Optimized aqueous Kinugasa reactions for bioorthogonal chemistry applications

Article abstract of DOI:10.1039/c9cc09473c

Kinugasa reactions hold potential for bioorthogonal chemistry in that the reagents can be biocompatible. Unlike other bioorthogonal reaction products, β-lactams are potentially reactive, which can be useful for synthesizing new biomaterials. A limiting factor for applications consists of slow reaction rates. Herein, we report an optimized aqueous copper(i)-catalyzed alkyne-nitrone cycloaddition involving rearrangement (CuANCR) with rate accelerations made possible by the use of surfactant micelles. We have investigated the factors that accelerate the aqueous CuANCR reaction and demonstrate enhanced modification of a model membrane-associated peptide. We discovered that lipids/surfactants and alkyne structure have a significant impact on the reaction rate, with biological lipids and electron-poor alkynes showing greater reactivity. These new findings have implications for the use of CuANCR for modifying integral membrane proteins as well as live cell labelling and other bioorthogonal applications.

Full text of DOI:10.1039/c9cc09473c

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Optimized Aqueous Kinugasa Reactions for Bioorthogonal
Chemistry Applications
Didier A. Bilodeau, Kaitlyn D. Margison, Noreen Ahmed, Miroslava Strmiskova, Allison R. Sherratt,
John Paul Pezacki*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
reaction,28 although notoriously sensitive and challenging,
Kinugasa reactions hold potential for bioorthogonal chemistry in
that the reagents can be biocompatible. Unlike other bioorthogonal
reaction products, β-lactams are potentially reactive, which can be
useful for synthesizing new biomaterials. A limiting factor for
applications consists of slow reaction rates. Herein we report an
represents a unique prototype reaction for development and
optimization for bioorthogonal chemistry applications. Since its
initial discovery, the Kinugasa reaction has received considerable
recognition as a reaction that produces -lactams in a variety of
conditions.28,29 -lactams are of significant interest because of their
use as antibiotics,30,31 and their general properties as
electrophiles.32 The Kinugasa reaction has been developed from
original reports using pyridine as a base and solvent,28,33 to using
polar aprotic solvents as well as polar protic/aprotic solvent
mixtures,29 and reactions in aqueous conditions,34,35 with the
optimized
aqueous
copper(I)-catalyzed
alkyne-nitrone
cycloaddition involving rearrangement (CuANCR) with rate
accelerations made possible by the use of surfactant micelles. We
have investigated the factors that accelerate the aqueous CuANCR
reaction and demonstrate enhanced modification of a model
membrane-associated
peptide.
We
discovered
that
potential to be applied to biological systems for bioorthogonal
labelling.2,3,21,36 Yet, examples of the Kinugasa reaction in aqueous
lipids/surfactants and alkyne structure have a significant impact on
the reaction rate, with biological lipids and electron-poor alkynes
conditions are few, with our group previously reporting one of the
showing greater reactivity. These new findings have implications first examples.34 Thus, it can be considered a prototype reaction
for the use of CuANCR for modifying integral membrane proteins as that still needs development and optimization in order to operate as
well as live cell labelling and other bioorthogonal applications.
a useful bioorthogonal reaction.
Bioorthogonal chemistry provides invaluable tools to chemical
biology research for studying specific biological processes inside
cells that are otherwise challenging to probe.1–6 A number of
bioorthogonal reactions have been developed and are now used as
techniques to efficiently, selectively and covalently link two reactive
groups.7–13 One such reaction is copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) (Fig. 1a),14,15 which has been and continues
to be utilized extensively in a myriad of biological labelling
applications and is used to perform labelling of cellular targets.16–20
(
a) Bioorthogonal CuAAC and CuANCR
R'
O
N
Cu(I)
R3
Cu(I)
NR2
N
N
R2
R'
R1
R3
R1
N
O
N3
R3
(b) This work: Micelle-Assisted Aqueous Kinugasa reaction
R2
R1
N
O
R3
Cu(I)
NR2
O
Concerns around copper toxicity are addressable with careful
choice of copper(I) ligand,21 through traceless reagents bearing
R3
R1
catalytic copper,22 or development of alternative copper-catalyzed
reactions.23,24 However, alternatives are still desired to expand the Fig 1. a) Comparing Kinugasa/CuANCR with CuAAC bioorthogonal chemistry. b)
variety of reacting partners and bring additional reactions for use in Lipid acceleration through micelles.
bioorthogonal applications, allowing for the discovery of more
efficiently catalyzed reactions.25–27 The classical Kinugasa
In addition to applications of biological labelling,
bioorthogonal chemistry allows for the production and
functionalization of nanoparticles and biomaterials.37,38 CuAAC
chemistry has been used extensively for modification of material
surfaces,39 attachment of proteins to materials40 and nanomaterial
synthesis.41 The Kinugasa reaction could be applied to similar
applications, while producing potentially bioactive β-lactam
products directly on biomaterial surfaces. The development of
Department of Chemistry and Biomolecular Sciences, University of Ottawa
150 Louis-Pasteur, Ottawa, Ontario, Canada, K1N 6N5
Correspondence to: john.pezacki@uottawa.ca
Supporting information for this article is given via a link at the end of the
document.
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biomaterials with antibacterial properties is vital due to bacterial surfactants/micelles. Additionally, the β-lactam yieldsVidewoAnrtoictlereOpnloinret
colonization being a major cause of implant failure.42 β-lactam on other potential covalently linked side pr oD dOu I c: 1t s0 . w1 0 h3 i 9c /hC 9w Co Cu 0l d9 4a 7l 3s Co
antibiotic doping has been proposed to produce such materials ; lead to successful bioconjugation.36 Thus, percent conversion is
43
thus the Kinugasa reaction could allow for direct functionalization thought to be more representative of reaction progression.
of such bioorthogonal implant materials.
Given difficulties in product isolation and quantification due to use
Nitrones are excellent tunable bioorthogonal reactants, which
of surfactants, the determination of reaction kinetics proves
have already been demonstrated in strain-promoted alkyne-nitrone
challenging. Therefore, to further evaluate the reaction efficiency
cycloaddition (SPANC) reactions.44,45 We recently discovered a
and ultimately relative rates, we performed studies utilizing our
version of the Kinugasa reaction, copper(I)-catalyzed alkyne-
recently reported alkyne-tagged magnetic bead assay.46 The latter
nitrone cycloaddition involving rearrangement (CuANCR), that can
represents a simple and convenient approach to assessing
be used as a bioorthogonal reaction for live-cell labelling.46
reaction progression under biological lipid/micellar catalysis, which
However, reaction rates were slow. Herein, we optimize reaction
otherwise interferes with most conventional spectroscopic
conditions for the aqueous CuANCR and use surfactant micelles47
approaches. Briefly, we employed a biotin-tagged cyclic nitrone
to accelerate aqueous reaction rates and yields.
(
biotin-CMPO) that was reacted with alkyne-functionalized
magnetic beads in the presence of CuSO and sodium ascorbate,
4
Table 1. Screen of alkynes in micelle-assisted Kinugasa reactions
while varying ligand type and presence of different biomimetic
lipids. After magnetic separation, covalently linked biotin
conjugated to alkyne-tagged magnetic beads via CuANCR, were
detected by FITC-streptavidin staining and fluorescence
microscopy. An initial comparison between L-histidine and L-
proline as bioorthogonal copper ligands led us to choose L-proline
to perform further on-bead assays of a variety of lipid micelles, due
to higher observed labelling efficiency (Fig. 2, S1) and due to L-
proline having been previously shown to be a competent copper
ligand.49,50

Cu, L-proline
Ph
Ph
Ph
Ph
N+
O-
+
R
N
H2O, surfactant, Py
sodium ascorbate
R
O
Conversiona
Isolated Yield %)
Entry
1
Alkyne
Ph
(
38 (22b)
76 (16b)
2
CF3
OEt
OEt
3
4
5
85 (20b)
90 (32b)
95 (69)
CN
O
OMe
Ph
N
H
O
6
7
81 (64)
91 (65)
N
H
O
OEt
a
Reactions were conducted in 20 mL Argon degassed H
SDS. Sodium ascorbate (0.2 mmol), pyridine (0.1mmol), L-proline (0.05 mmol)
and CuSO (0.025 mmol) were added successively. Following the addition of
alkyne (0.05 mmol), C,N-diphenylnitrone (0.05 mmol) was added and stirred for
2
O containing 10 mM
Fig. 2 Effect of ligand on CuANCR labelling of alkyne-tagged magnetic
beads. Alkyne beads were incubated for 30 minutes at 37 C in PBS, PBS with
biotin-CMPO or PBS containing 100 µM CuSO , 200 µM ligand, 2 mM sodium
4
4
ascorbate and 50 µM biotin-CMPO. Beads were washed in PBS then stained with
FITC-streptavidin prior to imaging. Scale bars = 2 µm.
3
0 minutes at 25 C. NMR yields are reported following extraction with 3x20 mL
b
EtOAc, using 1,4-Dimethoxybenzene as internal standard. Conducted with 3.5
mM CTAB in the absence of L-proline.
Next, we sought to evaluate the effects of biomimetic lipids on the
efficiency of CuANCR reactions. We have previously reported that
CuANCR is an effective approach for cell labelling.46 We
hypothesized that cell surface components, such as cellular lipids,
glycans, glycoproteins and the membrane itself increase the
efficiency of the CuANCR labelling reaction, as previously seen by
others.51 To test this hypothesis, we performed a screen of
biomimetic lipids with a hydrophilic sugar group or with quaternary
ammonium and phosphate groups (Table S3). We chose an initial
lipid concentration of 1.5 mM, corresponding to the highest critical
micellar concentration (CMC) amongst all screened lipids (Table
Initially, we sought to optimize the alkyne portion of the reaction.
Alkynes have been activated towards [3+2] cycloaddition by
conjugation to electron-withdrawing groups, such as esters,
through lowering of the energy of their LUMO.48 Therefore, other
alkynes bearing electron withdrawing groups were tested (Table 1,
Entries 2-7). The use of propiolamides (Table 1, Entries 5-6) and
propiolic esters (Table S1, Entry 7) led to increases in observed
yield, as did the inclusion of micellar catalysts.30 The scope of
acyclic nitrones was also evaluated and the reaction was found to
be amendable to a wide range of aryl substitution (See Table S2).
Furthermore, it should be noted that the reported yields of β-lactam
products after extraction might not fully represent the total reaction
yield, given potential product loss in the extraction process with
S3), to ensure that CuANCR is conducted in
a micellar
environment. The background fluorescence, determined for beads
incubated without biotin-CMPO and with Fos-choline 16 (arbitrarily
chosen amongst screened surfactant), was subtracted from all
2
| Chem. Commun., 2019, 00, 1-3
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samples. All examined lipids contributed to a more efficient roughly as efficient as in 10-fold or 100-fold excess oVfieβw-TArDticMle O(Fnliinge.
CuANCR labelling of alkyne beads (Fig. 3), and maltopyranoside- S3, S4, S5). However, in absence of β-T DD OM I: 1a 0n . 1d 0 3i n9 / sC u9 bC -Cm0 i9c 4e 7l 3l aC r
functionalized lipids increased the efficiency about 2-fold. These concentrations of β-TDM, the labelling efficiency drops to or below
results confirmed our hypothesis that biomimetic lipids can 50% in comparison, showing the importance of the presence of β-
significantly accelerate CuANCR reactions.
TDM micelles for optimal CuANCR labelling. This suggests that a
micellar environment, and not individual lipid species are
responsible for catalysis.
****
1
000
***
ns
ns
****
**
Next, we investigated the kinetics of CuANCR labelling in micellar
β-TDM, seeking insight in the possible mechanism of micelle
catalysis of CuANCR (Fig. S6). After 5 minutes of CuANCR
labelling, there is a significant difference between micelle-assisted
CuANCR, and surfactant-free reaction. Under pseudo-first order
kinetics with 50 µM biotin-CMPO nitrone in 10-fold excess over
alkyne, we were able to estimate a kobs-micelle/kuncat ratio of 100,
suggesting that presence of micelles contributes to a faster rate of
CuANCR, possibly by solubilizing hydrophobic reactants and
effectively increasing their local concentration, or orienting the
reactants for optimal reaction trajectory. We envision membrane
and biological lipids to play a similar role for bioorthogonal
applications; allowing for greater labelling efficiencies and
enhanced reaction rates on cells.
8
6
4
2
00
00
00
00
0
**
*
Fig. 3 Lipid screen for micelle-assisted Kinugasa/CuANCR reaction. Alkyne-
tagged magnetic bead assay was used. Beads were labelled via CuANCR
reaction in 100 µM CuSO4, 2 mM sodium ascorbate, 200 µM L-proline and 50 µM
biotin-CMPO, in presence of 1.5 mM of indicated lipid. Reaction was carried out
To demonstrate the applicability of micelle-assisted CuANCR
in PBS for 30 minutes at 37 °C, after which beads were washed in PBS and chemistry, we performed biotin labelling of a small membrane-
incubated with 5 µg/mL streptavidin-FITC, then washed. Above background
fluorescence for at least five beads from two separate field views (10 beads total)
was used to determine the average fluorescence of beads for each condition. *
associated peptide from the class of cell-penetrating peptides, E5-
TAT.52,53 Membrane proteins represent an important class of
protein acting as membrane receptors, integral structural
p<0.1, ** p< 0.01, *** p< 0.001, **** p< 0.0001.
components and transporters, amongst other functions. Their
isolation and functionalization can be challenging requiring
hydrophobic environments to maintain proper structure and
function. Micellar conditions represent a suitable system in which
these membrane proteins can be solubilized and modified.54 E5-
TAT is a small membrane-associated peptide that contains a fusion
between HIV-TAT protein transduction domain (PTD) and a variant
of the influenza HA2 domain that inserts into lipid membranes and
destabilizes them at low pH.52,53 It was chosen as a model peptide
to which an alkyne handle could be appended through lysine
residues on the cationic TAT sequence (KKRRQRRR). To
accomplish labelling, the peptide samples were treated with an N-
hydroxysuccinimide ester alkyne for
1 hour under micellar
conditions (Fig. 4). The alkyne modified peptides were purified by
FPLC and modification of expected lysine residues (K26, K27) was
confirmed by mass spectrometry analysis (see Fig. S8 and Tables
S4-S5). The modified peptides were then submitted to CuANCR
conditions for 1 hour to conjugate a biotin moiety, using Biotin-
CMPO as the nitrone reacting partner. After the two-step labelling
protocol, the peptide samples were analyzed by western blot with
Fig. 4 Biotin conjugation of E5-TAT membrane peptide using CuANCR. (A)
Schematic representation depicting the two step biotin conjugation of E5-TAT
peptide under micellar assistance. Alkyne functionalization of E5-TAT (20 μM)
was carried out using N-hydroxysuccinimide ester alkyne (300 μM). Biotin
4 2
labelling was achieved by treating modified peptide with CuSO ∙H O (20 μM), results showing a significant difference in the relative biotin
sodium ascorbate (300 μM), L-proline (40 μM) and biotin-CMPO (300 μM). (B)
Top panel represents the western blot analysis of biotin labelled E5-TAT samples
under varying percentages (%W/V) of detergent (FC12) ranging from (0.01-0.2%,
CMC=0.05%). Bottom panel represents the TGX-Stain free blot as a loading
conjugation between the micelle-assisted CuANCR conditions
≥CMC) and the surfactant-free conditions (>250 fold) (See Fig. S7
for non-modified peptide control). These results emphasize the
(
control. The presented relative biotin conjugation values consist of densitometry importance of micellar conditions not only for peptide solubility, but
analysis normalized to protein loading.
as well for achieving robust peptide modification.
To further optimize CuANCR reactions for future bioorthogonal Conclusions
applications, we selected an optimal lipid to examine the effects on
In conclusion we have found that lipids, biological ligands and
the rates of reactions. We chose β-TDM, which has the lowest CMC
amongst the three maltopyranoside lipids, for further CuANCR
condition optimization. We then focused on optimal conditions of
this biological lipid for CuANCR, focusing on sub-micellar and
micellar conditions. A concentration screen showed that at CMC of
β-TDM in water (10 µM), the CuANCR labelling of alkyne beads is
electron poor alkynes help accelerate and increase the yield of the
CuANCR reaction developed from the prototype Kinugasa reaction.
The use of micelle catalysis consists of a cost-effective platform on
which bioorthogonal reaction development can take place. We
have also shown that membrane protein modification is possible
through CuANCR chemistry, highlighting the bioorthogonal nature
This journal is © The Royal Society of Chemistry 20xx
Chem. Commun., 2019, 00, 1-3| 3
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Acknowledgements
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We thank Mariya Chigrinova for technical support and help with 36
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Keywords: Kinugasa Reaction • Micelle catalysis • Bioorthogonal
•
Copper-catalyzed alkyne-nitrone cyclization involving
rearrangement • β-lactam • Membrane-associated peptide
modification
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ChemComm
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DOI: 10.1039/C9CC09473C
We present optimized micelle-assisted aqueous copper(I)-catalyzed alkyne-nitrone cycloaddition
involving rearrangement (CuANCR) reactions applicable to bioorthogonal applications, namely
membrane-associated peptide modification.