Synthesis and characterisation of poly (L-lactic acid) galactosyl derivatives; access to functionalised microspheres

Article abstract of DOI:10.1016/S0040-4039(99)02216-9

A new series of galactosyl-derived polymers has been used for the preparation of microspheres. The strategy is based on the modification of the terminal carboxylic group L-PLA (73.000) by coupling to a galactosyl antenna in the presence of the peptide coupling agents: DCC/HOBT. The degree of functionalisation varies between 60 and 70%, and antenna density between 1.74 and 2.78. The characterisations of the new products were carried out using ¹H NMR, gel permeation chromatography, and acid base titration; the size of the functionalised microspheres was determined to be 210-270 μm by DLS. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(99)02216-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 877–881
Synthesis and characterisation of poly (L-lactic acid) galactosyl
derivatives; access to functionalised microspheres
Rima Kassab,a Bernard Fenet,b Hatem Fessi c and Hélène Parrot-Lopez a,∗
aEquipe Reconnaissance et Organisation Moléculaire et Biomoléculaire, associée au CNRS, Université Claude Bernard,
Bât. 305, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
bCentre de RMN UCB Lyon-ESCPE, associé au CNRS, Université Claude Bernard, Bât. 308, 43 Bd du 11 Novembre 1918,
69622 Villeurbanne Cedex, France
cLaboratoire de Génie Pharmaceutique, associé au CNRS, Faculté de Pharmacie, 8 Avenue Rockefeller, 69373 Lyon Cedex,
France
Received 1 October 1999; accepted 24 November 1999
Abstract
A new series of galactosyl-derived polymers has been used for the preparation of microspheres. The strategy is
based on the modification of the terminal carboxylic group L-PLA (73.000) by coupling to a galactosyl antenna in
the presence of the peptide coupling agents: DCC/HOBT. The degree of functionalisation varies between 60 and
70%, and antenna density between 1.74 and 2.78. The characterisations of the new products were carried out using
1H NMR, gel permeation chromatography, and acid base titration; the size of the functionalised microspheres was
determined to be 210–270 µm by DLS. © 2000 Published by Elsevier Science Ltd. All rights reserved.
Keywords: poly L-lactic acid; galactosyl antennae; covalent linkage; microspheres.
Biodegradable polymeric drug delivery systems possess several advantages compared to conventional
drug therapies.1,2 The use of poly L-lactic acid (L-PLA), a non hydrosoluble biodegradable polymer, in
controlling the release of drugs has become important in pharmaceutical applications. Encapsulation of
drugs in microspheres based on PLA has already been investigated by several groups.3–7 However, the
main problem in using conventional microspheres is that they are rapidly eliminated by the reticulo-
endothelial system or macrophages. One method to circumvent this problem of macrophage lies in the
formation of large microspheres. A second method lies in the targeting of specific bio-receptors via
surface modification involving coupling the carbohydrate antennae to the vectors. Numerous biological
recognition processes are regulated by protein–carbohydrate interactions.8 To this end, Roy et al. describ-
ed the synthesis and applications of neoglycoconjugates,9 especially the synthesis of glycopolymers,
by the incorporation of simple and complex oligosaccharide sequences into polymer.10 In the case
of graft polymerisation, the desired carbohydrate is directly coupled into polymers having reactive
∗
Corresponding author. E-mail: h.parrot@cdlyon.univ-lyon1.fr (H. Parrot-Lopez)
0040-4039/00/$ - see front matter © 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(99)02216-9
tetl 16144

878
functionalities: polyacrylamides,11 polyacrylates,11 poly-L-glutamic acid12 and poly (acrylamide-co-
allylamine).13
In this context, the coupling of carbohydrate antennae to the L-PLA and this microsphere organisation
provided a new class of vector for molecular recognition and controlled the time-release of the drugs,
thus presenting the advantages both of large size and of biorecognition. In this paper we report the
synthesis and characterisation of novel poly (L-lactic acid) galactosyl derivatives and their formation of
microspheres. The synthetic strategy consists in the modification of the terminal L-PLA carboxylic group
by coupling to a galactosyl antenna. We have previously demonstrated that coupling galactosyl antennae
to cyclodextrins leads to good molecular recognition by the Kluveromyces bulgaricus cell wall lectins
(Kb CWL).14
The galactosyl antennae 4a–c were synthesised from the corresponding tetra-O-acetyl-β-D-galactosyl
isothiocyanate15 1 in two steps (Scheme 1). Compound 1 (2.28 mmol) was condensed with the N-
protected amino acid derivative N-Boc-4-aminobutyric acid 2a, N-Boc-6-aminohexanoic acid 2b or N-
Boc-11-aminoundecanoic acid 2c (3.20 mmol) in dry toluene (14 mL) in the presence of triethylamine
(0.23 mmol) for 2 days at 20°C. After purification by flash chromatography on silica gel (toluene:acetone
8:2), complete deprotection of the derivatives 3a, 3b or 3c16 is achieved using methanolic sodium
hydroxide (1 M) and trifluoroacetic acid (TFA).
Scheme 1. Synthesis of galactosyl derivatives
The L-PLA derivatives 6a, 6b and 6c were synthesised, respectively, by condensation at room
temperature of β-amido-D-galactopyranosides 4a, 4b or 4c17 (2 equiv.) with L-PLA185 (Mw: 73.000)
(1 g, 2.10−4 function COOH) in dry DMF/CH2Cl2 mixture (20 mL) using the standard peptide coupling
reagents: dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBT 2 equiv./2 equiv.) (Scheme 2).
The characterisation of the L-PLA Gal derivatives was carried out according to three methods:
(i) Quantification of the galactosyl antennae. Before coupling acid–base titration using 0.01N methan-
olic sodium solution against bromothymol blue as indicator shows the presence of 10−5 carboxylic
acid functions per 100 mg of L-PLA dissolved in 20 mL of mixture CH2Cl2/CH3OH (1/1). Titration
of the L-PLA Gal derivatives 6a, 6b and 6c shows, respectively, the presence of 3.5 10−6, 3.0 10−6
and 4.0 10−6 carboxylic acid functions per 100 mg. The quantity of antennae functions coupled are
thus 6.5 10−6 moles (1.74 mg) for 6a, 7.0 10−6 moles (2.04 mg) for 6b and 6.0 10−6 moles (2.78
mg) for 6c, the figures in parentheses being weights per 100 mg. The degree of functionalisation
thus varies between 60 and 70%, with density of antennae being 1.74, 2.04 and 2.78 for 6a, 6b and
6c, respectively.
1
(ii) H NMR spectra. NMR analysis at 500 MHz of functionalised PLA is difficult due to the am-
phiphilic nature of the polymer. Thus, in CDCl3 the galactosyl antennae are folded back into

879
Scheme 2. Functionalisation of L-PLA
the interior of the polymer and are not observed in the NMR experiment. By modifying the
hydrophobic/hydrophilic balance of the solvent mixture, for example in CDCl3/CD3OD (70/30),
1H NMR can be used to detect the galactosyl antennae.19
The signals arising from the polymers occur as broad doublet at 1.4 ppm (CH3) and a quadruplet
at 5.1 ppm (CH). But the best results are obtained in pyridine-d5 (Table 1) and a COSY experiment
gives the proof that the antennae are linked covalently with the L-PLA. For 6c the NH amide proton
of Gal detected at 9.43 ppm (d, J=9.6 Hz) is correlated to the H-1 Gal proton at 5.9 ppm (t, J=9.5
Hz).
Table 1
1H NMR of the L-PLA functionalised in C5D5N
The NH amide signal characteristic for covalent linkage onto Gal antenna and L-PLA is observed
at 8.2 ppm. This signal is correlated to the CH2 terminal of the spacer at 3.4 ppm.
(iii) Measurement of molecular weight. The molecular weight of derivatives 6a, 6b and 6c was measured
by gel permeation chromatography (GPC).20 The number-average molecular weight (Mn), the
weight-average molecular weight (Mw) and the polydispersity (I=Mw/Mn) of polymers were
calibrated using monodisperse polystyrene as the standard. The variation in Mw may be explained
by the functionalisation of the polymer. The increase in Mn is possibly due to hydrolysis during the
analytical process.
Microspheres derived from these new amphiphilic polymers are fabricated by the ‘solvent
evaporation’ method.21 L-PLA Gal derivative (0.5 g) 6a, 6b or 6c was dissolved in CH2Cl2 (20
mL). This organic phase was added to an aqueous phase (250 mL) containing 4 g of Tween 80
(surfactant). The resulting biphasic system was stirred for 8–9 h at 700 rpm. The microspheres were
filtered, washed with water and finally dried under reduced pressure. The size of the microspheres
was determined using dynamic light scattering (DLS). Comparison of the blank microspheres (204
µm diameter) with those possessing the galactosyl antenna (207 µm for 6a, 210 µm for 6b and 271

880
µm for 6c) shows clearly that the antennae do not strongly modify the properties of L-PLA for the
formation of such structures.
1
In conclusion, by H NMR spectroscopy the functionalisation of the terminal carboxylic group of
the L-PLA by covalent linkage of galactosylated antennae has been confirmed. Given the amphiphilic
character of these modified polymers, it was possible to formulate microspheres having 207 µm to 271
µm diameter. A preliminary study carried out on the microsphere recognition by galactose specific lectins
confirms the activity of the galactosyl antennae on the surface of the microspheres and anticipates a
promising future for the use of these L-PLA functionalised microspheres in the field of vectorisation of
bioactive molecules.
Acknowledgements
We would like to thank Mrs. Nicole Marshall for correcting the manuscript.
References
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10. Roy, R. Trends in Glycoscience and Glycotechnology 1996, 8, 79–99.
11. Bovin, N. V.; Korchagina, E. Y.; Zemlyanukhina, T. V.; Byramova, N. E.; Galanina, O. E. Glycoconjugate J. 1993, 10,
142–151.
12. Kobayashi, K.; Tawada, E.; Akaike, T.; Usui, T. Glycoconjugate J. 1995, 12, 459–465
13. Klein, J.; Krauss, M.; Tich, M.; Zelezn, B.; Jonkov, V.; Kocourek, J. Glycoconjugate J. 1995, 12, 51–54.
14. Attioui, F.; Al-Omar, A.; Leray, E.; Parrot-Lopez, H.; Finance, C.; Bonaly, R. Biol. Cell. 1994, 82, 161–167.
15. Kassab, R.; Felix, C.; Parrot-Lopez, H.; Bonaly, R. Tetrahedron Lett. 1997, 38, 7555–7558.
16. 1N-(tert-Butoxycarbonyl-4-aminobutanoyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosylamine 3a. 1H NMR COSY
δH (ppm) CDCl3: 1.48 (s, 9H, CH3); 1.79 (t, 2H, CH2 βCO, J=7 Hz); 2.07; 2.14; 2.16; 2.17 (m, 12H, OAc); 2.23–2.24
(m, 2H, CH2 α/C_O); 3.11–3.14 (q, 2H, CH2 α/NH, J=7 Hz); 4.11–4.14 (m, H6 and H60); 4.25 (m, 1H, H-5); 4.62 (m, 1H,
NH-C_O); 5.17 (dd, 1H, J2,3=9.5 Hz; J2,1=9 Hz, H-2); 5.21 (dd, 1H, J3,2=9.5 Hz; J3,4=3.8 Hz, H-3); 5.25 (dd, 1H, JH1,NH=10
Hz; J1,2=9 Hz, H-1); 5.50 (dd, 1H, J4,3=3.8 Hz; J4,5=1 Hz, H-4); 6.62 (d, 1H, JNH,H1=10 Hz, NHGal). Rf: 0.163; yield: 56%;
mp: 77–78°C; ES-MS: m/z, 555.3[M+Na]+, 571.4[M+K]+. 1N-(tert-Butoxycarbonyl-6-aminohexanoyl)-2,3,4,6-tetra-O-
acetyl-β-D-galactopyranosylamine 3b. 1H NMR COSY δH (ppm) CDCl3: 1.3 (m, 2H, CH2); 1.48 (s, 9H, CH3); 1.62 (t,
2H, CH2 α/CO, J=7 Hz); 1.85 (m, 2H, CH2); 2.10; 2.12; 2.15; 2.16 (m, 12H, 4 OAc); 2.11 (m, 2H, CH2 β/C_O); 3.11 (q, 2H,
CH2 α/NH-CO); 4.09–4.11 (m, 2H, H6 and H60); 4.15–4.35 (m, 1H, H-5); 4.57 (m, 1H, NH-C_O); 5.12 (dd, 1H, J2,3=9.5
Hz; J2,1=9 Hz, H-2); 5.19 (dd, 1H, J3,2=9.5 Hz, J3,4=3.7 Hz, H-3); 5.25 (dd, 1H, JH1,NH=9.7 Hz; J1,2=9 Hz, H-1); 5.48 (dd,
1H, J4,3=3.7 Hz; J4,5=1 Hz, H-4); 6.62 (d, 1H, JNH,H1=9.7 Hz, NH Gal). Rf: 0.388; yield: 78%; ES-MS: m/z, 583.5[M+Na]+,
599.3[M+K]+. 1N-(tert-Butoxycarbonyl-11-aminoundecanoyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosylamine 3c.
1H NMR COSY δ (ppm) CDCl3: 1.25 (s, 12H, 6 CH2); 1.45 (s, 9H, CH3); 1.6 (t, 2H, CH2 β/C_O); 2.03; 2.10; 2.15;
2.19 (m, 12H, 4 OAc); 2.21 (t, 2H, CH2 α/C_O; J=7 Hz); 3.1 (q, 2H, CH2 /NH, J=7 Hz); 4.09–4.11 (m, 2H, H6 and H60);
4.12–4.14 (m, 1H, H-5); 4.61 (m, 1H, NH-C_O); 5.08 (dd, 1H, J2,3=9 Hz, J2,1=9.2 Hz, H-2); 5.19 (dd, 1H, J3,2=9 Hz, J3,4=3.5
Hz, H-3); 5.28 (dd, 1H, JH1,NH=9.6 Hz, J1,2=9.2 Hz, H-1); 5.5 (dd, 1H, J4,3=3.5 Hz, J4,5=0.8 Hz, H-4); 6.38 (d, 1H, NH-C_O,
J=9.6 Hz). Rf: 0.573; yield: 63%; ES-MS: m/z, 653.4 [M+Na]+; 597.4 [M-C(CH3)3+H+Na]+.

881
17. The products are hygroscopic and should be manipulated directly. 1-N-(4-Aminobutanoyl)-β-D-galactopyranosylamine
4a. 1H NMR δH (ppm) DMSO-d6: 1.7 (m, 2H, CH2); 2.2 (t, 2H, CH2-C_O); 2.8 (t, 2H, CH2-NH2); 2.9–3.1 (m, 1H, H-2);
3.6 (m, 4 OH); 3.6–3.7 (m, 1H, H-3); 4.7 (t, 1H, H-1, J=10 Hz); 8.5 (d, 1H, NH-C_O, J=10 Hz). Yield: 58%. 1-N-(6-
1
Aminohexanoyl)-β-D-galactopyranosylamine 4b. H NMR δH (ppm) DMSO-d6: 1.3 (m, 2H, CH2); 1.5 (m, 2H, CH2);
2.1 (t, 2H, CH2-C_O); 2.65 (t, 2H, CH2-NH2); 2.9–3.1 (m, 1H, H-2); 3.6 (m, 4 OH); 3.7–3.8 (m, 1H, H-3); 4.7 (t, 1H, H-1,
J=9.4 Hz); 8.4 (d, 1H, NH-C_O, J=9.5 Hz). Yield: 52%. 1N-(11-Aminoundecanoyl-β-D-galactopyranosylamine 4c. 1H
NMR δH (ppm) DMSO-d6: 1.3 (s, 12H, 6CH2); 1.5 (m, 4H, 2CH2); 2.1 (t, 2H, CH2-C_O); 2.75 (m, 2H, CH2-NH2); 3.1–3.2
(m, 1H, H-2); 3.6 (m, 4 OH); 3.7–3.8 (m, 1H, H-3); 4.7 (t, 1H, H-1, J=9 Hz); 8.4 (d, 1H, NH-C_O, J=9 Hz). Yield: 55%.
18. L-PLA 100 (Mw=73000) was obtained from PHUSIS (Grenoble, France). Ring-opening polymerisation of L-lactide was
conducted using metallic Zn as catalyst (0.05%) at 140°C.
19. L-PLA Gal 6a. 1H NMR COSY δH (ppm) (CDCl3:CD3OD 7:3): 1.4 (d, CH3 L-PLA); 1.8 (m, 2H, CH2); 2.2 (t, 2H, CH2-
C_O); 3.15 (m, 2H, CH2-NH); 3.4 (dd, 1H, H-2 Gal); 3.5 (d, 1H, H-4 Gal); 3.6–3.65 (dd, 2H H6,60 Gal); 3.7 (m, 1H,
1
H-5 Gal); 3.8 (dd, 1H, H-3 Gal); 4.8 (d, 1H, H-1 Gal); 5.1 (q, CH L-PLA). L-PLA Gal 6b. H NMR COSY δH (ppm)
(CDCl3:CD3OD 7:3): 1.1 (m, 4H, 2CH2 βC_O); 1.6 (d, CH3 L-PLA); 2.0 (m, 2H, CH2δC_O); 2.2 (t, 2H, CH2αC_O); 3.2
(m, 2H, CH2-NH); 3.5 (dd, 1H, H-2 Gal); 3.6 (d, 1H, H-4 Gal); 3.62–3.75 (dd, 2H, H-660 Gal); 3.8 (m, 1H, H-5 Gal); 3.9
1
(dd, 1H, H-3 Gal); 4.9 (d, 1H, H-1 Gal); 5.2 (q, CH L-PLA). L-PLA Gal 6c. H NMR COSY δH (ppm) (CDCl3:CD3OD
7:3): 1.05 (m, 16H, 8 CH2); 1.3 (d, 3H, CH3 L-PLA); 1.9 (t, 2H, CH2-C_O); 3.2 (d, 1H, H-2 Gal); 3.15 (d, 1H, H-3 Gal);
3.3 (d, 1H, H-5 Gal); 3.5 (dd, 2H, H-6,60 Gal); 3.6 (d, 1H, H-4 Gal); 3.9 (t, 2H, CH2-NH); 4.6 (d, 1H, H-1 Gal); 5.0 (q, 1H,
CH L-PLA).
20. GPC measurements were carried out using a Waters chromatograph system equiped with trible columns using a differential
refractometer for detection. Tetrahydrofuran (THF) was used as solvent. Mw is 73.000 for L-PLA 5, 76.200 for 6a, 77.800
for 6b and 76.600 for 6c; Mn is 32.000 for L-PLA 5, 48.600 for 6a, 44.300 for 6b and 42.500 for 6c; I 2.4 for L-PLA 5, 1.56
for 6a, 1.75 for 6b and 1.80 for 6c.
21. Kwong, A. K.; Chou, S.; Sun, A. M.; Sefton, M. V. J. Controlled Release 1986, 4, 47–62.