Benzophenoxazine-Based Fluorescent Dyes for Labeling Biomolecules

Tetrahedron 62 (2006) 11021–11037

Tetrahedron report number 775 Benzophenoxazine-based ﬂuorescent dyes for labeling biomolecules

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 ...... 11021 2. Meldola’s Blue ...... 11022 2.1. Syntheses ...... 11022 2.2. Photophysical properties ...... 11022 3. Nile Red derivatives ...... 11022 3.1. Introduction ...... 11022 3.2. Syntheses ...... 11023 3.3. Spectroscopic properties ...... 11025 4. Nile Blue ...... 11027 4.1. Introduction ...... 11027 4.2. Syntheses ...... 11027 4.3. Spectroscopic properties ...... 11030 5. Other benzophenoxazine dyes ...... 11033 5.1. Introduction ...... 11033 5.2. Syntheses ...... 11033 6. Conclusions ...... 11034 Acknowledgements ...... 11035 References and notes ...... 11035 Biographical sketch ...... 11037

1. Introduction limitations for others. None of these dyes are significantly soluble in aqueous media, and their quantum yields are dra- Meldola’s Blue 1, Nile Red 2, and Nile Blue 3 have some de- matically reduced. One of the big challenges in the produc- sirable attributes as fluorescent probes.3 Dyes 2 and 3 have tion of fluorescent dyes is to produce water-soluble probes reasonably high fluorescence quantum yields in apolar sol- that fluoresce strongly in aqueous media, particularly above vents and they fluoresce at reasonably long wavelengths. 600 nm or at even longer wavelengths. Motivation for Nile Red, in particular, fluoresces far more strongly in apolar research in this area is drawn from needs for intracellular, media than in polar ones, and the fluorescent emission shows tissue, and whole organism imaging where near-IR dyes a large bathochromic (i.e., red-) shift in polar media, hence are far more conspicuous than ones emitting at 550 nm or it can be used as a probe for environment polarity;4–6 these less.7 characteristics may be attributes for some applications, but The phenoxazine skeleton may be extended by adding fused benzene rings to the a–c or h–j faces. Benzophenoxazines of 1 * Corresponding author. Tel.: +1 979 845 4345; fax: +1 979 845 1881; this type are ‘angular’ or ‘linear’ depending on the orienta- e-mail: [email protected] tion of the ring fusion, as illustrated in Figure 1a.

a H conditions and the yield were not given. Subsequently, Mel- j a N dola’s Blue has been made with a variety of counter ions via i b reactions involving different Lewis acids (Scheme 1b).15 O h c Counter ion modiﬁcations have been used to modulate the phenoxazine electronic properties of solid materials for applications not directly related to labeling biomolecules. linear angular 2 1 3 H 11 H H a N N 12 4 N 10 NO CH3COOH 9 5 O O ° 8 O 6 110 C 7 Me2N HO

benzo[b]phenoxazine benzo[a]phenoxazine benzo[c]phenoxazine N b Cl- + N Me2 N O Cl- + 1 Me2 N O EtOH 1 max abs 540 nm Meldola's Blue max emiss 568 nm

N N (i) NaNO2 / H2O Cl- EtOH/HCl, 0-3 °C, 1 h + Et2N O O Et2N O N H2 (ii) OH Me2N 2 3 Nile Red Nile Blue ZnCl , EtOH, 70 ° C Figure 1. (a) Phenoxazines and benzophenoxazines; (b) structures of the 2 three best known ﬂuorescent dyes in this class.

N Substituents that freely donate and/or accept electron den- Cl- + sity on benzophenoxazine cores can, in some orientations, Me2 N O give fluorescent compounds. The first notably fluorescent 1 compound to be discovered in this class was Meldola’s Meldola's Blue 2 2 Blue 1, but Nile Red 2 and Nile Blue 3 are far more fre- Scheme 1. (a) Original synthesis of Meldola’s Blue; (b) a more recent quently used in contemporary science. approach.

The objective of this review is to summarize the state of the 2.2. Photophysical properties art of benzophenoxazine dyes in such a way that all readers can understand quickly what has been done to develop useful Phenoxazines without strongly electron withdrawing or probes in this category. Inspired readers should also be able donating substituents have unexceptional absorbance char- to use this summary to design research approaches that are acteristics, and they are not particularly fluorescent com- not yet explored but would lead to useful endpoints. pounds. Fusion of a benzene ring onto the heterocycle does not alter this situation dramatically. Meldola’s Blue has one dimethylamino substituent and the heterocyclic 2. Meldola’s Blue core is oxidized. These changes in the composition of the heterocycle shift its absorption and emission characteristics This dye is used in textiles, paper, and paints, mainly as a pig- into a useful range: lmax abs 540 nm, lmax emiss 568 nm EtOH. ment. It is not a particularly useful fluorescent dye for label- This dye is not noted for its solvatochromic properties.17 ing proteins because of its poor water solubility. Further, its fluorescence is weak in all common media (e.g., EtOH) and does not give a clear indication of the surrounding polarity. 3. Nile Red derivatives However, Meldola’s Blue has been used as a component in redox sensors16 for detection of materials such as NADH,8 3.1. Introduction pyruvates,9 hydrogen peroxide,10 glucose,11 and 3-hydroxy- butyrate.12 It has also been used in electrochemical experi- Nile Red has a neutral oxidized phenoxazine system, i.e., it ments involving DNA wherein the dye mediates electron is a phenoxazinone. The 9-diethylamino substituent is able transport.13 to donate electron density into the carbonyl group across the ring; this electronic arrangement probably accounts for 2.1. Syntheses its highly fluorescent properties (Fig. 2).

The original (1879) synthesis of Meldola’s Blue involved The water-solubility of Nile Red is extremely poor, but in condensation of a nitroso compound with 2-naphthol at ele- other solvents its ﬂuorescence maxima and intensity are vated temperatures (Scheme 1a).14 Details of the reaction good indicators of the dye’s environment-polarity. As J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037 11023

2 a de novo approach was used to prepare the 6-carboxyethyl 1 3 11 derivatives 4 (reaction 1). These are potentially interesting N 4 N 10 since hydrolysis of the ester would give a carboxylic acid for attachment to biomolecules; however, this does not N O O N+ O O- 8 6 appear to have been attempted yet.19 The patent literature also describes a synthesis of the 2-carboxy Nile Red derivative.20 Nile Red 2 MeOH n-heptane max abs 554 nm max abs 484 nm max emiss 638 nm max emiss 529 nm 0.38 NO 0.48 ethanol, reflux R1 Figure 2. Electron delocalization in Nile Red. N HO OH 3 h 2 R COOC2H5 mentioned above, this solvatochromic effect is such that polar media cause a red-shift but decreased fluorescence N intensity. This decreased fluorescence intensity is probably R1 R1, R2 = H and simple linear alkyl due to self-quenching of the dye in face-to-face aggregates. N O O Consequently, this dye is particularly useful for studying R2 COOC H 2 5 OH , NO lipids and events that involve impregnation of the dye in 4 apolar media. Surprisingly, very few water-soluble analogs 24 - 60 % of Nile Red have been reported, and only limited fluores- MeOH max abs 545 +/- 25 nm cence data have been given for those. max emiss 620 +/- 5 nm ð1Þ 3.2. Syntheses

The first synthesis of Nile Red was a condensation reaction De novo syntheses of 1- and 2-hydroxy Nile Red, com- of a nitrosophenol (Scheme 2a). The patent literature indi- pounds 5 and 6, respectively, have also been performed, cates that other solvent systems can be used. It is also possi- and the products are easily modified via other reactions.21,6 ble to prepare Nile Red via hydrolysis of Nile Blue as Thus, Briggs and co-workers at Amersham prepared the par- indicated in Scheme 2b. The product is usually isolated via ent hydroxy compounds as shown in Scheme 3. Some of the chromatography on silica. reactions used to derivatize these materials are also shown. In some ways the hydroxyl group of the hydroxy Nile Reds 5 and 6 is an inconvenience because the spectroscopic a properties of the dye under physiological conditions become OH highly pH dependent. However, this phenol is useful as NO CH3COOH a functional group to incorporate a handle for attachment 70 °C, 1 h to biomolecules (Scheme 3d). Et2N OH A series of fluorinated phenoxazines (not shown) and benzophenoxazines (Scheme 4) have been prepared via sequential N 22 SNAr substitutions reactions of fluorinated aromatics. The 6-fluoro substituent in the compound shown below changes Et N O O 2 its spectroscopic properties slightly (see below) and, pre- 2 10 % sumably its pH dependence.

b The so-called ‘FLAsH dyes’ feature bisarsenic(3+)-based dye precursors that are non-ﬂuorescent, presumably due to

N 0.5 % H2SO4 rapid quenching of the excited state via intramolecular energy transfer. However, the disposition of the arsenic + reflux, 2 h Et2N O N H2 dyes is such that they are thought to react with dicysteine Cl- 3 units engineered into modified proteins that are expressed within cells. This type of reaction has at least two effects, it: (i) alters the oxidation potential of the arsenic centers N and (ii) restricts rotations about the C–As bonds. For what- ever reason, structural changes such as this render the dye- Et2N O O protein complex fluorescent. Only proteins within the cell 2 22 % that have the special arrangement of Cys-residues are likely Scheme 2. (a) Original synthesis of Nile Red; (b) synthesis from Nile Blue. to become labeled in this way, so the approach allows for highly selective visualization of the engineered protein (Fig. 3). Substituted or modified Nile Red derivatives may be prepared via variations of the syntheses above, i.e., de novo The original FLAsH dyes were fluorescein derivatives.23 A methods, or by functionalizing Nile Red itself. For instance, range of bisarsenic derivatives on different dye skeletons 11024 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037

a HO HO

NO DMF, reflux N 4 h

Et2NOH OH Et2N O O 5 70 % MeOH max abs 565 nm max emiss 636 nm

b OH OH

NO N DMF, reflux 5 h Et2NOH OH Et2N O O 6 65 % MeOH max abs 551 nm max emiss 632 nm

c OH

OH NO N DMF, reflux Et 5 h Et N OH HO N O O

HO3S HO3S 7 79 % MeOH max abs 545 nm max emiss 618 nm

d O HO C 2 ( ) HO C OH 4 O 2 ( ) O 4 cat. DMAP (i) Br(CH2)5CO2Bn adipic anhydride K2CO3, MeOH N CH2Cl2, 25 °C, 12 h N reflux 48 h N (ii) PLE Et N O O NH H PO Et2N O O Et N O O 2 4 2 4 2 37 oC 6 62 % 7 d 60 %

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 and Scheme 6 describes syntheses of two more classical thiol- Nile Red derivatives have emerged as useful. This is because selective dyes, ones that rely on SN2 displacement of iodide several parameters must be controlled tightly if this type of from iodoacetyl groups.25 Thus probes 9 and 10 were pre- experiment will work. For instance, the As-to-As distance pared from a Nile Red derivative and from a 2-hydroxy must match the disposition of the target thiols, the ethane- Nile Red compound, respectively. Curiously, when these dithiol (EDT) concentration in the cell is critical, and the were complexed to a particular Cys-residue in maltose bind- dye must permeate into the cell. ing protein, dye 9 showed a three-fold enhancement of fluorescence, while the emission from 10 was reduced by The FLAsH Nile Red derivative 8 was prepared as indicated a factor of five. The authors explain this by proposing that in Scheme 5. This involves a standard condensation reaction 10 is less constrained when bound to protein. to form the benzophenoxazine core, but without the N,N-di- ethyl substituents of Nile Red. These were omitted to allow Two interesting questions arise from the work on Nile Red more space for manipulations at the 6- and 8-positions. derivatives that is described above. First, would water-solu- FLAsH dye 8 gives less fluorescent enhancement on binding ble derivatives of Nile Red have high quantum yields in than similar fluorescein dyes, but it emits at a longer wave- aqueous media? As already stated, most Nile Red deriva- length (604 nm) and that, as mentioned before, is a more tives do not fluoresce strongly in polar media, but this could transparent region of the spectrum for intracellular imaging. be due to aggregation effects that might be avoided if the Calcium-induced conformational changes of appropriately dye has some intrinsic water solubility. Second, do water- modified, intracellular calmodulin have been followed using soluble Nile Red derivatives also show the pronounced FLAsH dye 8.24 bathochromic shift in polar media, that is, observed for J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037 11025

F F NH O NaOH, pyridine 2 80 % AcOH + 100 °C, 6 h 100 °C, 12 h Et2N OH F O2N OH HO O F

F N N F (i) NaNO2, H2SO4 H2, Pd/C ° 40 C, 1 h MeOH ° O2N O O H2N O O Et2N O (ii) H2O, O2, 65 C, 1 h F 8 % 46 % 48 %

O- N Hg(OAc)2 N+ HCl, Zn dust ° ° AcOH, 50 C C6H6, 25 C, 3 h H2N O O Et2N O O HgOAc HgOAc F intermediate used 23 % as crude material (i) AsCl , Pd(OAc) , DIEA 3 2 N NMP, 25 °C, 12 h N (ii) EDT, acetone, 25 °C, 12 h H2N O O As As Et2N O O SS SS F 7 8 43 % 7 % EtOH λ max abs 561 nm Scheme 5. Preparation of the FLAsH system 8. Scheme 4. Preparation of 6-ﬂuoro Nile Red. would be to prepare iodo-, bromo-, or chloro-substituted protein surfaces compounds then elaborate them via organometallic couplings. There have been reports of attempted halogenation S S SH HS As "dye" As of Nile Red, but the regioselectivity and extent of the halo- S S genations proved hard to control and mixtures were pro- non-fluorescent duced.26–28 SH HS 3.3. Spectroscopic properties

protein surfaces Table 1 summarizes spectroscopic data for Nile Red in different solvents, all taken from the same source.4 These S S 604 nm data show decreased fluorescence intensity of Nile Red cor- for dye 8 As dye As relates much more with hydrogen bonding than with solvent S S polarity, and the magnitude of this difference is best appre- ciated from the graphical presentation of only the emission wavelengths and intensities that is given in Figure 4.How- Figure 3. Conceptual basis of fluorescent arsenic dyes. ever, the bathochromic shift seems to be a function of solvent polarity, consistent with stabilization of relatively polar compounds in the series with little or no significant aqueous excited states. solubility? A similar study featuring emission and absorption maxima, No significantly water-soluble Nile Red derivatives have quantum yields, and solvent polarities was performed for been prepared to answer the questions posed above until re- 18 cent work from our laboratories. Classical condensation Table 1. Solvent dependency of emission intensities and wavelengths for routes were used to prepare the three Nile Red derivatives Nile Red 2 11–13 (Scheme 7). These were designed to be water soluble, but, surprisingly, the diol/phenol 11 was not, even in aqueous Solvent lmax abs lmax emiss Relative fluorescence (nm) (nm) intensity base. The other two compounds do have very good water solubilities. Their spectroscopic properties are discussed in the Water 591 657 18 next sub-section; but the data shown in Table 3 show that EtOH 559 629 355 Acetone 536 608 687 the answers to both these questions were affirmative for CHCl3 543 595 748 compounds 12 and 13. iso-Amyl acetate 517 584 690 Xylene 523 565 685 Some researchers may be interested in access to more n-Dodecane 492 531 739 n sophisticated analogs of Nile Red. One obvious approach -Heptane 484 529 585 11026 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037

800 0.8

CHCl3 acetone toluene 700 n-dodecane 0.7 xylene acetone dibutylether 600 0.6 n-heptane iso-amylacetate EtOH 500 0.5 cyclohexane

400 0.4 EtOH 1,2-ethanediol 300 quantum yield relative intensity 0.3

200 0.2 CF3CH2OH 100 0.1 water (CF ) CHOH 0 3 2 500 600 700 0.0 wavelength of emission (nm) 500 600 700 wavelength of emission (nm) Figure 4. Solvent dependency of emission intensities and wavelengths for Nile Red 2. Figure 5. Solvent dependency of quantum yield and wavelengths for 2-hydroxy Nile Red 6.

Table 2. Solvent dependency of quantum yield and wavelengths for 2-hydroxy Nile Red 6 Table 3. Spectroscopic properties of water-soluble Nile Red derivatives 11–13 in different solvents

Solvent lmax abs (nm) lmax emiss (nm) Quantum yield (F) Dye lmax abs lmax emiss Quantum Solvent (nm) (nm) yield (F) Cyclohexane 514 528 0.51 Dibutyl ether 514 555 0.66 11 542 631 0.56 EtOH Toluene 525 568 0.76 12 520 632 0.43 EtOH 12 560 648 0.33 Phosa 7.4 Acetone 528 608 0.78 b EtOH 544 634 0.58 12 556 647 0.07 Bor 9.0 13 548 632 0.42 EtOH 1,2-Ethanediol 584 655 0.40 a CF CH OH 587 653 0.24 13 558 652 0.37 Phos 7.4 3 2 13 556 650 0.18 Borb 9.0 (CF3)2CHOH 612 670 0.09 a pH 7.4 Phosphate buffer. b 2-hydroxy Nile Red 6 (Table 2). Selected data from this pH 9.0 Borate buffer. study are shown in Figure 5. Just as with Nile Red, polar hydrogen-bonding solvents correlate with reduced quantum a single photon of higher energy. This is a way to excite in- yields and significant bathochromic shifts. tracellular or tissue samples at a wavelength that is more transparent to these media. The dependence of two-photon Fluorescence lifetimes contribute to two important physical absorption on the intensity of laser beam allows for high spa- parameters of fluorescent dyes. Dyes with long fluorescent tial selectivity by focusing the laser beam on the target cell lifetimes can emit strongly because non-radiative processes and thus preventing any damage to adjacent cells. Relatively are relatively slow, and because intersystem crossing to trip- few dyes are suitable for practical experiments using let states is less competitive. two-photon excitation because most do not absorb two long wavelength photons efficiently, i.e., they have poor Fluorescence lifetimes for Nile Red in different solvents two-photon cross-sections. Unfortunately, Nile Red has a have been measured (Table 3). These data show that the relatively poor two-photon cross-section.31 fluorescence lifetime of Nile Red is 3.65 ns (EtOH)29 in comparison to fluorescein (4.25 ns, EtOH)30 and tetramethyl- One issue with two-photon excitation experiments is that the rhodamine (i.e., rhodamine 6-G, 3.99 ns, EtOH).30 The emitted light is of a short wavelength compared to the exci- fluorescent lifetime of Nile Red does not seem to vary much tation source, and this might not be in a convenient region to with solvent polarity, but it is extremely sensitive to H-bond- permeate out of cells of other tissues, and for detection. One ing. In hydrogen-bonding solvents the fluorescence lifetime strategy to circumvent this problem is to arrange a fluores- of Nile Red decreases dramatically. cence energy transfer system (FRET) featuring a donor with a large two-photon cross-section and an acceptor with a As stated above, the bias of this review is to highlight poten- more convenient, longer wavelength, and emission maxima. tial applications in biotechnology, especially for intracellu- 2-Hydroxy Nile Red derivatives are being used in such lar imaging. There are two common approaches to long systems. Thus a series of compounds (of which 14 and 15 wavelength dyes for imaging in tissues. The first, and most are two of the simplest; Fig. 6) have been prepared toward obvious, is to prepare analogs of known dyes with extended this end; and donor-to-acceptor energy transfer efficiencies conjugated systems. Secondly, two-photon absorption can of more than 70% were observed (Fig. 5).32,33 These be used,31 in which two long wavelength photons absorbed compounds presumably do not have the solubility and size by a molecule promote it to an excited state that then emits characteristics that render them suitable for a range of J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037 11027

a HO HO NO would be less ﬂuorescent than Nile Red itself; this dye itself NaNO /HCl 2 has not been prepared. 5 °C, 2 h N N

40 % HO3S O

N O I O N O O HO N 2 HO OH DMAP EtOH, reflux, 4 h pyridine, 2 h N O O 16 more or less fluorescent than Nile Red? 50 %

N 4. Nile Blue

O I N O O 4.1. Introduction O 9 25 % Nile Blue has a positively charged, oxidized, phenoxazine system, i.e., it is a phenoxazinium; the conspicuous differ- b OH ence between this and Nile Red 2 is that the latter is neutral. Nphth Both dyes have a 9-diethylamino substituent to donate elec- N Br tron density across the ring, but Nile Red 2 and Blue 3 have

K2CO3, DMF different electron acceptors, a carbonyl and an iminium reflux, 4.5 h Et2N O O group, respectively (Fig. 7).

Nphth One obvious consequence of the difference in charges for O Nile Red 2 and Blue 3 is that water-solubility of Nile Blue is significantly better. Like its Red cousin, the fluorescence (i) MeNH , MeOH N 2 reflux, 2.5 h maxima and intensity of Nile Blue are good indicators of (ii) O the polarity of the dye’s environment. This solvatochromic Et2N O O I effect gives a red-shift in polar media, as would be expected O 85 % 2 for stabilization of a more charged excited state. The inten- CH2Cl2, 40 min sity of the fluorescence of 3 in water is about 0.01; this is a small value but, in comparison with the lack of fluores- H N cence of Nile Red in aqueous media, this is significant. O I O 4.2. Syntheses N The original synthesis of Nile Blue2 involved condensation

Et2N O O of 5-amino-2-nitrosophenol with 1-aminonaphthalene in 10 acetic acid, but the yield was only 3%. However, use of 61 % perchloric acid in ethanol gives a signiﬁcantly higher yield (reaction 2).35 Scheme 6. Two thiol-selective dye electrophiles: (a) a Nile Red derivative; (b) a 2-hydroxy Nile Red derivative.

NH2 NO applications in biotechnology, so further developments in 70 % HClO4 this area would be opportune. EtOH, reflux, 5 min Et2N OH

Finally, there is the possibility that the ﬂuorescence of Nile ð2Þ Red is somewhat attenuated if the excited state of the benzo- N phenoxazinone core is reduced via electron transfer from the - ClO4 9-amino substituent. We mention this because the possibility + Et2N O N H2 has been explored via AM1 calculations.34 These gauge the 3 degree of charge transfer in a planar state relative to a twisted 31 % one in which the amine lone pair is not disposed to electron donation to the heterocycle. The degree of electron transfer from the 9-amino substituent will be dependent on the con- De novo syntheses of Nile Blue derivatives involve use of formation and on the reduction potential of the heterocycle similar, but substituted starting materials. Scheme 8 de- in its excited state. If intramolecular charge transfer was scribes syntheses of dyes 17–20 that incorporate 8-hydroxy a major pathway for quenching the ﬂuorescence of Nile julolidine.35 This amine is a ‘privileged fragment’ in dye Red derivatives, it would be expected that compound 16 syntheses because it holds the nitrogen lone pair in 11028 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037

COOH MeOH, H+ LiAlH ,THF HOOC MeO C 4 H NOH ° 2 2 H2O, 70 C, 3 h NOHreflux, 24 h NOH25 °C, 2 h

COOH CO2Me 80 % 63 % OH OH OH OH OH NO N NaNO2, HCl HO NOH0-5 °C, 2.5 h NOHDMF, 130 °C, 5 h N O O

HO HO HO 11 90 % 74 % 54 % EtOH; Φ 0.56 λmax abs 542 nm λmax emiss 631 nm

b OH OH

MeO2C MeO2C NO MeO2C N NaNO2, HCl HO NOH0-5 °C, 2.5 h N OH MeOH, H+, reflux, 5 h N O O

CO2Me CO2Me CO2Me 74 % 53 % OH

HO2C N K2CO3, MeOH/H2O 12 h, 40 °C N O O

CO2H 12 60 %

EtOH; Φ 0.43 pH 7.4 buffer; Φ 0.33 λ λmax abs 520 nm max abs 560 nm λ λmax emiss 632 nm max emiss 648 nm

c O

S SO3H SO3H O O NO COOH DMF NaNO2, HCl H NOH ° HN OH ° ° N OH 2 H2O, 70 C, 12 h 130 C, 12 h NOH0 - 5 C, 2.5 h

COOH COOH COOH

40 % 48 % 70 % OH OH

SO3H N HO DMF, reflux, 5 h N O O

COOH 13 53 %

EtOH; Φ 0.42 pH 7.4 buffer; Φ 0.37 λ λmax abs 548 nm max abs 558 nm λ λmax emiss 632 nm max emiss 652 nm

Scheme 7. Synthesis of 2-hydroxy Nile Red derivatives featuring the following functionalities: (a) a diol; (b) a dicarboxylic acid; and (c) a carboxylic acid sulfonic acid combination. J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037 11029

two photons in at 815 nm

O O O O N N N N N N N N

D1 part donor N N

FRET

OH O 1 D acceptor CHCl3 λ N max abs 405 nm λ max emiss 510 nm Et2N O O one photon 14 out at 595 nm THF λ max abs 405 nm, 540 nm λ max emiss 595 nm

b two photons in at 810 nm

S N N S

S O O N N N O O N

OH donor D2 O

CHCl3 λ max abs 400 nm O λ 2 FRET max emiss 510 nm D part

N acceptor

Et2N O O one photon out at 595 nm 15 THF λ max abs 390 nm, 540 nm λ max 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 reactivity Nile Blue derivatives with enhanced water-solubilities and/ of the starting material; in fact, nitrosylated 8-hydroxy- or groups for bioconjugation can be made from amines julolidine was insufficiently reactive in this synthesis so with appropriate N-substituents. For instance, Scheme 9 Hartmann and co-workers modified the synthon to include shows preparations of dyes 21,36,37 22/23,38,39 and 24/2540 the reactive azo-functionality as shown. Unfortunately, the in which sulfonic acid groups enhance water solubilities julolidine fragment is quite hydrophobic too, so the product and carboxylic acid groups could potentially be activated dyes have very limited solubility in aqueous systems. and reacted with amines on biological molecules. The syntheses of 24 and 25 are shorter and higher yielding than other An early synthesis of Nile Red featured hydrolysis of a Nile syntheses of water-soluble Nile Blue derivatives. Blue derivative (see Scheme 2b). This implies that Nile Blue derivatives have finite hydrolytic stability, and this could be Compounds 22 and 23 are the ‘EVO Blue’ dyes.38,39 These a drawback in some situations. Dyes 17–20 are much less have been claimed to be more stable than rhodamine and vulnerable to this mode of decomposition because of their BODIPY dyes under acidic conditions. This enhanced acid fused cyclic structures that hold the amines in place. stability was suggested to be useful in the context of high 11030 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037

neutral molecule These photosensitizers promoted to the excited singlet state decays to the triplet state and generates singlet oxygen, 2 1 3 which is considered to be responsible for its potent activity 11 N 4 N toward cancer cells. 10

N O O N+ O O- 4.3. Spectroscopic properties 8 6

carbonyl Nile Red 2 Like Nile Red 2, Nile Blue shows progressively longer electron absorption and emission maxima as the solvent polarity is acceptor increased. However, the Stokes’ shifts observed for the red MeOH n-heptane λ λ probe 2 in different solvents is far greater; this parameter max abs 554 nm max abs 484 nm λ λ max emiss 638 nm max emiss 529 nm for the Blue compound can still be exceptionally high Φ 0.38 Φ 0.48 (almost 100 nm), but in polar solvents it is reduced to around 40 nm (Table 4).44 charged molecule 2 1 3 Table 4. UVabsorption and fluorescence emission maxima of Nile Blue 3 in 11 N 4 N 10 different solvents + Solvent l (nm) l (nm) N O NH N+ O NH max abs max emiss 8 6 2 2 Toluene 493 574 iminium Nile Blue 3 4-Chlorobenzene 503 576 electron Acetone 499 596 acceptor DMF 504 598 CHCl3 624 647 H2O MeOH n-hexane 1-Butanol 627 664 λ 635 nm λ 626 nm λ max abs max abs max abs 473 nm 2-Propanol 627 665 λ 674 nm λ 668 nm λ max emiss max emiss max emiss 546 nm EtOH 628 667 Φ 0.01 Φ 0.27 MeOH 626 668 increasing polarity Water 635 674 1.0 N HCl, pH 1.0 457 556 Figure 7. Structures of Nile-Red and Blue contrasted. 0.1 N NaOH, pH 11.0 522 668 NH4OH, pH 13.0 524 668 throughput screening and solid phase syntheses that involve cleavage from resins by acids. Table 5. Study of effect of different anions on spectral properties of Nile Blue in different solvents Other approaches to water-soluble Nile Blue derivatives have Anion Solvent l (nm) l (nm) involved modification of the parent dye after the hetero- max abs max emiss cyclic framework was assembled. For instance, Scheme 10a Chloride EtOH 628 689 shows a patent procedure for alkylation of the 5-amino/ O O 632 — iminium substituent; we note that chlorosulfonic acid is an O unusual choice for the ester hydrolysis reaction. In the second example, Scheme 10b, an amino anthracene was used to pre- MeO OH pare a derivative of Nile Blue that has an extended aromatic 634 694 system. This modification gave a probe with absorbance and O fluorescence emission shifted to the red. Unfortunately, the material 27 was probably not a pure compound since the de- Acetate EtOH 628 689 gree of sulfonation, the regiochemistry, and the chemical/ O O quantum yields were not given.41 632 687 O

Direct iodination of Nile Blue is possible, and the 6-iodo de- MeO OH rivative 28 can be prepared from this (Scheme 11). However, 634 700 chromatography was required, the yield of the iodinated O product was low, and slight variation of the conditions can lead to formation of other regioisomers. Similar consider- Benzoate EtOH 628 691 ations apply to the corresponding bromination reaction, ex- O O cept the product yield was better. Conversely, the de novo 632 690 synthesis of the 2-iodo derivative 30 is unambiguous with O respect to the regioisomer formed, but column chromatography is still required and the yield is also low. The diiodide MeO OH 634 702 31 can be formed by a second iodination of the 2-iodide. O These halogenated derivatives are potentially useful for forming more sophisticated derivatives, but they were iso-Butanoate EtOH 628 691 originally prepared as possible photosensitizers to initiate processes that are destructive to carcinogenic cells.42,43 Hydroxide EtOH 515 661 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037 11031

Ar N N OH N N ClN2Ar + N OH MeOH, 0 °C, 30 min cat.HClO , DMF NON N OH 4 ClO - reflux, 5 min 4

8-hydroxyjulolidine 17 Ar = 4-ClC6H4 57 % Φ CH2Cl2; 1.0 λmax abs 671 nm λmax emiss 688 nm

Ar N OH N ClN2Ar ° MeOH, 0 C, 30 min cat.HClO4, DMF + NH NH reflux, 5 min NON H - ClO4

18 Ar = 4-ClC6H4 10 % Φ CH2Cl2; 0.35 λmax abs 675 nm λmax emiss 712 nm

Ar N N OH N N ClN2Ar

MeOH, 0 °C, 30 min cat.HClO4, DMF + NH NH NON H - reflux, 5 min ClO4

Ar = 4-ClC H 19 6 4 10 % Φ CH2Cl2; 0.42 λmax abs 668 nm λmax emiss 677 nm

Ar N OH N N ClN2Ar SO N 2 SO2 MeOH, 0 °C, 30 min cat.HClO , DMF N+H SO2 4 NO NH reflux, 5 min - NH ClO4

Ar = 4-ClC6H4 20 1 % Φ CH2Cl2; 0.42 λmax abs 668 nm λmax emiss 677 nm

Scheme 8. Syntheses of Nile Blue derivatives from 8-hydroxyjulolidine.

Table 4 also reveals that the spectroscopic characteristics of corresponding value of Nile Red (see above; 3.65 ns). Nile Blue are pH dependent. This is because under basic The lifetime of Nile Blue is relatively invariant at dilute conditions the iminium group will be deprotonated, whereas concentrations (103–108 mol dm3) but changes as the under strongly acidic conditions the 5-amino might even be- concentration is increased, and in different solvents. come protonated.45 Curiously, the spectroscopic properties This is probably an indication of the interdependence of Nile Blue are somewhat dependent on the counter ion of the spectroscopic properties and the degree of aggre- used.46 We speculate that this could even be a reflection on gation in solution. In support of this assertion, we note intimate ion pairing influencing the solvent sphere of the that the lifetimes do not seem to be significantly im- dye (Table 5). pacted by the viscosity of the medium, but they are temperature dependent.47 As far as we are aware, the The fluorescence lifetime of Nile Blue 3 in ethanol two-photon cross-section of Nile Blue has not been has been measured at 1.42 ns. This is shorter than the reported. 11032 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037

R NO R N H+/ MeOH or EtOH 1 R H R1 H N OH N reflux N O N 2 R R3O R2 R3O

O 21 O R1, R2 = H, linear alkyl 7-90 % R = H, Et, R3 = H, Me, Et EtOH pH 7.4 buffer λ max abs 570 +/- 60 nm λmax abs 570 +/- 15 nm λ max emiss 640 +/- 30 nm λmax emiss 638 +/- 12 nm

b - SO3

- NO SO3 N CH3COOH reflux, 30 min Et N O N+H Et2N OH 2 NH COOH 22 COOH EVO blue 90 pH = 7.4 buffer λmax abs 649 nm λmax emiss 673 nm

c (i) CH3COOH, reflux 30 min ON N (ii) cat. HCl N OH H2O/acetone NH reflux, 8 h N O NH+

OO - - SO3 OOH SO3 23 EVO blue 100 10 % pH = 7.4 buffer λmax abs 634 nm λmax emiss 674 nm

d SO3H + - (i) ArN2 Cl SO3H MeOH, 25 °C, 30 min N (ii)

NH2 + - Et2N O N H2 ClO4 Et2N OH 24 DMF, HClO4 55 % 155-160 °C, 15 min pH 7.4 buffer; Φ 0.03 λmax abs 637 nm λmax emiss 677 nm

e O O SO3H SO3H + - S (i) ArN2 Cl N ° - O MeOH, 25 C, 30 min ClO4 + H2N OH DMF, 130 °C N OH (ii) NON H2 2 h

HO3S HO3S NH2 77 % DMF, HClO 4 25 155-160 °C, 15 min 64 % pH 7.4 buffer; Φ 0.10 λmax abs 633 nm λmax emiss 675 nm

Scheme 9. Syntheses of Nile Blue derivatives with water solubilizing N-substituents. J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037 11033

a O ( ) Br N NaOH, H2O N O 5 N - O Cl 65 °C, 30 min K2CO3, toluene + reflux, 18 h ( ) Et2NON H2 Et2NONH Et2NONO4

71 %

ClSO3H, Na2SO4 N O ° CH2Cl2, 70 C, 22 h ( ) Et2N O NOH4

26 90 % pH = 7.4 buffer λmax abs 643 nm λmax emiss 680 nm

O NO (i) EtOH, HCl Br reflux, 18 h O ( )5 Me NOH N N 2 (ii) NaOH, H2O K2CO3, toluene O reflux, 18 h NH2 ( ) Me2N O NH Me2NOO N 4

SO3H ClSO3H, Na2SO4 SO3H ° N CH2Cl2, 70 C, 22 h O ( ) Me2N O N 4 OH

27 pH = 7.4 buffer λmax abs 650 nm λmax emiss 698 nm

Scheme 10. Preparation of water-soluble Nile Blue derivatives: (a) with only carboxylic side chain; (b) with carboxylic acid side chain and two additional sulfonic acid groups.

5. Other benzophenoxazine dyes ON ZnCl2, CH3COOH 25 °C, 24 h 5.1. Introduction OH OH

There is no obvious reason why benzo[a]phenoxazines N should be more ﬂuorescent than similar compounds with ð3Þ different ring fusion patterns. Our interpretation of the liter- O O ature is that other phenoxazines with appropriate substitu- 32 ents have certainly been less well studied as ﬂuorescence 2-3 % probes, and are probably less synthetically accessible. EtOH max abs 505 nm 5.2. Syntheses

The benzo[c]phenoxazine 32 has been prepared via a route Benzo[b]phenoxazines are linear. Substituted derivatives that is similar to those used for the Nile compounds 2 and of these are numbered according to the system shown 3. 1-Naphthol is used in this synthesis (reaction 3)46 rather below. than 2-naphthol, the isomer used for 2 and 3. This change is necessary to obtain the different ring fusion, but it also H 1 2 11 N may account for the very poor yield. This is because of the 10 3 well-known reduced reactivity for 1-napthol at the 2-posi- 4 9 O tion in electrophilic substitution reactions, relative to the 8 6 5 greater tendency for 2-napthol to react at the 1-position. To the best of our knowledge, the fluorescent properties of 32 have not been reported in any depth; indeed, the molecule The linear system 33 has been prepared via the high temper- lacks an electron donor in conjugation with the carbonyl to ature condensation process shown in reaction 4.48 This give it the type of extended oscillating dipole that seems to has absorption and fluorescence emission properties that be common for fluorescent molecules. are characteristic of an extended aromatic heterocycle. 11034 J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037 Like 32, this compound does not have substituents that would allow it to be reduced to a phenoxazinone or phenox- a azinium form. N NIS, CH3COOH ° CF3CH2OH, 90 C, 80 min + Et2NON H2 NH HO 2 220 °C CO atmosphere N OH HO 2

+ - H Et2NON H2 CH3COO N I ð4Þ 28 O 22 % 33 MeOH 55% λmax abs 642 nm EtOH; 0.24 toluene; 0.28 max abs 365 nm max abs 361 nm max emiss 430 nm max emiss 420 nm b

N Br2, CH3COOH - ° Cl CF3CH2OH, 25 C, 20 min + Some nitro and amino derivatives of benzo[b]phenoxazines Et2NON H2 have been reported in literature, and Scheme 12 shows 49 them. Parts a and b show nitrosylation/oxidation reactions N that can be used to prepare nitro-substituted derivatives 35 Cl- + and 36. Predictably, these are not particularly fluorescent Et2NON H2 compounds. However, the 1,9-diaminobenzo[b]phenoxazi- Br 29 nium 37 has the potential to be strongly fluorescent. Unfor- 48 % tunately, it was neutralized to 38 then the fluorescence MeOH λ properties were recorded. max abs 643 nm

c 6. Conclusions I

NO Current knowledge of fluorescent benzophenoxazine- CH3COOH, cat. HCl derived probes is based almost on the benzo[a]phenoxazine 120 °C, 2.5 h ring fusion series. Almost all the syntheses feature high Et2N OH NH2 temperature condensation methods, and not contemporary I synthetic methods like, for example, Buchwald–Hartwig couplings to introduce amine substituents. In fact, many of the synthetic methods reported are based on the procedures N Cl- that are now over a century old. This, and the prevalence + of patent literature in this area, mean that many of the experi- Et2NON H2 mental procedures presented are difficult to follow, and 30 complete spectroscopic data is rarely recorded. 11 % MeOH λmax abs 633 nm Nile Red and its derivatives have some interesting spectroscopic properties (long wavelength emissions, large Stokes’ d shifts) but most compounds in this series have limited water I solubilities. There are, however, some recent efforts to make NIS, CH COOH modified compounds to redress this. Nile Blue and its deriv- 3 N ° atives tend to be more water soluble. CF3CH2OH, 25 C, 30 min Cl- + Et2NON H2 Relatively little work has been done to modify benzo- I phenoxazine dyes so that they emit even further to the red: the longest wavelength emission in the existing probes is about 700 nm. To this end, it would be useful to have access N to more functionalized compounds that can be prepared Cl- Et NON+H easily on a gram scale, like 2-hydroxy Nile Red 6. Other 2 2 31 I modifications might be used to give derivatives with en- 38 % hanced extinction coefficients (these tend to be 10,000 or MeOH λ less), or improved two-photon cross-sections. These types max abs 650 nm of developments would be facilitated by more detailed stud- Scheme 11. Preparation of halogenated Nile Blue derivatives: (a) 6-iodo; ies of fundamental reactions that can be used to modify these (b) 6-bromo; (c) 2-iodo; and (d) 2,6-diiodo. compounds, e.g., halogenation and nitration. J. Jose, K. Burgess / Tetrahedron 62 (2006) 11021–11037 11035

a Acknowledgements H N NaNO2, CH3COOH Support for this work was provided by The National Insti- DMF, 70 °C, 10 min. O tutes of Health (HG 01745 and GM72041) and by The 33 Robert A. Welch Foundation. H N References and notes O2N O 34 34 % 1. Okafor, C. O. Dyes Pigm. 1986, 7, 103–131. 2. Mohlau, R.; Uhlmann, K. Justus Liebigs Ann. Chem. 1896, 289, EtOH toluene; Φ 0.05 λ 94–128. λmax abs 436 nm max abs 438 nm λ λmax emiss absent max emiss 500-530 nm 3. Wichmann, C. F.; Liesch, J. M.; Schwartz, R. E. J. Antibiot. 1989, 42, 168–173. b 4. Greenspan, P.; Fowler, S. D. J. Lipid Res. 1985, 26, 781–789. H N 5. Okamoto, A.; Tainaka, K.; Fujiwara, Y. J. Org. Chem. 2006, 71, NaNO2, CH3COOH 25 °C, 3 h 3592–3598. O 6. Briggs, M. S. J.; Bruce, I.; Miller, J. N.; Moody, C. J.; 33 O Simmonds, A. C.; Swann, E. J. Chem. Soc., Perkin Trans. 1 NO 1997, 7, 1051–1058. H 2 N N 7. Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123– H N 128.

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Biographical sketch

Jiney Jose was born in Kerala, India. He received his B.Tech in Chemistry Kevin Burgess is a professor at Texas A & M University, where he has been (2002) from U.I.C.T., Mumbai, India. He joined the group of Prof. Kevin since September 1992. His research interests focuses on peptidomimetics Burgess at Texas A & M University, Department of Chemistry in 2003. for mimicking or disrupting protein–protein interactions via combinatorial His research is focused on syntheses, derivatization and photophysical chemistry and via asymmetric catalysis, and ﬂuorescent dyes for multiplex- studies of water-soluble benzophenoxazine dyes. 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.