Kinetics studies of rapid strain-promoted [3+2] cycloadditions of nitrones with bicyclo[6.1.0]nonyne

Article abstract of DOI:10.1139/cjc-2013-0577

Strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions represent a bioorthogonal labeling strategy that is both very rapid and at the same time efficient and selective. Nitrones provide increased reaction rates as well as greater susceptibility to

Full text of DOI:10.1139/cjc-2013-0577

337
ARTICLE
Kinetics studies of rapid strain-promoted [3+2] cycloadditions
of nitrones with bicyclo[6.1.0]nonyne
Douglas A. MacKenzie and John Paul Pezacki
Abstract: Strain-promoted alkyne−nitrone cycloaddition (SPANC) reactions represent a bioorthogonal labeling strategy that is
both very rapid and at the same time efficient and selective. Nitrones provide increased reaction rates as well as greater
susceptibility toward stereoelectronic modification when compared with organic azides. We find that strain-promoted cy-
cloadditions of cyclic nitrones with bicyclo[6.1.0]nonyne react with second-order rate constants as large as 1.49 L mol−1 s−1 at 25 °C.
These reactions display rate constants that are up to 37-fold greater than those of the analogous reactions of benzyl azide with
bicyclo[6.1.0]nonyne. We observed that reactions of nitrones with bicyclo[6.1.0]nonyne showed a stronger dependence on
substituent effect for the reaction, as evidenced by a larger Hammett ␳ value, than that for biaryl-aza-cyclooctanone. We
demonstrate the ability to stereoelectronically tune the reactivity of nitrones towards different cyclooctynes in SPANC reactions.
This ability to introduce selectivity into different SPANC reactions through substituent provides the opportunity to perform
multiple SPANC reactions in one reaction vessel and opens up potential applications in multiplex labeling.
Key words: metal-free bioothrogonal cycloadditions, 1,3-dipolar cycloadditions, nitrones, click chemistry, bicyclo[6.1.0]nonyne,
strain-promoted alkyne−nitrone cycloaddition (SPANC).
Résumé : La réaction de cycloaddition par catalyse forcée de nitrones a` un alcyne (« strain-promoted alkyne−nitrone cy-
cloaddition » ou SPANC) constitue une méthode de marquage a` la fois efficace, sélective et très rapide. Les nitrones augmentent
les taux de réaction amélioré et la sensibilité aux modifications stéréoélectroniques par comparaison avec les azotures orga-
niques. Nous constatons que les réactions de cycloaddition par catalyse forcée de nitrones cycliques au bicyclo[6.1.0]nonyne ont
lieu avec des taux de second ordre élevé de 1,49 L mol−1 s−1 a` 25 °C. Ces réactions montrent des valeurs de taux jusqu’a` 37 fois plus
élevées que celles des réactions similaires de l’azoture de benzyle avec le bicyclo[6.1.0]nonyne. Nous avons observé que les
réactions de nitrones avec le bicyclo[6.1.0]nonyne dépendaient davantage de substituant sur la réaction, comme l’a montré
l’augmentation de la valeur de Hammett ␳, que celles avec la biaryl-aza-cyclooctanone. Nous avons démontré qu’il était possible
d’ajuster stéréoélectroniquement la réactivité des nitrones avec différents cyclooctynes dans des réactions de SPANC. Cette
aptitude a` introduire une sélectivité dans différentes réactions de SPANC par l’intermédiaires de substituants rend possible la
réalisation de multiples réactions de SPANC dans un réacteur unique et pourrait éventuellement être appliquée au marquage
multiple. [Traduit par la Rédaction]
Mots-clés : cycloadditions bioorthogonales sans métal, cycloadditions 1,3-dipolaires, nitrones, chimie « click », bicyclo[6.1.0]nonyne,
cycloaddition par catalyse forcée de nitrones a` un alcyne (SPANC).
fabrication of micron-scale surface chemical gradients,7 in func-
Introduction
tionalizing poly(cyclic olefin) surfaces,8 and in organometallic
The study of complex biological systems at the molecular level
chemistry for the post-synthetic modification of metal organic
requires the use of a powerful and highly selective nonperturbing
probe or reporter reactions to produce coherent results with min-
imum alteration of the native chemical environment. To this end,
researchers have developed a number of rapid conjugation meth-
frameworks,9 for example. However, intracellular chemistry and
biomolecular labeling and capture often demand the use of less
perturbing labeling strategies, such as copper-free cycloaddition
reactions.10–12
ods that are inert to naturally occurring functional groups and
Staudinger ligation provides a means to covalently link an azide-
physiological conditions, thereby satisfying the requirements of
containing molecule to a triarylphosphine through an amide bond
bioorthogonality.1 Copper(I) catalyzed azide alkyne cycloaddition
under physiological conditions. Despite a comparatively slow rate
(CuAAC) has been used for site-specific labeling of cell surface or
constant (0.0077 L mol−1 s−1),13 this strategy has been used to de-
purified proteins, primarily because of the synthetically accessi-
ble and cost-effective reagents and rapid kinetics.2 Lowering the
LUMO energy of the alkyne has lead to enhanced reactivity in
CuAAC, while careful selection of ligands alters the cellular con-
sequences resulting from the copper catalyst, which have limited
the applications of CuAAC in living systems.3–5 CuAAC has found
widespread use in materials research where it has been employed
as a grafting approach in polymer and dendrimer science,6 in the
liver photo-crosslinking diazirene groups14 and azobenzene pho-
toswitches15 to azide functionalized biomolecules. To circumvent
the toxicity issues associated with copper catalysis in CuAAC re-
actions, copper-free strategies have been developed. Increased re-
action kinetics have been obtained by the use of strain-promoted
azide−alkyne cycloadditions (SPAAC).16,17 The Bertozzi group has
designed a number of cyclooctynes displaying rapid kinetics with
benzyl azide, most of which make use of electron-withdrawing
Received 17 December 2013. Accepted 24 February 2014.
D.A. MacKenzie and J.P. Pezacki. Life Sciences Division, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada;
Department of Chemistry, University of Ottawa, 10 Marie-Curie, Ottawa, ON K1N 6N5, Canada.
Corresponding author: John Paul Pezacki (e-mail: John.Pezacki@nrc-cnrc.gc.ca).
Can. J. Chem. 92: 337–340 (2014) dx.doi.org/10.1139/cjc-2013-0577
Published at www.nrcresearchpress.com/cjc on 5 March 2014.

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Can. J. Chem. Vol. 92, 2014
Fig. 1. (A) Overview of strain-promoted cycloadditions of
cyclooctynes with azides and nitrones. (B) Overview of strain-
promoted cycloadditions of electron-rich BCN (1) with
stereoelectronically tuned endocyclic nitrones.
Table 1. Kinetics of SPANC reactions of acyclic nitrones and BCN.
Nitrone
k2 (L mol−1 s−1
)
2a
2b
2c
2d
2e
2f
R1 = p-OMeC6H4
R1 = p-MeC6H4
R1 = Ph
R1 = p-BrC6H4
R1 = p-CO2MeC6H4
R1 = p-CNC6H4
R1 = p-NO2C6H4
0.0011
0.0023
0.0029
0.0065
0.011
0.021
0.029
2g
Note: Stock solutions of nitrones 2a–2g were mixed in equimolar ratios with
a stock solution of 1 at 25 0.1 °C in CD3OD. Rate constants k2 were determined
by 1H NMR under second-order conditions.
Fig. 2. Hammett plot depicting the substituent effects on SPANC
reactions of acyclic nitrones 2a−2g and BCN.
fluorine substituents or benzanulation to increase reaction
rates.18 Dommerholt et al. designed the relatively electron-rich
bicyclo[6.1.0]nonyne (BCN) (1), making use of a fused cyclopropane
ring to increase strain, leading to increased SPAAC rates.19 Chin
et al. have incorporated BCN as a dieneophile in unnatural amino
acids for site-specific protein labeling in rapid and fluorogenic
[4+2] cycloadditions with tetrazines.20 Fluorogenic cycloadditions
of tetrazoles and alkenes have also been demonstrated in live
mammalian cells by Lin et al. using two-photon excitation to drive
a “photoclick” conjugation.21 Given the stereoelectronic differ-
ences in the current strained cycloalkynes that are applicable to
bioorthogonal applications such as SPAAC reactions, there is an
opportunity for developing new reactions by tuning the reactivity
in copper-free ligation using stereoelectronically different dipoles
other than alkyl azides.
Results and discussion
SPANC have been presented as an alternative labeling strategy
and have displayed rapid kinetics with strained cyclooctynes,
showing second-order rate constants up to 58.8 L mol−1 s−1.22,23
SPANC reactions have proven useful in protein modification24,25
and cell surface labeling of human breast cancer cells.26 The use of
endocyclic nitrones rather than the linear azide allows for the
introduction of ring strain as well as three potential sites for
stereoelectronic modification and (or) incorporation of a handle
or linker to the dipolar component of the cycloaddition. With
these possibilities for modification and the variation of the calcu-
lated LUMO energy levels in known strained cycloalkynes that are
bioorthogonal, we envisioned that appropriate alkyne−nitrone
pairs could be designed to provide mutually exclusive reactivity
allowing for simultaneous duplex or multiplex SPANC chemis-
try.27 Herein, we have studied SPANC reactions with the electron-
rich bicyclo[6.1.0]nonyne and have shown that these reactions are
more sensitive to substituent effects than those involving more
electron-poor cyclooctynes. We demonstrate that SPANC reac-
tions can be tuned so that they can be reacted simultaneously
with minimal crossover products. These represent a proof of con-
cept for the stereoelectronic tuning of SPANC reactions using
endocyclic nitrones for the simultaneous duplex and possibly
multiplex SPANC labeling reactions.
To evaluate the sensitivity of SPANC toward different cyclooctynes,
we have studied the reactions of different acyclic and endocyclic
nitrones with BCN (1) (Fig. 1). To study the reactivity, we conducted
a Hammett study by changing the substituents on the nitrone
component systematically using a homologous series of ␣-aryl ni-
trones containing functional groups of varying electron-withdrawing
or -donating ability. The second-order rate constants of these acyclic
nitrones were measured with BCN (1)by1HNMRin deuterated meth-
anol. The rate constants for these reaction reactions are presented
in Table 1. For methods and kinetics data, see the supporting
information (“Supplementary material” section). Nitrones 2a and
2c–2g were prepared by micelle-catalyzed condensation of benzyl-
N-hydroxylamine with the appropriate aldehyde,22 while 2b
was prepared by condensation without catalysis.28
SPANC reactivity has largely been explored with electron-deficient
alkynes containing high levels of conjugation and delocalized elec-
tron density, as is the case with biaryl-aza-cyclooctanone (BARAC)
and dibenzocyclooctynol (DIBO). BCN (1) was chosen for this study
based on the localization of the alkyne ␲-electrons (calculated
alkyne LUMO energy of 38.2 kcal mol−1) and the lack of electron-
withdrawing functional groups.27 Figure 2 shows the Hammett
plot comparing the observed bimolecular rate constants with
Published by NRC Research Press

MacKenzie and Pezacki
339
Table 2. Kinetics of SPANC reactions of cyclic nitrones with BCN.
Nitrone
Product
k2 (L mol−1 s−1
)
krel
4a
5a
0.65
16.2
4b
4c
5b
5c
0.05
1.49
1.24
37.0
Note: Nitrones 4a–4c and 1 were mixed in 100:1 molar ratio in methanol at 25 0.1 °C; the concen-
tration of the excess alkyne was varied by ≥10% for an additional four trials. k2 was determined by UV-Vis
spectroscopy under pseudo-first-order conditions. krel is the second-order rate constant relative to the
reaction of BCN with benzyl azide.
Fig. 3. Stereoelectronically tuned alkyne−nitrone pairs in a one-pot competition experiment displaying potential for mutually exclusive
reactivity with minimal cross-reactivity.
␴
p values. Interestingly, we observed a Hammet ␳ value of 1.28
involving BCN (1) are more sensitive to electronic changes than
BARAC and the transition state is more strongly stabilized by
electron-withdrawing groups appended to the nitrone compo-
nent of the cycloaddition.23
0.01, which is significantly larger than the previously measured
␳ value of 0.25 0.04 observed with an analogous series of ␣-aryl
nitrones reacted with BARAC. This suggests that SPANC reactions
Published by NRC Research Press

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Can. J. Chem. Vol. 92, 2014
Next, we sought to determine whether SPANC reactions involv-
Supplementary material
ing BCN (1) and endocyclic nitrones would display a similar reac-
tivity and sensitivity to substitution pattern. To this end, nitrones
4a–4c were synthesized as previously described (see supporting
information) and the bimolecular rate constants were measured
in reactions with 1 under pseudo-first-order conditions by UV-
visible absorption spectroscopy as described in the Materials and
methods (see supporting information).29,30 4a was prepared by
metal-free oxidation of the secondary amine using oxone in a
biphasic medium,29 4b was commercially available, and nitrone
4c was prepared by oxidation of the secondary amine using m-CPBA.30
Table 2 summarizes the results of these kinetic studies, with
nitrone 4b showing a 30-fold rate enhancement over 4c and a
37-fold rate enhancement over benzyl azide in reactions with 1.
Strained BCN (1) displays a bimolecular rate constant with electron-
deficient 4b that is suitable for biological labeling applications
(1.49 L mol−1 s−1) while showing sluggish kinetics with electron-rich
4c (0.05 L mol−1 s−1), which may provide further opportunity for
selective one-pot reactions involving different nitrones and strained
cycloalkynes in simultaneous SPANC reactions.
Supplementary data for this paper are available on the journal
web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjc-2013-
0577.
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Conclusion
Strain-promoted cycloadditions between alkynes and nitrones
provide a rapid and bioorthogonal labeling strategy. Reactions
of nitrones with BCN (1) can achieve rate constants significantly
higher than those involving the corresponding alkyl azide. Exploi-
tation of the variety in stereoelectronic properties of bioorthogo-
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pairs that can undergo SPANC chemistry in one pot with minimal
cross-reactivity. Efforts to obtain higher selectivity and reduced
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