The Journal of Organic Chemistry
Note
referenced to the CFCl3 signal (0.00 ppm), used as an internal
standard. GC-MS experiments were run using a J&W DB-5 ms column
(30 m × 0.25 mm id, film thickness 0.25 μm). High-resolution mass
spectra (ESI-TOF) were recorded with the electrospray ion source in
positive-ion mode; the following instrument settings were used:
nebulizer gas, nitrogen at 1.5 L/min; dry gas, nitrogen at 102 MPa and
250 °C; collision gas, argon. The LC-HRMS analyses were carried out
using a Eurospher II 100−3 C18 column (100 × 2 mm ID);
conditions: water (A)−methanol (B) mobile phase; gradient elution,
90−100% B in 10 min, 0.15 mL/min. Unless noted otherwise,
semipreparative HPLC separations were carried out in normal phase
by employing an Ascentis, Si coulmn (250 × 10 mm, 5 μm); elution
conditions: isocratic 3:1 diethyl ether/hexane, flow rate 2.5 mL/min,
UV detector 220, 254 nm. Cyclo[(D-α-hydroxyisovaleryl-D-valyl-L-
lactyl-L-valyl)2(D-α,β-dihydroxyisovaleryl-D-valyl-L-lactyl-L-valyl)] (ana-
log 2) was prepared from commercially available valinomycin (1),
following our previously described procedure.5 N-Boc-glycine
pentafluorophenyl ester (3a) was obtained from commercial Boc-
Gly-OH upon esterification with pentafluorophenol, following a
described procedure.19 Bis(N,N-dimethylformamide)dichlorodioxo-
moybdenum(VI), MoO2Cl2(dmf)2 was obtained from commercial
dichlorodioxomoybdenum(VI), MoO2Cl2, according to a reported
procedure.20
means of a syringe, and stirring was continued at r.t. until TLC analysis
(silica gel, eluent diethyl ether/hexane 3:1, iodine/1% aqueous starch
solution) revealed complete substrate consumption (60 h). After
evaporating CH2Cl2 by a stream of N2, the crude product was diluted
with anhydrous diethyl ether, filtered through a silica gel pad, and
concentrated under reduced pressure. Purification by semipreparative
normal-phase HPLC afforded 19.5 mg of 4 (14.0 μmol, yield 75%,
1
purity ≥92% by HPLC); H NMR (acetone-d6, 500 MHz): δ 7.97−
7.93 (m, 3H, NH), 7.70 (d, J = 8.2 Hz, 1H, NH), 7.67 (d, J = 7.9 Hz,
1H, NH), 7.63 (d, J = 7.4 Hz, 1H, NH), 6.87 (br m, 1H, Gly NH),
5.58 (s, 1H, D-βGlyHyi CαH), 5.39−5.32 (m, 3H, L-Lac CαH), 5.01 (d,
J = 7.7 Hz, 1H, D-Hyi CαH), 5.00 (d, J = 7.5 Hz, 1H, D-Hyi CαH), 4.35
(m, 3H, Val CαH), 4.31 (d, J = 5.5 Hz, 2H, Gly CαH2), 4.24 (m, 1H,
Val CαH), 4.21−4.16 (m, 2H, Val CαH), 2.37−2.33 (m, 5H, D-Hyi and
Val CβH), 2.27−2.18 (m, 3H, Val CβH), 1.63 (s, 3H, D-βGlyHyi
CγH3), 1.55 (s, 3H, D-βGlyHyi Cγ′H3), 1.42−1.40 (m, 9H, L-Lac
CβH3), 1.09−0.97 (m, 48H, Val and D-Hyi Cγ,γ′H3) ppm; 13C{1H}
NMR (acetone-d6, 125 MHz): δ 172.4, 172.33, 172.26, 172.24, 172.18,
171.9, 171.12, 171.08, 171.0, 170.4, 170.3, 168.2, 167.2 (Gly CO),
1
156.1 (carbamate CO), 142.1 (m, JCF = 253 Hz, CF), 140.5 (m,
1
1JCF = 248 Hz, CF), 138.9 (m, JCF = 249 Hz, CF), 125.8 (pFP CO),
81.0 (D-βGlyHyi Cβ), 79.2 (D-Hyi CαH), 78.5 (D-βGlyHyi CαH), 71.1
and 71.0 (L-Lac CαH), 60.5, 60.3, 60.0, 59.1, 59.0, and 58.97 (Val
CαH), 42.6 (Gly CαH2), 31.21, 31.19, and 30.6 (D-Hyi and Val CβH),
23.8 (D-βGlyHyi Cγ′H3), 23.7 (D-βGlyHyi Cγ′H3), 19.72, 19.70, 19.63,
19.60, 19.53, 19.44, 19.41, 19.35, 19.24, 19.1, 19.02, 19.00, 17.43,
17.40, 17.3, and 17.1 (L-Lac CβH3 and Val, D-Hyi Cγ,γ′H3) ppm; 19F
NMR (acetone-d6, 470 MHz): δ −153.51 (d, J = 17 Hz, 2F, ortho-F),
−159.15 (t, J = 21 Hz, 1F, para-F) −163.63 (dd, J1 = 21 Hz, J2 = 17
Hz, 2F, meta-F) ppm; HRMS (ESI-TOF) m/z: [M + K]+ Calcd for
C63H92F5N7O22K+ 1432.5847; Found 1432.5856.
Pentafluorophenyl N-Carbonyl Glycinate (3). Dry HCl(g) was
bubbled into a stirred solution of Boc-Gly-OpFP 3a (0.40 g, 1.17
mmol) in anhydrous diethyl ether (20 mL), kept at 0 °C, for ca. 30
min. Stirring was continued at r.t. until TLC analysis (silica gel, eluent
CH2Cl2) revealed complete substrate consumption (4 h). The white
solid that precipitated during the reaction was collected by filtration,
washed with anhydrous diethyl ether, and dried under vacuum,
providing 0.32 g (1.15 mmol, yield 98%) of glycine pentafluorophenyl
ester hydrochloride (3b); HRMS (ESI-TOF) m/z: [M]+ Calcd for
LC-HRMS Studies on Analog 4 Derivatization. Transforma-
tions 1 and 2: 0.5 mg of 4 was dissolved in 1 mL of the appropriate
solvent (40% aqueous acetone for 1 and anhydrous CH3OH for 2),
and the solution left standing at r.t. until the LC-HRMS analysis
revealed that 4 had consumed (60 h); cbx-4: HRMS (ESI-TOF) m/z:
+
C8H5F5NO2 242.0235; Found 242.0255. Under a N2 atmosphere, a
three-necked flask was charged with freshly prepared glycine
pentafluorophenyl ester hydrochloride 3b (0.29 g, 1.05 mmol) and
anhydrous toluene (10 mL). The resulting suspension was then heated
at reflux. A neck of the flask was connected to a refrigerant kept at −10
°C, while a glass needle, dipping into the reaction suspension, was
jointed to the other neck. The glass needle was connected by a tube to
a Schlenk tube surmounted by a funnel for addition of solids charged
with solid triphosgene. The Schlenk tube contained 1 mg of Cu-
phtalocyanine in 2 mL of anhydrous toluene at 90 °C. Small portions
of triphosgene were added to the Schlenk tube every 10 min, allowing
a gentle stream of COCl2 to bubble into the reaction flask until glycine
pentafluorophenyl ester hydrochloride (3b) dissolved, and the reaction
mixture turned clear (ca. 3 h). Then, the reaction mixture was cooled
to r.t., and the excess COCl2 and HCl removed from the reaction flask
by a stream of N2 (collecting the outflowing gas into a flask containing
ethanol). The resulting solution was filtered under N2, to remove
traces of unreacted substrate, and concentrated under reduced
pressure to provide pentafluorophenyl N-carbonyl glycinate (3, 0.25
+
[M + NH4]+ Calcd for C57H97N8O22 1245.6717; Found 1245.6714;
+
met-4: HRMS (ESI-TOF) m/z: [M + NH4]+ Calcd for C58H99N8O22
1259.6874; Found 1259.6870.
Transformations 3 and 4: 0.5 mL of a 0.72 mM stock solution of 4
in anhydrous CH2Cl2 was mixed with 50 μL of a 11 mM solution of n-
pentylamine (for 3) or p-methoxyaniline (for 4) in anhydrous CH2Cl2.
LC-HRMS analysis of the mixture revealed consumption of the
substrate in a few minutes; pen-4: HRMS (ESI-TOF) m/z: [M +
NH4]+ Calcd for C62H108N9O21+ 1314.7660; Found 1314.7624; anis-4:
+
HRMS (ESI-TOF) m/z: [M + NH4]+ Calcd for C64H104N9O22
1350.7296; Found 1350.7282.
Transformation 5: To 1 mL of a 0.72 mM stock solution of 4 in
anhydrous CH2Cl2, 0.2 mg (1.5 equiv) of leucine methyl ester
hydrochloride, followed by 0.26 mg (3 equiv) of DMAP were added.
LC-HRMS analysis of the mixture revealed consumption of the
substrate within 2 h; leu-4: HRMS (ESI-TOF) m/z: [M + NH4]+
1
g, 0.94 mmol, yield 90%, purity ≥98% by H NMR) as a white solid,
+
1
Calcd for C64H110N9O23 1372.7715; Found 1372.7733.
mp 48.5−49.2 °C; H NMR (CDCl3, 500 MHz): δ 4.38 (s, CH2)
1
ppm; 13C{1H} NMR (CDCl3, 125 MHz): δ 165.7, 140.9 (m, JCF
=
Mitochondrial Depolarization Assays. cbx-4 and met-4,
obtained as described above, were purified by semipreparative
HPLC (column: Supelcosil LC-18, 250 × 10 mm ID, 5 μm;
conditions: isocratic elution methanol−water 9:1, UV detector 220
nm). The collected fractions, concentrated under reduced pressure
and then lyophilized, contained each product with purity ≥94%
(HPLC). Rat-liver mitochondria were isolated in mannitol-sucrose
medium as previously described,14 and the oxidative phosphorylation
efficiency was assayed by measuring the respiratory control index.14
Incubations were carried out at 25 °C in a standard medium
containing 125 mM KCl and 20 mM HEPES-Tris, pH 7.4. ΔΨm
variations were followed fluorimetrically, according to the safranine O
method,14 with the spectrofluorimeter set at the excitation and
emission wavelengths of 520 and 580 nm, respectively. Typically, to a
cuvette containing 3.0 mL of the standard medium, rat-liver
mitochondria (1 mg/mL), 6 μM rotenone, and 10 μM safranine O
were added. After equilibration, required to dissipate the ΔΨm
251 Hz, CF), 140.0 (m, 1JCF = 254 Hz, CF), 137.9 (m, 1JCF = 252 Hz,
CF), 126.8 (NCO), 124.4 (pFP CO), 43.7 ppm; 19F NMR
(CDCl3, 470 MHz): δ −152.8 (d, J = 17 Hz, 2F, ortho-F), −156.8 (t, J
= 22 Hz, 1F, para-F), −162.4 (dd, J1 = 22, J2 = 17 Hz, meta-F) ppm;
FT-IR (KBr): ν 2259 cm−1; GC-MS (EI, 70 eV) m/z (r.i.): 267 (M+,
+
0.5), 239 (M+ − CO, 8.6), 184 (pFPOH+, 2), 56 (OCNCH2 , 100);
HRMS (EI) m/z: [M]+ Calcd for C9H2F5NO3 266.99548; Found
+
266.99414.
Cyclo[(D-α-hydroxyisovaleryl-D-valyl-L-lactyl-L-valyl)2(D-α-hy-
droxy-β-(oxycarbonylglycine pentafluorophenyl ester)isovaleryl-D-
valyl-L-lactyl-L-valyl)] (4). N2 was flowed through a 5 mL round-
bottomed flask by means of a stainless-steel needle inserted into a
septum. Analog 2 (21 mg, 18.6 μmol), pentafluorophenyl N-carbonyl
glycinate (3, 99.4 mg, 372 μmol, 20 equiv), and MoO2Cl2(dmf)2 (6.4
mg, 18.6 μmol) were sequentially added, and the septum secured to
the flask. Under stirring, 100 μL of anhydrous CH2Cl2 were added by
D
J. Org. Chem. XXXX, XXX, XXX−XXX