Synthesis, Structural Characterization, and Biological Activities of Organically Templated Cobalt Phosphite (H2DAB)[Co(H2PO3)4]·2H2O

A novel hybrid cobalt phosphite, (H2DAB)[Co(H2PO3)4]·2H2O, has been synthesized by using slow evaporation method, in the presence of cobalt nitrate, phosphorous acid and 1,4diaminobutane (DAB= 1,4diaminobutane) as a structure-directing agent. Single crystal X-ray diffraction analysis showed that the compound crystallizes in the P-1(n.2) triclinic space group, with the following unit cell parameters (Å, °) a = 5.4814 (3), b = 7.5515 (4), c = 10.8548 (6), α = 88.001 (4), β = 88.707 (5), γ = 85.126 (5), and V= 447.33 (4) Å3. The crystal structure was built up from corner-sharing [CoO6] octahedra, forming chains parallel to [001], which are interconnected by H2PO3 pseudotetrahedral units. The deprotonated cations, residing between the parallel chains, interacted with the inorganic moiety via hydrogen bonds leading thus to the formation of the 3D crystal structure. The Fourier transform infrared spectrum showed characteristic bands corresponding to the phosphite group and the organic amine. The thermal behavior of the compound consisted mainly of the loss of its organic moiety and the water molecules. The biological tests exhibited significant activity against Candida albicans and Escherichia coli strains in all used concentrations, while less inhibitory activity was pronounced against Staphylococcus epidermidis and Saccharomyces cerevisiae, and in the case of multi-cellular organisms, no activity against the nematode model Steinernema feltiae was detected.

In the next section, we shall lay out the experimental setting regarding the synthesis, characterization and biological evaluation. In section 3, we opted to conjoin the results and the discussion. The section reports and discusses the structure of the organically templated cobalt phosphite synthesized, the results from the infrared spectroscopy, thermal behaviors of the compound and, finally the biological activity against the different organisms mentioned above. In the last section we conclude.

Materials and Instrumentation
All reagents were acquired from commercial sources and used without further purification. The infrared spectrum of the compound was recorded on a VERTEX 70 FTIR Spectrometer in the range 4000-400 cm −1 using the ATR technique at 4 cm −1 resolutions. Thermogravimetric analysis (TGA) data were recorded on an SDT-Q600 analyzer from TA Instruments (Eschborn, Germany). The temperature varied from RT to 1273.15 K at a heating rate of 10°/min −1 . Measurements were carried out on samples in open platinum crucibles under air flow.

Crystal Structure Determination
Single-crystal X-ray diffraction measurement was carried out at room temperature using an Agilent Gemini S diffractometer equipped with a CCD detector and molybdenum (Mo) radiation source. Acquired data was processed with the CrysAlisPro software [23]. Using Olex2 [24], the structure was solved with the olex2.solve [25] structure solution program using Charge Flipping and refined with the olex2.refine [25] refinement package using Gauss-Newton minimization. All non-hydrogen atoms were refined anisotropically, and hydrogen atoms were included in the model at calculated positions, refined with a rigid model with their Uiso value fixed at 1.2Ueq of their parent atoms. Table 1 reports the crystallographic data and experimental details of data collection and structure refinements. The structural graphics were created using both DIAMOND program [26] and Mercury [27].

Biological assays 2.4.1. The Antimicrobial Activity
The activity of (H2DAB)[Co(H2PO3)4]·2H2O compound against Escherichia coli, Staphylococcus epidermidis, Candida albicans, and Saccharomyces cerevisiae was investigated in routine microbial growth assays based on optical density and recorded in the form of growth curves. Fresh cultures of S. epidermidis, E. coli, C. albicans, and S. cerevisiae were prepared on bacterial tryptic soy broth, Luria-Bertani broth (LB), Sabouraud Dextrose Agar (SDA), and Yeast Peptone Dextrose (YPD) agar media, respectively. After 18-24 h of incubation, the microbial colonies from these Agar plates were then transferred into 10 mL solution of 0.9% w/v NaCl (saline), and the turbidity of the suspension was adjusted to 0.5 of McFarland standard. These microbial suspensions were then exposed to the samples as described below. Bacterial and yeast culture with growth medium were employed as negative control, sterile distilled water was utilized as solvent control, while the positive control consisted of a mixture of 10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of amphotericin B. The sample was evaluated at various dilutions (of 250, 500, and 1000 µM), and the plates were incubated at 37 °C for 24 h. Microbial growth was monitored by recording the optical density of the samples at 0 h and 24 h, using a Micro Plate Reader E800 at 593 nm. These absorbance values were converted into percentages and compared to the negative control whose absorbance values were normalized to 100% and served as references at each time interval. All experiments were carried out in triplicate at three different occasions (n = 9). Results are represented as mean ± SD, and statistical significances were calculated by one-way ANOVA using GraphPad Prism (Version 5.03, GraphPad Software, La Jolla, CA, USA) with p < 0.05 considered to be of statistical significance.

Nematocidal Activity
The model nematode Steinernema feltiae was purchased from Sautter und Stepper GmbH (Ammerbuch, Germany) in the form of powder and stored at 4 °C in the dark. Fresh samples were utilized prior to each experiment. A homogeneous mixture was prepared by dissolving 200 mg of nematode powder in 50 mL distilled water. Later, the nematode suspension was placed for 15 min at room temperature with shaking and in moderate light. Viability was examined under a light microscope at four-fold magnification (TR 200, VWR International, Leuven, Belgium). The viability of nematodes above 80% in each sample was considered a prerequisite for each experiment. Ten microliters of nematode suspension were added to each well of a 96-well plate. The hybrid cobalt phosphite (H2DAB)[Co(H2PO3)4]·2H2O was then added into the wells to achieve final concentrations of 250, 500, and 1000 µM. Afterward, the final volume in each well was adjusted to 100 µL by adding Phosphate Buffered Saline (PBS pH = 7.4). PBS and ethanol (10 µL per well) were employed as negative and positive controls, respectively, and sterile distilled water was the solvent control. Each experiment was performed independently on three different occasions and in triplicate (n = 9). Living and dead nematodes were counted under the microscope prior to treatment, and the viability fraction (V0) was calculated (usually >0.9). Then, 50 µL of lukewarm water (40 °C) was added to each well to stimulate the nematodes prior to counting. After 24 h, the V24 fraction was calculated, by once more counting the living and dead nematodes, and expressed as a percentage of initial viability V0 according to the equation: Results are represented as mean ± SD, and GraphPad Prism (Version 5.03, GraphPad Software, La Jolla, CA, USA) was used to calculate the statistical significances by one-way ANOVA. p < 0.05 was statistically significant.

Structural Description
As shown in Figure 1, there is one crystallographically distinct Co located at special position (½ 0 ½) ( Table 1). The asymmetric unit of (H2DAB)[Co(H2PO3)4]·2H2O, which contains 13 non-hydrogen atoms located on general positions (  [29]. All (H2PO3) -units adopt pseudo-tetrahedral coordination geometry. P(5) shares two oxygen with adjacent Co atoms, while P (7) Table 4). The 1,4-butanediammonium templates, made up by N12, C11, C10 atoms and their symmetry-related N12 i , C11 i and C10 i , which resides between the parallel chains, are deprotonated. They are further ensuring, together with the free-standing water molecules, the stability of the three-dimensional network.

Infrared Spectroscopy
The infrared spectrum of (H2DAB)[Co(H2PO3)4]·2H2O ( Figure 4) exhibits bands corresponding to the vibration modes of the organic template, phosphite groups and water molecules. The stretching vibration of NH2 in 1,4-butanediammonium cation is observed in the high frequencies 3090-3200 cm −1 , while its bending appears at 1600 cm −1 [32]. The values pointing between 2600-2736 cm −1 correspond to the symmetric and asymmetric stretching of (NH3) + , and it also confirms the protonation form of the organic amine molecule. The two large vibration at around 2813 and 2923 are boil down to the stretching mode of υ(-CH2-), while the medium band at 1314 cm −1 coincide with the stretching vibration of υ(C-N). The characteristic bond of phosphite groups is manifested by a small and medium vibration at around 2420 and 2430 cm −1 and it corresponds to the stretching vibration of υ(P-H), as well as the bands from 990 to 1030 cm −1 are assigned to the bending mode of δ(P-H). The vibration modes centered at 1057 and 1164 cm −1 are ascribed to the symmetric and asymmetric stretching of PO3 group, while its symmetric and asymmetric bending are observed at 670 and 460 cm −1 , respectively. The broad vibration located at 916 cm −1 is attributed to the stretching vibration of P-OH bond [20]. The set of bands related to the stretching vibration and deformation of the OH group and belonging to water molecules is observed at around 3000 and 1645 cm −1 [33,34].

Thermal Behavior
Thermal analysis was performed under air atmosphere, and as depicted in figure 5, the experimental data of (H2DAB)[Co(H2PO3)4]·2H2O showed four separated stages of weight loss in a total of 52.75% from 25 to 1000 °C. The first experimental mass loss (5.45%), occurred between 150 and 270 °C, corresponds to the easy departure of one and half molecule of the two structural water molecules (Calculated value 5.30%). This quick dehydration of the hybrid cobalt-phosphite, may be explained by the engagement of the water molecules in a weak network of hydrogen bonds. This variation is coupled with a sharp exothermic signal in the differential thermal analysis trace at 174 °C. The second weight loss of 5.35%, ranging from 271 to 358 °C on TG curve, was related to the departure of the remaining half water molecule and the release of NH3 unit from the organic moiety (Calculated value 5.11%). This degradation was manifested by an exothermic intense peak at 292 °C. The experimental value of 8.95%, pointing at 385 °C on TG profile and highlighted by a small set of signals on DTA measurement, coincided with the continuous volatilization of the organic moiety (Calculated value 8.85%). The last and final experimental mass loss (33%), observed between 497 and 973 °C, can be associated with the total degradation of the organic moiety and the formation of the Cobalt metaphosphate compound Co(PO3)2 through a condensation reaction of phosphite groups (Calculated value 37%). This formation was mainly characterized by a small exothermic heat flow at 632 °C.

Biological Activities
The hybrid cobalt phosphite compound was tested for its antimicrobial activity against two bacteria (Escherichia coli and Staphylococcus epidermidis) and two Fungi (Saccharomyces cerevisiae and Candida albicans) strains. A mixture of 10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of Amphotericin B was used as a control at three different concentration 250, 500, 1000 µg/mL. Ethanol was used as a positive control.The percentage inhibition and minimum inhibitory concentration (MIC) values of the compound based on the growth of microorganisms are shown in Figure 6. albicans at an MIC value of 500 and 1000 µg·mL −1 . The inhibition of growth, however, was much less in the case of S. cerevisiae. The compound showed lower activity as antibacterial agent. The E. coli media demonstrated higher inhibition that S. epidermidis. The nematode assay showed that the compound had almost no impact on viability.

Conclusions
A novel organically templated cobalt phosphite (H2DAB)[Co(H2PO3)4]·2H2O has been synthesized using wet chemistry. Single crystal structure analysis revealed that the framework displays a chain-like structure, containing vertex sharing four-membered rings formed by the connectivity between CoO6 octahedrons and [H2PO3]-pseudo-tetrahedral units bound through their edges. The diprotonated 1,4-butanediammonium acts as a stabilizer of the inorganic network through hydrogen bonds. The thermogravimetric analysis showed that the dehydration of the hybrid phosphite takes place in three steps, resulting mainly from the loss of the organic moiety and water molecules. The antimicrobial investigation showed significant activity against all the microorganisms utilized except for S. feltiae. Further studies on hybrid phosphite compound are needed to assess their safety and applicability in the fields of medicine and agriculture.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: title, Table S1: title, Video S1: title. Informed Consent Statement: Not applicable. Data Availability Statement: All the data generated from this study is available upon request. Please contact the corresponding author.