IE903042A1 - Process for the enzymatic synthesis of galactosylated glycoprotein units - Google Patents

Abstract

Synthesis of Gal- beta (1->4)-GlcNAc- beta (1->N)-Asn and Gal- beta (1->4)-GlcNAc- beta (1->4)-GlcNA- beta (1->N)-L-Asn from the precursors aspartyl-N-acetylglucosamine and aspartylchitobiosylamine in the presence of a galactosyl donor is possible in vitro with the aid of galactosyl- transferase. The galactosyl donor UDP-Gal can be synthesised in situ from UDP-Glc with catalysis by UDP-galactose 4-epimerase.

Description

HOECHST AKTIENGESELLSCHAFT HOE 89/F 274 Dr.KH/fe Description: Process for the enzymatic synthesis of galactosylated glycoprotein units Glycoproteins play a central part in biological recognition processes, such as tumorigenesis, bacterial and viral infection, cell/cell recognition, cell growth and cell differentiation. They form the basis of blood group classification and are responsible for the internaliz10 ation of various macromolecular substances and medicaments. Thus, an important area of use of glycoproteins is, for example, the selective directing of pharmaceuticals to the target organ and the protection of medicaments from proteolytic breakdown [N. Sharon, H. Lis, Chem. Eng. News 59 (13) (1981) 21; H. Schachter. Clinical Biochemistry 17, (1984) 3].

Glycoproteins are categorized as O-glycosidic and Nglycosidic glycoproteins depending on the kind of linkage between the peptide moiety and the carbohydrate moiety.

The N-glycosidic glycoproteins almost exclusively contain N-acetylglucosamine linked to asparagine as the central coupling unit. With respect to the stated functions of glycoproteins, the synthesis of glycosylated aspartyl-Nacetylglucosamines deserves particular attention.

The synthesis of such glycoprotein units using enzymes which follow the biosynthetic pathway of these substances in vitro has the advantage of a high stereoselectivity and regioselectivity and also of avoiding laborious chemistry involving protecting groups, whereby a good yield is ensured in most cases in comparison with the multi-step chemical synthesis.

Galactosyltransferase (EC 2.4.1.22) regiospecifically and stereospecifreally transfers galactose to N-acetylglucosamine with a >9(l-*4) linkage being formed. Natural - 2 10 substrates of galactosyltransferase are glucose, which, however, is accepted by the enzyme only in the presence of Q-lactalbumin, and N-glycoproteins having N-acetylglucosamine on the terminal end. Reactions of the enzyme with unnatural substrates are described in the following publications: 1. H.A. Nunez, R. Barker; Biochemistry 19, 489 (1980) 2. J. Thiem, W. Treder, T. Wiemann; Dechema Biotechnology Conferences, Vol. 2, 189 (1988), ed.: D. Behrens, VCH-Verlagsgesellschaft, Weinheim 3. C. Aug6, S. David, C. Mathieu, C. Gautheron, Tetrahedron Lett. 25, 1467 (1984) 4. U. Zehavi, (1984) M. Herchman; Carbohydr. Res. 128, 160 5. U. Zehavi, S. Sadeh, M. Herchman; Carbohydr. Res . 124. 23 (19831 6. U. Zehavi, M. Herchman; Carbohydr. Res. 133, 339 (1984) 7. C. Aug6. C. Mathieu, C. Merienne; Carbohydr. Res. 151, 147 (1986) 8. M.M. Palcic, O.P. Srivastava, 0. Hindsgaul; Carbohydr. Res. 159, 315 (1987) In this case, saccharides or lower oligosaccharides having glucose or else N-acetylglucosamine on their ter25 minal end are converted with the aid of the enzyme.

Glyco conjugates amino acids and carbohydrates, in particular corresponding asparagine derivatives, have hitherto not been used as substrates.

It has now been found that galactosyl transferase also allows the synthesis of glycoasparagines of the formulae I and II, it being possible to synthesize chemically the non-galactosidic part beforehand by a combination of known processes.

I Gal-0 (l-*4)-GlcNAc-0 (1-»N)-Asn (I) occurs in its completely, i.e. including the amino acid moiety, unblocked form in the urine of patients who suffer from aspartylglucosaminuria [R.J. Pollitt, K.M. Pretty, Biochem. J. 141 (1974) 141].

Gal-0 (1-4)-GlcNAc-0 (l-*4)-GlcNAc-0 (1->N)-L-Asn (II) is found in erythrocyte membranes. It has been speculated that it represents the receptor site for Robinia lectin there. [A.M. Leseney, R. Bourrillon, S. Kornfeld, Arch. Biochem. Biophys. 153 (1972) 831].

The invention thus relates to a process for the enzymatic preparation of the compound of the abovementioned formula I or the compound of the abovementioned formula II, which comprises incubating the compound of the formula III or the compound of the formula IV NHAc § NHAc co2ch3 III with a galactosyltransferase in the presence of a 20 galactosyl donor.

Hereinafter, the invention is explained in detail and defined in its patent claims.

Galactosyltransferase, for example EC 2.4.1.22, accepts - 4 the glycoasparagines of the formulae III and IV as substrates, which in this process are galactosylated in the 0(l->4) position on the terminal N-acetylglucosamine unit. The enzyme requires uridine-5'-diphosphate-galactose (UDP-Gal) as galactose donor. However, this nucleotide sugar is very expensive and it is therefore more advantageous to produce it in situ from uridine-5'-diphosphateglucose (UDP-Glc) in a reaction which is catalyzed by the enzyme uridine-5'-diphosphate-galactose 4-epimerase (EC .1.3.2).

For the preparation of the compound I or II the respective starting substrates III or IV mentioned are treated with galactosyltransferase in aqueous solution together with a galactosyl donor, such as for example UDP-Gal. The starting substrates and the galactosyl donor are each employed in equimolar concentration, preferably in 10 to 15 millimolar quantity. Commercially available galactosyltransferase can be used in soluble or immobilized form. If immobilization is carried out, silica gel is preferred as carrier, in particular for economic reasons.

The reaction is advantageously carried out in a buffered solution. Sodium cacodylate in particular is used as buffer. The incubation temperature can vary in wide ranges according to the temperature/activity range of the enzyme. The reaction is preferably carried out at 32-37°C.

The reaction according to the invention can, as already mentioned above, start with the preparation of UDP-Gal from UDP-Glc with the aid of uridine-5'-diphosphate30 galactose 4-epimerase in a so-called one-pot process.

A one-pot process is understood to mean that all participating substrates and all participating enzymes are put together in one reaction mixture. The mentioned reactions, the preparation of UDP-Gal from UDP-Glc and the prepara35 tion of the compounds I or II, can, of course, also be carried out sequentially. However, a one-pot process proved to be more economic when the above-described - 5 reaction parameters were maintained for the whole mixture.

Detection of the progress of reaction is carried out by thin layer chromatography. The final products are separa5 ted off after the nucleotide phosphates have been removed with ion exchange chromatography, such as, for example, on Dowex 1-X8 (counter-ion: C1‘), and gel chromatography with, for example, Bio-Gel P2, and are obtained in a yield of 25 - 35 %.· The galactosyl acceptors III and IV can be chemically synthesized by methods derived from the papers of various authors [D.E. Cowley, L. Hough, C.M. Peach, Carbohydr. Res. 19 (1971) 231; M. Spinola, R.W. Jeanloz, J. Biol.

Chem. 245 (1970) 4158; H. Kunz, H. Waldmann, Angew. Chem. 97 (1985) 885].

In the case of aspartyl-N-acetylglucosamine III, Nacetylglucosamine is converted with acetyl chloride into the peracetylated α-chloride which is then converted to the /9-azide by phase-transfer catalysis in water/chloro20 form. After reduction to the £-amine with hydrogen over palladium, coupling to α-methyl N-acetylaspartate can be carried out in an ethyl 2-ethoxy-l,2-dihydroquinoline-lcarboxylate (EEDQ)-assisted condensation. After de-Oacetylation with sodium methanolate, the galactosyl acceptor of the formula III is obtained and must be purified by gel chromatography before the enzymatic galactosylation.

For the synthesis of the aspartylchitobiosylamine IV, chitin is first cleaved by acetolysis into an oligomer mixture from which chitobiose octaacetate can be isolated by flash chromatography. This is converted into the peracetylated α-chitobiosyl chloride by suspending in acetyl chloride and passing in gaseous hydrogen chloride. Corresponding to the synthesis of the compound of the formula III, the α-chloride is converted into the /3-azide - 6 with sodium azide, reduced to the 0-amine, coupled to amethyl N-acetylaspartate and finally de-O-acetylated. The aspartylchitobiosylamine of the formula IV thus obtained can, after purification by gel chromatography, be used for the described enzymatic galactosylation.

The invention is illustrated in more detail hereinafter with the aid of examples.

Example 1. Procedure for the galactosylation of the glycosyl10 asparagines III and IV a. The reaction is carried out in sodium cacodylate buffer (25 mM, pH 7.7) in the presence of manganese chloride (5 mM) . After the addition of UDP-galactose (13 mM), glycosylasparagine (III or IV, 13 mM) and bovine serum albumin (1 mg/ml) and deoxygenation with nitrogen, galactosyltransferase (EC 2.4.1.22, 10 U) are added. After two days at 37°C, the mixture is worked up using an anion exchanger (Dowex 1-X8, chloride form) and is subsequently purified by gel chromatography on Bio-Gel P2. b. The reaction is carried out in sodium cacodylate buffer (25 mM, pH 7.7) in the presence of manganese chloride (5 mM) . After the addition of UDP-glucose (13 mM), glycosylasparagine (III or IV, 13 mM) and bovine serum albumin (1 mg/ml) and deoxygenation with nitrogen, galactosyltransferase (EC 2.4.1.22, 1 U) and UDP-galactose 4-epimerase (EC 5.1.2.2, 10 U) are added. After two days at 37°C, the mixture is worked up using an anion exchanger (Dowex 1-X8, chloride form) and subsequently purified by gel chromatography on Bio-Gel P2. 1.1) 2-Acetamido-4-0-(0-D-galactopyranosyl) -1-N-(Nacetyl-l-methyl-4-L-aspartyl) -2-deoxy-0-D-glucoIE 903042 - 7 pyranosylamine (I) Mixture: 64.5 mg (100 pmol) of UDP-Gal mg (100 pmol) of compound III TLC checks: mobile phase n-propanol/acetic acid/water 5 85:12:3 Rf values: GlcNAc-Asn (III) 0.22 Gal-GlcNAc-Asn (I) 0.08 Yield: 20 mg (35 %j Selected XH-NMR data (300 MHz, D20): δ = 5.06 (d, IH, H10 1), 4.40 (d, IH, H-l'), 2.95 (dd, IH, β-ksn), 2.82 (dd, IH, £'-Asn); Jx 2 = 9.4, J1>>2. = 7.6, Ja-β = 5.55, JB e. = 17.1, Ja-β’ = 7.63 Hz. 1.2) 2-Acetamido-4-[2-acetamido-2-deoxy-4-(0-D-galactopyranosyl)) -j3-D-glucopyranosyl )-1-N-(N-acetyl-115 methyl-4-L-aspartyl) -2-deoxy-^-D-glucopyranosylamine (II) Mixture: 32.0 mg (50 pmol) of UDP-Gal 29.0 mg (50 pmol) of compound IV TLC checks: mobile phase n-butanol/acetic acid/water 20 2:4:1 Rf values: GlcNAc-GlcNac-Asn (IV) 0.31 Gal-GlcNAc-GlcNAc-Asn (II) 0.19 Yield: 10 mg (25 %) Selected hl-NMR data (330 MHz, D20): δ = 5.09 (d, IH, H25 1), 4.62 (d, IH, H-l'), 4.47 (d, IH, H-l), 2.06, 2.02, 2.01 (each s, each 3H, each NAc); Jx 2 = 9.6 Hz, Jx. 2. = 8.1 Hz, Jj.,,2» = 7.7 Hz.

Claims (6)

1. Patent Claims HOE 89/F 274 A process for the enzymatic preparation of compounds of the formula I or II N Y~r 0 NH, COjCH, NHAc which comprises incubating the compound of formula III or the compound of the formula IV the HO AA NHAc C0,CH 3 NHAc III 0 NH with galactosyltransferase in the presence of a galactosyl donor.

2. The process as claimed in claim 1, wherein UDP-Gal is used as galactosyl donor.

3. The process as claimed in claim 1 or 2, wherein UDPGal is synthesized from UDP-Glc with catalysis by UDP-galactose 4-epimerase.

4. The process as claimed in one or more of claims 1 to

5. 5.

6. 6. 3, wherein the reaction is carried out in a buffered solution. The process as claimed in one or more of claims 1 to 4, wherein the reaction is carried out at 32 to 37°C. A process according to claim 1 for the enzymatic preparation of a compound of the formula I or II given therein, substantially as hereinbefore described and exemplified. A compound of the formula I or II given in claim 1, whenever obtained by a process claimed in a preceding claim.