Enzymatic Synthesis of Thioesters from Thiols and Vinyl Esters ... - MDPI

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Enzymatic Synthesis of Thioesters from Thiols and Vinyl Esters in a Continuous-Flow Microreactor Nani Zhou 1 1

2

*

ID

, Le Shen 1

ID

, Zhen Dong 1

ID

, Jiahong Shen 1

ID

, Lihua Du 1, * and Xiping Luo 2, *

College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, China; [email protected] (N.Z.); [email protected] (L.S.); [email protected] (Z.D.); [email protected] (J.S.) Department of Environmental Science and Technology, Zhejiang A&F University, Hangzhou 311300, China Correspondence: [email protected] or [email protected] (L.D.); [email protected] (X.L.); Tel.: +86-571-189-6906-9399 (L.D.)

Received: 16 May 2018; Accepted: 14 June 2018; Published: 16 June 2018

Abstract: The preparation of thioesters through the lipase-catalysed transesterification reaction of thiols with vinyl carboxyl esters is described. The reactions were carried out by Lipase TL IM from Thermomyces lanuginosus as a catalyst and performed under a continuous flow microreactor. We first found that lipase TL IM can be used in the reaction of thioester synthesis with high efficiency. Various reaction parameters were investigated including substrate molar ratio, reaction time, and temperature. Maximum conversion (96%) was obtained under the optimal condition of a substrate molar ratio of 1:2 (4-methylbenzyl mercaptan:vinyl esters) at 50 ◦ C for about 30 min. Compared with other methods, the salient features of this work include mild reaction conditions (50 ◦ C), short reaction times (30 min), high yields, and environment-friendliness. Keywords: enzymatic synthesis; thioester synthesis; flow chemistry; microreactor

1. Introduction Flow chemistry [1–7] has attracted significant attention from researchers from both academia and industry. Various benefits over conventional batch processes include increased controllability, safety, and selectivity because of improved heat and mass transfer and shorter residence times. In particular, utilizing immobilized enzyme catalysts in microreactors is recognized as the most prioritized consideration of key green engineering areas for chemical synthesis [8–14]. In recent years, there has been demand for enzymatic microreaction devices in several fields, especially for biotransformation. Several enzymatic syntheses in microreactors have been reported [15–20]. Thioesters are important building blocks for organic synthesis [21] and chemical biology [22]. They are valuable intermediates in food, medicinal, and cosmetic chemistry, and in the production of new materials [23–25]. Many works on the synthesis of thioesters have been reported [26–31]. The most common catalysts reported for organic transformation of thioesters are triflates [32], Lewis acids [33], or lanthanide catalysts [34], etc. Unfortunately, with very few exceptions, all of these methods implicate toxic and hazardous reagents, harsh conditions, or uncommon starting materials. Therefore, it is important to find a new, simple, and environmentally friendly method for the preparation of thioesters. Biocatalysis as an efficient and green biotransformation tool in organic synthesis that has attracted much attention of chemists and biochemists [35–38]. Especially, catalytic promiscuity in biocatalysis, which means using old enzymes to form new bonds and follow new pathways, was greatly extended and expanded rapidly [39–41]. Some enzymes, such as D-amino acylase from Escherichia coli (DA), have been applied to the synthesis of thioesters, but it requires a longer reaction time (48 h) to achieve the desired result [42]. Additinoally, only amino acylase was suitable for the synthetic reaction of Catalysts 2018, 8, 249; doi:10.3390/catal8060249

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time (48 h) to achieve the desired result [42]. Additinoally, only amino acylase was suitable for the time (48 h) to achieve the desiredwhile resultlipase [42]. Additinoally, amino acylase wasthe suitable for to the synthetic reaction of thioesters, from Candidaonly antarctica catalysed reaction synthetic reaction of thioesters, while lipase from Candida antarctica catalysed the reaction to thioesters, while lipase from Candida antarctica catalysed mild the reaction to Markovnikov In of the Markovnikov addition. In the interest of developing methodologies for theaddition. synthesis Markovnikov addition.mild In the interest developing mildofmethodologies for themicroreactor synthesis of thioesters, envisaged modifying our of procedure to achieve a continuous flow interest ofwe developing methodologies for the synthesis thioesters, we envisaged modifying thioesters, we envisaged modifying our procedure to achieve a continuous flow microreactor protocol for thetosynthesis thioesters.flow Specifically, we protocol directed for ourtheattention the our procedure achieve a of continuous microreactor synthesistowards of thioesters. protocol forwe ofmicroreactor thioesters. we directed our attention towards the development ofthe ansynthesis enzymatic strategy involving TL IM from Thermomyces Specifically, directed our attention towardsSpecifically, the development of lipase an enzymatic microreactor strategy development anIM enzymatic microreactor lipase TL IM aaim from lanuginosus as a of catalyst (Scheme 1). The aim of strategy this paper is to investigate, under continuous-flow involving lipase TL from Thermomyces lanuginosus as ainvolving catalyst (Scheme 1). The ofThermomyces this paper is lanuginosus as a catalyst (Scheme 1). The aim of this paper is to investigate, under a continuous-flow microreactor, the effectaof various reaction parameters the on the reaction conversion. to investigate, under continuous-flow microreactor, effect of various reaction parameters on the microreactor, the effect of various reaction parameters on the reaction conversion. reaction conversion.

Scheme 1. Enzymatic synthesis of thioesters from thiols and vinyl esters in a continuous flow microreactor. Scheme Scheme 1. 1. Enzymatic Enzymatic synthesis synthesis of of thioesters thioesters from from thiols thiols and and vinyl vinyl esters esters in in aa continuous continuous flow flow microreactor. microreactor.

2. Results and Discussion 2. 2. Results Results and and Discussion Discussion 2.1. Experimental Setup 2.1. 2.1. Experimental Experimental Setup Setup The enzymatic synthesis of thioesters from thiols and vinyl esters in a continuous-flow The enzymatic synthesis of thioesters from thiols and vinyl in aacontinuous-flow continuous-flow The enzymatic synthesis thioesters from thiols and vinyl in aesters continuous-flow microreactor microreactor is described in of Figure 1. We first examined theesters reactions using microreactor is described in Figure 1. We first examined the reactions using a continuous-flow is described in Figure 1. We first examined the reactions using a continuous-flow microreactor system microreactor system composed of a Y-shaped micromixer (Ф = 1.8 mm; M) and a microtube reactor microreactor system composed of a Y-shaped micromixer (Ф = 1.8 mm; M) and a microtube reactor composed a Y-shaped micromixer = 1.8 coil mm;(2M) andI. aD.) microtube reactor (R) shown in Figure (R) shown inofFigure 1. A 3.1 mL PFA (Φ reactor mm was constructed and reagents were1. (R)3.1 shown in two Figure 1. A (2 3.1 mL reactor coilfeed (2 mm D.) with waswere constructed andbyreagents were A mL PFA reactor coil mm I.PFA D.) was constructed introduced tworeagent separate introduced by separate feed streams. Reagent 1and (10I.reagents mL) the thiol solution and introduced by two separate feed streams. Reagent feed 1 (10 mL) with the thiol solution and reagent feed2streams. feedesters 1 (10 mL) the thiol and reagent feed 2 (10 mL) vinyl feed (10 mL)Reagent with vinyl werewith mounted in solution DMSO, respectively. Lipozyme TL with IM from feed (10 mL) withinvinyl esters were mounted in respectively. Lipozyme TL filled IM from esters2were mounted DMSO, respectively. Lipozyme IM from Thermomyces lanuginosus (catalyst Thermomyces lanuginosus (catalyst reactivity: 250 IUN· g−1DMSO, ,TL particle diameter: 0.3–1.0 mm) was in −1 − 1 Thermomyces lanuginosus (catalyst reactivity: IUN· g was , temperature particle mm) filled in reactivity: 250(R). IUN g , particle diameter: mm) filleddiameter: in the (R). water bath the microtube A ·water bath was applied 0.3–1.0 to250 control the of microtube this0.3–1.0 reaction byAwas immersion the microtube (R). A water bath was applied to control the temperature of this reaction by immersion applied temperature of this reaction by found immersion in water. Aftercould initial ofwas reactor coiltoincontrol water.the After initial optimization, it was that of thereactor targetcoil thioesters (3a–l) reactor coil water. After initial optimization, it was found that the (~86%) target thioesters (3a–l) could optimization, itinwas found thatminutes the target thioesterstime, (3a–3l) could be obtained, after onlyseparation a thirty minutes beof obtained, after only a thirty residence in excellent yield after and be obtained, after only a thirty minutes residence time, in excellent yield (~86%) after separation and residence time, in excellent yield (~86%) after separation and purification (Table 1). purification (Table 1). purification (Table 1).

Figure1.1.Experimental Experimentalsetup setupforforthe thesynthesis synthesisofofthioesters thioesterscatalysed catalysedbybyLipozyme LipozymeTL TLIM IMfrom from Figure Thermomyces lanuginosus in a continuous-flow microreactor. Figure 1. Experimental setup for the synthesis of thioesters catalysed by Lipozyme TL IM from Thermomyces lanuginosus in a continuous-flow microreactor. Thermomyces lanuginosus in a continuous-flow microreactor.

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Table 1. Shaker flow synthesis of thioesters catalysed by Lipozyme TL IM. Table 1. Shakerand and continuous continuous flow synthesis of thioesters catalysed by Lipozyme TL IM. Catalysts 2018, 8, x FOR PEER REVIEW

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Table 1. Shaker and continuous flow synthesis of thioesters catalysed by Lipozyme TL IM. Table 1. Shaker and continuous flow synthesis of thioesters catalysed by Lipozyme TL IM.


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Catalysts 2018,1.8,Shaker x FOR PEER REVIEW flow synthesis of thioesters catalysed by Lipozyme TL IM. Table and continuous

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Catalysts 2018, 8, x FOR PEER REVIEW a b a b Catalysts 2018,1.8,Shaker x FOR PEER REVIEW flow synthesis Table and continuous of thioesters catalysed by Lipozyme TL IM.

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Table 1. Shaker and continuous flow synthesis of thioesters catalysed by Lipozyme TL IM.

Entry Entry

Product Product

c c Time 3 of 12Yield Yield(%) (%) MethodMethod Time a b c Catalysts 2018,1. 8, x FOR PEER REVIEW 3 of 12 Table Shaker and continuous flow synthesis of thioesters catalysed by Lipozyme TL IM. Entry Product Method Time BYield (%) 30 min 65 Table Shaker and continuous flow synthesis of thioesters catalysed by Lipozyme TL IM. Catalysts 2018,1. 8, x FOR PEER REVIEW 3 of 12 a b c Entry Product Method Time B 30 min Yield 65 (%) 30 min 3 of 12 65 Catalysts 2018,1.8,Shaker x FOR PEER REVIEW Table and continuous flow synthesis of thioesters catalysedBby Lipozyme TL IM. B b 30 min Yield 65c (%) a Entry Product Method Time 1 Table 1. Shaker and continuous flow synthesis of thioesters catalysed by Lipozyme TL IM. Catalysts 2018, 8, x FOR PEER REVIEW 3 of 12 1 1 A 24 h 60 A 24 h 60 B b by30 min Table 1. Shaker and continuous flow synthesis of thioesters catalysed Lipozyme TL 65 IM. c (%) h 1 Entry Product a Method Yield A Time 60 A 24 h 60 24

Table 1. Shaker and continuous flow synthesis of thioesters catalysed by Lipozyme TL IM.

1 Entry Entry 1 Entry 2

12 2 2Entry

Entry 1 2 1 Entry Entry 312 1 213 3 132 3 2 423 4 32 243 23 4 4 543 435 354 34 5 64 5 5 546 4 5 465 56 57 657 6 6 56 76 6 7 68 768 67 7 87 7 78 79 879 798 8 8 89 8 10 8 98 10 89 10 9 9 10 99 9 11 10 11 9 10 9 11 10 10 11 10 10 10 11 1110 10 11 11 11 11 11 11

Product a Product a Product a Product a Product a Product a Product a

11

B b 30 min Yield 65c Method Time B 30 88 (%) A 24min h B 60 30 min b c (%) Method Time B min 65 B bB 30 30 min Yield 8830 c (%) min Method Time Yield A 24min h 60 B 30 65 A 24min h 78c B 88 Method Time Yield B b 30 30 min 65 (%) A 24 h 60 A 78c (%)24 h Method Time B bA 30 min Yield 65 24 h B b 30 30 min 88 A 24min h 60c (%) B 81 Method Time Yield A 24min h 78 B 30 65 A 24 h 60 b 81 B 30 min 88 Method Time B 30 min Yield 65c (%) B 30 min A 24 h 60 78 A h 76 B 3024min min 88 B 65 81 B 30 30 min A 24 h 60 B 30 min 88 A 24 h 76 A 24 78 A 24 h h 60 B 30 min A 91 88 81 24 h 78 A 24 h h 76 A 60 B 3024 min 88 91 A A 24 h 78 24 h B 30 min 81 A h 85 B 3024 min 88 76 A h 78 B 3024min 91 81 A 24min h B 85 30 min B 30 88 A 24 h 78 B 3024 min 81 A h 76 B 30 min 80 A 24 h 78 B 30 min 91 B 30 min A h 85 B 3024min 81 A h 76 B 3024min 80 24 h A h A 78 76 81 B 3024min 91 85 A 24 h 75 80 B 81 A h 76 B 3024min min 91 24 h A 30 A 24 h 75 A h B 85 76 91 B 3024min 81 B 30 min 80 30 min A h 76 85 91 B 3024 94 A 24min h 75 B 30 80 85 91 A h 76 B 3024min min 94 B 30 min 75 A 24min h A 85 B 3024 91 80 24 h A h 90 B 30 min 94 B 3024 min 80 A 24min h 85 B 30 91 A h 75 A 24 h 90 A h 85 B 3024 min 80 A 30 B min 94 A 24min h 75 24 h B 3024 88 A 24 h 90 B 30 min 80 A h 85 A 24min h B 75 30 min B 30 94 88 B 30 min 80 A 24 h 75 A h 90 B 3024min min 94 B 80 8630 min B 30 B 30 88 A 24min h 75 B 3024 min 94 A h 90 A 24 h A 75 86 24 h B 3024min min 94 B 30 88 A h 90 A h 75 96 A 24 h 86 B min 94 A 3024 24 h A 24 h 90 B 30 88 B 3024min min 94 96 A h 90 A h B 86 B 3024min min 88 30 min B 30 94 95 A 24 h 90 B 30 96 B 30 min min 88 A 24 86 B 30 min A 24 h h 90 95 B 30 min 88 A 24min h 86 B 30 min 96 A 24 h 90 B 30 62 B 30 min 88 A 24 h 95 A 24 h A 24 h 86 B 96 62 24 h 88 A 3024min A h 86 A 24 h 95 B 3024min min 96 B 30 88 A h 58 B 30 62 A 24min h 86 B 3024min 96 A 95 A 24 h h B 58 30 min 86 B 30 96 B 3024min min 62 A h 95 B 30 A h 86 B min 7530 min A 24min h 58 B 3024 96 A h 95 B 3024min 62 75 B 30 min 96 A h 95 h A 24min h A 58 B 3024 62 2424 A 30 h B 96 67 A 24min h 95 B 30 min 75 B 3024 min 62 A h 58 67 A h 95 B 3024min 62 A h B 58 B 3024min 75 A h 95 B 3024 min 67 30 min 62 A 24 h A 24 h 5830 min B 30 B min 75 62 B 30 min 67 A 24 h 58 A 24min h 67 B 3024 75 B 30 62 A h A 24min h 58 B 30 min 67 B 30 min 75 A A 67 A 24 h h 62 2424 A 24 h h 58 B 30 75 B 30 min 67 A 24min h A 24 h 58 62 B 30 min 75 A h 67 B 3024min 67 B min 75 min A 24 h 67 B 30 3030 min A 24min h B 62 B 30 67 B 30 min 75 A 24min h 67 B 30 67 A 24 62 A 24 h h 67 B 67 A 24min h 62 2424 A 3024 h h A h A 67 B 30 min 67 A 24 h 62 B 30 min 67 A 24 h 62 B 67 A h B 623030 min B 3024min min A 24 h 62 A 24 h 62

A

B

12 12

A

B 30 min A

2424 h h

A


24 h

8230 79

4 of 12

min

24 h

88 78 81 76

88 78 81 76

91 85 80 75

91 85 80 75

94 94

90 90

88 88

86 96 95 62

86 96 95 62

58 58 75 75 67 67 67 67 62 62 82 79

Reactions and the structure of the products (3a–3l) see Scheme 1. b Method A: Shaker reactor, benzyl a Reactions and the structure of the products (3a–3l) see Scheme 1. b Method A: Shaker reactor, benzyl mercaptan mercaptan (1 mmol) and vinyl esters (3 mmol) were added to 5 mL DMSO at 50 °C. Lipozyme TL IM ◦ C. −1 feed (0.22 g, 44 mgesters mL−1), 24(3 h. mmol) Method B:were continuous flow to microreactor, 10.4 µ L min 1 (4 mmol thiol TL IM (0.22 g, 44 mg mL−1 ), (1 mmol) and vinyl added 5 mL DMSO at 50 Lipozyme 1 feed derivatives in 10 mL DMSO) andmicroreactor, 10.4 µ L min−1 feed10.4 2 (8 mmol vinyl − esters in 101mL 50 °C derivatives in 10 mL DMSO) 24 h. Method B: continuous flow µL min (4DMSO) mmolatthiol −1 c Isolated yield. (residence 30 2 min), Lipozyme TL IMesters (0.87 g, in 44 mg and 10.4 µL min−1time feed (8 mmol vinyl 10 mL mL).DMSO) at 50 ◦ C (residence time 30 min), Lipozyme TL IM a

(0.87 g, 44 mg mL−1 ). c Isolated yield.

2.2. Molar Ratio (Thiols:Vinyl Esters) Effect We begin to explore the effects of various reaction parameters on the lipase-catalysed transesterification reaction of thiols with vinyl esters performed under a continuous flow microreactor. In our initial investigation, in order to ascertain the effect of molar ratio (thiols:vinyl esters) on the thioesters synthesis reaction in a microreactor, the thioester synthesis reaction of benzyl mercaptan and vinyl laurate was used as a model reaction. We tried substrate molar ratios (benzyl mercaptan:vinyl laurate) from 1:1 to 1:4. As we can see from Figure 2, the reaction conversion is 61% when the substrate molar ratio is 1:1. With the increase of vinyl laurate, the reaction conversion increases, too. The best result was obtained when the molar ratio reached 1:2. Considering the optimal

Reactions and the structure of the products (3a–3l) see Scheme 1. b Method A: Shaker reactor, benzyl mercaptan (1 mmol) and vinyl esters (3 mmol) were added to 5 mL DMSO at 50 °C. Lipozyme TL IM (0.22 g, 44 mg mL−1), 24 h. Method B: continuous flow microreactor, 10.4 µL min−1 feed 1 (4 mmol thiol derivatives in 10 mL DMSO) and 10.4 µL min−1 feed 2 (8 mmol vinyl esters in 10 mL DMSO) at 50 °C Catalysts 2018, 8, 249 4 of 12 (residence time 30 min), Lipozyme TL IM (0.87 g, 44 mg mL−1). c Isolated yield. a

2.2. Molar Esters) Effect Effect 2.2. Molar Ratio Ratio (Thiols:Vinyl (Thiols:Vinyl Esters) We begin begin to to explore explore the the effects effects of of various various reaction reaction parameters parameters on on the the lipase-catalysed lipase-catalysed We transesterification reaction reactionof thiols of thiols vinyl performed esters performed under a flow continuous flow transesterification with with vinyl esters under a continuous microreactor. microreactor. In our initial investigation, in order to ascertain the effect of molar ratio (thiols:vinyl In our initial investigation, in order to ascertain the effect of molar ratio (thiols:vinyl esters) on the esters) on synthesis the thioesters synthesis reaction in athe microreactor, the thioester synthesis reaction of benzyl thioesters reaction in a microreactor, thioester synthesis reaction of benzyl mercaptan and mercaptan and vinyl was used as a We model reaction. Wemolar triedratios substrate molar ratios (benzyl vinyl laurate was usedlaurate as a model reaction. tried substrate (benzyl mercaptan:vinyl mercaptan:vinyl laurate) 1:1 see to 1:4. AsFigure we can Figure 2, the reaction conversion is 61% laurate) from 1:1 to 1:4. Asfrom we can from 2, see the from reaction conversion is 61% when the substrate when the molar ratio is 1:1. Withlaurate, the increase of vinyl laurate, the reaction conversion molar ratiosubstrate is 1:1. With the increase of vinyl the reaction conversion increases, too. The best increases, The best result obtained when the molar ratio reached 1:2. Considering optimal result wastoo. obtained when thewas molar ratio reached 1:2. Considering the optimal reaction the conversion reaction conversion and economy of the reaction, we decide to use the substrate molar ratio of as and economy of the reaction, we decide to use the substrate molar ratio of 1:2 as the best molar1:2 ratio the the bestnext molar ratio for the next experiment. for experiment.

S-benzyl thiododecanoate

conversion (%)

100 80 60 40 20 0

1:1

1 : 1.5

1:2

1:3

1:4

Molar ratio of benzylmercaptan: vinyl laurate Figure 2. 2. The The influence influence of of substrate substrate molar molar ratio ratio (benzyl (benzyl mercaptan:vinyl mercaptan:vinyl laurate) laurate) on on the the enzymatic enzymatic Figure 1 feed synthesis ofofthioesters thioesters a continuous flow microreactor: min1−1(4 feed (4 mmol thiol synthesis in aincontinuous flow microreactor: 10.4 µL10.4 min−µL mmol1thiol derivatives −1 feed 2 (4–16 mmol vinyl esters in 10 mL − 1 ◦ derivatives in 10 mL DMSO) and 10.4 µL min DMSO) at in 10 mL DMSO) and 10.4 µL min feed 2 (4–16 mmol vinyl esters in 10 mL DMSO) at 50 C (residence − 1 −1 50 °C30 (residence time 30 TL min), time min), Lipozyme IMLipozyme (0.87 g, 44 TL mgIM mL(0.87 ). g, 44 mg mL ).

2.3. Reaction Effect 2.3. Reaction Temperature Temperature Effect The temperature temperatureisisanother anotherimportant importantfactor factorfor for the enzymatic reactions, to their effects on The the enzymatic reactions, duedue to their effects on the the enzyme stability and reaction rate, especially when the reaction conducted in a microreactor. enzyme stability and reaction rate, especially when the reaction conducted in a microreactor. After we Afterthe weoptimum find the substrate optimummolar substrate of the continuethe to examine the effect of find ratiomolar of theratio reaction, wereaction, continuewe to examine effect of temperature temperature on the lipase-catalysed thioester synthesis under a continuous flow microreactor. on the lipase-catalysed thioester synthesis under a continuous flow microreactor. We adjusted We the adjusted the from temperature °C to °C to the effect on the reaction temperature 40 ◦ C to from 60 ◦ C 40 to find the60effect of find temperature onofthetemperature reaction conversion. As we conversion. we can see from Figure was 3, when theout reaction carried outconversion at 40 °C, the reaction can see fromAs Figure 3, when the reaction carried at 40 ◦was C, the reaction is only 65%. conversion is only 65%. With the increase of reaction temperature, the reaction conversion has also With the increase of reaction temperature, the reaction conversion has also been obviously improved.

When the reaction is controlled at 50 ◦ C, the thioester synthesis can achieve the optimal conversion of 88%. Considering the optimal reaction conversion and the safety and operability of the reaction, we choose 50 ◦ C as the most suitable reaction temperature for the following study of the enzymatic thioester synthesis in a continuous flow microreactor.

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been obviously improved. When the reaction is controlled at 50 °C, the thioester synthesis can achieve the optimal conversion of 88%. Considering the optimal reaction conversion and the safety and operability of the reaction, we choose 50 °C as the most suitable reaction temperature for the Catalysts 2018, 8, 249 5 of 12 following study of the enzymatic thioester synthesis in a continuous flow microreactor.


conversion (%)

100 80 60 40 20 0

40

45 50 55 60 Reaction temperature (°C)

◦ C) on Figure 3. 3. The The influence influence of of reaction reaction temperature temperature ((°C) (°C) on the the enzymatic enzymatic synthesis synthesis of of thioesters thioesters in in aaa Figure 3. The influence of reaction temperature on the enzymatic synthesis of thioesters in Figure −11 feed 1 (4 mmol thiol derivatives in 10 mL DMSO) and − −1 continuous flow microreactor: 10.4 µL min continuous flow microreactor: 10.4 µL min feed 1 (4 mmol thiol derivatives in 10 mL DMSO) and continuous flow microreactor: 10.4 µL min feed 1 (4 mmol thiol derivatives in 10 mL DMSO) and −11 feed 2 (8 mmol vinyl esters in 10 mL DMSO) at different reaction temperature (residence 10.4µL µLmin min−−1 10.4 µL min feed 10.4 feed22(8 (8mmol mmolvinyl vinylesters estersin in10 10mL mLDMSO) DMSO)at atdifferent different reaction reaction temperature temperature (residence (residence −11). time 30 30 min), min),Lipozyme LipozymeTL TLIM IM(0.87 (0.87g, g,44 44mg mgmL mL−−1 time 30 min), Lipozyme TL IM (0.87 g, 44 mg mL ).). time

2.4. Reaction Reaction Time/Flow Time/Flow Rate Rate Effect Effect 2.4. Reaction time/flow time/flow rate enzymatic reaction reaction performed performed in in aa Reaction rate often often play play an an important important role role in in enzymatic continuous flow microreactor. The lipase-catalysed thioester synthesis from benzyl mercaptan and continuous flow microreactor. The lipase-catalysed thioester synthesis from benzyl mercaptan and vinyl laurate laurate under under aa continuous continuous flow flow microreactor microreactor was was conducted conducted in in 20–40 20–40 min min and and the the results results are are vinyl shown in Figure 4. It was found that the best conversion can be reached in 30 min, at a flow rate of shown in Figure 4. It was found that the best conversion can be reached in 30 min, at a flow rate of −11. Thus, we chose 30 min (flow rate 20.8 µL min− −11) as the optimum reaction time for the −1 −1 − 20.8 µL min 20.8 µL min . Thus, we chose 30 min (flow rate 20.8 µL min ) as the optimum reaction time for the following study study of of enzymatic enzymatic thioester thioester synthesis synthesis in in aa continuous continuous flow flowmicroreactor. microreactor. following


conversion (%)

100 80 60 40 20 0

20

25 30 35 Reaction time (min)

40

Figure4. 4. The The influence influence of of reaction reaction time time (min) (min) on on the the enzymatic enzymatic synthesis synthesis of of thioesters thioesters in in aaa continuous continuous Figure 4. of reaction time (min) on the enzymatic synthesis of thioesters in continuous Figure The influence flow microreactor. flow microreactor. microreactor. flow

2.5. Benzyl Mercaptan Donor Structure Effect Having obtained the favorable results given above, we then investigated the substrate structure effect on the enzymatic thioesters synthesis reaction in a microreactor. The effect of different substituted


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2.5. Benzyl Mercaptan Donor Structure Effect Catalysts 2018, 8,obtained 249 Having

6 of 12 the favorable results given above, we then investigated the substrate structure effect on the enzymatic thioesters synthesis reaction in a microreactor. The effect of different substituted groups on the benzyl mercaptan was examined, as shown in Figure 5. Reaction of 4groups on the benzyl mercaptan was examined, as shown in Figure 5. Reaction of 4-methylbenzyl methylbenzyl mercaptan (1b) to vinyl laurate (2b) afforded a higher yield (94%, entry 6) than benzyl mercaptan (1b) to vinyl laurate (2b) afforded a higher yield (94%, entry 6) than benzyl mercaptan mercaptan (88%, entry 2) (Scheme 1) in a short time, indicating that an electron-donating group (88%, entry 2) (Scheme 1) in a short time, indicating that an electron-donating group improves the improves the transesterification reactivity of the benzyl mercaptan. Oppositely, the reaction of 4transesterification reactivity of the benzyl mercaptan. Oppositely, the reaction of 4-chlorobenzyl chlorobenzyl mercaptan (1c) and vinyl esters afforded a lower yield (entries 9–12). Under the same mercaptan (1c) and vinyl esters afforded a lower yield (entries 9–12). Under the same condition, condition, the thioesters synthesis of benzyl mercaptan and vinyl laurate was more rapid than that the thioesters synthesis of benzyl mercaptan and vinyl laurate was more rapid than that using using 4-chlorobenzyl mercaptan as the reactant, while lower than that using 4-methylbenzyl 4-chlorobenzyl mercaptan as the reactant, while lower than that using 4-methylbenzyl mercaptan as mercaptan as the donor. the donor.

S-benzyl thiododecanoate derivatives

conversion (%)

100 80 60 40 20 0

an an an ap t ap t apt c c c r r r e e e zy lm zylm zylm n n n e e e b y lb rob eth hlo 4-c 4- m

Mercaptan derivatives

Figure 5. The The effect effect of of different different substituted substituted groups groups on on benzyl benzyl mercaptan mercaptan on on the the enzymatic enzymatic synthesis synthesis Figure 5. −1 − 1 of 1 (4 mmol thiol derivatives in 10 of thioesters thioesters in in aa continuous continuous flow flowmicroreactor. microreactor.10.4 10.4µL µLmin min feed feed 1 (4 mmol thiol derivatives in −1 − 1 feed ◦ C(residence mLmL DMSO) and 10.4 µLµLmin feed 2 (8 mmol vinyl 10 DMSO) and 10.4 min 2 (8 mmol vinylesters estersinin1010mL mLDMSO) DMSO)at at50 50 °C (residence time time −11 30 min), Lipozyme Lipozyme TL TL IM IM (0.87 (0.87 g, g, 44 44 mg mg mL mL− ).). 30 min),

2.6. 2.6. Vinyl Vinyl Ester Ester Acceptor Acceptor Structure Structure Effect Effect We structure effect on on thethe enzymatic thioester synthesis and We have have also alsoinvestigated investigatedthe theacceptor acceptor structure effect enzymatic thioester synthesis found the longer the the vinyl ester carboxyl group chain, benzyl and found the longer vinyl ester carboxyl group chain,the thehigher higherthe the conversion. conversion. Using Using benzyl mercaptan as the donor, the decrease of conversion was detected with the increase of carboxyl mercaptan as the donor, the decrease of conversion was detected with the increase of carboxyl group group chain. chain. The The conversion conversion yield yield was was less less than than 65% 65% in in the the reaction reaction of of benzyl benzyl mercaptan mercaptan and and vinyl vinyl acetate acetate (Figure 6). 6). (Figure

Finally, to limitations of this new new synthetic synthetic approach approach in in aa continuous-flow continuous-flow Finally, to explore explore the the scope scope and and limitations of this microreactor, three three thiol thiol derivatives, derivatives,benzyl benzylmercaptan mercaptan(1a), (1a),4-methylbenzyl 4-methylbenzyl mercaptan (1b), and microreactor, mercaptan (1b), and 44-chlorobenzyl mercaptan (1c),and andfour fourvinyl vinylesters esters(2a–d), (2a–d),were were subjected subjected to to the the general general reaction chlorobenzyl mercaptan (1c), reaction conditions, using using both both aa single-mode single-mode shaker shaker reactor reactor and and aa continuous-flow continuous-flow microreactor microreactor (Scheme (Scheme 1). 1). conditions, For the hh oror more to to obtain ideal yields (Method A). For the shaker shaker experiments, experiments,reaction reactiontime timeneeded neededabout about2424 more obtain ideal yields (Method Using lipase-catalysed thioesters synthesis reaction of thiol under continuous-flow conditions, A). Using lipase-catalysed thioesters synthesis reaction of thiol under continuous-flow conditions, 12 12 thioesters were synthesized in parallel a single experiment at same the same rate (Method B). thioesters were synthesized in parallel in ain single experiment at the flowflow rate (Method B). The The results better with flow microreactor processing than withthe thesingle-mode single-modeshaker shaker(Table (Table 1, 1, results werewere better with flow microreactor processing than with entries 1–12). Importantly, applying flow microreactor processing, yielded a conversion of thioester entries 1–12). Importantly, applying flow microreactor processing, yielded a conversion of thioester derivatives to to 82% 82% or or more. This allows allows us us to to reduce reduce the derivatives more. This the reaction reaction time time and and simplify simplify the the purification purification of products. of products.

Catalysts 2018, 8, 249 Catalysts 2018, 8, x FOR PEER REVIEW

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S-benzyl thioalkanoate derivatives

conversion (%)

100 80 60 40 20 0 e te te te itat i pa eta ura d c m a l l a a l l a l ny lp viny viny divi viny

Acceptor of acylation Figure 6. The effect of acceptor structures on the enzymatic synthesis ofreaction thioesters in a continuous − 1 flow microreactor. 10.4 µL min feed 1 (4 mmol thiol derivatives in 10 mL DMSO) and 10.4 µL min−1 Figure 6. The effect of acceptor structures on the enzymatic synthesis of thioesters in a continuous feed 2 (8 mmol vinyl esters in 10−1mL DMSO) at 50 ◦ C (residence time 30 min), Lipozyme TL IM (0.87 g, flow microreactor. 10.4 µL min feed 1 (4 mmol thiol derivatives in 10 mL DMSO) and 10.4 µL min−1 44 mg mL−1 ). feed 2 (8 mmol vinyl esters in 10 mL DMSO) at 50 °C (residence time 30 min), Lipozyme TL IM (0.87 g, 44 mg mL−1).

3. Materials and Methods

3. Materials and Methods 3.1. Materials

Unless otherwise stated, all chemicals were obtained from commercial sources and used without 3.1. Materials further purification. Lipase TL IM from Thermomyces lanuginosus was purchased from Novo Nordisk Unless otherwise stated, all chemicals were obtained from commercial sources and used without (Copenhagen, Denmark). Vinyl acetate (>99%), vinyl laurate (>99%), vinyl palmitate (>96%), and further purification. Lipase TL IM from Thermomyces lanuginosus was purchased from Novo Nordisk divinyladipate (>96%) were all purchased from Tokyo Chemical Industry Co., LTD. (Tokyo, Japan). (Copenhagen, Denmark). Vinyl acetate (>99%), vinyl laurate (>99%), vinyl palmitate (>96%), and Benzyl mercaptan (98%), 4-methylbenzyl mercaptan (97%), and 4-chlorobenzyl mercaptan (98%) were divinyladipate (>96%) were all purchased from Tokyo Chemical Industry co., LTD. (Tokyo, Japan). all purchased from Aladdin (Shanghai, China). Harvard Apparatus PHD 2000 syringe pumps were Benzyl mercaptan (98%), 4-methylbenzyl mercaptan (97%), and 4-chlorobenzyl mercaptan (98%) purchased from Harvard Apparatus (Cambridge, MA, USA). were all purchased from Aladdin (Shanghai, China). Harvard Apparatus PHD 2000 syringe pumps were purchased from Harvard 3.2. Thioester Synthesis OperatingApparatus Conditions (Cambridge, USA). 3.2. Thioester Operating Conditions 3.2.1. GeneralSynthesis Procedure for Thioesters Synthesis under Shaker Conditions

Method A: Benzyl mercaptan (1 mmol) and vinyl laurate (3 mmol) were added to 5 mL DMSO. 3.2.1. General Procedure for Thioesters Synthesis under Shaker Conditions The biocatalyst lipozyme TL IM (44 mg mL−1 , 0.22 g) was then added and the suspension maintained ◦ C for 24A: mercaptan (1 mmol)The andmixture vinyl laurate (3 mmol) added to 5 evaporated mL DMSO. at 50 Method h Benzyl under Shaker Conditions. was cooled andwere filtered. Then −1 The biocatalyst TL IM mg mLwas , 0.22 g) was then added and the suspensionon maintained under reduced lipozyme pressure and the(44 residue submitted to column chromatography silica gel at 50 °C for 24 h under Shaker Conditions. The mixture was cooled and filtered. Then evaporated (200–300 mesh). The products were eluted with a gradient of normal petroleum ether/ethyl acetate underby reduced pressure and thewas residue was submitted column chromatography onmain silicaproducts gel (200– (20:1, vol). The purification monitored by TLC.to The fractions containing the 1 H NMR, 13 300 mesh). The were elutedand with a gradient of normal ether/ethyl acetate (20:1, were pooled, theproducts solvent evaporated, the residue analysed bypetroleum C NMR, and ESI-MS. by vol). The purification was monitored by TLC. The fractions containing the main products were 13C NMR, and ESI-MS. 3.2.2. General Procedure for Thioester Synthesis Continuous Microreactors pooled, the solvent evaporated, and the residue in analysed by 1HFlow NMR, Method B: Four millimoles of the benzyl mercaptan were dissolved in 10 mL DMSO (feed 1) 3.2.2. General Procedure for Thioester Synthesis in Continuous Flow Microreactors and 8 mmol vinyl laurate were dissolved in 10 mL DMSO (feed 2). Lipozyme TL IM (0.87 g) was Method Fourthe millimoles of the were in 101 mL weighed, thenB:filled PFA reactor coilbenzyl (innermercaptan diameter ID: 2.0dissolved mm, length: m). DMSO Feeds 1(feed and 1) 2 −1 in ◦ C, and the were at a flow rate of 10.4 µL a Y-mixer and 8mixed mmoltogether vinyl laurate were dissolved in min 10 mL DMSO (feedat 2).50Lipozyme TLresulting IM (0.87 stream g) was −1 )filled weighed, then the PFA reactor coil (inner ID:used 2.0 mm, length: m). Feeds 1 and 2 were (20.8 µL min was connected to a sample vial diameter which was to collect the1final mixture. The final mixed together at a flow rate of 10.4 µL min−1 in a Y-mixer at 50 °C, and the resulting stream (20.8 µL min−1) was connected to a sample vial which was used to collect the final mixture. The final mixture


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mixture was then evaporated, and the residue was submitted to column chromatography on silica gel (200–300 mesh). The products were eluted with a gradient of normal petroleum ether/ethyl acetate (20:1, by vol). The purification was monitored by TLC. The fractions containing the main products were pooled, the solvent evaporated, and the residue analysed by 1 H NMR, 13 C NMR, and ESI-MS. In order to examine the reproducibility of the method, we repeated the reaction five times, the result are illustrate in Figure S1. 3.3. Analytical Methods 3.3.1. Thin-Layer Chromatography Analytical TLC was performed on silica gel 60 plates (Yantai Jiangyou Silicone Development Co., LTD., Yantai, China) using petroleum ether/ethyl acetate (20:1, by vol) as the eluent. Spots were detected by ultraviolet irradiation at 254 nm. 3.3.2. High-Performance Liquid Chromatography (HPLC) The reaction was monitored by HPLC analysis using a 4.6 × 250 mm, 5 µm XBridge C18 column with a gradient of methanol/water. For the analysis of products, methanol/water: 85/15 (v/v) was used as the mobile phase (flow rate, 0.6 mL min−1 ), thiol and thioester derivatives were detected at 254 nm. The conversion yield was defined as the ratio between the molar concentration of thioester derivatives and the initial molar concentration of the thiol derivatives used. 3.3.3. Nuclear Magnetic Resonance (NMR) Analysis After purification of the synthesized products by column chromatography, the chemical structures of thioesters were determined by 1 H NMR, 13 C NMR and ESI-MS. S-Benzyl thioacetate (3a): Light yellow oil; 1 H NMR (500 MHz, CDCl3 ): δ 7.32–7.26 (m, 5H), 4.15 (s, 2H), 2.37 (s, 3H). 13 C NMR (125 MHz, CDCl3 ): δ 195.0, 137.6, 128.8, 128.6, 127.2, 33.4, 30.2. ESI-MS: m/z = 189.1 [M + Na]+ . S-Benzyl thiododecanoate (3b): White crystals; 1 H NMR (500 MHz, CDCl3 ): δ 7.36–7.27 (m, 5H), 4.17 (s, 2H), 2.61 (t, J = 7.5 Hz, 2H), 1.73 (m, 2H), 1.35 (m, 16H), 0.98 (t, J = 6.0 Hz, 3H). 13 C NMR (125 MHz, CDCl3 ): δ 198.2, 137.7, 128.6, 128.4, 127.0, 43.6, 32.9, 31.8, 29.5, 29.4–29.1, 28.8, 25.5, 22.6, 14.0. ESI-MS: m/z = 329.2 [M + Na]+ . S-Benzyl thiohexadecanoate (3c): White solid; 1 H NMR (500 MHz, CDCl3 ): δ 7.34–7.25 (m, 5H), 4.14 (s, 2H), 2.58 (t, J = 7.5 Hz, 2H), 1.66 (m, 2H), 1.47–1.23 (m, 24H), 0.91 (t, J = 7.0 Hz, 3H). 13 C NMR (125 MHz, CDCl3 ): δ 198.9, 137.8, 129.3, 129.0, 128.8, 128.6, 127.2, 43.9, 33.1, 31.9, 29.7, 29.6–29.1, 29.0, 25.6, 22.7, 14.1. ESI-MS: m/z = 385.2 [M + Na]+ . 6-oxo-6-((Benzyl)thio)-hexanoate vinyl ester (3d): Yellow oil; 1 H NMR (500 MHz, CDCl3 ): δ 7.35–7.24 (m, 6H), 4.89 (ddd, J = 14.0, 3.4, 1.6 Hz, 1H), 4.59 (ddd, J = 6.3, 2.4, 1.7 Hz, 1H), 4.14 (s, 2H), 2.59 (m, 2H), 2.43 (m, 2H), 1.72 (m, 4H). 13 C NMR (125 MHz, CDCl3 ): δ 198.1, 170.1, 141.1, 137.5, 128.7, 128.6, 127.2, 97.6, 43.2, 33.4, 33.1, 24.7, 23.8. ESI-MS: m/z = 301.1 [M + Na]+ . S-(4-Methylbenzyl) ethanethioate (3e): Light yellow oil; 1 H NMR (500 MHz, CDCl3 ): δ 7.19 (d, J = 7.6 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H), 4.10 (s, 2H), 2.35 (s, 3H), 2.33 (s, 3H). 13 C NMR (125 MHz, CDCl3 ): δ 195.0, 137.9, 134.2, 129.8, 129.7, 33.4, 30.2, 21.1. ESI-MS: m/z = 203.1 [M + Na]+ . S-(4-Methylbenzyl) thiododecanoate (3f): Yellow liquid oil; 1 H NMR (500 MHz, CDCl3 ): δ 7.18 (d, J = 7.6 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H), 4.10 (s, 2H), 2.57 (t, J = 7.5 Hz, 2H), 2.33 (s, 3H), 1.64 (m, 2H), 1.31 (m, 16H), 0.89 (t, J = 7.1 Hz, 3H). 13 C NMR (125 MHz, CDCl3 ): δ 199.1, 137.0, 134.7, 129.4, 128.8, 43.9, 32.9, 32.0, 29.7, 29.6–29.3, 29.0, 25.7, 22.8, 21.1, 14.2. ESI-MS: m/z = 343.2 [M + Na]+ .


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S-(4-Methylbenzyl) thiohexadecanoate (3g): Light yellow oil; 1 H NMR (500 MHz, CDCl3 ): δ 7.19 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 7.9 Hz, 2H), 4.10 (s, 2H), 2.56 (t, J = 7.5 Hz, 2H), 2.34 (s, 3H), 1.68 (m, 2H), 1.32–1.27 (m, 24H), 0.90 (t, J = 7.0 Hz, 3H). 13 C NMR (125 MHz, CDCl3 ): δ 199.0, 136.9, 134.7, 129.3, 128.7, 43.9, 32.9, 32.0, 29.7, 29.6–29.3, 29.0, 25.6, 22.7, 21.1, 14.1. ESI-MS: m/z = 399.3 [M + Na]+ . 6-oxo-6-((4-Methylbenzyl)thio)-hexanoate vinyl ester (3h): Light yellow oil; 1 H NMR (500 MHz, CDCl3 ): δ 7.31–7.20 (m, 5H), 4.87 (ddd, J = 14.0, 3.3, 1.7 Hz, 1H), 4.56 (ddd, J = 6.2, 2.0, 1.7 Hz, 1H), 4.11 (s, 2H), 2.59 (m, 2H), 2.41 (m, 2H), 2.39 (s, 3H), 1.71 (m, 4H). 13 C NMR (125 MHz, CDCl3 ): δ 198.0, 170.1, 141.0, 137.5, 128.7, 128.5, 127.2, 97.6, 43.1, 33.4, 33.1, 24.7, 23.8, 23.7. ESI-MS: m/z = 315.1 [M + Na]+ . S-(4-Chlorobenzyl) ethanethioate (3i): Light yellow oil; 1 H NMR (500 MHz, CDCl3 ): δ 7.27 (dd, J = 8.6, 6.6 Hz, 2H), 7.23 (dd, J = 8.5, 6.1 Hz, 2H), 4.08 (s, 2H), 2.36 (s, 3H). 13 C NMR (125 MHz, CDCl3 ): δ 195.0, 135.7, 132.7, 130.8, 128.8, 33.1, 30.5. ESI-MS: m/z = 223.0 [M + Na]+ . S-(4-Chlorobenzyl) thiododecanoate (3j): Yellow liquid oil; 1 H NMR (500 MHz, CDCl3 ): δ 7.27 (dd, J = 8.5, 6.5 Hz, 2H), 7.23 (dd, J = 8.5, 6.0 Hz, 2H), 4.08 (s, 2H), 2.57 (t, J = 7.2 Hz, 2H), 1.66 (m, 2H), 1.29 (m, 16H), 0.90 (t, J = 6.0 Hz, 3H). 13 C NMR (125 MHz, CDCl3 ): δ 198.7, 141.2, 136.5, 133.0, 130.2, 128.7, 43.8, 34.0, 32.4, 31.9, 29.6–29.0, 28.9, 25.6, 24.6, 22.7, 14.1. ESI-MS: m/z = 363.2 [M + Na]+ . S-(4-Chlorobenzyl) thiohexadecanoate (3k): Yellow oil; 1 H NMR (500 MHz, CDCl3 ): δ 7.27 (dd, J = 8.4, 6.3 Hz, 2H), 7.23 (dd, J = 8.3, 6.0 Hz, 2H), 4.23 (s, 2H), 2.58 (t, J = 7.5 Hz, 2H), 1.78 (d, J = 7.1 Hz, 2H), 1.58–1.32 (m, 24H), 0.89 (t, J = 6.8 Hz, 3H). 13 C NMR (125 MHz, CDCl3 ): δ 199.0, 135.9, 132.8, 131.9, 131.7, 129.2, 128.8, 44.2, 33.6, 32.9, 30.1, 29.8–29.2, 28.0, 26.5, 23.4, 14.9. ESI-MS: m/z = 419.2 [M + Na]+ . 6-oxo-6-((4-Chlorobenzyl)thio)-hexanoate vinyl ester (3l): Yellow oil; 1 H NMR (500 MHz, CDCl3 ): δ 7.32–7.20 (m, 5H), 4.89 (ddd, J = 14.0, 3.4, 1.6 Hz, 1H), 4.59 (ddd, J = 6.3, 2.4, 1.6 Hz, 1H), 4.08 (s, 2H), 2.61 (m, 2H), 2.41 (m, 2H), 1.72 (m, 4H). 13 C NMR (125 MHz, CDCl3 ): δ 198.0, 170.2, 141.1, 136.3, 133.1, 130.2, 128.8, 97.7, 43.2, 33.4, 32.5, 24.8, 23.8. ESI-MS: m/z = 335.1 [M + Na]+ . 4. Conclusions In conclusion, we describe here the enzymatic synthesis of thioesters from thiols and vinyl esters in a continuous-flow microreactor. The reaction conditions including reaction temperature, reaction time/flow rate, substrate molar ratio and the substrate structure effect on the reaction were examined. The scope of the reaction was tested by varying the thiols and vinyl esters. Compared to traditional batch processes, continuous-flow microreactor technology offers a variety of advantages, such as reduced reaction times, improved operational safety and much lower process costs and increased process efficiency. The salient features of this method include mild reaction conditions (50 ◦ C), short reaction times (30 min), and high yields that make our methodology a valuable contribution to the thioester derivatives synthesis. The method of enzymatic synthesis in a microreactor environment described here may have general applications to synthetic organic chemistry by enzymatic catalysis in the future. Thioester derivatives synthesis of thiophenol, furanthiol, thienyl mercaptan, and other sulphur nucleophiles to vinyl esters catalysed by lipase TL IM from Thermomyces lanuginosus in a continuous-flow microreactor are in progress. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/8/6/249/s1, Figure S1: The reproducibility of the reaction on the conversion of S-Benzyl thiododecanoate catalysed by Lipozyme TL IM in a continuous flow microreactor. Author Contributions: L.D. and L.S. conceived and designed the experiments. L.S., Z.D., and N.Z. performed the experiments. L.D., L.S., and J.S. analysed the data. L.D. and X.L. contributed reagents/materials/analysis tools. L.D., L.S., J.S., Z.D., and N.Z. wrote or reviewed the manuscript. Funding: This research was funded by the Natural Science Foundation of Zhejiang Province grant number [LY17B020010], the National Science and Technology Support Project grant number [2015BAD14B0305], the International Cooperation Project 948 grant number [2014-4-29], the National Natural Science Foundation of China grant number [2130617], the Science and Technology Research Program of Zhejiang Province grant number


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[2014C32094], as well as the Natural Science Foundation of Zhejiang University of Technology grant number [116004029] and the APC was funded by Zhejiang Province grant number [LY17B020010]. Acknowledgments: This work was supported by the Natural Science Foundation of Zhejiang Province (LY17B020010), the National Science and Technology Support Project (2015BAD14B0305), the International Cooperation Project 948 (2014-4-29), the National Natural Science Foundation of China (21306172), the Science and Technology Research Program of Zhejiang Province (2014C32094), as well as the Natural Science Foundation of Zhejiang University of Technology (116004029) for financial support. Conflicts of Interest: The authors declare no conflict of interest.

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