Synchronized synthesis of peptide-bnased macrocycles by digital microfluidics

Article abstract of DOI:10.1002/anie.201001604

Digital synthesis has been applied to the formation of peptide-based macrocycles and their analogues with side chains appended during late-stage aziridine ring-opening. Discrete nanoliter- to microliter-sized droplets of samples and reagents are controlled in parallel by applying a series of electrical potentials to an array of electrodes coated with a hydrophobic insulator.

Full text of DOI:10.1002/anie.201001604

Angewandte
Chemie
DOI: 10.1002/anie.201001604
Microfluidic Synthesis
Synchronized Synthesis of Peptide-Based Macrocycles by Digital
Microfluidics**
Mais J. Jebrail, Alphonsus H. C. Ng, Vishal Rai, Ryan Hili, Andrei K. Yudin,* and
Aaron R. Wheeler*
There has been considerable interest in peptide-based macro-
cycles,[1–5] as their topology allows them to resist digestion by
exopeptidases while retaining high affinities for their bio-
chemical targets.[4,6,7] We recently described a novel macro-
cyclization strategy that is enabled by amphoteric aziridine
aldehydes (Scheme 1).[8] Peptide macrocycles can now be
The most common format for miniaturized synthesis is
enclosed microchannels in planar platforms. Such systems
have been used for conventional organic synthesis,[9–13]
polymerization reactions,[14,15] formation of biomolecules,
such as peptides and DNA,[16–18] and generation of nano-
particles and colloids.[19–21] However, there are some chal-
lenges that limit the scope of their use for parallel chemical
synthesis. For example, many microchannel platforms are
formed from poly(dimethylsiloxane), a material that is
susceptible to degradation in common organic solvents.[22,23]
Furthermore, control of many reagents simultaneously (a
feature required to implement parallel synthetic reactions) in
microchannels requires pumps, tubing, valves, and/or three-
dimensional channel networks that can be difficult to
fabricate and operate.[9,24] This has prompted researchers to
develop specialized techniques[25] to overcome this limitation.
Another disadvantage associated with the microchannel
format is the challenge inherent in the removal of solvents
and re-dissolution of solids that are common steps in syn-
thesis. Solid reagents and products can clog microchannels,
making targeted reagent delivery difficult to control. Finally,
the small volumes of samples in microchannels makes it
difficult to recover them in sufficient quantities for off-line
analysis techniques, such as NMR spectroscopy.
In need of a platform capable of generating a) peptide
macrocycles for downstream transfer onto functionalized
surfaces, and b) spatially resolved macrocyclic peptide prod-
ucts in the solid state, we chose an alternative format for
automation of synthesis, called digital microfluidics.[26] In
digital microfluidics, discrete nL to mL sized droplets of
samples and reagents are controlled in parallel by applying a
series of electrical potentials to an array of electrodes coated
with a hydrophobic insulator (Figure 1). Digital microfluidics
has become popular for biological and chemical applications,
including cell culture and assays,[27–29] enzyme assays,[30–32]
immunoassays,[33,34] protein processing,[35–40] clinical sample
processing and analysis,[41] and synthesis of anisotropic
Scheme 1. Synthesis of peptide-based macrocycles and their structur-
ally modified derivatives.
made with high chemoselectivity from amino acids or linear
peptides, isocyanides, and amphoteric aziridine aldehydes in a
one-step process. Importantly, the resulting products possess
useful structural features that allow specific modification at
defined positions. In our initial work, we formed serial
batches of peptides using conventional macroscale synthetic
techniques. The utility of this method is limited, however,
without a high-throughput approach for generating focused
libraries of peptide macrocycles. Such a method would be
automated and would enable multistep reactions to be
handled in parallel. Herein, we present a new miniaturized
technique for synchronized on-chip synthesis of peptide
macrocycles and related products.
[*] M. J. Jebrail, Dr. V. Rai, Dr. R. Hili, Prof. A. K. Yudin,
Prof. A. R. Wheeler
Department of Chemistry, University of Toronto
Toronto, ON M5S3H6 (Canada)
Fax: (+1)416-946-7676
Fax: (+1)416-946-3865
E-mail: ayudin@chem.utoronto.ca
aaron.wheeler@utoronto.ca
A. H. C. Ng, Prof. A. R. Wheeler
Institute of Biomaterials and Biomedical Engineering
University of Toronto (Canada)
[**] We acknowledge Natural Science and Engineering Research Council
(NSERC) and Canadian Institutes of Health Research (CIHR) for
financial support. We thank Timothy Burrow, Dmitry Pichugin, and
Daniel Majonis for NMR analysis.
Figure 1. A digital microfluidic platform. Each droplet acts as a micro-
vessel in which parallel reactions can take place with no cross-
contamination.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201001604.
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particles and tetrahydroquinolines.[42,43] The initial synthesis
studies[42,43] used simple one-plate devices that are capable of
carrying out only a single, serial reaction, with no reagent-
supply reservoirs, dispensing, splitting, or active mixing. Our
goal was to implement a fully integrated platform with
reagent supply reservoirs, precise control over reagents (i.e.,
dispensing, splitting, and active mixing), and the capability to
precipitate peptide macrocycles for hierarchical modification
and analysis.
Herein, we introduce the first two-plate digital micro-
fluidic platform for chemical synthesis that is suitable for
control of many different multi-component, multi-step reac-
tions in parallel. We used this system to carry out synchron-
ized synthesis of peptide macrocycles from three different
components. The resulting products contain aziridines as sites
that are primed for specific, late-stage modification by
nucleophilic ring-opening. Using the same chemistry we
demonstrate the synthesis of a nine-membered macrocycle,
a ring size that is associated with considerable synthetic
difficulties in conventional cyclic peptide synthesis.[44] The
new method is fast, amenable to automation, compatible with
a wide range of solvents, liberated from external hardware
(e.g., pumps, tubing, etc.), and is particularly well-suited for
parallel processing.
Parallel synthesis of peptide-based macrocycles was
implemented on a new digital microfluidic system (Fig-
ure 2a). The design features ten reagent reservoirs and
eighty-eight actuation electrodes dedicated to dispensing,
merging, and mixing droplets of reagents and products. The
devices were designed to handle ten different reagents,
including five amino acids, an aziridine aldehyde, tert-butyl
isocyanide (tBuNC), and thiobenzoic acid (PhCOSH) as
stock solutions in trifluoroethanol (TFE) or water. As shown
in Figure 2b, the digital microfluidic device facilitated the
implementation of synchronized synthesis of five different
macrocycles in three steps. First, five 900 nL droplets
containing one of five amino acid substrates (Val, Met, Ile,
Thr, and Pro) were dispensed from their respective reservoirs.
Second, five 900 nL droplets containing aziridine aldehyde
were dispensed and merged with the amino acid droplets and
mixed. Third, five 900 nL droplets of tert-butyl isocyanide
were dispensed and merged with the droplets containing the
amino acids, and incubated. Finally, macrocyclic peptide
products were isolated by allowing the solvent to evaporate.
Some macrocycles were further modified in three addi-
tional steps to form structurally modified products (Figure 3).
First, each macrocyclic peptide product was re-dissolved in
fresh trifluoroethanol. Second, five 900 nL droplets contain-
ing thiobenzoic acid were dispensed, and merged with
macrocycle droplets, and the reactions were allowed to
incubate. Third, the aziridine ring-opened products were
isolated by allowing the trifluoroethanol to evaporate,
delivering precipitated macrocycyclic peptide thioesters at
defined positions. In all, this method constituted thirty
processing steps—six steps each for five reactions in parallel.
Mass spectrometry (MS) and NMR were used to evaluate
the effectiveness of on-chip peptide macrocycle and aziridine
ring-opened product synthesis. Figure 4 shows representative
mass spectra generated from unreacted methionine (Met) and
Figure 2. a) Top and side views of the digital microfluidic device used
for peptide-based macrocycle (PM) synthesis. b) Sequence of frames
from a movie illustrating digital microfluidic-based synthesis of PM. In
frames 1–3, droplets (each 900 nL) containing amino acid (AA) sub-
strates and aziridine aldehyde were dispensed from their respective
reservoirs, merged, and mixed. In frames 4,5, droplets of tBuNC were
dispensed and merged with the droplets of AA substrates and aziridine
aldehyde, and the reaction was allowed to proceed for 1 h at room
temperature. Finally, in frame 6, PM products were isolated by allowing
the solvent to evaporate. The insets in frame 6 are magnified images
of the dried products. In these frames, the top plate is not visible, as it
is formed from transparent ITO glass.
also peptide-based macrocycle-containing Met and its azir-
idine ring-opened derivative synthesized on a digital micro-
fluidic device, with peaks at m/z 150, 342, and 480, respec-
tively (mass spectra of the other peptide-based macrocycles
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(Supporting Information, Figure S5a,b). Complementing the
MS data, NMR spectra of the peptide-based macrocycle
containing methionine (Supporting Information, Figure S6)
and its aziridine ring-opened derivative (Supporting Infor-
mation, Figure S7) synthesized on the digital microfluidic
platform and on the macroscale had similar chemical shifts
that correlated well with all the protons. The reaction progress
was monitored as a function of Met conversion over time on
the digital microfluidic platform (Figure 4d) and on the
macroscale (Supporting Information, Figure S5c). The reac-
tion kinetics of the two methods were similar, with about 70%
(digital microfluidic) and about 90% (macroscale) depletion
of the initial reagent within one hour; we anticipate that
future systems integrated with continuous, rapid mixing[45,46]
will improve the kinetics of the microfluidic technique.
To further show the application of the technique, a nine-
membered macrocycle was synthesized on-chip (Figure 5a).
In this method, a Pro–Leu-derived macrocycle was prepared
Figure 3. Sequence of frames from a movie illustrating the key steps in
digital microfluidic-based synthesis of aziridine ring-opened (RO)
products. Peptide-based macrocycles (PM) are solubilized in trifluor-
oethanol (frames 1,2), then merged (frames 3,4) with droplets con-
taining thiobenzoic acid (PhCOSH), and then mixed and incubated for
1 h at room temperature, followed by isolation of RO products
(frame 5) by allowing the trifluoroethanol solvent to evaporate. The
insets in frame 5 are magnified images of the dried products.
Figure 5. a) Synthesis of a nine-membered macrocycle. TFE=trifluoro-
ethanol. b) ESI-MS spectrum of 8.
in three steps. 900 nL droplets containing dipeptide, aziridine
aldehyde, and isocyanide were dispensed from respective
reservoirs, merged, mixed, and incubated. Figure 5b shows a
mass spectrum from a Pro–Leu-derived macrocycle synthe-
sized on a digital microfluidic device, which further demon-
strates the reaction selectivity. Of note is an exciting
possibility offered by this new on-chip conjugation strategy:
thioesters are well-known precursors to native chemical
ligation.[47] We anticipate applications that will take advant-
age of S-to-N acyl transfer from the thioester to a nucleophilic
residue within the macrocycle.
The new digital microfluidic method is capable of
synthesizing peptide-based macrocycles and aziridine ring-
opened derivatives that are analogous to the macroscale
method, as shown by MS and NMR results. In comparison
with other miniaturized fluidic technologies, digital micro-
fluidics is particularly well-suited for applications in synthesis,
as it allows precise control over multiple reagent phases.
Importantly, it supports the critical step of solvent removal
Figure 4. ESI-MS spectra of a) methionine (Met), and products synthe-
sized by digital microfluidics, including a b) peptide-based macrocycle
containing Met and c) the aziridine ring-opened derivative. d) Reaction
progress by digital microfluidics as percentage conversion of Met over
time. Each data point represents the meanÆstandard deviation of
4 samples.
and their aziridine ring-opened products are given in the
Supporting Information, Figures S1–S4). The mass spectra of
products from the microscale synthesis were nearly identical
to those of the same products from macroscale synthesis
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[4.1.0] macrocycles. The substrate in this reaction was proline–leucine
(0.1m in TFE, 90 nmol), and the reaction required longer incubation
(3 h, RT) and frequent mixing (20 s, every 30 min). For reagents and
materials, device fabrication and operation, mass spectrometry, NMR
spectroscopy, macroscale synthesis, and conversion analysis, see the
Supporting Information.
and re-dissolution of product for further processing (Figure 3,
frames 1,2). This highlights the flexibility of digital micro-
fluidics: there are no limits on the volume of solvent used to
re-dissolve a particular solid (for example, in this method,
four droplets of trifluoroethanol representing a combined
volume of 3.5 mL were dispensed to facilitate dissolution of
each solid macrocycle). The salient features of digital micro-
fluidics for synthesis include individual addressing of all
reagents with no need for complex networks of micro-
valves,[9,24] a chemically inert Teflon-based device surface
that diversifies the scope of compatible reagents to include
organic solvents and corrosive chemicals, and easy access to
reasonably large amounts of products for off-chip analysis
(such as simply removing the top plate on a device). Last but
not least, the technique will likely be well-suited for evaluat-
ing macrocyclic libraries if the solvent is removed by
evaporation.[48]
Received: March 17, 2010
Revised: June 10, 2010
Published online: August 16, 2010
Keywords: automated synthesis · digital microfluidics ·
.
macrocycles · microreactors · peptides
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