Development of Spectrophotometric Method to Assay Sulfadiazine in Pure and in Pharmaceutical Dosage form through Diazotization and Coupling Reaction

This study involves the development of a sensitive accurate and low-cost spectrophotometric procedure to analyze sulfadiazine as pure and in its dosage forms. In this method sulfadiazine was diazotized with an equimolar solution of sodium nitrite in the presence of hydrochloric acid to form corresponding diazonium salt followed by coupling with phloroglucinol reagent in basic solution of NaOH to produce a yellow water-soluble azo dye that has maximum absorption at 417 nm versus reagent blank. Under optimum conditions. The linearity of the method obeyed Beer’s law in the concentration range 0.25-15 μg/ml with an excellent determination coefficient (R 2 = 0.9992) and molar absorptivity 3.81×10 4 l/mol.cm. The detection limit (LOD) and quantification limit (LOQ) have also been estimated and their values were found to be 0.192 and 0.563 μg/ml, respectively. A relative error% was calculated and found in the range -2.75% to 4.02%, while the precision (RSD%) was estimated as ≤ 1.36%. The stoichiometry of the resulting azo dye was found to be 1:1 sulfadiazine: with stability constant 0.37×10 7 l/mol. The suggested procedure was applied to the analysis of sulfadiazine in cream and veterinary drugs (powder and injection).

INTRODUCTION
Sulfonamides are a class of drugs that have been commonly used as antibacterial agents in veterinary practice and in medicine especially in the treatment of eye infections and urinary tract infections as a prophylaxis of rheumatic fever (Petri, 2001). Sulfadiazine (SDZ), is one of the common sulfonamide's groups in clinical practice, chemically it is [4-amino-N -pyrimidin-2-ylbenzenesulfonamide], (John and John, 2004), whereas its chemical structure is: C 10 H 10 N 4 O 2 S M.Wt. =250.3g/mol SDZ is a synthetic agent that has been used in human and veterinary therapy about 60 years (Pecorelli et al., 2004) . It eliminates bacteria that cause infections by inhibiting the formation of folic acid inside the cell of bacteria (Vandana et al., 2011). SDZ has been employed to those prophylaxis furthermore in the medication of contamination for parasitic protozoa called toxoplasmosis gondii and also in the medicine for contamination brought about because of pneumocystis carinii, an extracellular lung pathogen, which makes pneumonia for immunecompromised hosts. (Vree et al., 1995). The literature survey revealed that several methods have been reported for direct or indirect estimation of SDZ, they include: potentiometry (Ayman et al., 2009), Nanozyme-labeled biomimetic immunoassay (He et al., 2020), capillary zone electrophoresis (Berzas et al., 2001), flow injection-flame atomic absorption (FI-FAAS) device (Haji Shabani et al., 2011), tandem mass spectrometric detection, (Forti and Scortichini, 2009), ICPatomic emission spectroscopy (Xiao-Ling et al., 2010) and immune-chromatographic assay (Wang et al., 2007). However, most of these methods either require several manipulation steps, sophisticated, cloud point extraction and expensive instruments which may not be available in every laboratory or they had high detection limit.
Several important spectrophotometric methods have also been employed to assay of SDZ in bulk and in its pharmaceutical preparations. Some of these methods based on the diazotization coupling reaction by using various organic coupling reagents such as, histidine (Othman and Kadder, 2006), oxine (Naik et al., 2007), pyrogallol (Naser et al., 2018), 2,6dihydroxybenzoic acid (Mohammed and Zebary, 2013) and 4-amino-2-hydroxy acetophenone (Al-Rufaie, 2016). Other methods are either based on the oxidative coupling reactions of SDZ with p-N,N-diethylphenylenediamine sulphate using KIO 4 as oxidizing agent (Nagaraja et al. , 2010) . In addition, determination of SDZ via charge transfer complex formation with phenosaphranine (Al-Attas, 2003) have been also reported. SDZ was also estimated through the oxidation of SDZ with permanganate solution in alkaline medium producing bluish-green colour of manganate MnO 4 2appeared absorption peak at 610 nm (Siddiqui et al., 2013). In the present investigation, a phloroglucinol (Phl) reagent has been employed to develop a simple , fast and inexpensive visible spectrophotometric method for the assay of SDZ in pure form and in its pharmaceutical drugs. This method was based on the reaction of Phl reagent with diazotized SDZ to yield a water-soluble yellow dye in a basic solution that has been employed to assay of SDZ in burn cream, powder and veterinary injectable solution.

EXPERIMENTAL Apparatus
A double beam UV-visible spectrophotometer (JASCOV-630) with 1.0-cm quartz cells was used for all absorbance measurements and absorption spectra. While a professional Benchtop pH meter BP3001 was used for the pH measurements.

Chemical Reagents
All chemical substances used were of the highest purity available and were obtained from Fluka, BDH and Merk companies. The standard material of SDZ was provided from the state company for drug industries and medical appliances (SDI), Samarra-Iraq.
-SDZ Stock Standard Solution 500 μg/ ml: A 0.0500 g of pure SDZ was dissolved in about 5 ml of ethanol and the volume was then made up to 100 ml with distilled water in a 100 ml calibrated flask. This solution was kept in a dark bottle. Similarly, a working standard solution (50 μg /ml = 1.9976 × 10 -4 M) of SDZ was freshly prepared by diluting 10 ml of the stock solution with distilled water in a 100 ml calibrated flask (Mohammed and Zebary, 2013). Phloroglucinol (Phl) Solution 0.1% w/v: A 0.100 g of Phl reagent was dissolved in small portions of distilled water and the volume was made to 100 ml in a calibrated flask with the same solvent. The solution was then transferred to a dark bottle. Sodium nitrite stock solution 1.9565 × 10 -3 M: This solution was prepared by dissolving 0.0135 g of NaNO 2 in 100 ml distilled water using a volumetric flask. Similarly, a working solution 1.9976 ×10 -4 M of NaNO 2 was then prepared by diluting 10.2 ml of the stock solution with distilled water in a 100 ml calibrated flask.

Recommended Procedure and Calibration Curve
Into a series of 20 ml calibrated flasks, 0.1 to 7 ml of 50 μg/ml pure SDZ solution, an equimolar of 1.9976×10 -4 M NaNO 2 and 1ml of 1M hydrochloric acid solutions were added. The contents were mixed thoroughly and kept constant at room temperature for 5 min then followed by adding 1.5 ml of 0.1% Phl reagent and 2 ml of 1M sodium hydroxide solutions. The contents were Shaked well and made up to the mark with distilled water. The absorbance of the azo dye formed was recorded at 417 nm against blank solution that was prepared similarly but without drug. A linear relationship between absorption and SDZ concentration in the range 5 300 μg of SDZ/20 ml was obtained Fig. (1). The apparent molar absorptivity and Sandell's sensitivity were found to be 3.81 ×10 4 l/mol.cm. and 0.00657 μg/cm 2 , respectively.

Recommended Procedure to Assay SDZ in Drug Injection solution:
The injection container (each ml of injection contains 200 mg SDZ and 40 mg trimethoprim, TMP) was mixed well to get a homogeneous solution. A 1ml of the injection solution was diluted with distilled water in 100 ml calibrated flask to prepare 2000 μg/ml SDZ solution and similarly a 10 ml of it was pipetted into a 100 ml calibrated flask and with distilled water made up to the mark (this is stock solution). A working solution of injection containing 50 μg/ml of SDZ was prepared by diluting 25 ml of the stock solution with distilled water using 100 ml calibrated flask. An aliquot of the diluted drug solution was then treated as done in recommended procedure. Flamalife Cream (each 100 g cream contains 1g silver SDZ): A 1.0 g of flamalife cream was dissolved in 5 ml of ethanol and transferred to 50 ml ether, shake well and moved to 50-ml isolating channel. SDZ was then removed multiple times with 25 ml refined water. The watery layer was gathered, separated and finished to 100 ml with refined water in utilizing volumetric carafe (United States Pharmacopeia, 1995). Each mL of this solution containing 100 μg SDZ. An aliquot of diluted solution of the drug was then analyzed using the recommended procedures.

Powder:
An accurately weighed 0.025g of the drug powder (each 1g powder contains 400 mg SDZ and 80 mg TMP) was dissolved in about 10 ml of distilled water. The resulting solution was then mixed well, filtered and diluted to 100 ml with distilled water in a calibrated flask. Each ml of this solution contains 100 μg of SDZ. The recommended procedure was then followed for the determination of SDZ by taking an aliquot of diluted solution of the drug.

RESULTS AND DISCUSSION Preliminary conditions
Under the response condition, SDZ was reacted with an equimolar of NaNO 2 in acidic solution to give the relating diazonium salt, which goes through a diazotization procedure with Phl as a coupling agent to produce the azo dye (Scheme 1): sulphadiazine diazotized sulfadiazine

Scheme 1
The azo dye gave maximum absorption at 417 nm against blank solution. The dye intensity has been found to be proportional to the SDZ amount that originally present in the solution.

Optimum Conditions for the Reaction Effect of Different Amounts of Various Acids on the Absorbance
The reaction of diazotisation requires acid solution. Therefore, the effect of different amounts of various acidic solutions (1M) such as, HCl, H 2 SO 4 , HCOOH, HNO 3 and CH 3 COOH has been examined on the dye colored. The experimental results are shown in Fig. (2). The data in Fig. (2) illustrate that HCl was convenient as acidic medium for a maximum stability and sensitivity and 1ml of 1M HCl with occasionally shaking for 5 and 6 min were found to be suitable, therefore 5 min was selected for the subsequent investigation. The results are explained in Fig. (3).

Effect of Phl Amount
The influence of various amounts (0.5-2.0) ml of Phl reagent (0.1%) on the azo dye absorbance has been investigated. The results in Fig. (4) indicated that 1.5 ml of Phl (0.1%) are the most suitable volume to give high absorbance and good determination coefficient value (R 2 =0.9995) therefore, it was selected for coupling reaction. Absorbance µg of SDZ /20 ml

Fig. 4: Effect of reagent amounts on absorbance
The effect of time on the completion of the coupling reaction of diazotized SDZ with Phl reagent has been studied by allowing the solution of the reactants after the addition of Phl solution to stand at room temperature for different times Fig. (5) then the other reagents were added and the absorbance was recorded at 417 nm against the reagent blank.  5) reveals that the coupling reaction was complete immediately after mixing the diazotized SDZ with Phl reagent and the absorbance remained maximum and stable for at least 7 min. at room temperature.

Effect of alkaline solution
The effect of different amounts 0.5-3 ml of various alkaline solutions (1M) such as, sodium hydroxide, sodium carbonate, sodium bicarbonate and potassium hydroxide has been examined for the purpose of producing azo dye with intense colour and lower blank value. The results in Fig. (6) revealed that maximum sensitivity and stability were obtained only when the reaction was carried out in the presence of sodium hydroxide solution. While, sodium carbonate and sodium bicarbonate exhibited low sensitivity which may be due to pH variation. Therefore, 2.0 ml of 1M sodium hydroxide at (pH=12.1) was found to be optimum.

Development Time and Stability Period
Under the optimal experimental conditions, the influence of time on the stability of the colored azo dye at 417 nm has been carried out by preparing two different amounts 50 and 150 μg of SDZ. The absorbance was measured at dissimilar pauses of time up to 60 minutes. The results in Fig.(7) illustrate that the absorbance of azo dye reached maximum value after the reaction mixture solution was mixed and the absorbance remained stable for at least 60 minutes at room temperature.

Absorption Spectrum
Under the optimum reaction conditions, SDZ was reacted with equimolar of NaNO 2 solution in the presence of HCl solution to give the corresponding diazonium salt. The diazonium salt was then coupled with Phl reagent in a basic solution of NaOH. A yellow water-soluble azo dye was obtained which showed maximum absorption at 417 nm versus the colorless reagent blank Fig. (8). The intensity of the azo dye formed was found to be proportional to the amount of SDZ originally present in the solution.

Fig. 8: Absorption spectra of 50 ppm SDZ treated according to the recommended procedure versus (A) reagent blank , (B) distilled water and (C) blank solution measured versus distilled water Quantification
The limits of Beer′s law, molar absorptivity (ε max ), accuracy (recovery%), precision (RSD%), conditional stability constant (Hargis, 1988), LOD and LOQ values were calculated and the data are summarized in (Table 1), which indicates that the proposed method is sensitive, precise and accurate. Linearity was represented by the regression equation and the corresponding determination coefficient (R 2 ) for SDZ determined by the proposed method represents the excellent linearity.

Composition of the Azo Dye
The stoichiometry of the product was investigated under the established conditions by applying the continuous variation (Job's) and the mole ratio methods (Delevie, 1997). In continuous variation method, volumes 0.5-4.5ml of 1.9976×10 -4 M portions of SDZ (VS) were diazotized using equimolar of 1.9976×10 -4 M sodium nitrite and 1 ml of 1M HCl and coupled according to the recommended procedure with the corresponding complementary volume of 1.9976×10 -4 M Phl solution (VR) to give a total volume of 5 ml for (VS+VR) in 2 ml of 1M NaOH and diluted to 20 ml with distilled water. In mole ratio method, increased volumes 1-4 ml of 1.9976×10 -4 M Phl solution (VR) were added to a 1 ml of 1.9976×10 -4 M of SDZ (VS) which was diazotized by using 1 ml of 1.9976×10 -4 M sodium nitrite in presence of 1ml of 0.8 M HCl, 2 ml of 1M NaOH were added and the absorbance was recorded at 417 nm after dilution to the mark with distilled water. The results obtained in Fig. (9) are revealed that the azo dye was formed by a 1:1 combining ratio of diazotized SDZ to Phl. The structure of the azo dye according to the results obtained in Fig. (9), can be represented as follows Fig. (10):

Application of the Method
The present method was applied successfully to the analysis of SDZ in various pharmaceutical preparations containing SDZ (cream, veterinary powder and injection). The results are summarized in (Table 2) reveal that the proposed procedure is in good agreement and with the declared content.

Evaluation of the suggested procedure
Standard additions method was followed to check the validity of the suggested procedure which proves that the recommended method can be successfully applied for determining SDZ without interferences. The data are shown and listed in Fig. (11) and (Table 3).  Table 3: The results of standard addition methods for analysis of SDZ CONCLUSION The proposed method for the determination of sulfadiazine in pharmaceutical samples have the advantage to be sensitive, inexpensive and simple since it does not need neither temperature control nor solvent extraction step. The method is also accurate and precise enough to be successfully adopted as an alternative to the existing spectrophotometric method and evaluation of drugs in pharmaceutical preparations.


s2fieldsofstudy: Chemistry Biology

text:
Bromoditerpenes from the Red Seaweed Sphaerococcus coronopifolius as Potential Cytotoxic Agents and Proteasome Inhibitors and Related Mechanisms of Action

Seaweeds are a great source of compounds with cytotoxic properties with the potential to be used as anticancer agents. This study evaluated the cytotoxic and proteasome inhibitory activities of 12R-hydroxy-bromosphaerol, 12S-hydroxy-bromosphaerol, and bromosphaerol isolated from Sphaerococcus coronopifolius. The cytotoxicity was evaluated on malignant cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, and SK-MEL-28) using the MTT and LDH assays. The ability of compounds to stimulate the production of hydrogen peroxide (H2O2) and to induce mitochondrial dysfunction, the externalization of phosphatidylserine, Caspase-9 activity, and changes in nuclear morphology was also studied on MCF-7 cells. The ability to induce DNA damage was also studied on L929 fibroblasts. The proteasome inhibitory activity was estimated through molecular docking studies. The compounds exhibited IC50 values between 15.35 and 53.34 µM. 12R-hydroxy-bromosphaerol and 12S-hydroxy-bromosphaerol increased the H2O2 levels on MCF-7 cells, and bromosphaerol induced DNA damage on fibroblasts. All compounds promoted a depolarization of mitochondrial membrane potential, Caspase-9 activity, and nuclear condensation and fragmentation. The compounds have been shown to interact with the chymotrypsin-like catalytic site through molecular docking studies; however, only 12S-hydroxy-bromosphaerol evidenced interaction with ALA20 and SER169, key residues of the proteasome catalytic mechanism. Further studies should be outlined to deeply characterize and understand the potential of those bromoditerpenes for anticancer therapeutics.

Introduction
Marine natural products (MNP) have been revealed to possess uncommon and diverse chemical structures with a great ability to interact with different biological targets, making maintenance of the intracellular protein homeostasis, and its dysfunction has been associated with the development of several diseases, such as cancer [24].
The bromoditerpenes displayed moderate cytotoxicity on the studied malignant cell lines, which seems to be mediated by an increase in H2O2 levels or DNA damage and apoptosis induction.

Interaction with Proteasome-Molecular Docking Studies
The ability of the three bromoditerpenes (bromosphaerol, 12S-hydroxybromosphaerol, and 12R-hydroxy-bromosphaerol) to inhibit the 20S proteasome was assessed through molecular docking calculations. The crystal structure with the PDB code 4R67 (human constitutive 20S proteasome in complex with carfilzomib) and the chymotrypsin-like (CT-L) catalytic site composed of the β5 catalytic subunit (and the β6 complementary subunit) were prepared for the docking calculations. The interaction profile showed that the three bromoditerpenes are positioned distantly from the Thr1 Nterminal γ hydroxyl group (Thr1Oγ), which acts as a nucleophile in the catalytic mechanism [25][26][27][28]: bromosphaerol and 12R-hydroxy-bromosphaerol are both at a distance of 10.87 Å from the Thr1Oγ, the 12S-hydroxy-bromosphaerol being at 9.26 Å.

Interaction with Proteasome-Molecular Docking Studies
The ability of the three bromoditerpenes (bromosphaerol, 12S-hydroxy-bromosphaerol, and 12R-hydroxy-bromosphaerol) to inhibit the 20S proteasome was assessed through molecular docking calculations. The crystal structure with the PDB code 4R67 (human constitutive 20S proteasome in complex with carfilzomib) and the chymotrypsin-like (CT-L) catalytic site composed of the β5 catalytic subunit (and the β6 complementary subunit) were prepared for the docking calculations. The interaction profile showed that the three bromoditerpenes are positioned distantly from the Thr1 N-terminal γ hydroxyl group (Thr1Oγ), which acts as a nucleophile in the catalytic mechanism [25][26][27][28]: bromosphaerol and 12R-hydroxy-bromosphaerol are both at a distance of 10.87 Å from the Thr1Oγ, the 12S-hydroxy-bromosphaerol being at 9.26 Å.
Mar. Drugs 2022, 20, x 3 of 18 maintenance of the intracellular protein homeostasis, and its dysfunction has been associated with the development of several diseases, such as cancer [24]. The bromoditerpenes displayed moderate cytotoxicity on the studied malignant cell lines, which seems to be mediated by an increase in H2O2 levels or DNA damage and apoptosis induction.

Interaction with Proteasome-Molecular Docking Studies
The ability of the three bromoditerpenes (bromosphaerol, 12S-hydroxybromosphaerol, and 12R-hydroxy-bromosphaerol) to inhibit the 20S proteasome was assessed through molecular docking calculations. The crystal structure with the PDB code 4R67 (human constitutive 20S proteasome in complex with carfilzomib) and the chymotrypsin-like (CT-L) catalytic site composed of the β5 catalytic subunit (and the β6 complementary subunit) were prepared for the docking calculations. The interaction profile showed that the three bromoditerpenes are positioned distantly from the Thr1 Nterminal γ hydroxyl group (Thr1Oγ), which acts as a nucleophile in the catalytic mechanism [25][26][27][28]: bromosphaerol and 12R-hydroxy-bromosphaerol are both at a distance of 10.87 Å from the Thr1Oγ, the 12S-hydroxy-bromosphaerol being at 9.26 Å.

Isolation and Identification of Bromoditerpenes
Sphaerococcus coronopifolius freeze-dried samples were subjected to a sequential extraction and fractionation process by vacuum liquid chromatography, resulting in five fractions (F1-F5). The F3 fraction was purified through successive chromatographic techniques, affording three pure compounds. The analysis of their NMR 1D/2D spectra and the comparison of the chemical shifts and structural assignments (Supplementary Material, Tables S1-S3) with the literature data allowed for the unequivocal identification of the purified compounds known as the bromoterpenes 12R-hydroxy-bromosphaerol, 12S-hydroxy-bromosphaerol, and bromosphaerol ( Figure 1).

Isolation and Identification of Bromoditerpenes
Sphaerococcus coronopifolius freeze-dried samples were subjected to a sequential ex traction and fractionation process by vacuum liquid chromatography, resulting in five fractions (F1-F5). The F3 fraction was purified through successive chromatographic tech niques, affording three pure compounds. The analysis of their NMR 1D/2D spectra and the comparison of the chemical shifts and structural assignments (Supplementary Mate rial, Tables S1-S3) with the literature data allowed for the unequivocal identification o the purified compounds known as the bromoterpenes 12R-hydroxy-bromosphaerol, 12S hydroxy-bromosphaerol, and bromosphaerol ( Figure 1).

Cytotoxic Activities on Malignant Cells
The cytotoxicity of the compounds was tested on malignant cell lines derived from distinct tissues (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, and SK MEL-28), and the results are displayed in Figure 3.  The 12R-hydroxy-bromosphaerol and bromosphaerol displayed a range of IC 50 values between 18.28/27.76 µM and 33.78/50.37 µM, the lowest value observed on MCF-7 cells and the highest one observed on CACO-2 cells, respectively ( Figure 3). 12S-hydroxybromosphaerol exhibited IC 50 values between 15.35 µM and 53.34 µM, the smallest value on cells derived from prostate adenocarcinoma (PC-3), and the highest on CACO-2 cells. Overall, the higher IC 50 values were observed on malignant cells derived from colorectal adenocarcinoma (CACO-2), these being the most resistant to the bromoditerpenes treatment. In some cases, the compounds exhibited effects more marked than those of the anticancer drugs, especially in colorectal cancer cells, when tested in these experimental conditions (Supplementary Material, Figures S1-S3). Since the compounds presented broad anti-cancer activity in several cell lines, further insights into the mechanisms underlying the observed effects were sought. The MCF-7 cell line was used because breast cancer leads the number of diagnoses in women and is the second most deadly cancer after lung cancer [6].

Cytotoxicity and Mitochondrial Function
To understand if the effects mediated by the compounds were linked to mitochondrial activity, the lactate dehydrogenase (LDH) release was used as an indicator of cell survival, while the mitochondrial function was assessed by the MTT assay. MCF-7 cells were exposed to compounds (0.1-100 µM) for 24 h, and the results are presented in Figure 4. tervals for 95%.
The 12R-hydroxy-bromosphaerol and bromosphaerol displayed a range of IC50 values between 18.28/27.76 µM and 33.78/50.37 µM, the lowest value observed on MCF-7 cells and the highest one observed on CACO-2 cells, respectively ( Figure 3). 12S-hydroxybromosphaerol exhibited IC50 values between 15.35 µM and 53.34 µM, the smallest value on cells derived from prostate adenocarcinoma(PC-3), and the highest on CACO-2 cells. Overall, the higher IC50 values were observed on malignant cells derived from colorectal adenocarcinoma (CACO-2), these being the most resistant to the bromoditerpenes treatment. In some cases, the compounds exhibited effects more marked than those of the anticancer drugs, especially in colorectal cancer cells, when tested in these experimental conditions (Supplementary Material, Figures S1-S3). Since the compounds presented broad anti-cancer activity in several cell lines, further insights into the mechanisms underlying the observed effects were sought. The MCF-7 cell line was used because breast cancer leads the number of diagnoses in women and is the second most deadly cancer after lung cancer [6].

Cytotoxicity and Mitochondrial Function
To understand if the effects mediated by the compounds were linked to mitochondrial activity, the lactate dehydrogenase (LDH) release was used as an indicator of cell survival, while the mitochondrial function was assessed by the MTT assay. MCF-7 cells were exposed to compounds (0.1-100 µM) for 24 h, and the results are presented in Figure  4.  Overall, the MCF-7 cells treated with the compounds revealed a similar profile on both assays; it was more marked on the MTT assay ( Figure 4). Through the MTT assay, all compounds at 25 µM showed significant differences when compared to the vehicle treatment. However, this was not observed on the LDH assay. Furthermore, when tested at 10 and 25 µM of 12S-hydroxy-bromosphaerol and bromosphaerol, respectively, the results revealed by the MTT assay displayed significant differences when compared with the effects observed on the LDH assay.

Hydrogen Peroxide Levels
Hydrogen peroxide (H 2 O 2 ) levels were estimated on MCF-7 cells in real-time following the treatment with the aforementioned compounds (IC 50 ) for 1, 3, and 6 h. The results are presented in Figure 5.
Mar. Drugs 2022, 20, x 6 of Overall, the MCF-7 cells treated with the compounds revealed a similar profile both assays; it was more marked on the MTT assay ( Figure 4). Through the MTT assay, compounds at 25 µM showed significant differences when compared to the vehicle tre ment. However, this was not observed on the LDH assay. Furthermore, when tested at and 25 µM of 12S-hydroxy-bromosphaerol and bromosphaerol, respectively, the resu revealed by the MTT assay displayed significant differences when compared with the fects observed on the LDH assay.

Hydrogen Peroxide Levels
Hydrogen peroxide (H2O2) levels were estimated on MCF-7 cells in real-time follo ing the treatment with the aforementioned compounds (IC50) for 1, 3, and 6 h. The resu are presented in Figure 5. The treatment with 12R-hydroxy-bromosphaerol and 12S-hydroxy-bromosphae significantly stimulated the production of hydrogen peroxide (H2O2) on MCF-7 cells af 1 h of treatment. However, the highest increase was mediated by 12S-hydroxy-brom sphaerol on MCF-7 cells after exposure for 3 h. Conversely, bromosphaerol was not ab to induce H2O2 generation.

Depolarization of Mitochondrial Membrane Potential
Alterations in the mitochondrial membrane potential were studied following MCF cells' exposure to the aforementioned compounds (IC50) for 15, 30, and 60 min (Figure The treatment with 12R-hydroxy-bromosphaerol and 12S-hydroxy-bromosphaerol significantly stimulated the production of hydrogen peroxide (H 2 O 2 ) on MCF-7 cells after 1 h of treatment. However, the highest increase was mediated by 12S-hydroxybromosphaerol on MCF-7 cells after exposure for 3 h. Conversely, bromosphaerol was not able to induce H 2 O 2 generation.

Depolarization of Mitochondrial Membrane Potential
Alterations in the mitochondrial membrane potential were studied following MCF-7 cells' exposure to the aforementioned compounds (IC 50 ) for 15, 30, and 60 min ( Figure 6). The treatment of MCF-7 cells with the isolated bromoditerpenes promoted ch in MMP after 15, 30, and 60 min ( Figure 6). 12R-hydroxy-bromosphaerol and br sphaerol induced a significant depolarization after 15 and 30 min, while 12S-hyd bromosphaerol induced the same effect after 30 and 60 min as compared to the v situation. However, the highest effect was mediated by 12R-hydroxy-bromosphaero bromosphaerol after 15 min (294.6 ± 34.7%) and 30 min (306.9 ± 23.5%) of treatmen spectively.

Externalization of Phosphatidylserine, Caspase-9 Activity, and Nuclear Condensation a Fragmentation
Apoptosis is characterized by key biological events, including the externalizat phosphatidylserine, caspases activation, and nuclear condensation and fragment Those events were studied on MCF-7 cells following treatment with the compounds at different exposition times (Figure 7). The treatment of MCF-7 cells with the isolated bromoditerpenes promoted changes in MMP after 15, 30, and 60 min ( Figure 6). 12R-hydroxy-bromosphaerol and bromosphaerol induced a significant depolarization after 15 and 30 min, while 12S-hydroxy-bromosphaerol induced the same effect after 30 and 60 min as compared to the vehicle situation. However, the highest effect was mediated by 12R-hydroxy-bromosphaerol and bromosphaerol after 15 min (294.6 ± 34.7%) and 30 min (306.9 ± 23.5%) of treatment, respectively.

Externalization of Phosphatidylserine, Caspase-9 Activity, and Nuclear Condensation and/or Fragmentation
Apoptosis is characterized by key biological events, including the externalization of phosphatidylserine, caspases activation, and nuclear condensation and fragmentation. Those events were studied on MCF-7 cells following treatment with the compounds (IC 50 ) at different exposition times (Figure 7).
Under controlled conditions, MCF-7 cells were mostly viable, with a small percentage in late apoptosis ( Figure 7A). All of the bromoditerpenes promoted a significant decrease in viable cells and cells in the late apoptosis stage compared to the vehicle treatment. As for the apoptosis and necrosis levels, there were no differences between the treated cells and the vehicle. The compounds induced Caspase-9 activity following 6 h exposure, bromosphaerol being the most efficient one ( Figure 7B). By analyzing Figure 7C, it is possible to observe changes in MCF-7 cells' nuclear morphology. After 24 h and 48 h of exposition to 12R-hydroxy-bromosphaerol, nuclear condensation seemed to be induced; however, the effects were much more expressive with 72 h of exposition. 12S-hydroxybromosphaerol treatment induced nuclear condensation following 24 h of exposure and extensive nuclear fragmentation for prolonged exposures, i.e., 48 h and 72 h. Similarly, bromosphaerol induced nuclear condensation after 24 h and nuclear fragmentation after 48 and 72 h.

DNA Damage on L929 Fibroblasts
The ability of bromoditerpenes (25 and/or 50 µM) to affect the DNA integrity was studied on L929 fibroblasts following 3 h of exposure ( Figure 8).
7C, it is possible to observe changes in MCF-7 cells' nuclear morphology. After 24 h and 48 h of exposition to 12R-hydroxy-bromosphaerol, nuclear condensation seemed to be induced; however, the effects were much more expressive with 72 h of exposition. 12S-hydroxy-bromosphaerol treatment induced nuclear condensation following 24 h of exposure and extensive nuclear fragmentation for prolonged exposures, i.e., 48 h and 72 h. Similarly, bromosphaerol induced nuclear condensation after 24 h and nuclear fragmentation after 48 and 72 h.

DNA Damage on L929 Fibroblasts
The ability of bromoditerpenes (25 and/or 50 µM) to affect the DNA integrity was studied on L929 fibroblasts following 3 h of exposure ( Figure 8).   (Figure 8). At levels 1, 2, and 3, only EMS displayed significant differences compared to the vehicle. Particularly, bromosphaerol (50 µM) was the only compound that induced level 4 DNA damage on fibroblasts. However, when tested at 25 µM, this compound exhibited a similar behavior to that of the other tested compounds.

Discussion
In the last decades, marine natural products have been shown to be key players in the development of innovative anticancer therapeutic strategies, which is easily proved by the currently approved MNP-derived drugs. The ongoing clinical pipeline presents thirty-three marine-derived molecules, from which twenty-seven are being tested for the treatment of different oncologic diseases [7]. Marine natural products display a great ability to act as biological modulators, exhibiting dissimilar modes of action within typical classes of drugs and establishing new avenues for cancer therapy [33,34]. Despite the efforts, the anticancer potential of several MNPs remains poorly explored, evidencing the relevance of performing studies with molecules that are already described but whose mechanisms affecting malignant cell lines are not fully understood. Thus, the present study aimed to evaluate the potential of three bromoditerpenes isolated from the red seaweed Sphaerococcus coronopifolius to interact with the proteasome core and induce cytotoxicity on different cancer cell lines, as well as study their possible mechanisms of action.
Proteasome plays a critical role in the regulation of intracellular protein levels, it being responsible for the degradation of most cellular proteins (80%) in the cytoplasm and nucleus after being tagged with ubiquitin. The malfunction of this system can lead to the degradation of normal proteins while the abnormal ones cannot be degraded, resulting in proteasome-related diseases such as cancer [35]. Furthermore, plasma-increased levels of the 20S proteasome catalytic core have been observed in cancer patients, particularly in the case of solid tumors, which can present values that are 1000-fold higher compared with those of healthy individuals [36,37]. These clinical findings suggest that the 20S proteasome is a hot and challenging target in cancer research. Proteasome inhibitors are able to induce apoptosis in several cancer cells, showing low cytotoxicity in normal cells [38]. Regarding the interaction pattern of the three bromoditerpenes with the proteasome CT-L catalytic site, molecular docking calculations showed that the compounds do not interact with the catalytic THR1. However, 12S-hydroxy-bromosphaerol interacts with ALA20 and SER169, which are key residues of proteasome catalytic activity. These interactions may be explained by the distinct conformation adopted by the three compounds, suggesting that the presence of an additional hydroxyl group in derivatives of bromosphaerol, as well as the spatial configuration of 12S and 12R, can be relevant in the interaction. However, considering these results, the interaction pattern of the three compounds, observed through molecular docking studies, is not very likely to lead to effective proteasome inhibition. Similarly, the marine bromoditerpene sphaerococcenol A also did not establish interactions with key amino acids that are essential for recognition and proteasome inhibition [39]. Nevertheless, several other reports attest to the ability of terpenes obtained from plants and marine organisms (e.g., triptolide, celastrol, pristimerin, withaferin A, petrosapongiolide M, and heteronemin) to interact with key residues of proteasome by molecular modelling, which was confirmed by in vitro and in vivo assays [40][41][42][43].
Regarding the cytotoxic effects, the data gathered revealed that those three bromoditerpenes induced a moderate loss of viability in several malignant cell lines with IC 50 values ranging from 15.35 to 53.34 µM. Regarding A549, PC-3 and SK-MEL-28 tumor cells, the observed IC 50 range is similar to the IC 50 values previously reported for those cells (9-35 µM) [23]. By looking to the results attained by the MTT and LDH assays, it is possible to conclude that the effects mediated by 12S-hydroxy-bromosphaerol and bromosphaerol were more marked in the MTT assay. Furthermore, despite the treatment with 12R-hydroxybromosphaerol not promoting significant differences between the assays, the effects on MCF-7 cells' viability were also more noticeable when estimated by the MTT assay compared to the LDH assay at a concentration near their IC 50 value (25 µM). Those data are particularly interesting since, although the MTT and LDH assays evaluate the effects on cells' viability, the cellular target of each method is distinct. In fact, while the MTT assay evaluates the cells' dehydrogenases activity (both mitochondrial and cytoplasmatic), LDH evaluates the membrane integrity [44]. The results suggest that the compounds may target mitochondrial activity and that their similar effects can be related to the presence of a bromine group in their structures, which is rare in nature and has been associated with their biological activities [18]. Furthermore, 12R-hydroxy-bromosphaerol and 12Shydroxy-bromosphaerol are diastereoisomers-specifically, epimers on position 12-which means that the compounds have the same molecular formula and constitutions around the carbon atoms, and the spatial arrangement of the groups around those atoms differ only in terms of the configuration of the stereocenter C12. These spatial differences are enough to mediate distinct pharmacological activities-as observed with thalidomide, a teratogenic drug used in pregnant women in the late 1950s-due to the simultaneous presence of both enantiomers, R and S, in its composition, a fact that was unknown at the time. The (R)enantiomer was responsible for the sedative effects, the therapeutic properties of interest, while the (S)-isomer induced teratogenic activities, provoking a range of severe birth defects [45]. However, despite 12R-hydroxy-bromosphaerol and 12S-hydroxy-bromosphaerol displaying different spatial arrangements around C12, this seemed to not influence the cytotoxic activities, since those compounds exhibited similar activities on all cell lines.
Apoptosis has been a mainstay and a goal of innovative oncology therapeutics to ensure an effective elimination of cancer cells. Apoptotic signaling pathways are triggered by multiple factors, including cellular stress and DNA damage [46]. Several chemotherapeutics, such as cisplatin and doxorubicin, can stimulate the formation of reactive oxygen species (ROS), leading to cancer cells' death by apoptosis [47] and demonstrating that the induction of ROS production could be an appropriate therapeutic strategy. On the other hand, the mechanism of action of other anticancer drugs is associated with the induction of high levels of DNA damage that trigger cell cycle checkpoints, leading to cell cycle arrest and/or cell death [48]. This strategy is based on two fundamental assumptions. The first relies on the fact that cancer cells multiply more often than normal cells; secondly, normal cells have a highly efficient DNA damage response and DNA repair mechanisms, giving normal cells the ability to stop the cell cycle to repair the DNA. Therefore, they have the ability to arrest proliferation for more efficient DNA repair and to avoid the damage mediated by the drug [48,49]. Herein, the ability of compounds to stimulate the production of hydrogen peroxide (H 2 O 2 ) and DNA damage was studied, as well as the capacity to modulate different biomarkers related to apoptosis. The compounds 12R-hydroxy-bromosphaerol and 12S-hydroxy-bromosphaerol stimulated the production of H 2 O 2 , and bromosphaerol induced DNA damage, suggesting that the presence of an additional hydroxyl group in the derivatives 12S and 12R induces a distinct effect between the derivatives and bromosphaerol. These effects were accompanied by changes in mitochondrial membrane potential, more cells in late apoptosis, increased Caspase-9 activity, and nuclear fragmentation/condensation. The ability of marine terpenes to modulate these intracellular signaling pathways has already been reported. The marine sesterterpenoid heteronemin isolated from the sponge Kyrgios sp. induced apoptosis and ferroptosis triggered by the production of ROS on several in vitro cancer cellular models [50,51]. Furthermore, previous studies conducted with the bromoditerpenes sphaerococcenol A and sphaerodactylomelol also displayed increased H 2 O 2 levels, modulating different apoptotic hallmarks [39,52]. However, further studies need to be accomplished to fully understand the involvement of oxidative stress in MCF-7 cells' death and loss of cell viability. For instance, a pre-treatment with N-acetylcysteine (NAC), known as an ROS scavenging molecule, can be used to understand if the cytotoxic effects mediated by 12R-hydroxybromosphaerol and 12S-hydroxy-bromosphaerol are related to the increase in H 2 O 2 levels. Despite the mechanisms of action underlying the activities of these compounds being unexplored, a potential cytostatic effect of 12S-hydroxy-bromosphaerol was suggested on U373 GBM cells. Smyrniotopoulos and co-workers [23] observed a marked increase in mitosis length through the computer-assisted phase-contrast microscopy approach. Accordingly, this evidence and our data suggest that 12S-hydroxy-bromosphaerol may have the capacity to arrest the cell cycle and trigger apoptosis, two independent physiological processes regulated by a complex network of pathways, including cell cycle regulators, such as P53, that are essential for tissue homeostasis [53].
On the other hand, according to the findings observed following bromosphaerol treatment, it is possible to suppose that its effects may be associated with the induction of DNA damage, leading to the activation of cell death by apoptosis. Among the tested compounds, bromosphaerol was the only one that induced significant DNA damage on L929 cells. However, when tested at a similar concentration (25 µM, the IC 50 value for MCF-7 cells, 3 h), L929 cells were not affected. However, it is possible that the longer treatment of MCF-7 cells triggered DNA damage repair mechanisms, leading to cells' survival by recovering the effects mediated by bromosphaerol observed on L929 cells after 3 h. Nevertheless, more studies are needed to validate the different hypotheses, including the evaluation of the genotoxic effects over time on L929 cells, carrying out those assays on MCF-7 cells, as well as studying the cell cycle. Although several NPs induce DNA damage, leading to cell cycle arrest and apoptosis [54][55][56], the present study discloses, for the first time, the potential mechanisms of action underlying the cytotoxic effects of the marine bromoditerpenes 12R-hydroxy-bromosphaerol, 12S-hydroxy-bromosphaerol, and bromosphaerol, which may be related to increased H 2 O 2 levels and DNA damage, leading to cell death by apoptosis. Further experiments are required to understand the involvement of oxidative stress and DNA damage in the cytotoxic activity of these compounds, as well as studies on their effects on normal cells (proliferating or quiescent) in order to define their therapeutic index and understand their antitumor potential.

Collection, Extraction, and Purification
Red seaweed specimens of Sphaerococcus coronopifolius were collected in the Berlengas Nature Reserve (39 • 24 44.8 N 9 • 30 29.5 W), Peniche, Portugal by scuba diving and immediately transported to MARE-Polytechnic of the Leiria laboratory, where they were identified by Prof. Teresa Mouga, a biologist with vast experience in the taxonomic identification and ecology of marine seaweeds. The samples were cleaned to remove detritus, sand, and epibionts and freeze-dried. The dry algal material was powdered and then sequentially extracted with methanol and dichloromethane (1:4) for 12 h. The dichloromethane extract was concentrated to dryness in a rotary evaporator and subjected to normal phase vacuum liquid chromatography on silica gel 60 (0.06-0.2 mm), using cyclohexane (VWR, Fontenay-sous-bois, France) with increasing amounts (25%) of ethyl acetate (EtOAc) (VWR, Fontenay-sous-bois, France) as the mobile phase (five fractions). . This step afforded 12R-hydroxy-bromosphaerol (f5 to f9 fraction). The structural elucidation of compounds was attained by NMR spectroscopy analysis, 1D ( 1 H and 13 C Attached Proton Test (APT)) and 2D (COSY, HMBC, HSQC-ed) techniques, and by comparison with previously reported data. For biological evaluation, the compounds were dissolved in DMSO (concentration below 0.2%) and evaluated through a set of in vitro assays. The control situation was always treated with the highest concentration of DMSO as the vehicle.

In Silico Docking Studies
The receptor structure was retrieved from the Protein Data Bank (www.rcsb.org) through the PDB code 4R67 (Human constitutive 20S proteasome in complex with carfilzomib, 2.89 Å) [32]. To prepare the protein structure for docking calculations, all atoms (i.e., ligand, salts, water, other chains) other than the receptor β5 and β6 subunits (chains L and M, respectively-CT-L active site) were deleted from the X-ray structure using the MOE software package (v.2019.0102) (Molecular Operating Environment; Chemical Computing Group ULC, 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2021). The AMBER99 forcefield was used to assign atom types and charges to each atom in the receptor. Hydrogen atoms were added, and the appropriate protonation states were assigned using the Protonate-3D tool within the MOE software package (pH 7.4 and T = 310 K). Structures of bromoditerpenes were built and energy-minimized using MOE. Molecular docking simulations were performed using GOLD 5.4 software [43]. The binding site was defined to be centered in the catalytic Thr1Oγ (β5 catalytic subunit) with a 15 Å search radius. Noncovalent docking calculations were performed, with the number of genetic algorithm (GA) runs set to 1000 and the search efficiency set to 100%, and the ten top-ranked solutions were selected. For the other settings, the default parameters were used. First, an initial docking validation step was carried out by performing self-docking calculations using GOLD (scoring functions: GoldScore, Chemscore, ChemPLP, and ASP) and 1000 exhaustive search runs. All protein amino acid residues were kept rigid, whereas all single bonds of the ligands were treated as fully flexible. The docking parameters (scoring function-ChemPLP-and protein 3D structure) selected were able to successfully reproduce the experimental pose (RMSD < 2 Å between the experimental and predicted pose). The docking procedure was subsequently used for the docking calculations. For the analysis of protein-ligand interactions, the top docking poses were submitted to the detection of residue contacts using the docker implementation of PLIP [57]. Images of the compound and the PDB structures were produced using PyMOL v.1.8.4.0.

Cell Culture Conditions
The cell lines were obtained from the DSMZ and ATCC biobanks. HTB-72) were grown in EMEM medium (Sigma, USA) supplemented with 10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin. The cells were dissociated with trypsin (Sigma-Aldrich, USA) and then neutralized with the respective fresh medium and centrifuged at 290 g for 5 min at room temperature. After that, the cells were resuspended in a fresh medium (1:10), seeded in 25 cm 2 T-Flasks, and cultivated in 5% CO 2 and a humidified atmosphere at 37 • C.

Translocation of Phosphatidylserine and Membrane Integrity
MCF-7 cells (1.0×10 6 cells/mL) were seeded in 6-well plates and treated with the compounds at the IC 50 concentration and staurosporine (1 µg/mL) (Sigma, Rehovot, Israel) for 24 h, and the effects were analyzed by flow cytometry using the Apoptosis Detection Kit (Immunostep, Salamanca, Spain). Ten thousand events were recorded with the AMNIS imaging flow cytometer using the AMNIS INSPIRE™ software. The data were analyzed using the AMNIS IDEAS™ software (Amnis Corporation v6.0, Luminex Corp, Austin, TX, USA).

DAPI Staining
DNA nuclear morphology was evaluated using the DAPI fluorescent probe [58]. MCF-7 cells (1.0×10 6 cells/mL) were seeded in 6-well plates and treated with compounds at the IC 50 concentration for 24, 48, and 72 h. DNA morphology was analyzed using a fluorescence inverted microscope (ZEISS Axio, VERT. A1, equipped with an AxioCam MRC-ZEISS camera, München, Germany), and a representative image of each treatment was presented.

DNA Damage
L929 mouse fibroblasts (2.0×10 4 cells/mL) were seeded in 12-well plates and cultured overnight. The fibroblasts were then treated with compounds (25 and/or 50 µM) and ethyl methanesulfonate (200 µg/mL) as a positive control for 3 h. The DNA damage was determined according to the protocol established by Singh et al. [59], with slight modifications [52]. One-hundred cells were randomly chosen, analyzed, visually scored, and classified into five levels according to the tail size formed by breaks in the DNA. Non-overlapping was performed.

Data and Statistical Analysis
At least three independent experiments were carried out in triplicate, and the results were presented as the mean ± standard error of the mean (SEM) and the half-maximal inhibitory concentration (IC 50 ). ANOVA with Dunnett's multiple comparison of group means analysis was accomplished, and the Tukey's test was applied for multiple comparisons. When applicable, Student's t-test was used. Differences were considered significant at the level of 0.05 (p < 0.05). The IBM SPSS Statistics 24 (IBM Corporation, Armonk, NY, USA) and GraphPad v5.1 (GraphPad Software, La Jolla, CA, USA) software were used to accomplish the analysis.


Chemical kinetics and promoted Co-immobilization for efficient catalytic carbonylation of ethylene oxide into methyl 3-hydroxypropionate

The carbonylative transformation of ethylene oxide (EO) into methyl 3-hydroxypropionate (3-HPM) is a key process for the production of 1,3-propanediol (1,3-PDO), which is currently viewed as one of the most promising monomers and intermediates in polyester and pharmaceuticals industry. In this work, a homogeneous reaction system using commercial Co2(CO)8 was first studied for the carbonylation of EO to 3-HPM. The catalytic behavior was related to the electronic environment of N on aromatic rings of ligands, where N with rich electron density induced a stronger coordination with Co center and higher EO transformation. A reaction order of 2.1 with respect to EO and 0.3 with respect to CO was unraveled based on the kinetics study. The 3-HPM yield reached 91.2% at only 40°C by Co2(CO)8 coordinated with 3-hydroxypyridine. However, Co-containing colloid was formed during the reaction, causing the tough separation and impossible recycling of samples. Concerning the sustainable utilization, Co particles immobilized on pre-treated carbon nanotubes (Co/CNT-C) were designed via an in situ reduced colloid method. It is remarkable that unlike conventional Co/CNT, Co/CNT-C was highly selective toward the transformation of EO to 3-HPM with a specific rate of 52.2 mmol·gCo-1·h-1 , displaying a similar atomic efficiency to that of coordinated Co2(CO)8. After reaction, the supported Co/CNT-C catalyst could be easily separated from the liquid reaction mixture, leading to a convenient cyclic utilization.

Introduction
Since the ring-opening carbonylation of epoxide in alcohol solvent has high atomic economy, it is very attractive to prepare downstream high value-added products using epoxide as the reactant via catalytic technique (Ocampo and Dolbier, 2004). Among the most commonly used epoxides, ethylene oxide (EO) could be obtained as a petrochemical product in excess capacity by industrial production. It also becomes a promising raw material to produce methyl 3-hydroxypropionate (3-HPM) through carbonylative conversion, with the subsequent hydrogenation product 1,3-propanediol (1,3-PDO) as one of the most expected industrial products for new polyester (PTT) and medical intermediates (Lv et al., 2010;Rajendiran et al., 2018;Wen et al., 2018;Xu and Wu, 2019). No matter how, the industrial path for perspective 3-HPM and 1,3-PDO required the key carbonylative transformation of EO with high efficiency and selectivity, which was, however, greatly challenging until current days.
In the past decades, carbonyl metallic salts have been used as operatable homogeneous catalysts for carbonylation of epoxides. In fact, researchers have reported in detail on the ring expansion and ring-opening carbonylation of epoxides (Suresh et al., 2021). However, complicated homogeneous reaction systems were dominantly used with metastable metal carbonyls with unsatisfied product yields. The early work of Lee and Alper (2004) designed a new Co 2 (CO) 8 /A (6,7-dihydro-5,8dimethyldibenzo [b,j]-1,10-phenanthroline)/PhCH 2 Br catalyst to synthesize aliphatic polyesters via alternating copolymerization of propylene oxide and CO, with 55% yield of atactic polyhydroxybutrate (PHB). While homogeneous reactions were carried out using phosphorus ligands in the carcinogenic benzene under 900 psi, Rowley et al. (2007) reported a special bimetallic [(CITPP)Al(THF) 2 ] + [Co(CO) 4 ] − catalyst efficient in transforming epoxides to β-lactone and subsequently succinic anhydrides via one-pot double carbonylation. The rates of both epoxide and lactone carbonylation were suggested independent of CO pressure and are first-order in the catalyst concentration. They also confirmed an opposite solvent effect for the different carbonylation processes.
In fact, Lewis acids as a kind of special ligands coordinating with metal centers were also frequently applied in the epoxiderelated reactions. Dunn et al. (2016) reported an improved synthesis of poly (3-hydroxypropionate) (P 3 HP) from ethylene oxide and CO through the intermediate β-propiolactone. The bimetallic [Lewis acid] + [Co(CO) 4 ] − catalyst was confirmed to be suitable for an optimized carbonylation yield. Maruyama et al. (2022) also reported a selective and quantitative cyclodimerization of an epoxide with a carbonyl compound by Lewis acid-catalyzed processes, where the ring opening of EO was involved as the first step. In a recent work, Zhao et al. (2021) used a series of N,P-ligand-cooperated [LCo(CO) 3 ] + [Co(CO) 4 ] − to catalyze EO (hydromethoxy carbonylation) into 3-HMP and proposed a cooperative reaction mechanism by [Co(CO) 4 ] − and its counter cation [LCo(CO) 3 ] + (L = Ph 2 PN(iPr)PPh 2 ). The yield of 3-HPM largely depended on the type of ligands forming complexes with Co 2 (CO) 8 , and the ion-pair complexes behaved more excellently than the neutral ones.
No matter how, the research progress of Co-based catalysts has stagnated in the past 5 years, and relevant reports still focus on the carbonyl cobalt in a homogeneous reaction system. Although the selectivity for 3-HPM is better, the reaction condition is harsh and in need of thoughtful operation due to CO dissociation and the oxidation of Co 0 . Co 2 (CO) 8 used as a catalyst is metastable in air and needs to be properly preserved during preparation, storage, and transportation. On the other hand, the separation process is complex, and the homogeneous metal catalyst cannot be fully recovered and reutilized in a simple way, which is not conducive to the sustainable and recycling of resources. Therefore, it is necessary to develop a more stable and durable catalyst system to replace Co 2 (CO) 8 in advance. In addition, the reaction kinetics of carbonylation of EO to 3-HPM lacks researches in depth and requires further discussion.
Over the years, many studies have been carried out on the carbonylation reaction via heterogeneous reactions, such as hydroformylation of olefins and hydroesterification of epoxides by supported noble metals (Li et al., 2003a;Li et al., 2003b;Liu et al., 2009;Liu et al., 2013). Compared with other supports, carbon materials have attracted more attention because of their easily controlled surface specialty and chemical structure. In addition to its outstanding electronic conductivity, carbon materials also exhibit the strong adsorption of CO with manually adjusted surface acidity acting as Lewis acid and Brönsted acid (Kainulainen et al., 1997), which possibly facilitated the immobilization of Co centers as a potential substitution of coordinated Co 2 (CO) 8 .
In this work, Co 2 (CO) 8 was first used to catalyze the carbonylative transformation of EO to 3-HPM. The reaction parameters including the dosages of reactants; the cooperated ligands and CO pressure were optimized to understand the chemical kinetics of EO carbonylation. In order to promote separation efficiency and sustainable utilization, Co particles supported by pre-treated carbon nanotubes (Co/CNT-C) were synthesized via an in situ reduced colloid method. It is necessary to mention that the Co/CNT-C sample was selective toward the transformation of EO to 3-HPM with a similar atomic efficiency to that of Co 2 (CO) 8 cooperated with 3-hydroxypyridine. The key factors for the enhanced catalytic performances were interpreted by combining the initial properties of Co/CNT by using the conventional impregnation method.

Materials and experimental methodology Materials
Co 2 (CO) 8 , pyrazole, 3-hydroxypyridine, and other ligands were purchased from Macklin (A.R.). Ethylene oxide (EO) in tetrahydrofuran (THF) (3.0 mol/L) was purchased from Energy Chemical Co., Ltd. Carbon monoxide with a purity of 99.99% was commercially available and supplied by Shandong Dazhan Nano Materials Co., Ltd. All other reagents were of analytical grade and used as received.
Pre-treated carbon nanotubes (abbreviated as CNT) were used as the supporting material and were preliminarily treated by concentrated HNO 3 aqueous solution at 120°C for 2 h. The treated CNT was washed with distilled water until the solution was neutral and used as supporting materials after drying overnight.
A solution of 20 wt% Co/CNT-C was synthesized by an in situ reduced colloid method, and the process was as follows. A certain amount of sodium lauryl sulfate (SDS), Co(NO 3 ) 2 ·6H 2 O, and oleic acid with a molar ratio of 10:100:1 were added in water in the presence of 1 g CNT at room temperature. The Co mass loading was kept at 20 wt%. After stirring for 30 min, fresh NaBH 4 aqueous solution (1 mg/ml) was dropped into the aforementioned mixture with a molar ratio of NaBH 4 to Co 2+ as 1:1. The sample powder was obtained after centrifugation, washed, and dried at 90°C. The supported sample went through two-step thermal treatment: calcination at 400°C in air for 3 h and subsequently in H 2 /Ar (v/v = 20/50) at 350°C for 3 h and denoted as Co/CNT-C. An additional Co/CNT-I reference was prepared by incipient wetness impregnation method by impregnating the treated CNT in the aqueous solution of Co(NO 3 ) 2 and aging for 16 h. The following synthesis process (centrifugation, drying, and two-step thermal treatment) was similar to that of Co/CNT-C.

Characterizations
Transmission electron microscopy (TEM) was performed using a FEI Tecnai G2 F30 electron microscope at an acceleration voltage of 120 KV. Fourier transform infrared (FT-IR) spectroscopy was carried out on a Bruker Vertex 80v FT-IR spectrometer. The specific surface area and pore structure of the samples were investigated using N 2 physical adsorption/ desorption at −196°C using an automatic volumetric sorption analyzer (Quantachrome, Autosorb-iQ-C). X-ray diffraction (XRD) was carried out on a Shimadzu 7000S diffractometer with Cu Kα radiation (λ = 1.5418 Å), with a scanning rate of 3 o / min in range of 10 o -80 o . X-ray photoelectron spectroscopy (XPS) was operated on the ESCALAB ™ 250Xi (Thermo Fisher Scientific, United States) using the X-ray excitation source of Al Kα. The charge correction is based on C1s peak at 284.6 eV. H 2 temperature-programmed reduction (H 2 -TPR) was carried out using Quantachrome ChemBET 3000 Chemisorber with the gas flow rate of 30 ml/min. The reduction process was carried out by increasing the temperature from 50°C to 800°C at a heating rate of 10°C/min.

Carbonylation of ethylene oxide
The carbonylative transformation of EO was carried out in a 50 ml stainless steel autoclave equipped with a magnetic stirrer, temperature control system, and pressure sensing device. In a typical experiment, a certain amount of Co 2 (CO) 8 and a ligand were mixed with the methanol and EO solution in the autoclave. The aforementioned operation was done in a homemade glove box filled with N 2 to keep the Co 2 (CO) 8 stable. The autoclave was then tightened under N 2 and purged with carbon monoxide three times to evacuate the residual N 2 in the reactor. After filling the autoclave with CO to a desired pressure, the mixture was heated to a certain temperature (30°C-100°C) under stirring at 800 rpm min −1 . After maintaining the reaction for a certain period of time, the reactor was cooled down to room temperature and the CO gas was vented off in a fume hood. The reaction mixture was homogeneous and transparent in redbrown. All the catalyst was dissolved in the solution after reaction. It is impossible to separate the catalyst from the reaction mixture by centrifugation. Herein, before further analyzing the reaction mixture by GC, two independent steps were necessary for the separation of catalyst and products. First of all, the reaction mixture was treated continuously by flowing O 2 at room temperature for 2 h. Purple deposition was observed after the oxidation treatment, and the supernatant liquid became light orange. In the second step, column chromatography was used. An internal standard method was used for quantitative analysis with toluene as the standard. The product mixture was analyzed by gas chromatography using a GC (Agilent 7890B) equipped with a hydrogen flame ion detector (FID) and a capillary column HP-5 (30 m × 0.32 mm × 0.25 μm). Products were also identified using a gas chromatography-mass spectrometer (Thermo Fisher Scientific Trace1300-ISQ LT, United States). The conversion rate of EO and the selectivity of methyl 3hydroxypropionate (3-HPM) are calculated by the following equations: where n 0,EO represents the initial mole number of EO; n t,EO represents the molar amount of EO after reaction for a certain time t; and n i is the produced mole number of i (product).
Frontiers in Chemistry frontiersin.org

Results and discussion
Carbonylative transformation of ethylene oxide The carbonylative transformation of ethylene oxide (EO) was catalyzed by using the cobalt catalyst in the presence of CO. Methanol was used in excess as both solvent and reactant. Compared with Co 2 (CO) 8 as catalyst, inorganic salts such as Co(OAc) 2 and Co(NO 3 ) 2 displayed poor EO conversion of only 2.3% and 2.9% (Table 1, Entry 1-2), respectively. Also, no methyl 3-hydroxypropionate (3-HPM) was detected in the reaction mixture. The invalid catalytic performances of the inorganic salts should be resulted from the failure coordination between Co and the ligand (Xu and Wu, 2019). If the Co 2 (CO) 8 was used as the catalyst, the EO conversion easily reached 56.5% at 40°C in 6 MPa CO pressure (Table 1, Entry 3) with 95.8% selectivity toward 3-HPM, in the absence of any ligand. For better catalytic activity, the influences of ligands were studied at first, as listed in Table 1 (Entry 4 and 8-13). A variety of N-donor ligand were used as doping ligands with Co 2 (CO) 8 for the carbonylative transformation of EO. Among all the tested ligands, 3-hydroxypyridine could be used as a most efficient one with 98.5% selectivity toward 3-HPM at 92.6% EO conversion under only 40°C.
In fact, Co 2 (CO) 8 coordinated with the N-donor ligands to form Co-centered complexes in different paths depending on the Frontiers in Chemistry frontiersin.org specific electronic environment. Cobalt has an empty d-orbital and is easily complexed with the ligand by lone pair electrons on N. The nucleophilicity of a particular N atom in these ligands determines their interaction with Co as well as the subsequent catalytic performances. Compared to pyridine, the -OH group on the pyridine ring of 3-hydroxypyridine resulted in rich electron density on the aromatic ring due to p-π conjugated effect. The lone pair electrons on N may accounts for a stronger interaction between N and Co atoms in the complexes. The N-groups on pyrazole and imidazole with the π 6 5 structure are similar to that on the pyridine ring, and the position of an additional −NH group further influences the electronic environment on the pyrrole ring. It can be revealed that the catalytic performances of Co 2 (CO) 8 largely depended on the electronic environment of N atoms on the aromatic rings. In fact, proton transfer between cobalt carbonyl and the N-containing compounds was commonly reported by researchers (Kristjansdottir et al., 1991). Zeng et al. (2021) also assumed that an acid-base neutralization easily happened between the HCo(CO) 4 with 3hydroxypyridine. It was thus inferred that a positive interaction may exist between the N-donor with rich electron density and the Co center, correlated to the better EO transformation. The much lower EO conversion via Co 2 (CO) 8 in the presence of 1,10phenanthroline may be caused by the steric hindrance to coordinate with the Co center atoms. Triphenylphosphine was also used as a typical P-donor for comparison, which was less active and selective for 3-HPM compared to most of the N-donor ligands. Liu et al. (2007) suggested that appropriate N-containing compound easily interrupted the Co-Co bond in Co 2 (CO) 8 to form the active Co(CO) − 4 anion. Other research work also confirmed that acidic compound generated from the alcoholysis of carbonyl cobalt salt easily resulted in undesired ethers. Herein, N-donor ligand, especially the 3hydroxypyridine, acted as base additives stabilized the catalyst and partially neutralized the catalyst acidity, inhibiting the etherification by-products (Liu et al., 2006;Gorbunov et al., 2019;Zeng et al., 2021). Under such circumstance, transformation of EO was further optimized by Co 2 (CO) 8 in the presence of 3-hydroxypyridine. The experimental data suggested that the increase of CO pressure under the same temperature correlated with very slightly increases of EO conversion and 3-HPM selectivity (Table 1, Entry 5-7). The EO conversion and product selectivity change as a function of the Co/EO molar ratio was also studied, as shown in Figure 1A. The EO conversion was only 2.4% with the Co/EO ratio of 0.1/15, and most of the product was 1,2-dimethoxyethane (selectivity > 99%) as a result of nucleophilic addition without any participation of CO molecules (Kim et al., 2006). Both the EO conversion and 3-HMP selectivity grew logarithmically along with the Co/EO ratio. The EO conversion reached 92.6% with 98.5% 3-HPM selectivity at Co/EO of 1/15. Further increasing the ratio of cobalt catalyst, the selectivity of 3-HPM decreased while 1,2-dimethoxyethane (2.8%) was produced with trace amount of 1,1-dimethoxyethane (1.0% selectivity), although the EO conversion was maintained. It was suggested that CO molecules could be inserted into the skeleton of carbon chains to form 3-HPM only if the catalyst dosage kept in an appropriate Co/EO range.
The influence of reaction temperature was also investigated, as illustrated in Figure 1B. The EO conversion displayed a
Frontiers in Chemistry frontiersin.org parabolic trend with the temperature, and 84.6% EO could be successfully transformed at only 30°C under 6 MPa CO with 96.8% selectivity toward 3-HPM. The catalytic performance maximized at 40°C, while the subsequent increasing of temperature induced the steadily decease of both the EO conversion and 3-HPM selectivity. The selectivity of byproduct 1,2-dimethoxyethane increased to 20.9% at 100°C. At lower temperature (<60°C), the nucleophilic reaction was facilitated between EO and MtOH with the increasing temperature (Zeng et al., 2021). However, it is a common sense that the catalytic performance increases until the kinetic equilibrium, only if a stable catalyst, was in use (except for a third-order reaction). Herein, it is inferred that the as-formed complexes by Co 2 (CO) 8 and N-donor ligand may encounter thermal decomposition under higher reaction temperature.

Kinetics of ethylene oxide carbonylation
To the best of our knowledge, few researches were reported concerning the chemical kinetics for EO carbonylation, which is essential to sufficiently understand the catalytic performances. In order to understand the key reaction factors and the kinetics of EO carbonylation, the concentration of EO changing as a function of reaction time at different temperature was studied, as shown in Figure 2A. between 1/c EO and reaction time according to the second-order reaction kinetics, (C) linear relationship between lnr EO and lnc EO at 40°C of the EO carbonylation, and (D) linear relationship between lnr EO and lnp CO for the EO carbonylation using Co 2 (CO) 8 . Reaction condition: 35°C-50°C, 6.0 MPa CO, 800 rpm for 4 h, Co/3hydroxypyridine = 1:2, and Co/EO = 0.2/15, balanced with 16 ml methanol, if not specified.

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The conversion of EO was kept at 10%-30% by adjusting the conditions to make a precise estimation of the reaction order and other kinetic parameters. The EO was steadily consumed during the reaction and its concentration monotonously decreased with the reaction time. A higher reaction temperature in the range of 35°C-50°C resulted in the faster transformation of EO as indicated by the lower EO concentration detected under the same reaction time. The reaction data was subsequently fitted by the zero-order, first-order, and second-order reaction models in Figure 2B and Supplementary Figure S1 (supporting information). The corresponding R 2 and rate constant (k A ) for each linear relationship are listed in Supplementary Table S1. A much higher R 2 approaching 1 based on the experimental data suggested the reaction order concerning EO was approximately 2. The reaction constant rapidly increased with the temperature, confirming the much accelerated reaction rate under higher temperature in the range of 35°C-50°C. For better interpretation, the reaction rate equation can be written in terms of EO concentration and CO pressure as Eq. 1. The influences of methanol on the reaction rate could be ignored in Eq. 1, since methanol is used as both reactant and solvent in excessive quantity.
The parameter k A is the rate constant with respect to EO, while α and β represent the reaction orders with respect to EO and CO. To obtain the reaction order of EO (α), CO pressure was kept sufficient and consistent at 6.0 MPa. Herein, the term concerning the CO pressure (P β CO ) can be viewed as a constant value during the reaction. The simplified Eq. 2 can be expressed as follows: The reaction order (α) in terms of EO was thus obtained by the slope of a linear relationship between the logarithm of EO concentration and the logarithm of the reaction rate ( Figure 2C). The mathematics fitting result suggested an apparent reaction order of 2.1 with respect to EO, further confirming the previous fitting results.
The reaction order (β) in terms of CO can be detected based on Eqs 4,5 under low EO conversion (superfluous EO concentration with nearly consistent quantity during reaction). As revealed by the slope of lnr CO vs. lnp CO in Figure 2D, the reaction order with respect to CO was 0.3. The low reaction order of CO suggested the weak dependency of the reaction rate with CO pressure. The pressure of CO in process was found to have a limited effect on the catalytic activity for carbonylation compared with the EO concentration. Kramer et al. (2006) reported the excellent production of β-lactones produced via carbonylation at only atmospheric CO pressure. Even for the CO inserting into the cobalt-alkyl bond process, Church et al. (2006) reported an independent reaction rate of CO pressure over the range considered. These phenomena may account for the very slight variation of EO conversion as the CO pressure changes (Table 1, Entry 4-7).
According to the aforementioned investigation on kinetics, the differential rate equation could be written in the following form: The activation energy (E a ) of the catalytic system reflected the necessary energy barrier to activate the reactant molecules and can be viewed as a parameter describing the catalytic efficiency (Joshi et al., 2018;Nazimov et al., 2021). According to the Arrhenius equation, the Ea based on Co 2 (CO) 8 using 3-hydroxypyridine as a ligand can be estimated. The linear relationship between lnk A and the reciprocal of T in K is shown in Supplementary Figure S2. The Ea value calculated by the slope of the Arrhenius curve is 62.9 kJ/mol. It is necessary to mention that such value was occasionally similar to the reported energy barrier for disproportionation of Co 2 (CO) 8 to [Co(MeOH) 6 ][Co(Co) 4 ] 2 in methanol under carbonylation reactions (Mirbach and Mirbach, 1985). It is inferred that the formation of Cocentered complex may become an indispensable course for an efficient EO transformation.
For better confirmation, the solid catalysts before and after reaction were characterized by FT-IR spectra. It has to be noted that separation of the catalyst from the reaction mixture was indeed a tough task before any other analysis of the products. The cobalt complex was uniformly dissolved in the liquid and a seemingly homogeneous solution was obtained after reaction, where metal colloid was generated as confirmed by the Tyndall phenomenon (showing a characteristic light path) in Figure 3A. Colloidal Co-containing particles may exist in the reaction mixture.
It is impossible to separate the catalyst directly by centrifugation or column chromatography. Herein, an additional re-oxidation step was operated to recycle the cobalt-containing composition. The fresh liquid after reaction was subsequently treated by the flowing O 2 for 2 h continuously to oxidize the Co colloid into Co-containing deposition ( Figure 3B). The TEM images of the transparent solution in Figure 3A displayed the well dispersed particles as shown in Figure 3B-I, indicating the as-formed Co colloidal particles protected by organic compounds. The under-layer deposition was also collected and tested by TEM and EDX analysis. Amorphous CoO x was detected as a main composition ( Figures 3B-II,C).
Frontiers in Chemistry frontiersin.org 07 FT-IR spectra of the fresh mixture after reaction without and with oxidation treatment were displayed in Figure 3D. Infrared spectroscopy is one of most useful methods for characterizing the presence of different bands. The dominant peaks of -OH stretching and -CH deformation vibration at approximately 3,400 cm −1 and 692 cm −1 were observed in all samples AlOmar et al., 2016). The adsorption peaks at 1,644 cm −1 and 1,724 cm −1 originated from the aromatic skeleton vibration and the stretching vibration of the C=O bond in the coordinated cobalt catalyst (Xie and Wan, 2018), while the peak at 1,888 cm −1 suggests the presence of Co(CO) − 4 species, which disappeared rapidly after 1-h oxidation. After oxidation, ] C-O = 1,050 cm −1 attributed to the C-O stretching vibration (Qiu et al., 2021) became apparent while peak situated at 1,888 cm −1 disappeared, confirming the possible decomposition of Co(CO) − 4 species and the variation of functional groups after oxidation and separation procedures.
No matter how, the high efficiency of Co 2 (CO) 8 collaborated with 3-hydroxypyridine happened at the expense of pseudohomogeneous reaction with untoward separation of the reaction liquid. It makes the separation of the metal catalyst and the recycle more difficult to be operated with considerable cost, which is also a big concern for the industrial production unit. Research works focusing on the carbonylative transformation of different chemical compounds by supported catalysts were also studied in recent years. However, tough reaction conditions including high temperature and pressure were requisite for better EO conversion and 3-HPM selectivity. The leaching of metal species was also one of the challenges during the liquidphase heterogeneous reactions. Considering the in situ formed Co colloid within the reaction process as well as the potential advantages of the easily controlled carbon surface, a specific sample with cobalt active centers supported by CNT were herein synthesized via an in situ reduced Colloid method and denoted as Co/CNT-C. A reference synthesized by conventional Impregnation was also synthesized for comparison and denoted as Co/CNT-I.
It is remarkable that compared to the poor EO conversion by Co/CNT-I (7.2%), EO conversion of 33.7% could be reached using Co/CNT-C by the in situ reduced colloid method. The EO conversion was 77.5% in the presence of Co 2 (CO) 8 with 3hydroxypyridine as co-catalyst at the same condition. The  Frontiers in Chemistry frontiersin.org 09 selectivity toward 3-HPM was 92.5% by Co/CNT-C and slightly higher than that using Co 2 (CO) 8 . Concerning the excessive Co content used in the Co 2 (CO) 8 system, the specific rate based on per gram Co per hour was calculated, as displayed in Figure 4A. The Co/CNT-I synthesized by conventional impregnation was only 13.3 mmol · g −1 Co · h −1 , while Co 2 (CO) 8 and Co/CNT-C had close specific rates. The specific rate by supported Co/CNT-C was 52.2 mmol.g −1 Co .h −1 and even higher than that by Co 2 (CO) 8 (49.6 mmol.g −1 Co .h −1 ). The better capacity to transform EO molecules by supported Co/CNT-C in terms of the same amount of Co centers could be disentangled. It has to be mentioned that the Co/CNT-C after reaction could be conveniently separated by centrifugation as illustrated in Figure 4B. The up-layer liquid was transparent with very light yellow, and colorless solution could be obtained and injected into GC for precise analysis after simple filtration. Meanwhile, the reaction mixture using Co/CNT-I by conventional impregnation method displayed a common metal leaching phenomenon as revealed by the yellow solution in Figure 4C. The advantageous stability of Co/CNT-C during reaction could be unraveled preliminarily.
For better information, the FT-IR spectra of different Co/ CNT-C and precursors during the synthesis are studied in Figure 5A to understanding the formation of surface functional groups on these samples. Nitric acid-treated CNT was used as the supporting material. The adsorption peak at 1,640 cm −1 was ascribed to the aromatic skeleton vibration in the main structure of CNT. After HNO 3 treatment, large amount of carboxyl species was generated as revealed by the C-O stretching vibration and O-H deformation vibration at with adsorption peak at 1,400 cm −1 . The deposition of Co particles caused the obvious decreased ratio of the later adsorption peak, suggesting the decreasing number of oxygenated groups during metal loading. Our previous research confirmed the possible consumption of surface oxygenated groups during tight metal loading (Luo et al., 2017;Luo et al., 2020). After the two-step thermal treatment, the adsorption peaks in the reduced Co/CNT-C were further exhausted.
The N 2 adsorption-desorption curves of CNT and supported Co/CNT ( Figure 5B) displayed the typical type IV isotherms, suggesting the maintained mesoporous structure after Co loading. Compared with the initial surface area of CNT (310.14 m 2 /g), the surface areas of Co/CNT-I and Co/CNT-C decreased to 221.8 and 204.6 m 2 /g, respectively. It could be attributed to the blocking of pores during the loading and growth of Co particles. Concerning the greatly differed catalytic performances of Co/CNT-I and Co/CNT-C, the XRD patterns in Figure 5C gave a clear evidence of the distinctive structures by different synthesis. Large amount of Co and CoO coexisted in Co/CNT-I, while both the diffraction peaks of Co and CoO became broadened in the Co/CNT-C, suggesting the smaller crystal sizes of Co-containing particles in such sample. The TEM image of Co/CNT-C is displayed in Supplementary   Figure S3 to understand the surface dispersion of Co. Co particles existed as small black dots (Supplementary Figure S3A) on the surface of CNT. There was not any clear aggregation of Co particles. The HAADF-STEM EDX-elemental mapping of the Co/CNT-C also suggested the high dispersion of Co species on the detected area. It is also remarkable to note that Co 0 was more readily enriched on the surface of Co/CNT-C and accounted for 44.3% of the total surface Co species by the Co 3d XPS core level ( Figure 5D). In the Co/CNT-I reference, only 20.6% Co 0 dispersed on the surface. The much lower H 2 consumption by Co/CNT-C (2,500 μmol/g) than Co/CNT-I (3815 μmol/g) during H 2 -TPR ( Figure 5E) further confirmed the accumulation of oxidative Co species in the Co/CNT-I. The combination results of XRD and XPS suggested a higher dispersion of Co particles with more Co 0 sites exposed on the surface, which may correlated with the faster reaction rate via Co/CNT-C samples as reported by many carbonylation research works using supported metal catalysts (Xu et al., 2011;Zhang, 2012;Ibrahim and Denmark, 2018;Wang et al., 2021). The Co/CNT-C was further tested as a recycle catalyst for the EO carbonylation. After three reaction cycles, the conversion of EO in Figure 5F were still kept at 27.4% with the stabilized high selectivity of 3-HPM (92.1%). The discussion about Co/CNT-C suggested that it is a stable catalyst with nice specific rate based on the same Co atomic efficiency of Co 2 (CO) 8 while avoiding the costly and complicated separation/post-treatments based on the metastable Co 2 (CO) 8 system.

Conclusion
In this work, carbonylative transformation of EO was investigated using commercial Co 2 (CO) 8 in a homogeneous system to produce 3-HPM. The reaction parameters including dosages of reactants, cooperated ligands, and CO pressure were optimized to understand the chemical kinetics of EO carbonylation. The reaction rate was interpreted as secondorder in EO concentration and less sensitive to the CO pressure. However, Co-containing colloid was generated during the reaction, causing the tough separation process before any further chemical analysis. Concerning the impressive separation and sustainable utilization problems caused by the homogeneous reaction process, colloidal Co particles were directly supported on pre-oxidized CNT (Co/CNT-C) via an in situ reduced colloid method. Greatly improved EO conversion was observed via Co/CNT-C, if compared with conventional Co catalyst. The better activity was ascribed to the high dispersion of Co particles with good Co 0 surface exposure and suitable amount of Co 3+ as Lewis acid sites. The specific rate based on per gram Co per hour via Co/ CNT-C was 52.2 mmol.g −1 Co .h −1 , although the macroscopical EO conversion still needs work in progress. The Co/CNT-C sample was highly selective toward 3-HPM with selectivity of Frontiers in Chemistry frontiersin.org 92.5% and a similar atomic efficiency to that of Co 2 (CO) 8 cooperated with 3-hydroxypyridine. Most important of all, the immobilized Co particles on Co/CNT-C enabled the facile separation and recycling for potential applications of EO carbonylation.

Data availability statement
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.