Kinetics studies of rapid strain-promoted [3 + 2]-cycloadditions of nitrones with biaryl-aza-cyclooctynone

Article abstract of DOI:10.1039/c2ob07165g

Strain-promoted cycloadditions of cyclic nitrones with biaryl-aza- cyclooctynone (BARAC) proceed with rate constants up to 47.3 M^-1 s^-1, this corresponds to a 47-fold rate enhancement relative to reaction of BARAC with benzyl azide and a 14-fold enhancement over previously reported strain promoted alkyne-nitrone cycloadditions (SPANC). Studies of the SPANC reaction using the linear free energy relationship defined by the Hammett equation demonstrated that the cycloaddition reaction has a rho value of 0.25 ± 0.04, indicating that reaction is not sensitive to substituents and thus should have broad applicability. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob07165g

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PAPER
Kinetics studies of rapid strain-promoted [3 + 2]-cycloadditions of nitrones
with biaryl-aza-cyclooctynone†
Craig S. McKay,‡a,b Mariya Chigrinova,‡a,b Jessie A. Blakea and John Paul Pezacki*a,b
Received 22nd December 2011, Accepted 7th February 2012
DOI: 10.1039/c2ob07165g
Strain-promoted cycloadditions of cyclic nitrones with biaryl-aza-cyclooctynone (BARAC) proceed
with rate constants up to 47.3 M−1 s−1, this corresponds to a 47-fold rate enhancement relative to reaction
of BARAC with benzyl azide and a 14-fold enhancement over previously reported strain promoted
alkyne–nitrone cycloadditions (SPANC). Studies of the SPANC reaction using the linear free energy
relationship defined by the Hammett equation demonstrated that the cycloaddition reaction has a rho
value of 0.25 0.04, indicating that reaction is not sensitive to substituents and thus should have broad
applicability.
The CuAAC reaction has broad applicability and rapid
Introduction
kinetics.9,21–24 The CuAAC reaction is also very popular since
Reactions that do not interfere with biological functionality,
termed bioorthogonal, have emerged as powerful tools for site-
specific modification of biomolecules in cells and within living
organisms.1–4 Despite the multitude of developed organic reac-
tions, only a handful of these are ideally suited for applications
in biology.5 Not only must the bioorthogonal reactive groups be
mutually reactive, bio-inert and ideally non-toxic, the reaction
must proceed with fast reaction rates to ensure formation of the
product at low concentrations typically required in biological
labelling experiments. A number of cycloaddition reactions have
been developed that show fast kinetics, and high selectivity for
functionalizing biomolecules. Cycloadditions that have been suc-
cessfully applied to bioconjugation include the copper(I)-cata-
lyzed azide–alkyne cycloaddition (CuAAC),6–9 strain-promoted
azide alkyne cycloaddition (SPAAC),4 strain-promoted nitrone–
alkyne cycloaddition (SPANC),10–12 and strain-promoted
alkyne–nitrile oxide cycloaddition (SPANOC).13,14 Diazo com-
pounds have also been shown to react with cyclooctynes rapidly
via strain promotion.15 Also, cycloadditions of strained alkenes
with tetrazines,16 photochemical reactions of unstrained alkenes
with tetrazoles,17 olefin cross-metathesis,18 and palladium-cata-
lyzed cross-coupling reactions19,20 have been reported specifi-
cally for bioorthogonal coupling or labelling reactions.
the azide and terminal alkyne moieties are small and non-pertur-
bative of biological function and these reactions are very rapid
and high yielding. Observed rate constants for CuAAC have
been reported as high as 1 ×105 M−1 s−1 per mole of copper cat-
alyst,22 however, not all applications of CuAAC are tolerant to
high concentrations of copper catalyst.25 Also, CuAAC reactions
can display complex dependencies on copper concentrations
depending on what ligand is used and what solvent system is
chosen.22 Despite the fast kinetics of CuAAC, many of the
copper catalysts used for CuAAC reactions are toxic in living
systems and also have effects on cellular metabolism.8,25–27 In
order to improve upon the biocompatibility of azide–alkyne
cycloadditions, Bertozzi and co-workers developed the copper-
free variant, SPAAC, involving rate acceleration due to strain
energy release in the alkyne moiety. A number of different
cyclooctynes have been developed including difluorinated
cyclooctyne (DIFO)28 and derivatives,29 Dibenzocyclooctynol
(DIBO),30 tetramethoxy-dibenzocyclooctynol (TMDIBO),31 aza-
dibenzocyclooctyne
(DIBAC),32
biaryl-aza-cyclooctynone
(BARAC)33,34 and bicyclononyne (BCN).35
Copper-free bioorthogonal reactions are bimolecular and bio-
conjugate formation can be represented by the following
equation: [bioconjugate] = k2 × [biomolecule] × [reagent] × t,
where k2 is the second-order rate constant, and the bioconjugate
yield is proportional to the reagent concentration as well as k2.5
From a practical point of view, reactions with larger k2 values
and thus faster observed kinetics of bioorthogonal coupling reac-
tions lead to shorter reaction times in biological systems. Also,
larger k2 values enable the reactions to reach completion at lower
reagent concentrations as compared to slower reactions over the
same reaction times. Thus, larger k2 values can allow bioortho-
gonal labelling reactions to take place efficiently in a wider
aSteacie Institute for Molecular Sciences, National Research Council
Canada, 100 Sussex Drive, Ottawa, Canada. E-mail: John.Pezacki@
nrc-cnrc.gc.ca; Fax: +1 613-952-0068; Tel: +1 613-993-7253
bDepartment of Chemistry, University of Ottawa, 10 Marie-Curie,
Ottawa, Canada
†Electronic supplementary information (ESI) available: Experimental
procedures and kinetic data. See DOI:10.1039/c2ob07165g
‡These authors contributed equally to the work presented in this paper.
3066 | Org. Biomol. Chem., 2012, 10, 3066–3070
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Table 1 Kinetics of SPANC reactions of cyclic nitrones with BARACa
Nitrone
Product
% Conv.b
>99
k2c (M−1 s−1
)
1a
3a
22.4
Scheme 1 Strain-promoted alkyne–nitrone cycloaddition (SPANC).
variety of living systems including live animals where the
amounts of reagents required may be limiting.
1b
1c
3b
3c
>99
>99
41.0
39.2
Nitrones have been shown to be useful dipoles in strain-pro-
moted nitrone–alkyne cycloadditions (SPANC).10–12 We recently
reported the utility of cyclic nitrones in strain-promoted cyclo-
additions with dibenzocyclooctyne and 4-dibenzocyclooctynol
(DIBO) and demonstrated that cyclic nitrones can serve as
alternatives to azides with larger k2 values generally observed
1d
3d
>99
47.3
with absolute values up to 3.38 M−1 s−1 11
We also showed that
.
cyclic nitrones were generally more stable than their acyclic
counterparts under aqueous conditions and could be employed
to directly functionalize protein targets in vitro as well as on the
surface of live human cancer cells.11 The fast kinetics of SPANC
reactions involving cyclic nitrones enables biological labelling
at lower reagent concentrations. Recently, a more reactive
aza-cyclooctyne, biaryl-aza-cyclooctynone (BARAC) was
reported.33 BARAC contains the highest number of sp2-hybri-
dized carbon atoms within the cyclooctyne ring, giving rise to
greater ring strain than other reported cyclooctynes. Rate con-
stants for SPAAC reactions involving BARAC were found to be
significantly larger than for other cyclooctynes. However,
BARAC has not yet been used for cycloaddition reactions with
nitrones. Herein, we study reaction kinetics of SPANC reactions
of BARAC33 with cyclic and acyclic nitrones to illustrate the
kinetic benefits of further ring strain to the SPANC reaction
(Scheme 1).
a Nitrone (1a–1d) and 2a were mixed in a molar ratio 100 : 1 in 99%
MeCN–H2O at 25 0.1 °C, the concentration of the excess nitrone was
varied by ≥10% for an additional four trials. b Nitrone (1a–1d) and 2a
were mixed 1 : 1 at 25 mM final concentration, % conversion was
determined by HPLC based on the amount of BARAC remaining after
20 min reaction. c k2 was determined by UV-visible spectroscopy under
pseudo-first order conditions.
conversion to products in cycloadditions with azides. Our expec-
tation was that we would observe similar high conversions with
cyclic nitrones that were shown previously to be stable and to
work well in SPANC reactions. We performed reactions of com-
pounds 1a–d (∼1 mM) with 2a (∼1 mM) in 99% MeCN–H2O
at r.t. We observed high yields of cycloaddition products and
greater than 99% conversion after 20 min of reaction (Table 1),
consistent with the previously reported rapid kinetics of SPAAC
reactions involving BARAC. Next we sought to determine the
bimolecular rate constants for SPANC reactions of a variety of
nitrones with BARAC.
Results and discussion
Cyclic nitrones, 1a and 1b, were prepared by metal-free oxi-
dation of the secondary amine using oxone in a biphasic
medium,36 5,5-Dimethyl-1-pyrroline N-oxide (DMPO), 1c was
commercially available, and nitrone 1d was prepared by an intra-
molecular alkyl-N-hydroxylamine/aldehyde condensation.37
Acyclic nitrones, 1e–1l, were efficiently prepared by micelle-pro-
moted intermolecular condensation of alkyl-N-hydroxylamine
and aldehyde.38 Biaryl-aza-cyclooctynone (BARAC, 2a) was
synthesized according to literature protocol.33
Kinetics
In order to establish a kinetic profile for SPANC reactions of
nitrones with BARAC, the associated bimolecular rate constant,
k2 was measured by UV-visible absorption spectroscopy under
pseudo first-order reaction conditions (see ESI†). Firstly, we
determined bimolecular rate constants of SPANC reactions of
cyclic nitrones with BARAC (Table 1). Reactions of cyclic
nitrones, 1a–d, with 2a yielded cycloadducts 3a–d with excel-
In order to assess the utility of BARAC in SPANC, we first
examined the efficiency of conversion to products. Previously,
BARAC had been shown to react rapidly and with high
lent
percent
conversions
within
20
min.
While
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Table 2 Kinetics of SPANC reactions of acyclic nitrones with
BARACa
Nitrone
k2b (M−1 s−1
)
1e
1f
1g
1h
1i
1j
1k
1l
R1 = Ph
R1 = Bn
R1 = Me
R1 = Me
R1 = Me
R1 = Me
R1 = Me
R1 = Me
R2 = Ph
R2 = Ph
R2 = Ph
R2 = p-OMeC6H4
R2 = p-MeC6H4
R2 = p-ClC6H4
R2 = p-CNC6H4
R2 = p-NO2C6H4
58.8
2.8
6.8
4.9
6.6
7.9
9.2
10.1
Fig. 1 Hammett plot depicting the substituent effects on the SPANC
reactions of acyclic nitrones 1g–l with BARAC.
Structure–reactivity relationships
Having established 1g as optimal in terms of reactivity and stab-
ility we varied the substituents of the α-aryl groups in nitrones
1h–l, to determine if electron withdrawing groups at this position
would lead to rate enhancements. Modest rate accelerations were
observed for acyclic nitrones containing electron withdrawing
α-aryl substituents (Table 2).
a Nitrone 1e–f and 2a were mixed in a molar ratio 1 : 100 in 99%
MeCN–H2O at 25 0.1 °C. Nitrone (1g–l) and 2a were mixed in a
molar ratio 100 : 1 in MeCN at 25 0.1 °C. b k2 was determined by UV-
visible spectroscopy under pseudo-first order conditions.
To probe the mechanism of SPANC, we investigated substitu-
ent effects of acyclic nitrones on the relative rates of SPANC
reaction with BARAC using a Hammett plot (Fig. 1). The best
fit was obtained using σP parameters owing to the direct reson-
ance interactions between the para substituents and the nitrone
functional group as illustrated for electron donating groups in
eqn (1) and for electron withdrawing groups in eqn (2). Plotting
log(kX/kH) against σP parameters, gives a reaction constant, ρ,
value of 0.25 0.04 (R = 0.94). The small value of ρ suggests
either an early transition state or a concerted transition state with
no charge build-up at the α-position of the nitrone during the
transition state.
dihydroisoquinoline N-oxide, 1a reacted with 2a with a rate con-
stant of 22.4 M−1 s−1, the five-membered pyrroline-N-oxides
1b–d proceeded with rate constants of 41.0, 39.2 and 47.3 M−1
s−1, respectively. Measuring rate constants allows one to infer
effective rates at any concentrations of reactants. The reaction
concentrations employed for measuring rate constants here are
similar to the concentrations used in biological labelling exper-
iments.29,33 The five-membered nitrones demonstrated two-fold
rate enhancement over the six-membered nitrones in reactions
with 2a. Cyclic nitrones reacted with BARAC up to 48 times
faster than analogous reactions of benzyl azide, and 14 times
faster than previously reported SPANC.
ð1Þ
In order to examine the substrate scope and generality of
SPANC reactions of nitrones with BARAC, we measured linear
free energy relationships by changing substituents on the
nitrones. We tested a series of acyclic nitrones bearing electron
withdrawing and electron donating groups at the para position of
the α-aryl substituent. We also varied the substitution at the N-
position. Reaction of electron withdrawing N-phenyl substituted
nitrone 1e, with 2a proceeded with an exceptionally fast rate
(k2 = 58.8 M−1 s−1), whereas more electron donating methylene
or methyl groups at the N-positions of nitrones 1f and 1g were
well tolerated in reactions with 2a, and demonstrated rate con-
stants of 2.8 and 6.8 M−1 s−1, respectively (Table 2). The pres-
ence of the N-phenyl electron withdrawing group in the
cycloadduct 3e, resulted in a thermally labile N–O bond, and it
is well known that similar structures are prone to thermal
rearrangement leading to azomethine ylides.39,40 We find that
smaller electron donating N-substituted nitrones proceed with
sufficiently fast kinetics and provide stable products.
ð2Þ
Previously we investigated the effects of substituents on the
micelle-catalyzed cycloadditions of nitrones with cyclopente-
none, according to eqn (3).41 A ρ value of −0.94 was determined
from the corresponding Hammett plot for these nitrone–alkyne
cycloadditions. In this study the cycloadditions were slow, and
relative rate constants were determined from the ratios of pro-
ducts for reactions in competition. The value of ρ for nitrone-
cyclopentenone is consistent with a transition state that has
modest positive charge buildup at the nitrone carbon atom in the
transition state and is also consistent with a concerted rather than
a stepwise mechanism for the micelle catalyzed cycloaddition.
Since cyclopentenone can act as a Michael acceptor, it is poss-
ible that a transition state that is slightly non-synchronous with
3068 | Org. Biomol. Chem., 2012, 10, 3066–3070
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Fig. 2 Proposed transition states for cycloaddition reactions of substi-
tuted nitrones with BARAC and nitrones with cyclopentenone. The
charge build-up in the transition states is depicted and is consistent with
the differences in magnitude of the slopes of the Hammett plots for
these two reactions.
more bond formation between the nitrone oxygen and the
β-carbon of the α,β-unsaturated ketone and less bonding is
formed between the nitrone α-carbon and the alkene exists. This
would give rise to a modest positive charge build up in the tran-
sition state that would be stabilized by electron donating
substituents.
Fig. 3 Plots of nitrone α-H proton chemical shift values vs. σP con-
stants with linear least squares fit showing positive correlation (R = 0.94)
and nitrone α-H proton chemical shift values vs. log(kX/kH) values
for the cycloaddition reactions between nitrones 1g–l and BARAC
(R = 0.95).
for HO· depending on solvent and from 1.2 to 310 M−1 s−1
− 49,50
˙
depending on solvent for O2
.
Since the magnitudes of the
ð3Þ
rate constants for addition of superoxide radical anions and for
SPANC reactions involving reactive nitrones and BARAC are
comparable, we expect that SPANC labelling should not be ham-
pered by the presence of superoxide. Given that these free rad-
icals are present at very low concentrations and also react rapidly
with other cellular components, the rapid rates of cycloadditions
between nitrones and cyclooctynes such as BARAC are expected
to be sufficient to dominate so long as the concentration of
BARAC is significantly higher than the concentrations of the
reactive oxygen species, especially for hydroxyl radicals and
alkoxyl radicals.
Unlike the reactions of nitrones with cyclopentenone, the reac-
tions of nitrones with BARAC are slightly accelerated by elec-
tron withdrawing substituents and show even less sensitivity to
substituent effects. This suggests that the reactions of nitrones
with BARAC have an even more synchronous transition state
and follow a concerted mechanism. The ρ value for cycloaddi-
tions between nitrones and BARAC has the opposite sign to that
for reactions of nitrones with cyclopentenone. This suggests that
there is more bonding between the nitrone α-carbon and the
cyclooctyne and less net positive charge on that α-carbon at the
transition state for the reaction relative to the starting materials.
Given that this is the case, the transition states for the two reac-
tions can be represented by the structures in Fig. 2.
Structure–reactivity relationships for addition reactions onto
nitrones have been studied extensively.42–47 Generally, addition
reactions that do not give rise to significant charge build up on
the nitrone α-carbon atom have shown Hammett ρ values
ranging from −1.0 to 0.76, consistent with the ρ value deter-
mined here. Interestingly, Durand et al. recently showed that
Hammett σP values correlate with computed nitrone charge
density as well as nitrone α-H NMR chemical shift values,
suggesting that these parameters may also be predictive of reac-
tivity of other nitrones.48 For the nitrones tested here, we also
observe a correlation between α-H chemical shift values and the
corresponding σP constants, Fig. 3.
Overall, we observe a positive ρ value that is close to zero for
cycloadditions of 1e–l with 2a and this indicates that the SPANC
reactions with BARAC are not sensitive to substituent at the
nitrone α-aryl carbon so that no significant rate enhancement can
be obtained through substitution at this centre. The lack of sensi-
tivity of SPANC to substituents suggests that the reaction is toler-
ant to a broad range of functionalized nitrones, which is desired
for bioorthogonal labelling reactions. The nitrone α-carbon is
therefore an ideal position for incorporation of bio-recognition
elements, labels, or affinity tags. On the other hand, significant
rate enhancements can be obtained by changing the substituents
at the nitrone nitrogen atom (R1 in Table 2). However, substitu-
ents at the nitrone nitrogen atom that accelerate the cycloaddition
unfortunately also destabilize the reaction product by weakening
the N–O bond. The optimal combination of rate enhancement
and reactant and product stabilities is found with cyclic nitrones
such as 1a–d (Table 1).
Nitrones are also often used as spin traps for free radicals and
tend to react with hydroxyl and alkoxyl radicals as well as super-
oxide radical anions. Therefore, applications of SPANC reactions
in living systems, particularly when those systems are under oxi-
dative stress, the rates of cycloaddition would have to out-
compete reactions with toxic free radicals. The rate constants for
the spin trapping by DMPO, range from (2.7–3.6) × 109 M−1 s−1
Conclusion
We have demonstrated that both acyclic and cyclic nitrones serve
as rapid alternatives to azides in strain-promoted cycloadditions
with BARAC. SPANC reactions of cyclic nitrones proceeded
with rate constants up to 47.3 M−1 s−1, which corresponds to a
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47-fold enhancement relative to reaction of benzyl azide and a
14-fold enhancement relative to previously reported SPANC.
According to our studies of the effects of substituents, the
SPANC reaction is sensitive to substituents on the nitrone nitro-
gen but not to substituents on the nitrone α-carbon atom. Thus,
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