Rapid Microwave-Assisted Solid Phase Peptide Synthesis

1592
Rapid Microwave-Assisted Solid Phase Peptide Synthesis
Rapid Máté Microwave-Assisted Solid Phase Peptide SynthesisErdélyi,
a,b Adolf Gogoll* a
a Department of Organic Chemistry, Uppsala University, Box 531, 751 21 Uppsala, Sweden
Fax +46(18)512524; E-mail: mate@kemi.uu.se, adolf@kemi.uu.se
b Department of Medicinal Chemistry, Uppsala University, Box 574, 751 23 Uppsala, Sweden
Fax +46(18)4714474
Received 20 May 2002
Dedicated to Prof. Hans-J. Schäfer on the occasion of his 65 th birthday
Abstract: A microwave-assisted, rapid solid phase peptide synthesis
procedure is presented. It has been applied to the coupling of
sterically hindered Fmoc-protected amino acids yielding di- and
tripeptides. Optimized conditions for a variety of coupling reagents
are reported. Peptides were obtained rapidly (1.5–20 min) and without
racemization.
Key words: SPPS, microwave, amino acids, peptide coupling, solid-phase
Since Merrifield’s pioneering work on solid phase peptide
synthesis (SPPS), peptide preparation has almost exclusively
been performed on resin. 1 The generation of combinatorial
libraries has caused a renaissance and growth of
interest in solid supported chemistry. Parallel to the developments
in combinatorial chemistry, it has been shown
that the use of microwave heating can be advantageous in
a large variety of organic reactions. 2 However, there have
been only very few reports on the use of microwave heating
in combination with solid phase synthesis, 3 possibly
due to the requirement of special heavy-walled vials for
microwave irradiation that makes resin handling rather
complicated, and problems to control reaction conditions.
In particular, enhancement of SPPS by the use of microwave
heating has so far received little attention. Peptide
synthesis performed in a kitchen microwave oven was
published in 1991. 4 However, the procedure described by
Wang et al is not easily reproducible, because of the use
of a commercial microwave oven for irradiation, and the
lack of temperature control. The couplings were made using
symmetric amino acid anhydrides or pre-formed Nhydroxybenzotriazole
activated esters, yielding 2–4 fold
reaction rate enhancements. In general, peptide chemistry
is today limited to room temperature conditions, originating
from the general belief of the heat-sensitivity of peptide
coupling reagents (Table).
Here, we describe a microwave-enhanced, rapid (1.5–20
min) procedure for the coupling of sterically hindered
amino acids on solid phase. The optimized conditions for
a variety of common coupling reagents yielded a significant
rate increase. Single mode irradiation with monitoring
of temperature, pressure and irradiation power versus
Synthesis 2002, No. 11, Print: 22 08 2002.
Art Id.1437-210X,E;2002,0,11,1592,1596,ftx,en;C01802SS.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0039-7881
SPECIAL TOPIC
Table Coupling Times and Temperatures for Peptide Synthesis on
Rink’s Amide Resin Employing a Variety of Coupling Reagents a
Coupling Reagent PyBOP Mukaiyama’s
Reagent
TBTU HATU
Reaction time (min) 20 10 10 1.5
Temperature (°C) 110 130 110 110
Solvent DMF CH 2Cl 2 DMF DMF
a The average pressure for reactions performed in DMF and CH2Cl 2
was 1–2 bar and 6–8 bar, respectively.
time was used throughout, making the procedure highly
reproducible.
Our goal was to investigate the compatibility of HATU, 5a
TBTU, 5b PyBOP5c and Mukaiyama’s reagent5d mediated
couplings at high temperatures. Reaction conditions for
the synthesis of a small tripeptide containing the three
most hindered natural amino acids (Fmo-Thr-Val-Ile-
NH2), and the Fmoc-Ala-Ile-NH2 or Fmoc-Thr-Ile-NH2 dipeptides were optimized. The coupling of Fmoc-protected
amino acids on polystyrene resin using Rink amide
linker was performed. No degradation of the solid support
was observed. Fast Fmoc-deprotection steps (15 minutes)
were conducted at room temperature, and the coupling
steps were performed using microwave irradiation. All
coupling steps were monitored by qualitative ninhydrin
test6 and by LC-MS investigation of peptides cleaved
from small amounts of the resin. Our optimized conditions
are shown in the Table.
The azobenzotriazole derivatives have shown an increased
coupling efficiency with increasing temperature
up to 110 °C. At higher temperatures the decomposition of
the reagents was indicated by the colour change of the reaction
mixtures. In contrast with the usual SPPS procedures
where double or triple coupling steps are needed for
completion, under microwave conditions, coupling was
complete already in a few minutes after a single coupling
step.
The absence of racemization during the high temperature
treatment of amino acids in the presence of a base (i-
Pr2NEt) was investigated by LC-MS and 1H NMR. The
presence of only one peak in the chromatogram (Figure 1)
of the synthesized peptides suggested the absence of dias-

SPECIAL TOPIC Rapid Microwave-Assisted Solid Phase Peptide Synthesis 1593
Figure 1 The LC-MS chromatogram of Fmoc-Ala-Ile-NH 2 prepared by PyBOP mediated couplings at 110 °C.
tereomeric compounds. The 1H NMR spectrum of this
material containing a single set of signals of the oligopeptide
suggested the presence of only one diastereomer. As
an example, the aliphatic region of the 1H NMR spectrum
of a synthesized dipeptide is shown in Figure 2.
The efficiency of the solid phase methodology was limited
by the need of transfer between different reaction vessels
to perform the coupling and washing steps. The high
pressure generated by volatile components during reac-
tions carried out with microwave irradiation makes the
use of special heavy-walled vials highly recommendable.
However, these vials are not suitable for filtration. Therefore,
the reaction mixtures were transferred for washing
and deprotection steps into plastic columns equipped with
a polypropylene frit, causing some loss of resin. Moderate
yields could therefore be obtained for the synthesis of the
di- (PyBOP: 60%; Mukaiyama’s reagent: 35%; TBTU:
41%; HATU 24%) and tripeptides (PyBOP: 48%; Mu-
Figure 2 The aliphatic region of the 1 H NMR spectrum of Fmoc-Ala-Ile-NH 2 showing the presence of one diastereomer after microwave
assisted synthesis (PyBOP mediated couplings at 110 °C).
Synthesis 2002, No. 11, 1592–1596 ISSN 0039-7881 © Thieme Stuttgart · New York

1594 M. Erdélyi, A. Gogoll SPECIAL TOPIC
kaiyama’s reagent: 65%; TBTU: 31%; HATU 42%). The
efficiency of the solid phase methodology including the
loading (PyBOP, i-Pr2NEt, DMF, 110 °C), cleavage (95%
TFA in CH2Cl2) and purification (preparative LC-MS)
steps was determined by attachment and cleavage of a single
Fmoc-Ile to the resin, yielding 60% of the expected
product. The efficiency of the methodology might be increased
by instrumental improvements, e.g., by the development
of heavy-walled vials more compatible with the
handling of resins used in solid phase synthesis.
This synthetic method will be of considerable interest for
incorporation of sterically hindered and deactivated nonnatural
amino acids in peptide synthesis on solid phase.
All reactions were conducted in heavy-walled glass Smith Process
Vials sealed with aluminum crimp caps fitted with a silicon septum.
The inner diameter of the vial filled to the height of 3.5 cm was 1.3
cm. The microwave heating was performed in a Smith Synthesizer single mode microwave cavity producing continuous irradiation at
2450 MHz (Personal Chemistry AB, Uppsala, Sweden). Reaction
mixtures were stirred with a magnetic stir bar during the irradiation.
The temperature, pressure and irradiation power were monitored
during the course of the reaction. The average pressure during the
reaction run in DMF was 1�2 bar, for the reactions run in CH2Cl2 it
was 6–8 bar. After completed irradiation, the reaction tube was
cooled with high-pressure air until the temperature had fallen below
39 °C (ca. 2 min). 1H NMR spectra were recorded for CD3CN solutions
at 400 MHz (Jeol JNM EX400 spectrometer) at r.t. Chemical
shifts are referenced indirectly to TMS via the residual solvent signal
(� =2.10). The 1H NMR spectra of all synthesized compounds
have been fully assigned using data from phase-sensitive DQF-
COSY7 and NOESY8 experiments. The ESI-MS spectra of the peptides
were obtained with a Finnigan ThermoQuest AQA mass spectrometer
(ESI 30eV, probe temperature 100 °C) equipped with a
Gilson 322-H2 GradientPump system, an SB-C18 analytical and an
SB-C8 (5 �m, 21.2 mm, 150 mm) preparative column. A H2O– MeCN–formic acid (0.05%) mobile phase was used with a gradient
of 20%�80% MeCN during 7 minutes on the analytical column,
and 30–80 minutes on the preparative column.
The starting materials were purchased from commercial suppliers
and were used without purification with the exception of CH2Cl2 which was freshly distilled over calcium hydride. Fmoc-Ala, Fmoc-
Ile, Fmoc-Val, PyBOP, and Rink amide MBHA resin were obtained
from Novabiochem, Fmoc-Thr(t-Bu)-OH from Alexis Biochemicals
(USA). TBTU was from Richelieu Biotechnologies (Canada).
HATU was purchased from PE Biosystems (United Kingdom). Trifluoroacetic
acid (99%), N,N-diisopropylethylamine (redistilled
99.5%), and piperidine (99%) were from Aldrich (Germany). DMF
was obtained from Fluka (Denmark).
Fmoc-Thr-Val-Ile-NH 2 (1); Method A
Procedure I
Rink amide resin (300 mg, 0.78 mmol/g, 0.234 mmol) was treated
with 20% piperidine in DMF (5 mL for 10 and 5 minutes) in a column
equipped with polypropylene frit placed in an overhead mixer.
The soln was then drained, and the resin was washed with DMF (3
� 5 mL) and CH 2Cl 2 (3 � 5 mL).
Procedure II
The resin from procedure I was transferred into a Smith Process
Vial and Fmoc-Ile (248 mg, 0.70 mmol), PyBOP (343 mg, 0.70
mmol), i-Pr2NEt (0.25 mL, 1.40 mmol) and DMF (3 mL) was added.
The mixture was irradiated in a microwave cavity at 110 °C for
20 min. Then, the mixture was transferred into a column equipped
Synthesis 2002, No. 11, 1592–1596 ISSN 0039-7881 © Thieme Stuttgart · New York
with a polypropylene frit using a Pasteur pipette. The soln was
drained and the resin was washed with DMF (3 � 5 mL) and CH 2Cl 2
(3 � 5 mL). The completion of the coupling was confirmed by Kaisers
test.
Thereafter, Fmoc-deprotection was performed using procedure I as
described above.
Procedure III
The resin from procedure I was transferred into a Smith Process
Vial and Fmoc-Val (238 mg, 0.70 mmol), PyBOP (343 mg, 0.70
mmol), i-Pr2NEt (0.25 mL, 1.40 mmol) and DMF (3 mL) was added.
The coupling was performed using procedure II as described
above. The completion of the coupling was confirmed by Kaisers
test and by cleavage of a small amount of the resin with 95% TFA
in CH2Cl2 and analytical LC-MS investigation of the cleaved peptide.
Thereafter, Fmoc-deprotection was performed using procedure
I as described above.
The resin was transferred into a Smith Process Vial and Fmoc-
Thr(t-Bu) (279 mg, 0.70 mmol), PyBOP (343 mg, 0.70 mmol), i-
Pr2NEt (0.23 mL, 1.32 mmol) and DMF (3 mL) was added. The
mixture was irradiated using procedure II, the completion of reaction
was confirmed by procedure III.
Procedure IV
Finally, the tripeptide was cleaved from the resin using 95% TFA in
CH2Cl2 (2 hours). The soln was separated from the resin by filtration,
the resin was washed with additional CH2Cl2 (5 mL) and the
combined phases were concentrated on a rotatory evaporator. The
residue was then dissolved in MeCN (1–2 mL) and was purified on
preparative LC-MS yielding 1 as a white solid (63 mg, 0.11 mmol,
48%).
Method B
Rink amide resin (300 mg, 0.78 mmol/g, 0.234 mmol) was deprotected
using procedure I. The resin was transferred into a Smith Process
Vial and Fmoc-Ile (248 mg, 0.70 mmol), 2-fluoro-1-methylpyridinium
tosylate (210 mg, 0.70 mmol), i-Pr 2NEt (0.25 mL, 1.40
mmol) and DMF (3 mL) was added and the subsequent synthesis
steps followed the procedure described for Method A, with the exception
of the coupling times and temperature (10 minutes at 130
°C), yielding as a white solid (83 mg, 0.15 mmol, 65%) 1.
Method C
Rink amide resin (300 mg, 0.78 mmol/g, 0.234 mmol) was deprotected
using procedure I. The resin was transferred into a Smith Process
Vial and Fmoc-Ile (248 mg, 0.70 mmol), TBTU (225 mg, 0.70
mmol), i-Pr 2NEt (0.25 mL, 1.40 mmol) and DMF (3 mL) was added
and the subsequent synthesis steps followed the procedure described
for Method A, with exception of the coupling times (10
minutes at 110 °C), yielding 1 as a white solid (41 mg, 0.075 mmol,
31%).
Method D
Rink amide resin (301 mg, 0.73 mmol/g, 0.232 mmol) was deprotected
using procedure I. The resin was transferred into a Smith Process
Vial and Fmoc-Ile (233 mg, 0.66 mmol), HATU (156 mg, 0.66
mmol), i-Pr 2NEt (0.23 mL, 1.32 mmol), DMF (3 mL) was added
and the subsequent synthesis steps followed the procedure described
for Method A, with exception of the coupling times (1.5
min. at 110 °C), yielding 1 as a white solid (129 mg, 0.098 mmol,
42%).
1 H NMR (400 MHz, CD3CN): � = 7.83 (d, J = 7.2 Hz, 2 H, Fmoc),
6.67 (br d, J = 7.6 Hz, 2 H, Fmoc), 7.42 (ddd, J = 1.2, 7.6, 7.6 Hz, 2
H, Fmoc), 7.34 (ddd, J = 1.2, 7.2, 7.6 Hz, 2 H, Fmoc), 7.15 (br d, 1
H, NH-Val), 6.80 (br d, 1 H, NH-Ile), 6.35 (br s, 1 H, NH), 6.08 (br
d, 1 H, NH-Thr), 5.79 (br s, 1 H, NH), 4.33 (dd, J = 5.9, 7.3 Hz, 1
H, CH �-Val), 4.31 (d, J = 6.6 Hz, 2 H, CH 2-Fmoc), 4.24 (t, J =6.6

SPECIAL TOPIC Rapid Microwave-Assisted Solid Phase Peptide Synthesis 1595
Hz, 1 H, CH-Fmoc), 4.17 (dd, J = 6.8 Hz, 1 H, CH�-Ile), 4.06–4.14
(m, 2 H, CH�-Thr, CH�-Thr), 1.7–1.81 (m, 2 H, CH�-Val, CH�-Ile), 1.44 (m, 1 H, CH2�(a)-Ile), 1.26 (d, J =Hz, 3 H, CH3-Thr),1.10 (m,
1 H, CH2�(b)-Ile), 0.8–0.9 (m, 12 H, CH3�-Ile, CH3�-Ile, CH3�-Val). ESI-MS: m/z = 553 (M + 1) + , 536, 522, 423, 179.
Fmoc-Ala-Ile-NH 2 (2); Method A2
Rink amide resin (300 mg, 0.73 mmol/g, 0.235 mmol) was deprotected
using procedure I. Then, the resin was transferred into a
Smith Process Vial and Fmoc-Ile (233 mg, 0.66 mmol), PyBOP
(343 mg, 0.70 mmol), i-Pr 2NEt (0.23 mL, 1.32 mmol), DMF (3 mL)
was added and the loading of resin was made following procedure
II (110 °C, 15 min), the confirmation of the completion of the reaction
was made following procedure III. Fmoc-deprotection (procedure
I) was followed by the coupling of Fmoc-Ala (205 mg, 0.66
mmol) in the presence of PyBOP (343 mg, 0.70 mmol), i-Pr 2NEt
(0.23 mL, 1.32 mmol) and DMF (3 mL) following procedure III
(110 °C, 15 min). This step was repeated due to the incomplete coupling
observed by the Kaisers test. The resin was washed with portions
of DMF (3 � 5 mL), portions of CH 2Cl 2 (3 � 5 mL) and the
dipeptide was cleaved following procedure IV yielding 2 as a white
solid (56 mg, 0.132 mmol, 60%).
Method B2
Rink amide resin (301 mg, 0.78 mmol/g, 0.235 mmol) was deprotected
using procedure I. The resin was transferred into a Smith Process
Vial and Fmoc-Ile (233 mg, 0.66 mmol), 2-fluoro-1-methylpyridinium
tosylate (210 mg, 0.70 mmol), i-Pr 2NEt (0.23 mL, 1.32
mmol), DMF (3 mL) was added and the subsequent steps were
made following Method A2 with the exception of the reaction times
and temperature (110 °C, 10 min) yielding 2 as a white solid (35 mg,
0.083 mmol, 35%).
Method C2
Rink amide resin (299 mg, 0.78 mmol/g, 0.233 mmol) was deprotected
using procedure I. Then, the resin was transferred into a
Smith Process Vial and Fmoc-Ile (233mg, 0.66 mmol), TBTU (225
mg, 0.70 mmol), i-Pr 2NEt (0.23 mL, 1.32 mmol) and DMF (3 mL)
was added and the subsequent steps were made following Method
A2 with the exception of the reaction times and temperature (110
°C, 10 min) yielding 2 as a white solid (41 mg, 0.097 mmol, 41%).
1 H NMR (400 MHz, CD3CN): � = 7.83 (d, J = 7.7 Hz, 2 H, Fmoc),
7.67 (br d, J = 7.3 Hz, 2 H, Fmoc), 7.42 (ddd, J = 1.2, 7.7, 7.7 Hz, 2
H, Fmoc), 7.33 (ddd, J = 1.2, 7.3, 7.7 Hz, 2 H, Fmoc), 6.78 (br d,
J = 7.7 Hz, 1 H, NH-Ile), 6.34 (br s, 1 H, NH), 6.08 (br d, J =5.9
Hz, 1 H, NH-Ala), 5.75 (br s, 1 H, NH), 4.32 (d, J = 7.0 Hz, 2 H,
CH2-Fmoc), 4.24 (t, J = 7.0 Hz, 1 H, CH-Fmoc), 4.16 (dd, J =5.8,
8.0 Hz, 1 H, CH�-Ile), 4.09 (dq, J = 5.9, 7.0 Hz, 1 H, CH�-Ala), 1.77
(m, 1 H, CH�-Ile), 1.42 (m, 1 H, CH2�(a)-Ile), 1.39 (d, J =7.0 Hz, 3
H, CH3-Ala), 1.09 (m, 1 H, CH2�(b)-Ile), 0.87 (d, J = 6.8 Hz, 3 H,
CH3�-Ile), 0.84 (t, J = 7.4 Hz, 3 H, CH3�-Ile). ESI-MS: m/z = 424 (M + 1) + , 407, 379, 179.
Fmoc-Thr-Ile-NH 2 (3); Method D3
Rink’s amide resin (300 mg, 0.78 mmol/g, 0.234 mmol) was deprotected
using procedure I. Then, the resin was transferred into a
Smith Process Vial and Fmoc-Ile (248 mg, 0.70 mmol), HATU (166
mg, 0.70 mmol), i-Pr 2NEt (0.25 mL, 1.40 mmol), DMF (3 mL) was
added and the loading of the resin was made following procedure II
(110 °C, 1.5 min), the confirmation of the completion of the reaction
was made following procedure III. Fmoc-deprotection (procedure
I) was followed by the coupling of Fmoc-Thr(t-Bu) (279 mg,
0.70 mmol) in the presence of HATU (343 mg, 0.70 mmol), i-
Pr 2NEt (0.23 mL, 1.32 mmol) and DMF (3 mL) following procedure
III (110 °C, 1.5 min). The resin was washed with portions of
DMF (3 � 5 mL), portions of CH 2Cl 2 (3 � 5 mL) and the dipeptide
was cleaved following procedure IV yielding 3 as a white solid
(25.4 mg, 0.06 mmol, 24%).
1 H NMR (400 MHz, CD3CN): � = 7.83 (d, J = 7.5 Hz, 2 H, Fmoc),
7.68 (br d, J = 7.0 Hz, 2 H, Fmoc), 7.42 (ddd, J = 1.2, 7.5, 7.5 Hz, 2
H, Fmoc), 7.33 (ddd, J = 1.2, 7.0, 7.5 Hz, 2 H, Fmoc), 6.87 (br d,
J = 8.4 Hz, 1 H, NH-Ile), 6.44 (br s, 1 H, NH), 5.99 (br d, J =5.9
Hz, 1 H, NH-Thr), 5.85 (br s, 1 H, NH), 4.36 (d, J = 7.5 Hz, 2 H,
CH2-Fmoc), 4.25 (t, J = 7.5 Hz, 1 H, CH-Fmoc), 4.22 (dd, J =5.9,
8.4 Hz, CH�-Ile), 4.7�4.15 (m, 2 H, CH�-Thr, CH�-Thr), 1.83 (m, 1
H, CH�-Ile), 1.45 (m, 1 H, CH2�(a)-Ile), 1.14 (m, 1 H, CH2�(b)-Ile), 1.08 (d, J = 6.0 Hz, 3 H, CH3�-Thr), 0.90 (d, J = 7.0 Hz, 3 H, CH3�- Ile), 0.87 (t, J = 7.7 Hz, 3 H, CH3�-Ile). ESI-MS: m/z = 454 (M + 1) + , 437, 409, 255, 214, 199.
Efficiency of the Solid Phase Methodology
Rink amide resin (297 mg, 0.78 mmol/g, 0.232 mmol) was deprotected
using procedure I. The resin was then transferred into a Smith
Process Vial, Fmoc-Ile (251 mg, 0.70 mmol), PyBOP (343 mg, 0.70
mmol), i-Pr2NEt (0.25 mL, 1.40 mmol), and DMF (3 mL) was added.
The mixture was irradiated in a microwave cavity at 110 °C for
15 minutes. Then, the mixture was transferred into a column
equipped with a polypropylene frit and was washed following procedure
II. The completion of the coupling was confirmed by Kaisers
test. The resin was cleaved and purified following procedure IV
yielding Fmoc-Ile-NH2 as a white solid (49 mg, 0.14 mmol, 60%).
1 H NMR (400 MHz, CDCl3) � = 7.76 (d, J = 7.5 Hz, 2 H, Fmoc),
7.57 (br d, J = 7.3 Hz, 2 H, Fmoc), 7.40 (dd, J = 7.3, 7.3 Hz, 2 H,
Fmoc), 7.31 (dd, J = 7.3, 7.5 Hz, 2 H, Fmoc), 5.89 (br s, 1 H, NH),
5.66 (br s, 1 H, NH), 5.36 (br d, J = 8.4 Hz, 1 H, NH-Ile), 4.42 (d,
J = 6.7 Hz, 2 H, Fmoc-CH2), 4.20 (t, J = 6.7 Hz, 1 H, CH-Fmoc),
4.07 (dd, J = 8.4, 8.4 Hz, 1 H, CH�-Ile), 1.89 (m, 1 H, CH�-Ile), 1.52
(m, 1 H, CH�(a)-Ile), 1.13 (m, 1 H, CH�(b)-Ile), 0.8�1.0 (m, 6 H,
CH3�-Ile, CH3�-Ile). ESI-MS: m/z = 353 (M+1) + .
Acknowledgements
We would like to thank Personal Chemistry AB for access to the
Smith Synthesizer TM and Uppsala University for financial support.
References
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Synthesis 2002, No. 11, 1592–1596 ISSN 0039-7881 © Thieme Stuttgart · New York

1596 M. Erdélyi, A. Gogoll SPECIAL TOPIC
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