232
`
`Bioconjugate Chem. 1991, 2, 232-241
`Chemical Modification of Hyaluronic Acid by Carbodiimides
`Jing-wen Kuo,* *-* David A. Swann,* and Glenn D. Prestwich*·*
`Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, and
`MedChem Products, Inc., 232 West Cummings Park, Woburn, Massachusetts 01801. Received February 14,1991
`
`Hyaluronic acid (HA) is a linear polysaccharide with repeating disaccharide units of glucuronic acid
`and IV-acetylglucosamine and is found in the extracellular matrix of connective tissues. Reaction of
`2 X 106) with EDC at pH 4.75, either in the
`high molecular weight sodium hyaluronate (NaHA, MW ~
`presence or absence of a primary diamine, gave the N-acylurea and O-acylisourea as NaHA-carbodi-
`imide adducts. None of the expected intermolecular coupling with the amine component was observed.
`On the basis of this new observation, this method for chemical modification of HA was used in conjunction
`with new synthetic carbodiimides to prepare HA derivatives bearing lipophilic, aromatic, cross-linked,
`and tethered functional groups. The degree of conversion to NaHA-acylurea products appears to
`depend upon both the characteristics of various carbodiimides and the conformational structure of
`NaHA.
`
`INTRODUCTION
`Hyaluronic acid (HA), a naturally occurring linear
`polysaccharide, is composed of repeating disaccharide units
`of glucuronic acid and IV-acetylglucosamine (2,2). Both
`monosaccharides have the /3-D-anomeric configuration at
`C-l. The linkage from glucuronic acid to N-acetylglu-
`cosamine is (l-»3) and the linkage from N-acetylglu-
`cosamine to glucuronic acid is (l-*-4). The nomenclature
`for this repeating disaccharide unit is [-*4)-0-(/3-D-glu-
`copyranuronosyl)-(l->3)-0-(2-acetamido-2-deoxy-/3-D-glu-
`copyranosyl)-(l->] (2) (Figure 1). HA is widely distributed
`in animal tissues, present in high concentrations in syn-
`ovial fluid and the vitreous body of the eye, and in loose
`connective tissues of rooster comb, umbilical cord, and
`dermis (3). All HA molecules are thought to have the
`same primary structure, but differences occur
`in the degree
`of polymerization of the HA polysaccharide chains ob-
`tained from different tissues (4). Sodium hyaluronate
`(NaHA) is characterized by its large hydrodynamic volume
`(5). NaHA with a molecular weight of 1 million has an
`It absorbs water,
`intrinsic viscosity of 3000 mL/g (6).
`cushions cells, and lubricates the soft tissues of joints.
`The wide application of the purified, high molecular weight
`HA in eye surgery is an example of the utility of HA as
`a noninflammatory, viscoelastic biomaterial (7).
`Studies on the chemical modification of hyaluronic acid
`have been mainly concerned with its cross-linking and
`coupling. Divinyl sulfone, bisepoxides, formaldehyde, and
`bishalides have been used to cross-link HA to produce
`highly swollen gels or virtually insoluble, plastic materials,
`depending upon the degrees of cross-linking (So,b, 9,10,
`11a). Coupling reactions, on the other hand, can also alter
`the properties of HA. For example, extensive esterifica-
`tion of HA with monofunctional organic halides can
`produce water-insoluble films (lib). These chemically
`modified HA are thought to have surgical and medical
`value as long-lasting biomaterials, and as potential drug-
`delivery vehicles.
`The generation of a free amino group on HA for further
`coupling reaction has been a subject of much interest, both
`at the polymer and oligosaccharide level. Preparation of
`an alkylamine derivative of HA oligosaccharide modified
`
`* Author to whom correspondence should be addressed.
`f State University of New York.
`* MedChem Products.
`
`Figure 1. Hyaluronic acid is a linear polysaccharide, with
`repeating disaccharide units of glucuronosyl-ß-1,3-N-acetylglu-
`cosamine linked by 0-1,4-glycosidic bonds.
`at its reducing end was used for radioactive labeling (22).
`For high molecular weight HA, alkaline N-deacetylation
`of its glucosamine moiety produced a free amino group on
`HA polymer chain, but concomitant degradations of HA
`via /3-elimination in the glucuronic acid moiety was
`observed (23). Hydrazinolysis of high molecular weight
`HA (>2 x 106 Da) was performed at 100 °C, to give a
`partially N-deacetylated HA, but the molecular weight
`was decreased by 1 order of magnitude (14).
`It was reported that the carbodiimide-catalyzed reaction
`of HA with monofunctional amines such as glycine meth-
`yl ester led to the formation of an amide linkage (25).
`Since carbodiimide-carboxylate reactions could be per-
`formed in aqueous solutions under mild conditions, we
`chose to explore carbodiimide-promoted coupling of HA
`carboxylic acid group with simple aliphatic diamines.
`When we began our studies using HA, difunctional amines,
`and carbodiimides, we expected to obtain an undegraded
`HA derivative with a free amino group. This was not
`realized, however, and only products from coupling of the
`carbodiimide to HA were observed. We now report the
`evidence for this reaction and the exploitation of this
`unexpected observation to prepare functionalized and
`cross-linked derivatives of HA.
`EXPERIMENTAL PROCEDURES
`General Experimental.
`Infrared spectra were taken
`on a Perkin-Elmer 1430 spectrophotometer. Only im-
`portant, diagnostic peaks are reported. UV-vis spectra
`were taken on a Perkin-Elmer Lambda 4B spectropho-
`tometer. Gas chromatography (GC) was performed on a
`Varían 3700 gas chromatograph (programmable temper-
`ature control) equipped with a flame ionization detector
`(FID), connected to helium carrier gas and interfaced to
`a HP 3380A integrator. The column used was DB-5 meg-
`abore (15 m x 0.5 mm) fused silica, at 30 psi He pressure.
`Thin-layer chromatography (TLC) was performed with
`
`1043-1802/91/2902-0232302.50/0
`
`© 1991 American Chemical Society
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`

`

`Modlficaton of Hyaluronic Acid by Carbodiimldes
`Macherey-Nagel Polygram Sil G/UV254 (40 x 80 mm, 0.25-
`mm silica gel) plastic TLC plates. Spots were visualized
`under UV light and / or under visible light by spraying the
`TLC plate with a ninhydrin spray solution (0.5% w/v of
`ninhydrin in water-saturated 1-butanol) or by dipping the
`TLC plate into a phosphomolybdic acid (10% w/w solution
`in EtOH) followed by heating with a heat gun. Flash
`column chromatography was performed under nitrogen
`pressure on Merck silica gel G (400-230 mesh). Low-
`resolution electron-impact mass spectra were obtained by
`using a Hewlett-Packard Model 5980A mass spectrometer
`interfaced to a HP 5710A GC. Only molecular ions (M+)
`are listed. NMR spectra were obtained on a GE QE-300
`spectrometer operating at 75 MHz for 13C and 300 MHz
`for  . Unless otherwise specified the chemical shifts for
`compounds dissolved in organic solvents are reported as
`ppm ( ) with CDCI3 as standard. Chemical shifts of
`modified HA are reported as ppm ( ) with 3-(trimeth-
`ylsilyl)-l-propanesulfonic acid sodium salt as the standard.
`The pH of the HA or modified HA samples was raised to
`about 14 by adding NaOD. The following abbreviations
`are used for peak multiplicities: s, singlet; d, doublet; t,
`triplet; q, quartet; m, multiplet. Concentration of HA
`was measured by the carbazole assay using glucurono-
`Intrinsic viscosity (IV) of HA
`lactone as standard (16).
`and modified HA was measured with Cannon-Ubbelohde
`semimicro dilution type viscometer (size 75) at 37 °C. Hy-
`aluronic acid was provided by MedChem Products, Inc.,
`as its sodium salt, NaHA, which was purified from rooster
`comb by modifications of procedures described elsewhere
`(17). Unless otherwise stated, the molecular weight of
`HA used was from 1.5 to 2 million. Denatured ethanol
`containing 5% of methanol and 5% of 2-propanol was
`used in the purification of HA and its derivatives. The
`ninhydrin test was used to quantify the free primary amino
`groups of the amine-functionalized HA and D-glucosamine
`was used as the standard. The ratios of the reagents were
`the ratios of the molar equivalents based on the reacting
`In the case of NaHA, it was the molar
`functional groups.
`equivalents of its dissacharide units.
`Procedures for Chemical Reactions JV-(Benzylox-
`ycarbonyl)- 1,6-hexanediamine (8). Toa solution of 1,6-
`hexamethanediamine (11.6g, 100 mmol) inSOmLofCHCls
`was added dropwise at room temperature a solution of
`benzyl chloroformate (1.83 g, 10 mmol) in 50 mL of CHCI3.
`The addition proceeded for 1 h, during which a white
`precipitate of
`formed. The
`the amine chloride was
`agitation was continued at room temperature for 5 h, the
`reaction mixture filtered, and the filtrate washed with
`water. The removal of the excess unreacted diamine was
`monitored by silica gel thin-layer chromatography (CHCI3/
`MeOH/EtsN = 90/10/5). The ninhydrin spray showed
`the disappearance of the purple spot at the bottom line
`on TLC plate, indicative of the complete removal of the
`unreacted 1,6-hexanediamine. The dried residue was
`weighed as 2.0 g. The crude product (1.2 g) was purified
`by column chromatography (silica gel, 0.8% MeOH, 5%
`EtsN in CHCI3) to provide 486 mg of the pure product
`  1.26-1.43 (8  ), 2.60 (t, J
`  NMR (CDCI3):
`(40.5%).
`= 6 Hz, 2 H, CH2NH2), 3.11 (m, 2 H, -CH2NHCOO-), 5.08
`(s, 2 H, Ctf2Ph), 7.35 (m, 5 H, Ph). 13C NMR (CDCI3):
`  26.48 and 29.93 (-CH2-) 33.60 (CH2NH2), 41.03 (-CH2-
`NHCOO-), 66.58 (CH2Ph), 128.03,128.47, and 136.70 (Ph),
`156.38 (C=0).
`JV-[3-(Dimethylamino)propyI]-jV-[6-[(benzyloxy-
`carbonyl)amino]hexyl]thiourea (9). To a solution of
`N-(benzyloxycarbonyl)-l,6-hexamethylenediamine (8) (50
`mg, 0.20 mmol) in 5 mL of CHCI3 was added isothiocy-
`
`233
`Bioconjugate Chem., Vol. 2, No. 4, 1991
`anate 7 (40 mg, 0.28 mmol) predissolved in 5 mL of CHCI3.
`After stirring for 6 hat room temperature, the solvent was
`evaporated to give 77 mg of the crude product (91 %, GC).
`  NMR (CDCI3):
`  1.27-1.68 (10 H, -CH2-), 2.19 (6 H,
`R-NAÍe2), 2.30-2.39 (2 H, CH2NMe2), 3.11 (2 H, -CHr
`NHCOO-), 3.13-3.52 (4 H, -CH2NH-), 5.00 (s, 2 H, CH2-
`Ph), 7.27 (m, 5 H, Ph). 13C NMR (CDC13):
`  26.02,28.64,
`and 29.54 (-CH2-), 40.59,44.31 (NMe2), 44.41,44.47,66.20
`(CH2Ph), 127.70,127.75,127.84,128.22, and 136.45 (Ph),
`156.24 (C=0), 181.80 (CHS).
`JV-Ethy 1-JV-octylthiourea (5). The experimental pro-
`cedure of the synthesis of 9 was adopted. Thus octylamine
`(402 mg, 3 mmol) and ethyl
`isothiocyanate (270 mg, 3
`mmol) gave 660 mg of thiourea 5, which was 97.5%
`  NMR (CDC13):
`  0.86 (t, J = 6.2
`homogenous (GC).
`Hz, 3 H, (CH2)7Me), 1.24 (t, 3 H, CH^),
`1.35-1.47 (10
` ), 1.58 (m, 2 H, -CH2CH2NH-), 3.38-3.45 (4 H, -CH2Cff2-
`NH-), 5.77-5.80 (2  , NH). 13C NMR (CDC13):
`  13.97,
`14.20,22.51, 26.81, 28.91, 29.05,29.11,39.08,44.34,44.42,
`181.12 (CHS).
`JV-Ethyl-JV-[6-[(benzyloxycarbonyl)amino]hexyl]-
`thiourea (12). The experimental procedure of the
`synthesis of 9 was adopted. Thus N- (benzyloxycarbonyl)-
`1,6-hexamethylenediamine (8) (100 mg, 0.4 mmol) and
`ethyl isothiocyanate (40 mg, 0.44 mmol) were combined
`in CHCI3 to give 150 mg of the crude product with a purity
`  NMR (CDC13)   1.18 (t, J = 7.2 Hz, 3
`of 86% (GC).
`H, CH3), 1.32 (4  ), 1.45-1.57 (4  ), 3.14 (t, J = 6.6 Hz,
`2 H, -CH2NHCOO-), 3.41 (4 H, -Ctf2NH-), 4.96 (2 H,
`Ctf2Ph), 7.26-7.33 (5H,Ph). 13C NMR (CDC13):   14.17,
`25.81, 25.94, 28.64, 29.56, 38.94, 40.47, 43.91,66.39 (CH2-
`Ph), 127.66,127.90,128.30, and 136.28 (Ph), 156.52 (C=0),
`181.19 (CHS). MS: m/e 337.1 (M+).
`JV-Ethyl-)V’’-(6-cyanohexyl)thiourea (15). The ex-
`perimental procedure of the synthesis of 9 was adopted.
`Thus 6-aminohexanenitrile (2.856 g, 22.8 mmol) and ethyl
`isothiocyanate (2.05 g) were combined in CHCI3 to give
`  NMR
`4.90 g of thiourea (15) (89.2% purity by GC).
`(CDCI3):   1.23 (t, J = 7.2 Hz, 3 H, CH3), 1.52-1.73 (6 H),
`2.37 (t, J = 7.0 Hz, 2 H, Ctf2C=N), 3.41-3.51 (4 H, -CHr
`NH-), 5.77-5.80 (2 H,-NJf-). 13C NMR (CDC13):   13.68,
`13.73,16.39,24.30,25.19,27.64,38.45,43.27,119.32 (CssN),
`180.51 (O-S).
`l,6-Hexamethylenebis(ethylthiourea) (23). To a
`solution of 1.351 g of ethyl isothiocyanate (15 mmol) in 10
`mL of CHClg was added dropwise 874 mg of 1,6-diami-
`nohexane (7.5 mmol) predissolved in 10 mL of CHCI3.
`The agitation continued for 2 h at room temperature as
`the reaction mixture gradually turned milky and viscous.
`The formed white solid was not soluble in hexane or Et20,
`poorly soluble in ethyl acetate and CHCI3, and freely
`soluble in acetone and MeOH. Recrystallization of the
`crude product in MeOH improved its purity from 40% to
`over 75% (GC), and it was identified by 1H NMR as bisthio-
`  NMR (CDC13):   1.19 (6 H, CH3), 1.22 (4 H),
`urea 23.
`I. 56 (4 H), 3.46 (8 H, -CH2NH-), 6.26-6.32 (4  , NH),
`This material was used without further purification.
`p-Phenylenebis(ethylthiourea) (26). To a solution
`of 811 mg of p-phenylenediamine (7.5 mmol) in 15 mL of
`acetone was added 1.351 g of ethyl
`isothiocyanate (15
`mmol). The reaction mixture was stirred 12 hr at room
`temperature and the formed precipitate was isolated by
`filtration, washed with cold methanol, and dried. The
`light brown precipitate was soluble in MeOH and DMSO,
`but not soluble in acetone, benzene, or CHCI3. The solid
`product (510 mg) showed UV absorbance on silica gel TLC
`(Rf 0.6, CHsCl/MeOH/EtsN = 90/10/5), and was homo-
`  NMR (DMSO-de):
`  1.10 (t, J = 7.2
`geneous by GC.
`
`

`

`234 BloconJugate Chem., Vol. 2, No. 4, 1991
`Hz, 6 H, CH3), 3.45-3.49 (q, J = 6.1 Hz, 4 H, -CH2NH-),
`7.31 (s, 4 H, Ph).
`Ethyl[6-[(benzyloxycarbonyl)amino]hexyl]carbo-
`diimide (13). To a suspension of mercuric oxide (100 mg)
`in dry acetone (15 mL) was added thiourea 12 (77 mg)
`predissolved in 5 mL of dry acetone and refluxed for 3 h.
`Black mercuric sulfide was formed and was removed by
`filtration through Celite, dried (MgSOJ, and concentrated
`to give 60 mg of the product with a purity of 93% (GC).
`  1.11-1.45 (8  ), 3.06-3.16 (6 H,
`  NMR (CDCla):
`-CH2N=C=N- and CH2NHCOO), 4.98 (2 H, Cff2Ph),
`7.24 (5 H, Ph). 13C NMR (CDCI3):
`  16.58, 26.13, 26.29,
`29.73, 31.00, 40.79, 41.34, 46.44, 66.38 (Cif2Ph), 127.91,
`128.34, and 136.50 (Ph), 156.30 (C=0) IR: 2120 (s,
`-N=C=N-) cm-1. MS: m/e 303.2 (M+).
`[3-(Dimethylamino)propyl][6-[(benzoyloxycarbon-
`yl)amino]hexyl]carbodiimide (10) was prepared as
`described for carbodiimide 13, except that the crude
`product was chromatographed with a column packed with
`silica gel (CHCla/EtaN = 95/5). Thus, 77 mg of the
`thiourea 3 gave 15 mg of pure carbodiimide 6. 1H NMR
`  1.24-1.74 (10 H), 2.22 (s, 6 H, N(CH3)2, 2.34
`(CDCla):
`(t, J = 7.5 Hz, 2  ), 3.16-3.26 (6 H, -CH2N=C=N- and
`CH2NHCOO), 5.08 (s, 2 H, benzyl methylene), 7.34 (5 H,
`benzene). 13C NMR (CDC13):   26.23,26.42,29.28,29.89,
`31.11,44.80,45.47,46.51,66.55 (CH2Ph), 128.04 and 128.46
`(Ph), 156.38 (C=0). IR: 2126 (s, -N=C=N-) cm"1. MS:
`m/e 360 (M+).
`Ethyltridecylcarbodiimide (3) was prepared as de-
`scribed for carbodiimide 13. Thus 320 mg of N-ethyl-
`N'-tridecylthiourea (2) gave 180 mg of carbodiimide 3,
`which was 97.4 % homogeneous by GC. 1H NMR (CDC13):
`  0.87 (t, 3 H, CH3(CH2)i2), 1.21-1.33 (20  , 1.57 (2 H,
`-CH2CH2N=C=N-), 3.17-3.24 (4 H, -CH2N=C=N-).
`IR: 2120.1 cm'1 (s, -N=C=N-).
`Ethyl-3-octylcarbodiimide (6) was prepared as de-
`scribed for carbodiimide 13. Thus 600 mg of N-ethyl-
`N'-octylthiourea (5) gave 434 mg of carbodiimide 6, which
`  NMR (CDC13):
`  0.87 (t, 3
`was 97.2% homogeneous.
`H, CH3(CH2)7), 1.20-1.26 (12  ),
`1.56 (2 H, -CH2-
`CH2N=C=N-), 3.19-3.24 (4 H, -CH2N=C=N-).
`IR:
`2128.5 cm’1 (s, -N=C=N-).
`Ethyl[6-(trifluoroacetamido)hexyl]carbodiimide
`(18) was prepared as described for carbodiimide 13. Thus
`108 mg of jV-ethyl-N'-[6-(trifluoroacetamido)hexyl]thio-
`urea (17) gave 90 mg of carbodiimide (18). GC showed the
`compound decomposed at elevated temperature. 1H NMR
`(CDC13):   1.23 (t, J = 7.2 Hz, 3 H, CHs), 1.25-1.58 (8 H),
`3.19-3.26 (m, 4 H, -CH2N=C=N-), 3.35-3.77 (2 H, CHr
`IR: 2128.5 cm"1 (s, -N=C=N-).
`NHCOCFs).
`l,6-Hexamethylenebis(ethylcarbodiimide) (24). The
`dehydrosulfurization of bisthiourea 23 with mercuric oxide
`in acetone was performed for 2 h, with the oil bath at 72
`°C. Higher temperature and longer reaction time may
`cause polymerization. Thus, the crude product after the
`reflux of 76.8 mg of bisthiourea 23 and 192 mg of HgO (in
`acetone) was purified by extracting with cold hexane to
`  NMR (CDC13):   0.87 (6  ), 1.21—
`give 22.5 mg of 24.
`I. 41 (4  ), 1.54-1.61 (4  ), 3.19-3.27 (m, 8 H,
`IR: 2124 cm-1 (s,-N=C=N-).
`-CH2N=C=N-).
`p-Phenylenebis(ethylcarbodiimide) (27) was pre-
`pared following the method above for biscarbodiimide 24,
`using 108 mg of the aromatic bisthiourea 26. The crude
`product was filtered through a silica gel column to give 45
`mg of aromatic biscarbodiimide 27, which was 97.3%
`homogeneous (GC). The compound was stored in a
` 
`refrigerator and used within 3 days of preparation.
`NMR (CDC13):
`  1.33 (t, 6 H, methyls), 3.41-3.44 (4 H,
`
`Kuo et al.
`
`-CH2N=€=N-), 6.98 (s, 4 H,-C4H4-).
`IR: 2120.1 cm"1
`(s, -N=C=N-). MS: m/z 214.1 (M+), intensity 100.
`General Procedure of Carbodiimide Reactions with
`NaHA. Sodium hyaluronate was dissolved in water to a
`concentration of ca. 4 mg/mL. For some reactions, as
`indicated below, amine was mixed with NaHA. The pH
`of the mixture was adjusted to 4.75 by 0.1 N HC1. The
`designed carbodiimide or commercially available carbo-
`diimide was dissolved in water or 2-propanol, depending
`upon its solubility. After mixing HA and carbodiimide
`solutions, a pH increase was immediately observed. The
`reaction was monitored by pH meter and 0.1 N HC1 was
`added dropwise to keep the pH at 4.75. The reaction was
`allowed to proceed for 2 h at room temperature. Then
`NaCl was added to make 5% w/vof the reaction mixture.
`Ethanol equal to 3 volumes of the reaction mixture was
`added and a stringy precipitate was obtained. The
`precipitate was redissolved for a second and third pre-
`cipitation, thus removing all the unreacted or produced
`small organic compounds. The final precipitate was
`dissolved in deionized water to a concentration of no more
`than 6 mg/mL and was then lyophilized. The NMR
`sample solution of NaHA or modified NaHA was prepared
`by dissolving 5-10 mg of the lyophilized product into 1
`mL of D20. The dissolution could be expedited by vor-
`texing the sample and adding NaOD (sample pH —14).
`The ratio of the reagents is defined as the molar equiv-
`alency ratio based upon the reaction of the carboxyl (HA)
`and carbodiimide functional groups.
`IV-Acylurea Products of HA and [3-(Dimethy!ami-
`no)propyl]ethylcarbodiimide (EDC) (la-e). The above
`procedure was adopted in making these JV-acylurea
`derivatives. The amount of reactants used and the
`analytical results are summarized as follows.
`la: The ratio of 1,6-diaminohexane/EDC was 3/1. The
`ratio of EDC/HA was 1/10. Thus NaHA (247.9 mg, 0.618
`mequiv) and EDC (11.98 mg, 0.062 mmol) gave 221 mg of
`the lyophilized product (85 % recovery). No amide linkage
`between HA and diaminohexane was formed. The purified
`product gave a negative ninhydrin test result. 1H NMR
`  1.08 (t, J = 7.2 Hz, 3 H, CH3CH2N), 1.64 (m, 2
`(D20):
` ), 2.18 (s, 6 H, (Ctf)3N), 2.34 (t, J = 7.2 Hz, 2  ), 3.11
`(m, 4 H, CH2N).
`lb: The ratio of 1,6-diaminohexane/EDC was 5/1. The
`ratio of EDC/HA was 1/10. Thus NaHA (246.4 mg, 0.615
`mequiv) and EDC 11.78 mg, 0.062 mmol) gave 226 mg of
`the lyophilized product (88% recovery). The analytical
`results of ninhydrin and   NMR are identical with those
`of la.
`lc: The ratio of 1,6-diaminohexane/EDC was 10/1. The
`ratio of EDC/HA was 1/10. Thus NaHA (247.7 mg, 0.618
`mequiv) and EDC (11.84 mg, 0.062 mmol) gave 232 mg of
`the lyophilized product (89.5% recovery). The analytical
`results of ninhydrin and 1H NMR are identical with use
`of la.
`Id: The ratio of 1,6-diaminohexane/EDC was 100/1.
`The ratio of EDC/HA was 1/10. Thus NaHA (122 mg,
`0.304 mequiv) and EDC (11.78 mg, 0.062 mmol) gave 114
`mg of the lyophilized product (85.4 % recovery). The nin-
`hydrin test result was negative.
`le: Thermally degraded NaHA with molecular weight
`of 60 000 was used in this reaction. The ratio of NaHA/
`1,6-diaminohexane/EDC was 1/1/1. Thus NaHA (264
`mg, 0.66 mequiv) and EDC (126.5 mg, 0.66 mmol) gave
`735 mg of the product which included le and NaCl co-
`precipitate. No amide linkage between NaHA and di-
`aminohexane was detected from NMR. The purified
`product showed less than 1% of the free amino group
`
`

`

`Modificaton of Hyaluronic Acid by Carbodllmktes
`  NMR showed no amine
`during the ninhydrin test.
`  NMR (D20):
` -methylene protons expected at   2.6.
`  1.12 (t, J = 7.2 Hz, CHs of N-acylurea), 1.21 (t, CHs of
`O-acylisourea) (  1.12/  1.21 > 4/1), 2.21 (s, (CH3)2N),
`2.37, 3.15 (m,  -CH2 of acylureas).
`O-Acylurea Product of NaHA and EDC (If). The
`above procedure was also followed. The reaction condition
`was the same as for le, except that NaHA was not thermally
`degraded. Thus NaHA (250.8 mg, 0.627 mequiv) and EDC
`(120.2 mg, 0.627 mmol, in 20 mL of water) gave 873 mg
`of the product which included If and NaCl coprecipitate.
`No amide linkage between NaHA and diaminohexane was
`detected from NMR. The purified product showed less
`than 1% of the free amino group during the ninhydrin
`  NMR (D20):   1.12 (t, CH3 of N-acylurea), 1.21
`test.
`(t, J = 7.2 Hz, CHs, of O-acylisourea) (  1.21/  1.12 > 5/1),
`2.21 (s, (CH3)2N), 2.38 (t, J = 7.2 Hz), 3.15 (m, ct-CH2 of
`acylureas).
`Acylurea Product of NaHA and Carbodiimide 6 (7a-
`c). The above procedure was followed. 2-Propanol (3
`mL) was used to dissolve the carbodiimide.
`7a: The ratio of carbodiimide/NaHA was 1/20. Thus
`NaHA (454 mg, 1.13 mequiv) and carbodiimide 6 (10.82
`mg, 0.056 mmol) gave 440 mg of 7a (94.6% recovery). The
`Intrinsic viscosity (IV):
`proton uptake was 0.02 mmol.
`2866 mL/g (starting NaHA, 2354 mL/g).
`7b: The ratio of carbodiimide/NaHA was 1/10. Thus
`NaHA (381 mg, 0.95 mequiv) and carbodiimide 6 (18.2
`mg, 0.095 mmol) gave 388 mg of 7b (95.6 % recovery). The
`IV: 2730 mL/g (starting
`proton uptake was 0.04 mmol.
`NaHA, 2354 mL/g).
`7c: The ratio of carbodiimide/NaHA was 1/5. Thus
`NaHA (477 mg, 1.19 mequiv) and carbodiimide 6 (45.6
`mg, 0.238 mmol) gave 465 mg of 7c (89.0% recovery). The
`IV: 2534 mL/g (starting
`proton uptake was 0.1 mmol.
`NaHA, 2354 mL/g).
`Acylurea Product of HA and Carbodiimide 3 (4a-
`c). The above procedure was followed. 2-Propanol (3
`In all three
`mL) was used to dissolved the carbodiimide.
`reactions, the proton uptake was less than 0.005 mmol.
`4a: The ratio of carbodiimide/HA was 1/20. Thus
`NaHA (444 mg, 1.1 mequiv) and carbodiimide 3 (15.4 mg,
`IV:
`0.055 mmol) gave 397 mg of 4a (89.5% recovery).
`2401 mL/g (starting NaHA, 2354 mL/g).
`4b: The ratio of carbodiimide/HA was 1/10. Thus
`NaHA (400 mg, 1.0 mequiv) and carbodiimide 3 (25.8 mg,
`IV: 2591
`0.1 mmol) gave 386 mg of 4b (90.8% recovery).
`mL/g (starting NaHA, 2354 mL/g). Additional proton
`to HA from 4b was not observed, but acid hy-
`resonance
`drolysis of 4b (pH = 1, boiling water bath, 4 h) produced
`  NMR signals as follows.
`  NMR (D20):   1.34,1.37,
`and 2.14 (br band, aliphatic protons), 3.12 (a-CH2 of ac-
`ylureas).
`4c: The ratio of carbodiimide/HA was 1/5. Thus NaHA
`(529 mg, 1.32 mequiv) and carbodiimide 3 (66 mg, 0.264
`IV: 2651
`mmol) gave 573 mg of 4c (96.3% recovery).
`mL/g (starting NaHA, 2354 mL/g).
`Acylurea Products of NaHA and Carbodiimide 10
`(11a,b). The above procedure was followed.
`11a: The ratio of carbodiimide/NaHA was 1/10. Thus
`NaHA (252 mg, 0.63 mequiv) and carbodiimide 10 (25.2
`mg, 0.063 mmol) gave 232 mg lyophilized product 1 la (92 %
`IV: 2760 mL/g (starting NaHA, 2354 mL/g).
`recovery).
`  NMR (D20):   1.25,1.58,2.12 (N(CJ¥3)2), 3.07 (a-CH2
`of acylureas), 7.39 (Ph).
`lib: The ratio of carbodiimide/NaHA was 1/5. Thus
`NaHA (126 mg, 0.315 mequiv) and carbodiimide 10 (25.2
`mg, 0.063 mmol) gave 119.1 mg of the lyophilized product
`
`236
`Bioconjugate Chem., Vol. 2, No. 4, 1991
`lib (94.5% recovery). The aqueous solution of lib was
`accidentally left stirring overnight without cooling. The
`IV:
`temperature of the solution was measured as 42 °C.
`2323 mL/g (starting NaHA, 2354 mL/g).   NMR (D20):
`  1.26,1.42,1.62,2.21,3.09 (a-CH2 of acylureas), 7.41 (Ph).
`Acylurea Product of NAHA and Carbodiimide 13
`(14). The above procedure was followed. The ratio of
`carbodiimide/NaHA was 1/5. Thus NaHA (90.6 mg, 0.225
`mequiv) and carbodiimide 13 (18 mg, 0.045 mmol) gave
`105 mg of the lyophilized product 14. The degree of
`coupling was measured as 16% by UV (240 nm). The
`IV: 2734 mL/g (starting
`proton uptake was 0.035 mmol.
`NaHA,2354mL/g). xHNMR(D20): 60.93(Cff3ofN-ac-
`ylurea), 1.33, 1.56, 3.15 (a-CH2 of acylureas), 7.43 (Ph).
`Acylurea Product of HA and Carbodiimide 18 (19).
`The above procedure was followed. The ratio of carbo-
`diimide/NaHA was 1/5. Thus NaHA (400 mg, 1.0 me-
`quiv) and carbodiimide 18 (60 mg, 0.2 mmol) gave 392 mg
`of 19 (85.2 % recovery). The proton uptake was 0.06 mmol.
`  NMR
`IV: 3115 mL/g (starting NaHA, 2966 mL/g).
`(D20):   1.11 (t, CHs of JV-acylurea), 1.21 (CHs of O-acyl-
`isourea) (  1.11/  1.21 > 5/1), 1.36, 1.49, 2.61 (CH2NH2
`from decomposition of CH2NHCOCF3 in NaOD), 3.12 (a-
`CH2 of acylureas).
`NaHA Cross-Linked by Biscarbodiimide 24 (25a,b).
`The general procedure of the carbodiimide reaction with
`NaHA was adopted. The ratio of carbodiimide/NaHA
`was 1/10.
`25a: The NaHA concentration was adjusted to 3.2 mg/
`mL and the carbodiimide concentration in 2-propanol was
`2.75 mg/mL. Thus NaHA (423 mg, 1.05 mequiv) and bis-
`carbodiimide 24 (11 mg, 0.05 mmol) gave 382 mg of 25a
`(88% recovery). The proton uptake was 0.04 mmol.
`During the purification process, the precipitated NaHA
`  NMR
`derivative was only partially soluble in water.
`  1.11 (t, CHs of N-acylurea), 1.36, 1.51, 3.12 (a-
`(D20):
`CH2 of N-acylurea).
`25b: The NaHA concentration was adjusted to 5.2 mg/
`mL and the carbodiimide concentration in 2-propanol was
`0.92 mg/mL. Thus NaHA (416 mg, 1.04 mequiv) and bis-
`carbodiimide 24 (11 mg, 0.05 mmol) gave 400 mg of 25b
`(93.6 % recovery). The proton uptake was 0.03 mmol. IV:
`2554 mL/g (starting NaHA, 2354 mL/g).
`NaHA Cross-Linked by Biscarbodiimide 27 (28a,b).
`The general procedure of the carbodiimide reaction with
`NaHA was adopted.
`28a: The NaHA concentration was adjusted to 4.2 mg/
`mL and the carbodiimide concentration in 2-propanol was
`1.5 mg/mL. The ratio of carbodiimide/HA was 0.18. Thus
`sodium hyaluronate (386 mg, 0.96 mequiv) and biscar-
`bodiimide 27 (18 mg, 0.084 mmol) gave 389 mg of 28a
`(96.4 % recovery). The proton uptake was 0.12 mmol. The
`dried polymer from the first precipitation swelled in 200
`volumes of water at 4 °C to form a cross-linked insoluble
`gel. The absorption of water appeared to reach an equi-
`librium after 5 days. Part of the gel was cut and stored
`in water at room temperature.
`28b: The NaHA concentration was adjusted to 4.2 mg/
`mL and the carbodiimide concentration in 2-propanol was
`0.89 mg/mL. The ratio of carbodiimide/HA was 0.10.
`Thus NaHA (404 mg, 1.01 mequiv) and biscarbodiimide
`27 (10.7 mg, 0.05 mmol) gave 393 mg of 28b (95 % recovery).
`IV: 3073 mL/g
`The proton uptake was 0.08 mmol.
`(starting NaHA, 2354 mL/g). XH NMR (D20):
`  1.09 (t,
`C/f3CH2 of N-acylurea), 1.19 (t, CH3CH2 of O-acylisourea)
`(  1.19/  1.09 > 2/1), 1.62, 3.04 (a-CH2 of acylureas).
`jV-Ethyl-JV-(6-aminohexyl)thiourea (16). To a so-
`lution of 1.327 g of nitrile 15 (6.6 mmol) in 50 mL of dried
`
`

`

`236 Bioconjugate Chem., Vol. 2, No. 4, 1991
`THF was added 600 mg of borane-dimethyl sulfide (7.92
`mmol). The reactor was heated to reflux, and dimethyl
`sulfide was distilled off. After 3 h, the mixture was cooled
`to room temperature and quenched with 3 mL of 1N HC1.
`Then pH was adjusted to 9 with 1N NaOH and saturated
`with K2CO3. Et20 was added to separate the layers. The
`aqueous layer was extracted twice with CHCI3, and the
`combined organics were concentrated to give crude thio-
`urea 16 (52%). The   NMR spectrum showed the ratio
`of the amine  -methylene protons over nitrile a-methyl-
`  1.21 (t, J
`ene protons as over 10/1. 1H NMR (CDCI3):
`= 7.2 Hz, 3 H, C/i3CH2NH), 1.24-1.64 (8 H, 2.66 (t, J =
`6.6 Hz, 2 H, CH2NH2), 3.44 (4 H, -CH2NH-), 5.92 (2 H,
`NH). 13C NMR (CDCI3):   14.29 (CH3CH2), 26.37,26.58,
`28.95, 33.34, 39.02, 41.87 (-CH2NH-), 44.20 (CH2NH2),
`181.25 (C=S).
`JV-Ethyl-jV'-[6-(trifluoroacetamido)hexyl]thio-
`(17). To an aqueous solution of 350 mg of thiourea
`urea
`16 (1.5 mmol) mixed with 5 mL of 0.2 M NaHCOs was
`added 480 mg of ethyl thiotrifluoroacetate (3 mmol). The
`reaction was continued at room temperature overnight
`and a white precipitate was formed. The product was
`isolated by ether extraction to give 317 mg of a white solid,
`which was 84.4 % homogeneous by GC.   NMR (CDCI3):
`  1.24 (t, J = 7.2 Hz, 3 H, CH3CH2), 1.37-1.40 (m, 4 H),
`1.58-1.64 (m, 4 H), 3.38 (t, J=6.6 Hz, 2 H, Ctf2NHCOCF3),
`3.47 (4 H, -CH2NH-). 13C NMR (CDC13):
`  14.18 (CH3-
`CH2) 25.99, 28.62,28.77,39.06, 39.65,44.08, 65.79,117.74
`(CF3), 157.22 (C=0), 181.22 (C=S).
`Detrifluoroacetylation of HA Derivative 19 to
`Amine-Functionalized HA (20a). HA-trifluoroaceta-
`mide (19) (12.6 mg) was treated for 14 h at room tem-
`perature and pH 11. Then 0.1 N HC1 was added to adjust
`the pH to 7.0-7.5. NaHA reaction products were pre-
`cipitated with ethanol as described above. The precipitate
`was collected after centrifugation. The ethanol precipi-
`repeated three times and the final aqueous
`tation was
`solution was lyophilized to give 11.9 mg of a white, fibrous
`material. To test the amount of free amino group attached
`to the NaHA polymer chain, an aqueous solution of the
`NaHA-trifluoroacetamide (19) (1 mg/mL) was prepared
`and a quantitative ninhydrin test was performed. The
`percentage ratio of the detected free amine over NaHA
`IV: 2865 mL/g (starting material (19), 3115
`was 3.1%.
`mL/g).
`Detrifluoroacetylation of HA Derivative 19 to
`Amine 20b. The experimental procedure for 20a was
`adopted except that the pH of the reaction mixture was
`adjusted to 11.5-12.0. Fourteen milligrams of the starting
`material (19) was used. The precipitate of 20b was less
`stringy than that of 20a. The lyophilized solid weighed
`19.3 mg. Apparently, NaCl coprecipitated with and
`adhered to the NaHA derivative and was therefore also
`collected. The percentage ratio of the detected free amine
`IV: 2286 mL/g (starting material
`over NaHA was 4.2%.
`(19), 3115 mL/g).
`RESULTS AND DISCUSSION
`Commercially available carbodiimides have been widely
`used for carboxyl activation, such as in protein modification
`(18, 20). EDC, [3-(dimethylamino)propyl]ethylcarbodi-
`imide, for example, is a water-soluble carbodiimide. The
`following mechanism of the carbodiimide reaction in
`protein modifications is generally accepted: carbodiim-
`ide reacts with carboxyl groups to form an unstable,
`intermediate O-acylisourea, which, in the absence of nu-
`cleophiles, rearranges to a stable N-acylurea “by way of
`In the presence of
`a cyclic electronic displacement” (21).
`
`RCOOH
`
`RCOCT + H+
`
` ·
`. R2"’
`o^n'H
`IR1"2
`
`r>n.c.nr2
`
`0—*N migration
`
`0
`
`Rl~.
`
`NH HN
`
`Kuo et al.
`
` 
`
`O
`
`/R,0,i
`
`N
`
`O-acylurea
`
`0
`
`,
`
`/R2 R/A„/ R
`
`  R
`
`NH
`
`amide
`N-acylurea
`Figure 2. Mechanism of reaction between carboxylic acid and
`carbodiimide with and without primary amines.
`a primary amine as nucleophile, the O-acylisourea for-
`mation is followed by a nucleophilic attack, forming an
`amide linkage between the amine and the acid (18) (Figure
`2). An acidic environment is needed to catalyze the
`reaction, presumably through the protonation of the car-
`bodiimide nitrogen. The reduced electron density at the
`carbodiimide central carbon is favorable to the nucleo-
`philic attack of the carboxylate anion. At pH 4.75, car-
`bodiimide nitrogens appear to be sufficiently protonated,
`while HA mainly exists as the carboxylate. The proton
`is not only a catalyst, but also a reagent. The stoichi-
`ometry of this process shows that one proton is consumed
`to form the urea compound. Thus the reaction can be
`monitored by the observed increase of pH and the
`consumption of hydrochloric acid during the reaction
`necessary to keep the reaction pH at 4.75.
`Amidation reactions of glycosaminoglycans and primary
`amines catalyzed by carbodiimides have been reported.
`One example by Danishefsky and Siskovic is the reaction
`between glycine methyl ester (the amine component) and
`hyaluronic acid. The mechanism of the reaction was
`reported to be the same as mentioned above, i.e., the
`“condensation of the uronic acid moiety with EDC to form
`the O-acylurea followed by the displacement of the
`substituted urea by the amino group” (15). Similarly, we
`attempted to use diaminohexane and EDC to build an
`amide linkage between HA and the diamine.
`The concentration of NaHA aqueous solution was
`adjusted to ca. 4 mg/mL, so that the solution would not
`be too viscous for thorough mixing with the added
`chemicals. A preliminary study without primary amine
`showed that carbodii