ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 5, pp. 472–477. © Pleiades Publishing, Inc., 2006. Original Russian Text © I.Yu. Ponedel’kina, V.N. Odinokov, E.S. Lukina, T.V. Tyumkina, L.M. Khalilov, U.M. Dzhemilev, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 5, pp. 524–529.
Chemical Modification of Heparin I. Yu. Ponedel’kina,1 V. N. Odinokov, E. S. Lukina, T. V. Tyumkina, L. M. Khalilov, and U. M. Dzhemilev Institute of Petrochemistry and Catalysis, Russian Academy of Sciences, pr. Oktyabrya 141, Ufa, 450075 Bashkortostan, Russia Received July 27, 2005; in final form, December 21, 2005
Abstract—Heparin was modified at carboxyl groups by reaction with several pharmacologically important amino-containing compounds in aqueous medium in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. In dependence on the nature of the amine and the ratio of reagents, conjugates containing 36–100% amide and 0–25% isoureidocarbonyl groups were synthesized. Isoureidoarylamide groups are present, along with amide moieties, in the products of heparin modification by hydroxyl-containing aromatic amines. The conjugate of heparin with p-aminobenzoic acid contained oligomeric arylamide. Key words: heparin, chemical modification; conjugates; amino-containing compounds; 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide DOI: 10.1134/S1068162006050104
INTRODUCTION Heparin (I), a linear N,O-sulfated negatively charged glycosaminoglycan, is widely known for its anticoagulant and antithrombotic properties. The antibacterial, antiviral, anti-inflammatory, and antitumor properties of heparin [1, 2] and its inhibiting action on human immunodeficiency virus [3] were revealed. In recent years, a great interest arises in the chemical modification of heparin. Epoxyheparin [4] and heparins of various sulfation degrees [5] have been synthesized. Conjugates of heparin at its carboxyl groups with steroids were obtained; they were shown to have a unique ability to inhibit angiogenesis [6]. RESULTS AND DISCUSSION A promising approach to the modification of glycosaminoglycans is their conjugation with primary amines in the presence of water-soluble carbodiimides. We had earlier reported the synthesis of hyaluronic acid conjugates with aromatic amino acids [7]. Here we studied the interaction of heparin [(I), unit A; scheme] with several pharmacologically significant amino-containing compounds [8, 9]: p-(IIa) and o-aminophenols (IIb), 5-aminosalicylic (IIc), 4-aminosalicylic (IId), anthranilic (IIe), and p-aminobenzoic (IIf) acids, p-aminobenzenesulfamide (Streptocid, sulfanilamide) (IIg), p-aminobenzenesulfacetamide-sodium (Sulfacyl-sodium) (IIh), p-aminobenzoic acid ethyl ester 1
Corresponding author; phone/fax: (3472) 31-2750; e-mail:
[email protected].
(anesthesin; benzocaine) (IIi), p-aminobenzoic acid β-diethylaminoethyl ester (Novocaine) (IIj), 1-phenyl2,3-dimethyl-4-aminopyrazolone-5 (4-aminoantipyrine) (IIk), and isonicotinic acid hydrazide (isoniazide) (IIl). The reactions were carried out in aqueous medium in the presence of 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (III). As in the case of hyaluronic acid [7], carbodiimide (III) should be added to a mixture of heparin (I) with the corresponding amino-containing compound (IIa)–(IIl) to achieve a high degree of conversion of carboxyl to amide groups. This order of addition of reagents is dictated by the fact that carbodiimide (III) is readily hydrolyzed by heparin (I). For example, the reaction of heparin with carbodiimide (0.75 mol equiv) is accompanied with only 20% carboxyl groups of heparin converted to isoureidocarbonyl groups [conjugate (V) whose structure corresponds to units A + C; see the scheme], whereas carbodiimide is completely exhausted (as determined by the method [10]). The content of isoureidocarbonyl groups in conjugate (V) was determined from the ratio of intensities of 1H NMR signals at (δ ~3 and 5.1–5.5 ppm, which correspond to the protons of åÂ2N-groups of isoureide residues and anomeric protons, respectively. Conjugate (V) does not react with amines (II‡)–(IIl), gives a positive hydroxamic color test to esters [11], and is reduced by sodium borohydride in aqueous medium with the conversion of isoureidocarbonyl (ester) groups into hydroxymethyl groups (cf. [7, 12]) and the formation of conjugate (VI) (A + E). Units E are detected and quantitatively determined by the anthrone reaction [13].
472
CHEMICAL MODIFICATION OF HEPARIN OSO3H O
O O COOH HO
OSO3H O
O
O HO
O CONHR1 HO
O
OSO3H
NHR
O HO
O
OSO3H
n
Heparin, unit A
NHR B
OSO3H O
O O 2 HO R
473
O HO
O CONHR3 HO
O
OSO3H
OSO3H O
O O HO
O
OSO3H
NHR
C, E
NHR D
O C O N C C R2 =
H3C N H3C
NH
CH3
NH
CH3 C N
and/or H3C N H3C
, E (R2 = CH2OH)
O C O
R = SO3H or Ac, SO3H/Ac = 70–80/20–30 CH3
N C N
(I) + R1NH2 (II‡–l)
H3C N H3C
(V) A+C
(I) + (III)
R1 =
NaBH4
(III) pH 4.7–4.8, H2O NaBH4
(IV‡–l); (IV‡–l) A+B+C+D A+B+C+D
(VII‡–l); A+B+E+D
(VI) A+E
OH (II‡, IV‡),
HOOC
(IIb, IVb),
OH (IIc, IVc),
HO (IIe, IVe),
COOH (IId, IVd),
COOH COOH (IIf),
OH COOH (IVf),
CONH
0–2
SO2NH2 (IIg, IVg),
SO2NNaCOCH3 (IIh, IVh),
COOC2H5 (IIi, IVi),
CH3 COOCH2CH2N(C2H5)2 (IIj, IVj),
O
N
N
CH3
CONH (IIk, IVk),
(IIl, IVl) N
R3 =
OR4 (IV‡),
OR4 (IVc),
(IVb), R4O
NH R4
= H3C N H3C
OR4
COOH CH3
C N
COOH (IVd), where
N C and/or H3C N H3C
NH
CH3
Structure of heparin [(I), unit A] and the units formed in the reaction of heparin with amino-containing compounds (IIa)–(IIl) in the presence of carbodiimide (III). Schemes of reactions of the formation of heparin derivatives (IVa)–(IVl) and O-acylisoureas (V), as well as the products of their reduction by sodium borohydride, compounds (VI) and (VIIa)–(VIIl) are given. The qualitative monosaccharide compositions (units A–E) of products (IVa)–(IVl), (V), (VI), and (VIIa)–(VIIl) are shown. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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The addition of carbodiimide (III) to a mixture of heparin (I) and the corresponding compound (IIa)– (IIl) at a molar ratio of 0.75 : 1 : 1 (per disaccharide repeating unit), pH 4.7–4.8, and room temperature leads to formation of the corresponding conjugates (IVa)–(IVl), which is completed for 1–3 min. In this case, the conversion of carboxyl groups of heparin into carboxamide groups reaches 36–75% (units B; see the scheme and table). The resonances in the region of 7.1– 8.9 ppm in the 1H NMR spectra of products (IVa)–(IVl) respond to aromatic protons in the residues of (IIa)– (IIl) in units B of the modified heparin. The content of units B in conjugates (IVa)–(IVl) was determined from the ratio of total intensities of the signals of aromatic (δ 7.1–8.9 ppm) and anomeric (δ 5.1–5.5 ppm) protons calculated per one proton (table). The ratio of the intensities of signals in the region of δ values of ~3 and 5.1– 5.5 ppm in 1H NMR spectra suggests that the units C are practically absent from (IVa)–(IVl); they are present only in (IVg)–(IVj), where their content is 3– 5% (table). The use of 1.5-fold molar excess of carbodiimide (III) relative to the heparin (I) unit and amine (IIa)– (IIl) taken at equimolar ratio enabled us to substantially increase the content of B units in conjugates (IVd)– (IVl); in (IVk) and (IVl), it reached 100%. The content of C units in conjugates (IVg)–(IVj), substantially increases under these conditions (table). The content of B units in conjugates (IVa) and (IVb) resulting from the modification of heparin by aminophenols (IIa) and (IIb) markedly decreases. By analogy with the known reaction of phenols with carbodiimides [14], it may be assumed that carbodiimide (III) is attached to phenol hydroxyl groups of amide residues in conjugates (IVa) and (IVb). This results in the formation of units D containing O-isoureidophenylamide groups. Units D are also present in the heparin conjugates with aminosalicylic acids (IIc) and (IId); however, their content in compounds (IVc) and (IVd) is substantially lower (table), which is probably due to the presence of carboxyl group, which reduces the reactivity of phenol hydroxyl toward carbodiimide (III). The 1H NMR spectra of conjugates (IVa)–(IVd) in the region of δ ~3 ppm exhibit two singlets corresponding to different åÂ2N-groups, which can be located in both C and D units. The total content of C and D units in conjugates (IVa)–(IVd) was determined from the ratio of total intensities of signals from methyl protons of åÂ2Ngroups (δ ~3 ppm) and anomeric protons (δ 5.1–5.5 ppm) calculated per one proton. To assign the resonances of åÂ2N-groups to either C or D units, conjugates (IVa)–(IVd) were treated with an aqueous solution of sodium borohydride. The conjugates (VIIa)– (VIId) with units E substituted for units C were obtained; the contents of E units in them were determined by the anthrone method. As a result, it was established that conjugates (IVa), (IVc), and (IVd) do not contain C units and both signals of Me2N-groups corre-
spond to two regioisomeric O-isoureidophenylamide groups in units D. On the other hand, (VIIb) was found to contain 27% E units, which corresponds to the same content of C units in conjugate (IVb). Consequently, one of the two signals from methyl protons of the Me2N-groups in the 1H NMR spectra of conjugate (IVb) should be assigned to O-isoureidocarbonyl groups. The interaction of heparin with a threefold excess of amine and carbodiimide allowed us to increase the content of B units only in conjugates (IVe), (IVf), and (IVj); in this case, the content of D units in conjugates (IVa)–(IVd) increased (table). An anomalously high value (140%) for the content of arylamide groups (units B) in conjugate (IVf) is probably explained by the formation of oligomeric arylamide groups. This follows from an enhanced intensity of signals of aromatic protons in the 1H NMR spectrum, an increased number of signals from the carbon atoms of aryl groups in the 13C NMR spectrum of conjugate (IVf) (δ 123.7, 124.0, 131.2, 132.5, 142.5, 143.8 ppm), an increased solubility in ethanol, and a bathochromic shift in the absorption maxima in UV spectra with increase in the content of B units in the conjugates (IVf) (cf. [15], λmax 291 nm for the conjugate with 53% B units, λmax 298 nm for the conjugate with 83% B units, and λmax 317 nm for the conjugate with 140% B units; see table). This suggests that, along with p-carboxyphenylamide groups, B units contain carboxy(oligophenylcarboxamido)phenylamide groups with the number of p-phenylcarboxamide groups 1 and 2. Note that, in addition to conjugate (IVf), the reaction of heparin with compound (IIf) is accompanied with the formation of oligomers of p-aminobenzoic acid (λmax of 284 nm). The reaction of hyaluronic acid with p-aminobenzoic and aminosalicylic acids was not accompanied with the formation of oligomeric arylcarboxamide residues and O-isoureidoarylamide groups in the corresponding conjugates [7], and, therefore, it may be suggested that the formation of these groupings in modified heparins is caused by the presence of strongly acidic sulfate groups in the polysaccharide. EXPERIMENTAL NMR and 13C NMR spectra were recorded in D2O on a Bruker AMX-300 spectrometer (working frequency 300.13 MHz for 1H NMR and 75 MHz for 13C NMR) using sodium 3-(trimethylsilyl)-1-propanesulfonate as internal standard. UV spectra were measured on a Specord M-40 spectrophotometer. Solution pH values were monitored by a pH-340 pH meter. o-Aminophenol, 5-aminosalicylic, anthranilic, and p-aminobenzoic acids were of analytical purity grade; Streptocid, Anesthesin, and Novocaine were of pharmacopoeial purity grade; Sulfacyl-sodium and isoniazide were isolated from aqueous solutions of the corresponding preparations; anthrone was purified by recrystallization from ethanol; and 1-ethyl-3-[3-(dime1H
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317 (301)
268 and 306 (265 and 299)
259 and 303 (320)
(IVc)
(IVd)
(IVe)
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* ** *** ****
–
(IVl)
8.2 br s, 8.9 br s
7.5 br s, 7.7 br s
7.8 br s, 8.1 br s
7.8 br s, 8.1 br s
7.8 br s, 8.0 br s
7.9 br s, 8.0 br s
7.8 br s, 8.0 br s
7.3 br s, 7.6 br s, 8.1 br s, 8.5 br s
55
25
21
25
47
61
47
48
62
52
25
25
A**
45
75
66
70
48
36
53
52
36
48
63
72
B
0
0
3
5
5
3
0
0
0
0
0
0
C
–
–
–
–
–
–
–
–
2
0
12
3
D
(I) : (II) : (III) = 1 : 1 : 0.75
0
0
10
0
0
0
17
25
29
36
0
4
A**
100
100
81
90
75
83
83
75
60
44
46
44
B
0
0
9
10
25
17
0
0
0
0
27
0
C
–
–
–
–
–
–
–
–
11
20
27
52
D
(I) : (II) : (III) = 1 : 1 : 1.5
0
0
0
0
0
0
0
14
25
4
0
4
A**
100
100
90
90
75
82
140***
86
61
45
0
24
B
0
0
10
10
25
18
0
0
0
0
30
0
C
–
–
–
–
–
–
–
–
14
41
70
72
D
(I) : (II) : (III) = 1 : 3 : 3
In parentheses are λmax values for compounds (IIa)–(IIl) (pH 7). The content of units A is defined as difference (100% minus total content of units B, C, and D). The anomalously high content of units B is due to the formation of oligomeric phenylenecarboxamides. The 1H NMR spectrum of conjugate (IVh) also shows a characteristic signal with δ 2.0 ppm (s, CH3Co), that of conjugate (IVi), signals with δ 1.5 (t, J 14.1 and 7.0 Hz, CH3CH2O) and 4.5 ppm (m, CH3CH2O), that of conjugate (IVj), a signal with δ 1.4 ppm (t, J 14.1 and 7.0 Hz, CH2CH3), and that of conjugate (IVk), signals with δ 2.4 (s, C=CCH3) and 3.4 ppm (s, NCH3).
266 (263)
–
(IVk)**** 262 (245 and 281)
2.94 s
(IVj)**** 273 (291)
(IVh)**** 263 (260) 2.97 s
2.95 s
258 (259)
(IVg)
(IVi)**** 271 (285)
2.94 s
291, 298, 317 (272)
(IVf)
–
–
2.95 s, 7.2 br s, 7.3 br s, 7.9 br s 3.00 s
2.94 s, 7.1 br s, 7.7 br s, 8.0 br s; 7.1 br s, 3.02 s 7.6 br s, 7.9 br s
281 and 308 (284) 2.99 s, 7.1 br s, 7.3 br s, 7.6 br s, 7.7 br s; 3.04 s 7.6 br s, 7.7 br s
(IVb)
HAr
3.02 s, 7.1 br s, 7.4 br s; 7.1 br s, 7.5 br s, 3.06 s 7.9 br s
Me2N
Chemical shifts, ppm
260 (297)
UV spectra: (H2O, λmax, nm)*
(IVa)
Product
Spectral characteristics of conjugates (IVa)–(IVl) and the content (%) of units A–D in their structures
CHEMICAL MODIFICATION OF HEPARIN 475
476
PONEDEL’KINA et al.
thylamino)propyl]carbodiimide, 4-aminosalicylic acid, p-aminophenol, and 4-aminoantipyrine were supplied by Aldrich. Heparin (I) from a pharmacopoeial preparation was additionally purified by column anion-exchange chromatography using a highly permeable fibrous DEAE cellulose. Impurities were eluted with 0.6 N NaCl solution; their complete elimination was monitored by a decrease in the intensity of eluate absorption at 220 nm to a minimum value. Heparin was eluted with 0.9–1 N NaCl and precipitated from the eluate with a fourfold volume of ethanol. The precipitate was separated by centrifugation, washed with ethanol and then with diethyl ether, and dried at a temperature no higher than 60°C and a reduced pressure. Heparin was obtained as a white powder; the content of sulfur was 13.9–14.6%. The test for protein (according to Lowry) was negative. The ratio of units with NHSO3ç and NHAc-groups in heparin was 70–80/20–30 [determined from the ratio of intensities of signals from methyl protons of N-acetyl groups (δ 2.2 ppm) and anomeric protons of heparin]. 1H NMR*:4 2.2 (MeCON), 3.4 (H2 N), 3.8 (H3 N), 3.9 (H4 N), 4.1 (H5 N), 4.2 (H4 I), 4.3 (H3 I), 4.5 (H2 I), 4.4-4.6 (H6 N), 4.9 (H5 I), 5.3 (H1 I), 5.5 (H1 N); 13C NMR: 177.2 (C6 I), 102.0 (C1 I), 99.4 (C1 N), 78.7 (C2 I, C4 I and C4 N), 72.3-71.9 (C3 N, C3 I, C5 I, C5 N), 69.1 (C6 N), 60.6 (C2 N), 24.6 (MeCON) (cf. [16]). Modification of heparin. a. The pH value of a mixture of heparin (I) (91.8 mg, 0.15 mmol per disaccharide unit) with the corresponding compound (IIa)– (IIh) or (IIj)–(IIl) (0.15 mmol) in H2O (15 ml) was adjusted to 4.7–4.8 with 0.1 N NaOH [for the reaction with (IIc) and (IId)] or 0.1 N HCl [for the reaction with (IIa), (IIb), (IIe)–(IIh) and (IIj)-(IIl)]. Carbodiimide (III) (21.5 mg, 0.11 mmol) was then added at 20–22°C under vigorous stirring; the pH 4.7–4.8 was maintained by titration with 0.1 N HCl. After 0.5 h, 0.1 N NaOH (to pH 7), a saturated NaCl solution (2-3 ml), and ethanol (60−65 ml) cooled to 0°C were successively added to the reaction mixture cooled to 0°C. The precipitate was separated by centrifugation and dissolved in 6% NaCl (10 ml), and ethanol (40 ml) was added. The precipitate was centrifuged, washed with ethanol (10 ml × 3) and diethyl ether (10 ml × 3), and dried at ≤60°C and a reduced pressure. The corresponding conjugates (IVa)– (IVh) or (IVj)–(IVl) (90–95 mg) were obtained as white water-soluble powders with a rose or yellow tint. b and c. Similarly, the reaction was carried out at the ratio of reagents (b) (I) : (II) : (III) 1 : 1 : 1.5 and (c) (I) : (II) : (III) 1 : 3 : 3 to give the corresponding conjugates (IIa)–(IIh) and (IIj)–(IIl). d. Compound (IIi) (24.8 mg, 0.15 mmol) was dissolved in ethyl alcohol (3–5 ml), poured into a solution 4 Designations in 1H NMR and 13C NMR spectra of heparin (I): (H
N) and (H I) and (C N) and (C I) are the signals of hydrogen and carbon atoms in 2-deoxy-2-sulfamido-D-glucopyranose 6-sulfate (N) and L-idopyranosyluronic acid 2-sulfate (I) residues.
of heparin (91.8 mg, 0.15 mmol per disaccharide unit) in 10–12 ml of water, and 0.1 N HCl was added to adjust pH to 4.7–4.8. Then carbodiimide (III) (21.5 mg, 0.11 mmol) was added; the pH value was maintained at 4.7-4.8 with 0.1 N HCl. The mixture was kept for 0.5 h at 20–22°C and treated as described above to give 92 mg of conjugate (IVi). e and f. Similarly, the reaction was carried out at the ratio of reagents (e) (I) : (IIi) : (III) 1 : 1 : 1.5 and (f) (I) : (IIi) : (III) 1 : 3 : 3 to give the corresponding conjugates (IVi). g. A solution of heparin (I) (91.8 mg, 0.15 mmol per disaccharide unit) in (I) (15 ml) was adjusted to pH 4.7–4.8 with 0.1 N ICl. Carbodiimide (III) (21.5 mg, 0.11 mmol) was then added under vigorous stirring at 20–22°C. The pH value of the reaction mixture was maintained at 4.7–4.8 with 0.1 N HCl. After 0.5 h, 0.1 N NaOH (to pH 7), a saturated NaCl solution (4– 5 ml), and ethanol (60 ml) were successively added to the reaction mixture, which was then cooled to 0°C. The precipitate was separated by centrifugation, dissolved in 6% NaCl (10 ml), and ethanol (40 ml) was added. The precipitated solid was centrifuged, successively washed with ethanol (10 ml × 3) and ether (10 ml × 3), and dried at ≤60°C and a reduced pressure. Adduct (V) (90 mg) was obtained as a water-soluble white powder; the content of A and C groups was 80 and 20%, respectively. The 1H NMR spectrum corresponded to that reported in [12]. 13C NMR: 177.2, 172.8, 157.6, 102.0, 100.0, 77.7, 72.4–71.9, 68.4, 60.3, 57.5, 45.5, 38.6, 26.7, 25.7, 16.2. Reduction of (IVa)–(IVl) and (V) with sodium borohydride. Dry NaBH4 (70–80 mg) was added in portions to a solution of the corresponding conjugate (IVa)–(IVl) or (V) (2–3 mg) in water (2 ml) at room temperature under vigorous stirring on a magnetic stirrer. The pH value of the solution was maintained at 7– 8 with 4 N HCl and monitored using a universal indicator paper. After 1.5 h, the reaction mixture was quantitatively transferred into a pycnometer of 3–4-ml volume; the volume was brought to mark by water. An aliquot (1 ml) was taken and transferred to a test tube, and 3 ml of the anthrone reagent (20 mg of anthrone in 100 ml of 80% H2SO4) was added. The mixture was heated for 5 min on a boiling water bath, cooled to room temperature, and the absorption of the solution of the corresponding product (VIIa)–(VIIl) or (VI) at 630 nm was measured (cf. [13]). A sample of the corresponding conjugate (IVa)–(IVl) or (V) that was not subjected to borohydride reduction was used as a reference solution to exclude the influence of uronic acids (cf. [17]). The content of E units in compounds (VIIa)– (VIIl) and (VI) was determined from a calibration plot obtained for galactose solutions of known concentration.
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CHEMICAL MODIFICATION OF HEPARIN
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