Efficient Synthesis of Tetrasubstituted Furans via Lipase-Catalyzed One-Pot Sequential Multicomponent Reaction

Abstract

Tetrasubstituted furans and their derivatives represent a versatile class of important heterocyclic frameworks widely distributed in natural products. These scaffolds also demonstrate significant potential in pharmaceutical chemistry, materials science, and organic synthesis methodologies. In this study, we successfully established a synergistic catalytic system utilizing benzoylacetonitriles, aldehydes, and benzoyl chlorides as substrates, facilitated by tributylphosphine and immobilized lipase (Novozym 435), to achieve efficient synthesis of cyano-containing tetrasubstituted furans. Under optimized conditions, we obtained a series of target products exhibiting exceptional substrate tolerance with good to excellent isolated yields ranging from 80% to 94%. Additionally, we proposed a reasonable reaction mechanism and verified it through controlled experiments. This methodology not only expands the synthetic utility of lipase in non-natural transformations but also establishes a paradigm of green chemistry for the construction of tetrasubstituted furans.

1. Introduction

The advancements in molecular biology and microbiology have facilitated the use of enzymes to catalyze organic reactions. Enzyme catalysis is now regarded as a more appealing approach for organic synthesis compared to traditional small molecule and metal catalysis [1,2,3]. Lipase, a well-established nonmetallic enzyme, exhibits excellent stability and activity in both organic solvents and aqueous solutions. Notably, most lipase-catalyzed reactions occur without the need for exogenous coenzyme supplementation [4]. Therefore, lipase has been widely used in the synthesis of pharmaceutical intermediates [5,6], the food industry [7,8], and the resolution of chiral compounds [9]. Lipases’ inherent hydrolase functionality enables catalytic versatility across multiple reaction modalities, encompassing hydrolysis, alcoholysis, esterification, and transesterification [10]. In addition, catalytic hybrids of lipases expand their capabilities in synthetic organic chemistry; representative transformations include epoxidation [11], the Dakin reaction [12], the aldol reaction [13,14], the Michael addition [15] and the Mannich reaction [16].

Furan-derived heteroaromatic frameworks represent a significant class of five-membered aromatic compounds with oxygen-containing rings. These functionalized furan derivatives exhibit remarkable biological and pharmacological activities [17,18], with prominent representations in natural products and therapeutic agents (Figure 1). Notable examples include prenylated coumarins (furanocoumarins) and terpene-derived furans (furanoterpenes) as plant secondary metabolites [19,20], α-guaiaconic acid from guaiac resin [21], the furan fatty acid metabolite CMPF [22], the histamine H2-receptor antagonist ranitidine [23], and the nitrofuran antibiotic furazolidone [24]. Beyond their pharmaceutical applications, functionalized furans play critical roles in synthetic materials [25], food chemistry [26], and as key intermediates in industrial chemical synthesis [27]. The strategic incorporation of polysubstituted furan frameworks continues to drive innovations in polymer materials through precise manipulation of functional group positioning and electronic effects [28].

Tetrasubstituted furans have attracted considerable research interest due to their versatile application potential, with synthetic methodologies demonstrating distinct features. In 2023, Trofimov et al. synthesized tetrasubstituted furans via the dimerization of alkynones catalyzed by t-BuOK in DMSO solution [29]. In 2022, Torres-Ochoa’s group developed a copper-mediated cyclization protocol, enabling the synthesis of 3-cyanofurans via high-temperature reactions between oxime esters and β-ketonitriles [30]. In 2023, Tadigoppula and coworkers reported a multimetallic catalytic system that facilitates oxidative cyclization of substrates at 120 °C using Ru, Cu, and Ag catalysts for polysubstituted furan production [31]. In 2010, Lin’s group selected triethylamine as the catalyst and developed a general procedure for synthesizing tetrasubstituted furans in mild conditions [32] (Scheme 1). Despite these developments, the reported synthetic methods exhibit notable limitations. Common challenges include substrate compatibility issues arising from bulky substituent requirements, energy-intensive high-temperature reaction conditions incompatible with thermally labile substrates, and the use of multiple catalytic systems that increase operational complexity and costs. These constraints highlight the continued need for developing sustainable synthetic routes to tetrasubstituted furans that combine mild reactions, excellent energy efficiency, and enhanced environmental compatibility.

The enzyme-catalyzed synthesis of tetrasubstituted furans presents significant advantages, including mild reaction conditions, environmental friendliness, superior energy efficiency, and enzyme reusability through immobilization technology. These advantages align closely with the principles of green chemistry, especially when compared to conventional chemical methodologies. In this study, we successfully synthesized monocyano-substituted tetrasubstituted furans through a three-component reaction system comprising benzoylacetonitriles, aldehydes, and benzoyl chlorides, employing Novozym 435 (a commercially available immobilized lipase) in conjunction with tributylphosphine as a co-catalyst. To our knowledge, this study presents the first example of lipase-catalyzed tetrasubstituted furan synthesis (Scheme 1).

2. Results and Discussion

2.1. Optimization of Conditions for the Reaction

Benzoylacetonitrile (1a), benzaldehyde (2a), and benzoyl chloride (3a) were selected as model substrates to screen for the optimal reaction conditions. Preliminary screening of enzymatic catalysis was conducted in ethanol at 37 °C (

Table 1. Effect of lipase types on the synthesis of 4a.

Entry	Lipase a	Yield (%) b
1	-	N.D. c
2	CSL	29
3	PSL	33
4	MML	42
5	PPL	70
6	CALB	82
7	Novozym 435	90
8	Novozym 435 d	N.D. c
9	Novozym 435 e	N.D. c
10	BSA	N.D. c

Reaction conditions: 1a (0.2 mmol), 2a (0.2 mmol), 3a (0.25 mmol), PBu₃ (0.25 mmol), lipase (2 mg, protein content), EtOH (3.0 mL), 37 °C, 12 h. ^a CSL (Candida sp. lipase); PSL (lipase from Pseudomonas sp.); MML (Mucor miehei lipase); CALB (C. antarctica lipase B); PPL (porcine pancreatic lipase); Novozym 435 (a commercial immobilized lipase B from C. antarctica); BSA (albumin from bovine serum); ^b Isolated yields. ^c Not detected. ^d Denatured Novozym 435 was obtained by heating Novozym 435 to 100 °C for 12 h in water. ^e Denatured Novozym 435 was obtained by treating Novozym 435 with phenylmethanesulfonyl fluoride (PMSF).

). Control experiments confirmed that no detectable formation of product 4a occurred under non-catalytic conditions (Table 1, entry 1). This result indicates the non-spontaneous nature of this multicomponent transformation at 37 °C. Comparative analysis of various lipase catalysts revealed distinct catalytic efficiencies. Among the tested lipases, CSL, PSL, and MML exhibited low reactivity, providing the corresponding 2,4,5-triphenylfuran-3-carbonitrile (4a) with low yields (Table 1, entries 2–4). In contrast, PPL demonstrated moderate reactivity, producing a 70% yield of the target product (Table 1, entry 5). Notably, the yield of 4a improved obviously when CALB and Novozym 435 were employed as catalysts (Table 1, entries 6–7). Novozym 435 (a commercial immobilized CALB) presented the best catalytic efficiency, achieving a 90% isolated yield of 4a (Table 1, entry 7). In addition, when the three-component reaction was catalyzed with thermally or chemically inactivated Novozym 435, no target product was produced (Table 1, entries 8–9), paralleling the inert behavior observed with BSA (Table 1, entry 10). These collective results substantiate that the observed catalytic activity is contingent upon the unique active site and spatial conformation of Novozym 435.

Solvent selection plays a crucial role in governing enzymatic reactions. Many enzymes are susceptible to structural denaturation and functional inactivation in organic solvents. Moreover, the physicochemical properties of the solvent medium critically influence the solubility of substrates or products, which directly determines both reaction kinetics and thermodynamic equilibria [33]. We employed Novozym 435 as the optimal lipase and conducted the screening of reaction solvents. Poor yields were obtained in nonpolar or low-polar solvents, including n-hexane and toluene, possibly due to the limited solubility of substrates in these solvents (

Table 2. Effect of solvents on the synthesis of 4a.

Entry	Solvent	Yield (%) a
1	n-Hexane	Trace
2	Toluene	10
3	Dichloromethane	35
4	Tetrahydrofuran	40
5	Ethyl acetate	48
6	Acetonitrile	57
7	Dimethyl sulfoxide	70
8	Ethanol	90
9	Water	N.D. b

Reaction conditions: 1a (0.2 mmol), 2a (0.2 mmol), 3a (0.25 mmol), PBu₃ (0.25 mmol), Novozym 435 (20 mg), solvent (3.0 mL), 37 °C, 12 h. ^a Isolated yields. ^b Not detected.

, entries 1–2). On the contrary, a higher yield of product 4a was acquired when polar solvents, such as dichloromethane, ethyl acetate, dimethyl sulfoxide, and ethanol, were selected as the reaction medium (Table 2, entries 3–8), with 4a achieving the highest isolated yield in ethanol (Table 2, entry 8). Notably, aqueous systems were excluded from consideration owing to the hydrolysis of benzoyl chloride in water, which precluded product formation (Table 2, entry 9). In addition, we optimized the co-catalyst and determined that PBu₃ was the best co-catalyst (Table S1).

Temperature is another critical factor influencing enzymatic reactions. According to Arrhenius theory, as temperature increases, the collision probability between reactant molecules and enzymes rises, leading to an increase in reaction rate. However, elevated temperatures also induce adverse effects, such as reduced enzyme stability and conformational changes in active sites, ultimately decreasing catalytic efficiency [34]. Systematic temperature gradient analysis (Figure 2) revealed that the yield of product 4a gradually increased with a rise in the temperature from 0 °C to 37 °C, demonstrating a progressive correlation between thermal energy input and biosynthetic output. However, as the temperature continues to rise, the yield of the target product 4a adversely decreases, and a sharp decline was observed at 60 °C. These results suggest that enzyme inactivation predominates at high temperatures. Therefore, 37 °C was selected as the optimal reaction temperature.

The dosage of enzymatic catalysts significantly influences reaction dynamics. When enzyme levels fall below optimal thresholds, substrate conversion rates decline due to reduced catalytic activity. Conversely, employing enzyme concentrations beyond stoichiometric requirements escalates operational expenditures. Therefore, we explored the effect of enzyme dosage. The optimization of Novozym 435 dosage was systematically investigated using benzoylacetonitrile (1a, 0.2 mmol), benzaldehyde (2a, 0.2 mmol), benzoyl chloride (3a, 0.25 mmol), and PBu₃ (0.25 mmol) in ethanol at 37 °C. As shown in Figure 3, within the enzyme dosage range of 0–30 mg, the yield of product 4a increased with the addition of enzyme. Notably, the yield enhancement became less pronounced between 20 and 30 mg, indicating that saturation conditions were being approached. When the dosage exceeded 30 mg (up to 50 mg), the yield of product 4a declined, likely due to enzyme aggregation resulting from excessive enzyme loading within the fixed solvent volume. Based on these findings, 20 mg was determined to be the optimal enzyme dosage for maximizing reaction efficiency.

2.2. Substrate Scopes for the Reaction

With the optimal conditions in hand, we investigated the scope of substrates for the Novozym 435-catalyzed one-pot three-component reaction. The enzymatic system demonstrated exceptional functional group tolerance, accommodating diverse aromatic and aliphatic components including benzoylacetonitriles, aldehydes, and benzoyl chlorides bearing varied substituents (

Table 3. Substrate scope for the Novozym 435-catalyzed synthesis of 4 [30,32].

Reaction conditions: 1 (0.2 mmol), 2 (0.2 mmol), 3 (0.25 mmol), PBu₃ (0.25 mmol), Novozym 435 (20 mg), EtOH (3.0 mL), 37 °C, 12 h. Yields were isolated yields.

). Surprisingly, electronic effects at the meta- and para-positions exhibited negligible influence on reaction efficiency, as evidenced by consistently high product yields (4b–4e, 4g–4l, 4r–4s, 83–94%). Steric hindrance emerged as the primary determinant for reactivity reduction. Due to the high steric, the reactivity of the substrate with substituents at the ortho-position (4f, 4m–4n, 4t, 80–82%) and 2-naphthalaldehyde (2p) was slightly reduced. Furthermore, both heteroaromatic aldehyde (2o) and aliphatic aldehyde (2q) participated effectively in the catalytic cycle, generating target tetrasubstituted furans with satisfactory isolated yield (4o, 92%; 4q, 80%). When acetyl chloride was used as the substrate in the reaction, the corresponding product was also obtained in a satisfactory yield (4u, 87%). These results underscore the remarkable versatility of Novozym 435 in facilitating the synthesis of tetrasubstituted furans contributing to the advancement of green chemistry and sustainable catalysis. The reusability of Novozym 435 was also investigated using the model reaction under the optimized conditions. The yield of 4a remained unchanged during the first four cycles. This finding aligns with the initial catalytic process results. The fifth to sixth cycles showed a gradual decrease in the yield of 4a. However, the reduction was still within an acceptable range (Figure S1).

2.3. Control Experiments and Mechanism Study of the Reaction

A series of control experiments were conducted to provide a more comprehensive elucidation of the mechanism and potential pathways involved in this one-pot three-component reaction. Without the participation of Novozym 435, benzoylacetonitrile (1a) and benzaldehyde (2a) could not spontaneously form (E)-2-benzoyl-3-phenylacrylonitrile III in ethanol at 37 °C (Figure 4a). In contrast, the intermediate III was generated with 93% yield in the presence of Novozym 435, highlighting its indispensable catalytic contribution to this transformation (Figure 4b). In a subsequent step, we found that the generation of the target product (4a) required a synergistic action between PBu₃ and Novozym 435. Neither PBu₃ nor Novozym 435 alone was sufficient to promote the completion of the corresponding process (Figure 4c,d). Conversely, the concomitant employment of PBu₃ and Novozym 435 successfully yielded 4a with a 90% yield (Figure 4e). These results provide compelling evidence for the essential role of cooperative catalysis involving PBu₃ and Novozym 435 within the proposed catalytic cycle.

Based on the experimental results, we propose a plausible mechanism for the Novozym 435-catalyzed one-pot three-component reaction (Figure 5). Initially, under the catalytic triad of Novozym 435, benzoylacetonitrile (1a) is deprotonated to form the corresponding carbon anion intermediate I. Subsequently, the nucleophilic carbon anion I attacks benzaldehyde (2a), generating intermediate II. Intermediate II undergoes dehydration, facilitated by the catalytic triad, yielding intermediate III. In the next step, zwitterionic adduct IV is formed from intermediate III and PBu₃ through Michael addition. Following this, the oxygen anions of adduct IV attack benzoyl chloride (3a), resulting in chloride ion migration and the formation of intermediate V. Intermediate V is then deprotonated again with the assistance of the Asp–His dyad of Novozym 435, leading to the generation of ylide intermediate VI. Finally, intermediate VI undergoes an intramolecular Wittig reaction, resulting in the formation of a tetrasubstituted furan product (4a) through cyclization.

3. Materials and Methods

3.1. General Information

CSL (Candida sp. lipase, ≥5000 LU/g), PSL (lipase from Pseudomonas sp., ≥20,000 U/g), MML (Mucor miehei lipase, ≥4000 U/mg), PPL (porcine pancreatic lipase, 15–35 U/mg), CALB (C. antarctica lipase B, 5000 LU/g), and BSA (albumin from bovine serum, >98%) were purchased from Shanghai Yuan Ye Biological Technology Company (Shanghai, China). Novozym 435 (10% protein content, ≥5000 U/g) was purchased from Sigma-Aldrich China Co. (Beijing, China). All other chemical reagents were purchased from commercial suppliers (Bide Pharmatech (Shanghai, China), Aladdin (Shanghai, China), and Energy Chemical (Beijing, China)). All commercially available reagents and solvents were used without further purification (the purity of all reagents ranged from 95% to 99%). Proton nuclear magnetic resonance (¹H NMR) spectra were recorded on a 400 MHz spectrometer in CDCl₃. Chemical shifts for protons are reported in parts per million downfield from tetramethyl silane (TMS) and are referenced to residual protium in the NMR solvent (CDCl₃ = δ 7.26 ppm). The NMR data are presented as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant in Hertz (Hz), integration. A 100 MHz spectrometer in CDCl₃ (δ 77.1 ppm) was used to report ¹³C NMR spectra. Mass spectra were recorded on the Bruker MicrOTOF Q II and an Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Scientifc, San Jose, CA, USA) coupled with HESI ion source. The experiments were performed in triplicate, and all data were obtained based on the average values.

3.2. Experimental Procedure for Lipase-Catalyzed Synthesis of 4

Novozym 435 (20 mg) was added into a mixture of benzoylacetonitrile (1, 0.2 mmol), aldehydes (2, 0.2 mmol), benzoyl chlorides (3, 0.25 mmol), and PBu₃ (0.25 mmol) in ethanol (3 mL) at 37 °C for 12 h. The progress of the reaction was monitored by TLC. After the reaction was completed, the reaction mixture was then concentrated in vacuo and purified by flash column chromatography (ethyl acetate/petroleum ether = 1/10–1/8) on silica gel to give products (4). All the isolated products were well characterized by their NMR and EI-MS.

4. Conclusions

A novel enzymatic co-catalytic methodology has been developed for synthesizing tetrasubstituted furans through a one-pot three-component reaction comprising benzoylacetonitriles, aldehydes, and benzoyl chlorides in an ethanol solution. The protocol employs Novozym 435 in conjunction with PBu₃ as dual catalytic systems, achieving good to excellent isolated yields of products (80–94%). This innovative approach demonstrates distinctive advantages including mild reaction conditions, environmentally sustainable characteristics, and high operational efficiency. Comprehensive control experiments established the indispensable roles of both Novozym 435 and PBu₃ in facilitating the transformation process. Through detailed mechanistic investigations, a plausible catalytic cycle involving sequential nucleophilic additions and the intramolecular Wittig reaction has been elucidated. These findings underscore the significant potential of integrating enzymatic catalysis with transition-metal-free strategies for developing sustainable methodologies in complex polyfunctionalized heterocycle synthesis.