Exploring Thermomyces lanuginosus Lipase (TLL)‐PdNPs Nanohybrid as Suitable Catalyst for One‐pot Synthesis of Bis(3‐indolyl)phenylmethane

Palladium nanohybrids were synthesized and applied to the one‐pot synthesis of bis(3‐indolyl)methanes by selective C−C bond reaction from benzyl alcohol and indole. A T. lanuginosus lipase‐palladium nanoparticles hybrid (Pd@TLL) was synthesized, yielding PdNPs with an average diameter size of 5 nm. This heterogeneous catalyst was first tested in the selective oxidation of benzyl alcohol to benzaldehyde in different solvents. Then, the direct formation of bis(3‐indolyl)methane, by in situ oxidation and C−C coupling, was successfully evaluated under different conditions, obtaining >99 % conversion at 80 °C in toluene, with a TOF value of 9 min−1 and 89 % in pure water, demonstrating the versatility of these biohybrids.


Introduction
Among the N-heterocycles, indole is one of the most ubiquitous bioactive scaffolds, and considered a privileged structure in medicinal chemistry.Functionalization of indole is extremely attractive for drug discovery programs, [1] and in this context, bis(indolyl)methanes, C-3 alkylated indoles, have attracted considerable attention. [2]Bis (3-indolyl)methanes are an important subgroup of indoles, consisting of two indole units linked by a single methylene bridge, and are present in several natural products such as trisindoline, 3,3-bis(3'-indolyl)propane-1,2diol,2-(2,2-di(1H-indol-3-yl)ethyl)aniline and dalesindole (Figure 1). [3]is(indolyl)methanes (BIMs) are commonly prepared by reacting indole with aldehydes or ketones, under acid catalysis, to give an azafulvenium salt, which then undergoes an addition reaction with a second indole molecule.[6][7] X. Vu's group [4] reported the alkylation of indoles with alcohols catalysed by an air-stable magnetically recycled CuFe 2 O 4 heterogeneous catalyst, the alkylation of indoles with alcohols catalysed by a Cu(OAc) 2 developed by P. Langers's group, [5] the application of N,N,N-Mn(II)pincer complexes to the synthesis of symmetrical and unsymmetrical bis(indolyl)methanes derivatives using tBuOK as a base, [6] or more recently, the C-3 functionalization of indoles via borrowing hydrogen catalysed by NNSÀ Mn(I) complexes, in the presence of tBuOK. [7]n addition, the Pd-catalyzed synthesis of bis-indolylmethanes has also proved to be an efficient way of synthesizing this type of compound.In particular, the use of readily available starting materials, such as benzyl alcohol and the use of greener solvents such as water.Y. Yokoyama and co-workers have reported the synthesis of non-activated bis(indolyl)methanes using an in situ generated (η3-benzyl)palladium system in the presence of water.The authors have performed mechanistic studies suggesting that the indole reacts with the palladium(II) complex via CÀ H activation in the C-3 position.They have also noted the role of water in this particular case, allowing activation of the sp3 CÀ O bond and facilitating the nucleophilic attack of indoles on benzyl alcohols. [8]ther examples have been reported by the same author, highlighting the role of water in this type of CÀ H activation in the presence of Pd(OAc) 2 -TPPMS. [9]lternatively, bis(indolyl)methanes can also be obtained in the absence of a metal catalyst, thus D. Sharma and co-workers have reported the use of a readily commercially available additive, K 2 S 2 O 8 in the presence of water to synthesize several BIMs at room temperature.The authors, proposed that the mechanism form BIMs formation involves synthesis of the corresponding aldehydes from benzylic alcohols, followed by condensation with indoles via a radical mechanism, where SO 4 À species acts as initiator. [10]13][14] Nanoparticles are remarkably interesting due to their intrinsic properties characterized by very small size and an extremely large surface-to-volume ratio, which translates into unique optical, physical, and chemical properties.[17][18][19][20] Recently, the development of a new sustainable synthetic strategy of metal nanoparticles embedded on enzymes as network scaffold has been described and successfully applied in different CÀ C or CÀ H reactions. [21][22][23][24] The advantages of these methods compared to others are the direct use of a heterogeneous catalyst; the control of the nanoparticle size, where the formation is directly induced by the enzyme; the nanoparticles are homogeneously dispersed on the protein network, avoiding aggregation.Thus, the synthetic process of the formation occurs by an initial coordination of the metal ions in solution with specific amino acid residues on the protein structure (i.e., hydrophobic or negatively charged side chains).This binding process results in the precipitation of cross-linked metal ionprotein complexes.The second role of the protein is based on the in situ reduction of Pd 2 + to Pd(0).This process is mediated by certain amino acid residues that are close to the binding sites of the same peptide chain or that are almost threedimensional located, and have a strong reducing capacity (i.e., amino acids with hydrophobic or hydroxyl side chains).After reduction, metal particles are formed which is the final step in the growth of the nanoparticle.This step is controlled by the protein network previously formed and is an important aspect in the final size of the nanoparticles. [22]herefore, these nanocatalysts provide accessible active sites, and the synthesis of metal nanoparticles allows a control of the nanoparticle size, which together result in higher catalytic efficiency.In addition, these nanocatalysts are compatible with both aqueous and organic solvents.
Here we describe the synthesis of a robust enzyme-PdNPs biohybrid using thermostable Thermomyces lanuginosus lipase (TLL) in aqueous media at room temperature.To highlight the practical utility, these Pd nanohybrids, were successfully applied to the synthesis of bis(3-indolyl)phenylmethane (Scheme 2).

Results and Discussion
Thermomyces lanuginosus lipase (TLL) was combined with Pd(OAc) 2 salt in an aqueous solution with 20 % (w/w) MeOH at 25 °C, yielding heterogeneous hybrid Pd@TLL as described in the experimental section.By this strategy, this hybrid contained two different Pd species, metallic Pd and PdO (around 40 %), which was confirmed by X-ray diffraction pattern (XRD) analysis (Figure 2a).A reflection peak at 2θ = 33.75corresponding to the pure tetragonal phase of PdO, and could be indexed to the, (101) plane (Joint Committee on Powder Diffraction Standards code: JCPDS#85-0713). [25]Other peaks corresponding to the (111), ( 200) and (220) planes of Pd(0) were observed (JCPDS #46-1043).XPS analysis confirmed the presence of PdO on the nanoparticles (Figure S1).Transmission electron microscopy (TEM) showed the formation of crystalline spherical nanoparticles around the protein matrix with an average diameter size of 5 nm (Figure 2c, Figure S2).The final content of Pd in the hybrid was 35 % (determined by ICP-OES).
The Pd@TLL hybrid was firstly tested as catalyst in the oxidation of benzyl alcohol to benzaldehyde (Table 1), to assess its potential applicability for the final synthesis of bis(indolyl)methane.
Mechanistically, the synthesis of bis(indolyl)methane is reported to occur by condensation of an indole with an aldehyde, or by reaction of the indole with an alcohol.When an alcohol is used, the reaction can proceed a borrowing hydrogen Table 1.a] Entry T (°C) Solvent Time (h) Conversion (%) [b] TOF (min À 1 ) reaction, [18] via oxidation [26] or dehydrogenation [27] of the alcohol followed by reaction with the indole, or directly via an activating agent, such as I 2 . [28]owever, the mechanism for the transformation of alcohols using palladium bionanohybrids is not well understood, and the reaction could occur via in situ formation of the aldehyde or direct reaction with the alcohol.Therefore, preliminary experiments consisted of using benzyl alcohol 1, in the presence of Pd@TLL at different temperatures, using different solvents and evaluating the effect of additives, such as tBuOK (Table 1).tBuOK, was chosen as the preferred additive during the screening of reaction conditions, as this strong base has been reported to play a valuable role in the synthesis of several BIMs, which may involve prior benzaldehyde formation, followed by reaction with indole.The reaction was first tested at 50 °C in THF in the presence of potassium tert-butoxide as an additive, giving 78 % conversion after 48 h (Table 1, entry 1).The reaction was also carried out without an additive by increasing the initial amount of benzyl alcohol, giving 89 % conversion after 72 h (Table 1, entry 2).At this point, the TOF value of Pd@TLL was 4 times higher in the second experimental conditions.The temperature was then increased to 80 °C in toluene.Under these conditions, Pd@TLL transformed 1 into benzaldehyde with 92 % conversion after 3 h, with a TOF value of 134 min À 1 (Table 1, entry 3).The soluble enzyme or immobilized derivative showed no activity (data not shown).
The first experiments, using the best experimental conditions obtained in the previous oxidation step, consisted of the reaction of the indole with pre-oxidized benzyl alcohol (benzaldehyde) (Table 2, entry 1) catalysed by Pd@TLL.The desired product 3 was obtained with 95 % conversion after 120 h.When indole was directly mixed with benzyl alcohol (Table 2, entry 2), Pd@TLL catalyzed the formation of 3 faster, with > 99 % conversion after 72 h, with a TOF value 8 times higher (Table 2, entries 1-2).This may indicate that the mechanism of reaction with benzyl alcohol is different from that with benzylaldehyde.Indeed, a reaction was carried out with benzylaldehyde and 0.5 eq of benzyl alcohol with the indole and mostly bisindole was formed from benzylaldehyde.Thus, the mechanism of formation of the product could be by a similar approach where a coordination between Pd and alcohol was observed in the rapid reaction of benzyl alcohol in water. [26]n order to evaluate the efficiency of the Pd and PdO active sites in the catalyst, a Pd hybrid was synthesized using C. antartica lipase (CALB) as the enzyme, which exclusively produced Pd(0)NPs, was synthesized (Figure S4). [22]The synthesis of bis(indoyl)methane with Pd@CALB was quite similar, slightly lower, to that observed with Pd@TLL (Table S1).
Next, the efficiency of the Pd@TLL catalyst was tested under different solvents and temperatures (Table 2, entries 3-6).Performing the reaction in THF at 50 °C was detrimental to the reaction outcome as the conversion decreased to 33 % (Table 2, entry 3).The efficiency of the catalyst was lower in polar solvents such as DMF, THF or dioxane.However, in pure water, the catalyst was also efficient, with 89 % conversion after 72 h at a TOF value of 6 min À 1 (Table 2, entry 6).This is a very interesting result in terms of sustainability and demonstrates the versatility of these biohybrids for metal-catalysed applications.
To broaden the scope, different benzyl alcohols and indoles were used, in some cases giving good yields of the bis-indole products.In some cases, the reaction was slower, and the

Proposed Reaction Mechanism
A mechanism can be proposed based on the use of palladium nanoparticles carrying Pd and PdO as metal species.It is proposed that the Pd nanohybrid can catalyse the alcohol oxidation via dehydrogenative coupling, followed by the reaction of the preformed benzaldehyde with the indole moiety, promoted by the nanohybrid (Scheme 3).According to Table 1, the oxidation of benzyl alcohol proceeds smoothly in toluene at 80 °C.However, from Table 2 we can see that under these conditions the reaction is more efficient when benzyl alcohol is used, than when benzaldehyde is used directly.This is due to the role of palladium in the dehydrogenative oxidation of benzyl alcohol, which leads to the activation of benzaldehyde, forming a more reactive complex (I).
Furthermore, H. Hikawa reported the influence of water in the reaction's mechanism.Although an enzyme is used as a support for the synthesis of these nanoparticles, enzymatic mechanisms are usually strictly attributed to water-based reactions. [29]

Conclusions
In conclusion, TLL-based palladium nanoparticles (PdNPs) biohybrid was prepared and resulted in the formation of small spherical Pd and PdO nanoparticles in the protein network, with an average size of 5 nm.The application of this nanohybrid to the synthesis of BIMs using indole and benzyl alcohol was successful with excellent conversion values for Pd@TLL (higher than 99 %), avoiding the use of any additives.Thus, Pd@TLL is versatile and able to generate the desired product 3 in a wide range of solvents with moderate to high yields, reinforcing the strength of its catalytic activity.This approach represents a major improvement in terms of the use of enzyme-based metal nanoparticles and efficiency in the synthesis of BIMs, as most metal-catalyzed methods used to synthesize BIMs rely on the use of an additive.a] Entry Catalyst T (°C) Solvent Time (h) Conversion (%) [d] TOF (min -1 ) [e] 1 [b] Pd@TLL 80 Toluene 120 95 1.6 [a] Conditions: 1 (0.037 mmol), 2 (2 mg, 0.017 mmol), solvent (1 mL), Pd@TLL (2 mg), [b] benzaldehyde (0.037 mmol) was used, [c] 100 °C for 48 h, [d] Conversion of the product 3 was quantified by HPLC, e TOF value was calculated considering the conversion between 20 to 50 % in each case.Conditions: alcohol (0.037 mmol), indole (0.017 mmol), toluene (1 mL), 80 °C, Pd@TLL (2 mg).Conversion of the products were quantified by HPLC.
product 4 was observed using Pd@TLL and the methodology showed compatibility with the use of water as the sole solvent.Furthermore, all reactions were carried out in an air atmosphere, highlighting the stability of this palladium bionanohybrid.

Structural Characterization
Inductively coupled plasma -optical emission spectrometry (ICP-OES) was performed on an OPTIMA 2100 DV instrument (PerkinElmer, Waltham, MA, USA).X-Ray diffraction (XRD) patterns were obtained using a Texture Analysis D8 Advance Diffractometer (Bruker, Billerica, MA, USA) with Cu Kα radiation.Transmission electron microscopy (TEM) and high-resolution TEM microscopy (HR-TEM) images were obtained on a 2100F microscope (JEOL, Tokyo, Japan).Scanning electron microscopy (SEM) imaging was performed on a TM-1000 microscope (Hitachi, Tokyo, Japan).To recover the biohybrids, a Biocen 22 R (Orto-Alresa, Ajalvir, Spain) refrigerated centrifuge was used.X-ray photoelectron spectroscopy (XPS) spectra were determined through a SPECS GmbH electronic spectroscopy system with a UHV system (pressure approx.10-10 mbar), with a PHOIBOS 150 9MCD energy analyser, monochromatic X-ray sources.The analysis of the same was carried out using the CasaXPS program.

Analytical Characterization
The HPLC analysis was performed in a JASCO HPLC equipment, a HPLC pump PU-4180 coupled with a UV-4075 UV-Vis detector.Samples (100 μL) were centrifuged and then 50 μL were diluted in 1 mL of 50 : 50 ACN:water before the injection.The analysis conditions were achieved with a Kromasil-C8 (150×4.6 mm and 5 μmø), at a flow of 1.0 mL/min; λ: 270 nm (unless otherwise noted); and 50 % (v/v) ACN in MilliQ water as the mobile phase.The conversion was determined by HPLC using samples standards.

Synthesis of Pd@TLL biohybrid
Pd(OAc) 2 (20 mg) was dissolved in 4 mL of methanol (MeOH).At the same time, 242 μL of Thermomyces lanuginosus (Lipozyme TL; 29.8 mg/mL) was dissolved in 16 mL of distilled water.The palladium solution was added to the enzyme solution.The mixture was kept under gentle stirring for 24 h at 25 °C.After that, the final suspension was separated by centrifugation (8000 rpm, 10 min) and the resulting solid was washed with the same solution (20 % v/v MeOH in distilled water) and, finally, with distilled water (twice).
After the last washing, 2 mL of water was added to the solid and the suspension was frozen in liquid nitrogen and lyophilized, obtaining a dry solid called Pd@TLL (14.2 mg).

General protocol for the oxidation of benzyl alcohol by Pd@TLL hybrid
To a flask equipped with stirring bar was added benzyl alcohol (4 μL, 0.037 mmol) and the THF or Toluene (1 mL).Next, 2 mg of Pd@TLL was added.The mixture was kept at 50 °C or 80 °C for the indicated time.The resulting reaction mixture was monitored by HPLC analysis of the reaction's samples collected at different times.

General protocol for the synthesis of bis-(indolyl)methane by Pd@TLL hybrid
To a flask equipped with stirring bar was added benzyl alcohol (4 μL, 0.037 mmol) and indole (2 mg, 0.017 mmol), followed by THF, Toluene, DMF, 1,4-Dioxane or water (1 mL).Next, 2 mg of Pd@TLL was added.The mixture was kept at 50 °C, 80 °C or 100 °C for the indicated time.The resulting reaction mixture was monitored by HPLC analysis of the reaction's samples collected at different times.Samples (100 μL) were centrifuged and then 50 μL of the supernatant were diluted in 1 mL of 50 : 50 ACN:water before the injection.The analysis conditions were achieved with a Kromasil-C8 (150×4.6 mm and 5 μmø), at a flow of 1.0 mL/min; λ: 270 nm and 50 % (v/v) ACN in MilliQ water as the mobile phase.The conversion was determined by HPLC using samples standards.

Scheme 1 .
Scheme 1.Selected reports for synthesis of BIMs via metal-catalyzed reactions.

Scheme 2 .
Scheme 2. Recent examples using metal nanoparticles and this work: synthesis of BIMs catalysed by Pd nanohybrids.