An easy and simple me hod o he immobiliza ion
o dyes h ough click eac ions: ac i a ed alkyne,
coppe no needed
Julia S´
anchez-Bod´
on,
a
Ane Ga c´
ıa-Ga c´
ıa,
ab
Ma ia Diaz-Galba ia u,
a
Jos´
e Luis Vilas-
Vilela *
ab
and Isabel Mo eno-Ben´
ı ez *
c
The coppe - ee azide–alkyne click eac ion has shown o be a success ul al e na i e o immobilize
co alen ly a fluo escen e compound on o poly(-L-lac ic) acid (PLLA) su aces. P oceded by basic
hyd olysis and amida ion eac ion, ypical su ace cha ac e iza ion echniques ha e alida ed each
unc ionaliz ion s ep and he success o he conjuga ion. This me hod offe s a ca alys - ee op ion o
a ious su ace conjuga ions, ex emely demanded in biomedical and biosenso y fields.
In oduc ion
The e m “click chemis y”, s coined by Ba y Sha pless and
cowo ke s in 2001, desc ibes he o ma ion o new C–Co C–
he e oa om bonds by chemical eac ions, which p o ide p od-
uc s in high yields and excellen selec i i ies.
1,2
The click eac-
ion allows he ab ica ion o complex s uc u es om a se ies o
smalle molecules by a selec numbe o e y efficien eac ions.
These building blocks con ain a high-ene gy con en ha d i es
a spon aneous and i e e sible linkage eac ion. A eac ion
mus ull ce ain equi emen s o be conside ed as a click
eac ion. So, p oduc s mus be ob ained wi h high yields and
high selec i i ies om easily accessible compounds and
eagen s. In addi ion, he seconda y p oduc s gene a ed mus
be able o be elimina ed wi hou using ch oma og aphic ech-
niques, as well as hey can no be ha m ul o oxic. Mo eo e ,
employed eagen s and eac ion condi ions mus ole a e
oxygen and wa e media.
3,4
The cycloaddi ion be ween azides and alkynes o gi e ise o
1,2,3- iazoles, s ly desc ibed by Huisgen and cowo ke s in
he la e 1960s, can be conside ed he p o o ype among all click
eac ions.
5,6
In his s e sion o he me hodology, he need
o high empe a u es and p essu es, mainly due o he poo
abili y o azides o ac as 1,3-dipola accep o s, cons i u ed he
mos impo an d awbacks, which limi ed i s applica ion in
di e se elds. Yea s la e , in 2001, Meldal and cowo ke s
success ully employed he s e sion o coppe ca alys , CuI,
and DIPEA as base o he click eac ion. Addi ionally, he
eac ion was conduc ed in o ganic sol en s and he use o
coppe ca alys allowed he eac ion o be ca ied ou a oom
empe a u es, making i as one o he pionee ing success ul
a emp s a his ype o eac ion.
5,6
This me hodology was la e
modied by Sha pless and co-wo ke s, who inco po a ed
coppe sulpha e pen ahyd a e along wi h a educ an , sodium
asco ba e, allowing he eac ion o be pe o med in wa e ,
which o e come p e iously men ioned incomes and became
a majo scien ic b eak h ough. This disco e y no only made
C–Co C–he e oa om bond o ma ion eac ions a eali y, bu
also ans o med chemis y in o a limi less ool.
7,8
By addi ion
o a ca alys , he eac ion conduc ed a single egioisome ,
pa icula ly, 1,4- egioisome o 1,2,3- iazole when coppe (I)is
employed.
9,10
Since he in oduc ion o Cu(I), azide–alkyne
cycloaddi ion eac ion has been ad anced ema kably o e he
las decade, and has now sp ead o almos all a eas o chem-
is y, applied sciences and e en biomedical elds.
11–15
Indeed, i
was a b eak h ough in d ug disco e y, such as he gene a ion o
lead compounds by combina o ial me hods, o applica ions
such as p o eomics o nucleomics, whe e a new e m, bio-
conjuga ion, appea ed. In ecen yea s, a p ima y ocus o ou
esea ch g oup has been he bioconjuga ion o d ugs o a ious
polyme ic su aces h ough click eac ions, aiming o enhance
hei biocompa ibili y and he eby imp o e hei sui abili y o
biomedical applica ions such as implan s o s en s.
16
Howe e ,
he scope o his me hodology has no been limi ed o he
bioconjuga ion o d ugs, bu has been ex ended o o he
domains, including (bio)senso y o conduc o ma e ials. In ac ,
he esul an su ace mus no only deli e selec i e, epea able,
and sensi i e elec ochemical esponses bu also enable s able
immobiliza ion o he ecogni ion elemen .
17
Despi e i s
excep ional ea u es, he CuAAC me hodology p esen s a majo
d awback ega ding he comple e emo al o he me allic
a
G upo de Qu´
ımica Mac omolecula (LABQUIMAC), Depa amen o de Qu´
ımica F´
ısica,
Facul ad de Ciencia y Tecnolog´
ıa, Uni e sidad del Pa´
ıs Vasco UPV/EHU, 48940 Leioa,
Spain
b
BCMa e ials, Basque Cen e o Ma e ials, Applica ions and Nanos uc u es, UPV/
EHU Science Pa k, 48940 Leioa, Spain
c
G upo de Qu´
ımica Mac omolecula (LABQUIMAC), Depa amen o de Qu´
ımica
O g´
anica e Ino g´
anica, Facul ad de Ciencia y Tecnolog´
ıa, Uni e sidad del Pa´
ıs
Vasco UPV/EHU, 48940 Leioa, Spain. E-mail: ma iaisabel[email p o ec ed]
Ci e his: RSC Ad .,2024,14, 14289
Recei ed 7 h Ma ch 2024
Accep ed 25 h Ap il 2024
DOI: 10.1039/d4 a01776e
sc.li/ sc-ad ances
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ca alys , in ac , aces o me als ions, a ppm le els, ha e been
ound ae p oduc pu ica ion. Indeed, in some cases ela ed
o biological o biomedical applica ions he CuAAC is no
a sui able choice due o he cy o oxici y o he me al, which
limi s hese eac ions in li ing sys ems.
18
Addi ionally,
ega ding luminescence p ope ies, he CuAAC eac ion p es-
en s challenges when applied o he conjuga ion o semi-
conduc o QDs like CdSe due o he quenching o QDs'
pho oluminescence by Cu ions.
19
While some s udies demon-
s a e he u ili y o CuAAC o QDs conjuga ion, his me hod
needs p ecise adjus men o Cu ca alys concen a ion and
o he eac ion pa ame e s o sui diffe en QDs samples.
20
Cu en ly, coppe - ee e sions o he eac ion ep esen
p omising al e na i es o QDs conjuga ion due o i s ela i e
simplici y and he absence o Cu ions.
In 2004, Be ozzi e al. in oduced he e m s ain p omo ed
[3 + 2] azide alkyne cycloaddi ion (SPAAC) eac ion, which p e-
sen ed a no el s a egy excluding he use o coppe ca alys .
Unlike linea ace ylenes, he de o ma ion o he ca bon–ca bon
iple bond o a cyclooc yne (163°) allows he spon aneous
eac ion wi h azide compound despi e i s poo abili y o ac as
1,3-dipola accep o .
21,22
This me hodology was used by he
same au ho s o label diffe en biomolecules in cellula en i-
onmen s, demons a ing no only he comple e bio-
o hogonali y o he p ocess bu also he su i al o he cells in
he eac ion media.
23,24
This eac ion has es ablished i sel as
a obus and adap able chemical p ocess wi h b oad applica-
ions in bo h academic and comme cial en u es. I s e sa ili y
is e iden in a ious elds, including bioconjuga ion
p ocesses,
25
he de elopmen o hyb id and block polyme s, as
well as he p oduc ion o high-pe o mance and sel -healing
ma e ials. Addi ionally, i nds u ili y in me abolic engi-
nee ing o biological sys ems and he ab ica ion o conduc o
ma e ials, among o he s.
8,26–28
Howe e , due o he high cos o
hese ma e ials and complexi y in syn hesis, al e na i e s a e-
gies a e pa amoun in o de o add ess he commen ed chal-
lenges effec i ely.
In his con ex , his wo k p esen s he use o ac i a ed
alkynes ha a ou he eac ion be ween alkyne and azide
a oiding he need o complex alkyne chemical s uc u es o
coppe ca alys . In his p oposal, he p e iously men ioned poo
abili y o azide compounds o pa icipa e as dipola ophiles
would be compensa ed, no by s ain as in Be ozzi's p oposal,
bu by elec onic effec s.
29
The e o e, modi ying alkyne g oups
and becoming mo e eac i e can be a p omising al e na i e. The
use o alkyne de i ed compounds wi h elec on-a ac ing
g oups has been shown o be a apid, efficien and cos -
effec i e me hod o acili a ing he eac ion be ween azide
and alkynes, educing he ac i a ion ene gy equi ed o he
eac ion. This no el me hodology was s ly s udied by Ju e al.
30
in solu ion, whe e hey epo ed he s 1,3-dipola cycload-
di ion eac ion be ween azides and elec on-decien alkynes in
wa e . When he alkyne (ei he e minal o in e nal) had a leas
one neighbou ing elec on-wi hd awing unc ional g oup, such
as p opiolic acid, he iazole o ma ion was achie ed e en in
wa e a oom empe a u e wi hou any ca alys s. Despi e he
g ea e sa ili y o his p omising al e na i e o SPAAC, he e a e
only ew examples whe e his ype o coppe ee eac ions a e
used.
30–32
Fo ins ance, i has been employed in he syn hesis o
hyd ogels, oams o polyme s among o he s.
33–36
Al hough i s
g ea po en ial, he use o alkyne de i ed compound wi h
elec on-a ac ing g oups in bioconjuga ion o immobiliza ion
o compounds on ma e ials is s ill no widely known. While i
has been shown o be a simila s a egy o o he coppe - ee
click eac ions, such as SPAAC, i s applica ion in hese elds
is s ill ela i ely new and unexplo ed. Howe e , as mo e
esea ch is conduc ed and he me hodology is u he op i-
mized, i has he po en ial o become aluable ool in he
modica ion o polyme ic su aces o biomedical use o (bio)
senso y,
37–39
among o he s.
Tha is why, in his a icle we p opose he use o an ac i-
a ed ace ylene wi h an elec on-a ac ing g oup. Fo his
pu pose, p opiolic acid was employed as ac i a ed alkyne,
which is comme cially a ailable and easily modiable. The
aimo hecu en s udywas ode elopame hod o he
conjuga ion o uo opho e molecule on o polyme su aces
by employing alkyne ac i a ed-azide “click eac ion”.Poly-
L-
lac ic acid (PLLA) was employed and syn hesized as subs a e,
whe e diffe en chemical unc ionaliza ion we e pe o med in
o de o acili a e he co alen a achmen o p opiolic
de i a i e. These alkyne ac i a ed subs a e was epo ed o
eac wi h de i a ized uo opho e compound offe ing
acon enien way oimmobilizeany ypeo moleculesby
asimplemodica ion. The su ace modica ion p ocedu e
was con med employing diffe en known echniques such as
ATR-FTIR,XPS,wa e con ac angleanduo escence
mic oscopy and spec oscopy.
Resul s and discussion
Fluo opho e compound Dns-N
3
was syn hesized ollowing
a p ocedu e simila o ha desc ibed by Demillo e al.
40
The
chemical eac ion mainly in ol ed in he sul onyl moie y o
dansyl chlo ide and he amino g oup o p e iously syn hesized
3-azidop opan-1-amine o o m a sul onamide, which con-
ained a e minal azido g oup o u he eac ion wi h he
e minal alkyne o modied PLLA su aces, as shown in Scheme
1. Al hough, in his wo k dansyl de i a i e has been used only
o he con ol o he conjuga ion owing o i s uo escence. I is
wo h men ioning ha sul onamides a e known as b oad
spec um an imic obial agen s, which can inhibi he g ow h o
bo h G am-posi i e and G am-nega i e bac e ia. Thus, he
p oposed me hodology would be a sui able al e na i e o ca y
ou he immobiliza ion o his ype o compounds.
Scheme 1 Syn hesis o dansyl de i i a i e Dns-N
3
.
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Hyd olysis eac ion o PLLA was ca ied ou in basic media,
employing NaOH 1 M du ing 30 min a 50 °C. As a esul ,
ca boxylic acids we e gene a ed on he su ace o PLLA and we e
hen amida ed using e hylendiamine (ETDA), ob aining an
amide wi h NH
2
as ending g oup (PLLA-NH
2
as can be seen in
Scheme 2 ae 1. amida ion). Then, u he unc ionaliza ion o
he e minal amine was pe o med in o de o inco po a e he
alkyne g oup on PLLA su ace, which is essen ial o he
conjuga ion o he uo opho e compound. This is, in ac , he
main diffe ence be ween his wo k and he p e ious one ca ied
ou in ou esea ch g oup.
16
Indeed, he s amida ion o PLLA
wi h a diamine, allowed ca ying ou a second amida ion wi h
p opiolic acid, hus achie ing an elec on-decien alkyne,
which could be used o accomplish he 1,3-dipola cycloaddi-
ion wi h he azide moie y o he uo opho e compound in he
absence o ca alys . Finally, p e iously syn hesized dansyl
de i a i e was immobilized on o PLLA by coppe - ee azide
alkyne cycloaddi ion eac ion (Scheme 2).
The ATR-FTIR spec a o p is ine PLLA, hyd olyzed PLLA,
bo h amida ed PLLA (PLLA-NH
2
and PLLA-alkyne) and clicked
PLLA (PLLA-Dns) a e shown in Fig. 1. Ae 30 min o being
subme ged on NaOH solu ion, hyd olyzed PLLA samples p e-
sen ed a band a 3250 cm
−1
, co esponding o he s e ching
ib a ions o O–H bonds, as well as he peak a 2900 cm
−1
ela ed o s e ching C
sp
3
–H bond sligh ly inc eased. These
changes in ATR-FTIR spec um e idenced he success o
hyd olysis eac ion on PLLA su ace. Ae u he g aing wi h
e hylendiamine, he in ensi y o he band a ound 3250–
3300 cm
−1
enhanced due o he p esence o NH and NH
2
on he
su ace, which indica ed ha amida ion eac ion was ca ied
ou success ully. The inco po a ion o alkyne g oup h ough
a second amida ion eac ion wi h p opiolic acid was alida ed
by obse ing wo dis inc i e s e ching bands a 2090 cm
−1
and
3350 cm
−1
in PLLA-alkyne samples. These bands di ec ly co -
esponded o he s e ching ib a ion o C
sp
–C
sp
and C
sp
–H
bonds, espec i ely. Addi ionally, he p esence o a peak a
1700 cm
−1
co esponding o he s e ching o C]O was sligh ly
shied o 1650 cm
−1
. This signal aligned wi h he common
appea ance o C]O in amides, p o iding s ong e idence ha
he second amida ion eac ion occu ed success ully. Finally,
ae dansyl de i a i e (Dns-N
3
) immobiliza ion ia coppe - ee
azide-yne click eac ion, he C
sp
–C
sp
and C
sp
–H s e ching
signals disappea ed, concluding ha dansyl de i a i e was
effec i ely immobilized on o PLLA.
Chemical composi ions o unc ionalized su aces we e
measu ed by XPS and he esul s a e esumed in Fig. 2 and
Table 1. As expec ed, PLLA-COOH only exhibi ed wo majo
peaks a 284 eV and 535 eV a ibu ing o C 1s and O 1s,
espec i ely. In addi ion, he elemen composi ion o C and O
we e 69.9% and 29.8%, espec i ely. Meanwhile, high esolu-
ion spec a o C 1s showed h ee peaks co esponding o C–C/
C–H (284.6 eV), C–O (286.3 eV) and C]O (288.8 eV) bonds as i
is an es e based polyme and also ag eed wi h li e a u e.
41–43
Ae amida ion wi h e hylendiamine and p opiolic acid, a new
peak a 400 eV a ibu ing o N 1s appea ed in he spec um o
PLLA-alkyne. Decon olu ion spec a o N 1s p esen ed only
a majo con ibu ion ela ed o C–N species. Meanwhile in he
same sample, high esolu ion spec a o ca bon exhibi ed ha
he peak a 285.8 eV (C–O/C–N) enhanced compa ed o he o he
Scheme 2 PLLA unc ionaliza ion s eps and dansyl de i a i e
conjuga ion.
Fig. 1 ATR-FTIR spec a o p is ine PLLA and unc ionalized PLLA
samples.
Fig. 2 C, O, N and S decon olu ion XPS spec a o PLLA-COOH,
PLLA-NH
2
, PLLA-alkyne and PLLA-Dns samples.
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peaks due o he in oduc ion o amines/amides on he su ace.
Ae u he unc ionaliza ion wi h dansyl de i a i e, he p es-
ence o new peak a ound 169 eV was obse ed, co esponding o
S 2p elemen . Addi ionally, high esolu ion spec a o sulphu
exhibi ed wo con ibu ions a 164 eV and 168 eV, co espond-
ing o C–NH/S–NH and S]O species, which ag eed wi h li e -
a u e.
44,45
Fu he mo e, high esolu ion spec a o ni ogen also
con med he p esence o sulphu a 402 eV.
46
These esul s
con med again he immobiliza ion o dansyl de i a i e on o
PLLA su ace.
The mo phology and we abili y o he su ace we e de e -
mined by SEM and wa e con ac angle measu emen s espec-
i ely. As can be seen in Fig. 3a, he hyd olysis and subsequen
unc ionaliza ion eac ions esul ed in no able changes in he
hyd ophobici y o he su aces which is eec ed in he changes
in he wa e con ac angles. P is ine PLLA p esen ed high s a ic
wa e con ac angle alue (111 ±4.7°) due o i s lack o pola
unc ional g oups on he op o he su ace, which implied ha
he su ace was hyd ophobic. Ae hyd olysis, he wa e con ac
angle dec eased o 72.4 ±13.5°, which indica ed ha ca boxylic
acid and hyd oxyl g oups we e success ully o med on he
su ace. The amida ion eac ion wi h ETDA supposed only
a sligh change in he con ac angle (54.2 + 8.7°). In ac , simila
pola i ies o in ol ed moie ies, such as amine o hyd oxyl
g oup, implied simila in e ac ions wi h wa e . Ne e heless,
he second amida ion, once he alkyne g oup is inco po a ed,
esul ed in a d as ic dec ease in he wa e con ac angle (18.7 ±
4.6°). Al hough he pola i y o he a ached unc ional g oups
diminished, he signican ly impo an oughening o he
su ace could explain he decline in he con ac angle alue in
his case.
47,48
Mo eo e , ae he click eac ion, once he uo-
opho e compound was conjuga ed on o PLLA, he wa e
con ac angle inc eased again o 100 ±8.7°, his esul could be
connec ed o he inc ease in hyd ophobici y due o he p esence
o a oma ic ing o dansyl de i a i e, specially, he nayl g oup.
SEM images indica ed he su ace mo phologies o diffe en
PLLA samples, as can be seen in Fig. 3b. Fo p is ine PLLA, he
su ace was smoo h wi h no ob ious de ec . Ne e heless, ae
hyd olysis, PLLA-COOH sample showed some clea sc a ches,
e en aking, which can be ela ed o he deg ada ion o he
su ace on he s s ep o unc ionaliza ion. Ae s amida-
ion, he mo phology con inued changing, becoming mo e
he e ogeneous wi h ound a eas. Once he alkyne was inco -
po a ed, he su ace oughness inc eased, which is closely
ela ed wi h he abo e men ioned wa e con ac esul s, since
a oughe su ace wi h a la ge a ea is mo e hyd ophilic.
47,48
Finally, e ica ion o dansyl de i a i e immobiliza ion ia
coppe - ee azide–alkyne click eac ions was conduc ed using
bo h a uo escen mic oscope and uo escence spec oscopy,
ocusing in he cha ac e is ic emission peak a 500 nm o he
uo opho e. P is ine PLLA and dansyl de i a i e clicked PLLA
(PLLA-Dns) we e compa ed in his s udy. As can be seen in
Fig. 4a, as expec ed, PLLA-Dns su ace exhibi ed a uo escence
maximum peak a 495 nm, specically a ibu ed o he dansyl
compound. Meanwhile, p is ine PLLA did no p esen any
maximum a he same emission wa eleng h. On he o he hand,
con ocal mic oscopy analysis (Fig. 4b) indica ed ha p is ine
PLLA p esen ed no uo escence emission ac oss a ious wa e-
leng hs, e en when exci ed a dansyl de i a i e's op imal exci-
a ion poin a ound 340 nm. Howe e , PLLA-Dns displayed
Table 1 C, O, N and S con en o PLLA modified su aces
Sample C/O C (%) O (%) N (%) S (%)
PLLA 2.4 69.9 29.8 ——
PLLA-COOH 2.6 71.6 27.5 ——
PLLA-NH
2
2.0 63.1 31.5 1.8 —
PLLA-alkyne 2.3 61.0 26.5 3.2 —
PLLA-Dns 2.6 67.2 26.0 3.8 0.5
Fig. 3 (a) Con ac angle alues o unc ionalized PLLA su aces.
S a is ical significance *(p< 0.05), (b) SEM images o p is ine PLLA,
PLLA-COOH, PLLA-NH2, PLLA-alkyne, PLLA-Dns.
Fig. 4 (a) S eady s a e fluo escence o PLLA and PLLA-Dns; (b) fluo-
escence mic oscopy images o p is ine PLLA and PLLa-Dns.
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a g een uo escence, a ibu ing his emission o he uo es-
cence egion o he dansyl de i a i e. These esul s deni i ely
con med he success ul immobiliza ion o he uo opho e
compound on o PLLA su aces.
Ma e ials and me hods
Poly-L-lac ide (PLLA) (Co bion, Ams e dam, The Ne he lands)
we e employed as subs a es. Chlo o o m (CHCl
3
, >98% Mac on
Fine Chemicals, Gliwice, Poland) and me hanol (MeOH, des-
illed om Pan eac) we e used o he p epa a ion o PLLA
lms. Sodium hyd oxide (99%, Pan eac, Ba celona), hyd o-
chlo id ic acid (HCl, 37%, Pan eac, Da ms ad, Ge many), N-(3-
dime hylaminop opyl)-N-e hylca bodiimide hyd ochlo ide
(EDC$HCl, 98%, Sigma Ald ich, USA), N-hyd oxysuccinimide
(NHS, 98%, Sigma Ald ich, USA), e hylendiamine (99%, Sigma
Ald ich, USA), p opiolic acid (95%, Sigma Ald ich, USA) ace ic
acid (99%, Sigma Ald ich, USA) and MiliQ wa e we e used as
sol en and eagen s o he p ocess o p e unc ionaliza ion. Fo
he syn hesis o he uo opho e, dansyl chlo ide (99%, Sigma
Ald ich, Swi ze land), ime hylamine (E 3N, 98%, Sigma
Ald ich, USA), sodium azide (NaN
3
, 99%, Sigma Ald ich, USA),
3-b omop opylamine hyd ob omide (98%, Sigma Ald ich, USA),
ace oni ile (99%, Pan eac, Da ms ad , Ge many), dichlo o-
me hane (CH
2
Cl
2
, 98%, Mac on Fine Chemicals, Poland),
sodium hyd oxide (NaOH, 99%, Pan eac, Da ms ad , Ge many)
and sodium sul a e anhyd ous (Na
2
SO
4
, 96%, Pan eac, Da m-
s ad , Ge many) we e employed. E hanol (E OH, 98%, Pan eac,
Ba celona) was used as sol en in coppe ee click eac ion. Fo
he cha ac e iza ion o syn hesized dansyl de i a i e and 3-
azidop opan-1-amino, deu e a ed chlo o o m (CDCl
3
, 99.8%,
Sigma Ald ich, USA) was employed as sol en .
Gene al p ocedu e o he syn hesis o 3-azidop opan-1-amine
The syn hesis o 3-azidop opan-1-amine p ecu so was ca ied
ou employing eac ion condi ions simila o hose ou lined by
Ca boni e al.
49
A de ailed e sion o he me hodology can be
ound in ou p e ious wo k.
16
Syn hesis o dansyl de i a i e (Dns-N
3
)
Dansyl chlo ide (2.0 g, 7.4 mmol) was dissol ed in CH
2
Cl
2
(20
mL) and 3-azidop opan-1-amine (0.75 g, 7.4 mmol) and E 3N
(0.75 g, 7.4 mmol) we e added. The eac ion was euxed o
48 h unde s i ing. Ae cooling o oom empe a u e, wa e
was added and hen he solu ion was ex ac ed wi h CH
2
Cl
2
(3 ×
10 mL) and washed wi h a sa u a ed solu ion o NaCl (2 ×10
mL). O ganic laye s we e collec ed, d ied o e Na
2
SO
4
anhy-
d ous and he sol en was e apo a ed unde acuum o affo d
N-(3-azidop opyl)-5-(dime hylamino)naph halene-1-
sul onamide (Dns-N
3
) as a b ownish oil (1.8 g, 94%).
1
H-NMR
(CDCl
3
) 2.86 (s, 6H, 3xCH
3
), 3.05 ( , J=5.7 Hz, 2H, CH
2
), 3.27
( , J=5.7 Hz, 2H, CH
2
), 4.15 (s, 2H, NH
2
), 7.16 (d, J=7.5 Hz, 1H,
CH
a om
), 7.52 (dd, J=7.5 Hz, J=7.8 Hz, 2H, CH
a om
), 8.27 (dd, J
=8.5 Hz, J=7.5 Hz, 2H, CH
a om
), 8.53 (d, J=8.5 Hz, 1H,
CH
a om
);
13
C-RMN (CDCl
3
) 26.8 (CH
2
), 39.4 (CH
2
), 44.1 (CH
3
),
48.4 (CH
2
), 115.3 (C
a om
–H), 118.7 (C
a om
–C), 123.2 (C
a om
–H),
128.5 (C
a om
-H), 129.5 (Ca om-H), 129.9 (C
a om
-C), 130.6 (C
a om
-
H), 134.5 (C
a om
-S, C4), 152.0 (C
a om
-N).
Func ionaliza ion o PLLA lms
The ab ica ion and hyd olysis o PLLA was ca ied ou unde
he same condi ions as in ou p e ious wo k.
16
Hyd olyzed PLLA
(PLLA-COOH) subs a es we e submi ed o amida ion eac ion
ollowing wo s eps. Fi s ly, EDC$HCl (0.025 g, 0.13 mmol) and
NHS (0.015 g, 0.13 mmol) we e dissol ed in ace ic/ace a e buffe
solu ion o pH 5. Then, hyd olyzed PLLA lms we e in oduced
in o he p epa ed solu ion o 4 h unde cons an s i ing a
oom empe a u e. Ae , hey we e imme sed in a new
EDC$HCl (0.028 g, 0.15 mmol) ace ic/ace a e buffe solu ion o
2 h a 40 °C. Then, lms we e le o eac a oom empe a u e
o 24 h. Ae 24 h, lms we e in oduced in e hylendiamine (88
mL, 1.32 mmol) PBS buffe solu ion o 24 h a oom empe a-
u e. Finally, samples we e d ied unde acuum o 24 h be o e
second s ep. Fo he inco po a ion o he alkyne g oup,
a p e ious modica ion o p opiolic acid is equi ed. P opiolic
acid (83 mL, 1.33 mmol) is mixed wi h EDC$HCl (0.29 g, 1.50
mmol) and NHS (0.17 g, 1.50 mmol) in wa e (10 mL) o 4 h.
Then, unc ionalized PLLA lms we e in oduced in o he
solu ion o 24 h. Finally, samples we e washed wi h wa e and
d ied unde acuum o 24 h.
Dansyl de i a i e immobiliza ion by coppe - ee azide–alkyne
click eac ion
Dansyl de i a i e (0.20 g, 0.61 mmol) was dissol ed in E OH :
H
2
O (1 : 1 ol/ ol, 10 mL) and p e unc ionalized PLLA lms we e
in oduced on o he solu ion unde cons an s i ing o 124 h
a oom empe a u e. Finally, dansyla ed PLLA (PLLA-Dns) lms
we e washed wi h E OH and d ied unde acuum o 24 h.
Nuclea magne ic esonance (NMR)
The cha ac e iza ion o syn hesized 3-azidop opan-1-amino and
dansyl de i a i e was pe o med by p o on (
1
H-NMR) and
ca bon hi een (
13
C-NMR) nuclea magne ic esonance. The
analysis was ca ied ou a oom empe a u e in an AV-300
spec ome e (300 MHz o
1
H and 75.4 MHz o
13
C) (B uke ,
Rheins e en, Ge many) using deu e a ed chlo o o m as
sol en . Chemical shis(d) a e exp essed in pa s pe million
(ppm) ela i e o TMS using he esidual signal o he sol en
[7.26 ppm (
1
H) and 77.0 (
13
C)] as in e nal e e ence. Addi ion-
ally, coupling cons an s (J) a e exp essed in He z (Hz).
A enua ed o al eec ance-Fou ie ans o m in a ed (ATR-
FTIR)
ATR spec oscopy, combined wi h Fou ie ans o m, has been
used o analyze he ib a ional bands o diffe en bonds o all
he modied PLLA su aces, in o de o isualize he unc ional
g oups gene a ed ae each unc ionaliza ion. Fo his pu pose,
NICOLET Nexus FT-IR spec opho ome e (The mo Elec on
Co po a ion) has been used and he condi ions used we e as
ollows: 32 scans, be ween 4000 and 500 cm
−1
wa enumbe , y
4cm
−1
esolu ion.
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Scanning elec on mic oscopy (SEM)
SEM images o he su ace o p is ine and unc ionalized PLLA
subs a es we e ob ained by HITACHI S-4800 Scanning Elec on
Mic oscope. The mic oscope wo ked a 5 kV wi h an elec on
op ical magnica ion o 100 ×100.
X- ay pho oelec on spec oscopy (XPS)
Elemen al analysis o modied PLLA lms was ca ied ou by X-
ay pho oelec on spec oscopy SPECS sys em (XPS, SPECS
Su ace Nano Analysis, Be lin, Ge many) using ocus mono-
ch oma ic adia ion sou ce 500 wi h dual anode Al/Ag and i is
equipped wi h a 150 1D-DLD analyze (Phoibos, Be lin, Ge -
many). PLLA samples we e xed wi h s ainless s eel holde s and
ca bon ape du ing de measu emen s. Mo eo e , a ca bon
e e ence was used o do he measu emen s.
Con ocal uo escence mic oscopy
An Olympus FluoView FV500 con ocal lase scanning wi h 20
imes magnica ion (20×) was used o analyze he uo escence
o dansyla ed PLLA su aces ( o Dansyl l
exci a ion
z335 nm and
l
emission
z518 nm). Con ocal uo escence mic oscopy mic o-
g aphs we e aken wi hou any p e ious special sample
p epa a ion.
Fluo escence spec oscopy
Emission spec a o he PLLA lms we e eco ded exci ing in he
UV egion using an Edinbu gh Ins umen s spec ouo ime e
(FLSP920 model, Li ings on, U.K.) in on - ace congu a ion.
Samples we e posi ioned a 40° and 50° angles o he exci a ion
and emission beams, espec i ely, and we e il ed a a 30° angle
ela i e o he plane o med by he di ec ion o incidence and
de ec ion.
Conclusions
This s udy de eloped a su ace ea men s a egy o he
conjuga ion o uo escen dyes o biological compounds on o
polyme su ace using coppe ee click chemis y. The success
o su ace p e unc ionaliza ion was con med h ough ATR-
FTIR analysis, which showed he p esence o alkyne on he
su ace. Addi ionally, XPS, wa e con ac angles, SEM and
uo escence analysis we e employed o con m ha he
p oposed me hodology was ca ied ou success ully and he
uo escen compound was effec i ely immobilized. This wo k
p o ides a new ou e o conjuga e biological compounds by
coppe ee azide alkyne click eac ion, which o e come he
d awbacks o he cy o oxic me al. This new and e sa ile
app oach o bioconjuga ion using la es coppe - ee click
chemis y could ha e g ea po en ial in he biomedical eld. By
combining simple amida ion eac ions and ad anced coppe -
ee click eac ions, i is possible o c ea e bioac i a ed ma e-
ials ha can be used o a wide ange o applica ions, such as
implan s coa ings, biosenso s o issue enginee ing. In ac , his
s udy highligh s he po en ial o coppe - ee click chemis y in
de eloping no el conjuga ion s a egies and pa es he way o
u u e esea ch o explo e he p ac ical applica ions o his
echnique in biomedical esea ch.
Au ho con ibu ions
Concep ualiza ion, J. S.-B. and I. M.-B.; me hodology, J. S.-B., A.
G.-G. and M. D.-G.; in es iga ion, J. S.-B., A. G.-G., M. D.-G. and
I. M.-B.; w i ing—o iginal d a, J. S.-B.; w i ing— e iew and
edi ing, I. M.-B.; supe ision, I. M.-B. and J. L. V.-V.; unding
acquisi ion, J. L. V.-V. All au ho s ha e ead and ag eed o he
published e sion o he manusc ip .
Conflic s o in e es
The e a e no conic s o decla e.
Acknowledgemen s
This esea ch was unding by Basque Go e nmen (ELKARTEK
p og am, Depa men o De elopmen and In as uc u es o
he Basque Coun y, KK-2021-00040, KK-2021-00082 and KK-
2022-00057; G upos Consolidados IT1756-22). Mo eo e ,
au ho s hank o echnical and human suppo p o ided by
SGIke (UPV/EHU/ERDF, EU).
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