1450 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Be´lec et al.
66.3, 66.8, 66.9, (118.2), 118.9, 119.8, (119.9), 125.1, (125.2),
(126.7), 126.8, 127.0, 127.6, (127.6), (127.9), 128.0, 129.4,
(129.5), (131.5), 131.8, (141.1), 141.2, (143.5), 143.9, 144.4,
(143.7), 154.8, (155.7), 171.0, (171.4), (171.8), 172.3; HRMS
calcd for C49H51N2O5S1 (MH+) 779.3519, found 779.3476.
mL, 600 mol %), and HOBt (410 mg, 300 mol %) in DMF (20
mL, 10 mL/g of resin) and agitated for 1 h when a negative
ninhydrin test was observed.30 The resin was sequentially
washed with 10 mL/g of the following solutions: DMF (2 × 2
min), piperidine in DMF (20% v/v, 1 × 2 min, 1 × 3 min and
1 × 10 min) and DMF (4 × 2 min). The resin was agitated
with a solution of 4-[(R,S)-R-[1-(9H-fluoren-9-yl)methoxyform-
amido]-2,4-dimethoxybenzyl]phenoxyacetic acid (Knorr linker;31
826 mg, 150 mol %), TBTU (600 mg, 150 mol %), HOBt (300
mg, 150 mol %) and DIEA (600 µL, 300 mol %) in DMF (10
mL/g of resin) for 1 h, rinsed with DMF (10 mL/g of resin, 3 ×
2 min) and capped by agitating with Ac2O (30 µL) and DIEA
(30 µL) in DMF (10 mL/g of resin) for 30 min.
P ep tid e Syn th esis. Synthesis was conducted, after resin
deprotection using piperidine in DMF (10 mL/g of resin, 20%
v/v, 1 × 2 min, 1 × 3 min and 1 × 10 min) followed by washing
with DMF (10 mL/g of resin, 4 × 2 min), by agitation with the
Fmoc-protected amino acid (150 mol %), TBTU (150 mol %),
HOBt (100 mol %, except in the case of Gly) and DIEA (300
mol %) in DMF for 1 h. The resin was agitated with N2 bubbles
during coupling, rinsing and deprotection sequences. Elonga-
tion of the linear peptides was performed on a semiautomatic
peptide synthesizer. Ninhydrin tests were performed after each
coupling reaction.30 In cases where a ninhydrin test of the resin
showed incomplete coupling, the resin was resubmitted to the
same conditions for an additional 0.5 h. This process was used
with N-Fmoc-Gly, N-Fmoc-Leu, dipeptide 6, N-Fmoc-Asn(Trt),
N-Fmoc-Pro, N-Fmoc-Gln(Trt), N-Fmoc-Ile, N-Fmoc-Tyr(O-t-
Bu), N-Fmoc-S-(Trt)Cys, S-(Trt)Mpa, and S-(4-MeOBn)dPen.
[Cys(H)1,Cys(H)6,5-t-Bu P r o7]oxytocin . The N-Fmoc group
was removed from the resin-bound nonapeptide with piperi-
dine (as described above). Cleavage from the resin (1.97 g, 0.26
mmol/g) with simultaneous side chain deprotection was con-
ducted by treating the resin with 10 mL/g of a cocktail
containing TFA (90%), thioanisole (5%), ethanedithiol (3%) and
anisole (2%) and agitating with a mechanical shaker for 2 h
at room temperature. Subsequent filtration, rinsing with TFA
(2 × 2 mL) and precipitation in Et2O (100 mL/g of resin) at 0
°C, followed by filtration through a membrane filter afforded
the unprotected linear nonapeptide. Lyophilization gave 210
mg of a white powder that was shown to be 72% pure by RP-
HPLC (tR ) 8.3) using an eluant of 20-60% B in A over 20
min with a flow rate of 1.5 mL/min and the detector centered
at λ ) 230 nm; LRMS calcd for C47H76N12O12S2 (MH+) 1065,
found 1065. Half of the material was taken to the next step.
[5-t-Bu P r o7]oxytocin (1). A solution of Cys-Tyr-Ile-Gln-
Asn-Cys-5-t-BuPro-Leu-Gly-NH2 (104 mg, of crude post-cleav-
age material) in DMSO (40 mL) was adjusted to a 0.2 mg/mL
concentration with a pH 6 buffer (7% CH3CO2H and (NH4)2-
CO3 in H2O)32 and agitated for 20 h. On the observation of a
negative Ellman test,33 the maximum quantity of DMSO was
removed by vacuum distillation and the peptide was precipi-
tated on the addition of CH3CN and Et2O and collected on a
membrane filter. Purification on preparative C18 RP-HPLC,
pooling of purest fractions and subsequent lyophilization gave
1 as a white powder (30.5 mg, 29 µmol, 11% overall yield from
initial loading on resin; an additional 12 mg of 87% purity was
N-F m oc-S-t r it yl-L-cyst ein yl-(2S,5R)-5-ter t-b u t ylp r o-
lin e (6). A solution of N-Fmoc-S-trityl-L-cysteinyl-(2S,5R)-5-
tert-butylproline allyl ester (5; 1.79 g, 2.30 mmol) in freshly
distilled and degassed CH2Cl2 (23 mL) was treated with Pd-
(PPh3)2Cl2 (82 mg, 115 µmol, 5 mol %), followed by a solution
of Bu3SnH (3.75 mL, 13.9 mmol, 600 mol %) in CH2Cl2 (2-3
mL) which was added dropwise until analysis by TLC showed
complete consumption of the allyl ester (Rf ) 0.24, 1:24:75
AcOH:EtOAc:hexanes) after 10-15 min. The mixture was
evaporated to a residue that was partitioned between hexanes
(25 mL) and CH3CN (25 mL) containing acetic acid (1.2 mL).
The CH3CN phase was washed with hexanes (4 × 25 mL),
evaporated and purified by chromatography on silica gel using
a gradient of 0-2% ethanol in CHCl3. Evaporation of the
collected fractions gave 1.69 g (96% yield) of 6 as a foam: [R]20
D
-26.2 (c 0.6, MeOH); TLC Rf ) 0.3 (1:24:75 AcOH:EtOAc:
hexanes); 1H NMR δ (CDCl3) 0.88 (s, 9 H), 1.53-1.90 (m, 2
H), 1.93-2.24 (m, 2 H), 2.39-2.67 (m, 2 H), 4.02-4.40 (m,
4.5H), 4.45-4.57 (m, 1 H), 4.62 (dd, 0.5 H, 7.8, 13.2), 4.87 (d,
0.57 H, 8), [5,63 (s, 0.43 H)], 7.23 (m, 3 H), 7.28 (m, 8H), 7.40
(m, 8H), 7.55 (m, 2 H), 7.75 (m, 2H); 13C NMR δ (CDCl3) 25.4,
(25.5), 26.1, (26.7), (27.2), 27.3, 27.7, (29.6), 33.4, (34.0), 35.2,
(35.9), (46.8), 46.9, 51.0, (52.3), (60.5), 62.0, 66.9, (67.2), (67.4),
68.0, (119.8), 120.0, 124.8, 125.0, (125.0), (125.2), (126.9), 127.0,
127.7, (127.8), 128.0, 129.4, 141.2, (141.3), (143.3), 144.1, 155.6,
171.3, 175.3; LRMS calcd for C46H47N2O5S1 (M+) 738.3, found
761.1 (MNa+) and 777.1 (MK+); HRMS calcd for C46H47N2O5S1-
Na (MNa+) 761.3025, found 761.3021.
S-(Tr ityl)m er ca p top r op ion ic a cid (Tr t-Mp a ) was pre-
pared according to the literature procedure for the tritylation
of cysteine.29 A solution of mercaptopropionic acid (0.3 mL, 3.46
mmol, 100 mol %), triphenylmethanol (900 mg, 3.46 mmol, 100
mol %; recrystallized from Et2O:hexanes), glacial acetic acid
(3.36 mL, 58.8 mmol, 1700 mol %) and boron trifluoride
etherate (0.48 mL, 3.8 mmol, 110 mol %) was stirred for 1 h
at 100 °C and for an additional hour at 22 °C. The mixture
was treated with Et2O (6 mL) and water (5 mL) followed by
sodium acetate (1.5 g) and additional water (20 mL). A white
precipitate was formed that was filtered and rinsed with water
to give 464 mg (1.33 mmol, 39% yield) of S-(trityl)mercapto-
propionic acid: 1H NMR δ (CDCl3) 2.24 (t, 2 H, 7.3), 2.47 (t, 2
H, 6.9), 7.20-7.44 (m, 15 H); 13C NMR δ (CDCl3) 26.4, 32.9,
67.0, 126.6, 127.8, 129.4, 144.4, 175.5.
S-(p -Met h oxyb en zyl)-â,â-d im et h ylm er ca p t op r op ion -
ic a cid (p-MeOBn -d P en ) was prepared by a modification of
the literature procedure for the synthesis of S-(p-methylben-
zyl)-â,â-dimethylmercaptopropionic acid.18 A solution of 3,3-
dimethylacrylic acid (1.4 g, 14 mmol, 100 mol %) and 4-methoxy-
R-toluenethiol (1.8 mL, 14 mmol, 100 mol %) in piperidine (2.1
mL, 21 mmol, 150 mol %) was heated at a reflux for 16 h,
cooled to room temperature and treated with 2 N HCl (20 mL).
The mixture was extracted with Et2O (3 × 20 mL) and the
combined Et2O fractions were extracted with saturated NaH-
CO3 (3 × 20 mL). The combined aqueous fractions were
acidified to pH ≈ 2 with 2 N HCl (25 mL) and extracted with
Et2O (3 × 15 mL). The organic phases were combined, dried
and evaporated to a residue that was purified by crystallization
in Et2O:hexanes (1:1) to give 1.36 g (5.3 mmol, 38% yield) of
S-(p-methoxybenzyl)-â,â-dimethylmercaptopropionic acid: 1H
NMR δ (CDCl3) 1.50 (s, 6H), 2.67 (s, 2 H), 3.79 (s, 2 H), 3.79
(s, 3 H), 6.84 (d, 2 H, 8.5), 7.27 (d, 2H, 8.4); 13C NMR δ (CDCl3)
28.6, 32.6, 43.7, 46.8, 55.1, 113.9, 129.3, 130.0, 158.5, 176.9;
HRMS calcd for C13H17O3S1 (M+ - H) 253.0898, found 253.0908.
also obtained from the less pure fractions): RP-HPLC (tR
)
13.06) using an eluant of 20-40% B in A over 20 min and a
flow rate of 1.0 mL/min with the detector centered at λ ) 230
nm; LRMS calcd for C47H75N12O12S2 (MH+) 1063, found 1063;
amino acid composition Asp 1.02 (1), Glu 1.01 (1), Gly 0.98
(1), Ile 1.03 (1), Leu 1.00 (1), Tyr 0.96 (1).
[Mp a (H)1,Cys(H)6,5-t-Bu P r o7]oxytocin . The nonapeptide
was cleaved from the resin (500 mg, 0.26 mmol/g) according
to the procedure described above to afford 26 mg of linear
peptide that was shown to be 63% pure by RP-HPLC (tR
)
13.4) using an eluant of 5-65% B in A over 15 min and a flow
rate of 1.5 mL/min with detection centered at λ ) 220 nm;
LRMS calcd for C47H76N11O12S2 (MH+) 1050.5, found 1050.4.
P r ep a r a tion of Solid Su p p or t. Benzhydrylamine resin
hydrochloride (2 g, loading ≈ 0.51 mmol/g, 1% DVB, 100-200
mesh) was washed for 1 min three times with 10 mL/g of each
of the following reagents: 5% DIEA/CH2Cl2; CH2Cl2; DMF. The
resin was treated with a solution of N-(Fmoc)aminocaproic acid
(400 mg, 100 mol %), TBTU (980 mg, 300 mol %), DIEA (1
[Mp a 1,5-t-Bu P r o7]oxytocin (2). A solution of Mpa-Tyr-Ile-
Gln-Asn-Cys-5-t-BuPro-Leu-Gly-NH2 (26 mg, of crude post-
cleavage material) in DMSO was oxidized to disulfide accord-
ing to the procedure described above and the reaction mixture