Tetrazine ligation: Fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity

Article abstract of DOI:10.1021/ja8053805

Described is a bioorthogonal reaction that proceeds with unusually fast reaction rates without need for catalysis: the cycloaddition of s-tetrazine and trans-cyclooctene derivatives. The reactions tolerate a broad range of functionality and proceed in high yield in organic solvents, water, cell media, or cell lysate. The rate of the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is very rapid (k₂ 2000 M^-1 s^-1). This fast reactivity enables protein modification at low concentration. Copyright

Full text of DOI:10.1021/ja8053805

Published on Web 09/18/2008
Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand
Diels-Alder Reactivity
Melissa L. Blackman, Maksim Royzen, and Joseph M. Fox*
Brown Laboratories, Department of Chemistry and Biochemistry, UniVersity of Delaware, Newark, Delaware 19716
Received July 11, 2008; E-mail: jmfox@udel.edu
Scheme 1. Diels-Alder Reactions of Tetrazines with
trans-Cyclooctene
Described herein is a bioorthogonal reaction that proceeds with
unusually fast reaction rates without need for catalysis: the
cycloaddition of s-tetrazine and trans-cyclooctene derivatives. The
reactions tolerate a broad range of functionality and proceed in high
yield in organic solvents, water, cell media, or cell lysate. The rate
of the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-
s-tetrazine is very rapid (k2 2000 M-1 s-1). This fast reactivity
enables protein modification at low concentration.
Bioorthogonal reactions, unnatural transformations that are unaf-
fected by biological functionality, are broadly useful tools with
applications that span synthesis, chemical biology, and materials
science.1 The utility of bioorthogonal reactivity has been augmented
by recent developments in post-translational strategies for incorporating
bioorthogonal functionality into proteins.2 Bioorthogonal reactions must
be exceptionally fast to be useful at the low concentrations relevant to
many biological applications. Recently, Bertozzi and co-workers
described strain-driven click reactions that take advantage of the
intrinsic reactivity of cyclooctyne toward organic azides,3,4 and work
by Bertozzi4a-d and by Boons4e has shown that click reactions of
cyclooctyne derivatives are fast (up to k2 2.3 M-1 s-1).4 Importantly,
this method avoids Cu-catalysts, which are cytotoxic, and the enhanced
reactivity enables applications for dynamic in ViVo imaging.4 Recently,
Lin and co-workers elegantly described bioconjugation based on a
photoinducible 1,3-dipolar cycloaddition reaction that proceeds with
fast rates (k2 11 M-1 s-1) with acrylamide.5 Faster ligation chemistry
will allow for the assembly of complex biomaterials under dilute
conditions. Ultimately, fast bioconjugation reactions should facilitate
the intracellular assembly of molecular structures that are too large to
cross cell membranes.
Scheme 2. Fast Reactivity at Low Micromolar Concentrations
with 1b (100 mM), but these had no effect on the efficiency of the
Diels-Alder reaction. As more stringent tests of tolerance of
biological functionality, it was shown that the reaction of 1b and
trans-cyclooctene can be carried out in cell media (DMEM +5%
FBS) or in an aqueous solution containing 10% untreated rabbit
reticulocyte lysate. The reactions were carried out at rt for 1 h with
50 µM 1b and 500 µM trans-cyclooctene and monitored by ESI-
MS. The yields in cell lysate and media were estimated to be >80%
(vs internal MS standards).
The second-order rate constant for 1b + trans-cyclooctene at
25 °C is k2 2000 (( 400) M-1 s-1 in 9:1 methanol/water. As
anticipated,10 there is a hydrophobic effect for the Diels-Alder
reaction: slower rates were observed for reactions in pure methanol
[k2 1140 (( 40) M-1 s-1] and in THF [k2 400 (( 20) M-1 s-1].
The reaction to form 2 is complete within 40 min at 25 °C at 5 µM
in THF without using an excess of either 1b or trans-cyclooctene.
The half-life is 7 s when 1b (20 µM) is reacted with excess trans-
cyclooctene (200 µM) in 9:1 methanol/water at 25 °C. The reaction
between 1b and trans-cyclooctene is much faster than background
reactivity toward water or exogenous nucleophiles.9,11 Further, 1a
also displays fast reactivity toward trans-cyclooctene [k2 3.1 ((
0.1) M-1 s-1 in THF] and does not display background reactivity
toward BuSH or BuNH2 after 8 h at rt.11,12
Unlike normal-electron-demand Diels-Alder chemistry,6 inverse-
electron demand Diels-Alder reactions have not previously been
applied to bioconjugation. Tetrazines are voracious dienes for inverse-
electron-demand Diels-Alder reactions, and N2 is produced as the
only byproduct upon subsequent retro-[4 + 2] cycloaddition.7 In 1990,
Sauer described the kinetics of electron-deficient tetrazines (Scheme
1, structure 1, where R ) CO2Me or CF3) with a number of dienophiles
and quantitatively demonstrated that their reactions with strained
alkenes are exceptionally fast.8 The most reactive dienophile is trans-
cyclooctene, which is 7 orders of magnitude more reactive than cis-
cyclooctene toward these tetrazines.8 In protic solvents, the 4,5-
dihydropyridizine 2 rapidly rearranges to isomeric 3.
We surmised that such fast and selective reactivity could form
the basis of a powerful bioorthogonal reaction. However, Sauer’s
conditions could not be directly applied to bioconjugation, as the
tetrazines that he studied immediately react with water.9 From a
survey of substituted s-tetrazines, 3,6-diaryl-s-tetrazines were
identified as suitable derivatives for bioorthogonal reactivity.10
As a model study, it was shown that 1b and trans-cyclooctene
combine to give 3 in quantitative yield after epimerization (Scheme
2). In separate experiments, EtSH (100 mM) and BuNH2 (100 mM)
were introduced to trans-cyclooctene (120 mM) before combination
The practicality of the tetrazine ligation is augmented by facile
access to starting materials. trans-Cyclooctene derivative 5 is readily
accessible from cis-cyclooctene 4 by a photochemical protocol that
we described recently (Scheme 3a).13 Functionalized analogues of
1a are known.14 Unsymmetrical tetrazine 8 can be prepared in gram
quantities from the reaction of hydrazine with commercially
9
13518 J. AM. CHEM. SOC. 2008, 130, 13518–13519
10.1021/ja8053805 CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
In summary, a new method for bioconjugation based on inverse-
electron demand Diels-Alder chemistry has been described. The
reaction proceeds with very fast rates and tolerates a broad range
of biological functionality.
Acknowledgment. This work was supported by NIH Grant
GM068640-01. We thank Colin Thorpe for donating Trx and Danny
Ramadan for HPLC assistance. We thank Ryan Mehl for insight
into the aqueous stability of 1b. We thank Colin Thorpe and John
Koh for insightful discussions.
Supporting Information Available: Experiments in which HPLC
was used to monitor bioconjugation reactions are described. Full
experimental details and 1H,13C NMR spectra are provided. This
material is available free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004. (b) Kohn, M.; Breinbauer, R. Angew. Chem., Int. Ed. 2004,
43, 3106. (c) Chen, I.; Ting, A. Y. Curr. Opin. Biotechnol. 2005, 16, 35.
(d) Antos, J. M.; Francis, M. B. Curr. Opin. Chem. Biol. 2006, 10, 253.
(e) Hahn, M. E.; Muir, T. W. Trends Biochem. Sci. 2005, 30, 26. (f)
Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2006,
128, 8820. (g) Lin, F. L.; Hoyt, H. M.; van Halbeek, H.; Bergman, R. G.;
Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 2686. (h) Kolakowski, R. V.;
Shangguan, N.; Sauers, R. R.; Williams, L. J. J. Am. Chem. Soc. 2006,
128, 5695.
(2) Recent examples: Duckworth, B. P.; Xu, J. H.; Taton, T. A.; Guo, A.;
Distefano, M. D. Bioconjugate Chem. 2006, 17, 967. (b) Gauchet, C.;
Labadie, G. R.; Poulter, C. D. J. Am. Chem. Soc. 2006, 128, 9274. (c)
Esser-Kahn, A. P.; Francis, M. B. Angew. Chem., Int. Ed. 2008, 47, 3751.
(3) (a) Wittig, G.; Krebs, A. Chem. Ber. 1961, 94, 3260.
(4) (a) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 2004,
126, 15046. (b) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard,
N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793. (c) Codelli, J. A.; Baskin,
J. M.; Agard, N. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2008, 130, 11486.
(d) Sletten, E. M.; Bertozzi, C. R. Org. Lett. 2008, 10, 3097. (e) Ning,
X. H.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Angew. Chem., Int. Ed. 2008,
47, 2253.
Figure 1. (a) Rapid reactivity to form 12 was monitored by ESI-MS and
HPLC. (b,c) Crude ESI-MS data for 11 and 12 in experiments that began
with 15 µM Trx.
Scheme 3. Synthesis of trans-Cyclooctene and Tetrazine
Derivatives
(5) Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew. Chem., Int.
Ed. 2008, 47, 2832. (b) Song, W.; Wang, Y.; Qu, J.; Lin, Q. J. Am. Chem.
Soc. 2008, 130, 9654.
(6) (a) Dantas de Araujo, A.; Palomo, J. M.; Cramer, J.; Seitz, O.; Alexandrov,
K.; Waldmann, H. Angew. Chem., Int. Ed. 2006, 45, 296. (b) Latham-
Timmons, H. A.; Wolter, A.; Roach, J. S. Nucleosides, Nucleotides Nucleic
Acids 2003, 22, 1495. (c) Yousaf, M. N.; Houseman, B. T.; Mrksich, M.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992. (d) Yousaf, M. N.; Mrksich,
M. J. Am. Chem. Soc. 1999, 121, 4286. (e) Seelig, B.; Jaschke, A.
Tetrahedron Lett. 1997, 38, 7729.
(7) Boger, D. L. Chem. ReV. 1986, 86, 781.
(8) Thalhammer, F.; Wallfahrer, U.; Sauer, J. Tetrahedron Lett. 1990, 31, 6851.
(9) Hunter, D.; Nielson, D. G.; Tweakley, T., Jr. J. Chem. Soc., Perkin Trans.
1 1985, 2709, and references therein.
(10) For Diels-Alder reactions of styrene and 1b in water: Wijnen, J. W.;
Zavarise, S.; Engberts, J. B. F. N.; Charton, M. J. Org. Chem. 1996, 61,
2001.
(11) No reaction was observed by 1H NMR after 8 h when 1a (2 × 10-2 M)
was combined with BuNH2 (2 × 10-2 M) or EtSH (2 × 10-2 M) in
CD3OD. When a CD3OD solution of 1b (2 × 10-2 M) was combined with
BuNH2 (2 × 10-2 M), 50% of 1b had reacted after 4 h. In a similar
experiment with EtSH (2 × 10-2 M), 50% of 1b had reacted after 10 min.
When 1b (2 × 10-2 M) was allowed to stir in pure water, 20%
decomposition of 1b was noted after 2 h.
(12) The product from trans-cyclooctene and 1a aromatizes upon workup. See:
Padwa, A.; Rodriguez, A.; Tohidi, M.; Fukunaga, T. J. Am. Chem. Soc.
1983, 105, 933.
available 5-amino-2-cyanopyridine (7) and 2-cyanopyridine (6)
(Scheme 3b). The amino group of tetrazine 8 provides a handle
for functionalization via acyl transfer (e.g., 9).15
To illustrate compatibility of the tetrazine ligation with proteins,
we functionalized thioredoxin (Trx) with trans-cyclooctene deriva-
tive 10. Trx is a 11.7 kDa protein that contains a single disulfide.
Upon reduction, the solvent exposed cysteine can be selectively
functionalized by maleimides.16 Thus, Trx (15 µM) was reduced
with tri(3-hydroxypropyl)phosphine (THP, 1 mM) and combined
with 10 (15 µM) in acetate buffer (pH 6). ESI mass spectral analysis
(Figure 1b) indicated that most of the Trx had been consumed and
that the conjugate 11 had formed. Subsequent combination of 11
with 1b (30 µM) indicated that the formation of 12 was complete
within 5 min. A control experiment with the cis-cyclooctene
analogue of 10 gave the analogue of 11, but reaction with 1b did
not give the analogue of 12 even after 24 h.
(13) Royzen, M.; Yap, G. P. A.; Fox, J. M. J. Am. Chem. Soc. 2008, 130, 3760.
(14) Soloducho, J.; Doskocz, J.; Cabaj, J.; Roszak, S. Tetrahedron 2003, 59,
4761.
(15) Acyl transfer to 8 was most effective when aromatic acylating agents were
employed. When aliphatic acylating agents were used, undesired reactivity
(presumably via ketene formation) competes with the rate of acylation. It
is likely that the rate of acyl transfer to 8 is slow because it is a very
electron-deficient aniline. Sugars and peptides can be appended to aromatic
acylating agents via terephthaloyl linkers, e.g.: (a) Angelastro, M. R.; Baugh,
L. E.; Bey, P.; Burkhart, J. P.; Chen, T.-M.; Durham, S. L.; Hare, C. M.;
Huber, E. W.; Janusz, M. J.; Koehl, J. R.; Marquart, A. L.; Mehdi, S.;
Peet, N. P. J. Med. Chem. 1994, 37, 4538. (b) Kanie, O.; Grotenbreg, G.;
Wong, C.-H. Angew. Chem., Int. Ed. 2000, 39, 4545. (c) Czifra´k, K.;
Hadady, Z.; Docsa, T.; Gergely, P.; Schmidt, J.; Wessjohann, L.; Somsa´k,
L. Carbohydr. Res. 2006, 341, 947.
HPLC was also used to monitor the bioconjugation reactions
that gave 11 and 12 and to demonstrate that the tetrazine ligation
to form 12 was fast and high yielding (see Supporting Information).
(16) Kallis, G.-B.; Holmgren, A. J. Biol. Chem. 1980, 255, 10261.
JA8053805
9
J. AM. CHEM. SOC. VOL. 130, NO. 41, 2008 13519