Synthesis of Conjugates of hyaluronic and nicotinic acids

Article abstract of DOI:10.1007/s10600-012-0201-9

Conjugates with nicotinic acid of hyaluronic acid carboxylic and hydroxyl groups were synthesized and exhibited polyampholyte properties.

Full text of DOI:10.1007/s10600-012-0201-9

Chemistry of Natural Compounds, Vol. 48, No. 2, May, 2012 [Russian original No. 2, March-April, 2012]
SYNTHESIS OF CONJUGATES OF HYALURONIC
AND NICOTINIC ACIDS
*
I. Yu. Ponedelꢀkina, E. V. Salꢀnikova, E. S. Lukina,
UDC 547.995.15:547.826.2:547.827
T. V. Tyumkina, and V. N. Odinokov
Conjugates with nicotinic acid of hyaluronic acid carboxylic and hydroxyl groups were synthesized and
exhibited polyampholyte properties.
Keywords: hyaluronic acid, nicotinic acid, conjugates, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide,
polyampholytes.
Hyaluronic acid (HA) has recently been more widely applied in various areas of medicine and cosmetics as a
macromolecular matrix for drugs and biocompatible material with reparative-regenerative properties. HA is modified chemically
by compounds of natural origin in order to change its physicochemical properties and expand its spectrum of biopharmacological
activity [1, 2]. Nicotinic acid, which is known for its unique physiological and pharmacological properties [3], is interesting
in this respect. Incorporation of it into the HA structure can impart new properties to HA conjugates (including polyampholyte
+
[4]) and lead to the creation of high-molecular-weight models of NAD and NADH [5, 6].
We modified HA (1) at both the carboxylic and hydroxyl groups. Conjugation at the carboxylic groups of HA was
carried out through diamine spacers of D,L-lysine and hydrazine.
COOH
HN
C
NH
2
OH
COOH
OH
O
O
HO
O
O
O
HO
D,L-lysine (2)
O
HO
O
O
O
HO
OH
NHAc
O
1
OH
NHAc
3
NH
6
O
NH
2
N
C
8
COOH
O
N
HN
NH
HN
N
OH
O
C
N
O
C
OH
O
HO
O
O
O
O
HO
O
O
O
HO
HO
OH
NHAc
NHAc
OH
9
7
O
-
C
+
Cl
N
HN
NH
O
O
O
O
OH
O
C
N
OH
O
HO
O
O
O
NOH
O
HO
+
O
N
N
O
OH
NHAc
4
6
10
5
Scheme 1
Institute of Petroleum Chemistry and Catalysis, Russian Academy of Sciences, 450075, Ufa, fax: (347) 284 27 50,
e-mail: ink@anrb.ru; ponedelkina@rambler.ru. Translated from Khimiya Prirodnykh Soedinenii, No. 2, March–April, 2012,
pp. 177–180. Original article submitted August 16, 2011.
©
0009-3130/12/4802-0189 2012 Springer Science+Business Media, Inc.
189

Conjugate 3 (Scheme 1) was obtained via reaction of 1 with D,L-lysine (2) in aqueous medium in the presence of
1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and N-hydroxybenzotriazole (HBT). The reaction did not occur
without HBT. Conjugate 3 was purified of low-molecular-weight reagents by dialysis and represented the amide of HA
bonded to lysine through its ꢁ-amine. This was consistent with PMR spectral data. Resonances of the terminal lysine methylene
were located in the HSQC spectrum (ꢂ 2.91 ppm, ꢂ 39.2 ppm) in the same region as in the spectrum of ꢁ-N-acetyllysine and
H
C
indicated that the ꢃ-NH was free. The ratio of intensities for the proton resonances of this methylene (ꢂ 2.91 ppm) and the
2
methyl protons of the acetamide (ꢂ 1.92 ppm) suggested that the content of lysine-modified units in 3 reached 30% (calculated
per 100 disaccharide units of HA).
The carboxylic group of nicotinic acid (4) was activated by conversion to N-succinimidyl 6 by reaction with
N-hydroxysuccinimide (5) in the presence of dicyclohexylcarbodiimide (DCC) in anhydrous DMF in order to conjugate 3 to
4. Combination of 3 and 6 in aqueous DMF in the presence of NaHCO gave the desired conjugate 7 in which HA and
3
nicotinic acid were linked through a lysine bridge. Resonances of the ꢃ-CH NH methylene protons (ꢂ 2.91 ppm) were
2
2
missing in the PMR spectrum of 7, indicating that it had been exhaustively acylated. Therefore, 7, like lysine-modified
product 3, contained 30% modified units, which was confirmed by the ratio of total intensity for resonances of Py protons
(ꢂ 7.5–9.0 ppm) and the acetamide singlet (ꢂ 1.92 ppm).
The conjugate of HA (1) with nicotinic acid hydrazide (8) was synthesized by the action of EDC and HBT (Scheme
1). Conjugates 9a and 9b with 20 and 52%, respectively, modified units were obtained depending on the ratio of reagents. The
extent of modification was determined from the relative total intensity for resonances of Py protons (ꢂ 7.54–8.90 ppm) and
CH CON methyl protons (ꢂ 1.92 ppm).
3
Conjugates 9a and 9b were converted to N-benzylpyridinium chlorides (10a and b) by benzylchloride in DMF at
80°C (Scheme 1). PMR spectra of 10a and b contained resonances for benzyl methylene (ꢂ 5.85 ppm) and aromatic (ꢂ 7.41
H
and 7.45 ppm) protons (in a 2:5 ratio). Resonances of Py protons were observed at weaker field (ꢂ 8.1–9.4 ppm) than the same
resonances in the spectra of conjugates 9a and 9b. The singlet for the acetamide methyl protons was split by the influence of
the magnetically anisotropic benzyl group that was close to the neighboring N-acetylglucopyranose moiety. A multiplet
consisting of seven resonances appeared. The absorption maximum of 10a and b in the UV spectrum was shifted to long
wavelength (ꢄ
276 and a shoulder at 290 nm) relative to conjugates 9a and 9b (ꢄ
263 nm). This was due to an increase
max
max
of the conjugation chain in the N-benzylpyridinium moiety.
The acid-chloride method was the most effective one of those studied by us for synthesizing O-esters of HA with
nicotinic acid [7]. HA nicotinate (13) was obtained by reaction of the tetrabutylammonium salt of HA (11) with freshly
prepared nicotinic acid chloride (12) (Scheme 2).
_
O
C
+
OH
OR
COOBu N
COOH
4
O
O
HO
O
O
RO
O
O
O
HO
O
RO
Cl
HCl
+
1
.
N
OH
NHAc
OR
NHAc
11
12
13
O
C
R =
or
H
N
Scheme 2
The PMR spectrum of 13 in the region of the Py protons (ꢂ 7.50–9.50 ppm) exhibited resonances of different intensity
and corresponded apparently to nicotinoyl substituents on all four OH groups of the disaccharide unit. The region of methyl
protons contained a singlet for MeCON at ꢂ 1.92 ppm and a strong-field resonance with ꢂ 1.26 ppm that correlated, like the
first singlet, with a resonance with ꢂ 25 ppm in the HSQC spectrum and; therefore, corresponded to MeCON methyl protons.
The strong-field shift was most probably due to the shielding effect of the magnetically anisotropic Py ring [8], which was
located close to the MeCON group, i.e., on C-2 of the glucuronic acid pyranose ring. The degree of substitution in 13 was
found to be 0.93 calculated for all four OH groups of the disaccharide unit (or the HA disaccharide unit) (the maximum
possible degree of substitution taking into account all four OH groups in the HA disaccharide unit is four) based on the ratio
of integrated intensities of the Py protons and both MeCON resonances adjusted to one proton.
190

D500
0.16
0.12
0.08
0.04
2
4
6
8
10
12
pH
Fig. 1. Absorption of solutions of conjugate 9b
at 500 nm as a function of pH (c = 4 mg/mL).
The ability of Py bases to give salts with acids could produce conjugates 7, 9a, 9b, 10a, 10b, and 13 with properties
of polyampholytes. Polyampholytes in aqueous solutions are known to be sensitive to a change of ionic strength and pH.
They form dispersions of colloidal particles under certain conditions. The absorption of these can be measured
spectrophotometrically at 500 nm [4]. It can be seen using 9b as an example (Fig. 1) that the absorption of the solutions
changed as a function of pH (with maxima in the regions of pH 4 and 7). This was indicative of the formation of a complex salt
of the Py base of nicotinic acid with a non-conjugated glucuronic acid and confirmed the properties of 9b as a polyampholyte.
EXPERIMENTAL
13
PMR and C NMR spectra (ꢂ, ppm) were recorded in D O solutions with acetone internal standard on a Bruker
2
1
13
Avance 400 spectrometer (operating frequency 400.13 MHz for H and 100.58 MHz for C). Electronic spectra of aqueous
solutions were taken on a Specord M-40 spectrophotometer. Solution pH values were monitored using a pH-340 pH-meter.
We used pharmaceutical grade nicotinic acid (India); thionylchloride (Becton); and tetrabutylammonium
iodide, N-hydroxybenzotriazole, N-hydroxysuccinimide, N,N-dimethylaminopyridine, D,L-lysine, ꢁ-N-acetyllysine,
dichclohexylcarbodiimide, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, and hydrazine hydrate (Aldrich). Benzylchloride
was purified by distillation. HA was prepared as before [8]. Nicotinic acid hydrazide was obtained from the reaction with
hydrazine hydrate [9]; the chloride (as the hydrochloride), from the reaction with thionylchloride [10].
Tetrabutylammonium Hydroxide (Bu NOH, prepared by the literature method [11] with our modification). A
4
solution of AgNO (2 g, 11.77 mmol) in H O (10 mL) was treated with NaOH solution (1 N) untilAg O stopped precipitating.
3
2
2
The precipitate was filtered off, washed with distilled H O, suspended in H O (20 mL), and treated with Bu NI (4.34 g,
2
2
4
11.77 mmol). The mixture was stirred at room temperature until the black Ag O solid changed to yellow AgI, which was
2
filtered off. Then, the aqueous solution of Bu NOH was used to prepare tetrabutylammonium salt 11.
4
Conjugate 3, ratio of reagents 1:2:EDC:HBT 1:5:2:2. A mixture of HA (1, 60.0 mg, 0.15 mmol), D,L-lysine (2,
137.0 mg, 0.75 mmol), and HBT (40.5 mg, 0.3 mmol) in H O (15 mL) was treated with NaOH solution (1 N) until the pH was
2
6.8, heated to 40°C, stirred vigorously, and treated with dry EDC (57.5 mg, 0.3 mmol). The mixture was heated for 2 h and left
for 24 h at room temperature. When the reaction was finished, the mixture was dialyzed against H O (24 h) and then treated
2
with EtOH (45–50 mL). The resulting precipitate was separated by centrifugation, washed with EtOH (3 ꢅ 5 mL) and Et O
2
(2 ꢅ 5 mL), and dried at <60°C and reduced pressure to afford an amorphous white powder (62 mg) that was soluble in H O
2
ꢆ
and contained 30% lysine-modified units. PMR spectrum (characteristic resonances ): ꢂ 1.2–1.8 (6H, , ꢇ, ꢂ-CH ), 1.92 (3H,
2
s, MeCON), 2.91 (2H, m, CH NH ), 3.3–4.5 (12H in GlcA and GlcNAc units, H-1–H-6).
2
2
N-Succinimidyl Nicotinic Acid (6) [12]. Acid 4 (86.1 mg, 0.70 mmol) and N-hydroxysuccinimide (80.5 mg,
0.70 mmol) in DMF (1.5 mL) was stirred vigorously, treated with ground dry DCC (158.6 mg, 0.77 mmol), and stirred at
20–25°C for 3–4 h. The course of the reaction was monitored by TLC on Sorbfil plates (Sorbpolimer, Krasnodar), R 0.87
f
(EtOAc:petroleum ether, 2:1). Compound 6 was used without isolation from the reaction mixture in the reaction with lysine-
modified HA 3.
ꢈꢈꢈꢈꢈꢈ
ꢆ
Here and henceforth characteristic resonances that are important for determining the content of modified units in the conjugates
are given.
191

Conjugate 7. A solution of 6 (154 mg, 0.70 mmol) in DMF (5 mL) was stirred, treated dropwise with a solution of
conjugate 3 (60 mg, 0.14 mmol) in aqueous NaHCO (5 mL, 0.1 N) at pH 8.5, stirred at 20–25°C for 10–12 h, and dialyzed
3
against H O for 24 h. Then the dialysate was treated with a three-fold excess of MeOH. The resulting precipitate was
2
separated by centrifugation, washed with MeOH (3 ꢅ 5 mL) and Et O (2 ꢅ 5 mL), and dried at <60°C and reduced pressure to
2
afford an amorphous white powder (54 mg) that was soluble in H O. PMR spectrum: ꢂ 1.2–1.8 (6H, , ꢇ, ꢂ-CH ), 1.92 (3H,
2
2
s, MeCON), 3.15 (2H, m, CH NH), 3.3–4.5 (12H in GlcA and GlcNAc units, H1-H6), 7.5–9.0 (4H, H ).
2
Py
Conjugate 9a, ratio of reagents 1:8:EDC:HBT 1:1.2:0.75:1. Compound 1 (60 mg, 0.15 mmol) and nicotinoylhydrazide
(8, 24.7 mg, 0.18 mmol) were dissolved in H O (15 mL), stirred vigorously, and treated with EDC (21.6 mg, 0.11 mmol) (20–
2
25°C). The pH was adjusted to 4.75 using HCl solution (0.1 N). After 2 h HCl (1 N, 2–3 drops) and EtOH (45 mL) were
added. The resulting precipitate was worked up as above for conjugate 7 to afford a product (56 mg) containing 20% modified
units.
Conjugate 9b, ratio of reagents 1:8:EDC:HBT 1:1.5:1.5:1. A mixture of HA (60 mg, 0.15 mmol) and 8 (30.8 mg,
0.23 mmol) in H O (15 mL) was stirred and treated with EDC (43.1 mg, 0.15 mmol). The reaction was carried out and worked
2
up as for 9a to afford a product (59 mg) containing 52% modified units. UV spectrum (H O, ꢄ , nm): 263. PMR spectrum:
2
max
ꢂ 1.92 (3H, s, MeCON), 3.3–4.5 (12H in GlcA and GlcNAc units, H1-H6), 7.5–8.9 (4H, H ).
Py
Conjugates 10a and b. The appropriate conjugate 9a or 9b (60 mg, 0.14 mmol) was suspended in anhydrous DMF
(4.5 mL), treated with an excess (0.05–0.10 mL) of benzylchloride, and stirred for 20 h at 80°C. When the reaction was
finished the DMF and benzylchloride were removed by Et O. The product was dissolved in H O and dialyzed against H O for
2
2
2
24 h in order to purify it further of the reagents. The conjugates were precipitated from the aqueous solutions by adding a
three-fold volume of EtOH to afford a water-soluble yellow powder (56–60 mg). UV spectrum (H O, ꢄ , nm): 276 and a
2
max
shoulder at 290. PMR spectrum: ꢂ 1.85, 1.86, 1.89, 1.92, 1.93, 1.94, 1.95 (3H, m, MeCON), 3.3–4.5 (12H in GlcA and
GlcNAc units, H1-H6), 5.85 (2H, m, CH Ph), 7.4–7.5 (5H, H ), 8.1–9.3 (4H, H ).
2
Ph
Py
Tetrabutylammonium Salt of HA (11). Compound 1 (300 mg, 0.75 mmol) was dissolved in H O (15–20 mL),
2
+
placed onto a column (300 ꢅ 25 mm) with Dowex 50WX4 in the H -form, and eluted by H O. The fraction with an acidic pH
2
value was collected in a plastic beaker and neutralized by Bu NOH. Salt 11 was dried at 80°C. The resulting film was
4
dissolved in MeOH (5 mL) and treated with Et O (15 mL). The resulting precipitate was separated by centrifugation, washed
2
with Et O (2 ꢅ 3 mL), and dried at reduced pressure (60°C) to afford 11 (410 mg), 88% yield.
2
HA Nicotinate (13). A solution of 11 (87.4 mg, 0.14 mmol) in anhydrous DMF (5 mL) was stirred, treated with
freshly prepared nicotinoyl chloride hydrochloride (12, 199.4 mg, 1.12 mmol, two-fold excess relative to each HA OH group)
in Py (3 mL) and a catalytic amount of N,N-dimethylaminopyridine, heated at 60°C for 2 h, and left at room temperature
overnight. The mixture was worked up as for conjugate 3 to afford a product (80 mg) with 0.93 degree of substitution by
nicotinic units. UV spectrum (H O, ꢄ , nm): 267. PMR spectrum: ꢂ 1.26, 1.92 (3H, MeCON), 3.0–4.5 (12H in GlcA and
2
max
GlcNAc units, H1-H6), 7.5–9.5 (4H, H ).
Py
Polyampholyte Properties of Conjugate 9b. Several aqueous solutions of the conjugate at concentration 4 mg/mL
were prepared. The pH of each solution was adjusted using NaOH (0.1 N) or HCl (0.1 N) to a certain value (from 2 to 11). The
absorption at 500 nm was measured in a 1-cm cuvette [4].
ACKNOWLEDGMENT
The work was supported financially by the Division of Chemistry and Materials Science, RAS, through the program
“Molecular Design of Physiologically Active Compounds and Drugs,” project “Conjugates of Glycosaminoglycans with
Nicotinic Acid Derivatives as Biocompatible Polyampholytes.”
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