Review BODIPY- and -Based Sensors for Recognition of Amino Acids and Their Derivatives

Marco Farinone, Karolina Urba ´nska and Miłosz Pawlicki *

Wydział Chemii, Uniwersytet Wrocławski, F. Joliot-Curie 14, 50-383 Wrocław, Poland; [email protected] (M.F.); [email protected] (K.U.) * Correspondence: [email protected]; Tel.: +48 (71)375-7649

 Academic Editor: M. Salomé Rodríguez-Morgade  Received: 30 August 2020; Accepted: 29 September 2020; Published: 2 October 2020

Abstract: Molecular recognition is a specific non-covalent and frequently reversible interaction between two or more systems based on synthetically predefined character of the receptor. This phenomenon has been extensively studied over past few decades, being of particular interest to researchers due to its widespread occurrence in biological systems. In fact, a straightforward inspiration by biological systems present in living matter and based on, e.g., bonding is easily noticeable in construction of molecular probes. A separate aspect also incorporated into the molecular recognition relies on the direct interaction between host and guest with a covalent bonding. To date, various artificial systems exhibiting molecular recognition and based on both types of interactions have been reported. Owing to their rich optoelectronic properties, chromophores constitute a broad and powerful class of receptors for a diverse range of substrates. This review focuses on BODIPY and porphyrin chromophores as probes for molecular recognition and chiral discrimination of amino acids and their derivatives.

Keywords: molecular recognition; covalent recognition; chiral discrimination; BODIPY; porphyrin tweezers

1. Introduction For several reasons, the visible light became an important aspect of research focusing on specific interactions with the matter, which can find a wide scope of applications. The inexhaustible access to visible light opens many paths for searching for cost-efficient light-activated catalysts or systems properly interacting with specific wavelength (λ), essential for optoelectronic applications. The generality of the light also drives the activity towards formation of systems that can involve, e.g., intramolecular transformation leading to substantial change in absorbance or emission indicating a presence of specific factor. The analytical tools based on application of the visible light are constantly explored because of straightforward detection of the change even with the eye. The construction of skeleton responding to specific factors requires precise planning and designing of structural motifs that would react with and eventually modify the observed outcome, starting to behave as a molecular probe. Covalent recognition is the ability of designed molecular probes to react towards target skeletons with the formation of a covalent bond. As a result, a different behaviour, especially the optical response, can be used to monitor the presence of the target in the medium that is tested. Molecular recognition is a specific tendency of compounds to selectively bind to each other through non-covalent interactions [1–3], such as metal coordination [4], hydrogen bonding [5], hydrophobic interactions [6], van der Waals interactions [7], π–π stacking [8] or electrostatic interactions [9]. The term “chiral recognition” refers to an event of molecular recognition, in which a receptor exhibits higher affinity towards one of existing enantiomers of guest molecule. If the host molecule is chiral, the formed

Molecules 2020, 25, 4523; doi:10.3390/molecules25194523 www.mdpi.com/journal/molecules MoleculesMolecules 20202020, 25, 25, x, 4523FOR PEER REVIEW 2 2of of 28 28 molecule is chiral, the formed host-guest complexes are diastereoisomeric and therefore possess sets ofhost-guest different complexesproperties, are enabling diastereoisomeric their easy and separation, therefore possess as well sets as of differentiation different properties, in 1H enabling NMR spectroscopytheir easy separation, (chiral discrimination). as well as differentiation in 1H NMR spectroscopy (chiral discrimination). InIn the the past past decades decades the the BODIPY BODIPY chromophores chromophores (Scheme(Scheme1 1,, top)top) as as well well as as porphyrins (Scheme (Scheme1, 1,bottom) bottom) or or porphyrinoids porphyrinoids have have found found widespread widespread applications applications as sensors as sensors for detectionfor detection of a broadof a broad scope scopeof substrates of substrates based on based several on straightforward several straight detectionforward channels, detection including channels, UV /Vis,including fluorescence UV/Vis, and fluorescencecircular dichroism and circular (CD) spectroscopies. dichroism (CD) These spectr techniquesoscopies. provideThese techniques a quick and provide accurate a responsequick and in accurateaddition response to requiring in addition minimal to amounts requiring of analyte.minimal Itamounts usually needsof analyte. a properly It usually defined needs optic a properly response definedfollowed optic by highresponse extinction followed coeffi bycients high and extinction effective coefficients fluorescence and with effective high quantum yield. with The linearhigh quantumconstruction yield. of BODIPYThe linear leads construction to sharp absorption of BODIPY and leads highly to esharpfficient absorption emission observed,and highly what efficient opens emissiona potential observed, for application what opens of those a potential skeletons for appl as opticalication probes. of those On skeletons the other as hand,optical owing probes. to On the theunique other properties hand, owing of porphyrinoids, to the unique properties they are interesting of porphyrinoids, candidates they for are molecular interesting [10 ]candidates and chiral for [11 ] molecularrecognition [10] with and multi-channel chiral [11] andrecognition straightforward with multi-channel UV/Vis, fluorescence and straightforward and CD detection UV/Vis, [12,13 ]. fluorescenceThe macrocyclic and CD character detection of porphyrin [12,13]. The (Scheme macrocyclic1, bottom) character shows of another porphyrin aspect (Scheme of oligopyrroles. 1, bottom) showsStrongly another conjugated aspectπ -cloudsof oligop withyrroles. a diatropic Strongly circuit conjugated operating π within-clouds the with porphyrins a diatropic 18π electroncircuit operatingpath can bewithin also the applied porphyrins in NMR 18 detection,π electron eventually path can be showing also applied a significant in NMR di detection,fference in eventually registered showingchemical a shifts.significant On thedifference other hand, in registered their rich chemic opticalal properties,shifts. On the such other as intense hand, their absorption rich optical in the properties,Soret region such (400–430 as intense nm) absorption and fluorescence, in the Sore givest region further (400–430 possibilities nm) and for fluorescence, using them gives as probes further as possibilitiestheir strong for and using narrow them absorption as probes and as their fluorescence strong and bands narrow allow absorption detecting even and afluorescence slight perturbation bands allowcaused detecting by a guest even binding. a slight perturbation caused by a guest binding.

SchemeScheme 1. 1. RepresentationRepresentation of ofthe the difference difference in the in thetarget target recognition recognition between: between: BODIPY BODIPY probe probe (1a); and (1a ); [18]Tetraphyrin(1.1.1.1)and [18]Tetraphyrin(1.1.1.1) probe probe (1b) ( (porphyrin1b)(porphyrin). The). yellow The yellow triangle triangle represents represents the target the target molecule, molecule, the lightthe lightblue bluetriangle triangle represents represents a leaving a leaving group. group.

TheThe abundance abundance of ofmolecular molecular recognition recognition phenomena phenomena in living in living organisms organisms [14] is [ 14one] isof onethe many of the reasonsmany reasonsthis field this of fieldinterest of interestdraws attention draws attention of many of researchers. many researchers. Herein, we Herein, present we recent present reports recent onreports BODIPY- on BODIPY- and porphyrin-based and porphyrin-based sensors sensors for the for recognition the recognition of amino of amino acids acids and and discuss discuss their their respectiverespective applications. applications. The The importance importance behind behind the the recognition recognition of of amino amino acids acids lies lies on on the the fact fact that that thosethose molecules molecules are are essential essential in in the the human human body body for for many many physiological physiological processes processes such such as as protein protein synthesis,synthesis, detoxification, detoxification, metal metal bi bindingnding and and many many more more [15]. [15]. The The ov over-er- or or under-expression under-expression of of those those naturalnatural derivatives derivatives is is usually usually strictly strictly related related to to di differentfferent pathologies, pathologies, hence hence the the ability ability to to detect detect such such abnormalitiesabnormalities is is very very useful useful especially especially for for the the scr screeningeening of of diseases diseases in in patients. patients. Moreover, Moreover, recognition recognition ofof amino amino acids acids and and other other natural natural derivatives derivatives is is a apowerful powerful tool tool that, that, if if combined combined to to the the peculiar peculiar optical optical propertiesproperties of of BODIPY BODIPY molecules molecules [16,17], [16,17], finds finds app applicationslications in in bioimaging bioimaging which which is is a a non-invasive non-invasive diagnosticdiagnostic tool tool of of extr extremeeme importance importance [18]. [18]. TheThe detection detection of of other other natural natural derivatives derivatives base basedd on on amino amino acids, acids, such such as as G-proteins G-proteins [19], [19], neurofibrillaryneurofibrillary tangles tangles [20] [20 and] and bio-thiols bio-thiols [21], [21 are], arestrongly strongly related related with with many many cellular cellular functions functions and pathologiesand pathologies and are and also are mentioned also mentioned in this in work. this work.

2. BODIPY Probes

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Molecules2. BODIPY 2020, Probes25, x FOR PEER REVIEW 3 of 28

BODIPYBODIPY (BOron(III) (BOron(III) DIPYrromethene)DIPYrromethene) is a class class of of chromophore chromophore formed formed from from two two pyrrolic pyrrolic units units linkedlinked byby aa methylene methylene bridge bridge (Schemes (Schemes1 and 12 )and in addition 2) in addition to coordinating to coordinating electron-deficient electron-deficient boron(III). boron(III).The planarity The of planarity those molecules of those jointly molecules with jointly the presence with the boron(III) presence give boron(III) structures give with structures extraordinary with extraordinaryoptical properties optical that properties are fascinating that are the fascinatin researchg world the research for many world decades. for many Extensive decades. investigation Extensive investigationand studies onand the studies synthetic on the aspects synthetic of those aspects derivatives of thosehave derivatives been carried have been out andcarried allowed out and the alloweddesign ofthe many design chromophores of many chromophores with versatile with electronicversatile electronic properties. properties. Those skeletons Those skeletons have found have foundapplications applications as probes as probes since they since have they a peculiarhave a peculiar opticalbehaviour optical behaviour presenting presenting very sharp very emission sharp emissionbands with bands a quantum with a quantum yield close yield to a close unit andto a moreunit and importantly more importantly even small even changes small in changes the molecular in the molecularstructure canstructure trigger can drastic trigger shifts drastic in the shifts fluorescence in the fluorescence spectrum spectrum [22–24]. Because[22–24]. ofBecause this, BODIPY of this, BODIPYprobes are probes widely are used widely as molecular used as molecular recognisers recognisers for the target for the with target covalent with interaction;covalent interaction; however, however,the involvement the involvement of non-covalent of non-covalent interaction was interact also reportedion was asalso an reported efficient process as an allowingefficient process efficient allowingrecognition. efficient The recognition. most important The most features important of those features molecules of those rely molecules on the fact rely that on theythe fact present that theynarrow present absorption narrow and absorption emission and bands emission together bands with together high quantum with high yields quantum approaching yields aapproaching quantitative afluorescence quantitative [25 fluorescence] forming skeletons [25] forming potentially skeletons applicable potentially in several applicable fields includingin several the fields recognition including of thespecific recognition targets. of The specific BODIPY targets. core wasThe extensivelyBODIPY core explored was extensively with an involvement explored with of specific an involvement positions of(Scheme specific2), positions eventually (Scheme leading 2), to eventually extraordinary leadin behaviourg to extraordinary including tuningbehaviour of the including optical properties tuning of thewith optical a modulation properties of with absorption a modulation and emission of absorption wavelength and emission in both directions–bathochromically wavelength in both directions– [26] bathochromicallyand hypsochromically [26] [an22d]. hypsochromically Most of the modifications [22]. Most showing of the themodifications skeletal dependence showing ofthe the skeletal optical dependenceproperties are of basedthe optical on covalent properties interaction are based but on the covalent probing interaction behaviour needsbut the a responseprobing behaviour to specific needsinitiator a response or target to potentially specific initiator available or withtarget the potentially applied structure. available with the applied structure.

SchemeScheme 2. 2. ExamplesExamples of of possible possible modification modification on on the the BODIPY BODIPY core. core.

OneOne of of the the most most fascinating fascinating ability ability for for those those chro chromophoresmophores is is the the change change in in the the optical optical response response triggeredtriggered by by simple simple initiators initiators such such us us pH, pH, presence presence of of anions and other factors [27]. [27]. Because Because of of this this behaviour,behaviour, BODIPY molecules are very useful optical tools, finding finding many applications including including the the probing of, e.g., metals [28] [28] or ni nitratetrate [29]. [29]. One One other other field field where where BODIPY BODIPY derivatives derivatives are are involved involved is is thethe bioimaging bioimaging and and detection detection of of specific specific targets targets fo foundund in in the the human human body body for for many many different different reasons reasons andand applications applications [18]. [18]. TheThe detection of thosethose componentscomponents can can be be achieved achieved in in di differentfferent ways ways but, but, in general,in general, a shift a shift or theor theON ON/OFF/OFF switch switch of absorption of absorption/emission/emission is the is finalthe final outcome outcome in the in recognitionthe recognition of a of target. a target. AA recent work published by Farinone et al. in in 2020 2020 showing showing the the optical behaviour behaviour for for BODIPY BODIPY derivativesderivatives connected connected to to different different amino acids is is th thee example example to to introduce introduce the the topic topic of of BODIPY-amino BODIPY-amino acidacid derivativesderivatives [ 23[23].]. The The hybrid hybrid derivatives derivatives were obtainedwere obtained through nucleophilicthrough nucleophilic aromatic substitution aromatic substitutionin the meso-position in the meso of the‑position BODIPY of the chromophore, BODIPY chromophore, as shown in Schemeas shown3. in Scheme 3.

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BODIPY (BOron(III) DIPYrromethene) is a class of chromophore formed from two pyrrolic units linked by a methylene bridge (Schemes 1 and 2) in addition to coordinating electron-deficient boron(III). The planarity of those molecules jointly with the presence boron(III) give structures with extraordinary optical properties that are fascinating the research world for many decades. Extensive investigation and studies on the synthetic aspects of those derivatives have been carried out and allowed the design of many chromophores with versatile electronic properties. Those skeletons have found applications as probes since they have a peculiar optical behaviour presenting very sharp emission bands with a quantum yield close to a unit and more importantly even small changes in the molecular structure can trigger drastic shifts in the fluorescence spectrum [22–24]. Because of this, BODIPY probes are widely used as molecular recognisers for the target with covalent interaction; however, the involvement of non-covalent interaction was also reported as an efficient process allowing efficient recognition. The most important features of those molecules rely on the fact that they present narrow absorption and emission bands together with high quantum yields approaching a quantitative fluorescence [25] forming skeletons potentially applicable in several fields including the recognition of specific targets. The BODIPY core was extensively explored with an involvement of specific positions (Scheme 2), eventually leading to extraordinary behaviour including tuning of the optical properties with a modulation of absorption and emission wavelength in both directions– bathochromically [26] and hypsochromically [22]. Most of the modifications showing the skeletal dependence of the optical properties are based on covalent interaction but the probing behaviour needs a response to specific initiator or target potentially available with the applied structure.

Scheme 2. Examples of possible modification on the BODIPY core.

One of the most fascinating ability for those chromophores is the change in the optical response triggered by simple initiators such us pH, presence of anions and other factors [27]. Because of this behaviour, BODIPY molecules are very useful optical tools, finding many applications including the probing of, e.g., metals [28] or nitrate [29]. One other field where BODIPY derivatives are involved is the bioimaging and detection of specific targets found in the human body for many different reasons and applications [18]. The detection of those components can be achieved in different ways but, in general, a shift or the ON/OFF switch of absorption/emission is the final outcome in the recognition of a target. A recent work published by Farinone et al. in 2020 showing the optical behaviour for BODIPY derivatives connected to different amino acids is the example to introduce the topic of BODIPY-amino acid derivatives [23]. The hybrid derivatives were obtained through nucleophilic aromatic Moleculessubstitution2020, 25in, 4523the meso‑position of the BODIPY chromophore, as shown in Scheme 3. 4 of 28

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Scheme 3. The amino acids used in this work are alanine, glycine, , p-amino-phenylalanine, Scheme 3. The amino acids used in this work are alanine, glycine, lysine, p-amino-phenylalanine, tyrosine and cysteine. tyrosine and cysteine.

Based on the the type type of of heteroatom heteroatom involved involved in inthe the mesomeso bondbond (3x (),3x hence), hence what what type type of amino of amino acid acidwas used, was used, a difference a difference in the in electronic the electronic properties properties was recorded was recorded with shifts with in shifts both inemission both emission (range, λmax = 452–535 nm) and absorption (range, λmax = 407–519 nm). The most red-shifted for both (range, λmax = 452–535 nm) and absorption (range, λmax = 407–519 nm). The most red-shifted for both absorption and emission was observed for 3f and the biggest hipsochrom hipsochromicic shift was recorded for 3d.. ThoseThose observationsobservations show show how how the the optical optical properties properti ofes BODIPY of BODIPY skeleton skeleton can be can modulated be modulated by a simple by a modificationsimple modification making making this chromophore this chromophore a versatile a versatile example example of the optical of the probe.optical probe.

2.1. Covalent Interaction (Recognition) The sensingsensing of thethe targettarget moleculesmolecules cancan bebe achievedachieved towardstowards the formationformation of a directdirect bondbond between the target and the BODIPY sensor with invo involvementlvement of different different positions of the fluorophore corecore and usually follows the chemistry of of a a nucleophilic nucleophilic aromatic aromatic substitution. substitution. It Itusually usually requires requires a aprecise precise designing designing of of the the mother mother skeleton skeleton and and spec specificific location location of of good good leaving leaving group group either either at at mesomeso-‑ or αα‑-positionposition.. After After the the substitution substitution (Schemes (Schemes 33 andand4 ),4), a a novel novel BODIPY-target BODIPY-target hybrid hybrid structure structure is is obtained withwith opticaloptical propertiesproperties strongly strongly di differenfferent fromt from the the starting starting materials materials making making the the detection detection of theof the target target molecule molecule easy easy and and reliable. reliable. This This type type of of interaction interaction between between the the probe probeand and thethe targettarget cancan triggertrigger didifferentfferent type type of of responses, responses, which which were were divided divided into into two two main main categories: categories: the the activation activation in the in emissionthe emission of the of the probe probe (or, (or, in some in some cases, cases, the the deactivation deactivation in thein the probe’s probe’s fluorescence) fluorescence) and and the the shift shift in emissionin emission and and absorption absorption of theof the probe probe (Scheme (Scheme4). 4).

Scheme 4.4. Representation of the two approaches in thethe recognitionrecognition throughthrough covalentcovalent interactioninteraction

observed forfor BODIPYBODIPY chromophore.chromophore. R11,, group with fluorescencefluorescence quenchingquenching properties;properties; LG,LG, leavingleaving group suchsuch asas -Cl-Cl oror -SMe.-SMe.

2.1.1. Emission Triggering Depending on the planned recognition, specific response modulating optic properties are required. The potential application to biological tissues very often requires a long emission wavelength available for specifically modified BODIPY skeletons. In 2011, Shao et al. reported on a BODIPY-based molecular probe with specific detection of cysteine among the biological thiols [30]. The structure of the chromophore was designed as a sensor 4 (see Scheme 5) with a 2,4- dinitrobenzenesulfonyl (DNBS) protecting group connected to the phenol ring present on the α position of the BODIPY through an ethylene bridge giving a mute in emission skeleton 4.

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2.1.1. Emission Triggering Depending on the planned recognition, specific response modulating optic properties are required. The potential application to biological tissues very often requires a long emission wavelength available for specifically modified BODIPY skeletons. In 2011, Shao et al. reported on a BODIPY-based molecular probe with specific detection of cysteine among the biological thiols [30]. The structure of the chromophore was designed as a sensor 4 (see Scheme5) with a 2,4-dinitrobenzenesulfonyl (DNBS) protecting group connected to the phenol ring present on the α position of the BODIPY through anMolecules ethylene 2020, bridge25, x FOR giving PEER REVIEW a mute in emission skeleton 4. 5 of 28

Scheme 5. Preparation of the BODIPY probe specific for cysteine and recognition. Scheme 5. Preparation of the BODIPY probe specific for cysteine and recognition. The recognition of the target molecule results in the unmasking of the structure 4 and eventually The recognition of the target molecule results in the unmasking of the structure 4 and eventually an appearance of intense fluorescence in the red region at 590 nm [30]. The derivative 5 presents a red an appearance of intense fluorescence in the red region at 590 nm [30]. The derivative 5 presents a shifted emission because of the specific extension of delocalisation along the α position. red shifted emission because of the specific extension of delocalisation along the α position. Another example based on the enhancement of the emission toward the elimination of a protecting Another example based on the enhancement of the emission toward the elimination of a group with quenching properties was reported by Wang et al. in 2018 [31]. They designed a BODIPY protecting group with quenching properties was reported by Wang et al. in 2018 [31]. They designed probe (6) (Scheme6) for the specific recognition of glutathione (GSH). The sensor was functionalised a BODIPY probe (6) (Scheme 6) for the specific recognition of glutathione (GSH). The sensor was with a 2,4-dinitrobenzenesulphonyl group as a fluorescent quencher but more importantly as a good functionalised with a 2,4-dinitrobenzenesulphonyl group as a fluorescent quencher but more leaving group reactive toward thiols. As expected, 6 presents very low emission (with a quantum yield importantly as a good leaving group reactive toward thiols. As expected, 6 presents very low of Φ = 0.03) at 656 nm. The probing behaviour of 6 was tested in a THF/PBS 1:1 solution by reaction emission (with a quantum yield of Φ = 0.03) at 656 nm. The probing behaviour of 6 was tested in a with various natural amino acids: lysine, asparagine, histidine, phenylalanine, tryptophan, glutamic THF/PBS 1:1 solution by reaction with various natural amino acids: lysine, asparagine, histidine, acid, asparagine and cysteine, as well as homocysteine and glutathione. Cys and Hcy enhanced the phenylalanine, tryptophan, glutamic acid, asparagine and cysteine, as well as homocysteine and fluorescence response with a quantum yield reaching 24% and 12%, respectively, at 659 and 657 nm glutathione. Cys and Hcy enhanced the fluorescence response with a quantum yield reaching 24% while with GSH it reached 48% at 656 nm. Considering the differences in the intracellular concentrations and 12%, respectively, at 659 and 657 nm while with GSH it reached 48% at 656 nm. Considering the of those natural derivatives, the response of the probe is remarkably selective toward GSH when tested differences in the intracellular concentrations of those natural derivatives, the response of the probe on MCF-7 cells. All other amino acids did not affect the efficiency of the fluorescence emission. is remarkably selective toward GSH when tested on MCF-7 cells. All other amino acids did not affect The mechanism of recognition of GSH by the BODIPY probe is shown in Scheme6 displaying the the efficiency of the fluorescence emission. deprotection of the 2,4-dinitrobenzenesulphonyl moiety. A covalent interaction between the BODIPY probe and the target molecules resulting in the activation of the chromophore emission was also presented in 2017 by Wang et al. [32]. In this work, they prepared a probe that can distinguish cysteine and homocysteine over GSH. They designed and synthetised a probe (8) featuring p-aminophenylthio and cyano groups attached to the BODIPY moiety. The decision on the presence of those groups was inspired by previous documented works that pointed out how specifically a p-aminophenylthio moiety can detect natural thiols.

Scheme 6. Recognition of GSH from the BODIPY probe with deprotection of quenching group.

The mechanism of recognition of GSH by the BODIPY probe is shown in Scheme 6 displaying the deprotection of the 2,4-dinitrobenzenesulphonyl moiety. A covalent interaction between the BODIPY probe and the target molecules resulting in the activation of the chromophore emission was also presented in 2017 by Wang et al. [32]. In this work, they prepared a probe that can distinguish cysteine and homocysteine over GSH. They designed and synthetised a probe (8) featuring p-aminophenylthio and cyano groups attached to the BODIPY

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Scheme 5. Preparation of the BODIPY probe specific for cysteine and recognition.

The recognition of the target molecule results in the unmasking of the structure 4 and eventually an appearance of intense fluorescence in the red region at 590 nm [30]. The derivative 5 presents a red shifted emission because of the specific extension of delocalisation along the α position. Another example based on the enhancement of the emission toward the elimination of a protecting group with quenching properties was reported by Wang et al. in 2018 [31]. They designed a BODIPY probe (6) (Scheme 6) for the specific recognition of glutathione (GSH). The sensor was functionalised with a 2,4-dinitrobenzenesulphonyl group as a fluorescent quencher but more importantly as a good leaving group reactive toward thiols. As expected, 6 presents very low emission (with a quantum yield of Φ = 0.03) at 656 nm. The probing behaviour of 6 was tested in a THF/PBS 1:1 solution by reaction with various natural amino acids: lysine, asparagine, histidine, phenylalanine, tryptophan, glutamic acid, asparagine and cysteine, as well as homocysteine and glutathione. Cys and Hcy enhanced the fluorescence response with a quantum yield reaching 24% and 12%, respectively, at 659 and 657 nm while with GSH it reached 48% at 656 nm. Considering the differences in the intracellular concentrations of those natural derivatives, the response of the probe is remarkably selective toward GSH when tested on MCF-7 cells. All other amino acids did not affect Molecules 2020, 25, 4523 6 of 28 the efficiency of the fluorescence emission.

Molecules 2020, 25, x FOR PEER REVIEW 6 of 28 moiety. The decision on the presence of those groups was inspired by previous documented works Scheme 6. RecognitionRecognition of GSH from the BODIPY probe with deprotection of quenching group. that pointed out how specifically a p-aminophenylthio moiety can detect natural thiols. The freemechanismfree probe probe presents ofpresents recognition an absorptionan ofabsorption GSH peak by the atpeak 546 BO nmDIPYat with546 probe substantiallynm iswith shown subs diminishedin tantiallyScheme 6 fluorescence.diminished displaying fluorescence.Thethe deprotection presence ofThe sulphur of presence the 2,4-dinitrobenzenesulphonyl from of sulphur cysteine from attached cysteine to the attached moiety.meso-position to the meso of BODIPY-position skeleton of BODIPY was reportedskeleton wasas bathochromically Areported covalent as interaction bathochromically shifting between factor [shiftingthe23], BODIPY but, fa afterctor probe treatment[23], andbut, the withafter target Cystreatment molecules (10), thewith absorptionresulting Cys (10 in), peak the absorptionshiftedactivation hypsochromically of peak the chromophore shifted hypsochromically by ~60 emission nm to was 480 also nmby ~60 withpres entednm a dramatic to in 480 2017 nm change by Wangwith in aet fluorescence dramatical. [32]. In changethis effi ciencywork, in fluorescenceeventuallythey prepared observed efficiency a probe at thateventually 549 can nm. distinguish Theobserved hypsochromic cysteine at 549 nm. and shiftThe homocysteine hypsochromic of BODIPY over absorption shift GSH. of BODIPY They was designed reported absorption and for wasmesosynthetised -nitrogenreported a substitutedprobefor meso (8)-nitrogen featuring skeletons substituted ofp-aminophenylthio chromophores skeletons [33 ]and furtherof cyanochromophores shifted groups by deprotonation attached[33] further to the ofshifted mesoBODIPY-NH by deprotonationgroup [22]. A similarof meso behaviour-NH group was [22]. detected A similar in behaviour the case of was Hcy de withtected the in formation the case of of Hcy9 (Scheme with the7). formationIn contrast, of upon 9 (Scheme treatment 7). In with contrast, GSH, upon only atreatment slight enhancement with GSH, only in the a fluorescenceslight enhancement was detected in the withfluorescence an increase was indetected intensity with of 18%an increase when compared in intensity to Cysof 18% and when Hcy, compared indicating to that Cys the and probe Hcy, is indicatingsuccessfully that discriminating the probe is Cys successfully and Hcy overdiscri GSH.minating The proposedCys and mechanismHcy over GSH. of interaction The proposed of the mechanismBODIPY with of cysteineinteraction and of homocysteine the BODIPY with (Scheme cyst7eine) incorporates and homocysteine formation (Scheme of 5- and 7) 6-memberedincorporates formationrings obtained of 5- upon and 6-membered nucleophilic attackrings obtained of the thiol upon groups. nucleophilic In the case attack of glutathione, of the thiol the groups. formation In the of casesuch of derivatives glutathione, is possible the formation but not of favoured such derivatives since the moleculeis possible is but much not bulkier favoured when since compared the molecule to the istwo much others. bulkier when compared to the two others.

SchemeScheme 7. Proposed 7. Proposed mechanism mechanism for for the the recognition recognition of cysteineof cysteine and and homocysteine homocysteine by BODIPY-basedby BODIPY-based probe. probe. One recent example of BODIPY-based amino acid sensor was presented in 2020 by Jeon et al. [34]. TheyOne designed recent and example developed of BODIPY-based a BODIPY sensor amino with acid high sensor affinity was for presented amino groups in 2020 by introducingby Jeon et al. at [34].the meso They-position designed a good and leaving developed group a withBODIPY an active sensor esters with type high of bondaffinity well for known amino from groups peptide by introducingsynthesis (Scheme at the 8meso). -position a good leaving group with an active esters type of bond well known from peptide synthesis (Scheme 8).

Scheme 8. Mechanism of recognition of the amine target.

Two similar probes are available, one bearing a pentafluorophenyl group (12) and the other one with a hydroxysuccinimidyl substituent (13). They both present very high recognition ability toward volatile amines such as ammonia, methylamine and dimethyl amine. While 12 was demonstrated to

Molecules 2020, 25, x FOR PEER REVIEW 6 of 28 moiety. The decision on the presence of those groups was inspired by previous documented works that pointed out how specifically a p-aminophenylthio moiety can detect natural thiols. The free probe presents an absorption peak at 546 nm with substantially diminished fluorescence. The presence of sulphur from cysteine attached to the meso-position of BODIPY skeleton was reported as bathochromically shifting factor [23], but, after treatment with Cys (10), the absorption peak shifted hypsochromically by ~60 nm to 480 nm with a dramatic change in fluorescence efficiency eventually observed at 549 nm. The hypsochromic shift of BODIPY absorption was reported for meso-nitrogen substituted skeletons of chromophores [33] further shifted by deprotonation of meso-NH group [22]. A similar behaviour was detected in the case of Hcy with the formation of 9 (Scheme 7). In contrast, upon treatment with GSH, only a slight enhancement in the fluorescence was detected with an increase in intensity of 18% when compared to Cys and Hcy, indicating that the probe is successfully discriminating Cys and Hcy over GSH. The proposed mechanism of interaction of the BODIPY with cysteine and homocysteine (Scheme 7) incorporates formation of 5- and 6-membered rings obtained upon nucleophilic attack of the thiol groups. In the case of glutathione, the formation of such derivatives is possible but not favoured since the molecule is much bulkier when compared to the two others.

Scheme 7. Proposed mechanism for the recognition of cysteine and homocysteine by BODIPY-based probe.

One recent example of BODIPY-based amino acid sensor was presented in 2020 by Jeon et al. [34]. They designed and developed a BODIPY sensor with high affinity for amino groups by introducing at the meso-position a good leaving group with an active esters type of bond well known fromMolecules peptide2020, 25 synthesis, 4523 (Scheme 8). 7 of 28

Scheme 8. Mechanism of recognition of the amine target. Scheme 8. Mechanism of recognition of the amine target. Two similar probes are available, one bearing a pentafluorophenyl group (12) and the other one withMoleculesTwo a hydroxysuccinimidyl 2020 similar, 25, x FORprobes PEER are REVIEW av substituentailable, one (13 bearing). They a both pentafluorophenyl present very high group recognition (12) and ability the other toward7 oneof 28 volatilewith a hydroxysuccinimidyl amines such as ammonia, substituent methylamine (13). They and both dimethyl present amine.very high While recognition12 was demonstratedability toward mostly find application in detecting amines in organic solutions and their vapours in solid states, 13 volatileto mostly amines find application such as ammonia, in detecting methylamine amines in and organic dimethyl solutions amine. and While their 12 vapours was demonstrated in solid states, to was investigated in water solutions toward the recognition of biological amines such as lysine 13 was investigated in water solutions toward the recognition of biological amines such as lysine residues on proteins, antibodies and nucleic acids. The recognition tests were carried out in PBS residues on proteins, antibodies and nucleic acids. The recognition tests were carried out in PBS buffer (10 mM, pH = 7.4) containing 1% acetonitrile at room temperature. The probe presented an buffer (10 mM, pH = 7.4) containing 1% acetonitrile at room temperature. The probe presented absorption peak at 622 nm, while the emission, recorded at 648 nm, was very weak (Φ = 0.001) because an absorption peak at 622 nm, while the emission, recorded at 648 nm, was very weak (Φ = 0.001) of aggregation-caused quenching. Upon stepwise addition of L-lysine, the absorption band rises to because of aggregation-caused quenching. Upon stepwise addition of l-lysine, the absorption band 517 nm with a blue shift of 105 nm because of the less electron withdrawing character of the meso rises to 517 nm with a blue shift of 105 nm because of the less electron withdrawing character of the amide specie obtained (14d). The emission, after addition of the specific amino acid, is drastically meso amide specie obtained (14d). The emission, after addition of the specific amino acid, is drastically turned on and shifted to 547 nm. A similar behaviour was observed for , demonstrating that turned on and shifted to 547 nm. A similar behaviour was observed for arginine, demonstrating that the guanidine group and the ε-amino group of lysine react very efficiently in this recognition process. the guanidine group and the ε-amino group of lysine react very efficiently in this recognition process. Other amino acids were tested but resulted in a very weak response with longer incubation times for Other amino acids were tested but resulted in a very weak response with longer incubation times for proline, histidine and cysteine while for the other amino acids the response was even weaker. proline, histidine and cysteine while for the other amino acids the response was even weaker. Aza-BODIPY probe 15 (Scheme 9) reported by Xiao and co-workers selectively responds to Aza-BODIPY probe 15 (Scheme9) reported by Xiao and co-workers selectively responds to lysine lysine with a drastic decrease of fluorescence [35]. with a drastic decrease of fluorescence [35].

Scheme 9. The recognition of the amino acid (lysine) shows a PET effect. Scheme 9. The recognition of the amino acid (lysine) shows a PET effect. The aza-BODIPY probe 15 presents an absorption maximum at 732 nm and a strong emission in the The aza-BODIPY probe 15 presents an absorption maximum at 732 nm and a strong emission in NIR region at 753 nm in dichloromethane. After incubating the probe with lysine in a 1:4 DMSO/water the NIR region at 753 nm in dichloromethane. After incubating the probe with lysine in a 1:4 mixture (pH = 7.3), the fluorescence intensity strongly decreased because of the photo-induced electron DMSO/water mixture (pH = 7.3), the fluorescence intensity strongly decreased because of the photo- transfer (PET) shown in Scheme9 from structure 16. The selectivity for the target was demonstrated induced electron transfer (PET) shown in Scheme 9 from structure 16. The selectivity for the target testing different amino acids and bio-thiols, which, after incubation with the probe, did not show was demonstrated testing different amino acids and bio-thiols, which, after incubation with the a change in the fluorescence intensity. Because of this specific behaviour, the BODIPY-based probe probe, did not show a change in the fluorescence intensity. Because of this specific behaviour, the was eventually used in a bioimaging application to sense lysine in PC3 cells showing good and BODIPY-based probe was eventually used in a bioimaging application to sense lysine in PC3 cells promising results. showing good and promising results. A similar recognition behaviour was presented in 2016 by Das and co-workers. In their work, A similar recognition behaviour was presented in 2016 by Das and co-workers. In their work, they synthetised a BODIPY-based probe (17, Scheme 10) which exhibits specific recognition abilities for they synthetised a BODIPY-based probe (17, Scheme 10) which exhibits specific recognition abilities lysine over other amino acids [36]. for lysine over other amino acids [36].

Scheme 10. The recognition of lysine from probe 17 shows the formation of an imine bond.

The optical behaviour of the probe consists on a sharp absorption band at 503 nm and a strong green emission with maximum at 515 nm; however, after addition of lysine in a 1:9 DMSO/HEPES

Molecules 2020, 25, x FOR PEER REVIEW 7 of 28 mostly find application in detecting amines in organic solutions and their vapours in solid states, 13 was investigated in water solutions toward the recognition of biological amines such as lysine residues on proteins, antibodies and nucleic acids. The recognition tests were carried out in PBS buffer (10 mM, pH = 7.4) containing 1% acetonitrile at room temperature. The probe presented an absorption peak at 622 nm, while the emission, recorded at 648 nm, was very weak (Φ = 0.001) because of aggregation-caused quenching. Upon stepwise addition of L-lysine, the absorption band rises to 517 nm with a blue shift of 105 nm because of the less electron withdrawing character of the meso amide specie obtained (14d). The emission, after addition of the specific amino acid, is drastically turned on and shifted to 547 nm. A similar behaviour was observed for arginine, demonstrating that the guanidine group and the ε-amino group of lysine react very efficiently in this recognition process. Other amino acids were tested but resulted in a very weak response with longer incubation times for proline, histidine and cysteine while for the other amino acids the response was even weaker. Aza-BODIPY probe 15 (Scheme 9) reported by Xiao and co-workers selectively responds to lysine with a drastic decrease of fluorescence [35].

Scheme 9. The recognition of the amino acid (lysine) shows a PET effect.

The aza-BODIPY probe 15 presents an absorption maximum at 732 nm and a strong emission in the NIR region at 753 nm in dichloromethane. After incubating the probe with lysine in a 1:4 DMSO/water mixture (pH = 7.3), the fluorescence intensity strongly decreased because of the photo- induced electron transfer (PET) shown in Scheme 9 from structure 16. The selectivity for the target was demonstrated testing different amino acids and bio-thiols, which, after incubation with the probe, did not show a change in the fluorescence intensity. Because of this specific behaviour, the BODIPY-based probe was eventually used in a bioimaging application to sense lysine in PC3 cells showing good and promising results. A similar recognition behaviour was presented in 2016 by Das and co-workers. In their work, theyMolecules synthetised2020, 25, 4523 a BODIPY-based probe (17, Scheme 10) which exhibits specific recognition abilities8 of 28 for lysine over other amino acids [36].

Molecules 2020, 25, x FOR PEER REVIEW 8 of 28 Scheme 10. TheThe recognition recognition of of lysine lysine from probe 17 shows the formation of an imine bond. buffer (pH = 7.4), absorption gradually shifts blue to 497 nm, together with the formation of a broad The optical behaviour of the probe consists on a sharp absorption band at 503 nm and a strong bandThe at 550 optical nm, behaviourwhile emission of the is probe effectively consists quench on a ed.sharp This absorption behaviour band relies at on 503 the nm specific and a designstrong green emission with maximum at 515 nm; however, after addition of lysine in a 1:9 DMSO/HEPES greenof the emissionprobe which with presents maximum an aldehydeat 515 nm; group however, highly after reactive addition toward of lysine lysine; in indeed, a 1:9 DMSO/HEPES after addition buffer (pH = 7.4), absorption gradually shifts blue to 497 nm, together with the formation of a broad of the amino acid, an imine bond is formed, as shown in structure 18, Scheme 9. An 1H NMR titration band at 550 nm, while emission is effectively quenched. This behaviour relies on the specific design of of the probe with lysine supports the proposed mechanism of recognition with the formation of the the probe which presents an aldehyde group highly reactive toward lysine; indeed, after addition of Schiff base, the aldehyde proton is gradually decreasing and substituted by a new imine proton. the amino acid, an imine bond is formed, as shown in structure 18, Scheme9. An 1H NMR titration of Because of those characteristics, the probe was tested for bioimaging. It was incubated with MDA- the probe with lysine supports the proposed mechanism of recognition with the formation of the Schiff MB 231 cells and showed a gradual quenching of fluorescence upon addition of lysine. base, the aldehyde proton is gradually decreasing and substituted by a new imine proton. Because of those2.1.2. Emission characteristics, Shift the probe was tested for bioimaging. It was incubated with MDA-MB 231 cells and showed a gradual quenching of fluorescence upon addition of lysine. As presented above, the fluorescence on/off behaviour of specific covalent modification applied 2.1.2.for BODIPY Emission skeleton Shift can be used for detecting the presence of specific analytes. Slightly different approach for detection is also possible and is based on easily detectable change in As presented above, the fluorescence on/off behaviour of specific covalent modification applied absorbance/emission wavelength triggered by interaction between probe and detected molecule (see for BODIPY skeleton can be used for detecting the presence of specific analytes. Slightly different Scheme 4). approach for detection is also possible and is based on easily detectable change in absorbance/emission One example using this specific method to selectively recognise cysteine over other bio-thiols wavelength triggered by interaction between probe and detected molecule (see Scheme4). such as homocysteine and glutathione (GSH) was reported by Liu et al. in 2015 [37]. An 8-alkynyl- One example using this specific method to selectively recognise cysteine over other bio-thiols such BODIPY fluorescent probe was designed having an activated alkynyl unit connected to the meso as homocysteine and glutathione (GSH) was reported by Liu et al. in 2015 [37]. An 8-alkynyl-BODIPY position, 19. The recognition of the targets follows the reactivity of dual Michael addition, as shown fluorescent probe was designed having an activated alkynyl unit connected to the meso position, 19. in Scheme 11. The recognition of the targets follows the reactivity of dual Michael addition, as shown in Scheme 11.

Scheme 11. The dual Michael addition reaction proposed for this cysteine recognition [29]. Scheme 11. The dual Michael addition reaction proposed for this cysteine recognition [29]. The probe presents two absorption bands at 410 and 538 nm and one emission band in the yellow The probe presents two absorption bands at 410 and 538 nm and one emission band in the yellow region at 560 nm. Upon addition of cysteine in a CH3CN/PBS buffer (pH = 7.4) at 25 ◦C, the absorption region at 560 nm. Upon addition of cysteine in a CH3CN/PBS buffer (pH = 7.4) at 25 °C, the absorption bands were replaced by a single absorbance at 489 nm, while, for the emission, a hypsochromic shift bands were replaced by a single absorbance at 489 nm, while, for the emission, a hypsochromic shift was observed to 505 nm responsible for the formation of 8-methyl-BODIPY derivative as product (20) was observed to 505 nm responsible for the formation of 8-methyl-BODIPY derivative as product (20) of the retro-aza-aldol process triggered by the dual Michael addition. of the retro-aza-aldol process triggered by the dual Michael addition. The specificity towards recognition of cysteine over homocysteine and glutathione is presented The specificity towards recognition of cysteine over homocysteine and glutathione is presented in Scheme 12. The structural similarities (nucleophilic thiol and amino group) in the architecture in Scheme 12. The structural similarities (nucleophilic thiol and amino group) in the architecture of of Hcy and GSH with Cys should not preclude a cascade reaction eventually leading to an optical Hcy and GSH with Cys should not preclude a cascade reaction eventually leading to an optical response. However, the limiting step for Hcy and GSH is the formation of the cyclic transition state, which will generate six- and ten-membered rings, respectively. This kinetically unfavoured step makes the cascade reaction very slow for Hcy and impossible for GSH, allowing a high specificity for cysteine.

Molecules 2020, 25, x FOR PEER REVIEW 8 of 28 buffer (pH = 7.4), absorption gradually shifts blue to 497 nm, together with the formation of a broad band at 550 nm, while emission is effectively quenched. This behaviour relies on the specific design of the probe which presents an aldehyde group highly reactive toward lysine; indeed, after addition of the amino acid, an imine bond is formed, as shown in structure 18, Scheme 9. An 1H NMR titration of the probe with lysine supports the proposed mechanism of recognition with the formation of the Schiff base, the aldehyde proton is gradually decreasing and substituted by a new imine proton. Because of those characteristics, the probe was tested for bioimaging. It was incubated with MDA- MB 231 cells and showed a gradual quenching of fluorescence upon addition of lysine.

2.1.2. Emission Shift As presented above, the fluorescence on/off behaviour of specific covalent modification applied for BODIPY skeleton can be used for detecting the presence of specific analytes. Slightly different approach for detection is also possible and is based on easily detectable change in absorbance/emission wavelength triggered by interaction between probe and detected molecule (see Scheme 4). One example using this specific method to selectively recognise cysteine over other bio-thiols such as homocysteine and glutathione (GSH) was reported by Liu et al. in 2015 [37]. An 8-alkynyl- BODIPY fluorescent probe was designed having an activated alkynyl unit connected to the meso position, 19. The recognition of the targets follows the reactivity of dual Michael addition, as shown in Scheme 11.

Scheme 11. The dual Michael addition reaction proposed for this cysteine recognition [29].

The probe presents two absorption bands at 410 and 538 nm and one emission band in the yellow region at 560 nm. Upon addition of cysteine in a CH3CN/PBS buffer (pH = 7.4) at 25 °C, the absorption bands were replaced by a single absorbance at 489 nm, while, for the emission, a hypsochromic shift was observed to 505 nm responsible for the formation of 8-methyl-BODIPY derivative as product (20) of the retro-aza-aldol process triggered by the dual Michael addition. The specificity towards recognition of cysteine over homocysteine and glutathione is presented inMolecules Scheme2020 12., 25 The, 4523 structural similarities (nucleophilic thiol and amino group) in the architecture9 of of 28 Hcy and GSH with Cys should not preclude a cascade reaction eventually leading to an optical response. However, the limiting step for Hcy and GSH is the formation of the cyclic transition state, response. However, the limiting step for Hcy and GSH is the formation of the cyclic transition state, which will generate six- and ten-membered rings, respectively. This kinetically unfavoured step which will generate six- and ten-membered rings, respectively. This kinetically unfavoured step makes makes the cascade reaction very slow for Hcy and impossible for GSH, allowing a high specificity for the cascade reaction very slow for Hcy and impossible for GSH, allowing a high specificity for cysteine. cysteine.

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Scheme 12. Specificity of the recognition toward cysteine is explained according to structural differences. Scheme 12. Specificity of the recognition toward cysteine is explained according to structural differences. InIn 2019, Avellanal-Zaballa et al.al. presented presented a a BODIPY BODIPY probe probe designed with functionalities responsiveresponsive to to thiol containing amino acids [38]. [38]. The The sensor sensor presents presents two α,β-unsaturated esters esters on both α positionspositions of the the BODIPY BODIPY core core ( 22,, see see Figure Figure 11)) asas thethe reactivereactive pointspoints thatthat areare ableable toto reactreact withwith thethe nucleophilic nucleophilic groups groups of the sensed targets. Th Thee formation formation of of the the probe probe was possible starting starting from from a di-formylated derivative sequentially converted to the unsaturated ester through Wittig reaction.reaction.

Figure 1. Structure of the BODIPY sensor 22 specific for detection of cysteine, homocysteine Figure 1. Structure of the BODIPY sensor 22 specific for detection of cysteine, homocysteine and and glutathione. glutathione. Because of the extension of delocalisation through the α positions of the sensor, an absorption Because of the extension of delocalisation through the α positions of the sensor, an absorption band is present at 590 nm and an emission band is recorded at 600 nm. After treatment with any of band is present at 590 nm and an emission band is recorded at 600 nm. After treatment with any of cysteine, homocysteine or glutathione, the absorption band is gradually replaced by two blue shifted cysteine, homocysteine or glutathione, the absorption band is gradually replaced by two blue shifted peaks at 550 and 515 nm; the emission is similarly shifted to shorter wavelengths at 560 and 525 nm. peaks at 550 and 515 nm; the emission is similarly shifted to shorter wavelengths at 560 and 525 nm. The tests were carried out in a 1:1 mixture of ethanol/HEPES to mimic the physiological media. Even at The tests were carried out in a 1:1 mixture of ethanol/HEPES to mimic the physiological media. Even low concentrations, the detection of targets (micromolar) was possible by monitoring the fluorescence; at low concentrations, the detection of targets (micromolar) was possible by monitoring the hence, the detection of bio-thiols is easily accessible using the described probe. fluorescence; hence, the detection of bio-thiols is easily accessible using the described probe. Intermolecular Displacement Intermolecular Displacement In some specific cases where a shift in absorption and emission is detected upon covalent interactionIn some between specific the cases probe where and the a target,shift in the absorp mechanismtion and for theemission change is in de thetected electronic upon properties covalent interactionis based on anbetween intermolecular the probe displacement. and the target, the mechanism for the change in the electronic propertiesIn 2018, is based Zhao andon an co-workers intermolecular reported displacement. a BODIPY chromophore applicable for specific recognition for cysteineIn 2018, in humanZhao and plasma co-workers [39]. In thisreported case, it a presented BODIPY a strongchromophore emission applicable and with nofor protecting specific recognitiongroups in the for architecture. cysteine in human The detecting plasma [39]. approach In this starts case, from it presented formation a strong of cysteine emission derivative and with24 no(Scheme protecting 13) that groups undergoes in the intermoleculararchitecture. The transformation detecting approach changing starts the from optical formation behaviour of withcysteine the derivativeformation of2425 (Scheme. 13) that undergoes intermolecular transformation changing the optical behaviour with the formation of 25.

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Scheme 13. Proposed mechanism for the cysteine recognition with resulting formation if 18 and Scheme 13. Proposed mechanism for the cysteine recognition with resulting formation if 18 and limitation over GSH recognition. limitation over GSH recognition. The probe 23 was chosen because of its stability in buffer solutions and the presence of a good The probe 23 was chosen because of its stability in buffer solutions and the presence of a good leaving group at the meso position. Moreover, the structure was designed as an asymmetric molecule leaving group at the meso position. Moreover, the structure was designed as an asymmetric molecule with steric substituents favouring a ratiometric response. The fluorescence spectrum was recorded with steric substituents favouring a ratiometric response. The fluorescence spectrum was recorded in in a CH3CN/PBS buffer (pH = 7.4) at 25 ◦C displaying main absorption and emission at 491 and a CH3CN/PBS buffer (pH = 7.4) at 25 °C displaying main absorption and emission at 491 and 515 nm 515 nm for probe 23. After addition of cysteine, a nucleophilic aromatic substitution took place in the for probe 23. After addition of cysteine, a nucleophilic aromatic substitution took place in the meso- meso-position and a ratiometric response was observed with a dramatic shift both on absorption and position and a ratiometric response was observed with a dramatic shift both on absorption and emission with maxima recorded at 412 and 470 nm. The strong shift in the emission is not explainable emission with maxima recorded at 412 and 470 nm. The strong shift in the emission is not explainable with the presence of a sulphur at the meso position; according to other reports presenting dissimilar with the presence of a sulphur at the meso position; according to other reports presenting dissimilar properties [23,33]. Indeed, due to the intramolecular displacement described in Scheme 13, the amino properties [23,33]. Indeed, due to the intramolecular displacement described in Scheme 13, the amino group replaced the sulphur function giving and explanation to the strong shift. group replaced the sulphur function giving and explanation to the strong shift. The specificity of this probe relies on the fact that upon treatment with another thiol rich natural The specificity of this probe relies on the fact that upon treatment with another thiol rich natural derivative, glutathione (GSH), the absorption peak at 491 nm rapidly decreased and a red-shifted peak derivative, glutathione (GSH), the absorption peak at 491 nm rapidly decreased and a red-shifted at λmax = 509 nm was formed. In addition, a new emission wavelength was measured at 537 nm and peak at λmax = 509 nm was formed. In addition, a new emission wavelength was measured at 537 nm both optical shifts were not ratiometric and support the formation of a thioether intermediary, 26; and both optical shifts were not ratiometric and support the formation of a thioether intermediary, indeed, the intramolecular displacement is not available for GSH, as shown on Scheme 13. 26; indeed, the intramolecular displacement is not available for GSH, as shown on Scheme 13. Another example was presented in 2012 by Niu et al. [40]. The focus of their work was the ability Another example was presented in 2012 by Niu et al. [40]. The focus of their work was the ability to selectively detect the presence of glutathione (GSH) over cysteine and homocysteine. To achieve to selectively detect the presence of glutathione (GSH) over cysteine and homocysteine. To achieve this, they designed a BODIPY-based probe (27, Scheme 14) substituted with a chlorine atom on the this, they designed a BODIPY-based probe (27, Scheme 14) substituted with a chlorine atom on the α α position as a good leaving group and a triazole on the other α position to enhance the reactivity position as a good leaving group and a triazole on the other α position to enhance the reactivity toward nucleophilic aromatic substitution because of the strong electron withdrawing character of toward nucleophilic aromatic substitution because of the strong electron withdrawing character of this substituent. Moreover, the BODIPY derivative presents good in water and a defined this substituent. Moreover, the BODIPY derivative presents good solubility in water and a defined emission peak at 556 nm. The fluorescence response of the probe was tested in an aqueous HEPES emission peak at 556 nm. The fluorescence response of the probe was tested in an aqueous HEPES buffer (20 mM, pH = 7.4) containing 5% acetonitrile at 37 ◦C with stepwise addition of GSH. As a result, buffer (20 mM, pH = 7.4) containing 5% acetonitrile at 37 °C with stepwise addition of GSH. As a the λem = 556 nm emission peak was gradually replaced by a new band at 588 nm with a fluorescence result, the λem = 556 nm emission peak was gradually replaced by a new band at 588 nm with a ratio proportional to the concentration of GSH and to the formation of derivative 28. fluorescence ratio proportional to the concentration of GSH and to the formation of derivative 28.

Molecules 2020, 25, x FOR PEER REVIEW 11 of 28 Molecules 2020, 25, 4523 11 of 28 Molecules 2020, 25, x FOR PEER REVIEW 11 of 28

Scheme 14.14. Recognition process process for for the BODIPY probe 25 toward GSHGSH andand proposedproposed molecularmolecular Schemedisplacement 14. Recognition for cysteine. process for the BODIPY probe 25 toward GSH and proposed molecular displacement for cysteine. The behaviour of the probe was tested with other bio-thiols such as cysteine and homocysteine. WithWithThe Cys,Cys, behaviour afterafter the the initial ofinitial the addition probeaddition was of of thetested the nucleophile, nucleophile, with other a newbio-thiolsa new emission emission such band as band cysteine appeared appeared and at homocysteine. around at around 590 590 nm Withbutnm rapidly butCys, rapidlyafter decreased, the decreased,initial favouring addition favouring another of the nucleophile, bandanother at 564band a nm new at according emission564 nm to bandaccording the proposedappeared to intermolecularatthe around proposed 590 nmdisplacementintermolecular but rapidly shown displacement decreased, in Scheme shownfavouring 12 andin Scheme resultinganother 12 and inband the resulting formationat 564 in nmthe of formation29according. In case of to of29 . Hcy, theIn case proposed similar of Hcy, to intermolecularGSH,similar an to emission GSH, displacement an bandemission at 587 bandshown nm at appeared in587 Scheme nm appear but 12 it and wased resultingbut followed it was in byfollowed the a strongformation by decrease a strong of 29. inIndecrease thecase intensity, of in Hcy, the similarfollowingintensity, to GSH,following the same an emission reactivitythe same band asreactivity proposed at 587 asnm forproposed appear cysteine.ed for but cysteine. it was followed by a strong decrease in the intensity, following the same reactivity as proposed for cysteine. 2.2. Molecular Recognition (Non (Non-Covalent)‑Covalent) 2.2. Molecular Recognition (Non‑Covalent) A didifferentfferent approach approach for for the the recognition recognition by BODIPY by BODIPY probes probes can be can used be especially used especially for the detection for the ofdetection aminoA different acids of amino and approach smallacids peptides.and for small the For peptides.recognition example, For preciseby exam BOple,DIPY functional precise probes groups functional can canbe used begroups placed especially can in be the placed BODIPYfor the in detectionarchitecturethe BODIPY of amino soarchitecture that acids it will and so specifically thatsmall it peptides.will interactspecifically For inexama interact non-covalentple, precise in a non-covalent functional fashion with gr fashionoups the can target with be molecule,theplaced target in theeventuallymolecule, BODIPY eventually resulting architecture in resulting a changeso that it ofin will thea change spectralspecifically of profiles. th interacte spectral Few in reportsa profiles.non-covalent following Few fashionreports this mechanism withfollowing the target havethis molecule,beenmechanism published eventually have for been BODIPY resulting published probes in for a andBODIPYchange amino probesof acidthe derivatives,anspectrald amino profiles. acid and derivatives, it Few is possible reports and to followingit find is possible examples this to mechanismwithfind examples other natural have with been derivative other published natural such for derivative as BODIPY G-proteins. such probes as G-proteins.and amino acid derivatives, and it is possible to find examplesOne interesting with other work natural focused derivative on the rapid such detection as G-proteins. of aspartic acid and glutamic acid in water waswas One recently interesting reported work by Guria focused et onal. al. the[41]. [41 ].rapid In In their their detection approach, approach, of aspartic a aBODIPY BODIPY acid probeand probe glutamic was was designed designed acid in withwater with a wasaspecific specific recently architecture architecture reported bearing by bearing Guria a diethyl aet diethyl al. [41]. amino amino In their substituted substituted approach, moiety moietya BODIPY connected connected probe to was tothe the mesodesignedmeso position,position, with asa specificasshown shown inarchitecture inFigure Figure 2, 230 ,bearing.30 . a diethyl amino substituted moiety connected to the meso position, as shown in Figure 2, 30.

FigureFigure 2. 2.The The BODIPYBODIPY sensorsensor thatthat presents a photo-inducedphoto-induced electron electron transfer transfer (PET) (PET) process process in in water. water. Figure 2. The BODIPY sensor that presents a photo-induced electron transfer (PET) process in water.

Molecules 2020,, 25,, 4523x FOR PEER REVIEW 12 of 28

Because of the specific construction of the probe, a photo-induced electron transfer (PET) is observedBecause with of only the a specific very weak construction emission ofdetectable the probe, at 527 a photo-induced nm in water. Upon electron addition transfer of (PET)aspartic is observedacid (Asp) with or onlyglutamic a very acid weak (Glu) emission (0–600 detectable μM amino at 527acid, nm 3:97 in water. DMSO/H Upon2O, addition pH 6.5), of the aspartic emission acid (Asp)maximum or glutamic was recorded acid (Glu) to rapidly (0–600 µ increaseM amino as acid, a consequence 3:97 DMSO/ Hof2 O,the pH interaction 6.5), the emissionbetween maximumthe probe wasand recordedthe amino to acid, rapidly which increase is effectively as a consequence blocking ofthe the PET interaction process. betweenIn addition, the probethe absorbance and the amino was acid,strongly which influenced is effectively upon blocking addition the of PET the process.target molecule; In addition, the the main absorbance absorption was band strongly at 514 influenced nm was uponhypsochromically addition of theshifted target to molecule; 503 nm. theThe maintype absorptionof interaction band between at 514 nmthe wasprobe hypsochromically and the target shiftedmolecule to was 503 nm.ascribed The typeto be ofof interactionhydrogen bonding between with the probetheoretical and the (DFT) target and molecule NMR evidence; was ascribed indeed, to besignificant of hydrogen changes bonding in the with chemical theoretical shifts (DFT) of the and protons NMR in evidence; the pyridine indeed, moiety significant were detected. changes in the chemicalThe shiftshighly of selectivity the protons of in the the system pyridine for moiety Asp and were Glu detected. was demonstrated by comparing the behaviourThe highly with all selectivity the other of amino the system acids that, for Aspwhen and added Glu to was thedemonstrated probe, did not by significantly comparing alter the behaviourthe fluorescent with profile all the of other the aminoBODIPY. acids Because that, whenof those added specific to the behaviours, probe, did the not probe significantly was used alter for theintracellular fluorescent imaging profile and of theshowed BODIPY. good Because response of and those high specific stability behaviours, in the cellular the probe systems. was used for intracellularRecently, imaging Guler and and co-workers showed good presented response an and interesting high stability work inabout the cellulara BODIPY systems. probe based on a pillar[5]areneRecently, Guler structure and co-workerswith sensing presented ability for an asparagine. interesting The work probe about (31 a in BODIPY Figure 3) probe was basedobtained on aby pillar[5]arene click chemistry structure between with an sensing azido BODIPY ability for precur asparagine.sor and The a pillar[5]arene probe (31 in Figure having3) terminal was obtained alkynes by click[42]. chemistry between an azido BODIPY precursor and a pillar[5]arene having terminal alkynes [42].

Figure 3. The pillar [5]arene BODIPY sensor presented in Guler’s work.

The cycliccyclic structurestructure presents presents absorption absorption and and emission emission maxima, maxima, respectively, respectively, at 325, at 325, 505 505 and and 520 nm.520 Upon addition of thirty different amino acids and derivatives in a DMF/H O 1:1 mixture, the only nm. Upon addition of thirty different amino acids and derivatives in a DMF/H2 2O 1:1 mixture, the l appreciableonly appreciable changes changes were recorded were recorded in the emission in the emission spectrum spectrum when -asparagine when L-asparagine was used. Specifically,was used. theSpecifically, emission the intensity emission was intensity enhanced was and enhanced a slight and blue a slight shift ofblue 6 nmshift was of 6 recorded.nm was recorded. The type The of interactiontype of interaction between between the probe the and probe the sensed and the specie sens wased specie described was from described the authors from tothe be authors non-covalent to be withnon-covalent the formation with the of anformation inclusion of type an inclusion of complex. type The of complex. host-guest The nature host-guest of interaction nature of is interaction supported byis supported NMR experiments, by NMR whichexperiments, show how which all theshow protons how all of the protons amino acid of the are amino reasonably acid are upfield reasonably shifted becauseupfield shifted of the richbecause electron of the environment rich electron of environment the cavity. of the cavity. Kennedy et al. designed designed and and synthetized a a GD GDPP analogue integrating a BODIPY moiety ( 32) (Figure(Figure4 4)) which which presents presents a a fluorescence fluorescence emission emission with with maximum maximum at at 510 510 nm nm [ 19[19].].

Molecules 2020, 25, x FOR PEER REVIEW 12 of 28

Because of the specific construction of the probe, a photo-induced electron transfer (PET) is observed with only a very weak emission detectable at 527 nm in water. Upon addition of aspartic acid (Asp) or glutamic acid (Glu) (0–600 μM amino acid, 3:97 DMSO/H2O, pH 6.5), the emission maximum was recorded to rapidly increase as a consequence of the interaction between the probe and the amino acid, which is effectively blocking the PET process. In addition, the absorbance was strongly influenced upon addition of the target molecule; the main absorption band at 514 nm was hypsochromically shifted to 503 nm. The type of interaction between the probe and the target molecule was ascribed to be of hydrogen bonding with theoretical (DFT) and NMR evidence; indeed, significant changes in the chemical shifts of the protons in the pyridine moiety were detected. The highly selectivity of the system for Asp and Glu was demonstrated by comparing the behaviour with all the other amino acids that, when added to the probe, did not significantly alter the fluorescent profile of the BODIPY. Because of those specific behaviours, the probe was used for intracellular imaging and showed good response and high stability in the cellular systems. Recently, Guler and co-workers presented an interesting work about a BODIPY probe based on a pillar[5]arene structure with sensing ability for asparagine. The probe (31 in Figure 3) was obtained by click chemistry between an azido BODIPY precursor and a pillar[5]arene having terminal alkynes [42].

Figure 3. The pillar [5]arene BODIPY sensor presented in Guler’s work.

The cyclic structure presents absorption and emission maxima, respectively, at 325, 505 and 520 nm. Upon addition of thirty different amino acids and derivatives in a DMF/H2O 1:1 mixture, the only appreciable changes were recorded in the emission spectrum when L-asparagine was used. Specifically, the emission intensity was enhanced and a slight blue shift of 6 nm was recorded. The type of interaction between the probe and the sensed specie was described from the authors to be non-covalent with the formation of an inclusion type of complex. The host-guest nature of interaction is supported by NMR experiments, which show how all the protons of the amino acid are reasonably upfield shifted because of the rich electron environment of the cavity.

MoleculesKennedy2020, 25 ,et 4523 al. designed and synthetized a GDP analogue integrating a BODIPY moiety13 of(32 28) (Figure 4) which presents a fluorescence emission with maximum at 510 nm [19].

Molecules 2020, 25, x FOR PEER REVIEW 13 of 28

FigureFigure 4. 4. TheThe GDP–BODIPY GDP–BODIPY hybrid hybrid sensor sensor in investigatedvestigated in in Kennedy’s Kennedy’s work. work.

Interestingly,Interestingly, the the incubation incubation of of the the probe probe with with G-protein’s G-protein’s αα subunitssubunits in in a a Tris Tris buffer buffer (192 (192 mM mM 2 glycine,glycine, 1 1 mM mM EDTA, EDTA, 10 mM CaCl 2,, pH pH == 8.5)8.5) resulted resulted in in a a fluore fluorescencescence anisotropy, anisotropy, indicating indicating the the formationformation of of a a complex. complex. As As control, control, the the probe probe was was tested tested with with trypsin trypsin inhibitor inhibitor and and no no change change in in anisotropyanisotropy was was noticeable. AnotherAnother example example reporting reporting a a non-covalent non-covalent interaction interaction between between the the probe probe and and the the target target was was documenteddocumented in 20092009 byby I. I. Hamachi Hamachi and and co-workers co-workers [20 [20].]. In theirIn their work, work, they they developed developed a BODIPY-based a BODIPY- basedprobe probe able to able interact to interact and identifyand identify the Neurofibrillary the Neurofibrillary Tangles Tangles presents presents in patients in patients affected affected by theby theAlzheimer’s Alzheimer’s disease. disease. They They designed designed a a probe probe with with bipyridyl-type bipyridyl-type Zn(II)Zn(II) complexcomplex moieties, i.e., i.e., 3333, , whichwhich can easily interact with hy hyperphosphorylatedperphosphorylated tau tau proteins proteins and and that that presents absorption and and emissionemission bands bands at at 520 520 and and 547 547 nm ( Φ = 0.31),0.31), respectively respectively (see (see Figure Figure 55).).

Figure 5. BODIPY probe with bipyridyl-type Zn(II) complex moieties. Figure 5. BODIPY probe with bipyridyl-type Zn(II) complex moieties. Upon addition of the probe in HBS buffer (100 nM) to a suspension of p-Tau (8 µM, overexpressed Upon addition of the probe in HBS buffer (100 nM) to a suspension of p-Tau (8 μM, in Alzheimer’s disease) in HBS buffer containing 10% DMSO, an increase of intensity in the emission overexpressed in Alzheimer’s disease) in HBS buffer containing 10% DMSO, an increase of intensity was recorded and a dissociation constant of 7.5 µM was calculated. When the sensor was mixed in the emission was recorded and a dissociation constant of 7.5 μM was calculated. When the sensor with mono- or non-phosphorylated tau, no changes in the emission could be appreciated, showing was mixed with mono- or non-phosphorylated tau, no changes in the emission could be appreciated, a specificity of the probe for the hyperphosphorylated tau proteins. showing a specificity of the probe for the hyperphosphorylated tau proteins. 3. Porphyrin Probes 3. Porphyrin Probes The structure of porphyrin, namely [18] tetraphyrin(1.1.1.1), provides three types of modification sites:The eight structureβ positions, of porphyrin, four meso namelypositions [18]tetraphyrin(1.1.1.1), and the coordination cavityprovid (Figurees three6 ).types Porphyrin of modification hosts for sites:amino eight acid β derivatives positions, typicallyfour meso make positions useof and both the the coordination peripheral alterationscavity (Figure and 6). metal Porphyrin coordination, hosts forthe amino latter being acid ofderivatives key importance typically for make such applications.use of both Thethe mesoperipheral-substituents alterations typically and include metal coordination, the donors latter (e.g.,being hydroxyl of key importance groups) and for acceptors such applications. (e.g., methoxyl The meso groups)-substituents and bulky typically aromatic includerings (e.g., hydrogen naphthyl). bondFormation donors (e.g., of stablehydroxyl supramolecular groups) and complexesacceptors (e.g., is achieved methoxyl via groups) three-point and bulkyfixation, aromatic consisting rings of (e.g., coordination naphthyl). of aminoFormation acid ofN -terminusstable supramolecular to the metal centre,complexes as well is achieved as hydrogen via three-pointbonding and fixation, electrostatic, consisting hydrophobic of coordination or steric of interactions. amino acid N-terminus to the metal centre, as well as hydrogen bonding and electrostatic, hydrophobic or steric interactions.

Figure 6. Porphyrin modification sites.

Porphyrin’s N4 coordination cavity allows binding of numerous metal ions. A careful choice of metal centre allows coordination of axial ligands, for instance amines [43] or alcohols [44]. Such coordination causes slight perturbation to the optical properties, easily detected with UV/Vis

Molecules 2020, 25, x FOR PEER REVIEW 13 of 28

Figure 4. The GDP–BODIPY hybrid sensor investigated in Kennedy’s work.

Interestingly, the incubation of the probe with G-protein’s α subunits in a Tris buffer (192 mM glycine, 1 mM EDTA, 10 mM CaCl2, pH = 8.5) resulted in a fluorescence anisotropy, indicating the formation of a complex. As control, the probe was tested with trypsin inhibitor and no change in anisotropy was noticeable. Another example reporting a non-covalent interaction between the probe and the target was documented in 2009 by I. Hamachi and co-workers [20]. In their work, they developed a BODIPY- based probe able to interact and identify the Neurofibrillary Tangles presents in patients affected by the Alzheimer’s disease. They designed a probe with bipyridyl-type Zn(II) complex moieties, i.e., 33, which can easily interact with hyperphosphorylated tau proteins and that presents absorption and emission bands at 520 and 547 nm (Φ = 0.31), respectively (see Figure 5).

Figure 5. BODIPY probe with bipyridyl-type Zn(II) complex moieties.

Upon addition of the probe in HBS buffer (100 nM) to a suspension of p-Tau (8 μM, overexpressed in Alzheimer’s disease) in HBS buffer containing 10% DMSO, an increase of intensity in the emission was recorded and a dissociation constant of 7.5 μM was calculated. When the sensor was mixed with mono- or non-phosphorylated tau, no changes in the emission could be appreciated, showing a specificity of the probe for the hyperphosphorylated tau proteins.

3. Porphyrin Probes The structure of porphyrin, namely [18]tetraphyrin(1.1.1.1), provides three types of modification sites: eight β positions, four meso positions and the coordination cavity (Figure 6). Porphyrin hosts for amino acid derivatives typically make use of both the peripheral alterations and metal coordination, the latter being of key importance for such applications. The meso-substituents typically include hydrogen bond donors (e.g., hydroxyl groups) and acceptors (e.g., methoxyl groups) and bulky aromatic rings (e.g., naphthyl). Formation of stable supramolecular complexes is achieved via three-point fixation, consisting of coordination of amino acid N-terminus to the metal centre, as well Molecules 2020, 25, 4523 14 of 28 as hydrogen bonding and electrostatic, hydrophobic or steric interactions.

Figure 6. Porphyrin modification sites. Figure 6. Porphyrin modification sites.

Porphyrin’s N4 coordination cavity allows binding of numerous metal ions. A careful choice Porphyrin’s N4 coordination cavity allows binding of numerous metal ions. A careful choice of Moleculesof metal 2020 centre, 25, x FOR allows PEERcoordination REVIEW of axial ligands, for instance amines [43] or alcohols14 of [44 28]. metal centre allows coordination of axial ligands, for instance amines [43] or alcohols [44]. Such Such coordination causes slight perturbation to the optical properties, easily detected with coordination causes slight perturbation to the optical properties, easily detected with UV/Vis spectroscopy.UV/Vis spectroscopy. Examples of Examples sensors ofdescribed sensors in described this section in involve this section zinc(II) involve and ruthenium(II) zinc(II) and porphyrins. ruthenium(II) porphyrins. Binding of a chiral molecule to an achiral porphyrinporphyrin results in chirogenesis-process of transfer of chiral information from the chiral partner (guest) to achiral host [[45].45]. This leads to induction of supramolecular chiralitychirality with with characteristic characteristic strong strong Cotton Cotton effects, effects, carrying carrying information information about about the guest the guestabsolute absolute configuration configuration (Scheme (Scheme 15)[46]. 15) Formation [46]. Formation of supramolecular of supramolecular host-guest host-guest complexes complexes described describedin this paper in this is therefore paper is easilytherefore detected easily with detected circular with dichroism circular dichroism spectroscopy. spectroscopy.

Scheme 15. Schematic presentation of supramolecular chirogenesis in porphyrin-amino acid Scheme 15. Schematic presentation of supramolecular chirogenesis in porphyrin-amino acid host-guest systems. host-guest systems. 3.1. Monomeric Hosts 3.1. Monomeric Hosts 3.1.1. Early Reports 3.1.1. Early Reports The use of porphyrins as probes for chiral recognition of amino acids dates back to the early 1990s,The when use Ogoshi of porphyrins and co-workers as probes published for chiral a series recognition of papers of describingamino acids the dates design back and to application the early 1990s,of several when hosts Ogoshi employing and co-workers porphyrin zincpublished complexes a series with peripheralof papers substituentsdescribing the tailored design to bindand applicationα-aminoester of guests. several hosts employing porphyrin zinc complexes with peripheral substituents tailoredThree to bind hosts α with-aminoester various bindingguests. capability were examined (Figure7) as zinc(II) complexes open for coordinationThree hosts ofwith a guest various and binding an auxiliary capability binding were site: examined a hydrogen (Figure bond 7) donoras zinc(II) (34) complexes and a hydrogen open forbond coordination acceptor (35 of)[ a47 guest]. Thus, and thean auxili constructionary binding of macrocyclic site: a hydrogen host wasbond predefined donor (34) forand formation a hydrogen of bonda doubly acceptor bound (35 system) [47]. Thus, with nitrogen the construction coordinated of macrocyclic to zinc(II) centre host was and predefined a hydrogen for bond formation interaction of a doublyof a C-terminus bound system of an with amino nitrogen acid. The coordinated UV/Vis titration to zinc(II) with centre LeuOMe and a revealedhydrogen formation bond interaction of a 1:1 ofcomplex a C-terminus34 LeuOMe of an upon amino addition acid. ofThe large UV/Vis excess titration of the guest with (FigureLeuOMe7) manifested revealed formation by the Soret of banda 1:1 · complexbathochromic 34·LeuOMe shift treated upon asaddition diagnostic of large of formation excess of ofthe host-guest guest (Figure system. 7) manifested1H NMR experimentsby the Soret band bathochromic shift treated as diagnostic of formation of host-guest system. 1H NMR experiments consistently support the participation of hydrogen bonding in host-guest formation. Titration of 34 with a solution of L-LeuOMe results in downfield shift of the naphthyl hydroxy proton signal (marked red in Figure 7) from δ ~5.2 ppm to δ ~6.4 ppm, indicating the presence of hydrogen bond incorporating the hydroxyl group. Further experiments performed for methyl esters of valine, phenylalanine, tryptophan, asparagine or glutamic acid showed that bulkier side chains of amino acids positively influence stability of final complexes. In addition, the presence of aromatic substituents increases the stability of final host–guest systems obtained for 35 where hydrogen bond character is inverted while carboxylic groups has no influence. The association constants for 34 (1.7 × 103–1.1 × 104 M−1) are 3.8–40.0 times larger than those obtained for 35. These observations led to the conclusion that hydrogen bonds are formed between naphthyl hydroxyl and aminoester carbonyl groups (Figure 7).

Molecules 2020, 25, 4523 15 of 28 consistently support the participation of hydrogen bonding in host-guest formation. Titration of 34 with a solution of l-LeuOMe results in downfield shift of the naphthyl hydroxy proton signal (marked red in Figure7) from δ ~5.2 ppm to δ ~6.4 ppm, indicating the presence of hydrogen bond incorporating the hydroxyl group. Further experiments performed for methyl esters of valine, phenylalanine, tryptophan, asparagine or glutamic acid showed that bulkier side chains of amino acids positively influence stability of final complexes. In addition, the presence of aromatic substituents increases the stability of final host–guest systems obtained for 35 where hydrogen bond character is inverted while carboxylic groups has no influence. The association constants for 34 (1.7 103–1.1 104 M 1) × × − are 3.8–40.0 times larger than those obtained for 35. These observations led to the conclusion that Molecules 2020, 25, x FOR PEER REVIEW 15 of 28 hydrogenMolecules 2020 bonds, 25, x FOR are PEER formed REVIEW between naphthyl hydroxyl and aminoester carbonyl groups (Figure15 of7 ).28

Figure 7. Ogoshi’sOgoshi’s mesomeso-naphthyl-naphthyl sensorssensors forfor aminamin aminoo acid acid methyl methyl esters. Ogoshi and co-workers designed also a condensed α,α,β,β-atropoisomer of Ogoshi and co-workers designed also a condensed α,,α,,β,,β-atropoisomer-atropoisomer ofof meso-tetra(-tetra(oo-- meso-tetra(o-amino)phenyl porphyrin with un- and substituted isophthaloyl chlorides to yield doubly amino)phenyl porphyrin with un- and substituted isophthaloyl chlorides to yield doubly bridged bridged chirality probes 36–39 (Figure8). Both optically pure enantiomers were examined, and they chiralitychirality probesprobes 36–39 (Figure(Figure 8).8). BothBoth opticallyoptically purepure enantiomersenantiomers werewere examined,examined, andand theythey exhibitedexhibited 1 1 significantexhibited significantenantioselectivity, enantioselectivity, from 4.1 for from al 4.1anine for to alanine 7.5 for to valine 7.5 for methyl valine methylester. Upon ester. Upon1H NMRH significant enantioselectivity, from 4.1 for alanine to1 7.5 for valine methyl ester. Upon H NMR NMR monitored coordination experiment, all1 guest H signals shift upfield to negative δ values, monitored coordination experiment, all guest 1H signals shift upfield to negative δ values,values, duedue toto thethe guestdue to molecule the guest being molecule placed being in porphyrin’s placed in porphyrin’s magnetic anisotropy magnetic anisotropycone [48]. In cone the case [48]. of In L the-ValOMe, case of guest molecule being placed in porphyrin’s magnetic anisotropy cone [48].α In the case of L-ValOMe, thel-ValOMe, most dramatic the most shift dramatic difference shift diis ffobservederence is for observed NH2 and for NHCαH2 protonsand C H (Δδ protons = −6.04 (∆ ppmδ = and6.04 Δδ ppm = the most dramatic shift difference is observed for NH2 and CαH protons (Δδ = −6.04 ppm− and Δδ = −and6.21∆ ppm,δ = 6.21respectively) ppm, respectively) as they appear as they at δ appear = −4.57 at ppmδ = and4.57 δppm = −2.90 and ppmδ = in2.90 the complex, ppm in the indicating complex, −6.21 ppm,− respectively) as they appear at δ = −4.57 ppm− and δ = −2.90 ppm −in the complex, indicating indicating that both groups of protons come in close contact with the porphyrin plane [49]. thatthat bothboth groupsgroups ofof protonsprotons comecome inin clclose contact with the porphyrin plane [49].

Figure 8. DoublyDoubly bridged bridged chirality chirality probes. probes.

The O‑‑nitro substituents play an important role in the recognition process. It was found that (-)-(-)- 35 bindsbinds thethe samesame guestsguests withwith associationassociation constantsconstants 1212 andand 2121 timestimes largerlarger thanthan 37 andand 38,, indicatingindicating thatthat thethe hydrogenhydrogen bondbond donatingdonating abilityability ofof thethe amideamide groupgroup remainingremaining inin thethe orthoortho positionposition toto thethe nitro substituent is increased by the electronic effect of NO2 group. nitro substituent is increased by the electronic effect of NO2 group.

3.1.2. Achiral Hosts Commercially available ruthenium(II) porphyrin complex 40,, withwith aa strongstrong LewisLewis acidacid character,character, waswas employedemployed asas aa chiralitychirality probeprobe forfor aminoamino acidacid estersesters andand hydrazideshydrazides [49].[49]. TheThe strongstrong binding of amines to the Ru centre is reflected in the high association constant determined for coordination of L-ValOMe as Kass = 1.7 × 107 M−1. The difference in coordination abilities to Ru and Zn coordination of L-ValOMe as Kass = 1.7 × 107 M−1. The difference in coordination abilities to Ru and Zn centrescentres isis reflectedreflected byby thethe bidingbiding constantconstant forfor 40,, whichwhich isis three-foldthree-fold higherhigher thanthan thosethose ofof Zn-basedZn-based

Molecules 2020, 25, 4523 16 of 28

The O-nitro substituents play an important role in the recognition process. It was found that (-)-35 binds the same guests with association constants 12 and 21 times larger than 37 and 38, indicating that the hydrogen bond donating ability of the amide group remaining in the ortho position to the nitro substituent is increased by the electronic effect of NO2 group.

3.1.2. Achiral Hosts Commercially available ruthenium(II) porphyrin complex 40, with a strong Lewis acid character, was employed as a chirality probe for amino acid esters and hydrazides [49]. The strong binding of amines to the Ru centre is reflected in the high association constant determined for coordination 7 1 ofMoleculesl-ValOMe 2020, 25 as, x KFORass PEER= 1.7 REVIEW10 M . The difference in coordination abilities to Ru and Zn centres16 of 28 × − is reflected by the biding constant for 40, which is three-fold higher than those of Zn-based probes, suchprobes, as 33such. Amino as 33 acid. Amino hydrazides acid hydrazides were obtained were uponobtained hydrazinolysis upon hydrazinolysis of corresponding of corresponding amino acid 1 methylamino acid esters, methyl and their esters, coordination and their coordination to 40 was monitored to 40 was with monitored1H NMR. with Exposing H NMR. such Exposing hydrazide such to 1hydrazide eq. of 40 yieldsto 1 eq. two of 40 1:1 yields complexes, two 1:141 complexes,and 42, diff 41ering and by 42 the, differing donor forby coordinationthe donor for tocoordination ruthenium. Furtherto ruthenium. addition Further to the addition macrocyclic to the host macrocyclic leads to formation host leads of sandwichto formation complex of sandwich43, in which complex the two 43, terminalin which the amino two groups terminal coordinate amino groups to ruthenium coordinate centres to ruthenium (Figure 9centres). In such (Figure magnetic 9). In such environment, magnetic 1 allenvironment,1H signals of all the H guest signals move of the to negative guest move values to negati of chemicalve values shifts of (from chemicalδ = shifts0.5 ppm (from to δ δ= = −6.40.5 ppm). ppm − − Protonsto δ = −6.4 attached ppm). Protons to the two attached terminal to the amino two groupsterminal show amino substantial groups show upfield substantial shifting toupfieldδ = 2.8shifting ppm α − andto δ =δ −=2.83.8 ppm ppm and (hydrazide δ = −3.8 ppm NH (hydrazide) and δ = NH5.42 ppm) and and δ = δ−5.4= ppm6.4 ppm and (Cδ =α -NH−6.4 ppm). (C -NH2). − 2 − − 2

Figure 9. Stoichiometry-dependent host-guest complex formation. Figure 9. Stoichiometry-dependent host-guest complex formation. The presence of chiral guest results in formation of a sandwich complex that exhibits a distinct The presence of chiral guest results in formation of a sandwich complex that exhibits a distinct CD effect in the Soret region (405 and 412 nm). When l isomers were used (valine and phenylalanine), CD effect in the Soret region (405 and 412 nm). When L isomers were used (valine and phenylalanine), the shorter wavelength band was negative and the other positive, while with d-phenylalanine the shorter wavelength band was negative and the other positive, while with D-phenylalanine derivative the CD image was reversed. Some other commercially available ruthenium(II) porphyrins derivative the CD image was reversed. Some other commercially available ruthenium(II) porphyrins ([RuII(TPP)CO], [RuII(TMP)CO] and [RuII(F20-TPP)CO]) were tested as potential chirality probes and ([RuII(TPP)CO], [RuII(TMP)CO] and [RuII(F20-TPP)CO]) were tested as potential chirality probes and gave corresponding results [49]. gave corresponding results [49]. 3.1.3. Chiral Hosts 3.1.3. Chiral Hosts Continuing their research on thermodynamic aspects of host–guest interactions between porphyrinsContinuing and aminotheir research acid esters, on Ogoshithermodynamic and co-workers aspects designedof host–guest a set ofinteractions chiral hosts between based onporphyrins a zinc(II) and coordination amino acid centreesters, andOgoshi auxiliary and co-wor hydroxylkers groupsdesigned used a set as of hydrogen chiral hosts bond based donors on a (Figurezinc(II) coordination10). Methyl cent acetatere and groups auxiliary were hydroxyl introduced groups for aused steric as repulsionhydrogen andbond hydrogen donors (Figure bond 10). Methyl acetate groups were introduced for a steric repulsion and hydrogen bond acceptance [50]. While the combination of coordination and hydrogen bonding in the positive isomer gave results corresponding to those reported previously for 35 [47], the presence of ester groups in 44 resulted in slightly repulsive behaviour towards the side chain of L-aminoesters. The effect was even more pronounced in a corresponding series of D-aminoesters. A significant exception was observed for D- SerOBz where the carbonyl group of methyl acetate acted as a hydrogen bond acceptor for serine side chain hydroxyl. Comparing association constants for binding of L- and D-aminoesters, it was concluded that (+)-44 shows significant enantioselectivity for L-aminoesters, except for D-SerOBz, where the enantioselectivity is reversed, probably due to the noteworthy contribution from a second hydrogen bond. The extent of steric repulsion towards L-aminoesters was independent of hydrogen bonding, but in the case of D-aminoesters the repulsion was larger in complexes of 44 (complexes with two-point fixation, i.e., coordination and hydrogen bonding) than 45, where no hydrogen

Molecules 2020, 25, 4523 17 of 28

acceptance [50]. While the combination of coordination and hydrogen bonding in the positive isomer gave results corresponding to those reported previously for 35 [47], the presence of ester groups in 44 resulted in slightly repulsive behaviour towards the side chain of l-aminoesters. The effect was even more pronounced in a corresponding series of d-aminoesters. A significant exception was observed for d-SerOBz where the carbonyl group of methyl acetate acted as a hydrogen bond acceptor for serine side chain hydroxyl. Comparing association constants for binding of l- and d-aminoesters, it was concluded that (+)-44 shows significant enantioselectivity for l-aminoesters, except for d-SerOBz, where the enantioselectivity is reversed, probably due to the noteworthy contribution from a second hydrogen bond. The extent of steric repulsion towards l-aminoesters was independent of hydrogen bonding, but in the case of d-aminoesters the repulsion was larger in complexes of 44 (complexes with Molecules 2020,, 25,, xx FORFOR PEERPEER REVIEWREVIEW 17 of 28 two-point fixation, i.e., coordination and hydrogen bonding) than 45, where no hydrogen bonding bondingoccurs (one-point occurs (one-point fixation by fixation coordination). by coordination). When d isomers When were D isomersisomers used, the werewere interaction used,used, thethe energies interactioninteraction were energiessensitive were to the sensitive character to (size the character and functional (size and groups) functional of the groups) guest. of the guest.

FigureFigure 10. 10. Three-pointThree-point fixation fixation of of aminoesters aminoesters inin thethe cavitycavity ofof porphyrinporphyrin host.host.

2-Hydroxynaphthyl2-Hydroxynaphthyl substituentsubstituent was was also also employed employ ined the in design the design of a chiral of ABCD-typea chiral ABCD-type porphyrin porphyrinreported by reported Yang et al.by StericYang hindrance et al. Steric of 2-hydroxylhindrance groupof 2-hydroxyl and 9-hydrogen group and atom 9-hydrogen prevents rotation atom 1 1 l preventsalong the rotation 5–5 bond along (Figure the 5–5 11).1 bondbond Enantioselectivity (Figure(Figure 11).11). EnEn ofantioselectivity(R)-48 was 1.6, of favouring (R)-48 waswas-PheOMe 1.6,1.6, favouringfavouring over itsL- PheOMechiral counterpart. over its chiral counterpart.

FigureFigure 11.11.Supramolecular Supramolecular complexes complexes of isomers of isomers of ABCD-type of ABCD-type porphyrin porphyrin sensor and sensorl-phenylalanine and L- phenylalaninemethyl ester. methyl ester.

TheThe phenomenon of chiral discrimination of porphyrin-aminoester pairs pairs was was employed for for NMR-assistedNMR-assisted configuration configuration assignment, assignment, where where phenylalanine phenylalanine methyl methyl esters esters were were used used as as chiral solvatingsolvating agents.agents. Formation Formation of host–guestof host–guest complexes complexes with two-point with two-point fixation via fixation NH2-Zn via coordination NH2-Zn coordinationand C=O HO and hydrogen C=O···HO bonding hydrogen led to bonding decrease inled freedom to decreasedecrease of motion inin freedomfreedom of the host ofof molecules,motionmotion ofof resultingthethe hosthost ··· molecules,in resolution resulting of NMR in signals resolution in thus of NMR obtained signals diastereoisomeric in thus obtained complexes diastereoisomeric [51]. complexes [51].

3.2. Dimeric Hosts Due to their 18π electronelectron circuitcircuit andand abilityability toto accommodateaccommodate variousvarious metalmetal ionsions insideinside theirtheir cavity, porphyrins have been used asas anan attractiveattractive buildingbuilding blockblock forfor molecularmolecular tweezerstweezers [52].[52]. AA molecular tweezer is a host molecule consisting of two, usually flat and aromatic subunits, designed to “recognise” specific guest molecules by means of metal coordination, π–π interactions,interactions, hydrogenhydrogen bonding, hydrophobic interactions or van der Waals forces. Such subunits are connected via a linker, which can be a spacer of greater or smaller flexibility. Since the first synthesis of a caffeine dimer with diyne linker was reported in 1978 by Chen and Whitlock [53], many molecular tweezers were designed to serve various purposes. Zinc complexes of porphyrins tend to bindbind neutralneutral ligandsligands through nitrogen, oxygen, phosphorus and sulphur, formingforming five-coordinatedfive-coordinated 1:11:1 adductsadducts [54].[54]. ThisThis property has been widely exploited in porphyrin tweezer applications, including those designed for chiral recognition.

Molecules 2020, 25, 4523 18 of 28

3.2. Dimeric Hosts Due to their 18π electron circuit and ability to accommodate various metal ions inside their cavity, porphyrins have been used as an attractive building block for molecular tweezers [52]. A molecular tweezer is a host molecule consisting of two, usually flat and aromatic subunits, designed to “recognise” specific guest molecules by means of metal coordination, π–π interactions, hydrogen bonding, hydrophobic interactions or van der Waals forces. Such subunits are connected via a linker, which can be a spacer of greater or smaller flexibility. Since the first synthesis of a caffeine dimer with diyne linker was reported in 1978 by Chen and Whitlock [53], many molecular tweezers were designed to serve various purposes. Zinc complexes of porphyrins tend to bind neutral ligands through nitrogen, oxygen,Molecules 2020 phosphorus, 25, x FOR PEER and sulphur,REVIEW forming five-coordinated 1:1 adducts [54]. This property has18 been of 28 widely exploited in porphyrin tweezer applications, including those designed for chiral recognition. In the the early early 2000s 2000s Borovkov Borovkov et et al. al. reported reported a chirality a chirality induction induction in achiral in achiral ethane-bridged ethane-bridged syn- synbis-porphyrins-bis-porphyrins 49–4950– via50 viaan aninteraction interaction with with chiral chiral amines amines and and amino amino acids. acids. Based Based on on the the reported dependence ofof CD CD signal signal sign sign and and intensity intensity on the on steric the bulksteric of bulk aminoester of aminoester side chain, side they chain, suggested they thatsuggested coordination that coordination of the enantiopure of the enantiopure ligand induced ligand such induced a transformation, such a transformation, where the achiralwhere synthe conformerachiral syn 49conformer(Figure 12 49) e(Figureffectively 12) transforms effectively intotransforms a chiral into variant a chiral49 (l variant-AlaOMe) 49·(.L-AlaOMe) In a subsequent2. In a · 2 study,subsequent the mechanism study, the of mechanism chirality induction of chirality was investigatedinduction was and investigated tweezers with and one tweezers or two coordinating with one or centrestwo coordinating were employed centres (Figure were employed12)[55]. (Figure 12) [55].

Figure 12. Achiral ethane-bridged syn-bis-porphyrins and their interactioninteraction with aminoester guests. In nonpolar solvents, 49 adapts a cofacial, syn conformation, due to strong π–π interactions In nonpolar solvents, 49 adapts a cofacial, syn conformation, due to strong π–π interactions between the two porphyrin rings. Depending on the number of coordination sites, two different between the two porphyrin rings. Depending on the number of coordination sites, two different anti- anti-conformers were observed: 1:2 for two zinc(II) centres and 1:1 for single coordination of zinc(II) as conformers were observed: 1:2 for two zinc(II) centres and 1:1 for single coordination of zinc(II) as documented spetroscopically. documented spetroscopically. In UV/Vis, host–guest complexes of 49 and amino acid esters show bathochromic shift of Soret In UV/Vis, host–guest complexes of 49 and amino acid esters show bathochromic shift of Soret band, as well as its splitting (at 435–437 and 424–426 nm), and a set of two Q bands, among which band, as well as its splitting (at 435–437 and 424–426 nm), and a set of two Q bands, among which the the higher energy one is significantly more intense than the other. In CD, these systems show strong higher energy one is significantly more intense than the other. In CD, these systems show strong bisignate couplets in the Soret region—with maxima corresponding to those observed in UV/Vis—and bisignate couplets in the Soret region—with maxima corresponding to those observed in UV/Vis— an additional blue-shifted lower intensity Cotton effect. and an additional blue-shifted lower intensity Cotton effect. The factors influencing the handedness of the screw are as follows: right-handed screw is formed The factors influencing the handedness of the screw are as follows: right-handed screw is formed when the C-terminus substituent is bulkier than amino acid side chain; in the opposite relation, when the C-terminus substituent is bulkier than amino acid side chain; in the opposite relation, such such arrangement is unfavourable and left-handed screw is induced. Therefore, it was found that arrangement is unfavourable and left-handed screw is induced. Therefore, it was found that the CD the CD response depends not only on the absolute configuration of amino acid guest, but also on response depends not only on the absolute configuration of amino acid guest, but also on the mutual the mutual steric relation between the Cα substituents. Thus, the Cotton effects in the complex only steric relation between the Cα substituents. Thus, the Cotton effects in the complex only correspond correspond to the guest absolute configuration when the side chain is less bulky than the COOR group. to the guest absolute configuration when the side chain is less bulky than the COOR group. Among L isomers, positive chirality corresponds to AlaOMe, IleOMe, ValOMe, ProOMe and ProOBn and negative sign was observed in the case of LeuOMe, LeuOBn, PheOMe, PheOBn, PhgOMe, TyrOBn, Tyr(Bn)OMe, Glu(OMe)OMe, GlnOtBu, CysOMe, Lys(Z)OMe and TrpOMe. Investigation of a 1:1 host-guest complex 50·L-AlaOMe (Figure 12) provided insight into the mechanism of chirality induction in such supramolecular complexes. It was demonstrated that steric interaction of 3- and 7′-ethyl groups in 49 and 50 with aminoester guests is the driving force behind chirogenesis (Figure 12), as it locks guest molecule in position. It was also found that the intensity of induced Cotton effects depends on the number of possible chirality-generating conformations, which increases with the number of accommodated aminoester molecules. Moreover, 50 is a suitable host for chiral recognition of bidentate guests, i.e., chiral diamines or amino alcohols, which form 1:1 CD- silent complexes with 49. For example, a 1:1 complex of 49 and 1,2-DACH (1,2-diaminocyclohexane) is so strong, that 1:2 cannot be formed even upon addition of large excess of ligand (Kassoc > 107 M−1).

Molecules 2020, 25, 4523 19 of 28

Among l isomers, positive chirality corresponds to AlaOMe, IleOMe, ValOMe, ProOMe and ProOBn and negative sign was observed in the case of LeuOMe, LeuOBn, PheOMe, PheOBn, PhgOMe, TyrOBn, Tyr(Bn)OMe, Glu(OMe)OMe, GlnOtBu, CysOMe, Lys(Z)OMe and TrpOMe. Investigation of a 1:1 host-guest complex 50 l-AlaOMe (Figure 12) provided insight into the · mechanism of chirality induction in such supramolecular complexes. It was demonstrated that steric interaction of 3- and 70-ethyl groups in 49 and 50 with aminoester guests is the driving force behind chirogenesis (Figure 12), as it locks guest molecule in position. It was also found that the intensity of induced Cotton effects depends on the number of possible chirality-generating conformations, which increases with the number of accommodated aminoester molecules. Moreover, 50 is a suitable host for chiral recognition of bidentate guests, i.e., chiral diamines or amino alcohols, which form 1:1 MoleculesCD-silent 2020 complexes, 25, x FOR withPEER 49REVIEW. For example, a 1:1 complex of 49 and 1,2-DACH (1,2-diaminocyclohexane)19 of 28 7 1 is so strong, that 1:2 cannot be formed even upon addition of large excess of ligand (Kassoc > 10 M− ). Jiang etet al.al. designed designed three three achiral achiral Zn(II) Zn(II) triphenylporphyrin triphenylporphyrin dimers dimers with amidewith amide linkers linkers of various of variousrigidity: rigidity:51–53 for 51– the53 for purpose the purpose of elucidating of elucidating the mechanism the mechanism of chiral of chiral recognition recognition in systems in systems with withtwo pointtwo point fixation. fixation. First, First, oxalic oxalic amide-linked amide-linked tweezer tweezer51 51was was investigated investigated as as a a hosthost forfor nonpolar amino acid ethyl esters. Crystal Crystal structure structure of of a 1:2 complex of 51 and rac-PheOEt was solved and the following structural features were brought to light: With With a a linker of of this flexibility, flexibility, the dimer adapts an anti-configuration-configuration in in the complex, and both ligand molecules coordinate to zinc from inside the tweezer cavity. Contrary Contrary to to prior prior reports, reports, it it was found that in 5151·(lL-PheOEt)(-PheOEt)(dD-PheOEt) hydrogen bonds are formed between aminoester NH2 and linker carbonyl group· (Figure 13). For comparison, bonds are formed between aminoester NH2 and linker carbonyl group (Figure 13). For comparison, in their their account account on on mesomeso-naphthyl-naphthyl probe probe 3434, Ogoshi, Ogoshi and and co-workers co-workers sugg suggestedested that that the naphthyl the naphthyl OH groupOH group acts as acts a hydrogen as a hydrogen bond bonddonor, donor, and amino and amino acid carbonyl acid carbonyl group oxygen groupoxygen is the acceptor is the acceptor (Figure (Figure7) [48]. 7)[48].

Figure 13. Porphyrin tweezer with oxalic amide linker and its 1:2 complex with an aminoester guest. Figure 13. Porphyrin tweezer with oxalic amide linker and its 1:2 complex with an aminoester guest. Formation of 1:2 complexes with 51 was documented by UV/Vis titration with solutions of amino Formation of 1:2 complexes with 51 was documented by UV/Vis titration with solutions of amino acid ethyl esters. Two sets of isosbestic points were detected at 426 and 424 nm. K2 was reported as acid ethyl esters. Two sets of isosbestic points were detected at 426 and4 4241 nm. K2 was reported4 as1 significantly lower than K1 for all guests employed (K1: 2.0–7.2 10 M− ;K2: 1.0–1.2 10 M− ), × 4 −1 × 4 −1 significantlyconsistently withlower morethan diK1ffi forcult all biding guests ofemployed the second (K1: guest2.0–7.2 due × 10 to M the; diKff2: erent1.0–1.2 coordination × 10 M ), consistentlygeometry [56 with]. The more intense difficult CD signalsbiding of 51the( lseco-LeuOEt)nd guestcomplexes due to the were different diagnostic coordination of guest geometry absolute · 2 [56].configuration The intense as the CD accommodation signals of 51·( ofL-LeuOEt) bulky guests2 complexes (e.g., LeuOEt) were diagnostic results in inductionof guest ofabsolute strong configurationpositive bisignate as the Cotton accommodation effect in the Soretof bulky region. guests The (e.g., positive LeuOEt) Cotton results effects in induced induction in host-guest of strong positivesystems indicatebisignate that Cotton the tweezereffect in adaptsthe Soret a clockwise region. The twist. positive In such Cotton conformation, effects induced the aminoester in host-guest side systemschain faces indicate outside that of the the tweezer intraporphyrin adapts a cavity—the clockwise twist. opposite In such conformer conformation, would the not aminoester be favoured side for chainsteric reasonsfaces outside (Figure of 13the). intraporphyrin cavity—the opposite conformer would not be favoured for stericIn reasons the efforts (Figure to provide 13). a more sensitive chirality probe, which would accommodate chiral ligands withIn higher the efforts CD amplitude to provide values, a more the groupsensitive designed chirality a bis probe,-porphyrin which tweezer would with accommodate slightly less chiral rigid ligandslinker, that with is higherm-phthalic CD amplitude diamide (Figure values, 14 the, 52 group). designed a bis-porphyrin tweezer with slightly less rigid linker, that is m-phthalic diamide (Figure 14, 52).

Figure 14. Bis-porphyrin tweezers with m-phthalic diamide and malonamide linkers.

Molecules 2020, 25, x FOR PEER REVIEW 19 of 28

Jiang et al. designed three achiral Zn(II) triphenylporphyrin dimers with amide linkers of various rigidity: 51–53 for the purpose of elucidating the mechanism of chiral recognition in systems with two point fixation. First, oxalic amide-linked tweezer 51 was investigated as a host for nonpolar amino acid ethyl esters. Crystal structure of a 1:2 complex of 51 and rac-PheOEt was solved and the following structural features were brought to light: With a linker of this flexibility, the dimer adapts an anti-configuration in the complex, and both ligand molecules coordinate to zinc from inside the tweezer cavity. Contrary to prior reports, it was found that in 51·(L-PheOEt)(D-PheOEt) hydrogen bonds are formed between aminoester NH2 and linker carbonyl group (Figure 13). For comparison, in their account on meso-naphthyl probe 34, Ogoshi and co-workers suggested that the naphthyl OH group acts as a hydrogen bond donor, and amino acid carbonyl group oxygen is the acceptor (Figure 7) [48].

Figure 13. Porphyrin tweezer with oxalic amide linker and its 1:2 complex with an aminoester guest.

Formation of 1:2 complexes with 51 was documented by UV/Vis titration with solutions of amino acid ethyl esters. Two sets of isosbestic points were detected at 426 and 424 nm. K2 was reported as significantly lower than K1 for all guests employed (K1: 2.0–7.2 × 104 M−1; K2: 1.0–1.2 × 104 M−1), consistently with more difficult biding of the second guest due to the different coordination geometry [56]. The intense CD signals of 51·(L-LeuOEt)2 complexes were diagnostic of guest absolute configuration as the accommodation of bulky guests (e.g., LeuOEt) results in induction of strong positive bisignate Cotton effect in the Soret region. The positive Cotton effects induced in host-guest systems indicate that the tweezer adapts a clockwise twist. In such conformation, the aminoester side chain faces outside of the intraporphyrin cavity—the opposite conformer would not be favoured for steric reasons (Figure 13). In the efforts to provide a more sensitive chirality probe, which would accommodate chiral ligands with higher CD amplitude values, the group designed a bis-porphyrin tweezer with slightly lessMolecules rigid2020 linker,, 25, 4523 that is m-phthalic diamide (Figure 14, 52). 20 of 28

Figure 14. Bis-porphyrin tweezers with m-phthalic diamide and malonamide linkers. Figure 14. Bis-porphyrin tweezers with m-phthalic diamide and malonamide linkers. The major feature of this tweezer is its aggregation in solution. It was found that even at 7 concentrations as low as 10− M, an aggregated form remains in equilibrium with single molecules. The nature of such aggregates was elucidated by means of UV/Vis spectroscopy—52 shows a Soret band at 418 nm and its shoulder at 427 nm, which increases the intensity at higher concentrations. This shoulder was assigned to a form in which Zn centres are five-coordinated, which is manifested by a bathochromic shift in the Soret region [57]. Host-guest complexes obtained with 52 and a series of nonpolar amino acid esters, including Ala, Val, Leu, Phe and Phg, exhibit bisignate Cotton effects in the Soret region with amplitudes around 10 times larger than those reported earlier for their oxalyl amide linker [47,56]. The signs of CD responses obtained for guests of the same handedness were also opposite to those reported previously. It was concluded that different mechanisms govern formation of host-guest complexes of those two sensors. Indeed, based on a comparative study with an aminoalcohol (α-leucinol), it was found that, in 52, hydrogen bonds are formed between linker NH as a donor and guest carbonyl oxygen as an acceptor. Similar results were obtained for an even more flexible malonamide-linked bis-porphyrin tweezer 53 (Figure 14)[58]. Host-guest complexes of this sensor and nonpolar amino acid ethyl esters exhibit CD features closely related to those obtained with a m-phthalic diamide linked tweezer, namely the sign of induced Cotton effects and their amplitude. Positive sign of induced Cotton effects indicates that tweezers 52 and 53 adapt into a right-handed screw. Hence, it was concluded that chiral recognition is governed by the same principles in those two types of chirality sensors [49,58]. A set of significantly more rigid bis-porphyrin tweezers was reported by Ogoshi and co-workers (Figures 15 and 16)[59]. Porphyrin subunits were connected via chiral 2,20-dihydroxy- (54) and 2,20-dimethoxy-1,10-binaphthyl (55) as well as achiral 3,30-biphenyl (56) and 5,50-dimethoxycarbonyl- 3,30-biphenyl (57) bridges. Such sensors were applied in chiral recognition of lysine derivatives (Figure 15) and cystine diesters (Figure 16), manifested by induction of strong CD response in the Soret region of the host. The bisignate Cotton effects were diagnostic of guest absolute configuration. Association constants for formation of 1:1 complexes were determined by means of UV/Vis spectroscopic titration and remained in the ranges of 1.1 104–1.6 105 and 9.0 104–2.4 106 M 1, for binaphthyl- × × × × − and biphenyl-bridged hosts, respectively. Molecules 2020, 25, x FOR PEER REVIEW 20 of 28

The major feature of this tweezer is its aggregation in solution. It was found that even at concentrations as low as 10−7 M, an aggregated form remains in equilibrium with single molecules. The nature of such aggregates was elucidated by means of UV/Vis spectroscopy—52 shows a Soret band at 418 nm and its shoulder at 427 nm, which increases the intensity at higher concentrations. This shoulder was assigned to a form in which Zn centres are five-coordinated, which is manifested by a bathochromic shift in the Soret region [57]. Host-guest complexes obtained with 52 and a series of nonpolar amino acid esters, including Ala, Val, Leu, Phe and Phg, exhibit bisignate Cotton effects in the Soret region with amplitudes around 10 times larger than those reported earlier for their oxalyl amide linker [47,56]. The signs of CD responses obtained for guests of the same handedness were also opposite to those reported previously. It was concluded that different mechanisms govern formation of host-guest complexes of those two sensors. Indeed, based on a comparative study with an aminoalcohol (α-leucinol), it was found that, in 52, hydrogen bonds are formed between linker NH as a donor and guest carbonyl oxygen as an acceptor. Similar results were obtained for an even more flexible malonamide-linked bis-porphyrin tweezer 53 (Figure 14) [58]. Host-guest complexes of this sensor and nonpolar amino acid ethyl esters exhibit CD features closely related to those obtained with a m-phthalic diamide linked tweezer, namely the sign of induced Cotton effects and their amplitude. Positive sign of induced Cotton effects indicates that tweezers 52 and 53 adapt into a right-handed screw. Hence, it was concluded that chiral recognition is governed by the same principles in those two types of chirality sensors [49,58]. A set of significantly more rigid bis-porphyrin tweezers was reported by Ogoshi and co-workers (Figures 15 and 16) [59]. Porphyrin subunits were connected via chiral 2,2′-dihydroxy- (54) and 2,2′- dimethoxy-1,1′-binaphthyl (55) as well as achiral 3,3′-biphenyl (56) and 5,5′-dimethoxycarbonyl- 3,3′- biphenyl (57) bridges. Such sensors were applied in chiral recognition of lysine derivatives (Figure 15) and cystine diesters (Figure 16), manifested by induction of strong CD response in the Soret region of the host. The bisignate Cotton effects were diagnostic of guest absolute configuration. Association constants for formation of 1:1 complexes were determined by means of UV/Vis spectroscopic titration and remained in the ranges of 1.1 × 104–1.6 × 105 and 9.0 × 104–2.4 × 106 M−1, for binaphthyl- and Molecules 2020, 25, 4523 21 of 28 biphenyl-bridged hosts, respectively.

Molecules 2020, 25, x FOR PEER REVIEW 21 of 28

Binaphthyl-bridged tweezers 54 and 55 show remarkable enantioselectivity for lysine derivatives, reaching 12 for D/L-Lys-NMe2, (S)-54 favouring its L isomer (11.5) and (R)-54 its D isomer. It was found that the methoxy groups in the cavity of 54 play a crucial role in chiral differentiation, as corresponding sensor 55 exhibited negligible enantioselectivity towards the same set of guests. Figure 15. Binaphthyl-bridged porphyrin tweezers as sensors for lysine derivatives. Figure 15. Binaphthyl-bridged porphyrin tweezers as sensors for lysine derivatives.

Figure 16. Biphenyl-bridged porphyrin tweezers as sensors for cysteine diesters.

Binaphthyl-bridgedAnother example of tweezers chiral hosts54 and was55 recentlyshow remarkable reported by enantioselectivity Wang et al. [60], for who lysine used derivatives, a fairly d l S l R d reachingflexible tartaric 12 for acid-based/ -Lys-NMe linker2, ( )-54 in favouringthe design its of theirisomer sensor (11.5) for and nonpolar( )-54 its aminoisomer. acid It ethyl was foundesters that the methoxy groups in the cavity of 54 play a crucial role in chiral differentiation, as corresponding (Figure 17). Compound 58 was found to form 1:2 complexes with both L and D isomers, with K1 in sensor 55 exhibited negligible enantioselectivity towards the same set of guests. the range of 4.3 × 103–6.8 × 104 M−1 and K2: 6.6 × 102–3.0 × 103 M−1. Notably, while initial formation of 1:1 complexesAnother exampleseems to ofbe chiralequally hosts easy was as in recently the case reported of oxalic byamide-linked Wang et al. tweezer [60], who reported used a by fairly Hu flexibleet al. [47,56], tartaric association acid-based constants linker determined in the design for of subsequent their sensor coordination for nonpolar of aminoanother acid guest ethyl molecule esters significantly lower in this case. Formation of diastereoisomeric host-guest complexes results in a noticeable bathochromic shift, from 419 nm for 58 to 428 nm for 58·L2.

Figure 17. Porphyrin tweezer with tartaric acid-based linker.

This chiral sensor shows a negative CD couplet at 416 and 425 nm; coordination of L-amino acid ethyl esters gives rise to a new negative Cotton effect at 426–428, 434 and 436 nm, while their chiral counterparts induce a positive Cotton effect at 424–426 and 430–433 nm. Compound 58 was reported

Molecules 2020, 25, x FOR PEER REVIEW 21 of 28

Binaphthyl-bridged tweezers 54 and 55 show remarkable enantioselectivity for lysine derivatives, reaching 12 for D/L-Lys-NMe2, (S)-54 favouring its L isomer (11.5) and (R)-54 its D isomer. It was found that the methoxy groups in the cavity of 54 play a crucial role in chiral differentiation, as corresponding sensor 55 exhibited negligible enantioselectivity towards the same set of guests.

Figure 16. Biphenyl-bridged porphyrin tweezers as sensors for cysteine diesters.

Molecules 2020, 25, 4523 22 of 28 Another example of chiral hosts was recently reported by Wang et al. [60], who used a fairly flexible tartaric acid-based linker in the design of their sensor for nonpolar amino acid ethyl esters

(Figure(Figure 1717).). Compound 5858 waswas foundfound to to form form 1:2 1:2 complexes complexes with with both bothl and L andd isomers, D isomers, with with K1 inK1the in 3 4 1 2 3 1 therange range of 4.3of 4.310 × 10–6.83–6.8 10× 10M4 M−1and andK K2:: 6.66.6 × 10102–3.0–3.0 × 10103 MM−1. Notably,. Notably, while while initial initial formation formation of × × − 2 × × − 1:1of 1:1complexes complexes seems seems to be to equally be equally easy easyas in asthe in case the of case oxalic of oxalicamide-linked amide-linked tweezer tweezer reported reported by Hu etby al. Hu [47,56], et al. [association47,56], association constants constants determined determined for subsequent for subsequent coordination coordination of another of guest another molecule guest significantlymolecule significantly lower in lowerthis case. in this Formation case. Formation of diastereoisomeric of diastereoisomeric host-guest host-guest complexes complexes results results in a noticeablein a noticeable bathochromic bathochromic shift, shift, from from 419 nm 419 for nm 58 for to58 428to nm 428 for nm 58 for·L582. L . · 2

FigureFigure 17. 17. PorphyrinPorphyrin tweezer tweezer with with tartaric tartaric acid-based acid-based linker. linker. This chiral sensor shows a negative CD couplet at 416 and 425 nm; coordination of l-amino acid This chiral sensor shows a negative CD couplet at 416 and 425 nm; coordination of L-amino acid ethyl esters gives rise to a new negative Cotton effect at 426–428, 434 and 436 nm, while their chiral ethyl esters gives rise to a new negative Cotton effect at 426–428, 434 and 436 nm, while their chiral counterparts induce a positive Cotton effect at 424–426 and 430–433 nm. Compound 58 was reported counterparts induce a positive Cotton effect at 424–426 and 430–433 nm. Compound 58 was reported to show enantioselectivity towards d-aminoesters, reaching 8.4, which, according to the authors, is the highest value obtained to date for this class of sensors. Optimised structure of 58 revealed two types of hydrogen bonds—both between linker amino and guest carbonyl groups and the other way around. Peptoids constitute a class of peptidomimetics, with the amino acid side chain attached to the nitrogen instead of the α-carbon. In other words, a peptoid is a synthetic N-substituted glycine oligomer [61]. Lately, several porphyrin–peptoid conjugates have been designed as energy transfer systems [62]. These hybrids have been of particular interest owing to the simplicity of backbone modification, which is a powerful tool to control the change in inter-chromophore interactions [63,64]. Recently, Seo et al. [65] reported bis-porphyrin tweezers with chiral and achiral peptoids as flexible linkers (Figure 18). Those tweezers were shown to accommodate achiral diamines of various sizes and basicities, but also served the purpose of chiral recognition of lysine stereoisomers, which was monitored with UV/Vis and CD spectroscopies. This bidentate ligand was found to bind to achiral host 59, giving rise to a bisignate Cotton effect in the Soret region (at 425 and 432 nm) reflecting guest absolute configuration. Coordination with l-LysOMe induced a negative Cotton effect, indicating that the complex formed a left-handed screw, while—as expected—d-LysOMe gave a mirror CD image, with a right-handed screw. When a chiral tweezer 60 was employed, accommodation of chiral guests led to diastereoisomeric supramolecular structures. Upon CD monitored titration of 60 with d-LysOMe, a slight bathochromic shift of the spectrum was observed, from 422 and 430 nm to 425 and 435 nm. A complex with l isomer gave a diminished “M”-shaped CD response (425, 430 and 435 nm), probably owing to the presence of multiple CD-active conformers in the solution. Molecules 2020, 25, x FOR PEER REVIEW 22 of 28 to show enantioselectivity towards D-aminoesters, reaching 8.4, which, according to the authors, is the highest value obtained to date for this class of sensors. Optimised structure of 58 revealed two types of hydrogen bonds—both between linker amino and guest carbonyl groups and the other way around. Peptoids constitute a class of peptidomimetics, with the amino acid side chain attached to the nitrogen instead of the α-carbon. In other words, a peptoid is a synthetic N-substituted glycine oligomer [61]. Lately, several porphyrin–peptoid conjugates have been designed as energy transfer systems [62]. These hybrids have been of particular interest owing to the simplicity of backbone modification, which is a powerful tool to control the change in inter-chromophore interactions [63,64]. Recently, Seo et al. [65] reported bis-porphyrin tweezers with chiral and achiral peptoids as flexible linkers (Figure 18). Those tweezers were shown to accommodate achiral diamines of various sizes and basicities, but also served the purpose of chiral recognition of lysine stereoisomers, which was monitored with UV/Vis and CD spectroscopies. This bidentate ligand was found to bind to achiral host 59, giving rise to a bisignate Cotton effect in the Soret region (at 425 and 432 nm) reflecting guest absolute configuration. Coordination with L-LysOMe induced a negative Cotton effect, indicatingMolecules 2020 that, 25, 4523the complex formed a left-handed screw, while—as expected—D-LysOMe gave23 of 28a mirror CD image, with a right-handed screw.

Figure 18. PorphyrinPorphyrin tweezers with achira achirall and chiral peptoid linkers. 3.3. Aqueous Phase When a chiral tweezer 60 was employed, accommodation of chiral guests led to diastereoisomericStudies on chiral supramolecular recognition struct of biomoleculesures. Upon CD o ffmonitoreder insight titration into corresponding of 60 with D-LysOMe, host-guest a slightphenomena bathochromic in biological shift systems.of the spectrum Therefore, was investigationobserved, from of such422 and interactions 430 nm to in 425 water and has 435 been nm. ofA complexparticular with interest, L isomer as mimicking gave a diminished physiological “M”-shaped conditions CD provides response more (425, precise 430 and information 435 nm), probably useful to owingelucidate to the specific presence mechanisms of multiple governing CD-active chiral conformers recognition in thein solution. vivo. The vast amount of porphyrin receptors has brought to light some of the most important factors influencing the process of chiral 3.3.recognition Aqueous ofPhase amino acids in nonpolar solvents, however the environment affects those interactions in a non-negligibleStudies on way.chiral In recognition organic solutions, of biomolecules hydrogen bondingoffer insight has beeninto foundcorresponding to play a crucialhost-guest role phenomenain two- (or three-)pointin biological fixation systems. of Therefore, guest molecule investig ontoation the of porphyrin such interactions plane, yetin water it is considerably has been of weakenedparticular interest, in water as due mimicking hydration physiological [66]. In this paragraphconditions we provides report somemore ofprecise the recent information water-soluble useful toporphyrin elucidate sensors specific for mechanisms chiral recognition governing of aminochiral recognition acids and elucidation in vivo. The of vast factors amount influencing of porphyrin such receptorsinteractions has in brought aqueous to media. light some of the most important factors influencing the process of chiral recognitionAmong of early amino reports acids is ain water-soluble nonpolar solvents, porphyrin however host for the chiral environment recognition affects of amino those acids interactions reported by Imai et al. [11,67]. Their sensor bears two quaternary ammonium groups above and below porphyrin plane, bound to adjacent meso-phenyl substituents (Figure 19). At the two remaining meso-phenyls, benzamide moieties were embedded for host-guest hydrophobic attractions. Enantiomers of 60 were tested for binding amino acids in water solutions at basic pH values, where their carboxylic group is deprotonated. In this study, charged (Ser, Asp and Glu) and nonpolar (Gly, Ala, Val, Leu, Phe and Trp) amino carboxylates were compared, together with di- and tripeptides (GG, GA, GV, GL, GF, GW, GP, GS, GE, GGG and GGW). Notably, association constants for formation of such host-guest complexes in aqueous solutions are 1–3-fold lower than those reported for organic-phase systems [47] and were in the range of 2.4 101–1.5 103 M 1. In aqueous solution, guest molecules compete with H O × × − 2 coordinated to the zinc centre. Compound 61 showed slight enantioselectivity towards enantiopure ligands, ranging from 1.2 to 3.3. Molecules 2020, 25, x FOR PEER REVIEW 23 of 28 in a non-negligible way. In organic solutions, hydrogen bonding has been found to play a crucial role in two- (or three-)point fixation of guest molecule onto the porphyrin plane, yet it is considerably weakened in water due hydration [66]. In this paragraph we report some of the recent water-soluble porphyrin sensors for chiral recognition of amino acids and elucidation of factors influencing such interactions in aqueous media. Among early reports is a water-soluble porphyrin host for chiral recognition of amino acids reported by Imai et al. [11,67]. Their sensor bears two quaternary ammonium groups above and below porphyrin plane, bound to adjacent meso-phenyl substituents (Figure 19). At the two remaining meso-phenyls, benzamide moieties were embedded for host-guest hydrophobic attractions. Enantiomers of 60 were tested for binding amino acids in water solutions at basic pH values, where their carboxylic group is deprotonated. In this study, charged (Ser, Asp and Glu) and nonpolar (Gly, Ala, Val, Leu, Phe and Trp) amino carboxylates were compared, together with di- and tripeptides (GG, GA, GV, GL, GF, GW, GP, GS, GE, GGG and GGW). Notably, association constants for formation of such host-guest complexes in aqueous solutions are 1–3-fold lower than those reported for organic-phase systems [47] and were in the range of 2.4 × 101–1.5 × 103 M−1. In aqueous solution, guest molecules compete with H2O coordinated to the zinc centre. Compound 61 showed slight Molecules 2020, 25, 4523 24 of 28 enantioselectivity towards enantiopure ligands, ranging from 1.2 to 3.3.

Figure 19. IonicIonic porphyrin porphyrin probes probes for detection of amino acids.

Coordination of guest NH to host zinc(II) centre and Coulomb interactions between cationic Coordination of guest NH2 to host zinc(II) centre and Coulomb interactions between cationic meso substituentssubstituents and and deprotonated deprotonated carboxy carboxy groups groups of of the the amino amino acids acids were were found found to tocontribute contribute to theto the binding binding constant constant together together with with hydrophobic hydrophobic attraction attraction between between amino amino acid acid side side chain chain and andthe benzamidethe benzamide phenyl phenyl ring ring or steric or steric repulsion repulsion between between them. them. The The latter latter depends depends on onthe the structure structure of aminoof amino carboxylate carboxylate in the in thefollowing following way. way. Among Among the themost most tightly tightly bound bound guests guests are tryptophan are tryptophan and phenylalanine;and phenylalanine; less hydrophobic less hydrophobic residues, residues, such suchas serine, as serine, aspartic aspartic acid or acid glycylserine, or glycylserine, do not do show not anshow increase an increase in association in association constant, constant, yet yettheir their stereoisomers stereoisomers are are bound bound to tothe the host host selectively. selectively. Dependence of association constant on ionic strength of the solution was determined, supporting the contribution ofof electrostaticelectrostatic interactions interactions to to formation formation of host-guestof host-guest complexes complexes (n-butylamine (n‑butylamine was usedwas Molecules 2020, 25, x FOR PEER REVIEW 24 of 28 usedas reference). as reference). Recognition Recognition ability ability towards towards GX dipeptidesGX dipeptides (where (where X = G,X = A, G, V, A, L, V, F, L, W, F, P, W, S orP, E)S or was E) wasretained, retained, even even though though the asymmetric the asymmetric centre cent is separatedre is separated from from the coordination the coordination site. site. Chiral recognition of nonpolar amino acids by ruthenium Halterman porphyrins in chloroform, Chiral recognition of nonpolar amino acids by ruthenium Halterman porphyrins in chloroform, methanol andand waterwater was was reported reported by by Nicolas Nicolas et et al. al [68. [68].]. In thisIn this study, study, a rigid a rigid porphyrin porphyrin sensor sensor62 with 62 Ru(CO)with Ru(CO) centre centre was employedwas employed to investigate to investigate ligation ligation of the of metal the metal centre centre in trans in positiontrans position to the to other the axialother ligandaxial ligand (Figure (Figure 20). 20).

Figure 20. Ruthenium porphyrin complexes as amino acid sensors for organic and aqueous phase. Figure 20. Ruthenium porphyrin complexes as amino acid sensors for organic and aqueous phase. The application of a chiral host for recognition enabled the differentiation and quantification of diastereoisomericThe application complexes of a chiral formed host for upon recognition addition ofenabled racemic the ligands differentiation in 1H NMR. and Among quantification the chiral of diastereoisomeric complexes formed upon addition of racemic ligands in 1H NMR. Among the chiral amines investigated were alanine, phenylalanine and valine as acids (as guests for 63 in aqueous phase) and methyl esters (for 62 in organic phase studies). In all environments, D-phenylalanine was favoured over its enantiomeric counterpart, while Val and Ala showed the opposite preference. In the case of phenylalanine, a drastic decrease of enantioselectivity was observed while changing the environment from organic to aqueous; it was therefore concluded that hydrophobic interactions play a significant but not crucial role in the process of chiral recognition. Based on previous studies on the origin of chiral recognition and its dependence on different association and dissociation constants in the formed complexes [69,70], the latter were examined with saturation transfer NMR technique, exploiting the slow axial ligand exchange in ruthenium porphyrins [45]. The data obtained from these experiments reveal that dissociation rate constants were lower for isomers, towards which selectivity was observed (i.e., kL-PheOMe = 0.36 s−1 and kD-PheOMe= 0.14 s−1), indicating that the rate of ligand dissociation affects the process of chiral recognition.

4. Conclusions We highlight the importance of the detection of natural derivatives, specifically focusing on amino acids, by two different molecular probes, BODIPY and porphyrins. The two probes essentially work with two different recognition approaches: BODIPY probes rely on the direct interaction with the target, hence a covalent interaction. Porphyrins probes, on the other hand, mostly interact with the target in the fashion of molecular recognition, which is mediated by non-covalent interactions— a synthesis of coordination, hydrogen bonding, hydrophobic interactions, steric repulsion and/or electrostatic attraction. To this day, sensors for both molecular recognition and chiral discrimination are designed. The development of such systems is of significant importance for the scientific community since there are already many specific applications, especially in the fields of biomedicine and molecular devices.

Funding: This work was supported by the National Science Centre, Poland with the grant No. 2016/23/B/ST5/01186.

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Persch, E.; Dumele, O.; Diederich, F. Molecular Recognition in Chemical and Biological Systems. Angew. Chem. Int. Ed. 2015, 54, 3290–3327, doi:10.1002/anie.201408487. 2. Baron, R.; McCammon, J.A. Molecular Recognition and Ligand Association. Annu. Rev. Phys. Chem. 2013, 64, 151–175, doi:10.1146/annurev-physchem-040412-110047. 3. Ariga, K.; Ito, H.; Hillab, J.P.; Tsukube, H. Molecular recognition: From solution science to nano/materials technology. Chem. Soc. Rev. 2012, 41, 5800–5835, doi:10.1039/C2CS35162E.

Molecules 2020, 25, 4523 25 of 28 amines investigated were alanine, phenylalanine and valine as acids (as guests for 63 in aqueous phase) and methyl esters (for 62 in organic phase studies). In all environments, d-phenylalanine was favoured over its enantiomeric counterpart, while Val and Ala showed the opposite preference. In the case of phenylalanine, a drastic decrease of enantioselectivity was observed while changing the environment from organic to aqueous; it was therefore concluded that hydrophobic interactions play a significant but not crucial role in the process of chiral recognition. Based on previous studies on the origin of chiral recognition and its dependence on different association and dissociation constants in the formed complexes [69,70], the latter were examined with saturation transfer NMR technique, exploiting the slow axial ligand exchange in ruthenium porphyrins [45]. The data obtained from these experiments reveal that dissociation rate constants 1 were lower for isomers, towards which selectivity was observed (i.e., kl-PheOMe = 0.36 s− 1 and kd-PheOMe= 0.14 s− ), indicating that the rate of ligand dissociation affects the process of chiral recognition.

4. Conclusions We highlight the importance of the detection of natural derivatives, specifically focusing on amino acids, by two different molecular probes, BODIPY and porphyrins. The two probes essentially work with two different recognition approaches: BODIPY probes rely on the direct interaction with the target, hence a covalent interaction. Porphyrins probes, on the other hand, mostly interact with the target in the fashion of molecular recognition, which is mediated by non-covalent interactions—a synthesis of coordination, hydrogen bonding, hydrophobic interactions, steric repulsion and/or electrostatic attraction. To this day, sensors for both molecular recognition and chiral discrimination are designed. The development of such systems is of significant importance for the scientific community since there are already many specific applications, especially in the fields of biomedicine and molecular devices.

Funding: This work was supported by the National Science Centre, Poland with the grant No. 2016/23/B/ST5/01186. Conflicts of Interest: The authors declare no conflict of interest.

References

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