Modulating Thiol p Ka Promotes Disulfide Formation at Physiological pH: An Elegant Strategy to Design Disulfide Cross-Linked Hyaluronic Acid Hydrogels

Article abstract of DOI:10.1021/acs.biomac.8b01830

The disulfide bond plays a crucial role in protein biology and has been exploited by scientists to develop antibody-drug conjugates, sensors, and for the immobilization other biomolecules to materials surfaces. In spite of its versatile use, the disulfide chemistry suffers from some inevitable limitations such as the need for basic conditions (pH > 8.5), strong oxidants, and long reaction times. We demonstrate here that thiol-substrates containing electron-withdrawing groups at the β-position influence the deprotonation of the thiol group, which is the key reaction intermediate in the formation of disulfide bonds. Evaluation of reaction kinetics using small molecule substrate such as l-cysteine indicated disulfide formation at a 2.8-fold higher (k₁ = 5.04 × 10^-4 min^-1) reaction rate as compared to the conventional thiol substrate, namely 3-mercaptopropionic acid (k₁ = 1.80 × 10^-4 min^-1) at physiological pH (pH 7.4). Interestingly, the same effect could not be observed when N-acetyl-l-cysteine substrate (k₁ = 0.51 × 10^-4 min^-1) was used. We further grafted such thiol-containing molecules (cysteine, N-acetyl-cysteine, and 3-mercaptopropionic acid) to a biopolymer namely hyaluronic acid (HA) and determined the pK_a value of different thiol groups by spectrophotometric analysis. The electron-withdrawing group at the β-position reduced the pK_a of the thiol group to 7.0 for HA-cysteine (HA-Cys); 7.4 for N-acetyl cysteine (HA-ActCys); and 8.1 for HA-thiol (HA-SH) derivatives, respectively. These experiments further confirmed that the concentration of thiolate (R-S^-) ions could be increased with the presence of electron-withdrawing groups, which could facilitate disulfide cross-linked hydrogel formation at physiological pH. Indeed, HA grafted with cysteine or N-acetyl groups formed hydrogels within 3.5 min or 10 h, respectively, at pH 7.4. After completion of cross-linking reaction, both gels demonstrated a storage modulus G′ ≈ 3300-3500 Pa, which indicated comparable levels of cross-linking. The HA-SH gel, on the other hand, did not form any gel at pH 7.4 even after 24 h. Finally, we demonstrated that the newly prepared hydrogels exhibited excellent hydrolytic stability but can be degraded by cell-directed processes (enzymatic and reductive degradation). We believe our study provides a valuable insight on the factors governing the disulfide formation and our results are useful to develop strategies that would facilitate generation of stable thiol functionalized biomolecules or promote fast thiol oxidation according to the biomedical needs.

Full text of DOI:10.1021/acs.biomac.8b01830

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Article
Modulating thiol pKa promotes disulfide formation at physiological pH: An
elegant strategy to design disulfide crosslinked hyaluronic acid hydrogels
Daniel Bermejo-Velasco, Alice Azemar, Oommen P. Oommen, Joens Hilborn, and Oommen P Varghese
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01830 • Publication Date (Web): 06 Feb 2019
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Modulating thiol pKa promotes disulfide formation at physiological pH: An
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elegant strategy to design disulfide crosslinked hyaluronic acid hydrogels
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Daniel Bermejo-Velasco,1 Alice Azémar,1 Oommen P. Oommen,2 Jöns Hilborn,1 Oommen P.
Varghese*1
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1Translational Chemical Biology Laboratory, Division of Polymer Chemistry, Department of
Chemistry-Ångstrom, Uppsala University, Uppsala, Sweden
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2Bioengineering and Nanomedicine Lab, Faculty of Medicine and Health Technologies and
BioMediTech Institute, Tampere University, Korkeakoulunkatu 3, Tampere-33720, Finland
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12 Abstract
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The disulfide bond plays a crucial role in protein biology and has been exploited by scientists
to develop antibody-drug conjugates, sensors and for the immobilization other biomolecules
to materials surfaces. In spite of its versatile use, the disulfide chemistry suffers from some
inevitable limitations such as the need for basic conditions (pH > 8.5), strong oxidants and
long reaction times. We demonstrate here that thiol-substrates containing electron-
withdrawing groups at the β-position influence the deprotonation of the thiol group, which is
the key reaction intermediate in the formation of disulfide bonds. Evaluation of reaction
kinetics using small molecule substrate such as L-cysteine indicated disulfide formation at a
2.8-fold higher (k1 = 5.04 x 10-4 min-1) reaction rate as compared to the conventional thiol
substrate, namely 3-mercaptopropionic acid (k1 = 1.80 x 10-4 min-1) at physiological pH (pH
7.4). Interestingly, the same effect could not be observed when N-acetyl-L-cysteine substrate
(k1 = 0.51 x 10-4 min-1) was used. We further grafted such thiol-containing molecules
(cysteine, N-acetyl-cysteine, and 3-mercaptopropionic acid) to a biopolymer namely
hyaluronic acid (HA) and determined the pKa value of different thiol groups by
spectrophotometric analysis. The electron-withdrawing group at the β-position reduced the
pKa of the thiol group to 7.0 for HA-cysteine (HA-Cys); 7.4 for N-acetyl cysteine (HA-
ActCys) and 8.1 for HA-thiol (HA-SH) derivatives respectively. These experiments further
confirmed that the concentration of thiolate (R-S-) ions could be increased with the presence
of electron-withdrawing groups, which could facilitate disulfide cross-linked hydrogel
formation at physiological pH. Indeed, HA grafted with cysteine or N-acetyl groups formed
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hydrogels within 3.5 minutes or 10 hours, respectively at pH 7.4. After completion of
crosslinking reaction both gels demonstrated a storage modulus G’ ≈3300–3500 Pa, indicating
comparable levels of crosslinking. The HA-SH gel, on the other hand, did not form any gel at
pH 7.4 even after 24 h. Finally, we demonstrated that the newly prepared hydrogels exhibited
excellent hydrolytic stability but can be degraded by cell-directed processes (enzymatic and
reductive degradation). We believe our study provides a valuable insight on the factors
governing the disulfide formation and our results are useful to develop strategies that would
facilitate generation of stable thiol functionalized biomolecules or promote fast thiol oxidation
according to the biomedical needs.
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44 Introduction
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Disulfide formation is crucial in many biological processes, such as protein folding,1, 2 redox
signaling,3 and the regulation of protein function.4 Disulfide linkages are of particular interest
for bioconjugation, controlled drug delivery, and cell encapsulation, because of the ability of
cells to cleave disulfide bonds by secreting natural reductants such as glutathione.5-7 The
strategy most commonly used in organic chemistry to form disulfide bonds is the oxidation of
thiol compounds in presence of oxygen (Equation 1). Thiol-oxidation, in general is a very
slow process under physiological conditions (pH 7.4) because it involves the deprotonation of
the thiol groups to form thiolate ions (R-S-) that react with the molecular oxygen to generate
the reactive radical species. Since thiols have a pKa of ≈8-10, it requires basic conditions (pH
> 8.5) to drive the forward reaction.8 Other strategies involve the use of radical initiators such
as peroxides that yield disulfides following a complex multistep reaction intermediates.9
Therefore, the rate of disulfide formation through thiol oxidation can be improved by
increasing the reaction pH9, 10 or by the addition of oxidants such as hydrogen peroxide or
iodine.9, 11
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R-SH + H2O ⇌ R-S- + H3O+
4R-S- + O2 + 4H3O+ ⇌ 4R-S˙ + 6H2O
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2R-S˙ → R-S-S-R
(1)
Another strategy to form disulfide bonds is the disulfide exchange reaction, which also
involves the deprotonation of the thiol group, followed by nucleophilic substitution reaction
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with other disulfides having labile thiol groups (Equation 2). The use of reactive pyridyl
disulfide compounds highly improves the disulfide exchange reaction rate at neutral
conditions and is extensively used for conjugation reactions.7, 12
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R-SH + H2O ⇌ R-S- + H3O+
R-S- + R’-SS-R’ ⇌ R-SS-R’ + R’-S-
(2)
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Even though disulfide chemistry is slow at physiological pH, it is intriguing to note that most
of the proteins form three-dimensional (3D) structure employing cysteine dimerization with
high fidelity. It was also reported previously by Lutolf et al. that cysteine pKa could be
increased or decreased by changing the neighboring amino acid in a peptide sequence that
regulate the rate of Michael addition reactions.13 This prompted us to investigate the role of
thiol-pKa in driving disulfide formation at physiological pH using different thiol derivatives.
We subsequently validated our finding by developing hyaluronic acid (HA) based injectable
hydrogels under physiological conditions.
Hydrogels are water containing 3D polymeric networks with highly tunable physical and
chemical properties14 and have been extensively used for a range of biomedical applications,
including tissue engineering,15-17 and drug delivery18-20 owing to their ability to mimic the
native extracellular environment. Hydrogels based on natural polymers such as HA, that is
present in the extracellular matrix (ECM), have defined interactions with cells that support
cellular activities, has inherent biocompatibility and biodegradability and can preserve cell-
secreted sensitive proteins.21 In addition, cells cultured on HA-hydrogels secrete factors that
regulate cell response and function.22 HA is composed of β-1,4-D-glucuronic acid-β-1,3-D-N-
acetylglucosamine disaccharide repeat units and represents the only non-sulfated
glycosaminoglycan present in the ECM. It is known to play a diverse biological role because
of its ability to hold large amounts of water, lack of immunogenicity and degradability by
ubiquitous enzymes.23 Therefore, HA-based hydrogels have been applied as tissue fillers,24
drug delivery systems,25, 26 and is intensively studied for tissue regeneration.17, 27 We have
previously developed hydrazone crosslinked HA-hydrogel to deliver clinically relevant
protein (recombinant human bone morphogenetic protein-2 or rhBMP-2) to induce bone
formation in vivo.28, 29
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Disulfide cross-linked HA hydrogels have been also developed by several groups but it
requires either a high degree of chemical modifications (≈50%), basic pH or oxidants.30-32 The
use of pyridyl disulfide exchange reaction, though significantly improves reaction rates, it
liberates 2-mercaptopyridine as a side-product, which may not be suitable for in vivo
applications.33, 34 Recently, we have shown the 1,2-aminothiols such as cysteine could form
stable thiazolidine linkages at pH 7.4 when mixed with reactive aldehydes.35 Unlike other
chemistries, disulfide cross-linking reaction has an added advantage for cell-based therapies
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because the hydrogels could be cleaved enzymatically or chemically by cell-secreted
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reductants such as glutathione.5,
We, therefore, envisioned developing disulfide
crosslinked HA hydrogels by tuning the pKa of the thiol group. We anticipated that by
reducing the thiol pKa, we could promote thiolate anion formation at physiological pH, which
could favor oxygen catalyzed disulfide formation. For this purpose, we explored the influence
of electron withdrawing groups at the β-position of thiol (cysteine and N-acetyl-L-cysteine) to
drive the disulfide bond formation and compared the reaction with conventional thiol
derivative without any electron-withdrawing groups having a similar degree of substitution on
HA. The hydrogels were developed by performing reaction at basic and physiological pH and
were characterized by evaluating their rheological properties, hydrolytic stability, and dual
degradability using enzymatic and non-enzymatic conditions.
119 Experimental section
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General
Hyaluronic acid (HA) sodium salt (200 kDa) was purchased from Lifecore Biomedical
(Chaska, MN, USA). All reagents were of the highest purity available from Sigma-Aldrich
(Steinheim, Germany). All the organic solvents were purchased from Fisher Scientific
(Loughborough, UK). Dialysis membranes Spectra/Por® 6 (3500 g/mol cut-off) were from
VWR International (Compton, CA, USA). All NMR-spectra were recorded on a JEOL JNM-
ECP Series FT-NMR spectrometer at magnetic field strength 9.4 T, operating at 400 MHz for
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1H-NMR and 100 MHz for C-NMR. The residual solvent peak was used as the internal
reference when possible. Chemical shifts were expressed in ppm. The pH measurements were
carried out on Mettler Toledo pH meter. The dihydrazide reagent for the synthesis of thiol-
modified HA (HA-SH) derivative (3,3'-dithiodi(propanehydrazide) (6)37 and the consequent
HA-SH derivative30 were synthesized according to previously reported procedures.
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NMR-spectroscopy kinetic study of disulfide formation
L-Cysteine (1), N-acetyl-L-cysteine (2) or 3-mercaptopropionic acid (3) were dissolved in
deuterated phosphate buffer saline (dPBS) at 20 mM concentration. The resulting solutions
pD (pH + 0.4) was adjusted to 7.4 or 9 by adding NaOD or HCl 0.1 M in D2O. The oxygen
concentration was kept constant by bubbling air in the reaction mixture. Disulfide formation
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was followed by periodically recording H-NMR spectra of the reaction mixture during 24
hours. Resonances of the newly formed disulfides were compared with the resonances from
the original thiol compounds and expressed as % of disulfide formation. Pseudo-first-order
reaction constant (k1) was defined as the slope of the line obtained by representing the
ln(disulfide concentration/thiol initial concentration) as a function of reaction time.
Synthesis of the dihydrazide used for the modification of HA with cysteine groups
The reagent used in the synthesis of HA-Cys (3,3'-dithiodi(2-aminopropanehydrazide)) (4)
was synthesized following a three-step synthetic procedure (Scheme 1).
Dimethyl
3,3'-dithiodi(2-(tert-butoxycarbonyl)aminopropanoate)
(8).
N-[(tert-
butoxy)carbonyl]-L-cysteine methyl ester (7) (2.66 g, 11.31 mmol) was dissolved in EtOAc
(32 mL). Solid sodium iodine (14.7 mg, 0.098 mmol) was added followed by hydrogen
peroxide 30% (1.6 mL, 12.44 mmol). The reaction mixture was stirred at room temperature.
The reaction was followed by TLC on silica gel plate revealed by ninhydrin. When the
reaction was completed, a saturated aqueous solution of sodium sulfite (40 mL) was added to
quench the reaction. The product was extracted with EtOAc (3 x 50 mL) and the combined
organic phases were washed with brine and dried over MgSO4. The solvent was evaporated
and the product was dried under vacuum. Yield: 2.57 g (97%). 1H-NMR (CD3OD, 400 MHz)
δ (ppm): 4.46 (dd, J = 8.3, 4.9 Hz, -CH-NHCO-, 2H), 3.75 (s, CH3O-, 6H), 3.19 (dd, J = 14.0,
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4.8 Hz, -CH2-S-, 2H), 2.96 (dd, J = 13.9, 8.8 Hz, -CH2-S-, 2H), 1.45 (s, (CH3-)3,18H). C-
NMR (CD3OD, 100 MHz) δ (ppm): 173.1 (-CO-OCH3), 157.8 (-CO-OC(CH3)3), 80.9 (-C-
(CH3)3), 54.3 (-CH-NHCO-), 52.9 (CH3O-), 41.1 (-CH2-S-), 28.7 ((CH3)3-).
3,3'-dithiodi(2-(tert-butoxycarbonyl)aminopropanehydrazide) (9). Hydrazine hydrate
80% (5.6 mL, 76.73 mol) was added to a stirred solution of Compound 8 (2.57 g, 5.48 mmol)
in ethanol (55 mL). The reaction mixture was stirred for one hour at room temperature. The
obtained white precipitate was filtered, washed with cold ethanol and dried under vacuum.
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Yield: 1.49 g (58%). H-NMR (400 MHz, CD3OD) δ (ppm): 4.42 – 4.35 (m, -CH-NHCO-,
2H), 3.12 (dd, J = 13.8, 5.5 Hz, -CH2-S-, 2H), 2.91 (dd, J = 13.6, 8.6 Hz, -CH2-S-, 2H), 1.45
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(s, (CH3-)3, 18H). 13C-NMR (100 MHz, CD3OD) δ (ppm): 172.2 (-CO-NHNH2), 157.6 (-CO-
OC(CH3)3), 80.9 (-C-(CH3)3), 54.1 (-CH-NHCO-), 42.0 (-CH2-S-), 28.7 ((CH3)3-).
3,3'-dithiodi(2-aminopropanehydrazide) (4). Hydrochloric acid 37% (13 mL) was added to
a stirred clear solution of Compound 9 (767.2 mg, 1.64 mmol) in 1,4-dioxane (25 mL). The
reaction mixture was stirred for one hour at room temperature. The obtained white precipitate
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was filtered, washed with cold ethanol and dried under vacuum. Yield: 458.8 mg (76%). H-
NMR (400 MHz, D2O) δ (ppm): 4.46 (dd, J = 8.1, 5.4 Hz, -CH-NHCO-, 2H), 3.41 (dd, J =
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15.1, 5.4 Hz, -CH2-S-, 2H), 3.21 (dd, J = 15.1, 8.2 Hz, -CH2-S-, 2H). C-NMR (100 MHz,
D2O) δ (ppm): 167.2 (-CO-NHNH2), 51.3 (CH-NH2), 37.4 (-CH2-S-).
Synthesis of the dihydrazide used for the modification of HA with N-acetyl cysteine
groups
The reagent used in the synthesis of HA-ActCys (3,3'-dithiodi(2-acetamidopropanehydrazide)
(5) was synthesized following a three-step synthetic procedure (Scheme 1).
Methyl 3-sulfanyl-2-acetamidopropanoate (11). N-acetyl-L-cysteine (2) (10.67 g, 65.37
mmol) was dissolved in methanol (130 mL). Sulfuric acid was added to the solution (30
drops) and the reaction mixture was refluxed overnight. The solvent was evaporated and the
transparent oil obtained was dissolved in ethyl acetate (150 mL). The organic phase was
washed with water (3 x 20 mL) and dried over MgSO4. The solvent was evaporated and the
product was dried under vacuum. Yield: 7.11g (61%). 1H-NMR (400 MHz, CD3OD) δ (ppm):
4.63 (dd, J = 6.6, 5.0 Hz, -CH-NHCO-, 2H), 3.75 (s, CH3O-, 6H), 2.91 (qd, J = 14.0, 5.8 Hz, -
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CH2-SH, 4H), 2.02 (s, CH3-, 6H). C-NMR (100 MHz, CD3OD) δ (ppm): 173.3 (-CO-CH3),
172.1 (-CO-OCH3), 56.2 (-CH-NHCO-), 52.9 (CH3O-), 26.6 (-CH2-SH), 22.3 (CH3-).
Dimethyl 3,3'-dithidi(2-acetamidopropanoate) (12). Compound 11 (7.1 g, 40.07 mmol)
was dissolved in EtOAc (125 mL). Sodium iodide (66.3 mg, 0.44 mmol) was added followed
by hydrogen peroxide 30% (5 mL, 44.08 mmol). The reaction mixture was stirred at room
temperature. The reaction was followed by TLC on silica gel plate revealed by ninhydrin.
When the reaction was completed, a saturated aqueous solution of sodium sulfite (40 mL) was
added to quench the reaction. The product was extracted with EtOAc (3 x 20 mL) and the
combined organic phases were dried over MgSO4. The solvent was evaporated and the
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obtained white product was dried under vacuum. Yield: 5.16 g (73%). H-NMR (400 MHz,
CD3OD) δ (ppm): 4.74 (dd, J = 8.6, 5.0 Hz, -CH-NHCO-, 2H), 3.75 (s, CH3O-, 6H), 3.23 (dd,
J = 14.5, 5.3 Hz, -CH2-S-, 2H), 2.97 (dd, J = 13.8, 8.6 Hz, -CH2-SH, 2H), 2.00 (s, CH3-, 6H).
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13C-NMR (100 MHz, CD3OD) δ (ppm): 173.4 (-CO-CH3), 172.4 (-CO-OCH3), 53.0 (-CH-
NHCO-), 49.0 (CH3O-), 40.5 (-CH2-S-), 22.3 (CH3-).
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3,3'-dithiodi(2-acetamidopropanehydrazide) (5). Hydrazine hydrate 80% (15 mL, 204.6
mmol) was slowly added to a stirred solution of compound 12 (5.15 g, 14.61 mmol) in
ethanol (120 mL). The white precipitate obtained was filtered, washed with cold ethanol and
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dried under vacuum. Yield: 4.68 g (91%). H-NMR (D2O, 400 MHz) δ (ppm): 4.60 (dd, J =
8.5, 5.6 Hz, -CH-NHCO-, 2H), 3.16 (dd, J = 14.3, 5.6 Hz, -CH2-S-, 2H), 2.93 (dd, J = 14.2,
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8.5 Hz, -CH2-S-, 2H), 2.03 (s, CH3-, 6H). C-NMR (100 MHz, CD3OD) δ (ppm): 174.3 (-
CO-CH3), 170.8 (-CO-NHNH2), 51.6 (-CH-NHCO-), 38.6 (-CH2-S-), 21.8 (CH3-).
HA modification with different thiol groups (HA-Cys, HA-ActCys, HA-SH)
Sodium hyaluronate was dissolved in deionized water at a concentration of 9 mg/mL. The
correspondent hydrazide reagent (4, 5 or 6) was added to the clear solution at the molar ratio
specified in Table 1. The pH of the solution was adjusted to 4.5 by adding NaOH (1 M). The
coupling reaction was initiated by the addition of solid 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) (see Table 1 for EDC/HA disaccharide ration). EDC was added portion-
wise during 1 hour while maintaining the pH at 4.5 by adding HCl (2M). The reaction mixture
was stirred overnight at room temperature. DTT (see Table 1) was then added and the pH was
adjusted to 8.5 by adding NaOH (5M). The reaction mixture was stirred overnight at room
temperature. The solution was diluted to 5 mg/mL, acidified to pH 3 by adding HCl (2M) and
transferred to a dialysis membrane. After exhaustive dialysis against dilute HCl (pH 3)
containing NaCl (0.1M) for 24 h (3 x dialyzing solvent change) and dilute HCl (pH 3) for 24
h (3 x dialyzing solvent change). The solution was lyophilized to give the corresponding thiol-
modified HA derivatives (HA-Cys, HA-ActCys, HA-SH). The incorporation of different thiol
functionality was verified by 1H-NMR. Specifically, the signals corresponding to the methine
(-CHNH2, 4.36 ppm) and methylene (-CH2SH, 2.88 ppm) protons of HA-Cys, the methine (-
CHNHCO-, 4.59 ppm), methylene (-CH2SH, 2.97 ppm) and methyl (CH3CONH-, 2.07 ppm)
protons of HA-ActCys and the methylene (-CH2CH2SH, 2.87 ppm and 2.72 ppm) protons of
HA-SH. The degree of substitution (DS) was determined by the Ellman’s test38 (see Table 1).
pKa Determination
The determination of the pKa values for the thiol groups in HA-Cys, HA-ActCys, and HA-SH
was conducted spectrophotometrically based on previously reported procedures.30 Briefly,
thiol-modified HA derivatives were dissolved in HCl (1 mM) containing NaCl (0.1M) (to
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provide stable ionic strength) at a concentration of 0.5 mg/L. Different amounts of NaOH
(0.01 M) were added to the freshly prepared solutions and the absorbances (242 nm) were
immediately measured on Perkin Elmer Lambda 35 UV/VIS spectrometer. The pH of the
resulting solutions was determined after the absorbance measurements. The pKa value was
obtained by representing the -log[(Amax – Ai)/Ai] as a function of the pH. The pKa values are
displayed where the linear fit crosses the abscissa.
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Hydrogels formation and characterization
HA derivatives were dissolved in acidified (pH 5) and degassed PBS (2% w/v) to avoid
disulfide formation during solubilization. The hydrogel formation was initiated by raising the
pH of the solutions to 7.4 or 9 with NaOH (1 M). The solutions (200 μL) were transferred to a
cylindrical mold, sealed with parafilm and allowed to cross-link overnight. The viscoelastic
properties of the hydrogels were evaluated on a Discovery Hybrid Rheometer 2 (DHR2) from
TA Instruments. The cylindrical hydrogels were carefully transferred from the molds to the
rheometer plate and the gap was adjusted to reach a normal force of 30 mN. The rheological
properties were determined with frequency 0.1-20 Hz and 1% strain at 25°C using 8 mm
parallel plate geometry. The gelation time was determined using the same rheometer. All the
gelation kinetics measurements were performed inside a humidity chamber to avoid hydrogel
desiccation. The solutions of the HA derivatives (350 μL) were immediately transferred to the
rheometer after pH adjustment and the gap was lowered to a size of 100 μm. The storage (G')
and loss modulus (G") were recorded over time using 20 mm parallel plate geometry (strain:
1%, frequency: 0.5 Hz). The time at which G’ cross G” was defined as the gelation time.
Hydrogel hydrolytic stability and degradation
Newly formed hydrogels were removed from the mold, weighed (m0) and immersed in 5 mL
Eppendorf tube containing 2 mL of degradation media. At specific time intervals, the
remaining hydrogels were weighed (mt) after removing the excess solution. After measuring
their mass, the hydrogels were immersed in fresh degradation media. All experiments were
performed in triplicates. The hydrolytic stability was studied in PBS (pH 7.4) as the
degradation media. The swelling ratio was calculated from the equation [(mt/m0)x100]-100.
The enzymatic and reductive degradation was studied using PBS containing hyaluronidase
from bovine testes (Type IV-S, 0.3 mg/mL, 210 U/mL) or DTT (1 mM) as the degradation
media, respectively. The degradation ratio was express as (mt/m0)x100.
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Kinetics of disulfide formation by 1H NMR spectroscopy
In order to determine the effect of an electron-withdrawing substituent at the β-position of the
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thiol residue on disulfide formation, we first determined the reaction kinetics by H-NMR
analysis using a small molecule model (Figure 1). We performed the reaction by dissolving
L-cysteine (1), N-acetyl-L-cysteine (2) and 3-mercaptopropionic acid (3) in deuterated
phosphate buffer at 20 mM concentrations. The reaction was performed at physiological (pD
7.4) and basic (pD 9) conditions. Since molecular oxygen concentration is a key factor for
such reactions, we constantly bubbled air in the reaction mixture.
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Figure 1. a) Chemical structure of L-cysteine (1), N-acetyl-L-cysteine (2) and 3-mercaptopropionic acid (3). b)
Disulfide formation of the above molecules followed by 1H-NMR at pD 9 and pD 7.4.
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Monitoring the reaction by H-NMR showed complete conversion of L-cysteine to disulfide
at pD 9 within 24 hours, as the cysteine resonances were completely disappeared (Figure S1,
in the Supporting Information). At pD 7.4, disulfide formation considerably slowed down
and only 49.8% conversion was observed after 24 hours. The rates of reaction were calculated
using pseudo-first-order conditions, which indicated that the rate of reaction at pD 9 (k1 =
15.1 x 10-4 min-1) was 3-fold higher than at pD 7.4 (k1 = 5.04 x 10-4 min-1; Figure S2 and
Figure S3, in the Supporting Information). The observed increase in reaction rate at higher
pH corroborates with the hypothesis that the rate of disulfide formation is directed by the
thiolate anion concentration. Since the electron-withdrawing group at the β-position
(protonated amine) of L-cysteine favors the deprotonation of the thiol group, we anticipated
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an increase in reaction rate as compared to the unmodified thiol substrate (3-
mercaptopropionic acid). 1H-NMR analysis of the disulfide formation with 3-
mercaptopropionic acid (Figure S4, in the Supporting information) revealed that the
reaction rate of such a substrate indeed was ≈2-fold (k1 = 7.68 x 10-4 min-1) lower at pH 9 and
≈2.8-fold (k1 = 1.80 x 10-4 min-1) lower at pD 7.4, as compared to the L-cysteine substrate
(Figure S5 and Figure S6, in the Supporting Information). Since the N-acetylation of
cysteine eliminates the protonation of nitrogen atom we anticipated a reduction in the reaction
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rate. H-NMR based study of the reaction kinetics with N-acetyl-L-cysteine group indeed
demonstrated reduction of the reaction rate by ≈5–10 folds at pH 9 (k1 = 3.30 x 10-4 min-1)
and pH 7.4 (0.51 x 10-4 min-1) respectively, as compared to the L-cysteine substrate (Figure
S7-S9, in the Supporting Information). Such a significant drop in reaction rate could be
attributed to the drop in nitrogen pKa and also due to the steric hindrance of the N-acetyl
group that slows down the reaction. Taken together, the observed reduction of the reaction
rates corroborates with our hypothesis that electron-withdrawing substituent favors the
oxidation of thiols to disulfides by increasing the acidity of the thiol group, though steric
factors could also affect the reaction rate. These experiments clearly demonstrated the pH
dependence on the reaction and the influence of the thiol electronic environment on the
reaction kinetics.
HA modification with different thiol groups (HA-Cys, HA-ActCys, HA-SH)
Encouraged by the results obtained with small molecule analogs, we prepared HA derivatives
modified with cysteine (HA-Cys), N-acetyl-cysteine (HA-ActCys) and mercaptopropionic
acid moieties (HA-SH). For this purpose, we first synthesized the dihydrazide derivatives of
the corresponding carboxylic acids molecules with a dimeric structure (Scheme 1). Such a
modification enabled incorporation of the pendant functional groups using carbodiimide-
mediated hydrazide coupling chemistry, as optimized previously in our group.29
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Scheme 1. Schematic representation of the synthesis of disulfide-containing dihydrazide compounds. a) 3,3'-
dithiodi(2-aminopropanehydrazide) (4) used in the preparation of HA-Cys. b) 3,3'-dithiodi(2-
acetamidopropanehydrazide) (5) used in the preparation of HA-ActCys. c) 3,3'-dithiodi(propanehydrazide) (6)
used in the preparation of HA-SH.
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Reaction conditions: i) cat. NaI, H2O2, EtOAc; ii) NH2NH2·H2O, EtOH; iii) HCl, 1,4-dioxane; iv) cat. H2SO4,
MeOH.
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It is important to note that the amine functionality of the dihydrazide 4 was not protected
during the EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) mediated coupling
reaction as such a reaction was performed at pH 4.5 that prevents the participation of free
amines that remain primarily as protonated species. The progress of the reaction was evident
by a visible increase in the viscosity of the reaction mixture, indicating that the dihydrazide
molecules acted as cross-linkers between HA macromolecules (Figure 2a). However, the
subsequent reduction of the central disulfide bonds with 1,4-dithiothreitol (DTT) liberated the
desired free thiol groups yielding the soluble form of thiolated HA derivatives. The excess of
reagent and DTT was removed by dialyzing against acidified water (pH 3) that prevented the
formation of new disulfide bridges during the purification process. The dialyzed product was
lyophilized to obtain the thiolated HA derivatives as a white fluffy material.
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Figure 2. a) Synthetic scheme and chemical structure of thiolated hyaluronic acid (HA) derivatives (HA-Cys,
HA-ActCys, and HA-SH). b) 1H-NMR spectra of thiolated HA derivatives.
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The chemical characterization of the obtained HA derivatives was carried out using 1H-NMR
analysis (Figure 2b and Figure S10-S13, in the Supporting Information). Specifically,
new resonances appeared at 4.36 ppm (-CHNH2) and 2.88 ppm (-CH2SH) for HA-Cys
derivative, whereas a downfield shift at 4.59 ppm (-CHNHCO-), 2.97 ppm (-CH2SH) and 2.07
ppm (CH3CONH-) was observed for the acetylated product (HA-ActCys). For the
mercaptopropionic acid derivative we observed a chemical shift at 2.87 ppm and 2.72 ppm for
the -CH2CH2SH signals. The degree of substitution (DS) could not be determined by
integrating the proton signals due to overlapped with the HA signals. The DS was therefore
determined by the modified Ellman’s method, which is a colorimetric assay for the
determination of free thiol groups.38 The colorimetric analysis indicated that the DS of
different thiolated HA derivatives were 11% for HA-Cys, 11% for HA-ActCys and 9% for
HA-SH. Additionally, HA-SH derivative with higher DS (19%) was also prepared. The DS
was controlled by regulating the molar ratios between HA and the coupling reagent EDC
(Table 1).
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Table 1. The molar ratio of the reagents used for the synthesis of thiol-modified HA derivatives and the obtained
degree of substitution (DS). The values were expressed based on HA disaccharide repeating units.
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HA sample
HA-Cys
Molar ratio
DS
EDC dihydrazide DTT
0.3
0.9a
0.525b
0.45c
0.9c
4.5
11%
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HA-ActCys 0.175
2.625 11%
HA-SH
0.15
0.3
2.25
4.5
9%
HA-SH
19%
a
b
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Compound 4 was the hydrazide reagent used for the synthesis of HA-Cys. Compound 5 was the hydrazide
reagent used for the synthesis of HA-ActCys. c Compound 6 was the hydrazide reagent used in the synthesis of
HA-SH.
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Determination of the thiol pKa in the HA derivatives
Since the concentration of thiolate anion (R-S-) has been found to be the key intermediate for
disulfide formation, we determined the pKa of different thiolated derivatives using UV
spectrophotometric method. This was performed by dissolving the HA derivatives in an
acidified (1 mM of HCl) aqueous media with a stable ionic strength (NaCl 0.1M). The
absorbance at 242 nm was recorded after pH adjustment, following a previously reported
procedure.30 The absorbance increased with the pH indicating the formation of thiolate ions.
Figure 3a shows the sigmoidal curves of the absorbance versus pH. The absorbance of the
HA-Cys solution showed an abrupt increase within a very short range of pH, while the
absorbance of HA-ActCys and HA-SH solution increased in a larger range of pH. This
difference may be explained by the chemical microenvironment near the thiol group that
strongly influences the formation of thiolate ions. The graphical representation of -log[(Amax
–
Ai)/Ai] versus pH (Figure 3b) displays the pKa values of the modified HA where the linear fit
crosses the abscissa. Interestingly, the pKa of the HA-Cys was found to be 7.0, which is
significantly lower than what is known in cysteine-containing peptides (≈8.3). Such a
decrease in pKa is presumably due to salt formation between the amino residue of cysteine
and carboxylate backbone of HA. We recently demonstrated that electrostatic interactions
between HA and a biologically active protein (recombinant human bone morphogenetic
protein-2) result in a retained molecular interaction between the two biopolymers even after
shifting to physiological pH.28 The N-acetyl derivative (HA-ActCys) on the other hand,
indicated a pKa value of 7.4, which is anticipated as the N-acetyl protection of primary amine
will limit the amino protonation and as a result will reduce the electron-withdrawing effect as
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compared to the parent molecule. The pKa value of the HA-SH was found to be 8.1, which is
closer to the reported pKa of HA-thiol.30 Of note, the reported pKa of mercaptopropionic acid
is ≈10.839 while the HA derivative has a significantly lower pKa indicating the role of the
molecular structure of HA in increasing the acidity of the thiol group.
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Figure 3. a) Absorbance of the thiol-modified HA derivatives (HA-Cys, HA-ActCys, and HA-SH) as a function
of pH. b) Logarithmic representation of the normalized absorbance (-log[(Amax-Ai)/Ai]) as a function of the pH.
The pKa values correspond to the interception with the abscissa.
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Hydrogel preparation and characterization
The observed decrease in thiol pKa encouraged us to test disulfide crosslinked HA-hydrogel
formation at physiological pH and at basic pH without any addition of oxidants. In earlier
works, the degree of thiol modification is kept very high (for oxidant-free hydrogel
preparation),30, 31 due to the low reactivity of thiol groups but we aimed to keep the
modification degree to ≈10%. The higher degree of chemical modification is expected to
influence the HA interaction with cells as it is known that a minimum of ten saccharide units
is needed for HA-CD44 interaction.40 To prepare HA-hydrogels with 2% solid content, the
three thiol-modified HA derivatives (HA-Cys, HA-ActCys, and HA-SH) were first dissolved
in an acidified (pH 5) phosphate buffer and then the pH was carefully adjusted to 7.4 or 9.0.
The gelation kinetics was determined using rheological measurements and the gel point was
determined by measuring the crossover point between the storage modulus (G’) and the loss
modulus (G’’) (Table 2 and Figure S14, in the Supporting information).
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Table 1. The gelation time of all thiol-modified HA derivative (HA-Cys, HA-ActCys, and HA-SH). The gelation
was performed at pH 7.4 and pH 9.0.
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DS
Sample
HA-Cys
HA-ActCys
HA-SH
pH 7.4
pH 9.0
3,5 min
6,5 h
-
11%
11%
9%
3,5 min
10 h
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-
19%
HA-SH
5 h
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We first observed the hydrogel formation with HA-SH derivative having 9% or 19% of thiol
groups at physiological pH (7.4) and surprisingly we did not observe any hydrogel formation
within the 24 h experiment. The absence of gelation at pH 7.4 was presumably due to the low
concentration of thiolate anions (16.6% according to the Henderson-Hasselbalch equation) in
the reaction mixture (pKa = 8.1). For this reason, most of the reported procedures employ a
very high degree of modification (≈50%) or a supplement to the reaction with additional
oxidants to obtain hydrogels within a reasonable time.30, 31 By increasing the pH of the
reaction to 9.0, the concentration of thiolate anions increased (88.8%) which resulted in
hydrogel formation with HA-SH derivative having 19% modification, however, no gelation
was observed with 9% modified HA-SH derivative. We anticipated that HA-Cys with a pKa
of 7.0 should form hydrogels at physiological pH because of the higher abundance of the
reactive thiolate anions. Indeed, HA-Cys formed hydrogels within ≈3.5 minutes at pH 7.4.
Surprisingly, the gelation time, in this case, was not significantly affected by increasing the
pH to 9.0, presumably due to the pKa of HA-Cys is lower than the experimental conditions
(pH 7.4 and pH 9.0). This will allow more than 70% free thiolate anions (71.5% of R-S- at pH
7.4 and 99% of R-S- at pH 9) at both the pH’s, resulting in high reaction rate. The most
interesting case of pH dependence on disulfide formation was seen for HA-ActCys having a
pKa of 7.4 (50% of R-S- at pH 7.4 and 97.6% of R-S- at pH 9). The sol to gel transition took
over 10 h for HA-ActCys at pH 7.4 but the gel time could be reduced to ≈6.5 h when the pH
was raised to 9.0. In spite of the close pKa values between HA-Cys and HA-ActCys, the
observed large differences in their gelation time could be attributed to steric factors with the
acetyl group being in close proximity to reactive thiol moiety. Nevertheless, the short gel time
of HA-Cys at both pHs demonstrates the significance of introducing electron-withdrawing
groups near the thiol residue that decrease the pKa of the thiol groups and promote disulfide
formation at physiological pH.
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Table 3. Mechanical properties of disulfide cross-linked HA hydrogels.
pH 7.4
G’ (Pa)
pH 9.0
G’ (Pa)
5
6
7
DS
Sample
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9
11%
11%
19%
HA-Cys
3312.3
3523.5
-
2261.2
1734.4
1910.6
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HA-SH
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We further evaluated the rheological properties of the HA-Cys and HA-ActCys hydrogels and
compared it with HA-SH hydrogels (Table 3). The average storage modulus (G’) of HA-Cys
and HA-ActCys hydrogels prepared at pH 7.4 gave comparable results (≈3300 Pa)
demonstrating that in spite of the different reaction rates, the hydrogels reached a similar
degree of cross-linking upon completion. The G’ was considerably reduced for hydrogels
prepared at pH 9.0, suggesting that the high concentration of thiolate ions favored the
formation of intramolecular loops reducing the effective intermolecular cross-links.
Additionally, HA-SH hydrogels containing 19% modification showed nearly similar G’ at pH
9.0 as obtained from HA-Cys and HA-ActCys with approximately half a degree of
functionalization (11%), demonstrating that the introduction of electron-withdrawing groups
not only improved the reaction kinetics but also improved the efficiency of the cross-linking
reaction. Such an observation could be due to the dynamic disulfide exchange reaction as a
result of a higher degree of thiol substitution.
Hydrogel hydrolytic stability and degradation
Finally, we evaluated the hydrolytic stability of the hydrogels at physiological pH (7.4) by
quantifying the change in hydrogel mass after incubation in phosphate buffer saline (PBS).
The most interesting aspect of our swelling studies was that both HA-Cys and HA-ActCys
hydrogels showed excellent stability and exhibited only limited swelling (Figure 4a). In fact,
HA-Cys hydrogels showed moderate shrinking during the 8 days of study. This atypical
shrinking behavior is attributed to the presence of free thiolate ions that spontaneous oxidation
reaction to form new cross-linkages. The HA-ActCys hydrogels demonstrated a moderate
swelling (≈15%) within the first 24 h followed by moderate shrinking of the gels indicating
the formation of new cross-links after incubation in PBS. The initial increase of mass could be
explained by the stretching of the polymeric network that allowed an increasing influx of
water (hydrogel swelling).29 It should be noted that even though there was shrinking of the
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gels, the hydrogels were intact even after 30 days incubation in the swelling medium. We did
not perform swelling studies with the HA-SH hydrogels as such gels could not be formed at
pH 7.4 but required pH over 9.0 to form hydrogels.
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Figure 4. a) Gravimetric swelling study of HA disulfide hydrogels at physiological conditions (pH 7.4). b)
Enzymatic degradation of HA hydrogels studied using hyaluronidase (0.3 mg/mL) solution. c) Reductive
degradation of HA hydrogels studied using DTT (1 mM) solution.
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Since hydrogel swelling characteristics and degradation are closely related we evaluated the
enzymatic and non-enzymatic degradation properties of the HA hydrogels. For enzymatic
degradation, we incubated different hydrogels with hyaluronidase (0.3 mg/mL), a ubiquitous
enzyme known to cleave glycosidic linkages of the HA backbone, and measured the weight
loss with time (Figure 4b). These experiments indicated that both the hydrogels showed a
similar degradation profile, which is expected for hydrogels with similar cross-linking
density. We further performed degradation experiments with DTT, a well-known reductant
known to cleave disulfide bonds. Interestingly, these experiments revealed that HA-Cys and
HA-ActCys hydrogels showed different reductive degradation profiles (Figure 5c). The
sterically hindered acetyl group of HA-ActCys hydrogel retarded the DTT-mediated disulfide
cleavage reaction slowing down the hydrogel degradation. The HA-Cys hydrogel, on the
other hand, responded to the added reductant resulting in hydrogel degradation at a higher
rate. Since cell-produced reductant such as glutathione is known to degrade disulfide bonds,
we believe the two types of hydrogels will behave differently in presence of cells. However,
the quick gelation time of HA-Cys hydrogels in less than 5-minutes will favor this gel in vivo
applications over the HA-ActCys hydrogel or HA-SH hydrogel.
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504 Conclusions
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Oxidation of thiols requires thiolate anion and molecular oxygen to form disulfide bonds
exploiting a radical chemistry. We demonstrate here that incorporation of an electron-
withdrawing group at the β-position as in cysteine results in significant increase in reaction
rate for disulfide formation at physiological pH. We exploited this observation to develop the
first disulfide-linked HA-based hydrogel system that forms crosslink at physiological pH in
just 3.5 minutes. To tailor such highly reactive thiol containing biopolymer, we designed the
HA-thiol derivative having cysteine units with free thiols. Incorporation of the cysteine
moiety onto HA reduced the pKa of thiol group from ≈8.3 (as in peptides) to 7.0, which
promoted thiolate anion formation at physiological pH. Acetylation of the amino residue of
cysteine reduced the protonation ability of the amino group that resulted in an increase in thiol
pKa to 7.4. To determine the effect of electron-withdrawing group at the β-position, we
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utilized the HA-thiol derivative that does not contain any substituent at the β-position using
hydrazide modified 3-mercaptopropionic acid unit. Such a thiol moiety displayed a pKa of 8.1.
Such differences in pKa of different thiols were reflected in disulfide-crosslinked hydrogel
formation at pH 7.4 without any addition of external oxidants. The HA-Cys derivative formed
hydrogel is just 3.5 minutes, however, the HA-ActCys derivative required over 10 h to form
crosslinks. The HA-SH derivative, on the other hand, did not form the hydrogel network at
pH 7.4 even after increasing the degree of chemical modification to 19% with respect to the
disaccharide units. The swelling studies of the HA-Cys and HA-ActCys hydrogels indicated
shrinking of the hydrogel without any visible change in gel structure. The hyaluronidase
mediated enzymatic degradation of the HA-Cys and HA-ActCys hydrogels were identical
however hydrogel degradation by the cleavage of disulfide bonds with DTT indicated a clear
difference between the two materials. The presence of the acetyl group near the disulfide
linkage hindered the cleavage of the disulfide resulting in slower degradation of HA-ActCys
hydrogels. We believe that the understanding of the factors that regulate disulfide formation
will provide new insight to design materials with stable thiol groups or fast disulfide
formation for different applications. Moreover, the thiol-modified HA developed in this study
undergo fast gelation under physiological pH without any need for catalysts. Such materials
will allow developments of clinically translatable hydrogels for cellular delivery or for
delivering therapeutic drugs.
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537 Associated content
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Supporting information
1H-NMR spectra showing the disulfide conversion of small molecule substrates,
representative plots of time-dependent disulfide conversion, representative linear least-squares
1
fits for disulfide formation, H-NMR spectra of thiolated HA derivatives, and rheological
evaluation of the gelation kinetics of the different thiolated HA derivatives.
545 Acknowledgment
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The present work was supported by Swedish Strategic Research grant “StemTherapy”
(139400126), Swedish foundation for strategic research (SSF) grant (139400127) and EU
Framework Program-7 project FP7/2007-2013/607868 (Marie Curie Actions- iTERM).
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