Pyrroloindole-Based Dynamic Combinatorial Chemistry

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Article Pyrroloindole–basedS S symmetry Dynamic Combinatorial Chemistry Article Tiberiu-MariusPyrroloindole-Based Gianga 1,2, Dora-Maria R Dynamicăsădean 1 and G. Dan Pantoș 1,* 1 Department of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK; tiberiu- [email protected] Chemistry (T.-M.G.); [email protected] (D.-M.R.) 2 Current address: Beamline B23, Diamond Light Source, Ltd., Chilton, Didcot OX11 0DE, UK 1,2 1 1, * Correspondence:Tiberiu-Marius [email protected]; Gianga , Dora-Maria Tel.: R ă+44-1225-384-376sădean and G. Dan Pantos, * 1 Received:Department 2 April 2020; of Chemistry, Accepted: University 16 April of2020; Bath, Published: Claverton date Down, Bath BA2 7AY, UK; [email protected] (T.-M.G.); [email protected] (D.-M.R.) 2 Beamline B23, Diamond Light Source, Ltd., Chilton, Didcot OX11 0DE, UK Abstract:* Correspondence: We report a [email protected];new class of building Tel.: +blocks44-1225-384-376 for Dynamic Combinatorial Chemistry (DCC) L based on the pyrroloindole scaffold. The attachment of -cysteine on the α, α′ positions of the core makesReceived: the molecule 2 April 2020; suitable Accepted: for 16 disulfide April 2020; exchange Published: 3in May aqueous 2020 dynamic combinatorial libraries (DCLs). The synthesis of the core follows a modified version of the Knoevenagel–Hemetsberger Abstract: We report a new class of building blocks for Dynamic Combinatorial Chemistry (DCC) based approach. The new building block (L-PI) is fluorescent (Φ = 48%) and relatively stable towards on the pyrroloindole scaffold. The attachment of l-cysteine on the α, α positions of the core makes thermal and photodegradation. The chirality of the cysteine is transferred0 to the electron-rich the molecule suitable for disulfide exchange in aqueous dynamic combinatorial libraries (DCLs). pyrroloindole core. Homo- and heterochiral DCLs of L-PI with electron-deficient L- and D- The synthesis of the core follows a modified version of the Knoevenagel–Hemetsberger approach. naphthalenediimideThe new building (NDI) block (leadl-PI) to is fluorescentsimilar library (Φ = di48%)stributions and relatively regardless stable of towards the enantiomer thermal and used. Whenphotodegradation. no salt is present, The the chirality major of component the cysteine is is a transferred dimer, while to the dimers electron-rich and tetramers pyrroloindole are obtained core. at increasedHomo- and ionic heterochiral strength. DCLs of l-PI with electron-deficient l- and d-naphthalenediimide (NDI) lead to similar library distributions regardless of the enantiomer used. When no salt is present, the major Keywords:component dynamic is a dimer, combinatorial while dimers chemistry; and tetramers pyrroloindoles; are obtained atchirality; increased disulfide ionic strength.

Keywords: dynamic combinatorial chemistry; pyrroloindoles; chirality; disulﬁde

1. Introduction 1.Dynamic Introduction combinatorial chemistry (DCC) has emerged in the last decades as a powerful tool in the developmentDynamic of combinatorial (bio)sensors chemistry [1–3], self-replicating (DCC) has emerged materials in the [4–7], last decades receptors as a [8–11], powerful etc. tool It inis also a well-establishedthe development approach of (bio)sensors in making [1–3], interlocked self-replicating molecular materials architectures [4–7], receptors (e.g., [8– 11catenanes], etc. It is and also knots) a usingwell-established simple precursors approach [12–31]. in making DCC interlocked involves reactions molecular under architectures thermodynamic (e.g., catenanes control and knots)in which startingusing molecules simple precursors (building [12 –blocks)31]. DCC combine involves to reactions generate under a complex thermodynamic mixture control of species in which called dynamicstarting combinatorial molecules (building library blocks) (DCL) combine [1,32,33]. to generate The building a complex blocks mixture have of species moieties called that dynamic allow for reversiblecombinatorial exchanges library until (DCL) the equilibrium [1,32,33]. The is buildingreached blocksand the have precursors moieties thatare completely allow for reversible consumed. Representativeexchanges until examples the equilibrium [1,34] isof reached reversible and the reactions precursors areinclude completely ester consumed. [35–37], Representativeimine [21,23,26], examples [1,34] of reversible reactions include ester [35–37], imine [21,23,26], hydrazone [38,39], hydrazone [38,39], hydrogen-bond [40–42], acetal [43] and disulfide exchanges [15,44,45]. Among hydrogen-bond [40–42], acetal [43] and disulﬁde exchanges [15,44,45]. Among these, disulfide exchange these,is disulfide of particular exchange interest asis itof is partic prevalentular ininterest biological as it systems is prevalent (e.g., cystines) in biological [46–48]. systems The reaction (e.g., works cystines) [46–48].close The to the reaction physiological works conditions close to (aqueousthe physiologi media andcal conditions pH 8), and it (aqueous stops when media all the and thiolate pH anions 8), and it stopsare when consumed all the (Scheme thiolate1). anions are consumed (Scheme 1).

SchemeScheme 1. 1.GeneralGeneral mechanism mechanism ofof disulﬁdedisulfide exchange. exchange.

SymmetrySymmetry 2020,2020 12, ,x;12 doi:, 726; FOR doi: 10.3390PEER REVIEW/sym12050726 www.mdpi.comwww.mdpi.com/journal/symmetry/journal/symmetry Symmetry 2020, 12, 726 2 of 11

Disulﬁde exchange reactions for DCC have been largely explored by Sanders and Pantos, groups [12,13,15,16,45,49]. Their work has mainly focused on naphthalenediimide (NDI) and dialkoxynaphthalene (DN) derivatives to synthesizing interlocked molecules. Other building blocks with thiol groups allowed for the synthesis of receptors and replicators [4–6,8]. The present work expands this pool by introducing a new electron-rich building block with thiol linkages. Its behaviour as a partner for electron-deﬁcient NDIs in DCLs is studied. The role played by chirality on the outcome of DCLs (mixtures of L and L vs. L and D enantiomers) is also explored.

2. Materials and Methods All reagents were purchased from commercial suppliers: Acros Organics, Alfa Aesar, Fluorochem, Merck, TCI Europe and used without further purification. 1H and 13C NMR spectra were recorded on 500 MHz Agilent Propulse or 500 MHz Bruker Avance II+ (1H NMR spectra at 500 MHz, 13C NMR spectra at 125 MHz) instruments. Chemical shifts (δ) are reported in parts per million (ppm). Coupling constants are reported in Hertz (Hz), and signal multiplicity is denoted as singlet (s), doublet (d), doublet of doublets (dd), doublet of doublets of doublets (ddd), triplet (t), quartet (q), dt (doublet of triplets), td (triplet of doublets), multiplet (m) and broad (br). All spectra were acquired at 25 ◦C, unless otherwise stated, and were referenced to the solvent residual peaks. The common solvent 1 13 impurities present in small amounts in H and C NMR spectra were water, acetone, CH2Cl2 or DMF. The microwave reactions were carried out in either CEM Discover or CEM Explorer 12 instruments. LC-MS studies were carried out on a Thermo Surveyor PDA Plus LC and LCQ classic ESI MS. LC-MS data were processed using the XCalibur software. The HRMS spectra were either acquired at the National Mass Spectrometry Facility at Swansea University. The HRMS and MS/MS spectra for the library components were done on a Bruker MaXis HD ESI-QTOF mass spectrometer for high mass accuracy, coupled to a Thermo Scientific Dionex Ultra High Performance Liquid Chromatography (UPLC) unit. Circular dichroism (CD), absorption and fluorescence data were acquired on an Applied Photophysics Chirascan spectrophotometer equipped with a Peltier temperature controller. DCL set-up: 5 mM total concentrations (the total volume of each library was 1 mL; single component libraries contained 5 10 6 moles; two-component libraries contained 2.5 10 6 moles × − × − per component) libraries were prepared by dissolving the building blocks in 10 mM aqueous NaOH, followed by titration with 100 mM NaOH/100 mM HCl (aqueous solutions) to adjust the pH to 8. The DCL solutions were stirred in closed-capped vials for at least three days at room temperature. LC-MS settings for ESI-MS spectra (Thermo Surveyor PDA Plus LC and LCQ classic ESI MS; negative ion) were acquired with a drying temperature of 250 ◦C, spray current 0.5 µA, sheath gas flow of 40 arb, spray voltage 4.5 kV, capillary voltage 13 V and tube lens 15.0 V. The mass range − was set from m/z 150–2000, the number of microscans in scan time was 5, and the maximum injection time was 150.0 ms. The HPLC (High Performance Liquid Chromatography) method is reported in Supplementary Materials Section3. LC-MS settings for HRMS data (Bruker MaXis HD ESI-QTOF): ESI-MS spectra (negative ion) were acquired with drying temperature of 320 C, collision energy 4 eV and dry gas 12 L/min. ◦ − The mass range was set from m/z 350–3500. MS/MS settings for HRMS data (Bruker MaXis HD ESI-QTOF): parent Mass (m/z): dependant on the species, ionisation width (m/z): 20.0, collision energy: 20 eV, drying temperature 240 ◦C and dry gas 12 L/min. CD and absorbance settings: experiments were performed in a 10 mm pathlength quartz cuvette, wavelength range: 250–450 nm, scan mode: 1 point/nm, time-per-point: 1 sec, bandwidth: 2 nm and temperature 23 ◦C. Settings for variable temperature (VT) studies: temperature range: 5–55 ◦C and return ramp, increment: 10 ◦C and setting time 45 sec. Fluorescence settings: fluorescence spectra were collected for samples with an absorbance below 0.1 AU. Experiments were performed in a 10 mm pathlength quartz cuvette. Emission spectra: wavelength range: 250–450 nm, scan mode: 1 point/nm, time-per-point: 1 sec, excitation wavelength: Symmetry 2020, 12, 726 3 of 11 Symmetry 2020, 12, x FOR PEER REVIEW 3 of 11

340 nm, bandwidth 8 nm, PMU: 1000 V and SEM: 2 4.65 nm. Excitation spectra: wavelength range: 250–400 nm, scan mode: 1 point/nm, time-per-point:× 1 sec, emission wavelength: 393 nm, bandwidth 6250–400 nm, PMU: nm, 1000 scan V mode: and SEM: 1 point 1.29/nm, × 4.65 time-per-point: nm. 1 s, emission wavelength: 393 nm, bandwidth 6 nm, PMU: 1000 V and SEM: 1.29 4.65 nm. Computational studies. Geometry× optimisation was performed using Avogadro [50] (Force field: ComputationalUFF, Algorithm: studies Conjugate. Geometry gradients). optimisation This was was followed performed by MOPAC using Avogadro 2016 [51] (Version [50] (Force 18.117 field: M)UFF, PM7 Algorithm: semiempirical Conjugate optimisation gradients). using This the wasCOSMO followed water by model MOPAC with 2016 a 1 convergence [51] (Version factor, 18.117 and M) GabeditPM7 semiempirical 2.5.0 [52] as interface. optimisation Qzz calculations using the COSMOwere performed water modelwith GAMESS with a at 1 convergenceMP2 level. factor, and Gabedit 2.5.0 [52] as interface. Qzz calculations were performed with GAMESS at MP2 level. 3. Results and Discussion 3. Results and Discussion 3.1. Building Block Design and Properties 3.1. Building Block Design and Properties

3.1.1.3.1.1. Synthesis Synthesis TheThe pyrrolo[2,3- pyrrolo[2,3-f]indolef ]indole scaffold scaﬀold consists consists of of a apyrrole pyrrole ring ring condensed condensed with with the the benzene benzene ring ring of anof anindole. indole. This This is the is the so-called so-called “type “type I pyrroloin I pyrroloindole”dole” found found in in many many natural natural alkaloids alkaloids and and also also widelywidely used used in in medicinal medicinal chemistry chemistry re researchsearch [53,54]. [53,54]. It It is is aromatic aromatic (14 (14 ππ electrons)electrons) and and bears bears cysteine cysteine appendagesappendages on the the αα,, αα’’ positions positions that that confer confer it it water water solubility solubility and and ability ability to to be be involved involved in in disulfide disulﬁde exchangeexchange (Figure (Figure 11).).

planar surface for aromatic interactions SH O N HN HOOC COOH NH N O HS amino-acid for free thiol for water solubility disulfide exchange

Figure 1. Structural features of the new building block designed in this work. Figure 1. Structural features of the new building block designed in this work. The initial synthesis of the core followed a previously published procedure [55]. The first step involvedThe initial the formation synthesis of of ethyl the azidoacetatecore followed2 ,a which previously was used published in a Knoevenagel-type procedure [55]. The condensation first step involvedwith terephthaldehyde the formation toof yieldethyl compoundazidoacetate4. 2 The, which next was step used was thein a Hemetsberger Knoevenagel-type reaction, condensation giving the withpyrroloindole terephthaldehyde5. This was to obtainedyield compound in low yield 4. The because next moststep was of the the product Hemetsberger polymerised—a reaction, common giving theissue pyrroloindole in pyrrole and5. This pyrroloindole was obtained chemistry. in low yield Other because substrates most of (1,4-thiophene-dialdehyde the product polymerised—a and common1,4-dimethoxy-terephthaldehyde) issue in pyrrole and pyrroloindole were used, butchemistry. the Knoevenagel Other substrates step worked (1,4-thiophene-dialdehyde in low yields (products and11 and 1,4-dimethoxy-terephthaldehyde)13; further information in Supplementary were used, Materials but the SectionKnoevenagel 1 and Schemesstep worked S1 and in low S2). yields (productsThe synthetic11 and 13 protocol; further wasinformation then optimised in Supplementary (Scheme2) Materials and the Hemetsberger Section 1 and stepSchemes carried S1 and out S2).in more diluted conditions to prevent polymerization. Pyrrole chemistry shows that the free -NH unitsThe lead synthetic to extensive protocol polymerization was then optimised [56]. Thus, (Scheme the -NH 2) and groups the Hemetsberger were alkylated, step which carried allowed out in morethe synthesis diluted conditions of pure pyrroloindole to prevent polymerization6. The next steps. Pyrrole included chemistry hydrolysis shows of that the esterthe free group -NH to units give lead7, followed to extensive by NHS polymerization activation, and [56].S-trityl-L-cysteine Thus, the -NH attachmentgroups were and alkylated, deprotection which via allowed previously the synthesisreported methodsof pure pyrroloindole [45]. The ﬁnal 6 building. The next block stepsl-PI includedwas obtained hydrolysis in 8 steps,of the none ester of group which to required give 7, followedlaborious by puriﬁcation NHS activation, (experimental and S details-trityl-L in-cysteine Supplementary attachment Materials and deprotection Section 1). via previously reported methods [45]. The final building block L-PI was obtained in 8 steps, none of which required laborious purification (experimental details in Supplementary Materials Section 1).

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EtO O O NaN3 3 N3 water:acetone EtOH, EtONa H O O O 1:1 ETFA o-dichlorobenzene O N Br N3 OEt OEt 1 hr N O overnight, r.t. o O o 30 min, 150 C H 0 C r.t. N3 92% 1 2 42% 5 O OEt 4

O N O N MeI, K CO MeOH EDC, NHS O 2 3 O OH DMF O N 2 M NaOHaq O N DMF N O O 5 N O o N O overnight, r.t. overnight O N O overnight, 74 C HO N , r.t. 88% O 8 2 90% 7 6 46% over the last 2 steps

SH L-Trityl-S-Cysteine STrt CH Cl , TFA 2 2 N HN Et3N O N HN Et SiH O COOH 3 HOOC COOH DMF HOOC N O 8 N O 30 min, r.t. NH overnight, N2, r.t. NH TrtS HS 9 24% quant. L-PI l SchemeScheme 2. 2.Optimised Optimised protocol protocol forfor thethe synthesissynthesis of the new building building block block L-PI-PI. The. The reagents reagents and and conditionsconditions used used for for each each step step as as well well as as the the yieldsyields areare indicatedindicated on on the the reaction reaction arrows. arrows. 3.1.2. Optoelectronic and structural properties 3.1.2. Optoelectronic and structural properties The trityl-protected pyrroloindole, compound 9, is soluble in common organic solvents, which The trityl-protected pyrroloindole, compound 9, is soluble in common organic solvents, which allowedallowed us us to to carry carry out out spectroscopic spectroscopic studiesstudies inin chloroform;chloroform; the the main main properties properties are are summarised summarised in in TableTable1 (for 1 (for molar molar extinction extinction coe coefficientﬃcient ε εcalculation, calculation, seesee FigureFigure S14).

TableTable 1. 1.Summary Summary of of the the main main optical optical propertiesproperties of trityl-cysteine pyrroloindole pyrroloindole 9 9inin chloroform. chloroform.

AbsorbanceAbsorbance & & CD CD Emission Emission λmax (nm) ε (L × mol−1 × cm−1) Molar Ellipticity (deg × cm2 × dmol−1) λmax (nm) Φ (%) λmax Molar Ellipticity λmax 327 ε (L 21,600mol ±1 1.8 cm 1) 2.11 × 104 394 Φ48 (%) (nm) × − × − (deg cm2 dmol 1) (nm) × × − The optoelectronic327 and 21,600 structural1.8 properties of the2.11 trityl-protected104 cysteine394 pyrroloindole 48 9 were assessed by UV-vis and fluorescence± spectroscopy. The× absorbance spectrum shows two bands centered at 280 nm and 327 nm (with a small shoulder at 345 nm), which correspond to the pyrroloindoleThe optoelectronic core. The and emission structural spectrum properties is characterised of the trityl-protected by a broad, intense cysteine band pyrroloindole centered at9 394were assessednm, with by UV-visa large Stokes and fluorescence shift of 67 nm. spectroscopy. The spectral The features absorbance of 9 in spectrum the excitation shows mode two are bands similar centered to at 280those nm displayed and 327 nmby (withUV-vis. a smallThe quantum shoulder yield at 345 (Φ nm),) of which9 is 48%, correspond and has tobeen the pyrroloindolecalculated using core. Theanthracene emission as spectrum standard is (Φ characterised of anthracene byin chloroform a broad, intense = 11% band[57]). centered at 394 nm, with a large Stokes shiftThe presence of 67 nm. of The the spectralcysteine featuresmoiety allows of 9 in us the to excitation explore an mode important are similar feature: to thosechirality. displayed The bypyrroloindole UV-vis. The quantumunit is intrinsically yield (Φ) ofachiral,9 is 48%, but the and chiral has been information calculated is transferred using anthracene from the as cysteine standard (Φontoof anthracene the core. We in have chloroform used circular= 11% dichroism [57]). (CD) spectroscopy to analyze the chirality of the new buildingThe presence block precursor. of the cysteineThe CD spectrum moiety allows shown usin Figure to explore 2a displays an important a bisignate feature: Cottonchirality. effect Thewith pyrroloindole relatively large unit response is intrinsically for a small achiral, molecule. but This the chirality chiral information transfer observed is transferred is not a result from of the cysteineaggregation onto the as indicated core. We by have a linear used Lambert–Beer circular dichroism plot shown (CD) spectroscopy in Figure S14. to analyze the chirality of the newThe building thermal block stability precursor. and photodegradation The CD spectrum of shown9 over 5–55 in Figure °C temperature2a displays range a bisignate and return Cotton effectramp with were relatively assessed large by variable response temperature for a small molecule.(VT) CD, UV-vis This chirality and fluorescence transfer observed studies is(Figure not aresult 2). The VT CD and UV-vis spectra show minor changes, indicating a weak temperature dependence of of aggregation as indicated by a linear Lambert–Beer plot shown in Figure S14. the chirality transfer. The CD melting profile exhibits isosbestic points, indicating a monophasic The thermal stability and photodegradation of 9 over 5–55 C temperature range and return transition across the temperature range. The VT emission and excitation◦ spectra display irreversible ramp were assessed by variable temperature (VT) CD, UV-vis and fluorescence studies (Figure2). thermal and photodegradation. The emission intensity drops by roughly 30%, while the excitation by The VT CD and UV-vis spectra show minor changes, indicating a weak temperature dependence of the almost 70%. chirality transfer. The CD melting profile exhibits isosbestic points, indicating a monophasic transition

Symmetry 2020, 12, 726 5 of 11 across the temperature range. The VT emission and excitation spectra display irreversible thermal and photodegradation.SymmetrySymmetry 20202020,, 1212,, xx FORFOR The PEERPEER emission REVIEWREVIEW intensity drops by roughly 30%, while the excitation by almost55 70%. ofof 1111

FigureFigure 2.2. Thermal and photodegradation photodegradation studies studies of of 999 ininin chloroformchloroform chloroform atat at thethe the specifiedspecified speciﬁed temperaturestemperatures temperatures (r(r (rdenotes denotes return return temperature temperature ramp) ramp) in in the the legend: legend: ( (aa)) variable variable temperaturetemperature (VT)(VT)(VT) circularcircularcircular dichroismdichroismdichroism 5 5 6 (CD)(CD)(CD) spectra spectraspectra (5 (5(5 ××10 1010−−−55 M);M);M); ( ((bb))) VT VTVT absorption absorptionabsorption spectra spectraspectra (5 (5(5 ××10 1010−−−55 M); M);M);( (c(cc))) VT VTVT emission emissionemission spectra spectraspectra (2.5 (2.5(2.5 ××10 1010−−−66 MM);M);); × 6 × × (d(d)) VT VT excitation excitation spectra spectra (2.5 (2.5 ×10 10−−−66 M). The sample concentration usedused inin eacheach experimentexperimentis isgiven givenin in (d) VT excitation spectra (2.5× × 10 M). The sample concentration used in each experiment is given in brackets.brackets. TheThe samesame colorcolor codecode isis usedused forfor eacheach graph.graph.

1 The first indication of an electron-rich aromatic core for l-PI comes from its H1 NMR spectrum The first indication of an electron-rich aromatic core for L-PI-PI comescomes fromfrom itsits 1H NMR spectrum δ (Figure(Figure(Figure S13), S13),S13), which whichwhich shows showsshows peaks peakspeaks in inin thethethe aromaticaromaticaromatic regionregionregion atatat relativelyrelativelyrelatively lowlowlow chemicalchemicalchemical shiftsshiftsshifts ((δ 7.357.35 ppm).ppm). TheThe electronic electronic nature nature of of organic organic molecules molecules can can be be determined determined fromfrom the the quadrupolequadrupole momentmoment onon thethe zz axis (Qzz) calculations. The lower the number in the negative regime, the more electron-rich a molecule axis (Qzzzz)) calculations.calculations. TheThe lowerlower thethe numbernumber inin thethe negativenegative regime,regime, thethe moremore electron-richelectron-rich aa is. The reverse applies to electron-deficient molecules. The calculated Qzz for the pyrroloindole core is molecule is. The reverse applies to electron-deficient molecules. The calculated Qzzzz forfor thethe 22.7 B (Buckinghams), meaning it is an electron-rich molecule. To put this number in perspective, it −pyrroloindole core is −22.7 B (Buckinghams), meaning it is an electron-rich molecule. To put this should be compared with the Qzz of other building blocks previously used for disulfide DCC. The Qzz number in perspective, it should be compared with the Qzzzz ofof otherother buildingbuilding blocksblocks previouslypreviously usedused of one of the electron-rich building blocks extensively used in DCC (2,6-DN) is 15.1 B, while the forfor disulfidedisulfide DCC.DCC. TheThe QQzzzz ofof oneone ofof thethe electron-richelectron-rich buildingbuilding blocks extensively− used in DCC (2,6- electron-deficient NDI has a Qzz of 14.2 (Figure3). DN) is −15.1 B, while the electron-deficient NDI has a Qzzzz ofof 14.214.2 (Figure(Figure 3).3).

Figure 3. General structures of two main building blocks for dynamic combinatorial chemistry (DCC) Figure 3. General structures of two main building blocks for dynamic combinatorial chemistry (DCC) and the new one designed in this work arranged in order of increased electron-richness. andand thethe newnew oneone designeddesigned inin thisthis workwork arrangarrangeded inin orderorder ofof increaincreasedsed electron-richness.electron-richness.

3.2. Behaviour of L-PI-PI inin DCLsDCLs

3.2.1. DCLs of L-PI-PI One of the driving forces behind DCLs in aqueouss systemssystems isis thethe hydrophobichydrophobic effecteffect [58].[58]. ThisThis refersrefers toto reorganizationreorganization ofof waterwater moleculesmolecules arouaround a hydrophobic unit formed within the system.

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3.2. Behaviour of l-PI in DCLs

3.2.1. DCLs of l-PI One of the driving forces behind DCLs in aqueous systems is the hydrophobic effect [58]. SymmetryThis refers 2020 to, 12 reorganization, x FOR PEER REVIEW of water molecules around a hydrophobic unit formed within the system.6 of 11 The hydrophobic effect is generally enhanced by adding inorganic salts [15]. Previous work in the field The hydrophobic effect is generally enhanced+ + by+ adding +inorganic salts [15]. Previous2 work in the shows that changing either the cation (Li , Na ,K and Cs ) or anion (NO3−, SO4 −, Br− and Cl−) has field shows that changing either the cation (Li+, Na+, K+ and Cs+) or anion (NO3−, SO42−, Br− and Cl−) led to similar DCLs distribution, which is due to an increasing in medium polarity with minimum has led to similar DCLs distribution, which is due to an increasing in medium polarity with minimum template contribution [15,45]. DCLs of l-PI with and without 1 M NaNO3 (added to enhance the template contribution [15,45]. DCLs of L-PI with and without 1 M NaNO3 (added to enhance the hydrophobic effect) were prepared according to the method described in Section2. Once equilibrated, hydrophobic effect) were prepared according to the method described in Section 2. Once equilibrated, the libraries have been analyzed by HPLC, MS and MS/MS; the chromatograms are shown in Figure4 the libraries have been analyzed by HPLC, MS and MS/MS; the chromatograms are shown in Figure (cartoon representations are used for clarity). The distribution of both libraries shows one major species 4 (cartoon representations are used for clarity). The distribution of both libraries shows one major identified as l-PI homodimer by MS and MS/MS analyzes (Figures S15–S17). species identified as L-PI homodimer by MS and MS/MS analyzes (Figures S15–S17).

SH O N HN 5 m M HOOC COOH NH N O pH 8 HS

L-PI

L-PI homodimer no salt

1 M NaNO3 10 11 12 13 14 15 16 17 18 19 20 Time (m in )

l FigureFigure 4. 4. Reverse-phaseReverse-phase HPLC HPLC analysis analysis of of L-PI-PI (5(5 mM mM total total concentration) concentration) libraries libraries without without salt salt (top) (top) and in the presence of 1 M NaNO (bottom). Absorptions recorded at 324 nm. The unlabeled peaks did and in the presence of 1 M NaNO3 3 (bottom). Absorptions recorded at 324 nm. The unlabeled peaks didnot not ionise ionise and and could could not not be identified. be identified. 3.2.2. Homochiral DCLs of l-PI and l-NDI 3.2.2. Homochiral DCLs of L-PI and L-NDI Another driving force behind DCLs is the donor–acceptor (D–A) interaction between electron-rich Another driving force behind DCLs is the donor–acceptor (D–A) interaction between and electron-deficient aromatic cores. A representative example of an electron-deficient building block electron-rich and electron-deficient aromatic cores. A representative example of an electron-deficient widely used in disulfide DCC is the cysteine-functionalised NDI. Homochiral libraries (i.e., same building block widely used in disulfide DCC is the cysteine-functionalised NDI. Homochiral libraries chirality of each component) of l-PI and l-NDI in 1:1 molar ratio without and with 1 M NaNO3 have (i.e., same chirality of each component) of L-PI and L-NDI in 1:1 molar ratio without and with 1 M been prepared (Figure5). The library with no salt contains the homochiral heterodimer ( l-PI and NaNO3 have been prepared (Figure 5). The library with no salt contains the homochiral heterodimer l-NDI) as dominant species and a homochiral heterotrimer. The distribution of the DCL with 1 M (L-PI and L-NDI) as dominant species and a homochiral heterotrimer. The distribution of the DCL NaNO3 is more diverse, showing the formation of a homochiral heterotetramer at the expense of the with 1 M NaNO3 is more diverse, showing the formation of a homochiral heterotetramer at the other components. This can be due to more favorable donor–acceptor interactions between the PI and expense of the other components. This can be due to more favorable donor–acceptor interactions NDI cores in the structure of the tetramer while in media with high salt concentration. The sequence of between the PI and NDI cores in the structure of the tetramer while in media with high salt tetramer (i.e., DADA or DDAA) could not be determined based on the obtained MS/MS fragmentation concentration. The sequence of tetramer (i.e., DADA or DDAA) could not be determined based on (Figures S26 and S27); however, this was assessed using computational studies. Complete MS and the obtained MS/MS fragmentation (Figures S26 and S27); however, this was assessed using MS/MS analyses are given in Figures S18–S27. computational studies. Complete MS and MS/MS analyses are given in Figures S18–S27.

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SH O SH O SH N COOH 5 m M SH N COOH 5 m M O N HN O (to ta l co n ce n tra tio n ) O N HN COOH O O HOOC COOH O (to ta l co n ce n tra tio n ) HOOC NH N O HOOC N NH N O HOOC N pH 8 HS O pH 8 HS HS O HS

L-PI L-NDI L-PI L-NDI

92% 92% homochiral homochiral 8% homochiral heterodimer 8% homochiral no salt heterodimer heterotrimer no salt heterotrimer 65% 65%

30% homochiral 5% 30% homochiral 1 M NaNO3 5% heterotetramer 1 M NaNO3 heterotetramer 10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 14 15 16 17 18 19 20 Time (m in ) Time (m in )

Figure 5. Reverse-phase HPLC analysis of l-PI:l-NDI (1:1 molar ratio, 5 mM total concentration) Figure 5. Reverse-phase HPLC analysis of L-PI:L-NDI (1:1 molar ratio, 5 mM total concentration) Figure 5. Reverse-phase HPLC analysis of L-PI:L-NDI (1:1 molar ratio, 5 mM total concentration) libraries without salt (top) and in the presence of 1 M NaNO3 (bottom). Absorptions recorded at 385 nm. libraries without salt (top) and in the presence of 1 M NaNO3 (bottom). Absorptions recorded at 385 libraries without salt (top) and in the presence of 1 M NaNO3 (bottom). Absorptions recorded at 385 Thenm. unlabeled The unlabeled peaks peaks did not did ionise not ionise and could and could not be not identified. be identified. nm. The unlabeled peaks did not ionise and could not be identified. 3.2.3. Heterochiral DCLs of l-PI and d-NDI 3.2.3. Heterochiral DCLs of L-PI and D-NDI 3.2.3. Heterochiral DCLs of L-PI and D-NDI The chirality influence on the library distribution was explored by setting up heterochiral DCLs. The chirality influence on the library distribution was explored by setting up heterochiral DCLs. The D enantiomerThe chirality of influence NDI [59 on] was the synthesisedlibrary distribution using was the reportedexplored by microwave-assisted setting up heterochiral method DCLs. for The D enantiomer of NDI [59] was synthesised using the reported microwave-assisted method for l-NDIThe [D60 enantiomer,61]. The l-PI: of NDId-NDI [59]mixture was synthesised mainly leads usin tog the the reported heterochiral microwave-assisted heterodimer when method no salt for is L-NDI [60,61]. The L-PI:D-NDI mixture mainly leads to the heterochiral heterodimer when no salt is presentL-NDI (Figure [60,61].6). The The L addition-PI:D-NDI of mixture salt changes mainly the leads library to the distribution heterochiral in heterodimer a similar trend when tothat no salt of theis presentpresent (Figure (Figure 6). 6). The The addition addition of of salt salt changes changes the the library library distribution distribution in in a a similar similar trend trend to to that that of of homochiralthe homochiral DCL withDCL NaNOwith NaNO3: a heterochiral3: a heterochiral heterotetramer heterotetramer is formed is formed (all (all MS MS and and MS MS/MS/MS spectra spectra are the homochiral DCL with NaNO3: a heterochiral heterotetramer is formed (all MS and MS/MS spectra provided in Figures S28–S32). The 1:1 molar ratio PI:NDI system does not show any chiral recognition, areare providedprovided inin FiguresFigures S28–S32).S28–S32). TheThe 1:11:1 molarmolar ratioratio PI:NDIPI:NDI systemsystem doesdoes not not showshow anyany chiralchiral producing similar outcomes regardless of whether homo- or heterochiral mixture of building blocks is recognition,recognition, producing producing similar similar outcomes outcomes regardless regardless of of whether whether homo- homo- or or heterochiral heterochiral mixture mixture of of used. Literature reports just one example of homo- and heterochiral DCLs based on disulfide exchange, buildingbuilding blocks blocks is is used. used. Literature Literature reports reports just just one one example example of of homo- homo- and and heterochiral heterochiral DCLs DCLs based based but no chiral recognition is observed either [13]. onon disulfide disulfide exchange, exchange, but but no no chiral chiral recognition recognition is is observed observed either either [13]. [13].

SH O SH O SH N COOH SH 5 m M O N HN O N COOH 5 m M O N HN COOH O O (to ta l co n ce n tra tio n ) HOOC COOH O HOOC NH N O HOOC N (to ta l co n ce n tra tio n ) N O HS NH HOOC N pH 8 HS O pH 8 HS O HS

L-PI D-NDI L-PI D-NDI

100% 100% heterochiral heterochiral heterodimer* no salt heterodimer* no salt

74% 74% 26% 26% heterochiral heterochiral 1 M NaNO3 heterotetramer 1 M NaNO3 heterotetramer 10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 14 Time15 (min)16 17 18 19 20 Time (min)

Figure 6. Reverse-phase HPLC analysis of Ll-PI:Dd-NDI (1:1 molar ratio, 5 mM total concentration) FigureFigure 6. Reverse-phase6. Reverse-phase HPLC HPLC analysis analysis of of L-PI:-PI:D-NDI-NDI (1:1(1:1 molarmolar ratio, 5 5 mM mM total total concentration) concentration) libraries without salt (top) and in the presence of 1 M NaNO3 (bottom). Absorptions recorded at 385 librarieslibraries without without salt salt (top) (top) and and in in thethe presencepresence of of 1 1 M M NaNO NaNO3 (bottom).3 (bottom). Absorptions Absorptions recorded recorded at 385 at nm. The unlabeled peaks did not ionise and could not be identified. * Additionally, contains L-PI 385nm. nm. The The unlabeled unlabeled peaks peaks did did not not ionise ionise and and coul couldd not notbe identified. be identiﬁed. * Additionally, * Additionally, contains contains L-PI homodimer. l-PIhomodimer.homodimer.

Symmetry 2020, 12, 726 8 of 11

The formation of macrocycles rather than interlocked molecules in pyrroloindole-based DCLs is supported by computational models. We have calculated the heat of formation of diﬀerent PI-NDI cyclic oligomers (homochiral heterodimer, DDAA and DADA tetramers, and DAAD [2]catenane). These have indicated that the DDAA tetramer arrangement is the most stable among the tetrameric and catenated species.

4. Conclusions This work reports the design and characterisation of a new building block for disulfide DCC. It is a pyrroloindole core functionalised with L-cysteine, and it respects all criteria for DCC in aqueous systems. The synthetic route is straightforward and does not require any column purification. A key step during the synthesis is the alkylation of the free -NH groups, which are prone to polymerization. The attachment of cysteine allows the chiral information to be transferred onto the otherwise achiral core, as proved by CD studies. The trityl-protected cysteine version of l-PI is relatively stable towards thermal and photodegradation. The new building block is also fluorescent, with a quantum yield of 48%, thus expanding its potential applications to (bio)sensing area. Quadrupole moment calculations for the pyrroloindole core have identified it as a more electron-rich aromatic than DN ( 22.7 B vs. 15.1 B). Homo- and heterochiral DCLs of l-PI and l- and − − d-NDIs have showed similar library distributions: homo- and heterodimers. The increase of ionic strength in the system by adding NaNO3 has led to the formation of tetramers besides dimers. The pyrroloindole core is versatile as α, β positions on the pyrrole ring and NH groups can be further functionalised to make new building blocks. The use of templates and other partners in DCLs can also led to different library distributions.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-8994/12/5/726/s1, Scheme S1: The reaction of 11, Scheme S2: The reaction of 13, Figure S1: The 1H NMR spectrum of 11, Figure S2: The 13C NMR spectrum of 11, Figure S3: The 1H NMR spectrum of 13, Figure S4: The 13C NMR spectrum of 13, Figure S5: The 1H NMR spectrum of 6, Figure S6: The 13C NMR spectrum of 6, Figure S7: The 1H NMR spectrum of 7, Figure S8: The 13C NMR spectrum of 7, Figure S9: The 1H NMR spectrum of 8, Figure S10: The 13C NMR spectrum of 8, Figure S11: The 1H NMR spectrum of 9, Figure S12: The 13C NMR spectrum of 9, Figure S13: The 1H NMR spectrum of l-PI, Figure S14: Plot of the absorbance data versus concentration used for molar extinction coefficient (ε) calculation. The data were fitted to a linear equation, Figure S15: MS (-ve) of l-PI homodimer, Figure S16: Isotope pattern of l-PI homodimer (top) and its calculated isotope pattern (bottom), Figure S17: MS/MS (-ve) of l-PI homodimer, Figure S18: MS (-ve) of homochiral heterodimer, Figure S19: Isotope pattern of homochiral heterodimer (top) and its calculated isotope pattern (bottom), Figure S20: MS/MS (-ve) of homochiral heterodimer, Figure S21: MS (-ve) of homochiral heterotrimer, Figure S22: Isotope pattern of homochiral heterotrimer (top) and its calculated isotope pattern (bottom), Figure S23: MS/MS (-ve) of homochiral heterotrimer, Figure S24: MS (-ve) of homochiral heterotetramer, Figure S25: Isotope pattern of homochiral heterotetramer (top) and its calculated isotope pattern (bottom), Figure S26: MS/MS (-ve) of homochiral heterotetramer, Figure S27: Expansion of region MS/MS (-ve) of homochiral heterotetramer, Figure S28: MS/MS (-ve) of heterochiral heterodimer, Figure S29: Isotope pattern of heterochiral heterodimer (top) and its calculated isotope pattern (bottom), Figure S30: MS/MS (-ve) of heterochiral heterodimer, Figure S31: MS (-ve) of heterochiral heterotetramer, Figure S32: Isotope pattern of heterochiral heterotetramer (top) and its calculated isotope pattern (bottom), Figure S33: Simulated structure of homochiral heterodimer, Figure S34: Simulated structure of DAAD heterotetramer, Figure S35: Different views of simulated structure of DADA heterotetramer, Figure S36: Different views of simulated structure of DAAD [2]catenane, Figure S37: Plot of calculated heat of formation versus dielectric constant of different simulated structures of PI-NDI species as described in the legend. Sections: Section 1: Synthesis and characterisation of precursors and building blocks, Section 2: Characterisation of library components, Section 3: HPLC method for DCL analyses, Section 4: Computational studies of PI-NDI systems. Author Contributions: Conceptualization, G.D.P. and T.-M.G.; writing—original draft preparation, D.-M.R. and T.-M.G.; writing—review and editing, G.D.P.; synthesis, T.-M.G.; analytical data acquisition and processing, T.-M.G. and D.-M.R.; molecular modelling: T.-M.G. and G.D.P.; supervision, G.D.P.; project administration, G.D.P.; funding acquisition, G.D.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by EPSRC-DTP studentships to T.-M.G. and D.-M.R., grant numbers EB-CH1264 and EB-BB1250, respectively. Acknowledgments: We acknowledge Shaun Reeksting from Material and Chemical Characterisation Facility at the University of Bath for MS and MS/MS analyses as well as the EPSRC UK National Mass Spectrometry Symmetry 2020, 12, 726 9 of 11 at Swansea University for high resolution mass spectrometry analyses. T.-M.G. thanks Yuto Kage for useful discussions about synthesis. Conflicts of Interest: The authors declare no conflict of interest.

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