Phospho ogenic Dipy ina o-I idium(III) Complexes as
Pho osensi ize s o Pho odynamic The apy
A. P ie o-Cas añeda, A. Lé ida-Viso, E. A ellanal-Zaballa, R. Sola-Llano, J.
Bañuelos, A. R. Aga abei ia, R. Ma ínez-Máñez and M. J. O iz
h ps://doi.o g/10.1016/j.dyepig.2021.109886
Dyes and Pigmen s 197 (2022) 109886
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Dyes and Pigmen s 197 :
(2022) // A icle ID 109886, which has been published in inal o m a h ps://doi.o g/10.1016/
j.dyepig.2021.109886. © 2021 Else ie unde CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/
by-nc-nd/4.0/)
1
Phospho ogenic Dipy ina o-I idium(III) Complexes as Pho osensi ize s o 1
Pho odynamic The apy. 2
A. P ie o-Cas añeda,a A. Lé ida-Viso,b,c E. A ellanal-Zaballa,d R. Sola-Llano,d J. Bañuelos,d* A. R. 3
Aga abei ia,a R. Ma ínez-Máñez,b,c,e, * and M. J. O iza*
4
aDepa amen o de Química O gánica, Facul ad de Ciencias Químicas, Uni e sidad Complu ense de Mad id, Ciudad 5
Uni e si a ia s/n, 28040, Mad id, Spain. 6
bUnidad Mix a de In es igación en Nanomedicina y Senso es, IIS La Fe, Uni e si a Poli ècnica de València, A da de 7
Fe nando Ab il Ma o ell, n.106 46026, Valencia, Spain. 8
cUnidad Mix a UPV-CIPF de In es igación en Mecanismos de En e medades y Nanomedicina, Uni e sidad Poli écnica de 9
Valencia, Cen o de In es igación P íncipe Felipe, Ca e d’Edua do P imo Yú e a 3, 46012, Valencia, Spain. 10
dDepa amen o de Química Física, Uni e sidad del País Vasco-EHU, Apa ado 644, 48080, Bilbao, Spain. 11
eIns i u o In e uni e si a io de In es igación de Reconocimien o Molecula y Desa ollo Tecnológico (IDM), Uni e si a 12
Poli ècnica de València, Uni e si a de València, Camino de Ve a s/n, 46022 Valencia, Spain. 13
CIBER de Bioingenie ía, Bioma e iales y Nanomedicina, Mad id, Spain. 14
15
ABSTRACT:
We ha e designed and syn hesized a amily o I (III) me al complexes coo dina ed
16
wi h wo ciclome alla ed bis-phenylpy idine ligands and an ancilla y dipy ome hene which is
17
unc ionalized wi h a mesi yl g oup (
I (dipy)-1
), an
α
-chlo oace yl es e (
I (dipy)-2
) o a chain
18
con aining an ammonium ca ion (
I (dipy)-3
). The I (III) complexes ea u e a high iple s a e
19
popula ion enabling ed phospho escence and e icien single oxygen gene a ion.
I (dipy)-2
20
and
I (dipy)-3
a e demons a ed o s ain cells in bo h one-pho on and wo-pho on con ocal
21
imaging. Mo eo e ,
I (dipy)-2
and
I (dipy)-3
p oduce ROS in cells upon i adia ion, inducing
22
cell dea h by apop osis. Colocaliza ion s udies in SK-Mel-103 cells show ha
I (dipy)-3
23
pa ially accumula ed in mi ochond ia and induces upon i adia ion a dis up ion in hei
24
mo phology. O e all ou s udies demons a e ha he p epa ed I (III) ac as pho osensi ize s
25
able o kill cells unde i adia ion, being sui able candida es o pho odynamic he apy
26
applica ions.
27
Keywo ds: Dipy ome henes, I idium(III) complexes, pho osensi ize s, single oxygen, pho odynamic 28
he apy 29
30
1. In oduc ion 31
Albei he he apeu ic p ope ies o ligh a e known long ago [1], he concep o
32
pho o he apy was no de eloped un il he end o he 19 h cen u y [2], and i was no un il
33
1960 ha he mode n e a o Pho odynamic The apy (PDT) eme ged [3-5], being necessa y
34
hi y mo e yea s o i s clinical app o al. PDT is a minimally in asi e ea men ha equi es
35
a simul aneous combina ion o i) a pho osensi ize (PS), ii) ligh o a speci ic wa eleng h, and
36
iii) molecula oxygen. These h ee elemen s a e no oxic by hemsel es, bu hei
37
combina ion igge s a oxic e ec based on he gene a ion o eac i e oxygen species (ROS),
38
which p omo e oxida i e cellula damage and des uc ion [6-9]. Ac ually, PDT is a sui able
39
medical ool, al e na i e o classical he apies based on su ge y, chemo he apy o
40
adio he apy, and i has shown e ec i eness o he ea men o age- ela ed macula
41
degene a ion and ac inic ke a osis among o he disseases [6]. Howe e , PDT is especially
42
sui ed o cance ea men , since PDT enables no only he elimina ion o cance ous cells bu
43
also he des uc ion o he ascula u e su ounding umou cells, and he ac i a ion o an
44
immunological esponse [6,9]. Besides, PDT can display a dual selec i i y, i.e., p e e en ial
45
accumula ion o he pho osensi ize in diseased issues, and oxici y ige ed by ligh ha can
46
addi ionally be localized o con ine he damage a ound a selec ed a ge egion, lessening
47
side-e ec s. In addi ion, PDT-based ea men s can be epea ed wi hou inducing esis ance.
48
Howe e , in spi e o ecen ad ances in his ield, pho odynamic he apy needs u he
49
de elopmen o become a i s -choice ea men . Ac ually, PDT is applied in a e y limi ed
50
numbe o cases in clinic. In his scena io, in ensi e esea ch is cu en ly ongoing o imp o e
51
PDT and o enhance i s applica ion. In his ega d, one o he challenges is he design o new
52
PSs wi h imp o ed capabili ies [7-10].
53
3
One o he s a egies o he syn hesis o e icien PSs consis s in he heigh ening o he
54
popula ion o hei iple exci ed s a e by means o hea y-a om induced in e sys em
55
c ossing. In his app oach, halogens (b omine o iodine) o ansi ion me als a e combined
56
wi h sui able ch omopho ic co es boos ing he gene a ion o cy o oxic ROS (like single
57
oxygen) media ed by he key iple mani old [10,11]. Among ansi ion-me al complexes,
58
hose based on i idium(III) ha e a ac ed a en ion as he apeu ically ac i e compounds,
59
hence eme ging as p omising al e na i es o design PSs. Ce ain I (III) complexes a e ine ,
60
biocompa ible, s able, non- oxic, ea u e la ge S okes shi s, display long-li ed ed
61
phospho escence, ha e ou s anding colo - uning capabili y, good esis ance o
62
pho obleaching, and can in e ac wi h speci ic cellula a ge s [11-12]. In addi ion I (III)
63
complexes can exhibi s ong wo-pho on phospho escence and ha e been applied
64
success ully in biosensing and bioimaging [12,13].
65
Ano he no ewo hy ad an age o i idium(III) complexes is he chemical e sa ili y o hei
66
molecula s uc u e. The oc ahed al coo dina ion geome y o e s a la ge numbe o
67
possibili ies, allowing o eadily modula e hei pho ophysical p ope ies by means o he
68
linkage o sui able ligands. Thus, homolec ic and he e olep ic I (III) complexes wi h ligands
69
based on phenylpy idines, dipy idines o phenan h olines ha e been widely es ed and
70
applied in di e en a eas, such as dye-sensi ized sola cells, o ganic ligh -emi ing diodes
71
(OLED), pho osensi ize s and/o bio-imaging [14-26].
72
Dipy in (o dipy ome hene) is an a ac i e complexa ing ligand owing o hei easy p e-
73
o pos - unc ionaliza ion [27]. Indeed, dipy in has been in ensi ely exploi ed in he
74
cons uc ion o sup amolecula o coo dina ion polyme ic a chi ec u es [28] o a numbe o
75
applica ions [29-31]. Ne e heless, i is no ewo hy ha he e a e ew s udies desc ibing
76
he e olep ic sys ems o med by phenylpy idine and ancilla y dipy ome hene ligands [32-34]
77
and, o he bes o ou knowledge, hi he o he e is only one epo in he li e a u e dealing
78
wi h hei applica ion as PDT agen s [35].
79
In his scena io, and aking in o accoun ou in e es in he de elopmen o new PSs o
80
PDT [21,36-38], he ein we epo a new s a egy o access h ee new he e olep ic dipy ina o-
81
i idium(III) complexes (i.e.
I (dipy)-1
,
I (dipy)-2
and
I (dipy)
-
3
) h ough a s aigh o wa d and
82
cos -e ec i e syn he ic p o ocol (Figu e 1). The pho onic pe o mance o hese me al
83
complexes is s udied paying special a en ion o cha ac e ize hei phospho escence emission
84
(de ec able a oom empe a u e and in ae a ed solu ions) and hei abili y o gene a e
85
single oxygen. The in i o pho ocy o oxic e ec o he i idium complexes unde i adia ion
86
is in es iga ed in he human melanoma cance cell line SK-Mel-103.
I (dipy)-2
and
I (dipy)-3
87
unde isible-ligh i adia ion (λ > 475 nm) esul in high ROS p oduc ion leading o apop o ic
88
cell dea h a low concen a ion. Mo eo e ,
I (dipy)-2
and
I (dipy)-3
also demons a e wo-
89
pho on abso p ion unde 900 nm i adia ion. S udies o colocaliza ion in o ganelles o
90
I (dipy)-2
and
I (dipy)-3
and cell apop osis/nec osis assays by low cy ome y a e also ca ied
91
ou .
92
O
OCl
ON
R2R2
R
1
N
N
F
F
F
F
I
N
N
I (dipy)-2: R
1
=
I (dipy)-3: R
1
=
I (dipy)-1: R1 = R2 = Me
; R2 = H
; R2 = H
I
93
Fig. 1.
No el PSs based on dipy ina e-i idium(III) complexes.
94
95
2. Expe imen al sec ion
96
2.1. Gene al me hods
97
5
Anhyd ous sol en s we e p epa ed by dis illa ion o e s anda d d ying agen s acco ding o
98
common me hods. All o he sol en s we e o HPLC g ade and we e used as p o ided. S a ing
99
chemical subs a es and eagen s we e used as comme cially p o ided unless o he wise
100
indica ed. Flash ch oma og aphy was pe o med using silica gel (230-400 mesh). NMR spec a
101
we e eco ded using CDCl
3
a 20
o
C.
1
H NMR and
13
C NMR chemical shi s (
δ
) we e e e enced
102
o in e nal sol en CDCl
3
(
δ
= 7.260 and 77.03 ppm, espec i ely) o ace one-d6. DEPT 135
103
expe imen s we e used o de e mine he ype o ca bon nucleus (C s. CH s. CH
2
s. CH
3
).
104
FTIR spec a we e ob ained om nea samples using he ATR echnique. High- esolu ion mass
105
spec ome y (HRMS) was pe o med using MALDI-TOF.
106
2.2. Syn hesis and cha ac e iza ion
107
2,4,6-T ime hylbenzaldehyde (
1a
) and 4-hyd oxybenzaldehyde (
1b
) we e pu chased om
108
Sigma-Ald ich. In e media es dipy ome hanes
2a
[39] and
2b
[40], dipy ome henes
3a
[41]
109
and
3b
[40], and complex
[I (d ppy)
2
Cl]
2
[42] we e syn hesized by he co esponding
110
desc ibed me hods.
111
2.2.1. Gene al p ocedu e o he syn hesis o dipy ina o-i idium(III) complexes
112
To a solu ion o complex
[I (d ppy)
2
Cl]
2
(1 equi ) and dipy ome hene (2 equi ) in CH
3
CN
113
(5 mL), sodium ace a e ihyd a e (3 equi ) was added unde an ine a gon a mosphe e, and
114
he eac ion was e luxed un il he comple e disappea ance o he s a ing ma e ial. The
115
eac ion mix u e was hen cooled o oom empe a u e ( ) and he sol en was e apo a ed
116
unde acuum. The esidue was pu i ied by column ch oma og aphy on silica gel.
117
2.2.2. Syn hesis o I (dipy)-1 118
Acco ding o he gene al p ocedu e, [I (d ppy)2Cl]2 (50 mg, 0.04 mmol), dipy ome hene 3a (21.6 119
mg, 0.08 mmol) and sodium ace a e ihyd a e (16.8 mg, 0.12 mmol) in CH3CN (5 mL) we e e luxed 120
o 18 h. Flash ch oma og aphy using hexane/CH2Cl2 (90:10) a o ded I (dipy)-1 (28 mg, 41%) as an 121
o ange solid. 1H NMR (700 MHz, CDCl3)
δ
8.24 (d, J = 8.4 Hz, 2H, 2CH), 7.87 (dd, J = 5.6 and 2.1 Hz, 2H, 122
2CH), 7.67 ( d, J = 8.4 and 2.1 Hz, 2H, 2CH), 6.92-6.90 (m, 4H, 4CH), 6.72 (s, 2H, 2CH), 6.44 (ddd, J = 123
12.6, 9.1 and 2.8 Hz, 2H, 2CH), 6.40 (dd, J = 4.2 and 1.4 Hz, 2H, 2CH), 6.20 (dd, J = 4.2 and 1.4 Hz, 2H, 124
2CH), 5.83 (dd, J = 8.4 and 2.1 Hz, 2H, 2CH), 2.35 (s, 3H, CH3), 2.02 (s, 6H, 2CH3) ppm. 13C NMR (176 125
MHz, CDCl3)
δ
165.4 (d, JCF = 7.0 Hz, C), 163.5 (dd, JCF = 255.2 and 12.0 Hz, CF), 161.4 (dd, JCF = 260.0 126
and 12.3 Hz, CF), 161.0 (d, JCF = 4.9 Hz, C), 151.5 (CH), 149.6 (CH), 147.7 (C), 137.1 (CH), 137.0 (C), 127
136.1 (C), 135.8 (C), 133.4 (C), 130.1 (CH), 128.3 (C), 127.5 (CH), 123.0 (CH), 122.8 (CH), 121.9 (CH), 128
117.4 (CH), 114.0 (d, JCF = 16.0 Hz, CH), 97.3 ( , JCF = 26.8 Hz, CH), 21.1 (CH3), 19.7 (CH3) ppm. FTIR
ν
129
2923, 2854, 1601, 1547, 1476, 1403, 1376, 1344, 1291, 1248, 1162, 1106, 1026, 987, 831 cm-1. HRMS-130
MALDI-TOF m/z 834.1946 (calcd. o C40H29F4I N4: 834.1958). 131
2.2.3. Syn hesis o complex 4 132
Acco ding o he gene al p ocedu e, [I (d ppy)2Cl]2 (50 mg, 0.04 mmol), dipy ome hene 3b (19.4 133
mg, 0.08 mmol) and sodium ace a e ihyd a e (16.8 mg, 0.12 mmol) in CH3CN (5 mL) was e luxed 134
o 3 h. Flash ch oma og aphy using CH2Cl2/CH3OH (97:3) a o ded 4 (16 mg, 24%) as a ed solid. 1H 135
NMR (700 MHz, CDCl3)
δ
8.23 (d, J = 8.4 Hz, 2H, 2CH), 7.77 (d, J = 5.6 Hz, 2H, 2CH), 7.67 ( d, J = 7.7 136
and 0.7 Hz, 2H, 2CH), 7.31 (d, J = 8.4 Hz, 2H, 2CH), 6.94 ( d, J = 6.7 and 0.7 Hz, 2H, 2CH), 6.86 (d, J = 137
8.4 Hz, 2H, 2CH), 6.78 (s, 2H, 2CH), 6.57 (dd, J = 4.4 and 0.7 Hz, 2H, 2CH), 6.43 (ddd, J = 9.8, 9.1 and 138
2.1 Hz, 2H, 2CH), 6.26 (dd, J = 4.2 and 1.4 Hz, 2H, 2CH), 5.78 (dd, J = 9.1 and 2.1 Hz, 2H, 2CH), 4.96 (s, 139
1H, OH) ppm. 13C NMR (176 MHz, CDCl3)
δ
165.3 (d, JCF = 7.0 Hz, C), 163.5 (dd, JCF = 255.6 and 12.0 140
Hz, CF), 161.3 (dd, JCF = 260.1 and 12.0 Hz, CF), 160.1 (d, JCF = 5.8 Hz, C), 155.6 (C), 151.9 (CH), 149.6 141
(CH), 148.5 (C), 137.1 (CH), 134.4 (C), 132.0 (CH), 131.6 (CH), 128.3 (C), 122.9 (CH), 122.8 (CH), 122.1 142
(CH), 117.2 (CH), 114.0 (CH), 113.9 (d, JCF = 16.7 Hz, CH), 97.3 ( , JCF = 26.4 Hz, CH) ppm. FTIR
ν
3358, 143
7
2925, 2852, 1601, 1543, 1476, 1405, 1378, 1344, 1290, 1246, 1200, 1106, 1031, 989, 819 cm-1. HRMS-144
MALDI-TOF m/z 808.1428 (calcd. o C37H23F4I N4O: 808.1437). 145
2.2.4. Syn hesis o I (dipy)-2 146
To a solu ion o 4 (27 mg, 0.03 mmol) and E 3N (5 d ops) in CH2Cl2 (5 mL), chlo oace yl chlo ide 147
(0.004 mL, 0.05 mmol) was added unde ine a mosphe e o a gon, and he eac ion mix u e was 148
s i ed a 50 oC o 2 h. The eac ion was hen cooled o and he sol en was e apo a ed unde 149
acuum. The esidue was pu i ied by column ch oma og aphy on silica gel (hexane/CH2Cl2, 30:70) o 150
a o d I (dipy)-2 (21 mg, 71%) as an o ange- ed solid. 1H NMR (700 MHz, CDCl3)
δ
8.24 (d, J = 9.1 Hz, 151
2H, 2CH), 7.79 (d, J = 5.6 Hz, 2H, 2CH), 7.68 ( d, J = 8.4 and 0.7 Hz, 2H, 2CH), 7.46 (d, J = 8.4 Hz, 2H, 152
2CH), 7.20 (d, J = 8.4 Hz, 2H, 2CH), 6.94 ( d, J = 7.7 and 0.7 Hz, 2H, 2CH), 6.80 (s, 2H, 2CH), 6.52 (d, J = 153
4.2 Hz, 2H, 2CH), 6.46-6.44 (m, 2H, 2CH), 6.26 (dd, J = 4.2 and 0.7 Hz, 2H, 2CH), 5.78 (dd, J = 8.4 and 154
2.1 Hz, 2H, 2CH), 4.35 (s, 2H, CH2) ppm. 13C NMR (176 MHz, CDCl3)
δ
165.7 (COO), 165.3 (d, JCF = 7.0 155
Hz, C), 163.5 (dd, JCF = 255.9 and 12.1 Hz, CF), 161.3 (dd, JCF = 259.4 and 12.5 Hz, CF), 160.6 (d, JCF = 156
6.2 Hz, C), 152.4 (CH), 150.2 (C), 149.5 (CH), 147.2 (C), 137.4 (C), 137.2 (CH), 134.0 (C), 131.6 (CH), 157
128.3 (C), 123.0 (CH), 122.8 (CH), 122.2 (CH), 119.9 (CH), 117.6 (CH), 114.0 (d, JCF = 15.8 Hz, CH), 97.4 158
( , JCF = 26.9 Hz, CH), 40.9 (CH2) ppm. FTIR
ν
2925, 2855, 1778, 1601, 1545, 1474, 1404, 1378, 1344, 159
1286, 1247, 1201, 1165, 1138, 1105, 1030, 989, 818 cm-1. HRMS-MALDI-TOF m/z 808.1138 (calcd. o 160
C39H24ClF4I N4O2: 884.1153). 161
2.2.5. Syn hesis o I (dipy)-3 162
A solu ion o 4 (16 mg, 0.02 mmol) and K2CO3 (8.2 mg, 0.06 mmol) in ace one (5 mL) was e luxed 163
unde ine a mosphe e o a gon o 2 h. The eac ion was cooled o , and hen 2-chlo o-N,N-164
dime hyle hanamine hyd ochlo ide (3.4 mg, 0.024 mmol) was added. The eac ion mix u e was 165
e luxed o 16 h, cooled o and il e ed o emo e ino ganic sal s. The sol en was e apo a ed 166
unde acuum and he esidue was pu i ied by column ch oma og aphy on silica gel (CH2Cl2/CH3OH, 167
98:2) o a o d complex 5 (13 mg, 73%) as an o ange- ed solid. 1H NMR (300 MHz, CDCl3)
δ
8.23 (d, J 168
= 8.4 Hz, 2H, 2CH), 7.77 (dd, J = 6.0 and 0.3 Hz, 2H, 2CH), 7.70-7.64 (m, 2H, 2CH), 7.35 (d, J = 8.7 Hz, 169
2H, 2CH), 6.97-6.93 (m, 4H, 4CH), 6.78 ( , J = 1.5 Hz, 2H, 2CH), 6.56 (dd, J = 4.2 and 1.5 Hz, 2H, 2CH), 170
6.47-6.39 (m, 2H, 2CH), 6.26 (dd, J = 4.2 and 1.5 Hz, 2H, 2CH), 5.77 (dd, J = 8.4 and 2.4 Hz, 2H, 2CH), 171
4.19 ( , J = 5.7 Hz, 2H, CH2O), 2.89 ( , J = 5.4 Hz, 2H, CH2N), 2.49 (s, 6H, 2CH3) ppm. 172
A solu ion o 5 (12.7 mg, 0.014 mmol) and iodome hane (1.0 mL) in CHCl3 (1.5 mL) was s i ed 173
unde ine a mosphe e o a gon a o 1 h. E 2O was added o p ecipi a ion. The p ecipi a e was 174
il e ed, hen washed wi h E 2O and d ied unde acuum o a o d I (dipy)-3 (10 mg, 65%) as an 175
o ange- ed solid. 1H NMR (700 MHz, CDCl3)
δ
8.31 (d, J = 8.4 Hz, 2H, 2CH), 7.97-7.93 (m, 4H, 4CH), 176
7.42 (d, J = 8.4 Hz, 2H, 2CH), 7.25 ( d, J = 6.3 and 1.4 Hz, 2H, 2CH), 7.16 (d, J = 8.4 Hz, 2H, 2CH), 6.80 177
( , J = 1.4 Hz, 2H, 2CH), 6.60 ( d, J = 9.1 and 2.1 Hz, 2H, 2CH), 6.50 (dd, J = 4.9 and 1.4 Hz, 2H, 2CH), 178
6.29 (dd, J = 4.9 and 1.4 Hz, 2H, 2CH), 5.81 (dd, J = 8.4 Hz and 2.1 Hz, 2H, 2CH), 4.79-4.78 (m, 2H, 179
CH2O), 4.24 ( , J = 4.9 Hz, 2H, CH2N), 3.61 (s, 9H, 3CH3) ppm. 13C NMR (176 MHz, CDCl3)
δ
164.8 (d, JCF 180
= 6.7 Hz, C), 163.4 (dd, JCF = 255.2 and 12.1 Hz, CF), 161.4 (d, JCF = 5.6 Hz, C), 161.3 (dd, JCF = 258.7 and 181
12.8 Hz, CF), 157.9 (C), 151.5 (CH), 149.8 (CH), 148.5 (C), 138.2 (CH), 134.3 (C), 132.6 (C), 131.8 (CH), 182
131.5 (CH), 128.5 (C), 123.0 (CH), 122.9 (CH), 122.8 (CH), 117.4 (CH), 113.6 (d, JCF = 15.8 Hz, CH), 113.4 183
(CH), 97.1 ( , JCF = 26.9 Hz, CH), 65.3 (CH2N), 62.3 (CH2O), 54.0 (CH3) ppm. FTIR
ν
1603, 1542, 1477, 184
1404, 1378, 1345, 1247, 1201, 1030, 989 cm-1. HRMS-MALDI-TOF m/z 894.2403 (calcd. o 185
C42H35F4I N5O+: 894.2401). 186
2.3. Pho ophysical p ope ies and single oxygen gene a ion
187
The pho ophysical p ope ies we e egis e ed using qua z cu e es wi h op ical pa hways
188
o 1 cm in dilu ed solu ions (a ound 2·10
-6
M), p epa ed by adding he co esponding sol en
189
15
dipy ome hene de i a i es) o comple e he I (III) me al coo dina ion sphe e (Fig. 1). The
328
elec onega i i y o he luo ine a oms u he s abilizes he chela ion o he me allic ca ion
329
by phenylpy idine. This elec ion o luo ina ed ligands is backed up by ou own p e ious
330
expe ience [21], as well as by he esul s epo ed by o he esea ch g oups, which suppo
331
he iabili y o i idium-(III) complexes wi h lu ophenylpy idine based ligands as sui able
332
agen s o PDT and pho o he agnosis [46-48]. On he o he hand, dipy ome hene is a key
333
chela ing ligand in ou design since di e en unc ional g oups can be appended a i s meso
334
posi ion o enhance he pho onic pe o mance o he me al complex and/o guide he
335
complex owa ds a ge cells o o ganelles [27,29,35]. Acco dingly, we designed he I (III)
336
complexes
I (dipy)-1
,
I (dipy)2
and
I (dipy)-3
(Fig.1) con aining a mesi yl g oup a he meso-
337
posi ion o he dipy ina o ligand (
I (dipy)-1
), an
α-chlo oace yl es e (
I (dipy)-2
) o a chain
338
con aining an
ammonium ca ion (
I (dipy)-3
). The
mesi yl g oup has been inco po a ed o
339
enhance he pho os abili y
[49,50], he α-chlo oace yl goup has been epo ed o eac wi h hiol 340
g oups on p o eins [51], whe eas ammonium de i a i es can accumula e in mi ochond ia h ough 341
elec os a ic in e ac ion wi h he nega i e mi ochond ial memb ane [52].
342
3.2. Syn hesis
343
The syn he ic ou es ollowed o p epa e he dipy ome hene-i idium(III) complexes a e
344
ou lined in Scheme 1. Following a usual syn he ic p ocedu e o dipy ome henes,
aldehydes
345
1a
o
1b
and py ole we e eac ed in acid medium o ob ain dipy ome hanes
2a
[39] and
2b
346
[40], which we e u he oxidized wi h DDQ o dipy ome henes
3a
[41] and
3b
[40]. By a
347
complexa ion eac ion ollowing a ep esen a i e syn he ic p ocedu e [33] be ween
3a
o
3b
348
and
[I (d ppy)
2
Cl]
2
[42], he complex
I (dipy)-1
and he in e media e complex
4
we e
349
ob ained, espec i ely, in 41 and 24% yield. F om his poin , he binding o
4
wi h chlo oace yl
350
chlo ide in he p esence o a base (E
3
N) a o ded
I (dipy)-2
in 71% yield, while
I (dipy)-3
was
351
ob ained in wo-s eps om
4
ha i s was eac ed wi h 2-chlo o-N,N-dime hyle hanamine in
352
a basic medium, o ob ain
5
(73%) ha was hen ea ed wi h an excess iodome hane esul ing
353
in
I (dipy)-3
in 65% yield. All he complexes we e ully cha ec e ized by
1
H and
13
C NMR
354
spec oscopy (see ESI), FTIR and HRMS-MALDI-TOF.
355
R1
H
O
+N
H
NH HN
R1
N HN
R1
i ii
3a
[I (d ppy)2Cl]2
iii
R2R2
1a, 2a, 3a: R1 = R2 = Me
1b, 2b, 3b: R1 = OH; R2 = H
1 2 3
R2R2R2R2
i
N
N
I
F
F
F
F
Cl
Cl
N
N
I
F
F
F F
N
O
N N
F
F
F
F
I
N
N
OH
N N
F
F
F
F
I
N
N
iii
3b
I (dipy)-1
4
I (dipy)-2 I (dipy)-3
5
i
356
Scheme 1.
Syn hesis o
I (dipy)-1
,
I (dipy)-2
and
I (dipy)-3
. Reac ion condi ions: i)
1a
, H
2
O/HCl (98.5:1.5), , 12
357
h;
1b
, TFA, , 12 h. ii) DDQ, CH
2
Cl
2
, , 30 min. iii) sodium ace a e ihyd a e, CH
3
CN, e lux, 3-18 h. i ) α-
358
chlo oace yl chlo ide, E
3
N, CH
2
Cl
2
, e lux, 2 h. ) 2-chlo o-N,N-dime hyle hanamine, K
2
CO
3
, ace one, e lux, 18
359
h. i) iodome hane, CHCl
3
, , 1 h.
360
361
3.3. Pho ophysical P ope ies
362
The spec oscopic signa u es o he h ee I (III)-based o ganome allic complexes a e e y
363
simila , being almos independen o he pa a- unc ionaliza ion. Thus, he abso p ion p o ile
364
o
I (dipy)-1
,
I (dipy)-2
and
I (dipy)-3
ea u ed a band a ca. 475-485 nm wi h mola
365
abso p ions up o 30000 M
-1
cm
-1
(Fig. 2). The p o ile and posi ion o he abso p ion bands
366
esemble hose eco ded o he isola ed dipy ome hene co e. Besides,
I (dipy)-1
,
I (dipy)-2
367
17
and
I (dipy)-3
shows a low nega i e sol a och omism; a adema k o dipy ome hene
368
delocalized π-sys ems (Fig. S1). Indeed, heo e ical simula ions p edic ed ha he elec onic
369
densi y o he molecula o bi als in ol ed in such elec onic ansi ions is exclusi ely loca ed
370
a he dipy ome hene amewo k (Fig. 2 and S2). Op imized geome ies esul ed in an
371
expec ed i idium-cen e ed oc ahed al con o ma ion, whe e he dipy ome hene ligand
372
adop s a plana geome y owing o he igid coo dina ion a o ded by he i idium chela ion
373
o he py olic ni ogens. The meso-a yl moie y is wis ed a ound 70
o
wi h ega d o he
374
dipy ome hene plane, eaching an o hogonal a angemen o he s e ically hinde ed
375
mesi yl in
I (dipy)-1
. The luo ina ed phenylpy idine ligands a he i idium cen e a e axially
376
disposed a away om each o he and do no ha e con ac wi h he dipy ome hene.
377
The pho oluminescence spec a o
I (dipy)-1
,
I (dipy)-2
and
I (dipy)-3
displayed a esidual
378
and almos negligible sho wa eleng h emission (530-550 nm), ollowed by a b oad and
379
dominan long-wa eleng h emission (670-675 nm), which endows a la ge S okes shi (6000
380
cm
-1
) and alls wi hin he biological window (Fig. 2 and S1). The i s emission likely owes o
381
he s ongly quenched luo escence emission om he locally exci ed s a e o he
382
dipy ome hene g oup, while he second one is iden i ied as a phospho escence signal. The
383
low luo escence om hese compounds can be a ionalized by he enhanced in e sys em
384
c ossing induced by he hea y a om e ec p omo ed by he i idium chela ing he ancilla y
385
dipy ome hene, and mainly by he abili y o hese kind o cyclome ala ed compounds o
386
induce me al-ligand cha ge ans e (MLCT) and ligand-ligand cha ge ans e (LLCT)
387
p ocesses. Conside ing such CT phenomena, he second and main long-wa eleng h emission
388
could be en a i ely assigned o hei own emission a a i s sigh . Howe e , he eco ded
389
emission is almos insensi i e o he sol en pola i y. No e ha he wa eleng h and
390
luo escence in ensi y o CT-like emissions a e expec ed o ma kedly depend on he
391
en i onmen , owing o he gene a ed cha ge sepa a ion in such s a e, and his e ec is no
392
obse ed o
I (dipy)-1
,
I (dipy)-2
no
I (dipy)-3
(see pho oluminescence spec a in Fig. S1).
393
The li e ime associa ed o such long-wa eleng h emission was e y long (hund eds o
394
nanoseconds, Table 1). Mo eo e , bo h he emission in ensi y and he li e ime (up o ens o
395
mic oseconds, Table 1) g ea ly enhanced in deae a ed (oxygen- ee) solu ions (Fig. S3 and Fig.
396
S4, epec i ely) and when cooling he samples down o 77 K (Fig. S5 and Fig. 2, espec i ely).
397
Indeed, he ime esol ed emission spec a unde an a gon a mosphe e eco ded o
I (dipy)-
398
1
, display such long-li ed emission de ec able e en a e delay imes o ens o mic oseconds
399
(Fig. S6). In suma y, all hese indings suppo ha he long-li ed iple s a e is in ol ed in
400
he eco ded emission and hence pinpoin o phospho escence a he han luo escence as
401
he sou ce o he ed-emission (Fig. 2). A quick su ey o he bibliog aphy e eals ha ela ed
402
cyclome ala ed i idium complexes a e able o display phospho escence e en unde ad e se
403
condi ions, such as oom empe a u e and ae a ed solu ions [22,29]. Indeed, he emissi e
404
iple s a es popula ed om hose MLCT and LLCT a e sugges ed as he sou ce o such weak
405
(e iciency lowe han 0.3% in ae a ed solu ions, inc easing up o 8% upon emo al o oxygen,
406
Table 1) ed emissions [53]. In o he wo ds, i hese compounds a e able o s ain cells ( ide
407
in a), he acking would be isualized hanks o a long-li ed ed phospho escence.
408
Table 1.
Pho ophysical p ope ies o he cyclome ala ed i idium-based o ganome allic complexes in dilu ed
409
ae a ed solu ion o wo ep esen a i e sol en s a oom empe a u e; chlo o o m (CHCl
3
) and ace oni ile (ACN).
410
See able S1 o pho ophysical da a in mo e sol en s. The pho oluminescence quan um yield and li e ime in
411
a gon-pu ged solu ions o chlo o o m a e also added in i alics.
412
413
λ
ab
(nm)
ε
max
(10
4
M
-1
cm
-1
)
λ
pl
(nm)
∆ν
S
(cm
-1
)
φ
τ
(µs)
φ
∆
I (dipy)-1
CHCl
3
484.0
3.4
675.0
5840
0.0025
0.085
0.475
22.3
0.92
ACN
480.0
3.1
676.0
6050
0.0014
0.327
0.92
I (dipy)-2
CHCl
3
482.5
3.8
675.0
5950
0.0023
0.070
0.375
10.4
0.75
ACN
478.5
3.3
668.0
5970
0.0015
0.215
1.00
19
I (dipy)-3
CHCl
3
483.0
2.8
676.0
5920
0.0025
0.045
0.376
15.3
0.84
ACN
479.0
2.6
673.0
6120
0.0013
0.286
0.94
414
Abso p ion (
λ
ab
) and pho oluminescence (
λ
pl
) wa eleng h, mola abso p ion (
ε
max
), S okes shi (
∆ν
S
),
415
pho oluminescence quan um yield (
φ
) and li e ime (
τ
), single oxygen gene a ion quan um yield (
φ
∆
).
416
417
418
Fig. 2.
Abso p ion and pho oluminescence spec a o he cyclome ala ed i idium-based complexes in dilu ed
419
solu ions o chlo o o m. The heo e ically simula ed (wb97xd/lanl2dz) op imized g ound s a e geome y and he
420
co esponding molecula o bi als in ol ed in he main isible abso p ion ansi ion a e also depic ed o he
421
ep esen a i e o ganome allic compound
I (dipy)-2
. See Fig. S1 o spec a in mo e sol en s and Fig. S2 o he
422
op imized geome ies and molecula o bi als o he es o o ganome allic compounds. Inse :
423
Pho oluminescence decay cu e o ep esen a i e
I (dipy)-1
a oom empe a u e and liquid ni ogen cooled
424
in e hanol.
425
426
This high iple s a e popula ion, allowing e en he eco ding o phospho escence unde
427
oom condi ions, pa es he way o an e icien single oxygen gene a ion. Indeed, all
428
compounds display a s ong phospho escence emission om single oxygen in he nea
429
in a ed ega dless o he sol en (Table 1 and S1). Thus, he h ee compounds in all he es ed
430
media yield high e iciencies o single oxygen gene a ion (highe han 70%), which each
431
100% in many o hem (Table 1 and S1). The e o e, hese i idium complexes a e expec ed o
432
beha e as op imal pho osensi ize s o kill e icien ly cells unde ae obic condi ions ( ide in a).
433
Fu he mo e, he la gely ed-shi ed emission o he compounds wi h espec o hei ligh -
434
abso p ion egion (S okes shi a ound 6000 cm
-1
, Table 1) would allow sensing and
435
bioimaging wi h s ongly educed backg ound in e e ences ( ide in a).
436
437
3.4.
In i o PDT expe imen s
438
PDT wi h complexes
I (dipy)-1
,
I (dipy)-2
and
I (dipy)-3
we e in es iga ed agains he
439
human melanoma cell line SK-Mel-103 using he WST-1 assay. SK-Mel-103 cells we e ea ed
440
wi h inc easing doses o he i idium complexes o 24 h and hen i adia ed o 30 min wi h a
441
36W LED sou ce a 10 cm o dis ance h ough a il e ha allowed passage o ligh o
442
wa eleng hs > 475 nm. The ob ained esul s a e depic ed in Figu e 3 ha shows he
443
pho oinduced cy o oxici ies in SK-Mel-103 cance cells ea ed wi h
I (dipy)-2
and
I (dipy)-3
.
444
Mo eo e ,
I (dipy)-1
up o 50 µM was no pho o oxic in he es ed condi ions (Fig. S7) and
445
was no conside ed o u he PDT s udies.
446
447
Fig. 3.
Pho oinduced cy o oxici ies in SK-Mel-103 cance cells wi h i idium(III) complexes,
I (dipy)-2
(A) and
448
I (dipy)-3
(B). T ea men was applied a inc easing concen a ion up o 2 µM o 24 h be o e i adia ion wi h
449
isible-ligh (λ>475) o 30 min. Cell iabili y was de e mined 24 h a e i adia ion s ep and in da k wi h WST-1
450
me hod. Values a e exp essed as mean ± SEM, and s a is ical signi icance was assessed by one-way ANOVA and
451
Tukey's pos - es (n=18 da a poin s). ** <0.010; **** <0.0001 indica e s a is ically signi ican changes.
452
453
21
As shown in Figu e 3, a e 24 h o ea men wi h PS
I (dipy)-2
and
I (dipy)-3
a all in
454
cul u es iabili y in a concen a ion-dependen way was ound. These educ ions in cell
455
iabili y a e in ag eemen wi h he high single oxygen gene a ion quan um yield obse ed
456
o hese i idium complexes (Table 1). In con as
I (dipy)-1
, also displaying a high single
457
oxygen gene a ion, was no able o induce cell dea h upon i adia ion. The lack o
458
pho oci o oxici y induced by
I (dipy)-1
, along wi h he lack o e iciency o s ain cells unde
459
con ocal imaging, sugges ha he p esence o he mesi yl g oup a he meso-posi ion o he
460
dipy ina o ligand p e en s he in e naliza ion o he compound in cells, and he e o e no PDT
461
e ec was obse ed. Se e al easons can accoun o his unexpec ed inding. On he one
462
hand, he s e ical hind ance o he mesi yl leads o a bulkie molecule (see i s o hogonal
463
disposi ion in Fig. S2) ha migh hampe i s in e naliza ion. On he o he hand, he absence
464
o pola /ionic a ge able g oups (as es e o ammonium in
I (dipy)-2
o
-3
, espec i ely) makes
465
I (dipy)-1
mo e hyd ophobic and wi h less p obabili y o in e ac wi h cells, hinde ing he
466
up ake.
467
Table S2 shows EC
50
alues de e mined o SK-Mel-103 cells wi h
I (dipy)-2
and
I (dipy)-
468
3
. EC
50
alues, calcula ed om he sigmoidal i ing o concen a ion-cell iabili y cu es o
469
I (dipy)-2
and
I (dipy)-3
(Fig. S8) we e 0.11 µM and 0.39 µM, espec i ely, indica ing ha
470
compound
I (dipy)-2
is mo e e ec i e killing cells han
I (dipy)-3
unde i adia ion condi ions.
471
Besides, he in insic cy o oxici y o
I (dipy)-2
and
I (dipy)-3
was also e alua ed in he absence
472
o ligh i adia ion (da k con ol).
Al hough
I (dipy)-2
and
I (dipy)-3
show sligh ly da k
473
cy o oxici y,
he pho o oxici y index (PI) (PI = EC50da k/EC50ligh ) o I (dipy)-2 and I (dipy)-3 is 45.45 474
and 23.7, espec i ely, which demons a es hei po en ial applica ion o PDT as hey
e ec i ely kill
475
cells by pho oac i a ion.
476
477
3.5. Cellula up ake and o ganelles accumula ion
478
The in acellula dis ibu ion o PSs has an impo an ole in PDT ou come, as he
479
localiza ion de e mines he mechanism o cell dea h and he cellula esponse o
480
pho odamage [54]. Cellula up ake o compounds
I (dipy)-2
and
I (dipy)-3
in SK-Mel-103 cells
481
we e demons a ed by one-pho on con ocal imaging (Fig. 4, A-D) o which a clea
482
cy oplasmic signal was obse ed a e 24 h incuba ion.
I (dipy)-2 and I (dipy)-3 also displayed 483
ed phospho esce unde wo-pho on exci a ion (TPE) when i adia ed a 900 nm (Fig. 4, E-H). 484
485
Fig. 4.
Cellula up ake o compounds
I (dipy)-2
and
I (dipy)-3
in SK-Mel-103 cells. (i) (A-D) One-pho on con ocal
486
images o SK-Mel-103 in he absence o p esence o 2.5 µM o
I (dipy)-2
(A, B), espec i ely, and in he absence
487
o p esence o 2.5 µM
I (dipy)-3
(C, D), espec i ely. One-pho on images we e acqui ed by using one-pho on
488
con ocal mic oscope (Leica TCS SP8 HyVolu ion II). Exci a ion a 488 nm and emission collec ed a 640-780 nm.
489
(E-H) Two-pho on con ocal images o SK-Mel-103 in he absence o p esence o 2.5 µM o
I (dipy)-2
(E, F),
490
espec i ely, and in he absence o p esence o 2.5 µM
I (dipy)-3
(G, H), espec i ely. Two-pho on images we e
491
acqui ed by using mul ipho on con ocal mic oscope (Olympus FV1000MPE). Exci a ion a 900 nm and emission
492
was collec ed in he spec a o 650-740 nm. (ii) Quan i ica ion o he in eg a ed luo escence in ensi y ela i e
493
o cell numbe o SK-Mel-103 cells in he absence o p esence o
I (dipy)-2
and
I (dipy)-3
wi h (A) one-pho on
494
con ocal imaging and (B) wo-pho on con ocal imaging. Values a e exp essed as mean ± SEM, and s a is ical
495
signi icance was assessed by he wo- ailed S uden ’s - es . * <0.05; *** p <0.001.
496
497
Once demons a ed he in e naliza ion o he I (III) complexes, we s udied mo e in de ail
498
he p e e en ial localiza ion o
I (dipy)-2 and I (dipy)-3
in he cells.
The subcellula localiza ion o 499
23
hese i idium complexes was s udied in
SK-Mel-103 cells
by con ocal mic oscopy using comme cially 500
a ailable luo escen ma ke s o subcellula o ganelles (including nuclei, mi ochond ia, lysosomes 501
and endoplasmic e iculum) ollowing he manu ac u e ecommenda ions. S udies we e pe o med 502
wi h I (dipy)-3
as he esul s ob ained in con ocal o
I (dipy)-2
we e oo weak o pe o m a
503
co ec colocaliza ion assay (Fig. S9).
504
Fig. 5 shows he subcellula colocaliza ion o I (dipy)-3 ( ed) in SK-Mel-103 s ained wi h 505
o ganelle ma ke s (g een). Colocaliza ion analysis we e pe o med on a pixel-by-pixel based analysis. 506
The ed emission o
I (dipy)-3
colocalized wi h he g een luo escence o he acke s o
507
mi ochond ia (Fig. 5B), o lysosome (Fig. 5C), and o a lesse ex en o
endoplasmic e iculum
508
(Fig. 5D). F om hese da a, i can no be concluded a speci ic colocaliza ion wi h any o he
509
o ganelles. Mo eo e , i is no ewo hy ha compa ed o he only Mi o acke con ol (Fig
510
5A), a change in he mo phology o he mi ochond ia a e ea men wi h
I (dipy)-3
was
511
clea ly obse ed. A simila beha io was also ound o
I (dipy)-2
(Fig. S9). This e ec was
512
e ec i e e en a low concen a ions o
I (dipy)-3
, al hough in his case he mi ochond ia was
513
less a ec ed han a high concen a ion o he i idium complex (Fig. S10). All his da a
514
sugges ed ha he e is in ac an accumula ion o
I (dipy)-3
in mi ochond ia which p omo es
515
he des uc ion o hei na i e mo phology in a dose-dependen manne . This is in ag eemen
516
wi h p e iously epo ed da a ha sugges ed ha luo opho es
con aining
a chain-end 517
ammonium ca ion
can ha e p e e ence o a ge mi ochond ia [55]. Ta ge ing mi ochond ia is
518
a desi able p ope y o enhance PDT as his o ganelle is he cell ene gy supplie and a sui able
519
a ge o o e come esis ance o apop osis in an icance he apies [52]. Mo eo e ,
520
mi ochond ia play a cen al ole in malignan umo p og ession, and i s biogenesis is many
521
imes up egula ed in cance s.
522
523
524
Fig. 5.
Subcellula colocaliza ion o
I (dipy)-3
. SK-Mel-103 ollowing 24 h incuba ion wi h
I (dipy)-3
( ed) a 2.5
525
µM and s ained wi h he g een acke s Mi o acke (B), Lyso acke (C), and ER- acke (D). Images we e
526
acqui ed by using one-pho on con ocal mic oscope (Leica TCS SP8 HyVolu ion II).
I (dipy)-3
was exci ed a 488
527
nm and he emission was collec ed a 640-780 nm. G een acke s we e exci ed a 488 and he emission was
528
collec ed a 490-530 nm. The 2D sca e plo diag am ep esen s he deg ee o colocaliza ion. Scale ba : 10 µm.
529
530
3.6. ROS gene a ion in cells and cell dea h mechanism
531
I is well-known ha PDT p ocesses in ol e se e al s eps in which he e is an exci ed
532
iple s a e (T
1
) in he pho osensi ize ha is able o eac wi h molecula oxygen esul ing in
533
he o ma ion o (ROS). This eac ion happens h ough wo mechanisms; one induces he
534
gene a ion o supe oxide anion, hyd ogen pe oxide and hyd oxyl adicals (Type I mechanism),
535
whe eas he o he gene a es single oxygen (
1
O
2
in Type II). I is mainly he gene a ion o
536
31
[53] Langdon-Jones EE, Halle , AJ, Rou ledge JD, C ole DA, Wa d BD, Pla s JA, Pope SJA. Using subs i u ed cyclome ala ed quinoxaline 724
ligands o inely une he luminescence p ope ies o i idium(III) complexes. Ino g Chem 2013;52:448-456. 725
[54] L W, Zhang Z, Zhang KY, Yang H, Liu S, Xu A, Guo S, Zhao Q, Huang W. A Mi ochond ia-Ta ge ed Pho osensi ize Showing 726
Imp o ed Pho odynamic The apy E ec s Unde Hypoxia. Angew Chem In Ed Engl 2016;55: 9947-9951. 727
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