An Expedient Synthesis of Cationic Rhodamine Fluorescent Probes Suitable for Conjugation to Amino Acids and Peptides

Article abstract of DOI:10.1055/s-2003-42475

Rhodamine 19 benzyloxycarbonylmethyl ester bromide 8 and rhodamine 19 4-chloromethyl-1-phenylmethyl ester chloride 12 act as precursors to cationic fluorescent probes. Molecules containing free amine or carboxylate functional groups, respectively, can be attached in a one-step procedure, yielding the desired probes without the need for chromatographic purification. As a proof of concept the method was applied to the attachment of amino acid and dipeptide residues through either the N- or C-termini. The precursor molecules 8 and 12 are readily synthesized from the inexpensive, commercially available dye rhodamine 6G.

Full text of DOI:10.1055/s-2003-42475

PAPER
2647
An Expedient Synthesis of Cationic Rhodamine Fluorescent Probes Suitable
for Conjugation to Amino Acids and Peptides
An
E
xpedient Sy
a
C
r
a
tionic
R
hodam
o
ine Fluoresc
s
ent Probe A. M. Afonso,a V. Santhakumar,b Alan Lough,b Robert A. Batey*b
a
Department of Chemistry, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2825 Monte de Caparica,
Portugal
E-mail: cma@dq.fct.unl.pt
b
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, M5S 3H6, Canada
Fax +1(416)9785059; E-mail: rbatey@chem.utoronto.ca
Received 8 November 2002; revised 10 January 2003
Abstract: Rhodamine 19 benzyloxycarbonylmethyl ester bromide
8 and rhodamine 19 4-chloromethyl-1-phenylmethyl ester chloride
12 act as precursors to cationic fluorescent probes. Molecules con-
taining free amine or carboxylate functional groups, respectively,
can be attached in a one-step procedure, yielding the desired probes
without the need for chromatographic purification. As a proof of
concept the method was applied to the attachment of amino acid and
dipeptide residues through either the N- or C-termini. The precursor
molecules 8 and 12 are readily synthesized from the inexpensive,
commercially available dye rhodamine 6G.
Key words: fluorescent probes, rhodamines, amino acid deriva-
tives, peptides
The application of fluorescent molecular probes in high-
throughput assays, cellular imaging and for single mole-
cule detection is of growing importance,1 particularly with
the advent of fluorescent resonance energy transfer, fluo-
rescence correlation spectroscopy and fluorescence polar-
ization techniques. Of the various types of fluorescent
dyes that are routinely used, the rhodamine family is a
particularly important class of long-wavelength visible
dye. The use of fluorescent rhodamine containing mole-
cules as probes is of importance in several different fields,
including molecular biology and medicine, combinatorial
chemistry, polymer chemistry, chromatography, as well
as for single molecule detection in cells.2–5 Conjugation of
biologically relevant molecules to rhodamine probes typ-
ically occurs through reaction of a nucleophilic functional
group on the molecule of interest with an activated ester,
sulfonyl chloride or isothiocyanate functionality present
on the lower ring of the rhodamine dye 1. Although sev-
eral such rhodamine probe molecules and their precursors
are commercially available, many of these are quite ex-
pensive. We now report an alternative attachment strategy
for the formation of cationic dyes 3, using inexpensive
rhodamine 6G 2 as a precursor (Figure 1).
Figure 1
The pyrolysis can be achieved on up to 20 g scale by heat-
ing 2 for 4–5 hours at 265–275 °C under vacuum. Initial-
ly, conjugation to the nitrogen atoms of 4 was attempted
through amide or carbamate linkages. Such molecules
could potentially be used as mechanism-based probes,
only displaying high fluorescence on amide/carbamate
bond cleavage. Several attempts were performed for func-
tionalization of the amino group on the rhodamine ring
system, but clean conversions were generally not ob-
served, and only the N-acetyl derivative 5 was prepared
cleanly in low yield. Once protected the second free amine
group of 5 it is more reactive, and functionalization as the
N-CBz derivative 6, for instance, was achieved in high
yield. A much cleaner system for carbamate or amide at-
tachment (e.g., for the C-terminal attachment of simple
peptides through an amide linkage) utilizes the more reac-
tive, and also more expensive, rhodamine 110 as a precur-
sor (which contains free NH2 rather than NHEt groups on
the rhodamine ring).7 Given the poor reactivity profile of
We envisaged that commercially available rhodamine 6G
2 would serve as a useful precursor for fluorescent probes
after hydrolysis of the ethyl ester. Lactone 4 was prepared
by pyrolysis of 2 using a reported procedure (Scheme 1).6
SYNTHESIS 2003, No. 17, pp 2647–2654
x
x.
x
x
.
2
0
0
3
Advanced online publication: 21.11.2003
DOI: 10.1055/s-2003-42475; Art ID: M04802SS
© Georg Thieme Verlag Stuttgart · New York

2648
C. A. M. Afonso et al.
PAPER
4 shown in these exploratory studies, we next turned our
attention to conjugation through the carboxylate residue
of rhodamines 1 (Y = H).
2 (Rhodamine 6G)
R1
N
R2
N
O
Et
Et
(a) 86 %
Me
Me
The formation of cationic probe molecules 3 can in prin-
ciple be achieved through the conjugation of biologically
relevant molecules to rhodamine dyes through the 2-car-
boxylate group in 1 (Y = H). Rhodamine 6G 2 constitutes
a trivial example of such a probe, and it is used for in-
stance as a cationic cell permeant lipophilic probe.8 Reac-
tion of 4 with activated alkyl halides ( -halo esters or
benzyl halides), i-Pr2NEt and a catalytic quantity of sodi-
um iodide in refluxing acetonitrile, resulted in lactone
ring-opening and alkylation to form esters 7, 8, 11 and 12
in high yields (Schemes 1 and 2).9 The preparation of
these compounds could also be accomplished, albeit in
lower yield, through initial hydroxide induced hydrolysis
of 2 followed by direct alkylation. For example, reaction
of 2 with LiOH in THF–H2O and alkylation with ethyl 2-
bromoacetate under the above conditions afforded 7 in
36% overall yield. Hydrogenolysis of benzylic ester 8 af-
forded the carboxylic acid derivative 9, which serves as a
useful precursor for conjugation of molecules containing
amine, or potentially thiol, functionality. For example,
DCC/N-hydroxysuccinimide promoted amide bond for-
mation between 9 and L-phenylalanine methyl ester gave
derivative 10.10 Such a strategy could be further extended
for the conjugation of more complex amines or peptides.11
An alternative recent report by Adamczyk outlines a
method for the direct attachment of reactive primary
amines through their reaction with 2 using Hünig’s base in
DMF.12 There are also some reports in the patent literature
in which compounds of type 1 (Y = H) are directly conju-
gated to amines at the carboxylate group to form amides.13
O
4 (R1, R2 = H)
O
4-6
(b) 21 %
(c) 85 %
5
(R1 = Ac; R2 = H)
(R1 = Ac; R2 = Cbz)
6
X
H
N
H
N
O
Et
Et
O
Me
Me
O
R3
O
7-10
(d) 80 %
7 (R3 = OEt)
(e) 77 %
8 R3 = OCH2Ph
(f) 79 %
9 R3 = OH
(f),(g) 69 %
CH2Ph
OCH3
10 R3
=
HN
O
Scheme 1 Conditions: (a) reported method:6 265–275 °C, 5 h, va-
For the attachment of the carboxylic acid group of peptide cuum; (b) AcCl (1.1 equiv), DMAP (cat.), Et3N (1.1 equiv), CH2Cl2,
reflux, overnight; (c) NaH (3.9 equiv), THF, BnOCOCl (3.9 equiv),
reflux, 3 h; (d) i-Pr2NEt (1.2 equiv), BrCH2CO2Et (1.0 equiv), NaI
(cat.), MeCN, reflux, 24 h; (e) i-Pr2NEt (1.2 equiv), BrCH2CO2CH2Ph
(1.1 equiv), NaI (cat.), MeCN, reflux, 2.5 h; (f) H2, 10% Pd/C, EtOH,
derivatives and other compounds to rhodamines an alter-
native strategy is required. We envisaged that a benzyl
chloride functionalized rhodamine derivative would serve
as an excellent electrophile for the alkylation of carboxy-
lic acids. Such a compound could be formed by the 1:1
coupling of 4 with a bis-benzylic halide. Preliminary ex-
periments using i-Pr2NEt and a catalytic quantity of sodi-
um iodide in refluxing acetonitrile, using benzylbromide
as a model electrophile afforded benzyl ester 11
(Scheme 2). Reaction of 4 with 20 equivalents of , -
dichloro-p-xylene similarly gave the key benzyl chloride
functionalized rhodamine derivative 12 in 88% yield. An
excess of the electrophile was employed to prevent bis-es-
terification by , -dichloro-p-xylene. Compound 12
could then be cleanly converted to the ester linked com-
pounds 13–18 in high yield through reaction with the ap-
propriate carboxylic acid using cesium carbonate and
sodium iodide in DMF.14 This method was applied to the
attachment of the C-termini of N-Boc, N-Fmoc and N-Cbz
protected -amino acids and a dipeptide to give 13–18.
During the course of this study we observed that these
compounds were insoluble in diethyl ether which allowed
the development of a simple purification procedure, by
washing the crude solid residues with diethyl ether and/or
r.t., 7 h; (g) DCC (1.1 equiv), N-hydroxysuccinimide (1.1 equiv), i-
Pr2NEt (1.1 equiv), RNH2·HCl (1.1 equiv), DMF, r.t., overnight
crystallization using ethanol (or ethyl acetate)–diethyl
ether. In addition to the reaction of 12 with carboxylates,
we anticipate that the benzyl chloride residue in 12 may
also serve as a useful molecular probe for reaction with
thiols and other highly nucleophilic functional groups.
Determination of comparative fluorescent properties of
some of the compounds prepared above, in relation to
rhodamine 6G, were also undertaken. Derivative 6 is not
fluorescent by comparison with the strong fluorescence of
rhodamine 6G or the derivatives 4, 5, 10, 13 and 15.15 X-
ray crystallographic analysis of 14 (Figure 2) revealed a
clear -stacked orientation of the xanthenylium ring sys-
tem with the aromatic ring of the linker.16,17
In conclusion, we have demonstrated the use of
rhodamine 6G as an inexpensive precursor for the creation
of model fluorescent molecular probes. Attachment of the
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An Expedient Synthesis of Cationic Rhodamine Fluorescent Probe
2649
Figure 2 Thermal ellipsoid plot of 14. Ellipsoids are drawn at the
30% level. Hydrogen atoms and the iodide ion have been omitted for
clarity.
ing point apparatus. Microanalyses were carried out at Canadian
Microanalytical Service Ltd. Low resolution mass spectra (MS)
were recorded on a Bell and Howell 21-490 spectrometer. High res-
olution mass spectra (HRMS) were recorded on a AEI MS3074
spectrometer. Infrared spectra (IR) were recorded on a Perkin-Elm-
er FT-IR spectrophotometer, with samples loaded as neat films on
NaCl plates. 1H and 13C NMR were recorded on a Varian Unity 200
and 400 spectrometers as solutions in CDCl3 or CD3OD. Chemical
shifts are expressed in (ppm) values. The following abbreviations
are used: s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, and br = broad. Fluorescence spectra (excitation at
495 nm) were recorded on a Spex Fluorolog Mod. F 111 spectro-
photometer in CH2Cl2 Spectranal® (Fluka). The comparative quan-
tum yields relative to Rhodamine 6G were determined according to
standard procedures.18
Scheme 2 Conditions: (a) PhCH2Br (1.0 equiv), NaI (cat.), i-Pr2NEt
(1.2 equiv), MeCN, reflux, 4 h; (b)
, -dichloro-p-xylene (20 mol
equiv), NaI (20 mol%), i-Pr2NEt (1.2 equiv), MeCN, reflux, 2.5 h; (c)
Cs2CO3 (0.65 mol equiv), RCO2H (1.06 equiv), NaI (1.1 equiv),
DMF, r.t., 24 h
Pyrolysis of Rhodamine 6G6
Rhodamine 6G (7.0 g, 12.5 mmol, Sigma-Aldrich) was taken in a
250 mL round bottom flask and fitted to a rotary evaporator in the
fume hood connected to high vacuum. The flask was immersed in a
large oil bath and heated with rotation to 265–275 °C (caution: de-
composition was observed if heated to more than 285 °C). After 4–
5 h the flask was cooled to give 4 (5.0 g, 86%) as a red powdered
solid.
rhodamine nucleus to either the N- or C-termini of amino
acids or peptides can be accomplished. The syntheses are
short and high yielding and the method developed should
be applicable to the formation of a variety of probe mole-
cules and fluorescent reporters.
1H NMR (200 MHz, CDCl3): = 1.32 (t, 6 H, J = 7.0 Hz, 2 × CH3),
1.93 (s, 6 H, 2 × ArCH3), 3.23 (m, 4 H, 2 × NCH2), 3.60 (br, 2 H,
2 × NH), 6.35 and 6.40 (2 × s, 2 × 2 H, ArH), 7.16 (d, 1 H, J = 7.7
Hz, ArH), 7.61 (m, 2 H, ArH), 8.02 (d, 1 H, J = 6.2 Hz, ArH).
All reagents were of commercial quality. Anhydrous CH3CN
(CaH2), DMF (BaO), Et3N (NaOH), CH2Cl2 (P2O5) were distilled.
Anhydrous benzene, THF and Et2O were prepared by distillation
from Na/benzophenone ketyl under argon. All other solvents and
reagents were used as obtained, except hexanes, which were dis-
tilled prior to use. All reactions under anhydrous conditions were
performed under an atmosphere of nitrogen. Flash column chroma-
tography was performed using silica gel (60 Å, 230-400 mesh, ob-
tained from Whatman Company). Analytical thin-layer
chromatography (TLC), was performed on pre-coated silica gel
plates, (Alugram SIL G/UV254 obtained from Rose Scientific Lim-
ited). Solvent systems used to determine Rf values and for chroma-
tography are reported as v/v ratios. Melting points (uncorrected)
were determined on a Electrothermal Mod. IA 6304 capillary melt-
N-Acetyl-rhodamine 5
Acetyl chloride (1.25 g, 15.9 mmol) was added drop-wise to a
stirred suspension of 4 (6.0 g, 14.5 mmol), Et3N (1.61 g, 15.9 mmol)
and DMAP (0.178 g, 0.15 mmol) in anhyd CH2Cl2 (150 mL) at r.t.
under nitrogen. The reaction mixture was refluxed overnight and,
after allowing to cool to r.t., Et2O (200 mL) was added and the re-
action mixture filtered. The filtrate was concentrated in vacuo, and
the residue purified by flash chromatography (Et2O–hexane, 4:1) to
afford 5 (1.36 g, 21%) as pale yellow needles, mp 268–270 °C.
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C. A. M. Afonso et al.
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IR (nujol): 3410, 2923, 2853, 1760, 1651, 1634, 1617, 1524, 1497,
1463, 1376, 1274, 1220, 1106, 1088, 1014, 876, 749 cm–1.
7.5 Hz, ArH), 7.77 (dt, 1 H, J = 1.0, 7.5 Hz, ArH), 7.84 (dt, 1 H,
J = 1.0, 7.5 Hz, ArH), 8.42 (dd, 1 H, J = 1.0, 7.5 Hz, ArH).
1H NMR (200 MHz, CDCl3): (hindered rotation) = 1.13 and 1.31
(2 × t, 6 H, J = 7.0 Hz, 2 × CH3), 1.76 and 1.78 (2 × s, 3 H, CH3CO),
1.93 and 2.02 (2 × s, 6 H, 2 × ArCH3), 3.13–3.27 (m, 3 H, NCH and
NCH2), 3.72 (br s, 1 H, NH), 4.09 (m, 1 H, NCH), 6.36 and 6.39
(2 × s, 2 H, ArH), 6.65 (d, 1 H, J = 2.5 Hz, ArH), 7.00 (s, 1 H, ArH),
7.20 (m, 1 H, ArH), 7.62–7.68 (m, 2 H, ArH), 8.06 (d, 1 H, J = 8.0
Hz, ArH).
13C NMR (50 MHz, CDCl3): = 13.73, 18.70, 38.20, 61.30, 93.52,
113.20, 126.12, 128.23, 128.86, 130.00, 130.19, 131.41, 133.18,
134.42, 155.82, 156.05, 157.02, 164.13, 166.65.
MS (EI): m/z (%) = 500 (M+, 75), 485 (100), 458 (6), 415 (15), 399
(17), 370 (35), 355 (99), 341 (5), 326 (13), 312 (11).
HRMS: m/z calcd for C30H32N2O5: 500.2311; found: 500.2315.
13C NMR (100 MHz, C6D6): (hindered rotation) = 13.35, 13.39,
14.48, 16.60, 16.67, 22.38, 22.44, 38.37, 43.06, 43.20, 83.00, 83.11,
96.79, 106.49, 117.92, 118.04, 119.39, 119.42, 120.30, 124.14,
125.24, 127.32, 127.55, 128.94, 128.98, 129.81, 130.67, 130.75,
131.68, 131.72, 134.96, 144.12, 148.94, 148.96, 150.63, 150.66,
151.68, 151.76, 154.59, 154.78, 168.44, 168.85, 169.44, 169.54.
MS (EI): m/z (%) = 456 (M+, 13), 441 (2), 412 (70), 397 (98), 383
(78), 369 (13), 353 (21), 342 (53), 326 (100), 299 (19), 282 (10),
170 (10), 70 (71).
Preparation of 7 from Rhodamine 6G
To a stirred solution of rhodamine 6G 2 (502 mg, 1.05 mmol, from
Aldrich) and LiOH.H2O (88 mg, 2.1 mmol) in THF (4 mL) and H2O
(4 mL) was refluxed for 4 h under nitrogen. The solvent was evap-
orated to dryness in vacuo and acetonitrile (11 mL), NaI (catalytic
amount), ethyl diisopropylamine (430 L, 2.5 mmol) and ethyl 2-
bromoacetate (270 L, 2.4 mmol) were added successively. The re-
action mixture was refluxed for 20 h, then cooled to r.t., concentrat-
ed in vacuo and purified as described earlier to give 7 (221 mg,
36%).
HRMS: m/z calcd for C28H28N2O4: 456.2049; found: 456.2066.
Rhodamine 19 Benzyloxycarbonylmethyl Ester Bromide 8
Preparation of benzyl 2-bromoacetate: To a stirred solution of ben-
zyl alcohol (2.01 g, 18.6 mmol) and 2,6-lutidine (2.4 mL, 20.4
mmol) in anhyd CH2Cl2 (80 mL) at 0 °C under nitrogen was added
drop-wise (5 min) bromoacetyl bromide (1.6 mL, 18.6 mmol) and
the mixture stirred for 2 h. The solvent was evaporated from the re-
action mixture, Et2O (100 mL) added, filtered and the filtrate was
concentrated in vacuo and chromatographed on silica gel column
(CH2Cl2) to afford benzyl 2-bromoacetate (3.87 g, 91%), as an col-
orless liquid. The material had identical spectral data to those re-
ported,19 and should be stored in the fridge and used within one
week.
N-Acetyl-N -benzyloxycarbonyl-rhodamine 6
Benzyl chloroformate (0.29 g, 1.75 mmol) was added drop-wise to
a stirred suspension of 5 (0.20 g, 0.44 mmol) and NaH (80% mineral
oil, 0.053 g, 1.75 mmol) in anhyd THF (4 mL) at r.t. under nitrogen.
The reaction mixture was refluxed for 3 h and then cooled to r.t.
H2O (15 mL) was carefully added, followed by extraction with Et2O
(2 × 50 mL). The combined organic layers were dried (MgSO4), fil-
tered, concentrated in vacuo and chromatographed on silica gel col-
umn (Et2O) to afford 6 (0.22 g, 85%) as colorless needles, mp 125–
127 °C.
IR (nujol): 2923, 2853, 1769, 1704, 1660, 1613, 1490, 1462, 1376,
1273, 1216, 1149, 1084, 748 cm–1.
To a stirred mixture of 4 (351 mg, 0.85 mmol), NaI (catalytic
amount) in anhyd CH3CN (8.5 mL) under nitrogen at r.t. was added
successively ethyl diisopropylamine (177 L, 1.0 mmol) and benzyl
2-bromoacetate (204 mg, 0.89 mmol) and then refluxed for 2.5 h.
The reaction mixture was cooled to r.t., concentrated in vacuo and
chromatographed on silica gel column (EtOAc–EtOH, 8:2 to 7:3) to
afford 8 (418 mg, 77%) as gold needles, mp 220–224 °C (EtOAc–
EtOH).
1H NMR (200 MHz, CDCl3): = 1.13 (m, 6 H, 2 × CH3), 1.15 and
1.16 (2 × s, 3 H, CH3CO), 1.75 and 2.04 (s and m, 6 H, 2 × ArCH3),
3.17–4.12 (4 × m, 4 H, 4 × NCH), 5.05–5.16 (m, 2 H, OCH2Ph),
6.66 and 6.72 (2 × s, 2 H, ArH), 7.04–7.50 (m, 8 H, ArH), 7.68 (br
s, 2 H, ArH), 8.08 (d, 1 H, J = 6.5 Hz, ArH).
13C NMR (50 MHz, CDCl3): (hindered rotation) = 13.36, 13.42,
14.25, 17.42, 17.56, 22.88, 43.36, 45.48, 67.69, 117.34, 118.01,
118.33, 119.47, 124.36, 125.85, 128.40, 128.86, 129.93, 130.61,
131.96, 132.85, 135.84, 137.09, 143.08, 144.02, 149.85, 150.17,
150.27, 153.24, 155.26, 169.55, 169.66, 170.00, 170.22.
IR (film): 3226, 3024, 2976, 1731, 1650, 1606, 1564, 1527, 1503,
1447, 1366, 1305, 1186, 1132, 1091, 1022, 816, 733, 698, 667 cm–1.
1H NMR (400 MHz, CDCl3): = 1.39 (t, 6 H, J = 7.0 Hz,
NCH2CH3), 2.26 (d, 6 H, J = 1.0 Hz, ArCH3), 3.53 (m, 4 H, NCH2),
4.63 (s, 2 H, OCH2COCH2Ph), 5.05 (s, 2 H, OCH2Ph), 6.62 (s, 2 H,
ArH), 6.73 (d, 2 H, J = 1.0 Hz, ArH), 6.94 (br s, 2 H, NH), 7.19–
7.22 (m, 2 H, ArH), 7.26–7.29 (m, 4 H, ArH), 7.75 (dt, 1 H, J = 1.5,
7.5 Hz, ArH), 7.82 (dt, 1 H, J = 1.5, 7.5 Hz, ArH), 8.39 (dd, 1 H,
J = 1.1, 7.5 Hz, ArH).
13C NMR (50 MHz, CD3OD): = 14.08, 17.60, 39.43, 62.55, 67.71,
94.87, 114.86, 126.83, 128.72, 129.20, 129.44, 130.04, 130.59,
131.47, 131.77, 132.49, 134.56, 135.70, 136.86, 157.62, 158.83,
158.92, 166.04, 168.59.
MS (EI): m/z (%) = 590 (M+, 21), 546 (73), 531 (17), 517 (65), 455
(98), 386 (63), 342 (18), 91 (100).
HRMS: m/z calcd for C36H34N2O6: 590.2417; found: 590.2405.
Rhodamine 19 Ethoxycarbonylmethyl Ester Bromide 79
To a stirred mixture of 4 (153 mg, 0.37 mmol), NaI (catalytic
amount) in anhyd CH3CN (3.7 mL) under nitrogen at r.t. was added
successively ethyl diisopropylamine (77 L, 0.44 mmol) and ethyl
2-bromoacetate (41 L, 0.37 mmol) and then refluxed for 24 h. The
reaction mixture was cooled to r.t., concentrated in vacuo and chro-
matographed on a silica gel column (EtOAc–EtOH, 8:2 to 7:3), to
afford a red amorphous solid (191 mg) which was crystallized from
EtOH–hexane to give 7 (173 mg, 80%) as red plates, mp 253–
256 °C (decomposition), (ethanol–hexane).
MS (EI): m/z (%) = 562 (M+, 31), 547 (52), 415 (14), 370 (32), 369
(31), 355 (100), 341 (5), 339 (15), 326 (15), 311 (12), 297 (6), 149
(18), 107 (27), 91 (87).
HRMS: m/z calcd for C35H34N2O5: 562.2468; found: 562.2466.
IR (film): 3224, 2978, 1729, 1649, 1562, 1528, 1502, 1448, 1367,
1306, 1244, 1211, 1186, 1133, 1092, 1022, 884, 816, 780, 733 cm–1.
Anal. Calcd for C35H35BrN2O5: C, 65.32; H, 5.48; N, 4.35. Found:
C, 64.98; H, 5.42; N, 4.29.
1H NMR (400 MHz, CDCl3): = 1.15 (t, 3 H, J = 7.5 Hz,
OCH2CH3), 1.40 (t, 6 H, J = 7.5 Hz, NCH2CH3), 2.29 (s, 6 H,
ArCH3), 3.55 (qui, 4 H, J = 7.5 Hz, NCH2CH3), 4.09 (q, 2 H, J = 7.5
Hz, OCH2CH3), 4.59 (s, 2 H, OCH2CO2Et), 6.28 (s, 2 H, ArH), 6.74
(s, 2 H, ArH), 7.03 (t, 2 H, J = 5.5 Hz, NH), 7.30 (dd, 1 H, J = 1.0,
Rhodamine 19 Hydroxycarbonylmethyl Ester Bromide 9
A mixture of 8 (197 mg, 0.31 mmol) and 10% palladium on carbon
(58 mg) in absolute EtOH (8 mL) was vigorously stirred under hy-
Synthesis 2003, No. 17, 2647–2654 © Thieme Stuttgart · New York

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An Expedient Synthesis of Cationic Rhodamine Fluorescent Probe
2651
drogen atmosphere (balloon) for 24 h at r.t. The mixture was fil-
tered, concentrated in vacuo and crystallized from EtOH–EtOAc to
afford 9 (134 mg, 79%) as green plates, mp 106–360 °C (slow de-
composition), (EtOH–EtOAc).
graphed on silica gel column (EtOAc–EtOH, 9:1 to 8:2) to afford 11
(145 mg, 66%) as red solid; mp 264-285 °C (decomposition)
(EtOH–hexane, red plates).
IR (film): 3233, 3024, 2976, 1718, 1649, 1607, 1561, 1528, 1500,
1449, 1367, 1306, 1187, 1130, 1088, 1022, 815, 733, 698 cm–1.
IR (film): 3270, 2970, 1725, 1647, 1606, 1529, 1501, 1446, 1305,
1266, 1242, 1185, 1134, 1089, 1021 cm–1.
1H NMR (400 MHz, CDCl3): = 1.46 (t, 6 H, J = 7.0 Hz,
NCH2CH3), 2.30 (s, 6 H, ArCH3), 3.62 (quintet, 4 H, J = 7.0 Hz,
NCH2CH3), 4.96 (s, 2 H, OCH2Ph), 6.57 (s, 2 H, ArH), 6.72 (d, 2 H,
J = 1.0 Hz, ArH), 6.93 (dd, 2 H, J = 1.5, 7.5 Hz, ArH), 7.15 (t, 2 H,
J = 5.5 Hz, NH), 7.21 (dd, 1 H, J = 1.5, 7.5 Hz, ArH), 7.22 (dd, 1 H,
J = 1.5, 7.5 Hz, ArH), 7.26–7.32 (m, 2 H, ArH), 7.76 (dt, 1 H,
J = 1.5, 7.5 Hz, ArH), 7.81 (dt, 1 H, J = 1.5, 7.5 Hz, ArH), 8.36 (dd,
1 H, J = 1.5, 7.5 Hz, ArH).
1H NMR (400 MHz, CD3OD): = 1.36 (t, 6 H, J = 7.5 Hz,
NCH2CH3), 2.12 (d, 6 H, J = 1.0 Hz, ArCH3), 3.52 (q, 4 H, J = 7.5
Hz, NCH2CH3), 4.88 (s, 2 H, OCH2CO2H), 6.87 (d, 2 H, J = 1.0 Hz,
ArH), 6.91 (s, 2 H, ArH), 7.42 (dd, 1 H, J = 1.5, 7.5 Hz, ArH), 7.83
(dt, 1 H, J = 1.5, 7.5 Hz, ArH), 7.89 (dt, 1 H, J = 1.5, 7.5 Hz, ArH),
8.41 (dd, 1 H, J = 1.5, 7.5 Hz, ArH).
13C NMR (50 MHz, CD3OD): = 14.09, 17.66, 39.43, 62.37, 94.85,
114.83, 126.79, 130.02, 130.79, 131.44, 131.59, 131.72, 131.84,
132.49, 134.46, 135.60, 157.55, 158.86, 166.05, 170.26.
13C NMR (50 MHz; CDCl3) = 13.88, 18.77, 38.34, 67.37, 93.63,
113.26, 126.15, 128.12, 128.26, 128.38, 129.99, 131.38, 132.76,
133.79, 134.21, 155.81, 156.28, 156.88, 164.91.
MS (EI): m/z (%) = 472 (M+, 55), 457 (80), 444 (15), 429 (20), 415
(60), 413 (100), 399 (45).
MS (EI): m/z (%) = 504 (M+, 35), 489 (38), 413 (13), 370 (42), 369
(42), 355 (100), 339 (12), 326 (15), 311 (12).
HRMS: m/z calcd for C28H28N2O5: 472.1998; found: 472.1983.
HRMS: m/z calcd for C33H32N2O3: 504.2413; found: 504.2431.
Rhodamine 19 (L-Methyl-phenylalaninyl)-carbonylmethyl Es-
ter Bromide 1010
Rhodamine 19 4-Chloromethyl-1-phenylmethyl Ester Chloride
12
To a stirred solution of the crude solid residue of 9 [prepared as re-
ported above starting from 8 (62.0 mg, 0.10 mmol)] in anhyd DMF
(2.5 mL) at r.t. under nitrogen, was successively added N-hydrox-
ysuccinimide (12.6 mg, 0.11 mmol) and a solution of 1,3-dicyclo-
hexylcarbodiimide (22.6 mg, 0.11 mmol) in anhyd CH2Cl2 (1 mL).
After 30 min ethyl diisopropylamine (19 L, 0.11 mmol) and L-phe-
nylalanine methyl ester hydrochloride (23.7 mg, 0.11 mmol) were
successively added and the mixture was stirred overnight. The reac-
tion mixture was diluted with CH2Cl2 (80 mL), washed with H2O
(2 × 50 mL), dried (MgSO4), concentrated in vacuo and chromato-
graphed on silica gel column (CH2Cl2–MeOH, 9:1) to afford 10
(47.3 mg, 69%) as red solid, mp 145–169 °C (decomposition),
(EtOH–EtOAc–Et2O; red plates).
To a stirred mixture of 4 (1.12 g, 2.70 mmol), NaI (81 mg, 0.54
mmol) in anhyd CH3CN (20 mL) under nitrogen at r.t. was added
successively ethyl diisopropylamine (565 L, 3.24 mmol) and , -
dichloro-p-xylene (9.47 g, 54.1 mmol) and then refluxed for 2.5 h.
The reaction mixture was warmed to r.t., concentrated in vacuo and
the solid residue washed successively with Et2O (5 × 30 mL),
EtOAc (2 × 20 mL) and Et2O (1 × 20 mL), and crystallized from
EtOH–EtOAc–Et2O to give 12 (1.40 g, 88%) as a red powdered sol-
id, mp > 345 °C (decomposition).
IR (film): 3222, 3024, 2976, 1717, 1649, 1606, 1566, 1528, 1501,
1448, 1367, 1306, 1186, 1130, 1087, 1022, 821, 733 cm–1.
1H NMR (400 MHz, CDCl3): = 1.42 (t, 6 H, J = 7.0 Hz,
NCH2CH3), 2.25 (s, 6 H, ArCH3), 3.56 (quintet, 4 H, J = 7.0 Hz,
NCH2CH3), 4.57 (s, 2 H, ArCH2Cl), 4.91 (s, 2 H, OCH2Ar), 6.54 (s,
2 H, ArH), 6.67 (s, 2 H, ArH), 6. 86 (d, 2 H, J = 8.0 Hz, ArH), 7.19
(d, 2 H, J = 8.0 Hz, ArH), 7.23–7.24 (m, 1 H, ArH), 7.32 (br t, 2 H,
J = 7.0 Hz, NH), 7.72 (dt, 1 H, J = 1.5, 7.5 Hz, ArH), 7.77 (dt, 1 H,
J = 1.5, 7.5 Hz, ArH), 8.32 (dd, 1 H, J = 1.5, 7.5 Hz, ArH).
13C NMR (100 MHz, CD3OD): = 14.15, 17.66, 39.46, 46.59,
67.97, 94.91, 114.63, 126.81, 129.66, 129.75, 129.80, 131.44,
131.51, 132.42, 134.12, 134.80, 135.97, 139.33, 157.43, 158.57,
158.66, 166.58.
IR (film): 3241, 3024, 2976, 2931, 2853, 1732, 1650, 1607, 1529,
1501, 1448, 1367, 1306, 1186, 1140, 1090, 1022, 814, 733, 701,
667 cm–1.
1H NMR (400 MHz, CDCl3): = 1.35 (q, 6 H, J = 7.0 Hz,
NCH2CH3), 2.22 (s, 3 H, ArCH3), 2.24 (s, 3 H, ArCH3), 3.04 (dd, 1
H, J = 6.0, 13.5 Hz, PhCH2CHN), 3.09 (dd, 1 H, J = 6.0, 13.5 Hz,
PhCH2CHN), 3.44–3.52 (br, 4 H, NCH2CH3), 3.69 (s, 3 H,
CO2CH3), 4.50 (d, 1 H, J = 15.5 Hz, OCH2CO), 4.54 (d, 1 H,
J = 15.5 Hz, OCH2CO), 4.78 (dd, 1 H, J = 6.0, 13.5 Hz,
PhCH2CHN), 6.59 (s, 1 H, ArH), 6.62 (s, 1 H, ArH), 6.66 (s, 1 H,
ArH), 6.69 (s, 1 H, ArH), 6.68–6.71 (m, 1 H, ArH), 6.92 (br s, 1 H,
NH), 7.00–7.03 (m, 3 H, ArH, NH), 7.20–7.21 (m, 3 H, ArH), 7.29
(d, 1 H, J = 7.0 Hz, ArH), 7.73 (t, 1 H, J = 7.5 Hz, ArH), 7.84 (t, 1
H, J = 7.5 Hz, ArH), 8.20 (d, 1 H, J = 7.5 Hz, ArH).
MS (EI): m/z (%) = 552 (M+, 58), 537 (70), 516 (34), 501 (28), 413
(25), 399 (17), 369 (30), 355 (70), 339 (28), 326 (23), 312 (19), 174
(26), 139 (100).
13C NMR (100 MHz, CDCl3): = 13.89, 15.25, 18.41, 18.53, 24.94,
25.58, 33.87, 37.45, 38.47, 49.00, 52.55, 52.84, 63.42, 65.84, 93.89,
113.39, 113.41, 126.06, 126.11, 127.16, 128.29, 128.46, 128.56,
129.23, 130.28, 130.39, 131.32, 133.58, 134.87, 135.49, 155.83,
155.89, 156.28, 157.04, 157.09, 163.46, 165.90, 171.47.
MS (EI): m/z (%) = 633 (M+, 20), 618 (37), 574 (6), 413 (70), 399
(21), 369 (24), 355 (72), 339 (15), 326 (17), 224 (16), 187 (17).
HRMS: m/z calcd for C34H33ClN2O3: 552.2179; found: 552.2183.
Attachment of Carboxylic Acid Derivatives to Rhodamine De-
rivative 12; General Procedure14
Rhodamine 19 4-(2 -Methyl-propionyl)-oxymethyl-1-phenyl-
methyl Ester Iodide 13
A suspension of isobutyric acid (162 mg, 0.18 mmol) and Cs2CO3
(36.1 mg, 0.11 mmol) in anhyd DMF (2 mL) was stirred at r.t. for 1
h, and then evaporated at 40–45 °C under reduced pressure. The res-
idue was dissolved in anhyd DMF (2 mL) and NaI (27.6 mg, 0.18
mmol) and 12 (102.0 mg, 0.17 mmol) were successively added. Af-
ter 24 h the mixture was partitioned between CH2Cl2 (70 mL) and
H2O (50 mL). The organic layer was washed with H2O (4 × 50 mL),
dried (MgSO4), concentrated in vacuo and crystallized from EtOH–
Et2O (1:10, 22 mL) to give 13 (100.1 mg, 79%) as red plates, mp
136–138 °C.
HRMS: m/z calcd for C38H39N3O6: 633.2839; found: 633.2809.
Rhodamine 19 Benzyl Ester Bromide 11
To a stirred mixture of 4 (157 mg, 0.38 mmol), NaI (catalytic
amount) in anhyd CH3CN (3.8 mL) under nitrogen at r.t. was added
successively ethyl diisopropylamine (79 L, 0.45 mmol) and benzyl
bromide (45 L, 0.38 mmol) and then refluxed for 4 h. The reaction
mixture was cooled to r.t., concentrated in vacuo and chromato-
Synthesis 2003, No. 17, 2647–2654 © Thieme Stuttgart · New York

2652
C. A. M. Afonso et al.
PAPER
IR (film): 3244, 3024, 2974, 2935, 2874, 1721, 1649, 1605, 1559,
1527, 1499, 1448, 1367, 1306, 1186, 1129, 1077, 1021, 885, 815,
732 cm–1.
6.70 (d, 2 H, J = 1.0 Hz, ArH), 6.94 (d, 2 H, J = 8.0 Hz, ArH), 7.17
(d, 2 H, J = 8.0 Hz, ArH), 7.23 (d, 1 H, J = 8.0 Hz, ArH), 7.72 (dt,
1 H, J = 1.5, 7.5 Hz, ArH), 7.78 (dt, 1 H, J = 1.5, 7.5 Hz, ArH), 8.32
(dd, 1 H, J = 1.0, 7.5 Hz, ArH).
13C NMR (100 MHz, CDCl3): = 13.75, 14.97, 17.30, 18.82, 18.91,
18.95, 27.97, 30.91, 38.06, 58.35, 65.52, 66.05, 66.63, 79.55, 93.51,
113.02, 125.81, 128.03, 128.11, 128.16, 129.35, 129.85, 129.93,
131.17, 132.84, 133.56, 134.29, 135.40, 155.36, 155.43, 155.45,
156.57, 156.70, 164.59, 171.95.
1H NMR (400 MHz, CDCl3): = 1.16 [d, 6 H, J = 7.0 Hz,
CH(CH3)2], 1.37 (t, 6 H, J = 7.0 Hz, NCH2CH3), 2.22 (s, 6 H,
ArCH3), 2.58 (heptet, 1 H, J = 7.0 Hz, CHMe2), 3.56 (quintet, 4 H,
J = 7.0 Hz, NCH2CH3), 4.88 (s, 2 H, ArCO2CH2Ar ), 5.06 (s, 2 H,
ArCH2OCOCHMe2), 6.53 (s, 2 H, ArH), 6.65 (s, 2 H, ArH), 6.82 (br
t, 2 H, J = 5.5 Hz, NH), 6.87 (d, 2 H, J = 7.5 Hz, ArH), 7.13 (d, 2 H,
J = 7.5 Hz, ArH), 7.19 (d, 1 H, J = 7.5 Hz, ArH), 7.69 (t, 1 H,
J = 7.5 Hz, ArH), 7.75 (t, 1 H, J = 7.5 Hz, ArH), 8.27 (d, 1 H,
J = 7.5 Hz, ArH).
13C NMR (100 MHz, CDCl3): = 13.87, 18.84, 19.01, 33.84, 38.21,
65.31, 66.87, 93.63, 113.18, 125.90, 127.83, 128.24, 128.28,
129.50, 129.93, 130.03, 131.32, 132.91, 133.67, 133.99, 136.46,
155.54, 156.69, 156.80, 164.72, 176.72.
MS (EI): m/z (%) = 733 (M+, 4), 676 (5), 644 (5), 533 (9), 413 (9),
370 (40), 369 (42), 355 (100), 326 (14), 311 (12), 264 (48).
HRMS: m/z calcd for C44H51N3O7: 733.3727; found: 733.3714.
Anal. Calcd for C44H52IN3O7: C, 61.32; H, 6.08; N, 4.88. Found: C,
61.19; H, 6.00; N, 4.83.
Rhodamine 19 4-(N-t-Butoxycarbonyl-l-isoleucinyl-glycinyl-
oxymethyl)-1-phenylmethyl Ester Iodide 16
Prepared as described for 13 using N-t-Boc-L-isoleucine-glycine
(63.3 mg, 0.22 mmol). Compound 16 (146.7 mg, 76%) was ob-
tained as red plates, mp >110 °C (decomposition, gas liberation,
EtOH–hexane).
MS (EI): m/z (%) = 604 (M+, 18), 589 (32), 533 (42), 413 (11), 370
(41), 369 (41), 355 (100), 326 (15), 311 (12), 191 (52), 104 (30).
HRMS: m/z calcd for C38H40N2O5: 604.2937; found: 604.2956.
Rhodamine 19 4-(N-t-Butoxycarbonyl-glycinyl-oxymethyl)-1-
phenylmethyl Ester Iodide 14
Prepared as described for 13 using N-t-Boc-Glycine (38.5 mg, 0.22
mmol). Compound 14 (148.5 mg, 88%) was obtained as red plates,
mp >113 °C (decomposition, gas liberation, EtOH–Et2O).
IR (film): 3254, 2973, 2928, 1717, 1650, 1607, 1558, 1528, 1500,
1449, 1366, 1306, 1185, 1129, 1089, 1021, 814, 733 cm–1.
1H NMR (400 MHz, CD3OD): = 0.87 (t, 3 H, J = 7.5 Hz,
CHCH2CH3), 0.94 (d, 3 H, J = 6.5 Hz, CHCH3), 1.12–1.57 (m, 2 H,
CHCH2CH3), 1.38 (t, 6 H, J = 7.5 Hz, NCH2CH3), 1.42 [s, 9 H,
C(CH3)3], 1.81 (m, 1 H, CHMe), 2.07 (s, 6 H, ArCH3), 3.54 (q, 4 H,
J = 7.5 Hz, NCH2CH3), 3.99 (d, 1 H, J = 7.0 Hz, CHNHBoc), 4.03
(d, 1 H, J = 17.5 Hz, CH2NH), 4.13 (d, 1 H, J = 17.5 Hz, CH2NH),
4.86 (s, 2 H, ArCO2CH2Ar ), 5.17 (s, 2 H, ArCH2OCOCH2NH),
6.77 (s, 6 H, ArH), 7.14 (d, 2 H, J = 8.0 Hz, ArH), 7.35 (d, 1 H,
J = 6.5 Hz, ArH), 7.80 (dt, 1 H, J = 1.5, 7.5 Hz, ArH), 7.85 (dt, 1 H,
J = 1.5, 7.5 Hz, ArH), 8.31 (dd, 1 H, J = 1.0, 7.5 Hz, ArH).
13C NMR (100 MHz, CD3OD): = 11.67, 14.24, 15.95, 17.77,
25.67, 28.70, 38.46, 39.49, 42.08, 60.57, 67.22, 68.00, 80.53, 95.04,
114.60, 126.51, 129.09, 129.59, 129.76, 129.89, 131.38, 131.43,
131.49, 132.35, 134.10, 134.76, 135.77, 137.40, 157.38, 157.78,
158.53, 158.56, 166.54, 170.86, 174.96.
IR (film): 3252, 3024, 2976, 2928, 1715, 1651, 1607, 1558, 1529,
1504, 1448, 1367, 1307, 1186, 1089, 1022, 815, 733 cm–1.
1H NMR (400 MHz, CDCl3): = 1.37 (t, 6 H, J = 7.0 Hz,
NCH2CH3), 1.37 [s, 9 H, C(CH3)3], 2.20 (s, 6 H, ArCH3), 3.56
(quintet, 4 H, J = 7.0 Hz, NCH2Me), 3.95 (d, 2 H, J = 5.5 Hz,
NCH2CO), 4.89 (s,
2 H, ArCO2CH2Ar ), 5.11 (s, 2 H,
ArCH2OCOCH2NHBoc), 5.20 (t, 1 H, J = 5.5 Hz, NHBoc), 6.56 (s,
2 H, ArH), 6.64 (s, 2 H, ArH), 6.82 (br t, 2 H, J = 5.0 Hz, NH), 6.87
(d, 2 H, J = 7.5 Hz, ArH), 7.12 (d, 2 H, J = 7.5 Hz, ArH), 7.19 (d, 1
H, J = 7.5 Hz, ArH), 7.69 (dt, 1 H, J = 1.0, 6.5 Hz, ArH), 7.75 (dt,
1 H, J J = 1.0, 6.5 Hz, ArH), 8.27 (d, 1 H, J = 7.5 Hz, ArH).
13C NMR (100 MHz, CDCl3): = 13.90, 18.93, 28.15, 38.26, 42.37,
66.22, 66.76, 79.85, 93.68, 113.20, 125.92, 128.08, 128.28, 128.35,
129.58, 129.96, 130.05, 131.33, 132.92, 133.66, 134.43, 135.52,
155.59, 155.68, 156.71, 156.85, 164.76, 170.14.
MS (EI): m/z (%) = 516 (2), 413 (7), 370 (45), 369 (46), 355 (100),
340 (9), 326 (18), 311 (16), 230 (30).
MS (Electrospray): m/z (%) = 806 (MH+, 20), 539 (8), 467 (5), 215
(30), 150 (55), 136 (100).
MS (EI): m/z (%) = 691 (M+, 15), 676 (7), 645 (7), 636 (18), 617 (7),
602 (12), 590 (8), 533 (17), 517 (98), 501 (100), 473 (13), 442 (23).
HRMS: m/z calcd for C41H45N3O7: 691.3258; found: 691.3239.
Anal. Calcd for C47H57IN4O8: C, 60.51; H, 6.16; N, 6.01. Found: C,
60.20; H, 6.10; N, 5.54.
Anal. Calcd for C41H46IN3O7: C, 60.07; H, 5.66; N, 5.13. Found: C,
60.32; H, 5.79; N, 4.90.
Rhodamine 19 4-(N-Fluorenylmethoxycarbonyl-l-valinyl-
oxymethyl)-1-phenylmethyl Ester Iodide 17
Prepared as described for 13 using N-Fmoc-L-valine (76.1 mg, 0.22
mmol). Compound 17 (174.8 mg, 86%) was obtained as red plates
by crystallization from EtOAc–Et2O), mp 129–140 °C (slow de-
composition).
Rhodamine 19 4-(N-t-Butoxycarbonyl-l-valinyl-oxymethyl)-1-
phenylmethyl Ester Iodide 15
Prepared as described for 13 using N-t-Boc-L-valine (47.8 mg, 0.22
mmol). Compound 15 (148.8 mg, 84%) was obtained as red plates,
mp 158–161 °C (EtOH–Et2O).
IR (film): 3241, 2963, 2932, 2874, 1723, 1651, 1607, 1557, 1526,
1504, 1449, 1367, 1306, 1186, 1129, 1075, 1022, 815, 739 cm–1.
IR (film): 3243, 3024, 2972, 1716, 1650, 1606, 1557, 1526, 1504,
1449, 1368, 1305, 1186, 1129, 1089, 1022, 814, 735 cm–1.
1H NMR (400 MHz, CDCl3): = 0.87 [d, 3 H, J = 7.0 Hz,
CH(CH3)2], 0.95 [d, 3 H, J = 7.0 Hz, CH(CH3)2], 1.40 [s, 9 H,
C(CH3)3], 1.415 (t, 3 H, J = 7.0 Hz, NCH2CH3), 1.422 (t, 3 H,
J = 7.0 Hz, NCH2CH3), 2.15 (m, 1 H, CHMe2), 2.21 (s, 3 H,
ArCH3), 2.22 (s, 3 H, ArCH3), 3.59 (quintet, 4 H, J = 7.0 Hz,
NCH2CH3), 4.23 (dd, 1 H, J = 5.0, 9.0 Hz, CHNHBoc), 4.91 (d, 1
H, J = 12.0 Hz, ArCO2CH2Ar ), 4.97 (d, 1 H, J = 12.0 Hz,
ArCO2CH2Ar ), 5.01 (d, 1 H, J = 9.0 Hz, NHBoc), 5.08 (d, 1 H,
J = 12.5 Hz, ArCH2OCOCHNHBoc), 5.18 (d, 1 H, J = 12.5 Hz,
ArCH2OCOCHNHBoc), 6.62 (s, 2 H, ArH), 6.68 (br s, 2 H, NH),
1H NMR (400 MHz, CD3OD): = 0.97 [d, 3 H, J = 7.0 Hz,
CH(CH3)2], 0.98 [d, 3 H, J = 7.0 Hz, CH(CH3)2], 1.33 (t, 3 H,
J = 7.0 Hz, NCH2CH3), 1.34 (t, 3 H, J = 7.0 Hz, NCH2CH3), 1.99 (s,
3 H, ArCH3), 2.00 (s, 3 H, ArCH3), 2.20 (octet, 1 H, J = 7.0 Hz,
CHMe2), 3.47 (m, 4 H, NCH2CH3), 3.98 (t, 1 H, J = 7.0 Hz,
CHCH2OCOCHNH), 4.17 (dd,
1
H, J = 7.5, 10.5 Hz,
CHCH2OCOCHNH), 4.20 (d, 1 H, J = 6.0 Hz, CHNHFmoc), 4.28
(dd, 1 H, J = 7.0, 10.5 Hz, CHCH2OCOCHNH), 4.74 (s, 2 H,
ArCO2CH2ArCH2OCOCHNHFmoc), 5.08 (d, 1 H, J = 12.5 Hz,
ArCH2OCOCHNHFmoc), 5.18 (d,
1
H, J = 12.5 Hz,
Synthesis 2003, No. 17, 2647–2654 © Thieme Stuttgart · New York

PAPER
An Expedient Synthesis of Cationic Rhodamine Fluorescent Probe
2653
ArCH2OCOCHNHFmoc), 6.63–6.74 (m, 6 H, ArH), 7.09 (d, 2 H,
J = 8.0 Hz, ArH), 7.14 (d, 1 H, J = 7.5 Hz, ArH), 7.16 (d, 1 H,
J = 7.5 Hz, ArH), 7.20 (d, 1 H, J = 7.0 Hz, ArH), 7.24 (d, 1 H,
J = 7.5 Hz, ArH), 7.26 (d, 1 H, J = 7.5 Hz, ArH), 7.49 (d, 1 H,
J = 7.5 Hz, ArH), 7.53 (d, 1 H, J = 7.5 Hz, ArH), 7.64 (d, 1 H,
J = 2.0 Hz, ArH), 7.65 (d, 1 H, J = 2.0 Hz, ArH), 7.75 (t, 1 H,
J = 7.5 Hz, ArH), 7.80 (t, 1 H, J = 7.5 Hz, ArH), 8.26 (dd, 1 H,
J = 1.5, 7.5 Hz, ArH).
13C NMR (100 MHz, CD3OD): = 14.28, 14.36, 17.82, 18.71,
19.76, 31.80, 39.43, 39.48, 61.42, 67.20, 67.91, 94.91, 95.08,
114.48, 114.56, 120.80, 120.83, 126.03, 126.13, 126.63, 128.00,
128.04, 128.63, 128.67, 129.32, 129.40, 129.46, 129.59, 129.72,
131.27, 131.34, 131.43,. 132.32, 134.07, 134.66, 135.69, 137.28,
142.23, 142.33, 144.78, 145.23, 157.14, 157.22, 158.27, 158.26,
158.32, 158.63, 166.42, 173.42.
References
(1) Valeur, B. Molecular Fluorescence: Principles and
Applications; Wiley-VCH: Weinheim, 2002.
(2) (a) Haugland, R. P. Handbook of Fluorescent Probes and
Research Products; Molecular Probes, Inc.: Eugene, 2001,
and references cited therein. (b) Probe Design and
Chemical Sensing, In Topics in Fluorescence Spectroscopy,
Vol. 4; Lakowicz, J. R., Ed.; Plenum Press: New York,
1994. (c) Scala-Valéro, C.; Doizi, D.; Guillaumet, G.
Tetrahedron Lett. 1999, 40, 4803.
(3) (a) Rahavendran, S. V.; Karnes, H. T. Anal. Chem. 1996, 68,
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(4) (a) Finney, N. S. Curr. Opin. Drug Discovery Dev. 1998, 1,
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Eritja, R.; Beeumen, J. V.; Herdewijn, P. Chem.–Eur. J.
1998, 4, 425.
MS (EI): m/z (%) = 618 (6), 533 (5), 413 (7), 371 (7), 355 (76), 326
(11), 178 (100).
MS (Electrospray): m/z (%) = 856 (MH+, 10), 538 (7), 215 (15), 150
(5) (a) Mitchison, T. J.; Sawin, K. E.; Theriot, J. A.; Gee, K.;
Mallavarapu, A. Caged Compounds, In Methods in
Enzymology, Vol. 291; Marriott, G., Ed.; Academic Press:
New York, 1998, 63. (b) Boturyn, D.; Boudali, A.;
Constant, J.-F.; Defrancq, E.; L’homme, J. Tetrahedron
1997, 53, 5485. (c) Harapanhalli, R. S.; Roy, A. M.;
Adelstein, S. J.; Kassis, A. J. Med. Chem. 1998, 41, 2111.
(d) Mayer, A.; Neuenhofer, S. Angew. Chem., Int. Ed. Engl.
1994, 33, 1044. (e) Kojima, H.; Hirotani, M.; Urano, Y.;
Kikuchi, K.; Higuchi, T.; Nagano, T. Tetrahedron Lett.
2000, 41, 69.
(50), 136 (100).
Anal. Calcd for C54H54IN3O7: C, 65.92; H, 5.53; N, 4.27. Found: C,
65.57; H, 5.84; N, 4.36.
Rhodamine 19 4-(N-Benzyloxycarbonyl-l-phenylalaninyl-
oxymethyl)-1-phenylmethyl Ester Iodide 18
Prepared as described for 13 using N-Cbz-L-phenylalanine (28.7
mg, 0.10 mmol). Compound 18 (71.2 mg, 84%) was obtained as red
plates, mp 122–125 °C (EtOH–EtOAc–Et2O).
IR (film): 3248, 3029, 2975, 1716, 1650, 1606, 1503, 1448, 1367,
1306, 1186, 1130, 1083, 1022, 886, 815, 734, 700 cm–1.
(6) Davie, E.; Morris, J. H.; Smith, E. Org. Mass Spectrom.
1974, 763.
1H NMR (400 MHz, CDCl3): = 1.38 (t, 6 H, J = 7.0 Hz,
NCH2CH3), 2.19, 2.22 and 2.23 (3 × s, 6 H, ArCH3), 3.10 (m, 2 H,
CHCH2Ph), 3.55 (br s, 4 H, NCH2CH3), 4.65 (dd, 1 H, J = 6.5, 13.5
Hz, CHNHCbz), 4.85–5.14 (m, 6 H, ArCH2 and NHCH2OBn), 5.30
(d, 1 H, J = 8.0 Hz, ArCH2), 6.57–6.60 (m, 2 H, ArH), 6.66 (d, 2 H,
J = 13.0 Hz, ArH), 6.79–6.85 (m, 2 H), 6.93 (d, 2 H, J = 7.5 Hz),
7.00 (d, 2 H, J = 7.0 Hz), 7.09 (d, 2 H, J = 7.5 Hz), 7.17–7.26 (m, 9
H), 7.71 (t, 1 H, J = 7.5 Hz, ArH), 7.77 (t, 1 H, J = 7.5 Hz, ArH),
8.30 (d, 1 H, J = 7.5 Hz, ArH).
13C NMR (100 MHz, CDCl3): = 13.93, 15.17, 18.74, 18.85, 18.91,
38.00, 38.36, 45.83, 54.85, 65.75, 66.58, 66.81, 66.87, 93.76, 93.79,
113.31, 125.90, 125.94, 127.06, 127.89, 128.09, 128.33, 128.39,
128.54, 128.63, 128.76, 129.13, 129.61, 130.07, 130.11, 131.36,
133.00, 133.81, 134.68, 135.29, 135.33, 135.94, 155.61, 155.64,
155.67, 156.79, 156.91, 156.93, 164.78, 171.33.
(7) Cai, S. X.; Zhang, H.-Z.; Guastella, J.; Drewe, J.; Yang, W.;
Weber, E. Bioorg. Med. Chem. Lett. 2001, 11, 39.
(8) (a) Bain, A. J.; Chandna, P.; Butcher, G.; Bryant, J. J. Chem.
Phys. 2000, 112, 10435. (b) Byassee, T. A.; Chan, W. C.;
Nie, S. Anal. Chem. 2000, 72, 5606. (c) Fang, X.; Tan, W.
Anal. Chem. 1999, 71, 3101. (d) Mandala, M.; Serck-
Hanssen, G.; Martino, G.; Helle, K. B. Anal. Biochem. 1999,
274, 1. (e) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994,
266, 1018. (f) Matsumoto, Y.; Sasaoka, N.; Tsuchida, T.;
Fujiwara, T.; Nagao, S.; Ohmoto, T. J. Neurooncol. 1992,
13, 217. (g) Choi, K. J.; Turkevich, L. A.; Loza, R. J. Phys.
Chem. 1988, 92, 2248. (h) Kuzela, S.; Joste, V.; Nelson, B.
D. Eur. J. Biochem. 1986, 154, 553.
(9) Heilporn, S.; Broeders, F.; Daloze, D.; Braekman, J. C.;
Boeynaems, J. M. Bull. Soc. Chim. Belg. 1994, 103, 309.
(10) Gallo, E. A.; Gellman, S. H. J. Am. Chem. Soc. 1993, 115,
9774.
(11) For a similar linking strategy for peptides to fluoresceins and
their application as fluorescent reporters, see: Chen, C.-A.;
Yeh, R.-H.; Lawrence, D. S. J. Am. Chem. Soc. 2002, 124,
3840.
(12) (a) Adamczyk, M.; Grote, J. Bioorg. Med. Chem. Lett. 2000,
10, 1539. (b) See also: Cincotta, L.; Foley, J. W. U.S. Patent
4,290,955, 1981.
(13) (a) Mayer, U.; Oberlinner, A. U.S. Patent 4,647,675, 1987.
(b) Arnost, M. J.; Meneghini, F. A.; Palumbo, P. S.; Stroud,
S. G. U.S. Patent 4,900,686, 1990. (c) Mayer, U.;
Oberlinner, A. U.S. Patent 4,935,059, 1990. (d) Haugland,
R. P.; Singer, V. L.; Yue, S. T. U.S. Patent 6,399,392, 2002.
(14) Wakamiya, T.; Saruta, K.; Yasuoka, J.-I.; Kusumoto, S.
Bull. Chem. Soc. Jpn. 1995, 68, 2699.
MS (EI): m/z (%) = 501 (6), 370 (18), 369 (19), 355 (60), 326 (9),
139 (13), 108 (27), 91 (100).
MS (Electrospray): m/z (%) = 817 (MH+, 100), 645 (20), 553 (30).
Acknowledgment
The Natural Science and Engineering Research Council (NSERC)
of Canada supported this work. C. A. M. Afonso acknowledges fi-
nancial support from Fundação para a Ciência e Tecnologia (FCT,
project PRAXIS XXI PCEX/C/QUI/53/96 and grant BLS 44),
Fundação Calouste Gulbenkian and INVOTAN. R.A.B. gratefully
acknowledges additional support through a Bio-Méga/Boehringer
Ingelheim Young Investigator Award and a Premier’s Research Ex-
cellence Award. We would like to thank Professors F. Pina, J. Paro-
la, Scott Taylor and Dr. Nicole Dinault for performing the
fluorescent experiments and helpful discussions. We also thank Dr.
A. B. Young for mass spectrometric analyses.
(15) The relative quantum yields at 495 nm of derivatives 10, 13
and 15 were 1.26, 1.03 and 0.95 × that of rhodamine 6G
respectively (see Ref.18 for the method used).
(16) The crystals of compound 14 were obtained by
crystallization from Et2O–EtOH: C41H46IN3O7, M = 819.71,
Synthesis 2003, No. 17, 2647–2654 © Thieme Stuttgart · New York

2654
C. A. M. Afonso et al.
PAPER
triclinic, space group P–1, a = 12.6230(2), b = 13.4724(3),
c = 13.5600(3) Å, = 101.865(1), = 107.003(1),
reflections with I>2 (I)) and wR2 (all unique data) = 0.0970.
Atomic coordinates and further crystallographic details have
been deposited at the Cambridge Crystallographic Data
Centre, deposition number CCDC–191818, and copies of
this data can be obtained in application to CCDC, 12, Union
Road, Cambridge CB2 1EZ, UK. [Fax: +44(1223)336033;
E-mail: deposit@ccdc.cam.ac.uk].
= 113.602(1)°, V = 1878.77(7) Å3, Z = 2, Dc = 1.449
gcm–3, = 0.907 mm–1, F(000) = 844, crystal dimensions
0.35 × 0.32 × 0.28 mm3. The intensities of 28224 reflections
were measured on a Nonius Kappa-CCD diffractometer
(MoK radiation, T = 100.0(1) K, 4.19 < < 30.48, 10822
unique reflections). The structure was solved and refined
using the SHELXTL package (see Ref.17). Non-hydrogen
atoms were assigned anisotropic thermal parameters.
Hydrogen atoms were included in calculated positions and
treated as riding atoms. The refinement which included 469
parameters, converged with R1[I>2 (I)] = 0.0375 (for 8449
(17) Sheldrick, G. M. SHELXTL/PC Version 5.1, Windows NT
Version, Bruker AXS Inc., Madison, USA.
(18) Parker, C. A. Photoluminescence of Solutions; Elsevier:
Amsterdam, 1968.
(19) Kohno, Y.; Narasaka, K. Bull. Chem. Soc. Jpn. 1995, 68,
322.
Synthesis 2003, No. 17, 2647–2654 © Thieme Stuttgart · New York