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Asymmetric synthesis of strongly fluorescent spirooxazino derivatives via multi-enzymatic telescopic reactions
Accepted Manuscript Title: Asymmetric synthesis of strongly fluorescent spirooxazino derivatives via multi-enzymatic telescopic reactions Authors: Xiao-Yang Chen, Yu-Jing Hu, Tao Hu, Xian-Fu Lin, Qi Wu PII: DOI: Reference:
S1381-1177(17)30014-0 http://dx.doi.org/doi:10.1016/j.molcatb.2017.01.014 MOLCAB 3513
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
8-11-2016 4-1-2017 20-1-2017
Please cite this article as: Xiao-Yang Chen, Yu-Jing Hu, Tao Hu, Xian-Fu Lin, Qi Wu, Asymmetric synthesis of strongly fluorescent spirooxazino derivatives via multi-enzymatic telescopic reactions, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2017.01.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Asymmetric synthesis of strongly fluorescent spirooxazino derivatives via multi-enzymatic telescopic reactions Xiao-Yang Chen‡, Yu-Jing Hu‡, Tao Hu, Xian-Fu Lin, Qi Wu*
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China *Author for correspondence (Fax: +86-571-87952618; E-mail: [email protected] )
‡ These authors contributed equally to this work.
1
Graphic abstract:
2
Highlights • PPL-catalyzed asymmetric aldol reactions and CALB-initiated MCRs were combined. • Complex spirooxazino derivatives with moderate to good ee values were provided. • Most synthesized spiro-products have moderate to high fluorescent quantum yields.
3
Abstract: Herein we reported an unprecedented two-lipase-catalyzed telescopic reaction, combining PPL (lipase from porcin pancreas)-catalyzed asymmetric aldol reactions with CALB (lipase B from Candida antarctica)-catalyzed multi-component reactions (MCRs), for the asymmetric synthesis of complicated spirooxazino derivatives with strong fluorescence emission ability.
Keywords: Multi-enzymatic reaction, spiro compounds, asymmetric synthesis, fluorescence, lipase
1. Introduction Spirocompounds, are widely found in some natural products, synthesized pharmacological agents, agricultural products, and also some new ligands or catalysts such as spirobisoxazolines, SPINOL, SPINOL-derived phosphoric acids [1-3]. It is, therefore, more interesting to consider the introduction of stereochemistry in spirocycles, which may provide a handle in modulating their superior properties relative to nonchiral spirocompounds. However, the synthesis of chiral spirocompounds is still an enormous challenge for chemists. Most synthetic chiral spirocompounds were prepared by organometallic and organocatalytic methodologies [4]. Clearly, though in most of the examples, the levels of stereoselectivity achieved are excellent, the catalysts have encountered environmental problems. Therefore, the development of cleaner and more efficient synthesis processes has become one of the major goals of chemical research in recent years. Biocatalysts are attractive alternatives to conventional 4
chemical catalysts because of their ability to operate under mild conditions, great environmental acceptability and the outstanding stereochemical specificity [5]. Recently, enzymatic promiscuity [6-14], a property of enzymes which endow one enzyme with new catalytic characters, contributes to the popularity of cascade reactions catalyzed by one enzyme with the method of multicomponent reactions (MCRs) [15-17]. For example, Wu developed a single-enzyme, ‘one-pot’ operation, for the synthesis of spirooxazino derivatives starting from readily available aldehydes, activated olefins, cyclohexanone and acetamide in moderate to high yields [18]. In order to increase the diversity of reaction products, multi-enzymatic cascade MCRs [19-21], which combines two or more enzymatic transformations in one reaction vessel, is also well exploited in organic synthesis [22-26]. Rother reported a two-enzyme-catalyzed reaction for the synthesis of nor(pseudo)ephedrine from benzaldehyde and pyruvate in one pot [27]. Turner developed a regio- and stereoselective multi-enzymatic cascade reaction for the synthesis of chiral 2,5-disubstituted pyrrolidines [28]. Many cases showed that the method of multi-enzymatic cascade MCRs which could simplify the synthesis, minimize waste and increase the productivity of the system, is considered to be an important strategy for establishing environmentally benign and sustainable chemical process. In some cases, however, it is not feasible to operate the cascade MCRs under the same conditions such as temperature and solvent. In addition, the by-product, such as H2O of the previous reaction can cause adverse effect to the next. In those situations, the bottlenecks in the reaction system can still be resolved by the so-called ‘telescopic’ processes [29-31], where the first step is completed, followed by the addition of reagents for the second step, and the second reaction is run under conditions that are independent on the previous. Turner designed a 5
chemo-enzymatic telescopic approach for the synthesis of L-arylalanines in high yield and optical purity, starting from commercially available and inexpensive substituted benzaldehydes [29]. Later, Turner developed a one-pot telescopic route to afford L-pyridylalanine analogues in high conversions, good isolated yields, and excellent purity [30]. Chanda reported a facile and efficient microwave assisted telescopic synthesis of diverse 3,5-disubstituted isoxazoles in green reaction medium [31]. In this work, we report on our attempts to develop an asymmetric synthesis of chiral spirooxazino derivatives via multi-enzymatic telescopic reactions in one pot, which combines a PPL-catalyzed asymmetric aldol condensation and a CALB-catalyzed multi-component reaction. Since the by-product H2O by PPL (lipase from porcin pancreas)-catalyzed asymmetric aldol condensation can cause adverse effect to a CALB (lipase B from Candida antarctica)-catalyzed multi-component reaction, the possibility of combining them as a cascade system is ruled out, but we envisaged that a telescopic process could be feasible. Through this multi-enzymatic telescopic reaction, 10 various spirocompounds with different substitutions could be prepared in moderate yields with moderate to good stereoselectivity. It is particularly exciting to obtain most of products with strong fluorescence emission ability and high quantum yields, providing great application potential in photoactive materials.
2. Experimental 2.1 Materials Lipase from Candida antarctica (CALB) immobilized on acrylic resin (≥10,000 U/g, recombinant, expressed in Aspergillus oryzae) and Lipase from porcin pancreas (PPL) 6
(30-90U/mg protein, one unit will hydrolyze 1.0 μequiv. of triacetin in 1 h at pH 7.7 at 37 oC) were purchased from Sigma (Steinheim, Germany). All reagents used in the experiments were obtained from commercial sources and used without further purification.
2.2. Analytical methods The 1H and
13
C NMR spectra were recorded with TMS as internal standard using a Bruker
AMX-400 MHz spectrometer. Chemical shifts were expressed in ppm and coupling constants (J) in Hz. Analytical HPLC was performed using a Agilent 1100 series with a reversed-phase Shim-Pack VP-ODS, AD-H, OJ-H, AS-H columns and a UV detector (211 or 254 nm). IR spectra were measured with a Nicolet Nexus FTIR 670 spectrophotometer. Fluorescence spectra were recorded on Shimadzu RF-5310PC Spectrofluorophotometer.
2.3. General procedure Aldehyde (0.5 mmol), water (50 μL), PPL (20 mg) and 1 mL cyclohexanone were stirred at 37 oC for 96h or 72h. Then, the reaction media was dried with 20mg MgSO4 and filtered, and then nitroalkene (2 mmol), acetamide (1 mmol), cyclohexanone (1 mL) and CALB (100 mg) were added. The whole reaction was terminated by filtering off CALB. The crude residue was purified by silica gel column chromatography with an eluent consisting of petrol ether/acetone (20/1 v/v). Product-contained fractions were combined, concentrated, and dried to give chiral products. The dr was determined by 1H NMR, and the ee value was determined by chiral HPLC.
3. Results and discussion 7
In the previous work, we discovered a novel CALB-initiated multi-component reaction from readily available aldehyde, nitrostyrene, cyclohexanone and acetamide, for the synthesis of spirooxazinos [18]. However, the substrate scope and stereoselectivity were not satisfactory. The following study used a chemically prepared aldol intermediate as one starting molecule instead of aldehydes and cyclohexanones, and remarkable improvements of substrate scope and reaction efficiency were observed [32]. Considering the reaction process, we found that the first step (aldol reaction) where the stereo-centers of final spiro-products were produced, failed to provide any enantio-selectivity, thus it would be not expected to have good stereoselectivity in the whole process. Therefore, we designed a multi-enzymatic system as a part of our continuing research. The first step was enzymatic aldol reaction between aromatic aldehydes and cyclic ketones, which has been reported previously [33-37]. Considering the stereoselectivity and yield, we found that PPL was the best catalyst for the first step. Then the reaction time was investigated under the optimized conditions according to the reference [37] (cyclic ketone as solvent, 5% water, 37 oC) (Table 1). Several aldol products could be obtained in good to excellent yields with good stereoselectivity. The satisfactory result inspired us to go forward to the second enzymatic step. Some reaction conditions, such as enzyme loading, amount of acetamide and reaction time were screened based on the model reaction of chiral aldol intermediate, olefin, acetamide and cyclohexanone to see whether it was possible to improve the CALB-catalyzed multi-component reaction. The reaction was found to proceed with 1.5 M nitroalkene, 0.25 M aldol intermediate, 0.75 M acetamide, and 50 mg CAL-B in cyclohexanone at 50 oC giving spiro product in the highest 43% yield (Table 2). Table 1. PPL-catalyzed adol reaction (the first step) a
8
a
Entry
Aldehydes
Time
Product
ee(%)
dr
Yield (%)
1
4-CF3C6H4
3d
6a
79
75/25
90
2
4-CNC6H4
3d
6b
76
85/15
71
3
4-ClC6H4
4d
6c
86
88/12
98
4
C6H5
4d
6d
85
78/22
84
5
3-NO2C6H4
3d
6e
81
84/16
99
Experimental conditions: 0.5 mmol aldehyde, 0.95 mL cyclohexanone, 0.05ml water, 30mg PPL, 37oC, 3d or 4d. All yields
and ee were detected by HPLC.
Table 2 CALB-catalyzed multi-component reaction (the second step) a
Entry
a
Enzyme
Amount of
Time/d
Yield/%
loading/mg
acetamide/mmol
1
30
0.5
3
15
2
40
0.5
3
19
3
50
0.5
3
24
4
60
0.5
3
25
5
70
0.5
3
25
6
50
0.75
3
32
7
50
1
3
30
8
50
1.25
3
26
9
50
0.75
4
40
10
50
0.75
5
43
Experimental conditions: 0.25 M aldol intermediate, 1 mL cyclohexanone, 50oC. All yields were detected by HPLC.
As mentioned above, one of the greatest challenges for bi-enzymatic system in organic solvent was conditions compatibility between two enzymatic steps. The media of both reactions was cyclohexanone, and actually it was one of the most important reasons for us to combine these two reactions in one pot. But a small quality of water would promote the first PPL-catalyzed aldol 9
reaction, while inhibiting the next CALB-catalyzed multi-component reaction, due to a dehydration step among the reaction sequence. Therefore, a drying process was necessary between these two reactions. We found that filtering off PPL powder before the second step was beneficial to the final product, in addition to the drying process (Table S1, see Supplementary data). With the optimized reaction conditions for separated steps in hand, a series of aldehydes and olefins were used to expand the generality and substrate scope of this double-enzymatic reaction. The results were summarized in Scheme 1, and showed that most of the corresponding products could be obtained in moderate yields with moderate to good stereoselectivity. Substituted nitrostyrenes bearing thienyl afforded the spirocompound in better yield and stereoselectivity, compared with other nitrostyrenes. The highest total yield of 51%, the best diastereoselectivity of 83/17 dr (anti/syn), and the best enantioselectivity of 86% ee (for anti-adducts) were achieved. Although the total yields are not very high, as a domino process simultaneously constructing six new C-C/N bonds and two rings in only two steps, they are competitive and more efficient than generally tedious asymmetric chemical synthesis.
10
Scheme 1 The scope of substrate
The discovery of novel unnatural activities of existing enzymes (enzymatic promiscuity) widened the applicability of enzymes in organic chemistry significantly. But the stereoselectivity 11
was hardly satisfying, because enzymes evolved well and the active site for natural reactions was possibly not suitable for the alternative reactions. Among the reported hydrolase-catalyzed nucleophilic addition reactions, aldol reaction was one of the most satisfying cases for stereoselectivity. The continuous research based on enzymatic asymmetric aldol reaction was important to academic and industrial areas. Unfortunately, although several conditions were screened for this multi-enzymatic reaction, the yield and stereoselectivity could not be improved to higher level. In order to develop a single-enzymatic method for preparing this series of spirooxazino derivatives with better stereoselectivity, directed evolution and site-mutagenesis of CALB for asymmetric aldol reaction are necessary and in progress in our lab. Considering the uniquely rigid structures of these prepared spirocompounds, we were interested in their fluorescent properties. Normalized emission spectra for some representative spiro-products in cyclohexane and their detailed fluorescence data are shown in Figure S1 (see Supplementary data) and Table 3, respectively. Maximum emission wavelength of most products were in the range of 402-434nm, showing purple colour, while the emission of three products with NO2 substitution at R1 (5a, 5g, 5h) were red-shifted to 498-540nm, showing green colour. Similar phenomena were observed in such solvents as chloroform, ether, tetrahydrofuran, ethanol, and acetone (data not shown). These results clearly show that the substitutions at the aromatic conjugated system have an important effect on the fluorescence emission. Furthermore, the corresponding
fluorescent
quantum
yields
were
calculated
based
on
9,10-bis(phenylethynyl)-anthracene [38] (Table 3). It was very exciting that most products have moderate to high fluorescent quantum yields, especially the quantum yield of spirocompound 5d reaches 0.87. Preliminary results of the optical properties of these novel spirooxazino derivatives 12
strongly imply their important application potential in photoelectric materials and devices. Table 3. Fluorescence data for some spirocompounds in cyclohexane. Compound
λmax(nm)
λem(nm)
Φem(%)
5a
454
510,540
38
5c
364
404,420
73
5d
376
416,434
87
5e
350
410
23
5g
444
498,528
47
5h
448
502,532
50
4. Conclusion In conclusion, we have developed a novel double-enzymatic telescopic reaction for the synthesis of chiral spirooxazino derivatives. This double-enzymatic reaction combines two steps: a PPL-catalyzed asymmetric aldol reaction and a CALB-catalyzed multi-component reaction, and cyclohexanone acts as reaction media and one of the substrates in both steps. The double-enzymatic reaction has wide substrate scopes and generality, and 10 various spirocompounds with different substitutions could be prepared in moderate yields with moderate to good stereoselectivity. We believe that these results can not only highlight the biocatalytic promiscuity but also afford a facile access to chiral spirooxazino derivatives.
Acknowledgements The financial support from the National Natural Science Foundation of China (No. 21272208), the Zhejiang Provincial Natural Science Foundation (No. LY14B020006) and Ph.D. Programs Foundation of Ministry of Education of China (20110101110008) is gratefully acknowledged.
Supplementary data 13
Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/...
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16
S1381-1177(17)30014-0 http://dx.doi.org/doi:10.1016/j.molcatb.2017.01.014 MOLCAB 3513
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
8-11-2016 4-1-2017 20-1-2017
Please cite this article as: Xiao-Yang Chen, Yu-Jing Hu, Tao Hu, Xian-Fu Lin, Qi Wu, Asymmetric synthesis of strongly fluorescent spirooxazino derivatives via multi-enzymatic telescopic reactions, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2017.01.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Asymmetric synthesis of strongly fluorescent spirooxazino derivatives via multi-enzymatic telescopic reactions Xiao-Yang Chen‡, Yu-Jing Hu‡, Tao Hu, Xian-Fu Lin, Qi Wu*
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China *Author for correspondence (Fax: +86-571-87952618; E-mail: [email protected] )
‡ These authors contributed equally to this work.
1
Graphic abstract:
2
Highlights • PPL-catalyzed asymmetric aldol reactions and CALB-initiated MCRs were combined. • Complex spirooxazino derivatives with moderate to good ee values were provided. • Most synthesized spiro-products have moderate to high fluorescent quantum yields.
3
Abstract: Herein we reported an unprecedented two-lipase-catalyzed telescopic reaction, combining PPL (lipase from porcin pancreas)-catalyzed asymmetric aldol reactions with CALB (lipase B from Candida antarctica)-catalyzed multi-component reactions (MCRs), for the asymmetric synthesis of complicated spirooxazino derivatives with strong fluorescence emission ability.
Keywords: Multi-enzymatic reaction, spiro compounds, asymmetric synthesis, fluorescence, lipase
1. Introduction Spirocompounds, are widely found in some natural products, synthesized pharmacological agents, agricultural products, and also some new ligands or catalysts such as spirobisoxazolines, SPINOL, SPINOL-derived phosphoric acids [1-3]. It is, therefore, more interesting to consider the introduction of stereochemistry in spirocycles, which may provide a handle in modulating their superior properties relative to nonchiral spirocompounds. However, the synthesis of chiral spirocompounds is still an enormous challenge for chemists. Most synthetic chiral spirocompounds were prepared by organometallic and organocatalytic methodologies [4]. Clearly, though in most of the examples, the levels of stereoselectivity achieved are excellent, the catalysts have encountered environmental problems. Therefore, the development of cleaner and more efficient synthesis processes has become one of the major goals of chemical research in recent years. Biocatalysts are attractive alternatives to conventional 4
chemical catalysts because of their ability to operate under mild conditions, great environmental acceptability and the outstanding stereochemical specificity [5]. Recently, enzymatic promiscuity [6-14], a property of enzymes which endow one enzyme with new catalytic characters, contributes to the popularity of cascade reactions catalyzed by one enzyme with the method of multicomponent reactions (MCRs) [15-17]. For example, Wu developed a single-enzyme, ‘one-pot’ operation, for the synthesis of spirooxazino derivatives starting from readily available aldehydes, activated olefins, cyclohexanone and acetamide in moderate to high yields [18]. In order to increase the diversity of reaction products, multi-enzymatic cascade MCRs [19-21], which combines two or more enzymatic transformations in one reaction vessel, is also well exploited in organic synthesis [22-26]. Rother reported a two-enzyme-catalyzed reaction for the synthesis of nor(pseudo)ephedrine from benzaldehyde and pyruvate in one pot [27]. Turner developed a regio- and stereoselective multi-enzymatic cascade reaction for the synthesis of chiral 2,5-disubstituted pyrrolidines [28]. Many cases showed that the method of multi-enzymatic cascade MCRs which could simplify the synthesis, minimize waste and increase the productivity of the system, is considered to be an important strategy for establishing environmentally benign and sustainable chemical process. In some cases, however, it is not feasible to operate the cascade MCRs under the same conditions such as temperature and solvent. In addition, the by-product, such as H2O of the previous reaction can cause adverse effect to the next. In those situations, the bottlenecks in the reaction system can still be resolved by the so-called ‘telescopic’ processes [29-31], where the first step is completed, followed by the addition of reagents for the second step, and the second reaction is run under conditions that are independent on the previous. Turner designed a 5
chemo-enzymatic telescopic approach for the synthesis of L-arylalanines in high yield and optical purity, starting from commercially available and inexpensive substituted benzaldehydes [29]. Later, Turner developed a one-pot telescopic route to afford L-pyridylalanine analogues in high conversions, good isolated yields, and excellent purity [30]. Chanda reported a facile and efficient microwave assisted telescopic synthesis of diverse 3,5-disubstituted isoxazoles in green reaction medium [31]. In this work, we report on our attempts to develop an asymmetric synthesis of chiral spirooxazino derivatives via multi-enzymatic telescopic reactions in one pot, which combines a PPL-catalyzed asymmetric aldol condensation and a CALB-catalyzed multi-component reaction. Since the by-product H2O by PPL (lipase from porcin pancreas)-catalyzed asymmetric aldol condensation can cause adverse effect to a CALB (lipase B from Candida antarctica)-catalyzed multi-component reaction, the possibility of combining them as a cascade system is ruled out, but we envisaged that a telescopic process could be feasible. Through this multi-enzymatic telescopic reaction, 10 various spirocompounds with different substitutions could be prepared in moderate yields with moderate to good stereoselectivity. It is particularly exciting to obtain most of products with strong fluorescence emission ability and high quantum yields, providing great application potential in photoactive materials.
2. Experimental 2.1 Materials Lipase from Candida antarctica (CALB) immobilized on acrylic resin (≥10,000 U/g, recombinant, expressed in Aspergillus oryzae) and Lipase from porcin pancreas (PPL) 6
(30-90U/mg protein, one unit will hydrolyze 1.0 μequiv. of triacetin in 1 h at pH 7.7 at 37 oC) were purchased from Sigma (Steinheim, Germany). All reagents used in the experiments were obtained from commercial sources and used without further purification.
2.2. Analytical methods The 1H and
13
C NMR spectra were recorded with TMS as internal standard using a Bruker
AMX-400 MHz spectrometer. Chemical shifts were expressed in ppm and coupling constants (J) in Hz. Analytical HPLC was performed using a Agilent 1100 series with a reversed-phase Shim-Pack VP-ODS, AD-H, OJ-H, AS-H columns and a UV detector (211 or 254 nm). IR spectra were measured with a Nicolet Nexus FTIR 670 spectrophotometer. Fluorescence spectra were recorded on Shimadzu RF-5310PC Spectrofluorophotometer.
2.3. General procedure Aldehyde (0.5 mmol), water (50 μL), PPL (20 mg) and 1 mL cyclohexanone were stirred at 37 oC for 96h or 72h. Then, the reaction media was dried with 20mg MgSO4 and filtered, and then nitroalkene (2 mmol), acetamide (1 mmol), cyclohexanone (1 mL) and CALB (100 mg) were added. The whole reaction was terminated by filtering off CALB. The crude residue was purified by silica gel column chromatography with an eluent consisting of petrol ether/acetone (20/1 v/v). Product-contained fractions were combined, concentrated, and dried to give chiral products. The dr was determined by 1H NMR, and the ee value was determined by chiral HPLC.
3. Results and discussion 7
In the previous work, we discovered a novel CALB-initiated multi-component reaction from readily available aldehyde, nitrostyrene, cyclohexanone and acetamide, for the synthesis of spirooxazinos [18]. However, the substrate scope and stereoselectivity were not satisfactory. The following study used a chemically prepared aldol intermediate as one starting molecule instead of aldehydes and cyclohexanones, and remarkable improvements of substrate scope and reaction efficiency were observed [32]. Considering the reaction process, we found that the first step (aldol reaction) where the stereo-centers of final spiro-products were produced, failed to provide any enantio-selectivity, thus it would be not expected to have good stereoselectivity in the whole process. Therefore, we designed a multi-enzymatic system as a part of our continuing research. The first step was enzymatic aldol reaction between aromatic aldehydes and cyclic ketones, which has been reported previously [33-37]. Considering the stereoselectivity and yield, we found that PPL was the best catalyst for the first step. Then the reaction time was investigated under the optimized conditions according to the reference [37] (cyclic ketone as solvent, 5% water, 37 oC) (Table 1). Several aldol products could be obtained in good to excellent yields with good stereoselectivity. The satisfactory result inspired us to go forward to the second enzymatic step. Some reaction conditions, such as enzyme loading, amount of acetamide and reaction time were screened based on the model reaction of chiral aldol intermediate, olefin, acetamide and cyclohexanone to see whether it was possible to improve the CALB-catalyzed multi-component reaction. The reaction was found to proceed with 1.5 M nitroalkene, 0.25 M aldol intermediate, 0.75 M acetamide, and 50 mg CAL-B in cyclohexanone at 50 oC giving spiro product in the highest 43% yield (Table 2). Table 1. PPL-catalyzed adol reaction (the first step) a
8
a
Entry
Aldehydes
Time
Product
ee(%)
dr
Yield (%)
1
4-CF3C6H4
3d
6a
79
75/25
90
2
4-CNC6H4
3d
6b
76
85/15
71
3
4-ClC6H4
4d
6c
86
88/12
98
4
C6H5
4d
6d
85
78/22
84
5
3-NO2C6H4
3d
6e
81
84/16
99
Experimental conditions: 0.5 mmol aldehyde, 0.95 mL cyclohexanone, 0.05ml water, 30mg PPL, 37oC, 3d or 4d. All yields
and ee were detected by HPLC.
Table 2 CALB-catalyzed multi-component reaction (the second step) a
Entry
a
Enzyme
Amount of
Time/d
Yield/%
loading/mg
acetamide/mmol
1
30
0.5
3
15
2
40
0.5
3
19
3
50
0.5
3
24
4
60
0.5
3
25
5
70
0.5
3
25
6
50
0.75
3
32
7
50
1
3
30
8
50
1.25
3
26
9
50
0.75
4
40
10
50
0.75
5
43
Experimental conditions: 0.25 M aldol intermediate, 1 mL cyclohexanone, 50oC. All yields were detected by HPLC.
As mentioned above, one of the greatest challenges for bi-enzymatic system in organic solvent was conditions compatibility between two enzymatic steps. The media of both reactions was cyclohexanone, and actually it was one of the most important reasons for us to combine these two reactions in one pot. But a small quality of water would promote the first PPL-catalyzed aldol 9
reaction, while inhibiting the next CALB-catalyzed multi-component reaction, due to a dehydration step among the reaction sequence. Therefore, a drying process was necessary between these two reactions. We found that filtering off PPL powder before the second step was beneficial to the final product, in addition to the drying process (Table S1, see Supplementary data). With the optimized reaction conditions for separated steps in hand, a series of aldehydes and olefins were used to expand the generality and substrate scope of this double-enzymatic reaction. The results were summarized in Scheme 1, and showed that most of the corresponding products could be obtained in moderate yields with moderate to good stereoselectivity. Substituted nitrostyrenes bearing thienyl afforded the spirocompound in better yield and stereoselectivity, compared with other nitrostyrenes. The highest total yield of 51%, the best diastereoselectivity of 83/17 dr (anti/syn), and the best enantioselectivity of 86% ee (for anti-adducts) were achieved. Although the total yields are not very high, as a domino process simultaneously constructing six new C-C/N bonds and two rings in only two steps, they are competitive and more efficient than generally tedious asymmetric chemical synthesis.
10
Scheme 1 The scope of substrate
The discovery of novel unnatural activities of existing enzymes (enzymatic promiscuity) widened the applicability of enzymes in organic chemistry significantly. But the stereoselectivity 11
was hardly satisfying, because enzymes evolved well and the active site for natural reactions was possibly not suitable for the alternative reactions. Among the reported hydrolase-catalyzed nucleophilic addition reactions, aldol reaction was one of the most satisfying cases for stereoselectivity. The continuous research based on enzymatic asymmetric aldol reaction was important to academic and industrial areas. Unfortunately, although several conditions were screened for this multi-enzymatic reaction, the yield and stereoselectivity could not be improved to higher level. In order to develop a single-enzymatic method for preparing this series of spirooxazino derivatives with better stereoselectivity, directed evolution and site-mutagenesis of CALB for asymmetric aldol reaction are necessary and in progress in our lab. Considering the uniquely rigid structures of these prepared spirocompounds, we were interested in their fluorescent properties. Normalized emission spectra for some representative spiro-products in cyclohexane and their detailed fluorescence data are shown in Figure S1 (see Supplementary data) and Table 3, respectively. Maximum emission wavelength of most products were in the range of 402-434nm, showing purple colour, while the emission of three products with NO2 substitution at R1 (5a, 5g, 5h) were red-shifted to 498-540nm, showing green colour. Similar phenomena were observed in such solvents as chloroform, ether, tetrahydrofuran, ethanol, and acetone (data not shown). These results clearly show that the substitutions at the aromatic conjugated system have an important effect on the fluorescence emission. Furthermore, the corresponding
fluorescent
quantum
yields
were
calculated
based
on
9,10-bis(phenylethynyl)-anthracene [38] (Table 3). It was very exciting that most products have moderate to high fluorescent quantum yields, especially the quantum yield of spirocompound 5d reaches 0.87. Preliminary results of the optical properties of these novel spirooxazino derivatives 12
strongly imply their important application potential in photoelectric materials and devices. Table 3. Fluorescence data for some spirocompounds in cyclohexane. Compound
λmax(nm)
λem(nm)
Φem(%)
5a
454
510,540
38
5c
364
404,420
73
5d
376
416,434
87
5e
350
410
23
5g
444
498,528
47
5h
448
502,532
50
4. Conclusion In conclusion, we have developed a novel double-enzymatic telescopic reaction for the synthesis of chiral spirooxazino derivatives. This double-enzymatic reaction combines two steps: a PPL-catalyzed asymmetric aldol reaction and a CALB-catalyzed multi-component reaction, and cyclohexanone acts as reaction media and one of the substrates in both steps. The double-enzymatic reaction has wide substrate scopes and generality, and 10 various spirocompounds with different substitutions could be prepared in moderate yields with moderate to good stereoselectivity. We believe that these results can not only highlight the biocatalytic promiscuity but also afford a facile access to chiral spirooxazino derivatives.
Acknowledgements The financial support from the National Natural Science Foundation of China (No. 21272208), the Zhejiang Provincial Natural Science Foundation (No. LY14B020006) and Ph.D. Programs Foundation of Ministry of Education of China (20110101110008) is gratefully acknowledged.
Supplementary data 13
Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/...
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