Biomimetic catalytic aerobic oxidation of C–sp(3)–H bonds under mild conditions using galactose oxidase model compound CuIIL

Developing highly efficient catalytic protocols for C–sp(3)–H bond aerobic oxidation under mild conditions is a long-desired goal of chemists. Inspired by nature, a biomimetic approach for the aerobic oxidation of C–sp(3)–H by galactose oxidase model compound CuIIL and NHPI (N-hydroxyphthalimide) was developed. The CuIIL–NHPI system exhibited excellent performance in the oxidation of C–sp(3)–H bonds to ketones, especially for light alkanes. The biomimetic catalytic protocol had a broad substrate scope. Mechanistic studies revealed that the CuI-radical intermediate species generated from the intramolecular redox process of CuIILH2 was critical for O2 activation. Kinetic experiments showed that the activation of NHPI was the rate-determining step. Furthermore, activation of NHPI in the CuIIL–NHPI system was demonstrated by time-resolved EPR results. The persistent PINO (phthalimide-N-oxyl) radical mechanism for the aerobic oxidation of C–sp(3)–H bond was demonstrated.


Introduction
Oxidation is one of the most useful chemical transformations in organic synthesis and the modern chemical industry. 1 Oxidation of the C-sp(3)-H bond is extremely challenging due to the high bond energy of the C-H bond. 2 Various catalysts have been used for the conversion of hydrocarbons, ranging from heterogeneous 3 to homogeneous catalytic systems, 4 as well as bio-and enzymatic catalysts. 5 In heterogeneous catalytic systems, alkanes are usually rst converted to more active alkenes via oxidative dehydrogenation. 6 To achieve acceptable conversion and selectivity, these processes are oen carried out under harsh conditions (e.g., high temperatures 500-900 C). 7 In the presence of O 2 or other peroxide oxidants, C-H bonds could be oxyfunctionalized by transition metals in homogeneous catalytic systems. 1a Usually, assistance of light, 8 electricity, 9 or other additives 10 is necessary. Therefore, a more sustainable and environmentally benign oxidative protocol for the chemical conversion of hydrocarbons is desirable.
Copper-containing metalloprotein galactose oxidase (GO) is an enzyme which selectively catalyzes the aerobic oxidation of alcohols to aldehydes under mild conditions. 11 From the structure, it could be known that GOase contains two oneelectron redox centers, a mononuclear copper center and a tyrosyl radical ligand (Tyr 272). 12 The Tyr 272 radical ligand in GOase abstracts H from the a-carbon of alcoholate and undergoes intramolecular electron transfer to form aldehyde and a Cu I species. Finally, the reduced GOase is converted to its original form by O 2 along with the formation of 1 equiv. of H 2 O 2 (Scheme 1A). 13 In the past, some GOase model compounds have been synthesized by employing redox-active non-innocent ligands (Scheme 1B). 14 Kitajima reported a salen type copper(II) complex as a possible GOase model. 15 Stack 16 and Pierre's 17 functional models were developed. Subsequently, a mononuclear Cu IIphenoxyl complex was synthesized by Chaudhuri and Wieghardt. 13a, 18 These model compounds are capable of oxidizing primary/secondary alcohols to the corresponding ketones in the presence of O 2 . However, GOase has insufficient activity to oxidize hydrocarbons due to the high BDE (bond dissociation energy) of the C-H bond in most hydrocarbons (85-105 kcal mol À1 ). 2b, 19 N-Hydroxyphthalimide (NHPI) is an efficient and low-cost Noxyl precursor capable of producing a highly active phthalimide-N-oxyl (PINO) radical, which is widely used in the aerobic oxidation of hydrocarbons. 20 Ishii and co-workers innovatively introduced metal salts and NHPI for the oxidation reaction of isobutane with molecular oxygen (100 C). 21  developed PdCl 2 /NHPI for C-sp(2)-H hydroxylation (100 C). 22 However, PINO readily self-decomposes at high temperature (>80 C) and under alkaline conditions. 19a,23 In addition, the application of NHPI was restricted because of the long initiation period and short existence time of PINO. 20d In response to these challenges, an electrochemical pathway was developed to activate NHPI under mild conditions by Jensen and co-workers. 24 Wang described the approach of PINO radical generation through the irradiation of a-Fe 2 O 3 and NHPI using 455 nm light. 25 It has also been reported that ionic liquids 26 and uorine-containing solutions 27 were utilized to stabilize PINO.
Inspired by the structure of galactose oxidase, the oxidation protocol of the C-sp(3)-H bond was developed, in which the Cu II L catalyst (ligand: N,N 0 -bis(3,5-di-tert-butyl-2-hydroxyphenyl)-1,2-phenylenediamine) 18 and NHPI were introduced. Through the proton coupling electron transfer (PCET) process, two electrons and protons were transferred from NHPI to Cu II L to form Cu II LH 2 and the PINO radical. The PINO radical can abstract H from the C-H bond of the substrate to produce a high-activity carbon-centered radical through hydrogen atom transformation (HAT). Subsequently, in situ generated Cu II LH 2 reacts with O 2 to produce H 2 O 2 and Cu II L (Scheme 1C). In addition, the detailed mechanism was investigated with the assistance of in situ UV-vis spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, X-ray absorption near-edge structure (XANES), as well as thermodynamic and kinetic analysis combined with density functional theory (DFT).

Results and discussion
We initiated aerobic oxidation of the C-sp(3)-H bond by using isobutane as a model substrate (Table 1). Aer an extensive survey of reaction conditions, including the amount of catalyst (Cu II L) (Fig. S1 †), the amount of NHPI (Fig. S2 †) and reaction temperature ( Fig. S3 †), the optimized conditions were obtained (isobutane 21 mmol, O 2 1.0 MPa, 70 C, Cu II L 0.8 mol%, NHPI 4.0 mol%, CH 3 CN solvent 20 mL). Under the optimized conditions, isobutane conversion was up to 75%, and the desired hydroxylation product tert-butanol (1) was obtained in 80% selectivity ( Table 1, entry 1).
The control experiments indicated that Cu II L and NHPI were indispensable for the success of this oxidation. Especially, the selectivity of tert-butanol was closely related to Cu II L or NHPI (Table 1, entries 2 and 3). When using other Cu-based catalysts such as CuCl 2 and CuCl, a lower yield of 1 and conversion of isobutane was observed ( Table 1, entries 4 and 5). The results of solvent screening showed that CH 3 CN was the optimal solvent for this reaction (Table 1, entries 6 and 7). Surprisingly, when the oxidation was conducted in air (2.0 MPa), the selectivity of 1 increased (84%) and the conversion slightly decreased (68%) ( Table 1, entry 8). Gratifyingly, aer 5 cycles of the reaction, the catalytic system still showed excellent catalytic activity (Fig. S4 †). Poor efficiency and selectivity were obtained when using the typical Co(OAc) 2 /NHPI as the catalyst under the same conditions as previously reported 21 (Table 1, entry 9). Addition of butylated hydroxytoluene (BHT, radical scavenger) resulted in oxidation being completely suppressed (Table 1, entry 10).
The effect of steric hindrance was observed from the oxidation of propylbenzene and dphenylmethane ( Table 2, entries 5 and 6). Cyclopentane was oxidized to cyclopentanone with 83% selectivity and cyclopentanol (17% selectivity) ( Table 2, entry 7). For cyclohexene containing both C-sp(3)-H and C]C bonds, the total selectivity of C-sp(3)-H bond oxidation to alcohols and ketones was 83%, and the selectivity of epoxide was 17% ( Table  2, entry 8). It indicated that the biomimetic catalytic system exhibited high regioselectivity.
To gain insight into the reaction pathway, the reaction between Cu II L and NHPI was originally investigated. The Xband EPR spectrum of the nearly square-planar (D 4h ) Cu II L in CH 3 CN recorded at 90 K displayed a typical Cu(II) signal with an unpaired electron in the d x 2 Ày 2 orbital (Fig. S5 †). 29 From the optimal geometric conguration of Cu II L (B3LYP/6-31G(d,p)/ IEF-PCM (solvent model) level with CH 3 CN as a solvent), the d x 2 Ày 2 orbital of the central Cu(II) atom was perpendicular to the p orbitals in the aromatic ring ( Fig. S6 †). This orbital orientation minimized the spatial overlap between the orthogonal d x 2 Ày 2 orbital of Cu(II) and the ligand's p orbitals, resulting in a difficulty for ligand to metal Cu(II) charge transfer (LMCT) to occur. 30 Cu II L has characteristic bands at 435 nm and 1167 nm with a black-green color in CH 3 CN solution (Fig. 1A, orange line). To determine the type of absorption peak, the electronic UV-vis-NIR absorption spectra of Cu II L were recorded using the TD-B3LYP/6-31G(d,p) method and IEF-PCM solvent model for CH 3 CN. The obtained absorption spectra, the FMO pictures, and the vertical excitation energies are given in Fig. S6 †, S7 and Table S1, † respectively. The obtained peaks at 435 nm (calcd. l ¼ 529 nm, f ¼ 0.0581) and 1167 nm (calcd. l ¼ 1022 nm, f ¼ 0.1249) of Cu II L were attributed to b-SOMO (the highest singly occupied molecular orbital)-4 to b-SUMO (the lowest singly unoccupied molecular orbital) and b-SOMOÀ1 to b-SUMO transitions, respectively. These are mainly intraligand chargetransfer (ILCT) transitions. 31 Another intense band of Cu II L in the UV region (<380 nm) was attributed to the p to p* transition within the ligand's aromatic ring (a-SOMO to a-SUMO+2, 32 It indicates that it is difficult for an intramolecular redox reaction to occur in planar structured Cu II L. As shown in the cyclic voltammograms ( Fig. S9 †), the Cu II L complex in CH 3 CN showed two reversible oxidations (E ox1 1/2 ¼ À0.17 V and E ox2 1/2 ¼ 0.30 V vs. Fc +/0 , the same below) and two reversible reductions (E red1 1/2 ¼ À0.77 V and E red2 1/2 ¼ À1.46 V). For comparison, NHPI has an oxidation potential E ¼ 1.04 V, which means that the single electron transfer (SET) reaction could not occur between NHPI and Cu II L. However, aer adding NHPI (0.20 mM) to an argon-saturated CH 3 CN solution of Cu II L (0.10 mM) at 25 C, the absorption band at 1167 nm disappeared, and the absorption intensity decreased at 435 nm. A new absorption BHT (10 mol%) as a radical scavenger n.r. band appeared at 998 nm, and the color of the solution changed from black-green to yellow (Fig. 1A). A blue-shi (1167 nm to 998 nm) occurred in the longwavelength region, which indicated a decrease in the electron density of the conjugated system. 33 In addition, the characteristic absorption peak of the resulting solution was consistent with the directly synthesized Cu II LH 2 and DFT computed UVvis/NIR spectrum (Fig. S10 †). The EPR spectrum of the resulting solution was also consistent with that of the directly synthesized Cu II LH 2 (Fig. S11 †). The presence of Cu II LH 2 in the resulting solution was also indicated by high resolution mass spectrometry (Fig. S12 †). Furthermore, when NHPI-d was used instead of NHPI (see Fig. S13 † for the synthesis and characterization of NHPI-d), the CSI-MS (cold-spray ionization mass spectrometry) spectrum of the resulting solution exhibited a prominent ion peak at m/z ¼ 579.3006, whose mass and isotope distribution patterns corresponded to Cu II LD 2 (calcd m/ z ¼ 579.3002) (Fig. 1A, inset). In addition, EPR showed that the PINO radical (A N ¼ 4.6, g ¼ 2.0073) was produced from homolysis of the O-H bond of NHPI (Fig. S14 †). 20a, 34 These results demonstrate unambiguously that Cu II L reacted with the hydrogen donor (NHPI) by H-atom transfers to form Cu II LH 2 .
Compared with 25 C, the in situ generated Cu II LH 2 shows a new absorption peak (464 nm) at 70 C (Fig. 1B). This absorption is attributed to Cu(II) d-d bands and/or ligand to copper charge transfer transitions (LMCT). 32 This led us to speculate whether this phenomenon was caused by structural changes of Cu II LH 2 caused by temperature changes.
The ligand of Cu II LH 2 is a closed shell and subsequently EPR-silent (radical silent). Surprisingly, when the reaction temperature was increased to 70 C, the in situ generated Cu II LH 2 in an argon atmosphere showed a typical S ¼ 1/2 organic radical signal (g ¼ 2.0043) with hyperne coupling to two N donors (I ¼ 1) and four H atoms (I ¼ 1/2), which was not observed at room temperature (Fig. 1C). The EPR spectra of PINO may be covered due to the peak location (g ¼ 2.0073) and total width (12 G).
Interestingly, the hyperne splitting of the two N atoms (A N(1) ¼ 7.8 G, A N(2) ¼ 3.5 G) were unequal and nearly twofold, which means the two N atoms of the ligand have different effects on the spin center. 35 For organometallic radical complexes, when heteroatoms and spin centers are in different spatial positions, the values of hyperne splitting are different (Table S2 †). 36 Simultaneously, the small hyperne splitting of the N atom indicates that the EPR signal is caused by the ligand-based delocalization of the p-electron. 37 Similar results were observed at 70 C using directly synthesized Cu II LH 2 (Fig. S15 †). These results suggest that Cu II LH 2 was distorted and led to a ligand-based radical at 70 C.
The Cu K-edge XANES spectra of in situ generated Cu II LH 2 show that the oxidation states of copper decreased (Fig. S16 †). 38 Furthermore, the variable-temperature EPR experiment of in situ generated Cu II LH 2 was conducted. Fig. 2 shows that the signal of Cu(II) is gradually enhanced with the decrease in temperature. The results indicated that some Cu(I) (3d 10 , EPR silent) species were present in the solution at 70 C, but changed to Cu(II) with the decrease in temperature. 11d,39 Based on the above experimental evidence, this phenomenon can be explained by the intramolecular redox reaction of Cu II LH 2 at 70 C, which results in the ligand-based unpaired p-electron and Cu(I). 39c For convenience, we express Cu II LH 2 at 70 C as a Cu I -radical species, Cu I LH 2 c. Subsequently, the cause of an intramolecular redox reaction in Cu II LH 2 was explored. For electron transfer to occur between the ligand and the central Cu, the ligand's p orbital needs to overlap with the Cu d x 2 Ày 2 orbital. 39b However, the optimized molecular structure of Cu II LH 2 is still planar, and the electronic spectral peaks in the long-wavelength region primarily corresponded to ILCT (Fig. S17 and Table S3 †). Obviously, intramolecular electron transfer cannot occur in the Cu II LH 2 plane structure because the ligand p orbital is perpendicular to the Cu d x 2 Ày 2 orbital. Therefore, the molecular structure of Cu II LH 2 needs to be distorted to achieve antiferromagnetic coupling between the d x 2 Ày 2 orbital of Cu(II) and the p z orbital of the ligand. Due to the change in the N atom hybridization from sp 2 to sp 3 (Cu II L to Cu II LH 2 ), the exibility of Cu II LH 2 increased. 30 This increases the possibility of changes in the ligand's spatial position. The speculated geometry of distorted Cu II LH 2 (Cu I LH 2 c) is shown in Fig. S18. † The absorption spectrum obtained by excited state calculations is similar to that of in situ generated Cu II LH 2 at 343 K (Fig. 1B), which proves that the conjecture is reasonable (Fig. S19 †). The long-wavelength bands of Cu I LH 2 c were attributed to ILCT and ligand-to-metal charge transfer (LMCT)  Table S4 †). 16 In addition, the singlepoint energy calculations showed that the energy of Cu I LH 2 c is 7.4 kcal mol À1 higher than that of Cu II LH 2 , which explained the different molecular structures caused by the temperature (Fig. 1E).
Aer the formation of Cu I LH 2 c, the addition of O 2 caused a new EPR signal (g ¼ 2.0043, Fig. 1D). Combined with the hyperne coupling to a N atom and four H atoms on the ligand, we assigned this EPR spectrum to the Cu II LHc radical (Fig. 1E). Furthermore, the spin density plot of Cu II LHc also showed that electrons were mainly concentrated in the conjugated system, which is consistent with the EPR data (Fig. 1E). Simultaneously, the EPR spectra of the HOOc radical (A N ¼ 14.3, A H(b) ¼ 11.7, g ¼ 2.0061) was captured using DMPO (dimethyl pyridine N-oxide) as a trapping agent (Fig. S21 †). 40 This indicates that an electron and a proton were transferred from Cu I LH 2 c to O 2 . In contrast, O 2 cannot be activated at room temperature by Cu II LH 2 .
The reaction between Cu II L and NHPI involves the transfer of two electrons and two protons, and a stepwise thermochemical analysis was conducted. In the process, an electron and a proton of NHPI are transferred to Cu II L to form Cu II LHc; the Hc transfer in both stepwise electron transfer-proton transfer (ET-PT) (DG ¼ 24.0 kcal mol À1 ) and PT-ET (DG ¼ 65.6 kcal mol À1 ) processes involved high-energy intermediates (Scheme S1A †). In contrast, the PCET pathway (DG ¼ 5.6 kcal mol À1 , per Hess's Law) has obvious thermodynamic advantages. 41 Similarly, the PCET pathway of the process, where an electron and a proton are transferred from NHPI to Cu II LHc to form Cu II LH 2 (DG ¼ 23.2 kcal mol À1 ), is energetically favorable (Scheme S1B †). Furthermore, the total energy (DG ¼ 28.8 kcal mol À1 ) of the transfer of two electrons and protons from NHPI to Cu II L is relatively low, which indicates that PCET is a possible reaction pathway.
The radicals involved in oxidation were conrmed by the complete inhibition of oxidation by the addition of 10 mol% BHT (free radical scavenger) (Table 1, entry 10); therefore, the identication of reactive oxygen species was carried out by spin trapping. During isobutane oxidation, the EPR spectrum of the t Bu-alkoxy radical captured by DMPO (A N ¼ 14.5, A H(b) ¼ 10.5, g ¼ 2.0062) was detected. 42 However, this signal was broadened, which resulted in other radical signals being covered (Fig. S22 †). However, the adducts of DMPO and the t Bu-alkoxy radical (m/z ¼ 186.1494) and DMPO and cOH radicals (m/z ¼ 137.0857) are identied by mass spectrometry (Fig. 3A, Fig. S23 and S24 †). This indicates the presence of cOH radicals in the reaction system. H 2 O 2 was also detected in the oxidation of isobutane by the probe reaction (Fig. S25 †). In the contrast experiment, Cu II L efficiently decomposed H 2 O 2 to produce cOH radicals (Fig. S26 †). To further conrm the existence of cOH radicals in the reaction system, Fig. S27A † shows the EPR spectrum of the reaction solution using BMPO (5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide) 43 as a capture agent, which is consistent with the cOH radical generated by the Fenton reaction captured by BMPO (Fig. S27B †). In addition, cOH radical can be quenched by ethanol. 44 The reaction solution of oxidation of isobutane was quenched with ethanol, and the cC 2 H 4 OH radicals were captured by DMPO ( Fig. S28A and S28B †). This phenomenon was also consistent with that of the Fenton reaction (Fig. S28C †). These results indicated that an cOH radical is the oxygen species in the reaction process.
Nitroxyl radicals are widely used in the aerobic oxidation of C-H bonds, but they have some limitations such as a long initiation period, a short existence time and self-decomposition. 20d In the Cu II L-NHPI catalytic system, time-dependent experiments were conducted to investigate the PINO radical by EPR. The PINO radicals are rapidly produced and accumulated to maximum concentrations within 60 min, which are attributed to the bionic catalyst Cu II L superior hydrogen extraction ability (Fig. 3B). Furthermore, the concentration of the PINO radical decreased slowly, lasting up to 480 minutes (Fig. 3B). This indicated the existence of persistent PINO radicals in the Cu II LÀNHPI system, which is desirable for efficient reactions. 23b,45 Combined with the time curves of isobutane oxidation, the initiation period and rapid reaction phase were synchronized with a change in the PINO radical (Fig. 3B). This further illustrates the importance of the PINO radical. In addition, when isobutane was replaced with cyclohexane-d 12 , the recovered NHPI contained deuterium products NHPId (Fig. S29 †). This phenomenon suggests that PINO radicals activate substrates by extracting a hydrogen atom from the C-H bond of the substrate. 20e To obtain direct evidence about the carbon-centered radical, radical capture experiments were performed in an Ar atmosphere. As shown in Fig. 3C, the spin-trap experiments with DMPO revealed the presence of a carbon-centered tert-butyl radical (Sim-C, A N ¼ 14.3, A H(b) ¼ 21.8, g ¼ 2.0048) and a nitrogen-centered radical (Sim-N, 42 To our knowledge, this is also the rst report of the capture of a tert-butyl radical (C4) during homogeneous catalytic oxidation. However, this signal was unstable due to its high activity. When isobutane was replaced by cyclohexane, we captured cC 6 H 11 , C 6 H 11 Oc, and C 6 H 11 OOc radicals under an O 2 atmosphere (Fig. S30 †). In addition, 1- Fig. 2 Variable temperature X-band EPR spectra of in situ generated Cu II LH 2 . Cu II L (0.1 mM) and NHPI (0.2 mM) reacted in an argon atmosphere at 70 C for 2 hours, and then were immediately placed in a 210 K environment for the EPR test.
bromo-2-methylpropane and 2-bromo-2-methylpropane were identied by adding 50 mol% CBr 4 (Fig. 3C). This indicates that in the Cu II LÀNHPI system, C-H bond activation mainly occurred via the formation of a carbon-centered radical.
According to previous studies, the C-H activation approach via the HAT process was likely to be involved in the ratedetermining step (RDS). 46 The kinetic isotope effect (KIE) experiment was carried out by in situ infrared spectroscopy. Cyclohexane oxidation was used as a model reaction. The reaction kinetics was studied by monitoring the absorption peak of the oxidation product cyclohexanone at 1704 cm À1 (C] O bond) over time ( Fig. 3D and S31 †). A KIE value of 1.5 (K H /K D ) was obtained for the oxidation of cyclohexane and cyclohexaned 12 by the Cu II LÀNHPI system. Thus, the C-H activation step should not be the rate-determining step. 47 However, when NHPI-d was used for oxidation, the value of KIE was 3.3, which indicates that the activation of NHPI is the rate-determining step. 48 Based on the experimental results and thermochemical calculations, a possible reaction pathway was outlined (Fig. 3E). First, Cu II L reacts with NHPI (electron and proton donor) through two successive PCET processes to form Cu II LH 2 . This process may be the rate-determining step of the reaction through the KIE experiment. Subsequently, Cu II LH 2 is converted to a Cu I -radical species Cu I LH 2 c through an intramolecular redox reaction at 70 C. Cu I LH 2 c is a highly reactive radical intermediate that reacts with O 2 to produce Cu II LHc and HOOc radicals. Next, the catalytic cycle returns to the starting point, with the formation of H 2 O 2 by the transfer of electrons and proton from Cu II LHc to HOOc. H 2 O 2 is rapidly decomposed into reactive oxygen species cOH under the reaction conditions. Activation of O 2 by the Cu II L-NHPI system is similar to the oxidation of alcohol by galactose oxidase. More importantly, this process is also accompanied by the production of a persistent PINO radical for substrate C-H bond activation by HAT. Subsequently, the carbon-centered radical reacts with reactive oxygen species in the reaction system to generate oxidation products.

Conclusions
In summary, we developed the protocol for C-sp(3)-H efficient catalytic aerobic oxidation under mild conditions based on a GOase model compound Cu II L and NHPI. In particular, the Cu II L-NHPI system showed excellent performance for the oxidation of light alkanes (C2-C4). Cu II L can activate NHPI efficiently to generate PINO radicals and Cu II LH 2 through the PCET pathway. Based on the EPR tests and theoretical calculations, the intramolecular redox process of Cu II LH 2 has been proved. Throughout the catalytic cycle, O 2 is reduced to its active form and the C-sp(3)-H bond of the substrate is converted to a highly active carbon-centered radical by PINO through the HAT pathway. Kinetic experiments showed the activation of NHPI was the rate-determining step. Meanwhile, the generation of PINO radicals was demonstrated by timeresolved EPR tests. It indicated the persistent radicals played an important role in oxidation.

Data availability
Data for this paper, including reaction screening and optimization experiments, detailed experimental procedures, spectral data, and computational study details, are provided in the ESI. † Author contributions X. L. and X. Z. developed the concept, designed these experiments, analyzed the experimental data and wrote the paper. J. S. analyzed the EPR experimental data. H. Y., J. H., C. X., Y. H., Q. H., D. X., and C. X. contributed to catalyst synthesis, catalytic experiments and analyzed the experimental data. X. Z. and H. J. directed the project. All authors discussed the results and provided comments during the manuscript preparation.

Conflicts of interest
There are no conicts to declare.