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Comparison of two thioxopeptide bond photoswitches in insect kinin
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Chinese Chemical Letters 21 (2010) 476–479 www.elsevier.com/locate/cclet
Comparison of two thioxopeptide bond photoswitches in insect kinin Zhi Yuan Cong, Long Fei Yang, Ling Jiang, Dan Ye, Shou Liang Dong * Institute of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000, China Received 24 June 2009
Abstract The photoisomerization abilities of secondary thioxopeptide bond (CS–NH) and thioxo prolyl bond (CS–N) incorporated into the C-terminal pentapeptide of insect kinin were compared. H-Phe-Phe-c[CS–NH]-D-Ala-Trp-Gly-NH2 and H-Phe-Tyr-c[CS–N]Pro-Trp-Gly-NH2 were studied by UV–vis absorption. The isomerization energy barriers of the two segments, Ac-Phe-c[CS–NH]D-Ala-NH2 and Ac-Tyr-c[CS–N]-Pro-NH2 picked from the two peptides, were calculated using ab initio method. The cis isomer of CS–N is more stable than that of CS–NH due to higher energy barrier, so the former is more suitable in peptide structure–activity relationship studies. # 2009 Shou Liang Dong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Photoswitch; Thioxopeptide bond; Energy barrier
Recently, thioxopeptide bond has attracted considerable interest for its ability of converting the stable trans conformation into the thermodynamically unfavorable cis conformation by irradiation. The carbonyl sulfur substitution leads to red-shifted p–p* and n–p* absorption, and lowers the excitation energy of photoisomerization. Thus it can induce conformational change to peptide backbone via trans/cis conversion by irradiation [1–4]. Since the cis/trans isomerization of prolyl bond (CO-N) and secondary peptide bond (CO–NH) are both essential to peptides and proteins [5,6], both of the corresponding thioxopeptide bonds are worth study. Frank et al. have shown that thioxo prolyl bond (CS–N) could switch between cis and trans isomers when it is present in endomorphins [1]. With the marked differences of the two isomers’ UV–vis absorption spectra, they investigated the isomerization of thioxo prolyl bond. The cis/trans isomerization constant (kc/t) of endomorphin-2 is (5.23 0.03) 10 4 s 1 at 313 K. Zhao et al. studied the photoisomerization of secondary thioxopeptide bonds (CS–NH) in dipeptides [2,3], a dual-directional photoswitching effect was detected, whose thermal relaxation constant is (9.78 0.02) 10 4 s 1 for H-Phe-c[CS– NH]-Ala-OH at 289 K. Ala4-c[CS–NH]-Ala5 segment was also incorporated into the S-peptide of RNase S [7], and its kc/t is (5.70 0.005) 10 3 s 1 at 283 K. In our previous studies, CS–N photoisomerization was investigated in insect kinin analog H-Phe-Tyr-c[CS–N]-Pro-Trp-Gly-NH2 (c[CS–N]2-kinin) [8]. The results showed that the kc/t is (2.90 0.1) 10 4 s 1 at 293 K and the trans conformation before irradiation was also confirmed by 2D-NMR [9].
* Corresponding author. E-mail address: [email protected] (S.L. Dong). 1001-8417/$ – see front matter # 2009 Shou Liang Dong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.12.033
Z.Y. Cong et al. / Chinese Chemical Letters 21 (2010) 476–479
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Fig. 1. UV–vis absorbance spectra of the two thioxopeptides: ground state (—), after 5 min of irradiation at 254 nm (- - - -), re-equilibrated after four cycles of irradiation–equilibration ( ). (a) c[CS–NH]2-kinin, 8.0 10 5 mol/L, 293 K. (b) c[CS–N]2-kinin, which is cited from Ref. [8], is presented here for comparing.
Fig. 2. The time-dependent cis/trans isomerization curve. The solid lines represent the first-order fit. (a) c[CS–NH]2-kinin, 8.0 10 5 mol/L 293 K. kc/t is (8.77 0.16) 10 3 s 1. (b) c[CS–N]2-kinin, which is cited from Ref. [8], is presented here for comparing. kc/t is (2.90 0.1) 10 4 s 1.
In order to explore the photoisomerization ability of CS–N and CS–NH under the same conditions, a new insect kinin thioxo analog H-Phe-Phe-c[CS–NH]-D-Ala-Trp-Gly-NH2 (c[CS–NH]2-kinin) containing a CS–NH, was synthesized. Insect kinins share a highly conserved C-terminal pentapeptide, Phe-Xaa-Xbb-Trp-Gly-NH2, in which Xaa and Xbb tolerate a broad range of amino acids [10]. The peptide bond between them was modified by thioxylation. The UV–vis absorption spectra, kc/t and the photoswitching effects of the two analogs (c[CS–N]2-kinin and c[CS– NH]2-kinin) were compared. To explore the mechanism, the thioxopeptide bond cis/trans isomerization energy barriers were calculated in two model peptides, Ac-Phe-[CS–NH]-D-Ala-NH2 and Ac-Tyr-[CS–N]-Pro-NH2 picked from the two analogs using ab initio method. The UV–vis spectrum of c[CS–N]2-kinin has been described in our previous study [8]. For c[CS–NH]2-kinin, all measurements were performed at 293 K. c[CS–NH]2-kinin was 8.0 10 5 mol/L in sodium phosphate buffer (0.01 mol/L, pH 7.2). The photostationary state was achieved by 5 min irradiation under 254 nm. The results are shown in Fig. 1. c[CS–NH]2-kinin red-shifted at 270 nm and showed a slightly reduced maximum intensity upon irradiation, which is a representative of the increased CS–NH cis/trans ratio [2,3]. There is a well-anchored isosbestic point at 273 nm, which means that only two conformers were involved [1,2]. And after four cycles of irradiation– equilibration, the spectrum of c[CS–NH]2-kinin can fully restore to the ground state. All these results indicate that the cis/trans photoswitching is reversible and occurs without decomposition. For c[CS–N]2-kinin [8], an increased maximum intensity at 277 nm and an isosbestic point at 252 nm were found after irradiation, showing that there is an increase of CS–N cis/trans ratio [1]. After irradiation, the UV absorption of c[CS–NH]2-kinin was monitored immediately at 293 nm. As shown in Fig. 2, kc/t is (8.77 0.16) 10 3 s 1, which is larger than that of c[CS–N]2-kinin (Its kc/t is (2.90 0.1) 10 4 s 1, 30 times lower than that of c[CS–NH]2-kinin) [8]. The smaller constant shows that the cis isomer of CS–N is much more stable than that of CS–NH upon UV irradiation. With the different stabilities of the
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Z.Y. Cong et al. / Chinese Chemical Letters 21 (2010) 476–479
Table 1 Relative energy of the cis isomers, transition states (TS) and energy barriers (Ebarrier) respect to the trans isomers of c[CS–N]-YP and c[CS–NH]F(D)A (kcal/mol). Dipeptides
c[CS–N]-YP c[CS–NH]-F(D)A
STO-3G
6-31G(d, p)
trans
cis
TS
Ebarrier
trans
cis
TS
Ebarrier
0.00 0.00
1.71 5.46
25.29 22.50
23.58 17.04
0.00 0.00
4.75 5.28
27.73 24.97
22.98 19.69
cis isomers, the photo-induced affection on cockroach hindgut myotropic activities were also compared between the two analogs. As the cis isomer of the thioxylation site is more bioactive than the trans, the activity of c[CS–N]2-kinin increased significantly due to the cis isomer rising to 47.7% after irradiation [8]. For c[CS–NH]2-kinin, no significant photo-induced activity change was found with a constant EC50 of (6.80 0.2) 10 7 mol/L. In addition, no antagonist effect was found for c[CS–NH]2-kinin, which was designed due to the inspiration from reported antagonistic insect kinin tetrazole analog [11]. The instability of cis CS–NH prevented the potential antagonist effect, further investigation will be needed if it is designed for practical use. To explain the mechanism of the different photoisomerization abilities, two sulfur modified dipeptides Ac-Tyrc[CS–N]-Pro-NH2 (c[CS–N]-YP) and Ac-Phe-c[CS–NH]-D-Ala-NH2 (c[CS–NH]-F(D)A) from c[CS–N]2-kinin and c[CS–NH]2-kinin, with acetyl in N-terminal and acylamide in C-terminal were studied. The bond rotational energy barriers were calculated by GAMESS [12]. In order to approach the real experiment conditions, the calculation was performed in water and the temperature was set as 298.15 K. The cis isomers, trans isomers and their transition states were optimized at HF/STO-3G level [13]. The initial molecular structure coordinates of cis isomers and trans isomers were used as GAMESS input files. The proposed probable structures were optimized to get the adjacent rotational transition states. By the successful location of the equilibrium geometry and root mean square (RMS) gradient convergence, all stationary points were confirmed as either minima or transition states, single point energy calculations were performed to obtain a more accurate energy value at HF/6-31G(d, p) level [14]. The calculation results are shown in Table 1. It is shown in Table 1 that at HF/STO-3G level the cis/trans isomerization energy barrier of c[CS–N]-YP is 6.54 kcal/mol higher than that of c[CS–NH]-F(D)A. From the results of single point energy calculation at HF/631G(d, p) level, a 3.29 kcal/mol higher energy barrier is also found in c[CS–N]-YP. These results indicate that to transfer from cis to trans, c[CS–N]-YP needs more energy. After irradiation, the increased cis isomer of c[CS–N]2kinin is more stable than that of c[CS–NH]2-kinin. With the relatively long isomerization half-life, different bioactivities of c[CS–N]2-kinin after/before irradiation were successfully investigated. But in the case of c[CS– NH]2-kinin, because of the very short half-life of cis isomer, the bioactivity was not affected by CS–NH. In summery, the differences between the photoisomerization abilities of CS–NH and CS–N, have been studied by introducing them into insect kinin C-terminal pentapeptide and theoretical calculations. The results showed that the cis isomer of thioxo prolyl bond has high cis/trans energy barrier and long half-life. While the cis isomer of secondary thioxopeptide bond is much less stable and its half-life is too short for application. Therefore the thioxo prolyl bond can be used for structure–activity relationship studies. Acknowledgments This work was partially supported by the grant from National Natural Science Foundation of China (No. 30870526) and SRF for ROCS, SEM. References [1] [2] [3] [4] [5] [6]
R. Frank, M. Jakob, F. Thunecke, et al. Angew. Chem. Int. Ed. 39 (2000) 1120. J.Z. Zhao, D. Wildemann, M. Jakob, et al. Chem. Commun. 22 (2003) 2810. J.Z. Zhao, J.C. Micheau, C. Vargas, et al. Chem. Eur. J. 10 (2004) 6093. J. Helbing, H. Bregy, J. Bredenbeck, et al. J. Am. Chem. Soc. 126 (2004) 8823. J.F. Brandts, H.R. Halvorson, M. Brennan, Biochemistry 14 (1975) 4953. A. Jabs, M.S. Weiss, R. Hilgenfeld, J. Mol. Biol. 286 (1999) 291.
Z.Y. Cong et al. / Chinese Chemical Letters 21 (2010) 476–479 [7] [8] [9] [10] [11] [12] [13] [14]
D. Wildemann, C. Schiene-Fischer, T. Aumuller, et al. J. Am. Chem. Soc. 129 (2007) 4910. Y. Huang, Z.Y. Cong, L.F. Yang, et al. J. Pept. Sci. 14 (2008) 1062. Z.Y. Cong, Y. Huang, L.F. Yang, et al., Protein Pept. Lett. 17 (2010). G.M. Holman, R.J. Nachman, G.M. Coast, Peptides 20 (1999) 1. R.J. Nachman, J. Zabrocki, J. Olczak, et al. Peptides 23 (2002) 709. M.W. Schmidt, K.K. Baldridge, J.A. Boatz, et al. J. Comput. Chem. 14 (1993) 1347. W.J. Hehre, R.F. Stewart, J.A. Pople, J. Chem. Phys. 51 (1969) 2657. P.C. Hariharan, J.A. Pople, Theor. Chim. Acta 28 (1973) 212.
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Chinese Chemical Letters 21 (2010) 476–479 www.elsevier.com/locate/cclet
Comparison of two thioxopeptide bond photoswitches in insect kinin Zhi Yuan Cong, Long Fei Yang, Ling Jiang, Dan Ye, Shou Liang Dong * Institute of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000, China Received 24 June 2009
Abstract The photoisomerization abilities of secondary thioxopeptide bond (CS–NH) and thioxo prolyl bond (CS–N) incorporated into the C-terminal pentapeptide of insect kinin were compared. H-Phe-Phe-c[CS–NH]-D-Ala-Trp-Gly-NH2 and H-Phe-Tyr-c[CS–N]Pro-Trp-Gly-NH2 were studied by UV–vis absorption. The isomerization energy barriers of the two segments, Ac-Phe-c[CS–NH]D-Ala-NH2 and Ac-Tyr-c[CS–N]-Pro-NH2 picked from the two peptides, were calculated using ab initio method. The cis isomer of CS–N is more stable than that of CS–NH due to higher energy barrier, so the former is more suitable in peptide structure–activity relationship studies. # 2009 Shou Liang Dong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Photoswitch; Thioxopeptide bond; Energy barrier
Recently, thioxopeptide bond has attracted considerable interest for its ability of converting the stable trans conformation into the thermodynamically unfavorable cis conformation by irradiation. The carbonyl sulfur substitution leads to red-shifted p–p* and n–p* absorption, and lowers the excitation energy of photoisomerization. Thus it can induce conformational change to peptide backbone via trans/cis conversion by irradiation [1–4]. Since the cis/trans isomerization of prolyl bond (CO-N) and secondary peptide bond (CO–NH) are both essential to peptides and proteins [5,6], both of the corresponding thioxopeptide bonds are worth study. Frank et al. have shown that thioxo prolyl bond (CS–N) could switch between cis and trans isomers when it is present in endomorphins [1]. With the marked differences of the two isomers’ UV–vis absorption spectra, they investigated the isomerization of thioxo prolyl bond. The cis/trans isomerization constant (kc/t) of endomorphin-2 is (5.23 0.03) 10 4 s 1 at 313 K. Zhao et al. studied the photoisomerization of secondary thioxopeptide bonds (CS–NH) in dipeptides [2,3], a dual-directional photoswitching effect was detected, whose thermal relaxation constant is (9.78 0.02) 10 4 s 1 for H-Phe-c[CS– NH]-Ala-OH at 289 K. Ala4-c[CS–NH]-Ala5 segment was also incorporated into the S-peptide of RNase S [7], and its kc/t is (5.70 0.005) 10 3 s 1 at 283 K. In our previous studies, CS–N photoisomerization was investigated in insect kinin analog H-Phe-Tyr-c[CS–N]-Pro-Trp-Gly-NH2 (c[CS–N]2-kinin) [8]. The results showed that the kc/t is (2.90 0.1) 10 4 s 1 at 293 K and the trans conformation before irradiation was also confirmed by 2D-NMR [9].
* Corresponding author. E-mail address: [email protected] (S.L. Dong). 1001-8417/$ – see front matter # 2009 Shou Liang Dong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.12.033
Z.Y. Cong et al. / Chinese Chemical Letters 21 (2010) 476–479
477
Fig. 1. UV–vis absorbance spectra of the two thioxopeptides: ground state (—), after 5 min of irradiation at 254 nm (- - - -), re-equilibrated after four cycles of irradiation–equilibration ( ). (a) c[CS–NH]2-kinin, 8.0 10 5 mol/L, 293 K. (b) c[CS–N]2-kinin, which is cited from Ref. [8], is presented here for comparing.
Fig. 2. The time-dependent cis/trans isomerization curve. The solid lines represent the first-order fit. (a) c[CS–NH]2-kinin, 8.0 10 5 mol/L 293 K. kc/t is (8.77 0.16) 10 3 s 1. (b) c[CS–N]2-kinin, which is cited from Ref. [8], is presented here for comparing. kc/t is (2.90 0.1) 10 4 s 1.
In order to explore the photoisomerization ability of CS–N and CS–NH under the same conditions, a new insect kinin thioxo analog H-Phe-Phe-c[CS–NH]-D-Ala-Trp-Gly-NH2 (c[CS–NH]2-kinin) containing a CS–NH, was synthesized. Insect kinins share a highly conserved C-terminal pentapeptide, Phe-Xaa-Xbb-Trp-Gly-NH2, in which Xaa and Xbb tolerate a broad range of amino acids [10]. The peptide bond between them was modified by thioxylation. The UV–vis absorption spectra, kc/t and the photoswitching effects of the two analogs (c[CS–N]2-kinin and c[CS– NH]2-kinin) were compared. To explore the mechanism, the thioxopeptide bond cis/trans isomerization energy barriers were calculated in two model peptides, Ac-Phe-[CS–NH]-D-Ala-NH2 and Ac-Tyr-[CS–N]-Pro-NH2 picked from the two analogs using ab initio method. The UV–vis spectrum of c[CS–N]2-kinin has been described in our previous study [8]. For c[CS–NH]2-kinin, all measurements were performed at 293 K. c[CS–NH]2-kinin was 8.0 10 5 mol/L in sodium phosphate buffer (0.01 mol/L, pH 7.2). The photostationary state was achieved by 5 min irradiation under 254 nm. The results are shown in Fig. 1. c[CS–NH]2-kinin red-shifted at 270 nm and showed a slightly reduced maximum intensity upon irradiation, which is a representative of the increased CS–NH cis/trans ratio [2,3]. There is a well-anchored isosbestic point at 273 nm, which means that only two conformers were involved [1,2]. And after four cycles of irradiation– equilibration, the spectrum of c[CS–NH]2-kinin can fully restore to the ground state. All these results indicate that the cis/trans photoswitching is reversible and occurs without decomposition. For c[CS–N]2-kinin [8], an increased maximum intensity at 277 nm and an isosbestic point at 252 nm were found after irradiation, showing that there is an increase of CS–N cis/trans ratio [1]. After irradiation, the UV absorption of c[CS–NH]2-kinin was monitored immediately at 293 nm. As shown in Fig. 2, kc/t is (8.77 0.16) 10 3 s 1, which is larger than that of c[CS–N]2-kinin (Its kc/t is (2.90 0.1) 10 4 s 1, 30 times lower than that of c[CS–NH]2-kinin) [8]. The smaller constant shows that the cis isomer of CS–N is much more stable than that of CS–NH upon UV irradiation. With the different stabilities of the
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Z.Y. Cong et al. / Chinese Chemical Letters 21 (2010) 476–479
Table 1 Relative energy of the cis isomers, transition states (TS) and energy barriers (Ebarrier) respect to the trans isomers of c[CS–N]-YP and c[CS–NH]F(D)A (kcal/mol). Dipeptides
c[CS–N]-YP c[CS–NH]-F(D)A
STO-3G
6-31G(d, p)
trans
cis
TS
Ebarrier
trans
cis
TS
Ebarrier
0.00 0.00
1.71 5.46
25.29 22.50
23.58 17.04
0.00 0.00
4.75 5.28
27.73 24.97
22.98 19.69
cis isomers, the photo-induced affection on cockroach hindgut myotropic activities were also compared between the two analogs. As the cis isomer of the thioxylation site is more bioactive than the trans, the activity of c[CS–N]2-kinin increased significantly due to the cis isomer rising to 47.7% after irradiation [8]. For c[CS–NH]2-kinin, no significant photo-induced activity change was found with a constant EC50 of (6.80 0.2) 10 7 mol/L. In addition, no antagonist effect was found for c[CS–NH]2-kinin, which was designed due to the inspiration from reported antagonistic insect kinin tetrazole analog [11]. The instability of cis CS–NH prevented the potential antagonist effect, further investigation will be needed if it is designed for practical use. To explain the mechanism of the different photoisomerization abilities, two sulfur modified dipeptides Ac-Tyrc[CS–N]-Pro-NH2 (c[CS–N]-YP) and Ac-Phe-c[CS–NH]-D-Ala-NH2 (c[CS–NH]-F(D)A) from c[CS–N]2-kinin and c[CS–NH]2-kinin, with acetyl in N-terminal and acylamide in C-terminal were studied. The bond rotational energy barriers were calculated by GAMESS [12]. In order to approach the real experiment conditions, the calculation was performed in water and the temperature was set as 298.15 K. The cis isomers, trans isomers and their transition states were optimized at HF/STO-3G level [13]. The initial molecular structure coordinates of cis isomers and trans isomers were used as GAMESS input files. The proposed probable structures were optimized to get the adjacent rotational transition states. By the successful location of the equilibrium geometry and root mean square (RMS) gradient convergence, all stationary points were confirmed as either minima or transition states, single point energy calculations were performed to obtain a more accurate energy value at HF/6-31G(d, p) level [14]. The calculation results are shown in Table 1. It is shown in Table 1 that at HF/STO-3G level the cis/trans isomerization energy barrier of c[CS–N]-YP is 6.54 kcal/mol higher than that of c[CS–NH]-F(D)A. From the results of single point energy calculation at HF/631G(d, p) level, a 3.29 kcal/mol higher energy barrier is also found in c[CS–N]-YP. These results indicate that to transfer from cis to trans, c[CS–N]-YP needs more energy. After irradiation, the increased cis isomer of c[CS–N]2kinin is more stable than that of c[CS–NH]2-kinin. With the relatively long isomerization half-life, different bioactivities of c[CS–N]2-kinin after/before irradiation were successfully investigated. But in the case of c[CS– NH]2-kinin, because of the very short half-life of cis isomer, the bioactivity was not affected by CS–NH. In summery, the differences between the photoisomerization abilities of CS–NH and CS–N, have been studied by introducing them into insect kinin C-terminal pentapeptide and theoretical calculations. The results showed that the cis isomer of thioxo prolyl bond has high cis/trans energy barrier and long half-life. While the cis isomer of secondary thioxopeptide bond is much less stable and its half-life is too short for application. Therefore the thioxo prolyl bond can be used for structure–activity relationship studies. Acknowledgments This work was partially supported by the grant from National Natural Science Foundation of China (No. 30870526) and SRF for ROCS, SEM. References [1] [2] [3] [4] [5] [6]
R. Frank, M. Jakob, F. Thunecke, et al. Angew. Chem. Int. Ed. 39 (2000) 1120. J.Z. Zhao, D. Wildemann, M. Jakob, et al. Chem. Commun. 22 (2003) 2810. J.Z. Zhao, J.C. Micheau, C. Vargas, et al. Chem. Eur. J. 10 (2004) 6093. J. Helbing, H. Bregy, J. Bredenbeck, et al. J. Am. Chem. Soc. 126 (2004) 8823. J.F. Brandts, H.R. Halvorson, M. Brennan, Biochemistry 14 (1975) 4953. A. Jabs, M.S. Weiss, R. Hilgenfeld, J. Mol. Biol. 286 (1999) 291.
Z.Y. Cong et al. / Chinese Chemical Letters 21 (2010) 476–479 [7] [8] [9] [10] [11] [12] [13] [14]
D. Wildemann, C. Schiene-Fischer, T. Aumuller, et al. J. Am. Chem. Soc. 129 (2007) 4910. Y. Huang, Z.Y. Cong, L.F. Yang, et al. J. Pept. Sci. 14 (2008) 1062. Z.Y. Cong, Y. Huang, L.F. Yang, et al., Protein Pept. Lett. 17 (2010). G.M. Holman, R.J. Nachman, G.M. Coast, Peptides 20 (1999) 1. R.J. Nachman, J. Zabrocki, J. Olczak, et al. Peptides 23 (2002) 709. M.W. Schmidt, K.K. Baldridge, J.A. Boatz, et al. J. Comput. Chem. 14 (1993) 1347. W.J. Hehre, R.F. Stewart, J.A. Pople, J. Chem. Phys. 51 (1969) 2657. P.C. Hariharan, J.A. Pople, Theor. Chim. Acta 28 (1973) 212.
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