CYCLIC PEPTIDES

PEPTIDES CYCLIQUES

English Abstract

ABSTRACT OF THE DISCLOSURE
A novel peptide represented by formula (I):
Cyclo-(Cys-Val-d-Phe-Pro)2 (I)
wherein Cys indicates a cysteine group, Val indicates a
valine group, d-Phe indicates a d-phenylalanine group,
Pro indicates a proline group, and Cyclo indicates a
cyclic form. This peptide is formed by way of active
esterification or azidation of an intermediate:
Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OME
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Acm indicates an SH group
protected by an acetamidomethyl group, and OMe indicates
a methoxylated carboxyl group.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A novel peptide represented by formula (I):
<IMG> (I)
wherein Cys indicates a cysteine group, Val indicates a
valine group, d-Phe indicates a d-phenylalanine group,
Pro indicates a proline group, and Cyclo indicates a
cyclic form.
2. A method of making the cyclic peptide defined
in claim 1, said method comprising the steps of:
repeatedly protecting and unprotecting a polar
group of a constitutive amino acid to form, by a
sequential elongation method, an intermediate peptide, in
which cysteines are protected, represented by the
following formula:
<IMG>
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
Val indicates a valine group, d-Phe indicates a d-
phenylalanine group, Pro indicates a proline group, Acm
indicates an SH group protected by an acetamidomethyl
group, and OMe indicates a methoxylated carboxyl group;
and
then dimerizing said intermediate peptide by an
active esterification to form said cyclic peptide.
3. A method as defined in claim 2, which
comprises the steps of forming intermediates represented
by the following formulas:
<IMG>
-33-

<IMG>
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
Val indicates a valine group, d-Phe indicates a d-
phenylalanine group, Pro indicates a proline group, Acm
indicates an SH group protected by an acetamidomethyl
group, OMe indicates a methoxylated carboxyl group, H
indicates a freed amino group, ONSu indicates an N-
oxysuccinimido group, and Cyclo indicates a cyclic form.
4. A method of making the cyclic peptide defined
in claim 1, said method comprising the steps of:
repeatedly protecting and unprotecting a polar
group of a constitutive amino acid to form, by a
sequential elongation method, an intermediate peptide, in
which cysteines are protected, represented by the
following formula:
<IMG>
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
Val indicates a valine group, d-Phe indicates a d-
phenylalanine group, Pro indicates a proline group, Acm
-34-

indicates an SH group protected by an acetamidomethyl
group, and OMe indicates a methoxylated carboxyl group;
azidating said intermediate peptide;
reacting the azidated peptide with a peptide
represented by the following formula:
<IMG>
to form said cyclic peptide.
5. A method as defined in claim 4, which
comprises the steps of forming intermediates represented
by the following formulas:
<IMG>
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
Val indicates a valine group, d-Phe indicates a d-
phenylalanine group, Pro indicates a proline group, Acm
indicates an SH group protected by an acetamidomethyl
group, NHNH2 indicates a hydrazidated carboxyl group, and
H indicates a freed amino group.
6. A novel peptide represented by the following
formula:
<IMG>
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Val indicates a valine group, Cys
indicates a cysteine group, Acm indicates an SH group
-35-

protected by an acetamidomethyl group, and OMe indicates
a methoxylated carboxyl group.
7. A novel peptide represented by the following
formula:
H-Val-Cys(Acm)-OMe
wherein H indicates a freed amino group, Val indicates a
valine group, Cys indicates a cysteine group, Acm
indicates an SH group protected by an acetamidomethyl
group, and OMe indicates a methoxylated carboxyl group.
8. A novel peptide represented by the following
formula:
Boc-Cys(Acm)-Val-Cys(Acm)-OMe
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
Val indicates a valine group, Acm indicates an SH group
protected by an acetamidomethyl group, and OMe indicates
a methoxylated carboxyl group.
9. A novel peptide represented by the following
formula:
Boc-Cys(Acm)-Val-Cys(Acm)-OH
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
Val indicates a valine group, and Acm indicates an SH
group protected by an acetamidomethyl group.
10. A novel peptide represented by the following
formula:
Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe
-36-

wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
Val indicates a valine group, d-Phe indicates a d-
phenylalanine group, Pro indicates a proline group, Acm
indicates an SH group protected by an acetamidomethyl
group, and OMe indicates a methoxylated carboxyl group.
11. A novel peptide represented by the following
formula:
<IMG>
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
Val indicates a valine group, d-Phe indicates a d-
phenylalanine group, Pro indicates a proline group, and
Acm indicates an SH group protected by an acetamidomethyl
group.
12. A novel peptide represented by the following
formula:
<IMG>
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
Val indicates a valine group, d-Phe indicates a d-
phenylalanine group, Pro indicates a proline group, Acm
indicates an SH group protected by an acetamidomethyl
group, and ONSu indicates an N-oxysuccinimido group.
13. A novel peptide represented by the following
formula:
<IMG>
- 37-

wherein H indicates a freed amino group, Cys indicates a
cysteine group, Val indicates a valine group, d-Phe
indicates a d-phenylalanine group, Pro indicates a
proline group, Acm indicates an SH group protected by an
acetamidomethyl group, and ONSu indicates an N-
oxysuccinimido group; or a hydrochlorate of said peptide.
14. A novel peptide represented by the following
formula:
<IMG>
wherein Cys indicates a cysteine group, Val indicates a
valine group, d-Phe indicates a d-phenylalanine group,
Pro indicates a proline group, Acm indicates an SH group
protected by an acetamidomethyl group, and Cyclo
indicates a cyclic form.
15. A novel peptide represented by the following
formula:
<IMG>
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
Val indicates a valine group, d-Phe indicates a d-
phenylalanine group, Pro indicates a proline group, Acm
indicates an SH group protected by an acetamidomethyl
group, and NHNH2 indicates a hydrazidated carboxyl group.
16. A novel peptide represented by the following
formula:
<IMG>
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
-38-

Val indicates a valine group, d-Phe indicates a d-
phenylalanine group, Pro indicates a proline group, and
Acm indicates an SH group protected by an acetamidomethyl
group.
17. A novel peptide represented by the following
formula:
Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Cys indicates a cysteine group,
Val indicates a valine group, d-Phe indicates a d-
phenylalanine group, Pro indicates a proline group, Acm
indicates an SH group protected by an acetamidomethyl
group, and NHNH2 indicates a hydrazidated carboxyl group.
18. A novel peptide represented by the following
formula:
H-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2
wherein H indicates a freed amino group, Cys indicates a
cysteine group, Val indicates a valine group, d-Phe
indicates a d-phenylalanine group, Pro indicates a
proline group, Acm indicates an SH group protected by an
acetamidomethyl group, and NHNH2 indicates a
hydrazidated carboxyl group; or a hydrochlorate of said
peptide.
19. A novel peptide represented by the following
formula:
Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-Phe-Pro]2
wherein Cys indicates a cysteine group, Val indicates a
valine group, d-Phe indicates a d-phenylalanine group,
-39-

Pro indicates a proline group, and Cyclo indicates a
cyclic form.
20. A novel metal or heavy metal cluster complex
of the novel cyclic peptide in accordance with claim 1
formed by a reaction of said novel cyclic peptide with a
cluster of a metal or heavy metal selected from the group
consisting of mercury, cadmium, zinc, molybdenum, cobalt,
nickel, iron, titanium, palladium, manganese, silicon,
and calcium.
-40-

Description

~046~24
NOVEL CYCLIC PEPTIDES
B~CKGROUND OF THE INVENTION
Field of the Invention
This invention relates to a novel peptide, a
method of making the same, and intermediates used for
making the same, and metal cluster complexes formed with
the same.
Description of the Prior Art
Recently, the peculiarities of metal complexes
have been attracted considerable attention in wide fields
such as medicals and functional materials. For example,
they are widely used as means for transferring metals in
organisms. Also, in the field of functional materials,
characteristics of complexed-metals are used to develop
new materials and their enzyme-like functions are used to
develop catalysts. Thus, the metal complexes contribute
to various technical fields.
Further, peptides themselves are widely used as
diagnostic and therapeutic agents. For example,
tuberactinomycin and gramicidin S are strongly
antibacterial and known as antibiotics.
Also, there are many peptides which exhibit
physiological activities with respect to plants of the
higher orders and insects. For example, destruxin and
beauvelicin show activity with respect to insects, which
is based on antibacterial effects against filamentous
fungi.

6~
It has been known that metals exist in organisms
while being held by peptides, namel~, as a cluster. E'or
example, iron exists as an active center of an iron-
sulfur protein having the structure of [Fe4S4(S-Cys)4]n-
and contributing to electron transport. So far, variousstudies have been conducted with respect to materials
similar to the clusters existing in organisms, i.e.
synthetic clusters. However, the conventional synthetic
clusters exhibit oxidation-reduction potentials lower
than those of the clusters in organisms and functions
similar to those of the latter cannot be expected from
the former.
SUMMARY OF THE INVENTION
It is thus an object of the present invention to
provide a novel peptide whic~ can form clusters which are
functionally close to those in organisms.
It is another object of the present invention to
provide a method of making the novel peptide.
It is further object of the present invention to
provide novel intermediates for making the novel peptide.
It is still another object of the present
invention to provide a novel metal cluster complex.
In order to provide the novel metal cluster-
complex which satisfies the forgoing demands, the
inventor has conducted deligent studies and found it
necessary to meet the following conditions:
l)As described in C. W. Carter, Jr., et al, Proc.
Natl. Acad. Sci. USA, 69, p.3526 (1972), the cluster

which exists at the active center of the iron sulfur
protein is covered with a peptide chain formed by
hydrophobic amino acids and their hydrophobic environment
stabilizes the cluster. Accordingly, a hydrophobic
environment is necessary.
2) The whole sturcture of the cluster which
coordinates the ligand or at least a local structure
thereof near the metal is distorted.
3) Since it is difficult for materials other than
peptides to synthesize a large ligand having four
coordinating sulfur molecules within its molecule, a
novel peptide is necessary.
4) When a peptide having amino and carboxyl groups
at its ends is used as a ligand, there is a possibility
that they may contribute to coordination. Accordingly,
the peptide should not have polar groups such as amino
group and carboxyl group at its end.
5) ~hen a peptide has a relatively large structure,
it becomes flexible and may form complicated mixtures
during complexing. In this case, the evaluation of its
function becomes difficult. Accordingly, the peptide
should have a certain hardness.
The inventor has found that a cyclic peptide is
quite excellent in satisfying the foregoing conditions.
Gramicidin Sl which is an antibiotic, is a well-known
cyclic peptide. However, since it does not have any
coordinating amino acid slde chain, it cannot coordinate
metals. However, the inventor has found that, while

~6~
maintaining its maln chain structure, four cysteines can
be introduced thereinto to form a novel cyclic peptide
which can coordinate metals and satisfy the above-
mentioned conditions. As the result of diligent studies,
the present invention has been achieved.
Namely, the present invention provides:
1) a novel cyclic peptide represented by ~ormula
(I)
Cyclo-(Cys-Val-d-Phe-Pro)2 (I)
wherei.n Cys indicates a cysteine group, Val indicates a
valine group, d-Phe indicates a d-phenylalanine group,
Pro indicates a proline group, and Cyclo indicates a
cyclic form, whose constitutional formula is represented
by formula (I'):
0 CH2Ph
C - NH - CH - C - N
HCCH2SH '1--
HN C = O
O=C NH
HCCHCH3 HSCH2CH
HN CH3 C = O
O=C NH
HCCH2SH 3HCCHCH
HN 3HC -C = O
O=C NH
~ HSCH2CH
N -C -CH -NH -C
/ 11 1 11
' O CHzPh S:)
2) methods of making the same;

3) novel intermediates used for making the same as
follows:
i) Boc-Val-Cys(Acm)-OMe
wherein Boc indicates an amino group protected by a t-
butyloxycarbonyl group, Acm indicates an SH groupprotected by an acetamidomethyl group, and OMe indicates
a methoxylated carboxyl group, which also apply to the
following:
ii) H-Val-Cys(Acm)-OMe
wherein H indicates a freed amino group, which also
applies to the following:
iii) Boc-Cys(Acm)-Val-Cys(Acm)-OMe
iv) Boc-Cys(Acm)-Val-Cys(Acm)-OH
v) Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe
vi) Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OH
vii) Boc-Cys(Acm)-Val-CyslAcm)-d-Phe-Pro-ONSu
wherein ONSu indicates an N-oxysuccinimido group, which
also applies to the followin~:
viii) H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-ONSu
or hydrochlorates thereof
ix) Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2
x) Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-NHNH2
wherein NHNH2 indicates a hydrazidated carboxyl group
xi) Boc-[Cys(Acm)-Val-CyS(Acm)-d-phe-pro]2
xii) Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2
xiii) H-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2
or hydrochlorates thereof
xiv) Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-Phe-Pro] 2

Z~ ~6
and
4) a metal or heavy metal cluster complex of the
above-mentioned novel cyclic peptide (I) formed by a
reaction of said novel cyclic peptide with a cluster of a
metal or heavy metal selected from the group consisting
of mercury, cadmium, zinc, molybdenum, cobalt, nickel,
iron, titanium, palladium, manganese, silicon, and
calcium.
The novel cyclic peptide (I) in accordance with
the present invention exhibits an antibacterial activity
and can be useful in the fields of medicines and
agricultural chemicals.
Also, the cluster complexes obtained from the
novel cyclic peptide (I) can exhibit high oxidation-
reduction potentials which are closer to those of the
clusters in organisms as compared with the conventional
synthetic clusters. Accordingly, they can be useful as
active agents in organisms in the fields of medicines and
functional materials.
The novel cyclic peptide (I) in accordance with
the present invention can be made by various methods.
Industrially, the following methods are advantageous.
1) Method (a)
While protection and unprotection of polar groups
of amino acids are repeated, a sequential elongation
method is used to form a peptide in which cysteines are
protected, which peptide is represented by the following
formula:

Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe
Then, its methoxy group is changed to an OH group.
Thereafter, a free acid and N-oxysuccinimide are used so
that the peptide is subjected to actlve esterification.
An active esterification method is used to effect
dimerization of thus modified peptide (cf. Figure 1).
2) Method (b)
~he above-mentioned peptide:
Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe
is subjected to an azidation method, namely, is reacted
with hydrazine monohydrate to yield hydrazide and then is
reacted with isoamyl nitrite under a weakly acid
condition so as to be azidated. The azidated peptide is
reacted with a free amino acid of the other synthesizing
block, namly, the peptide represented by the following
formula: -
H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe HCl
(Cf. Figure 2.)
In both cases, since the SH-group of Cys in the
cyclic peptide is protected by Acm, an HgCl-adduct is
formed and then HgCl2 is removed to form the novel cyclic
peptide (I).
The cluster complex in accordance with the present
invention can easily be formed when an organic solvent
solution of the novel
cyclic peptide (I) is mixed with an organic solvent
solution of a metal or heavy metal cluster.

~ ~4~a~
BRIEF DESCRIPTION CF THE DRA~INGS
Figure 1 is a flow sheet showing a scheme for
synthesizing the novel cyclic peptide in accordance with
the present invention, Cyclo-~Cys-Val-Cys-d-Phe-Pro) 2
(I), by an active esterification;
Figure 2 is a flow sheet showing synthesizing
steps of that peptide by an azidation method;
Figure 3 is a graph showing the results of
measurement of molecular weight of an intermediate in
accordance with the present invention, Cyclo-[Cys(Acm)-
Val-Cys(Acm)-d-Phe-Pro]2 (14), by FD-MS;
Figures 4A and 4B are charts showing 500MHz lH-NMR
spectral data of the above-mentioned intermediate;
Figure 5 is a chart showing the results of
measurement of 1H-lHCOSY2D-N~R of the above-mentioned
intermediate;
Figure 6 is a chart showing 500MHz lH-NMR spectral
data of the novel cyclic peptide in accordance with the
present invention;
Figure 7 is a chart showing an absorption spectrum
of the four-iron cluster itself used in Example 3;
Figure 8 is a graph showing titration curves of
the four-iron cluster and peptide in Example 3; and
Figure 9 is a chart showing the results of
measurment of ultraviolet-visible light absorption
spectra of solutions E, F, and G in Experiment 1.
DESCRIPTION OF THE PREFERRED EMBODIMENT

12~
The following examples wil] explain the present
invention in detail.
Example 1
I) Making of Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-
Pro~2 (14) by Active Esterification Method (Cf.
Figure 1)
1) Synthesis of Boc-Cys(Acm)-OH (1)
H-Cys(Acm)-OH HCl (4.58g, 20mmol) was dissolved in
12ml of water.
To this solution, 4.20ml (20mmol) of triethylamine
(abridged as "TEA" in the following) was added, while
being stirred and cooled with ice. Then, the mixture was
stirred at room temperature. Thereafter, dioxane
solution (12ml) of 5.42g (20mmol) of 2-tertiary
butyloxycarbonyloxyimino-2-phenylacetonitrile (abridged
as "BOC-ON" in the following) was added dropwise thereto
and the resulting mixture was stirred at room temperature
for 3 hours. After the reaction, TEA was further added
thereto to dissolve the insolubles. Then, the solvent
was evaporated under a vacuum to leave a dried product.
It was dissolved in ethyl acetate and then was washed
with water. The water layer was fùrther acidified and
extracted with ethyl acetate. The extracted layer was
dried with sodium sulfate anhydride. After the solvent
was evaporated under a vacuum, the dried product was
recrystallized in ether to yield the aimed white
amorphous product (1).
Yield: 3.50g (59.9~)

2) Synthesis of H-Cys(Acm)-Me HCl (2)
26.75g (0.30mmol) of N-(hydroxymethyl)acetamide
(abridged as "Acm" in the following) and 46.33g
(0.27mmol) of H-Cys-OMe.HCl were dissloved in 70ml of
S water. While the solution was cooled with ice, llml of
35% hydrochloric acid was added thereto. After the
mixture was reacted at 0C for 2 hours, the gas in the
reaction vessel was replaced with argon. Then the
reaction vessel was left for five days. Vacuum
evaporation was repeated with the addition of 20ml of
ethanol anhydride to remove water from the mixture.
After 200ml of ethanol was added
thereto and the insolubles were removed by filtration,
the aimed product ~2) of a white crystalline powder form
was recrystallized from ether.
Yield: 28.72g (43.8%)
The physical properties of the product thus
obtained were as follows:
lH-NMR(DMso-d6)
~DSS(Ppm)=l-88 (s, 9H, Acm CH3)
3.13 (d, 2H, Cys H~)
3.76 (s, 3H, OCH3)
3.82-4.56 (m, 3H, Cys Ha, Acm CH2)
8.20-9.20 (m, 2H, Acm HN, Cys HN)
3) Synthesis of Boc-Val-Cys(Acm)-OMe (3)
4.85g (20mmol) of the above-mentioned product (2)
was dissolved in 50ml of N,N-dimethylformamide (abridged
as "DMF" in the following). While the solution was cooled
- 1.0-

with ice and stirred, 2.19ml (20mmol) of N~
methylmorpholine (abridged as "NMM" in the following) was
added thereto. After the mixture was stirred at room
temperature for one hour, 4.35g (20mmol) of Boc-Val-OH,
2.97g (22mmol) of N-hydroxybenzotriazol (abridged as
"HO~t" in the following), 4.13g (20mmol) of
dicyclohexylcarbodiimide (abridged as "DCC" in the
following) were added thereto in this order. Then the
resulting mixture was sequentially stirred at -3C for
5.5 hours, at 0C for 12 hours, and at room temperature
for 5.5 hours. After DMF was evaporated under a vacuum,
50ml of ethyl acetate was added to the resulting product.
Then the crystal d~eposited therefrom was separated by
filtration, while the filtrate was sequentially washed
with lM sodium hydrogencarbQnate aqueous solution, water,
lM citric acid aqueous solution, and water. Thereafter,
the washed filtrate was dried with sodium sulfate
anhydride. The solvent was evaporated therefrom under a
vacuum to leave a crude product. Then it was purified by
a column chromatography to yield the aimed product (3)
of a white powder form.
Yield: 4.52g (55.7~)
The physical properties of the product thus
obtained were as follows:
Elementary Analysis (~ by weight): Cl7H3lO6N3S
Empirical Value C:50.21, X:7.44, N:10.56
Calculated Value C:50.34, H:7.72, N:10.36
lH-NMR ( DMSO-d6 )
- 11 -

~04~
(Ppm)-o-86 (m, 6H, Val HY)
1.40 (s, 9H, Boc CH3)
1.72-2.12 (m, 4H, Acm CH3, Val HP)
2.92 (m, 2H, Cys H~)
3.64 (m, 3H, OCH3)
3.82 (m, lH, Val H~)
4.00-4.62 (m, 4H, Cys Ha, Acm CH2)
6.60 (d, lH, Val HN)
8.00-8.68 (m,2H, Cys HN, Acm HN)
4) Synthesis of H-Val-Cys(Acm)-OCH3 (4)
To 1.62g (4mmol) of the above-mentioned product
(3), 30ml of 4M hydrochloric acid/dioxane was added. The
resulting mixture was stirred at room temperature for 1.5
hours. Dioxane was evaporated therefrom under a vacuum
to leave the aimed product (4~) of a white powder form.
5) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-OMe (5)
The above-mentioned product (4) was dissolved in
30ml of DMF. While the solution is cooled with ice and
stirred, NMM (0.44g, 4mmol) was added thereto. Twenty
minutes thereafter, Boc-Cys(Acm)-OH (1.17g, 4mmol), HOBt
(0.59g, 4.4mmol), and DCC (0.83g, 4mmol) were added
thereto in this order. Then the mixture was stirred at
-3C for 5 hours, at 0C ~or 11 hours, and at room
temperature for 5 hours. After DMF was evaporated under
a vacuum, chloroform was added to the remaining product.
After the insolubles were separated by filtration, the
filtrate was sequentially washed with lM sodium
hydrogencarbonate aqueous solution, water, lM citric acid
-12-

aqueous solution, and water. The extracted layer
obtained after the washing was dried with sodium sulfate
anhydride and the solvent was evaporated to leave a crude
product. Then it was purified by a column chromatography
to yield the aimed white amorphous product (5).
Yield: 1.67g (71.8%)
The physical properties of the product thus
obtained were as follows:
Elementary Analysis (% by weight): C23H4leN5S2
Empirical Value C:46.95, H:6.92, N:12.03
Calculated Value C:46.91, H:7.20, N:11.90
Remarks: Since 0.5mol of water of crystallization
is considered to be contained therein, the value is that
of C23H4lOgNsS2 0-5H2O-
lH-NMR(DMso-d6)
(PPm)=0-86 (m, 6H, Val Hr)
1.40 (s, 9H, Boc CH3)
1.60-2.20 (m, 7H, Acm CH3, Val H~)
2.46-3.00 (m, 4H, Cys H~)
3.64 (m, 3H, OCH3)
3.80-4.60 (m, 7H, Val Ha, Cys Ha, Acm CH2)
7.06, 7.54 (d, 2H, HN)
8.32-8.68 (t, 3H, Cys HN, Acm HN)
6) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-OH (6)
1.47g (2.53mmol) of the above-mentioned product
(5) was dissolved in 25ml of methanol. After lM sodium
hydroxide aqueous solution was added thereto, the mixture
was stirred at room temperature for 3 hours. Then, after
-13-

15ml of water was added thereto, methanol was evaporated
from the mixture under a vacuum. After unreacted esters
were removed by 20ml of ether, 30ml of lM citric acid
aqueous solution was added to the water layer to weakly
acidify the latter. Then, extraction was effected with
ethyl acetate. The extracted material was dried with
sodium sulfate anhydride and then the solvent was
evaporated therefrom under a vacuum to leave the aimed
white amorphous product (6).
Yield: 1.15g (80.2%)
The physical properties of the product thus
obtained were as follows:
[ a ]D=-25 . 7 (c 0.65, MeOH 23C)
Elementary Analysis (% by weight): C22H39O8N5S2
Empirical Value -C:44.65, H:6.64, N:10.24
Calculated Value C:44.63, H:7.35, N:10.85
Remarks: The calculated value of
C22H3gO8N5S2-0.5AcOEt-2H2o-
lH-NMR(DMSO-d6)
SDSS(Ppm)=0-88 (m, 6H, Val HY)
1.40 (s, 9H, Boc CH3)
1.60-2.20 (m, 7H, Acm CH3, Val H~)
2.56-3.00 (m, 4H, Cys H~)
3.80-4.60 (m, 7H, Val Ha, Cys Ha, Acm CH2)
7.08, 7.52 (d, 2H, HN)
8.16-8.68 (m, 4H, Cys HN, Acm HN)
12.2 (br, lH, OH)
7) Synthesis of Boc-d-Phe-OH (7)
-14-

9.90g (60mmol) of d-Phe-OH was added to 180ml of a
dioxane-water (2:1) mixed solvent. Then, 60ml (60mmol)
of lM sodium hydroxide aqueous solution was added thereto
to dissolve d-Phe-OH. After 14.43g (66mmol) of di-
(tertiary butyl)-dicarbonate ~abridged as "(BOC)2O" in the
following] was added thereto, the mixture was stirred at
room temperature for 3 hours. After dioxane was
evaporated under a vacuum, 80ml of ethyl acetate was
added to the remaining product which was being cooled
with ice. After being acidified with 20ml of 5~
potassium hydrogensulfate aqueous solution, the mixture
was separated. The resulting water layer was further
extracted with ethyl acetate. The extracted layers were
combined together and dried with sodium sulfate
anhydride. The solvent was^evaporated therefrom under a
vacuum to leave a crude product. Then, when it was
recrystallized in ether-petroleum ether, the aimed
product (7) of a white powder form was obtained.
Yield: 10.83g (68.1%)
The physical properties of the product thus
obtained were as follows:
H-NMR(CDCl~)
S(Ppm)=l-4o (s, 9H, Boc CH3)
3.12 (d, 2H, Phe H~)
4.26-4.76 (m, lH, Phe Ha)
4.76-5.12 (m, lH, Phe OH)
6.88-7.56 (m, 5H, Phe C6H5)
8.72 (s, lH, Phe HN)
-15-

~6~2~
8) Synthesis of Boc-d-Phe-Pro-OMe (8)
3.31g (20mmol) of Pro-OMe.HCl was dissolved in
50ml of DMF. While the solution was cooled with ice and
stirred, 2.19ml (20mmol) of NMM was added thereto. Ten
minutes thereafter, 5.31g (20mmol) of the above-mentioned
product (7), 2.97g (22mmol) of HOBt, 4.13g (20mmol) of
DCC were added to the mixture in this order. The
resulting mixture was sequentially stirred at -3C for 5
hours, at 0C for 10 hours, and at room temperature for 9
hours. Then, after DMF was evaporated from the mixture,
50ml of ethyl acetate was added thereto. After cooling,
deposited crystals were separated by filtration. The
filtrate was sequentially washed with lM sodium
hydrogencarbonate aqueous solution, water, lM citric acid
aqueous solution, and water. ~Then, the washed product
was dried with sodium sulfate anhydride. Thereafter, the
solvent was evaporated from the dried product to leave a
crude product. Then, it was purified by a column
chromatography to yield the aimed white amorphous
product.
Yield: 6.73g (89.5%)
The physical properties of the product thus
obtained were as follows:
lH-NMR(DMSO-d6)
~DSS ( ppm)=1.32 (s, 9H, Boc CH~)
1.44-2.28 (m, 4H, Pro H~Y)
2.60-2.92 (m, 2H, Pro H~)
3.00-3.40 (m, 2H, Phe H~)
-16-

2046~4
3.60 (s, 3H, OCH3)
3.84-4.60 (m, 2H, Phe H~, Pro Ha)
6.60-7.60 (m, 6H, Phe C6H5, Phe HN)
9) Synthesis of H-d-Phe-Pro-OMe HCl (9)
To 0.75g (2.Oml) of the above-mentioned product
(8), 30ml of 4M hydrochloric acid/dioxane was added and
the mixture was stirred at room temperature for 1.5
hours. Dioxane was repeatedly evaporated from the
mixture under a vacuum, while several milliliters of
ether was repeatedly added thereto. Thus, the aimed
product (9) of a white powder form was quantitatively
obtained.
10) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-
OMe (10)
0.67g (2.Ommol) of the above-mentioned product (9)
was dissolved in 30ml of DMF. While the solution was
cooled with ice and stirred, 220 ~1 of NMM was added
thereto. Ten minutes thereafter, a DMF solution of 1.13g
(2.0mmol) of the above-mentioned product (6), 0.30g
(2.2mmol) of HOBt, 0.41g (2.Ommol) of DCC were added to
the mixture in this order. The resulting mixture was
sequentially stirred at -3C for 5 hours, at 0C for 12.5
hours, and at room temperature for 7 hours. Thenr after
DMF was evaporated under a vacuum and the residue was
dissolved in chloroform, the impurities were separated by
filtration. The filtrate was sequentially washed with lM
sodium hydrogencarbonate aqueous solution, water, lM
citric acid aqueous solution, and water. Then, the

;~0461~4
organic layer was dried with sodium sulfate anhydride.
Thereafter, the solvent was evaporated from the dried
product to leave a crude product. Then, it was purified
by a column chromatography to yield the aimed yellowish
white amorphous product.
Yield: 0.86g (52.3%)
The physical properties of the product thus
obtained were as follows:
Elementary Analysis (~ by weight): C37H57OloN7S2
Empirical Value C:54.16, H:6.88, N:11.34
Calculated Value C:53.92, H:6.99, N:11.90
lH-NMR ( DMso-d6 )
(pPm)=o-88 (m, 6H, Val Hr)
1.40 (s, 9H, Boc CH3)
1.48-2.24 ~m, llH, Acm CH3, Val H~,
~ro H~, 9r
2.60-3.00 (m, 6H, Pro H~, Cys H~)
3.00-3.40 (m, 2H, Phe H~)
3.60 (s, 3H, OCH3)
3.80-5.00 (m, 9H, Val Ha,
Cys Ha, Phe Ha,
Pro Ha, Acm CH2)
6.80-7.40 (m, 5H, Phe C6H5)
7.40-8.60 (m, 6H, Cys HN, Acm HN,
Val HN, Phe HN)
11) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-
OH (11)
:;

21~4~12a~
l.OOg (1.2mmol) of the above-mentioned product
(10) was dissolved in lOml of methanol. After 1.44ml
(1.44mmol) of lM sodium hydroxide aqueous solution was
added thereto, the solution was stirred at room
temperature for 5 hours. lOml of water was added to the
resulting reaction solution and methanol was evaporated
therefrom under a vacuum. The remaining aqueous solution
was separated with addition of ether. While being cooled
with ice and stirred, the water layer was weakly
acidified with addition of 5ml of lM citric acid aqeous
solution and then subjected to extraction with ethyl
acetate. The extracted layer was dried with sodium
sulfate anhydride. Then, the solvent was evaporated
under a vacuum to leave the aimed yellowish white
amorphous product (11).
Yield: 0.85g (86.5~) -
The physical properties of the product thus
obtained were as follows:
Elementary Analysis (% by weight): C36H55OloN7S2
Empirical Value C:53.71, H:6.86, N:11.02
Calculated Value C:53.48, H:7.08, N:10.91
Remarks: The calculated value as
C36HssOloN7S2 AcOEt
lH--NMR(DMSO--d6)
~Dss(PPm)=0-86 (m, 6EI, Val HY)
1.44 (s, 9H, Boc CH3)
1.52-2.28 (m, llH, Acm CH3, Val H~,
Pro H~
- 19-

Z046~24
2.60-3.80 ~m, 8H, Pro H~,
Cys H~, Phe H~)
3.80-5.00 (m, 9H, Val HQ,
Cys Ha, Phe Ha,
Pro H~)
6.80-7.36 ~m, 5H, Phe C6H5)
7.~0-8.60 (m, 6H, Cys HN, Acm H
Val HN, Phe HN)
12) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-
ONSu (12)
0.74g (0.9mmol) of the above-mentioned product
(11) was dissolved in 10ml of DMF. While the solution
was cooled with ice, 0.21g (1.8mmol) of HONSu and 0.359
(1.8mmol) of 1-ethyl-3-(3-dimethylaminopropyl)-
1~ carbodiimide hydrochlorate (abridged as "EDC.HCl" in the
following) were added thereto in this order. The
resulting mixture was stirred at 0C for 12 hours. After
DMF was evaporated under a vacuum from the mixture, the
residue was dissolved in 20ml of chloroform. Then, the
solution was washed with lM sodium hydrogencarbonate and
water. The organic layer obtained thereby was dried with
sodium sulfate anhydride. Thereafter, the solvent was
evaporated under a vacuum to leave a crude product.
Then, it was purified by a column chromatography to yield
the aimed reddish white amorphous product (12).
Yield: 0.61g (73.6%)
The physical properties of the product thus
obtained were as follows:
-20-

Z 1:)4~
Elementary Analysis (% by weight): C40H58Ol2N8S2
Empirical Value C:50.61, H:6.24, N:10.96
Calculated Value C:50.30, H:6.11, N:11.59
Remarks: The calculated value as
C40H58Ol2N8S2 0.5CHCl3
lH-NMR(DMSO-d6)
ss(PP~n)=0-86 (m, 6H, Val Hr)
1.40 (s, 9H, Boc CH3)
1.52-2.20 (m, llH, Acm CH3, Val Ha,
Pro H~r)
2.60 (s, 4H, ONSu)
2.60-3.80 (m, 6H, Pro Hr,
Cys Ha, Phe Ha)
3.80-5.00 (m, 9H, Val Ha,
Cys Ha, Phe Ha,
Pro Ha ~ Acm CH2)
6.80-7.36 (m, 5H, Phe C6H5)
7.40-8.60 (m, 6H, Cys HN, Acm H~,
Val HN, Phe HN)
13) Synthesis of H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-
ONSu HCl (13)
To l.llg (1.22mmol) of the above-mentioned product
(12), 20ml of 4M hydrochloric acid/dioxane solution was
added. After the mixture was stirred at room temperature
for 1 hour, dioxane was repeatedly evaporated under a
vacuum with addition of several milliliters of ether.
After this evaporation process was repeated for several
-21-

261~6~4
times, the aimed product of a pale reddish purple powder
form was quantitatively obtained.
Yield: 1.079
14) Synthesis of Cyclo~[Cys(Acm~-Val-Cys(Acm)-d-Phe-
Pro]2 (14)
560mg (0.62mmol) of the above-mentioned product
(13) was dissolved in lOml of DMF. While being
vigorously stirred at room temperature, the mixture was
added dropwise to 200ml of pyridine for 1.5 hours. After
the mixture was further stirred at room temperature ~or
28.5 hours, pyridine was repeatedly evaporated therefrom
under a vacuum, while the residue was repeatedly
dissolved in chloroform, to leave a crude brown amorphous
product. The crude yield was 1.069.
0.53g of the crude produt was dissolved in a mixed
solvent of methanol-water. After being purified by a
column filled with an ion-exchange resin, the solution
was repeatedly purified by a column chromatography to
yield the aimed yellowish white amorphous product.
Yield: 20.2mg (3.8%)
Figure 3 is a graph showing the results of
measurement of molecular weight of this product by FD-MS.
Figures 4A and 4B are charts showing 500MHz lH-NMR
spectral data thereof. Figure 5 is a chart showing the
results of measurement of 1H-lHCOSY2D-NMR thereof.
In FD-MS, the molecular weight of the aimed
product (M.W. 1383.76) is indicated as a peak of 1383.
While Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 has 8

2~:)461~4
amide protons and 4 NH protons of the Acm groups, only 4
and 2 of them are respectively observed. It indicates
that the product is a symmetrical cyclic peptide.
II) Method of Making Cyclo-(Cys-Val-Cys-d-Phe-Pro)2
From Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2
(14)
15) Synthesis of Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-
Phe-Pro]2 (15)
55.8g (4.0 x 10-5mol) of the above mentioned
product (14) was dissolved in 2ml of dimethyl sulfoxide
(abridged as "DMSO" in the following). After 87.2mg (3.2
x 10-4mol) of mercury chloride was added thereto, the
solution was stirred at room temperature for 13 hours.
When 20ml of water was added thereto, a white precipitate
was formed. It was then cooled with an ice bath and
separated by filtration. The resulting solid was dried
under a vacuum to yield the aimed product of a white
powder form (15).
Yield: 60mg (70%)
16) Synthesis of Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-
Phe-Pro]2 (15)
[Alternative Method To 15)]
28.5mg (2.1 x 10-5mol) of the above-mentioned
product (14) was dissolved in lml of DMSO. 44.7mg (1.7 x
10-4mol) of mercury chloride was added to the solution and
the mixture was stirred at room temperature for 21 hours.
DMSO was evaporated therefrom under a vacuum and then
20ml of dichloromethane was added thereto to form a white
-23-

L2~
precipitate. The precipitate was washed with 50ml of
dichloromethane. After dichloromethane was washed with
water, the remaining product was dried with sodium
sulfate anhydride and then the solvent was evaporated
therefrom under a vacuum to leave the aimed product in a
pale yellow oil form. The yield thereof was 30.Omg
(71%). Chloroform was used to recover the compound from
the drying agent and the water layer. The recovered
compound was assembled with the white precipitate. The
total yield was 55.8mg.
17) Synthesis of Cyclo-(Cys-Val-Cys-d-Phe-Pro)2 (I)
In an argon atmosphere, not more than lOml of
deaerated methanol was added to 0.06g of the above-
mentioned product (15). The latter was not dissolved but
dispersed in the former. To~~the resulting white turbid
solution, hydrogen sulfate was introduced for 15 minutes.
After the solution was deaerated, the precipitate of
mercury sulfide was separated by filtration. Then,
methanol was evaporated therefrom under a vacuum to leave
the aimed product (I) of a white powder form. The yield
was 7.2mg (22~).
Figure 6 is a chart showing 500MHz lH-NMR spectral
data of the product.
18) Synthesis of Cyclo-(Cys-Val-Cys-d-Phe-Pro)2 (I)
[Alternative Method To 17)]
The synthesis was conducted in the same way as
17) except that distilled DMSO was used as the reaction
-24-

2al~6~2:4
solvent. The aimed product (I) was obtained with the
same yield.
Example 1
I) Making of Cyclo[Cys(Acm)-Val-Cys(Acm)-d-Phe-Prol2
(14) by Azidation Method (Cf. Figure 2)
1) Synthesis of Boc-Cys(Acm)-Val(Cys)-d-Phe-Pro-
NHNH2 (16)
209.7mg (0.254mmol) of the product (10) of Example
1 was dissolved in 5.2ml of methanol. 0.62ml (12.8mmol)
of hydrazine monohydrate was added thereto and the
mixture was stirred at room temperature for 31.5 hours.
Then, methanol was evaporated under a reduced pressure.
The residue was subjected to evaporation under a vacuum
with addition of distilled water. Then, the evaporation
under a vacuum was sequentially repeated with addition of
methanol and ether. Thereby, the aimed product of a
yellow powder form (16) was obtained.
Yield: 197.2mg (94.0%)
Hydrazide Test; positive
2) Synthesis of Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-
Pro]2 (17)
197.2mg (0.24mmol) of the above-mentioned product
(16) was dissolved in 1.22ml of DME' and then cooled to
-50C. To this solution, 180 ~1 (0.72mmol) of 4M
hydrochloric acid/dioxane was added. Thereafter, 34 ~1
(0.24mmol) of isoamyl nitrite was added thereto and the
mixture was stirred for 1.5 hours. Further, 8 ~1 of
isoamyl nitrite was added thereto and the mixture was
-2~-

20~6~4
stirred for 0.5 hour. The reaction solution was cooled
to -70C. To the cooled solution, 101 ~1 (0.72mmol) of
TEA, 1.22ml of a DMF solution of 187.3mg (0.25mmol) of
H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe.HCl cooled to
-70C, and 26 ~1 (0.24 mmol) of NMM were added in this
order. About 30 minutes thereafter, the mixture was
transferred to an ice bath of 0C. While 9 ~1 of NMM was
added thereto and the pH is adjusted to 7, the mixture
was stirred. Then, DMF was evaporated therefrom under a
vacuum. The evaporation was sequentially repeated with
addition of methanol and ether. After 3ml of chloroform
was added thereto, the remaining product was sequentially
washed with water, lM sodium hydrogencarbonate aqueous
solution, water, lM citric acid aqueous solution, and
water. After the organic layer was dried with sodium
sulfate anhydride, the solvent was evaporated under a
vacuum to leave 251.6mg of a crude product. The crude
yield was 69.lmg.
The crude product was purified by a column
chromatography to yield the aimed product (17) in a pale
brown amorphous form.
Yield: 168.Omg (46.1~)
3) Synthesis of Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-
Pro]2NHNH2 (18)
10.3mg (6.79 x 10-6mol) of the above-mentioned
product (17) was dissolved in 140 ~1 of methanol. 16.5
~1 (3.40 x 10-4 mol) of hydrazine monohydrate was added
thereto at room temperature and the mixture was stirred
-26-

Z046~24
at the same temperature for 41 hours. Then, methanol was
evaporated under a vacuum. The residue was subjected to
evaporation under a reduced pressure with addition of 100
~1 of methanol and 500 microliters of water. Further,
the vacuum evaporation was effected with addition of
ether to leave the aimed product of a white powder form
(18).
Yield: 10.4mg (100%)
4) Synthesis of H-[Cys(Acm)-Val-Cys(Acm)-d-Phe-
Pro]2-NHNH2 HC1 (19)
10.4mg (6.86 x 10-6mol) of the above-mentioned
product (18) was dissolved in 0.3ml of methanol. O.lml
of 4M hydrochloric acid/dioxane was added dropwise
thereto and the mixture was stirred at room temperature
for 1.5 hours. Then, the solvent was evaporated
therefrom under a reduced pressure and the remaining
product was washed with ether for several times.
Thereafter, the washed product was dried under a vacuum
to quantitatively yield the aimed product (19) of a
yellowish white powder. The yield was 10.2mg.
5) Synthesis of Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-
Pro]2 (14)
10.2mg (6.86 x 10-6mol) of the above-mentioned
product (19) was dissolved in 200 micoliters of DMF and
cooled to -30C. Then, 6 ~1 (3.5 equivalent weights) of
4M hydrochloric acid/dioxane solution was added thereto.
10 minutes thereafter, 10 ~1 (7.06 x 10-6mol) of isoamyl
nitrite which had been diluted to 1/10 by DMF was added
-27-

Z~)~6~
to the mixture. 1.5 hours thereafter, 2 microliters of
diluted isoamyl nitrite was added thereto and the mixture
was stirred for 6.5 hours. The reaction solution was
cooled to -60C. Then, DMF cooled to -60C was added
thereto. Thereafter, 6 ~1 of NMM (8 equivalent weights~
was added thereto and the p~ was adjusted to 7.30 minutes
thereafter, the mixture was stirred at -20C for 44
hours. Then, the solvent was evaporated therefrom under
a vacuum and the remaining product was sequentially
washed with methanol and ether. Thereafter, the solvent
was evaporated thereform under a vacuum to leave the
aimed product of a yellowish white powder form. The
crude yield was 14.1mg. It was then purified by a column
chromatography to yield the aimed product (14).
II) Method of Making Cyclo-(Cys-Val-Cys-d-Phe-Pro~2
From Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 (14)
The method same as that shown in Example 1 II) was
used to obtain the aimed product (I).
Example 3
Synthesis of Cyclic Peptide-4-Iron Cluster Complex
by Reaction Between Cyclo-(Cys-Val-Cys-d-Phe-Pro)2 (I)
And (NEt4)2[Fe4S4-(S-t-Bu)4] (abridged as "4-iron
cluster" in the following)
The cyclic peptide (I) was dissolved in DMSO to
form 0.8mM solution. Then, the 4-iron cluster was
dissolved in DMSO to form 0.66mM solution. To Xml of the
solution of (I), (2-X)ml of the 4-iron cluster solution
was added. After the mixture was shaken for 10 minutes,

the ultraviolet-visible light absorption spectrum thereof
was measured and the absorption maximums at 305nm and
419nm of each spectrum were plotted. Figure 7 shows the
absorption spectrum of the 4-iron cluster alone. Figure
3 shows their titration curves.
In view of the titration curves of Figure 8, it
was found that the 4-iron cluster and the cyclic peptide
were reacted at a ratio of about 1:1.2 to 1.3.
~ccordingly, the generation of the aimed complex was
confirmed.
Experiment 1
Core Extraction From Cyclic Peptide-4-Iron Cluster
Using Thiophenol
2mg of the cyclic peptide was dissolved in 1.2ml
of DMSO (solution A, whose concentration corresponds to
1.52 x 10-3M). Then, 0.2ml of solution A was sampled and
0~31ml of DMSO was added thereto to form a solution
(solution B, whose-concentration corresponds to 5.96 x
10-4M).
19.4mg of the 4-iron cluster was dissolved in
16.06ml of DMSO (solution C, whose concentration
corresponds to 1.25 x 10-3M). lml of solution C was
sampled and 1.53ml of DMSO was added thereto to form a
solution (solution D, whose conentration corresponds to
4.9 x 10-4M).
0.51ml of solution D was sampled and 0.51ml of
DMSO was added thereto to form solution E. Solution D
was added to solution B to form solution F. Further, 3
-29-

;2~41~ 4
~1 of thiophenol which had been diluted to 1/40 by DMSO
was added to solution F (1.2 equivalent weights per the
cyclic peptide) for core extraction (solution G).
I~ltraviolet-visible light absorption was measured
for each of solution E (containing the 4-iron cluster
alone), solution F (containing the cyclic peptide-4-iron
cluster complex), and solution G (in which the 4-iron
complex was core-extracted from the cyclic peptide-4-iron
complex by thiophenol). Their spectra are shown in
Figure 9. In Figure 9, spectra (a), (b), and (c)
correspond to solutions E, F, and G, respectively.
The maximum of 457nm in spectrum (c) is specific
to [Fe4S4(Sph)4]2-. Accordingly, it was found that the 4-
iron cluster maintained its structure in the cyclic
peptide which was a ligand. ~
Experiment 2
Measurement of Oxidation-Reduction Potential of
Cyclic Peptide-4-Iron Cluster Complex by
Derivative Pulse Polarography
lml of the 4-iron cluster solution C was added to
the cyclic peptide solution A of Experiment 1 and the
mixture was concentrated to about lml to form colution H.
34mg of tetra-n-butylammonium perchlorate (n-Bu4NClO4) was
added thereto as an electrolyte. After the base line of
a blank (DMSO lml + n-~u4NClO4 34mg) was measured,
solution H was subjected to a derivative pulse
polarography, by which the oxidation-reduction potential
was measured. As results, it was found that, although
-30-

2~
the 4-iron cluster by itself exhibited a low oxidation-
reduction potential, it had an oxidation-reduction
potential higher than that of the 4-iron cluster alone,
in fact, the highest oxidation-reduction potential in the
peptide-4-iron clusters which had been synthesized so
far, when the cyclic peptide in accordance with the
present invention coordinated therewith.
Table 1 shows the oxidation-reduction potential of
the peptide-4-iron cluster complex in accordance with the
present invention. For comparison, the oxidation-
reduction potentials of the 4-iron cluster itself and
conventionally synthesized peptide-4-iron clusters are
also shown therein.

Z~4~
Table 1
Measurement of Oxidation-Ruduction Potentials
Oxidation-Reduction Potential
El/2V (vs. Ag~/Ag)
_
Comparative Example 1
[Fe4S4(S-t-Bu4)4]Z~ -1.37 (in DMF)
Comparative Example 2
[Fe4S4(SPh)4]2- -0.99 (in DMF)
Comparative Example 3
[Fe4S4(Boc-(Gly-Cys-Gly)4-NH2] 2- -O . 86 (in DMSO)
Comparative Example 4
[Fe4S4(z-Cys-Gly-Aly-Ala-OMe)2]2- -0.79 (CH2C12, 233K)
Cyclic Peptide-4-Iron Cluster
Complex of Experiment 2 -0.70 (in DMSO)
_ _
-32-