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PII: OO32-3861 97)00189-4

Polymer Vol. 38 No. 25, PP. 62356242, 19910 1997 Elsevier Science Ltd

Printed n Great Britain. All rights reserved0032.3861/97/$17.00+0.00

A new class of polymerizable dextrans with

hydrolyzable groups: hydroxyethylmethacrylated dextran with and withoutoligolactate spacer

W. N. E. van Dijk-Wolthuisa, S. K. Y. Tsanga, J. J. Kettenes-van den Boschb andW. E. Hennink,*aFaculty of Pharmacy, Department of Pharmaceutics, Utrecht Institute forPharmaceutical Sciences UPS), Utrecht University, P.O. Box 80 082,3508 TB, Utrecht,The NetherlandsbFaculty of Pharmacy, Department of Pharmaceutical Analysis, Utrecht University,Utrecht, The Netherlands

Received 12 December 7996; revised 17 February 1997)

In this paper a synthetic procedure is described towards a new class of methacrylated dextrans which are sensitiveto hydrolysis. These methacry lated dextrans, which can be used for the development of biodegradable hydrogels,were obtained by grafting L-lactide onto 2-hydroxyethyl methacrylate (HEM A), followed by activation of theterminal hydroxyl group with N,N-carbonyldiimidazole (CDI), yielding HEM A-IactateCI. Coupling of thiscompound to dextran in dimethyl sulfoxide in the presence of dimethylamin opyridine resultedin dex-IactateHEMA. In the same way, dex-HEMA was obtained by coupling of CDI-activated HEMA to dextran. Thedegree of substitution, as determined by H-N MR , could be controlled by varying the molar ratio of HEM A-CI orHEMA-IactateCI to dextran.The incorporation efficiency was 6045 %. The dextran derivatives w ere obtained inhigh yield (85-90% ) and characte rized by NM R, FTIR and GPC. 0 1997 ElsevierScience Ltd.

(Keywords: dextran; biodegradable; hydrogel)

INTRODUCTION

Hydrogels are currently under investigation as deliver?systems for pharmaceutically active proteins and peptides-.Recently we reported on the release of proteins fromhydrogels4 obtained by polymerization of aqueous solutionsof glycidyl methacrylate derivatized dextran (dex-MA)5*6 .Although these gels contain m ethacrylate esters in theircrosslinks, the hydrolysis of these groups is very slow underphysiological conditions. The dex-M A hydrogels could berendered degradable by incorporation of the enzymedextranase, which w as also an effective route to modulate

the release of an entrapped protein from these matrices7. Analternative approac h to degradable, interpenetrating net-works of dextran and polymethacrylate is incorporation ofhydrolytically labile space rs between the polymerizedmethacrylate groups and dextran. It has been reported thatintroduction of lactate esters induces degradability underphysiological conditions in hydrogels derived from acyl-ated poly(ethylene glycol)-polylactate blockcopolymers .

In the present paper we report on the synthesis of HEM A-lactate- and HEM A derivatized dextran, a new class ofpolymerizable dextrans. In addition to methacryla te groups,dex-1ac tateHE MA contains carbonate and lactate estergroups, whereas dex-HEM A contains only additionalcarbonate ester groups. As shown in recent studies c arriedout by our group, hydrogels obtained by polymerization ofthese compounds degrade under physiological conditions by

*To whom correspondence should be addressed

chemical hydrolysis of the labile ester groups present in thecrosslinks,. Moreo ver, these hydrogels ar e assume d toposses s a good biocompatibility since the degradationproducts are lactate (endogenous compound), dextran, usedas plasma expander ,and poly(Zhydroxyethy1 metbacry-late), a well known polymer used in many biome dicalproducts and used for pharmace utical applicationsI *.

EXPERIMENTAL

C h e m i c a l s

Dextran (from Leuconos toc m esent ero i des, T40 , M , =15 000, M , = 32 500 g/mol, as determined by GPC analysis),dimethyl sulfoxide (D MSO , < 0.01% water), 2-hydroxyethylmethacryla te (HEM A, 2-hydroxyethyl methylpropenoate,less than 1% methacrylic acid (HPL C analysis)), andhydroquinone monomethyl ether ( > 98% by HPL C)were obtained from Fluka Chemie AG, Buchs, Sw itzerland.4-(NJ-dimethylamino)pyridine (DM AP, 99%) and N,N-carbonyldiimidazole (CDI, 98%) were from Acres Chimica,Geel, Belgium. L-Lac tide ((3S-cis)-3,6-dimethyl- 1 Cdioxane-2,5-dione, > 99.5%) was purchased from Purac BiochemBV (Gorinchem, The Netherlands) and used without pre-treatment. Stannous oc toate (tin(I1) bis(2_ethylhexanoate),SnOct2, 95%) (Sigma Chemical Co., St. Louis, MA, USA)was used as received.

Toluene was distilled from sodium/henzophenone, andstored on molecular sieves. Tetrahydrofuran (THF) wasdistilled from LiAlH4 immediately before use. Other

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A new class of polymerizable dextrans with hydrolyzable groups: W. N. E. van Dijk-Wolthuis et al.

solvents (p.a.) were obtained from Me rck, Darm stadt,Germany. Dialysis tubes (cellulose, MW cut off (based onproteins) = 12 000- 14 000) were purchased from MedicellInternational Ltd, London, UK. PD- 10 columns containingSephadex G-25 M, were supplied by Pharmacia Biotech,Uppsala, Sweden.

CharucterizationNM R spectra were recorded with a Gemini 300 MHz

spectrom eter (Varian Associates Inc. NM R Instruments,Palo Alto, CA, USA). Approxima tely 30 mg of material wasdissolved in 0.8 ml solvent. For measurem ents in HZ0(99.8% *H, Merck) the HOH signal at 4.8 ppm was used asthe reference line, whereas in DMSO -d6 (99.8% H. Merck)containing *H20, the central DM SO line was set at 2.50ppm. Chloroform (99.6 + % H, Acres) was set at 7.26 ppm.For H-NMR, a pulse length of 4.5 ps (PWgo 12 ps) wasused with a relaxation delay o f 15 s. The degree ofsubstitution (DS; the amount o f methacrylate groups per 100dextran glucopyranose residues) of the methacryla ted

dextrans was calculated as (I,.1 OO)/(1.04./n, ), in which I,is the average integral of the protons (H,) at the double bond(around 6 ppm), and /HI is the integral of the anomericproton (Hi) at 64.95-5.1 ppm; the correction factorI .04 isfor the 4% of (Y - I,3 linkages in dextran.

For the C-NM R spectra, the pulse length wa s set at4.5 ps (PWgo 12 ps), and the relaxation delay at 2 s. The(CH3)$ i resonance (0 ppm) of sodium 2,2-dimethyl-silapentane sulfonate (DSS ) was used as the referencelineI

FTIR spectra were recorded with a Bio-Rad FTS-25spectrom eter (Bio-Rad Laboratories Inc., Cambridge , M A.USA). The dry materials were powdered, ground w ith dryKBr powde r and pressed into pellets under vacuum . For

each sample, 16 scans were recorded between 4000 and450 cm-, with a resolution of 2 cm-.

The molecular weights and molecular weight distribu-tions of dextran and dex-HE MA were determined by gelpermeation chroma tography (GPC ) with a system consist-ing of a Wate rs 510 HPL C pump, and a 410 differentialrefractometer (Waters Associates Inc., Milford, MA, USA)with three thermostated (35C) Shodex KW series columns(OH pack KB SOOP 8 X 300 mm, precolumn; OH pack K B802 6 mm X 50 mm, exclusion limit 4*103; OH pack KB80M 8 mm 300 mm, exclusion limit 2*107; Showa Denko.Tokyo, Japan). The mobile phase was degassed watercontaining 10 mM NaCl. The flow rate was 1 O ml/min.The columns were calibrated with dextran standard s o fknown molecular weight and narrow molecular weightdistribution (Fluka). The chroma tograms were analysedwith Millennium 2010@ , Version 2 .0 softwa re (W atersAssociates Inc.).

Synthesis qf HEMA-imidazolyl carbamute (HEMA-Cl)CD1 (1.62 g; IO mmol) was dissolved in about 15 ml

anhydrous THF in a nitrogen atmosp here, and HEM A(1.30 g; 10 mmol) was added. The reaction mixture wasstirred for 16 h at ambient temperature . After addition ofa small amount of hydroquinone monomethyl ether (50-60 mg), the solvent was evaporate d, yielding a slightlyyellow liquid (yield 2.93 g). The crude product wasdissolved in ethyl acetate, e xtracted with water to removeimidazole, unreacted CD1 and HEM A, and dried onanhydrous M gS04. After filtration, hydroquinone mono-methyl ether (50-60 mg) was added once more, the solventevaporate d, yielding 2.33 g of HEM A-CI ( > 10 0%). Purity

6236 POLYMER Volume 38 Number 25 1997

(by H-NMR): 88% w/w (contains 2% (w/w) dimer, andresidual solvents). H-N MR (CDCll): 68.12 (m, lH,H,),7.40 (m, IH, H,), 7.06 (m, IH, Hf), 6.1 1 (broad-s. IH,H,),5.60 (bs, IH, Hi,), 4.64 (m, 2H, H,,), 4.48 (m, 2H, H,), 1.92(m, 3H, Hh) (Figure 6B). FTIR (KBr, in cm-): 3133(w. ~c=c_n), 1765 (s, vcYo lactate and carbonate ester), 172 1(s, v(~,(~methacrylate ester), 1636 (m, v,--), 8 16(w, Y~=~_~).jC-NMR (CDC13): 6 166.7 (H$ = C(CH3) - C = 0),148.3 (0 - (C= 0) - Im), 135.4 (H2C = C(CH3) - C = 0),130.6 (C,), 126.3 (C,), 117.0 (C, and C,), 65.6 (Cd ), 61.5(C,). 18.0 (C,).

Kinetics of HEMA-lactate formationA mixture of L-lactide (8.64 g; 60 mmol) and HEMA

(2.60 g; 20 mmol) was stirred at 110C in a nitrogenatmosp here until the lactide was molten. Subsequently, thereaction was started by adding SnOctz (81, 203 or 405 mg;I. 2.5 or 5 mol% with respect to HEMA, respectively),dissolved in about 0.5 ml toluene. Over a 1 h period,samples were taken periodically from the reaction mixture,

cooled w ith liquid nitrogen and analysed with H-NM R-spectrosc opy. The conversion of L-lactide to HEM A-lactatewas calculated from the relative intensities of the methinequartet at 5.04 (L-lactide) and 5.17 ppm (oligo-L-lactide,H,in Figure 4A)lJ.

Synthesis of HEMA-lactuteA mixture of L-lactide (4.32 g; 30 mmol) and HEM A

(3.90 g; 30 mmol) was stirred at 110C in a nitrogenatmosph ere until the lactide was molten. Subsequently, acatalytic amount of SnOctz (121.5 mg, 1 mol% with respectto HEM A) dissolved in about 0.5 ml toluene was added. Theresulting mixture was stirred for 1 h, and allowed to cool toroom temperatur e. The reaction mixture w as dissolved in

THF (20 ml) and added dropwise to ice-cold water( 180 ml).The formed precipitate was collected by centrifugation,dissolved in ethyl acetate (40 ml), and centrifuged toremove remaining solids. The supernatant was dried overMgS O+ filtered, and concentrated under reduced pressu re,yielding a viscous oil (3.74 g, 45%) , consisting mainly ofHEM A-lactate with 2 and 4 lactyl residues (average degreeof polymerization, DPAv. of 3) and residual HEMA(3 mol%).

The average length of the lactate space r(DPAv) can beincreased by increasing the molar ratio of L-lactide toHEM A. HEM A (5 mmol) and varying amounts of L-lactide( 10, 15. 20 or 25 mmol) were stirred at 110C in thepresence of 1 mol% (with respect to HEMA ) of SnOct2 in0.5 ml toluene for 60 minutes. After cooling of the reactionmixture the products w ere analysed with H-NM R. Theworkup procedure could be omitted, since at a lactide/HEMA ratio of 2 and higher, no residual HEM A wasdetected by H-NMR ( < 0.5%).

H-NMR (CDCl& interpretation based on HH-CO SYand the literature .: 6 6.11 (s, IH, H,), 5.59 (s, IH, H,),5.12-5.24 (m, H,), 4.47-4.26 (m, 5H, H,, Hd en H3.86 (m, Hd of residual HEMA ), 2.80 (broad-s, OH), 1.94(s, 3H, Hh), 1.39-1.59 (m, Hf and Hh), 0.90 (m, SnOctz)(Figure 4A).

C-NMR (CD C13): 6 175.4/175.0/174.7 (HO-CH(CH ,) -C = 0), 169.9-l 69.5 (cluster of five signals, RO -CH(C H7) -C = 0), 167.0 (HzC = C(CH1) - C = 0), 135.8 (H2C =C(CH I) - C = 0), 126.2 (C,), 69.2/69.0/66.7 (C, and C,),63.1 and 62.0 (C, and C,), 20.4 (C,,), 18.2 (C,,), 16.8/16.7(C,).

FTIR (K Br. in cm-): 3510 (w, vo.n), 175 4 (s, ~c,o

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HEMA 1 L ide. 2

CDI. 4 HEMA -lactate , 5 imidazole. 6

7, dextran dex-IactateHEMA, 8

Figure 1 Reaction scheme for the synthesis of dex-1actateHEMA

lactate ester), 1723 (s, vc=o methacrylate ester), 1638(m, Vc=c), 816 (w, C-H).

Synt hesis of HEM A-1actat eCI

to neutralize the DM AP and imidazole, transferred to adialysis tube, and dialyzed for 2-3 days against deminer-alized water at 4C. After lyophilization dex-HE MA had aDS of 20 as determined with NMR (80% incorporation).

CD1 (1.76 g, 10.8 mmol) was dissolved in 100 ml THF atca. 40 in a nitrogen atmosp here and added to HEM A-lactate, synthesized as described above (3.74 g, 10.8 mmol,DPAv 3). The clear solution was stirred for 16 h at roomtemperature . The solvent was evapora ted under reducedpressure, yielding a viscous oil. The product HEM A-lactate-CI (5.50 g) was used without further purification. Composi-tion by H-NMR: 72% w/w HEMA-lactateC1, 8% w/wHEMA-lactate, and 20% w/w imidazole.

General procedure for t he synt hesis of dex-HEM A and dex-

1actateHEM A

H-NMR (CDCls): 6 9.56 (broad-s, lH, NH), 8.16 (m, lH,Hi), 7.68 (m, lH, H,), 7.44 (m, lH, HI), 7.10 (s, 2H, H , andH,), 7.07 (m, lH, Hk), 6.08 (m, lH, H,), 5.58 (m, lH, H,),5.36 (m, lH, H,), 5.23-5.12 (m, H,), 4.64 (dd, CH2 - O-C1of residual HEM A-CI), 4.48-4.32 (m, 4H,H , and Hd), 1.91(m, 3H, Hb), 1.73-1.49 (m, H f and H ,,), 0.92 (m, SnOctz)Figw e 4B).

C-NM R (CDCls): 6 169.8-168.7 (cluster of six signals,RO-CH(CH 3) - C = 0), 166.9 (H2C = C(CH3) - C = 0),147.9 (0 - (C = 0) - Im), 135 .7 (H.&Z= C(CH3) - C = 0),130.7 (Ci), 126.1 (C,), 121.8 (C,), 117.2 (C, and Cl), 71.7171.5 (C,), 69.4-69.0 (cluster of six signals, C,), 63.U63.0and 62.0 (C, and C,), 1 8.1 (C,), 16.6/16.5 (Cr and C,).

Dextran (10.0 g) was dissolved in DM SO (90 ml) in a dry,stoppere d 250 ml round bottom flask in a nitrogenatmosp here. After dissolution of DM AP (2.0 g), a calculatedamount of HEMA-CI (purity 88%) or HEMA-lactateC1,DPAv = 3, purity 72% ) was added. The solution was stirred

at room temperature for 4 days, after which the reaction was

stopped by adding 2 ml of concentrated HCl to neutralizeDM AP and imidazole. The reaction mixture was transferredto a dialysis tube and dialyzed for 2-3 days againstdemineralized water at 4C. The methacryla ted dextran waslyophilized and the white fluffy product (yield 85-90%)was stored at - 20C until use. The DS was determined byH-NMR spectroscopy. The average incorporation ofHEMA was 85% (DS < 20), and of HEMA-lactate 60%(DS < 11).

FTIR (KB r, in cm-): 3133 (m, ~~-~-n), 1759 (s, vcZolactate a nd carbonate ester), 1722 (s, vc-o methacry lateester), 1637 (m, vc,& 817 (w, vCZC_n).

Dex-HEM A. H-NMR (*H20): 66.18 (s, H, ), 5.77(s, H,), 5.35 (broads, Hr of (Y - 1,3 branch), 5.00(broads, lH, HI), 4.54 and 4.48 (broads,H , and Hd),4.00-3.55 (m, 6H, H Z, H3, Hq, Hs, Hg and H61)), 1.95(s, Hb) Figure 6C).

Ki neti cs of dex-HEM A formation

Dextran (10.0 g) was dissolved in DM SO (90 ml) in a dry,stoppere d 250 ml round b ottomed flask in a nitrogenatmosphere. After dissolution of DMA P (2.0 g), HEMA-CI(88% w /w pure, 3.45 g; 15.4 mmol) was added, correspond-ing with a molar ratio of 0.25 of HEM A-CI to glucopyr-anose residues in dextran. Samples (1 ml) were takenperiodically from the reaction mixture and neutralized with1.5 ml of 0.1 M HCl. The dex-HEMA was separated fromunreacted HEMA-CI by elution with water over a SephadexPD-10 column. The first 3 ml, containing the methacrylate ddextran, were collected and lyophilized. The DS wasdetermined by H-N MR and used to calculate theincorporation efficiency of HEM A. The remaining reactionmixture was worked up by adding 2 ml of concentrated HCl

13C-NMR (*H20): 6171.9 (H& = C(CH3) - C = 0),158.3 and 157.5 (CH20-(C = 0) - OR), 138.3 (CH; = C -CH3), 130.0 (C,), 100.4 (C,), 76.1 (C,), 74.1 (C,), 72.9(CJ, 72.2 (Cd), 68.3 (C,), 69 .0 and 65.7 (C, and C,), 20.2(cb>.

FTIR (KBr, in cm-): 3424 (s, vO_n), 1752 (s, vcZolactate and carbonate ester), 1718 (s, vcZo methacryla teester), 1636 (m, vcZc), 816 (w, vC=c-n).

Dex-1actat eHEM A. H-NMR (12.5% *H20/DMSO-dh): 6 5.99 (s, H,), 5.67 (s, H,), 5.14-4.95 (broad m, resi-dual OH, H , and H,), 4.67 (s, lH, H,), 4.27 (m,H , and Hd),3.78 (water), 3.91-3.11 (m, 6H, Hz, H3, Hq, Hs, H6, H6)1),2.50 (DMSO ), 1.83 (s, Hb), 1.41 (m, HrandHh) Figure4C).

FTIR (KBr, in cm-): 3425 (s , vO_n), 1752 (s, vcco lactateand carbonate ester), 1718 (shoulder of 1752, vccomethacrylate ester), 1644 (m, vc=,-), 816 (w, ~~=~-n).

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HEMA. 1 CDL 4 HEMA-CI. 9 imidazole. 6

9 + H

dexuan, 7 dex-HEMA, 10

0-O

9

Figure 2 Reaction scheme for the aynthesls oldex-HEMA

:110 20 40 50 50

t ime minY

Figure 3 Conve rsion of L-lactide to oligolactate in the reactiori w ithHEMA at LL molar ratio of SnOctJHEMA of 0.05 (A). 0.02.5 (B). and0.01 K)

RESULTS AND DISCUSSION

Synthetic approach

In the synthesis of dex-IactateHEMA three steps can bedistinguished Figure I). First, L-lactide (2) is grafted ontoHEMA l), yielding HEM A-lactate (3). After activationwith NJ-carbonyldiimidazole (CD L 4). the resultingHEM A-lactate C1 (5) is coupled to dextran (7) to yielddex-1actateHEMA (8).

A compara ble dextran derivative without lactate space r

between the methacrylate ester and dextran was alsosynthesized. For this compound Figure 2), HEMA wasactivated with CDI, and the resulting HEM A-CI (9) is thencoupled to dextran, yielding dex-HE MA (10).

Synthesis o HEMA-lactutr

The s nthesisY

of HEMA-lactate has been describedbefore Is, 6. This compound was obtained by solutionpolymerization of L-lactide onto HEM A in toluene, usingtriethyl aluminium as catalyst. This procedure, howeve r, issensitive to the presence of traces o f water. Another catalyst,which is often used for the grafting of L-lactide ontohydroxy compounds, such as benzyl alcoho l7 or poly(ethylene glycol) is stannous octoa te (SnOctz). W einvestigated wheth er this approa ch would be successfulfor the synthesis of HEM A-lactate. The polymerizationtemperature was set atI 10C to ensure melting of L-Iactideand to prevent therm al polyme rization of HEM A. First, a

di(methacryloyloxyethyl)c ubonate, 11

kinetic study was done to establish the optimum in the

amount of SnOct? and the reaction time. InFigure 3, thelactide conversion is plotted as a function of time for threemolar ratios of SnOctdHEMA, with a lactide/HEMA ratioof 3 (mol/mol). This figure shows that decreasing the molarratio from 0.05 to 0.01 resulted in a lower reaction rate.However, at the lowest ratio of SnOctz/HEMA investigated(O.Ol), the conversion of L-lactide was still almost complete(95%) in 60 min. In a separa te experiment, with equimolaramounts of L-lactide and HEM A the conversion was shownto be more than 99% w ithin an hour. Therefor e, for thestandard preparation of HEM A-lactate (molar ratio in thefeed l/l) the reaction time was set at 1 h, with a molarratio of SnOct?/H EMA of 0.0 1. The homopolymerization ofL-lactide is assumed to be negligible in the presence of an

alcohol under the polymerization conditions selected, sincestannous alkoxide, the coordination product of SnOctz withthe alcohol, is the actual initiating species . After thisfirst coordination step, the stannous alkoxide polarizesthe carbonyl group in L-lactide, resulting in insertion ofL-lactide between tin and the alkoxy group. Th ese ste psare repeate d until all L-Iactide is consumed.

When equimolar amounts of HEMA and L-lactide wereused, about 15% of unreacted HEM A remained in thereaction mixture. In HEM A-lactate a secondary hydroxylgroup is present which can react with lactide. This w illresult in a mixture of mainly HEM A-lactates with 2 and 4lactyl residues per HEM A, and unreacted HEM A. Sincecontamination of HEM A-lactate with HEM A would resultin a mixed dextran derivative, with and without lactatespacer, HEMA had to be removed from the product. First,removal of HEMA by extraction with water layer was tried.This resulted in the formation of quite stable em ulsions,with a considerable loss of material. Therefore , precipitationfrom THF into cold water wa s used, resulting in a reductionof the amount of HEMA from 15 to 3%. A side-effect of thismethod w as that the isolated HEMA-lactate (yield 45%) hada slightly higher ave rage degree of polymerization of thespacer (DPAv,) than theDP,, of the raw product (3.0versus2.3, respectively). Probably, HEM A-lactate with 2 lactylresidues has a higher wa ter solubility than HEM A-lactatewith longer lactate grafts.

Other methods which were unsuccessful in reducing theamount of HEM A in the product, were carrying outthe reaction in solution (THF or toluene) to better controlthe reaction of HEM A with L-lactide, or adding a slightexcess of L-lactide (e.g. 1.I equivalent). Separation of

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1:I

I

a a e cd& iI I 1 1 I I I 1 I(

9 8 7 6 5 4 3

L.

-r

2

mQPN qimidazole

LI I

1 PPm

b,f__~o/fif~k ,

) HEMA-lactate-cl

9 8 7 6 5

dex-IactateHEMA

4 3 I 2 1 pm

r I ~~I~I--:--~--I --IIIII IfII9 8 7

6 5 4 3 2 1 mmFigure 4 300 MHz H-NMR spectra of HEMA-lactate in CDCI? (A), HEMA-IactateCI in CDCI, (B), and dex-IactateHEMA in 12.5 Hz0 in DMSO (C)

HEMA from HEMA-lactate by silica column chromato-graphy with ethylacetate/hexa ne was successful, but thismethod is labourious, and has a low capacity, and istherefore not useful for preparations on a larger scale.

H-NMR analysis of HEMA-lactate Figure 4A) shows,in addition to signals of the HEMA residue (If,,H , ,, H, andHd), also signals from the lactyl residues at 1.4- 1.6 ppm (Hrand H,,) and at 5.2 ppm (H,). H, only s hows up in theintegral; this signal coincides withH , and H , . The spectrumshows tha t HEM A is indeed esterified with the lactate graf t,

since the chemical shift of Hd in HEM A is at 3.8 ppmFigure 6A), whereas in HEM A-lactate this shift is at4.4 ppm. Unreac ted HEM A (3%) is still visible at 3.8 ppm.The D PA v of the lactate gra ft in HEM A-lactate canbe calculated from the ratio of the integrals of H, and

H , , increased with one for the lactyl end group (the NM Rsignal of H , is maske d by the 2-hydroxyethyl group at4.4 ppm).

Figure 5 shows the relationship between the molar ratioof L-lactide to HEM A in the reaction mixture and theamount of lactyl residues per HEM A molecule (whichequals DPAv) in the product. It demonstrates that the lengthof the lactate spacer can be controlled by varying the ratio ofL-lactide to HEMA in the feed. At a lactide/HEMA ratio of4 and higher, the average space r length is slightly less

than calculated from the feed ratioFigure 5, dashed line).This is caused by incomplete polymerization of L-lactidewithin 60 min (95%) . It is therefore recomm ended toincrease the reaction time for HEM A-lactate with longerlactate grafts.

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0 2 3fLd ratio I_-IactideiHEt&

5

Figure 5 Relationship between the average amount of lactyl residue5 perHEMA in the product and the molar feed ratio of L-IactidekIEMA.

calculated from the feed ratio (dashed line), and obtained from H-NMR(solid line)

Activation of HEM A and HEMA-lactate with CD1

HEMA and HEMA-lactate, in the following referred to asHEM A(-lactate), can be coupled to a hydroxyl group ofdextran via a mixed c arbonate ester. This linkage can beconveniently introduced with N,N-carbonyldiimidazole(CD1 )20. First, HEM A(-lactate) reacts with an equimolar

amount of CDI, yielding the imidazolyl carbam ate HEM A-(1actate)C I. The activation of the hydroxyl group wasestablished by H-NMR.

Comparison of Figure 6A with 6B shows that theHd ofHEMA after reaction with CD1 has shifted from 3.8 to 4.6ppm, and that the imidazole signals hav e appeared at 7.0,7.2 and 8.2 ppm. Initially, when the synthesis of HEM A-CIwas carried out in dichloromethane, the product was con-taminated with a considerable amount (up to 20 mol%) of

0b

.tiHed

a HEMA/

J,a a c d e

B

8 7 6 5 4 3 2 wm

bLo

;l f

d-? a HEMA-Cl

9 8 7 6 5

b, _,+~i;,a

dex-HEMA

I

R.dLcrJa-8 I 1 I

4.6 4.5 4.4 4.3

a a HI cd

4

L

b I ( I 1 I ( 1 i 1 1 I I I I a I 1 I I 1 s 1 r 1 I t 9 8 7 6 5 4 3 2 1 wm

Figure 6 300 MHz H-NMR spectra of HEMA in CDCI i (A). HEMA-Cl In CDCI 1 B) and dex-HEMA in Hz0 (C)

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0 2 6 6 10t?tne (days)

Figure 7 Incorporation of HEMA-CI in dextran as a function of time. Thecalculated DS from the molar feed ratio is 25

calculated DS

Figure 8 Relationship between the molar feed ratio of HEMA-(lactate)CIto glucopyranose residues in the reaction mixture ( = calculated DS) andthe DS of the product, dex-HEMA (A) and dex-1actateHEMA (B)

di(methacryloyloxyethy1) carbonate (Figure 2, 11).H, andHd in this compound give a signal at 4.4 ppmFigure 6B,inset). Since 11 ormed by reaction of a second molecule ofHEMA with HEMA-CI, is not reactive towards dextran,care was taken to minimize this side reaction. Changing thesolvent from dichloromethane to THF resulted in almostquantitative formation of HEM A-CI, with about 1 mol% of11. The HEM A-CI was obtained in almost quantitative yieldafter removal of imidazole by solvent extrac tion.

HEM A-lactate was also activated with CDI, yieldingHEMA-lactateC1. Figure 4B shows the H-NMR spectrumof HEM A-lactate C1, in which the methine protonH ,appears at 5.35 ppm, with a concomitant reduction of theintegral of the peak a t 4.4 ppm. From the integral ratio ofH ,and H , the extent of activation can be calculated, which isabout 90%. The residual amount (about 10% ) is probablypresent as unactivated HEM A-lactate, with its distinctivesignals in the H-NM R sp ectrum Figure 4B) overlappingwith the hydroxyethyl group at 4.4 ppm.

HEM A-1actate CI could not be purified by extraction withwater , because stable emulsions were formed with severalorganic solvents. Since imidazole probably does notadversely affect the coupling of the HEMA-1actateCI todextran in DM SO, as demonstrated before in the couplingreaction of glycidyl m ethacrylate to dextran, HEM A-lactateC 1 including the by-product imidazole, wasused forthe derivatization of dextran.

Coupli ng of HEM A- 1actat e)CI to dextr an

For the synthesis of dex-(lactate )HEM A, essentially theprocedure for dex-MA in DMS O in the presence of DMA Pwas used. First, the incorporation of HEM A-CI in dextran

was studied as a function of time to determine the optimalreaction time. Fi gure 7 shows that in 4 days theincorporation reache d 85% and after that only slowlyincreased to 95% in 10 days. The degree of substitution didnot significantly decrea se with longer reaction times, whichindicates that under these circumstances the incorporatedspacer is not eliminated by attack of a neighbouringhydroxyl group in dextran, as observed for 4-nitrophenyl-carbonate esters in dextran2.

Fi gure 8 show s the relationship between the molar ratioof HEMA-CI and HEMA-1actateCI DPAv = 3) toglucopyranose residues in dextran in the reaction mixture(i.e. the calculated DS), and the DS of the products, asdetermined by NMR -analysis after 4 days reaction time. It isshown that the degree of substitution of dex-(1ac tate)HE MAcan be tailored by the molar ratio of HEM A-(1acta te)CI todextran in the reaction mixture. For dex-HE MA theincorporation efficiency was 85%, where as for dex-1actateHEMA about 60% of HEMA-1actateCI was incor-porated in the product.

Although for dex-HEMA the DS could be increased to17, attempts to synthesize dex-1actateHEMA DPAv = 3)with a DS higher than 11 were not successful. When theincorporation of HEM A-lactate in dextran reached thislevel, the viscosity of the reaction mixture increased andresulted in solidification, despite the presence of hydro-quinone monomethyl ether as a radical inhibitor. FTIRanalysis of the dialyzed and freeze-dried , water insolubleproduct showe d that double bonds were still present*,indicating that the solidification was probably not due tochemical crosslinking of the methacrylate groups. Thissuggests that specific interactions between the HEM A-lactate spacer s m ay be responsible for the solidification,since this phenomenon was not observed in dex-HE MA

with a comparable DS.GPC analysis of dex-HEMA (DS from 3 to 17) showed

that the elution profile w as not significantly different fromdextran (results not shown), indicating that the hydrody-namic radii of dextran and dex-HE MA are equal.

Figure 4C and Figure 6C show the H-NMR spectra ofdex-1actateHEMA DPAv = 3) and dex-HEMA, respec-tively. Since dex-1ac tateHE MA is poorly soluble in water,NMR analysis was done in DMSO , w ith 12.5% 2H20 toexchange the hydroxylic protons. In addition to the lactategroup a t 1.41 and at 4.95-5.15 ppm in dex-1ac tateHE MAFigure 4C), the hydroxyethyl group was visible around

4.5 ppm in both derivatives, in a 1: 1 molar ratio with respe ctto the methacrylate ester Figur e 4C and Figur e 6C). In arecent study we determined the position of the methacryla teester at the glucopyranose ring in dex-MA with C-NM R6.This method could not be applied for dex-HE MA and dex-lactateH EMA , since the DS could not be increased to avalue high enough to obtain a spectrum, in which the 13Csignals of the substituted glucopyranose could be distin-guished. Howe ver, since the reaction of HEM A-(lactate)C Iwith dextran also is a SN2 substitution, we assume that thesubstituents are attache d to the 2- and 3-hydroxyl group inthe glucopyranose ring.

CONCLUSIONS

A universal method has been developed to couple hydroxyl

*The ratio of the height of the peaks at 813 cm- (double bond of meth-acrylate ester) and 763 cm- (dextran) is an indication for the DS of theproduct, as determined with H-NMR (see )

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A new class of polymerizable dextrans with hydrolyzable groups: W. N. E. van Dijk-Wolthuis et al.

containing compounds to dextran. This method is based onthe activation of a hydroxyl group w ith CDI, followed by thecoupling of the activated compound to dextran. With thisprocedure, both HEM A and HEMA-lactate were coupledto dextran, yielding dex-HE MA and dex-lactateH EMA ,respectively. The degree of substitution can be fullycontrolled by the molar feed ratio of activated hydroxycompound to dextran, with 60-850/o incorporation. Thecompounds can be polymerized in aqueous solution to formhydrogels, which hydrolyze under physiological conditions,due to the presence of hydrolytically labile crosslinksO .

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