From the molecule to the mole: improving heterogeneous copper catalyzed click chemistry using single molecule spectroscopy

Article abstract of DOI:10.1039/C6CC08905D

Single molecule spectroscopy (SMS) inspired the optimization of a heterogeneous 'click' catalyst leading to enhanced yields of the Cu-catalyzed reaction of azides with terminal alkynes. Changes in SMS data after optimization confirm the improvements in catalyst performance.

Full text of DOI:10.1039/C6CC08905D

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Cite this: DOI: 10.1039/x0xx00000x
From the Molecule to the Mole: Improving
Heterogeneous Copper Catalyzed Click Chemistry
using Single Molecule Spectroscopy
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
Bowen Wang,a,b Javier Durantini,a Matthew Decan,a Jun Nie,b Anabel E. Lanterna,*a
and Juan C. Scaiano*a
www.rsc.org/
The formation of 1,2,3-triazole by copper(I)-catalyzed
cyclization between azides and terminal alkynes (CuAAC) is
Single molecule spectroscopy (SMS) inspired the
optimization of a heterogeneous ‘click’ catalyst leading to
enhanced yields of the Cu-catalyzed reaction of azides with
terminal alkynes. Changes in SMS data after optimization
confirm the improvements in catalyst performance.
among all the click chemistry reactions the best known and
26
widely used (Scheme 1).25,
Few examples of Cu-based
heterogeneous catalysts have been reported in the past,27, 28 in
order to avoid incorporation of toxic copper complexes in the
end products. We have recently published the first example on
a heterogeneous Cu photocatalyst that can generate Cu(I)
species on demand.29 Previously in our group, we were able to
demonstrate, using Total Internal Reflection Fluorescence
Microscopy (TIRFM) and a system suited for single molecule
spectroscopy (vide infra), that click chemistry catalysed by
copper nanoparticles is a truly heterogeneous process.30 Later
Advanced microscopy techniques, including single molecule
spectroscopy and super-resolution techniques have led to
spectacular advances in our ability to monitor reactions under
conditions where the most intimate mechanistic details can be
revealed.1-3 ‘Seeing’ a single molecule, particularly as it
undergoes chemical reaction, may have belonged in the realm
of science fiction as we moved into the new millennium, yet,
today this can be done in carefully designed systems.
Biologists were first in recognizing the power of these tools in
the understanding of cellular processes, where the sensitivity
and spatial resolution of modern techniques offered an
unprecedented level of information.4-7 Chemists have been
somewhat slower8 in discovering how these microscopy tools
can help them, although a number of groups have made
excellent progress in this direction.9-18
work at the single-molecule level, performed with
a
commercial copper-on-charcoal (Cu@Charcoal) catalyst with
3% of Cu loading and particle sizes in the tens of micrometers
indicated that only 0.003% of the catalyst surface was active in
click chemistry.23 Cu@Charcoal is known as a very good
catalyst, although these results suggest there is plenty of room
for improvement. Here we present the first attempt to use
catalysis studies at the single molecule-single catalytic site
level to guide work at the bench scale.
In our group we have published several contributions,
sometimes labelled as ‘from the mole to the molecule’ where
we take advantage of advanced microscopy to visualize single
molecules as they reach and react at single catalytic sites and
depart once the process is complete.19-24 Yet, it is really ‘from
the molecule to the mole’ where the real advantages in
catalysis and organic chemistry can be expected. In other
words: can we use knowledge acquired using single molecule
techniques (‘the molecule’) to improve processes at the bench
and manufacturing level (the mole)? In this communication we
report one example as a proof of concept where single
molecule techniques inspired laboratory work that led to a
dramatic (>x10) improvement of the click reaction at the
bench level.
N
Y
N
Cu+
N
Y
+
X
N
N
N
X
Scheme 1. Formation of 1,2,3-triazole by copper(I)-catalysed cycloaddition
between azides and terminal alkynes (CuAAC).
Inspired by the previous results about the low efficiency of
Cu@Charcoal at the catalytic site level, we decided to try to
improve the catalyst efficacy by using different catalyst pre-
treatments. XPS analysis of this catalyst (Figure 1) reveals the
presence of CuO species, as confirmed by the characteristic
satellite peaks of Cu(II) between 945-940 eV.31 Thus, the
reason of the low catalytic efficiency found at single molecule
level could lay on the deficit of Cu(I) centres and therefore
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DOI: 10.1039/CJ6oCuCr0n8a9l05NDame
pre-treatment strategies involving reduction of the material Energy Transfer (FRET) as a probe for successful reaction,
were studied.32 Briefly, method 1 involves sonication of the and are the same that proved suitable in an earlier
material in ethanol for 2 h while method 2 uses a solution of publication.23 Thus, both functional groups required for click
NaBH4 in ethanol during sonication. After treatment, the chemistry (alkyne and azide) are attached to
a dye,
catalyst was tested in both bench and single molecule scale to AlexaFluor488 (AF488) and AlexaFluor596 (AF596),
evaluate its efficiency. The XPS analysis (Figure 1) shows respectively. AF488 acts as a donor chromophore, capable of
almost no changes in the nature of the Cu species after the transferring energy in a non-radiative process to the acceptor
catalyst is treated by method 1. Instead, more reduced Cu chromophore, AF596.
species are found when treated by method 2, as can be
appreciated by the shift toward lower energies in the Cu 2p3/2
region, suggesting Cu(I) or Cu(0) species are now predominant
100
0.02 weight %
80
60
in the material (differentiation between Cu(I) and Cu(0) is not
accurate by this technique).33
40
20
0
934.8 eV 931.8 eV
0
100
0.2 wt%
80
60
40
20
0
Untreated
Method 1
Method 2
Cu(II) satellites
Figure 2: Yields obtained for reaction in Scheme 2 using different concentration
of catalysts: 0.02 wt% (top) or 0.2 wt% (bottom) of untreated and pre-treated
Cu@Charcoal catalyst after 3 h (grey) and 12 h (black) of reaction.
970
960
950
940
930
Binding Energy (eV)
Figure 1. Comparison between Cu 2p HR-XPS spectra for Cu@Charcoal
untreated (black) and treated by Method 1 (red) and Method 2 (blue).
The efficiency of this FRET is inversely proportional to the
sixth power of the distance between donor and acceptor and
therefore at the concentrations of ~100 pM used FRET is
expected when they are part of the same molecule after
reaction as shown in Scheme 3. The selected dyes show
absorption and emission spectra overlap that fit with the
requirements of selective excitation of the donor and selective
detection of the acceptor emission (See figure S1).
Once treated, the catalysts were tested at the bench level using
the reaction shown in Scheme 2. Figure 2 shows the yields of
the reaction ran under the same conditions using untreated and
treated Cu@Charcoal by method 1 and 2. Clearly, the pre-
treated catalysts have improved their efficiency over the
untreated one. At least 10 times improvement in the catalytic
activity is reached when the catalyst is pre-treated with
NaBH4, and is attributed to the presence of more reduced Cu
species (Figure 1).
-
-
SO3
SO3
+
NH2
N
O
N
H2N
O
O-
OH
-
-
SO3
SO3
O
O
N
N
Cu@Charcoal
(0.02 - 0.2 wt%)
H
N
N
N3
N3
+
O
1 eq. Et3N
O
N
H
Air, THF, RT
1 eq.
1 eq.
AF594 (acceptor)
AF488 (donor)
Scheme 2: CuAAC used at bench top experiments.
-O3S
+
NH2
N
O
N
-O3S
H2N
FRET
O
These results illustrate how data from single molecule
microscopy can be used to inspire and guide experiments in
the laboratory that lead to dramatic improvements of the
catalytic process with modest effort. The improvements
observed at the bench scale could, in theory, be reproduced by
single molecule microscopy. However, it is important to note
that single molecule experiments are usually done in the 10-10
to 10-11 M concentration range, while bench experiments were
typically around 0.3 M. In addition to the 9 or 10 orders of
magnitude change in concentration, product formation always
involves interaction of two organic molecules (azide and
alkyne) and one catalytic site, albeit one of them (the alkyne)
pre-associates with the catalyst.30, 34-36
OH
-
-
SO3
-O
O
SO3
O
H
N
N
N
O
NH
N
O
Scheme 3: Click reagents employed for click chemistry with fluorescent
reporters. The product likely undergoes to FRET.
During TIRFM experiments the FRET events can be detected
as a burst in the intensity of the emission recorded versus time
(bursting events), Figure 3. Many fluorescence trajectories
showed multiple bursting events, indicative of localized
reaction, a typical indication of heterogeneous chemistry,30
Figure 3. However, we noted that most of these emission
events did not occur where the relatively large carbon particles
were detectable using optical microscopy, as they had been in
TIRFM studies would give more insights of the improvement
at the catalytic site level but different reagents need to be
chosen in order to follow the reaction by this fluorescence
spectroscopy technique (Scheme 3). The reagents were
carefully selected in order to use the Fluorescence Resonance
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earlier work23 with untreated catalyst, in fact, events shown are the same at the single molecule level; these results are not
dominated by the poputation of small particles.
surprising given the reaction order and the drastic decrease in
the reactant concentrations used for regular single molecule
studies (vide supra). No significant differences are found at
the single molecule level when untreated catalyst is compared
with the catalyst treated with ethanol (method 1), as can be
noticed in Figure 4. These results can account for the less
drastic improvement (~2x or less) found at the bench scale
(Figure 2). A simple, semi-quantitative, approach to this issue
is to consider the reaction as reminiscent of a Michaelis-
Menten mechanism as illustrated in Scheme 4. The initial
alkyne association equilibrium (Keq) likely is displaced largely
to the left under single molecule conditions (i.e, sub-
nanomolar concentrations), while at the high bench
concentrations its likely totally converted to the Alkyne-
Cu@Charcoal complex and thus at some point further
increases in concentration are not reflected in the overall
kinetics. Additionally, flow systems are a convenient tool to
keep the local reactant concentrations as low as possible and
minimize catalytic site poisoning.
500 counts
a
b
c
d
0
10
20
30
40
50
Time (s)
Figure 3: Intensity trajectories showing multiple bursting events within the 50 s
(500 frames) recording time of the video (excitation wavelength: 488 nm;
emission filter: 575 nm long pass). Traces a (single event), b (single or double
events) and c (double events) have been selected to be truly representative of
the many traces analysed. In particular, b and c illustrate the challenges
establishing the number of events. Trace d is one of the few rare cases where 3
events are clearly distinguishable. The black line is a 4-point smoothing function
and is provided for visual guidance and is not strictly required to decide on the
number of bursts per trajectory.
250
# events
3
200
x3
2
1
x2
150
100
50
Examination of the treated and untreated catalyst samples
using Scanning Electron Microscopy (SEM) revealed that in
addition to the very large particles on which we reported single
molecule studies earlier, there were numerous particles with
sizes of 100 nm or less, as illustrated in Figure S2 and noted in
earlier work.23 Particles of this size are not visible by optical
microscopy, but they provide a straightforward rationalization
for the observation of bursting at locations where the large
(micrometer) particles are absent. It is likely that these
particles are also responsible for increased light scattering
compared with large or colloidal particles examined before.
This is likely the reason for the increased background level
noticeable in Figure 3 compared to those in earlier reports.23, 30
With treated catalysts there are several possible ways to
analyse the data. The simplest, albeit rather qualitative
approach consists in counting successful events, such as those
illustrated in Figure 3 and adding them up for each type of
catalyst. For this purpose a trace that shows two events is
counted as two instances of success, and similarly for any
occurrence of multiple events. Here it is important to note that
successful events are those that show fluorescence bursts
within a 50 s recording of 500 images over an area of 34.5 µm
x 34.5 µm. There can be other catalytic sites that while active
showed no successful catalytic events (i.e., fluorescence
bursts) within the 50 s recording (see SI for Poisson type
analysis). We note also that while identical procedures are
used to prepare samples of different catalysts this does not
ensure that the number of sub-diffraction particles deposited
(and thus not observable by transmission optical microscopy)
are identical in the area monitored. Figure 4 summarizes these
results. Note that with the untreated catalyst (and treated by
method 1) triple events were rare, and their frequency was
only ~3% among all the events detected, and their numbers are
too small for meaningful statistical analysis. It is evident that
the number of successful events increased upon reductive
treatment, in particular with NaBH4 (triple events frequency
~7%). Although the improvement factor reached at the bench
scale (10x or greater after treatment with NaBH4) is clearly not
x1
0
Untreated
Method 1
Catalyst
Method 2
Figure 4: Cumulative successful events from analysis of an average of 4 videos
per catalyst. Different colours correspond to intensity trajectories showing
single event (grey), double events (blue) and triple events (red).
R1
R1
R2
N
N
N
Keq
R2-N3
+
+
kf
R1
Scheme 4: Schematic representation of the catalytic click reaction resembles
the Michaelis-Menten mechanism, except for the need of the participation of a
second reactant (the azide) in the final step leading to products.
The clearest indication of the improved catalyst is that in
Figure 4 about 50% of the bursts (i.e., catalytic events) occur
in trajectories showing multiple events, while in the untreated
sample this fraction is reduced to about 20%. Such analysis is
independent of possible deposition differences for different
catalysts. Thus, not only the number of catalytic events
changes, but their distribution of single and multiple events
also changes. Looking at the fraction of single and double
events observed with treated catalyst, a Poisson distribution
analysis (see SI) predicts that triple events will be rare, just as
observed. Further, this statistical analysis suggests that for
NaBH4 treated samples about 28% of viable catalytic sites
show no activity during any 50 s video recording.
It is worth noting that signal amplitude in this and previous
work are similar, the background level is higher in this work,
presumably due to significant increases in light scattering due
to particles of ~100 nm size. Some of these low signal-to-noise
bursts were likely present but not observed in earlier work
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DOI: 10.1039/CJ6oCuCr0n8a9l05NDame
with large Cu@Charcoal particles. Reductive treatment also
made their presence evident.
Exploratory studies included in the SI suggest that treated
catalysts show excellent reusability.
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The Natural Sciences and Engineering Research Council of
Canada supported this work through its Discovery program,
while the Canada Foundation for Innovation enabled the
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Electronic Supplementary Information (ESI) available: Experimental
details, SEM images, Poisson statistics, additional fluorescence
trajectories and reusability data. See DOI: 10.1039/c000000x/
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