Microwave Peptide Synthesis
FULL PAPER
with a 5 mm BBO probe; the spectra were processed with MestReNova
version 6. Chemicals were purchased from Sigma–Aldrich, Fluka, Nova-
Biochem, Iris Biotech GmbH, or Cambridge Isotope Laboratories. N -
TFA/TES/H
tate with Et
m/z calcd for C39
2
O (95:2.5:2.5) for 2 h at RT. The crude peptide was precipi-
2
O and analyzed by LCMS with a C18 column. MS (ESI):
a
+
62 10
H N O12 (average): 863.0 Da [M+H] ; found: 863.0.
Fmoc amino acids contained the following side-chain protecting groups,
Variable-temperature NMR experiments
unless otherwise stated: Fmoc-Arg
Asp(OtBu), Fmoc-Glu(OtBu), Fmoc-Gln
Fmoc-Lys Boc)-OH, Fmoc-Ser(tBu), Fmoc-Thr
(Boc)-OH, and Fmoc-Tyr(tBu)-OH. A standard Fmoc-RAM-TG resin
Rapp Polymer GmbH; loading: 0.23–0.24 mmolg ) was used as the pre-
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13
Sample preparation: C-labeled Boc-Ala-OH (5 mg, 0.026 mmol) and the
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N
ACHTUNGTRENNUNG
7
coupling reagent (0.029 mmol) were dissolved in [D ]DMF (1 mL) and
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ACHTUNGTRENNUNG
transferred into a 5 mm NMR tube. The samples were placed in the
NMR spectrometer and heated to the appropriate reaction temperature.
Each sample was locked, tuned, matched, and shimmed manually before
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ACHTUNGTRENNUNG
À1
(
ferred resin. Peptide synthesis was performed on a fully automated pep-
13
acquiring a C NMR spectrum that represented time point t, while these
TM
tide synthesizer from Biotage AB (Syro Wave ).
solution were stable. The sample was subsequently ejected from the
NMR instrument and DIEA (8.9 mL, 0.052 mmol) was added. The time
was started upon the addition of DIEA and the sample was re-inserted
into the magnet and allowed a short time for temperature equilibration
and shimming. The total time for ejection of the sample, addition of
DIEA, and re-insertion was about 30–45 s; thus, the reaction temperature
Solution stability studies
NMR spectroscopy: The stability of HBTU, HATU, and COMU were
1
measured as 0.13m solutions in [D
7
]DMF by H NMR spectroscopy. The
open-vial solutions were stored in 4 mL HPLC vials (no stirring) between
measurements and the closed-vial solutions were stored in the NMR tube
between measurements, both at RT.
1
3
was considered to be relatively stable during this short interval. C-spec-
tra were acquired with 64 transients and fixed receiver gain for all sam-
2
General procedure for the synthesis of H-WFTTLISTIM-NH (JR se-
1
3
ples, by using a C experiment with power-gated decoupling, 308 flip
angle, 64k spectral width, and 3 s repetition time to allow for full relaxa-
tion. Experiments were repeated with intervals of 3, 5, 30, 60, or 120 min
quence, Table 2): In all cases, the peptide syntheses were performed on
Fmoc-RAM-TG resin on a 0.05 mmol scale with different coupling re-
a
agents, as shown in Table 2. N -Fmoc deprotection was performed in two
(
starting with shortest values at the beginning of the reaction) to allow
stages: 1) piperidine in DMF (2:3) for 2 min at 608C and 2) piperidine in
DMF (1:4) for 2 min at 608C. This deprotection was followed by washing
the resin with NMP (4ꢂ45 s). The coupling reactions were performed by
for the acquisition of sufficient data points to characterize the reaction.
a
using N -Fmoc amino acids (5.2 equiv, 0.5m), coupling reagents HBTU,
HATU, COMU, or DIC (5 equiv, 0.44m) in DMF, and DIEA (10.4 equiv,
2
m) in NMP. Additives, such as HOBt, HOAt, and Oxyma, were added
to the amino acids stock solution (5.2 equiv, 0.5m) when required. In all
cases, the coupling reactions were performed for 5 min at 758C, followed
by washing the resin with NMP (3ꢂ45 s). The overall synthesis time was
Acknowledgements
We gratefully acknowledge support from Biotage AB, Sweden.
4
h and 33 min for each peptide. The peptide was released from the resin
by using TFA/TES/H O (95:2.5:2.5) for 2 h at RT. The crude peptide was
precipitate with Et O (when possible) and analyzed by LCMS with a C18
column. MS (ESI): m/z calcd for C58 14S (average): 1211.5 Da
2
2
[
1] Abbreviations for the coupling reagents: N-[(1H-benzotriazol-1-yl)-
(dimethylamino)methylene]-N-methylmethanaminium hexafluoro-
phosphate N-oxide (HBTU), N-[(dimethylamino)-1H-1,2,3-triazolo-
4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluoro-
phosphate N-oxide (HATU), 1-hydroxybenzotriazole (HOBt), 1-hy-
droxy-7-azabenzotriazole (HOAt), diisopropylcarbodiimide (DIC),
-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-
morpholinomethylene)]methanaminium hexafluorophosphate
COMU), 1-[(dimethylamino)-(morpholino)methylene]-1H-benzo-
triazolium hexafluorophosphate 3-oxide (HDBA), 1-[(dimethylam-
ino)(morpholino)-methylene]-1H-[1,2,3]triazolio-[4,5-b]pyridinium
hexafluorophosphate 3-oxide (HDMA), O-[cyano(ethoxy-carbonyl)-
methylidene]amino-1,1,3,3-tetramethyluronium
phate (HOTU).
90 12
H N O
+
ACHTUNGTRENNUNG
[
M+H] ; found: 1211.6.
General procedure for the synthesis of H-YGGFL-NH
2
: The peptide syn-
[
thesis was performed on Fmoc-RAM-TG resin on a 0.05 mmol scale on
a
a Biotage SyroWaveꢃ. The first N -Fmoc deprotection was performed
manually with piperidine/DMF (1:4) for 2+10 min followed by washing
1
with NMP. The resin was then placed inside the microwave cavity of the
TM
Syro Wave and the automated peptide synthesis was started with a cou-
(
a
pling time of 3 min and N -Fmoc deprotection for 2+2 min at 608C with
piperidine/DMF (1:4). The coupling reactions were performed by using
AHCTUNGTRENNUNG
a
N -Fmoc amino acids (5.2 equiv, 0.5m), COMU (5 equiv, 0.44m) in DMF,
and DIEA (10.4 equiv, 2m) in NMP. All of the coupling reactions were
performed by using a stock solution of COMU, which was dissolved at
the start of the first synthesis and placed in an open vial. The resin was
replaced before the start of each synthesis: t=0, 2, 4, 6, and 24 h after
COMU was dissolved in DMF. Each peptide synthesis took 1 h and
hexafluorophos-
[
[
6] A. El-Faham, F. Albericio, J. Pept. Sci. 2010, 16, 6–9.
7] R. Subirꢄs-Funosas, R. Prohens, R. Barbas, A. El-Faham, F. Alberi-
cio, Chem. Eur. J. 2009, 15, 9394–9403.
[9] L. Malik, A. P. Tofteng, S. L. Pedersen, K. K. Sørensen, K. J. Jensen,
J. Pept. Sci. 2010, 16, 506–512.
2
4 min to complete. A similar experiment was performed in which the
[
[
stock solution of COMU was placed in a closed vial. The experiment was
then started at t=0, 2, 4, 6, 24, 48 h, and 6 days after COMU was dis-
solved in DMF. The peptides were released from the resins by using
TFA/TES/H
cipitate with Et
m/z calcd for C28
Aib coupling reactions: The relative conversion of Aib onto resin-bound
H-Aib-Ile-Asp(OtBu)-Tyr(OtBu)-Ile-Asn(Trt)-Gly was performed in a Bi-
otage Initiator at 758C for 20 min. The resin-bound H-Aib-Ile-Asp-
(OtBu)-Tyr(OtBu)-Ile-Asn(Trt)-Gly peptide was synthesized by using
2
O (95:2.5:2.5) for 2 h at RT. The crude peptides were pre-
[
[
2
O and analyzed by LCMS with a C18 column. MS (ESI):
H N O (average): 554.6 Da [M+H] ; found: 555.5.
38 6 6
+
A
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ACHTUNGTRENNUNG
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ACHTUNGTRENNUNG
conventional SPPS. Fmoc-Aib-OH was coupled by using either HATU,
COMU, or DIC-Oxyma (1.1 or 4 equiv) on a 0.025 mmol scale. During
the HATU- and COMU-activated coupling reactions, DIEA (4.4 or
[12] L. A. Carpino, H. Imazumi, A. El-Faham, F. J. Ferrer, C. Zhang, Y.
Lee, B. M. Foxman, P. Henklein, C. Hanay, C. Mꢅgge, H. Wenschuh,
8
equiv) was used. The DIC-Oxyma coupling reactions were performed
without the use of base. Subsequently, the resin was washed (3ꢂNMP, 3ꢂ
CH Cl , and 3ꢂNMP), the Fmoc group was removed by using 20% pi-
2
2
peridine in DMF (2ꢂ5 min), and finally another wash cycle was per-
formed as above. The peptides were released from the resin by using
[13] T. Redemann, G. Jung, Proc. 24th Eur. Pept. Sym. 1996, 749–750.
Chem. Eur. J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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