4236
Macromolecules 1991,24, 4236-4241
Synthetic Glycoconjugates. 2.l n-Pentenyl Glycosides as Convenient Mediators for the Syntheses of New Types of Glycoprotein Models Shin-Ichiro Nishimura,’ Koji Matsuoka, Tetsuya Furuike, Shigeru Ishii, and Keisuke Kurita’ Department of Industrial Chemistry, Seikei University, Musashino-shi, Tokyo 180, Japan Keiko M. Nishimura Department of Cellular Immunology, National Institute of Health, Shinagawa-ku, Tokyo 141, Japan Received December 31, 1990 ABSTRACT The efficacy of n-pentenyl glycosides as excellent carbohydrate monomers in the syntheses of pseudoglycoproteins has been systematically demonstrated. A facile procedure for the preparation of novel glycoprotein models having pendant N,”-diacetylchitobioee [&mGlcpNAc-(1--4)-&mGlcpNAc] and N-acetyllactosamine[b-mGalp-(1+4)-@-mGlcpNAc]was established on the basis of radical copolymerization of the n-pentenylatedderivativeswith acrylamide. These syntheticglycoconjugatesexhibited good solubility in water and had high molecular weights. The sugar contents in the macromolecules could be regulated at will as the needs of the case demand. Specificadhesion of rat hepatocyteson these matrices was also examined to show that the matrix containing N-acetyllactosaminehad especially high potentials. Introduction Cell-cell interactions can be regarded as one of the cellular dynamic processes to regulate differentiation, aging, and malignant alterations in ectobiology. Oligosaccharide chains of cell surfaces and extracellular matrices are indispensable components of biopolymers as carriers of information in the biological systems. Although the structural versatility of these oligosaccharide chains of glycoconjugates has been suggested, each carbohydrate molecule plays an important role as a branching structure of polypeptide main chains (glycoproteins or proteoglycans) or a partial structure exposed on the surface of a plasma membrane (glycolipids). Thus, in general, the biologically significant carbohydrates exist as hybrid-type functional molecules. Synthetic carbohydrate polymers having pendant sugar residues are of great interest not only as simplified models of the biopolymers bearing oligosaccharidesbut as artificial glycoconjugates from the point of view of biochemistry and medical science. In fact, as a result of increasing needs for synthetic carbohydrate polymers for immunological use such as synthetic antigens containing sugar haptens, there are several examples of preparation of copolymers having saccharide However, rather complicated procedures for the syntheses of polymerizable carbohydrate derivatives have often made effective applications difficult. Moreover, polymerizability of the carbohydrate monomers is generally restricted due to the steric hindrance by the bulky carbohydrate residues, suggestingthe usefulness of spacer arms between the sugar moiety and terminal double bond. Our recent attention has been mainly focused on design and facile syntheses of new types of pseudoglycoproteins related to the asparaginelinked-type glycoproteins containing the appropriate spacer function in order to mimic and utilize the unique molecular recognition nature of oligosaccharide chains more efficiently.’ The introduction of suitable spacer arm moieties will increase flexibility of carbohydrate branches and thereby accessibility to the active sites and allow regulation of the sugar contents and molecular weights of the resulting macromolecules. OO24-9297191/2224-4236$02.50/0
Recently, Fraser-Reid et al. have reported that n-pentenyl glycosidesare remarkably chemospecific substrates for selective reactions occurring a t the anomeric center.8 They have also found the usefulness of n-pentenyl glycosides in the glycosidation reactionssJOand the asymmetric synthesis of monosubstituted chiral tetrahydrofurans.” In this paper, we describe the availability of the n-pentenyl group as a simple and efficient polymerizable aglycone with high reactivity and wide applicability. The n-pentenyl group is demonstrated to be useful in the syntheses of versatile artificial glycoconjugates,including the water-soluble glycoprotein models and biocompatible affinity gels obtained by radical copolymerization of pentenyl glycosides of N,”-diacetylchitobiose and N-acetyllactosamine with acrylamide. The applicability of these affinity gels to cell cultivation systems is also preliminarily demonstrated by specificadhesion of rat liver parenchymal cells, especially on the polyacrylamide gel in which N-acetyllactosamine residues were incorporated by means of the new method. The work is of growing interest since oligosaccharidesequences containing N-acetyllactu” ‘ e are widespread in the glycoproteins and glycolipids of cell surfaces and serve as recognition markers for cellular differentiation.12 Moreover, the macromolecules prepared in the present study will be available as new polymeric substrates for several glycosyltransferases.13 Results and Discussion Spacer-Armed Carbohydrate Monomers Containing N-Acetyl-D-glucosamine. Prior to the design of synthetic glycoconjugatea containing oligowxharide chains, evaluations of several aglycones having a C-C double bond at the w-position were carried out with N-acetyl-Dglucosamine derivatives 6-9 to clarify the effect of the chain length of the spacer arm moieties on polymerization behavior. These glycosides were derived from oxazoline derivative 1, readily prepared by the trimethylsilyl trifluoromethanesulfonate (TMSOTf) method“ from 2-acetamido-1,3,4,6-tetra-0-acetyl-2-deoxy-t-~-glucopyranose through glycosidation reactions with alcohols such as 2-propen-l-ol,3-buten-l-ol, 4-penten-1-01, and 10-un0 1991 American Chemical Society
Synthetic Glycoconjugates. 2 4237
Macromolecules, Vol. 24, No. 15, 1991 Scheme 1. A AGO
c AcO
O
8
e
0 (CH2)nCH=CH2 NHAc 2; n.1 3; n.2 4; n=3 5; n=9
Me
1
-CH-
IC
CONH2
Ht
$h;c o N H~
8 8
O*:'
8
NHAc l C H ~ l r $ , H y
/OH I/
" dHO =&o
(cH2InCH=CH2
NHAC 6; n*l 71 n=2 81 n=3 91 n.9
Reagents and conditions: i, HO(CHdnCH=CH2, CSA, C1CH2CH,C1,90 O C ; ii, NaOMe/MeOH.
a
Table I
Polvmerizations of Monomers Derived from NIAcetyl- glucosamine with Acrylamide total sugar carbohydr monomer yield, polym content, [a]D, * MW,' monomer ratie % compo!@ wt % deg J u g kDa 6 6 7 7
1:4 1:lO 1:4 1:lO 1:4 1:lO
46.1 57.4 61.9 59.0 65.8 75.6
1:12 1:42 1:9 1:31 1:8 1:28
23.5 8.0 29.6 11.0 34.6 12.5
-6.2 -4.1 -9.6 -4.5 -11.4 -7.6
0.27 230 1.15 >300 0.43 180 1.26 >300 0.34 190 1.03 >300
ChaRk8l Shlfl
(Ppl
Figure 1. 13C NMR spectrum of the copolymer from N-acetylD-glucosamine and acrylamide measured in DzO at 50 OC. a indicates 2,2-dimethyl-2-silapentane-5-sulfate (DSS). Table I1 Polymerizations of Monomers Derived from N,N'-Diacetylchitobiose and N-Acetyllactosamine with Acrylamide total
sugar
GS-510 column [pullulans (5.8,12.2, 23.7, 48.0, 100, 186, and 380 kDa, Shodex Standard P-82) were used as standards]. Insoluble in water.
carbohydr monomer yield, polym content, u o h p Mw: monomer ratid % c o m p o wt % deg &/g kDa 12 1:4 54.8 1:8 46.1 -11.9 0.31 170 12 1:lO 76.0 1:23 23.1 -6.4 1.02 >300 55.9 1:14 30.6 16 1:4 -6.6 0.48 180 23.0 76.5 1:21 -5.6 1.26 >300 16 1:lO Ratio of carbohydrate monomer to acrylamide. * In water at 25 OC. MWs were determined as described in Table I.
decen-1-01 in the presence of 10-camphorsulfonic acid (CSA) as the promoter and subsequent deacetylation by the Zemplen method (Scheme I). The copolymerization of these monomers with acrylamide in water was examined according substantially to the method previously reported by Kochetkov et al.15 for the preparation of carbohydrate-containing antigens from allyl glycosides of the oligosaccharidedeterminants having the group specificity of Salmonella. The results of copolymerization of the simple models are shown in Table I together with physical data. As can be seen from the data, the effect of the spacer arm length on polymerization behavior was evident, and the content of incorporated N-acetyl-D-glucosamineincreased with increasing spacer arm length from allyl (n = 1)to n-pentenyl (n = 3) glycoside except in the case of n-undecylenyl glycoside 9 (n = 91, which was insoluble in water. Since copolymerization of n-pentenyl glycoside 8 with acrylamide gave polymer containing 34.6 wt % of the N-acetyl-D-glucosamine unit in the highest yield, commercially available 4-penten-1-01 proved to be an excellent and convenient polymerizable aglycone for this type of carbohydrate monomer in view of the easiness and versatility of the monomer synthesis and high polymerization yields. As anticipated, the sugar contents in the macromolecules could be easily controlled by the feed ratio of carbohydrate monomers and acrylamide.16 A fully assigned 13C NMR spectrum of the polymer from 8 with acrylamide is shown in Figure 1. The spectrum shows characteristic signals attributed to the ring carbons of sugar residues (58.2104.3 ppm) as well as the methylene (35.7-38.8 ppm) and methine carbons (44.1-45.1 ppm) of polyacrylamide. Polyacrylamide Containing N,N-Diacetylchitobi088. We recently reported the facile preparation and regioselectivemodificationsof N,"-dia~etylchitobiose,~7an amino disaccharide, and subsequent chemical conversions
into biologically significant oligosaccharide s e q u e n c e ~ ~ ~ J ~ from the properly protected chitobiose derivatives as key intermediates. As an extention of derivatization studies on chitobiose, we have planned the syntheses of macromolecules having N,"-diacetylchitobiose branches in the same manner as in the case for N-acetyl-D-glucosaminecontaining polyacrylamide. Since asparagine-linked-type glycoproteins have been known to contain the invariable chitobiose core structure,20 this type of copolymer is of interest as a simple and available model from structure analyses and biochemical aspects. n-Pentenyl glycoside 12 was successfully prepared in high yield from chitobiose octaacetate through the reaction of oxazoline derivative 1017 with 4-penten-1-01. Intermediate 11 was deacetylated by the Zemplen method. Copolymerization of derivative 12 with acrylamide proceeded smoothly and gave the water-soluble polymers having pendant N,"diacetylchitobiose as shown in Table 11. Fully assigned 13C NMR spectra of n-pentenyl derivative 12 and the copolymer from 12 and acrylamide are shown in Figure 2 (Scheme 11). Polyacrylamide-Containing N-Acetyllactosamine. Versatility of this procedure was also demonstrated in the preparation of novel copolymers from n-pentenylated N-acetyllactosamine 16 and acrylamide. N-Acetyllactosamine peracetate 13, prepared from lactose in moderate yield through the azidonitration of the lacta121*22followed by hydrogenation and acetylation, was converted into oxazoline derivative 14 by treating with TMSOTf. 14 was then allowed to react with 4-penten-1-01in the presence of CSA as the promoter, giving the corresponding @-glycoside 15 in high yield. After 0-deacetylation by the Zemplen method, carbohydrate monomer 16 was copolymerized with acrylamidein a similar way. The resulting watersoluble polymers exhibited high viscosity and the N-acetyllactosamine content could be regulated as shown
a
8
9d a
Ratio of carbohydrate monomer to acrylamide. In water at 25
"C. MWs were estimated by the GPC method with an Asahipak
Macromolecules, Vol. 24, No. 15, 1991
4238 Nishimura et al.
bH IN
CH-CHl
Me
..
c-0
I 5'
C-0
3'
I' 1.1'
IC* Chen1c.l
m
rhlfl lppm)
Figure 2. 'Bc NMR spectra of carbohydrate monomer 12 (A) and the copolymer of 12 and acrylamide (B) measured in DzO at 50 O C . a indicates DSS. Scheme 11' Me
HAC 10
'OAC
11
Reagents and conditions: i, HO(CH2)&H=CHz, CSA, ClCHICHZCl, 90 "C, 5 h; ii, NaOMe/MeOH.
a
in Table 11. The structure of the copolymer from 16 and acrylamide was supported by 13C NMR spectra in DzO (Figure 3, Table 111, and Scheme 111). Selective Adhesion of Hepatocytes. Specific interactions between cell surface receptors and extracellular artificial signals are useful to study the long-term regulation of cell behavior or functions, but the approach requires chemically well-defined, stable surfaces, which should be supportive of cell adhesion and growth. Schnaar et al., for instance, reported that derivatizable anionically charged polyacrylamide surfaces supported adhesion and long-term growth of fibroblasts at a rate and to an extent comparable to those on tissue culture plastics.23 With respect to the use of specific interactions of sugar and hepatocytes, Lee et al. showed carbohydrate-dependent (specific) adhesion and growth of rat or fowl liver parenchymal cells on the surfaces of polyacrylamide gel containing 8-Dgalactopyranose or &-acetamido-&-deoxy-@-~glucopyranose, re~pectively.~'-~ Recently, Akaike and Kobayashi reported an efficient method for the preparation of a new type of styrene homopolymers carrying lactose and recognition by rat hepatocytes.2Tm
Chemical shill lppml
NMR spectra of carbohydrate monomer 16 (A) Figure 3. and the copolymer of 16 and acrylamide (B)measured in D20 at 50 O C . a indicates DSS.
Attention has, thus, been directed to biological evaluations of the novel polyacrylamide gel having pendant N,"-diacetylchitobiose and N-acetyllactosamine as specific ligands for rat liver lectins. As a preliminary examination, we elucidated short-term adhesion behavior of rat hepatocytes on the surfaces of the polyacrylamide matrices containing N,"-diacetylchitobiose and N-acetyllactosamine residues comparing with that of the simple polyacrylamide gel (Figure 4A). As shown in Figure 4C, highly selective adhesion of hepatocytes on the surface of N-acetyllactosamine-containinggel was clearly observed. The affinity of the cells with the N,"-diacetylchitobiose structure, however, appeared to be weaker (Figure 4B). Further biochemical and medical evaluations including long-term adhesion and growth are under way, and the results will be reported in the near future.
Experimental Section General Procedures. Unless otherwise stated, all commercially available solvents and reagents were used without further purification. Acetonitrile, 1,2-dichloroethane,ethyl acetate,and pyridine were stored over molecular sieves (3A) for several days before use. Melting points were determined with a Laboratory Devices melting point apparatus and are uncorrected. Optical rotations were determined with a JASCO DIP-370 digital polarimeter at 23 O C . IR spectra were recorded with a JASCO IR-700. 1H and proton-decoupled carbon NMR spectra were recorded at 270 and 67.8 MHz, respectively, with a JEOL JNM(3x270 spectrometer in chloroform-d, dimethyl-de sulfoxide, or deuterium oxide, using tetramethylsilane (TMS) or 3-(trimethylsily1)propanesulfonic acid sodium salt (DSS) as internal standards. Elemental analyses were performed with a Yanaco MT-3 CHNcorder on the samples extensively (ca. 24 h) dried in vacuo (50 O C , 0.1 Torr) over phosphorus pentoxide. Reactions were monitored by thin-layer chromatography (TLC) on a precoated plate of silica gel 60Fm (layer thickness, 0.26 mm; E. Merck, Darmmstadt, Germany).
Macromolecules, Vol. 24, No.15, 1991
compds c-3 c-4 c-5 C-6
6 102.7 58.2 76.5 73.1 78.5 63.4
c-2' c-3' c-4' c-5' C-6' CHpCH CHyCH CHz
136.0 120.8 72.6
c-1 c-2
c-1'
CH
c=o
177.2
C'=O
CONHg CHs C'H3 0
24.8
Synthetic Glycoconjugates. 2 4239
Table I11 Chemical Shifts of Glycorides and Copolymers (in ppm from DSS) carbohydr monomers copolymers 7 8 12 16 6 7 8 103.6 104.3 103.7 104.3 103.7 103.8 103.7 58.2 58.2 57.7 57.8 58.2 58.2 58.3 76.6 76.4 75.1 76.4 76.5 76.5 75.2 81.2 72.8 72.7 72.8 82.2 72.6 72.6 78.5 78.5 78.5 78.9 78.5 78.0 77.1 63.5 63.5 62.8 63.5 63.4 63.4 62.8 105.6 104.2 73.6 58.3 77.4 76.2 71.2 72.4 75.2 78.6 63.7 63.2 141.4 141.4 138.0 141.4 117.5 117.5 119.2 117.5 72.4 72.8 70.3 73.2 72.0 72.3 72.4 37.70 37.50 37.50 32.0 31.9 31.9 37.5 30.5 30.6 30.5 44.50 44.50 44-50 176.8 177.1 176.8 176.9 177.1 177.1 177.1 177.3 182.00 182.1' 182.00 25.0 25.0 25.0 24.8 24.9 24.9 24.9 24.9
12 103.7 57.7 75.2 82.3 77.2 62.9 104.1 58.3 76.2 72.5 78.6 63.3
16 103.6 57.8 74.9 81.5 78.0 62.9 105.6 73.6 77.4 71.2 75.0 63.6
73.4 37.5"
73.2 37.5'
44.5" 176.8 177.1 182.00 24.8 25.0
44.5" 176.8 182.00 25.0
Signals were observed as multiplets owing to the macromolecular structure. Scheme 111. 2
,
AcO bAc
AcO AcO -
OAc W
C
NHAc O
~
~
~
p
e
16
Reagents and conditions: i, TMSOTf, ClCH&H&l, 50 "C, 15 h; ii, HO(CH2)&H=CHg, CSA, 90 "C, 2 h; iii, NaOMe/MeOH.
Allyl 2-Acetamido-3,4,6-trii-Oacetyl-2-deo.y-B-~glucopy- CH=CHg), 3.71 (m, 1H, H-5h3.73 (m, 2 H, CHzOCHd, 3.83 (q, ranoside (2). A solution of oxazoline derivative 1 (2.1 g, 6.38 1H, J 10.0 Hz, H-2), 4.13 (dd, 1 H, J
