability in the B configuration. The binding energy of the B configuration is 43.28 kJ/mol lower than that in the A configuration ( Table 3). The significant difference between stabilization energies leads to a rather big difference between molecular structures. Thus, while in the more stable B configuration the PPH + is included with the azole ring in the β-CD cavity, at the level of the wider rim (Figure 9a), in the A configuration, the PPH + molecule is completely outside of the cavity (Figure 9b). Both AM1 and PM3 calculations indicate a strong correlation between the stabilization energy and the electric dipole moments (Table 3).
Modeling the inclusion pathway of PP and PPH + by means of semi-empirical PM3 and AM1, a strong correlation between the values of binding energy and electric dipole moments is revealed, which indicates a major contribution of dipole-dipole coupling to the molecular stability of the inclusion complexes.
The energy minimization of the β-CD/PP and β-CD/PPH + inclusion compounds with the PM3 and AM1 methods leads to equilibrium geometries with the guest molecules partially inserted in the β-CD cavity for both A and B orientations of the guests. When the protonated form of PP is considered, the process minimization generates very different molecular architectures for the A and B starting configurations. While the complex in the A configuration contains the PPH + molecule completely outside of the β-CD cavity, in the case of the B configuration, PPH + is deeply inserted in the cavity with the proto- nated azole ring entering through the narrow rim. By protonating the PP, a regional selectivity of the inclusion process relative to the β-CD cavity was noticed, with the complexation path through the narrow rim being the favored one.
As Table 4 shows, PP and PPH + experience some conformational transformations during the complexation process. The variation of molecular parameters is not significant, but it seems that the PP molecule changes its shape in order to augment the complex stability. The magnitude of the variation is higher for the β-CD/PP complex than for the β-CD/PPH + one, and is significant at the level of dihedral angles of the molecules. In the case of the β-CD/PP complex, the magnitude of the variation of the molecular parameters is higher for the A orientation, while in the case of β-CD/PPH + the trend is reversed. Since for both PP and PPH + , the most stable complex occurs in the B orientation, and because the magnitude of the conformational change is not correlated with any of the two configurations, we suppose that regional selectivity of cyclodextrin complexation is not governed by steric driving forces or by conformational effort of the PP during the inclusion process.
The effect of solvent on the orientation of a guest molecule toward the β-CD cavity Since all the previously discussed molecular geometries were optimized in vacuo, it is important to know if the orientation of the guest molecules toward the β-CD cavity remains the same in the presence of water molecules. In this respect, the geometries of the complexes were optimized in aqua, by combining techniques of molecular mechanics and quantum mechanics [23].
The results given in Table 5 shows that, as solvent, water does not affect the most stable orientation of PP and PPH + toward the β-CD cavity, and that the binding energy permanently remains negative. Likewise with the case of in vacuo calculations, the B orientations of the guests are favored for both β-CD/PP and β-CD/PPH + inclusion complexes. It is important to note that both in vacuo and in aqua, the guest molecules are deeply inserted in the β-CD cavity, according to the B configurations ( Figure 10), and no hydrogen bonds can be evidenced. The decrease of the global dipole moment by the increase of binding energy, suggests again that dipole-dipole interactions are the major contributor to the stabilization of the B structure.

Conclusion
The molecular geometry of the inclusion complexes of β-CD with PP and PPH + as guests was studied by using the MM+, AM1 and PM3 methods. The results have revealed that the PP and PPH + azole rings were included into the cavity of the β-CD through the narrower hydroxy rim. A strong correlation between the binding energy and the global dipole moments was proved, pointing to the fact that dipole-dipole coupling acts as a major force in stabilizing the complexes.
The presence of water molecules as a solvent does not affect the orientation of PP and PPH + toward the β-CD cavity: both guests penetrating through the narrow rim is always favored. The strong correlation between the binding energy and the global dipole moment is obviously maintained, no matter if the inclusion process is simulated in vacuo or in the presence of water molecules.

Experimental Computational method
The starting molecular conformations of β-CD, PP and PPH + were built by using the graphical tool of the HyperChem 7.52 software application [24]. β-CD was built up starting from α-Dglucopyranose residues (found in HyperChem data base) by interconnection with α-(1,4)-glycosidic oxygen bridges. The resulting molecular geometries where fully optimized by AM1 and PM3 quantum-mechanics semi-empirical methods, under HyperChem software application. DFT single-point calculations were performed using GAUSSIAN 09 software package [25], at the level of B3LYP/6-31G(d)+. The equilibrium molecular geometries were validated by comparing the obtained molecular parameters with those reported in the literature [10].
It is known that the calculation of the entire potential surface of CD inclusion complexes requires large computational resources and is time consuming. Because the three entering pathways of the PP molecule into the molecular cavity of the β-CD take place according to complexation mechanisms, several methods were developed to prove the compatibility of the molecular residue of the guest with the host cavity [26][27][28][29].
A molecular coordinate system was defined having the glycosidic oxygen atoms in the xOy plane, and with the z axis orthogonal to xy plane, so that z axis becomes the cavity axis. The primary hydroxy groups of cyclodextrin are therefore oriented toward the negative sense of z, while the secondary ones are directed toward the positive sense of the axis. In our approximation, the inclusion processes take place with the guest molecule moving along the cavity axis. To determine which PP residue has the highest affinity for the β-CD cavity, the PP molecule was placed on the cavity axis with one of the residues (either aromatic, aliphatic or azole ring) in front of the β-CD cavity. As a first stage, a MM+ calculation was applied to the guest PP molecule, while keeping the β-CD conformation frozen. The result was a deep insertion of the PP into the cavity, along with the advance of the calculation process. In the next stage, the whole system was fully optimized by using the PM3 quantum method, without any conformational constraints.
The molecular equilibrium geometries of the β-CD inclusion compounds with PP and PPH + were calculated by placing the drug molecule on the cavity axis. To find the favorable angular orientation, the guest molecule was placed in the center of the cavity and a complete rotation was performed, optimizing the molecular geometry by PM3 methods at equal intervals of 20°, in the absence of any conformational constraints. While keeping the favorable angular orientation constant, the guest was moved along the cavity axis through the β-CD cavity, and complete geometry-optimization calculations were performed for equal intervals of 1 Å, by the PM3 method.
To compare the results delivered by the two semi-empirical methods, the molecules with initial coordinates corresponding to their PM3 minimum energy were subjected to AM1 calculations and also to B3LYP/6-31G(d)+ single-point investigations.
The most stable inclusion complexes found by PM3 computations were placed in a cubic box with 36 × 36 × 36 Å, containing 1541 water molecules. The systems were firstly equilibrated by optimizing water molecules with the MM+ method, keeping the inclusion complexes frozen. Then a complete optimization of the whole system was performed for each system, without any constraints [23]. The resulting molecular geometries were finally subjected to singlepoint calculation by the PM3 method, in order to extract the heat of formation, after the removal of water molecules. A full optimization of the equilibrium geometries after removing water molecules would have led to a complete loss of information concerning the solvent effect.


extfieldsofstudy: Chemistry Medicine

text:
Elucidating the structural and conformational factors responsible for the activity and substrate specificity of alkanesulfonate monooxygenase

The mechanism and substrate specificity of alkanesulfonate monooxygenase (SsuD) was investigated by combining molecular dynamics simulations, docking, and a comprehensive quantitative structure activity relationships (QSAR) analysis. The FMNH2 dependent monooxygenase undergoes a dynamic conformational change of the active site, passing from a closed to an open state. As a consequence, substrates have access to the active site and the cofactor is then regenerated by the associated oxidoreductase FMN reductase SsuE.. Computational analysis of the interaction of SsuD with FMNH2 based on molecular docking and multiple 20 ns molecular dynamics simulations pointed out that the conformational change is mainly driven by salt bridge formation between Arg297 and Glu20 or Asp111. A set of substrates accepted by SsuD were described by means of ALMOND chemical descriptors and a partial least square (PLS) mathematical model was constructed. The PLS model correlates the structure of substrates and enzyme activity, namely kinetic properties (k cat/K M). Therefore, information coming from the PLS analysis goes beyond the simple ability of the enzyme to recognize the substrate, but includes the factors that affect the capacity of the enzyme to reduce the activation energy of the rate determining step of the reaction. The two principal components of the model are able to describe both steric and electronic factors and, more importantly, their interactions. Indeed, interactions of factors appear to affect significantly the ability of SsuD of transforming efficiently a substrate.

Introduction
The work reported here aims at elucidating the structural and conformational factors responsible for the activity and substrate specificity of alkansulfonate monooxygenase (SsuD). In bacteria, sulfur is mainly assimilated from inorganic sulfate via the cysteine biosynthetic pathway. In nature, where the levels of inorganic sulfate may be low, bacteria have to rely on organosulfur compounds such as sulfate esters, sulfamates, and sulfonates as sulfur sources. Under conditions of sulfate starvation, Escherichia coli synthesizes two systems, which cover the full range of uptake and desulfonation activities. These activities enable the growth of the microorganism with taurine (TauABCD proteins) and with a variety of aliphatic sulfonates (SsuEADCB proteins) as sole sources of sulfur (van der Ploeg, Eichhorn, & Leisinger, 2001). Sulfite liberated inside the cells from taurine and alkanesulfonates serves as sulfur for cell growth.
The structure of the substrate-free SsuD from E. coli has been solved previously (Eichhorn, Davey, Sargent, Leisinger, & Richmond, 2002), but for a detailed mechanistic study the structure of an enzyme-substrate complex is crucial. SsuD enzymes are of great relevance in diverse bacteria for organosulfur assimilation. This is demonstrated by the numerous protein sequences having high identity with E. coli SsuD and that can be derived also from open reading frames deposited in databases. Therefore, understanding substrate recognition and cofactor interaction is of high interest in order to clarify the distinctive role of this widespread enzyme in C-S bond cleavage.
In the attempt of elucidating the catalytic mechanism, previous studies of SsuD and SsuE from E. coli addressed the transfer of the reduced flavin from the SsuE reductase to the oxygenase SsuD. Spectroscopic experiments pointed out interactions between SsuE and SsuD, suggesting a channeling mechanism for the transfer of the reduced flavin (Abdurachim & Ellis, 2006). Moreover, the alteration of the cofactor affinity of the SsuE in the presence of SsuD was also reported (Eichhorn et al., 1999). Zhan and co-workers proposed the formation of a C(4a)-hydroperoxyflavin intermediate once FMNH 2 reacts with O 2 (Zhan, Carpenter, & Ellis, 2008). The complex would be directly involved in the desulfonation of alkanesulfonate, which leads to the release of sulfite and the aldehyde. The same intermediate was also observed in the mechanism of some flavindependent hydroxylases (Ballou, Entsch, & Cole, 2005).
Further contributions to the understanding of SsuD catalytic mechanism came from the comparison of a series of enzymes belonging to the same family of SsuD, namely the class C flavoprotein monooxygenases (van Berkel, Kamerbeek, & Fraaije, 2006). Flavoproteins of this class include enzymes involved in diverse biochemical reactions, like the biosynthesis of the antibiotics pristinamycin (Thibaut et al., 1995) and actinorhodin (Valton, Mathevon, Fontecave, Nivière, & Ballou, 2008), the degradation of natural aromatic compounds (Chaiyen, Suadee, & Wilairat, 2001), desulfurization of dibenzothiophene (Oldfield, Pogrebinsky, Simmonds, Olson, & Kulpa, 1997), nitrilotriacetate hydroxylation, (Uetz, Schneider, Snozzi, & Egli, 1992) and ethylenediaminetetraacetic acid (EDTA) degradation (Witschel, Nagel, & Egli, 1997). Among them, luciferase was studied extensively, both at biochemical and structural levels (Fisher, Thompson, Thoden, Baldwin, & Rayment, 1996) and such information was exploited for the interpretation of the structure of SsuD (Eichhorn et al., 2002). This enzyme presents a structural core with a TIM-barrel fold. The location of the active site was proposed at the end of the β-barrel (Eichhorn et al., 2002). The TIM-barrel fold is adopted by many enzymes for flavin binding and it has been found in the structure of all luciferases solved so far (Farber & Petsko, 1990). As a matter of fact, luciferase LuxAB, (Fisher et al., 1996) long-chain alkane monooxygenase LadA (Li et al., 2008), F 420 -dependent N 5 ,N 10 methylenetetrahydromethanopterin reductase Mer (Shima et al., 2000), and F 420 -dependent secondary alcohol dehydrogenase Adf from methanogenic Archaea (Aufhammer et al., 2004) use reduced flavin or the flavin analog F 420 as cofactors. All these enzymes show high structural similarity (Li et al., 2008). In SsuD, luciferase, and LadA there are highly conserved amino acid residues. SsuD presents Cys54, His228, Arg297, and Tyr331, which correspond to Cys106, His44, Arg291, and Tyr110 in LadA and Cys14, His311, and Tyr63 in luciferase. The importance of these conserved residues for the mechanism of action was confirmed by mutagenesis studies: mutation of His44 (Fisher et al., 1996) in luciferase causes the loss of activity, whereas LadA loses its activity upon single mutations either of His311, Tyr63, or Cys14 (Li et al., 2008).
It must be underlined that SsuD enzymes are synthesized under sulfate-starvation conditions and they show a very low content in sulfur-containing amino acids. The observation that Cys54 of E. coli SsuD is remarkably conserved, suggests a relevant role of this amino acid in the catalytic mechanism. Recent mutagenesis studies indicate that Cys54 of E. coli SsuD might contribute to the stabilization of the C(4a)-hydroperoxyflavin intermediate formed during catalysis (Carpenter, Zhan, & Ellis, 2010). This is in agreement with the observation that the thiol of Cys106 of luciferase points towards position C (4a) of the isoalloxazine ring of the cofactor in the crystal structure of the FMN bound enzyme (Campbell, Weichsel, Montfort, & Baldwin, 2009). It was also demonstrated that Hg complexation of Cys54 in SsuD leads to an inactive enzyme (Eichhorn et al., 2002).
A study concerning substrate specificity of SsuD reported that the pure enzyme catalyzed the conversion of pentanesulfonic acid to sulfite and pentaldehyde and was able to desulfonate a wide range of sulfonated substrates including C-2-C-10 unsubstituted linear alkanesulfonates, substituted ethanesulfonic acids, and sulfonated buffers (Eichhorn et al., 1999).
Since crystals of a SsuD-alkanesulfonate complex have not yet been obtained (Eichhorn et al., 2002), in the present study the mechanism of SsuD was investigated by computational methods. The analysis included the interactions of the enzyme with the substrates and with the reduced cofactor. The aim was to verify whether the preliminary binding of FMNH 2 is required for the subsequent recognition of the alkanesulfonate by a catalytically active conformation. Molecular docking and molecular dynamics were used for the calculation of enzyme-alkanesulfonate complexes, which were subsequently used for calculating structure activity relationships (QSAR) (Braiuca, Boscarol, Ebert, Linda, & Gardossi, 2006;Braiuca, Ebert, Basso, Linda, & Gardossi, 2009;Braiuca, Knapic, Ferrario, Ebert, & Gardossi, 2009). The statistical analysis allowed pointing out the structural basis of the substrate specificity of the enzyme.

Materials and methods
The protein structures used for this study were retrieved from the Protein Data Bank (PDB) (Berman et al., 2000) (Id: 1m41 Eichhorn et al., 2002 for SsuD, 1luc (Gao & Ellis, 2005) for bacterial luciferase, 3b9o (Li et al., 2008) for LadA, and 1z69 (Aufhammer et al., 2005) for Mer). These initial structures were pretreated in Molecular Operating Environment (MOE) by removing the crystallographic water molecules present in the PDB file. Hydrogen atoms were added and their positions were optimized with an energy minimization procedure in the Amber99 force field in its MOE implementation. Subsequently, a minimization of the side chains was performed keeping the backbone atoms fixed.
Superimpositions of protein structures were performed using the MOE Superpose tool fixing the spatial position of the most preserved residues (His, Trp, Arg) in all the superimposed structures. The alignment was performed using the blosum30 matrix (Henikoff & Henikoff, 1992).

GRID
The GRID program (Goodford, 1985) is a computational procedure that calculates the interaction energies between the substrate and a small chemical probe (e.g. a functional group). Energies are calculated in all the nodes of a three-dimensional grid, which spans the structure of the substrates considered. The output is called Molecular Interaction Field (MIF), which can be visualized as an isopotential surface. The GRID analysis was performed on the SsuD protein, choosing a cage big enough to include the whole protein. The grid nodes were set every 0.5 Å. The probes used for the calculation of the MIFs were DRY (hydrophobic probe), WATER (H 2 O probe), and OS (sulfonic acid mimic probe).

Docking
The Docking procedure was performed using the docking module of MOE (2006).
In the case of FMNH 2 , an area of 1 nm radius was selected surrounding the active site, taking as references the coordinates of active His, Trp, and Arg residues. The force field used for the docking was MMFF94x, the partial charges of the atoms were calculated at the PM3 semi-empirical level, by means of the MOPAC7 program and the different poses were placed with the alpha PMI method and scored by means of the London dG scoring function (MOE, 2006). The final FMNH 2 pose was finally located by evaluation of the scoring function (London dG) and the pose similarity with the FMN cocrystallized in the PDB structure of LadA (PDB 3b9o).
The docking simulations of the substrates were performed in the same way considering a 1.2 nm radius selected area surrounding the FMN; the initial positions of the substrates were manually set in order to meet the three different criteria: (i) sulfonic group located in a OS probe interacting area; (ii) sulfonic group distance from FMNH 2 ; and (iii) London dG scoring function. The substrate molecules were inserted by building each molecule with the MOE builder tool and subsequently minimized. For each substrate, the conformation presenting the highest score and fulfilling the structural requirements for the initiation of the enzymatic catalysis was chosen.

Molecular dynamics
The molecular dynamics simulations were performed using the software GROMACS 4 (Berendsen, van der Spoel, & van Drunen, 1995) with the GROMOS-96 53a6 force field (Oostenbrink, Villa, Mark, & van Gunsteren, 2004). The molecular dynamics simulations were performed using the software GROMACS 4 with the GROMOS-96 53a6 force field. The SsuD PDB crystal structure (PDB code 1m41) was implemented in the force field in gro file format by using the tool pdb2gmx of the GROMACS software package, which also adds the necessary hydrogens. The protein dimer was solvated with explicit SPC water in a virtual box of 1331 nm 3 . All the dynamics were performed in a NPT environment simulating the temperature of 300 K and keeping the pressure constant (Berendsen-thermostat and pressure (Berendsen et al., 1995)), cut-off for electrostatic interaction was set to 1.4 nm and the limit for the van der Waals interactions set to 1.4 nm. For the minimization procedures, the Particle Mesh Ewald (PME) algorithm (Essmann et al., 1995) was used for the calculation of the electrostatic interactions setting the limit at 1.0 nm. The FMNH 2 and the substrate molecules were parameterized in the GROMOS-96 53a6 force field by using the Dundee PRODRG2 server (Shuettelkopf & van Aalten, 2004) and manually refined in order to meet the correct force field definition. The FMNH 2 and the substrate molecules were manually added in the gro file taking the coordinate from the docking results. The system was previously minimized with 10,000 step of steep descendent algorithm before every molecular dynamics calculation.
Molecular dynamics analysis were performed with GROMACS tools; distances measurements were calculated using g_mindist and computing the minimum distance between two residues; the results were visualized using Grace software (http://plasma-gate.weizmann.ac.il/ Grace).
Each substrate conformation chosen for the construction of the data-set for the QSAR analysis (Cruciani, Clementi, & Baroni, 1993) was the one characterized by the lowest potential energy out of all the frames saved in the dynamics trajectories.

ALMOND
Four different probes were used for the calculation of the MIFs, namely the WATER, the DRY, the Ca++, and the TIP probes. The WATER probe describes and quantifies the dipolar interactions and the hydrogen bond formation; the DRY probe considers all the hydrophobic interactions; the Ca++ probe takes into account the interactions with charged groups (e.g. sulfonic group); and the TIP probe generates a MIF that is strictly dependent on the shape of the molecule (ALMOND v. 3.3.0).
The GRid INdependent Descriptors (GRIND) descriptors (ALMOND v. 3.3.0) are vectors connecting those points of the MIFs representing the most favorable interactions and they are generated under the condition of having the highest energy products.
The GRIND vectors can connect points belonging to the same MIF or to different MIFs and the ensemble of the vectors generates auto-correlograms and cross-correlograms, respectively.
The four probes used for the GRIND analysis generated four auto-correlograms and six cross-correlograms, corresponding to 600 independent variables (vectors). The GRIND vectors were further selected on the basis of their ability to discriminate between objects by using the tools of the ALMOND software, and consequently by discarding those vectors containing insufficient information in terms of ability to discriminate among substrates.
This operation resulted in the deletion of all the vectors of the Ca++/Ca++ auto-correlogram, because the Ca ++ probe generates MIFs only in correspondence of the sulfonic group which is present in every object of the data-set. A further variable selection was accomplished by using the FFD variable selection algorithm (ALMOND v. 3.3.0), which allowed retaining only 217 vectors, corresponding to the most informative variables.
It must be underlined that before this procedure the predictivity of the model was <0.10.
Partial least square (PLS) models with 5 principal components were computed and validated by leave-oneout (LOO) method (ALMOND v. 3.3.0). QSAR substrate predictivity model is expressed in terms of experimental vs. predicted (on two principal components) k cat /K M .

Hardware and software
The MD simulations have been performed on a dual Opteron workstation, on a 16 CPUs cluster and on the IBM Blade Center HS21 Linux Cluster provided by the High Performance Computing Center Stuttgart (HLRS), utilizing 256 cores per simulation.
The QSAR was calculated on a dual Xeon machine. The following software has been used: MOE 2008.10 distributed by Chemcomp, Montreal, Canada; GRO-MACS version 4 (Gromacs 4.0.6 in single precision for the HPCC simulations); GRID version 22c; and ALMOND version 3.3.0.

Analyzes of interactions between SsuD and cofactor
The crystal structure of SsuD was taken from the PDB (Berman et al., 2000). The structure is available since July 2002 under the entry code 1m41 and it has resolution of 2.3 Å. SsuD appears as a homodimer (subunits A and B), where each subunit consists in an eight-stranded α/β-barrel motif. This fold classifies SsuD as a member of the big (β/α) 8 TIM-barrel family (Eichhorn et al., 2002). A GRID (Goodford, 1985) analysis of the enzyme surface indicated a prevalence of hydrophilic regions, which is in agreement with the cytosolic origin of the protein (see supplementary material Figure S1). Several hydrophobic areas are spread on the surface. The most relevant one is located at the entrance of the active site and it might facilitate the approach of the substrate to the catalytic site, while reducing the entropic penalty caused by the expulsion of water generated during catalysis SsuD is a FMNH 2 -dependent enzyme, but in the 1m41 (Eichhorn et al., 2002) PDB structure the reduced cofactor is not co-crystallized. Although the overall structure of the two subunits is similar, there are major conformational changes due to interactions of Arg297 with either Asp111 or Glu20. The switching of these interactions corresponds to a conformational transition from the closed to the open conformation and suggests a dynamic mechanism mediated by Arg297 (see supplementary material Figures S2 and S3). As a consequence, subunit A is in a closed conformation where the active site is not accessible by the solvent, while subunit B is in an open conformation.
Because the factors responsible for the conformational transition cannot be directly elucidated from the analysis of the cofactor-free apoprotein structures, a more detailed study of enzyme-cofactor interactions was performed. To model the structure of the SsuD-FMNH 2 complex, SsuD was compared to the structures of two highly homologous FMNH 2 -dependent enzymes belonging to the (β/α) 8 TIM-barrel family: bacterial luciferase co-crystallized with FMN (PDB 1luc) (Gao & Ellis, 2005) and long-chain alkane monooxygenase (LadA) cocrystallized with FMN (PDB 3b9o) (Li et al., 2008). Conversely, similarities of sequence and tertiary structure were analyzed. The three enzymes share a homologous organization of the active site and are expected to have similar catalytic mechanisms (Fisher et al., 1996;Li et al., 2008). In particular, the superimposition of structures 1luc, 3b9o, and 1m41 showed three highly conserved residues: His111, Trp196, and Arg127 for SsuD; His44, Trp194, and Arg107 for bacterial lucifer-ase; and His311, Trp348, and Arg157 for LadA. The spatial position of the Arg residue is the most conserved (see supplementary material Figure S4) and previous studies on bacterial luciferase suggest a relevant role of Arg107 in coordinating and stabilizing the phosphate group of the cofactor (Campbell et al., 2009).
It is interesting to note that the analysis of the methylenetetrahydromethanopterin reductase (Mer) co-crystallized with its cofactor F 420 (PDB 1z69) (Aufhammer et al., 2005) revealed the absence of the corresponding Arg residue, even though Mer belongs to the same (β/α) 8 TIM-barrel family. As a matter of fact, the bulkier F 420 cofactor can fit into the cofactor binding domain of Mer thanks to the bending of a loop that increases the space available (see supplementary material Figure S5).
The conserved Trp residue is located at the center of the active sites of all four enzymes considered and it creates a hydrophobic pocket to accommodate the cofactors (see supplementary material Figure S6).
In bacterial luciferase, it is evident that the indolic ring of Trp194 stabilizes the cofactor by interacting with its isoalloxazine ring (Fisher et al., 1996). Moreover, it is also known that the hydrophobicity of the cofactor accommodation cavity, mainly due to the Trp residue, is necessary for the creation of a water-free environment that prevents rapid breakdown of unstable flavin intermediate (Li et al., 2008).
The position of the conserved His differs in the three superimposed enzymes, which can be ascribed to their different substrate specificities. In bacterial luciferase and LadA, the conserved His is positioned near the cofactor and mutations of this residue causes the loss of activity (Fisher et al., 1996;Li et al., 2008).
The study of SsuD interactions with the cofactor was performed by docking the cofactor into the active sites of subunits A and B (Lengauer & Rarey, 1996), followed by MD simulations (Alder & Wainwright, 1959)   Finally, the best pose was selected by comparing three-dimensional coordinates of LadA co-crystallized with FMN with each of the 20 poses of the FMNH 2 docked into subunit A.

MD simulations of the interactions between cofactor and active site
The interactions between cofactor and active site were investigated by molecular dynamics simulations to analyze the role of the cofactor in stabilizing the two conformations of SsuD. Two initial structures for the MD simulations were created: an enzyme-cofactor complex where the cofactor was placed in the subunit A (closed conformation) and a free enzyme based on the structure of subunit B (open conformation).
The force field GROMOS-96 53a6 (Oostenbrink et al., 2004) was assigned, the protein was solvated with explicit water, minimized using the PME (Essmann et al., 1995) algorithm and a 20 ns molecular dynamics simulation was performed using GROMACS software (Berendsen et al., 1995). The trajectory of Arg297 and its interactions, either with Asp111 or Glu20, underlines the dynamic behavior of the enzyme active site. From the comparison of the two subunits, it appears that this movement is related to the presence of FMNH 2 . In subunit A (closed conformation with docked cofactor), the distance between Arg297 and Asp111 reaches the equilibrium at the distance of 0.22 nm in approximately 10 ns. Afterwards, the subunit maintains that conformation for the rest of the simulation. The salt bridge between Arg297 and Asp111 in subunit A prevents the expulsion of the cofactor by moving a protein loop (from Thr108 to Glu114) and thus locking the cofactor inside the cavity (see supplementary material Figure S7). On the contrary, the distance between Arg297 and Glu20 fluctuates during the entire period of the simulation, changing between 0.2 and 1.0 nm (Figure 1), and indicating an absence of a salt bridge between these two amino acids.
In contrast, the dynamics of the cofactor-free subunit B (open conformation) shows a stable distance of 0.2 nm between Arg297 and Glu20 after the first 200 ps, which  is compatible with a salt bridge (Figure 2). Conversely, the distance between Arg297 and Asp111 fluctuates between 0.5 and 0.8 nm throughout the entire simulation, without reaching a constant value.
These observations suggest that the presence of the cofactor induces the stabilization of the subunit towards the closed conformation, whereas the cofactor-free subunit assumes a stable open conformation.
To confirm the two simulations of subunits A and B, 10 additional simulations of 20 ns were performed for each subunit (full details are reported in the supplementary material Table SI). In all simulations of subunit A (with the docked cofactor and in closed conformation) the distance between Arg297 and Asp111 was compatible with salt bridge formation, while the distance between Arg297 and Glu20 increased sporadically. This observation suggests that the opening mechanism can still occur although the closed state is favored. On the contrary, the distance between Arg297 and Asp111 remained below 1 nm. The 10 simulations of the cofactor-free subunit B (open conformation) showed a different behavior. The two reference distances changed in such a way that both the closed and open conformations occur, but there was no favored conformation. Figure 3 reports four typical conformations obtained in the 10 MD simulations. In one simulation (indicated as MD 1 in Figure 3 and Table SI) the distance between Arg297 and Glu20 is about 0.3 nm, while the distance between Arg297 and Asp111 is above 1.0 nm. This is in accordance with the previous simulation, where the absence of cofactor stabilizes the open conformation and a salt bridge between Arg297 and Glu20 was formed. In a second simulation (indicated as MD 2 in Figure 3 and Table SI) the distance between Arg297 and Asp111 is about 0.3 nm, while between Arg297 and Glu20 it is above 1.0 nm, indicating that the active site is in the closed conformation and a salt bridge between Arg297 and Asp111 is formed. In a third simulation (indicated as MD 3 in Figure 3 and Table SI) both distances are about 0.3 nm showing that the subunit is in an intermediate state generated by a contraction of the crevice due to the absence of the bulky cofactor. A fourth simulation (indicated as MD 4 in Figure 3 and Table SI)  The global evaluation of all dynamic simulations suggests that the cofactor has a relevant effect on the stabilization of the closed active site state, although the opening mechanism can still happen. This can be related to the release of the oxidized cofactor, which needs to be regenerated after catalysis. When the active site is not binding the cofactor, there is no predominant stabilized conformation and the enzyme switches between open and closed states, ready to accept the cofactor. Such kind of a breathing motion is very common in complex proteins, (Chakravarty & Varadarajan, 1999;Merlino et al., 2005). Figure 4 shows three different conformations of subunit B observed during the 20 ns simulations. The salt bridge between Arg297 and Glu20 induces the open conformation (A), whereas the progressive approaching of Arg297 and Asp111 (C) translates into the closed conformation (B).
These simulations point out that Arg297, Asp111, and Glu20 are important residues for the enzymatic activity because they are involved in the stabilization of the active site in the open and the closed conformations through formation of salt bridges. Furthermore, their movements have an essential role for cofactor binding and release. In conclusion, the presence of FMNH 2 cofactor stabilizes the closed state, while the empty active site showed to be in a dynamic equilibrium between the two states.
It must be underlined that during all the simulations the cofactor was never expelled from the active site, confirming the reliability of the structural model constructed by superimposing the SsuD and the LadA cofactor complexes. Therefore, the same model was used for the investigation of enzyme-substrate interactions ( Figure 5).

Understanding and modeling enzyme-substrate interactions
SsuD catalyzes the oxidation of a broad range of structurally different aliphatic sulfonates having as general structure R-CH 2 -SO 3 H (Eichhorn et al., 1999). In order to study the structural basis of enzyme-substrate recognition, docking and MD simulations were performed on selected substrates reported in the literature (Eichhorn et al., 1999). The substrates and their experimentally measured k cat /K M are reported in Table I. The data-set meets the necessary requirements in terms of structural diversity, k cat /K M range and homogeneity of distribution, which are crucial for the generation of a consistent QSAR model (ALMOND v. 3.3.0). Data were then used for calculating a preliminary 3D-QSAR model (Braiuca et al., 2006;Braiuca, Knapic, et al., 2009b), which was exploited for extracting information on the structural features affecting enzyme specificity. The analysis was performed by means of the PLS method, which is based on principal components analysis.
Enzyme-substrate interactions were investigated by docking the substrates into the active site of the subunit A complexed with FMNH 2 , which was obtained after 20 ns equilibration as described above ( Figure 5). The generated poses were selected on the basis of the London dG scoring function (MOE, 2006).
Further on, the GRID sulfone/sulfoxide probe (OS probe) was applied to map the affinity of the active site for the sulfonate group ( Figure 6) and to select only those poses, where the sulfonic group binds in the areas delineated by the OS probe MIFs ( Figure 6). However, since the distance between sulfonic group and the FMNH 2 must be compatible with the catalysis, His11 appears to be the only basic residue able to interact with the sulfonic group while meeting all the other scoring criteria (see supplementary material Figure S8).
Following this scheme, one single pose for each substrate was chosen. The systems comprising the subunit A-FMNH 2 complex and the docked substrates were solvated with explicit water and energy minimized using the PME (Essmann et al., 1995) algorithm implemented in GROMACS (Berendsen et al., 1995) and the GRO-MOS-96 53a6 force field (Oostenbrink et al., 2004). Subsequently, short 300 ps molecular dynamics simulations were carried out for each enzyme-cofactor-substrate complex. Each MD simulation was analyzed and the substrate conformers with the lowest potential energy were selected and subsequently used for extracting information on the structural features that determine the substrate specificity of SsuD. For this purpose, the structures of the substrates were correlated with the respective activity by means of PLS. The mathematical model was calculated on the basis of molecular descriptors obtained by a GRID derived method named GRIND (GRID Independent Descriptors) (Pastor, Cruciani, Mc Lay, Pickett, & Clementi, 2000), implemented in the software ALMOND (ALMOND v. 3.3.0). This software was selected because it allows the use of a wide array of chemical probes, including the Ca++ one. Unlike the classical 3D-QSAR approaches (e.g. CoMFA) (Todeschini & Consonni, 2009), the GRIND method presents the major advantage of being independent from any alignment of molecules/substrates, which is usually a primary source of error in this kind of studies (Puzyn, Leszczynski, & Cronin, 2010). The GRIND method transforms the information included in the MIFs into alignment independent descriptors (vectors) able to describe the relative spatial position of chemical groups throughout the molecules. A PLS model was built up by correlating the GRIND descriptors with the experimental k cat /K M values of the training set. The mathematical model explains 62% of the whole variance and has a predictive coefficient q 2 (calculated by means of the leave-one-out cross-validation procedure) of 0.86 on the second principal component.
Since the QSAR model here described is based on alignment free descriptors (GRIND), any direct connection between the structural/chemical features of molecules and their absolute position in the Cartesian space is lost. Nevertheless, the comparison of the different molecules of the data-set is feasible thanks to analysis of the PLS scores plot and the extraction of relevant information is Table 1. Structures of substrates accepted by the SsuD and corresponding kinetic data taken from Eichhorn et al. (1999).

Compound
General formula: R-CH 2 -SO 3 H, with R-Experimental k cat /K M (min À1 μM À1 ) 1 6.7  still possible by analyzing the loadings plots, which provide information on how the variables contribute to each principal component (Figures 7 and 8).
Variables with larger loading values better describe the activity of interest. It is important to underline that a positive loading indicates that the variable is directly correlated with k cat /K M . Conversely, variables negatively correlated with the activity are characterized by negative loadings.
The loadings plot for the first two principal components clearly shows that the first principal component discriminates the substrates mainly on the basis of their shape (TIP/TIP) (Figure 7). Indeed, the variance explained by the first PC is only the 24% of the whole variance and a PLS model based only on the first PC has a q 2 as low as 0.48.
The second principal component explains 38% of the whole variance and the loading plot (lower plot of Figure 7) indicates that all different types of descriptors contribute to this second PC and thus to substrate description and discrimination. The overall analysis of the PLS loadings plot clearly demonstrates how a satisfactory description of the substrates can be achieved only by accounting two principal components and conversely the largest part of all the relevant steric, electronic, and hydrophobic contributions and especially their interactions.
The analysis of the PLS score plot illustrates how each component is able to describe the different molecules ( Figure 8).
The first principal component ( Figure 8) is able to describe how enzyme specificity depends on the shape and length of substrate structure (TIP-TIP descriptors, see supplementary material Figure S9). When the only variability between molecules is the aliphatic chain length (as for molecules 1, 2, 7, and 9) the first principal component well indicates that longer aliphatic molecules are better substrates. On the other hand, substrates 8 and 10 are grouped because of their cyclic and polar structure (described by WATER/TIP variables, Figure 7 upper plot) and substrates 4, 5, and 6 are considered similar because of their globular shape (Figure 8). It is interesting to note that the second principal component includes the contributions of different variables describing not only the shape, but rather electronic properties and especially their interactions (confirmed by the high loading of all the correlograms in Figure 7). This latter point makes the interpretation more complex but it is evident that, differently from the first component, the aromatic substrates 3, 4, 5, and 6 are well described and discriminated by the second component on the basis of their ability of establishing H-bonds. As a consequence, the highly hydrophobic substrate 6 results to be the poorest one. On the other side, aliphatic substrates 1, 2, 9, and 7 are considered as a homogenous set of molecules since they do not differ in terms of electronic properties. Therefore, globally the two components are able to describe both steric and electronic factors and, more importantly, those interactions that affect the ability of SsuD of transforming efficiently a substrate. It must be underlined that molecular descriptors were correlated to kinetic properties (k cat /K M ) and consequently such information goes beyond the simple ability of the enzyme of recognizing the substrate, but includes the factors that affect the ability of the enzyme to stabilize the relevant transition state of the reaction.

Conclusions
Molecular simulation methods have been applied to the study of SsuD action at molecular level, shedding light to enzyme-cofactor and enzyme-substrate interaction mechanisms. By combining docking and molecular dynamics, a comprehensive illustration of the structural features of SsuD was accomplished. The enzyme showed a two-state dynamic mechanism, switching between an open and a closed conformations of its active sites. Arg297, Asp111, and Glu20 govern the dynamical switch between the inactive and active conformations. The presence of FMNH 2 cofactor stabilizes the closed state, while the empty active site showed to be in a dynamic equilibrium between the two states. This mechanism has never been observed before and its function seems to regulate substrate access and cofactor regeneration. This represents a further example of a "breathing motion mechanism," observable in many complex proteins.
The role of residues directly involved in the catalytic mechanism has been confirmed and clarified. His11 and Trp196 are crucial for stabilizing the sulfonic group of the substrates and the cofactor, respectively. The conformational analysis of a set of substrates performed by MD simulations was used for constructing a PLS model correlating substrate structures and enzyme activity. The model provided general guidelines for describing the physical-chemical features of substrates for being accepted by this enzyme. The output of the statistical analysis of the PLS model allows to withdraw information in a more rational and systematic procedure and to focus the attention also to the interactions of variables.
The analysis of the model suggests that the interaction between the substrate and the FMNH 2 molecule is crucial for the catalytic activity, whereas the nature of the substrate can influence the strength of this interaction and the correct substrate placement. So far, the lack of knowledge of SsuD mechanism and specificity has restricted the application of this enzyme. The substrates listed in Table 1 represent the only available data-set in the literature that meets the requirement necessary for the PLS construction. A more detailed elucidation of the mechanism of SsuD and the rational description of structural requirement of SsuD substrates provide new basis for a more extended exploitation of the catalytic potential of this class of enzymes, both for synthetic and environmental applications (Buhler, Witholt, Hauer, & Schmid, 2002). In this context, information here reported can be used as a reference also for substrate and enzyme engineering.


text:
Synthesis of Oligosilanyl Compounds of Group 4 Metallocenes with the Oxidation State +3

Recently, we showed that titanocene silyls are much more stable with Ti in the oxidation state +3. The current study demonstrates that analogous Zr and Hf compounds can also be obtained by reaction of a suitable metalate precursor with an oligosilanyl dianion. As the obtained complexes formally possess a d1 electron configuration, they were investigated using EPR spectroscopy. The corresponding spectra indicate that the compounds can be considered to also exhibit some cyclosilanyl radical anion character. In order to understand the strong preference of disilylated titan(IV)ocenes for reductive elimination, a theoretical study of the thermodynamics of these reactions was conducted, revealing that this behavior is essentially caused by the weak Si–Ti(IV) bond.

■ INTRODUCTION
Over the last few decades alkyl-substituted group 4 metallocenes have become a very well established class of compounds, for which numerous applications have been found. 1 Analogous compounds with higher group 14 substituents have received much less attention. However, silylated metallocenes are another interesting class of compounds which can serve as catalysts or catalyst precursors for a number of important chemical transformations. Prominent among these reactions is the dehydrogenative coupling polymerization of hydrosilanes, 2−5 an important synthetic alternative to the Wurtz type coupling of chlorosilanes, which still is the most widely used method for the formation of Si−Si bonds in general and the synthesis of polysilanes 6 in particular. The reaction was discovered by Harrod and co-workers as a process where PhSiH 3 reacts to give polyphenylsilane, H(PhSiH) n H, in the presence of Cp 2 TiMe 2 . 7 Harrod's initial mechanistic explanation involved the formation of the dinuclear Ti(III) complex [Cp 2 TiSiH 2 Ph] 2 as a source of the silylene PhSiH, which upon release could oligomerize to longer chains. 8,9 Subsequently, Tilley and coworkers showed that the polymerization of phenylsilanes can also be catalyzed by zirconocenes and hafnocenes of the type CpCp′M(X)H (Cp = C 5 H 5 , Cp′ = C 5 H 5 , C 5 Me 5 ; M = Zr, Hf; X = Cl, Me) starting with catalyst precursors such as CpCp′M-(X)Si(SiMe 3 ) 3 . For these systems, featuring the metal in the oxidation state +4, it was conclusively demonstrated that the catalytic cycle consists of two σ-bond metathesis steps. 2,10 While Harrod later expressed his belief that also in the titanium-catalyzed reaction "the key catalyst species is Cp 2 Ti-(H)SiH 2 R", 11 it is interesting to note that still no examples of stoichiometric σ-bond metathesis reactions between silicon hydrides and bis(silyl)titanocenes or silylhydridotitanocenes are known. Even the syntheses of the disilylated titanocenes or CpCp′Ti(Cl)Si(SiMe 3 ) 3 have never been reported.
A survey of known silylated titanocenes reveals that while there are a fair number of Ti(III) complexes 8,12−16 and even some Ti II −SiH σ-complexes, 17−19 less than a handful of examples of silylated titanocenes with Ti(IV) have been reported. Among these compounds Cp 2 Ti(Cl)SiMe 3 (I), 20 prepared by Rosch et al. utilizing the reaction of Cp 2 TiCl 2 with Al(SiMe 3 ) 3 ·Et 2 O, is well characterized with 1 H and 13 C NMR and IR spectra and a low-quality crystal structure (R = 16%). The titanacyclosilanes Cp 2 Ti(SiPh 2 ) n (n = 4 (II), 5 (III)) 21−23 were reported as products of the reaction of Cp 2 TiCl 2 with Li(Ph 2 Si) n Li. While for Cp 2 Ti(SiPh 2 ) 4 21,22 1 H and 13 C NMR spectra in acetone were reported, the formation of Cp 2 Ti-(SiPh 2 ) 5 23,24 was substantiated by multinuclear (including 29 Si) NMR 24 and a crystal structure analysis. 23 The reported synthesis of the silylene-bridged complex [Cp 2 Ti(SiH 2 )] 2 obtained by Weiss from the reaction of Cp 2 TiCl 2 with H 3 SiK 25 was later questioned by Harrod and co-workers, who repeated the published procedure and obtained only [Cp 2 Ti-(SiH 3 )] 2 . 26 One further example of a well-characterized disilylated titanocene was reported by our group. The reaction of Cp 2 TiCl 2 with (Me 3 Si) 2 KSiSiK(SiMe 3 ) 2 was found to give a titanacyclotrisilane (IV). 27 This compound, however, can be interpreted either as containing Ti(IV) with silyl groups or as Ti(II) with a disilene ligand.
For analogous germylated titanocenes with Ti(IV) the situation is quite similar. Cp 2 Ti(Cl)GeMe 3 (V) was prepared by Rosch in a manner analogous to that described for Cp 2 Ti(Cl)SiMe 3 utilizing Al(GeMe 3 ) 3 ·Et 2 O. 20 The triphenylgermylated compound Cp 2 Ti(Me)GePh 3 (VI) 28 was obtained by Harrod and co-workers from the reaction of Cp 2 TiMe 2 with Ph 3 GeH. Related studies from Razuvaev's group showed that Cp 2 Ti(GePh 3 ) 2 (VII) and Cp 2 Ti(Cl)GePh 3 are relatively stable at room temperature, while Cp 2 Ti(GeEt 3 ) 2 decomposes rapidly at room temperature to Et 6 Ge 2 . 29,30 They also reported reactions of digermylcadmium or digermylmercury with Cp 2 TiCl 2 and Cp 2 TiCl to give small amounts of germylated titanocenes. 31 Our own attempts to react Cp 2 TiCl 2 with (Me 3 Si) 3 SiM (M = K, Li, MgBr, MgSi(SiMe 3 ) 3 ) met with no success until recently. 32 While observation of the reaction of Cp 2 TiCl 2 with (Me 3 Si) 3 SiK·(18-crown-6) by means of NMR spectroscopy did not indicate formation of any type of metalated tris(trimethylsilyl)silyl compound, it was possible to isolate another product: Cp 2 TiCl 2 ·K (18-crown-6). The presence of this paramagnetic compound indicated the formation of paramagnetic, NMR-silent Ti(III) species in the reaction. 33 Continued attempts finally allowed the isolation of Cp 2 Ti(Cl)-Si(SiMe 3 ) 3 ·K(18-crown-6), which was formed in a reaction that was not very clean. Changing from (Me 3 Si) 3 SiK to the dimetalated compound K(Me 3 Si) 2 Si(Me 2 Si) 2 Si(SiMe 3 ) 2 K (1) 34 led to a much cleaner course of reaction (Scheme 1). It was found that half of 1 was converted cleanly to 1,2bis(trimethylsilyl)tetramethylcyclotetrasilane, which was presumably formed by reductive elimination of an initially formed titanacyclosilane. The concurrently formed titanocene "Cp 2 Ti" likely underwent a comproportionation with Cp 2 TiCl 2 to Cp 2 TiCl, which in the presence of KCl·(18-crown-6) reacted further to Cp 2 TiCl 2 ·K(18-crown-6) (3). 32 The latter could then react with the remaining 1 to give the final product, a titanacyclosilane (2) with titanium in the oxidation state +3. 32 The current study deals with further investigations concerning this reaction and, in particular, with the question whether this chemistry is also possible for the heavier metals of group 4.

■ RESULTS AND DISCUSSION
Synthesis. Dichlorinated Metalates. Knowing that Ti(III) was involved in the reaction of Cp 2 TiCl 2 with silyl anions, we set out to use another titanocene precursor to avoid wasting the metalated oligosilanes as reducing agents. While several methods for the synthesis of Cp 2 TiCl and related complexes with reducing metals (such as Zn, Al, and Mn) are known, 35,36 we thought to use comproportionation chemistry to obtain the Cp 2 Ti III species in a way similar to that outlined above. Thus, the well-known "Cp 2 Ti" 37 source Cp 2 Ti(btmsa) 38 was reacted with Cp 2 TiCl 2 (Scheme 2). This reaction turned out to be a valuable source of "salt free" (Cp 2 TiCl) 2 .  (18-crown-6) or with LiCl in the presence of tmeda to obtain the respective base adducts of Cp 2 TiCl with KCl (3) or LiCl (3a) (Scheme 2). This way the required precursors could be obtained without wasting oligosilane precursors, in a satisfying quantitative yield over two steps.
Reaction of 1 with Cp 2 ZrCl 2 and Cp 2 HfCl 2 was reported to lead to the formation of the respective metallacyclosilanes with the metals in the oxidation state +4, without any sign of reductive elimination. 34,39 Therefore, attempts directed to the preparation of zirconium and hafnium analogues of 2 required metal salts of zirconocene and hafnocene in the oxidation state +3. The synthesis of the respective K[Cp 2 ZrCl 2 ]·(18-crown-6) (4) and K[Cp 2 HfCl 2 ]·(18-crown-6) (5) was thus accomplished in an even more straightforward fashion by reduction of the respective metallocene dichlorides with potassium in the presence of 18-crown-6 (eq 1).
Disilylated Metalates. As anticipated, reactions of titanates 3 and 3a with the dianion 1 led to the formation of the titanacyclopentasilane 2 in a cleaner way. Using 3a, reactions with the respective 1,5-dipotassiopentasilane 6 and 1,3dipotassiotrisilane 8 were also carried out. From the reaction of 3a with 6 the expected anionic titanacyclohexasilane 7 could be obtained. The same anion could already be isolated from the reaction of 6 with Cp 2 TiCl 2 . 32 However, reaction of 6 with Cp 2 TiCl 2 gave the anionic part of 7 along with the complex counterion [(K·(18-crown-6)) 2 Cp] + , indicating a reaction course more complex than was anticipated, involving abstraction of a cyclopentadienide. The use of 3a instead of Cp 2 TiCl 2 provides a way to 7 with the simpler counterion [(K·(18-crown-6))·2THF] + ( Figure S1, Supporting Information).
Disilanide 6 can be regarded as an elongated version of 1, and in the same way 8 might be seen as the analogously shortened congener of 1. As expected, the reaction of 8 with 3a gave a complex anion comprised of a titanacyclotetrasilane ring (9) (Scheme 3). In this case, however, single-crystal structure analysis showed that the counterion consists again of the complex [(K·(18-crown-6)) 2 Cp]. + While the formation of 2, 9, and 7 utilizing 3a proceeded in an expected way, 32 it was also interesting to test whether the same reaction could also be accomplished for the analogous zirconium and hafnium cases. Therefore, the reactions of 1 with 4 and 5, respectively, were carried out and found to proceed much in the same way as was observed for the titanium case (Scheme 4). This was striking, as the obtained products 10 and 11 contained the same metallacyclopentasilane units with a d 1 electron configuration that were previously obtained as d 0 fragments. 34,39 As it was possible to analyze the solid-state structures of all four compounds, a structural comparison allows some interesting conclusions about the relationship between geometry and electronic state (vide infra).
The subsequently arising question of whether the d 1 complex could be obtained also by direct reduction of the neutral metallacyclosilane was addressed by the reaction of the respective zirconacyclopentasilane 12 with 1 equiv of potassium in the presence of crown ether. It was found that this reaction yielded the d 1 complex directly (Scheme 4). However, while The successful preparation of the d 1 complexes of zirconaand hafnacyclopentasilanes 10 and 11 and the strange behavior of the titanocenes forced us to revisit some chemistry that we found rather puzzling some years ago. While we found clean conversion of 1,4-dipotassiotetrasilane 1 with Cp 2 ZrCl 2 and Cp 2 HfCl 2 , 34,39 attempts to achieve the same conversion with the respective 1,4-dipotassiocyclohexasilane 13 40 with Cp 2 ZrCl 2 and Cp 2 HfCl 2 did not give any indication of the formation of the expected bicyclic metal-bridged compounds. Considering the course of the reaction of 1 with Cp 2 TiCl 2 32 and the fact that our judgment of the reactions was based primarily on NMR spectroscopic analysis, it seemed reasonable to reinvestigate the reaction of 13 with Cp 2 HfCl 2 . Again, NMR spectroscopic observation gave no indication for the formation of the respective disilylated metallocene but provided evidence for the decomposition of this compound by reductive elimination, which gives the respective coupled cyclosilane that is already known. 40 However, crystallographic analysis revealed that the disilylated metallocene 14 was formed with the metal being in the oxidation state +3 (Scheme 5). A similar course of reaction had already been found for the titanocene case. 32 A simple explanation as to why it is possible to prepare some disilylated zirconocenes and hafnocenes with the metal in the oxidation state +4, while others that are seemingly very similar cannot be formed, is difficult to give. The question certainly seems to be related to the equally puzzling behavior of silylated titanocenes. It seems reasonable to assume that in the reaction of two silyl anions with Cp 2 MCl 2 the expected disilylated metallocenes form initially but then undergo reductive elimination as the first step of the sequence, concluding with the formation of disilylated metallocenes in the oxidation state +3. This leads directly to the question of why certain reductive elimination processes are more facile than others.
Computational Study. To understand the outlined unexpected reductive elimination behavior of disilylated titanocenes, we set out to conduct a computational study of these compounds and their zirconocene and hafnocene counterparts at the density functional MPW1PW91/SDD (Ti, Zr, Hf) and 6-311G(d,p) (H, C, Si) level of theory. 41,42 In order to obtain some initial insights, the thermodynamics involved in the reaction depicted in eq 2 were studied. In this reaction the facility of reductive cyclosilane elimination from cyclic disilylated metallocene(IV) compounds is investigated as a function of metal and different ring size. The results of the calculations given in Table 1 correlate qualitatively with the observed experimental situation. While the reductive elimination of a disilene from the formal metallacyclotrisilane is endothermic (only slightly though for the Ti case) for all three metals, the elimination is strongly favored for all other ring sizes of titanacyclosilanes and it is more favorable for large ring sizes. This calculated trend for the ease of the reductive elimination process from titanacyclosilanes follows the experimental observation. That is, we were able to synthesize and isolate a titanacyclotrisilane 27 but no larger titanacycles. For the zirconaand hafnacyclosilanes the same trend is found: the thermodynamic stability decreases with increasing ring size of the metallasilacycles. However, the situation is more delicate, as in all investigated cases the reductive elimination is predicted to be endothermic. In the experiment, however, reductive elimination is not an isolated process but is followed by subsequent reactions; therefore, the trend visible through the data summarized in Table 1 provides a rationalization for the experimental facts that the respective metallacyclotetrasilanes and -pentasilanes are stable while the metallocyclohexasilanes undergo reductive elimination. While the study of the reaction of eq 2 reflects the real systems qualitatively well, it does not provide an intuitive explanation. For this reason it was decided to vary the parameters of the system in order to obtain further insight.
The first variation applied was the replacement of the trimethylsilyl groups at the positions β to the metals by methyl groups (eq 3). The calculated free enthalpies ΔG 298 according to these reactions are given in Table 2. In principle the picture is the same as was found for eq 2, but all results are shifted to more endothermic values. For this system also the three-and four-membered titanacyclosilanes are predicted to be stable and reductive elimination becomes only a feasible process for fivemembered and larger rings. For the heavier metals zirconium and hafnium six-membered rings should be stable. Although no calculations were carried out for phenyl substituents at the silicon atoms in α positions, the stability of this substitution pattern might be even higher, given the fact that a titanaoctaphenylcyclopentasilane 21,22 and even a titanadecaphenylcyclohexasilane are known. 23,24 The trend outlined here also provides an explanation for the fact that Cp 2 Ti(Cl)SiMe 3 was found to be a stable compound while the synthesis of the analogous tris(trimethylsilyl)silyltitanocene chloride failed. 32 In order to eliminate the potential influence of ring strain of the formed cyclosilane the reductive elimination of two silyl substituents to give a disilane was studied computationally for comparison (eq 4). The steric requirements and the silylation degree of the silyl substituents were varied from trimethylsilyl to tris(trimethylsilyl)silyl, and the results of the calculations are shown in Table 3. All disilylated titanocenes(IV) were found to be prone to facile reductive elimination.  42 Less clear is the trend revealed by the calculation upon α-silylation. For titanium a higher silylation degree was found again to correspond with a more favored elimination. For zirconium and hafnium, however, the stability of the metal−silicon bond does not decrease monotonically with increasing silylation degree, which seems to suggest that a higher silylation degree stabilizes the bond but becomes detrimental once steric interactions between the silyl groups or between the silyl and Cp ligands arise. For the disilylated metallocenes (M = Zr, Hf) this situation occurs for (trisilyl)silyl substituents.
As the results of this reaction (eq 4, Table 3) are clearly different from those of the metallacycle reductive eliminations (eqs 2 and 3, Tables 1 and 2), a profound influence of ring strain can be assumed. However, as in the actual reaction both the starting material and the product are cyclic in nature, the thermodynamics of the isodesmic reaction 43 depicted in eq 5 were studied in order to assess the influence of ring strain of the metallacycle ( Table 4). The results indicate that only the threemembered zircona-and hafnacycles are somewhat strained and,  even more important, that there is no evident correlation among ring strain, metal, and ring size for cycles with more than three constituent atoms. The fact that the results given in Table 3 substantially differ from those in Table 1 indicates that ring strain is effectively involved as a factor for the reductive elimination of metallacyclosilanes. However, when it is not the strain that is effective in the metallacycles (as can be concluded from the results given in Table 4), the strain of the cyclosilane products needs to be taken into consideration. This was attempted by studying the isodesmic reaction 43 outlined in eq 6, and the results are given in Table 5.
While the calculations might not reproduce the actual system accurately because the additional steric interaction involved in the formation of the 1,1,2,2-tetrakis(trimethyl)disilanylene unit is not accounted for, it gives a good idea that fiveand sixmembered rings essentially experience no or only little ring strain. 44 This tells us that for the cases of the three-, four-, and five-membered metallacycles a ring strain component works against a facile reductive elimination to give cyclic products. The behavior of the respective zirconium and hafnium compounds is consistent with this.
EPR Spectroscopy. While the d 1 configuration of the studied complexes does not permit NMR spectroscopic investigations, it allows the use of EPR spectroscopy. Especially for the study of titanocene and to some extent zirconocene d 1 complexes this is a very established technique. In fact, there exists even a prior EPR study of radical anions of group 4 metallacyclhexasilanes, namely titana-and zirconadecaphenylcyclohexasilane, 24 which addressed the question of electron delocalization within the cyclosilane framework.
In the course of this study we investigated compounds 2, 4, 10, and 11. The dichlorinated metalate 4 exhibited a signal at g = 1.970 with the expected hyperfine coupling to 91 Zr (a(I = 5 / 2 ) ≈ 27 G), which is in excellent agreement with the values reported by Samuel et al. for [Cp 2 ZrCl 2 ] −. (g = 1.970, a(I = 5 / 2 ) ≈ 29.7 G). 45 In contrast to this, the EPR spectrum of 10 shows a slightly more complex signal with coupling also to the α-silicon atoms (Figure 1). The smaller value of the hyperfine splitting (hfs) constant to 91 Zr, a(I = 5 / 2 ) = 19.4 G, indicates a delocalization of the electron between the metal and the attached silicon atoms. A hfs of 10.2 G to 29 Si can be compared to the value of 7.1 G measured for the coupling to trimethylsilyl groups in (Me 3 Si) 3 Si • . 46 Due to the presence of 47 Ti (I = 5 / 2 ) and 49 Ti (I = 7 / 2 ), which both display a hfs of 8.8 G, the spectrum of 2 ( Figure 2) is more complicated; still, the hfs to the α-29 Si atoms is 12.6 G, of the same magnitude as observed for 10. In addition, the simulation of the spectrum provides a hfs owing to another spin 1 / 2 nucleus with the double natural abundance of silicon with a magnitude of 4.8 G. For reasons of symmetry as well of geometry, we assign this coupling to the SiMe 2 groups in β positions, which lie in the plane of the fivemembered ring. In contrast to the well-resolved spectra of 2 and 10, the spectrum of 11 displayed an ESR signal at a g value of 1.986 with very broad lines, likely indicating unresolved hyperfine coupling to 177 Hf and 179 Hf as well as to 29 Si.
Structural Analysis. Compounds 3a, 4, 7, 9, 10, 11, 11a, and 14 were subjected to single-crystal structure analysis ( Table  6). The quality of the structures of compounds 7, 9, and 11a was not sufficient to allow a detailed structural discussion but nevertheless unambiguously proved the bond connectivity of    Dichlorotitanate 3a crystallizes in the tetragonal space group P4 3 2 1 2. Its structure (Figure 3) is similar to that already reported for 3. 32 The titanium, chlorine, and lithium atoms form a planar four-membered ring with a tmeda molecule coordinating to lithium, thus extending its coordination number to 4. The length of the two equivalent Ti−Cl bonds is, at 2.54 Å, even slightly longer than that for 3 32 but comparable to that for [Cp 2 TiCl] 2 (2.54−2.56 Å). 47 This is, however, considerably longer than what was found for Cp 2 TiCl 2 (2.36 Å). 48 Also the decrease in bond angle from 94°for Cp 2 TiCl 2 48 to 80°for [Cp 2 TiCl] 2 47 is reflected in 3a (86°). The situation for compound 4, which crystallizes in the monoclinic space group P2 1 /c with some disorder in the crown ether affecting atoms O1 and C11 (Figure 4), is similar to that for 3. 32 In contrast to the lithium titanate 3a, the potassium metalates 3 and 4 do not feature a planar arrangement of the four-membered ring K−Cl−M−Cl but show puckerings of 37 and 39°, respectively. Crystal structures of binuclear Cp 2 Zr III Cl species are quite rare. 49−52 All known compounds of the type display Zr−Cl distances of 2.55−2.60 Å, which is consistent with the 2.58 Å found for 4. This is again substantially longer than the Zr−Cl distance of 2.44 Å found for Cp 2 ZrCl 2 . 53 The structure analysis of compounds 10 and 11 is interesting, as in these cases a comparison of the same structural units with the metal in the oxidation state +4 is possible. Structural differences reflect therefore most likely the different oxidation states.
The zircona(III)cyclopentasilane 10 ( Figure 5) crystallizes in the monoclinic space group P2 1 /c, whereas the related hafna(III)cyclopentasilanes 11 and 11a crystallize in the triclinic space group P1̅ . The asymmetric unit of 10 contains the halves of a potassium crown ether unit coordinated by two THF molecules.  The crystals for the structures of 11 ( Figure 6) and 11a ( Figure S3, Supporting Information) were grown in the same solution and were separated under the microscope. For 11 the potassium crown ether unit is coordinating to a SiMe 3 group, whereas in 11a two potassium crown ether units are coordinating to a cyclopentadienide, thus forming the complex cation we found also frequently in oligosilylated titanocenes in the oxidation state +3. 32 For 11a there are two additional toluene molecules in the asymmetric unit. The comparably small Si−Hf−Si bond angle of 89°for 11 is again diminished compared to a value of 96.4°for the compound with Hf(IV). 39 The hafnate 11 features Hf−Si bond distances of 2.85/2.86 Å, which are again elongated in comparison to the neutral Hf(IV) case (2.79/2.83 Å). 39 For 11 and 11a relatively high residual electron density was located close to the Hf atoms.
Compound 14 (Figure 7) crystallizes in the monoclinic space group P2 1 /c with one toluene in the asymmetric unit. The structure of the anionic 7-metalla[2.2.1]bicycloheptasilane was already found for the analogous titanocene. 32 However, while the titanocene featured the complex cation [(K·(18-crown-6)) 2 Cp] + , we found for 14 a plain K·(18-crown-6) moiety which coordinates to a Cp of the metallocene. In comparison to 11 the Hf−Si bonds of 14 are much shorter (2.785/2.790 Å), even shorter than those found for the neutral hafnacyclopentasilane. 39 As a consequence of the bicyclic nature of 14 the Si− Hf−Si angle is only 76°.
For compound 14 parts of the geometries of the anionic moieties and some solvent molecules are strongly disordered. Attempts to treat these disorders did not result in diminished residual electron density. As the concerned parts of the structures are not involved in the discussion of structural parameters, it was decided to refrain from the introduction of restraints which would only result in brightened pictures without real improvement of the model.

■ CONCLUSION
The current study attempts to generalize the chemistry of silylated titanocenes. It was shown that chloride adducts of Cp 2 TiCl can simply be obtained by comproportionation of Rosenthal's Cp 2 Ti(btmsa) with Cp 2 TiCl 2 in the presence of either LiCl or KCl and an additional coordinating base (tmeda or 18-crown-6). The lithium or potassium salts of [Cp 2 TiCl 2 ] − thus obtained were used for further reactions with oligosilanyl dianions. Analogous dichlorinated metalates of zirconium and hafnium could be prepared by reduction of the respective metallocene dichloride with potassium in the presence of 18crown-6. Also, these metalates could be reacted with oligosilanyl dianions to give metallacyclosilanes with Zr and Hf in a d 1 electron configuration.
In order to gain some insight into the question as to why cyclic dioligosilylated titanocenes seem to be highly unstable and why analogous zircono-and hafnocenocenes are stable only with certain ring sizes, a theoretical study was conducted. The results show that the strong preference for the reductive elimination of disilylated titanocenes is mainly caused by the rather weak Si−Ti bond. The thermodynamic driving force of the reductive elimination of the titanacyclopentasilane in comparison to the analogous Zr and Hf compounds is substantial (171 kJ mol −1 (vs Zr) and 199 kJ mol −1 (vs Hf)). The reductive elimination to form cyclosilanes is also feasible for zircona-and hafnacyclosilanes. However, for these cases the energetic situation is more delicate, as the ring strain in the formed cyclosilanes constitutes a decisive contribution as to whether the reaction is thermodynamically favorable or not.
EPR spectroscopy of a set of d 1 metallacyclopentasilanes was particularly interesting, because these compounds can be regarded either as group 4 d 1 metal complexes or alternatively also as cyclosilanyl radical anions. While for the d 1 metal the unpaired electron would be considered to be largely localized at the metal, such electrons are considered to be delocalized along the ring atoms in cyclosilanyl radical anions. A comparison of the EPR spectra of the Zr(III) compounds 4 and 10 shows that, while no hfs to the attached chlorine atoms in 4 was observed, coupling to the attached silicon atoms indicates delocalization.
■ EXPERIMENTAL SECTION General Remarks. All reactions involving air-sensitive compounds were carried out under an atmosphere of dry nitrogen using either Schlenk techniques or a glovebox. All solvents were dried using a column solvent purification system. 54 Chemicals from different suppliers were used as received without further purification. Elementary analyses were carried out using a Heraeus VARIO ELEMENTAR instrument. EPR spectra were recorded on a Bruker ELEXSYS spectrometer at X-band. Simulation of EPR spectra was done with WinSim2002. 55 X-ray Structure Determination. For X-ray structure analyses the crystals were mounted onto the tip of glass fibers, and data collection was performed with a Bruker AXS SMART APEX CCD diffractometer using graphite-monochromated Mo Kα radiation (0.710 73 Å). The data were reduced to F o 2 and corrected for absorption effects with SAINT 56 and SADABS, 57,58 respectively. The structures were solved by direct methods and refined by full-matrix least-squares methods (SHELXL97). 59 If not noted otherwise, all non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogen atoms were located at calculated positions to correspond to standard bond lengths and angles. All diagrams were drawn with 30% probability thermal ellipsoids, and all hydrogen atoms were omitted for clarity. Crystallographic data (excluding structure factors) for the structures of compounds 3a, 4, 7, 9, 10, 11, 11a, and 14 reported in this paper have been deposited with the Cambridge Crystallographic Data Center as supplementary publications CCDC 825205 (3a), 767184 (4), 854110 (7), 825206 (9), 767185 (10), 767187 (11), 767189 (11a), and 705715 (14). Copies of the data can be obtained free of charge at http://www.ccdc.cam.ac.uk/products/csd/request/.
The respective alkali-metal chloride adducts could be obtained by adding equimolar amounts of LiCl/tmeda or KCl/18-crown-6 directly to the THF solutions of [Cp 2 TiCl] 2 and then following the same workup procedure. Single crystals of blue Cp 2 TiCl·LiCl(tmeda) (3a) were obtained by slow diffusion of pentane into a saturated THF solution. The corresponding Cp 2 TiCl·KCl(18-crown-6) was crystallized as green crystals from toluene. Anal. Calcd for C 16  General Procedure for the Reduction of Group 4 Metallocene Dichlorides with Potassium/18-crown-6. To a mixture of the respective metallocene dichloride (0.49 mmol), 18-crown-6 (130 mg, 1 equiv), and potassium (19 mg, 1 equiv) was added toluene (5 mL). After 1 h the color started to change and after stirring for 24 h a deeply colored solution was obtained. The reaction mixture was treated with pentane (10 mL), whereupon the product was isolated by filtration.