Application of microwave method to the solid phase synthesis of pseudopeptides containing ester bond

Article abstract of DOI:10.1016/j.tetlet.2007.11.004

A new procedure was developed for reducing the reaction time and improving the yield of esterification reaction in solid phase synthesis of pseudopeptides containing an ester bond by utilizing microwave irradiation. We selected a pseudodipeptide (Fmoc-LysΨ[COO]Leu-NH₂) and optimized the microwave-assisted esterification reaction in solid phase synthesis using Fmoc chemistry. For this, microwave-assisted esterification reactions with different reaction time, temperature, and solvents were performed using 1,3-diisopropylcarbodiimide (DIC) as the coupling reagent. We synthesized several pseudodipeptides containing an ester bond by using the optimized microwave irradiation method. The purity and yield of the pseudodipeptides synthesized in this way were better than those obtained without microwave irradiation. Furthermore, we applied this methodology for synthesizing pseudopeptides (6- and 12-mer) corresponding to the α helical peptide. The microwave-assisted esterification reaction afforded the target pseudopeptides with high yield (～80%) and purity within 12 min, whereas the reaction without microwave irradiation afforded the target compound with poor yield (～45%) and low purity.

Full text of DOI:10.1016/j.tetlet.2007.11.004

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 98–101
Application of microwave method to the solid phase synthesis
of pseudopeptides containing ester bond
Bishnu Prasad Joshi, Jun-won Park, Joung-min Kim, Chuda Raj Lohani,
Hyeongjin Cho and Keun-Hyeung Lee*
Department of Chemistry, Bio-organic Chemistry Lab, 253-Yunghyun-Dong, Nam-Gu, Incheon, 402-751, South Korea
Received 24 September 2007; revised 28 October 2007; accepted 1 November 2007
Available online 4 November 2007
Abstract—A new procedure was developed for reducing the reaction time and improving the yield of esterification reaction in solid
phase synthesis of pseudopeptides containing an ester bond by utilizing microwave irradiation. We selected a pseudodipeptide
(Fmoc-LysW[COO]Leu-NH2) and optimized the microwave-assisted esterification reaction in solid phase synthesis using Fmoc
chemistry. For this, microwave-assisted esterification reactions with different reaction time, temperature, and solvents were
performed using 1,3-diisopropylcarbodiimide (DIC) as the coupling reagent. We synthesized several pseudodipeptides containing
an ester bond by using the optimized microwave irradiation method. The purity and yield of the pseudodipeptides synthesized in
this way were better than those obtained without microwave irradiation. Furthermore, we applied this methodology for synthesizing
pseudopeptides (6- and 12-mer) corresponding to the a helical peptide. The microwave-assisted esterification reaction afforded
the target pseudopeptides with high yield (ꢀ80%) and purity within 12 min, whereas the reaction without microwave irradiation
afforded the target compound with poor yield (ꢀ45%) and low purity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Solid phase synthesis has been extensively used for the
preparation of peptides, pseudopeptides, and organic
compounds.1 This technique offers many advantages
over conventional synthesis in terms of efficiency, conve-
nient work up and purification procedures. However,
the main problems with this method are the difficulties
for the reagents to reach the active sites located in the
solid phase, thereby slowing the reaction and degrading
the resin under long reaction conditions, and the need
for excess reagents. Recently, significantly increased
reaction rate, improved yield and raised product purity
have frequently been observed in solid phase synthesis
with microwave irradiation.2 Several successful attempts
for the solid phase synthesis of peptides, peptoids and
beta polymers using microwave irradiation have been
published.3 However, the microwave irradiation method
has not been extensively applied for the solid phase syn-
thesis of pseudopeptides.
development of non-peptide drugs. Thus, many classes
of pseudopeptides have recently been reported.3,4 We
are interested in the solid phase synthesis of pseudopep-
tides containing an ester bond for the following reasons.
The amide and ester bonds are very similar in terms of
structural and conformational preferences.5 Thus,
replacement of the amide bond with an ester bond is a
well-known strategy for investigating the role of the
hydrogen bonding of the amide bond in proteins and
peptides for their structures and biochemical inter-
actions.6 In addition, pseudopeptides containing an ester
bond have better bioavailability, due to their higher
resistance against protease and increasing hydrophobic-
ity. Even though pseudopeptides containing an ester
bond exhibited unique properties, pseudopeptides con-
taining an ester bond were rarely synthesized because
the synthesis of the pseudopeptides is not straight-
forward and very often possesses a synthetic challenge
due to the low nucleophilicity of the hydroxyl group
compared to the amino group. Pseudopeptides contain-
ing ester bonds were synthesized by using solid phase
synthesis with a-hydroxy acids or unnatural a-hydroxy
acids.5b,6a,7 Compared to the solid phase peptide synthe-
sis, the reaction time was relatively long (4–16 h) and
even double coupling reaction afforded low yield
(70%).5b,6a,7 In the era of proteomics, the time required
Pseudopeptides have received much attention due to
their better bioavailability than peptides and the easy
Keywords: Microwave; Irradiation; Pseudopeptides; Ester bond; Solid
phase synthesis; Peptide.
*
Corresponding author. Tel.: +82 32 860 7674; fax: +82 32 867
5604; e-mail: leekh@inha.ac.kr
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.11.004

B. P. Joshi et al. / Tetrahedron Letters 49 (2008) 98–101
99
for the synthesis of the target pseudopeptides has
become an important issue and it would be beneficial
to increase the yield and reaction rate for the pseudo-
peptide synthesis. Considering this, we have developed
a microwave irradiation procedure to reduce the reac-
tion time and increase the reaction yield for the synthesis
of pseudopeptides containing an ester bond in solid
phase synthesis.
tion was repeated until no color was observed in nin-
hydrin test.10 Then, the microwave-assisted esterification
reactions were performed for solid phase synthesis of
the pseudopeptide (Fmoc-LysW[COO]Leu-NH2), as
mentioned.11 The coupling yield of the esterification
reaction was measured by Fmoc quantitation assay.12
DCM has frequently been used as a solvent in esterifica-
tion reaction. However, DCM was not considered as a
solvent for the microwave irradiation method due to
its low boiling point (40 ꢁC) and because some Fmoc-
protected amino acids have poor solubility in DCM.
As shown in Table 1, the esterification reaction with
microwave irradiation was performed in NMP as the
solvent due to its high boiling point. However, the yield
was not significantly improved13 and Fmoc-deprotec-
tion of the conjugated Fmoc amino acid to the resin
was observed in this condition, perhaps due to high tem-
perature in the presence of the base. We selected DMF
as the solvent and investigated the esterification reaction
with microwave irradiation. Table 1 indicates that the
reaction reached a level of 80% at 12 min. To improve
the coupling yield, we increased the reaction time but
the yield was not improved. We performed the esterifica-
tion reaction in DMF solvent with microwave irradia-
tion (150 W) at high temperature (120 ꢁC) but the
yield (80%) was not improved. In conventional solid
phase synthesis, a different DCM/DMF ratio was
frequently employed as the solvent for the coupling
reaction.14 The esterification reaction with microwave
irradiation was also attempted using DMF/DCM (1:1,
v/v); however, the obtained yield was low (56%). Thus,
the reaction condition with microwave irradiation (temp
90 ꢁC, power 150 W, time 12 min) was kept constant for
further synthesis of pseudopeptides in this study.
We chose a pseudodipeptide containing ester bond
(Fmoc-LysW[COO]Leu-NH2) as a model system and
optimized the microwave irradiation procedures for so-
lid phase synthesis. (S)-2-Hydroxy-4-methylpentanoic
acid (a-hydroxy-Leu) was synthesized by diazotization
method in acidic condition with sodium nitrite from
Leu.8 Previous research indicated that among various
coupling reagents, 1,3-diisopropylcarbodiimide (DIC)
afforded the best yield in the esterification reaction for
pseudopeptides in solid phase synthesis.2b,6,9 Thus, we
chose DIC as the coupling reagent and DMAP/NEM
as the base and investigated the esterification reaction
with microwave irradiation at various temperatures
and solvents for the optimization of this model reaction
(Scheme 1). First, a-hydroxy-Leu was coupled to Rink-
amide MBHA resin by the activation of alpha hydroxy
acid (3 equiv) with the same equivalent of DIC and
HOBt with microwave irradiation. The coupling reac-
2
2
1
1
°
Additionally, several coupling reagents were reconsid-
ered for the esterification reaction by applying this
microwave irradiation condition. The coupling yield
with various coupling reagents is summarized in Table 2.
Scheme 1. Introduction of ester bond in the resin using microwave
irradiation method.
Table 1. Coupling yield of the model pseudodipeptide at different time
As shown in Table 2, the DIC-mediated coupling reac-
tion afforded the higher yield. The activation of an
Fmoc-amino acid with coupling reagents 2, 4, and 5
resulted in the formation of the corresponding OBt ester
that was known to provide low racemic product. How-
ever, the OBt ester afforded low yield in this condition.
and different solvent under microwave irradiationa
Solvent
Power (W)
Temp (ꢁC)
Time (min)
Yield (%)
NMP
DMF
200
150
150
90
12
7
10
12
15
20
58
50
61
80
81
78
The microwave-assisted method by using DIC as the
coupling reagent was applied for the synthesis of various
dipseudopeptides (Table 3). Their yields and purities
a The reaction was performed under a closed vessel using microwave
irradiation.
Table 2. Microwave-assisted yields of Fmoc-LysW[COO]Leu-NH2 with various coupling reagentsa
No.
Coupling reagent
Base
Yield (%)
1
2
3
4
5
DIC (3 equiv)
DIC (3 equiv)/HOBt (3 equiv)
TFFH (3 equiv)
PyBop (3 equiv)/HOBt (3 equiv)
DCC (3 equiv)/HOBt (3 equiv)
NEM (1.2 equiv)/DMAP (0.l equiv)
NEM (1.2 equiv)/DMAP (0.l equiv)
DIEA (8 equiv)
DIEA (6 equiv)
DIEA (3 equiv)/DMAP (0.l equiv)
80
72
62
50
54
a The resin (0.025 mmol) containing a-hydroxy-Leu was mixed with Fmoc-Lys(Boc)-OH (3 equiv) in the presence of the coupling reagent and the
reaction was performed under microwave irradiation (temp 90 ꢁC, power 150 W, time 12 min).

100
B. P. Joshi et al. / Tetrahedron Letters 49 (2008) 98–101
Table 3. Coupling yield of various pseudodipeptides containing an
ester bond
microwave irradiation. The HPLC and mass spectra of
6-mer pseudopeptide (Figs. S6 and S7) indicated that
both methods afforded the target pseudopeptide as a
major product, whereas the purity obtained using micro-
wave irradiation methods was better than that obtained
without microwave irradiation.
No.
Sequence
Yield (%)
Microwave
Normal
(12 min)
(480 min)
1
2
3
4
5
Fmoc-KW[COO]L-NH2
Fmoc-EW[COO]L-NH2
Fmoc-SW[COOlL-NH2
Fmoc-FW[COO]L-NH2
Fmoc-AW[COO]L-NH2
80
83
78
78
78
75
72
68
68
64
We did not use HOBt in this reaction. Racemic product
may have been generated in the microwave-assisted
reaction. Even though only one peak was observed in
the HPLC spectrum of the product obtained using the
microwave method, the peak seemed to be broad. Thus,
we synthesized the D-form of 6-mer pseudopeptide
(Fmoc-D-LeuW[COO]Lys-Lys-Leu-Leu-Lys-NH2) and
mixed L- and D-forms and analyzed them by HPLC.
Two distinct peaks were observed with different reten-
tion time in this HPLC (Fig. S8). This result clearly indi-
cates that no racemic product formation occurred
during the microwave reaction.
were compared with those obtained by the esterification
reaction without microwave irradiation.15
The coupling yield of each reaction was measured by
Fmoc quantitation assay.12 After completion of the
reaction, the pseudopeptides were cleaved and deprotec-
ted by treatment of TFA/H2O (95:5) solution. The
resulting product was analyzed by using C18 reverse
phase HPLC and an ESI mass spectrometer. HPLC
and ESI mass analyses of the product mixtures indicated
that both methods afforded the target pseudopeptide as
the major product (Figs. S1–S5). The presence of only
one peak in the chromatogram suggested that no race-
mization occurred during the microwave-assisted reac-
tion. Table 3 shows that the coupling yields for the
various pseudodipeptides containing an ester bond syn-
thesized were higher in the microwave irradiation meth-
od than those achieved without microwave irradiation.
Specifically, the reaction time was significantly reduced
by applying this microwave irradiation method, as com-
pared to that achieved without microwave irradiation.
The 12-mer pseudopeptide was also successfully synthe-
sized by using the microwave-assisted procedure
(Fig. 1). The HPLC and MALDI TOF mass spectrum
(Figs. S9 and S10) indicated that the microwave method
afforded the target molecule as the sole product, whereas
the method without microwave irradiation afforded two
major products. The mass spectrum (Fig. S10) indicated
that the product corresponding to peak 1 in Figure 1B
was the unreacted product (HO-LLL-KWLK-KLLK-
NH2), while the product corresponding to peak 2 was
the target pseudopeptide. The presence of only one ma-
jor peak in the HPLC chromatogram indicated that no
detectable racemic product was synthesized in this
microwave-assisted reaction.
We chose the a helical peptide, which is known to be a
difficult sequence,16 and synthesized the corresponding
pseudopeptides containing an ester bond by using the
microwave irradiation procedure. The 6- and 12-mer
pseudopeptides containing a hydroxy group at their N-
terminus were synthesized on Rink-amide MBHA resin
in solid phase synthesis. The esterification reaction was
performed using the microwave-assisted esterification
method.11 The same pseudopeptides were synthesized
by using the method without microwave irradiation at
room temperature with continuous stirring for 8 h and
the yields were compared with those achieved using
microwave irradiation methods.
In conclusion, we successfully developed a fast and effi-
cient procedure for the solid phase synthesis of pseudo-
peptides using microwave irradiation. In a direct
As shown in Table 4, the coupling yield achieved using
the microwave-assisted method was about twice as high
as that obtained by the esterification reaction without
Table 4. Coupling yield of 6- and 12-mer pseudopeptide containing an
ester bond by using microwave irradiation method
No. Sequence
Yield (%)
Microwave Without microwave
(12 min)
(480 min)
1
2
Fmoc-LW[COO]K-
KLLK-NH2
KW[COO]LLL-KWLK- 79
86
45
Figure 1. HPLC of 12-mer pseudopeptide KW[COO]-LLL-KWLK-
KLLK-NH2: (A) with microwave irradiation, (B) without microwave
irradiation.
44
KLLK-NH2

B. P. Joshi et al. / Tetrahedron Letters 49 (2008) 98–101
101
R.; Providence, L. L.; Schmutzer, S. E.; Shobana, S.;
Greathouse, D. V.; Andersen, O. A.; Koeppe, R. E.
Biochemistry 2001, 40, 1460–1472.
comparison of our optimized protocol with normal
methods, the pseudodipeptides synthesized by the
microwave irradiation method, 6- and 12-mer, were
afforded at high yield and significant purity. Considering
the recent demand for pseudopeptides in the era of pro-
teomics, this microwave-assisted procedure has shown
promising potential for the synthesis of pseudopeptides
containing an ester bond.
8. Deechongkit, S.; You, S. L.; Kelly, J. W. Org. Lett. 2004,
6, 497–500.
9. (a) Stawikowski, M.; Cudic, P. Tetrahedron Lett. 2006, 47,
8587–8590; (b) Fmoc Solid Phase Peptide Synthesis; Chan,
W. C., White, P. D., Eds.; Oxford University: Oxford,
2000; (c) Albericio, F.; Burger, K.; Ruiz-Rodriguez, J.;
Spengler, J. Org. Lett. 2005, 7, 597–600.
10. Kaiser, E.; Colescott, R. E.; Bossinger, C. D.; Cook, P. I.
Anal. Biochem. 1970, 34, 595–598.
Acknowledgments
11. The coupling reaction was accomplished as follows.
Activated Fmoc-amino acid with DIC/DMAP in DMF
(3 mL) was mixed with the hydroxy Leu attached resin
followed by NEM. The reaction mixture was placed in a
microwave vial and irradiated at 90 ꢁC, supplying 150-W
power for 12 min in the Biotage Microwave Initiator
System. The reaction was cooled with N2 gas and the
resin was washed with DMF and MeOH, respectively
(3 mL, three times each). Deprotection was achieved by
treatment with a mixture of trifluoroacetic acid: water
(95:5, v/v) at room temperature for 4 h. The crude peptide
was analyzed by HPLC with a waters C18 column using a
water (0.1% TFA) acetonitrile (0.1% TFA) gradient. The
products were characterized by ESI mass spectrometry
(Mass1200L Quadruple LC/MS system, Varian) and
MALDI TOF mass spectrometry (Voyager-DE STR,
Applied Biosystem).
12. The dry resin was weighed (3–5 mg) in an eppendorf tube
and added to a 50% piperidine/DMF solution. The
mixture was incubated for 30 min at room temperature
with continuous stirring. Then, the absorbance of the
Fmoc-piperidine adduct was measured at 301 nm by using
a UV–vis spectrophotometer (Lambada 40, Perkin Elmer,
UK). The blank titrations were performed without resin
containing supernatant. The loading yield of the esterifi-
cation reaction was compared to that of the loading yield
of Fmoc-protected a-hydroxy leucine. The degree of
substitution was calculated by using the following equa-
tion: (mmol/g) = absorbance at 301 nm · volume (mL)/
7800 · weight (mg).
This work was supported by a grant (R01-2006-000-
10956-0) from the Basic Research Program of the Korea
Science and Engineering Foundation and a grant
(C00344) from the basic research program of the Kor-
ean Research Foundation. B. P. Joshi, Jun-won Park,
Joung-min Kim, and Chuda Raj Lohani were recipients
of a BK21 (II) fellowship.
Supplementary data
HPLC condition and characterization data for pseudo-
peptides by HPLC and mass spectroscopy. Supplemen-
tary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2007.11.004.
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