- Email: [email protected]
Fatty acyl-gramicidin S derivatives with both high antibiotic activity and low hemolytic activity
Bioorganic & Medicinal Chemistry Letters 22 (2012) 106–109
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Fatty acyl-gramicidin S derivatives with both high antibiotic activity and low hemolytic activity Makoto Tamaki a, Kenta Fujinuma a, Takuji Harada a, Kazumasa Takanashi a, Mitsuno Shindo b, Masahiro Kimura b, Yoshiki Uchida b,⇑ a b
Department of Chemistry, Toho University, Funabashi, Chiba 274-8510, Japan Department of Health and Nutrition, Osaka Shoin Women’s University, Higashi-Osaka, Osaka 577-8550, Japan
a r t i c l e
i n f o
Article history: Received 27 September 2011 Accepted 17 November 2011 Available online 25 November 2011 Keywords: Fatty acyl-gramicidin S analogue High antibiotic activity Low hemolytic activity Structure–activity relationship
a b s t r a c t In the present study, novel eight GS derivatives having the octanoyl-(Lys)n- moieties, cyclo{-Val-Orn-LeuD-Phe-Pro(4b-NH-X)-Val-Orn-Leu-D-Phe-Pro-} {X = H (1), and -(Lys)n-CO(CH2)6CH3 n = 0 (2), 1 (3), 2 (4), and 3 (5)} and cyclo{-Val-Orn-Leu-D-Phe-Pro(4a-NH-X)-Val-Orn-Leu-D-Phe-Pro-} {X = H (6), and (Lys)n-CO(CH2)6CH3 n = 1 (7), and 2 (8)} were synthesized. Among them, 4, 5 and 8 result the high antibiotic activity against both Gram-positive and Gram-negative microorganisms tested. In addition, 4 and 5 showed very low hemolytic activity compared with that of GS. Thus, the introduction of the excess amino groups and the fatty acyl moiety to the c-NH2 group of Pro5 residue in GS molecule lowered the unwanted hemolytic activity and enhanced the desired antibiotic activity. Ó 2011 Elsevier Ltd. All rights reserved.
A remarkable increase of multidrug-resistant bacteria has become a serious threat to public health threat.1 The amphiphilic antibiotics that attack bacterial membranes are promising agents for combating bacterial pathogens. Among them, cyclic decapeptide gramicidin S (GS), cyclo(-Val-Orn-Leu-D-Phe-Pro)2, are attractive targets for drug discovery.2–4 GS possesses the strong antibiotic activity against Gram-positive bacteria and weak antibiotic activity against Gram-negative bacteria. In addition, no resistance has been found for the antibiotics, because it requires significant alteration of the lipid composition of the cell membrane.5 However, the high antibacterial potency of GS is intimately associated with high hemolytic activity,6 preventing the direct use in combating the microbial resistance. The efforts to increase its therapeutic index, namely, minimizing its hemolytic activity while maintaining its high antibiotic activity, have been devoted.3,4,7,8 On the other hand, polymyxin B (PMB), octanoyl-Dab-Thr-Dabcyclic-(Dab⁄-Dab-D-Phe-Leu-Dab-Dab-Thr⁄) (⁄Amide bond between the ⁄c-NH2 of Dab and ⁄a-COOH of Thr), which is also an amphiphilic, membrane-active cyclic peptide antibiotic, is strongly active against Gram-negative bacteria and inactive against Grampositive bacteria.9,10 The polycationic property and the fatty acyl moiety of PMB plays very important rule for exhibiting the strong activity against Gram-negative bacteria.11 In addition, PMB shows little hemolytic activity.12 In the present account, we synthesized novel analogues of GS having octanoyl-(Lys)n- moiety, cyclo{-Val-Orn-Leu-D-Phe-Pro(4b⇑ Corresponding author. E-mail address: [email protected] (Y. Uchida). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.11.062
NH-X)-Val-Orn-Leu-D-Phe-Pro-} {X = H (1), –CO(CH2)6CH3 (2), -Lys-CO(CH2)6CH3 (3), -(Lys)2-CO(CH2)6CH3 (4), and -(Lys)3CO(CH2)6CH3 (5)}, and examined the biological activities, in order to find drug candidates with high antimicrobial against both Gram-positive and Gram-negative bacteria, and low hemolytic activities. (Fig. 1) Octanoyl-(Lys)n- moiety was introduced into GS, because the polycationic property and the fatty acyl moiety of PMB plays very important rule for exhibiting the strong activity against Gram-negative bacteria.11 In addition, cyclo{-Val-Orn-LeuD-Phe-Pro(4a-NH-Y)-Val-Orn-Leu-D-Phe-Pro-} {Y = H (6),-LysCO(CH2)6CH3 (7) and -(Lys)2-CO(CH2)6CH3 (8)} (Fig. 1) were synthesized, in order to investigate the cis and trans substitution effect of c-position of Pro residue for the biological activity. The syntheses of 1–8 were performed as shown in Scheme 1. The protected linear precursors were prepared by using t-butoxycarbonyl(Boc)-solid phase peptide synthesis on oxime resin (loading of oxime group: 0.35 mmol g1 resins).13–15 Leu residue as a C-terminal amino acid residue was used based on the propensity of the biosynthetic precursor of GS, tyrocidin(TA) and gratisin(GR) to form a conformation highly favorable for head-tail cyclization.3,4 The formations of the cyclic peptides by the cyclization-cleavage of the linear precursors on oxime resin were performed in 1,4-dioxane with 2 equiv each of triethylamine(NEt3) and acetic acid for 1 day at room temperature. Complete deprotection of the cyclic products, using the hydrogenolysis by triphenylphosphine(TPP)16 in water, and 25% HBr in acetic acid, furnished [Pro(4b-NH2)5]-GS (1) and [Pro(4a-NH2)5]-GS (6). In the synthesis 0 of 2–5, 7 and 8 (Fig. 1), the fully-protected [Orn(Z)2,2 , Pro(4a/b0 5 N3) ]-GS(Z = benzyloxycarbonyl) was changed into [Orn(Z)2,2 ,
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Figure 1. Primary structures of GS and 1–8.
Table 1 Antibiotic activities of 1–5 and GSa
GS 1 2 3 4 5
A
B
C
D
E
F
3.13 6.25 12.5 6.25 3.13 6.25
3.13 6.25 6.25 6.25 3.13 12.5
3.13 6.25 6.25 3.13 6.25 6.25
3.13 12.5 6.25 3.13 3.13 3.13
25 50 50 6.25 12.5 12.5
25 25 50 6.25 6.25 6.25
a Minimum inhibitory concentration(MIC) value in mg ml1 was determined by a microplate dilution method with 106 organisms per ml medium. Antibiotic activity tests were performed 7–10 times for each peptide and we obtained results within the same limits of error for each peptide. A: Bacillus subtilis NBRC 3513, B: Bacillus megaterium ATCC 19213, C: Staphylococcus epidermidis NBRC 12933, D: Staphylococcus aureus NBRC 12732, E: Pseudomonas aeruginosa NBRC 3080, F: Escherichia coli NBRC 12734. A–D are Gram-positive bacteria. E and F are Gram-negative bacteria.
Scheme 1. Synthesis of 1 and 2. Other GS analogues 3–8 were synthesized by a similar method to that of 2. Reagents and conditions: (a) Boc-amino acid (3 equiv), BOP (3 equiv), HOBt (3 equiv) and NEt3 (6.5 equiv) in DMF for 90 min. Deprotection by 25% TFA/DCM for 30 min; (b) NEt3 (2 equiv) and AcOH (2 equiv) in 1,4-dioxane for 24 h. TPP (2 equiv) and ultra-pure water (4 equiv) in THF for 24 h; (c) 25% HBr/ AcOH for 6 h; (d) octanoic acid (2 equiv), HOBt (2 equiv), NEt3 (2 equiv) and EDCI (2 equiv) in DMF for 24 h. Deprotection by 25% HBr/AcOH for 6 h.
Pro(4a/b-NH2)5]-GS using the hydrogenolysis by TPP in water. Next, Boc-Lys(Z) and octanoic acid were coupled by the step-wise 0 elongation methods to the free amino group of [Orn(Z)2,2 , Pro(4a/ 5 b-NH2) ]-GS. All the masking groups of the precursor peptides of 2–5, 7 and 8 obtained were removed by 25% HBr in acetic acid. The cyclic products obtained were purified by means of sephadex LH-20 column chromatography, followed by recrystallization. The purity and identity assessment of the products were confirmed by thin-layer chromatography, high performance liquid chromatography, elemental analysis and fast-atom bombardment mass spectrometry before determination of the biological activities. First, cyclo{-Val-Orn-Leu-D-Phe-Pro(4b-NH-X)-Val-Orn-Leu-DPhe-Pro-} {X = H (1), –CO(CH2)6CH3 (2), -Lys-CO(CH2)6CH3 (3), -(Lys)2-CO(CH2)6CH3 (4), and -(Lys)3-CO(CH2)6CH3 (5)} were examined, in order to find novel antibiotic candidates with high antimicrobial activity against both Gram-positive and Gram-negative bacteria and low hemolytic activities. The results are summarized in Table 1 and Figure 2. Peptide 1 with the additional one amino group at 4b-position of Pro5 residue showed about 1/2 antibacterial activities of GS against all bacteria tested and showed low hemolytic activity. On the other hand, 2 with octanoyl-NH- group at 4b position of Pro5 residue showed similar antibacterial activities to that of 1, but showed higher hemolytic activity comparison with that of GS. The results indicated that the additional 4b-NH2 group of Pro5 residue
Figure 2. Dose dependence curves of hemolysis (%) against sheep erythrocytes induced by 1–5 and GS. The experiments were carried out three times for each peptide.
decreases the interaction with sheep cell membrane, while the octanoyl group binding at 4b-NH2 of Pro5 residue increase the interaction with sheep erythrocytes. Therefore, simultaneous introduction of both amino groups and hydrophobic groups to GS seemed to be a promising strategy for obtaining GS analogues possessing both high antibiotic activity and low hemolytic activity. Next, novel analogues of GS (3–5), carrying -(Lys)nCO(CH2)6CH3 (n = 1, 2 and 3) on the amino groups of the Pro5 (4b-NH2) residue of 1, were, therefore, synthesized. Peptides 3 with Pro5 {4b-NH-Lys-CO(CH2)6CH3} showed similar to that of GS against Gram-positive bacteria, and four times higher than that of GS toward Gram-negative bacteria tested. However, 3 showed similar high hemolytic activity to that of GS. Compound 4 with Pro5{4b-NH-Lys-Lys-CO(CH2)6CH3} showed the similar level of
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antimicrobial activity as that of 3 against Gam-positive and Gram-negative bacteria tested. In addition, 4 possessed very low hemolytic activity comparison with that of 3 and GS. The peptide concentration of 4 required for 50% hemolysis of sheep red blood cells was about 100 lM. Further, 5 with Pro5{4b-NH-Lys-Lys-LysCO(CH2)6CH3} showed slightly lower antibiotic and hemolytic activity than that of 4. The results indicated that the hydrophobic–hydrophilic balance of -(Lys)n-CO(CH2)6CH3 group on the amino groups of the Pro5(4b-NH2) residue is very important for exhibiting both high antibiotic activity and low hemolytic activity. Next, CD spectra of 1–5 and GS were measured in methanol, in order to investigate the structure–activity relationship of 1–5 (Fig. 3). Peptides 1–5 exhibited similar CD spectra to that of GS but with slight weaker intensity. As a negative maximum around 207 nm with a shoulder around 220 nm are known to be good criteria for the pleated b-sheet structure of GS, these analogues were assumed to adopt GS-like b-sheet conformations, but the conformation was distorted or destabilized compared with the parent GS. These results suggested that the introduce of -NH-(Lys)nCO(CH2)6CH3 (n = 0, 1, 2 and 3) moiety at 4b-H of Pro5 residue of GS may be partially effective through a structural change in its biological activity of 1–5. Further, 6–8 with Pro5(4a-NH2) and Pro5{4a-NH-(Lys)nCO(CH2)6CH3} (n = 1 and 2), respectively, were synthesized, in order to investigate the effect of the diastereomeric difference of 4 position of Pro5 residue for the biological activity and secondary structure. The results showed in Table 2, and Figures 4 and 5. The antibiotic activity of 6–8 was similar to that of the corresponding analogues 1, 3 and 4 with Pro5(4b-NH2) and Pro5{4b-NH-(Lys)nCO(CH2)6CH3} (n = 1 and 2), respectively. (Table 2) On the other hand, the hemolytic activities of 6–8 increased significantly compared with those of the corresponding analogues 1, 3 and 4. (Fig. 4) On the other hand, CD spectra of 6–8 showed similar curves to those of 1–5 and GS, indicating that these analogues were assumed to adopt similar conformation to those of 1–5 and GS. (Fig. 5) The results indicated that the diastereomeric difference of (Lys)n-CO(CH2)6CH3 moiety on Pro5 residue of GS analogues affect largely the hemolytic activity against sheep red blood cells. In present studies, we synthesized novel hybrid analogues (1–8) of GS and PMB with the strong activity against both Gram-positive and Gram-negative bacteria, which have Pro5(4a/b-NH2) and Pro5{4a/b-NH-(Lys)n-CO(CH2)6CH3} (n = 0, 1, 2 and 3). Among them, we found that 4 and 5, carrying 4b-NH-(Lys)n-CO(CH2)6CH3 groups (n = 2 and 3) in Pro5 of GS, respectively, have differential ionic interaction against the prokaryotic membrane and eukaryotic
Table 2 Antibiotic activities of 6–10 and GSa
GS 6 7 8
A
B
C
D
E
F
3.13 6.25 6.25 3.13
3.13 6.25 3.13 6.25
3.13 6.25 6.25 6.25
3.13 12.5 6.25 6.25
25 50 12.5 12.5
25 25 6.25 6.25
a Minimum inhibitory concentration(MIC) value in mg ml1 was determined by a microplate dilution method with 106 organisms per ml medium. Antibiotic activity tests were performed 7–10 times for each peptide and we obtained results within the same limits of error for each peptide. A: Bacillus subtilis NBRC 3513, B: Bacillus megaterium ATCC 19213, C: Staphylococcus epidermidis NBRC 12933, D: Staphylococcus aureus NBRC 12732, E: Pseudomonas aeruginosa NBRC 3080, F: Escherichia coli NBRC 12734. A–D are Gram-positive bacteria. E and F are Gram-negative bacteria.
Figure 4. Dose dependence curves of hemolysis (%) against sheep erythrocytes induced by 6–8 and GS. The experiments were carried out three times for each peptide.
Figure 5. CD spectra of 6–8 and GS in methanol.
Figure 3. CD spectra of 1–5 and GS in methanol.
membrane. In other words, the dissociations of high antimicrobial and low hemolytic activities are caused by the additional 4b-NH(Lys)n-CO(CH2)6CH3 groups (n = 2 and 3) in Pro5 of GS. In addition, we found that the diastereomeric difference of (Lys)n-CO(CH2)6CH3 moiety on Pro5 residue of GS analogues affect largely the hemolytic activity against sheep red blood cells. Our findings should be helpful in finding drug candidates with high antimicrobial and low hemolytic activities that are capable of combating microbial resistance. Currently, further synthetic studies of GS peptides with both strong antibiotic and low hemolytic activities are carrying on.
M. Tamaki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 106–109
Supplementary data Supplementary data (physical data and MS data of 1–8) associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2011.11.062. References and notes 1. Travis, J. Science 1994, 264, 360. 2. Battersby, A. R.; Craig, L. C. J. Am. Chem. Soc. 1951, 73, 1887. 3. Izumiya, N.; Kato, T.; Aoyagi, H.; Waki, M.; Kondo, M. Synthetic Aspects of Biologically Active Cyclic Peptide-Gramicidin S and Tyrocidines; Wiley: New York, 1979. Kodansha, Tokyo. 4. Waki, M.; Izumiya, N. Biochemistry of Peptide Antibiotics. In Fed. Rep. Ger.; Kleinkauf, H., Dhren, H. Von., de Gruyter, Eds., 1990; pp 205–244. Berlin. 5. Hancock, R. E. W. Lancet 1997, 349, 418.
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6. Kawai, M.; Yamamura, H.; Tanaka, R.; Umemoto, H.; Ohmizo, C.; Higuchi, S.; Katsu, T. J. Pept. Res. 2005, 65, 98. 7. Kondejewski, L. H.; Farmer, S. W.; Wishart, D. S.; Kay, C. M.; Hancock, R. E.; Hodges, R. S. J. Biol. Chem. 1996, 271, 25261. 8. Solanas, C.; de la Torre, B. G.; Fern´andez-Reyes, M.; Santiveri, C. M.; Jime´nez, M. A´.; Rivas, L.; Jime´nez, A. I.; Andreu, D.; Cativiela, C. J. Med. Chem. 2009, 52, 664. 9. Ainsworth, G. C.; Brown, A. M.; Brownlee, G. Nature 1947, 160, 263–264. 10. Benedict, R. G.; Langlykke, A. F. J. Bacteriol. 1947, 54, 24. 11. Sakura, N.; Itoh, T.; Uchida, Y.; Ohki, K.; Okimura, K.; Chiba, K.; Sato, Y.; Sawanishi, H. Bull. Chem. Soc. Jpn. 2004, 77, 1915. 12. Zorko, M.; Majerle, A.; Sarlah, D.; Keber, M.; Mohar, B.; Jerala, R. Antimicrob. Agents Chemother. 2005, 49, 2307. 13. Nakagawa, S. H.; Lau, H. S. H.; Kezdy, F. J.; Kaiser, E. T. J. Am. Chem. Soc. 1985, 107, 7087. 14. Nishino, N.; Xu, M.; Mihara, H.; Fujimoto, T.; Ueno, Y.; Kumagai, H. Tetrahedron Lett. 1992, 33, 1479. 15. Tamaki, M.; Honda, K.; Kikuchi, S.; Ishii, R. Tetrahedron Lett. 2006, 47, 8475. 16. Tamaki, M.; Han, G.; Hruby, V. J. J. Org. Chem. 2001, 66, 1038.
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Fatty acyl-gramicidin S derivatives with both high antibiotic activity and low hemolytic activity Makoto Tamaki a, Kenta Fujinuma a, Takuji Harada a, Kazumasa Takanashi a, Mitsuno Shindo b, Masahiro Kimura b, Yoshiki Uchida b,⇑ a b
Department of Chemistry, Toho University, Funabashi, Chiba 274-8510, Japan Department of Health and Nutrition, Osaka Shoin Women’s University, Higashi-Osaka, Osaka 577-8550, Japan
a r t i c l e
i n f o
Article history: Received 27 September 2011 Accepted 17 November 2011 Available online 25 November 2011 Keywords: Fatty acyl-gramicidin S analogue High antibiotic activity Low hemolytic activity Structure–activity relationship
a b s t r a c t In the present study, novel eight GS derivatives having the octanoyl-(Lys)n- moieties, cyclo{-Val-Orn-LeuD-Phe-Pro(4b-NH-X)-Val-Orn-Leu-D-Phe-Pro-} {X = H (1), and -(Lys)n-CO(CH2)6CH3 n = 0 (2), 1 (3), 2 (4), and 3 (5)} and cyclo{-Val-Orn-Leu-D-Phe-Pro(4a-NH-X)-Val-Orn-Leu-D-Phe-Pro-} {X = H (6), and (Lys)n-CO(CH2)6CH3 n = 1 (7), and 2 (8)} were synthesized. Among them, 4, 5 and 8 result the high antibiotic activity against both Gram-positive and Gram-negative microorganisms tested. In addition, 4 and 5 showed very low hemolytic activity compared with that of GS. Thus, the introduction of the excess amino groups and the fatty acyl moiety to the c-NH2 group of Pro5 residue in GS molecule lowered the unwanted hemolytic activity and enhanced the desired antibiotic activity. Ó 2011 Elsevier Ltd. All rights reserved.
A remarkable increase of multidrug-resistant bacteria has become a serious threat to public health threat.1 The amphiphilic antibiotics that attack bacterial membranes are promising agents for combating bacterial pathogens. Among them, cyclic decapeptide gramicidin S (GS), cyclo(-Val-Orn-Leu-D-Phe-Pro)2, are attractive targets for drug discovery.2–4 GS possesses the strong antibiotic activity against Gram-positive bacteria and weak antibiotic activity against Gram-negative bacteria. In addition, no resistance has been found for the antibiotics, because it requires significant alteration of the lipid composition of the cell membrane.5 However, the high antibacterial potency of GS is intimately associated with high hemolytic activity,6 preventing the direct use in combating the microbial resistance. The efforts to increase its therapeutic index, namely, minimizing its hemolytic activity while maintaining its high antibiotic activity, have been devoted.3,4,7,8 On the other hand, polymyxin B (PMB), octanoyl-Dab-Thr-Dabcyclic-(Dab⁄-Dab-D-Phe-Leu-Dab-Dab-Thr⁄) (⁄Amide bond between the ⁄c-NH2 of Dab and ⁄a-COOH of Thr), which is also an amphiphilic, membrane-active cyclic peptide antibiotic, is strongly active against Gram-negative bacteria and inactive against Grampositive bacteria.9,10 The polycationic property and the fatty acyl moiety of PMB plays very important rule for exhibiting the strong activity against Gram-negative bacteria.11 In addition, PMB shows little hemolytic activity.12 In the present account, we synthesized novel analogues of GS having octanoyl-(Lys)n- moiety, cyclo{-Val-Orn-Leu-D-Phe-Pro(4b⇑ Corresponding author. E-mail address: [email protected] (Y. Uchida). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.11.062
NH-X)-Val-Orn-Leu-D-Phe-Pro-} {X = H (1), –CO(CH2)6CH3 (2), -Lys-CO(CH2)6CH3 (3), -(Lys)2-CO(CH2)6CH3 (4), and -(Lys)3CO(CH2)6CH3 (5)}, and examined the biological activities, in order to find drug candidates with high antimicrobial against both Gram-positive and Gram-negative bacteria, and low hemolytic activities. (Fig. 1) Octanoyl-(Lys)n- moiety was introduced into GS, because the polycationic property and the fatty acyl moiety of PMB plays very important rule for exhibiting the strong activity against Gram-negative bacteria.11 In addition, cyclo{-Val-Orn-LeuD-Phe-Pro(4a-NH-Y)-Val-Orn-Leu-D-Phe-Pro-} {Y = H (6),-LysCO(CH2)6CH3 (7) and -(Lys)2-CO(CH2)6CH3 (8)} (Fig. 1) were synthesized, in order to investigate the cis and trans substitution effect of c-position of Pro residue for the biological activity. The syntheses of 1–8 were performed as shown in Scheme 1. The protected linear precursors were prepared by using t-butoxycarbonyl(Boc)-solid phase peptide synthesis on oxime resin (loading of oxime group: 0.35 mmol g1 resins).13–15 Leu residue as a C-terminal amino acid residue was used based on the propensity of the biosynthetic precursor of GS, tyrocidin(TA) and gratisin(GR) to form a conformation highly favorable for head-tail cyclization.3,4 The formations of the cyclic peptides by the cyclization-cleavage of the linear precursors on oxime resin were performed in 1,4-dioxane with 2 equiv each of triethylamine(NEt3) and acetic acid for 1 day at room temperature. Complete deprotection of the cyclic products, using the hydrogenolysis by triphenylphosphine(TPP)16 in water, and 25% HBr in acetic acid, furnished [Pro(4b-NH2)5]-GS (1) and [Pro(4a-NH2)5]-GS (6). In the synthesis 0 of 2–5, 7 and 8 (Fig. 1), the fully-protected [Orn(Z)2,2 , Pro(4a/b0 5 N3) ]-GS(Z = benzyloxycarbonyl) was changed into [Orn(Z)2,2 ,
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Figure 1. Primary structures of GS and 1–8.
Table 1 Antibiotic activities of 1–5 and GSa
GS 1 2 3 4 5
A
B
C
D
E
F
3.13 6.25 12.5 6.25 3.13 6.25
3.13 6.25 6.25 6.25 3.13 12.5
3.13 6.25 6.25 3.13 6.25 6.25
3.13 12.5 6.25 3.13 3.13 3.13
25 50 50 6.25 12.5 12.5
25 25 50 6.25 6.25 6.25
a Minimum inhibitory concentration(MIC) value in mg ml1 was determined by a microplate dilution method with 106 organisms per ml medium. Antibiotic activity tests were performed 7–10 times for each peptide and we obtained results within the same limits of error for each peptide. A: Bacillus subtilis NBRC 3513, B: Bacillus megaterium ATCC 19213, C: Staphylococcus epidermidis NBRC 12933, D: Staphylococcus aureus NBRC 12732, E: Pseudomonas aeruginosa NBRC 3080, F: Escherichia coli NBRC 12734. A–D are Gram-positive bacteria. E and F are Gram-negative bacteria.
Scheme 1. Synthesis of 1 and 2. Other GS analogues 3–8 were synthesized by a similar method to that of 2. Reagents and conditions: (a) Boc-amino acid (3 equiv), BOP (3 equiv), HOBt (3 equiv) and NEt3 (6.5 equiv) in DMF for 90 min. Deprotection by 25% TFA/DCM for 30 min; (b) NEt3 (2 equiv) and AcOH (2 equiv) in 1,4-dioxane for 24 h. TPP (2 equiv) and ultra-pure water (4 equiv) in THF for 24 h; (c) 25% HBr/ AcOH for 6 h; (d) octanoic acid (2 equiv), HOBt (2 equiv), NEt3 (2 equiv) and EDCI (2 equiv) in DMF for 24 h. Deprotection by 25% HBr/AcOH for 6 h.
Pro(4a/b-NH2)5]-GS using the hydrogenolysis by TPP in water. Next, Boc-Lys(Z) and octanoic acid were coupled by the step-wise 0 elongation methods to the free amino group of [Orn(Z)2,2 , Pro(4a/ 5 b-NH2) ]-GS. All the masking groups of the precursor peptides of 2–5, 7 and 8 obtained were removed by 25% HBr in acetic acid. The cyclic products obtained were purified by means of sephadex LH-20 column chromatography, followed by recrystallization. The purity and identity assessment of the products were confirmed by thin-layer chromatography, high performance liquid chromatography, elemental analysis and fast-atom bombardment mass spectrometry before determination of the biological activities. First, cyclo{-Val-Orn-Leu-D-Phe-Pro(4b-NH-X)-Val-Orn-Leu-DPhe-Pro-} {X = H (1), –CO(CH2)6CH3 (2), -Lys-CO(CH2)6CH3 (3), -(Lys)2-CO(CH2)6CH3 (4), and -(Lys)3-CO(CH2)6CH3 (5)} were examined, in order to find novel antibiotic candidates with high antimicrobial activity against both Gram-positive and Gram-negative bacteria and low hemolytic activities. The results are summarized in Table 1 and Figure 2. Peptide 1 with the additional one amino group at 4b-position of Pro5 residue showed about 1/2 antibacterial activities of GS against all bacteria tested and showed low hemolytic activity. On the other hand, 2 with octanoyl-NH- group at 4b position of Pro5 residue showed similar antibacterial activities to that of 1, but showed higher hemolytic activity comparison with that of GS. The results indicated that the additional 4b-NH2 group of Pro5 residue
Figure 2. Dose dependence curves of hemolysis (%) against sheep erythrocytes induced by 1–5 and GS. The experiments were carried out three times for each peptide.
decreases the interaction with sheep cell membrane, while the octanoyl group binding at 4b-NH2 of Pro5 residue increase the interaction with sheep erythrocytes. Therefore, simultaneous introduction of both amino groups and hydrophobic groups to GS seemed to be a promising strategy for obtaining GS analogues possessing both high antibiotic activity and low hemolytic activity. Next, novel analogues of GS (3–5), carrying -(Lys)nCO(CH2)6CH3 (n = 1, 2 and 3) on the amino groups of the Pro5 (4b-NH2) residue of 1, were, therefore, synthesized. Peptides 3 with Pro5 {4b-NH-Lys-CO(CH2)6CH3} showed similar to that of GS against Gram-positive bacteria, and four times higher than that of GS toward Gram-negative bacteria tested. However, 3 showed similar high hemolytic activity to that of GS. Compound 4 with Pro5{4b-NH-Lys-Lys-CO(CH2)6CH3} showed the similar level of
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antimicrobial activity as that of 3 against Gam-positive and Gram-negative bacteria tested. In addition, 4 possessed very low hemolytic activity comparison with that of 3 and GS. The peptide concentration of 4 required for 50% hemolysis of sheep red blood cells was about 100 lM. Further, 5 with Pro5{4b-NH-Lys-Lys-LysCO(CH2)6CH3} showed slightly lower antibiotic and hemolytic activity than that of 4. The results indicated that the hydrophobic–hydrophilic balance of -(Lys)n-CO(CH2)6CH3 group on the amino groups of the Pro5(4b-NH2) residue is very important for exhibiting both high antibiotic activity and low hemolytic activity. Next, CD spectra of 1–5 and GS were measured in methanol, in order to investigate the structure–activity relationship of 1–5 (Fig. 3). Peptides 1–5 exhibited similar CD spectra to that of GS but with slight weaker intensity. As a negative maximum around 207 nm with a shoulder around 220 nm are known to be good criteria for the pleated b-sheet structure of GS, these analogues were assumed to adopt GS-like b-sheet conformations, but the conformation was distorted or destabilized compared with the parent GS. These results suggested that the introduce of -NH-(Lys)nCO(CH2)6CH3 (n = 0, 1, 2 and 3) moiety at 4b-H of Pro5 residue of GS may be partially effective through a structural change in its biological activity of 1–5. Further, 6–8 with Pro5(4a-NH2) and Pro5{4a-NH-(Lys)nCO(CH2)6CH3} (n = 1 and 2), respectively, were synthesized, in order to investigate the effect of the diastereomeric difference of 4 position of Pro5 residue for the biological activity and secondary structure. The results showed in Table 2, and Figures 4 and 5. The antibiotic activity of 6–8 was similar to that of the corresponding analogues 1, 3 and 4 with Pro5(4b-NH2) and Pro5{4b-NH-(Lys)nCO(CH2)6CH3} (n = 1 and 2), respectively. (Table 2) On the other hand, the hemolytic activities of 6–8 increased significantly compared with those of the corresponding analogues 1, 3 and 4. (Fig. 4) On the other hand, CD spectra of 6–8 showed similar curves to those of 1–5 and GS, indicating that these analogues were assumed to adopt similar conformation to those of 1–5 and GS. (Fig. 5) The results indicated that the diastereomeric difference of (Lys)n-CO(CH2)6CH3 moiety on Pro5 residue of GS analogues affect largely the hemolytic activity against sheep red blood cells. In present studies, we synthesized novel hybrid analogues (1–8) of GS and PMB with the strong activity against both Gram-positive and Gram-negative bacteria, which have Pro5(4a/b-NH2) and Pro5{4a/b-NH-(Lys)n-CO(CH2)6CH3} (n = 0, 1, 2 and 3). Among them, we found that 4 and 5, carrying 4b-NH-(Lys)n-CO(CH2)6CH3 groups (n = 2 and 3) in Pro5 of GS, respectively, have differential ionic interaction against the prokaryotic membrane and eukaryotic
Table 2 Antibiotic activities of 6–10 and GSa
GS 6 7 8
A
B
C
D
E
F
3.13 6.25 6.25 3.13
3.13 6.25 3.13 6.25
3.13 6.25 6.25 6.25
3.13 12.5 6.25 6.25
25 50 12.5 12.5
25 25 6.25 6.25
a Minimum inhibitory concentration(MIC) value in mg ml1 was determined by a microplate dilution method with 106 organisms per ml medium. Antibiotic activity tests were performed 7–10 times for each peptide and we obtained results within the same limits of error for each peptide. A: Bacillus subtilis NBRC 3513, B: Bacillus megaterium ATCC 19213, C: Staphylococcus epidermidis NBRC 12933, D: Staphylococcus aureus NBRC 12732, E: Pseudomonas aeruginosa NBRC 3080, F: Escherichia coli NBRC 12734. A–D are Gram-positive bacteria. E and F are Gram-negative bacteria.
Figure 4. Dose dependence curves of hemolysis (%) against sheep erythrocytes induced by 6–8 and GS. The experiments were carried out three times for each peptide.
Figure 5. CD spectra of 6–8 and GS in methanol.
Figure 3. CD spectra of 1–5 and GS in methanol.
membrane. In other words, the dissociations of high antimicrobial and low hemolytic activities are caused by the additional 4b-NH(Lys)n-CO(CH2)6CH3 groups (n = 2 and 3) in Pro5 of GS. In addition, we found that the diastereomeric difference of (Lys)n-CO(CH2)6CH3 moiety on Pro5 residue of GS analogues affect largely the hemolytic activity against sheep red blood cells. Our findings should be helpful in finding drug candidates with high antimicrobial and low hemolytic activities that are capable of combating microbial resistance. Currently, further synthetic studies of GS peptides with both strong antibiotic and low hemolytic activities are carrying on.
M. Tamaki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 106–109
Supplementary data Supplementary data (physical data and MS data of 1–8) associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2011.11.062. References and notes 1. Travis, J. Science 1994, 264, 360. 2. Battersby, A. R.; Craig, L. C. J. Am. Chem. Soc. 1951, 73, 1887. 3. Izumiya, N.; Kato, T.; Aoyagi, H.; Waki, M.; Kondo, M. Synthetic Aspects of Biologically Active Cyclic Peptide-Gramicidin S and Tyrocidines; Wiley: New York, 1979. Kodansha, Tokyo. 4. Waki, M.; Izumiya, N. Biochemistry of Peptide Antibiotics. In Fed. Rep. Ger.; Kleinkauf, H., Dhren, H. Von., de Gruyter, Eds., 1990; pp 205–244. Berlin. 5. Hancock, R. E. W. Lancet 1997, 349, 418.
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