tetrahedron report number 775 benzophenoxazine … · tetrahedron report number 775...

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.

mailto:[email protected]

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


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


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.26–28
3.3. 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


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.


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


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


520 nmmax emiss

632 nmλ

λ

pH 7.4 buffer; Φ 0.33max abs

560 nmmax emiss

648 nmλ

λ


548 nmmax emiss

632 nmλ

λ


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.


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


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


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


668 nmmax emiss

677 nmλ

λ


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.


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 abs 570 +/- 60 nmλ

λ

pH 7.4 buffer


max abs 570 +/- 15 nmλ

λ


637 nmmax emiss

677 nmλ

λ

pH = 7.4 buffermax abs

634 nmmax emiss

674 nmλ

λ


649 nmmax emiss

673 nmλ

λ


633 nmmax emiss

675 nmλ

λ

Scheme 9. Syntheses of Nile Blue derivatives with water solubilizing N-substituents.


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


643 nmmax emiss

680 nmλ

λ


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


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.


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


570 nmmax emiss

760-790 nm λ

λ


438 nmmax emiss

500-530 nm λ

λ


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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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.

tetrahedron report number 775 benzophenoxazine … · tetrahedron report number 775...

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