Andrei P Kroitor

Alexander A Dmitrienko

Alexander G Martynov

Yulia G Gorbunova

Alexander B Sorokin

Substitution pattern in ruthenium octa-n-butoxyphthalocyanine complexes influences on their reactivity in the N-H carbene insertion

Ruthenium phthalocyanine complexes bearing n-OBu substituents in the peripheral or non-peripheral positions are efficient catalysts for the selective double or single carbene insertion to the amine N-H bonds. This complementary reactivity of two Ru complexes can be used for the synthesis of asymmetric tertiary amines and diamines bearing different substituents that was demonstrated by two examples of readily available primary amines using different carbene precursors in successive reactions.

Catalytic X-H (X = N, C, O, S…) insertion of carbenes represents a powerful strategy for the construction of C-X bonds. 1 In particular, porphyrin complexes 2 and engineered hemoproteins 3 are remarkably efficient in the cyclopropanation and C-X carbene insertion reactions. However, it should be noted that carbene insertion into amine N-H bonds is more demanding compared to other C-X bonds because of the catalyst poisoning due to amine coordination to the metal sites. The selectivity to single insertion N-H products is also often limited. In recent years, a variety of transition metal complexes 4 and engineered hemoproteins 5 have been reported for the insertion of carbenes to the N-H bonds of amines. Along with Rh, Ir, Fe and Co complexes, considerable attention has been devoted to Ru compounds. 6 Among them, Ru porphyrins have been extensively studied as efficient catalysts for carbene transfer reactions, 7 principally in cyclopropanation of olefins. 8 Noteworthy, other tetrapyrrolic complexes, e.g. Ru phthalocyanines have been rarely studied in these reactions. 9 In general, the nature of substituents of phthalocyanine ligand determines the electronic properties of the corresponding complexes and hence their catalytic activity. 10 Our previous study showed that peripherally substituted Ru octa-nbutoxyphthalocyanine (BuO) 8 PcRu(CO) (1) was efficient catalyst for the carbene insertion into amine N-H bonds under practical reaction conditions. 11 A large variety of structurally divergent amines were selectively converted to monosubstituted glycine derivatives using a 0.05 mol% catalyst loading, a high amine concentration (1 M) and 1.1 equiv. of ethyl diazoacetate (EDA) with up to quantitative yields and turnover numbers achieving 2000. In course of our studies, we have found that the catalytic properties of Ru octa-nbutoxyphthalocyanine complex depend on the position of the substituents. Therein, we report that peripherally and nonperipherally substituted complexes (BuO) 8 PcRu(CO) and (BuO) 8 PcRu(CO) (1) exhibit different properties in carbene insertion to amine N-H bonds providing selectively either double or single N-H insertion products, respectively. We demonstrate how the complementary catalytic properties of 1 and 1 can in principle be used for the synthesis of elaborated amine derivatives with various substitution pattern. The peripherally substituted complex 1 was prepared by refluxing the metal-free phthalocyanine H 2 [β-(BuO) 8 Pc] with Ru 3 (CO) 12 in o-dichlorobenzene (o-DCB) according to optimized published procedure 9b with a 85 % yield (Scheme 1). Our attempts to synthesize 1 from non-peripherally substituted phthalocyanine H 2 [α-(BuO) 8 Pc] and Ru 3 (CO) 12 using the same protocol led to the mixture of unidentified products as was indicated by MALDI TOF MS analyses. The replacement of o-DCB with pure PhCN distilled in vacuum over P 2 O 5 then over K 2 CO 3 led to a high yield of the target 1complex (Scheme 1). The PhCN purity plays a crucial role since even the traces of benzamide in the solvent result in a very low yield of 1. Contrary to the preparation of peripherally substituted Ru phthalocyanines by the treatment with Ru 3 (CO) 12 in o-DCB accompanying by the formation of µ-carbido dimers, 9b no corresponding µ-carbido dimer was formed during the synthesis of 1 in PhCN. The 1 H NMR spectrum of 1 confirmed its diamagnetic nature. The presence of the axial carbonyl ligand was evidenced by the strong CO stretching band at 1948 cm -1 in the IR spectrum. The comparison of UV-vis spectra of the solutions of both Ru phthalocyanines shows the significant redshift of Q band from 655 nm for the peripheral 1 complex to 717 nm for non-peripherally substituted species 1(Fig. S4). This means a difference in their electronic structures which, in turn, can induce a difference in the catalytic properties. The catalytic properties of the complexes were evaluated in the reaction of 2.1 equiv. EDA with aromatic amines 2a-2j (1 M) in CH 2 Cl 2 at 40°C using 0.05 mol% catalyst loading (Table 1). Worth noting that carbene transfer reactions catalyzed by metal complexes are typically carried out under inert atmosphere. [1][START_REF] Intrieri | Handbook of Porphyrin Science[END_REF][3] Importantly, the carbene insertion to amines catalyzed by 1 and 1 can be performed under air without decreasing the product yields which represents a significant practical advantage. Aniline derivatives bearing electron-donating (Table 1, entries 1-3,8) or electron-withdrawing groups (Table 1,entries 4,5) were converted to the N-H double insertion products 4 with almost quantitative yields. Two strong electronwithdrawing groups of 3,5-ditrifluoromethylaniline 2f decrease its nucleophilic properties resulting in 1:1 ratio of products double and single N-H insertion, d-and s-products, 4f and 3f, respectively (Table 1, entry 6). The presence of methyl and isopropyl substituents in ortho-positions is well tolerated and corresponding d-products 4g and 4h were obtained with 89 and 97 % yield, respectively (Table 1,entries 7,8). However, bulky tert-butyl o-substituent prevents the second N-H insertion (Table 1, entry 9). The 1 -EDA system shows a clear preference for N-H insertion compared to the cyclopropanation of double bond. The products of single and double carbene insertion to paminostyrene 3j and 4j as well as cyclopropanation product of 4j were obtained with 10, 62 and 18 % yields, respectively (Table 1, entry 10). The 1 complex exhibited the lower catalytic activity in the reaction of aniline with EDA under conditions used for 1 providing a 35 % conversion to the s-product 3a. We optimized the protocol towards the quantitative substrate conversion (Table S1). The replacement of CH 2 Cl 2 with MeCN did not improve the aniline conversion. A slow addition of EDA/aniline mixture to the solution of 1 in CH 2 Cl 2 resulted in the sharp drop of conversion. However, increasing of the catalyst amount to 0.1 and 0.15 mol% allowed for improvement of the aniline conversion to 71 and 96 %, respectively (Table S1). Importantly, only the s-product 3a was selectively formed. Thus, the substrate scope was explored using 0.15 mol% catalyst and 2.1 equiv. EDA in CH 2 Cl 2 at 40°C (Table 2). Although the s-products can be obtained with 1.1 equiv. EDA, we used EDA excess in order to compare the catalytic properties of the two complexes under the same conditions except the catalyst loading.

In sharp contrast to 1 selectively providing d-products, 1 shows a very high selectivity to s-products even in the presence of 2.1 equiv. EDA. Quasi-quantitative yields of monosubstituted glycine derivatives were obtained from aniline with electron-donating (Table 2, entries 1-3,8) and electronwithdrawing (Table 2, entries 4,5) substituents. While 2,6diisopropylaniline 2h afforded a 97 % yield of 3h, less hindered highly nucleophilic 2,6-dimethylaniline afforded 80:20 ratio of s-and d-products 3g and 4g (Table 2, entries 7,8). Noteworthy, a highly selective single N-H insertion was observed in the reaction with p-aminostyrene, no double insertion and no Please do not adjust margins

Please do not adjust margins transformation to imine derivatives which could not be separated from the target 5ss product.

The 2h reaction of 2,6-dichloro-1,4-diaminobenzene 6 with 4.1 equiv. EDA mediated by 1 led to the formation of 6s, 6ss, 6sd and 6dd products with 5 %, 45 %, 47 % and 3 % yields, respectively (Scheme 2d). Owing to their similar polarity this mixture was difficult to separate and 6ss could be isolated in a 9 % yield. When the reaction was performed for 3h, the 6sd product was formed with 75 % yield and was isolated in a 50 % yield. In the presence of 1, the 6s, 6ss, 6sd and 6dd products were formed with 9 %, 5 %, 13 % and 66 % yields (Scheme 2c).

The target 6dd product was isolated in a 61 % yield. This example indicates that optimization of the reaction conditions depending on the properties of diamine can allow obtaining desired product with good yield. Indeed, under standard conditions, 1 catalyzed the reaction of 2,6diaminotoluene 7 with 4.1 equiv. EDA forming a mixture of 7dd (57 %) and 7sd products (42 %) which could not be completely separated. When 5.46 equiv. EDA were added in 4 portions during 3 h, a quantitative formation of 7dd was observed and the target product was isolated in 82 % yield (Scheme 2e). In a complementary fashion, 1 provided 85 % yield of 7ss accompanied by 7sd with 15 % yield (Scheme 2f). The desired 7ss was isolated in a 77 % yield. Such a different reactivity of two Ru phthalocyanines opens up new opportunities for the synthesis of elaborated amine derivatives, e.g., unsymmetrical tertiary amines bearing three different groups. In the first step, readily available primary amines and diamines can be converted to s-or ss-products using the 1 catalyst. Next, 1 can catalyze N-H insertion of another carbene to s-product to afford unsymmetrical NR 1 R 2 R 3 amine. The feasibility of this strategy was first evaluated using diazoacetonitrile as a carbene precursor and Nmethylaniline 8 as a model substrate. N 2 CHCN is a particularly useful compound to introduce nitrile groups into organic molecules. 8c,12 Compared to EDA, the use of N 2 CHCN as carbene precursor has been still rather limited 13 because of explosion propensity of neat compound. 12 Recently, the safe generation of N 2 CHCN in situ or the use of diluted solutions were proposed. 12 The high yields of the tertiary amine 9 were obtained from N-methylaniline using both protocols (Fig. S123). Among the organic solvents tested, the highest yield of 9 (83 %) was obtained using 1,2-dichloroethane. The use of 0.57 M N 2 CHCN solution in DCE (1.1 equiv.) allowed obtaining the target unsymmetrical tertiary amine with 97 % yields in the presence of 0.05 mol% 1β instead of 0.1 mol% catalyst charge applied for in situ protocol (Fig. S123).

Having in the hands the efficient protocols for the selective single carbene insertion from EDA to primary amines catalyzed by 1 and for the efficient functionalization of secondary amines using N 2 CHCN in combination with 1β, we have prepared unsymmetrical tertiary amines starting from the simple aromatic amine and diamine (Scheme3). First, ethyl p-tolylglycinate 3b was obtained from ptoluidine and EDA in the presence of 1 in 93 % isolated yield (Scheme 3a). The subsequent treatment of 3b with N 2 CHCN and 1 afforded the target unsymmetrical tertiary amine 10 in 87 % isolated yield. This approach was further extended to primary diamine exemplified by 2,6-diaminotoluene 7 converted to 7ss with 77 % yield through the reaction with EDA and 1 (Scheme 3b). The second step was accomplished using N 2 CHCN and 0.2 mol% 1 providing 56 % isolated yield of the asymmetric tertiary diamine 11. Asymmetric tertiary diamine can in principle be prepared in two steps using the same peripheral complex but the product yields were lower because of the formation of the side single-double insertion product and lower substrate conversion. Furthermore, the isolation of the target product is much more difficult in this case. Thus, the two-steps strategy involving two catalysts is particularly useful to access the asymmetric tertiary amines.

In conclusion, the Ru octa-n-butoxyphthalocyanine complexes are efficient catalysts for the carbene insertion to amine N-H bonds under practical reaction conditions. Using low loading of these air tolerant catalysts and near-equivalent substrate/carbene precursor ratio, a wide range of simple amines and diamines was converted to different products with high yields. Importantly, the position of the substituents in Ru octa-n-butoxyphthalocyanine complexes controls their catalytic properties: non-peripherally and peripherally substituted complexes 1 and 1 afford single and double N-H insertion products, respectively. Due to the complementary catalytic properties of the two Ru complexes, a novel synthetic strategy was proposed, which can provide access to asymmetric tertiary amines and diamines starting from simple readily available compounds. This approach was validated using ethyl p-tolylglycinate and 2,6diaminotoluene as model substrates. Since N-aryl amines are important building blocks in natural products and synthetic compounds, this approach might be useful to access to elaborated functionalized amines with interesting biological and pharmacological properties.

Conflicts of interest

There are no conflict of interest to declare.

Notes and references

Scheme 3. Preparation of unsymmetrical tertiary amines from primary amines by successive carbene transfer reactions catalyzed by 1 and 1.

Table 1 .

 1 Scheme 1. Synthesis of peripherally and non-peripherally substituted ruthenium octa-n-butoxyphthalocyanines 1 and 1.

  0.5 mmol amine, 1.05 mmol EDA, 0.15 mol% catalyst, 0.5 mL CH2Cl2, air, 40°C. b Yield determined by 1 H NMR (isolated yield). c Yields of diethylmaleate (DEM) and diethylfumarate (DEF) determined by 1 H NMR are based on the initial amount of EDA.

Scheme 2 .

 2 Scheme 2. Functionalization of the aromatic diamines with EDA in the presence of 1 and 1. a Yields were determined from 1 H NMR spectra. cyclopropanation of the double bond was observed in this case (Table2, entry 10). The synthetic utility of Ru octa-n-butoxyphthalocyanines 1 and 1 due to their different catalytic properties was demonstrated in the derivatization of three aromatic diamines with different electronic properties and substitution patterns (Scheme 2). The carbene insertion to diamines leads to the formation of 4 products: single insertion (s), single-single insertion (ss), singledouble insertion (sd) and double-double insertion (dd). Thus, selective preparation of desired product is more challenging than in the case of simple amines. Using 0.05 mol% of 1 and 4.1 equiv. EDA, p-phenylenediamine 5 was converted to ddproduct 5dd with quantitative yield (TON = 8000) (Scheme 2a). During its isolation by column chromatography with SiO 2 5dd underwent a partial elimination of one CH 2 COOEt group affording a double insertion-imine product 5s'd identified by GC-MS and 1 H NMR. Purification using neutral Al 2 O 3 prevented the degradation and 5dd was obtained with a 92 % isolated yield. As expected, the 1 complex was selective to the formation of ss-product 5ss from p-phenylenediamine. In the presence of 0.15 mol% 15ss was formed in 50 % yield (Scheme 2b). Under standard conditions with 0.3 mol% 1 (0.15 mol% per NH 2 group) 5ss and 5sd were obtained in 85 % and 4 % yields, respectively. Upon chromatographic purification either with SiO 2 or Al 2 O 3 these compounds underwent a partial

  14

  This work was supported by CNRS and RFBR through International Emerging Action 2021 and research project 21-53-15004.

Table 2 .

 2 Selective single carbene N-H insertion into aromatic amines mediated by the non-peripheral 1 complex. a

	Entry Substrate	Conversio n, %	Single N-H yield, b % insertion,	Double N-H % insertion, yield,	Yield of DEM (DEF), % c


Wei Liao

Abdelhamid Elaïssari

Emilie Dumas

Adem Gharsallaoui

Abdelhamid Elaissari

Effect of Interactions trans-Cinnamaldehyde and Citra on sodium caseinate: interfacial rheological and fluorescence quenching properties

Keywords: Sodium caseinate (CAS), trans-Cinnamaldehyde (TCA), Citral, Interfacial properties, Fluorescence quenching

published or not. The documents may come     Effect of trans-cinnamaldehyde or citral on sodium caseinate: Interfacial rheology and fluorescence quenching properties

Introduction:

Many food products are based on emulsions systems (O/W and W/O) (X. Zhang et al., 2021) which require the use of emulsifying agents to stabilize the interface between different phases.

Nowadays, food industries prefer to use more nature emulsifiers like proteins rather than chemical synthetic emulsifiers [START_REF] Felix | Effect of cinnamaldehyde on interfacial rheological properties of proteins adsorbed at O/W interfaces[END_REF], since these proteins can providing extra nutritional added values and are more acceptable by consumers. Some proteins such as soy, pea, gelatin, dairy protein have been reported as emulsifiers with good surface activity at the interface, which can adjust the interfacial properties of emulsion systems by partially spreading their hydrophobic groups to the oily phase [START_REF] Fuller | Complex fluid-fluid interfaces: rheology and structure[END_REF][START_REF] Ribeiro | Protein-and polysaccharide-based particles used for Pickering emulsion stabilisation[END_REF]. During the absorption process, protein molecular general unfold and expose numbers of hydrophobic residues which may physically or chemically interact with other functional compounds, thereby altering their functional performance [START_REF] Dickinson | Protein adsorption at liquid interfaces and the relationship to foam stability[END_REF]. Different active compounds introduced to protein based systems to tailor their solubility, hydrophobicity, dispersibility and surface charge by using of some enzyme or crosslinking agents [START_REF] Fischer | Rheology of food materials[END_REF][START_REF] Glusac | Enzymatic and chemical modification of zein for food application[END_REF][START_REF] Tann | Enzyme design by chemical modification of protein scaffolds[END_REF]. However, these treatments may introduce a new function group of components which is generally limited in the food section due to toxicity and absence of GRAS status.

Therefore, more natural compounds for protein functionalization should be search and investigated.

Among flavor compounds, aldehydes have an vital role in flavor development of food products (Wang et al., 2021). It was reported that certain aldehydes are able to interact with various functional residues of amino groups, like ε-amino groups of lysine, the sulfhydryl group of cysteines, the imidazole ring of histidines and the phenol group of tyrosines, etc [START_REF] Azeredo | Crosslinking in polysaccharide and protein films and coatings for food contact-A review[END_REF][START_REF] Habeeb | Reaction of proteins with glutaraldehyde[END_REF]. Specific cases include the some aromatic aldehydes such cinnamaldehyde, salicylaldehyde, syringaldehyde, vanillin and anisaldehyde, have been introduced in food and pharmaceutical industries [START_REF] Banerjee | Vanillin biotechnology: the perspectives and future[END_REF][START_REF] Boeve | Food-grade strategies to increase stability of whey protein particles: Particle hardening through aldehyde treatment[END_REF]. These aromatic aldehydes have gained considerable attention since their various health benefits like anti-inflammatory, antioxidant, antimicrobial agents and anticancer properties [START_REF] Banerjee | Vanillin biotechnology: the perspectives and future[END_REF]. Trans-cinnamaldehyde (3-phenyl-2-propenal, TCA, FEMA 2286) is a major component of volatile cinnamon essential oil (Adams et al., 2004). It showed broad-spectrum antimicrobial activity against bacteria, yeast, and molds [START_REF] Shen | Effects of cinnamaldehyde on Escherichia coli and Staphylococcus aureus membrane[END_REF]. Citral (3,7-dimethyl-2, 6-octadienal, FEMA 2303) is main component of citrus fruit's peel oil which is widely applied as a flavoring additive in foods and beverages (Zhang et al., 2020). As these two active compounds are extremely low solubility in water, it is difficult to prepare food formulations containing these nature aldehydes in solubilized form. There are two main approaches to overcome the insolubility issues: 1) mixing them with carrier oils and then producing it in the form of oil-water systems by some food component emulsifiers since these aldehydes show well solubility in oil; 2) introducing them with a food component may provide a way to deliver nature aldehydes since several reports showed that some aldehydes are properly able to bond with proteins [START_REF] Asghari | Study of the Interaction of Cinnamaldehyde with Alpha-lactalbumin: Spectroscopic and Molecular Docking Investigation[END_REF][START_REF] Felix | Effect of cinnamaldehyde on interfacial rheological properties of proteins adsorbed at O/W interfaces[END_REF][START_REF] Weibel | Interaction of cinnamaldehyde (a sensitizer in fragrance) with protein[END_REF]. A combination of van der Waals forces, electrostatic forces, hydrogen binding and/or hydrophobic binding may contribute to protein-aldehydes interactions and this should be further analyzed. Several studies reported that aldehydes can bind with amino acid residues of proteins by irreversible covalent binding [START_REF] Guo | Effects of concentration of flavor compounds on interaction between soy protein isolate and flavor compounds[END_REF], hydrophobic interaction [START_REF] Wang | Binding of selected volatile flavour mixture to salt-extracted canola and pea proteins and effect of heat treatment on flavour binding[END_REF] or hydrogen bonding [START_REF] Tan | Modeling bovine serum albumin binding of flavor compounds (alcohols, aldehydes, esters, and ketones) as a function of molecular properties[END_REF][START_REF] Weel | Effect of whey protein on the in vivo release of aldehydes[END_REF], which depend on the specific chemistry and structure of the interacting molecules. Meanwhile, the binding between aldehydes and proteins could be also used in oil in water interface to adjust the interfacial properties. However, the relative reports about the mechanisms of interaction between aromatic aldehydes and protein are still scarce.

Sodium caseinate (CAS) is heterogeneous and usually consists of four different polypeptide proteins chains: aα(s1)-and α(s2)-caseins, β-casein, and κ-casein [START_REF] Walstra | Understanding interactions among aldehyde compounds and porcine myofibrillar proteins by spectroscopy and molecular dynamics simulations[END_REF], which have outstanding amphiphilic properties [START_REF] Luo | Physical, chemical and biochemical properties of casein hydrolyzed by three proteases: partial characterizations[END_REF]. CAS can endow food with certain functionalities, such as water solubility, emulsification, foamability and encapsulation ability. This is really useful because dairy products are considered to be good carriers of biologically active ingredients as they are an integral part of many people's diets. Additionally, CAS can adsorb at O/W interfaces and have ability to fast reduce interfacial tension during emulsification process. Eventually, a densely packed interface can be formed which may exhibit viscoelastic behavior. The characteristics of the interfaces are essential for understanding the stability of O/W emulsion systems. To better understanding the interaction mechanism between protein and nature aldehydes, the first objective the first objective is to investigate the effect of aldehydes on the interfacial and oscillatory dilatational properties of CAS stabilized droplet. Moreover, fluorescence quenching was used to explore the binding properties among selected aldehydes and CAS. The effects of temperature and different concentration of aldehydes on CAS were carried out. In short, understanding the binding properties between nature aldehydes and dairy proteins will contribute food industries to formulate dairy-based products with more health benefit and functional properties.

Material and methods

Materials

Trans-cinnamaldehyde (TCA, 99%) and citral (99%) were purchased from Sigma-Aldrich (St Quentin Fallavier, France). Sodium caseinate (CAS) was purchased from Fisher Scientific (United Kingdom) and its protein content measured by the Kjeldahl method was 93.20% (nitrogen conversion factor N = 6.38). A medium chain triacylglycerol oil (MCT) was purchased from Gattefossé (Saint-Priest, France). Analytical grade imidazole (C 3 H 4 N 2 ), pure ethanol acetic acid, sodium hydroxide (NaOH), and hydrochloric acid (HCl), were purchased from Sigma-Aldrich Chimie (St Quentin Fallavier, France). All the solutions were prepared in deionized water.

Interfacial properties

Sample preparation

The aqueous phase for interfacial measurements was prepared by dispersing CAS powder (0.01 wt.%) into 100 mL of buffer solution (5 mmol/L acetic acid-imidazole, 0.05 wt.% sodium benzoate, pH 7). The protein solution was stirred at room temperature until it was completely dissolved. Oily phases were prepared by mixing TCA or citral (5 wt.%) with MCT under stirring until they are completely homogeneous. Pure MCT was used as control.

Droplet tension measurement

The O/W interfacial properties were measured by a Tracker automatic droplet tensiometer equipped with an oscillating drop accessor (Civrieux d'Azergue, France) at room temperature. The droplet profile was recorded every 0.02s by a connected digital camera. The values of interfacial tension were calculated by the Laplace equation [START_REF] Castellani | Hydrocolloids with emulsifying capacity. Part 2-Adsorption properties at the n-hexadecane-Water interface[END_REF]. According to the density of the oil phase, the rising drop method were chosen. Three different oil compositions (MCT, TCA/MCT, Citral/MCT). An initial droplet with a volume of 10 μL and a surface area of ~20 mm 2 was generated at the vertical needle, which was connected to an automatic syringe. The specific low absorbance glass cuvette (8 ml) was filled with the aqueous phase contained 0.01 wt.% CAS.

Interfacial tension was performed over a period of 10,800 s to monitor the protein adsorption kinetics. The oscillatory dilatational measurements (five cycles) were carried out at 10% amplitude and at a frequency of 0.1 Hz, which was tested to be within the linear viscoelastic regime. Further dilatational analysis was carried out during the protein adsorption phase, every 100 s.

Binding properties

Sample preparation

The CAS stock solution (0.048 wt.%, 20 μmol/L) was prepared by dispersing CAS powder into 100 mL of pH 7 acetic acid-imidazole buffer solution. The protein solution was stirred at room temperature until it was completely dissolved. The stock solution (160 μmol/L) of TCA and citral were prepared by dissolving 2 μL and 2.8 μL, respectively, into 100 mL of 10% ethanol pH 7 buffers. Stirring was continued until all of aldehydes were dissolved. Samples for ultraviolet fluorescence quenching test were prepared by mixed required volume of the stock solution of CAS (20 μmol/L), aldehydes and pH 7 buffer to the final volume of 4 mL. Therefore, each sample for fluorescence experiments has the fixed concentration (10 μmol/L) of CAS and various concentrations of TCA or citral (0,5,10,20,30,40,50,60 and 80 μmol/L). Meanwhile, the highest resulting concentration of ethanol in each sample was 5 wt.% and it have been as control which does not affect the fluorescence intensity of the protein.

Ultraviolet (UV) Spectroscopy

To study the interaction of both aldehydes and CAS, changes in absorption of CAS in the absence and presence of various concentrations of two aldehydes was determined by an UV spectrophotometer (Jenway 3705, Villepinte, France) at room temperature and the wavelength range of 230 -350 nm. Before each measurement, quartz cuvettes (1 cm path length) were filled with different samples in the dark for 30 min. Sample blank was buffer only.

Intrinsic fluorescence

Intrinsic fluorescence intensity of CAS was carried out in the absence and in the presence of various concentration of TCA or citral at room temperature by multimode microplate reader equipped with a temperature-controlled 96-wells microplate (Thermo Scientific™ Varioskan). The excitation wavelength was set at 280 nm and the emission spectrum was recorded in the wavelength range of 298-550 nm with excitation and emission slit width each of 5 nm. Different samples were placed in the wells as the order of increasing the concentration of aldehydes molar ratio range from 0 to 80 μmol/L. The measurements were performed at 25, 30, 35, 40 °C, separately. Before each measurement, the mixture of CAS and aldehydes was incubated at room temperature in the dark for 30 minutes. The fluorescence intensity of acetate-imidazole buffer was used as a control.

Statistical analysis

All measurements were performed at least in triplicate. The results were then reported as averages and standard deviations of these measurements. Statistical analysis was performed by one-way ANOVA using SPSS software (IBM, version 19.0, USA).

Results and discussion

Impact of aldehydes on the interfacial behavior

The shape tensiometer has been used for the characterization of complex interfaces since it can provide dynamic information at the O/W interface. In the formation of O/W, hydrophobic groups in CAS will gradually adsorb to the surface of the oil droplets and then after subsequent rearrangements, become an interfacial layer. Fig. 1A showed the plot of interfacial tension as a function of time (t < 10,800 s) during the protein adsorption time in the different oily composition.

In all three cases, the values of interfacial tension decreased with the time, at least during the initial adsorption period, which is related to different stages of protein adsorption (from protein diffusion to protein penetration, unfolding and rearrangement) [START_REF] Felix | Viscoelastic properties, microstructure and stability of high-oleic O/W emulsions stabilised by crayfish protein concentrate and xanthan gum[END_REF]. Pure MCT shows that the equilibrium interfacial tension was relatively high, about 13.56 mN/m, and when mixed with 5 wt.% TCA or citral, the equilibrium interfacial tension was significantly reduced to 9.12 and 11.27 mN/m, respectively. A similar result was observed by the study of cinnamaldehyde and whey protein [START_REF] Felix | Effect of cinnamaldehyde on interfacial rheological properties of proteins adsorbed at O/W interfaces[END_REF]. one possibility is that these two aldehydes are able to interact with proteins, leading to the additional rearrangements of CAS in the O/W interfaces, thereby resulting in a more densely packed structure, with lower interfacial tension [START_REF] Dickinson | Adsorbed protein layers at fluid interfaces: interactions, structure and surface rheology[END_REF]. Interestingly, the image of oil droplets during measurement was recorded in 0, 3 and 15 h as shown in Fig. 1B. After 3 or 15 hours of protein adsorption, the pictures of oil droplet containing TCA showed obvious more turbid than others. This phenomenon can further support the possibility which TCA may have chemically reacted with the adsorbed CAS due to the presence of an aldehyde group in TCA molecules, thereby changing the structure and adsorbed CAS.

The change in interfacial tension with absorption time can be fitted with by the equation of Ward and Tordai equation (1) with slightly modification [START_REF] Ward | Time-dependence of boundary tensions of solutions I. The role of diffusion in time-effects[END_REF].

(1) Where C 0 is the concentration of continuous phase, K B is the Boltzmann constant, T is the absolute temperature, and D is the diffusion coefficient. The initial process of adsorption was assumed to be mainly controlled by the protein diffusion [START_REF] Xiong | Characterization and interfacial rheological properties of nanoparticles prepared by heat treatment of ovalbumin-carboxymethylcellulose complexes[END_REF]. The plot of interfacial tension as function of t 1/2 is linear, and the diffusion rate (K diff ) can be calculated by the slope of this plot as shown in Table 1. The highest K diff rate was observed in the TCA/MCT oil phase, while there no obvious different observed in other two oily phases at the same CAS solution. These results suggested that for CAS stabilized interfaces, introducing citral into oily phase has only a marginal effect on protein diffusion. In addition, a small amount of TCA into oil phase can significantly improve the dispersibility and diffusion properties of CAS at the O/W interfaces. Several studies have indicated that TCA have the ability to interact with proteins, resulting in the formation of larger protein species and additional rearrangements in the O/W interface [START_REF] Chen | Enhancement of physicochemical properties of whey protein-stabilized nanoemulsions by interfacial cross-linking using cinnamaldehyde[END_REF][START_REF] Chen | Influence of pH and cinnamaldehyde on the physical stability and lipolysis of whey protein isolate-stabilized emulsions[END_REF].

After the initial diffusion stage, the adsorption kinetics become visibly slow which was caused by the energy barrier during the adsorption and rearrangement [START_REF] Graham | Proteins at liquid interfaces: III. Molecular structures of adsorbed films[END_REF].

Moreover, the rate of penetration and rearrangement of adsorbed proteins at O/W interfaces can be analyzed by the first-order equation with slightly modification [START_REF] Perez | Interfacial dynamic properties of whey protein concentrate/polysaccharide mixtures at neutral pH[END_REF][START_REF] Suttiprasit | The surface activity of α-lactalbumin, β-lactoglobulin, and bovine serum albumin: I. Surface tension measurements with single-component and mixed solutions[END_REF]:

(2) Where π 0 , π f and π t are the interfacial tension at the initial, final, and at any time, respectively, t is adsorption time and K i is the first-order rate constant. Fig. 2 displayed a typical fitting curve of ln[(π f -π t )/(π f -π 0 )] as a function of the time. All of these curves include in two linear region which represent the first-order rate constant of penetration (K p ) and molecular reorganization (K r ) at the O/W interface, respectively. As shown in Table 1, the relative highest K p value was found in the pure MCT and TCA/MCT oily phases, which suggested that TCA only have ability to promote the adsorption speed at the initial processing of protein absorption. Similar K p values (Table 1) were found in pure MCT and TCA/MCT oil phase, which indicates that TCA does not have the ability to promote protein adsorption during the protein penetration process at O/W interface. This was probably attributed that TCA interact with adsorbed CAS molecular entanglement by forming a network structure, and then hindered the protein penetration at the interfacial layer. However, a relatively low K p value was found in Citral/MCT oil and the lower R-value was obtained by a linear fit. This result may cause by the low density of citral (0.888 g/L). The low density of the mixed oily phase gradually changed the shape of droplets during the protein adsorption. On the other hand, the highest K r value was found in the pure MCT oil phase, which be interpreted the fact that TCA and Citral were introduced into the oily phase and then decrease the polarity of the oily phase [START_REF] Liao | Effect of carrier oil on the properties of sodium caseinate stabilized O/W nanoemulsions containing Trans-cinnamaldehyde[END_REF], and lower hydrophobicity of oil phases will reduce the ability to attract emulsifiers.

Dilatational viscoelastic properties

Measuring the viscoelastic properties of the layers adsorbed at the O/W interface is an effective ways to evaluate the performance of the performance of the emulsion system [START_REF] Chen | Impact of soy proteins, hydrolysates and monoglycerides at the oil/water interface in emulsions on interfacial properties and emulsion stability[END_REF]. The dilatational elastic dilatational modulus were calculated by the first harmonic of the Fourier transform of the oscillating interfacial tension signal [START_REF] Bagley | A theoretical basis for the application of fractional calculus to viscoelasticity[END_REF]. Fig. 3 displayed the elastic module of oil-water interface with three different oily phases. After adsorption for 3 hours, the dilatational elastic modulus of all samples was significantly greater than the viscosity modulus (data not shown), indicating that the interface absorbed layer mainly exhibited the elastic behavior. In all cases, the value of elastic module gradually increased as a function of time due to the protein adsorption and the interaction between protein and protein. At the initial process of protein adsorption, the growth of elastic module was obvious and rapid and then it became gradually flattened. This tendency has been previously reported by other proteins, such as soy protein [START_REF] Wang | Soy protein isolated-soy hull polysaccharides stabilized O/W emulsion: Effect of polysaccharides concentration on the storage stability and interfacial rheological properties[END_REF], bovine serum albumin [START_REF] Tang | Dynamic adsorption and dilatational properties of BSA at oil/water interface: Role of conformational flexibility[END_REF], and pea protein [START_REF] Sha | Interfacial dilatational and emulsifying properties of ultrasound-treated pea protein[END_REF]. Interesting, the results confirmed that the addition of both aldehydes, especially TCA, can increase the elastic modules and result in a stronger protein network. These findings suggested that the interaction between protein and aldehydes had a vital influence on the protein adsorption at the O/W interface.

Ultraviolet Spectroscopy

UV spectroscopy is one of the generally used techniques for the illumination of the binding mode of small molecules to proteins, by measuring the changes in absorption and shift in the wavelength [START_REF] Alam | In vitro DNA binding studies of therapeutic and prophylactic drug citral[END_REF]. The effect of various TCA and Citral concentrations (0 -80 μmol/L) on the CAS solution (10 μmol/L) was shown in Fig. 4. Pure TCA and Citral (40 μmol/L of, buffer) were used as the control. Fig. 4A showed that the mixture of CAS and TCA exhibited a maximum absorbance in the range of 280-290 nm and a distinct red shift was observed.

Meanwhile, the intensity of the maximum absorbance was significantly improved with the increasing TCA concentration, indicating that there was interaction between TCA and CAS.

Moreover, the adsorption peak at 280 nm of the mixture suggested that high concentrated TCA may cause the tryptophan, tyrosine and phenylalanine residues become more accessible and therefore causes conformational changes in the protein [START_REF] Sudha | Exploring the binding mechanism of 5-hydroxy-3′, 4′, 7-trimethoxyflavone with bovine serum albumin: Spectroscopic and computational approach[END_REF]. As shown in Fig. 4B, pure citral exhibited a maximum absorbance around 245 nm and the mixture of CAS and TCA exhibited a similar maximum intensity around 280 nm, which suggesting there is no obvious interaction between CAS and citral.

Fluorescence spectra

The fluorescence spectra of CAS with the various concentrations of TCA and Citral at 25 °C are shown in Fig. 5. The CAS solution with the presence and absence of aldehydes possessed different fluorescence intensities which indicated the changes of environmental conditions. As several studies reported [START_REF] Acharya | Binding of resveratrol with sodium caseinate in aqueous solutions[END_REF], pure CAS solution exhibited an obvious emission band around 350 nm when the extraction band was 280 nm. After mixing with TCA (Fig. 5A), the fluorescence intensity of the maximum emission band decreased, which further indicated the occurrence of fluorescence quenching and the formation of complexes by TCA binding to aromatic residues of CAS. This fluorescence quenching have been supported by many previous studies [START_REF] Mohammadian | Enhancing the aqueous solubility of curcumin at acidic condition through the complexation with whey protein nanofibrils[END_REF][START_REF] Shi | Binding interaction of sorafenib with bovine serum albumin: Spectroscopic methodologies and molecular docking[END_REF][START_REF] Wu | Insight into the effect of charge regulation on the binding mechanism of curcumin to myofibrillar protein[END_REF]. With the subsequent increase in the TCA concentration, the maximum intensity further decreases with a gradual red shift, which may cause by the presence of binding sites near the fluorophores in the protein [START_REF] Soltanabadi | Spectroscopic analysis, docking and molecular dynamics simulation of the interaction of cinnamaldehyde with human serum albumin[END_REF]. However, as shown in Fig. 5B, the fluorescence intensity of CAS and citral only shows a very weak quenching phenomenon, which suggested CAS molecular could not provide a suitable environmental structure to bind citral molecular.

The quenching of CAS induced by TCA can be further analyzed by the Stern-Volmer equation (3), which can be dynamic, a collision between the fluorophore and quencher, or static, the formation of a ground-state complex between the fluorophore and quencher [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF].

(3)

Where F 0 and F are the fluorescence intensities at 280 nm excitation wavelength before and after mixing with the quencher, respectively; k q is the bimolecular quenching constant, τ 0 is the average lifetime of the fluorophore in the absence of quencher; [Q] is the concentration of the quencher, and K sv is the Stern-Volmer quenching constant which can be calculated by linear regression of a plot of F 0 /F against [Q]. The curve of F 0 /F as a function of quencher concentration [Q] at different temperatures was shown as Fig. 6A. It is worth to note that, at high TCA concentration, an upward deviation from linearity can be observed, which suggested that both dynamic and static quenching may occur in the fluorescence quenching mechanism. However, this equation is still not enough to distinguish the dynamic quenching form static [START_REF] Acharya | Binding of resveratrol with sodium caseinate in aqueous solutions[END_REF]. An effective way to distinguish between dynamic quenching and static quenching can be to observe their bonding performance at different temperatures. The temperature increase can increase the quencher diffusion constant, which is usually accompanied by an increase in collisional quenching. For static quenching, the temperature have different effects [START_REF] Acharya | Binding of resveratrol with sodium caseinate in aqueous solutions[END_REF]. Typically, this process will result in a decrease in the binding constant of the fluorophore quencher, and if the complexation reaction is exothermic, it will result in a decrease in the quenching of the static quencher. The modified Stern-Volmer equation ( 4) [START_REF] Lakowicz | Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale[END_REF]) can be used to analysis when both static and dynamic quenching occur.

(4)

Where f is the available binding sites. When f is 1, the equation become the simple quenching process equation (3). The curve of F 0 /(F 0 -F) as a function of quencher concentration [Q] -1 at different temperatures was shown as Fig. 6B. At 25 °C, the quenching of CAS by TCA, K sv is 2.25 × 10 -4 M -1 . The fraction of the binding sites available for complexation was 1.63 higher than 1.0, which was probably attributed the occurrence of both dynamic and static quenching simultaneously. In addition, more binding sites could also be related with CAS which are a mixture of different casein protein, such as αs 1 , αs 2 , β and κcasein and there caseins differ in the number of tryptophan residues and their location in the hydrophobic regions [START_REF] Fu | Interactions of buttermilk with curcuminoids[END_REF][START_REF] Yazdi | Heating of milk alters the binding of curcumin to casein micelles. A fluorescence spectroscopy study[END_REF].

The fluorescence lifetime of tryptophan has been reported around 3 ns [START_REF] Lakowicz | Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale[END_REF]. The K q , calculated by the equation (3), is 7.5 ×10 12 M -1 per second at 25 °C, which is highly greater than the maximum dynamic quenching constant (~10×10 10 M -1 s -1 ). This indicated that there was a specific complex formation between CAS and TCA. For further analysis of quenching interaction, the binding constant and binding sites can be calculated by following modified Stern-Volmer equation ( 5).

(5) and n (binding sites) at different temperatures. The large values of K were obtained at 25 and 30 °C which indicated the binding of TCA with CAS is highly favorable. As increasing temperatures, the binding constants gradually decreased which suggested that the complexation becomes weak at higher temperatures. Similar results was also found in the study of resveratrol and whey proteins by fluorescence spectroscopy [START_REF] Hemar | Investigation into the interaction between resveratrol and whey proteins using fluorescence spectroscopy[END_REF]. The n values for binding of CAS and TCA were close to 1.0 for different temperatures.

To a better understanding of the thermodynamic of the interaction between CAS and TCA, Van't Hoff equation ( 6) was followed to calculate the change of enthalpy ΔH and entropy ΔS.

(6)

Where K is binding constant, R is the gas constant (8.314 J mol -1 k -1 ) and T is the absolute temperature (K). Thereby, thermodynamic parameters ΔH and ΔS can be calculated by the slope and intercept of the curve of lnK as a function of 1/T. Fig. 7 showed the current Vant's Hoff fitting curve and it gives ΔH = -55.76 kJ mol -1 , ΔS= -86.05 J mol -1 . The free energy change (ΔG) can be then calculated from the equation ( 7) as follow:

(7) Table 2 summarized the thermodynamic parameters of binding between CAS and TCA. ΔG values at different temperatures are all negative, indicating the binding process of TCA and CAS is spontaneous and exothermic. The negative ΔH and ΔS values suggested that binding would be mainly driven by hydrogen bonds, which would promote a more ordered configuration of the solvent molecules around the complexes. This similar interaction was also found for other protein-small molecular combinations, such as zein proteins-resveratrol [START_REF] Joye | Fluorescence quenching study of resveratrol binding to zein and gliadin: Towards a more rational approach to resveratrol encapsulation using water-insoluble proteins[END_REF], crosslinked caseinate-quercetin, caseinate-kaempferol (Y.-J. Zhang et al., 2021) and bovine serum albumin-theaflavin-3,3′-digallate [START_REF] Yu | Studies on the interactions of theaflavin-3,3′-digallate with bovine serum albumin: Multi-spectroscopic analysis and molecular docking[END_REF]. Moreover, the high absolute value of ΔH even indicates that a covalent bond may be formed between CAS and TCA. Other complementary technologies are needed in the future to fully explore the exact type and strength of interaction between CAS and TCA. Nevertheless, the present work showed that TCA, a common flavoring in food, may be used as an interesting ingredient to improve the surface rheological properties of protein-based interfaces.

Conclusion

The interaction between aldehydes and CAS was investigated in the current study. Both aldehydes, especially TCA, have pronounced abilities to decrease the interfacial tension at O/W interface during adsorption process. Small amount of TCA introduced into oil phase can significantly improve the dispersibility and diffusion properties of CAS at the O/W interfaces. The results of dilatational measurements indicated the use of TCA into oily phase resulted in the development of stronger and more elastic interfaces. Furthermore, the fluorescence quenching test proved that TCA have more obvious quenching effect on CAS than citral. The large binding constants (1.78 × 10 5 M -1 ) was calculated by Stern-Volmer equation which suggest that TCA has strong affinity with CAS. The negative free energy change for the binding between TCA and CAS indicated that complexation process is spontaneous. The negative enthalpy and negative entropy change suggested that the binding is mainly driven by formation of hydrogen. Entropy is unfavorable suggesting there is a loss of conformational freedom associated with binding. In summary, these nature aldehydes, could be a useful ingredient to improve the functionality of food protein-based products. Table 1. the apparent diffusion rate (K diff ), constants of penetration and structural rearrangement at the interface (K p and K r ), and interfacial pressure at the end of adsorption (π 10800 ) for different oily phase in 0.01% CAS solution.

Samples

Kdiff (mN/m/s

Fig

  Fig. 6C exhibited the curves of log [(F 0 -F)/F] against log [TCA] for the quenching of CAS

Fig. 1 .

 1 Fig. 1. (A) Interfacial tension over protein adsorption at O/W interface with or without TCA or Citral for 0.01% CAS solution at pH 7; (B) Visual image of oil droplets during measurement at different time.

Fig. 2 .

 2 Fig. 2. The profile of the molecular penetration and configurational rearrangement steps over protein adsorption at the O/W interface with and without TCA or Citral for 0.01% CAS solution. Kp and Kr represents the first-order rate constants of penetration and rearrangement, respectively.

Fig. 3 .Fig. 4 .Fig. 5 .

 345 Fig. 3. Dilatational elasticity over protein adsorption at O/W interface with and without TCA or Citral for 0.01% CAS solution.

Fig. 7 .

 7 Fig. 7. The Van't Hoff plot of ln K as a function of 1/T (K) for the binding of TCA with CAS solution.

  Table 2 displayed the quenching parameter K (binding constant)

Table 2 .

 2 Variation of binding constant K and the number of binding site n as a function of temperature and thermodynamic parameters of binding calculated using Van't Hoff equation.

		1/2 )	Kp × 10 4	Kr × 10 4	π10800 (mN/m)
	MCT	-0.090 ± 0.016	-2.151 ± 0.005	-8.543 ± 0.005	13.56
	TCA/MCT	-0.122 ± 0.006	-2.178 ± 0.001	-7.046 ± 0.009	9.12
	Citral/MCT	-0.081 ± 0.010	-1.820 ± 0.002	-5.626 ± 0.002	11.27

ACKNOWLEDGEMENTS

Wei Liao greatly thanks Chinese Scholarship Council for support.

CONFLICTS OF INTEREST

The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported