Porphyrin–Nanodiamond Hybrid Materials—Active, Stable and Reusable Cyclohexene Oxidation Catalysts

: The quest for active, yet “green” non-toxic catalysts is a continuous challenge. In this work, covalently linked hybrid porphyrin–nanodiamonds were prepared via ipso nitro substitution reaction and characterized by X-ray photoelectron spectroscopy (XPS), fluorescence spectroscopy, infrared spectroscopy (IR) and thermogravimetry-differential scanning calorimetry (TG-DSC). The amine-functionalized nanodiamonds (ND@NH 2 ) and 2-nitro-5,10,15,20-tetra(4-triﬂuoromethylphenyl)porphyrin covalently linked to nanodiamonds (ND@ β NH-TPP p CF 3 ) were tested using Allium cepa as a plant model, and showed neither phytotoxicity nor cytotoxicity. The hybrid nanodiamond–copper(II)–porphyrin material ND@ β NH-TPP p CF 3 -Cu(II) was also evaluated as a reusable catalyst in cyclohexene allylic oxidation, and displayed a remarkable turnover number (TON) value of ≈ 265,000, using O 2 as green oxidant, in the total absence of sacriﬁcial additives, which is the highest activity ever reported for said allylic oxidation. Additionally, ND@ β NH-TPP p CF 3 -Cu(II) could be easily separated from the reaction mixture by centrifugation, and reused in three consecutive catalytic cycles without major loss of activity.


Introduction
The recent advances of nanotechnology are remarkable. Nanostructured materials are part of our lives and a driving force towards the global economy of modern society. Among the many examples examples available, the nanoallotropes of carbon are a unique family, among which nanodiamonds (NDs) are included. These carbon nanomaterials were first prepared by detonation in the USSR in the 1960s [1], and have been an exquisite research subject for many groups ever since, attracting tremendous interest, given their specific optical and physical/chemical properties [2].
It is expected that the synergism between ND properties, namely, biocompatibility, unique optical features, good thermal conductivity, electrical resistivity, resistance to harsh environments, and chemical stability [2] and the properties of tetrapyrrolic macrocycles, may pave the way for multiple additional applications. In this regard, only a few reports have been published on the preparation of hybrid ND-porphyrin materials, through the formation of either electrostatic bonds [32] or amide bonds between carboxy-phenyl-porphyrin and amino-functionalized NDs [19,25] (Scheme 1). Scheme 1. Strategies for preparation of porphyrin-ND hybrid materials: (a) Previous works [19,25,32]; (b) this work.
The relatively low stability of these linkages against pH alterations or hydrolysis prompted us to devise and further establish an innovative synthetic strategy to immobilize porphyrins onto aminated functionalized NDs, via ipso-nitro substitution reaction with β-nitro substituted porphyrins, allowing the formation of much more stable covalent amine linkages. These new materials were fully characterized by X-ray photoelectron spectroscopy (XPS), thermogravimetrydifferential scanning calorimetry (TG-DSC), fluorescence spectroscopy, infrared spectroscopy (IR) and ultraviolet-visible spectroscopy (UV-Vis).
Furthermore, the new hybrid nanodiamond-copper(II)-porphyrin ND@βNH-TPPpCF3-Cu(II) was used as a catalyst in the oxidation of cyclohexene, using O2 as a green oxidant. The oxidation of cyclohexene is of great interest, as its products (key C6 chemicals) are largely used as intermediates in the pharmaceutical/chemical industries [33,34]. A wide variety of catalysts have been used for the oxidation of cyclohexene with O2 as an oxidant, such as ionic liquids; metal complexes, including metalloporphyrins; noble metals; and metal complexes immobilized on solid supports (carbon nanotubes, magnetic nanoparticles, titanium dioxide), as reported in a recent review [35]. However, controllable oxidation reactions that can selectively yield 7-oxabicyclo [4.1.0] heptane, trans/ciscyclohexane-1,2-diol, cyclohex-2-en-1-ol, cyclohex-2-en-1-one and adipic acid, using O2 as a green Scheme 1. Strategies for preparation of porphyrin-ND hybrid materials: (a) Previous works [19,25,32]; (b) this work.
The relatively low stability of these linkages against pH alterations or hydrolysis prompted us to devise and further establish an innovative synthetic strategy to immobilize porphyrins onto aminated functionalized NDs, via ipso-nitro substitution reaction with β-nitro substituted porphyrins, allowing the formation of much more stable covalent amine linkages. These new materials were fully characterized by X-ray photoelectron spectroscopy (XPS), thermogravimetry-differential scanning calorimetry (TG-DSC), fluorescence spectroscopy, infrared spectroscopy (IR) and ultraviolet-visible spectroscopy (UV-Vis).
Furthermore, the new hybrid nanodiamond-copper(II)-porphyrin ND@βNH-TPPpCF 3 -Cu(II) was used as a catalyst in the oxidation of cyclohexene, using O 2 as a green oxidant. The oxidation of cyclohexene is of great interest, as its products (key C6 chemicals) are largely used as intermediates in the pharmaceutical/chemical industries [33,34]. A wide variety of catalysts have been used for the oxidation of cyclohexene with O 2 as an oxidant, such as ionic liquids; metal complexes, including metalloporphyrins; noble metals; and metal complexes immobilized on solid supports (carbon nanotubes, magnetic nanoparticles, titanium dioxide), as reported in a recent review [35]. However, controllable oxidation reactions that can selectively yield 7-oxabicyclo [4.1.0] heptane, trans/cis-cyclohexane-1,2-diol, Catalysts 2020, 10, 1402 3 of 13 cyclohex-2-en-1-ol, cyclohex-2-en-1-one and adipic acid, using O 2 as a green oxidant, in solvent-free conditions, and using a stable, reusable and non-toxic catalyst, are still a challenge.
In this regard, the development of more sustainable practices is imperative in chemistry. Catalytic systems with negligible effects on the surrounding environments are desirable, but attention is usually paid to the substrate-product binomial, leaving catalysts out of the ecological concerns. Despite the increasing applications of NDs in bio-and nanomedicine [11,36], studies regarding their ecotoxic and genotoxic effects are still limited [37][38][39][40][41]. Herein, we also present a study on the cytotoxic and genotoxic evaluation of amine-functionalized nanodiamonds (ND@NH 2 ) and 2-nitro-5,10,15,20tetra(4-trifluoromethylphenyl)porphyrin covalently linked to nanodiamonds (ND@βNH-TPPpCF 3 ) using Allium cepa as a plant model, as we aimed to obtain non-fitotoxic "green" catalysts-that is, materials that will not harm the environment (even if, accidentally, not properly discarded). A. cepa is largely used in genotoxicity studies, since it has a reduced number of large chromosomes, where changes are easy to detect. Additionally, the effects observed in its cells show good correlations with those of mammal (including human) cells [42].
Catalysts 2020, 10, x FOR PEER REVIEW 3 of 13 oxidant, in solvent-free conditions, and using a stable, reusable and non-toxic catalyst, are still a challenge.
In this regard, the development of more sustainable practices is imperative in chemistry. Catalytic systems with negligible effects on the surrounding environments are desirable, but attention is usually paid to the substrate-product binomial, leaving catalysts out of the ecological concerns. Despite the increasing applications of NDs in bio-and nanomedicine [11,36], studies regarding their ecotoxic and genotoxic effects are still limited [37][38][39][40][41]. Herein, we also present a study on the cytotoxic and genotoxic evaluation of amine-functionalized nanodiamonds (ND@NH2) and 2nitro-5,10,15,20-tetra(4-trifluoromethylphenyl)porphyrin covalently linked to nanodiamonds (ND@βNH-TPPpCF3) using Allium cepa as a plant model, as we aimed to obtain non-fitotoxic "green" catalysts-that is, materials that will not harm the environment (even if, accidentally, not properly discarded). A. cepa is largely used in genotoxicity studies, since it has a reduced number of large chromosomes, where changes are easy to detect. Additionally, the effects observed in its cells show good correlations with those of mammal (including human) cells [42].
The hybrid materials were fully characterized by XPS, UV-Vis, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and thermogravimetry-differential scanning calorimetry (TG-DSC). The quantification of immobilized-nitro porphyrin (1 and 2) on the functionalized NDs (primary average particle size of 5 nm) was done by XPS. The atomic percentages The hybrid materials were fully characterized by XPS, UV-Vis, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and thermogravimetry-differential scanning calorimetry (TG-DSC). The quantification of immobilized-nitro porphyrin (1 and 2) on the functionalized NDs (primary average particle size of 5 nm) was done by XPS. The atomic percentages (%) of carbon, nitrogen, Catalysts 2020, 10, 1402 4 of 13 oxygen, fluorine and copper are presented in Table 1. Results for pristine-i.e., non-functionalized-ND are shown for comparison, together with ND@NH 2 .
Figures S1 and S2 of Supplementary Information show the XPS results for the samples ND@βNH-TPPpCF 3 -Cu(II) and ND@βNH-TPPpCF 3 , respectively. The C1s, N1s, O1s and F1s XPS spectra were similar for both samples. Detailed analysis of the XPS spectra is given in the SI. The results confirm the anchoring of the complexes.
From the compositional analysis shown in Table 1, a Cu percentage of 0.06 at.% in ND@βNH-TPPpCF 3 -Cu(II) and a F percentage of 3.2 at.% at in ND@βNH-TPPpCF 3 were determined. These results indicate that amine groups from ND materials reacted with the nitro-porphyrin derivatives, displaying 0.050 mmol of metalloporphyrin 1 per gram of ND@βNH-TPPpCF 3 -Cu(II) and 0.215 mmol of porphyrin 2 per gram of ND@βNH-TPPpCF 3 .
In Figure 1, the normalized fluorescence emission spectra of ND@βNH-TPPpCF 3 (blue line), nitro-porphyrin 2 (black line) and ND@NH 2 (grey line) are displayed, with excitation at 420 nm using N,N-dimethylformamide (DMF) as solvent. The fluorescence spectrum of nitro-porphyrin 2-immobilized nanodiamonds (ND@βNH-TPPpCF 3 ) is similar to that of the non-immobilized nitro-porphyrin 2 over the whole emission wavelength range (575 to 775 nm). In particular, two strong emission bands at 650 and 720 nm are present. Thus, qualitative analysis of the fluorescence emission spectrum of ND@βNH-TPPpCF 3 evidences that the energy of the excited state of porphyrin 2 is not changed by the presence of the material, probably due to the presence of a non-conjugated linker.  [40].
Detailed analysis of the XPS spectra is given in the SI. The results confirm the anchoring of the complexes.
From the compositional analysis shown in Table 1, a Cu percentage of 0.06 at.% in ND@βNH-TPPpCF3-Cu(II) and a F percentage of 3.2 at.% at in ND@βNH-TPPpCF3 were determined. These results indicate that amine groups from ND materials reacted with the nitro-porphyrin derivatives, displaying 0.050 mmol of metalloporphyrin 1 per gram of ND@βNH-TPPpCF3-Cu(II) and 0.215 mmol of porphyrin 2 per gram of ND@βNH-TPPpCF3.
In Figure 1, the normalized fluorescence emission spectra of ND@βNH-TPPpCF3 (blue line), nitro-porphyrin 2 (black line) and ND@NH2 (grey line) are displayed, with excitation at 420 nm using N,N-dimethylformamide (DMF) as solvent. The fluorescence spectrum of nitro-porphyrin 2immobilized nanodiamonds (ND@βNH-TPPpCF3) is similar to that of the non-immobilized nitroporphyrin 2 over the whole emission wavelength range (575 to 775 nm). In particular, two strong emission bands at 650 and 720 nm are present. Thus, qualitative analysis of the fluorescence emission spectrum of ND@βNH-TPPpCF3 evidences that the energy of the excited state of porphyrin 2 is not changed by the presence of the material, probably due to the presence of a non-conjugated linker.   The normalized UV-Vis absorption spectra of ND@NH 2 (grey line), ND@βNH-TPPpCF 3 (blue line) and nitro-porphyrin 2 (black line) dispersed in N,N-dimethylformamide are presented in Figure 2. ND@βNH-TPPpCF 3 clearly shows the typical Soret band at 425 nm with a blue shift of 14 nm relatively to the nitro-porphyrin 2 free base. This blue shift is an unequivocal confirmation that the ipso-nitro substitution reaction occurred and an amine covalent linkage between the porphyrin and the NDs is present.
Catalysts 2020, 10, x FOR PEER REVIEW 5 of 13 2. ND@βNH-TPPpCF3 clearly shows the typical Soret band at 425 nm with a blue shift of 14 nm relatively to the nitro-porphyrin 2 free base. This blue shift is an unequivocal confirmation that the ipso-nitro substitution reaction occurred and an amine covalent linkage between the porphyrin and the NDs is present.      2. ND@βNH-TPPpCF3 clearly shows the typical Soret band at 425 nm with a blue shift of 14 nm relatively to the nitro-porphyrin 2 free base. This blue shift is an unequivocal confirmation that the ipso-nitro substitution reaction occurred and an amine covalent linkage between the porphyrin and the NDs is present.     Additionally, the quantification of the nitro-porphyrin (1 and 2) content for all three different functionalized NDs was performed using TG-DSC measurements between 30 and 800 • C ( Figure S4a-c). By analysis and comparison with the TG-DSC curves of ND@NH 2 , calculated weight losses of ND@βNH-TPPpCF 3 -Cu(II) and ND@βNH-TPPpCF 3 were 5.6% and 6.3%, respectively, corresponding to 0.059 mmol/g ND of metalloporphyrin 1 and 0.071 mmol/g ND of porphyrin 2.

Cytotoxic and Genotoxic Evaluation of Porphyrin-Functionalized Nanodiamonds in Allium Cepa Assay
Despite previous findings that NDs are non-cytotoxic for different types of cells and have higher biocompatibility when compared with all other nanocarbon-based materials (for instance, carbon nanotubes, fullerenes, graphene oxide, etc.) [36,48,49], recent studies showed that the cytotoxicity activity of NDs is correlated with their surface functionalities [50][51][52]. As an example, Marcon et al. demonstrated that ND functionalized with -COOH was highly toxic to Xenopus frog embryos, even at a low concentration (2 mg/L), in comparison to the embryotoxicity potential of ND with -OH or -NH 2 [50].
Our results, described in Supplementary Information, showed that the cytotoxicity and genotoxic potential of ND were not impacted by functionalizing them with the TPPpCF 3 porphyrin, and consequently, a non-phytotoxic porphyrin-nanodiamond hybrid nanomaterial was successfully prepared ( Figure S5a-e).

Catalytic Oxidation Experiments
As stated above, cyclohexene oxidation products are of great interest in industry [33,34]. Therefore, to assess the application of copper-based ND@βNH-TPPpCF 3 -Cu(II) as potential reusable catalysts, this new ND hybrid material was evaluated as catalyst in cyclohexene oxidation, using O 2 as a green oxidant (Scheme 3).
Catalysts 2020, 10, x FOR PEER REVIEW 6 of 13 Additionally, the quantification of the nitro-porphyrin (1 and 2) content for all three different functionalized NDs was performed using TG-DSC measurements between 30 and 800 °C ( Figures  S4a-c). By analysis and comparison with the TG-DSC curves of ND@NH2, calculated weight losses of ND@βNH-TPPpCF3-Cu(II) and ND@βNH-TPPpCF3 were 5.6% and 6.3%, respectively, corresponding to 0.059 mmol/g ND of metalloporphyrin 1 and 0.071 mmol/g ND of porphyrin 2.

Cytotoxic and Genotoxic Evaluation of Porphyrin-Functionalized Nanodiamonds in Allium Cepa Assay
Despite previous findings that NDs are non-cytotoxic for different types of cells and have higher biocompatibility when compared with all other nanocarbon-based materials (for instance, carbon nanotubes, fullerenes, graphene oxide, etc.) [36,48,49], recent studies showed that the cytotoxicity activity of NDs is correlated with their surface functionalities [50][51][52]. As an example, Marcon et al. demonstrated that ND functionalized with -COOH was highly toxic to Xenopus frog embryos, even at a low concentration (2 mg/L), in comparison to the embryotoxicity potential of ND with -OH or -NH2 [50].
Our results, described in Supplementary Information, showed that the cytotoxicity and genotoxic potential of ND were not impacted by functionalizing them with the TPPpCF3 porphyrin, and consequently, a non-phytotoxic porphyrin-nanodiamond hybrid nanomaterial was successfully prepared (Figures S5a-e).

Catalytic Oxidation Experiments
As stated above, cyclohexene oxidation products are of great interest in industry [33,34]. Therefore, to assess the application of copper-based ND@βNH-TPPpCF3-Cu(II) as potential reusable catalysts, this new ND hybrid material was evaluated as catalyst in cyclohexene oxidation, using O2 as a green oxidant (Scheme 3). Appropriate amounts of cyclohexene and ND@βNH-TPPpCF3-Cu(II) catalyst were added in a reactor. After reaction at 100 °C, with O2 (4 bar), for 24 h, cyclohexene conversion and product selectivity were determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), using aliquots from the reaction mixture. The results of conversion of cyclohexene and product selectivity are shown in Figure 4.
A cyclohexene conversion of 53% was obtained, with major products being 2-cyclohexen-1-one (B-39%) and 2-cyclohexen-1-ol (C-56%). This trend may be attributed to cyclohexene allylic products [53,54]. It is also worth mentioning the remarkable turnover number (TON) ≈ 265,000 achieved, using O2 as green oxidant, in the total absence of sacrificial additives, whereas in the literature only TONs from 1210 to ≈200,000 were reported for the same oxidation reaction [55][56][57]. The ≈200,000 TON value refers to a previous publication of Pereira and co-workers, also using a copper metalloporphyrin but immobilized in magnetic nanoparticles [55]. A larger value was reported by other authors, but for the epoxidation reaction [58]. In this work, we obtained mainly the Appropriate amounts of cyclohexene and ND@βNH-TPPpCF 3 -Cu(II) catalyst were added in a reactor. After reaction at 100 • C, with O 2 (4 bar), for 24 h, cyclohexene conversion and product selectivity were determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), using aliquots from the reaction mixture. The results of conversion of cyclohexene and product selectivity are shown in Figure 4.
A cyclohexene conversion of 53% was obtained, with major products being 2-cyclohexen-1-one (B-39%) and 2-cyclohexen-1-ol (C-56%). This trend may be attributed to cyclohexene allylic products [53,54]. It is also worth mentioning the remarkable turnover number (TON) ≈ 265,000 achieved, using O 2 as green oxidant, in the total absence of sacrificial additives, whereas in the literature only TONs from 1210 to ≈200,000 were reported for the same oxidation reaction [55][56][57]. The ≈200,000 TON value refers to a previous publication of Pereira and co-workers, also using a copper metalloporphyrin but immobilized in magnetic nanoparticles [55]. A larger value was reported by other authors, but for the epoxidation reaction [58]. In this work, we obtained mainly the double bond products 2-cyclohexen-1-one and 2-cyclohexen-1-ol, as referred to above-that is, allylic oxidation products.
double bond products 2-cyclohexen-1-one and 2-cyclohexen-1-ol, as referred to above-that is, allylic oxidation products. Additionally, nanodiamond-immobilized porphyrin (ND@βNH-TPPpCF3-Cu(II)) could be separated from the reaction mixtures by centrifugation, and its recyclability was evaluated. The solid materials (ND@βNH-TPPpCF3-Cu(II)) were washed three times with CH2Cl2 and three times with CH3CN, then dried at 60 °C for 4 h and reused in the next catalytic cycle. The results are presented in Figure 5. The recycling experiments revealed good stability of the catalyst up to three cycles. Given the high catalyst dispersibility in cyclohexene, the observed slight decrease in the activity over the three cycles may be attributed to catalyst loss after decantation in each run.   Additionally, nanodiamond-immobilized porphyrin (ND@βNH-TPPpCF 3 -Cu(II)) could be separated from the reaction mixtures by centrifugation, and its recyclability was evaluated. The solid materials (ND@βNH-TPPpCF 3 -Cu(II)) were washed three times with CH 2 Cl 2 and three times with CH 3 CN, then dried at 60 • C for 4 h and reused in the next catalytic cycle. The results are presented in Figure 5. The recycling experiments revealed good stability of the catalyst up to three cycles. Given the high catalyst dispersibility in cyclohexene, the observed slight decrease in the activity over the three cycles may be attributed to catalyst loss after decantation in each run. oxidation products. Additionally, nanodiamond-immobilized porphyrin (ND@βNH-TPPpCF3-Cu(II)) could be separated from the reaction mixtures by centrifugation, and its recyclability was evaluated. The solid materials (ND@βNH-TPPpCF3-Cu(II)) were washed three times with CH2Cl2 and three times with CH3CN, then dried at 60 °C for 4 h and reused in the next catalytic cycle. The results are presented in Figure 5. The recycling experiments revealed good stability of the catalyst up to three cycles. Given the high catalyst dispersibility in cyclohexene, the observed slight decrease in the activity over the three cycles may be attributed to catalyst loss after decantation in each run.

Characterization of Chemicals and Materials
Reagents and solvents were purchased from Fluorochem (Derbyshire, UK) and/or Sigma-Aldrich (Lisbon, Portugal). All solvents and chemicals were purified and/or dried by methods from literature [59]. Amine functionalized nanodiamonds (ND@NH 2 , UDiamond, Amine P) were purchased from Carbodeon, Finland. Nanodiamonds (NDs) with a primary average particle size of 5 nm were obtained through detonation synthesis using a mixture of 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) high explosives as precursors. Boiling acid treatment was used to remove the amorphous carbon and impurities from the detonation soot to create the purified ND powder. Amine functionalization was subsequently done by heating the ND material for several hours in a gas atmosphere comprising ammonia gas.
XPS analyses were carried out in a Kratos AXIS Ultra HSA spectrometer (CEMUP, Porto, Portugal). All analysis was performed using the following conditions: monochromatic AlKα X-ray source (1486.7 eV), at 15 kV (90 W), in fixed analyzer transmission mode. Data acquisition was carried out using a pressure lower than 1 × 10 −6 Pa. Survey and multi-region spectra were obtained at C 1s, O 1s, N 1s, F 1s and Cu 2p (this one only for the Cu containing sample) photoelectron peaks. The modelling of the spectra was performed using the CASA XPS program (Clearwater, FL, United States).
Fluorescence spectra were acquired on a Horiba-Jobin-Ivon Fluorolog 322 spectrofluorometer, using a quartz cuvette and N,N-dimethylformamide as solvent. All fluorescence emission spectra were normalized according to maximum intensity (from 0 to 1). FT-IR spectra were recorded on a Cary 630 FT-IR spectrometer using ≈20-25 mg of each sample, with Microlab PC software and ATR sampling unit with a resolution of 8 cm −1 and scan range of 4000 to 700 cm −1 . Gas chromatography (GC) analysis was carried out on a HP-Agilent 6890 chromatograph with Agilent GC software (Santa Clara, CA, United States), equipped with ionization detector (FID) (Santa Clara, CA, United States), and a HP-5 chromatographic column, non-polar (5% diphenyl, 95% dimethylpolysiloxane (30 m × 0.32 mm). GC-MS analysis was performed on a Hewlett-Packard 5973 MSD spectrometer (Santa Clara, CA, United States) (using EI = 70 eV), coupled to a Hewlett-Packard Agilent 6890 chromatograph (Santa Clara, CA, United States), and equipped with a HP-5 MS chromatographic column (30 m × 0.25 mm). The analyses were performed using the following chromatographic conditions: initial temperature 40 • C, rate 10 • C min −1 , final temperature 200 • C and gas flow of 1.41 mL.min −1 . Conversion and selectivity were calculated based on peak areas of GC. The identification and qualification of the peaks was confirmed by comparison of the mass spectra and retention times against p-xylene as external standard. Thermogravimetric analyses were performed on a Perkin-Elmer STA 6000 (Shelton, CT, United States) simultaneous TG-DSC instrument in a range between 25 • C and 800 • C.

Cytotoxic and Genotoxic Evaluation of Porphyrin-Functionalized Nanodiamonds in Allium Cepa Assays
Pesticide-free A. cepa seeds were purchased from Isla (Isla Sementes Ltd.a., Porto Alegre, RS, Brazil). The A. cepa assays were carried out according to literature [42,60]. The seeds were placed to germinate at 23 ± 2 • C under a photoperiod of 12 h light/12 h dark for 96 h. 30 seeds were continuously exposed to an aqueous solution (pure or containing NDs at 10, 50 and 100 mg.L −1 ). All tests were performed in triplicate, totaling 90 seeds per group. The germination index (GI) was calculated after 96 h. The root length index (RLI) was measured using a digital caliper (Digimess, São Paulo, SP, Brazil) after 96 h of the germination. Then, the roots were collected and prepared for the mitotic index (MI), nuclear abnormality index (NAI) and micronucleus index (MNI) determination. After the collection, the roots were fixed in Carnoy Solution 3:1 (alcohol/acetic acid) during 8 h and then stored at 4 • C. After that, the fixed roots were washed with tap water and hydrolyzed in 1 N HCl at 60 • C for 10 min. Next, they were washed again and stained with Schiff's reagent for 2 h. Subsequently, the root meristems were slightly smashed in a drop of 45% acetic carmine. Finally, 5 slides were prepared per group and imaged using an optical microscope (Nikon, Tokyo, Japan) at a 400× magnification. 1000 cells were evaluated per slide, resulting in 5000 cells checked per treatment. All experiments were performed in triplicate and the GI, REI, MI, NAI, and MNI values were submitted to Paired Sample t Test (p ≤ 0.05) for comparison of pairs of means.

Procedure for Oxidation of Cyclohexene Using ND@βNH-TPPpCF 3 -Cu(II) as Catalyst
ND@βNH-TPPpCF 3 -Cu(II) (4 mg; 19.8 × 10 −8 mol of Cu) as catalyst and cyclohexene (10 mL; 0.099 mol) as substrate were added to a reactor and then charged with oxygen (O 2 , 4 atm). The reaction mixture was maintained at 100 • C, during 24 h, under stirring. After this time, the reactor was depressurized and an aliquot from the crude reaction was analyzed by GC.
In the reutilization tests, after each catalytic cycle, the copper(II) porphyrin-functionalized nanodiamonds (ND@βNH-TPPpCF 3 -Cu(II)) were separated from the reaction mixture by centrifugation and the supernatant solution by decantation. Then, the nanomaterials (ND@βNH-TPPpCF 3 -Cu(II)) were washed using acetonitrile (three times, using 4 mL) and dichloromethane (three times, using 4 mL), dried at 60 • C, during 4 h, and used in the next catalytic cycle. No catalyst adjustment was made after each run.

Conclusions
We demonstrated that the β-ipso-nitro-aromatic nucleophilic substitution of amine-functionalized NDs with β-nitro substituted porphyrins is an innovative and efficient approach to prepare covalently linked hybrid porphyrin-nanodiamond materials. The nanocomposites' characterization by XPS, fluorescence, UV-Vis spectroscopy, IR spectroscopy and TGA-DSC unequivocally corroborated the effective grafting of the nitro-porphyrins (1 and 2) onto the amine-functionalized NDs. Moreover, our study revealed the hybrid nanodiamond-copper(II)-porphyrin as the best ever reported allylic cyclohexene oxidation catalyst (TON ≈ 265,000), using only O 2 as a "green" oxidant, in the absence of reductants/solvents. Furthermore, the material could be recovered by centrifugation and used again in three consecutive catalytic cycles without major loss of activity. Moreover, this porphyrin-nanodiamond catalyst, under similar reaction conditions, may be able to oxidize other substrates, namely, α-pinene, β-pinene and thiophenol derivatives. In addition, no ecotoxic and genotoxic effects were observed for ND@NH 2 and ND@βNH-TPPpCF 3 materials, using Allium cepa as the plant model.
The findings described herein allow us to foresee the significance of materials of this type in oxidative catalysis, and their potential in biomedical applications.