Synthesis, characterization, and POM-protein interactions of a Fe-substituted Krebs-type Sandwich-tungstoantimonate

Abstract The novel iron-substituted Krebs-type polyoxotungstate (C12N4H11)4Na2H5[(Fe(H2O)3)2((FeO2)0.5(WO2)0.5)2(β-SbW9O33)2] (Fe-1) has been synthesized using ortho-phenylenediamine (opda) as a precursor for the in situ formation of the counter cation 2,3-diaminophenazinium (C12N4H11)+ (2,3-DAP). Fe-1 has been thoroughly characterized in the solid state by single-crystal X-ray diffraction (SXRD), powder X-ray diffraction (PXRD), IR spectroscopy, and elemental analysis as well as in solution by UV–Vis spectroscopy. The crystal structure of Fe-1 reveals π–π-interactions between the aromatic systems of the unconventional 2,3-DAP counter cation. POM-protein interaction studies using SDS-PAGE revealed a non-proteolytic behavior of Fe-1 towards Human Serum Albumin (HSA) as a model protein. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00706-019-2381-5) contains supplementary material, which is available to authorized users.


Introduction
Polyoxometalates (POMs) [1] represent a broad class of anionic clusters, which are composed of metal ions in high oxidation states and linked by oxygen atoms resulting in a vast variety of unique structures. Depending on their size, charge, and composition, POM frameworks exhibit numerous different properties opening potential applications in different research fields of catalysis [2], materials science [3], and biological chemistry [4,5] including protein crystallography [6,7].
Among the POM family, transition metal substituted POMs (TMSPs) represent the largest group, mainly counting the subgroup of Sandwich-type POMs. Sandwich-type POMs, which are generally composed of two lacunary building blocks linked by a belt of heteroatoms, can be further divided into various subgroups, among them the Krebs-archetype. Dedicated to Prof. Dr. Heinz Falk on the occasion of his 80th birthday.
Krebs-type POMs comprise two lone-pair cont a i n i n g β -Ke g g i n l a c u n a r y f r a g m e n t s , e . g .
Inspired by the use of opda as a precursor for the in situ generation of the unconventional 2,3-DAP counter cation, the novel iron-substituted Krebs-type Sandwich POM (C 12 N 4 H 11 ) 4 Na 2 H 5 [( Fe( H 2 O) 3 ) 2 (( FeO 2 ) 0 .5 (W -O 2 ) 0 .5 ) 2 (β-SbW 9 O 33 ) 2 ] (Fe-1) has been prepared. Herein, we report on the synthesis and thorough characterization of the novel Fe-substituted Krebs-type Sandwich tungstoantimonate Fe-1. Regarding the scarce number of studies on the POM-protein interactions of the Krebs-POM archetype [10] and the potential use of non-proteolytic POM clusters as additives in POM-assisted protein crystallography, the POM-protein interactions of Fe-1 with Human serum albumin (HSA) as a model protein were investigated using SDS-PAGE to assess whether Fe-1 shows any proteolytic activity towards HSA.

Synthesis of Fe-1
An aqueous solution of Na 9 [SbW 9 O 33 ] contains a mixture of [α-SbW 9 O 33 ] and [β-SbW 9 O 33 ] in equilibrium. It is well documented that the latter species [β-SbW 9 O 33 ] dominates the equilibrium at pH values lower than 6.0 [8]. As a matter of fact, the reaction was carried out in an acetate buffer at pH 4.8. Upon addition of opda to a warm aqueous acidic reaction mixture of Na 9 [SbW 9 O 33 ] and FeCl 3 , the initially yellow solution gradually turned dark red indicating the oxidation of opda to 2,3-diaminophenazine (2,3-DAP) catalyzed by the in situ formed Fe-1 Krebs-POM. Cooling of the reaction mixture to room temperature resulted in the formation of dark red crystal plates consisting of polyanion Fe-1 (Scheme 1).

Crystal structure of Fe-1
Single crystal X-ray diffraction (SXRD) studies were performed on Fe-1 revealing a Krebs-type structure which crystallizes in the triclinic space group P-1. The crystal structure of Fe-1 exhibits two [β-SbW 9 O 33 ] lacunary species linked by two Fe(III) metal centers at the peripheral sites and two W(VI) centers which show a 50:50 disorder with Fe(III) at the inner position of the linking belt. Regarding the synthetic conditions of Fe-1, which include the use of an acidic buffer (pH = 4.8), the disorder with tungsten is in accordance with the results for the disordered alpha-arsenotungstate compounds observed at lower pH values, reported by Kortz et al. in 2001 [12] as well as the disordered Krebs-type tungstoantimonates recently reported by our group [10]. The peripheral iron centers exhibit a distorted octahedral coordination environment with one acetate ligand and one H 2 O molecule coordinated to the metal center and Fe-O bond lengths ranging from 1.9271(1) at the inner site of the belt to 2.139(1) Å between the peripheral iron centers and the H 2 O ligand at the peripheral belt positions (Fig. 1).

UV-Vis spectrum of Fe-1
The UV-Vis spectrum of Fe-1 exhibits two major peaks, one at 271 nm corresponding to the pπ(O b ) → dπ*(W) ligand-tometal charge-transfer transition typical for the Keggin-type framework [13], whereas a second absorption maximum at 423 nm can be attributed to the aromatic transitions of the 2,3-DAP counter cations present in the structure [14] (Fig. 3).

POM-protein interactions
Considering the known catalytic activity of Fe(III) as a Lewis acid, the POM-protein interactions of the peripheral Fe(III) metal centers of Fe-1 with human serum albumin (HSA) as a model protein were investigated to assess whether Fe-1 exhibits any proteolytic activity. SDS-PAGE was performed on reaction mixtures of HSA and Fe-1 in a NaOAc buffer [10 mM] pH 5.5 to ensure a stable more accessible acidic conformation of the model protein [15]. The results revealed no hydrolytic activity of Fe-1 towards the peptide bonds of the model protein even at 65 °C and 100-fold excess of the POM compound indicated by intact protein bands at 66 kDa (Fig. 4). This is in good accordance with our previous results reported for the isostructural manganese-and zinc-substituted DAP-POM derivatives [10].

Conclusion
In conclusion, the synthetic pathway presented in this work may open new perspectives for the preparation of novel Krebs-POM archetypes exhibiting unconventional counter cations. The interactions of the Krebs-POM compound Fe-1 with HSA as a model protein have been investigated and the non-proteolytic behavior of Fe-1 may be interesting for further POM-protein interaction studies ultimately perhaps opening novel perspectives in the field of POM-assisted protein crystallography.

Experimental
All reagents were obtained commercially from Aldrich, of high-purity grade and were used as purchased without further purification. Na 9 [B-α-SbW 9 O 33 ] was prepared according to the literature procedure reported by Bösing et al. [8]. X-ray intensity data were measured on a Bruker X8 APEX2 diffractometer equipped with a multilayer monochromator, Mo K/α INCOATEC micro focus sealed tube and Oxford cooling device. The following software was used: Bruker SAINT software package [16] using a narrow-frame algorithm for frame integration, OLEX2 [17] for structure solution, refinement, molecular diagrams and graphical user-interface, Shelxle [18] for refinement and graphical user-interface SHELXS-2013 [19] for structure solution, SHELXL-2013 [20] for refinement. Experimental data and the CCDC-Code are provided in Table S1. Crystal data, data collection parameters, and structure refinement details are given in Tables S2 and S3 of the electronic supporting information. X-ray powder diffraction measurements were performed on a Bruker D8 ADVANCE diffractometer, Cu Kα radiation, λ = 1.54,056 Å, LYNXEYE silicon strip detector and SolX energy dispersive detector, variable slit aperture with 12 mm, 5° ≤ 2θ ≤ 40°. Attenuated total reflection Fourier-transform Infrared Spectroscopy: all spectra were recorded on a Bruker Tensor 27 IR Spectrometer equipped with a single-reflection diamond-ATR unit. Frequencies are given in cm −1 , intensities denoted as w = weak, m = medium, s = strong. Elemental analysis (C, H, N, O) was performed at Mikroanalytisches Laboratorium, Fakultät für Chemie, Universität Wien using the 2400 CHN Elemental Analyzer and the EA 3000, respectively. UV-Vis spectra were collected on a Shimadzu UV 1800 spectrophotometer. The spectra were recorded in 10 mM NaOAc buffer pH 5.5. SDS-PAGE was performed according to a standard procedure [21] using Precision Plus Protein Standard Dual Color (Bio-Rad) as molecular weight marker. Samples were applied to 14% polyacrylamide gels under reducing conditions. The sample amount loaded onto the gel was 5 μg. Gels were stained with Coomassie Brilliant Blue. Imaging of the gels was applied with Gel Doc™ XR of BIO-RAD. Human serum albumin (HSA) (5 µg) was mixed with 1, 10, and 100 equivalents of