Artificial Arthropod Exoskeletons/Fungi Cell Walls Integrating Metal and Biocatalysts for Heterogeneous Synergistic Catalysis of Asymmetric Cascade Transformations

A novel and sustainable tandem‐catalysis system for asymmetric synthesis is disclosed, which is fabricated by bio‐inspired self‐assembly of artificial arthropod exoskeletons (AAEs) or artificial fungi cell walls (AFCWs) containing two different types of catalysts (enzyme and metal nanoparticles). The heterogeneous integrated enzyme/metal nanoparticle AAE/AFCW systems, which contain chitosan as the main structural component, co‐catalyze dynamic kinetic resolution of primary amines via a tandem racemization/enantioselective amidation reaction process to give the corresponding amides in high yields and excellent ee. The heterogeneous AAE/AFCW systems display successful heterogeneous synergistic catalysis at the surfaces since they can catalyze multiple reaction cycles without metal leaching. The use of natural‐based and biocompatible structural components makes the AAE/AFCW systems fully biodegradable and renewable, thus fulfilling important green chemistry requirements.


Introduction
The arthropod exoskeleton is constituted of two layers. An outer layer, the epicuticle, is composed of lipoproteins and chains of fatty acids embedded in a protein-polyphenol complex. An inner layer, the procuticle, is formed by crossbonding chitin-protein chains which provides strength to the skeletal material. [1] The fungi cell wall is composed mainly of glucans, chitin, and glycoproteins and serves many functions, including providing cell rigidity and shape. [2] Interestingly, for both the arthropods and the fungis, the presence of a chitinprotein interwoven matrix constitutes the main structural element of these living organism.
The design of novel green industrial processes able to harmonize both reaction conditions and environmental impact is a great challenge for the new millennium. The research on eco-compatible feed stocks to replace oil-based synthetic derivatives, has directed the attention of the scientific community toward the valorization of biomass as source for novel compounds. [3] Polysaccharides are the most abundant macromolecules on the planet. They have found a wide range of applications in chemical and pharmacological industry. [4] The versatility of these compounds is due to their thermal and chemical stability and to the ease of modification and functionalization for the synthesis of a variety of derivatives. [5] In this context, chitin is the second most abundant polysaccharide present in nature and it is the main constituent of the exoskeleton of crustaceans and of the cell walls in fungi. Chitin is produced by direct chemical [6] or enzymatic [7] treatment of crustacean, fungi or insect waste. [8a] The global production is estimated to be more than 155 thousand metric tons by 2022 for a market value of 4.2 billion US dollars. [8b] Among chitin derivatives, chitosan is the most interesting product demonstrated by the high production growth and the continuous demand for reliable source of high-quality material. The market value for 2022 is predicted to be of 1088 million US dollars with an overall production of 63.7 kilotons. [9] Chitosan is a linear polysaccharide directly produced by deacetylation of chitin. [10] Chitosan is structurally similar to cellulose but contains both glucosamine and acetylglucosamine units. The free primary amino groups are a perfect starting point for linking functional groups, to create polyvalent cellulosic materials, or to act as support for metal nanoparticles. [11] The low price [12] and its great versatility have attracted attention from various research and industrial fields. Chitosan is largely used in agriculture as biopesticide against fungal infection [13] and in the food industry as an antimicrobial component of packaging. [14] In paper industry chitosan is used as modulator of strength and oxygen permeability, [15] and in the pharmaceutical industry it is used for drug delivery to target molecules with specific receptors on the cell surface. [16] Strategies for construction of hybrid catalysts and systems are very important since they not only increase performance (e. g. activity, yields, selectivity, lifetime and recyclability) to one of the components but offer extra functions such as tandem or cascade catalysis for synthesis of complex and/or optical active products in a highly selective fashion. [17] Hence, the use of multifunctional heterogeneous catalysts permits to combine various steps in a synergistic mode avoiding the isolation of intermediates and thus optimizing reaction time and consumption parameters. The number of reagents and solvents is directly correlated to the production of hazardous wastes. In our laboratory, we are inspired by nature and its interplay between homogeneous and heterogeneous catalyst systems. In this context, Córdova and coworkers disclosed in 2012 the synergistic combination between organocatalysis and heterogeneous metal catalysis for use in asymmetric cascade synthesis, [18] which was further followed up to a concept on enantioselective diversity-oriented synthesis and integrated multiple relay catalysis. [19] Biocatalysts are useful tools for catalyzing non-natural chemical reactions. Usually free enzymes show very high reactivity in aqueous solutions, which drastically decreases in organic solvents. [20] Two approaches to circumvent this problem are: (i) to use an nonpolar dry organic solvent in which the conformation of the enzyme remains unchanged or (ii) to stabilize the biocatalyst by immobilization on a solid support such as silica or polymeric resins. [21] The development of heterogeneous metallo-enzyme biohybrids had attracted con-siderable attention but is still in its infancy. Different synthetic strategies were evaluated to build efficient dual bio-metal catalysts such as direct introduction of the transition metal into the active site of the enzyme [22] or replacement of the natural metal of the heme enzyme by another metal. [23] Cross-linked enzyme aggregate (CLEA), where the enzyme itself constitute the heterogeneous support, was used as matrix to incorporate transition metal nanoparticle. [24] A hybride catalyst obtained from immobilization of enzyme and metal nanoparticles into the cavities of siliceous mesocellular foam was used for dynamic kinetic resolution (DKR) of amines. [25] However, in most of the cases, partial deactivation of the enzyme and leaching of the metal catalyst limited the recycling of the "metallo-enzyme" heterogeneous catalyst. Fossil-based polymers and metal organic frameworks have also been added as supports for hybrid enzyme/metal catalysis but there is a need for readily available, scalable and sustainable solutions. [26] We recently disclosed the construction of artificial plant cell walls where a cellulose and protein matrix constitute a multilayer architecture for integrating heterogeneous enzyme and metal-catalysis. [27] Intrigued by these results, we decided to continue our research in the emulation of natural biomaterials structures as support materials in heterogeneous catalysts focusing our attention on chitin/chitosan-based organisms. The peculiar chemical characteristics of chitosan make this polysaccharide an ideal heterogeneous support ready to be used, as scaffold for metal nanoparticles, without the need of extra timeconsuming functionalization steps. The self-assembly of mixtures of chitosan, proteins and surfactants could create an artificial fungi cell wall (AFCW) or artificial arthropod exoskeleton (AAE), which if contained enzyme/metal catalysts could be used as a heterogeneous catalyst for asymmetric synthesis ( Figure 1). It is worth noting that the presence of chitosan, in the AFCW or AAE, confers to higher shelf-life stability (due to the antimicrobial intrinsic characteristics) and robustness (due to the structural rigidity) as compared to a cellulose-based heterogeneous catalyst system. Herein, we disclose the construction of AAEs, which integrates both enzyme and metal nanoparticle catalysts, by self-assembly and their use in the highly enantioselective synthesis of amides through concurrent tandem racemization and enantioselective acylation of racemic amines, leading to a DKR.

Results and Discussion
We began our investigation by self-assembling the AAE catalyst using Candida antartica Lipase B (CALB) as the enzyme of choice without including metal nanoparticles ( Figure 2). Next, the constructed AAE1 was used to catalyze the kinetic resolution of racemic 1-phenylethylamine (rac-1a) in toluene. After 53 h, the conversion was 42 % to the corresponding amide (R)-3a with an enantiomeric excess (ee) of 93 %. In comparison, pure CALB did not form any product amide under the same reaction conditions. Thus, chitosan as other polysaccharides plays a crucial role for enzyme activation in organic solvents ( Figure 2). [27] With this result in hand, we further increased the complexity of our catalytic system by integrating metal nanoparticles to the AAE. This was accomplished by immobilizing palladium nanoparticles directly on the chitosan surface by first dispersing chitosan in an aqueous solution of Li 2 PdCl 4 to furnish, after isolation, Chitosan-Pd II . The amino-stabilized Pd II species were subsequently reduced by NaBH 4 to generate Pd 0 nanoparticles and Chitosan-Pd 0 (see Supporting Information). The AAE hybrid catalyst was readily prepared by self-assembly of the following components: Chitosan-Pd 0 , surfactant polyethylene glycol hexadecyl ether (Brij) and CALB, which were mixed in a phosphate buffer solution. Next, freeze drying gave the corresponding AAE3 as a solid grey foam (Figure 3).
Scanning transmission electron microscopy bright field (STEM-BF) and energy dispersive X-ray spectroscopy (EDS) map of Pd L-edge showed well dispersed palladium nanoparticles in the AAE matrix (Figures 4a and 4b, and Figure S1). X-ray photoelectron spectroscopy (XPS) was used to obtain information about the oxidation state of the Pd nanoparticles. XPS showed that there is a mixture of Pd 0 and Pd II with > 85 % of Pd(0) ( Figure 5). The oxidation state of the Pd nanoparticles incorporated into AAE3 was predominantly Pd 0 as in the starting chitosan-Pd 0 but the Pd 0 /Pd II ratio was lower as determined by XPS (Figure 5b).
The loading of Pd on Chitosan-Pd 0 and AAE3 was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) to 5.8 wt. % and 3.8 wt. %, respectively.
Our studies continued with the investigation of the AAEcatalyzed deracemization of racemic amine 1 a. An initial solvent screen showed that toluene was the best reaction medium as it gave a higher conversion rate as compared to 1,4dioxane or 2,4-dimethyl-3-pentanol (Table 1, entries 1-4). Knowing that the perfect balance between the structural components of the AAE is crucial for the optimal reaction outcome, in terms of reaction kinetics and stereoselectivity, we modified the ratio of the constituents increasing the amount of Chitosan-Pd 0 in the catalyst. Thus, AAE3 was assembled by mixing chitosan-Pd 0 , enzyme and surfactant in buffer followed by lyophilization (See Supporting Information). We continued screening several reaction conditions by tuning the ratio between the reagents (Table 1, entries 5-11) such as increasing the base (Table 1, entry 9) or the ester donor 2 ( Table 1, entries 10-11) equivalents. The results were quite similar. Thus, we decided to use the reaction parameters shown in entry 7 to test the versatility of the AAE3 dual-catalytic enzyme/metal system for the deracemization of different racemic amines 1. As shown in Table 2, the heterogeneous dual-catalyst system AAE3 catalyzed the conversion of racemic 1 to the corresponding amides (R)-3, which were isolated in high yields and excellent   enantiomeric excess, via a DKR process. In addition, we evaluated the stability and the efficiency of AAE3 catalyst conducing recycling experiments (Table 3, Supporting information). Catalyst AAE3 was recycled several times without showing any significant loss in product yield and selectivity as compared to other hybrid deracemizing catalytic systems, which display deactivation after a few cycles. [25] However, the reaction becomes slower during the recycling but is significantly better than other systems. [25,26c] In comparison, integrated chemical dual catalyst systems for enantioselective heterogeneous synergistic catalysis failed in recycling due to catalyst inhibition. [19c] Thus, this important feature for the development of enantioselective heterogeneous synergistic catalysis for asymmetric transformations is successfully accomplished when using AAE3. The increased amount of AAE3 to substrate rac-1 a, due to easier handling of the catalyst during its recovery, led to the formation of 4 a in different ratios. A leaching test was performed by filtering off the catalyst after completion of the reaction. No leaching of palladium could be detected in solution by inductively coupled plasma optical emission spectroscopy (ICP-OES).

Conclusions
In conclusion, herein we have reported the concept of how to fabricate artificial fungi cell walls or artificial arthropod exoskeletons (AAE), which integrates different types of catalysts, by self-assembly. The fabricated sustainable and bioinspired AAEs are excellent heterogeneous synergistic catalysts for cascade reaction and dynamic asymmetric transformations, [28] as demonstrated by the deracemization of primary amines, proceeding via a tandem racemization-kinetic resolution process mediated by integrated palladium nanoparticle/enzyme catalysis. The cooperative interplay between the metal and enzyme catalyst within the solid chitosan network (heterogeneous synergistic catalysis) allowed for the highly enantioselective synthesis of amides in high yields and 99 % ee. In addition, the heterogeneous AEE can be recycled for multiple reaction cycles to give the corresponding product amine in high yield and excellent  ee. The use of natural-based and biocompatible components makes our heterogeneous catalysts possible to fulfill several green chemistry criteria (renewable starting material, high selectivity, recyclability etc.). Further investigation on heterogeneous bio-inspired catalyst and multi-catalytic systems for selective organic synthesis using chitosan as a structural component are ongoing in our laboratories.

Experimental Section
Chitosan-Pd II synthetic procedure

AAE1 Chitosan/CALB/Brij/buffer assembling procedure
In a plastic beaker was added chitosan (60 mg), sodium phosphate buffer (6 mL, 0.1 M, pH 7.2) and Brij C10 (20 mg). The suspension was stirred with a spatula until completely solubilization of Brij C10. Next CALB (20 mg) was added, the mixture was stirred with a spatula until completely solubilization of the enzyme and rapidly frozen in liquid nitrogen. The catalyst was lyophilized for 70 hours to give a solid white foam (185 mg).

AAE2 assembling procedure
In a plastic beaker was added Chitosan-Pd 0 (60 mg), sodium phosphate buffer (6 mL, 0.1 M, pH = 7.2) and Brij C10 (20 mg). The solution was stirred with a spatula until completely solubilization of Brij. Next CALB (20 mg) was added and the mixture was rapidly frozen in liquid nitrogen. The catalyst was lyophilized for 70 hours to give a solid grey foam (200 mg).

AAE3 assembling procedure
In a plastic beaker was added Chitosan-Pd 0 (180 mg), sodium phosphate buffer (6 mL, 0.1 M, pH = 7.2) and Brij C10 (20 mg). The solution was stirred with a spatula until completely solubilization of Brij. Next CALB (20 mg) was added and the mixture was rapidly frozen in liquid nitrogen. The catalyst was lyophilized for 70 hours to give a solid grey foam (304 mg).

General procedure for the DKR of rac-1 in toluene using AAE3
In a microwave vial were added AAE3 (9 mg, 1.29 mol % Pd) and dry Na 2 CO 3 (80 mg, 0.75 mmol, 3 equiv). The vial was flushed with nitrogen and hydrogen. Next, toluene (1 mL), rac-1 (0.25 mmol, 1 equiv) and 2 (59 mg, 0.5 mmol, 2 equiv) were added. The vial was capped, evacuated and a hydrogen balloon was connected to the reaction vessel. The reaction was stirred at 90°C for 23 hours. The crude reaction mixture was directly purified by column chromatography (hexane/ ethyl acetate 9 : 1 to 2 : 1) to afford the corresponding product (R)-3.

Recycling procedure of AAE3 (Pd 0 2.58 mol %) for the DKR of rac-1 a
In a microwave vial were added AAE3 (36 mg, 2.58 mol % Pd 0 ) and dry Na 2 CO 3 (265 mg, 2.5 mmol, 5 equiv). The vial was flushed with nitrogen and hydrogen. Next, toluene (2 mL), rac-1a (60.5 mg, 0.5 mmol, 1 equiv) and 2 (118 mg, 1.0 mmol, 2 equiv) were added. The vial was capped, evacuated and a hydrogen balloon was connected to the reaction vessel. The reaction was stirred at 90°C for the time reported in the table. Next, dry toluene was added to the vial and the reaction mixture was centrifuged 3 times collecting the supernatant after each cycle. After the last centrifuge cycle, 2 mL of toluene was left in the vial and fresh Na 2 CO 3 (52 mg, 0.5 mmol, 1 equiv) and fresh substrates were added. The collected supernatant was concentrated under reduced pressure and the crude reaction mixture was directly loaded on a silica-gel column and chromatography (hexane/ ethyl acetate 2 : 1) afforded (R)-3a in the reported yield and ee. The cycle was repeated.