Continuous photochemical cleavage of linkers for solid-phase synthesis

Article abstract of DOI:10.1021/ol500530q

Photolabile linkers are an attractive alternative for solid-phase synthesis because they can be cleaved using light. However, irradiation in a classical batch photoreactor results in incomplete cleavage of the photolabile linkers. It is demonstrated that a continuous flow photoreactor is superior to a batch photoreactor for the cleavage of a linker from polystyrene resin.

Full text of DOI:10.1021/ol500530q

Letter
pubs.acs.org/OrgLett
Continuous Photochemical Cleavage of Linkers for Solid-Phase
Synthesis
Mattan Hurevich,† Jeyakumar Kandasamy,† Bopanna M. Ponnappa,†,‡ Mayeul Collot,† Daniel Kopetzki,†
D. Tyler McQuade,†,§ and Peter H. Seeberger*,†,‡
†Max Planck Institute of Colloids and Interfaces Am Muhlenberg 1, 14476 Potsdam Germany
̈
‡Institute of Chemistry and Biochemistry, Freie Universitat Berlin, Arnimallee 22, 14195 Berlin Germany
̈
§Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, United States
S
* Supporting Information
ABSTRACT: Photolabile linkers are an attractive alternative for solid-
phase synthesis because they can be cleaved using light. However,
irradiation in a classical batch photoreactor results in incomplete
cleavage of the photolabile linkers. It is demonstrated that a continuous
flow photoreactor is superior to a batch photoreactor for the cleavage of
a linker from polystyrene resin.
olid-phase organic chemistry (SPOC) relies on the
presence of readily cleavable linkers to join the resin and
cleavage mechanism in both reactors. Our study demonstrates
the advantage of using a flow reactor for photocleavage
reactions and highlights the limitations of the classic batch
reactor.
Two photoreactors were constructed to compare cleavage
efficiencies in batch and in flow (Figure 1). The batch reactor
we used is representative of a popular apparatus used for
photocleavage. The continuous-flow setup was a modified
version of the previously reported photoreactor regularly used
for continuous-flow photosynthesis.3
S
molecule of interest.1 Once the sequence of reactions on solid
support is complete, the product must be cleaved from the
resin without destroying the synthesized compound. Photo-
labile groups are often stable to many reagents and reaction
conditions and can be cleaved under mild conditions.
Photocleavable linkers consist of a photolabile functional
group which, when exposed to light of an appropriate
wavelength, undergoes photoinduced fission to liberate the
linker with the desired synthesized substrate.
While many photocleavable linkers have been reported, their
application in SPOC is not common.2 A factor limiting their
use is that cleavage in traditional batch photoreactors is
inefficient because of the presence of a fixed light source
resulting in low penetration of light to the entire sample and
the presence of an insoluble solid support solution, resulting in
a heterogeneous mixture. In addition, cleavage in a batch
photoreactor requires an extended reaction time and cannot
easily be scaled up.
On the other hand, continuous flow reactors have proven
efficient in performing challenging photochemical reactions.3
The use of flow photoreactors to induce cleavage from a solid
support is an attractive alternative to batch synthesis because
the solid support will receive a more homogeneous exposure to
the light and scaling up reactions in flow is typically
straightforward. Indeed, we have previously observed that
irradiation in flow, where beads are pumped through tubes
wrapped around a light source, results in efficient cleavage of
the product from the resin.4
A number of mechanistic models can be suggested to explain
the improved yields observed for flow photocleavage, but
systematic experimental evidence has not yet been described.
Here, cleavage efficiency in batch and continuous flow reactors
was compared. Polystyrene beads, labeled with either Fmoc or
fluorescein, were used to study the factors influencing the
Figure 1. Resin distribution in photoreactors: (A) in batch
photoreactors resin packs as a thick rim around the light source as
there is no movement; (B) constant flow helps to distribute the resin
in flow photoreactors.
Received: February 18, 2014
Published: March 11, 2014
© 2014 American Chemical Society
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To evaluate irradiation in batch, a classical photoreactor was
constructed from a double-jacketed glass reactor with a
constant cooling water circulation as the basic setup. To cleave
the compound from the support, the polystyrene resin
(suspended in DCM) was placed in the reactor central well.
To block irradiation below 300 nm, a Pyrex filter was placed
between the light source and reaction well. A UV pencil lamp
(365 nm) was inserted into the Pyrex filter and adjusted to
reach the level of the resin (as shown in Figure 1A). The entire
reactor was covered with aluminum foil and placed on an oval
shaker. Irradiation was performed for 30 min with constant
shaking of the resin to achieve maximal exposure. In order to
understand the effect of shaking on the reaction, the same
photocleavage procedure was repeated without shaking. After
irradiation, the light source was turned off, and the aluminum
foil was taken off. The Pyrex filter and the lamp were removed,
and the solid support was taken out using a Pasteur pipet and
filtered through a polypropylene filter tube equipped with
polyethylene frit (PE filter) to give the crude compound.
The flow reactor was built in a very different fashion (Figure
1B). The entire setting was placed in a sealed aluminum box
equipped with entry and exit holes for the fluorinated ethylene
propylene (FEP) tubing, the electrical cords, and the water
Scheme 1. Synthesis and Labeling of the Photolabile Linker
supply lines. Based on a previous setup by Hook et al.,3a
a
medium-pressure mercury lamp (450 W) was covered with a
Pyrex filter, and both were inserted into a double-jacket
immersion well with water circulation. FEP tubing was wrapped
around the jacket so that the entire length of the lamp was
covered. Both ends of the FEP tubing were connected outside
the box, with one end connected to a syringe pump and the
other end to a flask fitted with a PE filter, to separate the resin
from the filtrate (see pictures in the Supporting Information).
In a typical experiment, the solid support was preswollen in
DCM and pushed through the FEP tubing using a syringe
pump (usually in a vertical position so that the floating resin
can easily pass into the tube) with a residence time of 30 min
before being filtered through the PE frit. The tubing was
cleaned by injection of a fresh solution of DCM through the
system before the next use.
The o-nitrobenzyl photolabile group was used as the basis for
a model linker to study the cleavage mechanism.5 An amine-
functionalized o-nitrobenzyl-based linker (1) was synthesized
on the basis of a previous procedure.4b
The photolabile linker 1 was synthesized by forming first a
Schiff base between 5-hydroxy-2-nitrobenzaldehyde and N-Boc-
1,6-hexanediamine. Reduction of the imine with NaBH4 gave a
secondary amine that was later protected using CbzCl under
basic conditions to give 1 (Scheme 1).
Merrifield resin (chloromethyl polystyrene) was function-
alized using 1 in the presence of tetrabutylammonium iodide
and CsCO3 in N,N-dimethylformamide (DMF). The Boc
protecting group was subsequently removed using TFA to give
the solid support PSLin1. PSLin1-Fmoc and PSLin1-FTU
(FTU, fluorescein thiourea) were synthesized by coupling the
free amine of PSLin1 under basic conditions with Fmoc-Cl and
Fluorescein isothiocyanate (FITC), respectively (Scheme 1).
PSLin1-Fmoc was cleaved in flow, and the product was
characterized to demonstrate that irradiation does not affect the
cleaved compound (see the Supporting Information). Although
the photocleavage reactions preceded efficiently, variable
quantities of impurities where always present in the crude
sample (10−30%). Even though these impurities could be
easily removed by chromatography (see the Supporting
Information), their presence made evaluation of the cleavage
efficiency by crude mass yield inaccurate and too prone to
errors, especially when small amounts of resin were used. To
overcome the above limitations, the cleavage efficiency of flow
and batch reactors was measured using more sensitive optical
methods (UV spectrophotometry for PSLin1-Fmoc and
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confocal microscopy for PSLin1-FTU) that are accurate even
for small amounts of resin. By using optical methods we
decreased the error associated with the impurities as we
examined only the presence or absence of the fluorophore
marker.
Standard Fmoc quantification6 of PSLin1-Fmoc beads
before and after irradiation in batch (with shaking) showed
that 70% of the Fmoc protecting group was still on the beads
and only 30% was cleaved and found in solution. On the other
hand, irradiation in flow resulted in a greater than 70% decrease
in Fmoc loading of the PSLin1-Fmoc beads. Based on these
results, we assumed that the efficiency of the cleavage was
related to the overall exposure of the beads to the light source
during irradiation. This method provided us with an indication
that flow cleavage was more efficient than batch. However,
additional experiments were designed in an effort to obtain
visual evidence that explains the mechanism of cleavage.
A fluorescein thiourea (FTU)-labeled solid-supported photo-
linker PSLin1-FTU (Scheme 1) was used to study the impact
of the irradiation methods on the cleavage pattern. Confocal
laser scanning microscopy was used to measure and visualize
bead fluorescence before and after irradiation.
To ensure irradiation does not influence the fluorescein
moiety, aminomethylated polystyrene (commercially available)
was loaded with FITC, and the fluorescence was measured
before and after irradiation (see the Supporting Information).
Our results indicate that irradiation did not significantly quench
the fluorescence of the labeled beads when no photolabile
linker is used.
Conversely, when fluorescein-labeled beads PSLin1-FTU
(which has photolabile linker) were irradiated, a measurable
decrease in fluorescence was visible. Mean fluorescence
intensity (MFI) measurements were used to quantify the loss
of fluorescence due to photocleavage of the linker-fluorescein
moiety (results summarized in Figure 2). Unlabeled beads
(PSLin1) were imaged to measure bead autofluorescence as a
negative control (Figure 2A), whereas labeled beads (PSLin1-
FTU) exhibit strong fluorescence and served as a positive
control (Figure 2B).
Four different irradiation methods were compared. Beads
were irradiated in a standard batch photoreactor with or
without shaking the photoreactor using an orbital shaker
(Figure 2D ,C) to evaluate the effect of mixing on batch
cleavage. Beads were also injected through a small (1.5 mm
tube, inner diameter 0.8 mm, Figure 2E) or large diameter tube
(3.2 mm tube, and inner diameter 1.5 mm, Figure 2F) to study
the contribution of tube diameter on the flow assisted
photocleavage.
Batches that were irradiated without shaking (Figure 2C)
afforded a heterogeneous mixture where some beads
experienced almost complete photocleavage (beads with a
dark center and only a rim of fluorescence) while others
showed only a slight decrease in fluorescence. We assumed that
irradiation without shaking resulted in a nonhomogeneous bead
population because those beads positioned in proximity to the
light source absorbed most of the energy and shielded others
that were located further from the source. In contrast, batch
irradiation with shaking (Figure 2D) resulted in a population of
beads that mostly displayed partial cleavage of the Lin-FTU and
overall significantly decreased fluorescence. Clearly, more beads
were exposed to the light source when the reactor was shaken
in comparison to the previous irradiation experiment without
shaking. Hence, the mixing increased the absolute number of
Figure 2. Confocal microscopy images of fluorescein labeled beads
(PSLin1-FTU): (A) unlabeled beads (PSLin); (B) fluorescein-labeled
beads (PSLin1-FTU); (C) PSLin1-FTU beads after irradiation in
batch; (D) PSLin1-FTU beads after irradiation in batch using an
orbital shaker; (E) PSLin1-FTU beads after irradiation in flow-reactor
using a narrow bore tube; (F) PSLin1-FTU beads after irradiation in a
flow-reactor using a large bore tube. Mean fluorescence intensity
(MFI) analysis (bottom graph) shows that standard batch irradiation
of the beads results in minimal cleavage of fluorescein (C) while
irradiation in flow using the large bore tube results in almost complete
cleavage of fluorescein (F).
photocleavage events from the resin and at the same time
increased the homogeneity of cleavage by ensuring that each
bead experienced similar light exposure. From these batch
experiments, we conclude that both proximity and homoge-
neous exposure to the light source are crucial for efficient
cleavage.
The batch cleavage system incorporating shaking is an
improvement to the classic batch reactor but is technically
challenging and more dangerous to handle. In addition, it does
not address the problem of scaling up the reactions.
To examine how flow affects cleavage, we performed
photocleavage experiments in the photoreactor using small
and large bore tubes as the flow-through channel.
When PSLin1-FTU beads were pumped through small
dimension tubing (0.8 mm inner diameter), a heterogeneous
population of irradiated beads resulted (Figure 2E). Many
beads exhibited fluorescent intensity similar to the positive
control while others showed almost no fluorescence. These
results indicate that the beads pass through the narrow tubing
as a multilayer slug that cannot move freely along the diameter
of the tubing. As a result, beads closer to the light source
undergo near-complete photocleavag,e while those beads that
are on the outside (away from the light source) are shielded.
When a wider tubing (1.5 mm inner diameter) was used,
irradiated PSLin1-FTU beads exhibit a much more uniform
low fluorescence intensity (Figure 2F). Visual inspection of the
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(3) (a) Hook, B. D. A.; Dohle, W.; Hirst, P. R.; Pickworth, M.; Berry,
M. B.; Booker-Milburn, K. I. J. Org. Chem. 2005, 70, 7558.
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beads flowing through the larger tube indicated that the beads
undergo turbulent mixing and tumbling (see the movie in the
Supporting Information). We propose that the mixing results in
similar exposure of each bead to the light source and thus
results in uniform, highly efficient cleavage of the fluorophore
from the resin.
In addition to providing a more efficient cleavage, the flow
reactor is easier to use than the batch system. In a standard
batch reactor, manual placement and removal of beads and
reagents from the reactor requires turning off the light source
each time and thus limits throughput. The setup of the flow
reactor allows for continuous performance without turning off
the system. A simple wash between resin deliveries is sufficient
to enable multiple runs in the same reactor.
(6) (a) Gude, M.; Ryf, J.; White, P. D. Lett. Pept. Sci. 2002, 9, 203.
(b) Meienhofer, J.; Waki, M.; Heimer, E. P.; Lambros, T. J.; Makofske,
R. C.; Chang, C. D. Int. J. Pept. Prot. Res. 1979, 13, 35.
We have demonstrated that a combination of factors unique
to the flow photoreactor leads to improved photocleavage. The
flow reactor is an alternative to classical photoreactors and
offers high throughput and efficient cleavage in a short amount
of time. Flow photocleavage renders photocleavable linkers
useful for the solid-phase synthesis of many classes of
compounds.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed description of the reactors and cleavage procedure,
linker preparation, and solid support functionalization, methods
of analysis, NMR and HPLC characterization of cleaved
compounds, and a movie demonstrating bead movement in
flow. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: peter.seeberger@mpikg.mpg.de.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Max-Planck Society for generous financial
support. We also Mark Schlegel from Glycouniverse GMBH for
helping with editing the manuscript. M.H. thanks the Minerva
Foundation for a postdoctoral fellowship
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