Gels 2025, 11, 164 h ps://doi.o g/10.3390/gels11030164
A icle
T ipep ides Fea u ing Dehyd ophenylalanine and
Homophenylalanine: Homo- Ve sus He e o-Chi ali y
and Sequence E ec s on Sel -Assembly and Gela ion
And é F. Ca alho 1, Te esa Pe ei a 1, Ca los Oli ei a 1, Ped o Figuei edo 2,3, Alexand a Ca alho 2,4, Da id M. Pe ei a 5,
Loic Hilliou 6, Manuel Bañob e-López 7, Bing Xu 8, Paula M. T. Fe ei a 1,* and José A. Ma ins 1,*
1 Cen e o Chemis y, Uni e si y o Minho, 4710-057 B aga, Po ugal; [email p o ec ed] (C.O.)
2 CNC—Cen e o Neu oscience and Cell Biology, Ins i u e o In e disciplina y Resea ch (IIIUC), Uni e si y
o Coimb a, 3004-504 Coimb a, Po ugal
3 PhD P og amme in Expe imen al Biology and Biomedicine, Ins i u e o In e disciplina y Resea ch (IIIUC),
Uni e si y o Coimb a, Casa Cos a Alemão, 3030-789 Coimb a, Po ugal
4 Almac Sciences, Depa men o Bioca alysis and Iso ope Chemis y, Almac House, 20 Seagoe Indus ial
Es a e, C aiga on BT63 5QD, UK
5 REQUIMTE/LAQV, Labo a ó io de Fa macognosia, Depa amen o de Química, Faculdade de Fa mácia,
Uni e sidade do Po o, R. Jo ge Vi e bo Fe ei a, n 228, 4050-313 Po o, Po ugal
6 Ins i u e o Polyme s and Composi es, Uni e si y o Minho, 4800-058 Guima ães, Po ugal;
[email p o ec ed]
7 In e na ional Ibe ian Nano echnology Labo a o y (INL), A . Mes e José Veiga s/n, 4715-330 B aga,
Po ugal; manuel.ba[email p o ec ed] (MB)
8 Depa men o Chemis y, B andeis Uni e si y, 415 Sou h S ee , Wal ham, MA 02453, USA
* Co espondence: pm[email p o ec ed] (P.M.T.F.); [email p o ec ed] (J.A.M.)
Abs ac : O e he yea s, ou esea ch g oup de eloped dehyd odipep ides N-capped
wi h a oma ic moie ies as p o ease- esis an e icacious hyd ogela o s, a o ding sel -as-
sembled hyd ogels a low (c i ical) concen a ions. Dehyd o ipep ides, wi h di e en di-
pep ide sequences and (D,L) s e eochemis y, open a wide chemical space o he de el-
opmen o sel -assembled so nanoma e ials. In his wo k, a small lib a y o N-succinyl-
a ed dehyd o ipep ides con aining a C- e minal dehyd ophenylalanine (∆Phe) esidue
and a sc ambled dipep ide sequence wi h phenylalanine (Phe) and homophenylalanine
(Hph) (L-Phe-L,D-Hph and L,D-Hph-L-Phe) was syn hesized and cha ac e ized as a po-
en ial hyd ogela o . Two pai s o dias e eome ic ipep ides we e syn hesized, bo h as C-
p o ec ed me hyl es e s and as dep o ec ed dica boxylic acids. Pep ides wi h he sequence
Hph-Phe-Phe we e ob ained as a pai (D,L,Z)/(L,L,Z) o dias e eome s. Thei sc ambled
sequence analogues Phe-Hph-Phe we e ob ained also as a dias e eome ic (L,D,Z)/(L,L,Z)
pai . The e ec o s e eochemis y (homo- s. he e o-chi ali y) and sequence (Phe-∆Phe
s. Hph-∆Phe mo i ) on he sel -assembly, biocompa ibili y, gela ion and heological p op-
e ies o he hyd ogels was s udied in his wo k. Accessible, bo h as C-p o ec ed me hyl
es e s and as dica boxylic acids, N-succinyla ed dehyd o ipep ides a e in e es ing mo-
lecula a chi ec u es o he de elopmen o sup amolecula nanoma e ials. In e es ingly,
ou esul s do no comply wi h he well-documen ed p oposi ion ha he e ochi al pep-
ides display much highe sel -assembly p opensi y and gela ion abili y han hei homo-
chi al coun e pa s. Fu he s udies will be necessa y o ully unde s and he in e play
be ween pep ide sequence and homo- and he e o-chi ali y on pep ide sel -assembly and
on he p ope ies o hei sup amolecula ma e ials.
Keywo ds: homophenylalanine; dehyd ophenylalanine; N-succinyldehyd o ipep ides;
homochi al; he e ochi al; sel -assembly; hyd ogels
Academic Edi o s: Dipanka Ghosh,
Ga e h O. Lloyd and Elina I.
Sie anen
Recei ed: 12 Janua y 2025
Re ised: 14 Feb ua y 2025
Accep ed: 19 Feb ua y 2025
Published: 24 Feb ua y 2025
Ci a ion: Ca alho, A.; Pe ei a, T.;
Oli ei a, C.; Figuei edo, P.;
Ca alho, A.; Pe ei a, D.M.; Hilliou, L.;
Bañob e-López, M.; Xu, B.;
Fe ei a, P.M.T.; e al. T ipep ides
Fea u ing Dehyd ophenylalanine
and Homophenylalanine:
Homo- Ve sus He e o-Chi ali y and
Sequence E ec s on Sel -Assembly
and Gela ion. Gels 2025, 11, 164.
h ps://doi.o g/10.3390/gels11030164
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 con-
di ions o he C ea i e Commons A -
ibu ion (CC BY) license (h ps://c e-
a i ecommons.o g/licenses/by/4.0/).
Gels 2025, 11, 164 2 o 24
1. In oduc ion
Sup amolecula pep ide-based so nanoma e ials a e eme ging as po en ial he a-
peu ics, d ug deli e y nanoca ie s, he agnos ic pla o ms and as model sys ems o s udy
cellula p ocesses elying on sel -assembly [1–3]. In opposi ion o classical chemical
(bio)syn hesis, elying on s epwise cons uc ion o s ong co alen bonds, sel -assembly is
d i en by coope a i e ac ion o a (massi e) collec ion o non-co alen weak in e molecu-
la in e ac ions: hyd ophobic and a oma ic π-π s acking in e ac ions, elec os a ic, pola
and hyd ogen bonding [4,5]. Na u e c ea es s uc u al di e si y and (eme ging) unc ion-
ali y h ough epu posing, combina ion and sel -assembly o a limi ed pool o building
blocks: amino acids, suga s, lipids and nucleobases [6]. Sel -assembly is as , economical
and modula . Pep ides a e p i ileged monome s o he bo om-up sup amolecula as-
sembly o complex nanos uc u es and a chi ec u es. Twen y canonical amino acids, con-
aining side chains wi h di e en unc ional g oups, size and shape, pola i y and cha ge
can gene a e an eno mous a ie y o s uc u es. Pep ide s uc u al di e si y can be u he
ampli ied by side chain elabo a ion, e.g., phospho yla ion, glycosyla ion, e c. Sel -assem-
bled pep ide-based hyd ogels (SAPHs) embody he success o sel -assembly as an ena-
bling echnology. SAPHs b ing oge he a unique se o p ope ies: high wa e con en ,
esul ing om a sel -assembled ib illa , a highly hyd a ed po ous 3D ne wo k ha sup-
po s cell homing, di e en ia ion, g ow h and mig a ion; unable s uc u al, heological
and unc ional p ope ies h ough chemical syn hesis; and esponsi eness o en i onmen-
al s imuli [7]. Resembling he ex acellula ma ix (EC), SAPHs a e a che ypical ma e ials
o in i o applica ions, as d ug deli e y [8] and he agnos ic pla o ms and as sca olds
o cell he apies and issue enginee ing. Besides biocompa ibili y, he p ime equisi e o
in i o applica ions o SAPHs, i is also essen ial o ma ch hyd ogel’s elas ici y o ha o
a ge li ing issues, while en o cing injec able p ope ies, o minimally in asi e hyd o-
gel adminis a ion. C ea ing an endu ing EC-like en i onmen , SAPHs p omo e cell hom-
ing, di e en ia ion, g ow h and mig a ion [9]. Howe e , in ime, hyd ogels mus be de-
g aded and eplaced by EC, hus p omo ing issue in eg a ion and emodeling. Pep ides
a e highly suscep ible o p o eolysis by endogenous p o eases. P ema u e deg ada ion o
pep ide pha maceu ics esul s in sho in i o li e- ime and o en ailu e in a aining p e-
dic ed pha maceu ical ou comes [10]. Replacing L-amino acids by hei unna u al mi o
image D-amino acids is he simples , mos logical app oach o enhance he p o eoly ic
s abili y o pep ides [11]. The inco po a ion o nonp o einogenic amino acids in o pep-
ides, e.g., β-amino acids[12] o α,α-disubs i u ed amino acids [13], is an e ec i e solu ion
o enhancing pep ide s abili y. In ou esea ch endea o s, we (and o he s) ound ha N-
capped dehyd opep ides, di- and ipep ides con aining dep o ec ed C- e minal dehyd o-
phenylalanine (∆Phe), dehyd oaminobu y ic acid (∆Abu) o dehyd oalanine (∆Ala), and
a canonical amino acid esidue a e e y e ec i e gela o s, a o ding SAPHs a low (c i i-
cal) concen a ions. Besides enhancing p o eoly ic s abili y, he dehyd oamino acid esi-
due also limi s he con o ma ional eedom o he pep ide backbone, enhancing he sel -
assembly p opensi y and gela ion [14,15]. S uc u al di e si ica ion o he dehyd odipep-
ides was a ained h ough a ia ion o he dehyd oamino acid esidue (∆Phe, ∆Abu,
∆Ala,), inco po a ion o di e en canonical amino acid and by a ia ion o he na u e o
he a oma ic N-capping g oup (e.g., Nap oxen-Npx, ca boxybenzyl-Cbz; 2-Naph-2-naph-
hylace yl; Naph- naph haloyl). Bolaamphiphile- ype and bis-N-capped dehyd opep ide
a chi ec u es e ealed also powe ul gela o s [16–20]. Meaning ul co ela ions be ween
dehyd opep ide’s s uc u e, sel -assembly p opensi y, gela ion abili y and heological
p ope ies o he hyd ogels we e de eloped om ou s udies. Mo e ecen ly, we s a ed
Gels 2025, 11, 164 3 o 24
explo ing dehyd o ipep ides as building blocks o he cons uc ion o sup amolecula
so nanoma e ials [21]. Dehyd o ipep ides, con aining an ex a (canonical o nonp o ein-
ogenic) amino acid allow he explo a ion o a wide chemical space (di e en dipep ide
sequences) and s e eochemical (enan iome ic/dias e eome ic ela ionships) e ec s on
pep ide sel -assembly, gela ion and hyd ogels’ p ope ies. In his wo k, we en isaged ha
me hyl es e C-p o ec ed dehyd o ipep ides could be ende ed wa e soluble, o pH
d op- igge ed gela ion, by deploying a succinic acid N- e minal capping g oup, ins ead
o con en ional a oma ic N-capping. Mo eo e , C-dep o ec ion by saponi ica ion c ea es di-
ca boxylic acid analogues o he C-p o ec ed dehyd o ipep ide hyd ogela o s. Inspi ed by he
excep ional sel -assembly p opensi y o he diphenylalanine mo i (Phe-Phe), we assembled a
small lib a y o N-succinyla ed dehyd o ipep ides con aining a C- e minal ∆Phe esidue and
a sc ambled dipep ide sequence (L-Phe-L,D-Hph and L,D-Hph-L-Phe) [22–24]. Changing he
con igu a ion (D o L) and he posi ion o he homophenylalanine (Hph) esidue gene a ed
wo pai s o dis e eome ic hyd ogela o s. The N-succinyla ed dehyd o ipep ides we e ully
cha ac e ized ega ding sel -assembly, gela ion and cy o oxici y.
2. Resul s and Discussion
2.1. Syn hesis
A small lib a y o N-succinyla ed dehyd o ipep ides was syn hesized by he Boc so-
lu ion me hodology, epo ed p e iously by he esea ch g oup (Scheme 1) [15]. Boc-p o-
ec ed dehyd odipep ide 4, ea u ing he L-Phe-Z-∆Phe mo i , and enan iome ic dehyd o-
dipep ides 5 and 6, con aining he L- and D-Hph-Z-∆Phe mo i , espec i ely, we e de-
signed as en y poin s o s uc u al and s e eochemical lib a y di e si ica ion. Syn hons
4, 5 and 6 we e p epa ed by a one po wo s eps dehyd a ion p ocedu e disclosed be o e:
ea men o he co esponding Boc-p o ec ed β-hyd oxydipep ides 1–3 wi h (Boc)2O/DMAP
ollowed by TMG [25]. S a ing β-hyd oxydipep ides 1–3 we e p epa ed unde s anda d
HBTU/TEA amide linkage condi ions. Following TFA dep o ec ion, dehyd odipep ide 4 was
elonga ed wi h ei he , D- o L-Boc-p o ec ed Hph, gi ing di ec access o he pai o dias e eo-
me ic dehyd o ipepides 7 and 8 (D,L,Z and L,L,Z, espec i ely) wi h he sequence Hph-Phe-
Phe. Elonga ion o dehyd odipep ides 5 and 6 wi h L-Phe a o ded he pai o dias e eome ic
ipep ides 9 and 10 (L,D,Z and L,L,Z, espec i ely) ea u ing he sc ambled sequence Phe-
Hph-Z-∆Phe (Scheme 2). Boc-dep o ec ion (TFA) o compounds 7–10, ollowed by eac ion
wi h succinic anhyd ide in d y py idine a o ded N-succinyla ed dehyd o ipep ides 11–14 as
C-p o ec ed me hyl es e s. Alkaline hyd olysis o he C- e minal me hyl es e s 11–14 a o ded
hei dica boxylic acid analogues 15–18.
Gels 2025, 11, 164 4 o 24
Scheme 1. Syn he ic pa hway o N-succinyl dehyd o ipep ides 11–18. (a) (i) Boc2O, DMAP, d y
MeCN, (ii) TMG; (b) Boc-AA-OH, HBTU, E 3N, MeCN; (c) (i) TFA, (ii) succinic anhyd ide, py idine,
N2 a mosphe e; (d) (i) NaOH (1 M), 1,4-dioxane, (ii) HCl (1 M). TMG = 1,1,3,3-Te ame hyguanidine;
HBTU = Hexa luo ophospha e Benzo iazole Te ame hyl U onium.
This s e eochemical and s uc u al di e si ica ion s a egy a o ded a se o N-suc-
cinyla ed C-p o ec ed dehyd o ipep ides (11–14) and a co esponding se ies o dica box-
ylic acids (15–18) wi h he same s e eochemical con igu a ion (Scheme 2).
Scheme 2. Summa y o he s e eochemical ela ionship be ween dehyd o ipep ides 11–18.
Gels 2025, 11, 164 5 o 24
Dis inc i e 1H NMR (400 MHz, DMSO-d6) spec al ea u es, chemical shi s and sig-
nal mul iplici y we e obse ed o he α-NH amide backbone p o ons o he dehy-
d o ipep ides (Figu e 1).
Figu e 1. (A) De ail o he 1H NMR (400 MHz, DMSO-d6) amide egion, α-NH amide backbone p o-
ons, o dehyd opep ides 11–18. (B) Table wi h cha ac e is ic 1H NMR signals o he α-NH p o ons
o dehyd o ipep ides 11–18.
The signals assigned o he ∆Phe α-NH amide, as single s, display cha ac e is ically
high chemical shi , signi ican ly highe han hose a ibu ed o he α-NH amide p o ons
o Phe and Hph, p esumably owing o conjuga ion wi h he α, β-double bond. The ∆Phe
α-NH amide p o ons o he C-p o ec ed dias e eome ic pai 11/12, con aining he Phe-
ΔPhe mo i , display he highes chemical shi . C-dep o ec ion o he dias e eome ic pai s
11/12→15/16 and 13→17 esul s in signi ican down ield shi o he ΔPhe α-NH eso-
nance, owing o highe elec on wi hd awing e ec s o he ca boxylic acid g oup. I is
no ewo hy ha o he dias e eome ic dehyd opep ide pai s 11/12 and 15/16, con aining
he mo i Phe-ΔPhe, he he e ochi al (D,L,Z) dias e eome s 11 (me hyl es e ) and 15 (dica -
boxylic acid) display a simila pa e n o signals o he α-NH amide o he Phe and Hph
esidues, di e en om ha displayed by he homochi al (L,L,Z) 12/16 pai . The he e o-
chi al es e /dica boxylic acid 13/17 pai displays also a simila pa e n o esonances o
he α-NH Phe and Hph esidues, di e en om ha exhibi ed by he homochi al pai
14/18. This esul sugges s ha he pep ide backbone o he he e o-chi al and homo-chi al
pep ides sample di e en con o ma ions in he non-agg ega ing sol en DMSO. Owing
o highe solubili y in DMSO and DMSO/wa e mix u es, dica boxylic acid pep ides 15,
16, 17, and 18 we e selec ed o a iable- empe a u e 1H NMR spec oscopy s udies (Fig-
u es 2 and S1 and Tables S1 and S2 Suppo ing In o ma ion) [26].
Gels 2025, 11, 164 6 o 24
Figu e 2. Va iable empe a u e 1H-NMR analysis o he α-NH backbone amide signals o pep ides:
(A) 17; (B) 18 in DMSO-d6 and (C) 17; (D) 18 in DMSO-d6 con aining 10% / D2O.
The chemical shi empe a u e g adien s ∆δ/∆T indica e ha he NH backbone am-
ide g oups a e no likely o be in ol ed in in amolecula hyd ogen bonds, such as α-
helical seconda y s uc u es. Signi ican ly, addi ion o 10% wa e ( / ) o he DMSO sol-
en does no induce any signi ican change o he amides chemical shi empe a u e g a-
dien s ∆δ/∆T, sugges ing ha he backbone amides emain exposed o he sol en and do
no expe ience signi ican s uc u al o en i onmen al changes upon wa e addi ion [26].
Ne e heless, hese esul s in (ap o ic) DMSO mus be in e p e ed wi h cau ion. The s a-
bilizing in a- and in e molecula na i e hyd ogen bonding ne wo k o pep ides in (p o-
ic) wa e is likely o be e y di e en om ha seen in DMSO.
2.2. Molecula Dynamics Simula ions
A omis ic Molecula Dynamics (MD) simula ions we e ca ied ou o in es iga e he
sel -assembly dynamics, o ma ion o agg ega es, and he pa e n o ep esen a i e non-
co alen in e ac ions be ween dehyd o ipep ide molecules. A o al numbe o 15 sol a ed
pep ide molecules was andomly dis ibu ed wi hin an o ho hombic wa e box. The sys-
em was subjec ed o minimiza ion, ollowed by hea ing and equilib a ion, and was le
o un o 250 ns unde an iso he mal–isoba ic ensemble. The andomly dispe sed pep ide
ensembles e ol ed du ing he simula ion ime in o la ge single molecula clus e s (Figu e
3). The Sol en Accessible Su ace A ea (SASA) and olume o he sel -assembled pep ide
agg ega es was calcula ed as agg ega ion p opensi y indica o s (Figu e 3 and Suppo ing
In o ma ion Figu e S2).
Gels 2025, 11, 164 7 o 24
Suc-L-Phe-L-Phe-Z-ΔPhe-OH (SASA = 9418.0 Å2; Volume = 5105.2 Å3)
Suc-D-Hph-L-Phe-Z-ΔPhe-OMe, 11
(SASA = 8476.9 Å2; Volume = 5044.9 Å3)
Suc-L-Hph-L-Phe-Z-ΔPhe-OMe, 12
(SASA = 9653.2 Å2; Volume = 7583.8 Å3)
Suc-D-Hph-L-Phe-Z-ΔPhe-OH, 15
(SASA = 9130.7 Å2; Volume = 5113.2 Å3)
Suc-L-Hph-L-Phe-Z-ΔPhe-OH, 16 (SASA
= 9172.8 Å2; Volume = 6032.7 Å3)
Suc-L-Phe-D-Hph-Z-ΔPhe-OMe, 13
(SASA = 9653.2 Å2; Volume = 5625.6 Å3)
Suc-L-Phe-L-Hph-Z-ΔPhe-OMe, 14
(SASA = 9616.7 Å2; Volume = 6846.5 Å3)
Suc-L-Phe-D-Hph-Z-ΔPhe-OH, 17
(SASA = 9210.9 Å2: Volume = 5831.2 Å3)
Suc-L-Phe-L-Hph-Z-ΔPhe-OH, 18
(SASA = 9165.2 Å2; Volume = 5744.3Å3)
Figu e 3. Sel -assembled clus e s uc u es ob ained by MDS o dehyd opep ides 11–18. The pa-
ame e s SASA and olume a e indica ed (inside pa en hesis). Dehyd opep ide Suc-L-Phe-L-Phe-
Z-ΔPhe-OH was included in he simula ion o con ol pu poses.
Gels 2025, 11, 164 8 o 24
Wi h he excep ion o pep ide 11, o igina ing he mos compac agg ega e, he pep-
ide agg ega es o med by C-p o ec ed pep ides 12–14 display highe SASA and olume
han hei dep o ec ed dica boxylic acid analogues 16–18, indica ing less compac ness and
mo e exposed s uc u es, likely esul ing om sol en exposu e o he me hyl es e g oup.
The he e ochi al pep ides 11 and 15 (D,L,Z), con aining he Phe-ΔPhe mo i , display lowe
SASA and olume han he homochi al 12 and 16 (L,L,Z) dias e eome s, sugges ing mo e
compac agg ega es, i.e., highe sel -assembly p opensi y. Mo eo e , he he e ochi al pep-
ide 13 (L,D,Z), con aining he Hph-ΔPhe mo i , also displays lowe SASA and olume
han he homochi al dias e eome 14 (L,L,Z). Snapsho s o he s able pep ide agg ega es
gi e insigh in o he pa e n o in e molecula in e ac ions esponsible o pep ide sel -
assembly. In he agg ega es o pep ide Suc-L-Phe-L-Phe-Z-ΔPhe-OH he e a e some in e -
molecula hyd ogen bonds, be ween he backbone amide g oups and he e minal ca box-
ylic acid, and T-shaped π a oma ic s acking in e ac ions, mo e equen ha pa allel in-
e ac ions (Figu e 4A). In he agg ega es o he he e ochi al dica boxylic acid 15 (D,L,Z),
con aining he Phe-ΔPhe mo i , he in oduc ion o he Hph esidue, esul s in ewe in-
e molecula hyd ogen bonds. The C- e minal ca boxylic acid g oup engages in in amo-
lecula hyd ogen bonding wi h he Phe backbone amide owing o close spa ial p oximi y.
The a oma ic ings o he Hph esidue es ablish an in e molecula ne wo k (clus e s) o T-
shaped π-π s acking in e ac ions. The Phe a oma ic esidues engage p edominan ly in
in amolecula T-shaped π-π s acking in e ac ions (Figu e 4C). In he agg ega es o pep-
ide 11, he C-p o ec ed coun e pa o dica boxylic acid 15, he es e g oup p edominan ly
aces he sol en . The a oma ic esidues, especially he Hph, es ablish a clus e o π-π
s acking in e ac ions simila o ha seen o pep ide 15 (Figu e 4B). The agg ega es o he
dica boxylic acid he e ochi al pep ide 17 (L,D,Z) con aining he Hph-ΔPhe mo i , display
p edominan ly clus e s be ween he Hph side chains, ia T-shaped π-π s acking in e ac-
ions, and u he in e molecula a oma ic s acking in e ac ions be ween he Hph and Phe
and Phe Phe esidues. The agg ega es o es e 13 show a pa e n o in e molecula in e -
ac ions e y simila o hose desc ibed o pep ide 11, wi h he me hyl es e g oup acing
he sol en . In e es ingly, he agg ega es o he homochi al pep ides 16 and 18, con aining
he Hph-ΔPhe mo i , seem o o m ewe clus e s be ween he Hph ings han he agg e-
ga es o pep ides 15 and 17, bu a highe numbe o in amolecula Hph-Phe in e ac ions
esul ing in he ansi ion om a compac sphe ical agg ega e shape ( o pep ide agg e-
ga es 15/17) o a mo e elonga ed less compac shape o pep ide agg ega es 16/18. Rega d-
ing he e ec o he C- e minal on he o e all a ay o in e molecula in e ac ions, i seems
ha in he C-p o ec ed pep ides he es e g oup p edominan ly aces he sol en . Fo he
dep o ec ed dica boxylic acid pep ides he C- e minal ca boxylic acid s ays in a mo e bal-
anced posi ion acing bo h he ex e nal zone o he agg ega e as well as he inside, whe e
some hyd ogen bonding was obse ed. O e all, he MD simula ions sugges ha he sel -
assembly o he dehyd o ipep ides is mos ly d i en by an in e molecula ne wo k (clus-
e s) o T-shaped π-π s acking in e ac ions. Hyd ogen bonding seems o play a mino ole
in sel -assembly, as indica ed also by he NMR a iable empe a u e expe imen s. Es ab-
lishing a s onge ne wo k o clus e s be ween he Hph side chains, he he e ochi al pep-
ides a e likely o display highe sel -assembly p opensi y.
A
Gels 2025, 11, 164 9 o 24
B
C
D
Figu e 4. Selec ed ep esen a i e in e molecula non-co alen in e ac ions ound in pep ide agg e-
ga es: Suc-L-Phe-L-Phe-Z-ΔPhe-OH (A), Suc-D-Hph-L-Phe-Z-ΔPhe-OMe (11) (B), Suc-D-Hph-L-
Phe-Z-ΔPhe-OH (15) (C) and Suc-L-Phe-D-Hph-Z-ΔPhe-OH (17) (D). π-in e ac ions a e ep esen ed
as dashed lines. Hyd ogen bonds a e ep esen ed as do ed lines.
2.3. Hyd ogela ion
The C i ical Agg ega ion Concen a ion (CAC) was de e mined o dehyd o ipep-
ides 11–18 om he concen a ion-dependence o he luo escence in ensi y emission a-
io a 368 nm (agg ega es) and 308 nm (monome ) (I368/I308) (Figu e 5 and Table 1) (Figu e
S2 Suppo ing In o ma ion) [27].
Figu e 5. Illus a i e example, o dehyd opep ide 11, o he p ocedu e used o de e mina ion o
he c i ical agg ega ion concen a ion (CAC) o dehyd o ipep ides 11–18: (A) S eady-s a e luo es-
cence spec a (λexc = 280 nm) o dehyd opep ide 11 in he concen a ion ange 0.6 o 156 μM; (B)
semiloga i hmic g aphical ep esen a ion o he concen a ion dependence o he luo escence in-
ensi y a io (I368/I308).
Gels 2025, 11, 164 16 o 24
highe han G″ and he a io G′/G″ is a ound 10, indica ing a dominan elas ic beha io
o all hyd ogels. Mo eo e , he majo i y o he 0.6 w % hyd ogels display, a ound an
o de o magni ude, highe elas ici y (G′) han he 0.2 w % gels. Highe pep ide concen-
a ions a e likely o esul in ib il ne wo ks wi h highe densi y and en anglemen c oss-
linking ex en . Hyd ogels 12 and 18 a e excep ions o his end: he 0.2 w % hyd ogels
show a ound an o de o magni ude highe elas ici y han he 0.6 w % hyd ogels. The
TEM images ob ained a 0.6 w %, e eal ha hyd ogels 12 and 18 a e made o hicke
ibe s, wi h lowe ex en o en anglemen , compa ing o he o he hyd ogels (Figu e 7).
Fo mos hyd ogels he e is a egion o linea elas ici y and iscosi y s ain dependence,
be ween 0.001–1%. Fu he inc easing s ain esul s in a educ ion o bo h G′ and G″. E en-
ually, a c osso e poin is eached, indica ing ansi ion om elas ic (gel) o iscous (liq-
uid-like) beha io ma king gel b eakup (yielding), associa ed o b eakdown o he hyd o-
gel ne wo k. Hyd ogel 11 shows an ea ly c osso e o G′ and G″, especially he 0.6 w %
gel, indica ing ha i is mo e p one o so ening unde s ain (Figu e 10A). Gel 12 exhibi s
delayed c osso e and main ains highe G′ alues o e a wide s ain ange, sugges ing a
s onge gel s uc u e a bo h concen a ions (Figu e 10B). Hyd ogels 13 (Figu e 10C) and
14 (Figu e 10D) display simila ends, wi h hyd ogel 13 showing sligh ly highe ini ial G′
alues compa ed o 14. Hyd ogels 15 (Figu e 10E) and 16 (Figu e 10F) ha e high G′ alues
a lowe s ains, bu compound 16 shows a sha p d op in G′ a e he c osso e poin ,
indica ing easie ne wo k b eakdown a highe s ains. Finally, gels 17 (Figu e 10G) and
18 (Figu e 10H) show s ong gel-like beha io , wi h hyd ogel 17 showing mo e p o-
nounced s ain so ening han hyd ogel 18, sugges ing a weake ne wo k s uc u e o
hyd ogel 17. Hyd ogel 17 weake ne wo k s uc u e (as e idenced in he TEM images by
less wis ing and c oss-linking ex en ) easonably explains i s p onounced s ain so ening
compa ed o he mo e obus ne wo k o hyd ogel 18. Rega ding gels’ elas ici y (G′), wi h
he excep ion o pep ide 12, he C-p o ec ed pep ides (11–14), o igina e hyd ogels display-
ing highe elas ici y, by a ound one o de o magni ude, han hei dica boxylic acid coun-
e pa s (15–18), p esumably owing o highe hyd ophobici y (Table 2). Acco dingly, he
TEM images e eal ha , in gene al, he me hyl es e hyd ogels 11–14 o m signi ican ly
dense ibe ne wo ks han hei co esponding dica boxylic acid de i a i es 15–18 (Fig-
u e 7). The dis inc i e ne wo k o hyd ogel 12, made o hick ibbon like nanos uc u es,
may esul in educed en anglemen and low elas ici y. In e es ingly, in he me hyl es e
pep ide’s se ies 11–14, nei he he pep ide sequence, Phe-ΔPhe s. Hph-ΔPhe, no he con-
igu a ion (D s. L) o he Hph esidue, seem o ha e a signi ican impac on he hyd ogels’
elas ici y. The same gene al end is also obse ed o dica boxylic acid gels 15–18. In gen-
e al, all hyd ogels eco e o some ex en ollowing mechanical b eak up. A 0.6 w %, only
he homo-chi al C-p o ec ed hyd ogels 12 and 14 show highe eco e y. Mos gels, show
li le eco e y (<10% om ini ial alue), p esumably as esul o incomple e e o ma ion
o he ib illa 3D ne wo k. In con as , a 0.2 w % mos hyd ogels show highe eco e y
(>30% om ini ial alue). The homochi al C-p o ec ed/dica boxylic acid hyd ogel’s pai
14 and 18, shows a subs an ial inc ease in elas ici y upon e o ma ion, sugges ing injec a-
ble p ope ies sui able o in i o applica ions.
Table 2. G′ o hyd ogels a di e en concen a ions, be o e (1s sweep) and a e b eaking (2nd
sweep) and eco e y pe cen age om ini ial G′ alue.
Hyd ogel
Gela ion Concen a ion w %
Be o e B eaking
G′ (Pa)
A e B eaking
G′ (Pa)
Reco e y (%)
Suc-D-Hph-L-Phe-ΔPhe-OMe, 11
0.2
6.15 × 102
2.15 × 102
35%
0.6
3.19 × 105
2.88 × 104
9%
Suc-L-Hph-L-Phe-ΔPhe-OMe, 12
0.2
4.66 × 104
2.28 × 104
49%
0.6
5.79 × 103
6.92 × 103
120%
Suc-L-Phe-D-Hph-ΔPhe-OMe, 13
0.2
3.62 × 104
1.98 × 103
5%
Gels 2025, 11, 164 17 o 24
0.6
3.15 × 105
3.80 × 103
1%
Suc-L-Phe-L-Hph-ΔPhe-OMe, 14
0.2
3.31 × 104
3.82 × 105
1154%
0.6
2.73 × 105
1.25 × 105
46%
Suc-D-Hph-L-Phe-ΔPhe-OH, 15
0.2
3.35 × 103
1.05 × 103
31%
0.6
3.02 × 104
2.66 × 103
9%
Suc-L-Hph-L-Phe-ΔPhe-OH, 16
0.2
1.75 × 104
1.39 × 103
8%
0.6
1.27 × 105
2.02 × 104
16%
Suc-L-Phe-D-Hph-ΔPhe-OH, 17
0.2
1.47 × 103
2.09 × 103
142%
0.6
3.20 × 104
2.00 × 103
6%
Suc-L-Phe-L-Hph-ΔPhe-OH, 18
0.2
2.09 × 106
5.44 × 106
260%
0.6
8.73 × 104
5.28 × 103
6%
2.7. Biological Assays
Hyd ogela o biocompa ibili y is a undamen al equisi e o biological applica ions,
e.g., as d ug deli e y nanoca ie s and he apeu ic he agnos ic pla o ms. The sa e y p o-
ile o hyd ogela o s 11–18, ega ding po en ial biological applica ions, was e alua ed
wi h he non-cance ous human bone ma ow s omal cell line (HS-5) by he MTT assay
(Figu e 11) [33].
Figu e 11. MTT e alua ion o he impac o dehyd o ipep ides 11–18 (A–H), in he concen a ion
ange 10–200 μM, o e 24 h incuba ion pe iod, owa ds he iabili y o non-cance ous human bone
ma ow s omal cell line (HS-5).
Gels 2025, 11, 164 18 o 24
The main end eme ging om he MTT assays poin s o highe oxici y o he C-
p o ec ed hyd ogela o s 11–14 compa ing o hei dica boxylic acid coun e pa s 15–18.
Amongs he me hyl es e s (11–14), dehyd opep ides 12 and 13 show concen a ion-inde-
penden cy o oxici y, while pep ides 11 and 14 display concen a ion dependen cy o ox-
ici y. Concen a ion-dependen cy o oxici y sugges s in e ac ion wi h a molecula a ge .
The me hyl es e s, displaying highe hyd ophobici y han hei coun e pa dica boxylic
acids, a e likely o be in e nalized by cells in highe ex en han he dica boxylic acids.
Toxici y mechanisms linked o memb ane dis up ion a e also mo e likely o he am-
phiphile-like me hyl es e dehyd opep ides 11–14. The me hyl es e /dica boxylic acid
couple 12/16, bea ing he Phe-ΔPhe mo i and he L-Hph enan iome , e ealed he mos
oxic compounds. Mo eo e , dehyd opep ides 12 and 16 display essen ially concen a-
ion-independen cell oxici y, sugges ing a physical cell dea h mechanism. In e es ingly,
cell oxici y s a s a concen a ions (10 μM) bellow he dehyd opep ides CAC (40.74 and
38.64 μM, o dehyd opep ides 12 and 16, espec i ely) excluding oxici y om agg e-
ga es. Dehyd opep ides 15, 17 and 18 a e essen ially non- oxic in he concen a ion ange
10-200 μM. As he dica boxylic acids display CAC alues o he o de o magni ude 30–
40 μM, bo h he non-agg ega ed (monome ic) and agg ega ed pep ide dica boxylic acid
o ms a e essen ial non oxic. The e o e, he dica boxylic acid-based hyd ogels 15, 17 and
18 a e po en ially sui able nanoca ie s o d ug deli e y applica ions. O e all, a he
lowe concen a ion ange es ed, 10 μM and 20 μM, mos dehyd opep ides did no show
a signi ican e ec on he cell iabili y.
3. Conclusions
In his wo k we syn hesized a ocused lib a y o succinic acid N-capped dehy-
d o ipep ides ea u ing a C- e minal dehyd ophenylalanine (∆-Phe) esidue and a sc am-
bled dipep ide sequence (L-Phe-L,D-Hph-Phe and L,D-Hph-L-Phe-Phe). Changing he
con igu a ion (L o D) and swapping he posi ion o he Hph esidue a o ded ini ially wo
pai s o me hyl es e -p o ec ed dias e eome s. N-succinila ed C-p o ec ed ipep ides o -
mally display in e sion o pola i y compa ing o con en ional C-dep o ec ed pep ides N-
capped wi h (nonionizable) a oma ic g oups. Besides p omo ing solubili y, o gela ion
pu poses, saponi ica ion o he C-p o ec ed pep ides gene a ed a subse o dica boxylic
acid pep ides wi h he same sequence and s e eochemis y as hei C-p o ec ed p ecu -
so s. Pep ides wi h he sequence Hph-Phe-Phe 11/15 (D,L,Z) and 12/16 (L,L,Z) we e ob-
ained as a pai o dias e eome s. Thei sc ambled sequence analogues Phe-Hph-Phe
13/17 (L,D,Z) and 14/18 (L,L,Z) we e ob ained also as a dias e eome ic pai . Bo h he C-
p o ec ed and he dica boxylic acid pep ides showed simila , sequence and s e eochemis-
y-independen gela ion abili y, a o ding hyd ogels a low concen a ions, 0.2 w %, us-
ing he GdL pH d opping me hodology and he empi ical in e sion ube es . Simila c i -
ical agg ega ion concen a ions CAC alues we e de e mined o he dehyd o ipep ides,
sugges ing equi alen sel -assembly p opensi y. MD simula ions indica ed ha he sel -
assembly o he dehyd o ipep ides is mos ly d i en by an in e molecula ne wo k (clus-
e s) o T-shaped π-π s acking in e ac ions, p edominan ly be ween Hph esidues. Hy-
d ogen bonding seems o play a mino ole in he sel -assembly o he dehyd o ipep ides,
as indica ed also by he NMR a iable empe a u e expe imen s. The CD s udy con i ms
a a iable deg ee o chi al π-π s acking o a oma ic sides chains o all dehyd opep ides.
The sligh ly highe sel -assembly p opensi y, seen in he sel -assembly s udies, o he
he e ochi al pep ides, does no ansla e in o s ikingly highe gela ion abili y, as epo ed
in he li e a u e o dias e eome ic all-canonical di- and i-pep ides. TEM images o he
hyd ogels show ib illa ne wo ks, wi h a iable ibe hickness, densi y and en angle-
men . O e all, he dica boxylic acid hyd ogela o s display ne wo ks wi h highe ibe
densi y han hei me hyl es e s analogues. Bo h pep ide sequence and s e eochemis y
Gels 2025, 11, 164 19 o 24
seem o go e n he hyd ogels’ ne wo k a chi ec u e. Rega ding gels’ elas ici y C-p o ec ed
pep ides o igina e hyd ogels displaying signi ican ly highe elas ici y han hei dica box-
ylic acid coun e pa s, in acco dance wi h he TEM images, showing dense ibe ne -
wo ks. All gels display sel -healing p ope ies, lending hemsel es o biological applica-
ions elying on non-in asi e adminis a ion. In gene al, he C-p o ec ed hyd ogela o s
display highe oxici y han hei dica boxylic acid coun e pa s. The pep ides bea ing he
Phe-ΔPhe mo i and he L-Hph enan iome , a e especially oxic, displaying essen ially
concen a ion-independen cell oxici y, which sugges s a physical cell dea h mechanism.
The he e ochi al dica boxylic acid pep ides and he homochi al pep ide con aining he
Hph- ΔPhe mo i a e essen ially non- oxic.
In his wo k we disclosed a syn he ic pa hway o N-succinyla ed dehyd o ipep-
ides. Accessible, bo h as C-p o ec ed me hyl es e s and as dica boxylic acids, N-succinyl-
a ed dehyd o ipep ides a e in e es ing molecula a chi ec u es o de elopmen o su-
p amolecula nanoma e ials. In e es ingly, ou esul s do no comply wi h he well docu-
men ed p oposi ion ha he e ochi al pep ides display much highe sel -assembly p open-
si y and gela ion abili y han hei homochi al coun e pa s. Fu he s udies will be nec-
essa y o ully unde s and he in e play o pep ide sequence and homo- he e o-chi ali y
on pep ide sel -assembly and on he p ope ies o hei sup amolecula ma e ials.
4. Ma e ials and Me hods
4.1. Gene al p ocedu es
Chemicals, analy ical g ade eagen s and sol en s we e acqui ed om Ac os (Geel,
Belgium) and Sigma-Ald ich (S . Louis, MO, USA)and used as ecei ed. Whene e e-
qui ed, sol en s we e d ied by s anda d me hodologies. Aqueous solu ions we e made
using dis illed wa e . Thin Laye Ch oma og aphy (TLC), Me ck-Kieselgel pla es 60 F254,
was used o moni o eac ions wi h iodine e ela ion and examina ion and unde UV.
Anhyd ous magnesium sul a e (Riedel) was used o d y o ganic phases.
The 1H and 13C NMR spec a, elucida ed h ough Dis o ionless Enhancemen by Po-
la iza ion T ans e (DEPT), He e onuclea Single Quan um Cohe ence (HSQC), and He -
e onuclea Mul iple-Bond Co ela ion (HMBC) echniques, we e acqui ed u ilizing a
B uke A ance III 400 (Bille ica, Massachuse s, USA) spec ome e . The ins umen ope a ed
a equencies o 400.13 MHz and 100.62 MHz o 1H and 13C NMR, espec i ely. Spec a we e
eco ded a 25 °C, e e encing he esidual sol en signals. Deu e a ed dime hyl sul oxide
(DMSO-d6) se ed as he sol en , and chemical shi s a e epo ed in pa s pe million (ppm)
wi h coupling cons an s in He z (Hz). The elemen al analysis was ob ained in a LECO ins u-
men (S . Joseph, MI, USA). The mass spec ome y se ice CACTI, a he Uni e si y o Vigo,
Spain, p o ided he HRMS da a. Mass spec a, Elec osp ay Ioniza ion (ESI), we e acqui ed
wi h a The mo Finnigan LxQ (Linea Ion T ap, San Jose, CA, USA) mass spec ome e . All
syn hesis p ocedu es a e desc ibed in he suppo ing in o ma ion.
Fluo escence measu emen s we e conduc ed a oom empe a u e using a Shimadzu
RF5301PC (Kyo o, Japan) spec ome e . The c i ical agg ega ion concen a ion (CAC) o
pep ides was de e mined in phospha e-bu e ed saline (PBS) wi h a pH = 7.4. The expe i-
men al p o ocol in ol ed pep ide solu ion concen a ions anging om 0.6 o 156 μM. The
sample was exci ed a a wa eleng h o λex = 280 nm and he esul ing luo escence spec-
um was eco ded wi hin he wa eleng h ange o λem = 300–550 nm, using exci a ion and
emission bandwid hs o 1.5 and 3 nm, espec i ely. The luo escence in ensi y a io
(I368/I308) was plo ed agains he loga i hm o pep ide concen a ion. The concen a ion
co esponding o he in lec ion poin in he plo was iden i ied as he C i ical Agg ega ion
Concen a ion (CAC).
The molecula s uc u e o compounds 11–18 was designed wi h he USCF Chime a
so wa e 1.18 [34], and geome y op imized in Gaussian09 [35] using B3LYP [36] wi h he
Gels 2025, 11, 164 20 o 24
6-31++G(d,p) basis se and he Pola izable Con inuum Model sol en desc ip ion [37]. The
a omic pa ial cha ges we e calcula ed om he op imized s uc u e eso ing o he Re-
s ained Elec os a ic Po en ial (RESP) me hod [38] om he HF/6-31++G(d,p) single-poin
ene gy calcula ions. Fo each sys em, he ini ial con igu a ion was gene a ed using he
PACKMOL p og am [39], by andomly dis ibu ing 15 molecules in a 40 Å cubic box wi h
a dis ance ole ance o 8 Å. The MD simula ions we e ca ied ou using compounds 11–
18 as p o ona ed ca boxylic acids. The sys ems we e hen sol a ed wi h an o ho hombic
box wi h app oxima ely 8500 OPC wa e molecules [40] using he LEaP module om Am-
be molecula dynamics p og am (AMBER18) [41]. No coun e ions we e equi ed o neu-
alize he sys ems.
The GPU-accele a ed PMEMD module implemen ed in AMBER18 [41] wi h he
GAFF2 o ce ield [42] as used o un he Molecula Dynamics simula ions. Two ini ial
ene gy minimiza ions and 500 ps o equilib a ion we e ca ied ou in an NVT ensemble,
using Lange in dynamics wi h small es ain s o 10 kcal mol−1 o hea he sys em. P o-
duc ion simula ions we e achie ed by unning 250 ns a 310 K in an NPT ensemble using
he Lange in dynamics wi h a collision equency o 1 ps–1. Cons an p essu e pe iodic
bounda y condi ions we e imposed wi h an a e age p essu e o 1 a m. Iso opic posi ion
scaling was used o main ain p essu e wi h a elaxa ion ime o 2 ps and he ime s ep was
se o 2 s. SHAKE cons ain s we e applied o all bonds in ol ing hyd ogen a oms [43].
The Pa icle Mesh Ewald me hod [44] was used o calcula e he elec os a ic in e ac ions
wi h a cu -o dis ance o 10 Å. The MD ajec o ies we e cen ed back o he p ima y box
eso ing o he CPPTRAJ [45] module implemen ed in AMBER18 [41].
The clus e s uc u e ep esen a ions we e acqui ed h ough he DBScan clus e ing
algo i hm [46] wi h a minimum o 2 poin s o o m a clus e and 0.7–1.0 Å as cu -o dis-
ances conce ning he oo -mean-squa e de ia ion o he Cα. The las 20 ns ajec o y o
he co esponding a oms was ex ac ed and analyzed using 500 ames o each sys em.
The olume and SASA o he agg ega e o con ol dehyd opep ide Suc-L-Phe-L-Phe-
Z-ΔPhe-OH and dehyd o ipep ides 11–18 we e compu ed using a cus om py hon sc ip ex-
ecu ed wi hin he PyMOL [47] en i onmen . To compu e he SASA, he buil -in ge a ea unc-
ion was used. The sc ip se he do sol en and do densi y pa ame e s o 1 and 3, espec-
i ely, o ensu e accu a e SASA calcula ions. The esul ing SASA alues we e s o ed in he B-
ac o column o he PDB ile. The sol en -excluded olume was hen de e mined by sum-
ming hese B- ac o alues, p o iding a measu e o he agg ega e’s olume.
4.2. Hyd ogel P epa a ion
A 1.0 M NaOH solu ion was inc emen ally added o a mix u e o hyd ogela o s (2
mg) in 1.0 mL o wa e un il pH 10 was a ained. This mix u e unde wen sonica ion o
app oxima ely 1 min un il comple e dissolu ion, esul ing in gela o concen a ions o 0.2
w %. D-glucono-δ-lac one (GdL) (2 o 3 mg) was hen added, ollowed by ho ough mix-
ing o 1 min. The solu ions we e le undis u bed o e nigh , and he C i ical Gela ion
Concen a ion (CGC) was de e mined h ough ube in e sion es s, whe e gels we e iden-
i ied in ubes exhibi ing ee-s anding ma e ial a e 5 min o in e sion.
4.3. T ansmission Elec on Mic oscopy (TEM)
Coppe g ids wi h a 400-mesh s uc u e, which we e coa ed wi h a ca bon ilm which
we e glowed discha ged p e ious usage. Subsequen ly, sample solu ions, each comp is-
ing 5 μL, we e applied o he g ids. A e a du a ion o 60 s, he sample solu ions we e
ca e ully emo ed, and he g ids unde wen s aining wi h a 2% ( / ) u anyl ace a e solu-
ion. The s ained g ids we e hen allowed o ai -d y. T ansmission elec on mic oscopy
images we e acqui ed using a Mo gagni 268 (Hillsbo o, O egon, USA) ansmission elec-
on mic oscope ope a ing a a high ol age (HV) o 80 kV, wi h a ilamen se ing o 2.
Gels 2025, 11, 164 21 o 24
4.4. Ci cula Dich oism (CD) Spec oscopy
CD spec a we e eco ded unde a cons an low o N2 using a Jasco model J-1500
(JASCO, Tokyo, Japan) spec opola ime e a 25 °C. Solu ions o hyd ogela o s (0.01 w %)
and GdL (0.01 w %) we e loaded in o 0.1 mm qua z cells.
4.5. Rheological S udies
Gel- o ming solu ions we e injec ed in o he Coue e cell o a s ess-con olled o a-
ional heome e (MCR300, An on Paa GmbH, G az, Aus ia). Viscoelas ic cha ac e iza-
ion was pe o med a 25 °C, and gels unde wen dynamic s ain sweep es ing a 1 Hz up
o 100% o assess gel b eak-up.
4.6. Cell Cul u e and MTT Assay
The human ma ow s omal (HS-5) cell line was ob ained om he Ame ican Type
Cul u e Collec ion (ATCC, Manassas, VA, USA). Cells we e cul u ed in Dulbecco’s Mod-
i ied Eagle’s Medium (DMEM) supplemen ed wi h 10% e al bo ine se um (FBS), 100
U/mL penicillin and 100 μg/mL s ep omycin a 37 °C in a humidi ied a mosphe e o 5%
CO2. Cells we e seeded in 96-well pla es a 1 × 104 cells/well o 24 h o allow a achmen .
The cul u e medium was emo ed and eplaced by esh cul u e medium con aining di -
e en concen a ion o he es compounds. A e 24 h incuba ion, 10 μL o MTT solu ion
(5 mg/mL) (ACROS O ganics) was added o each well ollowed by incuba ion a 37 °C o
4 h. Nex , 100 μL o SDS-HCl solu ion was added o s op he educ ion eac ion and dis-
sol e he o mazan. The abso bance o each well a 595 nm was measu ed by a DTX880
Mul imode De ec o . The pe cen age o cell iabili y, in ela ion o un ea ed cells, was
calcula ed om he abso p ion da a. The MTT assay was pe o med in iple (n = 3) and
he a e age alue o he h ee measu emen s was aken.
Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a :
www.mdpi.com/xxx/s1, Figu e S1: Va iable empe a u e 1H-NMR analysis o he α-NH backbone
amide signals o pep ides; Figu e S2: De e mina ion o he c i ical agg ega ion concen a ion (CAC)
o dehyd o ipep ides 12 (A,B), 13 (C,D) , 14 (E,F), 15 (G,H), 16 (I,J), 17 (K,L) and 18 (M,N):
A,C,E,G,I,K,M - S eady-s a e luo escence spec a (exc = 280 nm) o dehyd opep ide in he concen-
a ion ange 0.6 o 156 μM; B,D,F,H,J,L,N – semiloga i hmic g aphical ep esen a ion o he con-
cen a ion dependence o he luo escence in ensi y a io (I368/I308); Figu e S3: Da a o cha ac e isa-
ion o hyd ogel 12 (0.2w %) a e shown he e as illus a i e example; Figu e S4: Da a o cha ac e i-
sa ion o hyd ogel 13 (0.2w %) a e shown he e as illus a i e example; Figu e S5: Da a o cha ac-
e isa ion o hyd ogel 14 (0.2w %) a e shown he e as illus a i e example; Figu e S6: Da a o cha -
ac e isa ion o hyd ogel 15 (0.2w %) a e shown he e as illus a i e example; Figu e S7: Da a o
cha ac e isa ion o hyd ogel 16 (0.2w %) a e shown he e as illus a i e example; Figu e S8: Da a o
cha ac e isa ion o hyd ogel 17 (0.2w %) a e shown he e as illus a i e example; Figu e S9: Da a o
cha ac e isa ion o hyd ogel 18 (0.2w %) a e shown he e as illus a i e example; Figu e S10: Da a
o cha ac e isa ion o hyd ogel 11 (0.6w %) a e shown he e as illus a i e example; Figu e S11: Da a
o cha ac e isa ion o hyd ogel 12 (0.6 w %) a e shown he e as illus a i e example; Figu e S12:
Da a o cha ac e isa ion o hyd ogel 13 (0.6w %) a e shown he e as illus a i e example; Figu e S13:
Da a o cha ac e isa ion o hyd ogel 14 (0.6w %) a e shown he e as illus a i e example; Figu e S14:
Da a o cha ac e isa ion o hyd ogel 15 (0.6w %) a e shown he e as illus a i e example; Figu e S15:
Da a o cha ac e isa ion o hyd ogel 16 (0.6w %) a e shown he e as illus a i e example; Figu e S16:
Da a o cha ac e isa ion o hyd ogel 17 (0.6w %) a e shown he e as illus a i e example; Figu e S17:
Da a o cha ac e isa ion o hyd ogel 18 (0.6w %) a e shown he e as illus a i e example; Table S1:
Chemical shi empe a u e g adien (∆δ/∆T, ppb) alues o backbone amides o pep ides 15-18 in
DMSO-d6; Table S2: Chemical shi empe a u e g adien (∆δ/∆T, ppb) alues o backbone amides
o pep ides 17 and 18 in DMSO-d6 and 10% D2O ( / ); Table S3: Op imized gela ion condi ions o
Gels 2025, 11, 164 22 o 24
dehyd o ipep ides 11-18; Table S4: P edominan seconda y s uc u e elemen s o hyd ogela o s
11-18 deduced om ci cula dich oism s udies; Syn hesis me hods.
Au ho Con ibu ions: Concep ualiza ion, M.B.-L., J.A.M. and P.M.T.F.; me hodology, M.B.-L.,
J.A.M., P.M.T.F., B.X., D.M.P., L.H. and A.C.; in es iga ion, A.F.C., T.P., C.O. and P.F.; w i ing—
o iginal d a p epa a ion, A.F.C., J.A.M. and P.M.T.F.; w i ing— e iew and edi ing, M.B.-L., J.A.M.
and P.M.T.F. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding: FCT: FEDER: PORTUGAL2020 and COMPETE2020 a e acknowledged o unding unde
esea ch p ojec s UID/QUI/00686/2019, UIDP/CTM/05256/2020, UIDB/05256/2020 and
UIDB/50006/2020. L.H. acknowledges g an CEECINST/00156/2018. And é Ca alho acknowledges
FCT o PhD G an 2020.07743.BD. Te esa Pe ei a acknowledges FCT o PhD G an FCT
2021.07290.BD. Ca los Oli ei a acknowledges FCT o PhD G an 2023.01012.BD.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : The o iginal con ibu ions p esen ed in his s udy a e included in he
a icle/supplemen a y ma e ial; u he inqui ies can be di ec ed o he co esponding au ho s.
Con lic s o In e es : The au ho s decla e no con lic s o in e es .
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ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
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