水相中脂肪酶Lipase lipoprotein催化的Aldol縮合反應(yīng)

丁 雁, 黃 和,2, 吳素文, 李 霜,2, 胡 燚,2

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水相中脂肪酶Lipase lipoprotein催化的Aldol縮合反應(yīng)

丁 雁1, 黃 和1,2, 吳素文1, 李 霜1,2, 胡 燚1,2

(1. 南京工業(yè)大學(xué) 生物與制藥工程學(xué)院, 江蘇 南京 210009;2. 材料化學(xué)工程國(guó)家重點(diǎn)實(shí)驗(yàn)室 南京工業(yè)大學(xué), 江蘇 南京 210009)

Aldol縮合是有機(jī)化學(xué)中最重要的C-C鍵形成反應(yīng)之一，在化工、醫(yī)藥領(lǐng)域有著重要的應(yīng)用。實(shí)驗(yàn)結(jié)果表明水相中Lipase lipoprotein脂肪酶(LPL)與咪唑添加劑能夠協(xié)同催化芳香醛與酮的Aldol縮合反應(yīng)，考察了添加劑種類及用量、水含量、溫度、時(shí)間等因素對(duì)反應(yīng)的影響。在優(yōu)化的反應(yīng)條件下，即在反應(yīng)體系中添加水30%(v/v)，咪唑60%(mol)，酶0.2 g，35℃反應(yīng)48 h，對(duì)于有吸電子取代基的芳香醛底物，能與脂肪酮、環(huán)酮、雜環(huán)酮等不同結(jié)構(gòu)的酮以51.8%~92.5%的收率發(fā)生Aldol縮合反應(yīng)。相比文獻(xiàn)報(bào)道方法，具有環(huán)境友好、底物適用范圍廣、產(chǎn)率高、反應(yīng)時(shí)間短等優(yōu)點(diǎn)。

脂肪酶；咪唑；Aldol縮合；水相

1 前 言

酶在有機(jī)與生物合成中是一類重要的催化劑，因其具有高選擇性、反應(yīng)條件溫和、環(huán)境友好、副產(chǎn)物少等優(yōu)點(diǎn)，被廣泛應(yīng)用于醫(yī)藥、化工、食品、紡織、洗滌劑及能源等工業(yè)領(lǐng)域[1~4]。脂肪酶(lipase, E.C.3.1.1.3)主要是指一類能夠催化甘油三酯水解生成脂肪酸、甘油和甘油單酯或二酯的酶，廣泛存在于動(dòng)植物和微生物體中[5]，已成功應(yīng)用于Aldol縮合、Knoevenagel縮合、Michael加成、Henry反應(yīng)等多種C-C鍵形成反應(yīng)[6~8]。

Aldol縮合被認(rèn)為是有機(jī)化學(xué)中最重要的C-C鍵形成反應(yīng)之一[9]，可用于多種天然或非天然有機(jī)化合物的合成中。在有機(jī)合成中有許多關(guān)于用酸[10]、堿[11]、有機(jī)小分子[12]催化的Aldol縮合報(bào)道，但發(fā)現(xiàn)來源更廣泛、成本更加低廉、更加綠色高效的新型Aldol縮合反應(yīng)催化劑仍然具有重要意義。2003年，Branneby 等[13]首次發(fā)現(xiàn)了脂肪酶具有催化Aldol縮合反應(yīng)的活性，他們用天然南極假絲酵母脂肪酶(lipase, CALB)催化此反應(yīng)，但僅限于丙醛和己醛等簡(jiǎn)單的脂肪醛且收率只有6%~12%。Li等[14]報(bào)道了水相中豬胰脂肪酶(Porcine Paoceneas Lipase, PPL)催化的不對(duì)稱Aldol反應(yīng)并提出了反應(yīng)機(jī)理，收率為11.7%~96.4%，值為9.6%~43.6%，然而論文僅考察了硝基取代的苯甲醛與丙酮的反應(yīng)。Guan等[15]發(fā)現(xiàn)PPL能催化雜環(huán)酮與各種有吸電子取代基的芳醛的不對(duì)稱Aldol反應(yīng)，在乙腈溶劑中反應(yīng)120 h以上，最高收率為56%，此時(shí)值為46%。Xie[16]等發(fā)現(xiàn)了在酸性緩沖溶液中牛胰脂肪酶(lipase，BPL)能催化多種芳香醛與環(huán)酮類化合物的不對(duì)稱 Aldol 反應(yīng)，但該法不能應(yīng)用于直鏈酮底物，如與丙酮反應(yīng)時(shí)，反應(yīng)沒有選擇性，收率僅為26%。

圖式1 LPL催化4-硝基苯甲醛與丙酮Aldol縮合反應(yīng)

Scheme 1 Aldol condensation of acetone and 4-nitrobenzaldehyde catalyzed by LPL

研究表明在酶催化反應(yīng)中加入合適的有機(jī)小分子作為添加劑，可以改善酶的催化性能。Itoh等[17]研究了洋蔥假單胞脂肪酶(lipase，PCL)催化2-氰基-1-甲基-乙酸乙酯的水解反應(yīng)中，加入冠醚可以顯著的改變其反應(yīng)速率和立體選擇性。Liu等[18]報(bào)道了在褶皺假絲酵母脂肪酶(lipase, CRL)催化酮洛芬酯的水解反應(yīng)時(shí)，加入8%(w/v)吐溫80可以使酶活性提高13倍，對(duì)映體選擇率E值從1.2提高到6.7。Chen等[19]研究了N-雜環(huán)化合物添加對(duì)酶促反應(yīng)的調(diào)控作用，發(fā)現(xiàn)咪唑可以促進(jìn)D-氨基酰化酶(D-aminoacylase, DA)催化的Claisen-Schmidt反應(yīng)。

Lipase lipoprotein脂肪酶(LPL)來源于黑曲霉，相比與BPL、CALB等成本要低得多。本文首先以對(duì)硝基苯甲醛與丙酮的Aldol縮合反應(yīng)為模型，考察了添加劑種類和添加量、水含量、時(shí)間、溫度等因素對(duì)反應(yīng)的影響(Scheme 1)，并對(duì)反應(yīng)底物的適用范圍進(jìn)行了研究，希望發(fā)展一種成本更低、效率更高、底物適應(yīng)范圍更寬的綠色Aldol縮合反應(yīng)新方法。

2 實(shí)驗(yàn)(材料和方法)

2.1 儀器與試劑

Lipase lipoprotein脂肪酶(LPL)購(gòu)自寧夏夏盛實(shí)業(yè)集團(tuán)有限公司，其它藥品和試劑均為市售分析純。熔點(diǎn)測(cè)定使用WRS-1B數(shù)字熔點(diǎn)儀，溫度未經(jīng)校正；核磁共振用BRUKERAC-P400型儀測(cè)定。

2.2 實(shí)驗(yàn)方法

將2 mmol對(duì)硝基苯甲醛和1 mL去離子水、2.3 mL丙酮加入10 mL反應(yīng)瓶中，然后加入0.2 g LPL、60%(mol)咪唑，35℃下攪拌反應(yīng)，TLC(石油醚/乙酸乙酯1/1，v/v)監(jiān)測(cè)反應(yīng)進(jìn)行情況，反應(yīng)完畢后過濾除酶，濾紙和濾液分別用CH2Cl2和水洗滌三次[16]。濾液用無水MgSO4干燥后過濾，減壓旋蒸，粗產(chǎn)物經(jīng)柱層析[石油醚/乙酸乙酯，9/1~2/1，v/v]分離即可得到純化的目標(biāo)產(chǎn)物。

2.3 部分產(chǎn)物結(jié)構(gòu)表征

(3i)1H-NMR (400 MHz, CDCl3) δ 8.23 (d,= 8.5 Hz, 2H), 7.64~7.46 (m, 2H), 5.48 (s, 0.68H), 4.97 (d,= 7.6 Hz, 0.28H), 4.18 (s, 1H), 3.83 (d,= 12.4 Hz, 1H), 3.27 (s, 2H), 2.75 (s, 1H), 2.51 (dd,= 10.1, 5.8 Hz, 2H), 1.38 (t,= 20.0 Hz, 10H)。

(3j)1H-NMR (400 MHz, CDCl3) δ 8.22 (d,= 8.8 Hz, 2H), 7.52 (d,= 8.6 Hz, 2H), 5.54 (s, 0.67H), 5.00 (d,= 8.1 Hz, 0.38H), 4.29~4.16 (m, 1H), 3.85 (dd,= 6.4, 1.3 Hz, 1H), 3.82~3.68 (m, 2H), 3.46 (dd,= 11.4, 9.9 Hz, 0.38H), 3.04 (s, 0.61H), 2.93 (dd,= 5.7, 4.8 Hz, 1H), 2.80~2.63 (m, 1H), 2.58~2.45 (m, 1H)。

3 結(jié)果與討論

3.1 不同添加劑對(duì)反應(yīng)的影響

從表1中可以看出添加劑的選擇對(duì)于LPL催化Aldol反應(yīng)有著重要的影響，當(dāng)加入咪唑類添加劑時(shí)對(duì)反應(yīng)有明顯的促進(jìn)作用，如以咪唑?yàn)樘砑觿r(shí)(Entry 5)，反應(yīng)48 h后，收率可達(dá)71.6%。而18冠醚-6、-環(huán)糊精以及表面活性劑不能輔助LPL催化Aldol反應(yīng)(Entry1-3)。對(duì)比單獨(dú)以LPL和咪唑?yàn)榇呋瘎r(shí)反應(yīng)的收率(Entry6~7)，可以看出咪唑添加劑與LPL對(duì)此反應(yīng)具有顯著的協(xié)同催化作用，這與Chen等[19]報(bào)道的結(jié)果相似。

表1 不同種類添加劑對(duì)LPL催化4-硝基苯甲醛與丙酮Aldol縮合反應(yīng)的影響

aReaction conditions: LPL (0.2 g), (30%(mol)), 4-nitrobenzaldehyde (2 mmol), acetone (2.3 mL), deionized water 1 mL, 35℃, reaction time (48 h);bIsolated yield;cWithout additive, LPL (0.2 g);dWithout LPL, 30 %(mol) imidazole;eWithout LPL, without additive.

3.2 咪唑添加量對(duì)反應(yīng)的影響

圖1考察了水含量為30%時(shí)，添加劑咪唑用量對(duì)反應(yīng)的影響。隨著咪唑用量的增加，該反應(yīng)的產(chǎn)物收率也逐漸增加。當(dāng)咪唑用量達(dá)到60%(mol)時(shí)，收率達(dá)到最大，此時(shí)再增加咪唑用量，產(chǎn)物收率基本不變。在此反應(yīng)條件下，單獨(dú)用60%(mol)咪唑催化收率僅為40%，再次證明了咪唑與LPL共同作用催化了Aldol縮合。因此，本文選擇咪唑用量為60%(mol)對(duì)反應(yīng)進(jìn)行后續(xù)探討。

3.3 水含量對(duì)反應(yīng)的影響

反應(yīng)中水含量會(huì)影響酶的活性中心、穩(wěn)定性，甚至引起酶分子三維構(gòu)象發(fā)生改變[20,21]，因此體系中水含量是影響反應(yīng)的重要因素之一。圖2考察了水含量對(duì)反應(yīng)的影響。如圖所示水含量對(duì)反應(yīng)的影響較大——不加水時(shí)，反應(yīng)基本不進(jìn)行，產(chǎn)物收率僅為0.9%；隨著反應(yīng)體系中水含量的增加，產(chǎn)物收率也逐漸的提高，在水含量為30%(v/v)時(shí)，收率達(dá)到92.7%；繼續(xù)提高反應(yīng)體系中的水含量，產(chǎn)物收率會(huì)逐漸下降，可能是反應(yīng)體系中過多的水導(dǎo)致底物的溶解度降低。因此，本文選擇最佳水含量為30%(v/v)。

3.4 反應(yīng)溫度的影響

通常，反應(yīng)溫度對(duì)酶的活性構(gòu)象、催化活性和穩(wěn)定性有較大的影響[22]。由圖3可見，在一定的溫度范圍內(nèi)，隨著溫度的升高，反應(yīng)收率會(huì)增加；當(dāng)溫度高于35℃時(shí)，收率會(huì)隨溫度的升高而降低，這是由于在較高溫度下使酶發(fā)生變性導(dǎo)致。

3.5 反應(yīng)時(shí)間的影響

圖4考察了反應(yīng)時(shí)間對(duì)反應(yīng)收率的影響。由圖可見產(chǎn)物收率隨反應(yīng)時(shí)間的增加而升高；反應(yīng)進(jìn)行到48 h時(shí)，反應(yīng)基本達(dá)到平衡，收率為92.7%，再延長(zhǎng)反應(yīng)時(shí)間，產(chǎn)物收率基本不變。

綜合以上實(shí)驗(yàn)結(jié)果，得到優(yōu)化后的條件：水含量為30%(v/v)，添加劑咪唑?yàn)?0%(mol)，催化劑LPL用量為0.2 g，在此條件下在35℃反應(yīng)48 h，對(duì)硝基苯甲醛與丙酮的Aldol縮合反應(yīng)收率可達(dá)92.7%。

圖式2 LPL催化芳醛與酮類化合物Aldol縮合反應(yīng)

Scheme 2 LPL-catalyzed Aldol condensation of ketones and other aromatic aldehydes

3.6 脂肪酶LPL催化Aldol縮合的底物擴(kuò)展

在優(yōu)化的反應(yīng)條件下，對(duì)底物進(jìn)行了擴(kuò)展，結(jié)果見表2。從實(shí)驗(yàn)結(jié)果可以看出，對(duì)于芳香醛來說，取代基的電子效應(yīng)對(duì)反應(yīng)有著顯著的影響。當(dāng)取代基為強(qiáng)吸電子的4-硝基苯甲醛、3-硝基苯甲醛及2-硝苯甲醛時(shí)，收率分別為92.7%、90.6%、87.2%(表2,3a~3c)；而具有相對(duì)吸電子效應(yīng)較弱基團(tuán)的對(duì)氰基苯甲醛、2,4-二氯苯甲醛和丙酮反應(yīng)時(shí)，收率分別為60.1%，68.6%(表2,3d、3e)。除此之外，還發(fā)現(xiàn)不同結(jié)構(gòu)的直鏈酮、環(huán)酮以及四氫吡喃酮、四氫噻喃-4-酮等雜環(huán)酮與4-硝基苯甲醛在此反應(yīng)條件下也能獲得較好的收率(表2，3f~3k)。

表2 LPL催化芳醛與酮類化合物Aldol縮合反應(yīng)

aReaction conditions: LPL (0.2 g), imidazole (60 %(mol)), aromatic aldehyde (2 mmol), ketone (2.3 mL), deionized water 1 mL, 35℃, reaction time (48 h).bIsolated yield after silica gel chromatography.

為了凸顯本方法的催化效率，以對(duì)硝基苯甲醛與丙酮以及環(huán)己酮的反應(yīng)為代表，與文獻(xiàn)報(bào)道的方法作對(duì)比，反應(yīng)時(shí)間得到大幅度減少，且不需要調(diào)控pH或加入乙腈等其他有機(jī)溶劑，結(jié)果見表3。

表3 不同種類酶催化的Aldol縮合

Note: Yield for reaction catalyzed by PPL at 30℃ for 48 h[14]. Yield for reaction catalyzed by BPL at 30℃, pH 5.6 for 108 h[16].a.Yield for reaction catalyzed by chymopapain at 30℃, pH 4.91 for 120 h in MeCN;b.Yield for reaction catalyzed by chymopapain at 30℃, pH 4.91 for 240 h in MeCN[31]. Yield for reaction catalyzed by pepsin at 30℃ for 96 h[32]. Yield for catalyzed by trypsin at 30℃ for 144 h in water[33]

4 結(jié) 論

研究首次發(fā)現(xiàn)了脂肪酶LPL與咪唑協(xié)同作用下，可以高效地催化吸電子取代的芳香醛與脂肪酮、環(huán)酮、雜環(huán)酮等不同結(jié)構(gòu)酮的Aldol縮合反應(yīng)。相比文獻(xiàn)報(bào)道的BPL、CALB等其他種類的脂肪酶，LPL成本更加低廉，且具有環(huán)境友好、底物適用范圍廣、產(chǎn)率高、反應(yīng)時(shí)間短等優(yōu)點(diǎn)。

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Aldol Condensation Catalyzed by Lipase Lipoprotein in Aqueous Phase

DING Yan1, HUANG He1,2, WU Su-wen1, LI Shuang1,2, HU Yi1,2

(1. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 210009, China;2. State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 210009, China)

Aldol condensation is one of the most important carbon-carbon bond-forming reactions in organic synthesis, which is widely used in the preparation of chemicals and pharmaceuticals. The experiment results in this study demonstrate that lipase lipoprotein (LPL) and imidazole can efficiently co-catalyze the Aldol reaction of aromatic aldehyde and ketones in aqueous phase. The influence of different additives, additive loading, water content, temperature, reaction time on the enzymatic reaction was investigated. The optimum conditions were found as 30% (v/v) water content, 60%(mol) imidazole, lipase 0.2 g, reaction temperature 35℃ for 48 h. Aromatic aldehydes with electron-withdrawing substituents can react with aliphatic ketones, cyclic ketones and heterocyclic ketones with good to excellent yields (51.8%~92.5%). Compared with previously reported methods, this new procedure offers advantages including environmental friendliness, wider substrate scope, higher yields and shorter reaction time.

lipase; imidazole; Aldol reaction; aqueous phase

1003-9015(2016)01-0121-06

O621.3

A

10.3969/j.issn.1003-9015.2016.01.018

2014-08-14；

2015-01-17。

國(guó)家高技術(shù)研究發(fā)展計(jì)劃(863)(2011AA02A209)；杰出青年科學(xué)基金(21225626)；國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(973)(2011CB710800)資助項(xiàng)目。

丁雁(1990-)，女，江蘇蘇州人，南京工業(yè)大學(xué)碩士生。通訊聯(lián)系人：胡燚，E-mail：huyi@njtech.edu.cn