Far-Red Emitting Fluorescent Dyes for Optical Nanoscopy: Fluorinated Silicon-Rhodamines (SiRF Dyes) and Phosphorylated Oxazines

Article abstract of DOI:10.1002/chem.201501394

Far-red emitting fluorescent dyes for optical microscopy, stimulated emission depletion (STED), and ground-state depletion (GSDIM) super-resolution microscopy are presented. Fluorinated silicon-rhodamines (SiRF dyes) and phosphorylated oxazines have absorption and emission maxima at about λ≈660 and 680 nm, respectively, possess high photostability, and large fluorescence quantum yields in water. A high-yielding synthetic path to introduce three aromatic fluorine atoms and unconventional conjugation/solubilization spacers into the scaffold of a silicon-rhodamine is described. The bathochromic shift in SiRF dyes is achieved without additional fused rings or double bonds. As a result, the molecular size and molecular mass stay quite small (<600 Da). The use of the λ=800 nm STED beam instead of the commonly used one at λ=750-775 nm provides excellent imaging performance and suppresses re-excitation of SiRF and the oxazine dyes. The photophysical properties and immunofluorescence imaging performance of these new far-red emitting dyes (photobleaching, optical resolution, and switch-off behavior) are discussed in detail and compared with those of some well-established fluorophores with similar spectral properties. Edge of vision: Fluorinated silicon-rhodamines (SiRF dyes) and phosphorylated oxazines are applied in stimulated emission depletion (STED) and ground-state depletion microscopy (GSDIM) as new far-red-emitting fluorophores (see figure). Their photophysical properties and immunofluorescence imaging performance with a redshifted STED beam are discussed in detail and compared to those of some well-established near-IR emitting dyes.

Full text of DOI:10.1002/chem.201501394

DOI: 10.1002/chem.201501394
Full Paper
&
Dye Design |Hot Paper|
Far-Red Emitting Fluorescent Dyes for Optical Nanoscopy:
Fluorinated Silicon–Rhodamines (SiRF Dyes) and Phosphorylated
Oxazines
Kirill Kolmakov,* Elke Hebisch, Thomas Wolfram, Lars A. Nordwig, Christian A. Wurm,
Haisen Ta, Volker Westphal, Vladimir N. Belov,* and Stefan W. Hell*[a]
Abstract: Far-red emitting fluorescent dyes for optical mi-
croscopy, stimulated emission depletion (STED), and ground-
state depletion (GSDIM) super-resolution microscopy are
presented. Fluorinated silicon–rhodamines (SiRF dyes) and
phosphorylated oxazines have absorption and emission
maxima at about lꢀ660 and 680 nm, respectively, possess
high photostability, and large fluorescence quantum yields
in water. A high-yielding synthetic path to introduce three
aromatic fluorine atoms and unconventional conjugation/
solubilization spacers into the scaffold of a silicon–rhod-
amine is described. The bathochromic shift in SiRF dyes is
achieved without additional fused rings or double bonds. As
a result, the molecular size and molecular mass stay quite
small (<600 Da). The use of the l=800 nm STED beam in-
stead of the commonly used one at l=750–775 nm pro-
vides excellent imaging performance and suppresses re-exci-
tation of SiRF and the oxazine dyes. The photophysical prop-
erties and immunofluorescence imaging performance of
these new far-red emitting dyes (photobleaching, optical
resolution, and switch-off behavior) are discussed in detail
and compared with those of some well-established fluoro-
phores with similar spectral properties.
Introduction
biological objects and reduce photobleaching of the dye
probe. As a result, with a STED beam of longer wavelengths
(l>775 nm), better imaging performance can be achieved.
However, STED microscopy with such a redshifted depletion
beam has not been reported so far. Additionally, the redshift
of the emission and absorption bands can expand the
spectral window for dual and multicolor STED imaging
schemes that utilize two or more dyes. Also, the bathochromic
and bathofluoric shifts in some newly developed red-emitting
dyes that are potentially applicable in living cells should re-
Near-IR emitting dyes are widely used in biology-related opti-
cal microscopy, modern super-resolution microscopy (e.g.,
stimulated emission depletion (STED) and ground-state deple-
tion microscopy (GSDIM)), sensing, and materials science.[1] Red
light is noninvasive, provides good tissue penetration depth,
reduces scattering, and does not cause protein autofluores-
cence. It is also important that a variety of inexpensive, con-
venient, and powerful commercial lasers are available for this
optical region (e.g., continuous-wave (CW) laser diodes, HeÀ quire shifting of the STED wavelength as well. This would
Ne, or krypton ion lasers). In STED microscopy, a laser beam se-
lectively switches off the fluorescence of the molecules ex-
posed to this depletion laser, so that the emitting area can be
“squeezed” to a spot with dimensions no longer limited by dif-
fraction.[2] Currently, a limited number of STED wavelengths are
available in commercial STED microscopes: l=592–595, 660,
and 765–775 nm.[3] Shifting the STED wavelength further to
the near-IR spectral region is expected to cause less harm to
avoid the re-excitation of the dye fluorescence caused by the
STED light.
New, efficient tools of dye design, “modular” approaches,
and flexible synthetic routes to fluorophores with required
spectral properties have been devised in recent years.[4] Due at-
tention was paid to the solubility of the dyes in water, their
net charge, and the hydrolytic stability of N-hydroxysuccinimid-
yl esters.[4,5] However, it still remains unclear how far one can
shift the absorption and emission bands to the red spectral
region, while still maintaining good performance under STED
conditions and a sufficiently high fluorescence quantum yield
(associated with the so-called brightness) of a dye. Rhodamine
dyes based on a xanthene core with an oxygen atom at posi-
tion 10 have found wide use as fluorophores due to their high
photostability and large fluorescence quantum yields.[4,5] Our
previous studies describe the synthesis, properties, and appli-
cations of red-emitting STED dyes with absorption and emis-
sion maxima at l=635–640 and 660 nm, respectively. To
[a] Dr. K. Kolmakov, E. Hebisch, Dr. T. Wolfram, L. A. Nordwig, Dr. C. A. Wurm,
Dr. H. Ta, Dr. V. Westphal, Dr. V. N. Belov, Prof. S. W. Hell
Department of NanoBiophotonics
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Gçttingen (Germany)
E-mail: Kirill.Kolmakov@mpibpc.mpg.de
vbelov@gwdg.de
shell@gwdg.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201501394.
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achieve these values, we used donor substituents at nitrogen
atoms, additional double bonds and cycles, rigidized frame-
works, and fluorinated phenyl rings at C-9.[5a–e] A further red-
shift for rhodamines is very difficult to achieve.[5g,6] An easier
option to move to the IR region is to use carbopyronine dyes,
in which the oxygen atom at position 10 of the xanthene frag-
ment is replaced by a geminal dimethyl group (C(CH3)2).[7] This
structural modification is known to cause a large bathochromic
shift in the absorption and emission bands. Therefore, in this
particular case (for carbopyronine dyes depleted by a l=
775 nm STED laser), there is no need for four fluorine atoms in
the o-substituted benzoic acid residue attached to C-9. One of
the most practically important and commercially available car-
bopyronine dyes with a proven good performance in STED mi-
croscopy, ATTO 647N, absorbs at l=644 nm and emits at l=
669 nm.[8] In general, the fluorine-free dyes are far less lipophil-
ic, more soluble in aqueous buffers, possess high fluorescent
quantum yields (even without additional polar groups), and
perform well in STED microscopy.[5f] Unfortunately, the chemi-
cal structures of some commercially available red-emitting
dyes remain undisclosed. The absence of these data compli-
cates the proper choice of fluorescent dye for certain biochem-
ical or imaging applications and hinders the design of new
dyes. To create new photostable fluorophores for (multicolor)
STED microscopy with variable STED wavelengths and to
achieve further redshifts, new tools in dye design or new dye
classes are required. A very efficient approach for a much
larger bathochromic shift in xanthene dyes was reported in
2008.[9a] Thus,
a family of silicon-containing rhodamines
(known as SiR dyes or silarhodamines) was introduced.[9] They
have a Si(CH3)2 residue (instead of oxygen) in position 10 of
the (former) xanthene fragment, as seen in Scheme 1; and in
this respect they are analogues of carbopyronines. The first
striking feature of SiR dyes is a very large bathochromic and
bathofluoric shift relative to conventional rhodamines
(+90 nm) and carbopyronines (+45 nm). In particular, Si-TMR,
which is the simplest N,N,N’,N’-tetramethyl derivative of sili-
con–rhodamine, has an absorbance maximum at l=645–
650 nm.[9b,h] 6-Carboxy-Si-TMR, which is usually called SiR-CO2H
or SiR for brevity, is a fluorogenic dye. Its monosubstituted de-
rivatives exist in equilibrium between a nonfluorescent spiro-
lactone (“OFF” state, closed form) and a fluorescent zwitterion
(“ON” state, opened form; Scheme 1).[9h,k] Derivatives of SiR
Scheme 1. Far-red emitting dyes: SiRF dyes (1a,b), phosphorylated oxazine
(2), and the reference red-emitting fluorescent dyes SiR-COOH (6-carboxy-Si-
TMR), ATTO 655, KK 114L, Abberior STAR RED, and SiR-methyl. NHS=N-hy-
droxysuccinimide.
SiR-COOH) used with common STED wavelengths of l=765
and 775 nm.[3]
were successfully used for labeling SNAP-, CLIP-, and Halo-Tag Results and Discussion
fusion proteins in living cells.[9g,h,k,m] These and also actin and
Motivation and general strategy
tubulin conjugates were successfully used in super-resolution
imaging of living cells.[9g,h,k,m] In contrast, the SiR-methyl dye is
nonfluorogenic (Scheme 1). A further redshift was not required
for these dyes because STED was performed at l=775 nm.[9h,m]
No additional methylene bridges or fused rings were attached
to maintain a smaller molecular mass, size, and cell permeabili-
ty. Notably, when the absorption and fluorescence maxima are
shifted too far to the IR region (l>700 nm), the fluorescence
quantum yield of dyes normally decreases.[9b] Therefore, the
newly emerging dye candidates for STED microscopy should
not have a very large bathochromic shift relative to those of
well-established dyes (e.g., ATTO 647N, Abberior STAR RED or
Pursuing our interest in exploring and optimizing new dyes for
optical nanoscopy (STED and GSDIM[10]) in the far-red spectral
region, we obtained and studied new silicon–rhodamines and
oxazine dyes.[11] With regard to most biolabeling applications
(conjugation) and imaging performance (brightness, photosta-
bility, absence of fluorescent background), additional proper-
ties of a dye are required. First, a versatile and generally appli-
cable fluorescent marker needs to have a readily modifiable re-
active group. If a dye can be decorated with a hydrolytically
stable amino-reactive group, which allows prolonged storage
in the solid state, then it can be easily transformed into other
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reactive forms, such as azides and maleimides. In some cases,
polar functional groups or additional solubilizing residues are
highly desirable. They provide good solubility in water, in-
crease the fluorescence quantum yield through disrupting ag-
gregates, and reduce the undesired background caused by un-
specific labeling. However, “compact” fluorophores with limited
molecular mass (<800 Da) are most advantageous because
they generally do not require additional polar groups for water
solubility. The small size of the molecule is also beneficial for
membrane permeability.[9h,k] It is known that the fluorescence
quantum yield of a dye decreases dramatically if its absorption
maximum lies beyond l=700 nm.[9b] Taking into account the
spectral data of the simplest Si-TMR (absorption and emission
maxima at l=645 and 661 nm, respectively; see Table 1),[9h]
we decided to prepare related dyes with a moderate batho-
chromic shift. The introduction of several fluorine atoms into
the phenyl ring attached to C-9 (meso position of the fluoro-
phore) is one of the simplest ways to achieve a redshift in the
xanthene scaffold.[5b,c,e] Multiple fluorine substituents in this
phenyl ring are quite reactive towards nucleophiles,[5b,c] so it
was a priori not clear if their presence was compatible with
the multistep synthetic routes we had to follow. An additional
redshift can be provided by amidation of the o-carboxyl group
in the phenyl ring attached to the meso position (C-9) of the
fluorophore.[5e] Rhodamines,[4,5] carbopyronines,[7] and silicon–
rhodamines[9] usually have this group. Exceptions include the
dyes SiR650, SiR680, SiR700, SiR720, and SiR-methyl,[9b,h] in
which the carboxyl group is replaced by a methyl group, and
an additional carboxyl group in the phenyl ring is used for
conjugation. This approach blocks cyclization, but, on the
other hand, does not provide an additional bathochromic shift.
However, as a better and simpler solution, one can try amida-
tion (or esterification) of the o-carboxyl group with a proper bi-
functional substrate. That would fulfill all three tasks at the
same reactive site of the dye: block cyclization into the non-
fluorescent, closed, spirolactone form (see Scheme 1); provide
a linker for conjugation through another reactive functional
group at its terminus; and, additionally, produce a noticeable
redshift. Additional double bonds or fused rings thus become
unnecessary. Despite providing fluorogenicity,[9h,k] the ability of
silicon–rhodamines to cyclize can be also a drawback: the col-
orless form might become dominating in the equilibrium
inside a nonpolar medium. As a result, the fluorescent signal
would drop dramatically. Generally, amidation or esterification
of the sterically hindered o-carboxyl group represents a classical
tool to create a spacer in dye chemistry. However, none of
these reactions have been reported for silicon–rhodamine dyes
so far.
Oxazine fluorescent dyes,[11] such as the dye ATTO 655
(Scheme 1),[8,12a] were shown to perform especially well in
single-molecule localization microscopy.[10,12b] ATTO 655 has
a sulfonic acid residue as a polar group, which provides good
solubility in water. Highly polar dyes are known to have better
fluorescence quantum yields in aqueous solutions and in con-
jugates as well. Also, they bind more specifically with the
target structures, produce less background fluorescence, and
yield bright and contrast-rich images with higher optical reso-
lution.[5] Despite interesting spectral properties (Table 1), ATTO
655 has not yet found wide use in STED microscopy. As shown
previously, phosphorylated rhodamines[5b–d] and coumarins[13a,b]
could be even “brighter” dyes than the corresponding sulfo-
nated analogues. The phosphonylation of cyanines, boron–di-
pyrromethene (BODIPY) fluorophores and perylenes was also
used as a tool to prevent dye aggregation and improve the
physicochemical properties.[13c–h] Therefore, another part of this
research work focused on the synthesis of a new oxazine dye
with a primary phosphate group (compound 2-H in Scheme 1)
and its application in STED and GSDIM.
Table 1. Spectral properties of free SiRF dyes (1a,b-H, 15a), phosphorylated oxazine 2-H, and the reference dyes (SiR-methyl, SiR-CO2H, ATTO 655, KK 114L,
Abberior STAR RED), as well as related data for their conjugates with antibodies used for immunolabeling in STED and GSDIM.
Dye
Additional
polar group
q[a]
Absorption[b]
l max[nm]
Emission
lmax [nm]
e”10À5
[mÀ1 cmÀ1
Ffl
[%][c]
t
Dye–antibody conjugates
]
[ns]
DOL[d]
Ffl
t
STED:[l] resolution [nm],
[%]
[ns]
switch-off, re-excitation
1a-H
1b-H
13a (13-O)[f]
15a
SiR-methyl
SiR-6-COOH
2-H
ATTO 655
KK 114L
–
+1
0
+1
À1
+1
0
662
663
657
657
648[h]
645[h]
661
663
637
636
680
680
677
676
662[h]
661[h]
678
684
660
659
1.18
0.90
0.53[g]
0.4
1.0 h
1.0[h]
0.60
1.25[i]
0.92
1.0
66
70
63
40
39[h]
39[h]
28
30[i]
55[i]
66[k]
2.4
2.5
–
–
–
2.6
1.8
1.8[i]
3.6
3.6
2.3
2.7
–
–
–
3.7
1.4
0.8
5.0
3.1
35
30
–
–
–
22
11
12
67
65
2.4
2.4
–
–
–
2.5
1.8
1.8
3.5
3.5
63, +, –
57, +, +
–
–
SO3H
–
SCH2CO2H
–
CO2H
OPO(OH)2
SO3H
2”SO3H
2”SO3H
–
60, +, + + +
69, + +, +
65, + +, –
67, + + +, + + +
64, + + +, + +
À1
0
À1
À1
Abberior STAR RED
[a] Net charge in the conjugated state; for free dyes, this value is one unit lower. [b] All spectroscopic measurements for free dyes were recorded in water.
[c] Determined for free dyes in solutions with the reference dye Atto AZ 237, unless otherwise stated. [d] DOL=degree of labeling; an average number of
dye residues attached to one protein molecule with Mr ꢀ150 000 Da in sheep antimouse antibodies; see the Supporting Information for details. [e] Optical
resolution (in nm), evaluation of the relative “switch-off” efficiency and relative re-excitation degree (see the main text for definitions, details, and discus-
sion). [f] Because it did not have a conjugation site, dye intermediate 13a was not used for immunolabeling (see Schemes 3 and 4 for the structure).
[g] The lower extinction value was due to the equilibrium between the closed (colorless) and open (zwitterionic) forms in solutions of 13a. [h] see ref. [9h].
[i] www.atto-tec.com. [j] See ref. [5c] and structure 2b therein. [k] See ref. [5b], www.abberior.com and Scheme 1 for structures of the dyes. [l] With 800 nm
light; see text for comparison of switch-off and re-excitation properties.
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SiRF dyes
work at all (with sodium acetate as a weaker base). Finally, we
managed to perform the ring-closure reaction of amides 4a,b
to form oxazolines 6a,b by using KHSO4 (a moderately acidic
heterogeneous dehydrating agent) in 1,2-dichlorobenzene at
elevated temperatures. Disappointingly, all attempts to per-
form the required addition of o-lithiated oxazoline 8a with
four fluorine substituents (Scheme 2) to ketone 10a
(Scheme 3) failed. We established that nBuLi and tBuLi only
Initially, we tried to introduce four fluorine atoms into Si-TMR
dye 1a (Scheme 1) by using a known approach that worked
well for non-fluorinated dye precursors. It was based on lithi-
um–bromine exchange in 2-(2-bromophenyl)-4,4-dimethyl-2-
oxazoline followed by reaction with the corresponding cyclic
diaryl ketone.[5e] The chemistry related to the preparation of
lithium reagents, starting from fluorine-substituted benzoic
acids 3a,b, is given in Scheme 2. We planned to use cyclic
diaryl ketones (compounds 10a[9c,h] and 10b[5e] in Scheme 3) as
Scheme 3. Rhodamine lactone 13a as the key building block for SiRF dyes:
a) aryl oxazoline 7 (Scheme 2), Li-TMP, THF, hexane, À788C to RT; b) aryl oxa-
zoline 6b (Scheme 2), 1.5m tBuLi in hexanes, THF, À788C; c) HOAc in MeOH,
À108C; d) 20% aqueous HCl, 808C, 3 h; e) aq. NaHCO3, RT. *: 30 h at 808C.
caused complete reductive debromination of oxazoline 6a.
Thus, after aqueous workup, bromine was lost (and compound
7 was isolated), but no further transformation occurred. How-
ever, the fact that all four fluorine atoms withstood the lithia-
tion reaction was reassuring. This result indicated that the
“benzine-type” intermediate with a triple bond had probably
not been formed (because it would lead to the loss of a fluo-
rine atom and a complex mixture of products) and ketone 10a
simply did not react with the quite stable lithiated oxazoline
8a. An alternative synthetic pathway involved the lithiation of
the corresponding “bromine-free” oxazoline 7 with Li-TMP fol-
lowed by addition of ketone 10a (Scheme 3).
This approach did not work either. Compound 7 and a new
colored substance (10a-“dimer”; Scheme 3) were isolated from
the reaction mixture. The formation of the dimer of ketone
10a may be explained by the presence of a very strong base
(excess Li-TMP was used). Li-TMP regioselectively deprotonates
the aromatic ketone 10a and the lithiated intermediate reacts
with the keto group of another molecule. The intermediate
lithiated ketone 10a is stabilized by the keto group in the o-
Scheme 2. Fluorinated oxazolines 6a,b and their precursors: a) (COCl)2,
CH2Cl2, RT; b) 2-amino-2-methyl propanol, Et3N, 08C; c) SOCl2, RT; d) KHSO4,
1,2-dichlorobenzene, 1808C; e) tBuOK in THF or NaOAc in EtOH; f) tBuLi,
THF, À788C; g) lithium 2,2’,6,6’-tetramethylpiperidide (Li-TMP; prepared from
2.5m nBuLi in hexanes and 2,2’,6,6’-tetramethylpiperidine in THF), À788C to
+58C.
the carbonyl substrates for further reactions with aryl lithium
reagents. The starting amides 4a,b with one bromine and
three or four fluorine atoms in the aromatic ring were ob-
tained with no difficulties (see the Supporting Information).
However, the known cyclization assisted by thionyl chloride[9h]
failed to convert amides 4a,b into oxazolines 6a,b (Scheme 2).
Instead of oxazolines, w-chloromethyl derivatives 5a,b were
formed. A base-induced cyclization of these undesired prod-
ucts caused substitution of fluorine (with tBuOK) or did not
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position to the CÀLi site. The dimer of compound 10a has
a brown color and also belongs to the silicon-rhodamine (SiR)
dyes. The unexpected color (not blue!) indicates that the sub-
stituent at C-9 strongly influences the spectral properties of
SiR dyes. An analogous lithiation of 3,4,5-trifluorobromoben-
zene with LiN(iPr)2 and the subsequent reaction of 2-lithio-
3,4,5-trifluorobromobenzene with carbon dioxide were used
for the preparation of 6-bromo-2,3,4-trifluorobenzoic acid (3b;
Scheme 2).[14] Acid 3b was used as a valuable intermediate to
prepare oxazoline 6b and check if its lithiated derivative (8b in
Scheme 2) would react with cyclic diarylketones (10a,b in
Scheme 3). The preparation of the building block 6b involved
amidation and cyclization steps (see the Supporting Informa-
tion) and proceeded smoothly. Similarly, so did the one-pot re-
action with the silicon-containing ketone substrate 10a (see
Scheme 3). The workup and product isolation were performed
exactly as described for carbopyronine dyes[5f] to give the
amino ester 11 (Scheme 3). The
the keto group in compound 10b than in ketone 10a. As
a result, the reactivity of 10b towards nucleophiles (i.e., aryl
lithium reagents) is reduced, compared with that of ketone
10a, in which amino groups can rotate freely.
In the neat state and even in solutions (alcohols, DMF,
MeCN), compound 13a exists predominantly in its “closed”
(colorless and nonfluorescent) lactone form (see Scheme 3, the
relevant part of the Supporting Information, and photographs
therein). It only develops a blue color in dilute aqueous solu-
tions or when adsorbed on silica gel, as witnessed in the
course of TLC or preparative column chromatography. With
regard to spectral properties, the open form of SiRF 13a (13-
OÀ in Scheme 4) met our expectations. A sufficient bathochro-
mic shift, relative to the previously studied red-emitting rhod-
amines and carbopyronine dyes (+20 nm; see Table 1),[5b–f] and
to non-fluorinated SiR-CO2H, was observed (+12 nm;
Scheme 1).[9h] A relatively low extinction coefficient at the ab-
acidic deprotection of 11 gave
the free silicon–rhodamine 13a.
This synthesis, as a whole, was
straightforward and proceeded
with high yields. Some impor-
tant details are worth mention-
ing. First, one should avoid
sodium bicarbonate and other
bases during workup. Otherwise,
the intermediate amino ester 11
rapidly undergoes cyclization
into a very stable colorless spiro-
lactame 12 (Scheme 3). This cy-
clization, which is clearly caused
by an intramolecular nucleophil-
ic attack, proved to be irrevers-
ible. Moreover, different to our
previously explored carbopyro-
nine spiroamide analogue,[5f]
amide 12 is much more resistant
to acidic hydrolysis (see the Sup-
porting Information). Also, inter-
estingly, polycyclic diaryl ketone
10b did not react with o-lithiat-
ed oxazoline 8b, but was known
to react with compound
9
(Scheme 2).[5f] These observa-
tions support our assumption
that o-lithiated oxazolines are
readily formed and their reactivi-
ty increases in the following
order: 8a<8b<9 (for struc-
tures, see Scheme 2). The very
low reactivity of the polycyclic
Scheme 4. Synthesis and transformations of SiRF dyes 1a,b, which are fluorinated esters of silicon rhodamines:
a) (COCl)2, 1,2-dichloroethane, RT; b) propanol-2, RT; c) CH2Cl2, RT; d) 0.05m NaOH in THF/H2O (1:2), RT; e) aqueous
HF in MeCN, RT; f) 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), DMF;
g) HCl·CH3NH(CH2)3COOCH3, Et3N, CH2Cl2, DMF, RT; h) N,N’-disuccinimidyl carbonate (DSC), EtNiPr2, CH2Cl2, MeCN,
diaryl ketone 10b (Scheme 3) RT; i) H3N+CH(CH2SO3À)CONH(CH2)2CO2H, Et3N, HATU, DMF; j) HSCH2CO2H, Et3N, MeCN, RT; k) allyl alcohol, N,N’-di-
cyclohexyl carbodiimide (DCC), Et3N, 4-dimethylaminopyridine (DMAP); l) [Pd(Ph3P)4], HCOOH. *: the opened form
can be explained by the inability
of the silicon–rhodamine 13a (see Scheme 3), which irreversibly forms the acid chloride upon treatment with
of the amino groups to rotate.
(COCl)2; **: the major isomer; ***: the disubstituted product is formed with an excess of HSCH2CO2H and/or at
Their lone electron pairs are
longer exposure times. TBDMS=tert-butyldimethylsilyl. For details of the syntheses and related analytical data,
much better conjugated with see the Supporting Information.
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sorption maximum (53000mÀ1 cmÀ1 at l=645 nm in water;
see Table 1) is clearly due to the equilibrium between the
closed (13a) and opened forms (13-OÀ). The tendency of sili-
con–rhodamine 13-OÀ with three fluorine substituents (see
Scheme 4) to cyclize into the colorless lactone form 13a is so
strong that it has to be accounted for during planning of its
transformation into the “non-fluorogenic” and “fully colored”
fluorescent dye. Thus, it was necessary to modify (block) the
carboxylic group in such a way that it could no longer form
the stable five-membered lactone ring. In particular, it proved
impossible to perform aminolysis of lactone 13a by using sec-
ondary aliphatic amines and HATU as a coupling reagent; this
always worked well for rhodamine dyes.[5] The reason for this
compound being unreactive is undoubtedly the presence of
a base, which shifts the equilibrium fully to the closed form of
13a (see Scheme 3 and Figure S2 in the Supporting Informa-
tion). Thus, conversion to the corresponding acid chloride (13-
Cl, R=Cl) was required.[15] Even less expected difficulty oc-
curred when we converted compound 13a into the acid chlo-
ride, attached the linker (CH3NH(CH2)3COOCH3 in Scheme 4),
and isolated the amido ester 14-Me. Although the amide bond
is known to be quite stable toward hydrolysis over a wide
range of pH, amide 14-Me partly decomposed upon standing
in a neutral organic solution at +58C. Attempted saponifica-
tion to the acid 14-H, even with a very dilute aqueous alkali
(0.05m), immediately produced the starting rhodamine 13a in
its spirolactone form,[16] as observed by a complete loss of
color. Therefore, we had to use an alternative type of a linker
or reconsider the whole synthetic scheme to avoid a carboxyl
group in the ortho position of the phenyl ring.
13-OiPr (Table 1 and the Supporting Information) and the hy-
drolytic stability was even better. For the conjugation proce-
dures, the amino-reactive form of a dye is required. Therefore,
we prepared the NHS carbonate of 1a-CONHS (Scheme 4). To
this end, alcohol 1a-H reacted with DSC in the presence of
iPr2NEt as a base. Despite the expected partial cleavage of the
linker in basic medium and the formation of the symmetrical
carbonate, the amino-reactive marker 1a-CONHS was obtained
in moderate to good yields (up to 60%). Luckily, this product
demonstrated very good hydrolytic stability and good reactivi-
ty towards amines. Importantly, in the test reactions with
a large excess of dilute aqueous ammonia, triethylamine, and
NaHCO3, no cleavage of the linker was observed. Furthermore,
bioconjugation reactions with antibodies proceeded with suffi-
cient degrees of labeling (DOL), even though they were per-
formed at relatively high pH values (8–8.5) in a NaHCO3 buffer
(see the Supporting Information). Also, being soluble in both
water and nonpolar solvents, dye 1a-H demonstrated amphi-
philic properties, as seen in Figure S6 in the Supporting Infor-
mation. To further improve dye 1a-H by increasing its polarity
and solubility in aqueous solutions, we attached a special solu-
bilizing moiety—the so-called “universal hydrophylizer” (H3N+
CH(CH2SO3À)CONH(CH2)2CO2H).[5a,18] Such postsynthetic modifi-
cations, also introduced by Romieu and co-workers, were
shown to drastically improve the solubility of dyes in
water.[4l,m,r] Most importantly for immunolabeling, the polarity
of dye residues prevents precipitation in the labeled proteins,
reduces their “stickiness”, unspecific binding, and the fluores-
cent background. As an overall result, the imaging per-
formance of the hydrophilic dyes is often greatly improved.
Thus, we subjected 1a-CONHS to a reaction with a presynthe-
sized peptide-like spacer that contained a sulfonic acid group
(Scheme 4). As expected, the solubility of the reaction product
1b-H in water became much better, while the fluorescence
quantum yield in aqueous solutions increased (relative to the
starting alcohol 1a-H). The polar, free-dye 1b-H proved to be
quite stable in basic solutions (e.g., bicarbonate buffer and
aqueous Et3N), probably because of its zwitterionic character.
The NHS ester of the modified dye 1b-NHS demonstrated ex-
cellent hydrolytic stability and was isolated and handled in the
pure state with no difficulties. Good hydrolytic stability of 1b-
NHS was in agreement with our previous observations on the
stability of NHS ester dyes with a zero net charge: they were
generally more stable than the negatively charged N-hydroxy-
succinimidyl esters.[5b,c] Antibody staining with compound 1b-
NHS as a marker also proceeded with a high DOL.
An ester group represents an alternative to the amide func-
tionality. The hydrolytic stability of esters is generally far lower
than that of amides, but it can be considerably improved by
steric hindrance. As a simple model compound, we obtained
the isopropyl ester 13-OiPr (from the acid chloride; see
Scheme 4). Ester 13-OiPr demonstrated the required batho-
chromic shift and high quantum yield of fluorescence in water,
despite the absence of polar groups (see Table 1). Fortunately,
the compound proved sufficiently stable against weak bases
(triethylamine in water). However, we established that the iso-
propyl ester 13-OiPr was cleaved within an hour, even with
a 0.025m solution of NaOH in water. Tertiary amines are in-
volved as reagents in the preparation of N-hydroxysuccinimidyl
esters or carbonates. The latter are more stable (yet react read-
ily with primary amines)[5b] and their syntheses do not require
the alkaline saponification of a methyl ester group attached to
the linker. The risk of cleaving off the linker, as occurred with
amide 14-Me, was thus avoided. As a bifunctional linker, we
chose a commercially available 1,4-penthanediol with the pro-
tected primary hydroxyl group. By using a large excess of the
alcohol (HOCH(CH3)(CH2)3OTBDMS), the ester 1a-TBDMS was
obtained in a high yield (Scheme 4). The cleavage of the
TBDMS protecting group occurred partially during workup of
the reaction mixture and was completed by a conventional
method (dilute aqueous HF in MeCN).[17] The dye 1a-H demon-
strated sufficient solubility in water due to its CH2OH group.
The absorption and emission spectra were identical to those of
An alternative way to modify the SiRF dyes was also ex-
plored. This involved aromatic nucleophilic substitution of the
fluorine atom(s) with
a thioglycolic acid residue (see
Scheme 4). This approach was used, for example, in the syn-
thesis of some red-emitting rhodamines and “masked” fluores-
cent dyes.[5a–c,19] The silicon–rhodamine substrate 13a (13-OÀ)
did react with thioglycolic acid in the presence of triethyl
amine. However, unlike tetrafluoro- or tetrachlorophenyl deriv-
atives of xanthene dyes,[19] this reaction proved unselective. A
3:1 mixture of two regioisomers (15a and 15b) was formed
(see Scheme 4 and the Supporting Information for details). In
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general, this derivatization method is potentially useful be-
cause compounds 15a and 15b are expected to be fluorogen-
ic. These interesting dyes are difficult to separate (preparative
HPLC is necessary). However, after isolation, each of them can
be used as an analogue of SiR with slightly redshifted absorp-
tion and emission spectra. For example, 4-SCH2COOH isomer
15a (major product) has absorption and emission maxima at
l=657 and 676 nm, respectively, a fluorescence quantum yield
of 40%, and eꢀ40000 (in water; see Table 1). The low e value
indicates that (even in aqueous solutions), compound 15a
exists mostly in the closed (colorless) form, in which one of the
carboxylic groups is protected by the formation of a five-mem-
bered lactone ring. Organic solutions of both dyes 15a and
15b are nearly colorless. With this intrinsic protection, we first
converted the SCH2COOH group in 15a into an allyl ester and
then tried to transform another carboxyl group into an isopro-
pyl ester by using the same methodology as that used for
compound 13-OiPr. Finally, we planned to cleave the allyl pro-
tective group and obtain a dye (15a-H-iPr) with a single, free
carboxyl group available for conjugation (Scheme 4, bottom).
However, the allyl ester (15a-All) formed neither the acid chlo-
ride nor the isopropyl ester (remained colorless), despite the
large excess of oxalyl chloride. We can assume that a stable
cyclic lactone ring remains intact under these reaction condi-
tions. Additionally, we subjected the isopropyl ester (13-OiPr)
to an SNAr reaction with triethylammonium thioglycolate
under mild conditions. Disappointingly, this only led to the
cleavage of the ester group, while the fluorine atoms remained
unsubstituted (unlike in the case of “unprotected” rhodamine
13a).
Scheme 5. Oxazine dyes with a primary phosphate group; condensation of
the building blocks followed by phosphorylation: a) 4-O2NC6H4N2(+)·BF4(À),
dilute H2SO4 in aqueous MeOH, RT; b) 4m HCl in EtOH, 858C; c) POCl3, THF,
1,2-dichloroethane, 08C to RT; d) Et3N, aqueous MeCN; e) 0.05m aqueous
NaOH; f) AcOH; g) N,N,N’,N’-tetramethyl-O-(N-succinimidyl)uronium tetra-
fluoroborate (TSTU), Et3N, DMF, RT. *: an average yield over two synthetic
steps in 2–3 runs.
tively (Table 1). Ethyl ester 20-Et,H was saponified with a dilute
aqueous solution of NaOH and converted into the correspond-
ing carboxylic acid 20-H,H. Compounds 20-Et,H and 20-H,H are
spectrally almost identical to the SiRF dyes described above
(see the Supporting Information and Table 1 for the spectral
data); only the solubility in water is very limited. In our prelimi-
nary experiments, the STED-imaging performance of dye 20-
H,H proved unsatisfactory. As usual, to obtain the antibody
conjugates, carboxylic acid 20-H,H was converted into the cor-
responding N-hydroxysuccinimidyl ester 20-NHS,H (com-
pounds 20-H,H and 20-NHS,H are not shown in Scheme 5).
This drawback is because the single hydroxyl group, as a polar
residue, is unable to prevent aggregation of the bulky and in-
herently hydrophobic oxazine molecules. To improve the dye
brightness and overall imaging performance, we phosphorylat-
ed the alcohol 20-Et,H by means of a straightforward proce-
dure. As a reagent, we used POCl3, and the intermediate phos-
phoric acid dichloride was hydrolyzed without purification (see
the Supporting Information for details). As shown in Scheme 5,
the phosphorylation of dye 20-Et,H was followed by the sapo-
nification of ethyl ester 20-Et,P(O)(OH)2, which gave the free
acid 20-H,P(O)(OH)2 (or 2-H for brevity; see also Scheme 1).
This dye demonstrated greatly improved imaging performance
in STED and GSDIM (see Figures 1 and 4, below) relative to its
non-phosphorylated precursor (20-H,H). As a proper reference
dye for dye 2-H, we chose ATTO 655 (see Scheme 1), which
Phosphorylated oxazine dyes
Oxazines were shown to be very useful red-emitting fluores-
cent dyes.[11] In the design of dyes with tailored properties, it is
always better to create a scaffold with multiple variable posi-
tions. We applied the oxazine-based scaffold of the kind (struc-
ture 20-R1,R2) depicted in Scheme 5. Taking advantage of both
variable positions in 20-R1,R2, one can obtain dyes with vari-
able net charges and functional groups. In this respect, the re-
cently published results of Pauff and Miller were especially
helpful because they enabled the creation of a general, most
efficient, and high-yielding synthetic route to diverse oxazine
dyes.[11b] Our optimized approach (Scheme 5) utilizes two key
building blocks (18 and 19) obtained from a single precursor
(compound 16). Additionally, the hydroxyl group in the 3-hy-
droxypropyl residue offers further opportunities for modifica-
tion of dyes.[5b,c,16,19b] In particular, it opens up an entry to fluo-
rescent dyes with a primary phosphate group. The latter is
very hydrophilic, polar, and brings a larger negative net charge
than the conventional sulfonate. The presence of primary
phosphate groups was shown to substantially improve the
fluorescence quantum yields and solubility of rhodamines and
coumarins in aqueous solutions.[5b–c,13] The cationic oxazine dye
20-Et,H demonstrated a sufficient bathochromic shift relative
to most commonly used red-emitting rhodamines; it has ab-
sorption and emission maxima at lꢀ660 and 676 nm, respec-
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comparison with the reference non-fluorinated dyes SiR-
methyl and SiR-CO2H.[9h] The positions of the absorption and
emission maxima (as well as other parameters) were deter-
mined for aqueous solutions of dyes. All new dyes (1a-H, 1b-
H, and 2-H) possess very similar spectral properties (Table 1).
The absorption and emission maxima were found at l=661–
663 and 678–680 nm, respectively. The Stokes shifts are rela-
tively small (17 nm). Remarkably, the fluorescence quantum
yields of SiRF dyes (1a,b-H) in aqueous solutions are high (66
and 70%) and independent of the presence of polar groups
(SO3H in 1b-H). These values are higher than the fluorescence
quantum yields of the reference dyes (SiR-methyl and SiR-
CO2H),[9h] compound 15a, both oxazines (2-H and ATTO 655[8]),
as well as the benchmark polar dyes KK 114L[5c] and Abberior
STAR RED.[5b] Solutions of the new and reference dyes (Table 1)
in water and organic solvents (alcohols, acetonitrile, DMF,
DMSO) are blue. (Compounds 15a and SiR-CO2H give nearly
colorless solutions in organic solvents.) The reference rhod-
amine dyes KK 114L and Abberior STAR RED show red fluores-
cence that is visible by the naked eye, whereas all other dyes
emit invisible, near-IR light (see Figure S5 in the Supporting In-
formation). Conjugates with antibodies were prepared accord-
ing to the standard protocol. Moderate DOL values (0.8–2.7;
Table 1) prevent aggregation and precipitation of the labeled
proteins. Earlier we established that the STED imaging per-
formance in the immunolabeled cell substrates weakly de-
pended on the DOL values over quite a wide range.[5c,d] The
fluorescence quantum yields of the conjugates varied from 11
to 35%. The highest values of Ffl in the conjugates (65–67%)
were observed for highly hydrophilic and negatively charged
rhodamine dyes (KK 114L and Abberior STAR Red) that pos-
sessed two sulfonic acid residues. However, even at the small-
est values of Ffl (11–12%) for the oxazine dyes (ATTO 655 and
2-H; Figure 1), it is possible to obtain good STED images. As
expected, less hydrophilic and redshifted SiRF dyes demon-
strated somewhat lower values of the fluorescence quantum
yields in the conjugates: 22, 35, and 30% for SiR-CO2H, 1a-H,
and 1b-H, respectively. Remarkably, the fluorescence lifetimes
of free dyes and their antibody conjugates were always con-
stant (see Table 1). According to the values of the fluorescence
lifetimes, all dyes can be separated into three distinct groups,
which correspond to three dye classes: 1a,b-H and SiR-CO2H
(2.4–2.5 ns); 2-H and Atto 655 (1.8 ns; the lowest value); KK
114L and Abberior STAR RED (3.8 ns; the highest value). These
differences enable the fluorescence decay of two dyes to be
registered separately,[20] and may even be three dyes (with very
similar emission spectra), which present simultaneously as
labels in a sample. Therefore, a set of two or three dyes from
Table 1 can be applied in fluorescence lifetime imaging (FLIM).
The new SiRF dyes (1a,b-H) and phosphorylated oxazine 2-H
were compared with each other and the reference dyes (SiR-
CO2H,[9h] Atto 655,[8] KK 114L,[5c] Abberior STAR RED[5b]) in STED
microscopy by using l=640 nm excitation and l=800 nm de-
pletion beams.
Figure 1. Far-red oxazine dyes in STED microscopy. STED and confocal
images of the immunolabeled cytoskeletal protein vimentin in mammalian
vero cells. The STED images were acquired by using excitation and depletion
beams at l=640 and 775 nm, respectively. Confocal images of the same
sample spot were acquired subsequent to the STED images by using an ex-
citation beam at l=640 nm. A) Vimentin labeled with dye 2-H; B) the corre-
sponding confocal image of the same spot. C) Vimentin labeled with ATTO
655 dye; D) the corresponding confocal image (see the Supporting Informa-
tion for details).
also belongs to the oxazine family. This comparison is reason-
able because Atto 655 also has an anionic polar group, a sul-
fonic acid residue, which is the classical solubilizing moiety in
dye chemistry. The spectral properties of ATTO 655 are almost
identical to those of compound 2-H (see Scheme 1 and
Table 1). However, in STED microscopy (with a standard deple-
tion laser of l=775 nm), dye 2-H demonstrated a noticeable
improvement over ATTO 655 (see Figure 1). Confocal images
obtained with these dyes were indistinguishable. Both dyes
have the same fluorophore and the only difference between
them is that the phosphate group in 2-H provides an addition-
al negative net charge in conjugates (q=À1 at pH>6). The
overall polarity therefore appears much larger and this may
result in better optical resolution and contrast, while the unde-
sired background fluorescence is reduced (due to stronger re-
pulsion of the dye residues from each other). These observa-
tions are consistent with our previous results on red-emitting
rhodamine dyes with polar groups.[5b–f]
Spectral properties of SiRF dyes (1a,b-H) and phosphorylat-
ed oxazine 2-H, and their performance in STED microscopy
Table 1 contains data on the spectral properties of free SiRF
dyes (1a,b-H, 15a), phosphorylated oxazine 2-H, and reference
dyes (SiR-methyl, SiR-CO2H,[9h] ATTO 655,[8] KK 114L,[5c] Abberior
STAR RED[5b]), as well as related data for their conjugates with
antibodies applied in STED and GSDIM. As expected, due to
the presence of three fluorine atoms and an ester group, com-
pounds 1a-H and 1b-H demonstrated bathochromic and
bathofluoric shifts (+14–17 nm and +19 nm, respectively) in
The vimentin cytoskeleton was stained in mammalian cells
by indirect immunofluorescence labeling with the dyes from
Table 1 (Figure 2). The labeling protocols, sample preparation,
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a STED beam represents another important parameter. Re-exci-
tation is undesirable because it strongly compromises the
image quality. The STED beam is usually strongly redshifted,
even relative to the maximum of the emission band. Therefore,
is not supposed to overlap with the absorption band of the
dye. However, when the Stokes shift is small, a dye may have
a residual absorption at the STED wavelength. In this case,
a very strong STED pulse can excite the fluorescence of this
dye to some extent. The STED beam may be represented as
a doughnut (or other diffraction-limited pattern)[2b–d] with zero
intensity only in the very center, so the fluorescence signal
that originates in the course of re-excitation is “broad” (diffrac-
tion-limited) and registers as a relatively weak diffuse emission
around the sharp elements of a STED image. The re-excitation
phenomenon was not critical for these dyes, except probably
compounds 1a-H and ATTO 655. In this respect, it is worth
mentioning that ATTO 655 is widely used in single-molecule
studies, including photoactivated localization microscopy
(PALM) and stochastic optical reconstruction microscopy
(STORM; for additional references, see www.atto-tec.com),[12]
but seldom in STED microscopy.[21] Dyes 1a,b-H differ from the
rest of the compounds in several respects (Table 1). Although
they provide a lower switch-off ratio than other dyes, the opti-
cal resolutions observed with these dyes are the best (see the
Supporting Information for resolution determination details).
Second, they bear a positive (1a-H) or zero (1b-H and SiR-
CO2H) electric net charge on the dye residue in (bio)conju-
gates. Therefore, conjugates with small molecules (recognition
units for the tagged proteins in living cells) are expected to be
cell permeable. Further studies are necessary to clarify whether
compounds 1a,b-H represent viable markers for living cells.
However, these new dyes (1a,b-H and SiR-CO2H) turned out to
be least photostable under STED conditions (Figure 3). The
lower photostability of the silicon–rhodamines can be attribut-
ed to their nature: the tetramethyl silicon–rhodamine fluoro-
phore is probably inherently less photostable than the oxazine
core (2-H and ATTO 655) or the rigidized rhodamine fluoro-
phore (KK 114L, Abberior STAR RED) additionally stabilized by
the negatively charged polar residues.
Figure 2. STED microscopy images of the immunolabeled cytoskeletal pro-
tein vimentin in mammalian cells. Images were acquired by using an excita-
tion beam at l=640 nm and a depletion beam at l=800 nm for the dyes
Abberior STAR RED, SiR-CO2H, ATTO 655, 1a-H, 1b-H, and 2-H. The images
were taken according to the STED image acquisition procedure described in
the main text and in the Supporting Information. For imaging performance
parameters, see Table 1; for dye structures, see Scheme 1.
and preparation of the reactive markers (i.e., 1a-CONHS, SiR-
CONHS, 1b-NHS, and 2b-NHS) are described in Supporting In-
formation.
The dyes ATTO 655, 1a-H, 1b-H, and 2-H have an emission
maximum at lꢀ680 nm (redshifted by 20–25 nm relative to
the most well-established near-IR emitting dyes), and there-
fore, better fit to a long-wavelength STED beam at l=800 nm.
The applied power of the STED laser was 200 mW (at the back
focal plane of the objective) at 80 MHz repetition rate (see the
Supporting Information for more details). All new dyes enable
us to obtain high-quality images by using redshifted excitation
and depletion wavelengths (l=640 and 800 nm, respectively).
This feature makes them especially valuable for staining bio-
logical samples because the near-IR STED light (l=800 nm in-
stead of conventional l=775 nm) even further reduces the
phototoxicity and enables deeper tissue penetration. The STED
performance of dyes (as conjugates with antibodies) was eval-
uated and compared with respect to optical resolution, switch-
off efficiency, and re-excitation by the very powerful l=
800 nm light (Table 1). All dyes provided an optical resolution
in the range of 57–69 nm (see the Supporting Information for
the resolution determination details and an exemplary plot).
The switch-off efficiency correlates with the ability of a dye to
undergo the transition from the excited to the ground state
when exposed to STED light. A better switch-off efficiency is
thus expected to provide better optical resolution. The bench-
mark dyes KK 114L and Abberior STAR RED were only used up
to now with l=775 nm STED laser, but they displayed the
best switch-off ratios with l=800 nm STED light. Indeed, with
l=775 nm STED light, these dyes were afforded the best opti-
cal resolution of 30–40 nm.[5c,d]
Oxazine dyes 2-H and ATTO 655 in GSDIM
We decided to take a closer look at the performance of the ox-
azine dyes 2-H and ATTO 655 (for structures, see Scheme 1) in
GSDIM.[10,12b] We labeled nuclear pore complex subunits (in the
central channel of the complex) by indirect immunofluores-
cence staining by using primary antibodies and secondary anti-
bodies coupled with dyes 2-H and ATTO 655. The labeled sam-
ples were mounted in GSDIM buffer with glucose oxidase
(Glox) enzyme or in phosphate-buffered saline (PBS) solution
(without additives of reducing agents) and observed in
a GSDIM microscope (LEICA Microsystems). As seen in Figure 4,
in GSDIM buffer, nanoscopy images can be generated by using
the dyes Atto 655 and 2-H. Importantly, in PBS solution, com-
pound 2-H provides much better images than those in Glox
buffer. If we evaluate Figure 4C and D, we can see that com-
pound 2-H can be advantageously compared with dye ATTO
The oxazine dyes 2-H and Atto 655 did not switch-off as
readily as rhodamines KK 114L and Abberior STAR RED. The
SiRF dyes 1a,b-H, as well as SiR-CO2H, switched-off even less
efficiently than the oxazine dyes. The extent of re-excitation by
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that show such properties in PBS are rare. Also, the presence
of thiols in high concentrations or Glox may be prohibitive in
the application of GSDIM to living cells.
Conclusion
Far-red emitting dyes are widely used in life sciences as fluo-
rescent markers. The present study fully describes the synthesis
and properties of SiRF dyes and phosphorylated oxazines with
absorption and emission maxima at lꢀ660 and 680 nm, re-
spectively. In particular, a high-yielding synthetic pathway to
introduce three “aromatic” fluorine atoms and additional con-
jugation/solubilization spacers into the scaffold of a silicon-
containing tetramethylrhodamine (Si-TMR) and the related
transformations are described in full detail. We introduced 2-
(2-bromo-4,5,6-trifluorphenyl)-4,4-dimethyloxazoline (6b) as
a new valuable building block for far-red emitting fluoro-
phores. The bathochromic shift in the new Si-TMR derivatives
is so large that it enables these dyes to be used in STED mi-
croscopy with l=775 or 800 nm depletion lasers (without ad-
ditional structural modifications of the dyes, such as fused
rings or extra double bonds). As a result, the size and molecu-
lar weight remain quite small (ꢀ570 Da), which is very inter-
esting in terms of cell permeability and the possibility of creat-
ing a “compact” fluorescent marker for lipids, carbohydrates,
and other relatively small molecules. The new SiRF dyes are de-
signed in such a way that cyclization to the colorless closed
form is completely avoided. At the same time, both a batho-
chromic shift and conjugation linker are provided by proper
modifications at the single reactive site of the silicon–rhod-
amine o-carboxyl group in the phenyl ring (attached to the C-9
meso position of the fluorophore). In these respects, SiRF dyes
are alternatives to the dyes SiR680, SiR700, SiR720,[9b] and SiR-
methyl.[9h] One should note that the equilibrium between the
fluorescent open and nonfluorescent closed forms may appear
as an undesired phenomenon. Some hydrophobic xanthene
dyes with the “unprotected” ortho-carboxyl group may exist
predominantly in the closed colorless form when the dye con-
jugate is placed into a nonpolar medium. In this regard, SiRF
dyes are complimentary to fluorogenic silicon–rhodamines[9b,h,k]
in which the very strong tendency to cyclize into the nonfluor-
escent lactone form is the main advantage. As we established,
rhodamine carboxylic acid esters obtained from secondary al-
cohols could be good alternatives to classical amide-based
spacers. Moreover, as a useful addition to the well-established
dye SiR-CO2H, we synthesized (presumably fluorogenic) com-
pounds 15a,b that contained the SCH2COOH group as
a second linking site. These bifunctional dyes were prepared
by means of nucleophilic aromatic substitution (SNAr) of one
fluorine atom in the SiRF dyes. In the solid state and in nonpo-
lar media these compounds also exist predominantly in the
closed colorless form. Therefore, their derivatives might be se-
lective fluorescent markers for living cells. Importantly, these
bifunctional dyes are spectrally redshifted by 10–12 nm relative
to SiR-CO2H (see Scheme 1 for structures).
Figure 3. Bleaching behavior under STED conditions. Bleaching curves were
obtained by using samples of the antibody-labeled protein vimentin in
mammalian cells, as described in the Supporting Information. One sample
spot was scanned 10 times by using both excitation and depletion beams at
l=640 and 800 nm, respectively. Pixel intensities per frame were added and
plotted against the number of frames. The resulting curves were normalized
to their maximum value and plotted in one graph for comparison of the
bleaching rates. For dye structures, see Scheme 1.
Figure 4. GSDIM images obtained with ATTO 655 and 2-H dyes. Nuclear
pore complexes of mammalian cells were labeled by indirect immunofluor-
escence with ATTO 655 (A, C) and 2-H (B, D) dyes (see the Supporting Infor-
mation for details). To compare the performance of the dyes in different buf-
fers, the samples were then embedded in Glox/2-mercaptoethyl amine
(MEA) buffer (A, B) and in PBS (C, D). Panels A) and B) contain images of the
middle planes, whereas panels C) and D) represent images of the top planes
of the nucleus. In both buffers, the average number of photons detected
per event was about 450.
655: the image in Figure 4D is brighter, possesses better locali-
zation precision, and provides a more realistic (uniform) distri-
bution of nuclear pore complexes (as confirmed by super-reso-
lution STED microscopy). The good GSDIM performance of oxa-
zine 2-H in PBS is especially noteworthy because fluorophores
Oxazine dyes also provide a sufficient redshift (absorption
maximum at l=660 nm), yet the dye core is bulky and hydro-
Chem. Eur. J. 2015, 21, 13344 – 13356
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Full Paper
phobic by nature. To improve the STED imaging performance
of oxazine dyes, they were decorated with a phosphate group.
The hydroxyl-containing dye substrate was subjected to
a straightforward phosphorylation with POCl3 in appropriate
solvents (followed by hydrolysis in basic media). This simple
phosphorylation protocol might be very useful in the long
term because it completely avoids the formation of phospho-
nates (undesired side products; see ref. [13a]) and can be ap-
plied to a wide range of substrates with free hydroxyl alkyl
groups. The phosphorylated zwitterionic dye (2-H) demonstrat-
ed high polarity, high solubility in water, and a negative net
charge. It performed very well in GSDIM and demonstrated
a greater improvement in STED images (especially with l=
755 nm depletion laser) relative to its analogue ATTO 655.
A predictable and constant fluorescence lifetime (which
does not change upon conjugation with various biological tar-
gets) is also a very important parameter of a fluorophore. If
the fluorescence lifetimes of two or more spectrally similar
emitters differ by about 0.6–0.8 ns, they can be detected sepa-
rately by using a gated detector.[20] Oxazine dyes have not
been used for such “lifetime separation” schemes so far, al-
though they seem to have the shortest fluorescence lifetime
among the microscopically useful fluorescent dyes. In the case
of the oxazine dyes, the lifetime proves to be exactly the same
(1.78–1.79 ns) with a very high precision for two different dyes
in solution (dye 2-H and Atto 655) and in their conjugates with
different substrates as well. This remains true for other dyes
such as silicon–rhodamines (dyes 1a-H, 1b-H, and SiR-CO2H)
and the “conventional” rhodamine KK114L (see Scheme 1 for
structures). As a whole, we have a set of three lifetime values
with noticeable differences: 1.8, 2.4, and 3.6 ns for oxazines, sil-
icon–rhodamines, and conventional rhodamines, respectively
(see also Table 1). If two detection channels (windows) for the
“short” and “long” lifetimes are used, one can expect that oxa-
zine and rhodamine dyes will be registered mainly in one
channel (for the markers with short and long lifetimes, respec-
tively), whereas silicon–rhodamines will appear in both chan-
nels significantly. This approach may help to discriminate be-
tween three markers by using only two detection channels.[23]
The photophysical properties and imaging performance of
the new far-red emitting fluorophores are discussed in detail
and compared with certain well-established “near-IR” dyes: SiR-
CO2H,[9h] ATTO 655 (see www.atto-tec.com), as well as bench-
mark dyes KK114L and Abberior STAR RED (www.abberior.com),
although the last two dyes were designed for STED with l=
775 nm laser.[22] This study has demonstrated that one can per-
form STED imaging with longer wavelength STED beams (l=
800 nm) by using spectrally fitted far-red emitting dyes and
the best near-IR rhodamine dyes. In the long term, the advan-
tages of a red-shifted STED irradiation may become crucially
important. Photodamage, protein autofluorescence, and light
scattering would be reduced, while achieving deeper light
penetration. The chemistry, scope, and applications of silicon–
rhodamines and oxazines have been broadened and the great
potential of these two important dye families has been shown.
Acknowledgements
We are grateful to Marianne Pulst, Kurt Müller, Jürgen Bienert
(MPI-BPC Gçttingen), Reinhard Machinek, Dr. Holm Frauendorf,
and their co-workers (Institut für Organische und Biomolekula-
re Chemie, Georg-August-Universität Gçttingen) for recording
the spectra. S.W.H. acknowledges a grant by the Bundesminis-
terium für Bildung und Forschung (BMBF 513) within the pro-
gram Optische Technologien für Biowissenschaften und Ge-
sundheit (FKZ 13N11066). Highly appreciated are the assistance
of Dr. Ellen Rothermel for protein labeling, the dye photos by
Maksim Sednev, and the manuscript proofreading by Jaydev
Jethwa.
Keywords: dyes/pigments · fluorescence · structure–activity
relationships · super-resolution microscopy · synthesis design
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[22] The dye KK114L (previously reported as KK1119; see ref. [5d]) has the
same fluorophore as KK114 (see refs. [5d,e], except the linker with a car-
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these two rhodamines are more photostable than SiR-CO2H or SiRF
dyes, despite having additional conjugated double bonds (as seen in
Scheme 1). The latter may undergo photoinduced reactions that cause
partial bleaching (so-called “bluing”, that is, hypsochromic and hypso-
fluoric shifts). No less importantly, derivatives of KK114 are far less sensi-
tive to changes in the excitation/depletion conditions and other setup
settings and develop far less re-excitation artifacts.
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Received: April 9, 2015
Revised: June 16, 2015
Published online on August 13, 2015
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim