Diastereomeric selective effects for growth inhibition of synthesized mini parallel double-stranded peptides on Escherichia coli and Staphylococcus aureus

Article abstract of DOI:10.1248/cpb.52.204

The synthesis of a series of mini double-stranded peptides containing chiral-x-Phe-y-Phe-peptide residues and the diastereomeric selective effects of these compounds on Escherichia coli NIHJ JC-2 and Staphylococcus aureus FDA209P growth are described. In

Full text of DOI:10.1248/cpb.52.204

204
Chem. Pharm. Bull. 52(2) 204—213 (2004)
Vol. 52, No. 2
Diastereomeric Selective Effects for Growth Inhibition of Synthesized
Mini Parallel Double-Stranded Peptides on Escherichia coli and
Staphylococcus aureus
Shigeki KOBAYASHI, ,a Nahomi ATUCHI,a Hiroki KOBAYASHI,a Akiko SHIRAISHI,b
*
a
Hajime HAMASHIMA,b and Akira TANAKA
a Department of Analytical Chemistry of Medicines, Showa Phamaceutical University; and b Department of Microbiology,
Showa Phamaceutical University; 3–3165 Higashi-tamagawagakuen, Machida, Tokyo 194–8543, Japan.
Received September 8, 2003; accepted November 7, 2003
The synthesis of a series of mini double-stranded peptides containing chiral–x-Phe–y-Phe–peptide residues
and the diastereomeric selective effects of these compounds on Escherichia coli NIHJ JC-2 and Staphylococcus
aureus FDA 209P growth are described. In the case of bis(y-Phe–x-Phe)-N,N-ethane-1,2-diamine, bis(y-Phe–x-
Phe)-N,N-buthane-1,4-diamine, bis(y-Phe–x-Phe)-N,N-hexane-1,6-diamine, and bis(y-Phe–x-Phe)-N,N-dodecane-
1,12-diamine, etc., the four configurations, (L-, L-), (D-, L-), (L-, D-) and (D-, D-), where the symbols x- and y- repre-
sent optical isomers with L- and D- forms, were used to investigate the relationship between chirality and antibac-
terial activity. The level of activity increased in the following order: (L-, L-)Ͻ(D-, D-)Ͻ(L-, D-)Ͻ(D-, L-). The data
show that (D-, L-) chilarity is more potent than (L-, L-) chilarity. Then, these results suggest that the –y-Phe–x-Phe
Phe–sequence in the double-stranded peptide has anti-bacterial activity and the chirality of –y-Phe–x-Phe affects
the anti-bacterial activity. Our results show that the uptake by penetration through the membrane of bis(y-
Phe–x-Phe)2-Spacers is a first step in the expression of anti-bacterial activity. This study provides new insights in
the chirality-antibacterial activity relationships of a series of mini double-stranded peptides.
Key words peptide; diastereomer; chirality; antibacterial activity; log P; circular dichroism
The function of proteins and enzymes is much related to ingly, the activity increased in the following order: bis(L-
the tertiary structure formed with the folding of secondary Phe–L-Phe)-N,N-hexane-1,6-diamineϽbis(D-Phe–D-Phe)-
structures, e.g., the a-helix, b-sheet, or b-turn. Although b- N,N-hexane-1,6-diamineϽbis(L-Phe–D-Phe)-N,N-hexane-
sheet or b-turn structures are found in peptides and proteins, 1,6-diamineϽbis(D-Phe–L-Phe)-N,N-hexane-1,6-diamine.
their biological functions are poorly understood yet com- Compound bis(D-Phe–L-Phe)-N,N-hexane-1,6-diamine is
pared with the a-helix. In an effort to clarify the chemical about 32 times more active than bis(L-Phe–L-Phe)-N,N-
properties, functions, and stereochemistry of b-turn (or b- hexane-1,6-diamine. A similar trend was seen in the order of
hairpin) and antiparallel b-sheet structures, the design and the activity for double-stranded peptides with a spacer of car-
synthesis of peptides have been reported by a number of re- bon number nϭ2, 4, 8, and 12. This result shows that the an-
search groups.1—6) Model compounds which mimic b-sheet tibacterial activity is affected by the diastereomeric selectiv-
and b-turn structures have been shown to adopt b-hairpin ity of Phe in double-stranded peptides. The biological po-
structures in an aqueous solution, and hydrogen bonding can tency of D-Phe–L-Phe– or –L-Phe–D-Phe– sequences may
stabilize the b-turn structure. Recently, we have reported the play an important role in antibacterial activity or cytotoxicity.
synthesis and chemical properties of mini parallel double- What is the biological role of the configurations (D-, L-) and
stranded peptides, (L-AA3–L-AA2–L-AA1)2-spacer(S) (AAϭ (L-, D-) in the y-Phe–x-Phe sequence? The mechanisms of ac-
amino acids; Sϭethano- and dodecano-, etc.) conjugated two tion may be similar to those of antibacterial peptides such as
peptide strands to a mini loop (ϭspacer).7) Especially, the gramisidine A. In order to elucidate the mechanism of inter-
chemical properties of bis(L-Leu–L-Phe–L-Phe)-N,N-ethane- action of these double-stranded peptides with cell membrane,
1,2-diamine and bis(L-Leu–L-Phe–L-Phe)-N,N-dodecane- we synthesized a new fluorescence probe bis(Dns–L-Phe)-
1,12-diamine mediated b-sheet-like conformation were de- N,N-ethane-1,2-diamine (Dnsϭdansyl group) conjugated to
scribed in the previous paper because the sequences like dansyl chloride to the N-terminus of bis(L-Phe)-N,N-ethane-
–Phe–Phe–, –Leu–Phe–Phe–, and –Val–Phe–Phe– which are 1,2-diamine. Fluorescence microscopy showed that the probe
little existence in natural peptides and proteins. Moreover, was taken up into the cytosol and passed through the mem-
the –Val–Phe–Phe–, interestingly, is contained in Alzheimer’s brane of A 431 cells. Bis(D-Phe–L-Phe)-N,N-hexane-1,6-di-
disease related b-amyloid peptide (Ab).
amine, etc. may also form pores which pass ions. This indi-
The aims of the present study are the synthesis of double- cates that double-stranded peptides pass through the cell
stranded peptides containing chiral –x-Phe–y-Phe– se- membrane (or bacterial cell wall). It suggests that com-
quences and elucidation of the structure–activity relationship pounds bis(D-Phe–L-Phe)-N,N-hexane-1,6-diamine, etc. form
between antibacterial activity and chirality of the –Phe–Phe– chiral pores on the membrane of bacteria or cells.
sequence. We synthesized four chiral types, –L-Phe–L-Phe–,
Finally, we found that the major and minor action groups
–D-Phe–L-Phe–, –L-Phe–D-Phe– and –D-Phe–D-Phe– of bis(y- can be seen in double-stranded peptides, bis(y-Phe–x-Phe)-
Phe2–x-Phe1)-N,N-hexane-1,6-diamine and examined the ef- N,N-buthane-1,4-diamine and bis(y-Phe–x-Phe)-N,N-hexane-
fect of chirality on the growth inhibition of Escherichia coli 1,6-diamine, etc., and the (D-, L-) or (L-, D-) forms of the
NIHJ JC-2 and Staphylococcus aureus FDA 209P. Interest- minor action group act as diastereomeric selective inhibitors
To whom correspondence should be addressed. e-mail: kobayasi@ac.shoyaku.ac.jp
© 2004 Pharmaceutical Society of Japan

February 2004
205
Reagents: (i) TFA at 4 °C. (ii) CDI, Boc-AA-OH (AAϭamino acid), dry CHCl3. (iii) TFA at 4 °C (or 5% Pd/C, H2, DMF/CH3OH for Z-deprotection). b) 14cϭZ protect com-
pound.
Chart 1
of the growth of E. coli NIHJ JC-2 and S. aureus FDA 209P.
Thus this is the first investigation of the diastereomeric selec-
tive effects and structure–activity relationship for the antibac-
terial activity of a series of new mini double-stranded pep-
tides.
Results
Synthesis and Characterization by CD and NMR
A
series of double-stranded peptides, bis(y-Phe–x-Phe)-N,N-
ethane-1,2-diamine (16a—d) and bis(y-Phe–x-Phe)-N,N-do-
decane-1,12-diamine (20a—d), were synthesized by the
methods described in the previous paper.7) The four opti-
cal isomers of bis(y-Phe–x-Phe)-N,N-buthane-1,4-diamine
(17a—d) and bis(y-Phe–x-Phe)-N,N-hexane-1,6-diamine
(x, yϭL- or D-form) (18a—d), were prepared as shownin
Chart 1 and their chemical properties and circular dichroism
(CD) were compared. bis(L-Phe)-N,N-hexane-1,6-diamine
(8a) was treated with C-terminal activated Boc–L-PheOH
Reagents: (i) Dansyl chloride, Na2CO3, acetone at 4 °C.
Chart 2
prepared using N,NЈ-carbonyldiimidazole (CDI) in dry butane-1,4-diamine 17b (Rfϭ0.16) and bis(L-Phe–D-Phe)-
CHCl3 to provide bis(Boc–y-Phe–x-Phe)-N,N-hexane-1,6-di- N,N-butane-1,4-diamine 17c (Rfϭ0.16), as listed in Table 1.
amine (13a). The crude reaction mixtures were purified by The Rf values in these compounds are proportional to the
silica gel column chromatography. The Boc- and Z-protect- carbon number (n) of the polyamines spacer and the chirality.
ing groups were removed with trifluoroacetic acid (TFA) in Compounds 17b and 17c, etc. have higher polarity than the
an ice bath and by hydrogenation in the presence of 5% Pd/C diastereomers, 17a and 17d, moreover, similar trends were
in DMF/MeOH solution, respectively. The resulting free observed for the other double-stranded peptides.
bases were purified with an overall yield of about 30 to 40%
In order to clarify the chemical properties, a novel fluo-
by recrystallization or silica gel column chromatography, and rescence probe, bis(Dns–L-Phe)-N,N-ethane-1,2-diamine 21
satisfactory results were obtained by thin layer (TLC, silica conjugated dansyl group (ϭDns) to N-terminus of bis(L-
gel) or reverse-phase high performance liquid chromatogra- Phe)-N,N-ethane-1,2-diamine 6a was synthesized as shown
phy (HPLC). Interestingly, the Rf values of Boc forms on in Chart 2. In fluorometry, compound 21 emits a siganal at
TLC, contrary to expectation, increased in the following about 530 nm with an excitation wavelength of 349 nm in
order: 6aϽ7aϽ8aϽ9aϽ10a. Compounds bis(L-Phe)-N,N- 85% methanol. Table 2 shows the chemical shift (d ppm) and
buthane-1,4-diamine (7a) (Rfϭ0.06) and bis(L-Phe)-N,N-oc- 3JaH,NH coupling constants of two protons, aH1 and aH2, of
tane-1,8-diamine (9a) (Rfϭ0.16) have larger Rf values than 17a—d, 18a—d, and 19a, b, d with the numbering shown in
bis(L-Phe–L-Phe)-N,N-butane-1,4-diamine 17a (Rfϭ0.41) Chart 1. The chemical shifts of aH1 and aH2 of 18a showed
and bis(L-Phe–L-Phe)-N,N-octane-1,8-diamine (19a) (Rfϭ dd (or ddd) and dd patterns at 4.64 and 3.51 ppm, and that of
0.43), respectively. Moreover, the Rf of compound 17a is 19a appeared at 4.63 and 3.51 ppm, respectively in DMSO-
larger than that of the diastereomers, bis(D-Phe–L-Phe)-N,N- d6/CDCl3 (volume ratioϭ0.2 ml/0.5 ml). However, the chemi-

206
Vol. 52, No. 2
cal shifts of aH1 and aH2 of the long molecule 20a appear-
ed at 4.50 and 4.09 ppm, respectively. The CD profiles of
the bis(y-Phe–x-Phe)-N,N-hexane-1,6-diamine, 18a—d (a),
bis(x-Phe)-N,N-octane-1,8-diamine, 9a—b, and (y-Phe–x-
Phe)-N,N-octane-1,8-diamine, 19a and 19d exhibit a mirror
image as shown in Fig. 1, and they are pure stereo-chemi-
cally. The molar ellipticity ([q]) of compounds 9a and 9b is
smaller in intensity than that of 19a or 19d conjugated to
four Phe. The CD curve of 18b, a diastereomer of 18a, is ob-
viously a mirror image of 18c, and the [q] intensity is small
compared with 18a or 18d. The positive maxima of CD at
228 and 234 nm of 18b differ from the positive maximum at
224 nm of 18a, which may be related to the difference in
electronic state of –L-Phe–L-Phe– and –D-Phe–L-Phe– se-
quences.
Table 1. TLC Rf Values of Some Double-Stranded Peptides Conjugated
–y-Phe–x-Phe– Sequence to Spacer
Compounds Carbon number (n) of spacer Configuration
Rf a,b)
16a
16c
17a
17c
18a
18c
19a
19c
20a
20c
2
2
4
4
6
6
8
8
12
12
(L-, L-)
(L-, D-)
(L-, L-)
(L-, D-)
(L-, L-)
(L-, D-)
(L-, L-)
(L-, D-)
(L-, L-)
(L-, D-)
0.36
0.15
0.41
0.16
0.42
0.21
0.43
0.31
0.51
0.41
Conformation in Solution We have reported that CD
data, NMR data from nuclear Overhauser enhancement and
exchange spectroscopy (NOESY) experiments, and J values
of 16a and 20a, bis(L-Leu–L-Phe–L-Phe)-N,N-dodecane-
1,12-diamine were consistent with
a folded b-sheet
structure.7) Here, the experimental dihedral angles in 17a
3
were calculated with JaH,NH values based on the coupling
constant between aH and amido proton –NHCO–. In order
to characterize the conformations, the values of sequential
dNN NOE and dNH,aH connectivities, especially, were used.
a) Mean values. b) CHCl3–MeOH (10 : 1) solvent was used for TLC development.
3
While JaH,NH values for b-sheet forms fall in the range of 8
to 10 Hz, the values for a-helical forms distributed between
4 and 5 Hz.8,9) The values indicate that 17a, 18a, and 19a
also adopt a b-sheet-like conformation. Figure 2 shows NOE
cross peaks involving the side chains of Phe1 and Phe2
(ortho-, meta-, and para-), amido proton (–NHaCO–), and
spacer protons of 18a and 18c in DMSO-d6/CDCl3 (volume
ratioϭ0.2 ml : 0.5 ml) solution. The amido proton, NHa, in
18a is observed at d 7.53. The a proton, aH1, in 18a
is observed at d 4.64. The coupling constants determined
at 21 °C are listed in Table 2. The dihedral angles
–CO–C1a–Nb–CO– in 18a estimated using the NMR data
Table 2. Chemical Shifts of Alpha Protons and Amido Protons for Several
Double-Stranded Peptides
Chemical shift, d (ppm)a
Compounds
aH1
aH2
–NHaCO– –NHbCO– JaH ,NH (Hz)
3
1
b
16a
16b
16c
17a
17b
18a
18b
18c
19a
20a
20b
20c
4.58
4.53
4.52
4.63
4.61
4.64
4.62
4.63
4.63
4.50
4.59
4.61
3.52
3.94
3.71
3.50
3.49
3.51
3.50
3.50
3.51
4.09
4.05
3.49
7.68
7.80
7.83
7.58
7.55
7.53
7.53
7.52
7.54
7.61
7.78
7.45
7.89
8.79
8.54
7.94
8.00
7.95
7.99
8.00
7.95
8.90
8.73
7.95
8.2
7.9
7.6
8.6
8.2
8.9
8.6
8.5
8.9
8.2
8.5
8.2
are about Ϫ110° since the coupling constants of NHa and
5)
aH1 protons in Phe1 are 3JNH ,aH ϭ8.6 Hz, respectively.
a
1
3
1
b
The JaH ,NH data listed in Table 2 clearly show that 17a,
18a, and 19a have a very similar average conformation in
DMSO-d6/CDCl3 solution and have a b-sheet-like structure.
However, 3JaH ,NH data for bis(D-Phe–L-Phe)-N,N-ethane-1,2-
diamine 16b and 16c of the short spacer, C2, support a fold-
1
b
a) In DMSO-d6/CDCl3 (0.2/0.5ϭ0.7 ml).
Fig. 1. Circular Dichroism Spectra for Mirror Images of Bis(y-Phe–x-Phe)-N,N-hexane-1,6-diamines 18a—d, Bis(x-Phe)-N,N-octane-1,8-diamines 9a, b
and Bis(y-Phe–x-Phe)-N,N-octane-1,8-diamines 19a, d
Concentration, 18a (6.150ϫ10Ϫ4 M), 18b (7.462ϫ10Ϫ4 M), 18c (6.580ϫ10Ϫ4 M), 18d (5.036ϫ10Ϫ4 M), 9a (1.370ϫ10Ϫ3 M), 9b (1.187ϫ10Ϫ3 M), 19a (9.016ϫ10Ϫ4 M), and 19b
(7.514ϫ10Ϫ4 M) in 85% aqueous methanol employing a path length quartz cell at 22 °C. x-axis, l (nm); y-axis, molar ellipticity [q].

February 2004
207
Table 3. Dipole Moment and Log P of Some Double-Stranded Peptides
Conjugated –x-Phe–y-Phe– Sequence to Spacer
Compounds
Dipole moment (DM)a) (debye)
Log Pb)
16a
16c
17a
17c
18a
18c
19a
19c
20a
20c
6.46
5.39
5.20
2.98
1.34
8.92
5.53
7.73
2.94
8.32
2.52
2.52
3.08
3.08
3.91
3.91
4.75
4.75
6.42
6.42
a) PM3 hamiltonian level. b) By Ghose–Crippen method.10)
1,6-diamine 18a, are obvious, the folding conformation is
confirmed. Therefore, the conformations obtained using the
MONTECARLO calculations are consistent with the NOE
data.
Log P is a measure of the hydrophobicity of chemicals,
and was evaluated according to the method of Ghose–Crip-
pen,10) in our study. The results are listed in Table 3. We de-
scribed in the section “Synthesis and Characterization by CD
and NMR” that the Rf values of 16a, c—20a, c on silicagel
TLC increase in the following order: 16aϽ17aϽ18aϽ
19aϽ20a and 16cϽ17cϽ18cϽ19cϽ20c. Interestingly, the
results support that the order of the Rf values is nearly di-
rectly proportional to the Log P, that is, the hydrophobicity of
16a, c—20a, c.
MIC Determination of Spacers C4 and C6 An assay
of activity for bacteria, E. coli NIHJ JC-2 and S. aureus FDA
209P, was used to examine the difference in activity caused
by the change in the configuration of the amino acid in dou-
ble-stranded peptides, 17a—d and 18a—d. Bacteria were
cultured for 24 h at 37 °C in 96-well micro plates.11) Tables 4
and 5 show the minimum inhibitory concentration (MIC,
mg/ml) for the growth of E. coli NIHJ JC-2 and S. aureus
FDA 209P measured by treatment with the broth dilution
method of double-stranded peptides. The MIC values show
that these compounds were able to effectively inhibit the
growth of E. coli NIHJ JC-2 and S. aureus FDA 209P. For the
growth inhibition of E. coli NIHJ JC-2, compound 17b is 16
times stronger than 17a. In order to investigate the relation-
ship between configuration patterns of the compounds and
inhibitory activity, the antibacterial activity of compounds 18
was compared by a similar method. Similarly, the (D-, L-) iso-
mer 18b and (L-, D-) isomer 18c were significantly more ac-
tive than the (L-, L-) isomer 18a, and the potency increases in
the following order; (L-, L-) 18aϽ(D-, D-) 18dϽ(D-, L-) 18bϽ
and nearly equal to (L-, D-) 18c. The antibacterial activities of
compounds 17a—d against E. coli NIHJ JC-2 and S. aureus
FDA 209P also led to similar trends as shown in Fig. 3.
In the spacers with carbon numbers, nϭ4 and 6, the di-
astereomers 17c and 18c have a MIC of 16 and 8 mg/ml, re-
spectively, while the enantiomers 17a, 17d, 18a, and 18d
have MIC values in excess of about 200 mg/ml (Fig. 3b).
Then, it is suggested that the antibacterial activity of double-
stranded peptides is closely related with the difference in the
mirror image and diastereomeric selective effects exist.
MIC Determination for Spacers C2 and C12 Our re-
sults also show a structure–activity relationship between anti-
Fig. 2. NOESY Dataa) in CDCl3/DMSO-d6 (0.5 ml/0.2 ml) and Lowest En-
ergy Conformationsb) of 18a and 18c
a) Showing NOEs involving the side chains of Phe1 and Phe2 (ortho-, meta-, para-),
amido proton (–NHaCO–), and spacer protons (C2ϫ2, C3ϫ2). b) Lowest Energy
Conformation of 18a (a) and 18c (b) obtained by a montecarlo calculation using the
MMFF (molecular mechanics) Method.
ing b-sheet-like conformation more than 16a. The NOEs be-
tween spacer protons-aromatic side chain of Phe1 and Phe2 in
16—20 also show that their distance is about 3.5—4.5 Å in
the intramolecule. This suggests that the confomation is con-
sistent with the folding structure.7)
Calculated Conformations and Log P It may be diffi-
cult to determine which conformation is predominant in
strand1 and strand2 of the double-stranded peptides in the in-
tramolecule, because the compounds have an axial symmet-
ric structure for the spacer. To understand the conformations
in solution, we searched for the most stable conformation
using computational chemistry. The minimum energy con-
formation of the double-stranded peptides was calculated by
making out a conformational search with the MONTE-
CARLO method using a MMFF94 force field (molecular
mechanics). With the several conformations obtained, the
minimum energy conformation was optimized with a single
point energy calculation using PM3 Hamiltonian, and Log P
was also calculated. Figure 2 shows several low energy con-
formations and the minimum energy conformation of bis(L-
Phe–L-Phe)-N,N-hexane-1,6-diamine 18a and bis(L-Phe–D-
Phe)-N,N-hexane-1,6-diamine 18c. The conformations sup-
port the experimental data obtained from the NOESY mea-
surements. As the NOE cross peaks among Phe1, Phe2, and
the hexano-spacer protons in bis(L-Phe–L-Phe)-N,N-hexane-

208
Vol. 52, No. 2
Table 4. Comparative Biological Activities of Bis(AA2–AA1)-N,N-butane-1,4-diamine Analog in Vitro
In vitro MIC and MBC values (mg/ml)
E. coli NIHJ JC-2 S. aureus FDA 209P
Compound
Residues
AA2
AA1
MIC
MBC
ra)
MIC
MBC
ra)
17a
17b
17c
17d
L-Phe
D-Phe
L-Phe
D-Phe
L-Phe
L-Phe
D-Phe
D-Phe
512
64
64
1024
64
64
2
1
1
1
128
32
16
256
32
16
2
1
1
2
1024
1024
256
512
a) rϭMBC/MIC.
Table 5. Comparative Biological Activities of Bis(AA2–AA1)-N,N-hexane-1,6-diamine Analog in Vitro
In vitro MIC and MBC values (mg/ml)
Compound
Residues
E. coli NIHJ JC-2
S. aureus FDA 209P
AA2
AA1
MIC
MBC
ra)
MIC
MBC
rb)
18a
18b
18c
18d
L-Phe
D-Phe
L-Phe
D-Phe
L-Phe
L-Phe
D-Phe
D-Phe
1024
32
32
Ͼ1024
64
32
Ͼ1024
1
2
1
2
256
8
8
512
16
16
2
2
2
4
512
256
1024
a) rϭMBC/MIC.
Fig. 3. Chirality Effect of Bis(y-Phe–x-Phe)-N,N-butane-1,4-diamines 17a—d and Bis(y-Phe–x-Phe)-N,N-hexane-1,6-diamines 18a—d on Activity
against E. coli NIHJ JC-2 (a) and S. aureus FDA 209P (b)
Each bar show the mean of MIC.
bacterial activity and spacer length besides chirality. Here, Phe)-N,N-ethane-1,2-diamine 6b, gave 4 times more activity
we examined antibacterial activity for E. coli NIHJ JC-2 and than the L-configuration 6a for anti-S. aureus FDA 209P pro-
S. aureus FDA 209P by stimulation of double-stranded pep- liferation. The antibacterial activity increases in the follow-
tides 16a—d and 20a—d which have a spacer length of Cϭ2 ing order, (D-, D-) 20dϽ(L-, L-) 20aϽ(L-, D-) 20cϽ(D-, L-)
and 12. In order to investigate the effect of the stereochemi- 20b. The compound 20b as diastereomers of 20a and 20d
cal configuration on bacterial proliferation, the activity showed the most anti-S. aureus FDA 209P activity in this se-
against E. coli NIHJ JC-2 and S. aureus FDA 209P of four ries, and has a MIC of 4 mg/ml. This trend is similar to the
isomers of bis(y-Phe–x-Phe)-N,N-dodecane-1,12-diamine activity of 17a—d and 18a—d against the growth of S. au-
20a—d was tested. Figure 4 shows histograms of the data. reus FDA 209P and E. coli NIHJ JC-2 (Fig. 4b). However,
Compound bis(L-Phe)-N,N-dodecane-1,12-diamine 10a (car- the four optical isomers 16a—d were less active than 17, 18,
bon numberϭ12) has 64 times the activity of 6a (carbon and 20a—d, and had a weaker chirality effect than 20a—d.
numberϭ2). It was interesting that the D-configuration, bis(D-
Fluorescence Microscopy In order to examine the inter-

February 2004
209
Fig. 4. Chirality Effect of Bis(y-Phe–x-Phe)-N,N-ethane-1,2-diamines 16a—d and Bis(y-Phe–x-Phe)-N,N-dodecane-1,12-dimaines 20a—d on Activity
against E. coli NIHJ JC-2 (a) and S. aureus FDA 209P (b)
Each bar show the mean of MIC.
Fig. 5. Fluorescence Microscopic Images of A431 Cells after Treatment with New Fluorescent Labeling Bis(Dns–L-Phe)-N,N-ethane-1,2-diamine (21)
Probe
A 431 cells were incubated with 27.5 mg/1000 ml (a) and 3.5 mg/1000 ml (b) of the fluorescent label 21 in DMEM containing 10% FBS at 37 °C for 24 h.
lexϭ349 nm,
l
emϭ530 nm, in 85% MeOH. Compound 21 was localized to the cell membrane and cytosol ((ii) and (iv)). Photographs (i) and (iii) without fluorescence are comparison of (ii) and
(iv) with fluorescence. Magnification ϫ100 and ϫ200.
action between double-stranded peptide and cell membrane, spond to compound 21 at 27.5 mg/1000 ml and 3.5 mg/
the fluorescence probe bis(Dns–L-Phe)-N,N-ethane-1,2-di- 1000 ml, respectively. A431 cells are stained at 3.5 mg/
amine 21 conjugated to a known fluorescence reagent, dansyl 1000 ml. Moreover, photograph (iv) shows that compound 21
(ϭDns), to N-terminus of 6a was synthesized. After 3ϫ102 has a stained cytosol and membrane but not nucleus. The re-
cells/ml of A431 cells seeded to 12-well plates were incu- sults imply that the double-stranded peptide, (L-Phe2–L-
bated for 48 h in a CO2 incubater, 20 ml of each solution Phe1–)2–spacer(S), also passes through the cell membrane,
(27.5 mg/1000 ml and 3.5 mg/1000 ml) of fluorescence probe and is taken up into the cytosol.
21 was added and the mixture was incubated for 24 h. Figure
5 shows fluorescence photographs of A431 cells penetrated Discussion
by 21 in the cytosol. Photographs (i), (ii) and (iii), (iv) corre-
Peptides like gramicidines A and S, well known as anti-

210
Vol. 52, No. 2
shown in Fig. 6.
The membrane and cell wall are located on the bacterial
envelope, and the chemicals, hormones, and small proteins
pass through porin to the inner cell. The double-stranded
peptides are more active against S. aureus (gram-positive
bacteria) than E. coli (gram-negative bacteria) which have
porins located in the membrane. In gram-negative bacteria,
E. coli, the most active bis(L-Phe–D-Phe)-N,N-dodecane-
1,12-diamine 20c would be taken up into the cytosol through
porin, which suggests antibacterial activity. The data sug-
gests that the passage of porin is not always related to the an-
tibacterial activity. When the double-stranded peptide is
taken up into the cytosol, the structure is changed. As an-
tibacterial peptides generally form a peptide-lipid complex
pore, a fluorescence probe 21 conjugated to the dansyl group
of 6a was synthesized to examine which of the double-
stranded peptides passes through the membrane. Figure 5
shows fluorescence photographs of A431 cells treated with
21. It shows that compound 21 entered the cytosol, but not
the nucleus. It suggests, therefore, that double-stranded pep-
tides also would be taken up by the cytosol or form double-
stranded peptides channels (or pores) on the memberane, and
express antibacterial activity. Recent our investigations
showed that the –D-Phe–L-Phe– sequence increases the mem-
brane permeability of Ca2ϩ ion by Fura-214) analysis as com-
pared with the –D-Phe–L-Phe– sequence. This evidence sup-
ports that compounds 18b and 20b, etc. form channels or
pores via double-stranded peptides.
Replacement of the –L-Phe–L-Phe– by –D-Phe–L-Phe– (or
–L-Phe–D-Phe–) sequence in compounds 1 led to a more ac-
tive antibacterial effect. From these results, the diastere-
omeric selective antibacterial activities clearly indicate that
the membrane permeability is linked to the chiral plane of
17b, c, 18b, c, and 20b, c, etc. Because, the Rf value of 18c
conjugated –D-Phe–L-Phe– (or –L-Phe–D-Phe–) is smaller
than that of 18a conjugated –L-Phe–L-Phe– as listed in Table
1. The Rf values represent the polarity of compounds, i.e. 18c
has higher polarity than 18a. When high log P and polar di-
astereomers, 16b—c, 17b—c, 18b—c, and 20b—c, etc. pass
through the cell membrane, it is suggested that the polarity of
the membrane increases. At present, we can not explain the
order of potency of double-stranded peptides against, E. coli
and S. aureus.
Fig. 6. Major and Minor Active Groups of (y-Phe–x-Phe)2–Spacer Pep-
tides for Diastereomeric Selective Antibacterial Activity
Symbols x and y show L- and D-configuration of Phe. Diastereomers (D-, L-) and
(L-, D-) increase the growth inhibition for bacteria.
bacterial substances, are characterized by a wide antibacterial
spectrum. It is suggested that gramicidine and other peptidal
antibiotics act on the lipid matrix of the cell membrane, in-
crease the membrane’s permeability, and cause channels or
pores to form.12,13) However, the mechanism and structure–
activity relationships of antibacterial peptides are still un-
known. In this study, we demonstrated a structure–activity
relationship between chirality and antibacterial activity based
on values of MICs. In order to elucidate the effect of chirality
on the antibacterial activity of artificial b-sheet-like double-
stranded peptides, we examined the growth inhibition of E.
coli and S. aureus. The effect, particularly on diastereomeric
selective activity, was found in the MICs of four optical iso-
mers, 17a—d, 18a—d, and 20a—d, conjugated –y-Phe–x-
Phe– sequences to the two N-terminals of the spacer. In the
series of bis(y-Phe–x-Phe)-N,N-hexane-1,6-diamine 18a—d,
for instance, the antibacterial activity for gram-negative E.
coli NIHJ JC-2 increase of within 32 from 1024 mg/ml in the
following order: (L-, L-)Ͻ(D-, D-)Ͻ(L-, D-)ϳ(D-, L-). For
gram-positive S. aureus FDA 209P proliferation, although
the order of antibacterial activity was similar, the activity of
compounds 18a—d was greater to gram-positive bacteria
than to gram-negative one. It is concluded that the potency of
the (L-, D-) and (D-, L-) forms are stronger than that of (L-, L-)
and (D-, D-) forms. Then, the diastereomeric (D-, L-) and (L-,
D-) forms for the (L-, L-) form, interestingly, may be the best
antibacterial configuration (ϭbioactive structure).
Conclusions
In this paper, we described the characterization and con-
formation of mini parallel double-stranded peptides and
demonstrated a structure–activity relationship for micro bac-
terial growth inhibition. Here our study showed that the (D-,
L-) or (L-, D-) forms are the most active configurations in
bis(y-Phe–x-Phe)-N,N-ethane-1,2-diamine, -N,N-butane-1,4-
diamine, -N,N-hexane-1,6-diamine, and -N,N-dodecane-1,12-
diamine. This suggests that the configurations, (D-, L-) or
(L-, D-), are probably bio-active conformations in double-
stranded peptides (L-Phe2–L-Phe1)2–spacer(S). Measured dif-
ferences of the activity show the highly stereospecific phar-
macokinetic phenomena of the double-stranded peptides for
E. coli or S. aureus and may relate to the penetration or pore
forming ability of mini double-stranded peptides in the mi-
crobacterial membrane. We are interested in designing and
synthesizing better anti-methicillin-resistant Staphylococcus
The molecular size of the spacer also may relate to an-
tibacterial activity since our data support that bis(x-Phe)-
N,N-dodecane-1,12-diamine and bis(y-Phe–x-Phe)-N,N-do-
decane-1,12-diamine are more active than bis(x-Phe)-N,N-
ethane-1,2-diamine and bis(y-Phe–x-Phe)-N,N-ethane-1,2-di-
amine. The analog 18a is less active than 16a. The activity of
double-stranded peptides increases in the following order:
C(6)ϽC(8)ϽC(4)ϽC(2)ϽC(12) (the number shows the
number of carbons in the spacer). These results suggest that
the spacer length and chirality are related to the activity of
double-stranded peptides against E. coli and S. aureus.
Changing the chirality and spacer length, therefore, is impor-
tant to the design of more active double-stranded peptides.
The results suggest that there are two action groups in dou-
ble-stranded peptides: first, spacer length, and second, the
chirality of the side chain of the phenylalanine residue, as

February 2004
211
aureus (MRSA) drugs by changing the chirality and spacer (A) 0.384; mp 159—162 °C; FAB-MS (NBA) m/z 933 (MϩH)ϩ. Bis(Z-L-
Phe–D-Phe)-N,N-octane-1,8-diamine 14c: 85% yield: Rf (A) 0.423; mp
length. Clinical bed sides are exposed to the menace of
MRSA, and the molecular design and synthesis of anti-
197—199 °C. FAB-MS (NBA) m/z 933 (MϩH)ϩ. Bis(Boc–D-Phe–D-Phe)-
N,N-octane-1,8-diamine 14d: 68% yield: Rf (A) 0.384; mp 157—160 °C;
MRSA drugs are expected in the field of chemotherapy. In
antibacterial peptidal drug design, our results show that spec-
ified chirality, (D-, L-) or (L-, D-), in double-stranded peptides
plays an important role in anti-bacterial activity. Further
studies are in progress to elucidate the diastereomeric selec-
tive effects on growth inhibition of several cells.
FAB-MS (NBA) m/z 933 (MϩH)ϩ. Anal. Calcd for C42H52N6O4·1/2H2O: C,
68.84; H, 7.81; N, 8.92. Found: C, 68.90; H, 7.84; N, 9.11.
The General Procedure for the Synthesis of Bis(L-Phe)-N,N-butane-
1,4-diamine (7a) A mixture of 1.00 g (1.14 mmol) of 2a and 10 ml of TFA
was stirred for 1 h in an ice bath. After a 1 M NaOH/5% NaHCO3 workup,
the solution was extracted with CHCl3. The organic layers were combined,
dried over Na2SO4, filtrated, concentrated under vacuum, and dried to give
colorless crystals of 7a: 81.0% yield, and the product was purified by col-
umn chromatography on silica gel (35.0 g) eluted with CHCl3 and 3%
MeOH/CHCl3 (stepwise elution): Rf (A) 0.056, mp 126—128 °C (from
Experimental
Melting points were taken on a Yanako (MP-21) apparatus and are uncor-
rected. IR spectra were recorded with a JASCO A-100 spectrophotometer
and UV–visible spectra with a JASCO U-best 30 spectrophotometer. 1H- and
13C-NMR spectra were recorded with a JNM GX-270 (270 MHz) or a-500
(500 MHz) spectrometer with TMS (tetramethyl silane) as an internal stan-
dard. FAB mass spectral data were recorded with a JEOL JMS-HX110 spec-
trometer, and relevant data are tabulated as m/z. TLC was performed using
Silica gel 60 F254 (Merck) plates and the following solvent systems:
CHCl3–MeOH (20 : 1) (A) and CHCl3–MeOH (10 : 1) (B). The peptides
were detected on the TLC plates using iodine vapor or UV absorption. Silica
gel column chromatography was performed on Wako gel C-200 (100 mesh)
or Merck Silica gel 60N (100 mesh). Analytical reverse phase HPLC was
carried out on a TOSO CCPD system equipped with a Tskgel (ODS-120T)
column. Elemental analyses were performed by the Laboratory Center of El-
emental Analysis, University of Ehime (Japan). For all UV–vis and CD
spectra, commercial solvents of the highest purity available were used.
Synthesis A series of double-stranded peptides were synthesized as de-
scribed in our previous paper. New compounds 2—4, 7—9, 12—14, and
17—19 were prepared from the corresponding protected peptide analogs by
a reaction with protected amino acids of the spacer according to previously
published procedures.7)
The General Procedure for the Synthesis of Bis(Boc–L-Phe)-N,N-
butane-1,4-diamine (2a) tert-Butoxycarbonyl phenylalanine (Boc–L-
PheOH) (3.20 g, 12.1 mmol) and CDI (2.20 g, 13.5 mmol) were dissolved in
dry CHCl3 and were stirred at room temperature for 1 h. To this a solution
1,4-diaminobutane (0.48 g, 5.5 mmol) was added, and the resulting mixture
was stirred overnight, and the solvent was evaporated. After the addition of
cooled aq. MeOH, the solid was collected, and washed with aq. MeOH, 5%
citric acid, 5% sodium hydrogen carbonate and water, and dried in vacuo.
Further purification by silicagel column chromatography (eluted with step-
wise: CHCl3 and 2% MeOH/CHCl3) gave the product 2a as a colorless crys-
tal in 75% yield: Rf (A) 0.463; mp 197 °C; FAB-MS (NBAϭnitrobenzyl al-
cohol) m/z 583 (MϩH)ϩ. Anal. Calcd for C32H46N4O6 C, 65.96; H, 7.96; N,
9.61. Found: C, 65.83; H, 7.88; N, 9.38.
The Other Boc Double-Stranded Peptides Bis(Boc–D-Phe)-N,N-bu-
tane-1,4-diamine 2b: 78% yield: Rf (A) 0.463; mp 197—198 °C; FAB-MS
(NBA) m/z 583 (MϩH)ϩ. Bis(Boc–L-Phe–L-Phe)-N,N-butane-1,4-diamine
12a: 70% yield: Rf (A) 0.337; mp 222—224 °C; Anal. Calcd for
C50H64N6O8·1/2H2O: C, 67.77; H, 7.39; N, 9.48. Found: C, 67.87; H. 7.29:
N, 9.55. Bis(Boc–D-Phe–L-Phe)-N,N-butane-1,4-diamine 12b: Rf (A) 0.395;
mp 213—214 °C; HR-FAB-MS (NBA) Found 877.4868, Calcd for
C50H64N6O8 877.4864 (MϩH)ϩ. Bis(Boc–L-Phe–D-Phe)-N,N-butane-1,4-di-
amine 12c: Rf (A) 0.395; mp 215—217 °C; HR-FAB-MS (NBA) Found
877.4882, Calcd for C50H64N6O8 877.4864 (MϩH)ϩ. Bis(Boc–D-Phe–D-
Phe)-N,N-butane-1,4-diamine 12d: Rf (A) 0.337; mp 200—201 °C (aq.
MeOH); FAB-MS (NBA) m/z 877 (MϩH)ϩ. Bis(Boc–L-Phe)-N,N-hexane-
1,6-diamine 3a: Rf (A) 0.585; mp 171—172 °C; FAB-MS (NBA) m/z 611
(MϩH)ϩ. Bis(Boc–D-Phe)-N,N-hexane-1,6-diamine 3b: 85% yield: Rf (A)
0.58; mp 172—173 °C; FAB-MS (NBA) m/z 611 (MϩH)ϩ. Bis(Boc–L-
Phe–L-Phe)-N,N-hexane-1,6-diamine 13a: Rf (A) 0.386; mp 210—212 °C;
FAB-MS (NBA) m/z 905 (MϩH)ϩ. Bis(Boc–D-Phe–L-Phe)-N,N-hexane-1,6-
diamine 13b: 87% yield; Rf (A) 0.390; mp 214—215 °C; FAB-MS (NBA)
m/z 905 (MϩH)ϩ. Bis(Boc–L-Phe–D-Phe)-N,N-hexane-1,6-diamine 13c:
80% yield; Rf (A) 0.390; mp 216—217 °C; HR-FAB-MS (NBA) Found
905.5156, Calcd for C52H68N6O8 905.5177. Bis(Boc–D-Phe–D-Phe)-N,N-
hexane-1,6-diamine 13d: 72% yield; Rf (A) 0.386; mp 211—213 °C; HR-
FAB-MS (NBA) Found 705.4133, Calcd for C52H68N6O8 705.4128
(MϩH)ϩ. Bis(Boc–L-Phe)-N,N-octane-1,8-diamine 4a: 71% yield: Rf (A)
0.654; mp 175—178 °C; FAB-MS (NBA) m/z 639 (MϩH)ϩ. Bis(Boc–D-
Phe)-N,N-octane-1,8-diamine 4b: 75% yield: Rf (A) 0.654; mp 174—
177 °C. Bis(Boc–L-Phe–L-Phe)-N,N-octane-1,8-diamine 14a: 85% yield: Rf
1
ether/CHCl3). H-NMR (signal assignments from the spectra of H–H and
H–C COSYs at 500 MHz, CDCl3/DMSO-d6 (0.5 ml/0.2 ml)) d: 1.35—1.38
(m, 4H, –CH2–ϫ2), 2.66 (dd, 2H, Jϭ8.2, 13.4 Hz, Phe–CH–ϫ2), 3.01 (dd,
2H, Jϭ4.9, 13.4 Hz, Phe–CH–ϫ2), 3.10 (m, 4H, –CH2–NHCO–ϫ2), 3.44
(dd, 2H, Jϭ4.9, 8.2 Hz, –CaH–ϫ2), 7.17—7.28 (m, 10H, Ar-Hϫ2), and
7.72 (t, 2H, Jϭ5.6 Hz, –NHCO–ϫ2). 13C-NMR (signal assignments from
the spectra of C–H COSY at 500 Mz, CDCl3/DMSO-d6 (0.5 ml/0.2 ml)) ppm
26.53 (C2), 38.18 (C1), 41.24 (Phe–CH–), 56.32 (–Ca–), 126.12 (p-), 128.09
(m-), 129.18 (o-), 138.43 (j-), and 174.13 (–NHCO–). IR (KBr) cmϪ1; 3270
and 1640. FAB-MS (NBA) m/z 383 (MϩH)ϩ.
Bis(L-Phe)-N,N-butane-1,4-diamine 7b: Rf (A) 0.056; mp 126—127 °C.
FAB-MS (NBA) m/z 383 (MϩH)ϩ.
Bis(L-Phe–L-Phe)-N,N-butane-1,4-diamine (17a) 70% yield, Rf (B)
0.41: mp 161—162 °C (from ether/CHCl3). 1H-NMR (signal assignments
from the spectra of H–H COSY at 500 MHz, CDCl3/DMSO-d6 (0.5 ml/
0.2 ml)) d: 1.33—1.36 (m, 4H, –CH2–ϫ2), 2.48 (dd, 2H, Jϭ8.85, 13.7 Hz,
Phe2–CH–ϫ2), 2.92 (dd, 2H, Jϭ7.6, 13.7 Hz, Phe1–CH–ϫ2), 2.97 (dd, 2H,
Jϭ4.3, 13.7 Hz, Phe2–CH–ϫ2), 3.03 (dd, 2H, Jϭ6.1, 13.8 Hz,
Phe1–CH–ϫ2), 3.08 (m, 2H, –CH2–NHCO–ϫ2), 3.50 (dd, 2H, Jϭ4.3,
8.85 Hz, –Ca2H–ϫ2), 4.63 (ddd, 2H, Jϭ6.1, 7.6, 8.5 Hz, –Ca1H–ϫ2),
7.12—7.28 (m, 5H, Ar-H), 7.58 (t, 2H, Jϭ5.5 Hz, –NHaCO–ϫ2), and 7.94
(d, 2H, Jϭ8.5 Hz, –NHbCO–ϫ2). 13C-NMR (signal assignments from the
spectra of C–H COSY at 500 Mz, CDCl3/DMSO-d6 (0.5ml/0.2 ml)) ppm
26.32 (C2), 38.34 (Phe–CH–, –CH2–NH–), 40.59 (Phe–CH–), 53.34
(–Ca1–), 56.09 (–Ca2–), 126.13 (p1-), 126.21 (p2-), 127.91 (o1-), 128.58 (o2-),
129.17 (m1-), 129.23 (m2-), 137.33 (j1-), 138.27 (j2-), 170.54 (–NHbCO–),
and 173.77 (–NHaCO–). IR (KBr) cmϪ1; 3270 and 1640. Anal. Calcd for
C40H48N6O4·1/2H2O: C, 70.05 H, 7.20; N, 12.25. Found: C, 70.17; H. 7.11:
N, 12.28.
Bis(D-Phe–L-Phe)-N,N-butane-1,4-diamine (17b) Compound 17b was
prepared in a similar manner to 7a and obtained as a colorless solid in 86%
1
yield: Rf (B) 0.16, mp 192—194 °C (from ether/CHCl3). H-NMR (signal
assignments from the spectra of H–H COSY at 500 MHz, CDCl3/DMSO-d6
(0.5 ml/0.2 ml)) d: 1.32—1.36 (m, 4H, –CH2–ϫ2), 2.57 (m, 2H,
Phe2–CH–ϫ2), 2.91 (dd, 2H, Jϭ7.9, 13.7 Hz, Phe1–CH–ϫ2), 3.01 (m, 2H,
Phe2–CH–ϫ2), 3.03 (dd, 2H, Jϭ6.1, 13.7 Hz, Phe1–CH–ϫ2), 3.08—3.09
(m, 4H, –CH2–NHCO–ϫ2), 3.49 (dd, 2H, Jϭ4.6, 8.2 Hz, –Ca2H–ϫ2), 4.6
(ddd, 1H, Jϭ6.4, 7.9, 8.2 Hz, –Ca1H–ϫ2), 7.15—7.19 (m, 12H, o-, p-),
7.20—7.26 (m, 8H, m-), 7.56 (t, 2H, Jϭ5.5 Hz, –NHaCO–ϫ2), and 8.01 (d,
2H, Jϭ8.2 Hz, –NHbCO–ϫ2). 13C-NMR (signal assignments from the spec-
tra of C–H COSY at 500 Mz, CDCl3/DMSO-d6 (0.5 ml/0.2 ml)) ppm 26.27
(C2), 38.23 (Phe1–CH–), 38.58 (–CH2–NH–), 40.93 (Phe2–CH–), 53.99
(–Ca1–), 56.20 (–Ca2–), 126.33 (p1-), 126.39 (p2-), 128.09 (m1-), 128.26
(m2-), 129.18 (o1-), 129.22 (o2-), 137.29 (j1-), 138.06 (j2-), 170.86
(–NHbCO–), and 173.89 (–NHaCO–). IR (KBr) cmϪ1; 3270 and 1640, FAB-
MS (NBA) m/z 677 (MϩH)ϩ.
Bis(L-Phe–D-Phe)-N,N-butane-1,4-diamine (17c) Compound 17c was
prepared in a similar manner to 17a and obtained as a colorless solid in 78%
yield: Rf (B) 0.16; mp 190—193 °C; FAB-MS (NBA) m/z 677 (MϩH)ϩ.
Bis(D-Phe–D-Phe)-N,N-butane-1,4-diamine (17d) Compound 17d was
prepared in a similar manner to 17a and obtained as a colorless solid in 70%
yield: Rf (B) 0.41, mp 160—163 °C; FAB-MS (NBA) m/z 677 (MϩH)ϩ.
Bis(L-Phe)-N,N-hexane-1,6-diamine (8a) Compound 8a was prepared
in a similar manner to 3a and obtained as a colorless solid in 65% yield: Rf
(B) 0.108, mp 122—123 °C (from ether/CHCl3). 1H-NMR (signal assign-
ments from the spectra of H–H and H–C COSYs at 500 MHz,
CDCl3/DMSO-d6 (0.5 ml/0.2 ml)) d: 1.275—1.289 (m, 2H, –CH2–), 1.452—
1.479 (m, 2H, –CH2–), 2.72 (dd, 1H, Jϭ8.85, 13.7 Hz, Phe–CH–), 3.17 (dd,
1H, Jϭ4.6, 13.7 Hz, Phe–CH–), 3.20 (m, 2H, –CH2–NHCO–), 3.57 (dd, 1H,
Jϭ4.6, 8.85 Hz, –CaH–), 7.21—7.31 (m, 5H, Ar-H), and 7.48 (m, 1H,

212
Vol. 52, No. 2
–NHCO–). 13C-NMR (signal assignments from the spectra of C–H COSY at
500 Mz, CDCl3/DMSO-d6 (0.5 ml/0.2 ml)) ppm 26.25 (C3), 29.26 (C2),
38.69 (C1), 41.04 (Phe–CH–), 56.35 (–Ca–), 126.53 (p-), 128.44 (m-),
129.28 (o-), 138.02 (j-), and 174.05 (–NHCO–). IR (KBr) cmϪ1; 3270 and
1640; FAB-MS (NBA) m/z 411 (MϩH)ϩ. Anal. Calcd for C24H34N4O2: C,
70.21; H, 8.35; N, 13.65. Found: C, 70.03; H, 8.26; N, 13.57.
Bis(D-Phe)-N,N-hexane-1,6-diamine (8b) Compound 8b was prepared
in a similar manner to 8a and obtained as a colorless solid in 80% yield: Rf
(B) 0.108; mp 121—122.5 °C; FAB-MS (NBA) m/z 411 (MϩH)ϩ.
–CH2–NHCO–ϫ2), 3.27 (dd, 2H, Jϭ3.96, 13.7 Hz, Phe–CH–ϫ2), 3.59 (dd,
2H, Jϭ3.96, 9.2 Hz, –CaH–ϫ2), 7.21—7.33 (m, 12H, 5Hϫ2, –NHCO–ϫ2).
13C-NMR (signal assignments from the spectra of C–H COSY at 500 Mz,
CDCl3/DMSO-d6 (0.5 ml/0.2 ml)) ppm 26.58 (C4), 28,90 (C3), 29.26 (C2),
38.76 (C1), 41.06 (Phe–CH–), 56.27 (–Ca–), 126.34 (p-), 128.77 (m-),
129.20 (o-), 138.02 (j-), and 173.97 (–NHCO–). IR (KBr) cmϪ1; 3270 and
1640.
Bis(D-Phe)-N,N-octane-1,8-diamine (9b) Compound 3b was prepared
in a similar manner to 3a and obtained as a colorless solid in 70% yield: Rf
(B) 0.16, mp 104—106 °C; FAB-MS (NBA) m/z 439 (MϩH)ϩ.
Bis(L-Phe–L-Phe)-N,N-octane-1,8-diamine (19a) Compound 19a was
prepared in a similar manner to 3a and obtained as a colorless solid in 70%
Bis(L-Phe–L-Phe)-N,N-hexane-1,6-diamine (18a) Compound 18a was
prepared in a similar manner to 3a and obtained as a colorless solid in 72%
1
yield: Rf (B) 0.42, mp 190—191 °C (from ether/MeOH). H-NMR (signal
1
assignments from the spectra of H–H COSY at 500 MHz, CDCl3/DMSO-d6
(0.5 ml/0.2 ml)) d: 1.19—1.23 (m, 4H, –CH2–ϫ2), 1.34—1.40 (m, 4H,
–CH2–ϫ2), 2.49 (dd, 2H, Jϭ8.85, 13.7 Hz, Phe2–CH–ϫ2), 2.93 (dd, 2H,
Jϭ7.6, 13.0 Hz, Phe1–CH–ϫ2), 2.98 (dd, 2H, Jϭ4.5, 14.0 Hz, Phe2–
CH–ϫ2), 3.03 (dd, 2H, Jϭ6.0, 14.0 Hz, Phe1–CH–ϫ2), 3.04—3.11 (m, 4H,
–CH2–NHCO–ϫ2), 3.51 (dd, 2H, Jϭ4.5, 8.85 Hz, –Ca2H–ϫ2), 4.64 (ddd,
2H, Jϭ6.0, 7.6, 8.85 Hz, –Ca1H–ϫ2), 7.14—7.17 (m, 8H, 2Hϫ4, o-),
7.18—7.28 (m, 12H, 3Hϫ4, m-, p-), 7.53 (t, 2H, Jϭ5.8 Hz, –NHaCO–ϫ2),
and 7.95 (d, 2H, Jϭ8.85 Hz, –NHbCO–ϫ2). 13C-NMR (signal assignments
from the spectra of C–H COSY at 500 Mz, CDCl3/DMSO-d6 (0.5 ml/
0.2 ml)) ppm 25.79 (C3), 28.97 (C2), 38.37 (Phe1–Cb–), 38.71 (C1), 42.67
(Phe2–Cb–), 53.81 (Phe1–Ca–), 56.17 (Phe2–Ca–), 126.52 (p1-), 126.58 (p2-),
128.17 (m1-), 128.45 (m2-), 129.20 (o1-), 129.33 (o2-), 137.12 (j1-), 137.68
(j2-), 170.82 (–NHbCO–), and 173.79 (–NHaCO–). IR (KBr) cmϪ1; 3270
and 1640. Anal. Calcd for C42H52N6O4·1/2H2O: C, 70.66; H, 7.48; N, 11.77.
Found: C, 71.19; H. 7.37; N, 11.78.
yield: Rf (A) 0.43, mp 170—172 °C (from ether/CHCl3). H-NMR (signal
assignments from the spectra of H–H COSY at 500 MHz, CDCl3/DMSO-d6
(0.5 ml/0.2 ml)) d: 1.14—1.23 (m, 8H, –CH2–CH2–ϫ2), 1.34—1.41 (m,
4H, –CH2–ϫ2), 2.53 (dd, 2H, Jϭ8.85, 13.4 Hz, Phe2–CH–ϫ2), 2.92 (dd,
2H, Jϭ7.6, 13.7 Hz, Phe1–CH–ϫ2), 2.98 (dd, 2H, Jϭ4.3, 13.4 Hz,
Phe2–CH–ϫ2), 3.03 (dd, 2H, Jϭ7.6, 13.7 Hz, Phe1–CH–ϫ2), 3.06—3.11
(m, 4H, –CH2–NHCO–ϫ2), 3.51 (dd, 2H, Jϭ4.3, 8.85 Hz, Phe2–Ca2H–ϫ2),
4.63 (ddd, 2H, Jϭ7.3, 7.6, 8.85 Hz, Phe1–Ca1H–ϫ2), 7.13—7.19 (m, 8H, o1-,
o2-, 4Hϫ2), 7.21—7.26 (m, 12H, m-, p-, 6Hϫ2), 7.54 (t, 2H, Jϭ5.5 Hz,
–NHaCO–ϫ2), 7.95 (d, 2H, Jϭ8.85 Hz, –NHbCO–ϫ2). 13C-NMR (signal
assignments from the spectra of C–H COSY at 500 Mz, CDCl3/DMSO-d6
(0.5 ml/0.2 ml)) ppm 26.49 (C4), 28.84 (C3), 29.07 (C2), 38.41 (Phe1–Cb–),
38.97 (C1), 40.63 (Phe2–Cb–), 53.52 (Phe1–Ca–), 56.17 (Phe2–Ca–), 126.35
(p1-, p2-), 128.00 (m1-), 128.28 (m2-), 129.17 (o1-), 129.29 (o2-), 137.17 (j2-),
137.97 (j1-), 170.62 (–NHbCO–) and 173.90 (–NHaCO–). IR (KBr) cmϪ1
;
3270 and 1640; Anal. Calcd for C44H48N6O4: C, 72.10; H, 7.70; N, 11.47.
Found: C, 71.80; H. 7.72: N, 11.43.
Bis(D-Phe–L-Phe)-N,N-hexane-1,6-diamine (18b) Compound 18b was
prepared in a similar manner to 18a and obtained as a colorless solid in 68%
Bis(L-Phe–D-Phe)-N,N-octane-1,8-diamine (19c) 70% yield: Rf (B)
0.31, mp 164—166 °C (from ether/CHCl3). 1H-NMR (signal assignments
from the spectra of H–H COSY at 500 MHz, CDCl3/DMSO-d6 (0.5 ml/
0.2 ml)) d: 1.14—1.26 (m, 8H, –CH2–CH2–ϫ2), 1.35—1.40 (m, 4H,
–CH2–ϫ2), 2.53 (dd, 2H, Jϭ8.85, 13.4 Hz, Phe2–CH–ϫ2), 2.86 (dd, 2H,
Jϭ7.6, 13.7 Hz, Phe1–CH–ϫ2), 2.94 (dd, 2H, Jϭ4.6, 13.4 Hz,
Phe2–CH–ϫ2), 2.98 (dd, 2H, Jϭ6.1, 13.7 Hz, Phe1–CH–ϫ2), 3.02—3.13
(m, 4H, –CH2–NHCO–ϫ2), 3.46 (dd, 1H, Jϭ4.6, 8.85 Hz, Phe2–Ca2H–ϫ2),
4.57 (ddd, 1H, Jϭ7.6, 8.5, 14.0 Hz, Phe1–Ca1H–ϫ2), 7.12—7.21 (m, 8H,
4Hϫ2, o1-, o2-), 7.17—7.18 (m, 4H, 2Hϫ2, p-), 7.21—7.24 (m, 8H, 4Hϫ2,
m-), 7.70 (t, 2H, Jϭ5.8 Hz, –NHaCO–ϫ2), and 8.03 (d, 2H, Jϭ8.5 Hz,
–NHbCO–ϫ2). 13C-NMR (signal assignments from the spectra of C–H
COSY at 500 Mz, CDCl3/DMSO-d6 (0.5 ml/0.2 ml) ppm 26.45 (C4), 28.81
(C3), 29.04 (C2), 38.34 (Phe1–Cb–), 38.81 (C1), 40.95 (Phe2–Cb–), 53.80
(Phe1–Ca–), 56.11 (Phe2–Ca–), 126.12 (p1-), 126.24 (p2-), 127.94 (m1-),
128.10 (m2-), 129.18 (o1-), 129.21 (o2-), 137.41 (j2-), 138.33 (j1-), 170.60
(–NHbCO–) and 173.89 (–NHaCO–). IR (KBr) cmϪ1; 3270 and 1650; FAB-
MS (NBA) m/z 725 (MϩH)ϩ.
1
yield: Rf (B) 0.21, mp 161—162 °C (from ether/MeOH). H-NMR (signal
assignments from the spectra of H–H COSY at 500 MHz, CDCl3/DMSO-d6
(0.5 ml/0.2 ml)) d: 1.18—1.21 (m, 4H, –CH2–ϫ2), 1.35—1.41 (m, 4H,
–CH2–ϫ2), 2.58 (dd, 2H, Jϭ8.85, 13.4 Hz, Phe2–CH–ϫ2), 2.92 (dd, 2H,
Jϭ7.9, 13.7 Hz, Phe1–CH–ϫ2), 3.03 (dd, 2H, Jϭ6.4, 13.7 Hz,
Phe1–CH–ϫ2), 3.05 (dd, 2H, Jϭ4.6, 13.4 Hz, Phe2–CH–ϫ2), 3.08—3.14
(m, 4H, –CH2–NHCO–ϫ2), 3.50 (dd, 2H, Jϭ4.6, 8.85 Hz, –Ca2H–ϫ2), 4.64
(ddd, 2H, Jϭ6.4, 7.9, 8.5 Hz, –Ca1H–ϫ2), 7.14—7.24 (m, 20H, 10Hϫ2),
7.52 (t, 2H, Jϭ5.5 Hz, –NHaCO–ϫ2), and 8.00 (d, 2H, Jϭ8.5 Hz,
–NHbCO–ϫ2). 13C-NMR (signal assignments from the spectra of C–H
COSY at 500 Mz, CDCl3/DMSO-d6 (0.5 ml/0.2 ml)) ppm 25.90 (C3), 28.95
(C2), 38.32 (Phe1–Cb–), 38.67 (C1), 40.88 (Phe2–Cb–), 53.98 (Phe1–Ca–),
56.16 (Phe2–Ca–), 126.32 (p1-), 126.38 (p2-), 128.06 (m1-), 128.25 (m2-),
129.17 (o1-), 129.24 (o2-), 137.28 (j1-), 138.04 (j2-), 170.82 (–NHbCO–),
and 173.86 (–NHaCO–). IR (KBr) cmϪ1; 3270 and 1640. Anal. Calcd for
C42H52N6O4·1/2H2O: C, 70.66; H, 7.48; N, 11.77. Found: C, 71.08; H, 7.38;
N, 11.77.
Bis(L-Phe–D-Phe)-N,N-hexane-1,6-diamine (18c) Compound 18c was
prepared in a similar manner to 18a and obtained as a colorless solid in 70%
yield: Rf (B) 0.21, mp 160—161 °C (from ether/MeOH). H-NMR (signal
Bis(D-Phe–D-Phe)-N,N-octane-1,8-diamine (19d) 72% yield: Rf (B)
0.43; mp 171—171.5 °C; FAB-MS (NBA) m/z 725 (MϩH)ϩ.
1
Bis(Dns–L-Phe)-N,N-ethane-1,2-diamine (21) Compound 6a (0.128 g,
assignments from the spectra of H–H COSY at 500 MHz, CDCl3/DMSO-d6
(0.5 ml/0.2 ml)) d: 1.18—1.23 (m, 4H, –CH2–ϫ2), 1.36—1.39 (m, 4H,
–CH2–ϫ2), 2.57 (dd, 2H, Jϭ8.5, 13.4 Hz, Phe2–CH–ϫ2), 2.92 (dd, 2H,
Jϭ7.6, 13.4 Hz, Phe1–CH–ϫ2), 3.00 (dd, 2H, Jϭ4.27, 13.4 Hz,
Phe2–CH–ϫ2), 3.03 (dd, 2H, Jϭ6.4, 13.4 Hz, Phe1–CH–ϫ2), 3.05—3.15
(m, 4H, –CH2–NHCO–ϫ2), 3.49 (dd, 2H, Jϭ4.27, 8.5 Hz, –Ca2H–ϫ2), 4.63
(ddd, 2H, Jϭ6.4, 7.6, 8.5 Hz, –Ca1H–ϫ2), 7.11—7.14 (m, 8H, 2Hϫ4, o-),
7.16—7.26 (m, 12H, 3Hϫ4, m-, p-), 7.52 (t, 2H, Jϭ5.5 Hz, –NHaCO–ϫ2),
and 8.00 (d, 2H, Jϭ8.5 Hz, –NHbCO–ϫ2). 13C-NMR (signal assignments
from the spectra of C–H COSY at 500 Mz, CDCl3/DMSO-d6
(0.5 ml/0.2 ml)) ppm 25.88 (C3), 28.95 (C2), 38.32 (Phe1–Cb–), 38.67 (C1),
40.84 (Phe2–Cb–), 54.01 (Phe1–Ca–), 56.15 (Phe2–Ca–), 126.36 (p1-),
126.40 (p2-), 128.08 (m1-), 128.28 (m2-), 129.17 (o1-), 129.24 (o2-), 137.27
(j1-), 137.98 (j2-), 170.80 (–NHbCO–), and 173.82 (–NHaCO–). IR (KBr)
cmϪ1; 3270 and 1640; FAB-MS (NBA) m/z 705 (MϩH)ϩ.
0.362 mmol) and dansyl chloride (Dns–Cl) (0.200 g, 0.742 mmol) was dis-
solved in 20 ml of acetone at room temperature and was stirred overnight.
The solution was evaporated and the residue was extracted with ethyl ac-
etate. The organic layer was washed with 5% NaHCO3 and was combined,
dried over MgSO4, filtered, and concentrated under vacuum. The residue was
purified by column chromatograhy on silica gel (100 mesh): Rf (B) 0.70, mp
114—117; HR-FAB-MS (NBA) Found 821.3163, Calcd for C44H48N6O2S2
821.3155.
CD Measurements The CD spectra of 18a—d were measured on a
JASCO J-600 spectrophotometer. Measurements were made in a 1 cm path
length in 85% methanol, circular quartz cell at ambient temperature. The
CD spectra are shown in Fig. 1 with molar ellipticity, [q], versus wave-
length, l (nm).
Fluorescence Microscopy A431 cells (3ϫ102 cells/ml) were cultured
on 12-well plates and incubated for 24 h with 20 ml of 27.5 mg/ml and
3.5 mg/ml fluorescent labeling compound 21 in DMEM containing 10% fetal
bovine serum (FBS) at 37 °C under a humidified 5% CO2 atmosphere. At the
end of the incubation, the cells were washed with PBS (Ϫ) and examined
using an Olympus IX70 fluorescence microscope. At an excitation wave-
Bis(D-Phe–D-Phe)-N,N-hexane-1,6-diamine (18d) Compound 3b was
prepared in a similar manner to 3a and obtained as a colorless solid in 75%
yield: Rf (B) 0.42, mp 193—194 °C; FAB-MS (NBA) m/z 705 (MϩH)ϩ.
Bis(L-Phe)-N,N-octane-1,8-diamine (9a) Compound 9a was prepared
in a similar manner to 3a and obtained as a colorless solid in 75% yield: Rf
length of 349 nm (lex), compound 21 emits near wavelength of 530 nm (lem
)
1
(B) 0.16; mp 105—106.5 °C (from ether/CHCl3). H-NMR (signal assign-
in 85% methanol.
ments from the spectra of H–H and H–C COSYs at 500 MHz, CDCl3) d:
1.28—1.31 (m, 8H, –CH2–CH2–ϫ2), 1.45—1.50 (m, 4H, –CH2–ϫ2), 2.69
(dd, 2H, Jϭ9.2, 13.7 Hz, Phe–CH–ϫ2), 3.21—3.24 (m, 4H,
MIC and MBC Determination The minimum concentration for inhibi-
tion (MIC, mg/ml) was determined by the two-fold broth dilution method
recommended by the Japan Society of Chemotherapy with a cation-supple-

February 2004
213
mented Mueller-Hinton broth (CSMHB, pHϭ7.4).15) A 50% dimethyl sul-
foxide (DMSO) solution of the compound was diluted with CSMHB to an
appropriate concentration. To 96-well plates was added 100 ml of the dilute
compound solution. Bacterial suspensions for inocula, prepared by diluting
overnight cultures to give a final concentration of 107 CFU/ml, and 5 ml of an
inoculum, corresponding to about 5.0ϫ104 CFU, was inoculated on the
drug-containing wells. The 96-well microplates were incubated for 24 h at
37 °C. MIC was defined as the lowest concentration of the compound that
prevented visible growth of the bacteria.
6) Raghothama S. R., Awasthi S. K., Balaram P., J. Chem. Soc., Perkin
Trans. 2, 1998, 137—143 (1998).
7) Kobayashi S., Kobayashi H., Yamaguchi T., Nishida M., Yamaguchi
K., Kurihara M., Miyata N., Tanaka A., Chem. Pharm. Bull., 48,
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Nature (London), 318, 480—487 (1985).
10) Ghose A. K., Pritchett A., Crippen G. M., J. Comput. Chem., 9, 80—
90 (1988).
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