Volume 50 Number 60 4 August 2014 Pages 8079–8258
ChemComm Chemical Communications
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COMMUNICATION Helma Wennemers et al. Peptide catalysis in aqueous emulsions
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Peptide catalysis in aqueous emulsions† Cite this: Chem. Commun., 2014, 50, 8109
Jo ´, Sonja Kohrt and Helma Wennemers* ¨ rg Duschmale
Received 8th March 2014, Accepted 3rd April 2014 DOI: 10.1039/c4cc01759e www.rsc.org/chemcomm
Amphiphilic tripeptides catalyze conjugate addition reactions of aldehydes to nitroolefins in water with high yields and stereoselectivities. The amphiphilic nature shields the peptidic catalyst from the water and stabilizes the emulsion formed by the reactants in the aqueous environment. The findings have intriguing implications for the chemical evolution of enzymes.
The use of peptides as asymmetric catalysts has attracted growing attention over the last decade.1 Since they consist of the same building blocks as natural enzymes but have low molecular weights, catalytically active peptides are in between the realms of catalysis with small molecules and enzyme-catalysis. This also raises the intriguing question of a potential role of peptides in the chemical evolution of enzymes.2 However, unlike enzymes, the vast majority of the developed peptidic catalysts perform best in organic solvents.1,3 Our group has recently developed tripeptidic catalysts of the type Pro-Pro-Xaa (Xaa = acidic amino acid) for aldol and related reactions.4–7 For example, the peptide H-D-Pro-Pro-Glu-NH2 (1) is an excellent catalyst for conjugate addition reactions of aldehydes to b-nitroolefins.5,7 This tripeptide has several features that are reminiscent of small molecule based organocatalysts, e.g. a low molecular weight (M = 340 g mol 1), highest reactivity and stereoselectivity in organic solvents, and a broad substrate scope. Other features are typical of enzymes, e.g., high catalytic efficiency at low catalyst loadings as well as high chemo- and stereoselectivity.5b Mechanistic studies showed that excess water significantly reduces the reaction rate, which is fastest when dried solvents and reagents are used.7 We were now intrigued by the question whether peptide 1 can nevertheless catalyze reactions in aqueous environments, a hallmark of enzyme catalysis. We envisioned that this could be achieved by equipping the peptide with alkyl moieties that should shield the organic substrates and the catalyst from the aqueous media and provide a hydrophobic microenvironment resembling Laboratory of Organic Chemistry, ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland. E-mail: [email protected] † Electronic supplementary information (ESI) available: Synthesis and analytical data of peptides 1a–1g, general procedures for the stereoselective reactions and analytical data of the 1,4-addition products. See DOI: 10.1039/c4cc01759e
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that of active sites within enzymes.8,9 Herein, we present that the introduction of an alkyl chain at the C-terminus of H-D-Pro-Pro-GluNH2 allows for performing conjugate addition reactions between aldehydes and nitroolefins in water with high stereoselectivities. We also show that the formation of an emulsion between the catalyst and the substrates is critical for effective catalysis in the aqueous environment. We started our investigations by evaluating the reactivity of H-D-Pro-Pro-Glu-NH2 (1) in water and allowed catalytic amounts of the peptide to react with butanal and trans-b-nitrostyrene. Since the peptide is water-soluble but the substrates are not, the resulting reaction mixture was a heterogeneous slurry of the substrates in the aqueous medium. As expected, only sluggish conversion was observed (28% within a reaction time of 96 h, Table 1, entry 1). The resulting g-nitroaldehyde was isolated with an excellent diastereomeric syn : anti ratio of 97 : 3 but only a moderate enantiomeric excess of 73%. In contrast, in the organic solvent CHCl3 : iPrOH (9 : 1), a conjugate addition product forms in the presence of 1 mol% of 1 with a yield of 98%, 40 : 1 dr, and 97% ee within 12 hours.5b These initial results showed that peptide catalysed conjugate addition reactions in water
Fig. 1
Amphiphilic derivatives of H-D-Pro-Pro-Glu-NH2 (1).
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Table 1 Conjugate addition reactions between butanal and nitrostyrene catalysed by peptides 1 and 1a–1g in an aqueous environment
Entry
Catalyst
Time [h]
Conversiona [%]
dra
eeb [%]
1 2 3 4 5 6 7 8
1 1a 1b 1c 1d 1e 1f 1g
96 8 19 18 14 18 20 20
28 495 495 495 495 20 495 495
97 : 3 98 : 2 97 : 3 96 : 4 97 : 3 95 : 5 97 : 3 97 : 3
73 91 80 85 87 86 87 71
a b
Determined by 1H-NMR analysis of the crude reaction mixture. Determined by chiral-stationary phase HPLC analysis.
are challenging due to the low solubility of the substrates in water and the lower enantioselectivity of the catalyst as compared to that in an organic environment. We envisioned that amphiphilic derivatives of peptide 1 should not only retain the catalytic activity but also form an emulsion with the substrates in the aqueous reaction medium, and hence address the observed challenges. Thus, analogues 1a–1g bearing hydrophobic and hydrophilic elements were synthesised and tested as catalysts in the conjugate addition reaction (Fig. 1). Whereas the amphiphilic tripeptide H-D-Pro-Pro-Glu-NH-C12H25 (1a) bears a single alkyl chain at the C-terminus, peptides 1c and 1d are functionalized with two C-terminal alkyl chains. Within peptide 1b the hydrophobic chain is attached to the middle proline residue; peptides 1e–1g bear additional hydrophilic polyethylene glycol moieties. When peptides 1a–1g were dissolved in water, foam formation was observed, underscoring their amphiphilic nature (Fig. 2, left). Upon addition of the substrates, an emulsion formed that remained stable throughout the reaction (Fig. 2, right). Thus, the amphiphilic nature of the peptides allowed for the formation and stabilization of a highly concentrated organic microenvironment in water, within which the conjugate addition reaction was readily catalyzed (Table 1). Most of the amphiphilic catalysts provided the product in a significantly shorter time and with higher enantiomeric excess than parent peptide 1, demonstrating that the introduction of hydrophobic elements into the catalyst is generally beneficial for reactions carried out in water. Derivative 1a bearing a single n-dodecyl chain at the C-terminus, turned out to be the best catalyst. In the presence of
Fig. 2 Peptidic catalyst 1a in water before (left) and after (right) the addition of butanal and trans-b-nitrostyrene.
8110 | Chem. Commun., 2014, 50, 8109--8112
3 mol% of 1a, nitrostyrene was quantitatively converted to the g-nitroaldehyde with the highest diastereoselectivity (dr = 98 : 2) and enantioselectivity (91% ee, Table 1, entry 2). Experiments in which aqueous buffers of different pH or solutions of inorganic salts were used as reaction media demonstrated that strongly acidic environments (e.g. 1 M NaHSO4) inhibited, as expected, the reaction completely.7b,10 Under basic conditions (pH 4 8), a decreased enantioselectivity was observed, presumably due to a base catalyzed background reaction (see the ESI† for details). To understand the factors that led to the lower enantioselectivity observed with H-D-Pro-Pro-Glu-NH-C12H25 (1a) in water (91% ee) compared to that of parent peptide H-D-Pro-Pro-Glu-NH2 (1) in CHCl3 : iPrOH (9 : 1) (97% ee),5b we reacted butanal and nitrostyrene in the presence of 1 in a concentrated solution of n-hexane as a hydrophobic solvent that resembles the alkyl chains within 1a. Under these conditions, the g-nitroaldehyde was obtained with an enantiomeric excess of 92%, which is comparable to that achieved with 1a in water. Thus, not the aqueous but the hydrophobic environment within the aqueous emulsion led to the loss in stereoselectivity. We therefore hypothesised that the addition of organic additives should lead to an increase in the enantioselectivity of reactions catalysed by 1a in water and performed the reaction in the presence of different additives (Table 2). Whereas the addition of polar protic and aprotic organic solvents such as ethanol or acetone to the aqueous reaction mixture did not significantly affect the stereochemical course of the reaction, addition of chloroform or toluene improved the enantioselectivity to 93% or 95% ee (Table 2, entries 7–9). This finding is in agreement with the enantiomeric excess of 95% obtained when the reaction was performed in the presence of 1a in pure toluene or chloroform.11 Next, we investigated the substrate scope of the peptide catalyzed conjugate addition reaction between aldehydes and nitroolefins in aqueous medium and allowed different combinations of aldehydes and aromatic as well as aliphatic nitroolefins to react with each other in the presence of the amphiphilic peptide catalyst 1a (Table 3). Reactions were performed under purely aqueous conditions (Table 3, conditions A) and in water containing 15% v/v of chloroform as an Table 2 Conjugate addition reaction between butanal and nitrostyrene catalysed by peptide 1a in water and organic solvents as additives
Entry
Additive
v/v [%]
Conversiona [%]
dra
eeb [%]
1 2 3 4 5 6 7 8 9
None EtOH Acetone DMEc EtOAc MeCN CHCl3 Toluene CHCl3
— 10 10 10 10 10 10 10 15
495 495 77 495 495 495 495 495 495
98 : 2 99 : 1 97 : 3 99 : 1 96 : 4 96 : 4 96 : 4 97 : 3 97 : 3
91 92 90 92 91 90 93 93 95
a Determined by 1H-NMR analysis of the crude reaction mixture. b Determined by chiral-stationary phase HPLC analysis. c 1,2-Dimethoxyethane.
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Table 3 Scope of conjugate addition reactions between aldehydes and nitroolefins catalyzed by peptide 1a in pure water (conditions A) and 15% v/v of CHCl3 in water (conditions B)
Conditions
Yield [%]
dra
eeb [%]
1
A B
82 95
98 : 2 97 : 3
91 95
2
A B
92 88
99 : 1 99 : 1
92 94
3
A B
97 86
95 : 5 98 : 2
93 95
4
A B
87 90
97 : 3 97 : 3
94 95
5
A B
81 95
99 : 1 95 : 5
88 93
6
A B
66c 95
93 : 7 92 : 8
89 95
7
A B
89 81
93 : 7 97 : 3
88 92
8
A B
99 99
99 : 1 98 : 2
84 94
A B
76d 61d
99 : 1 97 : 3
89 91
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Entry
9
Product
a
Determined by 1H-NMR analysis of the crude reaction mixture. Determined by chiral-phase HPLC analysis. c 70% conversion. d The yield was affected by the volatility of the g-nitroaldehyde. b
organic additive (Table 3, conditions B). In general, high yields and excellent levels of stereoselectivity were observed with all of the substrate combinations examined. As expected, the enantioselectivities were generally slightly higher in the presence of 15% v/v of chloroform in water than in pure water (Table 3). Interestingly, and in contrast to the analogous reactions in organic solvents, the observed rate of product formation was found to not only depend on the reactivity of the nitroolefin but to a large extent on its ability to form a stable emulsion with the aldehyde and peptide 1a. For example, conjugate addition reactions of aldehydes to 4-bromo nitrostyrene are typically completed within half the reaction time compared to reactions with the less electrophilic and therefore less reactive 4-methoxy nitrostyrene.5a,12,13 However, under aqueous conditions in the presence of 1a, 4-bromo nitrostyrene does not form a stable colloidal dispersion and, as a consequence, only 70% of this nitroolefin was converted to the g-nitroaldehyde within 24 h (Table 3, entry 6, conditions A), whereas full conversion of the less reactive 4-methoxy nitrostyrene was observed under identical reaction conditions (Table 3, entry 5, conditions A). Similarly, aliphatic nitroolefins, generally considered to be challenging b-substituted nitroolefins in reactions in organic
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solvents, formed stable emulsions and reacted readily under the aqueous inhomogeneous conditions and provided the addition products in high yields and stereoselectivities (Table 3, entries 8 and 9). Thus, the use of aqueous emulsions can provide for complementary reactivity to that in organic solvents. Finally, we investigated the peptide catalyzed conjugate addition reaction in the aqueous colloidal dispersion on a 10 mmol scale. In the presence of 3 mol% of the amphiphilic catalyst 1a, 1.50 g of nitrostyrene were reacted with a twofold excess of butanal. The crude conjugate addition product was obtained without the need for extraction with an organic solvent by saturation of the reaction mixture with sodium chloride followed by centrifugation and phase separation. Filtration through a short plug of silica gel and eluting with cyclohexane and ethyl acetate provided 1.57 g (71%) of the desired g-nitroaldehyde together with 361 mg (24%) of unreacted nitrostyrene. This experiment demonstrated that separation of the organic components from the aqueous reaction mixture can be achieved in a highly efficient manner and without the need for extraction with organic solvents.14 In summary, we have shown that the amphiphilic peptide H-D-Pro-Pro-Glu-NH-C12H25 is a highly active and stereoselective catalyst for conjugate addition reactions in aqueous medium. On a gram scale, the reaction product was isolated by simple centrifugation without the need for extraction with an organic solvent. The reaction takes place in an emulsion in which the alkylated peptide serves not only as the catalyst for the reaction but also as a detergent that stabilizes the colloidal dispersion. These results are particularly remarkable since the reactivity and stereoselectivity of the parent catalyst H-D-Pro-Pro-Glu-NH2 are significantly reduced by the addition of excess water. The results demonstrate that the catalytically active peptide is shielded from the surrounding water within a hydrophobic microenvironment, which is reminiscent of the hydrophobic pockets often found within the active sites of enzymes.15 This suggests that hydrophobic compartments containing reactants and catalytically active short peptides have likely played a critical role in the evolution of enzymes. We thank Prof. Peter Walde for useful discussions and the Swiss National Science Foundation for financial support.
Notes and references 1 For reviews, see: (a) H. Wennemers, Chem. Commun., 2011, 47, 12036–12041; (b) E. A. C. Davie, S. M. Mennen, Y. Xu and S. J. Miller, Chem. Rev., 2007, 107, 5759–5812; (c) J. Duschmale, Y. Arakawa and H. Wennemers, in Science of Synthesis, Asymmetric Organocatalysis, ed. B. List and K. Maruoka, Georg Thieme Verlag, Stuttgart, 2012, vol. 2, pp. 741–786. 2 (a) J. Soding and A. N. Lupas, BioEssays, 2003, 25, 837–846; (b) G. Danger, R. Plasson and R. Pascal, Chem. Soc. Rev., 2012, 41, 5416–5429. 3 For examples of asymmetric catalysis with peptides in water, see: (a) A. L. Weber and S. Pizzarello, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 12713–12717; (b) X. H. Chen, S. W. Luo, Z. Tang, L. F. Cun, A. Q. Mi, Y. Z. Jiang and L. Z. Gong, Chem. – Eur. J., 2007, 13, 689–701; (c) M. Lei, L. Shi, G. Li, S. Chen, W. Fang, Z. Ge, T. Cheng and R. Li, Tetrahedron, 2007, 63, 7892–7898; (d) K. Akagawa and K. Kudo, Org. Lett., 2011, 13, 3498–3501; (e) K. Akagawa and K. Kudo, Angew. Chem., Int. Ed., 2012, 51, 12786–12789; ( f ) K. Akagawa, R. Suzuki and K. Kudo, Adv. Synth. Catal., 2012, 354, 1280–1286. 4 (a) P. Krattiger, R. Kovasy, J. D. Revell, S. Ivan and H. Wennemers, Org. Lett., 2005, 7, 1101–1103; (b) J. D. Revell and H. Wennemers, Tetrahedron, 2007, 63, 8420–8424; (c) J. D. Revell and H. Wennemers, Adv. Synth.
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6 7
8
9
Catal., 2008, 350, 1046–1052; (d) M. Messerer and H. Wennemers, Synlett, 2011, 499–502. (a) M. Wiesner, J. D. Revell and H. Wennemers, Angew. Chem., Int. Ed., 2008, 47, 1871–1874; (b) M. Wiesner, M. Neuburger and H. Wennemers, Chem. – Eur. J., 2009, 15, 10103–10109; (c) M. Wiesner, J. D. Revell, S. Tonazzi and H. Wennemers, J. Am. Chem. Soc., 2008, 130, 5610–5611; (d) M. Wiesner and H. Wennemers, Synthesis, 2010, 1568–1571; (e) Y. Arakawa, M. Wiesner and H. Wennemers, Adv. Synth. Catal., 2011, 353, 1201–1206; ( f ) Y. Arakawa and H. Wennemers, ChemSusChem, 2012, 6, 242–245. ´ and H. Wennemers, Chem. – Eur. J., 2012, 18, (a) J. Duschmale 1111–1120; (b) R. Kastl and H. Wennemers, Angew. Chem., Int. Ed., 2013, 52, 7228–7232. (a) M. Wiesner, G. Upert, G. Angelici and H. Wennemers, J. Am. ´, J. Wiest, M. Wiesner Chem. Soc., 2010, 132, 6–7; (b) J. Duschmale ¨chle, and H. Wennemers, Chem. Sci., 2013, 4, 1312–1318; (c) F. Ba ´, C. Ebner, A. Pfaltz and H. Wennemers, Angew. Chem., J. Duschmale Int. Ed., 2013, 52, 12619–12623. For reviews on organocatalysis in water, see: (a) N. Mase and C. F. Barbas, 3rd, Org. Biomol. Chem., 2010, 8, 4043–4050; (b) M. Raj and V. K. Singh, Chem. Commun., 2009, 6687–6703; (c) M. Gruttadauria, F. Giacalone and R. Noto, Adv. Synth. Catal., 2009, 351, 33–57; (d) J. Paradowska, M. Stodulski and J. Mlynarski, Angew. Chem., Int. Ed., 2009, 48, 4288–4297. For selected examples of organocatalysis in water, see: (a) T. J. Dickerson and K. D. Janda, J. Am. Chem. Soc., 2002, 124, 3220–3221; (b) N. Mase, K. Watanabe, H. Yoda, K. Takabe, F. Tanaka and C. F. Barbas, 3rd, J. Am. Chem. Soc., 2006, 128, 4966–4967; (c) S. Luo, X. Mi, S. Liu, H. Xu and J.-P. Cheng, Chem. Commun., 2006, 3687–3689; (d) L. Zu, J. Wang, H. Li and W. Wang, Org. Lett., 2006, 8, 3077–3079; (e) Y. Hayashi, S. Aratake, T. Okano, J. Takahashi, T. Sumiya and M. Shoji, Angew. Chem., Int. Ed., 2006, 45, 5527–5529; ( f ) V. Maya, M. Raj and V. K. Singh, Org. Lett., 2007, 9, 2593–2595; (g) C. Palomo, A. Landa, A. Mielgo, M. Oiarbide, A. Puente
8112 | Chem. Commun., 2014, 50, 8109--8112
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10 11 12
13 14 15
and S. Vera, Angew. Chem., Int. Ed., 2007, 46, 8431–8435; (h) E. Alza, X. C. Cambeiro, C. Jimeno and M. A. Pericas, Org. Lett., 2007, 9, 3717–3720; (i) S. Zhu, S. Yu and D. Ma, Angew. Chem., Int. Ed., 2008, 47, 545–548; ( j) M. Lombardo, M. Chiarucci, A. Quintavalla and C. Trombini, Adv. Synth. Catal., 2009, 351, 2801–2806; (k) Z. Zheng, B. L. Perkins and B. Ni, J. Am. Chem. Soc., 2010, 132, 50–51; (l ) S. K. Ghosh, Y. Qiao, B. Ni and A. D. Headley, Org. Biomol. Chem., 2013, 11, 1801–1804. Strong acids protonate the secondary amine, the reactive center, within the catalyst and therefore reduce the catalytic activity, see ref. 7b. Analogues of 1 bearing a secondary instead of a primary amide at the C-terminus had been found to have lower stereoselectivity, ref. 5b. F. J. Vicario, Organocatalytic enantioselective conjugate addition reactions: a powerful tool for the stereocontrolled synthesis of complex molecules, Royal Society of Chemistry, Cambridge, 2010. For selected examples in organic solvents, see: (a) Y. Hayashi, H. Gotoh, T. Hayashi and M. Shoji, Angew. Chem., Int. Ed., 2005, 44, 4212–4215; (b) C. Palomo, S. Vera, A. Mielgo and E. Gomez-Bengoa, Angew. Chem., Int. Ed., 2006, 45, 5984–5987; (c) Y. Chi, L. Guo, N. A. Kopf and S. H. Gellman, J. Am. ´pe ˆche, Chem. Soc., 2008, 130, 5608–5609; (d) P. Garcı´a-Garcı´a, A. Lade R. Halder and B. List, Angew. Chem., Int. Ed., 2008, 47, 4719–4721; (e) H. Ishikawa, T. Suzuki and Y. Hayashi, Angew. Chem., Int. Ed., 2009, 48, 1304–1307; ( f ) H. Uehara and C. F. Barbas, Angew. Chem., Int. Ed., 2009, 48, 9848–9852; (g) D. Enders, C. Wang and A. Greb, Adv. Synth. ´. Madara ´sz, I. Pa ´pai and Catal., 2010, 352, 987–992; (h) H. Rahaman, A P. M. Pihko, Angew. Chem., Int. Ed., 2011, 50, 6123–6127. I. Zenz and H. Mayr, J. Org. Chem., 2011, 76, 9370–9378. Often the products can only be isolated from an aqueous reaction medium by extraction with organic solvents, see for examples ref. 8 and 9. For dendrimers as enzyme mimicks, see: J.-L. Reymond and T. Darbre, Org. Biomol. Chem., 2012, 10, 1483–1492.
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ChemComm Chemical Communications
www.rsc.org/chemcomm
ISSN 1359-7345
COMMUNICATION Helma Wennemers et al. Peptide catalysis in aqueous emulsions
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Peptide catalysis in aqueous emulsions† Cite this: Chem. Commun., 2014, 50, 8109
Jo ´, Sonja Kohrt and Helma Wennemers* ¨ rg Duschmale
Received 8th March 2014, Accepted 3rd April 2014 DOI: 10.1039/c4cc01759e www.rsc.org/chemcomm
Amphiphilic tripeptides catalyze conjugate addition reactions of aldehydes to nitroolefins in water with high yields and stereoselectivities. The amphiphilic nature shields the peptidic catalyst from the water and stabilizes the emulsion formed by the reactants in the aqueous environment. The findings have intriguing implications for the chemical evolution of enzymes.
The use of peptides as asymmetric catalysts has attracted growing attention over the last decade.1 Since they consist of the same building blocks as natural enzymes but have low molecular weights, catalytically active peptides are in between the realms of catalysis with small molecules and enzyme-catalysis. This also raises the intriguing question of a potential role of peptides in the chemical evolution of enzymes.2 However, unlike enzymes, the vast majority of the developed peptidic catalysts perform best in organic solvents.1,3 Our group has recently developed tripeptidic catalysts of the type Pro-Pro-Xaa (Xaa = acidic amino acid) for aldol and related reactions.4–7 For example, the peptide H-D-Pro-Pro-Glu-NH2 (1) is an excellent catalyst for conjugate addition reactions of aldehydes to b-nitroolefins.5,7 This tripeptide has several features that are reminiscent of small molecule based organocatalysts, e.g. a low molecular weight (M = 340 g mol 1), highest reactivity and stereoselectivity in organic solvents, and a broad substrate scope. Other features are typical of enzymes, e.g., high catalytic efficiency at low catalyst loadings as well as high chemo- and stereoselectivity.5b Mechanistic studies showed that excess water significantly reduces the reaction rate, which is fastest when dried solvents and reagents are used.7 We were now intrigued by the question whether peptide 1 can nevertheless catalyze reactions in aqueous environments, a hallmark of enzyme catalysis. We envisioned that this could be achieved by equipping the peptide with alkyl moieties that should shield the organic substrates and the catalyst from the aqueous media and provide a hydrophobic microenvironment resembling Laboratory of Organic Chemistry, ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland. E-mail: [email protected] † Electronic supplementary information (ESI) available: Synthesis and analytical data of peptides 1a–1g, general procedures for the stereoselective reactions and analytical data of the 1,4-addition products. See DOI: 10.1039/c4cc01759e
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that of active sites within enzymes.8,9 Herein, we present that the introduction of an alkyl chain at the C-terminus of H-D-Pro-Pro-GluNH2 allows for performing conjugate addition reactions between aldehydes and nitroolefins in water with high stereoselectivities. We also show that the formation of an emulsion between the catalyst and the substrates is critical for effective catalysis in the aqueous environment. We started our investigations by evaluating the reactivity of H-D-Pro-Pro-Glu-NH2 (1) in water and allowed catalytic amounts of the peptide to react with butanal and trans-b-nitrostyrene. Since the peptide is water-soluble but the substrates are not, the resulting reaction mixture was a heterogeneous slurry of the substrates in the aqueous medium. As expected, only sluggish conversion was observed (28% within a reaction time of 96 h, Table 1, entry 1). The resulting g-nitroaldehyde was isolated with an excellent diastereomeric syn : anti ratio of 97 : 3 but only a moderate enantiomeric excess of 73%. In contrast, in the organic solvent CHCl3 : iPrOH (9 : 1), a conjugate addition product forms in the presence of 1 mol% of 1 with a yield of 98%, 40 : 1 dr, and 97% ee within 12 hours.5b These initial results showed that peptide catalysed conjugate addition reactions in water
Fig. 1
Amphiphilic derivatives of H-D-Pro-Pro-Glu-NH2 (1).
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Table 1 Conjugate addition reactions between butanal and nitrostyrene catalysed by peptides 1 and 1a–1g in an aqueous environment
Entry
Catalyst
Time [h]
Conversiona [%]
dra
eeb [%]
1 2 3 4 5 6 7 8
1 1a 1b 1c 1d 1e 1f 1g
96 8 19 18 14 18 20 20
28 495 495 495 495 20 495 495
97 : 3 98 : 2 97 : 3 96 : 4 97 : 3 95 : 5 97 : 3 97 : 3
73 91 80 85 87 86 87 71
a b
Determined by 1H-NMR analysis of the crude reaction mixture. Determined by chiral-stationary phase HPLC analysis.
are challenging due to the low solubility of the substrates in water and the lower enantioselectivity of the catalyst as compared to that in an organic environment. We envisioned that amphiphilic derivatives of peptide 1 should not only retain the catalytic activity but also form an emulsion with the substrates in the aqueous reaction medium, and hence address the observed challenges. Thus, analogues 1a–1g bearing hydrophobic and hydrophilic elements were synthesised and tested as catalysts in the conjugate addition reaction (Fig. 1). Whereas the amphiphilic tripeptide H-D-Pro-Pro-Glu-NH-C12H25 (1a) bears a single alkyl chain at the C-terminus, peptides 1c and 1d are functionalized with two C-terminal alkyl chains. Within peptide 1b the hydrophobic chain is attached to the middle proline residue; peptides 1e–1g bear additional hydrophilic polyethylene glycol moieties. When peptides 1a–1g were dissolved in water, foam formation was observed, underscoring their amphiphilic nature (Fig. 2, left). Upon addition of the substrates, an emulsion formed that remained stable throughout the reaction (Fig. 2, right). Thus, the amphiphilic nature of the peptides allowed for the formation and stabilization of a highly concentrated organic microenvironment in water, within which the conjugate addition reaction was readily catalyzed (Table 1). Most of the amphiphilic catalysts provided the product in a significantly shorter time and with higher enantiomeric excess than parent peptide 1, demonstrating that the introduction of hydrophobic elements into the catalyst is generally beneficial for reactions carried out in water. Derivative 1a bearing a single n-dodecyl chain at the C-terminus, turned out to be the best catalyst. In the presence of
Fig. 2 Peptidic catalyst 1a in water before (left) and after (right) the addition of butanal and trans-b-nitrostyrene.
8110 | Chem. Commun., 2014, 50, 8109--8112
3 mol% of 1a, nitrostyrene was quantitatively converted to the g-nitroaldehyde with the highest diastereoselectivity (dr = 98 : 2) and enantioselectivity (91% ee, Table 1, entry 2). Experiments in which aqueous buffers of different pH or solutions of inorganic salts were used as reaction media demonstrated that strongly acidic environments (e.g. 1 M NaHSO4) inhibited, as expected, the reaction completely.7b,10 Under basic conditions (pH 4 8), a decreased enantioselectivity was observed, presumably due to a base catalyzed background reaction (see the ESI† for details). To understand the factors that led to the lower enantioselectivity observed with H-D-Pro-Pro-Glu-NH-C12H25 (1a) in water (91% ee) compared to that of parent peptide H-D-Pro-Pro-Glu-NH2 (1) in CHCl3 : iPrOH (9 : 1) (97% ee),5b we reacted butanal and nitrostyrene in the presence of 1 in a concentrated solution of n-hexane as a hydrophobic solvent that resembles the alkyl chains within 1a. Under these conditions, the g-nitroaldehyde was obtained with an enantiomeric excess of 92%, which is comparable to that achieved with 1a in water. Thus, not the aqueous but the hydrophobic environment within the aqueous emulsion led to the loss in stereoselectivity. We therefore hypothesised that the addition of organic additives should lead to an increase in the enantioselectivity of reactions catalysed by 1a in water and performed the reaction in the presence of different additives (Table 2). Whereas the addition of polar protic and aprotic organic solvents such as ethanol or acetone to the aqueous reaction mixture did not significantly affect the stereochemical course of the reaction, addition of chloroform or toluene improved the enantioselectivity to 93% or 95% ee (Table 2, entries 7–9). This finding is in agreement with the enantiomeric excess of 95% obtained when the reaction was performed in the presence of 1a in pure toluene or chloroform.11 Next, we investigated the substrate scope of the peptide catalyzed conjugate addition reaction between aldehydes and nitroolefins in aqueous medium and allowed different combinations of aldehydes and aromatic as well as aliphatic nitroolefins to react with each other in the presence of the amphiphilic peptide catalyst 1a (Table 3). Reactions were performed under purely aqueous conditions (Table 3, conditions A) and in water containing 15% v/v of chloroform as an Table 2 Conjugate addition reaction between butanal and nitrostyrene catalysed by peptide 1a in water and organic solvents as additives
Entry
Additive
v/v [%]
Conversiona [%]
dra
eeb [%]
1 2 3 4 5 6 7 8 9
None EtOH Acetone DMEc EtOAc MeCN CHCl3 Toluene CHCl3
— 10 10 10 10 10 10 10 15
495 495 77 495 495 495 495 495 495
98 : 2 99 : 1 97 : 3 99 : 1 96 : 4 96 : 4 96 : 4 97 : 3 97 : 3
91 92 90 92 91 90 93 93 95
a Determined by 1H-NMR analysis of the crude reaction mixture. b Determined by chiral-stationary phase HPLC analysis. c 1,2-Dimethoxyethane.
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Table 3 Scope of conjugate addition reactions between aldehydes and nitroolefins catalyzed by peptide 1a in pure water (conditions A) and 15% v/v of CHCl3 in water (conditions B)
Conditions
Yield [%]
dra
eeb [%]
1
A B
82 95
98 : 2 97 : 3
91 95
2
A B
92 88
99 : 1 99 : 1
92 94
3
A B
97 86
95 : 5 98 : 2
93 95
4
A B
87 90
97 : 3 97 : 3
94 95
5
A B
81 95
99 : 1 95 : 5
88 93
6
A B
66c 95
93 : 7 92 : 8
89 95
7
A B
89 81
93 : 7 97 : 3
88 92
8
A B
99 99
99 : 1 98 : 2
84 94
A B
76d 61d
99 : 1 97 : 3
89 91
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Entry
9
Product
a
Determined by 1H-NMR analysis of the crude reaction mixture. Determined by chiral-phase HPLC analysis. c 70% conversion. d The yield was affected by the volatility of the g-nitroaldehyde. b
organic additive (Table 3, conditions B). In general, high yields and excellent levels of stereoselectivity were observed with all of the substrate combinations examined. As expected, the enantioselectivities were generally slightly higher in the presence of 15% v/v of chloroform in water than in pure water (Table 3). Interestingly, and in contrast to the analogous reactions in organic solvents, the observed rate of product formation was found to not only depend on the reactivity of the nitroolefin but to a large extent on its ability to form a stable emulsion with the aldehyde and peptide 1a. For example, conjugate addition reactions of aldehydes to 4-bromo nitrostyrene are typically completed within half the reaction time compared to reactions with the less electrophilic and therefore less reactive 4-methoxy nitrostyrene.5a,12,13 However, under aqueous conditions in the presence of 1a, 4-bromo nitrostyrene does not form a stable colloidal dispersion and, as a consequence, only 70% of this nitroolefin was converted to the g-nitroaldehyde within 24 h (Table 3, entry 6, conditions A), whereas full conversion of the less reactive 4-methoxy nitrostyrene was observed under identical reaction conditions (Table 3, entry 5, conditions A). Similarly, aliphatic nitroolefins, generally considered to be challenging b-substituted nitroolefins in reactions in organic
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solvents, formed stable emulsions and reacted readily under the aqueous inhomogeneous conditions and provided the addition products in high yields and stereoselectivities (Table 3, entries 8 and 9). Thus, the use of aqueous emulsions can provide for complementary reactivity to that in organic solvents. Finally, we investigated the peptide catalyzed conjugate addition reaction in the aqueous colloidal dispersion on a 10 mmol scale. In the presence of 3 mol% of the amphiphilic catalyst 1a, 1.50 g of nitrostyrene were reacted with a twofold excess of butanal. The crude conjugate addition product was obtained without the need for extraction with an organic solvent by saturation of the reaction mixture with sodium chloride followed by centrifugation and phase separation. Filtration through a short plug of silica gel and eluting with cyclohexane and ethyl acetate provided 1.57 g (71%) of the desired g-nitroaldehyde together with 361 mg (24%) of unreacted nitrostyrene. This experiment demonstrated that separation of the organic components from the aqueous reaction mixture can be achieved in a highly efficient manner and without the need for extraction with organic solvents.14 In summary, we have shown that the amphiphilic peptide H-D-Pro-Pro-Glu-NH-C12H25 is a highly active and stereoselective catalyst for conjugate addition reactions in aqueous medium. On a gram scale, the reaction product was isolated by simple centrifugation without the need for extraction with an organic solvent. The reaction takes place in an emulsion in which the alkylated peptide serves not only as the catalyst for the reaction but also as a detergent that stabilizes the colloidal dispersion. These results are particularly remarkable since the reactivity and stereoselectivity of the parent catalyst H-D-Pro-Pro-Glu-NH2 are significantly reduced by the addition of excess water. The results demonstrate that the catalytically active peptide is shielded from the surrounding water within a hydrophobic microenvironment, which is reminiscent of the hydrophobic pockets often found within the active sites of enzymes.15 This suggests that hydrophobic compartments containing reactants and catalytically active short peptides have likely played a critical role in the evolution of enzymes. We thank Prof. Peter Walde for useful discussions and the Swiss National Science Foundation for financial support.
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