An efficient and versatile approach for the preparation of a rhodamine B ester bioprobe library

Article abstract of DOI:10.1016/j.dyepig.2012.01.005

A general approach for the preparation of a library consisting of reactive rhodamine B (RhB) bioprobes based on ester or thioester linkages is described. The synthesis of this library proceeds fast and efficiently in one reaction step. Pure RhB ester chromophores are readily obtained directly from the reaction mixture following a simple and straight forward workup procedure without further HPLC purification required. A variety of functional groups are attached to the RhB scaffold yielding the functional chromophores in moderate to high yields with particular focus on introducing bioorthogonal substituents suitable for protein and peptide labeling. The approach reported herein provides a concise and practical route to access a variety of reactive RhB fluorophores that could be applied for various bioconjugation chemistries.

Full text of DOI:10.1016/j.dyepig.2012.01.005

Dyes and Pigments 94 (2012) 296e303
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
An efficient and versatile approach for the preparation of a rhodamine B ester
bioprobe library
a
,
b
a
a
a
a,b,c,
*
Xi Chen , Qianzhen Wu , Lars Henschke , Günther Weber , Tanja Weil
a
Institute of Organic Chemistry III, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany
Department of Chemistry, National University of Singapore, 3 Science Drive 3, 117543 Singapore, Singapore
Max-Planck-Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 November 2011
Received in revised form
A general approach for the preparation of a library consisting of reactive rhodamine B (RhB) bioprobes
based on ester or thioester linkages is described. The synthesis of this library proceeds fast and efficiently
in one reaction step. Pure RhB ester chromophores are readily obtained directly from the reaction
mixture following a simple and straight forward workup procedure without further HPLC purification
required. A variety of functional groups are attached to the RhB scaffold yielding the functional chro-
mophores in moderate to high yields with particular focus on introducing bioorthogonal substituents
suitable for protein and peptide labeling. The approach reported herein provides a concise and practical
route to access a variety of reactive RhB fluorophores that could be applied for various bioconjugation
chemistries.
9
January 2012
Accepted 11 January 2012
Available online 20 January 2012
Keywords:
Rhodamine
Bioprobe
Bioorthogonal groups
Protein labeling
Ó 2012 Elsevier Ltd. All rights reserved.
2D-NMR
“
Click” chemistry
1. Introduction
However additional functional handles need to be introduced to
broaden their application spectrum [13,14]. Although there are
An emerging area of growth in dye chemistry has involved
colorants that are utilized for distinct applications [1,2]. Such dyes
are named functional dyes and their design is not merely directed
toward color-tuning [1]. For instance, they have been used in biology
to trace intracellular events [3], to determine ion concentrations
a few functionalized rhodamine dyes commercially available, they
are usually extremely expensive (typically > 40,000 Euro/g) and
therefore organic synthesis is required to obtain the respective
rhodamine derivative of choice in larger amounts.
However, there are several challenges associated when aiming
at preparing RhB derivatives as bioprobes. Purification of func-
tionalized RhB derivatives is tedious and often not feasible due to
the high polarity of the tetraethyl-RhB scaffold. Additionally, RhBs
are either prone to lactone formation under basic condition [15] or
rapidly cyclize to form a non-fluorescent spirolactam when it reacts
with primary amines or hydrazines [16]. These byproducts in
a mixture with the unreacted RhB chromophore share very similar
polarities and thus cannot be readily separated from the desired
product by conventional column chromatography. Previously,
a general approach for the preparation of RhB probes based on
a tertiary amide linkage via an RhB-piperazine amide intermediate
[17] has been reported. This three step synthetic route is generally
applicable for the preparation of functionalized RhB chromophores
but it often requires highly reactive reagents, e.g. trimethylalumi-
num as well as tedious purification steps.
[
4,5] like sensing Cu2 for live cell imaging [6], to develop photo-
þ
chromic switches [7] to estimate the shape of a molecule via fluo-
rescence anisotropy study [8], and to investigate protein dynamics
via fluorescence correlation spectroscopy (FCS) [9]. Additionally,
fluorophore pairs have been investigated as a molecular ruler to
measure the distance of biomolecule assembles via Förster reso-
nance energy transfer (FRET) [10]. More recently, they have been
applied to prepare fluorescent nanoparticles in nanoscience [11].
Rhodamine B (RhB) represents a low cost and widely used flu-
orophore known for its relatively high photostability and accept-
able water-solubility [12]. It features a conjugated fluorescent
xanthene ring and a free benzoic acid moiety for derivatization.
These characteristics render RhBs attractive for various uses.
*
Corresponding author. Institute of Organic Chemistry III, University of Ulm,
Albert-Einstein-Allee 11, 89069 Ulm, Germany. Tel.: þ49 731 5022870;
fax: þ49 731 50 22883.
Herein, we report an alternative and broadly applicable
approach for the preparation of RhB probes featuring an ester bond
E-mail address: tanja.weil@uni-ulm.de (T. Weil).
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2012.01.005

X. Chen et al. / Dyes and Pigments 94 (2012) 296e303
297
linkage. In this way, no spirolactam byproducts are formed and the
corresponding ester product could be purified via an optimized,
simple workup procedure in combination with an optional column
chromatography. A series of RhB ester probes has been synthesized
and this general reaction scheme has been further broadened
facilitating also the preparation of RhB thioesters. Although there
have been few different RhB esters reported previously [18e20],
the general and concise synthesis of pure RhB esters, in particular
those carrying reactive groups suitable for biomolecule labeling,
has yet to be developed. Herein, we report the systematic and
straight forward synthesis of an RhB ester library bearing reactive
functionalities that allow bioconjugation to native peptides and
proteins as well as engineered proteins bearing bioorthogonal
ethynyl or azido groups. In addition, optical properties and 2D-
NMR of these compounds are discussed, providing new insights
and structural information of this important class of chromophores.
2.2.2. RhB ethyl ester (2, from 479 mg of RhB)
351 mg dark violet solid was obtained as the product in a yield of
1
69%. H NMR (CDCl
¼ 0.36 Hz, 1H), 7.80 (td, J
(td, J ¼ 1.04 Hz, 1H), 7.30 (td, J
¼ 6.24 Hz, J
1H), 7.07 (d, J ¼ 7.6 Hz, 2H), 6.90 (dd, J ¼ 7.6 Hz, J
6.82 (d, J ¼ 2.00 Hz, 2H), 4.07 (q, J ¼ 5.72 Hz, 2H), 3.64 (q, J ¼ 5.76 Hz,
3
, 400 MHz):
d
8.28 (ddd, J
¼ 6.04 Hz, J
¼ 6.08 Hz, J
¼ 2.00 Hz, 2H),
1
¼6.32 Hz, J
¼ 1.08 Hz, 1H), 7.73
¼ 0.88 Hz,
2
¼1.04 Hz,
J
3
1
2
1
2
1
2
1
2
1
3
8H),1.32 (t, J ¼ 5.72 Hz,12H),1.07 (t, J ¼ 5.72 Hz, 3H); C NMR (CDCl
3
,
400 MHz): 165.00, 158.92, 157.72, 133.44, 132.94, 131.28, 131.26,
d
130.32, 130.14, 130.14, 114.20, 113.54, 96.34, 61.52, 46.14, 13.79,
þ
12.63; LC-MS (ESI): t
R
98.1% purity (254 nm), C30
H
l
35
N
2
O
3
calcd.
þ
471.21, found 471.14 [M] ; UVeVis (pH 7.2, 50
mM):
max ¼ 559 nm,
4
ꢂ1
ꢂ1
3
¼ 8.6 ꢁ10 M cm ; Fluorescence (pH 7.2, 50
m
M):
l
max ¼ 591 nm
(ex. 530 nm); V (pH 7.2, 50
F
mM) ¼ 0.34.
2.2.3. RhB propargyl ester (3, from 479 mg of RhB)
20 mg dark violet solid was obtained as the product in a yield of
3
1
6
1
3%. H NMR (CDCl
H), 7.79 (td, J
¼ 7.56 Hz, J
¼ 1.24 Hz, 1H), 7.28 (dd, J
d, J ¼ 9.52 Hz, 2H), 6.87 (dd, J
3
, 400 MHz):
d
8.26 (dd, J
1
¼7.92 Hz, J
2
¼ 1.04 Hz,
2
. Experimental
1
2
¼ 1.28 Hz, 1H), 7.70 (td, J
1
¼ 7.80 Hz,
J
2
1
¼ 7.56 Hz, J
2
¼ 1.04 Hz, 1H), 7.01
¼ 2.40 Hz, 2H), 6.76
2.1. Chemicals and instruments
(
1
¼ 9.48 Hz, J
2
(
2
4
d, J ¼ 2.36 Hz, 2H), 4.56 (d, J ¼ 2.44 Hz, 2H), 3.60 (q, J ¼ 7.12 Hz, 8H),
All chemical reagents were purchased from SigmaeAldrich,
Merck or Regent and were used without further purification
13
.36 (t, J ¼ 2.44 Hz, 1H), 1.27 (t, J ¼ 7.04 Hz, 12H); C NMR (CDCl
3
,
00 MHz): 164.08, 158.09, 157.65, 155.43, 133.59, 133.36, 131.37,
d
unless otherwise mentioned. Anhydrous DMF was dried over
1
31.05, 130.30, 130.18, 129.02, 114.21, 113.40, 96.21, 76.31, 75.40,
1
13
freshly activated 3 Å molecular sieves. H NMR, C NMR, DEPT 135,
HSQC and HMBC spectra were recorded on Bruker AC 300, AC 400,
AMX 500 or DRX500 NMR spectrometers operating at 400 MHz for
þ
33 3 2
H O N , calcd. 481.2486, found
52.75, 46.04,12.53; HRMS (ESI): C31
81.2471; EA: C31 Cl$2H O, calcd. C 67.32%, H 6.74%, N 5.06%,
4
33
H O
3
N
2
2
Cl 6.41%, found C 67.46%, H 6.71%, N 5.14%, Cl 6.94%; LC-MS (ESI):
6.3% (254 nm); UVeVis (pH 7.2, 50 M):
max ¼ 560 nm,
¼ 8.3 ꢁ10 M cm ; Fluorescence (pH 7.2, 50 M): max ¼ 561 nm
ex. 530 nm); V (pH 7.2, 50
M) ¼ 0.36.
1
13
ꢀ
H and 75.48 MHz for C NMR at 25 C. Chemical shifts were re-
ported in ppm ( scale) relative to the solvent signal (CDCl 7.26,
77.0), and coupling constant (J) values were reported in hertz
Hz). High-resolution mass spectra (HRMS) were recorded on
9
3
m
l
d
3 H
: d
4
ꢂ1
ꢂ1
m
l
d
C
(
F
m
(
a Finnigan MAT95XL-T mass spectrometer by direct infusion of the
solution of each compound by using electrospray ionization (ESI) in
the positive mode. Low resolution ESI-MS spectra were determined
using a Finnigan LCQ quadrupole ion trap mass spectrometer.
MALDI-ToF-MS spectra of protein samples were recorded on
Autoflex MALDI-ToF (Bruker Daltonics) mass spectrometer using
sinapinic acid solution as matrix. Purity studies by LC-MS (ESI) were
2
.2.4. RhB allyl ester (4, from 479 mg of RhB)
2
95 mg dark violet solid was obtained as the product in a yield of
1
5
(
6
7%; H NMR (CDCl
t, J ¼ 7.42 Hz, 1H), 7.59 (t, J ¼ 7.64 Hz, 1H), 7.16 (d, J ¼ 7.44 Hz, 1H),
.92 (d, J ¼ 9.48 Hz, 2H), 6.77 (dd, J ¼9.44 Hz, 2H), 6.64 (d, 2H), 5.51
3
, 400 MHz):
d
8.14 (d, J ¼ 7.76 Hz, 1H), 7.67
1
(
8
m, 1H), 4.98 (m, 2H), 4.34 (d, J ¼ 5.64 Hz, 2H), 3.50 (q, J ¼ 7.12 Hz,
13
H), 1.17 (t, J ¼ 6.96 Hz, 12H); C NMR (CDCl
3
, 400 MHz): d 164.25,
achieved on
a
Shimadzu LC-20AD/SPD-20A/SIL-20AC/LCMS-
158.24, 157.29, 155.10, 133.11, 132.73, 130.88, 130.81, 130.66, 130.00,
2
010EV instrument equipped with a C-18 or C-8 column using
129.78, 129.47, 118.60, 113.88, 113.07, 95.83, 77.21, 65.60, 45.74,
2
MeCN/H O as eluent. UVeVis and fluorescence spectra were
recorded by TECAN Microplate Reader at the concentration of
þ
12.25; HRMS (ESI): C31
H N
35 2
O
3
, calcd. 483.2642, found 483.2638;
M):
cm ; Fluorescence (pH 7.2,
max ¼ 592 nm (ex. 530 nm); V (pH 7.2, 50 M) ¼ 0.37.
LC-MS (ESI): 97.0% purity (254 nm); UVeVis (pH 7.2, 50
m
5
0 mM in PBS buffer (0.1 M, pH 7.2).
4
ꢂ1
ꢂ1
l
max ¼ 559 nm,
3
¼ 7.9 ꢁ 10
M
5
0
mM):
l
F
m
2
2
.2. Synthesis and characterization
2
.2.5. RhB-NHS ester (5, from 479 mg of RhB)
.2.1. The general procedure for the preparation of RhB (thio)ester
330 mg dark violet solid was obtained as the product in a yield of
1
probes
In a typical reaction, RhB (479 mg, 1.0 mmol, 1.0 equiv.), the
57%. H NMR (CDCl
1H), 7.97 (td, J
¼ 7.64 Hz, J
¼ 1.40 Hz, 1H), 7.47 (dd, J
(d, J ¼ 9.4 Hz, 2H), 6.87 (dd, J
3
, 400 MHz):
d
8.41 (dd, J
1
¼7.92 Hz, J
2
¼ 0.88 Hz,
¼ 7.18 Hz,
1
2
¼ 1.24 Hz, 1H), 7.82 (td, J
1
corresponding (thio)alcohol (1.1 mmol, 1.1 equiv.), EDC$HCl
J
2
1
¼ 7.68 Hz, J
2
¼ 0.88 Hz, 1H), 7.07
¼ 2.44 Hz, 2H), 6.84
0
(
N-(3-dimethylaminopropyl)-N -ethyl carbodiimide hydrochloride
1
¼ 9.40 Hz, J
2
salt) (211 mg, 1.1 mmol) and DMAP (4-dimethylaminopyridine)
24.4 mg, 0.2 mmol, 0.2 equiv.) were combined in a Schlenk tube
(d, J ¼ 2.40 Hz, 2H), 3.69 (q, J ¼ 7.60 Hz, 8H), 2.76 (s, br, 4H), 1.32
13
(
(t, J ¼ 7.12 Hz, 12H); C NMR (400 MHz, CDCl
3
): d 168.57, 160.57,
equipped with a stir bar. DCM (5 mL) was injected and the reaction
flask was wrapped with aluminum foil to exclude light. The resul-
tant reaction mixture was stirred at RT (room temperature) under
Ar for 4 h. DCM (10 mL) was added and the reaction mixture was
157.60, 155.57, 155.43, 134.80, 134.23, 131.58, 130.83, 130.81, 130.57,
125.19, 114.29, 113.21, 96.30, 46.05, 25.48, 12.50; LC-MS (ESI): 96.2%
purity (254 nm); UVeVis (pH 7.2, 50
m
M):
¼ 8.1 ꢁ10 M cm ; Fluorescence (pH 7.2, 50
(ex. 530 nm); V (pH 7.2, 50
M) ¼ 0.31.
l
max ¼ 563 nm,
4
ꢂ1
ꢂ1
3
m
M):
l
max ¼ 595 nm
washed with 10 mL of DI-H
four times by DCM and all organic layers were combined, washed
with 0.1 M HCl (10 mL), brine (5 mL), dried over anhydrous Na SO
filtrated, concentrated and purified via silica gel chromatography
MeOH:CHCl 1:5, R w 0.5) to afford the corresponding RhB (thio)
2
O. The aqueous layer was extracted
F
m
2
4
,
2.2.6. RhB 4-iodobenzyl ester (6, from 240 mg of RhB, 8 h)
400 mg reddish violet solid was obtained as the product in a yield
1
(
3
f
of 58%. H NMR (CDCl
3
, 400 MHz):
¼ 7.52 Hz, J
¼ 1.32 Hz, 1H), 7.46 (dm, J
d
8.25 (dd, J
¼ 11.32 Hz, 1H), 7.69
¼ 8.28 Hz, 2H), 7.23
1
¼ 7.80 Hz,
esters in a moderate to good yield between 57 and 79%. The purity
of each compound was assessed by LC-MS analysis at 254 nm.
J
2
¼ 1.12 Hz, 1H), 7.76 (td, J
1
2
(td, J
1
¼ 7.76 Hz, J
2
1

298
X. Chen et al. / Dyes and Pigments 94 (2012) 296e303
(
(
(
1
1
dd, J
dd, J
1
1
¼ 7.56 Hz, J
2
2
¼ 1.08 Hz, 1H), 6.99 (d, J ¼ 9.48 Hz, 2H), 6.84
d
169.39, 164.60, 158.66, 157.69, 155.49, 133.71, 133.21, 131.32, 131.19,
¼ 9.52 Hz, J
¼ 2.40 Hz, 2H), 6.68 (d, J ¼ 2.44 Hz, 2H), 6.67
130.34,130.28,129.53,114.23,113.47, 96.30, 81.18, 61.19, 46.11, 34.73,
27.99, 12.62; HRMS (ESI): C35
571.3163; LC-MS (ESI): 95.2% purity (254 nm); UVeVis (pH 7.2,
50
50
þ
d, J ¼ 8.2 Hz, 2H), 4.87 (s, 2H), 3.63 (qd, J
1
¼7.24 Hz, J
2
d
¼1.64 Hz, 8H),
H N
43 2
O
5
calcd. 571.3167, found
13
.30 (t, J ¼ 7.12 Hz,12H); C NMR (CDCl
3
, 400 MHz):
164.78,158.03,
4
ꢂ1
ꢂ1
57.40, 155.35, 137.43, 134.04, 133.10, 133.07, 131.34, 131.11, 130.33,
m
m
M):
M):
l
max ¼ 560 nm,
3
¼ 8.1 ꢁ10 M cm ; Fluorescence (pH 7.2,
1
1
30.10, 130.06, 129.60, 114.18, 113.30, 96.06, 94.14, 66.56, 46.13,
2.61; HRMS (ESI): C35
l
max ¼ 591 nm (ex. 530 nm); V
F
(pH 7.2, 50
m
M) ¼ 0.44.
þ
H
36IN
2
O
3
, calcd. 659.1765, found 659.1757;
M):
cm ; Fluorescence (pH 7.4):
(pH 7.2, 50
M) ¼ 0.42.
LC-MS (ESI): 95.8% purity (254 nm); UVeVis (pH 7.2, 50
l
m
2.2.10. RhB benzyl thioester (12)
4
ꢂ1
ꢂ1
max ¼ 561 nm,
3
¼ 6.7 ꢁ 10
M
RhB (479 mg,1.0 mmol), benzyl mercaptan (136.4 mg,1.1 mmol),
EDC$HCl (211 mg, 1.1 mmol) and DMAP (24.4 mg, 0.2 mmol) were
combined in a Schlenk flask equipped with a stir bar. DCM (5 mL)
was injected under Ar atmosphere and the reaction flask was
wrapped with aluminum foil to exclude light. The resultant solu-
tion was stirred under Ar at RT for 4 h. The reaction mixture was
partitioned in DCM/H O and the organic layer was separated. The
2
aqueous layer was extracted four additional times by DCM. All
organic layers were combined, washed with 0.1 M HCl (10 mL),
l
max ¼ 590 nm (ex. 530 nm); V
F
m
2
.2.7. RhB 2-azidoethyl ester (7, from 144 mg of RhB)
9
6.8 mg dark violet solid was obtained as the product in a yield
1
of 60%. H NMR (500 MHz, CDCl
td, J ¼ 1.1 Hz, 1H), 7.75 (td, J
¼ 7.6 Hz, J
.31 (dd, J
3
):
d
8.30 (dd, J
1
¼ 7.6 Hz, 1H), 7.82
¼ 1.3 Hz, 1H),
¼9.5 Hz,
(
7
1
2
1
¼ 8.2 Hz, J
2
1
¼7.6 Hz,1H), 7.07 (d, J ¼ 9.5 Hz, 2H), 6.91 (dd, J
1
J
2
¼ 2.6 Hz, 2H), 6.82 (d, J ¼ 2.5 Hz, 2H), 4.16 (t, J ¼ 4.04 Hz, 2H), 3.65
q, J ¼ 6.56 Hz, 8H), 3.36 (t, J ¼ 3.52 Hz, 2H), 1.32 (t, J ¼ 6.56 Hz,
(
1
1
9
brine, dried over anhydrous Na
purified via silica gel chromatography (MeOH:CHCl
yield 463 mg dark violet crystal as the product in a yield of 79%.
2
SO
4
, filtered, concentrated and
13
2H); C NMR (500 MHz, CDCl
3
):
d
164.74, 158.36, 157.79, 155.60,
3
1:5, R 0.5) to
f
33.63, 133.34, 131.49, 131.22, 130.46, 130.29, 129.43, 114.34, 113.55,
6.30, 63.72, 49.53, 46.15, 12.62; HRMS (ESI): C30
12.2656, found 512.2639; LC-MS (ESI): 97.0% (254 nm); UVeVis
þ
1
H
34
N
5
O
3
, calcd.
H NMR (CDCl
3
, 400 MHz):
¼ 1.16 Hz, 1H), 7.72 (td, J
d
8.14 (d, J ¼ 7.88 Hz, 1H), 7.81 (td,
5
J
1
¼7.56 Hz, J
2
1
¼7.72 Hz, J ¼ 1.12 Hz, 1H),
2
4
ꢂ1
ꢂ1
(
pH 7.2, 50
mM):
l
max ¼ 559 nm,
3
¼ 8.3 ꢁ 10
M
cm ; Fluo-
(pH 7.2,
7.33 (d, J ¼ 7.52 Hz, 1H), 7.14e12 (m, 3H), 7.08e03 (m, 4H), 6.85e81
(m, 4H), 4.03 (s, 2H), 3.64 (q, J ¼ 7.12 Hz, 8H),1.33 (t, J ¼ 7.0 Hz,12H);
rescence (pH 7.2, 50
m
M):
l
max ¼ 591 nm (ex. 530 nm); V
F
13
5
2
6
0
mM) ¼ 0.44.
3
C NMR (CDCl , 400 MHz): d 190.99, 157.62, 157.61, 155.48, 137.27,
1
36.68, 132.96, 131.16, 130.56, 130.53, 129.07, 128.63, 128.40, 127.23,
þ
37 2 2
H N O S
.2.8. RhB N-Boc aminoethyl ester (8, from 479 mg of RhB)
114.15, 113.48, 96.33, 46.10, 33.62, 12.63; HRMS (ESI): C35
calcd. 549.2571, found 549.2566; LC-MS (ESI): 95.2 purity
4
10 mg dark violet solid was obtained as the product in a yield of
1
6%. H NMR (CDCl
¼ 8.64 Hz, J ¼ 1.16 Hz, 1H), 7.69 (td, J
H), 7.25 (d, J ¼ 7.72 Hz, 1H), 7.03 (d, J ¼ 9.48 Hz, 2H), 6.87
¼9.52 Hz, J ¼ 2.40 Hz, 2H), 6.74 (d, J ¼ 2.36 Hz, 2H), 5.17 (s, br,
H, NH), 4.07 (t, J ¼ 5.52 Hz, 2H), 3.60 (qd, J ¼ 7.34 Hz, J ¼ 1.40 Hz,
H), 3.24 (m, 2H), 1.36 (s, 9H), 1.29 (t, J ¼ 7.14 Hz, 12H); C NMR
, 400 MHz): 164.69, 157.61, 155.46, 133.03, 132.99, 131.53,
3
, 400 MHz):
d
8.31 (d, J ¼ 7.68 Hz, 1H), 7.76
(254 nm); UVeVis (pH 7.2, 50
m
M):
¼ 6.4 ꢁ 10 M cm ; Fluorescence (pH 7.2, 50
(ex. 530 nm); V (pH 7.2, 50
M) ¼ 0.42.
l
max
m
¼
564 nm
max ¼ 595
4
ꢂ1
ꢂ1
(
td, J
1
2
1
¼ 7.76 Hz, J ¼ 1.36 Hz,
2
3
M):
l
1
(
1
8
(
F
m
dd, J
1
2
1
2
2.2.11. RhB 2-aminoethyl ester trifluoroacetic acid salt (9)
13
RhB N-Boc aminoethyl ester (190 mg, 31.5 mmol) was dis-
solved in DCM (5 mL). TFA (2.5 mL) was injected dropwise to this
stirring solution. The resultant solution was stirred in the dark at
RT for 30 min. DCM and TFA were removed under high vacuum to
CDCl
3
d
1
5
31.24, 130.34, 130.01, 129.62, 114.21, 113.42, 96.20, 77.21, 64.71,
0.33, 46.03, 28.26,12.54; HRMS (ESI): C35H N O , calcd. 586.3276,
þ
44 3 5
1
found 586.3270; LC-MS (ESI): 95.6% purity (254 nm); UVeVis (pH
7
yield the red solid as the product in a quantitative yield. H NMR
4
ꢂ1
ꢂ1
.2, 50
m
M):
m
l
max ¼ 560 nm
3
¼ 8.2 ꢁ 10 M cm ; Fluorescence
(CDCl
8.16 (s, br, 2H), 7.75 (t, J ¼ 7.32 Hz, 1H), 7.70 (t, J ¼ 7.20 Hz, 1H),
.23 (d, J ¼ 6.72 Hz, 1H), 7.07 (t, J ¼ 9.44 Hz, 2H), 6.80
(dd, J ¼ 2.04 Hz, 2H), 6.76 (d, J ¼ 2.12 Hz, 2H), 4.32
¼ 9.44 Hz, J
(t, br, 2H), 3.56 (q, J ¼ 6.36 Hz 8H), 3.27 (s, br, 2H), 1.29
3
, 400 MHz):
d
12.88 (m, br, 3H), 8.45 (d, J ¼ 7.32 Hz, 1H),
(
pH 7.2, 50
M):
l
max ¼ 591 (ex. 530 nm); V
F
(pH 7.2, 50
m
M) ¼ 0.37.
7
2
RhB)
.2.9. RhB 3-tert-butoxycarbonylethyl ester (10, from 479 mg of
1
2
13
4
37 mg violet solid was obtained as the product in a yield of 72%.
, 400 MHz):
8.23 (d, J ¼ 7.88 Hz, 1H), 7.81
¼1.16 Hz,1H), 7.71 (td, J ¼7.68 Hz, J ¼1.2 Hz,1H),
.29 (d, J ¼ 7.56 Hz, 1H), 7.04 (d, J ¼ 9.48 Hz, 2.00 Hz), 6.89
dd, J ¼ 2.36 Hz, 2H), 6.79 (d, J ¼ 2.36 Hz, 2H), 4.26
¼ 9.48 Hz, J
t, J ¼ 6.40 Hz, 2H), 3.63 (q, J ¼ 7.12 Hz, 8H), 2.46 (t, J ¼ 6.32 Hz, 2H),
(t, J ¼ 6.92 Hz, 12H); C NMR (CDCl
3
, 400 MHz): d 164.40, 159.20,
1
H NMR (CDCl
¼7.56 Hz, J
3
d
157.75, 155.55, 133.75, 133.10, 132.05, 131.30, 130.54, 129.76,
(
td, J
1
2
1
2
128.76, 114.02, 113.48, 96.13, 61.45, 45.87, 38.90, 12.32; HRMS
(ESI): C30
þ
7
(
(
H
36
N
3
O
3
, calcd. 486.2751, found 486.2749; LC-MS (ESI):
1
2
95.5% purity; UVeVis (pH 7.2, 50
m
M):
¼ 7.9 ꢁ 10 M cm ; Fluorescence (pH 7.2, 50
(ex. 530 nm); V (pH 7.2, 50
M) ¼ 0.45.
l
max
¼
560 nm,
max ¼ 590
4
ꢂ1
ꢂ1
3
mM):
l
13
1
.39 (s, 9H), 1.31 (t, J ¼ 7.04 Hz, 12H); C NMR (CDCl
3
, 400 MHz):
F
m
Fig. 1. a) Model reaction between RhB (1) and ethanol to afford the RhB ethyl ester (2) for evaluating RhB derivatization via Steglich esterification (above); three byproducts are
formed according to LC-MS, including a decarbonyl etherification byproduct, RhB ethyl ether (below). (b) The LC-MS chromatograph of the final RhB ethyl ester (2) reveals a purity
of 98.1% (m/z ¼ 471).

X. Chen et al. / Dyes and Pigments 94 (2012) 296e303
299
Table 1
Summary of the yields, maximal absorption (labs) and emission (
lem) wavelengths, molar extinction coefficients (
3
) and quantum yields (V
F
) of RhB and its (thio)ester
derivatives. Within this chromophore library, functional RhB ester 6, 8, 9, 10, 11 and 12 represent novel compounds that have not been reported elsewhere.
OH
R
O
1. EDC, DMAP, DCM, RT, 4h
. DCM/ HCl (0.1 M)
O
+
HR
2
N
O
N
N
O
N
(57 ~ 79 % Yield)
RhB (1)
RhB (Thiol)Esters (2-12)
> 95 % purity)
(
ꢂ1
ꢂ1
)
Compound
R
Yield/%
Purity/%
lab/nm (
3
/k M cm
lem/nm
V
F
1
2
RhB
e
e
554 (85)
584
0.32 [21]
6
9
98.1
96.3
559 (86)
591
0.34
O
3
4
63
560 (83)
591
0.36
O
5
7
97.0
96.2
559 (79)
563 (81)
592
595
0.37
0.31
O
O
O
5
6
57
N
O
O
58
60
95.8
97.0
561 (67)
559 (83)
590
591
0.42
0.44
I
7
8
N3
O
NHBoc
66
99
95.6
95.5
560 (82)
560 (79)
591
590
0.37
0.45
O
O
NH2
9
·
TFA
O
1
1
1
1
0
1
2
3
72
99
79
0
95.2
95.4
95.5
e
560 (81)
559 (84)
564 (64)
e
591
591
595
e
0.44
0.36
0.42
e
O
OtBu
O
O
S
OH
S
2.2.12. RhB 2-carboxyethyl ester (11)
dissolved in DMSO and freeze dried in order to remove traces of
RhB 2-tert-butoxycarbonylethyl ester (422 mg, 0.695 mmol)
TFA. 379 mg of a dark violet solid was obtained in quantitative
ꢀ
1
was dissolved in DCM (5 mL) and cooled down to 0 C by an ice-
water bath. TFA (5 mL) was added and the resultant solution was
wrapped with aluminum foil and stirred at RT overnight. DCM and
TFA were removed under high vacuum. The resultant residue was
yields. H NMR (CDCl
3
, 400 MHz):
d
8.28 (dd, J
1
¼ 7.00 Hz,
J
J
2
¼ 1.88 Hz, 1H), 7.73e7.71 (m, 2H), 7.23 (dd, J
1
¼ 6.88 Hz,
2
¼ 1.52 Hz, 1H), 7.04 (d, J ¼ 9.4 Hz, 2H), 6.91 (d, 2H), 6.81
(dd, J
1
¼ 9.1 Hz, 2H), 4.19 (t, J ¼ 5.24 Hz, 2H), 3.60 (q, J ¼ 7.0 Hz, 8H),

300
X. Chen et al. / Dyes and Pigments 94 (2012) 296e303
2
4
.27 (t, J ¼ 5.16 Hz, 2H), 1.32 (t, J ¼ 6.84 Hz, 12H); 13C NMR (CDCl
3
,
obtained in a straight forward fashion without further purification
steps required. Noteworthy, even though RhB benzyl thioesters
such as (12) could be achieved easily, all attempts to synthesize RhB
4-methylphenyl thioester (13) failed (Scheme 1).
00 MHz):
d 171.52, 165.41, 157.93, 157.80, 155.55, 132.94, 132.49,
131.69, 130.99, 130.50, 130.20, 129.85, 113.99, 113.50, 96.90, 61.18,
þ
4
5.99, 32.83, 12.51; HRMS (ESI): C31H N O calcd. 515.2451, found
35 2 5
5
15.2537; LC-MS (ESI): 95.4% purity (254 nm); UVeVis (pH 7.2,
These reactive RhB ester chromophores summarized in Table 1
represent attractive probes for targeting natural (probe 5, 9, 11
and 12) as well as unnatural peptides and proteins bearing e.g.
unnatural amino acids (probe 3, 4, 6 and 7, Fig. 2). In particular, RhB
propargyl ester (3, Table 1) is attractive for Huisgen 1,3-Dipolar
cycloaddition reactions (click labeling) [22] to e.g. proteins
bearing the unnatural amino acid azidohomoalanine (Aha) [23] and
Sonogashira labeling to proteins carrying iodo groups [24]. RhB
allyl ester (4, Table 1) allows labeling via olefin metathesis [25] and
the amine-reactive RhB-NHS ester (5, Table 1) reacts with lysine
residues [18]. RhB 4-iodobenzyl ester (6, Table 1) could be applied
for Sonogashira reactions [26] and RhB 2-azidoethyl ester
(7, Table 1) offers click labeling [22] or labeling via Staudinger
ligation [27]. Additionally, RhB benzyl thioester (12, Table 1) offers
the potential for native chemical ligation (NCL) labeling at
N-terminal cysteines [28]. Furthermore, there are two RhB deriv-
atives (9 and 11, Table 1) carrying amino or carboxylic group suit-
able for bioconjugation reactions on proteins via EDC-mediated or
dimethylglycine (DMG) ester-catalyzed [29] modifications. These
reactions usually proceed smoothly on proteins [22e29] and
labeling of proteins using these synthesized RhB ester probes is not
within the focus of this work. However, a representative protein
labeling experiment is given in the following section to underline
the suitability of these chromophores for such purposes.
4
ꢂ1
ꢂ1
5
0
m
M):
m
l
max ¼ 559 nm,
3
¼ 8.4 ꢁ 10 M cm ; Fluorescence (pH
7.2, 50
M):
l
max ¼ 591 nm (ex. 530 nm); V
F
(pH 7.2, 50
m
M) ¼ 0.36.
3
. Results and discussions
3.1. Development of the preparation and purification procedure of
RhB esters
Ethanol was chosen to react with RhB in the model reaction. The
coupling was mediated by EDC under the catalysis of DMAP in DCM
at RT. After 4 h, the LC-MS revealed the formation of the target RhB
ethyl ester (2) withm/z 471.15 [M ] together withimpurities(Fig.1a).
þ
Column chromatography failed to efficaciously remove the impuri-
ties. Apart from 1-ethyl-3-(3-dimethylaminopropyl)urea (EDU) and
DMAP, two major impurities were isolated and identified as the RhB
precursor and RhB base (the lactone form of RhB, Fig. 1a) based on
their mass spectra and 1H NMR/ C NMR spectra. Unfortunately,
these impurities could not be suppressed by altering the reaction
conditions, e.g. extending the reaction time during synthesis.
Since these two impurities together with EDU and DMAP
represent amines derivatives, we envisioned that they could be
removed simply by washing with acid solution, e.g. aq. HCl. As the
RhB ethyl ester also contains a cationic amine which could be
removed by adding aq. HCl, different organic phases in combination
with varying concentrations of aq. HCl solution were screened.
Finally, the combination of DCM as organic phase in combination
with 0.1 M HCl was identified to be ideally suited to separate the
reaction product from the educts and byproducts. After dissolving
the reaction mixture in DCM followed by washing with 0.1 M HCl,
most of the RhB, RhB base, EDU and DMAP impurities were
removed in one purification step only, while most of the RhB ethyl
ester (2) product remained in the organic layer. The application of
other organic solvents such as EtOAc instead of DCM proceeded less
efficiently since the RhB ethyl ester (2) is lost during washing.
Additionally, applying more concentrated HCl caused undesired
loss of the RhB ethyl ester (2) while usage of more diluted HCl
solution did not remove the impurities efficiently. After removal of
DCM under high vacuum, the RhB ethyl ester product (2) was ob-
tained with a purity above 90%. An optional column chromatog-
13
3.3. Reactivity of RhB propargyl ester (4) for labeling azido-
RNase A (14)
One of the most widely used bioorthogonal labeling reactions
for protein modifications represents the Huisgen 1,3-dipolar
cycloaddition reaction, also known as click reaction. As a repre-
sentative demonstration of the reactivity of the synthesized
rhodamine bioprobes, RhB propargyl ester (4) is reacted with the
azido-containing protein, bis-Aha-RNase A (14) under the catalysis
þ
of Cu at physiological pH in aqueous solution (Fig. 3a). Bis-Aha-
RNase A (14) with two surface-exposed
L-azidohomoalanine
(L-Aha) groups (Fig. 3b) at the position of Met 1 and Met 41 has
been obtained by selective pressure incorporation [30] using
methionine auxotroph Escherichia coli strains by replacing the
natural amino acid
Aha-RNase (14) is given in the Supporting Information
Figure S1). According to MALDI-ToF-MS analysis, bis-Aha-RNase A
L-methionine by L-Aha. The full sequence of bis-
raphy (MeOH:CHCl
to 98.1% (t 7.76 min, m/z 471) according to LC-MS (Fig. 1b) and the
pure RhB ethyl ester (2) was isolated in an acceptable yield of 69%.
3 f
1:5, R 0.5) allows to further increase the purity
A
R
(
(
14) displays a M.W. of around 15.8 kDa.
3.2. Synthesis of an RhB ester-based bioprobe library
After establishing the preparation and purification protocol, this
S
SH
approach was applied to prepare a variety of RhB ester probes,
including an RhB benzyl thioester (Table 1, 12), which is very
valuable for native chemical ligation (NCL) reactions to proteins and
peptides. Acceptable yields between 57 and 79% were achieved for
different chromophore derivatives in high purities according to LC-
MS (Table 1).
The preparation of RhB 2-aminoethyl ester (Table 1, 9) and RhB
-carboxyethyl ester chromophores (Table 1,11) requires a different
reaction protocol since they carry reactive and ionizable groups.
Therefore, a protection/deprotection strategy is applied. Briefly,
their protected precursors, RhB N-Boc-2-aminoethyl ester and RhB
O
EDC, DMAP
9 %
N
O
N
7
(
12)
RhB (1)
SH
S
2
O
N
O
N
EDC, DMAP
2
-tert-butoxycarbonylethyl ester (Table 1, 8 and 10) were synthe-
0
%
(13)
sized in high purities using the established protocol. After treat-
ment with TFA and vacuum drying, the unprotected bioprobes were
Scheme 1. Esterification of RhB using 4-thiocresol and benzyl mercaptan.

X. Chen et al. / Dyes and Pigments 94 (2012) 296e303
301
HS
4
1
2
Olefin Metathesis Labeling
Staudinger Ligation Labeling
NCL Labeling
S
H N
2
7
9
S
DMG-Catalyzed Labeling
EDC-Mediated Labeling
NHS Labeling
HOOC
PAr2
N3
3
6
1
1
Click Labeling
H N
2
5
Sonogashira Labeling
H N
2
Natural Protein/Peptides
Unnatural Protein/Peptides
Fig. 2. The potential of labeling native proteins (left) or proteins decorated with unnatural residues applying the synthesized RhB ester probes.
4
ꢂ1
ꢂ1
In order to further demonstrate that RhB propargyl ester (4) is
an efficient click labeling probe for modifying azido proteins, probe
559e560 nm and 8.0e8.5 ꢁ 10
with a red shift of 5e6 nm relative to RhB (1) (black dotted line,
max 554 nm). Interestingly, RhB-NHS ester (5, bright green) and
RhB benzyl thioester (12, blue) reveal an obviously larger red shift
of 9 nm ( max 563 nm) and 10 nm ( max 564 nm), respectively,
M
cm , respectively (Fig. 4a)
(
4) is reacted with bis-Aha-RNase A (14) in PBS buffer in the pres-
l
þ
ence of Cu catalyst (15). Gel-electrophoresis of three different
solutions has been performed: (1) the click reaction mixture (Fig. 3-
d1), (2) a reaction mixture consisting of 14 and 4 but in the absence
l
l
originating from the conjugated electron-donating effects of the
NHS group or the sulfur atom. RhB 4-iodobenzyl ester (6, bright
blue) and RhB benzyl thioester (12, blue) bear comparatively
þ
of the Cu catalyst (15) (Fig. 3-d2) as control to prove the absence of
any non-specific interactions between the azido protein (14) and 4,
as well as (3) pure bis-Aha-RNase A (14). Fig. 3-d4 reveals the
protein ladder. According to Fig. 3-d1 (above), a highly fluorescent
band is observed at w16 kDa whereas pure bis-Aha-RNase A (14)
and the control reaction gave only non-fluorescent gel bands.
Therefore, this study successfully demonstrates the reactivity of the
representative chromophore, RhB propargyl ester (4), as an
attractive bioprobe for labeling azido-functionalized proteins.
4
ꢂ1 ꢂ1
lower extinction coefficients below 7 ꢁ 10
M
cm , which
might be due to the presence of aromatic groups on RhB.
Furthermore, RhB benzyl thioester (12, blue curve) exhibits
a characteristic shoulder peak at 607 nm which is absent in all
other RhB ester derivatives.
For most of RhB esters, a similar maximal fluorescence emission
wavelength at around 591 nm (Fig. 4b) is recorded, which is 7 nm
shifted bathochromically compared with RhB (1, dotted black line).
Interestingly, the emission maxima of RhB-NHS ester (5, blue) and
RhB benzyl thioester (12, bright blue) reveal a bathochromic shift of
about 11 nm compared with RhB and their emission maxima are
found at 595 nm. A similar stroke shift of about 31 nm was recorded
for all RhB (thio)esters, including RhB, suggesting that this feature
3.4. Optical and NMR characteristics of RhB (thio)ester probes
Most synthesized RhB esters, regardless of the introduced
functional group, display similar maximum absorption wave-
lengths ( max) and molar extinction coefficients ( ) of around
l
3
Fig. 3. a) The reaction scheme of labeling of bis-Aha-RNase A (14) with RhB propargyl ester (4) via click reaction; (b) the chemical structure of the unnatural amino acid, L-azi-
dohomoalanine (L-Aha); (c) the chemical structure of probe (4); (d) the gel-electrophoresis image (above: fluorescence; below: Coomassie stain) of the click reaction solution
þ
(
lane 1), the control reaction solution in the absence of the Cu catalyst (15, lane 2), pure bis-Aha-RNase A (14) (lane 3) and the protein ladder (lane 4).

302
X. Chen et al. / Dyes and Pigments 94 (2012) 296e303
Fig. 4. a) The UV absorption spectra of RhB and RhB (thio)esters (pH 7.2, 50
30 nm).
mM); (b) The fluorescence emission spectra of RhB and RhB (thio)esters (pH 7.2, 50 mM, excitation at
5
of RhB is independent of the respective functional group. Finally,
the quantum yield (V ) of each ester derivative in PBS buffer (pH
.2, 50 M) was also calculated (Table 1) by comparison with the
reported V
Previously, most reported optical data (e.g.
Acknowledgment
F
7
m
We thank NUS Cross Faculty Grant (Grant No. WBS-R-143-000-
393-123) and the Deutsche Forschungsgemeinschaft (SFB 625,
P3246 029 DFG) for the financial support of this work and J. Wil-
helmi for developing a protocol for the preparation of bis-Aha-
RNase A (14).
F
value of the zwitter-ion form of RhB (V
F
l
¼ 0.32) [21].
max, and
l
abs
,
em
,
3
F
V ) of RhB ester derivatives were determined in organic solvents,
e.g. EtOH or THF [31]. In this study, all the data were collected at
physiological pH in PBS buffer and therefore, they will be invaluable
for the selection of an ideal chromophore for either in vivo or in
vitro biomolecule labeling.
Appendix. Supplementary information
Since RhB esters carry multiple tertiary and quaternary carbons,
a full interpretation of typical RhB ester necessitates combined
H NMR, C NMR, DEPT 135, HSQC and HMBC (Figure S2) spectra
Supplementary data related to this article can be found online at
doi:10.1016/j.dyepig.2012.01.005.
1
13
analysis which is exemplified for RhB propargyl ester (3). This
interpretation will serve as a general guideline to interpret all other
RhB esters since they all share a very similar pattern in the NMR
spectra. All RhB esters reveal a characteristic cluster of seven carbon
signals in the region of d 129e134 ppm, which will be referred to as
the RhB Ester Carbon Heptet (Figure S2a). Noteworthy, the chemical
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