CA2217741A1 - Synthesis of glycopolymers - Google Patents

Abstract

Neoglycoconjugates and new intermediates for the synthesis thereof are synthesized by use of Endo-.beta.-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) in a medium containing organic solvent. Such methods allow for the synthesis of novel glycoconjugates including high mannose glycopolymers.

Description

~YNl~SIS OF G~YCOPOLYMERS

R~r~,RQUND OF T~E lNV~NllON

O 1. Field of the Invention The present invention relates to glycopolymers and to methods and compositions for synthesi~ing glycopolymers. More particularly, it relates to use of Endo-~-N-acetylglucosaminidase from Arthrobacter protorhormiae, to produce neoglycoconjugates containing high-mannose type ch~ ; n .~ , 2. Backqround Information Carbohydrates possess important biological functions, such as cell-cell recognition (Wassarman, 1991; Patankar et al, 1993; and Lasky et al., 1992), lectin binding (Lee, 1988; and Lee et al., 1991, Pure & Appl.
Chem.), viral infection (Glick et al., 1991; and Toogood et al., 1991). Studies of carbohydrate functions require structurally well-defined and highly pure compounds which are usually difficult to obtain from the natural sources. Consequently, synthesis and construction of neoglycoconjugates (proteins, lipids and other types of compounds that have been derivatized with mono- or oligosaccharides) have rapidly gained attention during the past decade ~Lee, 1994). Chemical syntheses of neoglycoconjugates have been aggressively developed, but they usually involve multiple, laborious steps. Synthesis of high mannose type oligosaccharides has proven to be especially difficult, even with enzymatic methods.
Endo-$-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) is a glycosidase which performs both hydrolytic and W 096137502 PCTrUS96/06382 transglycosylation functions. This enzyme cleaves the glycosidic bond in the core GlcNAc~1,4GlcNAc residues of high mannose type and hybrid type N-linked sugar ch~;n~ in glycoprotein (Takegawa et al., 1989) and also transfers oligosacharide to some mono- and disaccharides ~Takegawa et al., l991a, l991b). (High mannose type compounds are compounds with only 2-acetylglucosamine residues immediately adjacent to the asparagine, with the r~m~; n~er of the chain being branched and usually consisting of mannose only, although further modifications with xylose and fucose are sometimes seen. A complex type compound is one consisting of N-acetylglucosamine, galactose, and sometimes lS fucose and sialic acids. A hybrid type compound is a hybrid of the two.) The efficiency of the transglycosylation reaction can be markedly increased by addition of organic solvents such as acetone, dimethyl sulfoxide (DMSO) and N,N-dimethyl formamide (DMF), to the reaction solution. For example, when transglycosylation activity o~ Endo-A is measured using Mang-GlcNAc2Asn as the donor and GlcNAc as acceptor, the ratio of transglycosylation to hydrolysis is 1:2 in aqueous medium, but when 30 acetone is added, transglycosylation will be performed to near completion. This characteristic makes it possible to synthesize novel glycosides and neoglycoconjugates with high efficency and purity.
Using this method we have synthesized several functional intermediates for neoglyco-conjugates, one of which was converted into a glycopolymer with pendant MangGlcNAc2 chains. The glycopolymer thus prepared displays a drastically greater inhibition of binding by mannose-binding protein from liver over the monomer oligo-W O 96/37502 PCTrUS96106382 saccharide.

SUMMARY OF THE INVENTION

This invention provides neoglyco-conjugates and new ~unctional intermediates for neoglycoconjugates synthesized by means of Endo-A
in reaction mixtures cont~;n;ng organic solvent.
This invention further provides synthetic high mannose type and hybrid type glycopolymers that have a high degree of purity.
This invention further provides a glycopolymer with pendant MangGlcNAc2 ch~;n~ with much greater inhibition of mannose binding protein (MBP) from liver than the monomer oligosaccharide.
This invention still further pro~ides a method of synthesizing neoglycocongugates and their functional intermediates.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Optimization of Endo-A
transglycosylation conditions. The optimum levels of the enzyme (A) and the acetone content (B) for transglycosylation were determined by the reactions carried out in a mixture of 11.6 nmol MangGlcNAc2Asn (donor), 4 ~mol GlcNAc-NAP
(acceptor) and various amounts of the enzyme (A) or 2.2 mU enzyme ~B) in 10 ~1 of 10 mM ammonium acetate buffer (pH 6.0) containing 30~ acetone (A) or different concentrations of acetone (B). The reaction mixtures were incubated at 37 C for 15 min and the products were analyzed with HPAEC-PAD.
(O): substrate; (-): transglycosylation product;
(~): hydrolytic product.
Fig. 2. Synthesis of MangGlcNAc2-NAP by Endo-A transglycosylation. The reaction was with W 096/37502 PCTrUS96/06382 5.75 ~mol MangGlcNAc2Asn, 2 mmol GlcNAc-NAP and 1.1 U of the enzyme in 5 ml of 10 mM ~3mlroI~; um acetate buffer (pH 6.0) containing 35~ acetone at 37~C for 15 min. After lyophilization, a sample equivalent to 0.7 nmol of MangGlcNAc2 oligosaccharide was injected into the HPAEC-PAD system for analysis.
The elution was performed with 100 mM NaOH and a linear gradient of NaOAc: 0 to 10~ in 20 min. A:
transglycosylation product, MangGlcNAc2-NAP; B:
hydrolytic product, Man9GlcNAc; C: remaining substrate, MangGlcNAc2Asn.
Fig. 3. lH-NMR (300 MHz) Analysis of GlcNAc-NAP (A) and MangGlcNAc2-NAP (B). The labile hydrogens in sample were exchanged with deuterium by repeating a cycle of dissolving in D2O followed by lyophilization three times before measurement.
The analyses were done in D2O using acetone (2.225 ppm) as internal standard at 25~C.
Fig. 4. Gel filtration of the glycopolymer on Sephadex G-50. The sample (1 ml) was applied onto a Sephadex G-50 column (2.5 x 90 cm), and eluted with water. The flow rate was approximately 30 ml/hr, and 4 ml fractions were collected. The neutral sugar was determined by the phenol-H2SO4 method (dotted line, absorbance at 480 nm), and GlcNAc was monitored by the absorbance at 220 nm (solid line). a: Indicates the fractions combined as the glycopolymer.
Fig. 5. 1H-NMR (600 MHz) Analysis of the glycopolymer. The chemical shifts measured in D2O
at 60~C were based on the HDO signal at 4.441 ppm.
*: Denotes the signals from the polymer back bone.
Fig. 6. Glycopolymer having MangGlcNAc2 sugar chain.
Fig. 7. Petermination of molecular weight of the glycopolymer by HPGFC. HPGFC was performed with a size exclusion column (7.5 x 600 W 096/37502 PCTrUS~G/~3~?
mm) and 0.1 M phosphate buffer (pH 7.0) containing 0.3 M NaCl as an eluent at a flow rate of 1.0 ml/min. Effluent was monitored by absorbance at 220 nm. O: glycopolymer; O: reference compounds;
~ 5 1: Blue dextran (2,000,000); 2: B-amylase (200,000); 3: alcohol dehydrogenase (150,000); 4:
albumin bo~ine serum (66,000) and 5: carbonic anhydrase (29,000).
Fig. 8. Inhibition of binding by serum-and liver-MBP-CRDs by the glycopolymer. The fitted curves were obtained using the program A~FIT (De ~ean et al., 1978). Concentrations of SBA and glycopolymer are expressed on the bases of MangGlcNAc2. ~: SBA + serum MBP-CRD; ~: SBA +
li~er MBP-CRD; ~: glycopolymer + serum MBP-CRD;
o: glycopolymer + liver MBP-CRD.

DETAILED DESCRIPTION OF THE lNv~NlION

MATERIALS AND METHODS
The following abbreviations are used in the specification:
Bn: benzyl; BSA: bovine serum albumin; CRD:
carbohydrate recognition domain; DMF: N,N-dimethyl formamide; DMSO: dimethyl sulfoxide; Endo-A:
Endo-~-N-acetyl-D-glucosaminidase from Arthrobacter protophormiae; GlcNAc:
N-acetyl-D-glucosamine; lH-NMR: 1H-nuclear magnetic resonance spectroscopy; HPAEC-PAD: high performance anion exchange chromatography with pulsed amperometric detector; HPLC: high performance liquid chromatography; HPGFC: high performance gel filtration chromatography; Man:
mannose; MBP: mannose-binding protein; 4mU:
4-methylumbelliferyl; NAP: 3- (N-acryloylamino)-propyl; pNP: p-nitrophenyl; SBA: soybean agglutinin.

W O 96/37502 PCTrUS9GIC~.?, All monosaccharides used are of the D-configuration.

Experimental Procedures Materials Endo-A was purified as described by Takegawa et al. (1989). MangGlcNAc2Asn was prepared from soybean agglutinin by exhaustive Pronase digestion, followed by gel filtration on Sephadex G-50 and further HPLC purification using a graphitized carbon column (Fan et al., 1994).
Glycoamidase A was from Seikagaku A~erica, Inc.
(Rockville, MD). GlcNAc was purchased from Pfanstiehl Laboratories, Inc. (Waukegan, IL).

3-(N-acryloylamino)-propyl ~-D-GlcNAc (GlcNAc-NAP) and GlcNAc-O-(CH2)3CH=CH2 were gifts from Dr.
Shin-Ichiro Nishimura of Hokkaido University, Japan. These can be synthesized using the procedures described by Nish;m~lra et al. (1990) and Nishimura et al. (1994a). Benzyl ~-GlcNAc, 4-methylumbelliferyl ~-GlcNAc, p-nitrophenyl ~-GlcNAc, GlcNAc-S-(CH2)6NH2, GlcNAc-_-CH2CH=CH2, GlcNAc-O(CH2)3NHCOCH=CH2, GlcNAc-S-CH2CN, GlcNAc-S-(CH2)3CH3, (GlcNAc-S-CH2CH2CH2) 2 and GlcNAc-S-CH2CONHCH2CH(OMe) 2 were synthesized in this laboratory as described by Lee et al. (1992).
Recombinant rat MBP-CRDs from serum and liver were expressed and purified according to the method of Quesenberry and Drickamer (1992) using expression plasmid-bearing bacterial strains which were gifts from Dr. Kurt Drickamer of Columbia University.

Methods Enzvmatic reaction.
A typical enzyme reaction for transglycosylation was performed in a mixture of 3 nmol MangGLcNAc2Asn, 4 ~mol acceptor and 0.9 mU of Endo-A in a total volume of 20 ~l with 25 mM
ammonium acetate buffer (pH 6.0) containing 30~
acetone. (Other organic solvents such as DMSO and DMF can also be used, with concentrations adjusted by routine experimentation to optimize the reaction.) After incubation at 37 C for 15 min, the reaction was terminated by boiling for 3 min.
in a water bath. The buffer was removed with a Speedvac using a vacuum pump. The reaction mixture was analysized using an HPAEC-PAD system (see below).

Hiqh ~erformance anion exchanae chromatoara~hy (HPAEC).
An HPAEC system consisting of a Bio-LC
(Dionex Corp., Sunnyvale, CA) equipped with a pulsed amperometric detector (PAD-II) was used for analysis of the reaction products. The chromatographic data were analyzed using an AI-450 chromatography software (Dionex). The Endo-A
reaction products were separated using a Dionex CarboPac PA-I column (4 x 250 mm) eluted at a flow rate of 1.0 ml/min with 100 mM sodium hydroxide and a gradient of sodium acetate from 30 mM to 80 mM developed in 30 min. Between runs the column was washed for 5 min. with a solution of 100 mM
sodium hydroxide/200 mM sodium acetate and allowed to equilibrate for 15 min. The PAD sensitivity was set at lK. The quantitative determination of MangGlcNAc and MangGlcNAc2 was carried out by comparison with standard materials obtained by complete digestion of MangGlcNAc2Asn by Endo-A and Man9GlcNAc2AsnPhe by Glycoamidase A. The quantity of transglycosylation products using acceptors other than N-acetyl-glucosamine was estimated by subtraction of the remaining substrate and hydrolysis product from the starting substrate.

W 096/37502 PCTrUS96/06382 TransqlycosYlation by Endo-A usinq GlcNAc-NAP as accePtOr A mixture consisting of 5.8 ~mol of MangGlcNAczAsn, 2 mmol of GlcNAc-NAP and 1.1 U of enzyme in 5 ml of 10 mM NH40Ac buffer (pH 6.0) containing 35~ of acetone was incubated at 37-C
for 15 min. After stopping the reaction by placement in a boiling water bath for 3 min., the sample was applied to a Sephadex G-25 column (2 x 140 cm), and eluted with 0.1 M acetic acid. The effluent was monitored by uv absorption at 229 nm, and the neutral sugar was determined by the phenol sulfuric acid method (McKelvy and Lee, 1969). The fractions containing high molecular weight materials were combined and lysophilized to yield 10.5 mg white powder.

PreParation of qlYcoPolvmer ha~inq Pendant chains of hiah mannose tv~e oliqosaccharide The white powder obtained from gel filtration was used as starting material for polymerization without further purification. A
small amount of the white powder (7.2 mg, ca. 3.25 ~mol MangGlcNAc2-NAP) was dissolved in 0.3 ml H20, followed by deaeration with a water aspirator for 30 min. Acrylamide (8.4 mg, 118 pmol), ammonium persulfate (APS, 0.14 ~mol) and N, N, N', N~-tetramethylethylenediamine (TEMED, 6.6 ~Lmol) were added, and the mixture was stirred at room temperature for three days, during which time, the same amounts of APS and TEMED were added to the reaction mixture daily for 2 days, and the reaction was finally completed by incubation of the mixture at 55 C for 3 hr. The reaction mixture was applied to a column (2.5 x 90 cm) of Sephadex G-50 and eluted with H20. The fractions containing the glycopolymer were combined and ~ = = ~ =

W O 96/37502 PCTrUS96/06382 lyophilized to obtain 5.3 mg of white powder.

~stimation of molecular weiqht of the qlyco~ol~mer by HPGFC
The HPGFC was performed with a Gilson HPLC system equipped with a size exclusion column (TSK-Gel G2000SW, 7.5 x 600 mm, TosoHaas, ND and a W detector (Model V4 ~ ISCO). The eluent was 0.1 M
phosphate buffer (pH 7.0) containing 0.3 M NaCl and the effluent was monitored at 220 nm. The standard compounds for molecular weight estimation were i) blue dextran (MW = 2,000,000); ii) ~-amylase (MW = 200,000); iii) alcohol dehydrogenase (MW = 150,000); iv) bovine serum albumin (MW = 66,000) and v) carbonic anhydrase (MW = 29,000).

MBP bindina of the alycoPolYmer The solid-phase binding studies were carried out essentially as described by Quesenberry and Drickamer (1992), with some minor modifications. All steps were carried out at 4 C.
Briefly, CRD (50 ~l) was coated onto individual polystyrene wells (Immulon 4 Removawell Strips by Dynatech, from Fisher Scientific). After incubating overnight, a blocking solution of 1~
BSA in 1.25 M NaCl/25 mM CaC12~25 mM Tris (pH 7.8) was added and allowed to react for 2 hours.
Ligands and inhibitors were in 0.5~ BSA in the above Tris buffer for binding and inhibition. The reference ligand used was 12sI-[mannose30-BSA] (ca.
2000 cpm/~g), radiolabelled by the Choloramine T
method (Lee et al., l991, J. Biol . Chem. ) Approximately 500 cpm/well reference ligand was incubated for 20 hr with or without inhibitors at various concentrations. The well contents were then removed, washed, and counted in a Packard -W 096/37502 PCTrUS~6/06~2 M;n;lX; gamma counter. Counts were corrected for background (counts remaining in a blocked well which was not coated with CRD), and the data were analyzed using the program ALLFIT (De Lean et al., 1978) to determine I50 values (concentration of test ligand required for 50~ inhibition) using a logistic e~uation for curve fitting.

H Nuclear maqnetic resonance spectroscopy 300 MHz NMR spectra were recorded on a Bruker AMX 300 spectrometer and measurement of a 600 MHz NMR was performed on a Bruker AM-600 spectrometer. The chemical shifts were based on acetone (~ = 2.225 ppm) as an internal standard.
The samples were prepared by three cycles of dissolving in D20 and lyophilizing followed by dissolving the residue in O.S ml of high purity D20 (99.96~ D) immediately before measurement. The 300 MHz data were recorded at 25-C and the 600 MHz data at 60-C.
Results Transal~cosylation of Endo-A to water-miscible alcohols The transglycosylation by Endo-A using MangGlcNAc2Asn as donor to various water-miscible alcohols was tested. The reactions in Table l were carried out in 20 ~l of 25 mM ammonium acetate buffer (pH 6.0) with 30~ of alcohol (v/v) containing 3 nmol MangGlcAc2Asn (donor) and 3 mU
enzyme at 37~C for 10 min. The products were determined by HPAEC using MangGlcNAc and MangGlcNAc2 as reference compounds.

W O 96/37502 PCT~US~G/0~82 Table 1. Tran~glycosylation of MangGlcNAc to alcohols by endo-A

Alcohol Hydrolysis') TranQglycosylation'~
(30% ~/~) (~) (%) ~O 94.1 0.0 MeOH 33.2 . 64.0 EtOH 45.5 46.9 PrOH 4.5 8.0 iPrOH 72.4 9.6 Allyl alcohol 0.0 0.0 Glycerol 27.8 56.5 ') Ba~ed on the starting donor substrate.

As can be seen in the Table, the enzyme transferred oligosaccharide to MeOH and EtOH with 64~ and 47~ yield, respectively, with hydrolysis levels of 33~ and 46~. The anomeric configuration of the product with MeOH was found to be ~ by lH-NMR (data not shown). PrOH (8~ yield) and iso-PrOH (10~ yield) could also serve as acceptors of transglycosylation, but allyl alcohol could not function as an acceptor. The enzyme appeared stable in 30~ MeOH and EtOH, but unstable in 30 PrOH and allyl alcohol, (the total enzyme activities [combined hydrolysis and ~ transglycosylation activities] in MeOH and EtOH
were shown to be similar to that in H2O, but much lower in the higher alcohols.) Glycerol was found to be as good an acceptor as MeOH or EtOH, with W O 96/37502 PCTrUS~G/06~82 the transglycosylation yield as high as 57~.

Transalycosylation of Endo-A to various GlcNAc qlycosides.
The transglycosylation of Endo-A to some functionalized GlcNAc glycosides was efficient as shown in Table 2. When acceptor concentration was 0.2 M, Endo-A transferred MangGlcNAc to GlcNAc-O-(CH2)6NH2 (93~ of the converted substrate), GlcNAc-O-CH2CH=CH2 (99~), GlcNAc-O-(CH2)3CH=CH2 (90~) and GlcNAc-O-(CH2)3NHCOCH=CH2 (78~) with yields of 81~, 81~, 84~ and 70~ of the starting substrate,.
respectively. The reactions were performed in 20 ~l of 25 mM ~mmop~um acetate buffer (pH 6.0) with 30~ acetone containing 3 nmol MangGlcNAc2Asn, 0.88 mU enzyme and the designated acceptor at 37~C for 15 min. The analyses were by HPAEC using 100 mM
NaOH with a linear gradient of NaOAc increasing from 30 to 80 mM in 30 min.

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W 096137~02 PCTAUS96/06382 Because of the low solubility, the concentration of benzyl ~-GlcNAc used was 0.05 M, and 4mU
B-GlcNAc and pNP ~-GlcNAc were used under saturating conditions (below 0.05 M). Even at these concentrations, the enzyme could transfer 67~, 66~ and 33~, respectively, of the starting oligosaccharide chain to them and the transglycosylation indices (the percentage of transglycosylation product to digested substrate) were found to be 82~, 77~ and 42~, respectively.
The thio-glycosides of GlcNAc are good acceptors for Endo-A transglycosylation. When GlcNAc-S-CH2CN, GlcNAc-S(CH2)3CH3 and GlcNAc-S-CH2CONHCH2CH(OMe) 2 were used as acceptors at 0.2 M, the transglycosylation indices were 88~, 86~ and 95~, with yields of 83~, 78~ and 81~, respectively. A divalent thio-glycoside of GlcNAc, (GlcNAc-S-CH2CH2CH2) 2l could be also used as acceptor for Endo-A transglycosylation at low concentratiou (below 0.05 M) with 50~
transglycosylation index and 43~ yield.

Optimization of the reaction conditions for a laraer scale transalYcosylation bv Endo-A.
In order to perform the transglyco-sylation on a larger scale, optimum levels o~ the enzyme and acetone content were examined for the transglycosylation at higher concentrations of W 096/37502 PCTrUS96/06382 substrate. As shown in Fig. lA, the hydrolytic product increased in proportion to the amount of enzyme. The yield of transglycosylation product increased upon addition of the enzyme up to 2.2 mU, then decreased as more enzyme was added. When 2.2 mU of enzyme was used, only 5.6~ substrate remained. On the other hand, the transglycosylation product increased and the hydrolytic product decreased as the acetone content was increased up to 35~ (Fig. lB). In 35 acetone, 86~ transglycosylation and 7~ hydrolysis were observed by HPAEC analysis. Although no hydrolytic product was found in the 40~ acetone medium, the efficiency of the reaction was lower compared with those in other media, because a greater amount of the substrate (64~ of starting substrate) remained.

Svnthesis of MangGlcNAc2-NAP bY transalvcosvlation activity of Endo-A.
To prepare MangGlcNAc2-NAP in a quantity useful for polymerization, the reaction scale was raised 500-fold over that in the optimum conditions described above. Transglycosylation product, MangGlcNAc2-NAP, was more than 90~ by HPAEC (Fig. 2), and the hydrolysis product as well as the starting donor substrate were barely detected. The unreacted acceptor was recovered by W 096/37502 PCT~US96/OÇ~X2 gel filtration on a Sephadex G-25 column and the MangGlcNAc2-NAP was analyzed by lH-NMR analysis and used for polymerization without further purification.
lH-NMR was used to indentify the transglycosylation product. As shown in Fig. 3A, the signals of the acceptor were completely assigned by the decoupling technique. The H-4 signal of GlcNAc was found at 3.436 ppm and the anomeric proton signal was around 4.495 ppm. On the other hand, the lH-NMR analysis of the transglycosylation product showed ten new anomeric proton signals, suggesting that the high mannose type sugar chain was transfered to the acceptor.
The lH-NMR assignments based on the reference values (Vliegenthart et al., 1983) are listed in Table 3. The lH-NMR data for GlcNAc-NAP and MangGlcNAc2-NAP were recorded on a 300 MHz spectrometer in D2O at 25~C using acetone as internal standard (~=2.225 ppm). The chemical shifts o~ MangGlcNAc2-polymer were recorded on a 600 MHz spectrometer in D2O at 60~C and relative to HDO (~=4.441 ppm).
-CA 022l774l l997-l0-08 W 096/37502 PCT~US96/06382 Ta'ole 3. ~H-NMR Dat~ of GlCNAc-NAP, M-n,~ -NAP ~nd the glycopolymer h~ving ~n,Ol r~~~ pendant chains.

Residue ~n~ -GlcNAc-NAP M-n t~ n t~
No . ') A8nb) NAPpolymer 5~-1 of 1 5.092 4.~95 4.4754.510 2 4.610 - 4.5794.611 NAc o~ 1 2.015 2.032 2.0212.041 2 2.067 - 2.0602.070 ~-1 of 3 ~4.77 - 4.7444 763 0 4 5.334 - 5.3245.322 4~ 4.869 - ~.8594.874 A 5.404 - 5.3955.379 B 5.143 - 5.1355.122 C 5.308 - 5.3005.Z90 15 D, 5.049 - 5.0345.057 D2 5.061 - 5.0345.073 D, 5.042 - 5.0345.057 C~ of a - 6.163 5.7391.701 a' - 5.748 6.1611.701 b - 6.27B 6.262--2.307 c - 3.301 3.2913.136 d _ 1.802 1.791-1.701 e - 3.944u.k.''u.k.
e' - 3.630 u.k. u.k ~I The number were the same as described in Fig. 3 b~ Cited from the published report (17).
'' u k : Unknown v W 096/37502 PCTrUS96106382 The anomeric signals agreed with those found from MangGlcNAc2Asn except two GlcNAc anomeric protons which appeared at higher field than those from the reference compound. This is because the linkages between GlcNAc and the aglycon in the former is an N-amide bond, and in the latter, an O-glycosidic bond. The coupling constant of GlcNAc-2 anomeric proton was 7.8 Hz, indicating that the linkage newly formed by Endo-A
transglycosylation is in the ~-configuration. The H-4 signal of GlcNAc at the "reducing end" at 3.436 ppm could no longer be seen, in agreement with results obtained with methyl ~-GlcNAc and indicating that the linkage occurs at the 4-OH of the GlcNAc. Mass spectrometry analysis showed the expected molecular weight of the transglyco-sylation product.

Polymerization of MangGlcNAc2-NAP with acrYlamide.
A glycopolymer was obt~;ne~ from MangGlcNAc2-NAP and acrylamide using TEMED and ASP
as catalysts. The fractions containing the polymer eluted at the void volume of the Sephadex G-50 column (Fig. 4) were pooled and lyophilized.
Completion of the polymerization was indicated by lH-NMR analysis (Fig. 5) which revealed disappearance of the signals at 6.2 ppm and 5.7 ppm, attributable to the unsaturated bond o~ the aglycon and the acrylamide monomer. The NMR also showed the existence of 11 anomeric proton signals, and the chemical shifts were similar to those found from the monomer (Table 3), confirming that the polymer contains MangGlcNAc2-sugar chains.
The sugar content of the polymer was estimated to be 37~ by the phenol-H2SO4 method using mannose as standard. There~ore, the ratio o~ sugar side ch~in.~: to acrylamide residues is estimated to be W 096/37S02 PCTrUS96/06382 1:44 as shown in Fig. 6.
Other compounds having a double bond at the terminal position (e.g. GlcNAc-O-CH2CH=CH and other representative compounds shown in Table 2) can be polymerized in essentially the same way.
In addition to acrylamide, other monomers (including, for example, styrene derivatives, vinyl, epoxide and ethylen;m;ne type compounds and other compounds with unsaturated bonds) can also ~10 be polymerized.

Determination of the molecular weiqht of the qlvcopolymer The molecular weight of the glycopolymer was estimated by HPGFC using blue dextran, B-amylase. alcohol dehydrogenase, bovine serum albumin and carbonic anhydrase as reference compounds. The polymer appeared near the void volume, and the retention volume was slightly greater than blue dextran (molecular weight =
2,000,000). According to the calibration curve (Fig. 7), the molecular weight is between 1,500,000 and 2,000,000.

Inhibition of mannose-bindinq ~roteins bY the alycopolymer.
A solid-phase binding assay was carried out on serum- and liver-MBP-CRDs, using the MangGlcNAc2glycopolymer and soybean agglutinin (SBA), which contains the same MangGlcNAc2. The results of the assay are shown in Fig. 8. In the concentration range of SBA tested, no significant inhibition of the serum-MBP-CRD was observed. For the liver-MBP-CRD, however, an Iso value of 13.2 ~M
based on MangGlcNAc2 or 0.4 mg/ml of SBA was obtained. However, the glycopolymer showed an Iso of 3.5 ~M for the serum-MBP-CRD, and an Iso of 74.5 W 096/37502 PCT~US~6/06~2 nM for the liver-~3P-CRD. In terms of the whole glycopolymer, the I50 values would be approximately 2.0 x 10-2 mg/ml for the serum-MBP-CRD and 3.8 x 10-4 mg/ml for the liver-MBP-CRD, respectively.
The magnitude of inhibitory potency enhancement of the glycopolymer over the precursor cannot be calculated with certainty for the serum form of MBP-CRD, because MangGlcNAc2 hardly inhibits this MBP-CRD. However, for the liver form, an enhancement was about 18o-fold based on the MangGlcNAc2, and ca. 1,000-fold based on the moleculars, although the sugar content of the glycopolymer was only 5.6-fold higher than SBA.

Discus~ion Endo-A demonstrates an efficient transglycosylation activity (~ 90%) in 30%
acetone, much higher than the 10-30% reported for other glycosidases (Bardales et al., 1989; Sakai et al., 1992; Cantacuzene et al., 1991; Nilsson, 1987 and 1989; Usui and Murata, 1988; and Usui et al., 1994). This finding has been utilized to synthesize neoglycoconjugate intermediates which are ~men~hle to further reactions.
Endo-A also transfers MangGlcNAc to alcohols such as MeOH, EtOH and PrOH. The transglycosylation to MeOH (64~ yield) and EtOH
(47% yield) compares favorably with those by ~-xylosidase, ~- and ~-glucosidase and ~-galactosidase (20-60~) from various sources (Shinoyama et al., 1988; and Shinoyama and Yasai, 1988). However, transglycosylations to PrOH and iPrOH were not as effective as to MeOH and EtOH.
Interestingly, although the total enzyme activity was lower in PrOH than in iPrOH, transglyco-sylation to PrOH was greater than to iPrOH.
Glycerol was also a good acceptor for Endo-A

W O 96/37502 PCT~US9610G382 transglycosylation. Endo-B-N-acetylglucosaminidase F (Trimble et al., 1986) and Endo-~-N-acetylgalactosaminidase from Di pl ococcus pneumoniae ~Bardales and Bhav~n~n~An, 1989) have been reported to transfer an oligosaccharide to the Cl(3) hydroxyl o~ glycerol.
Several GlcNAc derivatives having functionalized aglycons useful for synthesis of neoglycoconjugates were tested as acceptors for Endo-A transglycosylation. The yields based on the starting donor substrate were found to be greater than 80~ with 0.2 M acceptor and about 50 when 0.05 M or less was used in our system. The yield of transglycosylation can be further improved if higher acceptor concentrations are employed.
Endo-A transglycosylation is also effective at higher concentrations of reactants, as shown in Table 2. In the larger-scale transglycosylation to GlcNAc-NAP, transglycosylation yield (~ 90~) was even hiyher than those at the analytical scale reaction. A
similar yield (89~) can be obtained from the transglycosylation to GlcNAc~-OMe on a similar scale (4 ~mol).
An Endo-A transglycosylation product, MangGlcNAc2-NAP, was further polymerized with acrylamide to form a glycopolymer. Glycopolymers having di- or trisaccharide have been synthesized by chemical or chemo-enzymatic method recently (Kochetkov, 1984; Nishimura et al, 1991; Nishimura et al., 1994a and 1994b; Kobayashi et al., 1994;
and Fukase et al., 1994), but to our knowledge this is the first time glycopolymers with highly complex sugar chains have been synthesized. The high efficiency of Endo-A transglycosyla~ion provides an easier way to synthesize such W 096/37502 PCTÇUS96/06382 neoglycoconjugates.
Clustering of monosaccharides by attachment to a simple branched peptides enhances inhibitory potencies for some C-type lectins (Lee and Lee, 1987; and Lee et al., 1992). An affinity enhancement achieved by multi~alent ligands over monovalent ones that is greater than would be expected from a simple effect of a local concentration increase is termed the "glycoside cluster effect". Formation of glycopolymers is convenient way to provide glycoside clustering (Lee and Lee, 1994). In the instant invention, a dramatic increase in the inhibition of MBP-CRDs in comparison with that by the native glycoprotein (SBA) which contains the same MangGlcNAc2 oligosaccharide is demonstrated. In the case of the liver MBP-CRD, an approximately 180-fold enhancement of inhibitory potency over the native glycoprotein (SBA) was attained by the glycopolymer. Similarly, although no significant inhibition of the serum MBP-CRD was observed for SBA, the glycopolymer derived from its oligosaccharide demonstrated a surprisingly strong inhibitory potency (Iso=3.5 ~M). This is a good example of "macro-" vs. "micro-clustering" (Lee, 1993). ["Micro-clustering" describes a condition where the spatial arrangement of the target sugars is such that the distances between combining sites are small--e.g. 1.5 to 3.0 nM; "macro-cluster"
describes a condition where the spatial arrangement is such that the distances are much greater (e.g. 50-100 nM, as here)]
It will be apparent that the compounds described herein have many potential uses. In addition to their utility in the study of carbohydrate function and metabolism, the various compounds may also be used for diagnostic and therapeutic purposes, for example as antigens or for the measurement or isolation o~ specific carbohydrate binding proteins.
Measurement of MBP in a serum sample MBP is one of the acute phase proteins produced by liver in response to invading microorganisms or other foreign agents (Reid, 1983, Sastry et al., 1991). MBP binds to these agents, leading to their destruction either directly or through the participation of macrophages. The MangGlcNAc2 glycopolymer of the present invention has a much greater binding affinity than natural products containing mannose, and should thus be useful for diagnosis in a manner similar to that of C-reactive protein (Oyamada et al, 1992; Ohtake, 1993).
To measure the amount of MBP in a serum sample, the following procedure can be used:
1) The MangGlcNAc2 glycopolymer of Fig. 6 is conjugated to an enzyme commonly used for ELISA assays, for example alkaline phosphatase or ~-galactosidase.
2) A monoclonal antibody against MBP which does not affect its ability to bind mannose is placed in a well to coat its sur~ace. Such antibodies can be made using standard techniques known to the skilled practitioner, for example as described by Quesenberry and Drickamer (1992). A sample of serum to be tested is placed in the coated well and incubated under conditions favorable for binding of MBP to the antibody.
3) The unbound material is removed by suitable washing, and the glycopolymer-phosphatase complex of (1) is placed in the well. The MBP bound to the antibody W 096/37502 PCT/U~5''OG382 now binds the glycopolymer, acquiring the phosphatase activity. Upon addition of a suitable substrate, the level of phosphatase activity is a measure of the MBP in the serum sample.
Alternatively, the well can be coated with the unconjugated glycopolymer, the serum sample added, and bound MBP reacted with anti-MBP
conjugated to phosphatase or another ~uitable enzyme. The level of MBP can then be determined, as before, by the bound enzymatic activity.
While the invention has been described in connection with what is presently considered to be the most practical and perferred embodiment, it is to be understood that the invention is not to be limited to the disclosed enbodiment, but is intended to cover various modifications included within the spirit and scope of the appended claims.
The references earlier mentioned are more fully identified hereafter, and are hereby incorporated by reference and relied upon.

W 096/37502 PCTrUS96/06382 Bardales, R. M., and Bhav~n~n~n, V. P. (1989) ~.
Biol. Chem. 264, 19893-19897.
Cantacuzene, D., Attal, S., and Bay, S. (1991) Biomed. Biochim. Acta 50, S231-S236.
De Lean, A., Munson, P.J. and Rodbard, D. (1978) Am. J. Physiol. 235, E97-E102.
- Fan. J.-Q., Kondo, A., Kato, I., and Lee, Y.C.
(1994) Anal. Biochem. 219, 224-229.
Fukase, K., Nakayama, H., Kurosawa, M., Ikegaki, T., Kanoh, T., Hase, S., and Kusumoto, S. (1994) J. Carbohydr. Chem. 13, 715-736.
Glick, G. D., Toogood, P. L., Wiley, D. C., Skehel, J. J., and Knowles, J. R. (1991) J. Biol.
Chem. 266, 23660-23669.
Kobayashi, K., Kakishita, N., Okada, M., Akaike, T., and Usui, T. (1994) J. Carbohydr. Chem . 13, 753 -766.
Kochetkov, N. K. (1984) Pure & Appl. Chem. 56, 923-938.
Lasky, L. A. (1992) Science 258, 964-969.
Lee, Y. C. (1988) in "The Molecular Immunology of Complex Carbohydrates", (Wu, A. M., Ed.), Series Plenum Publishing Corporation, pp. 105-121.
Lee, Y. C. (1993) Biochem. Soc. Trans. 21, 460-463.
Lee, Y. C. (1994) in "Neoglycoconjugates:
Preparation and Applications" (Lee, Y. C. and Lee, R. T., Eds) Academic Press, San Diego, pp. 3-21.
Lee, R. T., Ichikawa, Y., Kawasaki, T., Drickamer, K. and Lee, Y.C. (1992) Arch. Biochem. Biophys.
299, 129-136.
Lee, R. T., Ichikawa, Y., Fay, M., Drickamer, M.
C., Shao, M. C., and Lee, Y. C. (1991) J. Biol.
Chem. 266, 4810-4815.
Lee, R. T. and Lee, Y.C. (198 7) Methods Enzymol.
138, 424-429.
Lee, Y. C., and Lee, R. T. (1992) in ~ "Glycoconjugates: Composition, Structure, and Function~ ~Allen, H. J., and Kisalius, E. C., Eds.), Marcel Dekker, Inc., New York, pp. 121-165.

W 096/37502 PCT~US9''~382 Lee, R. T., and Lee, Y. C. (1994) in ~Neoglycoconjugates: Preparation and Applications"
(~ee, Y. C. and Lee, R. T., Eds.) Academic Press, San Diego, pp. 23-50.
Lee, Y. C., Lee, R. T., Rice, K., Ichikawa, Y., and Wong, T.-C. (1991) Pure & Appl. Chem. 63, 499-506.
McKelvy, J. F., and Lee, Y. C. (1969) Arch.
Biochem. Biophy. 132, 99-110.
Nilsson, K. G. I. (1987) Carbohydr. Res. 167, 95-103.
Nilsson, K. G. I. (1989) Carbohydr. ~es. 188, 9-17.
Ni~h;mllra S., Furuike, T., Matsuoka, K., Murayama, S., Nagata, K., Kurita, K., Nishi, N., and Tokura, S. (1994a) Macromolecules 27, 4876-4880.
Nishimura, S., Matsuoka, K., Furuike, T., Ishii, S., Kurita, K., and Nishimura, K. M. (1991) Macromolecules 24, 4236-4241.
Nishimura, S., Matsuoka, K., and Kurita, K.
(1990) Macromolecules 23, 4182-4184.
Nishimura, S., Matsuoka, K., Furuike, T., Nishi, N., Tokura, S., Nagami, K., Murayama, S., and Kurita, K. (1994b) Macromolecules 27, 157-163.
Ohtake, T. (1993) Med. Technol. 721, 287-293.
Oyamada, H., Nakagomi, O., and Usugi, S. (1992) Jap. J. Clin. Path. 40, 9-15.
Patankar, M. S., Oehninger, S., Barnett, T., Williams, R. L., and Clark, G. F. (1993) J. Biol.
Chem. 268, 21770-21776.
Quesenberry, M. S. and Drickamer, K. (1992) J.
Biol. Chem. 267, 10831-10841.
Reid, K. B. M. (1983) Bioc~em. Soc. Trans. 11, 1-12.
3S Sakai, K., Katsumi, R., Ohi, H., Usui, T, and Ishido, Y. (1992) J. Carbohydr. Chem. 11, 553-565.
Sastry, K, Sahedi, K., Lelias, J.-M., Whitehead, A. S. and Ezkowitz, R. A. B. (1991) J. Immunol.
147: 692-697.
Shinoyama, H., Kamiyama, Y., and Yasui, T. (1988) CA 022l774l l997-l0-08 W 096/37S02 PCTrUS96/06382 Agric. Biol. Chem. 52, 2197-2202.
Shinoyama, H., and Yasui, T. (1988) Agric. Biol.
Chem. S2, 2375-2377.
Stowell, C.P., and Lee, Y.C. (1993) In "Methods in Carbohydrate Chemistry, Vol. IX", (J.N. BeMiller, R.L. Whistler, and D.H. Shaw, eds.) Wiley & Sons, Inc., pp. 173-178.
Takegawa, K., Nakoshi, M., Iwahara, S., Yamarnoto, K., and Tochikura, T. (1989) Appl . Environ .
Microbiol. 55, 3107-3112.
Takegawa, K., Tabuchi, M., Yamaguchi, S., Kondo, A., Kato, I., and Iwahara, S. (1995) J. Biol.
Chem. 270, 3094-3099.
Takegawa, K., Yamaguchi, S., Kondo, A., Iwamoto, H., Nakoshi, M., Kato, I., and Iwahara., S.
(199la) Biochem. Int. 24, 849-855.
Takegawa, R., Yamaguchi, S., Kondo, A., Kato, I., and Iwahara, S. (199lb) Biochem. I~t. 25, 829-835.
Toogood, P. L., Galliker, P. K., Glick, G. D., Knowles, J. R. (1991) ~. Med. Chem. 34, 3140-3143.
Trimble, R. B., Atkinson, P. H., Tarentino, A. L., Plummer, T. H., Jr., Maley, F., and Tomer, K. B.
(1986) J. Biol. Chem. 261, 12000-12005.
Usui, T., and Murata, T. (1988) ~J. Biochem. 103, 969-972.
Usui, T., Suzuki, M., Sato, T., Kawagishi, H., Adachi, K., and Sano, H. (1994) Glycoconjugate J.
11, 105 - 110.
Vliegenthart, J. F. G., Dorland, L, and van Halbeek, H., (1983) Adv. Carbohydr. Chem. Biochem.
(Tipson, R. S., and Horton, D., Eds.), Vol. 41, pp. 209-373.
Wassarman, P. M. (lssl) Development 108, 1-17.

Claims (11)

WHAT IS CLAIMED IS:

1. A neoglycoconjugate prepared by a method which includes the step of synthesizing a glycoside in a reaction mixture comprising Endo-.beta.-N-acetylglucosaminidase from Arthrobacter protophormiae and an organic solvent.

2. A monomer or intermediate for neoglycoconjugate synthesis which is synthesized by means of Endo-.beta.-N-acetylglucosaminidase from Arthrobacter protophormiae in a reaction mixture containing an organic solvent, said monomer or intermediate being selected from the group consisting of R-G1cNAc-O-(CH2)3NHCOCH=CH2 , R-G1cNAc-O-CH2CH=CH2, R-GlcNAc-O-(CH2) 3 CH=CH2 , R-G1CNAC-S -CH2CN, R-G1cNAc-S-CH2CONHCH2CH(OMe) 2 and R-G1cNAc-O-(CH2)6NH2 , where R represents ManxG1cNAc and x is an integer between 3 and 12, inclusive.

3. The monomer of claim 2 wherein the organic solvent is acetone.

4. The monomer of claim 2, wherein x is an integer between 6 and 9, inclusive.

5. The monomer of claim 3, wherein x = 9.

6. A glycopolymer that contains high mannose type sugar chains and whose synthesis comprises i) combining a donor and an acceptor in a reaction mixture containing Endo-.beta.-N-acetylglucosaminidase from Arthrobacter protophormiae and organic solvent, ii) incubating the reaction mixture under suitable conditions to produce a transglycosylation product, iii) purification of said transglycosylation product of the reaction mixture on a Sephadex column, and iv) polymerization of said transglycosylation product.

7. The glycopolymer of claim 6 where polymerization is achieved by use of acrylamide.

8. A glycopolymer with pendant Man9GlcNAc2 chains which inhibits mannose binding protein from liver.

9. The glycopolymer shown in Figure 6.

10. A method of synthesizing a glycopolymer containing mannose comprising the steps of i) combining a donor and an acceptor in a reaction mixture containing Endo-.beta.-N-acetylglucosaminidase from Arthrobacter protophormiae and organic solvent, ii) incubating the reaction mixture under suitable conditions such that a transglycosylation product is produced, and iii) polymerization of said transglycosylation product.

11. The method according to claim 10 where said glycopolymer is a glycopolymer with pendant Man9GlcNAc2 chains.