Superacid-resistant macrocyclic BODIPYs

Domain: biology
Published: 2026-03-19
Editorial outcome: major_revision

Abstract
Boron-dipyrromethenes(BODIPYs)areversatilefluorophoreswithintense
fluorescenceandbroadapplicationsinbioimagingandsensing.However,they
undergodeboronationunderacidicconditions,whichcausesfluorescence
degradation.Herein,wedesignedexceptionallyacid-stableBODIPYsbyhar-
nessingthesynergisticboron-chelationeffectofcalix[3]pyrrole-likemacro-
cycles.Weshowthattheircharacteristicopticalpropertiesareretainedin
stronglyacidicmedia,includingsuperacids,withoutundergoingdeborona-
tion.MacrocyclicBODIPYsexhibitsharpabsorptionandprotonation-induced
fluorescenceswitching,withquantumyieldsofupto0.90andnarrowStokes
shifts.Notably,nodeboronationwasobservedeveninnon-dilutedfluor-
osulfuricacid,andvisiblefluorescencewassustainedforoveraday.Beyond
theirunusualacidresistance,themacrocyclicBODIPYshadhigherthermal-
andphotostabilitycomparedwithconventionalBODIPYs.Peripheralsub-
stitutionallowedthemodulationofabsorptionandemissionwavelengths,and
fluorous-taggingthroughaxialligandexchangeenabledfluorescenceswitch-
inginresponsetoperfluorooctanoicacidinafluoroussolvent.Weused
superacid-resistantBODIPYsasacidindicatorsforthefluorescencestainingof
Nafionbeadsandsulfonylatedgels,whicharetooacidictosustainthefluor-
escenceemissionofconventionalBODIPYs.Ourfindingsexpandthescopeof
BODIPYsintostronglyacidic,non-aqueousenvironments,openingopportu-
nitiesforfluorescenceimagingandsensinginmaterialsandbiological
systems.

Introduction
Boron-dipyrromethene (BODIPY) dyes1 have firmly established
themselves as biological markers2–4, chemosensors5,6, and
photosensitizers7,8 owing to their distinctive optical properties, i.e.,
narrow Stokes shifts and high-emission quantum yields. Since their
absorption/emission wavelengths can be tuned by peripheral
substitutions9, various BODIPY analogs have been used to stain cell
proteins, lipids, and metabolites10,11, and to detect molecules and
ions12,13.However,acommonandcriticaldrawbackofBODIPYsisthat
theyreadilyreleasetheboronatominthepresenceofBrønstedacids,
resultinginfluorescencedegradation(Fig.1)14–16.Consequently,their
usefulnessinacidicmediaislimited.Moreover,althoughseveralacid-
stableBODIPYanalogshavebeendevelopedforuseinaqueoussolu-
tionsdowntopH~117,theirdeploymentinstronglyacidicandnon-
aqueousenvironmentsremainsparticularlychallengingowingtorapid
acid-induceddeboronation.
Among pyrrole-based boron complexes, boron(III)-sub-
phthalocyanines (SubPcs) and -subporphyrins (SubPors)18–21 are
remarkably resistant to acid-mediated deboronation. Under acidic
conditions,protonationorLewisacidcoordinationatthemeso-azo-
methine units is commonly observed for subphthalocyanines22,
whereasacidsoftenpromoteboron-axialligandexchangeattheboron
center of hydroxo- or alkoxo-substituted subporphyrins23. None-
theless, deboronation yielding free-baseSubPcsor SubPors hasnot
beenobservedtodate.EvenupontreatmentwithastrongLewisacids,
suchasasilyliumreagent,boronchelationismaintainedasthecoor-
dinativelyunsaturatedboreniumcationforms24,25.WhileSubPcsand
SubPorsusea14π-aromaticmacrocycleforboronchelation,asimilar
chelationeffecthasrecentlybeenobservedinaboron(III)complexof
calix[3]pyrrole (C3P), which has a globally non-aromatic peripheral
macrocycle26,27. C3P is a porphyrinogen-like tripyrrolic macrocycle
thatundergoesacid-mediatedmacrocyclicringcleavageinitsboron-
free form28,29. In contrast, the boron(III)-C3P (1) resists both macro-
cyclic ring cleavage and deboronation in the presence of tri-
fluoroacetic acid, whereas most BODIPYs release the boron atom
under similar conditions (Fig. 1). Given the synergistic relationship
betweentheboron(III)atomandC3P,weenvisionedthatanin-depth
understanding of the crucial effect of tripyrrolic macrocycle 1 on
boron(III)-chelationwouldprovideadesignstandardforacid-tolerant
BODIPYdyes.Herein,wereporttheamphotericnatureofboron(III)-
C3P 1, which maintains stable chelation of the boron atom under
stronglyacidicconditionsinexchangeforprotonation-induceddear-
omatizationofthepyrroleunits.Notably,thisacidtoleranceisasso-
ciated with the formation of a tetracoordinated boronium cation
species, in contrast to the borenium cation-based stabilization
observedinSubPcsandSubPors.Weexploitedthisphenomenonto
developexceptionallyacid-stablemacrocyclicBODIPYsthatretainthe
boron atom even in concentrated superacids, such as tri-
fluoromethanesulfonicacidorfluorosulfonicacid,andexhibitfluor-
escenceswitchinguponprotonation.Boronaxialligandexchangeand
peripheral substitution reactions can change their solubility and
absorption/emissionwavelengthofresultingBODIPYs.Thesuperacid-
stable BODIPY analogs complement the poor acid stability of con-
ventionalBODIPYsandenablefluorescencestainingofvariousacidic
media,includingNafion,sulfonatedorganogels,andcationexchange
resins,asacidindicators.

Results
Boron(III)–tripyrrolicmacrocyclesynergyforacidstability
Toexploretheoriginoftherobustboronchelationobservedinbor-
on(III)-C3P 1 under acidic conditions, we examined its solution
behavior in the presence of trifluoroacetic acid (TFA). The proton
nuclear magnetic resonance (1H NMR) spectrum of 1 recorded in
dehydratedCDCl (at6.0mM)revealedonlytwosingletsignalsat5.95
3
and1.69partspermillion(ppm),whichwereassignabletopyrroleβ-
CHandmethylprotons,respectively.The11BNMRsignalof1at22.81
ppmindicatedatrigonal-planarboron(III)center,asconfirmedbyits
singlecrystalX-raystructure.When3equivalentsofTFAwereaddedto
asolutionof1at25°C,anewsetofprotonsignalsattributabletothe
TFA-adduct 2 appeared in a molar ratio of 1:2=1/2. Adduct 2 was
formedthroughanequilibriumprocess;whereasthesignalratioof2
decreasedwithincreasingtemperature(51%and40%at40and50°C,
respectively), 2 was predominant (>90%) at 0°C (Supplementary
Fig.2).Adduct2showedgeminallycoupledmethyleneprotonsignals
at3.94and3.68ppmalongwithsixdifferentmethylprotonsandfive
pyrroleβ-CHsignals,reflectingitsC molecularsymmetry.The11BNMR
1
signal of 2 was observed at –2.18 ppm, indicating a tetrahedrally
coordinatedboroncenter.
Slow evaporation of the solvent from a CH Cl /TFA solution
2 2
furnisheddiffraction-gradesinglecrystalsof2.AsindicatedbyNMR
spectroscopy, the crystal structure of 2 revealed an axially
trifluoroacetoxy-bound boron center in a tetrahedral coordination
geometry(Fig.2).One ofthethreepyrroleunitshadasignificantly
longerN–Bbondlength(1.531(5)Å)thantheothers(1.490(4)–1.493(4)
Å).TheN–C2,C2–C3,C3–C4,andC4–C5distancesofthispyrrolering
were1.374(4),1.436(15),1.516(11),and1.389(16)Å,respectively,clearly
indicatinga3H-pyrrole-typebondlengthalternation.Thesestructural
analyses showed the amphoteric nature of 1 during TFA addition;
namely,thepyrroleunitbehavesasabasetobeprotonated,andthe
three-coordinatedboroncenteractsasaLewisacid.
In the presence of 25 equivalents of TFA, 2 underwent further
protonationtoyieldboroniumcation3exclusivelyatroomtempera-
ture. The 1H NMR spectrum of 3 recorded in CDCl featured a C-
3 s
symmetricsignalpatternwiththreepyrrolicβ-protonsignalsat8.14,
7.16,and6.08ppmasacoupleofdoublets(J=5.5Hz)andasinglet,
respectively.Inaddition,abroadsignalattributabletothepyrroleα-
CHwasobservedat5.90ppm.The11BNMRsignalat–2.81ppmsug-
gestedthepresenceofatetracoordinatedboroncenter.Furtherhet-
eronuclear multiple bond coherence measurements revealed the
structureof3(SupplementaryFig.7).While2containeda3H-pyrrole
moiety, the doubly protonated species 3 exhibited two 2H-pyrrole
rings resulting from α-protonation. Density functional theory (DFT)
calculationsalsosupportedthatstructure3wasmorestablethanthe
otherstructuralisomers,namely,theβ,β-andα,β-protonatedforms
(SupplementaryTable.1).
When1equivalentofmethanesulfonicacid(MSA)wasaddedto1
inCDCl ,theexpectedmonoadduct4wasnotdetectedby1HNMR
3
spectroscopyevenat–60°C.Instead,thedoublyprotonatedspecies5
was observed in the equilibrated mixture. A characteristic proton
signal at 6.14 ppm indicated the presence of 2H-pyrrole rings in 5.
Adduct5wassufficientlystabletoretainboththeboronatomandthe
macrocyclicframeworkeveninneatMSA.Whenaceticacid(AcOH)
wasusedtoprotonate1,themonoprotonatedform6wasobserved
below 0°C, but boronium cation species such as 3 and 5 were not
detected,eveninthepresenceofalargeexcess(>100equivalent)
of
AcOH.Similaradditionreactionswithlessacidicwaterandmethanol
werealsoconfirmedbyNMRspectroscopy.
InaD O-saturatedchloroformsolution,1exhibitedH/Dexchange
2
ofthepyrroleβ-protonsthroughformationofhydratedform7.When
excessD OwasaddedtoaCDCl solutionof1,theintensityofthe
2 3
pyrroleβ-protonsignalat5.95ppmwasattenuatedby92%after5min
at298K.Incontrast,boron-freeC3PexhibitedH/Dexchangeonlyat
theNHprotonunderthesameconditions.Thevan’tHoffplotanalysis
of the equilibrium constants between 1 and hydrated 7 at various
temperatures gave physical parameters of ΔH=–56.5kJmol–1,
ΔS=–151Jmol–1K–1,andΔG=–11.46kJmol–1at298K.Althoughoneof
thepyrrolemoietiesistobedearomatized,thefavorableformationof
7atroomtemperatureisattributabletotheenhancedbasicityofthe
pyrroleunituponcoordinationofthefourthligandtotheboronatom.

Synthesisandpropertiesofsuperacid-resistantmacrocyclic
BODIPYs
Inspiredbytheremarkableboron-chelatingbehaviorof1,wedesigned
anacid-resistantmacrocyclicBODIPYframework,designatedasbor-
on(III)-calix[1]dipyrrin[1]pyrrole(compound11,Fig.3).Thistripyrrolic
macrocycle features a ring size and boron-chelation environment
similartothoseofC3PandSubPor,andincorporatesdipyrromethene-
typeπ-conjugationalongwithanisolatedpyrroleunit.Toconstruct
macrocycle11,tripyrrane8wasreactedwithborane-triethylamineto
giveboron(III)-tripyrrane9in98%yield30,31.Macrocyclizationinexcess
trimethylorthoformateaffordedstableboron(III)complex10in46%
yield after treatment with methanol. Although boron complex 9
readily released the boron atom to give starting material 8 in the
presenceofwaterinchloroform,nodeboronationwasobservedfor
macrocycle10undereitheraqueousconditionsorinthepresenceofa
Lewisacid.Sincetheaxial-alkoxogroupontheboronwassusceptible
toaxialligandexchange27,32,33,fluorideligandwasintroducedtogive11
asamorestableanalog.Substitutionofthefluoridewasconfirmedbya
doubletsignalat–2.69ppm(J B–F =40.0Hz)inthe11BNMRspectrum
(Supplementary Fig. 63). Single crystal X-ray diffraction analysis
revealedthatthecentralboronatomof11adoptsatetrahedralcoor-
dinationgeometrywithanaxiallycoordinatedfluoroligand(Fig.3b).
The tripyrrolicmacrocycle acts as a dianionictridentate ligand in a
mannersimilartoSubPor,whileitsπ-conjugationissplitintodipyr-
rometheneandpyrrolefragmentsbytwosp3-hybridizedcarbonatoms
atthemeso-positions.
MacrocyclicBODIPY11exhibitedprotonationbehavioranalogous
tothatof1.The1HNMRspectrumof11inCDCl at6.0mMshoweda
3
coupleofdoubletsat6.94and6.26ppmandasingletat5.88ppm,
attributabletoβ-protonsofthedipyrrometheneandisolatedpyrrole
moieties, respectively. Upon addition of 20 equivalents of TFA, the
singletsignalbroadened,whilethetime-averagedC-symmetricsignal
s
pattern remained intact. However, desymmetrization of the signal
patternwasobservedat–50°C,withapairofdoubletsappearingat
4.44and4.16ppm,assignable tothe methylene protonsofthe3H-
pyrrole moiety in 11(cid:129)H+ (Supplementary Fig. 21). Similar spectral
changes were also observed upon addition of MSA and tri-
fluoromethanesulfonic acid (TfOH), without any loss of the boron
atom.1HNMRtitrationof11withMSAdeterminedtheaciddissocia-
tionconstantoftheprotonatedform11(cid:129)H+aspK =3.22inacetoni-
trile-d (SupplementaryFig.22).
3
Compound 11 exhibited acid-responsive, BODIPY-like visible
fluorescencewithasmallStokesshift.Indichloromethane,11showed
the lowest energy absorption band at 540nm with an absorption
coefficient of ε=3.3×104M–1 cm–1 due to the π–π* transition of the
boron(III)-dipyrromethenemoiety(Fig.3d).However,nofluorescence
emissionwasobserveduponexcitationatthelowestenergyabsorp-
tionbandunderneutralconditions.Thisisattributabletointramole-
cularphoto-inducedelectrontransfer(PET)fromthepyrrolefragment
tothedipyrrometheneunit,assimilarphenomenahavebeenreported
for BODIPY analogs bearing electron-donating moieties34,35. Upon
addition of TFA, a dichloromethane solution of 11 emitted yellow
fluorescenceat556nm(Stokesshift:397cm–1),whereastheabsorption
spectrum remained virtually unchanged (Fig. 3e). These spectral
changes indicated the contribution of the protonated form 11(cid:129)H+,in
whichtheelectrontransferfromthe3H-pyrrolemoietytotheBODIPY
coreisunlikelytooccur.Indichloromethanecontaining5vol%TFA,
thefluorescencequantumyieldandemissionlifetimeof11were0.86
and10.9ns,respectively.
The time-dependent DFT calculations together with electro-
chemical analysis based on the Rehm–Weller approximation sup-
portedthefluorescenceswitchingbehaviorofthemacrocyclicBODIPY
system. In the neutral state 11, the lowest-energy absorption band
appearedat443.2nm,withanoscillatorstrength(f)of0.3876,corre-
spondingtoaπ–π*transitionlocalizedwithintheBODIPYcore.This
transitioninvolvedexcitationfromHOMO–1totheLUMO.However,
theHOMOwasprimarilylocalizedontheisolatedpyrroleunit,which
actedasanelectrondonor,resultinginfluorescencequenchingdueto
efficient non-radiative decay pathways (Supplementary Fig. 17).The
protonated form 11(cid:129)H+ exhibited a comparable absorption band at
449.3nm (f=0.3281),which also corresponded to a π–π* transition
withintheBODIPYcore.Theprotonatedstateexhibitedadistinctive
lowering of the energy level of the pyrrole unit below that of the
HOMO of the BODIPY moiety, thus suppressing the intramolecular
electrontransferfromthepyrroleunit.Electrochemicalanalysisfur-
thersupportedtheoccurrenceofPETof11underneutralconditions.
The first oxidation and reduction potentialsof 11 wereobserved at
0.70and–1.53V(vs.ferrocene/ferroceniumioncouple),respectively,
by cyclic voltammetry in CH Cl (Supplementary Fig. 26). The free
2 2
energychangefortheelectrontransferprocess,estimatedusingthe
Rehm–Wellerequation36,was–0.46eV,indicatingthatthePETprocess
isthermodynamicallyfavorable.Incontrast,thefirstoxidationpeakat
0.70V was significantly attenuated in the presence of 5v/v% TFA,
presumablyduetoprotonationatthepyrrolemoiety.Thisobservation
is consistent with the emergence of fluorescence emission under
acidicconditions,whereprotonationresultedintheweakeningofthe
electron-donatingcharacterofthepyrrolemoiety.Thecalculationand
electrochemicalanalysesindicatedthat11functionsasaturn-ontype
fluorescentBODIPYanalogunderacidicconditions,inwhichthepyr-
rolefragmentisprotonated.
Stability tests with various acids confirmed the exceptional
superacid tolerance of macrocyclic BODIPY 11 against acid-induced
deboronation.Wemeasuredtheabsorptionandfluorescencespectra
of11invariousmedia,includingstrongacids,suchasMSAandfluor-
osulfonicacid(Fig.4a).Theabsorptionspectraof11remainedessen-
tially unchanged, regardless of the solvent polarity and acidity.
However, fluorescence emission was not observed in neutral and
weaklyacidicsolvents,suchastetrahydrofuran,acetonitrile,dimethyl
sulfoxide,andaceticacid(SupplementaryFig24).Incontrast,when
thesolventacidityincreasedtoaHammettacidityfunction(H )37of
0
≤–2.2,11exhibitedpronouncedfluorescenceattributabletothepro-
tonatedform11(cid:129)H+.Thefluorescencequantumyieldsinformicacid
(H =–2.2), TFA (H =–3.0), 42% aqueous tetrafluoroboric acid
0 0
(H =–4.2), 55% aqueous hexafluorophosphoric acid, and MSA
0
(H =–7.9)weredeterminedtobe0.46,0.81,0.56,0.56,and0.75with
0
corresponding Stokes shifts of 372, 371, 342, 307, and 338cm–1,
respectively.Remarkably,intensefluorescencewasretained even in
concentrated sulfuric acid (H =–12.0), chlorosulfonic acid
0
(H =–13.8) and fluorosulfonic acid (H =–15.1), with fluorescence
0 0
quantumyieldsof0.85,0.68,and0.90,respectively,indicatingthat
theboroncenterremainsintactinsuperacids(Fig.4bandTable1).
Notably,afluorescencequantumyieldof0.90wasobservedinfluor-
osulfonic acid 12h after dissolving 11, and continuous monitoring
revealedthattheintensefluorescencewasmaintainedforoveraday.It
isnoteworthythattheabsorptionspectraof11intheseacidsexhibited
a shoulder around 550nm. Matrix-assisted laser desorption/ioniza-
tion-time-of-flightmassspectrometric analysis of a solutionof11in
fluorosulfonicacidrevealedpartialfluorosulfonylationoftheBODIPY
coreasasidereaction.Theseresultssuggestthatdirectsulfonylation
of 11 under harsh conditions using concentrated sulfonic acids can
proceed without deboronation, whereas conventional BODIPYs
typically require mild conditions for sulfonylation38. In tri-
fluoromethanesulfonic acid (TfOH),11 underwent multiple protona-
tionatboththeBODIPYcoreandthemono-pyrrolefragment,leading
to sequential changes in the absorption and fluorescence spectra
depending on the TfOH concentration (Supplementary Fig. 25).
Despitetheharshacidicconditions,boroncomplex11wasrecovered
upon neutralization of the solution, demonstrating the exceptional
superacid resistance oftheBODIPYcoretodeboronation.
Todemonstratetheenhancedacidstabilityof11,weperformed
comparative experiments with conventional BODIPY 12 and B–CN
analog13(Fig.4c,SupplementaryFig.27),whichhasbeenreportedto
exhibitnotableacidresistance39–41.Whilemostreportsonacid-tolerant
BODIPYshaveevaluatedfluorescenceemissioninaqueousmediawith
pH≥ 0,macrocyclicBODIPY11remainedfluorescentevenatpH=–1.1
(SupplementaryFigs.30,31).Undertheseconditions,mostreported
analogs,including12,losttheiremission,whereasB–CNanalog13still
exhibiteddetectablefluorescence.Therefore,wefurtherevaluatedthe
stabilityof11–13undermorestronglyacidicconditions.Whenfluor-
escencespectrawererecordedimmediatelyafterdissolutioninTFA/
H O(95/5,v/v)at10µMconcentration,BODIPYs11–13exhibitedvisi-
2
ble emission in the 500–650nm region. However, the fluorescence
intensityof12graduallydecreasedandwasalmostcompletelyquen-
ched within 1h. Compound 13 also underwent slight degradation
duringthesameperiod,withanapproximate15%decreaseinfluor-
escenceintensity.InthemoreacidicmediumMSA/HO(95/5,v/v),the
2
fluorescenceemissionof13wascompletelyquenchedwithin15minat
roomtemperature.Incontrast,macrocyclicBODIPY11exhibitednei-
therfluorescencequenchingnoranychangeinspectralshape,high-
lighting its remarkable acid stability relative to that of previously
reportedBODIPYs.WhentheseBODIPYs11–13wereheatedat180°C
for 48h in o-dichloromethane, the B–CN analog 13 underwent
remarkablethermaldecomposition,leadingtoa71%lossofabsorption
intensity at 504nm (Supplementary Fig. 28). Compound 12 also
exhibited a 14% decrease in absorbance, whereas the absorption
spectrum of cyclic BODIPY 11 remained unchanged after heating.
Continuous excitation in o-dichlorobenzene at 502nm with an LED
light(29.4mWmm–2)for2hcaused66%and24%lossofabsorption
intensity for 12 and 13, respectively, whereas the spectrum of 11
remainedvirtuallyunchanged,demonstratingnotablephotostability
(SupplementaryFig.29).
Peripheralfunctionalizationandaxialligandexchange
Acid-stableBODIPY11isamenabletobothperipheralandaxialfunc-
tionalization,offeringtunabilityofemissionwavelengthandsolubility.
When 11 was treated with an excess of bromine, all the pyrrole β-
positions werebrominatedtoafford14in98%yield.Single-crystalX-
raydiffractionanalysisconfirmedthatthemeso-CHgroupremained
intact, while it engaged in C-H(cid:129)(cid:129)(cid:129)F hydrogen-bonding interactions
owingtotheincreasedaciditybytheinductiveeffectsofthebromine
atoms(SupplementaryFig.15).Notably,thebowl-depth33,definedas
the distance from the boron atom to the mean plane of the six pyrrole-
βcarbonatoms,wasshallowerin14(1.14Å)comparedto10and11,
whichexhibited greater depth of1.58and 1.49Å, respectively (Sup-
plementaryFig.16).Fluorescencefromcompound14wasremarkably
attenuated,exhibitingaquantumyieldof1.4%inH SO ,whichcanbe
2 4
attributed to the internal heavy-atom effect. Instead, photophysical
processes associated with the triplet excited state, arising from
intersystem crossing, become prominent. In a degassed
2-methyltetrahydrofuran solution containing 5v/v% MSA,phosphor-
escenceemissionwasobservedat727nmuponfreezingthesampleat
100K, exhibiting a multiexponentialdecay with lifetimes of 1.7 and
5.9ms,whereasnoemissionwasdetectedatroomtemperature.Fur-
thermore,irradiationof14at510nminthepresenceof1,3-dipheny-
lisobenzofuran (DPBF) resulted in a gradual decrease in the
characteristic absorption band of DPBF. Comparative analysis using
5,10,15,20-tetraphenylporphyrinasareferencephotosensitizergavea
singletoxygengenerationquantumyieldof27%for14.
Peripherallyaryl-substitutedanalogs15–19werepreparedusing
several synthetic strategies. β-Hexaphenylated analog 15 was
obtained by Suzuki–Miyaura cross-coupling of 14, whereas meso-
phenyl-substituted 16 was synthesized by condensation of 9 with
trimethyl orthobenzoate in the presence of benzoic anhydride to
give16in4%yield.Althoughthesyntheticyieldsweremodest(~3%),
the use of acryl chlorides enabled the introduction of 4-methox-
yphenyl, 4-cyanophenyl, and 5-methylthiophene-2-yl groups at the
meso-position of the BODIPY core, affording 17, 18, and 19,
respectively.Theperipherallyaryl-substitutedderivatives15–19also
exhibited protonation-induced turn-on fluorescence behavior
(Table 2 and Supplementary Figs. 33–37). While these compounds
were non-fluorescent in neutral organic solvents, including tetra-
hydrofuran,acetonitrile,andethanol,distinctsubstituenteffectson
fluorescence emission were observed in acidic media. Figure 5c
showstheabsorptionandfluorescencespectraof15–19recordedin
97%sulfuricacid.Amongthisseries,β-hexaphenylanalog15exhib-
itedadistinctred-shiftoftheabsorptionband(574nm),whilethe
lowest energybandsofotheranalogs11and16–19remainedinthe
rangeof505–522nm.Consistentwiththeabsorptionbehavior,the
fluorescence emission of 15 wasalso red-shifted to595nm,witha
fluorescencequantumyieldof47%.Thethienyl-substitutedanalog19
exhibitedapronouncedredshiftoftheemissionspectrumto605nm
withalargeStokesshift,accompaniedbyspectralbroadeninganda
low-energy tail extending beyond 750nm. This behavior can be
attributed to intramolecular charge-transfer interactions between
the thienyl moiety and the BODIPY core42. Consistent with this
interpretation, a relatively large slope was observed for 19 in the
Lippert–Mataga plot analysis compared with analogs 15–18 (Sup-
plementaryFig.38).Whilethesubstituenteffectsatthe4-positionof
themeso-phenylringonabsorptionandemissionwavelengthwere
modest,thefluorescence quantum yieldsof meso-substituted ana-
logs 16–19 decreased considerably. Such meso-aryl substituent
effects have been reported for conventional BODIPYs, in which
rotationofthearylgroupsintheexcitedstatecontributestonon-
radiative decay pathways43,44. Table 2 shows that fluorescence
quantumyieldstendtoincreaseinmoreviscousacids,suchassul-
furicacid,comparedwithTFAandformicacid.Basedonthistrend,
wemeasuredfluorescencequantumyieldsof15–19infrozenMSA
solutionsandfoundthatthevaluesincreasedmarkedlyto0.37,0.79,
0.85,0.80,and0.34,respectively,comparedwiththosemeasuredin
liquid solution (0.09, 0.01, 0.04, <0.01, and 0.05). These observa-
tions are attributable to the restricted aryl ring rotation in frozen
MSA,whichsuppressesnon-radiativedecaypathways.Takentoge-
ther, these results demonstrated that macrocyclic BODIPYs allow
tuningofemissionwavelengthandfluorescencequantumyieldsby
substituenteffectsofperipheralarylgroupswithoutdeboronationin
highlyacidicenvironments.
Axialligandexchangeontheboronatomoffersafacile,ortho-
gonal functionalization strategy for macrocyclic BODIPYs, enabling
themodulationofsolubilitywithoutalteringtheπ-electronicstructure
oftheBODIPYcore.Axiallymethoxy-substitutedanalog10underwent
ligandexchangereactionwithcarboxylicacidsinamannersimilarto
subporphyrins45.Azeotropicremovalofmethanolinthepresenceof
tridecafluorooctanoic acid in chloroform afforded axially fluorous-
taggedderivative20in86%yield.AlthoughmacrocyclicBODIPYs10
and11wereinsolubleinfluoroussolvents,20wassufficientlysoluble
in perfluoromethylcyclohexane (49mg/L), enabling fluorescence
switching in the fluorous phase in response to acids (Fig. 5). We
examined perfluorooctanoic acid (PFOA), which is a per- and poly-
fluoroalkylsubstance(PFAS)46,47,asafluorous-solubleacidtoserveasa
fluorescence trigger. Compound 20 was non-fluorescent in per-
fluoromethylcyclohexane.UponadditionofPFOAtoa72µMsolution
of 20, visible fluorescence from in situ-generated 20(cid:129)H+ became
observableatapproximately1mM.Althoughthedetectionsensitivity
of20wasmodestcomparedwiththatofknownPFOAsensors,this
resultprovidesapotentialscaffoldforthedevelopmentoffluorescent
sensorsinfluorousphases.

Potentialapplications
Superacid-resistantBODIPY11servesasapromisingfluorescentindi-
catorforstainingstronglyacidicmaterials.Nafionisaperfluoroalk-
ylsulfonic acid-based polymer widely used as a solid polymer
electrolyteandacidcatalyst,butitsfluorescentstainingwithconven-
tionalBODIPYsisoftenunsuccessfulowingtoitsstrongacidity.When
Nafionbeads(ϕ:4.0mm)wereimmersedina0.25mMtoluenesolu-
tionof11for2days,thebeadschangedfromcolorlesstoorange,and
intensereddish-orangefluorescencearisingfrom11(cid:129)H+wasobserved
(Fig.6).Underthesamestainingconditions,conventionalBODIPY12
resultedinonlyaslightcolorchange,butitsfluorescenceemissionwas
barelydetectableduetodeboronation.The11-stainedNafionbeads
remainedfluorescentformorethan1weekwithoutsignificantlossof
intensity, whereas exposure to triethylamine vapor quenched the
emissionduetothedeprotonationof11(cid:129)H+toneutral11.
BODIPY 11 also functions as an acid indicator for sulfonyl-
functionalized cation exchange resins. When the resin in its -SO H
3
form was soaked in an acetonitrile solution of 11, it turned reddish-
orange,indicatingtheimmobilizationof11.Aleachingtest,performed
bypassingdistilledwaterthroughacolumnpackedwiththe11-stained
resin, confirmed that 11 was firmly retained, because there was no
detectable leaching. In the -SOH form, the 11-stained resin emitted
3
fluorescenceunderUVirradiationowingtoinsitu-generated11(cid:129)H+.The
fluorescence disappeared when a 1.0M aqueous sodium hydroxide
solutionwaspassedthrough,convertingtheresinintoits-SONaform.
3
Subsequenttreatmentwith1.0Mhydrochloricacidrestoredthefluor-
escencewithoutdetectableleachingof11,demonstratingitsreversible
acid-indicating capacity. Notably,compound 11also remained stable
underbasicconditionsanddidnotundergodeboronation.
The interconversion between fluorescent 11(cid:129)H+ and non-
fluorescent11wasutilizedtovisualizethepenetrationofacontact-
ingsolutionintoacidicgelmaterials.Doublenetworkgelsarewidely
recognized for their versatile applications, such as self-growing
material48 and artificial cartilage49, and also include strongly acidic
sulfonylated analogs50. A sulfonated double network organogel
(approximately 20mm×15mm×5mm) was successfully stained by
immersingitina0.25mMethanolsolutionof11for12h.UnderUV
light, the stained gel emitted orange fluorescence uniformly
throughoutthegel.Whenthestainedgelwassoakedina0.1Methanol
solutionoftriethylamineatroomtemperature,thefluorescencewas
gradually quenched from its edges, and completely disappeared
within 90min. This enabled clear visualization of the penetration
processofthebaseintotheorganogel.Theresultsdescribedabove
demonstrate that staining with a superacid-resistant macrocyclic
BODIPYs complements conventional BODIPY-based staining by
enabling bright emission and reversible acid–base switching in
strongly acidic materials. This broadens the scope of fluorescent
detectionandsensingwithBODIPYs.

Methods
Solvents and reagents were purchased from WAKO Pure Chemical
Industries Ltd., TCI Co., Ltd., Kanto Chemical Co., Inc., or Sigma-
AldrichCo.,andwereusedwithoutfurtherpurificationunlessother-
wisementioned.Boron(III)-calix[3]pyrrolecomplex126andtripyrrane
851werepreparedaccordingtoareportedprocedure.All1Hand13C
NMRspectrawererecordedusingJEOLJMN-ECS400orJMN-ECZ600R
spectrometers.Chemicalshiftswerereportedinppmrelativetothe
internal standard tetramethylsilane (δ=0.00 ppm for 1H NMR in
CDCl ), the external standard boron trifluoride ethyl etherate
3
(δ=0.00ppm for 11B NMR in CDCl ), the external standard hexa-
3
fluorobenzene(–162.9ppmfor19FNMRinCDCl ),orasolventresidual
3
peak(δ=77.16ppmfor13CNMRinCDCl ).Thin-layerchromatography
3
wasperformedona silicagelsheet,MERCKsilicagel60F254.Pre-
parativescaleseparationswereperformedbymeansofgravitycolumn
chromatography over silica gel (Wakosil® 60. 64–210µm). Infrared
spectraweremeasuredusingaJASCOCo.FT/IR-4600spectrometer.
ESI-TOF-MSspectrawererecordedonaThermoScientificExecutive
spectrometer.Elemental analyses werecarriedoutusing an Exceter
Analytical, Inc. CE440 or MICRO CORDER JM10. UV/Vis absorption
spectrawere recorded on a JASCO V-770, V-670 or SHIMADZU UV-
1800 spectrophotometers. Fluorescence spectra were recorded on
HITACHIF-7000orJASCOFP-8550spectrometers.Fluorescencelife-
times were measured using an Edinburgh FLS1000 photo-
luminescence spectrometer. Fluorescence quantum yields were
measured using a Hamamatsu Photonics Quantaurus QY C11347-01
systemoranEdinburghFLS1000photoluminescencespectrometer.
Phosphorescence spectra and decay curves were recorded using a
JASCO FP-8550 spectrometer with a cryostat (Oxford Instruments,
OptistatDN) and a temperature controller (Oxford Instruments,
ITC502S).Single crystalX-raydiffractiondatawereobtainedusinga
Rigaku XtaLAB P200 diffractometer equipped with a PILATUS200K
detector,whichusesamultilayermirror(MoKαradiationλ=0.71073Å)
oraRigakuXtaLABSynergy-R/DWinstrumentequippedwithaHyPix-
6000HE detector, whichuses a monochromated mirror. Cyclic vol-
tammogramsweremeasuredwithanALSModel660Eelectrochemical
analyzerusingathree-electrodesystem.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This work presents a class of macrocyclic BODIPYs that utilize a tripyrrolic macrocycle for synergistic coordination to the\nboron center, with a focus on maintaining coordination without deboronation in superacidic environments and\nachieving\nprotonation-triggered luminescence switching. The authors report high quantum yields and long-term\nstability in superacids\nsuch as fluorosulfonic acid, and they demonstrate application scenarios including the staining of Nafion and sulfonated gels.\nThe overall novelty and visibility are good. However, for submission to Nature Communications, the mechanistic depth and\nthe persuasiveness of the applications need to be strengthened. My major concern is that the overall synthetic strategy\nshows significant limitations in its generality, restricting access to key classes of BODIPY derivatives such as meso-aryl\nsubstituted and aza-BODIPY analogues. The manuscript's text could also be further condensed and more organized. Thus,\nmajor revisions are recommended before reconsideration. Specific recommendations and comments that would improve the\nmanuscript are listed below:\n1. The manuscript attributes the fluorescence quenching of compound 11 to a PET mechanism, supported solely by TD-DFT\ncalculations. While the calculated orbital distributions are consistent with this proposal, experimental validation is crucial for\nsuch a central mechanistic claim. I recommend that the authors provide experimental evidence to substantiate the PET\nprocess. Techniques such as cyclic voltammetry should be used to obtain donor and acceptor redox potentials, then\nestimate ΔG(PET) with the Rehm–Weller approximation for the neutral and protonated states.\n2. Hexabrominated 14 is reported to be non-fluorescent. Please examine phosphorescence and quantify singlet oxygen\ngeneration. The internal heavy atom effect from six bromine substituents should enhance intersystem crossing and populate\na long-lived triplet, which could make 14a useful photosensitizer.\n3. The fluorescence spectrum of 15 in concentrated H2SO4 (in Figure 5c) clearly shows a complex, broad emission band\nwith a distinct dual-emissive character (peaks at approx. 575 nm and 600 nm). The current description as merely \"red-\nshifted\" is insufficient and overlooks this important feature.\n4. The hexaphenyl derivative 15, with its six rotatable peripheral phenyl groups, is a prime candidate for exhibiting AIE. The\nrotation of these phenyls can serve as a non-radiative decay channel in solution, which would be blocked upon aggregation.\nThe fluorescence observed in highly viscous sulfuric acid (QY= 0.16) may already hint at this restriction of intramolecular\nmotion.\nMinor points:\n1. To significantly improve the clarity and accessibility of the results, I strongly recommend that the authors compile the\nspectroscopic and photophysical data for all new compounds into a summary table.\n2. Supplementary Table 2 should be included in the TEXT or shown as a plot.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "The manuscript describes the synthesis and characterization of a new family of contracted pyrrolic macrocycles that\nstructurally resemble macrocyclic BODIPY analogues with a tridentate cavity. The authors show that these frameworks can\ntightly coordinate boron, imparting exceptional acid resistance — a feature of high relevance since BODIPY dyes are\nnotoriously prone to deborylation under acidic conditions. The reported systems are stable even at pH ≈ 1 and can be\npost‑functionalized at both the meso and peripheral positions, allowing for subsequent derivatization. The photophysical\nbehavior is explored and potential applications are demonstrated in polymer staining, pH sensing, and imaging in acidic\nmedia. The work is well executed, and the data are of high experimental quality.\nHowever, I do not believe the manuscript meets the novelty and impact threshold required for Nature Communications. The\nstudy, while solid, represents a specialized development within the already well‑established field of boron–pyrrole\nchromophores rather than a conceptual or technological breakthrough of broad interest. I would therefore not recommend\npublication in its current form. Other more chemistry specialized journals are recommended instead.\nMajor Comments\n1. Limited novelty relative to existing acid‑resistant fluorophores: The claim of exceptional acid stability is not unique.\nSeveral acid‑tolerant fluorescent probes and chromophores have been previously reported (e.g., Anal. Chim.\nActa 2014, 816, 1; Chem. Soc. Rev. 2014, 43, 4629; Biosens. Bioelectron. 2024, 116638). The authors should position their\ncompounds explicitly against these precedents and demonstrate in quantitative terms why their system is superior or\nconceptually distinct. Without this comparison, the contribution appears incremental.\n2. Inconsistencies in the mechanistic rationale and background: e.g., the introduction states that “acid accelerates axial\nexchange in subporphyrinoids.” This is not generally the case; protonation at meso positions or ring opening are far more\ncommon acid‑induced processes (see New J. Chem. 2018, 42, 10478). Clarification is needed, as this assertion currently\nundermines the mechanistic logic.\n3. Redundancy with previous work on boron–tridentate macrocycles: The notion of embedding boron within a tridentate\npyrrolic environment lacks strong novelty, particularly given the authors’ own prior studies on C₃P frameworks and the\nestablished chemical robustness of subporphyrinoids. The manuscript would benefit from a clearer statement of what\ngenuinely new coordination or photophysical behavior arises here beyond what is already known for SubPcs, SubPors, or\nrelated contracted systems.\n4. The proposed “acid‑induced fluorescence switching” mechanism—arising from suppression of intramolecular quenching\n—is a recurring concept in chromophore design and has been widely documented. The authors should better\ncontextualize their findings within this literature and specify whether any mechanistic feature here is truly unprecedented.\n5. Limited optical tunability and modest spectral range: the emission remains confined to the 500–600 nm region. Extension\ninto the near‑infrared, which is the current frontier in bioimaging, would represent genuine novelty and impact. Similarly, the\nobserved red‑shifts upon peripheral aryl substitution are small (ompared with subporphyrinoids or subporphyrazines (e.g., J.\nOrg. Chem. 2020, 85, 1152)), and the substantial decrease in quantum yield upon functionalization raises questions about\nthe practical value of these derivatives.\nAdditional Remarks\n1. Literature coverage\nSeveral seminal works are missing. For instance, the comprehensive Chem. Rev. 2007, 107, 4891–4932 on BODIPYs\nshould be cited, as well as the most recent reviews on subporphyrinoids (Chem. Soc. Rev. 2022, 51, 9482; Trends\nChem. 2023, 5, 279). The current bibliography underrepresents key prior art, weakening the contextual framing.\n2. Line 227: “solubile” → “soluble”. Please also perform a careful language and consistency revision\nIn summary, the manuscript presents technically competent work and interesting synthetic chemistry, but it falls short of the\nconceptual advance and broad significance expected for Nature Communications. The acid stability and\npost‑functionalization are incremental improvements within an already mature class of compounds. A more comprehensive\ncomparison with known acid‑resistant fluorophores, clarification of the mechanistic novelty, and demonstration of distinctive\noptical performance (e.g., NIR emission, tunability) would be required for reconsideration.", "reviewer_4": "This is a very interesting paper that describes the synthesis, characterization and application of superacid resistant cyclic\nBODIPYs. I believe the work presented is significant to the field of BODIPYs and their practical applications; a clear\ncomparison between the new reported acid-stable cyclic BODIPYs and non-cyclic BODIPYs is investigated by the authors.\nAll compounds are adequately characterized by NMR and HRMS. The methodologies used for the characterization of the\nneutral and positively charged species are all adequate and the conclusions are supported by the results obtained. The\nFigures are adequate and informative. I suggest that the authors correct a few typos in the manuscript, such as in lines 227\nand 248. In addition, the Discussion section is more of a Conclusions section."}, "author_response": "Point-by-Point Response to the Reviewers’ comments\n(blue: original comments by reviewers, black: our response)\nReviewer 1\nThis work presents a class of macrocyclic BODIPYs that utilize a tripyrrolic macrocycle for synergistic coordination\nto the boron center, with a focus on maintaining coordination without deboronation in superacidic environments and\nachieving protonation-triggered luminescence switching. The authors report high quantum yields and long-term\nstability in superacids such as fluorosulfonic acid, and they demonstrate application scenarios including the staining\nof Nafion and sulfonated gels. The overall novelty and visibility are good. However, for submission to Nature\nCommunications, the mechanistic depth and the persuasiveness of the applications need to be strengthened. My major\nconcern is that the overall synthetic strategy shows significant limitations in its generality, restricting access to key\nclasses of BODIPY derivatives such as meso-aryl substituted and aza-BODIPY analogues. The manuscript's text could\nalso be further condensed and more organized. Thus, major revisions are recommended before reconsideration.\nSpecific recommendations and comments that would improve the manuscript are listed below:\nWe thank the reviewer for the careful evaluation and for recognizing the novelty and\nrelevance of our study, as well as for providing constructive comments that helped us\nimprove the mechanistic discussion and overall clarity. In response to the reviewer’s major\nconcern regarding the synthetic generality of macrocyclic BODIPYs, we have expanded the\nscope to include additional meso-aryl derivatives, which were successfully synthesized using\nthe corresponding acyl chlorides. The successful use of acyl chlorides represents a notable\nsynthetic advance, enabling broad access to meso-aryl-substituted derivatives of this\nmacrocyclic BODIPY framework, despite the modest yields. Introduction of a meso-thienyl\nmoiety resulted in a remarkable red-shift of the emission band owing to intramolecular\ncharge-transfer interactions, a behavior commonly observed in conventional BODIPYs and\nsupported by Lippert–Mataga plots. To summarize the effects of peripheral substitution, the\nphotophysical data of peripherally aryl-substituted analogues 15–19 are shown in Table 2,\nenabling direct comparison with the detailed data for the parent compound 11 summarized\nin Table 1. Although attempts to synthesize aza-BODIPY analogues using precursor 9 were\nunsuccessful at this stage, we believe that the demonstrated expansion to meso-aryl\nderivatives effectively addresses the reviewer’s concern regarding synthetic scope and\nwavelength tunability.\nOur detailed responses to the other comments are given below.\n1. The manuscript attributes the fluorescence quenching of compound 11 to a PET mechanism, supported solely by\nTD-DFT calculations. While the calculated orbital distributions are consistent with this proposal, experimental\nvalidation is crucial for such a central mechanistic claim. I recommend that the authors provide experimental evidence\nto substantiate the PET process. Techniques such as cyclic voltammetry should be used to obtain donor and acceptor\nredox potentials, then estimate ΔG(PET) with the Rehm–Weller approximation for the neutral and protonated states.\nThe free energy change for the electron transfer process (DG(PET)) in BODIPY 11 under\nneutral conditions, estimated using the Rehm–Weller equation, was –0.46 eV, indicating that\nthe PET process is thermodynamically favorable (Supplementary Fig. 26). While the\nDG(PET) value was obtained based on the 1st oxidation (0.70 V) and reduction (–1.53 V)\npotentials of 11 measured by cyclic voltammetry in CH Cl , the addition of 5 v/v% TFA\n2 2\nresulted in near-complete attenuation of the 1st oxidation peak, presumably due to the\nprotonation at the pyrrole moiety. Consequently, the apparent DG(PET) estimated under the\nacidic conditions became positive, indicating suppression of the PET process. These results\nare consistent with the DFT and spectroscopic analyses, thereby supporting the proposed\nPET mechanism. We have added these results and discussions in the main text.\n2. Hexabrominated 14 is reported to be non-fluorescent. Please examine phosphorescence and quantify singlet oxygen\ngeneration. The internal heavy atom effect from six bromine substituents should enhance intersystem crossing and\npopulate a long-lived triplet, which could make 14a useful photosensitizer.\nPhosphorescence from brominated BODIPY 14 was observed at 727 nm in a frozen 2-\nmethyltetrahydrofuran matrix containing 5 v/v% MSA at 100 K under degassed conditions.\nThe phosphorescence decay of 14 was fitted with a multiexponential function, giving lifetimes\nof 1.7 and 5.9 msec (Supplementary Fig. 32). Although we initially described compound 14\nas non-fluorescent, careful analysis of the fluorescence spectrum in the same solvent\nrevealed that its fluorescence quantum yield, while low, was measurable and determined to\nbe 1.4% at room temperature.\nSinglet oxygen generation efficiency was also evaluated by monitoring the photooxidation\nof 1,3-diphenylisobenzofuran (DPBF), using 5,10,15,20-tetraphenylporphyrin as a standard\nphotosensitizer and was determined to be 27%.\n3. The fluorescence spectrum of 15 in concentrated HSO (in Figure 5c) clearly shows a complex, broad emission\n2 4\nband with a distinct dual-emissive character (peaks at approx. 575 nm and 600 nm). The current description as merely\n\"red-shifted\" is insufficient and overlooks this important feature.\n4. The hexaphenyl derivative 15, with its six rotatable peripheral phenyl groups, is a prime candidate for exhibiting AIE. The\nrotation of these phenyls can serve as a non-radiative decay channel in solution, which would be blocked upon aggregation.\nThe fluorescence observed in highly viscous sulfuric acid (QY= 0.16) may already hint at this restriction of intramolecular\nmotion.\nThank you for these important suggestions. We carefully re-examined the fluorescence\nmeasurements of 15 and found that, due to its limited solubility in sulfuric acid, the compound\nwas not fully dissolved at the time of previous measurement, resulting in undissolved solid\nmaterial remaining suspended in the sample solution. The revised spectrum of 15 in sulfuric\nacid exhibited a single fluorescence emission band at 595 nm with a quantum yield of 0.47,\nwhich appears as a mirror image of the most intense absorption band at 574 nm, consistent\nwith the behavior observed for 11. Although we analyzed the absorption and fluorescence\nspectra of 15 in various acidic media (see Table 2 and Supplementary Fig. 33), no clear\nspectroscopic evidence for aggregation was observed under the conditions examined.\nInstead, we observed the distinct red-shifts in both the absorption and fluorescence bands\ncompared to 11.\nAll peripherally aryl-substituted analogues 15–19 exhibited enhanced fluorescence\nquantum yields in concentrated sulfuric acid (viscosity at 298 K, h = 24.2 mPa·s) compared\nwith those in formic acid (h ~ 1.8) and TFA (h ~ 0.86). Furthermore, freezing the MSA\nsolutions of 15–19 also led to remarkably increased fluorescence quantum yields. These\nresults indicate that non-radiative decay pathways are suppressed as a result of restricted\nintramolecular rotation of the aryl groups.\nMinor points:\n1. To significantly improve the clarity and accessibility of the results, I strongly recommend that the authors compile\nthe spectroscopic and photophysical data for all new compounds into a summary table.\n2. Supplementary Table 2 should be included in the TEXT or shown as a plot.\nSupplementary Table 2 has been incorporated into the main text as Table 1, with additional\ninformation on absorption maxima and molar extinction coefficients. The photophysical\nproperties of the peripherally aryl-substituted analogues, including newly synthesized meso-\naryl derivatives, are also summarized in Table 2 of the main text to facilitate direct\ncomparison.\nReviewer 2\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.\nWe thank the reviewer for taking part in the peer-review process and for the valuable time\nand effort. We appreciate the inclusion of early-career researchers in the review, as such\nmulti-perspective evaluations enhance the rigor of the review process and help us further\nimprove the quality of our work.\nReviewer 3\nThe manuscript describes the synthesis and characterization of a new family of contracted pyrrolic macrocycles that\nstructurally resemble macrocyclic BODIPY analogues with a tridentate cavity. The authors show that these frameworks\ncan tightly coordinate boron, imparting exceptional acid resistance — a feature of high relevance since BODIPY dyes\nare notoriously prone to deborylation under acidic conditions. The reported systems are stable even at pH ≈ 1 and can\nbe post-functionalized at both the meso and peripheral positions, allowing for subsequent derivatization. The\nphotophysical behavior is explored and potential applications are demonstrated in polymer staining, pH sensing, and\nimaging in acidic media. The work is well executed, and the data are of high experimental quality.\nHowever, I do not believe the manuscript meets the novelty and impact threshold required for Nature Communications.\nThe study, while solid, represents a specialized development within the already well-established field of boron–pyrrole\nchromophores rather than a conceptual or technological breakthrough of broad interest. I would therefore not\nrecommend publication in its current form. Other more chemistry specialized journals are recommended instead.\nWe appreciate the reviewer’s careful assessment of our work and the important concerns\nraised regarding its novelty and conceptual significance. Although the central advance of this\nstudy lies in the discovery of a previously unrecognized protonation behavior in pyrrole–\nboron complexes bearing a π-conjugationally isolated pyrrole unit, we recognize that our\ninitial manuscript did not sufficiently clarify how this behavior differs mechanistically from the\nwell-studied acid responses of conventional BODIPYs as well as subporphyrins and\nsubphthalocyanines. As a result, the conceptual novelty of the present system may not have\nbeen clearly conveyed, as also pointed out in the reviewer’s major comments.\nThe striking difference in the protonation behavior of the tripyrrolic macrocycle in the\npresent work, compared with other tripyrrolic boron macrocycles such as subporphyrins and\nsubphthalocyanines, is the completely reversible, dearomative protonation at the π-\nconjugationally isolated pyrrole unit. While subporphyrins retain boron chelation within a 14π-\naromatic macrocycle via formation of a tricoordinated borenium cation under acidic\nconditions, globally non-aromatic systems such as calix[3]pyrroles and macrocyclic\nBODIPYs exhibit a fundamentally different response. In these systems, protonation leads to\na tetracoordinated boronium cation, thereby maintaining stable chelation of the boron atom.\nImportantly, the boronium-forming pathway enabled by a π-conjugationally isolated pyrrole\nunit represents a distinct and previously unrecognized strategy for achieving superacid\nresistance in BODIPY chromophores. Although boron–calix[3]pyrrole complex 1 was known\nto tolerate acidic conditions, the present study clarifies the underlying protonation behavior\nand demonstrates how this mechanism can be translated into the rational design of\nsuperacid-resistant BODIPY dyes.\nTo clarify this conceptual distinction, and in direct response to the reviewer’s major\ncomments, we have revised the Introduction and Discussion sections to more clearly\narticulate the unique protonation mechanism and its implications for the design of superacid-\nresistant BODIPY chromophores (please see the yellow-highlighted revisions in the\nmanuscript).\nMajor Comments\n1. Limited novelty relative to existing acid-resistant fluorophores: The claim of exceptional acid stability is not unique.\nSeveral acid-tolerant fluorescent probes and chromophores have been previously reported (e.g., Anal. Chim.\nActa 2014, 816, 1; Chem. Soc. Rev. 2014, 43, 4629; Biosens. Bioelectron. 2024, 116638). The authors should position\ntheir compounds explicitly against these precedents and demonstrate in quantitative terms why their system is superior\nor conceptually distinct. Without this comparison, the contribution appears incremental.\nThis important suggestion prompted us to strengthen the quantitative comparison with\npreviously reported acid-tolerant fluorophores. We added Supplementary Fig. 31, which\nsummarizes representative BODIPY structures reported in the literature and the most acidic\npH conditions under which fluorescence emission has been observed for each analogue. As\nshown in the figure, most BODIPY analogues have been evaluated in aqueous media at pH\n≥ 0. In contrast, our BODIPY 11 remains fluorescent even in 36% hydrochloric acid (pH < –\n1; Supplementary Fig. 30). Among the BODIPY derivatives surveyed in the literature, B–CN\nanalogues have been reported to exhibit particularly high acid tolerance. Consistent with\nthese reports, we also confirmed that compound 13 retained BODIPY-based fluorescence in\n36% hydrochloric acid, while conventional BODIPYs such as 12 could not retain fluorescence\nunder these conditions. Therefore, we further investigated the acid stability of these dyes\nthrough comparative experiments (Fig. 4c). We have also incorporated this discussion into\nthe main text to clarify the quantitative and conceptual distinction from previously reported\nsystems.\n2. Inconsistencies in the mechanistic rationale and background: e.g., the introduction states that “acid accelerates\naxial exchange in subporphyrinoids.” This is not generally the case; protonation at meso positions or ring opening are\nfar more common acid-induced processes (see New J. Chem. 2018, 42, 10478). Clarification is needed, as this assertion\ncurrently undermines the mechanistic logic.\nWe thank the reviewer for pointing out this important issue. We agree that the acid responses\nof subphthalocyanines and subporphyrins should not be generalized, and that our original\nwording was misleading. We have revised the Introduction to clarify that, as follows:\n“Under acidic conditions, protonation or Lewis acid coordination at the meso-azomethine\nunits is commonly observed for subphthalocyanines, whereas acids often promote boron-\naxial ligand exchange at the boron center of hydroxo- or alkoxo-substituted subporphyrins.”\nThis statement is now supported by an appropriate literature citation (New J. Chem. 2018,\n42, 1622).\n3. Redundancy with previous work on boron–tridentate macrocycles: The notion of embedding boron within a\ntridentate pyrrolic environment lacks strong novelty, particularly given the authors’ own prior studies on C₃P\nframeworks and the established chemical robustness of subporphyrinoids. The manuscript would benefit from a clearer\nstatement of what genuinely new coordination or photophysical behavior arises here beyond what is already known\nfor SubPcs, SubPors, or related contracted systems.\nA key advance that distinguishes the present work from previous studies is the identification\nof a distinct protonation mode in globally non-aromatic tripyrrolic macrocycles, which leads\nto the formation of a tetracoordinated boronium cation rather than a borenium species.\nAlthough the boron–tripyrrolic framework itself may appear similar to previously reported\nsystems, this work reveals that similarity at the structural level does not translate into\nequivalence in acid response, and that the newly identified protonation behavior is the origin\nof the superacid-resistance in the macrocyclic BODIPYs.\nIn particular, while the boron–calix[3]pyrrole complex was previously reported to retain the\nboron atom in trifluoroacetic acid, this earlier work described only the phenomenological acid\ntolerance, and the underlying protonation mechanism remained unexplored. Since our initial\nreport on the synthesis of boron–C3P complexes in 2021, we have systematically\ninvestigated this behavior and identified a reversible, dearomative protonation pathway as\nthe key stabilizing mechanism under acidic conditions. Elucidation of this mechanism directly\nenabled the rational design of the present superacid-resistant BODIPYs.\nAccordingly, the current study does not represent a simple extension of prior boron–\ntripyrrole chemistry, but rather reveals a previously unrecognized synergy between boron\ncoordination and tripyrrolic macrocycles, and demonstrates how this synergy can be\nexploited to achieve exceptional acid resistance in BODIPY chromophores.\nTo make this conceptual distinction explicit, we have revised the Discussion section to\nclearly articulate the mechanistic difference in protonation behavior and to emphasize how\nthis insight underpins the conceptual advance of the present study.\n4. The proposed “acid-induced fluorescence switching” mechanism—arising from suppression of intramolecular\nquenching—is a recurring concept in chromophore design and has been widely documented. The authors should better\ncontextualize their findings within this literature and specify whether any mechanistic feature here is truly\nunprecedented.\nThe Rehm–Weller analysis of DG(PET) indicates that photoinduced intramolecular electron\ntransfer is thermodynamically favorable under neutral conditions (please also see our\nresponse to Reviewer 1 and Supplementary Fig. 26), supporting a PET-based fluorescence\nquenching mechanism that is conceptually similar to those widely employed in acid-\nresponsive fluorescent sensors. We therefore agree that the basic “acid-induced\nfluorescence turn-on” mechanism itself is not unprecedented. However, in previously\nreported systems, such switching is typically limited to moderately acidic conditions, as\nstrong acids often induce irreversible degradation of BODIPY chromophores.\nIn the present system, the key mechanistic distinction lies in the unique acid response of\nthe macrocycle-embedded pyrrole unit, which proceeds via formation of a tetracoordinated\nboronium cation. This stabilization mode preserves the BODIPY framework even under\nsuperacidic conditions, thereby enabling PET-based fluorescence switching in an acidity\nregime that has been inaccessible to conventional BODIPYs.\n5. Limited optical tunability and modest spectral range: the emission remains confined to the 500–600 nm region.\nExtension into the near-infrared, which is the current frontier in bioimaging, would represent genuine novelty and\nimpact. Similarly, the observed red-shifts upon peripheral aryl substitution are small (compared with subporphyrinoids\nor subporphyrazines (e.g., J. Org. Chem. 2020, 85, 1152)), and the substantial decrease in quantum yield upon\nfunctionalization raises questions about the practical value of these derivatives.\nTo demonstrate the optical tunability of the superacid-resistant BODIPYs, we added a new\nsynthetic route to meso-aryl-substituted analogues using acyl chlorides, and summarized the\noptical data for peripherally aryl substituted analogues 15–19 in Table 2. As also discussed\nin our response to Reviewer 1, although the red-shift observed for compound 15 is modest,\ncareful re-examination revealed that it retains a moderate fluorescence quantum yield (47%)\nin sulfuric acid. While rotation of the meso-aryl ring in the excited state accelerates non-\nradiative decay pathways, as commonly observed for conventional BODIPYs, intramolecular\ncharge-transfer interactions between the meso-thienyl moiety and the BODIPY core resulted\nin a pronounced red-shift of the fluorescence spectrum, approaching the near-infrared region\n(ca. 750–800 nm) in highly acidic media. In addition, the fluorescence quantum yields of\nthese analogues were enhanced upon restriction of intramolecular rotation of the aryl groups\nin viscous solvents or frozen solutions. Notably, meso-phenylated analogues 16–18 in frozen\nMSA solutions exhibited fluorescence quantum yields of 79–85%, which are comparable to\nthat of compound 11, despite their low quantum yields (<5%) in non-viscous acids.\nThe revised data demonstrate that macrocyclic BODIPYs retain optical tunability through\nperipheral substitution in a manner analogous to conventional BODIPYs. Importantly, the\naryl-substituted analogues 15–19 did not undergo deboronation even in superacidic media,\nthereby enabling fluorescence tuning under conditions that are inaccessible to conventional\nBODIPY systems.\nAdditional Remarks\n1. Literature coverage\nSeveral seminal works are missing. For instance, the comprehensive Chem. Rev. 2007, 107, 4891–4932 on BODIPYs\nshould be cited, as well as the most recent reviews on subporphyrinoids (Chem. Soc. Rev. 2022, 51, 9482; Trends\nChem. 2023, 5, 279). The current bibliography underrepresents key prior art, weakening the contextual framing.\nWe thank the reviewer for this suggestion. We have added the recommended review articles\nas refs. 9, 20, and 21.\n2. Line 227: “solubile” → “soluble”. Please also perform a careful language and consistency revision\nWe thank the reviewer for pointing this out. We have corrected “solubile” to “soluble” and\nhave carefully revised the manuscript for language accuracy and consistency. In particular,\nthe substrate for the synthesis of BODIPY 11 is not “triethyl orthoformate” but “trimethyl\northoformate”, and we have corrected this accordingly.\nIn summary, the manuscript presents technically competent work and interesting synthetic chemistry, but it falls short\nof the conceptual advance and broad significance expected for Nature Communications. The acid stability and\npost-functionalization are incremental improvements within an already mature class of compounds. A more\ncomprehensive comparison with known acid-resistant fluorophores, clarification of the mechanistic novelty, and\ndemonstration of distinctive optical performance (e.g., NIR emission, tunability) would be required for reconsideration.\nWe again appreciate the reviewer’s careful evaluation and the opportunity to address the\nconceptual advance of our work.\nIn the revised manuscript, (1) we explicitly articulate that dearomative protonation at a\npyrrole unit leading to the formation of a boronium cation represents a previously\nunrecognized protonation mode in tripyrrole-based macrocyclic chemistry and constitutes a\nkey mechanism underlying the observed superacid resistance, (2) the comparison of acid\ntolerance with existing BODIPYs clearly demonstrated the substantial expansion of the\naccessible acidity range for BODIPY dyes, and (3) implementation of an acyl chloride-based\nsynthetic route to meso-aryl-substituted macrocyclic BODIPYs expanded the scope of\nderivatization and enabled further tunability of emission wavelengths through intramolecular\ncharge-transfer interactions. We believe that these revisions clarify that the present work\ngoes beyond an incremental extension of boron–tripyrrole chemistry and identifies a new\nmechanistic principle for designing superacid-resistant BODIPY chromophores.\nReviewer 4\nThis is a very interesting paper that describes the synthesis, characterization and application of superacid resistant\ncyclic BODIPYs. I believe the work presented is significant to the field of BODIPYs and their practical applications; a\nclear comparison between the new reported acid-stable cyclic BODIPYs and non-cyclic BODIPYs is investigated by\nthe authors. All compounds are adequately characterized by NMR and HRMS. The methodologies used for the\ncharacterization of the neutral and positively charged species are all adequate and the conclusions are supported by\nthe results obtained. The Figures are adequate and informative.\nWe thank the reviewer for the positive and encouraging evaluation of our work.\nI suggest that the authors correct a few typos in the manuscript, such as in lines 227 and 248. In addition, the Discussion\nsection is more of a Conclusions section.\nWe also thank for pointing this out. We have corrected the typographical errors:\nFor line 227: “solubile” ® “soluble”\nFor line 248: “these” ® “there”\nWe have also revised the Discussion section to clarify its purpose and structure.\nResponse to Editorial Requirements\n• In accordance with the editorial policy, we have prepared a Source Data file containing the\nraw data underlying the figures and tables, and this has been uploaded with the revised\nmanuscript.\n• During the revision process, the author list was updated to reflect substantial contributions\nmade to the additional experiments. The completed author list change approval form, signed\nby all authors, has been submitted."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have appropriately addressed the major concerns. However, to ensure reproducibility, the methodological\ndetails in the Supporting Information needs to be expanded in detail prior to acceptance. The newly conducted experimental\nprocedures, such as phosphrorensence and singlet oxygen measurements, were wrapped in the captions of Figures.\nThey should have a more detailed experimental section. Other than that, this excellent manuscript is recommended for\nacceptance.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "The authors have clarified all the concerns raised by this referee. With these changes, this referee is now convinced that the\nwork is suitable for Nat. Comm, and therefore recommends its publication.", "reviewer_4": "[Editor's Note: This reviewer left remarks to the editor indicating all technical concerns have been addressed]"}, "author_response": "Point-by-Point Response to the Reviewers’ comments\n(blue: original comments by reviewers, black: our response)\nReviewer 1\nThe authors have appropriately addressed the major concerns. However, to ensure reproducibility, the methodological\ndetails in the Supporting Information needs to be expanded in detail prior to acceptance. The newly conducted\nexperimental procedures, such as phosphrorensence and singlet oxygen measurements, were wrapped in the captions\nof Figures. They should have a more detailed experimental section. Other than that, this excellent manuscript is\nrecommended for acceptance.\nWe thank the reviewer for re-evaluating our revised manuscript and for the positive\nassessment and recommendation for acceptance. To ensure reproducibility, detailed\nexperimental procedures for the phosphorescence and singlet oxygen measurements have\nbeen added to the supporting information (page S52).\nReviewer 2\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.\nWe also thank this reviewer.\nReviewer 3\nThe authors have clarified all the concerns raised by this referee. With these changes, this referee is now convinced\nthat the work is suitable for Nat. Comm, and therefore recommends its publication.\nWe thank the reviewer for the careful re-evaluation of our manuscript and for the positive\nrecommendation.\nResponse to Editorial Requirements\nIn accordance with the editorial checklist, we have revised the main manuscript and\nSupporting Information to comply with all formatting and presentation requirements. For ease\nof review, we have also provided versions of the revised manuscript and Supporting\nInformation in which all changes are highlighted in yellow. No scientific content or data have\nbeen altered during this editorial revision."}]

Decision letter