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Cite this: Org. Biomol. Chem., 2012, 10, 8200

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Photochemical generation of oxa-dibenzocyclooctyne (ODIBO) for metal-freeclick ligations†

Christopher D. McNitt and Vladimir V. Popik*

Received 9th August 2012, Accepted 28th August 2012DOI: 10.1039/c2ob26581h

Oxa-dibenzocyclooctynes (ODIBO, 2a–c) are prepared byphotochemical decarbonylation of corresponding cycloprope-nones (photo-ODIBO, 1a–c). While photo-ODIBO does notreact with azides, ODIBO is one of the most reactive cyclo-octynes exhibiting rates of cycloaddition over 45 M−1 s−1 inaqueous solutions. ODIBO is stable under ambient con-ditions and has low reactivity towards thiols. Photo-ODIBOsurvives heating up to 160 °C and does not react with thiols.

Numerous methods for the efficient immobilization, labeling, orligation of various substrates are based on Cu(I)-catalyzed acety-lene-azide cycloaddition (CuAAC).1 Use of cytotoxic coppercatalyst, however, somewhat limits the utility of CuAAC.2

Recently discovered strain-promoted cycloaddition (SPAAC) ofazides to cyclooctynes,3 dibenzocyclooctynes,4 azadibenzo-cyclooctynes,5 and thia-cycloalkynes6 offers a bio-compatiblecatalyst-free version of the azide click reaction.7

The rate of the 1,3-dipolar cycloaddition of the parentcyclooctyne8 is too slow (∼0.001 M−1 s−1)3c for the majority ofpractical applications and the search for cycloalkynes withhigher reactivity is one of the major goals in the development ofSPAAC technology. Electronic activation of cyclooctynes can beachieved by placing electron-withdrawing groups in the pro-pargylic position. While one fluorine substituent has limitedeffect on reactivity (e.g., MOFO, k ∼ 4.3 × 10−3 M−1 s−1),3c

difluorinated cyclooctyne (DIFO)3c reacts 40–60 times fasterthan the parent compound. Alternatively, the cyclooctyne reac-tivity can be enhanced by the modification of its structuralparameters, 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 thecombination of both approaches in difluorobenzocyclooctyne(DIFBO, k ∼ 0.22 M−1 s−1), is, unfortunately, accompanied bylow stability, as DIFBO undergoes trimerization in solution or insolid state.11 Introduction of a sulfur atom in DIFBO structure

enhances the stability of cycloalkynes but also decreases thereactivity of thia-DIFBO (1.4 × 10−2 M−1 s−1).6 Azacycloalk-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 cycloalkynesavailable. 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 testedin our lab underwent rapid hydration in PBS buffer at 60 °C.Cyclooctynes containing electron-withdrawing substituents arealso known to react with cytosolic thiols and cysteine residues inproteins.12 Thus, DIFO reacts with cysteine at a rate similar tothat observed in cycloaddition with azides.12a

Photochemical generation of reactive cyclooctynes permitsspatial and temporal control of the click reaction. Cycloprope-nones are especially suitable for this purpose as they do not reactwith azides and possess excellent thermal stability.10,13 Wereport here the use of a cyclopropenone route for the first everpreparation of oxa-dibenzocyclooctyne (ODIBO, 2, Scheme 1).

Preparation of cyclooctynes used in SPAAC usually requireslengthy multistep syntheses. ODIBO, on the other hand, is pre-pared in two steps starting from the appropriately substitutedphenyl benzyl ether. The Friedel–Crafts alkylation of the latterwith tetrachlorocyclopropenone followed by hydrolysis givescyclopropenone (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 compoundsthat have a long shelf life if stored protected from light. Cyclo-propenone 1a melts at 160–163 °C without decomposition andsurvives 6 h refluxing in o-dichlorobenzene. 1a was also quanti-tatively recovered after 2 h incubation at 90 °C in aqueoussolutions, as well as in biphosphate or TRIS buffers.

Scheme 1 Synthesis of ODIBO.

†Electronic supplementary information (ESI) available: Experimentalprocedures; synthesis and characterization of new compounds. See DOI:10.1039/c2ob26581h

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

8200 | Org. Biomol. Chem., 2012, 10, 8200–8202 This journal is © The Royal Society of Chemistry 2012

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Cyclopropenones 1a–c do not react with organic azides or thiolsunder ambient conditions. UV spectra of methanol solutions ofcyclopropenones 1a–c contain three close-lying intense bands(λmax = 308, 330 and 343 nm, log ε ∼ 4.5, Fig. 1). Irradiation ofphoto-DIBO 1a–c with 350 nm light results in efficient decarbo-nylation of the starting material (Φ = 0.16), which could beobserved 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 tocyclopropenone precursor (λmax = 290, 308 and 322 nm, log ε ∼4.2, Fig. 1). This feature allows for convenient monitoring of thedecarbonylation reaction of 1. ODIBO 2a–c can be stored formonths in a neat form (yellowish oil) at temperatures ∼4 °C andare stable in aqueous PBS solutions below 50 °C. At highertemperatures ODIBO undergoes slow hydration, e.g., at 75 °C inPBS : DMSO (7 : 3) half-lifetime of 2a is 1.5 h. ODIBO has rela-tively low reactivity towards endogenous thiols: the half lifetimeof 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, theproducts of head-to-head and head-to-tail cycloadditions. Thisphenomenon often complicates spectral analysis of the resultingproducts. According to chromatographic data, cycloaddition ofazides to t-butyl substituted ODIBO derivatives 2a,c producesonly one regioisomer of triazole 3 (Scheme 2). The triazole pro-duced in a preparative scale reaction of 2a with benzyl azideshows only one set of signals supporting the regioselectivity ofthe addition. We assume that the less sterically hindered isomerof triazole 3 is produced (Scheme 2).

The accurate rate measurements of ODIBO 2a–c reaction withorganic azides were conducted by UV spectroscopy at 25 ±0.1 °C in methanol and in aqueous solutions. Reactions wereconducted under pseudo-first order conditions, using 20 fold or

higher excess of azides. The consumption of starting materialwas monitored by following the decay of the characteristic322 nm absorbance of 2a–c. The experimental data fits thesingle exponential equation well. Linear dependence of theobserved pseudo-first order rate constants on azide concentrationwas 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 timesfaster 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 ofthe cycloaddition reaction is commonly measured using inher-ently less accurate NMR technique

14

and solvents of differentpolarity (CD3CN for BARAC). Bulky t-butyl substituent incyclooctynes 2a,c does not significantly affect the rate of thecycloaddition as sterically less hindered analog 2b shows similarreactivity (Table 1).

While SPAAC ligation methods were specifically designed forbiological applications, which implies aqueous solutions, thereactivity of cyclooctynes is usually studied in organic solvents.In a handful of cases where the kinetics of SPAAC was measuredin mostly aqueous solutions, presence of water has significantlyenhanced 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).

Scheme 2 Regioselective reaction of ODIBO 2a,c with azides.

Table 1 Bimolecular rate constants for the reaction of ODIBO 2a–cwith benzyl azide in various solvents

ODIBO Solvent Azide Rate (M−1 s−1)

2a MeOH Benzyl azide 1.66 ± 0.042a MeOH n-Butyl azide 1.77 ± 0.022a MeOH TEG-azide 1.96 ± 0.032a Water–MeOH–THFa Benzyl azide 45.1 ± 2.62a Water–MeOH–THFa n-Butyl azide 28.6 ± 1.32a Water–THF (7 : 3) TEG-azide 7.2 ± 0.12b MeOH Benzyl azide 2.22 ± 0.01b

2c MeOH TEG-azide 1.7 ± 0.82c Water–MeOH (7 : 3) TEG-azide 8.91 ± 0.07b

2c Water–MeOH (95 : 5) TEG-azide 11.3 ± 0.2b

a 65% water, 20% MeOH, 15% THF. b Evaluated from ratemeasurements conducted at a single azide concentration.

Fig. 2 Reaction of 0.115 mM ODIBO 2a with 2.5 mM BzN3 inMeOH at 25 °C. The insert illustrates the linear dependence of theobserved rates on azide concentration.

This journal is © The Royal Society of Chemistry 2012 Org. Biomol. Chem., 2012, 10, 8200–8202 | 8201

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for DIBO.4 This rate-enhancing effect was also observed inpresent work for ODIBO. In 65–70% aqueous solutions15

ODIBO reacts 5–28 times faster with organic azides than inmethanol (Table 1). The reaction still follows the first order lawwell and the rate shows linear dependence on azide concentration(Fig. S1†14).

To explore the water concentration effect on the rate ofODIBO click reactions further, we have measured the rate of thedisappearance of ODIBO 2a (115 μM) in the presence of2.5 mM of benzyl azide in aqueous methanol with variablewater content. Second order rate constants calculated from theobserved 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 whollyaqueous solution azide cycloaddition to ODIBO could proceedwith the rates in excess of 2–3 × 102 M−1 s−1. The accelerationof the SPAAC reaction in aqueous solution is apparently causedby higher polarity and/or donor–acceptor interaction with thesolvent. However, it is also possible that rate enhancement iscaused by the formation of an inhomogeneous solution, such asthe microemulsion of azide or aggregation of ODIBO, at higherwater concentrations. While the smooth monotonic increase ofthe reaction rates shown in Fig. 3 and linear dependence ofpseudo-first order rate constant on azide concentration(Fig. S1†14) do not support such a hypothesis, we conductedtwo 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 theobserved rate of the reaction with benzyl azide in water–metha-nol–THF mixture (13 : 4 : 3) was virtually identical to thatrecorded at 150 times higher concentration of the substrate(insert in Fig. 3). This observation shows that solubility ofODIBO does not cause rate enhancement in aqueous solutions.Next, we explored the reaction of ODIBO 2c with water solubleazide, 1-amino-8-azido-3,5-dioxaoctane (TEG-Azide). The for-mation of triazole proceeded progressively faster on the wayfrom methanol solutions to almost neat water albeit effect wasless pronounced than for aromatic azide (Table 1).

Conclusions

Oxa-dibenzocyclooctyne (ODIBO, 2) exhibits unsurpassed reac-tivity in metal-free acetylene-azide cycloaddition. The rate of thereaction dramatically increases in aqueous solution, making thiscompound very suitable for rapid labeling applications in bio-chemistry. High reactivity of ODIBO towards organic azides iscombined with good aqueous stability and low susceptibility tonucleophilic attack. These properties should reduce or eliminatenon-specific binding, known for other SPAAC reagents. Thephotochemical precursor of ODIBO, in which the triple bond ismasked 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 possessesgreat thermal stability and can survive heating in excess of160 °C. Our current work is focused on the optimization of thepreparative procedures and conjugation of ODIBO with biotinand fluorescent dyes for cell-labeling experiments.

Notes and references

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2 L. M. Gaetke and C. K. Chow, Toxicology, 2003, 189, 147; C. J. Burrowsand J. G. Muller, Chem. Rev., 1998, 98, 1109; Q. Wang, T. R. Chan,R. Hilgraf, V. V. Fokin, K. B. Sharpless and M. G. Finn, J. Am. Chem.Soc., 2003, 125, 3192.

3 (a) J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang,I. A. Miller, A. Lo, J. A. Codelli and C. R. Bertozzi, Proc. Natl. Acad.Sci. U. S. A., 2007, 104, 16793; (b) E. M. Sletten and C. R. Bertozzi,Org. Lett., 2008, 10, 3097; (c) J. A. Codelli, J. M. Baskin, N. J. Agardand C. R. Bertozzi, J. Am. Chem. Soc., 2008, 130, 11486;(d) J. Dommerholt, S. Schmidt, R. Temming, L. J. A. Hendriks, F. P. J.T. Rutjes, J. C. M. van Hest, D. J. Lefeber, P. Friedl and F. L. van Delft,Angew. Chem., Int. Ed., 2010, 49, 9422.

4 X. Ning, J. Guo, M. A. Wolfert and G.-J. Boons, Angew. Chem., Int. Ed.,2008, 47, 2253.

5 (a) J. C. Jewett, E. M. Sletten and C. R. Bertozzi, J. Am. Chem. Soc.,2010, 132, 3688; (b) M. F. Debets, S. S. van Berkel, S. Schoffelen,F. P. J. T. Rutjes, J. C. M. van Hest and F. L. van Delft, Chem. Commun.,2010, 46, 97; (c) A. Kuzmin, A. Poloukhtine, M. Wolfert andV. V. Popik, Bioconjugate Chem., 2010, 21, 2076.

6 G. de Almeida, E. M. Sletten, H. Nakamura, K. K. Palaniappan andC. R. Bertozzi, Angew. Chem., Int. Ed., 2012, 51, 2443.

7 (a) D. H. Ess, G. O. Jones and K. N. Houk, Org. Lett., 2008, 10, 1633;(b) J. C. Jewett and C. R. Bertozzi, Chem. Soc. Rev., 2010, 39, 1272;(c) M. F. Debets, C. W. J. van der Doelen, F. P. J. T. Rutjes and F. L. vanDelft, ChemBioChem, 2010, 11, 1168; (d) M. F. Debets, S. S. van Berkel,J. Dommerholt, A. J. Dirks, A. F. P. J. T. Rutjes and F. L. van Delft, Acc.Chem. Res., 2011, 44, 805; (e) E. M. Sletten and C. R. Bertozzi, Acc.Chem. Res., 2011, 44, 666.

8 A. Krebs and J. Wilke, Top. Curr. Chem., 1983, 109, 189.9 B. R. Varga, M. Kállay, K. Hegyi, S. Béni and P. Kele, Chem.–Eur. J.,2012, 18, 822.

10 A. A. Poloukhtine, N. E. Mbua, M. A. Wolfert, G.-J. Boons andV. V. Popik, J. Am. Chem. Soc., 2009, 131, 15769.

11 E. M. Sletten, H. Nakamura, J. C. Jewett and C. R. Bertozzi, J. Am.Chem. Soc., 2010, 132, 11799.

12 (a) K. E. Beatty, J. D. Fisk, B. P. Smart, Y. Y. Lu, J. Szychowski,M. J. Hangauer, J. M. Baskin, C. R. Bertozzi and D. A. Tirrell, ChemBio-Chem, 2010, 11, 2092; (b) P. V. Chang, J. A. Prescher, E. M. Sletten,J. M. Baskin, I. A. Miller, N. J. Agard, A. Lo and C. R. Bertozzi, Proc.Natl. Acad. Sci. U. S. A., 2010, 107, 1821.

13 S. V. Orski, A. A. Poloukhtine, S. Arumugam, L. Mao, V. V. Popik andJ. Locklin, J. Am. Chem. Soc., 2010, 132, 11024.

14 ESI†15 Organic co-solvent was necessary for ensuring homogeneity of the azide

solution.

Fig. 3 Dependence of the rates constants for the reaction of ODIBO1a with benzyl azide on water contents in aqueous methanol. (Bimolecu-lar rate constants were evaluated from a rate measured at a 2.5 mM ofbenzyl azide and 115 μM of 1a). Insert shows the kinetic trace recordedat 800 nM of ODIBO 2a.

8202 | Org. Biomol. Chem., 2012, 10, 8200–8202 This journal is © The Royal Society of Chemistry 2012

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