FeII/III and MnII complexes based on 2,4,6-tris(2-pyridyl)-triazine: synthesis, structures, magnetic and biological properties

Reaction of the polypyridyl ligand 2,4,6-tris(2-pyridyl)-s-triazine (tpt) with mono- and polynuclear Fe(iii) or Mn(ii) precursors, specifically tri- or hexanuclear Fe(iii) pivalates (piv−), [Mn6O2(piv)10(Hpiv)4] and Mn(ii) isobutyrate (ib−), in various solvents and under various reaction conditions showcase the ligand’s surprising coordination characteristics. The reactions result in mononuclear [FeIII(tpt)(tptH)][FeIIICl4]4·2(thf)·0.23(H2O) (1), [FeIII(piv)(tpt)Cl2] (2), [FeII(tpt)Cl2]·2(H2O) (3a), dinuclear [FeIII2O(tpt)2Cl4] (3), and heptanuclear [FeIII7O4(piv)12(tpt-O)]·A [A = MeCN (4a) or 4(dioxane) (4b)] and [FeIII7O4(piv)11(tpt-O)(i-PrO)(i-PrOH)]·0.75(i-PrOH) (5), as well as the mononuclear compounds [MnII(tpt)(NO3)(H2O)2](NO3) (6) and [MnII(tpt)(ib)(Cl)(MeOH)]·MeOH (7). Single-crystal X-ray diffraction analyses identify tpt as a tridentate NNN donor ligand that forms two five-membered metallocycles in 1–3, 6 and 7, whereas in 4 and 5 five tpt N atoms form coordinative bonds accompanied by an unusual metal-induced oxidation of one of the carbon atoms of the central triazine core. Magnetic properties of Fe(iii)–tpt (2–5), Fe(ii)–tpt (3a), and Mn(ii)–tpt (7) compounds show dominant antiferromagnetic coupling for polynuclear coordination cluster compounds. The Mn(ii)–tpt complexes 6 and 7 exhibit efficient catalytic properties in the production of enzymes by microorganisms, which concerns the synthesis of exocellular proteases in Fusarium gibbosum CNMN FD 12 or Trichoderma koningii Oudemans CNMN FD 15 fungi strains. Thus, compounds 6 and 7 can be used for producing proteolytic enzymes with wide applications including in the food, detergents and pharmaceutical industries.


Introduction
Coordination compounds of polypyridyl derivatives have attracted great interest and enormous efforts since the discovery that such clusters can be used as potential model systems for studying and identifying fundamental biochemical processes 1 as well as individual objects for realizing nano-devices in molecular electronics, spintronics, and sensor technologies. [2][3][4] Among polypyridyl ligands, 2,4,6-tris(2-pyridyl)-s-triazine (tpt) has been widely utilized for the spectrophotometric determination of V(IV) and V(V), 5 Fe(II), 6 Ru(III), 7 Co(II), 8 Al(III) 9 and Ag(I) 10 ions or as a solvent extraction reagent. 11 Moreover, tpt shows various pharmaceutical properties such as antiviral, antitumor, analgesic, antioxidant, antidepressant, antibacterial, and antimicrobial activities. 12 This widely used ligand has a rich coordination chemistry and formed a variety of mono-, di-, tri-, tetra-, penta-, hexa-, and decanuclear metal complexes and coordination polymers. The reader is referred to one of the excellent reviews by B. Therrien 13 on the coordination properties of tpt. In coordination compounds this versatile ligand may adopt different coordination modes (Scheme 1 and Table S1 †). A detailed analysis of coordination modes of tpt in previously published examples of its metal complexes retrieved from the Cambridge Structural Database (CSD 2020.2) 14 reveals ten different coordination types: tridentate terpyridine-like (A), bidentate bipyridine-like (B), mixed tridentate terpyridine-like and bidentate bipyridine-like (C), mixed terpyridine-like and monodentate pyridine-like (D), bis-bipyridine-like (E and F), mixed bidentate bipyridine-like and monodentate pyridine-like (G), tris-bipyridine-like (H and K), and mixed bis-bipyridine-like and monodentate pyridine-like (J). Out of the 324 hits for tpt metal complexes in the latest version of the CSD database, 279 belong to the terpyridine-like coordination mode (A). Other dominant modes of the coordination of tpt are bidentate bipyridine-like (B, 14 hits), mixed tridentate terpyridine-like diffraction studies. In particular, single-crystal X-ray analysis revealed an unusual metal-induced oxidation at the carbon atom of the triazine ring of the bridging tpt ligand and subsequent coordination of the thus-formed O donor in the heptanuclear Fe(III) pivalate complexes. A selected range of Fe(III)/ Fe(II)-tpt and Mn(II)-tpt complexes have also been characterized via magnetometry.
Coordination compounds of tpt are known for their bioactivity, for example, an inhibition effect against the larial parasite Setaria cervi that is specic to Ru-tpt compounds. 23 A series of Co(II), Ni(II), Cu(II), Mn(II) and Rh(III) complexes with tpt exhibits antibacterial activity against both Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. 24 Polynuclear Pt(II)-tpt complexes show potent cytotoxic activity in various cancer cell lines and can been used as potential chemotherapeutic agents. 25 Beside the possible use of metal-tpt based complexes as antimicrobial, anticancer and antiparasitic agents, the employment of such compounds to increase the enzyme biosynthesis has not been reported. This is the rst study where the prepared Mn(II)-tpt compounds have been tested as biostimulators for protease synthesis by Fusarium gibbosum CNMN FD 12 and Trichoderma koningii Oudemans CNMN FD 15 fungi strains. Note that manganese is one of the most studied metal ions among other transition metals due to its signicant role in the various biological processes, 26 as well as antibacterial and anticancer activities exhibited by its complexes with various organic ligands. 27

Synthetic aspects and preliminary characterization
Among the multiple synthetic pathways toward novel polynuclear clusters, the most common approach relies on "serendipitous" self-assembly 28 where "ligands are used that display several different coordination modes, combined with metal centers that can vary their coordination geometries" in one-pot reactions. The source of metal ions can range from simple salts to preformed polynuclear metallic precursors, for example well-known trinuclear m 3 -oxo-bridged metal carboxylates that can lose their terminal ligands to create vacant coordination sites and aggregate to higher nuclear clusters under various reaction conditions. The numerous Fe(III)/Fe(II)-tpt and Mn(II)-tpt clusters introduced herein are synthesized via this route. The tri-or hexanuclear Fe(III) and Mn(II,III) carboxylate precursors are soluble in various organic solvents; moreover, their terminal and their carboxylate ligands frequently are sufficiently labile, allowing the assembly into higher-nuclearity clusters. These properties made it possible to realize a very substantial range of different reaction conditions, utilizing different synthetic procedures such as stirring at room temperature in different solvents or mixtures of solvents, reuxing, sonochemical irradiation, and solvothermal reactions under high temperature and pressure. In this way, the coordination of Fe(II,III) and Mn(II,III) ions with tpt could be systematically studied, resulting in six Fe(III), one Fe(II), and two Mn(II) clusters were prepared. The reaction pathways resulting in new Fe(III)/Fe(II) coordination compounds are summarized in Scheme 2.
Large red-brown block-shaped crystals of compound 1, [Fe(tpt)(tptH)][FeCl 4 ] 4 $2(thf)$0.23(H 2 O), in addition to a blue microcrystalline powder, were obtained aer several weeks of crystallization from a blue-green mixture of the hexanuclear Fe(III) precursor [Fe 6 O 2 (OH) 2 (piv) 12 ] and tpt in a thf/CCl 4 mixture (1 : 1, v/v) stirred at room temperature (Scheme 2). Further investigation of this synthesis revealed that visible light is critical for the formation of 1. If the reaction is carried out in the dark, only small, irregular shaped red crystals are formed from a red solution. The structure of these red crystals has not been resolved yet. The impact of the ratio of the reactants has also been examined. Doubling the amount of the tpt ligand led to an over-saturated solution and undissolved ligand that was ltered off. Aer several weeks only a blue precipitate was found. Another less concentrated synthesis with the same ratio of starting materials was repeated, which led to a yellow solution with a colorless precipitate. Hence, it was found that the solution needs to be highly concentrated and the 1 : 1 ratio of the reactants leads to more satisfying results. The blue and red crystals are well soluble in thf, acetone and chloroform, but are insoluble in CCl 4 , preventing the separation of two different kinds of crystals by re-crystallization. The crystals were separated manually under the microscope.
Solvothermal heating of the same hexanuclear Fe(III) pivalate precursors with tpt in acetone and 1,2-dichlorethane lead to another mononuclear complex, [Fe(piv)(tpt)(Cl) 2 ] (2) (Scheme 2). The synthesis was repeated with the ratio of the reactants according to the formula but it resulted in a worse yield. The source of Cl − anions in 1 and 2 was CCl 4 and 1,2-dichloroethane, respectively, which undergo dehalogenation and introduce chloride ions into the reaction solution.
Dark-blue rod-shaped crystals of a dinuclear Fe(III)-tpt cluster, [Fe 2 O(tpt) 2 Cl 4 ] (3), have also been received from solvothermal syntheses when using m-oxo trinuclear Fe(III) pivalate precursors with tpt in acetone and chloroform (Scheme 2). The yield of 3 was a very low, and we noted that the reactant ratio corresponding to the composition of the product ( (piv) 12 ] precursors in the reaction with tpt from a 1 : 1 mixture of n-hexane and MeCN (method B), or from a 1 : 1 mixture of acetone and MeCN (method C). The complex is well soluble in acetone, thf, ethanol and methanol and is slightly soluble in n-hexane but insoluble in acetonitrile. We also run the same reactions with the ratio of starting reactants corresponding to the composition of the nal product and found no improvement but a degradation of the yield. Stirring m-oxo trinuclear [Fe 3 O(piv) 6 (H 2 O) 3 ](piv)$2(Hpiv) precursors with tpt in 1,4-dioxane gave a structurally similar [Fe 7 O 4 (piv) 12 (tpt-O)]$ 4(dioxane) (4b) cluster but with solvent dioxane molecules instead of MeCN as in 4a. The yield of 16% was low, but repeating the synthesis with the starting materials in the ratio corresponding to the composition of the product gave no crystals. Finally, a further heptanuclear compound, [Fe 7 O 4 (piv) 11 (tpt-O)(i-PrO)(i-PrOH)]$0.75(i-PrOH) (5), has been prepared from the reaction of trinuclear Fe(III) pivalates with tpt in a mixture of iso-propanol and n-hexane (1 : 1) with a moderate yield of 45%. We also explored the same reaction but using only iso-propanol and found that this led to compound 5, too.
For the preparation of Mn(II)-tpt compounds, [Mn 6 O 2 (piv) 10 (Hpiv) 4 ] and Mn(ib) 2  acetonitrile does not coordinate to Mn(II), its presence is critical for the growth of diffraction-quality crystals of 7. All bulk Fe(III)/Mn(II)-tpt samples were further characterized by IR spectroscopy and TGA to conrm their identities.
The IR spectra of carboxylate free compounds (1, 3, and 6) show the characteristic bands of coordinated tpt ligand (n C]C and n C]N stretching vibrations of the pyridine and triazine rings) in the region of 1637-1441 cm −1 , which are shied compare to a free tpt ligand with a strong band appeared at 1522 cm −1 , and weak bands at 1469 and 1433 cm −1 . 29,30 In the spectra of the carboxylate-containing compounds 2, 4 and 7 these bands overlap with strong vibrations associated with methyl and carboxylate groups of pivalates and isobutyrates. The moderately intense bands in the region 3066-2872 cm −1 belong to the C-H asymmetric and symmetric stretching vibrations of the tpt ligand and methyl groups of the pivalates (2 and 4) or isobutyrates (7). In carboxylate containing compounds 4a and 7 these are accompanied by a strong single band at 1485 cm −1 (4a) and 1474 cm −1 (7) and a typical doublet at 1378 and 1362 (4a) and 1387 and 1377 cm −1 (7) arising from the asymmetric and symmetric bending vibrations of methyl groups. The strong and broad bands arising from asymmetric and symmetric stretching vibrations of the coordinated carboxylate groups are found in 4a at 1543 and 1424 cm −1 , in 7 in the 1544-1532 cm −1 region and at 1427 cm −1 . The strong bands at 1388 and 1377 cm −1 attributed to the uncoordinated and coordinated nitrate groups in 6. The broad bands between 3731 and 3068 cm −1 correspond to O-H [water molecules (6) and methanol (7)] stretching vibrations.
The solution behavior of 2-5 and 7 has been characterized by electrospray ionization mass spectrometry (  3 ] − fragments, respectively. The ESI+ patterns of the heptanuclear Fe(III) cluster compounds 4b and 5 reveal several series of peaks (Table S2 †). A difference between neighboring peaks in each series of Dm/z ¼ 70 can be identied as the gradual replacement of one negatively charged piv − moiety by one MeO − group in the cluster core of 4b and 5.  3 ] + fragments, respectively (see Table S2 † for more data). Their ESI-MS patterns in negative-ion mode reveal the following fragments: in 4b, a series of peaks at m/z 1487, 1417, 1347, and 1277 are assignable to [Fe III . The pattern also displays signals for the heptanuclear core plus a dioxane molecule at m/z 1699, 1629, and 1559, which arise from The negative ESI pattern of 5 also exhibit a series of peaks at m/z 1799, 1729, 1659, 1589, and 1519, which corresponds to sequential replacement of piv   (2). Single-crystal X-ray diffraction analysis reveals that the mononuclear compounds 1 and 2 crystallize in the monoclinic crystal system, space groups P2 1 /m (1) and P2 1 /c (2) (Table S3 †). The structures of 1 and 2 are depicted in Fig. 1, 2, S1 and S2, † and selected bond distances and angles are listed in Table S4  . Single-crystal X-ray diffraction analysis showed that dinuclear cluster compound 3 and mononuclear 3a crystallize in the triclinic crystal system, space group P 1. The asymmetric unit of 3 containing one Fe(III) atom (BVS: 2.95), one tpt ligand, two Cl − and one m-O 2− center (see Fig. 3 for the molecular structure of 3 and Table S4 † 33 In the crystal lattice the complexes stack along the c axis, and adjacent dimers are connected by p/p interactions between the uncoordinated pyridyl ring of one dimer and one coordinated pyridyl ring of another dimer (Fig. S3 †). The distances between the centroids of the rings range from 3.708 to 4.066Å. Within the dimeric   4) ). This unusual behavior of tpt has already been observed when the triazine ring of tpt was hydroxylated [15][16][17][18][19] or methoxylated 21 as mentioned above, but for the rst time, this O atom is coordinated to metal atoms.
Within the tpt structure, the saturated carbon atom is not in plane with the other ve atoms of the ring (0.195Å above the plane of the other ve atoms). The plane of the N6-pyridyl ring substituted to this carbon atom is almost perpendicular to the plane of the triazine ring (83.83 ), whereas the other two pyridyl rings are rotated against the triazine plane by 4.23 (N1) and 9.76 (N5). Two pivalates act as chelating ligands and complete the coordination sphere around Fe3 and Fe6.

Magnetic studies
Magnetic data were evaluated for the title compounds that exhibited unexpected features or comprised interesting spin connectivities.
[Fe(piv)(tpt)Cl 2 ] (2). The magnetic properties of 2 are shown in Fig. 7 as c m T vs. T and M m vs. B plots. They are determined by an isolated Fe(III) center in a C 2v -symmetric, approx. pentagonalbipyramidal ligand eld (see Fig. 2).
At 290 K and 1.0 T, the c m T value of 4.37 cm 3 K mol −1 is well within the expected 46 range 4.1-4.5 cm 3 K mol −1 of an isolated high-spin Fe(III) center (m eff ¼ 5.91 m B ). Upon cooling, c m T slightly decreases to 4.01 cm 3 K mol −1 at 50 K, and subsequently drops off reaching 0.64 cm 3 K mol −1 at 2.0 K. At this temperature, the eld dependent magnetization curve shows a nearly sigmoidal shape with an inection point at 2.5 T. At 5.0 T, the  curve shows a distinct slope and a value of M m ¼ 3.8 N A m B , both indicating that the saturation of M m,sat ¼ 5.0 N A m B is not reached. The observed data are remarkable, since the ground state of the free Fe(III) ion is (almost exclusively) 6 S 5/2 . Thus, the high-spin Fe(III) centers are isotropic and spin-like. Since the Fe(III) centers are widely separated in 2 (closest intermolecular Fe/Fe distance: ca. 8.2Å) and in the absence of alternative exchange pathways such as p/p stacking, the c m T vs. T plot should be almost constant above 10 K considering the magnitude of Zeeman effect at 1.0 T. In addition, the magnetization M m at 2.0 K should be a continuously growing linear or concave function of B, and approach the saturation value M m,sat at 5.0 T. However, the data below 50 K are in conict with this simple situation, which has yet to be resolved.
[Fe(tpt)Cl 2 ]$2(H 2 O) (3a). In compound 3a, the closest intermolecular Fe/Fe distances of ca. 4.4Å are shorter compared to 2, but there are again no signicant supramolecular interactions (such as p/p stacking) between nearest neighbor complexes that could mediate intermolecular coupling, limiting spin-spin interactions to dipole-dipole coupling. As such, the paramagnetic properties of 3a are essentially determined by individual Fe(II) centers. The plot of c m T vs. T (Fig. 8)   m B that should be approached at such temperatures and elds, in the absence of coupling interactions. The ground state is likely characterized by an total effective spin of either S total ¼ 3/2 or 1/2, since S total,max ¼ N centers Â S center ¼ 7 Â 5/2 ¼ 35/2, DS total ¼ AE1 (S total $ 0) and M m,step ¼ g eff S total N A m B (g eff z 2.00 for Fe(III) centers).
We therefore use the effective spin-only option of the computational framework CONDON 47 considering Heisenberg exchange interactions (using the '−2J ex ' notation of the Hamiltonian) and the Zeeman effect to t the data. We generate starting values of the exchange interaction parameters J ik between the seven isotropic S ¼ 5/2 (g eff ¼ 2.00) centers using wxJFinder. 48 The least-squares t yield the solid lines in Fig. 9. Note that we restricted the tting range of the magnetization to data below 2 T to avoid the regions of potential saturation effects, which are not comprehensively considered in the used effective spin model. The values of the t parameters are given in Table 1. The relevant exchange interactions between the seven Fe(III) centers are all antiferromagnetic with the exception of the coupling between Fe 1 and Fe 2 . However, the magnetic properties of 4a are barely affected by a larger variation of the corresponding parameter value, which is also represented by its rather large error margin. According to this set of parameters, the molecular ground state is characterized by S total ¼ 1/2. Magnetization M m vs. applied magnetic eld B at 2.0 K are shown in Fig. 9 for 4a. At 290 K, c m T amounts to 7.94 cm 3 K mol −1 , which is well below the range 28.4-31.5 cm 3 K mol −1 Fig. 7 Temperature dependence of c m T at 1.0 T of 2; inset: field dependence of the molar magnetization M m at 2.0 K. Fig. 8 Temperature dependence of c m T at 0.1 T of 3a; inset: field dependence of the molar magnetization M m at 2.0 K. expected 42 for seven isolated high-spin Fe(III) centers. Upon cooling the compound, c m T almost linearly decreases, showing a weak dent at about 9 K, and subsequently drops down to 0.81 cm 3 K mol −1 at 2.0 K.
The recorded SQUID magnetometry data of 4b are shown in Fig. 10. Due to the structural similarities of 4a and 4b, the magnetic properties of both compounds are also very similar: at 290 K and 1.0 T, c m T is again well below the expected range for seven isolated Fe(III) centers. The value of 8.65 cm 3 K mol −1 is slightly larger than the value of 4a at this temperature. Comparison of the c m T vs. T curves of both compounds reveals the same characteristics including the dent at ca. 9 K. At 2.0 K, the value of c m T is 0.86 cm 3 K mol −1 . With respect to the magnetization curve of 4b at 2.0 K, it is also very similar to the curve of 4a, yet reaching a value of 2.1 N A m B at 5.0 T. Fitting the data using the same effective spin model yields the parameters given in Table 1. The obtained values of the tting parameters of 4a and 4b are comparable within the error margins, with a slight difference in the parameters J 45 that describes the exchange pathway between the triangular (Fe5-Fe7) and rhombic units (Fe1-Fe4). According to the set of parameters, the ground state is characterized by . Whereas the arrangement of the Fe(III) centers in 5 is similar to those in 4a and 4b, the structures distinctly differ in detail as discussed above. In particular, the triangular unit of 5 (Fe5-Fe7) is rather acute (vs. almost equilateral) due to the isopropanolate bridging ligand. Therefore, we expect from magnetostructural correlations that the exchange interactions within the triangular unit distinctly differ from those in 4a and 4b. Consequently, the deviations of the c m T vs. T and M m vs. B curves of the former and 5 are substantial (see Fig. 11). At 290 K Fig. 9 Temperature dependence of c m T at 0.1 T of 4a; inset: field dependence of the molar magnetization M m at 2.0 K (data: symbols, least-squares fit: solid line). Table 1 Coupling energies (in cm −1 ) derived from least-squares fits of the magnetic data of 4a, 4b and 5 using an effective spin model with S ¼ 5/2 and g eff ¼ 2.00 per Fe III center ('−2J' notation). Indices of J ik according the labels Fe(i) and Fe(k) in Fig. 4 (4a, 4b) and 5 (5), respectively. Note that the exchange interaction parameters listed per row correspond to the comparable pathways  and 0.1 T, c m T ¼ 9.22 cm 3 K mol −1 . Upon cooling, c m T decreases with a decreasing rate and reaches an almost plateaulike value of about 4.3 cm 3 K mol −1 between 10 to 7 K. Subsequently, c m T drops to 4.0 cm 3 K mol −1 at 2.0 K. The molar magnetization rapidly increases in the eld range 0 to 2 T, and hints at a saturation step at larger elds, reaching 4.8 N A m B at 5.0 T and 2.0 K. Therefore, the structural changes from 4a and 4b involve a change in the exchange interactions, likely with respect to the magnitude of J ik . The ground state of 5 is potentially S total ¼ 5/2, which is indicated by the value of M m at 5.0 T and the plateau of c m T at 7-10 K.
Fitting these data, we nd the exchange interaction parameters shown in Table 1, corresponding to the solid lines in Fig. 11. Note that the deviation of data and t in the c m T vs. T plot at T < 5 K is due to the applied simple model. In comparison to 4a and 4b, the exchange interaction parameters of 5 are stronger within the triangular unit and weaker within the rhombic unit but do not change their signs. The total effective ground state spin is S total ¼ 5/2.
The c m T vs. T plot at 0.1 T and the M m vs. B plot at 2.0 K of 7 are shown in Fig. 12. At 290 K, c m T is 4.37 cm 3 K mol −1 , i.e. within the expected 42 range 4.0-4.7 cm 3 K mol −1 for an isolated high-spin Mn(II) center (m eff ¼ 5.91 m B ). This value stays almost constant down to 20 K, at which a value of 4.28 cm 3 K mol −1 is observed. Upon further cooling, c m T noticeably decreases to reach 4.14 cm 3 K mol −1 at 2.0 K. At this temperature, the molar magnetization continuously increases with decreasing slope without hinting at an inection point in the experimental data. At 5.0 T, the magnetization is signicantly affected by saturation effects and reaches 4.8 N A m B .
Therefore, the data for 7 are fully in line with a regular highspin Mn(II) center at probed temperatures, in apparent contrast to the similar iron(III) tpt complex 2. Note, however, that the decrease of c m T below 20 K is only marginally due to the Zeeman effect, which generates relevant contributions not until below 5 K at 0.1 T (about 0.02 cm 3 K mol −1 at 2.0 K). These observed data can be explained by a small zero eld splitting due to the ligand eld.

Biological studies
In view of the general bioactivity of Mn(II)-tpt compounds, we assessed the potential biostimulator functionality of compounds  (7). We found that the addition of 6 and 7 to a nutrient medium of fungal strain Fusarium gibbosum CNMN FD 12 in concentrations of 0.0005 to 0.0015% essentially increases fungal proteolytic activity by up to 24.8-355.4% (Table S7 †).
According to the obtained data, the control presented the maximal activity of neutral (33.01 m mL −1 ) and alkaline (21.77 m mL −1 ) proteases at the sixth day of growth. The activity of neutral proteases of samples cultivated in the presence of coordination compounds ranged from 21.05 to 63.41 m mL −1 , the highest values being revealed at the h and sixth day for compound 6 and 7, respectively. It has to be mentioned that in the case of compound 6, the activity of neutral proteases at the 5th day exceeds the value of maximal control activity achieved on the 6th day of cultivation by 104.4% (concentration 0.0010%) and 71.3% (0.0015%). The highest activity was observed at the complex concentration of 0.0010%. When the compound 7 was introduced into the nutrient medium, the maximal activity of neutral proteases (49.25-63.41 m mL −1 ) was revealed at 6th day, similar to the control. The activity increased by 49.2-92.1%, with the maximum established, also, at the concentration of 0.0010%.
The study indicated that both 6 and 7 showed positive effect on alkaline protease activity and accelerated the biosynthesis of enzymes, reducing the cycle of cultivation by 24 h. Thus, the highest values of protease activity were found at the 5th day of growth. Activity ranged from 59.96 to 88.57 m mL −1 , being by 208.3-355.4% and 175.4-306.8% higher than the control from 5th and 6th day, respectively. Similar to neutral proteases, the most effective concentration for enzyme production was 0.0010%. The results regarding the inuence of compounds 6 and 7 on proteolytic activity of micromycete Trichoderma koningii CNMN FD 15 are summarized in Table S8. † Maximum of enzymatic activity in control was observed at the 9th day of cultivation, constituting 51.03 and 23.55 m mL −1 for neutral and alkaline proteases, respectively. A more prolonged cultivation leads to a considerable decreasing of proteolytic activity (13.92 and 8.92 m mL −1 ).
A signicant difference was found in the effect of coordination compounds 6 and 7: the activity of neutral proteases is preferentially higher than the control, while the activity of alkaline proteases is mostly inhibited. The highest protease production was shown by samples grown on the medium supplemented with compound 6 in concentration of 0.0005% and 7 in concentration of 0.0005 and 0.0010%, respectively. Activity of neutral proteases under these conditions was 94.39 m mL −1 (6) and, respectively, 71.91 and 79.41 m mL −1 (7). The stimulating effect constituted 84.9%, 40.9% and 55.6%, respectively. Increasing of coordination compound concentration leads to a decrease in the activity of neutral proteases and to a corresponding decrease of stimulating effect. According to the obtained data, the optimal concentration for both complex compounds is 0.0005%, providing a maximal increase in the activity of neutral proteases.
The use of studied compounds in the process of T. koningii cultivation negatively affects the alkaline proteases, leading to a decrease of activity by 42.4-55.3% compared to the maximum control level revealed at the 9th day of growth. At the same time, in samples cultivated in the presence of coordination compounds during ten days, the activity of alkaline proteases was increased, reaching at a concentration of 0.0015% the maximal level of control achieved at the 9th day. Thus, under these conditions the activity of samples cultivated in the media supplemented with coordination compounds were 23.91 m mL −1 and 25.52 m mL −1 for compounds 6 and 7, respectively.
According to the data, the both examined coordination compounds showed a stimulator effects on proteases production by fungal strains Fusarium gibbosum CNMN FD 12 and Trichoderma koningii CNMN FD 15 and can be used to improve the proteolytic activity of micromycetes. The most notable result was the increase of proteolytic activity of F. gibbosum. Thus, the use of complex compounds 6 and 7 in the optimal concentration of 0.0010% during submerged cultivation of the producer for 5 and, respectively, 6 days provide an increase in the level of neutral protease activity by 104.4% (compound 6) and 92.1% (compound 7) and of alkaline proteases by 264.8% (6) and 306.8% (7) compared to the highest value of control. Also, the possibility to reduce the duration of cultivation of the producer by 24 hours and, respectively, to obtain enzymatic preparations with high activity over shorter periods of time and with less energy expenditure has been revealed. Regarding the strain Trichoderma koningii CNMN FD 15, the most promising assay is the use of compound 7 in concentration of 0.0005%, which ensures the greatest enhancement of the biosynthesis of neutral proteases.

Materials and methods
All reactions were conducted under aerobic conditions using commercially available chemicals and solvents as received without further purication. [Fe 3 O(piv) 6 (H 2 O) 3 ]piv$2(Hpiv), [Fe 6 O 2 (OH) 2 (piv) 12 ] and Mn(ib) 2 has been prepared as described elsewhere. 51,52 The precursor [Mn 6 O 2 (piv) 10 (Hpiv) 4 ] has been prepared with a slight modication 53 by the reaction of manganese(II) acetate with an excess of pivalic acid followed by adding MeCN solution to the reaction mixture. For solvothermal syntheses the reaction mixtures were placed in a sealed PTEE-lines steel autoclave. The temperature program started with heating from room temperature up to 120 C within 1 h, temperature was held constant for 4 h and then slowly cooled down to room temperature within 48 h. For elemental analysis the analyser Elementar Vario El was used. The IR spectra were recorded on a PerkinElmer IR spectrometer using KBr discs in the region 4000-400 cm −1 (1)(2)(3)(4)(5) or PerkinElmer Spectrum 100 FT-IR in the region 4000-650 cm −1 (6-7). ESI mass spectra were recorded on a ThermoFinnigan LCQ Deca XP plus mass spectrometer with a thf/MeOH/acetic acid gel. The sets of PXRD data (Fig. S8-S11 †) were collected at room temperature with the aid of a STOE StadiP diffractometer (Stoe&Cie, Darmstadt, Germany; area detector; Cu Ka radiation; l ¼ 1.54059Å). A WiseClean ultrasonic bath operating at 40 kHz with a maximum output of 140 W was used for the ultrasonic irradiation.  (piv) 12 ] (0.160 g, 0.099 mmol) and tpt (0.031 g, 0.099 mmol) in tetrahydrofuran (5 mL) and tetrachloromethane (5 mL) was stirred for 2.5 h, then ltered off and the ltrate was put for crystallization. Aer twenty days the ltrate had turned green and two different kinds of crystals were obtained: small branched, dark blue and big red-brown crystalline blocks which were suitable for X-ray diffraction analysis. Structure determination of the red-brown crystals revealed the title compound. All crystalline precipitate was ltered off, washed with CCl 4 and dried on air. For analysis the two kinds of crystals were separated manually. Yield: 0.007 g Crystallography X-ray data sets for 1-5 were collected on a Bruker APEX CCDdiffractometer, for 3a on a STOE STADIVARI diffractometer, and for 6 and 7 on an Oxford Xcalibur diffractometer equipped with a CCD detector using graphite monochromator utilizing MoKa radiation (l ¼ 0.71073Å). The structures were solved by direct methods and rene by full-matrix least-squares on weighted F 2 values for all reections using SHELXL suite of programs. 54 All non-H atoms in the compounds were rened with anisotropic displacement parameters except in the case of water molecule in 1 and disordered solvent molecules in 5. Some of [FeCl 4 ] − anions in 1 and some pivalate and isopropanol groups in 4a, 4b, and 5 were found to be disordered over two positions. Therefore, SIMU, DELU, SADI, and ISOR restrains were used to deal with the disordered moieties in the structures and to obtain reasonable geometrical parameters and thermal displacement coefficients. The positions of hydrogen atoms in the structures were located on difference Fourier maps or calculated geometrically and rened isotropically in the "rigid body" model with U ¼ 1.2 U eq or 1.5 U eq of corresponding O and C atoms. Some H atoms of solvent molecules (H 2 O in 1 and hydroxyl hydrogen of i-PrOH molecules in 5) have not been localized. Crystallographic data and structure renements of 1-7 are summarized in Table S2. † The gures were drawn using the Mercury program. CCDC reference numbers for the structures are CCDC reference numbers for the structures are 2108859 (1), 2108860 (2), 2108861 (3), 2108862 (3a), 2108863 (4a), 2108864 (4b), 2108865 (5), 210956 (6), and 2109057 (7). †

Biological studies
The biological properties of coordinative compounds were evaluated on the base of their effect on the proteolytic activity of micromycetes Fusarium gibbosum CNMN FD 12 and Trichoderma koningii CNMN FD 15 in the dynamic of submerged cultivation. The producers' strains were grown in Erlenmeyer asks with a capacity of 0.75 L, which contained 0.2 L nutrient medium with the following composition, %: corn our, 2.0; soy our, 1.0; CaCO 3 , 0.2; (NH 4 ) 2 SO 4 , 0.1; compounds 6 or 7, 0.0005-0.0015; the rest was a tap water. Initial pH of the medium was 6.25. The nutrient medium was inoculated with a suspension of spores and mycelium in an amount of 5% v/v, which were obtained by washing with sterile distilled water of the culture grown on sloping surfaces of malt-agar for 12-14 days. Cultivation was performed at 28 C under continuous stirring (180-200 rpm) for 5-6 days in the case of Fusarium gibbosum CNMN FD 12 strain and for 9-10 days for Trichoderma koningii Oudemans CNMN FD 15. Coordination compounds were introduced into a sterile nutrient medium, simultaneously with inoculum, in the concentration of 0.0005, 0.0010 and 0.0015%. A sample cultivated on nutrient medium without coordination compounds served as a control. The activity of proteolytic enzymes was essayed by the Anson method 55 based on the degree of sodium caseinate hydrolysis.

Conclusions
In conclusion, a series of Fe(III)/Fe(II)/Mn(II)-tpt compounds of different nuclearity ranging from mononuclear up to heptanuclear form in the reaction of Fe/Mn pivalates or isobutyrates and the tpt ligand. Solvothermal reactions lead to the precipitation of mono-or dinuclear Fe-tpt compounds (1-3), whereas stirring of precursor solutions at room temperature afforded the heptanuclear Fe-tpt compounds 4 and 5. 4 and 5 represent condensed coordination cluster compounds with unprecedented structural features, in which an unexpected metalinduced oxidation at the carbon atom of the triazine ring of the bridging tpt ligand and the coordination of the thusresulting O atom to metal ion were observed. Previously, a complexation-induced hydrolysis of tpt was only observed in Ru, Rh, Os, Pd, Pt or Cu complexes, in which the formed O center did however not engage in coordination. Thus, our research, to some extent, provides a new path for the design and synthesis of specic Fe(III)-(tpt-O) cluster materials under so conditions. The heptanuclear Fe(III) compounds (4a, 4b, 5), the iron-oxo core structures result in the anticipated dominant antiferromagnetic exchange coupling. Finally, the Mn-tpt compounds studied in this work exhibit promising catalytic properties in the production of enzymes by microorganisms.

Conflicts of interest
There are no conicts to declare.