Introduction

Copper-bound reactive oxygen adducts, such as Cu-superoxo, -peroxo, -hydroperoxo, and -oxyl radical species, have been proposed to be reactive intermediates in biological systems1,2. One such intermediate, specifically a Cu(II)-hydroperoxo complex, has received much attention since it has been hypothesized to play an important role in the catalytic cycle of certain copper-containing enzymes (Fig. 1). In peptidylglycine-α-hydroxylating monooxygenase (PHM) and dopamine-β-monooxygenase (DβM), it has been proposed that Cu(II)-hydroperoxo species were converted to Cu(II)-oxyl radicals and water after electron and proton transfers3,4,5. The reactive species in lytic polysaccharide monooxygenases (LPMOs) has also been proposed to be a Cu(II)-hydroperoxo intermediate that directly reacts with polysaccharides6,7,8,9.

Fig. 1
figure 1

Bioinorganic context. Representative mechanism of copper intermediates involved in dioxygen activation in metalloenzymes

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In synthetic chemistry, a number of Cu(II)-OOH species have been prepared and characterized by spectroscopic and crystallographic methods. Masuda’s group reported the first crystal structure of the Cu(II)-OOH complex bearing a trigonal bipyramidal ligand where the hydroperoxo bound Cu(II) core was stabilized through intramolecular hydrogen bonding interactions10. Most recently, stabilization of [Cu(biot-et-dpea)(OOH)]+ has been achieved in artificial proteins by using antigen-antibody interaction11. In general, the reaction of Cu(II)-OOH complexes with substrates occurs through O–O bond cleavage to give putative Cu(II)-oxyl radical or Cu(III)-oxo species. Detailed studies on the ligand hydroxylation by the putative intermediates have been reported by Karlin and co-workers12. Recently, it was also reported that the cleavage of Cu–O bond in Cu(II)-OOH species results in the N-dealkylation through the Fenton chemistry13.

Notably, to our knowledge no reports of direct reactions of Cu(II)-OOH species with external substrates, i.e., prior to O–O bond cleavage, have yet appeared in the literature, although numerous examples of electrophilic reactivity in Cu(II)-OOH complexes have been shown to occur via O–O bond cleaved intermediates14,15,16. By contrast the nucleophilic reaction of Cu(II)-alkylperoxo complexes was reported very recently17.

Herein, we report the nucleophilic reactivity of the Cu(II)-hydroperoxo complex, [Cu(iPr3-tren)(OOH)]+ (1, iPr3-tren = tris[2-(isopropylamino)ethyl]amine), which we find is capable of oxidation of organic carbonyl compounds directly. 1 was successfully characterized by various spectroscopic methods such as UV-vis, resonance Raman (rRaman), and electron paramagnetic resonance (EPR) spectroscopy together with cold spray ionization mass spectrometry (CSI-MS). The nucleophilic reactivity of 1 has also been investigated by kinetic studies including a Hammett analysis.

Results

Synthesis and characterization of copper complexes

As a starting material, the copper(II) complex, [Cu(iPr3-tren)(CH3CN)]2+, was synthesized by combining iPr3-tren and Cu(ClO4)2·H2O in acetonitrile and characterized using UV-vis, electrospray ionization mass spectrometry (ESI-MS) and X-ray crystallography (see the Supplementary Methods, Supplementary Tables 1 and 2, and Supplementary Figs. 1 and 2). The copper(II)-hydroperoxo complex, [Cu(iPr3-tren)(OOH)]+ (1), was generated by the addition of 5 equiv of H2O2 and 2 equiv of TEA to [Cu(iPr3-tren)(CH3CN)]2+ in CH3OH at –50 °C (Fig. 2). Spectrophotometric titration used to monitor the formation of 1 revealed that 5 equiv of H2O2 is required for full formation (Supplementary Fig. 3). The intermediate 1 is observed to be metastable (e.g., ca. 5% decay for 1 h at –50 °C). The color of the solution changed from blue to green, with the final solution showing an intense band at 360 nm (ε = 1300 M–1 cm–1) and two weak bands at 656 (ε = 230 M–1 cm–1) and 830 nm (ε = 270 M–1 cm–1) in the UV-vis spectrum, which are characteristic features of Cu(II)-hydroperoxo complexes (Fig. 3a)18,19,20,21,22,23,24. The former band has been assigned to a LMCT band of the Cu(II)-OOH species, and the latter bands to the d-d transitions of the Cu(II) ion bearing a trigonal bipyramidal structure.

Fig. 2
figure 2

Synthetic procedure for 1. DFT structure of 1 (Black, C; Cyan, H; Blue, N; Red, O; Green, Cu). Hydrogen atoms are omitted for clarity except for hydrogen atoms of N–H and O2–H groups

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Fig. 3
figure 3

Characterization of complex 1. a UV-vis spectrum of 1 in CH3OH at –50 °C. Inset shows the rRaman spectra of 1 (16 mM) prepared with H216O2 (red line), H218O2 (blue line), and the difference spectrum (black line), which were obtained upon excitation at 405 nm in CH3OH at –30 °C. b CSI-MS for 1-16O2H (red line) and 1-18O2H (blue line) in CH3OH at –40 °C, and for 1-O22H (green line) prepared with 2H2O2 in CH3O2H at –40 °C. Asterisk is assigned to thermally decomposed species of 1. c X-band EPR spectrum of 1 in frozen CH3OH at 113 K. Spectroscopic settings: frequency = 9.155 GHz, microwave power = 0.998 mW, modulation frequency = 100 kHz, and modulation amplitude = 0.6 mT

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The CSI-MS spectrum of 1 supported the formation of Cu(II)-hydroperoxo species, [Cu(iPr3-tren)(OOH)]+ (1-16O2H) at a mass-to-charge ratio (m/z) of 368.2 (calcd m/z 368.2; Fig. 3b, red line), together with some unidentified species presumably resulting from the thermal instability of 1 (Supplementary Fig. 4). When the reactions were carried out with isotopically labeled H218O2 in CH3OH and 2H2O2 in CH3O2H, mass peaks corresponding to [Cu(iPr3-tren)(18O18OH)]+ (1-18O2H) (Fig. 3b, blue line) and [Cu(iPr3-tren)(OO2H)]+ (1-O22H) (Fig. 3b, green line) were observed at m/z 372.2 (calcd m/z 372.2) and 369.2 (calcd m/z 369.2), respectively. The four-mass unit shift upon substitution of 16O with 18O demonstrates that 1 contains two oxygen atoms. The one-mass unit shift in a deuterium isotope experiment verifies the existence of a hydroperoxide ligand in 1. The rRaman spectrum of 1 was collected using 405 nm excitation in CH3OH at –30 °C (Fig. 3a, inset). 1-16O2H exhibits two isotope sensitive bands at 834 and 501 cm−1, which shifted to 790 and 482 cm−1, respectively, in 1-18O2H. The results support the assignments of these features as O–O and Cu–O stretching vibration on the basis of 16-18Δ value of 44 and 19 cm−1 (16-18Δ (calcd) = 48 and 23 cm−1 for diatomic harmonic oscillator), respectively18,19,20,21,22,23,24. Upon 2H-substitution in 1, the characteristic Cu–O and O–O stretching vibrational features exhibited a 2-cm−1 downshift each (Supplementary Fig. 5), which is comparable to those of a number of metal-hydroperoxo species (Supplementary Table 3)24,25,26,27,28,29,30.

The EPR spectrum of a frozen solution of 1 at 113 K shows a reverse axial signal with g┴ = 2.26 (A┴ = 108 G) and g║ = 2.06 (A║ = 70 G), which is typical for trigonal bipyramidal five-coordinate Cu(II) complexes (Fig. 3c; see also Supplementary Fig. 6)2,18,31,32,33,34,35,36,37. Spin quantification finds that the EPR signal corresponds to 91(6)% of the total copper content in the sample (see Supplementary Methods). All the spectroscopic results clearly show that 1 is assigned to be a Cu(II)-hydroperoxo complex in trigonal bipyramidal geometry, which is further supported by DFT calculations (vide infra).

Computational study

To investigate the geometric structure of 1, DFT calculations were conducted at the spin-unrestricted B3LYP level of theory (see Supplementary Methods). The optimized structure of 1 reveals the trigonal bipyramidal geometry whose axial site is occupied by the hydroperoxo group in the manner of end-on replacing an acetonitrile of [Cu(iPr3-tren)(CH3CN)]2+ (Fig. 2, and see also Supplementary Table 4). This result is in agreement with the result of EPR spectroscopy described above. The calculated O–O bond distance is determined to be 1.46 Å, which is very close to that determined crystallographically for Masuda’s Cu(II)-hydroperoxo complex (1.460 Å)10 and also that observed for homoprotocatechuate 2,3-dioxygenase (1.5 Å)38.

TD-DFT calculations were employed to investigate the electronic structure of 1. The calculated spectrum exhibits an intense peak at 368 nm and multiple weak peaks in the range from 650 to 850 nm which are seen in experimental UV-Vis spectrum (Supplementary Fig. 7). The peak at 368 nm was comprised of electron transitions from overlapped orbitals between the ligand and Cu d-orbitals to σ* orbital of the dz2 orbital in Cu and pz orbital in O2H. The minor peaks in the range from 650 to 850 nm are assigned as electron transitions between π* orbitals of Cu-OOH moieties. The singly occupied molecular orbital (SOMO) for 1 was observed to be σ* orbital whose spin density distribution was calculated indicating that about half a radical belongs to Cu center and the rest is contained in the O2H group and ligand in doublet state (Supplementary Fig. 8; Supplementary Table 5). Natural population analysis (NPA) depicted atomic charges for 1, which demonstrates that the proximal oxygen atom possesses more negative charge than the distal oxygen atom (Supplementary Fig. 9)39. Thus, overall calculations are indicative of a trigonal bipyramidal Cu(II)-hydroperoxo intermediate for 1.

Reactivity study of 1

The reactivity of 1 was examined in oxidation reaction with organic substrates. The electrophilic reactivity of 1 was tested in the oxidation of triphenylphosphine (i.e., oxygen atom transfer) and cyclohexadiene (i.e., hydrogen atom abstraction). Upon addition of the substrates to 1 in CH3OH at –50 °C, 1 remained intact without showing any spectral changes (Supplementary Fig. 10). Only trace amounts of products were detected in the product analysis of the reaction solutions. These results indicate that 1 is not capable of oxidizing substrates in electrophilic oxidation under the reaction conditions.

Nucleophilic reaction with acyl chloride

The nucleophilic character of 1 was established in oxidation of acyl chlorides, which are electrophile substrates. On the addition of 100 equiv of benzoyl chloride (PhCOCl) to 1 in CH3OH at –50 °C, the characteristic absorption bands of 1 disappeared through a first-order decay profile (Fig. 4a). After the reaction was completed, product analysis revealed the formation of benzoic acid in a quantitative yield (Supplementary Table 6). Reaction of 1 with a series of para-substituted benzoyl chlorides (para-X-PhCOCl, X = OCH3, CH3, F, H, Cl) gave a linear correlation of the Hammett plot of the first-order rate constant against σp+ resulting a ρ value of 2.0(3) (Fig. 4b). The positive ρ value indicates nucleophilic character for 1.

Fig. 4
figure 4

Reactions of 1 with benzoyl chloride derivatives in CH3OH at –50 °C. a UV-vis spectral changes of 1 (1 mM) upon addition of 100 equiv of benzoyl chloride. Inset shows the time course of the absorbance at 360 nm. b Hammett plot of logkobs against σp+ of para-X-Ph-COCl (X = OCH3, CH3, F, H, Cl)

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Aldehyde deformylation reaction

It is well known that metal-peroxo species are frequently capable of nucleophilic reactivity. Since it has been reported that the iron-hydroperoxo species is much more reactive than the corresponding iron-peroxo species in aldehyde deformylation40, the reactivity of 1 was further examined in the oxidation of 2-phenylpropionaldehyde (2-PPA) and cyclohexylcarboxaldehyde (CCA). Kinetic studies of 1 with CCA in CH3OH at –70 °C exhibit a pseudo-first-order reaction profile (Supplementary Fig. 11). A plot of the first-order rate constants against the concentration of CCA shows a linear correlation, giving a second-order rate constant (k2) of 6.5(3) M–1 s–1 (Fig. 5a). Similarly, the k2 value of 1.5(1)×10–1 M–1 s–1 was determined in the reaction of 1 and 2-PPA at –50 °C (Supplementary Fig. 12b). Cyclohexene (84(9)%) and acetophenone (93(7)%) were obtained in the oxidation of CCA and 2-PPA, respectively, as final products. Also 1 changed back to the Cu(II) precursor after completion of aldehyde deformylation reaction (Supplementary Fig. 13). The k2 values were dependent on the reaction temperature where the activation parameters for oxidation of aldehydes by 1 were determined to be ΔH‡ = 23(1) kJ mol–1 and ΔS‡ = −112(2) J mol–1 K–1 in the range of 193–223 K for CCA (Fig. 5b), and ΔH‡ = 31(1) kJ mol–1 and ΔS‡ = −118(7) J mol–1 K–1 in the range of 213–243 K for 2-PPA (Supplementary Fig. 12c). A bimolecular mechanism for the nucleophilic oxidative reaction by 1 is suggested by the observed negative entropy and the second-order kinetics. Compared with the reactivity of a previously reported iron(III)-hydroperoxo complex40 and copper(II)-alkylperoxo adducts17, the reactivity of 1 (k2 = 3.6 × 10–1 M–1 s–1 at –40 °C) is higher than that of the iron(III)-hydroperoxo species (k2 = 1.3 × 10–1 M–1 s–1 at –40 °C) and those of copper(II)-alkylperoxo complexes (k2 = 1.2–1.8 × 10–1 M–1 s–1 at –40 °C) in aldehyde deformylation of 2-PPA (Supplementary Table 7). It should be noted that the reactivity of 1 (k2 = 3.4 M–1 s–1 at –80 °C) with CCA is also higher than that of a Cu(II)-superoxo complex, [Cu(βDK)(O2)]– (k2 = 1.4 M–1 s–1 at –80 °C), which was a highly reactive oxidant in aldehyde deformylation reported so far (Supplementary Table 8)41. Very recently, it has been reported that an anionic copper(II)-superoxo complex acts as a base than a nucleophile in the aldehyde deformylation of wet 2-PPA42. Furthermore, nucleophilic reactivity has been observed for an Fe(III) porphyrin peroxo complex in the epoxidation of electron-deficient olefins43,44. Thus, we also explored epoxidation of olefins such as cyclohexene and 2-cyclohexene-1-one, which is an electron-deficient olefin, by 1 in CH3OH at –50 °C (Supplementary Figs. 14 and 15). Product analyses of the reaction solutions with olefins did not show oxidized products. These results suggest that 1 lacked reactivity in the epoxidation of electron-deficient olefins.

Fig. 5
figure 5

Reactions of 1 with CCA in CH3OH. a Plot of kobs against CCA concentration to determine a second-order rate constant for 1 at −70 °C. b Plot of second-order rate constants against 1/T to determine activation parameters

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Discussion

Based on the results of kinetic and product analyses, there are two possible initial steps for the nucleophilic reaction, a proximal oxygen attack and a distal oxygen attack by 1 (Fig. 6). The formation of a peroxyhemiacetal intermediate initially proceeds via an attack of the proximal oxygen to the carbonyl center of aldehydes, while the formation of a [Cu(iPr3-tren)]2+ bound peroxyhemiacetal-like species occurs through a proton transfer and a distal oxygen attack to the aldehydes. The calculated atomic charge distribution of the proximal oxygen (–0.60) from NPA is more negative than that of the distal oxygen (–0.49) (Supplementary Fig. 8), supporting the proximal oxygen attack mechanism. However, the negative charge difference is small. Thus, further detailed DFT studies such as reaction coordinate calculations are under investigation.

Fig. 6
figure 6

Proposed mechanism for the reaction of 1 and aldehydes. In both cases the Cu(II)-hydroperoxo complex is proposed to act as a nucleophile

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In this study, we have demonstrated the oxidation of organic carbonyl compounds by 1, giving the first, to our knowledge, example of nucleophilic reactivity of a copper(II)-hydroperoxo complex. 1 was characterized by various methods such as UV-vis, CSI-MS, rRaman and EPR. These results clearly indicate that 1 is an end-on Cu(II)-hydroperoxo complex with trigonal bipyramidal coordination geometry, which is also supported by isotope labeling experiments and DFT calculations. The nucleophilic character of 1 was confirmed by a positive slop in the Hammett plot for the reaction of 1 and para-substituted acyl chlorides. Kinetic studies represent that 1 is a very reactive nucleophilic oxidant in aldehyde deformylation. We believe the observed oxidative nucleophilic reactivity of 1 demonstrates a new class of reactivity for Cu(II)-hydroperoxo intermediates.

Methods

Preparation of [Cu(iPr3-tren)(CH3CN)](ClO4)2

[Cu(iPr3-tren)(CH3CN)](ClO4)2 was prepared by reacting Cu(ClO4)2·6H2O (0.185 g, 0.50 mmol) and tris[2-(isopropylamino)ethyl]amine (iPr3-tren) (176.42 μL, 0.50 mmol) in CH3CN (3.0 mL). The mixture was stirred for 12 h, giving a blue solution. Et2O (40 mL) was added to the resulting solution to yield a blue powder, which was collected by filtration, washed with Et2O, and dried in a vacuo. Yield: 97% (0.2799 g). X-ray crystallographically suitable crystals were obtained by slow diffusion of Et2O into a solution of the complex in CH3CN (Supplementary Table 1 and 2; Supplementary Fig. 1). ESI-MS in CH3CN (Supplementary Fig. 2): m/z 167.6 for [Cu(iPr3-tren)]2+, m/z 188.2 for [Cu(iPr3-tren)(CH3CN)]2+, m/z 208.7 for [Cu(iPr3-tren)(CH3CN)2]2+, and m/z 434.3 for [Cu(iPr3-tren)](ClO4)+. Anal. Calcd for C17H39CuN5O8: C, 35.45; H, 6.82; N, 12.16. Found: C, 35.54; H, 7.05; N, 12.16. The effective magnetic moment of μeff = 1.8 B.M. was determined by 1H NMR Evans method in CD3CN at 25 °C.

Generation of [Cu(iPr3-tren)(OOH)]+ (1)

Treatment of [Cu(iPr3-tren)(CH3CN)](ClO4)2 (1 mM) with 5 equiv of H2O2 in the presence of 2 equiv of triethylamine (TEA) in CH3OH at −50 °C afforded a green solution. [Cu(iPr3-tren)(18O18OH)]+ and [Cu(iPr3-tren)(OO2H)]+ were prepared by adding 5 equiv of H218O2 (36 μL, 95% 18O-enriched, 2.2% H218O2 in water) and 5 equiv of 2H2O2 (30% in 2H2O), respectively, to a solution containing [Cu(iPr3-tren)(CH3CN)](ClO4)2 (1 mM) and 2 equiv of TEA in CH3OH at −50 °C. UV-vis in CH3OH: λmax (ε) = 360 nm (1300 M−1 cm−1), 656 nm (230 M−1 cm−1) and 830 nm (270 M−1 cm−1).

X-ray crystallography

See Supplementary Methods, Supplementary Fig. 1, and Supplementary Tables 1 and 2.

Computational details

See Supplementary Methods, Supplementary Figs. 6, 7, and 8, and Supplementary Tables 4 and 5.

Reactivity studies

See Supplementary Methods, Supplementary Figs. 9–14, and Supplementary Tables 6–8.