Click Reac ions
Bioo hogonal Azide–Thioalkyne Cycloaddi ion Ca alyzed by
Pho oac i a able Ru henium(II) Complexes
Alejand o Gu i8 ez-Gonz#lez, Paolo Des i o,Jos8R. Coucei o,Cib an P8 ez-Gonz#lez,
Fe nando Llpez,* and Jos8L. Masca eÇas*
Abs ac : Tailo ed u henium sandwich complexes bea ing
pho o esponsi e a ene ligands can e icien ly p omo e azide–
hioalkyne cycloaddi ion (RuA AC) when i adia ed wi h UV
ligh . The eac ions can be pe o med in abioo hogonal
manne in aqueous mix u es con aining biological compo-
nen s.The s a egy can also be applied o he selec i e
modi ica ion o biopolyme s,such as DNAo pep ides.
Impo an ly, his u henium-based echnology and he s anda d
coppe -ca alyzed azide–alkyne cycloaddi ion (CuAAC)
p o ed o be compa ible and mu ually o hogonal.
In oduc ion
Bioo hogonal eac ions,byenabling he co alen modi-
ica ion o speci ic eac an s o biomolecula a ge s in
complex biological en i onmen s,ha e b ough apa adigm
shi on he po en ial o chemis y o in e oga ing o /and
al e ing biology.[1,2] Wi hin he “ oolbox” o bioo hogonal
eac ions, hose ha a e ca alyzed by ansi ion me als a e
especially a ac i e,owing o hei in insic me al-dependen
cha ac e is ics,and he possibili y o uning he eac i i y by
adjus ing he cha ac e is ics o he ca alys .[3] Howe e ,
p og ess in his ield has been slow,ing ea pa because o
he no ion ha ansi ion me al eagen s a e incompa ible
wi h aqueous and biological milieu, and ha hey can be
easily inac i a ed by biological componen s.Mo eo e ,while
he e has been an inc easing numbe o epo s on bioo -
hogonal me al-ca alyzed eac ions, hey usually p esen low
ca aly ic e iciencies,especially unde he dilu ed condi ions
usually equi ed o biological applica ions.[3,4]
Among all ansi ion-me al-media ed bioo hogonal e-
ac ions, he e is one ha s ands ou , namely, he coppe (I)-
p omo ed azide–alkyne cycloaddi ion (CuAAC).[5,6] The e-
ac ion engages o ganic azides and alkynes,which a e ideal
chemical en i ies in e ms o biological o hogonali y,and
ends o exhibi e y good a es.Howe e , his ans o ma ion
s ill p esen s impo an limi a ions such as i s low compa i-
bili y wi h hiols,i s es ic ion o e minal alkynes o he side
eac i i y and oxici y o coppe (I) ions in biological
con ex s.[7] Fu he mo e, o each e icien con e sions unde
ypically dilu ed condi ions, he eac i e coppe (I) species
need o be gene a ed in si u using excess amoun s o
acoppe (II) sou ce and sodium asco ba e,a educ an which
is no innocen in biological con ex s (Figu e 1).[8] The e o e,
he e is aclea need o disco e new, obus and aqueous-
compa ible me al-ca alyzed annula ions as al e na i es o he
CuAAC.[9,10]
In his con ex , in 2017, we epo ed he i s examples o
a u henium-ca alyzed azide–alkyne cycloaddi ion ha akes
place in aqueous and in biologically ele an milieu.[11] The
me hod makes key use o hioalkynes as eac ion pa ne s,
and o he comme cially a ailable complex [Cp*RuCl(COD)]
(Ru1)asca aly ic eagen (Figu e 1). Following ou epo ,
o he g oups de eloped al e na i e condi ions o achie e
Figu e 1. Me al-ca alyzed azide–( hio)alkyne cycloaddi ion eac ions.
Ad an ages a e ma ked wi h blue squa es, and limi a ions wi h ed.
[*] A. Gu i8 ez-Gonz#lez, D .P.Des i o, D .J.R.Coucei o,
D .C.P8 ez-Gonz#lez, D .F.Lkpez, P o . J. L. Masca eÇas
Cen o Singula de In es igaciknenQu&mica Biolkxica eMa e iais
Molecula es (CiQUS) and Depa amen o de Qu&mica O g#nica
Uni e sidade de San iago de Compos ela
15782 San iago de Compos ela (Spain)
E-mail:j[email p o ec ed]
D .F.Lkpez
MisiknBiolkgica de Galicia
Consejo Supe io de In es igaciones Cien & icas (CSIC)
36080 Pon e ed a (Spain)
E-mail: [email p o ec ed]
Suppo ingin o ma ion and he ORCID iden i ica ion numbe (s) o
he au ho (s) o his a icle can be ound unde :
h ps://doi.o g/10.1002/anie.202103645.
T2021 The Au ho s. Angewand e Chemie In e na ional Edi ion
published by Wiley-VCH GmbH. This is an open access a icle unde
he e ms o he C ea i e Commons A ibu ion License, which
pe mi suse, dis ibu ion and ep oduc ion in any medium, p o ided
he o iginal wo k is p ope ly ci ed.
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ela ed cycloaddi ions in wa e , using nickel,[12] hodium[13] o
i idium ca alys s,[14] albei only he la e has been shown o
ope a e in biological mix u es.
Impo an ly,all hese new me al-ca alyzed azide–alkyne
cycloaddi ion eac ions,including ou u henium-ca alyzed
p ocess,ha e demons a ed e ec i i y only when he con-
cen a ion o eagen s is in he mid o high millimola ange.
This ep esen s an impo an limi a ion in e ms o de eloping
biological applica ions,which usually equi e e y dilu ed
samples.Anaddi ional challenge in his ield has o do wi h
he possibili y o con olling he eac i i y using ex e nal
s imuli, as his could open new oppo uni ies o biological
egula ion.
He ein, we demons a e ha ca ionic RuII complexes such
as [Cp*Ru(MeCN)3]PF6(Ru2)a e excellen p eca alys s o
pe o m e icien , o mal cycloaddi ions be ween hioalkynes
and azides in aqueous media. Con a y o [Cp*RuCl(COD)]
(Ru1), his ca ionic complex (Ru2)is e y e ec i e unde
dilu ed mic omola condi ions,e en in PBS and biologically
complex media, such as DMEM, o HeLa cells lysa es.Mo e
impo an ly,wealso show ha he azide– hioalkyne cyclo-
addi ion can be ca alyzed by [Cp*RuIIa ene] sandwich com-
plexes,[15] p o ided ha hey a e pho oac i a ed by asho -
ime i adia ion wi h aLED lamp a 365 nm (Figu e 1). The
possibili y o con olling he gene a ion o he ca aly ically
ac i e RuII species wi h ligh opens in e es ing pe spec i es in
op obiology.[16] Finally,wealso demons a e ha his ech-
nology is ully o hogonal wi h he CuAACand, mo eo e ,i
can be used o he chemoselec i e modi ica ions o small
pep ides and ssDNAs, o ins ance, o he in oduc ion o
luo ogenic ags.
Resul s and Discussion
Ou i s expe imen s we e ca ied ou wi h he an h a-
cenyl azide 1a and he hioalkyne 2a,because he esul ing
iazole p oduc (3aa)is luo escen and, hus, he eac ion
can be eadily moni o ed. In consonance wi h p e ious
obse a ions, he neu al RuII complex [Cp*RuCl(COD)]
(Ru1)was signi ican ly mo e e icien han [Cp*Ru-
(MeCN)3]PF6(Ru2), when he eac ion was ca ied ou unde
anhyd ous condi ions in CH2Cl2(75 mM).[17] Speci ically, he
eac ion ga e a99%yield o he p oduc a e 0.5 hwi h Ru1
(Table 1, en y 1), bu jus a15% yield wi h Ru2,a e 1h
(30%yield a e 6h,en y 2). Se e al con ol expe imen s
and ca e ul analysis by NMR and ESI-MS allowed o disco e
ha he poo e pe o mance o Ru2 was likely due o he
o ma ion o seconda y u henium-con aining p oduc s.In
pa icula ,wecould iden i y Ru2’’,which esul s om an
unp eceden ed u henium-p omo ed ime iza ion o hioal-
kynes,ap ocess ha gene a es achela ing di hio ul ene
ligand (Figu e 2).[18] Thepe o mance o Ru2 in he RuA AC
could be pa ially imp o ed using E 3NCl as addi i e
(5 mol%), p obably by a o ing he in si u o ma ion o
aneu al Cp*– u henium(II) chlo ide species (en y 3), which
migh hampe he hioalkyne- o- ul ene ime iza ion.[19]
Rema kably,when he eac ion ca alyzed by Ru2 was
ca ied ou in wa e , in he p esence o his chlo ide sou ce,
he cycloadduc 3aa was ob ained in 99%yield, a e only
0.5 h(en y 4). We la e ound ha he use o E 3NCl is no
needed, as he eac ion p o ided he same yield wi hou any
addi i e (en y 5). Mos likely, he highe ac i i y o Ru2 in
wa e is pa ially ela ed o he o ma ion o ac i e RuII aquo
o oxo de i a i es,which a o he desi ed annula ion o e
al e na i e pa hways.[20] Indeed, we ha e de ec ed by ESI-MS
se e al u henium-oxygena ed species in he aqueous solu-
ions o Ru2.[21]
Impo an ly,using hese condi ions,wecould p omo e he
annula ion o a a ie y o azides and hioalkynes (Scheme 1).
Thioalkynes bea ing an e hyl g oup a he sul u a om we e
pa icula ly eac i e,bu o he alkyl g oups like benzyl (2c),
isop opyl (2d)o a oma ic subs i uen s (2b)a e also ole -
a ed, p o iding in all cases he expec ed p oduc s in good
yields.The o he subs i uen o he hioalkyne can also be
modi ied wi hou comp omising he yields o he desi ed
iazoles.
Ru2 p o ed o be much mo e selec i e han he p e iously
desc ibed ca alys Ru1 wi h espec o he ype o alkyne
pa ne used. Indeed, Ru1 p omo es he eac ion o egula
alkynes lacking he hioe he ,such as he 2 and 2g, ogi e
he co esponding iazoles in mode a e o good yields (3b ,
3bg,Scheme 1). Howe e , he ca ionic eagen Ru2 ailed o
induce any con e sion wi h hese alkynes,e en a e 7ha
Table 1: Viabili y o he RuA AC wi h he RuII ca ionic complex Ru2.[a]
En y [Ru] (%) Sol en [h] Yield [%][b]
1Ru1 (5) CH2Cl20.5 99
2Ru2 (5) CH2Cl2115(30)[c,d]
3[e] Ru2 (5) CH2Cl2128(72)[c]
4[e] Ru2 (5) H2O0.5 99
5Ru2 (5) H2O0.5 4
[a] Reac ion condi ions: 2a (150 mmol), 1a (75 mmol), sol en (1 mL)
and he u henium ca alys (5 mol%) we e added o a ial unde ai ,and
he mix u e s i ed o he indica ed ime. [b] Yield de e mined by
1HNMR spec oscopy o he eac ion c ude mix u e using 1,3,5-
ime hoxybenzene as an in e nals anda d. [c] The yield a e 6his
indica ed in pa en hesis. [d] The complex Ru2’’was de ec ed in he
eac ion mix u e (NMR and ESI-MS).[18] [e] The Ru complex Ru2 and
E 4NCl (5 mol%) we e p emixed in he co esponding sol en o 5min.
No e:Reac ion mix u es in wa e can be conside ed as suspensions
a he han solu ions.
Figu e 2. S uc u e and X- ay c ys allog aphic analysis o Ru2’’(speci ic
hyd ogen a oms and he coun e ion (PF6@)a e omi ed o cla i y).
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(Scheme 1). The e o e, Ru2 no only allows o ca y ou he
annula ion in aqueous media in an e icien manne ,bu also
in oduces ale el o chemoselec i i y ha was p e iously
una ainable wi h Ru1,allowing o ully dis inguish hioal-
kynes om alkynes (bo h mono- and disubs i u ed).
Conside ing his chemoselec i i y,wenex explo ed he
o hogonali y o he RuA ACand he CuAAC. When
amix u e o azide 1b (1 equi .) and alkynes 2a and 2h
(2 equi .o each one) was ea ed unde s anda d CuAAC
condi ions [ ha is,CuSO4·5H2O(5mol%) and sodium
asco ba e (NaAsc,10mol%)] he iazole 3bh, esul ing
om he eac ion o he e minal alkyne 1h was exclusi ely
o med in high yield (Table 2, en y 1). I ,a e his eac ion
has been comple ed (2 h), we add asecond equi alen o he
azide,and he ca alys Ru2 (5 mol%), he hioe he con ain-
ing adduc 3bais o med in agood 78%yield (en y 2). E en
mo e ele an , when he ini ial mix u e o he azide 1b and
alkynes 2a and 2h is i s ea ed wi h Ru2,only he sul u -
con aining iazole 3ba is obse ed (79%yield, en y 3),
whe eas he subsequen addi ion o 1a and he Cu ca alys
leads o 3bhin 95%yield (en y 4). Fu he mo e,when azide
1b was mixed wi h bo h 2a and 2h (1 equi .each) in he
p esence o bo h Cu and Ru ca alys s (5 mol%o each one),
an almos equimola mix u e o 3ba and 3bh is ob ained, in
an excellen yield (94%yield, en y 5). O e all, hese esul s
con i m as iking chemoselec i i y and mu ual o hogonali y
be ween bo h me hods and indica e ha bo h cycloaddi ions
sha e simila kine ic p o iles unde hese condi ions.This
mu ual o hogonali y p omises ele an applica ions,such as
o he dual agging o biomolecules.[22]
In iew o he excellen pe o mance o Ru2,weexplo ed
i s beha io unde mo e dilu ed condi ions.G a i yingly, he
annula ion be ween 1cand 2acan be e icien ly ca ied using
azide concen a ions o 1mM(99%yield), and e en a
250 mM, wi hou signi ican de e io a ion in he yield (86%
yield).[23]
A his poin we ques ioned he possibili y o enginee ing
u henium de i a i es ha could be ac i a ed using ex e nal
s imuli, owing o he ensuing possibili ies o in oducing
empo al con ol on he ac i i y.Conside ing ha he
p eca alys Ru2 can be made om RuII a ene sandwich
complexes o ype [Cp*Ru(a ene)]X by UV i adia ion in
ace oni ile,[15,24] we an icipa ed ha he ca aly ic species
esul ing om mixing Ru2 wi h wa e could be equally
gene a ed om hese RuII sandwich complexes,p o ided ha
he a ene ligand could be easily eleased wi h ligh unde
aqueous condi ions.This idea was a ac i e no only as
ameans o con ol eac i i y,bu also because o he high
s abili y o he u henium(II) sandwich p ecu so s,which
migh be especially use ul o a oid i s deac i a ion in
biological con ex s.
We he e o e syn he ized he naph halene de i a i e
[Cp*Ru(naph halene)]BPh4(Ru3), and wo analogs wi h
py ene ligands:[Cp*Ru(py ene)]PF6(Ru4)and [Cp*Ru(py -
ene-SO3Na)]PF6(Ru5).[24] Thei po en ial as pho oac i a able
ca alys s o he RuA ACwas es ed in amodel eac ion using
hioalkyne 2a and he p- olyl azide 1b (Table 3).[25] No
su p isingly, ea men o 1b and 2awi h 5mol%o Ru3,in
a9:1 wa e :CH3CN mix u e,did only p o ide aces o he
iazole p oduc (<3% yield, Table 3, en y 1).[23] Howe e ,
we we e glad o obse e ha when his mix u e was i adia ed
wi h a365 nm LED lamp o 10 min, he cycloaddi ion
Scheme 1. Scope o he annula ion using he complex Ru2.[a] The
coun e ion is p obably b omide, as in he s a ing ma e ial. [b] Yield o
he eac ion ca ied ou using aH
2O/DMSO (9:1) mix u e is shown in
pa en hesis. [c] Ca ied ou using Ru1 ins ead o Ru2.Fmoc= luo enyl-
me hoxyca bonyl.
Table 2: O hogonali y be ween RuA AC and CuAAC annula ions.[a]
En y Ca alys ( eac ion ime) Yield [%][b]
3ba3bh
1[Cu] (2 h) 078
2[c] [Cu] (2 h); hen [Ru] (2 h)[c] 79 78
3[Ru] (2 h) 79 0
4[c] [Ru] (2 h); hen [Cu] (2 h)[c] 78 95
5[d] [Ru] and [Cu] (2 h) 44 50
[a] Reac ion condi ions: Asolu ion o 2a (2.0 equi ), 2h (2.0 equi ), 1b
(1.0 equi ;0.75 mmolg@1)inDMSO was added o wa e (500 mL,
75 mM), ollowed by he co esponding ca alys :ei he [Ru] (co e-
sponds o Ru2, 5mol%) o [Cu] (co esponds oCuSO4·5H2O, 5mol%,
NaAsc, 10 mol%), in a ial open o ai .[b] Yield de e mined by 1HNMR
spec oscopy using 1,3,5- ime hoxybenzene as an in e nal s anda d.
[c] A e 2h,asecond equi alen o 1b was added, ollowed by he
second ca alys . [d] Bo h [Ru] and [Cu] we e p esen om he beginning.
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p oceeded smoo hly o a o d he desi ed iazole 3bain 90%
yield (en y 2). The ela ed py ene complex Ru4 was sligh ly
mo e e icien , p o iding a99% yield upon pho oac i a ion
(10 min/ 365 nm, en y 4). In absence o ligh he e is no
con e sion (en y 3). Thesul ona e de i a i e Ru5 also wo ks
unde i adia ion, al hough i is less e icien .
Wi h hese esul s in hand, we nex s udied he beha io o
p eca alys s Ru2, Ru4 and Ru5 unde mo e dilu ed condi-
ions.Toca y ou hese assays we used as subs a e he wa e
soluble iphenylphosphonium-con aining azide 1c,because
he phosphonium ag acili a es ahighly p ecise moni o ing
by LC–ESI-MS.Fo compa a i e pu poses,wealso analyzed
he pe o mance o ou i s gene a ion ca alys [Cp*RuCl-
(COD)] (Ru1), which had been poo ly e ec i e a he
mic omola ange.Reac ions we e ca ied ou in wa e , a
o 4h,using di e en concen a ions o he azide 1c(1 mM,
500 mM, 250 mMand 100 mM) and 50 mol%o he u henium
complexes (Figu e 3and Figu e S5). In he case o he
pho oac i a able complexes Ru4 and Ru5, he eac ion
mix u e in wa e , wi hou any cosol en ,[26] was i adia ed
o 15 min wi h a365 nm LED.G a i yingly,ascan be seen in
he Figu e 3, he h ee u henium complexes p o ided
quan i a i e yields o he p oduc when using azide concen-
a ions o 1mMand 500 mM(a e 4h), whe eas he chlo ide
u henium complex Ru1 p o ided only amodes 35%yield
wi h an azide concen a ion o 1mM, o e y poo yields a
highe dilu ions (2–7%yield). The esul s a 250 mMshowed
ha he py ene RuII complex Ru4 is he mos ac i e,
p oducing aquan i a i e yield o 3ca,while he analogue
complex bea ing asul ona e moie y (Ru5)led o amode a e
67%yield. The is(ace oni ile) u henium(II) ca alys (Ru2)
and he pho oac i a able py ene complex Ru4 we e he mos
e ec i e ca alys s when he concen a ion o he azide was
dec eased down o 100 mM(30 and 47%yield, espec i ely).
Theamoun o Ru2 and Ru4 can be dec eased up 15 mol%
wi hou signi ican ly e oding he e iciency(52 and 84%yield
a 250 mM, espec i ely wi h Ru2 and Ru4;see Figu es S6 and
S7).[27]
As indica ed in Scheme 2, he eac ion unde hese dilu ed
condi ions is no limi ed o 1c and 2a;o he azides and
hioalkynes a e also sui able eac an s,con i ming he po en-
ial o he me hodology,ei he using he p eca alys Ru2 o
he ligh -ac i a able p ecu so Ru4 (Scheme 2).
A his s age,wemo ed o mo e demanding,biologically
ele an en i onmen s,using a500 mMconcen a ion o he
azide and 50 mol%o he u henium complexes (Figu e 4).
G a i yingly, he ca ionic complex Ru2 p o ided e y good
esul s ega dless o he biological media used. Thus,yields
abo e 94%we e ob ained in PBS,cell cul u e milieu
(DMEM), bo h wi h and wi hou Fe al Bo ine Se um, and
e en in p esence o HeLa cell lysa es.The pho oac i a able
complex Ru4 also showed an excellen pe o mance unde
Figu e 3. Ca alys pe o mance in he mic omola ange. Reac ions
we e conduc ed in HPLC ials, and yields we e de e mined by UHPLC–
MS using couma in as an in e nal s anda d. Resul sa e he a e age o
h ee di e en eac ions. Reac ion mix u es in he p esence o Ru4 and
Ru5 we e i adia ed o 15 min a 365 nm o ac i a e he ca alys .
Con ols wi hou i adia ion o Ru4 and Ru5 p o ided yields <1%.
No e:The coun e ion in 1c is B @.
Scheme 2. Scope o he eac ion a mic omola concen a ions (1:
250 mM; 2:500 mM). Reac ionswe e conduc ed in HPLC ials open o
ai and yields we e de e mined by UHPLC–MS, using couma in as an
in e nal s anda d. Resul sa e he a e age o h ee di e en eac ions.
Reac ionsusing Ru4 we e i adia ed o 15 min a 365 nm.
Table 3: Viabili y o he cycloaddi ion wi h pho o oac i a ed Cp*Ru
a ene complexes Ru3,Ru4 and Ru5.[a]
En y [Ru] Sol en Con .[%][b] Yield [%][b]
1Ru3 H2O/MeCN (9:1) 33
2Ru3+hnH2O/MeCN (9:1) 90 90
3Ru4[c] H2O/MeCN (9:1) 10
4Ru4+hnH2O/MeCN (9:1) 99 99
5Ru5+hnH2O/MeCN (9:1) 80 70
[a] Condi ions o he eac ion unde i adia ion: 2a (2.0 equi ), 1b
(1.0 equi ), he sol en and [Ru] we e sequen ially added o a ial, which
was closed and i adia ed wi h a365 nm LED lamp o 10 min, and he
mix u e s i ed o 2h.[b] Yield and con e sion de e mined by 1HNMR
spec oscopy using 1,3,5- ime hoxybenzene as an in e nal s anda d.
[c] The eac ion was ca ied ou wi hou i adia ion.
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ligh i adia ion, p o iding quan i a i e yields in PBS as well
as in Hela cell lysa es,and good yields, om 59 o 70%, in cell
cul u e media. Cu iously, Ru5 (wi h ligh ) pe o med wo se
han Ru4 and Ru2.
To be e assess he po en ial o hese ca aly ic sys ems,we
compa ed hei pe o mance wi h ha o Rhodium and
I idium complexes p e iously de eloped o ela ed azide–
alkyne annula ions.[13,14] The e o e,we es ed he eac ion o
2aand 1c in wa e (a 250 mM), as well as in he p esence o
cell cul u e media (a 500 mM), in bo h cases using 50 mol%
o he me al complexes.Reac ions wi h Rh and I complexes
we e ca ied ou using he azide: hioalkyne a io ha had
been iden i ied as op imal o each o hese me al ca alys s
(azide: hioalkyne =1.5:1 o I and 1:2 o Rh).[23] As can be
deduced om Figu e 5, [I (COD)Cl]2a o ded he desi ed
p oduc in wa e in 46%yield, whe eas [Rh(CO2)Cl]2only
p o ided a24% yield o 3ca. Un o una ely, he pe o m-
ances o hese me als d opped d ama ically when used in
biologically complex media such as DMEM o HeLa Cell
lysa es (Figu e 5and Figu e S10). The e o e,ou u henium
complexes a e clea ly supe io in wa e and, especially unde
biologically demanding condi ions.
Apa icula ly appealing applica ion o bioo hogonal
chemis y is ela ed wi h he chemoselec i e modi ica ion o
biopolyme s.The e o e,weanalyzed whe he ou ca alys s
could also be used o bioconjuga ion eac ions wi h pep ides
o nucleic acids.Asindica ed in Scheme 3, he isace oni ile
RuII complex Ru2 is e ec i e in p omo ing he cycloaddi ion
be ween he hioalkyne 2a and an hep apep ide bea ing a6-
azidolysine a he N- e minal posi ion, o yield he co e-
sponding pep ide– iazole in 55%yield [Scheme 3, Eq. (1)].
Mo e impo an ly,when his eac ion was ca ied ou wi h he
pho oac i a able ca alys Ru4,unde 15 min i adia ion a
365 nm, he eac ion p oceeded e en mo e e icien ly,p o id-
ing he desi ed pep ide conjuga e in an excellen 84%yield.
In he case o assDNA, we ound ha oligonucleo ides
con aining an azide-modi ied adenine a i s 5’end [Scheme 3,
Eq. (2)],can be eadily labelled wi h a hioalkyne de i a i e
Figu e 4. Reac i i y o Ru2,Ru4 and Ru5 unde biologically ele an
condi ionsa 500 mM(1c). Reac ionswe e conduc ed in HPLC ials.
Reac ion yields we e measu ed by UHPLC–MS using an in e nal
s anda d (couma in). Reac ionswi h Ru4 and Ru5 we e ca ied ou by
i adia ing he mix u es o 15 min a 365 nm o ac i a e he ca alys .
PBS=phospha e bu e solu ion;DMEM=Dulbecco’s Modi ied Eagle
Medium;DMEM*=DMEM+10% e al bo ine se um+1% an ibio -
ics;DEMEM-HEPES=DMEM wi hou phenol ed and wi h HEPES (4-
(2-hyd oxye hyl)-1-pipe azinee hanesul onic acid);Lysa es=HeLa cell
lysa e 5mgmL@1.
Figu e 5. Compa ison o di e en me al ca alys s in he model eac-
ion. In all examples, 50 mol%o he me al complex was used. I
s ands o [I (COD)Cl]2;Rh s ands o [Rh(CO2)Cl]2.Reac ions we e
pe o med using he op imal azide/ hioalkyne a ios ound o each
me al ca alys (1.5:1 o I and 1:2 o Rh and Ru complexes). Yields
we e de e mined by UHPLC–MS using couma in as an in e nal
s anda d.
Scheme 3. DNA and pep ide labeling using Ru2 and Ru4 ca alys s.
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equipped wi h aRhodamine ag, using ei he Ru2 and Ru4
(wi h i adia ion), o gi e he expec ed p oduc s in 50 and
72%yields, espec i ely.The e o e,again, he pho oac i a -
able ca alys Ru4 is he one p esen ing he bes pe o mance.
As a inal es o he po en ial o ou new ca aly ic sys ems,
we checked he iabili y o he annula ions in cellula
en i onmen s,using he phosphonium azide 1c.The eac ion
could be moni o ed by LC–MS,owing o he sensi i i y
enhancemen o e ed by phosphonium ca ions in mass
spec ome y (Figu e 6).[23] Theexpe imen s we e ca ied
ou by mixing he u henium ca alys (Ru2 o Ru4), he
hioalkyne (2a,800 mM) and azide 1c (100 mM) in DMEM–
HEPES con aining HeLa cells (1 X106cellsmL@1). A e 2h,
he cells we e cen i uged, he supe na an collec ed, and he
cell pelle s ea ed wi h MeOH (80%aq.) o ex ac he
cellula con en . Bo h he ex acellula media and he
me hanol ex ac s we e analyzed by LC–MS.Ei he using
Ru2,o he pho oac i a able complex Ru4 (wi h i adia ion
o 15 min), we we e glad o de ec he expec ed eac ion
p oduc 3ca,bo h in he supe na an as well as in he
me hanolic ex ac .[28] Wo h o no e, he me hanolic ex ac
u ned ou o be pa icula ly ich in iazole p oduc 3ca,
whe eas he supe na an con ained conside able amoun s o
bo h azide (1c)and iazole (3ca). Howe e ,i islikely ha a
leas pa o he p oduc in e nalizes a e being o med.
O e all, bo h, he pho oac i a able complex Ru4 and he
ca ionic Ru2,we e capable o p omo e he annula ion unde
dilu ed condi ions in cellula suspensions.
Conclusion
We ha e disco e ed ha speci ically ailo ed ca ionic
u henium (II) complexes a e highly e ec i e p eca alys s o
pe o m Ru henium Ca alyzed Azide Thioalkyne Cycloaddi-
ions (RuA AC) in aqueous media. These p eca alys s allow
o ca y ou he annula ion unde dilu e condi ions,and also in
complex, bio ele an media.
Mo eo e , he eac ion is ully compa ible and mu ually
o hogonal o he s anda d CuAAC. Impo an ly, he u he-
nium eagen s can be enginee ed as a ene sandwich com-
plexes o wo k as ligh -ac i a able p eca alys s.This p o ides
no only o he empo al con ol o he eac i i y,bu also o
highe e iciencies,likely because o he in insic s abili y o
he me al complexes un il ecei ing he op ical s imulus.We
ha e also demons a ed ha he u henium complexes can be
used o he o hogonal modi ica ion o azide- agged pep ides
and oligonucleo ides,and ha he eac ion can be ca ied ou
in he p esence o cells.
Acknowledgemen s
This esea ch ecei ed inancial suppo om he Spanish
MINECO (SAF2016-76689-R, CTQ2017-84767-P and
PID2019-106184GB-I00, and aFPU p edoc o al Fellowship
o AGG), he Xun a de Galicia (ED431C 2017/19, 2015-
CP082, Cen o Singula de In es igacilndeGalicia acc ed-
i a ion 2019–2022, ED431G 2019/03), he ERDF,and he
ERC (Ad .G an No.340055). TheO eo-Cinqa ne wo k
(CTQ2016-81797-REDC) is also acknowledged. We hank J.
Miguel A ila and R. Menaya o hei assis ance wi h
expe imen s in he p esence o Hela cells.
Con lic o in e es
Theau ho s decla e no con lic o in e es .
Keywo ds: biocompa ible eac ions ·bioo hogonal eac ions ·
click chemis y · u henium · hioalkynes
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Ru2 in wa e allowed he de ec ion o molecula ions co e-
sponding o oxo de i a es,[Cp*Ru(O)]+,[Cp*Ru(O)(MeCN)2]
and [Cp*Ru(O)2(MeCN)2],as well as hyd oxo species [{Cp*Ru-
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Howell, N. F. Ash o d, D. T. Dixon, J. C. Kola, T. A. Alb igh ,
S. K. Kang, O ganome allics 1991,10,1852–1864.
[25] a) Theuse o an h acenyl azide 1b is also possible,bu he
i adia ion mus be pe o medimmedia ely be o e he addi ion
o 1b, oa oid he decomposi ion o he an h acenyl moie y by
he ligh . b) See Re .[16b] o he use o Ru4 in apho o-
igge ed N-allylca bama e clea age.
[26] Con ol expe imen s con i med ha , unde hese dilu e con-
di ions he p esence o ace oni ile as acosol en isde imen al
o he ac i i y o he pho oac i a able ca alys s Ru4 and Ru5,
p obably owing o compe ing coo dina ion o he Ru cen e ,
which slows down he a e o he p ocess.
[27] In all cases,con ol expe imen s wi h pho oac i a able ca alys s
Ru4 and Ru5,wi hou i adia ion, p o ided negligibleyields o
iazole p oduc s.
[28] Con ol expe imen s wi h Ru2 con i med i s low oxici y owa ds
HeLa cells (see he Suppo ing In o ma ion). Howe e , he use
o Ru4 and ligh unde he condi ionso he in i o expe imen s
(365 nm, 15 min) caused se e e damage o he cells.Thus,in i o
applica ions will equi e speci ic op imiza ion, he use o
al e na i e wo-pho oni adia ion se ings o he de elopmen
o isible-ligh -ac i a able Ru ca alys s.
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