A Novel Oxidation of Salicyl Alcohols Catalyzed by Lipase

Abstract

A novel and efficient oxidation of salicyl alcohols to the corresponding salicylaldehydes catalyzed by lipase is reported for the first time. Under the optimal reaction conditions, the method exhibited high yields (81–95%) and selectivities for salicylaldehydes. Moreover, this study expands the application of enzyme catalytic promiscuity in organic synthesis.

1. Introduction

Enzyme catalytic promiscuity refers to the properties of enzymes which can catalyze different types of chemical transformations in addition to their main physiological ones [1]. Thus as a side benefit of the enzyme, it may afford a new synthesis pathway and widen the utilization of the enzyme [2]. Lipases have attracted wide attention in this area due to their broad promiscuous catalytic abilities in many organic reactions [3,4,5,6,7]. Recently, lipase-catalyzed perhydrolysis of carboxylic acids or esters, resulting in the formation of reactive peroxycarboxylic acids, have been studied as interesting cases of lipase catalytic promiscuity [8]. The in situ generated peroxycarboxylic acids by perhydrolysis have been directly used to oxidize ketones, alkenes, sulfides, enaminones, and amines [9,10,11,12,13,14]. However, lipase-catalysis of other oxidative reactions has been less explored, especially with alcohols as starting substrates.

Salicylaldehyde is one of the most fundamental and widely used aldehydes being an ingredient in agricultural pesticides, chemicals, perfumes, polymers, and fibers. Beside the Reimer Tiemann process [15,16], salicylaldehydes can be obtained by oxidation of salicylic alcohols over metal catalysts, such as Au, Pd, Pt, CuCo/C, and alkaline hydroxide [17,18,19,20,21,22]. However, certain drawbacks have always existed for most of the reported methods, such as the utilization of metals, hazardous or toxic solvents, high temperature, and undesirable byproducts. Therefore, the oxidation of salicylic alcohols is still a big challenge from the environmental and economic point of view.

As part of our studies on the development of lipase catalytic promiscuity, we focused on the lipase-catalyzed oxidation of salicyl alcohols to salicylaldehydes (Scheme 1). To the best of our knowledge, the lipase-catalyzed oxidation of salicyl alcohols to salicylaldehydes has not yet been reported.

2. Results and Discussion

Based on previous reports [23], Novozym 435 (a commercial immobilized Candida antarctica lipase B) has usually displayed excellent activities in these lipase-catalyzed oxidations. Hydrogen peroxide, a useful oxidant in many biocatalytic reactions, can seriously inactivate enzymes. Many methods have been explored to avoid the enzyme inactivation [24,25]. In this study, urea-hydrogen peroxide complex (UHP) was adopted as oxidizing agent for its controllable liberation of hydrogen peroxide which could help to avoid the inactivation of the enzyme in lipase-catalyzed oxidations [26]. Initially, we selected salicyl alcohol (1a) as the substrate of the lipase-catalyzed oxidation and n-caprylic acid as the peracid precursor (in situ perhydrolysis catalyzed by lipase). As shown in

Table 1. Oxidation of salicyl alcohol to salicylaldehyde catalyzed by lipase. ^a mCPBA: meta-chloroperoxybenzoic acid; TEMPO: 2,2,6,6-tetramethyl-1-piperidinyloxy; BSA: Bovine serum albumin.

Entry	Catalyst	Additive	Yield of 2a (%) b
1	Novozym 435	None	18
2	mCPBA	None	5
3	TEMPO	None	ND c
4	Novozym 435	TEMPO	91
5	mCPBA	TEMPO	52
6	Novozym 435 d	TEMPO	ND
7	Novozym 435 e	TEMPO	ND
8	BSA	TEMPO	ND

^a Reaction conditions: 1a (1 mmol), acetonitrile (5 mL), UHP (1.1 mmol), n-caprylic acid (0.2 mmol), additive (0.05 mmol), Novozym 435 (200 U), room temperature, 2 h; ^b isolated yield; ^c ND: not detected; ^d Inactivated by PMSF (phenylmethanesulfonyl fluoride). ^e Pretreated Novozym 435 by heating it for 1 h in boiling water.

, the yield of the lipase-catalyzed oxidation was poor (Entry 1), though salicylaldehyde (2a) was obtained as the only product. Only 5% yield of 2a was achieved when mCPBA (meta-chloroperoxybenzoic acid, a commercial organic peracid, 1 equivalent) was used as the oxidant (Entry 2). TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) is always introduced for the oxidation of alcohols as a highly efficient oxidative catalyst or an additive combined with peracid [27,28,29]. It was observed that the reaction did not occur when TEMPO (1 equivalent) was used in this reaction (Entry 3). Meanwhile, the yield of salicylaldehyde was dramatically increased to 91% by using TEMPO (5 mol%) as the additive in the lipase-catalyzed oxidation (Entry 4). It is worth noting that the lipase-catalyzed oxidation presented a higher yield than that catalyzed by the combination of mCPBA and TEMPO (Entry 5). To certify the role of the active center of lipase in this oxidation, a serine-specific inhibitor (phenylmethanesulfonyl fluoride, PMSF) was utilized to inactivate Novozym 435 according to our previous report [9]. No oxidation of salicyl alcohol was observed when Novozym 435 inactivated by PMSF was used as catalyst (Entry 6). This result showed that the active center of lipase was directly involved in the reaction. Furthermore, the denatured Novozym 435 (Entry 7) and Bovine serum albumin (BSA, Entry 8) did not exhibit activity, which suggests that a specific conformation of lipase is necessary for the in situ generation of peracid.

Based on the experimental results, a possible reaction mechanism for this lipase-catalyzed oxidation of salicyl alcohol was proposed (Scheme 2). First, TEMPO bound to the oxyanion hole and was activated to form the active intermediate (TEMPO⁺). Secondly, TEMPO⁺ oxidized salicyl alcohol to salicylaldehyde. Finally, TEMPOH was formed along with the product, which was converted back to TEMPO by the peracid generated by the lipase-catalyzed perhydrolysis of n-caprylic acid. Then, the system was ready for another round of salicyl alcohol oxidation.

In this lipase-catalyzed system, two competitive reactions were involved in the conversion of salicyl alcohol. One reaction was the perhydrolysis of carboxylic acid catalyzed by lipase to generate peracid, where the in situ generated peracid oxidized salicyl alcohol to produce salicylaldehyde. The other reaction was the esterification of salicyl alcohol with the carboxylic acid catalyzed by lipase. Therefore, the carboxylic acid used as the peracid precursor plays a key role in the lipase-catalyzed reaction. As shown in

Table 2. The effect of carboxylic acid on the oxidation of salicyl alcohol ^a.

Entry	Carboxylic Acid	Yield of 2a (%) b	Yield of Ester (%) b
1	Acetic acid (0.2 mmol)	16	17
2	n-Butyric acid (0.2 mmol)	42	12
3	n-Hexanoic acid (0.2 mmol)	63	9
4	n-Caprylic acid (0.2 mmol)	91	ND c
5	n-Caprylic acid (0.1 mmol)	69	ND
6	n-Caprylic acid (0.3 mmol)	92	ND
7	n-Caprylic acid (0.4 mmol)	94	ND

^a Reaction conditions: 1a (1 mmol), acetonitrile (5 mL), UHP (1.1 mmol), TEMPO (0.05 mmol), Novozym 435 (200 U), room temperature, 2 h; ^b isolated yield; ^c ND: not detected.

, the yield of 2a was gradually increased when the alkyl chain of the carboxylic acid was elongated, and the carboxylic acids with shorter alkyl chains were preferred as acylation agents rather than the peracid precursors (Entries 1–4). In principle, water can inhibit the acylation in lipase catalyzed esterification. However, the conversion of 1a decreased gradually, and no apparent effect on the oxidative selectivity of 1a could be observed in this reaction system even when the water content varied from 0 to 50% v/v in acetonitrile (data not shown). Furthermore, the optimal amount of n-caprylic acid was also screened (Entries 4–7). When the amount of n-caprylic acid was 2 mmol, a satisfactory yield of 2a could be obtained. Further increasing the amount of n-caprylic acid did not affect the reaction obviously. More importantly, no esterification occurred when the amount of n-caprylic acid varied from 0.1 mmol to 0.4 mmol. Therefore, 2 mmol was selected as the optimal amount of n-caprylic acid in this study.

Generally, the reaction medium can influence the catalytic performance of an enzyme. In this study, we used several organic solvents and the results in Figure 1 indicated that acetonitrile was the best choice between dichloromethane, tetrahydrofuran, dimethyl sulfoxide, dioxane, and water. Some literature references reported that ethyl acetate could be adopted as reaction medium and peracid precursor in a lipase-catalyzed oxidation [26,30]. However, it was disappointing that only the acetate of 1a was obtained instead of 2a when ethyl acetate was used as the reaction medium. Therefore, acetonitrile was selected as the reaction medium for this lipase-catalyzed oxidation.

The dosage of TEMPO and lipase was also investigated in this study. The effect of the dosage of TEMPO is outlined in Entries 1–4 of

Table 3. The effect of dosage of TEMPO and lipase on the oxidation of salicyl alcohol. ^a

Entry	Dosage of TEMPO (mol%)	Dosage of Enzyme (U)	Yield of 2a (%) b
1	1	200	57
2	3	200	76
3	5	200	91
4	7	200	92
5	5	50	32
6	5	100	53
7	5	150	84
8	5	250	92

^a Reaction conditions: 1a (1 mmol), acetonitrile (5 mL), UHP (1.1 mmol), n-caprylic acid (0.2 mmol), room temperature, 2 h; ^b isolated yield.

. The yield of 2a was increased by increasing the dosage of TEMPO from 1 mol% to 5 mol%. However, the yield did not obviously improve by further increasing the dosage of TEMPO. In addition, the yield of 2a increased as the dosage of lipase was elevated from 50 to 200 U, followed by a slight change of yield at higher dosage of lipase. As for the oxidant, we fixed the amount of UHP at 1.1 mmol due to the instability of salicylaldehyde exposed to excessive peracid [31].

Generally, the immobilized enzyme is easy to recover and reuse, which makes the enzymatic process economically viable [32,33,34]. In this work, Novozym 435 can be easily recovered after each run by filtration. The recovered Novozym 435 was then reused directly for the oxidation of 1a under the same conditions. It is demonstrated in Figure 2 that 82% oxidation yield of 2a could be obtained even after six reaction cycles. Furthermore, with the optimized reaction conditions to hand, we scaled up the lipase-catalyzed oxidation 50-fold (1a (50 mmol), acetonitrile (250 mL), Novozym 435 (10,000 U), TEMPO (2.5 mmol), and UHP (55 mmol)) and the yield of 2a was up at 93% after 2 h. These results demonstrated that this lipase-catalyzed method has high potential for industrial production.

To explore the scope and limitation of the method, we used a panel of substituted salicylic alcohols as the substrates in the lipase-catalyzed oxidation (

Table 4. The oxidation of substituted salicylic alcohols catalyzed by lipase.

Reaction conditions: 1 (1 mmol), acetonitrile (5 mL), UHP (1.1 mmol), n-caprylic acid (0.2 mmol), TEMPO (0.05 mmol), Novozym 435 (200 U), room temperature, 2 h; isolated yield.

). It was found that all the substituted salicylic alcohols could be oxidized to the desired salicylaldehydes in high yields (81–95%), and the yields of salicylaldehydes were higher when salicyl alcohols bearing electron donating groups were adopted as the substrates (1b and 1f). It is noteworthy that no ester of salicyl alcohol was observed in the oxidation. Therefore, this method is a mild, efficient, and highly selective means for the synthesis of salicylaldehydes. Furthermore, some substituted benzyl alcohols were also investigated as substrates at the same reaction conditions. However, only benzyl alcohol could be totally converted to benzaldehyde, and other substituted benzyl alcohols afforded both corresponding benzaldehydes and esters (data not shown). Further investigation on the selective oxidation of other benzyl alcohols is currently ongoing in our laboratory.

3. Materials and Methods

3.1. Materials

Novozym 435 (15,000 U/g) and Bovine serum albumin were purchased from Sigma (Beijing, China). One unit (U) of enzyme activity was defined as the amount of enzyme that hydrolyzes 1 μmol of 4-nitrophenyl acetate per min at 30 °C. Silica gel was purchased from Qing Dao Hai Yang Chemical Industry Co. (Qingdao, China). The chemical reagents were of analytical reagent grade and purchased from J & K Scientific Ltd. (Beijing, China). NMR spectra were taken with an Inova 500 (500 MHz) spectrometer (Vernon Hills, IL, USA).

3.2. A Typical Lipase-Catalyzed Oxidation of Salicyl Alcohol

A mixture of UHP (1.1 mmol), n-caprylic acid (0.2 mmol), TEMPO (0.05 mmol), Novozym 435 (200 U), in acetonitrile (5 mL) was mixed for 5 min in a 25 mL round-bottom flask, and then salicyl alcohol (1 mmol) was added to the reaction system. The mixture was stirred for 2 h at room temperature. Then the reaction mixture was filtered and the residue was washed with acetonitrile. The combined organic phases were concentrated under vacuum, and the resulting residue was purified by flash column chromatography on silica gel with ethyl acetate /hexane (1/20) to afford the desired salicylaldehyde. All the isolated products were well characterized by the ¹H-NMR spectral analysis. The experiments were performed in triplicate, and all data were obtained based on the average values.

3.3. ¹H-NMR of Products

The spectra of products 2a–2g were displayed in the Supplementary Materials.

2a: ¹H-NMR (500 MHz, CDCl₃) δ: 6.97–7.02 (m, 2H), 7.50–7.55 (m, 2H), 9.87 (s, 1H), 11.03 (s, 1H).

2b: ¹H-NMR (500 MHz, CDCl₃) δ: 3.92 (s, 3H), 6.97 (t, J = 6.5 Hz, 1H), 7.12 (dd, J = 1.0 Hz, 6.5 Hz, 1H), 7.18 (dd, J = 1.0 Hz, 6.5 Hz, 1H), 9.91 (s, 1H), 11.1 (s, 1H).

2c: ¹H-NMR (500 MHz, CDCl₃) δ: 6.97 (t, J = 6.5 Hz, 1H), 7.17 (dd, J = 1.0 Hz, 6.5 Hz, 1H), 7.21 (m, 1H), 9.91 (s, 1H), 11.11 (s, 1H).

2d: ¹H-NMR (500 MHz, CDCl₃) δ: 6.98 (d, J = 7.5 Hz, 1H), 7.49 (dd, J = 2.0 Hz, 7.5 Hz, 1H), 7.55 (d, J = 7.5 Hz, 1H), 9.86 (s, 1H), 10.93 (s, 1H).

2e: ¹H-NMR (500 MHz, CDCl₃) δ: 7.00 (dd, J = 3.5 Hz, 7.5 Hz, 1H), 7.26–7.31 (m, 2H), 9.88 (s, 1H), 10.8 (s, 1H).

2f: ¹H-NMR (500 MHz, CDCl₃) δ: 2.36 (s, 3H), 6.91 (d, J = 7.5 Hz, 1H), 7.35 (s, 1H), 7.35–7.37 (m, 2H), 9.87 (s, 1H), 10.85 (s, 1H).

2g: ¹H-NMR (500 MHz, CDCl₃) δ: 7.15 (d, J = 7.5 Hz, 1H), 8.44 (dd, J = 2.0 Hz, 7.5 Hz, 1H), 8.59 (d, J = 2.0 Hz, 1H), 10.03 (s, 1H), 11.63 (s, 1H).

4. Conclusions

In summary, this is a first report on the efficient lipase-catalyzed oxidation of salicylic alcohols to salicylaldehydes under mild conditions. Under the optimal reaction conditions, the salicylic alcohols could be selectively converted to the salicylaldehydes in high yields (81–95%). Meanwhile, Novozym 435 could be reused six times without significant loss of activity which indicated that the method has great potential for the preparation of salicylaldehydes. Generally, immobilization is a powerful technique to enhance the reusability and stability of enzyme [35,36,37,38]. Further study to improve the enzyme properties (mainly stability) of this lipase-catalyzed oxidation by using other immobilization strategies or even coupling immobilization and chemical modification is underway and will be reported in due course. Moreover, this study provides a new case of lipase-catalyzed oxidation and expands the utilization of lipase in organic synthesis.