Tetrahedron Letters 53 (2012) 4959–4961

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Tetrahedron Letters

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Biocatalytic promiscuity: lipase-catalyzed asymmetric aldol reactionof heterocyclic ketones with aldehydes

Zhi Guan ⇑, Jian-Ping Fu, Yan-Hong He ⇑School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China

a r t i c l e i n f o

Article history:Received 1 April 2012Revised 19 June 2012Accepted 3 July 2012Available online 7 July 2012

Keywords:Biocatalytic promiscuityLipaseAldol reactionHeterocyclic ketonesAsymmetry

0040-4039/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.tetlet.2012.07.007

⇑ Corresponding authors. Fax: +86 23 68254091.E-mail addresses: guanzhi@swu.edu.cn (Z. Guan), h

a b s t r a c t

The new promiscuous activity of lipase from porcine pancreas, type II (PPL II), has been observed to cat-alyze the direct asymmetric aldol reaction of heterocyclic ketones with aromatic aldehydes. PPL IIshowed favorable catalytic activity and had a good adaptability to different substrates in the reaction.The enantioselectivities of up to 87% ee and diastereoselectivities of up to 83:17 (anti/syn) were achieved.It is interesting that PPL II possesses the function of aldolase in organic solvents.

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Biocatalysis is an efficient and green tool for modern organicsynthesis due to its high selectivity, mild reaction conditions, andpotential use of inexpensive regenerable resources.1 Especially,biocatalytic promiscuity, the flexibility of the reactions catalyzedby enzymes, using the same or different, or induced active sitebut performing overall different chemical transformations whichusually differ in the type of bond formation or cleavage and inthe catalytic mechanism of bond making or breaking,2 has at-tracted much attention and expanded rapidly in recent years.3 Itis very significative to profile the novel unnatural activities ofexisting enzymes systematically since it might lead to improve-ments in existing catalytic methods and provide novel synthesispathways which are currently not available.3b Some elegant worksof biocatalytic promiscuity including aldol condensations,4 Man-nich reactions,5 Michael additions,6 Markovnikov additions,7 andHenry reactions8 have been reported in the last decades.

Carbon–carbon bond-forming reactions are fundamental in or-ganic synthesis. The aldol reaction is recognized as one of the mostpowerful methods for the construction of new carbon–carbonbonds. Therefore, the controls of both the absolute and the relativeconfiguration of the aldol products are of paramount importancefor the synthesis.9 There are many reports about enantioselectivealdol reactions catalyzed by small organic molecules and metalcomplexes.10 Despite the plentiful variety of aldol acceptors, therange of donors has remained narrow. Meanwhile the aldol prod-ucts of heterocyclic ketone donors are important intermediates

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eyh@swu.edu.cn (Y.-H. He).

to construct heterocyclic natural products. However, only limitedreports about enantioselective aldol reactions of heterocyclic ke-tones with aldehydes catalyzed by organocatalysts are available.11

Moreover, more environmentally friendly and sustainable biocata-lytic methods are still to be explored. Wang and Yu and co-workersreported the first lipase-catalyzed asymmetric aldol reaction be-tween acetones and aldehydes in ‘wet’ acetone.12 Recently, ourgroup has reported direct asymmetric aldol reactions betweencyclohexanone and aromatic aldehydes catalyzed by Nuclease p1from Penicillium citrinum, alkaline protease from Bacillus lichenifor-mis, chymopapain from Carica papaya, and acidic protease fromAspergillus usamii respectively.13 However, to the best of ourknowledge, the enzymatic asymmetric aldol reactions of heterocy-clic ketones have not been reported yet. Herein we hope to reportthe first direct asymmetric aldol reaction of heterocyclic ketoneswith aldehydes catalyzed by PPL II.

In initial research, the aldol reaction of ketone 1a and 4-nitro-benzaldehyde 2a was used as the model reaction (Table 1). Eighthydrolases which are stable and commercially available had beeninvestigated to find the most suitable enzyme to catalyze the directaldol reaction of heterocyclic ketones. The best result, 59% ee and43% yield, was achieved by using PPL II as catalyst after 120 h inmixed solvents [H2O/(H2O+CH3CN) = 1:10, v/v] at 25 �C (Table 1,entry 1). Meanwhile, some tested hydrolases also showed catalyticactivities. Among them, protease from streptomces griseus gave ayield of 46% but no enantioselectivity was observed (Table 1, entry2). Lipase from wheat germ gave 28% yield and 12% ee (Table 1, en-try 3). Amano lipase A from Aspergillus niger provided 26% yieldand 13% ee (Table 1, entry 4). Trypsin from porcine pancreas gave

Table 1The catalytic activities and stereoselectivities of different hydrolases to the aldol reactiona

N

O

Boc

+CHO

NO2

hydrolase

25 °C, solvent/H2O N

O

Boc

OH

NO2

+

1a 2a anti-3a

syn-isomer

Entry Enzyme Time (h) Yieldb (%) drc (anti:syn) % eed (anti)

1 Lipase from porcine pancreas, type II (PPL II) 120 43 60:40 592 Protease from Streptomces griseus 120 46 32:68 03 Lipase from wheat germ 120 28 54:46 124 Amano Lipase A from Aspergillus niger 120 26 40:60 135 Trypsin from porcine pancreas 120 45 57:43 196 Lipase from Candida rugosa 144 Trace — —7 Lipase from Rhizopus niveus 144 Trace — —8 Protease from Bacillus sp. 144 Trace — —9 no enzyme 168 nr — —10 Bovine serum albumin (B.S.A.) 120 43 37:63 511 PPL II denatured with ureae 120 9 35:65 2012 PPL II inhibited with PMSFf 120 Trace — —13 NaHCO3

g 48 61 31:69 0

a The reaction was performed by employing ketone 1a (0.25 mmol), 4-nitrobenzaldehyde 2a (0.50 mmol), enzyme (50 mg) in 1 mL mixed solvents [H2O/(H2O+CH3CN) = 1:10, v/v] at 25 �C.

b Isolated yield after silica gel chromatography.c Determined by chiral HPLC.d Determined by chiral HPLC, and the absolute configuration was determined by comparison with literature.11a

e Pre-treated with 50 mg urea in 1 mL water (0.83 M) at 100 �C for 24 h.f Pre-treated with 50 mg PMSF in 1 mL THF (0.29 M) at 25 �C for 24 h.g The reaction was performed by employing ketone 1a (0.25 mmol), 4-nitrobenzaldehyde 2a (0.25 mmol), NaHCO3 (20 mg) in 1 mL EtOH.

Table 2Solvent screening of PPL II-catalyzed direct asymmetric aldol reactiona

Entry Solvent Yieldb (%) drc (anti:syn) % eec (anti)

1 CH3CN 43 60:40 592 MTBE 31 52:48 503 Xylene 21 56:44 434 1,4-Dioxane 30 59:41 405 THF 43 55:45 366 i-PrOH 39 51:49 187 DMSO 46 44:56 168 DMF 52 47:53 15

a The reaction was performed by employing ketone 1a (0.25 mmol), 4-nitro-benzaldehyde 2a (0.50 mmol), PPL II (50 mg) in 1 mL mixed solvents [H2O/(H2O+solvent) = 1:10, v/v] at 25 �C for 120 h.

b Isolated yield after silica gel chromatography.c Determined by chiral HPLC.

4960 Z. Guan et al. / Tetrahedron Letters 53 (2012) 4959–4961

45% yield and 19% ee (Table 1, entry 5). However, lipase fromCandida rugosa, lipase from Rhizopus niveus, and protease fromBacillus sp displayed almost no activity for the aldol reaction andjust trace aldol products were observed (Table 1, entries 6–8). Asseen from Table 1, in order to verify that enzyme was necessaryand the reaction took place at the active site of enzyme, some con-trol experiments were carried out, and the significative resultswere observed. In the absence of enzyme no detectable productswere obtained for the model aldol reaction even after 168 h (Ta-ble 1, entry 9), which indicated that catalyst was essential for thereaction. When the reactants were incubated with urea-denaturedPPL II, just 9% yield was obtained (Table 1, entry 11), which sug-gested the tertiary structure of the enzyme might also be neces-sary. Then PPL II was pre-treated with PMSF (phenylmethylsulfonylfluoride, an irreversible serine protein inhibitor), whichgave almost no product after 120 h (Table 1, entry 12). It mightbe concluded that the catalytic site of PPL II contributed to the bio-catalytic promiscuity.14 Moreover, the reaction catalyzed by non-enzyme protein bovine serum albumin (B.S.A.) gave product in43% yield with only 5% ee (Table 1, entry 10), which showed thatnon-enzyme protein also had the ability to catalyze aldol reaction,but it almost did not have the enantioselectivity for aldol products.These results implied that the reaction must take place in a specificfashion on the catalytic site of PPL II. Therefore, PPL II was chosenas a catalyst for the aldol reaction in our study.

The reaction medium has been recognized to be one of the mostimportant factors influencing the enzymatic reactions.15 Thus, wesurveyed the reaction in eight organic solvents in the presence ofwater and the results are shown in Table 2. The results clearly indi-cated that the catalytic activity and the stereoselectivity of PPL IIwere significantly influenced by the reaction media. Generally,the reaction in high-polarity solvents such as DMF and DMSO gavehigher yields (Table 2, entries 7 and 8) than in low-polarity sol-vents such as MTBE and xylene (Table 2, entries 2 and 3). The ste-reoselectivities including diastereo- and enantioselectivities hadno clear correlation with the polarities of solvents. It could be seen

that PPL II gave the best yield of 52% but the lowest enantioselec-tivity of 15% ee in DMF (Table 2, entry 8). However, the best diaste-reoselectivity (60:40/anti:syn) and enantioselectivity (59% ee) wereobtained in CH3CN (Table 2, entry 1). In order to pursue asymmet-ric aldol condensation, CH3CN was chosen as the reaction mediumfor further investigation.

In order to optimize the reaction conditions, the influences ofwater content in the reaction medium, the molar ratio of sub-strates, enzyme concentration, and temperature were investigated.The time course of PPL II-catalyzed aldol reaction was also plotted.The optimal temperature of 30 �C, the water content of 0.10 [H2O/(H2O+CH3CN) = 1:10, v/v], the 1:1 molar ratio of ketone to alde-hyde, and the enzyme concentration of 50 mg/ml were chosen asthe optimized reaction conditions. For details, please consult theSupplementary data.

Having established optimized reaction conditions, we probedthe generality of the PPL II-catalyzed aldol reaction. Various aro-matic aldehydes and heterocyclic ketones were investigated. Theresults were summarized in Table 3. It could be seen that thePPL-catalyzed aldol addition of heterocyclic ketones with aromatic

Table 3Substrate scope of PPL II-catalyzed direct asymmetric aldol reaction of aromatic aldehydes and heterocyclic ketonesa

X

O

+

O

H

X

O OH

PPL II

CH3CN/H2OR +

anti-31 2

syn-isomerR

30 °C

Entry R X Product Time (h) Yieldb (%) drc (anti:syn) % eed (anti)

1 4-NO2 NBoc 3a 120 49 62:38 622 3-NO2 NBoc 3b 120 45 32:68 453 2-NO2 NBoc 3c 120 36 53:47 874 3-Br NBoc 3d 120 38 47:43 655 4-NO2 O 3e 120 56 38:62 466 3-NO2 O 3f 120 50 37:63 507 2-NO2 O 3g 144 43 48:52 628 4-CN O 3h 144 35 34:66 629 4-NO2 S 3i 120 41 51:49 4310 3-NO2 S 3j 120 34 80:20 7211 2-NO2 S 3k 144 31 83:17 7412 4-CN S 3l 144 32 54:46 61

a The reactions were performed by employing aldehyde (0.25 mmol), ketone (0.25 mmol), PPL II (50 mg) in 1 mL mixed solvents [H2O/(H2O+CH3CN) = 1:10, v/v] at 30 �C.b Isolated yield after silica gel chromatography.c Determined by chiral HPLC.d Determined by chiral HPLC, and the absolute configurations were determined by comparison with literature (for details, please consult the Supplementary data).

Z. Guan et al. / Tetrahedron Letters 53 (2012) 4959–4961 4961

aldehydes gave the desired products in reasonable yields (31–56%)and enantioselectivities (up to 87% ee) (Table 3, entries 1–12). Weinvestigated the electronic effect and steric effect of the substitu-ents in aromatic aldehydes on the reaction. This procedure workedwell when benzaldehydes with electron-withdrawing groups wereemployed (Table 3, entries 1–12). On the contrary, only trace prod-ucts were obtained when benzaldehydes with electron-donatinggroups, such as methyl and methoxyl, were employed (data werenot shown). This could be explained that electron-withdrawinggroups enhance the electrophilicity of carbonyl carbons in alde-hydes which facilitates the reaction, while electron-donatinggroups lessen the electrophilicity. Otherwise, the stericallyhindered substituents in benzaldehydes had a great impact onthe stereoselectivity and yield of the reaction. For instance, amongthe 2-, 3-, and 4-nitrobenzaldehydes, the most sterically hindered2-nitrobenzaldehyde provided the best enantioselectivity but thelowest yield (Table 3, entries 3, 7, and 11), while the least stericallyhindered 4-nitrobenzaldehyde provided the best yield but rela-tively low enantioselectivity (Table 3, entries 1, 5, and 9). We spec-ulated that the large steric hindrance limited the attack direction ofketone.

In conclusion, we herein report a lipase (PPL II)-catalyzed directasymmetric aldol reaction of heterocyclic ketones with aromaticaldehydes in CH3CN/H2O. A series of substrates participated inthe reaction. Although the yields and stereoselectivities are notsatisfied, it is interesting that PPL II possesses the function of aldol-ase in organic solvents. This novel process also provides an exam-ple for exploring environmentally friendly enzyme-catalyzedsynthetic route for organic chemistry in nonaqueous media.

Acknowledgments

Financial support from Natural Science Foundation Project ofCQ CSTC (2009BA5051) is gratefully acknowledged.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.tetlet.2012.07.007.

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