Photoactivatable Rhodamine Spiroamides and Diazoketones Decorated with "universal Hydrophilizer" or Hydroxyl Groups

Article abstract of DOI:10.1021/acs.joc.8b00756

Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers applicable in multicolor super-resolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is often limited by aggregation, unspecific adhesion, and low reactivity caused by insufficient solubility and precipitation from aqueous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels decorated with a reactive group: attachment of 3-sulfo-l-alanyl-beta-alanine dipeptide (a "universal hydrophilizer") or allylic hydroxylation in photosensitive rhodamine diazoketones (and spiroamides). The super-resolution images of tubulin and keratin filaments in fixed and living cells exemplify the performance of "blinking" spiroamides derived from N,N,N′,N′-tetramethyl rhodamine.

Full text of DOI:10.1021/acs.joc.8b00756

Subscriber access provided by Universitaetsbibliothek | Johann Christian Senckenberg
Article
Photoactivatable Rhodamine Spiroamides and Diazoketones
Decorated with “Universal Hydrophilizer” or Hydroxyl Groups
Benoit Roubinet, Matthias Bischoff, Shamil Nizamov, Sergey Yan, Claudia Geisler, Stefan
Stoldt, Gyuzel Y. Mitronova, Vladimir N Belov, Mariano L. Bossi, and Stefan W. Hell
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00756 • Publication Date (Web): 11 May 2018
Downloaded from http://pubs.acs.org on May 11, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 13
1
The Journal of Organic Chemistry
Photoactivatable Rhodamine Spiroamides and Diazoketones Deco-
rated with “Universal Hydrophilizer” or Hydroxyl Groups
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
a
b
a
c
a
Benôit Roubinet, Matthias Bischoff, Shamil Nizamov, Sergey Yan, Claudia Geisler, Stefan Stoldt,
a
a,
a,
a
Gyuzel Y. Mitronova, Vladimir N. Belov *, Mariano L. Bossi, * and Stefan W. Hell
a
Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen,
Germany
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
Abberior GmbH, HansꢀAdolfꢀKrebsꢀWeg 1, 37077 Göttingen, Germany
c
Department of Optical Nanoscopy, LaserꢀLaboratorium Göttingen e.V., 37077 Göttingen, Germany
ABSTRACT: Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers appliꢀ
cable in multicolor superresolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is
often limited by aggregation, unspecific adhesion and low reactivity caused by insufficient solubility and precipitation from aqueꢀ
ous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels
decorated with a reactive group: attachment of 3ꢀsulfoꢀLꢀalanyl – betaꢀalanine dipeptide (a “universal hydrophilizer”) or allylic
hydroxylation in photosensitive rhodamine diazoketones (and spiroamides). The superresolution images of tubulin and keratin
filaments in fixed and living cells exemplify the performance of “blinking” spiroamides derived from N,N,N
′
,N
′
ꢀtetramethyl rhoꢀ
damine.
INTRODUCTION
1
,2
2
Colorless and nonꢀfluorescent spiroamides and spirocyclic
reactive group at the imide nitrogen is nonꢀsoluble in water. It
was attached to cellꢀpenetration peptides and applied in
STORM microscopy of actin in living BSCꢀ1 cells.
3ꢀ5
diazoketones (Scheme 1) are easily prepared from fluoresꢀ
cent rhodamine and carbopyronine dyes. Rhodamine spiroamꢀ
ides can be transformed by light into a colored and fluorescent
openꢀring isomer (Scheme 1A, 1) which cyclizes spontaneousꢀ
ly (in the dark) back into the colorless and nonꢀfluorescent
form. Photolysis of spirocyclic diazoketones obtained from
rhodamines (so called RhꢀNN) and carbopyronines is carꢀ
ried out in the presence of nucleophiles and gives fluorescent
homologs (accompanied by nonꢀfluorescent byꢀproducts).
These transformations may be induced by UVꢀlight (313–380
nm, Scheme 1A, 1), UVꢀ and violet light (405ꢀ440 nm,
Scheme 1B), or with powerful red light in a twoꢀphoton mode
15
Scheme 1. Photoactivation of rhodamine spiroamides (A, 1), spontaneous
blinking of rhodamine and siliconꢀrhodamine based spiroꢀcyclic ethers,
thioethers and amines (A, 2). A Wolff rearrangement of rhodamine and
carbopyronine diazoketones results in fluorescent homologs (B, the “dark”
byꢀproduct is not shown).
3
,4
5
(
650–800 nm, Scheme 1A, 1 and 1B). Another effect – sponꢀ
taneous blinking – was observed with spiroamides prepared
from aliphatic amines, as well as rhodamines and siliconꢀ
rhodamines having spiroꢀcyclic ether, thioether and amine
6ꢀ8
7
8
residues.
Along
with
bisꢀN,N′ꢀ(oꢀnitrobenzyloxy)ꢀ
9
carbonylated Siꢀanalog of Qꢀrhodamine, rhodamine spiroamꢀ
ides and diazoketones were applied as photoactivatable
labels in stochastic optical reconstruction microscopy
and related methods of optical superresoluꢀ
For example, Nꢀaryl derivatives of Rhodamine B
Scheme 1A) were used as small labels on live cell surfaces in
threeꢀdimensional superresolution imaging. An amide obꢀ
tained from Rhodamine B and 4ꢀaminophthalimide bearing a
2,7
3ꢀ5
10,11
(
STORM)
12,13
tion.
(
14
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 13
Interestingly, blinking inside the cells was observed without
activation with UVꢀlight; only 561 nm laser was used for
irradiation and excitation. Prepared from Rhodamine B,
and urethane 1ꢀFmoc converted into amide 2 in a oneꢀpot
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
fashion. For that, the carboxylic acid group in 1ꢀFmoc was
activated with 1,1′ꢀcarbonyldiimidazole (CDI), coupled with
methyl 3ꢀaminopropionate, and then the protecting group
removed with piperidine. Saponification of methyl ester with
aqueous NaOH followed by treating with a strong ionꢀ
1
5
spirocyclic Nꢀalkylamides have a shoulder at 320 nm (ε ~
−1
−1
5
600 M cm ). They do not absorb beyond 350 nm and reꢀ
quire very drastic activation with UV light of 312 nm or 302
16,17
+
nm.
Due to inefficient light activation, spirocyclic Nꢀ
exchange resin (H ꢀform) afforded the hydrophilic dipeptide 3
alkylamides prepared from rhodamines (with exceptions of
in an overall yield of 52%. Chromatography (on regular SiO )
2
6
,7
spontaneously blinking ones ) have not yet been applied in
biologyꢀrelated single molecule localization microscopy. Phoꢀ
was applied only once: for isolation of piperidinium salt 2. To
demonstrate the utility of compound 3 as an efficient polar
linker, we first attached it to a compact rhodamine spiroamide
derived from TMR (Scheme 3). Ester 4ꢀMe in Scheme 3 has
the same “light antenna” (based on 4ꢀaminophthalimide) as
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
ꢀ4
toactivatable RhꢀNN (prepared from carboxylic acid chloꢀ
rides and diazomethane) were attached to primary antibodies
and used as single molecules in superresolution imaging of
18
2,7,15
asymmetric Gꢀprotein coupled receptor oligomers. Hydroꢀ
phobic RhꢀNN were applied for imaging nanostructures by
singleꢀmolecule localization microscopy in organic solvents
spiroamides of Rhodamine B applied in earlier studies.
TMR has a somewhat higher fluorescence quantum yield than
22
23
Rhodamine B (0.41 vs. 0.31 in aqueous solutions). Addiꢀ
tionally, four carbon atoms can be “saved” by using TMR
1
9
(with addition of protic solvents); e.g., for the in situ visualiꢀ
2
0
2
zation of block copolymers’ selfꢀassembly.
instead of Rhodamine B. Using the published method, we
prepared ester 4ꢀMe, cleavedꢀoff the methyl group, activated
the carboxylic acid group in 4ꢀLi and coupled it with an amino
group in dipeptide 3 (Scheme 3). Saponification of ester 4ꢀMe
is accompanied with a ring opening of the phthalimide cycle
Most of photoactivatable rhodamine spiroamides and spiroꢀ
1
ꢀ
cyclic diazoketones are lipophilic and nonꢀwaterꢀsoluble.
5
,7,8,14,15,18ꢀ20
Their imaging performance is limited by aggregaꢀ
tion, unspecific adhesion and low reactivity caused by insuffiꢀ
cient solubility and precipitation from aqueous buffers. In
order to increase polarity of these promising fluorescent markꢀ
ers and stabilize their bioconjugates, we synthesized cysteic
acid – betaꢀalanine dipeptide (“universal hydrophylizer”;
Scheme 2) and attached it to a spiroamide obtained from
(it readily occurs already at pH 9). Fortunately, excess of LiI
in refluxing THF cleanly cleaves the methyl ester group in 4ꢀ
Me within 2 or 3 days. Although this reaction is more rapid in
2
ethyl acetate, the solvent also reacts with lithium iodide (lithꢀ
ium acetate is formed), and the isolation of the target salt 4ꢀLi
is complicated. Conversion of 4ꢀLi to 4ꢀSu was straightforꢀ
ward (Scheme 3). The reaction of an active ester 4ꢀSu with
amino acid 3 (1.4 equiv.) took place in DMF in the presence of
triethylamine and afforded the coupling product 5ꢀH (as triꢀ
ethylammonium salt) in moderate yield (29%). The greater
excess of dipeptide 3 (3ꢀ5 fold) provides a better conversion to
the product (>50%), but the chromatographic separation beꢀ
comes more difficult. The anionic centers in dipeptide 3 reꢀ
mained unprotected, and this provided the shortest synthetic
path. Finally, the relatively stable Nꢀhydroxysuccinimidyl
ester 5ꢀSu was prepared from carboxylic acid 5ꢀH. If cysteic
acid 1ꢀH is used as hydrophilizer without attaching βꢀalanine
“extender”, the stability of active esters becomes low. In one
of the recent applications, a 3D singleꢀmolecule based superꢀ
resolution method (singleꢀobjective selectiveꢀplane illuminaꢀ
tion microscopy) was realized with ester 5ꢀSu as a photoactiꢀ
N,N,N′,N′ꢀtetramethylrhodamine (TMR) (5ꢀH) and two spiroꢀ
cyclic diazoketones prepared from greenꢀ and redꢀemitting
rhodamines (8ꢀH and 9ꢀH). Besides that, we used allylic hyꢀ
droxylation as an “electrically neutral” tool for increasing
21
polarity and applied it to the redꢀemitting RhꢀNN with a
compact polycyclic and hydrophobic core decorated with a
reactive group.
Scheme 2. Cysteic acid – betaꢀalanine dipeptide 3 as a “universal
hydrophylizer”: a) CDI (1.1 equiv.), DMF, r.t., 1 h; Et
3
N (3 equiv.), r.t., 2
O, 0 °C, 1 h;
h; piperidine, r.t., 1 h; b) aq. NaOH (1 M, 3.3 equiv), H
2
+
2
Amberlite IRꢀ120 (H ꢀform), H O.
25
vatable marker conjugated with secondary antibodies. Strucꢀ
ture 3 relates to a similar, yet more complex, compound A in
Scheme 2 prepared by P. A. Grieco et al. in 9 steps starting
24
from Nꢀmethylcarprolactame. Being bound on both sides
with amide bonds, linker A is zwitterionic with a zero net
charge, while linker 3 is negatively charged. The negative
charge provided by hydrophilizer 3 in conjugates was intended
to neutralize the positive charge emerging upon photoactivaꢀ
tion of rhodamine spiroamides or diazoketones (Scheme 1).
RESULTS AND DISCUSSION
Synthesis. Hydrophilic dipeptide 3 (3ꢀsulfoꢀLꢀalanylꢀbetaꢀ
alanine, Scheme 2) was obtained from commercially available
Lꢀcysteic acid (1ꢀH). The amino group in acid 1 was protected,
2
ACS Paragon Plus Environment

Page 3 of 13
The Journal of Organic Chemistry
Scheme 3. Photoactivatable rhodamine spiroamide and diazoketones decorated with a hydrophilic peptide 3: a) LiI, THF, reflux, 2 – 3 d; b) TSTU (1.7
equiv.), Et N, DMF, rt, 4 h; c) 3 (2 equiv.), Et N, (3 equiv.), DMF, rt, 24 h; d) NHS (1.2 equiv.), HATU (3.3 equiv.), Et N (5.0 equiv.), DMF, rt, 16ꢀ19 h; e)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TSTU (40 equiv.), Et
3
N (70 equiv.), CH
3
CN, rt, 1 h; f) 3 (2 equiv.), Et
3
N (5 of 10 equiv), DMF, rt; g) 1 M aq. NaOH (5 equiv.), THF/H
2
O, rt, 23 h.
4
18,20
Scheme 4. Hydroxylated photoactivatable Rh590ꢀNN: a) Ac
1:4, v/v), rt, 4 h; b) Ac
(COCl) , CH Cl , 0 °C ꢀ rt, 6 h, 2. CH
d) aq. NaOH, MeOH, 0 °C, overnight; e) NHS, EDC, CH
overnight.
2
O, pyridine
masked greenꢀemitting dye 6ꢀH (Abberior Cage 500)
and
(
2
O, TMSOTf (9 equiv.), DCM, 0 °C, 1 h; c) 1.
, ether, CH Cl , 0 °C, overnight;
diazoketone 8ꢀH (Scheme 3) prepared from the rhodamine
2
2
2
2
N
2
2
2
4,21
emitting at about 610–620 nm, in a color channel compliꢀ
2 2
Cl , rt,
mentary to emission centered at 650 nm. The standard filter
settings in optical microscopy resolve these emission bands
with minimal crossꢀtalk. A very powerful 775 nm laser used
in stimulation emission depletion microscopyphotoactivates
21
dyes 8ꢀH and 8ꢀSO H in a two photon mode. In an earlier
3
report, the polycyclic hydrophobic framework 8ꢀH had been
decorated with two sulfonic acid groups (compound 8ꢀSO H
3
4
in Scheme 3). However, it is difficult to isolate compound 8ꢀ
SO H in a pure state, free from fluorescent impurities which
3
accumulate in the course of multistep synthesis and manipulaꢀ
tion with protecting groups on unstable intermediates. Thereꢀ
fore, a robust photoactivatable marker with the emission maxꢀ
imum in the range of 610–620 nm, complementary to “caged”
5
carbopyronines and a Siꢀanalog of Qꢀrhodamine (all emitting
9
at about 655 nm), would be an important addition to a set of
2
photoactivatable “masked” fluorescent dyes. As starting
materials
for
“hydrophilization”,
we
used
Nꢀ
Irreversibly photoactivatable rhodamines and carbopyronines
with lightꢀsensitive 3ꢀspiroꢀ2ꢀdiazoindanone fragment
hydroxysuccinimidyl esters 6ꢀSu and 8ꢀSu (Scheme 3). They
reacted with peptide 3 and gave coupling products 7ꢀH and 9ꢀ
H decorated with polar groups (yields 84% and 32%, respecꢀ
tively). Activation of the carboxylic acid residues in diazoꢀ
a
3ꢀ5
(
Scheme 1B) are structurally similar to rhodamine spiroamꢀ
1,2,6,7
ides,
“blinking” spiroꢀcyclic ethers, thioethers and
8
amines. All of them are polycyclic aromatic compounds with
only a few polar groups. For “hydrophilization”, we used a
3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 13
ketones 7ꢀH and 9ꢀH led to aminoꢀreactive Nꢀ based superresolution microscopies, is limited. Indeed, such
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hydroxysuccinimidyl esters 7ꢀSu and 9ꢀSu.
markers are applicable only in the pH range where the equiꢀ
librium is shifted towards the “dark” closedꢀring isomer. To
this end, compounds 4ꢀH (generated from 4ꢀLi in situ) and 5ꢀ
H were studied in aqueous buffered solutions in the range of
pH from 1 to 9. Absorption and emission changes for comꢀ
pounds 5ꢀH and 4ꢀH are given in Figures 1 and S1, respecꢀ
tively. We found that three species are necessary to explain
the observed behavior and a bellꢀshaped curve. In addition to
two species shown in Scheme 1, a third (protonated) form
accounts for decoloration at low pH:
Recently, hydroxylated rhodamines, carbopyronines, siliconꢀ
and germaniumꢀcontaining rhodamines have been prepared
and applied as cellꢀpermeant fluorescent markers in liveꢀcell
STED microscopy with optical resolution below 60 nm.
As alternatives to negatively charged “masked” rhodamines 8ꢀ
21,27
SO H and 9ꢀH (Scheme 3), we prepared new hydroxylated
3
diazoketones (12ꢀH,H and 13ꢀH,H in Scheme 4) and their Nꢀ
hydroxysuccinimidyl esters 12ꢀH,Su and 13ꢀH,Su. The spiroꢀ
diazoketone group was introduced by activation of the free
carboxylates in dyes 10ꢀAc,Et and 11ꢀAc,Me with oxalyl
chloride followed by reaction with diazomethane (39 and 47%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢄ
ꢄ
ꢂꢃ
→
←
ꢅꢃ
ꢂꢃ
→
←
ꢅꢃ
ꢆꢁꢃꢄ
ꢀ
ꢁ
ꢆꢁ
3ꢀ5
yields). The hydroxyl groups were protected as acetates,
ꢄ
ꢄ
and the “remote” carboxyl group – as an ester. These groups
are stable against HCl evolving in the course of the reaction
with oxalyl chloride. On the other hand, diazoketones are
stable in aqueous alkaline solutions. Therefore, all ester
groups in the intermediates 12ꢀAc,Et and 13ꢀAc,Me could be
hydrolyzed with aqueous NaOH in ethanol, and the desired
+
This protonated form (OFH ) does not absorb light at 550 nm.
The protonation site is yet undefined. Assuming that only the
OF absorbs and emits in the green/yellow spectral region, the
two equilibrium constants were calculated, in addition to the
absorption coefficient of the emissive OF (note that there is
no pH at which this form is present in more than 95% of the
total concentration). The parameters extracted from the best
fits (insets in Figures 1 and S1) are presented in Table 1.
“
4
masked” rhodamines 12ꢀH,H and 13ꢀH,H obtained (Scheme
). Prior to introduction of the diazoketone fragment, but after
formation of the dye core and separation of isomers, dye 10ꢀ
H,Et (precursor of 12ꢀAc,Et) had been decorated with hyꢀ
droxyl groups upon oxidation of allylic methyl group with
Figure 1. Absorption and emission of compound 5ꢀH in buffered
solutions (excitation with 530 nm light). The absorption intensity at the
maximum (558 nm) is plotted vs pH of the solution in the inset; data was
fit with a twoꢀequilibrium model. For the data related to compound 4-H /
28
selenium dioxide. To obtain dye 13-H,H with Nꢀ(2ꢀ
hydroxyethyl) residues, we used benzyl 2ꢀbromoethyl ether
2
9
for Nꢀalkylation of an aminophenol precursor (see Supportꢀ
ing Information for details). The relatively harsh conditions of
the alkylation reaction required robust protection of the hyꢀ
droxyl groups as benzyl ethers. After formation of the rhodaꢀ
4
ꢀLi, see Figure S1.
30
mine fluorophore, the benzyl groups were replaced with
acetyls, because cleavage of benzyl groups requires strongly
acidic or reducing conditions, incompatible with diazoketone
functionality. When compound 11ꢀBn,Me was treated with
BCl or BBr in CH Cl , the desired alcohols reacted further
3
3
2
2
to the corresponding chlorides or bromides. By using a large
excess of TMS triflate (9 equiv.) in acetic anhydride (a reaꢀ
gent and solvent) at 0 °C for 1 h, we managed to obtain dye
1
1ꢀAc,Me directly and with 48% yield.
PHOTOPHYSICAL PROPERTIES
Table 1. Photophysical properties of spiroamides 4ꢀH / 4ꢀLi and 5ꢀH in
aqueous solutions: Ka and Ka – acidity constants, Fl – emission effiꢀ
ciency, Fl – fluorescence lifetime.
Rhodamine spiroamides (RSA) 4-H and 5-H. The fluoresꢀ
cent form of rhodamine spiroamides can be generated by
irradiation with UVꢀlight (photoinduced activation). In addiꢀ
tion, they are also halochromic: the protonation of the colorꢀ
less “closed” form (CF) transform it to an “open” (colored
and fluorescent) form (OF; Scheme 1, Y = NH). The pKa
value of such equilibrium depends on the substituents atꢀ
tached to the “central” chromophore and to the lactam ring (R,
1
2
Φ
τ
4-H / 4-Li
5-H
MAX
λ
– Abs (OF), 558
558
nm
MAX
λ
– Em (OF), nm 583
583
31
558 nm
-1
-1
and Z in Scheme 1). Therefore, the pH range in which the
RSAs can be used as single molecule markers in localizationꢀ
ε
(OF), M cm
40 000
51 000
1.8±0.2
2
Ka *10
2.3±0.3
1
4
ACS Paragon Plus Environment

Page 5 of 13
The Journal of Organic Chemistry
5
Ka *10
3.0±0.4
0.21
2.6±0.3
0.22
absorption spectra of the separate peaks confirmed this asꢀ
2
1
2
3
4
5
sumption. Fluorescence quantum yields and emission lifeꢀ
times were calculated from solutions irradiated at low converꢀ
sions (ODMAX < 0.1).
ΦFl
τFl, ns
0.9
0.9
Table 2. Photolysis of diazoketones 8ꢀH, 9ꢀH and 12ꢀH,H in methanol: FP
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Equilibrium constants are consistent with the values obtained
previously for similar RSAs derived from 4ꢀ
– fluorescent product; DP –“dark” product, φ ꢀ quantum yields of the
photochemical reactions; for other definitions, see Table 1. Fluorescence
quantum yields and lifetimes correspond to FP.
2,7
aminophthalimide, with absorption coefficients being 40ꢀ
50% lower than the extinction of the parent tetramethyl rhoꢀ
damine, a tendency observed for (some) tertiary amides preꢀ
8
ꢀH
9ꢀH
595
618
5.7
12ꢀH,H
590
610
4.6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
MAX
594
617
4.8
pared from rhodamines. The emission quantum efficiencies
of the fluorescent forms are equal in compounds 4ꢀH and 5ꢀ
H.These results indicate that at pH > 6 (two units over pKa2),
both compounds are in the “closed” forms and can be used as
fluorescent photoactivable probes at biologically relevant
neutral pH (6–8). However, at higher pH (>8), the
phthalimide unit (light antenna) hydrolyses rapidly forming
monoamides of the phthalic acid, and the efficiency of photoꢀ
activation decreases strongly. Incorporation of the hydrophiꢀ
lizer has no significant effect on the photophysical properties
of RSAs. The improved polarity of compound 5ꢀH has a side
effect: amides obtained from this carboxylic acid are cell
impermeable (see next section with imaging results).
λ
– Abs (FP), nm
MAX
λ
– Em (FP), nm
3
φFP*10
3
φDP*10
1.3
1.2
0.8
φFP
/
φDP
3.6
4.9
5.5
ΦFl
0.82
4.0
0.86
4.1
0.85
4.0
τFl, ns
The photoꢀphysical properties are given in Table 2. We found
no significant differences among all compounds in Table 3.
All of them show high emission quantum yields and relatively
long lifetimes. Activation efficiencies are below 1% and allow
manipulations with dyes (isolation by flash chromatography,
preparation of derivatives, antibody staining and immunoꢀ
labeling) without rigorous exclusion of light. However, unꢀ
caging quantum yields are high enough for efficient activation
using relatively low light intensities, even with 405 nm light.
Spiro-cyclic diazoketones 8-H, 9-H, 12-H,H. While RSAs
can be reversibly photoactivated by irradiation with UV light,
spiroꢀcyclic diazoketons in Schemes 3 and 4 are masked
fluorescent dyes (rhodamines) that can be photoactivated with
UV and visible light (λ< 440 nm), or in a 2ꢀphoton mode with
4
very powerful red and near IR light (e.g., 775 nm laser).
Under these conditions, an irreversible extrusion of dinitrogen
33
is followed by Wolff rearrangement (carbene to ketene) and
Figure 2. Photoactivation of compounds 9ꢀH and 12ꢀH,H dissolved
methanol with 405 nm light. Black curves – initial state (diazoketones);
red curves – absorption and emission spectra (excitation with 530 nm
light) after photoactivation.
3
addition of methanol, water or another nucleophile. To deꢀ
termine the key parameters of this transformation − the quanꢀ
tum efficiency of the photolysis, product distribution and the
properties of the fluorescent dye − we irradiated solutions of
compounds 8ꢀH, 9ꢀH and 12ꢀH,H in methanol with 405 nm
2
light (∼100 mW/cm ; Figure 2). This wavelength is available
as an illumination source in commercial microscopes. To
calculate photoactivation efficiencies, the light intensity was
measured with a chemical actinometer (azobenzene in MeOH)
having the same geometry, as for photolysis. The progress of
the reaction was monitored by HPLC under conditions, where
the starting compound, the photoproduct (new fluorescent
dye) and a “dark” byproduct were clearly separated (Figure
S2; for structures of the fluorescent and “dark” products, see
refs. 3 and 4). The amount of fluorescent product was estiꢀ
mated assuming all dyes (8ꢀH, 9ꢀH and 12ꢀH,H) have the
Superresolution imaging. Rhodamine spiroamides prepared
2,7,14,15
from Rhodamine B
labels in STORM
superresolution
were applied as photoactivatable
and related methods of optical
10,11
12,13
based on single molecule switching
(SMS). Addition of solubilizing groups to the hydrophobic
core structure (rhodamine + light antenna) provided higher
solibility and sufficient concentration of the reactive marker
in aqueous solutions (in this study). In previous experiments
with polycyclic spiroamides prepared from Rhodamine B and
similar dyes, we often applied Nꢀhydroxysulfosuccinimdyl
5
ꢀ
1
ꢀ1
same absorption coefficient (61000 M cm at the maxiꢀ
4
mum). The amount of “dark” product was calculated from
the absorption at 660 nm, where the fluorescent product has
3ꢀ5
negligible absorption. The HPLC analyses of irradiated
solutions were performed using diode array detection, and the
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 13
2
,7
esters in immunolabeling. These reagents have a negatively
charged group and do not precipitate from aqueous buffers so
easily as more lipophilic Nꢀhydroxysuccinimidyl (NHS) esters
do. Unfortunately, Nꢀhydroxysulfosuccinimdyl esters are less
stable than their neutral analogs and have to be generated in
situ. In this respect, compound 5ꢀSu is advantageous: it is
based on TMR instead of Rhodamine B (see Synthesis) and
represents a relatively stable NHS ester which can be isolated
rhodochrous dehalogenase),which is able to selectively and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
rapidly form a covalent bond with the substrate. The images
of keratin in living Ptk2 cells were obtained with compounds
4ꢀHalo (Scheme 3) and TMRꢀHalo (commercially available
from Promega Corp. as amide obtained from 6ꢀcarboxyꢀTMR
and amine in Scheme 3). The overview image (Figure 4) was
obtained using
a
Leica DM 6000B epifluorescence
microscope (Leica Microsystems, Germany), and the superꢀ
3
4
and used in bioconjugation experiments in
a
wide
resolution image was taken in the same setup as in Figure 3.
concentration range. Higher degrees of labeling (up to 3ꢀ5 and
even more) can be achieved by using ester 5ꢀSu, without
excessive precipitation of the labeled protein or aggregation
of the unspecifically labeled objects. Figure 3 shows superꢀ
resolution and conventional images of αꢀtubulin in a Ptk2 cell
labeled by ester 5ꢀSu conjugated with a secondary antibody.
The superꢀresolution image was taken in a customꢀbuilt setup
(modified, ref. 36) implementing the principle of SMS in
optical nanoscopy.
Only in one case (Figure 3), a laser emitting at 375 nm was
used for the photoactivation. For imaging of keratin (Figure
4), UV light was not required, because the blinking density
was high enough. Thus, the keratin filaments are resolved in
the superresolution mode, and their morphology revealed.
However, due to highly unpolar nature of the probe 4ꢀHalo,
unspecific background is present in the SMS image (Figure
4).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. In vivo labelling of Keratin 6A-Halo fusion proteins with
Halo Tag TMR Ligand and 4-Halo probes. Transiently transfected
Ptk2 cells expressing KRT6AꢀHalo under the control of a CMV promotor
were incubated with Halo Tag TMR ligand (Promega Corporation, USA;
5 µM) or 4ꢀHalo (10 µM) for 30 min followed by a washing step (30
min); pH = 7.2. For excitation, the sample plane was illuminated with an
Figure 3. Superꢀresolution (left) and conventional (right) images of 5ꢀSu
labeled αꢀtubulin in a Ptk2 cell. The sample was mounted in Mowiol.
Reconstruction from a sequence of 40 000 single frames, each recorded
with a camera exposure time of 20 ms. A 532 nm laser was used for wideꢀ
2
field excitation with an average intensity of 2 kW/cm in the sample
2
average intensity of 1 kW/cm (532 nm laser). No UV light was used for
plane. For activation, a 375 nm laser was kept constant at an average
2
activation. Except for contrast stretching, no further image processing
was applied. The superꢀresolution image (inset) was reconstructed from a
sequence of 20 000 single frames, each recorded with a camera exposure
time of 10 ms. The SMS image contains ca.140 000 localized positions;
each plotted as a 2D Gaussian with FHWM of 30 nm. Scale bars: 1 µm.
intensity of 0.1 W/cm during the acquisition. The SMS image contains
6
ca. 2.3*10 localized positions; each plotted as a 2D Gaussian with full
width at half maximum (FHWM) of 20 nm. The conventional image is
the sum of the backgroundꢀcorrected single camera frames. Scale bars: 1
µm.
The left image in Figure 3 was acquired with superesolution;
it is bright and contrastꢀrich. Some diffuse background is seen
in the upper left corner, but the tubulin filaments are “smooth”
and do not have the explicit dotꢀlike pattern (often observed
with hydrophobic markers). The presence of the negatively
charged groups in spiroamides 5ꢀR makes them cellꢀ
impermeable. Therefore, they are generally not applicable
inside living cells. On the other hand, spiroamides 4ꢀR are
uncharged and “small” enough to penetrate through the outer
plasma membranes of live cells. For specific labeling of the
intracellular targets in living cells, we used a wellꢀestablished
Conclusion and outlook. Compounds 4ꢀR and 5ꢀR are introꢀ
duced as cellꢀpermeate and cellꢀimpermeable photosensitive
rhodamine spiroamides, respectively. The structure 5ꢀR inꢀ
corporates an amino sulfonic acid residue as a solubilizing
®
35
and robust procedure based on HaloTag fusion proteins. In
this technology, the protein of interest is genetically fused
36
(tagged) with an engineered enzyme (modified Rhodococcus
unit. Spiroꢀamides 4ꢀR and 5ꢀR were shown to be applicable
6
ACS Paragon Plus Environment

Page 7 of 13
The Journal of Organic Chemistry
described as follows: s = singlet, d = doublet, t = triplet, q =
in superresolution microscopy based on single molecule
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
switching. The intracellular performance of the probes 4ꢀR
can be improved by attaching neutral solubilizing groups; e. g.
oligo(ethylene glycol) residues. In this context, the use of
neutral polar groups is advantageous, because the negatively
charged residues are known to inhibit cellꢀpermeability, and
the positively charged – facilitate nonꢀspecific binding of the
probes with cell organelles. Photoactivatable dye 5ꢀH enabled
to realize singleꢀobjective selectiveꢀplane illumination miꢀ
quartet, quint = quintet, m = multiplet or overlap of nonꢀ
equivalent resonances. J values are given in Hz. Massꢀspectra
with electroꢀspray ionization (ESIꢀMS) were recorded on a
Varian 500ꢀMS spectrometer (Agilent). ESIꢀHRMS were
recorded on a MICROTOF spectrometer (Bruker) equipped
with an Apollo ion source and a direct injector with an LCꢀ
autosampler Agilent RR 1200. Analytical and preparative RPꢀ
HPLC were carried out with a Knauer Smartline system
equipped with a Dionex Ultimate 3000 detector or a UVꢀVIS
detector 2500. Automated flash chromatography on regular
silica gel and reversed phase (RPꢀC18) cartridges was perꢀ
formed with a Biotage Isolera One device. HPLC systems:
System A: HPLC (Kinetec C18 100 column, 5 ꢁm, 4.6 × 250
25
croscopy. The optical setup was constructed on the basis of
a conventional Nikon TiꢀE microscope, and photoactivation
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5
was achieved with 405 nm laser (focusable light), highlightꢀ
ing the importance of a lightꢀabsorbing “antenna” incorpoꢀ
rated into the spiroꢀxanthene fragment and its sensitivity not
only to 375 nm light (this report) but also to irradiation at 405
nm. Diazoketones 12ꢀH,R and 13ꢀH,R are intended for further
use in twoꢀ and multicolor superresolution imaging based on
stimulated emission depletion of SMS modalities. Taking into
mm) with CH CN and water (+0.05% v/v TFA) as eluents;
3
linear gradient from 30 to 100% of CH CN in 20 min, flow
3
rate 1.2 mL/min; detection at 260 nm. System B: HPLC (Euꢀ
rospherꢀ100 C18, 5 ꢁm, 250 × 4 mm) with CH CN and water
3
4
account the published data, we expect them to be photoactiꢀ
(+0.05% v/v TFA) as eluents; linear gradient from 30 to 100%
vatable by STED laser (775 nm) in a twoꢀphoton mode. Imꢀ
portantly, neither of these probes requires the use of “blinking
buffers” essential, for example, for cyanine dyes applied in
of CH CN in 25 min, flow rate of 1.0 mL/min; detection at
3
254 nm. System C: HPLC (Eurospherꢀ100 C18, 5 ꢁm, 250 × 4
mm with CH CN and water (+0.05% v/v TFA) as eluents;
3
11
SMS experiments.
linear gradient from 50 to 100% of CH CN in 25 min, flow
3
rate of 1.0 mL/min; detection at 254 nm. System D: automated
flash purification device Biotage Isolera One (ISOꢀ1EW);
cartridge PFꢀC ꢀHC, 30 ꢁM, with 20 g of RP silica gel; eluꢀ
EXPERIMENTAL SECTION
18
General Remarks. Flash column chromatography was perꢀ
formed using regular silica gel 60 (40ꢀ63 ꢁm) from Maꢀ
ent: H O / CH CN (linear gradient from 40 % to 60% in 10
2
3
column volumes), flow rate of 20 mL/min; detection at 254
nm. System E: HPLC (Kinetec C18 100 column, 2.6 ꢁm, 4.6 ×
chereyꢀNagel, or cartridges from Interchim (PFꢀSIHC, 15 ꢁM,
®
2
5 g or 40 g SiO ) or Teledyne Isco (RediSep Rf, 35 ꢁM, 24
2
7
5 mm) with CH CN and water with 0.05% v/v TFA as eluꢀ
3
g or 40 g SiO ). Filtration through BGB or Rotilabo syringe
2
ents; linear gradient from 20% to 100% in 10 min; flow rate
of 1 mL/min; detection at 254 nm.
filters (0.22 and 0.45 µm) removed silica gel from the prodꢀ
ucts, when acetonitrile – water mixtures were used as eluents
in column chromatography on regular SiO . Analytical TLC
General procedures A, B (GP A, B) for the preparation of
N-hydroxysuccinimidyl (NHS) esters. To a solution of
“masked” fluorescent dye (1 equiv.) in DMF (1 mL), NHS
(1.2 equiv.), HATU (3.3 equiv. [A] or 2.1 equiv. [B]) and
2
was performed on Merck Millipore readyꢀtoꢀuse plates with
silica gel 60 (F254). The spots were visualized by illumination
with a UV lamp (λ = 254 and 365 nm) and/or staining with
aq. KMnO solution. Anhydrous DMF and HPLC grade aceꢀ
NEt (5.0 equiv.) were added and the mixture was stirred at
3
4
tonitrile were purchased from SigmaꢀAldrich; DMF was
stored over molecular sieves (4 Å). Water for HPLC was
distilled in an ELGA system. The following starting material
r.t. for 16ꢀ19 h. After removal of volatile materials in vacuo,
the product was isolated by column chromatography on silica
gel.
were synthesized according to published procedures: 2ꢀ
Methyl N-(3-sulfo-L-alanyl)-3-aminopropanoate piperi-
dinium salt (2). To a solution of Lꢀcysteic acid 1 (1.00 g, 5.91
37
formylphthalic acid 4ꢀmethyl ester,
7ꢀ(tertꢀbutylꢀ
29
dimethylsilyl)oxyꢀ1,2ꢀdihydroꢀ2,2,4ꢀtrimethylꢀquinoline, 4ꢀ
aminoꢀNꢀ(methoxycarbonylmethyl)ꢀphthalimide, 6ꢀSu and 8ꢀ
mmol) in water (5 mL) and dioxane (5 mL), NaHCO (1.99 g,
3
2
2
3.7 mmol) was added followed by FmocꢀCl (1.68 g,
Et (compounds 2aꢀCONHS and 2a-CO Et in ref. [4]), and 10-
2
6.49 mmol), and the reaction mixture stirred at r.t. for 2 h.
Water (20 mL) was added, the aqueous phase was separated,
washed with EtOAc (20 mL), acidified to pH 1 with 1 M aq.
HCl and lyophilized to give the NꢀFmoc protected cysteic acid
and NaCl (total amount 3.31 g) as a colorless solid. This
material was suspended in DMF (20 mL), CDI (1.05 g, 6.50
mmol,) was added and the mixture was stirred at r.t. for 1 h.
Afterwards methyl 3ꢀaminopropanoate hydrochloride (866
28
H,Et. Instruments and methods: UV/Vis absorption and
fluorescence spectra were recorded in closed quartz cuvettes
with 1 cm path length on a Varian Cary 4000 UV/Vis and
1
13
Eclipse spectrophotometers, respectively. H and C NMR
spectra were recorded at 25 °C on Varian Mercury 300, Agꢀ
1
3
ilent 400ꢀMR and Inova 500 ( C) spectrometers. Chemical
shifts are given in parts per million (ppm) using the residual
solvent peak(s) as references. Multiplicities of the signals are
mg, 6.21 mmol) and NEt (2.47 mL, 17.7 mmol) were added,
3
7
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 13
and the mixture was stirred at r.t. for 2 h. Finally, piperidine
1 mL, 10 mmol) was added, and the mixture was stirred at
r.t. for 1 h. After evaporation of the solvent and volatile mateꢀ
rials under reduced pressure, the residue was subjected to
4 H, Hꢀ2,4,5,7), 6.64 (d, J = 8.1, 2 H, Hꢀ1,8), 7.09 (m, 1 H, Hꢀ
7′), 7.29 (dd, J = 8.2, 1.9, 1 H, Hꢀ5′′), 7.50 (m, 2 H, Hꢀ5′,6′),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
7
0
.59 (dd, J = 8.2 and 0.6, 1 H, Hꢀ6′′ ), 7.71 (dd, J = 1.9 and
.6, 1 H, Hꢀ3′′), 8.02 (m, 1 H, Hꢀ4′). CꢀNMR (100 MHz,
13
column chromatography on regular silica gel (MeCN/H O; a
2
CDCl ): δ = 38.9, 40.3, 52.8, 98.9, 106.6, 109.2, 121.0, 123.8,
3
linear gradient from 10:1 to 5:1). The product was detected on
1
24.0, 124.1, 128.3, 128.5, 128.6, 129.4, 130.1, 132.7, 133.9,
TLC with a ninhydrin stain; R = 0.33 (MeCN/H O, 5:1).
f
2
143.5, 151.6, 152.6, 153.8, 167.0, 167.1, 167.9, 168.3. HRMS
1
Ester 2 was isolated as a colorless solid (1.74 g, 77%). Hꢀ
(
ESI, QꢀTOF) m/z: [M+H]+ calcd for C H N O , 603.2238 ;
35 31 4 6
NMR (300 MHz, D O): δ = 1.78 (m, ~3H, CH2 in
2
found, 603.2223.
1
6
3
1
.5×C H N), 1.90 (m, ~6H, CH in 1.5×C H N), 2.77 (t, J =
5 11 2 5 11
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Lithium salt 4-Li. Ester 4-Me (966 mg, 1.60 mmol) was
dissolved in dry THF (8 mL) under Ar in a screwꢀcap testꢀ
tube, LiI (649 mg, 4.28 mmol, 2.7 equiv.) added, and the
.5 Hz, 2H, CH CO Me), 3.28 (m, ~6H, CH in 1.5×C H N),
2
2
2
5
11
A
B
.34 (dd, J = 14.8 and 8.3 Hz, 1H, CH H SO ), 3.45 (dd, J =
4.8 and 4.5 Hz, 1H, CH H SO ), 3.58 (m, 2H, CH N), 3.77
3
A
B
3
2
13
mixture refluxed at 90 ˚C (bath temp.) for 48 h. Then, Et O (6
2
(s, 3 H, CO Me), 4.31 (dd, J = 8.2 and 4.5 Hz, 1H). CꢀNMR
2
mL) was added, and the reaction mixture stirred for 5 min.
(
125 MHz, D O): δ = 24.1, 24.9, 35.8, 38.0, 47.2 (all CH ),
2
2
The precipitate was filtered off, washed with Et O and dried
2
5
2.9 (CH/CH ), 53.7 (CH /CH), 171.3, 177.2 (CO). HRMS
3 3
in vacuo to give 4-Li as a light yellow solid (stored in a
(ESI, QꢀTOF) m/z: [MꢀH]– 253.0500, calcd for C H N O S;
7
13
2
6
1
“
dark” flask; 945 mg, 99%). HꢀNMR (400 MHz, CD OD): δ
3
found, 253.0493.
N-(3-Sulfo-L-alanyl)-3-aminopropanoic acid (3). Ester 2
1.66 g, 4.36 mmol) was dissolved in H O (15 mL), the soluꢀ
=
2.89 (s, 12 H), 3.77 (s, 2 H), 6.42 (m, 4 H, Hꢀ2,4,5,7), 6.64
(d, J = 7.8 Hz, 2 H, Hꢀ1,8), 7.02 (m, 1 H, Hꢀ7′), 7.40 (dd, J =
(
2
8
.2 and 1.9, 1 H, Hꢀ5′), 7.57 (m, 3 H, Hꢀ5′/6′/6′′), 7.66 (d, J =
tion was cooled to 0 °C, 1 M aq. NaOH (15 mL) was added,
and the solution was stirred at 0°C for 1 h. The solution was
passed slowly through an Amberlite IRꢀ120 ionꢀexchange
13
8.2, 1 H, Hꢀ3′), 7.93 (m, 1 H, Hꢀ4′). CꢀNMR (100 MHz,
CD OD): δ = 40.6, 42.4, 70.0, 100.1, 107.6, 110.6, 122.0,
3
124.4, 124.5, 125.4, 129.4, 130.1, 131.0, 131.4, 132.2, 134.5,
+
resin (82 g, H ꢀform). Washing of the resin with water (3 × 50
1
35.3, 143.8, 153.4, 154.2, 155.1, 168.9, 169.0, 170.0, 174.3.
mL) and lyophilization afforded compound 3 as a colorless
solid (709 mg, 67%). R = 0.18 (MeCN/H O, 5:1, ninhydrin
HRMS (ESI, QꢀTOF) m/z: [M]– 587.1936, calcd for
f
2
C H N O ; found, 587.1937.
34
27
4
6
stain). Decomposition point: 238–240 °C (sealed capillary):
1
2
D
4
N-Hydroxysuccinimidyl ester 4-Su. To a solution of 4ꢀLi
400 mg, 0.67 mmol) in DMF (3 mL), TSTU (344 mg 1.14
mmol) and NEt (101 mg, 1.0 mmol) were added, and the
= +12.0° (c = 5.0, H O). HꢀNMR (300 MHz, D O): δ =
[α
]
2
2
(
2
8
.69 (t, J = 6.5 Hz, 2H, CH CO Me), 3.40 (dd, J = 14.9 and
2 2
A
B
3
.4 Hz, 1H, CH H SO ), 3.49 (dd, J = 14.9 and 4.4 Hz, 1H,
3
A
B
reaction mixture was stirred at r.t. for 4 h. All volatile materiꢀ
als were removed in vacuum; the residue was takenꢀup in
DCM and applied onto a plug of silica gel. Rapid elution with
EtOAc provided ester 4ꢀSu as a yellow solid (160 mg, 35%).
CH H SO ), 3.57 (m, 2H, CH N), 4.43 (dd, J = 8.4 and 4.4
Hz, 1H). CꢀNMR (125 MHz, D O): δ = 35.7, 38.1, 52.6,
3
2
13
2
5
2
2.9, 170.0, 178.5. HRMS (ESI, QꢀTOF) m/z: [MꢀH]–
39.0343, calcd for C H N O S; found, 239.0345.
6
11
2
6
R = 0.67 (EtOAc). HRꢀMS (ESI, positive mode): 708.2046
f
+
+
Methyl ester 4-Me. Dry 1,2ꢀdichloroethane (20 mL) was
added via syringe into a dry Schlenk flask charged with TMR
(1.09 g, 2.82 mmol), and the suspension was stirred under Ar.
(
found [M+Na] ), 708.2065 (calculated for C H N NaO ).
38 31 5 8
Amide 4-Halo. To 5.0 mg (7.3 µmol) of Nꢀ
hydroxysuccinimidyl ester 4ꢀSu in 0.2 mL DMF, 1.7 mg (7.7
µmol) of H N(CH ) O(CH ) O(CH ) Cl and of 10 µL Et N
POCl (2.0 mL, 22 mmol, 7.8 equiv.) was added, and the
3
2
2 2
2 2
2 6
3
reaction mixture was refluxed (bath temp. ~ 110 ˚C) for 5 h
under Ar. After distilling off the solvent and excess of POCl3
in vacuo, the residue was dissolved in a mixture of dry
were added, and the reaction mixture was stirred overnight at
rt. The product was isolated by chromatography on SiO (13
2
g) using hexane – ethyl acetate (1:2) mixture as eluent. Yield
– 3.0 mg (51%) of yellowish solid. HRMS (ESI, QꢀTOF) m/z:
[M+H]+ 794.3315, calcd for C H N O Cl; found, 794.3291.
CH CN and toluene (10 mL, 1:1) under Ar, 4ꢀaminoꢀNꢀ
3
1
[
(methoxycarbonyl)methyl]ꢀphthalimide (690 mg, 3.15
44
49
5
7
mmol, 1.1 equiv.) added, and the reaction mixture stirred at
0 ˚C overnight. After concentration under reduced pressure,
8
Spiroamide 5-H. To a solution of ester 4ꢀSu (150 mg, 0.22
the residue was dissolved in CHCl (100 mL), washed with
mmol) in DMF (3 mL), NEt (92 ꢁL, 0.66 mmol) and peptide
3
3
water (4 × 20 mL), dried over Na SO and evaporated in
3 (74 mg, 0.31 mmol) were added, and the reaction mixture
was stirred at r.t. for 24 h. After evaporation of the solvent
under reduced pressure, the residue was subjected to column
2
4
vacuo. Column chromatography on silica gel (100 g,
DCM/hexane/1,4ꢀdioxane, 4:3:1) gave compound 4-Me as a
yellow solid (983 mg, 58%) with m. p. 291–293°C (sealed
chromatography on regular silica gel (MeCN/H O, 10:1). The
2
1
capillary). HꢀNMR (400 MHz, CDCl ): δ = 2.95 (s, 12 H,
fractions containing spiroamide 5ꢀH were pooled and lyophiꢀ
3
2
×NMe ), 3.73 (s, 3 H, OCH ), 4.35 (s, 2 H, CH N), 6.36 (m,
lized to give a pink solid (52 mg, 29% yield). R = 0.11
2
3
2
f
8
ACS Paragon Plus Environment

Page 9 of 13
The Journal of Organic Chemistry
1
(
2
1
MeCN/H O, 10:1). HꢀNMR (300 MHz, DMSOꢀd ): δ =
N-Hydroxysuccinimidyl ester 7-Su. Compound 7-Su was
synthesized from 7ꢀH (10 mg, 14 ꢁmol) according to GP B,
and purified by column chromatography on silica gel
2
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
.26 (t, J = 7.2 Hz, 2H, CH CO H), 2.67 (dd, J = 13.8, 6.4Hz,
2 2
H, CH H SO H), 2.79 (dd, J = 13.8, 5.9 Hz, 1H,
CH H SO H), 2.90 (s, 12H, 4×NMe), 3.20 (m, 2H, NHCH
A
B
3
A
B
(MeCN/H O, 25:1 to 10:1). Lyophilization afforded comꢀ
3
2,
2
masked by water peak in DMSO), 4.08 (d, J = 16.5 Hz, 1H,
NCH H ), 4.19 (d, J = 16.5 Hz, 1H, NCH H ), 4.32 (q, J =
pound 7ꢀSu as a slightly brown solid (3 mg, 27% yield). R =
f
C
D
C
D
0.65 (MeCN/H O, 10:1). HRMS (ESI, QꢀTOF) m/z: [MꢀH]–
2
6
2
.4 Hz, 1H, CH), 6.42 (m, 4H, 2/4/5/7ꢀH), 6.64 (d, J = 9.5 Hz,
H, 1/8ꢀH), 7.01 (dd, J = 6.2 and 1.6 Hz, 1H, 7′ꢀH), 7.48 (dd,
880.1477, calcd for C H F N O S; found, 880.1482.
36
29
6
7
11
4
Carboxylic acid 8-H. To a solution of ester 8ꢀEt (10 mg, 16
J = 8.2, 1.9 Hz, 1H, 5′′ꢀH), 7.58 (m, 3 H, 5′/6′/3′′ꢀH), 7.72 (d,
J = 8.2 Hz, 1H, 6′′ꢀH), 7.79 (t, J = 6.0 Hz, 1H, CONHCH2),
ꢁmol) in a mixture of THF/H O (0.3 mL, 2:1, v/v), 1 M aq.
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NaOH (82 ꢁL, 5.0 eq.) was added, and the mixture was stirred
at r.t. for 23 h. After completion of the reaction (TLC control),
the solution was cooled in an ice bath and acidified carefully
7.94 (dd, J = 6.6, 2.3 Hz, 1H, 4′ꢀH), 8.33 (d, J = 6.4 Hz, 1H,
13
CONHCH). CꢀNMR (125 MHz, DMSOꢀd ): δ = 34.7, 35.4,
6
50.8, 52.3, 66.7, 98.3, 98.5, 106.0, 109.2, 109.3, 118.3, 123.3,
123.4, 123.6, 123.8, 127.8, 128.0, 128.3, 128.7, 132.3, 134.1,
142.8, 151.2, 151.6, 153.8, 165.5, 166.7, 166.8, 167.5, 170.1.
HRMS (ESI, QꢀTOF) m/z: [MꢀH]ꢀ 809.2247, calcd for
to pH = 2 – 3 with 1 M aqueous KHSO . The reaction mixture
4
was extracted with CH Cl (2×10 mL), organic solutions were
2
2
dried over Na SO and concentrated in vacuum to give acid 8ꢀ
2
4
1
H as a green solid (9 mg, 94%). R = 0.52 (EtOAc). HꢀNMR
f
C H N O S; found, 809.2245. HPLC (system A): t = 11.5
4
0
37
6
11
R
(400 MHz, CDCl ): δ = 1.26 (s, 6H, CH ), 1.29 (s, 6H, CH ),
3
3
3
min, peak area 92% (260 nm).
1
.70 (q, J = 1.8 Hz, 6H, CH C=), 2.81 (s, 6H, NCH ), 5.18 (d,
3 3
J = 2.0 Hz, 2H, CH=), 6.26 (d, J = 1.8 Hz, 2H), 6.40 (d, J =
N-Hydroxysuccinimidyl ester 5-Su. Compound 5ꢀSu was
synthesized from 5ꢀH (15.0 mg, 18.5 ꢁmol) according to GP
A, and purified by column chromatography on silica gel
1
8
.8 Hz, 2H), 7.18 (d, J = 8.1, 1.8 Hz, 1 H, 7′ꢀH), 8.17 (dd, J =
.1, 1.6 Hz, 1 H, 6′ꢀH), 8.56 (d, 1H, J = 1.8 Hz, 4′ꢀH). Cꢀ
1
3
NMR (100 MHz, CDCl ): δ = 18.7, 27.9, 28.0, 31.1, 56.7,
(MeCN/H O, with a gradient from 25:1 to 10:1). The pure
3
2
9
1
8.1, 106.8, 120.3, 122.0, 124.6, 125.8, 127.1, 129.2, 129.3,
30.3, 134.9, 136.0, 146.4, 152.0, 160.8, 170.1, 186.3. HRMS
fractions were lyophilized to give compound 5ꢀSu as a pink
solid (4.0 mg, 24%). R = 0.57 (MeCN/H O, 10:1). Isolation
f
2
(ESI, QꢀTOF) m/z: [MꢀH]– 585.2507, calcd for C H N O ;
with prep. HPLC provided higher yields (7–8 mg) of 5ꢀSu as
36 33
4
4
found, 585.2503.
a red solid. (Eurospherꢀ100 C18, 5 ꢁm, 250 × 16 mm, CH CN
3
and water (+0.05% v/v TFA) as eluents; linear gradient from
N-Hydroxysuccinimidyl ester 8-Su. Compound 8ꢀSu was
prepared from 8ꢀH (8.0 mg, 14 ꢁmol) according to GP B, and
isolated by column chromatography on silica gel (penꢀ
3
0 to 100% of CH CN in 20 min, flow rate of 10 mL/min;
3
detection at 254 nm. Analytical HPLC (system B): t = 8.8
R
min, HPLC area = 97% (254 nm). HRMS (ESI, QꢀTOF) m/z:
tane/EtOAc, 2:1) as a green solid (8.0 mg, 86%). R = 0.14
f
[
M+Na]+ 930.2375, calcd for C H N NaO S; found,
4
4
41
7
13
(pentane/EtOAc, 2:1). HRMS (ESI, QꢀTOF) m/z: [M+Na]+
9
30.2378.
7
06.2636, calcd for C H NaN O ; found, 706.2630.
40
37
5
6
4
Compound 7-H. To a solution of compound 6ꢀSu (10.0 mg,
Compound 9-H. To a solution of compound 8ꢀSu (8.0 mg, 12
mol) in DMF (0.5 mL), NEt (8.0 ꢁL, 59 ꢁmol) was added
15.2 ꢁmol) in DMF (0.5 mL) were added NEt (21 ꢁL, 0.15
3
ꢁ
3
mmol) and peptide 3 (7.3 mg, 31 ꢁmol), and the mixture was
stirred at r.t. for 16 h. The solvent was evaporated under reꢀ
duced pressure, and the residue was subjected to column
chromatography on silica gel. Elution with a mixture of
followed by peptide 3 (6.2 mg, 26 ꢁmol), and the mixture was
stirred at r.t. for 5 days. After removal of volatile materials in
vacuo, the residue was subjected to column chromatography
on silica gel (MeCN/H O, 10:1), and compound 9ꢀH was
2
MeCN/H O (10:1) followed by lyophilization afforded comꢀ
2
isolated as a green solid (3.0 mg, 32%). R = 0.64
f
pound 7ꢀH as a slightly brown solid (10 mg, 84%). R = 0.20
1
f
(
MeCN/H O, 10:1). HꢀNMR (300 MHz, DMSOꢀd ): δ = 1.24
2
6
1
(
MeCN/H O, 10:1). HꢀNMR (300 MHz, DMSOꢀd ): δ = 1.02
2
6
(
s, 6H, CH ), 1.28 (s, 6H, CH ), 1.63 (s, 6H, CH ), 2.31 (t, J =
3 3 3
(
t, J = 7.2, “1H”, Et N), 2.33 (t, J = 7.0 Hz, 2H CH CO H),
3
2
2
7
.2 Hz, 2H, CH CO H), 2.78 (s, 6H, NCH ), 2.90 (m, 1H,
2 2 3
2
3
4
8
2
.66 (q, “0.8H”, J = 7.2 Hz, Et N), 2.90 (m, 2H, CH SO ),
A
B
A
B
3
2
3
CH H SO H), 3.24 (m, 3H, NCH +CH H SO H), 4.48 (m,
3
2
3
.25 (t, J = 6.6 Hz, 2H, CH N), 3.94 (m, J = 10.0 and 7.2 Hz,
2
1
H, CH), 5.27 (s, 2H, CH=), 6.25 (s, 2H), 6.34 (s, 2H), 7.16
H, CH CF ), 4.49 (m, 1 H, J = 5.3 Hz, CH), 6.46 (dd, J =
2
3
(d, J = 8.1 Hz, 1H, 7′ꢀH), 7.95 (t, 1H, CONHCH ), 8.05 (dd, J
2
.6, 2.4 Hz, 2H), 6.54 (m, 4H), 6.64 (dd, J = 8.6 and 4.3 Hz,
H), 7.15 (d, J = 8.1 Hz, 1H, 7′ꢀH), 7.91 (t, J = 5.4 Hz, 1H,
= 8.1, 1.5 Hz, 1H, 6′ꢀH), 8.23 (d, J = 1.5 Hz, 1H, 4′ꢀH), 8.91
13
(d, J = 5.6 Hz, 1H, CONHCH). CꢀNMR (125 MHz, DMSOꢀ
CONHCH ), 8.04 (dd, J = 8.1, 1.6 Hz, 1H, 6′ꢀH), 8.23 (d, J =
2
d6; due to the presence of the chiral center, “right and “left”
parts of the masked fluorophore are inequivalent): δ = 17.9,
1
.6 Hz, 1H, 4′ꢀH), 8.88 (d, J = 5.7 Hz, 1H, CONHCH. HRMS
(ESI, QꢀTOF) m/z: [MꢀH]– 783.1313, calcd for
2
5
5.2, 27.66/27.71, 27.94/27.96, 30.8. 35.8, 45.7, 48.7,
1.5/51.8, 56.3, 74.6, 97.3, 106.1, 119.3, 120.7, 121.0, 125.3,
C H F N O S; found, 783.1317.
3
2
26
6
6
9
9
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 13
1
1
8
25.58/125.61, 129.4, 133.6, 134.4, 134.7, 145.8, 151.3,
58.1, 164.7, 170.3, 185.3. HRMS (ESI, QꢀTOF) m/z: [MꢀH]–
07.2818, calcd for C H N O S; found, 807.2885. HPLC
70:30) gave an orange oil (787 mg, 74%). Rf (nꢀ
1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hexane/EtOAc, 70:30) = 0.29. HꢀNMR (400 MHz, CDCl ): δ
3
= 1.26 (s, 6H), 1,92 (d, J = 1.5, 3 H, CH C=), 3.45 (t, J = 7.2,
4
2
43
6
9
3
(system C): t = 7.1 min, peak area 91% (254 nm).
2 H, CH N), 3.60 (t, J = 7.8, 2 H, CH O), 4.55 (s, 2 H,
R
2
2
CH Ph), 4.84 (br. s, 1 H, OH), 5.09 (q, J = 1.5, 1 H, CH=),
2
Compound 9-Su. Compound 9ꢀSu was synthesized from 9ꢀH
(3.0 mg, 3.7 ꢁmol) according to GP B, and purified by colꢀ
umn chromatography on silica gel (MeCN/H O, 25:1 to 10:1).
The pure fractions were lyophilized to afford ester 9ꢀSu as a
slightly green solid (1.3 mg, 39%). R = 0.77 (MeCN/H O,
5
.95 (d, J = 2.4, 1 H), 6.07 (dd, J = 8.1 and 2.3, 1 H), 6.90 (d,
13
J = 8.1, 1 H), 7.41ꢀ7.26 (m, 5 H). CꢀNMR (101 MHz,
2
CDCl ): δ = 18.9, 28.6, 43.7, 56.8, 67.8, 73.4, 98.0, 102.4,
3
1
1
16.6, 125.0, 126.8, 127.6, 127.9, 128.0, 128.6, 138.4, 145.5,
f
2
56.6. ESIꢀMS, positive mode, m/z(rel. int., %): 324.2 (100)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10:1). MS (ESI, negative mode) m/z: [MꢀH]– 904.3, calcd for
C H N O S; found, 904.9. HPLC (system C): t = 8.7 min,
+
[
M+H] , 324.4 (C H NO ).
21
26
2
4
6
47
7
11
R
peak area = 96% (254 nm).
Triester 10-Ac,Et. To a cold solution of compound 10-H,Et
74 mg, 0.12 mmol; see Scheme 4) in dry pyridine (2 mL),
Rhodamine 11-Bn,Me. In a sealed tube, 2ꢀformylphthalic
37
28
acid 4ꢀmethyl ester
(91 mg, 0.41 mmol), Nꢀ[2ꢀ
(benzyloxy)ethyl]ꢀ1,2ꢀdihydroꢀ2,2,4ꢀtrimethylquinolinꢀ7ꢀol
(
(370 mg, 1.15 mmol) and pꢀTsOH (14.1 mg, 0.08 mmol) were
Ac O (0.5 mL) was added at 0°C, and the reaction mixture
2
dissolved in propionic acid (1.6 mL), and the resulting mixꢀ
ture was heated at 80 °C overnight. Then, tetrachloroꢀ1,4ꢀ
benzoquinone (100 mg, 0.41 mmol) was added, and the reacꢀ
tion mixture was stirred at 80 °C for 2 h. After cooling down
to rt, concentration under reduced pressure and purification by
flash chromatography on silica gel (DCM/MeOH; step gradiꢀ
was stirred at r.t. for 4 h. Volatile materials were removed in
vacuo, the residue was dissolved in DCM (30 mL), washed
successively with aq. HCl (0.1 M, 10 mL) and sat. aq. Naꢀ
HCO (20 mL), dried over Na SO and concentrated in vacuo.
Column chromatography on silica gel (40 g, 15 ꢁM,
DCM/MeOH, with a linear gradient from 100:0 to 80:20)
3
2
4
ent from 100:0 to 90:0), rhodamine 11ꢀBn,Me was isolated as
afforded compound 10ꢀAc,Et as a blue solid (49 mg, 58%).
1
1
a blue solid (99 mg, 30%). HꢀNMR (400 MHz, CDCl
3
): δ =
HꢀNMR (400 MHz, CDCl ): δ = 1.35 (s, 6 H), 1.37 (s, 6 H),
3
1
7
2
.34 (s, 12 H), 1.64 (s, 6 H), 3.62 (t, J = 7.5, 4 H), 3.70 (t, J =
.9, 4 H), 3.89 (s, 3 H, OCH ), 4.59 (s, 4 H, OCH Ph), 5,23 (s,
H, Hꢀ3,10), 6.43 (s, 2 H, Hꢀ1,12), 6.47 (s, 2 H, Hꢀ6,7), 7.45ꢀ
1
.45 (t, J = 7.1, 3 H, CH CH ), 1.83 (s, 6 H, 2×CH CO), 2.87
3 2 3
3
2
(s, 6 H, CH N), 4.45 (q, J = 7.1, 2 H, CH CH O), 4.55 (s, 4 H,
3
3
2
CH OAc), 5.46 (s, 2 H, 2×CH=), 6.33 (s, 2 H, Hꢀ1,12), 6.35
2
7.26 (m, 10 H), 7.81 (s, 1 H, Hꢀ7′), 8.23 (m, 2 H, Hꢀ4′,5′).
(s, 2 H, Hꢀ6,7), 7.24 (d, J = 8.7, 1 H, Hꢀ7′), 8.29 (dd, J = 8.0
13
CꢀNMR (101 MHz, CDCl ): δ = 18.4, 29.2, 29.3, 44.5, 52.6,
13
3
and 1.5, 1 H, Hꢀ6′), 8,72 (d, J = 1.6, 1 H, Hꢀ3′). CꢀNMR
101 MHz, CDCl ): δ = 14.4, 20.8, 27.7, 28.2, 31.3, 57.1,
58.1, 67.4, 73.6, 96.9, 121.4, 122.7, 126.4, 127.1, 127.8,
127.9, 128.6, 130.1, 130.7, 134.3, 138.0, 148.6, 154.7.0,
(
6
1
3
1.8, 64.0, 97.7, 106.3, 117.3, 122.7, 124.6, 126.4, 127.2,
32.4, 132.7, 135.2, 148.1, 153.4, 165.3, 168.5, 170.5. HRMS
1
66.0, 168.6. HRMS (ESI, QꢀTOF) m/z: [M+H]+ 817.3847,
calcd for C H N O ; found, 817.3850.
52
52
2
7
(ESI, QꢀTOF) m/z: [M+H]+ 707.2963, calcd for C H N O ;
41 43 2 9
found, 707.2957.
Rhodamine 11-Ac,Me. To a solution of rhodamine 11ꢀ
Bn,Me (90 mg, 0,11 mmol) in acetic anhydride (2 mL), a
freshly prepared solution of TMSOTf in DCM (500 ꢁL, 50%
v/v, 0.96 mmol) was carefully added at 0 °C, and the reaction
mixture was stirred for 1 h at 0°C. Then, the reaction mixture
N-[2-(Benzyloxy)ethyl]-1,2-dihydro-7-hydroxy-2,2,4-
trimethylquinoline. In sealed tube, 7ꢀ(tertꢀ
butyldimethylsilyl)oxyꢀ1,2ꢀdihydroꢀ2,2,4ꢀtrimethylquinoline
1.0 g, 3.3 mmol), DIPEA (1.15 mL, 6.60 mmol,) and benzyl
ꢀbromoethyl ether (1.04 mL, 6.60 mmol) were combined and
a
29
(
2
was transferred into a saturated aq. solution of NaHCO (25
3
mL) and extracted with DCM (2×25 mL). Organic solutions
were dried over Na SO , evaporated, and the residue was
stirred at 110 °C for 3 days. More 2ꢀbromoethyl ether (0.5
mL, 3.30 mmol) was added, and the reaction mixture was
further stirred at 110 °C for 1 day. After cooling, it was dilutꢀ
ed with diethyl ether, washed with brine, dried over Na SO ,
2
4
separated by column chromatography on silica gel
DCM/MeOH, with a linear gradient from 100:0 to 80:20).
Compound 11ꢀAc,Me was isolated as a blue solid (37 mg,
(
2
4
evaporated and purified by flash chromatography on silica gel
nꢀhexane/EtOAc, with a step gradient from 100:0 to 90:0) to
1
4
6
8%). HꢀNMR (400 MHz, CDCl ): δ = 1.32 (s, 6 H), 1.33 (s,
3
(
H), 1.62 (d, J = 1.4 Hz, 6 H, CH C=CH), 2.12 (s, 6 H,
3
obtain a colorless oil (mixture of the product and starting
material). It was used in TBDMS deprotection without further
purification. The oil was dissolved in THF (10 mL) and added
to a stirred solution of TBAF×3H O (828 mg, 2.62 mmol) in
THF (10 mL) at ꢀ5 °C. After stirring for 10 min, the reaction
mixture was diluted with CHCl (20 mL), washed with brine,
dried over Na SO , and evaporated. Flash chromatography on
silica gel (nꢀhexane/EtOAc, with a step gradient from 100:0 to
OAc), 3.58 (t, J = 7.2, 4 H), 3.87 (s, 3 H, OCH ), 4.24 (t, J =
3
7
2
.0, 4 H), 5.20 (s, 2 H, Hꢀ3,10), 6.35 (s, 2 H, Hꢀ1,12), 6.49 (s,
H, Hꢀ6,7), 7.80 (dd, J = 1.4 and 0.7 Hz, 1 H, Hꢀ7′), 8.13 (dd,
2
J = 8.0 and 0.7 Hz, 1 H, Hꢀ5′), 8.24 (dd, J = 8.0 and 1.4 Hz, 1
13
H, Hꢀ4′). CꢀNMR (101 MHz, CDCl ): δ = 18.5, 21.0, 28.8,
3
3
2
1
9.1, 42.8, 52.7, 57.8, 61.1, 97.2, 107.5, 121.0, 122.8, 126.5,
29.6, 130.7, 132.9, 135.1, 147.4, 153.8, 165.9, 168.7, 171.1.
2
4
10
ACS Paragon Plus Environment

Page 11 of 13
The Journal of Organic Chemistry
HRMS (ESI, QꢀTOF) m/z: [M+H]+ 721.3120, calcd for
C H N O ; found, 721.3120.
N-Hydroxysuccinimidyl ester 12-H,Su. To a solution of
compound 12ꢀH,H (2.0 mg, 3.2 ꢁmol) in anhydrous CH Cl
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
2
44
2
9
2
(500 ꢁL), Nꢀhydroxysuccinimide (4.0 mg, 35 µmol) was
Diazoketone 12-Ac,Et. To a solution of diacetate 10ꢀAc,Et
added followed by a stock solution of N,N′ꢀdicyclohexyl
(
(
49 mg, 0.069 mmol) in dry DCM (5 mL), oxalyl chloride
625 ꢁL) was added dropwise at 0 °C, followed by 1 small
carbodiimide (DCC, 80 mg in 1.0 mL) which was added at
…+5°C in 10 µL portions. After addition of each portion and
0
drop of dry DMF. The reaction mixture was stirred for 6 h at
r.t. All volatile materials were distilled in vacuo into a trap
cooled with dry ice/acetone mixture, the residue was flushed
with Ar, dissolved in dry DCM (8 mL) at 0 °C, and a solution
stirring in the dark at 0…+5°C for several hours, HPLC analꢀ
ysis was performed. For that, dichloromethane was evapoꢀ
rated from an aliquote of several µL, the residue was disꢀ
solved in acetonitrile (+0.1% TFA v/v) and immediately after
that injected into a HPLC column. Addition of four 10 µL
aliquotes of DCC (15.6 µmol) at 0°C in 2 days provided the
full conversion of the starting material. No macrocyclic lacꢀ
tone was detected. After concentration of the reaction mixture
in vacuum, the residue was applied onto a column with reguꢀ
lar silica gel. Elution with a mixture of nꢀhexane and EtOAc
(80:20 to 0:100) afforded ester 12ꢀH,Su as green oil (1.9 mg,
82%). HRMS (ESI, QꢀTOF) m/z: [MꢀH]− 714.2569, calcd for
C H N O ; found, 714.2541. HPLC (system E): t = 7.8 min,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of diazomethane in Et O (0.2 M, 5 mL) was added. The reacꢀ
2
tion mixture was stirred overnight at 0 °C. The color of the
solution became less intense; a clear pink solution was
formed. Solvents were evaporated in vacuo, and the residue
was subjected to chromatography on silica gel (20 g, 15 ꢁM,
nꢀhexane/EtOAc, step gradient from 90:10 to 50:50). Diazoꢀ
1
ketone 12ꢀAc,Et was isolated as a green oil (24 mg, 47%). Hꢀ
NMR (400 MHz, CDCl ): δ = 1.32 (s, 6 H), 1.33 (s, 6 H), 1.40
3
(
t, J = 7.2, 3 H, CH CH ), 1.80 (s, 6 H, 2×Ac), 2.83 (s, 6 H,
3 2
40
37
5
8
R
2
×NCH ), 4.38 (q, J = 7.1, 2 H), 4.60 (ABꢀq, J = 12.6, 4 H,
3
peak area = 82% (254 nm).
CH OAc), 5.42 (s, 2 H, Hꢀ3,10), 6.27 (s, 2 H, Hꢀ1,12), 6.44
2
(s, 2 H, Hꢀ6,7), 7.15 (dd, J = 8.0 and 0.7, 1 H, Hꢀ7′), 8.14 (dd,
Diazoketone 13-Ac, Me. To a cold solution of 11ꢀAc,Me (40
mg, 0.055 mmol) in dry DCM (5 mL), oxalyl chloride (625
ꢁL) was added dropwise at 0 °C, followed by 1 drop of dry
DMF. The reaction mixture was stirred for 6 h at rt. After
concentration in vacuo (volatile materials were condensed
into a trap cooled with a mixture of dry ice with acetone), the
residue was flushed with Ar, dissolved in dry DCM (8 mL) at
J = 8.1 and 1.7, 1 H, Hꢀ6′), 8.51 (dd, J = 1.6 and 0.7 H, 1 H,
13
Hꢀ3′). CꢀNMR (101 MHz, CDCl ): δ = 14.4, 20.7, 27.6,
3
27.7, 31.1, 49.2, 56.7, 61.5, 64.0, 98.5, 107.3, 117.3, 122.7,
123.9, 125.7, 126.7, 131.1, 132.3, 135.1, 135.4, 146.5, 152.2,
159.9, 165.7, 170.4, 185.8. HRMS (ESI, QꢀTOF) m/z:
[M+H]+ 731.3075, calcd for C H N O ; found, 731.3059.
42
43
4
8
0
°C, and a solution of diazomethane in Et O (0.2 M, 5 mL)
2
was added. Upon stirring overnight at 0 °C, the color of the
reaction mixture vanished. The reaction mixture was evapoꢀ
rated in vacuo, and the residue was purified by column chroꢀ
matography on silica gel (20 g, 15 ꢁM, nꢀhexane/EtOAc, with
a step gradient from 90:10 to 70:30). Compound 13ꢀAc,Me
Compound 12-H,H. To a solution of compound 12ꢀAc,Et (24
mg, 33 µmol) in EtOH (20 mL) at 0 °C, a solution of aq.
NaOH (1 M, 2.8 mL) was added, and the mixture was keep in
the dark and stirred at 0 °C overnight. Then, the reaction
mixture was concentrated in vacuum, cooled down to 0 °C
and acidified carefully to pH = 2 – 3 with a 5% aq. citric acid.
After that, the mixture was extracted with CH Cl (2×15 mL),
1
was isolated as a green oil (16 mg, 39%). HꢀNMR (400
MHz, CDCl
H, CH C=CH), 2.13 (s, 6 H, OAc), 3.52 (t, J = 7.0 Hz, 4 H,
): δ = 1.29 (s, 6 H), 1.31 (s, 6 H), 1.68 (d, J = 1.4,
3
3
3
2
2
CH N), 3.84 (s, 3 H, OCH ), 4.23 (t, J = 7.2 Hz, 4 H, CH O),
2
3
2
and the green organic solutions dried over Na SO and conꢀ
2
4
5
.15 (q, J = 1.5, 2 H, CH=CMe), 6.36 (s, 2 H, Hꢀ1,12), 6.39
centrated in vacuo. The residue was subjected to an automated
flash chromatography on RPꢀC18 cartridge with
H O/CH CN (system D) as eluent. The fractions containing
(
=
s, 2 H, Hꢀ6,7), 7.72 (dd, J = 1.2 and 0.5, 1 H, Hꢀ7′), 7.88 (d, J
8.0, 1 H, Hꢀ4′), 8.08 (dd, J = 8.0, 1.4 Hz, 1 H, Hꢀ5′). Cꢀ
a
13
2
3
NMR (101 MHz, CDCl ): δ = 18.7, 21.1, 28.8, 42.6, 49.4,
3
the product were lyophilized to give compound 8ꢀH as a green
5
2.6, 57.1, 61.6, 98.2, 107.3, 120.3, 122.3, 122.8, 126.9,
1
amorphous solid (9.0 mg, 45%). HꢀNMR (400 MHz, DMSOꢀ
127.0, 128.8, 129.8, 135.7, 138.3, 144.8, 152.0, 156.1, 166.3,
171.2, 186.2. HRMS (ESI, QꢀTOF) m/z: [M+H]+ 745.3232,
d ): δ = 1.24 (s, 6H), 1.30 (s, 6H), 2.77 (s, 6H), 3.89 (dd, J =
6
A
B
calcd for C H N O ; found, 745.3232.
1
3.2 and 1.3 Hz, CH H O, 2H), 3.97 (dd, J = 13.2 and 1.3
Hz, CH H O, 2H), 5.43 (s, CH=, 2H), 6.27 (s, 2 H), 6.54 (s, 2
43 44
4
8
A
B
Carboxylic acid 13-H,H. To a solution of compound 13ꢀ
Ac,Me (24 mg, 32 µmol) in MeOH (10 mL), aq. NaOH (1 M,
H), 7.14 (d, J = 7.4 Hz, 1 H, 7′H), 8.11 (dd, J = 8.1, 1.7 Hz, 1
1
3
H, 6′H), 8.18 (d, J = 1.7 Hz, 1 H, 3′H). CꢀNMR (101 MHz,
2
.8 mL) was added at 0 °C, and the mixture was stirred in the
DMSOꢀd ): δ = 27.5, 27.9, 30.8, 48.9, 56.2, 60.5, 73.9, 97.6,
6
dark at 0 °C overnight. The reaction mixture was concentrated
in vacuum, cooled down to 0 °C and acidified carefully to pH
= 2–3 with 5% aq. citric acid. The mixture was extracted with
1
1
06.4, 117.4, 121.0, 122.7, 125.5, 128.2, 129.9, 131.3, 133.8,
35.7, 146.0, 151.2, 159.5, 166.4, 185.1. HRMS (ESI, Qꢀ
TOF) m/z: [MꢀH]− 617.2406, calcd for C H N O ; found,
36 33
4
6
CH Cl2 (2×15 mL), and the green organic solutions were
2
6
17.2398. HPLC (system E): t = 7.1 min, peak area = 87%
R
dried over Na SO . The solvent was evaporated in vacuum,
2
4
(254 nm).
and the residue subjected to an automated flash chromatogꢀ
raphy on a RPꢀC18 cartridge with H O/CH CN mixture (sysꢀ
2
3
11
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 13
tem D) as eluent. The fractions containing acid 13ꢀH,H were H,H (Figures S2 and S3), data on cloning, cell culture and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
lyophilized to give a green amorphous solid (8.0 mg, 39%
lasers, as well as copies of NMR spectra for all new comꢀ
pounds.
1
yield). HꢀNMR (400 MHz, DMSOꢀd ): δ = 1.22 (s, 6H), 1.26
6
(
s, 6H), 1.62 (s, 6H), 3.37 (m, 4H, masked by water peak in
DMSO), 3.49 (t, J = 7.2 Hz, 4H), 5.24 (s, 2H, CH=), 6.37 (s,
H), 6.38 (s, 2H), 7.47 (dd, J = 1.4, 0.6 Hz, 1 H, 7′H), 7.87 (d,
J = 8.0, 0.5 Hz, 1H, 4′H), 8.02 (dd, J = 8.0, 1.4 Hz, 1H, 5′H).
AUTHOR INFORMATION
2
Corresponding
Authors:
vbelov@gwdg.de,
mbossi@gwdg.de
1
3
CꢀNMR (101 MHz, DMSOꢀd ): δ = 185.1, 166.3, 155.9,
6
1
1
2
51.4, 144.8, 136.8, 136.5, 129.7, 129.1, 125.5, 125.4, 122.5,
21.5, 119.3, 106.1, 97.4, 74.8, 58.2, 56.5, 48.7, 46.0. 28.8,
8.6, 18.0. HRMS (ESI, QꢀTOF) m/z: [MꢀH]− 645.2719,
ORCID
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Gyuzel Y. Mitronova: 0000ꢀ0003ꢀ4376ꢀ6981
Vladimir N. Belov: 0000ꢀ0002ꢀ7741ꢀ4653
Stefan W. Hell: 0000ꢀ0002ꢀ9638ꢀ5077
Notes
calcd for C H N O ; found, 645.2710. HPLC (system E): t =
7.1 min, peak area = 97% (254 nm).
38 37
4
6
R
Ester 13-H,Su was prepared from carboxylic acid 13ꢀH,H
2.0 mg, 3.2 ꢁmol) in dry CH Cl (100 ꢁL) according to the
(
2
2
method given for compound 12ꢀH,Su. Column chromatogꢀ
raphy on silica gel (nꢀhexane/EtOAc, 80:20 to 0:100) afforded
13ꢀH,Su as green oil (1.9 mg, 83% yield). HRMS (ESI, Qꢀ
TOF) m/z: [M+Na]+ 766.2847, calcd for C H N NaO ;
VNB and SWH own shares in Abberior GmbH which producꢀ
es compounds 5ꢀSu and 12ꢀH,Su as photoactivatable dyes
FLIP 565 and Cage 590, respectively.
4
2
41
5
8
found, 766.2828.
ACKNOWLEDGMENTS
ASSOCIATED CONTENT
Supporting information
The financial support provided by the German Bundesministeꢀ
rium für Bildung und Forschung (grant FKZ 13N14122 for
SWH) is acknowledged. We thank J. Bienert (MPIBPC), Dr.
H. Frauendorf, Dr. M. John and coꢀworkers (Institut für Orꢀ
ganische und Biomolekulare Chemie, GeorgꢀAugustꢀ
Universität, Göttingen, Germany) for recording spectra. We
appreciate the assistance of E. Rothermel with the cell culture
and J. Jethwa for proofꢀreading the manuscript.
The supporting information is available free of charge on the
ACS Publications website at DOI: 101021/acs.joc. Supporting
information contains absorption and emission spectra of comꢀ
pound 4ꢀH / 4ꢀLi in buffered solutions (Figure S1), details of
the photolysis experiments with compounds 8ꢀH, 9ꢀH and 12ꢀ
REFERENCES
(1) Knauer, K.ꢀH.; Gleiter, R. Photochromism of Rhodamine Derivatives.
of threeꢀdimensional volumes by 4Pi detection of stochastically switched
Angew. Chem. Int. Ed. 1977, 16, 113–113; Angew. Chem. 1977, 89, 116–
fluorophores. Nat. Methods 2011, 8, 353–359.
117.
(
7) For example, rhodamine spiroamide 1a featuring a pHꢀdependent
(
2) Fölling, J.; Belov, V.; Kunetsky, R.; Medda, R.; Schonle, A.; Egner,
equilibrium with fluorescent acid 3ꢀOH: Belov, V. N.; Bossi, M. L.;
Fölling, J.; Boyarskiy, V. P.; Hell, S. W. Rhodamine spiroamides for
multicolor singleꢀmolecule switching fluorescent nanoscopy. Chem. – Eur
J. 2009, 15, 10762–10776.
A.; Eggeling, C.; Bossi, M.; Hell, S. W. Photochromic Rhodamines
Provide Nanoscopy with Optical Sectioning. Angew. Chem. Int. Ed. 2007,
4
6, 6266ꢀ6270; Angew. Chem. 2007, 119, 6382–6386.
(
3) Belov, V. N.; Wurm, C. A.; Boyarskiy, V. P.; Jakobs, S.; Hell, S. W.
(
8) Uno, S.; Kamiya, M.; Yoshihara, T.; Sugawara, K.; Okabe, K.; Tarhan,
Rhodamines NN: A Novel Class of Caged Fluorescent Dyes. Angew.
Chem. Int. Ed. 2010, 49, 3520–3523; Angew. Chem. 2010, 122, 3598–
3
M. C.; Fujita; H.; Funatsu, T.; Okada, Y.; Tobita, S.; Urano, Y. A
spontaneously blinking fluorophore based on intramolecular
spirocyclization for liveꢀcell superꢀresolution imaging. Nat. Chem. 2014,
602.
6
, 681–689.
(4) Belov, V. N.; Mitronova, G. Yu.;, Bossi, M. L.; Boyarskiy, V. P.;
Hebisch, E.; Geisler, C.; Kolmakov, K.; Wurm, C. A.; Willig, K. I.; Hell,
S. W. Masked Rhodamine Dyes of Five Principal Colors Revealed by
(
9) Grimm, J. B.; Klein, T.; Kopek, B.; Shtengel, G., Hess, H. F.; Sauer,
M.; Lavis, L. D. Synthesis of a Far‐Red Photoactivatable Silicon‐
Containing Rhodamine for Super‐Resolution Microscopy. Angew. Chem.
Int. Ed. 2016, 55, 1723–1727; Angew. Chem. 2016, 128, 1755–1759.
Photolysis of
a
2‐Diazo‐1‐Indanone Caging Group: Synthesis,
Photophysics, and Light Microscopy Applications. Chem. – Eur. J. 2014,
20, 13162–13173.
(10) Rust, M. J.; Bates, M.; Zhuang, X. Subꢀdiffractionꢀlimit imaging by
stochastic optical reconstruction microscopy (STORM). Nat. Methods
(
5) Kolmakov, K.; Wurm, C.; Sednev, M. V.; Bossi, M. L.; Belov, V. N.;
Hell, S. W. Masked redꢀemitting carbopyronine dyes with photosensitive
ꢀdiazoꢀ1ꢀindanone caging group. Photochem. Photobiol. Sci. 2012, 11,
522–532.
2
006, 3, 793–795.
2
(
11) Bates, M.; Huang, B.; Dempsey, G. T.; Zhuang, X. Multicolor superꢀ
resolution imaging with photoꢀswitchable fluorescent probes. Science
007, 317, 1749–1753.
(6) Aquino, D.; Schönle, A.; Geisler, C.; von Middendorff, C.; Wurm, C.
2
A.; Okamura, Y.; Lang, T.; Hell, S. W.; Egner, A. Twoꢀcolor nanoscopy
12
ACS Paragon Plus Environment

Page 13 of 13
The Journal of Organic Chemistry
(
12) Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.;
Electrophoresis Based on Side Chain Modifications of CyDye
Fluorophores. New Tools For Use in Proteomics and Diagnostics.
Bioconj. Chem. 2013, 24, 1552–1561.
Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; LippincottꢀSchwartz, J.;
Hess, H. F. Imaging Intracellular Fluorescent Proteins at Nanometer
Resolution. Science 2006, 313, 1642–1645.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(25) Galland, R.; Grenci, G.; Aravind, A.; Viasnoff, V.; Studer, V.;
Sibarita, J.ꢀB. 3D highꢀ and superꢀresolution imaging using singleꢀ
objective SPIM. Nat. Methods, 2015, 12, 641–644.
(13) Hess, S. T.; Girirajan, T. P. K.; Mason, M. D. UltraꢀHigh Resolution
Imaging by Fluorescence Photoactivation Localization Microscopy.
Biophys. J. 2006, 91, 4258–4272.
(26) Mlodzianoski, M. J.; Curthoys, N. M.; Gunewardene, M. S.; Carter,
(14) Lee, M. K.; Rai, P.; Williams, J.; Twieg, R. J.; Moerner, W. E. Smallꢀ
Molecule Labeling of Live Cell Surfaces for ThreeꢀDimensional Superꢀ
Resolution Microscopy. J. Am. Chem. Soc. 2014, 136, 14003–14006.
S.; Hess, S. T. SuperꢀResolution Imaging of Molecular Emission Spectra
and Single Molecule Spectral Fluctuations. PLoS ONE, 2016, 11(3):
e0147506. http://doi.org/10.1371/journal.pone.0147506.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(15) Pan, D.; Hu, Z.; Qiu, F.; Huang, Z.ꢀL.; Ma, Y.; Wang, Y.; Qin, L.;
Zhang, Z.; Zeng, S:, Zhang; Y.ꢀH. A general strategy for developing cellꢀ
permeable photoꢀmodulatable organic fluorescent probes for liveꢀcell
superꢀresolution imaging. Nat. Comm. 2014, 5:5573, DOI:
(27) Butkevich, A. N.; Mitronova, G. Yu.; Sidenstein, S. C.; Klocke, J. L.;
Kamin, D.; Meineke, D. N. H.; D'Este, E.; Kraemer, P.ꢀT.; Danzl, J. G.;
Belov, V. N., Hell, S. W. Fluorescent Rhodamines and Fluorogenic
Carbopyronines for Super‐Resolution STED Microscopy in Living Cells.
Angew. Chem. Int. Ed. 2016, 55, 3290–3294; Angew. Chem. 2016, 128,
1
0.1038/ncomms6573.
3
350–3355.
(
16) Wu; L.; Dai, Y.; Marriott, G. Optical Control of Calcium Affinity in a
Spiroamidoꢀrhodamine Based Calcium Chelator. Org. Lett. 2011, 13,
018–2021.
(28) Kolmakov, K.; Wurm, C. A.; Meineke, D. N. H.; Göttfert, F.;
Boyarskiy, V. P.; Belov, V. N.; Hell, S. W. Polar Red‐Emitting
Rhodamine Dyes with Reactive Groups: Synthesis, Photophysical
Properties, and Two‐Color STED Nanoscopy Applications. Chem. – Eur.
J. 2014, 20, 146–157.
2
(
17) Sou, K.; Chan, L. Y.; Ken Lee, C.ꢀL. J. Photoꢀswitchable and selfꢀ
erasable fluorescent nanoprobe. Photochem. & Photobiol. A 2017, 332,
5–31.
2
(29) Nizamov, S.; Willig, K. I.; Sednev, M. V.; Belov, V. N.; Hell, S. W.
Phosphorylated 3‐Heteroarylcoumarins and Their Use in Fluorescence
Microscopy and Nanoscopy. Chem. – Eur. J. 2012, 18, 16339–16348.
(18) (a) Jonas, K. C.; Fanelli, F.; Huhtaniemi, I. T.; Hanyaloglu, A. C.
Single Molecule Analysis of Functionally Asymmetric G Proteinꢀcoupled
Receptor (GPCR) Oligomers Reveals Diverse Spatial and Structural
Assemblies. J. Biol. Chem. 2015, 290, 3875–3892;
(30) Dwight, S. J.; Levin, S. Scalable Regioselective Synthesis of
Rhodamine Dyes. Org. Lett. 2016, 18, 5316–5319.
(b) Jonas, K. C.; Chen, S.; Virta, M.; Mora, J.; Franks, S.; Huhtaniemi, I.;
Hanyaloglu, A. C. Temporal reprogramming of calcium signalling via
crosstalk of gonadotrophin receptors that associate as functionally
asymmetric heteromers. Sci Rep. 2018; 8: 2239, doi: 10.1038/s41598ꢀ018ꢀ
20722ꢀ5.
(31) Montenegro, H.; Di Paolo, M.; Capdevila, D.; Aramendia, P. F.;
Bossi, M. L. The mechanism of the photochromic transformation of
spirorhodamines. Photochem. & Photobiol. Sci. 2012, 11, 1081–1086.
(19) Aloi, A.; Jentzsch, A. V.; Vilanova, N.; Albertazzi, L.; Meijer, E. W.;
(32) Nguyen, T.; Francis, M. B. Practical Synthetic Route to
Functionalized Rhodamine Dyes. Org. Lett. 2003, 5, 3245–3248.
Voets, I. K. Imaging Nanostructures by SingleꢀMolecule Localization
Microscopy in Organic Solvents. J. Am. Chem. Soc. 2016, 138, 2953–
2
956.
(33) Kirmse, W. 100 Years of the Wolff Rearrangement. Eur. J. Org.
Chem. 2002, 2193–2256.
(20) Boott, C. E.; Laine, R. F.; Mahou, P.; Finnegan, J. R.; Leitao, E. M.;
Webb, S. E. D.; Kaminski, C. F.; Manners, I. In Situ Visualization of
Block Copolymer Self‐Assembly in Organic Media by Super‐Resolution
Fluorescence Microscopy. Chem. – Eur. J. 2015, 21, 18539–18542.
(34) Egner, A.; Geisler, C.; von Middendorff, C.; Bock, H.; Wenzel, D.;
Medda, R.; Andresen, M.; Stiel, A. C.; Jakobs, S.; Eggeling, C.; Schönle,
A.; Hell, S. W. Fluorescence Nanoscopy in Whole Cells by Asynchronous
Localization of Photoswitching Emitters. Biophys. J. 2007, 93, 3285–
3290.
(21) Butkevich, A.N.; Belov, V. N.; Kolmakov, K.; Sokolov, V. V.;
Shojaei, H.; Sidenstein, S. C.; Kamin, D.; Matthias, J.; Vlijm, R.;
Engelhardt, J.; Hell, S. W. Hydroxylated Fluorescent Dyes for Live‐Cell
Labeling: Synthesis, Spectra and Super‐Resolution STED. Chem. – Eur. J.
(35) (a) Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.;
Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohana, R. F.; Urh,
M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.; Vidugiris, G.;
Zhu, J.; Darzins, A.; Klaubert, D. H.; Bulleit, R. F.; Wood, K. V.
HaloTag: A Novel Protein Labeling Technology for Cell Imaging and
Protein Analysis. ACS Chem. Biol. 2008, 3, 373ꢀ382;
2
017, 23, 12114–12119.
(22) Grimm, J. B.; English, B. P.; Chen, J.; Slaughter, J. P.; Zhang, Z.;
Revyakin, A.; Patel, R.; Macklin, J. J.; Normanno, D.; Singer, R. H.;
Lionnet, T.; Lavis, L. D. A general method to improve fluorophores for
liveꢀcell and singleꢀmolecule microscopy. Nat. Methods. 2015, 12, 244–
(b) http://www.promegaconnections.com/tag/halotag.
2
50.
(36) Grygorenko, O. O.; Biitseva, A. V.; Zhersh, S. Amino sulfonic acids,
peptidosulfonamides and other related compounds. Tetrahedron, 2018, 74,
1355–1421.
(23) Magde, D.; Rojas, G. E.; P. Seybold, P. Solvent Dependence of the
Fluorescence Lifetimes of Xanthene Dyes. Photochem. & Photobiol.
1999, 70, 737–744.
(37) Mudd, G.; Pi, I. P.; Fethers, N.; Dodd, P. G.; Barbeau, O. R; Auer, M.
A general synthetic route to isomerically pure functionalized rhodamine
dyes. Methods Appl. Fluoresc. 2015, 3, 045002.
(24) Epstein, M. G.; Reeves, B. D.; Maaty, W. S.; Fouchard, D.; Dratz, E.
A.; Bothner, B.; Grieco, P. A. Enhanced Sensitivity Employing
Zwitterionic and pI Balancing Dyes (ZꢀCyDyes) Optimized for 2DꢀGel
13
ACS Paragon Plus Environment