Biomimetic synthesis of a peptide antibiotic, gratisin

Article abstract of DOI:10.1039/a701537b

Recently, we reported the direct formation of a peptide antibiotic, gramicidin S (GS), cyclo(-D-Phe-Pro-Val-Orn-Leu-)₂, by the dimerization-cyclization of pentapeptide active esters, D-Phe-Pro-Val-Orn-Leu-ONSu (-ONSu: succinimide ester), having the sequence identical with that of the linear precursor pentapeptide in the biosynthesis of GS and no protecting group on the side-chain of the Orn residue. This biomimetic approach has been extended to the synthesis of a peptide antibiotic, gratisin (GR), cyclo(-D-Phe-Pro-D-Tyr-Val-Orn-Leu-)₂, isolated from Bacillus brevis Y-33. In the cyclization of six hexapeptide succinimide esters having a Val, Orn, Leu, D-Phe, Pro or D-Tyr residue at each C-terminus, only H-D-Phe-Pro-D-Tyr-Val-Orn-Leu-ONSu gave semi-GR and GR in yields of 31 and 8%, respectively. Other hexapeptide esters did not give semi-GR and GR. In both biomimetic syntheses of GS and GR, the amino acid sequences having a Leu residue at the C-terminus are essential. In addition, a change in the concentration of peptide and the polarity of reaction solvents influenced greatly the yields of cyclic monomer (semi-GR) and cyclic dimer (GR). However, the yield of GR by dimerization-cyclization of hexapeptide active ester was lower when compared with the direct formation of GS (38%). The difference may result from differences in the chain length and the configurations of amino acid residues around the Pro residue.

Full text of DOI:10.1039/a701537b

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Biomimetic synthesis of a peptide antibiotic, gratisin
Makoto Tamaki,*,a Seiji Komiya,a Sadatoshi Akaboria and
Ichiro Muramatsub
a
Department of Chemistry, Faculty of Science, Toho University, Funabashi, Chiba 274, Japan
b Tokyo Bunka Junior College, Hon-cho, Nakano-ku, Tokyo 164, Japan
Recently, we reported the direct formation of a peptide antibiotic, gramicidin S (G S), cyclo(-D -Phe-Pro-
Val-Orn-Leu-)2, by the dimerization–cyclization of pentapeptide active esters, D -Phe-Pro-Val-Orn-Leu-
ON Su (-ON Su: succinimide ester), having the sequence identical with that of the linear precursor
pentapeptide in the biosynthesis of G S and no protecting group on the side-chain of the Orn residue. This
biomimetic approach has been extended to the synthesis of a peptide antibiotic, gratisin (G R ), cyclo(-D -
Phe-Pro-D -Tyr-Val-Orn-Leu-)2, isolated from Bacillus brevis Y-33. In the cyclization of six hexapeptide
succinimide esters having a Val, Orn, Leu, D -Phe, Pro or D -Tyr residue at each C-terminus, only H -D -
Phe-Pro-D -Tyr-Val-Orn-Leu-ON Su gave semi-G R and G R in yields of 31 and 8%, respectively. Other
hexapeptide esters did not give semi-G R and G R . In both biomimetic syntheses of G S and G R , the amino
acid sequences having a Leu residue at the C-terminus are essential. In addition, a change in the
concentration of peptide and the polarity of reaction solvents influenced greatly the yields of cyclic
monomer (semi-G R ) and cyclic dimer (G R ). H owever, the yield of G R by dimerization–cyclization of
hexapeptide active ester was lower when compared with the direct formation of G S (38%). The difference
may result from differences in the chain length and the configurations of amino acid residues around the Pro
residue.
analogous to that of GS.6,7 To investigate the formation of GR
Introduction
by the dimerization–cyclization of hexapeptide active esters
having no protecting group on the side-chains of Orn and -Tyr
residues, the cyclizations of six linear hexapeptide-ONSus 1–6
having a Val, Orn, Leu, -Phe, Pro or -Tyr residue at each
C-terminus were examined. In addition, Z--Phe-Pro--
In synthetic studies of gramicidin S (GS),1,2 cyclo(--Phe-Pro-
Val-Orn-Leu-)2, and its analogues by dimerization–cyclization
of pentapeptide precursors having protecting groups on the side-
chains of the Orn residue, it was pointed out that the mode of
cyclization in the chemical synthesis of GS is significantly dif-
ferent from that of its biosynthesis,3 in which the C-terminal
Leu residue of the precursor is fastened onto the GS syn-
thetase.4 For example, the yields of the cyclic dimer (the di-Z
derivative of GS) in the reaction of H--Phe-Pro-Val-Orn(Z)-
Leu-N3 and -ONSu (-ONSu: succinimide ester) were lower
than those from precursors having a -Phe, Pro, Val or Orn(Z)
residue at the C-terminus.3
Tyr(BzlCl2)-Val-Orn-Leu-ONSu,
H-(--Phe-Pro--Tyr-Val-
Orn-Leu-)2-ONSu, and three hexapeptide-ONSus 7–9, which
possess -Phe-Pro-Tyr, Phe-Pro--Tyr and Phe-Pro-Tyr
sequences at the N-terminus, respectively, were cyclized, in
order to investigate the cyclization mode of H--Phe-Pro--
Tyr-Val-Orn-Leu-ONSu.
H-Val-Orn-Leu-D-Phe-Pro-D-Tyr-ONSu
H-Orn-Leu-D-Phe-Pro-D-Tyr-Val-ONSu
H-Leu-D-Phe-Pro-D-Tyr-Val-Orn-ONSu
H-D-Phe-Pro-D-Tyr-Val-Orn-Leu-ONSu
H-Pro-D-Tyr-Val-Orn-Leu-D-Phe-ONSu
H-D-Tyr-Val-Orn-Leu-D-Phe-Pro-ONSu
H-D-Phe-Pro-Tyr-Val-Orn-Leu-ONSu
H-Phe-Pro-D-Tyr-Val-Orn-Leu-ONSu
H-Phe-Pro-Tyr-Val-Orn-Leu-ONSu
1
2
3
4
5
6
7
8
9
Val-Orn-Leu-D-Phe-Pro-D-Tyr
D-Tyr-Pro-D-Phe-Leu-Orn-Val
gratisin (GR)
Val-Orn-Leu-D-Phe-Pro
Pro-D-Phe-Leu-Orn-Val
gramicidin S (GS)
[
]
[
]
Primary structures of GR and GS
Recently, we reported the direct formation of GS by the
dimerization–cyclization of pentapeptide active esters having
no protecting group on the side-chain of the Orn residue.5
Among the five succinimide esters having a Val, Orn, Leu,
-Phe or Pro residue at each C-terminus, only H--Phe-Pro-Val-
Orn-Leu-ONSu, having a sequence identical with that of the
linear precursor pentapeptide in the biosynthesis of GS,4 gave
semi-GS (cyclic monomer) and GS (cyclic dimer) in yields of 15
and 38%, respectively. Other pentapeptide active esters did not
give GS. It can be concluded from studies on the cyclization
mode of H--Phe-Pro-Val-Orn-Leu-ONSu that, both in bio-
logical and in chemical syntheses, the formation of the cyclic
peptides is subject to similar regiospecific control, although the
conditions of their cyclizations are different, especially in bio-
synthesis, the enzyme probably giving a steric environment
more favourable for the formation of GS.
Primary structures of hexapeptide active esters 1–9 related to GR
Results and discussion
The hexapeptide-ONSus 1–6 related to GR were synthesized
using a solution-phase methodology. These active esters 1–6
were cyclized in pyridine for 1 day at 25 ЊC (concentration of
peptide in pyridine: 3 mM). Purification of the main products
in the reaction mixture was performed by gel filtration using
Sephadex LH-20 and semipreparative high-performance liquid
chromatography (HPLC). The primary structure of the prod-
ucts was elucidated by amino acid analyses and fast-atom bom-
bardment (FAB) mass spectra, and was confirmed by a direct
comparison with authentic samples synthesized according to
conventional methods. The primary structures of the main
products are shown in Table 1.8
In the present study, this biomimetic approach has been
extended to the synthesis of a peptide antibiotic, gratisin (GR),
cyclo(--Phe-Pro--Tyr-Val-Orn-Leu-)2, which has a structure
Among the six active esters 1–6 having a Val, Orn, Leu,
J. Chem. Soc., Perkin Trans. 1, 1997
2045

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Table 1 Primary structures of the cyclic products isolated from the cyclization mixture of linear hexapeptide active esters 1–6
Cyclic products
Active
ester
a
b
1
2
3
H-Val-Orn-Leu--Phe-Pro--Tyr
H-Orn-Leu--Phe-Pro--Tyr-Val
H-Leu--Phe-Pro--Tyr-Val-Orn
[-Phe-Pro--Tyr-Val-Orn-Leu ]
Leu-Orn-Val--Tyr-Pro--Phe
-Phe-Pro--Tyr-Val-Orn-Leu
(semi-GR)
4
(GR)
5
6
H-Pro--Tyr-Val-Orn-Leu--Phe
H--Tyr-Val-Orn-Leu--Phe-Pro
Table 2 Effect of peptide concentration on the cyclization of
H--Phe-Pro--Tyr-Val-Orn-Leu-ONSu 4a,b
resulted in different ratios of semi-GR to GR. Thus, the pola-
rity of the solvent affects the yield of GR.
These results concerning the effects of concentration and
solvent on the cyclization of H--Phe-Pro--Tyr-Val-Orn-Leu-
ONSu are similar to those of the pentapeptide related to GS,
H--Phe-Pro-Val-Orn-Leu-ONSu.5
Z--Phe-Pro--Tyr(BzlCl2)-Val-Orn-Leu-ONSu was cyclized
in pyridine (concentration of the peptide: 3 × 10Ϫ3 M) at 45 ЊC
for 1 day to examine the reactivity between the δ-amino group
of the Orn residue and the C-terminal ester group. A cyclic
product was isolated by gel filtration using Sephadex LH-20,
followed by recrystallization. However, considerable amounts
of the starting material were recovered unchanged from the
reaction mixture. The cyclic product was identified as the cyclic
dimer,
Ratio of cyclic
products
semi-GR :GR
Concentration of peptide
in pyridine (×10Ϫ3 M)
Total yield (%) of
semi-GR and GR
0.3
3
30
100:0
79:21
58:42
42
39
13
The cyclization was carried out in pyridine at 25 ЊC for 1 day. b The
ratio and yield of cyclic products were determined by HPLC analysis.
a
-Phe, Pro or -Tyr residue at each C-terminus, only peptide 4,
H--Phe-Pro--Tyr-Val-Orn-Leu-ONSu, gave semi-GR (cyclic
monomer) and GR (cyclic dimer) in yields of 31 and 8%,
respectively. On the other hand, other hexapeptide active esters
1–3, 5 and 6 did not give any amount of semi-GR and GR.
Active esters 1–3, 5 and 6 gave exclusively the cyclic compounds
containing the amide bond formed between the ester group of
the C-terminal residue and the δ-amino group of the Orn
residue.
The present results are similar to those obtained on the
cyclization of pentapeptide active esters related to GS having
no protecting group on the side-chain of the Orn residue,5 and
indicate that for the direct formation of GR and GS by the
dimerization–cyclization of hexa- and penta-peptide active
esters having no protecting group on the side-chain of the Orn
residue, the amino acid sequences having a Leu residue at the
C-terminus, H--Phe-Pro--Tyr-Val-Orn-Leu-ONSu and H--
Phe-Pro-Val-Orn-Leu-ONSu, are essential. However, the yield
of GR (8%) by cyclization–dimerization of peptide 4 is lower
when compared with the direct formation of GS (38%). The
difference may result from differences in the chain length and
the configurations of amino acid residues around the Pro
residue between each precursor active ester.
Next, the mode of the cyclization of H--Phe-Pro--Tyr-Val-
Orn-Leu-ONSu 4 was investigated by several methods.
The effect of concentration of the hexapeptide active ester
4 on the cyclization yield was examined (concentrations of
peptide in pyridine: 0.3, 3 and 30 × 10Ϫ3 M. Other conditions
are similar to those described above) (Table 2). The proportion
of semi-GR to GR in the product mixture depended on the
concentration of the active ester in pyridine solution and indi-
cates that formation of the cyclic dimer competes with that of
the cyclic monomer.
The solvent effect on the cyclization of peptide 4 was investi-
gated in various solvents [1,4-dioxane, benzene, CHCl3, EtOH,
dimethylformamide (DMF) and water]. The cyclization of the
active ester in 1,4-dioxane gave semi-GR and GR in ~4:6 ratio,
and its total yield was 25%. On the other hand, cyclization in
water give mainly semi-GR in 20% yield and the production of
GR was not detected by HPLC. Cyclizations in other solvents
-Leu-Orn-R
|
|
R-Orn-Leu-
R = [Z-D-Phe-Pro-D-Tyr(BzlCl2)-Val]—
on the basis of its relative molecular mass, which was deter-
mined by FAB mass spectrometry. The yield of cyclic dimer was
7%. This result indicates that the formation of Z--Phe-Pro--
Tyr(BzlCl2)-Val-Orn-Leu , having a highly strained nine-
membered ring, is very difficult. This should be one reason for
the preferential formation of semi-GR or GR in the reaction of
peptide 4.
Recently, in a study of the contribution of the -Phe-Pro-Val
sequence in the direct formation of GS by the dimerization–
cyclization of H--Phe-Pro-Val-Orn-Leu-ONSu, it has been
reported that the change in the configurations of the Phe and
Val residues around the Pro residue greatly affected the cycliz-
ation.9 That is, the active ester with a -Phe-Pro--Val sequence
produced exclusively [-Val]-semi-GS in 58% yield. The active
esters having Phe-Pro--Val and Phe-Pro-Val sequences did
not yield any cyclic monomer or cyclic dimer. On the other
hand, H--Phe-Pro-Val-Orn-Leu-ONSu gave semi-GS (cyclic
monomer) and GS (cyclic dimer) in yields of 15 and 38%,
respectively.
Next, in order to investigate the effect of configuration of
Phe and Tyr residues on the cyclization of H--Phe-Pro--Tyr-
Val-Orn-Leu-ONSu 4, three hexapeptide-ONSus 7–9 related to
GR, and which possess -Phe-Pro-Tyr, Phe-Pro--Tyr and
Phe-Pro-Tyr sequences at the N-terminus, respectively, were
synthesized, and cyclized in pyridine for 1 day at 25 ЊC (concen-
tration of peptide in pyridine: 3 mM). Peptide 7 having the -
Phe-Pro-Tyr sequence produced cyclic monomer ([-Tyr]-semi-
GR) and cyclic dimer ([-Tyr]-GR) in yields of 57 and 15%,
respectively, and this ratio of cyclic monomer to cyclic dimer is
similar to that obtained from peptide 4 having the -Phe-Pro--
Tyr sequence. Peptide 8 having the Phe-Pro--Tyr sequence
2046
J. Chem. Soc., Perkin Trans. 1, 1997

View Online
Fig. 2 CD spectra of (a) GR (——), (b) H-(-Phe-Pro--Tyr-Val-Orn-
Leu)2-OEt (——), and (c) H--Phe-Pro--Tyr-Val-Orn-Leu-OEt (– – –)
in ethanol
CD spectrum of H--Phe-Pro--Tyr-Val-Orn-Leu-OEt [Fig. 1
(A)] showed a shoulder near 217 nm, and a negative trough at
202 nm, and its features are similar to that of H--Phe-Pro--
Val-Orn-Leu-OEt, which the corresponding active ester, H--
Phe-Pro--Val-Orn-Leu-ONSu, produced exclusively [-Val]-
semi-GS in 58% yield.9 In addition, from CD and NMR studies
of pentapeptide precursors related to GS, it was pointed out
that the negative band or shoulder near 217 nm reflects mainly
the N-terminus conformation in those molecules.9 The present
study suggests that the N-terminal part of H--Phe-Pro--Tyr-
Val-Orn-Leu-OEt has an ordered conformation similar to that
of H--Phe-Pro--Val-Orn-Leu-OEt, which makes it suitable
for the intramolecular cyclization. This should be one reason
for the preferential formation of semi-GR compared with that
of GR in the cyclization of H--Phe-Pro--Tyr-Val-Orn-Leu-
ONSu 4. On the other hand, the CD spectrum of semi-GR
showed a curve that slopes steadily without any trough at 200–
220 nm [Fig. 1 (B)], suggesting that semi-GR has a disordered
conformation in ethanol. Thus, no relationship between the CD
spectra of H--Phe-Pro--Tyr-Val-Orn-Leu-OEt and semi-GR
produced mainly by the cyclization of its active ester was found.
The CD spectrum of H-(-Phe-Pro--Tyr-Val-Orn-Leu)2-OEt
showed a shoulder near 220 nm and a trough near 200 nm,
and its features resembled those of GR. In addition, cycliz-
ation of H-(-Phe-Pro--Tyr-Val-Orn-Leu)2-ONSu under con-
ditions similar to those in the case of the hexapeptide active
ester gave GR in 42% yield, but no H--Phe-Pro--Tyr-Val-
Fig.
1
CD spectra of (A) H--Phe-Pro--Tyr-Val-Orn-Leu-OEt
(——) and H--Phe-Pro--Val-Orn-Leu-OEt (– – –), and (B) semi-GR
(——) in ethanol
gave cyclic monomer ([-Phe]-semi-GR) in 32% yield, but the
production of cyclic dimer was not observed. On the other
hand, peptide 9 with the Phe-Pro-Tyr sequence did not produce
the corresponding cyclic monomer or cyclic dimer. These
results indicate that the amino acid sequences having a -Phe
residue at the N-terminus are essential for the production of
cyclic dimer, and also that the configuration of the Tyr residue
following the Pro residue contributes to the cyclization mode of
these precursors.
The cyclization of H--Phe-Pro--Tyr-Val-Orn-Leu-ONSu 4
and H-(-Phe-Pro--Tyr-Val-Orn-Leu)2-ONSu in ethanol gave
cyclic products similar to those formed in pyridine. That is, the
modes of its cyclization in both solvents seem to be similar. To
investigate the cyclization mode of H--Phe-Pro--Tyr-Val-
Orn-Leu-ONSu 4, circular dichroism (CD) spectra of ethyl
esters corresponding to H--Phe-Pro--Tyr-Val-Orn-Leu-
ONSu and H-(-Phe-Pro--Tyr-Val-Orn-Leu)2-ONSu, and
semi-GR and GR were measured in ethanol (Figs. 1 and 2). The
Orn-Leu--Phe-Pro--Tyr-Val-Orn-Leu . These results indi-
cate that the dodecapeptide active ester formed by the inter-
molecular reaction of two molecules of H--Phe-Pro--Tyr-
Val-Orn-Leu-ONSu 4 has a conformation in pyridine similar to
that of GR, and cyclizes preferentially to afford the antibiotic.
On the basis of the above results, the mode of cyclization of
H--Phe-Pro--Tyr-Val-Orn-Leu-ONSu 4 is proposed as fol-
lows. In the intramolecular reaction, the C-terminal ester reacts
with the α-amino group of the -Phe residue to give the semi-
GR but does not react with the δ-amino group of the Orn
residue. The cyclization is more favourable for the formation
of semi-GR than for GR. On the other hand, in the inter-
molecular reaction, the dodecapeptide active ester formed by
the coupling of two molecules of hexapeptide active ester
cyclize to afford GR. Thus, the formation mechanism of GR by
dimerization–cyclization of H--Phe-Pro--Tyr-Val-Orn-Leu-
J. Chem. Soc., Perkin Trans. 1, 1997
2047

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Table 3 Physical properties and analytical data for intermediary products
[α]D25
Found (%) (required)
Mp
(c 1.0,
No.
1
Compound
(T/ЊC)
MeOH)
Formula
C
H
N
Boc-Val-Orn(Z)-Leu--Phe-Pro--Tyr(BzlCl2)-OBzl
Boc-Orn(Z)-Leu--Phe-Pro--Tyr(BzlCl2)-Val-OBzl
Boc-Leu--Phe-Pro--Tyr(BzlCl2)-Val-Orn(Z)-OBzl
Boc--Phe-Pro--Tyr(BzlCl2)-Val-Orn(Z)-Leu-OBzl
Boc-Pro--Tyr(BzlCl2)-Val-Orn(Z)-Leu--Phe-OBzl
Boc--Tyr(BzlCl2)-Val-Orn(Z)-Leu--Phe-Pro-OBzl
Boc-[-Phe-Pro--Tyr(BzlCl2)-Val-Orn(Z)-Leu]2-OBzl
Boc-Val-Orn-Leu--Phe-Pro--Tyr-OH
85–88
Ϫ51.2
Ϫ40.8
Ϫ60.8
Ϫ23.9
Ϫ25.4
Ϫ42.5
Ϫ37.5
Ϫ77.1
Ϫ64.2
Ϫ46.5
Ϫ40.2
Ϫ4.1
C66H81Cl2N7O12ؒ
2H2O
C66H81Cl2N7O12ؒ
0.5H2O
C66H81Cl2N7O12ؒ
0.5H2O
C66H81Cl2N7O12ؒ
0.5H2O
C66H81Cl2N7O12ؒ
H2O
C66H81Cl2N7O12ؒ
0.5H2O
C120H146Cl4N14O21ؒ
1.5H2O
C44H65N7O10ؒH2O
62.52
(62.35)
63.77
(63.71)
63.53
(63.71)
63.67
(63.71)
63.60
(63.25)
63.86
(63.71)
63.13
(62.96)
60.71
(60.74)
61.02
(60.74)
62.23
(62.02)
62.09
(62.02)
60.50
(60.74)
62.06
(62.02)
57.13
(57.03)
61.64
(61.23)
60.66
(60.66)
60.65
6.76
(6.74)
6.43
(6.64)
6.38
(6.64)
6.44
(6.64)
6.46
(6.67)
6.75
(6.64)
6.74
(6.56)
7.94
(7.76) (11.27)
7.67 11.48
(7.76) (11.27)
7.71 11.22
(7.69) (11.51)
7.93 11.36
(7.69) (11.51)
7.60 11.40
(7.76) (11.27)
7.33 11.54
(7.69) (11.51)
7.81 11.30
(7.96) (11.22)
8.01 9.81
(7.76) (10.20)
7.52 10.01
(7.79) (10.11)
8.01 9.99
(7.79) (10.11)
7.77 9.90
(7.76) (10.20)
7.82 9.92
(7.76) (10.20)
7.43
(7.71)
7.99
(7.88)
7.79
(7.88)
7.96
(7.88)
7.79
(7.88)
7.77
(7.88)
8.42
(8.56)
11.23
2
89–91
3
104–105
137–140
153–154
173–174
124–127
210–212
4
5
6
7
8
9
Boc-Orn-Leu--Phe-Pro--Tyr-Val-OH
200–201
(decomp.)
160–162
(decomp.)
167–169
(decomp.)
192–195
(decomp.)
192–196
(decomp.)
198–202
(decomp.)
144–148
(decomp.)
113–117
(decomp.)
169–172
(decomp.)
134–138
(decomp.)
235–238
(decomp.)
C44H65N7O10ؒH2O
C44H65N7O10
10
11
12
13
14
15
16
17
18
19
Boc-Leu--Phe-Pro--Tyr-Val-Orn-OH
Boc--Phe-Pro--Tyr-Val-Orn-Leu-OH
C44H65N7O10
Boc-Pro--Tyr-Val-Orn-Leu--Phe-OH
C44H65N7O10ؒH2O
C44H65N7O10
Boc--Tyr-Val-Orn-Leu--Phe-Pro-OH
Ϫ62.1
Boc-(-Phe-Pro--Tyr-Val-Orn-Leu)2-OH
Boc-Val-Orn(Boc)-Leu--Phe-Pro--Tyr-OH
Boc-Orn(Boc)-Leu--Phe-Pro--Tyr-Val-OH
Boc-Leu--Phe-Pro--Tyr-Val-Orn(Boc)-OH
Boc--Phe-Pro--Tyr-Val-Orn(Boc)-Leu-OH
Boc-Pro--Tyr-Val-Orn(Boc)-Leu--Phe-OH
Ϫ45.2
(c 0.5)
Ϫ66.7
C83H120N14O17ؒ9H2O
C49H73N7O12ؒ0.5H2O
C49H73N7O12ؒH2O
C49H73N7O12ؒH2O
C49H73N7O12ؒ0.5H2O
C49H73N7O12ؒ0.5H2O
Ϫ34.9
Ϫ52.9
Ϫ24.0
(60.66)
60.98
(61.23)
61.37
Ϫ36.3
(c 0.2,
DMF)
Ϫ45.6
(61.23)
20
21
22
23
24
25
26
27
Boc--Tyr-Val-Orn(Boc)-Leu--Phe-Pro-OH
Z--Phe-Pro-Tyr(BzlCl2)-Val-Orn(Boc)-Leu-OH
Boc-[-Phe-Pro--Tyr-Val-Orn(Boc)-Leu]2-OH
Boc--Phe-Pro-Tyr-Val-Orn(Boc)-Leu-OH
Boc-Phe-Pro--Tyr-Val-Orn(Boc)-Leu-OH
Boc-Phe-Pro-Tyr-Val-Orn(Boc)-Leu-OH
164–166
(decomp.)
109–113
C49H73N7O12ؒ0.5H2O
C59H75Cl2N7O12
61.43
(61.23)
61.58
(61.88)
60.09
(60.11)
61.39
(61.23)
61.38
(61.23)
60.18
(60.10)
56.67
(56.68)
54.97
(54.93)
7.61
(7.76) (10.20)
6.67
(6.60)
8.02
(7.81) (10.55)
7.74 10.41
(7.76) (10.20)
7.67 10.38
(7.76) (10.20)
7.88 9.99
(7.82) (10.01)
7.43 11.38
(7.31) (11.28)
7.59 10.89
(7.66) (11.21)
9.98
Ϫ19.1
Ϫ35.3
Ϫ57.1
Ϫ13.3
Ϫ39.8
8.54
(8.56)
10.46
154–158
145–148
128–130
141–144
158–162
204–206
C93H136N14O21ؒ4H2O
C49H73N7O12ؒ0.5H2O
C49H73N7O12ؒ0.5H2O
C49H73N7O12ؒ1.5H2O
C41H63Cl2N7O8ؒH2O
H--Phe-Pro--Tyr-Val-Orn-Leu-OEtؒ2HCla
H-(-Phe-Pro--Tyr-Val-Orn-Leu)2-OEtؒ3HCla
Ϫ60.4
(c 0.9)
Ϫ70.2
C80H119Cl3N14O15ؒ
7H2O
a
Compounds in experiments 26 and 27 were hexa- and dodeca-peptide ethyl esters used in the measurement of CD spectra.
ONSu 4 is similar to that of GS by the cyclization of H--Phe-
Pro-Val-Orn-Leu-ONSu.
produced on the synthetase, but its biosynthetic process has not
yet been estimated.6,10 As described above, the cyclization of
H--Phe-Pro--Tyr-Val-Orn-Leu-ONSu 4 affords GR, but the
other hexapeptide active ester having a different C-terminal
residue does not. In addition, the mechanism of formation
of GR by dimerization–cyclization of H--Phe-Pro--Tyr-
Val-Orn-Leu-ONSu 4 is similar to that in the cyclization of
H--Phe-Pro-Val-Orn-Leu-ONSu. From the results of our
investigation, it is quite possible that GR is produced via
dimerization–cyclization of a hexapeptide fragment having a
Leu residue at the C-terminus, --Phe-Pro--Tyr-Val-Orn-
Leu-, on the synthetase in a similar way to that of the GS
synthesis.
GS, which has a structure analogous to that of GR and was
isolated from a strain of Bacillus brevis ATCC 9999, is pro-
duced via the dimerization of a pentapeptide fragment (--Phe-
Pro-Val-Orn-Leu-) and the ensuing cyclization of the resulting
decapeptide on the GS synthetase.4 Recently, we found that
although the environment in the chemical synthesis of GS is
significantly different from that of the biosynthesis, H--Phe-
Pro-Val-Orn-Leu-ONSu, having a sequence identical with that
of the linear precursor pentapeptide in the biosynthesis of GS,
gave GS directly.5 On the other hand, GR, which is obtained
from Bacillus brevis Y-33, a mutant of the GS producer, is also
2048
J. Chem. Soc., Perkin Trans. 1, 1997

View Online
Table 4 Physical properties and analytical data for cyclic productsa
No.
1
Compound
Mp (T/ЊC)
[α]D25 (c 0.5, MeOH)
Ϫ66.7 (c 0.2)
FAB-MS m/z (required)
H-Val-Orn-Leu--Phe-Pro--Tyr ؒHCl
247–249
(decomp.)
734.4235
(M ϩ Hϩ, C39H56N7O7 requires m/z, 734.4194)
2
H-Orn-Leu--Phe-Pro--Tyr-Val ؒHCl
165–167
(decomp.)
Ϫ36.9
734.4216
(M ϩ Hϩ, C39H56N7O7 requires m/z, 734.4194)
3
4
H-Leu--Phe-Pro--Tyr-Val-Orn ؒTFA
H-Pro--Tyr-Val-Orn-Leu--Phe ؒTFA
H--Tyr-Val-Orn-Leu--Phe-Pro ؒTFA
108–111
(decomp.)
Ϫ13.3
734.4235
(M ϩ Hϩ, C39H56N7O7 requires m/z, 734.4194)
258–260
(decomp.)
Ϫ36.3 (c 0.5, DMF)
734.4234
(M ϩ Hϩ, C39H56N7O7 requires m/z, 734.4194)
5
6
189–192
Ϫ109.2
734.4252
(M ϩ Hϩ, C39H56N7O7 requires m/z, 734.4194)
1467
H--Phe-Pro--Tyr-Val-
Orn-Leu--Phe-Pro--Tyr-Val-Orn-Leu
ؒ2HClb
219–222
(decomp.)
Ϫ35.3 (c 0.2)
(M ϩ Hϩ, C78H111N14O14 requires m/z, 1467)
7
᎐Leu-Orn-Val--Tyr-Pro--Phe-H
|
1467
|
H--Phe-Pro--Tyr-Val-Orn-Leu᎐ ؒ2HCl 198–200
Ϫ53.4 (c 0.5, DMF)
(M ϩ Hϩ, C78H111N14O14 requires m/z, 1467)
(decomp.)
a
The results of amino acid analyses of these peptides agreed closely with the theoretical values. b The cyclic peptide 7 was synthesized by a similar
method described in the literature (ref. 11).
were prepared by liquid-phase methodologies. In the synthesis
Experimental
of Boc--Phe-Pro-Tyr-Val-Orn(Boc)-Leu-OH as an example,
Mps were measured on an Ishii melting point apparatus and
are uncorrected. Amino acid analysis of each hydrolysate of
the peptides was carried out with an Hitachi 835 amino acid
analyser. Relative molecular masses of the cyclic products
were determined by using FAB mass spectrometry on a JEOL
JMS-D-300 mass spectrometer (Asahi Chemical Industry
Company). CD spectra were obtained with a JASCO spectro-
polarimeter (model J-720) using 0.1 mm cells at room tem-
perature. CD spectra of semi-GR, GR and its ethyl esters
corresponding to active esters 1–6 were measured in ethanol
solutions at a concentration of 0.5 m.
the coupling of Boc--Phe-Pro-Tyr(Bzl)-Val-OH and H-
Orn(Boc)-Leu-OEt prepared by stepwise elongation was per-
formed using WSCDؒHCl and HOBt. The protected groups of
Boc--Phe-Pro-Tyr(Bzl)-Val-Orn(Boc)-Leu-OEt were removed
by saponification and hydrogenolysis. Other Boc-hexapeptides
were synthesized in a similar manner.
The peptides were characterized by elemental analyses, TLC,
HPLC, and amino acid analyses of their hydrolysates. The phys-
ical properties and analytical data of these peptides are shown
in Table 3.
Reaction of hexa- and dodeca-peptide-ONSus
HPLC was performed on an LC-800 series instrument
(JASCO, Japan) consisting of an 880 intelligent HPLC pump,
an 875-UV intelligent UV/visible detector, an 860-CD column
oven, a model 7125 syringe-loading sample injector (Rheodyne,
Cotati, CA, USA), and a Finepak SIL C18 column (4.6 × 250
mm; 10 µm particle size, JASCO, Japan). Chromatography was
carried out using a linear gradient of 50–80% MeOH–5% aq.
NaClO4 during 60 min with a flow rate of 1 ml minϪ1 at 30 ЊC.
The column eluent was monitored at 220 nm. The micro-
organisms employed in the assays were Staphylococcus aureus
MS353, Streptomyces pyogenes N.Y.5, Corynebacterium diph-
theriae P.W.8, Micrococcus luteus ATCC 9341, Bacillus subtilis
ATCC 6633, Escherichia coli NIHJ-JC2 and Proteus vulgaris
OX19. Minimum inhibitory concentrations (in µg mlϪ1) of the
compounds were determined by an agar dilution method with
106 organisms per millilitre.
Boc-hexapeptide-OHs (50–100 mg) were converted into the
corresponding succinimide esters by using HONSu and WSCDؒ
HCl. Boc-hexapeptide-ONSus were treated with trifluoroacetic
acid (TFA) to remove all Boc groups. Hexapeptide-ONSu tri-
fluoroacetates were dissolved in small amounts of DMF, and
the solutions were added dropwise to pyridine at 25 ЊC (concen-
tration of the active esters was 3 mM). After the mixture had
been stirred for 1 day at 25 ЊC the reaction mixtures were
analysed by HPLC. Main products from the reaction mixtures
of hexapeptide-ONSus were purified by gel filtration on a
Sephadex LH-20 column (1.5 × 150 cm) using ethanol as the
elution solvent, semipreparative HPLC, and by reprecipitation
from methanol–diethyl ether. The cyclic peptides were charac-
terized by TLC, HPLC, FAB mass spectra, and amino acid
analyses of their hydrolysates. The physical properties and
analytical data for these peptides are shown in Tables 4 and 5.
Syntheses of Boc-hexa- and -dodeca-peptides
Determination of the free amino group in cyclic peptides
A cyclic peptide isolated from reaction mixtures of -Phe-Pro-
-Tyr-Val-Orn-Leu-ONSu was treated with 2,4-dinitrofluoro-
benzene. The resulting dinitrophenyl cyclic peptide was hydro-
lysed in 6 M HCl for 24 h at 110 ЊC. The free amino group of
the peptide was confirmed by comparing the results of the
amino acid analyses of the hydrolysates of both the 2,4-
dinitrophenol (DNP)-treated peptide and the non-treated
peptide. The free amino group in other cyclic peptides was
determined in a similar manner.
Method 1. Boc-hexapeptides related to active esters 1–6 and
Boc-dodecapeptide, in which the δ-amino group of the Orn
residue and the N-terminal amino group were protected by the
Boc group, were prepared by liquid-phase methodologies. In
the synthesis of Boc-Val-Orn(Boc)-Leu-Phe-Pro-Tyr-OH as an
example, Boc-Phe-Pro-Tyr(Bzl)-Val-Orn(Z)-Leu-OBzl was
prepared by stepwise elongation using 1-ethyl-3-[3-(dimethyl-
amino)propyl]carbodiimide hydrochloride (WSCDؒHCl) and
N-hydroxy-1,2,3-benzotriazole (HOBt) from Leu-OBzl. The Z-
and -Bzl groups of Boc-Val-Orn(Z)-Leu-Phe-Pro-Tyr(Bzl)-
OBzl were removed by hydrogenolysis. Then the resulting Boc-
hexapeptide-OH was treated with (Boc)2O to give Boc-Val-
Orn(Boc)-Leu-Phe-Pro-Tyr-OH. Other Boc-hexapeptides were
synthesized in a similar manner.
Syntheses of cyclic peptides as authentic samples
These cyclic peptides were synthesized by one-pot cyclization
of the corresponding Boc-hexapeptide-OH (compounds 8–13
shown in Table 3) using WSCDؒHCl and HOBt, followed by
removal of the Boc-group by 4 M HCl–1,4-dioxane. Main
Method 2. Boc-hexapeptides related to active esters 7–9
J. Chem. Soc., Perkin Trans. 1, 1997
2049

View Online
Table 5 Physical properties and analytical data for cyclic productsa
Found (%) (required)
[α]D25
(c 1.0, MeOH) Formula
FAB-MS m/z
(M ϩ Hϩ)
No.
8
Compound
Mp (T/ЊC)
C
H
N
cyclo(--Phe-Pro--Tyr-Val-
Orn-Leu-)ؒHCl
cyclo(--Phe-Pro-Tyr-Val-Orn- 196–199
Leu-)ؒHCl
cyclo(-Phe-Pro--Tyr-Val-Orn- 228–231
Leu-)ؒHCl
cyclo(-Phe-Pro-Tyr-Val-Orn-
226–230
(decomp.)
Ϫ38.5
C39H55N7O7ؒHCl
734
61.18
7.38
12.59
(12.73)
12.42
(12.44)
12.66
(12.44)
12.62
(60.88) (7.33)
59.20 7.60
(59.42) (7.41)
59.12 7.14
(59.42) (7.41)
60.89 7.42
(60.88) (7.33)
9
Ϫ86.7 (c 0.2)
Ϫ45.5
C39H55N7O7ؒHClؒH2O 734
C39H55N7O7ؒHClؒH2O 734
C39H55N7O7ؒHCl 734
10
11
261–265
(decomp.)
Ϫ54.4
Leu-)ؒHCl
(12.73)
a
The results of amino acid analyses of these peptides agreed closely with the theoretical values.
Tyrocidines, Kodansha, Tokyo and Halsted Press, New York, 1979,
p. 49; M. Tamaki, M. Takimoto, M. Hayashi and I. Muramatsu,
Bull. Chem. Soc. Jpn., 1989, 62, 594; M. Tamaki, S. Akabori and
I. Muramatsu, Bull. Chem. Soc. Jpn., 1993, 66, 3113; Y. Minematsu,
M. Waki, K. Suwa, T. Kato and N. Izumiya, Tetrahedron Lett.,
1980, 21, 2179.
products from the reaction mixtures were purified by gel
filtration on a Sephadex LH-20 column (1.5 × 150 cm) using
ethanol as the elution solvent and by reprecipitation from
methanol–diethyl ether. The cyclic peptides were characterized
by TLC, HPLC, FAB mass spectra, and amino acid analyses
of their hydrolysates.
4 S. G. Laland, Ph. Frøyshov, C. Gilhuus-Moe and T. L. Zimmer,
Nature (London) New Biol., 1972, 239, 43.
5 M. Tamaki, S. Akabori and I. Muramatsu, J. Am. Chem. Soc., 1993,
115, 10 492.
Syntheses of hexa- and dodeca-peptide ethyl esters
Hexa- and dodeca-peptide ethyl esters used in the measurement
of CD spectra were prepared by the ethyl esterification of the
corresponding Boc-hexa- and -dodeca-peptide-OH groups by
using WSCDؒHCl in ethanol and the removal of the Boc group
by the action of 4 M HCl–1,4-dioxane. Analytical data for the
esters are shown in Table 3.
6 G. G. Zharikova, S. P. Myaskovskaya and A. B. Silaev, Vestn. Mosk.
Univ. Ser. Biol. Pochvoved. Geol. Geogr., 1972, 27, 110 (Chem.
Abstr., 1972, 77, 162982p); S. P. Myaskovskaya, G. G. Zharikova
and A. B. Silaev, Vestn. Mosk. Univ. Ser. Biol. Pochvoved. Geol.
Geogr., 1973, 28, 123 (Chem. Abstr., 1974, 80, 37446c); G. G.
Zharikova, A. P. Zarubina, D. M Kherat, S. P. Myaskovskaya and
V. N. Maksimov, Antibiot. IKh Prod., 1975, 163 (Chem. Abstr.,
1976, 84, 119884r).
7 M. Tamaki, Bull. Chem. Soc. Jpn., 1984, 57, 3210; M. Tamaki,
M. Takimoto and I. Muramatsu, Bull. Chem. Soc. Jpn., 1987, 60,
2101.
8 The cyclic monomers produced by the cyclization of the linear
hexapeptide active esters 1 and 3–6 were found to have no antibiotic
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific
Research (No. 06640702) from the Ministry of Education,
Science and Culture of Japan. We are grateful to the staff of the
Research Laboratories of Asahi Chemical Industry Co., Ltd.
for elemental analyses, microbiological assays, and measure-
ments of the FAB mass spectra.
activity. On the other hand, H-Orn-Leu--Phe-Pro--Tyr-Val ,
isolated from a reaction mixture of peptide 2, showed the activities
1/4–1/8 of that of GR against Bacillus subtilis ATCC 6633,
Streptomyces pyogenes N.Y.5, Staphylococcus aureus MS353 and
Micrococcus luteus ATCC 9341.
9 M. Tamaki, S. Komiya, S. Akabori and I. Muramatsu, Bull. Chem.
Soc. Jpn., 1997, 70, 899.
References and notes
1 C. F. Gause and M. G. Brazhnikov, Am. Rev. Sov. Med., 1944, 2,
134; D. C. Hodgkin and B. M. Oughton, Biochem. J., 1957, 65, 752;
S. Nozaki and I. Muramatsu, J. Antibiot., 1984, 37, 689; Bull. Chem.
Soc. Jpn., 1985, 58, 331.
10 H. Kleinkauf and H. von Dohren, Eur. J. Biochem., 1990, 192, 1.
11 M. Tamaki, S. Akabori and I. Muramatsu, Bull. Chem. Soc. Jpn.,
1991, 64, 583.
2 Amino acid residues with no prefix have the -configuration. The
abbreviations for amino acids and peptides are in accordance
with the rules of the IUPAC-IUB Commission on Biological
Nomenclature.
3 N. Izumiya, T. Kato, H. Aoyagi, M. Waki and M. Kondo, Synthetic
Aspects of Biologically Active Cyclic Peptides-Gramicidin S and
Paper 7/01537B
Received 4th March 1997
Accepted 10th April 1997
2050
J. Chem. Soc., Perkin Trans. 1, 1997