A common diaryl ether intermediate for the gram-scale synthesis of oxazine and xanthene fluorophores

Article abstract of DOI:10.1002/anie.201205369

Common ground: Copper-catalyzed coupling reactions can be used for the high-yielding preparation of widely used oxazine and xanthene fluorophores from a common diaryl ether intermediate on a gram-scale (see scheme). This general approach may facilitate th

Full text of DOI:10.1002/anie.201205369

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DOI: 10.1002/anie.201205369
Fluorescent Dyes
A Common Diaryl Ether Intermediate for the Gram-Scale Synthesis of
Oxazine and Xanthene Fluorophores**
Andrew V. Anzalone, Tracy Y. Wang, Zhixing Chen, and Virginia W. Cornish*
Recent advances in fluorescence spectroscopy have driven
the demand for dyes with improved photophysical and
fluorescence properties.[1] In addition to their development
as cellular and single-molecule imaging tools, engineered
fluorescent dyes have also been developed as environmental
sensors that can provide information on local viscosity,
pH value, solute concentration, and electrical potential.[2]
Current and future developments in this field, especially
those relevant to single-molecule and cellular imaging,
depend on the synthesis of customized fluorescent dyes that
emit in the red region of the visible spectrum, have high
extinction coefficients and quantum yields, and display high
photostability.[3]
Dyes from the oxazine and xanthene classes are among
the best for these purposes, exemplified by commercial
compounds ATTO-655 and Alexa Fluor-594, respectively.[3a,4]
In the synthesis of derivatized or customized fluorophores,
modification of commercially available dyes is often limited,
Scheme 1. Retrosynthetic analysis for oxazine and xanthene fluoro-
either because of cumbersome functionalization of the parent
compounds, or because of prohibitive costs to obtain suffi-
phores, highlighting the differences in approach between prior works
and our work. LG=leaving group, X=H or RCO2À
.
cient quantities of dye for carrying out the necessary synthetic
steps. Thus, the de novo synthesis of fluorescent dyes from
basic organic building blocks is an essential aspect of
technology development. Despite the obvious importance of
these molecules and the evident need for improved synthetic
methodologies, oxazines and xanthenes are still largely
synthesized by using methods reported decades or more ago
that do not take advantage of the efficiencies of modern
chemical transformations.[5]
Here, we report a novel and scalable synthetic approach
to widely used oxazine and xanthene fluorophores through
a common diaryl ether intermediate (Scheme 1). Taking
advantage of recent developments in transition-metal catal-
ysis, we prepared electronically activated diaryl ethers to
serve as tethered nucleophiles, reacting with a range of
substrates to undergo cyclization to cationic fluorescent
compounds (Scheme 1, our work). Final products were
provided in good overall yields, were amenable to purification
using standard silica-based normal-phase flash chromatogra-
phy, and, significantly, could be prepared on gram scale.
As part of our ongoing research program to develop
improved technologies for in vivo super-resolution imaging,
we sought to prepare derivatives of the commonly utilized
oxazine ATTO-655.[4b] Because of the expense associated
with obtaining large quantities of this dye, and the lack of
commercial availability of other desirable analogues, we set
out to synthesize the oxazines following previously described
methods.[5b,c] These prior works rely on coupling a pair of
aminophenol derivatives, one of which is substituted with an
electrophilic nitroso or diazo functionality, by heating the two
components in acidic medium (Scheme 1, prior works).
Though the aminophenol intermediates are readily prepared,
the final coupling and cyclization reactions frequently result
in low yields ( ꢀ 15%), and necessitate the use of preparative-
scale reverse-phase high-performance liquid chromatography
(prep-HPLC) in order to obtain material of satisfactory
purity. In our hands, the quantities obtained by this method
proved insufficient to reasonably carry out the remaining
steps in the synthesis of our final targets, and we concluded
that scaling up the process to obtain the desired quantities was
impractical. As a result, we concluded that the existing
approach was not a viable synthetic route and chose to pursue
alternative synthetic strategies.
[*] A. V. Anzalone, T. Y. Wang, Z. Chen, Prof. V. W. Cornish
Department of Chemistry, Columbia University
550 West 120thStreet, MC 4854, New York, NY 10027 (USA)
E-mail: vc114@columbia.edu
[**] Financial support was provided by the NIH (GM090126,
GM087519, GM091804). A.V.A. is supported by Columbia Univer-
sity College of Physicians and Surgeons Medical Scientist Training
Program (NIH T32 GM007367). We are grateful to Dr. D. Romanini
for editing the manuscript, Prof. S. A. Snyder and R. L. Gonzalez for
helpful discussions, and Prof. W. Min for graciously offering the use
of his spectroscopy equipment.
Within the oxazine core structure, we identified a previ-
ously unexplored diaryl ether disconnection, which is
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201205369.
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Table 1: Copper(I)-catalyzed couplings between phenols and aryl iodides
to furnish diaryl ethers.[a]
exploited in the retrosynthetic analysis shown in Scheme 1.
We envisioned that a step-wise coupling and cyclization
process would proceed in higher overall yield compared to the
classical coupling strategy and, more importantly, would lead
to fewer by-products in the final step. Thus, isolation and
purification could be carried out using standard flash-
chromatography techniques amenable to gram-scale opera-
tions. In pursuit of this synthetic strategy, it was necessary to
examine methods for synthesizing the diaryl ether in a versa-
tile and robust fashion.
Entry
1
Phenol
Aryl iodide
Diaryl ether
Yield
[%]
90
In addition to the more traditional dihydroquinoline and
tetrahydroquinoline derivatives, we also chose to explore an
indoline-based scaffold to study this synthetic sequence. The
precursor indoles provide a large number of building blocks
from which to start, and we were also interested in the
photophysical properties of the resulting oxazine products,
which are not well described for the indoline class. Prepara-
tion of the indoline coupling partners proved to be straight-
forward by Gribble reduction and alkylation of the commer-
cially available indole starting materials (see the Supporting
Information, Scheme S1).[6]
1a
2a
3a
2
82
1b
1c
1c
2a
2b
2c
3b
3c
3d
3
4
88
80
The Ullmann ether synthesis poses a potential route to the
diaryl ether structural motif by copper(I)-promoted reaction
between phenols and aryl halides.[7] Although attractive in
theory, the classical reaction conditions typically employ
strong base, stoichiometric quantities of copper, and heating
at temperatures higher than 2008C. Additionally, the reaction
is known to be highly substrate dependent and, even when
successful, commonly results in only modest yields. To
overcome these limitations and eliminate the harsh reaction
conditions, we chose to explore a more contemporary method
that utilizes palladium catalysis.[8] Unexpectedly, coupling
between phenol 1c and its corresponding aryl-triflate resulted
predominantly in reaction at C5 of the indoline in a Heck-
type manner, producing the biaryl phenol derivative as the
major product (Scheme S2). Thus, the palladium-catalyzed
coupling reaction does not appear to be a useful method for
synthesizing diaryl ethers when electron-rich carbon nucleo-
philes, such as those that exist in our system, are present.
We next turned to recent developments in ligand-assisted
copper-catalyzed coupling reactions.[9] Based on work
reported by Buchwald and co-workers, the coupling between
phenol 1b and 3-iodoaniline (2a) provided the diaryl ether 3b
in 82% yield when carried out in the presence of catalytic
copper iodide (10 mol%) and 2-picolinic acid (20 mol%) at
858C in DMSO (Table 1, entry 2). This catalytic system offers
the advantage of orthogonality with the aniline functional
group. As opposed to aryl iodides, aryl bromides were found
to be very poor substrates in our system, with nearly no
product formation, even at elevated temperatures (1808C)
and increased catalyst loadings (50 mol%). Nonetheless, aryl
iodides were readily prepared from the corresponding
commercially available aryl bromides by employing
a copper-promoted halogen exchange reaction.[10] Couplings
carried out under the aforementioned conditions provided
a range of substituted diaryl ethers in good yields (Table 1).
Following these coupling reactions, anilines 3a and 3b were
transformed to their dihydroquinoline derivatives through
5
6
77
80
1c
2d
3e
1c
2e
3 f
[a] Conditions: phenol (1.2 equiv), copper(I) iodide (10 mol%), 2-
picolinic acid (20 mol%), K3PO4 (2.0 equiv), DMSO, 858C, 24 h.
DMSO=dimethyl sulfoxide.
a modified Skraup reaction, and subsequently alkylated and
reduced where appropriate (see the Supporting Information).
Having established a reliable means of synthesizing the
critical diaryl ether intermediates, we next explored condi-
tions to convert these compounds to their corresponding
oxazine dyes. This transformation was readily accomplished
by reaction of diaryl ether 3d with one equivalent of 4-
nitrobenzene diazonium tetrafluoroborate, followed by heat-
ing the corresponding diazene 4d with p-toluenesulfonic acid
(TsOH) to 658C in ethanol (Scheme 2). The latter process
proceeded with near-quantitative conversion and, following
simple silica-based flash chromatography to remove the
liberated p-nitroaniline, we obtained the oxazine tosylate
salt in high purity and 90% yield. Implementing this strategy,
several substituted oxazine dyes with various spectroscopic
properties were synthesized in good to excellent yields from
the corresponding diazene compounds (Table 2). Of note,
over one gram of oxazine 6 f was prepared by this method in
94% yield, thus demonstrating the scalability of our approach
(Table 2, entry 6). Additionally, 6g, which is the sulfonated
analogue of 6c and resembles commercially available ATTO-
655, was synthesized in good yield by this method.
To further expand the scope of this methodology, we
examined other popular fluorescent dyes for similar diaryl
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Table 2: Synthesis of substituted oxazine dyes.[a]
Entry
Diazene[b,c]
Oxazine
Yield
[%]
1
74
85
87
Scheme 2. Reaction sequence for conversion of diaryl ethers to oxazine
dyes.
4a
6a
6b
2
3
ether disconnections, and identified xanthene dyes, exempli-
fied by rhodamine and rosamine, as potential targets
(Scheme 1). The classical method to synthesize these amino-
xanthenes requires heating two equivalents of an amino-
phenol with one equivalent of a carboxylic acid anhydride or
aldehyde under acidic conditions (ZnCl2 or H2SO4), often at
high temperatures (Scheme 1, prior works).[5a] When this
strategy is employed with derivatized analogues, the reactions
typically result in modest to poor yields, and isolation of the
products is difficult by conventional purification methods.[11]
We sought to prepare these fluorescent dyes from our diaryl
ethers in an analogous manner to the synthesis of the oxazine
dyes. After screening several Lewis acid catalysts and reaction
conditions, we found that Ga(OTf)3 catalyzed the tandem-
Friedel–Crafts acylation–cyclization reaction between the
aforementioned diaryl ethers and aromatic acid chlorides to
provide the corresponding xanthene dyes in modest to good
yields with substantial amounts of recoverable starting
materials (Table 3).[12]
Supplementing the reaction with additional Ga(OTf)3 did
not lead to further product formation, suggesting that a by-
product formed in the course of the reaction inhibited the
progression of the reaction. Possibly, this was due to substrate
inactivation by HCl, which is generated by the Friedel–Crafts
acylation and from hydrolysis of the acid chloride (water is
generated in the cyclization step). Interestingly, in the case of
the p-Me2N-substituted derivative (Table 3, entry 3), nearly
full consumption of starting material was observed, suggest-
ing that the p-Me2N group may be capable of buffering the
reaction to allow increased conversion. Higher conversions of
starting material could be obtained by resubjecting the crude
product mixture (following workup) to the initial reaction
conditions.
4b/5b
4c/5c
6c
6d
4
5
90
78
4d
4e/5e
4 f/5 f
6e
6[d]
94
83
6 f
7
4g/5g
6g
[a] Conditions: TsOH (3.0 equiv), EtOH, 658C, 4–8 h. [b] Ar=p-NO2Ph.
[c] Diazenes were obtained and used as regioisomeric mixtures to cyclize
to a single oxazine product. [d] Reaction conducted on a gram-scale.
Of note, this synthetic strategy allows the synthesis of
asymmetrically functionalized xanthene dyes, which are not
accessible by the classical coupling strategy. Additionally,
several dyes were prepared in significantly higher yields
compared to traditional syntheses. For example, compound
7b, an asymmetric xanthene, was synthesized in 83% yield
simply by reaction of the diaryl ether with 2.2 equivalents of
trifluoroacetic anhydride at room temperature without
a Lewis acid catalyst. By comparison, rhodamine 700, a similar
dye that bears the trifluoromethyl substituent at the meso
carbon atom, is reported to be prepared in only 5% yield
from aminophenol derivatives.[13] Although this current work
is limited to the synthesis of rosamines with fully substituted
anilines, it may be possible to synthesize mono- or non-
alkylated analogues by minor modification of this synthetic
approach.
Figure 1 shows the absorption and fluorescence spectra
for several of the oxazine and xanthene dyes synthesized.
Notably, the increased conjugation of oxazines 6a and 6b
results in significant red-shifted absorption and fluorescence.
Similarly, xanthenes 7b, 7e, and 7 f, which possess electron-
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Table 3: Tandem catalytic Friedel–Crafts acylation/cylization reaction for
the synthesis of xanthene fluorophores.[a]
Entry
R1
Et
R2
Prod. Yield [%][b]
1
7a
7b
56 (92)
83
2[c,e]
3[d]
Et
Et
7c
7d
77 (82)
48 (89)
4
5
Et
Et
7e
7 f
53 (93)
36 (81)
6[d]
[a] Conditions: acid chloride (8 equiv), Ga(OTf)3 (15 mol%), MeNO2,
608C, 4 ꢀ molecular sieves (M.S.), 16 h. [b] Yields in brackets based on
recovered starting material. [c] No Lewis acid catalyst required, TFAA
(2.2 equiv) in CH2Cl2 at RT for 12 h. [d] Acid chloride (5 equiv) was used.
[e] Reaction conducted on a gram-scale.
Figure 1. a) Absorbance and b) fluorescence spectra of various oxazine
and xanthene derivatives. Spectra were obtained in H2O, with the
exception of 7c, which was obtained in aqueous HCl (50 mm).
withdrawing side chains, also display red-shifted spectra. The
p-Me2N-substituted rosamine 7c displays pH-dependent
fluorescence, with a near 30-fold increase in fluorescence
intensity under acidic conditions (data not shown). Spectral
properties of the dyes in aqueous solution are provided in
Table 4.
oxazine- and xanthene-based fluorescent dyes from
a common diaryl ether intermediate. Compared to previously
existing synthetic methodologies, our work provides a versa-
tile means for preparing these fluorescent dyes and eliminates
the need for tedious and expensive purifications. Following
this synthetic approach, a number of oxazine and xanthene
fluorophores were synthesized and characterized. With
proper synthetic planning, we believe this to be a general
and widely applicable approach to the synthesis of derivatized
oxazine and xanthene dyes, thus possibly facilitating the
development of novel fluorophores and probes with unique
properties.
In summary, we have established a high-yielding, scalable
synthetic route for the preparation of the widely used
Table 4: Spectral properties of fluorescent dyes in H2O.[a]
Dye
lmaxabs
[nm]
emax
[mÀ1 cmÀ1
lfluor
[nm]
Fwhm[b]
[nm]
Ff
]
6a
6b
6c
6d
6g
7a
7b
7c
7d
7e
7 f
703
682
664
648
663
553
616
559
555
579
581
50000
69000
67000
66000
97000
58000
61000
79000
60000
68000
57000
717
696
681
661
677
576
643
585
575
602
600
42
44
45
43
43
49
90
53
47
44
42
0.08
0.09
0.24
0.11
0.20
0.19
0.08
0.14
0.21
0.27
0.24
Received: July 7, 2012
Revised: September 25, 2012
Published online: November 21, 2012
Keywords: copper · cyclization · domino reaction · dyes/
.
pigments · fluorescence
[a] Measurements were taken in H2O, with the exception of 7c, which
was measured in aqueous HCl (50 mm). [b] Full-width at half-maximum
height.
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