Azulene—A Bright Core for Sensing and Imaging

molecules

Review Azulene—A Bright Core for Sensing and Imaging

Lloyd C. Murﬁn * and Simon E. Lewis

Department of Chemistry, University of Bath, Bath BA2 7AY, UK; [email protected] * Correspondence: lloyd.murﬁ[email protected]

Abstract: Azulene is a hydrocarbon isomer of naphthalene known for its unusual colour and fluorescence properties. Through the harnessing of these properties, the literature has been enriched with a series of chemical sensors and dosimeters with distinct colorimetric and fluorescence responses. This review focuses specifically on the latter of these phenomena. The review is subdivided into two sec- tions. Section one discusses turn-on fluorescent sensors employing azulene, for which the literature is dominated by examples of the unusual phenomenon of azulene protonation-dependent fluorescence. Section two focuses on fluorescent azulenes that have been used in the context of biological sensing and imaging. To aid the reader, the azulene skeleton is highlighted in blue in each compound.

Keywords: ﬂuorescence; azulene; sensor; dosimeter; bioimaging; chemosensor; chemodosimeter

1. Introduction Azulene, 1, is an isomer of naphthalene, 2, composed of fused 5- and 7-membered ring systems (Figure1) and named for its vibrant blue colour. Unlike naphthalene, azulene is a non-alternant hydrocarbon, possessing nodal points at C-2 and C-6 of the HOMO and C-1 and C-3 of the LUMO [1]. The location of these nodes results in low electronic repulsion in the S1 singlet excited state, affording a relatively small HOMO-LUMO gap. Hence, the S0→S1 transition arises from absorption in the visible region. Conversely, in naphthalene, coefficient magnitudes remain consistent for each position in both the HOMO Citation: Murfin, L.C.; Lewis, S.E. and LUMO, affording a greater electronic repulsion; the greater S0–S1 energy gap sees Azulene—A Bright Core for Sensing naphthalene absorb in the UV region. In a similar relationship, there is little change in the and Imaging. Molecules 2021, 26, 353. coefficient magnitudes of the HOMO and LUMO+1 of azulene, creating a large S0–S2 gap https://doi.org/10.3390/ (and hence also a large S –S gap). This produces a system where the transition S →S is molecules26020353 1 2 2 0 the predominant mode of fluorescence [2,3], violating Kasha’s rule [4].

Received: 18 December 2020 By varying the nature of the functional group attached to the azulene core at positions Accepted: 8 January 2021 where electron density is greatest, the wavelength of light absorbed can be fine-tuned [5]. Published: 12 January 2021 Electron-withdrawing groups present at the C-1 and C-3 positions stabilise the HOMO and LUMO+1 (whilst having no effect on the LUMO), increasing the S0–S1 gap. The contrary Publisher’s Note: MDPI stays neu- argument is true for electron-donating groups at these positions: the HOMO and LUMO+1 tral with regard to jurisdictional clai- are destabilised, reducing the S0–S1 gap [6]. S2 emission predominates when the S2–S1 gap −1 −1 ms in published maps and institutio- is >10,000 cm (cf. 14,000 cm in azulene 1), whilst a mixture of S2 and S1 fluorescence −1 −1 nal affiliations. emission is present when the S2–S1 gap is 9000 cm –10,000 cm [7]. When the gap is −1 smaller than 9000 cm , the rate of internal conversion between S2 and S1 is greater than the rate of relaxation from S2, so fluorescence occurs from S1 [8]. Whilst the azulene core has been incorporated into a wide variety of colorimetric Copyright: © 2021 by the authors. Li- chemical probes (exploiting the vivid colours it is famous for) [9–30], this review will focus censee MDPI, Basel, Switzerland. on those published probes that exploit the fluorescent nature of azulene. The nature of This article is an open access article azulene fluorescence is highly dependent on substitution around the azulene core, which distributed under the terms and con- ditions of the Creative Commons At- affects whether emission is more dominant from the S2 or S1 singlet excited state (a notable tribution (CC BY) license (https:// exception from this can be found in the work of Zhu et al., who showed that emission of creativecommons.org/licenses/by/ azulene aldehydes can occur from the S3 or S2 excited state dependent on the nature of the 4.0/). hydrogen bonding present [31,32]).

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FigureFigure 1. 1.Structures, Structures, HOMOs HOMOs and LUMOs and LUMOs of azulene of andazulene naphthalene. and naphthalene. Asterisks (*) areAsterisks used to (*) are used to showshow the the alternant alternant nature nature of naphthalene of naphthalene and the non-alternant and the non-alternant nature of azulene. nature of azulene. A variety of fluorescent probes harnessing the azulene core have been reported. Such probesWhilst are traditionally the azulene composed core ofhas three been parts: incorporated a recognition into moiety a wide (that interactsvariety of colorimetric selectivelychemical with probes the desired (exploiting analyte), the a reportervivid colours moiety (thatit is famous gives a measurable for) [9–30], response this review will focus uponon those analyte published interaction—in probes this instancethat exploit the azulene the fluorescent core) and a nature linker that of connectsazulene. the The nature of az- two moieties. If the probe undergoes an irreversible, usually covalent, interaction with the analyte,ulene thefluorescence probe is described is highly as a chemodosimeter dependent on [33 substitution]. Reaction of the around recognition the groupazulene core, which andaffects the analyte whether usually emission results in is a more cleavage dominant of the linker from and the release S2 or of theS1 singlet response excited moiety state (a notable asexception a separate species.from this Chemodosimeters can be found are in highly the work selective of due Zhu to theiret al covalent., who interactionshowed that emission of with an analyte. In contrast, chemosensors interact reversibly and non-covalently with an azulene aldehydes can occur from the S3 or S2 excited state dependent on the nature of the analyte. The inherent reversibility of chemosensors often leads to reduced selectivity, but hydrogen bonding present [31,32]). allows for a real-time response to the analyte-binding event and can therefore be used to monitorA changes variety in of analyte fluorescent concentration probes over harnessing time [34]. the azulene core have been reported. Such probesFluorescent are traditionally probes offer a composed non-invasive of means three of analyteparts: detection.a recognition They aremoiety required (that interacts se- inlectively scenarios with where the a colorimetric desired analyte), response a is reporter unsuitable, moiety i.e., in an(that environment gives a measurable that is response alreadyupon analyte highly coloured, interaction—in such as a biologicalthis instance matrix the [35 azulene]. Upon analytecore) and interaction, a linker the that connects the reporter of a fluorescence probe will respond in one of three ways: two moieties. If the probe undergoes an irreversible, usually covalent, interaction with the 1. Begin fluorescing—a “turn-on” response. 2.analyte, Stop fluorescing—a the probe is “turn-off” describe response.d as a chemodosimeter [33]. Reaction of the recognition 3.groupAlter and emission the analyte wavelength—a usually ratiometric results in probe, a cleava wherebyge of responsethe linker is recordedand release as a of the response moietyratio as of thea separate emission ofspecies. the product Chemodosimeter over the emissions are of starting highly material. selective due to their covalent interactionWhen considering with an probesanalyte. for In biological contrast, applications, chemosensors probes thatinteract excite reversibly or emit in and non-cova- thelently red to with near-infrared an analyte. (NIR) The window inherent (650–900 reversibility nm) offer significant of chemosensors advantages, often namely leads to reduced non-invasive deep-tissue penetration (µm to cm) and reduced background absorption selectivity, but allows for a real-time response to the analyte-binding event and can there- from water and haemoglobin [36], whilst reducing the risk of sample damage and photo- bleachingfore be used common to monitor with higher changes energy in UV analyte light sources concentration [37]. Autofluorescence over time [34]. from endogenousFluorescent species should probes also offer be avoided, a non-invasive as such species means typically of exciteanalyte or emit detection. below They are re- 600quired nm [38 in]. scenarios where a colorimetric response is unsuitable, i.e., in an environment that is already highly coloured, such as a biological matrix [35]. Upon analyte interaction, the reporter of a fluorescence probe will respond in one of three ways: 1. Begin fluorescing—a ”turn-on” response. 2. Stop fluorescing—a “turn-off” response. 3. Alter emission wavelength—a ratiometric probe, whereby response is recorded as a ratio of the emission of the product over the emission of starting material. When considering probes for biological applications, probes that excite or emit in the red to near-infrared (NIR) window (650–900 nm) offer significant advantages, namely non-invasive deep-tissue penetration (µm to cm) and reduced background absorption from water and haemoglobin [36], whilst reducing the risk of sample damage and photo- bleaching common with higher energy UV light sources [37]. Autofluorescence from endogenous species should also be avoided, as such species typically excite or emit below 600 nm [38].

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2. Discussion 2. Discussion 2.1. Turn-on Azulene Fluorescent Sensors 2.1.2.1. Turn-OnTurn-on Azulene Fluorescent Sensors The most common fluorescence sensing motif within the literature is that of azulene TheThe mostmost commoncommon fluorescencefluorescence sensingsensing motifmotif withinwithin thethe literatureliterature isis thatthat ofof azuleneazulene proton probes. By considering azulene as a cyclopentadienyl anion fused to a tropylium protonproton probes.probes. ByBy consideringconsidering azuleneazulene asas aa cyclopentadienylcyclopentadienyl anionanion fusedfused toto aa tropyliumtropylium cation, it can be appreciated that electron density is increased at the 1- and 3-positions. cation,cation, itit cancan bebe appreciatedappreciated thatthat electronelectron densitydensity isis increasedincreased atat thethe 1-1- andand 3-positions.3-positions. Protonation at either of these positions is known to lead to a significant increase in fluo- ProtonationProtonation atat either either of of these these positions positions is is known known to to lead lead to to a significanta significant increase increase in fluores-in fluorescence (Scheme 1, in which the nomenclature [1 + H]+ denotes an azulene protonated on cencerescence (Scheme (Scheme1, in 1, which in which the the nomenclature nomenclature [1 +[1 H] + H]+ denotes+ denotes an an azulene azulene protonated protonated on on the 5-membred ring) [39]. thethe 5-membred5-membred ring)ring) [[39].39].

Scheme 1. Protonation mechanism of azulene 1. SchemeScheme 1.1. ProtonationProtonation mechanismmechanism ofof azuleneazulene1 1..

WhenWhen consideringconsidering synthetic synthetic access access to chemicalto chemical probes, probes, the electronicthe electronic nature nature of azulene, of az- asulene, shown as shown in Scheme in Scheme1, is often 1, is exploited. often expl Theoited. nucleophilicity The nucleophilicity of the of 5-membered the 5-membered ring isring known is known to facilitate to facilitate the additionthe addition of electrophiles of electrophiles to theto the azulene azulene core core at at the the 1- 1- and/or and/or 3-positions3-positions (e.g.,(e.g., halideshalides [[40],40], sulfoniumsulfonium saltssalts [[41]).41]). FunctionalityFunctionality cancan alsoalso bebe introducedintroduced toto thethe azuleneazulene corecore byby preferentialpreferential C-HC-H borylationborylation ofof thethe 2-position2-position [[42],42], amination aminationof of the the 6-position6-position byby vicariousvicarious nucleophilicnucleophilic substitutionsubstitution [[43]43] oror throughthrough thethe ground-upground-up synthesissynthesis ofof thethe azuleneazulene skeleton.skeleton. ForFor example,example, thethe workwork byby NozoeNozoe etetal. al. detailsdetails howhow substitutedsubstituted azulenesazulenes cancan easily be synthesised synthesised in in high high yield yield from from troponoids troponoids [44]. [44 ].The The following following se- selectionlection will will highlight highlight a arange range of ofazulene azulene moti motifsfs that that have have specifically specifically exploited exploited the the in- increasedcreased nucleophilicity nucleophilicity of ofthe the azulene azulene 1- and 1- and 3-positions 3-positions to protonate to protonate the theazulene azulene core core and and generate turn-on fluorescence species. generate turn-on fluorescence species. Protonation-dependence fluorescence of azulene has been reported multiple times in Protonation-dependence fluorescence of azulene has been reported multiple times in the azulene-oligomer field [45]. In 2009, Xu et al. synthesised a series of 1,3-difluorenyl the azulene-oligomer field [45]. In 2009, Xu et al. synthesised a series of 1,3-difluorenyl substituted azulene monomer (M1, M2) and polymer (P5, P6) macromolecules (Figure2 ) substituted azulene monomer (M1, M2) and polymer (P5, P6) macromolecules (Figure 2) that were shown to exhibit turn-on fluorescence upon titration with trifluoracetic acid that were shown to exhibit turn-on fluorescence upon titration with trifluoracetic acid (TFA) [46]. It was found that, upon protonation, the LUMO–LUMO+1 gap of the monomers (TFA) [46]. It was found that, upon protonation, the LUMO–LUMO+1 gap of the mono- shrinks,mers shrinks, affording affording a decrease a decrease in the S in1–S the2 gap. S1–S The2 gap. authors The authors attributed attributed the resulting the resulting change mers shrinks, affording a decrease in the S1–S2 gap. The authors attributed the resulting in the HOMO–LUMO gap as the cause of fluorescence. change in the HOMO–LUMO gap as the cause of fluorescence.

FigureFigure 2.2. MonomersMonomers andand polymerspolymers synthesisedsynthesised byby XuXu etet al.al. [[46].46].

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Similarly,Similarly, Hawker Hawker et et al.al. reportedreported aa ten-foldten-fold increaseincrease inin fluorescencefluorescenceintensity intensityof oftheir their azulene-thiopheneazulene-thiophene oligomeroligomer 3a3a uponupon treatmenttreatment withwith TFATFA (Figure(Figure3 3))[ [47].47]. UponUpon protona-protona- tiontion ofof thethe azuleneazulene core,core, bothboth thethe wavelengths wavelengths ofof maximummaximum absorbanceabsorbance andand maximummaximum fluorescencefluorescence emission emission underwent underwent a a bathochromic bathochromic shift. shift.Subsequent Subsequent efforts efforts of ofHawker Hawker and and co-workersco-workers revealedrevealed thatthat thethe protonation-basedprotonation-based fluorescencefluorescence ofof azuleneazulene waswas dependentdependent onon thethe nature,nature, positionposition andand connectivityconnectivity ofof thethe aromaticaromatic groupsgroups attachedattached toto thethe azuleneazulene corecore [[48].48]. OligomersOligomers 3b3b––3f3f remained non-fluorescentnon-fluorescent uponupon treatmenttreatment withwith TFA. However,However, (bis)furan(bis)furan3g 3gwas wasfound foundto toexhibit exhibita afluorescent fluorescent turn-on turn-on response response when when protonated. protonated.

FigureFigure 3.3. Azulene-thiopheneAzulene-thiophene oligomeroligomer synthesisedsynthesised byby HawkerHawker etet al.al. [[47,48].47,48].

InIn 2012,2012, VenkatesanVenkatesan etet al.al. reportedreported thethe synthesissynthesis ofof aa familyfamily ofof 2-alkynyl2-alkynyl azulenesazulenes ((4a4a––4k4k,, FigureFigure4 ),4), showing showing that that the the fluorescence fluorescence emission emission could could be be altered altered depending depending on on thethe protonationprotonation state state of of the the azulene azulene [49 [49].]. In particular,In particular, pentafluorophenyl pentafluorophenyl derivative derivative4d (the 4d most(the most fluorescent fluorescent species species synthesised synthesised in the in study) the study) was seenwas seen to undergo to undergo a significant a significant red- shiftred-shift in emission in emission when when treated treated with with TFA. TFA. Neutral Neutral4d was 4d foundwas found to absorb to absorb strongly strongly in the in visiblethe visible region region at 310 at and310 and 394 394 nm, nm, which which the authorsthe authors calculated calculated (Time-Dependent (Time-Dependent Density Den- → → Functionalsity Functional Theory Theory at the PBE1PBE/6-31+G9(d)at the PBE1PBE/6-31+G9(d) level) correspondslevel) corresponds to the S 0to Sthe4 and S0→ SS04 andS2 → transitionsS0→S2 transitions respectively respectively (notably, (notably, the theoretical the theoretical S0 S 1S0transition→S1 transition was was experimentally experimen- absent).tally absent). When When treated treated withMeSO with MeSO3H, a single,3H, a single, broad, broad, red-shifted red-shifted absorbance absorbance was observed was ob- for [4d + H]+ at 440 nm (corresponding to the S →S transition). Neutral species 4d was served for [4d + H]+ at 440 nm (corresponding to0 the1 S0→S1 transition). Neutral species 4d found to emit with low fluorescence intensity (λ = 394 nm, λ = 424 nm), which doubled was found to emit with low fluorescence intensityex (λex = 394em nm, λem = 424 nm), which when treated with TBAF, and increased 12-fold when treated with nPr NBF . Protonated doubled when treated with TBAF, and increased 12-fold when treated 4with n4Pr4NBF4. Pro- + speciestonated [4d species + H] [4dwas + H] found+ was tofound fluoresce to fluoresce ~250x as~250x intensely as intensely as the as neutral the neutral species, species, with awith broad a broad red-shifted red-shifted emission emission at 487 at nm. 487 The nm. red-shift The red-shift in absorbance in absorbance and emission and emission upon protonation was observed with all species tested, with fluorescence emissions occurring upon protonation was observed with all species tested, with fluorescence emissions oc- from 443 nm all the way to 750 nm. curring from 443 nm all the way to 750 nm.

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FigureFigureFigure 4.4. AlkynylAlkynyl azuleneazulene species speciesspecies develope developeddeveloped dby byby Venkatesan VenkatesanVenkatesan et et al.et al. inal. in 2012in 2012 2012 [49] [ 49[49]. ]. .

TheThe followingfollowing year, year, Venkatesan VenkatesanVenkatesan et etet al. al.al. published publishedpublished a follow-up aa follow-upfollow-up article articlearticle on ontheon thethestimuli- stimuli-stimuli- responsiveresponsiveresponsive behaviour behaviour of of of di(p di(p di(phenylethynyl)henylethynyl)henylethynyl) azulenes azulenes azulenes species species species 5a– 5a5d5a– (Figure5d–5d (Figure(Figure 5) [50]. 5) 5[50]. )[All 50 neu-All]. neu- All neutraltraltral species species showedshowed showed threethree three distinct distinct distinct absorbance absorbance absorbance regions: regions: regions: >500 >500 nm nm (S0 (S→(S0S0→1→), S500–3701),), 500–370500–370 nm nmnm (S(S(S000→→S22)) and and <370<370 <370 nmnm nm (S (S0 (S→0→0S→S3),3),S with3 with), with the the most the most most intense intense intense absorptions absorptions absorptions occurring occurring occurring at 409 at 409( at5d), 409( 5d418), (5d 418), 418((5b5b), ( 5b423), ( 4235a)) andand (5a )446 446 and nm nm 446 ( 5d(5d). nm). The The (5d fluorescence fluorescence). The fluorescence emissions emissions of emissions 5aof (4655a (465 nm) ofnm) and5a and 5b(465 (432 5b nm) (432 nm) and nm) 5bwerewere(432 found nm) to wereto bebe very very found weak, weak, to be with with very intensity intensity weak, withincreasing increasing intensity slightly slightly increasing for for5c (484 5c slightly (484 nm). nm). forConversely, 5cConversely,(484 nm). 2→ 0 Conversely,5d5d gavegave aa strong,strong,5d gave sharpsharp a strong, emission emission sharp at at 416 emission416 nm. nm. TF-DFT TF-DFT at 416 calculations nm. calculations TF-DFT suggested calculationssuggested S S suggested2 →asS 0the as the Sdominantdominant2→S0 as themode dominant ofof fluorescencefluorescence mode of in in fluorescence all all cases. cases. Upon Upon in allprotonation protonation cases. Upon all allspecies protonation species exhibited exhibited all speciesthe the exhibitedpreviouslypreviously the observedobserved previously red-shift red-shift observed in in absorbances, absorbances, red-shift in with absorbances,with maxima maxima at with 703,at 703, maxima689, 689, 525 525 atand 703, and 490 689, 490nm 525nm for [5a + H]+ to [5b + H]++, respectively.+ Absorbance intensities of [5c + H]+ and [5d + H]+ + andfor [5a 490 + nm H] for+ to [5a [5b + + H] H]+to, respectively. [5b + H] , respectively. Absorbance Absorbance intensities intensities of [5c + H] of+ [5cand + [5d H] +and H]+ were significantly+ greater than the other probes, which exhibited relatively low extinction [5dwere + H]significantlywere significantly greater than greater the other than prob the otheres, which probes, exhibited which exhibitedrelatively relativelylow extinction low coefficients. Fluorescent emissions of the protonated species also underwent a batho- extinctioncoefficients. coefficients. Fluorescent Fluorescent emissions emissionsof the protonated of the protonated species also species underwent also underwent a bathochromic shift relative to their neutral counterparts. Probes [5a + H]+ (722 nm) and [5b+ + H]+ achromic bathochromic shift relative shift relativeto their neutral to their counterparts. neutral counterparts. Probes [5a Probes+ H]+ (722 [5a nm) + H] and(722 [5b + nm) H]+ (733 nm) remained+ relatively weakly fluorescent, whilst [5c + H]+ (571 nm) and [5d+ + H]+ and(733 [5b nm) + H]remained(733 nm) relatively remained weakly relatively fluorescent, weakly whilst fluorescent, [5c + H] whilst+ (571 [5c nm) + H]and (571[5d + nm) H]+ and(537 [5d nm) + were H]+ (537strongly nm) fluorescent were strongly with fluorescent the former withnow emitting the former with now the emittinggreatest inten- with the (537 nm) were strongly fluorescent with2→ the0 former now emitting with the greatest+ inten- greatestsity. The intensity. authors commented The authors that commented the S S emission that the was S → dominantS emission for [5a was + H] dominant, whilst for 2→ 0 2 0 + sity.S1→S The0 emissions authors were commented dominant that for the otherS S three emission probes. was dominant for [5a + H] , whilst [5a + H]+, whilst S →S emissions were dominant for the other three probes. S1→S0 emissions were1 dominant0 for the other three probes.

Figure 5. Di(phenylethynyl) azulene species developed by Venkatesan et al. in 2013 [50]. FigureFigure 5.5. Di(phenylethynyl)Di(phenylethynyl) azuleneazulene speciesspecies developeddeveloped bybyVenkatesan Venkatesan et et al. al. in in 2013 2013 [ 50[50]]. . In 2015, Belfield et al. showed that 4-styrylguaiazulene derivates (6a–6l, Figure 6) were also able to show NIR fluorescent emission upon protonation of the guaiazulene core InIn 2015,2015, BelfieldBelfield etet al.al. showedshowed thatthat 4-styrylguaiazulene4-styrylguaiazulene derivatesderivates ((6a6a––6l6l,, FigureFigure6 )6) [51]. Similar to the work published by Venkatesan et al. in 2012, the nature of the absorp- werewere alsoalso able to to show show NIR NIR fluorescent fluorescent emissi emissionon upon upon protonation protonation of the of theguaiazulene guaiazulene core tion and emission of the guaiazulene derivatives was tuned by altering the nature of the core [51]. Similar to the work published by Venkatesan et al. in 2012, the nature of [51].attached Similar conjugated to the work aromatic published system. by Neutral Venkatesan species et 6a al.– 6lin were 2012, found the nature to display of the weak absorp- thetion absorption and emission and of emission the guaiazulene of the guaiazulene derivatives derivativeswas tuned by was altering tuned the by nature altering of thethe S1→S0 fluorescent emission in DCM. Protonation of all species afforded a fluorescence nature of the attached conjugated aromatic system. Neutral species 6a–6l were found to attachedturn-on response, conjugated with aromatic accompanying system. red-shift Neutral of species emission. 6a –Significan6l were foundt Stokes to shifts display were weak → displayS1→S0 fluorescent weak S1 Semission0 fluorescent in DCM. emission Protonatio in DCM.n of Protonation all species ofafforded all species a fluorescence afforded a turn-on response, with accompanying red-shift of emission. Significant Stokes shifts were

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Molecules 2020, 25, x fluorescence turn-on response, with accompanying red-shift of emission. Significant6 of Stokes19 shifts were observed, ranging from 67–182 nm. Fluorescence emission (S1→S0) of the compoundsobserved, ranging all occurred from 67–182 at relatively nm. Fluorescence long wavelengths emission (512–768(S1→S0) of nm), the compounds with compounds all + [6doccurred + H]+ –[6fat relatively + H]+ and long [6j wavelengths + H]+–[6l + H](512–768+ emitting nm), in with the compounds NIR region (650–950[6d + H] –[6f nm). + The + + + authorsH] and proposed[6j + H] –[6l that + H] upon emitting protonation in the NIR of the region guaiazulene (650–950 core,nm). The an intramolecular authors proposed charge transferthat upon (ICT) protonation state is generated. of the guaiazulene Computational core, an studies intramolecular have been charge performed transfer by (ICT) Belfield etstate al. is on generated. analogous Computational fluorescent guaiazulene studies have species. been performed The group by Belfield were able et al. to on refine anal- their ogous fluorescent guaiazulene species. The group were able to refine their computational computational model to obtain predicted fluorescence spectra which closely matched those model to obtain predicted fluorescence spectra which closely matched those obtained ex- obtained experimentally [52]. perimentally [52].

FigureFigure 6. 4-Styrylguaiazulene derivates derivates synthesised synthesised by by Belfield Belﬁeld et al. et al.in 2015 in 2015 [51] [.51 ].

InIn 2018, Gao Gao et et al. al. explored explored the the effect effect on on fluorescence fluorescence of substituting of substituting azulene azulene at either at either thethe 2-2- oror 6-position with with aromatic aromatic moieties moieties (7a (7a–f–, fFigure, Figure 7)7 [53].)[ 53 Similar]. Similar to the to theprevious previous observations,observations, fluorescence fluorescence was was dependent dependent on on th thee protonation protonation state state of the of theazulene azulene core. core. In In thethe neutralneutral state only only 7d7d waswas found found to to be be strongly strongly fluorescent fluorescent (λem (λ em= 386= 386 nm), nm), whilst whilst the the remainingremaining compounds emitted emitted with with weak weak intensities intensities (λ (emλ em= 408–447= 408–447 nm). nm). Upon Upon protona- protona- tion,tion, red-shifted emissions emissions with with stro strongng intensities intensities were were observed observed for 7a for (λem7a =( 451λem nm),= 451 7b nm), 7b(λem(λ =em 503= 503nm) nm)and and7f (λ7fem =(λ 500em =nm). 500 The nm). remaining The remaining compounds compounds were found were to foundbe rela- to be relativelytively non-fluorescent non-fluorescent in comparison. in comparison. Within the azulene protonation-dependent fluorescence field, there are multiple published examples reported by Shoji and co-workers [54]. A series of 2-arylazulenes were synthesised, of varying electronic nature, on the attached aryl moieties (Figure8). Azu- lenes 8a–8c and 9a–9h were all found to undergo fluorescence enhancement when in acidic solution. The presence of the isopropyl group within 8b and 8c gave minor in- creases in quantum yield and decreases in Stokes shift. The fluorescence properties were found to be dependent on the electronic nature of group at the para-position of the phenyl substituents. Electron-donating moieties (9a, 9b, 9e) afforded lower quantum yields, fluorescence emission at longer wavelengths and greater Stokes shifts. Conversely, the presence of electron-withdrawing moieties (9c) resulted in greater quantum yields and emission at shorter wavelengths. Figure 7. Substituted azulenes synthesised by Gao et al. in 2018 [53].

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observed, ranging from 67–182 nm. Fluorescence emission (S1→S0) of the compounds all occurred at relatively long wavelengths (512–768 nm), with compounds [6d + H]+–[6f + H]+ and [6j + H]+–[6l + H]+ emitting in the NIR region (650–950 nm). The authors proposed that upon protonation of the guaiazulene core, an intramolecular charge transfer (ICT) state is generated. Computational studies have been performed by Belfield et al. on analogous fluorescent guaiazulene species. The group were able to refine their computational model to obtain predicted fluorescence spectra which closely matched those obtained experimentally [52].

Figure 6. 4-Styrylguaiazulene derivates synthesised by Belfield et al. in 2015 [51].

In 2018, Gao et al. explored the effect on fluorescence of substituting azulene at either the 2- or 6-position with aromatic moieties (7a–f, Figure 7) [53]. Similar to the previous observations, fluorescence was dependent on the protonation state of the azulene core. In the neutral state only 7d was found to be strongly fluorescent (λem = 386 nm), whilst the remaining compounds emitted with weak intensities (λem = 408–447 nm). Upon protona- Molecules 2021, 26, 353 tion, red-shifted emissions with strong intensities were observed for 7a (λem = 451 nm),7 of 7b 19 (λem = 503 nm) and 7f (λem = 500 nm). The remaining compounds were found to be relatively non-fluorescent in comparison.

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Within the azulene protonation-dependent fluorescence field, there are multiple published examples reported by Shoji and co-workers [54]. A series of 2-arylazulenes were synthesised, of varying electronic nature, on the attached aryl moieties (Figure 8). Az- ulenes 8a–8c and 9a–9h were all found to undergo fluorescence enhancement when in acidic solution. The presence of the isopropyl group within 8b and 8c gave minor in- creases in quantum yield and decreases in Stokes shift. The fluorescence properties were found to be dependent on the electronic nature of group at the para-position of the phenyl substituents. Electron-donating moieties (9a, 9b, 9e) afforded lower quantum yields, fluorescence emission at longer wavelengths and greater Stokes shifts. Conversely, the pres- enceFigure of 7.electron-withdrawing Substituted azulenes synthesisedmoieties (9c byby) resulted GaoGao etet al.al. in ingreater 2018 [[53] 53quantum].. yields and emission at shorter wavelengths.

FigureFigure 8. 8.2-Arylazulenes2-Arylazulenes synthesised synthesised by Shoji by Shoji et al. et [54] al.. [ 54].

ShojiShoji and and co-workers co-workers have have also also shown shown that that fused-azulene fused-azulene ring ringsystems systems (Figure (Figure 9) can9 ) can undergoundergo protonation-mediated protonation-mediated fluore fluorescence.scence. Fused Fused azulenephthalimide azulenephthalimide 10 was10 wasshown shown to to undergoundergo a a “turn-on” “turn-on” fluorescence fluorescence response in acidic medium (λexex = 390 390 nm, nm, λλemem == 444 444 nm) nm) [55]. [55].The groupThe group attributed attributed this fluorescence this fluorescence enhancement enhancement to the formation to the offormation the tropylium of the cation— tropyliumthe potential cation—the quenching potential role of quenching the azulene role was of the inhibited, azulene allowingwas inhibited, for emission allowing from for the emissionphthalimide from core. the phthalimide Interestingly, core. the Interestin fluorescentgly, properties the fluorescent of non-fused properties azulenephthalimides of non-fused azulenephthalimideswere shown to undergo were only shown a small to undergo fluorescent only enhancement a small fluorescent in acidic solutionenhancement [56]. Ain series acidicof non-fused solution azulenephthalimides [56]. A series of non-fused were synthesised,azulenephthalimides varying thewere nature synthesised, of the substitution varying on thethe nature 5- and 7-memberedof the substitution rings of on azulene. the 5- Onlyand 7-membered compounds bearingrings of alkyl azulene. chains Only on thecom- azulene pounds5-membered bearing ring alkyl were chains shown on tothe undergo azulene a5-membered significant turn-onring were fluorescence shown to undergo response a upon significantprotonation turn-on (e.g., 11 fluorescence, whereby the responseamine of upon the phthalimide protonation moiety (e.g., becomes11, whereby protonated the amine, rather of than thethe phthalimide azulene core). moiety The becomes cause protonated of this fluorescence, rather than the behaviour azulene is core). unknown, The cause but of it hasthis been fluorescence behaviour is unknown, but it has been suggested that it is caused by perturb- suggested that it is caused by perturbing the nature of the donor–acceptor interaction between ing the nature of the donor–acceptor interaction between the azulene and phthalimide the azulene and phthalimide moieties. moieties. Unlike the fused azulenephthalimide counterpart, fused azulenethiophene 12 showed Unlike the fused azulenephthalimide counterpart, fused azulenethiophene 12 moderately weak fluorescence emission in acidic solvent [57]. It was suggested that a showed moderately weak fluorescence emission in acidic solvent [57]. It was suggested non-radiative decay pathway was present due to the rotational freedom of the phenyl that a non-radiative decay pathway was present due to the rotational freedom of the phe- moiety bound to the thiophene. The fluorescent properties of analogues of 10, 11 and 12 nyl moiety bound to the thiophene. The fluorescent properties of analogues of 10, 11 and 12are are reported reported in in thethe Electronic Supplement Supplementaryary Information Information (ESI) (ESI) of their of their corresponding corresponding publications,publications, which which show show the the fluore fluorescencescence trends trends described described above. above.

Molecules 2021, 26, 353 8 of 19

Molecules 2020, 25, x 8 of 19 MoleculesMolecules 2020 2020, 25, 25, x, x 8 of 819 of 19

FigureFigure 9. 9. FusedFused and unfused azuleneazulene systems systems synthesised synthesised by by Shoji Shoji et al. et al.[55–57] [[55–57]55–57. ].. Figure 9. Fused and unfused azulene systems synthesised by Shoji et al. [55–57]. TheTheThe mostmost recent recent entry entry inin in the the the literature literature literature on on protonation-dependent on protonation-dependent protonation-dependent fluorescence fluorescence fluorescence of az- of az- of The most recent entry in the literature on protonation-dependent fluorescence of az- azuleneuleneulene waswas was reportedreported reported by by Lewis,Lewis, Lewis, KannKann Kann and and and co-workers co-workers co-workers in in 2020 in2020 2020[29]. [29]. [They29 They]. Theyfound found found that that 1,3-di- that 1,3-di- 1,3- ulenefunctionalised was reported azulenes by 13aLewis,–e (Figure Kann 10)and protonated co-workers upon in 2020 the addition [29]. They of TFA, found for that which 1,3-di- difunctionalisedfunctionalised azulenes azulenes 13a13a––ee (Figure(Figure 10) 10 )protonated protonated upon upon th thee addition addition of TFA, forfor whichwhich afunctionaliseda strong strong turn-on turn-on azulenes fluorescence fluorescence 13a– responsee response(Figure was 10) was protonated reported reported in eachinupon each instance. th instance.e addition Of allOf of theall TFA, the compounds forcom- which a strong turn-on fluorescence response was reported in each instance. Of all the com- assayed,apounds strong azuleneassayed,turn-on 13c azulenefluorescencewas found13c was response to found undergo towas undergo the reported greatest the in fluorescencegreatest each instance.fluorescence enhancement Of enhance-all the when compounds assayed, azulene 13c was found to undergo the greatest fluorescence enhance- protonatedpoundsment when assayed, ( λprotonatedex = 266azulene nm, (λexλ 13cem= 266 =was336 nm, found nm), λem = for 336to whichundergo nm), for the whichthe tricarbonyliron(diene) greatest the tricarbonyliron(diene) fluorescence moiety enhance- did ment when protonated (λex = 266 nm, λem = 336 nm), for which the tricarbonyliron(diene) notmentmoiety lead when did to emission notprotonated lead to quenching. emission (λex = 266 quenching. nm, λem = 336 nm), for which the tricarbonyliron(diene) moiety did not lead to emission quenching. moiety did not lead to emission quenching.

Fe(CO)3 (OC)3Fe O Fe(CO)3 O 13a 13b Fe(CO)3 13c 13d (OC)3Fe O (OC)3Fe O 13a 13b O 13c 13d O 13a 13b 13c 13d

(OC)3Fe Fe(CO) (OC) Fe 3 3 Fe(CO)3 (OC) Fe 3 Fe(CO)3 (OC)3Fe Fe(CO) (OC)3Fe Fe(CO) (OC) Fe 3 O O 3 O 3 Fe(CO)O 3

O O O O O O O O O O O O O O O O O O O O 13e 13f Figure 10. The 1- and 3-substituted azulenes reported by Lewis, Kann and co-workers in 2020 [29]. 13e 13f 13e 13f Figure 10. The 1- and 3-substituted azulenes reported by Lewis, Kann and co-workers in 2020 [29]. FigureFigureAside 10. 10.The The from1- 1-and and protonation 3-substituted 3-substituted of the azulenes azulenes azulene reported reported core, byazulene by Lewis, Lewis, monoaldehydes Kann Kann and and co-workers co-workers have inlikewise in 2020 2020 [ 29[29]. ]. been shown to exhibit alterations in their fluorescence when protonated (whereby proto- AsideAside fromfrom protonationprotonation ofof thethe azuleneazulene core,core, azuleneazulene monoaldehydesmonoaldehydes havehave likewiselikewise nationAside occurs from on theprotonation aldehyde ofmoiety). the azulene Aldehydes core, 14azulene and 15 monoaldehydes (Figure 11), reported have by likewise Li, beenbeen shownshown toto exhibitexhibit alterationsalterations in their ﬂuorescencefluorescence when protonated (whereby proto- beenGao andshown co-workers to exhibit in 2008,alterations were shown in their to fluorescencegive considerable when redshifts protonated (> 100 (whereby nm) in their proto- nationnation occursoccurs onon thethe aldehydealdehyde moiety).moiety). Aldehydes 14 andem 15 (Figure(Figure 1111),), em reportedreported byby Li,Li, nationemissions occurs when on the the solvent aldehyde was moiety). changed Aldehydes from EtOH 14(14 and λ 15= 420 (Figure nm, 1511), λ reported = 428 nm) by Li, GaoGaoto HClO andand co-workers4co-workers (14 λem = 552 inin nm, 2008,2008, 15 wereλem = shown530shown nm) to or givegi TFAve considerable (14 λem = 537 nm, redshifts 15 λem (>100(> = 530100 nm)nm) [58]. inin theirtheir Gao and co-workers in 2008, were shown to give considerable redshifts (> 100 nm) in their emissionsemissionsThe change whenwhen in emission the the solvent solvent of dialdehyde was was changed changed 15 was from from similar EtOH EtOH enough (14 (14λem λ to=em 420monoaldehyde= 420 nm, nm,15 λ15em λ 14=em428 to= 428sug- nm) nm) to emissions when the solvent was changed from EtOH (14 λem = 420 nm, 15 λem = 428 nm) HClOtogest HClO that4 (144 only(14λem λ monoprotonationem= 552= 552 nm, nm,15 15λem λem occurs= = 530 530 nm) innm) the or or former TFA TFA (14 (species.14λ emλem= = 537 537nm, nm,15 15λ λemem = 530 nm) [[58].58]. to HClO4 (14 λem = 552 nm, 15 λem = 530 nm) or TFA (14 λem = 537 nm, 15 λem = 530 nm) [58]. TheThe change in in emission emission of of dialdehyde dialdehyde 1515 waswas similar similar enough enough to monoaldehyde to monoaldehyde 14 to14 sug-to The change in emission of dialdehyde 15 was similar enough to monoaldehyde 14 to sug- suggestgest that that only only monoprotonation monoprotonation occurs occurs in the in theformer former species. species. gest that only monoprotonation occurs in the former species.

Figure 11. Fluorescent azulene aldehydes synthesised by Li, Gao and co-workers [58].

Figure 11. Fluorescent azulene aldehydes synthesised by Li, Gao and co-workers [58]. FigureFigure 11. 11.Fluorescent Fluorescent azulene azulene aldehydes aldehydes synthesised synthesised by by Li, Li, Gao Gao and and co-workers co-workers [ 58[58]]. .

Molecules 2020, 25, x 9 of 19 Molecules 2021, 26, 353 9 of 19 Similarly, the 1,3-connected azulene imine ligands described by Jamali, Bagherzadeh Molecules 2020, 25, x 9 of 19 and co-workers (Figure 12) showed considerable changes in their fluorescence properties uponSimilarly,Similarly, protonation thethe 1,3-connected[59].1,3-connected Both monoimine azuleneazulene 16 imineimine and ligandsdiimineligands described described17 were found byby Jamali,Jamali, to be Bagherzadeh weaklyBagherzadeh emis- andsiveand co-workersco-workersin CH2Cl2 ( (Figure≈(Figure λem = 450 1212)) showednmshowed for both considerableconsiderable species). changesUponchanges addition inin theirtheir of fluorescencefluorescence TFA (10%), properties propertieslarge red- uponshiftsupon protonationin emission afforded[59]. [59]. Both Both strong monoimine monoimine fluorescence 16 16andand diimineat λ diimineem = 61017 were 17nmwere andfound λ foundem to = be 570to weakly nm be weaklyfor emis- mo- noimine 16 and diimine 17, respectively. For both species, 1H-NMR studies confirmed that emissivesive in CH in2 CHCl2 2(Cl≈ λ2em(≈ = λ450em =nm 450 for nm both for species). both species). Upon Upon addition addition of TFA of TFA(10%), (10%), large large red- protonation occurs on the imine nitrogen and that, unlike dialdehyde 15, diimine 17 un- redshiftsshifts in emission in emission afforded afforded strong strong fluorescence fluorescence at λ atemλ =em 610= 610nm nmand and λem λ=em 570= nm 570 for nm mo- for monoiminedergoesnoimine protonation16 and16 and diimine diimine at both 17, 17respectively. nitrogen, respectively. atoms. For For both both species, species, 1H-NMR1H-NMR studies studies confirmed confirmed that thatprotonation protonation occurs occurs on the on imine the imine nitrogen nitrogen and andthat, that, unlike unlike dialdehyde dialdehyde 15, diimine15, diimine 17 un-17 undergoesdergoes protonation protonation at atboth both nitrogen nitrogen atoms. atoms.

N N

N 16

N 16 17 Figure 12. The 1- and 3-connected azulenes described by Jamali, Bagherzadeh and co-workers [59]. 17 FigureFigure 12.12. TheThe 1-1- andand 3-connected3-connected azulenesazulenes describeddescribed byby Jamali,Jamali, BagherzadehBagherzadeh andand co-workersco-workers[ 59]. [59]. Fluorescence sensing for specific analytes, outside of azulene protonation, is scarce. In 2005,Fluorescence Eichen et sensing al. published for specific a “turn-on” analytes, fluorescent outside of azuleneazulene protonation,sensor for fluoride is scarce. [60]. In 2005,The Fluorescencesensor, Eichen et1,3-di(2-pyrrolyl)azulene al. publishedsensing for a specific “turn-on” analytes,18 fluorescent, was outsidefound azulene toof azulenebe sensor selective protonation, for fluoridetowards [ is60tetrabu- scarce.]. The sensor,Intylammonium 2005, 1,3-di(2-pyrrolyl)azulene Eichen (TBA) et al. fluoridepublished over a18 “turn-on” other, was TBAX found fluorescent salts to be in selective both azulene DMSO towards sensor and tetrabutylammo-DCMfor fluoride (where [60]. X = − − niumCl−, Br (TBA)−, I−, p fluoride-toluenesulfonate, over other BF TBAX4−, PF salts6−). Upon in both stoichiometric DMSO and DCM binding (where of fluoride, X = Cl ,a Br ten-, The− sensor, 1,3-di(2-pyrrolyl)azulene− − 18, was found to be selective towards tetrabu- Ifoldtylammonium, p -toluenesulfonate,increase in (TBA) fluorescence fluoride BF4 was, PFover6 observed ).other Upon TBAX ( stoichiometricλex = salts 355 nm,in both λem binding =DMSO 450 nm). of and fluoride, The DCM authors (where a ten-fold noted X = increasethat− the− inproposed− fluorescence S2→S was0 fluorescence observed− ( λresulted− = 355 from nm, λ a change= 450 nm).in conformation The authors of noted the thatpyr- Cl , Br , I , p-toluenesulfonate, BF4 , PF6ex). Upon stoichiometricem binding of fluoride, a ten- therole proposed rings. In the S → unboundS fluorescence species, resulted the rings from are expected a change to in be conformation in the plane ofof thethe pyrroleazulene fold increase in2 fluorescence0 was observed (λex = 355 nm, λem = 450 nm). The authors noted rings.ring. Mechanistically, In the unbound species,the authors the rings proposed are expected that fluoride to be in was the planehydrogen-bonded of the azulene to ring. the that the proposed S2→S0 fluorescence resulted from a change in conformation of the pyr- Mechanistically, the authors proposed that fluoride was hydrogen-bonded to the pyrrole rolepyrrole rings. moieties, In the unbound which in species,turn rotated the rings out ofare the expected plane ofto thebe inazulene the plane (Scheme of the 2).azulene With moieties, which in turn rotated out of the plane of the azulene (Scheme2). With the pyrrole ring.the pyrrole Mechanistically, rings out ofthe plane, authors the proposed excited statethat fluorideof the complex was hydrogen-bonded was altered, allowing to the rings out of plane, the excited state of the complex was altered, allowing greater emission. pyrrolegreater emission.moieties, which in turn rotated out of the plane of the azulene (Scheme 2). With the pyrrole rings out of plane, the excited state of the complex was altered, allowing greater emission.

SchemeScheme 2.2. FluorideFluoride sensorsensor 1818 andand thethe mechanismmechanism ofof fluorescencefluorescence proposedproposed byby EichenEichenet etal. al. [ 60[60]]. . 2.2. Fluorescent Azulenes in Biological Contexts 2.2. Fluorescent Azulenes in Biological Contexts SchemeA range 2. Fluoride of azulenes sensor have18 and been the employedmechanism inof biologicalfluorescence settings, proposed including by Eichen as et anti-cancer al. [60]. A range of azulenes have been employed in biological settings, including as anti- agents [61–63], antiretrovirals [64], anti-inflammatories [65,66], erectile dysfunction treat- cancer agents [61–63], antiretrovirals [64], anti-inflammatories [65,66], erectile dysfunction ments2.2. Fluorescent [67,68], anti-ulcer Azulenes in agents Biological [69– 72Contexts] and as a potential type II diabetes treatment [73]. treatments [67,68], anti-ulcer agents [69–72] and as a potential type II diabetes treatment The workA range by Pham of azulenes et al. highlighted have been howemployed an 18F-labelled in biological azulene settings, derivative including can beas usedanti- [73]. The work by Pham et al. highlighted how an 18F-labelled azulene derivative can be tocancer image agents cancer [61–63], cells [74 antiretrovirals]. By using COX2 [64], an asti-inflammatories a biomarker, they [65,66], were able erectile to gain dysfunction selective retentionusedtreatments to image of [67,68], the cancer probe anti-ulcer withincells [74]. breastagents By cancer using[69–72] tumoursCOX2 and asas aina potentialbiomarker, mice and type image they II diabetes themwere viaable treatment positron to gain emissionselective[73]. The tomography.retentionwork by Phamof the However, probeet al. highlighted within few biologically breast how cancer an relevant 18 tumoursF-labelled azulenes in azulene mice have and derivative beenimage tested them can forvia be theirusedpositron fluorescenceto image emission cancer properties. tomography. cells [74]. However,By using COX2few biologically as a biomarker, relevant they azulenes were able have to beengain selectivetestedThe for mostretention their prominent fluorescence of the probe entry properties. inwithin the field breast of azulenecancer tumours fluorescence in mice in biological and image settings them via is thepositron useThe of emissionmost the non-canonical prominent tomography. entry amino inHowever, the acid fieldβ-(1-azulenyl)- fewof azu biologicallylene Lfluorescence-alanine relevant (referred in azulenes biological to in have the settings litera- been tureistested the as foruseAal their ofand the fluorescenceAzAla non-canonicalinterchangeably), properties. amino acid commonly β-(1-azulenyl)- used asL a-alanine bioisostere (referred of tryptophan to in the (FigureThe 13 most). The prominent first enantiomerically entry in the field pure of synthesis azulene fluorescence of Aal was published in biological in 1999settings by is the use of the non-canonical amino acid β-(1-azulenyl)-L-alanine (referred to in the

Molecules 2020, 25, x 10 of 19 MoleculesMolecules2021 2020,,26 25,, 353 x 1010 of 1919 literature as Aal and AzAla interchangeably), commonly used as a bioisostere of trypto- phanliterature (Figure as 13). Aal The and first AzAla enantiomerically interchangeably), pure commonlysynthesis of used Aal aswas a bioisosterepublished inof 1999trypto- by phanMoroder (Figure et al. 13). through The first a key enantiomerically enantioselective pure deacetylation synthesis ofstep Aal using was thepublished enzyme in ac- 1999 ylaseMoroderby IMoroder [75]. etIt was al. et throughal. found through that a key aAal key enantioselective could enantioselective be excited deacetylation at deacetylation both 276 step and step using 339 using nm the to enzyme the emit enzyme at acylase 381 ac- nm.I[ylase The75]. Ilonger It[75]. was It excitation found was found that wavelengthAal thatcould Aal could bewas excited noted be excited atto bothbe atparticularly both 276 and 276 339and useful, nm 339 to allowingnm emit to emit at selec- 381 at nm.381 The longer excitation wavelength was noted to be particularly useful, allowing selective tivenm. excitation The longer over excitation tryptophan wavelength (λex = 280 wasnm, λnotedem = 381 to benm). particularly useful, allowing selec- excitationtive excitation over over tryptophan tryptophan (λex =(λ 280ex = nm,280 nm,λem λ=em 381 = 381 nm). nm). HO HO O O HO HO O O H2N H2N H2N H2N

N H N H L-Aal (AzAla) L-Trp L-Aal (AzAla) L-Trp Figure 13. Non-canonical amino acid Aal (AzAla). FigureFigure 13.13. Non-canonicalNon-canonical aminoamino acidacid AalAal (AzAla).(AzAla). In 2004, Pispisa et al. assessed the fluorescent properties of Aal in hexapeptides when placed atInIn different 2004, Pispisa Pispisa distances et et al. al. assessedfrom assessed the thefluorescent the fluorescent fluorescent quencher properties properties 2,2′ ,6,6of Aal′-tetramethyl-1-oxyl-4- of inAal hexapeptidesin hexapeptides when 0 0 amino-4-piperidinewhenplaced placed at different at different carboxylic distances distances acid from (TOAC) the from fluorescent the [76]. fluorescent Two quencher unique quencher hexapeptides 2,2′,6,6 2,2′-tetramethyl-1-oxyl-4-,6,6 were-tetramethyl-1- synthesised,oxyl-4-amino-4-piperidineamino-4-piperidine wherein the Aal groupcarboxylic was carboxylic acideither (TOAC) 1- acid or 2-amino (TOAC) [76]. Two [acid76 unique]. residues Two uniquehexapeptides away hexapeptides from were TOAC synthe- werein thesynthesised,sised, primary wherein structure wherein the Aal (A1T3 the groupAal and wasgroup A1T4 either was respectively, 1- either or 2-amino 1- or Figure 2-aminoacid residues 14a). acid It residuesaway was fromfound away TOAC that from in quenchingTOACthe primary in of the the primarystructure Aal fluorescence structure (A1T3 ( A1T3wasand moreA1T4and significantA1T4 respectively,respectively, when Figure the Figure TOAC 14a). 14 wasa). It It wasfurther was found found away thatthat in thequenchingquenching primary ofof structure, thethe AalAal fluorescencefluorescence due to the greater waswas moremore proximity significantsignificant of TOAC whenwhen theandthe TOAC TOACAal in was wasthe secondaryfurtherfurther awayaway Aal structureinin thethe primaryprimaryaffording structure,structure, a higher dueleveldue toto of the thethrough- greatergreaterspace proximityproximity quenching. ofof TOACTOAC The andfollowingand Aal inin year, thethe secondary secondaryPispisa structure affording a higher level of through-space quenching. The following year, Pispisa et al.structure went on affording to report a higher the use level of Aal of through- as a fluorescentspace quenching. biomarker The by following incorporating year, Pispisa the et al. went on to report the use of Aal as a fluorescent biomarker by incorporating the aminoet al. acid went into on a tosynthetic report theanalogue use of ofAal the as antibiotic a fluorescent peptide biomarker trichogin by GA incorporating IV [77]. The the amino acid into a synthetic analogue of the antibiotic peptide trichogin GA IV [77]. The fluorescentamino acid peptide into aanalogue, synthetic A3 analogue (Figure of 14b), the wasantibiotic used topeptide probe trichoginthe membrane GA IV activity [77]. The fluorescent peptide analogue, A3 (Figure 14b), was used to probe the membrane activity of of trichoginfluorescent GA peptide IV, for analogue, which the A3 ability (Figure to selectively 14b), was exciteused to Aal pr obeover the Trp membrane was exploited. activity trichoginof trichoginGA GA IV ,IV for, for which which the the ability ability to selectivelyto selectively excite exciteAal Aalover over Trp Trp was was exploited. exploited.

(a) (b) (a) (b) FigureFigure 14. Aal-bearing 14. Aal-bearing peptides peptides synthesised synthesised by Pispisa by Pispisa et al. in et (a al.) 2004 in [76] (a) 2004 and [(76b)] 2005.[77] and (b) 2005 [77]. Figure 14. Aal-bearing peptides synthesised by Pispisa et al. in (a) 2004 [76] and (b) 2005.[77] AibAib = 2-Aminoisobutyric = 2-Aminoisobutyric acid; acid; Dab Dab = 2,4-diaminobutyric = 2,4-diaminobutyric acid. acid. Aib = 2-Aminoisobutyric acid; Dab = 2,4-diaminobutyric acid. AalAal hashas also also been been used used as a as fluorescence a fluorescence quencher. quencher. In 2006, In 2006, Koh Koh and and Lee Lee incorporated incorporated Aal has also been used as a fluorescence quencher. In 2006, Koh and Lee incorporated fluoresceinfluorescein (as(as a fluorescence a fluorescence donor, donor, λexλ =ex 467= 467 nm, nm, λemλ em= 506–560= 506–560 nm) nm) and and AalAal (as (asa fluo- a fluo- rescencerescencefluorescein acceptor) acceptor) (as a into fluorescence into a synthetic a synthetic donor, oligomer oligomer λex =mimic 467 mimic nm, of ofnaturallyλem naturally = 506–560 occurring occurring nm) andβ-amyloidβ -amyloidAal (as (A a β (Afluo-) β) peptidespeptidesrescence [78]. [acceptor)78 Fluorescence]. Fluorescence into a decaysynthetic decay was was oligomer monitore monitored mimicd as function as of function naturally of increased of increasedoccurring peptide peptideβ-amyloid concen- concen- (Aβ) trationtrationpeptides and and subsequent [78]. subsequent Fluorescence aggregation, aggregation, decay forwas for which monitore which the the increased as increase function in the in theof extent increased extent of conformational of peptide conformational concen- changechangetration increased and increased subsequent the the efficiency efficiency aggregation, of the of the Förster for Förster which resonance resonance the increase energy energy in transfer the transfer extent (FRET) (FRET) of conformational quenching quenching mechanism.mechanism.change increased the efficiency of the Förster resonance energy transfer (FRET) quenching mechanism.AalAal waswas shown shown to to be be a auseful useful fluorophore fluorophore forfor monitoringmonitoring protein—protein protein–-protein interactions interac- tionsby by KorendovychAal Korendovych was shown et al. et to inal. be 2013 in a 2013useful [79]. [79]. Unlike fluorophore Unlike Trp, Trp,Aal for wasAal monitoring shownwas shown to protein–-protein be to insensitive be insensitive to localinterac- to en- localvironmenttions environment by Korendovych changes changes (e.g., et solvent (e.g.,al. in solvent 2013 exposure, [79]. exposure, Unlike local interactions) Trp,local Aalinteractions) was and shown therefore and to thereforebe proposed insensitive pro- tobe to posedmorelocal to sensitiveenvironment be more to sensitive weaker changes intrinsicto (e.g.,weaker solvent quenchers intrinsic exposure, (e.g.,quenchers methionine). local (e.g., interactions) methionine). Due to theand environmental thereforeDue to the pro- environmentalsensitivityposed to be of sensitivitymore Trp, the sensitive deconvolution of Trp, to theweaker deconvolution of theintrinsic effects quenchers of the environment effects (e.g., of environmentmethionine). changes and changes quenchingDue to the andonenvironmental quenching fluorescence on sensitivity isfluorescence difficult. of To Trp, is test difficult. the this deconvolution proposal, To test thisAal of proposal,was the effects incorporated Aal of environment wasinto incorporated the bindingchanges intopartnerand the quenchingbinding of the eukaryoticpartner on fluorescence of the protein eukaryotic calmodulinis difficult. protein (CaM),To calmodulin test a this 20-residue proposal, (CaM), peptide a Aal 20-residue ofwas smooth incorporated peptide muscle myosininto the lightbinding chain partner kinase of the (smMLCK). eukaryotic The protein ability calmodulin to selectively (CaM), excite a 20-residueAal over peptide Trp at

Molecules 2020, 25, x 11 of 19

of smooth muscle myosin light chain kinase (smMLCK). The ability to selectively excite Molecules 2021, 26, 353 Aal over Trp at 342 nm proved advantageous for monitoring the resultant protein–protein11 of 19 interaction for determining binding stoichiometry, without the interference of the environmentally sensitive Trp residue. Furthermore, the exchange of Aal for Trp within 342smMLCK nm proved was found advantageous to not significantl for monitoringy alter the the smMLCK–CaM resultant protein–protein interaction. interaction for determiningKorendovych binding et al. further stoichiometry, explored without the effects the interferenceof exchanging of theTrp environmentally for Aal in 2015, sensitiveassessing Trpif the residue. presence Furthermore, of Aal would the affect exchange native of ionAal chforannels Trp within[80]. Using smMLCK the trans- was foundmembrane to not domain significantly of the alterM2 proton the smMLCK–CaM channel (M2TM) interaction. of the influenza A virus as their model,Korendovych a key Trp residue et al. furtherwas replaced explored with the Aal effects (termed of exchanging M2TM Trp4 Trp1AzAla). for Aal Notably,in 2015, assessingthe ion channel if the presenceassembles of intoAal a wouldtetramer affect of M2TM. native In ion M2TM, channels the [channel80]. Using positions the trans- the membraneTrp residue domainone helical of theturn M2 below proton a key channel His residue (M2TM) responsible of the influenza for proton A virusconductance. as their model,Upon forming a key Trp a 3:1, residue M2TM: was M2TM replaced Trp41AzAla with Aal tetramer,(termed the M2TM protonation Trp41AzAla). state of Notably, the His theresidue ion channelcould be assembles determined into as aa tetramerfunction of fluorescence M2TM. In M2TM, of the theAal channel moiety ( positionsλex = 342 nm, the Trpλem = residue 380 nm), one whereby helical turnfluorescence below a intensity key His residue decreased responsible when pH for > 7proton and increased conductance. when UponpH < 7. forming a 3:1, M2TM: M2TM Trp41AzAla tetramer, the protonation state of the His residueAnother could instance be determined in which as aAal function has been of fluorescence used in place of of the TrpAal wasmoiety highlighted (λex = 342 by nm,the λworkem = 380by Kalesse nm), whereby et al. in fluorescence 2018 [81]. Th intensitye work decreased details the when total pH synthesis > 7 and of increased Argyrin when C, a pHbiologically < 7. relevant cyclic octapeptide and the subsequent synthesis of Aal argyrin C in whichAnother a Trp residue instance (highlighted in which Aal in hasred) been was usedreplaced in place with of Aal Trp (Figure was highlighted 15). When byincor- the workporated by into Kalesse the octapeptide, et al. in 2018 the [81 fluorescence]. The work intensity details the (λex total = 342 synthesis nm, λem = of 380Argyrin nm) ofC Aal, a biologicallywas reduced relevant (~50% of cyclic the free octapeptide Boc-Aal-OH and residue) the subsequent but was synthesis much greater of Aal than argyrin that of C inTrp, which which a was Trp residuealmost non-fluorescent (highlighted in atred th)e wassame replaced excitation with wavelength.Aal (Figure However, 15). When the incorporatedauthors noted into that the the octapeptide, circular dichroism the fluorescence (CD) spectrum intensity of the (λ exaltered= 342 peptide nm, λem differs= 380 dra- nm) ofmaticallyAal was to reduced that of the (~50% wild of type. the free The Boc- workAal notably-OH residue) reported but the was synthesis much greater of Boc- thanAal-OH that ofwith Trp, a 93% which yield was over almost two non-fluorescentsteps. at the same excitation wavelength. However, the authorsMost recently, noted that with the regard circular to dichroismAal, in 2019 (CD) Arnold spectrum et al. ofshowed the altered that by peptide using differsan en- dramaticallygineered tryptophan to that of synthase, the wild Aal type. could The workbe synthesised notably reported in the near the synthesisgram-scale of (965 Boc- Aalmg)- OHin a withsingle a step 93% from yield serine over two and steps. azulene [82].

Figure 15. Argyrin C and Aal argyrin C synthesised by Kalesse etet al.al. [[81]81]..

MostAzulenyl recently, squaraines with regard have found to Aal use, in in 2019 biological Arnold settings et al. showed as NIR thatFRET by quenchers using an engineered tryptophan synthase, Aal could be synthesised in the near gram-scale (965 mg) for use in peptides. In 2002 Tung et al. developed NIRQ750 (Figure 16), proposing that the inextended a single π step-system from of serine the molecule and azulene would [82 ].allow for long wavelength absorbance in the Azulenyl squaraines have found use in biological settings as NIR FRET quenchers for region of 650–900 nm [83]. It was found that NIRQ750 absorbed broadly in the NIR region useof 700–800 in peptides. nm. Absorbance In 2002 Tung was et found al. developed to vary withNIRQ solvent750 (Figure polarity, 16), whereby proposing more that polar the π extendedsolvents afforded-system a ofbathochromic the molecule shift would in ab allowsorbance. for long To assess wavelength the quenching absorbance feasibility in the region of 650–900 nm [83]. It was found that NIRQ750 absorbed broadly in the NIR region of NIRQ750, a fluorescent capase-3 peptide substrate was designed to incorporate NIRQ750 of 700–800 nm. Absorbance was found to vary with solvent polarity, whereby more polar at the N-terminus. At the opposite end of the peptide, attached to a cysteine residue, solvents afforded a bathochromic shift in absorbance. To assess the quenching feasibility of Alexa-680 C2 maleimide (λex = 679 nm, λem = 702nm) was used as a NIR-fluorescence do- NIRQ , a fluorescent capase-3 peptide substrate was designed to incorporate NIRQ nor. When750 tested against the capase-3 enzyme, a fluorescence response was reported.750 at the N-terminus. At the opposite end of the peptide, attached to a cysteine residue, Tung et al. further contributed to the field the following year with the addition of NIRQ700 Alexa-680 C maleimide (λ = 679 nm, λ = 702 nm) was used as a NIR-fluorescence (Figure 16) 2[84]. They proposedex that a shemorter wavelength NIR-fluorophore could be donor. When tested against the capase-3 enzyme, a fluorescence response was reported. achieved by removing the electron-donating isopropyl group in NIRQ750, which would Tung et al. further contributed to the field the following year with the addition of NIRQ700 (Figure 16)[84]. They proposed that a shorter wavelength NIR-fluorophore could be achieved by removing the electron-donating isopropyl group in NIRQ750, which would

Molecules 2021, 26, 353 12 of 19

Molecules 2020, 25, x 12 of 19

afford aa hypochromichypochromic shiftshift inin absorbance.absorbance. TheThe maximummaximum absorbanceabsorbance ofofNIRQ NIRQ700700 waswas found to be blue-shifted, withwith aa broadbroad rangerange inin absorbanceabsorbance ofof 600–750600–750 nm.nm.

O O O O

HO O HO O

O O

BuO BuO

NIRQ750 NIRQ700 Figure 16. NIR fluorescent ﬂuorescent quenchers developed by Tung etet al.al. [[83]83]. .

In 2017, Zhu etet al.al. published an exampleexample ofof aa fluorescent,fluorescent, NIR-emissiveNIR-emissive cyanostyrylcyanostyryl azulene that was used for cell imagingimaging [[85].85]. Using cyanostyryl azulenes 19 and 20,, itit waswas found that fluorescencefluorescence was dependentdependent on both thethe protonationprotonation statestate ofof thethe azuleneazulene andand (Z)-19 also the alkene geometry. geometry. Initial Initial studies studies were were performed performed on on (Z)-19 inin CHCl CHCl3. 3Attempts. Attempts at atphotoisomerisation photoisomerisation to tothe the corresponding corresponding (E ()-alkene,E)-alkene, using using both both UV UV and and visible visible light, proved unsuccessful.unsuccessful. UponUpon protonationprotonation ofof the the azulene azulene 5-membered 5-membered ring, ring, photoisomersa- photoisomer- + + tionsation of [(ofZ [()-19Z)-19 + H] + H]to+ [(toE )-19[(E)-19 + H] + H]was+ was achieved achieved byirradiation by irradiation at 365 atnm 365 (Scheme nm (Scheme3). Both 3). + → (BothZ)-19 (Zand)-19 [( andZ)-19 [( +Z)-19 H] +showed H]+ showed strong strong absorbances absorbances at 420 nm,at 420 corresponding nm, corresponding to a S0 toS 2a transition. Whilst (Z)-19 was found to be only very weakly fluorescent (λex = 330 nm), S0→S2 transition. Whilst (Z)-19 was found to be only very weakly fluorescent (λex = 330 [(Z)-19 + H]+ was found to be emissive at 500 nm at the same excitation wavelength. In nm), [(Z)-19 + H]+ was found to be emissive at 500 nm at the same excitation wavelength. contrast to the (Z)-isomers, the (E)-isomers showed a decrease in absorbance at 420 nm In contrast to the (Z)-isomers, the (E)-isomers showed a decrease in absorbance at 420 nm with a new, strong absorbance band at 680 nm suggesting an increase in favourability of the with a new, strong absorbance band at 680 nm suggesting an increase in favourability of S0→S1 transition (and subsequent decrease in the S0→S2 transition). Fluorescence emission the S0→S1 transition (and subsequent decrease in the S0→S2 transition). Fluorescence emis- of [(E)-19 + H]+ was found to be greater than [(Z)-19 + H]+, but emission intensity of (E)-19 sion of [(E)-19 + H]+ was found to be greater than [(Z)-19 + H]+, but emission intensity of (480 nm) was found to be the greatest of all. A new, weaker emission band at the NIR region (E)-19 (480 nm) was found to be the greatest of all. A new, weaker emission band at the of 700–900 nm was observed for (E)-19. B3LYP/6-31G studies of the photoisomerisation NIR region of 700–900 nm was observed for (E)-19. B3LYP/6-31G studies of the pho- process suggested a twisted intramolecular charge transfer in the cyanostyryl group in the excitedtoisomerisation state of the processZ isomer, suggested affording a atwisted non-radiative intramolecular decay pathway. charge Thetransfer increased in the steric cy- hindranceanostyryl group and rigidity in the excited of the E stateisomer of the are Z thought isomer, toaffording disfavour a non-radiative the non-radiative decay charge path- transferway. The decay increased pathway, steric affording hindrance fluorescence and rigidity emission. of the E isomer are thought to disfavour the non-radiativeBy changing thecharge nature transfer of the decay phenolic pathway, tail group affording from an fluorescencen-butyl chain emission. to the polyethy- lene glycol chain PEG2000-yl, a water-soluble probe (Z)-20 was generated. Identical results were obtained for (Z)-20 in water for the photoisomerisation process. Similarly, dual emission bands were also found for (E)-20. However, unlike (E)-19, the quantum yield of the NIR emission of (E)-20 (λem~800 nm) was found to be roughly equal to that of the quantum yield of the visible emission band (λex = 330 nm, λem~400 nm) when excited at 710 nm. The intense NIR emission was attributed to the favourability of the S0→S1 transition allowing emission from the S1 excited stated, as well as the hindered non-radiative decay pathway in the (E)-isomer. The dual emission bands of (E)-20 were exploited for cell imaging in MC3T3- E1 cell lines, which was contrasted with the (Z)-20 isomer (Figure 17). Cell endocytosis was mediated by vesicle formation of the probes, as a result of the PEG tails. When excited at 405 or 640 nm, the (E)-isomer was clearly observed in the cell images. When contrasted to (Z)-20, some fluorescence was observed at the shortest excitation wavelength, whilst no fluorescence was seen at the longer wavelength. The group have further utilised similar photoswitching platforms for acidic sensing [86,87]. It should also be noted that changes in

Molecules 2020, 25, x 13 of 19

H CN CN 19 : R = nBu + H

20 : R = PEG2000-yl OR OR (Z)-19 [(Z)-19+H]+ (Z)-20 [(Z)-20+H]+

hv (365 nm) 65 oC

OR OR

Molecules 2021, 26, 353 13 of 19 - H H

CN CN ﬂuorescent emission of azulene derivatives upon photoswitching have previously been (E)-19 [(E)-19+H]+ reported by Nica(E)-20 et al. [88]. + Molecules 2020, 25, x [(E)-20+H] 13 of 19

Scheme 3. Photoisomerisation pathway of (Z)-19 and (Z)-20 [85]. H CN CN 19 : R = nBu + H By changing the nature of the phenolic tail group from an n-butyl chain to the poly- 20 : R = PEG2000-yl ethylene glycol chain PEG2000-yl, a water-soluble probe (ZOR)-20 was generated. Identical re- + OR sults were obtained for (Z)-20 in water for the(Z)- 19photoisomerisation process. Similarly,[(Z)- dual19+H] (Z)-20 [(Z)-20+H]+ emission bands were also found for (E)-20. However, unlike (E)-19, the quantum yield of the NIR emission of (E)-20 (λem ~ 800 nm) was found to be roughly equal to that of the hv (365 nm) quantum yield of the visible emission band65 o C(λex = 330 nm, λem ~ 400 nm) when excited at 710 nm. The intense NIR emission was attributed to the favourability of the S0→S1 transi- 1 tion allowing emission from the S excited stated, ORas well as the hindered non-radiativeOR decay pathway in the (E)-isomer. The dual emission bands of (E)-20 were exploited for cell imaging in MC3T3-E1 cell lines, which was contrasted with the (Z)-20 isomer (Figure H 17). Cell endocytosis was mediated by vesicle formation of the -probes, H as a result of the PEG tails. When excited at 405 or 640 nm, the (E)-isomer was clearly observed in the cell images. When contrasted to (Z)-20, some fluorescenceCN was observed at the shortest exci- CN

tation wavelength, whilst no fluorescence wa(Es) -seen19 at the longer wavelength. The[( groupE)-19+H] + have further utilised similar photoswitching platforms(E)-20 for acidic sensing [86,87]. It should + [(E)-20+H] also be noted that changes in fluorescent emission of azulene derivatives upon pho- Scheme 3. Photoisomerisation pathway of (Z)-19 and (Z)-20 [85]. toswitching haveScheme previously 3. Photoisomerisation been reported pathwayby Nica ofet (alZ)-19. [88]. and (Z)-20 [85].

By changing the nature of the phenolic tail group from an n-butyl chain to the poly- ethylene glycol chain PEG2000-yl, a water-soluble probe (Z)-20 was generated. Identical results were obtained for (Z)-20 in water for the photoisomerisation process. Similarly, dual emission bands were also found for (E)-20. However, unlike (E)-19, the quantum yield of the NIR emission of (E)-20 (λem ~ 800 nm) was found to be roughly equal to that of the quantum yield of the visible emission band (λex = 330 nm, λem ~ 400 nm) when excited at 710 nm. The intense NIR emission was attributed to the favourability of the S0→S1 transition allowing emission from the S1 excited stated, as well as the hindered non-radiative decay pathway in the (E)-isomer. The dual emission bands of (E)-20 were exploited for cell imaging in MC3T3-E1 cell lines, which was contrasted with the (Z)-20 isomer (Figure 17). Cell endocytosis was mediated by vesicle formation of the probes, as a result of the PEG tails. When excited at 405 or 640 nm, the (E)-isomer was clearly observed in the cell Figure 17. FigureSelective 17. cell Selective imagingimages. cell of imaging( ZWhen)-20 and contrastedof ((EZ)-20)-20in and eitherto ((EZ)-20)-20 the in visible, some either orfluorescence the NIR visible region or inwas NIR MC3T3-E1 observed region in cells. at the Reproduced shortest exci- MC3T3-E1 cells. Reproduced with permission from ref. [85]. Copyright 2017 Wiley–VCH Ver- with permission from ref. [85tation]. Copyright wavelength, 2017 Wiley–VCH whilst no Verlag fluorescence GmbH & wa Co.s KGaA,seen at Weinheim. the longer wavelength. The group lag GmbH & Co. KGaA,have further Weinheim. utilised similar photoswitching platforms for acidic sensing [86,87]. It should also Azulenebe noted fluorescent that changes sensing in fluorescent for analyte emission detection isof relativelyazulene derivatives under-developed. upon pho- The Azulene fluorescent sensing for analyte detection is relatively under-developed. The azulenetoswitching proton have probe previously field has been mainly reported seen by use Nica inthe et al area. [88]. of functional materials. The azulene protonAal probefield field is rich has with mainly applications seen use in peptidethe area chemistryof functional but materials. this amino The acid has not been used for analyte detection. Similarly, whilst cyanostyryl probe 20 was successfully used for cell imaging, no application in detection of a specific analyte was reported. To the best of our knowledge, our probe AzuFluor® 483-Bpin is currently the only azulene probe that has been designed and employed for fluorescence bioimaging in response to specific analytes [89]. Chemodosimeter AzuFluor® 483-Bpin was developed for the detection of reactive oxygen species (ROS) and reactive nitrogen species (RNS), exploiting the use of a boronate ester as a receptor. Upon reaction with ROS/RNS species, AzuFluor® 483-Bpin was rapidly converted to 6-hydroxy species 21 (Scheme4). The maximum absorption was observed at 327 nm for AzuFluor® 483-Bpin, which was red-shifted to 350 nm in oxidized product 21. AzuFluor® 483-Bpin was found to be non-fluorescent, undergoing a fluorescent “turn-on” in the presence of ROS/RNS. Oxidized species 21 was found to emit at 483 nm Figure(λex = 35017. Selective nm), showing cell imaging a broad of Stokes(Z)-20 shiftand (E of)-20 133 in nm. either the visible or NIR region in MC3T3-E1 cells. Reproduced with permission from ref. [85]. Copyright 2017 Wiley–VCH Ver- lag GmbH & Co. KGaA, Weinheim.

Azulene fluorescent sensing for analyte detection is relatively under-developed. The azulene proton probe field has mainly seen use in the area of functional materials. The

Molecules 2020, 25, x 14 of 19

Aal field is rich with applications in peptide chemistry but this amino acid has not been used for analyte detection. Similarly, whilst cyanostyryl probe 20 was successfully used for cell imaging, no application in detection of a specific analyte was reported. To the best of our knowledge, our probe AzuFluor® 483-Bpin is currently the only azulene probe that has been designed and employed for fluorescence bioimaging in response to specific analytes [89]. Chemodosimeter AzuFluor® 483-Bpin was developed for the detection of reactive oxygen species (ROS) and reactive nitrogen species (RNS), exploiting the use of a boronate ester as a receptor. Upon reaction with ROS/RNS species, AzuFluor® 483-Bpin was rapidly converted to 6-hydroxy species 21 (Scheme 4). The maximum absorption was observed at 327 nm for AzuFluor® 483-Bpin, which was red-shifted to 350 nm in oxidized ® Molecules 2021, 26, 353 product 21. AzuFluor 483-Bpin was found to be non-fluorescent, undergoing a fluores-14 of 19 cent ”turn-on” in the presence of ROS/RNS. Oxidized species 21 was found to emit at 483 nm (λex = 350 nm), showing a broad Stokes shift of 133 nm.

Scheme 4. Dosimeter mechanism of AzuFluor® 483-Bpin483-Bpin withwith ROS/RNS [89] [89]..

DFT calculations (using the BP86 functional and 6-31G** basis set) showed a destabi- ® lization of the LUMOLUMO ofof 21 relativerelative toto AzuFluorAzuFluor® 483-Bpin, affordingaffording aa narrowingnarrowing ofof thethe LUMO–LUMO+1 gap. The The presence of the electronelectron donating 6-hydroxy group increasesincreases the overall ICT character ofof 21,, leadingleading toto fluorescencefluorescence emission.emission. The dosimeter dosimeter was was also also evaluated evaluated for for its itstwo-photontwo-photon fluorescence fluorescence properties.properties. Two-pho- Two- photonton microscopy microscopy (TPM) (TPM) [90] [90 allows] allows for for excitati excitationon in in the the NIR NIR region, region, enabling enabling deep tissue penetration and greater localization localization of of excitati excitationon [91]. [91]. As As such, such, it it has has been been exploited exploited for for a arange range of of bioimaging bioimaging applications applications [92]. [92 Impo]. Importantlyrtantly the the wavelengths wavelengths required required by byTPM TPM re- reduceduce the the risk risk of of autofluorescence autofluorescence from from en endogenousdogenous species. species. The maximum two-photon ® action cross-section of AzuFluor ® 483-Bpin upon addition of ROS was found to red-shiftred-shift from 700700 nmnm toto 810 810 nm, nm, affording affording a a two-photon two-photon absorption absorption cross-section cross-section of 320of 320 GM GM for for21. ® In vitro HeLa cell studies of AzuFluor 483-Bpin® highlighted that fluorescence emission 21. In vitro HeLa cell studies of AzuFluor 483-Bpin− highlighted that fluorescence emis- couldsion could be turned be turned on in on the in the presence presence of ONOOof ONOOto− to image image cells. cells. When When excited excited atat 800800 nm, the emission intensity of AzuFluor® 483-Bpin + ONOO− increased four-fold with respect the emission intensity of AzuFluor® 483-Bpin + ONOO− increased four-fold with respect to AzuFluor® 483-Bpin (Figure 18). The probe was also shown to successfully undergo to AzuFluor® 483-Bpin (Figure 18). The probe was also shown to successfully undergo turn on fluorescence, and subsequent TPM imaging, when introduced to RAW 264.7 cells turn on fluorescence, and subsequent TPM imaging, when introduced to RAW 264.7 cells pretreated with inducers of endogenous ROS species. AzuFluor® 483-Bpin was found to pretreated with inducers of endogenous ROS species. AzuFluor® 483-Bpin was found to be non-cytotoxic and to be photostable for 1 h with and without the presence of ONOO−. Molecules 2020, 25, x be non-cytotoxic and to be photostable for 1 h with and without the presence of ONOO15 of 19−.

Figure 18. ((a)) Two-photon excitation of HeLa cells stained with AzuFluor® 483-Bpin (5 µM)µM) with − with(lower) (lower) or without or without (control, (control, upper) upper) pre-treatment pre-treatment of exogenousof exogenous ONOO ONOO− (100 (100µ µM).M). Wavelengths Wave- lengthsshown shown are excitation are excitation wavelengths. wavelengths. Incubation Incuba timetion was time 30 min. was Images30 min. were Images obtained were atobtained400–600 nm at 400–600 nm emission windows. Scale bar = 50 µm. (b) Average fluorescence intensities at emission windows. Scale bar = 50 µm. (b) Average ﬂuorescence intensities at various excita- various excitation wavelengths. (c) Fluorescence emission spectrum of AzuFluor® 483-Bpin tion wavelengths. (c) Fluorescence emission spectrum of AzuFluor® 483-Bpin and AzuFluor® and AzuFluor® 483-Bpin + ONOO− at λex = 800 nm excitation, taken from the corresponding 483-Bpin + ONOO− at λ = 800 nm excitation, taken from the corresponding cell image. Figure cell image. Figure reproducedex from ref. [89]. AF483 = AzuFluor® 483-Bpin. reproduced from ref. [89]. AF483 = AzuFluor® 483-Bpin. 3. Conclusions This review highlighted areas of the literature in which the azulene core has been employed as a fluorescent reporter moiety in the field of chemical probes. A summary of each system is reported in Table 1.

Table 1. Summary of fluorescent azulene-based sensors, dosimeters and imaging agents with accompanying references.

Analyte or Imaging Purpose Author and Reference H+ (Polymers and oligomers) 1 Xu [46], Hawker [47,48] H+ (Alkynyl azulenes) 1 Venkatesan [49,50] H+ (Guaiazulene) 1 Belfield [51] H+ (Fused cyclic azulenes) 1 Shoji [55,57] Shoji [56], Li and Gao [58], Jamali and H+ (α-Atom protonation) Bagherzadeh [59] H+ (Misc. small molecule azulene proton Gao [53], Shoji [54], Lewis and Kann [29], probes) 1 Zhu [86,87] Fluoride (F−) Eichen [60] Moroder [75], Pispisa [76,77], Korendovych Biomarker (Aal) [79,80] Kalesse [81] Fluorescence quenching Koh and Lee [78], Tung [83,84] Cell imaging Zhu [85], Lewis [89] Reactive oxygen species (e.g., HOO−, Lewis [89] ONOO−) 1 Protonation occurs at the 1- and/or 3-position.

So far, the concept of azulene-based probes has been underutilised in the literature when compared with other common fluorophores (e.g., pyrene, coumarin), mainly being employed with regard to functional materials that are turn-on fluorescent protonation probes. One reason that may be hindering the growth of azulene sensors and dosimeters may be the relatively low quantum yield of azulene fluorophores (e.g., the quantum yield

Molecules 2021, 26, 353 15 of 19

3. Conclusions This review highlighted areas of the literature in which the azulene core has been employed as a ﬂuorescent reporter moiety in the ﬁeld of chemical probes. A summary of each system is reported in Table1.

Table 1. Summary of ﬂuorescent azulene-based sensors, dosimeters and imaging agents with accompanying references.

Analyte or Imaging Purpose Author and Reference H+ (Polymers and oligomers) 1 Xu [46], Hawker [47,48] H+ (Alkynyl azulenes) 1 Venkatesan [49,50] H+ (Guaiazulene) 1 Belﬁeld [51] H+ (Fused cyclic azulenes) 1 Shoji [55,57] H+ (α-Atom protonation) Shoji [56], Li and Gao [58], Jamali and Bagherzadeh [59] H+ (Misc. small molecule azulene proton probes) 1 Gao [53], Shoji [54], Lewis and Kann [29], Zhu [86,87] Fluoride (F−) Eichen [60] Biomarker (Aal) Moroder [75], Pispisa [76,77], Korendovych [79,80] Kalesse [81] Fluorescence quenching Koh and Lee [78], Tung [83,84] Cell imaging Zhu [85], Lewis [89] Reactive oxygen species (e.g., HOO−, ONOO−) Lewis [89] 1 Protonation occurs at the 1- and/or 3-position.

So far, the concept of azulene-based probes has been underutilised in the literature when compared with other common fluorophores (e.g., pyrene, coumarin), mainly being employed with regard to functional materials that are turn-on fluorescent protonation probes. One reason that may be hindering the growth of azulene sensors and dosimeters may be the relatively low quantum yield of azulene fluorophores (e.g., the quantum yield of 21 was determined to be 0.010). Therefore, the future of azulene probes may lie in increasing their emission efficiency. Various methods for achieving this have been reported for other classes of fluorophores, such as rigidification and the introduction of additional rings [93]; such approaches may prove to be equally applicable to azulenes. The azulene core provides a highly attractive reporter motif when considering the creation of a novel probe species. As well as offering a fluorescence response, azulene is highly colourful. Importantly, the colour of azulene compounds can easily be predicted based on the location and electronic nature of its substituents [6]. Therefore, in comparison to other common fluorophores, azulene provides to the synthetic chemist a platform with which to create “designer probes”, whereby the fluorescence and colorimetric response can be fine-tuned. Once access to greater quantum-yielding azulene cores becomes available, greater application of azulene in the world of sensing will follow. Examples such as the fluoride probe by Eichen et al. and the ROS/RNS probe by Lewis et al. prove that such applications are possible. We believe that the future is bright for azulene.

Funding: The EPSRC Impact Acceleration Account provided funding to L.C.M. under grant EP/R51164X/1. The APC was funded by UKRI. Conﬂicts of Interest: The authors are employees of the University of Bath, which owns the trademark “AzuFluor®”.

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