Photochemical generation of oxa-dibenzocyclooctyne (ODIBO) for metal-free click ligations

Article abstract of DOI:10.1039/c2ob26581h

Oxa-dibenzocyclooctynes (ODIBO, 2a-c) are prepared by photochemical decarbonylation of corresponding cyclopropenones (photo-ODIBO, 1a-c). While photo-ODIBO does not react with azides, ODIBO is one of the most reactive cyclooctynes exhibiting rates of cycloaddition over 45 M^-1 s ^-1 in aqueous solutions. ODIBO is stable under ambient conditions and has low reactivity towards thiols. Photo-ODIBO survives heating up to 160 °C and does not react with thiols.

Full text of DOI:10.1039/c2ob26581h

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COMMUNICATION
Photochemical generation of oxa-dibenzocyclooctyne (ODIBO) for metal-free
click ligations†
Christopher D. McNitt and Vladimir V. Popik*
Received 9th August 2012, Accepted 28th August 2012
DOI: 10.1039/c2ob26581h
enhances the stability of cycloalkynes but also decreases the
reactivity of thia-DIFBO (1.4 × 10−2 M−1 s−1).6 Azacycloalk-
Oxa-dibenzocyclooctynes (ODIBO, 2a–c) are prepared by
photochemical decarbonylation of corresponding cycloprope-
nones (photo-ODIBO, 1a–c). While photo-ODIBO does not
react with azides, ODIBO is one of the most reactive cyclo-
octynes exhibiting rates of cycloaddition over 45 M−1 s−1 in
aqueous solutions. ODIBO is stable under ambient con-
ditions and has low reactivity towards thiols. Photo-ODIBO
survives heating up to 160 °C and does not react with thiols.
ynes, e.g., azadibenzocyclooctyne (ADIBO/DIBAC,
k ∼
0.29–0.42 M−1 s−1 5b,c
)
and azadibenzocyclooctynone (BARAC,
k ∼ 0.96 M−1 s−1),5a are currently the most reactive cycloalkynes
available. It is important to note that enhancement of azide reac-
tivity in cycloalkynes is often accompanied by the increased sus-
ceptibility to nucleophilic attack. Most of the cyclooctynes tested
in our lab underwent rapid hydration in PBS buffer at 60 °C.
Cyclooctynes containing electron-withdrawing substituents are
also known to react with cytosolic thiols and cysteine residues in
proteins.12 Thus, DIFO reacts with cysteine at a rate similar to
that observed in cycloaddition with azides.12a
Numerous methods for the efficient immobilization, labeling, or
ligation of various substrates are based on Cu(I)-catalyzed acety-
lene-azide cycloaddition (CuAAC).1 Use of cytotoxic copper
catalyst, however, somewhat limits the utility of CuAAC.2
Recently discovered strain-promoted cycloaddition (SPAAC) of
azides to cyclooctynes,3 dibenzocyclooctynes,4 azadibenzo-
cyclooctynes,5 and thia-cycloalkynes6 offers a bio-compatible
catalyst-free version of the azide click reaction.7
Photochemical generation of reactive cyclooctynes permits
spatial and temporal control of the click reaction. Cycloprope-
nones are especially suitable for this purpose as they do not react
with azides and possess excellent thermal stability.10,13 We
report here the use of a cyclopropenone route for the first ever
preparation of oxa-dibenzocyclooctyne (ODIBO, 2, Scheme 1).
Preparation of cyclooctynes used in SPAAC usually requires
lengthy multistep syntheses. ODIBO, on the other hand, is pre-
pared in two steps starting from the appropriately substituted
phenyl benzyl ether. The Friedel–Crafts alkylation of the latter
with tetrachlorocyclopropenone followed by hydrolysis gives
cyclopropenone (photo-ODIBO, 1) in a moderate yield. Sub-
sequent irradiation of 1 with UV light results in quantitative for-
mation of ODIBO (2, Scheme 1).14
Cyclopropenones 1a–c are colorless crystalline compounds
that have a long shelf life if stored protected from light. Cyclo-
propenone 1a melts at 160–163 °C without decomposition and
survives 6 h refluxing in o-dichlorobenzene. 1a was also quanti-
tatively recovered after 2 h incubation at 90 °C in aqueous
solutions, as well as in biphosphate or TRIS buffers.
The rate of the 1,3-dipolar cycloaddition of the parent
cyclooctyne8 is too slow (∼0.001 M−1 s−1 3c
for the majority of
)
practical applications and the search for cycloalkynes with
higher reactivity is one of the major goals in the development of
SPAAC technology. Electronic activation of cyclooctynes can be
achieved by placing electron-withdrawing groups in the pro-
pargylic position. While one fluorine substituent has limited
effect on reactivity (e.g., MOFO, k ∼ 4.3 × 10−3 M−1 s−1),3c
difluorinated cyclooctyne (DIFO)3c reacts 40–60 times faster
than the parent compound. Alternatively, the cyclooctyne reac-
tivity can be enhanced by the modification of its structural
parameters, as was demonstrated on the examples of benzocy-
clooctyne (COMBO, k ∼ 0.24 M−1 s−1),9 dibenzocyclooctynes
(DIBO k ∼ 5.7 × 10−2 M−1 s−1 10
and bicycle[6.1.0]non-4-yne
)
(BCN, k ∼ 0.14 M−1 s−1).3d High reactivity achieved by the
combination of both approaches in difluorobenzocyclooctyne
(DIFBO, k ∼ 0.22 M−1 s−1), is, unfortunately, accompanied by
low stability, as DIFBO undergoes trimerization in solution or in
solid state.11 Introduction of a sulfur atom in DIFBO structure
Department of Chemistry, University of Georgia, Athens, GA 30602,
USA. E-mail: vpopik@uga.edu; Fax: +1 (706) 583-0342;
Tel: +1 (706) 542-1953
†Electronic supplementary information (ESI) available: Experimental
procedures; synthesis and characterization of new compounds. See DOI:
10.1039/c2ob26581h
Scheme 1 Synthesis of ODIBO.
8200 | Org. Biomol. Chem., 2012, 10, 8200–8202
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higher excess of azides. The consumption of starting material
was monitored by following the decay of the characteristic
322 nm absorbance of 2a–c. The experimental data fits the
single exponential equation well. Linear dependence of the
observed pseudo-first order rate constants on azide concentration
was analyzed by the least squares method to obtain the bimole-
cular rate constants (Table 1, Fig. 2 and Fig. S1†14).
In methanol ODIBO 2a,c react with azides almost two times
faster than the current most reactive SPAAC reagent BARAC5a
(Table 1). Direct comparison of the reactivity of various cyclo-
octynes, however, should be done with caution since the rate of
the cycloaddition reaction is commonly measured using inher-
ently less accurate NMR technique14 and solvents of different
polarity (CD3CN for BARAC). Bulky t-butyl substituent in
cyclooctynes 2a,c does not significantly affect the rate of the
cycloaddition as sterically less hindered analog 2b shows similar
reactivity (Table 1).
While SPAAC ligation methods were specifically designed for
biological applications, which implies aqueous solutions, the
reactivity of cyclooctynes is usually studied in organic solvents.
In a handful of cases where the kinetics of SPAAC was measured
in mostly aqueous solutions, presence of water has significantly
enhanced the rate of the reaction, from mere 16% for ADIBO/
DIBAC5b to more than 100% for BCN3d to a 13-fold increase
Fig. 1 UV spectra of 115 μM methanol solutions of photo-ODIBO
(1a, red line), ODIBO (2a, green line), and triazole (3a, blue line).
Cyclopropenones 1a–c do not react with organic azides or thiols
under ambient conditions. UV spectra of methanol solutions of
cyclopropenones 1a–c contain three close-lying intense bands
(λmax = 308, 330 and 343 nm, log ε ∼ 4.5, Fig. 1). Irradiation of
photo-DIBO 1a–c with 350 nm light results in efficient decarbo-
nylation of the starting material (Φ = 0.16), which could be
observed by the bleaching of the 343 nm band, and the quantitat-
ive formation of ODIBO 2a–c (Scheme 1).
Cyclooctyne 2 absorbance bands are blue-shifted in relation to
cyclopropenone precursor (λmax = 290, 308 and 322 nm, log ε ∼
4.2, Fig. 1). This feature allows for convenient monitoring of the
decarbonylation reaction of 1. ODIBO 2a–c can be stored for
months in a neat form (yellowish oil) at temperatures ∼4 °C and
are stable in aqueous PBS solutions below 50 °C. At higher
temperatures ODIBO undergoes slow hydration, e.g., at 75 °C in
PBS : DMSO (7 : 3) half-lifetime of 2a is 1.5 h. ODIBO has rela-
tively low reactivity towards endogenous thiols: the half lifetime
of ODIBO 2a in PBS solution in the presence of 10 mM of glu-
tathione is 8.2 h at room temperature. On the other hand,
ODIBO shows exceptional reactivity towards organic azides
(vide infra). SPAAC of cyclooctynes, with the exception of sym-
metric BCN, produces a mixture of two isomeric triazoles, the
products of head-to-head and head-to-tail cycloadditions. This
phenomenon often complicates spectral analysis of the resulting
products. According to chromatographic data, cycloaddition of
azides to t-butyl substituted ODIBO derivatives 2a,c produces
only one regioisomer of triazole 3 (Scheme 2). The triazole pro-
duced in a preparative scale reaction of 2a with benzyl azide
shows only one set of signals supporting the regioselectivity of
the addition. We assume that the less sterically hindered isomer
of triazole 3 is produced (Scheme 2).
Table 1 Bimolecular rate constants for the reaction of ODIBO 2a–c
with benzyl azide in various solvents
ODIBO
Solvent
Azide
Rate (M−1 s−1
)
2a
2a
2a
2a
2a
2a
2b
2c
2c
2c
MeOH
MeOH
MeOH
Benzyl azide
n-Butyl azide
TEG-azide
Benzyl azide
n-Butyl azide
TEG-azide
Benzyl azide
TEG-azide
TEG-azide
1.66 0.04
1.77 0.02
1.96 0.03
45.1 2.6
28.6 1.3
7.2 0.1
Water–MeOH–THFa
Water–MeOH–THFa
Water–THF (7 : 3)
MeOH
2.22 0.01b
1.7 0.8
MeOH
Water–MeOH (7 : 3)
Water–MeOH (95 : 5)
8.91 0.07b
11.3 0.2b
TEG-azide
a 65% water, 20% MeOH, 15% THF. b Evaluated from rate
measurements conducted at a single azide concentration.
The accurate rate measurements of ODIBO 2a–c reaction with
organic azides were conducted by UV spectroscopy at 25
0.1 °C in methanol and in aqueous solutions. Reactions were
conducted under pseudo-first order conditions, using 20 fold or
Fig. 2 Reaction of 0.115 mM ODIBO 2a with 2.5 mM BzN3 in
MeOH at 25 °C. The insert illustrates the linear dependence of the
observed rates on azide concentration.
Scheme 2 Regioselective reaction of ODIBO 2a,c with azides.
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Conclusions
Oxa-dibenzocyclooctyne (ODIBO, 2) exhibits unsurpassed reac-
tivity in metal-free acetylene-azide cycloaddition. The rate of the
reaction dramatically increases in aqueous solution, making this
compound very suitable for rapid labeling applications in bio-
chemistry. High reactivity of ODIBO towards organic azides is
combined with good aqueous stability and low susceptibility to
nucleophilic attack. These properties should reduce or eliminate
non-specific binding, known for other SPAAC reagents. The
photochemical precursor of ODIBO, in which the triple bond is
masked as a cyclopropenone functionality (Photo-ODIBO, 1)
does not react with azides, thus allowing for spatially and tem-
porally-resolved labeling. In addition, Photo-ODIBO possesses
great thermal stability and can survive heating in excess of
160 °C. Our current work is focused on the optimization of the
preparative procedures and conjugation of ODIBO with biotin
and fluorescent dyes for cell-labeling experiments.
Fig. 3 Dependence of the rates constants for the reaction of ODIBO
1a with benzyl azide on water contents in aqueous methanol. (Bimolecu-
lar rate constants were evaluated from a rate measured at a 2.5 mM of
benzyl azide and 115 μM of 1a). Insert shows the kinetic trace recorded
at 800 nM of ODIBO 2a.
Notes and references
for DIBO.4 This rate-enhancing effect was also observed in
present work for ODIBO. In 65–70% aqueous solutions15
ODIBO reacts 5–28 times faster with organic azides than in
methanol (Table 1). The reaction still follows the first order law
well and the rate shows linear dependence on azide concentration
(Fig. S1†14).
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To explore the water concentration effect on the rate of
ODIBO click reactions further, we have measured the rate of the
disappearance of ODIBO 2a (115 μM) in the presence of
2.5 mM of benzyl azide in aqueous methanol with variable
water content. Second order rate constants calculated from the
observed pseudo-first order rate constants show a smooth non-
linear increase with the rise in water concentration (Fig. 3). Con-
servative extrapolation of this trend suggests that in wholly
aqueous solution azide cycloaddition to ODIBO could proceed
with the rates in excess of 2–3 × 102 M−1 s−1. The acceleration
of the SPAAC reaction in aqueous solution is apparently caused
by higher polarity and/or donor–acceptor interaction with the
solvent. However, it is also possible that rate enhancement is
caused by the formation of an inhomogeneous solution, such as
the microemulsion of azide or aggregation of ODIBO, at higher
water concentrations. While the smooth monotonic increase of
the reaction rates shown in Fig. 3 and linear dependence of
pseudo-first order rate constant on azide concentration
(Fig. S1†14) do not support such a hypothesis, we conducted
two additional experiments to get further insight in the reactiv-
ity-enhancement effect of water. Under the extreme dilution con-
ditions, with the concentration of ODIBO 2a at 800 nM the
observed rate of the reaction with benzyl azide in water–metha-
nol–THF mixture (13 : 4 : 3) was virtually identical to that
recorded at 150 times higher concentration of the substrate
(insert in Fig. 3). This observation shows that solubility of
ODIBO does not cause rate enhancement in aqueous solutions.
Next, we explored the reaction of ODIBO 2c with water soluble
azide, 1-amino-8-azido-3,5-dioxaoctane (TEG-Azide). The for-
mation of triazole proceeded progressively faster on the way
from methanol solutions to almost neat water albeit effect was
less pronounced than for aromatic azide (Table 1).
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14 ESI†
15 Organic co-solvent was necessary for ensuring homogeneity of the azide
solution.
8202 | Org. Biomol. Chem., 2012, 10, 8200–8202
This journal is © The Royal Society of Chemistry 2012