Synthesis and reactivity comparisons of 1-methyl-3-substituted cyclopropene mini-tags for tetrazine bioorthogonal reactions

Article abstract of DOI:10.1002/chem.201304225

Substituted cyclopropenes have recently attracted attention as stable "mini-tags" that are highly reactive dienophiles with the bioorthogonal tetrazine functional group. Despite this interest, the synthesis of stable cyclopropenes is not trivial and their reactivity patterns are poorly understood. Here, the synthesis and comparison of the reactivity of a series of 1-methyl-3-substituted cyclopropenes with different functional handles is described. The rates at which the various substituted cyclopropenes undergo Diels-Alder cycloadditions with 1,2,4,5-tetrazines were measured. Depending on the substituents, the rates of cycloadditions vary by over two orders of magnitude. The substituents also have a dramatic effect on aqueous stability. An outcome of these studies is the discovery of a novel 3-amidomethyl substituted methylcyclopropene tag that reacts twice as fast as the fastest previously disclosed 1-methyl-3-substituted cyclopropene while retaining excellent aqueous stability. Furthermore, this new cyclopropene is better suited for bioconjugation applications and this is demonstrated through using DNA templated tetrazine ligations. The effect of tetrazine structure on cyclopropene reaction rate was also studied. Surprisingly, 3-amidomethyl substituted methylcyclopropene reacts faster than trans-cyclooctenol with a sterically hindered and extremely stable tert-butyl substituted tetrazine. Density functional theory calculations and the distortion/interaction analysis of activation energies provide insights into the origins of these reactivity differences and a guide to the development of future tetrazine coupling partners. The newly disclosed cyclopropenes have kinetic and stability advantages compared to previously reported dienophiles and will be highly useful for applications in organic synthesis, bioorthogonal reactions, and materials science.

Full text of DOI:10.1002/chem.201304225

DOI: 10.1002/chem.201304225
Full Paper
&
Bioorthogonal Mini-tags
Synthesis and Reactivity Comparisons of 1-Methyl-3-Substituted
Cyclopropene Mini-tags for Tetrazine Bioorthogonal Reactions
[b]
[a]
[b]
[a]
Jun Yang,[a, c] Yong Liang, Jolita Seckute, K. N. Houk,* and Neal K. Devaraj*
ˇ
ˇ
˙
Abstract: Substituted cyclopropenes have recently attracted
attention as stable “mini-tags” that are highly reactive dieno-
philes with the bioorthogonal tetrazine functional group.
Despite this interest, the synthesis of stable cyclopropenes is
not trivial and their reactivity patterns are poorly under-
stood. Here, the synthesis and comparison of the reactivity
of a series of 1-methyl-3-substituted cyclopropenes with dif-
ferent functional handles is described. The rates at which
the various substituted cyclopropenes undergo Diels–Alder
cycloadditions with 1,2,4,5-tetrazines were measured. De-
pending on the substituents, the rates of cycloadditions vary
by over two orders of magnitude. The substituents also
have a dramatic effect on aqueous stability. An outcome of
these studies is the discovery of a novel 3-amidomethyl sub-
stituted methylcyclopropene tag that reacts twice as fast as
the fastest previously disclosed 1-methyl-3-substituted cyclo-
propene while retaining excellent aqueous stability. Further-
more, this new cyclopropene is better suited for bioconju-
gation applications and this is demonstrated through using
DNA templated tetrazine ligations. The effect of tetrazine
structure on cyclopropene reaction rate was also studied.
Surprisingly, 3-amidomethyl substituted methylcyclopropene
reacts faster than trans-cyclooctenol with a sterically hin-
dered and extremely stable tert-butyl substituted tetrazine.
Density functional theory calculations and the distortion/in-
teraction analysis of activation energies provide insights into
the origins of these reactivity differences and a guide to the
development of future tetrazine coupling partners. The
newly disclosed cyclopropenes have kinetic and stability ad-
vantages compared to previously reported dienophiles and
will be highly useful for applications in organic synthesis, bi-
oorthogonal reactions, and materials science.
Introduction
drew several comparisons to the popular azide-alkyne cycload-
dition, commonly referred to as “click” chemistry.[6] However,
whereas azides and alkynes have the benefit of being low mo-
lecular weight “mini-tags” and are straightforward to synthe-
size, tetrazines and previously used strained dienophiles are
both larger in size and harder to access synthetically. In an
effort to overcome those drawbacks, our group recently devel-
oped a metal-catalyzed one-pot synthesis of unsymmetric
1,2,4,5-tetrazines.[7] However, this did not address the problem
of the larger size of the coupling partners. For instance,
a common application of bioorthogonal chemistry is to intro-
duce a small bioorthogonal reactive handle into a biologically
active molecule that is metabolically incorporated through pro-
miscuous enzymatic pathways.[8] These pathways are often ex-
tremely specific to the size of the handle. For instance, elegant
studies of the sialic acid salvage pathway have determined
that N-acetyl mannosamine derivatives can be accepted below
a threshold size limit and, based on these and other prior stud-
ies, it was clear that neither tetrazines nor high molecular
weight strained dienophiles would be expected to be incorpo-
rated.[9] Furthermore, the solubility and size of the bioorthogo-
nal dienophile tags influence the pharmacokinetics of the pro-
teins and small molecules appended, which is an important
consideration for in vivo applications.[4,10] Finally, despite their
proven applications, there have been stability concerns with
highly reactive dienophiles, such as cyclooctynes and trans-cy-
clooctenes. The former are known to react with biologically rel-
Tetrazine inverse-electron-demand Diels–Alder cycloadditions
are a recently introduced class of rapid reactions driven by the
high electrophilicity of the tetrazine and high exothermicity of
reaction with alkenes.[1] These reactions have found wide-
spread application in bioorthogonal chemistry,[2] materials sci-
ence,[3] and imaging research.[4] Initial efforts focused on the
coupling of substituted 1,2,4,5-tetrazines with highly strained
dienophiles, such as trans-cyclooctene, norbornene, and cyclo-
octyne.[5] These studies revealed that tetrazine cycloadditions
could occur extremely rapidly with high yield in mild and
aqueous conditions. The features of this reaction immediately
[a] Dr. J. Yang,+ Dr. J. Seckute, Prof. N. K. Devaraj
Department of Chemistry and Biochemistry
University of California, San Diego
9500 Gilman Dr., La Jolla, CA 92037 (USA)
E-mail: ndevaraj@ucsd.edu
[b] Dr. Y. Liang,+ Prof. K. N. Houk
˙
ˇ
ˇ
Department of Chemistry and Biochemistry
University of California, Los Angeles, CA 90095 (USA)
E-mail: houk@chem.ucla.edu
[c] Dr. J. Yang+
Current address: School of Chemistry and Chemical Engineering
Shaanxi Normal University, Xi’an, 710062 (P.R. China)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201304225.
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evant nucleophiles,[11] and the latter have the potential to iso-
merize to the very slow reacting cis-cyclooctene, especially in
the presence of biological functional groups, such as thiols.[12]
A possible solution to the challenge of large dienophiles
was to explore the use of smaller cyclopropenes as tetrazine
reactive partners. In his pioneering studies of tetrazine reactivi-
ty with dienophiles, Sauer described and measured the rapid
reaction of tetrazine with cyclopropene and 3-methylcyclopro-
pene.[13] However, development of a practical cyclopropene
mini-tag required balancing the rate of reaction, which de-
pends heavily on substitution pattern, and the poor stability of
unsubstituted cyclopropenes, which tend to react with each
other through ene chemistry.[14] Considering these require-
ments, we introduced two methylcyclopropene mini-tags as
fast and stable dienophiles for reaction with tetrazines.[12b] The
tags are capable of rapidly reacting with tetrazines, elicit fluo-
rogenic responses with quenched tetrazine-fluorophores, and
were shown to be suitable for live-cell labeling of lipid ana-
logues. Subsequent work utilized the small size and high reac-
tivity of cyclopropenes as coupling agents. For instance, the
Lin group developed cyclopropene tags as bioorthogonal
chemical reporters that can be labeled via photoclick chemistry
and introduced site-specifically on proteins.[15] The Prescher
group elegantly utilized the 3-carbamoyl substituted methylcy-
clopropene tag to introduce modified neuramic acids on gly-
cans and reveal them through tetrazine cycloaddition.[16] An
important aspect of methylcyclopropenes is their potential to
undergo tetrazine cycloaddition in parallel with traditional
azide–alkyne cycloaddition chemistry. This feature was sug-
gested based on theory[17] and has been verified experimental-
ly.[16,18] Furthermore, we recently demonstrated the ability of
methylcyclopropene modified N-acetyl mannosamines to be
used in metabolic imaging, an application that is highly sensi-
tive to the steric bulk of appended bioorthogonal probes.[18]
Thus, it appears that 1-methyl-3-substituted cyclopropenes are
acid sequences. Tetrazine structure can greatly affect biological
stability and reaction rate with dienophiles. As such, we com-
pared the reactivity of the 3-amidomethyl-1-methylcyclopro-
pene with several tetrazines to quantitate how tetrazine struc-
ture affects cyclopropene reaction rate. During this study, we
surprisingly found that the 3-amidomethyl-1-methylcyclopro-
pene reacts faster than a highly strained trans-cyclooctenol
with a sterically hindered tert-butyl substituted tetrazine. This
result is significant for future biological applications since tert-
butyl substituted tetrazines are extremely resistant to biologi-
cally relevant nucleophiles, more so than other more common-
ly used methyl- and hydrogen-terminated tetrazines.
In an effort to better understand the reactivity patterns ob-
served, we performed quantum mechanical simulations of the
cyclopropene–tetrazine cycloaddition. We found that the dis-
tortion energy required for methylcyclopropene to achieve the
transition-state geometry is dramatically smaller than that for
the acyclic alkene, greatly accelerating the reaction. We select-
ed several models of the synthesized methylcyclopropenes,
and computed the transition states with 3-methyl-6-phenyl-
1,2,4,5-tetrazine. The activation free energies were calculated
for water, as well as the relative rate constants, which show
good correspondence with the experimental data. These calcu-
lations reveal a correlation between the activation free energy
and the cyclopropene HOMO energy. In addition, calculations
support a previously overlooked advantage of 1-methyl-3-sub-
stituted cyclopropenes as mini-tags: their reactivities are not
sensitive to the size of tetrazines. Our study sheds significant
light on the reactivity of the methylcyclopropene mini-tag and
will likely guide future work aiming on improving the rate of
reaction with tetrazines, further promoting the use of methyl-
cyclopropene and related mini-tags in chemical transforma-
tions, biological research, and materials science.
suitable as chemical mini-tags, readily and rapidly modified by Results and Discussion
inverse-electron-demand Diels–Alder cycloaddition with tetra-
Synthesis of methylcyclopropenes
zines and alternative coupling partners.
Given their potential utility in a wide range of applications,
the development of novel methylcyclopropene handles would
be of great interest, particularly if improvements could be
made to their reaction kinetics with tetrazines and their stabili-
ty in aqueous media. With these considerations in mind, we
designed and synthesized several new methylcyclopropenes
with functional handles substituted at the C3 position. These
methylcyclopropenes are straightforward to synthesize from
commercially available precursors and show varied reactivity
and aqueous stability based on substitution pattern. An impor-
tant outcome of these studies was the discovery of a novel 3-
amidomethyl-1-methylcyclopropene that reacts significantly
faster than previously reported methylcyclopropenes. Addition-
ally, we have adapted the newly disclosed dienophile for bio-
conjugation applications. Compared to previous probes, the
novel amide-linked cyclopropene shows improved per-
formance in bioconjugation applications. This feature was
demonstrated by comparing cyclopropene probe stability and
performance during the DNA templated detection of nucleic
Although a wide variety of cyclopropenes are synthetically ac-
cessible, our primary concern was to create a series of cyclo-
propene mini-tags that were reactive with 1,2,4,5-tetrazines
while possessing adequate aqueous stability.[19] Additionally,
the appended tag should be of low molecular weight to mini-
mize steric perturbation. In his previous studies, Sauer demon-
strated that cyclopropene and 3-methylcyclopropene were
both highly reactive with 1,2,4,5-tetrazine, but the 3,3-dime-
thylcyclopropene lowered the rate of reaction by more than
three orders of magnitude.[20] This illustrated the importance of
restricting substitution at the C3 position. Cyclopropenes with-
out substituents at the C1 position showed rapid reactivity
with tetrazine, but were not amenable to overnight storage
due to rapid degradation. As a method to improve stability
while conserving reactivity, we appended a methyl group to
the alkene as a minimal steric perturbation. This successfully
led to stable yet reactive cyclopropene mini-tags, and based
on this previous experience, we limited our current study to 1-
methyl-3-substituted cyclopropenes. In our initial studies, we
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Figure 1. Previously studied methylcyclopropenes 1 and 2 react with tetra-
zine 3 in aqueous solution.
noted that the substituents on the C3 position greatly affect
reactivity in the Diels–Alder cycloaddition.[12b] For instance, the
rate of cycloaddition of 3-carbamoyloxymethyl-1-methylcyclo-
propene 2 is approximately one-hundred times faster than 3-
carbamoyl-1-methylcyclopropene 1 when reacting with tetra-
zine 3 in aqueous solution (Figure 1). This would be consistent
with prior work, which demonstrated that electron-rich dieno-
philes react more rapidly in inverse-electron-demand Diels–
Alder reactions.[21]
Scheme 1. Synthesis of cyclopropene 8. DPPA=diphenylphosphoryl azide;
DBU=1,8-diazabicyclo[5.4.0]undec-7-ene; TBAF=tetra-n-butylammonium
fluoride; TMS=trimethylsilyl.
amine 6 could readily react to form stable amides or secondary
amines. For instance, reaction with glutaric anhydride or
methyl bromoacetate formed the corresponding amide 7 and
secondary amine 9. Unlike 6, compounds 7 and 9 were amena-
ble to long-term storage. The introduced carboxyl handle
could also be further coupled with primary amines, such as
ethanolamine, and the subsequent desilylation by TBAF afford-
ed compounds 8 and 13 (Scheme 1 and Scheme 2).
That the reaction rate could be modified so dramatically mo-
tivated us to explore how modifying the C3 position of meth-
ylcyclopropenes affects the kinetics and aqueous stability. Si-
multaneously, to easily introduce these groups to molecules of
interest, we explored substitu-
ents possessing reactive handles
that could be used for further
modification. Herein, we report
the synthesis of these new
methylcyclopropene mini-tags.
The synthesis of minimally
substituted cyclopropenes is
challenging, as cyclopropenes
can be volatile and prone to
polymerization. Previously, we
accessed cyclopropene alcohol 4
through rhodium-catalyzed cy-
clopropenation of trimethylsilyl-
propyne with ethyldiazoacetate
followed by ester reduction
(Scheme 1).[12b] Using the cyclo-
Scheme 2. Synthesis of cyclopropenes 10 and 13. TBAF=tetra-n-butylammonium fluoride; TMS=trimethylsilyl;
Su=succinimidyl; Fmoc=fluorenyl-methyloxycarbonyl.
propene alcohol 4 as a starting material, we initially attempted
to transform the alcohol into an amine by substitution chemis-
try. The inclusion of an amine was expected to increase the re-
action rate while simultaneously providing a handle for later
functionalization. However, conversion of the hydroxyl into
a good leaving group leads to complex mixtures of degrada-
tion products.
We also explored whether cyclopropene alcohol 4 could be
converted into an aldehyde by Dess–Martin oxidation. This re-
action proved successful, although aldehyde 14 is volatile and
unstable, slowly degrading over 48 h when stored at À208C.
However, aldehyde 14 could be converted to a,b-unsaturated
ester 15 through Horner–Wadsworth–Emmons reaction. Simul-
taneous hydrolysis of the ester and deprotection of the trime-
thylsilyl group with potassium hydroxide afforded carboxylic
acid 16. This agent could react with amines forming stable
amide 17 (Scheme 3).
Previously, we have shown that eliminating an electron with-
drawing carbonyl group can improve the rate of cyclopro-
pene–tetrazine cycloaddition. To test the effect of the carbonyl
group, we synthesized cyclopropene 19 through first reducing
cyclopropene ester 15 followed by trimethylsilyl deprotection
(Scheme 3). However, it should be noted that compound 19
As an alternative approach, we attempted to form the
amine by first converting the alcohol directly to an azide fol-
lowed by reduction. To our delight, treatment with diphenyl-
phosphoryl azide (DPPA) and 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU) can convert alcohol 4 into azide 5 in high yield.
After simple purification, the azide is reduced with triphenyl-
phosphine yielding amine 6. Both cyclopropene azide 5 and
amine 6 are volatile and, although useful as synthetic inter-
mediates, were not amenable to long-term storage. However,
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Scheme 3. Synthesis of cyclopropenes 16, 17, and 19. DMP=Dess–Martin
periodinane; TBAF=tetra-n-butylammonium fluoride; TMS=trimethylsilyl.
was not stable to long-term storage at À208C, and was used
immediately following purification.
Kinetic measurements and stability studies
After synthesizing the new cyclopropene mini-tags, we next
sought to compare their reactivity with tetrazines. Additionally,
we assayed their aqueous stability, given the ultimate goal of
applications in bioorthogonal coupling reactions. In order to
monitor kinetics, we conducted reactions of eight representa-
tive 1-methyl-3-substituted cyclopropenes with tetrazine 20
under pseudo-first-order conditions, and tracked the Diels–
Alder reaction by monitoring the disappearance of the charac-
teristic tetrazine absorption at 520 nm, similar to previous re-
ports (Figure 2a).[5b,22] Our choice of tetrazine 20 was motivat-
ed by previous work demonstrating that methyl-terminated
tetrazines are more resistant to decomposition in biologically
relevant media when compared to hydrogen-terminated tetra-
zines.[22] For cyclopropene stability experiments, we incubated
methylcyclopropenes in aqueous deuterated solutions (D2O/
[D6]DMSO, 4:1) as indicated, and monitored cyclopropene sta-
bility using NMR spectroscopy. The results of these experi-
ments are summarized in Table 1, and the data are shown in
Figure 2.
Figure 2. Tetrazine–cyclopropene kinetics. a) Representative set of UV/Vis ab-
sorbance scans during the reaction of the fastest stable cyclopropene
(entry 2, Table 1) with tetrazine 20 after background subtraction. Tetrazine
absorbance peak at 520 nm decreases as a function of reaction progress.
b) Data points and fitted lines for the reactions shown in Table 1. Tetrazine
20 was treated with the reported cyclopropene entries at 1 and 10 mm, re-
spectively, in 50 mm MOPS, pH 7.5, 250 mm NaCl with specific conditions in-
dicated in Table 1.
to be stable. Finally, 3-carbamoyl-1-methylcyclopropene
(entry 8), was relatively sluggish with respect to tetrazine reac-
tivity, but also proved to be highly stable. For comparison, the
rate of tetrazine reaction with entry 8 was approximately 138-
times slower than that using the fastest stable cyclopropene
handle (entry 2).
One of the potential drawbacks of highly strained trans-cy-
clooctenes is their propensity for trans/cis isomerization in the
presence of nucleophiles, such as thiols.[12] Recent work from
several groups has demonstrated this property. Since the cy-
cloaddition between tetrazine and cis-cyclooctene is expected
to have a second-order reaction rate five orders of magnitude
slower than the reaction with trans-cyclooctene, the cis form is
expected to be significantly less active.[20] This could be a prob-
lem, particularly for long-term storage in complex media or for
biological studies that require lengthy dienophile incubation.
Our recent work with 1-methyl-3-substituted cyclopropenes in-
dicated that these species are resistant to degradation when
challenged with cysteine in aqueous conditions. As cyclopro-
pene 8 is more reactive than cyclopropene 2 (as shown in the
experiment above) as well as highly stable in aqueous deuter-
ated solutions (entry 2, Table 1), we used this compound for in-
cubation with l-cysteine in 4:1 D2O/[D6]DMSO at 378C, over-
night. No decomposition could be observed by NMR spectros-
copy. Given its rapid kinetics and excellent stability, we believe
that 3-amidomethyl substituted methylcyclopropenes should
be excellent mini-tags and superior to previously introduced
Of the stable cyclopropenes, the amide derived from 3-ami-
nomethyl-1-methylcyclopropene (entry 2, Table 1) is the fastest
s
with a second-order rate constant of 0.65mÀ1 À1. This rate is
approximately twice as fast as that of the previously synthe-
sized 3-carbamoyloxymethyl-1-methylcyclopropene (entry 3).
The methylcyclopropenes derived from aldehyde precursors
are slower, and the carboxylic acid (entry 4) proved to be un-
stable to incubation in aqueous solvent at 378C. However, con-
jugation with the primary amine to form an amide (entry 5)
greatly improved stability, but significantly lowered the rate of
reaction with tetrazine. As expected from prior studies, reduc-
tion of the ester to the alcohol greatly increased the reaction
rate (entry 1). However, the resulting compound was unstable
in aqueous solution, completely decomposing in 10.5 h when
dissolved in D2O/[D6]DMSO=4:1 at room temperature. The
secondary amines resulting from 3-aminomethyl-1-methylcy-
clopropene (entries 6 and 7) showed a reduced rate of reaction
with tetrazine in aqueous solvent, although they were found
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er is consumed. Although the
tetrazine probe is highly stable
over lengthy periods of time in
buffer and biological media,
methyl-cyclopropene probe 38
linked by a carbamate linkage is
susceptible to degradation over
extended periods and multiple
freeze-thaw cycles (as deter-
mined by HPLC, Figure 3d). This
prevents the long-term storage
of such constructs and limits
their biological application. How-
ever, attachment of the novel cy-
clopropene amide 35 to oligo-
nucleotide probes (Figure 3c)
dramatically reduced the decom-
position rate and the probes are
viable after long-term storage
and multiple freeze-thaw cycles
with minimal loss of reactivity. In
Table 1. Kinetic characterization of cyclopropene–tetrazine reactions.[a,b]
Entry
1[a]
Cyclopropene
t1/2 [s]
135.1
k2 [mÀ1 sÀ1
]
Stability
0.74Æ0.04
0.65Æ0.01
unstable[d]
2[a]
3[a]
4[a]
153.8
273.2
297.6
stable[e,f]
stable[e,f]
unstable[e]
0.366Æ0.005
0.336Æ0.006
5[a]
6[b]
7[b]
500.0
2571
0.20Æ0.01
stable[e]
stable[e]
stable[e]
0.0389Æ0.0007
0.030Æ0.002
3333
a
head-to-head comparison,
8[b]
21277
0.0047Æ0.0004
stable[e]
we submitted samples to five
freeze-thaw cycles over a one
week period and compared the
ability of amide-linked cyclopro-
pene 37 and carbamate-linked
[a] 1.0 mm 20, 10.0 mm cyclopropene, 50 mm MOPS buffer, pH 7.5, 250 mm NaCl, RT; [b] 1.0 mm 20, 10.0 mm
cyclopropene, 25% DMF in MOPS buffer, RT; [c] reaction of cyclopropene with tetrazine 20 leads to formation
of diazanorcaradiene isomers (only one regioisomer is depicted); [d] D2O/[D6]DMSO=4:1, RT; [e] D2O/
[D6]DMSO=4:1, 378C; [f] D2O/[D6]DMSO=4:1, 378C, mixture with l-cysteine.
cyclopropenes 38 to elicit
fluorogenic reaction from
a
a
quenched tetrazine probe in the
methylcyclopropenes. However, it is noteworthy that the
method of synthetic introduction should be taken into account
when deciding on the use of a specific tag, as certain reactive
handles will be easier to introduce onto molecular scaffolds
given the available functional groups.
presence of an appropriate DNA template (Figure 3e). After
storage, the amide 37 remained viable and a strong fluorogen-
ic response was elicited by the template. In contrast, the de-
graded carbamate 38 elicited a minimal turn-on response. We
believe that the newly disclosed amide linked methylcyclopro-
penes will find broad application for their long-term stability
when conjugated to biological molecules.
Biological application: improved stability and performance
of cyclopropene amide modified DNA ligation probes
Dependence of methylcyclopropene reaction rate on tetra-
zine structure
In addition to improved reaction rates, the amide linkage may
have greater biological stability compared to the previously in-
troduced carbamate linkage. Carbamates can be prone to de-
composition in the proximity of biologically relevant nucleo-
philes and enzymes, such as esterases.[24] To determine if cyclo-
propene amide possessed improved stability relevant for bio-
conjugation applications, we synthesized cyclopropene amide
NHS ester 35, which can be conveniently conjugated to pri-
Having identified amides derived from 3-aminomethyl-1-meth-
ylcyclopropene as highly reactive and stable dienophiles for in-
verse-electron-demand Diels–Alder reaction with tetrazines, we
next measured the reaction rates of the cyclopropene amide 8
with six different tetrazines to quantify how tetrazine structure
affects the rate of cyclopropene–tetrazine cycloaddition. The
mary amine containing biomolecules, such as lipids, antibod- functional group substitution at the C3 and C6 position of
ies, proteins, or DNA. We conjugated the NHS ester 35 to
a short oligonucleotide and used the resulting DNA–cyclopro-
1,2,4,5-tetrazines can dramatically affect the stability of tetra-
zines in biological media as well as their reactivity with dieno-
pene amide 37 to compare its stability to a previously de- philes. Tetrazines with electron-rich substituents are highly
scribed cyclopropene carbamate linkage 38 (Figure 3a).[23] stable while possessing slower reaction rates, whereas those
Such 3’ dienophile-modified oligonucleotide probes are ex-
pected to undergo DNA-templated cycloaddition with
quenched 5’ fluorescein–tetrazine oligonucleotide probes in
with electron-poor substituents have lower stability in biologi-
cal media but have faster reaction rates.[5a] We chose substitut-
ed tetrazines based on our previous experience of synthesizing
the presence of a complementary DNA template (Figure 3b).[25] aliphatic tetrazines.[7] Alkoxyltetrazines have also been shown
Reaction elicits a fluorogenic response as the tetrazine quench- to be easily accessible, highly stable, and useful for a variety of
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Figure 3. Stability of cyclopropene–DNA oligonucleotide probes. a) Synthesis scheme of the modified DNA cyclopropene probes. b) Modified DNA probe reac-
tion upon hybridization to a fully complimentary DNA template. Illustration depicts the placement of fluorescein (star) and tetrazine quencher that results in
fluorescence turn-on (green star) after reaction with the cyclopropene probe. c) After five freeze-thaw cycles, 13 nucleotide 3’ cyclopropene amide probe 37
shows lower than 1% degradation, based on a gradient HPLC trace of 260 nm absorbance peak containing expected probe product mass peaks. Initial and
final HPLC traces are shown overlaid. d) Similarly, after five freeze-thaw cycles, 13 nucleotide 3’ cyclopropene carbamate probe 38 shows 95% degradation,
estimated from the HPLC trace peak areas. e) Cyclopropene–DNA probes (37 in green trace, 38 in blue) were treated with the neighboring tetrazine probes
along a DNA template. Reaction progress was measured by the unquenching of fluorescein emission that is initially quenched by the unreacted tetrazine
(trace in black). Final reacted fluorescein emission traces using the cyclopropene probes after freeze-thaw cycles are shown in green and blue, respectively.
Inset: the reactions upon completion, visualized under a long range UV lamp. Stars correspond with measured fluorescence scan colors (cyclopropene 37 re-
actions in green, 38 in blue). Cyclopropene 38 reaction product was confirmed previously,[23] cyclopropene 37 reactant and the reaction product are con-
firmed in Figure S2 in the Supporting Information.
applications, particularly in materials science.[26] However, cal-
culations indicated that the electron-rich substituents, such as
methoxy and dimethylamino groups, would greatly decrease
the tetrazine reactivity in the inverse-electron-demand Diels–
Alder cycloaddition (for details, see Figure S4 in the Supporting
Information).
applications. The current work demonstrates that the small
size of the methylcyclopropene dienophile is also important
for enabling a rapid reaction rate with sterically hindered tetra-
zines, such as those substituted by tert-butyl groups.
This result has important implications for future biological
applications, as tert-butyl tetrazines are extremely resistant to
degradation in biologically relevant media. We have found that
sterically hindered tetrazines possessing a tert-butyl group are
significantly more stable in comparison to hydrogen- and
methyl-terminated tetrazines (Figure S1 in the Supporting In-
formation). Using the characteristic visible absorption of tetra-
zines at approximately 520 nm to monitor stability, tert-butyl
tetrazine 26 showed negligible degradation in the presence of
buffer over 48 h whereas hydrogen- and methyl-terminated
tetrazines degraded 3 and 10%, respectively. Tetrazines have
previously been shown to be susceptible to nucleophilic attack
by thiols, with degradation rates varying depending on the
substitution pattern.[5a] Remarkably, one equivalent of l-cys-
teine causes <1% degradation of tert-butyl modified tetrazine
26 over 3 h. However, equimolar l-cysteine results in 4% deg-
radation of methyl-terminated tetrazine 25 and 20% degrada-
tion of hydrogen-terminated tetrazine 24 over the same time-
scale (for full experimental details, see the Supporting Informa-
tion). Control reactions in the absence of nucleophiles (see the
Supporting Information) showed no loss of tetrazine signal, in-
dicating that the loss of absorption was not due to intrinsic
tetrazine decomposition or light induced degradation.[27] Since
methylcyclopropenes are also highly stable, even under condi-
tions in which trans-cyclooctene is known to isomerize, the
As such, the reactivity of monoaryl tetrazine 24 is the great-
est with methylcyclopropene 8, and additional alkyl or aryl
substitution on the tetrazine decreases the rates (Table 2). We
also tested the reactivity of a sterically hindered tert-butyl sub-
stituted tetrazine 26 (for the synthesis, see the Supporting In-
formation). It was found that tetrazine 26 reacts only two-
times slower than methyl substituted tetrazine 25 (entries 4
and 5, Table 2). We also compared the tetrazine cycloaddition
reaction rates of cyclopropene 8 with trans-cycloctene 30
(Table 2). Previous work had indicated that for sterically less en-
cumbered tetrazines, such as monosubstituted tetrazines,
trans-cyclooctene reacts 2–3 orders of magnitude faster than
methylcyclopropenes.[12b,22] Although we observed relative
rates in line with this observation for most of the studied tetra-
zines, we surprisingly found that methylcyclopropene 8 reacts
with tert-butyl modified tetrazine 26 faster than trans-cyclooc-
tene 30. This result might be due to the larger size of the
trans-cyclooctene dienophile, which may have limited accessi-
bility to tetrazines that are flanked by sterically bulky substitu-
ents, such as the tert-butyl group. In contrast to trans-cyclo-
ctene, methylcyclopropenes are mini-tags of similar size to
azides and alkynes. The low molecular weight of cyclopro-
penes has previously been used to enable metabolic labeling
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implementation of this reactive pair in bioconjugation applica-
tions.
Table 2. Kinetic characterization of cyclopropene 8 and trans-cyclooctene
30 with selected tetrazines.[a,b]
DFT calculations
Quantitative insights into the high rate of the cyclopropene–
tetrazine cycloaddition and the effect of substituents on the
reactions of 1-methyl-3-substituted cyclopropenes with 1,2,4,5-
tetrazines were obtained with density functional theory (DFT)
calculations.[30] M06-2X,[31] a density functional that gives rela-
tively accurate energetics for cycloadditions,[32] was used in this
computational study. First we compared the reactivities of
methylcyclopropene and its unstrained counterpart 2-methyl-
2-butene in the inverse-electron-demand Diels–Alder reactions
with 3-methyl-6-phenyltetrazine (Figure 5). The energy of the
highest occupied molecular orbital (HOMO) of 2-methyl-2-
butene is À9.00 eV, higher than that of methylcyclopropene
(À9.31 eV). Frontier molecular orbital (FMO) analysis would
predict that the acyclic alkene is electronically more reactive.
However, transition-state calculations showed that the activa-
tion free energy for the cycloaddition of methylcyclopropene
with tetrazine via TS2 is 7.3 kcalmolÀ1 lower than that of 2-
methyl-2-butene via TS1 (19.8 vs. 27.1 kcalmolÀ1, Figure 5).
This corresponds to an increase of rate by over five orders of
magnitude. Our distortion/interaction analysis[17,33] reveals the
origin of the high rate of the cyclopropene–tetrazine cycload-
dition: the distortion energy required for methylcyclopropene
to achieve the transition-state geometry is dramatically smaller
than that for 2-methyl-2-butene (8.8 vs. 14.6 kcalmolÀ1,
Figure 5). The highly strained three-membered ring of methyl-
cyclopropene results in very easy distortion of the CÀH and CÀ
C bonds out of the C=C bond plane,[34] which is a prominent
distortion in the transition state. Next, we located the Diels–
Entry
Tetrazine
k2,MCP [mÀ1 sÀ1
]
k2,TCO [mÀ1 sÀ1
>100
31.5Æ0.3
>100
>100
0.086Æ0.002
49Æ42
>100
]
1[b]
2[b]
3[b]
4[b]
5[b,c]
6[d]
7[d]
22
23
24
25
26
27
28
1.037Æ0.007
0.0810Æ0.0003
3.34Æ0.01
0.288Æ0.002
0.131Æ0.004
0.1755Æ0.0005
2.29Æ0.02
[a] Reactions of cyclopropene 8 and trans-cyclooctene 30 with tetrazine
lead to the formation of isomers (only one isomer is depicted);
[b] 1.0 mm tetrazine, 10.0 mm cyclopropene or (E)-cyclooct-4-enol, reac-
tion conditions: 50% DMF, 50% PBS, pH 7.4, RT; [c] experiment done in
triplicate; [d] 1.0 mm tetrazine, 10.0 mm cyclopropene or (E)-cyclooct-4-
enol, reaction conditions: 70% DMF, 30% PBS, pH 7.4, RT.
tert-butyl tetrazine and methyl-
cyclopropene amide functional
groups are a highly stable yet
mutually reactive bioorthogonal
coupling pair. In order to further
characterize this reaction, we
performed an LC-MS trace of
tert-butyl tetrazine 26 reacting
with methylcyclopropene amide
8 in 1:1 DMF/H2O (Figure 4). The
reaction resulted in isomers of
3,4-diazanorcaradiene derivatives
39 in 97% yield based on react-
ed started material through an
electrocyclic ring-opening and
closing process.[16,28] The prod-
ucts appear to be stable to hy-
drolysis over the 18 h reaction in
Figure 4. Reaction between cyclopropene amide 8 with tetrazine 26. HPLC trace of purified tetrazine 26 (blue) is
shown overlaid with the trace of the products (red) of the reaction between 8 (1.1 mm) and 26 (1.0 mm) in 1:1
DMF and H2O after 18 h. Inset: the MS trace of the reaction with the selected ion monitoring at m/z 457.2 is
line with previous reports of dia-
zanorcaradiene stability.[29] We
are currently investigating the shown in black (i.e., [M+H]+ peak). Only one isomer of 39 is depicted in the scheme above.
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We also analyzed the activation barriers of these reactions
using the distortion/interaction model.[17,33] The distortion ener-
gies are nearly identical, so the differences in reactivity arise
from differences in interaction energies, which are related to
the degree of charge transfer from the cyclopropene highest
occupied molecular orbital (HOMO) to the tetrazine lowest un-
occupied molecular orbital (LUMO).[35] There is a good linear
correlation between the activation free energy and the cyclo-
propene HOMO energy (Figure 6). The experimentally em-
Figure 6. Plot of activation free energy versus cyclopropene HOMO energy
for various model 1-methyl-3-substituted cyclopropenes
(Gwater =À5.72EHOMOÀ33.4, r2 =0.95).
ployed substituents are all inductively electron-withdrawing as
compared with hydrogen, and lower the HOMO energy, de-
creasing favorable charge-transfer interaction with the low-
lying tetrazine LUMO. Calculations show that the methyl sub-
stituent can serve as an inductively electron-donating group,
elevating the HOMO energy of cyclopropene. Therefore, it is
predicted that the cyclopropene with a pure alkyl (methyl) 3-
substituent (TS7, Figure 5) will have larger relative rate con-
stant compared to the cyclopropenes studied here. Indeed,
this is consistent with past observation that 3-methylcyclopro-
pene reacts more rapidly with 1,2,4,5-tetrazines compared to
cyclopropene.[20]
Figure 5. M06-2X/6-31G(d)-optimized transition-state structures for the
Diels–Alder reactions of 3-methyl-6-phenyltetrazine with 2-methyl-2-butene
and six methylcyclopropenes (distances in ꢁ) and M06-2X/6-311+G(d,p)//6-
31G(d)-computed activation free energies in water (Gwater, in kcalmolÀ1) and
relative rate constants (krel).
To better understand the effect of tetrazine substituents on
the cyclopropene and trans-cyclooctene reaction rates, we in-
vestigated the Diels–Alder reactions of 3-phenyltetrazine, 3-
methyl-6-phenyltetrazine, and 3-tert-butyl-6-phenyltetrazine
with both 3-formamidomethyl-1-methylcyclopropene and
trans-cyclooctene, using DFT calculations (Figure 7 and
Figure 8). Computational results indicated that, for the small
methylcyclopropene, the sterically bulky 3-tert-butyl-6-phenyl-
tetrazine reacts only four-times slower than 3-phenyltetrazine.
The distortion energies required for tetrazines are very close
despite the obvious difference in size (Figure 7). This supports
a significant advantage of 1-methyl-3-substituted cyclopro-
penes as mini-tags: their reactivities are not sensitive to the
size of tetrazines and other coupling partners. By contrast, for
the trans-cyclooctene–tetrazine cycloaddition, the change of
one substituent of tetrazine from hydrogen to tert-butyl group
leads to a decrease of rate by over four orders of magnitude
(Figure 8). In the transition state TS12, to avoid the steric re-
Alder transition states for reactions of 3-methyl-6-phenyltetra-
zine with five 1-methyl-3-substituted cyclopropenes to model
the variety of dienophiles studied experimentally (Figure 5).
The activation free energies in water and relative rate con-
stants at 298 K were determined and are shown below the
transition structures in Figure 5. Compared to the activation
barrier for methylcyclopropene (TS2), the experimentally
tested substituents at the C3 position of methylcyclopropenes
(TS3–6) increase the activation barriers for cycloadditions with
tetrazine. The reactivity trends found experimentally are repro-
duced by these calculations. The calculations also reveal that
the cycloaddition is always preferred on the face away from
the 3-substituent of the cyclopropene.
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7.28 as compared with that in TS10 (142.88 vs. 150.08,
Figure 8). This needs more distortion energy of tetrazine (20.1
vs. 14.0 kcalmolÀ1), resulting in a higher activation barrier (20.8
vs. 15.2 kcalmolÀ1, Figure 8).
Conclusion
We have synthesized and characterized the reactivity of several
novel 1-methyl-3-substituted cyclopropenes as “mini-tag” dien-
ophiles for bioorthogonal reactions with electron poor tetra-
zines. These reactions are irreversible, do not require a catalyst,
and can be performed in a mutually orthogonal manner with
azide-dibenzocyclooctyne cycloadditions. We have developed
synthetic protocols to access several stable 1-methyl-3-subsi-
tuted cyclopropenes that react with tetrazines at rates span-
ning over two orders of magnitude dependent on the sub-
stituent at the C3 position. Importantly, we have found that
amidomethyl substitution on the C3 position of methylcyclo-
propene leads to a new class of cyclopropene mini-tags that
react with tetrazines faster than previously reported cyclopro-
pene handles while maintaining stability in aqueous solution
in the presence of thiols. These new tags are significantly more
stable in bioconjugation applications, and we have demon-
strated this feature by using these probes for the fluorogenic
detection of DNA templates. Additionally, we compared the re-
activity of 3-amidomethyl-1-methylcyclopropene with several
different substituted tetrazines. Unexpectedly, we discovered
that amidomethyl-substituted cyclopropene reacts faster than
a trans-cyclooctene with a sterically hindered yet extremely
stable tert-butyl substituted tetrazine. This feature is significant
for future bioconjugation applications that require reactive bio-
orthogonal probes that are highly stable to degradation. In
order to better understand the origins of the reactivity differ-
ences, we performed DFT calculations and the distortion/inter-
action analysis of activation barriers. The high rate of the cyclo-
propene–tetrazine cycloaddition is because the distortion
energy required for methylcyclopropene to achieve the transi-
tion-state geometry is dramatically smaller than that for the
acyclic alkene. The computational results reproduced the ex-
perimentally observed reactivity trend of methylcyclopropenes
with various C3 substituents and show that the activation bar-
riers have a good correlation with the cyclopropene HOMO en-
ergies. Besides, calculations support a key advantage of cyclo-
propene mini-tags: their reactivities are not sensitive to the
size of tetrazines and other coupling partners. The mechanistic
information provides a guide to the future development of bi-
oorthogonal cycloadditions. Methylcyclopropenes have size
and stability advantages compared to previously reported tet-
razine reactive dienophiles and, for these reasons, will likely
find widespread use in bioorthogonal chemistry applications.
Figure 7. M06-2X/6-31G(d)-optimized transition-state structures for the
Diels–Alder reactions of 3-formamidomethyl-1-methylcyclopropene with
three tetrazines (distances in ꢁ, dihedral angles in deg) and M06-2X/6-
311+G(d,p)//6-31G(d)-computed activation free energies in water (Gwater, in
kcalmolÀ1) and relative rate constants (krel).
Figure 8. M06-2X/6-31G(d)-optimized transition-state structures for the
Diels–Alder reactions of trans-cyclooctene with three tetrazines (distances in
ꢁ, dihedral angles in deg) and M06-2X/6-311+G(d,p)//6-31G(d)-computed
activation free energies in water (Gwater, in kcalmolÀ1) and relative rate con-
stants (krel).
Experimental Section
Materials
pulsions between the trans-cyclooctene and the bulky tert-
All chemicals were obtained from commercial sources and used
without further purification. Thin layer chromatography (TLC) was
butyl group, the dihedral angle of tetrazine plane decreases by
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Acknowledgements
N.K.D. gratefully acknowledges discussions with Prof. Carlos
Guerrero and support from the National Institutes of Health
(K01EB010078) and the University of California, San Diego.
K.N.H. gratefully acknowledges the National Science Founda-
tion (CHE-1059084) for financial support of this research. Calcu-
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Keywords: bioconjugation · bioorthogonal cycloaddition ·
cyclopropene · density functional calculations · tetrazine
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