Syntheses of low-hemolytic antimicrobial gratisin peptides

Article abstract of DOI:10.1016/j.bmcl.2009.03.133

Antibiotic and hemolytic activities of gratisin (GR), cyclo(-Val¹-Orn²-Leu³-d-Phe⁴-Pro⁵-d-Tyr⁶-)₂, and fifteen GR analogues, which have various d-amino acid residues in place of

Full text of DOI:10.1016/j.bmcl.2009.03.133

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2856–2859
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Bioorganic & Medicinal Chemistry Letters
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Syntheses of low-hemolytic antimicrobial gratisin peptides
a
a
a
a
a
Makoto Tamaki a, , Manabu Kokuno , Ichiro Sasaki , Yumiko Suzuki , Michiko Iwama , Kenichi Saegusa ,
*
Yusuke kikuchi a, Mitsuno Shindo b, Masahiro Kimura b, Yoshiki Uchida b,
*
a Department of Chemistry, Toho University, Funabashi, Chiba 274-8510, Japan
b Department of Food Science and Nutrition, Osaka Shoin Women’s University, Higashi-Osaka, Osaka 577-8550, Japan
a r t i c l e i n f o
a b s t r a c t
Antibiotic and hemolytic activities of gratisin (GR), cyclo(-Val1-Orn2-Leu3-
D
-Phe4-Pro5-
D
-Tyr6-)2, and fif-
Article history:
0
-Tyr6,6 residues, were exam-
Received 16 December 2008
Revised 19 March 2009
Accepted 21 March 2009
Available online 28 March 2009
teen GR analogues, which have various
D
-amino acid residues in place of
D
0
0
0
ined. Among them, [
both Gram-positive and Gram-negative bacteria. In addition, the antibiotics showed significantly reduced
toxicity against human blood cells compared with gramicidin S, cyclo(-Val1-Orn2-Leu3- -Phe4-Pro5-)2.
Ó 2009 Elsevier Ltd. All rights reserved.
D D D
-Orn6,6 ]-GR, [ -Lys6,6 ]-GR and [ -Arg6,6 ]-GR showed the strong activity against
D
Keywords:
Gratisin
Fifteen analogues
High antibiotic activity
Low hemolitic activity
Structure–activity relationship
Gramicidin
tyrocidine
-Phe7-Asn8-Gln9-Tyr10-) and gratisin (GR).1b,4 cyclo(-Val1-Orn2-
Leu3- -Phe4-Pro5- -Tyr6-)2, are potent cyclopeptide antibiotics
S
(GS)1,2
,
,
cyclo(-Val1-Orn2-Leu3-
D
-Phe4-Pro5-)2,
A
(TA)1,3 cyclo(-Val1-Orn2-Leu3-
D
-Phe4-Pro5-Phel6-
D
D
D
with the amphiphilic b-sheet conformation. In view of widespread
antibiotic resistance that has become a serious threat to public
health,5 the amphiphilic antibiotics are attractive targets for drug
discovery. It has been proposed that the principal modes of the
antibiotic actions result from an interaction of these antibiotics
with the cell membrane of the target microorganisms. These anti-
biotics then adopt an antiparallel b-sheet conformation with amph-
iphilicty, which disrupts cell membrane.1–4 In addition, so far, no
resistance has been found for the antibiotics, because it requires
significant alteration of the lipid composition of the cell mem-
brane.6 However, GS and TA have the high hemolytic activity, pre-
venting their direct use in combating the microbial resistance.1–3 In
order to find drug candidates with high antimicrobial and low
hemolytic activities, many analogues of GS and TA have been de-
signed and synthesized.1–3 For example, single substitution of
Gln6 of the natural TA with a cationic amino acid residue results
in significant increase of a therapeutic index.3c On the other hand,
a therapeutic index of GR and its analogues has not been studied.
Figure 1. Dose dependence curves of hemolysis (%) induced by GS, GR and [
D
-
0
Ala6,6 ]-GR (2).
was very low compared with that of GS. Further, the substitution
0
of
of the hemolytic activity compared with that of GR. The results
D D-Ala residues results in decrease
-Tyr6,6 residues of GR with
0
indicated that the structural modifications at D
-Tyr6,6 residues of
GR are beneficial to identification of novel antibiotic candidates
without hemolytic activity. Therefore, we planed to synthesize
Recently, we measured the hemolytic activities against human
6,60
erythrocytes of GR and [D-Ala ]-GR (2), which has D-Ala residues
6,60
GR analogues that possess various
D
-amino acid residues in place
-Tyr6,6 residues to find other candidates with both high antimi-
crobial and low hemolytic activities.
In the present account, we designed and synthesized novel fif-
teen GR analogues (1–15), which have various -amino acid resi-
in place of
D-Tyr
residues. (Fig. 1) The hemolytic activity of GR
0
of
D
* Corresponding authors. Tel.: +81 47 472 4323; fax: +81 47 472 1855 (M.T.).
E-mail address: tamaki@chem.sci.toho-u.ac.jp (M. Tamaki).
D
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.133

M. Tamaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2856–2859
2857
Table 1
Antibiotic activity and HPLC retention times (RT) of GS, GR and GR peptides 1–7
No.
Peptides
MICa
C
(l
g/mL)
rtb (min)
A
B
D
E
F
GS
GR
3.13
6.25
100
12.5
12.5
6.25
6.25
6.25
6.25
3.13
6.25
100
12.5
12.5
6.25
6.25
6.25
6.25
3.13
12.5
>100
25
3.13
6.25
100
12.5
12.5
12.5
12.5
12.5
3.13
25
25
100
95.0
22.0
12.5
21.0
24.0
31.0
32.5
34.5
37.0
100
>100
>100
>100
>100
100
100
50
0
1
2
3
4
5
6
7
[Gly6,6 ]-GR
>100
>100
>100
>100
>100
>100
100
0
[D
[D
[D
[D
[D
[D
-Ala6,6 ]-GR
0
-Nva6,6 ]-GR
25
0
-Val6,6 ]-GR
12.5
12.5
12.5
6.25
0
-Leu6,6 ]-GR
0
-Phe6,6 ]-GR
0
-Nle6,6 ]-GR
a
MIC (Minimum Inhibitory concentration) was determined by microplate dilu-
tion method with 106 organisms per ml. The microorganisms employed in the
assays were Bacillus subtilis NBRC 3513 (A), Bacillus megaterium ATCC 19213 (B),
Staphylococcus epidemidis NBRC 12933 (C), Staphylococcus aureus NBRC 12732 (D),
Pseudomonas aeruginosa NBRC 3080 (E) and Escherichia coli NBRC 12734 (F).
Scheme 1. Syntheses of GR and Its Analogues (1–15). Reagents and conditions: (a)
Boc-Leu-OH (3 equiv), DCC (3 equiv) in DCM 24 h; (b) Boc-amino acid (3 equiv),
BOP(3 equiv), HOBt (3 equiv), NEt3 (6.5 equiv) in DMF 45 min Deprotection 25%
TFA/DCM 30 min; (c) NEt3 (2 equiv), AcOH (2 equiv) in 1,4-dioxane 24 h; (d) H2 10%
b
TSK-Gel C18 column (4.6 ꢀ 250 mm, 10 mm particle size, Tosoh Co., Japan) was
used; flow rate, 1 ml/min; solvent, MeOH-0.1%TFAaq (65:35); monitoring wave-
length, 220 nm.
Pd/C, HCl (2 equiv) in MeOH and 1,4-dioxane, 2 days. (X = Gly,
D
-Ala,
-His(Bzl),
-Phe, -Nle, D
D
-Nva,
-Lys(Z),
-Gln,
D-Val,
D
D
D
-Leu,
-Orn(Z), and
-Glu, -Thr,
D
-Phe,
D
-Nle,
-Arg(NO2). Y = Gly,
-Ser, -His, -Lys, -Orn, and D
D
-Gln,
D
-Glu(OBzl),
D
-Thr(Bzl),
-Nva, -Val,
-Arg).
D
-Ser(Bzl),
D
D
D
D-Ala,
D
D
D-Leu,
D
D
D
D
D
D
D
increased according to increasing of the degree of effective hydro-
phobicity of their molecular. (Fig. 2) Thus, the increase of the
hydrophobicity of GR peptide molecules is facilitating its binding
with the membrane of Gram-positive bacteria, while detrimental
to the interaction with mammalian cell membrane, which have
limited its therapeutic value to topical applications. (Table 1 and
Fig. 2).
dues (
D
-AA = Gly,
-Thr, -Ser,
-Tyr6,6 residues.
D
-Ala,
D
-Nva,
D
-Val, -Phe,
D
-Leu,
D
D-Nle, D-Gln,
D
-Glu,
D
D
D
-His,
D
-Lys,
D-Orn, and D-Arg) in place of
0
D
In the synthesis of gratisin and its analogues, a protected linear
precursor
(X = Gly,
oxime,
H-(-
-Val,
-Ser(Bzl),
D
-Phe-Pro-X-Val-Orn(Z)-Leu)2-oxime,
-Leu, -Phe, -Nle, -Gln,
-His(Bzl), -Lys(Z), -Orn(Z),
Next, GR peptides 8–15 having
ious functional hydrophilic side chains in place of
were synthesized, because the introduction to GR of
residues with hydrophilicity could expected to possess limited tox-
icity to erythrocytes.2e,3c The retention times of GR and GR pep-
tides 8–15 from ODS column showed in Table 2. The results
indicated that these GR peptides 8–15 have lower hydrophobicity
of their molecular than that of GR. As expected all of GR analogues
8–15 exhibited substantially reduced hemolytic activities. (Fig. 3)
D-amino acids residues with var-
0
D
-Tyr6,6 residues
-amino acid
D
-Ala,
D-Nva,
D
D
D
D
-D
D
D-
Glu(OBzl),
and
D
-Thr(Bzl),
D
D
D
D
D
D
-Arg(NO2)), were prepared by using Boc-solid phase peptide
synthesis on resin (Loading of oxime group: 0.6 mmol/g resins).7
(Scheme 1) Leu residue as a C-terminal amino acid residue was
used based on the propensity of the biosynthetic precursor of GS,
TA and GR to form a conformation highly favorable for head-tail
cyclization.1,2,4 The formation of the cyclic peptide by the cycliza-
tion-cleavage of H-(-D-Phe-Pro-X-Val-Leu-Orn(Z)-Leu)2-oxime on
Thus, a good correlation between the hemolytic activity and reten-
0
tion times of GR peptides 1–15 was found. (Fig. 4) [
D
-Gln6,6 ]-GR (8)
resin was performed in 1,4-dioxane with 2 equiv of triethylamine
and acetic acid for 1 day at room temperature.7 The cyclic product
obtained was purified by means of sephadex LH-20 column chro-
matography, followed by recrystallization. The cyclizations gave
with the neutral amide side chains showed 1/2 activity of GR
against Gram-positive bacteria and no activity toward Gram-nega-
0
0
tive bacteria. On the other hand, [
D
-Ser6,6 ]-, [
D
-Thr6,6 ]- and [
D-Glu
0
6,6 ]- GR (9–11) with the neutral hydroxyl and acidic side chains al-
cyclo[-Val-Leu-Orn(Z)-Leu-D-Phe-Pro-X-]2, in 50–70% yields. The
removal of all the masking groups by hydrogenolysis yielded the
corresponding antibiotics 1–15. The homogeneities of the antibiot-
ics were confirmed by thin-layer chromatography, high perfor-
mance liquid chromatography (HPLC), elemental analysis, and
fast-atom bombardment (FAB) mass spectrometry.
most showed the antibiotic activity against Gram-positive and
Gram-negative bacteria. Thus, the presence of D-amino acid resi-
dues with the neutral hydroxyl and the acidic side chains is almost
effective for the interaction with both bacterial membrane and
0
mammalian cell membrane. [
D
-His6,6 ]-GR (12) with weak basic
First, GR peptides 1–7 having
D-amino acids residues with
hydrophobic aliphatic and aromatic side chains in place of
D
-Tyr
6,60
residues were examined, in order to find novel antibiotic candi-
dates with high antimicrobial and low hemolytic activities. The re-
0
sults are summarized in Table 1 and Figure 2. [Gly 6,6 ]-GR (1)
almost showed antibacterial activities against both Gram-positive
and Gram-negative bacteria. On the other hand, GR peptides 2–7
having D-amino acids residues with hydrophobic aliphatic and aro-
matic side chains in 6, 60 position showed the strong against
Gram-positive bacteria, but almost showed the activity against
Gram-negative bacteria. Its activity against Gram-positive bacteria
increased according to increasing of the degree of effective hydro-
phobicity of their molecular, which was showed by the retention
times of each antibiotic from ODS column.8 (Table 1). The results
indicated that the presence of D-amino acid residues with hydro-
phobic side chains is effective for the interaction with Gram-posi-
tive bacterial membrane, while not for Gram-negative bacterial
membrane. However, the hemolytic values of GR peptides 1–7 also
Figure 2. Dose dependence curves of hemolysis (%) induced by GS, GR and GR-
peptides (1–7).

2858
M. Tamaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2856–2859
Table 2
addition, the antibiotics 13–15 showed the strong activity against
Gram-negative bacteria. The antibiotic activities against Pseudomo-
nas aeruginosa NBRC 3080 of 13, and Pseudomonas aeruginosa NBRC
3080 and Escherichia coli NBRC 12734 of 15 are eight times higher
than that of parent GR, respectively. It is interesting to note that
the antibiotic potency against both Gram-positive and Gram-nega-
tive bacteria can increase and the hemolytic potency for mamma-
Antibiotic activity and HPLC retention times (RT) of GR and GR peptides 8–15.
No.
Peptides
MICa
C
(l
g/mL)
D
rtb (min)
A
B
E
F
GR
6.25
12.5
>100
100
>100
12.5
6.25
6.25
6.25
6.25
12.5
>100
100
>100
12.5
6.25
6.25
3.13
12.5
25
>100
100
>100
25
6.25
6.25
6.25
6.25
25
>100
50
100
12.5
6.25
6.25
3.13
100
100
22.0
17.2
16.5
4.5
3.5
2.2
2.2
2.0
2.0
0
8
9
[
[
[
[
[
[
[
[
D
D
D
D
D
D
D
D
-Gln6,6 ]-GR
>100
>100
>100
>100
>100
12.5
25
>100
>100
>100
>100
>100
25
0
-Glu6,6 ]-GR
0
10
11
12
13
14
15
-Thr6,6 ]-GR
lian cell membrane can decrease when cationic
D
-amino acid
-Tyr6,6 of GR.
GR is an amphipathic peptide antibiotic with four hydropho-
0
0
-Ser6,6 ]-GR
0
residues are incorporated into positions of
D
-His6,6 ]-GR
0
-Lys6,6 ]-GR
1’
3’
bic side chains of Val1, and Leu3, residues on one side and
two cationic side chains cons of Orn2, 2’ residues on another side
of the antiparallel b-sheet structure.4e GR with two cationic side
chains is the strong antibacterial activity against Gram-positive
bacteria, but is very weakly active against Gram-negative bacte-
ria. On the other hand, polymixin B, which is an amphiliphic
structure with polycationic nature, is strongly active against
Gram-negative bacteria but is inactive or very weakly active
against Gram-positive bacteria.10 Polycationic antibiotics such
as polymyxin B are considered to bind to the outer membrane
of Gram-negative bacteria, leading to their disorganization and
permeabillization.11 In the present studies, we synthesized novel
GR peptides 13–15 with the strong activity against both Gram-
0
-Orn6,6 ]-GR
0
25
12.5
-Arg6,6 ]-GR
12.5
a
MIC (Minimum Inhibitory concentration) was determined by microplate dilu-
tion method with 106 organisms per ml. The microorganisms employed in the
assays were Bacillus subtilis NBRC 3513 (A), Bacillus megaterium ATCC 19213 (B),
Staphylococcus epidemidis NBRC 12933 (C), Staphylococcus aureus NBRC 12732 (D),
Pseudomonas aeruginosa NBRC 3080 (E) and Escherichia coli NBRC 12734 (F).
b
TSK-Gel C18 column (4.6 ꢀ 250 mm, 10 mm particle size, Tosoh Co., Japan) was
used; flow rate, 1 ml/min; solvent, MeOH-0.1%TFAaq (65:35); monitoring wave-
length, 220 nm.
positive and Gram-negative bacteria, which have
D-amino acid
0
residues with cationic side chains in place of
D
-Tyr6,6 residues.
In addition, we found that the GR peptides 13–15 have differen-
tial ionic interaction against the prokaryotic membrane and
eukaryotic membrane. In other words, the dissociations of high
antimicrobial and low hemolytic activities are caused by the
additional positive charges of 13–15.
In conclusion, we have found a novel position on the scaffold of
0
GR at
D
-Tyr6,6 residues whose modification will significantly lower
the unwanted hemolytic activity and simultaneously enhance the
desired antibiotic activity. Our findings should be helpful in finding
drug candidates with high antimicrobial and low hemolytic activ-
ities that are capable of combating microbial resistance. Currently,
further synthetic studies of GR peptides with both strong antibiotic
and low hemolytic activities are carrying on.
Figure 3. Dose dependence curves of hemolysis (%) induced by GR and GR-peptides
(8–15).
Supplementary data
Supplementary data (experimental procedures, and TLC, HPLC,
MS and elemental analyses data for 1–15) associated with this arti-
cle can be found, in the online version, at doi:10.1016/
j.bmcl.2009.03.133.
References and notes
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Figure 4. Correlation between hemolysis (%)a of GR peptides (1–15) and their
retention time (min) in HPLC analysis.b aPercentage hemolysis of the peptides
(100 l
M) in 10% DMSO-buffer solution against human erythrocytes. bTSK-Gel C18
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cationic amino acid residual
D
-Orn,
residues. [
GR (13–14) possessed the same activity as that of parent GR
D-Lys, and D-Arg is introduced
6,60
0
0
into positions of
D
-Tyr
D D
-Lys6,6 ]-,9 and [ -Orn6,6 ]-
0
against Gram-positive bacteria. Further, the activity of [D
-Arg6,6 ]-
GR (15) is two times higher than that of parent GR against Bacillus
megaterium ATCC 19213 and Staphylococcus aureus NBRC 12732. In

M. Tamaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2856–2859
2859
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