Silicon-rhodamine isothiocyanate for fluorescent labelling

Article abstract of DOI:10.1039/d0ob02016h

An efficient synthesis for silicon-rhodamines was developed, enabling the preparation and evaluation of silicon-rhodamine isothiocyanate (SITC) as a novel tool for facile fluorescent labeling. Ease of use in conjugation to amino groups, high stability and excellent photophysical properties are demonstrated. SITC-actin was found to be neutral to F-actin polymerization induction and well suited for high resolution fluorescence microscopy.

Full text of DOI:10.1039/d0ob02016h

Organic &
Biomolecular Chemistry
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Silicon-rhodamine isothiocyanate for fluorescent
labelling†
Cite this: Org. Biomol. Chem., 2021,
9, 574
1
a
b
c
a
Veselin Nasufović,
Christoph Kaether,
Hans-Dieter Arndt
Patrick Then, Fabian Dröge, Michael Duong,
Received 2nd October 2020,
Accepted 21st December 2020
d
b,c
b,c
aBenjamin Dietzek,
Rainer Heintzmann
and
*
DOI: 10.1039/d0ob02016h
rsc.li/obc
An efficient synthesis for silicon-rhodamines was developed, a particularly bright fluorophore with high extinction coeffi-
2
4
enabling the preparation and evaluation of silicon-rhodamine iso- cient and quantum yield. Its favourable “spiro” to “open”
thiocyanate (SITC) as a novel tool for facile fluorescent labeling. state equilibrium enhances cell permeability and can make
Ease of use in conjugation to amino groups, high stability and probes fluorogenic, rendering SiR a useful tool in super-resolu-
1
7,24–26
excellent photophysical properties are demonstrated. SITC-actin tion microscopy.
Although SiRs have been proven to be
was found to be neutral to F-actin polymerization induction and very useful fluorescent labels, syntheses of SiR-COOH (2) and
well suited for high resolution fluorescence microscopy.
its variants are challenging and are hampered by low yields.
Furthermore, additional approaches for introduction of groups
suitable for (bio)conjugation are desirable. Herein, we report
an improved, scalable protocol for silicon-rhodamine prepa-
ration, as well as a regioselective synthesis and first appli-
cations of the novel SiR isothiocyanate (SITC, 3) that suggests
diversified analogs to be easily accessible.
With its first synthesis by Adolf von Baeyer in 1871, fluorescein
1
was applied as a staining reagent. By employing amino
2
phenols instead of resorcinols, rhodamines were prepared.
Fluorescein-isothiocyanate (FITC, 1, Fig. 1) was subsequently
developed for specific labelling, combining ease of synthesis
3
–5
with ease of use. The spectral properties of FITC conjugates
Initially, SiRs have been synthesized from 10-sila-anthrones
5
mimic fluoreceine. FITC reacts with a variety of nucleophiles,
by nucleophilic addition of metallated terephthalic acid frag-
but in water it is fairly selective for free amino groups in pep-
27,28
ment surrogates.
Condensation chemistry similar to fluor-
6
tides and proteins. FITC has been the first fluorescent label
29
escein and rhodamine synthesis proved to be low yielding.
Enhanced yields were reported by inverting polarity in using
3
–5
that was successfully conjugated to antibodies and was used
7
–12
extensively.
It keeps to be widely applied in immuno-
labelling of biomolecules, surfaces, or in
1
3,14
15
fluorescence,
1
6
FRET couples. Unfortunately, the common synthesis of FITC
is not regioselective, but forms regioisomers (Fig. 1).
More recently, isoelectronic exchange of oxygen for other
heteroatoms has provided fluorophores with improved pro-
perties such as high absorptivity, red-shifted absorption and
1
7
emission, enhanced photostability, and cell permeability. In
1
8–22
cellulo and in vivo experiments were realized,
as well as
2
3
FRET experiments. Among them, silicon-rhodamine (SiR) is
a
Friedrich-Schiller-University, Institute of Organic Chemistry and Macromolecular
Chemistry, Humboldtstr. 10, 07743 Jena, Germany. E-mail: hd.arndt@uni-jena.de
b
Leibniz-Institut of Photonic Technologies (Leibniz-IPHT), Albert-Einstein-Str. 9,
0
7745 Jena, Germany
c
Friedrich-Schiller-University, Institute of Physical Chemistry, Helmholtzweg 4,
0
7743 Jena, Germany
d
Leibniz-Institute on Aging – Fritz Lipmann Institute (FLI), Beutenbergstr. 11,
0
7745 Jena, Germany
Fig. 1 Fluorescein isothiocyanates, silicon-rhodamine, and SITC: struc-
tures and synthesis planning. 5a, 6a: Z = 4,4-dimethyloxazolin-2-yl; 5b,
6b: Z = OTIPS.
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0ob02016h
574 | Org. Biomol. Chem., 2021, 19, 574–578
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3
0,31
dimetallated diarylsilanes electrophiles.
In order to access
The reactivity of SITC was briefly investigated. SITC is stable
the target compounds even more effectively, we envisioned to on storage and reacts cleanly with amine nucleophiles in
desymmetrize the para-substituted aryl oxazolines by organic solvents (DMF, CH Cl , THF) to give thiocarbamates in
directed ortho metalation and acylation (→5), which could good yield (Scheme S5, Fig. S3†). SITC is surprisingly stable in
6
2
2
3
1
directly lead to SiR derivatives from dibromide 4. Indeed, aqueous medium (pH 9 carbonate buffer, 24 h, 25 °C or 37 °C,
cheap terephthalaldehyde (7) smoothly underwent double N,O- Fig. S1 and 2†), and reacts with amines in water rather slowly,
acetalization followed by oxidation with NBS in one pot, to give even at 37 °C (Fig. S2†). Despite slow conversion, selective con-
3
2
bisoxazoline 6 in 91% yield (Scheme 1). ortho-Metalation by jugation with primary amines could be observed (Fig. S4†).
3
3,34
employing the TMPMgCl·LiCl reagent
followed by acyla- Importantly, SITC was found to be stable to thiols, DMAP, and
tion with benign diethyl pyrocarbonate provided ester 5a in TCEP in aqueous buffer (Fig. S5†).
very good yield. The 1,5-dilithium reagent derived by double
Spectral properties of SITC conjugates and the novel SiR
Li–Br exchange of dibromide 4 smoothly added to ester 5a. derivatives were investigated next. For this purpose, SITC was
Acidic dehydration and hydrolysis of the oxazoline protecting conjugated to methyl ε-aminocaproate (SITC-Ahx, Scheme S5†)
groups gave SiR-COOH (2). Importantly, in contrast to other and compared to SiRs 2, 8, 9, and 10 (Fig. 2, Table 1).
reported protocols, this procedure was robustly reproduced by Absorbance and emission were found maximal around pH 3
us on the >1 g scale (50% yield from 7, see ESI, Fig. S7†).
(Fig. 2c), where a xanthene monocation likely predominates in
Diacid 2 was then regioselectively converted to Boc-pro- its open form (ESI, Scheme S1†). Wavelength maxima of
tected aniline 8 by treatment with stoichiometric DPPA and absorption and emission lie in the deeply red region (around
tBuOH (63% yield). This regioselective transformation is likely 645 and 665 nm, respectively), only moderately influenced by
enabled by a spiro-lactone intermediate present at basic con-
ditions (cmp. Fig. 1). Acid-mediated deprotection gave aniline
9
(79% yield) that was transformed to isothiocyanate 3 (SITC)
by using thiophosgene (72% yield). This sequence (Scheme 1,
e–g) could be executed without chromatography of intermedi-
ates 8 and 9 in an enhanced overall yield (64% from 2).
Unfortunately, a more direct introduction of amino groups
failed (see ESI, Schemes S2 and 3†). However, intermediate 8
was alternatively accessed by preparing the phenol 10 from
TIPS-protected para-hydroxybenzaldehyde via oxazoline 6e,
similar to 2 (4 steps, see ESI, Scheme S4†). Triflation and
Buchwald–Hartwig-type carbamoylation then gave carbamate 8
by avoiding hazardous acyl azide intermediates. Either route
(azide-free or -containing) gave SITC as a single regioisomer.
Fig. 2 Normalized UV-Vis absorption (a) and fluorescence emission
spectra (b) of SiR compounds in aqueous phosphate-citrate buffer with
20 vol% DMSO at pH 3.14, aqueous TBS buffer with 20 vol% DMSO at
pH 7.4 (SiR-COOH, 2) or aqueous solution with 1.7 vol% DMSO adjusted
to pH 3 (SiR-OH, 10), using a concentration range from 0.1 µM to
100 µM; (c) pH dependent UV-Vis absorption and fluorescence emission
maxima of the SITC-Ahx conjugate in aqueous solutions with pH
ranging from 1.36 to pH 10.33 supplemented with 10 vol% DMSO (for
spectra see Fig. S6 in the ESI†).
Scheme 1 SiR-COOH and SITC synthesis. Reagents and conditions: (a)
-Amino-isobutanol, mol. sieves 4 Å, CH Cl , 25 °C, 16 h, then: NBS, 3 h,
1%; (b) TMPMgCl·LiCl, 25 °C, 4 h, then: DEPC, −15 °C to 25 °C, 3 h,
7%; (c) 4, t-BuLi, −78 °C, 30 min then: 6, to 25 °C, 16 h; (d) 6 M HCl,
6 h, 68% (2 steps); (e) DPPA, DIPEA, toluene, t-BuOH, 90 °C, 16 h, 63%;
f) TFA/CH Cl (3 : 7), 0 °C, 2 h, 79%; (g) Cl CvS, NEt , CH Cl , 25 °C,
h, 72%; (h) Tf O, NEt , CH Cl , 0 °C, 1 h; (i) Pd(dppf)Cl , Xantphos,
CO , H NCOOtBu, 1,4-dioxane, 100 °C, 18 h, 29% (2 steps).
2
9
7
3
(
1
Cs
2
2
2
2
2
3
2
2
2
3
2
2
2
2
3
2
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Table 1 Spectral properties of synthesized silicon–rhodamines
2
8
9
3
10
SITC-Ahx
λ
λ
abs (max)/nm
645 652
666 669
650
672
16
651 645 654
673 665 670
em (max)/nm
ε/10 L mol cm−1 67
3
−1
26
15
13
46
em
Φ
0.41 0.33
0.04
0.31 n.d. 0.39
pK
a
n.d. 2.4, 4.1 2.7, 4.4 n.d. n.d. 2.3, 4.4
the aryl substituent, with a small bathochromic shift found for
the SITC conjugate. With exception of (basic) aniline 9 and
phenol 10, absorption and quantum yield of emission were
4
−1
−1
em
high (ε = 2.6–6.7 × 10 L mol cm ; ϕ = 0.33–0.41; Table 1).
In very acidic medium (pH < 2) the absorbance of SITC
drops, suggesting the formation of a deconjugated, non-fluo-
rescent spiro-dication (ESI, Scheme S1, Fig. S6†). By contrast,
considerable absorption and emission is still found when the
medium gets less acidic (pH > 4). Here, the zwitterionic
neutral form predominates, equilibrating between fluorescent
open and non-fluorescent closed states, influenced by the
local environment. This property is believed to facilitate cell
permeability and fluorogenicity of SiR conjugates in cell
Fig. 4 Fluorescence microscopy images of fixed HeLa-cells. Cells were
stained with SITC-actin (a and b) or SiR-Actin (c and d), imaged in the
2
6
microscopy, and is retained for SITC. Notably, spectral pro- red channel, and with DAPI, imaged in the blue channel. a + c: widefield,
perties of SITC conjugates are constant at biologically relevant b + d: SIM images of the same area; c(probe) = 300 nM, 3 h; see ESI† for
pH (5–11), rendering them useful for a wide range of appli-
experimental conditions. Scalebar is 10 µm.
cations in biochemistry and biology.
Conjugation of SiR-COOH (2) to an optimized natural
over longer time (6–12 h, Fig. S9†), whereas SiR-Actin showed
product analog of jasplakinolide creates a cell permeable agent
good staining at higher concentration with shorter exposure
2
6
for staining the F-actin cytoskeleton. This SiR-Actin reagent 11
(
1 µM for 1–2 h). Interestingly, at elevated excitation intensity,
SITC-Actin displayed spontaneous blinking in PBS buffer
Fig. S11†). While this property may reduce the fluorescence
(
Fig. 3) has become a far-red emissive phalloidin alternative
that can be equally used in fixed or living cells. For a detailed
comparison, the conjugate 12 (Fig. 3) was synthesized (2 steps,
see ESI, Scheme S6†) and investigated in cell microscopy.
Generally, SITC-Actin (12) exhibited good fluorescence pro-
perties which were qualitatively similar to SiR-Actin in terms of
brightness and photostability (Fig. S10†). Staining of fixed, but
non-embedded HeLa cells along the protocols developed for
SiR-actin uncovered highly selective staining of actin filaments
(
signal in widefield fluorescence microscopy imaging, it could
potentially be exploited for single molecule localization
35
microscopy (SMLM) techniques such as dSTORM.
SITC-Actin is a fluorogenic compound (3.1-fold increase of
fluorescence upon binding to F-actin), slightly less so than
SiR-Actin (5-fold increase), but with similar binding kinetics
(
Fig. S8†). When tested in actin depolymerization assays (high
(
Fig. 4a), comparable to SiR-Actin (Fig. 4c), using a standard
salt), both SiR-Actin and SITC-Actin retarded F-actin dis-
solution, similarly to jasplakinolide (Fig. S9a†). When monitor-
ing G-actin polymerization at low salt conditions, jasplakinolide
strongly promoted this process, as expected. By contrast,
SITC-Actin performed completely neutral, whereas SiR-Actin
even slightly decelerated F-actin assembly (Fig. S9b and c†).
These data indicate that by introducing different labels, actin
tool compound properties and toxicity can be engineered, likely
by influencing individual compound folding and aggregation or
Zeiss Elyra microscope. For enhanced resolution and contrast,
the SIM-mode could be used in which both dyes showed com-
parable properties (Fig. 4b and d).
Despite their similarity, SITC-Actin and SiR-Actin display
somewhat distinct features. In practice, SITC-Actin showed
better staining when lower concentrations were used (150 nM)
36
by modulating G-actin binding, as suggested previously.
In terms of live cell application, both SITC- and SiR-Actin
feature no detectable toxicity at the concentrations used for
live imaging (≤1 μM), in contrast to the highly toxic jasplakino-
lide (Fig. S12†). This beneficial feature, which may correlate
with the much reduced propensity for F-actin polymerization
induction by SIR- and SITC-Actin, renders these tool com-
pounds essentially non-toxic in the low-μM range and hence
Fig. 3 SITC-actin thiourea conjugate 12 of an optimized jasplakinolide
analog.
2
6
well suited for undistorted live-cell imaging.
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