Preparation and metal ion-binding properties of gramicidin S derivatives carrying picolinoyl groups on the ornithine side chains

Article abstract of DOI:10.1039/a805468a

Derivatives of gramicidin S (GS) in which one or both of the two ornithine side chains were picolinoylated were prepared. The dipicolinoyl derivative [Om(PyCO)^2,2′]GS 1 formed a 1:1 complex with Cu²⁺ and Zn²⁺ in MeOH, whil

Full text of DOI:10.1039/a805468a

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Preparation and metal ion-binding properties of gramicidin S
derivatives carrying picolinoyl groups on the ornithine side chains
Keiichi Yamada, Hirotaka Ozaki, Naoki Kanda, Hatsuo Yamamura, Shuki Araki and
Masao Kawai*
Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku,
Nagoya 466-8555, Japan
Received (in Cambridge) 14th July 1998, Accepted 16th October 1998
Derivatives of gramicidin S (GS) in which one or both of the two ornithine side chains were picolinoylated were
prepared. The dipicolinoyl derivative [Orn(PyCO)2,2Ј]GS 1 formed a 1:1 complex with Cu2ϩ and Zn2ϩ in MeOH,
while in the case of the monopicolinoyl derivative [Orn(Boc)2,Orn(PyCO)2Ј]GS (2) stepwise formation of 1:1 and 2:1
(2:Cu2ϩ) complexes was observed. The formation constant of the Cu2ϩ-mediated dimeric species of compound 2 was
larger than those of the corresponding linear compounds possessing the partial structure of macrocycle 2. The
corresponding tetra-N-methyl derivative [MeOrn(Boc)2,MeOrn(PyCO)2Ј,-MePhe4,4Ј]GS 3 also showed lower
stability of the 2:1 complex compared with compound 2, which suggested the presence of a β-sheet-type
intermolecular H-bonding interaction between the two molecules of macrocycle 2 in the 2:1 complex.
[Orn(PyCO)2,2Ј]GS 1, while no formation of monopicolinoyl
Introduction
derivative [Orn(PyCO)2]GS was observed in the reaction mix-
A great number of studies on metal ion-induced self-assembly
ture. Reaction of GSؒ2HCl with the acid chloride PyCOCl
systems have been carried out to construct functional
yielded a mixture of di- and mono-acylated products and un-
supramolecules with well defined structures; e.g., metal ion-
reacted GS, from which [Orn(PyCO)2]GS was isolated in 32%
induced assembly of α-helical peptides which possess a metal
ion-binding site at N-termini was reported by Lieberman and
yield by column chromatographic separation. The monopicol-
inoyl derivative was then treated with (Boc)2O to afford [Orn-
Sasaki,1a and Ghadiri et al.1b,c However, studies using metal-
(Boc)2,Orn(PyCO)2Ј]GS 2 as summarized in Scheme 1.
binding peptides with β-structures are quite few. We attempted
The low yield of the monoacyl derivative 2 was not
to incorporate metal ion-binding site(s) into the antibiotic
cyclic decapeptide gramicidin S (GS), cyclo(Val-Orn-Leu--
unexpected, since modification of one of the two δ-amino
groups of the Orn residues in GS is usually quite difficult. For
Phe-Pro)2, whose conformation is established as an antiparallel
example, 2,4-dinitrophenylation of GS using one equivalent of
β-sheet possessing two Orn side chains on the same side of
the molecule. Nishino et al. reported the synthesis of the GS
analogue possessing 5,5Ј-bipyridylalanine residues in place of
1-fluoro-2,4-dinitrobenzene resulted in an equimolar mixture
of bis-2,4-dinitrophenyl derivative and unchanged GS, which
is quite analogous to the acylation using picolinoylimidazole
described above. Very recently, we have found that trifluoro-
the Orn residues, which was shown to bind a divalent metal
ion.2 Here, we have prepared analogues of GS possessing one
acetylation of GS with trifluoroacetic anhydride (TFAA)
or two picolinoylamido (pyridine-2-carbonylamino, PyCONH)
group(s) by chemical modification of the Orn residue(s) of nat-
ural GS. The PyCONH group is known to coordinate a divalent
metal ion and the bis(PyCO) derivatives of GS can be expected
afforded a ~70% yield of mono-TFA derivative, a versatile
intermediate for the preparation of mono-substituted GS.5
CD spectra of the GS derivatives 1 and 2, possessing one and
two PyCONH group(s), respectively, were very similar to that
of natural GSؒ2HCl, indicating that both peptides also adopt
the same β-sheet conformation as that of GS.
to form a tetradentate 1:1 complex with a metal ion such as
Cu2ϩ or Zn2ϩ. In the case of the Cu2ϩ complex, pyridine N
atoms and deprotonated amide N atoms constitute an N4 tetra-
As a derivative of compound 2 lacking α-NH groups re-
dentate ligand, while in the Zn2ϩ complex the pyridine N
quired for intermolecular inter-β-sheet H-bonding interaction,
atoms and amide O atoms are considered to coordinate Zn2ϩ
tetra-N-methyl derivative [MeOrn(Boc)2,MeOrn(PyCO)2Ј,
ion.3 The GS analogue containing only one PyCONH group is
-MePhe4,4Ј]GS 3 was also prepared, in which solvent-exposed
expected to undergo metal ion-mediated self assembly forming
α-NH groups of the Orn and -Phe residues of structure 2 were
methylated. As shown in Scheme 1, preparation of the tetra-N-
a 2:1 complex with a metal ion such as Cu2ϩ or Zn2ϩ. The metal
ion-binding properties of these analogues are described in
comparison with those of the corresponding linear peptides.
methylated derivative 3 made use of selective N-methylation
with MeI–Ag2O in DMF.6 N-Methylation of the di-Boc deriv-
ative [Orn(Boc)2,2Ј]GS yielded
a
tetramethyl derivative
[MeOrn(Boc)2,2Ј,-MePhe4,4Ј]GS, which was deprotected and
picolinoylated in essentially the same manner as the prepar-
ation of compound 2 to yield di- and mono-picolinoyl deriv-
atives, [MeOrn(PyCO)2,2Ј,-MePhe4,4Ј]GS and [MeOrn(PyCO)2,
MeOrn2Ј,-MePhe4,4Ј]GS, respectively. The latter compound
was then treated with (Boc)2O to furnish [MeOrn(Boc)2,
MeOrn(PyCO)2Ј,-MePhe4,4Ј]GS 3 possessing four N-methyl
groups instead of α-NH groups which could participate in
intermolecular H-bonding interaction. Since not only the
Results and discussion
Synthesis
Introduction of a picolinoyl group as an amino-protecting
group to afford α-amino acid esters was reported by Koul et al.4
using picolinic acid (pyridine-2-carboxylic acid, PyCO2H) and
1,1Ј-carbonyldiimidazole (CDI). Treatment of GSؒ2HCl with
picolinoylimidazole reagent prepared from PyCO2H and CDI
was found to afford only dipicolinoyl derivatives of GS, namely
J. Chem. Soc., Perkin Trans. 1, 1998, 3999–4004
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R3
D-Phe
Leu
Pro
O
O
R3
Val
N
N
N
H
N
Orn
HN
Orn
N
O
O
H
O
Val
Pro
Leu
D-Phe
H
O
O
O
H
N
R1NH
N
N
N
N
O
N
O
N
NH
R3
O
O
R3
Zn2+
R2NH
R1 = PyCO
R2 = PyCO
R3 = H
R1 = PyCO
R2 = H
R3 = H
Fig. 1 Proposed structure of 1:1 [Orn(PyCO)2,2Ј]GS 1–Zn2ϩ complex.
R1 = H
R2 = H
R3 = H
PyCOCl
+
Monopicolinoyl GS
(Boc)2O
Dipicolinoyl GS 1
GS
i) (Boc)2O
ii) MeI–Ag2O
R1 = Boc
R2 = Boc
R3 = Me
R1 = PyCO
R2 = Boc
R3 = H
Mono(Boc)-mono-
picolinoyl GS 2
i) TFA
ii) PyCOCl
MeI–Ag2O
Fig. 2 Absorption spectra of [Orn(PyCO)2,2Ј]GS 1 with the addition
of ZnCl2 (MeOH; 25 ЊC; [1] = 5.0 × 10Ϫ5 M, [ZnCl2] = 0–9.1 × 10Ϫ5 M).
R1 = PyCO
R2 = H
R3 = Me
R1 = PyCO
2 = PyCO
R3 = Me
R
1 = PyCO
(Boc)2O
R2 = Boc
R3 = Me
R
+
Metal ion-binding analysis
Complexation ability of the picolinoyl peptides 1–5 with Zn2ϩ
and Cu2ϩ ions was examined in MeOH at 25 ЊC using UV-
visible absorption spectral changes induced by complexation.
The following equilibria were assumed and the binding con-
stants, K1:1, K1:2 and K2:1, were defined according to equations
(1)–(3).
Tetra-N-methyl
derivative of 2
3
Scheme 1 Preparation of picolinoyl derivatives [Orn(PyCO)2,2Ј]GS 1,
[Orn(Boc)2,Orn(PyCO)2Ј]
2
and [MeOrn(Boc)2,MeOrn(PyCO)2Ј,-
MePhe4,4Ј]GS 3 from gramicidin S (GS).
L ϩ M = ML (1:1 complex) K1:1 = [LM]/[L][M] (1)
LM ϩ M = LM2 (1:2 complex) K1:2 = [LM2]/[LM][M] (2)
LM ϩ L = L2M (2:1 complex) K2:1 = [L2M]/[LM][L] (3)
BocNH group but also the PyCONH group in the Orn side
chain in GS was found to be methylation-resistant,7 [Orn-
(Boc)2,Orn(PyCO)2Ј]GS 2 was directly subjected to selective N-
methylation to yield compound 3, which was indistinguish-
able from the compound prepared from [Orn(Boc)2,2Ј]GS as
described above.
L: PyCO-carrying peptide derivatives.
M: metal ion (Cu2ϩ or Zn2ϩ).
Linear peptide derivatives corresponding to partial structures
of the picolinoylamide-containing GS, namely Ac-Orn(PyCO)-
NHMe 4 and Ac-Val-Orn(PyCO)-Leu-NHMe 5, were also pre-
pared to compare the dimeric Cu2ϩ-complex-forming ability.
Introduction of a PyCO group to the δ-amino group of the Orn
residue in these compounds was accomplished by treatment
with picolinoylimidazole reagent.4 Their structures were con-
Dipicolinoyl GS derivative 1. The pyridine N atoms and
amide O atoms of the two PyCONH groups of [Orn(Py-
CO)2,2Ј]GS 1 are assumed to participate in the coordination to
a Zn2ϩ ion forming a 1:1 complex3 as schematically shown in
Fig. 1. Upon addition of ZnCl2, the absorption maximum of
the dipicolinoyl derivative 1 at 264 nm (ε 11 200 dm3 molϪ1
cmϪ1) showed hyperchromic and bathochromic shifts, exhibit-
ing an isosbestic point at 253 nm (Fig. 2). Nonlinear least-
squares curve-fitting analysis8 based on the absorbance change
at 272 nm indicated the formation of a 1:1 complex with the
log K1:1 value of 5.28. The CD spectrum of compound 1 with a
weak positive maximum around 290 nm ([θ] ϩ 800) and a neg-
ative shoulder around 265 nm also changed upon the addition
of ZnCl2 to a distinct negative maximum at 273 nm ([θ] Ϫ 6200)
with an isoelliptic point at 270 nm (Fig. 3). The log K1:1-value
obtained from the change of ellipticity at 280 nm was in good
agreement with that obtained by absorption spectral analysis.
No evidence of formation of a 1:2 or 2:1 complex was
observed over the range of [1]:[Zn2ϩ] from 1:0.24 to 1:1.9.
Addition of CuCl2 to a solution of compound 1 was also
1
firmed by H NMR, liquid secondary-ion mass (LSIMS) and
UV-visible spectroscopy.
N
N
CO
CO
HN
HN
(CH2)3
CH(CH3)2 (CH2)3
CH2CH(CH3)2
AcNH
C
H
CONHCH3
C
H
CONH
AcNH
C
H
CONH
C
H
CONHCH3
Ac-Orn(PyCO)-NHMe
Ac-Val-Orn(PyCO)-Leu-NHMe
4
5
4000
J. Chem. Soc., Perkin Trans. 1, 1998, 3999–4004

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Table 1 The formation constants of Cu2ϩ complex with PyCONH-
containing peptides in MeOH at 25 ЊC
Peptide
log K1:1
log K2:1
log K1:2
1
2
3
4
5
7.64 0.2
5.22 0.1
5.18 0.1
5.15 0.15
5.06 0.1
5.72 0.15
3.08 0.05
2.67 0.1
2.53 0.1
2.62 0.05
Fig. 3 CD spectra of [Orn(PyCO)2,2Ј]GS 1 with the addition of
ZnCl2 (MeOH; 25 ЊC; [1] = 5.0 × 10Ϫ5 M, [ZnCl2] = 0–9.1 × 10Ϫ5 M).
shown to produce a 1:1 complex, but in the presence of an
excess of Cu2ϩ additional spectral change was observed indicat-
ing the formation of a 1:2 complex. The formation constants
of the 1:1 and 1:2 complexes with Cu2ϩ were determined simi-
larly by the curve-fitting analysis using equations (1) and (2),
and the log K1:1- and log K1:2-values were obtained as 7.64 and
5.72, respectively.
Thus, the dipicolinoyl derivative 1, as expected, formed a 1:1
complex both with Zn2ϩ and Cu2ϩ, indicating the coordination
of the two PyCONH groups to the divalent metal ions. In the
case of Cu2ϩ, however, the 1:2 species was also formed in which
each of the two picolinoylamido groups of 1 was considered to
bind a Cu2ϩ ion. The pyridine N atom and ionized amide N
atom of the picolinoylamide group are assumed to coordinate
Cu2ϩ ion3 although no structural study of these complexes was
undertaken.
Monopicolinoyl GS derivatives 2 and 3. In order to investigate
Cu2ϩ-mediated assembly of the monopicolinoyl derivative
[Orn(Boc)2,Orn(PyCO)2Ј]GS 2, the absorption spectral change
of the d–d* band of Cu2ϩ ion due to complexation was moni-
tored. Keeping the total concentration of Cu2ϩ species constant
([CuCl2] = 1 mM), aliquots of MeOH solution of compound 2
containing CuCl2 were added to the solution of CuCl2. As
shown in the difference spectra, complex-formation-induced
increase of the absorption around 650 nm was observed upon
the addition of compound 2 (Fig. 4a). The plots of the absorb-
ance change (∆A) at 800, 750, 700 and 650 nm vs. [2]/[CuCl2]
gave an inflection point around [2]/[CuCl2] = 1, suggesting the
stepwise formation of 1:1 and 2:1 complexes (Fig. 4b). Non-
linear least-squares curve-fitting analysis based on equations (1)
Fig. 4 Absorption spectral change of CuCl2 in MeOH with the add-
ition of the monopicolinoyl derivative 2 (25 ЊC; [CuCl2] = 1.0 × 10Ϫ3 M,
[2] = 0.26–5.0 × 10Ϫ3 M). (a) Difference spectra are given. ∆A refers to
the absorbance of the solution less the absorbances of the component
solutions of CuCl2 and compound 2 at the corresponding concen-
trations. (b) Plots of ∆A vs. [2]/[CuCl2] at 800, 750, 700 and 650 nm
are shown.
and (3) yielded the complex-formation constants as log K1:1
5.22 and log K2:1 = 3.08.
=
The reference compounds Ac-Orn(PyCO)-NHMe 4 and Ac-
Val-Orn(PyCO)-Leu-NHMe 5, possessing the partial structures
of compound 2, were also subjected to the Cu2ϩ ion-binding
analysis, and the results are summarized in Table 1. The K2:1
value of the GS derivative 2 is larger than those of the linear
compounds 4 and 5 (log K2:1 = 2.53 and 2.62, respectively). The
relative stability of the Cu2ϩ-mediated dimeric GS species could
be attributed to inter-β-sheet H-bonding interaction between
the two GS units in the complex. The ‘cross-β’ aggregation ten-
dency of the GS molecule in oriented poly(oxyethylene) was
indicated by an IR dichroism study.9 Seto and Whitesides also
reported intermolecular H-bonding-mediated aggregation of
the di-Boc derivative [Orn(Boc)2,2Ј]GS in CHCl3 solution stud-
ied by vapour pressure osmometry.10 The possible structure of
the 2:1 complex composed of the antiparallel β-sheet type
assembly of four Val-Orn(PyCO)-Leu strands is illustrated in
Fig. 5, the structure might also be stabilized by hydrophobic
interaction between iPr and iBu groups of Val and Leu residues.
In order to study the importance of the intermolecular H-
bonding interaction for the formation of the 2:1 complex, the
tetra-N-methyl derivative of compound 2, namely [MeOrn-
(Boc)2,MeOrn(PyCO)2Ј,-MePhe4,4Ј]GS 3, was prepared. Pre-
vention of aggregation of β-strands by the introduction of
N-methyl group(s) was reported for a three-stranded β-sheet
peptide11 and an interleukin monomer.12 It is known that tetra-
N-methylation does not affect the β-sheet conformation of GS.6
The tetra-N-methyl derivative 3 lacking α-NH groups of the
Orn and -Phe residues required for intermolecular H-bonding
interaction was subjected to the metal ion-binding analysis. As
J. Chem. Soc., Perkin Trans. 1, 1998, 3999–4004
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spectropolarimeter, respectively. SIMS were measured on a
Hitachi M-2000 spectrometer. Elemental analyses were done at
the Elemental Analysis Center of Kyoto University.
D-Phe
Leu
Pro
D-Phe
Leu
Orn
Val
Pro
Val
Val
Orn
Leu
Orn
Orn
[Orn(PyCO)2,2Ј]GS 1
Val
Leu
D-Phe
A solution of PyCO2H (372 mg, 3.0 mmol) and 1,1Ј-
carbonyldiimidazole (CDI, 487 mg, 3.0 mmol) in dry THF (10
cm3) was stirred at rt under argon for 30 min, to which was
added a solution of GSؒ2HCl (413 mg, 0.5 mmol) in dry THF
and reaction mixture was stirred at rt for 2 h. After removal of
precipitated imidazole hydrochloride by filtration the filtrate
was evaporated in vacuo and the residue was subjected to GFC.
The crude product was purified by SiO2 column chroma-
tography using CHCl3–MeOH (95:5 v/v) as eluent to afford
title compound 1 (512 mg, 76%) as a powder (lyophilized);
λmax(MeOH)/nm 264 (ε/dm3 molϪ1 cmϪ1 11 200); [θ]max(MeOH)/
nm 206 ([θ]/deg cm2 dmolϪ1 Ϫ4.4 × 105), 217 (Ϫ4.3 × 105), 265sh
(Ϫ9.5 × 103), 285 (0) and 292 (800) [Found: C, 62.3; H, 7.1; N,
14.0. C72H98N14O12ؒ2H2O requires C, 62.3; H, 7.4; N, 14.1%;
m/z (SIMS) 1351.7642 (M ϩ H)ϩ. (C72H98N14O12 ϩ H) requires
m/z, 1351.7544].
Pro
Pro
D-Phe
O
N–
N
N–
N
NH
HN
O
Cu2+
Boc
Boc
Fig. 5 Illustration of the proposed structure of 2:1 [Orn(Boc)2,Orn-
(PyCO)2Ј]GS 2–Cu2ϩ complex. Broken arrows indicate H-bonds consti-
tuting a four-strand antiparallel β-sheet. The intermolecular H-bonds
are impossible for the tetra-N-methylated derivative 3.
expected, compound 3 exhibited lower stability of its 2:1 com-
plex (log K2:1 = 2.67) than did analogue 2, which is in agree-
ment with the proposed antiparallel β-sheet-type assembly of
the two GS units in the case of compound 2. Very recently we
have synthesized dimeric analogues of GS which actually possess
a methylene-chain bridge between the δ-N atoms of the two GS
[Orn(Boc)2,Orn(PyCO)2Ј]GS 2
1
units.13 Preliminary H NMR spectral studies of the synthetic
To a stirred solution of GSؒ2HCl (242 mg, 0.2 mmol) in DMF
were added PyCOClؒHCl (76 mg, 0.4 mmol), Et3N (0.172 cm3,
1.2 mmol) and DMAP (28 mg, 0.2 mmol) at rt. After being
stirred for 2 days at rt the reaction mixture was lyophilized and
was subjected to gel filtration chromatography. The crude
product was then chromatographed over SiO2 using CHCl3–
MeOH (9:1 v/v) as eluent to yield 1 (31 mg, 12%) and [Orn(Py-
CO)2]GS (79 mg, 32%); λmax(MeOH)/nm 264 (ε/dm3 molϪ1 cmϪ1
6400); m/z (SIMS) 1248.1 (M ϩ H)ϩ]. The latter compound (34
mg, 0.027 mmol) was dissolved in CHCl3–MeOH (3:1 v/v) (4
cm3), to which were added (Boc)2O (41 mg, 0.19 mmol) and
Et3N (0.027 cm3, 0.19 mmol). After stirring the mixture at rt for
5 h, the solvent was evaporated in vacuo. The crude product was
purified by GFC and then SiO2 column chromatography using
CHCl3–MeOH (95:5 v/v) as eluent to afford title compound 2
(36 mg, 98%) as a powder (lyophilized); λmax(MeOH)/nm 264
(ε/dm3 molϪ1 cmϪ1 7000); [θ]max(MeOH)/nm 206 ([θ]/deg cm2
dmolϪ1 Ϫ5.2 × 105), 216 (Ϫ4.8 × 105) and 264sh (Ϫ2.3 × 103)
[Found: C, 61.3; H, 7.7; N, 13.25. C71H103N13O13ؒ2.5H2O
requires C, 61.3; H, 7.8; N, 13.1%; m/z (SIMS) 1346.7842
(M ϩ H)ϩ. (C71H103N13O13 ϩ H) requires m/z, 1346.7852].
trimethylene-bridged precursor molecules have indicated the
presence of β-sheet-type H-bonding interaction between the
two cyclic decapeptide modules. The dimeric derivatives can be
considered as synthetic models of the inter-Orn side chain
interaction-mediated assembly of GS molecules validating the
proposed structure of the 2:1 complex illustrated in Fig. 5.
Detailed studies on the conformational characteristics of these
δ-N(Orn)–δ-N(OrnЈ) covalently linked dimeric compounds are
in progress.
In the present study the GS derivative 2 carries a metal ion-
binding group on the δ-N atom of an Orn residue while another
Orn side chain is protected with a Boc group which can be
replaced with different functional groups to construct a func-
tional assembly system. However, although formation of the
expected metal ion-mediated dimeric complex was observed for
the GS derivative–Cu2ϩ system, the stability of the 2:1 complex
was not high enough compared with that of the 1:1 complex.
Thus, stepwise formation of the 1:1 and 2:1 complexes was
observed when the GS derivative was added to the metal ion
solution and the 2:1 complex became prominent only after the
addition of more than 1 equivalent of the peptide to Cu2ϩ. In
order to realize the system in which formation of the dimeric
species can be controlled by the addition of a metal ion, a
higher tendency to form a metal ion-mediated dimeric com-
plex is necessary. Studies on the construction of self-assembly
systems using metal ion-binding peptide derivatives which
carry pyridine ring-containing groups other than PyCO are
in progress.
[MeOrn(Boc)2,MeOrn(PyCO)2Ј,D-MePhe4,4Ј]GS 3
via selective N-methylation of [Orn(Boc)2,2Ј]GS. [MeOrn-
(Boc)2,2Ј,-MePhe4,4Ј]GS was prepared from [Orn(Boc)2,2Ј]GS as
described in ref. 5. The tetra-N-methylated Boc derivative (104
mg, 0.074 mmol) was treated with TFA (1 cm3) at 0 ЊC for 1 h.
TFA was evaporated off in vacuo and the residue was dissolved
in 1 M HCl (10 cm3) and lyophilized to give [MeOrn2,2Ј,-
MePhe4,4Ј]GSؒ2HCl (112 mg, ≈100%). Picolinoylation of the
tetra-N-methyl derivative (102 mg, 0.080 mmol) using PyCO-
ClؒHCl (29 mg, 0.16 mmol) in the same manner as described
above, followed by chromatographic separation (SiO2; CHCl3–
MeOH, 9:1 v/v), yielded [MeOrn(PyCO)2,2Ј,D-MePhe4,4Ј]GS
(20 mg, 23%) (Found: C, 63.6; H, 7.5; N, 13.3. C76H108N14O12ؒ
1.5H2O requires C, 63.5; H, 7.8; N, 13.6%) and [MeOrn2,
MeOrn(PyCO)2Ј,-MePhe4,4Ј]GS (20 mg, 19%). The monopi-
colinoyl derivative (40 mg, 0.030 mmol) was treated with
(Boc)2O and Et3N to afford [MeOrn(Boc)2,MeOrn(PyCO)2Ј,
D-MePhe4,4Ј]GS 3 (26 mg, 62%) [Found: C, 63.2; H, 7.8; N,
12.65. C75H111N13O13ؒH2O requires C, 63.4; H, 8.0; N, 12.8%. m/
z (SIMS) 1403.4 (M ϩ H)ϩ. C75H111N13O13 requires m/z,
1403.9].
Experimental
Mps were determined on a hot-plate apparatus and are uncor-
rected. Column chromatography was performed using SiO2
(Fuji Sylisia, FL60D). Gel filtration chromatography (GFC)
was performed using Sephadex LH-20 (Pharmacia Biotech)
with MeOH as eluent. Monitoring of reactions and analysis of
chromatographic fractions were undertaken by means of TLC
with precoated SiO2 plate (Merck, Silica gel 60F254). Purity of
all of the synthetic compounds was ascertained by HPLC
analysis (JASCO, Finepak-SIL, 4.6 × 50 mm, elution with
CHCl3–MeOH). 1H NMR spectra were recorded in [2H6]-
DMSO solutions at 25 ЊC on a Varian Gemini-200 (200 MHz)
or a JEOL α-500 (500 MHz) spectrometer. J-Values are in Hz.
UV-visible and CD spectra were recorded in MeOH solution
on a HITACHI U-3500 spectrometer and a JASCO J-600
via selective N-methylation of compound 2. To a stirred solu-
tion of compound 2 (68 mg, 0.050 mmol) in DMF were added
4002
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Ag2O (232 mg, 1.00 mmol) and methyl iodide (2.5 cm3, 40
mmol) at rt. The reaction mixture was stirred for 1 day and
lyophilized to remove the solvent. After GFC, the crude
product was chromatographed with aminopropyl-modified
SiO2 (Fuji Sylisia, Chromatorex NH-DM1020) using CHCl3–
MeOH (98:2 v/v) as eluent to afford compound 3 (48 mg, 68%),
which was indistinguishable from the compound prepared from
[MeOrn2,2Ј,-MePhe4,4Ј]GSؒ2HCl as described above.
NaCl and was then dried over Na2SO4. The solvent was evapor-
ated off and the residue was chromatographed over SiO2 using
CHCl3–MeOH as eluent to give Boc-Orn(Cbz)-Leu-NHMe as
a solid (1.52 g, 69%).
After treatment of the protected dipeptide methylamide (465
mg, 0.945 mmol) with TFA as described above, the resulting
H-Orn(Cbz)-Leu-NHMe was subjected to a coupling reaction
with Boc-Val-OH (294 mg, 1.35 mmol), using DCC (542 mg,
2.63 mmol) and HOBt (101 mg, 0.75 mmol) in CH2Cl2 (2 cm3)
in a similar manner to the preparation of Boc-Orn(Cbz)-
NHMe. After stirring of the reaction mixture for 4 h it was
treated similarly and the crude product was subjected to SiO2
column chromatography using CHCl3 as eluent to afford Boc-
Val-Orn(Cbz)-Leu-NHMe as crystals (467 mg, 84%), mp 176–
178 ЊC (from Et2O–MeOH).
The Cbz group of the protected tripeptide was replaced by a
PyCO group in a similar manner as described for the prepar-
ation of Boc-Orn(Cbz)-NHMe to give Boc-Val-Orn(PyCO)-
Leu-NHMe in 66% yield. The Boc group was then replaced by
an Ac group also in a similar manner as above to yield the
desired product 5 as a powder (66%), mp 256–256.8 ЊC (lyophil-
ized) [Found: C, 58.7; H, 7.8; N, 16.1. C25H40N6O5ؒ0.5H2O
requires C, 58.45; H, 8.05; N, 16.35%; m/z (SIMS) 505.0
(M ϩ H)ϩ. C25H40N6O5 requires M, 504.3].
Ac-Orn(PyCO)-NHMe 4
A mixture of Boc-Orn(Cbz)-OH (1.43 g, 3.69 mmol), MeNH2ؒ
HCl (621 mg, 9.20 mmol) and HOBt (450 mg, 3.33 mmol) in
CH2Cl2 (15 cm3) was neutralized by the addition of Et3N, to
which DCC (1.49 g, 7.21 mmol) was added under ice-cooling.
After stirring of the reaction mixture at rt for 23 h the sol-
vent was evaporated off in vacuo and to the residue were
added AcOEt and 10% aq. citric acid. After removal of pre-
cipitated dicyclohexylurea by filtration the AcOEt layer was
washed successively with 10% aq. citric acid, 5% aq. NaHCO3
and saturated aq. NaCl and was then dried over Na2SO4.
The solvent was evaporated off and the residue was subjected
to SiO2 column chromatography using CHCl3 as eluent to
afford Boc-Orn(Cbz)-NHMe as a solid, mp 142–143.5 ЊC
(1.00 g, 68%).
The N-methylamide (1.00 g, 2.65 mmol) as a solution in
MeOH (5 cm3) was hydrogenolysed under atmospheric H2
over Pd-black (0.35 g) for 2 h. Filtration, and evaporation off
of the solvent, yielded crude Boc-Orn-NHMe, which was
added to a solution of picolinoylimidazole reagent prepared
by stirring PyCO2H (0.509 g, 4.14 mmol) and CDI (0.559 g,
3.45 mmol) in dry THF (5 cm3) for 1 h. After stirring of the
mixture for 15 h at rt the solvent was evaporated off in vacuo
and the residue was chromatographed over SiO2 using CHCl3–
MeOH as eluent to give Boc-Orn(PyCO)-NHMe as an oil
(0.958 g, 94%).
The picolinoyl compound (0.956 g, 2.50 mmol) was treated
with TFA (3 cm3) at 0 ЊC for 2 h. After evaporation off of TFA
in vacuo the crude product was dissolved in dry pyridine (10
cm3) and excess of TFA was neutralized by addition of Et3N to
which Ac2O (4 cm3) had been added, and the mixture was
stirred for 1.5 h. The volatiles were removed in vacuo, and the
resulting crude product was purified by SiO2 column chrom-
atography using CHCl3 as eluent and crystallization from
MeOH–Et2O to yield title compound 4 as crystals (0.419 g,
57%), mp 190–192 ЊC; δH(200 MHz; [2H6]DMSO) 1.82–1.64
(4H, m), 1.84 (3H, s), 2.80 (3H, d, J 4.8), 3.45 (1H, m), 3.75
(1H, m), 4.65 (1H, m), 6.71 (1H, d, J 8.2), 6.82 (1H, q, J 4.8),
7.44 (1H, ddd, J 7.7, 4.8 and 1.2), 7.86 (1H, dt, J 7.7 and 1.7),
8.18 (1H, ddd, J 7.7, 1.2 and 0.9), 8.24 (1H, br t, J 6.6) and 8.55
(1H, ddd, J 4.8, 1.7 and 0.9) {Found: C, 57.65; H, 6.9; N, 19.1%;
[M ϩ H]ϩ (SIMS), 292.9. C14H20N4O3 requires C, 57.5; H, 6.9;
N, 19.2%; [M ϩ H]ϩ, m/z, 292.2}.
Metal ion-binding analysis
Analyses were repeated several times with varying concen-
trations of the initial solution and the added solution of the
peptide and metal ion. Typical examples are given below.
Dipicolinoyl derivative 1. To a MeOH solution of compound
1 (5.2 × 10Ϫ5 M; 2.5 cm3) were added aliquots (1.2 × 10Ϫ4 cm3)
of a MeOH solution of CuCl2 (2.8 × 10Ϫ3 M) containing com-
pound 1 (5.2 × 10Ϫ5 M) at 25 ЊC. Thus, while the concentration
of 1 was kept constant, that of CuCl2 was changed from 0.11 to
1.4 × 10Ϫ4 M. After each addition absorption spectrum was
recorded and the absorbance changes at 272 nm were subjected
to curve-fitting analysis.8 Essentially the same method was
applied to the analysis of CD spectral changes of compound 1
with the addition of the metal ion.
Monopicolinoyl derivatives 2 and 3. To a MeOH solution of
CuCl2 (1.00 × 10Ϫ3 M; 2.5 cm3) were added aliquots
(7.50 × 10Ϫ4 cm3) of a MeOH solution of a compound 2 or 3
(2.50 × 10Ϫ2 M) containing CuCl2 (1.00 × 10Ϫ3 M) at 25 ЊC. The
concentration of the peptide ranged from 0.26 to 4.99 × 10Ϫ3
M. The absorbance changes at 650, 700, 750 and 800 nm were
used for the analysis. Spectral changes upon the addition of
CuCl2 to the peptide solution as described for compound 1 were
also recorded (peptide 9.90 × 10Ϫ6 M, CuCl2 0.049–1.30 × 10Ϫ4
M). All these data were included for the estimation of the K1:1
and K2:1-values.
-
Ac-Val-Orn(PyCO)-Leu-NHMe 5
Acknowledgements
Boc-Leu-NHMe (1.10 g, 4.51 mmol), which was prepared in
90% yield from Boc-Leu-OH and MeNH2ؒHCl in a similar
manner to the preparation of Boc-Orn(Cbz)-NHMe as
described above, was treated with TFA (2.5 cm3) at rt for 2 h.
TFA was evaporated off in vacuo and the residue was dissolved
in CH2Cl2 (6 cm3) and neutralized by addition of Et3N.
We are grateful to Nikken Kagaku Co., Ltd. for the gener-
ous supply of GSؒ2HCl and to Prof. Ryoichi Katakai and
Ms Kyoko Kobayashi (Department of Chemistry, Gunma
University) for the measurement of 1H NMR spectra (500
MHz).
Boc-Orn(Cbz)-OH (2.03 g, 5.54 mmol) was added to the
solution of H-Leu-NHMe and the mixture was cooled to 0 ЊC,
to which HOBt (450 mg, 3.33 mmol) and 1-ethyl-3-(3-di-
methylaminopropyl)carbodiimide hydrochloride (1.13 g, 5.90
mmol) were added. After stirring of the reaction mixture at rt
for 21 h the solvent was evaporated off in vacuo and the residue
was dissolved in AcOEt. The solution was washed successively
with 10% aq. citric acid, 5% aq. NaHCO3 and saturated aq.
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