The effect of l-DOPA hydroxyl groups on the formation of supramolecular hydrogels

Article abstract of DOI:10.1039/c7ob01026e

Fmoc-l-DOPA-d-Oxd-OH was prepared starting from commercially available l-DOPA. Its gelation ability was tested by comparison with Fmoc-l-Tyr-d-Oxd-OH and Fmoc-l-Phe-d-Oxd-OH using ten different triggers. Among them, only GdL, CaCl₂ and ZnCl₂ form strong hydrogels with the three gelators. The analysis of the aerogels obtained by freeze drying the hydrogels show that the three gelators always induce the formation of dense networks, which strongly depend on the nature of the gelator. Rheological analysis of these samples demonstrates that stronger gels were obtained using the l-Tyr containing gelator, while the l-DOPA containing hydrogels were characterized by a storage modulus approximately one order of magnitude lower. Finally, the l-Phe containing gelators show a different trend with respect to the other samples depending on the trigger used. All the hydrogels show a thixotropic behaviour at the molecular level. These results indicate that hydrogel formation is sensitive to both the number of the hydroxyl moieties on the aromatic rings and trigger used.

Full text of DOI:10.1039/c7ob01026e

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DOI: 10.1039/C7OB01026E
Effect of the L-DOPA hydroxyl groups in the
formation of supramolecular hydrogels
a
a
a
Nicola Zanna,* Debora Iaculli, and Claudia Tomasini*
aDipartimento di Chimica “G. Ciamician” - Alma Mater Studiorum Università di Bologna - Via Selmi
, 40126 Bologna (Italy)
2
Abstract
Fmoc-L-DOPA-D-Oxd-OH was prepared starting from commercially available L-DOPA. Its gelation
ability was tested as a comparison with Fmoc-L-Tyr-D-Oxd-OH and Fmoc-L-Phe-D-Oxd-OH using
ten different triggers. Among them, only GdL, CaCl
2
and ZnCl form strong hydrogels with the
2
three gelators. The analysis of the aerogels obtained by hydrogels freeze drying show that the
three gelators induce always the formation of dense networks, that strongly depend on the
gelator nature. The rheological analysis of these samples demonstrate that the stronger gels are
obtained with the L-Tyr containing gelator, while the L-DOPA containing hydrogels are
characterized by a storage modulus approximately an order of magnitude lower. Finally, the L-Phe
containing gelator show a different trend respect to the other ones depending on the trigger. All
the hydrogels show a thixotropic behaviour at the molecular level. These results indicate that the
hydrogel formation is sensitive both to the number of the hydroxyl moieties on the aromatic rings
and on the trigger nature.

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DOI: 10.1039/C7OB01026E
Introduction
In order to understand aggregation phenomena, oligopeptides may be designed and prepared
either to mimicking1–10 or to interfere with11–13 these processes. The non-proteinogenic amino acid
3
,4-dihydroxyphenyl-L-alanine (L-DOPA) contains the catechol moiety which has the peculiar
1
4
property to form stable complexes with metal cations. This property is responsible of the marine
mussel ability to attach to hard surfaces in the sea by means of byssus, that is a mixture of
15
proteins rich in DOPA. The adhesion strength of byssus is also given by the ability to remove
interfacial water from the target surfaces.16
Although various synthetic DOPA containing hydrogels have been reported, they are constituted
by polymeric materials, whose gelation process is activated by high pressure,17 high
18
19
20
temperature, large change in the pH value, and redox reagents.
(4R,5S)-4-Methyl-5-carboxy-oxazolidin-2-one moiety (D-Oxd) can be successfully utilized in the
formation of several supramolecular materials,21–23 including hydrogels.24–26 This little molecule,
that mimics a proline group, may form oligomers having stable secondary structures in solution,
due to its ability to block the peptide bond always in the trans conformation.27–29 Recently, we
reported the gelation behavior of Fmoc-protected dipeptides, all containing the D-Oxd moiety and
we demonstrated that good results may be obtained with dipeptides containing L-Phe or L-Tyr.30,31
In this paper we describe the preparation of physical hydrogels 32–35 using as gelator the L-DOPA
containing pseudopeptide A (Figure 1). We also compare its ability to form supramolecular
hydrogels with L-Phe and L-Tyr containing gelators B and C.
Figure 1. Chemical structure of the three gelators A, B and C described in this work.

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DOI: 10.1039/C7OB01026E
Experimental Section
Materials - All chemicals and solvents were purchased by Sigma-Aldrich, VWR or Iris Biotech and
used as received. Acetonitrile was distilled under inert atmosphere before use. MilliQ water
(Millipore, resistivity = 18.2 mΩ.cm) was used throughout. Solvent were dried by distillation before
use. All reaction were carried out in dried glassware. The melting points of the compounds are
uncorrected. High quality infrared spectra (64 scans) were obtained with an ATR-FT-IR Bruker
Alpha System spectrometer (64 scans). The spectra were obtained in 3 mM solutions in
dichloromethane or as solids at 297 K. All compounds were dried in vacuo and all the sample
preparations were performed in a nitrogen atmosphere. NMR spectra were recorded with a
1
13
Varian Inova 400 spectrometer at 400 MHz ( H NMR) and 100 MHz ( C NMR). Chemical shifts are
reported in δ values relative to the solvent peak.
Boc-L-DOPA-OMe - The molecule was prepared and characterized according to ref. 36
Boc-L-DOPA(OBn)
Boc-L-DOPA(OBn)
2
2
-OH - The molecule was prepared and characterized according to ref. 36
-D-Oxd-OBn - To a stirred solution of Boc-L-DOPA(OBn) -OH (466 mg; 0.98
2
mmol) and HBTU (400 mg, 1.07 mmol) dissolved in dry acetonitrile (10 mL), under inert
atmosphere, H-D-Oxd-OBn (229 mg, 0.98 mmol) dissolved in dry acetonitrile (5 mL) was added at
room temperature, together with a solution of DIPEA (349 μL, 2.05 mmol). The reaction was
monitored by thin layer chromatography; when the reaction was complete, acetonitrile was
removed under reduced pressure. Then the crude mixture was dissolved in dichloromethane (30
mL) and washed with water (30 mL), 1 N aqueous HCl (30 mL) and 5% aqueous NaHCO
3
(30 mL),
dried over anhydrous Na SO , filtered and evaporated in vacuo. The obtained solid was dissolved
2
4
in methanol (10 mL), ultrasonicated for 15 minutes then filtered over Gooch flask. The solid was
washed with methanol (1 x 20 mL), dissolved in dichloromethane to recover it from the filter and
evaporated in vacuo to afford Boc-L-DOPA(OBn)
85 % yield).
M.p. = 171.6-172.0 °C; IR (3 mM in CH
400 MHz, CDCl ): δ 1.36 (d, J=6.4 Hz, 3H, CH
DOPA), 2.96–3.09 (m, 1H, CHHβ-DOPA), 4.25 (d, J=3.9 Hz, 1H, CHN Oxd), 4.43–4.56 (m, 1H, CHO
Oxd), 5.11 (s, 2H, CH Ph), 5.14 (s, 2H, CH Ph), 5.21 (s, 2H, CH Ph), 5.78 (s, 1H, CHα-DOPA), 6.69 (d,
J=8.2 Hz, 1H, CH Ar DOPA), 6.75–6.96 (m, 2H, CH Ar DOPA), 7.14–7.58 (m, 15H, ArH). C NMR (100
MHz, CDCl ): δ 21.1, 28.4, 38.8, 53.9, 61.9, 68.1, 71.3, 71.4, 73.6, 79.9, 115.1, 116.4, 122.5, 127.3,
27.6, 127.8, 127.9, 128.4, 128.5, 128.8, 129.1, 134.7, 137.3, 137.4, 148.2, 149.0, 151.2, 154.7,
2
-D-Oxd-OBn (576 mg, 0.83 mmol) as a white solid
(
-
1 1
2
Cl
2
): ν 3439, 1791, 1754, 1711, 1508, 1496 cm ; H NMR
Oxd), 1.40 (s, 9H, t-Bu), 2.81–2.94 (m, 1H, CHHβ-
(
3
3
2
2
2
1
3
3
1

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DOI: 10.1039/C7OB01026E
1
3
67.5, 172.7. Anal. Calcd. for C50
.39.
44
H N
2
O : C, 73.52; H, 5.43; N, 3.43. Found: C, 73.48; H, 5.41; N,
9
Fmoc-L-DOPA(OBn)
nitrogen atmosphere to a solution of Boc-L-DOPA(OBn)
dichloromethane (5 mL). After 4 hours the reaction was complete, dichloromethane was removed
under reduced pressure and H-L-DOPA(OBn) -D-Oxd-OBn∙CF CO H was obtained in quantitative
2
-D-Oxd-OBn - Trifluoroacetic acid (1.14 mL, 14.40 mmol) was added under
2
-D-Oxd-OBn (558 mg, 0.80 mmol) in dry
2
3
2
yield. The crude was dissolved in dichloromethane (20 mL), then Fmoc N-hydroxysuccinimmide
ester (270 mg, 0.80 mmol) and DIPEA (516 μL, 3.04 mmol) were added to the mixture, that was
stirred at room temperature for 24 hours. Then dichloromethane (30 mL) was added to the
mixture, that was washed with water (30 mL), 1 N aqueous HCl (30 mL) and 5% aqueous NaHCO
3
(
30 mL), dried over anhydrous Na SO , filtered and evaporated in vacuo. The obtained solid was
2
4
dissolved in methanol (10 mL), ultrasonicated for 15 minutes then filtered over Gooch flask. The
solid was washed with methanol (1 x 20 mL), dissolved in dichloromethane to recover it from the
filter and evaporated in vacuo to afford Fmoc-L-DOPA(OBn)
2
-D-Oxd-OBn (630 mg, 0.77 mmol) as a
Cl ): ν 3428, 1792, 1754, 1712, 1511
Oxd), 2.88–2.98 (m, 1H, CHHβ-DOPA),
-Fmoc), 4.23–4.33 (m, 2H, O-CH-CH
Fmoc), 4.33-4.43(m, 1H, CHN Oxd), 4.48–4.57 (m, 1H, CHO Oxd), 5.08 (s, 2H, CH Ph), 5.12 (s, 2H,
CH Ph), 5.19 (s, 2H, CH Ph), 5.41 (d, J=8.6 Hz, 1H, NH DOPA), 5.87 (dd, J=14.4, 8.6 Hz, 1H, CHα-
white solid (96% yield). M.p. = 160.1-160.3°C; IR (3 mM in CH
2
2
-
1 1
cm ; H NMR (400 MHz, CDCl
3
): δ 1.38 (d, J=6.4 Hz, 3H, CH
3
3
.02–3.13 (m, 1H, CHHβ-DOPA), 4.14–4.23 (m, 1H, O-CH-CH
2
2
-
2
2
2
DOPA), 6.69 (dd, J=8.1, 2.0 Hz, 1H, CH Ar DOPA), 6.83 (d, J=8.1 Hz, 1H, CH Ar DOPA), 6.87 (d, J=2.0
Hz, 1H, CH Ar DOPA), 7.18–7.50 (m, 19H, ArH), 7.55 (d, J=6.3 Hz, 2H, ArH Fmoc), 7.75 (d, J=6.3 Hz,
1
3
2
7
1
1
H, ArH Fmoc); C NMR (100 MHz, CDCl ): δ 21.2, 38.6, 47.2, 54.3, 61.9, 67.2, 68.2, 71.4, 71.4,
3
3.8, 115.1, 116.4, 120.0, 122.5, 125.3, 127.2, 127.3, 127.3, 127.5, 127.6, 127.8, 127.8, 127.9,
28.4, 128.5, 128.6, 128.8, 128.8, 134.6, 137.3, 137.4, 141.4, 144.0, 148.4, 149.1, 151.3, 167.4,
72.4. Anal. Calcd. for C40
H
42
N
2
O
9
: C, 69.15; H, 6.09; N, 4.03. Found: C, 69.18; H, 6.08; N, 4.07.
-D-Oxd-OBn (100 mg,
.12 mmol) in a mixture of TFA:methanol 5:95 (100 mL), Pd/C (10% w/w, 10 mg) was added, then
Fmoc-L-DOPA-D-Oxd-OH A - To a stirred solution of Fmoc-L-DOPA(OBn)
2
0
vacuum was created inside the flask using the vacuum line. The flask was then filled with hydrogen
using a balloon (1 atm). The solution was stirred for 90 minutes under hydrogen atmosphere. The
mixture was then filtered and evaporated in vacuo, the solid was dissolved in dichloromethane,
sonicated and filtrated over Gooch flask. The desired product Fmoc-L-DOPA-D-Oxd-OH was

Page 5 of 17
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dissolved in methanol to recover it from the filter and evaporated in vacuo and was obtained as a
DOI: 10.1039/C7OB01026E
white solid in 85% yield (56 mg, 0.104 mmol).
-
1
1
M.p. = 135.4-136.4°C; IR (ATR-IR): ν 3325, 1783, 1689, 1604, 1517 cm ; H NMR (400 MHz,
CD OD): δ 1.44 (d, J=6.4 Hz, 3H, CH Oxd), 2.75 (dd, J=13.5, 5.3 Hz, 1H, CH β-DOPA), 3.01 (dd,
J=13.5, 5.4 Hz, 1H, CH β-DOPA), 4.10–4.22 (m, 2H, O-CH-CH -Fmoc), 4.23–4.33 (m, 1H, O-CH-CH
3
3
2
2
2
2
-
Fmoc), 4.37 (d, J=3.7 Hz, 1H, CHN Oxd), 4.62–4.73 (m, 1H, CHO Oxd), 5.76 (dd, J=9.0, 5.3 Hz, 1H,
CHα-DOPA), 6.62 (d, J=8.3 Hz, 1H, CH-Ar DOPA), 6.67 (d, J=8.3 Hz, 1H, CH-Ar DOPA), 6.78 (s, 1H,
CH-Ar DOPA), 7.22–7.33 (m, 2H, ArH Fmoc), 7.33–7.41 (m, 2H, Ar-H Fmoc), 7.59 (d, J=7.6 Hz, 2H,
1
3
Ar-H Fmoc), 7.77 (d, J=7.7 Hz, 2H, A-rH Fmoc); C NMR (100 MHz, CD
3
OD): δ 21.2, 30.7, 38.9, 39.2,
5
1
5
6.1, 63.3, 68.1, 75.9, 116.2, 117.5, 120.7, 120.8, 122.1, 125.0, 126.2, 126.3, 127.9, 128.1, 128.6,
29.2, 142.4, 145.1, 145.2, 145.3, 146.1, 153.6, 157.9, 171.5, 174.1. Anal. Calcd. for C19
3.77; H, 5.70; N, 6.60. Found: C, 53.73; H, 5.75; N, 6.62.
H
24
N
2
9
O : C,
Conditions for the Gel Formation with pH Trigger - 20 mg of compound A, B or C was placed in a
test tube (diameter: 8 mm), then MilliQ water (≈0.95 mL) and aqueous NaOH 1 N (1 equiv.) were
added to get a final volume of 1 mL and the mixture was stirred and sonicated in turn for about 30
minutes, until sample dissolution. Then glucono-δ-lactone (GdL: 1.1 equiv.) was added in one
portion to the mixture. After a rapid mixing to allow the GdL complete dissolution, the sample was
allowed to stand quiescently until gel formation.
Conditions for Gel Formation with Inorganic Triggers - 20 mg of compound A, B or C was placed in
a test tube (diameter: 8 mm), then MilliQ water (0.5 mL) and a 1M aqueous NaOH (1 equiv.) were
added to get a final volume of 1 mL. The mixture was stirred until sample dissolution. The cationic
trigger (0.3 equiv.) was added to the solution under rapid stirring then the tube was allowed to
stand quiescently until gel formation.
Conditions for Gel Formation with Amino Acid Triggers - 20 mg of compound A, B or C was placed
in a test tube (diameter: 8 mm), then the selected amino acid (1 equiv.) was added. 1 mL of MilliQ
water was added to the test tube under stirring, then after 1 minute the magnetic stirrer removed
and the tube was allowed to stand quiescently until gel formation.
Conditions for Tgel Determination - Tgel was determined by heating some test tubes (diameter: 8
mm) containing the gel and a glass ball (diameter: 5 mm, weight: 165 mg) on the top of it. When
the gel is formed, the ball is suspended atop. The Tgel is the temperatures in which the ball start to
penetrate inside the gel. Some hydrogel samples melt, producing a clear solution, while in other
cases the gelator shrinks and water is ejected, as syneresis occurs.

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Aerogels Preparation - The aerogels were prepared with the following procedure: 0.5 mL of the
DOI: 10.1039/C7OB01026E
hydrogel was prepared directly into an Eppendorf test tube at room temperature. After the
hydrogel is formed (test tube inversion), the sample was deeped in liquid nitrogen for 10 minutes,
then it was freeze-dried for 24 hours in vacuo (0.02 mBar) at -50 °C using a BENCHTOP Freeze Dry
System CHRIST Alpha 1-2 LD Plus.
SEM Analysis - Scanning electron micrographs of the samples were recorded using a Hitachi 6400
field emission gun scanning electron microscope.
Rheology - Rheology experiments were carried out on an Anton Paar Rheometer MCR 102 using a
parallel plate configuration (25 mm diameter). Experiments were performed at constant
temperature of 25 °C controlled by the integrated Peltier system and a Julabo AWC100 cooling
system. To keep the sample hydrated, a solvent trap was used (H-PTD200). Amplitude and
Frequency sweep analysis were performed with fixed gap value of 1 mm on gel samples prepared
directly on the upper plate of the rheometer. The samples were prepared the day before the
analysis and left overnight at controlled temperature of 20 °C to complete the gelation process.
Oscillatory amplitude sweep experiments (γ: 0.01−100%) were carried out in order to determine
-
1
the linear viscoelastic (LVE) range at fixed frequency of 1 rad s . Once established the LVE of each
-
1
hydrogel, frequency sweep tests were performed (ω: 0.1-100 rad·s ) at constant strain within the
LVE region of each sample. To verify the thixotropic properties of the hydrogels strain values
within and over the crossover point region where consecutively applied to the hydrogels. The
values of the applied strain were selected on the basis of the crossover point value obtained from
amplitude sweep experiment for hydrogels 1-9.
Results and Discussion
Synthesis of Gelator A
The synthesis of gelator A started from the unprotected and commercially available L-DOPA that
was transformed in five steps into Boc-L-DOPA(OBn) -OH in multigram scale with excellent yields,
2
following a reported literature.36,37 Further steps allowed us to introduce the D-Oxd moiety, to
replace the Boc moiety with the Fmoc moiety and to remove the three benzyl protecting groups
(Scheme 1). The gelator A was obtained pure as a white solid in 67% overall yield from L-DOPA.
Compounds B and C have been efficiently prepared in multigram scale, following the reported
literatures.30,31

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DOI: 10.1039/C7OB01026E
Scheme 1. Reagents and conditions: (i) SOCl
2
(excess), MeOH, 0 °C, 24 h; (ii) Boc
2
O (2 equiv.),
NaHCO
3
(2 equiv.), THF/H
2
O, r.t., 18 h; (iii) BnBr (2.2 equiv.), K
2
CO
3
(2.2 equiv.), TBAB (0.2 equiv.),
NaI (0.2 equiv.), acetone, reflux, 4 h; (iv) 1M NaOH, MeOH/THF, r.t., 18 h; (v) 1M HCl; (vi) D-Oxd-
OBn (1 equiv.), HBTU (1.1 equiv.), DIEA (2 equiv.), AcCN, r.t., 4 h; (vii) TFA (18 equiv.), CH Cl , r.t., 4
, MeOH/TFA (95:5),
2
2
h; (viii) Fmoc-NHS (1 equiv.), DIEA (3 equiv.), r.t., 24 h; (ix) Pd/C (10% w/w), H
r.t., 90 min.
2
Hydrogels Formation
A systematic study on the ability of compounds A, B and C to form hydrogels was then undertaken
to check the importance of the cresol moiety, as a comparison with a phenol or a phenyl group.
The presence of two ortho- hydroxyl groups should in principle favour its interaction with metal
cations due to their chelation ability. The study of the ability to form metal:peptide complexes
with zinc could be of particular interest, as zinc is an essential biological cofactor and plays a
critical role in the central nervous system.38,39 The concentration of intracellular zinc typically
ranges between 180 and 250 mM in eukaryotic cells. However, very little zinc exists as ‘free’ ions
within the cell, usually only pico- to low nanomolar concentrations.40
We tested the behaviour of zinc cations as triggers to check if they can induce the formation of
strong hydrogels with gelators A, B and C. We also compared these results with the behaviour of
2
+
2+
2+
2+
3+
3+
other triggers: divalent and trivalent metal cations (Ca , Ba , Mg , Cu , Al and Fe ) and two

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basic amino acids (hystidine and arginine), as we recently demonstrated that they induce the
DOI: 10.1039/C7OB01026E
formation of physical hydrogels with selected gelators by coordination with the gelator
carboxylate anions.41,42 We analysed also the hydrogels obtained using the pH variation method as
trigger, by addition of a stoichiometric amount of δ-gluconolactone (GdL).43
All the hydrogels were prepared with a 2% w/w gelator concentration, that we recognized as the
optimal concentration for strong hydrogels formation.30,41,44 The general method for the hydrogels
preparation is to place a portion (20 mg) of gelator A, B or C in a test tube with 1 mL of MilliQ
water (for GdL and metal cations triggers a stoichiometric amount of 1M NaOH is also needed).
The mixture is stirred until sample dissolution, then the trigger is added to the solution under
rapid stirring and the tube is allowed to stand quiescently until gels formation. While a
stoichiometric amount of GdL or of a given amino acid is needed for strong gel formation, a
4
5
stoichiometric trigger/gelator ratio of a metal cation not always provides the best results. We
recently demonstrated that a 0.3 trigger/gelator ratio affords strong hydrogels, using calcium
chloride as trigger.42
We checked if hydrogels are formed under thirty different conditions using A, B or C and ten
triggers, but we were able to prepare only eighteen hydrogels. Although we are aware that the
modification of the trigger/gelator ratio may afford interesting results, in this preliminary study we
chose to screen the triggers only under given conditions to focus the investigation on the gelation
efficiency as a function of the hydroxyl groups number. All the trials are summarized in Table 1 and
the formed hydrogels have been numbered. All the details are reported in Table S1.
Table 1. Summary of the conditions for hydrogel formation using gelators A, B and C. Only the
formed hydrogels have been numbered for clarity.
Gelator → Fmoc-L-Dopa-D-Oxd-OH Fmoc-L-Tyr-D-Oxd-OH Fmoc-L-Phe-D-Oxd-OH
Trigger ↓
A
B
C
GdL
1
2
3
CalCl
2
4
5
6
ZnCl
BaCl
2
7
8
9
-
-
18
21
-
2
10
13
-
19
-
11
14
17
-
-
-
L-Hys
MgCl
CuCl
Al (SO
Fe(NO
2
2
2
4
3
)
)
2
3
-
-
L-Arg
-
-
30

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DOI: 10.1039/C7OB01026E
GdL, CaCl
2
and ZnCl
2
allow the formation of hydrogels with any gelator. In contrast BaCl
2
and L-Hys
result in the formation of hydrogels only with the gelators A and B, while MgCl
2
allow the gelation
of only B and C. Finally, Al (SO , Fe(NO ) and L-Arg furnish unsatisfactory results. Several
2
4
)
2
3
3
hydrogels show a thixotropic behaviour at macroscopic scale, as they become liquid if a shear
stress is applied then they quickly recover the solid form on resting.46,47
Hydrogels Characterization
We have recently demonstrated that good rheological properties are usually associated with
3
0
higher Tgel, so a first screening of the optimal conditions for hydrogel formation may be spotted
using this method. We measured the Tgel for all the eighteen formed hydrogels by the dropping
ball method (see Experimental and Table S1 for the details), getting results ranging between 40 °C
and 98 °C. Hydrogels 1-9, obtained with GdL, CaCl
2
or ZnCl
2
have always a Tgel higher than 60 °C.
Among them, the ZnCl containing hydrogels 7-9 afford the best results, with Tgel always higher
2
than 89 °C. In contrast, no considerable Tgel variation may be ascribed to the gelator.
Another interesting parameter that needs to be checked in the hydrogels formation is the final pH,
as a neutral pH is very important for the preparation of biocompatible hydrogels. Among the three
most interesting triggers, GdL produces the hydrogels 1-3 with a pH ranging between 4.0 and 4.5,
too low to be biocompatible, although these hydrogels may be very useful for other applications.
In contrast, all the other hydrogels have a final pH ranging between 6.5 and 7.5, so they are all
good candidates for biological applications.
After this preliminary screening, we focused our attention on hydrogels 1-9 that are formed using
GdL, CaCl
2
or ZnCl as gelators. These triggers allow the hydrogels formation with any gelator. The
2
photograph of these hydrogels is shown in Figure 2, while the photographs of all the other
hydrogels are reported in Figure S1.
Figure 2. Photographs of hydrogels 1-9 (from left to right).

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Some more information on the nature of hydrogels 1-9 were obtained by SEM analysis of the
DOI: 10.1039/C7OB01026E
aerogels prepared by freeze-drying these samples (Figure 3). All the L-DOPA containing aerogels 1,
4
and 7 are organized in platelets, the L-Tyr containing aerogels 2, 5 and 8 are organized in dense
fibrous networks, finally the L-Phe containing aerogels 3, 6 and 9 are organized in locally oriented
long strips that cross on the large scale. In any case the fast formation of all the hydrogels suggest
that the self-assembly occurs under kinetic rather than under thermodynamic conditions.48,49
Figure 3. SEM images of samples of xerogel obtained by freeze drying samples of hydrogel 1-9. Bar
=
20 µm.
Another piece of information on the supramolecular interactions involved in the formation of
hydrogels 1-9 may be obtained by the ATR-IR spectra of some aerogels samples. The more ordered
fibers obtained with gelator C (Figure 4C) produce very reproducible IR spectra, all containing a
-1
band at 3330 cm typical of hydrogen bonded NH bonds, another strong stretching band at 1687
-
1
-1
cm , typical of hydrogen bonded CO bonds, together with a weaker band around 1067 cm that

Page 11 of 17
Organic & Biomolecular Chemistry
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may be attributed to the amide band II. The dense fibrous networks, typical of aerogels 2, 5 and 8
DOI: 10.1039/C7OB01026E
-
1
show a more complex pattern (Figure 4B), where prevails either band I at about 1690 cm or band
-
1
II at about 1600 cm . Finally the L-DOPA containing aerogels 1, 4 and 7 (Figure 4A) have IR spectra
where both amide band I and amide band II are present, together with the NH stretching band at
-
1
3
325 cm .
C
1687
1
,0
0,8
,6
0,4
,2
0,0
3500
3
6
9
A
1692
B
1691
1609
1
0
0
0
0
0
,0
,8
,6
,4
,2
1
4
7
1,0
0,8
0,6
0,4
0,2
0,0
2
5
8
1
605
1607
1720
1
736
1740
0
3
325
3322
3330
0
,0
3
500
3400
3300
1900 1800 1700 1600
3500
3400
3300
1900 1800 1700 1600
-1
Wavelength (cm )
3400
3300
1900 1800 1700 1600
-1
-
1
Wavelength (cm )
Wavelength (cm
)
Figure 4. Selected regions of ATR-IR spectra of aerogels 1-9: (A) L-DOPA containing aerogels 1, 4
and 7; (B) L-Tyr containing aerogels 2, 5 and 8; (C) L-Phe containing aerogels 3, 6 and 9.
Rheological analyses have been carried out to evaluate the viscoelastic properties of hydrogels 1-9
in terms of storage and loss moduli (G′ and G″ respectively) (Table 2, Figures S3-S5). All the
obtained hydrogels are characterized by a storage modulus approximately an order of magnitude
higher than the loss component, indicating their “solid-like” attitude. Frequency sweep analysis
pointed out that for all the obtained hydrogels both G′ and G″ were almost independent from the
−1
frequency in the range from 0.1 to 100 rad·s (with G′ always greater than G″) confirming the
solid-like” rheological behaviour.
“
Table 2. Rheological properties of hydrogels 1-9.
Hydrogel Gelator Trigger (equiv.) G’(Pa) G’’(Pa) γ (%)
1
2
3
4
5
6
7
8
9
A
B
C
A
B
C
A
B
C
GdL (1.1)
GdL (1.1)
GdL (1.1)
3400
430
9.64
52600 5000 11.55
48800 3440
8500 1050
17000 2500
6400
2144
3.22
6.65
5.42
12.76
3.88
4.69
6.00
CaCl
CaCl
CaCl
ZnCl
ZnCl
ZnCl
2
2
2
2
2
2
(0.3)
(0.3)
(0.3)
(0.3)
(0.3)
(0.3)
430
366
74271 7280
700 61

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Rheological studies indicate that the strength of the hydrogels is strongly affected both by the
DOI: 10.1039/C7OB01026E
aromatic amino acid and by the trigger. The general trends demonstrate that, maintaining a 0.3
trigger/gelator ratio, the strongest gels are obtained with the L-Tyr containing gelator B, as
hydrogels 2, 5 and 8 are characterized by storage modulus G’ values ranging between 10 and 105
4
Pascal, with the hydrogels strength following the trend ZnCl
2
> GdL > CaCl . The L-DOPA containing
2
hydrogels 1, 4 and 7 are characterized by storage modulus approximately an order of magnitude
3
4
lower, always ranging between 10 and 10 Pascal, with the hydrogels strength following the trend
CaCl > GdL > ZnCl . This unexpected result may be ascribed to the trigger/gelator ratio that could
2
2
be more suitable for gelator B rather than for the cresol containing gelator A. We are planning to
study the effect of the variation of the trigger/gelator ratio on the hydrogel properties when
gelator A is used.
Finally, the L-Phe containing gelator C, that does not possess hydroxyl groups, is very sensitive
towards the trigger, as the hydrogels strength follow the trend GdL > CaCl
2
> ZnCl . These results
2
indicate that there is not a universal trigger able to induce the formation of a hydrogel endowed
with the best mechanical properties.
Step strain experiments were performed to check the thixotropic behaviour of hydrogels 1-9 at
the molecular level. Strain values within and over the crossover point region where consecutively
applied to the hydrogels, that lose their “solid-like” behavior (G’<G”) when the strain applied is
over their crossover point region and quickly go back to a “solid-like” state (G’>G”) if the strain is
in the LVE region the hydrogels (Figure 5).

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DOI: 10.1039/C7OB01026E
4
3
2
1
5
4
3
2
5
4
3
2
1
1
1
1
0
10
10
10
10
10
10
10
10
1
2
3
0
0
0
0
500
1000
1500
2000
0
500
1000
1500
2000
500
1000
Time(s)
1500
2000
Time(s)
Time(s)
4
5
5
10
10
10
10
10
10
10
10
6
4
5
4
3
2
10
10
10
4
3
3
2
2
1
10
10
500
1000
1500
2000
500
1000
Time(s)
1500
2000
500
1000
Time(s)
1500
2000
Time(s)
4
3
2
1
0
5
10
4
10
10
10
10
10
7
8
9
4
3
2
3
2
1
1
0
0
10
10
10
1
10
0
500
1000
1500
2000
0
500
1000
1500
2000
0
500
1000
1500
2000
Time(s)
Time(s)
Time(s)
Figure 5. Values of storage moduli G’ (black) and loss moduli G” (red) recorded during a step strain
experiment performed on hydrogels 1-9.
Although only hydrogels 3, 6, 7, 8 and 9 show a thixotropic behaviour at macroscopic scale (Table
S1), the results observed for hydrogels 1-9 show that they are all characterized by a great
capability to recover the gel-like behaviour, thus showing their thixotropic properties at the
molecular level.
Conclusions
We prepared the new gelator Fmoc-L-DOPA-D-Oxd-OH A and we tested its gelation ability as a
comparison with the already reported gelators Fmoc-L-Tyr-D-Oxd-OH B and Fmoc-L-Phe-D-Oxd-OH
C under several gelation conditions, to check the effect of the cresol moiety.

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As an overall observation, the different gelators behaviour may be attributed to the increased
DOI: 10.1039/C7OB01026E
water solubility of the L-DOPA and L-Tyr derivatives compared with the L-Phe containing gelator.
The results obtained with the ten different triggers indicate that the hydrogel formation is very
sensitive both to the number of the hydroxyl moieties linked to the aromatic rings and to the
trigger nature, so there is not a universal trigger able to produce the hydrogel with the best
mechanical properties in any case.
GdL, CaCl
2
and ZnCl form strong hydrogels endowed with thixotropic behaviour at the molecular
2
level with all the three gelators. The analysis of the aerogels obtained by freeze drying the
hydrogels show that the three gelators induce the formation of dense networks, as the A aerogels
show a platelet pattern, the B aerogels display an unordered dense fibrous network and the C
aerogels are organized in locally oriented long strips. The rheological analysis of these samples
demonstrate that the strongest hydrogels are obtained with the L-Tyr containing gelator B, while
the L-DOPA containing hydrogels 1, 4 and 7 are characterized by a storage modulus approximately
an order of magnitude lower. Finally, the strength of L-Phe containing hydrogels show a different
trigger dependency respect to the other ones.
Although the fast formation of all the hydrogels suggest that the self-assembly occurs under
kinetic rather than under thermodynamic conditions, more studies are planned to have a deeper
understanding in the hydrogelation process that could be responsible for the formation of
stronger hydrogels with the L-Tyr gelator B compared to the gels formed by the L-DOPA gelators
A. Moreover, the variation of the trigger/gelator ratio may modify these results, as a different
2
+
2+
amount of Ca or Zn could be more suitable for hydrogels formation in the presence of the
cresol moiety.
Finally, the strong ZnCl
propensity to form complexes with this metal. Further studies are currently ongoing to find the
optimal gelator/ZnCl ratio for the formation of strong and thixotropic hydrogels for biological and
2
containing hydrogels 7-9, suggest that the three gelators have a good
2
cell growth applications.
Acknowledgements
We gratefully acknowledge Ministero dell’Università e della Ricerca (PRIN 2015 project
2
0157WW5EH) and Alma Mater Studiorum Università di Bologna for financial support.

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DOI: 10.1039/C7OB01026E
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