Optimized triazine-mediated amidation for efficient and controlled functionalization of hyaluronic acid

Article abstract of DOI:10.1016/j.carbpol.2014.04.012

Triazine-based coupling agents have the potential to replace carbodiimides in the functionalization of hyaluronic acid (HA) giving derivatives with high degrees of substitution (DS) under mild conditions with excellent efficiency. Kinetics of the triazine

Full text of DOI:10.1016/j.carbpol.2014.04.012

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Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Optimized triazine-mediated amidation for efficient and controlled
functionalization of hyaluronic acid
Tina Borkea, Franc¸ oise M. Winnika,b,c, Heikki Tenhua, Sami Hietalaa,∗
a Laboratory of Polymer Chemistry, Department of Chemistry, P.O. Box 55, 00014 University of Helsinki, Finland
b Faculty of Pharmacy and Department of Chemistry, Universite de Montreal, CP 6128 Succursale Centre Ville, Montreal, QC H3C 3J7, Canada
c World Premier International (WPI) Research Center Initiative, International Center for Materials Nanoarchitectonics (MANA), National Institute for
Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxx
Triazine-based coupling agents have the potential to replace carbodiimides in the functionalization of
hyaluronic acid (HA) giving derivatives with high degrees of substitution (DS) under mild conditions
with excellent efficiency. Kinetics of the triazine-mediated amidation of HA in aqueous solution were
investigated to understand the reaction mechanism and the role of the amine reagent. The DS decreased
with increasing basicity of the amine. The water soluble coupling agent was stable under the reaction
conditions (t1/2 = 10 days) in the absence of amines. The activation of HA proceeded quantitatively. The
stoichiometry of amine was the limiting factor in the substitution. Functional HA derivatives with DS
up to 55% were obtained by the triazine-mediated amidation. They were used successfully to prepare
well-defined HA conjugates via the maleimide-thiol and the azide-alkyne “click” reactions.
© 2014 Elsevier Ltd. All rights reserved.
Keywords:
Hyaluronic acid
Triazine
Carbodiimide
Maleimide-thiol
Azide-alkyne
Click reaction
1. Introduction
it is best to select reactions that proceed under mild conditions, and
ideally in neutral aqueous solution at room temperature.
Hyaluronic acid (HA), a naturally occurring glycosaminoglycan
found ubiquitously in the extracellular matrix, is an attractive scaf-
fold for various medical applications. It is readily modified to form
hydrogels of unique viscoelastic and hygroscopic properties useful
for applications in tissue engineering or drug delivery (Coviello,
Matricardi, Marianecci, & Alhaique, 2007; Garg & Hales, 2004;
Huerta-Angeles et al., 2012; Owen, Fisher, Tam, Nimmo, & Shoichet,
2013; Xu, Jha, Harrington, Farach-Carson, & Jia, 2012). The chemical
modification of HA poses several obstacles related to its solubility
and reactivity characteristics. Sodium hyaluronate is soluble readily
in water, but it scarcely dissolves in most solvents commonly used
in synthesis. Also, HA is prone to degradation and depolymeriza-
tion under harsh reaction conditions, such as strongly alkaline,
acidic or oxidative solutions, excessive heat, shear, and ultra-
sound or microwave irradiation (Drˇímalová, Velebny´, Sasinková,
Hromádková, & Ebringerová, 2005; Maleki, Kjøniksen, & Nyström,
2008). To preserve the chemical integrity and the molar mass of HA,
When designing a particular HA derivative the targeted degree
of substitution (DS) is dictated by the envisaged application. For
applications in the area of cell growth within a hydrogel scaf-
fold (Seidlits et al., 2010) or targeted drug/gene delivery vehicles
(Takei et al., 2004), low degrees of substitution are needed to main-
tain the biological functionality of HA. Oh et al. observed that HA
derivatives with DS less than 25% could be efficiently taken up by
cells via receptor-mediated endocytosis (Oh et al., 2010). In con-
trast, if a derivative with long residence time in the body, such as
a hydrogel for tissue augmentation (Oh et al., 2008; Yeom et al.,
2010), is designed the carboxylic acid groups are preferentially
masked by a high degree of substitution to inhibit the recogni-
tion by hyaluronidase and thus slow down the degradation rate
(C. Schanté, Zuber, Herlin, & Vandamme, 2011; Yeom et al., 2010).
Chemical modification of HA can be accomplished by ami-
dation or esterification of the carboxylic acid substituent of
the d-glucuronic acid moiety of the HA disaccharide repeat-
ing unit (Bulpitt & Aeschlimann, 1999; Crescenzi, Cornelio, Di
Meo, Nardecchia, & Lamanna, 2007; Di Meo et al., 2006; Dong
et al., 2012) as well as by substitution of the primary alco-
hol of the N-acetyl-glucosamine (see a review by C.E. Schanté,
Zuber, Herlin, & Vandamme, 2011). Several methods have been
devised to facilitate the amidation of the HA carboxylate groups
(see Table 1). They usually involve “activation” of the carboxylate
∗
Corresponding author. Tel.: +358 9 191 50333; fax: +358 9 1915 0330.
E-mail addresses: tina.borke@helsinki.fi (T. Borke),
francoise.winnik@umontreal.ca (F.M. Winnik), heikki.tenhu@helsinki.fi (H. Tenhu),
sami.hietala@helsinki.fi (S. Hietala).
http://dx.doi.org/10.1016/j.carbpol.2014.04.012
0144-8617/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Borke, T., et al. Optimized triazine-mediated amidation for efficient and controlled functionalization
of hyaluronic acid. Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.04.012

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groups with reagents, such as N-(3-dimethylaminopropyl)-Nꢀ-
ethylcarbodiimide (EDC) or 2-chloro-4,6-dimethoxy-1,3,5-triazine
(CDMT) (C.E. Schanté et al., 2011). EDC reacts with HA carboxylic
acid groups to form an O-acylisourea intermediate, which can then
be attacked by the nucleophilic amine. Unfortunately this interme-
diate is prone to rapid rearrangement to an unreactive N-acylurea
group linked covalently to HA (Kuo, Swann, & Prestwich, 1991;
Nakajima & Ikada, 1995). This side reaction can be reduced by
activation of the carboxylates with N-hydroxysuccinimide (NHS)
or 1-hydroxybenzotriazole (HOBt) to form active esters with HA,
which undergo further aminolysis (Bulpitt & Aeschlimann, 1999).
Nevertheless the efficiency of carboxylic acid activation by car-
bodiimides is low and the DS are poorly controlled. The coupling
reagents, additives and amines have to be applied in large molar
excess to achieve DS ranging typically from 5 to 30 per 100 disac-
charide units causing high cost and waste production (see Table 1).
The triazine-mediated amidation of polysaccharides has been
reported more recently. It was shown to proceed with high effi-
ciency and without side reactions (Bergman, Elvingson, Hilborn,
Svensk, & Bowden, 2007; Farkasˇ & Bystricky´, 2007; C. Schanté et al.,
2011). Here, HA is activated by formation of an acyloxytriazine
(Kamin´ ska, Kamin´ ski, & Góra, 1999; Kunishima, Kawachi, Hioki,
Terao, & Tani, 2001), which is subsequently attacked by the amine to
form a tetrahedral intermediate (Kamin´ ski, 1994). The latter prefer-
entially forms the amide derivative. By this method, it is possible to
obtain HA amide derivatives with a high DS, up to 75%, with reduced
amounts of coupling reagents (see Table 1). These results represent
a great improvement compared to the EDC-mediated substitution,
in view of the reagent amounts needed, the mild conditions, and the
minimal degradation of the polysaccharide chain (Bergman et al.,
2007).
In addition, triazine coupling agents are milder than carbodi-
imide compounds, less allergenic, and safer (Kamin´ ski, 1985; Rayle
& Fellmeth, 1999). They are easily synthesized from inexpen-
sive commodity chemicals, such as cyanuric acid (Cronin, Ginah,
Murray, & Copp, 1996). These excellent features motivated us to
investigate in detail the use of the triazine-mediated amidation to
functionalize HA. The results of this study are presented here. We
start with a series of experiments aimed at understanding the ami-
dation mechanism, in particular the effect of the pKa of the amine.
Although ratios of coupling agent and amine to HA-carboxylate
groups are commonly varied to obtain different DS (see Table 1),
little is known on the specific influence of the amine concentration
or the carboxylate concentration on the outcome of the reaction.
To further the understanding of the triazine mediated amidation of
HA, we investigated the kinetics of the steps involved in the reac-
tion and monitored the stability of the coupling agent under various
reaction conditions. The amidation procedure was validated with
five functional amines selected with a view on further modifica-
tions of HA. Each amine carries a functional group, stable under
the conditions imposed by the amidation, but reactive upon imple-
mentation of common “click” coupling reactions. We illustrate the
use of HA derivatives obtained via triazine-mediated amidation
as starting materials for azide-alkyne and maleimide-thiol “click”
reactions.
2. Experimental
2.1. Materials
High molar mass sodium hyaluronate (HA, 752 kg/mol accord-
ing to manufacturer, research grade) was obtained from Lifecore
Biomedical (U.S.) and used as received. Low molar mass HA
(∼1 kg/mol) was obtained by enzymatic hydrolysis of the former
according to a published procedure (Yang, Kataoka, & Winnik,
Please cite this article in press as: Borke, T., et al. Optimized triazine-mediated amidation for efficient and controlled functionalization
of hyaluronic acid. Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.04.012

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3
2005). The reagents: allylamine (98%), N-(2-aminoethyl)maleimide
trifluoroacetate (≥95%), 2-aminoethyl-methacrylate hydrochlo-
ride (90%, containing ∼500 ppm phenothiazine as stabilizer),
2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 97%), cysteamine
hydrochloride (≥97%), ethylenediaminetetraacetic acid (EDTA,
≥99.4%), 1-mercapto-11-hydroxy-3,6,9-trioxaundecane (97%), N-
methylmorpholine (NMM, 99%), propargylamine hydrochloride
(95%) and (+)-sodium l-ascorbate (≥98%) as well as Dowex®
50WX8 ion exchange resin (hydrogen form, 100-200 mesh) were
purchased from Sigma–Aldrich (Finland) and used as received.
1-Azido-11-hydroxy-3,6,9-trioxaundecane and 4-(4,6-dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM)
were synthesized according to published procedures (Bertozzi &
Bednarski, 1991; Kunishima, Kawachi, Monta, et al., 1999). Cop-
per(II) sulfate pentahydrate (puriss.) was obtained from Merck
(Germany) and used as received. Acetonitrile (HPLC gradient grade,
VWR, Finland) and distilled water were used as solvents. Dialy-
sis was conducted in CelluSep T2 regenerated cellulose tubular
membranes (Membrane Filtration Products, U.S.) with a molecu-
lar weight cut off (MWCO) of 6000–8000 g/mol. Samples for 1H
NMR spectroscopy were prepared in deuterium oxide (D2O, 99.96%
D) and dimethyl sulfoxide-d6 (DMSO-d6, 99.80% D) obtained from
Euroiso-Top (France).
HA-alkynyl: ı(D2O/DMSO-d6 (1:1, v/v)) = 2.12 (–NH–C O–CH3,
3.0 H, s), 3.06 (–C CH, 0.5 H, m), 3.35-4.10 (sugar rings, 12.9 H, br),
4.12 (–CH2–C CH, 0.9 H, d, J = 17.7 Hz), 4.25 (–CH2–C CH, 0.8 H, d,
J = 16.6 Hz), 4.63 (anomeric protons, 1.9 H, br) ppm. DS = 55%.
HA-maleimide:
ı(D2O/DMSO-d6
(1:1,
v/v)) = 2.11
(–NH–C O–CH3, 3.0 H, s), 3.10–4.20 (sugar rings + others, 11.7 H,
br), 4.67 (anomeric protons, 1.3 H, br), 7.08 (–C O–CH CH–C O–,
0.6 H, s) ppm. DS was calculated to be 1.4 times higher than indi-
cated by the maleimide ring protons at 6.88 ppm, as the spectrum
of the N-(2-aminoethyl)maleimide trifluoroacetate precursor
in D2O exhibited only 1.5 protons at this position instead of 2.
DS = 40%.
HA-methacrylate: ı(D2O/DMSO-d6 (1:1, v/v)) = 1.40 (0.3 H, s),
2.09 (–NH–C O–CH3, 3.0 H, s), 3.18 (0.4 H, s), 3.20–4.10 (sugar
rings + other, 10.0 H, br), 4.33 (–NH–CH2–CH2–O–, 1.05 H, br), 4.62
(anomeric protons, 1.0 H, br), 5.91 (–C(CH3) CH2, 0.4 H, s), 6.31
(–C(CH3) CH2, 0.4 H, s) ppm. DS = 41%.
HA-allyl: ı(D2O/DMSO-d6 (1:1, v/v)) = 2.13 (–NH–C O–CH3, 3.0
H, s), 3.20–4.20 (sugar rings, 12.1 H, br), 4.65 (anomeric protons, 1.3
H, br), 5.37 (–CH CH2, 0.6 H, br), 6.03 (–CH CH2, 0.4 H, br) ppm.
DS = 28%.
HA-thiol: ı(D2O/DMSO-d6 (1:1, v/v)) = 2.11(–NH–C O–CH3, 3.0
H, s), 3.04 (–CH2–SH, 0.1 H, s), 3.20–4.10 (sugar rings, 13.9 H, br),
4.66 (anomeric protons, 1.8 H, br) ppm. DS = 4%.
2.2. Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR)
2.3.2. Hydrolysis of DMT-MM
1H NMR spectra (500.13 MHz) of the samples (5 mg/mL in
D2O/DMSO-d6 (1:1, v/v) unless otherwise stated) were recorded on
a Bruker Avance III 500 spectrometer at 298 K. The chemical shifts
are given in parts per million (ppm) and are calibrated relative
to the residual solvent protons (HDO: 4.80 ppm). The multiplici-
ties of the signals are indicated by: s singlet, d doublet, t triplet,
p pentet, m multiplet, br broad signal and coupling constants (J)
are expressed in Hz. Degrees of substitution (DS) were calculated
from comparing the integrals of the methyl protons (2.2 ppm) of
the N-acetylglucosamine moiety of HA and distinctive peaks of the
introduced side groups and are given in % per 100 disaccharide
units.
The hydrolysis of DMT-MM was followed by 1H NMR of a solu-
tion of DMT-MM (∼5 mg) in 700 ␮L D2O at specific time intervals.
2.4. Active ester formation between HA and DMT-MM
The reaction of HA and DMT-MM to form the active 2-acyloxy-
4,6-di methoxy-1,3,5-triazine compound was studied by 1H NMR.
To a solution of HA (1.0 eq., 8.7 ␮mol) in 500 ␮L D2O was added a
solution of DMT-MM (1.1 eq., 9.6 ␮mol) in 200 ␮L D2O. The mixture
was transferred to a NMR tube and spectra were recorded at specific
time intervals.
2.4.1. Kinetics of HA amidation in water
2.3. Reactions
The coupling reaction of HA and propargyl amine in water was
investigated by 1H NMR. Low molar mass HA (ca. 1 kg/mol) was
used to facilitate the study. HA (1.0 eq., 8.7 ␮mol) was dissolved in
500 ␮L D2O. To this solution were added stock solutions of propar-
gyl amine (1.1 eq., 9.6 ␮mol in 100 ␮L D2O) and DMT-MM (1.1 eq.,
9.6 ␮mol in 100 ␮L D2O). The mixture was transferred into a NMR
tube and spectra were recorded at specific time intervals.
2.3.1. Amidation of HA in water/acetonitrile
Following a published procedure (Bergman et al., 2007; C.
Schanté et al., 2011), HA (752 kg/mol) was coupled with various
“clickable” amines. In a typical reaction, HA (1.0 eq., 200.0 mg,
0.50 mmol –COO−) was dissolved in 40 mL water under gentle
stirring. The solution was cooled in an ice bath and 27 mL ace-
tonitrile were added dropwise. CDMT (1.0 eq., 87.5 mg, 0.50 mmol)
and NMM (1.5 eq., 82 ␮L, 0.75 mmol) were added to the cold solu-
tion and the mixture was subsequently allowed to come to room
temperature and was stirred for 1 h. Thereafter the amine com-
pound (1.5 eq., 0.75 mmol) was added and the mixture was stirred
at room temperature overnight. The crude mixture was incubated
with Dowex® 50WX8 ion exchange resin (ca. 2 g) for 1 h, then fil-
tered and dialyzed against aqueous NaCl (0.1 M) for 2 days and
against water for 2 days and lyophilized. The product yields were
typically around 80% based on the average molecular weight of the
disaccharide repeating units considering the degree of substitution.
The pH values of the reaction mixtures were measured after
addition of all reactants using a MeterLab PHM 210 standard
2.5. Copper-mediated azide-alkyne-cycloaddition (CuAAC)
In a typical procedure, HA-alkynyl (1.0 eq., 12.5 ␮mol of propar-
gyl groups, 2.5 g/L) and 1-azido-11-hydroxy-3,6,9-trioxaundecane
(1.1 eq., 13.7 ␮mol) were dissolved in water. The solution was
bubbled with argon gas for 15 min to remove oxygen. Solutions
of copper(II) sulfate pentahydrate (CuSO4·5H2O, 0.1 eq., 1.7 ␮mol,
3.4 g/L) and sodium ascorbate (1.3 eq., 16.8 ␮mol, 26.6 g/L) in water
were prepared separately and also deoxygenated by bubbling with
argon gas for 10 min. Subsequently, the copper and ascorbate solu-
tions were added to the first mixture under an inert atmosphere
via a syringe. The mixture was stirred under constant argon bub-
bling at room temperature overnight. The cycloaddition product
was purified by dialysis of the crude mixture against ethylene-
diaminetetraacetic acid (EDTA)-containing water for 1 day and
against distilled water for 2 days and then lyophilized. The pure
product was obtained in 80% yield.
pH meter (Radiometer, Copenhagen) equipped with
a VWR
662-1767 semi-micro pH electrode calibrated in aqueous buffer
solutions. The values were converted to real pH values for the
water/acetonitrile mixture according to Gagliardi, Castells, Ràfols,
Rosés, and Bosch (2007).
HA-alkynyl-azide click: ı(D2O) = 2.00 (–NH–C O–CH3, 3.0 H, s),
3.23–3.30 (–CH2–OH, 0.3 H, br), 3.30–3.41 (–(CH2–O–CH2)3–, 1.0
1H NMR spectral characteristics of the derivatives:
Please cite this article in press as: Borke, T., et al. Optimized triazine-mediated amidation for efficient and controlled functionalization
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H, p, J = 8.4 Hz), 3.41–4.08 (sugar rings, 11.1 H, br), 4.27–4.37
(–N–CH2–CH2–O–, 0.3 H, br), 4.44–4.68 (anomeric protons, 2.6 H,
m), 8.00 (triazole, 0.2 H, s) ppm.
2.6. Maleimide-thiol Michael addition reaction
To an ice-cold solution of HA-maleimide in water (1.0 eq.,
11.2 ␮mol, 2.5 g/L) was added 1-mercapto-11-hydroxy-3,6,9-
trioxaundecane (1.0 eq., 11.2 ␮mol). The mixture was stirred for
1 h at 0 ◦C. Subsequently it was warmed to room temperature, dia-
lyzed against water for 2 days and lyophilized to yield 76% of the
pure product.
HA-maleimide-thiol click: ı(D2O) = 2.04 (–NH–C O–CH3,
3.0 H, s), 2.74–2.87 (–S–CH2–CH2–O–, 0.4 H, m), 2.95–3.12
(O C–CH2–CH(C O)–S–, 0.6 H, br), 3.20–4.10 (sugar rings + others,
13.9 H, br), 4.11–4.19 (O C–CH2–CH(C O)–S–, 0.4 H, br) 4.45–4.69
(anomeric protons, 1.9 H, br) ppm.
Fig. 1. 1H NMR spectra of sodium hyaluronate and its amidation products (see
Table 2) in D2O/DMSO-d6 (1:1, v/v). The signals used to calculate DS, are indi-
cated by the letters as follows: (from below) Unmodified HA: methyl protons of
the N-acetylglucosamine moiety at ∼2.2 ppm (*); HA-alkynyl: propargyl protons
–CH2–C H at ∼4.1 (a) and –C CH at ∼3.1 ppm (b); HA-maleimide: maleimide
ring protons at ∼7.1 ppm (c); HA-methacrylate: double bond protons –C(CH3) CH2
at ∼5.9 and ∼6.3 ppm (d); HA-allyl: double bond protons –CH CH2 at ∼5.4 and
∼6.0 ppm (e); HA-thiol: methylene protons –CH2–SH at ∼3.0 ppm (f).
3. Results and discussion
3.1. Triazine-mediated amidation of HA with functional amines
in water/acetonitrile
We set out first to confirm that, in our hands, the triazine-
mediated amidation proceeded well under the conditions reported
previously (Bergman et al., 2007; C. Schanté et al., 2011) using five
amines ranging in pKa from 8.2 to 10.8 (see Table 2). The same ratio
of [–COO−]:[CDMT]:[NMM]:[–NH2] was used in all reactions. The
resulting HA derivatives were purified, isolated, and their DS values
were determined by 1H NMR spectroscopy in D2O/DMSO-d6 (see
Fig. 1). The measurements were conducted in a mixed solvent to
ensure the solubility of hydrophobically modified HA-derivatives,
thus obtaining the real DS values. However, DS values calculated
from pure aqueous solutions were essentially the same. The results
of this survey were disheartening (see Table 2): the DS values
ranged from 55% at best to 4% at worst. Interestingly, the DS values
correlated with the pKa of the amine: the higher the pKa, the lower
the DS.
Scheme 1 d and e, respectively). Therefore the rate determining
step in the amide bond formation is the nucleophilic attack on the
carbonyl carbon by the amine, which is only possible if the amine
is not protonated. The pH of the reaction mixtures was measured
to be 7.61 0.02 at the beginning of the reaction. It decreased to
7.17 0.05 in the course of the reaction, due to the elimination of
protons from the coupled amines upon collapse of the tetrahedral
intermediate (see Scheme 1 d). Under these conditions, hyaluronic
acid and the amines exist as salts (Kunishima et al., 2013). The
amines are involved in acid-base equilibria with the liberated N-
methylmorpholinium and the weakly basic triazine leaving group
(Scheme 1c). Thus, as the basicity of the amine increases, the amine
exists more and more in its inactive protonated form, and conse-
quently, the yield of the amidation decreases
To understand the crucial role of the amine basicity on the out-
come of the amidation, it is important to review the mechanism and
to monitor the kinetics of the triazine-mediated amide bond forma-
tion. The mechanism, as described by Kamin´ ski (Kamin´ ski, 1994), is
shown in Scheme 1. The first step of the reaction involves the in situ
generation of the reactive coupling compound 4-(4,6-dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM)
starting from 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and
4-methylmorpholine (NMM) (Kamin´ ski, Paneth, & Rudzin´ ski, 1998;
Kunishima, Kawachi, Iwasaki, Terao, & Tani, 1999), see Scheme 1a.
According to the mechanism suggested by Kamin´ ski (Kamin´ ski,
1994) the 2–acyloxy-4,6–dimethoxy–1,3,5–triazine intermediate,
formed from the attack of HA on DMT-MM, is the active species
in the coupling reaction (Scheme 1 b). This “superactive” ester,
as Kamin´ ski named it, subsequently undergoes aminolysis. The
collapse of the tetrahedral intermediate is fast and driven by
the tautomeric rearrangement of the triazine leaving group (see
3.2. Triazine-mediated amidation of HA in water
3.2.1. Kinetics of activated HA in water
Having established the validity and limitations of the pub-
lished procedure, we set out to improve it in order to provide
a viable alternative method to the EDC/NHS mediated amida-
tion. First, we addressed the mixed solvent issue. It is necessary
to use a mixed solvent system of water and acetonitrile in the
published procedures to ensure the dissolution of the CDMT
precursor. It is interesting to note that DMT-MM, which is water-
soluble, can be prepared and isolated in high yields (Kunishima,
Kawachi, Monta, et al., 1999). We envisaged the possibility of
using DMT-MM obtained ex situ, as activating agent for amida-
tion in water in the absence of co-solvent. To check the feasibility
of the approach, we monitored by 1H NMR spectroscopy the
kinetics of DMT-MM hydrolysis in water and in an aqueous HA
Table 2
Degree of substitution of “clickable” hyaluronic acid derivatives prepared from amines with different basicity.
Derivative
Amine reagent
pKa ofAmine
DSa [%]
Ref. for pKa values
HA-alkynyl
HA-maleimide
HA-methacrylate
HA-allyl
Propargyl amine·HCl
8.2
8.4b
8.8
9.5
10.8
54.5 4.4
39.5 2.6
41.3 6.1
28.2 2.2
3.7 0.7
(Jarman et al., 1993)
–
(Riauba, Niaura, Eicher-Lorka, & Butkus, 2006)
(Thompson, Read, & Armes, 2008)
(Houson, 2011)
N-(2-Aminoethyl)-maleimide·CF3COOH
2-Aminoethyl-
Allylamine
methacrylate·HCl
HA-thiol
Cysteamine·HCl
a
Number of functionalized side groups per 100 disaccharide units as determined by 1H NMR. Average value of three integrations standard deviation.
Calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02 (© 1994–2013 ACD/Labs).
b
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5
Scheme 1. Mechanism of the triazine-mediated coupling of hyaluronic acid and propargyl amine in aqueous solution.
solution (see Fig. 2 and Fig. S1 in SI). The overall half-life (t1/2) of
DMT-MM in D2O at room temperature was found to be 10 days.
The decomposition of DMT-MM involved several steps. DMT-MM
was first hydrolyzed to N-methylmorpholinium (NMM-H+) and
2-hydroxy-4,6-dimethoxy-1,3,5-triazine (DMT-OH), which rear-
ranged to its tautomer, 4,6-dimethoxy-1,3,5-triazin-2-one, and
finally underwent isomerization to 6-methoxy-3-methyl-1,3,5-
triazine-2,4-dione by O→N migration (see Scheme 2a and b)
(Kamin´ ski, 2000; Tosato, 1979). Isomerization of the triazine by-
product was previously observed when the reaction was carried
out at 100 ◦C in an organic solvent. In this case, both methyl groups
migrated during the rearrangement (Kamin´ ski, 2000). The isocya-
nuric acid derivative obtained here results from the migration of
a single methyl group. It is stable in water at room temperature.
De-methylation and formation of chloromethane was reported
to occur when the reaction is carried out in organic solvents
(Kunishima, Kawachi, Iwasaki, et al., 1999) It was not observe in
this study, as indicated by the constant total intensity, over as
long as 63 days, of the –N+–CH3 signals originating from DMT-
MM (ı 3.54 ppm) and NMM-H+ (ı 2.96 ppm), respectively (see
Fig. S2 in SI). All hydrolysis products as well as the coupling
agent remained water-soluble. They were easily removed by dial-
ysis.
formation of the “superactive” ester. The disappearance of DMT-
MM in the presence of HA was found to be about 4 times faster
(t1/2 = 2.7 days) than the hydrolysis of DMT-MM alone (Fig. 2d). The
formation of the ester was indicated by a shift in the 1H NMR signal
Scheme 2. Slow hydrolysis of DMT-MM in aqueous solution to DMT-OH and NMM-
H+ (t1/2 = 10 days). DMT-OH rearranges and then isomerizes by O→N migration of
the methyl group.
Next, we monitored the consumption of DMT-MM in the pres-
ence of HA, [COO−]:[DMT-MM] = 1.0:1.5, which should lead to the
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Fig. 2. (a) Hydrolysis of DMT-MM in D2O over time followed by 1H NMR. Diamonds: Consumption of DMT-MM; circles: appearance of hydrolysis product, DMT-OH;
triangles: formation of isocyanuric acid derivative by O→N migration of one methyl group; squares: for comparison, consumption of DMT-MM in presence of HA. (b) Active
ester formation between HA and DMT-MM in D2O and hydrolysis over time followed by 1H NMR. diamonds: Consumption of DMT-MM by reaction with HA and hydrolysis;
circles: appearance of active ester and hydrolysis product, DMT-OH; triangles: formation of isocyanuric acid derivative from isomerization of DMT-OH. (c) First-order kinetic
plot of DMT-MM consumption [ln(ct /c0) = −k·t] by hydrolysis (diamonds) or in presence of HA (squares) with respective apparent rate constants determined from the slope
of the curves. (d) Formula of the half-life (t1/2) of a reaction following first order kinetics and t1/2 of DMT-MM in aqueous solution and in presence of HA.
and amine (kapp = 5 × 10−6 s−1) was comparable to the rate of con-
of the methoxy protons of DMT-MM from 4.15 to 4.02 ppm. The
intensity of the new signal reached a plateau value of 70% after
sumption of DMT-MM in presence of HA only (kapp = 3 × 10−6 s−1),
4 days. This value corresponds to a near quantitative reaction of
DMT-MM and HA, given that the initial ratio of [COO−]:[DMT-MM]
was 1.0:1.5 (see Fig. 2b, Tab. S1 in SI). Unfortunately the methoxy
signals of the “superactive” ester and of the hydrolysis product,
DMT-OH, have the same chemical shift (4.02 ppm) and cannot be
distinguished by 1H NMR spectroscopy. Nonetheless, we can con-
clude from these experiments that the kinetics of ester formation
are much faster than the hydrolysis of DMT-MM. Also, the inten-
sity of the methoxy signal in the 1H NMR spectrum of the mixture
remained constant for at least 10 days, which suggests the presence
and stability of the “superactive” ester. This has also been observed
by Kunishima, Kawachi, Monta, et al. (1999), who suggested ear-
lier that the 2-acyloxy-4,6-dimethoxy-1,3,5-triazine derivative is
more stable than the quaternary ammonium compound, DMT-MM.
In addition, it was shown here that the “superactive” ester is more
stable than the hydrolysis product, DMT-OH, which does not accu-
mulate in the reaction mixture, but quickly isomerizes into the
partially saturated isocyanuric acid derivative (compare Fig. 2a,
circles and triangles).
Figs. 3b and 2c, respectively, suggesting that the amine has no sig-
nificant effect on the formation of the “superactive” ester. Some
researchers have reported an acceleration of the reaction between
DMT-MM and the acid in the presence of an amine (Hasani &
Westman, 2007; Kamin´ ski et al., 2005; Kunishima et al., 2013); this
effect was not observed in this study (see Tab. S1 in SI). The forma-
tion of the 2-acyloxy-1,3,5-triazine intermediate is only possible
if the carboxylic acid is deprotonated. Hence, commercial sodium
hyaluronate can be used, without special pre-treatment.
After about 1 day, the amidation of HA by propargyl amine
slowed down. The decrease in reaction rate may be due to the shift
of the acid-base equilibrium towards the more protonated amine
species as a result of the pH change in the reaction mixture. It could
be due also to the increased bulkiness of HA as a consequence of the
amidation or the formation of hydrogen bonds between the newly
formed amide groups and the carboxyl groups of the unreacted d-
glucuronic acid moieties. Eventually, the reaction levels off when
the DS reached 40%.
The DS value obtained during the kinetics studies is lower
than the value we obtained in our initial assessment of the
triazine-activated amidation with in situ generation of DMT-MM
in water/acetonitrile mixture (see Section 3.1). It is difficult, based
on the kinetic studies, to compare the two experimental condi-
tions. The reaction in water/acetonitrile was conducted with high
molar mass HA and 1.5 molar excess of propargyl amine to [–COO−]
under stirring for 20 h. The kinetics study was done in D2O with low
molar mass HA and a 1.1 molar excess of amine. The coupling effi-
ciency however was the same in both cases, viz. 36.7 and 36.4% DS
per molar equivalent of amine to carboxylate group, respectively,
which implies that the amine stoichiometry is the limiting factor
controlling the ultimate DS of HA.
3.3. Kinetics of HA amidation in water
The kinetics of HA amidation with propargyl amine in D2O in the
presence of DMT-MM ([–COO−]:[–NH2]:[DMT-MM] = 1.0:1.1:1.1)
were monitored by 1H NMR spectroscopy. The consumption of the
coupling agent, monitored via the signal at 4.15 ppm, the degree of
substitution, monitored by the appearance of a signal at 2.70 ppm,
and the liberation of the triazine leaving group, DMT-OH, moni-
tored by the signal at 4.02 ppm, proceeded linearly and with equal
rates, during the first 10 h of the reaction (see Fig. 3a). The appar-
ent rate constant of DMT-MM consumption in the presence of HA
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Scheme 3. Model “click” reactions conducted on the HA-derivatives: (a) copper-mediated azide-alkyne click reaction; (b) maleimide-thiol Michael addition reaction.
3.4. Model “Click” reactions
proceed under mild conditions in an orthogonal, fast and quanti-
tative manner giving stable, regiospecific linkages in high yields
without appreciable amounts of side products (Lutz & Börner,
2008; Nandivada, Jiang, & Lahann, 2007). Besides the well-known
copper-mediated azide-alkyne click (CuAAC) reaction, based on the
Huisgen 1,3-dipolar cycloaddition, several thiol-ene addition reac-
tions have proven to be successful. They typically employ allyl-,
(meth)acrylate- or maleimide-derivatives to which thiols easily
attach via either free radical or nucleophilic reaction (Dong et al.,
2012; Jing et al., 2013; Pounder, Stanford, Brooks, Richards, & Dove,
2008; Yu, Chan, Hoyle, & Lowe, 2009). To demonstrate the util-
ity of the aziridine mediated amidation method, two model “click”
reactions, i.e. the CuAAC and the maleimide-thiol Michael addition
(Scheme 3), were conducted starting from modified HA bearing,
respectively, propargyl and maleimide moieties. Michael addition
of 1-mercapto-11-hydroxy-3,6,9-trioxaundecane to the maleimide
groups of HA proceeded rapidly in water at 0 ◦C reaching comple-
tion after 1 h as judged by NMR spectroscopy (see Fig. S3 in SI).
The CuAAC reaction of 1-azido-11-hydroxy-3,6,9-trioxaundecane
with HA-alkynyl, carried out in water in the presence of Cu(II) ions
and ascorbic acid, was complete after 20 h (see Fig. S3 in SI). The
degree of modification of HA-alkynyl, estimated from the integral
of the 1H NMR signal of the triazinyl proton, was ∼23%, which is
significantly lower than the DS of HA-alkynyl (55%), possibly due
to the poor solubility of 1-azido-11-hydroxy-3,6,9-trioxaundecane
in water. These initial results confirm that the products of the
triazine-mediated amidation of HA serve their intended purpose
and that they are poised to enable the preparation of well defined
HA-conjugates.
The HA derivatives prepared can be reacted with various
biomolecules, drugs, polymers or probes via “click” reactions for
the formation of bioconjugates. Reactions of the “click family”
4. Conclusions
The triazine-based coupling agent, DMT-MM was used to carry
out the amidation of HA in aqueous solution under mild condi-
tions with excellent efficiency. Compared to the commonly used
EDC/NHS coupling reaction, the method described here is more
economical, yields higher degrees of substitution (up to 55%) while
using almost stoichiometric amounts of reagents. The activating
agent, DMT-MM, was prepared ex situ and used on demand. It
Fig. 3. (a) DMT-MM mediated coupling reaction between hyaluronic acid and
propargyl amine in D2O at room temperature followed by 1H NMR spectroscopy.
Diamonds: Consumption of DMT-MM; circles: formation of the “superactive” ester
or triazine leaving group, DMT-OH, respectively; triangles: degree of amidation of
HA. (b) First-order kinetic plot of DMT-MM consumption [ln(ct /c0) = −k·t]. Reaction
slows down after about 10 h as indicated by different slopes of the fitted lines.
Please cite this article in press as: Borke, T., et al. Optimized triazine-mediated amidation for efficient and controlled functionalization
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was stable upon storage in the dry form at ambient conditions
and in aqueous solution of neutral pH. Kinetic studies showed
that DMT-MM activates the carboxylic acid groups of HA fast and
quantitatively, yielding a stabilized intermediate. Furthermore the
amine basicity and stoichiometry were found to be the limiting fac-
tors for controlling DS. During the reaction the coupling reagent
is converted into soluble by-products that can be removed eas-
ily. 6-Methoxy-3-methyl-1,3,5-triazine-2,4-dione, an isocyanuric
acid derivative with one migrated methyl group, was found to be
the stable by-product in aqueous solution at RT. Understanding
the role of the amine was critical for establishing effective syn-
thetic parameters. The triazine-mediated synthesis of functional
HA derivatives followed by conjugation by “click” chemistry of suit-
ably functionalized molecules provides a mild and efficient route
towards well-defined bioconjugates.
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Acknowledgements
Kamin´ ski, Z. J. (1985). 2-Chloro-4,6-disubstituted-1,3,5-triazines
a
novel
The authors gratefully acknowledge the project funding by the
Academy of Finland, grant number 263573, and the Finnish Fund-
ing Agency for Technology and Innovation (Tekes), grant number
40339/12. Furthermore we thank Mr. Fabian Pooch for enzymati-
cally hydrolyzed hyaluronic acid.
group of condensing reagents. Tetrahedron Letters, 26(24), 2901–2904.
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http://dx.doi.org/10.1055/s-1999-3448
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found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.
2014.04.012.
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