3202
H. Konno et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3199–3202
12. Synthesis of cyclic peptide (11): To
1.60 mmol/g) in DMF (2.0 mL) were added Fmoc-
0.240 mmol) and DIPEA (27.4 L, 0.160 mmol). The mixture was stirred for 2 h
a
suspension of 2-CTC resin (50.0 mg,
200 lg/mL (entry 2). D-LAP-containing Bk-analogues (11), (13), and
D
-Asn(Trt)-OH (143.2 mg,
(15)–(17) showed moderate MIC values (entries 4–8). These D-rich
Bk-analogues seemed to have digestion resistance and conse-
quently, antifungal activities were observed. Interestingly, the lin-
l
at 25 °C. To a resultant resin was added 20% piperidine in DMF and the mixture
was stirred for 30 min at 25 °C. Fmoc-Ser(tBu)-OH (92.0 mg, 0.240 mmol),
Fmoc-Asn(Trt)-OH (143.2 mg, 0.240 mmol),
0.240 mmol), Fmoc- -Asn(Trt)-OH (143.2 mg, 0.240 mmol), Fmoc-
OH (110.3 mg, 0.240 mmol), Fmoc- -Ser(tBu)-OH (92.0 mg, 0.240 mmol) were
successively condensed to this resin using DIPC (37.2 L, 0.240 mmol)/HOBt–
H2O (36.8 mg, 0.240 mmol)/DIPEA (27.4 L, 0.160 mmol) for 2 h. Fmoc- -LAP-
OH (125.4 mg, 0.240 mmol) were successively condensed to this resin using
DIPC (49.8 L, 0.320 mmol)/HOBt–H2O (49.1 mg, 0.320 mmol)/DIPEA (54.8 L,
Fmoc-Gly-OH (71.4 mg,
ear-type peptide (36) showed an MIC value of 50 lg/mL despite no
D
D-Tyr(tBu)-
antifungal activity of linear-type peptides (29)–(35) (entry 9).
These results suggest that the sequences with stereochemistry
strictly are important factor to access the target enzyme (Table 5).
The molecular force fields for the selected Bk-type cyclic pep-
tides were calculated by SPARTAN. Stable conformers were indi-
cated and the Bk-1097 (1) and the Bk-analogue (2) with potent
antifungal activities showed similar conformational structures.
Especially, intramolecular hydrogen bonding between the hydroxy
group of Ser (position B) and amide side chain of Asn (position F)
were observed, and therefore fixed stereo-structures were formed
to show moderate antifungal activities. For this reason, we propose
that the peptide motif of the linear peptide (36) binds on the target
enzyme with a high degree of flexibility and hydrocarbon acts as
the hydrophobic anchor to the membrane. Other linear peptides
and cyclic heptapeptides showed no antifungal activities because
the sequences and conformation were not preferred. Thus, in order
to examine the potential of the linear peptides as novel agents for
pharmaceutical and agrochemical use, it seems worthwhile to
design and study simple analogues of the Bk-type peptides.
Taking into account our hypothesis and active conformation of
the target enzyme, a simplified analogue design will be attempted.
In conclusion, we synthesized 35 cyclic Bk-1097 analogues by
ordinary Fmoc-SPPS and macrolactamization and global deprotec-
tion in solution phase. However, antifungal activities of these cyclic
peptides were very weak against S. cerevisiae and A. oryzae in com-
parison with the Bk-analogue (2). In contrast, we found the linear
peptide (36) has moderate antifungal activity. For antifungal
agents, we will investigate simple structures and produce non-
peptide molecules in due course.
D
l
l
D
l
l
0.320 mmol) for 3 h. The resulting resin was treated with HFIP/CH2Cl2 (1:4,
5 mL) for 2.5 h and the mixture was filtrated. After evaporation of the HFIP,
ether was added, and the resulting precipitate was purified by preparative
HPLC (CH3CN/H2O = 89:11) to yield octapeptide (110.0 mg 0.0575 mmol, 72%)
as a white powder. To a solution of octapeptide (110.0 mg 0.0575 mmol) in
DMF (6.4 mL) were added DIPC (44.6
lL, 0.288 mmol), HOBt–H2O (44.1 mg,
0.288 mmol) and DIPEA (29.6 L, 0.173 mmol) and the mixture was stirred for
l
24 h at room temperature. After adding AcOEt and H2O, the mixture was
separated. The organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (CHCl3/MeOH = 98:2) to give the cyclic peptide as
a white powder. Cyclicpeptide (23.5 mg, 0.0123 mmol) was added to TFA/TIPS/
H2O (95:2.5:2.5; 2 mL) and the mixture was stirred for 2 h at room
temperature. After evaporation of TFA, ether was added, and the resulting
precipitate was purified by preparative HPLC (CH3CN/H2O = 48:52) to yield a
cyclicoctapeptide (11) (7.6 mg, 0.00746 mmol, 61%) as
Analytical HPLC using
a white powder.
Cosmosil 5C18 (10 Â 250 mm) column: linear
a
peptide precursor of 11: tR 18.86 min (CH3CN gradient; 70–100% in 30 min).
ESIMS Calcd 1932.04 for C114H139N12O16; found: 1932.90 for [M+H]+. Cyclic
peptide (11) with protecting groups: tR 35.31 min (CH3CN gradient; 70–100%
in 30 min and then 100% CH3CN for 10 min). ESIMS Calcd 1914.02 for
C
114H137N12O15; found: 1914.76 for [M+H]+. Cyclic peptide (11): 1H NMR
(500 MHz, DMSO-d6) d 9.16 (s, 1H), 8.41 (br s, 1H), 8.20 (d, J = 9.0 Hz, 1H), 8.10
(d, J = 7.0 Hz, 1H), 7.87 (d, J = 7.0 Hz, 1H), 7.81 (m, 2H), 7.74 (d, J = 8.5 Hz, 1H),
7.61–7.60 (m, 1H), 7.43 (s, 1H), 7.30 (s, 2H), 7.01–6.99 (m, 4H), 6.90 (s, 1H),
6.87 (s, 1H), 6.61 (d, J = 8.0 Hz, 2H), 5.10 (s, 1H), 4.94 (t, J = 6.0 Hz, 1H), 4.53–
4.41 (m, 3H), 4.25 (m, 1H), 4.15 (m, 2H), 3.83 (dd, J = 16.5, 6.0 Hz, 1H), 3.56–
3.45 (m, 5H), 3.25 (overlap, 1H), 3.00 (m, 1H), 2.91–2.90 (m, 1H), 2.76 (t,
J = 10.5 Hz, 1H), 2.70–2.54 (m, 5H), 2.35 (m, 2H), 1.35 (m, 2H), 1.19 (m, 18H),
0.82 (t, J = 6.5 Hz, 3H). tR 4.01 min (CH3CN gradient; 40–70% in 30 min).
HRESIMS calcd 1019.5162 for C45H71N12O15; found: 1019.5176 for [M+H]+.
13. Synthesis of linear peptide (36): To a suspension of 2-CTC resin (50.0 mg,
1.60 mmol/g) in DMF (2.0 mL) were added Fmoc-Asn(Trt)-OH (143.2 mg,
0.240 mmol) and DIPEA (27.4 lL, 0.160 mmol). The mixture was stirred for 2 h
at 25 °C. To a resultant resin was added 20% piperidine in DMF and the mixture
was stirred for 30 min at 25 °C. Fmoc-Ser(tBu)-OH (92.0 mg, 0.240 mmol),
Acknowledgement
Fmoc-Asn(Trt)-OH (143.2 mg, 0.240 mmol),
0.240 mmol), Fmoc- -Asn(Trt)-OH (143.2 mg, 0.240 mmol), Fmoc-
OH (110.3 mg, 0.240 mmol), Fmoc- -Ser(tBu)-OH (92.0 mg, 0.240 mmol) were
successively condensed to this resin using DIPC (37.2 L, 0.240 mmol)/HOBt–
H2O (36.8 mg, 0.240 mmol)/DIPEA (27.4 L, 0.160 mmol) for 2 h. Fmoc- -LAP-
OH (125.4 mg, 0.240 mmol) were successively condensed to this resin using
DIPC (49.8 L, 0.320 mmol)/HOBt–H2O (49.1 mg, 0.320 mmol)/DIPEA (54.8 L,
Fmoc-Gly-OH (71.4 mg,
This work was supported in part by Yamagata University
Research Fund.
D
D-Tyr(tBu)-
D
l
l
L
Supplementary data
l
l
0.320 mmol) for 8 h. The resulting resin was treated with HFIP/CH2Cl2 (1:4,
5 mL) for 2.5 h and the mixture was filtrated. After evaporation of the HFIP,
ether was added, and the resulting precipitate was purified by preparative
HPLC (CH3CN/H2O = 88:12) to yield octapeptide (90.9 mg 0.0470 mmol, 59%)
as white powder. Octapeptide (90.9 mg, 0.0470 mmol) was added to TFA/TIPS/
H2O (95:2.5:2.5; 2 mL) and the mixture was stirred for 2 h at room
temperature. After evaporation of TFA, ether was added, and the resulting
precipitate was purified by preparative HPLC (CH3CN/H2O = 48.0:52.0) to yield
a linear octapeptide (36) (5.9 mg, 0.00569 mmol, 33%) as a white powder.
Analytical HPLC using a Cosmosil 5C18 (10 Â 250 mm) column: Linear peptide
(36) with protecting groups: tR 11.50 min (CH3CN gradient; 80–100% in
30 min). ESIMS Calcd 1932.04 for C114H139N12O16; found: 1932.63 for [M+H]+.
Linear peptide (36): 1H NMR (500 MHz, DMSO-d6) d 9.14 (s, 1H), 8.29 (d,
J = 7.0 Hz, 1H), 8.20 (t, J = 5.5 Hz, 1H), 8.14 (t, J = 8.0 Hz, 1H), 8.04 (d, J = 8.0 Hz,
1H), 8.00 (d, J = 8.0 Hz, 1H), 7.94 (t, J = 5.5 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.41
(s, 1H), 7.33 (s, 1H), 7.30 (s, 1H), 6.98 (d, J = 8.5 Hz, 2H), 6.94 (s, 1H), 6.90 (s,
1H), 6.88 (s, 1H), 6.59 (d, J = 9.0 Hz, 2H), 4.57 (q, J = 7.5 Hz, 1H), 4.49 (q,
J = 7.5 Hz, 1H), 4.42 (q, J = 7.5 Hz, 1H), 4.37 (ddd, J = 6.0, 5.5, 5.0 Hz, 1H), 4.31 (q,
J = 6.0 Hz, 1H), 4.22 (dt, J = 7.5, 5.0 Hz, 1H), 3.89–3.92 (m, 1H), 3.74 (dd, J = 16.5,
6.0 Hz, 1H), 3.59 (dd, J = 16.5, 5.0 Hz, 1H), 3.56 (d, J = 5.5 Hz, 2H), 3.48–3.51 (m,
2H), 3.15 (overlap, 1H) 3.06–3.01 (m, 2H), 2.93–2.89 (m, 1H), 2.68–2.63 (m,
2H), 2.60–2.51 (m, 4H), 2.44–2.38 (m, 2H), 1.35 (m, 2H), 1.24–1.20 (m, 18H),
0.82 (t, J = 7.0 Hz, 3H). tR 5.36 min (CH3CN gradient; 35–60% in 30 min).
HRESIMS calcd 1037.5268 for C45H73N12O16; found: 1037.5240 for [M+H]+.
Supplementary data associated with this article can be found, in
References and notes
Konno, Hiroyuki
