Strain-promoted cycloadditions of cyclic nitrones with cyclooctynes for labeling human cancer cells

Article abstract of DOI:10.1039/c1cc13808a

Strain-promoted cycloadditions of cyclic nitrones with cyclooctynes proceed with rate constants up to 3.38 ± 0.31 M^-1 s^-1 in CD₃CN, or 59 times faster than the analogous reaction of azides. This highly specific modular labeling strategy can be applied for direct labeling of proteins and for live cell imaging of cancer cells. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1cc13808a

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COMMUNICATION
Strain-promoted cycloadditions of cyclic nitrones with cyclooctynes for
labeling human cancer cellsw
Craig S. McKay,ab Jessie A. Blake,ab Jenny Cheng,a Dana C. Danielsonac and
John Paul Pezacki*abc
Received 26th June 2011, Accepted 21st July 2011
DOI: 10.1039/c1cc13808a
Strain-promoted cycloadditions of cyclic nitrones with cyclooctynes
proceed with rate constants up to 3.38 ꢀ 0.31 Mꢁ1 sꢁ1 in CD3CN,
or 59 times faster than the analogous reaction of azides. This highly
specific modular labeling strategy can be applied for direct labeling
of proteins and for live cell imaging of cancer cells.
with photochemically generated nitrile imines25 as well as
olefin metathesis.26 Strain promoted reactions of cyclooctynes
with other 1,3-dipoles, including diazo compounds and nitrile
oxides, have also been reported.27,28
In pursuit of fast and efficient metal-free bioorthogonal
cycloadditions as a means for reducing the concentrations of
labeling reagents, we have shown that cyclic nitrones display
exceptionally fast kinetics in strain-promoted cycloadditions
with cyclooctynes, proceeding up to 25 times faster than
analogous reactions involving azides.29 We attributed this rate
increase to the additional strain associated with the cyclic
nitrone moiety. A one-pot N-terminal modification of peptides
via strain-promoted alkyne–nitrone cycloaddition (SPANC)
was independently reported thereafter.30 Here we demonstrate
strain-promoted cycloadditions of cyclic nitrones with cyclo-
octynes, as well as direct protein modification and live-cell
labeling of cell surface proteins. In addition to rapid reactivity, the
synthetic accessibility and ease of functionalization of nitrones
make them particularly attractive 1,3-dipoles for strain-promoted
[3 + 2] cycloadditions with cyclooctynes. Nitrones can also
undergo Kinugasa reactions with terminal alkynes.31,32
Incorporation of fluorescent probes into proteins, DNA,
RNA, lipids and glycans within their native cellular environ-
ments provides opportunities for imaging and understanding
their roles in vivo. Labeling of specific functional groups on
live cells via bioorthogonal chemical reporter strategies1 have
become increasingly powerful applications in cell biology.2–4
Cycloaddition reactions are particularly attractive for this
purpose because they are usually very selective, high yielding
and proceed rapidly in aqueous media. Azide-based reactions5
have been applied broadly to bioconjugation in vivo since the
azide 1,3-dipole is stable and easily introduced into biomolecules
or probe molecules without changing functional properties.
The copper-catalyzed azide–alkyne cycloaddition (CuAAC)6,7
has been applied mainly to cell lysates8 but recently has also
been applied to labeling the exterior of cell surfaces in vivo.9,10
To avoid using potentially toxic copper catalysts and reducing
agents,11,12 Staudinger ligation with phosphine esters13 and
strain-promoted azide–alkyne cycloaddition (SPAAC)14 have
been implemented for labeling of biomolecules on the exterior
of live cells15 and within living animals8,16 with minimal
cytotoxicity. A number of cyclooctyne reagents have been
synthesized for SPAAC applications including difluorinated
cyclooctynes (DIFO) and derivatives,15 4-dibenzocyclooctynol
(DIBO),17 photochemically generated dibenzocyclooctynes,18
aza-dibenzo-cyclooctyne (DIBAC),19 biarylcyclooctynone
(BARAC),20 and bicyclononynes.21 Other examples of
bioorthogonal cycloadditions include reactions of strained
alkenes with nitrile oxides,22 tetrazines,23,24 unstrained alkenes
To probe the feasibility of SPANC reactions of cyclic nitrones
as a tool for direct biological labeling, we examined the reaction
scope and kinetics for reactions of a series of cyclic nitrones with
dibenzocyclooctyne (2a).29 In order to optimize the tradeoff
between cyclic nitrone reactivity and stability for bioconjugation,
we tested a series of cyclic nitrones differing in ring size, sterics
and stereoelectronics to determine their respective contributions
to nitrone reactivity in SPANC reactions.
Aldonitrones 1a–e reacted significantly faster than the
ketonitrones 1g–h in SPANC reactions with 2a. All nitrones
employed yielded isoxazoline cycloadducts in excellent yields.
The pyrroline N-oxides, 1a–e, were found to be optimal,
blending fast kinetics with enhanced stability under ambient
conditions. The presence of the gem-disubstitution adjacent to
nitrogen in nitrones 1a and 1b resulted in second-order rate
constants of 0.63 ꢀ 0.12 Mꢁ1 sꢁ1 and 7.69 ꢀ 0.19 Mꢁ1 sꢁ1
respectively. Prolinol nitrone 1c reacted with 2a in good yield
with a rate constant of 1.95 ꢀ 0.60 Mꢁ1 sꢁ1 (Table 1). The
presence of the N-Boc group adjacent to the a-position in 1d
resulted in a slower reaction with 2a relative to the analogous
reaction of 1c. Attempts to further activate the cyclic nitrone
using gem-difluoro groups at the a-position in nitrone 1e
a Steacie Institute for Molecular Sciences, National Research Council
Canada, Ottawa, Canada. E-mail: John.Pezacki@nrc-cnrc.gc.ca;
Fax: +1 613-952-0068; Tel: +1 613-993-7253
b Department of Chemistry, University of Ottawa, Ottawa, Canada
c Department of Biochemistry, Microbiology and Immunology,
University of Ottawa, Ottawa, Canada
w Electronic supplementary information (ESI) available: Experimental
procedures, spectroscopic characterization for all new compounds and
kinetic data. See DOI: 10.1039/c1cc13808a
c
10040 Chem. Commun., 2011, 47, 10040–10042
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Table
1
Kinetics for strain-promoted cycloadditions of cyclic
Table 2 Kinetics of strain-promoted cycloadditions of cyclic nitrones
1a–c, 1f with 2ba
nitrones 1a–f with 2aa
Nitrone
Product
Yieldb (%)
k2c/Mꢁ1 sꢁ1
1a
1b
1c
1f
3i
3j
3k
3l
97
96
92
95
0.75 ꢀ 0.04
3.38 ꢀ 0.31
1.04 ꢀ 0.17
0.46 ꢀ 0.01
a
b
Reagents were mixed 1 : 1 at 25 mM in CD3CN at 25 1C. Yield %
was determined by 1H NMR. Rate constants, k2, was determined by
c
1H NMR under second order reaction conditions.
Nitrone
Product
Yieldb (%)
t/min
k2c/Mꢁ1 sꢁ1
of benzyl azide with DIFO,15 a 59-fold enhancement relative
to reaction with DIBO18 or a 3-fold enhancement relative to
reaction with BARAC.20 We also tested the prolinol nitrone,
1c in reaction with 2b and the rate constant was found to be
1a
1b
1c
1d
1e
1f
3a
3b
3c
3d
3e
3f
91
96
88
90
87
84
89
97
15
o2
5
18
180
3
0.63 ꢀ 0.12
7.69 ꢀ 0.19
1.95 ꢀ 0.60
0.62 ꢀ 0.08
0.0215 ꢀ 0.00038
1.41 ꢀ 0.19
n.d.
1.04 ꢀ 0.17 Mꢁ1
s
ꢁ1, this corresponds to an 18-fold enhance-
1g
1h
3g
3h
1380
1440
ment over analogous reaction of benzyl azide with 2b. The rate
constant for reaction of 1f with 2b determined by UV-visible
spectroscopy under pseudo first-order conditions was found to
be 0.56 ꢀ 0.01 Mꢁ1 sꢁ1 (see ESIw). This value agrees with that
n.d.
a
b
Reagents were mixed 1 : 1 at 25 mM in C6D6 at 25 1C. Isolated
yield. Rate constant, k2, was determined by H NMR under second
order conditions.
c
1
1
determined by H NMR. Attempts to measure rate constants
for the remaining nitrones in Table 2 by UV-visible spectro-
scopy were complicated by overlapping absorption spectra.
The hydrolytic stability of cyclic nitrone 1a was compared
with an acyclic nitrone, 2-(methyloxidoimino)-N-(phenylmethyl)-
acetamide29 under acidic and basic conditions to determine
whether the cyclic nitrones could offer enhanced stability toward
hydrolysis (see ESIw). In 100 mM HCl solution, the acyclic
nitrone underwent rapid hydrolysis to the corresponding
aldehyde and N-methylhydroxylamine within 15 min, and in
100 mM NaOH solution, the acyclic nitrone was significantly
hydrolyzed over 2 h. Alternatively, cyclic nitrone 1a remained
intact under both acidic and basic conditions.
resulted in slower rates for cycloaddition. This indicates that
nitrones with electron withdrawing amide groups at the
a-position react faster than those with inductively withdrawing
fluoro-groups. Employing a six membered nitrone, 1f containing
the electron withdrawing a-amide group reacted with 2a in
96% yield within 3 min at 25 1C; this corresponded to a rate
constant of 1.41 ꢀ 0.19 Mꢁ1
s
ꢁ1. The rate constant for
reaction of 1f with 2a in toluene under pseudo first-order
conditions was found to be 1.21 ꢀ 0.06 Mꢁ1 sꢁ1 by UV-visible
spectroscopy, this value agrees with that determined by 1H
NMR. Despite its fast reactivity, nitrone 1f was less stable and
prone to dimerization at room temperature. Attempts to
synthesize additional six membered nitrones that would be
electronically and sterically similar to the five membered
nitrones gave similar dimerization products as 1f (Table 1).
Having established the five membered cyclic nitrones as
optimal in terms of reactivity and stability in SPANC reactions
with unfunctionalized 2a in benzene, we measured the rate
constants for the most reactive cyclic nitrones with 2b in
acetonitrile (Table 2). All cyclic nitrones tested gave excellent
yields of isoxazoline products with comparable rates upon
moving from benzene to acetonitrile. The rate constant for
reaction of 1a was 0.75 ꢀ 0.04 Mꢁ1 sꢁ1. Although the value for
reactions of 1a with 2b in acetonitrile is within experimental
error with that obtained for its reaction with 2a in benzene, it
is still 13 times faster than analogous reactions of benzyl azide
with 2b.18 Cyclic nitrone 1b reacted with 2b in 91% yield in 3 min
with a rate constant of 3.38 ꢀ 0.31 Mꢁ1 sꢁ1. This rate constant
corresponds to a 44-fold enhancement relative to the reaction
To demonstrate direct protein functionalization by SPANC,
we labeled bovine serum albumin (BSA) protein in vitro
(see ESIw). An NHS-activated prolinol nitrone, NHS-1c, was
prepared by desilylation of 1c with TBAF and subsequent
NHS-activation with N,N0-disuccinimidyl carbonate, and was
coupled with lysine residues on BSA. BSA was modified with
an average of four nitrone groups as confirmed by MALDI-MS.
The nitrone modified BSA was treated with 2b functionalized
Alexa Fluors 488 cadaverine for 0 to 60 min. Highly specific
time-dependent labeling was detected by in-gel fluorescence
scanning (Fig. S4, ESIw).
To illustrate the utility of SPANC for cellular imaging, we
labeled epidermal growth factor receptors (EGFRs) that are
overexpressed on the surface of human breast cancer cells
(MDA-MB-468) (Fig. 1). In this approach, the human recombi-
nant epidermal growth factor (EGF) was coupled with NHS-1c
and targeted to EGFRs on the cell surface. Incorporation of two
nitrone groups was confirmed by MALDI-MS (see ESIw).
c
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Chem. Commun., 2011, 47, 10040–10042 10041

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Notes and references
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Fig. 1 In situ labeling of EGF–EGFR interactions via SPANC in
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bright field (bottom) image of in situ SPANC as described in (a). (c)
Negative control, cells pretreated with EGF followed by treatment
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In summary, we have shown that five membered cyclic
nitrones blend fast kinetics with enhanced stability in SPANC
reactions with dibenzocyclooctynes. Reactions of cyclic nitrones
proceeded with rate constants up to 3.38 ꢀ 0.31 Mꢁ1 sꢁ1 in
CD3CN, or 59 times faster than the analogous reaction of
benzyl azide with DIBO. We have demonstrated highly specific
direct labeling of BSA and EGF in vitro and have shown
efficient labeling of EGFRs on the surface of cancer cells via
SPANC. The SPANC bioconjugation approach presented
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c
10042 Chem. Commun., 2011, 47, 10040–10042
This journal is The Royal Society of Chemistry 2011