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Tetrahedron 62 (2006) 11021–11037
Tetrahedron report number 775
Benzophenoxazine-based fluorescent dyes for labelingbiomolecules
Jiney Jose and Kevin Burgess*
Department of Chemistry, Texas A & M University, College Station, TX-77841, USA
Received 28 July 2006
Available online 27 September 2006
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110212. Meldola’s Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11022
2.1. Syntheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110222.2. Photophysical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11022
3. Nile Red derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110223.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110223.2. Syntheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110233.3. Spectroscopic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11025
4. Nile Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110274.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110274.2. Syntheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110274.3. Spectroscopic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11030
5. Other benzophenoxazine dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110335.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110335.2. Syntheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11033
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11034Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11035References and notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11035Biographical sketch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11037
1. Introduction
Meldola’s Blue 1, Nile Red 2, and Nile Blue 3 have some de-sirable attributes as fluorescent probes.3 Dyes 2 and 3 havereasonably high fluorescence quantum yields in apolar sol-vents and they fluoresce at reasonably long wavelengths.Nile Red, in particular, fluoresces far more strongly in apolarmedia than in polar ones, and the fluorescent emission showsa large bathochromic (i.e., red-) shift in polar media, henceit can be used as a probe for environment polarity;4–6 thesecharacteristics may be attributes for some applications, but
* Corresponding author. Tel.: +1 979 845 4345; fax: +1 979 845 1881;e-mail: [email protected]
0040–4020/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.tet.2006.08.056
limitations for others. None of these dyes are significantlysoluble in aqueous media, and their quantum yields are dra-matically reduced. One of the big challenges in the produc-tion of fluorescent dyes is to produce water-soluble probesthat fluoresce strongly in aqueous media, particularly above600 nm or at even longer wavelengths. Motivation forresearch in this area is drawn from needs for intracellular,tissue, and whole organism imaging where near-IR dyesare far more conspicuous than ones emitting at 550 nm orless.7
The phenoxazine skeleton may be extended by adding fusedbenzene rings to the a–c or h–j faces. Benzophenoxazines ofthis type are ‘angular’1 or ‘linear’ depending on the orienta-tion of the ring fusion, as illustrated in Figure 1a.
11022 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
Substituents that freely donate and/or accept electron den-sity on benzophenoxazine cores can, in some orientations,give fluorescent compounds. The first notably fluorescentcompound to be discovered in this class was Meldola’sBlue 1, but Nile Red 22 and Nile Blue 32 are far more fre-quently used in contemporary science.
The objective of this review is to summarize the state of theart of benzophenoxazine dyes in such a way that all readerscan understand quickly what has been done to develop usefulprobes in this category. Inspired readers should also be ableto use this summary to design research approaches that arenot yet explored but would lead to useful endpoints.
2. Meldola’s Blue
This dye is used in textiles, paper, and paints, mainly as a pig-ment. It is not a particularly useful fluorescent dye for label-ing proteins because of its poor water solubility. Further, itsfluorescence is weak in all common media (e.g., EtOH) anddoes not give a clear indication of the surrounding polarity.However, Meldola’s Blue has been used as a component inredox sensors16 for detection of materials such as NADH,8
pyruvates,9 hydrogen peroxide,10 glucose,11 and 3-hydroxy-butyrate.12 It has also been used in electrochemical experi-ments involving DNA wherein the dye mediates electrontransport.13
2.1. Syntheses
The original (1879) synthesis of Meldola’s Blue involvedcondensation of a nitroso compound with 2-naphthol at ele-vated temperatures (Scheme 1a).14 Details of the reaction
a
O
HN a
b
c
i
j
h
phenoxazine
O
HN
O
HN
benzo[b]phenoxazine
O
HN
linear angular
benzo[a]phenoxazine benzo[c]phenoxazine
12
3
4
5678
9
1110
12
b
N
OMe2+N
Cl-
1
Meldola's Blue
O
N
OEt2N O
N
N+H2Et2NCl-
2
Nile Red
3
Nile Blue
Figure 1. (a) Phenoxazines and benzophenoxazines; (b) structures of thethree best known fluorescent dyes in this class.
conditions and the yield were not given. Subsequently, Mel-dola’s Blue has been made with a variety of counter ions viareactions involving different Lewis acids (Scheme 1b).15
Counter ion modifications have been used to modulate theelectronic properties of solid materials for applications notdirectly related to labeling biomolecules.
a
Me2N
NO CH3COOH
110 °CHO
N
OMe2+N
Cl-
Me2+NCl-
1
EtOHmax abs
540 nmmax emiss
568 nm
b
Me2N
(i) NaNO2 / H2OEtOH/HCl, 0-3 °C, 1 h
(ii) OH
ZnCl2, EtOH, 70 °C
O
N
1
Meldola's Blue
Scheme 1. (a) Original synthesis of Meldola’s Blue; (b) a more recentapproach.
2.2. Photophysical properties
Phenoxazines without strongly electron withdrawing ordonating substituents have unexceptional absorbance char-acteristics, and they are not particularly fluorescent com-pounds. Fusion of a benzene ring onto the heterocycledoes not alter this situation dramatically. Meldola’s Bluehas one dimethylamino substituent and the heterocycliccore is oxidized. These changes in the composition of theheterocycle shift its absorption and emission characteristicsinto a useful range: lmax abs 540 nm, lmax emiss 568 nm EtOH.This dye is not noted for its solvatochromic properties.17
3. Nile Red derivatives
3.1. Introduction
Nile Red has a neutral oxidized phenoxazine system, i.e., itis a phenoxazinone. The 9-diethylamino substituent is ableto donate electron density into the carbonyl group acrossthe ring; this electronic arrangement probably accounts forits highly fluorescent properties (Fig. 2).
The water-solubility of Nile Red is extremely poor, but inother solvents its fluorescence maxima and intensity aregood indicators of the dye’s environment-polarity. As
11023J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
mentioned above, this solvatochromic effect is such thatpolar media cause a red-shift but decreased fluorescenceintensity. This decreased fluorescence intensity is probablydue to self-quenching of the dye in face-to-face aggregates.Consequently, this dye is particularly useful for studyinglipids and events that involve impregnation of the dye inapolar media. Surprisingly, very few water-soluble analogsof Nile Red have been reported, and only limited fluores-cence data have been given for those.
3.2. Syntheses
The first synthesis of Nile Red was a condensation reactionof a nitrosophenol (Scheme 2a). The patent literature indi-cates that other solvent systems can be used. It is also possi-ble to prepare Nile Red via hydrolysis of Nile Blue asindicated in Scheme 2b. The product is usually isolated viachromatography on silica.
a
NO
OHEt2N
OH
1 h
O
N
OEt2N
2 10 %
b
O
N
N+H2Et2NCl-
0.5 % H2SO4
reflux, 2 h
3
O
N
OEt2N
2 22 %
CH3COOH
70 °C,
Scheme 2. (a) Original synthesis of Nile Red; (b) synthesis from Nile Blue.
Substituted or modified Nile Red derivatives may be pre-pared via variations of the syntheses above, i.e., de novomethods, or by functionalizing Nile Red itself. For instance,
MeOHmax abs 554 nm
max emiss
max abs
max emiss 638 nm 0.38
n-heptane484 nm 529 nm
0.48
O
N
ON
Nile Red 2
1
2
6
3
4
8
11
10
O
N
O-N+
Figure 2. Electron delocalization in Nile Red.
a de novo approach was used to prepare the 6-carboxyethylderivatives 4 (reaction 1). These are potentially interestingsince hydrolysis of the ester would give a carboxylic acidfor attachment to biomolecules; however, this does notappear to have been attempted yet.19 The patent literature alsodescribes a synthesis of the 2-carboxy Nile Red derivative.20
HO OHCOOC2H5
NO
N
ethanol, reflux
3 hR1
R2
O
N
OCOOC2H5
NR1
R2
R1, R2 = H and simple linear alkyl
OH N O,4
24 - 60 %MeOH
max abs545 +/- 25 nm
max emiss 620 +/- 5 nm
ð1Þ
De novo syntheses of 1- and 2-hydroxy Nile Red, com-pounds 5 and 6, respectively, have also been performed,and the products are easily modified via other reactions.21,6
Thus, Briggs and co-workers at Amersham prepared the par-ent hydroxy compounds as shown in Scheme 3. Some of thereactions used to derivatize these materials are also shown.In some ways the hydroxyl group of the hydroxy NileReds 5 and 6 is an inconvenience because the spectroscopicproperties of the dye under physiological conditions becomehighly pH dependent. However, this phenol is useful asa functional group to incorporate a handle for attachmentto biomolecules (Scheme 3d).
A series of fluorinated phenoxazines (not shown) and benzo-phenoxazines (Scheme 4) have been prepared via sequentialSNAr substitutions reactions of fluorinated aromatics.22 The6-fluoro substituent in the compound shown below changesits spectroscopic properties slightly (see below) and, pre-sumably its pH dependence.
The so-called ‘FLAsH dyes’ feature bisarsenic(3+)-baseddye precursors that are non-fluorescent, presumably due torapid quenching of the excited state via intramolecularenergy transfer. However, the disposition of the arsenicdyes is such that they are thought to react with dicysteineunits engineered into modified proteins that are expressedwithin cells. This type of reaction has at least two effects,it: (i) alters the oxidation potential of the arsenic centersand (ii) restricts rotations about the C–As bonds. For what-ever reason, structural changes such as this render the dye-protein complex fluorescent. Only proteins within the cellthat have the special arrangement of Cys-residues are likelyto become labeled in this way, so the approach allows forhighly selective visualization of the engineered protein(Fig. 3).
The original FLAsH dyes were fluorescein derivatives.23 Arange of bisarsenic derivatives on different dye skeletons
11024 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
a
Et2N OH
NO
HO
OH
DMF, reflux4 h
O
N
O
HO
Et2N5
70 %MeOH
max abs 565 nm
max emiss 636 nm
b
OH
DMF, reflux
5 h
OH
Et2N OH
NO
O
N
OEt2N6
65 %MeOH
max abs 551 nm
max emiss 632 nm
OH
c
OH
NO
NEt
OH
HO
DMF, reflux
5 h
HO3S7
79 %MeOH
max abs 545 nm
max emiss 618 nm
O
N
O
OH
NEt
HO3S
d
O
N
OEt2N
OH
O
N
OEt2N
O
O
N
OEt2N
O(i) Br(CH2)5CO2Bn
K2CO3, MeOHreflux 48 h
(ii) PLENH4 H2PO4
37 oC7 d60 %
62 %
( )4 cat. DMAPadipic anhydride
CH2Cl2, 25 °C, 12 h
HO2CO HO2C( )4
6
Scheme 3. (a) Synthesis of 1-hydroxy Nile Red; (b) synthesis of 2-hydroxy Nile Red; and (c) some derivatization reactions of 1-hydroxy Nile Red.
has been prepared for evaluation, but only fluorescein andNile Red derivatives have emerged as useful. This is becauseseveral parameters must be controlled tightly if this type ofexperiment will work. For instance, the As-to-As distancemust match the disposition of the target thiols, the ethane-dithiol (EDT) concentration in the cell is critical, and thedye must permeate into the cell.
The FLAsH Nile Red derivative 8 was prepared as indicatedin Scheme 5. This involves a standard condensation reactionto form the benzophenoxazine core, but without the N,N-di-ethyl substituents of Nile Red. These were omitted to allowmore space for manipulations at the 6- and 8-positions.FLAsH dye 8 gives less fluorescent enhancement on bindingthan similar fluorescein dyes, but it emits at a longer wave-length (604 nm) and that, as mentioned before, is a moretransparent region of the spectrum for intracellular imaging.Calcium-induced conformational changes of appropriatelymodified, intracellular calmodulin have been followed usingFLAsH dye 8.24
Scheme 6 describes syntheses of two more classical thiol-selective dyes, ones that rely on SN2 displacement of iodidefrom iodoacetyl groups.25 Thus probes 9 and 10 were pre-pared from a Nile Red derivative and from a 2-hydroxyNile Red compound, respectively. Curiously, when thesewere complexed to a particular Cys-residue in maltose bind-ing protein, dye 9 showed a three-fold enhancement of fluo-rescence, while the emission from 10 was reduced bya factor of five. The authors explain this by proposing that10 is less constrained when bound to protein.
Two interesting questions arise from the work on Nile Redderivatives that is described above. First, would water-solu-ble derivatives of Nile Red have high quantum yields inaqueous media? As already stated, most Nile Red deriva-tives do not fluoresce strongly in polar media, but this couldbe due to aggregation effects that might be avoided if thedye has some intrinsic water solubility. Second, do water-soluble Nile Red derivatives also show the pronouncedbathochromic shift in polar media, that is, observed for
11025J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
compounds in the series with little or no significant aqueoussolubility?
No significantly water-soluble Nile Red derivatives havebeen prepared to answer the questions posed above until re-cent work18 from our laboratories. Classical condensationroutes were used to prepare the three Nile Red derivatives11–13 (Scheme 7). These were designed to be water soluble,but, surprisingly, the diol/phenol 11 was not, even in aqueousbase. The other two compounds do have very good water sol-ubilities. Their spectroscopic properties are discussed in thenext sub-section; but the data shown in Table 3 show thatthe answers to both these questions were affirmative forcompounds 12 and 13.
Some researchers may be interested in access to moresophisticated analogs of Nile Red. One obvious approach
protein surfaces
"dye"S
AsS
SAs
SSH HS
SH HSnon-fluorescent
protein surfaces
S S
S S
dye AsAs
604 nmfor dye 8
Figure 3. Conceptual basis of fluorescent arsenic dyes.
FF
FF
Et2N OH+
NaOH, pyridine100 °C, 6 h
Et2NF
O
FF
(i) NaNO2, H2SO440 °C, 1 h
(ii) H2O, O2, 65 °C, 1 h
48 %
O
N+
OEt2N
O-
F
HCl, Zn dustC6H6, 25 °C, 3 h
23 %
EtOHmax abs
561 nm
7
43 %
O
N
OEt2NF
λ
Scheme 4. Preparation of 6-fluoro Nile Red.
would be to prepare iodo-, bromo-, or chloro-substitutedcompounds then elaborate them via organometallic cou-plings. There have been reports of attempted halogenationof Nile Red, but the regioselectivity and extent of the halo-genations proved hard to control and mixtures were pro-duced.26–283.3. Spectroscopic properties
Table 1 summarizes spectroscopic data for Nile Red in dif-ferent solvents, all taken from the same source.4 Thesedata show decreased fluorescence intensity of Nile Red cor-relates much more with hydrogen bonding than with solventpolarity, and the magnitude of this difference is best appre-ciated from the graphical presentation of only the emissionwavelengths and intensities that is given in Figure 4. How-ever, the bathochromic shift seems to be a function of solventpolarity, consistent with stabilization of relatively polarexcited states.
A similar study featuring emission and absorption maxima,quantum yields, and solvent polarities was performed for
NH2
OHO2N
O
OHO
80 % AcOH100 °C, 12 h
O
N
OO2N
8 %
H2, Pd/CMeOH O
N
OH2N
46 %
Hg(OAc)2
AcOH, 50 °C O
N
OH2NHgOAc HgOAc
intermediate used
as crude material
(i) AsCl3, Pd(OAc)2, DIEANMP, 25 °C, 12 h
(ii) EDT, acetone, 25 °C, 12 hO
N
OH2NAs As
8
7 %
S SS S
Scheme 5. Preparation of the FLAsH system 8.
Table 1. Solvent dependency of emission intensities and wavelengths forNile Red 2
Solvent lmax abs
(nm)lmax emiss
(nm)Relative fluorescenceintensity
Water 591 657 18EtOH 559 629 355Acetone 536 608 687CHCl3 543 595 748iso-Amyl acetate 517 584 690Xylene 523 565 685n-Dodecane 492 531 739n-Heptane 484 529 585
11026 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
2-hydroxy Nile Red 6 (Table 2). Selected data from thisstudy are shown in Figure 5. Just as with Nile Red, polarhydrogen-bonding solvents correlate with reduced quantumyields and significant bathochromic shifts.
Fluorescence lifetimes contribute to two important physicalparameters of fluorescent dyes. Dyes with long fluorescentlifetimes can emit strongly because non-radiative processesare relatively slow, and because intersystem crossing to trip-let states is less competitive.
Fluorescence lifetimes for Nile Red in different solventshave been measured (Table 3). These data show that thefluorescence lifetime of Nile Red is 3.65 ns (EtOH)29 in com-parison to fluorescein (4.25 ns, EtOH)30 and tetramethyl-rhodamine (i.e., rhodamine 6-G, 3.99 ns, EtOH).30 Thefluorescent lifetime of Nile Red does not seem to vary muchwith solvent polarity, but it is extremely sensitive to H-bond-ing. In hydrogen-bonding solvents the fluorescence lifetimeof Nile Red decreases dramatically.
As stated above, the bias of this review is to highlight poten-tial applications in biotechnology, especially for intracellu-lar imaging. There are two common approaches to longwavelength dyes for imaging in tissues. The first, and mostobvious, is to prepare analogs of known dyes with extendedconjugated systems. Secondly, two-photon absorption canbe used,31 in which two long wavelength photons absorbedby a molecule promote it to an excited state that then emits
0
100
200
300
400
500
600
700
800
500 600 700wavelength of emission (nm)
re
la
tiv
e in
te
ns
ity
water
EtOH
n-heptane
xylene
iso-amylacetate
acetonen-dodecane
CHCl3
Figure 4. Solvent dependency of emission intensities and wavelengths forNile Red 2.
Table 2. Solvent dependency of quantum yield and wavelengths for2-hydroxy Nile Red 6
Solvent lmax abs (nm) lmax emiss (nm) Quantumyield (F)
Cyclohexane 514 528 0.51Dibutyl ether 514 555 0.66Toluene 525 568 0.76Acetone 528 608 0.78EtOH 544 634 0.581,2-Ethanediol 584 655 0.40CF3CH2OH 587 653 0.24(CF3)2CHOH 612 670 0.09
a single photon of higher energy. This is a way to excite in-tracellular or tissue samples at a wavelength that is moretransparent to these media. The dependence of two-photonabsorption on the intensity of laser beam allows for high spa-tial selectivity by focusing the laser beam on the target celland thus preventing any damage to adjacent cells. Relativelyfew dyes are suitable for practical experiments usingtwo-photon excitation because most do not absorb twolong wavelength photons efficiently, i.e., they have poortwo-photon cross-sections. Unfortunately, Nile Red has arelatively poor two-photon cross-section.31
One issue with two-photon excitation experiments is that theemitted light is of a short wavelength compared to the exci-tation source, and this might not be in a convenient region topermeate out of cells of other tissues, and for detection. Onestrategy to circumvent this problem is to arrange a fluores-cence energy transfer system (FRET) featuring a donorwith a large two-photon cross-section and an acceptor with amore convenient, longer wavelength, and emission maxima.2-Hydroxy Nile Red derivatives are being used in suchsystems. Thus a series of compounds (of which 14 and 15are two of the simplest; Fig. 6) have been prepared towardthis end; and donor-to-acceptor energy transfer efficienciesof more than 70% were observed (Fig. 5).32,33 Thesecompounds presumably do not have the solubility and sizecharacteristics that render them suitable for a range of
EtOH
acetone
(CF3)2CHOH
CF3CH2OH
1,2-ethanediol
toluene
cyclohexane
dibutylether
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
500 600 700wavelength of emission (nm)
qu
an
tu
m y
ie
ld
Figure 5. Solvent dependency of quantum yield and wavelengths for2-hydroxy Nile Red 6.
Table 3. Spectroscopic properties of water-soluble Nile Red derivatives11–13 in different solvents
Dye lmax abs
(nm)lmax emiss
(nm)Quantumyield (F)
Solvent
11 542 631 0.56 EtOH12 520 632 0.43 EtOH12 560 648 0.33 Phosa 7.412 556 647 0.07 Borb 9.013 548 632 0.42 EtOH13 558 652 0.37 Phosa 7.413 556 650 0.18 Borb 9.0
a pH 7.4 Phosphate buffer.b pH 9.0 Borate buffer.
11027J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
applications in biotechnology, so further developments inthis area would be opportune.
Finally, there is the possibility that the fluorescence of NileRed is somewhat attenuated if the excited state of the benzo-phenoxazinone core is reduced via electron transfer from the9-amino substituent. We mention this because the possibilityhas been explored via AM1 calculations.34 These gauge thedegree of charge transfer in a planar state relative to a twistedone in which the amine lone pair is not disposed to electrondonation to the heterocycle. The degree of electron transferfrom the 9-amino substituent will be dependent on the con-formation and on the reduction potential of the heterocyclein its excited state. If intramolecular charge transfer wasa major pathway for quenching the fluorescence of NileRed derivatives, it would be expected that compound 16
a
N
HO
N
HO NONaNO2/HCl
40 %
OHHO
EtOH, reflux, 4 h O
N
ON
50 %
DMAPpyridine, 2 h
HO
IO
O
2
O
N
ONO
OI
9
25 %
b
O
N
OEt2N
OH
NphthBr
K2CO3, DMFreflux, 4.5 h
O
N
OEt2N
O
85 %
(i) MeNH2, MeOHreflux, 2.5 h
IO
O
2
Nphth
(ii)
CH2Cl2, 40 min
O
N
OEt2N
O
10
61 %
OI
HN
5 °C, 2 h
Scheme 6. Two thiol-selective dye electrophiles: (a) a Nile Red derivative;(b) a 2-hydroxy Nile Red derivative.
would be less fluorescent than Nile Red itself; this dye itselfhas not been prepared.
O
N
ON
16
more or less fluorescent than Nile Red?
OHO3S
4. Nile Blue
4.1. Introduction
Nile Blue has a positively charged, oxidized, phenoxazinesystem, i.e., it is a phenoxazinium; the conspicuous differ-ence between this and Nile Red 2 is that the latter is neutral.Both dyes have a 9-diethylamino substituent to donate elec-tron density across the ring, but Nile Red 2 and Blue 3 havedifferent electron acceptors, a carbonyl and an iminiumgroup, respectively (Fig. 7).
One obvious consequence of the difference in charges forNile Red 2 and Blue 3 is that water-solubility of Nile Blueis significantly better. Like its Red cousin, the fluorescencemaxima and intensity of Nile Blue are good indicators ofthe polarity of the dye’s environment. This solvatochromiceffect gives a red-shift in polar media, as would be expectedfor stabilization of a more charged excited state. The inten-sity of the fluorescence of 3 in water is about 0.01; this isa small value but, in comparison with the lack of fluores-cence of Nile Red in aqueous media, this is significant.
4.2. Syntheses
The original synthesis of Nile Blue2 involved condensationof 5-amino-2-nitrosophenol with 1-aminonaphthalene inacetic acid, but the yield was only 3%. However, use ofperchloric acid in ethanol gives a significantly higher yield(reaction 2).35
NO
OHEt2N
NH2
70 % HClO4
EtOH, reflux, 5 min
O
N
N+H2Et2NClO4
-
3
31 %
ð2Þ
De novo syntheses of Nile Blue derivatives involve use ofsimilar, but substituted starting materials. Scheme 8 de-scribes syntheses of dyes 17–20 that incorporate 8-hydroxyjulolidine.35 This amine is a ‘privileged fragment’ in dyesyntheses because it holds the nitrogen lone pair in
11028 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
N OHHOOC
COOH
H2N OHCOOH
H2O, 70 °C, 3 h
H2O, 70 °C, 12 h
80 %
N OHMeO2C
CO2Me
MeOH, H+
reflux, 24 hLiAlH4,THF
25 °C, 2 h
63 %
NaNO2, HCl
0-5 °C, 2.5 h
0-5 °C, 2.5 h
N OH
HO
N OH
HO
NO
90 % 74 %
OH OHOH
HODMF, 130 °C, 5 h
130 °C, 12 h
O
N
ON
OH
HO
OH
11
54 %
b
a
N OH OH
NONaNO2, HCl
CO2Me CO2Me
MeO2C
N
MeO2C
74 %
O
N
O
OHOH
HOMeOH, H+, reflux, 5 h
CO2Me 53 %
N
MeO2C
K2CO3, MeOH/H2O
12 h, 40 °C O
N
O
OH
CO2H12
60 %
N
HO2C
c
HN OH
COOH
H2N OHCOOH DMF
OS
O
O
40 %
N OHNaNO2, HCl
0 - 5 °C, 2.5 h
COOH
48 % 70 %
SO3H
OH
COOH
N
SO3HNO
O
N
O
OHOH
HODMF, reflux, 5 h
COOH13
53 %
N
SO3H
EtOH; Φ 0.56max abs
542 nmmax emiss
631 nmλ
λ
EtOH; Φ 0.43max abs
520 nmmax emiss
632 nmλ
λ
pH 7.4 buffer; Φ 0.33max abs
560 nmmax emiss
648 nmλ
λ
EtOH; Φ 0.42max abs
548 nmmax emiss
632 nmλ
λ
pH 7.4 buffer; Φ 0.37max abs
558 nmmax emiss
652 nmλ
λ
Scheme 7. Synthesis of 2-hydroxy Nile Red derivatives featuring the following functionalities: (a) a diol; (b) a dicarboxylic acid; and (c) a carboxylic acidsulfonic acid combination.
11029J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
a
N
NN
OON
N
OHD
1
N
NN
OON
N
O
N
O OEt2N
14
D1 part
donor
acceptor
FRET
two photons in at 815 nm
two photons in at 810 nm
b
N
OH
N
S
D2 O
OO
O
N
O OEt2N
15
NO
N S
N O
NS
donor
acceptor
FRET
one photon
out at 595 nm
one photon
out at 595 nm
D2 part
CHCl3λmax abs 400 nm
λmax emiss 510 nm
CHCl3λmax abs 405 nm
λmax emiss 510 nm
THFλmax abs 390 nm, 540 nm
λmax emiss 595 nm
THFλmax abs 405 nm, 540 nm
λmax emiss 595 nm
Figure 6. Two-photon-donor acceptor FRET cassettes: (a) D1 is a two-photon donor used to make cassette 14; (b) cassette 15 is made from D2.
conjugation with the aromatic rings. This alters the reactivityof the starting material; in fact, nitrosylated 8-hydroxy-julolidine was insufficiently reactive in this synthesis soHartmann and co-workers modified the synthon to includethe reactive azo-functionality as shown. Unfortunately, thejulolidine fragment is quite hydrophobic too, so the productdyes have very limited solubility in aqueous systems.
An early synthesis of Nile Red featured hydrolysis of a NileBlue derivative (see Scheme 2b). This implies that Nile Bluederivatives have finite hydrolytic stability, and this could bea drawback in some situations. Dyes 17–20 are much lessvulnerable to this mode of decomposition because of theirfused cyclic structures that hold the amines in place.
Nile Blue derivatives with enhanced water-solubilities and/or groups for bioconjugation can be made from amineswith appropriate N-substituents. For instance, Scheme 9shows preparations of dyes 21,36,37 22/23,38,39 and 24/2540
in which sulfonic acid groups enhance water solubilitiesand carboxylic acid groups could potentially be activatedand reacted with amines on biological molecules. The syn-theses of 24 and 25 are shorter and higher yielding than othersyntheses of water-soluble Nile Blue derivatives.
Compounds 22 and 23 are the ‘EVO Blue’ dyes.38,39 Thesehave been claimed to be more stable than rhodamine andBODIPY dyes under acidic conditions. This enhanced acidstability was suggested to be useful in the context of high
11030 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
throughput screening and solid phase syntheses that involvecleavage from resins by acids.
Other approaches to water-soluble Nile Blue derivatives haveinvolved modification of the parent dye after the hetero-cyclic framework was assembled. For instance, Scheme 10ashows a patent procedure for alkylation of the 5-amino/iminium substituent; we note that chlorosulfonic acid is anunusual choice for the ester hydrolysis reaction. In the secondexample, Scheme 10b, an amino anthracene was used to pre-pare a derivative of Nile Blue that has an extended aromaticsystem. This modification gave a probe with absorbance andfluorescence emission shifted to the red. Unfortunately, thematerial 27 was probably not a pure compound since the de-gree of sulfonation, the regiochemistry, and the chemical/quantum yields were not given.41
Direct iodination of Nile Blue is possible, and the 6-iodo de-rivative 28 can be prepared from this (Scheme 11). However,chromatography was required, the yield of the iodinatedproduct was low, and slight variation of the conditions canlead to formation of other regioisomers. Similar consider-ations apply to the corresponding bromination reaction, ex-cept the product yield was better. Conversely, the de novosynthesis of the 2-iodo derivative 30 is unambiguous withrespect to the regioisomer formed, but column chromato-graphy is still required and the yield is also low. The diiodide31 can be formed by a second iodination of the 2-iodide.These halogenated derivatives are potentially useful forforming more sophisticated derivatives, but they wereoriginally prepared as possible photosensitizers to initiateprocesses that are destructive to carcinogenic cells.42,43
carbonyl
electron
acceptor
neutral molecule
O
N
ON
Nile Red 2
12
6
3
4
8
1110
O
N
O-N+
O
N
NH2N
Nile Blue 3
12
6
3
4
8
1110
O
N
NH2N+
iminium
electron
acceptor
charged molecule
increasing polarity
+
MeOHλ
max abs 554 nm
λmax emiss
638 nmΦ 0.38
n-heptaneλ
max abs 484 nm
λmax emiss
529 nmΦ 0.48
MeOHλ
max abs 626 nm
λmax emiss
668 nmΦ 0.27
H2Oλ
max abs 635 nm
λmax emiss
674 nmΦ 0.01
n-hexaneλ
max abs 473 nm
λmax emiss
546 nm
Figure 7. Structures of Nile-Red and Blue contrasted.
These photosensitizers promoted to the excited singlet statedecays to the triplet state and generates singlet oxygen,which is considered to be responsible for its potent activitytoward cancer cells.
4.3. Spectroscopic properties
Like Nile Red 2, Nile Blue shows progressively longerabsorption and emission maxima as the solvent polarity isincreased. However, the Stokes’ shifts observed for the redprobe 2 in different solvents is far greater; this parameterfor the Blue compound can still be exceptionally high(almost 100 nm), but in polar solvents it is reduced to around40 nm (Table 4).44
Table 5. Study of effect of different anions on spectral properties of NileBlue in different solvents
Anion Solvent lmax abs (nm) lmax emiss (nm)
Chloride EtOH 628 689
O
OO632 —
O
MeO OH
634 694
Acetate EtOH 628 689
O
OO632 687
O
MeO OH
634 700
Benzoate EtOH 628 691
O
OO632 690
O
MeO OH
634 702
iso-Butanoate EtOH 628 691
Hydroxide EtOH 515 661
Table 4. UVabsorption and fluorescence emission maxima of Nile Blue 3 indifferent solvents
Solvent lmax abs (nm) lmax emiss (nm)
Toluene 493 5744-Chlorobenzene 503 576Acetone 499 596DMF 504 598CHCl3 624 6471-Butanol 627 6642-Propanol 627 665EtOH 628 667MeOH 626 668Water 635 6741.0 N HCl, pH 1.0 457 5560.1 N NaOH, pH 11.0 522 668NH4OH, pH 13.0 524 668
11031J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
a
Ar = 4-ClC6H4
OHN OHN
N
MeOH, 0 °C, 30 min
MeOH, 0 °C, 30 min
MeOH, 0 °C, 30 min
MeOH, 0 °C, 30 min
8-hydroxyjulolidine
NAr
ClN2Ar
cat.HClO4, DMFreflux, 5 min
N O
N
N+ClO4
-
17
57 %CH2Cl2; Φ 1.0max abs
671 nmmax emiss
688 nm
N OH
b
NH NH
Ar
Ar = 4-ClC6H4
ClN2Ar
cat.HClO4, DMFreflux, 5 min N O
N
N+HClO4
-
18
10 %
N OH
c
Ar = 4-ClC6H4
NH NH
NN
Ar
ClN2Ar
cat.HClO4, DMFreflux, 5 min N O
N
N+HClO4
-
19
10 %
N OH
d
Ar = 4-ClC6H4
Ar
NHSO2
NHSO2
NClN2Ar N
N O
N
ClO4-
N+HSO2
20
1 %
cat.HClO4, DMFreflux, 5 min
N OH
λλ
CH2Cl2; Φ 0.35max abs
675 nmmax emiss
712 nmλ
λ
CH2Cl2; Φ 0.42max abs
668 nmmax emiss
677 nmλ
λ
CH2Cl2; Φ 0.42max abs
668 nmmax emiss
677 nmλ
λ
Scheme 8. Syntheses of Nile Blue derivatives from 8-hydroxyjulolidine.
Table 4 also reveals that the spectroscopic characteristics ofNile Blue are pH dependent. This is because under basicconditions the iminium group will be deprotonated, whereasunder strongly acidic conditions the 5-amino might even be-come protonated.45 Curiously, the spectroscopic propertiesof Nile Blue are somewhat dependent on the counter ionused.46 We speculate that this could even be a reflection onintimate ion pairing influencing the solvent sphere of thedye (Table 5).
The fluorescence lifetime of Nile Blue 3 in ethanolhas been measured at 1.42 ns. This is shorter than the
corresponding value of Nile Red (see above; 3.65 ns).The lifetime of Nile Blue is relatively invariant at diluteconcentrations (10�3–10�8 mol dm�3) but changes as theconcentration is increased, and in different solvents.This is probably an indication of the interdependenceof the spectroscopic properties and the degree of aggre-gation in solution. In support of this assertion, we notethat the lifetimes do not seem to be significantly im-pacted by the viscosity of the medium, but they aretemperature dependent.47 As far as we are aware, thetwo-photon cross-section of Nile Blue has not beenreported.
11032 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
a
NR2
R1OH
NOR
NH
R3O
O
H+/ MeOH or EtOH
reflux O
N
NNR2
R1 H
R3O
O
R
R1, R2 = H, linear alkylR = H, Et, R3 = H, Me, Et
21
7-90 %
b
OHEt2N
NO SO3-
NH
CH3COOHreflux, 30 min
COOH
O
N
N+HEt2N
SO3-
COOH22
EVO blue 90
c
OHN
O O
NH
ON
(i) CH3COOH, reflux 30 min
(ii) cat. HClH2O/acetone
reflux, 8 h
SO3-
O
N
NH+N
O OH SO3-
23
EVO blue 100
10 %
d
NH2
SO3H
(ii)
DMF, HClO4155-160 °C, 15 min
OHEt2N
(i) ArN2+Cl-
MeOH, 25 °C, 30 min
O
N
N+H2
SO3H
Et2N
24
55 %
ClO4-
e
OHH2N
SO O
OOHNDMF, 130 °C
2 h
77 %HO3S
-(i) ArN2+Cl
MeOH, 25 °C, 30 minN O
N
N+H2
25
64 %
ClO4-
SO3HSO3H
HO3S
(ii)
NH2
DMF, HClO4155-160 °C, 15 min
EtOH
max emiss 640 +/- 30 nm
max abs 570 +/- 60 nmλ
λ
pH 7.4 buffer
max emiss 638 +/- 12 nm
max abs 570 +/- 15 nmλ
λ
pH 7.4 buffer; Φ 0.03max abs
637 nmmax emiss
677 nmλ
λ
pH = 7.4 buffermax abs
634 nmmax emiss
674 nmλ
λ
pH = 7.4 buffermax abs
649 nmmax emiss
673 nmλ
λ
pH 7.4 buffer; Φ 0.10max abs
633 nmmax emiss
675 nmλ
λ
Scheme 9. Syntheses of Nile Blue derivatives with water solubilizing N-substituents.
11033J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
a
Et2N O
N
N+H2
NaOH, H2O
65 °C, 30 minCl-
Et2N O
N
NHK2CO3, toluene
reflux, 18 h
BrO
O ( )5
71 %
Et2N O
N
N O
O
( )4
ClSO3H, Na2SO4
CH2Cl2, 70 °C, 22 h
CH2Cl2, 70 °C, 22 h
Et2N O
N
N OH
O
( )4
26
90 %
b
Me2N OH
NO (i) EtOH, HClreflux, 18 h
(ii) NaOH, H2O
NH2 O
N
NHMe2N
K2CO3, toluenereflux, 18 h
BrO
O ( )5
O
N
NMe2N O
O
( )4
ClSO3H, Na2SO4
O
N
NMe2N
SO3H
SO3H
OH
O
( )4
27
pH = 7.4 buffermax abs
643 nmmax emiss
680 nmλ
λ
pH = 7.4 buffermax abs
650 nmmax emiss
698 nmλ
λ
Scheme 10. Preparation of water-soluble Nile Blue derivatives: (a) with only carboxylic side chain; (b) with carboxylic acid side chain and two additionalsulfonic acid groups.
5. Other benzophenoxazine dyes
5.1. Introduction
There is no obvious reason why benzo[a]phenoxazinesshould be more fluorescent than similar compounds withdifferent ring fusion patterns. Our interpretation of the liter-ature is that other phenoxazines with appropriate substitu-ents have certainly been less well studied as fluorescenceprobes, and are probably less synthetically accessible.
5.2. Syntheses
The benzo[c]phenoxazine 32 has been prepared via a routethat is similar to those used for the Nile compounds 2 and3. 1-Naphthol is used in this synthesis (reaction 3)46 ratherthan 2-naphthol, the isomer used for 2 and 3. This changeis necessary to obtain the different ring fusion, but it alsomay account for the very poor yield. This is because of thewell-known reduced reactivity for 1-napthol at the 2-posi-tion in electrophilic substitution reactions, relative to thegreater tendency for 2-napthol to react at the 1-position. Tothe best of our knowledge, the fluorescent properties of 32have not been reported in any depth; indeed, the moleculelacks an electron donor in conjugation with the carbonyl togive it the type of extended oscillating dipole that seems tobe common for fluorescent molecules.
Benzo[b]phenoxazines are linear. Substituted derivativesof these are numbered according to the system shownbelow.
O
HN 1 2
34
5689
1011
The linear system 33 has been prepared via the high temper-ature condensation process shown in reaction 4.48 Thishas absorption and fluorescence emission properties thatare characteristic of an extended aromatic heterocycle.
OH
ON
OH
ZnCl2, CH3COOH 25 °C, 24 h
O
N
O
32
2-3 %EtOH
max abs 505 nm
ð3Þ
11034 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
Like 32, this compound does not have substituents thatwould allow it to be reduced to a phenoxazinone or phenox-azinium form.
Some nitro and amino derivatives of benzo[b]phenoxazineshave been reported in literature, and Scheme 12 showsthem.49 Parts a and b show nitrosylation/oxidation reactionsthat can be used to prepare nitro-substituted derivatives 35and 36. Predictably, these are not particularly fluorescentcompounds. However, the 1,9-diaminobenzo[b]phenoxazi-nium 37 has the potential to be strongly fluorescent. Unfor-tunately, it was neutralized to 38 then the fluorescenceproperties were recorded.
6. Conclusions
Current knowledge of fluorescent benzophenoxazine-derived probes is based almost on the benzo[a]phenoxazinering fusion series. Almost all the syntheses feature hightemperature condensation methods, and not contemporarysynthetic methods like, for example, Buchwald–Hartwigcouplings to introduce amine substituents. In fact, many ofthe synthetic methods reported are based on the proceduresthat are now over a century old. This, and the prevalenceof patent literature in this area, mean that many of the experi-mental procedures presented are difficult to follow, andcomplete spectroscopic data is rarely recorded.
Nile Red and its derivatives have some interesting spectro-scopic properties (long wavelength emissions, large Stokes’shifts) but most compounds in this series have limited watersolubilities. There are, however, some recent efforts to makemodified compounds to redress this. Nile Blue and its deriv-atives tend to be more water soluble.
Relatively little work has been done to modify benzo-phenoxazine dyes so that they emit even further to the red:the longest wavelength emission in the existing probes isabout 700 nm. To this end, it would be useful to have accessto more functionalized compounds that can be preparedeasily on a gram scale, like 2-hydroxy Nile Red 6. Othermodifications might be used to give derivatives with en-hanced extinction coefficients (these tend to be 10,000 orless), or improved two-photon cross-sections. These typesof developments would be facilitated by more detailed stud-ies of fundamental reactions that can be used to modify thesecompounds, e.g., halogenation and nitration.
OH
NH2 HO
HO
220 °CCO2 atmosphere
O
HN
33
55% EtOH; 0.24max abs
365 nmmax emiss
430 nm
toluene; 0.28max abs
361 nmmax emiss
420 nm
ð4Þ
a
Et2N O
N
N+H2
NIS, CH3COOHCF3CH2OH, 90 °C, 80 min
Et2N O
N
N+H2I
CH3COO-
28
22 %MeOH
b
Et2N O
N
N+H2
Br2, CH3COOHCF3CH2OH, 25 °C, 20 min
CF3CH2OH, 25 °C, 30 min
Cl-
Et2N O
N
N+H2Br
Cl-
29
48 %MeOH
c
NO
OHEt2N NH2
I
CH3COOH, cat. HCl120 °C, 2.5 h
Et2N O
N
N+H2
I
Cl-
30
11 %MeOH
d
Et2N O
N
N+H2
I
3NIS, CH COOH
Cl-
Et2N O
N
N+H2
I
Cl-
I31
38 %MeOH
max abs 642 nmλ
max abs 643 nmλ
max abs 633 nmλ
max abs 650 nmλ
Scheme 11. Preparation of halogenated Nile Blue derivatives: (a) 6-iodo;(b) 6-bromo; (c) 2-iodo; and (d) 2,6-diiodo.
11035J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
As far as we can see, there is no good reason why virtually allfluorescent dyes in this series are benzo[a]phenoxazine de-rivatives, and not any other ring fusion. This appears to bemerely a question of synthetic availability.
Overall, benzophenoxazine-based probes are an intriguingsubset of the fluorescent dye toolbox. They have some obvi-ous drawbacks for applications in biotechnology, but the de-velopments that need to be made to make more useful labelsof this type are reasonably well defined.
a
O
HN NaNO2, CH3COOH
DMF, 70 °C, 10 min.
33
O
HN
34
34 %
O2N
b
O
HN NaNO2, CH3COOH
25 °C, 3 h
33
O
HN
O2N
NO2
O
HN
O2N
N
O
35
30 %36
9 %
c
SnCl2, HCl
EtOH, reflux, 5 hO
HN
O2N
NO2
35
O
N
H2+N
NH2
37
51 %
KOH, EtOHCl-
O
N
HN
NH2
38
83 %
toluene; Φ 0.05max abs
470 nmmax emiss
500-610 nm λ
λ
EtOH; Φ 0.05max abs
570 nmmax emiss
760-790 nm λ
λ
toluene; Φ 0.05max abs
438 nmmax emiss
500-530 nm λ
λ
toluene; Φ 0.30max abs
412 nmmax emiss
515 nmλ
λ
EtOHmax abs
436 nmλmax emiss
absent λ
Scheme 12. Syntheses of benzo[b]phenoxazine derivatives: (a) 9-nitro;(b) 1,9-dinitro and 9-nitro-1,12-bis(benzo[b]phenoxazine); and (c) 1-amino-9-iminobenzo[b]phenoxazine.
Acknowledgements
Support for this work was provided by The National Insti-tutes of Health (HG 01745 and GM72041) and by TheRobert A. Welch Foundation.
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11037J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037
Biographical sketch
Jiney Jose was born in Kerala, India. He received his B.Tech in Chemistry
(2002) from U.I.C.T., Mumbai, India. He joined the group of Prof. Kevin
Burgess at Texas A & M University, Department of Chemistry in 2003.
His research is focused on syntheses, derivatization and photophysical
studies of water-soluble benzophenoxazine dyes.
Kevin Burgess is a professor at Texas A & M University, where he has been
since September 1992. His research interests focuses on peptidomimetics
for mimicking or disrupting protein–protein interactions via combinatorial
chemistry and via asymmetric catalysis, and fluorescent dyes for multiplex-
ing in intracellular imaging of protein–protein interactions. Motivation to
write this review came from a need for water-soluble Nile Red derivatives
in the design and syntheses of through-bond energy transfer cassettes for
use in the imaging project.