Isomeric cyclopropenes exhibit unique bioorthogonal reactivities

Article abstract of DOI:10.1021/ja407737d

Bioorthogonal chemistries have provided tremendous insight into biomolecule structure and function. However, many popular bioorthogonal transformations are incompatible with one another, limiting their utility for studies of multiple biomolecules in tandem. We identified two reactions that can be used concurrently to tag biomolecules in complex environments: the inverse electron-demand Diels-Alder reaction of tetrazines with 1,3-disubstituted cyclopropenes, and the 1,3-dipolar cycloaddition of nitrile imines with 3,3-disubstituted cyclopropenes. Remarkably, the cyclopropenes used in these transformations differ by the placement of a single methyl group. Such orthogonally reactive scaffolds will bolster efforts to monitor multicomponent processes in cells and organisms.

Full text of DOI:10.1021/ja407737d

Communication
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Isomeric Cyclopropenes Exhibit Unique Bioorthogonal Reactivities
David N. Kamber,† Lidia A. Nazarova,† Yong Liang,⊥ Steven A. Lopez,⊥ David M. Patterson,†
Hui-Wen Shih,† K. N. Houk,*,⊥ and Jennifer A. Prescher*,†,‡,§
†
‡
§
Departments of Chemistry, Molecular Biology & Biochemistry, and Pharmaceutical Science, University of California, Irvine,
California 92697, United States
⊥Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, United States
S
* Supporting Information
ABSTRACT: Bioorthogonal chemistries have provided
tremendous insight into biomolecule structure and
function. However, many popular bioorthogonal trans-
formations are incompatible with one another, limiting
their utility for studies of multiple biomolecules in tandem.
We identified two reactions that can be used concurrently
to tag biomolecules in complex environments: the inverse
electron-demand Diels−Alder reaction of tetrazines with
1,3-disubstituted cyclopropenes, and the 1,3-dipolar cyclo-
addition of nitrile imines with 3,3-disubstituted cyclo-
Figure 1. Cyclopropene scaffolds undergo bioorthogonal cyclo-
additions. 1,3-Disubstituted cyclopropenes (top) react with tetrazines.
propenes. Remarkably, the cyclopropenes used in these
transformations differ by the placement of a single methyl
group. Such orthogonally reactive scaffolds will bolster
efforts to monitor multicomponent processes in cells and
organisms.
3,3-Disubstituted scaffolds (bottom) react with 1,3-dipoles to afford
covalent adducts.
We were intrigued by cyclopropene IED-DA and dipolar
cycloadditions for an additional reason: these reactions had the
potential to be orthogonal to one another and, thus, applicable
to multicomponent biomolecule labeling. In earlier work, we
observed that 1,3-disubstituted cyclopropenes react with
tetrazines at the less-hindered face of the three-membered
ring (i.e., the side bearing the C-3 H atom).14 Additional steric
bulk at this position (as in the case of 3,3-disubstituted
cyclopropenes) would, in theory, impede IED-DA reactivity but
not impact cycloadditions with less sterically encumbered
reactants (e.g., 1,3-dipoles).
he bioorthogonal chemical reporter strategy has been
Twidely used to interrogate glycans and other biopolymers
in living systems.1−5 This approach relies on the introduction of
a uniquely reactive functional group (i.e., a “chemical reporter”)
into a biomolecule of interest. The chemical reporter can be
ligated to probes for visualization or retrieval using highly
selective (i.e., “bioorthogonal”) chemistries.2,6 While powerful,
this two-step strategy has been largely limited to examining one
biological feature at a time in live cells and tissues. This is
because many bioorthogonal reactions are incompatible with
one another and cannot be used in tandem to monitor multiple
species.7−12
To predict whether cyclopropene reactivity could be tuned
with steric modifications at C-3, we examined the reactions of
1,3- and 3,3-dimethylcyclopropene (Cp(1,3) and Cp(3,3)) with
diphenyl-substituted nitrile imine (NI) and tetrazine (Tz) using
density functional theory (DFT) calculations.19 M06-2X,20,21
a
Our long-term goal is to identify transformations that can be
used concurrently to tag biomolecules in complex environ-
ments. As a starting point, we were drawn to the cycloaddition
reactions of cyclopropenes. Functionalized cyclopropenes are
stable in physiological environments, yet readily reactive with
dienes and other biocompatible motifs.13−17 We and others
have shown that 1,3-disubstituted cyclopropenes can be
metabolically incorporated into cellular glycans and selectively
ligated via inverse electron-demand Diels−Alder (IED-DA)
reactions with tetrazines (Figure 1, top).14,15,18 In related work,
Lin and colleagues demonstrated that 3,3-disubstituted cyclo-
propenes can be introduced into proteins and ultimately
detected via 1,3-dipolar cycloaddition with nitrile imines
(Figure 1, bottom).16 This reaction, similar to the cyclo-
propene−tetrazine ligation, proceeds readily in cellular environ-
ments.
density functional that provides relatively accurate energetics
for cycloadditions,22,23 was used to generate the transition-state
structures shown in Figure 2. We also analyzed activation
barriers using the distortion/interaction model,24,25 in which
the activation energy (Eact) is analyzed in terms of the
distortion energy (Edist) required for the reactants to achieve
their transition-state geometries, and the interaction energy
(Eint) arising from orbital overlap between the two distorted
reactants in the transition state. The computed activation free
energies in water (Gwater), relative rate constants (krel), and
distortion/interaction energies are provided in Figure 2.
Our calculations indicate that for the sterically less
encumbered nitrile imine, 1,3-dimethylcyclopropene reacts
Received: August 2, 2013
© XXXX American Chemical Society
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Scheme 1. Synthesis of Disubstituted Cyclopropenes
Table 1. Second-Order Rate Constants for the
Cyclopropene−Tetrazine Ligation
Figure 2. M06-2X/6-31G(d)-optimized transition-state structures for
the cycloadditions of 1,3- and 3,3-dimethylcyclopropene [Cp(1,3) and
Cp(3,3)] with diphenyl-substituted nitrile imine (NI) and tetrazine
(Tz). M06-2X/6-311+G(d,p)//6-31G(d)-computed energies and
relative rate constants (distances in Å, energies in kcal/mol, krel
based on Gwater at 298 K) are also shown.
only 2.8 times faster than 3,3-dimethylcyclopropene. The
distortion and interaction energies are very close, suggesting
that increased steric bulk at C-3 of the cyclopropene does not
dramatically influence reactivity with linear 1,3-dipoles.
However, placement of a single methyl group at C-3 reduces
cyclopropene reactivity by over 4 orders of magnitude in the
IED-DA reaction. In the transition state TS-Tz-Cp(3,3), to
avoid steric clashes between the C-3 methyl and tetrazine
nitrogens, the dihedral angle between the cyclopropene plane
and the C−C bonds-forming plane increases to 120°, about 15°
larger than the corresponding value in TS-Tz-Cp(1,3). In
Figure 2, note how the cyclopropene C-3 and methyl groups
are tilted away from the tetrazine. This results in increased
distortion energy (24.0 versus 22.0 kcal/mol) and less favorable
interaction energy (−13.7 versus −18.1 kcal/mol) due to
poorer orbital overlap. Similar reactivities were predicted for
more functionalized cyclopropenes and tetrazines (Figure S1).
Collectively, these data suggest that isomeric cyclopropenes
possess unique bioorthogonal reactivities: 3,3-disubstituted
cyclopropenes should react readily with nitrile imines, but not
tetrazines, under physiological conditions; 1,3-disubstituted
cyclopropenes, by contrast, should react readily with both.
To test these predictions, we synthesized a panel of
disubstituted cyclopropenes bearing methyl groups at either
C-1 or C-3. The scaffolds also comprise amide or carbamate
groups as these linkages mimic those found in numerous
bioconjugates. The amide-functionalized probes 1a,b were
synthesized similarly to previous reports (Scheme 1).14−16 In
brief, esters 3a,b were subjected to base-catalyzed hydrolysis.
The resulting acids (4a,b) were subsequently treated with PFP-
TFA, followed by isopropylamine to access the desired probes.
To prepare the carbamate scaffolds, esters 3a,b were first
reduced with DIBAL-H. The reaction with 3b was prone to
cyclopropane formation; over-reduction was avoided at −78
°C. Alcohols 5a,b were ultimately converted to the desired
carbamates (2a,b) via CDI coupling with isopropylamine,
followed by TMS removal.
a
All rate constants were measured in 15% DMSO/PBS. N/R*
indicates no reaction observed after 90 min.
With the desired cyclopropenes in hand, we analyzed their
reactivity with model tetrazines (Table 1). Tetrazines 7 and 8
were incubated with excess cyclopropene, and the cyclo-
additions were monitored by the change in tetrazine
absorbance over time. As shown in Figures 3 and S2, robust
IED-DA reactivity was observed with the 1,3-disubstituted
scaffolds 1a and 2a, while no reactivity was detected with their
3,3-disubstituted counterparts (1b and 2b). In fact, no reaction
between 1b or 2b and tetrazine 7 was observed in phosphate
buffer even after 24 h at elevated temperature (Figure S3).
Figure 3. Tetrazines react selectively with 1,3-disubstitued cyclo-
propenes. Cyclopropenes 1a,b (5 mM in 15% MeCN/PBS) were
treated with tetrazine 7 (10 mM) and monitored by HPLC. The initial
cycloadduct formed between 1a and 7 can undergo further
intramolecular cyclization in aqueous solution.14
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Minimal IED-DA reactivity with the 3,3-disubstituted probes
was observed only in the presence of large amounts of organic
solvent, and in these cases, the transformations were quite slow
(Table S2). It should also be noted that the tetrazine−
cyclopropene ligations revealed the expected trends, with the
more electron-rich carbamates and less sterically hindered
tetrazine exhibiting the fastest rates (Table 1).26,27
Despite their extremely sluggish reaction kinetics with
tetrazines, 3,3-disubstituted cyclopropenes react readily with
nitrile imines in “photo-click” reactions.16 Indeed, when
micromolar concentrations of 1b and 9 were subjected to UV
light (generating 10 in situ), the fluorescent cycloadduct 11 was
formed in less than 5 min (Scheme 2). The corresponding 1,3-
Scheme 2. Cyclopropenes React with Nitrile Imines To
Generate Stable Cycloadducts
Figure 4. Cyclopropenes can be selectively detected on model
proteins. (A) Cyclopropenes 13a,b were appended to BSA. The
modified proteins Cp(1,3) and Cp(3,3) were subsequently reacted
with either a tetrazine−rhodamine conjugate (Tz-Rho) or nitrile imine
10 (generated via photolysis of 9). (B) Gel analysis of Cp(1,3) or
Cp(3,3) incubated with Tz-Rho (100−750 μM) or no reagent ()
for 1 h. (C) Gel analysis of Cp(1,3) or Cp(3,3) treated with 9 (100−
1000 μM) and UV irradiation. (D) Analysis of samples treated with
Tz-Rho (100−750 μM) or no reagent (), followed by 9 (5 mM)
and UV irradiation (in gel). The gel was scanned at 532 nm (top) to
visualize rhodamine, and also illuminated with UV light (middle) to
visualize nitrile imine cycloadducts (green). The red color in the UV-
illuminated gel (middle) is due to rhodamine fluorescence. For B−D,
protein loading was assessed with Coomassie stain.
cyclopropene 1a also reacted rapidly with 10 to provide the
rearranged cycloadduct 12. Similar rearrangements have been
observed in cycloadditions with cyclopropenes and nitrile
oxides.28 Both ligation products 11 and 12 were found to be
stable in aqueous solution for over three days. Importantly,
nitrile imine 10 could also be generated in the presence of
tetrazine 7 with no observable side reactivity, highlighting the
compatibility of these reagents (Figure S4).
samples. The Cp(3,3) samples, along with unmodified scaffolds
on Cp(1,3), were detected following nitrile imine generation
(Figures 4D and S7).
The unique reactivity profiles of 1,3- and 3,3-disubstituted
cyclopropenes suggested that the probes could be used in
tandem for biomolecule labeling. To test this hypothesis, we
functionalized model proteins (BSA and lysozyme) with the
isomeric cyclopropenes 13a,b using standard coupling con-
ditions (Figure 4A). Mass spectrometry analysis was used to
verify that equivalent numbers of cyclopropenes were appended
to the biomolecules (Figure S5). When the proteins were
treated with a tetrazine−rhodamine conjugate (Tz-Rho), only
samples functionalized with 1,3-disubstituted cyclopropenes
(Cp(1,3)) showed robust dose- and time-dependent labeling,
in agreement with our kinetic data (Figures 4B, S6, and S7). No
labeling above background was observed with proteins outfitted
with 3,3-disubstituted cyclopropenes (Cp(3,3)). Both Cp(1,3)
and Cp(3,3) samples were covalently modified with nitrile
imines using “photo-click” conditions (Figures 4C and S7). The
fluorescent intensities of the Cp(1,3) adducts were somewhat
reduced, though, likely due to the decreased absorption
efficiency of the products (12 versus 11, Figure S8). When
conjugates Cp(1,3) and Cp(3,3) were subjected to both
cycloaddition reactions (treatment with Tz-Rho, followed by
9), tetrazine labeling was again only observed for Cp(1,3)
In sum, we have identified cyclopropenes that exhibit unique
modes of bioorthogonal reactivity. Computational analyses
predicted that 1,3-disubstituted cyclopropenes would undergo
facile IED-DA reactions, while 3,3-disubstituted scaffolds would
be minimally reactive with tetrazines. Upon synthesis and in
vitro characterization of a panel of modified cyclopropenes, we
discovered that scaffolds that differ in the placement of a single
methyl group (C-1 vs C-3) exhibit vastly different IED-DA
reaction profiles: 1-methylcyclopropenes can be selectively
ligated with tetrazine probes in the presence of 3-
methylcyclopropenes; the unmodified 3-methyl-substituted
scaffolds can be efficiently ligated via dipolar cycloaddition.
The ability to selectively modify isomeric cyclopropenesand
ultimately target them to discrete biomoleculeswill facilitate
multicomponent imaging studies in vitro and in live cells. The
cyclopropene scaffold also offers unique opportunities for
further biocompatible reaction development, including selective
nucleophilic additions and normal-demand Diels−Alder
reactions. An arsenal of such orthogonal reactions will continue
to provide insight into complex biological systems.29,30
C
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ASSOCIATED CONTENT
* Supporting Information
Experimental details, full spectroscopic data for all new
compounds, and additional computational data. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
■
Corresponding Authors
houk@chem.ucla.edu
jpresche@uci.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the UCI School of Physical
Sciences and the NSF (CHE-1059084). Calculations were
performed on the Hoffman2 cluster at UCLA and the Extreme
Science and Engineering Discovery Environment (XSEDE),
which is supported by the NSF (OCI-1053575). We thank
members of the UCI Chemistry Department for providing
reagents and equipment. We also thank Drs. John Greaves and
Beniam Berhane for assistance with mass spec analyses.
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