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Characterization and Biological Activity of a Hydrogen Sulfide-Releasing Red Light-Activated Ruthenium(II) Complex.
Communication
CiteThis:J.Am.Chem.Soc.XXXX,XXX,XXX−XXX pubs.acs.org/JACS
fi
Characterization and Biological Activity of a Hydrogen Sul de-
Releasing Red Light-Activated Ruthenium(II) Complex
Joshua J. Woods,†,§ Jian Cao,‡ Alexander R. Lippert,‡ and Justin J. Wilson*,§
†
Robert F. Smith School for Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States
§
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
‡
Department of Chemistry, Southern Methodist University, Dallas, Texas 75275, United States
*
S Supporting Information
thesesystemsrequireupconvertingnanoparticlestomediatethe
ABSTRACT: Hydrogen sulfide (H 2 S) is a biological low-energy photoactivation process.21,23 Because red light
gasotransmitterthathasbeenemployedforthetreatment effectively penetrates biological tissue and is nontoxic, the
of ischemia-reperfusion injury. Despite its therapeutic developmentofsmall-moleculeredlight-activatedH S-releasing
2
value, the implementation of this gaseous molecule for agents would be particularly valuable for both studying the
e th ff i e s ct p iv u e rpo in s t e rac h e a l s lul r a e r qu d i e re li d ver H y. 2 S T -r h e e leas m in a g jor p it r y od o ru f gs the fo se r b th io is lo g g a ic s a o l tr r a o n le s s m o it f t H er 2 .3 S 9 a I n n dl t e h v i e s ra r g e i p n o g rt t , he w t e he d ra e p sc e r u ib ti e ce t ff h e e ct fi s r o st f
prodrugs, however, spontaneously release H 2 S via prototype of this class of molecules and its implementation to
uncontrolled hydrolysis. Here, we describe a Ru(II)- protectagainstinvitroischemia-reperfusioninjury.
basedH 2 S-releasingagentthatcanbeactivatedselectively Our strategy to develop such a red light-activated H 2 S-
byredlightirradiation.Thiscompoundoperatesinliving releasing molecule invoked a combination of the established
cells,increasingintracellularH 2 Sconcentrationonlyupon H 2 S-releasing compound morpholin-4-ium 4-methoxyphenyl-
irradiation with red light. Furthermore, the red light (morpholino)phosphinodithioate(GYY4137,Chart1)40,41and
irradiation of this compound protects H9c2 cardiomyo-
blasts from an in vitro model of ischemia-reperfusion Chart1.GYY4137,andtheCompounds[1]+and[2]+That
injury.Theseresultsvalidatetheuseofredlight-activated WereExploredinThisStudy
H S-releasing agents as valuable tools for studying the 2
biology and therapeutic utility of this gasotransmitter.
T hebiologicalroleofthetoxicgashydrogensulfide(H S)as
2
aneuromodulatorwasfirstrecognizedin1996.1Sincethis
initialdiscovery,H Shasreceivedsignificantattentionbecause
2
of its therapeutic potential for treating inflammation,2
Parkinson’sdisease,3reproductivedysfunction,4braininjury,5−9
diabetes,10cancer,11andischemia-reperfusion(I/R)injury.12,13
The therapeutic implementation of H S in its gaseous state,
2
however, is challenged by difficulties in administering bio-
logically relevant and beneficial concentrations while avoiding
aphotolabileruthenium(II)scaffold[Ru(tpy)(biq)(L)]n+(tpy
problemsassociatedwithitsknowncytotoxicity,volatility,and
flammability.14−16
To circumvent this challenge, researchers
=2,2′:6′-2″-terpyridine;biq=2,2′-biquinoline,n=1,2).Ru(II)
compounds of this class possess low energy metal-to-ligand
havedevelopedsyntheticdonorsthatreleaseH Sinresponseto
stimuli such as pH,17 external light,18−25 2 reactive oxygen chargetransfer(MLCT)absorptionbandsthatextendintothe
species,26 andenzymatic activity.26−32 Amongthesestrategies, red region of the visible spectrum. After population of the
1MLCT state by absorption of red light, efficient intersystem
light-activatedH Sreleasehasbeenrecognizedasapromising
tool for biomed
2
ical and therapeutic applications.18,21 Light-
crossingtoadissociativetripletligandfield(3LF)stateejectsthe
activatedprodrugsareexcitingtherapeuticcandidatesthatallow
monodentateligandLwithhighquantumyields.42−45
In aqueous or wet organic solvent, GYY4137 releases H S
for localized and noninvasive treatment of serious medical 2
conditions,whilecircumventingtoxicsideeffectsthatarisefrom over a few hours.46 Sustained H
2
S release from GYY4137 has
traditionalchemotherapy.33−37 beenusedtherapeuticallytotreatinflammation,47inhibitcancer
cellgrowth,48andpreventischemia-reperfusioninjury.49,50The
The majority of light-activated H S-releasing agents require
2
UVlight,whichineffectivelypenetratesbiologicaltissueandcan hydrolysis of GYY4137, however, occurs spontaneously in
give rise to toxic effects.38 Efforts to move photoactivation solution, limiting spatiotemporal control of H 2 S release from
wavelengths to more biologically useful regions of the visible
spectrumhaveresultedinseveralsystemsthatcanbetriggered Received: August13, 2018
with visible and near-infrared light.21−23 Except in one case,22
©XXXXAmericanChemicalSociety A DOI:10.1021/jacs.8b08695
J.Am.Chem.Soc.XXXX,XXX,XXX−XXX
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JournaloftheAmericanChemicalSociety Communication
thismolecule.WehypothesizedthatcoordinationofGYY4137 Table1.AbsorptionMaxima(λ ),MolarExtinction
max
to the photoactive [Ru(tpy)(biq)(L)]n+ scaffold would inhibit Coefficients(ε)atλ ,andPhotosubstitutionQuantum
max
its spontaneous hydrolysis until it was released by irradiation Yields(Φ )of[1]Cland[2]Cl
626
withredlight.Thus,thecomplex[Ru(tpy)(biq)(GYY4137)]Cl
([1]Cl)(Chart1)wasdevelopedasthefirstredlight-activated Compound λ
max
(nm) ε(M−1cm−1) Φ
626
(%)
H S-releasingmolecule. [1]Cl 581 4050±140 0.85±0.03
2
Thedirectreactionbetween[Ru(tpy)(biq)Cl]Clandexcess [2]Cl 570 4000±200 1.02±0.11
GYY4137 in 50% aqueous acetone gave [1]Cl as the major
product in solution. Further purification by silica gel 1MLCTabsorptionbandsatλ=581nm([1]Cl)andλ=568nm
chromatography (90/10 CH CN/H O) afforded pure [1]Cl ([2]Cl), which both tail past 650 nm. Upon irradiation of the
3 2
in 24% yield. When [Ru(tpy)(biq)Cl]PF was sequentially complexes with 626 nm light (Figures S13 and S14, SI), the
treatedwithAgPF ,whichremovedtheinner 6 -spherechlorideas UV−vis spectraevolve,resulting in ablue shift of the1MLCT
6
insolubleAgCl,andexcessGYY4137inrefluxingmethanol,we band to a new maximum at 549 nm, characteristic of the
unexpectedly isolated [Ru(tpy)(biq)(GYYOMe)]Cl ([2]Cl), expectedphotoproduct[Ru(tpy)(biq)(OH 2 )]2+(Figures2and
where GYYOMe is O-methoxy 4-methoxyphenylphosphinodi-
thioate.Overthecourseofthisreaction,themorpholinegroup
ofGYY4137wasreplacedbythemethanolsolventgivingriseto
theGYYOMeligand(Chart1).51
In additiontocharacterizationbystandardtechniques,such
asNMRspectroscopy,massspectrometry,andIRspectroscopy
(FiguresS1−S12,SupportingInformation,SI),[1]PF wasalso
6
characterized by single-crystal X-ray diffraction (Figure 1).
Figure1.X-raycrystalstructureof[1]PF.Hydrogenatomsandthe
6
PF−counterionareomittedforclarity.Thermalellipsoidsareshownat
6
the50%probabilitylevel. Figure2.Top:Changesintheelectronicabsorptionspectrumof[1]Cl
in100mMMOPS(pH7.4)under626nmlightirradiation(photon
Crystallographicparametersandrelevantinteratomicdistances flux=2.01×10−8mols−1,t irr =15min,T=298K).Bottom:Electronic
andanglesarelistedinTablesS1andS2intheSI.TheRu(II) absorptionspectrumof[1]Clin100mMMOPSinthedarkafter5days
(T=298K).
center of [1]PF attains a distorted octahedral geometry with
6
GYY4137 coordinated trans to the biquinoline ligand. The
stericallydemandingtpyandbiqligandsperturbtheRu-ligand S15, SI).44,45 The photosubstitution quantum yields (Φ ) at
626
bond angles, which range from 77.31° to 99.23°, significantly roomtemperaturefor[1]Cland[2]Clwerefoundtobesimilar
from the 90° of an ideal octahedron. This steric strain to those measured for related Ru(II) complexes (Table
contributes to the efficient photosubstitution reactions of 1).42,44,45
thesecomplexesbyloweringtheenergyofthedissociative3LF The red light-induced photoreaction of [1]Cl was further
state, allowing for it to be thermally populated from the investigated by 1H and 31P{1H} NMR spectroscopy in 90/10
photogenerated3MLCTstate.52−55TheRu−S1distance(2.41
D O/DMSO-d .Uponirradiation,thecharacteristicdoubletof
2 6
Å) is similar to that found in a related organometallic Ru(II) [1]Clinthe1HNMRspectrumat9.97ppm,whichisassigned
complexbearingGYY4137asaligand(2.403Å).56 to the proton in the ortho position of the biq ligand directly
Thephotophysicalpropertiesof[1]Cland[2]Clinbuffered adjacent to the coordinated GYY4137, vanished while a new
aqueous solution (3-morpholinopropanesulfonic acid; MOPS; quartetat8.94ppm,assignedto[Ru(tpy)(biq)(OH )]2+,grew
2
pH 7.4) were investigated (Table 1). The complexes exhibit inovertime.Similarly,peaksoriginatingfrom[1]Clat8.80and
B DOI:10.1021/jacs.8b08695
J.Am.Chem.Soc.XXXX,XXX,XXX−XXX
JournaloftheAmericanChemicalSociety Communication
8.70 ppm were replaced with doublets corresponding to Ru(II)compoundscanbindtoDNAandinducecytotoxicity.54
[Ru(tpy)(biq)(OH )]2+ at 8.63 and 8.59 ppm, (Figure S16, As expected, GYY4137 is noncytotoxic (Figures S22 and S23,
2
SI).Inthe31P{1H}NMRspectrum,thesingletarisingfromthe SI).
coordinatedGYY4137ligandof[1]Clwasreplacedbyapeakat Given the promising toxicity profile of [1]Cl and its
98.93 ppm, which is conclusively assigned to free GYY4137 photoproducts,theH S-releasingcapabilitiesofthiscompound
2
(FigureS17,SI). were measured in living cells using the cell-trappable H S-
2
Fortheselight-activatedcomplexestobevaluableasstimuli- responsive fluorescent probe, SF7-AM.58 Lung cancer (A549)
responsive agents, they must not undergo thermal loss of the cellswereloadedwithSF7-AMandtreatedwiththecomplexes,
phosphinodithioate ligand. As such, the thermal stabilities of followed by irradiation with red light for 30 min prior to
thesecomplexeswereassessedinaqueousbuffer.Wefoundthat fluorescence microscopy imaging. Cells that were only treated
[2]Clisunstableinthedark,forming[Ru(tpy)(biq)(OH )]2+ with[1]Clorexposedtoredlightshowednosignificantincrease
2
andGYYOMeviaathermalaquationreaction(FigureS18,SI). influorescenceintensityofSF7-AM.Incontrast,whenboth[1]
In contrast, [1]Cl did not show appreciable changes in its Clandredlightwereadministeredtocells,asignificantincrease
absorption spectrum after 5 days in solution (Figure 2), in fluorescence intensity was observed, indicating that both
indicatingthatthiscompoundissufficientlystableforuseasa
components are critical for the intracellular release of H S
2
light-activated H S-releasing agent. Notably, the GYY4137 (Figure4).
2
liganddidnothydrolyzewhilecoordinatedtotheRu(II)center.
Given the promising thermal stability and efficient photo-
substitutionquantumyieldof[1]Cl,wefurtherinvestigatedits
H S-releasing properties. The formation of H S in aqueous
2 2
bufferwasevaluatedusingthefluorescentprobe1,5-dansylazide
(DNS-az).57 The azide functional group of this probe is
selectivelyreducedbyH Stoelicitalinear,fluorescentturn-on
2
response (Figure S19, SI). Using this probe, we observed
negligible release of H S from [1]Cl in the dark. By contrast,
2
upon irradiation with 626 nm light, a steady time-dependent
increaseinH Sconcentrationwasdetected,comparabletothat
2
offreeGYY4137(Figure3).BycoordinatingGYY4137tothe
ruthenium photocage in complex [1]Cl, we gain temporal
control over the otherwise continuous release of H S from
2 Figure4.ImagesofHSreleasefrom[1]Cl.A549cellswereincubated
GYY4137. with5μMSF7-AMfo 2 r30min,washedandthen(A)exposedtored
lightfor30min;(B)treatedwith60μM[1]Clandexposedtoredlight
for30min;(C)incubatedfor30mininthedark;(D)treatedwith60
μM [1]Cl for 30 min in the dark. (E) Mean pixel intensity of
fluorescence images for A549 cells in the experiments described in
panelsA−D.ErrorbarsareSEofn=5or6wellsfromtwobiological
replicates. Statistical significance was determined using a two-tailed
student’sttest.**p<0.005.
Because H S can protect against I/R
injury,59−65
we
2
investigated the ability of [1]Cl to give rise to cytoprotective
effects selectively upon red light irradiation in H9c2 cells
subjected to hypoxia/reoxygenation injury. Cells were in-
cubatedinapH6.4ischemiamimeticbuffer(seeSIfordetails)
in hypoxic conditions, followed by treatment with [1]Cl or
GYY4137inthedarkorunderredlightirradiationfor30min
prior to reoxygenation. Control cells were incubated in
Figure3.H 2 Sreleasefrom[1]Cl(200μM)withredlightirradiation normoxicconditionsforthedurationoftheexperiment.
(red circles), [1]Cl (200 μM) in the dark (black squares), and Theviabilityofuntreatedcells,measuredbythecolorimetric
GYY4137 (200 μM) (purple triangles) measured by DNS-az MTTassay,66subjectedtothisI/Rinjurymodelwasdecreased
fluorescence. Errors bars are the standard error (SE) of three
by approximately 80%, indicating that this model accurately
independenttrials. capturesthecytotoxiceffectsofthiscondition.Whencellswere
treatedwith[1]Clinthedark,therewasnosignificantchangein
For [1]Cl to be useful as a H S-releasing biological tool or cellviabilitycomparedtountreatedcells.Inthepresenceofred
2
therapeutic agent, it should not give rise to toxic effects. With light,however,cellstreatedwith[1]Clshowa75%increasein
short (4 h) incubation times, conditions that are favorable for viability relative to the untreated control cells. In comparison,
H S-releasing applications, [1]Cl is essentially nontoxic to the extent of the cytoprotective effect of GYY4137 does not
2
healthy cells (Table S3, Figures S20 and S21, SI). At longer changeappreciablywhencellsaretreatedeitherinthelightor
times,[1]Cldoesshowmildtoxicity(TableS4,FiguresS22and dark (Figure 5). These results demonstrate that [1]Cl can
S23,SI).Theaquatedphotoproduct[Ru(tpy)(biq)(OH )]2+is effectivelycageGYY4137andpreventH Sreleaseuntilselective
2 2
effectivelynontoxic,asindicatedbyitsIC valueof97±4μM, redlightactivationtopreventcelldeathinamodelofI/Rinjury.
50
consistent with values that have previously been measured for In summary, we have developed a novel red light-activated
this compound. These results are significant because related H S-donatingcomplex.Comparedtootherlight-activatedH S-
2 2
C DOI:10.1021/jacs.8b08695
J.Am.Chem.Soc.XXXX,XXX,XXX−XXX
JournaloftheAmericanChemicalSociety Communication
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t■reatmentandpreventionofseriousmedicalconditions. M.;Xian,M.Org.Lett.2013,15,2786−2789.
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ASSOCIATEDCONTENT
2281.
*
S SupportingInformation (20)Fukushima,N.;Ieda,N.;Kawaguchi,M.;Sasakura,K.;Nagano,
The Supporting Information is available free of charge on the T.; Hanaoka, K.; Miyata, N.; Nakagawa, H. Bioorg. Med. Chem. Lett.
ACSPublicationswebsiteatDOI:10.1021/jacs.8b08695.
2015,25,175−178.
(21)Chen,W.;Chen,M.;Zang,Q.;Wang,L.;Tang,F.;Han,Y.;Yang,
Complex characterization data, cell viability curves, C.;Deng,L.;Liu,Y.-N.Chem.Commun.2015,51,9193−9196.
crystal data tables, UV−vis spectra, NMR data of (22)Yi,S.Y.;Moon,Y.K.;Kim,S.;Kim,S.;Park,G.;Kim,J.J.;You,Y.
irradiation(PDF) Chem.Commun.2017,53,11830−11833.
■ Datafor[1]PF 6 (CIF) (23)Sharma,A.K.;Nair,M.;Chauhan,P.;Gupta,K.;Saini,D.K.;
Chakrapani,H.Org.Lett.2017,19,4822−4825.
AUTHORINFORMATION (24)Fukushima,N.;Ieda,N.;Sasakura,K.;Nagano,T.;Hanaoka,K.;
Suzuki,T.;Miyata,N.;Nakagawa,H.Chem.Commun.2014,50,587−
CorrespondingAuthor
589.
*jjw275@cornell.edu
(25) Xiao, Z.; Bonnard, T.; Shakouri-Motlagh, A.; Wylie, R. A. L.;
ORCID Collins, J.; White, J.; Heath, D. E.; Hagemeyer, C. E.; Connal, L. A.
JoshuaJ.Woods:0000-0002-6213-4093 Chem.-Eur.J.2017,23,11294−11300.
JustinJ.Wilson:0000-0002-4086-7982 (26)Zhao,Y.;Pluth,M.D.Angew.Chem.,Int.Ed.2016,55,14638−
14642.
Notes
(27)Steiger,A.K.;Marcatti,M.;Szabo,C.;Szczesny,B.;Pluth,M.D.
T■heauthorsdeclarenocompetingfinancialinterest. ACSChem.Biol.2017,12,2117−2123.
(28)Chauhan,P.;Bora,P.;Ravikumar,G.;Jos,S.;Chakrapani,H.Org.
ACKNOWLEDGMENTS Lett.2017,19,62−65.
ThisworkwassupportedbyCornellUniversityandtheNational (29)Zhao,Y.;Bhushan,S.;Yang,C.;Otsuka,H.;Stein,J.D.;Pacheco,
Science Foundation (NSF-GRFP for J.J.W.; award no. DGE- A.;Peng,B.;Devarie-Baez,N.O.;Aguilar,H.C.;Lefer,D.J.;Xian,M.
ACSChem.Biol.2013,8,1283−1290.
1650441).ThisworkmadeuseoftheNMRfacilityatCornell
(30) Foster, J. C.; Radzinski, S. C.; Zou, X.; Finkielstein, C. V.;
Universitywhichisfunded,inpart,bytheNSF(awardno.CHE-
Matson,J.B.Mol.Pharmaceutics2017,14,1300−1306.
1531632). This work was additionally supported by the NIH
(31)Zhao,Y.;Henthorn,H.A.;Pluth,M.D.J.Am.Chem.Soc.2017,
(■awardno.NIGMSR15GM114792toA.R.L.). 139,16365−16376.
(32)Zhao,Y.;Steiger,A.K.;Pluth,M.D.Chem.Commun.2018,54,
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