A Nanostructured Cu(II) Coordination Polymer Based on Alanine as a Trifunctional Mimic Enzyme and Efficient Composite in the Detection of Sphingobacteria

This research raises the potential use of coordination polymers as new useful materials in two essential research fields, allowing the obtaining of a new multiartificial enzyme with the capacity to inhibit the growth of bacteria resistance. The fine selection of the ligands allows the design of a new 2D coordination polymer (CP), with the formula [Cu2(IBA)2(OH2)4]n·6nH2O, by the combination of Cu (II) as the metal center with a pseudoamino acid (H2IBA = isophthaloyl bis β-alanine). Quantitative total X-ray fluorescence (TXRF) analyses show that the obtained CP can gradually release Cu (II) ions. Additionally, this CP can be nanoprocessed and transformed into a metal-organic gel (MOG) by using different Cu (II) salt concentrations and the application of ultrasounds. Considering its nanometric dimensions, the slow Cu (II) release and its simple processability, its performance as an artificial enzyme, and its antibacterial ability were explored. The results obtained show the first nanocoordination polymer acting as an artificial multienzyme (peroxidase, catalase, and superoxodismutase) exhibiting antibacterial activity in the presence of hydrogen peroxide, with selective behavior for three bacterium strains (S. spiritovirum, A. faecales, and B. cereus). Indeed, this CP shows a more robust inhibition capacity for Sphingobacterium. Going beyond that, as there are no comfortable and practically clinical tests capable of detecting the presence of Sphingobacteria, the compound can be easily embedded to form moldable gelatin that will facilitate the handling and low-cost commercial kits.


Introduction
Pathogenic intracellular bacteria have been proven to trigger numerous chronic or recurrent infectious diseases that pose substantial global public health threats. e main medical route for treating bacteria is antibiotics, but these microorganisms use many mechanisms to survive by becoming resistant [1]. For this reason, the discovery and development of alternative antimicrobial strategies are also critical [2][3][4][5][6]. Additionally, in the last decades, researchers have tried intensively to find new compounds capable of behaving as artificial enzymes, copying the functions of natural enzymes [7]. ese artificial enzymes will be able to produce essential chemicals on an industrial scale with a performance that rivals their natural counterparts. Moreover, if we are inspired by the use that the human body makes of enzymes to improve its defense mechanisms, see, for example, the xanthine oxidase that can generate superoxide anion (O − 2 ) and H 2 O 2 in the presence of O 2 , or the myeloperoxidase that can catalyze the conversion of H 2 O 2 into highly reactive oxygen species (ROS) that behave as natural antimicrobials, then artificial enzymes could also be used as alternative compounds in the fight against bacteria [8]. Investigations into these two subjects cover an immense variety of materials [9,10]. However, coordination polymers can be a possible solution to both aspects since they have fascinating characteristics and properties to achieve these objectives. From the chemical point of view, a recently published precedent shows how coordination polymers (CPs) can be highly interesting in manufacturing mimic enzymes with, at the same time, antimicrobial capacity as they can generate ROS in a controlled way [11]. Indeed, one exciting advantage of the CPs as possible artificial enzymes is their facile preparation, nanoprocessability, low cost, and superior stability [12]. Other advantages of the CPs are their tuneability, since the proper selection of the building blocks (metal ions and organic ligand/s) should allow the obtaining of a material with the desired properties [13][14][15][16]. us, the use of copper (II) as a metal center enables the creation of compounds with different antibacterial mechanisms [17][18][19]. One can be related to the metal ion release in a controlled manner, favoring obtaining compounds that improve wound healing without generating toxicity. e metal ion release is related to the electrostatic attraction between the positively charged Cu(II) or Cu(I) ions and microorganisms negatively charged cell membranes, resulting in bacterial cell death by damaging the cell wall and plasma membrane [17]. Indeed, considering this type of strategy, A. Lauf et al. have proved that the copper (II) release rate is crucial in antibacterial effectiveness, finding that this efficacy can be improved as the particle size is reduced [20,21]. Indeed, considering particle insertion into bacteria as a possible antibacterial mechanism, it has been demonstrated that Cu(II) coordination polymer nanofibers are more effective than the same compound in the form of microcrystals [17]. ese research studies show that the synthesis of new Cu(II)-based structures with control in the Cu (II) ion release rate is essential [11,17,19,22]. Additionally, the presence of this ion is critical to provide peroxidase [23], catalase, or superoxodismutase (SOD)-like activity. Organic ligands are also crucial to obtaining compounds of biological interest that when prepared with the appropriate synthetic conditions can generate metal-organic gels (MOGs) with three-dimensional network structures capable of harboring solvents. ese new phases have been highly interesting for real industrial applications since they facilitate the localized superficial administration of metal ions as Cu (II) in the antibacterial treatment of external wounds [24,25]. Other studies show that creating new composite materials by a combination of Cu (II) CPs and organic matrices such as polyacrylamide, cellulose, fiber, gelatin [26], chitosan (CS), or cotton [27][28][29][30], among others [31], provides improvements in antibacterial effectiveness in addition to new mechanical properties allowing their manufacture.
Despite these important discoveries, there are just a few examples of CPs combining multiple enzymatic actions, although none acting as a trifunctional mimic enzyme [32][33][34]. Here we present a study that combines Cu (II) with a pseudoamino acid (H 2 IBA � isophthaloyl bis β-alanine).
e synthesis in a single step and sustainable conditions (water and 25°C) allows obtaining a 2D CP that can be nanoprocessed and gelled by sonication of different Cu (II) metal salts. e obtained CP, with the chemical formula [Cu 2 (IBA) 2 (OH 2 ) 4 ] n ·6nH 2 O shows slow Cu (II) release and is the first example of a trimimic artificial enzyme having peroxidase, catalase, and superoxodismutase like activity, being effective for the resistant Sphingobacterium. In addition, the easy preparation of the corresponding metalorganic gel and also a composite material based on gelatin and the nanocoordination polymer (NCP) will allow the creation of easy-handling and low-cost commercial kits valid for detecting Sphingobacterium [35,36].

Chemical and Morphological
Characteristics of 1n. An essential advantage in the synthesis of this CP is the possibility of obtaining it in different phases and sizes by slightly modifying the synthetic conditions. at is, the direct reaction between H 2 IBA deprotonated with NaOH and Cu(II) can generate compound 1 as single microcrystals, as polycrystalline, as a colloid formed by nanofibers (1n), or as metal-organic gel (1n@MOG) depending on the starting copper (II) salt (Cu(NO 3 ) 2 or CuSO 4 ) and the application of different sonication times, as shown in Figure 1 (methods described in Supplementary Materials) [37]. e characterization of 1, 1n, and 1n@gelatin by IR ( Figure S1), PXRD ( Figures S2 and S3), and SEM ( Figure S4) has been carried out.
Structurally speaking, compound 1 is a 2D coordination polymer that crystallizes in the monoclinic Pc space group [38]. Its structure has been described previously by S. Lymperopoulou  As ultrasounds are used to assist sol-gel transitions [39,40], a bottom-up approach using sonication and CuSO 4 instead of Cu(NO 3 ) 2 results in the formation of a metalorganic gel (1n@MOG) which is transformed into a colloid formed by the corresponding nanocrystals (1n) with longer sonication times (Figure 1 (c) and (d)). As shown in Figure S4, the use of CuSO 4 instead of Cu(NO 3 ) 2 seems to enhance the reduction of particle size. e morphology and dimensions of both 1 and 1n were studied by SEM and AFM (Figures 3 and 4). e average width for 1 ribbons and 1n nanoribbons is 354 ± 170 nm and 170 ± 60 nm, respectively, while the average height for 1n is 17 ± 10 nm.
Compound 1n is stable at physiological pHs (between pHs 3 and 8) for long periods of time (    and S7). Additionally, Figure S8 shows thermal stability up to 254°C. It loses all the solvation water molecules at this temperature, and the coordinated ligands decompose mainly into CO 2 . At temperatures above 100°C, the blue color of compound 1n reversibly transforms into a new less crystalline green color phase due to the loss of the first solvated water molecules (Figures S9-S11). e magnetic properties of 1n show the presence of a metamagnetic behavior with a high critical field of ca. 5.5 T at 2 K, i. e., the Cu (II) dimer shows a weak antiferromagnetic coupling of −6.0 cm −1 that becomes ferromagnetic for applied magnetic fields above 5.5 T, as depicted in Figures S12 and S13.

Catalase Mimicking Activity of 1n.
Catalase is responsible for the catalytic decomposition of hydrogen peroxide through its disproportionation reaction into nontoxic dioxygen and water. e catalytic activity of 1n (methods in Supplementary Materials) [37] towards the decomposition of hydrogen peroxide has been investigated in water at 25°C. Initially, the suspension of 1n is dark blue, but after adding 30% (v/v) hydrogen peroxide, it became faded yellow and rapid evolution of gas was observed, such as in Figures 5(a)-5(d). It can be easily understood that the evolved gas is oxygen, and it comes from the catalytic decomposition of hydrogen peroxide solution [41].
Moreover, to quantitatively estimate the catalase activity of compound 1n, a colorimetric assay was performed by titration of excess unreacted H 2 O 2 . In this method, the decomposition of H 2 O 2 is estimated spectrophotometrically by a reaction with potassium dichromate/acetic acid reagent [42] (catalytic activity: methods in the Supplementary Materials). In the presence of H 2 O 2 , potassium dichromate in acetic acid is reduced to green-colored chromic acetate, which can be measured colorimetrically at 570 nm. As shown in Figure 5(e), when H 2 O 2 is added to the 1n, the absorbance signal decreases significantly from 1 to 0.43 after 2.5 hours of incubation. is result demonstrated that compound 1n presents catalase-like activity, directly proportional to the dissociation rate of the H 2 O 2 produced in the samples.

Peroxidase Mimicking Activity of 1n.
e excellent intrinsic peroxidase-like activity of 1n was evaluated by oxidizing chromogenic peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) to form a blue-colored product under the assistance of H 2 O 2 (catalytic activity: methods in Supplementary Materials) [37]. TMB itself is colourless and displays no absorbance ( Figure 6) [43]. As depicted in Figure 6(a), a strong absorption was observed in the group of TMB + 1n + H 2 O 2 , indicating that 1n possesses an excellent catalytic activity. Moreover, a time-dependent change in the TMB absorbance was recorded by a UV-vis spectrophotometer at 440 nm ( Figure 6(b)).

Superoxo-Dismutase (SOD) Mimicking Activity of 1n.
To determine if 1n can be a candidate as a mimic antioxidant enzyme useful, for example, in the treatment of disorders related to oxidative stress, we have studied its activity as a superoxodismutase (SOD) [34]. To evaluate this SOD-like activity, an assay with nitroblue tetrazolium (NBT) was performed to quantify the 1n enzymatic activity (catalytic activity: methods in the Supplementary Materials; Figures S14 and S15) [37]. e reaction kinetics between NBT and the xanthine/xanthine oxidase (x/xo) system were measured to establish when the absorbance reaches its maximum value at a wavelength of 560 nm. Triplicate NBT spectra were then performed with different concentrations of 1n in a stepwise manner (0, 0.005, 0.5, 1, 3, and 5 mg/mL), observing a continuous decrease of the signal as the concentration of 1n increases, which disappears entirely at a concentration of 3 mg/mL. Nearly 80% of the superoxides were removed from a concentration of 5 mg/mL of 1n, and IC 50 � 1.1 mg/mL was calculated (Figure 7) to establish the concentration of 1n that inhibits the rate of NBT reduction by 50%.

Antibacterial Assays.
To determine the copper concentrations produced when compound 1n is in suspension, release assays of the compound in deionized water were carried out ( Figure S16). Considering the system's slow Cu(II) release (45 ppm at 24 h), its nanometric dimensions, synthesis in the water a room temperature in just one step, and its ability to act as a multiartificial enzyme [11], its antimicrobial activity [44] was also explored. e antibacterial activity of 1n compound and 1n + H 2 O 2 was tested by agar diffusion against different microorganisms (antibacterial experiments in Supplementary Materials) [37] including Gram-positive (Bacillus, Deinococcus, and Lactococcus lactis) and Gram-negative (Escherichia, Pseudomonas, and Sphingobacterium, Alcaligenes). Furthermore, different concentrations of H 2 O 2 alone were checked against Sphingobacterium to discard the growth inhibition by oxygen peroxide's effect at the range of work concentrations, only observing a slight halo at 300 mM, as shown in Figure S17.
As well, the minimal inhibitory concentration (MIC) and growth curves of the compound 1n without and with H 2 O 2 versus different strains were assayed (Table S1 and Figure S18). e results obtained indicate that compound 1n did not show growth inhibition against any strain, which means that 1n is not an antimicrobial agent.
Most isolates from humans are Sphingobacterium spiritivorum which are generally resistant to kanamycin and ampicillin and are susceptible to the quinolones and trimethoprim-sulfamethoxazole.
Bacterial growth of Sphingobacteria was also measured by the optical density method at 600 nm (OD 600 ) overnight at 37°C. After 24 h in the presence of 1n + H 2 O 2, no bacterial growth was detected that performed as an antimicrobial agent (Table 1).
Possible changes in Sphingobacterium morphology were explored using FESEM. In Figure 9, the damages caused by the presence of 1n + H 2 O 2 upon 24 h to the bacterial surface were clearly observed regarding the control, probably due to the generation of ROS (O 2 , HO · ) that could oxidize the cell membranes as has been concluded using 3,3′,5,5′-tetramethylbenzidine (TMB) or catalase experiments [48].

Efficient Composite (1n@Gelatin) in the Detection of Sphingobacteria.
e prepared metal-organic gel of 1n (1n@ MOG) was not stable at physiological pH (only around pH     3). erefore, we have used gelatin as a biocompatible organic matrix in order to process 1n in a moldable and easy way creating a gel-like composite (Figure 10(a)). is new composite allows the release and diffusion of 1n during the incubation stage thanks to its excellent solubility in water at 37°C. With compound 1n in this format, we have achieved an improved manufacture and handling of the material, facilitating its application in vitro tests compared to bulk material. e antibacterial efficacy of 1n@Gelatin was checked against Sphingobacterium by the colony count method (antibacterial experiments in Supplementary Materials) [37]. e number of grown colonies of Sphingobacterium overnight at 37°C was 490 colonies, while in the presence of 1n@ Gelatin plus H 2 O 2 (25 mM) it was half (Figure 10(b)). From these results, the percentage of inhibition (K inhibition (%)) of 1n@Gelatin was evaluated versus Sphingobacterium and E. coli DH5α.
e results showed K � 57% for Sphingobacterium inhibition, while there was no decrease in the number of colonies for E. coli DH5α.

Conclusions
is research raises the potential use of coordination polymers as new useful materials in two significant fields of research, allowing the obtaining of new artificial enzymes with the capacity of inhibiting the growth of bacteria resistant to the usual antibiotics. e adequate selection of the ligands has enabled the design of the first artificial trienzyme model (peroxidase, catalase, and superoxodismutase) based on a Cu (II) CP with modified alanine. It is worth emphasizing that studies based on the search for enzyme mimics are focused on organic materials, but the study of artificial enzymes with CPs is practically nonexistent, despite the good characteristics that are present in CPs based on biologically relevant ligands. Remarkably, its excellent catalytic activity in the presence of minimal amounts of hydrogen peroxide is essential for its antibacterial effect on three resistant bacteria strains. Another critical aspect of this work is that Gram negative bacteria (Alcaligenes faecalis and Sphingobacterium) are usually involved in respiratory infection processes and present a vast antibiotic resistance, making this CP a powerful alternative for its use against these types of pathogens. e damages caused to the bacterial wall indicate indirect evidence of the generation of oxidizing hydroxyl species or reactive oxygen species due to the CP plus minimal concentrations of H 2 O 2 . Finally, the transformation of the CP into a metal-organic gel (MOG) could facilitate its use in the manufacture of new commercial Sphingobacteria detection kits. However, the MOG obtained does not present enough stability at physiological pH. For this reason, as a proof of concept, this compound has been homogeneously dispersed in soluble gelatin as an easily useable and moldable organic matrix with excellent antibacterial results which will allow the manufacture of lowcost, comfortable, easy-to-use, and affordable commercial Sphingobacteria kits for assessing the presence of microorganisms in a test sample employing the identification of the inhibition of the microorganism.
Data Availability e data underlying the findings of this paper are publicly available. All the obtained and used data are available and have been deposited in a public repository whose access link is: https://doi.org/10.21950/NR3KKL. Additionally, they are included in the article in the form of supplementary material (PDF file with the name: SI_bioinorg_chem_app_20_1_22). Supplementary Materials S1 experimental section: materials and instrumentation; S2 synthesis of 1, 1n, and 1n@Gelatin; S3 characterization of 1, 1n, and 1n@Gelatin; S4 solvent and thermal stability of 1n; S5 study of magnetic properties; S6 catalytic activity: methods; S7 copper release and antibacterial experiments; and S8 references. is material can be found in https://edatos.consorciomadrono.es/dataset.xhtml? persistentId�doi:10.21950/NR3KKL. (Supplementary Materials)