1134
Regular Article
Chem. Pharm. Bull. 60(9) 1134–1138 (2012)
Vol. 60, No. 9
Polycationic Gramicidin S Analogues with Both High Antibiotic Activity
and Very Low Hemolytic Activity
Makoto Tamaki,a Takuji Harada,a Kenta Fujinuma,a Kazumasa Takanashi,a Mitsuno Shindo,b
,b
Masahiro Kimura,b and Yoshiki Uchida*
b
a Department of Chemistry, Toho University; Funabashi, Chiba 274–8510, Japan: and Department of Health and
Nutrition, Osaka Shoin Women’s University; Higashi-Osaka, Osaka 577–8550, Japan.
Received April 2, 2012; accepted May 15, 2012
The substitution of each constituent amino acid residue of gramicidin S (GS), cyclo(-Val1,1′-Orn2,2′
-
Leu3,3′-D-Phe4,4′-Pro5,5′-)2 with Lys residue indicated that each side chain structure of the constituent amino
acid residues affect largely the antibiotic activity and hemolytic activity of GS. Further, the substitution of
D-Phe4,4′ and Pro5,5′ residues with basic amino acid residues as a Lys residue results the high antibiotic activ-
ity and the very low hemolytic activity. Thus, we have found novel positions on the scaffold of GS at D-Phe4,4′
and Pro5,5′ residues whose modification will significantly increase the therapeutic index.
Key words gramicidin S; polycationic analogue; high antibiotic activity; low hemolytic activity; structure–
activity relationship
In view of widespread antibiotic resistance that has be- linear precursors were prepared by using Boc-solid phase pep-
come a serious threat to public health,1) amphiphilic antibi- tide synthesis (Boc=t-butoxycarbonyl) on oxime resin (load-
otics are attractive targets for drug discovery. Gramicidin S ing of oxime group: 0.35mmol/g resins).19–23) Leu residue as a
(GS),2–8) cyclo(-Val1,1′-Orn2,2′-Leu3,3′-D-Phe4,4′-Pro5,5′-)2, tyro- C-terminal amino acid residue was used based on the propen-
cidine A (TA),2,3,9,10) cyclo(-Val1-Orn2-Leu3-D-Phe4-Pro5-Phel6- sity of the biosynthetic precursor of GS, TA and GR to form a
D-Phe7-Asn8-Gln9-Tyr10-) and gratisin (GR),3,11–15) cyclo(-Val1,1′
-
conformation highly favorable for head-tail cyclization.3,4) The
Orn2,2′-Leu3,3′-D-Phe4,4′-Pro5,5′-D-Tyr6,6′-)2 are potent cyclopep- formations of the cyclic peptides by the cyclization-cleavage
tide antibiotics with the amphiphilic β-sheet conformation. It of the linear precursors on oxime resin were performed in
has been proposed that the principal modes of the antibiotic 1,4-dioxane with 2eq of triethylamine (NEt3) and acetic acid
actions result from an interaction of these antibiotics with the (AcOH) for 1d at room temperature. Complete deprotection
cell membrane of the target microorganisms.2–15) In addition, of the cyclic products, using 25% HBr in AcOH, furnished
so far, no resistance has been found for the antibiotics, be- [Lys1,1′]-GS (1). The cyclic products obtained were purified by
cause it requires significant alteration of the lipid composition means of Sephadex LH-20 column chromatography, followed
of the cell membrane.16) However, GS, TA and GR have the by recrystalization. Peptides 2–10 were synthesized from
high hemolytic activity, preventing their direct use in com- Boc-Leu-oxime resin (514mg, 0.18mmol) by using a similar
bating the microbial resistance.1–3) In order to find drug can- manner to that of 1. The masking groups of the protected pre-
didates with high antimicrobial activity and low hemolytic ac- cursors of 7, 8 and 10 were removed by hydrogenolysis. The
tivity, many analogues of GS, TA and GR have been designed purity and identity assessment of the products were confirmed
and synthesized.2–15) For example, we reported in the studies by thin-layer chromatography, fast-atom bombardment mass
of GR using a lysine-scanning method, which is replaced by spectrometry and elemental analysis before determination of
Lys residue in place of each constituent amino acid residue of the biological activities.
GR, that the substitution of Pro5,5′ and D-Tyr6,6′ residues of the
natural product with a cationic amino acid results the signifi-
cant increase of the therapeutic index.17,18) Further, similar re-
sults were obtained in the studies of single substitution of Gln6
of the natural TA with a cationic amino acid residue by Qin
et al.10) However, the modifications of each constituent amino
acid residue of GS with cationic amino acid residue have not
been performed yet.
Circular Dichroism (CD) Spectra of 1–10 The CD spec-
In the present studies, we performed a systematic approach
using a lysine-scanning method to identify the structural de-
terminants of its antibiotic property and hemolytic property
that induced to dissociation of the two closely associated
properties.
Experimental
Preparation of GS Analogues 1–10 The syntheses of GS
analogues were performed as shown in Chart 1. The protected
Other GS analogues 2–10 (Figs. 1, 4) were synthesized a similar method to
that of 1. Reagents and conditions; (a) Boc-amino acid (3eq), BOP (3eq), HOBt
(3eq) and NEt3 (6.5eq) in DMF for 90min. Deprotection by 25% TFA/DCM for
30min; (b) NEt3 (2eq) and AcOH (2eq) in 1,4-dioxane for 24h; (c) 25% HBr/
AcOH for 6h.
The authors declare no conflict of interest.
Chart 1. Synthesis of 1
*To whom correspondence should be addressed. e-mail: uchida.yoshiki@osaka-shoin.ac.jp
© 2012 The Pharmaceutical Society of Japan