Synthesis of a new bifunctionalised fluorescent label and physical properties of the bound form on model peptide of troponin C

Article abstract of DOI:10.1039/b705704k

A new bifunctional fluorescent label, BRos, was synthesised in order to monitor protein dynamics using fluorescence microscopy, and the photophysical properties were compared with those of bifunctionalised rhodamine, BRho. In a labelling experiment with a model peptide of troponin C, which regulates muscle contraction and relaxation, it was found that BRos was bound to the peptide through two linkages and provided a homogeneous compound, whereas BRho gave a pair of diastereomers having different physical properties in NMR and HPLC analyses. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b705704k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of a new bifunctionalised fluorescent label and physical properties of
the bound form on model peptide of troponin C†
Tasuku Hirayama,a Shohei Iyoshi,a Masayasu Taki,a Yuichiro Maedab,c and Yukio Yamamoto*a
Received 16th April 2007, Accepted 17th May 2007
First published as an Advance Article on the web 4th June 2007
DOI: 10.1039/b705704k
A new bifunctional fluorescent label, BRos, was synthesised in order to monitor protein dynamics using
fluorescence microscopy, and the photophysical properties were compared with those of
bifunctionalised rhodamine, BRho. In a labelling experiment with a model peptide of troponin C,
which regulates muscle contraction and relaxation, it was found that BRos was bound to the peptide
through two linkages and provided a homogeneous compound, whereas BRho gave a pair of
diastereomers having different physical properties in NMR and HPLC analyses.
occur when a fluorescent probe bound in a single manner is used.
Introduction
Herein, we report a new bifunctionalised fluorescent dye, BRos, for
Recently, single molecule analysis by fluorescence microscopy has
protein labelling based on a rosamine platform (Scheme 1). Since
become a powerful method for monitoring and clarifying protein
rosamine has no substituent on the phenyl ring attached to the
dynamics.1 In order to reveal the orientation of the target domain
9-position of the xanthene ring, this fluorescent molecule should
of the protein by analysing that of the fluorescent dipole, not
not generate any diastereomers of the labelled form. Furthermore,
we performed a model peptide study8 to show the structural
differences between the two BRho-labelled peptide diastereomers,
prepared as a model of labelled-TnC.
only should the dye be fixed tightly but also the direction of
the dipole relative to the target a-helix of the protein should be
unambiguously determined. A number of fluorescent labels are
now available and have been applied to protein labelling,2 but
most of them are monofunctionalised, which have one reactive
linker arm to bind to a protein. It is impossible to estimate
a priori their dipole orientation against an a-helix, because of
free rotation around the linker, and thus the orientation should
be clarified by some experimental methods. In order to overcome
this problem, bifunctionalised rhodamine, BRho, was reported
by Corrie and co-workers.3 BRho binds to a protein through
two linkages and immobilizes its fluorescent dipole parallel to
the a-helix. They applied it to the myosin head domain and
troponin C (TnC),4,5 which play key roles in the regulation of
muscle contraction and relaxation,6 and discussed the mechanism
of the lever arm movement of myosin light chain and the TnC
orientation in the actin filament.5 In the double binding, however,
diastereomeric isomers are generated by the sterically restricted
rotation of the carboxyphenyl ring around the 1ꢀ–9 bond of
rhodamine.7 The energy barrier of the rotation was estimated to
be more than 20 kcal mol−1 by a DFT calculation.7 When it binds
to a chiral a-helix, the difference in the carboxyl group orientation
relative to the xanthene plane provides two diastereomers, and
their separation was reportedly difficult.4,5 This problem does not
Scheme 1 Structures of bifunctionalised fluorescent labels.
Results and discussion
Both BRos and BRho were synthesised as depicted in Scheme 2.
The aminophenol 5 is a key intermediate, as a common pre-
cursor for BRos and BRho. The previous reported synthesis of
aminophenol 5 took 8 steps with 12% overall yield.3 In this
study, we established a simple and efficient synthetic route for the
intermediate 5. The hydrochloride salt of 2, obtained by mono-
methylation of m-anisidine, was treated with 2-oxasolidinone to
yield the amine 3.9 Cleavage of O–CH3 with refluxing in HBr, fol-
lowed by acetylation provided 4, and selective hydrolysis afforded
5, which was purified by silica gel column chromatography. Thus,
we achieved the preparation of the aminophenol 5 in 25% overall
yield with only 5 steps.
By following general procedures, successive Friedel–Crafts
acylation of 5 with benzaldehyde and chloranil oxidation afforded
the rosamine-acetamide 6a in a yield of 12%. On the other hand,
the rhodamine-acetamide 6b was synthesised simply by heating
a mixture of 5 and phthalic anhydride, without any solvent or
catalyst. Although the rhodamine-acetamide 6b was synthesised
aGraduate School of Human & Environmental Studies, Kyoto University,
Yoshida, Sakyo-ku, Kyoto, 606–8501, Japan. E-mail: yamamoto_y@mbox.
kudpc.kyoto-u.ac.jp; Fax: +81 75 753 6833; Tel: +81 75 753 6833
bERATO Actin Filament Dynamics Project, Japan Science and Technology
Agency, c/o RIKEN, Harima SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo,
679-5148, Japan. E-mail: ymaeda@spring8.or.jp; Fax: +81 791 58 2836;
Tel: +81 791 58 2822
cGraduate School of Science, Nagoya University, Furo-cho, Nagoya, 464-
8602, Japan. E-mail: ymaeda@nucc.cc.nagoya-u.ac.jp; Fax: +81 52 789
2989; Tel: +81 52 789 3544
† Electronic supplementary information (ESI) available: MALDI-TOF
mass spectra and CD spectra. See DOI: 10.1039/b705704k
2040 | Org. Biomol. Chem., 2007, 5, 2040–2045
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Scheme 2 Synthesis of BRos and BRho. Reagents and conditions: (a) formaldehyde, NaOMe, NaBH4, 85 ◦C, overnight, quant.; (b) 6 M HCl, and then,
2-oxazolidinone, 150 ◦C, overnight; (c) conc. HBr, reflux, and then acetic anhydride, THF, 0 ◦C, 2 h, 28% for 2 steps; (d) KOH, MeOH, RT, 30 min, 91%;
(e) 6a: benzaldehyde, propionic acid, TsOH, 65 ◦C, overnight, and then chloranil, RT, 2 h, 12%; 6b: phthalic anhydride, 143 ◦C, 20%; (f) 1.5 M HCl,
reflux, 6 h, quant.; (g) chloroacetyl chloride, diisopropylethylamine, CH2Cl2, 0 ◦C, 2 h, 34%, (h) NaI, acetone, MeOH, CHCl3, RT, 3 days, 68%.
through two Friedel–Crafts acylations in previous studies, one-
step formation of the rhodamine platform was achieved in our
method. Finally, according to the reported procedure,3 the ac-
etamide group was converted to the thiol-reactive iodoacetamide
to give BRos and BRho. As a result, the overall yields were 0.8
and 1.4% for BRos and BRho, respectively, from the commercially
available compound 1.
The photophysical properties of the dyes were characterised
with the 2-mercaptoethanesulfonate (MES) derivatives, MES-
BRos and MES-BRho, respectively. The new fluorescent label,
BRos, showed almost the same spectroscopic properties as BRho
(Table 1). The quantum yields of MES-BRos and MES-BRho in
water are sufficiently high for fluorescense microscopy analyses.
We previously employed a chicken skeletal TnC mutant (S94C)
for labelling with the bifunctionalised spin labels.10 In the present
work, we chose an a-helical peptide, C8C15 (Ac-MKEDKAKCE-
EELANCFRIFDK-OH), as a model for the E-helix of TnC, to
confirm the structural homogeneity of the BRos-labelled protein.
The model peptide C8C15 has two cysteines, and the distance
between them is appropriate for the present fluorescent labels. In
this case, glycine-6 was replaced by alanine to stabilise the a-helical
structure (Fig. 1). Labelling C8C15 with BRos or BRho was per-
formed by the procedure reported previously,10 except for the final
purification using reverse phase HPLC. Fig. 2a shows part of the
HPLC profile of the BRos-labelled peptide in the final purification,
in which the major, single peak at 37 min, together with a broad
shoulder, appeared (monitored at 550 nm, solid line). A small peak
corresponding to unreacted peptide was also detected at 35 min
(monitored at 214 nm, dashed line). The MALDI-TOF mass spec-
trum of the main peak indicated the 1 : 1 adduct (BRos-pep, mass =
Fig. 1 Sequence and model of a labelled peptide.
3074) of the peptide labelled through two linkages. The formation
of BRos-pep was also confirmed by UV-vis and emission spectra,
in which the absorption and emission bands are similar to those
of BRos, together with a 214 nm band in the UV-vis spectrum,
due to the peptide absorption. By mass analysis, the shoulder
component (retention time 41 min) that appeared after the main
peak was assigned to the 1 : 2 adduct of the peptide with two dyes.
Another undesired 1 : 1 adduct, bound through only one linkage,
was not detected. In contrast to the labelling with BRos, the
reaction of the same peptide with BRho produced two distinctly
separated peaks with almost the same intensities (Fig. 2b). Their
mass spectra were identical to each other and were consistent with
the calculated mass of the 1 : 1 adduct (BRho-pep-1 and BRho-
pep-2, mass = 3118) with two linkages. The UV-vis spectra of these
two products showed the same features with absorption maxima
at 214 nm and 551 nm, due to the peptide backbone and the dye,
respectively. These results indicate that the reaction between BRho
and C8C15 produces almost equal amounts of each of the two
diastereomers, which are separable by HPLC. This suggests that
the configuration of the label is determined by the first of the two
consecutive reactions between the iodoacetamide groups and the
cysteine residues. Thus, it is evident that labelling with BRos yields
a structurally homogeneous product, while labelling with BRho
affords a pair of diastereomers with different hydrophobicities.
The CD spectra of the labelled peptides (BRos-pep, BRho-pep1,
and BRho-pep2) revealed Cotton effects at 194 nm (positive),
205 and 222 nm (negative), which are typical for an a-helix. The
intensities of their Cotton effects were almost the same as those
of the non-labelled peptide, and no spectral differences between
Table 1 Photophysical properties of MES-BRos and MES-BRho
kabs,max /nm
kem,max/nma
e/M−1cm−1b
Quantum yield
BRos
BRho
548
547
576
571
52 000
89 000
0.22
0.40
a Excited at 548 nm for BRos and at 547 nm for BRho. b Calculated at
548 nm for BRos and at 547 nm for BRho.
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other hand, the peaks corresponding to the aromatic hydrogens
of the xanthene and those of tyrosine-16 and -19 did not show
meaningful differences. These results indicate that the carboxylate
moieties of BRho on each labelled peptide are located in distinctly
different environments, while the peptide backbone remains the
same. The electrostatic interaction between the negatively charged
carboxylate of the dye and the positively charged ammonium
ion of lysine-7 is suggested to cause the down-field shifts of the
aromatic hydrogens in BRho-pep-2 (Fig. 3, blue line; Fig. 4).
Moreover, the fact that BRho-pep-2 elutes more slowly in the
reverse phase HPLC indicates its lower hydrophilicity compared to
that of the counterpart BRho-pep-1, and supports our interpreta-
tion (Fig. 2). The carboxylate group must be exposed to the solvent
in BRho-pep-1, making it more hydrophilic (Fig 4). Thus, the
HPLC and NMR results indicate that between the two diastere-
omers, the dye moiety must be in different environments which
must influence the direction of the fluorescent dipole of the dye.
Fig. 4 Models of the interaction between the amino group of Lys7 and the
carboxyl group of BRho, expected for BRho-pep-1 (left) and BRho-pep-2
(right).
Conclusions
In summary, we have synthesised a new bifunctionalised dye,
BRos, in which the synthetic route for the key intermediate
amino phenol 5, common to BRho, was improved. In addition,
a one-step synthesis of the rhodamine platform was achieved.
The photophysical properties of BRos and BRho were measured
and found to be almost the same. A peptide-labelling experiment
revealed that BRos provided the homogeneous 1 : 1 adduct (BRos-
pep) through two linkages, while a pair of diastereomers separable
by HPLC was obtained with BRho. On the other hand, we have
carried out labelling the TnC mutant (S94C) with BRho, and
the diastereomers could not be separated from each other. The
different behavior of the diastereomeric BRho-peptides in NMR
and HPLC analyses indicates that their physical properties are
not identical, and this could possibly have adverse consequences
for single molecular analyses. This problem may be avoided by
selecting BRho-labelling sites that are far from the cationic side
chains. As an alternative, BRos should be used, as this probe is
more predictable for the analyses. Now, the labelling of TnC with
BRos and its application in fluorescent analyses are in progress.
Fig. 2 HPLC charts of (a) BRos-peptide and (b) BRho-peptide at 550 nm
(solid line) and 214 nm (dashed line).
BRho-pep-1 and BRho-pep-2 were detected at the region of dye
absorption, despite their axial chirality. The structural difference
1
between these diastereomeric peptides was also confirmed by H
NMR experiments. Significant chemical shift differences (Dd) were
observed at the carboxylphenyl moieies of BRho-pep-1 and BRho-
ꢀ
pep-2 (Fig. 3). That is, a large difference of Dd3 = 0.08 ppm
ꢀ
ꢀ
and medium differences of Dd4 ,5 = 0.03 ppm were observed,
respectively, while the hydrogens at the 6ꢀ-position were overlapped
with other aromatic hydrogens and were not assigned. On the
Experimental
General
1H NMR spectra were recorded with a JEOL JNM-EX-270
spectrometer (at 270 MHz) and a JEOL alpha-500 spectrometer
Fig. 3 NMR spectra of BRho-pep-1 (red line) and BRh-pep-2 (blue line).
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(at 500 MHz) to 1H, using TMS as the internal standard. HRMS
was performed with a JEOL JMS-DX-300 spectrometer. BioRad
Duoflow was employed for HPLC. MALDI-TOF mass spectra
were measured by an Applied Biosystem Voyager-DE(tm) PRO
with a-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. UV-
vis spectra were measured with an Agilent 8453 spectrometer.
Circular dichroism (CD) spectra were obtained with a JASCO J-
715 spectropolarimeter. TLC analyses were carried out by using
silica gel 60 F254 (Merck). Flash chromatography was carried out
with Wakogel C-300 (silica gel, 100 ∼ 200 mesh, Wako Pure
Chemical Industries, Ltd.). Most of the reagents were purchased
from Wako Pure Chemical Industries, Ltd., Nacalai Tesque,
and Aldich Chemical Company, Inc. The model peptide (Ac-
MKEDKAKCEEELANCFRIFDK-OH) was purchased from
Tufts University Core Factory, Boston, USA.
270 MHz) d 1.86 (3 H, s, NHCOCH3), 2.26 (3 H, s, OCOCH3),
2.90 (3 H, s, NCH3), 3.42–3.43 (4 H, m, CH2CH2), 6.23–6.33 (3 H,
m, Ar), 7.06 (1 H, t, J = 8.1, Ar).
3-Hydroxy-N-(2-acetamidoethyl)-N-methylaniline
(5). The
acetamide 4 (500 mg, 2.0 mmol) was dissolved in MeOH (20 mL)
containing KOH (236 mg, 2.1 mmol), and the mixture was stirred
at room temperature under an argon atmosphere for 0.5 h. After
evaporation, the residue was diluted with 2 M aqueous potassium
phosphate (pH 8.5, 30 mL). The mixture was extracted with
CH2Cl2 (50 mL × 4). The combined organic extracts were dried
over MgSO4 and evaporated. The residual gum was azeotroped
with toluene (3 mL × 3) to afford 5 (379 mg, 91%), which was
1
used without further purification. TLC Rf = 0.21 (AcOEt); H
NMR (CDCl3, 270 MHz) d 1.94 (3 H, s, NHCOCH3), 2.91 (3 H,
s, NCH3), 3.42–3.43 (4 H, m, CH2CH2), 6.23–6.33 (3 H, m, Ar),
7.06 (1 H, t, J = 8.1, Ar).
Synthesis
Bis[N-(2-acetamidoethyl)-N-methyl)]-rosamine (6a). A mix-
ture of phenol 5 (417 mg, 2.0 mmol), benzaldehyde (106 mg,
1.0 mmol), and p-TsOH (18 mg, 0.10 mmol) was stirred at 65 ◦C for
21 h. After cooling to room temperature, the mixture was poured
into 3 M NaOAc (200 mL). The resulting suspension was extracted
with CHCl3 (100 mL × 3). The combined organic extracts were
dried over MgSO4 and then evaporated to give the crude dihydro-
compound. This was dissolved in a mixture of MeOH (25 mL) and
CHCl3 (25mL), andwas treated withchloranil (197 mg, 0.8 mmol).
After stirring vigorously for 2 h, the mixture was evaporated. The
residue was purified by flash chromatography (CHCl3–MeOH, 9 :
1) to give 6a (116 mg, 12%) as a purple solid. TLC Rf = 0.63
(CHCl3–MeOH, 2 : 1); 1H NMR (CDCl3, 270 MHz) d 1.95 (6 H,
s, NHCOCH3), 3.30 (6 H, s, NCH3), 3.55 (4 H, br s, CH2NH),
3.80 (4 H, br s, –CH2N), 7.10 (4 H, br s, Ar), 7.33–7.36 (4 H, m,
Ar), 7.62 (3 H, m, Ar).
3-Methoxy-N-methylaniline (2). To a suspension of NaOMe
(7.90 g, 146.2 mmol) in MeOH (37 mL) was added m-anisidine 1
(3.00 g, 24.4 mmol). The resulting brown solution was poured into
a suspension of paraformaldehyde (1.02 g, 34.2 mmol) in MeOH
(24 mL). After stirring at room temperature for 16 h, NaBH4
(923 mg, 24.2 mmol) was added to the mixture. After refluxing
for 2 h, the mixture was evaporated to 1/2 volume, treated with
1 M KOH (100 mL), and then extracted with ether (100 mL ×
2). The combined organic extracts were dried over MgSO4 and
evaporated to afford 2 (3.20 g, 96%), which was used without
1
further purification. TLC Rf = 0.41 (AcOEt–hexane, 1 : 1), H
NMR (CDCl3, 270 MHz) d 2.81 (3 H, s, –NCH3), 3.77 (3 H, s,
–OCH3), 6.15–6.16 (1 H, m, Ar), 6.20–6.29 (2 H, m, Ar), 7.08 (1H,
t, J = 8.1, Ar).
3-Acetoxy-N-(2-acetamidoethyl)-N-methylaniline (4). A mix-
ture of aniline 2 (2.20 g, 16.0 mmol) and 6 M HCl (20 mL)
was stirred at room temperature for 10 min, washed with ether
(50 mL), and evaporated to dryness. The residue was mixed with
2-oxazolidone (1.39 g, 16.0 mmol), and the mixture was heated at
150 ◦C for 19 h. After cooling to room temperature, 10% NaOH
(100 mL) was added to the mixture. The resulting solution was
extracted with CH2Cl2 (100 mL × 2). The combined extracts were
dried over MgSO4, and then evaporated. The residue was dissolved
in 48% HBr (40 mL), and the solution was refluxed under an argon
atmosphere for 2 h and evaporated. The residue was dissolved in
saturated aqueous sodium borate (138 mL). To the mixture was
added acetic anhydride (10.4 mL, 110.4 mmol) in THF (70 mL)
dropwise under an argon atmosphere at 0 ◦C. The mixture was
stirred at 0 ◦C for 30 min, while the pH of the mixture was
maintained at 9.0–9.5 by the addition of 2 M KOH, as required. A
further portion of acetic anhydride (5.52 mL, 58.6 mmol) in THF
(20 mL) was added to the mixture. Stirring was continued at 0 ◦C
for 1 h, while the pH was maintained as described above. A final
portion of acetic anhydride (3.45 mL, 36.6 mmol) in THF (12 mL)
was added, and the mixture was stirred at room temperature. After
stirring for 0.5 h, the mixture was extracted with CH2Cl2 (100 mL ×
3). The combined organic extracts were washed with saturated
NaHCO3 (150 mL), dried over MgSO4, and then evaporated. The
residual gum was azeotroped with toluene (3 mL × 3). Purification
by flash chromatography (AcOEt) afforded 4 (1.12 g, 28%) as
Bis[N-(2-acetamidoethyl)-N-methyl]-rhodamine (6b). The mix-
ture of phthalic anhydride (89 mg, 0.6 mmol) and phenol 5
(250 mg, 1.2 mmol) was heated at 150 ◦C for 19 h under an
argon atmosphere. The resultant gum was purified by flash column
chromatography (MeOH–CHCl3 1 : 1 → 1 : 0) to afford 64 mg
(20%) of 6b as a dark purple glass. TLC Rf = 0.21 (CHCl3–MeOH,
1 : 1); 1H NMR (CD3OD, 500 MHz) d 1.83 (6 H, s, NHCOCH3),
3.20 (6H, s, NCH3), 3.40 (4 H, t, J = 6.4, CH2NH), 3.66 (4H, t,
J = 6.4, –CH2N), 6.91 (d, 2 H, J = 2.4, 4-, 5-H), 6.99 (dd, J = 9.6
and 2.4 Hz, 2-,7-H), 7.17–7.23 (3H, m, 1-,8-,6ꢀ-H), 7.60–7.72 (2H,
m, 4ꢀ-,5ꢀ-H), 8.12 (d, J = 7.6 Hz, 1H, 3ꢀ-H).
Bis[N-(2-chloroacetamidoethyl)-N-methyl]-rosamine (8a).
A
mixture of the acetamide-rosamine 6a (116 mg, 0.24 mmol) and
1.5 M HCl (10 mL) was refluxed for 6 h and evaporated to give
the crude product 7a. It was azeotroped with toluene (5 mL) and
diluted with CH2Cl2 (10 mL). Chloroacetyl chloride (0.077 mL,
0.96 mmol) and diisopropylethylamine (0.33 mL, 1.9 mmol) were
◦
added to the solution with ice-cooling. After stirring at 0 C for
2 h and at room temperature for 1 h, a mixture of CHCl3 (135 mL)
and MeOH (15 mL) was added. The resulting solution was washed
with 0.5 M HCl (30 mL). The aqueous layer was extracted with
CHCl3–MeOH (9 : 1, 30 mL × 2), and the combined organic layers
were washed with 10% NaHCO3 (20 mL) and saturated NaCl
(20 mL). The solution was dried over MgSO4 and then evaporated.
1
a colorless gum. TLC Rf = 0.30 (AcOEt); H NMR (CDCl3,
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Purification by flash chromatography (MeOH–CHCl3, 9 : 1 → 4 :
1) afforded 9a (46 mg, 34%), TLC Rf = 0.70 (CHCl3–MeOH, 2 : 1);
1H NMR (CD3OD, 500MHz) d 3.27 (6 H, s, NCH3), 3.60 (4 H, br s,
CH2NH), 3.82 (4H, br s, –CH2N), 3.96 (4 H, s, –CH2Cl), 7.03 (4 H,
br s, Ar), 7.30–7.34 (4 H, m, Ar), 7.54–7.58 (3 H, m, Ar); 13C-NMR
(CD3OD, 126 MHz) : d 39.5, 42.8, 51.9, 53.7, 97.4, 129.0, 129.4,
130.5, 131.8, 131.9, 157.4, 158.0, 158.1, 167.7; HRMS (FAB) : m/z
calcd for C29H31Cl2N4O3 (M+) 553.1773, found 553.1775.
peptide was obtained, which was confirmed by its MALDI-TOF
mass spectrum (m/z 2589.1, calcd 2590.0). To a solution of 100 lM
purified model peptide, in 10 mM MOPS containing 0.1 M KCl
(pH 8.5), was added the BRos solution (10 mM stock solution in
DMF, final concentration of 100 lM). After a 1 h incubation, the
unreacted iodoacetamide groups were quenched by the addition
of 0.1 M aqueous sodium 2-mercaptoethanesulfonate (final conc.
2 mM). Removal of the unreacted dye by chromatography on
Sephadex G-25 (GE-Healthcare) provided the crude labelled
peptide. Reverse phase HPLC (Resource-RPC, GE-Healthcare)
with a linear gradient from 20% solvent A (0.1% TFA) and 80% B
(0.1% TFA, 90% acetonitrile) to 30% A and 70% B at 1 mL min−1
over 30 min, followed by lyophilisation, yielded BRos-pep (21 mg,
84%). The same procedure was applied to BRho-labelling and
afforded BRho-pep1 (10 mg, 40%) and BRh-pep2 (9.0 mg, 36%).
These peptides were all identified by MALDI-TOF mass spectra
(BRos-pep: m/z 3074.4, calcd for the 1 : 1 adduct 3072.4, BRho-
pep-1: m/z 3117.7 and BRho-pep-2: m/z 3118.1 calcd for the 1 :
1 adduct 3115.4).
Bis[N-(2-chloroacetamidoethyl)-N-methyl]-rhodamine (8b). The
rhodamine 8b was obtained from 105 mg (0.20 mmol) of 6b by
a similar procedure as for 8a in 37% yield (44 mg). TLC Rf =
0.79 (CHCl3–MeOH, 1 : 1); 1H-NMR (CD3OD, 500 MHz) d 3.23
(6H, s, NCH3), 3.51 (4 H, t, J = 6.4 Hz, CH2NH), 3.75 (4 H, t,
J = 6.4, –CH2N), 3.94 (4 H, s, –CH2Cl), 6.96 (2 H, d, J = 2.5, 4-,
5-H), 7.02 (2 H, dd, J = 9.6 and 2.5 Hz, 2-,7-H), 7.20–7.23 (3 H,
m, 1-,8-,6ꢀ-H), 7.63–7.67 (2 H, m, 4ꢀ-, 5ꢀ-H), 8.12 (1 H, d, J = 7.6,
3ꢀ-H); 13C-NMR (CD3OD, 126 MHz) : d 38.3, 39.6, 43.1, 52.3,
97.7, 97.8, 114.6, 114.7, 130.0, 130.7, 130.9, 131.1, 132.6, 135.5,
158.0, 158.1, 158.8, 172.8, 173.8.
NMR measurements of the labelled peptides
Bis[N-(2-iodoacetamidoethyl)-N-methyl]-rosamine, BRos. So-
dium iodide (2.08 mg, 13.9 mmol) was dissolved in a mixture of
dry acetone (13 mL), methanol (5 mL), and chloroform (2 mL).
The solution was stirred under an argon atmosphere for 10 min
and was added to 8a (46 mg, 0.083 mmol). The reaction vessel was
flushed with argon and was kept in the dark at room temperature
for 3 days. After adding CHCl3 (180 mL), the mixture was washed
with 5% aqueous sodium ascorbate (100 mL) and water (30 mL ×
3). The solution was dried over MgSO4 and evaporated to give
BRos (42 mg, 68%). The product was dissolved in DMF and stored
in aliquots (10 mM) at −80 ◦C.
NMR measurements of each labelled peptides were done with
the samples containing 2 mg of the labelled peptide (BRos-pep,
BRho-pep-1, BRho-pep-2) dissolved in 2 mL of D2O at 25 ◦C.
Fluorescent spectroscopy
Fluorescent spectra were recorded with a Hitachi F-4500 flu-
orophotometer with MES-BRos and MES-BRho in 25 mM
phosphate buffer (pH 7.2), prepared as described above. For
all measurements, the pathlength was 1 cm, with a cell volume
of 3.0 mL. The quantum yields for fluorescence were obtained
by comparison with fluorescein in 0.1 M NaOH, which has a
quantum efficiency of 0.95.11 The concentration of the reference
sample was adjusted to match the test sample. The quantum
efficiencies of MES-BRos and MES-BRho were obtained by using
diluted samples (absorbance <0.02) in 25 mM phosphate buffer
(pH 7.2).
Bis[N-(2-iodoacetamidoethyl)-N-methyl]-rhodamine, BRho.
A
similar procedure for BRos production yielded BRho (35 mg, 70%).
The product was dissolved in DMF and stored in aliquots (10 mM)
at −80 ◦C.
MES-BRos and MES-BRho
For photophysical measurements, BRos and BRho were converted
to the mercaptoethanesulfonate(MES) forms (MES-BRos and
MES-BRho), in order to inactivate the highly reactive iodoac-
etamide groups and to imitate a cysteine-bound form. To a mixture
of a portion of a 10 mM solution of the dye in DMF (40 lL) and
200 mM potassium phosphate buffer (pH 7.2, 0.35 mL) was added
200 mM aqueous sodium 2-mercaptoethanesulfonate (80 lL).
After stirring overnight in darkness at room temperature, the
solution was diluted with 25 mM phosphate buffer containing
150 mM NaCl (pH 7.2) to an appropriate concentration for
photophysical measurements.
Acknowledgements
This work was supported by Special Coordination Funds for pro-
moting Science and Technology from the Japanese Government
(No. 16655070) and by an ERATO grant from the Japan Science
and Technology Agency.
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