Solid-phase peptide synthesis in water using microwave-assisted heating

Article abstract of DOI:10.1021/ol901893p

An approach using water as a solvent (coupling and deprotection) was developed for the solid-phase synthesis of peptides using the most common Boc-amlno acid derivatives. Key aspects of this methodology are the use of a PEG-based resin, EDC-HONB as a coupling method, and microwave Irradiation as an energy source.

Full text of DOI:10.1021/ol901893p

ORGANIC
LETTERS
2
009
Vol. 11, No. 20
488-4491
Solid-Phase Peptide Synthesis in Water
Using Microwave-Assisted Heating
4
†
,‡
‡
Athanassios S. Galanis, Fernando Albericio, and Morten Grøtli*
,†
Department of Chemistry, Medicinal Chemistry, UniVersity of Gothenburg, SE-412 96
G o¨ teborg, Sweden and Institute for Research in Biomedicine and CIBER-BBN,
Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona
Science Park, 08028 Barcelona, Spain, and Department of Organic Chemistry,
UniVersity of Barcelona, 08028 Barcelona, Spain
grotli@chem.gu.se
Received June 8, 2009
ABSTRACT
An approach using water as a solvent (coupling and deprotection) was developed for the solid-phase synthesis of peptides using the most
common Boc-amino acid derivatives. Key aspects of this methodology are the use of a PEG-based resin, EDC-HONB as a coupling method,
and microwave irradiation as an energy source.
Solid-phase synthetic (SPS) methods have made peptide
synthesis simple, rapid, and easily subject to automation.
difficult peptide sequences have been obtained by performing
1
peptide coupling steps at elevated temperatures (30-80 °C)
6
The solid-phase method is now the principal method for
peptide synthesis, but it requires a large amount of organic
solvent. As the safe disposal of organic solvent waste is an
important environmental issue, a method for peptide synthesis
using thermal heating and more recently by MW irradia-
7
tion.
Herein we report the development of a water-based SPPS
method using low cost commercially available Boc-deriva-
tives and coupling reagents, without any preparation or
modification, and microwave (MW) technology for rapid and
efficient temperature elevation.
Initially, several coupling agents were tested and evaluated
for their coupling efficiency in water using MW irradiation.
Several protected/masked amino acids were tested at different
2
in water would be desirable. To carry out solid-phase peptide
synthesis (SPPS) in water, researchers have so far focused
on developing novel hydrophilic N-protecting groups for
amino acids in order to improve the water solubility of the
3
derivatives because the conventional protecting groups (e.g.,
Z-, Boc-, and Fmoc- groups) are hydrophobic. Alternatively,
more complicated synthesis methods have been reported such
4
(3) (a) Hojo, K.; Maeda, M.; Tanakamaru, N.; Mochida, K.; Kawasaki,
K. Protein Pept. Lett. 2006, 13, 189–192. (b) Hojo, K.; Maeda, M.;
Kawasaki, K. Tetrahedron Lett. 2004, 45, 9293–9295. (c) Hojo, K.; Maeda,
M.; Smith, T. J.; Kita, E.; Yamaguchi, F.; Yamamoto, S.; Kawasaki, K.
Chem. Pharm. Bull. 2004, 52, 422–427. (d) Hojo, K.; Maeda, M.; Kawasaki,
K. Tetrahedron. 2004, 60, 1875–1886. (e) Hojo, K.; Maeda, M.; Kawasaki,
K. Tetrahedron Lett. 2003, 44, 2849–2851. (f) Hojo, K.; Maeda, M.;
Kawasaki, K. J. Pept. Sci. 2001, 7, 615–618. (g) Royer, G. P.; Ananthar-
maiah, G. M. J. Am. Chem. Soc. 1979, 101, 3394–3396. (h) Kunz, H. Angew.
Chem., Int. Ed. Engl. 1978, 17, 67–69.
as enzymatic catalysis of coupling reactions and the use of
5
nanoparticulated derivatives.
Typically, all steps in the SPPS cycle are carried out at
room temperature. However, significant improvements for
†
University of Gothenburg.
University of Barcelona.
‡
(
(
1) Albericio, F. Curr. Opin. Chem. Biol. 2004, 8, 211–221.
2) Anastas, P. T.; Warner, J. C. Green Chemistry: Theory and Practice;
(4) (a) Schellenberger, V.; Jakubke, H.-D. Angew. Chem., Int. Ed. Engl.
1991, 30, 1437–1449. (b) Wong, C. H. Science 1989, 244, 1145–1152.
Oxford University Press: Oxford, 1989.
10.1021/ol901893p CCC: $40.75
Published on Web 09/16/2009
 2009 American Chemical Society

pHs and various reaction times using Rink Amide Tentagel
as solid support. In our hands, N-ethyl-N′-(3-dimethylami-
8
Table 1. Screening of Amino Acid Protecting Groups for the
MW-Assisted SPPS of Leu-Enk in Water
nopropyl)carbodiimide hydro-chloride (EDC hydrochloride)
in combination with N-hydroxy-5-norbornene-2,3-dicarboxy-
9
lic acid imide (HONB) rendered the best yields when
compared with other coupling agents such as N-hydroxy-
1
0
11
benzotriazole (HOBt), N-hydroxysuccinimide (HOSu),
1
2
2
-mercaptobenzothiazole, and ethyl cyanoglyoxalate-2-
13
oxime (Oxyma) (data not shown). However, the formation
of the active esters in water was a rapid reaction, as expected
decomposition occurred faster in water than in DMF, and it
was further enhanced at high temperature. Therefore, a one
minute short preactivation time and relatively short reaction
times (7 min) were used.
14
On the basis of the above results, Leu-Enkephalin (Leu-
Enk; H-YGGFL-OH, an endogenous opioid peptide neu-
rotransmitter found naturally in the brain of many animals)
was used as a model to evaluate MW-assisted SPPS in water.
R
Screening for the best protection/masking of the N -function
of the amino acids was carried out. The most common
protecting groups used in SPPS, Boc, and Fmoc, together with
R
15
the N -azido modified residues, were investigated. We
reasoned that azido acids should be particularly useful due to
their more hydrophilic character compared to Boc and Fmoc.
For these preliminary experiments, each amino acid derivative
was coupled (by classical SPPS using organic solvents at room
temperature) to a presynthesized resin-bound oligopeptide
(Table 1). Different commercially available supports (TentaGel,
ChemMatrix, polystyrene, and NovaGel) and linkers (Rink
Amide, hydrazinobenzoic, and HMBA) were compared.
Thus, each amino acid derivative was coupled twice to
the resin-bound peptide sequence (9 µmol scale) using a
5
-fold excess of the masked-AA/EDC/HONB coupling
mixture in combination with a CEM Discover MW reactor
with temperature control via a standard IR thermometer and
applying 70 W power of MW radiation for 7 min at 75 °C
(
Table 1; see Supporting Information).
Extending the reaction time did not improve yields, and a
a
(
5) Hojo, K.; Ichikawa, H.; Maeda, M.; Kida, S.; Fukumori, Y.;
Peptides were prepared using classical SPPS with organic solvents. P
Kawasaki, K. J. Pept. Sci. 2007, 13, 493–497.
(
in brown) ) Rink Amide TentaGel; (in gray): P (in brown) ) Rink Amide
(
6) (a) Rabinovich, A. K.; Rivier, J. E. Am. Biotechnol. Lab. 1994, 12,
ChemMatrix; (in cyan): P (in brown) ) Rink Amide Polystyrene; (in red):
4
1
1
8–51. (b) Tam, J. P.; Lu, Y.-A. J. Am. Chem. Soc. 1995, 117, 12058–
b
P (in brown) ) Hydrazinobenzoyl NovaGel. Yields were obtained by
2063. (c) Varanda, L. M.; Miranda, M. T. M. J. Pept. Res. 1997, 50, 102–
analytical RP-HPLC analysis and after proper workup and cleavage of the
08.
c
d
peptides. Unreacted A oligopeptide. Target synthesized oligopeptide.
Boc-Arg(Tos)-OH. Boc-Cys(Acm)-OH.
(
7) (a) Murray, J. K.; Gellman, S. H. Nature Protein 2007, 2, 624–631.
e
f
(
1
b) Sabatino, G.; Papini, A. M. Curr. Opin. Drug DiscoVery DeV. 2008,
1, 762–770. (c) Bacsa, B.; Horvati, K.; Bosze, S.; Andreae, F.; Kappe,
O. C. J. Org. Chem. 2008, 73, 7532–7542. (d) Caddick, S.; Fitzmaurice,
R. Tetrahedron 2009, 65, 3325–3355, and references therein.
temperature range of 70-80 °C was sufficient for complete
coupling (data not shown). Coupling reactions at a lower
temperature required an extended reaction time (>1 h), but
the prolonged reaction times resulted in formation of several
side-products. The reactions were performed in deionized
water and the pH changed upon adding the reagents
(approximately 3.5-4.5 initially, and 3.0-4.0 after the end
of the reaction). Coupling reactions at higher pH values
generated a variety of impurities.
(
8) Sheehan, J. C.; Cruickshank, P. A.; Boshart, J. A. J. Org. Chem.
1
961, 26, 2525–2528.
(
9) Fujino, M.; Kobayashi, S.; Obayashi, M.; Fukuda, T.; Shinagawa,
S.; Nishimura, O. Chem. Pharm. Bull. 1974, 22, 1857–1863.
(
(
10) K o¨ nig, W.; Geiger, R. Chem. Ber. 1970, 103, 788–798.
11) Dhaon, M. K.; Olsen, R. K.; Ramasamy, K. J. Org. Chem. 1982,
4
7, 1962–1965.
(
(
(
12) Evans, D. J. Speciality Chem. Mag. 2005, 25, 50–52.
13) Izdebski, J. Pol. J. Chem. 1979, 53, 1049–1057.
14) Approximately one minute after the addition of the coupling
reagents into the amino acid derivative solution, the solvent becomes
nebulous due to the formation of the active ester (see Supporting
Information).
Boc derivatives in combination with Rink Amide Tentagel
rendered high coupling efficiency (Table 1, entries 2, 5, 8,
and 17), providing products with a purity ranging from
(
15) The azido acids were synthesized by following a published
procedure: Lundquist, J. T., IV; Pelletier, J. C. Org. Lett. 2001, 3, 781–
83.
7
Org. Lett., Vol. 11, No. 20, 2009
4489

8
0-98% (yield 86-94%). Coupling reactions of Boc-
derivatives provided almost complete coupling reactions as
only traces of unreacted starting material (up to 0.9%) were
detected. The Fmoc-protected amino acids (Table 1, entries
1
, 4, 7, and 16) did not give such high coupling efficiency
as observed with the Boc-protected amino acids, probably
due to the bulky and more hydrophobic character of the Fmoc
group compared to the Boc group. On the other hand, the
azido acids (Table 1, entries 3, 6, 9, and 18) gave low
coupling yields (purity: 21-48%), and significant amounts
of unreacted starting material were detected (30-70%) in
the individual coupling steps.
In comparison, using Rink Amide (RAM) polystyrene
(
instead of Tentagel) as a solid support resulted in very low
coupling efficiency (Table 1, entries 13-15 and 22-24)
regardless of the protection/masking of the N-function of the
amino acids, while the RAM-ChemMatrix support (Table
Figure 1. Analytical HPLC chromatograms of crude Leu-Enk
synthesized by SPPS in water using: MW reactor, HZB-NovaGel
resin without (A) and with (B) 0.5% Triton-X100, (C) MW reactor,
HZB-ChemMatrix resin with 0.5% Triton-X100, (D) MW reactor,
HMBA-ChemMatrix resin with 0.5% Triton-X100, and (E) hot plate
at 83 °C, HZB-ChemMatrix resin with 0.5% Triton-X100. In all
cases, the reaction time was set to 2 × 7 min. Numbered peaks
1
, entries 10-12 and 19-21) gave couplig efficiencies
similar to those obtained with the use of RAM-Tentagel.
Using Hydrazinobenzoyl NovaGel (HZB-NovaGel) in com-
bination with Boc-protected amino acid derivatives (Table
correspond to: 1, Leu-Enk; 2, [desLeu]-Leu-Enk; 3, [Leu-Enk]MW
1
, entries 25 and 26) resulted in high coupling efficiency
18
+
135.0. HPLC system: see Supporting Information.
comparable with the results for RAM-Tentagel and RAM-
ChemMatrix.
To study whether other more lipophilic Boc-protected
amino acids could be solvated and give high coupling yields,
Boc-protected valine, arginine (Tos-protected side chain), and
cysteine (Acm-protected side chain) were coupled to a solid-
phase bound dipeptide (Gly-Phe-RAM-TentaGel) using the
same reaction conditions as described earlier (Table 1, entries
first introduced by Merrifield for the removal of the Boc-
group during SPPS using acetic acid instead of water as the
solvent. Diluted HCl in water can also be regarded as a
19
more environmentally friendly reagent compared to the
common use of TFA in the classical Boc -SPPS strategy.
Careful stirring was used in both coupling and deprotection
experiments, in order to avoid volumetric heating without
2
7-29). All the Boc-protected amino acids gave products
with a purity of 85% or better. These results demonstrate
that hydrophobic amino acids or derivatives with bulky side
chain protecting groups are compatible with microwave-
assisted SPPS synthesis in water when a hydrophilic solid
support is used.
Once the best coupling conditions, protecting group and
solid support were determined, the complete aqueous Boc
SPPS of Leu-Enk was carried out with MW irradiation (scale:
6,20
gradients.
The use of MW irradiation (2 × 7 min) at 75 °C (Figure
1
A) using hydrazine-AM-NoveGel gave the target peptide
at 67% purity. Small amounts of byproducts and failure
sequences were observed. The main byproduct is the result
of a Lossen rearrangement after the attack of a nucleophile
at the Boc-AA-ONB active ester (3 in Figure 1), similar to
the earlier proposed side reaction when HOSu is used in
0
.042 mmol). The TFA-labile Rink Amide linker is not
18
classical SPPS procedures.
suitable for the preparation of peptides using the Boc strategy.
Instead, hydrazinobenzoic acid (HZB) and 4-hydroxymethyl-
benzoic acid (HMBA) were used because both linkers are
compatible with Boc chemistry.
The same experiment was also repeated with the addition
of 0.5% Triton-X100 detergent in the coupling reactions, as
well as in the swelling and washing steps. This zwitterionic
detergent is known to improve coupling efficiency in classical
The synthesis was carried out using the commercially
available HZB-NovaGel resin or the preprepared HZB-
ChemMatrix as the solid support due to the required mild
21
3a,e
SPPS as well as in the aqueous environment, due to its
ability to improve both the aqueous solubility of the
derivatives as well as the swelling properties of the resin.
Indeed, the combination of MW-assisted heating with the
1
6
17
cleavage conditions. For comparison, an HMBA - func-
tionalized ChemMatrix solid support was also prepared and
used. Each amino acid derivative was coupled (twice) on
the solid phase using the MW reactor as described above
(
18) Proposed side product through a side reaction includes a Lossen
rearrangement according to similar reactions referred to by (a) Gross, H.;
Bilk, L. Tetrahedron 1968, 24, 6935. (b) Isidro-Llobet, A.; Just-Baringo,
X.; Ewenson, A.; Alvarez, M.; Albericio, F. Biopolymers (Pept. Sci.) 2007,
88, 733–737.
(
70 W MW power; 75 °C; 7 min).
The Boc group was removed using 1 N HCl in water (MW
(
19) Marshall, G. R.; Merrifield, R. B. Biochemistry 1965, 4, 2394–
heating; 70 W; 3 × 7 min; 70 °C). Hydrochloric acid was
2
401.
20) Herrero, A. M.; Kremsner, M. J.; Kappe, O. C. J. Org. Chem. 2008,
73, 36–47
(
(
16) (a) Millington, C. R.; Quarrell, R.; Lowe, G. Tetrahedron. Lett.
.
1
6
998, 39, 7201–7204. (b) Peters, C.; Waldmann, H. J. Org. Chem. 2003,
8, 6053–6055.
(21) Zhang, L.; Goldammer, C.; Henkel, B.; Zuhl, F.; Panhaus, G.; Jung,
G.; Bayer, E. In InnoVation and PerspectiVes in Solid Phase Synthesis;
Epton, R., Ed.; Mayflower Worldwide Ltd.: Birmingham, 1994, pp
711-716.
(
17) Atherton, E.; Sheppard, R. C. In Solid Phase Peptide Synthesis: A
Practical Approach; IRL Press: Oxford, 1989.
4490
Org. Lett., Vol. 11, No. 20, 2009

zwitterionic detergent enhanced the synthesis efficiency of
the pentapeptide to 78% purity (Figure 1B). Only traces of
complete absence of organic solvents; only small amounts
of MeOH are required for optimal washing after Boc-
removal.
side products were detected. The peptide was obtained at
1
7
2% isolated yield after HPLC purification. A H NMR
In conclusion, MW-assisted SPPS with water as a solvent
for coupling and deprotection, in combination with Boc-
protected amino acids, EDC/HONB as coupling reagents and
the use of zwitterionic detergent provided Leu-Enkephalin
at high yield and purity, and free of racemization. This
synthetic approach uses commercially available derivatives
and agents without any specific pretreatment at any step of
the procedure. This result is, to the best of our knowledge,
the first reported SPPS in which the entire peptide has been
synthesized using water as a solvent for coupling and
deprotection.
spectra of this sample showed no traces of racemization (see
Supporting Information).
MW-assisted SPPS of Leu-Enk using HZB- or HMBA-
ChemMatrix as the solid support and the addition of 0.5%
Triton-X100 detergent generated the target peptide at 77 and
8
1% purity, respectively (Figure 1C, D). This slightly
enhanced synthetic yield following the use of the HMBA
linker is the result of the attachment of the first Leu derivative
onto the hydroxyl-functioned linker by traditional esterifi-
cation using organic solvents (see Supporting Information).
In this case, the [desLeu]LeuEnk byproduct is absent.
Although the MW technology covers the requisites for the
rapid and efficient synthetic approach in water, the same
sequence was prepared using thermal heating (hot plate) in
order to investigate the generality of the strategy. Each amino
acid coupling was carried out for 7 min. The Boc group was
removed using 1 N HCl in water (hot plate; 3 × 7 min; 70
Table 2. Optimized Synthetic Procedure for the SPPS in Water
in the Presence of a Zwitterionic Reagent
step
reagents
time (min)
swelling
coupling
0.5% Triton X100 aq
Boc- aa/EDC/HONB (5 equiv)
5 × 1
2 × 7
°
C). The same type of vial and stirring was used in these
experiments as for the experiments using MW-assisted
heating.
0.5% Triton X100 aq,
MW; 75 °C; 70 W
The SPPS in water using thermal heating was first tested
at 75 °C using HZB-NovaGel resin without Triton X-100,
providing poor results compared to the relative synthesis by
MW technology. When the temperature was increased to 83
washing
H O
5 × 1
10 × 1
5 × 1
2
0.5% Triton X100 aq
H O
2
1N HCl aq, MW; 70 °C; 70 W
Boc-removal
washing
3 × 7
2
H O
5 × 1
°C (see Supporting Information, Figure S5) the target product
MeOH: H
2
O (1:1)
3 × 1
was obtained with similar purity as for MW at 75 °C.
Thus, the synthesis of Leu-Enk was repeated using the
HZB-ChemMatrix resin by thermal heating at 83 °C with
the addition of 0.5% TritonX-100, yielding the peptide at
H
2
O
10 × 1
4 × 5
neutralization
washing
2
10% DIEA in H O
H O
2
10 × 1
3 × 1
0.5% Triton X100 aq
H
2
O
10 × 1
76% purity (Figure 1E), comparable with the MW irradiation
at 75 °C (Figure 1C, 77%). The results demonstrate that the
SPPS strategy is compatible with both the MW and thermal
heating and are consistent with a previous study in which
nearly identical results were obtained for SPPS in organic
solvents when thermal heating and MW-assisted heating were
compared. The observed differences between the MW and
conventional heating are probably due to inaccurate tem-
perature measurements and/or the use of different temper-
ature monitoring systems. However, we find the MW reactor
more easy to use and it is a more rapid technique.
Acknowledgment. A.S.G. acknowledges a MARIE-
CURIE Intra-European Fellowship, project no. 040451. This
study was partially supported by CICYT (BQU 2006-03794),
CIBER-BBN, Generalitat de Catalunya, and the Barcelona
Science Park.
7c
Supporting Information Available: Experimental details
and characterization data. This material is available free of
charge via the Internet at http://pubs.acs.org.
The general protocol of the MW strategy is shown in Table
2
. The protocol differs from standard SPPS in the almost
OL901893P
Org. Lett., Vol. 11, No. 20, 2009
4491