The synthesis of peptides using micro reactors

Article abstract of DOI:10.1039/b102125g

We have demonstrated the first application of multi-step synthesis within a micro reactor and have shown that peptides may be prepared in quantitative yield in a period of 20 min, compared with batch reactions where only moderate yields (40-50%) were obta

Full text of DOI:10.1039/b102125g

The synthesis of peptides using micro reactors†
Paul Watts,a Charlotte Wiles,a Stephen J. Haswell,*a Esteban Pombo-Villarb and Peter Styringc
a Department of Chemistry, University of Hull, Cottingham Road, Hull, UK HU6 7RX.
E-mail: S.J.Haswell@chem.hull.ac.uk
b Nervous System Research, WSJ-386.07.15, Novartis Pharma Ltd., CH4002, Basel, Switzerland
c
Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield,
UK S1 3JD
Received (in Cambridge, UK) 6th March 2001, Accepted 19th April 2001
First published as an Advance Article on the web 15th May 2001
We have demonstrated the first application of multi-step
synthesis within a micro reactor and have shown that
peptides may be prepared in quantitative yield in a period of
20 min, compared with batch reactions where only moderate
yields (40–50%) were obtained in a 24 h period.
Commercially available Boc-b-alanine 1 was protected as the
Dmab ester using an EDCI [1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride] and DMAP coupling reac-
tion, to give the ester 2 in 92% yield in a bulk reaction (Scheme
1). Treatment of 2 with trifluoroacetic acid furnished the desired
amine 3 in 61% yield, which was subsequently reacted with
Fmoc-b-alanine 4 via a carbodiimide coupling reaction, to give
a synthetic sample of dipeptide 5.
During the past ten years, there has been a rapid growth in the
development of micro-Total Analytical Systems (m-TAS)1–3
which exploit electroosmotic flow (EOF).4 The development of
micro reactor devices for chemical synthesis based on com-
plementary technology is less common. However, recent
research has shown that Suzuki5 and Wittig6 reactions may be
performed using micro reactor systems.7
Peptides have been commonly prepared via solid supported
techniques since its introduction by Merrifield in 1963.8 Solid
phase peptide synthesis is based on the addition of a protected
amino acid residue to an insoluble polymeric support. The acid-
labile Boc group9 and base-labile Fmoc group10 have been
commonly used for N-protection. After removal of the protect-
ing group the next protected amino acid may be added using
either a coupling reagent or a pre-activated amino acid
derivative. If this dipeptide is the desired product, it may be
cleaved from the polymer support using various reagents, one of
the more common methods being treatment with 25–80% HF.11
If a longer peptide is required additional amino acids can be
added by repeating further coupling reactions.
Solid phase peptide synthesis has the disadvantage that a
fairly expensive polymer support is required. In addition, extra
steps are added to the synthesis as a result of initially linking the
amino acid to the support and finally having to remove the
peptide from the polymer. In this paper a micro reactor has been
used to prepare peptides using solution phase chemistry in an
attempt to overcome some of the current problems associated
with such syntheses.
The micro reactor devices used in this work were prepared
using standard procedures developed at Hull.12 A schematic of
a typical micro reactor produced using such fabrication
techniques is shown in the graphical abstract.† Microporous
silica frits13 were placed in the channels to prevent hydro-
dynamic flow occurring.
Having prepared dipeptide 5, it represented a synthetic target
for preparation using the micro reactor. Prior to synthesis, the
micro reactor channels were primed with anhydrous DMF to
remove any air and moisture from the channels and the
microporous silica frits. A standard solution of Fmoc-b-alanine
4 (50 ml, 0.1 M) in anhydrous DMF was added to reservoir A,
a solution of EDCI (50 ml, 0.1 M) was placed in reservoir B and
a solution of amine 3 (50 ml, 0.1 M) was placed in reservoir C.
Anhydrous DMF (40 ml) was placed in reservoir D, which was
used to collect the products of the reaction. Platinum electrodes
were placed in each of the reservoirs (A, B and C positive, D
ground) and an external voltage was applied to the channels
inducing electroosmotic flow of the reagents. The reactions
were conducted at rt for a period of 20 min, in order to acquire
sufficient volume of product to determine the yield of the
reaction. Analysis was achieved by high performance liquid
chromatography (Jupiter C18 10 mm, 4.6 3 250 mm, obtained
from Phenomenex), mobile phase composition: 0.1% TFA in
water and 0.1% TFA in acetonitrile, using a gradient system of
30% aqueous to 70% aqueous over 20 min, with a flow rate of
2.5 ml min21 at rt).
When stoichiometric quantities of the reagents were used
only ca. 10% conversion to peptide 5 was achieved when a
voltage of 700 V was applied to the reagents (A, B and C).
However, by using two equivalents of EDCI (0.2 M solution)
the yield of the reaction was increased to ca. 20%. By applying
a stopped flow technique (2.5 sec injection length with flow
stopped for 10 sec) the yield of the reaction was further
increased to 50%. Since the yield of the reaction appeared to
greatly depend on the number of equivalents of EDCI used, we
wished to further investigate the effect of carbodiimide
concentration on the reaction, however we found that EDCI was
insoluble in DMF above 0.2 M concentrations. In further
experiments DCC was used as the coupling reagent as it was
considerably more soluble in DMF. Using 5 eq. of DCC (0.5 M
solution in reservoir B) a 93% yield of dipeptide 5 was obtained
using the optimised conditions described above.
In the first instance a one-step reaction was considered in
which an N-protected b-amino acid was reacted with an O-
protected b-amino acid, to prepare the protected b-dipeptide. To
enable the methodology to be applicable to the synthesis of
more complex peptides, the use of orthogonal protecting groups
was clearly required. After careful consideration, the base-labile
Fmoc protecting group10 was selected for N-protection while
the Dmab ester14 was chosen for protection of the carboxylic
acid. Importantly, both protecting groups may be removed
under mild conditions, since electroosmotic flow is retarded if
the pH of the reaction is outside the range 3–10.
† Electronic supplementary information (ESI) available: schematic of a
borosilicate glass micro reactor. See http://www.rsc.org/suppdata/cc/b1/
b102125g/
Scheme 1 Synthesis of standard dipeptide derivative.
990
Chem. Commun., 2001, 990–991
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b102125g

Another common method utilised in peptide bond formation
involves the reaction of a pre-activated amino acid derivative,
such as a pentafluorophenyl ester, with an amine.15,16 Fmoc-b-
alanine 4 was activated as the pentafluorophenyl ester 6 via an
EDCI coupling reaction (Scheme 2). The pentafluorophenyl
ester 6 was stable and could be stored indefinitely in the freezer.
The ester 6 was subsequently reacted in bulk with amine 3 to
produce dipeptide 5.
Scheme 4 Multi-step peptide synthesis.
no peptide was evident. It was however found that the excess
piperidine used in the reaction was reacting with the penta-
fluorophenyl ester 7 to give amide 10.
As a result, an alternative method of Fmoc deprotection was
required that would not cause the aforementioned problem.
Using the micro reactor, the Dmab ester of Fmoc-b-alanine 9
was reacted with one equivalent of DBU to give the free amine
3 which was then reacted with the pentafluorophenyl ester of
Boc-b-alanine 7, in an attempt to form the dipeptide 8.
In this case, when the reagents were mixed using continuous
flow, with the reagents maintained at 700 V, product 8 was
observed in typically 25% yield. By comparing the flows of
each reagent at this stage we were able to optimise the reaction.
The Dmab ester of Fmoc-b-alanine 9 was maintained at 750 V
while reacted with DBU at 800 V. The deprotected amine was
then reacted, using continuous flow, with the pentafluorophenyl
ester of Boc-b-alanine 7 to give a conversion of 96%, based on
the amount of Dmab ester 9 present at the end of the reaction.
Having shown that more complex peptides could be produced
by removal of the N-protecting group we wished to determine if
we could remove the Dmab protecting group using hydrazine.
Hence, a solution of the Dmab ester of Fmoc-b-alanine 9 (50 ml,
0.1 M) in anhydrous DMF was added to reservoir A and a
solution of hydrazine (50 ml, 0.1 M) was placed in reservoir B.
Anhydrous DMF (40 ml) was placed in reservoir D, which was
used to collect the products of the reaction. Using continuous
flow of both reagents, maintained at 700 V, quantitative
deprotection was observed to give carboxylic acid 4.
Scheme 2 Preparation and reaction of pentafluorophenyl esters of Fmoc
protected amino acids.
Having prepared dipeptide 5 via the alternative pre-activated
strategy, we wished to investigate if the reaction could be
performed in a micro reactor. A standard solution of the
pentafluorophenyl ester of Fmoc-b-alanine 6 (50 ml, 0.1 M) in
anhydrous DMF was added to reservoir A, a solution of amine
3 (50 ml, 0.1 M) was placed in reservoir B and anhydrous DMF
(40 ml) was placed in reservoir D, which was used to collect the
products of the reaction. It was found that using continuous flow
of both reagents, where the ester 6 was maintained at 700 V and
the amine 3 was maintained at 600 V, dipeptide 5 was produced
in quantitative yield in just 20 min. This represented a
significant increase in yield compared with the traditional batch
synthesis where only 40–50% conversions were obtained.
Similarly, the reaction between the pentafluorophenyl ester 7
of Boc-b-alanine and amine 3 was also investigated in the micro
reactor (Scheme 3). In this case, when the reagents were mixed
under a continuous flow regime, with both reagents maintained
at 700 V, a quantitative yield of peptide 8 was observed.
Importantly, this result demonstrates that both Boc and Fmoc
protecting groups are suitable for use in the preparation of
peptides using micro reactors.
We wish to thank Novartis Pharmaceuticals (P. W. and
C. W.) for financial support. We are grateful to Dr Tom
McCreedy (University of Hull) for help in fabricating the micro
reactor devices.
Notes and references
1 S. J. Haswell, Analyst, 1997, 112, 1R.
Scheme 3 Reaction of pentafluorophenyl esters of Boc-b-alanine.
2 A. Manz, D. J. Harrison, E. Verpoorte and H. M. Widmer, Adv.
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3 D. J. Harrison, K. Fluri, K. Seiler, Z. H. Fan, C. S. Effenhauser and A.
Manz, Science, 1993, 261, 895.
4 P. D. I. Fletcher, S. J. Haswell and V. N. Paunov, Analyst, 1999, 124,
1273.
Having successfully demonstrated that peptide bonds could
be formed in micro reactors using two common methods, we
wished to show that we could extend the methodology to the
preparation of longer chain peptides. Consequently, we needed
to be able to conduct deprotection reactions in the micro reactor
and subsequently perform further peptide bond forming reac-
tions. Fmoc-b-alanine 4 was converted into the Dmab ester 9, in
a bulk reaction, using standard conditions (Scheme 4). It was
proposed to convert ester 9 into amine 3 by deprotection of the
Fmoc group in the micro reactor and subsequently react the
amine ‘in situ’ with pentafluorophenyl ester 7, to give the
dipeptide 8. Treatment of 9, with 10 eq. of piperidine in DMF
using the micro reactor, resulted in 60–70% deprotection over a
20 min period, to give amine 3.17
Subsequently, a standard solution of the Dmab ester of Fmoc-
b-alanine 9 (50 ml, 0.1 M) in anhydrous DMF was added to
reservoir A, a solution of piperidine (50 ml, 1.0 M, 10 eq.) was
placed in reservoir B and a solution of pentafluorophenyl ester
7 (50 ml, 0.1 M) was placed in reservoir C, in an attempt to
prepare dipeptide 8 using this multi-step approach. Anhydrous
DMF (40 ml) was placed in reservoir D, which was used to
collect the products of the reaction. The HPLC of the reaction
mixture showed that Fmoc deprotection had occurred, however
5 G. M. Greenway, S. J. Haswell, D. O. Morgan, V. Skelton and P.
Styring, Sensors & Actuators B, 2000, 63, 153.
6 V. Skelton, G. M. Greenway, S. J. Haswell, P. Styring, D. O. Morgan,
B. Warrington and S. Y. F. Wong, Analyst, 2001, 126, 7.
7 S. J. Haswell, R. J. Middleton, B. O’Sullivan, V. Skelton, P. Watts and
P. Styring, Chem. Commun., 2001, 391.
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11 D. B. Whitney, J. P. Tam and R. B. Merrifield, Tetrahedron, 1984, 40,
4237.
12 T. McCreedy, Anal. Chim. Acta, 2001, 427, 39.
13 P. D. Christensen, S. W. P. Johnson, T. McCreedy, V. Skelton and N. G.
Wilson, Anal. Commun., 1998, 35, 341.
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17 L. A. Carpino and G. Y. Han, J. Org. Chem., 1972, 37, 3404.
Chem. Commun., 2001, 990–991
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