Synthesis of rhodamines from fluoresceins using pd-catalyzed c-n cross-coupling

Article abstract of DOI:10.1021/ol202618t

A unified, convenient, and efficient strategy for the preparation of rhodamines and N,N′-diacylated rhodamines has been developed. Fluorescein ditriflates were found to undergo palladium-catalyzed C-N cross-coupling with amines, amides, carbamates, and other nitrogen nucleophiles to provide direct access to known and novel rhodamine derivatives, including fluorescent dyes, quenchers, and latent fluorophores.

Full text of DOI:10.1021/ol202618t

ORGANIC
LETTERS
2011
Vol. 13, No. 24
6354–6357
Synthesis of Rhodamines from
Fluoresceins Using Pd-Catalyzed
CꢀN Cross-Coupling
Jonathan B. Grimm and Luke D. Lavis*
Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive,
Ashburn, Virginia 20147, United States
lavisl@janelia.hhmi.org
Received September 28, 2011
ABSTRACT
A unified, convenient, and efficient strategy for the preparation of rhodamines and N,N0-diacylated rhodamines has been developed. Fluorescein
ditriflates were found to undergo palladium-catalyzed CꢀN cross-coupling with amines, amides, carbamates, and other nitrogen nucleophiles to
provide direct access to known and novel rhodamine derivatives, including fluorescent dyes, quenchers, and latent fluorophores.
Rhodamine dyes and their fluorogenic derivatives enjoy
widespread use as laser dyes, tracer agents, and biological
probes.1 This broad utility stems from the ability to modify
the optical properties of the dye by appending different
substituents on the rhodamine nitrogens. N-Alkyl rhoda-
mines are valuable fluorescent dyes where the absorption
and fluorescence emission can be tuned by altering the
number and type of alkyl groups. Attachment of aryl
functionalities yields strongly absorbing, nonfluorescent
lactone form; such compounds serve as useful latent
fluorescent compounds,1 including fluorogenic enzyme
substrates3 and photoactivatable “caged” fluorophores.4
Despite the utility and flexibility of rhodamines, the
established synthetic approach to this dye scaffold is
archaic and difficult. Rhodamines are typically prepared
through the acid-catalyzed condensation of an aminophe-
nol (1) with a phthalic anhydride (2) to yield 3 (Scheme 1,
Route A). Unfortunately, only a limited number of 3-amino-
phenols are compatible with this century-old route. Use of
phthalic anhydrides bearing a substituent (R2 in 3) for
bioconjugation yields products as intractable mixtures of
5- and 6-substituted regioisomers. Consequently, commer-
cially available functionalized rhodamines are expensive
and often sold as regioisomeric mixtures.1c Furthermore,
derivatization of the already elusive rhodamines into
fluorogenic derivatives via N-acylation is often inefficient,
due primarily to the low nucleophilicity of the rhodamine
nitrogens.3b,4c
€
dyes, which can serve as quenchers for Forster resonance
energy transfer (FRET) experiments.2 Acylation of the
rhodamine nitrogens locks the molecule in the nonfluorescent
(1) (a) Haugland, R. P.; Spence, M. T. Z.; Johnson, I. D.; Basey, A.
The Handbook: A Guide to Fluorescent Probes and Labeling Technolo-
gies, 10th ed.; Molecular Probes: Eugene, OR, 2005. (b) Lavis, L. D.; Raines,
R. T. ACS Chem. Biol. 2008, 3, 142. (c) Beija, M.; Afonso, C. A. M.;
Martinho, J. M. G. Chem. Soc. Rev. 2009, 38, 2410.
(2) (a) Ioffe, I. S.; Zal0manovich, M. Z. Zh. Obshch. Khim. 1962, 32,
1480. (b) Haugland, R. P.; Singer, V. L.; Yue, S. L. U.S. Patent 6,399,392
B1, June 4, 2002.
(3) (a) Hug, H.; Los, M.; Hirt, W.; Debatin, K.-M. Biochemistry
1999, 38, 13906. (b) Lavis, L. D.; Chao, T.-Y.; Raines, R. T. ACS Chem.
Biol. 2006, 1, 252.
(4) (a) Mitchison, T. J.; Sawin, K. E.; Theriot, J. A.; Gee, K.;
Mallavarapu, A. Methods Enzymol. 1998, 291, 63. (b) Puliti, D.;
Warther, D.; Orange, C.; Specht, A.; Goeldner, M. Bioorg. Med. Chem.
2011, 19, 1023. (c) Wysocki, L. W.; Grimm, J. B.; Tkachuk, A. N.;
Brown, T. A.; Betzig, E.; Lavis, L. D. Angew. Chem., Int. Ed. 2011, 50,
11206.
Given the difficulties with existing syntheses, we sought
an alternative route wherein the C(aryl)ꢀN bonds of
(5) High-temperature nucleophilic substitution of dihalofluorans
with amines has been used previously but is limited in scope. See ref 2
and: Song, X.; Johnson, A.; Foley, J. J. Am. Chem. Soc. 2008, 130,
17652.
r
10.1021/ol202618t
Published on Web 11/17/2011
2011 American Chemical Society

Table 1. Amination of Fluorescein Ditriflates
Scheme 1. Strategies for Rhodamine Synthesis
rhodamines are formed at a late stage.5 We envisoned
using the BuchwaldꢀHartwig cross-coupling of nitrogen
nucleophiles with fluorescein ditriflates (Scheme 1, Route
B). Pd-catalyzed cross-coupling has emerged as a powerful
method for CꢀN bond formation,6 yet these amination
reactions have seen little use in the preparation of xanthene
dyes. Lippard and co-workers reported a single example of
coupling pyrrolidinetoa dibromofluoran.7 Pengand Yang
prepared a rhodol library via cross-coupling of a fluo-
rescein monotriflate with various amines.8 We recently
used this strategy to construct a precursor for a photo-
activatable “caged” rhodamine.4c These examples inspired
us to further explore the CꢀN cross-coupling (Route B) as
a general strategy for the direct conversion of fluorescein
ditriflates to not only N-alkyl rhodamines but also N-aryl
(5, HNR3R4 = aniline) and N-acyl derivatives (HNR3R4 =
amide, carbamate, etc.). Most importantly, isomerically
pure 5- and 6-substituted fluorescein dyes are readily
synthesized on a multigram scale,9 making this approach a
convenient and divergent synthetic route to regioisomer-
ically pure rhodamine dyes.
We initially investigated the ability of the cross-coupling
to generate N-alkyl and N-aryl rhodamine dyes. Following
preparation of several fluorescein ditriflates by straight-
forward reaction of known fluoresceins with Tf2O,10 bis-
amination of the parent analog 6 or the 5-carboxyfluo-
rescein-derived 7 was explored with a variety of amines
(Table 1). In all cases, the primary competitive side reac-
tion was triflate hydrolysis to give fluorescein and/or
rhodols. The undesired detriflation;most notable at low
catalyst levels;was mitigated in a manner similar to that
of Yang8 by increasing loadings to 20 mol % Pd and
30 mol % ligand (10% and 15% per triflate, respectively).
a A: 20 mol % Pd(OAc)2, 30 mol % BINAP, toluene. B: 10 mol %
Pd2dba3, 30 mol % XPhos, dioxane. C: 10 mol % Pd2dba3, 30 mol %
Xantphos, dioxane. b See Supporting Information for optical spectros-
copy of rhodamine products.
Well-established BuchwaldꢀHartwig conditions6,11 using
Pd(OAc)2, BINAP, and Cs2CO3 in toluene at 100 °C (A)
were effective in coupling 6 and 7 with cyclic amines
(entries 1ꢀ7) and anilines (entries 8ꢀ12) to provide tetra-
lkyl rhodamines 8aꢀg and aryl rhodamines2 8hꢀl in good
to excellent yields. Surprisingly, these conditions resulted
in poor yields and low conversions for a number of secon-
dary acyclic and primary aliphatic amines. The use of Pd2dba3
with the active biaryl ligand XPhos12 in dioxane (B)
expanded the scope to include these types of amines
(entries 13ꢀ15) and provided convenient access to known
dyes such as rhodamine B (8o). The cross-coupling was
also tolerant of atypical, electronically diverse amination
substrates, including benzophenone hydrazone13 (entry 16)
(6) Reviews: (a) Hartwig, J. F. In Modern Arene Chemistry; Astruc,
D., Ed.; Wiley-VCH: Weinheim, 2002; p 107. (b) Muci, A. R.; Buchwald,
S. L. Top. Curr. Chem. 2002, 219, 131. (c) Jiang, L.; Buchwald, S. L. In
Metal-Catalyzed Cross-Coupling Reactions, 2nd ed.; de Meijere, A.,
Diederich, F., Eds.; Wiley-VCH: Weinheim, 2004; p 699.
(7) Woodroofe, C. C.; Lim, M. H.; Bu, W.; Lippard, S. J. Tetrahedron
2005, 61, 3097.
˚
(11) Ahman, J.; Buchwald, S. L. Tetrahedron Lett. 1997, 38, 6363.
(12) (a) Huang, X.; Anderson, K. W.; Zim, D.; Jiang, L.; Klapars, A.;
Buchwald, S. L. J. Am. Chem. Soc. 2003, 125, 6653. (b) Surry, D. S.;
Buchwald, S. L. Angew. Chem., Int. Ed. 2008, 47, 6338.
(13) (a) Wagaw, S.; Yang, B. H.; Buchwald, S. L. J. Am. Chem. Soc.
1999, 121, 10251. (b) Chen, A.; Gee, K.; Kang, H.-C. U.S. Patent Appl.
US 2010/0291547 A1, Nov. 18, 2010.
(8) Peng, T.; Yang, D. Org. Lett. 2010, 12, 496.
(9) (a) Coons, A. H.; Kaplan, M. H. J. Exp. Med. 1950, 91, 1. (b) Sun,
W.-C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. J. Org. Chem. 1997,
62, 6469. (c) Rossi, F. M.; Kao, J. P. Bioconjugate Chem. 1997, 8, 495.
(10) See Supporting Information for experimental details.
(14) Old, D. W.; Harris, M. C.; Buchwald, S. L. Org. Lett. 2000, 2,
1403.
Org. Lett., Vol. 13, No. 24, 2011
6355

and, under Xantphos14,15 conditions (C), nitrogen hetero-
aromatics (entries 17ꢀ18).16
Table 2. Cross-Coupling of Fluorescein Ditriflates with Car-
bamates and Other Nitrogen Nucleophiles
Carbamates, amides, and other N-acyl building blocks
were of particular interest as substrates in the CꢀN cross-
coupling of fluorescein ditriflates. In addition to providing
efficient access to fluorogenic rhodamine derivatives, such
compounds can also serve as surrogates for amines (e.g.,
ammonia) that are unsuitable for the direct amination
process due to volatility, poor reactivity, or difficult prod-
uct purification.17 Moreover, using protecting-group-
based carbamates (e.g., BocNH2) provides convenient
access to “lactone-locked” forms of the dyes that are easier
topurify and manipulate thanthe free rhodamines. Tothat
end, a thorough investigation of the ditriflate amidation
was undertaken (Table 2). In concurrence with the sub-
stantial precedent for palladium-catalyzed amidations,
Pd2dba3/Xantphos conditions, with Cs2CO3 as base and
dioxane as solvent at elevated temperature (80ꢀ100 °C),
were found to be effective for nearly all substrates
tested.6,18 Comparatively high catalyst loadings were once
again necessary to minimize triflate hydrolysis. As illu-
strated in entries 1ꢀ7, Boc-, Cbz-, and Teoc-masked
ammonia equivalents underwent smooth cross-coupling
with 6, 7, and 9ꢀ11 to afford dicarbamates 12aꢀg in
excellent yields (73ꢀ91%). These included rhodamine
110 (Rh110) derivatives with halide substituents on the
xanthene core (R1 = F, Cl) and the crucial carboxylhandle
on the bottom ring (R2 = CO2t-Bu). Gratifyingly, more
hindered secondary carbamates18,19 were also effectively
arylated under these conditions to provide several Boc-
protected rhodamines bearing N-alkyl groups (entries
8ꢀ11), including those with ester functionalities (12jꢀk).
The reactivity of several other types of related nitrogen
nucleophiles was also examined. The protected hydroxyl-
amine tert-butyl benzyloxycarbamate was found to couple
with 6 in excellent yield (entry 12, 91%). This result is
significant as few reports exist detailing the CꢀN cross-
coupling of hydroxylamines,20 and 12l represents the first
example involving a triflate. Primary and secondary sulfon-
amides were also viable substrates (entries 13ꢀ14), as was
a urea3b (entry 15). Hence, the robustness of this reac-
tion allows for the rapid preparation of lesser-known,
yet potentially useful rhodamine derivatives (e.g., N,N0-disul-
fonyl rhodamines).21
a Reaction performed at 80 °C for 18 h. b Reaction performed at
80 °C for 2ꢀ3 h. c Product resulted exclusively from coupling at primary
amide.
fluorophores. Rather than employ unreliable rhodamine
acylations, we hoped to achieve the direct preparation of
these fluorogenic molecules via the same ditriflate amida-
tion strategy. We found the appropriately functionalized
nucleophiles were well tolerated when coupled with fluo-
rescein ditriflates (entries 16ꢀ20). A carbamate containing
the photolabile ortho-nitroveratryloxycarbonyl (NVOC)
cage was reacted with 6 and 7 to conveniently afford
NVOC2-Rh110 (12p, entry 16, 68%) and the regioisomer-
ically pure 5-tert-butoxycarbonyl analog 12q (entry 17,
77%).4c Primary amides of amino acids were also cross-
coupled to 6 in excellent yields (entries 18ꢀ19). Rhoda-
mine-linked amino acids like 12s have seen significant use
as fluorogenic substrates for proteases.3a Finally, a rhoda-
mine 110 substrate bearing the esterase-labile trimethyl
lock3b moiety (12t) was easily prepared in high yield through
coupling of the trimethyl lock amide with 6 (entry 20).
To further illustrate the ease of preparing rhodamine
dyes via this strategy, the N,N0-di-Boc coupling prod-
ucts were deprotected (Table 3). Standard conditions
As mentioned earlier, more elaborate N,N0-diacyl
rhodamines possessing photolytically or enzymatically
labile acyl moieties are themselves valuable as latent
(15) Xantphos was found to be optimal for heteroaromatics; other
ligands (e.g., BINAP) were effective, albeit with diminished yield.
(16) Interestingly, bis(heteroaryl)xanthenes 8q and 8r are colorless
and nonfluorescent in solution and in the solid state, suggesting they
(like N-acyl derivatives) exist primarily in the lactone form.
(17) Bhagwanth, S.; Waterson, A. G.; Adjabeng, G. M.; Hornberger,
K. R. J. Org. Chem. 2009, 74, 4634 and references cited therein.
(18) Yin, J.; Buchwald, S. L. Org. Lett. 2000, 2, 1101.
(19) Hicks, J. D.; Hyde, A. M.; Cuezva, A. M.; Buchwald, S. L.
J. Am. Chem. Soc. 2009, 131, 16720.
(20) (a) Jones, K. L.; Porzelle, A.; Hall, A.; Woodrow, M. D.;
Tomkinson, N. C. O. Org. Lett. 2008, 10, 797. (b) Porzelle, A.;
Woodrow, M. D.; Tomkinson, N. C. O. Org. Lett. 2009, 11, 233.
(21) Shibata, A.; Furukawa, K.; Abe, H.; Tsuneda, S.; Ito, Y. Bioorg.
Med. Chem. Lett. 2008, 18, 2246.
6356
Org. Lett., Vol. 13, No. 24, 2011

In summary, we have developed a general, efficient, and
unified strategy for the synthesis of rhodamines and N,N0-
Table 3. Deprotection of Boc-Protected Rhodamines
Scheme 2. Cross-Coupling Route to Naphthorhodamines
a See Supporting Information for optical spectroscopy of rhodamine
products. b tert-Butyl esters also cleaved during Boc deprotection.
(TFA/CH2Cl2, room temperature) cleanly removed the
Boc groups and cleaved pendant tert-butyl esters, provid-
ing the free, deacylated rhodamines in excellent to nearly
quantitative yields (80ꢀ94%). Several rhodamine 110
analogs were prepared in this expedient manner (entries
1ꢀ5), including the extremely useful;and expensive22;
5-carboxy-Rh110 (13d, entry 4) and the 20,70-difluororhod-
amines 13b and 13e (entries 2 and 5) recently reported by
Hell and co-workers.23 N-Alkyl rhodamines (entries 6ꢀ7)
weresimilarly prepared ina straightforward fashion. Entry
8 is notable because it represents the first preparation of an
N-alkoxy rhodamine; exploration of the utility of this
novel chemotype is ongoing.
Finally, we explored the utility of the cross-coupling
strategy for other dye scaffolds. As shown in Scheme 2,
coupling of naphthofluorescein ditriflate (14) with tert-
butyl carbamate followed by deprotection with TFA
provided naphthorhodamine 16 in excellent yield (81%,
two steps).24 The chemistry proved flexible enough to
allow the preparation of the novel naphthorhodamine
derivative 17 bearing the esterase-labile trimethyl lock.
Thus, the facile coupling chemistry could prove useful
for generating a variety of novel nitrogenous dyes from
phenolic precursors, as also evidenced by recent work on
the coumarin system by Ting and co-workers.25
diacylated rhodamines. Fluorescein ditriflates, which are
easily prepared from readily available, regioisomerically
pure fluoresceins, were found to undergo palladium-
catalyzed CꢀN cross-coupling with amines, amides,
carbamates, and related nucleophiles. Amination with alkyl
and aryl amines allowed for convenient synthesis of fluor-
ophores and FRET quencher dyes. Where the synthesis of
rhodamine dyes by direct amination was impractical,
protecting-group-based carbamates were effectively em-
ployed. Appropriately functionalized carbamates and amides
were also coupled with ditriflates to efficiently assemble
rhodamine-based latent fluorophores, including fluorogenic
enzyme substrates and photoactivatable dyes. This process
constitutes a divergent strategy for the rapid synthesis of
many types of rhodamines and will enable the fine-tuning of
optical and chemical properties for specific applications.
Acknowledgment. This work was supported by the
Howard Hughes Medical Institute. We thank S. M. Sternson,
L. M. Wysocki, and P. H. Lee (Janelia Farm Research
Campus) for contributive discussions.
Supporting Information Available. Experimental pro-
cedures and spectral data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(22) Price for single regioisomer (5-carboxy-Rh110): $59 000/g (AAT
Bioquest).
(23) Mitronova, G. Y.; Belov, V. N.; Bossi, M. L.; Wurm, C. A.;
Meyer, L.; Medda, R.; Moneron, G.; Bretschneider, S.; Eggeling, C.;
Jakobs, S.; Hell, S. W. Chem.;Eur. J. 2010, 16, 4477.
(24) (a) Lee, L. G.; Berry, G. M.; Chen, C.-H. Cytometry 1989, 10,
151. (b) Furukawa, K.; Abe, H.; Wang, J.; Uda, M.; Koshino, H.;
Tsuneda, S.; Ito, Y. Org. Biomol. Chem. 2009, 7, 671.
(25) Jin, X.; Uttamapinant, C.; Ting, A. Y. ChemBioChem 2011,
12, 65.
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