Photochemical syntheses, transformations, and bioorthogonal chemistry of: Trans -cycloheptene and sila trans -cycloheptene Ag(i) complexes

Article abstract of DOI:10.1039/c7sc04773h

A photochemical synthesis of AgNO₃ complexes of trans-cycloheptene (TCH) and trans-1-sila-4-cycloheptene (Si-TCH) derivatives is described. A low temperature flow photoreactor was designed to enable the synthesis of carbocyclic TCH derivatives due to their thermal sensitivity in the absence of metal coordination. Unlike the free carbocycles, TCH·AgNO₃ complexes can be handled at rt and stored for weeks in the freezer (-18 °C). Si-TCH·AgNO₃ complexes are especially robust, and are bench stable for days at rt, and for months in the freezer. X-ray crystallography was used to characterize a Si-TCH·AgNO₃ complex for the first time. With decomplexation of AgNO₃in situ, metal-free TCO and Si-TCH derivatives can engage in a range of cycloaddition reactions as well as dihydroxylation reactions. Computation was used to predict that Si-TCH would engage in bioorthogonal reactions that are more rapid than the most reactive trans-cyclooctenes. Metal-free Si-TCH derivatives were shown to display good stability in solution, and to engage in the fastest bioorthogonal reaction reported to date (k₂ 1.14 × 10⁷ M^-1 s^-1 in 9:1 H₂O:MeOH). Utility in bioorthogonal protein labeling in live cells is described, including labeling of GFP with an unnatural tetrazine-containing amino acid. The reactivity and specificity of the Si-TCH reagents with tetrazines in live mammalian cells was also evaluated using the HaloTag platform. The cell labeling experiments show that Si-TCH derivatives are best suited as probe molecules in the cellular environment.

Full text of DOI:10.1039/c7sc04773h

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DOI: 10.1039/C7SC04773H.
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Photochemical Syntheses, Transformations, and Bioorthogonal
Chemistry of trans-Cycloheptene and Sila trans-Cycloheptene
Ag(I) Complexes
Received 00th January 20xx,
Accepted 00th January 20xx
Yinzhi Fang,a Han Zhang,a, † Zhen Huang,b, † Samuel S. L. Scinto,a Jeffrey C. Yang,b Christopher W.
Am Ende,c Olga Dmitrenko,a Douglas S. Johnson,b Joseph M. Foxa,*
DOI: 10.1039/x0xx00000x
www.rsc.org/
A photochemical synthesis of AgNO3 complexes of trans-cycloheptene (TCH) and trans-1-sila-4-cycloheptene (Si-TCH)
derivatives is described. A low temperature flow photoreactor was designed to enable the synthesis of carbocyclic TCH
derivatives due to their thermal sensitivity in the absence of metal coordination. Unlike the free carbocycles, TCH•AgNO3
complexes can be handled at rt and stored for weeks in the freezer (–18 °C). Si-TCH•AgNO3 complexes are especially
robust, and are bench stable for days at rt, and for months in the freezer. X-ray crystallography was used to characterize a
Si-TCH•AgNO3 complex for the first time. With decomplexation of AgNO3 in situ, metal-free TCO and Si-TCH derivatives can
engage in a range of cycloaddition reactions as well as dihydroxylation reactions. Computation was used to predict that Si-
TCH would engage in bioorthogonal reactions that are more rapid than the most reactive trans-cyclooctenes. Metal-free
Si-TCH derivatives were shown to display good stability in solution, and to engage in the fastest bioorthogonal reaction
reported to date (k2 1.14 x 107 M–1s–1 in 9:1 H2O:MeOH). Utility in bioorthogonal protein labeling in live cells is described,
including labeling of GFP with an unnatural tetrazine-containing amino acid. The reactivity and specificity of the Si-TCH
reagents with tetrazines in live mammalian cells was also evaluated using the HaloTag platform. The cell labeling
experiments show that Si-TCH derivatives are best suited as probe molecules in the cellular environment.
photoprotonation reactions of cyclic alkenes, including
cycloheptene, have shown that cis/trans equilibria could be
driven by selective addition reactions of trans-cycloalkenes.
Introduction
For nearly seventy years,1–3 the unusual bonding, reactivity and
planar chirality of trans-cycloalkenes have captured the imagination
of scientists. The unique reactivity of trans-cycloalkenes has
produced an impressive collection of applications in synthesis,
including reactions with dienes,4–6,1,3-dipoles7 and ketenes.8–10
Additionally, strained trans-cycloalkenes can serve as excellent
ligands for transition metals. In the field of bioorthogonal
chemistry,11–19 trans-cycloalkenes hold special significance due to
their particularly fast kinetics in cycloaddition reactions.20–23
Relative to trans-cyclooctene, the chemistry of the homolog trans-
cycloheptene is less explored. trans-Cycloheptene was first trapped
with diphenylisobenzofuran by Corey and Winter from trans-1,2-
cycloheptenethionocarbonate through treatment with P(OMe)3.24
Studies by Marshall,25 Kropp26 and Beauchemin27 on the
trans-Cycloheptene was first spectroscopically characterized
by Inoue via singlet sensitized photoisomerization of cis-
cycloheptene at –35 °C.28,29 Unlike trans-cyclooctene, which is
stable at room temperature, trans-cycloheptene undergoes
rapid isomerization under ambient conditions via a proposed
‘interrupted dimerization’ mechanism.30 trans-Cycloheptene
has also been prepared via ligand exchange from a trans-
cycloheptene•CuOTf complex.31 1-Phenyl-trans-cycloheptene
and trans-cycloheptenone derivatives are also known to be
thermally unstable at ambient temperature, but can be
trapped in situ.32–38
While the parent trans-cycloheptene is thermally labile, it has
been demonstrated in several studies that metal complexes
can be isolated. CuOTf has been proposed to catalyze
photodimerization reactions of cyclic olefins via photoinduced
cis-trans isomerization,39 with predominant formation of a
cyclotrimer from cycloheptene.40
A
stable trans-
cycloheptene•CuOTf complex has been prepared through
irradiation of cis-cycloheptene•CuOTf, but a yield for the
process was not reported.41 A pybox-RuCl2 complex of trans-
cycloheptene has been prepared by irradiation of the
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corresponding ethylene complex in the presence of cis- in situ directly in cell media for bioorthogonal protein labeling
cycloheptene under singlet sensitized conditions.42 Jendralla in live cells.
described the preparation of AgOTf and AgClO4 complexes of
3-methoxy-trans-cycloheptene
and
6-Methoxy-(Z),4(E)-
cycloheptadiene.43,44 These compounds can be prepared
through the Ag-mediated ring opening of a nitrosourea
derivative of bicyclo[4.1.0]heptane. In unspecified yields, the
AgClO4•3-methoxy-trans-cycloheptene complex was combined
with a number of dienes to give the products of metal
decomplexation and [4+2] cycloaddition.43,44
Results and discussion
For the photochemical synthesis of trans-cyclooctene
derivatives, the trans-cycloalkene is first scavenged on
AgNO3/SiO2 and then liberated through treatment with
aqueous or methanolic ammonia.21,53-55 However, our
attempts to directly apply this procedure to the synthesis of
carbocyclic TCHs was unsuccessful, most likely due to the
susceptibility of carbocyclic TCHs to readily isomerize to their
cis-isomers.30 Recently, we demonstrated that the shelf-life of
conformationally strained trans-cyclooctenes could be
enhanced by storing the cycloalkenes as their AgNO3
complexes, and that the free alkenes could be liberated in situ
through treatment with NaCl in aqueous solution or cell
media.56 We reasoned that TCH and Si-TCH derivatives may
also be isolable and better stored as their Ag-complexes, and
that the corresponding free-alkenes could be liberated at later
time points as required.
Because C-Si bonds are long, the inclusion of silicon into the
cyclic backbone can alleviate olefinic strain and impart stability
to trans-cycloalkenes.45–52 In 1997, (E)-1,1,3,3,6,6-hexamethyl-
1-sila-4-cycloheptene was synthesized, resolved, and
characterized crystallographically.46,47 Here, the exhaustive
allylic substitution imparts a high degree of stability to the
trans-alkene. Recent studies by Woerpel48-51 and Tomooka52
have provided demonstrations of the utility of backbone
heteroatom-containing trans-cycloalkene derivatives in
synthesis. Woerpel has elegantly developed a general method
for the preparation of trans-oxasilacycloheptenes—seven
membered rings that contain trans-alkenes and siloxy bonds in
the backbone.48-51
Photoisomerizations to form Si-TCH•AgNO3 derivatives were
carried out at rt using the previously described flow-
photoisomerization apparatus,21,53,54 with the modification
that Si-TCH•AgNO3 complexes were directly isolated from SiO2
without Ag-decomplexation. The metal complexes that were
obtained were stable in neat form for >1 month in the freezer.
However, it was necessary to alter our reactor design for the
synthesis of carbocyclic trans-cycloheptenes due to their
thermal lability (Figure 1). As with the standard photoreactor,
this system uses methyl benzoate as a sensitizer and a
There has been little investigation into the reaction chemistry
of trans-cycloheptene derivatives. In 1980, Jendralla showed
that silver complexes of 3-methoxy-trans-cycloheptene are
isolable, and can reversibly dissociate and in unspecified yields
undergo cycloaddition reactions.43,44 Woerpel has described
selective addition reactions and difunctionalization reactions
of trans-oxasilacycloheptenes, and recently has reported the
Diels-Alder reactions of trans-oxasilacycloheptenes with furan
and tetrazine derivatives.48-51 At 25 °C in benzene, an
oxasilacycloheptene was 7-fold more reactive toward 2,5-
diphenylisobenzofuran than
trans-cyclooctene (‘s-TCO’)
a
conformationally strained
derivative.48
An
oxasilacycloheptene derivative was shown to react with 3,6-
diphenyl-s-tetrazine in benzene at rt in less than 10 min and in
8
90% NMR yield.4
Our group has described a closed-loop flow reactor for the
synthesis of trans-cyclooctene derivatives, whereby selective
complexation with AgNO3 is used to drive the formation of
trans-isomer from cis-cyclooctene.21,53–55 Described herein is
an approach to the synthesis of trans-cycloheptene (TCH) and
trans-1-sila-4-cycloheptene (Si-TCH) derivatives via flow
photochemical synthesis. The derivatives described are
especially stable as their AgNO3 metal complexes, which can
be stored neat or in solution for long periods. With
decomplexation of AgNO3 in situ, metal-free TCH and Si-TCH
derivatives can engage in a range of cycloaddition reactions as
well as dihydroxylation reactions. Unlike the carbocycles, Si-
TCH derivatives display good stability in solution and are
shown to engage in the fastest bioorthogonal reaction
reported to date. Decomplexation of AgNO3 can be carried out
Figure 1
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the flowing mixture either before or just after the UV lamp.
The temperature was measured as 0 °C before entering the
Rayonet photoreactor, and as 20 °C after exiting the
photoreactor. With this apparatus, TCH•AgNO3 complexes
were eluted from the column, and isolated as semisolids that
are moderately stable at r.t. but stable for weeks in the
freezer.
Scheme 1. Flow-photochemical synthesis of AgNO3 complexes
of trans-cycloheptenes and trans-1-sila-4-cycloheptenes.
The scope of TCH and Si-TCH synthesis is shown in Scheme 1.
With the exception of cycloheptene itself, the carbocyclic and
sila-cycloheptene precursors were prepared in 2–7 steps using
olefin metathesis as a key step (Supporting information).
Silver nitrate complexes of trans-cycloheptene (1a) and trans-
5-hydroxymethylcycloheptene (1b) were prepared in 53% and
64% yields, respectively. These TCH•AgNO3 complexes are
stable enough to handle on the bench for modest periods
(hours), and to longer-term storage in the freezer (–18 °C).
NMR monitoring showed 90% fidelity for a CD3OD solution of
1a after 10 days storage in freezer (–18 °C), and 92% fidelity
for a CD3OD solution of 1a after 10 hours at rt on the bench.
The silver complexes of Si-TCH are much more stable. NMR
monitoring showed 93% fidelity for a CD3OD solution of 2a
after 8 days at rt, and 96% fidelity for 2a after storage for 1
month in the freezer (–18 °C, neat). The photoisomerization
method could be used to produce diphenyl (2a) or dialkyl (2b-
g
) substituted silacycles. Cyano (2b) and hydroxyl (2c-e
)
groups were tolerated, as were NHS ester (2f) and
chloroalkane (2g) groups that could be used to enable
conjugation to fluorophores and HaloTag58,59 fusion proteins,
respectively. TCH•AgNO3 and Si-TCH•AgNO3 complexes were
isolated as semisolids that contained 20–30% free AgNO3. The
isolated yields were corrected by measuring the 1H NMR
against an internal standard.
Previously, a crystal structure showed that trans-cyclooctene
coordinates with AgNO3 as a 1:1 complex.60 X-ray quality
crystals of silver complex 2a were grown from ethyl
acetate/methanol. Selected bond lengths and angles are
displayed in Figure 2. The coordination environment at silver
is distorted trigonal, with bridging coordination of nitrite in an
TCH•AgNO3 and Si-TCH•AgNO3 complexes were isolated as semisolids
that contained 20–30% free AgNO3. Yields are the average of two runs,
and were corrected by measuring the 1H NMR against an internal
standard. aPhotoisomerization of carbocycles were conducted at low
temperature using the flow apparatus described in Figure 1.
Photoisomerizations of silicycles were conducted at room temperature
using the flow apparatus previously described for trans-cyclooctene
synthesis.
extended polymeric structure in the solid state.
For
comparison, we also grew crystals of the silver (I) nitrate
complex of the equatorial diastereomer of 5-hydroxy-trans-
metering pump to pass a solution of substrate at a rate of 100
mL/min through a photowell and then through a column of
AgNO3•SiO2, where the trans-cycloalkene is selectively
captured as a AgNO3 complex. In the reactor for carbocyclic
TCH synthesis, a reservoir of solvent chilled in a cold bath (–50
°C) was positioned before the photowell, and the
photoisomerization was conducted in a coil of optically
transparent FEP tubing.57 The fluoropolymer tubing provides a
high surface area and minimal volume (only 30 mL for 8 m
tubing) thereby minimizing the residence time before product
adsorption on AgNO3/SiO2. In our standard setup, an inline
thermometer was included to measure the temperature for
cyclooctene
3 (Figure 2). Here, the coordination environment
at silver is distorted tetrahedral due to the ability of the
hydroxyl to serve as a second bridging ligand. The C-Ag bond
lengths and bond angles were similar for 2a and
expected, the C–C=C-C dihedral angle for (136.7 °) is smaller
than metal-free TCO
(139.1 °).53 Similarly, the C–C=C-C
dihedral angle for 2a (126.3 °) was smaller than that of metal
free Si-trans-cycloheptene
(130.9 °),46 but comparable to
3.
As
3
4
5
6
(126.1 °C)50— a compound with additional strain due to
relatively short C–O and Si–O bonds in the cyclic backbone.
Reflecting the low level of metal backbonding that is common
for Ag(I) alkene complexes, the C=C bond length for the Ag(I)
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complexes (1.329 Å for 2a, 1.366 Å for 3
) was very similar to complex 2d. This 65 cm–1 shift is consistent both in magnitude
that of the metal-free complexes 4–6 (1.331–1.335 Å).
and direction for a Ag(I) alkene complex.61 Finally, we noted
Figure
2 X-ray crystal structures of SiTCH•AgNO3 and
TCO•AgNO3 complexes, with comparisons to known metal-free
trans-cycloalkenes that have been crystallographically
characterized.
Figure 3 (a) Decomplexation of AgNO3 with NH4OH gives
metal-free Si-TCH 7a which is stable to overnight storage in
solution. Aq. NaCl can also be used to liberate metal from
SiTCH• AgNO3 complexes. The metal-free complexes are stable
in solution, but give variable amounts of isomerization when
concentrated to dryness. (b) FT-IR spectrum of the C=C
stretches of metal complex 2d and free alkene 7d. (c)
Diagnostic shifts in alkene peaks in the 1H and 13C NMR spectra
of metal complex 2b and metal-free 7b.
Silver-free Si-TCH compounds could be prepared by treating
their corresponding silver nitrate complexes with an excess of
aq. NH4OH or aq. NaCl, followed by extraction with organic
solvent. For example, silver complex 2a upon treatment with
aq. NH4OH was extracted with C6D6 to give a solution of Si-TCH
7a (98% trans isomer). Consistent with previous reports on a
biomolecular mechanism for TCH isomerization,30 variable
amounts (20-30%) of the cis-isomer of 7a was observed when
7a was concentrated to dryness on the rotovap. However, 7a
displayed high stability when maintained in solution, with only
8% isomerization observed for a 100 mM solution of 7a that
was stored for 24 hours at room temperature, and <5%
isomerization for a similar solution that was stored for 24
hours in a freezer (–20 °C).
that Ag(I)-complexation leads to signature shifts of alkene
resonances in both the 1H and 13C NMR spectra (Figure 3C). For
1
example, alkene resonances in the H NMR spectrum of metal
complex 2b were shifted downfield relative to metal-free 7b
by 0.15 ppm, while in 13C NMR spectra alkene resonances of
1
2b were shifted up field by ~16 ppm. In the H NMR spectra,
complexity arises due to the higher order effects for a ddd
couplet.
Ag(I)-complex 2d could also be freed of metal to give alkene 7d
as a mixture of diastereomers (Figure 3A). The allylic
substituents of 7d protect the alkene from biomolecular
chemistry, and unlike most other metal-free Si-TCH
compounds, 7d is stable when stored in neat form and can be
characterized by FT-IR (Figure 3B). The weak C=C double bond
stretch of 7d at 1624 cm–1 is shifted to 1559 cm–1 for the Ag(I)
TCH 1a and Si-TCH 2a were shown to engage in a range of
reactions as shown in Scheme 2. Metal complex 1a was
directly combined with 3,6-diphenyl-1,2,4,5-tetrazine to give
pyridazine
8 — the product of metal dissociation, Diels-
Alder/retro-Diels-Alder, and oxidation, in 98% yield.
Cyclopenta-1,3-diene was also used to trap trans-
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cycloheptene, delivering the [4+2] cycloaddition adduct
9 in We sought to demonstrate that Si-TCH cycloadducts could be
81% yield as a single diastereomer. We also investigated the oxidized to give formal cycloadducts of 1,2,-dialkylolefins—
vicinal dihydroxylation of 1a, and found that catalytic OsO4 and which are recalcitrant substrates in intermolecular Diels-Alder
NMO gave 10 in 82% yield as a single diastereomer. The reactions.62 With the cycloadduct 11, we demonstrated that
observation that the dihydroxylation of 1a is stereospecific is the diphenylsila- group could be oxidized to a diol-product.
in line with earlier observations by Cope with trans- Thus 11 was subjected to Tamao-Fleming reaction under
cyclooctene.2
Woerpel’s conditions63 to give the unknown diol product 15 in
76% yield (Scheme 3).
Si-TCH•AgNO3 complex 2a could be freed from silver by
treatment with NH4OH (Scheme 2), and subsequently
Scheme 3. Fleming-Tamao Oxidation
combined
with
cyclopentadiene,
diazomethane,
dichloroketene, and benzyl azide to provide the cycloadducts
11–14 in 76%–96% yields (Scheme 2). In each case, a single
diastereomer was obtained. Attempts to combine cis-1-
diphenylsila-4-cycloheptene (the cis-isomer of 7a
) with
dichloroketene, benzylazide or diazomethane only returned
unreacted starting material. An X-ray structure was obtained
for the dichloroketene adduct 13 (Supporting Information).
We further demonstrated that NHS-ester 2f could be modified
though coupling to a BODIPY-fluorophore conjugate. As shown
in Scheme 4, NHS ester 2f could be decomplexed from AgNO3
by treatment with brine and extraction into CH2Cl2. The
resulting free Si-TCH was then conjugated to an aminohexyl
BODIPY 16, and the resulting conjugate was isolated and
stored as a AgNO3 complex (AgSiTCH-BODIPY). As discussed
below, this fluorophore conjugate finds utility for
bioorthogonal labeling in live cells. We also demonstrated that
the equatorial allylic alcohol 17(eq), derived from Ag-complex
Scheme 2. Reactions of TCH 1a and Si-TCH 2a.
2e, could be elaborated to the carbamate 18 through
treatment with benzylisocyanate (Scheme 4). trans-
Cyclooctenes with allylic carbamate leaving groups have been
used by Robillard64,65 and Chen66,67 for the tetrazine-ligation
initiated decaging of doxorubicin and other cargo molecules.
The 7-membered analog 18 is particularly stable as the Ag-free
trans-cycloalkene, and can be handled neat and stored
without AgNO3 in the freezer for long periods. Efforts to
synthesize and explore the ability of analogs of 18 to function
for payload release is a topic of ongoing study.
Scheme 4. Transformations of Si-TCH derivatives
Anticipating that Si-TCH compounds may be useful in
bioorthogonal chemistry, computation was used to study the
aYields represent isolated yields as average of two runs
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structure and Diels-Alder reactivity of Si-TCH 19 (Scheme 5). The computations prompted us to conduct experimental
We compared 19 to the parent trans-cyclooctene and a investigations into the utilization of Si-TCH complexes for
conformationally strained ‘s-TCO’ derivative 20 that was applications in bioorthogonal chemistry.
previously studied in our labs.21,54 We reasoned that the 7-
Previously, we had studied the reaction of diphenyl-s-tetrazine
in MeOH at 25 °C with s-TCO,21,54 d-TCO,22,21 and trans-
membered ring in the backbone of 19 would augment the
olefinic strain of the trans-cycloalkene, and thereby increase
the reactivity in tetrazine ligation.
Previously, cyclooctene,69 with second order rate constants of 3100 (+/–
50), 520 (+/–3) and 19.1 (+/–0.2) M–1s–1, respectively. Si-TCH
7c was liberated from AgNO3, and in agreement with
computational prediction, was found to react with diphenyl-s-
tetrazine with a rate constant of 4360 (+/–430) M–1s–1— 1.4
times faster than sTCO, 8.4 times faster than dTCO, and 228
times faster than trans-cyclooctene itself (Scheme 6A).
thiacycloheptynes and dibenzoselenocycloheptynes have been
studied in bioorthogonal chemistry.67 As shown in Scheme 5A,
ground state calculations were carried out for Si-TCH 19 at the
M06L/6-311+G(d,p) level, and compared to previous
calculations on trans-cyclooctene and s-TCO 20 21,54
.
The
calculated C-C=C-C dihedral angle for 19 (128.8°) is smaller
than that for trans-cyclooctene (137.7°) or 20 (131.9°).
M06L/6-311+G(d,p) calculations were also carried out to
compare the Diels-Alder reactivity of Si- TCH 19 to trans-
cyclooctene and s-TCO 20 (Scheme 5B). These calculations
were carried out with diphenyl-s-tetrazine so that they could
be benchmarked against previous calculations.21,54 For Si-TCH
19, the calculated barriers relative to a pre-reaction complex
for the Diels-Alder reaction with 3,6-diphenyl-s-tetrazine are
ΔG‡ 12.48 kcal/mol; ΔE‡ 9.27 kcal/mol, ΔE‡(ZPE) 9.91 kcal/mol
and ΔH‡ 8.87 kcal/mol. The barrier is significantly lower than
that of trans-cyclooctene with 3,6-diphenyl-s-tetrazine (ΔΔG‡
–3.61 kcal/mol, ΔΔE‡ –4.02 kcal/mol, ΔΔE‡(ZPE) –3.99
kcal/mol, ΔΔH‡ 4.06 kcal/mol). The barrier is also lower than
that calculated for s-TCO 20 (ΔΔG‡ –0.26 kcal/mol, ΔΔE‡ –0.92
kcal/mol, ΔΔE‡(ZPE) –0.59 kcal/mol, ΔΔH‡ –0.72 kcal/mol).
Under aqueous conditions, tetrazine ligations are accelerated
due to the hydrophobic effect. We previously had studied the
Scheme 5. (A) Ground state calculations on Si-TCH 19 show
that the <C-C=C-C dihedral angle is more distorted than that of
trans-cyclooctene or the conformationally constrained 20. (B)
Transition state calculations predict that Si-TCH 19 would be
more reactive than trans-cyclooctene and 20.
Scheme 6. Second order rate constants (k2) were determined
with a stopped-flow spectrophotometer under pseudo-first
order conditions using excess trans-cycloalkene and limiting
tetrazine. (A) The kinetics of the cycloaddition of 3,6-diphenyl-s-
tetrazine in MeOH at 25 °C with Si-TCH 7c was measured by
stopped flow spectroscopy with UV-vis monitoring at 295 nm,
and compared to previously reported rate constants for s-TCO
21, d-TCO 22 and trans-cyclooctene. (B) Stopped flow kinetics
with
a
TAMRA-3,6-dipyridyl-s-tetrazine conjugate were
monitored in 9:1 H2O:MeOH with monitoring by fluorescence
(Ex: 556 nm; Em: 576 nm). Data points are shown in red, and
the fit is shown in blue. (C) Second order rate constants (k2)
were determined by plotting kobs vs. the concentration of Si-TCH
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reactions of s-TCO, d-TCO and trans-cyclooctene with a water on’70 to monitor reaction progress. The fluorogenic reaction
soluble dipyridyl-s-tetrazine derivative under stopped flow enabled reaction monitoring at much lower concentrations
21
conditions in water with UV-vis monitoring.
In the most (down to 3 µM in tetrazine). As shown in Scheme 6B and 6C,
rapid example, a rate constant of 3.3 x 106 M–1s–1 was the reaction of 23 with Si-TCH 7c in 9:1 water:MeOH proceeds
measured for an s-TCO derivative.21 However, our attempts to at 25 °C with a second order rate constant k2 1.14 x 107 (+/– 5
conduct
a
similar measurement using Si-TCH 7c were x 105) M–1s–1. This is the fastest rate constant reported to
complicated because the reaction was complete before we date for a bioorthogonal reaction.
could collect data even with stopped flow monitoring. To
We also studied the in vitro and in vivo cycloaddition of SiTCH
and a green fluorescent protein with an unnatrual tetrazine-
containing amino acid (sfGFP-150Tet-v.2.0, referred to as GFP-
Tet), encoded via the procedure of Mehl and coworkers.71
Thus, 4-(6-methyl-s-tetrazin-3-yl)phenylalanine was site-
specifically introduced into a C-terminally hexahistidine-tagged
GFP (sfGFP-150TAG-His6) via orthogonal translation using the
evolved aminoacyl-tRNA synthetase MjRS/tRNACUA pair. Co-
expression of these components in E. coli resulted in the
amino acid-dependent synthesis of full-length recombinant
GFP-Tet (Scheme 7A). The reaction of GFP-Tet with
dienophiles is fluorogenic, and it is therefore possible to
determine the reaction kinetics by monitoring the increase in
GFP fluorescence.
enable the measurement, we synthesized
a fluorescent
tetrazine-TAMRA conjugate 23, and used fluorescence ‘turn-
Although GFP-Tet is highly reactive, with in vitro rates as fast
as 87,000 M–1s–1 toward sTCO, GFP-Tet is not as rapid as 23
due to the less reactive nature of the tetrazine. Kinetic
measurements were carried out with silver free Si-TCH 7c
,
which was obtained by treating Ag-complex 2c with NH4OH
and extracting with ether. The second order rate constant of
the reaction between Si-TCH 7c and GFP-Tet was determined
to be 250,000± 15,000M–1s–1 in PBS at rt (Scheme 7B). The
reaction was quantitative under these conditions as
determined by ESI-MS (Scheme 7C), and is the fastest rate
measured to date for GFP-Tet
— 2.9 times faster than
previously measured rate constant for sTCO.71 Si-TCH 18 was
also shown to display very rapid labeling of GFP-Tet when
carried out in live bacteria. The kinetics of the in vivo tetrazine
ligation were monitored in a suspension (PBS) of E. coli
overexpressing GFP-Tet by measuring the increase in whole-
cell fluorescence upon addition of 7c. At room temperature, a
–1
second-order rate constant of 155,000 ± 20,000 M–1
S
was
measured for the in vivo reaction, which is 62% as rapid as the
in vitro ligation. The modest reduction in rate is in line with
previous observations with TCO-based dienophiles.21,71,72
Quantitative determination of the bioorthogonal reaction was
verified by cell washing, lysis, purification by IMAC, and
analysis by ESI-MS. Thus, Si-TCH 7c is capable of crossing the
bacterial cell membrane and engaging in rapid, high yielding
conjugation inside a living cell.
Scheme 7. (A) GFP-Tet reacts with 7c to give a Diels-Alder
adduct. Because the tetrazine quenches GFP fluorescence, the
reaction progress can be measured by monitoring fluorescence
increase. (B) Stopped-flow kinetic data for the reaction of GFP-
Tet (50 nM) with 7c (1.96 μM) at 25 °C in PBS with fluorescence
monitoring (Ex: 488 nm; Em: 506 nm). Data points are shown in
green, and the fit is shown in black. A second order rate constant
(k2)was determined by plotting kobs vs. the concentration of Si-
TCH 7c. (C) Quantitative determination of the cycloadduct was
confirmed by ESI-MS. Deconvoluted Mass spectra of GFP-Tet
(grey) and the cycloadduct (green) are displayed and show the
expected mass shift of 170 Da. Smaller peaks in each spectrum
correspond to proteins that are truncated by a methionine.
The reactivity and specificity of the SiTCH reagent with
tetrazines in live mammalian cells was evaluated using the
HaloTag platform56, which we have previously used to
benchmark the efficiency of various bioorthogonal reactions in
the cellular environment. We have previously shown that
trans-cycloalkene-AgNO3-complexes
liberate
AgNO3
immediately in cell media due to the high NaCl content, and
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perform identically to their metal-free analogs in cell labeling was quenched at the various time points by chasing with
experiments.56 Thus, we synthesized chloroalkane derivatives excess non-fluorescent tetrazine-amine (method 1) or TCO-
of SiTCH (AgSiTCH-Halo) and methyl-tetrazine (MeTz-Halo
)
amine (method 2) (Fig S27), and in-gel fluorescence was used
and used these clickable HaloTag ligands to covalently label to quantify conversion vs time.
HaloTag protein expressed in HEK 293T cells with the clickable
tag. In a competitive pulse-chase experiment, it was shown We found that the reaction of SiTCH-Halo with MeTz-BODIPY
that 10 µM of these HaloTag ligands completely blocked was complete within 15 minutes (Scheme 8B, Method 2).
incorporation of BODIPY-Halo substrate (Fig S25), and Interestingly, the reaction appeared to be slower with the
therefore this concentration was used for subsequent reverse pairing where SiTCH-BODIPY was reacted with MeTz-
experiments. Next we evaluated the tetrazine ligation of Halo and did not reach saturation until 90 minutes (Scheme
SiTCH in mammalian cells by reacting the AgSiTCH-Halo and 8B, Method 1). We believe this is due to the suboptimal
MeTz-Halo protein conjugates with the corresponding MeTz- permeability and nonspecific protein binding of AgSiTCH-
BODIPY or AgSiTCH-BODIPY fluorescent probes (300 nM) for BODIPY which results in lower free concentrations available
different times (2-90 min) (Scheme 8, Fig S26). The reaction for the reaction. Compared to MeTz-BODIPY or fluorescent 5-
Scheme 8. Reactions of AgSiTCH with 3-methy-6-s-tetrazine derivatives in live HEK 293T cells expressing HaloTag. (A) Cells were
treated with 10 µM MeTz-Halo (method 1) or AgSiTCH-Halo (method 2) for 30 min, followed by two 30 min washings and treatment
with 300 nM AgSiTCH-BODIPY (method 1) or 300 nM MeTz-BODIPY (method 2) for the indicated time. (B) Analyzed by in-gel
fluorescence and Western blot for each method: in-gel fluorescence is shown on the top panel, and Western blot with an anti-HaloTag
antibody is on the bottom panel. (C) Time course for the two reactions in B. In-gel fluorescence intensity from n = 3 independent
replicates was normalized by the corresponding Western blot signal, and was fit to a one-phase exponential equation. Data were
plotted as mean ± SEM.
8 | J. Name., 2012, 00, 1-3
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ARTICLE
hydroxy-trans-cyclooctenes,56 AgSiTCH-BODIPY also resulted
in more nonspecific protein labeling (Fig S26). However,
compared to in-gel fluorescence with
a TAMRA-labeled
bicyclononyne (BCN),56 AgSiTCH-BODIPY appears to be more
selective.
To investigate in vivo stability of incorporated SiTCH probes in
the intracellular environment, we compared HaloTag protein
tagging by AgSiTCH-Halo to the previously described Ag-sTCO-
Halo 56
Subsequently the TAMRA-Tz fluorescent probe was
.
attached via the tetrazine ligation (Scheme 9A). As a
benchmark, we also compared directly incorporated TAMRA-
Halo without
a
second bioorthogonal step.
In these
experiments, HaloTag-transfected cells were treated with 10
µM HaloTag ligands for 30 min, followed by two 30 min wash
periods. After this initial 90 min exposure to cells, time course
experiments were carried out to test the intracellular stability
of the sTCO- and SiTCH-labeled HaloTag proteins. Thus, cells
were allowed to incubate for up to 24 additional hours, and
cells initially tagged by AgSiTCH-Halo or Ag-sTCO-Halo were
then treated with TAMRA-Tz at the indicated time points. Loss
of fluorescence intensity relative to the TAMRA-Halo
benchmark indicated instability of the bioorthogonal protein
tag.
Compared to the TAMRA-Halo ligand, the Ag-sTCO-Halo
tagged protein displayed 84% fluorescence intensity when the
TAMRA-Tz probe was attached immediately after the 30 min
HaloTag-labeling and two 30 min wash periods (Scheme 9B, Fig
S28 and S29). By contrast, AgSiTCH-Halo showed only 50%
fluorescence intensity at this initial timepoint.
After
incubation for 7 h and 24 h, Ag-sTCO-Halo tagged protein
showed 61% and 43% fluorescence intensity—very similar to
the TAMRA-Halo benchmark. However, the AgSiTCH-Halo
tagged protein displayed only 10% fluorescence after 7 h, and
negligible fluorescence after 24 h. The reduced cellular
labeling efficiency with AgSiTCH-Halo could also be observed
in live cell images according to the published protocol (Scheme
9C, Fig S30).56 Specifically, HeLa cells overexpressing Halo-H2B-
GFP were labeled with AgSiTCH-Halo or Ag-sTCO-Halo, and
after two 30 min wash periods, treated with TAMRA-Tz for
imaging. In both cases, specific fluorescent signals were
observed in the nucleus with good colocalization with GFP
signals. When AgSiTCH-Halo was used, however, the
fluorescent intensity was only about half of that achieved with
Ag-sTCO-Halo, confirming that the reduced stability of SiTCH
in cells resulted in lower labeling efficiency. The lower
labelling efficiency with AgSiTCH-Halo is likely due to trans-to-
cis deactivation of the cycloalkane in the intracellular
environment. Previously, we have studied the isomerization of
trans-cyclooctenes under various conditions, and have shown
that isomerization of trans-cyclooctenes likely occurs via
radical mechanism that can be promoted by high thiol
Scheme 9. Live cell stability and imaging studies of SiTCH-
Halo. (A) In the stability experiment, HEK293T cells expressing
HaloTag were treated with 10 µM Ag-sTCO-Halo or AgSiTCH-
Halo for 30 min, followed by two 30 min wash periods. Control
cells were treated with 2 µM TAMRA-Halo. At 0, 7, and 24 h
time points, 2 µM TAMRA-Tz was added and allowed to react
for 1 h before analysis. (B) Cellular stability of Ag-sTCO-Halo and
AgSiTCH-Halo. In-gel fluorescent intensities were normalized by
the corresponding Western blot signals, and were subsequently
normalized by the value of TAMRA-Halo at time 0. Data were
plotted as mean ± SEM. (C) Live cell images of HeLa cells
expressing Halo-H2B-GFP. Cells were labeled with 10 µM Ag-
sTCO-Halo or AgSiTCH-Halo for 30 min, followed by two 30 min
wash periods, before the treatment with 1 µM TAMRA-Tz for 5
min. The reactions were quenched with 100 µM TCO-amine for
10 min, followed by two 30 min wash periods prior to addition
of 8 µM Hoechst 33342 to visualize the nuclei and confocal
imaging. Signals of Hoechst, GFP, and TAMRA were shown in
blue, green, and red, respectively. Colocalization was
demonstrated in composite images of GFP and TAMRA. Scale
bar =25 µm.
concentrations.
Consistent with the faster in cellulo
deactivation of SiTCH-Halo relative to sTCO-Halo, we observe
that SiTCH 7b (30 mM) completely isomerizes in 2 h at 22 °C in
CD3OD with mercaptoethanol (30 mM), whereas as control
sample without thiol was >98% stable under these conditions.
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There are no conflicts to declare
At –17 °C, 7b isomerizes more slowly in the presence of 30 mM
mercaptoethanol, with 47% isomerization after 24 h. By
comparison, the trans-cyclooctenes d-TCO, s-TCO, and oxo-
TCO are much more stable toward thiol promoted
isomerization. In CD3OD with mercaptoethanol (30 mM) at
room temperature, s-TCO (30 mM) isomerized only after an 8
hour induction period, with complete conversion to the cis-
isomer after 4 additional hours.54 In a similar experiment with
d-TCO (30 mM), the induction period was 10 hours. After the
induction period, there was 42% isomerization after 4 hours,
and 92% isomerization after 14 hours.56 5-OxoTCO (25 mM) in
the presence of mercaptoethanol (25 mM) showed only 8%
isomerization in CD3OD over a 22 hour period at room
temperature.72
Acknowledgements
This work was supported by NIH R01EB014354, R01DC014461,
and NSF DMR-1506613. Spectra were obtained with
instrumentation supported by NIH grants P20GM104316,
P30GM110758, S10RR026962, S10OD016267 and NSF grants
CHE-0840401, CHE-1229234, and CHE-1048367. We thank
Joshua Judkins for preliminary cellular bioorthogonal labeling
experiments, Dr. Glenn Yap and Casey Rowland for x-ray
crystallography, Shuyu Xu and He Zhang for IR(ATR)
characterization and Prof. Dr. Colin Thorpe for insightful
discussions.
Together, the labeling experiments in bacteria and HEK 293T
cells show that SiTCH derivatives can serve as useful probe
molecules in the cellular environment, as the unprecedented
speed of the bioorthogonal reactions of SiTCH are much more
rapid than competing deactivation pathways. However, the
utility of SiTCH derivatives as protein tagging molecules, where
extended incubation in the cellular environment takes place
prior to bioorthogonal reactivity, appears much more limited
in utility plausibly due to alkene isomerization in the cellular
environment.
Notes and references
‡ Footnotes relating to the main text should appear here. These
might include comments relevant to but not central to the
matter under discussion, limited experimental and spectral data,
and crystallographic data.
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Conclusions
In conclusion, AgNO3 complexes of trans-cycloheptene and
trans-1-sila-4-cycloheptene derivatives have been prepared via
a flow photochemical synthesis, using a new low temperature
flow photoreactor to enable the synthesis of carbocyclic TCH
derivatives. TCH•AgNO3 complexes can be handled for brief
periods at rt and stored for weeks in the freezer (–18 °C). Si-
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Conflicts of interest
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R
R'
Si
R
x-ray structure of silacycle
applications in synthesis
bioorthogonal reactions >107 M–1s–1
protein labeling in live cells
AgNO3
AgNO3
stable metal complexes