Rearrangements and addition reactions of biarylazacyclooctynones and the implications to copper-free click chemistry

Article abstract of DOI:10.1039/c3ob40683k

Highly strained biarylazacyclooctynone (BARAC) and analogous bioconjugation reagents were shown to undergo novel rearrangement and addition reactions leading to tetracyclic products. This may limit their practical applicability as bioorthogonal reporters

Full text of DOI:10.1039/c3ob40683k

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Rearrangements and addition reactions of biarylazacyclooctynones and
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the implications to copper-free click chemistry
Mariya Chigrinova,a,b Craig S. McKay, Louis-Philippe B. Beaulieua,b Konstantin A. Udachin, André M.
Beaucheminb and John Paul Pezacki*a,b
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Highly strained biarylazacyclooctynone (BARAC) and analogous bioconjugation reagents were shown to
undergo novel rearrangement and addition reactions leading to tetracyclic products. This may limit their
practical applicability as bioorthogonal reporters for imaging biomolecules within living systems.
10 Introduction
Results and discussion
Bioorthogonal reactions allow tracking of nucleic acids, lipids,
We originally sought to synthesize a simplified BARAC analogue
and glycans, and post-translational modifications that are not 55 containing an allyl linkage for kinetic studies of its reaction with
accessible using conventional genetically encoded reporters.1-4
Azide-based bioorthogonal reactions such as Staudinger ligations
cyclic nitrones, as we anticipated that the absence of the
additional stereocenter in the linker moiety of BARAC would
simplify characterisation of the resultant cycloadducts. However,
treatment of known intermediate 120 with CsF in MeCN for 1.5
6
15 with triaryl phosphines,5,
Cu(I)-catalysed azide-alkyne
8
cycloaddition (CuAAC),7,
and strain-promoted azide-alkyne
cycloaddition (SPAAC)9 have been most widely employed. 60 hrs, and purification following overnight storage at –20 ˚C did
SPAAC has been shown to be biocompatible,10 and has
undergone several mechanistic modifications to improve its
20 applicability for bioconjugation. Rate enhancements have
typically been achieved with modifications to the alkyne
not yield 11,12-didehydro-5-allyl-dibenz[b,f]azocin-6(5H)-one
(2). Rather, mixture of (E)-N-(5-allyl-6-oxo-5,6-
a
dihydrodibenzo[b,f]azocin-12-yl)acetamide (3) and 11-allyl-6H-
isoindolo[2,1-a]indol-6-one (4) was obtained in a 9:1 ratio,
component, through incorporation of propargylic gem-difluoro 65 respectively (Scheme 1).
12
groups (difluorocyclooctyne, DIFO, k = 0.076 M-1s-1),11,
benzannulation (dibenzocyclooctyne, DIBO, k = 0.057 M-1s-1),13
25 cyclopropyl fusion (bicyclononyne, BCN, k = 0.140 M-1s-1),14
increase in the number of sp2 hydridised carbon atoms in the ring
(keto-DIBO, k = 0.26 M-1s-1),15 or by reducing the strained alkyne
ring
size
through
heteroatom
substitution
(tetramethylthiacycloheptyne, TMTH, k = 4.0 M-1s-1).16 Another
30 important modification to SPAAC has been to increase the
hydrophilicity and pharmacokinetics of the cyclooctyne
component for in vivo applications within living organisms.
Alkynes such as dimethoxyazacyclooctyne (DIMAC, k = 0.0030
M-1s-1),17 azadibenzocyclooctynes (DIBAC or ADIBO, k = 0.31
35 M-1s-1),18, 19 and biarylazacyclooctynone (BARAC, 0.96 M-1s-1).20,
Scheme 1 Synthesis of 2 and first observation of its decomposition
into products 3 and 4.
21
contain additional nitrogen atoms embedded within the
cyclooctyne ring to increase solubility. Alternative 1,3-dipoles,
such as nitrones,22-24 diazoalkanes,25 and nitrile oxides have also
been employed to improve the kinetics of strain-promoted
40 cycloadditions with cyclooctynes.26, 27
The N-alkyl amide by-product 3 may have been formed by a
70 Ritter reaction,28 via protonation of the alkyne, electrophilic
addition with MeCN, and hydrolysis of the nitrilium ion by H2O
present. It is also noteworthy that an indole product analogous to
4, has been observed by others during the synthesis of DIBAC.18
It was presumed that indole formation resulted from 5-endo-dig
75 cyclisation to relieve ring strain during CBz-group removal.18
This observation further supports our findings with BARACs. To
determine if the rearrangement of 1 to 4 was reversible, 4 was
heated to 78 °C for 1 h, formation of 2 was not observed. Next,
CsF (1 equiv.) was added to solution of 4, to determine if CsF
80 had a role in the reverse reaction, no reaction was observed.
Lastly, upon heating 4 in the presence of CsF and TMS-Cl in
solution (CDCl3), no reaction was observed. To confirm that
One of the desired properties of bioorthogonal click reactions
is that they occur with sufficiently fast rates. However, in
developing reactions with higher rates and better reactivity the
possibility of reduced selectivity and competing side reactions
45 becomes problematic. Herein, we report the rearrangement of a
BARAC analogue and the kinetic studies thereof. We also
determine that the rearrangement is acid catalysed and propose a
mechanism for this process. Understanding the rearrangement of
azacyclooctynes now makes it possible to predict when BARAC
50 and related analogues can be used successfully for bioconjugation
in vivo, and why certain analogues may fail.
formation
4 resulted from rearrangement of the alkyne

intermediate 2, 1 was reacted with CsF in the presence of an
acyclic aldonitrone, the corresponding cycloadduct was isolated
46 % yield (see ESI). These data suggest that 4 was formed
irreversibly via rearrangement of 2.
View Article Online
1
5
Next we measured kinetics for the rearrangement of 2 by H
NMR in CDCl3 (rather than CD3CN) to avoid competing
nucleophilic addition of CD3CN to the strained alkyne. Scheme 2
1
shows stacked H NMR plots, which illustrate the progression of
rearrangement of
2 in neutralised CDCl3. Interestingly,
10 rearrangement of 2 under these conditions provided a mixture of
products 4 and 5 (5-allylindeno[1,2-b]indol-10(5H)-one), formed
in a ratio of 1.3:1. No additional by-products were observed by
1H NMR. Plotting the natural logarithm of the peak intensity of
the disappearance of 2 as a function of time provides the
15 unimolecular rate constant for the rearrangement (krear. = 4.54 x
10-6 s-1), corresponding to a half-life of ~42 hrs. This has
significance to the practical utility of 2 for bioorthogonal
35
Scheme 3 Rearrangement of 6 to 7. The ORTEP diagram of 7 is shown.
Having observed that BARAC derivatives 2 and 6 are prone to
rearrangement, we evaluated if the rearrangement of 2 can be
accelerated in the presence of acid. We hypothesised that
40 protonation at the amide oxygen or the alkyne would facilitate
rearrangement. Generally, proton transfers to heteroatoms are fast
(diffusion controlled),31 whereas transfers to carbon are slower,
with only a few exceptions.32 It was anticipated that amide
protonation at oxygen would accelerate the hydration of the
45 alkyne, whereas protonation of the alkyne would be expected to
accelerate the 5-endo-dig transannular cyclisation (vide infra).
Rearrangement of 2 in CDCl3 in the presence of varying
concentrations of TFA was monitored by 1H NMR at 25 ± 0.1 ºC.
In the presence of TFA (0-0.3 M), the rate of rearrangement was
50 accelerated as the concentration of acid increased, suggesting the
rearrangement is acid catalysed. The slope of the plot of kobs
versus [TFA] corresponded to a kH+ value of 8.18 x 10-3 M-1s-1
(Figure 2). We also observe that 6 undergoes acid catalysed
rearrangement with a rate constant comparable to that of 2.
reactions,
especially
in
biological
studies
where
azacyclooctynones must be incubated in living systems over
20 prolonged periods of time.29, 30 The structures of 3, 4 and 5 were
elucidated by X-ray crystallography (Figure 1) and NMR.
Scheme 2 Overlay of 1H NMR spectra showing time-dependant
rearrangement of 2 over time in CDCl3 at 25 ± 0.1 °C.
55
Figure 2 Rate constants for the kinetic studies of rearrangement of
BARAC correlated to the various concentrations of TFA in CDCl3 at 25
± 0.1 ºC.
25
The highly strained alkyne moiety in 2 is prone to various
60 reactions with nucleophiles present. We speculate that 4 was
Figure 1 ORTEP diagrams of 3, 4 and 5 with thermal ellipsoids shown.
We
also
observe
that
rearrangement
of
formed
by
an
intramolecular
cyclisation/aza-Claisen
biarylazacyclooctynones is not exclusive to analogues bearing the
allyl linker. It was noted that BARAC containing a 4,5-
30 didehydro-oxazole side chain (6) rearranged to the analogous
product (7). We estimated the half-life of compound 6 to be
approximately 5 days in CDCl3. The structure of 7 was elucidated
by X-ray crystallography. The ORTEP diagram is displayed in
Scheme 3 and in the ESI.
rearrangement sequence involving transannular 5-endo-dig
cyclisation of the endocyclic amide nitrogen (Scheme 4).

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Scheme 5 Plausible mechanism for the formation of 5 under TFA
catalysis.
In the literature, cyclooctynes have been reported to undergo
25 nucleophilic addition from cellular nucleophiles such as
38
glutathione37,
or spontaneous homotrimerisation.39 It is
important to consider that by increasing reactivity of BARAC
designed for strain-promoted 1,3-dipolar cycloadditions, there is
a risk that selectivity and bioorthogonality may be compromised.
Scheme 4 Plausible mechanism for the formation of 4 under TFA
catalysis.
This mechanism is supported by the computational study the
MM2 energy-minimised geometry of 2 using ChemBio3D Ultra,
which shows orbital overlap between the nitrogen lone pair and
the alkyne π* molecular orbital (Figure 3).
5
30 Conclusions
In summary, we have shown that BARAC derivatives are prone
to novel rearrangements and various addition reactions that could
have implications for their practical utility as bioorthogonal
reporter groups. We have shown that rearrangement of 2 is
35 accelerated in the presence of acid, and have found that BARAC
containing
a 4,5-didehydro-oxazole side chain linker (6)
underwent analogous rearrangement. The difference in rates of
rearrangement of these BARAC analogues has enabled us to
speculate that the linker group has profound effects on rates of
40 rearrangement. BARAC analogues containing linkers that can
form good leaving groups, likely rearrange more readily than
those bearing poorer leaving groups. The rearrangements and
addition reactions of biarylazacyclooctynones have particular
relevance to biological labelling via copper-free click chemistry.
45 At low concentrations of labelling reagents rearrangement could
become a competing process and may lower the efficiency of the
tagging. It is necessary to slow the rearrangement while keeping
the cycloaddition fast. This problem of BARAC rearrangement
can be minimised through linkers consisting of poor leaving
50 groups (i.e. primary alkyl groups, exocyclic amide, etc.).18, 20, 21
Also, the use of BARAC should be avoided in conditions where
acid catalysis can occur. The most reactive cyclooctynes will
inevitably be further tuned for animal studies where the
concentrations of reagents will be low and possibility of side
55 reactions higher. These studies emphasize the delicate balance
between reactivity and stability that must be achieved for optimal
applications of bioorthogonal reactions and suggests that
biarylazacyclooctynones may be reaching the limits of practical
applicability as bioorthogonal reporter tags.
Figure 3 a) MM2 energy-minimised force field results for 2 determined
10 using ChemBio3D Ultra. b) HOMO extended Hückel molecular model
calculation results. c) LUMO extended Hückel molecular model
calculation results.
This proposed mechanism is also corroborated by a previous
account of a Brønsted acid-catalysed 5-endo-dig cyclisation of
60 Experimental
All reagents and solvents were purchased from Sigma-Aldrich,
unless otherwise stated, and used without further purification.
Deuterated solvents were purchased from Cambridge Isotope
laboratories. Thin layer chromatography was performed on
65 Analtech Uniplate® silica gel plates (60 Å F254, layer thickness
250 μm). Flash chromatography was performed using silica gel
(60 Å, particle size 40–63 μm). LC-MS/MS spectra were
15 propargylglycine derivatives to afford the corresponding
34
pyrroles.33,
Furthermore, the room-temperature aza-Claisen
rearrangement of a quaternary N-allyl enammonium salt has been
36
disclosed.35,
reaction of the alkyne moiety with residual H2O present (Scheme
20 5).
The formation of 5 presumably resulted from
1
obtained using ESI+. All H and 13C NMR spectra and kinetic

studies were obtained on a Bruker-DRX-400 spectrometer using a
1.1, 0.6 Hz), 6.06 (1 H, tdd, J = 17.1, 10.2, 4.8 Hz), 5.29 (1 H,
1
frequency of 400.13 MHz for H and 100.61 MHz for 13C and
dtd, J = 10.5, 1.8, 0.5 Hz), 5.16 (1 H, dtd, J = 17.1, 1.8, 0.4 Hz),
View Article Online
processed using Bruker TOPSPIN 2.1 software. Chemical shifts 60 4.92 (2 H, td, J = 4.7, 1.8 Hz); 13C-NMR (100 MHz, CDCl3): δ
are reported in parts per million (δ) using residual CHCl3
resonance as an internal reference (7.26 and 77.0 ppm for 1H and
13C NMR, respectively). The following abbreviations were used
to designate chemical shift multiplicities: s = singlet, d = doublet,
t = triplet, m = multiplet or unresolved, br = broad signal and J =
coupling constants in Hz. The nitrone (8),40 intermediate (1) and
185.1, 158.6, 142.4, 141.2, 134.7, 132.0, 131.5, 129.7, 123.6,
123.3, 123.2, 123.0, 120.9, 118.6, 118.0, 115.4, 110.8, 47.4. ESI-
MS: Calculated for C18H14NO (M+) = 260.10, Found 260.1.
5
65 Rearrangement of 2 under acidic conditions
Precautions were taken to not introduce acid into the starting
material 2. Non-deuterated chloroform for azeotroping and
deuterated chloroform used as solvent in all reactions were
carefully neutralised by filtering through a short plug of solid
70 sodium bicarbonate. Fresh batch of neutralised solvent was used
for each reaction. A 1H-spectra was taken of each sample without
acid for assuring purity of starting material. A calculated amount
of stock solution of TFA required to achieve the desired final
concentration was added to the solution of 2 in NMR-tube. A 1H-
75 spectra was taken periodically over several hours until complete
disappearance of the starting material. All reaction were
performed at 25 ± 0.1 ºC using the NMR spectrometer built-in
temperature control system.
10 BARAC (6) 41 were prepared according to previously to literature
procedure.
Synthesis of 11,12-didehydro-5-allyl-dibenz[b,f]azocin-6(5H)-
one 2
15 To a mixture of 1 (150 mg, 0.31 mmol, 1 equiv.) and CsF (284
mg, 1.9 mmol, 6 equiv.) was added CH3CN (1.5 mL) all at once.
The solution was stirred vigorously for 1.5 h, and the solvent was
removed. The resultant crude oil was immediately purified by
silica gel column chromatography (Hx:EtOAc/9:1, Rf = 0.28).
20 The SiO2 was neutralised with 96:4/Hx:Et3N prior to purification
of 2. The title compound was obtained as a light yellow oil (59.3
mg, 0.23 mmol, 74 %) and was stored under argon at -80 ºC. 1H-
NMR (400 MHz, CDCl3): δ 7.59 (1 H, dd, J = 6.8, 1.9 Hz), 7.54
(1 H, dd, J = 6.4, 2.8 Hz), 7.4-7.5 (4 H, m), 7.34-7.39 (2 H, m),
25 5.83 (1 H, dddd, J = 17.1, 10.2, 7.2, 5.0 Hz), 5.07 (1 H, ddd, J =
10.2, 2.5, 1.25 Hz), 4.99 (1 H, ddd, J = 17.0, 2.9, 1.4 Hz), 3.78 (1
H, tdd, J = 15.2, 4.9, 1.6 Hz), 3.26 (1 H, tdd, J = 15.4, 7.2, 1.0
80 Kinetic data analysis
The data analysis was identical in all kinetic experiments. The
intensity of a characteristic peak of 2 was calibrated to 100 %.
The same peak in all spectra in the kinetic trial was integrated
using the initial calibration. Natural logarithms of intensity values
Hz); 13C-NMR (100 MHz, CDCl3): δ 176.7, 155.1, 149.2, 133.9, 85 were plotted against time (in seconds). Only the linear portion of
130.6, 129.5, 129.3, 128.7, 128.1, 127.5, 126.4, 126.0, 122.6,
30 122.4, 118.2, 109.8, 109.4, 54.7. ESI-MS: Calculated for
C18H14NO (M+) = 260.10, Found 260.1
the regression was used to fit a linear trend line, the negative
slope of which represented the rate constant for the given
experiment at the given concentration of TFA. The rate constants
were then plotted against the corresponding TFA concentrations.
90 The slope of the linear trend line fitted to the data points
represents the kcat for the rearrangement reaction. For detailed
kinetic results see ESI.
Procedures for kinetic studies of rearrangement of 2
Rearrangement of 2 under neutral conditions
35 Precautions were taken as to not introduce acid into the starting
material 2. Non-deuterated chloroform for azeotroping and
deuterated chloroform used as solvent in the reaction were
carefully neutralised by filtering through a short plug of solid
sodium bicarbonate. The NMR tube was sealed and kept in a
40 water bath at 25 ºC. Caution was also taken as to not expose the
reaction to light. A 1H-spectra was taken daily until 2 had
disappeared completely. The solvent was evaporated in vacuo
and crude was purified by silica gel column chromatography
(Hx:EtOAc/8:2, Rf (4) = 0.6, Rf (5) = 0.3) to yield 4 as light
45 yellow solid (10.7 mg, 0.041 mmol, 53 %) and 5 (8.2 mg, 0.0317
mmol, 41 %) as red solid. 11-allyl-6H-isoindolo[2,1-a]indol-6-
one (4): 1H-NMR (400 MHz, CDCl3): δ 7.89 (1 H, d, J = 8.0 Hz),
7.77 (1 H, d, J = 7.6 Hz), 7.51 (2 H, m), 7.42 (1 H, d, J = 7.8
Hz), 7.30 (2 H, m), 7.15 (1 H, m), 6.03 (1 H, tdd, J = 16.2, 10.1,
50 6.1 Hz), 5.20 (2 H, ddd, J = 13.6, 11.5, 1.6 Hz), 3.63 (2 H, td, J =
6.1, 1.6 Hz). 13C-NMR (100 MHz, CDCl3): δ 162.4, 135.1, 135.0,
134.8, 134.5, 134.0, 133.7, 133.6, 133.6, 128.3, 26.6, 125.4,
123.7, 121.6, 120.6, 117.3, 116.7, 113.4, 28.9. ESI-MS:
Calculated for C18H14NO (M+) = 260.10, Found 260.2; 5-
55 allylindeno[1,2-b]indol-10(5H)-one (5): 1H-NMR (400 MHz,
CDCl3): δ 7.81 (1 H, ddd, J = 8.0, 1.3, 0.7 Hz), 7.46 (1 H, ddd, J
= 7.0, 1.4, 0.6 Hz), 7.15-7.25 (4 H, m), 7.11 (1 H, ddd, J = 7.1,
Notes and references
95 a National Research Council of Canada, 100 Sussex Drive, Ottawa, ON,
KIA 0R6, Canada. Fax: (+) 613 941 8447; E-mail: John.Pezacki@nrc-
cnrc.gc.ca
b Department of Chemistry, University of Ottawa, 10 Marie-Curie,
Ottawa, ON, K1N 6N5, Canada.
100 [**] This work was supported by a grant from the NSERC. The authors
thank Donald M. Leek for NMR assistance.
† Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/b000000x/
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