Base‐free Oxidative Esterification of HMF over AuPd/nNiO‐TiO2. When Alloying Effects and Metal‐support Interactions Converge in Producing Effective and Stable Catalysts

Furan‐2,5‐dimethylcarboxylate (FDMC), along with ethylene glycol (EG), is the key monomer to produce (poly‐(ethylene‐furanoate) (PEF). Noble metal‐based catalysts can convert hydroxymethyl furfural (HMF) to FDMC in methanol through liquid phase catalytic oxidative esterification. In this work, the catalytic performance of Au, Pd and AuPd NPs supported on nanosized nickel oxide (nNiO) have been evaluated under base‐free conditions at 90 °C and 3 bar O2. Synergistic effects between Au and Pd imparted high activity and higher yield to FDMC compared to the monometallic counterparts. The role of support was also investigated by depositing AuPd NPs on TiO2 and nNiO‐TiO2. Remarkable yield to FDMC (85 % after 8 h) and high stability were observed over AuPd/ nNiO‐TiO2 catalyst. This peculiar catalytic behavior could be imputed to the formation of trimetallic AuPdNi particles offering highly active interfacial sites.

Introduction
The plastics industry is growing at a rapid pace. Plastic production reached nearly 370 million tons on a worldwide basis in 2019, according to a recent analysis. [1] The ever-morewidespread use in daily life, along with population growth and market expansion have contributed to the continuously rising demand for primary plastics. In addition, new specialty plastics are continually being developed to meet the need for emerging specific technical applications. Currently, petroleum-based polymers are still predominantly used in the plastics industry (lowdensity polyethylene LDPE, high-density polyethylene HDPE, polypropylene PP, polyvinylchloride PVC, polystyrene PS and polyethylene terephthalate PET). [2] From a sustainable and green perspective, these polymers have been criticized for their environmental issues and hazards related to present disposal methods, oil-dependency and difficult degradation. However, replacement of petroleum-based plastics is a challenging process. Under the green and circular economy stimuli, the plastic industry has been looking at using biomasses and their derivatives as potential sources for monomers and polymers. [3,4] Poly(ethylene 2,5-furandicarboxylate) or polyethylene furanoate (PEF) represents a furan-based polyester that can be sourced from biomass and therefore it is a valid bio-based alternative to PET. The replacement of PET by PEF would lead to several advantages in terms of energy use, greenhouse gas (GHG) emissions, degradability (PEF films can undergo faster enzymatic hydrolysis compared to PET films). Furthermore, PEF possesses improved mechanical (e. g. tensile module) and thermal properties (high glass transition temperature), superior gas (O 2 , CO 2 and H 2 O) barrier properties than PET, and for this reason it is gaining growing attention for use in the food and beverage packaging industry. [3][4][5] PEF can be effectively obtained by condensation of ethylene glycol and 2,5-furandicarboxylic acid (FDCA) units. FDCA is a highly versatile and functional molecule that has been included among the twelve building blocks derived from biomass. Specifically, it can be easily synthesized through selective oxidation of hydroxymethyl furfural (HMF), a valuable platform chemical extracted from lignocellulosic biomass. [6] Heterogeneously catalyzed oxidation of HMF with molecular oxygen (O 2 ) or air is preferred over the use of stoichiometric oxidants or homogeneous catalysts (e. g. Co(OAc) 2 and Mn-(OAc) 2 in AMOCO process [7] ) for a more sustainable, energy saving and environmentally friendly process. [8,9] Almost quantitative yields to FDCA have been achieved over supported noble-metal (Au, Ru, Pd, Pt) nanoparticles as catalysts under relatively mild conditions in the presence of an alkaline medium. [10][11][12][13][14] Intensive research has been devoted to gain advanced insights on the reaction mechanism and to disclose the role of metal phase, [9,13,[15][16][17][18][19][20][21] particle sizes, [22] support chemistry [19,[23][24][25][26][27][28][29] and promoter addition [14,30,31] with the aim to optimize catalyst formulation and process conditions, with particular emphasis to the adoption of base-free conditions. [16,[32][33][34][35][36] Nevertheless, the large scale production of FDCA under environmentally benign conditions and its use as monomer for PEF synthesis is still hindered by some drawbacks associated with the large polarity, high boiling point, poor solubility of FDCA together with its tendency to undergo decomposition during the polymerization procedure, which results in a PEF product with undesirable color. An optimal solution could involve the replacement of FDCA by its corresponding dimethyl ester, i. e. furan-2,5-dimethylcarboxylate (FDMC), which can be directly employed in the fabrication of PEF by transesterification reaction with benefits in terms of cost-efficiency and technological solution. Indeed, FDMC can be synthesized by oxidative esterification of HMF in methanol, a solvent with higher volatility than water and then more easily separable in the course of polycondensation process. In addition, FDMC is characterized by good solubility in a large variety of solvents, relatively low boiling point and improved stability under polycondensation conditions, leading to a colorless PEF product. For all these reasons, the oxidative esterification to FDMC has recently moved into the limelight of research on chemo-catalytic processes for HMF upgrading to valueadded chemicals. Although non-precious metal based catalysts, in particular Co-based and Mn-based systems, have been successfully proposed in the literature, [15,18,[37][38][39][40][41][42][43][44] noble-metal based catalysts, specifically supported Au NPs, still remain the most studied catalysts for the oxidative esterification of HMF to FDMC. [10,26,[45][46][47][48][49][50][51][52][53][54][55] According to the literature, the reaction would proceed through an acetalization-oxidation-esterification mechanism (Scheme 1), although an alternative pathway involving alcohol oxidation to 2,5-diformylfuran (DFF) has been rarely reported. [18,37] The acetalization-oxidation-esterification mechanism implies the initial formation of a hemiacetal intermediate (A), which is unstable and cannot be detected by a gas chromatographic analysis. The hemiacetal can rapidly react following two different routes. In the former the hemiacetal is directly oxidized into the monoester alcohol (C), whereas the latter route entails the hemiacetal transformation into the acetal alcohol (2-furaldehyde-dimethyl-acetal, B), which may be then converted to the monoester alcohol (C). The alcohol function of C is then oxidized to form the monoester aldehyde (D), a highly unstable intermediate that rapidly undergo acetalization to form the hemiacetal ester (E), that, similarly to the hemiacetal A, can be directly converted to diester (FDMC, G) or undergo acetalization to acetal ester (F).
As in the case of the catalytic HMF oxidation to FDCA, the optimization of product distribution in the oxidative esterification requires a fine tuning and combination of several factors, including catalyst properties (catalyst support, particle size and shape, the active sites exposure) as well as process conditions (oxidant pressure, pH and HMF concentration). Unprecedented yield to FDMC can be achieved in alkaline media, as revealed by Christensen et al., [10] who first proposed the use of Au/TiO 2 as catalysts for HMF oxidative esterification attaining 99 % yield to FDMC. However, high yields to FDMC have been observed also under base-free conditions, as demonstrated by Corma et al. using Au/nCeO 2 catalyst. [49] In this case the combination of Au catalytic oxidative function and Lewis acid sites of ceria support promoted the reaction, which demonstrated to occur according to the pathway depicted in Scheme 1, where the oxidation of alcohol function was identified as the rate-determining step. Few years later, Manzoli et al. [50] performed tailored FT-IR measurements on Au/ZrO 2 which confirmed the hypotheses by Corma. According to Manzoli et al., the oxidative esterification over Au-based catalysts involved an oxygen sensitive and cooperative mechanism: the support promoted acetalization reaction while negatively charged Au clusters chemisorbed oxygen forming basic oxygen atoms which activated HMF and methanol molecules, by extracting H to originate alcoholate species. Furthermore, the same authors proved that functionalities and structure of the support also influence the shape of Au NPs with significant implications in the selectivity of the studied reaction. [51] More recently, Yuan et al. [52] reported remarkable yield to FDMC over optimized bimetallic AuCu/γ-Al 2 O 3 catalyst and ascribed such results to the presence of strongly interacting AuÀ CuO x interfacial sites which might act as the active sites. Synergistic effects were observed also in bimetallic AuPd NPs supported on Fe 3 O 4 , which demonstrated to be active and selective catalysts under alkaline conditions. [55] Hence, based on previous evidences on the outstanding performances of Au, Pd and AuPd NPs supported on nanosized nickel oxide (nNiO) in the base-free oxidation of HMF to FDCA, [36] this work investigates the catalytic performance of Au, Pd and AuPd NPs on nNiO, TiO 2 and mixed nNiO-TiO 2 supports in the base-free oxidative esterification of HMF, with the aim to unveil the synergistic effects deriving from the AuÀ Pd alloy formation and from metal-support interactions.

Results and Discussion
Monometallic (Au and Pd) and bimetallic (AuPd, with optimized internal Au : Pd molar ratio of 6 : 4) nanoparticles were deposited on nNiO, TiO 2 and nNiO-TiO 2 supports by sol-immobilization procedure. X-ray photoelectron spectroscopy (XPS) data were collected to understand the surface chemistry and oxidation states of the catalyst species.
Evaluation of Au 4f data in Au and AuPd samples revealed the presence of one specie with binding energy (BE) 83.2-83.9 eV, which can be attributed to the presence of metallic Au (Table 1). [56] Evaluation of the Pd 3d for Pd and AuPd samples data showed the presence of two Pd 3d 5/2 species with binding energies of 335.1-335.7 and 336.9-337.4 eV attributed to Pd 0 and to oxidized Pd δ + respectively (Table 1). In all samples Pd is mainly present in the metallic form (76-88 %) with a minority of oxidized Pd (12-24 %) probably due to surface oxidation from the reaction with atmospheric O 2 . [57] After the addition of gold a slight negative shift in Au 0 binding energy from gold foil (84.0) is likely due to the interaction of AuÀ Pd due to the formation and presence of AuPd alloy nanoparticles with respect to the monometallic Au sample. [58,59] On the contrary, after the addition of gold no modifications in the Pd 3d 3/2 BE were detected, except when NiO-TiO 2 was used as the support. In this case also a shift of~0.5 eV in Pd BE was envisaged. Moreover, in this sample, a higher content of Pd 0 in AuPd/nNiO-TiO 2 (88 % and 12 % for Pd 0 and Pd δ + , respectively Table 1) compared to the other AuÀ Pd catalysts (77-78 % and 22-23 % for Pd 0 and Pd δ + , respectively Table 1) The Ni2p XPS data collected for the nNiO based catalysts reveals the presence of three Ni species (Table 1, Figure S1). The low energy Ni species (~854.5) can be attributed to the NiÀ O bonds of nNiO. The peak centered at~856 eV is due to the formation of NiÀ OH, whereas the peak centered around 857.5 eV can be due to the formation of NiOOH. nNiO based catalysts showed similar Ni features, with the main presence of nNiO and NiOH, species. On the contrary, when nNiO was deposited on TiO 2 a higher contribution of Ni 3 + in the form of NiOOH was observed (27 %).
The morphology of the prepared catalysts was analyzed by transmission electron microscopy (TEM). The detailed analysis of Au/nNiO, Pd/nNiO, AuPd/nNiO and AuPd/TiO 2 is reported in previous works; [36,60] nNiO nanoparticles were present in the form of 3-5 nm, with the presence of large, ordered agglomerates with a fraction of needle-like particles.. [61] Au, Pd and AuPd are present as well dispersed nanoparticles in most cases, with a comparable particle size in the range of 3-5 nm (Table 1). Au/ nNiO showed mean particle diameter of 4.5 nm (Table 1, Figure S.2) whereas smaller nanoparticles with a mean diameter of 3.6 nm were obtained for Pd/nNiO (Table 1, Figure S.3). [36] TEM analysis of AuPd/nNiO sample showed the presence of nanoparticles with a mean diameter of 3.3 nm (Table 1 The EELS map confirms the presence of bimetallic nanoparticles with an enrichment of Pd around AuÀ Pd core particle. (S)TEM-EDS analysis confirms that all metal nanoparticles analyzed are alloy structures. [36] Alloyed PdÀ Au particles with a mean size of 3.5 nm was also observed in the case of AuPd/TiO 2 . [62] When nNiO was deposited on TiO 2 , nNiO forms as nano crystalline thin sheets (the curled/folded edges of the sheets appear as dark needles in the TEM image) whereas the TiO 2 is primarily single crystal B20 nm cuboidal particles of both anatase and rutile crystallite. [63] EELS analysis showed the high density of Nickel particles distributed over the Titanium -rich crystal ( Figure 1). Interestingly, the majority of the AuPd nanoparticles (average particle size of 3.2 nm) seems to be preferentially associated with the nNiO (Figure 1). EELS has been used to study the composition and nanostructure of bimetallic catalysts. The corresponding elemental map of Pd and Au reveals clearly the presence of both elements within the particle. Small amounts of Ni were found in the same region, probably partially covering the surface of the particle as can be seen in the elemental map ( Figure 2). EELS line profiles of PdÀ M5,4 AuÀ M4,5 and NiÀ L3,2 taken across the nanoparticle are shown above. It can be noted the presence of two pronounced Ni signals at the center of the Np, whereas Au and Pd signals appears continuously across the entire particle, suggesting an alloy structure. The formation of a AuPdNi trimetallic system can be responsible of an electron transfer from Ni to AuPd and the shift in Pd BE.
In a previous work, some of the authors have demonstrated that supporting Au, Pd and AuPd NPs on nNiO resulted in effective catalysts for the base free oxidation of HMF under relatively low temperature (90°C). In particular, AuPd/nNiO emerged for the remarkable yield to FDCA (70 %) and the significant stability. These encouraging results suggested ex- ploring the potential of these catalysts also in the base-free oxidative esterification of HMF. The catalytic performance in the oxidative esterification process was evaluated at 90°C and under 3 bar oxygen pressure. The main results in terms of activity and selectivity are summarized in Table 2.
Focusing on the metal catalysts supported on nNiO, it is worth noting that all catalysts were active under the adopted experimental conditions, despite the absence of an alkaline environment. This evidence proves that nNiO enhances the catalytic properties of noble metal nanoparticles conferring activity even in base-free conditions, in a similar way to nanosized-CeO 2 [49] and ZrO 2 . [50] The initial catalytic activity, defined as the HMF conversion rate per total moles of metal and calculated after 15 minutes of reaction, decreased in the order AuPd/nNiO (336 h À 1 ) > Pd/nNiO (280 h À 1 ) > Au/nNiO (168 h À 1 ). The same trend was observed in the final conversion    Figure 3a.
Looking at the selectivity after 8 h, as expected in all cases highly reactive hemiacetal products (A and E) were not detected, however important differences emerged in product distribution among Au, Pd and AuPd catalysts. In the case of Au/nNiO the diester (FDMC, G in Scheme 1) was the main product with a selectivity of 38.5 %, which corresponds to a yield of 30.8 %. This value is comparable to those obtained in the literature over Au/ZrO 2 and Au/sulf-ZrO 2 at 130°C (Table S.1). Together with FDMC, no negligible amount of acetal alcohol (B, 21 %) and acetal ester (F, 27 %) were observed, while only 10 % of monoester (C) was present. Conversely, the monoester C was the main final product (65 %) when Pd/nNiO was used as catalyst. In this case the acetal alcohol (B) and the diester (G) were the main co-products with a selectivity of 16 and 13 % respectively. A deeper insight on the selectivity and mechanism over Pd/nNiO may be gained by analyzing the evolution of product distribution as a function of the reaction time ( Figure 3c). Actually, a close inspection of selectivity profiles revealed that the diester (G) and the acetal ester (F) were the main products with selectivity values around 30-40 %, within the first of reaction, when 60 % of HMF is converted according to data in Figure 3a. After the first hour, HMF is still converted (albeit at lower conversion rate, as suggested by the decreased slope of the red curve in Figure 3a), but selectivity to diester and acetal ester declines, whereas the selectivity to monoester (C) increases. Such a behavior could be ascribed to a partial deactivation of Pd sites during the reaction. Indeed, it is known that the metal sites are responsible for oxidative steps, while the support promotes the acetalization reaction. Thus, it could be proposed that in the course of reaction, Pd active sites undergo deactivation and are no longer able to activate the alcohol function of the monoester C, which consequently cannot be further transformed to aldehyde ester (D), hemiacetal ester (E), acetal ester (F) and diester (G) and then accumulate, becoming the main detected product. The deactivation of Pd sites could be caused by metal leaching and overoxidation, as already reported in the literature for analogous Pd-based systems. [13,64] Interestingly, in the presence of AuPd/nNiO the sum of diester, FDMC (G), and acetal ester (F) constituted more than 95 % of the detected products with selectivity of 54 % and 42 %, respectively. The good selectivity to FDMC and the high activity of AuPd/nNiO highlighted the beneficial impact of the alloy effects. Actually, the addition of Au to Pd imparted good activity and high resistance to deactivation. Such a synergistic effect has been already observed in the literature and has been tentatively ascribed to both geometric (e. g. site isolation) and electronic effects. [65] Regarding the formation of acetals (B and F), it should be emphasized that the origin and the role of acetals in the mechanism of oxidative esterification is still debated in the literature. Indeed, although most of literature studies agree in ruling out the involvement of acetals as intermediates for the formation of mono-and diester, some recent reports [47] hypothesized that also acetals could undergo transformation to esters. However, product distribution profiles of Au/nNiO and AuPd/nNiO in Figure 3, b and d, showed that on these catalysts the selectivity to acetals remained stable over the time, while selectivity to diester increased, thus suggesting that acetals are stable byproducts. In any case, the formation of stable acetals over AuPd/nNiO affected in some extent the yield to diester and for this reason their origin under reaction conditions deserved to be further investigated with the aim to minimize their formation. Several factors have been reported to contribute to acetal formation, such as particle size (acetal is formed on Au with size d > 4 nm), [66] basic site depletion [45] and presence of strong Lewis acid sites. [50] Considering that metal nanoparticle size in the studied catalysts in < 4 nm, this factor can be excluded. The formation of acetal could be then associated with peculiar acid/base surface properties of nNiO. Indeed, according to previous reports, nNiO has a pronounced basic character with basic site density of 0.21 mmol g À 1 , as determined by liquid phase titration. [61] To unravel the role of the support in promoting acetal formation, AuPd NPs were supported onto TiO 2 and nNiO-TiO 2 (10 : 90 wt/wt ratio) supports and the resulting catalysts were tested in the base-free oxidative esterification of HMF. Initial activity data in Table 2 provided important information on role of support. On one side, AuPd/TiO 2 exhibited initial activity value (72 h À 1 ) five times lower than AuPd/nNiO (336 h À 1 ). On the other side a modest percentage of nNiO (10 wt.%) is sufficient to lead the activity to be twice as high (144 h À 1 ), thus confirming the peculiar catalytic behavior of nNiO. Interestingly, despite its lower initial activity compared to AuPd/nNiO, AuPd/nNiO-TiO 2 assured almost complete HMF conversion after 8 h (92 %). However, the most striking fact is the unique selectivity to FDMC (92 %) on AuPd/ nNiO-TiO 2 . Actually, the yield to FDMC was significantly higher over AuPd/nNiO-TiO 2 (85 %) than over the other bimetallic catalysts (Figure 4a).
Although higher values of yield to FDMC have been reported in the literature over Au-based catalysts (Table S.1), the result here obtained on AuPd/nNiO-TiO 2 is of great importance, since it has been obtained under mild conditions (absence of base, 90°C, 3 bar O 2 ). Moreover, recycling tests proved that AuPd/nNiO-TiO 2 is also highly stable and maintains initial activity, conversion and selectivity over six runs (Figure 3b). The good stability of AuPd/nNiO-TiO 2 was confirmed by additional TEM and XPS characterization on the catalyst after use. The analyses (Table S.2) demonstrated that the metal phase was not modified after testing, since original particle size and oxidation states were maintained. Slight difference was observed in the electronic state of Ni: fitting of the Ni2p 3/2 XP spectrum showed a decrease in the fraction of low energy Ni species at 854.5 eV (from 35 % to 26 %) and simultaneous increase (from 27 to 41 %) of the component at 857.5 eV associated with Ni 3 + À (O)OH species. The oxidation of Ni 3 + can be ascribed to O atoms origination from O 2 dissociation, as already reported in previous works. [61] The unique catalytic performance of AuPd/nNiO-TiO 2 could be rationalized by considering the information from XPS and TEM-EELS characterization. In particular, EELS analyses evidenced the formation of Ni-decorated AuPd nanoparticles AuPd/nNiO-TiO 2 . The presence of dispersed Ni-phase in intimate contact with AuPd surface might result in a greater number of interface sites, which are known to be active sites in the Aucatalyzed oxidative esterification. In addition, the nickel phase migrated at the surface of AuPd NPs could be associated with NiOOH species, which are predominant in AuPd/nNiO-TiO 2 according to XPS characterization. Such NiOOH species have been demonstrated to be highly active in the electrocatalytic production of oxygen from water [67,68] and have been also predicted to promote the oxidative dehydrogenation of alkane. [69] Additionally, the presence of NiOOH has been associated with high yield to methyl lactate in the fructose esterification reaction over nNiO catalysts. [70] Based on these considerations, the high activity and selectivity to FDMC of AuPd/nNiO-TiO 2 can be ascribed to the interfacial sites where the complete oxidative esterification of HMF is promoted by the co-presence of NiOOH sites facilitating the acetalization reaction and metal sites responsible for hemiacetal oxidation (Scheme 2).

Conclusion
Monometallic (Au, Pd) and bimetallic (AuPd) were successfully deposited onto nNiO through the sol immobilization procedure. The catalytic oxidative esterification of HMF with methanol was performed in base-free conditions at 90°C with 3 bar O 2 . The synergistic effect between Au and Pd substantially enhanced the initial activity (336 h À 1 ), improved the resistance to deactivation and influenced the product distribution.
Bimetallic AuPd NPs were also supported onto TiO 2 and nNiO-TiO 2 to study the effect of support.
Catalytic tests revealed that the support markedly influenced the catalytic performance. In particular, best results (85 % yield to FDMC after 8 h, stability over 6 runs) were attained over AuPd/nNiO-TiO 2 catalyst. XPS and TEM-EELS characterization allowed to explore the structure-activity relationships. Results suggested that the presence of highly dispersed Ni-phase (likely in the form of NiOOH clusters) over AuPd NPs might generate active interfacial sites, responsible for the unique catalytic performance of AuPd/nNiO-TiO 2 catalyst. Although further investigation by in situ characterization and theoretical simulations are still needed for a full understanding of the mechanistic aspects and of the structure of catalytically active sites, the present study on bimetallic AuPd catalysts for the oxidative esterification of HMF may provide some guidance for the preparation of active, selective and stable catalysts for biomass upgrading.

Experimental Section
Support preparation nNiO: nNiO was prepared according to the procedure described elsewhere. Ni(NO 3 ) 2 · 6H 2 O(5 × 10 À 4 M) (Sigma-Aldrich, purity > 99.9 %) and urea (Sigma-Aldrich, purity > 99.5 %) (Ni/urea molar ratio 1 : 10 mol/mol) were mixed into 0.2 L of MilliQ water under magnetic stirring. The solution was maintained under stirring for 6 h at 80°C. The precipitated Ni(OH) 2 was separated from the solution by filtration and washed several times. The powder was dried at 60°C for 12 h and then calcined at 300°C for 3 h in static air.

Monometallic catalyst preparation
Au-nNiO: Solid NaAuCl 4 · 2H 2 O (51 μmol,) and polyvinyl alcohol (PVA, 1 wt %) solution (Au:PVA mass ratio of 2 : 1 wt/wt) were introduced in 0.1 L of MilliQ water. After 5 min, a fresh solution of NaBH 4 (0.1 M) (Au:NaBH 4 1 : 4 mol/mol) was added to the solution under stirring, resulting in the rapid formation of Au(0) sol. Within a few minutes of sol generation, colloidal nanoparticles were immobilized by adding the support under vigorous stirring. The amount of support was calculated to achieve a total final nominal metal loading of 1 wt %, as verified by ICP analysis. After 2 h the slurry was filtered, and the catalyst was washed thoroughly with distilled water and dried at 80°C for 4 h. 1 : 8 mol/mol) was added to the solution under stirring and a Pd(0) sol was generated. Within a few minutes of sol formation, the colloid was immobilized by adding the support under vigorous stirring. The amount of support was calculated to attain a total final nominal metal loading of 1 wt %, as verified by ICP analysis. After 2 h the slurry was filtered, and the catalyst was washed thoroughly with distilled water and dried at 80°C for 4 h.

Bimetallic catalyst preparation
AuPd-nNiO, AuPd/TiO 2 and AuPd/NiO-TiO 2 . Solid NaAuCl 4 · 2H 2 O (31 μmol) and Na 2 PdCl 4 · 2H 2 O (20 μmol) (Au : Pd molar ratio of 6 : 4 mol/mol) and 1 wt % PVA solution (Pd:PVA mass ratio of 2 : 1 wt/wt) were added to 0.1 L of MilliQ water. After 5 min, a fresh solution of NaBH 4 (0.1 M) (Pd:NaBH 4 molar ratio of 1 : 8 mol/mol) was added to the solution under stirring. Within a few minutes of sol generation, the colloid was immobilized by adding the support under vigorous stirring. The amount of support was calculated as having a total final nominal metal loading of 1 wt %, as verified by ICP analysis. After 2 h the solid was recovered from the slurry by filtration, washed thoroughly with distilled water and dried at 80°C for 4 h. After washing, the intensity of C 1s signal significantly decreased ( Figure S5), confirming the almost complete removal of the protecting agent.

Catalyst characterization
TEM: Samples for transmission electron microscopy (TEM) studies were prepared by depositing small amounts of dry catalyst powder onto holey carbon copper grids. Micrographs combined with analytical studies by energy-dispersive X-ray (XEDS) and electron energy loss spectroscopy were performed in High Annular Dark Field mode using a FEI Titan3 80-300 microscope operated at 80 kV. Digital Micrograph, TIA, and INCA software's were used for the analysis of the TEM micrographs and XEDS spectra. The PSD, Gaussian fit, average particle diameter, and metal dispersion were calculated assuming a truncated cubooctaedron particle shape using Gauss software. EELS data including drift correction base on the zero-loss peak, background subtraction, and principal component analysis (PCA), were processed using Gatan's Digital Micrograph and Hyperspy software's.
XPS: X-ray photoelectron spectroscopy (XPS) data were collected using a PHI 3056 XPS with an Al anode source operated at 15 kV and an applied power of 350 W. The energy scale calibration of the instrument is checked using a sputter cleaned piece of Ag. The lowest C1s BE species were shifted about 0.1 eV to 284.8 eV for comparison to literature values. Surface concentrations are calculated by integrating the peaks area and using standard atomic sensitivity factors supplied by the equipment manufacturer. The spectra were deconvoluted using Gaussian-Lorentzian functions and a Shirley-type background.
Composition: Atomic Absorption Spectroscopy (AAS). The metal content was checked by AAS analysis of the filtrate on a Perkin Elmer 3100 instrument. After the catalyst filtration, 10 ml sample of the filtered solution was collected and analyzed.

Catalytic tests
The catalytic tests were performed in a lab scale autoclave reactor (100 mL capacity from Parr), equipped with a mechanical stirrer and sensors for measuring temperature and pressure. The solution of HMF (0.3 M, 25 mL in methanol) and catalyst (HMF/total metal molar ratio = 200) were loaded in the reactor. HMF (purity > 99 %) was purchased from AVABiochem and used without any purification.
The autoclave reactor was purged 3 times with O 2 and then pressurized at 3 bar. The temperature was increased to the set point (90°C) and the reaction mixture was stirred at 1200 rpm for the whole duration of the experiment. Initial time (time zero) for the reaction was considered when the set point temperature was reached (after 10 min). At the end of the reaction, the reactor was cooled down to room temperature and the solution was filtered. Samples were removed periodically (0.2 mL) and HP 7820 A gas chromatograph equipped with a capillary column HP-5 30 m × 0.32 mm, 0.25 μm Film, by Agilent Technologies. Authentic samples were analyzed to determine separation times. Quantitative analyses with external standard method (dodecanol) were used. Products were identified using gas chromatograph-mass spectroscopy (GC-MS). Aliquots of the reaction mixture were analyzed using a Thermo Scientific ISQ QD, equipped with an Agilent VF-5 ms column, 60 m × 0.32 mm × 1 μm (inner diameter thickness). Recycling tests have been performed by reusing the recovered catalyst without any further cleaning procedure.


The Synthesis of Carbon Black-Loaded Pt Concave Nanocubes with High-Index Facets and Their Enhanced Electrocatalytic Properties toward Glucose Oxidation

Catalysts with high catalytic activity and good stability are desirable in the electrocatalytic oxidation of glucose. Herein, Pt concave nanocubes with high-index facets (HIFs) supported by carbon black (Pt CNC/CB) are prepared through a hydrothermal method. The experimental results demonstrate that the peak current densities in different potential regions on the Pt CNC/CB anode are 0.22, 0.20, and 0.60 mA cm−2. The catalytic process of the glucose oxidation reaction is investigated in electrolytes with different pH values. Better stability is achieved by Pt CNC/CB than by Pt concave nanocubes (Pt CNCs). Abundant surface defects with low-coordinated atom numbers, such as steps, kinks, and edges, served as active sites in the electrocatalytic oxidation of glucose. With the addition of carbon black, the catalytic activity can be improved by facilitating the full exposure of the active surface defects on the HIFs of the Pt CNCs. Moreover, to address the aggregation of Pt CNCs, caused by the high surface energy of HIFs, the introduction of carbon material is an effective way to preserve the HIFs and thus enhance the stability of the catalyst. Hence, the prepared Pt CNC/CB electrocatalyst has great potential to be applied in the electrooxidation of glucose.

Introduction
As the main source of energy for human metabolism, glucose is generally present in body fluids. Many favorable features, including stability, safety, and abundance, make it a good choice of fuel for implantable glucose fuel cells [1][2][3]. Electricity can be converted by the oxidation of glucose at the anode and the reduction of oxygen at the cathode [4][5][6]. This kind of glucose fuel cell is a promising power supply for various implantable medical devices, such as pacemakers, sensors, artificial heart devices, and electrodes to treat neurological diseases [7]. It is regarded as an alternative to lithium batteries, which are difficult to miniaturize and are restricted by a limited lifetime [8]. However, the oxidation of glucose is a difficult step, which is affected by slow kinetics [9]. To overcome this problem, an efficient catalyst for glucose oxidation has been largely studied.
Abiotic catalysts, especially noble metals and their alloys, are suitable candidates that have good stability in the in vivo environment [10,11]. It is indicated that the electrocatalytic oxidation of glucose on platinum (Pt) single crystals is a structure-sensitive reaction [7]. The reactivity of Pt-based catalysts can be governed by varying the exposure of crystal facets, tuning the shapes, and controlling the sizes [12,13]. A large number of studies have demonstrated that nanocrystals (NCs) enclosed by high-index facets (HIFs) exhibit high catalytic properties due to the defect structures with low coordination atoms, such as steps, kinks, and edges [14][15][16], and Pt-based NCs with HIFs have been widely researched for many catalytic applications [17][18][19][20]. However, due to the active unsaturated atoms and the high surface energy, the phenomena of atom migration and aggregation can easily Nanomaterials 2022, 12, 3761 2 of 10 occur on the surface of Pt-based NCs enclosed by HIFs, which may result in surface restructuring and the attenuation of the surface activity [21]. Hence, maintaining the high activity and preserving the HIFs of Pt-based catalysts are key issues to be addressed. A suitable material that can provide a large surface area for Pt distribution is recommended to avoid aggregation.
Carbon materials as typical electrical conductivity supports have attracted extensive attention. The characteristic of the high specific surface area makes it an appropriate support for different catalysts [22,23]. Generally, loading Pt-based NCs on the carbon supports (graphene, carbon nanotubes, carbon black, etc.) is an effective strategy for enhancing the catalytic stability and promoting the dispersion of the catalyst [24][25][26][27][28][29]. Zhou et al. investigated the catalytic properties of high-index faceted Pt NCs supported on carbon black (HIF-Pt/C) for ethanol electrooxidation. The HIF-Pt/C catalysts exhibited 2-3 times higher electrocatalytic activity than the commercial Pt/C catalysts for ethanol oxidation reaction, which can be attributed to their high density of atomic steps [30]. Zhao et al. prepared three-dimensional (3D) flowerlike Pt nanoparticle clusters on multiwalled carbon nanotubes (MWCNTs) through an electrochemical method. The resulting Pt/MWCNTs in 3D morphology exhibited significantly higher electrocatalytic activity and better stability than the dispersive morphology in the glucose oxidation reaction and oxygen reduction reaction [31]. Although numerous studies on carbon-supported Pt-based catalysts with HIFs have been reported, to our knowledge, they have rarely been studied in the electrocatalytic oxidation of glucose.
In this study, Pt concave nanocubes (Pt CNCs) with HIFs supported by Vulcan XC-72R carbon black (CB) were synthesized as catalysts for the oxidation of glucose and labeled as Pt CNCs/CB. The surface morphology and structure of Pt CNC/CB were characterized. The catalytic performances in the glucose oxidation reaction were measured under neutral conditions. The effect of the typical Vulcan XC-72R CB support in Pt CNC/CB was evaluated. It is expected that the aggregation of Pt CNCs can be alleviated by the addition of a carbon support with a sufficient surface area. Therefore, the surface defect structures derived from the HIFs of Pt CNCs can be fully exposed, which are generally considered to be the active sites in catalytic reactions.

Synthesis of Pt CNC/CB Catalysts
The Pt CNC/CB catalyst was synthesized by using a facile hydrothermal method ( Figure 1). A specific amount of CB (3 mg), PVP (MW = 28,000, 800 mg), and glycine (150 mg) were added to deionized water with stirring. After that, 2 mL of H 2 PtCl 6 ·6H 2 O solution (40 mM) was added to the above solution and stirred at room temperature for a few minutes. Then, the as-prepared homogeneous solution was transferred to a Teflon-lined stainless-steel autoclave. The sealed vessel was held at 200 • C for 6 h before it cooled down to room temperature. The products were separated by centrifugation at 10,000 rpm for 15 min and were then purified by washing with ethanol three times. Nanomaterials 2022, 12, x FOR PEER REVIEW 3 of 10

Characterization
The surface morphology of the samples was characterized by transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) on a Tecnai G2F30 STWIN (FEI Inc., Hillsboro, OR, USA), operating at an acceleration voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) measurements were conducted on a Thermo ESCALAB 250 spectrometer (Waltham, MA, USA) using a monochromic Al Kα X-ray source. The functional group information was identified by Fourier transformed infrared (FT-IR) on a Thermo Fisher 6700 (Waltham, MA, USA).

Electrochemical Measurements
Electrochemical measurements including cyclic voltammetry (CV) and chronoamperometry (CA) were performed on an M204 electrochemical workstation (Metrohm Autolab, Utrecht, Netherlands). The CV tests were conducted in 0.1 M PBS (pH 7.4) solution with and without the addition of 50 mM glucose. The potential ranged from 0.079 to 1.679 V, and the scan rate was 20 mV s −1 . Before the electrochemical measurements, the activation of the electrode was conducted by fast CV scanning from 0.042 to 1.242 V in a 0.5 M H2SO4 solution. The electrochemically active surface area (ECSA) of the catalyst was calculated based on the CV curves, measured in oxygen-free 0.5 M H2SO4 solution from 0.042 to 1.242 V at a scan rate of 50 mV s −1 . The correlations between the CV scan rate, pH value, and the current density were investigated. CA measurement was performed at a potential of 1.279 V in 0.1 M PBS (pH 7.4) solution with the addition of 50 mM glucose. The ink for electrochemical measurements was prepared by adding the catalyst (5 mg) to ethanol (1 mL), followed by sonication for 30 min. A working electrode was prepared by loading the ink (3 μm) on a glassy carbon electrode (GCE) at 25 °C and then drying naturally. The saturated calomel electrode (SCE) and a Pt sheet (1 cm 2 ) were used as the reference and counter electrodes, respectively. All of the potentials mentioned in this article were converted to the reversible hydrogen electrode (RHE) via the equation E RHE = E SCE + 0.242 + 0.0591 × pH.

Physicochemical Characterizations
Information related to surface morphology can be provided by TEM measurement. As shown in the low-resolution TEM images for the Pt CNC and Pt CNC/CB samples (Figure 2a,d), uniform nanocubes were successfully synthesized at high yield. It was clearly observed that Pt nanocubes were uniformly dispersed on the CB support, in comparison with the pure Pt CNC sample without catalyst support. The addition of catalyst support can greatly improve the dispersibility of catalysts such that the loss of catalytic activity caused by the aggregation of active sites can be largely avoided [30]. Sufficient exposure of the active surface is beneficial to enhance the catalytic efficiency and improve

Characterization
The surface morphology of the samples was characterized by transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) on a Tecnai G2F30 STWIN (FEI Inc., Hillsboro, OR, USA), operating at an acceleration voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) measurements were conducted on a Thermo ESCALAB 250 spectrometer (Waltham, MA, USA) using a monochromic Al Kα X-ray source. The functional group information was identified by Fourier transformed infrared (FT-IR) on a Thermo Fisher 6700 (Waltham, MA, USA).

Electrochemical Measurements
Electrochemical measurements including cyclic voltammetry (CV) and chronoamperometry (CA) were performed on an M204 electrochemical workstation (Metrohm Autolab, Utrecht, Netherlands). The CV tests were conducted in 0.1 M PBS (pH 7.4) solution with and without the addition of 50 mM glucose. The potential ranged from 0.079 to 1.679 V, and the scan rate was 20 mV s −1 . Before the electrochemical measurements, the activation of the electrode was conducted by fast CV scanning from 0.042 to 1.242 V in a 0.5 M H 2 SO 4 solution. The electrochemically active surface area (ECSA) of the catalyst was calculated based on the CV curves, measured in oxygen-free 0.5 M H 2 SO 4 solution from 0.042 to 1.242 V at a scan rate of 50 mV s −1 . The correlations between the CV scan rate, pH value, and the current density were investigated. CA measurement was performed at a potential of 1.279 V in 0.1 M PBS (pH 7.4) solution with the addition of 50 mM glucose. The ink for electrochemical measurements was prepared by adding the catalyst (5 mg) to ethanol (1 mL), followed by sonication for 30 min. A working electrode was prepared by loading the ink (3 µm) on a glassy carbon electrode (GCE) at 25 • C and then drying naturally. The saturated calomel electrode (SCE) and a Pt sheet (1 cm 2 ) were used as the reference and counter electrodes, respectively. All of the potentials mentioned in this article were converted to the reversible hydrogen electrode (RHE) via the equation E RHE = E SCE + 0.242 + 0.0591 × pH.

Physicochemical Characterizations
Information related to surface morphology can be provided by TEM measurement. As shown in the low-resolution TEM images for the Pt CNC and Pt CNC/CB samples (Figure 2a,d), uniform nanocubes were successfully synthesized at high yield. It was clearly observed that Pt nanocubes were uniformly dispersed on the CB support, in comparison with the pure Pt CNC sample without catalyst support. The addition of catalyst support can greatly improve the dispersibility of catalysts such that the loss of catalytic activity caused by the aggregation of active sites can be largely avoided [30]. Sufficient exposure of the active surface is beneficial to enhance the catalytic efficiency and improve the catalytic activity of the catalyst. The average sizes of the nanocubes in Pt CNC and Pt CNC/CB were 31.06 ± 0.6 nm and 28.92 ± 0.5 nm, respectively, determined by the edge length (Figure 2c,f).
projected Pt CNC and the {100} facets of an ideal cube was 11°. The theoretical value of the angle between high-index planes with Miller indices (510) and the {100} planes was reported to be 11.3° [15,32]. From the comparison between the measured angles and the ideal values, it is suggested that the as-synthesized Pt CNCs are mainly enclosed by highindex (510) facets. It was reported that the high-index (510) facets can be indicated as [5(100) × (110)] [33]. As shown in Figure 2h,i, the atomic steps on the edge of Pt CNC can be seen clearly, which is consistent with the atomic arrangement of the (510) facets. Figure  2j is an atomic model of the (510) planes in the Pt CNC/CB catalyst, which was established to illustrate the arrangement of atomic steps directly. Such HIFs possess a high density of step atoms, which generally constitute the active sites of electrocatalysts [18].   [15,32]. From the comparison between the measured angles and the ideal values, it is suggested that the as-synthesized Pt CNCs are mainly enclosed by high-index (510) facets. It was reported that the high-index (510) facets can be indicated as [5(100) × (110)] [33]. As shown in Figure 2h,i, the atomic steps on the edge of Pt CNC can be seen clearly, which is consistent with the atomic arrangement of the (510) facets. Figure 2j is an atomic model of the (510) planes in the Pt CNC/CB catalyst, which was established to illustrate the Nanomaterials 2022, 12, 3761 5 of 10 arrangement of atomic steps directly. Such HIFs possess a high density of step atoms, which generally constitute the active sites of electrocatalysts [18].
XPS analysis of Pt CNC/CB was carried out to estimate the chemical state of elements. The binding energy of the C 1s (284.6 eV) was used as the standard for calibration. As shown in Figure 3a, the full survey spectrum showed major peaks for Pt 4f, C 1s, and O 1s at 71.0, 284.6, and 531.4 eV, respectively. The O element mainly originated from the air. The chemical nature of Pt 4f was further analyzed using the deconvoluted XPS spectra as shown in Figure 3b. The Pt 4f peak could be resolved into two pairs of peaks, identified as Pt 4f 7/2 and Pt 4f 5/2 . The peaks located at around 71.11 and 74.44 eV represent metallic Pt. The peaks at around 72.37 and 76.35 eV can be assigned to the Pt (II) in PtO or Pt(OH) 2 species [34,35]. The FT-IR spectra of Pt CNC/CB, PVP, and glycine are shown in Figure 3c. The strong characteristic peaks in PVP and glycine could not be observed in Pt CNC/CB, which demonstrates that the redundant PVP and glycine were removed completely after the successful synthesis of the Pt CNC/CB catalyst. XPS analysis of Pt CNC/CB was carried out to estimate the chemical state of elements. The binding energy of the C 1s (284.6 eV) was used as the standard for calibration. As shown in Figure 3a, the full survey spectrum showed major peaks for Pt 4f, C 1s, and O 1s at 71.0, 284.6, and 531.4 eV, respectively. The O element mainly originated from the air. The chemical nature of Pt 4f was further analyzed using the deconvoluted XPS spectra as shown in Figure 3b. The Pt 4f peak could be resolved into two pairs of peaks, identified as Pt 4f7/2 and Pt 4f5/2. The peaks located at around 71.11 and 74.44 eV represent metallic Pt. The peaks at around 72.37 and 76.35 eV can be assigned to the Pt (II) in PtO or Pt(OH)2 species [34,35]. The FT-IR spectra of Pt CNC/CB, PVP, and glycine are shown in Figure 3c. The strong characteristic peaks in PVP and glycine could not be observed in Pt CNC/CB, which demonstrates that the redundant PVP and glycine were removed completely after the successful synthesis of the Pt CNC/CB catalyst.

Electrochemical Analysis
The ECSA of catalyst is associated with the charges in both the adsorption and desorption processes of hydrogen [36]. The ECSA value is evaluated by integrating the peak area of the CV scanning, performed in a nitrogen-saturated 0.5 M H2SO4 solution with a scan rate of 50 mV s −1 . The charge was normalized using a surface area-specific charge of an ideal one-electron transfer, which is 210 μC cm −2 [37,38]. As shown in Figure 4a, the peak area of Pt CNC/CB was larger than that of the Pt CNC catalyst. The calculated ECSA value of the Pt CNC/CB catalyst was 2.80 m 2 g −1 , which was approximately two times higher than that of Pt CNC (1.56 m 2 g −1 ). The higher specific ECSA of Pt CNC/CB implies a greater dispersion of Pt CNCs, which is beneficial to the exposure of the HIFs of Pt CNCs. The HIFs have a high density of low-coordinated atoms, which can serve as highly active sites to break C-H bonds in glucose oxidation reactions. Figure 4b shows the catalytic performance of the Pt CNC/CB anode in the glucose oxidation reaction. The CV test was evaluated in 0.1 M PBS (pH 7.4) with 50 mM glucose electrolyte. A CV test in 0.1 M PBS (pH 7.4) solution without the addition of glucose was also conducted for comparison. As shown in Figure 4b, obvious peaks could be observed from the CV curve conducted in the 0.1 M PBS (pH 7.4) with 50 mM glucose electrolyte, which demonstrates the oxidation reaction of glucose. During the oxidation process, the glucose is first electrooxidized to gluconolactone and then hydrolyzed to gluconic acid [39,40]. The electrocatalytic oxidation of glucose can be characterized by the peaks at three potential ranges. The oxidation peak in the hydrogen region (0.15-0.35 V) demonstrates the adsorption of glucose molecules on the surface of the Pt CNC/CB anode. The bond

Electrochemical Analysis
The ECSA of catalyst is associated with the charges in both the adsorption and desorption processes of hydrogen [36]. The ECSA value is evaluated by integrating the peak area of the CV scanning, performed in a nitrogen-saturated 0.5 M H 2 SO 4 solution with a scan rate of 50 mV s −1 . The charge was normalized using a surface area-specific charge of an ideal one-electron transfer, which is 210 µC cm −2 [37,38]. As shown in Figure 4a, the peak area of Pt CNC/CB was larger than that of the Pt CNC catalyst. The calculated ECSA value of the Pt CNC/CB catalyst was 2.80 m 2 g −1 , which was approximately two times higher than that of Pt CNC (1.56 m 2 g −1 ). The higher specific ECSA of Pt CNC/CB implies a greater dispersion of Pt CNCs, which is beneficial to the exposure of the HIFs of Pt CNCs. The HIFs have a high density of low-coordinated atoms, which can serve as highly active sites to break C-H bonds in glucose oxidation reactions. Figure 4b shows the catalytic performance of the Pt CNC/CB anode in the glucose oxidation reaction. The CV test was evaluated in 0.1 M PBS (pH 7.4) with 50 mM glucose electrolyte. A CV test in 0.1 M PBS (pH 7.4) solution without the addition of glucose was also conducted for comparison. As shown in Figure 4b, obvious peaks could be observed from the CV curve conducted in the 0.1 M PBS (pH 7.4) with 50 mM glucose electrolyte, which demonstrates the oxidation reaction of glucose. During the oxidation process, the glucose is first electrooxidized to gluconolactone and then hydrolyzed to gluconic acid [39,40]. The electrocatalytic oxidation of glucose can be characterized by the peaks at three potential ranges. The oxidation peak in the hydrogen region (0.15-0.35 V) demonstrates the adsorption of glucose molecules on the surface of the Pt CNC/CB anode. The bond breaking between the hemiacetal carbon and the attached hydrogen atom is considered as the rate-limiting step in the glucose electrooxidation reaction [9,41,42]. In this step, both the glucose molecule and the hydrogen atom are adsorbed on the electrode surface through the Pt CNC/CB catalyst (Equations (1)-(3)).
6 10 6 2 6 12 7 A peak located at the potential over 1.1 V was observed, which is regarded as the oxygen region. In this region, the active OHads species on the surface of Pt CNC/CB are stripped and replaced by the Oads species, which exhibit less activity than OHads. The Oads adsorbed on the surface of the Pt CNC/CB catalyst facilitate the formation of the PtO layer, which can oxidize the glucose molecule directly (Equations (7) and (8)) [40]. The peak current densities measured on the Pt CNC/CB anode were 0.22, 0.20, and 0.60 mA cm −2 . It exhibited higher catalytic activity than Pt CNC and commercial Pt/C catalysts compared with our previous study [29]. The improved catalytic activity can be attributed to the addition of CB support, which largely improved the dispersity of the Pt CNCs and allowed adequate exposure of the active HIFs of Pt CNCs. In the double-layer region (0.4-1.0 V), as the applied potential increases, abundant OH ads species are formed through the fast decomposition of water. According to the 'Incipient Hydrous Oxide Adatom Mediator' model (IHOAM) proposed by Burke, the incipient hydrous oxide layer of OH ads is generated through a premonolayer oxidation step on the surface of active metals, which can mediate the oxidation of glucose [43,44]. The active OH ads layer is more likely to generate on the active sites with low lattice coordination values and disconnected areas such as the edges. Thus, the concave structure in the Pt CNC/CB catalyst is considered to be a favorable structure that can serve as an active site for the electrocatalytic oxidation of glucose (Equations (4)- (6)).
A peak located at the potential over 1.1 V was observed, which is regarded as the oxygen region. In this region, the active OH ads species on the surface of Pt CNC/CB are stripped and replaced by the O ads species, which exhibit less activity than OH ads . The O ads adsorbed on the surface of the Pt CNC/CB catalyst facilitate the formation of the PtO layer, which can oxidize the glucose molecule directly (Equations (7) and (8)) [40]. The peak current densities measured on the Pt CNC/CB anode were 0.22, 0.20, and 0.60 mA cm −2 . It exhibited higher catalytic activity than Pt CNC and commercial Pt/C catalysts compared with our previous study [29]. The improved catalytic activity can be attributed to the addition of CB support, which largely improved the dispersity of the Pt CNCs and allowed adequate exposure of the active HIFs of Pt CNCs. As shown in Figure 5a, a series of CV curves of the fabricated Pt CNC/CB anode were measured by varying the scan rate from 10 mV s −1 to 200 mV s −1 in a mixing solution of 0.1 M PBS (pH 7.4) and 50 mM glucose. Based on the CV curves, as the scan rate increased, the peak current densities presented an upward trend. Figure 5b shows the linear relationship between the current density of the oxidation peak and the square root of the scan rate, which reveals the typical diffusion-controlled electrochemical behavior on the Pt CNC/CB anode [11].  As shown in Figure 5a, a series of CV curves of the fabricated Pt CNC/CB anode were measured by varying the scan rate from 10 mV s −1 to 200 mV s −1 in a mixing solution of 0.1 M PBS (pH 7.4) and 50 mM glucose. Based on the CV curves, as the scan rate increased, the peak current densities presented an upward trend. Figure 5b shows the linear relationship between the current density of the oxidation peak and the square root of the scan rate, which reveals the typical diffusion-controlled electrochemical behavior on the Pt CNC/CB anode [11]. The catalytic performance of the Pt CNC/CB electrode toward glucose oxidation in the mixing solution of 0.1 M PBS and glucose was detected at different pH values. The pH values 6, 7, and 10 were selected on behalf of the acidic, neutral, and basic conditions. From Figure 5c, the CV curve detected in the solution of pH 6 exhibited the highest peak current density among all pH values in the hydrogen region, which can be associated with adequate hydrogen ions in the acidic electrolyte. However, in the double-layer region, the peak current density under the basic conditions was much more obvious than that in the The catalytic performance of the Pt CNC/CB electrode toward glucose oxidation in the mixing solution of 0.1 M PBS and glucose was detected at different pH values. The pH values 6, 7, and 10 were selected on behalf of the acidic, neutral, and basic conditions. From Figure 5c, the CV curve detected in the solution of pH 6 exhibited the highest peak current density among all pH values in the hydrogen region, which can be associated with adequate hydrogen ions in the acidic electrolyte. However, in the double-layer region, the peak current density under the basic conditions was much more obvious than that in the acidic and neutral solutions. In the basic environment, abundant OH − are likely to be adsorbed on the surface of the Pt CNC/CB electrode, which can serve as active sites for the glucose oxidation reaction. With the increase in the applied potential, the active OH ads are replaced by O ads in the oxygen region. The peaks observed in the basic and neutral electrolyte in the oxygen region were higher than those under the acidic conditions. Therefore, the electrocatalytic process of the Pt CNC/CB electrode varies with different pH values of the electrolyte [44].
The stability of the Pt CNC/CB and Pt CNC electrodes in the glucose oxidation reaction were further evaluated by CA tests at 1.279 V for 60 s. As shown in Figure 5d, the CA curves demonstrated a fast decrease initially and then became stable. After 60 s, the steady current densities of glucose oxidation on the Pt CNC/CB and Pt CNC electrodes were 0.061 mA cm −2 and 0.0062 mA cm −2 , respectively. This reveals that the Pt CNC/CB electrode exhibited higher activity and stability compared to the Pt CNC electrode. The loading of Pt CNCs on the CB catalyst support may be beneficial, effectively facilitating the exposure of the active HIFs of Pt CNCs.
Compared with the conventional Pt nanoparticle catalysts, the catalytic activity of Pt CNC was greatly improved by a high density of atomic steps provided by the HIFs on the surface of Pt CNCs, which served as active sites in the glucose oxidation reaction. During the catalytic process, the discontinuous structures with low coordination numbers are more likely to adsorb or bond with the glucose, OH ads , O ads , and the intermediates. The addition of the CB catalyst support effectively increased the dispersion of Pt CNCs, which facilitated the full exposure of the active structures. The sufficient exposure of active sites improved the catalytic efficiency and thus enhanced the catalytic activity of Pt CNC/CB. Moreover, the stability of Pt CNC/CB was strengthened by coating the Pt CNCs on the CB catalyst support with a large surface area, which can effectively avoid the agglomeration of the Pt CNCs.

Conclusions
In summary, a Pt CNC/CB catalyst was synthesized using a facile hydrothermal method. The prepared catalyst presented high catalytic activity and good stability for the electrocatalytic oxidation of glucose under neutral conditions. Reasons for the enhanced catalytic properties can be expressed as follows. First, the CB catalyst support provided a large surface area for the sufficient dispersion of Pt CNCs, which facilitated the full exposure of the active HIFs. Second, the good chemical stability and remarkable mechanical properties of the carbon material helped to improve the stability of the Pt CNC/CB catalyst. Third, the high densities of atomic steps, edges, and kinks derived from the HIFs of Pt CNC were favorable for boosting the electrocatalytic activity of the Pt CNC/CB catalyst. This work provides an alternative catalyst for further application in implantable glucose fuel cells and electrochemical biosensors, which are capable of power supply and glucose detection in the human body.


H2S/Thiols, NO•, and NO–/HNO: Interactions with Iron Porphyrins

In the past decade, gasotransmitters NO• and H2S have been thoroughly studied in biological contexts, as their biosynthesis and physiological effects became known. Moreover, an additional intricate crosstalk reaction scheme between these compounds and related species is thought to exist as part of the cascade signaling processes in physiological conditions. In this context, heme enzymes, as modeled by iron porphyrins, play a central role in catalyzing the key interconversions involved. In this work, iron porphyrin interactions with sulfide and nitric-oxide-related species are described. The stability and reactivity of mixed ternary systems are also described, and future perspectives are discussed.

■ INTRODUCTION
The discovery of its biosynthesis and the assertion of its physiological effects have made nitric oxide (NO • ) a biologically relevant species. In mammals, this molecule acts as a mediator in the cardiovascular, nervous, and immune systems, mainly via interaction with its natural receptor, soluble guanylyl cyclase (sGC). This is a heme enzyme, displaying an iron-bound porphyrin active site just like many enzymes of the nitrogen cycle which deal with NO-related species: nitric oxide synthase (NOS, responsible for endogenous NO • production), fungal CytP450 nitric oxide reductase (P450nor, which converts it to N 2 O), and more. Other small nitrogen species also present biologically relevant reactivity, such as nitroxyl (NO − or HNO, the one-electron reduction products of NO • ), the nitrosonium cation (NO + ), and peroxynitrite (ONOO − ). In particular, nitroxyl has recently been established to produce  its own physiological effectsboth similar and distinct from those of NO • and though it could be produced from different metabolites, the question of its biosynthesis remains unsolved. 1 Biologically relevant sulfur species, on the other hand, consist mainly of thiols, as in cysteine (Cys, Figure 1a) and methionine (Met, Figure 1b) amino acids in proteins or as the antioxidant tripeptide glutathione (GSH, Figure 1c).
Despite the extent of studies regarding the biologically relevant reactivity of those sulfur-based ligands, across many subdisciplines from organometallics and bioinorganic chemistry to cluster science, studies on the hydrosulfide ion (HS − ) are significantly less developed. Biological roles for this ion, however, have revolved around its coordination with transition metal ions that produce a unique class of effects that differ from metal thiolates. Conventionally, dihydrogen sulfide (H 2 S) was considered toxic until more than 20 years ago when Kimura suggested its possible physiological role in the nervous system. Since then, there is evidence that H 2 S plays an important role in several mammalian processes, such as vascular contractility (and thus its function in blood pressure regulation) and neuronal activity, among others. 2 The endogenous H 2 S production in mammalian tissues is a consequence of the action of at least three enzymes: cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercaptopyruvate-sulfuryltransferase (MPST). The enzymes CSE and CBS use cysteine as the main substrate.
Both NO • and H 2 S are now considered major intracellular signaling molecules, part of the gasotransmitters family, along with carbon monoxide (CO). They are defined as endogenous, gaseous molecules whose metabolism is strictly regulated and have a defined biological role at low concentrations, although they are toxic at high concentrations. In particular, NO • and H 2 S display notable chemical reactivity because of their mutual involvement in numerous cellular processes. 3 After long speculation, the interaction between these two species has been demonstrated, and they were found to influence the production of each other as well as the expression and activity of the enzymes responsible for their biosynthesis. 4 In addition, the inhibition of vasorelaxant effects of NO • in the presence of H 2 S was attributable to a possible biochemical "crosstalk" between the two gases, even though other authors report that H 2 S reduces such activity. 4 As a result, interest in the possible link between H 2 S and NO • to better understand their biological functions has recently dramatically increased. It has also been suggested that nitroxyl could be endogenously generated via the reaction of these two gasotransmitters. 5 H 2 S is capable of transforming endogenous NO • to HNO, being involved in blood pressure and cardiac contractibility control. This is because HNO activates the HNO/transient receptor potential channel/calcitonin gene-related peptide cascade, via the formation of disulfide bonds, resulting in continuous calcium influx.
Reaction of H 2 S with S-nitrosothiols (RSNOs) forms thionitrous acid (HSNO), which is best described as a mixture of rapidly interconverting key intermediate isomers: SNO − (thionitrite) and perthionitrite (SSNO − ). HSNO is liposoluble and capable of crossing cell membranes and causing further trans-nitrosation of proteins. It is reactive in different competitive modes, with a half-life of a few seconds at pH 7.4 toward the homolytic cleavage of the N−S bond, and can be deprotonated at pH ≈ 10, giving SNO − . Moreover, it was proposed that NO • and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, each with in vitro and in vivo bioactivity ( Figure 2). 6 The reaction of thiols with NO-related species has also been studied. S-nitrosothiols are formed by the reaction of NO + with thiols (RSH) and are involved in nitric oxide storage and transport. They undergo transnitrosation reactions, in which NO + is transferred between different nucleophiles: HSNO can even act as an NO + shuttle between proteins in different cellular compartments. 4 This compound has also been shown to decompose to HNO by unclear pathways. Additionally, HNO can also be produced by reaction of thiols directly with NO • . In the reaction with cysteine, the first step comprises a proton-coupled nucleophilic attack of the thiol toward NO • , with an effective bimolecular rate k eff = 30 M −1 s −1 . 7 The direct reaction between NO • and H 2 S has been described as eq 1, and the observed rate constant for HSNO •− production (eq 1)

HS NO HSNO
(1) As has already been hinted, in biological systems, metalloproteins are deeply involved in the reactivity of all these small molecules. In this context, their relation with heme proteins, especially relevant for nitric-oxide-related species, is particularly interesting. Although the crosstalk between NO • and H 2 S in the presence of iron complexes has already been addressed, 3,4,8 in this work, we will focus on describing the biologically relevant reactivity of sulfide and nitrosyl species with hemeprotein models. We will revise the reactivity of inorganic sulfide species and nitric oxide derivatives with synthetic porphyrins acting as heme models, comment on research efforts focused on ternary systems involving Fe porphyrins, sulfide species, and NO • derivatives, and finally expose our thoughts and perspectives on this promising, albeit complicated, bioinorganic study topic.

■ REACTIVITY OF INORGANIC SULFIDE SPECIES
TOWARD IRON HEME PLATFORMS The coordination chemistry of H 2 S and its conjugated base to heme proteins is diverse. The interaction of hemoglobin (Hb) and myoglobin (Mb) with H 2 S in the presence of O 2 results in covalent modification of the heme periphery to generate sulfmyoglobin and sulfhemoglobin, respectively. 9 In the absence of O 2 , on the other hand, it is known that the reaction of H 2 S toward some heme proteins can form stable Fe III −sulfide complexes, as in the case of hemoglobin I of Lucina pectinata (HbI LP), a clam inhabiting sulfide-rich environments. In this case, the protein binds sulfide and forms a hexacoordinated low-spin complex with a very high affinity constant. 10 Under anaerobic conditions, more cases of sulfidecoordinated heme proteins have been detected and kinetically characterized in buffered aqueous solutions. 11 Additionally, it is also known that some heme proteins are reduced by sulfide. 9 The stabilization of the binding of sulfur species to ferric heme proteins has therefore been widely discussed in terms of the role of neighboring amino acids or other functional structures at the distal and proximal sites, the polarity of the local environment around the heme center, the accessibility of sulfur species to the heme active site, and many variables that cannot be isolated in the protein matrix. To isolate protein factors governing redox chemistry, model compounds are used as representatives of heme proteins. However, biorelevant sulfide coordination chemistry with heme model compounds is scarce, as will be described as follows. In general, free ferric porphyrinates are reduced by inorganic sulfide in aqueous solution, but in certain cases, sulfide binding is favored, as in the example cited in ref 10. In organic media, ferrous sulfide complexes have been   [Fe II (OEP)(SH)] − and [Fe II (T-p-OMePP)(SH)] − complexes were obtained, as well as that of the mixed-ligand species [Fe II (Por)(Im)(SH)] − . On the other hand, the attempt to obtain ferric complexes was not successful since sulfide reduced all ferric porphyrinates, resulting in the same products observed with ferrous porphyrins. 12 However, a low-spin Fe(III)−SH intermediate could be recently identified during the reduction of ferric porphyrins by sulfide at low temperature in dichloromethane, providing early evidence of this kind of compounds. 13 The reactivity of the hydrosulfide ion HS − toward several synthetic ferrous meso-tetraphenylporphine (TPP) derivatives has been examined. In all cases, HS − was found to bind to fivecoordinate high-spin ferrous centers following a 1:  14 In a further study, a picket-fence porphyrin model (TPivPP, (All-cis)-5,10,15,20-tetrakis[2-(2,2-dimethylpropionamido)phenyl]porphyrinate, Figure 3f) was used, and it was found that the sterically restricted porphyrin does not bind the neutral H 2 S species in either the ferric or ferrous state. In contrast, reaction of Fe III (P) with HS − reduces iron and eventually binds to the ferrous forms. 15 The sulfide binding affinities were similar to those obtained with nonhindered porphyrins, and so they concluded that the extra stabilization is related to kinetic rather than thermodynamic effects. The authors also mention the significance of hydrogen bonding interactions in facilitating reversible interaction between sulfide and ferric hemes. All mentioned porphyrin hydrosulfide complexes are shown in Figure 3. Hydrosulfide binding to semisynthetic heme systems has been investigated using bis-N-acetyl-microperoxidase undecapeptide (NAcMP11), a heme peptide derived from cytochrome c proteolysis that retains proximal histidine bound to Fe(III). Addition of H 2 S to a pH 6.8 solution of Fe III (NAcMP11) revealed the formation of a moderately stable low-spin ferric sulfide complex. The results suggest that delayed metal reduction is possible with the assistance of proximal mechanisms as a minimum requirement. 17 A joint study combining experimental and theoretical tools showed that both H 2 S and HS − species are capable of binding in the model system Fe III (MP11), where access to the active site is only restricted by the presence of a water molecule, and in Fe III (Mb) where the access of ligands to the active site is restricted by the topology of the protein matrix. HS − binding is favored over H 2 S, if the active site is accessible ( Figure 5). 12 AND NO − /HNO Due to their biological significance as models for heme enzymes of the nitrogen cycle, Fe-NO porphyrin complexes are of high interest, and have been thoroughly studied over the years. 18 Since the NO ligand is redox active and noninnocent, it is not always easy to establish the oxidation states involved, and so these complexes can be more conveniently represented as {FeNO} n using the Enemark-Feltham notation. Here, the Fe-NO fragment is considered as a covalent unit with n electrons, corresponding to the sum of electrons present in the metal's d orbitals and the π* orbital of the NO ligand. 18 For iron complexes, the limiting descriptions of {FeNO} 6 ,  Fe(II)−HNO complexes, in this context, also with n = 8, may be represented as {FeHNO}. 8 This notation is particularly useful because it allows us to predict the Fe−N−O angle merely as a function of n, as shown in Figure 6 for the {FeNO} 6/7/8 complexes of Fe II (OEP). 18 {FeNO} 6 Porphyrin Complexes. {FeNO} 6 complexes are biologically relevant since they constitute reactive intermediates in the enzymatic mechanisms of different proteins such as nitrophorins and P450nor. 18 These complexes show almost linear Fe−N−O angles and are preferentially described as Fe(II)−NO + species. This makes the NO fragment electrophilic and susceptible to nucleophilic attack by hydroxide, hydride, alkyl groups, and thiols. Historically, synthetic complexes of this kind were considered kinetically unstable, as they tend to lose NO • to give free ferric porphyrinates much faster than protein Fe(II)−NO + species. However, it has recently been established that the observed instability is caused by halide traces or other kind of impurities and is not an intrinsic property of these complexes. 18 In this context, the electrophilic reactivity of {FeNO} 6 porphyrin complexes with physiologically relevant species, such as thiols and other sulfur compounds, is an interesting feature to be explored.
In fact, reaction of the hexacoordinated {FeNO} 6 porphyrin complex [Fe(OEP)(NO)(5-MI)][OTf] (5-MI = 5-methylimidazole, OTf = triflate) with borohydride resulted in the formation of the corresponding {FeHNO} 8 complex, making this an interesting strategy for preparing this elusive kind of species, which are described below. 19 Interestingly, if the starting complex is pentacoordinated, hydride binds to the axial position instead.
Another special property of the {FeNO} 6 species is the lack of the so-called trans labilization effect, and so the Fe−L This reactivity is key for biologically relevant mechanisms, such as the fast activation of sGC. In this process, binding to NO • promotes the labilization of a proximal histidine, which in turn activates the enzymatic response. These complexes are common intermediates in enzymatic mechanisms of the nitrogen cycle. 21 Their general stability means that these species have to be activated, by a Lewis acid or via radical− radical coupling, for example, in order to be reactive in physiological conditions. 18 {FeNO} 6 and {FeNO} 7 porphyrin complexes have been the subject of numerous studies and reviews, and the focus will be put on the less explored {FeNO} 8 and {FeHNO} 8 species.
{FeNO} 8 and {FeHNO} 8 Porphyrin Complexes. As the biological activity of nitroxyl, the reduced form of NO • , became known, the interest of bioinorganic chemists in characterizing and studying the reactivity of its elusive coordination compounds has significantly increased. However, due to their tendency to reoxidize to their {FeNO} 7 counterparts, not many examples have been reported for {FeNO} 8 and {FeHNO} 8 model complexes (Figure 7). In fact, the most stable {FeHNO} 8 complexes have been obtained directly in protein environments, with myoglobin and other globins. 22 These species were stable for weeks and allowed  The presence of a protective scaffold is key in preventing the decay of {FeHNO} 8 complexes, as shown by the remarkable stability of a sterically hindered bis-picket fence porphyrin, shown in Figure 7c, whose half-life extended up to 5 h. 23 A bimolecular reaction with hydrogen production was proposed as the main decomposition pathway of these species (eq 4): This pathway had been proposed in early works, in which the first {FeNO} 8 porphyrin complexes were prepared by electrochemical reduction of the nitrosyl complexes of OEP and TPP. 18 Evidence for the {FeHNO} 8 adduct of OEP (Figure 7a) was later obtained in the presence of phenols acting as weak acids, 24 while a different preparation scheme involving hydride attack on the corresponding hexacoordinated {FeNO} 6 complex could even provide NMR and IR characterization. 19 In 2019, the {FeHNO} 8 complex of the water-soluble porphyrin TPPS (Figure 7d) could be obtained by chemical reduction of the {FeNO} 7 complex and characterized by UV− vis, decomposing completely only after a few minutes. 25 The fact that this species could be observed at room temperature using an unhindered porphyrin suggested that other factors apart from steric protection are relevant in the stabilization of these complexes, such as the ability to form strong hydrogen bonds with water. Interestingly, the decomposition reaction did not follow the expected bimolecular pathway but was first order in the complex. DFT calculations suggested a new possible decay route involving hydrogen atom migration to a porphyrin meso-carbon to form a transient phlorin intermediate. This species would rapidly react to give the starting {FeNO} 7 complex (Figure 8).
The nonprotonated {FeNO} 8 complex could also be obtained, and the pK a for the coordinated HNO could be estimated as 9.7 from spectroscopic and electrochemical experiments.

■ NO−SULFUR INTERPLAY WITH FE−PORPHYRINS
In the context of biologically relevant interactions, porphyrin complexes of NO • (or sibling molecules) and sulfur species are of special relevance. The RS−Fe−NO arrangement is commonly found since many heme proteins involved in the nitrogen cycle rely on thiols as the sixth axial ligand. 28 Nitric oxide synthase, the enzyme responsible for NO • biosynthesis, shows a deprotonated cysteine in the proximal position, which also binds the heme site to the protein structure. The same scheme is observed in nitric oxide reductase P450Nor (which reduces NO • to N 2 O) and other enzymes including certain kinds of nitrite reductase and nitrophorins. This is no coincidence since coordination to electron-rich sulfur species allows stabilization of oxidated iron states and subsequent substrate activation via a push ef fect. For example, during the enzymatic NOS production of NO • from arginine, the proposed Fe(IV)−O highly oxidizing intermediate is stabilized by electron donation from the cysteinate ligand. A similar situation is seen in the enzymatic cycle of P450Nor, in which an alleged Fe(IV)−NHOH key intermediate is formed from an Fe(II)−HNO complex. The double protonation of this complex can be achieved because of the enhanced basicity of the heme ligand, again due to the electron-donating ability of the trans-thiolate residue. 20 Moreover, in these complicated protein systems, linkage to thiolate species is found to be stabilized by hydrogen bonding interactions with neighboring amino acids, which also play protective roles and are central to fine-tuning their reactivity.
Given all this context, the study of RS−Fe−NO porphyrin model systems arises as a very valuable tool for examining the influence of all these factors in enzyme reactivity and broadening our understanding of the underlying mechanisms through inorganic chemistry techniques. In laboratory conditions, these systems are elusive, unstable, and not easy to characterize, and so there are not many examples in the literature. Although a few protein {FeNO} 6 thiolate-bound species could be prepared, 29 model complexes remained elusive.
The reactivity toward NO • (g) of a ferric heme thiolate complex, in which the thiol moiety is covalently linked to  porphyrin substituents and protected by bulky groups, was the first to be explored ( Figure 10). 30 The resulting {FeNO} 6 complex was characterized by UV−vis and EPR, and its conversion to the {FeNO} 7 form could be achieved by sodium borohydride reduction. NO • binding was reversible, and the reaction rate depended on the coordinating nature of the solvent, three times faster in toluene than in methanol, where a coordinated solvent molecule needs to be removed. This behavior correlates well with the reactivity observed for heme protein CytP450Cam and emphasizes the importance of the fine-tuning ability of the protein environment, which can determine whether a vacant position will be available or not.
A bent {FeNO} 6 thiolate complex was the firstand yet, onlyof its kind to be structurally characterized by reaction of crystalline Fe III (OEP)(thiolate) with NO • gas ( Figure 11). In solution, however, reaction led to the formation of the corresponding {FeNO} 7 complex and free thiolate. 31 Interestingly, hydrogen bonding between the thiolate sulfur and neighboring hydrogen atoms is evidenced: a very similar situation is observed in crystalline P450Nor and NOS, in which the Fe−S−R bond is stabilized by hydrogen bonding between the sulfur atom and HN moieties from nearby amide peptide groups and tryptophane residues, respectively.
Considering that a {FeNO} 6 complex is formed in the first step of the CytP450Nor enzyme, Lehnert and co-workers studied the reaction of different heme thiolate complexes with NO • gas in the hope of better understanding the functioning of this naturally occurring system and methodically evaluating the influence of the thiolate ligand. 18 In 2019, they reported the UV−vis characterization of a series of {FeNO} 6 complexes prepared by reaction of the corresponding heme thiolate precursors with nitric oxide, only achievable at very low temperatures ( Figure 12). Notably, for thiolates (1), (2), and (3), the spectral features of the products were similar to those observed in protein complexes. At −80°C, the complexes were stable for more than 2 h, but the authors observed that above −60°C the complexes inevitably start decomposing to give pentacoordinate {FeNO} 7 . The syntheses of the precursor [Fe(TPP)(SR)] complexes are also described in their work.
The electron-poor thiophenolates shown in Figure 12 were initially selected via DFT analysis as the best models for the local electronic structure of CytP450nor and were used as axial donors for FeTPP. Their reactivity proved to be sensitive to the electron-donating differences throughout the series. For instance, when thiols (4), (5), and (6) within this group were employed, NO • was also observed to react directly with the thiol to form nitrosothiols, giving only partial coordination to the Fe center. Not surprisingly, for the most donating thiol (7), this was the only observed outcome.
The complexes and their decomposition products were further characterized by FT-IR and Raman spectroscopy, and interestingly, a correlation was found between the donating strength of the thiolate ligands and the Fe−NO and N−O stretching frequencies in the thiolate {FeNO} 6 models. Less   electron-donating thiols resulted in higher stretching frequencies, in agreement with stronger bonds. This finding confirms the σ-donating effect of the thiol moiety and implies, as previously proposed, that fine-tuning of the electronic environment of heme thiolate proteins results in different degrees of activation of the FeNO fragment. DFT studies also showed that, accordingly, Fe−N−O stretching frequencies can act as probes for establishing the donating strength of a given thiolate ligand.
The effect of the proximal hydrogen bonding network is key since it also influences the donating properties of the thiolate ligands in Nature. This influence was the subject of an additional study with FeTPP, in which the intramolecular Hbond strength was modulated using different amide-based thiolates, shown in Figure 13. 32 Again, a correlation was found between H-bond strength and Fe−NO and N−O stretching frequencies: as expected, stronger H bonds resulted in weaker thiolate donation and stronger Fe−NO and N−O bonds (higher vibration frequencies).
The comparison between two thiolates with similar electrondonating properties, but differing in their H-bond availability, showed that the presence of H bonds helps stabilize the thiol moiety toward reactivity with other small molecules (such as NO • , which would result in nitrosothiol formation) and protonation. Moreover, the effect of multiple weak H bonds as can be found in a proximal protein environmentturns out to be comparable to the effect of a single stronger bond.
The reaction between {FeNO} 7 complexes and thiolates is even less explored. The formation of a weak Fe−S bond (as compared to Fe−N bonds for imidazole-like axial ligands) was evidenced after the reaction of the ferrous {FeNO} 7 model Fe(TPP)NO with both thiolates and thioethers, only at low temperature. 33 The resulting complexes could be characterized by EPR spectroscopy and showed a more pronounced radical character of the NO • fragment. The tetrahydrothiophene (THT) complex could be characterized by FT-IR and UV−vis spectroscopy at 183 K in solution and in the solid state.
There are no reports on the reactivity of sulfide species toward porphyrin {FeNO} 8 and {FeHNO} 8 complexes. However, it has been shown that iron porphyrins mediate nitrite reduction to HNO in the presence of H 2 S, in aqueous media. 34 In a later study, NO • and HNO production was verified in mitochondria (rich in heme proteins) by incubation with nitrite and H 2 S. 4 The reaction of a water-soluble ferric porphyrin with varying ratios of sulfide and nitrite was evaluated, resulting in the formation of {FeNO} 7 complexes, evidencing nitrite reductase activity from the iron porphyrin. This implied the formation of a ferrous porphyrin complex, which could be detected when H 2 S/NO 2 − was 2:1, and sulfide acts as the reducing agent. When the ratio was increased to 5:1, an intermediate ferric Fe(P)(SH) complex was detected by EPR, along with HS • formation from its decomposition to give again the ferrous porphyrin with the catalytic properties. When sulfide was present in 10-fold excess over nitrite (as physiological conditions are thought to be), HNO and N 2 O were also detected, making H 2 S a strong candidate for being responsible for endogenous nitroxyl generation from nitrite reduction.
Most remarkably, when sulfide was added to the ferric nitrosyl (formed from the reaction of the ferric porphyrin with nitrite), a reaction intermediate was detected, whose identity could be assigned as a rare Fe−HSNO complex by mass spectrometry at −20°C. This again evidences the possibility of a nucleophilic attack by a sulfide species on the electrophilic {FeNO} 6 fragment and posed a probable biosynthetic pathway for the strong nitrosating agent HSNO. The authors conclude their investigation by showing the generation of S-nitrosothiols in cells incubated with nitrite and hydrogen sulfide. A reaction scheme for all the involved processes is shown in Figure 14, which is adapted from ref 4.

■ PERSPECTIVES
The overall complex reactivity between iron porphyrins, NOrelated species, thiols, and hydrogen sulfide described throughout this work is represented in Figure 15. The chemistry involving these species is often particularly challenging since it involves the generation and manipulation of reactive and sensitive species, making their study not a straightforward task.
Although a great deal of progress has been achieved in this subject during the last decades, these systems are far from being understood, and many reactions remain to be studied. For example, there are very few examples of the reactivity between {FeNO} 7 porphyrin complexes and sulfur species in general, and in particular, there are almost no reports exploring the reactivity of water-soluble nitrosylated porphyrins with H 2 S, HS − , and thiols.
In this regard, the reactivity of the elusive {FeNO} 8 and {FeHNO} 8 nitroxyl porphyrin complexes toward hydrogen sulfide and related species is a completely unexplored subject. Taking advantage of the relative stabilization of these complexes in water using the [Fe(TPPS)] 3− complex, the study of their reactivity toward biologically relevant sulfur species arises as an exciting challenge which we are committed to undertake in the near future in our laboratory.
Probably, a good starting point would be to study the reaction between sulfides and iron porphyrins coordinated to nitroxyl. Also, isolation of the RSNO/porphyrin compounds and the study of their reactions and decomposition products will certainly give important data to understand the elusive {FeNO} 8 /RSH chemistry.
All in all, the rich interplay seen in solution and gas studies between NO and sulfur species shows a promising platform for research which will remain fruitful for years to come.

Notes
The authors declare no competing financial interest.