Scalable Regioselective Synthesis of Rhodamine Dyes

Article abstract of DOI:10.1021/acs.orglett.6b02635

A one-step, operationally simple protocol for the synthesis of isomerically pure rhodamine dyes from phthalaldehydic acids is reported. Using a mixture of 2,2,2-trifluoroethanol and water as reaction media allows for clean and efficient formation of vario

Full text of DOI:10.1021/acs.orglett.6b02635

Letter
pubs.acs.org/OrgLett
Scalable Regioselective Synthesis of Rhodamine Dyes
Stephen J. Dwight and Sergiy Levin*
Promega Biosciences LLC, 277 Granada Drive, San Luis Obispo, California 93401, United States
S
* Supporting Information
ABSTRACT: A one-step, operationally simple protocol for the synthesis of isomerically pure rhodamine dyes from
phthalaldehydic acids is reported. Using a mixture of 2,2,2-trifluoroethanol and water as reaction media allows for clean and
efficient formation of various rhodamines as a single isomer. This method was successfully applied to the synthesis of several
isomerically pure rhodamines, including 6-carboxytetramethylrhodamine and 6-carboxy-X-rhodamine (6-CXR) on gram scale. A
simple, one-step, Pd-catalyzed hydroxycarbonylation approach to phthalaldehydic acids from appropriately substituted
dihalobenzadehydes is also described.
ince the discovery of the first rhodamines1 by Swiss chemist
letter we report an operationally simple protocol for synthesis
of pure rhodamine dyes on a gram scale.
2
SMaurice Ceresole in 1887, this dye family has achieved
prominence in many applications, in part due to its photo-
chemical properties.3 These dyes are widely used as tracer
agents, laser dyes, labeling agents for biologics, etc. Remarkably,
even a century after their discovery, rhodamines are still largely
produced by variations of the original process reported by
Ceresole. However, the relatively simple Ceresole process
(Scheme 1 Route A) has significant drawbacks, e.g. harsh
reaction conditions, the generation of difficult to separate
isomers, and often low yields.4 These constraints significantly
limit the availability of isomerically pure dyes and pose a
challenge for the efficient synthesis of novel rhodamines. In this
In many applications, rhodamine dyes are chemically
attached to another molecule or biomolecule via a carboxylate
handle at the 5- or 6-position of the rhodamine core. Mixtures
of isomers not only complicate synthetic and purification
operations but also can give confounding results in some cases
due to differences in physicochemical properties and bioactivity
between two dye isomers.4,5 Thus, widespread adoption of the
classic synthesis combined with the broad use of rhodamines in
biological research sparked numerous efforts to develop
efficient separation and purification strategies for the isolation
of isomerically pure rhodamines. Although purification
approaches have achieved significant advances, they lack
generality, which significantly increases cost.6 Despite a dire
need for a generally robust and efficient regioselective synthesis
of this class of dyes, only recently have researchers started to
address this problem.4,7
Scheme 1. Historical Examples of Synthetic Approaches to
Rhodamines
To our knowledge, the earliest examples to produce
synthetically useful quantities of isomerically pure rhodamines
were reported in the patent literature. The patent sources
indicate that when 3-amino-1H-isoindol-1-one (5) is reacted
with 3-aminophenol (1), a single isomer of 6 is produced,8
which could be further elaborated to a single isomer of a
rhodamine dye (Scheme 1 Route B).9 The more recent report
by Lavis and co-workers achieves synthesis of isomerically pure
rhodamines from fluorescein ditriflate via Buchwald−Hartwig
coupling.10 Although this approach allows for efficient access to
Received: September 1, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b02635
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Organic Letters
Letter
many rhodamines,10,11 it is not applicable to rhodamines with
cyclic backbones (X-rhodamine for example).
phthalaldehydic acid (11) in 75% yield.20 Interestingly, we
noticed that, depending on conditions, 11 crystallized in both
closed and open forms. Lactone 11 crystallized out of a
heptane−EtOAc mixture, and carboxylate salt 11a formed X-
ray quality crystals from a water−acetonitrile mixture in the
presence of triethylamine (Scheme 3). This result is consistent
with prior evidence that a basic environment favors the ring-
opened form of phthalaldehydic acid.21
Attempted condensation of tricyclic phenol 7 with 4-
carboxyphthaladehydic acid (11) under rosamine synthesis
conditions (Scheme 1 Route C) produced only small quantities
of dye 10. The reaction produced mostly lactone 14, the
product of the initial Friedel − Crafts condensation (Scheme 4
Another approach utilizes phthalaldehydic acids (2-carboxy-
benzaldehydes) under rosamine synthesis conditions12 to
access the rhodamine core regioselectively (Scheme 1 Route
C). Considering that both benzaldehydes and 2-sulfobenz-
aldehydes are successfully used to synthesize single isomers of
rosamines12 and sulforadamines13 respectively, it would seem
surprising that phthalaldehydic acids have not been applied to
the synthesis of rhodamines until recently.7,14 Under rosamine
synthesis conditions, phthalaldehydic acids delivered isomeri-
cally pure rhodamines in low to moderate yields. Furthermore,
carboxyrhodamines such as 6-carboxytetramethylrhodamine
(17) required a final deprotection step.7
We encountered the aforementioned shortcomings when
attempting to scale up 6-carboxy-NCT (10).15 6-Carboxy-NCT
is a carbon-homologated analogue of 6-carboxy-X-rhodamine
(19). Due to its photophysical and biological properties, 10 is
an acceptor dye of choice for intracellular BRET applications.16
Isomerically pure 6-carboxy-NCT (10) was originally prepared
by a variation of a classic process of condensing terephthalic
anhydride (8) with tricyclic aminophenol 7, followed by HPLC
separation of the resulting isomers (Scheme 2).15 Considering
Scheme 4. Synthesis of 6-Carboxy-NCT from 4-
Carboxyphthalaldehydic Acid
Scheme 2. Classic Approach to 6-Carboxy-NCT via
Condensation of Terephthalic Anhydride with 7
a
Table 1. Solvent Screen for Rhodamine Synthesis
b
yield, %
entry
solvent
additive
14
10
15
c
c
1
EtCO2H
EtCO2H
EtCN
TSA
−
TSA
57(54)
48(52)
55
8
3
−
−
−
−
−
−
2
that the commercially unavailable phenol 7 requires a multistep
synthesis,15 using a nonregioselective route to access a 6-
carboxy-NCT (10) results in considerable loss of valuable
material through formation of the undesired 5-carboxy-NCT
(9).
To address this challenge, we investigated the rosamine
synthesis conditions7,12,14a with phthalaldehydic acids (Scheme
1 Route C). Although many methods exist for synthesis of
phthalaldehydic acids,14b,17 we opted for Pd-catalyzed hydroxy-
carbonylation18,19 of properly substituted, commercially avail-
able dihalobenzaldehydes (Scheme 3). Briefly, exposure of 2,5-
dibromobenzaldehyde (12) to carbon monoxide (balloon) in
wet THF in the presence of a base and catalytic amounts of
Pd(OAc)2/Xantphos yielded 11 in 89% yield. Similarly,
exposure of 2,5-dichlorobenzaldehyde (13) to carbon mon-
oxide (balloon) in wet DMSO in the presence of a base and
catalytic amounts of Pd(OAc)2/dcpp provided 4-carboxy-
d
3
trace
trace
−
d
4
EtCN
−
−
−
−
−
−
−
58
5
DMF
13
6
iPrOH
73
2
e
d
d
c
7
HFIP
22
trace
trace
66
76(73)
8
TFE
47
52
−
−
fe
9
HFIP ,
30
f
c
10
TFE
8
88(85)
a
Reactions were run open to air, [11] = 0.03 M at 80 °C for 20 h.
b
Determined by 1H NMR analysis of crude reaction mixture, using an
c
d
internal standard. Isolated yield. Observed in HPLC analysis of
crude reaction mixture only. Reaction at 60 °C. Reaction under O2
e
f
balloon.
and Table 1 entry 1). As we investigated the reaction
conditions, we found that, unlike rosamine synthesis with
benzaldehydes, no Brønsted acid was required to complete the
Friedel−Crafts reaction. When we attempted the reaction in
neat propionic acid without any catalytic p-toluenesulfonic acid
(TSA), the result was essentially the same (Table 1 entry 2).
Furthermore, propionic acid was not required, as lactone 14
also formed in propionitrile in 58% yield with no added acid
catalyst (Table 1 entry 4). We attribute these results to
intramolecular activation of the aldehyde group of the
phthalaldehydic open form (Scheme 5).22
Scheme 3. Pd-Catalyzed Hydroxycarbonylation of
Dihalobenzaldehydes
To push the reaction beyond lactone 14, we turned to the
1965 report by Reese and Sabet investigating the reactivity of
phthalaldehydic acid toward indoles.22 They reported that a
B
DOI: 10.1021/acs.orglett.6b02635
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
with commercial 3-dimethylaminophenol (22) varied greatly
depending on the impurity profile of commercial 22. When
additionally purified 2229 was employed, intermediate triaryl-
methane 24 precipitated out of TFE, preventing efficient
formation of dye 17 (Scheme 6). Through a cosolvent screen
Scheme 5. Mechanistic Hypothesis for Rhodamine
Formation from Phthalaldehydic Acid
Scheme 6. Synthesis of 6-Carboxy-TMR
base was required to achieve an addition of a second equivalent
of indole to the intermediate lactone. In our case, using a base
was not an option, as lactone 14 was unstable in the presence of
a base. On the other hand, a more recent report mentioned
formation of a small amount of bis-indolyl side-product when
phthalaldehydic acid was allowed to react with indole in neutral
H2O.23 Mindful of these reports, we hypothesized that solvent
may have an effect on this process by possibly stabilizing the
open form of the lactone via some form of benzhydrylium ion
pair 14a (Scheme 5).
The solvent screen (Table 1) revealed that fluorinated
alcohols, 2,2,2-trifluoroethanol (TFE) and hexafluoro-2-prop-
anol (HFIP), are excellent solvents for 6-carboxy-NCT
synthesis from phthalaldehydic acid (Scheme 4 and Table 1).
Both HFIP and TFE promoted two consecutive Friedel−Crafts
condensations of 11 with 2 equiv of 7, providing primarily
triarylmethane 15 along with a trace amount of dye 10 when
exposed to air (Table 1 entries 7 and 8). Due to the inefficient
and irreproducible air oxidation of 15, an oxygen atmosphere
was used, which cleanly provided 10 in either solvent (Table 1
entries 9 and 10). We hypothesized that the unique
physicochemical properties of the fluorinated alcohols,24 such
as high ionizing power,25 low nucleophilicity,26 and hydrogen-
bond donating ability,27 may promote the reactivity of the
benzhydrylium ion 14a (Scheme 5). For cost reasons (TFE is
much cheaper than HFIP)28 and its higher boiling point, we
chose TFE over HFIP for our further studies.
we discovered that addition of 10% to 20% water by volume
kept the reaction mixture homogeneous, providing 17 in 83%
yield on a gram scale. The intermediate triarylmethanes of dyes
9, 19, and 21 (Table 2) also showed poor solubility in TFE,
and addition of water was necessary to ensure efficient
oxidation by molecular oxygen and minimize the amount of
TFE needed in large-scale reactions. In general, reaction of
various 3-aminophenols with 4- or 5-substituted phthalaldehy-
dic acids in TFE or TFE−water under an oxygen atmosphere
(balloon) cleanly provides isomerically pure rhodamines in high
yields after simple silica column purification (Table 2).
Dyes (10, 17, and 19) crystallized from a DCM−MeOH
solvent mixture yielded deep colored, X-ray quality crystals in
the open zwitterionic form (Figure 1). The zwitterionic
The optimized synthetic processes scaled up smoothly,
providing 6-carboxy-NCT (10) in 85% isolated yield on gram
scale (Table 2, entry 2). The gram-scale synthesis of commonly
used dye 6-carboxytetramethylrhodamine (17) required addi-
tional optimization. The impurity profile for the reaction of 11
Figure 1. Crystal structures of some rhodamines.
structure allows for charge delocalization over the rhodamine
core, resulting in partial double bond character, thus shortening
the C−N bonds. Lengths of the aryl carbon−nitrogen bonds in
crystal structures of 10, 17, and 19 were measured as 1.35−1.36
Å, consistent with reported carbon−nitrogen partial double
bond length.30 On the other hand, 6-Cl-NCT (20) formed
colorless crystals from DMSO-d6. X-ray analysis revealed the
closed nonzwitterionic structure of 20 (Figure 1). The closed
structure prevents charge delocalization, resulting in a typical
aryl carbon−nitrogen single bond length of 1.42 Å.30
In summary, we developed a simple and efficient method for
synthesis of rhodamine dyes from phthalaldehydic acids. Using
TFE−water as a reaction medium and molecular oxygen as an
oxidant allowed for one-step, operationally simple, and efficient
access to a variety of rhodamines. We demonstrated the
efficiency of this approach through a gram scale synthesis of
a
Table 2. Synthesis of Selected Rhodamine Dyes
b
entry
dye
solvent
yield, %
1
2
3
4
5
6
7
8
5-carboxy-NCT (9)
6-carboxy-NCT (10)
5-carboxy-TMR (16)
6-carboxy-TMR (17)
5-CXR (18)
4:1 TFE−H2O
TFE
71
c
85
TFE
78
c
4:1 TFE−H2O
TFE
83
87
c
6-CXR (19)
4:1 TFE−H2O
TFE
82
6-Cl-NCT (20)
78
75
6-Br-NCT (21)
4:1 TFE−H2O
a
b
Dye structures are provided in Supporting Information. Isolated
yield after silica column purification. 18−48 h at 75−80 °C under
c
oxygen balloon. Gram scale.
C
DOI: 10.1021/acs.orglett.6b02635
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Organic Letters
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rhodamine in 83% yield as a single isomer.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b02635.
■
(14) (a) Deal, P. E.; Kulkarni, R. U.; Al-Abdullatif, S. H.; Miller, E. W.
J. Am. Chem. Soc. 2016, 138, 9085. (b) For application of
phthalaldehydic acids to the synthesis of Si-rhodamines: Wang, B.;
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2013078244, Nov 20, 2012.
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Experimental procedures, characterization data, H and
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X-ray crystal structure data for 10 (CIF)
X-ray crystal structure data for 11 (CIF)
X-ray crystal structure data for 11a (CIF)
X-ray crystal structure data for 19 (CIF)
X-ray crystal structure data for 20 (CIF)
X-ray crystal structure data for 14 (CIF)
X-ray crystal structure data for 17 (CIF)
X-ray crystal structure data for S05 (CIF)
X-ray crystal structure data for S09 (CIF)
(18) Martinelli, J. R.; Watson, D. A.; Freckmann, D. M.; Barder, T.
E.; Buchwald, S. L. J. Org. Chem. 2008, 73, 7102.
(19) Watson, D. A.; Fan, X.; Buchwald, S. L. J. Org. Chem. 2008, 73,
7096.
(20) On 50 g scale we prepeared 11 in three steps from commercial
5-carboxyphthalide (see the Supporting Information)
(21) Bernatek, E. Acta Chem. Scand. 1960, 14, 785.
(22) Rees, C. W.; Sabet, C. R. J. Chem. Soc. 1965, 680.
(23) Lin, H.; Sun, X.-W. Tetrahedron Lett. 2008, 49, 5343.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: Sergiy.Levin@promega.com.
Notes
(24) (a) Bonnet-Delpon, D.; Beg
́
ue,
́
J.-P.; Crousse, B. Synlett 2004,
18. (b) Borner, A.; Shuklov, I.; Dubrovina, N. Synthesis 2007, 2007,
̈
The authors declare no competing financial interest.
2925. (c) Khaksar, S. J. Fluorine Chem. 2015, 172, 51.
(25) Bentley, T. W.; Llewellyn, G. Prog. Phys. Org. Chem. 1990, 17,
121.
(26) Minegishi, S.; Kobayashi, S.; Mayr, H. J. Am. Chem. Soc. 2004,
126, 5174.
ACKNOWLEDGMENTS
■
We thank Dr. Thomas A. Kirkland and Dr. Dieter H. Klaubert
of Promega Biosciences for insightful discussions and Dr. Carla
Slebodnick of Virginia Tech for providing crystal structures.
Financial support by Promega Corporation is gratefully
acknowledged.
(27) Berkessel, A.; Adrio, J. A.; Huttenhain, D.; Neudorfl, J. M. J. Am.
̈
̈
Chem. Soc. 2006, 128, 8421.
(28) HFIP is $990 for 500 g and TFE is $186 for 500 g when
purchased from Sigma-Aldrich
(29) See the Supporting Information.
(30) Allen, F. H.; Kennard, O.; Watson, D. G.; Brammer, L.; Orpen,
A. G.; Taylor, R. J. Chem. Soc., Perkin Trans. 2 1987, S1.
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