A flow reactor process for the synthesis of peptides utilizing immobilized reagents, scavengers and catch and release protocols

Article abstract of DOI:10.1039/b612197g

A general flow process for the multi-step assembly of peptides has been developed and this procedure has been used to successfully construct a series of Boc, Cbz and Fmoc N-protected dipeptides in excellent yields and purities, including an extension of t

Full text of DOI:10.1039/b612197g

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COMMUNICATION
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A flow reactor process for the synthesis of peptides utilizing immobilized
reagents, scavengers and catch and release protocols{
Ian R. Baxendale, Steven V. Ley,* Christopher D. Smith and Geoffrey K. Tranmer
Received (in Cambridge, UK) 24th August 2006, Accepted 19th September 2006
First published as an Advance Article on the web 3rd October 2006
DOI: 10.1039/b612197g
process.8 Initially, we placed the starting materials in reagent loops,
A general flow process for the multi-step assembly of peptides
which were then connected in-line with the flow system via an
AFRICA1 reagent store. The contents of the reagent loops are
then introduced into the flow stream when needed using a manual
switch and pumped through the columns containing the supported
reagents, creating the peptides.
has been developed and this procedure has been used to
successfully construct a series of Boc, Cbz and Fmoc
N-protected dipeptides in excellent yields and purities, including
an extension of the method to enable the preparation of a
tripeptide derivative.
The synthesis of the Cbz and Boc protected dipeptides was
accomplished by first packing a glass Omnifit1 column9 with
polymer supported 1-hydroxybenzotriazole (HOBt, reagent 2), a
useful reagent for amide bond synthesis.10 This column was
washed with DMF and a solution of the Cbz or Boc protected
amino acid, DIPEA, and the phosphonium coupling reagent
PyBroP1 (bromo-tris-pyrrolidino-phosphonium hexafluoropho-
sphate11) in DMF was pumped through the column. This allowed
the PS-HOBt to sequester the activated amino acid, ‘‘catching’’ the
substrate for later use, while also allowing any by-products of the
reaction to be directed to waste. The column was then washed
thoroughly with DMF leaving an activated amino ester which was
covalently attached to the polymer support. The column was then
switched in-line with prewashed/swelled columns (in DMF) of
polymer supported dimethylaminopyridine (PS-DMAP, reagent 1)
and polymer supported sulfonic acid resin (MP-SO3H, reagent 3),
linked in series (DMAP–HOBt–SO3H, Scheme 1). A second
amino acid, in the form of the HCl salt of the protected ester, was
then passed through all three columns in DMF at a flow rate of
100 mL min21. The effect of this continuous flow process was to
make use of PS-DMAP to liberate the amine from its salt, which
then reacts with the HOBt-activated Cbz or Boc protected amino
ester, forming a new peptide bond. The product of this reaction
was then passed through the MP-SO3H column which scavenges
any unreacted amine. The resulting solution was then evaporated
The preparation of peptides is largely considered to be a solved
synthetic problem, yet one should remember that it took 25 years
to develop reliable procedures for the formation of multiple amide
bonds.1 A significant proportion of the methodology developed
has used solid-phase synthesis, as this is readily adapted to
automated synthesiser systems.2 Depending upon the length of the
sequence, however, costs can escalate rapidly and scale up becomes
an issue due to the linear nature of the processing. The inherent
simplicity of the machinery also renders it hard to incorporate
novel amino acids or adopt unusual chemical bond forming
reactions, as required for peptidomimetics, in an automated
fashion.3
Here we report an alternative method for the synthesis of di-
and tripeptides using a simple flow process that should be
extendable to give access to a variety of advanced amide products.
The process is conducted by progressing N-protected amino acids
through a microfluidic flow system that is comprised of various
packed columns that contain supported reagents, scavengers and
catalysts. The resulting peptide products can be collected upon
elution from the flow system, and isolated in high purity by merely
removing the solvent, thereby bypassing costly and time consum-
ing purification procedures.
Previously we have developed continuous flow processes for the
synthesis of the natural products grossamide4 and oxomaritidine.5
As part of the project on grossamide, an automated flow6 reactor
was developed and used to prepare a small focused library of
amides through the coupling of various amines with a particular
carboxylic acid. We have extrapolated upon this amide bond
forming process and applied it to the synthesis of small Boc, Cbz
and Fmoc N-protected dipeptides. In addition, we also report on
an elongated flow process for the synthesis of a tripeptide
derivative.{
Our process makes use of a commercially available Syrris
AFRICA1 micro/mesofluidic pumping system,7 however any
HPLC-grade pump would be adequate to facilitate this flow
Innovative Technology Centre, Department of Chemistry, University of
Cambridge, Lensfield Road, Cambridge, UK CB2 1EW.
E-mail: svl1000@cam.ac.uk;Fax:+441223336442;Tel: +44 1223336398
{ Electronic supplementary information (ESI) available: Experimental
section. See DOI: 10.1039/b612197g
Scheme 1 Synthesis of Boc and Cbz N-protected dipeptides.
Chem. Commun., 2006, 4835–4837 | 4835
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using a Vapourtec V-101 solvent evaporator,12 an apparatus that
can easily remove y 10 mL of DMF in 15 minutes, while only
heating the mixture to 38 uC. It should also be noted that to ensure
a uniform flow stream (y 9 bar back pressure), a readily available
and inexpensive back pressure regulator was also placed in-line at
the exit of the flow stream.
Table 1 displays the results of coupling the amino acids to create
the corresponding dipeptides. Typically, the Boc protected amino
acids were found to give the highest yields, . 80% isolated yield
(entries 1–3), when compared to analogous Cbz protected
dipeptides, 75–79% (entries 5–8). When the proline derived amino
acids were used (entries 4 and 9), the isolated yields were found to
be slightly lower at 66% and 61%, respectively, presumably due to
the steric effects of the more hindered secondary amine. For the
dipeptides which contained glycine derived amino acids, the optical
purities were found to be in accordance with literature values
(entries 2, 6 and 8), while all other products were found to be single
diastereoisomers. In general, all products were found to be of
. 95% purity by 1H NMR and LC-MS, and to display the same
physical properties as those previously published in the literature.
It is possible to achieve higher yields by loading the column with a
greater equivalence of HOBt, although the current method uses
the most practical flow rate and the most appropriate levels of
reagents to give the most practical yields of product.
Scheme 2 Synthesis of Fmoc N-protected dipeptides.
disconnected from the PS-HOBt column and prewashed/swelled
PS-DMAP and MP-SO3H columns were in turn connected in
series to generate a flow stream that matches Scheme 1. Initially,
the flow synthesis was carried out at room temperature and Fmoc-
Ala-Phe-OEt was only isolated in 19% yield. The low yield was
considered to be a result of incomplete generation of the anhydride
from the IIDQ step. When the IIDQ column was heated to 60 uC
using a Vapourtec R-41 column heater,12 much more efficient
activation of the HOBt column occurred and the product was
isolated in an improved yield of 75%, while Fmoc-Ala-Gly-OEt
was produced in a 71% isolated yield. As with previous examples,
the spectral and physical data of these compounds were found to
be consistent with those reported in the literature. Overall, this
alternate method establishes a modified flow process for the
synthesis of Fmoc protected dipeptides, which progresses with high
purity (. 95%) and generates products as single diastereomers.
Our next step was to develop a process for the flow synthesis of
tripeptides using the existing methodology, while also incorporat-
ing an additional deprotection step. We have previously reported
on a method for the reduction of imines using a flow
hydrogenator15 (Thales H-Cube2)16 and also for the deprotection
of Cbz protected amines.17 In an analogous fashion, it was our
desire to use this piece of equipment to deprotect the Cbz group of
the dipeptides. The resulting product could then be used in a
subsequent coupling reaction generating a continuous flow
process. Scheme 3 depicts how the flow process was implemented.
Firstly, two separate columns of PS-HOBt were similarly activated
with different Cbz protected amino acids, denoted HOBtI and
HOBtII. Prewashed/swelled columns of PS-DMAP and MP-SO3H
were then placed in series with the first activated HOBt column
(DMAP–HOBtI–SO3H). The HCl salt of an ester-protected amino
acid was then pumped through these columns (in DMF) and
directed towards the flow hydrogenator. Due to solvent incompat-
ibility, the DMF was removed using a Vapourtec V-101 solvent
evaporator and the crude product from the HOBtI reaction was
redissolved in a mixture of EtOAc : EtOH (1 : 1). The dipeptide
was then subjected to flow hydrogenation for the deprotection of
the Cbz group by the H-Cube18 and the solvent removed using the
Vapourtec V-101, then redissolved in DMF for the last coupling
step. This solvent switch procedure from DMF to EtOAc : EtOH,
This same method was also used to couple Fmoc N-protected
amino acids with the corresponding amino esters in an analogous
fashion to that shown in Scheme 1. The products, however, were
found to be of an unacceptable purity (y 80%) by LC-MS and 1H
NMR, most likely a result of the tertiary amine (DIPEA)
degrading the Fmoc functionality by deprotection, a common
process when using similar amine bases such as piperidine.13 In
order to improve the method with the aim of creating only high
purity products, a separate route was developed. Scheme 2 details
the flow process for the specific synthesis of Fmoc protected
dipeptides. In this case, polymer supported IIDQ (2-isobutoxy-1-
isobutoxycarbonyl-1,2-dihydroquinoline,14 reagent 4) was first
used to create an anhydride of the Fmoc protected amino acid
which was then passed onto a column containing the PS-HOBt.
This process acts as an activation step which allows for the
resulting anhydride to react with the supported HOBt, generating
an activated amino ester which is ready for the peptide coupling
step. The by-products of this initial activation step are only
2-methylpropan-1-ol, carbon dioxide, and the residual Fmoc
protected amino acid from the anhydride–HOBt reaction which
can easily be collected and recycled. The PS-IIDQ column was
Table 1 Yields of coupled dipeptides
Yield
(%)a,b
Entry
Pg
R
R1
R2
1
2
3
4
5
6
7
8
9
a
Boc
Boc
Boc
Boc
Cbz
Cbz
Cbz
Cbz
Cbz
Me
Me
Me
Me
CH2Ph
CH2Ph
Me
Me
Me
CH2Ph
H
CHMe2
[–CH2–]3
CHMe2
H
CH2Ph
H
[–CH2–]3
Et
Et
Me
Me
Me
Et
Et
Et
Me
80
81
83
66
79
76
75
78
61
b
Isolated yield. Purities measured as . 95% by 1H NMR and LC-
MS.
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Scheme 3 Flow synthesis of tripeptide.
1 For a general review on synthetic studies of cyclic peptides and
depsipeptides please see: Y. Hamada and T. Shiori, Chem. Rev., 2005,
105, 4441 and/or references cited therein.
2 For a general discussion on automated continuous flow peptide
synthesis please see: R. P. Andrews, Nature, 1986, 319, 429 and/or
references cited therein.
3 (a) D. Bang, N. Chopra and S. B. H. Kent, J. Am. Chem. Soc., 2004,
126, 1377; (b) I. Coin, R. Do¨lling, E. Krause, M. Bienert, M. Beyermann,
C. D. Sferdean and L. A. Carpino, J. Org. Chem., 2006, 71, 6171; (c)
C. Toniolo, F. Formaggio, B. Kaptein and Q. B. Broxterman, Synlett,
2006, 1295.
4 I. R. Baxendale, C. M. Griffiths-Jones, S. V. Ley and G. K. Tranmer,
Synlett, 2006, 427.
5 I. R. Baxendale, J. Deeley, C. M. Griffiths-Jones, S. V. Ley, S. Saaby
and G. K. Tranmer, Chem. Commun., 2006, 2566.
6 For additional information on flow chemistry, please see research
contained and cited in references 4 and 5, as well as W. Solodenko,
H. Wen, S. Leue, F. Stuhlmann, G. Sourkouni-Argirusi, G. Jas,
H. Scho¨nfeld, U. Kunz and A. Kirschning, Eur. J. Org. Chem., 2004,
3601.
7 Pumping of solvents through the reagent columns was performed using
the commercially available Syrris AFRICA1 system. Website: http://
www.syrris.com.
back to DMF is the only time product is handled by the user and
is a result of solvent incompatibility and engineering, rather than a
limitation of the flow process itself. Currently, we are working on
methods to perform this solvent switch in a purely automated
fashion.
In the final coupling process, the deprotected product was
passed through the last two columns connected in series (PS-
HOBtII and MP-SO3H) and the product collected and solvent
removed. The tripeptide Cbz-Phe-Ala-Gly-OEt was isolated in
59% yield as a single diastereoisomer, based on the longest linear
flow process (HCl?Gly-OEt as limiting reagent) in greater than or
equals to 95% purity.
One of the advantages of the flow process is that the starting
materials and products spend very little time being exposed to the
supported reagents while they are in solution. The products are
part of a moving, dynamic flow stream and are quickly pumped
beyond the reach of the reagents, making further chemical
transformation, degradation or racemization negligible. An
additional advantage of this flow process is the improvement in
terms of time over conventional batch techniques, which usually
take y 24 hours.19 A typical reaction for the flow synthesis of a
dipeptide can easily be performed in 3 to 4 hours, while the
synthesis of a tripeptide takes only 6 to 7 hours in total.
In this work we describe the development of several simplified
flow methods that can be applied to the synthesis of dipeptides and
tripeptides; we believe, however, that these procedures can be
readily applied to the synthesis of a wide array of small
polypeptides. This communication details only our preliminary
efforts in the development of a flow process for the synthesis of
peptides and we will eventually be extending our flow methodol-
ogy to include longer peptide chains, a wider array of natural and
unnatural amino acids, and peptidomimetics.
8 M. Baumann, I. R. Baxendale, S. V. Ley, C. D. Smith and
G. K. Tranmer, Org. Lett., 2006, accepted.
9 Commercially available Omnifit glass chromatography columns with
adjustable height endpieces (plunger). Typically, the polymer supported
reagent is placed in an appropriately sized Omnifit1 column, usually
6.6 mm bore by 150 mm length, or shorter, and the plungers are
adjusted to relevant bed heights and the polymer swelled/washed with
solvent. Website: http://www.omnifit.com.
10 I. E. Pop, B. P. De´prez and A. L. Tartar, J. Org. Chem., 1997, 62, 2594.
11 (a) J. Coste, E. Fre´rot and P. Jouin, Tetrahedron Lett., 1991, 32, 1967;
(b) E. Fre´rot, J. Coste, A. Pantaloni, M.-N. Dufour and P. Jouin,
Tetrahedron, 1991, 47, 259.
12 Vapourtec V-101 rapid solvent evaporator and R-41 flow reactor
heater available from Vapourtec Ltd, Place Farm, Ingham, Suffolk, UK
IP31 1NQ. Website: http://www.vapourtec.co.uk.
13 J. Lee, J. H. Griffin and T. I. Nicas, J. Org. Chem., 1996, 61, 3983.
14 E. Valeur and M. Bradley, Chem. Commun., 2005, 1164.
15 S. Saaby, K. R. Knudsen, M. Ladlow and S. V. Ley, Chem. Commun.,
2005, 2909.
We gratefully acknowledge financial support from the RS
Wolfson Fellowship (to IRB and SVL), the Natural Sciences and
Engineering Research Council of Canada for a Postdoctoral
Fellowship (to GKT), Syngenta for financial support (to CDS)
and the BP endowment and the Novartis Research Fellowship (to
SVL).
16 Commercially available from Thales Nanotechnology, H-1031
Budapest, Za´hony utca 7 (Graphisoft Park), Hungary. Website: http://
www.thalesnano.com.
17 V. Franckevicius, K. R. Knudsen, M. Ladlow, D. A. Longbottom and
S. V. Ley, Synlett, 2006, 889.
18 Conditions used available from the Thales Nanotechnology website in
an Adobe Acrobat1 PDF file, benchmarking.pdf. Website: http://
thalesnano.com/filerepository/download/file/applicationbenchmarking.
A manuscript is in preparation on the detailed investigation of Cbz-N
hydrogenolysis using the Thales H-Cube, K. R. Knudsen, M. Ladlow
and S. V. Ley, University of Cambridge, Cambridge, UK, personal
communication.
Notes and references
{ General methods for the synthesis of Boc and Cbz protected dipeptides,
for Fmoc protected dipeptides and a method for the synthesis of a
tripeptide are located in the supplemental information.
19 R. Chinchilla, D. J. Dodsworth, C. Na´jera and J. M. Soriano,
Tetrahedron Lett., 2000, 2463.
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Chem. Commun., 2006, 4835–4837 | 4837