Bioorthogonal double-fluorogenic siliconrhodamine probes for intracellular super-resolution microscopy

Article abstract of DOI:10.1039/c7cc02212c

A series of double-fluorogenic siliconrhodamine probes were synthesized. These tetrazine-functionalized, membrane-permeable labels allowed site-specific bioorthogonal tagging of genetically manipulated intracellular proteins and subsequent imaging using s

Full text of DOI:10.1039/c7cc02212c

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc
first methanofullerene derivatives of Sc
3
N@C2n (2n
=
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3
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Bioorthogonal Double-Fluorogenic Siliconrhodamine Probes for
‡
Intracellular Super-resolution Microscopy
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
b
b
a
E. Kozma, G. Estrada Girona, G. Paci, E. A. Lemke and P. Kele*
DOI: 10.1039/x0xx00000x
www.rsc.org/
5
,6
A series of double-fluorogenic siliconrhodamine probes was by the use of fluorogenic species.
synthesized. These tetrazine-functionalized, membrane-
Our continuing efforts to develop fluorescent probes that
permeable labels allowed site-specific bioorthogonal tagging of combine the above listed features have resulted in several
genetically manipulated intracellular proteins and subsequent bioorthogonally applicable fluorogenic probes with various
7
imaging using super-resolution microscopy.
excitation/emission characteristics. Lately, our attention
turned to tetrazine quenched fluorescent frameworks as
Recent years have brought substantial progress in the field of tetrazines can act both as bioorthogonal handles and quencher
7
d
super-resolution microscopy (SRM) enabling the exploration of moieties. Tetrazines can exert their quenching effect either
biomolecular processes in the sub-diffraction range. Although by Förster-resonance energy transfer (FRET) or through-bond
1
8,9
the emerged SRM techniques have revealed fine details of live energy transfer (TBET) mechanisms. FRET-based fluorogenic
organisms, there is still room for further improvements. probes are limited to tetrazine-fluorophore pairs where the
Nowadays, the unavailability of dyes suitable for site-specific absorption band of the tetrazine (typically between 520-540
tagging of intracellular biomolecules is considered as the nm) overlaps with the emission band of the fluorophore. TBET
2
biggest limitation of SRM techniques. Ideal small-sized organic based systems, on the other hand, are not constrained by the
fluorophores can be synthetically tailored in order to meet the emission band of the fluorescent unit and, in theory, any kind
criteria necessary for intracellular SRM imaging of live cells. of fluorophore-tetrazine pairs are suitable upon proper design.
Such criteria are: (a) both the labels themselves and the Moreover, TBET-based quenching is much faster and more
chemistry applied for tagging should be biocompatible, (b) efficient, thus enhancement of the fluorescence signal can
their photophysical characteristics should allow minimal reach up to 11,000-fold upon IEDDA reaction with
9
a
background and autofluorescence in order to result in high dienophiles. The tetrazine-based TBET-quenching approach
signal-to-noise (S/N) ratio, (c) their fluorescence should be has already been applied to various fluorophores, yet, it has
bright and not or minimally impaired by photobleaching and not been performed on SRM compatible dyes. However,
9
(d) they should be able to transit through membranes. As of techniques like SRM would greatly benefit from the high
the chemistry applied for installation of such probes, signal-to-noise ratio offered by TBET-mechanism.
bioorthogonal reactions are extremely useful as they are fully
Siliconrhodamine (SiR) is a widely used membrane-permeable
NIR dye for SRM applications. Besides the high photostability
and brightness, the unique environment-dependent
fluorescence of carboxy-SiRs due to a polarity dependent
lactone-formation offers an appealing opportunity to
3
biocompatible. Among these transformations, inverse
electron demand Diels-Alder (IEDDA) reactions between
tetrazines and strained unsaturated ring systems enable the
10
4
fastest reactions. In order to minimize phototoxicity and
autofluorescence, far-red/near-infrared (NIR) emitting
fluorophores (650-900 nm) are especially well suited, while
background fluorescence of unreacted probes bound non-
specifically to hydrophobic surfaces can be reduced efficiently
distinguish between specific and non-specific labeling,
(
10
polarity-based fluorogenicity). When bound to polar protein
surfaces, carboxyl-SiRs exist in a fluorescent zwitterionic form,
while upon non-specific adhesion to hydrophobic surfaces, it
closes to non-fluorescent spirolactone (Figure 1). SiR
derivatives were applied in voltage sensing, in vivo tumor
imaging, photodynamic therapy and multicolour imaging
1
1
studies as well. Despite their wide applications, there are
only a few examples of SiR-based probes for site-specific
1
0a,12,13
labeling of proteins.
carboxyl-SiR fluorogenic probes were reported yet.
Furthermore, no TBET-based
Herein we report the first set of bioorthogonally applicable,
NIR-emitting, membrane permeable, double fluorogenic
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probes suitable for SRM imaging with exceptionally high turn- and phenyl-linked SiR-tetrazine
on rates compared to previously reported tetrazine-based NIR moderate to good yields (11-48%). To improve reaction rate of
Journal Name
(
15), respectively, with
9
d
dyes. We demonstrate the power of the developed SiR-dyes alkene-linked SiR-tetrazine in IEDDA, we designed a derivative
in site-specific labeling of intracellular skeletal proteins and with trifluoromethyl-phenyl substituent. Electron-withdrawing
subsequent SRM imaging applications.
groups are known to markedly increase the reactivities of
tetrazines in IEDDA reactions. To this end, we synthesized
4
a
TBET-based fluorogenic probes are composed of a donor
fluorophore and an acceptor tetrazine unit that are linked
together with a conjugated but electronically decoupled
trifluormethyl-tetrazine (
Pinner-synthesis and subsequent O-mesylation.
reaction of tetrazine with compound 6 proceeded with
9
) in two steps via Zn(OTf)
2
catalyzed
1
5b
Heck-
1
4
9
(
twisted) linker. The tetrazine moiety can be installed onto
moderate yields (32%). It is known that different N-alkylation
patterns on the rhodamine core can markedly alter spectral
characteristics and brightness of probes. Rotation about the
the phenyl group of SiR directly or via alkene, alkyl and phenyl
linkers. Former examples showed that incorporation of the
tetrazine through direct conjugation or via alkyl-linkers to
1
6
9
c,d,f
C-N bond in -NMe substituted rhodamines leads to faster
2
decay of the excited state thus lowered quantum yield. Thus
fluorophores is less efficient.
Therefore we decided to
apply alkene and phenyl linkers to connect the tetrazine to
carboxy-SiR. Devaraj and co-workers and also our group have
recently reported Heck- and Suzuki-type cross-coupling
reactions to connect methyl-substituted tetrazines to
we synthesized -NEt
starting from Br-SiR derivative
bond rotation compared to –NMe
2
substituted derivatives (12
, which have restricted C-N
derivatives.
, 14, 16)
7
2
7
d,9b
fluorophores.
substituted intermediate
derivatives proceeds through a Si-xantone intermediate which containing 0.01% SDS to prevent aggregation of the dyes.
is reacted with aryl lithium reagents via nucleophilic Excitation maxima were found to be at around 645 nm and
To this end we have synthesized Br- Next, we characterized the spectral properties of SiR-
6. The general synthetic route for SiR tetrazines 11 16. Measurements were conducted in water
-
1
0a
1
0a
addition.
Through this route, however, introduction of 655 nm, and fluorescence emission was detected at around
halogen-substituted aromatic rings is problematic. Therefore 665 nm and 670 nm for the dimethylamino- and diethylamino-
we proceeded via an aldehyde condensation reaction derivatives, respectively (Figure S1, see ESI). We measured the
developed by Wang et al. (Scheme 1) and acquired Br-SiR fluorogenic properties of the probes in an IEDDA reaction with
1
.
5a
7d
derivative
6
OxTCO (17) (Figure 1, S2, Table 1). To our delight, all
derivatives showed much larger fluorescence enhancements
(
FE) than formerly reported fluorogenic tetrazine NIR dyes (cf.
9
.6x). We propose that this is attributed to the double
d
5
fluorogenic nature of our SiR dyes. Among dimethylamine
derivatives, phenyl-linked 15 showed the highest turn-on ratio
(
31x). As expected, ethyl substitution on the N-atom improved
turn-on rates of the dyes, and up to 49-fold increase was
observed. Although energy transfer occurs from the excited
state, interestingly, the extinction coefficients were measured
to be lower than ε of parent fluorophores (Table 1).
Table 1. Photophysical properties of SiR dyes 11-16.
-
1
-
λ
exc(max)
a
λ
[nm]
em(max)
a
εmax [M cm
a,c
F
d
F
e
Dye
1
a,b
φ
∆φ
FE
[
(
(
(
(
nm]
]
6
43
645)
53
652)
47
648)
55
654)
45
665
19,700
(19,200)
20,600
0.016
(0.34)
0.015
(0.80)
0.028
(0.26)
0.037
(0.27)
0.012
(0.34)
0.015
(0.53)
1
1
1
1
1
2
3
4
21
50
9
22
49
6
(667)
671
6
(672)
662
(19,800)
26,200
6
(665)
673
(22,500)
29,000
6
7
8
(672)
660
(25,600)
23,000
6
Scheme 1. Synthesis of SiR dyes 11
C, 2 h; ii) SiCl Me , rt, 16 h; b) CuBr
Cy) NMe, DMF, 50 °C, 40 min, MW; d) Pd
10 °C, 16 h; e) PdCl (dppf), Cs CO , 1,4-dioxane, 90 °C, 16 h.
-
16. Reaction conditions: a) i) n-BuLi, THF, -78
, 140 °C, 16 h; c) Pd (dba) , QPhos,
(dba) , QPhos, DIPEA, 1,4-dioxane, 90-
15
16
28
35
31
40
°
(
1
2
2
2
2
3
(644)
651
(660)
671
(21,600)
26,000
2
2
3
2
2
3
(652)
(670)
(23,900)
a
values refer to tetrazine-dyes while values in parenthesis are that of the
We positioned the Br-substituent at the 6-position as previous
reports showed superior fluorogenic properties of 6-
b
c
respective conjugates with 17; measured at absorption maxima of the species;
d
F
cresyl violet in MeOH was used as quantum yield reference (φ = 0.53); quantum
e
substituted 9d,rfhodamine derivatives compared to 5- yield ratios of conjugates with 17 and tetrazine-dyes in 0.1% SDS in water;
with fluorescence intensity enhancements upon reaction with 17 in 0.1% SDS in water
) and methyltetrazine at products emission maxima.
substitution. Pd-catalyzed cross-coupling reactions of
methyl vinyltetrazine precursor
phenylboronic acid pinacol ester (10) resulted in alkene- (11
6
(
8
)
2
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Figure 1. a) Fluorogenicity of SiR dyes: IEDDA reaction between 11 and 17 resulting in fluorescence turn-on upon the formation of conjugate 18 due to the
transformation of quenching tetrazine moiety into dihydropyridazine during reaction. b) Corresponding fluorescence emission spectra of free dye 11 (black) and
conjugate 18 (red) showing fluorescence turn-on upon reaction. c) Polarity-dependent fluorogenicity of SiR dyes: conjugate 18 is present in fluorescent open,
zwitterionic form in polar environment, and in non-fluorescent closed spirolactone form in apolar solvents. d) Normalized integral of fluorescence spectra of 18 in
water-dioxane mixtures as a function of dielectric constant. Conjugate 18 is in fluorescent zwitterionic form in solvents with dielectric constant >50.
The second order rate constants were also determined and dominated the spectrum (Figure S4). As a consequence, in
were in good agreement with k values of previously reported solvents with dielectric constants <50 conjugate 18 was non-
2
methyl-tetrazine derivatives with similarly reactive TCO* (for fluorescent while fluorescence gradually reinstated with
As expected, trifluoromethylphenyl increasing polarity (Figure 1).
1
7
values see ESI).
derivatives, 13 and 14 reacted much faster than methyl
derivatized congeners with OxTCO (17) (Figure S3). Due to the
lower electron density of the tetrazines in 13 and 14 IEDDA
reactions went to completion within minutes compared to
methyl substituted probes where reaction required ca. 1.5 h.
With the SiR-dyes in hand, showing exceptional fluorogenicity
in the NIR region, we were eager to evaluate their
performance in the cellular environment. We sought to utilize
genetic code expansion (GCE) technology for site-specific
labeling with the newly developed double-fluorogenic probes.
We and others have recently shown that non-canonical amino
acids (ncAA) with strained alkene and alkyne moieties can be
genetically encoded in mammalian cells using the pyrrolysine
Pyl
18
tRNA /PylRS pair from Methanosarcina species. We used
1
16TAG
the previously reported skeletal protein vimentin
mOrange to test the intracellular labeling ability of the
-
1
3,18e
Pyl
AF
dyes.
encoded commercially available cyclooctynylated-lysine (Lys(
Using orthogonal tRNA /PylRS pair we genetically
ε-
endo
116TAG
N-BCN ), Figure S5) into vimentin
performed live cell labeling experiments with dyes 11
mammalian cells. Clear live cell labeling was observed with dye
-mOrange and
-16 in
1
3
1
7
and 12 in concentrations as low as 1.5 µM within 10 min at
°C (Figure 2, for additional images at different
concentrations and labeling times see Figure S6, S7, and for a
comparison of different post-labeling washing time see Figure
S8). The labeling was specific with no strong background
fluorescence (as quantified by channel colocalisation in Figure
BCNendo
Figure 2. Confocal images of live cell SiR-labeling of vimentin
–mOrange S9). Less efficient labeling was observed with phenyl-linked
with dyes 11 (a,c,e) and 12 (b,d,f). Left to right: reference channel (mOrange- dyes 15 and 16, which is probably due to their slower kinetics
cyan) (a,b), labeling channel (c, d) and overlay (e,f). Labeling was performed at 3
(
3
Figure S10). Although CF -dyes 13 and 14 showed the fastest
µM for 10 min at 37 °C.
labeling properties in vitro, they did not give any specific
labeling in vivo (results not shown). We attribute this to the
low polarity of these dyes, which probably impairs their ability
to cross cell-membrane or to label the protein specifically.
We also investigated the polarity dependent absorbance of the
Diels-Alder product of alkene-SiR (18). In agreement with
previous findings, in solvents with lower dielectric constants,
absorption maximum, characteristic of the spirolactone form
at 290 nm was dominant. In polar solvents, however, the
zwitterionic form with a characteristic absorption at 645 nm
To date, there are only a few reports on bioorthogonally
applicable, fluorogenic tetrazine-mediated intracellular
labeling inside living cells and subsequent SRM. Among
them, to the best of our knowledge, there are no reports of
1
3,18e,19
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site-specific labeling with TBET-based fluorogens. We set out
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resolution compared to the reference channels (Figure 3A-D,
and Figure S11).
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siliconrhodamine-tetrazine probes with improved kinetics and
enhanced fluorescence turn-on ratios in the NIR region upon
inverse electron demand Diels-Alder reactions. We have
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of the SiR-tetrazine core on labeling efficiency by confocal
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Figure 3. SRM imaging of vimentin
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A,C (cyan) and B,D (magenta) show the mOrange and SiR reference channels for
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Notes and references
‡
Present work was supported by the Hungarian Scientific
Research Fund (OTKA, NN-116265) and the “Lendület” Program
of the Hungarian Academy of Sciences (LP2013-55/2013). E.K. is
grateful for the support of The New National Excellence Program
of The Ministry of Human Capacities (Hungary). EAL
acknowledges the SPP1623 and SFB1129 for funding.
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