Angewandte
Chemie
DOI: 10.1002/anie.201202122
Fluorogenic Mini-Tags
Live-Cell Imaging of Cyclopropene Tags with Fluorogenic Tetrazine
Cycloadditions**
ˇ
Jun Yang, Jolita Se cˇ kut e˙ , Christian M. Cole, and Neal K. Devaraj*
There is growing interest in the use of inverse Diels–Alder
tetrazine cycloadditions as rapid catalyst-free bioorthogonal
[
1–3]
reactions.
Fluorogenic tetrazines that increase in fluores-
cence after reaction with dienophiles are particularly useful
[
4]
for live-cell imaging applications. Fluorogenic tetrazines
have been recently used for live-cell imaging of small
molecules, biomolecules tagged enzymatically with dieno-
philes, and proteins modified by reactive unnatural amino
Figure 1. a) Inverse Diels–Alder reaction of cyclopropene with tetrazine
results in loss of nitrogen gas and formation of a diazanorcaradiene.
b) Examples of substituted cyclopropene handles that may be suitable
dienophiles for tetrazine cycloaddition.
[
4,5]
acids.
Although fluorogenic tetrazine probes hold great
potential for intracellular imaging of small molecules, pre-
vious approaches are limited by requiring a large strained
dienophile, such as trans-cyclooctene, cyclooctyne, or norbor-
[
1,6]
[9]
nene. This situation is in contrast to Staudinger ligations or
strain-promoted azide–cycloalkyne cycloadditions that utilize
tetrazines to form stable diazanorcaradienes (Figure 1a).
In seminal work, Sauer and co-workers demonstrated that
unsubstituted cyclopropene reacts with dimethyl 1,2,4,5-
tetrazine-3,6-dicarboxylate extremely rapidly, measuring
[
7,8]
a small azide functional group.
This requirement has
limited the use of tetrazine reactions in methods that require
tags with minimal steric impact or nominal effect on the
À1 À1
[10]
a rate constant of 448m
s
in dioxane at 208C. However,
[2]
partition ratio. The development of smaller dienophile
partners capable of reacting rapidly with tetrazines would
therefore represent a major advance. However, it has been
unclear whether small dienophiles could be developed that
react rapidly with tetrazines while maintaining their stability.
Herein, we demonstrate the applicability of methylcyclopro-
pene tags as dienophiles for reaction with fluorogenic
tetrazines. Through systematic synthetic modifications we
have optimized the stability, size, and reactivity of the
cyclopropene scaffold. We have developed methylcyclopro-
pene derivatives that react rapidly with tetrazines while
retaining their aqueous stability and small size. These cyclo-
propene handles elicit fluorescent responses from quenched
tetrazine dyes and are suitable for cellular imaging applica-
tions, which we demonstrate by imaging cyclopropene
phospholipids distributed in live human breast cancer cells.
The use of cyclopropenes offers a possible approach to
smaller dienophile partners for tetrazine cycloadditions. It has
long been known that cyclopropenes react rapidly with
unsubstituted cyclopropene is highly unstable, dimerizing and
[11,12]
polymerizing readily at room temperature.
Our challenge
was to create a stable cyclopropene handle while maintaining
rapid tetrazine reactivity and small size. The stability of
cyclopropenes can be dramatically increased by substitution
and we envisioned several substituted cyclopropenes that
could be suitable for cycloaddition with tetrazines (Fig-
[
11,13,14]
ure 1b).
Sauer and co-workers demonstrated that
substitutions do not necessarily diminish the reactivity of
cyclopropenes with tetrazine. In fact, the reaction of 3-
methylcycloprop-1-ene with tetrazine proceeded with
a slightly higher rate constant than the unsubstituted cyclo-
propene. However, 3,3-dimethylcycloprop-1-ene reacted with
the same tetrazine approximately 5766-times slower, illus-
[10]
trating the importance of balancing stability with reactivity.
Based on studies from Sauer and co-workers, we initially
synthesized carboxamide derivatives of cycloprop-2-enecar-
boxylic acid without substitution of the double bond. These
dienophiles reacted very rapidly with monoaryl tetrazines, but
the substituted cyclopropenes proved to be highly unstable
and could not be stored overnight at À208C without
degradation. Recently, Fox and co-workers have elegantly
demonstrated that N-acyloxazolidinone derivatives of cyclo-
prop-2-enecarboxylic acid are unusually stable and therefore
ˇ
[
*] Dr. J. Yang, Dr. J. Se cˇ kut e˙ , C. M. Cole, Prof. N. K. Devaraj
Department of Chemistry and Biochemistry
University of California, San Diego
9500 Gilman Drive, La Jolla, CA 92093 (USA)
[
15]
E-mail: ndevaraj@ucsd.edu
Homepage: http://devarajgroup.ucsd.edu/
valuable cyclopropene synthons.
Unfortunately, these
modifications require a significant increase in the size of the
reactive moiety and would defeat the purpose of using a small
cyclopropene tag. The addition of a methyl substituent on the
alkene offers an alternative method to improve stability with
[
**] We greatly acknowledge Ralph Mazitschek, Michael Hardy, and
Carlos Guerrero for helpful discussions. This work was funded in
part by NIH grant K01EB010078, the University of California, San
Diego, the NSF under CHE-0741968, and by NIH grant P30
NS047101 supported by Jennifer Santini.
[
14]
a less-dramatic steric impact.
Protected methylcyclopro-
pene 1 was generated by rhodium-catalyzed cyclopropenation
and used to synthesize 2-methylcycloprop-2-enecarboxamide
[
16]
(2). The synthesis of 2 required four steps from commer-
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
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