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3-Hydroxyflavone (3HF) as a model molecules for protontransfer processes

3-Hydroxyflavone (3HF) as a model molecules for protontransfer processes

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O



OH

5



O



Fig. 3 3-Hydroxyflavone (5, 3HF).



1

O

O–



1N



OH+



ESIPT

1T



1

1A



O

+

hνN



hν1



O–



hνT

OH



hνA



hν2

BPT



T



–H+

N

A



O



O

O–

O



OH

O



5



Scheme 1 Excited State and Ground State Proton Transfer occurring in 3-hydroxyflavone (5).



(BPT) from T to the initial N state. Furthermore, under specific conditions,

3HF can also undergo ground-state deprotonation, giving the corresponding anion A which absorbs at longer wavelength (hn2 about 400 nm)

than the neutral 3HF and gives an emission band (hnA) separated from

those of both normal and tautomeric states of 5.18a Since the presence of 1N,

1

T and 1A can be easily observed by monitoring their fluorescence (Fig. 4,

see below for further details), 3HF is considered an ideal model for the

study of both excited state and ground state proton transfer processes.

The process and the structure of the intermediates involved in ESIPT

have been the subject of both experimental18 and computational19 investigations. Among these extensive studies, the most important aspect of the

proton transfer mechanism is its dependence on the chemico-physical

properties of the microenvironment surrounding the 3HF molecule. Thus,

Photochemistry, 2012, 40, 295–322 | 297



Fig. 4 Fluorescence spectra of 5 in methanol recorded at a) 305 b) 345 and c) 410 nm

respectively. As hinted above, the presence of three well separated emission bands assigned to

1

N (a), 1T (b) and 1A (c) is observable. Reprinted with permission from P. K. Mandal and

A. Samanta, J. Phys. Chem. A 2003, 107, 6334. Copyright 2003 American Chemical Society.



(a)



1

1



O



O

ESIPT

O-



O

O



O



H



H



(b)



1



1

O



O



O

O

H



ESIPT



H

O

CH 3



OO+

H



H

O

CH 3



Scheme 2 Proposed mechanism for ESIPT taking place in 3HF when irradiated in a) apolar

and b) protic solvents.



ESIPT from 1N to 1T has been described as extremely fast and efficient20 in

apolar solvents (e.g. 2-methylbutane) at room and even at cryogenic temperatures (rigid glass, 77 K) and in argon matrix.21 Under these conditions,

green emission (with high quantum yield) from the tautomer is exclusively

observed (Scheme 2a). On the other hand, the intramolecular hydrogen

bond in 3-hydroxyflavone is perturbed by intermolecular hydrogen bonding

298 | Photochemistry, 2012, 40, 295–322



in polar or protic solvents such as ethers, esters or alcohols. Thus, in solvated 3HF molecule, proton transfer can not occur directly, allowing for a

radiative decay also from 1N. On the other hand, photoinduced tautomerization is not totally prevented in such solvents, and an alternative

mechanism for proton transfer must be involved. Woolfe and Thistlethwaite

suggested a solvent assisted proton transfer taking place (either via two

successive PTs or via a concerted double PT) in a seven-membered chelate

ring between 3HF and methanol (Scheme 2b).22

The extreme sensitivity of 3HF photophysics to the presence of protic

solvents has been demonstrated23a and used by Kasha et al. for checking the

purity grade (and, as consequence, the presence of even traces of protic

solvents such as water or alcohols) in alkanes.23b The photophysics of 3HF

has been investigated in different microenvironments, including acetonitrile/

apolar solvent mixtures,24 aqueous micelles,25 Aerosol OT (AOT) reverse

micelles26 and cyclodestrins.27

As for the ground-state deprotonation of 3HF in neat solvents to give the

corresponding anion A (Scheme 1), it has been first reported by Kasha

(1990),28 and rationalised by Mandal and Samanta in 2003.29 The last

Authors described the process in terms of quantitative chemico-physical

parameters of the solvent. They reported the formation of the anion in the

examined solvents (alcoholic solvents and formamide28) as the result of a

two-step mechanism with two hydrogen bonding interactions involved. In

the first step the formation of a complex between 3HF and the solvent

occurs, the latter acting as an H-bond donor towards the carbonyl moiety of

3HF thanks to its hydrogen bond donor acidity (HBA, a). In the second

step, proton abstraction from the hydroxy proton of 3HF takes place

thanks to the significant hydrogen bonding donor basicity (HBD, b) of the

solvent (Scheme 3a). On the other hand, ground state deprotonation of 3HF

takes place also in solvents with negligible HBA such as N,N-dimethylformamide (DMF),30 dimethyl sulfoxide (DMSO) and, even if in small

amounts, in tetrahydrofuran (THF),31,32 suggesting that anion formation

(a)

O



O

O-



O

H



O

H



O



O



H



CH3



H

+



O



CH3



(b)

O



O



O

O



H



O

O-



O

S+



H



O



S+



Scheme 3 Ground state deprotonation of 3HF in a) methanol and in b) DMSO.



Photochemistry, 2012, 40, 295–322 | 299



occurs also in media with zero HBD acidity but HDA basicity value sufficiently high to ensure the formation of a 5–solvent complex and therefore

deprotonation (Scheme 3b).

Interesting information comes from the investigation of 3HF anion

spectral properties. Indeed, both the absorption and the fluorescence

spectra seem very sensitive to the surrounding environment, especially to

the presence of hydrogen bonds,31 and TD-DFT calculations were shown to

be very useful to rationalize the sensitivity of 3HF anion spectral properties

in organic solvents.31 Taken all together, these observations significantly

widen the relevance of 3HF as a fluorescent probe: beside the environmentsensitive 3HF dual emission, under appropriate conditions the fluorescent

3HF anion can be formed. Furthermore, as seen above, anion emits in a

region well separated from the 1N and 1T emission bands, and its photophysical properties are strongly environment-sensitive. Ground-state

deprotonation has been also observed for 3HF in cyclodextrins33 and in

aqueous micelles.26b

3



Interaction of 3HF and natural flavonols with biomolecules



After the seminal paper by the group of Kasha,34 both ESIPT process and

ground-state anion formation have been largely used to investigate flavonol

interactions with biomolecules.35 In this section the main results for 3HF

and natural flavonols (such as quercetin (1), fisetin (6) and myricetin (3)) will

be discussed. The use of synthetic derivatives of 3HF as fluorophores in

biophysical investigations represents a separate topic and will be described

in the following section.

In 1996, Sytnik and Litvinyuk36 showed, in the investigation of 3HF

binding to Human Serum Albumin (HSA), the presence of emission bands

arising from both the neutral molecule and the anionic form of 3HF. This

led them to conclude that two binding sites are involved in the HSA-3HF

interaction. From a methodological point of view, this study demonstrated

that 3HF (and, more broadly speaking, flavonols) in specific biological

microenvironments can undergo deprotonation.

Several research groups have investigated the interaction of 3HF with

biomolecules through fluorescence spectroscopy approach.37 Apart from

the results obtained by using 3HF as fluorescent probe, a larger amount of

studies have been carried out on natural flavonols (see Fig. 5 and Tables 1

and 2 for a list of selected examples). Interestingly, flavonols often play the

role of both the fluorophore and the biologically-active molecule object of

the investigation. Furthermore, as described in details for the case of

quercetin, the emission properties are modulated by the interactions with

OH



OH

HO



O



OH HO

OH



O



HO



O



O



O



OH

OH 7



6



OH



HO



OH



OH

OH O



8



Fig. 5 Flavonols examined in this section: Fisetin (6), Robinetin (7), and Morin (8).



300 | Photochemistry, 2012, 40, 295–322



Table 1 Behaviour of non 5-OH substituted flavonols in the presence of biomolecules.

Examined

flavonols



Biomolecule



3-HF (5)



HSA



5



BSA



5



DNA



Fisetin (6)



HSA



6



DNA



Robinetin (7)



Hemoglobin



Observed behaviour



Ref.



1



ESIPT ( T) fluorescence upon binding. Anion formation (ground state proton transfer) and

enhancement of 1A fluorescence upon binding

ESIPT (1T) fluorescence enhanced upon binding;

Anion formation (ground state proton transfer)

enhancement of 1A fluorescence upon binding

Normal (1N) and ESIPT (1T) fluorescence modified

upon binding

Normal (1N) and ESIPT (1T) fluorescence modified

upon binding (1T enhanced) and possible anion

formation.

Normal (1N) and ESIPT (1T) fluorescence modified

upon binding (1T enhanced).

Normal (1N) and ESIPT (1T) fluorescence decrease

upon binding; 1N and 1T bands overlap to give a

broad peak.



36



37h



37a

51a



51a

54



Table 2 Behaviour of 5-OH substituted flavonols in the presence of biomolecules.

Examined

Flavonols



Biomolecule



Quercetin (1)



HSA



1



HSA, BSA



1



DNA



Myricetin (3)



HSA



Morin (8)



HSA



Observed behaviour



Ref.

1



1



Strong increase in Normal ( N) and ESIPT ( T)

fluorescence (dual fluorescence), due to the

perturbation of the hydrogen bond between the

OH in position 5 and the C¼O group. Presence

of a third emitting species attributed to a

ground-state complex formed in the protein

environment (ref. 50).

Strong emission peak at B530 nm interpreted as

partial formation of pyrilium-like (4-hydroxy)

form(s) (ref. 46).

Enhancement of (1T) fluorescence; possible

anion formation (as indicated by the observed

fluorescence peak at 465 nm ca.).

1

N fluorescence predominates. From UV-Vis

spectra Myricetin exists as an anion in the

binding pocket.

1

T fluorescence consciously enhanced upon

binding. From UV-Vis spectra Morin exists as

an anion in the binding pocket.



48, 50



46, 47



53b



55



42



the biomolecule (notably through H-bonds or protonation/deprotonation

reactions). These are also intrinsically related to the mode the flavonols bind

to the biomolecule. In other words, fluorescence studies (and to a lesser

extent, UV-Vis spectroscopy) can directly bring information at a submolecular scale on the interactions between biologically-active flavonols

and the biomolecule. In this framework, it should be noticed that the protonation state of the OH groups play also a key-role for the antioxidant

properties of flavonols, the anions being more effective.38

Photochemistry, 2012, 40, 295–322 | 301



The fluorescence enhancement of flavonols upon binding to biomolecules

has been recently exploited for the development of analytical methods39 and

in microfluorescence studies using flavonols as endogenous fluorophores.40

In particular, the use of quercetin and other flavonols as probes in vivo for

target proteins has been reported by Gutzeit et al.40a

A particularly interesting feature is represented by 5-OH substituted

flavonols such as quercetin (1) and morin (6, Fig. 5a). In these molecules,

the 5-OH group is able to hamper ESIPT from the 3-OH to the carbonyl

oxygen favouring internal conversion.41 These flavonols establish specific

interactions with their microenvironment, e.g. upon binding to biomolecules40b,42 or when placed in specific microenvironments such as Sodium

Dodecyl Sulfate (SDS) micelles43 and in lipid nanocapsules.44,45

This aspect has been investigated in details for 1. In particular, the

enhancement of quercetin fluorescence upon binding to Bovine Serum

Albumin (BSA)46 and to HSA47 has been attributed to the formation of a

fluorescent pyrilium-like structure, due to the strong similarity with the

fluorescence spectrum of quercetin dissolved in 1 M HBr in acetic acid. As

an alternative explanation, Sengupta et al. proposed that upon binding to

HSA the intramolecular hydrogen bond between the OH in position 5 and

the C¼O group is perturbed, favouring the ESIPT reaction.48 This phenomenon takes indeed place in EtOH glasses at 77 K under prolonged

irradiation.49 The same Authors, in a following paper, observed however a

second emitting species, which they attributed to a ‘‘ground-state complex

formed in the protein environment’’.50 Recently, Mezzetti and co-workers45

suggested, in an experimental and computational investigation, that a

deprotonation of an OH group induced by the protein binding pocket could

give a fluorescent quercetin anion, as commonly observed for hydroxyflavones upon binding to biomolecules.34,51 By comparison of fluorescence

and excitation spectra obtained at different pHs and in different solvents

(including solvents with sufficiently high HBD basicity to induce partial

deprotonation from one – or more – of the OH groups), quercetin anions

resulted strong emitters45 (whereas neutral quercetin is a weak one, characterized by a 3HF-like dual fluorescence). Taking into account experimental42 and literature38a values for pKa, the spectra recorded in aqueous

solutions at different pHs showed that the mono, the di- and the tri-anionic

form of quercetin are fluorescent.52 The paper thus suggested that the

deprotonation process (to give fluorescing anionic species) could also

account – at least partially - for other fluorescence enhancements of quercetin upon binding to biomolecules. Where available, literature molecular

modelling studies were exploited to propose a possible binding scenario.45

Modification of 1 fluorescence has been also reported upon binding to many

other biomolecules53 and in most cases seems to be related to protonation/

deprotonation processes or hydrogen bonding interactions.

4 Photophysical behavior of synthetic 3-hydroxyflavones

and their use as fluorescent probes

As mentioned above for the case of natural occurring flavonols, molecules

that undergo ESIPT processes are appealing for the development of

302 | Photochemistry, 2012, 40, 295–322



fluorescent probes.56 The connection between the absorption or emission

spectra of a molecule and the chemico-physical properties of its surrounding

microenvironment (especially in the case of complex and sometimes microheterogeneous systems such as solvent mixtures, micelles or biological

systems) can be fully explained only through a multiparametric approach

that takes into account different solvent properties such as the polarity and

electronic polarizability (that are function of the low-frequency dielectric

constant e and of the refractive index n respectively) as well as the presence

of specific intermolecular interactions such as hydrogen bonding.57 The

most important contribution to the development of fluorescent probes and

sensors58 able to respond to a slight modification of their surrounding

microenvironment by independently measured parameters has been offered

by Demchenko and co-workers56 with their innovative work on the

synthesis of flavonols with Excited State Intramolecular Charge Transfer

(ESICT) and Proton Transfer (ESIPT) coupled processes.59 Seminal studies

have been carried out on a prototypical system namely 4 0 -N,N-diethylamino-3-hydroxyflavones (9 in Scheme 4).

Irradiation of 9 (hn1) causes, after solvent stabilization of the normal

Frank Condon state, an ESICT taking place from the nitrogen lone pair of

the amino group to the carbonyl oxygen affording the excited 1N state.59 On

the other hand, analogously to other excited flavonols, ESIPT from 1N

could also take place giving the corresponding 1T state. Thus, whereas in

polar, aprotic solvents only the tautomer emission (1T) is significantly



Et

N+



O



Et

Et



OH

1N



ESIPT



O–



N+



1



Et



O

O–



1



T



OH

1. hν1,

2 ESICT



hνN



hνT

Et

N+

Et

N



Et



O

Et



O–



O



T



OH

OH



9



O

Scheme 4



Photochemistry, 2012, 40, 295–322 | 303



present for 3HF, dual emissions consisting of both CT (hnN) and PT (hnT)

bands can be observed for 9. The difference in the surrounding microenvironment strongly affects the relative energy of the involved excited

states resulting in a ESICT/ESIPT coupled process.59a,60 Due to their

peculiar photophysics, 9 and its derivatives have found a plethora of

applications in physical and organic chemistry as well as in biochemistry.

The photophysics of 9 in different organic solvents has been deeply investigated by Klymchenko and Demchenko with the aim of employing the

molecule as a multiparametric fluorescent probe.61 In particular, it has been

demonstrated that four spectroscopic parameters (absorption, 1N and 1T

emission maxima and the emission intensity ratio indicated as Log(I1N/I1T))

give a simultaneous estimation of the different properties of the microenvironment, including polarity, electronic polarizability and H-bond

donor ability. The photoprocesses occurring with 9 have been also investigated in imidazolium,62 ammonium and phosphonium63 based ionic

liquids, in supercritical carbon dioxide (scCO2)64 and in Aerosol OT (AOT)

Reverse Micelles.65 Different 4 0 -N,N-dialkyl-amino-3-hydroxyflavones (see

some examples in Fig. 6) have been employed to probe the different

microheterogeneous systems such as aqueous micelles generated by both

cationic surfactant (cethyltributyl ammonium bromide, CTAB) and neutral

Triton X-100. Interestingly, the examined dyes are incorporated into Triton

X-100 micelles hydrophobic core, while in CTAB micelles the fluorophore

has found to be located on the polar surface of the micelle with the 4-carbonyl group interacting with the solvent by means of hydrogen bond.66

The solvatochromogenitic properties of 9 have suggested its employment

as probe in spectroscopic analytical chemistry. The determination of water

in low concentrations in reagent-grade acetone by means of N,N-dimethylamino derivative 10 (Fig. 7) and of the corresponding 4 0 -N,N-dimethylaminoflavone-3-yl methacrylate 11 has been proposed by Liu et al.,67 and



R1

N



R1



O

OH



R = C4H9, C8H17, C12H25



O

Fig. 6



NMe2

O

OR



10, R = H

11, R = CH2=CH2C(CH3)CO–



O

Fig. 7



304 | Photochemistry, 2012, 40, 295–322



the results obtained are comparable to those ensured by traditional methods

(viz. gas-chromatography).

Due to the efficient response to the dramatic changes in local polarity

imposed by water, 10 has been efficiently employed as dual band ratiometric

probe (log I1N/I1T) for monitoring water uptake in thermo-responsive

hydrophilic films of poly(N-isopropylacrylamide (1–100 mm).68 The formation of a 1:2 associate between 10 and adenosine-5 0 -triphosphate (ATP) and

the consequent ground state deprotonation of 10 induced by the tetracharged ATP anion has been observed by Yushchenko et al. and exploited

for the development of a spectroscopic sensing of ATP.69 Furthermore, no

interference by other nucleotide triphosphates has been observed, making

10 a promising probe for the detection of ATP both in vitro and in vivo

conditions.

The photophysical properties (and as consequence, the sensing efficiency)

of the 3HF based dyes can be tuned by introducing different substituents

at the 2- and 7- position of the flavone core as well as by substituting the

2-phenyl ring for 2-(2-benzo[b]furanyl) ring (12, Fig. 8).70,71 In particular,

the presence of the electrondonating 7-methoxy group (13) caused a shift of

the 1N and 1T emission maxima in opposite directions, along with a strong

decrease of the I1N/I1T value and an increase of the fluorescent quantum

yield (with respect to parent 9).70

The observed behaviour pointed out the significant influence of substituent in position 7 on the dual emission properties of 4 0 -N,N-dialkylamino-substituted 3HF,72 and suggested to Yushchenko et al. the

development of ESIPT based switchers based on the variation of the electronic properties of the substituent at that position. The 7-isothiocyanate

derivative of 3-hydroxyflavone (14, Scheme 5) smoothly reacted with

amines to afford the corresponding thioureas 15. The conversion of electron



Et

Et



N Et



N

O



MeO



O



O

OH



OH

12



O



Et



13



O

Fig. 8



S



NEt2

C



N



O

R-NH2

OH



14



NEt2



R



–H+



O



H

N



HN



O



S



OH



15



O

Scheme 5



Photochemistry, 2012, 40, 295–322 | 305



Et

N



Et



O



OH

O



16



Fig. 9



acceptor substituent into an electron donor one involves a dramatic change

in both absorption and dual emission favouring ESIPT rather than emission

from 1N and making the molecule a suitable sensor for NH2 groups both in

synthetic compounds and in biomolecules.72

The presence of an additional benzene ring in 5,6-benzo-4 0 -N,N-diethylamino-3-hydroxyflavone (16, Fig. 9) inhibits intermolecular hydrogen bond

formation with protic solvents, yet allowing, at the same time, the photoinduced intramolecular proton transfer with the hydroxyl group. These

properties permitted to set up a linear correlation of the log (I1N/I1T) value

with the solvent polarity function (f(e)) that could be extended to all of the

examined solvents, contrary to the case of parent 9, where only a correlation

with aprotic media was found.73

The introduction of a charged group without p-electronic conjugation in

3HF chromophores lead to the modification of both the absorption and of

the dual emission spectra. The observed shift can be explained by the presence of an internal Stark effect due to the influence of the electric field

produced by the charged substituent.74 As described for compound 9,

irradiation of ammonium substituted flavone 17 (Scheme 6a) produces a 1N

state in which the chromophore induced a charge separation with the

negative charge located on the 4-carbonyl group and the positive charge

distributed between the pyran ring oxygen atom and the amino group.

Thus, due to the electrostatic interaction of the ammonium group and the

4-carbonyl, the 1N emission bands is shifted to the red and the I1N/I1T value

results higher than that of the neutral parent 9.75 When ESIPT occurs, the

charge distribution in the molecule changes, and the negative charge

migrates to the 3-hydroxy group. Whereas the electrostatic stabilization of

the 1T state is lacking, the new charge distribution causes a dipole oriented

against the field over the charged substituent. The resulting increase in the

energy of this state lead to a red-shift of the emission band assigned to the

1

T state.74

Interestingly, a slight modification in the dielectric constant of the surrounding microenvironment was found to induce a dramatic variation in

the relative intensities of both fluorescence emission bands, testifying the

strong influence of internal electric field on ESIPT process. As an example,

the irreversible substitution of the counter ion bromide with BPh4 À caused

by the addition of NaBPh4 to a chloroform solution of 17 lead to a system in

which the ammonium cation is less efficiently screened (Scheme 6b). Thus,

the increased interaction of the ammonium group with the carbonyl caused

a red shift in both the absorption and the emission maxima, together with a

306 | Photochemistry, 2012, 40, 295–322



(a)



Et

N+

Et





O



Br –



OH

N+



C8H17





O



ESIPT



Br –



1N



O–



C8H17



Et

N+

Et



O–

N+



1T



OH



hνT

hνN



hν1

NEt2

O



NEt2

O

Br –

C8H17



C8H17



OH

N+



O–



Br –



BT



N+



OH+



O

17



(b)



NEt2



NEt2

O

Br –

C8H17



NaPPh4

OH



N+



O

17



PPh4–



OH



CHCl3

-NaBr



O



C8H17



N+



O



Scheme 6



doubling of the I1N/I1T emission ratio. The dramatic change in the emission

color observed suggested its use as a new ultrasensitive electrochromic twowavelength emission probe for several applications.74

Zhao et al. carried out a spectroscopic investigation of differently

ammonium substituted 3-hydroxyflavones, finding a W100 fold increase in

quantum yield in organic solvents (chloroform, acetonitrile) and in dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles in comparison to acqueous Hepes buffered solution (pH=7.4). The obtained results

suggested the ability of ammonium derivatives to act as efficient probes

for changes in dipole potential occurring in biological systems such as

biomembranes.76 A set of 3-hydroxyflavones substituted with positively

charged groups (see some examples in Fig. 10) has been investigated as two

color-fluorescent probe to monitor the dipole potential cd value of both

phospholipids and cell plasma membranes.77,78 The use of these dyes allows

the generation, by a single wavelength excitation, of two separate emissions

that can be collected on two separate detectors. Furthermore, the high

sensitivity of the examined probes (in comparison to the traditional and

Photochemistry, 2012, 40, 295–322 | 307



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3-Hydroxyflavone (3HF) as a model molecules for protontransfer processes

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