Flexible Versus Rigid G-Quadruplex DNA Ligands: Synthesis of Two Series of Bis-indole Derivatives and Comparison of Their Interactions with G-Quadruplex DNA

Article abstract of DOI:10.1002/chem.201800078

Small molecules that target G-quadruplex (G4) DNA structures are not only valuable to study G4 biology but also for their potential as therapeutics. This work centers around how different design features of small molecules can affect the interactions with G4 DNA structures, exemplified by the development of synthetic methods to bis-indole scaffolds. Our synthesized series of bis-indole scaffolds are structurally very similar but differ greatly in the flexibility of their core structures. The flexibility of the molecules proved to be an advantage compared to locking the compounds in the presumed bioactive G4 conformation. The flexible derivatives demonstrated similar or even improved G4 binding and stabilization in several orthogonal assays even though their entropic penalty of binding is higher. In addition, molecular dynamics simulations with the c-MYC G4 structure showed that the flexible compounds adapt better to the surrounding. This was reflected by an increased number of both stacking and polar interactions with both the residues in the G4 DNA structure and the DNA residues just upstream of the G4 structure.

Full text of DOI:10.1002/chem.201800078

A Journal of
Accepted Article
Title: Flexible versus rigid G-quadruplex DNA ligands - synthesis
of two series of bis-indole derivatives and comparison of their
interactions with G-quadruplex DNA.
Authors: Bagineni Prasad, Jan Jamroskovic, Sudipta Bhowmik,
Rajendra Kumar, Tajanena Romell, Nasim Sabouri, and Erik
Chorell
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201800078
Link to VoR: http://dx.doi.org/10.1002/chem.201800078
Supported by

Chemistry - A European Journal
10.1002/chem.201800078
FlexibleꢀversusꢀrigidꢀG-quadruplexꢀDNAꢀligandsꢀ–ꢀsynthesisꢀofꢀtwoꢀseriesꢀofꢀ
bis-indoleꢀ derivativesꢀ andꢀ comparisonꢀ ofꢀ theirꢀ interactionsꢀ withꢀ G-
quadruplexꢀDNA.ꢀꢀ
ꢀ
BagineniꢀPrasad ,ꢀJanꢀJamroskovic ,ꢀSudiptaꢀBhowmik[a,ꢀc]#,ꢀRajendraꢀKumar[a],ꢀ
[
a]
[b]#
[
a]
[b]
[a]
TajanenaꢀRomell ,ꢀNasimꢀSabouri ,ꢀandꢀErikꢀChorell *ꢀ
ꢀ
[
a]ꢀDr.ꢀB.ꢀPrasad,ꢀDr.ꢀS.ꢀBhowmik,ꢀDr.ꢀR.ꢀKumar,ꢀT.ꢀRomell,ꢀandꢀDr.ꢀE.ꢀChorell.ꢀDepartmentꢀofꢀ
Chemistry,ꢀUmeåꢀUniversity,ꢀ901ꢀ87ꢀUmeå,ꢀSwedenꢀ
E-mail:ꢀerik.chorell@umu.seꢀ
[
b]ꢀDr.ꢀJ.ꢀJamroskovicꢀandꢀDr.ꢀN.ꢀSabouri.ꢀDepartmentꢀofꢀMedicalꢀBiochemistryꢀandꢀBiophysics,ꢀ
UmeåꢀUniversity,ꢀ901ꢀ87ꢀUmeå,ꢀSweden.ꢀ
c]ꢀDr.ꢀS.ꢀBhowmik.ꢀDepartmentꢀofꢀBiophysics,ꢀMolecularꢀBiologyꢀ&ꢀBioinformatics,ꢀUniversityꢀofꢀ
Calcutta,ꢀ700009,ꢀKolkata,ꢀIndiaꢀ
ꢀTheseꢀauthorsꢀcontributedꢀequallyꢀtoꢀthisꢀwork.ꢀ
[
#
Supportingꢀinformationꢀisꢀavailableꢀforꢀthisꢀarticle.ꢀ
ꢀ
Abstractꢀ
Smallꢀ moleculesꢀ thatꢀ targetꢀ G-quadruplexꢀ (G4)ꢀ DNAꢀ structuresꢀ areꢀ notꢀ onlyꢀ
valuableꢀtoꢀstudyꢀG4ꢀbiologyꢀbutꢀalsoꢀforꢀtheirꢀpotentialꢀasꢀtherapeutics.ꢀThisꢀworkꢀ
centersꢀaroundꢀhowꢀdifferentꢀdesignꢀfeaturesꢀofꢀsmallꢀmoleculesꢀcanꢀaffectꢀtheꢀ
interactionsꢀwithꢀG4ꢀDNAꢀstructures,ꢀexemplifiedꢀbyꢀtheꢀdevelopmentꢀofꢀsyntheticꢀ
methodsꢀtoꢀbis-indoleꢀscaffolds.ꢀOurꢀsynthesizedꢀseriesꢀofꢀbis-indoleꢀscaffoldsꢀareꢀ
structurallyꢀveryꢀsimilarꢀbutꢀdifferꢀgreatlyꢀinꢀtheꢀflexibilityꢀofꢀtheirꢀcoreꢀstructures.ꢀ
Theꢀflexibilityꢀofꢀtheꢀmoleculesꢀprovedꢀtoꢀbeꢀanꢀadvantageꢀcomparedꢀtoꢀlockingꢀtheꢀ
compoundsꢀinꢀtheꢀpresumedꢀbioactiveꢀG4ꢀconformation.ꢀTheꢀflexibleꢀderivativesꢀ
demonstratedꢀsimilarꢀorꢀevenꢀimprovedꢀG4ꢀbindingꢀandꢀstabilizationꢀinꢀseveralꢀ
orthogonalꢀ assaysꢀ evenꢀ thoughꢀ theirꢀ entropicꢀ penaltyꢀ ofꢀ bindingꢀ isꢀ higher.ꢀ Inꢀ
addition,ꢀmolecularꢀdynamicsꢀsimulationsꢀwithꢀtheꢀc-MYCꢀG4ꢀstructureꢀshowedꢀ
thatꢀtheꢀflexibleꢀcompoundsꢀadaptꢀbetterꢀtoꢀtheꢀsurrounding.ꢀThisꢀwasꢀreflectedꢀ
byꢀanꢀincreasedꢀnumberꢀofꢀbothꢀstackingꢀandꢀpolarꢀinteractionsꢀwithꢀ bothꢀ theꢀ
residuesꢀinꢀtheꢀG4ꢀDNAꢀstructureꢀandꢀtheꢀDNAꢀresiduesꢀjustꢀupstreamꢀofꢀtheꢀG4ꢀ
structure.ꢀ
ꢀ
Introduction:ꢀ
G-quadruplexꢀ(G4)ꢀDNAꢀstructuresꢀplayꢀimportantꢀrolesꢀinꢀnumerousꢀbiologicalꢀ
processesꢀbutꢀareꢀalsoꢀinvolvedꢀinꢀinducingꢀgenomeꢀinstability.ꢀForꢀtheseꢀreasons,ꢀ
G4ꢀDNAꢀstructuresꢀareꢀessentialꢀstructuresꢀtoꢀstudy.[1-2]ꢀꢀTheꢀfour-strandedꢀnucleicꢀ
acidꢀsecondaryꢀstructureꢀofꢀaꢀG4ꢀdifferꢀfromꢀtheꢀWatson-CrickꢀB-DNAꢀstructureꢀinꢀ
manyꢀaspects.ꢀItꢀisꢀconstructedꢀbyꢀplanarꢀguanosineꢀtetradsꢀthatꢀstackꢀonꢀtopꢀofꢀ
eachꢀotherꢀandꢀeachꢀguanosineꢀtetradꢀisꢀheldꢀtogetherꢀbyꢀHoogsteenꢀhydrogenꢀ
bonding.[ ꢀG4ꢀstructuresꢀexhibitꢀenormousꢀstructuralꢀpolymorphismꢀcomparedꢀ
toꢀdoubleꢀstrandedꢀDNA.ꢀTheseꢀstructuralꢀvariationsꢀinꢀG4sꢀdependsꢀonꢀtheꢀlength,ꢀ
number,ꢀ directionality,ꢀ andꢀ sequenceꢀ ofꢀ bothꢀtheꢀ DNAꢀ strandsꢀ formingꢀ theꢀG4ꢀ
structure,ꢀandꢀtheꢀloopsꢀthatꢀconnectꢀtheꢀguanosineꢀstrands.[ ꢀFurthermore,ꢀtheꢀ
3-4]
5-8]
cationsꢀthatꢀstabilizeꢀtheꢀguanosineꢀtetradsꢀareꢀalsoꢀimportantꢀforꢀtheꢀstructuralꢀ
variationsꢀofꢀG4ꢀstructures.[
7,ꢀ9-11] ꢀ
.Itꢀisꢀestimatedꢀthatꢀmoreꢀthanꢀ700,000ꢀpositionsꢀ
inꢀtheꢀhumanꢀgenomeꢀcanꢀformꢀG4ꢀstructures.[12]ꢀTheꢀlocationsꢀofꢀG4ꢀstructuresꢀinꢀ
theꢀ genomeꢀ areꢀ notꢀ random,ꢀ asꢀ theyꢀ clusterꢀ inꢀ specificꢀ regionsꢀ ofꢀ DNA.ꢀ Theseꢀ
regionsꢀincludeꢀpromoters,ꢀtelomeres,ꢀbothꢀ5’ꢀandꢀ3’ꢀuntranslatedꢀregionsꢀ(UTRs),ꢀ
ribosomalꢀDNA,ꢀandꢀoriginsꢀofꢀreplication.[1-2,ꢀ12-16]ꢀTheꢀfrequencyꢀofꢀG4ꢀmotifsꢀinꢀ
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Chemistry - A European Journal
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promotersꢀisꢀhighꢀ(>40%)ꢀandꢀespeciallyꢀthoseꢀofꢀoncogenesꢀandꢀregulatorꢀgenesꢀ
asꢀ comparedꢀ toꢀ housekeepingꢀ genes.[ ꢀ G4ꢀ structuresꢀ haveꢀ generatedꢀ aꢀ lotꢀ ofꢀ
13]
interestꢀinꢀrecentꢀyearsꢀbecauseꢀtheirꢀformationꢀisꢀwidelyꢀassociatedꢀwithꢀvariousꢀ
humanꢀdiseasesꢀsuchꢀasꢀcancer,ꢀandꢀneurodegenerativeꢀdiseases.[
17-21]
ꢀ
SmallꢀmoleculesꢀthatꢀbindꢀandꢀstabilizeꢀG4ꢀstructuresꢀhaveꢀgreatꢀpotentialꢀnotꢀonlyꢀ
asꢀresearchꢀtoolsꢀtoꢀunderstandꢀtheꢀcomplexꢀrolesꢀofꢀG4ꢀstructuresꢀinꢀbiology,ꢀbutꢀ
alsoꢀ forꢀ theirꢀ potentialꢀ toꢀ beꢀ furtherꢀ developedꢀ intoꢀ therapeutics.ꢀ Toꢀ furtherꢀ
advanceꢀtheꢀdevelopmentꢀofꢀtheseꢀsmallꢀmolecules,ꢀitꢀisꢀimportantꢀtoꢀlearnꢀmoreꢀ
aboutꢀhowꢀsmallꢀmoleculesꢀinteractꢀwithꢀG4ꢀstructuresꢀandꢀwhichꢀdesignꢀelementsꢀ
thatꢀareꢀimportantꢀforꢀaꢀstrongꢀandꢀselectiveꢀbinding.ꢀꢀ
ToꢀaddressꢀsomeꢀofꢀtheseꢀissuesꢀandꢀsimultaneouslyꢀdevelopꢀnewꢀG4ꢀbindingꢀandꢀ
stabilizingꢀ compounds,ꢀ weꢀ hereinꢀ establishedꢀ syntheticꢀ methodsꢀ ofꢀ twoꢀ
complementaryꢀ(flexibleꢀandꢀrigid)ꢀseriesꢀofꢀbis-indoleꢀstructures.ꢀThereꢀareꢀonlyꢀ
twoꢀpreviousꢀrecordsꢀofꢀbis-indolesꢀthatꢀwereꢀdesignedꢀtoꢀbindꢀandꢀstabilizeꢀG4ꢀ
structures.[
22-23]ꢀ
Oneꢀ ofꢀ theseꢀ examplesꢀ areꢀ theꢀ 2,2’-diindolylmethanes,ꢀ whichꢀ
possessedꢀequalꢀorꢀevenꢀimprovedꢀG4ꢀbindingꢀabilityꢀcomparedꢀtoꢀtheꢀfrequentlyꢀ
usedꢀandꢀhighlyꢀefficientꢀG4ꢀbindingꢀcompoundꢀPhen-DC3.[23-24]ꢀForꢀtheseꢀclassesꢀ
ofꢀcompounds,ꢀaꢀcrescentꢀshapeꢀhasꢀbeenꢀsuggestedꢀtoꢀbeꢀimportantꢀforꢀtheꢀcorrectꢀ
positioningꢀofꢀtheꢀsideꢀchainsꢀoverꢀtheꢀtopꢀG-tetradꢀinꢀtheꢀG4ꢀstructureꢀtoꢀallowꢀforꢀ
anꢀefficientꢀpi-piꢀstacking.[
24-27]
ꢀꢀ
Theꢀtwoꢀnewꢀbis-indoleꢀcentralꢀfragmentsꢀdevelopedꢀinꢀthisꢀworkꢀareꢀbasedꢀonꢀtheꢀ
,2’-diindolylmethanes.ꢀOneꢀofꢀtheꢀcentralꢀfragmentsꢀareꢀrigidifiedꢀ(scaffoldꢀ II)ꢀ
2
andꢀforcedꢀtowardsꢀtheꢀcrescentꢀshapedꢀbioactiveꢀG4ꢀconformation,ꢀwhereasꢀtheꢀ
otherꢀisꢀmoreꢀflexibleꢀ(scaffoldꢀI)ꢀandꢀhasꢀtheꢀabilityꢀtoꢀadoptꢀmanyꢀconformationsꢀ
thatꢀmayꢀnotꢀbeꢀoptimalꢀforꢀG4ꢀDNAꢀbindingꢀ(Figureꢀ1).ꢀInꢀaddition,ꢀitꢀisꢀwell-
knownꢀ thatꢀ positivelyꢀ chargedꢀ sideꢀ chainsꢀ onꢀ G4ꢀ bindingꢀ compoundsꢀ canꢀ
dramaticallyꢀ increaseꢀ theꢀ bindingꢀ affinity.[26]ꢀ Alongꢀ thisꢀ line,ꢀ methylatedꢀ
quinolinesꢀareꢀfrequentlyꢀusedꢀasꢀsideꢀchains.ꢀChungꢀet.ꢀal.ꢀrecentlyꢀreportedꢀanꢀ
NMRꢀ studyꢀ ofꢀ howꢀ theꢀ G4ꢀ stabilizingꢀ compound,ꢀ Phen-DC3,ꢀ bindꢀ throughꢀ
extensiveꢀpi-piꢀstackingꢀwithꢀtheꢀguanineꢀbasesꢀatꢀtheꢀtopꢀofꢀtheꢀG-tetrad.[28]ꢀTheꢀ
extensionꢀofꢀtheꢀN-methylꢀgroupsꢀwasꢀdescribedꢀasꢀaꢀpotentialꢀintroductionꢀpointꢀ
toꢀ identifyꢀ strongerꢀ bindingꢀ interactions.ꢀ However,ꢀ extensionꢀ ofꢀ theꢀ N-methylꢀ
groupsꢀ hasꢀ toꢀ theꢀ bestꢀ ofꢀ ourꢀ knowledgeꢀ neverꢀ beenꢀ presented.ꢀ Hence,ꢀ theꢀ
importanceꢀ ofꢀ theꢀ substituentꢀ thatꢀ quaternizeꢀ theꢀ sideꢀ chainsꢀ hasꢀ alsoꢀ beenꢀ
investigated.ꢀꢀ
ꢀ
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Chemistry - A European Journal
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Figureꢀ1.ꢀOutlineꢀofꢀtheꢀsynthesisꢀofꢀtheꢀflexibleꢀ(scaffoldꢀI)ꢀandꢀrigidꢀ(scaffoldꢀII)ꢀcentralꢀ
fragments.ꢀꢀ
ꢀ
TheꢀcompoundsꢀabilityꢀtoꢀbindꢀandꢀstabilizeꢀG4ꢀstructuresꢀwasꢀevaluatedꢀbyꢀusingꢀ
theꢀDNAꢀpolymeraseꢀstopꢀassayꢀandꢀtheꢀThioflavinꢀTꢀ(ThT)ꢀdisplacementꢀassay.ꢀ
Sixꢀofꢀtheꢀmostꢀinterestingꢀcompoundsꢀfromꢀtheꢀscreensꢀwereꢀfurtherꢀevaluatedꢀ
byꢀ circularꢀ dichroismꢀ (CD)ꢀ meltingꢀ studies,ꢀ CDꢀ titrations,ꢀ Fluorescenceꢀ
IntercalatorꢀDisplacementꢀ(FID)ꢀassay,ꢀfullꢀdose-responseꢀstudiesꢀwithꢀtheꢀDNAꢀ
polymeraseꢀstopꢀassay,ꢀandꢀmolecularꢀdynamicsꢀ(MD)ꢀsimulations.ꢀThisꢀrevealedꢀ
bothꢀ effectiveꢀ G4ꢀ bindingꢀ andꢀ stabilizingꢀ compoundsꢀ andꢀ displayedꢀ importantꢀ
designꢀ elementsꢀ andꢀ interactionꢀ pointsꢀ toꢀ considerꢀ inꢀ theꢀ optimizationꢀ ofꢀ G4ꢀ
ligands.ꢀ
ꢀ
ResultsꢀandꢀDiscussionsꢀ
ꢀ
SynthesisꢀofꢀflexibleꢀscaffoldꢀIꢀandꢀrigidꢀscaffoldꢀII
Theꢀtwoꢀbis-indoleꢀcentralꢀfragmentsꢀdescribedꢀinꢀthisꢀstudyꢀwereꢀdesignedꢀtoꢀbeꢀ
asꢀsimilarꢀasꢀpossibleꢀbutꢀstillꢀvaryꢀinꢀtheirꢀflexibility.ꢀꢀThereꢀareꢀmanyꢀplausibleꢀ
syntheticꢀroutesꢀtoꢀachieveꢀtheseꢀtargetꢀcentralꢀfragments.ꢀHowever,ꢀmanyꢀofꢀtheꢀ
initialꢀ routesꢀ thatꢀ wereꢀ testedꢀ provedꢀ toꢀ beꢀ problematic,ꢀ seeꢀ Figureꢀ S1.ꢀ Theꢀ
successfulꢀsynthesisꢀofꢀtheꢀrigidꢀandꢀflexibleꢀscaffoldsꢀwasꢀfinallyꢀachievedꢀstartingꢀ
fromꢀtheꢀaffordableꢀandꢀcommerciallyꢀavailableꢀindolineꢀ(1),ꢀwhichꢀwasꢀconvertedꢀ
toꢀ 6-nitroindolineꢀ (2)ꢀ (Schemeꢀ 1).ꢀ Thisꢀ wasꢀ achievedꢀ byꢀ aꢀ selectiveꢀ nitrationꢀ
reactionꢀusingꢀaꢀHNO
yield.ꢀSubsequentꢀoxidationꢀofꢀ2ꢀinꢀtheꢀpresenceꢀofꢀMnO
3)ꢀinꢀquantitativeꢀyield.ꢀReductionꢀofꢀtheꢀ6-nitroindoleꢀbyꢀhydrogenationꢀwithꢀ
Pd/Cꢀgaveꢀ6-amino-indole,ꢀwhichꢀwasꢀfurtherꢀprotectedꢀwithꢀBoc-anhydrideꢀ(di-
tert-butylꢀdicarbonate)ꢀinꢀpresenceꢀofꢀNaHCO ꢀtoꢀgiveꢀintermediateꢀ5ꢀinꢀ77%ꢀyieldꢀ
3 2
ꢀandꢀH SO4ꢀmixtureꢀatꢀroomꢀtemperatureꢀtoꢀgiveꢀ2ꢀinꢀ80%ꢀ
2
ꢀaffordedꢀ6-nitroindoleꢀ
(
3
overꢀtwoꢀsteps.ꢀNext,ꢀtheꢀkeyꢀcarbonylꢀinsertionꢀreactionꢀbetweenꢀtheꢀtwoꢀindolesꢀ
atꢀtheꢀN-1ꢀpositionsꢀwasꢀdeveloped,ꢀseeꢀFigureꢀS1.ꢀByꢀusingꢀtheseꢀconditions,ꢀtheꢀ
desiredꢀ carbonylꢀ insertionꢀ workedꢀ wellꢀ andꢀ gaveꢀ 6ꢀ inꢀ 64%ꢀ yield.ꢀ Theꢀ tert-
butyloxycarbonylꢀ(Boc)ꢀprotectingꢀgroupꢀwasꢀnextꢀremovedꢀinꢀtheꢀpresenceꢀofꢀ
TFAꢀtoꢀgiveꢀtheꢀbis(6-amino-1H-indol-1-yl)methanoneꢀ(7)ꢀinꢀquantitativeꢀyield.ꢀ
Theꢀ couplingꢀ reactionꢀ betweenꢀ theꢀ bis(6-amino-1H-indol-1-yl)methanoneꢀ andꢀ
quinoline-3-carboxylicꢀacidꢀwithꢀHATUꢀgaveꢀ96%ꢀyieldꢀofꢀscaffoldꢀIꢀꢀ(8).ꢀNext,ꢀtheꢀ
introductionꢀofꢀtheꢀcarbon-carbonꢀbondꢀbetweenꢀtheꢀtwoꢀC2ꢀandꢀC2’ꢀpositionsꢀwasꢀ
developed,ꢀseeꢀTableꢀS1.ꢀByꢀusingꢀtheseꢀconditionsꢀ(Pd(OAc)
aceticꢀacidꢀatꢀ100ꢀ°C),ꢀscaffoldꢀIIꢀ(9)ꢀcouldꢀbeꢀisolatedꢀinꢀ64%ꢀyieldꢀ(Schemeꢀ1).ꢀ
2
ꢀandꢀCu(OAc)
2
ꢀinꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀ
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Chemistry - A European Journal
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Schemeꢀ 1.ꢀ Synthesisꢀ ofꢀ N,N'-(6-oxo-6H-imidazo[1,5-a:3,4-a']diindole-3,9-diyl)bis(quinoline-3-
carboxamide).ꢀa) HNO , H SO , rt, 1 h, 80%; b) MnO , Dry DCM, rt, 12 h, 92%; c) Pd/C, H , MeOH,
rt, 3 h, 84%; d) (Boc) O Sat. NaHCO , THF, rt, 16 h, 92%; e) CDI, DMF, MWI, 150 °C, 1 h, 64% f)
TFA, DCM, rt, 3 h, 97%; g) quinoline-3-carboxylic acid, HATU, DIPEA, DMF, rt, 12 h, 96%; h)
3
2
4
2
2
2
3
Pd(OAc)
2
, Cu(OAc) , AcOH, 100 °C, 20 h, 64%.ꢀ
2
ꢀ
QuaternizationꢀofꢀtheꢀsideꢀchainsꢀonꢀscaffoldꢀIꢀandꢀII.ꢀ
Toꢀ investigateꢀ theꢀ importanceꢀ ofꢀ theꢀ substituentꢀ thatꢀ quaternizeꢀ theꢀ
aminoquinolineꢀ sideꢀ chains,ꢀ weꢀ developedꢀ syntheticꢀ methodsꢀ toꢀ variousꢀ
analoguesꢀofꢀquaternizedꢀsaltsꢀfromꢀscaffoldꢀIꢀandꢀII,ꢀTableꢀ1.ꢀAlkylꢀbromidesꢀandꢀ
iodidesꢀareꢀparticipatingꢀwellꢀinꢀthisꢀreactionꢀandꢀaffordedꢀgoodꢀyieldsꢀ(Tableꢀ1,ꢀ
entriesꢀ1-3,ꢀ5,ꢀ6,ꢀ11-14).ꢀNotably,ꢀsubstitutedꢀallylꢀbromideꢀandꢀbenzylꢀbromidesꢀ
alsoꢀproducedꢀmoderateꢀtoꢀgoodꢀyieldsꢀ(Tableꢀ1,ꢀentriesꢀ4,ꢀ7-10).ꢀTheꢀreactionꢀ
proceededꢀwellꢀforꢀbothꢀscaffoldꢀIꢀandꢀIIꢀresultingꢀinꢀtheꢀdesiredꢀproductsꢀinꢀ46-
8
ꢀ
8%ꢀyields.ꢀꢀ
ꢀ
a
Tableꢀ1.ꢀSynthesisꢀofꢀquaternizedꢀanaloguesꢀofꢀscaffoldꢀIꢀandꢀII. ꢀꢀ
ꢀ
Timeꢀ Yieldꢀ
(
1
Entryꢀ Reactantꢀ Productꢀ
R ꢀ
Xꢀ
min)ꢀ (%)bꢀ
1
2
3
ꢀ
ꢀ
ꢀ
8ꢀ
8ꢀ
8ꢀ
11aꢀ
11bꢀ
11cꢀ
Iꢀ 70ꢀ 68ꢀ
Iꢀ 30ꢀ 65ꢀ
Iꢀ 20ꢀ 82ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
4ꢀ
8ꢀ
11dꢀ
Brꢀ 70ꢀ 78ꢀ
ꢀ
5
ꢀ
ꢀ
8ꢀ
8ꢀ
11eꢀ
11fꢀ
Brꢀ 25ꢀ 88ꢀ
Brꢀ 40ꢀ 60ꢀ
6
ꢀ
7ꢀ
8ꢀ
11gꢀ
Brꢀ 60ꢀ 62ꢀ
ꢀ
8ꢀ
8ꢀ
11hꢀ
Brꢀ 20ꢀ 74ꢀ
ꢀ
9
ꢀ
8ꢀ
8ꢀ
11iꢀ
11jꢀ
Brꢀ 20ꢀ 75ꢀ
Brꢀ 40ꢀ 63ꢀ
ꢀ
10ꢀ
ꢀ
1
1
1
1ꢀ
2ꢀ
3ꢀ
9ꢀ
9ꢀ
9ꢀ
12aꢀ
12bꢀ
12cꢀ
Iꢀ 180ꢀ 55ꢀ
Iꢀ 180ꢀ 61ꢀ
Iꢀ 180ꢀ 65ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
14ꢀ
9ꢀ
12dꢀ
Brꢀ 240ꢀ 46ꢀ
aꢀ
Allꢀreactionsꢀwereꢀperformedꢀusingꢀ8ꢀorꢀ9ꢀ(0.016-0.041ꢀmmol)ꢀinꢀalkyl/benzylꢀhalidesꢀatꢀ140ꢀ°Cꢀ
microwaveꢀheating)ꢀforꢀ20ꢀminꢀtoꢀ240ꢀmin.ꢀꢀ
Isolatedꢀyield.ꢀ
(
bꢀ
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CompoundꢀscreeningꢀbyꢀTaqꢀDNAꢀpolymeraseꢀstopꢀassayꢀandꢀThioflavinꢀTꢀ
ThT)ꢀ displacementꢀ assayꢀ identifiesꢀ compoundsꢀ withꢀ efficientꢀ G4ꢀ
stabilizationꢀandꢀbinding.ꢀꢀ
(
G4ꢀstabilizationꢀcanꢀbeꢀassayedꢀbyꢀtheꢀTaqꢀDNAꢀpolymeraseꢀstopꢀassay.[29]ꢀInꢀthisꢀ
assay,ꢀaꢀlabeledꢀprimerꢀisꢀannealedꢀtoꢀaꢀDNAꢀtemplate,ꢀandꢀtheꢀabilityꢀofꢀtheꢀTaqꢀ
DNAꢀ polymeraseꢀ toꢀ synthesizeꢀ theꢀ templateꢀ isꢀ examinedꢀ onꢀ aꢀ denaturatingꢀ
polyacrylamideꢀgel,ꢀandꢀtheꢀamountꢀofꢀfull-lengthꢀproductꢀcanꢀbeꢀquantified.ꢀInꢀ
addition,ꢀifꢀtheꢀDNAꢀtemplateꢀcausesꢀpausingꢀorꢀarrestꢀofꢀtheꢀTaqꢀDNAꢀpolymerase,ꢀ
theꢀ positionꢀ ofꢀ pausingꢀ siteꢀ andꢀ amountꢀ ofꢀ pausingꢀ product,ꢀ andꢀ full-lengthꢀ
productꢀisꢀdetectedꢀonꢀtheꢀgel.ꢀWeꢀannealedꢀaꢀfluorescentꢀTET-labeledꢀprimerꢀtoꢀaꢀ
DNAꢀ templateꢀ thatꢀ includedꢀ aꢀ G4ꢀ motifꢀ fromꢀ theꢀ ribosomalꢀ DNAꢀ ofꢀ
Schizosaccharomycesꢀpombeꢀgenome.[ ꢀG4ꢀmotifsꢀareꢀenrichedꢀinꢀribosomalꢀDNAꢀ
andꢀthisꢀenrichmentꢀisꢀevolutionaryꢀconserved.[14-15,ꢀ30-31]ꢀTheꢀwell-characterizedꢀ
16]
S.ꢀpombeꢀrDNAꢀG4ꢀmotifꢀformsꢀaꢀstableꢀparallelꢀG4ꢀstructureꢀinꢀvitro,ꢀandꢀarrestꢀtheꢀ
Taqꢀ DNAꢀ polymerase.[
32-33]
ꢀ Asꢀ aꢀ control,ꢀ weꢀ alsoꢀ annealedꢀ aꢀ non-G4ꢀ formingꢀ
sequenceꢀfromꢀtheꢀS.ꢀpombeꢀgenomeꢀtoꢀtheꢀTET-labeledꢀprimer,ꢀwhichꢀdoesꢀnotꢀ
arrestꢀ theꢀ Taqꢀ DNAꢀ polymeraseꢀ (Figureꢀ 2A).[33-34]ꢀ Next,ꢀ weꢀ askedꢀ ifꢀ ourꢀ newꢀ
compoundsꢀ fromꢀ scaffoldꢀ Iꢀ andꢀ IIꢀ (Tableꢀ 1)ꢀ couldꢀ bindꢀ andꢀ stabilizeꢀ theꢀ G4ꢀ
structureꢀorꢀtheꢀnon-G4ꢀDNA.ꢀTheꢀreactionsꢀwereꢀrunꢀforꢀbothꢀtheꢀnon-G4ꢀtemplateꢀ
andꢀtheꢀribosomalꢀG4ꢀDNAꢀtemplateꢀforꢀtenꢀminutesꢀinꢀtheꢀpresenceꢀofꢀ1ꢀµMꢀofꢀ
compoundꢀ orꢀ DMSOꢀ (Figureꢀ 2B).ꢀ Mostꢀ ofꢀ theꢀ compoundsꢀ fromꢀ bothꢀ scaffoldsꢀ
efficientlyꢀstabilizedꢀtheꢀribosomalꢀG4ꢀDNAꢀstructureꢀandꢀanꢀenhancedꢀpausingꢀ
siteꢀeitherꢀoneꢀorꢀtwoꢀnucleotidesꢀbeforeꢀtheꢀfirstꢀG-tractꢀwasꢀdetectedꢀ(Figureꢀ2B).ꢀ
Inꢀaddition,ꢀtheꢀamountꢀofꢀfullꢀlengthꢀproductꢀwasꢀdecreasedꢀcomparedꢀtoꢀtheꢀ
DMSOꢀcontrolꢀ(Figureꢀ2C).ꢀTheꢀresultsꢀalsoꢀshowedꢀthatꢀallꢀcompoundsꢀwereꢀmoreꢀ
selectiveꢀ forꢀ theꢀ G4ꢀ DNAꢀ structureꢀ comparedꢀ toꢀ theꢀ non-G4ꢀ DNA.ꢀ However,ꢀ
althoughꢀallꢀcompoundsꢀdisplayedꢀG4ꢀselectivity,ꢀtheꢀrigidꢀscaffoldꢀIIꢀcompoundsꢀ
alsoꢀshowedꢀdecreasedꢀamountꢀofꢀsynthesizedꢀfull-lengthꢀproductꢀalsoꢀonꢀtheꢀnon-
G4ꢀDNAꢀtemplateꢀ(e.g.ꢀ12a,ꢀ12b,ꢀ12c,ꢀandꢀ12dꢀshowedꢀaboutꢀ70%,ꢀ45%,ꢀ45%,ꢀandꢀ
3
9
0%ꢀreductionꢀofꢀfull-lengthꢀproductꢀforꢀtheꢀnon-G4ꢀtemplateꢀcomparedꢀtoꢀaboutꢀ
5%,ꢀ85%,ꢀ80%,ꢀandꢀ70%ꢀreductionꢀofꢀfull-lengthꢀproductꢀforꢀtheꢀG4ꢀtemplate,ꢀ
respectively).ꢀAsꢀoutlined,ꢀtheꢀquaternizationꢀofꢀtheꢀquinolineꢀsideꢀchainsꢀofꢀtheꢀ
bis-indolesꢀprovedꢀhighlyꢀimportantꢀforꢀtheꢀcompoundsꢀabilityꢀtoꢀblockꢀtheꢀDNAꢀ
polymeraseꢀasꢀexemplifiedꢀbyꢀtheꢀneutralꢀcompoundꢀ8,ꢀwhichꢀshowedꢀnoꢀactivityꢀ
atꢀthisꢀconcentrationꢀandꢀwasꢀthusꢀnotꢀfurtherꢀtested.ꢀFurthermore,ꢀtheꢀvariationꢀ
ofꢀtheꢀquaternizationꢀsubstituentsꢀalsoꢀprovedꢀimportantꢀforꢀtheꢀactivity;ꢀsmallerꢀ
substituentsꢀlikeꢀinꢀ11a-dꢀandꢀ12a-dꢀ(Tableꢀ1,ꢀentryꢀ1-4ꢀandꢀ11-14)ꢀwereꢀhighlyꢀ
activeꢀ(Figureꢀ2BꢀandꢀC).ꢀAlso,ꢀtheꢀbenzylꢀsubstituentsꢀinꢀ11gꢀandꢀ11iꢀ(Tableꢀ1,ꢀ
entryꢀ7ꢀandꢀ9)ꢀandꢀtheꢀbutyronitrileꢀsubstituentꢀinꢀ11fꢀ(Tableꢀ1,ꢀentryꢀ6)ꢀwereꢀwellꢀ
toleratedꢀ(11f,ꢀ11g,ꢀandꢀ11iꢀreducedꢀtheꢀamountꢀofꢀfullꢀproductꢀbyꢀaboutꢀ60%,ꢀ
5
5%,ꢀandꢀ70%,ꢀrespectively)ꢀalthoughꢀtheꢀselectivityꢀforꢀG4ꢀoverꢀnon-G4ꢀDNAꢀwasꢀ
slightlyꢀdecreasedꢀforꢀ11iꢀ(aboutꢀ20%ꢀreductionꢀofꢀtheꢀamountꢀofꢀfullꢀproduct).ꢀ
Theꢀsubstitutionsꢀonꢀtheꢀbenzylꢀgroupꢀprovedꢀimportantꢀasꢀ11hꢀandꢀ11jꢀ(Tableꢀ1,ꢀ
entryꢀ8ꢀandꢀ10)ꢀwereꢀlessꢀactiveꢀ(30%ꢀandꢀ40%ꢀreductionꢀofꢀfullꢀproductꢀforꢀtheꢀ
G4ꢀtemplate,ꢀrespectively).ꢀOverall,ꢀtheꢀmostꢀactiveꢀandꢀselectiveꢀflexibleꢀscaffoldꢀ
Iꢀcompoundsꢀwereꢀinꢀtheꢀsameꢀrangeꢀofꢀactivityꢀandꢀselectivityꢀinꢀthisꢀassayꢀasꢀtheꢀ
mostꢀ activeꢀ 2,2´-diindolylmethaneꢀ (ECH22,ꢀ Figureꢀ S2)ꢀ usedꢀ asꢀ referenceꢀ
compound.ꢀ Theꢀ rigidꢀ scaffoldꢀ IIꢀ compoundsꢀ onꢀ theꢀ otherꢀ hand,ꢀ inꢀ generalꢀ
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displayedꢀ increasedꢀ activityꢀ comparedꢀ toꢀ theꢀ referenceꢀ butꢀ alsoꢀ reducedꢀ
selectivity.ꢀꢀ
ꢀ
Figureꢀ2.ꢀPrimaryꢀscreeningꢀofꢀtheꢀcompoundsꢀbyꢀTaq-polymeraseꢀstopꢀassay.ꢀ(A)ꢀSequencesꢀofꢀ
non-G4ꢀ DNAꢀandꢀ ribosomalꢀ G4ꢀDNAꢀ primer-templatesꢀ usedꢀ inꢀ thisꢀ assay.ꢀ Primerꢀ annealedꢀ toꢀ
complementaryꢀtemplateꢀDNAꢀisꢀinꢀred.ꢀTheꢀGGG-tractsꢀfromꢀtheꢀribosomalꢀG4ꢀDNAꢀareꢀmarkedꢀinꢀ
boldꢀ andꢀ numberedꢀ (1,ꢀ 2,ꢀ 3,ꢀ andꢀ 4)ꢀ withꢀ respectꢀ toꢀ theꢀ directionꢀ ofꢀ DNAꢀ synthesis.ꢀ Allꢀ DNAꢀ
sequencesꢀareꢀderivedꢀfromꢀS.ꢀpombeꢀgenome.ꢀRibosomalꢀDNAꢀisꢀtakenꢀfromꢀrDNAꢀregionꢀandꢀnon-
+
G4ꢀDNAꢀisꢀfromꢀade6 ꢀopenꢀreadingꢀframe.ꢀ(B)ꢀTaqꢀpolymeraseꢀstopꢀassayꢀforꢀribosomalꢀG4ꢀDNAꢀ
andꢀnon-G4ꢀDNAꢀinꢀtheꢀpresenceꢀofꢀ5ꢀ%ꢀ(v/v)ꢀDMSOꢀorꢀ1ꢀµMꢀcorrespondingꢀcompoundꢀ(labeledꢀ
aboveꢀeachꢀline).ꢀTheꢀreactionsꢀwereꢀstartedꢀbyꢀadditionꢀofꢀ0.025ꢀunitsꢀofꢀTaq-polymeraseꢀandꢀ
incubatedꢀ forꢀ 10ꢀ minutesꢀ atꢀ 50°C.ꢀ Allꢀ reactionsꢀ wereꢀ loadedꢀ onꢀ aꢀ denaturingꢀ 10ꢀ %ꢀ (v/v)ꢀ
polyacrylamideꢀsequencingꢀgel,ꢀandꢀvisualizedꢀbyꢀTyphoonꢀscanner.ꢀTheꢀblackꢀarrowsꢀindicateꢀtheꢀ
positionꢀofꢀtheꢀprimer.ꢀTheꢀopenꢀarrowsꢀindicateꢀfullyꢀextendedꢀprimer.ꢀGGG-tractsꢀareꢀshownꢀonꢀ
theꢀrightꢀsideꢀofꢀtheꢀgelꢀareꢀnumberedꢀandꢀshownꢀinꢀboldꢀaccordingꢀtoꢀtheꢀsameꢀpositionsꢀasꢀinꢀA.ꢀ
st
AsterisksꢀindicateꢀtheꢀpausingꢀsitesꢀofꢀTaq-polymeraseꢀoneꢀorꢀtwoꢀnucleotidesꢀupstreamꢀofꢀtheꢀ1 ꢀ
GGG-tract.ꢀ (C)ꢀ Relativeꢀ quantificationꢀ ofꢀ full-lengthꢀ productꢀ inꢀ eachꢀ reactionꢀ toꢀ DMSOꢀ treatedꢀ
sampleꢀfromꢀtwoꢀindependentꢀexperimentsꢀforꢀeachꢀDNAꢀtemplate:ꢀribosomalꢀG4ꢀDNAꢀandꢀnon-G4ꢀ
DNA.ꢀErrorꢀbarsꢀrepresentꢀabsoluteꢀerror.ꢀ
ꢀ
Theꢀcompounds’ꢀabilityꢀtoꢀbindꢀG4ꢀstructuresꢀwereꢀfurtherꢀevaluatedꢀthroughꢀtheꢀ
ThTꢀdisplacementꢀassay.ꢀꢀThTꢀboundꢀtoꢀaꢀG4ꢀstructure,ꢀbutꢀnotꢀtoꢀsingleꢀstrandedꢀ
[
35-
DNA,ꢀgivesꢀanꢀenhancedꢀfluorescenceꢀpeakꢀatꢀ480ꢀnmꢀwhenꢀexcitedꢀatꢀ440ꢀnm.
36]
ꢀ
ꢀCompoundsꢀwithꢀhigherꢀaffinityꢀtoꢀtheꢀG4ꢀstructureꢀthanꢀThTꢀwillꢀdisplaceꢀThTꢀ
fromꢀtheꢀG4ꢀstructure,ꢀwhichꢀisꢀmeasuredꢀbyꢀaꢀreducedꢀfluorescenceꢀintensity.[
23,ꢀ
33]ꢀ
Inꢀthisꢀassay,ꢀthreeꢀdifferentꢀtypesꢀofꢀG4ꢀformingꢀoligonucleotidesꢀwereꢀused;ꢀ
theꢀ ribosomalꢀ (parallel)ꢀ andꢀ telomericꢀ (mixtureꢀ ofꢀ parallelꢀ andꢀ antiparallelꢀ
structure,ꢀorꢀaꢀhybridꢀstructure)ꢀG4ꢀDNAꢀfromꢀS.ꢀpombe,[37]ꢀandꢀtheꢀhumanꢀbasedꢀ
Pu24Tꢀc-MYCꢀ(parallel)ꢀ(FigureꢀS3A).[ ꢀTheꢀcompoundsꢀwereꢀtestedꢀatꢀ1,ꢀ5,ꢀandꢀ
38]
1
5ꢀ µM.ꢀ Mostꢀ ofꢀ theꢀ testedꢀ compoundsꢀ efficientlyꢀ displacedꢀ ThTꢀ inꢀ aꢀ doseꢀ
dependentꢀmannerꢀonꢀallꢀthreeꢀG4ꢀsubstratesꢀ(FigureꢀS3B-D).ꢀCompoundsꢀ11gꢀandꢀ
1
1jꢀwereꢀlessꢀactiveꢀthanꢀtheꢀotherꢀtestedꢀcompounds,ꢀespeciallyꢀonꢀtheꢀPu24Tꢀc-
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MYCꢀG4ꢀDNA.ꢀThisꢀshowsꢀtheꢀimportanceꢀofꢀorthogonalꢀassaysꢀforꢀscreeningꢀG4ꢀ
ligandsꢀasꢀcompoundꢀ11gꢀdisplayedꢀgoodꢀactivityꢀinꢀtheꢀTaqꢀDNAꢀpolymeraseꢀstopꢀ
assay,ꢀwhichꢀcouldꢀnotꢀbeꢀconfirmedꢀwithꢀtheꢀThTꢀassay.ꢀꢀ
BasedꢀonꢀtheꢀresultsꢀobtainedꢀfromꢀtheꢀTaqꢀpolymeraseꢀstopꢀassayꢀandꢀtheꢀThTꢀ
displacementꢀassay,ꢀweꢀcontinuedꢀwithꢀmoreꢀdetailedꢀevaluationsꢀforꢀsixꢀofꢀtheꢀ
mostꢀpromisingꢀcompoundsꢀ(inꢀtermsꢀofꢀactivityꢀand/orꢀselectivity),ꢀthreeꢀfromꢀ
theꢀflexibleꢀscaffoldꢀIꢀ(11a,ꢀ11cꢀandꢀ11e)ꢀandꢀthreeꢀfromꢀtheꢀrigidꢀscaffoldꢀIIꢀ(12a,ꢀ
1
2bꢀandꢀ12d).ꢀꢀꢀ
ꢀ
CircularꢀDichroismꢀ(CD)ꢀshowsꢀenhancedꢀthermalꢀstability.ꢀ
Toꢀexamineꢀhowꢀwellꢀcompoundsꢀ11a,ꢀ11c,ꢀ11e,ꢀ12a,ꢀ12b,ꢀandꢀ12dꢀstabilizeꢀG4ꢀ
structures,ꢀweꢀperformedꢀCDꢀmeltingꢀexperimentsꢀusingꢀtheꢀribosomalꢀG4ꢀDNAꢀ
andꢀ telomericꢀ G4ꢀ DNAꢀ oligonucleotides.ꢀ Toꢀ calculateꢀ theꢀ meltingꢀ ofꢀ theꢀ
oligonucleotides,ꢀtheꢀCDꢀspectraꢀwereꢀmeasuredꢀbetweenꢀ25-90ꢀ°Cꢀatꢀ264ꢀnmꢀforꢀ
theꢀribosomalꢀG4ꢀDNAꢀandꢀ290ꢀnmꢀforꢀtheꢀtelomericꢀG4ꢀDNAꢀ(FigureꢀS4).ꢀAllꢀsixꢀ
compoundsꢀ stronglyꢀ stabilizedꢀ theꢀ ribosomalꢀ G4ꢀ structureꢀ andꢀ wereꢀ moreꢀ
effectiveꢀthanꢀtheꢀbestꢀpublishedꢀ2,2’-diindolylmethaneꢀ(ECH22,ꢀFigureꢀS2)ꢀusedꢀ
asꢀreferenceꢀcompoundꢀ(theꢀcompoundsꢀincreasedꢀtheꢀmeltingꢀpointꢀbyꢀ4ꢀtoꢀoverꢀ
1
1ꢀ°Cꢀmoreꢀthanꢀtheꢀreferenceꢀcompound).ꢀCompoundꢀ12aꢀincreasedꢀtheꢀmeltingꢀ
temperatureꢀtheꢀmostꢀ(overꢀ17ꢀ°C),ꢀwhereasꢀtheꢀrestꢀofꢀtheꢀcompoundsꢀincreasedꢀ
theꢀmeltingꢀtemperatureꢀbyꢀ10-13ꢀ°Cꢀ(FigureꢀS5,ꢀS6,ꢀandꢀTableꢀ2).ꢀ
Fiveꢀofꢀtheꢀsixꢀcompoundsꢀ(11a,ꢀ11c,ꢀ11e,ꢀ12a,ꢀandꢀ12d)ꢀalsoꢀefficientlyꢀstabilizedꢀ
theꢀ telomericꢀ G4ꢀ structureꢀ andꢀ alsoꢀ forꢀ thisꢀ G4ꢀ structure,ꢀ theseꢀ wereꢀ moreꢀ
effectiveꢀ inꢀ stabilizationꢀ thanꢀ theꢀ referenceꢀ compound.ꢀ Forꢀ theꢀ telomericꢀ G4ꢀ
structure,ꢀtheꢀmoreꢀflexibleꢀcompoundsꢀbasedꢀonꢀscaffoldꢀIꢀ(11a,ꢀ11c,ꢀandꢀ11e)ꢀ
wereꢀslightlyꢀmoreꢀefficientꢀinꢀstabilizingꢀtheꢀG4ꢀstructureꢀcomparedꢀtoꢀtheꢀmoreꢀ
rigidꢀscaffoldꢀIIꢀcompoundsꢀ(12a,ꢀ12b,ꢀandꢀ12d).ꢀHowever,ꢀtheꢀrigidꢀscaffoldꢀIIꢀ
compoundsꢀ hadꢀ aꢀ clearꢀ selectivityꢀ towardsꢀ theꢀ ribosomalꢀ G4ꢀ DNAꢀ overꢀ theꢀ
telomericꢀ G4ꢀ DNA.ꢀ Thisꢀ wasꢀ mostꢀ obviousꢀ forꢀ compoundꢀ 12b,ꢀ whichꢀ didꢀ notꢀ
increaseꢀtheꢀmeltingꢀtemperatureꢀforꢀtheꢀtelomericꢀG4ꢀDNA,ꢀbutꢀincreasedꢀtheꢀ
meltingꢀforꢀtheꢀribosomlꢀG4ꢀDNAꢀwithꢀ11ꢀ°Cꢀ(Tableꢀ2ꢀandꢀFigureꢀS6).ꢀꢀ
ꢀ
Tableꢀ2.ꢀTheꢀCDꢀmeltingꢀpointsꢀofꢀribosomalꢀandꢀtelomericꢀG4ꢀDNAꢀtreatedꢀwithꢀ11a,ꢀ11c,ꢀ
1
1e,ꢀ12a,ꢀ12b,ꢀandꢀ12dꢀ
ꢀ
ꢀ
DMSOꢀ Refaꢀ 11aꢀ
11cꢀ
79ꢀ
12ꢀ
69ꢀ
12ꢀ
11eꢀ
77ꢀ
10ꢀ
66ꢀ
9ꢀ
12aꢀ
84ꢀ
12bꢀ
78ꢀ
11ꢀ
57ꢀ
0ꢀ
12dꢀ
80ꢀ
13ꢀ
65ꢀ
8ꢀ
T
m
ꢀ(oC)ꢀ
67ꢀ
0ꢀ
73ꢀ
6ꢀ
78ꢀ
11ꢀ
68ꢀ
11ꢀ
Ribosomalꢀ
G4ꢀ
ΔT
m
ꢀ(oC)ꢀ
ꢀ(oC)ꢀ
>17bꢀ
66ꢀ
T
m
57ꢀ
0ꢀ
62ꢀ
5ꢀ
Telomericꢀ
G4ꢀ
ΔT
m
ꢀ(oC)ꢀ
9ꢀ
aTheꢀreferenceꢀcompoundꢀisꢀtheꢀbestꢀpublishedꢀ2,2´-diindolylmethaneꢀ(ECH22,ꢀFigureꢀS2).[23]ꢀꢀbTheꢀ
ribosomalꢀG4ꢀDNAꢀwasꢀnotꢀfullyꢀmeltedꢀevenꢀatꢀ90˚C,ꢀthereforeꢀtheꢀvalueꢀisꢀthusꢀanꢀestimationꢀ
basedꢀonꢀtheꢀcurveꢀ(FigureꢀS5).ꢀ
ꢀ
CDꢀtitrationꢀexperimentsꢀshowꢀbigꢀdifferencesꢀbetweenꢀtheꢀflexibleꢀscaffoldꢀ
IꢀandꢀrigidꢀscaffoldꢀIIꢀcompounds.ꢀ
ToꢀobtainꢀinformationꢀaboutꢀtheꢀinteractionsꢀbetweenꢀtheꢀcompoundsꢀandꢀtheꢀG4ꢀ
structures,ꢀ CDꢀ titrationꢀ experimentsꢀ wereꢀ performedꢀ withꢀ 10ꢀ μMꢀ ofꢀ G4ꢀ DNAꢀ
(ribosomalꢀandꢀtelomericꢀG4ꢀDNAꢀfromꢀS.ꢀpombe,ꢀandꢀtheꢀhumanꢀbasedꢀPu24Tꢀc-
MYCꢀ G4ꢀ DNA)ꢀ atꢀ varyingꢀ concentrationsꢀ ofꢀ allꢀ sixꢀ scaffoldꢀ Iꢀ andꢀ scaffoldꢀ IIꢀ
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10.1002/chem.201800078
compoundsꢀ(11a,ꢀ11c,ꢀ11e,ꢀ12a,ꢀ12b,ꢀandꢀ12d).ꢀPu24Tꢀc-MYCꢀG4ꢀDNAꢀgivesꢀaꢀwellꢀ
definedꢀpositiveꢀpeakꢀatꢀ264ꢀnmꢀcorrespondingꢀtoꢀaꢀparallelꢀconformation.ꢀForꢀallꢀ
compounds,ꢀthisꢀCDꢀpeakꢀatꢀ264ꢀnmꢀofꢀPu24Tꢀc-MYCꢀG4ꢀDNAꢀwasꢀunchangedꢀinꢀ
theꢀpresenceꢀofꢀoneꢀequivalentꢀofꢀcompoundꢀ(Figureꢀ3A,ꢀ3D,ꢀ3GꢀandꢀFigureꢀ4A,ꢀ4D,ꢀ
4
G).ꢀ Uponꢀ increasedꢀ compoundꢀ concentrationsꢀ (overꢀ twoꢀ equivalents),ꢀ aꢀ
reductionꢀinꢀtheꢀsignalꢀatꢀ264ꢀnmꢀisꢀobserved,ꢀwhichꢀwasꢀmoreꢀpronouncedꢀforꢀtheꢀ
flexibleꢀ scaffoldꢀ Iꢀ compoundsꢀ (2.5,ꢀ 3.1,ꢀ andꢀ 3.2ꢀ foldꢀ forꢀ 11a,ꢀ 11cꢀ andꢀ 11e,ꢀ
respectively),ꢀcomparedꢀtoꢀtheꢀrigidꢀscaffoldꢀIIꢀcompoundsꢀ(1.6,ꢀ1.5,ꢀandꢀ1.4ꢀfoldꢀ
forꢀ 12a,ꢀ 12b,ꢀ andꢀ 12d,ꢀ respectively).ꢀ Thisꢀ apparentꢀ unfoldingꢀ effectꢀ atꢀ higherꢀ
concentrationsꢀsuggestꢀthat,ꢀatꢀhigherꢀconcentrations,ꢀtheꢀcompoundsꢀpartiallyꢀ
disruptꢀtheꢀstackingꢀinteractionsꢀbetweenꢀtheꢀG-tetrads.[39]ꢀꢀTheꢀribosomalꢀG4ꢀDNAꢀ
givesꢀaꢀpositiveꢀpeakꢀatꢀ264ꢀnmꢀfollowedꢀbyꢀaꢀsmallꢀshoulderꢀatꢀ290ꢀnmꢀandꢀforꢀ
thisꢀG4ꢀstructureꢀthereꢀwasꢀaꢀveryꢀbigꢀdifferenceꢀbetweenꢀtheꢀflexibleꢀscaffoldꢀIꢀ
compoundsꢀandꢀtheꢀrigidꢀscaffoldꢀIIꢀcompounds.ꢀTheꢀlatterꢀinducedꢀalmostꢀnoꢀ
ligandꢀdependentꢀdecreaseꢀinꢀellipticityꢀofꢀtheꢀCDꢀpeaksꢀ(Figureꢀ3B,ꢀ3E,ꢀ3H,ꢀFigureꢀ
4
B,ꢀ4E,ꢀ4H,ꢀandꢀFigureꢀS7)ꢀwhereasꢀtheꢀflexibleꢀscaffoldꢀIꢀcompoundsꢀreducedꢀtheꢀ
signalꢀatꢀ264ꢀnmꢀandꢀinducedꢀaꢀnewꢀCDꢀpeakꢀatꢀ335ꢀnm.ꢀTheꢀinducedꢀCDꢀpeakꢀ
whichꢀ wasꢀ mostꢀ prominentꢀ forꢀ 11a)ꢀ suggestsꢀ that,ꢀ atꢀ higherꢀ compoundꢀ
(
concentrations,ꢀtheꢀcompoundsꢀmightꢀfindꢀadditionalꢀbindingꢀinteractionsꢀapartꢀ
fromꢀend-stackingꢀthatꢀthusꢀstartꢀtoꢀremodelꢀtheꢀG4ꢀsecondaryꢀstructure.[ ꢀTheꢀ
telomericꢀG4ꢀhasꢀ aꢀmixedꢀ hybridꢀ typeꢀ ofꢀ structureꢀ andꢀ alsoꢀinꢀ thisꢀ case,ꢀ aꢀ bigꢀ
structuralꢀtransformationꢀwasꢀinducedꢀwithꢀincreasingꢀconcentrationꢀofꢀscaffoldꢀIꢀ
compoundsꢀ (Figureꢀ 3C,ꢀ 3F,ꢀ 3I,ꢀ Figureꢀ 4C,ꢀ 4F,ꢀ 4I,ꢀ andꢀ Figureꢀ S7).ꢀ Again,ꢀ thisꢀ
structuralꢀ transformationꢀ wasꢀmoreꢀ prominentꢀ withꢀ theꢀ scaffoldꢀ Iꢀ compoundsꢀ
comparedꢀ toꢀ theꢀ scaffoldꢀ IIꢀ compounds.ꢀ Furthermore,ꢀ aꢀ relativelyꢀ weakꢀ signalꢀ
aroundꢀ 335ꢀ nmꢀ wasꢀ inducedꢀ whenꢀ theꢀ scaffoldꢀ Iꢀ compoundsꢀ interactedꢀ withꢀ
telomericꢀ G4ꢀ DNA,ꢀ whichꢀ wasꢀ notꢀ visibleꢀ withꢀ theꢀ scaffoldꢀ IIꢀ compounds.ꢀ
Altogether,ꢀtheꢀflexibleꢀscaffoldꢀIꢀcompoundsꢀhadꢀaꢀbiggerꢀimpactꢀonꢀtheꢀoverallꢀ
structuresꢀofꢀtheꢀG4sꢀuponꢀbinding,ꢀmaybeꢀbyꢀfindingꢀadditionalꢀinteractionsꢀwithꢀ
theꢀ DNAꢀ thatꢀ resultedꢀ inꢀ theꢀ observedꢀ structuralꢀ changesꢀ inꢀ ribosomalꢀ andꢀ
telomericꢀG4.ꢀTheseꢀstructuralꢀchangesꢀwereꢀhighlyꢀconcentrationꢀdependentꢀandꢀ
alsoꢀmuchꢀlessꢀpronouncedꢀforꢀtheꢀrigidꢀscaffoldꢀIIꢀcompounds,ꢀandꢀareꢀthusꢀlikelyꢀ
anꢀeffectꢀofꢀtheꢀdifferenceꢀinꢀflexibilityꢀbetweenꢀtheꢀtwoꢀscaffoldsꢀandꢀtheirꢀabilityꢀ
toꢀfindꢀadditionalꢀinteractionsꢀonꢀtheꢀDNA.ꢀImportantly,ꢀnoneꢀofꢀtheꢀcompoundsꢀ
giveꢀanyꢀfree-ligandꢀCDꢀsignaturesꢀ(FigureꢀS8),ꢀthusꢀconfirmingꢀthatꢀtheꢀobservedꢀ
changesꢀ inꢀ theꢀ CDꢀ spectraꢀ actuallyꢀ reflectsꢀ changesꢀ inꢀ theꢀ G4ꢀ structuresꢀ uponꢀ
additionꢀofꢀtheꢀcompounds.ꢀ
40]
ꢀ
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Chemistry - A European Journal
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ꢀ
Figureꢀ3.ꢀCDꢀtitrationꢀwithꢀPu24Tꢀc-MYCꢀG4ꢀDNA,ꢀribosomalꢀG4ꢀDNA,ꢀandꢀtelomericꢀG4ꢀDNAꢀwithꢀ
theꢀ flexibleꢀ scaffoldꢀ Iꢀcompoundsꢀ 11aꢀ (A,ꢀ B,ꢀ andꢀ C);ꢀ11cꢀ (D,ꢀ E,ꢀandꢀ F)ꢀandꢀ 11eꢀ (G,ꢀH,ꢀandꢀ I),ꢀ
respectively.ꢀLongerꢀwavelengthsꢀforꢀRibosomalꢀG4ꢀandꢀTelomericꢀG4ꢀcanꢀbeꢀfoundꢀinꢀFigureꢀS7.ꢀ
EquivꢀindicatesꢀtheꢀnumberꢀofꢀequivalentsꢀofꢀcompoundꢀcomparedꢀtoꢀDNA.ꢀ
ꢀ
Figureꢀ4.ꢀCDꢀtitrationꢀwithꢀPu24Tꢀc-MYCꢀG4ꢀDNA,ꢀribosomalꢀG4ꢀDNA,ꢀandꢀtelomericꢀG4ꢀDNAꢀwithꢀ
theꢀ rigidꢀ scaffoldꢀ IIꢀ compoundsꢀ 12aꢀ (A,ꢀ B,ꢀ andꢀ C);ꢀ 12bꢀ (D,ꢀ E,ꢀ andꢀ F)ꢀ andꢀ 12dꢀ (G,ꢀ H,ꢀ andꢀ I),ꢀ
respectively.ꢀEquivꢀindicatesꢀtheꢀnumberꢀofꢀequivalentsꢀofꢀcompoundꢀcomparedꢀtoꢀDNA.ꢀ
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Chemistry - A European Journal
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ꢀ
ꢀ
Bindingꢀ constantꢀ determinationꢀ throughꢀ Fluorescenceꢀ Intercalatorꢀ
Displacementꢀ(FID)ꢀassay.ꢀ
Theꢀ synthesizedꢀ compoundsꢀ haveꢀ absorptionꢀaroundꢀ 290ꢀ nm,ꢀ whichꢀ makesꢀ itꢀ
difficultꢀ toꢀ doꢀ spectroscopicꢀ titrationsꢀ asꢀ itꢀ overlapsꢀ withꢀ theꢀ DNAꢀ absorptionꢀ
(FigureꢀS9).ꢀInꢀsuchꢀcases,ꢀtheꢀFIDꢀassayꢀisꢀaꢀgoodꢀtoolꢀtoꢀdetermineꢀtheꢀbindingꢀ
constantꢀforꢀcompoundsꢀtoꢀG4ꢀDNA.ꢀImportantly,ꢀnoneꢀofꢀtheꢀflexibleꢀscaffoldꢀIꢀorꢀ
rigidꢀ scaffoldꢀ IIꢀ compoundsꢀ areꢀ fluorescentꢀ (dataꢀ notꢀ shown),ꢀ whichꢀ isꢀ aꢀ
prerequisiteꢀtoꢀperformꢀtheꢀFIDꢀassay.ꢀHence,ꢀtheꢀFIDꢀassayꢀwasꢀusedꢀwithꢀthiazoleꢀ
orangeꢀ(TO)ꢀasꢀaꢀG4ꢀbindingꢀfluorophore.[41]ꢀUponꢀadditionꢀofꢀtheꢀcompounds,ꢀtheꢀ
displacementꢀofꢀTOꢀfromꢀtheꢀstudiedꢀG4ꢀstructureꢀcanꢀbeꢀdeterminedꢀbyꢀobservingꢀ
theꢀdecreaseꢀofꢀtheꢀfluorescenceꢀintensityꢀ(FigureꢀS10,ꢀS11).ꢀFromꢀtheseꢀstudies,ꢀ
apparentꢀdissociationꢀbindingꢀconstantsꢀforꢀallꢀtheꢀsixꢀcompoundsꢀtowardsꢀPu24Tꢀ
c-MYC,ꢀ ribosomal,ꢀ andꢀ telomericꢀ G4ꢀ DNAꢀ couldꢀ beꢀ calculatedꢀ (Figureꢀ 5).ꢀ Theꢀ
fluorescenceꢀ spectraꢀ ofꢀ theꢀ interactionꢀ ofꢀ TOꢀ withꢀ allꢀ theꢀ G4ꢀ structuresꢀ areꢀ
providedꢀinꢀFigureꢀS12.ꢀAsꢀanticipatedꢀbasedꢀonꢀtheꢀcompoundꢀscreeningꢀandꢀtheꢀ
CDꢀ meltingꢀ andꢀ titrationꢀ data,ꢀ allꢀ compoundsꢀ hadꢀ aꢀ goodꢀ affinityꢀ towardsꢀ theꢀ
testedꢀG4ꢀstructures.ꢀAllꢀofꢀtheꢀscaffoldꢀIꢀcompoundsꢀ(11a,ꢀ11cꢀandꢀ11e)ꢀandꢀtheꢀ
scaffoldꢀIIꢀcompoundsꢀ12aꢀandꢀ12bꢀshowedꢀaꢀveryꢀstrongꢀaffinityꢀtowardsꢀG4ꢀDNAꢀ
(
(
K
K
d
ꢀvaluesꢀ£ꢀ1ꢀµM),ꢀwhereasꢀtheꢀscaffoldꢀIIꢀcompoundꢀ12dꢀdisplayedꢀlowerꢀaffinityꢀ
d
ꢀ valuesꢀ >ꢀ 1ꢀ µM).ꢀ Mostꢀ compoundsꢀ showedꢀ theꢀ highestꢀ affinityꢀ towardsꢀtheꢀ
humanꢀ parallelꢀ Pu24Tꢀ c-MYCꢀ G4ꢀ DNAꢀ andꢀ aꢀ bitꢀ lessꢀ toꢀ theꢀ S.ꢀ pombeꢀ parallelꢀ
ribosomalꢀG4ꢀDNA.ꢀꢀWhenꢀcomparingꢀtheꢀscaffoldsꢀwithꢀtheꢀsameꢀsubstituentsꢀasꢀ
1
1aꢀandꢀ12a,ꢀtheꢀflexibleꢀscaffoldꢀIꢀcompoundꢀ11aꢀshowedꢀslightlyꢀhigherꢀbindingꢀ
affinityꢀwithꢀallꢀtheꢀthreeꢀG4ꢀstructuresꢀcomparedꢀtoꢀ12a.ꢀThisꢀtrendꢀalsoꢀholdsꢀ
trueꢀforꢀtheꢀcyclopropylꢀquaternizedꢀquinolineꢀsideꢀchainsꢀinꢀtheꢀtwoꢀscaffolds,ꢀ
compoundsꢀ11eꢀversusꢀcompoundꢀ12dꢀ(FigureꢀS6).ꢀ
ꢀ
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Chemistry - A European Journal
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ꢀ
Figureꢀ5.ꢀFIDꢀassayꢀofꢀscaffoldꢀIꢀcompoundsꢀ11aꢀ(A),ꢀ11cꢀ(B),ꢀ11eꢀ(C)ꢀandꢀscaffoldꢀIIꢀcompoundsꢀ
1
2aꢀ(D),ꢀ12bꢀ(E),ꢀ12dꢀ(F)ꢀandꢀtheirꢀapparentꢀdissociationꢀbindingꢀconstantsꢀ(K )ꢀ(G)ꢀwithꢀPu24Tꢀ
d
G4ꢀDNA,ꢀribosomalꢀG4ꢀDNA,ꢀandꢀtelomericꢀG4ꢀDNA.ꢀ
ꢀ
ꢀ
TheꢀcompoundsꢀinhibitꢀDNAꢀsynthesisꢀinꢀaꢀdoseꢀdependentꢀmanner.ꢀ
Oneꢀofꢀtheꢀmanyꢀbenefitsꢀofꢀusingꢀsmallꢀmoleculesꢀtoꢀstudyꢀbiologicalꢀsystemsꢀisꢀ
thatꢀtheirꢀeffectꢀcanꢀbeꢀtunedꢀbyꢀvaryingꢀtheꢀdose.ꢀHence,ꢀweꢀagainꢀperformedꢀtheꢀ
TaqꢀpolymeraseꢀstopꢀassayꢀtoꢀconfirmꢀthatꢀtheꢀsixꢀcompoundsꢀfromꢀscaffoldꢀIꢀandꢀ
IIꢀstabilizedꢀtheꢀG4ꢀstructures,ꢀandꢀthatꢀthisꢀalsoꢀtranslatesꢀintoꢀaꢀdose-dependentꢀ
inhibitionꢀofꢀDNAꢀsynthesisꢀbyꢀperformingꢀtheꢀexperimentsꢀatꢀdifferentꢀcompoundꢀ
concentrations.ꢀTheꢀTaqꢀpolymeraseꢀstopꢀassayꢀwasꢀconductedꢀwithꢀtheꢀdifferentꢀ
compoundsꢀ(11a,ꢀ11c,ꢀ11e,ꢀ12a,ꢀ12b,ꢀandꢀ12d)ꢀatꢀdifferentꢀconcentrationsꢀ(5,ꢀ2,ꢀ
0
.8,ꢀ0.32,ꢀ0.13ꢀ0.05,ꢀ0.025ꢀandꢀ0.008ꢀµM)ꢀorꢀDMSOꢀwithꢀribosomalꢀG4ꢀDNAꢀfromꢀS.ꢀ
pombeꢀandꢀnon-G4ꢀDNAꢀasꢀcontrolꢀ(Figureꢀ6,ꢀS13,ꢀandꢀS14).ꢀFromꢀthis,ꢀtheꢀIC50
ꢀ
valuesꢀ wereꢀ determined,ꢀ whichꢀ correspondsꢀ toꢀ theꢀ halfꢀ maximumꢀ inhibitoryꢀ
concentrationꢀofꢀDNAꢀsynthesisꢀ(Figureꢀ6C).ꢀTheꢀresultsꢀshowedꢀthatꢀallꢀtheꢀtestedꢀ
compoundsꢀefficientlyꢀstabilizedꢀtheꢀG4ꢀstructures,ꢀandꢀthatꢀthisꢀstabilizationꢀalsoꢀ
inhibitedꢀDNAꢀsynthesisꢀatꢀveryꢀlowꢀcompoundꢀconcentrationsꢀ(IC50ꢀvaluesꢀdownꢀ
toꢀ 0.11ꢀ μM).ꢀ Aꢀ greatꢀ advantageꢀ ofꢀ thisꢀ assayꢀ isꢀ thatꢀ theꢀ IC50ꢀ valuesꢀ forꢀ theꢀ
compoundsꢀforꢀaꢀnon-G4ꢀDNAꢀcanꢀalsoꢀbeꢀdeterminedꢀandꢀcomparedꢀtoꢀtheꢀG4ꢀ
DNAꢀtemplate.ꢀAllꢀcompoundsꢀshowedꢀmuchꢀhigherꢀIC50ꢀvaluesꢀforꢀtheꢀnon-G4ꢀ
DNAꢀ(aboutꢀ7-14ꢀfoldꢀhigherꢀforꢀnon-G4ꢀDNAꢀcomparedꢀtoꢀribosomalꢀG4ꢀDNA).ꢀ
TheꢀspecificꢀG4ꢀbindingꢀofꢀtheꢀcompoundsꢀwasꢀfurtherꢀverifiedꢀbyꢀtheꢀincreaseꢀinꢀ
theꢀpausingꢀsitesꢀatꢀtheꢀG4ꢀstructureꢀ(FigureꢀS13ꢀandꢀS14,ꢀmarkedꢀwithꢀaꢀstar),ꢀ
whichꢀ indicateꢀ aꢀ lowꢀ unspecificꢀ bindingꢀ toꢀ otherꢀ componentsꢀ inꢀ theꢀ system.ꢀ
Comparingꢀ theꢀ rigidꢀ andꢀ flexibleꢀ compounds,ꢀ theꢀ rigidꢀ scaffoldꢀ IIꢀ compoundsꢀ
showedꢀslightlyꢀlowerꢀIC50ꢀvaluesꢀforꢀbothꢀtheꢀribosomalꢀG4ꢀDNAꢀandꢀtheꢀnon-G4ꢀ
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DNAꢀcomparedꢀtoꢀtheꢀflexibleꢀscaffoldꢀIꢀcompoundsꢀinꢀthisꢀassayꢀ(FigureꢀS6).ꢀꢀ
ꢀ
ꢀ
Figureꢀ6.ꢀDoseꢀresponseꢀstudiesꢀwithꢀtheꢀTaqꢀDNAꢀpolymeraseꢀstopꢀassayꢀofꢀribosomalꢀG4ꢀDNAꢀ
(A),ꢀandꢀnon-G4ꢀDNAꢀ(B)ꢀinꢀtheꢀpresenceꢀofꢀallꢀsixꢀflexibleꢀscaffoldꢀIꢀandꢀrigidꢀscaffoldꢀIIꢀcompounds,ꢀ
andꢀ theirꢀ correspondingꢀ IC50ꢀ valuesꢀ (C).ꢀ Errorꢀ barsꢀ inꢀ Aꢀ andꢀ Bꢀ representꢀ standardꢀ deviationꢀ
calculatedꢀfromꢀthreeꢀindependentꢀexperiments.ꢀ
ꢀ
Molecularꢀ dynamicsꢀ (MD)ꢀ simulationsꢀ ofꢀ flexibleꢀ 11aꢀ andꢀ rigidꢀ 12aꢀ
compoundsꢀwithꢀG4ꢀDNA.ꢀ
Ourꢀsynthesizedꢀbis-indoleꢀcompoundsꢀwereꢀdesignedꢀtoꢀstackꢀonꢀtopꢀofꢀtheꢀG4ꢀ
structure,ꢀwhichꢀhasꢀbeenꢀshownꢀwithꢀPhen-DC3ꢀ(FigureꢀS15)ꢀthatꢀshareꢀtheꢀsameꢀ
aminoquinolineꢀsideꢀchainsꢀasꢀ11aꢀandꢀ12a.[28]ꢀEnd-stackingꢀinteractionsꢀisꢀalsoꢀ
inꢀlineꢀwithꢀtheꢀstrongꢀbindingꢀandꢀstabilizationꢀobservedꢀforꢀtheseꢀcompounds,ꢀ
howeverꢀ theirꢀ bindingꢀ interactionꢀ atꢀ atomicꢀ resolutionꢀ isꢀ stillꢀ unknown.ꢀ Toꢀ
understandꢀtheꢀbindingꢀmodeꢀofꢀ11aꢀandꢀ12a,ꢀweꢀmodeledꢀthemꢀatꢀtheꢀtopꢀofꢀG4ꢀ
DNAꢀusingꢀtheꢀalreadyꢀavailableꢀPhen-DC3/Pu24Tꢀc-MYCꢀG4ꢀDNAꢀasꢀtemplate.[
ꢀ
28]
Subsequently,ꢀ5x200ꢀnsꢀMDꢀsimulationsꢀwereꢀperformedꢀtoꢀsampleꢀtheꢀcomplexesꢀ
conformations.ꢀDuringꢀtheꢀmicrosecondꢀsimulation,ꢀbothꢀcompoundsꢀremainedꢀatꢀ
theꢀ topꢀ ofꢀ theꢀ G4ꢀ structureꢀ withꢀ onlyꢀ smallꢀ conformationalꢀ fluctuations,ꢀ thusꢀ
supportingꢀthatꢀtheꢀcompoundsꢀstackꢀonꢀtopꢀofꢀtheꢀG4ꢀstructure.ꢀ
Toꢀ studyꢀ theꢀ bindingꢀ modesꢀ ofꢀ theseꢀ compounds,ꢀ weꢀ clusteredꢀ theꢀ ligandꢀ
conformationsꢀ(byꢀrootꢀmeanꢀsquareꢀdeviationsꢀafterꢀsuperimposingꢀDNA)ꢀandꢀ
obtainedꢀtwoꢀlargeꢀclustersꢀforꢀ11aꢀwhileꢀaꢀsingleꢀclusterꢀforꢀ12a.ꢀInꢀtheꢀfirstꢀ
clusterꢀofꢀ11a,ꢀbothꢀtheꢀbis-indoleꢀandꢀtheꢀquinolineꢀsideꢀchainsꢀareꢀstackedꢀaboveꢀ
theꢀ topꢀ G-tetradꢀ (Figureꢀ 7Aꢀ andꢀ 7B)ꢀ whileꢀoneꢀ quinolineꢀsideꢀ chainꢀ isꢀ slightlyꢀ
outsideꢀtheꢀtopꢀG-tetradꢀinꢀtheꢀsecondꢀcluster.ꢀInꢀcontrast,ꢀtheꢀsingleꢀclusterꢀforꢀ
1
2aꢀshowsꢀbothꢀtheꢀquinolineꢀsideꢀchainsꢀoutsideꢀofꢀtheꢀtopꢀG-tetradꢀ(Figureꢀ7Cꢀ
andꢀ 7D).ꢀ Interestingly,ꢀ bothꢀ compoundsꢀ wereꢀ sandwichedꢀ betweenꢀ theꢀ topꢀ G-
tetradꢀandꢀtheꢀguanineꢀnumberꢀ2ꢀandꢀadenineꢀnumberꢀ3ꢀresiduesꢀ(G-2ꢀandꢀA-3)ꢀ
fromꢀbelowꢀandꢀabove,ꢀrespectivelyꢀ(Figureꢀ7B).ꢀHowever,ꢀtheꢀoverallꢀcompoundꢀ
contactꢀwithꢀDNA,ꢀwhichꢀalsoꢀreflectsꢀtheꢀextentꢀofꢀsandwiching,ꢀwasꢀlargerꢀinꢀ11aꢀ
comparedꢀtoꢀ12aꢀ(moreꢀthanꢀ65%ꢀofꢀtheꢀsurfaceꢀareaꢀwereꢀinꢀcontactꢀwithꢀDNAꢀ
duringꢀ ~32%ꢀ ofꢀ theꢀ simulationꢀ timeꢀ forꢀ 11aꢀ asꢀ comparedꢀ withꢀ ~5%ꢀ ofꢀ theꢀ
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simulationꢀtimeꢀforꢀ12a,ꢀFigureꢀS16).ꢀTheꢀlargerꢀcontactꢀinꢀ11aꢀwasꢀdueꢀtoꢀtheꢀ
stackingꢀofꢀbothꢀitsꢀindolesꢀandꢀquinolinesꢀwithꢀDNAꢀfromꢀbelowꢀandꢀaboveꢀwhileꢀ
onlyꢀtheꢀindolesꢀformedꢀsuchꢀinteractionsꢀinꢀ12b.ꢀ
Figureꢀ 7.ꢀ Conformationꢀ ofꢀ compoundsꢀ bindingꢀ toꢀ G4ꢀ structureꢀ duringꢀ simulations.ꢀ (A)ꢀ Aꢀ
representativeꢀstructureꢀofꢀcompoundꢀ11aꢀ(greyꢀlicoriceꢀmodel)ꢀwithꢀtheꢀG4ꢀ(cartoonꢀmodel).ꢀ
(B)ꢀCompoundꢀ11aꢀ(greyꢀlicoriceꢀmodel)ꢀembeddedꢀbetweenꢀG4ꢀDNAꢀresiduesꢀfromꢀbelowꢀandꢀ
aboveꢀ(blueꢀsurfaceꢀview)ꢀinꢀtwoꢀdifferentꢀorientationsꢀ(leftꢀshowsꢀtop-viewꢀandꢀrightꢀshowsꢀ
side-view).ꢀ(C)ꢀAꢀrepresentativeꢀstructureꢀofꢀcompoundꢀ12aꢀ(greyꢀlicoriceꢀmodel)ꢀwithꢀtheꢀG4ꢀ
(cartoonꢀmodel).ꢀ(D)ꢀ12aꢀ(greyꢀlicoriceꢀmodel)ꢀinsideꢀG4ꢀDNAꢀ(leftꢀshowsꢀtop-viewꢀandꢀrightꢀ
showsꢀ side-view).ꢀ Theꢀ representativeꢀ conformationsꢀ wereꢀ selectedꢀ onꢀ theꢀ basisꢀ ofꢀ lowestꢀ
bindingꢀenergyꢀcalculatedꢀusingꢀMM/PBSAꢀmethod.[42]ꢀ
ꢀ
Toꢀ dissectꢀ theꢀ bindingꢀ interaction,ꢀ weꢀ calculatedꢀ theꢀ contactꢀ frequencyꢀ ofꢀ theꢀ
indoleꢀandꢀquinolineꢀring-systemsꢀwithꢀtheꢀDNAꢀresiduesꢀforꢀbothꢀcompounds.ꢀAsꢀ
shownꢀinꢀTableꢀ3,ꢀtheꢀtwoꢀindolesꢀ(IꢀandꢀI´)ꢀinꢀbothꢀ11aꢀandꢀ12aꢀwereꢀdominantlyꢀ
inꢀcontactꢀwithꢀguanineꢀnumberꢀ4ꢀandꢀ17ꢀ(G-4ꢀandꢀG-17).ꢀFurthermore,ꢀtheꢀindolesꢀ
wereꢀ inꢀ contactꢀ withꢀ G-2ꢀ andꢀ A-3ꢀ showingꢀ theꢀ extentꢀ ofꢀ sandwichꢀ duringꢀ theꢀ
simulations.ꢀ Moreover,ꢀ oneꢀ 11aꢀ quinolineꢀ sideꢀ chainꢀ (Q´)ꢀ wereꢀ dominantlyꢀ inꢀ
contactꢀwithꢀG-4ꢀandꢀG-8ꢀwhileꢀtheꢀotherꢀquinolineꢀsideꢀchainꢀ(Q)ꢀinteractedꢀwithꢀ
G-13.ꢀ Theꢀ quinolineꢀ sideꢀ chainsꢀ ofꢀ 12aꢀ interactꢀ withꢀ G-8ꢀ andꢀ G-13ꢀ butꢀ withꢀ
reducedꢀ frequencyꢀ duringꢀ theꢀ simulations,ꢀ suggestingꢀ thatꢀ theꢀ quinolineꢀ sideꢀ
chainsꢀofꢀ12aꢀareꢀmoreꢀdistantꢀfromꢀG-8ꢀandꢀG-13.ꢀToꢀestimateꢀtheꢀstackingꢀofꢀtheꢀ
sideꢀ chains,ꢀ weꢀ calculatedꢀ theirꢀ interactionꢀ energiesꢀ withꢀ theꢀ respectiveꢀ DNAꢀ
residuesꢀ(TableꢀS2).ꢀThisꢀclearlyꢀshowꢀthatꢀtheꢀinteractionꢀenergyꢀinꢀ11aꢀisꢀmuchꢀ
moreꢀfavorableꢀthanꢀthatꢀofꢀ12a.ꢀTheseꢀresultsꢀthusꢀsuggestꢀthatꢀtheꢀmoreꢀflexibleꢀ
1
1aꢀformꢀstackingꢀinteractionsꢀwithꢀtheꢀtopꢀG-tetradꢀ(G-4,ꢀG-8,ꢀG-13ꢀandꢀG-17)ꢀ
fromꢀbelow,ꢀandꢀisꢀfurtherꢀsandwichedꢀbyꢀG-2ꢀandꢀA-3ꢀfromꢀtheꢀtop.ꢀInꢀcontrast,ꢀ
2aꢀdoꢀ notꢀ haveꢀ theꢀ sameꢀ strongꢀ interactions,ꢀ theꢀ middleꢀbis-indoleꢀstructureꢀ
1
stronglyꢀstacksꢀwithꢀallꢀfourꢀbasesꢀofꢀtheꢀtopꢀG-tetrad,ꢀoneꢀofꢀtheꢀquinolineꢀsideꢀ
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chainsꢀweaklyꢀstackꢀwithꢀG-8ꢀandꢀtheꢀotherꢀsideꢀchainꢀremainsꢀoutsideꢀofꢀtheꢀtopꢀ
tetrad.ꢀ
ꢀ
Tableꢀ3:ꢀTheꢀcontactꢀfrequencyꢀbetweenꢀG4ꢀDNAꢀresiduesꢀandꢀindolesꢀ(I,ꢀI´)ꢀandꢀquinolineꢀsideꢀ
chainsꢀ (Q,ꢀ Q´)ꢀ ofꢀ compoundꢀ 11aꢀ andꢀ 12aꢀ duringꢀ MDꢀ simulations.ꢀ Theꢀ DNAꢀ residuesꢀ andꢀ
compounds’ꢀsideꢀchainꢀwereꢀconsideredꢀtoꢀbeꢀinꢀcontactꢀwhenꢀanyꢀofꢀtheꢀheavyꢀatomsꢀfromꢀbothꢀ
groupꢀwereꢀcomingꢀwithinꢀ4ꢀÅꢀdistanceꢀduringꢀtheꢀsimulations.ꢀ
ꢀ
Residuesꢀ
G-4ꢀ
11aꢀ
12aꢀ
Qꢀ
Iꢀ
I'ꢀ
Q'ꢀ
Q'ꢀ
I'ꢀ
Iꢀ
Qꢀ
0
ꢀ
ꢀ
6ꢀ
98ꢀ
10ꢀ
9ꢀ
72ꢀ
95ꢀ
41ꢀ
9ꢀ
1ꢀ
87ꢀ
2ꢀ
97ꢀ
94ꢀ
20ꢀ
47ꢀ
48ꢀ
87ꢀ
45ꢀ
21ꢀ
67ꢀ
99ꢀ
50ꢀ
88ꢀ
0ꢀ
G-8ꢀ
0
2ꢀ
0ꢀ
G-13ꢀ
G-17ꢀ
G-2ꢀ
8
7ꢀ
8ꢀ
35ꢀ
92ꢀ
68ꢀ
74ꢀ
68ꢀ
36ꢀ
35ꢀ
19ꢀ
2
91ꢀ
69ꢀ
95ꢀ
0ꢀ
3ꢀ
45ꢀ
11ꢀ
12ꢀ
2ꢀ
A-3ꢀ
4ꢀ
Qꢀ–ꢀquinoline,ꢀIꢀ–ꢀIndole.ꢀ
ꢀ
Apartꢀfromꢀtheꢀstacking,ꢀadditionalꢀinteractionsꢀwereꢀalsoꢀobservedꢀinꢀ11aꢀthatꢀ
wereꢀweakerꢀinꢀ12a.ꢀꢀOneꢀofꢀtheꢀsideꢀchainsꢀquaternizedꢀnitrogenꢀinꢀ11aꢀinteractedꢀ
withꢀtheꢀphosphateꢀgroupsꢀofꢀbothꢀG-7ꢀandꢀG-8ꢀ(FigureꢀS17AꢀandꢀS17B)ꢀwhileꢀtheꢀ
quaternizedꢀnitrogenꢀonꢀtheꢀotherꢀsideꢀchainꢀinteractedꢀwithꢀG-13ꢀdeoxyriboseꢀ
ringꢀoxygenꢀ(FigureꢀS17C).ꢀAdditionally,ꢀtheꢀamideꢀinꢀtheꢀlatterꢀsideꢀchainꢀalsoꢀ
interactedꢀwithꢀtheꢀphosphateꢀgroupꢀofꢀG-17ꢀ(FigureꢀS17D).ꢀTheseꢀinteractionsꢀareꢀ
notꢀasꢀpronouncedꢀforꢀtheꢀsideꢀchainsꢀinꢀ12a.ꢀTheꢀsimulationsꢀthusꢀsuggestꢀthatꢀ
theꢀflexibleꢀscaffoldꢀIꢀcompoundꢀ11aꢀshouldꢀhaveꢀaꢀstrongerꢀbindingꢀtoꢀtheꢀG4ꢀ
structureꢀasꢀcomparedꢀtoꢀtheꢀmoreꢀrigidꢀscaffoldꢀIIꢀcompoundꢀ12aꢀdueꢀtoꢀtheꢀ
strongꢀstackingꢀandꢀotherꢀpolarꢀinteractions.ꢀꢀ
ꢀ
Conclusionꢀ
Inꢀ here,ꢀ weꢀ haveꢀ designedꢀ andꢀ developedꢀ syntheticꢀ methodsꢀ toꢀ twoꢀ
complementaryꢀ(flexibleꢀandꢀrigid)ꢀseriesꢀofꢀbis-indoleꢀstructures.ꢀTheꢀaimꢀwithꢀ
thisꢀwasꢀtwofold;ꢀtoꢀidentifyꢀnewꢀefficientꢀG4ꢀbindingꢀandꢀstabilizingꢀcompounds,ꢀ
andꢀtoꢀfurtherꢀadvanceꢀtheꢀunderstandingꢀofꢀhowꢀtheseꢀtypesꢀofꢀsmallꢀmoleculesꢀ
interactꢀwithꢀG4ꢀstructureꢀandꢀthusꢀhowꢀtheyꢀcanꢀbeꢀfurtherꢀoptimized.ꢀInꢀadditionꢀ
toꢀthis,ꢀtheꢀimportanceꢀandꢀpossibilityꢀtoꢀvaryꢀtheꢀsubstituentꢀthatꢀquaternizeꢀtheꢀ
sideꢀchainsꢀhaveꢀalsoꢀbeenꢀinvestigated.ꢀTheꢀcompounds'ꢀ(11a-jꢀandꢀ12a-d)ꢀabilityꢀ
toꢀbindꢀandꢀstabilizeꢀG4ꢀstructuresꢀwereꢀscreenedꢀbyꢀtheꢀTaqꢀpolymeraseꢀstopꢀ
assayꢀ usingꢀ theꢀ parallelꢀ ribosomalꢀ G4ꢀ DNAꢀ andꢀ aꢀ non-G4ꢀ DNAꢀ asꢀ selectivityꢀ
control.ꢀInꢀaddition,ꢀtheꢀbindingꢀofꢀtheꢀcompoundsꢀwasꢀalsoꢀevaluatedꢀbyꢀscreeningꢀ
allꢀtheꢀcompoundsꢀusingꢀthreeꢀdifferentꢀG4ꢀstructuresꢀinꢀtheꢀThTꢀdisplacementꢀ
assay.ꢀTheseꢀinitialꢀevaluationsꢀshowedꢀthatꢀ itꢀ isꢀ crucialꢀ toꢀ quaternizeꢀtheꢀsideꢀ
chainsꢀ inꢀ theseꢀ derivativesꢀ andꢀ thatꢀ largeꢀ substituentꢀ variationsꢀ areꢀ tolerated.ꢀ
Theseꢀ dataꢀ openꢀ upꢀ forꢀ furtherꢀ optimizationsꢀ ofꢀ theꢀ bindingꢀ andꢀ selectivityꢀ
propertiesꢀofꢀtheseꢀandꢀsimilarꢀtypesꢀofꢀcompoundsꢀinꢀtheꢀfuture.ꢀꢀ
Theꢀsixꢀmostꢀinterestingꢀcompoundsꢀfromꢀtheꢀscreeningꢀevaluations,ꢀthreeꢀfromꢀ
eachꢀbis-indoleꢀscaffoldꢀ(11a,ꢀ11c,ꢀandꢀ11eꢀfromꢀtheꢀflexibleꢀscaffoldꢀIꢀandꢀ12a,ꢀ
1
2b,ꢀandꢀ12dꢀfromꢀtheꢀrigidꢀscaffoldꢀII)ꢀwereꢀfurtherꢀstudiedꢀandꢀevaluatedꢀbyꢀ
severalꢀ differentꢀ assays.ꢀ First,ꢀ byꢀ performingꢀ CDꢀ thermalꢀ meltingꢀ studies,ꢀ weꢀ
demonstratedꢀ thatꢀ allꢀ theseꢀ compoundsꢀ (exceptꢀ 12bꢀ withꢀ oneꢀ G4ꢀ structure)ꢀ
displayedꢀexcellentꢀstabilizationꢀ(8ꢀtoꢀoverꢀ17ꢀ°C)ꢀofꢀtwoꢀdifferentꢀG4ꢀstructuresꢀ
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Chemistry - A European Journal
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andꢀ stabilizedꢀ theꢀ G4ꢀ structuresꢀ muchꢀ moreꢀ thanꢀ theꢀ publishedꢀ referenceꢀ
compoundꢀECH22.[ ꢀSecondly,ꢀCDꢀtitrationꢀexperimentsꢀwereꢀusedꢀtoꢀprobeꢀhowꢀ
theꢀcompoundsꢀaffectꢀtheꢀG4ꢀstructuresꢀuponꢀbindingꢀbyꢀusingꢀthreeꢀdifferentꢀG4ꢀ
structures.ꢀ Theseꢀ dataꢀ clearlyꢀ showedꢀ thatꢀ theꢀ additionꢀ ofꢀ increasingꢀ
concentrationsꢀofꢀtheꢀmoreꢀflexibleꢀscaffoldꢀIꢀcompoundsꢀaffectedꢀtheꢀG4ꢀstructureꢀ
toꢀ aꢀ muchꢀ largerꢀ extentꢀ thanꢀ theꢀ rigidꢀ scaffoldꢀ IIꢀ compounds,ꢀ suggestingꢀ aꢀ
differenceꢀ inꢀ theꢀ compounds’ꢀ abilityꢀ toꢀ interactꢀ withꢀ theꢀ G4ꢀ structuresꢀ thatꢀ isꢀ
directlyꢀrelatedꢀtoꢀtheꢀcompounds’ꢀflexibility.ꢀThirdly,ꢀtheꢀFIDꢀassayꢀwasꢀusedꢀtoꢀ
23]
measureꢀtheꢀapparentꢀdissociationꢀbindingꢀconstantsꢀ(K
showedꢀ thatꢀ allꢀ compoundsꢀ haveꢀ aꢀ highꢀ affinityꢀ forꢀ theꢀ G4ꢀ structuresꢀ withꢀ
apparentꢀ K ꢀ valuesꢀ betweenꢀ 0.5-2ꢀ µMꢀ forꢀ theꢀ threeꢀ testedꢀ G4ꢀ structures.ꢀ Theꢀ
d
)ꢀforꢀtheꢀcompounds.ꢀThisꢀ
d
apparentꢀKdꢀvaluesꢀforꢀtheꢀflexibleꢀscaffoldꢀIꢀcompoundsꢀwereꢀinꢀgeneralꢀlowerꢀ
thanꢀ theꢀ rigidꢀ scaffoldꢀ IIꢀ compounds,ꢀ suggestingꢀ thatꢀ theꢀ flexibleꢀ scaffoldꢀ Iꢀ
compoundsꢀhaveꢀaꢀslightlyꢀhigherꢀaffinity.ꢀHowever,ꢀtheꢀstrikingꢀselectivityꢀseenꢀ
forꢀcompoundꢀ12bꢀinꢀtheꢀCDꢀthermalꢀstabilizationꢀwasꢀnotꢀtranslatedꢀintoꢀtheꢀFIDꢀ
assay.ꢀThisꢀsuggestꢀthatꢀalthoughꢀCDꢀmeltingꢀexperimentsꢀareꢀvaluableꢀtoꢀevaluateꢀ
G4ꢀ stabilizationꢀ effectsꢀ itꢀ needsꢀ additionalꢀ assaysꢀ forꢀ verificationꢀ ofꢀ binding.ꢀ
Fourthly,ꢀ weꢀ alsoꢀusedꢀ theꢀ Taqꢀ polymeraseꢀ stopꢀ assayꢀ inꢀ aꢀ fullꢀdose-responseꢀ
studyꢀtoꢀinvestigateꢀifꢀtheꢀstabilizationꢀofꢀtheꢀG4ꢀstructureꢀbyꢀourꢀcompoundsꢀcouldꢀ
beꢀ correlatedꢀ toꢀ doseꢀ dependentꢀ blockageꢀ ofꢀ DNAꢀ synthesis.ꢀ Indeed,ꢀ thisꢀ
confirmedꢀthatꢀallꢀcompoundsꢀefficientlyꢀ(IC50ꢀvaluesꢀfromꢀ0.1-0.4ꢀµM)ꢀblockedꢀtheꢀ
TaqꢀDNAꢀpolymeraseꢀjustꢀbeforeꢀtheꢀfirstꢀG-tract,ꢀwhichꢀleadꢀtoꢀinhibitionꢀofꢀDNAꢀ
synthesisꢀinꢀaꢀdoseꢀdependentꢀmanner.ꢀꢀ
Finally,ꢀ MDꢀ simulationsꢀ wereꢀ usedꢀ toꢀ understandꢀ theꢀ detailsꢀ ofꢀ howꢀ theꢀ
compoundsꢀinteractꢀwithꢀtheꢀPu24Tꢀc-MYCꢀG4ꢀstructure.ꢀThisꢀshowedꢀthatꢀtheꢀ
flexibleꢀscaffoldꢀIꢀcompoundsꢀcanꢀadaptꢀbetterꢀtoꢀtheꢀG4ꢀstructureꢀbyꢀfindingꢀmoreꢀ
favorableꢀinteractions,ꢀwhichꢀlikelyꢀisꢀtheꢀreasonꢀtoꢀtheirꢀslightlyꢀbetterꢀactivitiesꢀ
inꢀmostꢀofꢀtheꢀassaysꢀtestedꢀinꢀthisꢀstudy.ꢀTheseꢀresultsꢀwereꢀinꢀconflictꢀwithꢀoneꢀ
ofꢀtheꢀdesignꢀstrategiesꢀbehindꢀtheꢀscaffoldꢀIIꢀclassꢀofꢀcompounds,ꢀwhichꢀwasꢀtoꢀ
lockꢀthemꢀ inꢀtheꢀbioactiveꢀcrescentꢀshapedꢀconformation.ꢀThisꢀ showsꢀthatꢀitꢀisꢀ
difficultꢀtoꢀexactlyꢀdetermineꢀandꢀmimicꢀtheꢀG4ꢀbindingꢀconformationꢀwithꢀaꢀveryꢀ
rigidꢀ compound.ꢀ Furthermore,ꢀ rigidꢀ compoundsꢀ doꢀ notꢀhaveꢀ theꢀ possibilityꢀ toꢀ
moveꢀandꢀadaptꢀaccordingꢀtoꢀitsꢀsurroundings,ꢀwhichꢀbecameꢀevidentꢀfromꢀtheꢀMDꢀ
simulations.ꢀTheꢀmoreꢀflexibleꢀscaffoldꢀIꢀanaloguesꢀonꢀtheꢀotherꢀhand,ꢀhaveꢀtoꢀbeꢀ
forcedꢀintoꢀtheirꢀcrescentꢀshapeꢀuponꢀbindingꢀtoꢀtheꢀG4.ꢀHowever,ꢀtheꢀscaffoldꢀIꢀ
compoundsꢀstabilizedꢀtheꢀG4ꢀstructuresꢀsimilarlyꢀorꢀevenꢀbetterꢀthanꢀtheꢀrigidꢀ
scaffoldꢀIIꢀcompoundsꢀinꢀseveralꢀassaysꢀinꢀthisꢀstudy.ꢀThisꢀclearlyꢀshowꢀthatꢀtheꢀ
entropicꢀpenaltyꢀofꢀbindingꢀforꢀtheꢀflexibleꢀscaffoldꢀIꢀcompoundsꢀisꢀoutweighedꢀbyꢀ
theꢀpositiveꢀaspectsꢀofꢀtheꢀflexibilityꢀinꢀfindingꢀoptimalꢀinteractionsꢀwithꢀtheꢀG-
tetradꢀasꢀwellꢀasꢀotherꢀregionsꢀofꢀtheꢀDNA.ꢀTheꢀMDꢀsimulationsꢀalsoꢀdemonstratedꢀ
thatꢀitꢀisꢀnotꢀonlyꢀtheꢀstackingꢀinteractionsꢀwithꢀtheꢀG-tetradꢀthatꢀareꢀimportantꢀforꢀ
efficientꢀG4ꢀbinding,ꢀbutꢀalsoꢀadditionalꢀinteractionsꢀwithꢀtheꢀDNA.ꢀOneꢀofꢀtheꢀmostꢀ
strikingꢀadditionalꢀinteractionsꢀwasꢀtheꢀsandwichꢀinteractionꢀwithꢀtheꢀupstreamꢀ
sequence.ꢀ Theseꢀ additionalꢀ interactionsꢀ willꢀ beꢀ ofꢀ keyꢀ importanceꢀ inꢀ futureꢀ
developmentꢀ andꢀ optimizationsꢀ ofꢀ efficientꢀ andꢀ selectiveꢀ G4ꢀ bindingꢀ andꢀ
stabilizingꢀcompounds.ꢀꢀ
ꢀ
ExperimentalꢀSectionꢀ
ꢀ
ꢀ
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Chemistry - A European Journal
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Synthesisꢀ
ꢀ
Procedureꢀforꢀpreparationꢀofꢀtert-butylꢀ1H-indol-6-ylcarbamateꢀ(6):ꢀ
Theꢀmixtureꢀofꢀtert-butylꢀ1H-indol-6-ylcarbamateꢀ5ꢀ(0.8ꢀg,ꢀ3.44ꢀmmol)ꢀandꢀCDIꢀ
0.31ꢀ gꢀ 0.55ꢀ mmol)ꢀ dissolvedꢀ inꢀ dryꢀ DMFꢀ (10ꢀ mL).ꢀ Thenꢀ 10-20ꢀ mLꢀ MWꢀ vialꢀ
containingꢀreactionꢀmixtureꢀwasꢀirradiatedꢀunderꢀMWꢀconditionsꢀatꢀ150ꢀꢀCꢀforꢀ1ꢀh.ꢀ
(
̊
TheꢀprogressꢀofꢀtheꢀreactionꢀwasꢀmonitoredꢀbyꢀTLC.ꢀUponꢀcompletion,ꢀtheꢀreactionꢀ
mixtureꢀ wasꢀ extractedꢀ withꢀ ethylꢀ acetateꢀ (3ꢀ ×ꢀ 30ꢀ mL).ꢀ Theꢀ combinedꢀ organicꢀ
phasesꢀwereꢀwashedꢀwithꢀbrineꢀ(40ꢀmL),ꢀdriedꢀoverꢀanhydrousꢀNa
2
SO ,ꢀfiltered,ꢀ
4
andꢀconcentratedꢀunderꢀaꢀreducedꢀpressure.ꢀTheꢀresidueꢀwasꢀpurifiedꢀbyꢀcolumnꢀ
chromatographyꢀ overꢀ silicaꢀ gelꢀ usingꢀ ethylꢀ acetate–heptaneꢀ (1:9)ꢀ toꢀ giveꢀ theꢀ
desiredꢀproductꢀ6ꢀinꢀ64%ꢀyieldꢀasꢀwhiteꢀsolid.ꢀ
1
R
f
ꢀ(1%ꢀAcetoneꢀinꢀDCM)ꢀ0.82;ꢀ HꢀNMRꢀ(400ꢀMHz,ꢀCDCl
3
)ꢀδ:ꢀ8.03ꢀ(d,ꢀJꢀ=ꢀ1.9ꢀHz,ꢀ2H),ꢀ
7
6
CDCl
.51ꢀ(dd,ꢀJꢀ=ꢀ8.5,ꢀ2.5ꢀHz,ꢀ2H),ꢀ7.41(d,ꢀJꢀ=ꢀ8.5ꢀHz,ꢀ2H),ꢀ7.33ꢀ(dd,ꢀJꢀ=ꢀ3.ꢀ7,ꢀ2.4ꢀHz,ꢀ2H),ꢀ
13
.67ꢀ(d,ꢀJꢀ=ꢀ14.5ꢀHz,ꢀ2H),ꢀ6.62ꢀ(t,ꢀJꢀ=ꢀ3.0ꢀHz,ꢀ2H),ꢀ1.52ꢀ(s,ꢀ18H);ꢀ CꢀNMRꢀ(100ꢀMHz,ꢀ
)ꢀδ:ꢀ153.03,ꢀ148.57,ꢀ136.65,ꢀ135.92,ꢀ126.24,ꢀ125.95,ꢀ121.27,ꢀ116.03,ꢀ108.46,ꢀ
05.65,ꢀ80.5,ꢀ28.39;ꢀMSꢀ(ESꢀmass):ꢀm/zꢀ491.3ꢀ(M+1).ꢀ
3
1
Procedureꢀforꢀpreparationꢀofꢀbis(6-amino-1H-indol-1-yl)methanoneꢀ(7):ꢀ
Toꢀtheꢀsolutionꢀofꢀtert-butylꢀ1H-indol-6-ylcarbamateꢀ6ꢀ(0.8ꢀg,ꢀ1.63ꢀmmol)ꢀinꢀdryꢀ
DCMꢀ (25ꢀ mL)ꢀ wasꢀ addedꢀ TFAꢀ (2.5ꢀ mL).ꢀ Theꢀ mixtureꢀ wasꢀ stirredꢀ atꢀ roomꢀ
temperatureꢀunderꢀnitrogenꢀatmosphereꢀforꢀ4ꢀh.ꢀTheꢀprogressꢀofꢀtheꢀreactionꢀwasꢀ
monitoredꢀbyꢀTLC.ꢀUponꢀcompletion,ꢀtheꢀreactionꢀmixtureꢀwasꢀneutralizedꢀwithꢀ
ammoniaꢀsolutionꢀandꢀextractedꢀwithꢀethylꢀacetateꢀ(3ꢀ×ꢀ30ꢀmL).ꢀTheꢀcombinedꢀ
organicꢀphasesꢀwereꢀwashedꢀwithꢀbrineꢀ(50ꢀmL),ꢀdriedꢀoverꢀanhydrousꢀNa
filtered,ꢀandꢀconcentratedꢀunderꢀaꢀreducedꢀpressure.ꢀTheꢀresidueꢀwasꢀisolatedꢀinꢀ
7%ꢀ yieldꢀ asꢀ offꢀ whiteꢀ solidꢀ andꢀ wasꢀ usedꢀ inꢀ theꢀ nextꢀ stepꢀ withoutꢀ furtherꢀ
purification.ꢀ
ꢀ(80%ꢀEtOAcꢀinꢀn-heptane)ꢀ0.06;ꢀ HꢀNMRꢀ(400ꢀMHz,ꢀCDCl
H),ꢀ7.36ꢀ(d,ꢀJꢀ=ꢀ1.9ꢀHz,ꢀ2H),ꢀ7.25ꢀ(d,ꢀJꢀ=ꢀ3.7ꢀHz,ꢀ2H),ꢀ6.71ꢀ(dd,ꢀJꢀ=ꢀ8.4,ꢀ2.0ꢀHz,ꢀ2H),ꢀ
2
SO ,ꢀ
4
9
1
R
f
3
)ꢀδ:ꢀ7.38ꢀ(d,ꢀJꢀ=ꢀ8.3ꢀHz,ꢀ
2
6
1
1
3
.56ꢀ(d,ꢀJꢀ=ꢀ3.7ꢀHz,ꢀ2H);ꢀ CꢀNMRꢀ(150ꢀMHz,ꢀCDCl
3
)ꢀδ:ꢀ144.46,ꢀ137.67,ꢀ124.69,ꢀ
22.66,ꢀ121.71,ꢀ113.02,ꢀ108.60,ꢀ100.92;ꢀMSꢀ(ESꢀmass):ꢀm/zꢀꢀ291.ꢀ
Procedureꢀ forꢀ theꢀ preparationꢀ ofꢀ N,N'-(1,1'-carbonylbis(1H-indole-6,1-
diyl))bis-(quinoline-3-carboxamide)ꢀ(ECH47)ꢀ(8):ꢀ
Toꢀtheꢀsolutionꢀofꢀquinoline-3-carboxylicꢀacidꢀ(0.51ꢀg,ꢀ2.96ꢀmmol)ꢀinꢀaꢀdryꢀDMFꢀ(6ꢀ
mL)ꢀwasꢀaddedꢀHATUꢀ(1.15ꢀg,ꢀ3.03ꢀmmol)ꢀandꢀtheꢀreactionꢀmixtureꢀwasꢀstirredꢀ
untilꢀtheꢀsolidsꢀwereꢀdissolvedꢀcompletelyꢀatꢀroomꢀtemperature.ꢀThenꢀtheꢀamineꢀ
7
ꢀ(0.42ꢀg,ꢀ1.46ꢀmmol)ꢀinꢀDMFꢀ(4ꢀmL)ꢀwasꢀslowlyꢀaddedꢀtoꢀtheꢀreactionꢀmixtureꢀandꢀ
stirredꢀforꢀ10ꢀminꢀfollowedꢀbyꢀtheꢀadditionꢀofꢀDIPEAꢀ(0.56ꢀg,ꢀ4.34ꢀmmol).ꢀTheꢀ
mixtureꢀwasꢀstirredꢀatꢀroomꢀtemperatureꢀunderꢀnitrogenꢀatmosphereꢀforꢀ12ꢀh.ꢀ
TheꢀprogressꢀofꢀtheꢀreactionꢀwasꢀmonitoredꢀbyꢀTLC.ꢀUponꢀcompletion,ꢀtheꢀreactionꢀ
mixtureꢀwasꢀpouredꢀintoꢀiceꢀandꢀfilteredꢀthroughꢀsinteredꢀfunnel.ꢀTheꢀsolidꢀwasꢀ
driedꢀunderꢀvacuum.ꢀTheꢀpureꢀcompoundꢀ8ꢀwasꢀisolatedꢀinꢀ96%ꢀyieldꢀasꢀaꢀlightꢀ
yellowꢀsolid.ꢀ
1
R
f
ꢀ(80%ꢀEtOAcꢀinꢀn-heptane)ꢀ0.21;ꢀ HꢀNMRꢀ(600ꢀMHz,ꢀDMSO-d
6
)ꢀδ:ꢀ10.79ꢀ(s,ꢀ2H),ꢀ
9
4
6
1
.39ꢀ(d,ꢀJꢀ=ꢀ2.3ꢀHz,ꢀ2H),ꢀ9.00ꢀ(d,ꢀJꢀ=ꢀ2.3ꢀHz,ꢀ2H),ꢀ8.71ꢀ–ꢀ8.68ꢀ(m,ꢀ2H),ꢀ8.19ꢀ–ꢀ8.10ꢀ(m,ꢀ
H),ꢀ7.90ꢀ(td,ꢀJꢀ=ꢀ8.4,ꢀ1.5ꢀHz,ꢀ2H),ꢀ7.80ꢀ(dd,ꢀJꢀ=ꢀ8.5,ꢀ1.9ꢀHz,ꢀ2H),ꢀ7.76ꢀ–ꢀ7.67ꢀ(m,ꢀ6H),ꢀ
13
.86ꢀ(d,ꢀJꢀ=ꢀ3.7ꢀHz,ꢀ2H);ꢀ CꢀNMRꢀ(150ꢀMHz,ꢀDMSO-d )ꢀδ:ꢀ164.52,ꢀ149.63,ꢀ148.97,ꢀ
6
48.83,ꢀ136.54,ꢀ 136.49,ꢀ36.45,ꢀ 131.82,ꢀ 129.67,ꢀ 129.27,ꢀ 128.29,ꢀ128.19,ꢀ 127.97,ꢀ
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Chemistry - A European Journal
10.1002/chem.201800078
1
26.95,ꢀ126.91,ꢀ121.62,ꢀ117.55,ꢀ108.80,ꢀ107.28;ꢀMSꢀ(ESꢀmass):ꢀm/zꢀ601.3ꢀ(M+1);ꢀ
+
HRMS:ꢀm/zꢀcalcdꢀforꢀC37
24
H N
6
O [Mꢀ+ꢀH] 601.1983,ꢀfoundꢀ601.2003.ꢀ
3
Procedureꢀ forꢀ preparationꢀ ofꢀ N,N'-(1,1'-carbonylbis(1H-indole-6,1-
diyl))bis-(quinoline-3-carboxamide)ꢀ(ECH48)ꢀ(9):ꢀ
Toꢀaꢀsolutionꢀofꢀ8ꢀ(0.2ꢀg,ꢀ0.33ꢀmmol)ꢀinꢀaceticꢀacidꢀwasꢀaddedꢀPd(OAc)
2
ꢀ(7.5ꢀmg,ꢀ
0
.033ꢀmmol)ꢀandꢀCu(OAc)2ꢀ(66.5ꢀmg,ꢀ0.36ꢀmmol)ꢀunderꢀnitrogenꢀatmosphereꢀatꢀ
°
roomꢀtemperature.ꢀThenꢀtheꢀmixtureꢀwasꢀstirredꢀatꢀ100ꢀ Cꢀunderꢀnitrogenꢀforꢀ14ꢀ
h.ꢀ Theꢀ progressꢀ ofꢀ theꢀ reactionꢀ wasꢀ monitoredꢀ byꢀ TLC.ꢀ Uponꢀ completion,ꢀ theꢀ
reactionꢀ mixtureꢀ wasꢀ cooledꢀ andꢀ neutralizedꢀ withꢀ ammoniaꢀ solutionꢀ andꢀ theꢀ
mixtureꢀwasꢀtreatedꢀwithꢀultrasonicꢀwavesꢀforꢀ5ꢀhoursꢀbeforeꢀbeingꢀextractedꢀwithꢀ
ethylꢀacetateꢀ(3ꢀ×ꢀ30ꢀmL).ꢀTheꢀcombinedꢀorganicꢀphasesꢀwereꢀwashedꢀwithꢀbrineꢀ
(
50ꢀmL),ꢀdriedꢀoverꢀanhydrousꢀNa
2
SO ,ꢀfiltered,ꢀandꢀconcentratedꢀunderꢀaꢀreducedꢀ
4
pressureꢀtoꢀgiveꢀcompoundꢀ9ꢀinꢀ64%ꢀyieldꢀinꢀaꢀlightꢀyellowꢀcolorꢀsolid.ꢀ
1
R
f
ꢀ(80%ꢀEtOAcꢀinꢀn-heptane)ꢀ0.18;ꢀ HꢀNMRꢀ(600ꢀMHz,ꢀDMSO-d )ꢀδ:ꢀ10.82ꢀ(s,ꢀ2H),ꢀ
6
9
8
.40ꢀ(d,ꢀJꢀ=ꢀ2.2ꢀHz,ꢀ2H),ꢀ9.01ꢀ(d,ꢀJꢀ=ꢀ2.3ꢀHz,ꢀ2H),ꢀ8.48ꢀ(s,ꢀ2H),ꢀ8.17ꢀ(d,ꢀJꢀ=ꢀ8.1ꢀHz,ꢀ2H),ꢀ
.13ꢀ(d,ꢀJꢀ=ꢀ8.4ꢀHz,ꢀ2H),ꢀ7.91ꢀ(t,ꢀJꢀ=ꢀ7.7ꢀHz,ꢀ2H),ꢀ7.75ꢀ–ꢀ7.72ꢀ(m,ꢀ4H),ꢀ7.64ꢀ(d,ꢀJꢀ=ꢀ8.5ꢀ
1
3
Hz,ꢀ2H),ꢀ6.93ꢀ(s,ꢀ2H);ꢀ CꢀNMRꢀ(150ꢀMHz,ꢀDMSO-d
1
1
6
)ꢀδ:ꢀ164.07,ꢀ149.13,ꢀ148.54,ꢀ
43.72,ꢀ137.21,ꢀ136.05,ꢀ132.58,ꢀ131.41,ꢀ129.92,ꢀ129.79,ꢀ129.22,ꢀ128.81,ꢀ127.52,ꢀ
26.42,ꢀ122.67,ꢀ116.58,ꢀ103.55,ꢀ102.34;ꢀMSꢀ(ESꢀmass):ꢀm/zꢀ599.4ꢀ(M+1);ꢀHRMS:ꢀ
+ꢀ
m/zꢀcalcdꢀforꢀC37
H
22
N O3ꢀ[Mꢀ+ꢀH] 599.1826,ꢀfoundꢀ599.1840.ꢀ
6
Generalꢀ procedureꢀ forꢀ theꢀ preparationꢀ ofꢀ 3,3'-(((1,1'-carbonylbis(1H-
indole-6,1-diyl))bis(azanediyl))bis-(carbonyl))-bis(1-alkyl/benzyl-
quinolin-1-ium)ꢀbromides/iodidesꢀ(11):ꢀ
Toꢀ theꢀ compoundꢀ 8ꢀ (10-25ꢀ mg,ꢀ 0.016-0.041ꢀ mmol)ꢀ wasꢀ addedꢀ alkylꢀ
bromide/iodideꢀorꢀarylꢀbromideꢀinꢀaꢀmicrowaveꢀvialꢀandꢀtheꢀvialꢀwasꢀsealedꢀwithꢀ
°
aꢀcap.ꢀThenꢀreactionꢀmixtureꢀwasꢀirradiatedꢀunderꢀMWꢀconditionsꢀatꢀ150ꢀ Cꢀforꢀ20ꢀ
minꢀ toꢀ 70ꢀ min.ꢀ Theꢀ progressꢀ ofꢀ theꢀ reactionꢀ wasꢀ monitoredꢀ byꢀ LC-MS.ꢀ Uponꢀ
completion,ꢀtheꢀreactionꢀmixtureꢀwasꢀfilteredꢀthroughꢀsinteredꢀfunnelꢀandꢀwashedꢀ
withꢀdiethylꢀether,ꢀDCM,ꢀandꢀacetone.ꢀTheꢀsolidꢀwasꢀdriedꢀunderꢀvacuumꢀtoꢀobtainꢀ
pureꢀcompoundꢀ11ꢀinꢀ63-88%ꢀyield.ꢀ
Generalꢀ procedureꢀ forꢀ theꢀ preparationꢀ ofꢀ 3,3'-(((6-oxo-6H-imidazo[1,5-
a:3,4-a']diindole-3,9-diyl)bis-(azanediyl))bis-(carbonyl))bis(1-
alkylquinolin-1-ium)ꢀbromides/iodidesꢀ(12):ꢀ
Toꢀcompoundꢀ9ꢀ(15-20ꢀmg,ꢀ0.025-0.033ꢀmmol)ꢀwasꢀaddedꢀalkylꢀbromide/iodideꢀ
inꢀaꢀmicrowaveꢀvialꢀandꢀtheꢀvialꢀwasꢀsealedꢀwithꢀaꢀcap.ꢀTheꢀreactionꢀmixtureꢀwasꢀ
irradiatedꢀunderꢀMWꢀconditionsꢀatꢀ150ꢀ°Cꢀforꢀ3ꢀtoꢀ4ꢀh.ꢀTheꢀprogressꢀofꢀtheꢀreactionꢀ
wasꢀ monitoredꢀ byꢀ LC-MS.ꢀ Uponꢀ completion,ꢀ theꢀ reactionꢀ mixtureꢀ wasꢀ filteredꢀ
throughꢀcenteredꢀfunnelandꢀwashedꢀwithꢀdiethylꢀether,ꢀDCM,ꢀandꢀacetone.ꢀTheꢀ
solidꢀwasꢀdriedꢀunderꢀvacuumꢀtoꢀobtainꢀpureꢀcompoundꢀ12ꢀinꢀ46-68%ꢀyield.ꢀ
ꢀ
SeeꢀtheꢀSupportingꢀInformationꢀforꢀfullꢀexperimentalꢀdetails.ꢀ
ꢀ
FoldingꢀofꢀG4ꢀstructuresꢀ
5
0ꢀ µMꢀ Oligonucleotidesꢀ (MWGꢀ Operon,ꢀ Germany)ꢀ wereꢀ foldedꢀ inꢀ 10ꢀ mMꢀ
tris(hydroxymethyl)aminomethaneꢀhydrochlorideꢀ(Tris-HCl,ꢀpHꢀ7.5)ꢀandꢀ100ꢀmMꢀ
KClꢀbyꢀheatingꢀtoꢀ95ꢀ°Cꢀforꢀ5ꢀminꢀandꢀslowlyꢀcooledꢀdownꢀovernightꢀtoꢀroomꢀ
temperature.ꢀ
ꢀ
Taq-polymeraseꢀstopꢀassayꢀ
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Chemistry - A European Journal
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1
ꢀµMꢀTET-labeledꢀprimerꢀwasꢀannealedꢀtoꢀ1.25ꢀµMꢀtemplateꢀDNAꢀinꢀ50ꢀmMꢀKClꢀbyꢀ
heatingꢀtheꢀreactionꢀatꢀ95ꢀ°Cꢀforꢀ5ꢀminꢀandꢀslowlyꢀcoolꢀdownꢀovernightꢀtoꢀroomꢀ
temperature.ꢀIfꢀtheꢀtemplateꢀcontainedꢀtheꢀG4ꢀmotif,ꢀfoldingꢀofꢀtheꢀG4ꢀstructureꢀ
proceededꢀ simultaneouslyꢀ withꢀ primerꢀ annealing.ꢀ Forꢀ eachꢀ reactionꢀ inꢀ theꢀ
screeningꢀprocedure,ꢀ40ꢀnMꢀannealedꢀDNAꢀwasꢀincubatedꢀwithꢀ1ꢀµMꢀcompoundsꢀ
orꢀ5%ꢀ(v/v)ꢀDMSOꢀforꢀatꢀleastꢀ30ꢀminꢀinꢀ50ꢀmMꢀKCl,ꢀ10ꢀmMꢀTris-HClꢀ(pHꢀ7.5),ꢀ1.5ꢀ
mMꢀMgCl ,ꢀandꢀ200ꢀmMꢀdNTPsꢀatꢀroomꢀtemperature.ꢀDNAꢀsynthesisꢀwasꢀstartedꢀ
2
byꢀ theꢀ additionꢀ ofꢀ 0.025ꢀ Uꢀ ofꢀ recombinantꢀ Taqꢀ DNA-polymeraseꢀ (Thermoꢀ
Scientific)ꢀintoꢀtheꢀreactionꢀmixtureꢀ(10ꢀµL),ꢀandꢀincubatedꢀ10ꢀminꢀatꢀ50ꢀ°C.ꢀTheꢀ
reactionꢀwasꢀstoppedꢀbyꢀadditionꢀofꢀoneꢀvolumeꢀofꢀstopꢀsolutionꢀ(95%ꢀformamide,ꢀ
2
0ꢀ mmꢀ ethylenediaminetetraaceticꢀ acidꢀ (EDTA),ꢀ andꢀ 0.1%ꢀ bromophenolꢀ blue)ꢀ
intoꢀtheꢀreaction.ꢀTotalꢀofꢀ5ꢀµLꢀofꢀtheꢀmixtureꢀwasꢀloadedꢀintoꢀ10%ꢀpolyacrylamideꢀ
gelꢀ containingꢀ 8ꢀ Mꢀ urea,ꢀ 25%ꢀ formamide,ꢀ andꢀ 1xꢀ
tris/borate/ethylenediaminetetraaceticꢀacidꢀ(TBE).ꢀTheꢀgelꢀwasꢀvisualizedꢀwithꢀaꢀ
TyphoonꢀScannerꢀ9400ꢀ(GEꢀHealthcare)ꢀatꢀtheꢀAlexaꢀsettingꢀofꢀλꢀ=ꢀ532ꢀnmꢀandꢀ
quantifiedꢀwithꢀtheꢀImageꢀQuantꢀTLꢀ8.1ꢀsoftwareꢀ(GEꢀHealthcare).ꢀSampleꢀtreatedꢀ
withꢀ5%ꢀDMSOꢀwasꢀusedꢀasꢀaꢀcontrolꢀreaction.ꢀFullꢀproductꢀofꢀeachꢀreactionꢀwasꢀ
normalizedꢀ toꢀ theꢀ fullꢀ productꢀ ofꢀ DMSO-treatedꢀ sample.ꢀ Allꢀ reactionsꢀ wereꢀ
repeatedꢀtwiceꢀandꢀabsoluteꢀerrorꢀwasꢀcalculated.ꢀ
Forꢀ theꢀ determinationꢀ ofꢀ IC50ꢀ values,ꢀ theꢀ DNAꢀ wasꢀ incubatedꢀ withꢀ differentꢀ
concentrationsꢀ (5,ꢀ 2,ꢀ 0.8,ꢀ 0.32,ꢀ 0.13ꢀ 0.05,ꢀ 0.025ꢀ andꢀ 0.008ꢀ µM)ꢀ ofꢀ theꢀ testedꢀ
compoundsꢀ orꢀ 5%ꢀ (v/v)ꢀ DMSO.ꢀ IC50ꢀ valuesꢀ wereꢀ calculatedꢀ byꢀ plottingꢀ theꢀ
normalizedꢀfull-lengthꢀproductsꢀagainstꢀconcentrationꢀofꢀtheꢀcompoundsꢀinꢀOriginꢀ
8
.5ꢀsoftware.ꢀAllꢀreactionsꢀwereꢀindependentlyꢀrepeatedꢀthreeꢀtimesꢀandꢀstandardꢀ
deviationꢀwasꢀcalculated.ꢀꢀ
ꢀ
THTꢀassayꢀꢀꢀ
AllꢀcompoundsꢀusedꢀforꢀtheꢀTHTꢀassayꢀwereꢀdilutedꢀinꢀ100ꢀ%ꢀDMSOꢀandꢀfinalꢀ
concentrationꢀofꢀDMSOꢀinꢀallꢀsamplesꢀwereꢀ2.5ꢀ%.ꢀTheꢀTHTꢀexperimentsꢀwereꢀ
performedꢀasꢀdescribedꢀearlier.[23,ꢀ33]ꢀFoldedꢀoligonucleotidesꢀwereꢀdilutedꢀintoꢀ1ꢀ
µMꢀconcentrationꢀintoꢀ10ꢀmMꢀTris,ꢀ100ꢀmMꢀKCl,ꢀ0.5ꢀµMꢀThT,ꢀandꢀ1ꢀµM,ꢀ5ꢀµMꢀorꢀ15ꢀ
µMꢀofꢀeachꢀcompound.ꢀ2.5ꢀ%ꢀ(v/v)ꢀDMSOꢀwasꢀusedꢀasꢀtheꢀreference.ꢀ384-wellꢀ
Corningꢀblackꢀflat-bottomꢀplatesꢀwereꢀusedꢀforꢀmeasurements.ꢀThTꢀfluorescenceꢀ
wasꢀrecordedꢀatꢀλꢀ=ꢀ435/480ꢀnmꢀ(ex/em)ꢀbyꢀInfiniteꢀM200ꢀplateꢀreaderꢀ(Tecan).ꢀ
Fluorescenceꢀofꢀtheꢀbufferꢀ(noꢀDNA)ꢀsupplementedꢀwithꢀappropriateꢀcompoundsꢀ
orꢀDMSOꢀwasꢀsubtractedꢀfromꢀtheꢀdata.ꢀTheꢀrelativeꢀfluorescenceꢀwasꢀcalculatedꢀ
asꢀ aꢀ foldꢀ changeꢀ toꢀ theꢀ referenceꢀ sample.ꢀ Assayꢀ wasꢀ performedꢀ inꢀ technicalꢀ
triplicatesꢀandꢀstandardꢀdeviationꢀwasꢀcalculated.ꢀꢀ
ꢀ
CDꢀmeltingꢀexperimentꢀ
5
0ꢀµMꢀfoldedꢀoligonucleotidesꢀwereꢀdilutedꢀtoꢀ5ꢀµMꢀconcentrationꢀintoꢀ10ꢀmMꢀTrisꢀ
(pHꢀ7.5),ꢀ100ꢀmMꢀKClꢀandꢀ40ꢀµMꢀcompoundꢀorꢀ1.25%ꢀDMSO.ꢀQuartzꢀcuvetteꢀwithꢀ
aꢀ pathꢀ lengthꢀ ofꢀ 1ꢀ mmꢀ wasꢀ usedꢀ forꢀ theꢀ measurementsꢀ byꢀ JASCO-720ꢀ
spectropolarimeterꢀ(JascoꢀInternatiolꢀCo.ꢀLtd.).ꢀFirst,ꢀCDꢀspectraꢀwereꢀrecordedꢀatꢀ
2
5ꢀ°Cꢀoverꢀλꢀ=ꢀ230-350ꢀnmꢀwithꢀanꢀintervalꢀofꢀ1ꢀnmꢀandꢀscanꢀrateꢀofꢀ100ꢀnm/min.ꢀ
Correspondingꢀ bufferꢀ wasꢀ usedꢀ forꢀ baselineꢀ correctionꢀ ofꢀ eachꢀ measurement.ꢀ
Signalsꢀ atꢀ appropriateꢀ peaksꢀ forꢀ eachꢀ G4ꢀ DNAꢀ wereꢀ selectedꢀ forꢀ CDꢀ meltingꢀ
measurements.ꢀ Meltingꢀ curvesꢀ wereꢀ recordedꢀ betweenꢀ 25-90ꢀ °C.ꢀ Meltingꢀ
temperatureꢀ(Tm)ꢀisꢀdefinedꢀasꢀtheꢀtemperatureꢀatꢀwhichꢀ50%ꢀofꢀtheꢀG4ꢀstructuresꢀ
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Chemistry - A European Journal
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areꢀunfolded.ꢀTmꢀvaluesꢀwereꢀestimatedꢀbyꢀfittingꢀtheꢀnormalizedꢀmeltingꢀcurvesꢀ
intoꢀaꢀdoseꢀresponseꢀfunctionꢀusingꢀOriginꢀ8.5ꢀsoftware.ꢀꢀ
ꢀ
CDꢀtitrationꢀ
Circularꢀdichroismꢀ(CD)ꢀspectraꢀwereꢀrecordedꢀatꢀroomꢀtemperatureꢀonꢀaꢀJASCO-
7
20ꢀspectropolarimeterꢀꢀ(JascoꢀInternationalꢀCo.ꢀLtd.)ꢀusingꢀaꢀcuvetteꢀofꢀ1ꢀmmꢀpathꢀ
length.ꢀEachꢀspectrumꢀisꢀtheꢀaverageꢀofꢀthreeꢀscansꢀoverꢀλꢀ=ꢀ230-330ꢀnmꢀwithꢀanꢀ
intervalꢀofꢀ1ꢀnmꢀatꢀaꢀscanꢀrateꢀofꢀ100ꢀnm/min.ꢀInducedꢀCDꢀpeaksꢀwereꢀobservedꢀ
forꢀscaffoldꢀIꢀcompoundsꢀwithꢀribosomalꢀandꢀtelomericꢀG4ꢀDNAꢀandꢀtheꢀscanꢀrangeꢀ
wasꢀ thereforeꢀ extendedꢀ (230-360ꢀ nm).ꢀ Theꢀ concentrationꢀ ofꢀ DNAꢀ wasꢀ keptꢀ
constantꢀatꢀ10ꢀµMꢀwhileꢀvaryingꢀtheꢀconcentrationꢀofꢀcompoundsꢀfromꢀ10-100ꢀµM.ꢀ
Theꢀ concentrationsꢀ ofꢀ DNAꢀ andꢀ compoundsꢀ duringꢀ CDꢀ measurementsꢀ areꢀ
mentionedꢀinꢀtheꢀrelevantꢀdiscussion.ꢀTheꢀcorrespondingꢀbufferꢀblanksꢀwereꢀusedꢀ
toꢀcorrectꢀbackground.ꢀ
ꢀ
ꢀ
FIDꢀassayꢀ
FIDꢀassayꢀwasꢀcarriedꢀoutꢀwithꢀaꢀJascoꢀFP-8500ꢀfluorescenceꢀspectrometer.ꢀTheꢀ
FIDꢀassayꢀwasꢀperformedꢀusingꢀtheꢀprocedureꢀdescribedꢀearlier.[ ꢀ0.25ꢀμMꢀofꢀpre-
foldedꢀDNAꢀwasꢀmixedꢀwithꢀ0.50ꢀµMꢀThiazoleꢀorangeꢀ(TO)ꢀandꢀincubatedꢀforꢀ2ꢀ
minutes.ꢀ Compoundsꢀ wereꢀ addedꢀ toꢀ theꢀ reactionꢀ stepwiseꢀ withꢀ aꢀ 2ꢀ minꢀ
equilibrationꢀperiod,ꢀandꢀtheꢀfluorescenceꢀspectrumꢀwasꢀrecordedꢀ(λexꢀ=ꢀ501ꢀnm;ꢀ
41]
λemꢀ=ꢀ510ꢀ−ꢀ650ꢀnm)(FigureꢀS10ꢀandꢀS11).ꢀTheꢀpercentageꢀofꢀdisplacementꢀofꢀTOꢀ
wasꢀcalculatedꢀfromꢀtheꢀfluorescenceꢀintensityꢀ(ꢀ)ꢀatꢀtheꢀemissionꢀmaxima,ꢀusingꢀ
theꢀfollowingꢀequation:[41]
ꢀ
ꢀ
ꢀ
PercentageꢁofꢁTOꢁdisplacement = 100 −ꢁꢂ ꢁ× 100ꢄꢀ
ꢀ
ꢃ
ꢀ
withꢀꢀ ꢀbeingꢀ theꢀ fluorescenceꢀ intensityꢀ ofꢀ TOꢀ boundꢀ toꢀ DNAꢀ withoutꢀ addedꢀ
ꢃ
compound.ꢀTheꢀpercentageꢀofꢀTOꢀdisplacementꢀwasꢀplottedꢀasꢀaꢀfunctionꢀofꢀtheꢀ
concentrationꢀofꢀaddedꢀcompound.ꢀ
Theꢀ associationꢀ bindingꢀ constantꢀ (K
a
)ꢀ ofꢀ theꢀ ligandꢀ wasꢀ calculatedꢀ fromꢀ theꢀ
followingꢀequation:[
43]
ꢀ
ꢇꢈ
ꢋ
ꢀ
ꢀ
ꢀ
ꢅꢆ ꢁ×ꢁ[ꢉꢊ] =ꢁꢅ ꢁ×ꢁ[ꢌꢍꢎꢏꢃ]ꢀ
Where,ꢀꢀꢅꢆ ꢁ= ꢁꢐꢑꢑꢒꢓꢔꢕꢖꢔꢒꢗꢁꢘꢔꢗꢙꢔꢗꢚꢁꢓꢒꢗꢑꢖꢕꢗꢖꢁꢒꢛꢁꢉꢊꢁꢜꢔꢖℎꢁꢝ4ꢁꢞꢟꢐꢀ
ꢆ
ꢇꢈ
ꢋ
ꢀ
ꢀ
ꢀ
ꢀꢀꢅ ꢁ= ꢁꢐꢑꢑꢒꢓꢔꢕꢖꢔꢒꢗꢁꢘꢔꢗꢙꢔꢗꢚꢁꢓꢒꢗꢑꢖꢕꢗꢖꢁꢒꢛꢁꢠꢔꢚꢕꢗꢙꢁꢜꢔꢖℎꢁꢝ4ꢁꢞꢟꢐꢀ
ꢀꢀ[ꢉꢊ]ꢁ= ꢁꢡꢒꢗꢓꢢꢗꢖꢣꢕꢖꢔꢒꢗꢁꢒꢛꢁꢉꢊꢀ
ꢀꢀ[ꢌꢍꢎꢏꢃ]ꢁ= ꢡꢒꢗꢓꢢꢗꢖꢣꢕꢖꢔꢒꢗꢁꢒꢛꢁꢠꢔꢚꢕꢗꢙꢁꢛꢒꢣꢁ50%ꢁꢙꢔꢑꢤꢠꢕꢓꢢꢥꢢꢗꢖꢁꢒꢛꢁꢉꢊꢀ
ꢆ
Theꢀ apparentꢀ dissociationꢀ bindingꢀ constantꢀ (K )ꢀ wasꢀ nextꢀ calculatedꢀ fromꢀ theꢀ
d
followingꢀequation:ꢀ
1
ꢀ
ꢀ
ꢀ
ꢀ
ꢅ = ꢧ ꢋꢀ
ꢦ
ꢅꢆ
ꢇꢈ
Toꢀdetermineꢀꢅꢆ ,ꢀweꢀhaveꢀperformedꢀspectrofluorimetricꢀtitrationsꢀofꢀTOꢀwithꢀ
Pu24Tꢀc-MYC,ꢀribosomalꢀandꢀtelomericꢀG4ꢀDNAꢀsequencesꢀ(FigureꢀS12).ꢀWeꢀhaveꢀ
usedꢀ2ꢀμMꢀTOꢀandꢀaddedꢀtheꢀG4ꢀDNAꢀfromꢀ0ꢀtoꢀ5ꢀμM.ꢀTheꢀassociationꢀbindingꢀ
ꢇꢈ
1
constantꢀofꢀTOꢀ(ꢅꢆ = ꢧ )ꢀwasꢀcalculatedꢀfromꢀtheꢀplotꢀofꢀΔF/ΔFmaxꢀatꢀ540ꢀnmꢀ
ꢅ
ꢦ
ꢀ
againstꢀtheꢀconcentrationꢀofꢀDNAꢀusingꢀtheꢀfollowingꢀequation:
ꢨꢩ
ꢭ
ꢁ[ꢍꢮꢯ]
ꢪꢫꢬ
ꢀ
ꢀ
ꢀ
ꢀ
ꢁ=ꢁ
ꢀ
ꢰ ꢁꢲꢁ[ꢍꢮꢯ]
ꢨꢩꢪꢫꢬ
ꢱ
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201800078
Where,ꢀꢅ = ꢕꢤꢤꢕꢣꢢꢗꢖꢁꢙꢔꢑꢑꢒꢓꢔꢕꢖꢔꢒꢗꢁꢘꢔꢗꢙꢔꢗꢚꢁꢓꢒꢗꢑꢖꢕꢗꢖꢁꢀꢀ
ꢦ
ꢀ
ꢀ
ꢀ
ꢀ
∆ꢀꢁ = ꢓℎꢕꢗꢚꢢꢁꢔꢗꢁꢖℎꢢꢁꢢꢥꢔꢑꢑꢔꢒꢗꢁꢑꢤꢢꢓꢖꢣꢳꢥꢁꢕꢖꢁ540ꢁꢗꢥꢁꢕꢛꢖꢢꢣꢁꢞꢟꢐꢁꢕꢙꢙꢔꢖꢔꢒꢗꢀ
∆ꢀꢴꢆꢵ ꢁ= ꢥꢕꢶꢔꢥꢳꢥꢁꢓℎꢕꢗꢚꢢꢁꢔꢗꢁꢖℎꢢꢁꢢꢥꢔꢑꢑꢔꢒꢗꢁꢑꢤꢢꢓꢖꢣꢳꢥꢀ
ꢷꢴꢆꢵ ꢁ= ꢥꢕꢶꢔꢥꢳꢥꢁꢑꢤꢢꢓꢔꢛꢔꢓꢁꢘꢔꢗꢙꢔꢗꢚꢀ
Modelingꢀandꢀsimulationꢀofꢀ11aꢀandꢀ12aꢀwithꢀG4ꢀDNAꢀ
Toꢀmodelꢀtheꢀcomplexesꢀofꢀ11aꢀandꢀ12aꢀwithꢀG4ꢀDNA,ꢀweꢀconsideredꢀsolutionꢀ
structureꢀofꢀPhen-DC3/DNAꢀ(PDBꢀID:ꢀ2MGN)[28]ꢀandꢀusedꢀtheꢀfirstꢀconformationꢀ
asꢀ template.ꢀ Inꢀ placeꢀ ofꢀ Phen-DC3,ꢀ 11aꢀ andꢀ 12aꢀ wasꢀ builtꢀ separatelyꢀ usingꢀ
Avogadroꢀpackage.ꢀTheseꢀcomplexꢀstructuresꢀwereꢀconsideredꢀforꢀtheꢀmolecularꢀ
dynamicsꢀ(MD)ꢀsimulations.ꢀ
Forꢀ theꢀ MDꢀ simulations,ꢀ atꢀ firstꢀ molecularꢀ systemꢀ wasꢀ preparedꢀ byꢀ placingꢀ
complexꢀatꢀtheꢀcenterꢀofꢀaꢀdodecahedronꢀperiodicꢀbox,ꢀwhichꢀisꢀsubsequentlyꢀfilledꢀ
byꢀwaterꢀmolecules.ꢀNext,ꢀ0.150ꢀMꢀofꢀNaClꢀwasꢀaddedꢀwithꢀtheꢀexcessꢀofꢀsodiumꢀ
ionsꢀtoꢀcounterꢀtheꢀnegativeꢀchargeꢀofꢀtheꢀsystem.ꢀForꢀtheꢀDNA,ꢀAmber99sb[
ꢀ
44]
withꢀPARMBSC1[45]ꢀimprovementsꢀwereꢀtakenꢀasꢀtheꢀforce-fieldꢀparametersꢀ.ꢀForꢀ
theꢀcompoundsꢀ11aꢀandꢀ12a,ꢀatꢀfirst,ꢀpartialꢀatomicꢀchargesꢀwereꢀcalculatedꢀbyꢀ
[
46]
[47]
RESP ꢀmethodꢀusingꢀAmberToolꢀpackage ꢀafterꢀtheirꢀgeometryꢀoptimizationꢀ
andꢀESPꢀbyꢀHF/6-31G*ꢀbasisꢀsetꢀusingꢀGaussian16.[48]ꢀSubsequently,ꢀtheirꢀforce-
fieldꢀparametersꢀwereꢀgeneratedꢀfromꢀGAFF[ ꢀusingꢀAmberToolꢀandꢀconvertedꢀ
intoꢀGromacsꢀcompatibleꢀformatꢀusingꢀacpypeꢀscript.[50]ꢀTheꢀDNAꢀandꢀligandꢀforce-
fieldꢀ parametersꢀ wereꢀ mergedꢀ inꢀ accordanceꢀ withꢀ theꢀ standardꢀ Gromacsꢀ
procedure.ꢀ Forꢀ water,ꢀ TIP3P[ ꢀ parametersꢀ wereꢀ consideredꢀ duringꢀ theꢀ
simulations.ꢀ
49]
51]
InꢀtheꢀfirstꢀstageꢀofꢀMDꢀsimulation,ꢀenergyꢀofꢀtheꢀmolecularꢀsystemꢀwasꢀminimizedꢀ
-
1
-2
usingꢀsteepest-descentꢀwithꢀaꢀtoleranceꢀofꢀ1000ꢀkJꢀmol nm ꢀuntilꢀforceꢀconvergedꢀ
toꢀ machineꢀ precision.ꢀ Inꢀ theꢀ secondꢀ stage,ꢀ systemsꢀ wereꢀ heatedꢀ toꢀ 300ꢀ Kꢀ atꢀ
constantꢀvolumeꢀforꢀ100ꢀps.ꢀInꢀnextꢀstep,ꢀsystemsꢀwereꢀequilibratedꢀatꢀconstantꢀ
temperatureꢀ 300ꢀ Kꢀ andꢀ pressureꢀ 1ꢀ atmꢀ forꢀ 1ꢀ nsꢀ NPTꢀ simulation.ꢀ Duringꢀ bothꢀ
previousꢀstep,ꢀallꢀheavyꢀatomsꢀwereꢀrestrainedꢀatꢀtheꢀstartingꢀpositionꢀbyꢀ1000ꢀkJꢀ
-
1
-2
mol nm ꢀwhileꢀwaterꢀmoleculesꢀandꢀionsꢀwereꢀallowedꢀtoꢀmoveꢀfreelyꢀforꢀtheꢀ
solventꢀequilibration.ꢀInꢀtheꢀnextꢀstep,ꢀrestraintsꢀwereꢀgraduallyꢀremovedꢀduringꢀ
1
ꢀnsꢀNPTꢀsimulation.ꢀTheꢀtemperatureꢀandꢀpressureꢀwereꢀkeptꢀconstantꢀatꢀ300ꢀKꢀ
andꢀ1ꢀatmꢀinꢀbothꢀstagesꢀofꢀNPTꢀsimulationsꢀusingꢀBerendsenꢀtemperatureꢀandꢀ
pressureꢀcouplingꢀmethods.[52]ꢀInꢀtheꢀlastꢀphase,ꢀ5x200ꢀnsꢀMDꢀsimulationsꢀwereꢀ
performedꢀforꢀeachꢀsystem.ꢀBothꢀtemperatureꢀandꢀpressureꢀwereꢀmaintainedꢀatꢀ
3
00ꢀKꢀandꢀ1ꢀatmꢀusingꢀv-rescale[53]ꢀandꢀmodifiedꢀParrinello-Rahman[54]ꢀmethodsꢀ
duringꢀ theꢀ productionꢀ simulations.ꢀ Allꢀ MDꢀ simulationsꢀ wereꢀ performedꢀ usingꢀ
GROMACS-2016.[55]ꢀDuringꢀallꢀtheꢀaboveꢀsimulations,ꢀaꢀ1.4ꢀnmꢀcut-offꢀwasꢀusedꢀforꢀ
shortꢀrangeꢀvanꢀderꢀWaalsꢀandꢀelectrostaticꢀinteractions.ꢀExceptꢀduringꢀtheꢀaboveꢀ
heatingꢀsimulation,ꢀthisꢀcut-offꢀwasꢀappliedꢀusingꢀVerletꢀschemeꢀatꢀeveryꢀ40ꢀstepsꢀ
-
1
-1
withꢀ 0.005ꢀ kJꢀ mol ꢀ ps ꢀ energyꢀ driftꢀ perꢀ atom.ꢀ Forꢀ longꢀ rangeꢀ electrostaticꢀ
interactions,ꢀ PMEꢀ methodꢀ wasꢀ usedꢀ withꢀ aꢀ gridꢀ spacingꢀ ofꢀ 1.35ꢀ Åꢀ andꢀ fourthꢀ
interpolationꢀorder.[ ꢀAllꢀbondsꢀwereꢀconstrainedꢀusingꢀparallelꢀLINCSꢀalgorithmꢀ
withꢀfourthꢀorderꢀexpansionꢀofꢀconstraintꢀcouplingꢀmatrix.[57]ꢀꢀ
ꢀ
56]
Acknowledgementsꢀ
E.C.ꢀ gratefullyꢀ acknowledgesꢀ theꢀ Kempeꢀ foundationsꢀ (SMK-1632.2),ꢀ theꢀ Carlꢀ
Tryggerꢀ foundation,ꢀ theꢀ Åkeꢀ Wibergꢀ foundation,ꢀ Stiftelsenꢀ Olleꢀ Engkvistꢀ
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201800078
Byggmästare,ꢀ theꢀ Magnusꢀ Bergvallꢀ foundation,ꢀ andꢀ theꢀ Larsꢀ Hiertaꢀ Memorialꢀ
Foundationꢀ forꢀ financialꢀ support.ꢀ Theꢀ MDꢀ simulationsꢀ wereꢀ performedꢀ onꢀ
resourcesꢀprovidedꢀbyꢀtheꢀSwedishꢀNationalꢀInfrastructureꢀforꢀComputingꢀ(SNIC)ꢀ
atꢀHPC2NꢀUmeå,ꢀSweden.ꢀS.B.ꢀgratefullyꢀacknowledgesꢀDepartmentꢀofꢀScienceꢀandꢀ
Technologyꢀ
(DST),ꢀ
Govt.ꢀ
ofꢀ
Indiaꢀ
(DSTꢀ
INSPIREꢀ
Grant:ꢀ
DST/INSPIRE/04/2014/000217).ꢀWorkꢀinꢀtheꢀN.S.ꢀlabꢀwasꢀsupportedꢀbyꢀKnutꢀandꢀ
Aliceꢀ Wallenbergꢀ foundations,ꢀ theꢀ Swedishꢀ Societyꢀ forꢀ Medicalꢀ Research,ꢀ theꢀ
Kempeꢀfoundationsꢀ(SMK-1449),ꢀandꢀtheꢀMedicalꢀFacultyꢀatꢀUmeåꢀUniversity.ꢀ
ꢀ
Keywordsꢀ
Bis-indole,ꢀG-Quadruplexes,ꢀDrugꢀdesign,ꢀDNAꢀstructures,ꢀNitrogenꢀheterocyclesꢀ
ꢀ
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Chemistry - A European Journal
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FULL PAPER
ꢀ
ꢀ
G-quadruplex ligand design:
Bagineni Prasad, Jan
#
Synthesis, evaluations, and
molecular dynamics simulations
of two series of bis-indoles
show that ligand flexibility is a
key factor to consider in the
design of G-quadruplex
Jamroskovic , Sudipta
#
Bhowmik , Rajendra Kumar,
Tajanena Romell, Nasim
Sabouri, and Erik Chorell*
Page No. – Page No.
Flexible versus rigid G-
quadruplex DNA ligands –
synthesis of two series of
bis-indole derivatives and
comparison of their
interactions with G-
quadruplex DNA ꢀ
stabilizing ligands. Moreover,
the study also reveals G-
quadruplex DNA binding
interactions for the ligands that
can be used to further advance
G-quadruplex DNA ligand
efficiency.
ꢀ
ꢀ
ꢀ
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