WO2017147646A1

WO2017147646A1 - Dual emission fluorescent compounds

Info

Publication number: WO2017147646A1
Application number: PCT/AU2017/050170
Authority: WO
Inventors: Peter Karuso; Soumit CHATTERJEE
Original assignee: Macquarie University
Priority date: 2016-02-29
Filing date: 2017-02-28
Publication date: 2017-09-08

Abstract

The invention relates to a dual emission fluorescent compound comprising a chromophore suitable for exhibiting an emission at a first wavelength from an excited state; wherein the compound further comprises one or more groups suitable for inducing a structural rearrangement in the molecule on formation of the excited state, wherein the structural rearrangement stabilises at least a second electromagnetic emission at a second wavelength, such that dual emission is observable at room temperature. Such dual emission allows for the super sensitive ratiometric quantification of analytes.

Description

Dual emission fluorescent compounds

Field of the invention

The invention relates to fluorescent compounds, their synthesis, as well as uses thereof. In particular, the invention relates to dual fluorescent compounds capable of emitting at two different wavelengths that are suitable for use in biotechnological applications, for example, as sensors or probes.

Background

Fluorescent molecules are responsible for the recent increase in our capacity to sensitively monitor biological systems at the cellular and subcellular level. An ideal probe combines good photophysical properties (including high extinction coefficient, high quantum yield, long Stokes' shift and long lifetimes), emits in the visible or near infrared, has high sensitivity toward specific ligands or environments and low (ph ototoxicity. Another demanding feature is the capability to provide a quantitative fluorescence signal irrespective of excitation-light intensity or probe concentration.

Most fluorescent probes rely on changes in fluorescence intensity as the reporting mechanism, for example, an increase in the non-radiative decay rates with increasing temperature that leads to diminished luminescence. However, intensity-based sensors are susceptible to errors due to changes in probe concentration, excitation or detection efficiency, the concentration and location of quenchers, aggregation, pH, temperature or a lack of specificity. Furthermore, changes in the sensor local environment are difficult to control in complicated systems such as biological systems or devices and can cause inaccurate measurement. True quantitative measurements are thus challenging, since it is difficult or impossible to calibrate a sensor's dose-response in-situ.

In this respect, one of the most appealing strategies involves the design of "self-calibrating" (ratiometric) fluorophores that display dual emission by combining excited state intramolecular proton transfer (ESIPT) with intramolecular charge transfer (ICT). Such fluorophores have a unique sensitivity to subtle variations in the local environment and the two-colour emission allows for accurate and sensitive ratiometric measurements that are independent of the probe concentration. This makes them very much more useful than fluorophores that just shift their emission or increase/decrease their emission in response to the local environment because ratiometric measurements are self calibrating and every measurement relies on two data points (the intensity are the two emission lines) instead of just one. Excitonic, excimer, and energy transfer interactions can yield fluorescent dual emission colour readouts but this requires two fluorophores to be in close proximity. Doped semiconductor nanocrystals/nanoparticles of various types have also been observed to have dual emission but dual emission in single small molecules at room temperature in water is extremely rare. Dual emission can also be the result of a chemical reaction (e.g. protonation/deprotonation, cis-/trans-isomerization) but this is not really dual emission as the two emitting species are not the same molecule.

There is thus a need for new and ever more effective fluorescent probes sensitive to biologically and environmentally relevant parameters, particularly, fluorescent indicators that allow ratiometric measurements by virtue of dual emission. Green fluorescence protein (GFP) is a unique genetically encoded fluorescent marker that comes in a variety of colours, and is widely used in molecular biology and biotechnology as a genetically encoded fluorescent tag. Many GFP variants have been discovered from a variety of organisms. Furthermore, various engineered mutants provide specific tailorable characteristics and colours. GFP variants tend to exhibit good extinction (from 20 - 50K) and quantum yields (≤ 80%).

GFP's strong green fluorescence (Φ _f = 0.8) is ascribed to its chromophore 4-(4-hydroxybenzylidene)- 1 ,2-dimethyl-1 H-imidazol-5(4H)-one (p-HBDI) (shown below), which is anchored by covalent and hydrogen bonds inside the β-barrel structure of the protein.

The photophysics of GFP involves excited state proton transfer (ESPT) to the E222 residue, through a proton relay of water molecules. The resulting anionic excited state of p-HBDI is believed to be responsible for the intense green fluorescence. As the active chromophore is protected inside the β-barrel structure of the protein, it can be relatively insensitive to the local environment.

Furthermore, applications are limited by the size of the GFP tag, its lack of cell permeability and the fact that hydrogen peroxide is produced in the maturation of the GFP chromophore. Organic fluorescent dyes are unique in their ability to report on subtle changes in all the major types of weak non-covalent intramolecular interactions. Small molecule mimetics of the chromophore p-HBDI would be useful as more sensitive tags. However, the isolated chromophore was found to be essentially nonfluorescent in its neutral or anion form at room temperature as the molecule experiences cis-/trans- conformational isomerisation around the exocyclic C=C double bond that result in activation of a major non-radiative deactivation pathway involving internal conversion to the ground state. This has been attributed to facile photoisomerization of the isolated chromophore (see Figure 1), resulting in an internal conversion to the ground state within 1 ps. Addressing this problem is important because the GFP chromophore (p-HBDI) is small and with the correct design elements could find application in electronics, flow cytometry, environmental sensing, protein labelling and super high resolution microscopy for example.

Previous design elements used to enhance fluorescence include adding steric bulk, and moving the para-hydroxyl group (p-HBDI) into the ortho-position (o-HBDI) to hinder φ/τ rotation (see Figure 1). Introducing structural rigidity into the GFP chromophore mimetics has resulted in a number of weakly emissive analogues. In structurally locked GFP chromophores, a five membered ring stops φ rotation and a seven membered hydrogen bonded ring inhibits τ rotation. Together, these modifications result in a dramatic enhancement of fluorescence for o-LHBDI (see o-HBDI in Figure 1) at room temperature (Φ _f = 0.18 in toluene), opening up the possibility of using these compounds in organic light emitting diodes, due to an amplified spontaneous emission.

More specifically, relocating the para-hydroxyl group to the ortho-position to form o-HBDI enables an intramolecular hydrogen bond between -OH and a N of the imidazol-4-one ring and T rotation inhibition through a seven membered hydrogen bonded ring which facilitates ultrafast excited state intramolecular proton transfer (ESIPT) taking place from the hydroxyl proton to the nitrogen resulting an emissive excited state proton transfer tautomer. Likewise, the introduction of certain ring structures to restrict C(3)-C(4)-C(5) rotation improves the tautomer excited state emission significantly, particularly with regards to quantum yield.

Structure of the GFP chromophore and conformationally restricted analogues

Indeed, Hsu et al. (J. Am. Chem. Soc. 2014, 136, 1 1805-1 1812) teaches that analogues having structural planarity and rigidity can provide particularly high emission quantum yield. For example, o-LHBDI, in which both φ/ τ rotation is impeded, exhibits a single red emission at 600 nm attributable to the zwitterion formed via excited state intramolecular proton transfer ('ESIPT, phenol proton to imidazolone nitrogen transfer).

In WO2010/096584, a novel class of fluorescent dyes based on a five-membered heterocyclic ring conjugated with a substituted aromatic group is described for use as analytical reagents. Fluorescence output for the dyes can be increased by binding to aptamers. No φ rotation groups are included, nor is any importance placed on τ rotation blocking substituents. Finally, there is no suggestion of molecules that can emit fluorescence at two different wavelengths.

The photophysics of the GFP chromophore continues to be an area of intense research and conflicting hypotheses but with the aim of producing small molecule fluorophores, based on the GFP chromophore, that fluoresce at room temperature. However, further emissive compounds are desirable. Furthermore, to date, no room temperature dual emission GFP chromophore mimetic molecules have been realised. Although advances have been made in the design and application of new fluorophores for sensing applications, there still exists a need for new compounds, particularly compounds that can be used for absolute quantification by virtue of ratiometric dual emission, having high extinction coefficients, high quantum yields, long Stokes' shifts and long lifetimes, emit in the visible or near infrared, and have high sensitivity toward specific ligands or environments and low (photo)toxicity.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

Statements of the invention

According to a first aspect, the invention provides a compound having general formula,

salts, esters, conformational and configurational isomers thereof, including zwitterions and tautomers thereof, in which,

Q is S or O; Y is N or O;

D is a proton donating group;

optional Ar, when present, is an optionally substituted monocyclic aryl ring, an optionally substituted bicyclic aryl ring system, an optionally substituted polycyclic aryl ring system, an optionally substituted monocyclic heteroaryl ring, an optionally substituted bicyclic heteroaryl ring system, or an optionally substituted polycyclic heteroaryl ring system;

optional R¹ , when present, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyi, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH₂, and -NC(NH₂)₂, an optionally substituted aryl and an optionally substituted heteroaryl ring, wherein b is 0, 1 , 2 or 3;

L is an optionally substituted carbocycle or an optionally substituted heterocycle ring, optionally fused to another aromatic or heteroaromatic ring, wherein n is 1 , 2, 3 or 4, and A is independently selected from the group consisting of: -CH₂-, -CR₂ ^*-, =CH-, -CH=, -N=, =N-, -NR -, -0-, C₆H₄, a fused optionally phenyl, where the phenyl can be optionally substituted with hydroxyl, NR₂ ^**; wherein R^* is independently H or C₁-C₃ alkyi, where the alkyi is optionally substituted with hydroxyl, halogen, carboxylic acid or sulfonic acid, and wherein R^™ is alkyi or aryl;

R⁴, optionally present when Y is N, is selected from the group consisting of: H, optionally substituted alkyi, optionally substituted aryl and optionally substituted heteroaryl;

R⁵ is a bulky substituent having more than four non-hydrogen atoms; and

optional R⁶, when present on each Ar ring, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyi, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH₂ -NHC(0)NH₂ and -NHC(S)NH₂, optionally substituted carbocycle or optionally substituted heterocycle ring, wherein e is 0, 1 , 2, 3 or 4.

Optional substituents as described herein include methyl, chloro, methoxyl, nitro, cyano, hydroxy or NR₂, where R is independently selected from H, or C₁-C₄ alkyi.

The ring L is a locking ring for preventing rotation and isomerisation around the exocyclic double bond in the excited state that prevents φ rotation. Preferably, L is a 4, 5 or 6-membered ring, preferably, a carbocyclic or heterocyclic ring, which may be optionally substituted. When the compound is in the Z-isomer configuration, and D is a proton donating group, a 7- membered ring is formed through hydrogen bonding from the proton donating group to N* of the heterocyclic ring. The 7-membered ring prevents τ rotation and in general improves fluorescence.

In a preferred embodiment, when present, the optional Ar is phenyl, thereby providing a napthyl component to the compounds of the invention. However, in particularly preferred embodiments, the Ar group is absent entirely.

Suitably, the amine substituent on R¹ and R⁶ as described herein can be a primary amine (-NH₂), a secondary amine (-NHR) or a tertiary amine (-NR₂), wherein R is alkyl or aryl.

Suitably, the amide substituent on R¹ and R⁶ as described herein can be a primary amide (-CONH₂), a secondary amide (-CONHR) or a tertiary amide (-CONR₂), wherein R is alkyl or aryl.

The halo group as described herein is preferably selected from chloro, fluoro, bromo and iodo. However, fluoro is preferred.

Desirably, where described herein, preferably, alkyl is C_1-10 alkyl, alkoxy is C_1-10 alkoxy, alkenyl is ₁₀ alkenyl, alkynyl is C_1-10 alkynyl. Methyl, ethyl, propyl, butyl groups are preferred, and may be branched or unbranched. Preferably, the alkyl group is a branched alkyl group. Methyl groups are particularly preferred. Particularly preferred branched alkyl groups include t-butyl or neopentyl.

In a preferred embodiment, b is 0 such that R¹ is absent.

As explained above, D is a proton donating group. A suitable proton donating group is one capable of hydrogen bonding, to N* of the 5-membered ring, as well as intramolecular proton transfer. Preferably, D is selected from the group consisting of: -OH, or -NH₂, -SH, -NHC(NH)NH₂ -NHC(0)NH₂ and -NHC(S)NH₂. More preferably, D is -OH, -NH₂, or -SH. Most preferably, D is -OH.

When Ar is present, D is preferably located at the ortho phenyl ring position, wherein the phenyl is adjacent to Ar, and closest to L.

However, when the Ar group is not present, the D substituent preferably takes an ortho or meta position on the phenyl ring. In certain embodiments, D is located on the aromatic ring (Ar) and in these cases, D is always in a position, preferably ortho, of that aromatic ring such that a hydrogen bond can be formed to N*.

Likewise, when Ar is absent, the R¹ substituent can be in any unoccupied ortho, meta or para position on the phenyl ring, however, most preferably, R¹ is in the para position.

A preferred compound has general formula:

In particularly a preferred compound, Q is O, R⁴ is Me, and R⁵ is phenyl, thereby providing compounds having the following general structures:

As explained above, L is an optionally substituted carbocycle or an optionally substituted heterocycle ring, optionally fused to another aromatic or heteroaromatic ring, wherein n is 1 , 2, 3 or 4, and A is independently selected from the group consisting of: -CH₂-, -CR₂ ^*-, =CH-, -CH=, -N=, =N-, -NR -, -0-, C₆H₄, a fused optionally phenyl, where the phenyl can be optionally substituted with hydroxyl, NR₂ ^**; wherein R^* is independently H or C_rC₃ alkyi, where the alkyi is optionally substituted with hydroxyl, halogen, carboxylic acid or sulfonic acid, and wherein R^** is alkyi or aryl.

However, in a preferred embodiment, L is a 4, 5 or 6-membered ring, preferably, a carbocyclic or heterocyclic ring, more preferably, a saturated carbocyclic or heterocyclic ring, which may be optionally substituted. In a preferred embodiment, n is 1 , 2, 3 or 4. As explained above, A may be independently selected from the group consisting of: -CH₂-, -CR₂ ^*-, =CH-, -CH=, -N=, =N-, -NR -, -0-, C₆H₄, a fused optionally substitued phenyl, where the phenyl can be optionally substituted with hydroxyl, NR₂ ^**; wherein R^* is independently H or C₁-C₃ alkyi, halogen or carboxyl, and wherein R^™ is H, alkyi or aryl. In a further preferred embodiment, A and n are selected such that group (A)_n is -CHCH-, wherein the locking ring L is a 5-membered ring. In another embodiment, A and n are selected such that, (A)_n is - CH₂CH₂-, wherein the locking ring L is a 5 membered ring. In another embodiment, A and n are selected such that (A)_n is -CR₂CR₂CR₂-, wherein the locking ring L is a 6 membered ring. In this case, R of (A)_n can independently be chosen from H, alkyl or aryl such that at least 2 of the substituents are not H.

Suitably, L is a 4, 5 or 6 membered ring, which is optionally fused to another aromatic ring, for example, an optionally substituted aryl ring, preferably a phenol ring.

Preferably, Y is N, and R⁴ is present and is selected from the group consisting of: H, optionally substituted alkyl, optionally substituted aryl and optionally substituted heteroaryl.

Preferably, R⁵ is a bulky substituent having more than four non-hydrogen atoms, more preferably more than five non-hydrogen atoms, more than six non-hydrogen atoms, or more preferably more than ten non-hydrogen atoms. It will be appreciated that within the context of the present invention a bulky substituent is one that is of a sufficient size to slow down molecular structural and/or conformational rearrangements that take place in an excited state, particularly slow enough such that the lifetime of an associated Franck Condon emission is longer than the time required for formation of an emissive state, for example, an emissive state associated with formation of a ESIPT zwitterion. Preferably, R⁵ is selected from the group consisting of: an optionally substituted cycloalkyi ring, an optionally substituted heteroalkyi ring, an optionally substituted aromatic ring or ring system or an optionally substituted heteroaryl ring or ring system, or -C(R⁹)₃, wherein R⁹ is independently selected from optionally substituted alkyl, optionally substituted carbocycle or optionally substituted heterocycle. Preferably, R⁵ is -C(R⁹)₃, wherein R⁹ is independently selected from ₄alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyi or optionally substituted heterocycloalkyl. Desirably, -C(R⁹)₃ is selected from the group consisting of: a branched C_1-4alkyl, phenyl, napthyl, pydridyl, cyclohexyl. Preferably, R⁵ is selected from the group consisting of: butyl, pentyl. Most preferably, the branched C^alkyl is tert-butyl or neo-pentyl. More preferably, R⁵ is selected from an optionally substituted monocyclic aryl, fused bicyclic aryl ring system, fused polycyclic aryl ring system or a monocyclic heteroaryl ring, fused bicyclic heteroaryl ring system, or a fused polycyclic heteroaryl ring system. Preferably, R₅ is selected from the group consisting of: phenyl, napthyl, furanyl, thiophenyl, pyrrolyl, pyridinyl, imidazolyl, thiazolyl, pyrimidinyl, oxazolyl, isoxazolyl, tetrahydrofuran, pyrrolidinyl, pyranyl, piperidinyl, dioxanyl, morpholyl, pyrazolyl, pyridazinyl, Indolyl, isoindolyl, indolizinyl, quinolyl, isoquinolyl, purinyl, carbazolyl, dibenzofuranyl, 2H-chromenyl, and xanthenyl. Most preferably, R⁵ is unsubstitued phenyl.

In embodiments wherein A and n are selected such that (A)_n is -CH₂CH₂-, Y is N, and, R⁵ is not methyl.

The most preferred compounds of the invention may be selected from the group consisting of:

Suitably, the compound comprises one or more optional, linker groups, G, for attaching the compound to a target compound, wherein G is selected from C₃-C₂₀ alkyl, a polypeptide or polyethyleneglycol and wherein G is located at one or more of R¹ , R⁴ or R⁵.

The target compound may be selected from, biomolecules, organelles, cells, tissues, and other molecules such as, but not limited to metal ions, lipids and cofactors.

The compounds of the invention include a conjugated portion or conjugated functional group, typically involving a conjugated π-system comprising alternating single and double bonds having delocalised % electrons across aligned p-orbitals of the π-system, being capable of absorption and emission of electromagnetic radiation. In the context of absorption, the conjugated functional group is known as a chromophore, while for emission, the conjugated functional group in an excited state is a fluorophore.

Preferred compounds of the invention absorb electromagnetic radiation of at least one wavelength, however, dual, tri or multiple absorbing compounds are also encompassed by the invention. Particularly preferred compounds emit electromagnetic radiation of at least one wavelength after excitation. Indeed, the most preferred compounds are 'dual emission' compounds that emit electromagnetic radiation of at least two different wavelengths. Suitably, the compounds of the invention are modelled on the GFP chromophore.

It will be understood that the compounds include zwitterions and tautomer isomeric forms thereof. Suitably, D donates a proton to N* of the 5 membered ring to form a tautomer/zwitterion configurational isomers of the above compound. It will also be understood that conformational isomers of the compounds include E- and Z- isomers may exist around the exocyclic bond of the compounds described herein. While the above general formulas correspond to Z-isomers in particular, stable E-isomers are included within the scope of the invention.

Also described herein is a compound having the following general formula, salts, esters, conformational and configurations isomers thereof, including zwitterions and tautomers thereof:

in which,

Q is S or O; Y is N or O; Z is N; and X is N or CH, wherein c is 0 or 1 ;

optional D, when present, is a proton donating group;

optional Ar, is an optionally substituted monocyclic aryl ring, an optionally substituted bicyclic aryl ring system, an optionally substituted polycyclic aryl ring system or an optionally substituted monocyclic heteroaryl ring, an optionally substituted bicyclic heteroaryl ring system, an optionally substituted polycyclic heteroaryl ring system;

optional R¹ , when present, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyi, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH_2, -NHC(0)NH₂ and -NHC(S)NH₂, an optionally substituted aryl and an optionally substituted heteroaryl ring, wherein b is 0, 1 , 2 or 3;

R² is H, or optionally substituted alkyl;

R³ is optionally substituted alkyl; or

R² and R³ together with the shared bonds from the adjacent phenyl ring form a carbocycle or heterocycle ring, L, as described elsewhere herein;

R⁴, optionally present when Y is N, is selected from the group consisting of: H, optionally substituted alkyl, optionally substituted aryl and optionally substituted heteroaryl;

R⁵ is a bulky substituent having more than four non-hydrogen atoms; and

optional R⁶, when present on each Ar ring, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyi, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH_2, -NHC(0)NH₂ and -NHC(S)NH₂, optionally substituted carbocycle or optionally substituted heterocycle ring, wherein e is 0, 1 , 2, 3 or 4.

Preferably, R¹ is independently selected from the group consisting of: H, acetate, dialkylamine, halo, preferably fluoro. Suitably, R² is H or alkyl. Preferably the dialkylamine is dimethylamine. Suitably, the halo is fluoro. Suitably R⁴ is methyl. Desirably, R⁵ is phenyl. Preferably, R¹ is selected from the group consisting of: H, acetate (OAc), and fluoro.

However, in embodiments, where R² and R³ form a carbocyclic or heterocyclic ring and D is -OH in the ortho position, it is preferred that R⁴ is an alkyl group selected from the group consisting of: C₂ _ ₁₀ alkyl. Particularly preferred branched alkyl groups include t-butyl or neopentyl.

Preferred compounds include: (Ά) Unlocked oxazolines

In some embodiments, where the group R⁵ is phenyl, R³ may not be methyl. For example, one or more of the following compounds may be excluded from the compound per scope of the invention:

However, these compounds may be suitable for one or more applications of the invention as described herein.

Preferred compounds - emission and/or excitation

The preferred compounds of the invention absorb light of at least one wavelength and preferably transition from the ground state to one or more excited states. Particularly preferred compounds of the invention also emit light of at least one wavelength, preferably by a fluorescence process, on relaxation to the ground state. The most preferred compounds of the further emit light of at least a second wavelength. Indeed, the most preferred compounds of the invention are dual emission compounds. In this regard, it will be understood that the preferred compounds of the invention comprise a conjugated functional group capable of absorbing and emitting light, each of at least one wavelength and more particularly capable of emitting light of at least two wavelengths upon relaxation from an excited state to the ground state. It should be noted that in such compounds all emissions occur from the same excited chromophore (i.e., the conjugated functional group or fluorophore which is formed from the chromophore when in the excited state) which is also responsible for excitation by absorption. In other words, dual emission occurs from the same fluorophore. It will be understood that where dual emission occurs, the first and second emission wavelengths are different. Desirably, the dual emission occurs from the same fluorophore which arises from excitation of the ground state conjugated functional group or chromophore. Desirably, the molecule contains only a single conjugated functional group capable of behaving as a chromophore. However, if more than one chromophore is present, it will be understood that the dual or multiple emissions arise from the same (a single) chromophore. This effect is thereby distinguished from dual or multiple emission compounds in which the dual/multiple emission arises from a series of single emissions from several chromophores that may be present in the molecule.

Where the emission process is a fluorescence process, it will be understood that the fluorescence is associated with allowed transitions with a lifetime in the nanosecond range that arise from higher to lower excited singlet states of the molecule. Short relaxation times are advantageous for time-resolved dynamic processes and, for example, in super resolution microscopy.

Preferably, the conjugated functional group or chromophore of the preferred compounds of the invention is excitable upon absorption of suitable energy to promote an electronic transition from the ground state to a higher energy state, causing the molecule to reside for a short time in an excited state. It will be understood that where different wavelengths simultaneously supplied cause excitation, a given proportion of the molecules present can simultaneously be in different excited states.

Typically, the starting energy state corresponds to the ground state (S₀, the highest occupied molecular orbital (HOMO)) when the molecule is at room temperature. The higher energy state is preferably the first excited singlet state, the second excited state, S₂, or the third excited state, S₃, etc., depending on the wavelength of the light supplied. Any of the electronic energy levels, S₀, S₂, etc., may have one or more associated sublevels that arise from specific vibrational and/or rotational states possible for the molecule. Desirably, the first energy level (LUMO) is the first excited state (S^ associated with the compound.

To cause an electronic transition from a lower to higher energy state, the energy provided to the chromophore in the form of one or two nearly simultaneous photons must be of sufficient energy to promote at least one molecular orbital electron from a first starting energy state to a second or subsequent energy state ('S1 ', 'S2', 'S3', for example). In other words, the excitation energy absorbed by the chromophore must be at least equal to the energy difference between allowable energy levels/states (bandgap).

The energy of the light, in the form of a photon or multiple photons, is determined by the combined energy of the incident photons. The wavelength must match the energy gap of an allowed transition for the molecular to become excited. Indeed, the compound may absorb light of more than one wavelength if more than one transition is possible. It will be understood that the excitation wavelength can be of any wavelength in the range of from 300 to 1200 nm, depending on the degree of conjugation in the chromophore, any influencing substituents, and whether 2-photon excitation is used that can affect the bandgap and therefore the wavelength of emission and/or absorption. Preferably, the excitation may result from the absorption of visible light or other electromagnetic radiation, for example, UV, UV-VIS or near IR radiation of suitable wavelength to cause an allowable transition, for example, from at least S₀ - or S₀ - S₂.

Suitably, the excitation radiation has a wavelength > 300 nm. Below a wavelength of 300 nm, a photon can have sufficient energy to ionise a molecule and thereby absorption of light of such short wavelengths can result in photochemical decomposition. Molecules that absorb longer wavelengths of around 700 nm and above can be thermally and photochemically unstable, and tend to react with solvent, dissolved oxygen, impurities, etc., upon excitation. Therefore, the compounds of the invention preferably absorb radiation in the 300 nm to 700 nm range.

Likewise, the electromagnetic radiation emitted by the preferred compounds of the invention may be in the about 400 nm to about 800 nm wavelength range.

In preferred compounds of the invention, a significant change in the geometry of the chromophore in the ground state compared to that of the excited fluorophore preferably occurs. When the conformational changes that occur in going from the starting to final geometries are sufficient slow, emission from one or more further emissive states may become observable at room temperature. For example, in preferred compounds of the invention, a conformational change to a more planar structure can allow ESIPT but this takes some time. In the meantime normal Franck-Condon emission is observed resulting in dual emission (see Figure 30).

Room temperature dual emission compounds

A further aspect of the invention provides a compound comprising:

a conjugated functional group for emitting electromagnetic radiation on excitation to at least a first energy state, wherein upon excitation, the compound attains an emissive ST excited state associated with emission of a first wavelength;

wherein the compound is such that the excited state can undergo a structural and/or conformational rearrangement to a second excited state associated with emission of a second wavelength.

Suitably, the compound attains the emissive ST excited state through an internal conversion process.

More suitably still, the structural and/or conformational rearrangement which occurs in the exited state is a tautomerism rearrangement. A preferred compound of the invention is a compound comprising, in a ground state, a chromophore for absorption of light which causes excitation of the compound from the ground state to at least the first excited energy state associated with emission of electromagnetic radiation of a first wavelength,

wherein the compound is adapted such that upon excitation to at least the first excited energy state, electromagnetic radiation of a first wavelength is emitted and the compound undergoes a structural and/or conformational rearrangement that stabilises an additional energy state for a sufficient period to allow the emission of electromagnetic radiation of a second wavelength.

Desirably, the emission of the first wavelength is a cyan emission. Suitably, the emission of the second wavelength is a red emission

In a further aspect of the invention, there is provided a dual emission fluorescent compound comprising

a chromophore suitable for exhibiting an emission at a first wavelength from an excited state;

wherein the compound further comprises one or more groups suitable for inducing a structural rearrangement in the molecule on formation of the excited state, wherein the structural rearrangement stabilises at least a second electromagnetic emission at a second wavelength, such that dual emission is observable at room temperature. Suitably, the first and second wavelengths are different.

Thus, particularly preferred compounds of the invention represent a new class of fluorescent compounds that display dual emission at two wavelengths at room temperature. Desirably the dual emission occurs at room temperatures of from about 10 °C to about 40 °C.

Desirably, the compounds are based on the GFP chromophore or derivatives thereof. Suitable structural features of the GFP chromophore mimetic, as well as suitable groups for inducing dual emission have been described herein. Most preferably, the chromophore may be a derivative of the GFP chromophore that has been tailored to induce dual emission.

In a preferred embodiment, the compound comprises: at least one conjugated functional groups excitable from the ground state, S₀, to at least a first excited state, S^ wherein upon excitation to the first excited state, S^ the compound undergoes a structural and/or conformational rearrangement resulting in a further emissive state associated with S^ relaxation from which, produces emissions of electromagnetic radiation at two wavelengths. Desirably, the structural and/or conformational rearrangement to the further energy state associated with ST is sufficiently slow to stabilise the additional energy state associated with ST such that the emission at the second wavelength can be detected at room temperature.

Suitably, the structural and/or conformational rearrangement to the further energy state associated with ST results from an intramolecular rearrangement, preferably, ESIPT. Desirably, the additional energy state associated with ST is stabilised such that the emission at the second wavelength, preferably, a Franck Condon emission, can be observed.

Preferably, the one or more structural rearrangements are slow enough to stabilise the second energy states for a sufficient period to allow the emission of the electromagnetic radiation. It has been found that the speed of, or duration of time required from start to completion of each structural rearrangement is important for observation of room temperature dual emission. In this regard, the provision of bulky groups or substituents at certain positions on the molecule has been found to slow down the rate of certain structural and/or conformation rearrangements sufficiently to allow stabilisation of one or more further emissive states, particularly at room temperature.

In a preferred embodiment, the structural rearrangement arises from the inclusion of a bulky group which, on formation of the excited and/or subsequent excited states, slows down geometrical adjustment of the compound from the first geometry to the second substantially less planar geometry.

Desirably, the compound comprises one or more functional groups of such nature that they significantly slow down the structural rearrangement of the molecular for the desired duration of time to facilitate the otherwise unobservable room temperature emission of the electromagnetic radiation of the second wavelength.

Suitably, molecule further comprises one or more bulky groups as substituents on and/or near the chromophore. The bulky substituents are suitable for inducing a slow structural rearrangement in the molecule on formation of the excited and/or subsequent excited states. Bulky groups or substituent have been described herein.

Suitably, the chromophore of the invention can be repeatedly excited and detected and thus leads to a high sensitivity in luminescent detection techniques.

In the above preferred compounds, suitably, at least one emission, but more preferably, all of the emissions, occur at room temperature.

Suitably, the electromagnetic radiation emissions at the first and second wavelengths are at a longer wavelength (lower energy) than the one or more excitation wavelengths, resulting in a fluorescence process involving a Stokes' shift.

It will be understood that the conjugated functional group is excited to at least first energy state by absorption of light of at least one wavelength suitable to cause excitation, which it is understood included 2- photon excitation and multiple-photon excitation. Desirably, the excitation involves the transition of an electron from the ground state (S₀) of the compound to at least a first excited state (S^, although, where excitation by more than one wavelength is induced, there can be a further electronic transitions to a higher excited state, for example, S₂. In this case, internal conversion results in relaxation to S^

The structural and/or conformational rearrangement induced by excitation arises from the tendency of the compound to adopt a lowest energy configuration having a minimised potential energy. The structural and/or conformational rearrangement may involve one or more of the following rearrangement processes: isomerisation, intramolecular proton transfer which may lead to tautomer and/or zwitterion formation, bending, twisting of the fluorophore to accommodate geometrical changes, or bending and/or twisting of substituents attached thereto to further reduce the overall potential energy associated with the molecule in the excited state.

Thus the compound exhibits dual emission arising from the normal Franck-Condon ("FC") state and the excited-state intramolecular proton transfer ("ESIPT") state. The conformational changes required means the ESIPT state takes some time to attain and during the process the normal Franck Condon emission can be observed. The bulky group is selected such that attainment of the emissive ESIPT state is slow by virtue of a structural rearrangement during the excited state to form the emissive ESIPT state. During this time, normal room temperature Franck-Condon emission is observed. Suitably, slow reorientation of the bulky group allows both the FC and ESIPT emissions to be observed simultaneously. Such simultaneous dual emission has not been previously observed in GFP mimetic compounds. o-LHBPI has facile ESIPT and it is thought that the zwitterionic tautomer is formed on in a timescale that is so fast, the normal Franck Condon emission cannot be observed, at least at room temperature.

It will be appreciated that subtle changes in the local environment can result in dramatic changes in the proportion of FC and ESIPT emissions. Thus, these compounds advantageously allow very sensitive ratiometric, fluorescence measurements to be made based on these emissions. Ratiometric measurements are independent of the probe concentration or other variables that make quantitation of single emission fluorophores difficult.

It is preferred that the molecules are designed to undergo substantial structural rearrangement from the ground to first and/or further excited states. These may include the proton moving during ESIPT, but more notably, the bulky group must rotate to become more planar such that other parts of the molecule must also rotate to accommodate changes arising from the spatial arrangement of the bulky group. Indeed, the most preferred compounds of the invention involve an excited state in which the whole π-system has far from planar geometry. The non-planar nature of the excited state is quite unlike that observed for o-LHBDI.

It will be appreciated that subtle changes in the local environment can result in dramatic changes in the proportion of dual or multiple emissions observed using the compounds of the invention. Thus, these compounds advantageously allow very sensitive ratiometric, fluorescence measurements to be made based on the observable emissions.

Suitably, the conjugated functional group or chromophore, when the molecule is in the ground state, is a structurally locked GFP chromophore or derivative thereof. By 'structurally locked', it is meant that one or more portions of the molecule experience a high barrier to rotation around one or more bonds. In particular, where the compound comprises a GFP chromophore or derivative thereof, a structurally locked GFP chromophore means that rotation around the chromophore's exocyclic double bond is hindered. Advantageously, locking the GFP structure to prevent exocyclic double bond rotation (τ rotation) and single bond (Φ rotation) in preferred compounds has been found to result in room temperature dual fluorescence.

The structure may be locked by including the rotatable bond in a bond locking functional group, for example, a ring structure at the single bond between C4 and C5 (Φ rotation) of the molecule. By making the single bond part of a ring, this effectively locks either atom on each end of the rotatable bond into a non rotatable conformation, thereby preventing Φ rotation. Smaller rings are particularly preferred as they are less flexible that larger rings. By including a proton donating group in molecule, intramolecular H-bonding can lead to a 7-membered ring that stops τ rotation.

Suitably, preferred compounds of the invention have an associated quantum yield (Φ) that is 500 times greater than that of unlocked analogues in which bond rotation around the exocyclic bond can occur on suitable excitation. It will be appreciated that the quantum yield (Φ) corresponds to the number of emissions per photon absorbed by the system. Desirably, this quantum yield is achievable in water. In compounds having a chromophore comprising a double bond, it will be understood that conformational Z-isomer and E-isomers (in the ground state) are possible. Furthermore, one of the ground state Z-isomer or E isomer may be more stable than the other. For example, in certain GFP analogues of the invention, the Z-isomer (in which τ = 180°) has been found to be more stable than the E-isomer (τ = 0°). Thus, suitably, the Z-isomer may be preferred, for example, where dual emission is desirable, or vice versa, depending on which isomeric form is most stable. On excitation, one or other of the Z-isomer or E isomers may not be emissive, but may rearrange by a conformation isomerisation process initiated by excitation to an emissive isomer.

Desirably, the compounds of the invention exhibit a large increase in observed quantum yield in certain solvents, particularly, protic solvents, most preferably, water. The increased quantum yield is thought to occur where the excited state involves a conjugated functional group adopting a non-planar configuration (that is induced in the excited state) a proton relay can occur. This is in contrast to compounds that remain in a relatively planar conformation during excited state. Such a proton relay can be particularly favoured in water explaining the particularly efficient quantum yield (Φ) observed. This is demonstrated well by o-LHBPI in contrast with o-LHBDI, which do not comprise bulky groups at C2. The higher quantum yield of o-LHBPI in water indicates that H-bonding facilitated by the proton donating group (-OH) facilitates formation of the ESIPT state. Therefore, the compounds of the invention which demonstrate high quantum yields in water are expected to be particularly useful for applications in aqueous environments.

Furthermore, during use, where desired, one or more of dual emissions may be suppressed. For example, in the compound of GFP mimetic compounds of the invention, the ESIPT emission may be suppressed. For example, ESIPT emission suppression can result on application of conditions that result in deprotonation of the phenyl ring proton donating group. For example, at pH > 10, deprotonation occurs, thereby blocking the ESIPT emissive state as the necessary tautomer cannot form. It will be appreciated that suppression of at least one of the emissions allows the compound to behave as a switch, whereby the molecule can effectively be put into an On' or Off emission state.

An exemplary dual emission compound - o-LHBPI

In a particularly preferred embodiment, an exemplary dual emission compound is o-locked hydroxybenzylidene-p-phenylimidazolinone (o-LHBPI). This compound has been found to exhibit a particularly effective dual emission at room temperature. Room temperature dual emission has not been observed for o-HBDI or o-LHBDI.

The compound (o-LHBPI) has a phenyl group at position C2 of the imidazolone ring and a proton donating hydroxy group at the ortho position of the phenyl chromophore ring (providing a phenol functionality). Indeed, o-LHBPI has an associated very high value pKa of 10, which indicates that the phenol is involved in strong intramolecular H bond formation in o-LHBPI, providing a 7 membered locked ring that stops T rotation, in addition to the 5 membered locked ring that stops Φ rotation.

The compound o-LHBPI exhibits dual emission at approximately 475 (cyan) nm and approximately 610 (red) nm. The 610 nm band can be attributed to an emissive state arising from an ESIPT conformational and/or structural rearrangement. As the relative fluorescence quantum yield of the ESIPT band is greatest in water, water is a preferred solvent particularly for biological probe applications. o-LHBPI may be excited on absorption of the light of wavelength of approximately 370 nm and/or approximately 410 nm.

In o-LHBDI, the further energy state associated with ST arises from the formation of a zwitterion by ESIPT of the phenol proton to the heteroatom of the 5 membered ring, and is associated with a red emission of approximately 600 nm. Likewise, excitation of o-LHBPI from the ground state to the first energy state induce an ESIPT type structural and/or conformational rearrangement that produces an emissive zwitterion that is associated with a red emission of approximately 610 nm. The ESIPT structural and/or conformational rearrangement in o-LHBDI is so facile at room temperature, the second emissive state, for example, a Franck Condon emission, cannot be observed before formation of the emissive zwitterion. Therefore, emission of electromagnetic radiation at a second wavelength is not observed for o-LHBDI at room temperature. For example, the emission from the Franck Condon state in o-LHBDI may be in the cyan region, that is, at approximately 470 nm but is not observed without the appropriate design.

In contrast, in o-LHBPI, the structural and/or conformational rearrangement takes places slowly enough so that the lifetime of the Franck Condon emission is longer than the time required for formation of the emissive ESIPT zwitterion.

Unlike the known C2 methyl-substituted analogue, o-LHBDI, which has a single red emission at 600 nm arising from an excited-state intramolecular proton transfer ("ESIPT") state which results in the formation of a emissive zwitterion formed via ESIPT (phenol proton to imidazolone nitrogen), the bulky group at C2 of the imidazolone ring of o-LHBPI means the compound exhibits a dual emission, one red and the other cyan.

The dual emission arises from the presence of the bulky group at C2 in the Z-isomer. It is believed that the bulky group at C2, stabilises the duration of normal Franck-Condon ("FC") state, resulting the further cyan fluorescence, while the zwitterionic tautomer fluoresces red.

It will be appreciated that subtle changes in the local environment can result in dramatic changes in the proportion of FC and ESIPT emissions. Thus, these compounds advantageously allow very sensitive ratiometric, fluorescence measurements to be made based on these emissions.

In o-LHBPI, the bulky group is a phenyl group.

In the case of o-LHBPI, the Z-isomer (τ = 180°) has been found to be more stable than the E-isomer (τ = 0°). Suitably, the Z-isomer may be preferred, for example, where dual emission is desirable. In o-LHBPI, in the ground state, the conformation of the molecule is substantially planar, while the conformation in the first excited state arising from suitable excitation is non-planar. In other words, it is preferred that the molecules are designed to undergo substantial structural rearrangement from the ground to first and/or further excited states. These may include the proton moving during ESIPT, but more notably, the bulky group must rotate to become more planar such that other parts of the molecule must also rotate to accommodate changes arising from the spatial arrangement of the bulky group.

Indeed, the most preferred compounds of the invention involve an excited state in which the whole TT-system has far from planar geometry. The non-planar nature of the excited state is quite unlike that observed for analogue o-LHBDI.

o-LHBPI's ESIPT emission may be suppressed. For example, ESIPT emission suppression can result on application of conditions that result in deprotonation of the ring. (Figure 32, Figure 28, Figure 17). For example, at pH > 10, deprotonation occurs, thereby blocking the ESIPT emissive state as the necessary tautomer cannot form.

Designing dual/multiple emission compounds

In a further aspect, there is provided a method for preparing a dual emission fluorescent compound comprising the steps of:

(i) providing a compound having a first substantially nonplanar geometry in the ground state and an emissive first excited state; and

(ii) inducing a structural and/or conformational rearrangement in the compound from the first substantially nonplanar geometry to at least a second substantially planar geometry that is also emissive, wherein the duration of the structural rearrangement is such that a further emissive state is generated for a sufficient period to allow the emission of electromagnetic radiation at two wavelengths.

The invention also includes a method of designing a dual emission fluorescent compound comprising the steps of:

(i) providing a compound having a first substantially nonplanar geometry in the ground state and an emissive first excited state, preferably associated with an ESIPT emission; and

(ii) inducing a structural and/or conformational rearrangement in the compound from the first substantially nonplanar geometry to at least a second substantially planar geometry that is also emissive, wherein the rearrangement induced is such that the duration of the structural rearrangement is sufficient to generate a further emissive state, preferably associated with a Franck Condon emission, for a sufficient period of time to allow the emission of electromagnetic radiation at two wavelengths. It will be understood that the rearrangement may be facilitated by inclusion of certain, preferably bulky groups, as defined herein.

Suitably, the two (first and second) wavelengths are different. Desirably, dual emission occurs at room temperature. In a preferred embodiment, the structural and/or rearrangement arises from the inclusion in the compound of one more bulky groups which, on formation of the excited state, slows down geometrical adjustment of the compound from the first geometry to the second more planar geometry.

It will be appreciated that the one or more bulky groups are provided such that the time required for structural rearrangement is sufficient to stabilize the second emission at the at the second wavelength.

Preferably, the first electromagnetic emission at a first wavelength results from a normal FC excited state.

Desirably the substantially more planar configuration of the excited state is suitable for an ESIPT that allows observation of a second wavelength.

Preferably, the compound is a GFP chromophore mimetic. Suitable structural feature of the GFP chromophore mimetic, as well as suitable bulky groups for inducing the structural rearrangement have been described above.

Thus, the method preferably provides a means to design dual emission fluorescent probes that provide emission from the normal Franck-Condon ("FC") state and at least one excited-state intramolecular proton transfer ("ESIPT") state. It will be appreciated that the fluorescent probe is designed such that the ESIPT is slow by virtue of a spatial rearrangement of the excited state to form the one or more emissive ESIPT states. During the time required for the rearrangement, normal FC emission at room temperature is observed.

Method of synthesis of preferred compounds of the invention

In another aspect of the invention provides a method of preparing a compound comprising the steps of:

(i) condensing an arylketone having general formula (B) with an aryl oxazoline having general formula (C) in the presence of a Lewis acid, preferably TiCI₄ to form an oxazolinone having general formula (D):

in which:

optional D, when present, is a proton donating group;

optional Ar, when present, is an optionally substituted monocyclic aryl ring, an optionally substituted bicyclic aryl ring system, an optionally substituted polycyclic aryl ring system, an optionally substituted monocyclic heteroaryl ring, an optionally substituted bicyclic heteroaryl ring system, or an optionally substituted polycyclic heteroaryl ring system; optional R¹ , when present, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyi, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH_2, -NHC(0)NH₂ and -NHC(S)NH₂, an optionally substituted aryl and an optionally substituted heteroaryl ring, wherein b is 0, 1 , 2 or 3;

R⁵ is a bulky substituent having more than four atoms; and

optional R⁶, when present on each Ar ring, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyi, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH₂ -NHC(0)NH₂ and -NHC(S)NH₂, optionally substituted carbocycle and optionally substituted heterocycle ring, wherein e is 0, 1 , 2, 3 or 4;

(ii) performing a nucleophilic ring opening of oxazolinone (F) with an amine to form an arylacrylamine intermediate having general formula (E):

(iii) performing a dehydrative condensation initiated ring closing step on the arylacrylamine (E) to form the corresponding imidazolinone (A):

optionally, wherein the method further comprises one or more purification steps, for example, involving recrystallisation and/or chromatography.

Suitable proton donating groups have been discussed above.

It will be appreciated that where oxazolinone compounds having general formula (F), or arylacrylamine compounds (E) are required, the method terminating with each of the above respective steps (i) or (ii) alone may be utilised, with subsequent purification step if desired. Suitably, purification involves recrystallisation and/or chromatographic methods.

The proton donating group (D) may be protected with a suitable protecting group well known to those skilled in the art. For example, if D is -OH, an acetate may be used as a suitable protecting group.

Preferably, ring opening step (ii) is carried out by stirring a dichloromethane solution of oxazolinone (F) at room temperature in the presence of acetonitrile and dry methylamine, preferably for a period of from about 15 minutes to 2 hours, and/or wherein the ring closing step (iii) cyclises the arylacrylamine intermediate (E) through dehydrative cyclization of (E) by heating the intermediate, preferably to a temperature of from room temperature to 300 °C for a period of from 1 -10 minutes.

In preferred embodiments, a single conformation Z-isomer is formed. However, in some embodiments, a mixture of both Z- and E- isomers may be formed.

Preferably, R⁵ is optionally substituted phenyl, preferably unsubstituted phenyl. In this case, the synthesis thus starts with phenyl oxazolone.

Synthesis of o-LHBPI

In preferred embodiment, the imidazolinone has structure (A) and is derived from the arylketone (B) on condensation with phenyloxazoline in the presence of TiCI₄ to form an oxazolinone (C). In this case, arylacrylamine intermediate (D) is formed.

Intermediates

In a further embodiment, the invention provides for a use of a compound having general formula:

wherein

optional D, when present, is a proton donating group; optional Ar, is an optionally substituted monocyclic aryl ring, an optionally substituted bicyclic aryl ring system, an optionally substituted polycyclic aryl ring system or an optionally substituted monocyclic heteroaryl ring, an optionally substituted bicyclic heteroaryl ring system, an optionally substituted polycyclic heteroaryl ring system;

optional R¹ , when present, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH_2, -NHC(0)NH₂ and -NHC(S)NH₂, an optionally substituted aryl and an optionally substituted heteroaryl ring, wherein b is 0, 1 , 2 or 3;

R² is H, or optionally substituted alkyl;

R³ is optionally substituted alkyl; or

R² and R³ together with the shared bonds from the adjacent phenyl ring form a carbocycle or heterocycle ring, L;

R⁵ is a bulky substituent having more than four atoms; and

optional R⁶, when present on each Ar ring, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH_2, -NHC(0)NH₂ and -NHC(S)NH₂, optionally substituted carbocycle and an optionally substituted heterocycle ring, wherein e is 0, 1 , 2, 3 or 4,

as an intermediate in imidazolinone synthesis, preferably wherein the imidazolinone is a dual emission fluorescent compound.

Preferably, the intermediate is in accordance with general structure:

wherein

optionally, D when present, is a proton donating group;

optional R¹ , when present, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH₂ -NHC(0)NH₂ and -NHC(S)NH₂, an optionally substituted aryl and an optionally substituted heteroaryl ring, wherein b is 0, 1 , 2 or 3; L is an optionally substituted carbocycle or an optionally substituted heterocycle ring, optionally fused to another aromatic or heteroaromatic ring, wherein n is 1 , 2, 3 or 4, and A is independently selected from the group consisting of: -CH₂-, -CR₂ ^*-, =CH-, -CH=, -N=, =N-, -NR -, -0-, C₆H₄, a fused optionally phenyl, where the phenyl can be optionally substituted with hydroxyl, NR₂ ^**; wherein R^* is independently H or C₁-C₃ alkyl, where the alkyl is optionally substituted with hydroxyl, halogen, carboxylic acid or sulfonic acid, and wherein R^™ is alkyl or aryl;

R⁵ is a bulky substituent having more than four atoms; and

optional R⁶, when present on each Ar ring, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH_2, -NHC(0)NH₂ and -NHC(S)NH₂, optionally substituted carbocycle and an optionally substituted heterocycle ring, wherein e is 0, 1 , 2, 3 or 4.

Applications of the compounds

The results presented here open the door to using the compounds of the present invention in a broad range of environmental, biotechnological and diagnostic applications.

Thus, the present invention includes methods for ratiometric sensing and imaging using the compounds of the invention, particularly, those exhibiting dual emission. The sensing and imaging generally applies to in vivo and in vitro techniques using fluorescent probes with dual emission that are sensitive to their local environment. Furthermore, preferred dual emitting compounds provide "self-calibrating" (ratiometric) fluorophores that may function as probes that display a spectral modification upon interaction with a given biological target thereby providing a concentration-independent measurement through the ratio of fluorescence-intensity values measured at two selected wavelengths (dual emission).

Although, in general maximum emission occurs only for specific excitation and emission wavelength pairs, the magnitude of fluorescent intensity is dependent on both intrinsic properties of the compound and experimental parameters, including intensity of the absorbed light and concentration of the fluorophore in solution, as well as influence of local environmental conditions on the chromophore's behaviour.

Accordingly, preferred dual emitting compounds of the present invention may be used as sensors of biological and environmental signals such as, for example, viscosity, electric fields, temperature, pH, hydrophobicity and/or ionic strength, especially of specific ions by virtue of their emission wavelengths and relative strengths being influenced by said signals. Due to their small size and relative hydrophobicity, these compounds are ideally suited to studying biological structures and systems with minimal perturbations.

For example, it can be envisaged that o-LHPDI analogues can be used to report on the local environment through a comparison of the cyan and red emissions or to prepare chemically tuneable optoelectronic devices. In particular, the compounds of the present invention may be useful in areas of mixing and fluid dynamics where spectral modifications and/or fluorescence-intensity value fluctuations in a mixture may be indicative of a proportional and/or localised change viscosity, electric fields, temperature, pH, hydrophobicity and/or ionic strength, especially of specific ions.

As the compounds of the invention comprise fluorophores that absorb light energy of a specific wavelength and re-emit light at a longer wavelength, the compounds of the invention may be used as luminescent probes designed to respond to specific stimuli or to localise within a specific region of a biological specimen.

The compounds of the present invention may also be conjugated to targets including, but not limited to, enzymes and other proteins, biomolecules such as nucleic acids, lipids, organelles, cells, tissues, metal ions and co-factors in a manner suitable for a desired application. These targets conjugated to compounds of the present invention may be selectively exposed to light of particular wavelengths to excite the compound to induce a colour emission or a colour change that enable the target to be visualised with high contrast by virtue of ultrasensitive ratiometric fluorescence measurements.

Preferred dual emitting compounds of the present invention, such as o-LHPDI and its derivatives, demonstrate a novel dual emission; red (610 nm) and cyan (475 nm) colour at excitation by violet (410 nm) or ultraviolet (370 nm) light. The red emission (the ESIPT emission) of this particular compound may also be switched on and off. For example, ESIPT emission suppression can result from application of conditions that result in deprotonation of the proton donating group or a conformational change that puts the proton donating group far from N*.

The person skilled in the art would also understand that the single and dual emitting compounds of the present invention or the methods of the present invention may be used in a range of applications generally known for fluorescent proteins and compounds, for example, as physiological indicators and biosensors (cell marker for live cell or organelle labelling, in protein trafficking studies, in monitoring promoter activity as a photoactive genetic label, for monitoring protein dynamics). In this regard, after association of a compound of the invention with a ligand, the ratiometric fluorescence may be affected allowing quantitative measurement of subtle changes to the local environment, ranging from a stop-go assay where the probe switches between FC and ESIPT emissions to a continuous variation from 100% FC to 100% ESIPT. Sensitive and accurate measurements are facilitated by a large difference in Stokes' shift between the FC and ESIPT emissions, which for some examples is > 100 nm.

Dual emitting compounds, such as those of the present invention, can also be used in FRET, FLIM and FCS based applications as well as in super-resolution imaging.

The photoswitching and dual emission properties of the compounds of the present invention are particularly suited to super-resolution imaging applications enabling imaging of the localization and function of individual molecules at nano-scales that cannot be achieved using light microscopy. These super- resolution methods include Photo-Activation Localization Microscopy (PALM), Fluorescence Photo-Activation Localization Microscopy (FPALM) and Stochastic Optical Reconstruction Microscopy (STROM) and light sheet fluorescence microscopy (LSFM) for example. However, there is a need for more photoactive fluorescent compounds that can be used as super-resolution probes.

Accordingly, the dual emission and photoswitching properties of the compounds of the present invention provide new probes for super-resolution imaging. In particular, the cyan and red emission of o- LHPDI is distinct from the commonly used dual emitting molecules used in fluorescent and super-resolution imaging, enabling single, dual and tri-colour super-resolution imaging.

Accordingly, the invention provides use of a dual emitting compound in a method of super-resolution imaging.

Accordingly, the invention provides use of a dual emitting compound in a method of time-lapse imaging. Accordingly, the invention provides use of a dual emitting compound in a method of advanced multiparameter imaging.

Accordingly, the invention provides use of a dual emitting compound in a method of live cell imaging.

Accordingly, the invention provides use of a dual emitting compound in a method of Stochastic Optical Reconstruction Microscopy.

In particular, the compounds of the invention can be used in biotechnological applications. For example, the compounds can be used as probes to report on the local environment through a comparison of the dual emissions. Other applications involve ratiometric fluorescence measurements, sensing and/or imaging, a fluorescence switching, a fluorescent probe, a GFP mimetic, a chemically tuneable optoelectronic device, a two colour light emitting diode, etc. The compounds of the invention may be used for super high resolution microscopy (e.g. STORM) where the dual emission is switchable. The UV-excited fluorophores exhibit excited-state intramolecular proton transfer (ESIPT) fluorescence at > 520 nm and may be used in the manufacture of fluorescent probes, coatings, objects, scintillators, light sources, and the like.

The fluorophores emit two different colours upon excitation with light frequencies > 320 nm.

In a further embodiment, the invention provides a method of detecting a local change in an environment, comprising the step of: providing the environment with one or more dual fluorescent compounds; determining an initial level of ratiometric fluorescence arising from said compounds and associated with an initial set of environmental conditions; and determining a change in the local environment on the basis of observation of a degree of change in the ratiometric fluorescence.

Suitably, the method may further comprise: associating the one or more compounds with a ligand of interest; and determining a change in the ratiometric fluorescence resulting from the association of the ligand with the compound, preferably wherein the ligand is selected from enzymes, biomolecules, organelles, cells, tissues, and other molecules such as, but not limited to, metal ions, lipids and cofactors.

Furthermore after association of a compound of the invention with a ligand, the ratiometric fluorescence is affected allowing quantitative measurement of subtle changes to the local environment, ranging from a stop-go assay where the probe switches between FC and ESIPT emissions to a continuous variation from 100% FC to 100% ESIPT. Sensitive and accurate measurements are facilitated by a large difference in Stokes' shift between the FC and ESIPT emissions, which for some examples is > 200 nm for a number of the compounds.

Furthermore, during use, where desired, o-LHBPI's ESIPT emission may be suppressed. For example, ESIPT emission suppression can result on application of conditions that result in deprotonation of the proton donating group D. (Figure 28, Figure 14). For example, at pH > 10, deprotonation occurs, thereby blocking the ESIPT emissive state as the necessary tautomer cannot form. Thus, the invention also provides a method of switching off on emission mode of a dual emission fluorescent compound comprising the step of suppressing one emissive state associated with the compound, preferably an ESIPT emissive state. For example, an emissive state may be suppressed by subjecting the compound to conditions that result in deprotonation of a tautomer associated with the compound, for example, high pH > 10. Other compounds of this invention respond to different pH ranges and can be used for ultrasensitive, super resolution pH monitoring inside live cells, for example. The invention also provides for a use of a compound of the invention as a sensor of one or more biological and environmental signals such as viscosity, electric fields, temperature, pH, hydrophobicity, lipophilicity/micelle formation, ionic strength, and/or the determination of low concentrations of protein, lipids, DNA, glyocprotein and polysaccharides, such as starch.

Brief description of the drawings

Figure 1 illustrates the molecular structures of p-HBDI, o-HBDI, o-LHBDI and o-LHBPI and schematic representation of different mechanisms A) Hula-Twist, B) Internal nucleophilic attack of the excited state and C) TICT (twisted intramolecular charge transfer) mediated isomerization of p-DMPI, proposed for the ultrafast nonradiative relaxation of HBDI analogues.

Figure 2 illustrates the normalized absorption spectra of oxazolinones in acetonitrile.

Figure 3 illustrates the normalized absorption spectra of ring-opened acrylamides in acetonitrile.

Figure 4 illustrates the steady state absorption and emission spectra of imidazolinone (4a-4e, 4h) in water and ferf-butanol, 4f in water and 4g in acetonitrile. 4c is not fluorescent.

Figure 5 illustrates area normalized absorption spectra of 4a with varying pH.

Figure 6 illustrates area normalized absorption spectra of 4e with varying pH. (pH's less than 3 (for 4a) and 4 (for 4e) resulted in hydrolysis. This is evidenced by absorption curves that no longer go through the isosbestic point at ~425 nm.)

Figure 7 illustrates the area normalized absorption spectra of 4h with varying pH. (pH's less that 5 and greater than 1 1 resulted in spectra that did not run through the isobestic point, indicating an instability at low and high pH. This would most likely be non-specific acid/base catalyzed hydrolysis of the imidazolinone.)

Figure 8 illustrates the plot of pH vs. normalized absorption intensity for A) for 4a at 470 nm from Figure 5, B) 4e at 480 nm from Figure 6 and C) 4h at 480 nm from Figure 7.

Figure 9 illustrates the femtosecond upconversion decay traces of locked imidazolinones (4d, 4e, 4f and 4h).

Figure 10 illustrates the absorbance spectrum (blue) and calculated spectrum (red; DFT//bpe0/TZVPP- COSMO) of 4e in water.

Figure 11 illustrates the conformations of the tetralone HBDI analogue (4e) showing two possible chair conformations. Ball-and-stick atoms highlight the conformational changes that are possible in the excited state.

Figure 12 illustrates the DFT calculated ground state conformations of the indanone HBDI analogue (4h) showing ground state trans-(left) and cis-(right) isomers showing the coplanarity of the aromatic rings.

Figure 13 A) illustrates the normalized absorption (black) and emission spectra (red) and B) femtosecond fluorescence upconversion decay trace of 4h in water. λ_ex = 400 nm; λ_em = 490 nm.

Figure 14 illustrates the DFT//bp86/SV(P) calculated energies for E-/Z-isomerization (τ rotation) for 4e, showing the ground state (S₀; green diamonds), first excited state (S₁ red squares) and the ground state energy at the first excited state geometry (orange circles) showing a conical intersection at ~100°. Figure 15 A) illustrates normalized absorption spectra of o-LHBPI in six solvents. B) Normalized steady state absorption (black) and emission spectra for o-LHBPI. Emission spectra with excitation at 370 nm (blue) and 410 (red) are shown for six solvents.

Figure 16 illustrates two Gaussian fittings of o-LHBPI emissions from Figure 15. (A) λ_ex = 370 nm, (B) λ_ex = 410 nm.

Figure 17 illustrates a plot of relative area under 475 nm and 610 nm emission bands (after fitting the spectra as a sum of two-Gaussian functions from Figure S12) vs. pH at A) λ_ex = 370 nm. B) λ_ex = 410 nm. The fluorescence spectra at different pH values do not exhibit an isoemissive point, but upon area normalization, a pseudo-isoemissive point occurs at 557 nm (Fig. S13A), indicating a two state process. The ratio of the areas under the two Gaussian components is pH-independent in acidic media, but appears to approach a point of inflexion at very high pH (Fig. S14). With 410 nm excitation, a similar trend is observed (Fig. S14B). This is indicative of the pKa of the phenol, which is understandably high due to the strong H- bond to the imidazolone.

Figure 18 illustrates excitation spectra at (A) λ_em = 475 nm and (B) λ_em = 610 for different pH values in universal buffer using Horiba Fluoromax instrument at excitation/emission slit width of 5 nm.

Figure 19 illustrates emission intensity at 475 nm (Figure 19A at 370 nm) and at 610 nm (Figure 19B at 370 and 410 nm) are various pH values (universal buffer). The pKa of the excited state is 8.8-10.3 (95% confidence interval) in all cases.

Figure 20 illustrates femtosecond fluorescent transients of o-LHBPI in different solvents from upconversion experiments. λ_ex = 410 nm; λ_em = 475 nm (red) and 610 nm (blue).

Figure 21 illustrates (A) DFT//bp/SV(P) calculated ground state cis-trans isomerization of o-LHBPI. (B) Red emissive ESIPT state of o-LHBPI arises from the cyan emissive Franck Condon state.

Figure 22 illustrates calculated absorption lines for o-LHBPI in water (DFT//pbe0/TZVPP-COSMO) for the E, Z- and zwitterionic forms as well as the anion (10 nm SD). Calculated lines are narrowed to facilitate comparison of the different forms and comparison to the actual spectrum (dashed line) in water.

Figure 23 illustrates potential energy surface (DFT//bp/SV(P)) calculated for τ-bond rotation in o-LHBPI. The ground state is in blue (E- left and Z- right) and the first excited state (purple). Red is the ground state energy at the first excited state geometry.

Figure 24 illustrates ground state (GS; blue, left axis), first excited state (S1 ; red, right axis), second excited state (green, right axis) and third excited state (purple, right axis) energy calculations (DFT//bp/SV(P)) of the O— H bond length for Z-o-LHBPI.

Figure 25 illustrates global first excited state (S1 ) minimum energy structures (DFT//pbe0/TZVPP) for o- LHBDI (left) and o-LHBPI (right).

Figure 26 illustrates a potential energy surface (DFT//bp/SV(P)) calculated for τ-bond rotation in o-LHBPI at the second excited state (S2). The ground state is in blue (E- left and Z- right) and the first excited state (red) are calculated at the optimized S2 geometry. Figure 27 illustrates a schematic of the excited state dynamics associated with o-LHBPI; illustrates how reorientation of a bulky substituent allows both the FC and ESIPT emissions to be observed simultaneously. Subtle changes in the local environment result in dramatic changes in the proportion of FC and ESIPT emissions and allow very sensitive ratiometric, fluorescence measurements to be made.

Figure 28 illustrates that ratiometric fluorescence is sensitive to pH; this figure shows area normalized emission spectra of o-LHBPI in different pH solutions. (A) λ_ex=370 nm, (B) λ_ex = 410.

Figure 29 illustrates FC emission in acetonitrile (λ_ex = 370), as well as ESIPT emission in water (λ_ex = 370)..

Figure 30 illustrates a schematic of conformational changes that may occur on going from the planar ground state configuration to a non-planar excited state (ESIPT) whereby FC emission may be observed where the time required for the molecular to reach the non-planar excited state (ESIPT) is sufficiently slow.

Figure 31 illustrates NOE build-up curve for H3-H4 cross-peaks (0-400 ms) in the NOESY NMR spectrum of o-LHBPI. The Table shows the measured and calculated intramolecular distances (A). The observed NOE results are consistent with only the cis-o-LHBPI in solution.

Figure 32 illustrates ratiometric measurement of pH. A) Graphical fluorescence emission profile of o-LHBDI from pH 2-12. B) plot of the ratio between emission at 600 nm and 500 nm vs pH (normalised for absorption at 410 nm)

Figure 33 illustrates the absolute fluorescence of o-LHBDI at A) λ_ex 400, λ_em 500 nm and B) λ_ex 420, λ_em 600 nm in the presence of protein (BSA), C) a plot of the 600:500 nm emission ratio vs % BSA

Figure 34 illustrates the effect of detergent (SDS) on the fluorescence emission of o-LHBDI. A) Cyan emission vs log[SDS] λ_ex 400, λ_em 500 nm, B) Red emission vs log[SDS], λ_ex 420, λ_em 600 nm, C) Ratio of red:cyan (600/500) emission plotted against [SDS] in mM.

Figure 35 illustrates the effect of DNA on the fluorescence emission of o-LHBDI. A) Cyan emission vs log[DNA] λ_ex 400, λ_em 500 nm, B) Red emission vs log[DNA], λ_ex 420, λ_em 600 nm, C) Ratio of red:cyan emission above 0.1 % DNA.

Figure 36 illustrates the effect of glycoprotein (fetuin) on the fluorescence emission of o-LHBDI, A) Cyan emission vs log[fetuin] λ_ex 400, λ_em 500 nm, B) Red emission vs log[fetuin], λ_ex 420, λ_em 600 nm, C) Ratio of red:cyan emission from 0.004 to 0.5% fetuin.

Figure 37 illustrates the effect of starch on the fluorescence emission of o-LHBDI. A) Cyan emission vs log[starch] λ_ex 400, λ_em 500 nm, B) Red emission vs log[starch], λ_ex 420, λ_em 600 nm, C) Ratio of red:cyan emission of starch solution (20.0% - 0.07%). D) the same as Fig 37C except the fluorescence is normalised against absorption at 410 nm.

Figure 38 illustrates the effect of viscosity on the fluorescence emission of o-LHBDI. A) Cyan emission vs log(viscosity) λ_ex 400, A _em 500 nm, B) Red emission vs log(viscosity) λ_ex 420, λ_em 600 nm, C) Ratio of red:cyan emission of sucrose solutions (60%-0.1 %)

Detailed description of the invention

A series of GFP analogues, which are fluorescent in the solid state at room temperature, but weakly fluorescent in solution, have been synthesized via oxazolone formation. The oxazolone formation process involves a condensation reaction in the presence of a Lewis acid following a Knoevenagel condensation. A ring opened intermediate is formed which cyclizes readily upon heating to produce the imidazolinone. This method is faster, simpler and produces higher yields than alternative methods. A few of these analogues are locked GFP derivatives where the exocyclic single bond rotation has been stopped. Weak fluorescence even after stopping of the single bond rotation indicates that restriction of conformation is not effectively controlled and that the double bond rotation is solely responsible for the major non-radiative pathway. These methods assist in engineering the structure of the future derivatives of GFP chromophore to obtain room temperature fluorescence. Preferred compounds exhibit dual emission.

Table 1. Absorption maxima (λ_max ) and molar absorptivity (ε) of the compounds in acetonitrile.

Table 5. Ground state pK_a values for compounds 4a, 4e and 4h

Table 6. Temporal parameters from upconversion decay traces of imidazolinones

. Temporal parameters of o-LHBPI in various solvents from upconversion experiments, λ,

Table 8. Steady state absorption and fluorescence data of o-LHBPI

Synthesis of compounds

As explained above, the Green Fluorescent Protein (GFP) family is extensively used in cell biology as a genetically encoded, fluorescent marker. The chromophore in wild-type GFP is (2)-5-(4- hydroxybenzylidene)-3,5-dihydro-4/-/-imidazol-4-one (p-HBDI). However, the isolated chromophore is non- fluorescent (Φ _f < 10^"4) in contrast to the protein (Φ _f = 0.8). This has been found to be due to non-radiative pathways (Φ and τ rotation in the excited state) for the free fluorophore that is not available to the chromophore inside the constrained GFP β-barrel.

Although numerous structural analogues of the GFP chromophore have been prepared, only a few synthetic routes are utilized. In general, these routes start with Erlenmeyer azalactone synthesis, in which the reaction of /V-acylglycines with arylaldehydes under the influence of sodium acetate in acetic anhydride results in the formation of 4-arylidene-5-oxazolinones (Scheme 1). Direct condensation of the oxazolinones with primary amines in the presence of a base is the default approach for synthesizing the final 4-arylidene- 5-imidazolinones (Scheme 1 , route a). An indirect approach via a cinnamide (3a), followed by dehydration is generally higher yielding (Scheme 1 , route b). Route b was more efficient in time with more than double the yield when starting from p-hydroxybenzaldehyde despite the extra synthetic step. The reason for this is that the conversion of 2a to 3a is quantitative and requires no purification (other than filtration) and the second step simply requires column chromatography.

However, the Erlenmeyer method (first step in Scheme 1) fails with arylketones due to their poor reactivity compared to aldehydes. A number of variations were attempted, including catalytic lead acetate, bases and phosphorous oxychloride, but to no avail.

An alternate route was explored via Knoevenagel condensation of /V-acylglycines. This type of method, a TiCI₄ mediated aldol condensation, has been successfully implemented by some researchers and subsequently by further researchers to condense indanones with 2,3-dimethylimidazolin-4-one, going directly from 1 f to 4f for example.

Scheme 1 : Synthesis of phenyl substituted p-HBDI

However, 2,3-dimethylimidazolin-4-one is difficult to synthesize compared to 2-phenyloxazolinone. The former requires three steps starting from /V-methylacetamide and chloroacetyl chloride compared to the later which can be made on a gram scale from /V-acylglycines (e.g. hippuric acid) in one step without purification by simply heating in acetic anhydride for 30 min and then pouring into ice/water.

A better synthesis of GFP chromophores that would be especially useful for conformationally restricted HBDI analogues via oxazolinone formation is therefore of interest.

A series of oxazolinones (2) was synthesized by the condensation of the arylketones with phenyloxazolone in good yield (Scheme 2). NMR analysis showed that for a few oxazolinones (2), both Z- and E- isomers were formed. These were not separated keeping in mind that conversion to the final imidazolinone could lead to isomerization and that any isomers could be separated at the end. To our dismay, refluxing oxazolinones (2) in ethanol with aqueous methylamine did not form the corresponding imidazolinones (4) but rather, a white precipitate formed which was shown to be the ring opened /V-alkyl-2- acylamino-3-arylacrylamides (3b-3h). Formation of this type of compound has previously been reported by Stafforst et al. and Lee et al. The yield of ring-opened products (3) was optimized by stirring the oxazolinones (2) in dichloromethane at room temperature and adding acetonitrile saturated with dry methylamine (2 equivalents). This minimized formation of the corresponding carboxylic acids and the reactions were generally over within 15 min except for 2e and 2h, which required an hour because there is concomitant deprotection of the phenol required. The yields were found to be 98-100% in general and 80- 90% for 3e and 3h.

Scheme 2. Efficient synthesis of restrained p-HBDI analogues

Dehydrative cyclization of the acrylamide (3b) to imidazolinone (4b) proved more difficult than anticipated. Standard literature procedures failed. Fortuitously, it was discovered that by drying off a TLC plate with a hot-air gun turned the colorless acrylamide spot yellow. Elution of this spot separated it into two yellow bands that corresponded to 4b and 2b (ESI-MS). Optimization of this reaction revealed that heating of the solid acrylamides (3) in a furnace at 350 °C for 1 min (Scheme 2) led to the formation of the desired products in good to high yield (Table 3) and recovery of 2, which was recycled leading to a cumulative yield of over 90% for all compounds. Some 2 was always formed regardless of how carefully water was excluded. NMR spectroscopy indicated that only the Z-isomer is formed for all imidazolinones (4) except for 4b, where both were formed.

Steady state absorption and emission spectra were recorded for each compound. Compound 4c was non-fluorescent but 4f was 30x more fluorescent than p-HBDI and had the highest quantum yield of all the compounds synthesized here.

Table 9. Steady state absorption and emission maxima and quantum yields for imidazolinones (4)

All the conformationally restricted analogues (4d - 4h) had increased quantum yields compared to 4a - 4c (Table 1). Femtosecond fluorescent transients for 4d, 4e, 4f and 4h in water and ferf-butanol indicate a very fast relaxation process (< 5 ps) that is relatively solvent independent. This is in agreement with the observed low quantum yield (< 1 %) of all the compounds (Table 1). The faster component of < 1 ps predominates for all the compounds in both solvents.

To understand this rapid relaxation we conducted a series of DFT calculations. Scanning the τ angle (Figure 14) revealed that the Z-isomer of 4e is more stable by ~2.5 kcal/mol than the E-isomer but more importantly, that the activation barrier to rotation is ~34 kcal/mol indicating that there is no isomerism in the ground state. However, in the first excited state the potential energy surface (PES) is relatively flat with a conical intersection with the ground state at τ ~ 100 ° (Figure 14 (2)). The dip in energy of the ST state when the imidazolone and tetrahydronaphthalene ring are perpendicular is associated with concomitant flexing of the cyclohexene (R₂/R₃ bridge; Scheme 2). This would allow a facile return to the ground state without emission of a photon and could explain the low quantum yields observed (Table 1) and the very fast decay recorded in upconversion experiment (Figure 13 (1). This indicates that restricting the Φ angle is not sufficient for a high quantum yield and that the τ angle also needs to be physically restrained to generate room temperature HBDI analogues.

For the phenols (4a, 4e and 4h) pH variation studies of absorption spectra were used to calculate the ground state pK_a of the respective compounds. The pK_a of 4e (8.52) is an order of magnitude higher than that of the other two. This suggests that the conjugate base of 4e is relatively unstable perhaps due to the presence of a flexible six membered ring that can undergo facile conformational changes (chair-boat-chair), even in the ground state.

In conclusion, a new, simple, facile and high yielding route to GFP-chromophore analogues via oxazolinone formation is provided. The locked analogues are brightly fluorescent in the solid state but poorly fluorescent in solution, indicating either the restriction of conformation (Φ) is not sufficient or that other non- radiative pathways are involved. However, these studies provide valuable information on the design and synthesis of more fluorescent analogues and these will be reported in due course.

Synthesis of o-LHBPI A dual emission structurally locked GFP chromophore with a phenyl group at position C2 of the imidazolone ring of the chromophore has also been synthesized.

Rotation around the exocyclic double bond is hindered in this molecule, resulting in room temperature fluorescence. Unlike the C2 methyl-substituted analogue (o-LHBPI), the phenyl analogue exhibits a dual emission (cyan and red). The quantum yield in water is 500 times greater than that of unlocked analogues.

To explain this unexpected and surprising behaviour, DFT calculations were carried out along with fluorescence upconversion experiments. The E-isomer was found to be non-emissive, while the origin of the dual emission was dependent on the bulky phenyl group in the Z-isomer, which stabilizes the Franck- Condon state, resulting a cyan fluorescence, while the zwitterionic tautomer fluoresces red. These results bring important new insights into the photophysics of the GFP chromophore and suggest new utility for this class of fluorophore.

The synthesis and photophysics of o-LHBPI (o-locked hydroxybenzylidene-p-phenylimidazolinone), which has a dual emission at room temperature is reported herein.

This dual emission observed for o-LHBPI is remarkably different from o-LHBDI that has a single emission at 600 nm attributed to the zwitterions formed via ESIPT (phenol proton to imidazolone nitrogen).

The unique characteristics of o-LHBPI could be used to sense the local environment, two colour light emitting diodes or for super high resolution microscopy (e.g. STORM) if the dual emission observed can be switched.

Synthesis of o-LHBPI (Scheme 3) was non-trivial as the typical Erlenmeyer-Plochl azlactone synthesis used for HBDI analogues, which works well for aldehydes, failed. In accordance with the above method, TiCI₄ was used as a Lewis acid (Scheme 1) to make the intermediate oxazolinone. Deprotection and conversion to the imidazolinone was achieved in one step with aqueous methylamine. Alternatively a ring- opened intermediate can be isolated, with the reaction with methylamine in DCM medium, which can be readily cyclized upon heating to produce the final imidazolinone.

Reagents and conditions: (a) Ac₂0, NaOAc (2.0 equiv), μwave 300W, 140 °C, 3 min, 97%; (b) phenyloxazolinone (1.6 equiv.), TiCI₄ (1.0 equiv.)/THF, -10 °C, 20 min; then pyridine, 5 h, 40%; (c) aqueous MeNH₂ (40%; 4.0 equiv), K₂C0₃ (1.0 equiv), reflux, 3 h, 45%.

Scheme 3: synthesis of o-LHBPI

The absorption spectra of o-LHBPI exhibit two peaks at ~370 nm and ~410 nm in all the solvents studied (Figure 15). The small solvent effect suggests delocalized π-π* transitions. These are in good agreement with the absorption lines (360 and 400 nm) predicted for o-LHBPI by DFT calculations in water (Figure 22, red line). While the absorption spectra of o-LHBPI and o-LHBDI are qualitatively similar, the emission spectra are not. For o-LHBPI, a dual emission at ~475 (cyan) and 610 (red) nm is observed (Figure 15 and 16). The origin of the 475 and 610 nm bands can be attributed to Franck-Condon (FC) normal emission and an emissive state arising from ESIPT respectively. Surprisingly, the relative fluorescence quantum yield of the ESIPT band is greatest in water. This may seem to be counterintuitive, as ESIPT is usually hindered by the formation of "blocked" structures in protic solvents, but can be rationalised by the involvement of water in a proton relay, facilitating the ESIPT.

The absorption spectrum of o-LHBPI is not affected significantly by pH, except for the appearance of a 20 nm red shifted band at pH = 12, marking the formation of the anionic ground state of the molecule. The ESIPT emission band is suppressed above pH 10, due to deprotonation of the phenol. (Figure 28, Figure 17). This very high value of pKa indicates that the phenol is involved in strong intramolecular H bond formation in o-LHBPI.

Fluorescence transients at 475 nm are identical for excitation wavelengths of 370 nm and 410 nm and are complete within 2 ps, which is consistent with a short-lived Franck-Condon (FC) normal emission. For an excitation at 410 nm, the transients at 610 nm are significantly slower than those at 475 nm. No perceivable rise time is observed at 610 nm (Figure 20). The decays, except those in aqueous solution, are biexponential, with components in the picosecond and tens of picosecond regimes. An additional subpicosecond component is found only in aqueous solutions, possibly indicating the involvement of hydrogen bond dynamics in the excited state of the molecule.

The occurrence of an ESIPT gains support from the calculated PES shown in Figure 23. These calculations confirm that Z-o-LHBPI (τ = 180°) is more stable than the E-isomer (τ = 0°).

Higher level calculations in water (DFT//pbe0/TZVPP-COSMO) yield an estimate of 6.4 kcal/mol for the difference in energy between the two geometrical isomers. In the excited state there is a somewhat lower barrier between Z-/E-isomerization (Figure 23; purple). In addition there appears to be a conical intersection with the ground state at τ ~ 90°, which predicts that the E-isomer of o-LHBPI should be nonfluorescent due to a facile return to the GS at τ = 90, which falls back exclusively to the Z-o-LHBPI ground state.

DFT calculations are also used to show, for the global minimum (Z-o-LHBPI), that the O-H bond length is optimally 1 .08 A (Figure 24, blue) in the ground state. However, in the first excited state, the most stable form is with the proton on the imidazolone nitrogen, with an optimal N-H bond length of 1 .07 A (Fig. 5, red). Interestingly, for the second excited state, the zwitterion (proton on nitrogen) is more stable but there is a significant barrier to ESIPT of ~ 3.5 kcal/mol suggesting that SO - S2 excitation does not necessarily lead to facile ESIPT.

The ultrafast nature of the Franck-Condon normal emission at 475 nm indicates a facile ESIPT for the S1 state, which is similar to what is observed with o-LHBDI.

While the E- isomer is 6.4 kcal/mol higher in energy than the Z-isomer in the ground state (Figure 23, blue line) and there is a ~20 kcal/mol barrier to E to Z isomerization, in the excited state there is a much smaller barrier (~2 kcal/mol). In contrast, there is a 15 kcal/mol difference in energy in the first excited state between the Z to E isomers (Figure 23). We determined that only the Z-isomer exists in solution by NMR spectroscopy, and that the E-isomer should be non-emissive due to the presence of a conical intersection of the E-excited state and ground state PES (Figure 23, red line). Therefore, the dual emission must be due solely to the Z-isomer.

For the red emission, a Stokes' shift of > 200 nm unambiguously assigns this emission to the ESIPT tautomer (zwitterion). The cyan emission must therefore arise from the normal Franck Condon emission.

According to DFT calculations, for the methyl analogue (o-LHBDI), the ground state and first excited states are almost planar with virtually no skeletal rearrangements required during the ESIPT (Figure 25). In contrast, o-LHPDI undergoes substantial structural rearrangement from the ground to first excited states. These include the proton moving 0.64 A (c.f. 0.58 A for o-LHBDI) but most notably the phenyl group must rotate over 20° (Figure 30) to become more planar and consequently the /V-methyl must also rotate by 7.5° to accommodate this change. In addition, the whole π-system is now far from planar geometry (Figure 25), quite unlike o-LHBDI. This lack of planarity in o-LHPDI would favour a proton relay with water compared to o- LHBDI, explaining the large increase in quantum yield observed in water.

These data also suggest that the ESIPT state takes some time to attain and during the process the normal Franck Condon emission occurs for Z-o-LHPDI.

A second question is why excitation at 370 nm leads preferentially to Franck Condon emission in a few solvents while irradiation at 410 nm always favours ESIPT. This could be due to S₀ - S₂ excitation (370 nm) favouring normal emission. To test this we ran DFT calculation on the ground state and first two excited states along the τ (Z-/E-) and O-H bond length PES (Figures 24 and 26). DFT calculations of the second excited state (S₂) indicated that there is an intersystem crossing possible to S₁ with only a 20° movement in τ (Figure 26). Interestingly, for the S₂ state, there is no ESIPT as, even though the N-H form is more stable, there is a predicted activation barrier (Figure 24, green line) that would at least inhibit ESIPT.

As has been proposed for o-HBDI the ultrafast component found in aqueous solution could be assigned to the vibronic relaxation of the normal form reaching thermal equilibrium. Faster components like the 1 .4 ps and 1 .5 ps decays in ferf-butanol and methanol and the 6 ps decay in water can be assigned to rotation of the phenyl group. The slowest components of ~50 ps (25 ps in acetonitrile) can be assigned to the population decay time of the tautomer and is solvent dependent. But, unlike o-HBDI, no direct relation with solvent viscosity is found; rather the stability of the excited state depends on the interplay of solvent polarity and viscosity, suggesting this compound could be used for ultrasensitive ratiometric measurement in biological systems where local viscosity, polarity, and pH can vary considerably. Together, our results are in accordance to what has been proposed for o-HBDI; we have observed a > 9000 cm^"1 Stokes' shift and a barrierless proton transfer (ultrafast ESIPT), the onset of which was not observed due to instrumental limitations.

A plausible schematic of the excited state dynamics of o-LHBPI can be constructed (Figure 27) on the basis of above mentioned experimental results as well as the output from the computational studies. Upon S₀-S₂ electronic excitation, the non-fluorescent E- form of o-LHBPI, undergoes a facile E-/Z- isomerisation, followed by an S₂-S_! internal conversion. Subsequent rotation of the phenyl group by more than 20° as well as rotation of the N-methyl group by 7.5° are mandatory to make the molecule planar and thus to facilitate ESIPT. Hence, the molecule can spend a substantial amount of time in the non-planar form leading to a percentage of the population undergoing the normal FC emission at 475 nm. The rest, on the other hand, forms the zwitterion via ESIPT, and leads to the tautomer emission at 610 nm. In case of Z-o- LHBPI, similar processes take place except that Z-/E-isomerisation is not possible due to a higher activation barrier. From this scheme, it can also be understood why 370 nm leads preferentially to FC emission in some cases, while excitation at 410 nm favours ESIPT. In the case of 410 nm excitation, the molecules do not need to undergo extensive torsional rearrangement to attain planarity, resulting in the majority of the population undergoing ESIPT. While the in case of 370 nm excitation, loss in planarity causes a delay in the ESIPT, leading to the predominance of the FC emission, although this process depends on an interplay of factors in the local environment that affect H-bonding, viscosity and dielectric as evident from the steady state behaviour in different solvents (Figure 16). Thus we have found that by fine-tuning the rigidity and bulk of the substituents it is possible tune the ration of FC:ISEPT emissions and produce a range of useful ratiometric fluorophores.

In summary, the effect of phenyl substitution in the o-locked GFP chromophore analogue, Z-o- LHPDI, was found to be non-trivial. The fluorescence quantum yield is lower than the methyl-substituted analogue because rotation of the phenyl ring provides an additional nonradiative channel of deactivation.

Unlike the methyl-substituted analogue o-HBPI, dual emission is observed at room temperature, indicating a hindrance to the ESIPT process that leads to the formation of the red emissive phototautomer.

This observation is rationalized with high level DFT calculations that indicate that, unlike o-LHBDI, o- HBPI requires significant structural rearrangement in the excited state zwitterion leading to a mixture of Franck- Condon normal emission and emission from the ESIPT zwitterion. The higher quantum yield in water indicates that H-bonding facilitates the ESIPT and this would be a useful design motif for applications in aqueous environments.

The results presented here open the door to using GFP chromophore analogues in biotechnological applications. For example, it can be envisaged that o-LHPDI analogues can be used to report on the local environment through a comparison of the cyan and red emissions or to prepare chemically tunable optoelectronic devices using the compounds revealed here.

Example 1 - measurement of pH using ratiometric analysis

In this example, compound o-LHPDI was found to be very sensitive to the presence of [OH-] with the FC emission dominating at pH 12 and the ESIPT emission dominating at pH < 5. (See Figures 17-19, 32).

Example 2 - measurement of solvent effects

In this example, compound o-LHPDI was found to be very sensitive to the solvent and excitation wavelength. For example with irradiation at 370 nm in water the ESIPT emission dominates, whereas in acetonitrile, the FC emission dominates leading to a visible difference in colour. (See Figures 15-16). This effect is also observed in other solvents and is dependent on the irradiation frequency as well as the viscosity of the solvent.

Example 3 - measurement of protein concentrations

In this example, compound o-LHPDI was found to be very sensitive to the concentration of protein (BSA) in solution (See Figure 33). This effect is also observed for other proteins. Example 4 - measurement of hydrophobicity

In this example, compound o-LHPDI was found to be very sensitive to the hydrophobicity of its local environment. With increasing concentrations of SDS the ratio of red:cyan emission changes by 300x. (See Figure 34).

Example 5 - measurement of sugars

In this example, compound o-LHPDI was found to be very sensitive to the polysaccharides. (See Figure 36, 37). The dye was found to respond to fetuin (glycoprotein) and starch in a similar way, giving sensitive ratiometric readout of polysaccharide concentration.

Example 6 - measurement of viscosity

In this example, compound o-LHPDI was found to be sensitive to the viscosity of the local environment with both the cyan and red emissions increasing linearly against log(viscosity). More importantly, the ratio of red:cyan emission is very sensitive to viscosity (See Figure 38).

Materials and Experimental Procedure

Unless otherwise stated, all chemicals and reagents were received from Sigma-Aldrich (Castle Hill, Australia) and used without further purification. Dichloromethane, diethyl ether, ethanol, ethyl acetate, light petroleum, methanol, tetrahydrofuran and toluene were obtained from ChemSupply (Australia). HPLC grade acetonitrile was obtained from BDH/Merck (Germany), and was used without further purification. Spectroscopy grade acetonitrile from Spectrochem was distilled over CaH2 and the distillate passed through activated neutral alumina immediately prior to use. Spectroscopy grade tert-butanol from Spectrochem, Mumbai, India was used as received. 1 H NMR and 13C NMR spectra were recorded in 5 mm Pyrex tubes (Wilmad, USA) on either a Bruker Avance DPX-400 400 MHz or AVII-600 600 MHz spectrometer. All spectra were obtained at 25 °C, processed using Bruker Topspin 3.2 and referenced to residual solvent (CDCI3 7.26/77.16 ppm; DMSO-d6 2.49/39.8 ppm). Infrared spectra were taken on a Perkin Elmer paragon 1000PC FTIR spectrometer, or Nicolet iS90 FT-IR Spectrometer (Thermo Scientific, Australia). Low resolution mass spectrometry was performed by electrospray ionization (ESI) MS in positive or negative polarity mode as required on a Shimadzu LC-20A prominence system coupled to a LCMS-2010 EV mass spectrometer using LCMSsolution 3.21 software. LC-MS experiments were carried out on a Gemini C18 column (Phenomenex, Australia) 150.0 χ 2.00 mm, 1 10A, 3 μm High resolution mass data were obtained from ESI in positive polarity mode on a Waters Q-TOF Ultima Tandem Quadrupole/Time-of-Flight ESI mass spectrometer, performed by the Mass Spectrometry Unit at the University of Illinois, USA. Melting point was measured using DSC 2010 differential scanning calorimeter from TA instruments. pH was measured using (ISFETCOM, model S2K712, JAPAN) pH meter. Microwave reactions were carried out using a CEM Discover system.

Equipment

¹H NMR and ¹³C NMR spectra were recorded in 5 mm Pyrex tubes (Wlmad, USA) on either a Bruker Avance DPX400 400 MHz or AVII600 600 MHz spectrometer. All spectra were obtained at 25 °C, processed using Bruker Topspin 3.2 and referenced to residual solvent (CDCI₃ 7.26/77.16 ppm; DMSO-c/₆ 2.49/39.8 ppm). Infrared spectra were taken on a Perkin Elmer paragon 1000PC FTIR spectrometer, or Nicolet iS10 FT-IR Spectrometer (Thermo Scientific, Australia). UV-Vis absorption spectra are recorded on JASCO V-530 double beam absorption spectrophotometer with a slit width of 1 .0 nm at room temperature or on Varian Cary-eclipse spectrophotometer.

Low resolution mass spectrometry was performed by electrospray ionisation (ESI) MS in positive or negative polarity mode as required on a Shimadzu LC-20A prominence system coupled to a LCMS-2010 EV mass spectrometer using LCMSsolution 3.21 software. LC-MS experiments were carried out on a Gemini C18 column (Phenomenex, Australia) 150.0 x 2.00 mm, 1 10 A, 3 μm

High resolution mass data were obtained from ESI in positive polarity mode on a Waters Q-TOF Ultima Tandem Quadrupole/Time-of-Flight ESI mass spectrometer, performed by the Mass Spectrometry Unit at the University of Illinois, USA.

HPLC analysis was performed on a Shimadzu 10AD-VP system running Class- VP 7.4 SP1 software or a Waters manual 6000A pump. Analytical, semipreparative and preparative HPLC were performed on Gemini C18 HPLC columns (Phenomenex): Gemini-NX C18 250.0 x 4.6 mm, 1 10 A, 5 μm; Gemini C18 250.0 x 10.0 mm, 1 10 A, 10 μm; Gemini-NX C18 150.0 x 21 .2 mm, 1 10 A, 5 μm.

TLC was performed with Merck Kieselgel 60 F254 plates with viewing under ultraviolet light (254 nm and 365 nm). Flash column chromatography was performed on silica gel (60 A 0.040-0.063 mm, 230-400 mesh ASTM from Grace Chemicals, Melbourne).

After reverse phase HPLC, acetonitrile was evaporated and the residual water removed by lyophilization. The freeze dryer system was from Labconco (USA).

Melting point was measured using DSC 2010 differential scanning calorimeter from TA instruments.

Design and Synthesis o-LHBPI

The primary challenge of the synthesis was the relatively poor reactivity of the ketones compared to aldehydes in Erlenmeyer condensations. For aldehydes, simply refluxing with phenyloxazolinone in acetic anhydride/acetic acid for 5 hours resulted in the p-HBDI analogues in excellent yields. However, this method was not successful when using any ketone. However, TiCI₄ in THF and a stoichiometric amount of pyridine yielded (2)-3-(5-oxo-2-phenyloxazol-4(5/-/)-ylidene)-2,3-dihydro-1 /-/-inden-4-yl acetate (o-AcBPA) in a moderate yield (Scheme 1). o-LHBPI was obtained by deacylation with aqueous methylamine and potassium carbonate.

3-oxo-2,3-dihydro-1 H-inden-4-yl acetate. 7-Hydroxy-2,3-dihydro-1 H-inden-1 -one (0.5 g, 3.37 mmol) was added to acetic anhydride (5 mL) and 2 equivalent of sodium acetate (0.55 g, 7.74 mmol). The reaction mixture was irradiated in a microwave reactor for 3 min (300 W, 140 °C). The mixture was then poured in ice/water and extracted with ethylacetate. The organic layer was washed thoroughly with water (4 ^χ 25 mL) and concentrated in vacuo. Purification by flash chromatography gave the pure 7-acetoxyindanone (0.62 g, 97%), as white crystals, m.p. 76 °C [lit m.p. 75-77 °C]. ¹H NMR (400 MHz, CDCI₃) δ 7.58 (t, J = 7.80 Hz, 1 H), 7.33 (d, J = 8.00 Hz, 1 H), 6.97 (d, J = 7.70 Hz, 1 H), 3.13 (t, J = 5.90 Hz, 2H), 2.66 (t, J = 5.90 Hz, 2H), 2.39 (s, 3H).

2-phenyloxazol-5(4H)-one (phenyloxazolinone). Hippuric acid (5.0 g, 27.9 mmol) and EDCI HCI (7.0 g, 36.3 mmol) were dissolved in CH₂CI₂ (50 mL) and stirred at room temperature for 45 min under a N₂ atmosphere. The reaction was quenched with water. The organic layer was separated, and washed twice with water and once with brine, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to afford phenyloxazolinone as an off-white solid (4.1 g, 91 %), which was used for the next reaction without further purification. ¹H NMR (600 MHz, CDCI₃) δ 7.96 (dd, J = 5.2, 3.3 Hz, 2H), 7.55 (m, 1 H), 7.46 (m, 2H), 4.38 (s, 2H); ¹³C NMR (151 MHz, CDCI₃) δ 176.00, 163.54, 132.87, 129.09, 128.88, 128.66, 127.86, 125.94, 77.37, 77.16, 76.95, 55.04.

(Z)-3-(5-oxo-2-phenyloxazol-4(5H)-ylidene)-2,3-dihydro-1 H-inden-4-yl acetate (o-AcBPA). THF (10 ml.) was chilled under N₂ to -10 °C. To it, TiCI₄ (0.23 ml_, 2.20 mmol) in CH₂CI₂ (0.2 mL) was added and stirred for 10 min. To the stirring solution, 3-oxo-2,3-dihydro-1 H-inden-4-yl acetate (7-acetoxyindanone; 0.1 g, 0.53 mmol) was added and the mixture was stirred for 5 min, then 2-phenyloxazol-5(4H)-one (phenyloxazolinone, 0.13 g, 0.85 mmol) was added and stirred for a further 40 min. To the mixture, pyridine (0.17 mL, 2.20 mmol) was added dropwise. The black mixture was stirred for a further 5 hours and was monitored by TLC until the starting material had disappeared. The reaction was then quenched with saturated ammonium chloride solution (3 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layer was washed thoroughly with water (4 x 10 mL) then brine solution (2 x 10 mL) and concentrated under vacuum. The oxazolone was purified by flash chromatography and recrystallized from methanol/water to yield a yellow solid (0.071 g, 40%), m.p. 192 °C; UV (acetonitrile) λ_max 365 nm (ε = 36800); IR (ATR) v_max 1790, 1780, 1751 , 1640 cm^"1 ; ¹H NMR (400 MHz, CDCI₃) δ 8.07 (m, 2H), 7.53 (m, 4H), 7.30 (d, J = 7.50 Hz, 1 H), 7.06 (d, J = 7.87 Hz, 1 H), 3.49 (t, J = 5.90 Hz, 2H), 3.18 (t, J = 5.90 Hz, 2H), 2.24 (s, 3H); ¹³C NMR (125 MHz, CDCI₃) δ 169.33, 166.99, 153.59, 153.47, 148.19, 133.46, 132.80, 131 .56, 129.06, 128.07, 126.15, 123.06, 122.1 1 , 32.68, 31 .43, 22.12;MS (ESI) m/z 334; HRMS (ESI) calcd. for C₂₀H₁₆NO₄ m/z 334.1073 [M+H⁺], found m/z 334.1079

(Z)-4-(7-hydroxy-2,3-dihydro-1 H-inden-1-ylidene)-1-methyl-2-phenyl-1 H-imidazol-5(4H)-one (o-LHBPI).

To the stirred solution of (2)-3-(5-oxo-2-phenyloxazol-4(5/-/)-ylidene)-2,3-dihydro-1 /-/-inden-4-yl acetate (o- AcBPA; 0.02 g, 0.06 mmol) in DCM (1 mL) was added a solution of sat. methylamine in ACN (50 L L) at RT. The resultant reaction mixture was stirred at same temp for 30 min. The white precipitate was filtered and dried under vacuum. The solid was then heated at 300 °C for 1 min under high vacuum. The resultant residue was purified by HPLC using ACN/water (70:30) to obtain the final imidazolone (0.008 g, 44%) as a yellow solid, m.p. 192.5 °C; UV (acetonitrile) λ_max 370 nm (ε = 18600), 410 nm (ε = 21200) nm; IR (ATR) v_max 1686 cm^"1 ; ¹ H NMR (600 MHz, CDCI₃) δ 14.87 (s, 1 H, -OH), 7.78 (d, J = 7.25 Hz, 1 H, -CH_ar), 7.56 (m, 3H, - CH_ar), 7.35 (t, J = 7.73 Hz, 1 H, -CH_ar), 6.87 (d, J = 7.38 Hz, 1 H, -CH_ar), 6.80 (d, J = 8.20 Hz, 1 H, -CH_ar), 3.49

(t, J = 5.81 Hz, 2H, -CH₂), 3.45 (s, 3H, -NCH₃), 3.18 (t, J = 7.73 Hz, 2H, -CH₂); ¹³C NMR (150 MHz, CDCI₃) δ 167.92, 157.45, 156.87, 154.26, 153.42, 135.53, 131 .62, 129.27, 128.40, 128.31 , 127.55, 125.57, 1 16.06, 1 15.76, 31 .31 , 30.89, 29.14; MS (ESI) m/z 305; HRMS (ESI) calcd. for C₁₉H₁₇N₂0₂ m/z 305.1295 [M+H⁺], found m/z 305.1290.

Solution Structure:

The structure of the final product was confirmed by NMR spectroscopy and HRMS. The solution structure of o-LHBPI was investigated by 2D NOESY spectroscopy. An nOe build-up curve (Figure 31) was constructed to determine if there was any spin diffusion. The graph indicated that even with a mixing time (T_m) of 400 ms there was no evidence of spin diffusion. NOESY spectra for mixing times of 100, 200 and 400 ms were acquired, cross-peaks integrated and the distances calculated (integral a 1/r⁶) for all mixing times (Table in Figure 31).

In organic solvent (CDCI₃) o-LHBPI exists solely in the "cis" (Z)-form. This is based on integral # 4, which is the only diagnostic NOE that can differentiate the cis and trans- forms. The measured distance of 3.30 A ± 0.07, correlates well with the measured distance (3.55 A) for the cis- form but not the trans- form (r = 6.05 A). Synthetic data

GFP analogues and intermediates

(Z)-4-((5-oxo-2-phenyloxazol-4(5H)-ylidene)methyl)phenyl acetate (2a). To a solution of 4-formylphenyl acetate (1 , 0.13 ml_, 0.82 mmol) in acetic anhydride (5 ml_) was added hippuric acid (0.14 g, 0.82 mmol) and sodium acetate (0.067 g, 0.82 mmol) and the reaction mixture was refluxed for two hours, cooled and then poured into ice water (30 ml_) and stirred for 20 min. The precipitate was filtered, washed several times with ice

cold ethanol and dried in vacuo. The product was recrystallized from DCM/PE and to yield oxazolylidene (2) as a pale yellow solid (0.15 g, 60%) which was used immediately in the next step, m.p. 180.5 °C [lit. 179 °C]; UV (acetonitrile) λ_max 364 nm (ε = 95000); IR (ATR) u_max 1793, 1755, 1653 cm^"1 ; ¹H NMR (400 MHz, CDCI₃) δ 8.25 (d, J = 8.6 Hz, 2H), 8.18 (d, J = 7.5 Hz, 2H), 7.62 (t, J = 7.32 Hz, 1 H), 7.54 (t, J = 7.32 Hz, 2H), 7.23 (m, 3H), 2.34 (s, 3H); ¹³C NMR (100 MHz, CDCI₃) δ 170.8, 163.7, 133.1 , 132.9, 131 .1 , 129.2, 128.9, 128.9, 128.6, 127.9, 125.9, 125.9, 55.2.

(Z)-4-(4-hydroxybenzylidene)-1-methyl-2-phenyl-1H-imidazol-5(4H)-one (4a). To an ethanolic solution of

(Z)-4-((5-oxc-2-phenyloxazol-4(5H)-ylidene)methyl)phenyl acetate (2a, 0.1 g, 0.33 mmol) was added aqueous methylamine (40%; 0.1 ml_) and potassium carbonate (0.045 g, 0.33 mmol) and the solution was refluxed under N₂ for three hours, cooled, extracted with

ethyl acetate (20 ml_), washed with water (3 x 50 ml_) and concentrated in vacuo. The crude product was purified by flash chromatography to provide the imidazolone (3, 0.032 g, 35%) as a bright yellow solid (0.032 g, 35%), m.p. 203 °C; UV (acetonitrile) λ_max 297 nm (ε=30700); IR (ATR) u_max 3169, 1673, 1640 cm^-1 ; ¹H NMR (600 MHz, CDCI₃) δ 8.15 (d, J = 8.78 Hz, 2H), 7.84 (dd, J = 8.00, 1 .5 Hz, 2H), 7.55 (m, 3H), 7.24 (s, 1 H), 6.88 (d, J = 6.78 Hz, 2H), 3.36(s, 3H); ¹³C NMR (150 MHz, CDCI₃) δ 158.4, 135.0, 131 .6, 129.7, 128.9, 128.8, 127.3, 1 16.1 , 29.2; MS (ESI) m/z 279; HRMS (ESI) calcd. for C₁₇H₁₅N₂0₂ m/z 279.1 133 [M+H⁺], found m/z 279.1 134.

Alternatively, (Z)-4-((5-oxo-2-phenyloxazol-4(5H)-ylidene)methyl)phenyl acetate (2a, 0.1 g, 0.33 mmol) was dissolved in CH₂CI₂ and acetonitrile saturated with dry methylamine (0.15 ml_) was added and the mixture stirred for 1 hour. A white precipitate was formed, which was filtered and washed with cold CH₂CI₂ and dried to yield (Z)-N-(1-(4-hydroxyphenyl)-3- (methylamino)-3-oxoprop-1-en-2-yl)benzamide (3a, 0.097 g, 100%), m.p. 144 °C; UV

(acetonitrile) λ_max 389 nm (ε=16600); IR (ATR) u_max 3237, 1648 cm^"1 _; ¹H NMR (400 MHz, DMSO-d₆) δ 9.77 (s, 1 H), 8.0 (d, J = 7.13 Hz, 2H), 7.91 (quart, J = 4.57 Hz, 1 H), 7.47 (m, 3H), 7.37 (d, J = 8.6 Hz, 2H), 7.18 (s, 1 H), 6.70 (d, J = 8.6 Hz, 2H), 2.64 (d, J = 3.58 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 166.66, 166.53, 159.00, 134.72, 132.43, 132.03, 130.55, 129.13, 128.77, 127.82, 125.93, 1 16.23, 27.20; MS (ESI) m/z 297; HRMS (ESI) calcd. for C₁₇H₁₇N₂0₃ m/z 297.1234 [M+H⁺], found m/z 297.1239.

The benzamide (3a; 0.093 g) was heated in furnace at 350 °C for 60 seconds to give an orange solid that was purified by flash chromatography to yield the imidazolone 3 (0.074 g, 80%).

General procedure for the synthesis of oxazolones (2b-2h): THF (10 ml_) was chilled under N₂ to -10 °C. To it, TiCI₄ (1 .5 equiv) in CH₂CI₂ (200 μΙ_) was added and stirred for 10 min. To the stirring solution, the ketones 2b-2h were added and the mixture was stirred for 5 min., then 2-phenyloxazol-5(4H)-one (2 equiv) was added and stirred for a further 20 min. To this mixture, pyridine (2 equiv) was added dropwise. The mixture was stirred for a further 5 hours and was monitored by TLC until there were no starting materials left. The reaction was then quenched with saturated ammonium chloride solution (3 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed thoroughly with water (4 x 10 mL) and brine solution (2 x 10 mL) and concentrated in vacuo. The oxazolones were purified by flash chromatography and recrystallized from DCM/PE.

General procedure for the preparation of p-acetylated ketones (9'-10'): Compounds 1e and 1 h were added to acetic anhydride (5 mL) and 2 equivalent of sodium acetate. The reaction mixture was irradiated in a microwave reactor (CEM) for 3 min (300 W, 140 °C). The mixture was then poured in ice cold water and extracted with ethylacetate. The organic layer was washed thoroughly with water (4 x 25 mL) and concentrated in vacuo. Purification by flash chromatography gave the pure acetate 9' and 10'.

General procedure for the synthesis of ring opened acrylamides (3b-3h): Compounds 4a-10a were dissolved in CH₂CI₂ (2 mL) and acetonitrile saturated with dry methylamine (0.15 mL) was added to it and the mixture was stirred for 15 min. A white precipitate was formed. The reaction mixture was concentrated in vacuo, filtered and the solid washed with cold CH₂CI₂ (3 x 3 mL) and dried in vacuo to yield 3b-3h as a white powder that was used in the next step without further purification.

* please note that in some molecules the ¹⁵N signal for the imide nitrogen was not observed

Steady State Measurements

All experiments were carried out at 25 °C by using double distilled water or freshly distilled solvents. In aqueous solutions, pH was 7.1 , measured by pH meter (ISFETCOM, model S2K712, JAPAN). The baseline for absorbance was recorded using air as a reference. Steady state fluorescence spectra were recorded having the source and the detector at right angle. The source was a Xenon lamp pulsed at 80 Hz, with 2 με FWHM pulse with peak power equivalent to 75 kW and the Hamamatsu R928 photomultiplier tube, respectively. The excitation and emission slits were kept constant at 5 nm and the PMT voltage was 600 volt for all the experiments unless mentioned elsewhere.

All fluorescence spectra were corrected for changes in absorbance using equation 1 , where the absorbance is at the wavelength of excitation. This procedure nullifies the changes in intensity that might arise due to concentration¹

2

For the quantum yield measurements, the experiments were carried out back to back to maintain identical conditions. The relative quantum yield for the fluorophore can be calculated by integrating the fluorescence spectra by using equation 2, which gives the area under the curve

Here, is the area under the curve in fluorescence emission spectra of the fluorophore at a given excitation wavelength; The absolute quantum yield is given by equation 3²

where are the relative quantum yields of the fluorophore and the standard, is the absolute

quantum yield of the standard known from the literature and n corresponds to the refractive index of the solvent in which the measurements are done. The quantum yields for our experiments were calculated using Quinine sulphate in 0.5 M H₂S0₄ (Φ f = 0.546)³ as standard.

Femtosecond Upconversion Experiment

The output of femtosecond pulsed oscillator from the mode-locked Ti:sapphire laser (Tsunami, Spectra Physics, USA) pumped by 5 W DPSS laser (Millennia, Spectra Physics), centred at 800 nm and with a repetition rate of 80 MHz, was used as the gate pulse for the femtosecond fluorescence upconversion experiments. The second harmonic (400 nm) of this pulse was used as the source of excitation for the sample placed in a rotating cell for the experiments with epicocconone and its analogues. The power of the second harmonic light is restricted to 5 mW at the sample in order to minimize photobleaching. The fluorescence from the sample is upconverted in a nonlinear crystal (0.5 mm BBO, Θ = 38°, Φ = 90°) by mixing with the gate pulse, which consists of a portion of the fundamental beam. The upconverted light is dispersed in a monochromator and detected using photon counting electronics. A cross-correlation function obtained using the Raman scattering from ethanol has a FWHM of 300 fs. The femtosecond fluorescence decays have been fitted using a Gaussian function of the same FWHM as the excitation pulse. The fluorescence decays were recorded at the magic angle polarization with respect to the excitation pulse on FOG 100 fluorescence optically gated upconversion spectrometer (CDP Systems Corp., Russia). The resolution was in appropriate multiples of the minimum step size of the instrument, i.e. 0.78 fs/step. The decays were analyzed by iterative reconvolution using a homemade program.

Steady state and time resolved fluorescence

UV-Vis absorption spectra are recorded on JASCO V-530 double beam absorption spectrophotometer with a slit width of 1 .0 nm at room temperature or on Varian Cary-eclipse spectrophotometer. Emission spectra are recorded ion HORIBA FLUROMAX photon counting fluorimeter with slit width of 5.0 nm for both excitation and emission monochromators. All experiments were carried out at 25 °C by using double distilled water and solvents. The absorbencies of the solutions were kept at ~1 .0 for the upconversion experiments.

For femtosecond upconversion (FOG 100, CDP), the samples were excited at 370 or 410 nm using the second harmonic of a mode-locked Ti-sapphire laser (Tsunami, Spectra Physics) pumped by a 5W Millennia (Spectra Physics) laser. The fundamental beam (740/820 nm) was frequency doubled in a nonlinear crystal (1 mm BBO, Θ = 25°, φ = 90°). The fluorescence emitted from the sample was upconverted in a nonlinear crystal (0.5 mm BBO, Θ = 38°, φ = 90°) using the fundamental beam as a gate pulse. The upconverted light is dispersed in a monochromator and detected using photon counting electronics. A cross- correlation function obtained using the Raman scattering from ethanol displayed a full width at half-maximum (fwhm) of ~300 fs. The femtosecond fluorescence decays were fitted using a Gaussian function of the same FWHM as the excitation pulse. The fluorescence decays were recorded at the magic angle polarization (54.7°) with respect to the excitation pulse on a FOG 100 fluorescence optically gated upconversion spectrometer. The resolution was in appropriate multiples of the minimum step size of the instrument, i.e. 0.78 fs/step. The decays were analyzed by iterative reconvolution using a homemade program. In theory this allows interpolation up to 1 /10th the instrumental response function.

Experiments demonstrating applications as probe

The following examples of ratiometric quantification of various analytes with o-LHBDI demonstrate the functionality of the compounds of the invention. These examples are not limiting, as those skilled in the art will be able to immediately envisage further applications.

Ratiometric measurement of pH

Figure 32 shows the ratiometric measurement of pH using o-LHBDI as a probe. Graphically (A) it can be seen that the ratio between red (600 nm) and cyan (475 nm) fluorescence can be used to accurately measure pH in the range 6-12. There is a 20-fold difference between cyan and red emission over this range when the emissions are normalised against absorption at 410 nm.

Ratiometric measurement of protein concentration

Bovine serum albumin (BSA) was used as a model protein to determine if it is possible to measure the concentration of protein in solution ratiometrically using o-LHBDI as a probe. The cyan emission (at 500 nm; Figure 33A) is exponential whereas the red emission (600 nm) was found to be sigmoidal (Figure 33B). Importantly, the ratio between 600 nm and 500 nm emission was found to be strongly correlated when plotted against the protein concentration (Figure 33C). The ratio of red:cyan emission decreased from 25:1 to 1 :1 over the range 280 nM to 0.14 mM, indicating that this particular fluorophore can be used as a probe to accurately measure low concentration of protein in solution that is independent of many experimental variables.

Ratiometric measurement of lipophilicity

As the internal hydrogen bonding in o-LHBDI can be affected by the availability of external hydrogen bonding sources (e.g. water), it was important to look at the ratio between cyan and red fluorescence in a more hydrophobic environment. From a biological perspective, lipid membranes are very hydrophobic with ~4% water content and this can be mimicked with a detergent, such as SDS, which forms micelles at ~0.2% (CMC; Critical Micellular Concentration) in water. Membranes are also more viscous than water. The present results in Figure 34A show that below the CMC (log[SDS] < -0.5), there was virtually no cyan emission, only red (Figure 34B). In contrast, the red emission increases slightly above the CMC (log[SDS] > -1 ). The observed 300-fold difference between red and cyan emission with increasing SDS (Figure 34C) is dramatic.

Above the CMC, the lipophilicity of the micelles does not change but the viscosity does. Looking at the red:cyan emissions above the CMC revealed that there is a 4-fold increase in cyan emission with increasing viscosity (Figure 34D). These results, taken together suggest that this probe can be used in super resolution microscopy to measure membrane formation, fluidity and viscosity, characteristics that are difficult to measure by other means.

Ratiometric measurement of DNA concentration

In the presence of DNA (1 .0-0.0001 % annealed salmon sperm double stranded DNA in PBS buffer) the red emission of o-LHBDI did not change appreciably (25000 ± 10000) but the cyan emission increased exponentially at high DNA concentrations (Figure 35). This ratiometric behaviour (Figure 35C) is evident at high DNA concentrations as observed for SDS. See Figure 34.

Ratiometric measurement of glycoprotein concentration Glyocoproteins are important surface proteins on cells responsible for cell-cell recognition and integrity. The ability to specifically measure glycoproteins is an unmet need in biology. We have looked at bovine fetuin, a heavily glycosylated protein, that is mostly sugars (glycan). A serial dilution of fetuin from 1 % to 0.001 % resulted in a linear relationship between red and cyan emission with fetuin concentration (Figure 36C). This is seen as an exponential increase in fluorescence with log[fetuin] (Figure 36A and B). At low concentration, no cyan emission is observed (Figure 36A) but this increases exponentially with increasing glycoprotein. However, red emission also increases in the same way. Interestingly, at low concentration there is a systematic difference between the cyan and red emission such that it is possible to accurately quantify the amount of fetuin in solution using the ratio between emission at 500 nm and 600 nm over at least 2 orders of magnitude (Figure 36C).

Ratiometric measurement of starch concentration

The interesting results for a glycoprotein (fetuin) suggest the use of ratiometric quantification on other types of glycans. Thus a serial dilution of soluble starch was prepared. In a similar response to that observed with fetuin, higher concentrations of starch led to an exponential increase in cyan fluorescence (Figure 37A) but a much smaller increase in red fluorescence (Figure 37B). A log-log plot of the

concentration of starch vs the red:cyan emission ratio (Figure 37C) revealed an extraordinary linear plot over at least 3 orders of magnitude. The log-linear plot (Figure 37D) shows the expected exponential relationship between emission ration (500 nm:600 nm) vs log[starch]

The increase in red emission is likely due to an increase in viscosity of starch and fetuin solutions but the exponential increase in cyan emission resulting from the normal Franck-Condon emission of the fluorophore is thought to result from subtle interactions between the probe and the glycan that could be useful for the ultrasensitive ratiometric quantification of glycosylation.

Ratiometric measurement of viscosity (sucrose)

Typically bond rotation in the excited state is responsible for lower quantum yields and that the restriction of bond rotation by increasing steric bulk of the probe or increasing the viscosity of the solution can have dramatic effects on quantum yields. From the results (Figure 38A, B), cyan and red emission clearly increases with log(viscosity). The ratio between red and cyan emissions changes by 2-fold over the viscosity range tested indicating that both normal and ESIPT emission increase ~100 fold at higher viscosity. Summary of results

Solution pH (6-12) has been accurately measured using ratiometric fluorescence. This will be very useful in physiological measurements.

Additionally, there is a correlation between the ratio of red to cyan emission for protein, lipids, DNA, glyocprotein and starch. This relationship is not related to the local viscosity but rather subtle effects that relate to the interaction of the probe with biomolecules.

There is a 300-fold increase in cyan:red emission ratios in response to lipophilicity/micelle formation.

The exemplary probe of the present invention can therefore be used in applications including pH determination, lipophilicity/micelle formation, and the determination of low concentrations of protein, DNA, glyocprotein and polysaccharide (e.g. starch). In addition this probe can be used in environments were protein, lipids (e.g. detergents, membranes) DNA, glyocprotein and polysaccharides occur such as inside cells, in materials science and other analytical applications that require the quantification of protein, lipids, DNA, glyocprotein and polysaccharides.

Claims

1 . A compound having general formula,

Q is S or O; Y is N or O;

D is a proton donating group;

optional R¹ , when present, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyi, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH₂, and -NC(NH₂)2, an optionally substituted aryl and an optionally substituted heteroaryl ring, wherein b is 0, 1 , 2 or 3;

L is an optionally substituted carbocycle or an optionally substituted heterocycle ring, optionally fused to another aromatic or heteroaromatic ring, wherein n is 1 , 2, 3 or 4, and A is independently selected from the group consisting of: -CH₂-, -CR₂ ^*-, =CH-, -CH=, -N=, =N-, -NR -, -0-, C₆H₄, a fused optionally phenyl, where the phenyl can be optionally substituted with hydroxyl, NR₂ ^**; wherein R^* is independently H or C_rC₃ alkyi, where the alkyi is optionally substituted with hydroxyl, halogen, carboxylic acid or sulfonic acid, and wherein R^™ is alkyi or aryl;R⁴, optionally present when Y is N, is selected from the group consisting of: H, optionally substituted alkyi, optionally substituted aryl and optionally substituted heteroaryl;

R⁵ is a bulky substituent having more than four non-hydrogen atoms; and

optional R⁶, when present on each Ar ring, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyi, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH₂ -NHC(0)NH₂ and -NHC(S)NH₂, optionally substituted carbocycle and optionally substituted heterocycle ring, wherein e is 0, 1 , 2, 3 or 4.

2. A compound according to claim 1 , wherein Ar is absent, D is in an ortho or meta position on the phenyl ring, preferably D is in the ortho position.

3. A compound according to claim 1 or claim 2, wherein D is a proton donating group capable of hydrogen bonding, to N* of the 5-membered ring, as well as intramolecular proton transfer.

4. A compound according to any one of claims 1 to 3, wherein Ar is absent and R¹ is in any unoccupied ortho, meta or para phenyl ring position, preferably R¹ is in the para position.

5. A compound according to any one of the preceding claims, wherein Y is N, R⁴ is selected from the group consisting of: H, optionally substituted alkyl, optionally substituted aryl and optionally substituted heteroaryl.

6. A compound according to any one of the preceding claims, in which b is 0, or b is 1 to 3, and R¹ is independently selected from the group consisting of: H, acetate, dialkylamine, and halo, preferably R¹ is fluoro.

7. A compound according to any one of the preceding claims, wherein R⁵ is a bulky substituent of a sufficient size to slow down molecular structural and/or conformational rearrangements that take place in an excited state, such that the lifetime of an associated Franck Condon emission is longer than the time required for formation of an emissive state, for example, an emissive state associated with formation of a ESIPT zwitterion, wherein preferably said bulky substituent has more than six non-hydrogen atoms, or is - C(R⁹)₃, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl or optionally substituted heterocycloalkyl, wherein R⁹ is independently selected from optionally substituted alkyl, optionally substituted carbocycle, optionally substituted heterocycle, preferably R⁵ is optionally substituted phenyl, optionally substituted napthyl, optionally substituted pydridyl, optionally substituted cyclohexyl, optionally substituted methyl, optionally substituted t-butyl and optionally substituted neopentyl.

8. A compound according to any one of the preceding claims, having general formula:

A compound according to any one of the preceding claims, having general formula:

10. A compound according to any one of the preceding claims, wherein D selected from the group consisting of: -OH, or -NH₂, -SH, -NHC(NH)NH_2, -NHC(0)NH₂. and -NHC(S)NH₂

1 1 . A compound according to any one of the preceding claims, having general formula:

12. A compound according to any one of the preceding claims, wherein A and n are selected such that group (A)n is -CHCH-, wherein the locking ring L is a 5-membered ring, or A and n are selected such that (A)_n is -CH₂CH₂-, wherein the locking ring L is a 5 membered ring, A and n are selected such that (A)_n is - CR₂CR₂CR₂-, wherein the locking ring L is a 6 membered ring, whereby R of (A)_n can independently be chosen from H, alkyl or aryl such that at least 2 of the substituents are not H.

13. A compound according to any one of claims 1 to 10, having a structure selected from the group consisting of:

14. A compound according to any one of the preceding claims, having structure:

15. A dual emission fluorescent compound comprising

wherein the compound further comprises one or more groups suitable for inducing a structural rearrangement in the molecule on formation of the excited state, wherein the structural rearrangement stabilises at least a second electromagnetic emission at a second wavelength, such that dual emission is observable at room temperature.

16. A compound comprising:

a conjugated functional group for emitting electromagnetic radiation on excitation to at least a first energy state, wherein upon excitation, the compound attains an emissive ST excited state associated with emission of a first wavelength, preferably an ESIPT emission; wherein the compound is such that the excited state can undergo a structural and/or conformational rearrangement to a second excited state associated with emission of a second wavelength, preferably a Franck Condon emission.

17. The compound of claim 16 wherein the compound attains the emissive ST excited state through an internal conversion process and/or wherein the structural and/or conformational rearrangement which occurs in the exited state is a tautomerism rearrangement.

18. The compound according to claim 16, wherein the structural and/or conformational rearrangement is sufficiently slow that the lifetime of the second excited state associated with emission of a second wavelength is longer than the time required for formation of the emissive ST excited state associated with emission of a first wavelength.

19. A compound according to claim 16 to claim 8 wherein emission at the second wavelength is detectable at room temperature.

20. A compound according to claim 19, wherein the ESIPT rearrangement forms a tautomer and/or zwitterion.

21 . A compound according to claim 19 or claim 20, wherein the functional groups are one more bulky groups which, on formation of the excited and/or subsequent excited states, slows down geometrical adjustment of the compound from the first geometry to a second substantially more planar geometry.

22. A compound according to any one of claims 15 to 21 , having a structure as defined in any one of claims 1 to 14.

23. A compound according to any one of the preceding claims, comprising a conjugated functional group or chromophore, which is a structurally locked GFP chromophore or derivative thereof when the compound is in the ground state.

24. A compound according to any one of the preceding claims, further comprising one or more linker groups, G, for attaching the compound to a target compound, wherein G is selected from C₃-C₂₀ alkyl, a polypeptide or polyethyleneglycol and wherein G is located at one or more of R¹ , R⁴ or R⁵.

25. A compound according to claim 24 wherein the target compound is selected from, biomolecules, organelles, cells, tissues, and other molecules such as, but not limited to metal ions, lipids and cofactors.

26. A method of designing a dual emission fluorescent compound comprising the steps of:

(ii) providing a compound having a first substantially nonplanar geometry in the ground state and an emissive first excited state, preferably associated with an ESIPT emission; and

(ii) inducing a structural and/or conformational rearrangement in the compound from the first substantially nonplanar geometry to at least a second substantially planar geometry that is also emissive, wherein the rearrangement induced is such that the duration of the structural rearrangement is sufficient to generate a further emissive state, preferably associated with a Franck Condon emission, for a sufficient period to allow the emission of electromagnetic radiation at two wavelengths.

27. A method of preparing a compound comprising the steps of: (i) condensing an arylketone having general formula (B) with an aryl oxazoline having general formula (C) in the presence of a Lewis acid, preferably TiCI₄ to form an oxazolinone having general formula (F):

in which:

optional, D when present, is a proton donating group;

optional R¹ , when present, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyi, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH_2, -NHC(0)NH₂ and -NHC(S)NH₂, an optionally substituted aryl and an optionally substituted heteroaryl ring, wherein b is 0, 1 , 2 or 3;

L is an optionally substituted carbocycle or an optionally substituted heterocycle ring, optionally fused to another aromatic or heteroaromatic ring, wherein n is 1 , 2, 3 or 4, and A is independently selected from the group consisting of: -CH₂-, -CR₂ ^*-, =CH-, -CH=, -N=, =N-, -NR -, -0-, C₆H₄, a fused optionally phenyl, where the phenyl can be optionally substituted with hydroxyl, NR₂ ^**; wherein R^* is independently H or C_rC₃ alkyi, where the alkyi is optionally substituted with hydroxyl, halogen, carboxylic acid or sulfonic acid, and wherein R^™ is alkyi or aryl;

R⁵ is a bulky substituent having more than four atoms; and

(iv) performing a nucleophilic ring opening of oxazolinone (F) with an amine to form an

arylacrylamine intermediate having general formula (E):

(v) performing a dehydrative condensation initiated ring closing step on the arylacrylamine (E) to form the corresponding imidazolinone (A):

28. The method according to claim 27, wherein ring opening step (ii) is carried out by stirring a dichloromethane solution of oxazolinone (F) at room temperature in the presence of acetonitrile and dry methylamine, preferably for a period of from about 15 minutes to 2 hours, and/or wherein the ring closing step (iii) cyclises the arylacrylamine intermediate (E) through dehydrative cyclization of (E) by heating the intermediate, preferably to a temperature of from room temperature to 300 °C for a period of from 1 -10 minutes.

wherein

optional D, when present, is a proton donating group;

optional Ar, is an optionally substituted monocyclic aryl ring, an optionally substituted bicyclic aryl ring system, an optionally substituted polycyclic aryl ring system or an optionally substituted monocyclic heteroaryl ring, an optionally substituted bicyclic heteroaryl ring system, an optionally substituted polycyclic heteroaryl ring system; optional R , when present, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyi, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH_2, -NHC(0)NH₂ and -NHC(S)NH₂, an optionally substituted aryl and an optionally substituted heteroaryl ring, wherein b is 0, 1 , 2 or 3;

R² is H, or optionally substituted alkyl;

R³ is optionally substituted alkyl; or

R⁵ is a bulky substituent having more than four atoms; and

optional R⁶, when present on each Ar ring, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyi, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH_2, -NHC(0)NH₂ and -NHC(S)NH₂, optionally substituted carbocycle and optionally substituted heterocycle ring, wherein e is 0, 1 , 2, 3 or 4,

30. Use according to claim 29 wherein the intermediate has general structure:

wherein

D is a proton donating group;

optional R , when present, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyi, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH₂ -NHC(0)NH₂ and -NHC(S)NH₂, an optionally substituted aryl and an optionally substituted heteroaryl ring, wherein b is 0, 1 , 2 or 3;

L is an optionally substituted carbocycle or an optionally substituted heterocycle ring, optionally fused to another aromatic or heteroaromatic ring, wherein n is 1 , 2, 3 or 4, and A is independently selected from the group consisting of: -CH₂-, -CR₂ ^*-, =CH-, -CH=, -N=, =N-, -NR -, -0-, C₆H₄, a fused phenyl, where the phenyl can be optionally substituted with hydroxyl, NR₂ ^**; wherein R^* is independently H or C₁-C₃ alkyl, halogen or carboxyl, and wherein R^™ is alkyl or aryl;

R⁵ is a bulky substituent having more than four atoms; and

optional R⁶, when present on each Ar ring, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH₂ -NHC(0)NH₂ and -NHC(S)NH₂, optionally substituted carbocycle and optionally substituted heterocycle ring, wherein e is 0, 1 , 2, 3 or 4.

31 . Compound obtained by the method of any one of claims 26 to 28 or the use of claim 29 or claim 30.

32. Use of a compound according to any one of claims 1 to 25 or claim 31 in one or more of environmental, biotechnological and diagnostic applications involving ratiometric fluorescence measurements, sensing and/or imaging, fluorescence switching, a fluorescent probe, a GFP mimetic, a chemically tuneable optoelectronic device, a two colour light emitting diode, and/or in super high resolution microscopy (e.g. STORM) or in the manufacture of fluorescent probes, coatings, objects, scintillators, or light sources or in mixing and fluid dynamics where spectral modifications and/or fluorescence-intensity value fluctuations in a mixture may be indicative of a proportional and/or localised change viscosity, electric fields, temperature, pH, hydrophobicity and/or ionic strength, preferably of specific ions, FRET, FLIM and FCS based applications, in super-resolution imaging, including Photo-Activation Localization Microscopy (PALM), Fluorescence Photo-Activation Localization Microscopy (FPALM), Stochastic Optical Reconstruction Microscopy (STROM) and light sheet fluorescence microscopy (LSFM), for example, time-lapse imaging, advanced multiparameter imaging, live cell imaging, Stochastic Optical Reconstruction Microscopy or ultrasensitive, super resolution pH monitoring inside live cells.

33. Use of a compound according to any one of claims 1 to 25 or claim 31 as a self-calibrating (ratiometric) fluorophore probe that displays a spectral modification upon interaction with a given biological target thereby providing a concentration-independent measurement through the ratio of fluorescence- intensity values measured at two selected wavelengths (dual emission).

34. Use of a compound according to any one of claims 1 to 25 or claim 31 as a sensor of one or more biological and environmental signals such as viscosity, electric fields, temperature, pH, hydrophobicity, lipophilicity/micelle formation, ionic strength, and/or the determination of low concentrations of protein, lipids, DNA, glyocprotein and polysaccharides, such as starch.

35. A method of detecting a local change in an environment, comprising the step of:

providing the environment with one or more dual fluorescent compounds;

determining an initial level of ratiometric fluorescence arising from said compounds and associated with an initial set of environmental conditions; and

determining a change in the local environment on the basis of observation of a degree of change in the ratiometric fluorescence.

36. The method according to claim 35, further comprising: associating the one or more compounds with a ligand of interest; and determining a change in the ratiometric fluorescence resulting from the association of the ligand with the compound, preferably wherein the ligand is selected from enzymes, biomolecules, organelles, cells, tissues, and other molecules such as, but not limited to, metal ions, lipids and cofactors.

37. A method according to any one of claims 35 or 36 wherein the method is a stop-go assay whereby the one or more compounds are used as a probe capable of switching between FC and ESIPT emissions, or as a probe capable of a continuous transition from 100% FC to 100% ESIPT.

38. A method according to any one of claims 35 to 37, wherein the change in the ratiometric fluorescence corresponds to a Stokes' shift between FC and ESIPT emissions > 200 nm.

39. A method of switching off on emission mode of a dual emission fluorescent compound accordingly to any one of claims 1 to 25 or claim 31 , comprising the step of suppressing one emissive state associated with the compound, preferably an ESIPT emissive state.

40. A method according to claim 40 wherein the emissive state is suppressed by subjecting the compound to conditions that result in deprotonation of a tautomer associated with the compound.

41 . A compound having the following general formula, salts, esters, conformational and configurations isomers thereof, including zwitterions and tautomers thereof:

in which,

Q is S or O; Y is N or O; Z is N; and X is N or CH, wherein c is 0 or 1 ;

optional D, when present, is a proton donating group;

R² is H, or optionally substituted alkyl;

R³ is optionally substituted alkyl; or

R⁴, optionally present when Y is N, is selected from the group consisting of: H, optionally substituted alkyl, optionally substituted aryl and optionally substituted heteroaryl; R⁵ is a bulky substituent having more than four atoms; and

optional R⁶, when present on each Ar ring, is independently selected from the group consisting of: H, acetate, hydroxyl, amine, amide, thiol, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, halo, cyano, nitro, haloalkyi, alkylamine, dialkylamino, alkylthio, -NHC(NH)NH_2, -NHC(0)NH₂ and -NHC(S)NH₂, optionally substituted carbocycle and optionally substituted heterocycle ring, wherein e is 0, 1 , 2, 3 or 4.

42. The compound of claim 41 selected from the group consisting of:

Locked imidazolinones