Investigation of copper-free alkyne/azide 1,3-dipolar cycloadditions using microwave irradiation

Article abstract of DOI:10.1016/j.bmcl.2017.12.007

The prevalence of 1,3-dipolar cycloadditions of azides and alkynes within both biology and chemistry highlights the utility of these reactions. However, the use of a copper catalyst can be prohibitive to some applications. Consequently, we have optimized

Full text of DOI:10.1016/j.bmcl.2017.12.007

Accepted Manuscript
Investigation of copper-free alkyne/azide 1,3-dipolar cycloadditions using mi-
crowave irradiation
Lindsay E. Chatkewitz, John F. Halonski, Marshall S. Padilla, Douglas D.
Young
PII:
S0960-894X(17)31167-8
https://doi.org/10.1016/j.bmcl.2017.12.007
BMCL 25463
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
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29 November 2017
4 December 2017
Please cite this article as: Chatkewitz, L.E., Halonski, J.F., Padilla, M.S., Young, D.D., Investigation of copper-free
alkyne/azide 1,3-dipolar cycloadditions using microwave irradiation, Bioorganic & Medicinal Chemistry Letters
(2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.007
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Investigation of copper-free alkyne/azide 1,3-
dipolar cycloadditions using microwave
irradiation
Lindsay E. Chatkewitz, John F. Halonski, Marshall S. Padilla and Douglas D. Young *
Cu
N
N
N3
+
N
MW

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Investigation of copper-free alkyne/azide 1,3-dipolar cycloadditions using
microwave irradiation
Lindsay E. Chatkewitz, John F. Halonski, Marshall S. Padilla and Douglas D. Young *
Department of Chemistry, College of William & Mary, P.O. Box 8795, Williamsburg, VA 23187 (USA)
ARTICLE INFO
ABSTRACT
Article history:
Received
The prevalence of 1,3-dipolar cycloadditions of azides and alkynes within both biology and
chemistry highlights the utility of these reactions. However, the use of a copper catalyst can be
prohibitive to some applications. Consequently, we have optimized a copper-free microwave
assisted reaction to alleviate the necessity for the copper catalyst. A small array of triazoles was
prepared to examine the scope of this approach, and the methodology was translated to a protein
context through the use of unnatural amino acids to demonstrate one of the first microwave-
mediated bioconjugations involving a full length protein.
Revised
Accepted
Available online
Keywords:
bioconjugation
azide
2009 Elsevier Ltd. All rights reserved
.
alkyne
unnatural amino acid
microwave irradiation
The alkyne/azide 1,3-dipolar cycloaddition reaction has
become an indispensible tool for scientists as a mechanism to
conjugate an alkyne moiety with an azide (Scheme 1).1-4 While
the general class of pericyclic reactions have proven useful to
organic chemists,5,
6
the robust nature of this particular
alkyne/azide “click” reaction in particular has allowed it to
become ubiquitous in a plethora of fields such as drug design,
sensors, catalysis, materials chemistry, and bioconjugations.7-14
While this specific cycloaddition was first discovered in 1963,
the copper catalyzed variant has expanded its utility via
decreasing reaction times and temperatures, while increasing
regioselectivity.3 However, the addition of a copper catalyst has
some limitation within biological systems and in some materials
applications.15, 16 These issues have necessitated the development
of rapid copper-free conditions, which is typically accomplished
via the use of highly strained alkynes, many of which are
challenging to synthetically access.17-19 Alternatively,
heterogeneous reactions employing an immobilized catalyst, and
non-transition metal catalyzed reactions have been explored.20, 21
While these methodologies are extremely useful, we became
interested in exploring other options for rapid copper-free
reaction conditions using unstrained alkynes. Another
mechanism to accelerate the reaction has been the application of
microwave irradiation, which has been primarily examined under
Scheme 1. Standard copper-free 1,3-dipolar cycloaddition
Due to the extensive number of variables associated with
microwave irradiation, initial investigations involved the
optimization of reaction conditions with a model system.27 Based
on commercial availability of reagents and spectroscopic
properties, the reaction of benzyl azide (2) and phenylacetylene
(1) was selected to explore the copper-free microwave mediated
1,3-dipolar cycloaddition of alkynes and azides. The rapid nature
of the microwave was expected to facilitate productive reactions
in a short amount of time, without necessitating the use of a
copper catalyst, and ultimately making the reaction more useful
for biological settings or other applications where the use of
copper precludes the utilization of the reaction. Using a CEM
Discover, various temperatures, microwave powers and
microwave settings were explored (Table 1).
Standard
microwave conditions involve the input of power until a specific
temperature is obtained, followed by brief bursts of power to
maintain the temperature. Power mode involves the constant
input of a microwave power until a set temperature is reached,
followed by termination of reaction conditions. Finally, Pulsed
Power (SPS) mode involves a power cycling to maintain the
temperature within a specific range (δT). A delicate interplay
between power input and reactant decomposition was noted, as
higher product yields were observed with increased power
copper-catalyzed conditions.22-25 Previously,
a
microwave
mediated copper-free cycloaddition toward the preparation of a
complex polymer was reported; however, little experimental
optimization of the reaction was conducted and yields were
significantly low.26 We aim to significantly expand on this
approach, optimizing reaction conditions and expanding its utility
towards biological applications.

settings (300W); however, increased irradiation times began to
lead to decreased yields. Thus, the SPS setting was found to be
optimal as it afforded high power inputs, while reduced times at
elevated temperatures. Ultimately, maximizing power to 300W in
SPS mode for 20 min afforded a 96% yield and was employed in
further reactions. As expected, a mixture of the two regioisomers
(3a and 3b) was obtained under all conditions.
the free triazole ring after column chromatography. Control
reactions that mimicked the microwave temperature profile
without microwave irradiation did afford product, albeit in
dramatically lower yield < 20%. Thus, the dramatically increased
yields using the optimized microwave conditions demonstrate the
utility of this methodology. While these yields are sometimes
comparable to previously reported reactions, the combination of
decreased reaction times and absence of copper suggest that the
methodology may be useful in specific applications.28
Table 1. Optimization of the copper-free microwave assisted 1,3-dipolar
cycloaddition.
N3
TMS
N3
C6H13
N3
2
4
5
Ph
C6H13
H
N
Power Time Temperature
MW Setting
Yield
(%)
N
N
N
(oC)
100
N
N
(W)
100
100
100
100
200
300
300
200
300
(min)
10
10
20
10
2
1
2
20
20
Ph
N
Ph
N
N
Ph
Ph
Standard
Standard
Standard
Power
Power
Power
Power
SPS
21.9
26.0
31.4
23.0
26.0
49.2
21.9
48.1
96.2
125
125
168
168
168
168
1
3
96.2% 1: 1 a/b
10
68.0% 2:1 a/b
1 1
64.8%
Ph
C6H13
H
N
N
N
N
N
N
N
C4H9
N
168 (δT=15)
168 (δT=15)
N
C4H9
C4H9
C4H9
SPS
6
12
96.8% 1: 6 a/b
13
87.3% 1:7 a/b
14
94.8%
In order to assess the scope of the reaction, we next examined
a variety of alkynes and azides under the optimized reaction
conditions. In addition to benzyl azide (2), azidoheptane (4) and
trimethylsilyl azide (5) were examined due to commercial
availability and chemical functionality. This set of azides was
reacted with phenylacetylene (1), 1-hexyne (6), and propargyl
alcohol (7) under the previously optimized microwave
conditions. When reacting equimolar ratios of the propargyl
alcohol and any azide, very little product was recovered <5%,
obviating the further optimization of the reaction. We
hypothesized that the propargyl alcohol was susceptible to
decomposition under the microwave conditions as reactions
typically resulted in a very dark crude mixture, which was
unobserved with other alkynes. Two remedies were examined to
address the low yields, first trityl-protected propargyl alcohol (9)
and methyl propargylether (8) were employed to protect the
hydroxyl group, and second, excess of the alkyne was employed
to identify if decomposition was an issue. Ultimately, it appears
that alkyne decomposition under microwave conditions was the
primary factor, as increasing from 1 equivalent to 3 equivalents
dramatically increased the yield. While not ideal, eventual
translation to bioconjugations typically employ extreme excess of
the non-protein partner to drive reactions, so 3 equivalents is
relatively minimal. Consequently, the array of triazole products
(3, 10-23) was re-synthesized using an excess of alkyne (Figure
1). Conveniently, the volatility of the alkyne reactants allowed
for easy purification under vacuum or via column
chromatography. Overall, the reaction proceeded in moderate to
excellent yield (96-47%), with the benzyl azide being the most
reactive azide (96-84%), and the azidoheptane exhibiting lower
activity (94-46%). This may be a result of some solubility issues,
or simply the aliphatic nature of the azide. Trityl protected
propargyl alcohol reactions involved the lowest yields,
potentially due to decomposition. These yields could be
significantly increased via alteration of the protecting group to a
methyl substituent. Additionally, reactions performed with the
TMS-azide in the microwave resulted in desilylation and yielded
Ph
C6H13
H
N
N
N
N
N
N
N
RO
N
N
CH2OR
CH2OR
CH2OR
7: R=H
15: R=H
87.0% 1: 1.5 a/b
18: R=Me
84.0% 1: 1.5 a/b
21: R=Trt
32.7% 1: 3 a/b
16: R=H
92.5% 1.6: 1 a/b
19: R=Me
75.2% 1: 1.8 a/b
22: R=Trt
10.7% 1:3 a/b
17:R=H
<5%
20:R=Me
46.9%
23:R=Trt
12.1%
8: R=Me
9: R=Trt
Figure 1. Triazole array prepared to assess the scope of the microwave
assisted copper-free 1,3-dipolar cycloaddtion. Reactions were performed with
3 eq. alkyne, using SPS mode 300W, 20 min.
In order to further apply the microwave assisted copper-free
1,3-dipolar cycloadditions, we next investigated its use in a
biological context. This is especially relevant due to the
propensity of copper to generate radicals that degrade proteins,
and due to the general cytotoxicity of copper.15 To accomplish
this aim, an alkynyl unnatural amino acid was incorporated using
the Schultz amber suppresion technology into green fluorescent
protein (GFP).29-34 GFP was selected due to its nascent
fluorescent properties and well-documented use as a reporter
protein. The p-propargyloxyphenylalanine (24) was expressed at
residue 151 of GFP, which is located within the rigid β-barrel of
the protein (Figure 2).13, 35, 36 With the alkyne-containing protein
in hand, we next investigated the ability to translate our
previously optimized reaction to a biological context. The mutant
GFP was reacted with Alexafluor-488 azide to generate a
fluorescent bioconjugate that could be analyzed via SDS-PAGE.
Not surprisingly, when subjected to microwave irradiation in the
standard CEM Discover under a variety of conditions (including
the previously optimized SPS conditions), protein degradation
was observed at high temperatures, providing no observable
conjugated product (data not shown).

the absence of copper to prevent cytotoxicity. Attempts to
translate these Coolmate conditions to the previously optimized
small molecule reactions resulted in some coupling but only
around 20% for many of the reaction partners. We hypothesize
this is due to the significantly higher reagent concentrations
compared to the protein system, coupled with substantially
shorter reaction times. Thus, optimal conditions for the
microwave-mediated click reaction are dependent on the nature
of the reactants (proteins vs. small molecules).
H3N
COOH
A
B
O
24
TCEP
A
B
H2O/tBuOH
H2O
MW
MW
1
2
1
2
CuSO4
MW Power (W)
+
0
-
200
+
0
-
200
Figure 2. Incorporation and cycloaddition using an unnatural amino acid.
A) Structure of p-propargyloxyphenylalanine incorporated into GFP. B)
Proposed 1,3-dipolar cycloadditon reaction between the mutant GFP and an
azide-containing fluorophore in the microwave.
In order to prevent protein degradation, the reaction was
translated to a CEM Coolmate system, which utilizes a jacketed
reaction vessel to allow microwave transparent cooling fluid to
be flowed through to significantly reduce reaction temperatures
while still affording microwave irradiation.37 The SPS setting
previously employed is not feasible under Coolmate conditions,
and thus modulation of microwave power was the most logical
variable to examine. Reactions containing the mutant protein and
azide fluorophore were conducted at microwave powers of 100
W, 200 W, and 300 W. The coolant was pre-chilled to -50 oC and
used to cool the reaction to -30 oC prior to microwave irradiation,
and the reaction was then irradiated until the temperature reached
40 oC.
C
D
Fluor-Azide
MW Power (W)
+
0
-
200
+
0
-
200
Figure 3. Microwave mediated 1,3-dipolar cycloadditions on GFP. A)
SDS-PAGE Coomassie stain indicating the presence of GFP both with a
control reaction not utilizing the microwave (Lane 1) and under microwave
conditions in the absence of copper (Lane 2) . B) SDS-PAGE fluorescence
image, demonstrating the effective coupling between the protein and the azide
fluorophore to generate a triazole linkage. The difference in protein
concentration is potentially due to some ninor degradation under microwave
conditions. C) Copper-free control reactions in the absence of either
microwave irradiation or the fluorescently labelled reaction partner. While
GFP is present and non-degraded in both reactions when stained with
Coomassie blue. D) Fluorescence imaging indicates that no bioconjugation
occurs under the two control conditions as no fluorescence is observed
Following irradiation, the protein was denatured and analysed
by SDS-PAGE. Due to the covalent modification of the protein
with a fluorophore, successful reactions were expected to yield a
fluorescent product even after denaturation of the protein
fluorophore. Reaction success was determined by first examining
the fluorescence of the gel, and then staining the gel with
coomassie blue to ascertain the presence of protein. Reactions at
300 W displayed significant protein degradation, while reactions
at 100 W did not exhibit significant fluorophore coupling.
However, reactions at 200 W displayed significant coupling
without protein degradation. While other types of
bioconjugations have been performed in the microwave, we
believe this to be one of the first reported microwave-mediated
bioconjugations involving full length proteins.38 In order to
further optimize the coupling, the reaction was subjected to 2
pulses of microwave irradiation at 200 W prior to purification
(Figure 3). Based on both absorption spectroscopy of the
conjugates and densiometry measurements of the gels the
coupling yields are ~85%. Additionally, control irradiations in
the absence of the fluorophore were performed, demonstrating
that GFP was still fluorescent after irradiation, signifying that it
was not denatured as a result of microwave irradiation at low
In conclusion, we have demonstrated that it is feasible to
conduct alkyne/azide 1,3-dipolar cycloadditions in the
microwave without the requisite of a copper catalyst. This has
far-reaching applications within the realms of both biology and
materials science where copper may be prohibitive to specific
reactions. The microwave-mediated reaction was optimized to
afford high yields of triazole products and further applied to a
protein context via the utilization of unnatural amino acids.
Overall, we believe this to be one of the first reported protein
bioconjugations utilizing microwave irradiation. Moreover, the
methodology developed facilitates an extremely rapid method to
obtain bioconjugates and in the absence of potentially cytotoxic
copper.
Acknowledgments
temperature (see supplementary information).
This is an
important consideration when considering the utility of these
reactions within the context of the microwave. Reactions under
identical temperature profiles to the microwave yielded no
observable conjugate. It is important to note that GFP is a
relatively hearty protein and the presence of copper does not
necessarily lead to degradation; however, many other less stable
proteins may require the copper-free conditions, or the eventual
application of the protein in a biological setting may necessitate
The authors would like to thank the College of William &
Mary and the National Institute of General Medical Sciences of
the NIH (R15GM113203) and the Camille and Henry Dreyfus
Teacher-Scholar Award (TH-17-020) for financial support. We
would also like to thank Dr. Peter Schultz for providing the
pEVOL plasmid utilized in the research.

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