Total synthesis of quinaldopeptin and its analogues

Article abstract of DOI:10.1021/jo402267r

The first total synthesis of quinaldopeptin (1) was accomplished. Our approach to the synthesis of 1 includes the solid-phase peptide synthesis of the linear decapeptide 4 followed by macrocyclization and introduction of the quinoline chromophores 2 at a late stage of the synthesis. As for the preparation of 4, a fragment coupling approach was applied considering the C2 symmetrical structure of 1. Chromophore analogues 22 and 23 and desmethyl analogue 27 were also prepared in a manner similar to the synthesis of 1. Synthetic 1 exhibits a strong cytotoxicity with the IC₅₀ value of 3.2 nM. On the other hand, the activity of 23 and 27 was largely reduced.

Full text of DOI:10.1021/jo402267r

Article
pubs.acs.org/joc
Total Synthesis of Quinaldopeptin and Its Analogues
Satoshi Ichikawa,* Takuya Okamura, and Akira Matsuda
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
S
* Supporting Information
ABSTRACT: The first total synthesis of quinaldopeptin (1) was accomplished. Our
approach to the synthesis of 1 includes the solid-phase peptide synthesis of the linear
decapeptide 4 followed by macrocyclization and introduction of the quinoline
chromophores 2 at a late stage of the synthesis. As for the preparation of 4, a fragment
coupling approach was applied considering the C2 symmetrical structure of 1.
Chromophore analogues 22 and 23 and desmethyl analogue 27 were also prepared in a
manner similar to the synthesis of 1. Synthetic 1 exhibits a strong cytotoxicity with the
IC50 value of 3.2 nM. On the other hand, the activity of 23 and 27 was largely reduced.
1 and other C2-symmetric cyclic decapeptide natural products.
Total synthesis of sandramycin,6 luzopeptines,7 and quinox-
apeptines8 as well as its analogues9 has been accomplished via a
sequential peptide coupling approach in a solution phase. For
the synthesis of 1, we planned to apply solid-phase peptide
synthesis (SPPS).11 The strategy is applicable to parallel
synthesis which would enable access to a range of analogues. A
proposed retrosynthetic analysis of 1 is shown in Scheme 1.
Our approach to the synthesis of 1 includes the SPPS of the
linear decapeptide 4 followed by macrocyclization and the
introduction of the quinoline chromophores 2 at a late stage.
The macrocyclization site of 3 was planned at the less sterically
hindered Gly residue (as the N-terminus) and L-Pip (as the C-
terminus) that are derived from the linear decapeptide
precursor 4. A fragment coupling approach11d−f was applied
for the synthesis of 4, considering the C2 symmetrical structure
of 1. As a synthetic strategy, the pentapeptide 5 was assembled
on a solid support. 2-Chlorotrityl (2CT)12 was used as the solid
support. A part of the resin-bound pentapeptide 5 was cleaved
from the resin to give the pentapeptide carboxylic acid 7, and
the remainder was deprotected to give the amine 6. A coupling
reaction between 7 and the resin-bound amine 6 followed by
cleavage from the resin would yield the linear decapeptide 4.
Generally, the SPPS requires an excess amount (4−5 equiv) of
N-protected amino acid derivatives in coupling steps. In
addition, it is difficult to recover the excess or unused amino
acid, because the amino acid would convert to the active ester
in the reaction mixture. This is a severe drawback especially in
the case when the target peptide contains precious non-
proteinogenic amino acids that would require a number of steps
for the preparation. However, the unreacted amino acids from
INTRODUCTION
■
A class of C2-symmetric cyclic decadepsipeptides such as
sandramycin,1 luzopeptines,2 and quinoxapeptines3 are known
to exhibit a variety of promising biological properties including
antitumor, antibacterial, and antiviral activities (Figure 1). They
can bind to double-stranded DNA by the phenomenon called
bisintercalation.4 It is presumed that the binding of this class of
natural products to a gene suppresses the aberrant activation
and expression of the corresponding cis-element by inhibiting
the binding of transcription factors to the cis-element.
Quinaldopeptin (1),5 which was first isolated from the culture
broth of Streptoverticillium album (actinomycetes strain Q132-
6), constitutes one of the members of this class of natural
products. Quinaldopeptin has a strong activity against
melanoma B16 and Moser cells in vitro with IC50 values of
0.6 and 32 nM, respectively. Furthermore, in vivo antitumor
activity against lymphocytic leukemia P388 in mice is
promising, indicating that 1 can exhibit greater potency than
the chemotherapeutic agent, mitomycin C. Therefore, 1 could
be a potential candidate for therapeutic use in anticancer
chemotherapies. Although DNA binding properties as well as
the structure−activity relationship has been extensively
elucidated for sandramycin, luzopeptines, and quinoxapeptines
by Boger’s group,6,7a,b,8,9 those for 1 are completely unknown.
In order to investigate the structure−activity relationship of 1, it
is necessary to establish its synthetic strategy first. Herein, we
describe the first total synthesis of 1.
RESULTS AND DISCUSSION
■
Quinaldopeptin consists of glycin (Gly), sarcosin (Sar), and
two nonproteinogenic amino acids, L-pipecolic acid (L-Pip)10
and (2R,3R)-2,3-diaminobutanoic acid (Dab) residues, and all
of the amino acid components of 1 are linked with amide
bonds, which is one of the major structural differences between
Received: October 12, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jo402267r | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Figure 1. Structure of cyclic decapeptide bisintercalator natural products.
Scheme 1. Retrosynthetic Analysis of Quinaldopeptin (1)
the first step of the SPPS, which is the immobilization of the
substrate to the 2CT chloride resin, could be recovered because
the reaction is simply an alkylation of the carboxylate ion.
Therefore, we strategically immobilize the dipeptide containing
the Dab to yield 8, followed by sequential coupling of Fmoc
protected amino acids 913 and 10. Based on this strategy, we
synthesized a suitably protected Dab residue 18 as the starting
unit for the synthesis of 1 by the SPPS approach (Scheme 2).
To synthesize 18, tert-butyl crotonate (11) was converted to
the known α-azido-β-aminobutanoate derivative 12.14 The
azide group of 12 was reduced by a Staudinger reaction, and the
liberated amine was protected with a Boc group to give 13.
After the exchange of the Cbz group of 13 to the Fmoc,
deprotection of both the Boc and tert-butyl group of 14 with
80% aq. TFA followed by the protection of the α-amino group
by the Cbz group yielded the suitably protected α,β-
diaminobutylic acid 15. Compound 15 was then coupled
with the allyl ester of L-Pip 1615 by O-(7-azabenzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and
i-Pr2NEt to afford the dipeptide 17 in 73% yield. Deprotection
of the allyl group afforded the desired starting unit dipeptide
18. With the dipeptide 18 in hand, the total synthesis of 1 was
B
dx.doi.org/10.1021/jo402267r | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
was coupled with 7 by HBTU to yield the decapeptide on the
resin 20. Deprotection of the Fmoc group by piperidine and
the release from the resin by 5% TFA/CH2Cl2 afforded the
crude decapeptide 4, a precursor to the cyclization. The
cyclization of 4 was conducted with 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxy-
7-azabenzotriazole (HOAt), and NaHCO3 in CH2Cl2 (5 mM)
at rt. However, only a trace amount of the desired cyclic
peptide 3 was obtained even after a prolonged reaction time (a
week). Despite extensive efforts to obtain 3 with a variety of
conditions using various coupling reagents, additives, solvents,
or temperature, it was difficult to increase the reaction rate. A
prolonged reaction time caused a severe racemization at the C-
terminal Pip residue. The conditions using diphenylphosphoryl
azide (DPPA)16 and NaHCO3 in DMF (5 mM) for 6 days gave
the desired cyclic peptide 3 (18%) as well as its epimer (ca.
27%). The stereogenic center at the Pip residue of 3 was
confirmed by conventional amino acid analysis17 as shown in
Scheme 4. For the analysis, 3 was heated under reflux in 6 M
aq. HCl for 24 h, and the resulting mixture was treated with
Marfey’s reagent. The reaction mixture was analyzed by
reversed phase HPLC (ODS, 10−60% MeCN−H2O linear
gradient containing 0.1% TFA) and compared with a reference
sample 21 derived from L- or D-Pip18 (Figure S1 in Supporting
Information). The arylamine 21 obtained from the hydrolysis
products of 3 was identical to the reference L-21, whereas the D-
21 analogue was not detected. This analysis unambiguously
determined the stereochemistry at the stereogenic center of
four L-Pip residues. Finally, the Cbz groups of 3 were removed,
Scheme 2. Preparation of Dipeptide 18
pursued (Scheme 3). Compound 18 was immobilized to the
2CT chloride resin (19, 1.57 mmol/g resin). Elongation of the
L-Pip and Gly-Sar residues was achieved by a conventional
Fmoc-SPPS with 9 and 10 to give solid-supported pentapeptide
5. Five-sevenths of 5 was treated with 5% TFA/CH2Cl2 to give
the crude carboxylic acid 7. The Fmoc group of the remaining
two-sevenths of 5 was deprotected to give the amine 6, which
Scheme 3. Total Synthesis of Quinaldopeptin (1) and Its Analogues
C
dx.doi.org/10.1021/jo402267r | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Scheme 4. Amino Acid Analysis of 3 and 26
Table 1. Cytotoxic Activity of 1 and Its Analogues against
Human Cancer Cell Lines
a
IC50 (nM)
HCT-
118
SU-
SU-
RPMI8226
A431
12
RKO
DHL6
DHL10
1
3.2
41
11
19
5
42
11
12
91
22
23
27
42
>10 000
457
51
>10 000
213
8400
279
>10 000
171
9683
264
>10 000
307
a
HCT-118 and RKO: human colon cancer cells. A431: human
epidermal cancer cells. RPMI8226: human myeloma cells. SU-DHL6
and SU-DHL10: human diffuse large B-cell lymphoma cells.
and quinaldic acid derivative 219 was attached to the liberated
amines to successfully afford the quinaldopeptin (1) in 43%
over 2 steps. It was reported that natural 1 did not give clear
5
1
signals in H and 13C NMR spectra. Those of synthetic 1 as
well as 3 also exhibited complex and broadened NMR spectra
despite our efforts to obtain clear spectra by changing the
group at the chromophore moiety, retained cytotoxicity
although it reduced the activity by a factor of 1.7−13. On the
other hand, 3-quinoline analogue 23, which is an isomer of 22
at the chromophore moiety, almost lost the cytotoxicity. The
solution conformation of 23 was single and rigid in CD3OD
and was clearly different from that of 1 and 22. Presumably the
solution conformation of 23 would be disfavored in DNA
binding to nearly result in loss of function. The activity of 27
was also reduced by approximately 2 orders of magnitude
(171−457 nM). Although data are not shown, the analogues of
27, where two L-Pip residues on either the N- or C-terminal of
the diaminopropanoic acid residues or all the L-Pip residues
were replaced by L-Pro residues, were also synthesized as in
Scheme 5. The cytotoxicity of these analogues were largely
reduced (IC50 2000−20 000 nM). Truncation of the methyl
groups or ring contraction caused a conformational change of
the macrocycle, which would not be preferred in DNA binding.
The DNA binding property and structural analysis of 1 will be
reported elsewhere soon.
1
solvents and temperature. However importantly, the H NMR
spectrum of synthetic 1 as well as optical rotation [synthetic
[α]2D2 −120.8 (c 0.27, MeOH); natural [α]2D0 −129.3 (c 0.19,
MeOH)] and HPLC analysis (SiO2, MeOH/CHCl3 0−10%
linear gradient, 14.34 min for natural and synthetic 1,
respectively) as well as coinjection in HPLC of synthetic
material with natural 1 proved that synthetic 1 was identical to
natural 1 (Figures S2 and S3 in Supporting Information).
Similar to the synthesis of 1, two chromophore analogues 22
and 23 were also prepared. In contrast to 1 and 22, 23 gave
clear signals in 1H and 13C NMR spectra, which indicate that it
adopts a single and rigid solution conformation. The C2-
symmetrical cyclic structure was confirmed by the fact that only
a half of the peaks could be observed in both 1H and 13C NMR
spectra. In conjunction with amino acid and HPLC analyses as
well as the biological data (described later), the structure of 1
and that of the key intermediate 3 also were unambiguously
confirmed as shown in Scheme 3.
Scheme 5. Synthesis of Desmethyl Analogue 27
As a preliminary study, the synthesis of desmethyl analogue
27 was planned using a readily available diaminopropanoic acid
derivative 25 by following a strategy similar to that for the
synthesis of 1 in order to see the impact of the methyl groups at
the Dab residues on the biological activity. As for the
cyclization step to prepare 26, the conditions using EDCI,
HOBt, and NaHCO3 and CH2Cl2 (5 mM) gave a better result
(30%) than in the case of the synthesis of 3. However, the
corresponding epimer was also obtained (ca. 21%) at the L-Pip
residue. The 1H NMR spectra of 27 also exhibited more
complex and broadened NMR spectra than those of 1, and
efforts to obtain clear spectra were unsuccessful similar to the
case of 1 and 3. Due to the presence of multiple conformers, we
were unable to obtain the 13C NMR spectrum of the
compound, and an attempt to obtain the spectrum with a
concentrated solution only caused the precipitation of the
excess compound. It was confirmed by the amino acid analysis
that 26 contained only L-Pip residues and no epimers were
contaminated (Scheme 4, Figure S1g). This indicated that 27
existed as a mixture of multiple conformers including
asymmetric ones in the solution.
The cytotoxicity of 1, 22, 23, and 27 was tested against a
range of human cancer cell lines (Table 1). Synthetic 1
exhibited strong cytotoxicity with IC50 values ranging from 3.2
to 12 nM. The observed strong cytotoxicity also confirmed the
accomplishment of the total synthesis of 1. 2-Quinoline
derivative 22, which is an analogue of 1 with no hydroxyl
D
dx.doi.org/10.1021/jo402267r | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
× 2), 1.01 (d, 3H, γ-CH3, J = 8.6 Hz); 13C NMR (DMSO-d6, 100
MHz) δ 170.0, 155.6, 155.3, 137.1, 128.3, 127.8, 127.7, 80.5, 78.3,
65.2, 58.3, 47.3, 28.2, 27.6, 16.8; ESIMS-LR m/z 431 [(M + Na)+];
ESIMS-HR calcd for C21H32N2NaO6 431.2153, found 431.2154.
(2R,3R)-tert-Butyl 3-[(fluorenylmethoxycarbonyl)amino]-2-
[(tert-butoxycarbonyl)amino] Butanoate (14). A mixture of 13
CONCLUSION
■
The first total synthesis of quinaldopeptin (1) as well as its
analogues was accomplished employing the SPPS approach.
Synthetic 1 exhibited strong cytotoxicity against a range of
human cancer cell lines. On the other hand, the activity of 23
and 27 was largely reduced. Further studies on its structure−
activity relationship will be pursued and discussed soon.
EXPERIMENTAL SECTION
■
General Experimental Methods. 1H and 13C NMR chemical
shifts were reported in parts per million (δ) relative to
tetramethylsilane (0.00 ppm) as an internal standard unless otherwise
noted. Coupling constants (J) were reported in herz (Hz).
Abbreviations of multiplicity were as follows; s: singlet, d: doublet, t:
triplet, q: quartet, m: multiplet, br: broad. Data were presented as
follows: chemical shift (multiplicity, integration, coupling constant).
(2.42 g, 5.92 mmol) and 10% Pd/C (242 mg) in MeOH (30 mL) was
vigorously stirred under a hydrogen atmosphere at room temperature
for 30 min. The insoluble portion was filtered off through a Celite pad,
and the filtrate was concentrated in vacuo to give a crude amine (1.62
g), which was used in the next reaction without further purification. To
a stirred solution of the resulting amine in THF/H2O (30/10 mL) was
added NaHCO3 (1.09 g, 13.0 mmol) and FmocCl (1.68 g, 6.51
mmol). The mixture was stirred at room temperature for 1 h. The
reaction mixture was poured into H2O (200 mL) and extracted with
AcOEt. The combined organic phase was washed with H2O and
saturated aq. NaCl, dried (Na2SO4), filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatography
1
Assignment was based on H−1H COSY, HMBC, and HMQC NMR
spectra. The mass analyzer type used for the HRMS measurements
was TOF. Analytical thin layer chromatography (TLC) was performed
on Merck silica gel 60F254 plates. Normal-phase column chromatog-
raphy was performed on Merck silica gel 5715 or Kanto Chemical
silica gel 60N (neutral). Flash column chromatography was performed
on Merck silica gel 60. All reactions except that carried out in aqueous
phase were carried out under an argon atmosphere, unless otherwise
noted. Isolated yields were calculated by weighing products. The
weight of the starting materials and the products were not calibrated.20
6-[(9-Fluorenylmethoxycarbonyl)amino]glycylsarcocine (9).
A solution of glycylsarcosine (2.48 g, 17.0 mmol) and NaHCO3 (3.14
(SiO2, 29% AcOEt−hexane) to afford 14 (2.73 g, 93% over 2 steps) as
1
a white foam. [α]23 −15.9 (c 0.89, CHCl3); H NMR (DMSO-d6,
D
400 MHz) δ 7.89−7.29 (m, 9H, fluorenyl and NH), 7.06 (d, 1H, NH,
J = 10.3 Hz), 4.29−4.17 (m, 3H, OCH2-fluorenyl and H-fluorenyl),
3.99 (t, 1H, α-CH, J = 9.8 Hz), 3.88 (m, 1H, β-CH), 1.35 (s × 2, 18H,
tBu × 2), 1.02 (d, 3H, γ-CH3, J = 8.6 Hz); 13C NMR (DMSO-d6, 100
MHz) δ 170.0, 155.7, 155.3, 143.8, 140.7, 127.6, 127.1, 125.2, 120.1,
80.5, 78.3, 65.5, 58.4, 47.2, 46.7, 28.2, 27.6, 16.7; ESIMS-LR m/z 418
[(M + Na)+]; ESIMS-HR calcd for C28H36N2NaO6 519.2466, found
519.2466.
(2R,3R)-3-[(9-Fluorenylmethoxycarbonyl)amino]-2-[(benzyl-
oxycarbonyl)amino] Butanoic Acid (15). Compound 14 (2.73 g,
g, 37.4 mmol) in dioxane/H2O (125/125 mL) was treated with
FmocCl (4.84 g, 18.7 mmol) at 0 °C, and the mixture was stirred at
room temperature for 3 h. The mixture was diluted with AcOEt, and
the organic phase was washed with 1 M aq. HCl, and saturated aq.
NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue
was triturated from hexane to afford 9 (5.87 g, 94%) as a white solid.
1H NMR (DMSO-d6, 500 MHz, 2:1 mixture of rotamers, data for the
major rotamer) δ 7.25 (m, 9H, fluorenyl and NH), 4.27−4.20 (m, 3H,
OCH2-fluorenyl and H-fluorenyl), 3.98 (s, 2H, Gly-α-CH2), 3.89 (d,
2H, Sar-α-CH2, J = 6.3 Hz), 2.98 (s, 3H, NCH3); 13C NMR (DMSO-
d6, 125 MHz, 2:1 mixture of rotamers, data for the major rotamer) δ
170.7, 169.2, 156.5, 143.9, 140.7, 127.7, 127.1, 125.3, 120.1, 99.5, 65.7,
49.2, 46.6, 34.9; ESIMS-LR m/z 391 [(M + Na)+]; ESIMS-HR calcd
for C20H20N2NaO5 391.1270, found 391.1261.
5.51 mmol) was treated with 80% aq. TFA (40 mL), and the resulting
mixture was stirred at room temperature for 4 h. The mixture was
concentrated in vacuo. A mixture of the residue and NaHCO3 (2.31 g,
27.5 mmol) in THF/H2O (40/10 mL) was treated with benzyl
chloroformate (1.58 mL, 11.2 mmol) at 0 °C, and the resulting
mixture was stirred at room temperature for 12 h. The mixture was
diluted with AcOEt, and the organic phase was washed with H2O and
saturated aq. NaCl, dried (Na2SO4), filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatography
(SiO2, 5% MeOH−CHCl3) to afford 15 (2.12 g, 81% over 2 steps) as
a white solid. [α]21D −7.9 (c 0.91, DMSO); 1H NMR (DMSO-d6, 500
MHz) δ 7.86−7.26 (m, 13H, aromatic), 7.19 (s, 1H, NH), 6.85 (s, 1H,
NH), 4.98 (q, 2H, CH2Ph, J = 12.1, 13.2 Hz), 4.28−3.99 (m, 5H,
OCH2-fluorenyl, H-fluorenyl, α-CH and β-CH), 0.96 (d, 3H, γ-CH3, J
= 6.9 Hz); 13C NMR (DMSO-d6, 125 MHz) δ 156.4, 155.3, 144.1,
143.8, 140.7, 137.0, 128.4, 127.8, 127.7, 127.6, 127.1, 127.0, 125.5,
125.3, 120.1, 65.5, 58.7, 48.6, 46.7, 15.9; ESIMS-LR m/z 497 [(M +
Na)+]; ESIMS-HR calcd for C27H26N2NaO6 497.1689, found
497.1678.
(2R,3R)-tert-Butyl 3-[(benzyloxycarbonyl)amino]-2-[(tert-
butoxycarbonyl)amino] Butanoate (13). A solution of 1213
(2.49 g, 7.47 mmol) in THF/H2O (50/20 mL) was treated with
PPh3 (5.88 g, 22.4 mmol) at room temperature, and the mixture was
stirred at 40 °C for 2 h. The resulting mixture was treated with
(Boc)2O (1.81 mL, 7.84 mmol) and stirred at room temperature for
24 h. The mixture was diluted with AcOEt, which was washed with
H2O and saturated aq. NaCl, dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by silica gel column
chromatography (SiO2, 14% AcOEt−hexane) to afford 13 (2.42 g,
Allyl L-Pipecolate Hydrochloride (16). A solution of Boc-L-Pip-
OH (2.29 g, 10.0 mmol) and CsCO3 (3.58 g, 11.0 mmol) in DMF (50
80%) as a colorless oil. [α]23 −1.5 (c 0.59, CHCl3); 1H NMR
D
(DMSO-d6, 400 MHz) δ 7.36−7.25 (m, 6H, aromatic and NH), 7.05
(d, 1H, NH, J = 10.9 Hz), 4.98 (q, 2H, CH2Ph, J = 8.6, 16.0 Hz), 4.00
(t, 1H, α-CH, J = 9.7 Hz), 3.88 (m, 1H, β-CH), 1.36 (s × 2, 18H, tBu
E
dx.doi.org/10.1021/jo402267r | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
mL) was treated with allyl bromide (930 μL, 11.0 mmol) at 0 °C, and
the mixture was stirred at room temperature for 1 h. The mixture was
diluted with AcOEt, and the organic phase was washed with H2O (5
times) and saturated aq. NaCl, dried (Na2SO4), filtered, and
concentrated in vacuo. The compound was used in the next reaction
without further purification. The Boc-L-Pip-OAll was treated with 4 M
HCl in AcOEt for 30 min. The resulting solution was concentrated in
3H, Dab-γ-CH3, J = 6.9 Hz); 13C NMR (CD3OD, 125 MHz) δ 173.8,
171.9, 158.4, 158.3, 145.7, 145.1, 142.6, 142.5, 138.0, 129.4, 129.0,
128.9, 128.8, 128.7, 128.1, 128.0, 126.5, 126.2, 120.9, 67.9, 67.8, 55.0,
53.9, 50.1, 44.7, 27.9, 26.6, 22.0, 14.6; ESIMS-LR m/z 608 [(M +
Na)+]; ESIMS-HR calcd for C33H35N3NaO7 608.2373, found
608.2365.
Cyclo[Gly-Sar-L-Pip-Dab(Cbz)-L-Pip]2 (3). Solid-phase peptide
syntheses were performed in polypropylene syringes (10 mL) fitted
vacuo to afford 16 (2.01 g, 98% over 2 steps) as a white solid. [α]21
D
1
−12.1 (c 1.06, MeOH); H NMR (DMSO-d6, 500 MHz) δ 5.90 (m,
1H, CH2CHCH2O), 5.37 (dd, 1H, Ha, Jgem = 1.7 Hz, Jcis = 17.2 Hz),
5.23 (dd, 1H, Hb, Jgem = 1.7 Hz, Jtrans = 10.9 Hz), 4.66 (m, 2H, CH2
CHCH2O), 4.09 (d, 1H, α-CH, J = 8.0 Hz), 3.18 (d, 1H, ε-CHex, J =
12.6 Hz), 2.87 (m, 1H, ε-CHax), 2.05 (d, 1H, β-CHex, J = 10.3 Hz),
1.19 (m, 4H, γ-CH2 and δ-CH2), 1.56 (m, 1H, β-CHax); 13C NMR
(DMSO-d6, 125 MHz) δ 168.4, 131.8, 118.4, 65.8, 55.4, 43.3, 25.6,
21.2, 21.0; ESIMS-LR m/z 170 [(M + H)+]; ESIMS-HR calcd for
C9H16NO2 170.1181, found 170.1176.
Fmoc-Dab(Cbz)-L-Pip-OAll (17). A solution of 15 (20.0 mg,
i
0.042 mmol) and Pr2NEt (44 μL, 0.25 mmol) in DMF (1 mL) was
with polyethylene porous discs. Solvents and soluble reagents were
removed by suction. The Fmoc group was removed with piperidine/
DMF (1:4, 3 min, then 1:8, 12 min). Washings between deprotection,
couplings, and final deprotection steps were performed with DMF (7
mL × 3) and CH2Cl2 (7 mL × 3). Peptide synthesis transformations
and washes were performed at 25 °C. The 2CT chloride resin (19, 500
mg, 1.57 mmol/g) was placed in a 10 mL polypropylene syringe fitted
with a polyethylene filter disc. The resin was then washed with CH2Cl2
(7 mL × 3 min), and a solution of Fmoc-Dab(Cbz)-L-Pip-OH (18,
treated with HATU (32 mg, 0.084 mmol) at 0 °C, and the mixture was
stirred at the same temperature for 30 min. The amine 16 (13 mg,
0.063 mmol) was then added to the mixture, which was stirred at
room temperature for 2 h. The mixture was diluted with AcOEt, which
was washed with 1 M aq. HCl, saturated aq. NaHCO3, and saturated
aq. NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (SiO2, 20%
i
552 mg, 0.94 mmol) and Pr2NEt (575 μL, 4.8 equiv) in CH2Cl2 (5
mL) was then added. The mixture was stirred at 25 °C for 1 h. The
Fmoc-Dab(Cbz)-L-Pip-O-2CT resin 8 was subjected to the following
washings/treatments: filtration, CH2Cl2−MeOH−iPr2NEt (17:2:1, 7
mL × 3), DMF (7 mL × 3), and CH2Cl2 (7 mL × 3). The resin 8 was
dried over P2O5 overnight. The loading, calculated by measuring
absorbance at 290 nm, was 0.834 mmol/g.
AcOEt−hexane) to afford 17 (19.1 mg, 73%) as a white foam. [α]21
D
1
−40.7 (c 0.25, MeOH); H NMR (CD3OD, 500 MHz, a mixture of
rotamers, data for the major rotamer) δ 7.77−7.15 (m, 13H, fluorenyl
and Ph), 5.99 (m, 1H, CH2CHCH2O), 5.28 (dd, 1H, Ha, Jgem = 1.7
Hz, Jcis = 17.2 Hz), 5.18 (dd, 1H, Hb, Jgem = 1.7 Hz, Jtrans = 10.9 Hz),
5.16 (br s, 1H, Pip-α-CH), 5.05 (s, 2H, CH2Ph), 4.59 (m, 2H, CH2
CHCH2O), 4.45 (dd, 1H, OCH-fluorenyl, J = 6.3, 10.3 Hz), 4.33 (m,
1H, Pip-ε-CH), 4.21 (m, 2H, OCH-fluorenyl, Pip-ε-CH), 3.89 (m, 1H,
Pip-β-CH), 2.24 (d, 1H, CH-fluorenyl, J = 13.1 Hz), 1.74−1.27 (m,
6H, Pip-(CH2)3), 1.09 (d, 3H, Dab-γ-CH3, J = 6.9 Hz); 13C NMR
(CD3OD, 125 MHz) δ 172.2, 171.7, 158.4, 158.2, 145.7, 145.1, 142.6,
138.0, 133.2, 129.4, 130.0, 128.9, 128.7, 128.1, 128.0, 126.5, 126.2,
121.0, 118.6, 67.8, 66.8, 55.0, 54.1, 50.0, 44.8, 27.7, 26.4, 21.8, 14.7;
ESIMS-LR m/z 648 [(M + Na)+]; ESIMS-HR calcd for
C36H39N3NaO7 648.2686, found 648.2676.
The elongation of the peptide was achieved by sequential addition
of the corresponding Fmoc-protected amino acid 9 and 10 with either
HBTU (for the primary amine, 775 mg, 4.9 equiv) or HATU (for the
secondary amine, 777 mg, 4.9 equiv) and iPr2NEt (436 μL, 6.0 equiv)
as coupling reagents in DMF (3 mL) in preactivation mode. The
mixture was stirred for 1 h, and after filtration the corresponding
colorimetric test (Kaiser test for the primary amine and chloranil test
for the secondary amine) indicated the completion of the coupling.
Next, the peptide resin was washed with DMF (7 mL × 3) and
CH2Cl2 (7 mL × 3) and treated with a solution of piperidine/DMF
(1:4, 3 min, then 1:8, 12 min) to remove the Fmoc group, and the
coupling plus Fmoc removal was then measured. The peptide resin
was split into fractions (5/7 and 2/7). The former was cleaved from
the resin with 5% TFA in CH2Cl2 solution (5 mL × 3), and the
combined filtrates were concentrated in vacuo. The residue was
precipitated in ether to afford the crude pentapeptide 7 (867 mg) as a
pale yellow solid (ESIMS-LR (negative mode) m/z 823 [(M − H)−];
ESIMS-HR (negative mode) calcd for C44H51N6O10 823.3672, found
823.3685).
Fmoc-Dab(Cbz)-L-Pip-OH (18). A solution of 17 (100 mg, 0.16
mmol) and morpholine (41.5 μL, 0.48 mmol) in THF (2 mL) was
The remaining 2/7 fraction of 5 was treated with a piperidine/DMF
(1:4, 3 min, 1:8, 12 min) solution as described above to give the free
amine 6. The pentapeptide 7 was added to the free amine peptide
i
resin fraction, with HBTU (193 mg, 2.8 equiv) and Pr2NEt (554 μL,
treated with Pd(PPh3)4 (18.5 mg, 0.016 mmol) at room temperature
for 1 h. The mixture was diluted with AcOEt, which was washed with 1
M aq. HCl and saturated aq. NaCl, dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by silica gel column
6.0 equiv) as coupling reagents in DMF (3 mL) in preactivation mode.
The mixture was stirred at room temperature for 2 h. Then, the
peptide resin was washed with DMF (7 mL × 3) and CH2Cl2 (7 mL ×
3) and treated with a solution of piperidine/DMF (1:4, 3 min, then
1:8, 12 min). The resin 20 was treated with 5% TFA in CH2Cl2
solution (5 mL × 3), and the combined filtrates were concentrated in
vacuo to afford the crude decapeptide 4 (582 mg) as a pale yellow solid
(ESIMS-LR m/z 1187 [(M + H)+]; ESIMS-HR calcd for
C58H83N12O15 1187.6101, found 1187.6101).
chromatography (SiO2 and SH silica, 50% AcOEt−hexane) to afford 9
1
(67 mg, 72%) as a yellow foam. [α]21 −37.1 (c 0.78, MeOH); H
D
NMR (CD3OD, 500 MHz, a mixture of rotamers, data for the major
rotamer) δ 7.77−7.15 (m, 13H, fluorenyl and Ph), 5.16 (br s, 1H, Pip-
α-CH), 5.05 (s, 2H, CH2Ph), 4.84 (m, 1H, Dab-α-CH), 4.47 (dd, 1H,
OCH-fluorenyl, J = 6.3, 10.3 Hz), 4.33 (m, 1H, Pip-ε-CH), 4.21 (m,
2H, OCH-fluorenyl, Pip-ε-CH), 3.88 (m, 1H, Pip-β-CH), 2.27 (d, 1H,
CH-fluorenyl, J = 13.3 Hz), 1.77−1.28 (m, 6H, Pip-(CH2)3), 1.09 (d,
A sample of linear decapeptide 4 (20 mg, 0.017 mmol) and
NaHCO3 (5.6 mg, 0.067 mmol) in DMF (3.4 mL) was treated with
DPPA (15 μL, 0.067 mmol), and the reaction mixture was stirred at
F
dx.doi.org/10.1021/jo402267r | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
room temperature for 6 days. The reaction mixture was poured onto 1
M aq. HCl (ca. 20 mL) and extracted with AcOEt. The combined
organic phases were washed with saturated aq. NaHCO3 and saturated
aq. NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (SiO2, 1−2%
MeOH−CHCl3) to afford 3 (3.6 mg, 18%) as a colorless glass. Data
1
for 3: [α]20 −37.0 (c 0.31, MeOH); H NMR (CD3OD, 500 MHz,
D
selected peaks are listed because of multiple conformers) δ 7.45−7.21
(m, 10H), 5.35−4.90 (m, 6H), 4.84−3.99 (m, 6H), 3.82−3.35 (m,
4H), 3.19−3.03 (m, 6H), 2.94−2.89 (m, 4H), 2.26−2.10 (m, 2H),
1.62−1.23 (m, 20H), 1.30−1.11 (m, 6H); 13C NMR (CD3OD, 125
MHz, selected peaks are listed because of multiple conformers) δ
173.4, 172.5, 172.3, 172.2, 171.5, 171.0, 170.7, 170.2, 158.5, 158.4,
138.2, 129.5, 129.1, 79.5, 71.6, 67.8, 67.6, 67.5, 54.6, 45.1, 42.0, 41.7,
36.2, 35.9, 33.1, 30.8, 30.5, 28.7, 28.5, 27.6, 26.8, 26.4, 26.1, 25.6, 23.7,
21.8, 21.5, 21.4, 18.5, 14.4, 13.2; ESIMS-LR m/z 1191 [(M + Na)+];
ESIMS-HR calcd for C58H80N12NaO14 (M + Na)+ 1191.5815, found
1191.5798.
CH2), 3.22−2.80 (m, 4H, Pip-ε-CH2), 2.32 (d, 2H, Pip-β-CH2, J =
12.6 Hz), 2.23 (d, 2H, Pip-β-CH2, J = 12.6 Hz), 1.90−1.22 (m, 16H,
Pip-γ-CH2, Pip-δ-CH2, Pip-γ-CH2 and Pip-δ-CH2), 1.17 (d, 6H, Dab-
CH3, J = 6.9 Hz); 13C NMR (CDCl3, 125 MHz) δ 172.7, 172.6, 172.3,
172.0, 171.3, 166.4, 150.7, 147.8, 138.6, 131.4, 130.8, 130.6, 129.2,
128.9, 119.6, 79.5, 57.2, 54.6, 52.7, 52.1, 45.1, 42.2, 41.8, 36.2, 27.9,
+
26.9, 26.3, 25.3, 21.7, 21.6, 13.2; ESIMS-LR m/z 1233 [(M + Na) ];
ESIMS-HR calcd for C62H78N14NaO12 1233.5816, found 1233.5810.
3-Quinoline Analogue (23). In a manner similar to the synthesis
of 1, 23 (colorless glass, 11.9 mg, 43%) was prepared from 3 using 3-
Quinaldopeptin (1). A mixture of 3 (5.8 mg, 5.0 μmol) in MeOH
(1 mL) and 10% Pd(OH)2/C (1 mg) was vigorously stirred under a
quinolinecarboxylic acid (15.9 mg, 0.092 mmol), HOBt (18.6 mg, 0.14
mmol), and EDCI (17.6 mg, 0.092 mmol) after purification by silica
H2 atmosphere at room temperature for 1 h. The catalyst was filtered
off through a Celite pad, and the filtrate was concentrated in vacuo to
give the diamine. The residue in DMF (1 mL) was treated with HATU
gel column chromatography (SiO2, 1% MeOH−CHCl3). [α]20
D
1
−135.7 (c 0.32, MeOH); H NMR (CD3OD, 500 MHz) δ 9.33 (d,
2H, NH, J = 9.7 Hz), 8.34 (d, 2H, J = 1.7 Hz), 8.22 (d, 2H, J = 8.0
Hz), 8.06 (s, 2H), 7.99 (d, 2H, J = 8.6 Hz), 7.88 (dt, 2H, J = 1.7, 8.6
Hz), 7.68 (t, 2H, J = 8.0 Hz), 7.21 (d, 2H, Dab-NH-Pip, J = 8.0 Hz),
5.70 (m, 2H, Dab-α-CH), 5.13 (d, 2H, Pip-α-CHex, J = 4.6 Hz), 4.68
(dd, 2H, Pip-α-CH), 4.53 (m, 2H, Dab-β-CH), 4.37 (d, 2H, Gly-α-
CH2, J = 17.2 Hz), 3.91 (t, 2H, Pip-ε-CH), 3.74 (d, 2H, Gly-α-CH2, J
= 17.2 Hz), 3.53 (d, 2H, Sar-α-CH2, J = 15.5 Hz), 3.07 (d, 2H, Sar-α-
CH2, J = 15.5 Hz), 3.01 (d, 2H, Pip-ε-CH2), 2.71 (t, 2H, Pip-ε-CH2),
2.37 (d, 2H, Pip-β-CH2), 2.17 (d, 2H, Pip-β-CH2), 1.97 (m, 2H, Pip-γ-
CH2), 1.81 (m, 2H, Pip-δ-CH2), 1.55−1.15 (m, 12H, Pip-γ-CH2 and
Pip-δ-CH2), 1.49 (d, 6H, Dab-CH3, J = 6.9 Hz); 13C NMR (CD3OD,
125 MHz) δ 174.7, 173.4, 171.0, 170.8, 170.5, 166.2, 149.8, 149.4,
137.8, 133.0, 131.4, 128.7, 128.6, 128.1, 126.0, 79.5, 56.8, 54.7, 53.5,
51.6, 45.2, 43.9, 41.4, 37.1, 28.3, 26.2, 25.8, 25.5, 21.2, 20.7, 15.1;
ESIMS-LR m/z 1233 [(M + Na) +]; ESIMS-HR calcd for
C62H78N14NaO12 1233.5816, found 1233.5816.
i
(7.6 mg, 20 μmol), Pr2NEt (3.5 μL, 20 μmol), and chromophore 2
(3.8 mg, 20 μmol) at 0 °C, and the mixture was stirred at room
temperature for 17 h. The resulting mixture was partitioned between
AcOEt (30 mL) and 1 M aqueous HCl (10 mL), and the organic
phase was washed with saturated aqueous NaHCO3 (10 mL), H2O
(10 mL), and saturated aqueous NaCl, dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by flash silica gel
column chromatography (3% MeOH−CHCl3) to afford quinaldo-
peptin (1, 2.7 mg, 43%) as a yellow solid. Mp >300 °C (lit. >300 °C);
[α]22 −120.8 (c 0.27, MeOH); natural [α]20 −129.3 (c 0.19,
D
D
MeOH); 1H NMR (CD3OD, 500 MHz, selected peaks are listed
because of multiple conformers) δ 7.90 (br d, 1H, J = 5.8 Hz), 7.81−
7.26 (m, 4H), 5.86 (br s, 1H), 5.67 (br s, 1H), 5.30 (d, 1H, J = 4.1
Hz), 5.12 (d, 1H, J = 4.0 Hz), 4.70 (d, 1H, J = 18.3 Hz), 4.60 (d, 1H, J
= 10.3 Hz), 4.47 (d, 1H, J = 13.2 Hz), 4.30 (br s, 1H), 4.23 (m, 2H),
4.07 (d, 1H, J = 18.9 Hz), 3.95 (d, 1H, J = 17.2 Hz), 3.65 (m, 1H),
3.43 (m, 1H), 2.87 (m, 1H), 3.16 (br s, 1H), 3.22−2.99 (m, 2H), 2.96
(s, 3H), 2.89−2.69 (m, 4H), 2.30 (br d, 1H, J = 12.1 Hz), 2.20 (br d,
1H, J = 13.7 Hz), 1.87−1.37 (m, 16H), 1.21 (d, 3H, J = 7.5 Hz); 13C
NMR (CD3OD, 125 MHz, selected peaks are listed because of
multiple conformers) δ 172.7, 172.6, 172.4, 171.9, 170.1, 169.7, 154.4,
135.8, 133.2, 130.5, 129.9, 129.8, 129.5, 128.5, 128.2, 127.4, 121.0,
57.1, 54.5, 54.4, 53.7, 52.6, 45.2, 42.0, 41.9, 36.2, 33.1, 30.7, 30.5, 30.3,
28.1, 27.9, 27.0, 26.4, 25.2, 23.7, 21.6, 21.4, 21.3, 14.4, 13.2; ESIMS-LR
m/z 1265 [(M + Na)+]; ESIMS-HR calcd for C62H78N14NaO14 (M +
Na)+ 1265.5720, found 1265.5728.
(R)-3-(9-Fluorenylmethoxycarbonylamino)-2-(benzyloxy-
carbonylamino)propanoic Acid (25). A solution of (2R)-2-N-
benzyloxycarbonyl-2,3-diaminopropanoic acid (1.00 g, 4.20 mmol)
and NaHCO3 (776 mg, 9.24 mmol) in dioxane/H2O (38/12 mL) was
treated with FmocCl (1.20 g, 4.62 mmol) at 0 °C, and the mixture was
stirred at room temperature for 2 h. The mixture was diluted with
AcOEt, and the organic phase was washed with 1 M aq. HCl and
saturated aq. NaCl, dried (Na2SO4), filtered, and concentrated in
vacuo. The residue was triturated from hexane to afford 25 (1.74 g,
2-Quinoline Analogue (22). In a manner similar to the synthesis
of 1, 22 (colorless glass, 9.2 mg, 33%) was prepared from 3 using 2-
quinolinecarboxylic acid (15.9 mg, 0.092 mmol), HOBt (18.6 mg, 0.14
mmol), and EDCI (17.6 mg, 0.092 mmol) after purification by silica
gel column chromatography (SiO2, 1% MeOH−CHCl3). [α]21
D
1
−116.3 (c 0.67, CHCl3); H NMR (CD3OD, 500 MHz, a mixture
1
of rotamers, data for the major rotamer) δ 8.23 (d, 2H, J = 8.0 Hz),
8.08 (d, 2H, J = 8.6 Hz), 8.00 (d, 2H, J = 8.6 Hz), 7.83 (d, 2H, J = 7.5
Hz), 7.56 (t, 2H, J = 6.9 Hz), 7.60 (t, 2H, J = 6.9 Hz), 5.72 (br s, 2H,
Dab-α-CH), 5.30 (d, 2H, Pip-α-CHex, J = 5.2 Hz), 4.62 (m, 2H, Pip-α-
CH), 4.50−3.97 (m, 12H, Dab-β-CH, Gly-α-CH2, Pip-ε-CH, Sar-α-
90%) as a white solid. H NMR (DMSO-d6, 500 MHz) δ 7.88−7.29
(m, 14H, aromatic and NH), 7.09 (d, 1H, NH, J = 6.3 Hz), 4.98 (s,
2H, CH2Ph), 4.26−3.97 (m, 3H, OCH2fluorenyl, H-fluorenyl), 3.33
(m, 2H, α-CH and β-CH); 13C NMR (DMSO-d6, 125 MHz) δ 172.4,
156.2, 155.9, 143.9, 140.7, 137.0, 128.4, 127.8, 127.6, 125.3, 127.1,
G
dx.doi.org/10.1021/jo402267r | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
+
120.1, 65.6, 65.4, 54.7, 46.7, 42.4; ESIMS-LR m/z 483 [(M + Na) ];
ESIMS-HR calcd for C26H24N2NaO6 483.15266, found 483.15215.
Cyclo(Gly-Sar-L-Pip-Dap(Cbz)-L-Pip)2 (26). Solid-phase peptide
synthesis of 26 was performed in a manner similar to the synthesis of
127.7, 66.0, 53.1, 52.2, 50.6, 44.2, 43.4, 40.9, 36.7, 29.6, 25.5, 25.1,
24.9, 24.5, 19.6; ESIMS-LR m/z 1163 [(M + Na)+]; ESIMS-HR calcd
for C56H76N12NaO14 (M + Na)+ 1163.5502, found 1163.5500.
Desmethylquinaldopeptin (27). A mixture of 26 (20 mg, 0.017
mmol) and Pd/C (10 mg) in MeOH (5 mL) was vigorously stirred for
3 h under hydrogen atmosphere. The Pd/C was filtered off through a
Celite pad, and the filtrate was concentrated in vacuo to give the crude
diamine. A solution of 2 (12.7 mg, 0.068 mmol) and DMAP (8.3 mg,
0.068 mmol) in CH2Cl2 (1 mL) was treated with EDCI (13 mg, 0.068
mmol), and the reaction mixture was stirred at room temperature for
30 min. The mixture was added to the diamine, and the reaction
mixture was stirred at room temperature for 24 h. The reaction
mixture was poured onto 1 M aq. HCl (5 mL) and extracted with
AcOEt. The combined organic phases were washed with saturated aq.
NaHCO3 and saturated aq. NaCl (5 mL), dried (Na2SO4), filtered,
and concentrated in vacuo. Preparative TLC (thin SiO2, 20 × 20 cm,
10% MeOH−CHCl3 eluent) afforded 27 (8.0 mg, 39%) as a white
solid colorless glass. [α]21D −60.6 (c 0.49, MeOH); 1H NMR (CDCl3/
CD3OD = 9/1, 500 MHz, selected peaks are listed because of multiple
conformers) δ 7.46−7.35 (m, 10H), 5.74−4.93 (m, 6H), 4.56−3.50
(m, 14H), 3.05−2.89 (m, 4H), 2.45−2.22 (m, 4H), 1.87−1.12 (m,
16H); the 13C NMR spectrum is not able to be obtained because of
multiple conformers. Increasing the amount of the compound also
caused precipitation. ESIMS-LR m/z 1237 [(M + Na)+]; ESIMS-HR
calcd for C60H74N14NaO14 1237.5407, found 1237.5383.
3. A solution of Fmoc-L-Pip-OH (10, 551 mg, 0.79 mmol) and
iPr2NEt (1.09 mL, 4.0 equiv) in CH2Cl2 (5 mL) was then added. The
mixture was stirred for 1 h at 25 °C. The Fmoc-L-Pip-O-2CT resin was
subjected to the following washings/treatments: filteration, CH2Cl2−
MeOH−iPr2NEt (17:2:1, 7 mL × 3), DMF (7 mL × 3), and CH2Cl2
(7 mL × 3). The resin was dried over P2O5 overnight. The loading,
calculated by measuring absorbance at 290 nm, was 0.998 mmol/g.
The elongation of the peptide was achieved by sequential addition
of the Fmoc-protected amino acid with either HBTU (for the primary
amine, 548 mg, 2.9 equiv) or HATU (for the secondary amine, 550
mg, 2.9 equiv) and iPr2NEt (521 μL, 6.0 equiv) as coupling reagents in
DMF (4 mL) in preactivation mode. The mixture was stirred for 1 h,
and after filtration the corresponding colorimetric test (Kaiser test for
the primary amine and cloranil test for the secondary amine) indicated
the completion of the coupling. Next, the peptide resin was washed
with DMF (7 mL × 3) and CH2Cl2 (7 mL × 3) and treated with a
solution of piperidine/DMF (1:4, 3 min, then 1:8, 12 min) to remove
the Fmoc group, and the coupling plus Fmoc removal was then
measured. The peptide resin was split into fractions (1/3 and 2/3).
The former was treated with a piperidine/DMF (1:4, 3 min, 1:8, 12
min) solution as described above, while the latter was cleaved from the
resin with 5% TFA in CH2Cl2 solution (5 mL × 3), and the combined
filtrates were concentrated in vacuo. The residue was precipitated in
ether to afford the crude pentapeptide (278 mg) as a pale yellow solid
(ESIMS-LR (negative mode) m/z 833 [(M − Na)−]; ESIMS-HR
(negative mode) calcd for C43H50N6NaO10 833.34806, found
833.34782).
L-Ala-NH2-DNP-L-Pip (L-21). A solution of L-pipecolic acid (5 mg,
0.038 mmol) in acetone (1 mL) and saturated aq. NaHCO3 (1 mL)
The crude pentapeptide was added to the unprotected peptide resin
i
fraction with HBTU (104 mg, 1.7 equiv) and Pr2NEt (102 μL, 3.6
equiv) as coupling reagents in DMF (2 mL) in preactivation mode.
The mixture was stirred at room temperature for 2 h. Then, the
peptide resin was washed with DMF (7 mL × 3) and CH2Cl2 (7 mL ×
3) and treated with a solution of piperidine/DMF (1:4, 3 min, then
1:8, 12 min). The resin was treated with 5% TFA in CH2Cl2 solution
(5 mL × 3), and the combined filtrates were concentrated to dryness
under reduced pressure. The residue was precipitated in ether to afford
the crude decapeptide (121 mg, 64%) as a pale yellow solid (ESIMS-
LR m/z 1159 [(M + H) +]; ESIMS-HR calcd for C56H79N12O15
1159.57824, found 1159.57738).
A sample of linear decapeptide (14.8 mg, 0.013 mmol) in CH2Cl2
(2.6 mL) was treated sequentially with EDCI (7.5 mg, 0.039 mmol),
HOBt (5.3 mg, 0.039 mmol), and NaHCO3 (6.5 mg, 0.077 mmol),
and the reaction mixture was stirred at room temperature for 5 days.
The reaction mixture was poured onto 1 M aq. HCl (20 mL) and
extracted with AcOEt (20 mL). The combined organic phases were
washed with saturated aq. NaHCO3 (20 mL) and saturated aq. NaCl
(10 mL), dried (Na2SO4), filtered, and concentrated in vacuo.
Preparative TLC (thin SiO2, 20 × 20 cm, 10% MeOH−CHCl3
eluent) afforded 26 (4.7 mg, 31%) as a colorless glass. 1H NMR
(CDCl3/CD3OD = 9/1, 500 MHz, selected peaks are listed because of
multiple conformers) δ 7.25−7.04 (m, 10H), 5.12−4.88 (m, 8H),
4.58−4.51 (m, 6H), 4.13−4.04 (m, 4H), 3.23−2.81 (m, 10H), 2.06−
1.83 (m, 4H), 1.65−1.12 (m, 20H); 13C NMR (CDCl3/CD3OD = 9/
1, 125 MHz, selected peaks are listed because of multiple conformers)
δ 172.1, 170.5, 170.3, 168.2, 167.8, 156.3, 136.7, 128.4, 128.1, 127.9,
was treated with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (12.5 mg,
0.046 mmol) at 40 °C for 6 h. The reaction was quenched by 1 M aq.
HCl at 0 °C. The mixture was concentrated in vacuo, and the residue
was purified by silica gel column chromatography (SiO2, 20% MeOH−
1
CH2Cl2) to afford L-21 (10 mg, 69%) as a yellow powder. H NMR
(CD3OD, 500 MHz) δ 8.82 (s, 1H, Ha), 6.13 (s, 1H, Hb), 4.27 (q,
1H, Ala-α-CH, J = 6.9 Hz, J = 13.8 Hz), 4.09 (br s, 1H, Pip-α-CH),
3.45 (m, 1H, Pip-ε-CHax), 3.22 (d, 1H, Pip-ε-CHex, J = 12.6 Hz), 2.22
(d, 1H, Pip-β-CHex, J = 10.9 Hz), 1.78−1.28 (m, 4H, Pip-γ-CH2 and
Pip-δ-CH2), 1.57 (d, 3H, Ala-β-CH3, J = 6.9 Hz); 13C NMR (CD3OD,
125 MHz) δ 176.8, 174.5, 153.3, 147.7, 131.5, 129.4, 126.0, 103.0,
62.3, 53.5, 28.5, 25.9, 21.7, 19.2; ESIMS-LR m/z 380 [(M + H)+];
ESIMS-HR calcd for C15H18N5O7 380.12117, found 380.12135.
L-Ala-NH2-DNP-D-Pip (D-21). A solution of D-pipecolic acid (5 mg,
0.038 mmol) in acetone (1 mL) and saturated aq. NaHCO3 (1 mL)
H
dx.doi.org/10.1021/jo402267r | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
was treated with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (12.5 mg,
0.046 mmol) at 40 °C for 6 h. The reaction was quenched by 1 M aq.
HCl at 0 °C. The mixture was concentrated in vacuo, and the residue
was purified by silica gel column chromatography (SiO2, 20% MeOH−
(6) Boger, D. L.; Chen, J.; Saionz, K. W. J. Am. Chem. Soc. 1996, 118,
1629−1644.
(7) (a) Boger, D. L.; Schule, G. J. Org. Chem. 1998, 63, 6421−6424.
̈
(b) Boger, D. L.; Ledeboer, M. W.; Kume, M. J. Am. Chem. Soc. 1999,
121, 1098−1099. (c) Ciufolini, M. A.; Xi, N. J. Org. Chem. 1997, 62,
2320−2321. (d) Ciufolini, M. A.; Valognes, D.; Xi, N. J. Heterocycl.
Chem. 1999, 36, 1409−1419. (e) Valognes, D.; Belmont, P.; Xi, N.;
Ciufolini, M. A. Tetrahedron Lett. 2001, 42, 1907−1909. (f) Ciufolini,
M. A.; Swaminathan, S. Tetrahedron Lett. 1989, 23, 3027−3028.
(g) Ciufolini, M. A.; Valognes, D.; Xi, N. Tetrahedron Lett. 1999, 40,
3693−3696. (h) Ciufolini, M. A.; Valognes, D.; Xi, N. Angew. Chem.
Int. Ed. 2000, 39, 2493−2495.
CH2Cl2) to afford D-21 (7.2 mg, 50%) as a yellow powder. [α]20
D
−46.9 (c 0.23, MeOH); 1H NMR (CD3OD, 500 MHz) δ 8.80 (s, 1H,
Ha), 6.10 (s, 1H, Hb), 4.22 (q, 1H, Ala-α-CH, J = 6.9 Hz, J = 13.7
Hz), 4.00 (br s, 1H, Pip-α-CH), 3.59 (m, 1H, Pip-ε-CHax), 3.17 (d,
1H, Pip-ε-CHex, J = 12.6 Hz), 2.25 (d, 1H, Pip-β-CHax, J = 13.2 Hz),
1.75−1.29 (m, 4H, Pip-γ-CH2 and Pip-δ-CH2), 1.60 (d, 3H, Ala-β-
CH3, J = 6.9 Hz); 13C NMR (CD3OD, 125 MHz) δ 177.7, 153.4,
147.8, 131.7, 129.3, 125.7, 102.9, 63.3, 54.1, 28.8, 26.0, 21.9, 19.1;
ESIMS-LR m/z 380 [(M + H)+]; ESIMS-HR calcd for C15H18N5O7
380.12117, found 380.12142.
(8) Boger, D. L.; Ledeboer, M. W.; Kume, M.; Searcey, M.; Jin, Q. J.
Am. Chem. Soc. 1999, 121, 11375−11383.
Amino Acid Analysis of 3 and 26. Compound 3 or 26 (1.0 mg,
0.88 μmol) was treated with 6 M aq. HCl (1.0 mL) at 100 °C for 24 h.
The resulting mixture was concentrated in vacuo. The residue in
acetone (1 mL) and saturated aq. NaHCO3 (1 mL) was treated with
1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (4.90 mg, 0.018 mmol) at
40 °C for 6 h. The crude mixture was dissolved in DMSO (1.0 mL)
and analyzed by HPLC (YMC J′sphere ODS M80, 4.6 × 150 mm,
0.1% TFA 25−50% MeCN−H2O for 40 min, 9.2 min).
(9) (a) Boger, D. L.; Chen, J.-H.; Saionz, K. W.; Jin, Q. Bioorg. Med.
Chem. 1998, 6, 85−102. (b) Boger, D. L.; Chen, J.-H. Bioorg. Med.
Chem. Lett. 1997, 7, 919−922. (c) Boger, D. L.; Saionz, K. W. Bioorg.
Med. Chem. 1999, 7, 315−321.
(10) There are several cyclic peptide/depsipeptide natural products.
For example, see: (a) apicidin: Singh, S. B.; Zink, D. L.; Polishook, J.
D.; Dombrowski, A. W.; Darkin-Rattray, S. J.; Schmatz, D. M.; Goetz,
M. A. Tetrahedron Lett. 1996, 37, 8077−8080. (b) petriellin A: Lee, K.
K.; Gloer, J. B.; Scott, J. A.; Malloch, D. J. Org. Chem. 1995, 60, 5384−
ASSOCIATED CONTENT
́
5385. (c) friulimicins: Vertesy, L.; Ehlers, E.; Kogler, H.; Kurz, M.;
■
Meiwes, J.; Seibert, G.; Vogel, M.; Hammann, P. J. Antibiot. 2000, 53,
816−827. (d) pipecolidepsin A: Pelay-Gimeno, M.; Garcia-Ramos, Y.;
Martin, M. J.; Spengler, J.; Molina-Guijiarro, J. M.; Munt, S.;
Francesch, A. M.; Cuevas, C.; Tulla-Puche, J.; Albericio, F. Nat.
Commun. 2013, 4, 2352.
S
* Supporting Information
1H and 13C NMR spectra of all the new compounds except 13
C
NMR spectra of 27, and amino acid analysis of 3 and 26. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(11) Solid-phase synthesis of cyclic octadepsipeptide of this class of
́ ́
molecules: (a) Bayo-Puxan, N.; Fernandez, A.; Tulla-Puche, J.; Riego,
́
AUTHOR INFORMATION
Corresponding Author
*E-mail: ichikawa@pharm.hokudai.ac.jp.
Notes
E.; Cuevas, C.; Alvarez, M.; Albericio, F. Chem.Eur. J. 2006, 12,
9001−9009. (b) Malkinson, M. K.; Anim, M.; Zloh, M.; Searcy, M. J.
Org. Chem. 2005, 70, 7654−7661. (c) Dietrich, B.; Diederrichsen, U.
Eur. J. Org. Chem. 2005, 70, 147−153. (d) Garcia-Martin, F.; Cruz, L.
J.; Rodriguez-Mias, R. A.; Giralt, E.; Albericio, F. J. Med. Chem. 2008,
■
The authors declare no competing financial interest.
́
51, 3194−3202. (e) Puche-Tulla, J.; Bayo-Puxan, N.; Moreno, J. A.;
́
Francesch, A. M.; Cuevas, C.; Alvarez, M.; Albericio, F. J. Am. Chem.
Soc. 2007, 129, 5322−5323. (f) Marcucci, E.; Tulla-Puche, J.;
Albericio, F. Org. Lett. 2012, 14, 612−615.
ACKNOWLEDGMENTS
■
This research was supported by JSPS Grant-in-Aid for
Challenging Exploratory Research (SI, Grant Number
22659020) and Scientific Research (B) (SI, Grant Number
25293026). We thank Ms. S. Oka and Ms. A. Tokumitsu
(Center for Instrumental Analysis, Hokkaido University) for
measurement of the mass spectra and Taiho Pharmaceutical
Co., Ltd. for evaluation of cytotoxicity of 1, 22, 23, and 27.
(12) Barlos, K.; Gatos, D.; Kallitsis, J.; Papaphotiu, G.; Sotiriu, P.;
Yao, W.; Schaefer, W. Tetrahedron Lett. 1989, 30, 3943−3946.
(13) Preparation of the suitably protected amino acids was described
in the Supporting Information.
(14) Han, H.; Yoon, J.; Janda, K. D. J. Org. Chem. 1998, 63, 2045−
2048.
(15) Wu, X.-H.; Wang, L.-S.; Han, Y.-H.; Regan, N.; Li, P.-K.;
Villalona, M. A.; Hu, X.-C.; Briesewitz, R.; Pei, D.-H. ACS Comb. Sci.
2011, 13, 486−495.
(16) Yamada, S.; Yokoyama, Y.; Shioiri, T. J. Org. Chem. 1974, 39,
3302−3303.
(17) Bhuchan, R.; Bruckner, H. Amino Acids 2004, 27, 231−247.
(18) Aurelio, A. B.; Robert, T. C.; Brownlee, A.; Jason, A. C.;
Andrew, B.; Hughes, A. E.; Gideon, M.; Polya, D. Aust. J. Chem. 2006,
59, 407−414.
(19) Boger, D. L.; Chen, J.-H. J. Org. Chem. 1995, 60, 7369−7371.
(20) Tambade, P. J.; Patil, Y. P.; Bhanushali, M. J.; Bhange, B. M.
Synthesis 2008, 15, 2347−2352.
REFERENCES
■
(1) (a) Matson, J. A.; Bush, J. A. J. Antibiot. 1989, 42, 1763−1767.
(b) Matson, J. A.; Colson, K. L.; Belofsky, G. N.; Bleiberg, B. B. J.
Antibiot. 1993, 46, 162−166.
(2) (a) Ohkuma, H.; Sakai, F.; Nishiyama, Y.; Ohbayashi, M.;
Imanishi, H.; Konishi, M.; Miyaki, T.; Koshiyama, H.; Kawaguchi, H. J.
Antibiot. 1980, 33, 1087−1097. (b) Tomita, K.; Hoshino, Y.; Sasahira,
T.; Kawaguchi, H. J. Antibiot. 1980, 33, 1098−1102. (c) Konishi, M.;
Ohkuma, H.; Sakai, F.; Tsuno, T.; Koshiyama, H.; Naito, T.;
Kawaguchi, H. J. Antibiot. 1981, 34, 148−159.
(3) Lingham, R. B.; Hsu, A. H. M.; O’Brien, J. A.; Sigmund, J. M.;
Sanchez, M.; Gagliardi, M. M.; Heimbuch, B. K.; Genilloud, O.;
Martin, I.; Diez, M. T.; Hirsch, C. F.; Zink, C. D. L.; Liesch, J. M.;
Koch, G. E.; Gartner, S. E.; Garrity, G. M.; Tsou, N. N.; Salituro, G. M.
J. Antibiot. 1996, 49, 253−259.
(4) For reviews: (a) Dawson, S.; Malkinson, J. P.; Paumier, D.;
Searcey, M. Nat. Prod. Rep. 2007, 24, 109−126. (b) Zolova, O. E.;
Mady, A. S. A.; Garneau-Tsodikova, S. Biopolymers 2010, 93, 777−790.
(5) Toda, S.; Sugawara, K.; Nishiyama, Y.; Ohbayashi, M.; Ohkusa,
N.; Yamamoto, H.; Konishi, M.; Oki, T. J. Antibiot. 1990, 43, 796−
808.
I
dx.doi.org/10.1021/jo402267r | J. Org. Chem. XXXX, XXX, XXX−XXX