A. El-Faham, R. Subirós-Funosas, F. Albericio
FULL PAPER
trated to a small volume (1:4) under reduced pressure, and then
dry ether was added to afford the product as a white solid in high
purity.
Z-Aib-4-chloroanilide:[16] An authentic sample was prepared by the
reaction of Z-Aib-Cl (0.125 mmol) with 4-chloroaniline
(0.125 mmol) in the presence of Et3N (0.125 mmol) in DCM for
3 h at room temperature. The product was recrystallized from
DCM/hexane to afford the product in 66% yield; m.p. 169–170 °C.
1H NMR (400 MHz, CDCl3): δ = 1.61 (s, 6 H, 2 CH3), 5.13 (s, 2
H, CH2), 7.08 (dd, 2 H, CH ar), 7.11–7.34 (m, 6 H, 6 CH ar), 7.45
(d, 2 H, 2 CH ar) 8.56 (br. s, 1 H, NH) ppm.
N-[(2,2-Dimethyl-4,6-dioxo-1,3-dioxan-5-ylideneaminooxy)(di-
methylamino)methylene]-N-methylmethanaminium Hexafluorophos-
phate (HTMU, 6): The product was obtained as a white solid from
1
CH3CN and ether in 94% yield (3.9 g); m.p. 177–178 °C (dec.). H
NMR (400 MHz, [D6]acetone): δ = 1.89 (s, 6 H, 2 CH3), 3.3.37 (s,
12 H, 4 CH3) ppm. 13C NMR (100 MHz, [D6]acetone): δ = 27.78,
40.71, 107.73, 141.81, 150.14, 154.75, 162.32 ppm. C11H18F6N3O5P
(417.09): calcd. C 31.66, H 4.35, N 10.07; found C 31.89, H 4.47,
N 10.29.
Solid-Phase Synthesis of H-Tyr-Aib-Aib-Phe-Leu-NH2:[13] The syn-
thesis was carried out in a plastic syringe, attached to a vacuum
manifold so as to effect rapid removal of reagents and solvent. The
Fmoc-RinkAmide-AM-PS resin, with a loading of 0.63 mmolg–1
(100 mg), was washed with DCM and DMF (2ϫ10 mL each) and
then treated with 20% piperidine in DMF (10 mL) for 10 min. The
resin was then washed with DMF, DCM, and DMF (2ϫ10 mL
each) and acylated with a solution of Fmoc-Leu-OH (3 equiv.), the
corresponding coupling reagent (3 equiv.) and DIEA (6 equiv.) in
DMF (0.4 mL, previously preactivated for 1–2 min; 7-min preacti-
vation in the case of DIC/HONM). The mixture was added to the
resin and manually stirred slowly for 1 min and left to stand for
30 min (30-min double coupling only for Aib-Aib). After peptide
coupling, the resin was washed with DMF and then deblocked by
treatment with 20% piperidine in DMF for 7 min. The resin was
washed with DMF, DCM, and DMF (2ϫ10 mL each) and then
coupling with the next amino acid as explained before and deblock-
ing were repeated to obtain the pentapeptide. The peptide was
cleaved from the resin with TFA/H2O (9:1) at room temperature
for 2 h. TFA was removed in vacuo, and the crude peptide was
precipitated with ether. The weight of the crude peptide was re-
corded, and the ratio of the penta- and tetrapeptide was determined
by HPLC analysis by using a Sun fire C18 (4.6ϫ150 mm, 5 µm)
column, with a linear gradient of 0 to 100% of 0.036% TFA in
CH3 CN/0.045 %TFA in H2 O ove r 1 5 m in , f low rat e =
1.0 mLmin–1, detection at 220 nm: tR = 6.68 (penta), 6.78 (des-
Aib) min. HPLC–MS showed the expected mass for the penta at
m/z = 611.0, and also for des-Aib at m/z = 526. Neither des-Tyr
(m/z = 448) nor the tripeptide des-Aib,Tyr (m/z = 363) were ob-
served.
1-[1-(2,2-Dimethyl-4,6-dioxo-1,3-dioxan-5-ylideneaminooxy)dimeth-
ylaminomorpholinomethylene]methanaminium Hexafluorophosphate
(HMMU, 7): The product was obtained as a white solid from
CH3CN and ether in 90% yield (4.0 g); m.p. 200–202 °C (decomp.,
with eff.). 1H NMR (400 MHz, [D6]acetone): δ = 1.89 (s, 6 H, 2
CH3), 3.41 (s, 6 H, 2 CH3), 3.82–3.84 (m, 4 H, 2 CH2), 3.88–3.91
(m, 4 H, 2 CH2) ppm. 13C NMR (100 MHz, [D6]acetone): δ = 27.8,
40.77, 49.96, 66.07, 107.80, 141.54, 150.13, 154.78, 161.48 ppm.
C13H20F6N3O6P (459.10): calcd. C 34.00, H 4.39, N 9.15; found C
34.26, H 4.52, N 9.32.
1-{[1-(2,2-Dimethyl-4,6-dioxo-1,3-dioxan-5-ylideneaminooxy)-
dimethylaminopyrrolidinomethylene]}methanaminium Hexafluoro-
phosphate (HDmPyMU, 8): The product was obtained as an off-
white solid from CH3CN and ether in 96% yield (4.2 g); m.p. 164–
165 °C (decomp., with eff.). 1H NMR (400 MHz, [D6]acetone): δ =
1.89 (s, 6 H, 2 CH3), 2.06–2.11 (m, 4 H, 2 CH2), 3.36 (s, 6 H, 2
CH3), 3.94–3.98 (m, 4 H, 2 CH2) ppm. 13C NMR (100 MHz, [D6]-
acetone): δ = 25.09, 27.75, 40.39, 51.51, 107.65, 141.58, 150.13,
154.79, 159.58 ppm. C13H20F6N3O5P (443.10): calcd. C 35.22, H
4.55, N 9.48; found C 35.49, H 4.46, N 9.69.
Formation of Active Esters Using Distinct Coupling Reagent in
DMF: (Z-Aib-OH) was preactivated with the coupling reagent
HMMU (11), COMU (13), HDMB (14), or HDMA (15) for 3 min
in the presence of DIEA (2 equiv.) in DMF at room temperature.
Then, an aliquot (20 µL) of the solution was picked up and diluted
to 2 mL with a mixture of CH3CN/H2O (1:2), and 10 µL was in-
jected into a reverse-phase HPLC apparatus, equipped with a PDA
detector, using a Sun fire C18 (5 µm, 4.6ϫ150 mm) column, a linear
gradient 0 to 100% of 0.036% TFA in CH3CN/0.045%TFA in H2O
Solid-Phase Synthesis of H-Tyr-N-MeIle-N-MeIle-Phe-Leu-
NH2:[15] The synthesis was carried out in a plastic syringe, attached
to a vacuum manifold so as to effect rapid removal of reagents and
solvent. The Fmoc-RinkAmide-AM-PS resin, with a loading of
0.7 mmol g–1 (100 mg) was washed with DCM and DMF
(2ϫ10 mL each) and then treated with 20% piperidine in DMF
(10 mL) for 10 min. The resin was then washed with DMF, DCM,
and DMF (2ϫ10 mL each) and acylated with a solution of Fmoc-
Leu-OH (3 equiv.), the corresponding coupling reagent (3 equiv.),
over 8 min, flow rate = 1.0 mLmin–1, detection at 220 nm: tR
=
3.58 (HOAt), 3.85 (HOBt), 4.62 (HONM), 5.57 (Oxyma), 5.98 (Z-
Aib-OH), 7.19 (Z-Aib-Oxyma), 7.37 (Z-Aib-ONM), 7.25 (Z-Aib-
OAt), 7.84 (Z-Aib·OBt) min.
Reaction of Z-Aib-OH with 4-Chlororaniline Using Distinct Cou-
pling Reagents in DMF: Z-Aib-OH (0.125 mmol) was mixed with and DIEA (6 equiv.) in DMF (0.4 mL, previously preactivated for
the corresponding coupling reagent (0.125 mmol) and preactivated
for 3 min in the presence of DIEA (2 equiv.) in DMF (2 mL) at
room temperature. Then, 4-chloroaniline (0.125 mmol) was added
with stirring. The resulting solution (20 µL) was picked up and
diluted to 2 mL with a mixture of CH3CN/H2O (1:2), and 10 µL
was injected into reverse-phase HPLC apparatus, equipped with a
PDA detector, using a Sun fire C18 (5 µm, 4.6ϫ150 mm) column,
linear gradient 0 to 100% of 0.036% TFA in CH3CN/0.045%TFA
in H2O over 8 min, flow rate = 1.0 mLmin–1, detection at 220 nm:
tR = 3.58 (HOAt), 3.85 (HOBt), 4.62 (HONM), 5.57 (Oxyma), 5.98
1–2 min). The mixture was added to the resin and manually stirred
slowly for 1 min and allowed to stand for 30 min (1 h double cou-
pling in case of NMeIle-NMeIle). After peptide coupling, the resin
was washed with DMF and then deblocked by treatment with 20%
piperidine in DMF for 7 min. The resin was washed with DMF,
DCM, and DMF (2ϫ10 mL each) and then coupling with the next
amino acid as explained before and deblocking were repeated to
obtain the pentapeptide. The peptide was cleaved from the resin
with TFA/H2O (9:1) at room temperature for 2 h. TFA was re-
moved in vacuo, and the crude peptide was precipitated with ether.
(Z-Aib-OH), 7.19 (Z-Aib-Oxyma), 7.37 (Z-Aib-ONM), 7.25 (Z- The precipitate was redissolved in H2O and acetic acid (1:5) and
Aib-OAt), 7.84 (Z-Aib·OBt), 3.94 (4-chloroaniline), 7.77 (Z-Aib-4-
chloroanilide) min, as detected from injection of authentic samples.
The reaction rate was followed by the increase in the amount of
product and the decrease in the amount of active ester.
then lyophilized. The weight of the crude peptide was recorded,
and the ratio of the penta- and tetrapeptide was determined by
HPLC analysis by using a Sun fire C18 (4.6ϫ150 mm, 5 µm) col-
umn, with a linear gradient of 20 to 50 % of 0.036 % TFA in
3648
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Eur. J. Org. Chem. 2010, 3641–3649