Pepsin-catalyzed direct asymmetric aldol reactions for the synthesis of vicinal diol compounds

Pepsin-catalyzed direct asymmetric aldol reactions for the synthesis of vicinal diol compounds

Tetrahedron 71 (2015) 1659e1667 Contents lists available at ScienceDirect Tetrahedron journal homepage: www.elsevier.com/locate/tet Pepsin-catalyze...

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Tetrahedron 71 (2015) 1659e1667

Contents lists available at ScienceDirect

Tetrahedron journal homepage: www.elsevier.com/locate/tet

Pepsin-catalyzed direct asymmetric aldol reactions for the synthesis of vicinal diol compounds Ling-Yu Li, Da-Cheng Yang, Zhi Guan *, Yan-Hong He * Key Laboratory of Applied Chemistry of Chongqing Municipality, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 October 2014 Received in revised form 19 January 2015 Accepted 27 January 2015 Available online 31 January 2015

The catalytic promiscuity of pepsin from porcine gastric mucous was observed in catalysis of the direct asymmetric aldol reactions of aromatic aldehydes with acetones, which were substituted by hydroxy-, dihydroxy-, methoxy- and benzyloxy- for the synthesis of diol compounds in acetonitrile. This biocatalysis was also applicable to the aldol reactions of cyclic or hetereocyclic ketones with aromatic aldehydes. Yields of up to 87%, diastereoselectivities of up to >99/1 dr and enantioselectivities of up to 75% ee were achieved. Ó 2015 Elsevier Ltd. All rights reserved.

Keywords: Porcine pepsin Asymmetric aldol reaction Diol compound Biocatalysis

1. Introduction Aldol reaction is one of the most classical and powerful methods to construct CeC bonds in organic synthesis.1 Chiral 1,2-diols, which are very important building blocks are found in a vast array of natural and biologically active molecules (Fig. 1).2 1,2-Diols can be formed via aldol reactions of 1-hydroxypropan-2-one or 1,3dihydroxypropan-2-one to aldehydes. However, these compounds are still challenging in synthesis.2a,3 Especially, in asymmetric synthesis, there still exist some limitations such as the need for protected hydroxyl and dihydroxy-acetones.2a On the other hand, the

Fig. 1. 1,2-Diol units in natural products.

* Corresponding authors. Fax: þ86 23 68254091; e-mail addresses: [email protected] swu.edu.cn (Z. Guan), [email protected] (Y.-H. He). http://dx.doi.org/10.1016/j.tet.2015.01.061 0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

aldol reactions of 1-hydroxypropan-2-one or 1,3-dihydroxypropan2-one to aldehydes are crucial for the biosynthesis of sugars and their derivatives.4 Initially, the aldol condensations for the formation of carbohydrates were catalyzed by type I aldolase or type II aldolase through different mechanism, respectively in nature (Scheme 1).4 However, many of such reactions were only capable of 1,3-dihydroxypropan-2-one phosphate (DHAP) as a specific donor.5 This paper first report using hydrolase (pepsin from porcine gastric mucous) to catalyze the aldol reactions with unprotected 1hydroxypropan-2-one and 1,3-dihydroxypropan-2-one as electron donors. Enzymes as practical catalysts and enzymatic methods as part of green technologies for organic synthesis were considered to possess great potential, and they were exploited increasingly in asymmetric synthesis.6 Originally, enzyme catalytic reactions had a narrow scope of application since reactions were restricted to natural substrates of enzyme and aqueous reaction media.6c,7 In recent years, enzyme promiscuity including condition promiscuity, substrate promiscuity and catalytic promiscuity has emerged as a new frontier in bio-catalysis.8 The study of enzyme promiscuity not only provided novel research methods and tools for organic synthesis, but also formed a bond, which led to biology and organic chemistry moving forward together.6d,9 As promiscuous enzymes, hydrolases with high stability, multiple sources and a wide range of substrates, undoubtedly played a pivotal role in organic synthesis.3a,10 Some elegant works on hydrolase-catalyzed synthesis have been reported.6c,10e11 Among them, hydrolase-catalyzed aldol reactions have been successfully achieved. In 2003, Berglund and

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Scheme 1. Mechanism of type I and type II aldolases.

co-workers first used lipase from Candida Antarctica (CAL-B) to catalyze the self-aldol additions of propanal and hexanal in cyclohexane. They found that mutant CAL-B (mutants Ser105Ala and Ser105Gly) exhibited increased reaction rate than that of wild-type lipase.12 In 2008, Wang and co-workers reported the first lipasecatalyzed asymmetric aldol reaction, and the best ee of 43.6% was detected for the reaction of 4-nitrobenzaldehyde and acetone.13 Subsequently, some other asymmetric aldol reactions catalyzed by lipases from porcine and bovine pancreas were described.14 In addition, some proteases, such as pepsin15 and trypsin from porcine pancreas,16 chymopapain,17 ficin,18 Alcalase-CLEAÒ,19 alkaline protease from Bacillus licheniformis,20 acidic protease from Aspergillus usamii,21 and proteinase from Aspergillus melleus,22 were disclosed to show the promiscuous behavior in catalysis of the asymmetric aldol reactions of aromatic aldehydes with cyclic ketones, heterocyclic ketones or acetone. Besides lipases and proteases, some other hydrolases were also found to have the ability to catalyze asymmetric aldol reactions. For example, nuclease P1 from Penicillium citrinum could catalyze the asymmetric aldol reactions of aromatic

aldehdes with cyclic ketones, and isatin derivatives with cyclic ketones;23 thermophilic esterase from the archaeon Aeropyrum pernix K1 could promote the asymmetric aldol addition of 2butanone and 4-nitrobenzaldehyde.24 However, to date, no promiscuous hydrolase-catalyzed aldol reaction for the synthesis of vicinal diol compounds has been reported yet. Herein, we first report that pepsin from porcine gastric mucous, a member of the well-known hydrolases, can effectively catalyze the direct asymmetric aldol reactions to afford syn-selective 1,2-diols using unprotected 1-hydroxypropan-2-one as a donor and various substituted aromatic aldehydes as accepters. 2. Results and discussion In our initial study, we chose the reaction of 4nitrophenylaldehyde and 1-hydroxypropan-2-one as a model reaction. Originally, the model aldol reaction was performed in the absence of enzyme, and no detectable product was obtained after 120 h (Table 1, entry 1). After that, we investigated some proteases

Table 1 The screening of proteases for the catalysis of aldol reactiona

Entry 1 2 3 4 5 6 7 8 9 10 11 12

Enzyme None Pepsin from porcine gastric mucous Proteinase from Aspergillus melleus, type XXIII Protease from Rhizopus sp. Protease from Aspergillus saitoi, type XIII Trypsin, from porcine pancreas Protease from Bacillus sp. Rennet from Mucor miehei, type II Ficin from fig tree latex Bromelain from pineapple stem Papain from Carica papaya Protease from Streptomyces griseus

Time (h) 120 120 120 96 96 120 96 96 120 120 96 96

Yield (%)b n.d. 76 47 79 9 23 13 7 33 36 60 56

e

dr (syn/anti)c

ee (syn) (%)d

d 63/37 63/37 61/39 65/35 62/38 63/37 67/33 61/39 55/45 51/49 64/36

d 35 26 18 18 17 17 13 10 6 0 0

a The reactions were conducted using 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (2.5 mmol), enzyme (50 mg, lyophilized powder), MeCN (0.9 mL), deionized water (0.1 mL) at 25  C. b Yield of the isolated product after chromatography on silica gel. c Determined by HPLC analysis of the diastereomeric isomers. d Determined by HPLC analysis using a chiral column (AD-H); relative and absolute configurations of the products were determined by comparison with the known 1H NMR and chiral HPLC analysis. e n.d.: no product was detected.

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for the catalysis of the model aldol reaction (Table 1). The best yield of 79% was achieved using protease from Rhizopus sp. as a catalyst, but only 18% ee was obtained (Table 1, entry 4). Luckily, using pepsin from porcine gastric mucous as a catalyst, we obtained a good yield of 76%, moderate dr of 63/37 with the best ee of 35% (Table 1, entry 2). In addition, some other proteases investigated also exhibited certain catalytic activity toward the model reaction, respectively (Table 1, entries 3e12). In view of the above-mentioned results, pepsin from porcine gastric mucous was chosen as the catalyst. To confirm the specific catalytic effect of pepsin on the model aldol reaction, some control experiments were performed (Table 2). In the absence of enzyme, no reaction occurred (Table 2, entry 1). The model aldol reaction with the pepsin preparation gave the product in a good yield of 76% with 35% ee (Table 2, entry 2), which indicated that pepsin preparation indeed catalyzed the reaction in an asymmetric manner. To exclude the possibility that some nonenzyme components catalyzed the reaction, the pepsin preparation was pretreated at high temperature (100  C) for 24 h, and then used to catalyze the model reaction, which only gave the product in a low yield of 13% with 22% ee (Table 2, entry 3). At the same time, the natural activity of pepsin in hydrolyzing hemoglobin was also tested, and it showed that high temperature treatment caused serious denaturation of pepsin (the natural activity decreased from 451 U/mg protein to 41 U/mg protein after 100  C treatment). These results indicated that the observed catalysis effect arose from the enzyme itself instead of non-enzyme components, and the native fold of the enzyme was not only responsible for the natural activity but also for the promiscuous activity. To further confirm this speculation, metal ion Cu2þ was used as a denaturation agent to pretreat the pepsin preparation at 25  C for 24 h, and the Cu2þ pretreated pepsin was then used to catalyze the model aldol reaction, giving the product in a low yield of 9% with 34% ee (Table 2, entry 4). Meantime, a parallel experiment without Cu2þ was conducted, which gave the product in a good yield of 78% with 37% ee (Table 2, entry 5), indicating that the process itself (the treatment of pepsin

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at 25  C for 24 h) did not influence the enzyme. Moreover, Cu2þ alone was verified no effect on the model reaction (Table 2, entry 6). These results further confirmed that enzyme was responsible for the observed catalytic effect, and once the enzyme denatured, its catalytic ability in the aldol reaction nearly completely lost. In addition, pepstatin, a specific competitive peptide inhibitor of pepsin,25 was also used to inhibit the enzyme, and the decreases of 16% in yield and of 3% in ee were detected in the presence of pepstatin compared to the parallel experiment without pepstatin (Table 2, entries 7 and 8), demonstrating that the promiscuous enzymatic process may proceed in the active site. From the above control experiments, it could be deduced that the specific fold of pepsin played a decisive role for the asymmetric aldol reaction. Reaction medium has been considered as one of the most important factors influencing catalytic activity and the stability of an enzyme.7,26 Thus, we investigated the catalytic effects of pepsin in different solvents (Table 3). Using water as a solvent, a poor yield of 7% with enantioselectivity of 21% ee was obtained (Table 3, entry 12). The reaction in toluene gave the product in a good yield of 87% but with a low ee value of 26% (Table 3, entry 7). Among the tested solvents, the best product enantioselectivity of 35% ee in a moderate yield of 76% was achieved in acetonitrile (Table 3, entry 1). Therefore, we chose acetonitrile as the optimal solvent for the following studies. As water content in an organic solvent affects both stereoselectivity and activity of the enzymatic reaction,16,17 we investigated the effects of different amounts of water addition in acetonitrile on the pepsin-catalyzed model aldol reaction (Table 4). Obviously, the water addition had a great influence on the selectivity and activity of pepsin in the model aldol reaction. The best yield of 76% with 63/37 dr and 35% ee was obtained at the water addition of 10% [H2O/(H2OþMeCN), in vol.] (Table 4, entry 2), but the best ee value of 42% with 70/30 dr and 70% yield was obtained without adding water into the reaction system (Table 4, entry 1). The acetonitrile employed was A.R. grade, and it was used directly without drying treatment. Considering the selectivity of the

Table 2 Control experiments for the pepsin-catalyzed direct asymmetric aldol reactiona

Entry

Catalyst

Yield (%)b

dr (syn/anti)c

ee (syn) (%)d

1 2 3 4 5 6 7 8

No enzyme Pepsin Pepsin (pretreated Pepsin (pretreated Pepsin (pretreated CuSO4 (39.9 mg)h Pepsin (pretreated Pepsin (pretreated

n.d.k 76 13 9 78 n.d.k 83l 67l

d 63/37 57/43 61/39 60/40 d 59/41 61/39

d 35 22 34 37 d 22 19

at 100  C)e with 0.25 M Cu2þ at 25  C)f at 25  C)g in DMSO with 10% H2O)i with pepstatin in DMSO with 10% H2O)j

a Unless otherwise noted, the reaction was conducted using 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (2.5 mmol), pepsin (22.5 kU), MeCN (0.9 mL), deionized water (0.1 mL) at 25  C for 120 h. b Yield of the isolated product after chromatography on silica gel. c Determined by HPLC analysis of the diastereomeric isomers. d Determined by HPLC analysis using a chiral column (AD-H). e Pepsin (22.5 kU) in deionized water (1.0 mL) was stirred at 100  C for 24 h, and then water was removed under reduced pressure before use. f Pepsin (22.5 kU) in Cu2þ solution (0.25 M, 39.9 mg CuSO4 in 1.0 mL deionized water) was stirred at 25  C for 24 h and then water was removed under reduced pressure before use. g Pepsin (22.5 kU) in deionized water (1.0 mL) was stirred at 25  C for 24 h, and then water was removed under reduced pressure before use. h CuSO4 (39.9 mg) was used instead of pepsin. i Pepsin (2.25 kU) in the mixed solvents (0.09 mL DMSOþ0.01 mL H2O) was stirred at 25  C for 5 h, and then 4-nitrobenzaldehyde (0.05 mmol) and 1-hydroxypropan-2-one (0.25 mmol) were added. The mixture was stirred at 25  C for another 120 h. j Pepsin (2.25 kU) and pepstatin (5 mg) in the mixed solvents (0.09 mL DMSOþ0.01 mL H2O) were stirred at 25  C for 5 h, and then 4-nitrobenzaldehyde (0.05 mmol) and 1hydroxypropan-2-one (0.25 mmol) was added. The mixture was stirred at 25  C for another 120 h. k n.d.: no reaction was detected. l Yield was determined by HPLC.

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Table 3 The effect of solvents on the pepsin-catalyzed direct asymmetric aldol reactiona

Entry

Solvent

Yield (%)b

dr (syn/anti)c

ee (syn) (%)d

1 2 3 4 5 6 7 8 9 10 11 12 13

MeCN EtOAc EtOH THF Butyl acetate CH2Cl2 Toluene Cyclohexane CHCl3 MTBE Hexane H2O Organic solvent free

76 71 84 83 72 64 87 86 85 83 78 7 79

63/37 61/39 60/40 65/35 57/43 58/42 58/42 58/42 59/41 58/42 58/42 61/39 57/43

35 30 29 28 27 27 26 24 24 24 24 21 24

a Unless otherwise noted, the reaction conditions were as follows: 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (2.5 mmol), solvent (0.9 mL), deionized water (0.1 mL) and pepsin (22.5 kU) at 25  C for 120 h. b Yield of the isolated product after silica gel chromatography. c Determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers. d Determined by HPLC analysis using a chiral column (AD-H).

Table 4 Influence of water addition on the pepsin-catalyzed direct asymmetric aldol reactiona

Entry

Water addition (%)

Yield (%)b

dr (syn/anti)c

ee (syn) (%)d

1 2 3 4 5 6 7 8 9 10 11

0 10 20 30 40 50 60 70 80 90 100

70 76 66 66 54 43 34 35 19 13 7

70/30 63/37 63/37 63/37 63/37 60/40 60/40 60/40 61/39 66/34 61/39

42 35 28 22 22 20 19 19 19 19 21

a The reaction conditions were as follows: 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (2.5 mmol), deionized water (0e1.0 mL), MeCN (1.0e0 mL) and pepsin (22.5 kU) at 25  C for 120 h. b Yield of the isolated product after silica gel chromatography. c Determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers. d Determined by HPLC analysis using a chiral column (AD-H).

reaction, ultimately we selected acetonitrile (A.R.) as the optimized solvent without addition of water for further studies. After that, we examined the effect of molar ratio of substrates on the pepsin-catalyzed aldol reaction (Table 5). The results of the reaction were obviously influenced by the molar ratio of substrates. We fixed the amount of 4-nitrobenzaldehyde, and gradually increased the amount of 1-hydroxypropan-2-one. The yield, dr and ee values ascended with the increasing amounts of 1-hydroxypropan2-one. Among the performed experiments, the best ee value of 48% with yield of 77% and dr value of 72/28 was obtained when the molar ratio of 1-hydroxypropan-2-one to 4-nitrobenzaldehyde was 13:1 (Table 5, entry 6). Thus, the molar ratio of 13:1 (1-hydroxypropan-2one 6.5 mmol to 4-nitrophenylaldehyde 0.5 mmol) was identified as the optimal ratio.

Next, we surveyed the effect of enzyme loading on the pepsincatalyzed aldol reaction (Table 6). The yield and stereoselectivity of the reaction were affected by the enzyme loading. A growing ee value was observed with increasing enzyme loading from 4.5 kU to 22.5 kU. The best enantioselectivity of 48% ee was obtained with a yield of 77% when the reaction was performed with an enzyme loading of 22.5 kU (Table 6, entry 5). Thus, we chose the enzyme loading of 22.5 kU to continue investigation. Temperature is a crucial factor affecting stability of enzymes, and stereoselectivity and reaction rate of enzymatic reactions.20,27 Hence, investigating the influence of temperature on the pepsin-catalyzed aldol reaction was essential. We then conducted the model reaction at several different temperatures (Table 7). A relatively good result of 87% yield with 71/29 dr and

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Table 5 The effect of mole ratio of substrates on the pepsin-catalyzed direct asymmetric aldol reactiona

Entry

Molar ratio (1a/2a)

Yield (%)b

dr (syn/anti)c

ee (syn) (%)d

1 2 3 4 5 6 7 8

1/1 1/3 1/5 1/7 1/10 1/13 1/15 1/20

25 69 70 70 74 77 75 80

70/30 70/30 70/30 69/31 73/27 72/28 72/28 71/29

23 40 42 46 47 48 48 48

a b c d

The reaction conditions were as follows: 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (0.5e10 mmol), MeCN (1.0 mL) and pepsin (22.5 kU) at 25  C for 120 h. Yield of the isolated product after silica gel chromatography. Determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers. Determined by HPLC analysis using a chiral column (AD-H).

Table 6 Effect of enzyme loading on the pepsin-catalyzed direct asymmetric aldol reactiona

Entry

Enzyme loading (kU)

Yield (%)b

dr (syn/anti)c

ee (syn) (%)d

1 2 3 4 5 6

4.5 9.0 13.5 18.0 22.5 27

39 51 76 80 77 77

62/38 68/32 70/30 70/30 72/28 70/30

31 40 43 45 48 46

a b c d

The reaction conditions were as follows: 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (6.5 mmol), MeCN (1.0 mL) and pepsin (4.5e27 kU) at 25  C for 120 h. Yield of the isolated product after silica gel chromatography. Determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers. Determined by HPLC analysis using a chiral column (AD-H).

Table 7 Effect of temperature on the pepsin-catalyzed direct asymmetric aldol reactiona

Entry

Temperature ( C)

Yield (%)b

dr (syn/anti)c

ee (syn) (%)d

1 2 3 4 5 6 7 8

25 30 35 40 45 50 55 60

77 87 88 84 82 83 85 84

72/28 71/29 70/30 70/30 70/30 70/30 70/30 64/36

48 47 44 42 38 39 35 30

a The reaction conditions were as follows: a mixture of 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (6.5 mmol) and pepsin (22.5 kU) in MeCN (1.0 mL) was stirring at 25e60  C for 120 h. b Yield of the isolated product after silica gel chromatography. c Determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers. d Determined by HPLC analysis using a chiral column (AD-H).

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47% ee was exhibited at 30  C (Table 7, entry 2). Although temperature was greatly elevated, the yield was not improved. On the contrary, high temperature brought a decline in enantioselectivity. Accordingly, carrying out the reaction at 30  C was proven to be the best choice. Subsequently, time course of the pepsin-catalyzed asymmetric aldol reaction between 4-nitrobenzaldehyde and 1-hydroxypropan2-one was tested at 30  C, 40  C and 50  C, respectively (Table 8). Generally, the yield increased as the reaction time went on at the early stage and finally kept almost constant. A slight growth of ee value was detected at the early stage when the reaction was performed at 30  C or 40  C (Table 8, entries 1e7), while no significant change of dr was detected along the time course at each temperature. Obviously, increasing the temperature could accelerate the reaction, but the lower stereoselectivities were received at higher temperature. To test the generality and application scope of catalytic promiscuity embodied by pepsin, some other substrates including various aldehydes and ketones were investigated to expand upon the pepsin-catalyzed direct asymmetric aldol reaction. The results were summarized in Table 9 and Table 10. All the experiments showed that pepsin had the ability to tolerate different aromatic aldehydes as acceptors and various ketones as donors such as 1-hydroxypropan-2-one, protected-1-hydroxypropan-2-one, 1,3dihydroxypropan-2-one, acetone and cyclic-ketones. In these reactions, aromatic aldehydes exhibited excellent reactivity. The interaction of aromatic aldehydes with acyclic ketones catalyzed by pepsin mainly offered syn-products (Table 9), while anti-products were obtained from the reactions of aromatic aldehydes with cyclic ketones (Table 10). Substituents on the aromatic ring had a significant impact on both the yields and enantioselectivities. Generally, aromatic aldehydes with electron-withdrawing groups provided higher yields than those with electron-donating substituents (Table 9, entries 1e11 and 13). Of course, different ketones as substrates showed different effects on the aldol reaction. Acyclic ketones as substrates reacting with various aromatic aldehydes provided low to high yields and low to moderate enantioselectivities (Table 9). In general, 1-hydroxypropan-2-one showed better reactivity than dihydroxylacetone, acetone and protected 1-hydroxypropan-

2-one. For instance, the reaction of 4-nitrobenzaldehyde with 1-hydroxypropan-2-one provided product in a good yield of 87% with 69/31 dr (syn/anti) and 47% ee (Table 9, entry 1). The best enantioselectivity of 70% ee with 88/12 dr in a fair yield of 53% was obtained when 1-hydroxypropan-2-one reacted with 1-naphthaldehyde (Table 9, entry 14). However, dihydroxylacetone or acetone reacting with aromatic aldehydes only gave low yields of 11e16% with 30e52% ee (Table 9, entries 15, 16, 21 and 22). The reactions of methoxyacetone with nitrobenzaldehydes produced products in yields of 21e74% with 55/45->99/1 dr and 28e40% ee (Table 9, entries 17 and 18). The reactions of 1-(benzyloxy) propan2-one with aromatic aldehydes gave products in yields of 24e58% with 37e53% ee (Table 9, entries 19 and 20). Moreover, heteroaromatic aldehyde, 2-thenaldehyde, could also participate in the reaction with 1-hydroxypropan-2-one giving the desired product in a low yield of 7% with >99/1 dr and 30% ee (Table 9, entry 23). In addition, we attempted to use aliphatic aldehydes as aldol acceptors, such as using isobutyraldehyde and (R)-2,2-dimethyl-1,3dioxolane-4-carbaldehyde to react with 1-hydroxypropan-2-one and 1,3-dihydroxypropan-2-one, respectively, but no products were detected after 96 h (Table 9, entries 24 and 25). The reactions of cyclic ketones with aromatic aldehydes were listed in Table 10. Pepsin showed good substrate adaptability to different cyclic ketones including cyclohexanone, cyclopentanone and heterocyclic ketones containing nitrogen, oxygen or sulfur. The heterocyclic ketones as the aldol donors showed good reactivity. The best yield of 85% was obtained with 77/23 dr and 66% ee for the reaction of 4-nitrobenzaldehyde and tetrahydropyran-4-one (Table 10, entry 8). The best enantioselectivity of 75% ee with 80/20 dr and 40% yield was received when 3-nitrobenzaldehyde reacted with cyclohexanone (Table 10, entry 2). Generally, the reactions of aromatic aldehydes with cyclopentanone or heterocyclic ketones gave better yields than those with cyclohexanone. 3. Conclusion In summary, the pepsin from porcine gastric mucous displayed catalytic promiscuity in catalysis of direct asymmetric aldol reactions of aromatic aldehydes with acetones, which were substituted by

Table 8 The time course of the pepsin-catalyzed direct asymmetric aldol reaction at different temperaturesa

Entry

Time (h)

30  C

40  C

50  C

Yield (%)b

drc

ee (%)d

Yield (%)b

drc

ee (%)d

Yield (%)b

drc

ee (%)d

1 2 3 4 5 6 7 8 9 10 11 12

2 4 6 8 12 16 24 36 48 60 72 96

12 23 24 26 53 63 81 83 88 85 89 87

70/30 70/30 68/32 70/30 68/32 70/30 70/30 68/32 68/32 68/32 69/31 68/32

34 39 40 41 43 45 46 46 46 46 46 45

17 24 31 35 58 79 82 87 86 88 90 86

69/31 69/31 68/32 69/31 68/32 69/31 69/31 68/32 69/31 69/31 70/30 68/32

35 38 40 40 42 44 44 43 44 43 43 42

50 70 72 68 79 83 83 85 82 86 87 86

69/31 69/31 68/32 68/32 68/32 69/31 68/32 68/32 68/32 68/32 68/32 68/32

36 37 37 38 36 39 38 38 38 38 37 38

a The reaction conditions were as follows: a mixture of 4-nitrobenzaldehyde (0.5 mmol), 1-hydroxypropan-2-one (6.5 mmol) and pepsin (22.5 kU) in MeCN (1.0 mL) was stirred at 30  C, 40  C or 50  C for specified time. b Yield of the isolated product after silica gel chromatography. c dr of syn/anti determined by 1H NMR and chiral HPLC analysis of the diastereomeric isomers. d ee values of syn-isomer, determined by HPLC analysis using a chiral column (AD-H).

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Table 9 Substrate scope of the pepsin-catalyzed direct asymmetric aldol reactions of aldehydes with acyclic ketonesa

Entry

R1

R2, R3

Prod.

Time (h)

Yield (%)b

dr (syn/anti)c

ee (syn) (%)d

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

4-NO2C6H4 2-NO2C6H4 3-NO2C6H4 4-FC6H4 2-ClC6H4 4-ClC6H4 2,6-Cl2C6H3 3-BrC6H4 4-BrC6H4 4-CNC6H4 4-CF3C6H4 C6H5 4-MeC6H4 1-Naphthyl 2-NO2C6H4 4-NO2C6H4 2-NO2C6H4 4-NO2C6H4 4-NO2C6H4 4-ClC6H4 4-NO2C6H4 4-BrC6H4 2-Thienyl Isobutyl 2,2-Dimethyl-1,3-dioxolaneylf

H, OH H, OH H, OH H, OH H, OH H, OH H, OH H, OH H, OH H, OH H, OH H, OH H, OH H, OH OH, OH OH, OH H, OMe H, OMe H, OBn H, OBn H, H H, H H, OH H, OH OH, OH

3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o 3p 3q 3r 3s 3t 3u 3v 3w 3x 3y

96 120 90 144 96 120 120 96 144 120 120 108 144 120 120 120 168 144 168 168 120 144 120 96 96

87 80 86 34 46 39 66 70 54 82 62 55 23 53 14 11 21 74 58 24 16 11 7 n.d.e n.d.e

69/31 86/14 71/29 76/24 87/13 82/18 81/19 76/24 79/21 68/32 80/20 78/22 82/18 88/12 86/14 74/26 >99/1 55/45 60/40 74/26 d d >99/1 d d

47 64 53 49 59 45 24 54 44 11 44 66 59 70 52 30 40 28 37 53 32 46 30 d d

Reaction conditions: a mixture of 1 (0.5 mmol), 2 (6.5 mmol) and pepsin (22.5 kU) in MeCN (1.0 mL) at 30  C. Yield of isolated product after silica gel chromatography. c Determined by HPLC analysis of the diastereomeric isomers. d Determined by HPLC analysis using a chiral column; relative and absolute configurations of the products were determined by comparing with the known 1H NMR and chiral HPLC analysis. e n.d.: no reaction was detected. f The 2,2-dimethyl-1,3-dioxolane-4-carbaldehyde is R configuration. a

b

hydroxy-, dihydroxyl, methoxyl or benzyloxyl for the synthesis of diol compounds in acetonitrile. This biocatalysis was also applicable to the aldol reactions of cyclic ketones or hetereocyclic ketones with aromatic aldehydes. Yields of up to 87%, diastereoselectivities of up to >99/1 dr and enantioselectivities of up to 75% ee were achieved. Although the yields and stereoselectivities were not thoroughly satisfied, using hydrolase to catalyze the aldol reactions with unprotected 1-hydroxypropan-2-one and 1,3-dihydroxypropan-2-one as aldol donors to afford syn-selective 1,2-diols was first reported. This finding not only provides a novel case for the application of pepsin, but also offers an alternative method to prepare optically active 1,2-diols. 4. Experimental 4.1. Materials All enzymes were purchased from SigmaeAldrich, Shanghai, China. Pepsin from porcine gastric mucous [P-7000-25G, Lot#050M1304V, 920 units/mg protein (used for Tables 1e7 and 9); Lot#SLBC4920V, 420 units/mg solid (used for Tables 8 and 10). One unit will produce DA280nm of 0.001 per minute at pH 2.0 at 37  C measured as TCA-soluble products using hemoglobin as substrate (Final volume¼16 mL, Light path¼1 cm)]; Proteinase from A. melleus, type XXIII (P4032-25G, Lot#080M1456V, 4 units/mg solid); Protease from Aspergillus saitoi, type XIII (P2143-5G, Lot#074K0727V, 1.0 units/mg solid); Trypsin, from porcine pancreas

(93615-25G, Lot#1434759V, 1460 units/mg solid); Protease from Bacillus sp. (P0029-50G,119K1454,1.7 AU-NH/G); Rennet from Mucor miehei, type Ⅱ (R5876-10G, Lot#080M1388V); Ficin from fig tree latex (F4165e1KU, Lot#030M1280V, 0.1 units/mg solid); Bromelain from pineapple stem (B4882-10G, Lot#SLBB8535V, 4 units/mg protein); Papain from Carica papaya (76220-25G, Lot#BCBD3116V, 3.6 units/ mg solid); Protease from Streptomyces griseus, type XIV (P5147-1G, Lot#040M1163V, 6.1 units/mg solid). Unless otherwise noted, all reagents were purchased from commercial suppliers and used without further purification. 4.2. Analytical methods Reactions were monitored by thin-layer chromatography (TLC) with Haiyang GF254 silica gel plates (Qingdao Haiyang chemical industry Co Ltd, Qingdao, China) using UV light and vanillic aldehyde as visualizing agents. Flash column chromatography was performed using 100e200 mesh silica gel at increased pressure. 1H NMR and 13C NMR spectra were recorded on Bruker-AM 300 (300 MHz) (Bruker BioSpin AG Ltd., Beijing, China) and Bruker-AM 400 (400 MHz) (Bruker BioSpin AG Ltd., Beijing, China). Chemical shifts were reported in ppm from TMS with the solvent resonance as the internal standard. Data were reported as follows: chemical shifts (d) in ppm, coupling constants (J) in Hz, and solvent (CD3OD and CDCl3). The enantiomeric excesses (ee) of aldol products were determined by chiral HPLC analysis performed using Chiralcel ADH, Chiralpak AS-H and OD-H columns (Daicel Chiral Technologies

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L.-Y. Li et al. / Tetrahedron 71 (2015) 1659e1667

Table 10 Substrate scope of the pepsin-catalyzed direct asymmetric aldol reactions of aromatic aldehydes with cyclic ketonesa

Prod.

Time (h)

Yield (%)b

dr (anti/syn)c

ee (anti) (%)d

2-NO2C6H4

5a

120

19

>99/1

73

2

3-NO2C6H4

5b

120

40

80/20

75

3

4-NO2C6H4

5c

120

38

65/35

58

4

4-ClC6H4

5d

120

21

88/12

15

5

2-NO2C6H4

5e

96

72

67/33

70

6

4-NO2C6H4

5f

72

66

64/36

63

7

4-ClC6H4

5g

72

60

67/33

70

8

4-NO2C6H4

5h

120

85

77/23

66

9

4-NO2C6H4

5i

120

46

93/7

70

10

4-NO2C6H4

5j

120

69

76/24

56

Entry

Ar

1

Cyclic ketone

Reaction conditions: a mixture of 1 (0.5 mmol), 4 (6.5 mmol) and pepsin (22.5 kU) in MeCN (1.0 mL) at 30  C. Yield of isolated product after silica gel chromatography. c Determined by HPLC analysis of the diastereomeric isomers. d Determined by HPLC analysis using a chiral column; relative and absolute configurations of the products were determined by comparing with the known 1H NMR and chiral HPLC analysis. a

b

CO., LTD.; Shanghai, China). Relative and absolute configurations of the products were determined by comparing with the known 1H NMR, 13C NMR and chiral HPLC analysis. All the aldol products are known compounds (for details about references, please see the Supplementary data). 4.3. General procedure for the pepsin-catalyzed aldol reactions A round-bottom flask was charged with pepsin (22.5 kU), aldehyde (0.5 mmol), and ketone (6.5 mmol), to which MeCN (1.0 mL) was introduced. The resultant mixture was stirred at 30  C for the specified reaction time and monitored by TLC. The reaction was terminated by filtering out the enzyme (with 40 mm Buchner funnel and qualitative filter paper), and mixed solvents (CH3OH/ CH2Cl2, 1:4) was employed to wash the filter paper and the residue to assure that the products were dissolved in the filtrate. The solvents were then removed under reduced pressure. The residue was

purified by silica gel flash column chromatography with petroleum ether/ethyl acetate as eluent to afford the product. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21276211 and No. 21472152). Supplementary data Supplementary data (Characterization of all the aldol products, and 1H NMR, 13C NMR and Chiral HPLC spectra for the aldol products are available as Supplementary data) related to this article can be found at http://dx.doi.org/10.1016/j.tet.2015.01.061. References and notes 1. (a) Machajewski, T. D.; Wong, C. H. Angew. Chem., Int. Ed. 2000, 39, 1352; (b) Mestres, R. Green Chem. 2004, 6, 583.

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