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Determination of 2-Acetyl-1-pyrroline via a Color-Change Reaction Using Chromium Hexacarbonyl

At present, there is no colorimetric method for the quantitation of the aroma compound 2-acetyl-1-pyrroline (2AP). A novel colorimetric method was developed for the determination of 2AP content using chromium hexacarbonyl (Cr(CO)6) as a reagent. The reaction of synthetic 2AP with chromium hexacarbonyl reagent solution in the presence of light produced a green product with an absorption maximum (λmax) at 623 nm. GC–MS was used to confirm the color-change reaction, which showed the loss of 2AP after the addition of Cr(CO)6. This novel method enables facile and cost-effective determination of 2AP in fragrant rice. A comparative analysis of fragrant and nonfragrant rice grain extracts showed that no color-change reaction occurred with the nonfragrant rice sample. A limit of detection (LOD) of 2.00 mg L−1 was determined by method validation with an effective linear concentration ranging from 5.00 to 60.00 mg L−1 of 2AP. The results obtained using the developed colorimetric method were consistent with those obtained by automated static headspace gas chromatography with nitrogen-phosphorus detection (SHS-GC–NPD).

Introduction
2-Acetyl-1-pyrroline (2AP) is a prominent aroma compound found in many foods that is described as smelling 'popcorn-like'. 2AP is a five-membered N-heterocyclic ring compound, which makes the compound highly volatile and unstable with a low odor threshold. 2AP has a boiling point of 183 • C and a molecular weight of 111.14 [1]. 2AP contributes to the flavor of a diverse array of foods, such as cooked mushrooms, nuts, sweet corn, and lobster [2]. 2AP has many functions, for example, providing aroma in essential oils used as therapeutic fragrances and acting as a flavoring compound or food additive [3]. 2AP is biosynthetically metabolized in several plant species, including soybean (Glycine max), sorghum (Sorghum bicolor (L.) Moench), fragrant coconut (Cocos nucifera L.), winter melon (Benincasa hispida) and yellow dwarf coconut (Cocos nucifera L.) [4][5][6][7][8]. Fragrant or aromatic rice contains over 200 volatiles with 2AP as the major component for the pleasant aroma. This quality is highly regarded for its superior grain quality [1]. 2AP can also be found in wheat, brown and rye bread, where the 2AP content of the crust is reported to be 30-fold that of the crumbs [9].
The development of analytical methods for the determination of 2AP in food samples in recent years has been challenging because of the volatility and low concentration (in some samples) of 2AP, as well as the high cost of high-purity chemical standards [10].
The determination of 2AP content is usually carried out via gas chromatography (GC) employing various detection systems, such as nitrogen-phosphorus detectors (NPD), flame ionization (FID), and mass spectrophotometry (MS) [11,12]. Additionally, static headspace (SHS) or solid-phase microextraction (SPME) coupled with GC-MS methods has proven to be a powerful tool for the detection of 2AP at very low concentrations (ppb) [13]. However, GC analysis is extremely time consuming, expensive and requires trained personnel.
There have been no reports on the qualitative or quantitative analysis of 2AP via colorimetry to date. Nadaf et al. reported that spraying a 2,4-dinitrophenyl hydrazine reagent onto a TLC plate loaded with 2AP extracted from pandan leaves produced an orange-red compound [14]. The color-change reaction was identified as that between the reactive ketone group in 2AP and 2,4-dinitrophenyl hydrazine to produce the colored compound hydrazone derivative. Other detection methods based on colorimetric sensor assays have been developed and validated for the analysis of volatile compounds in rice samples. Data processing or principal component analysis is often used to classify volatile compounds to predict the desired outcome in an assay. These outcomes include the differentiation of different rice cultivars or rice storage times [15,16]. Chemically responsive dyes have been used to visualize volatile compounds by recording the change in the dye color before and after exposure to the volatile compounds. The aforementioned colorimetric sensors offer many advantages for qualitative analysis and quantitative discrimination, but their analytical power depends on information obtained using other methods. The qualitative and quantitative analytical capacities of these methods are therefore limited. Consequently, a simple colorimetric method was developed for the quick and easy quantitation of 2AP content via UV-Vis spectrophotometry.
Buttery and de Kimpe reported chemical synthesis methods for preparing 2AP for use as a standard compound [17,18]. In this study, 2AP was chemically synthesized through hydrogenation and oxidation reactions using commercially available 2-acetylpyrrole as a precursor. A colorimetric method was then developed by investigating the color change reactions for different types of organic compounds and organometallic complexes with 2AP. The developed colorimetric technique can be applied to analyze 2AP in fragrant rice samples without the use of advanced analytical instruments, such as GC or GC-MS. The proposed method is applicable and can easily be adapted to a diverse number of samples. The novel method exhibits comparable analytical performance to chromatographic-based methods, cost-effective and time-efficient analysis. Specialized training is not required to perform this colorimetric method, which can therefore be used by farmers and people in the private sector who are concerned with the aroma compound 2AP.

Synthetic 2AP and Its Reaction with Reagents
Synthetic 2AP dissolved in dichloromethane (DCM) was screened using several reagents, and the UV-Vis absorption spectra of the reagent and the colored products were recorded using a UV-Vis spectrophotometer. The λ max values of the reaction product and the colors produced are summarized in Table 1. Distinct absorption bands for the product of the reaction with 2AP were observed for some of the aromatic amine and aromatic hydrazine reagents, such as aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine. For example, λ max was 232 and 272 nm for phenylhydrazine hydrochloride but 368 nm for phenylhydrazine condensed with synthetic 2AP. It has been suggested that 2AP interacts more effectively with aromatic amines in salt form, such as aniline hydrochloride or aniline sulfate, than aniline and p-nitroaniline. Aromatic amines are more soluble in salt or acidic environments in which the protonation of -C=O increases the positive charge of C, thereby accelerating the nucleophilic attack of aromatic amines on the ketone functional group. The λ max values were 203-205 nm and 253 nm for aniline hydrochloride and aniline sulfate, respectively, and changed to 378 nm after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λ max of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λ max was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. after these compounds reacted with 2AP. The nitro group at the ortho and para positions of 2,4-dinitrophenylhydrazine stabilizes the overall structure, facilitating the reaction [19]. Furthermore, an λmax of 380 nm was observed for 2,4-dinitrophenylhydrazine reacted with 2AP, whereas an λmax was not detected for the pure reagent solution. Overall, aniline hydrochloride, aniline sulfate, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine exhibited low specificity for 2AP in rice grain extracts. An absorption band with an λ max of 430 nm was observed for 2-aminobenzaldehyde, whereas a distinct absorbance with an λ max of 623 nm was observed for Cr(CO) 6 and 2AP. Cr(CO) 6 exhibited the highest potential of the reagents in the organometallic group due to the formation of a bright green product after exposure to light, whereas no absorbance for Co 2 (CO) 8 was detected either for the reagent solution or upon reaction with synthetic 2AP. In comparison to the results for Cr(CO) 6 , the product obtained using 2-aminobenzaldehyde had a light yellow color that was difficult to detect with the naked eye. The reaction of 2-aminobenzaldehyde with the fragrant rice grain extract in DCM exhibited a similar absorbance (λ max = 423 nm) as the synthetic 2AP (λ max = 427 nm), as expected, but the intensity was low. Note that 2-aminobenzaldehyde is an expensive and unstable reagent that must be stored below −20 • C [20]. By contrast, Cr(CO) 6 is inexpensive, stable at room temperature, and dissolves effectively in various organic solvents. Therefore, Cr(CO) 6 was the reagent of choice for the colorimetric method.

Confirmation of the Interaction between 2AP and Cr(CO) 6 via GC-MS
A possible mechanism for the formation of the green product of the light-assisted reaction between Cr(CO) 6 and 2AP is shown in Figure 1. The compound 2AP could be considered a chelating ligand through the N and O atoms [21,22]. When activated by light, the CO ligands dissociate to expose vacant coordination sites on the chromium, facilitating the association of 2AP to produce a green Cr(CO) 6-n/2 (2AP) n complex. In this case, a substitution mechanism of CO by 2AP cannot be ruled out. Control experiments were carried out to elucidate complex formation. First, the green solution was not obtained in the absence of light, implying that light is crucial for complexation. Next, reacting Cr(CO) 6 with 2-acetylpyrrole failed to produce a green solution upon exposure to light. This result was attributed to the inability of the lone pair of electrons in pyrrolic N to serve as a good donor atom because of its participation in the aromatic ring. However, light-assisted complexation of Cr(CO) 6 and 2-acetylpyridine did produce a green product (λ max = 606 nm), albeit with low molar absorptivity, indicating that the pyridinic N is a less effective ligand than 2AP [23]. An absorption band with an λmax of 430 nm was observed for 2-aminobenzaldehyde, whereas a distinct absorbance with an λmax of 623 nm was observed for Cr(CO)6 and 2AP. Cr(CO)6 exhibited the highest potential of the reagents in the organometallic group due to the formation of a bright green product after exposure to light, whereas no absorbance for Co2(CO)8 was detected either for the reagent solution or upon reaction with synthetic 2AP. In comparison to the results for Cr(CO)6, the product obtained using 2-aminobenzaldehyde had a light yellow color that was difficult to detect with the naked eye. The reaction of 2-aminobenzaldehyde with the fragrant rice grain extract in DCM exhibited a similar absorbance (λmax = 423 nm) as the synthetic 2AP (λmax = 427 nm), as expected, but the intensity was low. Note that 2-aminobenzaldehyde is an expensive and unstable reagent that must be stored below −20 °C [20]. By contrast, Cr(CO)6 is inexpensive, stable at room temperature, and dissolves effectively in various organic solvents. Therefore, Cr(CO)6 was the reagent of choice for the colorimetric method.

Confirmation of the Interaction between 2AP and Cr(CO)6 via GC-MS
A possible mechanism for the formation of the green product of the light-assisted reaction between Cr(CO)6 and 2AP is shown in Figure 1. The compound 2AP could be considered a chelating ligand through the N and O atoms [21,22]. When activated by light, the CO ligands dissociate to expose vacant coordination sites on the chromium, facilitating the association of 2AP to produce a green Cr(CO)6-n/2(2AP)n complex. In this case, a substitution mechanism of CO by 2AP cannot be ruled out. Control experiments were carried out to elucidate complex formation. First, the green solution was not obtained in the absence of light, implying that light is crucial for complexation. Next, reacting Cr(CO)6 with 2-acetylpyrrole failed to produce a green solution upon exposure to light. This result was attributed to the inability of the lone pair of electrons in pyrrolic N to serve as a good donor atom because of its participation in the aromatic ring. However, light-assisted complexation of Cr(CO)6 and 2-acetylpyridine did produce a green product (λmax = 606 nm), albeit with low molar absorptivity, indicating that the pyridinic N is a less effective ligand than 2AP [23].   Figure 2B) before and after the addition of Cr(CO)6 ( Figure 2C). These chromatograms show that 6-methyl,5-oxo-2,3,4,5-tetrahydropyridine (6M5OTP) was not present in the synthetic 2AP sample, where 2AP was formed by the oxidation of 1-(pyrrolidin-2yl)ethan-1-ol with silver (I) carbonate on celite in toluene. In our experiment, 6M5OTP formed in relatively high quantities during acidic extraction to transfer 2AP from toluene   Figure 2C). These chromatograms show that 6-methyl,5-oxo-2,3,4,5-tetrahydropyridine (6M5OTP) was not present in the synthetic 2AP sample, where 2AP was formed by the oxidation of 1-(pyrrolidin-2yl)ethan-1-ol with silver (I) carbonate on celite in toluene. In our experiment, 6M5OTP formed in relatively high quantities during acidic extraction to transfer 2AP from toluene to DCM. The presence of the 6M5OTP compound has been reported to correlate strongly with the production of 2AP and is simultaneously produced with 2AP in rice, contributing to the aroma of fragrant rice [24]. Olfactory analysis of 6M5OTP via gas chromatography has provided evidence of a similar scent to 2AP. The results of our studies showed that 6M5OTP did not interfere with light-assisted complexation with Cr(CO) 6 . GC-MS analysis revealed that the isomer 6M5OTP was consumed following light-assisted complexation, implying that 6M5OTP isomerizes to 2AP via an open chain structure. Then, thermodynamics drive subsequent complexation of 2AP with Cr(CO) 6 . Overall, the highly selective complexation of 2AP with Cr(CO) 6 can be used to determine 2AP by a simple colorimetric method. The mass spectrum of 2AP, 6M5OTP and Cr(CO) 6 are shown in Figure 2D-F, respectively. to DCM. The presence of the 6M5OTP compound has been reported to correlate strongly with the production of 2AP and is simultaneously produced with 2AP in rice, contributing to the aroma of fragrant rice [24]. Olfactory analysis of 6M5OTP via gas chromatography has provided evidence of a similar scent to 2AP. The results of our studies showed that 6M5OTP did not interfere with light-assisted complexation with Cr(CO)6. GC-MS analysis revealed that the isomer 6M5OTP was consumed following light-assisted complexation, implying that 6M5OTP isomerizes to 2AP via an open chain structure. Then, thermodynamics drive subsequent complexation of 2AP with Cr(CO)6. Overall, the highly selective complexation of 2AP with Cr(CO)6 can be used to determine 2AP by a simple colorimetric method. The mass spectrum of 2AP, 6M5OTP and Cr(CO)6 are shown in Figure  2D-F, respectively.

Specificity of the Colorimetric Method for the Determination of 2AP
Fragrant rice samples extracted with 100.00, 150.00 and 200.00 g of rice grains showed similar absorbances at 623 nm. These results were consistent with those of the aforementioned experiment on synthetic 2AP at various concentrations. The GC-MS profiles of the fragrant rice extract before ( Figure 3A) and after ( Figure 3B) the addition of Cr(CO)6 confirmed that the reaction of Cr(CO)6 is specific to both 2AP and 6M5OTP. The reaction of Cr(CO)6 with fragrant and nonfragrant rice grain extracts was investigated. The absorption maximum at 623 nm was observed in the extracts of fragrant rice grains but not in the nonfragrant-cultivar extract. The specificity of the reaction was also determined by studying the reaction of Cr(CO)6 with other nitrogenous compounds with chemical structures similar to that of 2AP that are found in naturally occurring samples. These compounds include pyrrolidine, 2-acetylpyrrole, 2-acetylpyridine, 2-acetylthiosol and proline. The absorbance band of the nitrogen compounds, especially proline, did not occur in the λmax range of 620-670 nm that was expected from the reaction of 2AP and Cr(CO)6. Pyrrolidine, 2-acetylpyrrole and 2-acetylthiosol exhibited no absorbance between 400 and 700 nm. An λmax of 606 nm and very low molar absorptivity was observed only for 2-acetylpyridine. 2-Acetylpyridine has been detected in a scented Chinese fragrant rice, Xiangjing-8618 in only one study [25]. To the best of our knowledge, 2-acetylpyridine does not naturally occur in the seeds of fragrant or nonfragrant varieties of Thai rice. Two-dimensional gas chromatography (GC×GC) was used to confirm the presence of 2AP in fragrant and nonfragrant rice samples (Figure 4). GC×GC techniques can be a valuable tool to study the volatile fraction where every part of the sample is subjected to two individual separation

Specificity of the Colorimetric Method for the Determination of 2AP
Fragrant rice samples extracted with 100.00, 150.00 and 200.00 g of rice grains showed similar absorbances at 623 nm. These results were consistent with those of the aforementioned experiment on synthetic 2AP at various concentrations. The GC-MS profiles of the fragrant rice extract before ( Figure 3A) and after ( Figure 3B) the addition of Cr(CO) 6 confirmed that the reaction of Cr(CO) 6 is specific to both 2AP and 6M5OTP. The reaction of Cr(CO) 6 with fragrant and nonfragrant rice grain extracts was investigated. The absorption maximum at 623 nm was observed in the extracts of fragrant rice grains but not in the nonfragrant-cultivar extract. The specificity of the reaction was also determined by studying the reaction of Cr(CO) 6 with other nitrogenous compounds with chemical structures similar to that of 2AP that are found in naturally occurring samples. These compounds include pyrrolidine, 2-acetylpyrrole, 2-acetylpyridine, 2-acetylthiosol and proline. The absorbance band of the nitrogen compounds, especially proline, did not occur in the λ max range of 620-670 nm that was expected from the reaction of 2AP and Cr(CO) 6 . Pyrrolidine, 2-acetylpyrrole and 2-acetylthiosol exhibited no absorbance between 400 and 700 nm. An λ max of 606 nm and very low molar absorptivity was observed only for 2-acetylpyridine. 2-Acetylpyridine has been detected in a scented Chinese fragrant rice, Xiangjing-8618 in only one study [25]. To the best of our knowledge, 2-acetylpyridine does not naturally occur in the seeds of fragrant or nonfragrant varieties of Thai rice. Two-dimensional gas chromatography (GC×GC) was used to confirm the presence of 2AP in fragrant and nonfragrant rice samples (Figure 4). GC×GC techniques can be a valuable tool to study the volatile fraction where every part of the sample is subjected to two individual separation dimensions. The qualitative analysis of volatile components in seed extracts of more than twenty rice varieties using GC×GC-MS did not identify 2-acetylpyridine in the samples, while the presence of 2AP in fragrant rice KDML105 and PSL80 cultivars were confirmed (Figure 4). One limitation of this developed colorimetric method is that some other aromatic plants or foods contain compounds with the same active site as 2AP that can interact with the reagent and cause an inaccurate result. Therefore, a good understanding of the chemistry of the sample of interest is considered a prerequisite for accurate detection and evaluation of aroma quality.
dimensions. The qualitative analysis of volatile components in seed extracts of more than twenty rice varieties using GC×GC-MS did not identify 2-acetylpyridine in the samples, while the presence of 2AP in fragrant rice KDML105 and PSL80 cultivars were confirmed (Figure 4). One limitation of this developed colorimetric method is that some other aromatic plants or foods contain compounds with the same active site as 2AP that can interact with the reagent and cause an inaccurate result. Therefore, a good understanding of the chemistry of the sample of interest is considered a prerequisite for accurate detection and evaluation of aroma quality.

Analytical Performance and Validation
The effect of the following factors on the rate of the reaction between synthetic 2AP and the Cr(CO)6 reagent to produce a colored product was investigated: the light exposure time (5, 10, 15, 20, 25 and 30 min) and the distance from light source (10, 15 and 20 cm). The UV-Vis spectrum of the green product solution exhibited an λmax of 623 nm, which did not change significantly with the light exposure time. However, the absorbance of the

Analytical Performance and Validation
The effect of the following factors on the rate of the reaction between synthetic 2AP and the Cr(CO) 6 reagent to produce a colored product was investigated: the light exposure time (5,10,15,20,25 and 30 min) and the distance from light source (10, 15 and 20 cm). The UV-Vis spectrum of the green product solution exhibited an λ max of 623 nm, which did not change significantly with the light exposure time. However, the absorbance of the colored product increased with exposure time, where the green color appeared much darker at 30 min than at 5 min. The UV-Vis spectra at different 2AP concentrations are shown in Figure 5. The reaction between Cr(CO) 6 and synthetic 2AP at concentrations below 15.00 mg L −1 , the product solution appeared lighter in color than at concentrations above 15.00 mg L −1 . The optimal reaction time was determined at 5 min where the green product decomposed into a green precipitate for reaction times of more than 15 min. Additionally, the distance of 10 and 15 cm of the product solution from the light source showed that precipitate was formed. The optimal distance was determined at 20 cm. and yielded a coefficient of variation of 1.57 %RSD. The validation data showed lower sensitivity and precision for the colorimetric method than for automated headspace gas chromatography [11]; however, the data obtained using the colorimetric method are acceptable. Overall, although the multi-step sample preparation for the colorimetric method is time-consuming, the lack of sophisticated instrumentation results in a cost-effective analysis technique. Application of the colorimetric technique to the quantitation of 2AP in fragrant rice samples requires a properly designed experiment to ensure there is a sufficient quantity of extracted 2AP in DCM to react with Cr(CO)6 to yield a green product. An experiment was conducted using KDML105, the most popular Thai fragrant rice variety with a high content of 2AP. The optimal weight of the rice sample for extraction was found at 100 g.

Comparison of the 2AP Content Determined by SHS-GC-NPD and the Colorimetric Method
Extracts of fragrant rice grains and nonfragrant rice grains were quantitatively determined using the developed colorimetric method and SHS-GC-NPD ( Table 2). The addition of Cr(CO)6 to extracts of KDML105 and PSL80 fragrant rice grains resulted in a colorchange reaction, where a green solution was developed after exposing the solution to UV light. However, a color change was not observed for the nonfragrant rice grains PSL2 and RD79 upon addition of Cr(CO)6. These results were confirmed by SHS-GC-NPD, where the 2AP content of both cultivars could not be detected. SHS-GC-NPD indicated 3.68 ± 0.07 µg g −1 and the colorimetric method showed 3.83 ± 0.09 µg g −1 for KDML105 while those for PSL80 were 2.11 ± 0.14 µg g −1 and 1.95 ± 0.10 µg g −1 for colorimetric and SHS-GC-NPD, respectively. The PSL80 cultivar was reported as having been developed from KDML105 to contain less amylose but comparable aroma contents [26]. These results were obtained from a study published by the Rice Department of Thailand, in which the similarities between cultivars PSL80 and KDML105 were identified. The developed colorimetric method for 2AP quantitative analysis was validated with respect to the linearity range, sensitivity (limit of detection) and precision. The absorbance of the green product at 623 nm was linear in the synthetic 2AP concentration over the concentration range of 5.00-60.00 mg L −1 with a correlation coefficient (r 2 ) of 0.9934. The sensitivity is reflected by the limit of detection (LOD), which is defined as the concentration at which the signal-to-noise ratio (S/N) is more than 3. The LOD of 2AP was found to be 2.00 mg L −1 . The precision of the proposed method is expressed in terms of the repeatability (intraday) and reproducibility (interday) as a percentage of the relative standard deviation. A solution of synthetic 2AP at 40.00 mg L −1 in DCM was prepared and reacted with 1000 mg L −1 of Cr(CO) 6 ten times on the same day under optimal conditions. The recorded absorbances at 623 nm were averaged, and the %RSD was calculated to be 4.31. The reproducibility was determined on five different days with five replicates on each day and yielded a coefficient of variation of 1.57 %RSD. The validation data showed lower sensitivity and precision for the colorimetric method than for automated headspace gas chromatography [11]; however, the data obtained using the colorimetric method are acceptable. Overall, although the multi-step sample preparation for the colorimetric method is time-consuming, the lack of sophisticated instrumentation results in a cost-effective analysis technique.
Application of the colorimetric technique to the quantitation of 2AP in fragrant rice samples requires a properly designed experiment to ensure there is a sufficient quantity of extracted 2AP in DCM to react with Cr(CO) 6 to yield a green product. An experiment was conducted using KDML105, the most popular Thai fragrant rice variety with a high content of 2AP. The optimal weight of the rice sample for extraction was found at 100 g.

Comparison of the 2AP Content Determined by SHS-GC-NPD and the Colorimetric Method
Extracts of fragrant rice grains and nonfragrant rice grains were quantitatively determined using the developed colorimetric method and SHS-GC-NPD ( Table 2). The addition of Cr(CO) 6 to extracts of KDML105 and PSL80 fragrant rice grains resulted in a color-change reaction, where a green solution was developed after exposing the solution to UV light. However, a color change was not observed for the nonfragrant rice grains PSL2 and RD79 upon addition of Cr(CO) 6 . These results were confirmed by SHS-GC-NPD, where the 2AP content of both cultivars could not be detected. SHS-GC-NPD indicated 3.68 ± 0.07 µg g −1 and the colorimetric method showed 3.83 ± 0.09 µg g −1 for KDML105 while those for PSL80 were 2.11 ± 0.14 µg g −1 and 1.95 ± 0.10 µg g −1 for colorimetric and SHS-GC-NPD, respectively. The PSL80 cultivar was reported as having been developed from KDML105 to contain less amylose but comparable aroma contents [26]. These results were obtained from a study published by the Rice Department of Thailand, in which the similarities between cultivars PSL80 and KDML105 were identified.

Synthesis of 2-Acetyl-1-pyrroline
2-Acety-1-pyrroline was synthesized by the hydrogenation of 2-acetylpyrrole to yield 1-(pyrrolidin-2-yl)ethan-1-ol, which was then oxidized by silver (I) carbonate. The experimental method involved considerable modifications of methods reported in the literature [17,18] and is detailed below. Silver (I) carbonate on Celite was prepared using a method reported by Fétizon [27]. Celite (30.00 g) was added to a solution of AgNO 3 (30.00 g of AgNO 3 in 200 mL of distilled water) and vigorously stirred for 10 min. Then, a solution of Na 2 CO 3 (30.00 g of Na 2 CO 3 ·10H 2 O in 300 mL of distilled water) was slowly added to the suspension to yield a yellow-green precipitate of Ag 2 CO 3 on Celite. The product was recovered by filtration and washed several times with distilled water until the pH reached ca. 7. The product was dried in an oven at 110 • C for 24 h.

1-(Pyrrolidin-2-yl)ethan-1-ol
A 250-mL round-bottomed flask equipped with a magnetic stir bar was filled with 2-acetylpyrrole (0.700 g) and methanol (50.00 mL). Then, Rh/Al 2 O 3 (1.00 g) was added to the solution, followed by purging with H 2 using a balloon. The reaction progress was monitored by TLC and GC-MS to ensure complete consumption of 2-acetylpyrrole. After 48 h, the solution was filtered through Celite and evaporated to dryness to afford 1-(pyrrolidin-2-yl)ethan-1-ol in quantitative yield. 1 H-NMR and GC-MS analyses showed that the obtained compound was sufficiently pure to be used without further purification.

2-Acetyl-1-pyrroline (2AP)
A 250-mL round-bottomed flask equipped with a magnetic stir bar was filled with 1-(pyrrolidin-2-yl)ethan-1-ol (0.625 g), Ag 2 CO 3 on Celite (5.00 g) and toluene (150.00 mL) under an N 2 atmosphere. The reaction was heated to reflux for 1 h to obtain 2-acetyl-1-pyrroline. The suspension was filtered through Celite and transferred to a 250-mL volumetric flask to obtain a stock solution of 2AP in toluene. The 2AP in toluene was extracted twice with 100.00 mL of 0.1 M HCl. Then, the aqueous solution was basified with 1 M NaOH and extracted with DCM. A portion of the organic layers was transferred to a 1 L volumetric flask to obtain a stock solution of 250.00 mg L −1 2AP in DCM.

Plant Materials and Extraction
Fragrant and nonfragrant rice grains were used to validate the color-change reaction between the naturally occurring 2AP in plant samples and Cr(CO) 6 . The fragrant rice grains used were Oryza sativa L. cv. Khao Dawk Mali 105 (KDML105) and Phitsanulok 80 (PSL80), and the nonfragrant rice grains used were Oryza sativa L. cv. Phitsanulok 2 (PSL2) and Rice Department 79 (RD79). Samples were extracted using the acid-base method. Briefly, 100.00 g of rice grains ground in a blender and added to 200.00 mL 0.1 M HCl; the resulting mixture was shaken for 15 min and centrifuged at 6000 rpm for 15 min. The supernatant was basified with approximately 2.00 mL of 1 M NaOH and added to 200.00 mL of DCM. The solution was shaken and left to stand until it separated into layers. The DCM portion was collected and added to anhydrous Na 2 SO 4 to remove water molecules. The solution was dried using a rotary evaporator to obtain 2.00 mL of 2AP extract.

GC-MS Analysis
A Model 6850 gas chromatograph equipped with a Model 5973 mass spectrometer with dimensions of 30 m × 0.25 mm and a 0.25-µm nonpolar capillary column HP-5MS (5%-phenyl)-methylpolysiloxane (Agilent Technologies, Santa Clara, CA, USA) was used to analyze the synthetic 2AP, including its intermediate, byproducts and reaction product with Cr(CO) 6 . The GC carrier gas used was purified helium (99.99%) at a constant flow rate of 1 mL min −1 . The oven temperature was initially held at 45 • C and then increased at a rate of 3 • C min −1 to a final temperature of 150 • C. To analyze the rice sample extracts, the initial oven temperature was set at 45 • C, held for 5 min and increased to 200 • C at a rate of 4 • C min −1 . The injector temperature was 230 • C. The sample was injected with a split ratio of 10:1. The mass spectrometer was operated in electron impact (EI) mode with an electron energy of 70 eV. The ion source and quadrupole temperatures were set at 230 • C and 150 • C, respectively. The mass range (m/z) was 29-550 with a scan rate of 0.68 s.
The GC-MS transfer line was set to 280 • C. 2AP and Cr(CO) 6 were identified by matching their mass spectra with synthetic and standard compound reference spectra in the Wiley 7n Mass Spectral Library (Revision C.00.00). The NIST 17 Mass Spectral Library (Revision D.01.00/1.6d.) was also utilized.

Comprehensive Two-Dimensional Gas Chromatography-Mass Spectrometry (GC×GC-MS) Analysis
GC×GC-MS was carried out on an Agilent Technologies 6890 model GC system retrofitted with a longitudinally modulated cryogenic system (LMCS). The instrument was equipped with a headspace auto-sampler and a flame ionization detector. The column set-up consisted of primary capillary column dimensions of 30 m × 0.53 mm i.d. × 1.5 µm film thickness HP-5 phase (5% phenyl 95% methylpolysiloxane) coupled with a second capillary column dimensions of 1.0 m × 0.32 mm i.d.× 0.5 µm film thickness Innowax phase (Polyethylene glycol). The thermostatically controlled cryogenic trap was maintained at about −20 • C for the duration of each analysis. The modulation period was 6 s in all analyses. Temperature-programmed conditions were performed for all analyses, using an initial oven temperature of 45 • C (constant for 4 min), then increased at a rate of 3 • C per minute to a final oven temperature of 230 • C. The GC injector was operated in splitless mode, and the temperature was set at 230 • C. Purified helium gas was used as the carrier gas. The mass spectrometer was operated in electron impact (EI) mode with the same parameters as the above.

Chemical Reaction of 2AP with Reagents to form Colored Products
To identify reagents capable of forming colored products with 2AP, three groups of reagents were investigated that were classified according to their structure and reaction mechanisms. These groups included aromatic amines (aniline, aniline hydrochloride and aniline sulfate, p-nitroaniline, p-nitroaniline hydrochloride, 2-aminobenzoic acid, 2-aminobenzonitrile, and 2-aminobenzaldehyde); phenylhydrazines (phenylhydrazine, phenylhydrazine hydrochloride and 2,4-dinitrophenylhydrazine); and organometallic complexes (Co 2 (CO) 8 ) and Cr(CO) 6 ). Reagent solutions were prepared at a concentration of 1000 mg L −1 in distilled water or DCM. Then, 1.00 mL of each reagent was added to 100.00 mg L −1 of synthetic 2AP in DCM. The color-change reaction was allowed to occur under a solar light source (Hagen Inc., Baie-D'Urfe, QC, Canada) for 5 min, and the solution of the colored product was scanned using a spectrophotometer with a wavelength range of 400-700 nm. A 125-W Solar-Glo lightbulb was used as the solar light source and emitted UVA, UVB, and visible light.

Colorimetric Method
A series of 2AP standard solutions with concentrations of 5.00-60.00 mg L −1 in DCM was prepared by diluting from the stock solution of 250 mg L −1 2AP in DCM described in 3.2.3. Each of the synthetic 2AP solutions of different concentrations (1.00 mL) was added to 1.00 mL of 1000 mg L −1 Cr(CO) 6 reagent solution. The resulting mixture was shaken and exposed to the solar light source (125-W Solar-Glo lightbulb) for 5 min. Then, the absorbances at 623 nm were recorded. The colorimetric method was qualitatively validated in terms of specificity and sensitivity. Reagent specificity to the 2AP compound was assessed for fragrant and nonfragrant rice grain extracts, and other nitrogen-containing compounds, including 2-acetylpyrrole and 2-acetylpyridine, in terms of the presence of a colored product. The reagent sensitivity was assessed by the limit of detection (LOD) of the 2AP standard. GC-MS was used to monitor the formation of 2AP and its isomer 6-methyl, 5-oxo-2,3,4,5-tetrahydropyridine (6M5OTP), which can form during the acidic extraction of rice grains.

SHS-GC-NPD
The determination of 2AP in grains was carried out using the method of Boontakham [28] with some modifications. First, rice grains were ground into a powder that was sieved through an aperture with a 45-mesh size. Then, the rice powder (1.00 g) was weighed into a 20-mL headspace vial, to which 1.00 µL of 500.00 mg L −1 2,6-DMP was added as an internal standard. The headspace vial was then sealed immediately with PTFE/silicone septum and aluminum caps prior to analysis by SHS-GC-NPD. A static headspace autosampler (TurboMatrix 40, PerkinElmer, Inc., Waltham, MA, USA) connected to a PerkinElmer Clarus 690 Series GC system coupled to an NPD detector was used. Separation was carried out using an Elite-5MS column (with a 30 m × 0.25 mm i.d. and 0.25-µm film thickness; PerkinElmer, Inc., Waltham, MA, USA) with splitless injection at 250 • C. The column temperature was programmed to increase from 45 • C to 125 • C at a gradient of 7 • C min −1 . The headspace operating conditions for the rice grains were a 125 • C oven temperature and a 20 min vial equilibration time.

Statistical Analysis
Samples were extracted in triplicate (n = 3). The determination of the 2AP content via SHS-GC-NPD, GC-MS and colorimetric methods was carried out in triplicate, and the results were expressed as a mean ± standard deviation (SD). Statistical analysis was performed using SPSS software version 17.0 (SPSS Inc., Chicago, IL, USA) for the one-way analysis of variance (ANOVA). The significance level at 95% (p < 0.05) was determined by the post hoc Tukey's test.

Conclusions
In this study, we developed a colorimetric method for the quantitation of the aroma compound 2AP. Synthetic 2AP and extracted 2AP from plant samples were added with Cr(CO) 6 solution, and a bright green color change reaction occurred. A distinct absorbance with an λ max of 623 nm was observed for the green color product solution. The method was validated with fragrant and nonfragrant rice extracts, as well as other nitrogen-containing compounds to confirm the reaction specificity of 2AP. A color-change was not observed for the nonfragrant rice grains PSL2 and RD79 upon addition of Cr(CO) 6 . The quantitative results were confirmed by SHS-GC-NPD, where the good agreement was obtained for the concentrations of 2AP in the fragrant and nonfragrant rice samples. The colorimetric method proposed in the manuscript is simple and easy to perform by untrained personnel. The results obtained will be of interest for researchers in the related field who can develop our method further, including other food samples and on-field applications.


s2fieldsofstudy: Chemistry Biology Engineering

text:
Enhanced Catalytic Activity of a New Nanobiocatalytic System Formed by the Adsorption of Cytochrome c on Pluronic Triblock Copolymer Stabilized MoS2 Nanosheets

The formation of nanobiohybrids through the immobilization of enzymes on functional nanomaterials has opened up exciting research opportunities at the nanobiointerfaces. These systems hold great promise for a wide range of applications in biosensing, biocatalytic, and biomedical fields. Here, we report the formation of a hybrid nanobiocatalytic system through the adsorption of cytochrome c (Cyt c) on pluronic triblock copolymer, P123 (PEO-b-PPO-b-PEO), stabilized MoS2 nanosheets. The use of pluronic polymer has helped not only to greatly stabilize the exfoliated MoS2 nanosheets but also to allow easy adsorption of Cyt c on the nanosheets without major structural changes due to its excellent biocompatibility and soft protein-binding property. By comparing the catalytic activity of the Cyt c–MoS2 nanobiohybrid with that of the free Cyt c and as-prepared MoS2 nanosheets, we have demonstrated the active role of the nanobiointeractions in enhancing the catalytic activity of the hybrid. Slight structural perturbation at the active site of the Cyt c upon adsorption on MoS2 has primarily facilitated the peroxidase activity of the Cyt c. As the MoS2 nanosheets and the native Cyt c individually exhibit weaker intrinsic peroxidase activities, their mutual modulation at the nanobiointerface has made the Cyt c–MoS2 a novel nanobiocatalyst with superior activity.

INTRODUCTION
The discovery of a two-dimensional (2D) layered graphene nanomaterial opened up an area of intensive research due to its exceptional mechanical strength, high thermal and electrical conductivity, and optical properties. 1−4 Since then, graphenelike 2D nanosheet materials such as transition metal dichalcogenides (TMDCs), graphitic carbon nitride, MXenes, hexagonal boron nitride (h-BN), and transition metal oxides (TMOs) have become the center of attention. 5 2D layered materials (single or few layers) are more fascinating than their bulk form because of the high density of active surface sites available over a large area. 6 A broad range of electronic properties like metallic/semimetallic (e.g., graphene, VS 2 , TaS 2 ) to semiconducting (e.g., MoS 2 , WS 2 ) or insulating (e.g., h-BN) properties are exhibited by them. 4,5 Similarly, they have a broad range of optical properties like fluorescence quenching or emission behavior. 7,8 2D molybdenum disulfide (MoS 2 ) is one of the pioneers of TMDC materials with an excellent layer-dependent band structure. 9,10 A direct band gap of ∼1.8 eV possessed by the monolayer MoS 2 nanosheet makes it a good semiconducting material, resolving the gapless problems of graphene. 11 A single MoS 2 nanosheet is composed of a layer of Mo atoms sandwiched between two layers of S atoms. Strong covalent bonding between Mo and S atoms strengthens the trilayer MoS 2 nanosheet, and a weak Van der Waal force stacks them up. The MoS 2 nanosheet can have two distinct crystalline phases at room temperature, one with trigonal prismatic coordination, which is found to be semiconducting (2H), and the other one with octahedral coordination (1T), which is metallic. MoS 2 nanosheets have gained burgeoning interest in biosensor, biocatalytic and biomedical applications specifically due to their unique optoelectronic properties, high capacity of biomolecule loadings and good biocompatibility. 12−19 Moreover, their tailoring behavior to selectively respond to specific analytes with high sensitivity through different surface functionalizations has led to their extensive usage in biosensing and biocatalytic applications. 20,21 Although natural enzymes can perform catalytic activity with high efficiency and selectivity, they often require strict reaction conditions like specific temperature, pH, and ionic strength. The instability of natural enzymes under drastic conditions limits their industrial applications. 22 To overcome these limitations, researchers are now focusing on the development of new low-cost, stable, and active nanobiocatalytic systems by interfacing natural proteins/ enzymes on a wide variety of functional nanomaterials (or nanozymes). 23 In this work, we have demonstrated superior catalytic performance of a new Cyt c−MoS 2 nanobiocatalytic system developed by interfacing Cyt c on triblock copolymer P123 stabilized MoS 2 nanosheets (Scheme 1).
The MoS 2 nanosheets used in this work were synthesized via the ultrasonication-assisted liquid-phase exfoliation method. 10 This method provides a simple but effective route for obtaining sufficient quantities of thin-layered MoS 2 nanosheets with high structural quality. This method has previously been extensively employed using various types of surfactants and polymers as dispersants to yield MoS 2 nanosheets/nanoparticles of varying sizes and thicknesses. 10,24,25 Here we have used 1% (w/v) aqueous solution of a pluronic triblock copolymer, P123, as an effective dispersant for the exfoliation of bulk MoS 2 . P123 is a nonionic amphiphilic triblock copolymer composed of hydrophobic poly(propylene oxide) (PPO) and hydrophilic poly-(ethylene oxide) (PEO) blocks. The choice of P123 was made based on the biocompatible nature of the polymer and its preventive role toward aggregation or the major structural change of proteins after adsorption on nanomaterials. 26 The large molecular weight of the polymer and its long hydrophobic and hydrophilic chains also provides effective stabilization to the exfoliated MoS 2 nanosheets by capping on their surface. The hydrophobic PPO units can be adsorbed on the MoS 2 nanosheets, while the hydrophilic PEO polymer chains remain extended into the bulk solution (Scheme 1). These hydrophilic chains provide effective steric hindrance to prevent flocculation or aggregation of the nanosheets. 27−30 Apart from providing a biocompatible environment, the polymer chains on MoS 2 nanosheets also play an important role in anchoring more Cyt c on the nanosheets for enhanced catalytic activity of the hybrid.
Cytochrome c (Cyt c) is a globular heme protein which functions as an electron carrier for oxidative phosphorylation (or mitochondrial respiration) in the inner mitochondrial membrane of healthy cells. 31,32 The Fe center of Cyt c is six coordinated in its native state with methionine (Met 80) as a distal ligand. 33 The protein chain of Cyt c consists of 104 amino acids with an isoelectric point of ∼10 (due to the presence of a large number of basic amino acids like lysine). Hence, Cyt c bears positive surface charge at physiological pH. 32 Although Cyt c primarily functions as an electron carrier in its native state, under pro-apoptotic (programmed cell death) conditions it gains peroxidase activity in the presence of H 2 O 2 . 31,34,35 Certain conformational change of Cyt c induced by its interactions with membrane-bound cardiolipin is believed to be responsible for gaining in vivo peroxidase activity. 36 However, the exact nature of the conformation and the mechanism by which it gains peroxidase activity still remain elusive. Classical peroxidases have a five-coordinate heme structure, and the sixth coordination of the iron center is either vacant or occupied by a loosely bound water molecule. 37 This structural feature facilitates the interaction of H 2 O 2 with the iron center. Peroxidases generally follow a mechanism in which H 2 O 2 reacts with the Fe(III) resting state of heme to produce an oxyferryl compound (Fe(IV)O heme) known as Compound I along with an adjacent radical. The resting state of enzymes is regenerated with the abstraction of hydrogen from organic substrates, and the newly formed radical then yields stable oxidation products upon reactions with O 2 . 34 The native Cyt c becomes a potent peroxidase only after some conformational changes (or after the formation of a partially unfolded structure) with the weakening or rupturing of the Met80 S−Fe bond. 32,34 The improved peroxidase-like catalytic activity of Cyt c has been extensively investigated using a wide variety of supporting materials such as polymers, 38 graphene oxide nanosheets, 39,40 metal organic frameworks, 41 carbon dots, 42 and silica nanoparticles. 32 However, no such study has been reported using 2D MoS 2 nanosheets as supporting materials in spite of the outstanding catalytic performances exhibited by them. 19 In this work, we have used triblock copolymer P123-stabilized MoS 2 nanosheets as supporting materials for interfacing Cyt c and making a new nanobiocatalytic system with superior peroxidase-like catalytic activity. The MoS 2 nanosheets are of particular importance in this regard because: (1) they exhibit intrinsic peroxidase-like catalytic activity (therefore, they are often referred to as nanozymes) and (2) they can effectively interact with Cyt c, causing a conformational change to improve not only their own intrinsic catalytic activity but also the catalytic activity of Cyt c. 25 Therefore, the MoS 2 -nanozyme-activated synergistic amplification of the catalytic activity of our developed Cyt c−MoS 2 bionanohybrid catalyst is the major focus of the present work. Besides, we have also emphasized the understanding of the structural and conformational changes of the immobilized Cyt c on MoS 2 responsible for enhancing its catalytic efficiency. The peroxidase-like catalytic activity of the nanobiohybrid has been explored by probing the oxidation of a 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) substrate in the presence of another substrate H 2 O 2 . Cytochrome c from horse heart and sodium phosphate monobasic (anhydrous) and sodium phosphate dibasic (anhydrous) used to prepare phosphate buffer (PB) solution were purchased from Sisco Research Laboratories (SRL), India. All the reagents were used as received without further purification.
2.2. Preparation of MoS 2 Nanosheets. MoS 2 nanosheets were prepared by an ultrasonication-assisted centrifugation method. 10,43,44 In a typical experiment, 11 mg/mL of MoS 2 solution was prepared in 1% (w/v) aqueous pluronic P123 polymer solution, and the solution was sonicated for 5 h in a 100 W bath sonicator. Then the dispersed solution was subjected to centrifugation at 7000 rpm (in RM-12 Micro Centrifuge) to remove the bigger particles, and the supernatant was taken out as MoS 2 nanosheet dispersion. Nanosheet dispersions were found to be stable over several weeks.
2.3. Preparation of Cyt c−MoS 2 Nanobiohybrids. The Cyt c−MoS 2 nanobiohybrid was prepared through the spontaneous adsorption of Cyt c on P123 polymer functionalized MoS 2 nanosheets. First, we prepared stock solutions of MoS 2 (21 μg/mL) and Cyt c (100 μM) in aqueous 10 mM, pH 7.1 phosphate buffer. Appropriate amounts of the MoS 2 and Cyt c from their respective stock solutions were mixed in phosphate buffer to obtain a series of Cyt c−MoS 2 nanobiohybrid-containing solutions with increasing concentration (from 0.5 μg/mL to 3 μg/mL) of MoS 2 and a fixed 0.5 μM concentration of Cyt c. These solutions were used to study the MoS 2 concentration-dependent catalytic activity of the nanobiohybrids. The solutions were stirred for 5 min at room temperature before performing catalytic studies. The nanobiohybrids with 3 μg/mL of MoS 2 and 0.5 μM Cyt c were used for kinetic analysis by studying catalytic activities under varying substrate (ABTS/H 2 O 2 ) concentrations.
2.4. UV−vis Absorption, FTIR, and TEM Characterizations. UV−vis absorption studies of the MoS 2 nanosheets, Cyt c, and Cyt c−MoS 2 nanobiohybrids were performed using a spectrophotometer (model: LABINDIA-UV 3092). 44 The formation of the Cyt c−MoS 2 nanobiohybrid was confirmed by the observed shift of the Soret band position of Cyt c from 409 nm in the free native state to 400 nm in the bound state of the nanobiohybrids. The amount of Cyt c adsorbed on MoS 2 nanosheets was roughly estimated by spectrophotometric measurements of an aqueous buffer solution of Cyt c (10 μM) before and after addition of MoS 2 (10 μg/mL) into the solution. The decrease in absorbance of the free Cyt c at 409 nm (ε = 106 000 M −1 cm −1 ) due to the adsorption has been used to roughly estimate the concentration of the adsorbed Cyt c.
For FTIR studies of the MoS 2 nanosheets and the Cyt c− MoS 2 nanobiohybrids, the suspensions were centrifuged at higher rpms (around 12 000 rpm) multiple times and collected the solid particles. The solid particles were washed three to four times with distilled water to remove weakly adsorbed polymer molecules and dried at 56°C. The samples were analyzed with a Thermo Scientific-Nicolet iS5 FTIR spectrometer, in the frequency range of 500−4000 cm −1 . TEM measurement of the MoS 2 nanosheets was carried out using a JEM−2100 HRTEM (Make−JEOL, Japan) instrument. 25 2.5. Peroxidase Activity and Michaelis−Menten Kinetic Analysis. The peroxidase activity of immobilized Cyt c protein on MoS 2 was evaluated by measuring the initial rates of ABTS oxidation in the presence of H 2 O 2 . The formation of the oxidation product, the ABTS radical cation (ε 418 = 3.6 × 10 4 M −1 cm −1 ), was recorded by monitoring the time-dependent absorption increase at 418 nm. For the catalytic activity studies, a total 100 μL volume of the Cyt c−MoS 2 nanobiohybrid solution was taken in a cuvette (1 cm path length) and mixed with an appropriate concentration of ABTS (from a stock solution prepared in 10 mM phosphate buffer at pH 7.1). The absorption kinetics (at 418 nm) were started by the addition of H 2 O 2 . ABTS, H 2 O 2 , and nanobiohybrid samples were freshly prepared each time.
Peroxidase enzymatic kinetics of the Cyt c−MoS 2 nanobiohybrid (3 μg/mL of MoS 2 + 0.5 μM Cyt c) and MoS 2 (3 μg/mL) only systems as catalysts were analyzed using the Michaelis−Menten equation (eq 1), and the associated Lineweaver−Burk equation (eq 2) was used to obtain the kinetic parameters. In the Michaelis−Menten approach, the initial reaction rates (v 0 ) are measured at different initial substrate concentrations (S 0 ), and the kinetic parameters (V max , k cat , and K m ) are obtained from the linear fitting analysis of the Lineweaver−Burk plot (eq 2). In this work, the peroxidase-like catalytic cycle involves two substrates (H 2 O 2 and ABTS). To obtain the Michaelis−Menten kinetic parameters with respect to both the substrates, the Lineweaver−Burk plots have been constructed from a series of experiments in which the concentration of one of the substrate is held constant while varying the other one. The kinetic parameters obtained from this treatment are only apparent constants.
Here "v" is the initial reaction rate; "[S]" is the substrate concentration; "[E 0 ]" is the total enzyme concentration; k cat is the turnover rate constant; and K m is the Michaelis−Menten constant. 2.6. Molecular Docking Analysis. Docking analysis was carried out to observe any probable molecular interactions between the protein Cyt c and the PEO units of the triblock copolymer that are attached to the MoS 2 nanosheet surface. A ligand, 2-[2-(2-hydroxyethoxy)propoxy]ethanol, was docked on a horse heart Cyt c molecule using "Autodock Vina" software. Grid box dimensions were set to be 40 Å for X, Y, and Z conformations. To analyze the nature of substrate− protein binding interactions, the Cyt c molecule was also docked with ABTS and H 2 O 2 by the above procedure, in which the ligand was chosen as ABTS or H 2 O 2 .

RESULTS AND DISCUSSION
3.1. Spectroscopic and Microscopic Characterizations of MoS 2 Nanosheets. Figure 1a shows the absorption spectrum of MoS 2 nanosheet dispersion in aqueous P123 solution. The spectral characteristics share common features of the trigonal prismatic-coordinated 2H phase of MoS 2 . 45 The two characteristic absorption bands in the 600−700 nm region of the spectrum correspond to the excitonic transition of MoS 2 from the valence bands to the minima of the conductance band (at the K point of the Brillouin zone). The splitting of the valence band by spin−orbit and interlayer coupling interactions accounts for the two excitonic bands. 9,46 There are two additional higher energy absorption bands in the 350−450 nm region of the spectra corresponding to the transition of electrons to the higher density states of the Brillouin zone. 47 The concentration of the as-prepared MoS 2 nanosheet dispersion was calculated using the absorbance value at 345 nm, for which the extinction coefficient was previously reported as 6820 L g −1 cm −1 . 44 The concentration of the prepared nanosheet solution was found to be 21 μg/mL.
The FTIR spectrum of the P123 triblock copolymer coated MoS 2 nanosheets is given in Figure 1b. The corresponding spectra of the bulk MoS 2 material and the P123 polymer are given in Figure S1 of the Supporting Information. The FTIR results confirm that the surface of the MoS 2 nanosheets is functionalized with the P123 polymers. The presence of CH 3 groups of the central hydrophobic PPO blocks of the polymer on the MoS 2 nanosheets is confirmed by the two bands at ∼2965 and ∼1374 cm −1 . The 2965 cm −1 band can be assigned to the antisymmetric C−H stretching vibration of the methyl groups and the 1374 cm −1 band to the symmetric deformation band of the methyl groups. The FTIR peak at 2869 cm −1 corresponds to the antisymmetric methylene (CH 2 ) stretching modes. The 1093 cm −1 band is due to the C−O−C stretching vibration of the P123 polymer. These characteristic FTIR bands of the P123 polymer on the MoS 2 nanosheets are consistent with the FTIR spectrum of the pure P123 polymer ( Figure S1). 48 The other bands peaking at 1627 and 1260 cm −1 correlate well with the FTIR absorption bands of MoS 2 . 49 The TEM images of the P123 polymer stabilized MoS 2 nanosheets are given in Figure 1c,d. These images confirm the two-dimensional nanosheet structure of the synthesized MoS 2 . Moreover, the average dimension of the exfoliated individual nanosheets is found in the range of 90−100 nm.
3.2. Length and Thickness of the MoS 2 Nanosheets. The mean length and thickness of the MoS 2 nanosheets can be calculated using previously reported empirical relations based on the information at the characteristic A (at 660 nm) and B (at 610 nm) excitonic bands of the prepared MoS 2 nanosheets. The length of the MoS 2 nanosheet is related to the ratio of the extinction coefficient at the B-excitonic peak (E B ) to that at 345 nm (E 345 ) by the following relation: 44 The thickness of the nanosheets (referred to as number of MoS 2 layers in each nanosheet, N MoS 2 ) can be obtained from the peak position of the A-exciton band using the following relation: 44  Using the above relations, the calculated length and thickness of the MoS 2 nanosheets are found to be ∼90 nm and ∼3.4 layers/nanosheet, respectively. The calculated length of the MoS 2 nanosheets is found to be well correlated with the average dimension obtained from TEM results.
3.3. Characterization of the Cyt c−MoS 2 Nanobiohybrid System. The P123 stabilized MoS 2 nanosheets were used for the formation of a Cyt c−MoS 2 hybrid nanobiocatalytic system through the adsorption of Cyt c on the MoS 2 nanosheets. The formation of an interactive bionanointerface can be understood from UV−visible absorption spectral changes of Cyt c before and after adsorption on MoS 2 . Cyt c in its native oxidized state shows a highly intense Soret band at 409 nm and two other relatively low intense peaks around 522 and 360 nm named as Q and δ band, respectively (Figure 1a). The two visible region bands (Q and Soret bands) originate from the π to π* transitions of the porphyrin ring. 50 The Soret band is highly sensitive to the heme microenvironment of Cyt c. Any perturbations in and around heme will be reflected by the change in the peak position and/or intensity of the Soret band. 42 After being stirred with MoS 2 nanosheets, the Cyt c native Soret band position at 409 nm has been found to be blue-shifted to 400 nm. This clearly indicates possible interactions between Cyt c and the MoS 2 nanosheet template that might lead to some perturbations on the heme center suitable for peroxidase activity enhancement. Based on the decreased absorbance of the free native Cyt c at 409 nm upon adsorption on MoS 2 , the amount of adsorbed Cyt c on the MoS 2 nanosheets was estimated as 1.12 μM in 10 μg/mL of MoS 2 solution.
The influence of MoS 2 nanosheets on Cyt c is further supported by the FTIR analysis. The FTIR spectra of native Cyt c, the Cyt c−MoS 2 nanobiohybrid, and free MoS 2 nanosheets are given in Figure 2. The major characteristic amide I peak of the protein that arises from the carbonyl functionality of the peptide linkage has been shifted from 1654 cm −1 in the native state to 1650 cm −1 in the nanobiohybrid state. A similar shift (from 1545 cm −1 in the native state to 1541 cm −1 in the hybrid state) is also observed for the amide II region of the protein in the FTIR spectra (Figure 2b). Based on the correlation between the amide I band positions and the secondary structure of the proteins, the 1654 cm −1 band can be attributed to the α-helices, and the bands in the lower-and higher-frequency regions at 1629 and 1675 cm −1 correspond to the vibrations in β-sheet structure. Any major changes (position and intensity) of the amide I and II absorption bands are considered to be associated with the protein's secondary structural variations. 50 Ye et al. previously reported a similar FTIR spectral shift (from 1653 to 1649 cm −1 ) with a concomitant decrease in intensity of the amide I band of Cyt c upon increasing temperature. 51 This was assigned to the partial unfolding of the protein segment containing Met-80 which led to the disruption of the S−Fe bond at the heme coordination site. A similar structural perturbation can be expected for the Cyt c upon binding with MoS 2 . Such minor structural changes at the heme center are suitable for the activation of the peroxidase-like behavior of Cyt c. However, a major structural deviation or a complete unfolding would conversely affect protein's catalytic activity.
3 A binding energy of −3.7 kcal mol −1 was obtained for the best docking conformation of the protein with the ligand. The hydroxy-ethoxy moiety mainly exhibits polar hydrogen bonding interactions with the neighboring amino acids of the protein, such as arginine 38, glycine 23, histidine 33, glycine 34, and asparagine 31, either directly or through water molecules ( Figure S2). Some nonpolar interactions can be observed from a central propoxy moiety which is relatively hydrophobic in nature. Parray et al. 45 have recently indicated that the interaction of Cyt c with polyethylene glycol (with an interaction energy of −3.9 kcal mol −1 ) does not significantly perturb the Met 80-heme structure of Cyt c, meaning that the protein binds to PEG without major structural deviations. In the P123-supported MoS 2 system, the PEO (PEG) units provide a soft template for protein binding interactions and help to prevent major structural changes of Cyt c.  system. To understand the role of interactions between Cyt c and MoS 2 in modulating the catalytic activity of the hybrid system, we have also performed the catalytic oxidation of ABTS using free Cyt c and the MoS 2 nanosheets separately. The quantitative analysis of the catalysis was obtained from the time-dependent absorbance changes of the solutions at 418 nm, which is the characteristic absorption wavelength maximum of the ABTS radical cation formed during the reaction. The increase in absorbance (at 418 nm) with increasing time corresponds to an increase in concentration of the ABTS radical and thus provides kinetic information about the catalysis. Figure 3a shows the absorption kinetic curves recorded immediately after addition of 5 mM H 2 O 2 to a series of reaction solutions containing fixed concentrations of ABTS (0.5 mM) and Cyt c (0.5 μM) with an increasing concentration (0−3.0 μg/mL) of MoS 2 nanosheets in 10 mM phosphate buffer of pH 7.1. Before H 2 O 2 addition, the solutions were stirred and equilibrated for 5 min to ensure sufficient adsorption of Cyt c on MoS 2 nanosheets for effective catalysis reaction. The background absorptions of the reaction solutions before H 2 O 2 addition were subtracted to get the actual concentration of the ABTS radical cation formed at any given time of the reaction by converting the absorbance values into concentration using an extinction coefficient of 3.6 × 10 4 M −1 cm −1 of the radical cation at 418 nm. 39,52 In the absence of MoS 2 , free Cyt c resulted in only a slight increase in absorbance at 418 nm (over 300 s reaction time), suggesting weak peroxidase activity of free Cyt c in solution (Figure 3a). This is consistent with previous reports. 41 The peroxidase activity of the free Cyt c remains unchanged in the presence of 1% (w/v) P123 polymer, which has been used to stabilize the MoS 2 nanosheets ( Figure S3a). However, the presence of P123 polymer coated MoS 2 nanosheets as a supporting system significantly enhances the extent of absorbance increase (over the same reaction time) with a faster kinetics of ABTS radical cation formation. As the concentration of MoS 2 nanosheets increases from 0 to 3 μg/ mL, the absorbance at 418 nm at 300 s reaction time is increased linearly without leveling off (Figure 3b).
It is noteworthy to mention that, like graphene oxide, 53 MoS 2 nanosheets are also intrinsically active toward peroxidase catalysis, and the activity is highly dependent on the nature of surface modifications, as recently reported by Yu et al. 25 Therefore, we have studied the catalytic activity of our Weak peroxidase activity of native Cyt c can be attributed to the lower accessibility of the substrate to the six-coordinated structure with a low spin heme center protected by its native protein environment. 40,54 This is in contrast to that of a true peroxidase where a five-coordinated heme center with an almost vacant or loosely bound sixth coordination site allows easy access to the substrate to initiate peroxidase activity. 37 The enhanced peroxidase-like catalytic activity of Cyt c on MoS 2 can be due to the structure−function switching of Cyt c from its typical redox protein state into a well-performing peroxidase mimic. The interaction of Cyt c with the MoS 2 nanosheets probably led to partial unfolding of Cyt c with the slight perturbation of the heme structure, making the active site accessible to the substrates. The structural perturbation of the heme environment of Cyt c upon interactions with the MoS 2 nanosheets has already been confirmed from the UV− visible and FTIR analysis.
The MoS 2 nanosheets are known to have surface negative charge. Quinn et al. 43 previously reported a low negative surface charge (an approximate zeta potential of −13 mV) of the pluronic triblock copolymer supported MoS 2 nanosheets at pH 6.5. Based on this, the initial adsorption of the positively charged protein Cyt c on MoS 2 can be influenced by electrostatic attraction forces. However, as the MoS 2 nanosheet surface is covered with nonionic polymer P123, the hydrogen bonding and hydrophobic interactions between the polymer units (PEO and PPO moieties) and Cyt c will also play The influence of electrostatic forces on the peroxidase catalytic activity of the nanobiohybrid system was studied with the addition of 1 M NaCl into the reaction mixture ( Figure  S4a). The catalytic activity of the nanobiohybrid catalyst is found to be only slightly decreased in the presence of 1 M NaCl. For a better understanding, we have plotted the catalytic rate vs time curves in the absence and presence of 1 M NaCl as given in Figure S4b. From this figure, we can see that the decrease in catalytic activity is more pronounced at the initial reaction time (0−50 s), suggesting the influence of electrostatic forces at the initial stage of the reaction. At longer reaction time (50−300 s), the reactions in the absence and presence of NaCl converges to a state where the catalytic reaction progresses at a similar reaction velocity of 0.05 μM s −1 . Therefore, it can be inferred that increasing ionic strength reduces the initial activity through electrostatic forces, thereby leading to slightly lower concentration of the ABTS •+ formed at any equal time points. A strong influence of electrostatic forces on the catalytic activity was earlier reported for Cyt c on graphene oxide because of the presence of various ionizable polar groups on graphene oxide. 39,40 The effect of pH on the catalytic activity of the Cyt c−MoS 2 nanobiocatalyst was studied by preparing the reaction mixtures in phosphate buffers of pH 3.7, 7.1, and 10.3 ( Figure S5a). The catalytic activity of the Cyt c−MoS 2 nanobiohybrid significantly increases with decreasing pH of the medium. A similar trend is also observed for the control experiments using Cyt c (without MoS 2 ) and MoS 2 (without Cyt c) as catalysts ( Figure  S5b,c). Acidic pH enhances the peroxidase activity, whereas the basic pH significantly reduces the activity. However, in any particular pH condition, the catalytic activity of the Cyt c− MoS 2 nanobiohybrid is significantly higher than the activity of the individual components, MoS 2 and Cyt c ( Figure S5). The acidic pH condition facilitates the approach of the negatively charged substrate ABTS to the MoS 2 nanosheets and thereby improves the catalytic activity. Moreover, in the case of the nanobiohybrid, the acidic pH conditions probably provide more favorable conditions for the adsorption of Cyt c on the MoS 2 surface. 39 Both MoS 2 and Cyt c separately exhibit negligible catalytic properties in basic pH, while a weak activity is observed for their nanobiohybrid. The observed pH dependence is consistent with the previous reports on the peroxidase-like activity using a wide variety of catalytic systems. Gao et al. 55 earlier reported an optimal pH of 3.5 for the intrinsic peroxidase-like activity of Fe 3 O 4 nanoparticles. The activity of a peroxidase enzyme HRP was also shown to be optimum in similar acidic regions.
3.6. Time-Dependent Study of Catalytic Activity. Time-dependent variations in the catalytic activity of Cyt c− MoS 2 nanobiohybrids were studied by monitoring the H 2 O 2mediated ABTS oxidation kinetics at different incubation times of Cyt c with MoS 2 . For this, a solution containing the nanobiohybrids was prepared by mixing 0.5 μM Cyt c and 3 μg/mL of MoS 2 in aqueous buffer solution and its catalytic activity was assessed at different incubation times up to 48 h after mixing. With increasing incubation time, the catalytic activity of the nanobiohybrid was found to be decreased (Figure 4a). This is evidenced by the slower kinetics of ABTS oxidation with a lower extent of absorbance increase observed at a longer incubation period compared to that observed at a shorter incubation period. The reduced catalytic activity of the Cyt c−MoS 2 hybrid at longer incubation time is also supported by the time-dependent decrease in intensity of the Soret band of Cyt c upon adsorption on MoS 2 (Figure 4b). Immediately after mixing Cyt c with MoS 2 (within 10−15 min), the generation of the Cyt c−MoS 2 bionanointerface has caused a blue shift of the Soret band from 409 nm (characteristic of the native Cyt c) to 400 nm. As a result, the Cyt c gets activated for effective peroxidase like behavior. As the incubation time progresses, the intensity of the resultant 400 nm absorption band is gradually reduced, consistent with the observed loss of peroxidase activity (Figure 4). At 24 h of incubation, the absorbance of Cyt c is significantly reduced, and consequently the catalytic activity is dramatically decreased. This can be due to the degradation of the protein after a long incubation period (24 and 48 h).
3.7. Michaelis−Menten Kinetic Analysis. The catalytic activity of the Cyt c−MoS 2 bionanohybrid catalyst presented in this work can be viewed as a bisubstrate-based peroxidase-like catalysis reaction. The ABTS substrate is oxidized in the presence of another substrate H 2 O 2 , producing the ABTS radical cation (ABTS •+ ) as the product. Previously, multiple irreversible reaction steps were used to describe the peroxidase catalysis as shown in Scheme 2. 56 The first step of the peroxidase cycle involves a rapid two-electron oxidation of the catalyst by H 2 O 2 , producing an intermediate Compound I.
Compound I is converted back to the catalyst by two successive electron transfer reactions from the ABTS substrate (Scheme 2). The first step is reported to be highly rapid, and therefore, the rate of product formation is largely dependent on the reducing substrate ABTS. 56 In the case of horseradish peroxidase isoenzyme C, the rate constants follow the order k a > k b ≫ k c . 57 In the above model, the kinetic steps are considered as irreversible, assuming a linear kinetics of product formation. However, a large number of recent reports have suggested the reversible nature of the binding of H 2 O 2 to the enzyme before forming the intermediate compound I, justifying the applicability of the Michaelis−Menten kinetic model with evidence of saturated kinetics in peroxidase-like catalytic activities. 25,32,38,39,41 Here we have analyzed the kinetic data for the peroxidase activity of the Cyt c−MoS 2 nanobiohybrid catalyst with ABTS−H 2 O 2 as a dual substrate system using the Michaelis−Menten theory by applying eqs 1 and 2. To obtain the kinetic parameters using this model, the steady-state reaction kinetics were monitored by varying substrate concentration. The ABTS concentration variation (from 0.2 to 1 mM) kinetics assay was performed by keeping the H 2 O 2 fixed at an excess concentration of 5 mM (Figure 5a). Similarly, for the kinetics assay measurements with varying H 2 O 2 concentration (from 2 to 10 mM), the ABTS concentration was kept fixed at 0.5 mM ( Figure S6a). The absorbance kinetics were converted into the time-dependent concentration changes of the ABTS radical cation by using the molar extinction coefficient of the ABTS radical cation (ε 418 = 3.6 × 10 4 M −1 cm −1 ). The apparent steady-state reaction rates at the initial stage of the reaction at different substrate concentrations were obtained from the initial slope of the concentration kinetic curves. The initial reaction rates (v) vs substrate concentration plots were found to be hyperbolic (Figures 5b and S6b).
The Lineweaver−Burk plots ( Figure 6) exhibit linear characteristics, suggesting that the catalytic behavior of the Cyt c−MoS 2 nanobiohybrid follows the Michaelis−Menten enzymatic kinetics for both the ABTS and H 2 O 2 as substrates. The fitting analysis of the Lineweaver−Burk plots is provided by the Michaelis−Menten kinetic parameters, as given in Table   1. The Michaelis−Menten constant (K m ) is inversely related to the catalyst−substrate binding affinity. A lower K m value is desirable for a newly designed catalyst to obtain higher catalytic efficiency (k cat /K m ). 39 The K m values of the Cyt c− MoS 2 nanobiohybrid catalyst are found to be 1.20 and 2.94 mM for the ABTS and H 2 O 2 substrates, respectively. Similar low K m values were earlier reported for the peroxidase activity of Cyt c using graphene oxide, silica nanoparticles, and polymer scaffold as supporting systems. 32,38,39 The K m value of the Cyt c−MoS 2 nanobiohybrid is significantly lower than that of the free Cyt c (123 mM). 41 This suggests that Cyt c has a higher substrate binding affinity when adsorbed on the MoS 2 nanosheets.
Consistent with the literature reports, a lower K m value is observed for the nanobiocatalyst against the ABTS substrate compared to the H 2 O 2 substrate, indicating more profound catalyst−ABTS binding affinity than catalyst−H 2 O 2 binding. 39 This is primarily due to the electrostatic interactions between the negatively charged ABTS substrate and the positively charged Cyt c that are adsorbed on the surface of the P123 coated MoS 2 nanosheets at a physiological pH. This concentration fixed at 5 mM ( Figure S8). On the other hand, the H 2 O 2 concentration variation kinetic assay was obtained in the range 0.02−2 mM, keeping the ABTS concentration fixed at 0.5 mM ( Figure S9). The MoS 2 nanosheets (3 μg/mL) without Cyt c exhibit Michaelis−Menten kinetic behavior for both the substrates. This is evidenced from the hyperbolic plots of the initial rate vs substrate concentration as given in Figures S8b and S9b. The kinetic parameters obtained from the linear Lineweaver−Burk plots ( Figure S10) are given in Table 1. The catalytic efficiency (k cat /K m ) values of the P123 stabilized MoS 2 nanosheets alone are found to be around 109 and 106 for the ABTS and H 2 O 2 substrates, respectively. The catalytic efficiency of the MoS 2 nanosheets alone is significantly weaker than the corresponding Cyt c−MoS 2 nanobiohybrid. Therefore, free MoS 2 nanosheets alone only weakly contribute to the enhanced peroxidase activity of the Cyt c−MoS 2 nanobiohybrid.
3.8. Reaction Progress Kinetic Analysis. The peroxidase-like catalytic cycle involves two substrates (H 2 O 2 and ABTS) with multiple reaction steps. The kinetic analysis of such multistep-and multisubstrate-based catalytic reactions is complicated. The Michaelis−Menten kinetic model has been extensively applied to describe peroxidase-like catalytic activity for a wide variety of catalysts. However, it is important to note that the kinetic parameters (V max and K m ) obtained from this analysis for varying initial concentrations of a particular substrate will be dependent on the concentration of the other substrate. Thus, the obtained Michaelis−Menten kinetic parameters are considered to be only apparent constants. This can be better understood if we consider a simple possible mechanism for a bisubstrate-based catalytic cycle, as given in Scheme 3.
The rate of the reaction as derived under pseudo-steadystate conditions is given by (eq 5): Under the conditions of varying concentrations of both the substrates during the reaction, the reaction−progress−kinetic (RPK) analysis 58 provides an important understanding about the reaction mechanism and the orders of the reaction. In this graphical method, one needs to plot reaction rate vs substrate concentration by combining the substrate concentration vs time and the reaction rate vs time plots. In our peroxidase-like catalysis experiment, we record the accumulation of the product (ABTS •+ ) concentration (in terms of its absorbance) with increasing time. The rate vs time data were obtained by differentiating the product concentration vs time data. On the other hand, the substrate concentration vs time data were created by subtracting the product concentration vs time data  from the initial substrate concentration used in the reaction. By combining the rate vs time data and substrate concentration vs time data, we obtained the rate vs substrate concentration plot ( Figure S11) for the catalytic system with 1 mM ABTS, 5 mM H 2 O 2 , and 0.5 μM Cyt c supported on 3 μg/mL of MoS 2 as a catalyst. It is important to note that this plot describes the rate of the catalytic reaction as the ABTS substrate is consumed from right to left because the substrate concentration decreases from the initial concentration as the product forms over the course of the reaction. The plot shows a curved feature, indicating a complex relationship between the rate and the substrate ABTS as described in the rate equation (eq 5).
We have applied variable-time-normalization analysis (VTNA) following a reported procedure to find the order of the reaction with respect to the hybrid catalyst. 59 In this analysis, the time scale of the two reactions run with different catalyst loadings is substituted by the normalized time scale, ∑[C] γ Δt (eq 7).
where [C] is the concentration of the catalyst, and the value of "γ" that produces the overlay of the curves is the order in catalyst. The normalization term is based on the fact that the concentration of the catalyst is constant during the entire reaction without any deactivation. We have used this "constant normalization" of the time scale method to overlay the curves of two different kinetic profiles recorded using two different concentrations of the catalyst (3 and 2 μg/mL of MoS 2 with a fixed 0.5 μM of Cyt c). The overlay analysis of the curves in this concentration range of the MoS 2 gave the order of the reaction with respect to the catalyst as 2 ( Figure S12).

CONCLUSION
In conclusion, a new nanobiocatalytic system has been prepared through the spontaneous adsorption of Cyt c on a triblock copolymer, P123, stabilized MoS 2 nanosheet. The catalytic activity of these nanobiohybrid systems has been successfully demonstrated by studying H 2 O 2 -mediated oxidation of the ABTS substrate. The enhanced catalytic performance of the Cyt c−MoS 2 nanobiohybrid catalyst compared to that of the free Cyt c and MoS 2 nanosheets can be explained based on the minor structural perturbation of the active site of the Cyt c upon adsorption on MoS 2 nanosheets. The triblock copolymer support on the MoS 2 nanosheets plays an important role in protecting the proteins from undergoing major structural changes. Moreover, the polymer support attracts more proteins to be adsorbed on the MoS 2 nanosheets and thereby increases the catalytic performance. From the study of Michaelis−Menten kinetics analysis, it is revealed that the adsorption of Cyt c on P123 stabilized MoS 2 nanosheets significantly increases the substrate binding affinity as well as the catalytic efficiency. Such a nanobiocatalytic system exhibiting high stability with enhanced catalytic activity may find their potential applications in biosensing, biofuel cells, and biomedical fields.


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Cytotoxic and antileishmanial triterpenes of Tabebuia aurea (Silva Manso) leaves

Abstract The bioactivity-guided fractionation of the total ethanolic extract of the leaves of Tabebuia aurea revealed the cytotoxic and antileishmanial potency of the ethyl acetate fraction, in which its phytochemical investigation resulted in the isolation of five triterpenes; identified as oleanolic acid (1), ursolic acid (2), pomolic acid (3), tormentic acid (4), 3β,6β,19α-trihydroxy-urs-12-en-28-oic acid (5) in addition to one triterpenoid glucoside, spathodic acid 28-O-β-D-glucopyranoside (6). Whereas compound 1 showed cytotoxic activity against three different cell lines; A549, MCF-7 and HepG2 with IC50 values of 31.7 ± 1.2, 27.4 ± 1.8 and 28.8 ± 1.1 μg/mL, respectively (etoposide as a positive control: 28.1 ± 4.2, 22.5 ± 4.5, and 20.4 ± 0.8 μg/mL, respectively), while compounds 1 and 2 showed antileishmanial activity with IC50 values of 10.2 ± 0.9 μg/mL and 5.1 ± 0.4 μg/mL, respectively (miltefosine: 7.7 ± 2.1 μg/mL). Graphical Abstract

Introduction
Triterpenoids are a large group of natural products with structural diversity; they exhibit over 100 distinct skeletons (Xu et al. 2004;Isah et al. 2016). Pentacyclic triterpenes are a member of these wide-ranging isoprene-derived secondary metabolites (Isah et al. 2016). They are derived mainly as a result of the cyclization of oxidosqualene and squalene, involving the formation of carbocations through multiple enzymatic and redox stages (J€ ager et al. 2009). Triterpenes are well known for their many promising physiological and pharmacological activities, including anti-inflammatory (Banno et al. 2006), antibacterial (Cunha et al. 2007), besides their role in calming the nerves (Luo and Lin 2002). Additionally, the literature survey revealed their role in the immune regulation (Mart ın et al. 2012), regulation of blood sugar (Melo et al. 2010) and lowering of blood pressure (Somova et al. 2003). Furthermore, in vivo and in vitro studies revealed many antitumor effects of triterpenes (Kikuchi et al. 2011), such as ursolic acid which can clearly inhibit S180 (murine sarcoma), TSCC-a (human tongue cancer) and HL-60 cell (human promyelocytic leukemia) proliferation, likewise the induction of breast cancer MCF-7 cell apoptosis and blocking of tumor angiogenesis (Wang et al. 2012, Zhang et al. 2014. The oleanolic acid also exhibits a significant tumor inhibitory effect through a cell-cycle arrest on HCT15 (human colon carcinoma cell line) (Li et al. 2002). Therefore, these triterpenes are promising lead compounds for the development of new bioactive agents with low toxicity and high efficiency.
Likewise, many researches revealed the varied potency of the triterpenoids with different skeletons against cutaneous leishmaniasis that occurs 0.7-1.2 million cases each year worldwide (Alvar et al. 2012). On our continuous search to find effective cytotoxic and antileishmanial agents from natural sources, the cytotoxic and antileishmanial activity of the total extract, sub-fractions and isolated compounds from T. aurea were investigated.
Tabebuia genus (Silva Manso) Benth. & Hook. f. ex S. Moore (Bignoniaceae) is widely distributed in tropical and subtropical regions of the American continent (Reis et al. 2014). T. aurea [Synonym: T. argentea Britton (Bureau & K. Schum) and Bignonia aurea Silva Manso] (IPNI 2019) is considered an important species for the local population of the Brazilian Cerrado, for timber logging and for homemade recipes for medical purposes (Braga et al. 2006). Additionally, the ethnobotanical studies have revealed that T. aurea, known as 'paratudo', is widely used in traditional medicine, in the form of infusion or maceration with alcohol or chewing of stem bark during the day, to treat snake bites as an anti-inflammatory medication (Reis et al. 2014). Laboratory studies revealed the effect of T. aurea hydroethanolic extract in reducing inflammation, haemorrhage and myotoxic activities of the snake venom in experimental mice (Nocchi et al. 2020). Additionally, flavonoids, phenolic glycosides, lignans, and iridoids are reported in the leaves of T. aurea (De Abreu et al. 2014;Hamed et al. 2020;Mahmoud et al. 2021), likewise the exhibition of remarkable antioxidant and nephroprotective activities against carbon tetrachloride-induced nephrotoxicity in rats, as well as the exhibition of a prominent in-vitro antitrypanosomal activity against Trypanosoma brucei (Mahmoud et al. 2021). This study aims to evaluate the cytotoxic and antileishmanial activities of the leaves of T. aurea as a total extract, sub-fractions and consequently isolated phytoconstituents from the most promising fraction.

Results and discussion
The bioassay-guided fractionation of the total ethanolic extract (TEE) and sub-fractions of the leaves of T. aurea revealed the high potency of the ethyl acetate (EtOAc) fraction as a cytotoxic agent against different cell lines including A549, MCF-7, and HepG2 with IC 50 34.2 ± 3.5, 38.2 ± 2.8, and 42.3 ± 2.9 lg/mL, respectively, and showed noticeable antileishmanial activity with IC 50 value of 48.1 ± 0.6 lg/mL as shown in Table S1. The etoposide, a clinical antitumor drug developed from a plant constituent, was used as a positive control (IC 50 values: 28.1 ± 4.2, 22.5 ± 4.5, and 20.4 ± 0.8 lg/mL, respectively). Furthermore, the EtOAc fraction was subjected to column chromatography using normal and reversed phase silica gel and final purification using reversed phase high performance liquid chromatography (RP-HPLC) resulting in the isolation of six triterpenoidal compounds, which were identified based on intensive spectroscopic analyses; (Tables S2, S3 and Figures S1-S19) together with comparing their physical and chemical properties with the reported data. The isolated compounds include, a oleanane type compound, oleanolic acid (1) (Onoja and Ndukwe 2013), besides four ursane type compounds identified as ursolic acid (2) (4) (Taniguchi et al. 2002;Mahmoud et al. 2019) and 3b, 6 b, 19a-trihydroxy-urs-12-en-28-oic acid (5) in addition to one triterpenoidal glucoside, spathodic acid 28-O-b -D-glucopyranoside (6) (Zhang et al. 2005) (Figure 1). Compound 5 was isolated from T. aurea for the first time, while compounds 3 and 4 were obtained for the first time from the genus. In addition, compound 6 was reported in the family Bignoniaceae for the first time.
The identified compounds (1-6) were also investigated for their cytotoxicity against cancer cell lines and antileishmanial activity (Table S1). Whereas compound 1 exhibited noticeable cytotoxic activity against the three different cell lines; A549, MCF-7 and HepG2 with IC 50 values of 31.7 ± 1.2, 27.4 ± 1.8 and 28.8 ± 1.1 lg/mL, respectively, and antileishmanial activity with IC 50 value of 10.2 ± 0.9 lg/mL. Miltefosine was used as a control (IC 50 , 7.7 ± 2.1 lg/mL), which is the sole oral clinical agent for leishmaniasis and listed in the WHO model list of essential medicines which are necessary for a health care system. On the other hand, Compound 2 showed relatively selective activity against HepG2 cell line and leishmania, with IC 50 value of 26.7 ± 1.5 and 5.1 ± 0.4 lg/ mL, respectively. Compound 4 exhibited cytotoxicity against MCF-7 cell line with IC 50 value of 25.3 ± 1.4 lg/ml, but negligible antileishmanial activity.

Conclusion
The cytotoxic and antileishmanial assay of the different fractions of T. aurea leaves showed that the ethyl acetate-soluble fraction exhibited the highest potency. The use of different chromatographic techniques led to the isolation of six triterpenes 1-6, which exhibited varied cytotoxic activities against tumor cell lines and anti-leishmania activity. Compound 1 may be non-specific caused by general toxicity, but 2 and 4 showed weak selectivity against HepG2 and leishmania. At the present stage of our research, however, further investigations are needed to clarify their actual mechanism of action and selectivity.

Disclosure statement
The authors declare no conflict of interest.

Funding
This work was supported by Ministry of Higher education, Egypt through scientific mission system.


s2fieldsofstudy: Chemistry Biology

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Mechanistic insights into the C55-P targeting lipopeptide antibiotics revealed by structure–activity studies and high-resolution crystal structures

The continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity. Notable in this regard is the subset of CDAs comprising the laspartomycins and amphomycins/friulimicins that specifically target the bacterial cell wall precursor undecaprenyl phosphate (C55-P). In this study we describe the design and synthesis of new C55-P-targeting CDAs with structural features drawn from both the laspartomycin and amphomycin/friulimicin classes. Assessment of these lipopeptides revealed previously unknown and surprisingly subtle structural features that are required for antibacterial activity. High-resolution crystal structures further indicate that the amphomycin/friulimicin-like lipopeptides adopt a unique crystal packing that governs their interaction with C55-P and provides an explanation for their antibacterial effect. In addition, live-cell microscopy studies provide further insights into the biological activity of the C55-P targeting CDAs highlighting their unique mechanism of action relative to the clinically used CDA daptomycin.

Reagents and General Methods
All reagents employed were of American Chemical Society (ACS) grade or finer and were used without further purification unless otherwise stated. D-amino acids and 2-chlorotrityl resin was obtained from Iris Biotech GmbH, Egg PG and 0:6 PA was obtained from INstruchemie BV. C 10 -P lithium salt was obtained from Sigma Aldrich and lyophilized from warm t BuOH:H 2 O (1:1) to obtain a white powder with increased aqueous solubility.

Instrumentation for Compound Characterization
2D NMR experiments were performed on a 850 MHz instrument. HSQC, TOCSY and NOESY spectra were recorded for all peptides (5 mM in DMSO d6 ) and the parent compound laspartomycin C matched pervious recorded spectra reported by our group.
HRMS analysis was performed on a Shimadzu Nexera X2 UHPLC system with a Waters Acquity HSS

S4
The compounds were purified via preparative HPLC using a BESTA-
Chloramphenicol (130 µg mL -1 ) was added and after incubation for 15 minutes at 37°C, the culture was divided in 5 mL aliquots. Antibiotics were added at 10xMIC and one aliquot remained untreated. After 60 minutes, cells were separated from the medium and extracted with boiling d-H 2 O (1 mL) for 15 minutes.
The suspensions were spun down and the supernatant was lyophilized. The resulting material was analyzed by HPLC applying a gradient from 100% eluent A (50 mM NaHCO 3

Structure solution and refinement
The dataset of lipopeptide 5 was processed in the DIALS pipeline 2 , whereas autoPROC 3 was used for lipopeptide 7. The crystal of lipopeptide 7 was initially indexed in a hexagonal setting but based on the merging R-values the true symmetry appeared to be Primitive monoclinic with β = 120°. The reflection file was therefore re-indexed accordingly, and parameters for pseudo-merohedral twinning were included in the structure refinement. Additional anisotropic correction was done for the datasets of both analogues in STARANISO. 3 Structures were solved by molecular replacement using PHASER, 4 and one copy (lipopeptide 5) or one dimer (lipopeptide 7) of laspartomycin C in complex with geranyl phosphate (PDB: 5O0Z) 5 was used as a search model. Models were manually improved in Coot, 6 Figure S2. A) Structure of the ternary complex with lipopeptide 7 (green stick representation), two bound Ca 2+ ions (orange spheres), a bound water molecule (red sphere), and the C10-P ligand (lipid in grey). B) Lipopeptide 7 adopts a saddle-shaped conformation when complexed with two Ca 2+ ions and C10-P and forms a dimer in the crystal.  5 Asymmetric units are composed of one monomer for lipopeptide 5 (the dimer shown for lipopeptide 5 generated by applying two-fold crystallographic symmetry), six dimers for lipopeptide 7 and one dimer for laspartomycin C. RMSD between dimers is indicated in the table. Ca 2+ ions are represented by orange spheres and water molecules are represented by red spheres, C10-P is also indicated. Figure S4. In the crystal state lipopeptide 5 forms a higher-ordered assembly when complexed with Ca 2+ and C10-P consisting of alternating hydrophobic (grey), peptidic (green), and hydrophilic (red) layers. A similar lattice is also observed for lipopeptide 7. Notably, this higher ordered assembly is not seen for laspartomycin C.
Fluorescence microscopy was carried out using a Zeiss Axiovert 200M equipped with a Zeiss Neofluar 100x/1.30 Oil Ph3 objective, a Lambda S light source (Shutter Instruments), a Photometrics Coolnap HQ2 camera, and Metamorph 6 software (Molecular Devices). Images were analyzed using ImageJ (National Institutes of Health) v.1.52a.