Polyme Deg ada ion and S abili y 193 (2021) 109726
Con en s lis s a ailable a ScienceDi ec
Polyme Deg ada ion and S abili y
jou nal homepage: www.else ie .com/loca e/polymdeg ads ab
No el poly(azoamide iazole)s con aining win azobenzene uni s in
he backbone. Syn hesis, cha ac e iza ion, and in i o deg ada ion
s udies
Ad ián Suá ez-C uz, Inmaculada Molina-Pinilla, Khalid Hakkou, C is ian Rangel-Núñez,
Manuel Bueno-Ma ínez
∗
Depa amen o de Química O gánica y Fa macéu ica. Facul ad de Fa macia. Uni e sidad de Se illa, C/ P o eso Ga cía González 2, 41012 Se illa, Spain
a i c l e i n o
A icle his o y:
Recei ed 5 June 2021
Re ised 15 Sep embe 2021
Accep ed 19 Sep embe 2021
A ailable online 28 Sep embe 2021
Keywo ds:
Click polyme iza ion
Deg adable polyme
Azo polyme
Azobenzene
Di hioni e educ ion
Biodeg adable polyme
a b s a c
We desc ibe he syn hesis and cha ac e iza ion o ou new ligh and educ ion sensi i es poly(azoamide
iazole)s, in which he azobenzene uni s a e ound along he main chain o he mac omolecule. These
polyme s we e p epa ed by he azide-alkyne cycloaddi ion eac ion ca alyzed wi h coppe (I) (CuAAC).
They we e ob ained in high yield and wi h appa en molecula weigh s in he ange om 95 o 148 kDa.
All poly(azoamide iazole)s a e soluble in pola ap o ic sol en s, and wo o hem a e also soluble
in chlo o o m showing good coa ing and film- o ming p ope ies. They we e cha ac e ized by Fou ie
ans o m in a ed, nuclea magne ic esonance (NMR), ul a iole - isible spec oscopy and gel pe me-
a ion ch oma og aphy (GPC). The pho oisome iza ion s udy o he syn hesized polyme s has been ca ied
ou by UV–Vis spec oscopy, as well as hei ans - cis - ans e e sibili y beha io . Di e en ial scanning
calo ime y (DSC) and hemog a ime ic analysis (TGA) we e used o in es iga e hei he mal p ope ies.
Resul s show ha he polyme s we e amo phous and s able up o 300 °C unde ni ogen. The hyd oly ic
deg ada ion o films o hese polyme s has been s udied in i o unde a ious condi ions o pH and em-
pe a u e and was moni o ed by GPC. Fu he mo e, he p esence o azo uni s along he polyme backbone
as clea able g oups p o ides access o hei deg ada ion by educ ion. In his sense, he deg ada ion o
polyme s has also been s udied using sodium di hioni e as a mimic o he enzyme azo educ ase. The e-
sul s o hese s udies show ha he polyme s a e s able enough unde hyd oly ic physiological condi ions,
bu hey deg ade apidly when sodium di hioni e is used. A p elimina y s udy o biocompa ibili y o poly-
me s PAAT1 and PAAT4 has been ca ied ou . A hemolysis s udy wi h human ed blood cells (hRBC) and
a cy o oxici y s udy wi h human gingi al fib oblas s (HGnF) ha e been ca ied ou . The esul s ob ained
sugges ha hese polyme s could be good candida es o be used as d ug coa ing ma e ials.
©2021 The Au ho (s). Published by Else ie L d.
This is an open access a icle unde he CC BY-NC-ND license
( h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/ )
1. In oduc ion
The design and p epa a ion o d ug deli e y sys ems ha al-
low he elease o bioac i e compounds in a con olled and local-
ized way ha e ecei ed a g ea deal o a en ion in ecen decades.
These sys ems, which we e ini ially sensi i e o a pa icula s imu-
lus, such as ligh , pH, oxidizing o educing condi ions, ce ain en-
zymes, o empe a u e [1–9] , ha e been he basis o de elop sys-
ems ha espond o dual o mul iple signals, such as edox and
pH [10] , empe a u e and pH [11] , empe a u e and enzyme [12] ,
∗Co esponding au ho .
E-mail add ess: [email p o ec ed] (M. Bueno-Ma ínez).
educing condi ions and ligh [13] , enzyme and pH [14–16] ; ligh
and pH [17–19] ; ligh and enzyme [20] and o he s [21] .
Azo de i a i es ha e been widely used o se e al applica-
ions, o example, as su ac an s [22] , gela o s [23] , liquid c ys-
als [24] o biocides [25] . Azobenzene de i a i es ha e also been
used o he p epa a ion o s imulus-sensi i e d ug deli e y sys-
ems based on hei pho ochemical beha io [26] . As i is well
known, azobenzenes a e pho osensi i e ch omopho es ha ha e an
unique sensi i i y o ligh (o hea ), which causes e e sible ans -
cis pho oisome iza ion [27] leading o significan changes in hei
molecula size and dipole momen s [28] . This pho oisome iza ion
p ocess can also ha e an impac on o he cha ac e is ics o he
azo g oup, such as i s sensi i i y o educ ion. Boulègue and col-
labo a o s ha e obse ed an inc ease in he educ ion a e o cis
h ps://doi.o g/10.1016/j.polymdeg ads ab.2021.109726
0141-3910/© 2021 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license
( h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/ )
A. Suá ez-C uz, I. Molina-Pinilla, K. Hakkou e al. Polyme Deg ada ion and S abili y 193 (2021) 109726
azobenzene in ela ion o he ans isome [29] , which may allow
a g ea e con ol o he deg ada ion o his ype o ma e ial.
The e a e a wide a ie y o applica ions in biomedicine ha use
he sensi i i y o azo polyme s o ligh and educ ion [ 14 , 30–38 ].
Al hough azobenzene de i a i es can also be used in any umo
issue ha su e s om hypoxic condi ions o ha exp esses azo e-
duc ases, as o example, in he case o li e cance cells [39] , we
a e in e es ed in hei use in he design and p epa a ion o sys-
ems ha allow he ec o iza ion o d ugs o he colon h ough
o al adminis a ion. Many compounds con aining azobenzene in
hei s uc u e ha e been s udied o enabling o al adminis a ion
and colonic elease o specific d ugs. Thus, a ious non-polyme ic
p od ugs [40–44] , as well as mac omolecula p od ugs which con-
ain he azo g oup in he main o side chain, ha e been de eloped
[45–53] . The bioac i e compounds can be p o ec ed by polyme ic
coa ings o ma ixes based on azobenzene o each he colonic de-
li e y. In seminal wo k, Sa an and cowo ke s fi s used his ap-
p oach o o al adminis a ion o azo polyme -coa ed pep ide d ugs
[54] . In his way, d ugs a e p o ec ed om he ad e se en i on-
men al condi ions aced by hese bioac i e compounds in he gas-
oin es inal ac , delaying he elease un il he combina ion o
azo-de i a i e ma e ials and d ugs eaches he colon. Azo com-
pounds deg ade in he colon because mic obiome exc e es he en-
zyme azo educ ase, which can educe selec i ely he azobenzene
g oup o a oma ic amines [ 36 , 55–57 ]. Al hough azo educ ase en-
zymes ha e he mos sui able cha ac e is ics o ca y ou he e-
duc ion o azo g oup, managing his ype o enzyme a labo a o y
condi ions can be difficul and esul s hea ily depend on he gene
codifica ion o he enzyme. Fo his eason, o he non-biological
compounds such as hyd azine hyd a e [ 58 , 59 ] o sodium di hion-
i e [ 30 , 31 , 35 , 60 , 61 ] can be used o he same pu pose. In pa ic-
ula , i is common o find he use o sodium di hioni e since his
educing agen ac s in a gen le way, i is cheap and po en ially bio-
o hogonal [38] .
The design o azo polyme s ha exhibi adequa e sensi i i y un-
de biological and chemical condi ions is an impo an esea ch
objec i e wi h implica ions in di e en fields. We p e iously e-
po ed on he syn hesis o linea copoly(azoes e iazole)s con-
aining di e en amoun s o he p od ug olsalazine in he polyme
backbone [53] . He ein, we desc ibe he syn hesis and cha ac e iza-
ion o ou new ligh and educ ion sensi i e poly(azoamide ia-
zole)s, in which he azo uni s a e ound along he main chain o
he mac omolecule. These polyme s we e p epa ed by he azide-
alkyne cycloaddi ion eac ion ca alyzed wi h coppe (I) (CuAAC).
The hyd oly ic deg ada ion o hese polyme s has been s udied un-
de a ious condi ions o pH and empe a u e. Likewise, he deg a-
da ion using sodium di hioni e as a mimic o he enzyme azo e-
duc ase has also been s udied.
2. Ma e ial and me hods
2.1. Ma e ials
All sol en s and eagen s we e ob ained om Me ck and we e
used wi hou u he pu ifica ion. 96-wells pla es, Pe i dishes,
bo ine e al se um, Dulbecco’s Modified Eagle’s Medium, an ibi-
o ics, phospha e bu e ed saline (PBS) and ypsin we e ob ained
om The mo Fishe Scien ific (Massachuse s, USA). Human gingi-
al fib oblas s we e pu chased om Innop o (Vizcaya, Spain).
2.2. Measu emen s
Thin-laye ch oma og aphy (TLC) was pe o med on Silica Gel
60 F254 (E. Me ck) wi h de ec ion by UV ligh o cha ing wi h
H
2
SO
4
o phosphomolybdic acid. Flash column ch oma og aphy
was pe o med using E. Me ck Silica Gel 60 (230–400 mesh).
Fou ie ans o m in a ed (FTIR) spec a we e eco ded on a JASCO
FT/IR-4200 spec ome e in he wa enumbe ange om 650 o
40 0 0 cm
−1 using films o KB disks.
1
H and
13
C NMR spec a
we e eco ded in he CITIUS o he Uni e sidad de Se illa using
a B uke AV300, B uke AMX-500 o B uke AVIII-700 spec ome-
e s. Chemical shi s a e epo ed as pa s pe million (ppm) and
a e e e enced o he esidual sol en signals as he in e nal s an-
da d. Two-dimensional
1
H–1
H homonuclea and
13
C-
1
H he e onu-
clea shi co ela ion spec a we e eco ded wi h he COSY and
HETCOR pulse sequences, espec i ely.
Elemen al analyses we e ca ied ou in he Mic oanalysis Labo-
a o ies o he CITIUS Se ice a he Uni e sidad de Se illa. Chemi-
cal ioniza ion (CI) and as -a om bomba dmen mass spec a we e
pe o med on a Mic omass Au ospec spec ome e . FABMS spec-
a we e ob ained using hioglyce ol-NaI as a ma ix. The he mal
beha io o he polyme s was examined by di e en ial scanning
calo ime y (DSC) using a TA DSC Q200 Ins umen , calib a ed wi h
indium. Samples o abou 2–3 mg we e hea ed a a a e o 10
°C/min unde a ni ogen flow a e o 20 mL/min and cooled o
−35 °C. The mog a ime ic analyses (TGA) we e ca ied ou by a
SDT Q600 TA ins umen a a hea ing a e o 10 °C/min unde a
ni ogen flow o 100 mL/min, and he empe a u e ange was om
oom empe a u e o 600 °C. The size exclusion ch oma og aphy
ins umen consis ed o a Wa e s appa a us equipped wi h a Wa-
e s 2414 e ac i e-index de ec o and wo mS y agel HR columns
(7.8 mm ×300 mm) linked in se ies, he mos a ed a 60 °C. N,
N -dime hyl o mamide con aining 0.5 mg/mL LiB was used as he
eluen wi h a flow a e o 1.0 mL/min. Twel e polys y ene samples
o na ow molecula weigh dis ibu ion we e used o calib a e he
appa a us. Abso bances a 540 and 570 nm we e measu ed using a
Bio ek Syne gy HT pla e eade (Ve mon , USA). UV– isible spec a
we e eco ded using a spec opho ome e UV-1280 (Shimadzu) in
qua z cu e es wi h 10 mm leng h o he op ical pa hway. The UV
ligh sou ce consis ed o OSRAM Ul a i alux 300 W, se a 20 cm
(2 mW / cm
2
) om he sample. The isible ligh sou ce was a
Scho KL1500 LCD se a 1 cm om he sample.
2.3. Syn hesis o he monome s
E hyl 4-(4-hyd oxyphenyl)azobenzoa e (1) [62] . A solu ion o
NaNO
2
(4.8 g, 56.4 mmol) in wa e (30 mL) was added d opwise o
a s i ed suspension o e hyl 4-aminobenzoa e (9.3 g, 56.2 mmol)
in HCl 2 M (100 mL) p e iously cooled a ound 0–5 °C. A e wa ds,
phenol (6.34 g, 67.3 mmol) was added. The eac ion mix u e was
kep cooled and s i ed o 90 min. Then he eac ion mix u e was
neu alized wi h a sa u a ed solu ion o NaHCO
3
and fil e ed. Fi-
nally, he solid was ec ys allized in me hanol: wa e (1:1), ob ain-
ing 1 as a b ownish c ys alline solid. Yield: 9.4 g (63%), m. p. 162–
164 °C; IR:
max 3389 (OH), 1691 (CO), 1591 cm
−1 (A ); NMR da a
(300 MHz, CDCl
3
):
1
H, δ8.17 (d, 2H, H-b), 7.95–7.85 (2d, 4H, H-c,
H- ), 6.97 (d, 2H, H-g), 5.95 (bs, 1H, OH), 4.42 (q, 2H, C H
2
), 1.43 ( ,
3H, C H
3
).
E hyl 4-(4-p opynyloxyphenyl)azobenzoa e (2) [33] . To a
s i ed suspension o 1 (16 g, 59.2 mmol) and K
2
CO
3
(24 g,
173.6 mmol) in d y ace oni ile (572 mL), p opa gyl b omide
(240 mmol, 26 mL) was added d opwise and he eac ion mix-
u e was efluxed o 24 h. Then he eac ion mix u e was fil e ed
and he solid was ec ys allized in e hanol, ob aining an o ange
c ys alline solid. Yield: 14.2 g (78%), m. p. 108–110 °C; IR:
max
3252 (HC
≡), 2126 (C
≡C), 1702 (CO), 1599 cm
−1 (A ); NMR da a
(300 MHz, CDCl
3
): 1H, δ8.17 (m, 2H, H-b), 7.95 (m, 2H, H- ), 7.90
(m, 2H, H-c), 7.01 (m, 2H, H-g), 4.77 (d, 2H, J 2.0 Hz, H-i), 4.40 (q,
2H, J 7.12 Hz, C H
2
CH
3
), 2.57 ( , 1H, ≡C H ), 1.42 ( , 3H, C H
3
);
13
C,
δ166.12 (CO), 160.40 (C-h), 155.21 (C-d), 147.49 (C-e), 131.70 (C-a),
130.55 (C-b), 125.08 (C- ), 122.40 (C-c), 115.24 (C-g), 77.92 (C
≡C
–C),
76.14 (
≡C H), 61.21 ( C H
2
CH
3
), 56.04 (C-i), 14.36 (CH
3
).
2
A. Suá ez-C uz, I. Molina-Pinilla, K. Hakkou e al. Polyme Deg ada ion and S abili y 193 (2021) 109726
4-(4-p opynyloxyphenyl)azobenzoic acid (3) [33] . A suspen-
sion o 2 (1 g, 3.24 mmol) in 49 mL o e hanol was efluxed
o 10 min. A e wa ds, a solu ion o KOH (0.292 g, 5.2 mmol) in
25 mL o wa e was added, and he eac ion mix u e was hea ed a
85 °C o 2 h. The suspension was fil e ed and HCl 2 M was added
o he esul ing solu ion un il pH 3–4. The o med p ecipi a e was
fil e ed and d ied, ob aining an o ange solid. Yield: 815 mg (90%).
NMR da a (300 MHz, DMSO–d
6
):
1
H, δ8.13 (d, 2H, H-b), 7.98–7.85
(2d, 4H, H-c, H- ), 7.21 (d, 2H, H-g), 4.95 (d, 2H, H-i), 3.65 ( , 1H,
≡C H );
13
C, δ167.23 (CO), 160.86 (C-h), 154.84 (C-d), 147.09 (C-e),
132.77 (C-a), 131.06 (C-b), 125.32 (C- ), 122.75 (C-c), 116.06 (C-g),
79.23 (C
≡C
–C), 79.18 (
≡C H), 56.38 (C-i).
Succinimide 4-(4-p opynyloxyphenyl)azobenzoa e (4) [63] . To
a mix u e o 3 (1 g, 3.56 mmol) and N -hyd oxysuccinimide
(0.410 g, 3.56 mmol) in d y ace oni ile (15 mL) and dime hyl o -
mamide (5 mL), EDC
•HCl (0.85 g, 4.44 mmol) was added. The mix-
u e was s i ed unde a gon a mosphe e o 24 h. A e wa ds, he
eac ion mix u e was d opped in wa e , and he esul ing p ecipi-
a e was fil e ed and d ied o ob ain an o ange solid. Yield: 1.2 g
(87%), IR:
max 3271 (HC
≡), 2134 (C
≡C), 1765, 1729 (CO), 1597
cm
−1 (A ); NMR da a (300 MHz, CDCl
3
):
1
H, δ8.28 (d, 2H, H-b),
8.00–7.90 (2d, 4H, H-c, H- ), 7.12 (d, 2H, H-g), 4.80 (d, 2H, H-i),
2.93 (s, 4H, succ), 2.58 ( , 1H,
≡C H ).
2-(2-me hoxye hoxy)e hyl me hanesul ona e (5) [64] . To
a s i ed mix u e o die hylene glycol me hyl e he (2 g,
16.65 mmol), ie hylamine (2.53 g, 25 mmol) and d y
dichlo ome hane (40 mL) unde a gon a mosphe e a 0 °C, mesyl
chlo ide (1.5 mL, 18.7 mmol) was added d opwise. A e 1.5 h, he
eac ion mix u e was washed wi h wa e (10 mL), HCl 2 N (5 mL),
sa u a ed NaHCO
3
solu ion un il basic pH, and wa e . O ganic
phase was d ied wi h Na
2
SO
4
and concen a ed. The esidue was
pu ified by column ch oma og aphy (e hyl ace a e) ob aining a
colou less sy up. Yield: 2.9 g (87%). NMR da a (300 MHz, CDCl
3
):
1
H, δ4.38 (m, 2H, MsOC H
2
CH
2
), 3.77 (m, 2H, MsOCH
2
C H
2
),
3.66 (m, 2H, CH
2
C H
2
OCH
3
), 3.55 (m, 2H, C H
2
CH
2
OCH
3
), 3.38 (s,
3H, CH
3
), 3.07 (s, 3H, Ms);
13
C, δ71.83 ( C H
2
CH
2
OCH
3
), 70.61
(CH
2
C H
2
OCH
3
), 69.18 (MsO C H
2
C), 69.05 (MsOCH
2
C ), 58.98 (CH
3
),
37.66 (Ms).
1,12-Diazido-4,9-dioxadodecan-2,11-diol (6) [65] . A mix u e o
1,4-bu anediol diglycidyl e he (3.3 g, 16.0 mmol), e abu ylam-
monium sul a e (0.68 g, 2.0 mmol), and sodium azide (13 g,
200 mmol) in wa e -dioxane (1:1, 200 mL) was efluxed o 4 h.
Then, he eac ion mix u e was concen a ed o abou hal i s ol-
ume and ex ac ed wi h e hyl ace a e (3 ×100 mL). The com-
bined o ganic phase was d ied wi h anhyd ous sodium sul a e, fil-
e ed, and concen a ed o d yness unde educed p essu e o ob-
ain a sy upy esidue which was pu ified by column ch oma og a-
phy ( e –bu yl me hyl e he :hexane, 1:1), gi ing a e e apo a ion
o he sol en s 6 as a solid (3.2 g, 68%), m.p. 56–58 °C, IR:
max
3400 (OH), 2087 cm
−1 (N
3
); NMR da a (CDCl
3
):
1
H, δ3.99–3.88
(m, 2H, H-2), 3.55–3.30 (m, 12H, H-1, H-3, H-4), 2.72 (d, 2H, OH),
1.69–1.63 (m, 4H, H-5);
13
C, δ71.92, 71.30 (C-3, C-4), 69.65 (C-2),
53.41 (C-1), 26.27 (C-5).
Diazide monome 7. To a mix u e o 6 (2.4 g, 8.32 mmol), pow-
de ed KOH (2.3 g, 41 mmol), e abu ylammonium b omide (1.07 g,
3.32 mmol) and wa e (1 mL), a solu ion o 5 (4.0 g, 20.18 mmol)
in oluene (10 mL) was added. The eac ion mix u e was efluxed
o 24 h. A e wa ds dichlo ome hane was added, and he esul -
ing solu ion was fil e ed and concen a ed. The esidue was pu i-
fied by column ch oma og aphy (hexane: ace one 3:1), ob aining a
colou less sy up. Yield: 2.3 g (56%), IR:
max 2094 (N
3
), 1106 cm
−1
(C
–O); NMR da a (300 MHz, CDCl
3
):
1
H, δ3.85–3.72 (m, 4H, H-6),
3.69–3.64 (m, 10H, H-2,7,8), 3.68–3.44 (m, 12H, H-3,4,9), 3.40 (s,
6H, H-10), 3.39–3.33 (m, 4H, H-1), 1.6 (m, 4H, H-5);
13
C, δ78.49
(C-2), 71.94 (C-9), 71.33 (C-4), 70.80 (C-7), 70.57 (C-8), 70.12 (C-3),
69.86 (C-6), 59.03 (C-10), 51.94 (C-1), 26.29 (C-5). Anal. Calcd o
C
20
H
40
N
6
O
8
: C, 48.77; H, 8.19; N, 17.06. Found: C, 48.67; H, 8.351;
N, 16.96. HRMS: m/z 515.2796 (calcd. o [M]
+
: 515.2800).
1,12-Diamino-4,9-dioxadodecan-2,11-diol (8). Compound 6
(0.577 g, 2 mmol), Pd/C 10% (0.286 g) and me hanol (50 mL) we e
added o a hyd ogena ion flask. Hyd ogena ion was ca ied ou a
40 psi o 3 h. Then, he mix u e was fil e ed h ough a celi e pad,
and he sol en e apo a ed un il d yness, ob aining a waxy pu -
ple solid. Yield: 0.43 g (90%). NMR da a (300 MHz, DMSO–d
6
):
1
H,
δ3.53–3.40 (m, 2H, H-2

), 3.40–3.30 (m, 4H, H-4

), 3.30–3.24 (m,
4H, H-3

), 2.57 (dd, 2H, J
1’a,
1

b
12.8 Hz, J
1’a,
2
 4.4 Hz, H-1

a), 2.41
(dd, 2H, J
1’b,
2
 6.7 Hz, H-1

b), 1.51 (m, 4H, H-5

);
13
C, δ73.48 (C-
3

), 71.37 (C-2

), 70.75 (C-4

), 45.63 (C-1

), 26.45 (C-5

). HRMS: m/z
237.1809 (calcd. o [M]
+
: 237.1809).
2,11-bis(2-(2-me hoxye hoxy)e hoxy) −4,9-dioxadodecan-1,12-
diamine (9). Compound 7 (0.575 g, 1.17 mmol), Pd/C 10% (0.173 g)
and me hanol (30 mL) we e added o a hyd ogena ion flask.
Hyd ogena ion was ca ied ou a 40 psi o 3 h. Then, he mix u e
was fil e ed h ough a celi e pad, and he sol en e apo a ed un il
d yness, ob aining a colou less sy up. Yield: 0.460 g (89%). NMR
da a (300 MHz, CDCl
3
):
1
H, δ3.89–3.67 (m, 4H, H-6

), 3.67–3.59
(m, 8H, H-7

,8

), 3.59–3.50 (m, 4H, H-9

), 3.50–3.37 (m, 10H,
H-2

,3

,4

), 3.38 (s, 6H, H-10

), 2.90–2.63 (m, 4H, H-1

), 1.68 (bs,
4H, NH), 1.61 (m, 4H, H-5

);
13
C, δ80.67 (C-2

), 71.95 (C-9

), 71.38,
71.30, 70.87, 70.47 (C-3

, 4

, 7

, 8

), 69.52 (C-6

), 59.03 (C-10

),
43.45 (C-1

), 26.37 (C-5

). HRMS: m/z 441.3161 (calcd. o [M]
+
:
441.3170).
Dialkyne monome 10. To a solu ion o 4 (0.2 g, 0.53 mmol)
in d y dime hyl o mamide (1 mL) unde a gon a mosphe e, 3,6-
dioxaoc an-1,8-diamine (40 μL, 0.266 mmol,) and ie hylamine
(111 μL, 0.8 mmol) we e added. The esul ing mix u e was s i ed
o 24 h. Then wa e was added, p ecipi a e was fil e ed and pu i-
fied by column ch oma og aphy (dichlo ome hane-me hanol 20:1
o 1:1), ob aining an o ange solid. Yield: 0.152 g (85%), m. p. 193–
196 °C, IR:
max 3297 (HC
≡, NH), 3064 (A ), 2134 (C
≡C), 1625
(CO), 1596 cm
−1 (A ); NMR da a (300 MHz, DMSO–d
6
):
1
H, δ
8.72 ( , 2H, NH), 8.07 (d, 2H, H-b), 7.96 (d, 2H, H-c), 7.92 (d, 2H,
H- ), 7.23 (d, 2H, H-g), 4.98 (d, 4H, J 2.3 Hz, H-i), 3.69 ( , 2H,
≡C H ), 3.66–3.56 (m, 8H, H-2

, 3

), 3.56–3.44 (m, 4H, H-1

);
13
C, δ
166.08 (CO), 160.69 (C-h), 153.82 (C-d), 147.08 (C-e), 136.47 (C-a),
128.91 (C-b), 125.18 (C- ), 122.55 (C-c), 116.01 (C-g), 79.21 ( C
≡C ),
70.10 (C-3

), 69.34 (C-2

), 56.37 (C-i), 39.8 (C-1

). Anal. Calcd o
C
38
H
36
N
6
O
6
.H
2
O: C, 66.08; H, 5.55; N, 12.17. Found: C, 65.81; H,
5.55; N, 12.11.
Dialkyne monome 11. To a solu ion o 4 (0.438 g, 1.16 mmol)
in d y dime hyl o mamide (2.2 mL) unde a gon a mosphe e, 8
(0.137 g, 0.58 mmol) and ie hylamine (0.24 mL, 1.75 mmol) we e
added. The mix u e was s i ed o 24 h. Then wa e was added,
p ecipi a e was fil e ed and d ied o ob ain an o ange solid. Yield:
0.34 g (76%), m. p. 172–177 °C, IR:
max 3292 (HC
≡, OH), 2124
(C
≡C), 1625 (CO), 1600 cm
−1 (A ); NMR da a (300 MHz, DMSO–
d
6
):
1
H, δ8.56 ( , 2H, NH), 8.05 (d, 2H, H-b), 7.94 (d, 2H, H-c), 7.90
(m, 2H, H- ), 7.20 (d, 2H, H-g), 4.97 (d, 2H, J 5.2 Hz, OH), 4.94 (d,
4H, J 2.3 Hz, H-i), 3.82 (m, 2H, H-2

), 3.65 ( , 2H, ≡C H ), 3.51–3.16
(m, 12H, H-1

, 3

,4

), 1.56 (m, 4H, H-5

);
13
C, δ166.22 (CO), 160.70
(C-h), 153.82 (C-d), 147.11 (C-e), 136.67 (C-a), 128.98 (C-b), 125.19
(C- ), 122.51 (C-c), 116.04 (C-g), 79.23 ( C
≡C ), 73.71 (C-3

), 70.89 (C-
4

), 68.79 (C-2

), 56.38 (C-i), 43.92 (C-1

), 26.47 (C-5

). Anal. Calcd
o C
42
H
44
N
6
O
8
: C, 66.30; H, 5.83; N, 11.05. Found: C, 66.04; H,
6.174; N, 11.03.
Dialkyne monome 12. To a solu ion o 4 (0.168 g,
0.4 4 4 mmol) in d y dime hyl o mamide (0.84 mL) unde a gon
a mosphe e, 9 (0.098 g, 0.222 mmol) and ie hylamine (0.1 mL,
0.64 mmol) we e added. The mix u e was s i ed o 24 h. Then
wa e was added, he p ecipi a e was decan ed and d ied o ob ain
an o angish sy up. Yield: 0.19 g (89%). NMR da a (300 MHz, CDCl
3
):
1
H, δ8.07–7.85 (m, 6H, H-b,c, ), 7.31 ( , 2H, NH), 7.10 (d, 2H, H-g),
3
A. Suá ez-C uz, I. Molina-Pinilla, K. Hakkou e al. Polyme Deg ada ion and S abili y 193 (2021) 109726
Table 1
GPC
a
da a o poly(azoamide iazole)s.
Polyme Yield (%) M
w M
w
/M
n
PAAT1 99 148,500 1.24
PAAT2 93 138,000 1.31
PAAT3 85 131,000 1.27
PAAT4 84 95,000 1.46
a De e mined by GPC analysis wi h
polys y ene s anda ds. Measu ed in DMF-
LiB .
Table 2
The mal analysis da a o poly(azoamide iazole)s.
Polyme T
g
a
( °C) T
10%
b
( °C) T
dec
b
( °C)
PAAT1 35.5 318.3 319.5, 382.6
PAAT2 87.7 325.2 304.8, 371.3
PAAT3 2.1 323.6 322.9, 385.0
PAAT4 2.4 325.4 325.8, 390.8
a De e mined by DSC, second hea ing.
b Measu ed by TGA.
Table 3
Quali a i e solubili ies o poly(azoamide iazole)s.
Sol en PAAT1 PAAT2 PAAT3 PAAT4
TBME ––––
Hexane ––––
E hyl ace a e - ––±
Me hanol - ––±
Ace one –––++
Chlo o o m ++ ––++
Wa e ––––
DMF ++ ±+ ++
DMSO ++ ±++ ++
(-) insoluble, ( ±) sligh ly soluble, ( + ) soluble on wa ming,
( ++ ) soluble a oom empe a u e.
4.78 (d, 4H, J 2.4 Hz, H-i), 3.97–3.30 (m, 30H, H-1

−4

, H-6

−9

),
3.27 (s, 6H, H-10

), 2.57 ( , 2H, ≡C H ), 1.66 (m, 4H, H-5

);
13
C, δ
166.96 (CO), 160.26 (C-h), 154.25 (C-d), 147.49 (C-e), 136.07 (C-a),
128.16 (C-b), 124.96 (C- ), 122.55 (C-c), 115.23 (C-g), 77.94 (C
–C
≡C),
77.78 (C-2

), 76.08 (
≡C H), 71.90, 71.81, 71.45, 70.79, 70.35, 69.6 (C-
3

,4

,9

,8

,7

,6

), 58.90 (C-10

), 56,05 (C-i), 41.56 (C-1

), 26.38 (C-5

).
Anal. Calcd o C
52
H
64
N
6
O
12
: C, 64.71; H, 6.68; N, 8.71. Found: C,
62.60; H, 7.06; N, 8.49. HRMS: m/z 987.4467 (calcd. o [ M + Na]
+
:
987.4474).
2.4. Syn hesis o he polyme s
2.4.1. Gene al p ocedu e o he syn hesis o poly(azoamide iazole)s
(PAATn)
S oichiome ic amoun s o diazide and dialkyne we e mixed
and dissol ed in dime hyl sul oxide. Wa e was added un il a sligh
cloudiness appea ed. A e wa ds CuSO
4
pen ahyd a e (20%) and
sodium asco ba e (40%) we e added, and he eac ion mix u e was
s i ed and hea ed o 50 °C unde a gon a mosphe e o 24 h. Re-
ac ion mix u e was hen p ocessed in di e en ways depending on
he polyme o med. Polyme PAAT1 was eco e ed by p ecipi a-
ion in e –bu yl me hyl e he (TBME), ollowed by e-dissolu ion
in dichlo ome hane and p ecipi a ion in ace one. Polyme s PAAT2
and PAAT3 we e isola ed by fil a ion on a glass fil e and washed
wi h wa e and ace one. These polyme s we e pu ified by disso-
lu ion in dime hyl sul oxide and p ecipi a ion in TBME. Polyme
PAAT4 was also isola ed by fil a ion bu pu ified by dissolu ion
in CH
2
Cl
2
and p ecipi a ion in TBME. Tables 1–4 show he s ud-
ied cha ac e is ics o he p epa ed polyme s. The in a ed and NMR
spec oscopy da a a e lis ed below.
Table 4
UV Abso p ion da a o he
PAATn polyme s in DMSO.
Compound λ(nm)
10 429, 357, 255
11 437, 358, 255
12 449, 357, 255
PAAT1 426, 360, 255
PAAT2 432, 359, 255
PAAT3 432, 360, 255
PAAT4 442, 361, 256
Fig. 1. Syn hesis o diazides and diamines.
PAAT1 . IR:
max 3326 (NH), 1644 cm
−1 (CO); NMR da a
(500 MHz, CDCl
3
):
1
H, δ7.95 (s, 2H, H-j), 8.00–7.80 (m, 12H, H-
b,c, ), 7.10 (d, 4H, H-g), 6.90 (bs, 2H, NH), 5.30 (s, 4H, H-i), 4.64
(bd, 2H, H-1a), 4.42 (dd, 2H, J
1b,2
6.94 Hz, J
1a,1b
14.0 Hz, H-1b),
3.86 (bs, 2H, H-2), 3.80–3.30 (m, 36H, H-1

,2

,3

,3,4,6,7,8,9), 3.30
(s, 6H, H-10), 1.63 (bs, 4H, H-5);
13
C, δ166.96 (CO), 161.19 (C-
h), 154.37 (C-d), 147.20 (C-e), 143.18(C-k), 135.67 (C-a), 127.98 (C-
b), 125.08 (C- ), 124.90 (C-j), 122.63 (C-c), 115.16(C-g), 77.73 (C-
2), 71.87, 71.43, 70.65, 70.41, 70.31, 69.84, 69.68 (C-2

,3

,3,4,6,7,8,9),
62.21 (C-i), 58.95 (C-10), 51.62 (C-1), 39.88 (C-1

), 26.30 (C-5).
PAAT2 . IR:
max 3309 (OH, NH), 1630 cm
−1 (CO); NMR da a
(500 MHz, DMSO–d
6
):
1
H, δ8.67 (bs, 2H, NH), 8.19 (s, 2H, H-j),
4
A. Suá ez-C uz, I. Molina-Pinilla, K. Hakkou e al. Polyme Deg ada ion and S abili y 193 (2021) 109726
Fig. 2. Syn hesis o dialkyne monome s.
8.02 (d, 4H, H-b), 7.91, 7.87 (2d, 8H, H-c, ), 7.25 (d, 4H, H-g), 5.32
(d, J 5.1 Hz, OH), 5.26 (s, 4H, H-i), 4.48 (bd, 2H, H-1a), 4.31 (dd,
2H, J
1b,2
7.8 Hz, J
1a,1b
13.6 Hz, H-1b), 3.99 (bs, 2H, H-2), 3.62–
3.20 (m, 20H, H-1

,2

,3

,3,4), 1.56 (bs, 4H, H-5);
13
C, δ166.11 (CO),
161.65 (C-h), 153.83 (C-d), 146.80 (C-e), 142.27(C-k), 136.37 (C-a),
128.89 (C-b), 126.21 (C-j), 125.29 (C- ), 122.51 (C-c), 115.87 (C-g),
72.54, 70.94, 70.07, 69.31, 68.71 (C-2

,3

,3,4,2), 61.97 (C-i), 53.43 (C-
1), 40.84 (C-1

), 26.32 (C-5).
PAAT3 . IR:
max 3399 (OH, NH), 1644 cm
−1 (CO); NMR da a
(500 MHz, DMSO–d
6
):
1
H, δ8.54 (bs, 2H, NH), 8.23 (s, 2H, H-
j), 8.04 (bd, 4H, H-b), 7.92, 7.89 (2bd, 8H, H-c, ), 7.25 (d, 4H,
H-g), 5.28 (s, 4H, H-i), 4.96 (bs, 2H, OH), 4.58 (m, 2H, H-1a),
4.43 (m, 2H, H-1b), 3.84 (m, 4H, H-2

,2), 3.60–3.10 (m, 36H, H-
1

,3

,4

,3,4,6,7,8,9), 3.18 (s, 6H, H-10), 1.55 (bs, 8H, H-5

,5);
13
C, δ
166.23 (CO), 161.60 (C-h), 154.84 (C-d), 146.84 (C-e), 142.48(C-k),
136.58 (C-a), 128.97 (C-b), 126.21 (C-j), 125.27 (C- ), 122.46 (C-c),
115.93 (C-g), 77.39 (C-2), 73.71, 71.68, 70.93, 70.67, 70.30, 69.95,
69.26, 68.81 (C-2

,3

,4

,3,4,6,7,8,9), 61.99 (C-i), 58.47 (C-10), 51.09
(C-1), 43.92 (C-1

), 26.47, 26.32 (C-5

,5).
PAAT4 . IR:
max 3336 (NH), 1644 cm
−1 (CO); NMR da a
(500 MHz, CDCl
3
):
1
H, δ8.05–7.85 (m, 14H, H-j,b,c, ), 7.30 (bs, 2H,
NH), 7.14 (d, 4H, H-g), 5.31 (s, 4H, H-i), 4.68 (m, 2H, J
1a,2
2.9 Hz,
H-1a), 4.44 (m, 2H, J
1b,2
7.3 Hz, J
1a,1b
14.3 Hz, H-1b), 4.00–3.15
(m, 56H, H-1

,2

,3

,4

,2,3,4,6

,6,7

,7,8

,8,9

,9), 3.33, 3.28 (2 s, 12H,
H-10

,10), 1.67 (m, 8H, H-5

,5);
13
C, δ166.97 (CO), 161.15 (C-h),
154.29 (C-d), 147.25 (C-e), 143.19 (C-k), 136.02 (C-a), 128.12 (C-b),
125.01 (C- ), 124.83 (C-j), 122.52 (C-c), 115.16 (C-g), 77.81, 77.75 (C-
2

,2), 71.86, 71.79, 71.43, 70.77, 70.65, 70.40, 70.33, 69.68, 69.59 (C-
3

,3,4

,4,6

,6,7

,7,8

,8,9

,9), 62,23 (C-i), 58.93, 58.86 (C-10

,10), 51.64
(C-1), 41.57(C-1

), 26.36, 26.29 (C-5

,5).
2.5. Al e na ing i adia ion o he sample wi h ul a iole and isible
ligh
A solu ion o he polyme (0.028 mg/mL) in dime hyl sul oxide
was consecu i ely i adia ed wi h ul a iole ligh , o di e en pe-
iods o ime, ollowed by isible ligh o 1 min. The solu ion was
s udied by ul a iole - isible spec oscopy a e each i adia ion.
2.6. Deg ada ion o polyme s
2.6.1. Deg ada ion in bu e solu ion
The hyd oly ic deg ada ion s udy was ca ied ou on films p e-
pa ed by e apo a ions o polyme solu ions PAAT1 and PAAT4 in
dichlo ome hane (20 mg/mL). Films o med we e d ied unde ac-
uum un il no weigh loss was obse ed. The hickness o he films
ob ained was app oxima ely 70 μm. A e wa ds, 10 mL o bu e ed
solu ions a di e en pH we e added, and hey we e incuba ed a
ei he 37 o 70 °C. A e di e en pe iods o ime, abou 40 days,
he sample was eco e ed by fil a ion, and he film was washed
wi h dis illed wa e and d ied unde acuum. Finally, he emain-
ing film was analyzed by Gel Pe mea ion Ch oma og aphy.
2.6.2. Deg ada ion wi h di hioni e
2.6.2.1. Deg ada ion o monome 12 . To a solu ion o 12 in
me hanol (5 mg, 1.5 mL) wa e was added un il u bidi y (0.7 mL).
Then, sodium di hioni e (10 equi alen s) was added and s i ed
a 37 °C p o ec ed om ligh . The eac ion mix u e was moni-
o ed by TLC, adding mo e di hioni e un il educ ion was com-
ple e. A e ou days, he sol en was e apo a ed o d yness and
dichlo ome hane was added o ob ain a suspension. The solid
o med was fil e ed o and he fil a e was e apo a ed again in
acuo . Finally, he mix u e ob ained was d ied o cons an weigh ,
being used di ec ly o i s analysis by UV- isible spec oscopy, in
o de o e alua e he educ ion o he azo g oup.
2.6.2.2. Deg ada ion o polyme . Polyme s PAAT1 and PAAT4 we e
dissol ed in CH
2
Cl
2
(4 mg/mL), and he sol en was slowly e-
mo ed by e apo a ion a oom empe a u e o ob ain a film ha
was d ied unde acuum o cons an weigh . A e wa ds, a pH
6 bu e ed solu ion o sodium di hioni e (155 mM, 2.0 mL) was
added, and he ials we e hea ed a 37 °C wi h s i ing. Sam-
ples we e wi hd awn a 2, 4, 8 and 11 days, eplacing he sodium
di hioni e solu ion in he emaining samples. The film was washed
wi h dis illed wa e and d ied. Finally, he samples we e analyzed
by size exclusion ch oma og aphy.
2.7. Biocompa ibili y s udies
2.7.1. Sample p epa a ion
Polyme s PAAT1 and PAAT4 we e dissol ed in DMSO o cell
cul u e (4 mg/mL). Then, 200 μL o each solu ion was added o
a 96-well pla e, and DMSO was emo ed in a acuum o en un il
d yness, ob aining he wells coa ed wi h hin films o he samples
o be analyzed.
2.7.2. Hemolysis assay
Toxici y o human ed blood cells (hRBC) was es ed by ca e ully
p epa ing a mix u e o esh human blood and PBS. The mix u e
5

A. Suá ez-C uz, I. Molina-Pinilla, K. Hakkou e al. Polyme Deg ada ion and S abili y 193 (2021) 109726
was cen i uged a 700 G o 10 min, supe na an was disca ded,
and he p ocedu e was epea ed h ee imes. Then, PBS was added
o p epa e a 5% ( / ) suspension o hRBC. Aliquo s o his suspen-
sion (150 μL) was added o each coa ed well. PBS (150 μL) was
used as blank and hRBC suspension (150 μL) con aining 1% o T i-
on X-100 was used as posi i e con ol ( o al hemolysis). The 96-
wells pla e was incuba ed o 1 hou a 37 °C. Then, cen i uged
o 10 min a 700 G. Supe na an s we e placed in a new mic o i e
pla e and abso bance was measu ed a 540 nm. The expe imen
was pe o med h ee imes in iplica e, and he equa ion o calcu-
la e he hemoly ic ac i i y is:
2.7.3. Cell cul u e
DMEM was supplemen ed wi h 10% o FBS, 1% Penicillin and
s ep omycin, 1% l -glu amine, 1% sodium py u a e and 1% non-
essen ial aminoacids. A c yo ube con aining 0.5 million o Human
Gingi al Fib oblas s (HGnF) cells was hea ed o 37 °C, dilu ed wi h
9 mL o DMEM and cen i uged a 300 G o 5 min. Supe na an
was disca ded and pelle esuspended in 5 mL o supplemen ed
DMEM. Then, pla ed in a cell cul u e flask and incuba ed a 37 °C
wi h 5% CO
2
. A e 48 h, he medium was emo ed, he cul u e
was washed wi h PBS, and ea ed wi h ypsin-EDTA. HGnF we e
coun ed and dilu ed wi h DMEM o ob ain a suspension o 1 ×10
5
cells pe millili e . Then, 150 μL o he cell suspension we e added
o he wells o a 96-wells pla e o seed 1.5 10
4 HGnF pe well. 96-
wells pla e was incuba ed o 24 h a 37 °C wi h 5% CO
2
.
2.7.4. MTT assay
To e alua e he oxici y p oduced by PAAT1 and PAAT4 , a cell
iabili y es was ca ied ou using he MTT me hod, which con-
sis s o educing his e azolium dye o a wa e -insoluble c ys al
( o mazan). To pe o m his assay, a 96 wells mic o i e pla e was
p epa ed acco ding o he Sec ion 2.7.1 . A HGnF suspension (150
μL) con aining 1 ×10
5 cells pe millili e was added o each poly-
me coa ed well. HGnF suspension (150 μL) was dispensed o a
well which did no con ain any polyme as posi i e con ol. Wells
con aining only supplemen ed DMEM was used as nega i e con-
ol. Mic o i e pla e was incuba ed a 37 °C wi h 5% CO
2
. A e
24 h, 20 μL o a MTT solu ion (5 mg/mL) was added o each well
and mic o i e pla e was incuba ed. A e 4 h, supe na an was
ca e ully wi hd awn and 150 μL o DMSO was added o dissol e
he o mazan c ys als and films. This solu ion was ans e ed o
a new mic o i e pla e and abso bance o he solu ions was mea-
su ed a 570 nm using a mic opla e eade . This assay was e-
pea ed h ee imes in iplica e. Posi i e con ol was used as 100%
o iabili y, and esul s o he samples we e ela i e o his posi i e
con ol.
3. Resul s and discussion
3.1. Syn hesis and chemical s uc u e o he azo polyme s
In his wo k, we desc ibe he p epa a ion o ou no el linea
poly(azoamide iazole)s, e e ed o as PAATn , which we e p e-
pa ed om diazide ( 6, 7 ) and dialkyne unc ionalized monome s
( 10 –12 ) ( Figs. 1 and 2 , espec i ely), by CuAAC polyaddi ion eac-
ion in solu ion. Diazide monome 6 was eadily p epa ed om
comme cially a ailable 1,4-bu anediol diglycidyl e he by opening
o he epoxide ings wi h sodium azide as nucleophile [65] .
A e wa d, he alcohol unc ions ob ained in he opening o he
epoxides we e eac ed wi h 2-(2-me hoxye hoxy)e hyl me hane-
sul ona e ( 5 ), p e iously p epa ed acco ding o a p ocedu e de-
sc ibed in he li e a u e [64] , o ob ain monome 7 .
The syn hesis o dialkyne monome s 10, 11 and 12 we e con-
enien ly ca ied ou by eac ion o ac i e es e 4 , p epa ed as
displayed in Fig. 3 in ou s eps by p e iously desc ibed me hods
[63] , wi h h ee di e en diamines: comme cial 3,6-dioxaoc an-
1,8-diamine and diamines 8 and 9 , espec i ely ( Fig. 1 ). These di-
amines we e easily ob ained in high yield by educ ion o he di-
azide unc ions o he co esponding monome s 6 and 7 ( Fig. 1 ).
All hese compounds we e con enien ly cha ac e ized by in a ed,
1
H and
13
C NMR spec oscopies, elemen al analysis and/o high-
esolu ion mass spec ome y.
Fig. 3. Syn hesis o he azobenzene de i a i es.
6
A. Suá ez-C uz, I. Molina-Pinilla, K. Hakkou e al. Polyme Deg ada ion and S abili y 193 (2021) 109726
Fig. 4. Syn hesis o he PAATn polyme s.
The azo polyme s PAATn we e p epa ed by polyme iza ion o
he bis-azide and bis-alkyne monome s using coppe -ca alyzed
azide–alkyne cycloaddi ion (CuAAC) eac ions ( Fig. 4 ). These poly-
me s we e isola ed in yields g ea e han 80% ( Table 1 ) and we e
pu ified as desc ibed in he expe imen al pa .
The chemical composi ions an icipa ed o hese poly(azoamide
iazole)s we e confi med by FTIR and NMR spec oscopy, and
he co esponding da a a e de ailed in he expe imen al sec-
ion. All he FTIR spec a displayed he expec ed abso p ion
bands o he amide unc ions p esen s along he polyme chain.
No esidual bands co esponding o azide o alkyne unc ional
g oups o he s a ing monome s we e de ec ed in he co e-
sponding polyme ic ma e ials. The abso p ion bands appea ing
a ound 3300 and 1650 cm
−1 we e a ibu ed o he s e ch-
ing band o NH and ca bonyl g oup o he amide unc ions,
espec i ely.
As an example, he
1
H and
13
C NMR spec a ( Figs. 5 and 6 , e-
spec i ely) o poly(azoamide iazole) PAAT2 , eco ded in deu e -
a ed dime hyl sul oxide, a e displayed wi h he s uc u al co e-
la ion o he signals ha appea in bo h ypes o spec um. The
1
H NMR spec um o PAAT2 ( Fig. 5 ) shows fi e signals in he
egion o 8.80 o 7 ppm. The signals appea ing as single s a
app oxima ely 8.70 and 8.20 ppm we e a ibu ed o he amide
unc ion p esen in he bis-alkyne monome , and o he H-j p o-
ons o he 1,4-disubs i u ed iazole a oma ic ing ob ained in
he CuAAC eac ion, espec i ely. Likewise, he hyd oxyl g oups
p esen in he epea ing uni o his polyme gi e ise o he sig-
nal ha appea s a 5.30 ppm, as a double , displaying a coupling
cons an o abou 5 Hz. The p oposed s uc u e o he PAAT2
polyme is also confi med by i s
13
C NMR spec um ( Fig. 6 ). Ac-
co ding o he egioselec i i y expec ed in he CUAAC polyme -
iza ion eac ion, he polyme should ha e 1,4-disubs i u ed 1,2,3-
iazole ings in i s s uc u e. This ing p oduces wo signals in
he
13
C NMR spec um a 142.27 and 126.21 ppm ha we e
a ibu ed o he C-K and C-j ca bons o he iazole, espec-
i ely.
Fig. 5.
1
H NMR spec um o PAAT2 eco ded in deu e a ed dime hyl sul oxide.
3.2. Gel pe mea ion ch oma og aphy
The pu i y o all polyme s was confi med by size exclu-
sion ch oma og aphy, using μS y agel columns calib a ed agains
polys y ene s anda ds, finding ha he ch oma og ams o he
PAATn polyme s we e unimodal. Mass-a e aged mola masses
(Mw), measu ed using li hium b omide in dime hyl o mamide as
mobile phase, showed alues be ween 95,0 0 0 and 148,0 0 0 g/mol
( Table 1 ). The appa en mola masses o he azo homopolyme s
7
A. Suá ez-C uz, I. Molina-Pinilla, K. Hakkou e al. Polyme Deg ada ion and S abili y 193 (2021) 109726
Fig. 6.
13
C NMR spec um o PAAT2 eco ded in dime hyl sul oxide.
PAAT1, PAAT2 and PAAT3 a e qui e like each o he while PAAT4
has a clea ly lowe mola mass.
Howe e , he PAATn polyme s ha e di e en chemical s uc-
u es so hey a e expec ed o ha e di e en hyd odynamic ol-
umes o he same absolu e mola mass. This makes i difficul o
make compa isons be ween he appa en mola masses measu ed
by GPC o hese polyme s.
3.3. The mal analysis
The he mal s udy o he poly(azoamide iazole)s PAATn was
ca ied ou using a combina ion o DSC and TGA. The da a esul -
ing om his s udy a e shown in Table 2 . The DSC s udy e ealed
ha all he polyme s exhibi ed one glass ansi ion s ep (T g ) du -
ing he second hea ing ace measu ed wi h a hea ing a e o 10
°C/min. Annealing expe imen s we e ca ied ou o a o ha he
in e molecula in e ac ions could lead o he c ys alliza ion o he
samples. Howe e , unde he es ed condi ions he he mog ams
only showed he p esence o glass ansi ions. The PAAT2 polyme
has he highes T g alue (T g 87.7 °C) while he PAAT3 and PAAT4
polyme s ha e much lowe alues o T g , abou 2 °C. In iew o
hese da a ( Table 2 ), i seems ha he alues o he glass ansi-
ion could be ela ed o he sepa a ion o he azobenzene g oups,
which a e he mos igid segmen s along he mac omolecules main
chain. Thus, he PAAT1 and PAAT2 polyme s, whe e he azoben-
zene g oups a e sepa a ed by segmen s con aining 8 a oms, display
highe T g alues han he PAAT3 and PAAT4 polyme s whe e
hese segmen s always con ain 12 a oms. Likewise, he p esence o
absence o die hylene glycol side chains also a ec s he T g alues.
Thus, polyme PAAT1 ha e a T g alue lowe han PAAT2 due o
he die hylene glycol side chains o monome 7 , possibly exe ing
a plas icizing e ec .
The mog a ime ic analysis o he PAATn polyme s was pe -
o med by TGA, unde ine a mosphe e and hea ing he sample
om oom empe a u e o 600 °C. Table 2 shows he alues ob-
ained om he he mog a ime ic analysis o he polyme s. The
empe a u es a 10% weigh loss de e mined a a hea ing a e o
10 °C/min we e highe han 315 °C ( Table 2 ), which shows i s good
he mal s abili y. All polyme s he mally decompose in wo s ages,
as i is shown in Table 2 . Al hough he decomposi ion empe a-
u e alues a e qui e simila , i can be obse ed ha he polyme s
ha ha e ee alcohol unc ions in he main chain, PAAT2 and
PAAT3 , p esen somewha lowe decomposi ion empe a u e al-
ues han hei analogues wi h die hylene glycol side chains, PAAT1
and PAAT4 .
3.4. Quali a i e solubili ies
Table 3 shows he solubili ies [66] o PAATn polyme s in di -
e en sol en s. None o he syn hesized polyme s was soluble in
wa e , bu hey we e dissol ed in dime hyl sul oxide o dime hyl-
o mamide.
In gene al, hey we e also no soluble in common o ganic sol-
en s. I can be obse ed ha he PAAT2 and PAAT3 polyme s,
which con ain ee seconda y alcohol unc ions along he polyme
chain, ha e lowe solubili y han he o he polyme s in common
o ganic sol en s. Thus, o example, PAAT1 is easily soluble in chlo-
o o m o he polyme PAAT4 can be dissol ed in se e al o ganic
sol en s.
3.5. Pho ophysical p ope ies
Solu ions o he PAATn polyme s in dime hyl sul oxide, kep
in he da k o e nigh , we e used o ob ain hei ul a iole - isible
spec a. The alues co esponding o he maximum abso p ion a e
shown in Table 4 .
The abso p ion spec a o azobenzenes a e known o consis
o h ee main bands appea ing a app oxima ely 430, 320 and
230 nm, and which a e assigned o he n- π∗, π- π∗ ansi ions o
ans azobenzene and π- π∗in he phenyl ings, espec i ely.
Simila bands appea in he abso p ion spec a co esponding o
he PAAT1 - PAAT4 polyme s, which in u n a e e y simila o he
spec a exhibi ed by he espec i e monome s 10 –12 , om which
hey a e p epa ed. The polyme s we e i adia ed wi h an ul a i-
ole lamp placed a 20 cm om he sample. Fig. 7 shows how
he band ha appea s a 320–400 nm, co esponding o he ab-
so p ion o ans -azobenzene, p og essi ely dec eases as he i adi-
a ion ime wi h UV ligh inc eases due o i s isome iza ion o cis -
azobenzene. As can be seen, he PAAT1 polyme eached he pho-
os a iona y s a e a e 240 s ( Fig. 7 A), while he es o he poly-
me s needed only 180 s o each he maximum deg ee o pho oiso-
me iza ion ( Fig. 7 B-D). The deg ee o pho oisome iza ion achie ed
is no high, p obably because he azobenzene g oups a e o m-
ing pa o he main chain, which will hinde hei mo emen s.
The e e sibili y o pho oisome iza ion was s udied by ul a iole –
isible spec oscopy employing wo di e en me hods: he mally
and by i adia ing wi h isible ligh . Thus, i p e iously UV i adi-
a ed samples a e exposed o isible ligh , he abso p ion band a
320–400 nm inc eases ma kedly un il i almos eco e s i s ini ial
abso bance, which ep esen s he isome iza ion o cis -azobenzene
o ans -azobenzene. This p ocess o in e con e sion be ween he
cis- and ans -azobenzene emains e e sible when i adia ion cy-
cles wi h UV and isible ligh a e ca ied ou consecu i ely o all
he polyme s. Fig. 8 illus a ed he cis - ans - cis e e sibili y p ocess
o polyme PAAT1 as an example.
As p e iously men ioned, we also s udied he e e sibili y o
pho oisome iza ion by a he mal me hod. Thus, o example, when
a PAAT1 polyme solu ion in DMSO was i adia ed wi h ul a io-
le ligh , and subsequen ly kep in he da k a 60 °C, i was pos-
sible o e i y how he abso bance o he solu ion (band a abou
360 nm) inc eased wi h hea ing ime. A e 45 min o hea ing a
8
A. Suá ez-C uz, I. Molina-Pinilla, K. Hakkou e al. Polyme Deg ada ion and S abili y 193 (2021) 109726
Fig. 7. UV-Visible spec a illus a ing he pho oisome iza ion o PAAT1 (A), PAAT2 (B), PAAT3 (C) and PAAT4 (D). All spec a we e egis e ed in DMSO.
Fig. 8. Re e sibili y o pho oisome iza ion o PAAT1 solu ion o he ans - o- cis and
cis - o- ans p ocesses. The sample was al e na ely i adia ed wi h ul a iole ligh
o 300 s and isible ligh o 30 s.
60 °C he abso bance eached he ini ial alue. I his same expe -
imen is ca ied ou a oom empe a u e, i would ake ou days
o each a alue close o he ini ial one.
3.6. Deg ada ion o polyme s
The sensi i i y o hese polyme s o hyd olysis has been s ud-
ied unde di e en condi ions. As expec ed, he amide unc ions
p esen in he s uc u e o hese polyme s can hyd olyze unde
ela i ely d as ic condi ions, being s able in a physiological en i-
onmen . The hyd olysis o PAATn polyme s has been s udied a
pH 2.0, 7.4, and 10, and a di e en em pe a u es, 37 and 70 °C,
o se e al mon hs.
Likewise, aking ad an age o he ac ha , in addi ion o he
amide unc ions, he e a e also azo unc ions, a deg ada ion ex-
pe imen ha implied he b eaking o his bond and he e o e he
b eaking o he polyme chain was ca ied ou .
All hese deg ada ion expe imen s ha e been s udied by ollow-
ing he a ia ion o he mola masses o he samples subjec ed o
deg ada ion by means o size exclusion ch oma og aphy.
3.6.1. Bu e ed deg ada ion
The esul s o he hyd oly ic deg ada ion s udies a e shown in
Figs. 9 and 10 , whe e he dec ease in mola mass o he deg aded
samples is shown agains he deg ada ion ime. The hyd oly ic
deg ada ions o polyme s PAAT1 and PAAT4 ha e been s udied a
37 °C and in solu ions bu e ed a pH 7.4 and pH 10 o 10 mon hs.
I was obse ed ha unde hese condi ions bo h he numbe - and
mass-a e age mola masses a he end o s udy was p ac ically he
same as he ini ial one o PAAT4 ( Fig. 10 ), o i had sligh ly de-
c eased a he beginning o he deg ada ion o emain p ac ically
cons an un il he end o he s udy, as in he case o PAAT1 ( Fig. 9 ).
Howe e , i was ound ha i ins ead o ca ying ou he hyd oly ic
deg ada ion unde physiological condi ions, pH 7.4 and empe a-
u e 37 °C, i was done a a highe empe a u e, 70 °C, he deg a-
da ion o he polyme occu ed, as expec ed, as e . Unde hese
condi ions, he PAAT4 polyme , ha did no show deg ada ion un-
de physiological condi ions, now did show a con inuous dec ease
in i s numbe - and mass-a e age mola masses alues ( Fig. 10 ).
9