s2fieldsofstudy: Chemistry Biology

text:
First biphotochromic fluorescent protein moxSAASoti stabilized for oxidizing environment

Biphotochromic proteins simultaneously possess reversible photoswitching (on-to-off) and irreversible photoconversion (green-to-red). High photochemical reactivity of cysteine residues is one of the reasons for the development of “mox”-monomeric and oxidation resistant proteins. Based on site-saturated simultaneous two-point C105 and C117 mutagenesis, we chose C21N/C71G/C105G/C117T/C175A as the moxSAASoti variant. Since its on-to-off photoswitching rate is higher, off-to-on recovery is more complete and photoconversion rates are higher than those of mSAASoti. We analyzed the conformational behavior of the F177 side chain by classical MD simulations. The conformational flexibility of the F177 side chain is mainly responsible for the off-to-on conversion rate changes and can be further utilized as a measure of the conversion rate. Point mutations in mSAASoti mainly affect the pKa values of the red form and off-to-on switching. We demonstrate that the microscopic measure of the observed pKa value is the C–O bond length in the phenyl fragment of the neutral chromophore. According to molecular dynamics simulations with QM/MM potentials, larger C–O bond lengths are found for proteins with larger pKa. This feature can be utilized for prediction of the pKa values of red fluorescent proteins.

Since the decoding of the GFP gene, fluorescent proteins have become reliable and effective genetically encoded biological markers 1 . To date, a huge color palette of fluorescent proteins has been developed, covering the entire visible spectrum 2,3 . Phototransformations of proteins have also been discovered: photoactivation 4,5 , reversible photoswitching 6 , and irreversible photoconversion 7 . Biphotochromic proteins simultaneously possessing reversible photoswitching and irreversible photoconversion were genetically engineered [8][9][10] and discovered in nature 11 . In recent years, work has been actively carried out to increase the chemical inertness of proteins 12 , stability at different pH values 13 and oxidative environmental conditions 14 . These factors can disrupt stable folding and maturation of the chromophore or lead to rapid photobleaching.
High photochemical reactivity of cysteine residues is one of the reasons for these problems. Accordingly, amino acids such as alanine, valine, serine, threonine, and methionine were used as replacements for the amino acid residues of cysteine in several popular FPs (Table 1), generating MoxFP ("mox"-monomeric and oxidation resistant) proteins [14][15][16][17] .
To date, there are three types of phototransformations: irreversible photoactivation, reversible photoswitching, and irreversible photoconversion. Photoactivatible fluorescent proteins (PAFPs) upon proper wavelength light irradiation irreversibly change from a non-fluorescent to a fluorescent state. Some of the representatives are PA-GFP 19 , PAmCherry 20 , PATagRFP 21 .
Photoconvertible fluorescent proteins (PCFPs) constitute a group of fluorescent proteins, starting with Kaede 22 that, when exposed to proper light, bear the capability of irreversibly switching their emission color, generally from green to red. During this process, there is an irreversible break in the polypeptide chain, followed by rearrangement of conjugated bonds 23 (Fig. S1).
Reversible switching fluorescent proteins (RSFPs) can repeatedly switch between fluorescent and non-fluorescent state under the influence of certain wavelengths of light. For most RSFPs, this occurs as a result of cis-trans isomerization of the chromophore 6,24,25 , except for protein Dreiklang 26 , it is characterized by the mechanism of covalent attachment and detachment of a water molecule close to the imdazolinone ring.
First well-studied RSFP was Dronpa 27 . It demonstrated the influence of key residues in the chromophore environment-157, 159, 173-on the properties of phototransformations [28][29][30] . Mutagenesis of these residues in the photoconvertible EosFP and Dendra proteins led to the production of biphotochromic variants: IrisFP 8  www.nature.com/scientificreports/ and Dendra-M159A 9 .Another biphotochromic protein pcDronpa was obtained by introduction C62H mutation into RSFP Dronpa 10 . SAASoti is a protein that is unique in structure because it is ancestorily biphotochromic, without requiring mutagenesis of the corresponding, or any other, residues to obtain these properties 31,32 (Fig. S1,  Fig. 2). Phylogenetic analysis shows that, in terms of its primary structure, SAASoti is the most distant from similar phototransformable proteins, despite the fact that their sequences are more than 50% identical. (Fig. 1, Table S1).
In this work we set out to engineer a monomeric and cysteine-free variant of SAASoti (moxSAASoti). Notably, the replacement of each amino acid residue of cysteine led to unexpected effects, such as a shift in the pKa of the red form to the alkaline region, a change in the rate and depth of phototransformation 34 , and an attempt to combine all the substitutions turned out to be a nontrivial task.

Results and discussion
As described previously 34 , the mSAASoti-3C (C21N/C71G/C175A mSAASoti mutant, mSAASoti is a SAASoti with V127T substitution (Table 2)) form was characterized by the fastest rates of green-to-red photoconversion and green-form photobleaching, and highest extinction coefficient (green form). The next aim was to develop the "mox" SAASoti form by substituting all 5 cysteine residues.  www.nature.com/scientificreports/ The structural role of cysteine residues and the effect of their substitutions on the properties of fluorescent proteins have not yet been sufficiently studied. In some cases, mutation of cysteines to the other amino acid residues led to dim or dark proteins 15,17,35 .
Based on aligned sequence with similar mox PCFPs and biphotochromic FPs ( Fig. 2 and S2) we identified the most promising hotspots for cysteine replacement-residues 105 and 117.
mSAASoti-3C was used as a matrix. We applied site-saturated two-point mutagenesis for both substitutions simultaneously at both 105 and 117 residues, since the combination of single successful mutations led to the production of non-fluorescent proteins. According to the SAASoti 3D model (the 3D full atom model of the mSAASoti was previously obtained 34 from the crystal structure of the IrisFP (PDB ID: 2VVH 8 )), residues at positions 105 and 117 are located on adjacent beta-sheets of the barrel. We hypothesized that combining two 10 20  www.nature.com/scientificreports/ independently generated substitutions at these positions, acting alone, may disrupt beta-sheet interaction and disrupts the barrel structure, resulting in non-fluorescent protein variants. The brightest clones contained glycine at position 105 and threonine or valine at position 117, New SAASoti mutants were expressed in E. coli cells and purified as described by a standard procedure 32 . The new variants were named moxSAASoti-V and moxSAASoti-T according to the sole alternative at position 117-valine or threonine.
Since the a.a. in position 117 is exposed on the surface in original mSAASoti-3C, its replacement could lead to increased aggregation of the new protein form. Therefore, the oligomerization state of new mutants was analyzed by size-exclusion chromatography. The elution volume (Table S2) and elution profiles (Fig. S3) for both mutants corresponded to a molecular weight of 25.7 kDa, which corresponded to a monomeric form of SAASoti. This findings may suggest that the external 117 residue does not play any important role in the oligomerization of moxSAASoti variants.
The most important thing was to analyze how substitutions influenced photochemical and photophysical properties. On-to-off green photoswitching kinetics are described by a biexponential model (Eq. (1)): The bi-exponential decay model during photoswitching is typical of most of the SAASoti variants published earlier 31,34 . A similar pattern was also observed for Dronpa ancestors 36 . Previously, it was hypothesized that the fluorescent proteins could exist in two different emissive states 37 ; There are possible explanations for this phenomenon: (1) existence of several protein populations with different conformations of the protein environment of the chromophore 38 ; (2) parallel photooxidation reaction proceeding with photoswitching, leading to the appearance of an oxidized fraction of a protein with a different photoswitching kinetics 34 .
On-to-off switching rates for new variants are very similar and larger than observed for mSAASoti-3C and parental mSAASoti: additionally, less photodestruction than for mSAASoti-3C was highlighted. (Fig. 3, Table 3).
The moxSAASoti-V variant showed lower recovery after photoswitching than moxSAASoti-T (30% and 20% of the initial fluorescence intensity recovered after the first PS cycle in the case of moxSAASoti-T and moxSAASoti-V, respectively) and practically the same kinetic constants, indicating high rates of "on-to-off "switching (Table 3 and Fig. 3). The fluorescence recovery after the second PS cycle is more complete, which probably indicates some photodestruction reaction in the first PS cycle. moxSAASoti-T shows the highest ratio  We tested the green to red photoconversion capacity of new variants. Mox variants such as their closest ancestor mSAASoti-3C showed a high green-to-red photoconversion rate but a low degree of photoconversion (Fig. 4). We assume several possible reasons for this fact: (1) low brightness of the red form and (2) unstable red form. It certainly could be a combination of both phenomena, which is the subject of further research.
The process of photoconversion was detected by changes in the red form fluorescence intensity (λ em = 590 nm). It can be described by a bi-exponential model (Eq. (2)): where the first exponent is responsible for the red form formation, while the second exponent describes its photodestruction, c-background and residual signal. The corresponding kinetic parameters (Table 3) for mox variants are close to the parent mSAASoti-3C, which could indicate that residues in positions 105 and 117 do not affect the green-to-red photoconversion.
We hypothesize that changes in the chromophore environment leading to an increase in the photoswitching rate, in turn, also impairs photoconversion. This may be due to the fact that the additional space in the chromophore environment, which facilitates cis-trans isomerization, hampers the stabilization of the red form of the chromophore, which decreases the efficiency of photoconversion. Currently, work is underway to confirm this hypothesis for SAASoti.
Based on the photochemical and photophysical properties of the new mox forms of the SAASoti protein, we chose the moxSAASoti-T variant for further characterization, since its on-to-off photoswitching rate is higher, off-to-on recovery is more complete and photoconversion rates are higher than those of mSAASoti.
We tested off-to-on thermal relaxation kinetics for moxSAASoti-T by observing the fluorescence at 520 nm. The relaxation kinetic constant value is 0.021 ± 0.001 min −1 , which is up to 8 times greater than that for other variants with substituted cysteines and of the same order as mSAASoti, as previously published 34 .
We compared the main physicochemical properties (excitation/emission maxima λ ex /λ em , pK a values of the chromophore, molar extinction coefficient (ε) and quantum yield (ϕ)) of moxSAASoti-T and other mSAASoti mutants with cysteine substitutions ( Table 4). The excitation and emission maxima for the green and red forms did not change. Although the molar extinction coefficient decreased for the green form, however, it is still one of the highest among the cysteine variants of mSAASoti, and the reduced value of the extinction coefficient for the red form may be associated with a decrease in its stability. The fluorescence quantum yield of the green form also decreased by 0.1, and the molecular brightness of the new variant practically coincides with C117S and C21N/C175A mSAASoti. Interestingly, most impact new substitutions provided upon pK a of the moxSAASoti red form. Previously, all cysteine substitutions resulted in higher pK a values (except SAASoti C117S, which has the same mSAASoti pK a ). We suggest that it could be an allosteric effect from T117 because a previous mutant with a single C117S substitution showed the lowest pK a of the red form of all other mutants with single substitutions of cysteine residues.
Point mutations in mSAASoti mainly affect the pK a values (Table 4) of the red form and off-to-on switching rates of the green form (k off-to-on for mSAASoti variants with cysteines substitutions, except moxSAASoti-T, were obtained in work 34 . Notably, single and triple mutations that are present in moxSAASoti-T considerably change these two macroscopic properties. However, substitutions of all cysteine residues result in the recovery of the mSAASoti properties. It is not evident which particular microscopic structural features are responsible for the changes in the macroscopic parameters. Previously 34 , it was demonstrated that the flexibility of F177  www.nature.com/scientificreports/ determines the rate of off-to-on photoswitching and that a change in the C-O distance of the phenyl fragment of the chromophore is responsible for the pK a shift. Here, we test these notions on the set of five proteins of the mSAASoti family: the mSAASoti, its single mutants C21N and C175A and a triple mutant mSAASoti-3C with exactly the same point mutations as in the moxSAASoti, and moxSAASoti-T itself. We performed a 200 ns classical MD run for each model system and analyzed the conformational behavior of the F177 side chain. As a measure of conformational diversity, we chose the dihedral angle C-C α -C β -C γ (Fig. 5A,B). The mSAASoti and moxSAASoti-T variants predominantly demonstrate conformations with dihedral values between 140° and 180°. For slower proteins, single and triple mutants of mSAASoti, the dihedrals are distributed between 20° and 100°. This conformation is less favorable for off-to-on photoswitching, as the chromophore binding pocket is tighter in this case, which hinders isomerization (Fig. 5). Thus, the conformational flexibility of the F177 side chain could be the main responsible for the off-to-on photoswitching rate changes and can be further utilized as a measure of the photoswitching rate.
The C-O bond lengths in the substituted phenols are known to correlate with their pK a values 39 . Here, we utilize this approach for more complicated systems, the same chromophore in slightly different protein environments due to point mutations. We perform QM/MM MD runs of the models comprising neutral red chromophores and analyze d (C-O) distributions (Fig. 5C). Although the chromophores in fluorescent proteins have a large number of interatomic interactions, the elongation of the C-O distance, which is a calculated parameter, reproduces the experimentally observed increase in the pK a value (Fig. 5D).
This result is of great importance, as it demonstrates that even though we cannot distinguish the impact of each amino acid residue and its substitution, we can evaluate the d (C-O) values along the MD trajectories and use it as a calculated parameter to predict the pK a value.

Conclusions
In the present work, monomeric and cysteine-free moxSAASoti proteins were obtained. We hypothesize that these variants will have a more stable folding under the oxidative conditions of the cell.
MoxSAASoti was obtained from the triple mutant mSAASoti-3C by simultaneous random mutagenesis at positions 105 and 117, since, as we assume, due to the close location of these residues, working single substitutions disrupted the interaction of two adjacent β-sheets and destroyed the structure of the β-barrel. As a result of simultaneous random mutagenesis at two positions, two variants with bright fluorescence at 520 nm were obtained: moxSAASoti-T and moxSAASoti-V (containing substitutions of all cysteines, V127T and differing only in substitutions at position 117: C117T and C117V, respectively). Photoconversion and photoswitching properties were characterized for new variants in vitro in comparison with their ancestors, mSAASoti and mSAASoti-3C. The mox variants are characterized by a higher photoswitching rate compared to the mSAASoti and a decrease in the photobleaching rate compared to the mSAASoti-3C variant. The photoconversion rates are close to the mSAASoti variant and higher than that of mSAASoti-3C, however, the photoconversion efficiency is worse, due, as we assume, to a decrease in the stability of the red form. Since the kinetic parameters of moxSAASoti-V are slightly worse than those of moxSAASoti-T, the moxSAASoti-T variant was chosen as the basic moxSAASoti-T. www.nature.com/scientificreports/ New substitutions mostly affect on pKa value of the red form. Using QM/MM MD simulations we find correlation between length of the d(C-O) bond of the phenyl fragment of the chromophore and pKa, Main importance is that it could possibly allow to predict the effect of a particular substitution on the value of pKa. Also, using classical MD simulations, we confirmed that the substitutions indirectly affect the mobility of 177 phenylalanine, which is responsible for the photoswitching rate. Since the altered residues are not located in close proximity to Phe177, we assume that they change the mobility of the beta sheets, freeing up and reducing the space for the movement of this residue.
Thus, we suggest that moxSAASoti-T can be used to study the dynamics of proteins in the oxidative and secretory environment, and its biphotochromic properties make it an interesting object for pulse-chase experiments in combination with PALM (photoactivation localization microscopy) or combination of pcSOFI and PALM.

Materials and methods
Mutagenesis and colonies screening. Site-saturated mutagenesis was performed by overlapping PCR with degenerate primers (Table 5). PCRs were carried out sequentially with each pair of primers using Pfu DNA polymerase. DNA with random substitutions in two positions (105 and 117) was cloned into the pEt22b vector and transformed into E. coli BL21(DE3) cells. The resulting colonies were transferred to LB agar medium with IPTG and grown at 20 °C for 24 h. Colonies were analyzed for fluorescence on an Olympus CKX41SF microscope by irradiation with excitation light at 470 nm.
Protein expression and purification. moxSAASoti was expressed and purified as described previously 32 , with the exception that cells were disrupted by ultrasonication. Oligomerization analysis by size-exclusion chromatography was performed as described earlier 32 . Absorbance and fluorescence spectra were detected using Cary 60 and Cary Eclipse, respectively, as described earlier 32 . Green-to-red photoconversion and reversible photoswitching experiments were performed using a homemade setup based on an Olympus CX41 upright microscope with Thorlabs (USA) light sources, 400 nm (and 560 nm for red form excitation) for photoconversion, and 470 nm for photoswitching using an Avesta ASP-75 spectrometer for detection. LED light was passed through Thorlabs MF390/18 and Chroma ET470/24 m bandpass filters. We obtained 282. 4   www.nature.com/scientificreports/ also obtained in ref 34 . Here, we additionally prepared models of the triple mSAASoti-3C mutant and the mox form. The CHARMM36 40 force field parameters were utilized for protein and the CGenFF 41 force field parameters for the chromophore in the green form. The system was solvated in a rectangular water box with TIP3P 42 water molecules and neutralized by adding sodium ions. Classical molecular dynamics simulations were performed in the NAMD software package 43 . Each system was preliminarily equilibrated by 10,000 minimization steps and a 20 ns MD run. Production runs for the mSAASoti and C21N variants of SAASoti were performed for 200 ns with a 1 fs time step in the NPT ensemble at p = 1 atm and T = 300 K. The pressure and temperature were controlled by a Nosé-Hoover barostat and Langevin thermostat, respectively. To decrease the influence of error accumulation for such long trajectories, we randomly reassigned velocities every 40 ns. The cutoff distances were 12 Å for both electrostatic and van der Waals interactions with switching to the smoothing function at 10 Å.

MD simulations with QM/MM potentials.
The systems for the QM/MM (combined quantum mechanics/molecular mechanics) MD simulations were preliminarily equilibrated in classical MD runs as described above. The simulations were performed for the same set of five systems, and the chromophore was in the neutral red form. The MM subsystems were described similarly to the classical MD. The QM part was composed of the chromophore, the side chains of Gln42, The63, Arg70, Arg95, Ser146, His197, Glu213 (in the neutral form) and two water molecules. The green to red conversion was manually performed, and these coordinates were used as initial for the QM/MM MD runs. The system was preliminarily minimized for 100 steps. After that the 5 ps production runs were performed. The QM part was described at the PBE0-D3/6-31G** Kohn-Sham DFT level 44,45 . The QM/MM MD simulations were performed using the interface 46 for the classical MD software NAMD 43   www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Reversal of a Fluorescent Fluoride Chemosensor from Turn-Off to Turn-On Based on Aggregation Induced Emission Properties

Here we present a new approach for the development of fluoride chemosensors taking advantage of aggregation induced emission (AIE) properties. Although AIE-based chemosensors have been described, they rely primarily on the analyte causing aggregation and hence fluorescence. We propose a new concept in the use of AIE for the development of fluorescent sensors. Our hypothesis is based on the fact that a turn-off chemosensor in solution can be transformed into turn-on in the solid state if the properties of ACQ and AIE are properly combined between the fluorescent molecules involved. To demonstrate this hypothesis, we have selected a fluorescent chemosensor for the fluoride anion with a conjugated structure of bis(styryl)pyrimidine that, while showing turn-off behavior in solution, becomes turn-on when it is brought to the solid state. We have also combined it with the advantages of a detection system based on the microfluidic paper-based analytical devices (μPAD). The system is fully characterized spectroscopically both in solution and in the solid state, and quantum mechanical calculations were performed to explain how the sensor works. The prepared device presents a high sensitivity, with no interference and with an LoD and LoQ that allow determination of fluoride concentrations in water 2 orders of magnitude below the maximum allowed by WHO.

Apparatus, instruments and software
1 H NMR and 13 C NMR spectra were acquired at room temperature in VARIAN 400MHz. The chemical shifts () are given in ppm and are referenced to the residual protons of the deuterated solvent or carbon nuclei of chloroform ( 1 H,  = 7.27 ppm; 13 C,  = 77.0 ppm) or DMSO ( 1 H,  = 2.50 ppm; 13 C,  = 39.5 ppm). Acidic impurities in CDCl 3 were removed by treatment with anhydrous K 2 CO 3 . IR spectra were recorded on an FT-IR spectrophotometer equipped with an ATR accessory and the main peaks are given in cm -1 . MALDI-TOF mass spectrometry was carried out on an Autoflex maX (Bruker) and high-resolution mass spectrometry (HR-MS) was carries out on an LCT Premier XE.
The µPAD were fabricated using a craft-cutting technique by cutting the paper using a 12W CO 2 laser engraver (Rayjet, Barcerlona, Spain). Fluorescence spectrums were collected using a Varian Cary Eclipse luminescence spectrometer (Varian Ibérica, Madrid, Spain). For µPAD samples, excitation spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer in the range 550 -800 nm with a xenon lamp as excitation source.
Luminescence intensity was measured using the following conditions: λ ex = 530 nm and λ em = 617 nm, the photomultiplier voltage was set at 550 V and excitation and emission slits of 5 nm were used.
UV-Vis absorption spectra were acquired on a Cary 100 (Varian) spectrophotometer at room temperature using a slit width of 0.4 nm and scan rate of 600 nm/min. Steady state fluorescence were recorded on an FLS920 system (Edinburgh Instruments) equipped with a time correlated single photon counting (TCSPC) detector. A TLC 50 temperature-controlled cuvette holder (Quantum Northwest) was used for the measurements (temperature was controlled at 296 S3 K). Quartz cuvettes (Hellma Analytics) of 10 mm were employed for all the spectroscopic measurements. For SSF spectra, a Xe lamp of 450 W was used as the light source and the excitation and emission slits were both fixed at 1 nm. The step and dwell time were 1 nm and 0.1 s, respectively. The quantum yields of the compounds in solution and solid state were recorded on an FS5 system (Edinburgh Instruments) equipped with a time-correlated single-photon counting (TCSPC) detector and an integrating sphere module (SC-30). The quantum yields of solutions were measured using 10 mm quartz cuvettes (Hellma Analytics). In solid, the quantum yields were obtained from the corresponding drop cast samples over quartz slides and directly from the Pad paper and measuring both the direct and the indirect fluorescence emission. In all cases, the step and dwell time were 1 nm and 0.3 s, respectively.
The microstructure was evaluated by Scanning Electron Microscopy (SEM) using a Jeol 6490LV electron microscope equipped with SE and BSE detectors and an Oxford Link EDS Probe. The samples were prepared by placing a spatula tip of the organogel on top of a sampler stub and allowing them to dry for 6 h at room temperature. Specimens for SEM observation required coating (Au-Pt, Emitech) to avoid problems related to the surface charging-up.

Synthesis of SP-OSi
Compounds 1 and 3 were prepared according to literature 1 and compound 4 was prepared as follows: Cl Scheme S1. Synthesis of SP-OSi 4.

Computational details
The molecular geometry of the ground, anionic and first excited states were optimized using the density functional theory (DFT) method implemented in the Gaussian 16 programme package M06-2X functional was used along with the 6-31+G** basis set which has proven to be suitable to estimate the singlet excited state in previous works. 3 The vibrational frequencies were also calculated for both ground and excited stets to check the absence of imaginary frequencies. The energy barrier to rotation of the N-C-C-C dihedral angle was calculated to determine the most stable conformation (see Figure S3). The polarisable continuum model (PCM) was used to consider the effect of the solvent as implemented in the Gaussian package. 4 Time-dependent DFT calculations at the TD-M06-2X/6-31+G** were performed in dimethylsulfide. The reorganisation energy upon excitation, , was calculated using the programme DUSHIN developed by Reimers 5 according to: is the wavenumber associated with the vibrational mode i and S i is the Huang-Rhys  (HR) factor.   S10 Table S1. Calculated lowest-energy transition wavelengths (λ ab calc ), oscillator strengths (f) and orbital contributions for these transitions. Calculations were carried out at the TD-M062X/6-31+G** level of theory in DMSO solution. Calculated wavelengths (λ emis calc ) for the S 1 S 0 transition.    Figure S9 shows the titration of SP-OSi with acid, exhibiting a single equivalence point at pH=5.18. When a similar titration was conducted using an equivalent amount of water to the case of HCl, the quenching of fluorescence was negligible even after adding 7 times the volume usually employed for titrations (2.5 L). This experiment supports the fact that variation of fluorescence is not caused by the increase of polarity due to the addition of water. To study any effect of a polarity, increase due to the presence of ions, the averages of emission intensities for SP-OSi solutions in DMSO before and after the addition (2.5 L) of different inert salts at 1 mM concentrations were compared. Figure  S6b shows the average values and their standard deviations (n = 18). These averages were compared using a two-tailed t-test assay with Welch's correction at a 95% confidence level; obtained p-value was 0.3778, which stated that there were no significant differences between them, pointing to the absence of any polarity influence on the fluorescence using the reported volumes of aqueous solutions. Precision, % 1.5 Table S4. Analytical parameters of µPAD for fluoride ions determination.

Fabrication and preparation of the µPAD
Microfluidic paper-based analytical devices were fabricated using a paper sheet (Whatman nº1) by craft-cutting employed a CO 2 laser engraver. The pattern of the µPAD was first designed using Illustrator software (Adobe Systems) and the design was cut by CO 2 laser.
The µPAD consists of two layers prepared individually, as shown in Figure S7. The first layer is the detection area where the SP-OSi is immobilized. The second layer is made up of two separates areas, one for sampling and the second for transport of the sample. To prepare the µPAD, 4 µL of SP-OSi in 10 M THF solution was dispensed onto the detection area by drop casting and 2 µL of phosphate buffer 100 mM pH 7 was added on the transport channel, both pieces of paper were dried at room temperature for 2 min and stored in a dry environment at 4ºC in the dark.   S18 concentrations from 11.9 -125.0 µM, and the detection limit was evaluated to be 3.9 µM, indicating that SP-OSi has a very good sensitivity to the detection of fluoride. LOQ, µM 11.9 Precision, % 1.3 Table S5. Analytical parameters of µPAD for fluoride ions determination.

S19
In order to evaluate the operating applicability of the sensor in real samples it was carried out the analysis of samples of tap water, brick green tea infusion and commercial mouthwash. All the samples were collected in clean polyethylene bottle and were stored at -18ºC until they were analyzed. The sample of tap water and the commercial green tea were filtered using a filter of 0.45 µm to remove the impurities. Different concentrations of fluoride ions were added to the samples of water and green tea, then these samples were analyzed using the sensor. For the sample of the mouthwash solution, a dilution of 1:100 with distilled water was carried out and the fluoride ions concentration was determined using the PAD sensor such as the analytical protocol indicated. On the other hand, the sample of toothpaste (1g) was dissolved in 100 mL of distilled water heating for 10 minutes in a water bath. Later, 100 µL of propanol was added to reduce foaming and the sample was diluted 100-fold. 6 The concentration average of fluoride for each sample was obtained from three independent measurements and presented with the associated standard deviation.
For fluoride determination, 20 µL of a solution of different concentrations of fluoride is dropped onto the sampling area of the µPAD. After the solution flows by capillarity towards the areas where the reagents are placed, twenty minutes are needed to complete the reaction, when a blue or red colour appears in the white detection area, depends on the concentration of fluoride in the sample. Once the sensing area is allowed to react a fluorescence measurement is carried out using a luminescence spectrometer. For the luminescence measurement, the detection area of the µPAD was excited at 530 nm.


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Cytotoxic derivatives of dichloroacetic acid and some metal complexes

This study outlines a number of studies of dichloroacetic acid (DCA) and some of its derivatives. Although DCA has low cytotoxic potencies, various structural modifications are described which result in potent cytotoxins. In particular, hybrid molecules created from DCA and other bioactive molecules whose modes of action differ from DCA are particularly promising as candidate anticancer agents. Considerable emphasis in this review is placed on various series of compounds that incorporate both platinum and DCA into their structures. In addition, the importance of the formulation of some of the bioactive compounds described herein is revealed.

| INTRODUCTION
The therapeutic potential of dichloroacetate (DCA) was foreshadowed in 1960s for its interesting biological activities. [1] At that time, it was discovered that DCA can stimulate significant effects on carbohydrates and lipid metabolism in experimental diabetes [2] and soon thereafter, the historic report of its eliciting effect on the pyruvate dehydrogenase complex (PDC) was revealed. [3] These early findings led to the identification of DCA as a lipid-lowering and antidiabetic drug, [4] with the possibility of treating these diseases as well as myocardial and cerebrovascular ischemia [5] and acquired [6,7] and congenital [8,9] forms of lactic acidosis.
The anticancer properties of DCA were first discovered by Bonnet et al. They suggested that the metabolic alterations that occur in tumor cells can be manipulated to kill them. [10] Since then, more than 500 peer-reviewed publications have been published to establish DCA as a prototype of a new class of "metabolic modulators" acting to shift a cancer cell's metabolism away from cytoplasm and toward mitochondria, a consequence that, ironically, initiates the cancer cell's death. [11] To understand the interesting effects of DCA on cancer metabolism, the mechanism of action is briefly described below.
Over the last 10 years, a number of reviews have been written on DCA that provide further evidence of its potential usefulness in cancer chemotherapy. [12][13][14] Although an extensive body of literature demonstrates the antineoplastic properties of DCA, its effective clinical application for treating cancer is limited to clinical trials. [15] The clinical use of DCA is still restricted due to the existence of side effects such as neurotoxicity as well as the suspicion of carcinogenicity. [15,16] To avoid side effects that enhance drug bioavailability and efficacy, many researchers studied the synergistic effects of DCA with other potential or clinically used anticancer drugs. [17][18][19][20] In addition, over the last decade many scientists have designed and synthesized DCA derivatives that could be used as hybrids or prodrugs. A hybrid molecule is designed so that it can react at different receptor sites to increase cytotoxic potency while a prodrug is devoid of bioactivity until it releases one or more bioactive compounds. In this article, we have focused on compounds containing the DCA moiety in esters, amides, and metal complexes that showed antineoplastic properties and suggesting the employment of DCA in cancer therapy.

| MECHANISM OF ACTION OF DCA IN CANCER CELLS
Malignant cells have well-known properties including abnormal proliferation, deregulation of apoptosis, and cell cycle. Besides these properties, cancer cells also exhibit different metabolisms compared to normal cells. [21,22] In fact, to achieve increased energetic and anabolic needs, cancer cells follow reprogramming metabolic pathways for ATP, reduced coenzymes (e.g., NADPH), lipids, proteins, and nucleotides. [23] Therefore, one of the popular strategies for developing antineoplastic agents has been therapies that target the metabolic differences between cancers and normal cells. These therapeutic potentials arise from a discovery by Otto Warburg in the 1920s known as the "Warburg effect." According to the Warburg effect, for ATP production cancer cells incline to transform glucose into lactate via glycolysis even under aerobic conditions in place of the much more efficient oxidative phosphorylation (OXPHOS) pathway which is the preferred pathway for most other cells in the body. [24] Although ATP production via cytoplasmic glycolysis is 100 times faster than the mitochondrial OXPHOS, the energetic yield of glycolysis is very small since ATP production through glycolysis is 18 times lower than OXPHOS. [25] Because of this thermodynamic trade-off between the yield and rate of ATP production, cancer cells significantly increase glucose uptake by upregulating the glucose receptors in an attempt to "catch up." In fact, positron emission tomography imaging studies confirmed that most solid tumors showed markedly increased glucose uptake and metabolism, compared to noncancerous cells. [26] This bioenergetic difference between malignant and normal cells may be targeted therapeutically due to its unique importance in cancer cells. [27] Before OXPHOS can occur, pyruvate must first be converted to acetyl-CoA in a series of reactions facilitated by the PDC, named after pyruvate dehydrogenase (PDH), the first enzyme in the complex. PDC inhibition can occur if the E1α subunit of the PDH is phosphorylated by pyruvate dehydrogenase kinase-1 (PDK1), an enzyme commonly overexpressed in malignant cells. [28,29] PDC inhibition by PDK1's stops acetyl-CoA formation from pyruvate, which prevents oxidation from occurring in the TCA cycle and inhibits OXPHOS ( Figure 1).
A clear need is demonstrated for compounds which inhibit PDK activity since PDK inhibition can reverse the Warburg effect. A number of compounds such as DCA, a halogenated acetophenone (1), analogs of ATP, for example, adenosine 5′-[β,γ-imido]triphosphate (2), certain triterpenes such as 3 [30] and lactones, such as 4 and (R) −3,3,3-trifluoro-2-hydroxy-2-methylpropionamides such as 5 are known inhibitors that can inhibit PDK activity ( Figure 2). [28] Among these, DCA is the focus of this review. The molecular interactions between DCA and PDKs have been studied extensively. [31][32][33][34] In general, DCA appears to bind to a hydrophobic pocket in the N-terminal domain of PDK1 which promotes conformational changes at the active site and prevents phosphorylation of the E1α subunit of the PDC. [35] This frees up the PDH enzyme in the PDC and allows for full glucose oxidation through OXPHOS, inhibiting lactate fermentation (cytoplasmic glycolysis), and restoring apoptosis capability via mitochondrial depolarization ( Figure 3). [11] This cascade of events increases the interest in the use of DCA for its anticancer properties. Many different studies have shown that DCA and its derivatives reverse the glycolytic phenotype by activating a metabolic switch from aerobic glycolysis to OXPHOS; they also show suppression of tumor proliferation. [17,36,37] DCA combination with other chemotherapeutic drugs such as 5-fluorouracil or paclitaxel was more effective in killing resistant cancer cells than either chemotherapeutic drugs or DCA treatment alone. [20,38] Apoptosis capability F I G U R E 1 In the metabolic pathway preferred by cancer cells, pyruvate dehydrogenase kinase-1 (PDK1) phosphorylates a subunit of the pyruvate dehydrogenase complex (PDC) on the mitochondrial membrane and inhibits conversion of pyruvate to acetyl-CoA by blocking substrate entrance into the mitochondria is restored. DCA has also been approved as a treatment method for lactic acidosis. [9] 3 | LIMITATIONS OF THE USE OF DCA The clinical use of DCA has been related with reversible peripheral neurotoxicity, which is thought to be caused by uncompensated oxidative stress from increased reactive oxygen species (ROS) formed because of PDK inhibition by DCA. [15,39,40] In addition, it has been found that the catabolism of the amino acids phenylalanine and tyrosine and heme synthesis are interfered by the metabolism of DCA. As a result, the accumulation of reactive molecules capable of forming adducts with DNA and proteins are formed and resulted in oxidative stress. [15] Suspicion of carcinogenicity is also a restricting factor in the use of DCA and administration as an anticancer agent should be confined solely to clinical trials.
The low stability of DCA in a biological environment and also its low cellular membrane permeability are other major issues that are principally associated with the clinical use of DCA for cancer treatment. [11,41] In fact, due to its anionic charge DCA is unable to cross the cell membrane through passive diffusion and is unable to F I G U R E 2 Structures of different pyruvate dehydrogenase kinase inhibitors F I G U R E 3 Dichloroacetate (DCA) inhibits pyruvate dehydrogenase kinase-1 (PDK1) from phosphorylating the pyruvate dehydrogenase complex (PDC) on the mitochondrial membrane, which promotes the conversion of pyruvate to acetyl-CoA and subsequently facilitates the entrance of the substrate into the mitochondria HOSSAIN ET AL. | 3 of 20 enter inside mitochondria by the classical mitochondrial pyruvate carrier. [42] Therefore, high dosages of DCA are required to reach therapeutic effectiveness and clinical results. [43,44]

| DERIVATIVES OF DICHLOROACETIC ACID
Because of neurotoxicity, the clinical effectiveness, and toxic properties of DCA as an antineoplastic agent need to be carefully investigated in clinical trials. Nonetheless, promising clinical and translational research suggests a wide therapeutic window for DCA, provided it can be administered with adequate tolerability and safety. Thus, there is increasing interest in the development of new strategies aimed to engineer DCA derivatives with increased tolerability and safety and that will be able to cross the cellular membranes, enabling the cellular uptake. Toward this goal over the last decade many scientists designed and synthesized derivatives of dichloroacetic acid as potent cytotoxic agents. Therefore, in this study, we have focused on various types of compounds containing the dichloroacetyl group that showed promising anticancer activity. A major emphasis is given on the structure-activity relationships of these dichloroacetic acid derivatives in this study.
Different compounds are grouped together as derivatives of natural, synthetic organic and metal complexes, and their potencies as anticancer agents are discussed.

| DCA derivatives of natural compounds
Historically natural products and their derivatives play a significant role in drug development, especially in the area of cancer therapy.
A diverse source of new medicinal leads have been obtained from natural products and their derivatives. [45] Among them, triterpenes are a significant class of compounds for their anticancer properties.
Recently, a dichloroacetamide (6b) of a triterpenoid saponin albiziabioside A (6a) (Figure 4) was reported to show selective cytotoxicity against PDK-medium and PDK-high-expressed human cancer cells. [46] Previously, it was found that albiziabioside A (isolated from Albizia inundata) could induce apoptosis in A375 cells by adjusting mitochondrial function, involving a caspase cascade with an IC 50 value of 5.47 μM. [47] When the dichloroacetyl moiety was attached to albiziabioside A (6a) as an amide, the resultant compound 6b displayed greater cytotoxicity compared with 6a and DCA alone against all tested cancer cells (MCF-7, HCT116, A375, and 4T1). [46] Most importantly, the dichloroacetamide 6b exhibited the best cytotoxicity against the PDK-high-expressed human breast cancer cells (MCF-7) with IC 50 value 0.43 μM, which is enhanced 47.5-fold compared to albiziabioside A. This enhanced activity can be attributed to the more efficient PDK inhibition capacity of 6b.
The compound 6b could inhibit PDK activity and impact the energy metabolism reprogramming of tumor cells and turn cellular metabolism away from aerobic glycolysis to OXPHOS. [46] It was also reported that compound 6b induced a significant increase in intracellular ROS and reduced the accumulation of lactic acid in the tumor microenvironment (TME). Importantly, compound 6b caused apoptosis via a caspase dependent pathway, inhibited the glutathione peroxidase IV (GPX4) pathway and induced ferroptosis by the accumulation of lipid peroxidation. Additionally, in an antitumor experiment on nude mice containing MCF-7 tumors, compound 6b was found to be an effective and safe agent. It was also reported that 6b could inhibit both primary and distal tumor progression in a dual-4T1 tumor model in female BALB/c mice by remolding the tumor immunosuppression microenvironment via eliminating M2-TAMs (tumor-associated macrophages). [46] Tormentic acid (7a) (Figure 4), a triterpenoid, can be isolated from different plants including Myrianthus serratus, Perilla frutescens, Cotoneaster simsonsii, and Rubus sieboldii. [48] Tormentic acid (7a) possesses many biological activities such as in vitro inhibition of platelet aggregation, reduction of vascular smooth muscle cell proliferation, anti-inflammatory activity, and inhibition of αand β-DNA polymerases. Although only a weak cytotoxic activity has been established against different cancer cell lines for 7a, the F I G U R E 4 Structures of compounds 6 and 7 derivatives 7b-7d containing monochloroacetyl groups showed excellent cytotoxic activities against 518A2 (melanoma), 8505C (anaplastic thyroid), A253 (head), A2780 (ovarian), A549 (lung), DLD1 (colon), MCF7 (mamma). [48] Among all the tormentic acid derivatives, compound 7b exhibited excellent antitumor activity with IC 50 values ranging 1.1-1.6 μM and this anticancer property was found due to the induction of an apoptosis pathway. Doxorubicin (8a) ( Figure 5) is a natural compound isolated from a soil born bacteria, Streptomyces peucetius. It has been used as an efficient chemotherapeutic drug for many cancers, including bladder cancer, Kaposi's sarcoma, breast cancer, lymphoma, and acute lymphocytic leukemia. [49] However, because of several side effects such as vomiting and nausea, hepatotoxicity, cardiotoxicity, and myelosuppression, its clinical application is limited. To reduce side effects, enhance antitumor efficiency, and increase drug loading content, Yang et al. synthesized the amide 8b by coupling doxorubicin (8a) and DCA. [50] It was reported that compound 8b can be easily self-assembled into NPs with a small amount of PEGylated lipid DSPE- cancer cell lines. [51] To increase the potency of these compounds, Omran et al. developed a codrug of 14-hydroxytylophorine and dichloroacetic acid by combining the two molecules through an ester linkage. [52] The resultant DCA esters 9 ( Figure 5) were evaluated against two reference cell lines: human embryonic kidney cells (HEK293) and Chinese hamster ovary cells (HEK293) and against four cancer cell lines: neuroblastoma (SK-N-DZ), Burkitt's lymphoma, [53] hepatocarcinoma (HepG2), and colorectal adenocarcinoma (DLD-1).
The cytotoxic data of esters 9 were compared with the parent 14-hydroxytylophorine and with the reference chemotherapeutic drug, doxorubicin. Although the developed codrugs 9 maintained the cytotoxicity of the parent alkaloids, the codrug strategy did not improve their potency or selectivity. antioxidative, and antibacterial activities. [54] Recently, honokiol (10a) has also been found as a promising anticancer agent in preclinical studies because of its nontoxic properties. [55] However, honokiol has poor solubility and bioavailability. [56] To increase its cellular uptake and lipophilicity, honokiol (10a) was transformed into the corresponding bis-DCA ester derivative (10b). It was reported that both honokiol (10a) and its DCA derivative 10b could inhibit androgen receptor (AR) activity in prostate cancer cells, [57] showed in vivo activity against vemurafenib-resistant melanoma, [58] and are selective allosteric inhibitors of the mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1). [59] Both honokiol (10a) and its DCA derivative 10b were reported to inhibit the growth of an androgen-responsive prostate cancer cell line with wildtype p53 (LNCaP) and its androgen-independent variant (C4-2) in a time and dose-dependent manner. [57] The bis-DCA ester 10b was more potent than the parent compound 10a and other tested analogs. Like the cell viability data, compound 10b was relatively more efficient in reducing AR protein level than the parent compound 10a in prostate cancer cells. It was also reported that both proteasomal degradation and transcriptional repression mechanisms likely cause honokiol-mediated downregulation of the AR protein expression. In addition, the reasons for showing different effects of 10 on mRNA and AR protein between LNCaP and C4-2 cells were not yet clear.
Honokiol bis-DCA (10b) and a hexafluoro analog 11 ( Figure 6) were studied against aggressive melanoma such as LM36R and A375, and less aggressive LM36 cells in vivo. [58] Both 10b and 11 showed activity against A375 melanoma in vivo, but compound 10b was more effective. Honokiol 10b exhibited activity against LM36R (vemurafenib resistant) in vivo but not toward parental LM36. Both 10b and 11 inhibited the phosphorylation of dynamin-related protein 1 (DRP1), which stimulates a phenotype suggesting respiration via mitochondrial normalization. It was suggested that 10b possibly acts in vemurafenib-resistant melanomas by increasing both respiration and ROS production, which leads to show its activity against aggressive melanoma in vivo.
Honokiol bis-DCA (10b) was also found to display allosteric inhibition of TRAP1 chaperone activity which makes it a potential lead compound for a new generation of antineoplastic agents. [59] The molecular chaperones TRAP1 are the mitochondrial paralog of the heat shock protein 90 (Hsp90). It is necessary for tumor growth found in several tumor cell models. TRAP1 induces bioenergetic rewiring, maintenance of redox homeostasis, and orchestration of a hypoxia-inducible factor 1-alpha (HIF1α)-mediated pseudohypoxic program by inhibiting succinate dehydrogenase (SDH) activity. It was reported that compound 10b can bind to the allosteric site in TRAP1, which inhibited TRAP1 but not Hsp90 ATPase activity. In neoplastic cells, 10b was able to revert TRAP1-dependent downregulation of SDH, decrease proliferation rate, increase mitochondrial superoxide levels, and abolish tumorigenic growth. [59] A series of new DCA derivatives of phenstatin were reported recently that focused on the use of a single compound acting on two different biological targets thereby enhancing potency and lowering the toxicity of the chemotherapeutic agent. [60] The first point of action was inhibition of the tubulin which is involved in cell proliferation due to its ability to polymerize and form microtubules polymerization inhibitor, with mono-, di-and tri-chloroacetate groups targeting PDK1. Based on biological studies, all the synthesized compounds were grouped as: (i) inhibitors of tubulin polymerization, classic cytotoxic agents, for example, compounds 12a, 12b, 12d, 12e; (ii) inhibitors of PDK1, which are cytostatic, such as compounds 12f, 12g, 12h; (iii) dual inhibitors of tubulin/PDK1, for example, compounds 12c and 13; (iv) highly cytotoxic compounds. To efficiently deliver compound 16 inside the mitochondria in cancer cells, a biodegradable nanoparticle (NP) was constructed from poly-(lactic-co-glycolic acid) (PLGA)-block(b)-polyethylene glycol (PEG) functionalized using a terminal TPP cation. [63] The NP formulation of compound 16 was reported to reduce glycolysis by targeting PDK1 only in cancer cells, not affecting the immune cells. A syngeneic tumor model was used that stimulates pathways to instruct inhibitory effects on multiple immune checkpoint proteins while improving cytotoxic T cell infiltration.
3,5-Bis(benzylidene)−4-piperidones are known to possess promising cytotoxic potencies. These compounds were designed to undergo sequential interactions with cellular thiols leading to greater toxicity to tumors than normal cells. [22,64] To enhance tumorselective cytotoxicity of this piperidone scaffold, the dichloroacetyl group was attached to the cytotoxic warheads, 3,5-bis(benzylidene) −4-piperidones, to create a series of 15 novel hybrid molecules having the general structure 17 [65] (Figure 10). In general, these new was lower than that of 18b. It was suggested that the orthocompound may block the interaction between the pharmacophore and the receptor as the ortho-substituent is close to the pharmacophore (2,2-dichloroacetyl). The best potencies to inhibit A549 cells was found to be the meta-substituted structures, followed by the ones with para-substituents. For example, the IC 50

| Metal complexes of DCA
Metal complexes have been used in cancer chemotherapy for many years. Because of their unique molecular geometries as well as their catalytic, ligand exchange, photophysical, and redox reactions, metal complexes have the ability to react and interact with biomolecules in distinctive ways and by unique mechanisms of action. [72] Since the discovery of cisplatin as an anticancer drug in 1967 by Rosenberg, platinum-based metal complexes particularly have played an important role in chemotherapy. [73] Since then, more than 30 platinum- formation of DNA lesions leading to cell death. [75,76] Passive diffusion and/or active transport are the two predominant mechanisms known for cellular uptake for cisplatin and related compounds. [77] After cellular uptake, a ligand substitution event occurs in the cytoplasm in which a chloride ligand is replaced by a water molecule in cisplatin to form cis-[Pt(NH 3 ) 2 Cl(H 2 O)] + , known as aquation or activation. [76] Due to the positive charge on the platinum complex, it can be attracted to the negatively charged nuclear DNA, and subsequently coordinatively interact with DNA to form DNA adducts. The N-7 atoms of the purine residues of guanine and adenine are the most nucleophilic sites of DNA, and these are preferentially coordinated with the cis-[Pt(R-NH 2 ) 2 Cl] fragment.
Following this event, the remaining chloride ligand is substituted for a second guanine base, forming either an intrastrand or interstrand DNA cross-links. The formation of cross-link adducts distort the structure of DNA in a substantial manner leading to an impairment of DNA expression and stability of G quadruplexes, which are thought to be the crucial lesion for its biological effectiveness as an antitumor agent. [73,78,79] Besides platinum-based metal complexes, complexes with other metals such as Cu, Ru, Os, and Rh are also known in the literature that can act as anticancer agents.

| Platinum complexes of dichloroacetic acid
Although platinum-based complexes have entered clinical trials, the poor selectivity, severe side effects, drug resistance, and systemic toxicity have restricted the application to clinical use of these drugs. Therefore, a considerable amount of effort has been dedicated for developing new platinum-based antineoplastic agents having equal or higher antitumor activity but lower toxicity. [73] To obtain novel have been reported to be potential dual-targeting anticancer agents. [83] These four complexes exhibited potent anticancer activity against  Figure 15) containing DCA and biotin moiety as axial ligands. [80] It  [86] Cell lines that were used include cervical (A431), breast (MCF-7), lung (A549), pancreatic (BxPC3), and colon (HCT-15) cancers, as well as melanoma (A375).
Both compounds 32 and 33 displayed a better cytotoxicity toward neoplastic cells in comparison to nontumor cells. In particular, compound 32 exhibited the best cytotoxicity against human pancreatic carcinoma cells (BxPC3) among all the tested cells.
Generally, the presence of oxalate leaving ligand appears to decrease the cytotoxicity of kiteplatin derivatives. It was reported that the most active Pt(IV) complex 32 underwent very rapid hydrolysis in a solution at pH = 7.4 to release the axial DCA ligands. In addition, both complexes underwent rapid reduction (half times of minutes) by ascorbic acid to form the Pt(II) complex (kiteplatin). As a result, DNA-platination was observed due to the presence of Pt(II) and a significant increase of ROS generation, inhibition of OXPHOS, hypopolarization of the mitochondrial membrane, and caspase-3/7mediated apoptotic cell death were also observed due to the presence of DCA which was released by the Pt(IV) compounds. Finally, complex 32 was able to reduce the tumor mass of murine LLC solid tumor found in an in vivo study which was similar to that of cisplatin but it is less cytotoxic.
A series of novel Pt(IV) prodrugs with triple action capacity has been developed and synthesized by Petruzzella et al. for evaluating the hypothesis that compounds with multi-action capacity may have more cytotoxic effect than a single action compound for killing cancer cells. [87] The rationale behind this hypothesis was based on the assumption that different bioactive moieties may interact with different cellular processes. Thus, they developed Pt(IV) derivatives of cisplatin with "triple action" ability where they introduced PDK, inhibitors of cyclooxygenase, [25] or histone deacetylase (HDAC). were investigated against both Pt sensitive A2780 and Pt resistant SKOV-3 human cancer cell lines. [90] The amine complexes 42-43 were slightly more active against Pt sensitive A2780 cells than the showed the ability to overcome the resistance of cancer cells found with cisplatin. In addition, it was expected that complexes 48a-48b may cross the cell membrane more easily to enter the cells because of better lipophilicity of the molecules and increase the uptake of DCA into cells as compared to free DCA alone.

| Other metal complexes of dichloroacetic acid
Beside platinum, osmium and ruthenium complexes have been studied extensively as potential anticancer agents. [94,95] Their mechanisms of actions are different than the platinum-based drugs.
Therefore, their cytotoxicity profiles are different and they have the potency to overcome both the intrinsic and acquired resistance of various tumors against platinum-based drugs. Two half-sandwich complexes of Ru (60a) and Os (60b) ( Figure 23) containing a releasable DCA ligand have been reported to show good in vitro anticancer property against A2780 human ovarian carcinoma cells with IC 50 = 3.5 and 2.6 μM respectively, slightly higher than that of the clinically used platinum-based drug cisplatin (IC 50 = 5.9 μM). [96] However, they were much less toxic toward healthy human hepatocytes. Upon hydrolysis, the complexes 60a and 60b released the DCA ligand which is probably the reason for this cytotoxicity at (SKBR-3 and MCF-7). [97] Importantly, 60b displayed remarkable submicromolar cytotoxicity for killing CSCs, which is considerably more efficient than that of its Ru analog 60a and salinomycin, known for one of the most selective CSC-targeting compounds. Additionally, Os complex 60b has been reported to reduce the size, formation, and existence of three-dimensional mammospheres which is more closely related to the tumor microenvironment than cells in traditional two-dimensional cultures. Complex 60b can kill human breast CSCs primarily by a programmed form of necrosis, known as necroptosis. In contrast, Ru(II) complex 60a and salinomycin displayed different modes of action which involved blocking of both apoptosis and necroptosis in CSCs. Os complex 60b undergoes hydrolysis slower than the Ru complex 60a, therefore, it is possible that 60b has better ability to carry the biologically active DCA into the cell as compared to the Ru(II) complex and salinomycin, and thereby, they have differences in their biological effects.
A multifunctional rhenium (Re) complex 61a ( Figure 23) containing DCA has been developed and synthesized as an anticancer agent. [98] Complex 61a can alter cancer cell metabolism from glycolysis to glucose oxidation because of its ability to efficiently diffuse into cancer

ACKNOWLEDGMENT
There is no one to acknowledge for writing this review article.
Open Access Funding provided by Big Ten Academic Alliance hybrid -Indiana University Kokomo.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.


s2fieldsofstudy:
Chemistry
Materials Science

text:
Isolation and crystal and molecular structures of [(C5H2Br3)2Fe], [(C5HBr4)2Fe] and [(C5Br5)(C5Br4HgBr)Fe]

Hexa- and octabromoferrocene, as well as a mercury derivative of decabromoferrocene, were isolated from mixtures of polybromoferrocenes and characterized by X-ray diffraction. The crystals show halogen and hydrogen bonding, which act co-operatively.

Introduction
'Noncovalent interactions' are found in nearly all disciplines of chemistry, biochemistry and biology, and have been studied, at least in part, for quite a while (Hobza & Ř ezá č, 2016). This term brings together such apparently different interactions as hydrogen, halogen, lone-pair-, anion-, cation-andbonding, and these interactions can either act independently or co-operatively (Mahadevi & Sastry, 2016;Portela & Ferná ndez, 2021). Among these, halogen bond(ing) has been studied continuously at a high level since about 1995. The last comprehensive review dates back to 2016 (Cavallo et al., 2016). A look at SciFinder shows since then nearly 2000 new entries for the years 2021 and 2022, and already 492 entries with the concept 'Halogen Bonding' (accessed on May 26th, 2022). The vast majority of these studies are centred on organic or biological systems, with a focus on crystal engineering (Mukherjee et al., 2014). Relatively rarely studied were metal-containing systems (Brammer et al., 2008), in particular, organometallic systems have so far been restricted to a few metal carbonyls, ruthenium-complexed aryl iodides (Kelly & Holman, 2022) and one study on 1,1 0 -dihaloferrocenes (Shimizu & Ferreira da Silva, 2018). Our group has been working on polyhalogenated metallocenes for quite a while (Sü nkel & Motz, 1988;Sü nkel & Hofmann, 1992;Sü nkel et al., 1994Sü nkel et al., , 2015Sü nkel & Bernhartzeder, 2011), and some very recent reports on the synthesis and crystal structure determinations of [(C 5 H n Br 5-n )(C 5 H 2 Br 3 )Fe] (n = 1 or 2; Butler et al., 2021;Butler, 2021) and [(C 5 H n Br 5-n )(C 5 Br 5 )Fe] (n = 0 or 1; Rupf et al., 2022) prompted us to report on our synthetic and crystallographic studies of polybromoferrocenes. A special focus is made on the occurrence of halogen and hydrogen bonding in these systems.

Experimental
2.1. Synthesis and crystallization 2.1.1. Reaction of 1,1 0 0 0 ,2,2 0 0 0 -tetrabromoferrocene (1) with LiTMP in a 1:10 molar ratio and C 2 H 2 Br 4 . A solution of 1 (243 mg, 0.48 mmol) in tetrahydrofuran (THF; 2 ml) was added to a freshly prepared solution of LiTMP (4.8 mmol) in THF (4 ml) at À30 C. After stirring for 5 h, the temperature was lowered to À78 C and C 2 H 2 Br 4 (0.6 ml, 5.0 mmol) was added. With continuous stirring, the temperature was raised to ambient temperature over a period of 16 h. After this, water (10 ml) was added and the mixture was extracted with several 10 ml portions of CH 2 Cl 2 . The combined extracts were washed with water, then dried with MgSO 4 and completely evaporated in vacuo. The residue was taken up in the minimum amount of petroleum ether and chromatographed on an alumina column (20 Â 2 cm), using petroleum ether as eluent. 21 fractions were collected and examined by mass spectroscopy and selected fractions were examined by 1 H NMR spectroscopy (Figs. S1 and S2 in the supporting information). All fractions contained mixtures of polybromoferrocenes. While the first fraction consisted of a mixture of penta-, hexa-and heptabromoferrocene, the intermediate fractions contained hexa-, hepta-and octabromoferrocene, and the last fraction was a mixture of hepta-and octabromoferrocene with traces of nonabromoferrocene. Crystals of 1,1 0 ,2,2 0 ,3,3 0 -hexabromoferrocene (3) were obtained by slow evaporation of the sixth fraction in a refrigerator, and crystals of 1,1 0 ,2,2 0 ,3,3 0 ,4,4 0 -octabromoferrocene (5) were obtained from the last fraction by the same method. All other fractions were also recrystallized from different solvents (petroleum ether, Et 2 O and CH 2 Cl 2 ), but yielded neither crystals nor 'pure' powders (according to 1 H NMR spectra taken after redissolution).

Refinement
Compound 3: SHELXT (Sheldrick, 2015a) provided the complete molecule of 3 on the first run. The following difference Fourier synthesis (see Fig. S6 of the supporting information) showed two electron-density maxima (Q15 and Q16 in Fig. S6; d = 2.66 and 2.48 e Å À3 ) at radial distances of 1.54 and 1.40 Å from ring atoms C24 and C14, respectively. Despite these short distances (more typical for C-C bonds), we assigned these peaks to Br atoms (first named X1 and X2) with very low site-occupancy factors, since from the preceeding synthesis no other elements could have been present. The following refinement, however, showed rather short intermolecular distances (2.581/3.328 and 2.955/3.106 Å ) from these positions to atoms Br21 i /Br22 i and Br11 i /Br12 i , respectively [symmetry code: (i) x, y À 1, z] (Fig. S7). It was concluded that X1/X2 could not be present in the same molecule as Br11/Br12/Br21 (and eventually Br22 also) and therefore it was assumed that compound 3 (with Br11-Br13 and Br21-Br23) cocrystallized with very small amounts (ca 3%) of compound 2 [with Br13-Br14 (= X2) and Br22-Br24 (= X1)], and this model was used for the subsequent refinements. The refinement procedure was as follows: first, it was assumed that all Br atoms would have the same isotropic U values and then the site-occupation factors for X1 = Br24/H24 and X2 = Br14/ H14, as well as Br11/H11, Br12/H12, Br21/H21 and Br23/H23, were allowed to refine. The site-occupancy factors were then fixed at these values and the U values were allowed to refine freely for the main components even anisotropically. Any attempts to produce longer C14-Br14 and C24-Br24 'bonds' via the use of restraints met with failure. It should be noted at this point that the crystal structure of 1,1 0 ,2,4-tetraiodoferrocene showed a similar disorder and an apparent 'bond shortening', which the authors were able to resolve (Evans et al., 2021).
Compound 5: the measured crystal was recognized as a twin (two domains, rotated by 180 around 010) and a HKLF5 data file was created. The scale factor BASF refined to a final value of 0.17818. The refinement proceeded without any problems, and no signs of disorder were found.
Crystal data, data collection and structure refinement details of all compounds are summarized in Table 1.
high yields of 1,1 0 ,2,2 0 ,3,3 0 -hexabromoferrocene (3), contaminated, however, with heptabromoferrocene (4) and octabromoferrocene (5). All attempts to repeat this procedure on compound 3 met with failure, due to the very low solubility of this compound. On the other hand, the preparation of 'permercurated ferrocenes' followed by the addition of KBr 3 , first reported in 1977, then later in 1994, 1997 and 2022, suffered from difficulties due to solubility problems (Boev & Dombrovskii, 1977;Han et al., 1994;Neto et al., 1997;Rupf et al., 2022). For example, Han and co-workers showed that using Hg(O 2 CCF 3 ) 2 as the mercuration agent gave a 'mixture of at least four partially brominated ferrocenes'. When they used Hg(O 2 CCH 3 ) 2 as the mercuration agent, decabromoferrocene (7) could be isolated in 60% yield, contaminated, however, with at least two partially brominated ferrocenes. Rupf and coworkers repeated this latter experiment and showed that besides 7 also nonabromoferrocene (6) and nonabromo-(bromomercurio)ferrocene (8) were formed (based on 13 C NMR spectroscopy; a closer look at Fig. S20 of their supporting information shows the additional formation of octabromoferrocene 5 and the bromomercurioferrocenes [C 10 H n Br 9-n HgBrFe] with n = 1 and 2). When they used Hg(O 2 CC 3 H 7 ) 2 , they apparently obtained a mixture of 7 and 8 with no other contaminants (based on NMR and IR). Neto and co-workers reported the use of Hg(O 2 CCCl 3 ) 2 as the mercuration agent, the transformation of the apparently formed [C 10 (HgO 2 CCl 3 ) 10 Fe] to the decachloromercurioferrocene, followed by reaction with KBr 3 to give pure 7 [characterization by NMR and IR spectroscopy, and elemental analysis (C and Fe)].
We decided to look at the lithiation reactions with LiTMP as the lithiating reagent, C 2 H 2 Br 4 as the brominating agent and THF as the reaction medium at low temperatures again. We started with a solution of 1,1 0 ,2,2 0 -tetrabromoferrocene (1; purity > 95%) in THF and treated it with ten molar equivalents of LiTMP, followed by the addition of tetrabromoethane. After standard work-up, a chromatographic separation was attempted. Since no band formation was recognizable, 21 fractions with equal volume were collected. Fractions 1, 10, 12 and 21 were examined by mass spectrometry (Fig. S1), while fractions 4, 6, 8 and 21 were studied by NMR spectroscopy (Fig. S2). All fractions were left standing in open vials for slow evaporation of the solvent. From these crystallization attempts, fractions 1, 6 and 21 gave crystals. The observation that some compounds were present in nearly all fractions is most likely due to the low solubility in the eluting solvent, which led to 'smearing' over the length of the chromatography column. The use of different solvent mixtures for elution (PE/ Et 2 O, PE/THF and PE/CH 2 Cl 2 ; PE is petroleum ether) increased the solubility, but did not improve the resolution of the compounds. This problem might have been overcome by the use of high-performance liquid chromatography (HPLC); however, this was not available to us.
The mass spectrum of fraction 1 showed the presence of 2 (m/z = 579.7), 3 (m/z = 659.6) and 4 (m/z = 737.5), with 2 as the main component. In both of fractions 10 and 12, 4 was the main component, contaminated by 2 (traces), 3 and 5 (m/z = 817.5). Finally, the mass spectrum of fraction 21 showed 5 as the main component, contaminated by 4 and traces of 6 (m/z = 895.2). The 1 H NMR spectra of fractions 4, 6 and 8 showed different mixtures of compounds 3 ( = 4.47) and 4 ( = 4.72 and 4.43) [assignments based on Butler (2021)]. The 1 H NMR spectrum of fraction 21 (in dimethyl sulfoxide) showed three very weak signals at = 5.33, 5.20 and 4.76, which might be assigned to 4 and 5 by comparison with the mass spectra (no other NMR data in this solvent were available). The crystals obtained from fraction 1 suffered from disorder or cocrystallization effects, which could not be properly resolved. The crystals from fraction 6 also showed disorder, which could, however, be successfully modelled as cocrystallization of compounds 3 (ca 97% contribution) and 2. Fraction 21 yielded pure crystals of compound 5. Fig. 1 shows the structural formulae of the compounds discussed in this study.
We also repeated the permercuration of ferrocene according to Winter and co-workers, using Hg(O 2 CCH 3 ) 2 as the mercurating agent and dichloroethane as the solvent, followed by bromination with KBr 3 . Chromatography of the crude reaction product yielded two fractions (Han et al., 1994). The first contained, according to its mass spectrum (Fig. S3), a mixture of bromoferrocenes 5-7 (with compound 6 dominating), while the second consisted of a mixture of bromomercurioferrocenes [C 10 H n Br 10-n HgFe] (n = 0-2; Fig. S4). Fig. S5 shows the 1 H NMR spectrum of the first fraction, which apparently consists of four proton-containing substances, of which one dominates. Although we did not perform these sigma-hole interactions Acta Cryst. (2022). C78, 578-590 Blockhaus and Sü nkel Crystal and molecular structures of polybromoferrocene 581 Table 2 Overview of the CSD structures of polyhaloferrocenes substituted on both rings.  Rupf et al. (2022) experiments, it can be assumed that compounds 5-7 are formed by further bromination of [C 10 H n Br 10-n HgFe]. Therefore, we conclude that neither the permercuration nor the bromination reactions are complete. Although all fractions were used for crystallization attempts, only crystals of compound 8 could be obtained.

Molecular structures
An intensely debated topic since the very early days of ferrocene chemistry was the question of the relative stability of the eclipsed and staggered conformers of this molecule. While the very first crystal structure determination of ferrocene (Fischer & Pfab, 1952) hinted at a staggered geometry, the most recent low-temperature IR and XANES (X-ray absorption near edge structure) spectra, as well as DFT (density functional theory) calculations showed that the eclipsed conformation is the energy minimum (Bourke et al., 2016;Silva et al., 2014). For ferrocenes substituted on both rings, an additional conformational isomerism arises from the possibility of different relative positions of the substituents (Scheme 1).
While theoretical calculations on 1,1 0 -dibromoferrocene showed that the two C 2 isomers (II and III in Scheme 1) are minimum conformations (Silva et al., 2014), in the crystal structure, only the less favourable C 2v structure (I in Scheme 1) was obtained (Hnetinka et al., 2004). To obtain an overview of the 'realized' structures, a Cambridge Structural Database (CSD; Groom et al., 2016) search on ferrocenes with at least one halogen substituent on each ring was undertaken. This search delivered 40 hits, of which 14 contained only halogen substituents: all four FdX 2 , two FdX 3 , three FdX 4 , FdCl 6 , FdBr 9 and FdBr 10 ; FdCl 2 was determined twice and FdI 4 exists as two positional isomers; 'Fd' is a common abbreviation for ferrocenes with substituents on both rings, while 'Fc' symbolizes ferrocenes with substituents only on one ring; strictly speaking, 'Fc' stands only for the [C 10 H 9 Fe] residue, while 'Fd' symbolizes a [(C 5 H 4 ) 2 Fe] group. The four 1,1 0 -dihaloferrocenes have been discussed already with respect to supramolecular interactions in general and halogen bonding in particular (Shimizu & Ferreira da Silva, 2018  Top view of the molecular structure of compound 3 (major orientation), with displacement ellipsoids drawn at the 30% probability level.

Figure 3
Top view of the molecular structure of compound 5, showing a whole molecule, with displacement ellipsoids drawn at the 30% probability level.

Figure 4
Top view of the molecular structure of 8, with displacement ellipsoids drawn at the 30% probability level. compounds, except for FdI 2 , FdBr 9 and FdBr 10 , showed eclipsed conformations (or nearly eclipsed in the case of FdBr 3 and FdI 3 , with torsion angles of ca 14 ). Within this group of eclipsed structures, most showed the apparent 'higher energy' conformations I and IV, respectively. Only FdBr 3 , FdI 3 and FdI 4 crystallized in the most stable form VI. Table 2 gives an overview on these structures.
Compound 3 crystallizes in the triclinic space group P1, with one molecule in the asymmetric unit. Fig. 2 shows the major orientation of the disordered molecule. As in most polyhaloferrocene structures (see Table 2), the cyclopentadienyl (Cp) rings are nearly perfectly eclipsed, planar and parallel to each other. All Br atoms are shifted slightly to the distal side of the Cp rings with respect to the Fe atom.
Compound 5 also crystallizes in the triclinic space group P1, however, as a twin with half a molecule in the asymmetric unit and the Fe atom residing on an inversion centre (Fig. 3). As a consequence of this, the Cp rings are perfectly staggered, with the two C-H bonds in relative transoid positions. Both Cp rings are planar and parallel to each other and the Br atoms are all shifted to the distal sides of the Cp rings, however, to a smaller extent than in the eclipsed structures mentioned before. The iron-centroid distance (determined within PLATON) also seems to be more dependent on the relative orientation of the Cp rings than on the degree of bromination. Table 3 collects important geometrical parameters of several polybromoferrocenes from the literature, together with those of compounds 3, 5 and 8.
Compound 8 crystallizes in the monoclinic space group P2 1 /n, with one molecule in the asymmetric unit (Fig. 4). The C10-Hg1-Br10 bond deviates slightly from being linear [171.0 (2) ]. The Cp rings are planar and parallel to each other, while their relative orientation is staggered. The distances from Fe1 to both Cp ring centroids are identical within 1. Except for atoms Br5 and Hg1, which are within the Cp ring planes, all the ring substituents are shifted again to the distal sides of the Cp rings. In comparison with the structure of the ferricenium salt 8 + ÁAsF 6 À , the C-Br bonds are slightly longer, Hirshfeld surfaces of compounds 3 (left), 5 (middle) and 8 (right), together with the closest contact atoms. Red spots show very close contacts between atoms inside and outside the Hirshfeld surface. The Hg compound differs from the other two by the appearance of such a red spot over the plane of the Cp ring. while the iron-centroid distances are significantly shorter in 8, which is quite usual when comparing ferrocenes with their oxidized counterparts (Rupf et al., 2022). For all three compounds, an analysis with PLATON showed no residual solvent-accessible voids (Spek, 2020).

Hirshfeld analysis and intermolecular contacts
To gain some insight into the intermolecular interactions at work in these compounds, a Hirshfeld analysis was undertaken, using the program CrystalExplorer (Spackman et al., 2021). Fig. 5 shows the Hirshfeld surfaces of the three compounds, together with the closest contact atoms (within 3.8 Å ).
The so-called 'fingerprint plots', which summarize all contacts between atoms inside and outside the Hirshfeld surface (Spackman & McKinnon, 2002;Spackman & Jayatilaka, 2009), are shown in Fig. 6. A common feature of all three plots is the occurrence of a red stripe around the main diagonal, reaching from ca d e /d i = 1.8/1.8 to 2.2/2.2, which corresponds to a large number of BrÁ Á ÁBr contacts (d e + d i = 3.6 to 4.4 Å ; the sum of the van der Waals radii of two Br atoms is 3.70 Å ).
The very different appearance of these plots is mainly due to the decreasing number of H atoms present. Table 4 and Due to purely statistical effects (there are eight H atoms in FdBr 2 , four in 3, two in 5 and none in 8), the absolute numbers cannot be compared directly. However, it is quite obvious that the importance of CÁ Á ÁH and especially HÁ Á ÁH contacts decreases drastically with increasing bromine content, while the importance of BrÁ Á ÁBr contacts increases in the same direction. At the same time, it appears quite interesting that HÁ Á ÁBr contacts are very important in all compounds where H atoms are present.
To obtain a more detailed picture of the individual interactions, a Mercury analysis was undertaken (Macrae et al., 2020) 3.3.1. Hydrogen bonds. The structures of the known polyhaloferrocenes collected in Table 2 Table 3 Important geometrical parameters of compounds 3, 5 and 8 in comparison with literature data for closely related compounds.
FdBr 2 is 1,1 0 -dibromoferrocene; 'Ct' is the abbreviation for the 'centroid' of the Cp rings, as calculated by the corresponding feature in PLATON (Spek, 2020); (Br-Cp) is the distance of the Br atoms from the Cp plane.
When using the standard settings of Mercury, no hydrogen bonds are indicated for compound 3. However, when increasing the limit by 0.2 Å , four (obviously very weak) hydrogen bonds appear [ Fig. 7 (left) and Table 5].
As can be seen from Fig. 7 (left), the atom pairs H14/Br11 and H24/Br21 connect the individual molecules in the y direction, while the pairs H15/Br13 and H25/Br23 join them in the x direction. Atoms Br12 and Br22 are not involved in hydrogen bonding.
In compound 5, the standard settings of Mercury suffice to show that only atom H5 (and, of course, its inversionsymmetry-generated counterpart H5 0 ) engages in a symmetrical bifurcated hydrogen bond (Type A in Scheme 2) with atoms Br2 and Br3 [ Fig. 7 (right) and Table 5]. As the figure shows, these interactions join individual molecules in the y direction.

Halogen bonding and other BrÁ Á ÁBr interactions.
In the discussion of halogen bonding, a distinction is usually made between XB Type I and XB Type II. According to the IUPAC and IUCr classifications, Type I contacts are geometry based, arising from close-packing requirements, while Type II arise from interactions between an electron-rich region on one halogen atom and an electron-deficient region on the other. The distinction can be made on the basis of the angles Â1 and Â2, which occur at halogen atoms X and X 0 of R-XÁ Á ÁX 0 -R 0 , and their difference (Scheme 3).
Usually it is assumed that for 0 < |Â1 -Â2| < 15 , a Type I contact is formed, while for Type II contacts, 30 < |Â1 -Â2| < 105 is found, and only the latter are regarded as real halogen bonds (Cavallo et al., 2016). In a more recent article, a distinction into Types I-IV was suggested, but was still based on these angles (Ibrahim et al., 2022): Type I: 90 < Â1 ' Â2 < 180 ; Type II: Â1 = 180 and Â2 = 90 ; Type III: Â1 ' Â2 = 180 ; Type IV: Â1 ' Â2 = 90 (obviously, Types III and IV are only extrema of the more general Type I). The forces behind these attractions are either van der Waals (Type I), electrostatic (Type II) or dispersion (Types III and IV) forces. It was further found that 'the Type I interactions were more frequent at the shortest distances' (Cavallo et al., 2016). Table 6 Table 4 Individual contributions (%) of the different interactions present in the crystal structures of FdBr 2 , 3, 5 and 8.

Figure 8
Packing plot of compound 3, viewed along c. Colour codes as defined by Mercury: carbon dark grey, hydrogen light grey, iron orange and bromine brown; the red lines are unexpanded contacts and the cyan lines are expanded contacts. Table 5 Hydrogen-bond parameters (Å , ) in compounds 3 and 5. the structural parameters of compounds 3, 5 and 8, while Figs. S9-S11 show Mercury representations of these interactions. All the listed BrÁ Á ÁBr contacts in Table 6 are well below the sum of the van der Waals radii (3.70 Å ; Bondi, 1964). When using the |Â1 -Â2| criterion, most interactions classified as Type II are also 'real' halogen bonds. To look at the structural consequences of this halogen bonding, a visualization of the packing plots should be helpful. Fig. 8 shows how the BrÁ Á ÁBr interactions join the individual molecules of compound 3 in the direction of the xy diagonal (b-a vector). It can also be seen that there are two intramolmolecular BrÁ Á ÁBr contacts of Type IV, emphasized in italic in Table 5. Two Br atoms (Br12 and Br22) are not involved in BrÁ Á ÁBr interactions. Fig. 9 shows that in compound 5 the BrÁ Á ÁBr contacts join the individual molecules in the z direction. All eight Br atoms are involved in BrÁ Á ÁBr interactions. In addition, there is also somestacking in the x direction; the centroids of two adjacent Cp rings are only 3.773 Å apart, while the ring planes have an interplanar distance of 3.507 Å (corresponding to an angle of 21.6 between the Ct-Ct 0 vector and the plane normal).
In bromomercurio compound 8, matters are a bit more complicated. Fig. 10 shows that BrÁ Á ÁBr contacts join the individual molecules in all directions. All Br atoms, except for Br2, Br5 and Br8, are involved in BrÁ Á ÁBr contacts.

Figure 9
Packing plot of compound 5, viewed along b. Colour codes as defined by Mercury: carbon dark grey, hydrogen light grey, iron orange and bromine brown; the red lines are unexpanded contacts and the cyan lines are expanded contacts.

Figure 10
Packing plots of compound 8, viewed along a (left) and along b (right). Colour codes as defined by Mercury: carbon dark grey, hydrogen light grey, iron orange and bromine brown; the red lines are unexpanded contacts and the cyan lines are expanded contacts.
interactions (Schmidbaur & Schier, 2015) can be excluded. In the crystal of the ferricenium complex 8 + ÁAsF 6 À , there is also a Hg 2 Br 2 ring with significantly shortened intermolecular HgÁ Á ÁBr contacts of 3.061 Å and a HgÁ Á ÁHg distance of 3.993 Å (Rupf et al., 2022). Furthermore, there are BrÁ Á Á contacts of 3.543 Å to a close Cp ring, in addition to a weak interaction between two Cp rings (Fig. 12);stacking occurs between two inversion-related C 5 Br 5 rings. Since the difference between the Ct-Ct 0 distance of 3.756 Å and the perpendicular distance between the Cp ring planes (3.690 Å ) is rather small (corresponding to an angle of 10.8 between the Ct-Ct 0 vector and the plane normal), it can be regarded as a 'true'interaction (though rather weak).

Co-operativity between HÁ Á ÁBr and BrÁ Á ÁBr contacts.
The importance of co-operativity in noncovalent interactions in general (Mahadevi & Sastry, 2016) and for the interplay of halogen and hydrogen bonds (Decato et al., 2021;Portela & Ferná ndez, 2021) in particular has been recognized in recent years and has been modelled by DFT calculations. This interplay has also been discussed for the 1,1 0 -dihaloferrocenes (Shimizu & Ferreira da Silva, 2018). In the preceding sections, we have discussed the individual contributions in compounds 3 and 5, and a look at Fig. 13 (and Tables 4 and 5) shows that also in these compounds HB and XB work together on the same halogen atoms.

Energetics of the intermolecular interactions found
in compounds 3, 5 and 8. The program CrystalExplorer allows for the calculation of interaction energies using the DFT program TONTO at the HF/3-21G level (Mackenzie et al., 2017). Fig. 14 shows the results of calculations for compounds 3 and 5 (apparently, due to the presence of Hg, the program cannot calculate wavefunctions for compound 8).
Inspection of the numerical values shows that the total interaction energies are stronger for compound 3. This is apparently due to the larger repulsion terms for 5, because both the largest dispersion and the largest electrostatic terms are found in compound 5. Another graphical representation ('energy frameworks') of the individual contributions can be seen in Fig. 15.

sigma-hole interactions
Acta Cryst. (2022). C78, 578-590 Blockhaus and Sü nkel Crystal and molecular structures of polybromoferrocene 587 Figure 11 The Hg 2 Br 2 ring in compound 8. Colour codes as defined by Mercury: carbon dark grey, hydrogen light grey, iron orange and bromine brown; the red lines are unexpanded contacts and the cyan lines are expanded contacts. Generic atom labels without symmetry codes ahve been used.

Figure 12
Partial packing diagram of compound 8, showing the BrÁ Á Á andcontacts (Å ). Colour codes as defined by Mercury: carbon dark grey, hydrogen light grey, iron orange, mercury blue and bromine brown; the red lines are unexpanded contacts and the cyan lines are expanded contacts. Ct1 0 /Ct1 00 and Ct2 0 / Ct2 00 are the centroids of inversion-related cyclopentadienyl rings, with one Fe atom between Ct1 0 and Ct2 0 , and another between Ct1 00 and Ct2 00 .

Comparison with halogen bonding in other halofer-
rocenes FdX n with X 6 ¼ Br and n > 2. At this point, it seems worthwhile to look at the occurrence of halogen bonding in the other polyhaloferrocenes mentioned in Table 2. As mentioned already, this study has been performed for the 1,1 0dihaloferrocenes before, and therefore these structures will sigma-hole interactions 588 Blockhaus and Sü nkel Crystal and molecular structures of polybromoferrocene Acta Cryst. (2022). C78, 578-590

Figure 13
Co-operativity of hydrogen and halogen bonding in compounds 3 and 5. Colour codes as defined by Mercury: carbon dark grey, hydrogen light grey, iron orange and bromine brown; the red lines are unexpanded contacts and the cyan lines are expanded contacts.

Figure 14
Interaction energies (HF/3-21G) for compounds 3 (left) and 5 (right) (standard program settings). The colour codes in the images refer to the tables below them. not be considered here again. Instead, the structure of the homoannularly substituted pentabromoferrocene (FcBr 5 ; Sü nkel & Bernhartzeder, 2011) is included (Table 7). All the listed XÁ Á ÁX contacts are below the sum of the van der Waals radii and of Type II except for the chloro compound (0.004 Å longer than this sum and Type I). This result (the increasing importance of XÁ Á ÁX contacts when going from X = Cl to X = I) parallels the observations in the FdX 2 sytems. In addition to the XÁ Á ÁX interactions, C-HÁ Á ÁX hydrogen bonds are important for all compounds, especially the chloro compound.
interactions are very strong for FdI 4 (virtually no displacement of the Cp rings of different molecules), while in FdCl 4 , the shift between the perpendicular projection of one centroid to the centroid of a neighbouring molecule is quite substantial. In FdI 3 , C-HÁ Á Á interactions seem to be of some importance, while in FcBr 5 , a weak C-BrÁ Á Á interaction can be observed.

Table 6
Characteristics of the BrÁ Á ÁBr interactions found in compounds 1, 3, 5, 8 and FdBr 2 . io)ferrocene. Hexabromoferrocene shows an eclipsed conformation of the Cp rings, as was also found for the already known structures of 1,1 0 -dibromo-and 1,1 0 ,2,2 0 -tetrabromoferrocene. Ferrocenes with a higher bromine content apparently prefer a staggered conformation, as was observed before for nona-and decabromoferrocene. All three title compounds show a combination of halogen bonding with either hydrogen bonding or interactions. Dispersion interactions appear to be stronger than electrostatic interactions. Table 7 Characteristics of the XÁ Á ÁX interactions in FdCl 4 , FdI 3 , FdI 4 and FcBr 5 .  where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max = 0.001 Δρ max = 1.63 e Å −3 Δρ min = −1.24 e Å −3 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes.