Journal of Medicinal Chemistry
Article
cartridge, frit, and cap plugs were purchased from Applied Separations
(Allentown, PA). HTS Transwell 96-well plates (Product no.: 3381)
for Caco-2 permeability assay were purchased from Corning
(Corning, NY).
was added to the mixture, and the concentration of peptide was kept
at 1.0 mM. The mixture was stirred under microwave irradiation (75
°C, 300 W max power, ramp 2.5 min, hold 17.5 min, stirring level 3).
The mixture was then cooled to room temperature, and the reaction
was quenched by water. The solvent was evaporated under reduced
pressure (50 mbar) at 75 °C. The residue was dissolved in 1:1 (v/v)
water/acetonitrile, filtered by a 0.45 μm poly(tetrafluoroethylene)
(PTFE) syringe filter, and purified by preparative HPLC to obtain
CsO and CP1. Purity confirmed by analytical HPLC (220 nm) is
>98% for CsO and CP1. For the synthesis of CP2 and CP3, Fmoc-
Rink amide MBHA resin (250 mg, 0.13 mmol, loading 0.52 mmol/g)
was used. After Fmoc deprotection, the resin was treated with Fmoc-
Glu-OAll (160 mg, 0.39 mmol, 3.0 equiv), HATU (150 mg, 0.39
mmol, 3.0 equiv), and DIEA (130 mg, 1.04 mmol, 8.0 equiv) in DMF
(2 mL) and stirred under microwave irradiation (75 °C, 300 W max
power, ramp 2.5 min, hold 8.0 min, stirring level 3). After peptide
elongation, allyl deprotection was carried out by the addition of
Pd(PPh3)4 (15 mg, 0.013 mmol, 0.1 equiv) and PhSiH3 (340 mg, 3.1
mmol, 24 equiv) in CH2Cl2 (2 mL) at room temperature for 3 h. The
N-terminal Fmoc was removed after allyl deprotection by treatment
with 20% (v/v) piperidine/DMF solution (2 mL). The resin was
thoroughly washed between each reaction using the above conditions.
For the on-resin macrocyclization, the resin was swelled in CH2Cl2 (2
mL) for 20 min. PyBOP (270 mg, 0.52 mmol, 4.0 equiv), HOAt (70
mg, 0.52 mmol, 4.0 equiv), and DIEA (168 mg, 1.3 mmol, 10 equiv)
in CH2Cl2 (2 mL) were added to the resin, and the mixture was
stirred at room temperature for 2 h. The resin was washed, and the
cyclic peptide was cleaved from the resin using TFA/TIS/CH2Cl2
95:2.5:2.5 (v/v/v) at room temperature for 2 h. The cleavage solution
was concentrated using a stream of N2 gas. The residue was dissolved
in 1:1 (v/v) water/acetonitrile, filtered, and purified by preparative
HPLC to yield CP2 and CP3. Purities are confirmed by analytical
HPLC (220 nm) for CP2 and CP3 are 97 and 98%, respectively.
Electrospray ionization mass spectrometry (ESI-MS) m/z CsO: calcd
for C60H110N11O11 [M + H]+ 1160.8, found 1160.7; CP1: calcd for
C61H112N11O11 [M + H]+ 1174.9, found 1174.7; CP2: calcd for
C62H113N12O12 [M + H]+ 1217.9, found 1217.7; CP3: calcd for
C63H115N12O12 [M + H]+ 1231.9, found 1231.8.
Reversed-Phase HPLC. Analytical HPLC was performed on a
Waters HPLC system (Waters 2489 UV−visible detector, Waters
1525 binary HPLC pump, Waters 2707 autosampler, and Water 5CH
column oven) with a C18 column (SunFire, C18, 4.6 × 250 mm2, 5
μm). The column oven temperature was set at 40 °C. The binary
mobile phase was used as follows: A, water with 0.1% CF3COOH; B,
CH3CN with 0.1% CF3COOH; flow rate, 1 mL/min; (1) 0−5 min at
50% B, 5−25 min with a linear gradient to 100% B, and 25−30 min at
100% B for CsO; (2) 0−5 min at 70% B, 5−25 min with a linear
gradient to 100% B, 25−30 min at 100% B for CP1; and (3) 0−5 min
at 10% B, 5−25 min with a linear gradient to 100% B, and 25−30 min
at 100% B for CP2 and CP3. Peptides were monitored at 220 and 254
nm. Preparative HPLC was performed on a Waters HPLC system
(Waters prepLC system, Waters 2545 quaternary HPLC pump,
Waters 2489 UV−visible detector, Waters fraction collector III) with
a C18 column (SunFire, C18, 19 × 150 mm2, 5 μm) at a flow rate of
14 mL/min. Preparative HPLC was performed under the same
mobile phase conditions as analytical HPLC, and sample elution was
monitored at 220 and 254 nm by absorbance. Fractions with >97%
purity were collected, lyophilized, and stored at −80 °C.
LC-MS. LC-MS analysis was performed on an Agilent Technology
Infinity 1260 system (quaternary pump, autosampler, temperature
controller, and Hewlett Packard series 1100 detector) and Agilent
6120 quadrupole MS with a C18 column (Poroshell, 120 EC-C18, 4.6
× 50 mm2, 2.7 μm). The column heating temperature was set at 50
°C. A binary mobile phase system was used as follows: A, water with
0.1% CF3COOH; B, CH3CN with 0.1% CF3COOH; flow rate, 0.8
mL/min; 0−2 min at 5% B, 2−7 min with a linear gradient to 100%
B, and 7−10 min at 100% B. Peptides were monitored at 220 and 254
nm.
Peptide Synthesis. Cyclic peptides were synthesized manually via
a standard Fmoc/tBu solid-phase peptide synthesis (SPPS) protocol.
Reactions were accelerated by microwave heating, and a CEM MARS
multimodal microwave reactor equipped with a fiber-optic temper-
ature probe and magnetic stirrer was used (CEM Corp., Matthews,
NC). For the synthesis of CsO and CP1, 2-chlorotritylchloride resin
(110 mg, 0.13 mmol, loading 1.14 mmol/g) was swelled in CH2Cl2 (3
mL) at room temperature for 20 min. Fmoc-Ala-OH (120 mg, 0.39
mmol, 3.0 equiv) in CH2Cl2 (2 mL) was added, followed by DIEA
(140 mg, 0.78 mmol, 6.0 equiv), and the resin was stirred at room
temperature for 5 h. The resin was washed with CH2Cl2 (×2), DMF
(×2), MeOH (×1), DMF (×2), and CH2Cl2 (×2). For Fmoc
deprotection, the resin was treated with 20% (v/v) piperidine/DMF
solution (2 mL) at room temperature for 5 min and then washed
using the above sequence. Amino acid coupling was performed by the
addition of Fmoc-amino acid (0.39 mmol, 3.0 equiv), HATU (150
mg, 0.39 mmol, 3.0 equiv), and DIEA (130 mg, 1.04 mmol, 8.0 equiv)
in DMF (2 mL). The mixture was stirred under microwave irradiation
(75 °C, 300 W max power, ramp 2.5 min, hold 8.0 min, stirring level
3) and washed using the above sequence. Coupling reactions were
employed twice for MeLeu10−D-Ala8 to improve the total yield. Fmoc
deprotection and amino acid coupling were repeated until the desired
linear peptide was obtained. The coupling reaction of MeLeu10 onto
MeVal11 for CsO was performed by the addition of Fmoc-MeLeu-OH
(140 mg, 0.39 mmol, 3.0 equiv), HOAt (53 mg, 0.39 mmol, 3.0
equiv), DIC (49 mg, 0.39 mmol, 3.0 equiv), and DIEA (100 mg, 0.39
mmol, 3.0 equiv) in DMF (2 mL) and was repeated twice. The linear
peptide was cleaved with TFA/TIS/CH2Cl2 95:2.5:2.5 (v/v/v) at
room temperature for 10 min. The volatiles were evaporated using a
stream of N2 gas, and the crude peptide was dissolved in 1:1 (v/v)
water/acetonitrile solution, filtered, lyophilized, and used for the
cyclization reaction without further purification. The linear peptide
(∼0.13 mmol) was dissolved in DMF (125 mL) containing DIEA
(340 mg, 2.6 mmol, 20 equiv), and the solution was stirred at room
temperature for 30 min to facilitate free amine formation at the N-
terminus. HATU (250 mg, 0.65 mmol, 5.0 equiv) in DMF (5 mL)
NMR Spectroscopy. All NMR experiments were carried out using
a JEOL 400 MHz (GIST, Gwangju) or a Bruker 900 MHz NMR
1
spectrometer equipped with the cryoprobe (KBSI, Ochang). 1D H
spectra were collected at 298−318 K on a JEOL 400 MHz NMR
spectrometer, and 2D NMR spectra including heteronuclear single
quantum coherence (HSQC), heteronuclear multiple bond correla-
tion (HMBC), total correlated spectroscopy (TOCSY), and NOESY
were obtained at 298 K on a Bruker 900 MHz NMR spectrometer.
The samples for 2D NMR spectra were prepared at 2 mg/mL. 2D
NMR data processing and analysis were performed by Topspin
(Bruker) and SPARKY software, respectively.83 For variable-temper-
ature NMR spectroscopy, each cyclic peptide (3 mg) was dissolved in
1
CDCl3 (500 μL), and 1D H NMR spectra were measured at 5 K
intervals between 298 and 318 K. The chemical shift changes of amide
protons were recorded to calculate the temperature coefficients for
each amide proton.84 For hydrogen-deuterium exchange NMR
spectroscopy, each cyclic peptide (3 mg) was dissolved in 9:1 (v/v)
CDCl3/CD3OD (500 μL).26 CD3OD was used as a source of
1
exchangeable deuterium, and 1D H NMR spectra were measured at
298 K at eight time points (0, 30, 60, 120, 180, 360, 720, and 1440
min). The integration of peaks corresponding to amide protons was
carried out compared with a proton signal of tetramethylsilane as an
internal standard to calculate the H/D exchange rate of the amide
protons. For DMSO-titrated NMR spectroscopy, each cyclic peptide
1
(3 mg) was dissolved in CDCl3 (500 μL), and 1D H NMR spectra
were measured at 298 K with every 50 μL addition of DMSO-d6 up to
250 μL. The chemical shift changes of each amide proton were
recorded depending on the volume percentage of DMSO-d6.
Generation of Conformational Ensemble. The conformations
of cyclic peptides were predicted through a previously reported
method, Conformational Analysis from NMR and Density functional
prediction of Low-energy Ensembles (CANDLE),65 which employs
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J. Med. Chem. 2021, 64, 8272−8286
Lee, Dongjae
