From spirolactam mixtures to regioisomerically pure 5- and 6-rhodamines: A chemodosimeter-Inspired Strategy

Article abstract of DOI:10.1021/ol300523m

Inspired by the ring-open reaction of rhodamine spriolactams as typical chemodosimeters, a general strategy is proposed to conveniently and efficiently synthesize isomerically pure 5- and 6-R-tetramethylrhodamine on a larger scale.

Full text of DOI:10.1021/ol300523m

ORGANIC
LETTERS
2012
Vol. 14, No. 8
2014–2017
From Spirolactam Mixtures to
Regioisomerically Pure 5- and
6-Rhodamines: A Chemodosimeter-
Inspired Strategy
Haibo Yu, Yi Xiao,* and Haiying Guo
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian
116012, China
xiaoyi@dlut.edu.cn
Received February 29, 2012
ABSTRACT
Inspired by the ring-open reaction of rhodamine spriolactams as typical chemodosimeters, a general strategy is proposed to conveniently and
efficiently synthesize isomerically pure 5- and 6-R-tetramethylrhodamine on a larger scale.
Rhodamine dyes, bearing a linker on the 5 or 6 position
of the benzoic acid moiety, are well-known as fluore-
scent labels to covalently attach to many biomolecules
(oligonucleotides and proteins).1ꢀ3 Using one of the pure
5 or 6 regioisomerical rhodamines is highly recommended
in some advanced biomedical fields where use of their
mixture might be problematic.4 For example, in DNA
sequencing, an oligonucleotide primer attaching to a mix-
ture of 5/6-R-rhodamine isomers will cause significant
spreading of PAGE (PolyAcrylamide Gel Electrophoresis)
bands and give rise to multiple bands of identically sized
oligonucleotides, which leads to false sequence deter-
mination.5 In the typical synthesis of rhodamines, mix-
tures of 5/6 isomers are generated simultaneously by the
Scheme 1. Concept of the Chemodosimeter Based on Rhodamine-
Spirolactam: Metal-Induced Ring-Opening Reaction
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r
10.1021/ol300523m
Published on Web 04/03/2012
2012 American Chemical Society

Table 1. Approach to Regioisomerically Pure 5-R-TMR and 6-R-TMR
purity and
ratio of 5/6
isomera
total
yielde
5/6-R-TMR
5/6-Br-TMR
yields
74%
5/6-R1-TMRH
Rfb
yieldc
purityd
5/6-R-TMR
purityf
80% 3:2
5-Br-TMRH
6-Br-TMRH
5-CN-TMRH
6-CN-TMRH
5-NO-TMRH
6-NO-TMRH
0.21
0.12
0.36
0.48
0.32
0.21
53%
45%
48%
41%
44%
55%
>95%
>95%
>95%
>95%
>95%
>95%
5-Br-TMR
6-Br-TMR
5-CO-TMR
6-CO-TMR
5-NO-TMR
6-NO-TMR
39%
31%
37%
33%
38%
45%
98%
99%
99%
99%
98%
98%
5/6-CO-TMR
5/6-NO-TMR
80%
85%
97% 4:5
97% 1:2
a Determined by High Performance Liquid Chromatography (HLPC). b Eluent systems could be found in the Supporting Information. c Isolated
yields. d Determined by 1H NMR e Isolated yields f Determined by High Performance Liquid Chromatography (HLPC)
condensation of a monofunctionalized phthalic anhydride
with m-aminophenols.6 To date, column chromatography
is the main method for obtaining pure 5/6-R-rhodamine
isomers. However, owing to the extreme resemblance
between the two isomers and the characteristic of rhoda-
mine dyes as being cationic, the separation of them
through this method is cumbersome.7 Alternatively, a
stepwise synthesis has been used to produce a pure rhoda-
mine isomer, for each of the 5/6-R-rhodamine isomers, but
the total yields are not satisfactory.8,9 Hence, any regio-
isomerically pure 5- or 6-R-rhodamine from a commercial
source is extremely expensive ($37 000ꢀ40000/g).
further hydrolysis into rhodamine dyes induced by metal
ions and other analytes.10 Based on our previous studies,11
we found that neutral spirolactams had lower polarity and
better solubility in less polar solvent, compared with cationic
rhodamines, and could be easily purified through conven-
tional silica-gel column chromatography. Thus, as shown in
Table 1, our strategy involved converting 5/6-R-TMR into
the corresponding rhodamine spirolactam, namely 5/6-R-
rhodamine hydrazine (5/6-R-TMRH), and then purifying
two isomers of 5/6-R-TMRH through silica-gel column
chromatography. Finally, the two regioisomerical 5/6-R-
TMR would be obtained by hydrolysis of the Cu2þ ion,
respectively. To confirm the universality of our method,
three tetramethylrhodamine dyes 5/6-Br-TMR, 5/6-CO-
TMR, and 5/6-NO-TMR, important precursors for rhoda-
mine labeling reagents, were chosen in our experiments.
According toour strategy, three pairs of rhodamine dyes
have been produced and successfully separated to obtain a
pure isomer with high yields. The related data were
compiled in Table 1. Herein, 5/6-Br-TMR was used as an
example to illustrate the protocol of our approach. A
mixture of 1 equiv of 4-bromophthalic anhydride and
2 equiv of N,N-dimethylaminophenol, as starting materi-
als, was heated at 180 °C. During this process there are two
sequential FriedelꢀCrafts type electrophilic aromatic
To this end, we proposed an efficient and general
strategy to separate the mixture of 5/6-R-tetramethyl
rhodamine isomers (5/6-R-TMR), which was inspired
from a chemodosimeter of rhodamine spirolactams, as
shown in Scheme 1. These spirolactams widely utilized as
fluorescent sensors occur by a ring-opening process and
(7) Beija, M.; Afonso, C. A. M.; Martinho, J. M. G. Chem. Soc. Rev.
2009, 38, 2410.
(8) Corrie, J. E. T.; Craik, J. S. J. Chem. Soc., Prekin Trans. 1 1994,
2967.
(9) Kvach, M. V.; Stepanova, I. A.; Prokhorenko, I. A.; Stupak,
A. P.; Bolibrukh, D. A.; Korshun, V. A.; Shmanai, V. V. Bioconjugate
Chem. 2009, 20, 1673.
(10) (a) Dujols, V.; Ford, F.; Czarnik, A. W. J. Am. Chem. Soc. 1997,
119, 7386. (b) Du, P.; Lippard, S. J. Inorg. Chem. 2010, 49, 10753. (c)
Kim, H.; Lee, S.; Lee, J.; Tae, J. Org. Lett. 2010, 22, 5342. (d) Zheng, H.;
Shang, G.; Yang, S.; Gao, X.; Xu, J. Org. Lett. 2008, 12, 2357. (e) Kim,
H. N.; Lee, M. H.; Kim, H. J.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2008,
37, 1465.
(12) (a) Liu, J.; Diwu, Z.; Leung, W.-Y.; Lu, Y.; Patch, B.; Haugland,
R. P. Tetrahedron Lett. 2003, 44, 4355. (b) David, E.; Lejeune, J.;
Pellet-Rostaing, S.; Schulz, J.; Lemaire, M.; Chauvin, J.; Deronzier,
A. Tetrahedron Lett. 2008, 49, 1860. (c) Scala-ValEro, C.; Doiz, D.;
Guillaumet, G. Tetrahedron Lett. 1999, 40, 4803.
(13) (a) Wathen, S. P.; Czarnik, A. W. J. Org. Chem. 1992, 57, 6129.
(b) Attanasi, O.; Serra-Zanetti, F. Synthesis 1980, 4, 314.
(11) (a) Xue, L.; Li, G.; Yu, C.; Jiang, H. Chem.;Eur. J. 2012, 18,
1050. (b) Yu, H.; Xiao, Y.; Guo, H.; Qian, X. Chem.;Eur. J. 2011, 17,
3179.
Org. Lett., Vol. 14, No. 8, 2012
2015

substitution reactions for the formation of the xanthene
skeleton. Unfortunately, according tothe methodreported
previously,12 large amounts of benzophenone derivatives
were obtained and they failed ultimately to convert into
rhodamine dyes, evendespite prolonging the reaction time.
To improve the yield of rhodamine, dry DMF containing
polyphosphoric acid as a catalyst was particularly adopted
to promote intramolecular FriedelꢀCrafts reactions. A
mixture of 5/6-Br-TMR was obtained in 74% yield, and
the ratio of two isomers was approximately 3:2, analyzed
byHLPC(Supporting Information). Thenthe mixturewas
esterified with methanol in the presence of SOCl2. The
products of rhodamine ester, without further purification,
were directly converted into rhodamine hydrazide in high
carboxylic acid, respectively, which then reacted with an-
other equimolar quantity of m-aminophenol catalyzed by
polyphosphoric acid.
Scheme 2. Stepwise Synthesis of 5-Br-TMR and 6-Br-TMR
yield with hydrazine hydrate (NH2NH2 H2O) at room
3
temperature. Next, the mixture of 5/6-R-TMRH was sepa-
rated by column chromatography (silica gel 200ꢀ300 mesh,
15 cm column, petroleum ether/ethyl acetate 3:1, v/v).
Separation by chromatography afforded 53% isolated
yields for 5-Br-TMRH with Rf 0.32 and 45% isolated yields
for 6-Br-TMRH with Rf 0.21. Cu(II) has been known to
promote the hydrolysis of acylhydrizine and usually used as
a deprotection reagent in organic synthesis.13 Czarnik
reported that Cu(OAc)2 could enhance the hydrolysis of
rhodamine hydrazine into rhodamine B in acetronitrile.10a
In our experiment, extensive optimization was performed to
develop conditions for the hydrolysis of TMRH in high
yield. Finally, 1 equiv of CuCl2 exhibited a high efficiency of
TMRH hydrolysis in a solvent mixture of methanol and
tetrahydrofuran (1:9, v/v), and in order to remove Cu(II)
from this system, a sodium hydroxide solvent was added.
After purification by a short silica gel pad, we obtained a
31% total yield for 5-Br-TMR and 39% total yield for 6-Br-
TMR, respectively. Depending on the same strategy, we
also obtained 38% and 45% isolated yields for 5-NO-TMR
and 6-NO-TMR or 37% and 33% isolated yields for 5-CO-
TMR and 6-CO-TMR, respectively. Moreover, the purities
of these rhodamine dyes were more than 98%, determined
by HLPC, shown in Figures S3ꢀS5.
The regioisomerically pure 5-Br-TMR and 6-Br-TMR
were obtained in 4% and 20% total yields, respectively. As
described above, the yields of 5-Br-TMR and 6-Br-TMR
were 31% and 39% according to our strategy. Thus, we
again confirmed that our new spirolactam-based strategy for
5/6- substituted TMR was not only more straightforward but
also more efficient compared to the stepwise method.
The complete structural analyses of 5-Br-TMR and
6-Br-TMR were deduced using the 1D and 2D spectrum
of isomer 1, shown in Figure 1a: in the HMBC spectrum of
isomer 1, the downfield signal at 196.7 ppm corresponded
to the carbonyl group at the C-13 position, which showed
strong remote coupling effects of adjacent proton 3 (δ 7.52
ppm) and 12 (δ 6.88 ppm); the singlet signal of proton 3
indicated that the bromo group was located at the 4
position. Proton 6 was split by adjacent proton 5, and it
appeared downfield as a doublet (δ 7.89 ppm). Different
from isomer 1, proton 6 of isomer 2 displayed a single peak
at a lower field due to the bromo group locating at the 5
position, shown in Figure 1b. Based on the successful
structural analysis of isomers 1 and 2, the structure of
5-Br-TMR and 6-Br-TMR can be conveniently deter-
mined, shown in Figure 2. The deshielding effect of the
In previous reports, isomerically pure 5/6-NO-TMR and
5/6-CO-TMR had been synthesized by a stepwise method
aided by fractional crystallization of the intermediate.8,9
However, in the previous stepwise synthesis, the total yields
of the corresponding pure regioisomers were far lower than
those we obtained through the spirolactam-based strategy.
The reported yields of 5- and 6-CO-TMR were just 15% and
10%; nevertheless, in our experiment they were obtained in
37% and 33% yields, respectively. Herein, for further
comparison, we also synthesized pure 5- and 6-Br-TMR,
through a similar two-step method previously adopted for
5/6-NO-TMR and 5/6-CO-TMR. As shown in Scheme 2, a
mixture of 4-bromophthalic anhydride and an equimolar
quantity of N,N-dimethylaminophenol was used as starting
materials. A mixture of intermediate 1 and 2 was easily
obtained by silica gel column chromatography after the
esterification reaction. Fractional crystallization was suc-
cessfully applied to isolate isomer 1 with a 23% isolated
yield and isomer 2 with a 5% yield. Isomers 1 and 2 were
hydrolyzed in 10% NaOH to produce the corresponding
2016
Org. Lett., Vol. 14, No. 8, 2012

electron-withdrawing carboxyl group made the chemical
shifts of the phenylcarboxyl moiety appear at a lower field,
compared with xanthenes. For 5-Br-TMR, due to the
bromo group at the 5 position and the strong deshielding
effectofthe carboxyl group, thesingletsignalatδ 8.30ppm
was assigned for proton Ha. Two separated doublet signals
at δ 8.06 and 7.42 ppm were assigned for two adjacent aryl
protons Hb and Hc, respectively, with typical values of
coupling constants (J = 8.2 Hz). Moreover, there was a
weak vicinal coupling constant between proton Ha and Hc
multiplets, but rather as a broad single peak. The chemical
shift of the aliphatic protons (N(CH3)2) are equivalent, and
there was no difference between 5-Br-TMR and 6-Br-TMR.
Similar to 5-Br-TMR and 6-Br-TMR, the structural analy-
sis of 5/6-CO-TMR and 5/6-NO-TMR can be easily con-
firmed from the chemical shift and coupling patterns for the
aromatic proton in the downfield region of the H NMR
spectrum. The NMR spectra of 5/6-CO-TMR and 5/6-NO-
TMR are described in the Supporting Information.
1
Figure 2. Low-field section of 1H NMR of 5-Br-TMR (top) and
6-Br-TMR (bottom).
In summary, inspired by the design concept of a rhoda-
mine spirolactams-based chemodosimeter, we proposed a
novel approach to prepare a regioisomerical 5-R-TMR
and 6-R-TMR, e.g. 5/6-Br-TMR, 5/6-CO-TMR, and 5/6-
NO-TMR, in high yields and high purities. This approach
is more concise and more efficient, compared with stepwise
methods previously established. Moreover, it is worth
noting that this approach is also suitable for preparation
of regioisomerically pure 5- and 6- rhodamine dyes on a
larger scale. In a typical experiment, we successfully sepa-
rated a mixture of 5/6-NO-TMR on an 8.0 g scale. The
availability of a facile and inexpensive route to 5-R-TMR
and 6-R-TMR should cut down the cost of rhodamine-
labeling reagents and enable their adoption for a broad
range of applications in biomedical research.
Figure 1. (a) HMBC spectrum of isomer 1. (b) Low-field section
of 1H NMR of two isomers 1 (top) and 2 (bottom).
Acknowledgment. We thank the National Natural
Science Foundation of China (No. 21174022) and Specia-
lized Research Fund for the Doctoral Program of Higher
Education (No. 20110041110009) for financial support.
(J = 1.6 Hz). Compared with 5-Br-TMR, 6-Br-TMR
exhibited different proton chemical shifts. In the 1H
NMR spectrum of 6-Br-TMR, doublet signals at δ 8.13
0
0
and 8.02 corresponded to proton Ha and Hb , attributed to
the bromo group at the 6 position, with a large vicinal
coupling constant (J = 8.4 Hz). The single signal at δ 7.81
Supporting Information Available. Experimental de-
tails. This material is available free of charge via the
Internet at http://pubs.acs.org.
0
ppm was assigned for Hc and showed a weak correlation
0
with proton Hb . Different from 5-Br-TMR, the signal of
the xanthenes moiety of 6-Br-TMR did not appear as
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 8, 2012
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