Synthesis of near-IR fluorescent oxazine dyes with esterase-labile sulfonate esters

Article abstract of DOI:10.1021/ol202619f

Near-IR oxazine dyes are reported that contain sulfonate esters which are rapidly cleaved by esterase activity to unmask highly polar anionic sulfonates. Strategies for the synthesis of these dyes included the development of milder dye condensation conditions with improved functional compatibility and the use of an alkyl halide that allows for the introduction of esterase-labile sulfonates without the need for sulfonation of the target molecule.

Full text of DOI:10.1021/ol202619f

ORGANIC
LETTERS
2011
Vol. 13, No. 23
6196–6199
Synthesis of Near-IR Fluorescent Oxazine
Dyes with Esterase-Labile Sulfonate
Esters
Steven M. Pauff and Stephen C. Miller*
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts
Medical School, Worcester, Massachusetts 01655, United States
Stephen.miller@umassmed.edu
Received September 28, 2011
ABSTRACT
Near-IR oxazine dyes are reported that contain sulfonate esters which are rapidly cleaved by esterase activity to unmask highly polar anionic
sulfonates. Strategies for the synthesis of these dyes included the development of milder dye condensation conditions with improved functional
compatibility and the use of an alkyl halide that allows for the introduction of esterase-labile sulfonates without the need for sulfonation of the
target molecule.
Cells and organisms are most transparent to near-IR
light (650À900 nm), making this region of the electromag-
netic spectrum optimal for optical imaging.1 Unfortu-
nately, most organic near-IR dyes are not suitable for
live intracellular applications. Dyes that are capable of
diffusing across cell membranes typically give high back-
ground staining of cells because they accumulate within
intracellular membranes and organelles.2 Moreover, most
near-IR dyes are cationic, which favors accumulation in
the negatively polarized membranes of mitochondria.3 On
the otherhand, highlypolarwater-solublesulfonated near-
IR dyes (e.g., Alexa and Cy dyes) are unable to diffuse
across cell membranes.
One potential strategy to overcome this problem is to
design cell-permeable near-IR dyes that can be selectively
unmasked to a more water-soluble form upon cellular
entry. Toward this end, we recently reported a chemically
stable but esterase-labile sulfonate ester, AcOTFMB
(Figure 1), that enables the delivery and unmasking of
dansyl sulfonate within the cytoplasm of cells.4
Most sulfonate esters are potent electrophiles. Exceptions
include neopentyl sulfonates (steric block of reactivity) and
R-trifluoromethylated sulfonates (electronic deactivation).5
(1) Weissleder, R. Nat. Biotechnol. 2001, 19, 316–317.
(2) (a) Johnson, I. Histochem. J. 1998, 30, 123–140. (b) Cunningham,
C. W.;Mukhopadhyay, A.; Lushington, G. H.; Blagg, B. S. J.; Prisinzano,
T. E.; Krise, J. P. Molecular Pharm. 2010, 7, 1301–1310.
(3) Bunting, J. R.; Phan, T. V.; Kamali, E.; Dowben, R. M. Biophys.
J. 1989, 56, 979–993.
(4) Rusha, L.; Miller, S. C. Chem. Commun. 2011, 47, 2038–2040.
(5) Miller, S. C. J. Org. Chem. 2010, 75, 4632–4635.
r
10.1021/ol202619f
Published on Web 11/02/2011
2011 American Chemical Society

We have utilized these classes of sulfonate esters as plat-
forms for the design of protecting groups that can be
removed under specific conditions. In particular, we found
that the incorporation of an acetoxy group into trifluoro-
methylbenzyl (TFMB) sulfonate esters maintained stability
to nucleophiles but rendered these groups highly labile to
esterase activity (Figure 1).4
N-methylated and sulfonated to afford 3.7a Diazonium
coupling yielded 4, and subsequent acid-catalyzed conden-
sation of this arylazo compound with a m-aminophenol13
yielded the sulfonated dye 22, which exhibits maximal
absorption at 673 nm and emission at 689 nm in phos-
phate-buffered saline (PBS).
While this approach was facile for the small dansyl
fluorophore, it was unclear whether the AcOTFMB group
could be incorporated into near-IR dyes, which are con-
siderably larger and more challenging to synthesize. Here
we report the successful synthesis of oxazine near-IR
fluorophores bearing AcOTFMB esterase-labile sulfonate
esters. We demonstrate that these dyes are chemically
stable but are readily cleaved to the free sulfonate by
esterase activity, while the corresponding TFMB esters
are unaffected.
Scheme 1. Synthesis of a Sulfonated Oxazine Dye
With this sulfonated near-IR fluorophore in hand, the
next synthetic challenge was determining how and when to
incorporate the TFMB and AcOTFMB sulfonate esters.
Our initial attempts to directly introduce TFMB and
AcOTFMB sulfonate esters into 22 by formation and
reaction of the sulfonyl chloride were unsuccessful. We
therefore installed the sulfonate esters into the dye pre-
cursors (Scheme 2). Thus, the sodium sulfonate salt 3 was
converted into the allylic sulfonyl chloride 5 and subse-
quently treated with the corresponding alcohol and Et3N
at 0 °C to afford the sulfonate esters 6 and 7.
The sulfonate esters 6 and 7 were stable to diazonium
coupling conditions in acidic aqueous methanol to afford 8
and 9. Subsequent acid-catalyzed condensation of 8 with
N-ethyl-7-hydroxy-tetrahydroquinoline yielded the de-
sired TFMB-protected sulfonated oxazine 23. However,
the standard oxazine dye-formation conditions of HCl in
hot aqueous ethanol led to substantial deprotection of
AcOTFMB, presumably because carboxylic esters are
labile to these conditions. Gratifyingly, dye formation in
hot acetic acid readily afforded the desired oxazine dye 24
with the AcOTFMB sulfonate ester intact (Scheme 2).
To facilitate the synthesis of more dyes containing
a protected sulfonate, we constructed TFMB and
AcOTFMB-protected iodopropyl sulfonates 11 and 13
(Scheme 3). Treatment of 3-chlorosulfonyl chloride with
the corresponding alcohols and Et3N at 0 °C yielded the
respective chloropropyl sulfonates 10 and 12. The chlor-
ides were then selectively displaced with iodides. These
compounds are especially notable because (1) their synth-
esis clearly demonstrates the remarkable stability of
TFMB and AcOTFMB sulfonates to nucleophilic attack
(chloro is displaced selectively even after overnight reflux
with excess iodide) and (2) the iodide 13 can allow the
introduction of an AcOTFMB-protected sulfonate into
molecules with a suitable nucleophile, avoiding the need
for sulfonation.
Figure 1. Esterase-labile protecting groups for sulfonates. Yellow-
green fluorescent AcOTFMB dansyl sulfonate esters are stable
to nucleophilic attack due to the electron-withdrawing proper-
ties of the trifluoromethyl group. The acetoxy trigger can be
readily removed by esterase activity. Subsequent rapid 1,
6-elimination affords the blue-fluorescent dansyl sulfonate anion.
Examples of sulfonated near-IR fluorophores include
cyanines,6 oxazines,7 and some rhodamine8 and BODIPY9
derivatives. Among these dye classes, near-IR oxazines
have the advantages of a compact structure, excitation and
bright fluorescence in the 650À700 nm region,10 and high
photostability.11 To determine whether the AcOTFMB
protection strategy was amenable to this class of near-IR
fluorophores, we first synthesized the sulfonated oxazine
dye 22 as shown in Scheme 1. Reaction of m-anisidine with
acetone to form the dihydroquinoline 1 was facile in the
presence of 2 mol % In(OTf)3.12 This product was then
(6) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.;
Waggoner, A. S. Bioconjugate Chem. 1993, 4, 105–111.
(7) (a) Zilles, A.; Arden-Jacob, J.; Drexhage, K.-H.; Kemnitzer, N. U.;
Hamers-Schneider, M. US 2006/0179585 A1, 2006. (b) Toutchkine, A. WO
2009/152024 A1, 2009.
(8) Kolmanov, K.; Belov, V. N.; Bierwagen, J.; Ringemann, C.;
Muller, V.; Eggeling, C.; Hell, S. W. Chem.;Eur. J. 2010, 16, 158–166.
(9) Niu, S. L.; Ullrich, G.; Ziessel, R.; Kiss, A.; Renard, P.-Y.;
Romieu, A. Org. Lett. 2009, 11, 2049–2052.
(10) (a) Hintersteiner, M.; Enz, A.; Frey, P.; Jaton, A.-L.; Kinzy, W.;
Kneuer, R.; Neumann, U.; Rudin, M.; Staufenbiel, M.; Stoeckli, M.;
Wiederhold, K.-H.; Gremlich, H.-U. Nat. Biotechnol. 2005, 23, 577–583.
(b) Wakata, A.; Lee, H.-M.; Rommel, P.; Toutchkine, A.; Schmidt, M.;
Lawrence, D. S. J. Am. Chem. Soc. 2010, 132, 1578–1582.
(11) Eggeling, C.; Widengren, J.; Brand, L.; Schaffer, J.; Felekyan, S.;
Seidel, C. A. M. J. Phys. Chem. A 2006, 110, 2979–2995.
(12) Theoclitou, M.-E.; Robinson, L. A. Tetrahedron Lett. 2002, 43,
3907–3910.
(13) Kanitz, A.; Hartmann, H. Eur. J. Org. Chem. 1999, 923–930.
Org. Lett., Vol. 13, No. 23, 2011
6197

Scheme 2. Incorporation of TFMB and AcOTFMB Sulfonate
Esters into an Oxazine Dye
Scheme 4. Synthesis of Sulfonated Derivatives of MR121
Scheme 5. Intramolecular Nucleophiles Can Cause Self-Immo-
lation of AcOTFMB Sulfonates
Scheme 3. Synthesis of Iodopropyl Sulfonates
Because the TFMB-protected phenol 29 is stable, we
surmise that the phenol promotes intramolecular deacety-
lation and self-immolation of the AcOTFMB group
(Scheme 5). Indeed, the corresponding AcOTFMB-pro-
tected anisole 19 is stable and readily isolated. Thus,
derivatives of AcOTFMB bearing intramolecular nucleo-
philes may be prone to self-immolation.
In order to access symmetrical bis-sulfonated dyes with-
out the need for the isolation of a phenolic intermediate
such as 29, we constructed the silyl ether 30. We hypothe-
sized that the AcOTFMB group would be stable in the
presence of the silyl ether and that the acidic conditions of
dye synthesis would unmask the silyl ether and allow dye
formation (Scheme 6). We were gratified to find that the
silyl ether 32 was indeed stable and that the silyl ethers 31
and 32 readily formed the desired symmetric bis-sulfo-
nated oxazine dyes 33 and 34 when treated with 20 and 21
under acidic conditions (Scheme 6).
This method of oxazine dye synthesis has three key
advantages over the classical methods that utilize conden-
sation of one or more phenolic compounds.7,10a,13,14a First,
during the N-alkylation step, protection of the phenol
avoids any undesired O-alkylation (Scheme 6), which
improves yields and simplifies purification. Second, there
is no need to deprotect prior to dye synthesis, eliminating a
synthetic step. Finally, the use of silyl ethers expands the
To make use of 11and 13for the construction of near-IR
dyes, we synthesized sulfonated analogs of the oxazine dye
MR12114 (Scheme 4). The tetrahydroquinoline 15 was
dissolved in DMF and heated with 11 or 13 in the presence
of excess K2CO3 to yield 18 and 19. Diazonium coupling
followed by acid-catalyzed condensation with N-ethyl-7-
hydroxytetrahydroquinoline yielded the desired oxazine
dyes 26 and 27. The corresponding free sulfonate dye was
synthesized analogously by treatment of 15 with 1,3-
propanesultone to afford 16 which was subsequently
elaborated to yield the sulfonated dye 25 (Scheme 4). The
excitation and emission wavelengths for these dyes are
similar to those for MR121, at 659 and 671 nm respectively.
We next attempted to synthesize symmetrical oxazine
dyes with two protected sulfonates, which would generate
highly polar anionic molecules upon cleavage. Current
synthetic approaches to such dyes require a phenolic
intermediate such as 29 (Scheme 5).7,10a,13,14a While the
synthesis of the TFMB-protected sulfonate ester 29 was
facile, we were unable to isolate the corresponding
AcOTFMB-protected phenolic intermediate (Scheme 5).
(14) (a) Herrmann, R..; Josel, H.-P..; Drexhage, K.-H.; Marx, N.-J.
EP 757447, 1996. (b) Lieberwirth, U.; Arden-Jacob, J.; Drexhage, K.-H.;
Herten, D. P.; Muller, R.; Neumann, M.; Schultz, A.; Siebert, S.; Sagner, G.;
Klingel, S.; Sauer, M.; Wolfrum, J. Anal. Chem. 1998, 70, 4771–4779.
6198
Org. Lett., Vol. 13, No. 23, 2011

Scheme 6. Bypassing Phenols: Formation of Bis-Sulfonated
Oxazine Dyes from Silyl Ether and Anisole Starting Materials
Figure 2. AcOTFMB-oxazine dyes 24 and 27 were incubated in
PBS (P), 5 mM glutathione (G), 1 mg/mL ovalbumin (O), or 1U/
mL pig liver esterase (E) for 30 min, then separated by TLC
(10% MeOH/CH2Cl2), and imaged. The respective free sulfo-
nate dyes 22 and 25 are shown for reference.
range of functionality that is compatible with oxazine dye
synthesis. Typically, anisoles have been used for protection
of phenolic intermediates in dye synthesis,7,10a,14a and
the requirement for HBr or BBr3 deprotection greatly
limits functional group compatibililty. We anticipate
that this method of dye synthesis will also find use in
the preparation of rhodamine dyes bearing sensitive
chemical functionality.
The AcOTFMB-protected sulfonated oxazine dyes 24
and 27 are readily unmasked by pig liver esterase (PLE) to
afford the zwitterionic free sulfonate dyes 22 and 25,
respectively (Figure 2). Similarly, treatment of 34 with
PLE rapidly formed the highly polar bis-sulfonate (Figure
S1). In contrast, TFMB-protected dyes are stable to
esterase activity (Figure S1), and both TFMB and
AcOTFMB dyes are stable to treatment with biological
nucleophiles such as 5 mM glutathione and 1 mg/mL
ovalbumin (Figure 2, Figure S1). These findings are con-
sistent with our results with dansyl dyes4 and suggest that
the AcOTFMB group is generally suitable as a chemically
stable, esterase-labile protecting group for dyes containing
aryl, allyl, or alkyl sulfonates.
We have described complex near-IR fluorophores that
contain the esterase-labile sulfonate protecting group
AcOTFMB. Moreover, intermediates such as 7, 21, and
32 are basic building blocks for the incorporation of
esterase-labile sulfonates into oxazines and other dyes such
as rhodamines, and intermediate 13 can allow the incor-
poration of the esterase-labile AcOTFMB sulfonate into
an even wider variety of molecules. Further optimization
of this approach and cell-based applications of these
molecules are currently underway.
Acknowledgment. Thisworkwas supportedbythe NIH
(GM087460). We thank Anjan Bhunia (formerly Univer-
sity of Massachusetts Medical School) for preliminary
work on oxazine dye synthesis.
Supporting Information Available. Experimental pro-
cedures and full spectroscopic data for all new com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Org. Lett., Vol. 13, No. 23, 2011
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