Academic Edi o s: Ge ald An hony
Mu phy and Liudmil An ono
Recei ed: 13 May 2025
Re ised: 24 June 2025
Accep ed: 25 June 2025
Published: 28 June 2025
Ci a ion: Coelho, J.R.A.; Pacheco,
A.R.F.; Domingues, D.C.; Rod igues,
A.R.O.; Temi ope, A.A.; Cou inho,
P.J.G.; Fe nandes, M.J.G.; Cas anhei a,
E.M.S.; Gonçal es, M.S.T. New Ca ion
Senso s Based on Eugenol-De i ed
Azo Dyes. Molecules 2025,30, 2788.
h ps://doi.o g/10.3390/
molecules30132788
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
New Ca ion Senso s Based on Eugenol-De i ed Azo Dyes
José R. A. Coelho 1, Ana Ri a F. Pacheco 2,3, Diogo C. Domingues 1, Ana Ri a O. Rod igues 2,3 ,
Akani A. Temi ope 1, Paulo J. G. Cou inho 2,3 , Ma ia José G. Fe nandes 1, Elisabe e M. S. Cas anhei a 2,3
and M. Samei o T. Gonçal es 1,*
1Chemis y Cen e o he Uni e si y o Minho (CQ-UM), Depa men o Chemis y, Uni e si y o Minho,
Campus de Gual a , 4710-057 B aga, Po ugal; [email p o ec ed] (J.R.A.C.);
[email p o ec ed] (D.C.D.); [email p o ec ed] (A.A.T.);
[email p o ec ed] (M.J.G.F.)
2Physics Cen e o Minho and Po o Uni e si ies (CF-UM-UP), Depa men o Physics, Uni e si y o Minho,
Campus de Gual a , 4710-057 B aga, Po ugal; [email p o ec ed] (A.R.O.R.);
[email p o ec ed] (P.J.G.C.); [email p o ec ed] (E.M.S.C.)
3Associa e Labo a o y LaPMET, Uni e si y o Minho, Campus de Gual a , 4710-057 B aga, Po ugal
*Co espondence: msamei [email p o ec ed]
Abs ac
Eugenol-based azo dyes illus a e how bio-sou ced compounds like eugenol can be ans-
o med h ough syn he ic p ocesses in o unc ional and colo ul compounds. The main
pu pose o he p esen wo k was o de elop new esponsi e colo ime ic senso s o me al
ca ions based on eugenol-de i ed azo compounds. The inco po a ion o he azo g oup
in o he eugenol amewo k allows o s ong elec onic in e ac ions wi h me al ca ions,
leading o dis inc colo changes obse able o he naked eye. These azo-eugenol dyes
exhibi shi s in hei UV-Vis abso p ion spec a upon complexa ion wi h me al ca ions
such as coppe (Cu
2+
) and lead (Pb
2+
), making hem e ec i e senso s o en i onmen al
and analy ical applica ions. The eugenol-based azo dyes we e subjec ed o pho ophys-
ical s udies o unde s and selec i i y, esponse ime, and s abili y in ela ion o me al
ca ions, which will be a s a ing poin o he moni o ing o oxic me al con aminan s in
aqueous en i onmen s.
Keywo ds: eugenol; phenolic compounds; azo dyes; ca ion de ec ion; colo ime ic senso s
1. In oduc ion
Azo dyes, known o hei di e si y o appealing colo s and s abili y, ha e been widely
used in a ious applica ions such as ex iles, ood, and analy ical chemis y, due o hei
ease o syn hesis, s uc u al a iabili y, and unique ch omogenic onali ies o he naked
eye, whe e eds, o anges, and yellows p edomina e [
1
–
6
]. The exac colo a ion o he
espec i e dyes is based on he exis ence o a ch omopho e g oup (-N=N-), associa ed
wi h he na u e and posi ion o he subs i uen s connec ed o he a oma ic sys ems, whe e
he ampli ica ion o he deg ee o conjuga ion o he
π
sys em enhances he abso p ion
o ligh a longe wa eleng hs [
7
,
8
]. These dyes can unde go signi ican colo changes
in esponse o he binding o speci ic ca ions, enabling apid, isual de ec ion o me al
ions [
9
–
11
]. Among hese, azo dyes de i ed om na u al sou ces, like eugenol, may o e a
p omising eco- iendly al e na i e o ull syn he ic dyes, whe e i is possible o combine
he physicochemical and biological p ope ies inhe en in phenolic compounds, known o
ha e signi ican an ioxidan ac i i ies [12,13].
Molecules 2025,30, 2788 h ps://doi.o g/10.3390/molecules30132788
Molecules 2025,30, 2788 2 o 17
Eugenol, he majo seconda y me aboli e ound in clo e oil (Syzygium a oma icum) and
p esen in o he essen ial oils, p o ides a e sa ile basis o he syn hesis o azo dyes due o
i s eac i e unc ional g oups and a oma ic s uc u e. Azo dyes syn hesized om eugenol
a e no only po en ial en i onmen ally benign, bu also possess unique pho ophysical
p ope ies, making hem ideal candida es o colo ime ic sensing applica ions [
14
–
16
].
F om a s uc u al pe spec i e, eugenol is a phenolic compound, meaning ha he exis ence
o a hyd oxyl g oup makes i a sui able ligand o complexing wi h me als. Ano he c ucial
aspec is he ole o he me hoxyl g oup in he o ien a ion o he ligand in ela ion o
he me al cen e , which posi ions he dono a om in he me al’s coo dina ion sphe e [
17
].
As a ligand, eugenol can o m complexes wi h impo an biome als such as Fe
2+
and
Fe
3+
ca ions. The eugenol moie y has been al eady epo ed as a colo ime ic p obe o
i on(III), whe e i displays complexes ha ha e a blue-g een colo (
λmax
= 653 nm) [
18
].
Thus, eugenol-de i ed azo dyes p esen a no el, sus ainable, and e ec i e app oach o
de eloping selec i e and sensi i e colo ime ic senso s o ca ion de ec ion.
This ea u e is pa icula ly aluable in en i onmen al moni o ing, medical diagnos ics,
and indus ial p ocesses, whe e de ec ing oxic o essen ial ca ions wi h high sensi i i y
and selec i i y is c ucial. Me als p esen a dicho omy based on hei dose and hei bio-
logical impo ance [
19
]. Some me als a e essen ial in me abolic p ocesses, whe e excess o
de iciency can lead o se e e damage. O he me als a e eal poisons ha end o bioaccu-
mula e in he en i onmen . Coppe is a biologically ele an elemen ; howe e , p ominen
le els o Cu
2+
can con ibu e o he de elopmen o neu odegene a i e diseases (such as
Alzheime ’s disease, Menkes’ synd ome, o Wilson’s disease), p ion disease, hypoglycemia,
and dyslexia, and also o he p og ession o cance [
20
–
22
]. On he o he hand, lead (Pb
2+
)
is desc ibed as a poison ha can a ec many o gans in he human body, damaging he
b ain, ne ous sys em, blood, diges i e sys em, and also he ep oduc i e sys em, due
o i s abili y o mimic and, in some cases, inhibi he ac ion o calcium as a egula o o
cellula unc ion [23,24].
In ecen yea s, scien i ic esea ch e o s ha e been di ec ed owa d signi ican ad-
ances in he de ec ion o a ious me al ca ions, pa icula ly h ough he de elopmen o
chemosenso s o Cu
2+
and Pb
2+
ions, which ha e ecen ly been desc ibed in de ail [
25
,
26
].
O ganic colo ime ic chemical senso s, such as hiou eas, Schi bases (imines), py idines,
hiophenes, hiazoles, indoles, calixa enes, po phy ins, c own e he s, and BODIPYs, a e
p omising candida es [
27
–
31
]. Fu he mo e, he alo iza ion o na u al p oduc s has been
an eme ging ield in he design o new ligands o he de ec ion o me al ca ions.
Taking hese ac s in o conside a ion, he p esen wo k ocuses on b oadening he
scope o applica ion o eugenol-based azo dyes—including p e iously epo ed s uc u es
de eloped o o he pu poses and applica ions [
14
,
16
]—by e alua ing hei use in he
de ec ion o a ious me al ca ions, wi h p omising esul s in he colo ime ic sensing o
lead(II) and coppe (II) ions.
2. Resul s and Discussion
2.1. Syn hesis o Eugenol Azo Dyes 3a–e
Azo dyes inco po a ing eugenol (4-allyl-2-me hoxyphenol) in o hei s uc u es ha e
been syn hesized ollowing a con en ional me hodology. In his p ocess, eugenol was
coupled wi h diazonium sal s o aniline-based amines o o m he azo compounds.
Aniline 1a, 3-me hoxyaniline 1b, 3-b omoaniline 1c, 4-chlo oaniline 1d, and 3-amino-
2-b omobenzoic acid 1e we e eac ed in an acidic medium wi h sodium ni i e, a low
empe a u e, p oducing diazonium sal s, which we e added o eugenol 2in a basic
medium, o yield 4-allyl-2-me hoxy-6-(phenyldiazenyl)phenol 3a [
14
], 4-allyl-2-me hoxy-
6-((3-me hoxyphenyl)diazenyl)phe-nol 3b [
16
], 4-allyl-2-((3-b omophenyl)diazenyl)-6-
Molecules 2025,30, 2788 3 o 17
me hoxyphenol 3c, 4-allyl-2-((4-chlo ophenyl)diazenyl)-6-me hoxy-phenol 3d [
14
], and
3-((5-allyl-2-hyd oxy-3-me hoxyphenyl)diazenyl)-2-b omobenzoic acid 3e (Scheme 1).
Scheme 1. Syn hesis o eugenol azo dyes 3a–e.
Compounds 3a–ewe e ob ained as o ange and ed solids in mode a e yields (14–44%).
These yields esul due o he ac ha eugenol has e y limi ed solubili y in wa e , meaning
ha he eac ion ends no o be comple e and equi es pu i ica ion o he c ude p oduc
ob ained using ch oma og aphic echniques, which also leads o signi ican losses. Con-
side ing he elec onic na u e o he subs i uen g oups o eugenol, as well as he possible
s e ic impedimen s associa ed wi h he allyl side chain, he si e o azo- unc ionaliza ion
will occu in he o ho posi ion ela i e o he hyd oxyl g oup. The espec i e compounds
we e ully cha ac e ized by using he usual analy ical echniques:
1
H NMR,
13
C NMR, FTIR
spec oscopy, and HRMS (see Figu es S1–S22, Supplemen a y Ma e ials).
In he
1
H NMR spec a, i is possible o dis inguish di e en signals o alipha ic
p o ons, namely, me hylenic (
δ
3.37–3.43 ppm) and me hoxyl (
δ
3.84–3.95 ppm) g oups,
in addi ion o he expec ed p o ons o he eugenol’s double bond as mul iple s CH
2
(
δ
5.06–5.19 ppm) and CH (
δ
5.96–6.07 ppm). Wi h he occupa ion o he mos ac i a ed
posi ion o he occu ence o azo coupling, he espec i e a oma ic signals o eugenol
(H-3 and H-4 o H-4 and H-6, in he case o 3e) will co espond o wo double s (J1.6 o
2.0 Hz) o be included in a mul iple (H-5 in 2b) (
δ
6.80–7.47 ppm). Fo amines, he a oma ic
p o ons will appea a speci ic chemical shi s, depending on he elec onic na u e and
posi ion o he subs i uen s p esen .
The
13
C NMR spec a o all compounds showed signals o he alipha ic ca bons om
he CH
2
(
δ
38.81–39.55 ppm) and OCH
3
g oups (
δ
56.14–56.48 ppm), in addi ion o ca bons
o he eugenol’s double bond CH
2
(
δ
116.03–116.28 ppm) and CH (
δ
136.96–137.42 ppm).
The ca bons o he a oma ic ings we e also shown (
δ
112.15–160.38 ppm). Fo compound
3e, i is possible o assign he ca bon in he ca bonyl g oup o he ca boxylic acid unc ion
a
δ
168.04 ppm. The FTIR spec a also con i med he p esence o a CO
2
H g oup o
compound 3e, showing he co esponding C=O s e ching ib a ion bands a 1695 cm−1.
2.2. UV/Vis Abso p ion o he Eugenol Azo Dyes 3a–e
The eugenol de i a i es 3a–eexhibi abso p ion in he UV/ isible egion, and hei
abso p ion spec a a e illus a ed in Figu e 1, showing an in ense abso p ion band in he
UV ange, a a ound 345 nm, and ano he band in he blue egion, jus i ying he yellowish
colo . The s ong ed shi o he UV band ela i e o he eugenol UV/Vis abso p ion
spec um (maximum a ~280 nm) [
32
] poin s o an ex ended conjuga ion be ween he
a oma ic moie ies, including he azo g oup. The band in he isible egion is p obably due
o an n→π* ansi ion in ol ing N a oms.
Molecules 2025,30, 2788 4 o 17
Figu e 1. Abso p ion spec a o compounds 3a–ein neu al e hanolic solu ion (2 ×10−5M).
The compounds we e compa ed, and small a ia ions a e de ec ed in he UV band
posi ion and in ensi y, e lec ing he in luence o he subs i uen s in he phenyl ing linked o
he azo moie y. Maximum abso p ion wa eleng hs (
λmax
) and mola abso p ion coe icien s
(
ε
) a e indica ed in Table 1. Compound 3a, wi h no subs i uen s in he phenyl ing, display
he highes abso p ion coe icien . Compound 3e, possessing wo subs i uen s in his ing
(–B and –COOH g oups in he o ho posi ion ela i e o each o he ), p esen s a lowe
abso p ion coe icien and a signi ican ba hoch omic shi , p obably due o he ex ension
o he conjuga ion o he a oma ic ing o he ca bonyl g oup o –COOH.
Table 1. Maximum abso p ion wa eleng hs (
λmax
) and mola abso p ion coe icien s (
ε
) o com-
pounds 3a–ein neu al e hanolic solu ion.
Compound λmax (nm) ε(M−1cm−1)
3a 340 1.14 ×105
3b 338 8.27 ×104
3c 343 1.02 ×105
3d 345 9.40 ×104
3e 353 7.48 ×104
G ound s a e equilib ium geome ies o cis and ans con o me s o compound 3a,
as well as i s hyd azone au ome , a e shown in Figu e 2, oge he wi h elec on den-
si y a ia ion upon exci a ion o he i s exci ed s a e (Ca esian coo dina es in Table S4
o Supplemen a y Ma e ials). Vib a ional analysis con i med, h ough he absence o imagi-
na y equencies, ha no me as able molecula geome ies we e ob ained; his allowed us
o place he ee ene gies o he cis con o me and he hyd azone au ome , espec i ely, a
19.5 kcal/mol and 1.83 kcal/mol abo e he ans o m o compound 3a. This esul p edic s
ha 4.4% o compound 3a exis s in i s hyd azone o m a oom empe a u e (25 ◦C).
Table 2shows he ob ained ansi ion wa eleng hs and oscilla o s eng hs o he
i s eigh exci ed s a es o bo h con o me s and o he hyd azone au ome . Conside ing
he low oscilla o s eng h o he i s exci ed s a e and he elec on densi y a ia ions
in Figu e 2, i is possible o conclude ha , in bo h cis and ans con o me s, he i s elec onic
ansi ion is mainly o he n
→π
* ype, as a dec ease occu s along he azo backbone and an
inc ease occu s in he
π
elec on sys em abo e he molecula plane. The cis con o me shows
highe oscilla o s eng h as non-plana i y esul s in a be e o e lap be ween nand
π
*
molecula o bi als. The hyd azone au ome , which is plana like he azo au ome , clea ly
Molecules 2025,30, 2788 5 o 17
shows a
π→π
* ansi ion, co esponding o a highe oscilla o s eng h. Compa ing he
expe imen al spec um (Figu e 1) in he egion abo e 440 nm wi h he oscilla o s eng hs
in Table 2(7.4
×
10
−4
o he ans con o me s. 0.049 and 0.13 o he au ome ), i
is possible o conclude ha he abso p ion spec um mainly co esponds o he ans
con o me , as expec ed om i s lowe ee ene gy.
A
B
C
D
Figu e 2. G ound s a e equilib ium geome ies and elec on densi y a ia ion upon exci a ion o
he i s exci ed s a e o ans (A) and cis (B) con o me s, he hyd azone au ome (C), and he
Pb
2+
complex (D). The g een and pu ple a eas co espond o dec eases and inc eases in elec on
densi y, espec i ely.
Table 2. Resul s o TD-DFT calcula ions o compound 3a in i s ans and cis con o me s, as well as
i s hyd azone au ome ic o m and i s complex wi h Pb
2+
: di e ences in ene gy be ween elec onic
s a es and co esponding oscilla o s eng hs (see Sec ion 3.4.3 o calcula ion de ails).
T ansi ion Wa eleng h (nm) Oscilla o S eng h ( )
ans cis Tau ome Pb2+ Complex ans cis Tau ome Pb2+ Complex
S0→S1448 476 472 537
7.40
×
10
−4
4.02
×
10
−2
4.94
×
10
−22.51 ×10−3
S0→S2417 395 445 468
8.15
×
10
−2
6.80
×
10
−3
1.29
×
10
−11.30 ×10−1
S0→S3328 319 348 396 1.06
2.11
×
10
−2
9.02
×
10
−11.05 ×10−2
S0→S4302 288 313 347
1.76
×
10
−2
4.96
×
10
−3
4.05
×
10
−38.59 ×10−2
S0→S5296 280 311 335
3.26
×
10
−4
1.32
×
10
−1
1.50
×
10
−27.84 ×10−4
S0→S6278 275 287 325
5.96
×
10
−2
5.28
×
10
−3
9.03
×
10
−45.03 ×10−2
S0→S7256 271 278 324
1.97
×
10
−3
1.19
×
10
−1
8.14
×
10
−32.99 ×10−1
S0→S8253 258 257 318
1.10
×
10
−2
7.25
×
10
−3
8.93
×
10
−22.17 ×10−1
P edic ion o he abso p ion spec al shape equi es ib a ional analysis o bo h g ound
and exci ed s a es in hei equilib ium geome ies. The geome y op imiza ion o he
i s exci ed s a e esul ed in a e y high geome y a ia ion, so ha he S
0→
S
1
ene gy
di e ence became e y small (~1 eV). Thus, a simpli ied me hod was used, in which
each elec onic ansi ion is conside ed o be a Gaussian unc ion in wa enumbe , wi h a
ixed s anda d de ia ion co esponding o 0.4 eV (3226 cm
−1
). In Figu e 3, a easonable
acco dance is shown be ween he p edic ed spec um and he expe imen al one, al hough
he mola abso p i i y o he S
0→
S
2
band is lowe han he expe imen al alue. The highe
abso p i i y in ha egion could be due o he con ibu ion o he hyd azone au ome ha
has a signi ican mola abso p i i y in ha spec al egion.
Molecules 2025,30, 2788 6 o 17
Figu e 3. Calcula ed abso p ion spec a (in log scale) o compound 3a (3a calc), i s hyd azone
au ome (3a_ au ome calc), and i s complex wi h Pb2+ (3a_Pb calc).
2.3. Colo ime ic Assays o Compounds 3a–eas Ion Senso s
The compounds 3a–ewe e p elimina y es ed as senso s o se e al ca ions in o de
o ob ain a colo ime ic assay, which is especially use ul o de ec ing pollu an s and
poisoning me als. The ca ions (15 equi .) we e added o compounds’ solu ions, and
he colo s we e compa ed be ween he nea ca ion solu ions and he compound/ca ion
solu ions (Figu es 4and 5).
K+ Fe2+ Fe3+ Cs+ Zn2+ Cd2+ Ni2+ Cu2+ Al3+ Pb2+ Na+ Cd2+ Co2+
Figu e 4. Solu ions o he sal s o se e al ca ions. Cd
2+
was es ed as a pe chlo a e sal (le ) and a
ni a e sal ( igh ).
Conside ing he objec i e o ob aining a colo ime ic assay o he de ec ion o ca ions,
we ind, in Figu e 5, e idence ha he compounds can be use ul o he de ec ion o
coppe (II) and lead(II), which display a clea change in colo , despi e compound 3e seeming
no use ul o his de e mina ion.
Pa icula ly, compound 3c seems o be he mos use ul o Cu
2+
, demons a ing a
clea ly dis inguishable and in ense pink colo in he p esence o his ca ion. This colo
change can be obse ed a concen a ions as low as 10
µ
M o ca ions, while o he o he
compounds, highe ca ion concen a ions a e needed. He e, he possibili y o he de ec ion
o Pb
2+
is especially aluable, because lead is desc ibed as a poison ha can a ec a la ge
Molecules 2025,30, 2788 7 o 17
numbe o o gans in human body. The e o e, he i e compounds’ solu ions we e i a ed
wi h lead(II) ca ions.
Compound3aCompound3bCompound3cCompound3dCompound3e
Figu e 5. Solu ions o compounds 3a–ea e he addi ion o 15 equi . o he sal s. Cd
2+
was es ed as
a pe chlo a e sal (le ) and a ni a e sal ( igh ), and no di e ences we e ob ained.
Molecules 2025,30, 2788 8 o 17
2.4. Ti a ion o Compound 3c wi h Cu2+
Spec opho ome ic i a ions in e hanol we e pe o med o u he in es iga e he
abili y o compound 3c o de ec coppe (II) ion. The e o e, inc easing amoun s o he ca ion
we e added o he compound solu ion, and he co esponding abso p ion spec a we e
ob ained. A new abso p ion band, cen e ed a a ound 530 nm, is obse ed (Figu e 6), he
maximum abso p ion o his band being a ained a 10 equi alen s o he ca ion, poin ing
o a high sensi i i y. The p esence o h ee clea isosbes ic poin s indica es a complex
in e ac ion be ween he compound and Cu2+.
Figu e 6. Abso p ion spec a o compound 3c (1
×
10
−5
M) wi h he addi ion o Cu
2+
. The spec a o
10 and 12 equi . o Cu2+ a e supe posed.
In hese esul s, a linea ela ionship be ween he ca ion concen a ion and he ab-
so bance o he new band a 540 nm (whe e he abso p ion o he nea compound is
negligible) was obse ed up o 60
µ
M o ca ions (Figu e 7A). F om his plo , a limi o
de ec ion (LOD) as low as 0.16
µ
M was es ima ed. This alue shows ha compound 3c can
be e y use ul o he de ec ion o Cu
2+
in e luen s and o he samples, conside ing ha he
maximum le el o coppe in d inking wa e is 20.5
µ
M (WHO, 2008 [
33
]), well abo e he
LOD and in he linea ange o he calib a ion cu e (Figu e 7A). Mo eo e , he a e age
concen a ion o coppe in blood should no exceed 15.7–23.6
µ
M in no mal condi ions [
34
],
also in he de ec able ange. This LOD alue is lowe han he ones epo ed o o he
senso s o coppe (II) [
35
–
37
], making compound 3c highly ad an ageous o his pu pose.
To calcula e he binding cons an , he Benesi–Hildeb and equa ion was used, he K
a
alue being de e mined om he a io o he in e cep o he slope o he Benesi–Hildeb and
plo (Figu e 7B). A linea ela ionship was ob ained only o n= 2 (while o n= 1, he plo
is no linea ), indica ing a binding s oichiome y o 1:2 be ween he compound and he
coppe (II) ca ion, wi h a co esponding binding cons an o K
a
= 1.21
×
10
9
M
−2
. Azo dyes
based on eugenol (wi hou he subs i uen s R, R
1
, and R
2
) inco po a ed in silicone ilms
ha e also shown he capabili y o de ec Cu
2+
me al ions, ep esen ed by a change in colo
o he ilm, simila ly exhibi ing a 1:2 s oichiome y [38].
Molecules 2025,30, 2788 9 o 17
Figu e 7. (A) Linea plo o abso bance a 540 nm e sus Cu
2+
concen a ion. (B) Benesi–Hildeb and
plo o he spec opho ome ic i a ion o compound 3c wi h Cu2+ a 540 nm.
2.5. Ti a ion o Compounds 3a–ewi h Pb2+
Conside ing he p oo -o -concep ob ained wi h coppe (II) ca ions, ha ing a low LOD
alue, we explo ed he possibili y o he syn hesized compounds ac ing as lead(II) senso s.
Fo his pu pose, each o he compounds 3a–ewas i a ed wi h lead(II), aking in o accoun
he signi ican ha m ul en i onmen al and heal h e ec s om high concen a ions o
lead [
39
]. Lead exposu e can ha e se ious consequences o heal h, especially wi h child en.
The WHO has es ablished “In e na ional Lead Poisoning P e en ion Week” (e e y yea in
Oc obe ) o p e en childhood lead exposu e and o s op he use o lead in pain [40].
As p edic ed by he less p ominen a ia ion in colo upon Pb
2+
addi ion (compa ing
wi h coppe ), he spec al e ec s o he p esence o he ion a e much less p onounced,
compound 3e no being sensi i e o he p esence o Pb
2+
in he i a ion (as in e ed om
he colo in a iance in Figu e 5). Mo eo e , he i a ion o compound 3d also shows
e y small spec al changes wi h he addi ion o lead ca ions. The e o e, he i a ion o
compounds 3a, 3b, and 3c wi h Pb
2+
we e analyzed o de e mine he binding cons an s and
LOD alues (Figu es 8–10). The pa ame e s o he in e ac ion o each compound, 3a–c,
wi h lead ca ions a e summa ized in Table 3. I mus be poin ed ou ha o compounds
3a, 3b, and 3c, he Benesi–Hildeb and plo s (Figu es 8C, 9C and 10C) only display a good
co ela ion o n= 1, indica ing a 1:1 s oichiome y o he in e ac ion o lead(II) wi h
he azo compounds. In ac , he spec a o he i a ion o compounds wi h lead(II) a e
comple ely di e en om ha o compound 3c wi h coppe (II), poin ing o a di e en kind
o in e ac ion o he azo dyes wi h he wo ca ions.
Table 3. LOD alues and binding cons an s o he in e ac ion o compounds 3a–cwi h lead(II) ca ions.
Compound LOD (µM) Ka(M−1)
3a 0.19 2.80 ×104
3b 0.14 1.33 ×104
3c 0.16 1.02 ×104
Molecules 2025,30, 2788 16 o 17
6.
Ryzhko , A.I.; Fedo , V.; D ozdo , F.V.; Geo gij, V.; Che kae , G.V.; Buzin, M.I.; S idchenko, E.A.; Muza a o , A.M. Syn hesis and
p ope ies o new siloxane wi h e minal azo dyes unc ions based on eugenol. J. Appl. Polym. Sci. 2022,139, e52340. [C ossRe ]
7.
Ghana a ka , C.W.; Mish a, V.R.; Seka , N. Re iew o NLOpho ic azo dyes—De elopmen s in hype pola izabili ies in las wo
decades. Dyes Pigmen . 2021,191, 109367–109392. [C ossRe ]
8.
Ma inescu, M.; Popa, C.V.; Tănase, M.A.; Soa e, A.C.; Table , C.; Bala, D.; Cin eza, L.O.; Di¸ u, L.M.; Gi u, I.C.; Pe cu, C. Syn hesis,
cha ac e iza ion, DFT s udy and an i ungal ac i i ies o some no el 2-(phenyldiazenyl)phenol based azo dyes. Ma e ials 2022,
15, 8162–8182. [C ossRe ]
9.
Khanum, R.; Shouka Ali, R.A.; Rangaswamy, H.R.; San hosh Kuma , S.R.; Pashan ha, A.G.; Jagadisha, A.S. Recen e iew on
syn hesis, spec al s udies, e sa ile applica ions o azo dyes and i s me al complexes. Resul s Chem. 2023,5, 100890–100905.
[C ossRe ]
10.
Deghadi, R.G.; Mahmoud, W.H.; Mohamed, G.G. Me al complexes o e aden a e azo-dye ligand de i ed om 4,4
′
-oxydianiline:
P epa a ion, s uc u al in es iga ion, biological e alua ion and MOE s udies. Appl. O ganome . Chem. 2020,34, e5883. [C ossRe ]
11.
Al-Gabe , M.A.I.; Abd El-La ee , H.M.; Khala , M.M.; Shaaban, S.; Shawky, M.; Mohamed, G.G.; Abdou, A.; Gouda, M.; Abu-Die ,
A.M. Design, syn hesis, spec oscopic inspec ion, DFT and molecula docking s udy o me al chela es inco po a ing azo dye
ligand o biological e alua ion. Ma e ials 2023,16, 897–919. [C ossRe ] [PubMed]
12.
Zhu, Z.; Chen, R.; Zhang, L. Simple phenylp opanoids: Recen ad ances in biological ac i i ies, biosyn he ic pa hways, and
mic obial p oduc ion. Na . P od. Rep. 2024,41, 6–24. [C ossRe ] [PubMed]
13.
Zieli´nska-Błaje , M.; Fede -Kubis, J. Mono e penes and hei de i a i es– ecen de elopmen in biological and medical applica-
ions. In . J. Mol. Sci. 2020,21, 7078–7115. [C ossRe ] [PubMed]
14.
Dandge, S.V.; Nikume, S.R.; Bend e, R.S. An e icien syn hesis, cha ac e iza ion, an imic obial and an icance ac i i ies o azo
dyes de i ed om eugenol. Syn h. Commun. 2023,54, 282–292. [C ossRe ]
15.
Ca ado i, S.; Ammazzalo so, A.; Niccolai, S.; Tanini, D.; D’Agos ino, I.; Mel i, F.; Cappe ucci, A.; G ande, R.; Sis o, F. Na u e-
inspi ed compounds: Syn hesis and an ibac e ial suscep ibili y es ing o eugenol de i a i es agains H. pylo i s ains. Pha maceu-
icals 2023,16, 1317–1335. [C ossRe ]
16.
Mel i, F.; Fan acuzzi, M.; Ca ado i, S.; Ammazzalo so, A.; Menca elli, N.; Gallo ini, M.; Spano, M.; Agamennone, M.; Haji Ali, S.;
Al-Samydai, A.; e al. Azo de i a i es o mono e penes as an i-Helicobac e pylo i agen s: F om he syn hesis o a s uc u e-based
a ge in es iga ion. RSC Med. Chem. 2025,16, 346–366. [C ossRe ]
17.
Fe ei a, V.R.F.; Mili ani, I.A.; de Almeida, K.J.; Lunguinho, A.S.; Saczk, A.A.; Ion a, M.; da Sil a, G.A.F.; Felix, F.S.; Nelson,
D.L.; Ca doso, M.G. An ioxidan and cy o oxic ac i i y o essen ial oils and hei p incipal componen s: Spec opho ome ic,
ol amme ic, and heo e ical in es iga ion o he chela ing e ec o eugenol and ca ac ol. ACS Food Sci. Technol. 2023,3, 350–360.
[C ossRe ]
18.
Sadaphal, Y.R.; Gholap, S.S. Eugenol [2-Me hoxy-4-allylphenol (MAP)] a colo ime ic sensing p obe o selec i e de e mina ion o
I on(III). J. Ma e . En i on. Sci. 2022,13, 42–53.
19. Jomo a, K.; Valko, M. Ad ances in me al-induced oxida i e s ess and human disease. Toxicology 2011,283, 65–87. [C ossRe ]
20.
Ruiz, L.M.; Libedinsky, A.; Elo za, A.A. Role o coppe on mi ochond ial unc ion and me abolism. F on . Mol. Biosci. 2021,
8, 711227. [C ossRe ]
21.
Gup e, A.; Mumpe , R.J. Ele a ed coppe and oxida i e s ess in cance cells as a a ge o cance ea men . Cance T ea . Re .
2009,35, 32–46. [C ossRe ] [PubMed]
22.
Ka dos, J.; Héja, L.; Simon, A.; Jablonkai, I.; Ko ács, R.; Jemni z, K. Coppe signalling: Causes and consequences. Cell Commun.
Signal. 2018,16, 71–92. [C ossRe ] [PubMed]
23.
Texas Commission on En i onmen al Quali y (TCEQ). Lead Poisoning: Wha A e he Sou ces? Wha A e he Risks? TCEQ
Publica ion GI-069. 2004. A ailable online: h ps://www. ceq. exas.go /downloads/ oxicology/publica ions/lead-poisoning-
gi-69.pd (accessed on 1 Ma ch 2025).
24.
Collin, M.S.; Venka aman, S.K.; Vijayakuma , N.; Kanimozhi, V.; A baaz, S.M.; S acey, R.G.S.; Anusha, J.; Choudha y, R.; L o ,
V.; To a , G.I.; e al. Bioaccumula ion o lead (Pb) and i s e ec s on human: A e iew. J. Haza d. Ma e . 2022,7, 100094–100101.
[C ossRe ]
25.
T e ino, M.; Wagne , C.R.; Tamu a, E.K.; Ga cia, J.; Louie, A.Y. Small molecule senso s o he colo ime ic de ec ion o Coppe (II):
A e iew o he li e a u e om 2010 o 2022. Dyes Pigmen . 2023,214, 110881. [C ossRe ]
26.
Chen, S.-Y.; Li, Z.; Li, K.; Yu, X.-Q. Small molecula luo escen p obes o he de ec ion o lead, cadmium and me cu y ions.
Coo d. Chem. Re . 2021,429, 213691. [C ossRe ]
27.
Wang, Y.; Song, H.; Zhang, Y.; Gong, H.; Sun, R.; Mao, W.; Wang, D.; Chen, Y. A colo ime ic Pb
2+
chemosenso : Rapid naked-eye
de ec ion, high selec i i y, heo e ical insigh s, and applica ions. J. Pho ochem. Pho obiol. A Chem. 2018,355, 101–108. [C ossRe ]
28.
Za alishin, M.N.; Gamo , G.A.; Kisele , A.N.; Niki in, G.A. A luo escein conjuga e as colo ime ic and ed-emissi e luo escence
chemosenso o selec i e ecogni ion Cu2+ ions. Op . Ma e . 2024,153, 115580. [C ossRe ]
Molecules 2025,30, 2788 17 o 17
29.
Al-Saidi, H.M. Recen ad ancemen s in o ganic chemosenso s o he de ec ion o Pb
2+
: A e iew. Chem. Pap. 2023,77, 4807–4822.
[C ossRe ]
30.
Alyami, A.Y. Recen p og ess in o ganic luo escence and colo ime ic chemosenso s o Cu
2+
de ec ion: A comp ehensi e e iew
(2018–2023). Dyes Pigmen . 2023,220, 111740. [C ossRe ]
31.
Mohammed, G.I.; Nassa , S.; Fa ghaly, T.A. Spec opho ome ic me hod o de ec ing lead (II) ions using a chemosenso o he
hiosemica bazone-linked phenyl sulphone moie y. Resul s Chem. 2025,15, 102174. [C ossRe ]
32.
Ayoubi, R.; Wali, S.; Singh, G.B. The UV and FTIR inge p in o ocimum kilimandscha icum gue ke essen ial oil: A eugenol- ich
chemo ype. In . J. Inno . Res. Sci. S ud. 2022,5, 1–9. [C ossRe ]
33.
Wo ld Heal h O ganiza ion. Guidelines o D inking-Wa e Quali y: Second Addendum, 3 d ed.; Wo ld Heal h O ganiza ion: Gene a,
Swi ze land, 2008; Volume 1.
34. Linde , M.C.; Hazegh-Azam, M. Coppe biochemis y and molecula biology. Am. J. Clin. Nu . 1996,63, 797S–811S. [C ossRe ]
35.
Ghule, N.V.; Bhosale, R.S.; Puyad, A.L.; Bhosale, S.V. Naph halenediimide amphiphile based colo ime ic p obe o ecogni ion o
Cu2+ and Fe3+ ions. Sens. Ac ua o s B Chem. 2016,227, 17–23. [C ossRe ]
36.
Wang, J.; Wei, T.; Ma, F.; Li, T.; Niu, Q. A no el luo escen and colo ime ic dual-channel senso o he as , e e sible and
simul aneous de ec ion o Fe
3+
and Cu
2+
based on e hiophene de i a i e wi h high sensi i i y and selec i i y. J. Pho ochem.
Pho obiol. A Chem. 2019,383, 111982–111990. [C ossRe ]
37.
Pin o, S.C.S.; Gonçal es, R.C.R.; Cos a, S.P.G.; Raposo, M.M.M. Colo ime ic chemosenso o Cu
2+
and Fe
3+
based on a
meso- iphenylamine-BODIPY de i a i e. Senso s 2023,23, 6995–7007. [C ossRe ] [PubMed]
38.
Vasilye a, A.A.; Ryzhko , A.I.; Che kae , G.V.; D ozdo , F.V.; Muza a o , A.M. Silicone ilms wi h azo dyes moie ies based on
eugenol wi h esponse o Cu2+ me al ions. Ma e . Chem. Phys. 2024,318, 129248. [C ossRe ]
39.
Wo ld Heal h O ganiza ion. Exposu e o Lead: A Majo Public Heal h Conce n: P e en ing Disease Th ough Heal hy En i onmen s,
3 d ed.; Wo ld Heal h O ganiza ion: Gene a, Swi ze land, 2023.
40.
A ailable online: h ps://www.who.in /campaigns/in e na ional-lead-poisoning-p e en ion-week (accessed on 22 No embe
2024).
41.
Wo ld Heal h O ganiza ion. Guideline o Clinical Managemen o Exposu e o Lead; Wo ld Heal h O ganiza ion: Gene a, Swi ze land,
2021.
42. A ailable online: h ps://www.cdc.go /lead-p e en ion/abou / (accessed on 22 No embe 2024).
43.
Wang, L.; Fang, G.; Cao, D. A no el phenol-based BODIPY chemosenso o selec i e de ec ion o Fe
3+
wi h colo ime ic and
luo ome ic dual-mode. Sens. Ac ua o s B Chem. 2015,207, 849–857. [C ossRe ]
44.
Benesi, H.A.; Hildeb and, J.H. A spec opho ome ic in es iga ion o he in e ac ion o iodine wi h a oma ic hyd oca bons. J. Am.
Chem. Soc. 1949,71, 2703–2707. [C ossRe ]
45. Neese, F. So wa e upda e: The ORCA p og am sys em, e sion 5.0. WIRES Compu . Molec. Sci. 2022,12, e1606. [C ossRe ]
46.
Weigend, F.; Ahl ichs, R. Balanced basis se s o spli alence, iple ze a alence and quad uple ze a alence quali y o H o Rn:
Design and assessmen o accu acy. Phys. Chem. Chem. Phys. 2005,7, 3297–3305. [C ossRe ]
47.
Neese, F. An imp o emen o he esolu ion o he iden i y app oxima ion o he o ma ion o he Coulomb ma ix. J. Comp.
Chem. 2003,24, 1740–1747. [C ossRe ] [PubMed]
48. Weigend, F. Accu a e coulomb- i ing basis se s o H o Rn. Phys. Chem. Chem. Phys. 2006,8, 1057–1065. [C ossRe ] [PubMed]
49.
Caldeweyhe , E.; Ehle , S.; Hansen, A.; Neugebaue , H.; Spiche , S.; Bannwa h, C.; G imme, S. A gene ally applicable a omic-
cha ge dependen London dispe sion co ec ion. J. Chem. Phys. 2019,150, 154122. [C ossRe ] [PubMed]
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