DE19802748A1 - Synthesis of glycosamino acid derivatives from lactose and serine derivative, used for targeting therapeutic and diagnostic agents - Google Patents

Abstract

The synthesis of glycosamino acid derivatives (I), particularly galactosylamino acid derivatives (Ia) is new, and comprises using lactose as donor and serine derivatives as acceptor, in the presence of various beta -galactosidases. The reaction takes place in a medium containing 50 - 95 wt.% water and 5 - 50 wt.% organic solvent. (Ia) are of formula Y-(Gal beta 1)-Ser-OX: Y = allyloxycarbonyl, tert-butoxycarbonyl or benzyloxycarbonyl; X = hydrogen, Me or Et

Description

The invention relates to a process for the enzymatic synthesis of glycoamino acid derivatives, especially galactosylamino acid derivatives.

Have glycoamino acids as an essential structural element of glycopeptides essential importance as terminal components in cell receptors for specific Connection of viruses, blood group and tumor-recognizing antibodies, bacterial Toxins and a variety of lectins. The oligosaccharide chains from Glycoproteins continue to influence their solubility, charge distribution, Conformation and contribute to protection against proteolytic enzymes. The Linking a saccharide molecule to a biologically active peptide sequence, see above one hopes, made possible by specific interaction of the saccharide residue with the Cell surface the selective addressing of pharmaceuticals and their protection against proteolytic degradation. The goal of making glycosylated amino acids effective The ability to use components in diagnostics and therapeutics Demand for efficient syntheses. Because glycloproteins are relatively difficult to access are, one synthesizes partial structures, the glycopeptides or their Basic building blocks - the glycoamino acids.

The complex multi-stage synthesis of the classic chemical processes, which optimally adapted protective group combinations from the carbohydrate and peptide chemistry, have been promising enzymatic tech for some time niken to the side. The decisive advantage in enzyme-catalyzed Synthesis lies in the high stereo and regional specificity. Elaborate protective groups techniques are not necessary here.

In recent years, glycosidases have become glycosidic in their suitability To catalyze links, examined. Glycosidases allow in natural Systems hydrolytic cleavage of glycosides, e.g. B. the splitting of milk  sugar (lactose) in glucose and galactose by β-galactosidase, which by Intestinal bacteria (Escherichia coli) is formed. Instead of water, which in the enzymatic hydrolysis acts as a nucleophilic reaction partner, are theoretical other nucleophiles with suitable hydroxyl groups are also conceivable. Verifiable are some monosaccharides, primary and secondary alcohols or - like in naturally occurring structures - the hydroxylamino acids serine and threonine as easily connectable nucleophiles (glycosyl acceptor). An O-glycosidic Linking is possible in principle if the glycosyl acceptor to be linked with regard to its steric structure are accepted by the enzyme and a have sufficiently high nucleophilicity in relation to the reactant Water to compete.

Depending on the desired target product, an activated glycoside, e.g. As cellubiose or lactose, can be used as the substrate, which provides the saccharide to be linked by its cleavage (glycosyl donor). The enzymatic synthesis of amino acid glycosides with glycosidases is limited to the use of hydroxy amino acids. The following reaction scheme illustrates the glycosidase-catalyzed synthesis of β-O-linked glycoamino acid derivatives:

Y = tert-butyloxycarbonyl (boc), allyloxycarbonyl (Aloc), benzyloxycarbonyl (Z)
X = methyl, ethyl, -H.

Many glycosidases that are suitable for these syntheses are commercial available and compared to the glycosyltransferases - which in nature the Take over glycosylation of peptides - much cheaper.  

To date, numerous glycoamino acid derivatives such as Y- (Galβ1) Ser-OX, Y- (Galβ1) Thr-OX, Y- (NAcGalβ1) Ser-OX or Y- (Gluβ1) Ser-OX (Y, X-amino or carboxyl protecting groups) by chemo-enzymatic means be prepared, isolated and characterized (Cantacuzène, D. Attal, S. Bay, S. Bioorganic & Medical Chemistry Letters, Vol. 1, No. 4 pp 197-200, 1991, Nielsson, K- G., Scigelova, M. Biotechnology Letters, Vol. 16, No./(July 1994), pp 671-676).

The main disadvantage of this synthetic process is the low product content loot. When using commercially available glycosidases and their natural Substrates as glycosyl donors can be achieved in kinetically controlled reactions rarely more than 15%, in equilibrium controlled reactions less than 10%, Yield. The main reason for this is the parallel to the glycosidic linkage from ongoing secondary hydrolysis of the target product, since the glycoamino formed acid derivative is also a good substrate for the enzyme. Increases in Yields are obtained by using some unnatural substrates as Glycosyl donors reached (Holla, E. W., Schudock, M., Weber A., Zulauf, M., J. Carbohydrate Chemistry, 11 (5), 659-663, 1992). Here occur in the course of Synthesis temporarily (kinetically controlled reactions) yields of up to 40% on. However, these synthetic substrates are relatively expensive and expensive the toxicity of their fission products is not harmless.

The aim of the invention is a new process for the production of O-glycosidic Linkages of hydroxyamino acid derivatives - especially of serine derivatives with D-galactose by β-galactosidases of different origins. It should try be, the syntheses of the desired target compounds, in particular by Modeling of a suitable reaction environment to optimize a to allow convenient and economically viable access to this class of substance lichen.  

According to the invention, the process for the enzymatic synthesis of glycoamino acid derivatives, in particular galactosylamino acid derivatives of the form Y- (Galβ1) Ser-OX [NY-3-O-β-D-galactopyranosyl-L-serine-X-ester]

wherein
Y = Aloc (allyloxycarbonyl-), Boc (tert-butoxycarbonyl-) or Z (benzyloxycarbonyl-) and
X = methyl, ethyl or -H,
with lactose as galactosyl donor and serine derivatives of the type Y-Ser-OX as galactosyl acceptors in the presence of β-galactosidases of different origins so that the reaction in an aqueous-organic reaction medium with 50 to 95 wt .-% water and 50 to 5 % By weight of organic solvent.

The starting point was the fact that secondary hydrolysis was the desired goal products only from a water activity (pure water = water activity 1; by add other solvents, it is reduced, comparable to water vapor pressure) in the reaction system of <1 can be effectively suppressed. The Verrin reduction of water activity to a value <1 is principally through the use of organic solvents instead of water possible (Halling, P.J., Bell, G., Janssen, A., "Water activity... In polar organic solvents: interconversion of water concentrations and activites "). However, organic solvents influence the  Stability and activity of enzymes are often unfavorable by going through a Structural destabilization influence the catalytically active conformation and / or the bound water content on the enzyme surface - another Prerequisite for the enzymatic activity - reduce. One for certain A cheap alternative is the use of apolar solvents either alone or as cosolvents (Kuhl, P. Jakubke, H.-D., Die Pharmazie, Issue 6, June 1990, SS 393-400). In the enzymatic synthesis of glycoamino acid Derivatives with glycosidases in reaction media with a high proportion of (Especially polar) organic solvents decrease enzyme activity sometimes drastically.

It has now been shown that this disadvantageous effect by the inventive by adding certain organic solvents to a highly concentrated reak tion mixtures - producible by appropriate concentration of the educts used - is significantly less pronounced. Organic solvents proportions of up to 20% by weight in the reaction system result in only a relatively small amount Loss of enzyme activity. This may be due to a stabilization of the Enzyme due to the high substrate concentration, which the toxic influence of Solvent largely compensated (p. V. Sundaram, S. Shubhada, Int. Symposium, Stability and Stabilization of Enzymes, 1993 Elsevier Science Publishers).

In the syntheses, commercial β-galactosidases from Escherichia are preferably used coli and Aspergillus oryzae used.

In addition to the origin of the enzyme and the type or composition of the reaction medium used has the choice of amino and carboxyl protection group on the serine / threonine derivative is crucial for the success of the Synthesis. The decisive factor is the affinity of the aglycone - which strongly depends on the Geometry, charge distribution and solvation of the molecule in question were determined becomes - the respective binding site in the enzyme-substrate complex. Because of nature  the β-galactosidases can be an O-glycosidic link to the binding site in the Enzyme-substrate complex theoretically only via the free hydroxyl group in the side chain of hydroxyamino acid. Nevertheless, acceptable yields are only now possible if the amino and carboxyl function of the hydroxyamino acid also suitable protective groups is blocked. Very good results have been achieved by the Blocking of the amino function of the allyloxycarbonyl and by esterification of the Carboxyl group to the methyl ester reached.

When using the free, unprotected hydroxyamino acid as aglycon takes place no significant glycosylation takes place.

The linked serine and threonine derivatives (aglycone) are either commercially available or can be prepared by known methods ("Amino Acid Derivatives", 1, carboallyloxy Derivatives, by Carl M. Stevens and Ronald Watanabe; E. Schnabel, Liebigs Ann. Chem., 702, 188 (1967).

The molar ratio of substrate to acceptor fluctuates depending on the synthesis between 4: 1 and 2: 1. At least 150 U per mmol of substrate used Enzyme can be used.

Furthermore, it was found that a number of minor can form products that deteriorate the yield of the target product. So In the course of the reaction there is an oligomerization of the released Galactose. This effect only occurs at relatively high lactose concentrations and a response time of more than 15 hours.

The method for the enzymatic synthesis of glycoamino acid is useful derivatives, in particular of galactosylserine derivatives, carried out so that 1 mol Lactose used at least 2 moles, preferably 2 to 4 moles of serine derivatives become.  

With a donor / acceptor ratio greater than 1: 2, i. H. for example 1: 1 or 2: 1, another galactose residue on the galactosylamino acid derivative transferred to form an amino acid digalactoside. With a further increase of the donor portion, the monoglycosylated amino acid reacts almost quantitatively to that respective disaccharide compound. The yields on this product are rarely higher than 10 to 15%.

The enzymatic synthesis is preferably carried out by initially mixing made from lactose and serine derivatives, enzymes and aqueous buffer solutions, then added the organic solvent and finally the reaction itself is carried out. The synthesis result is strongly dependent on the type and purity of the ver used depending on the solvent.

As an organic component for the aqueous-organic reaction system preferably acetone, acetonitrile, ethyl acetate, 2-butanone or cyclohexane.

The composition of the aqueous-organic system should preferably be 75 to 90 % By weight of water and 25 to 10% by weight of organic solvent.

During e.g. B. ethanol in a reaction mixture of lactose and β-galactosidase Methanol forms from E. coli and acts as a reaction partner in the same place a deactivating influence on the enzyme. Already relatively small Amounts of dimethylformamide (DMF), dimethylformamide sulfoxide (DMSO) or pyridine lead to a sharp decline in activity and partially damage the enzyme irreversible.

The use of organic solvents in concentrations of 5 to 20% by weight promotes diffusion in the system, increases the solubility of the glycosyl acceptor and can reduce water activity, especially with water-soluble solvents.  

By adding acetone, 2-butanone or acetonitrile, the oligomerization of Largely suppressed galactose. When using ethyl acetate or Cyclohexane in amounts of 25 to 50 wt .-% is an increased oligomerization too observe.

Fig. 1 shows the influence of organic cosolvents on the course of the reaction and the yield in the enzymatic synthesis of Aloc- (Galβ1) Ser-OMe. The type and concentration of the organic solvent must always be adapted to the particular synthesis problem. Experience has shown that concentrations between 10 and 25% by weight are optimal.

Despite slight losses in activity of the glycosidase were among the specified Reaction conditions in almost all syntheses carried out up to 30% higher Yields than achieved with previously known processes.

The enzymatic reaction is advantageously carried out between 35 and 45 ° C.

To ensure optimal diffusion in the reaction mixture, there is a reaction temperature above 37 ° C required. Temperatures up to 55 ° C are at certain enzymes possible (Ogushi, S., Yoshimoto, T., Tsuru, D. J. Ferment Technol. Vol. 58, No. 2, pp 115-122, 1980).

As expected, the reaction time is longer than in aqueous homogeneous systems and is between 15 and 48 hours.

It is advantageous if the enzymes are immobilized, preferably cross-linked or microencapsulated.  

Immobilization of the enzyme is not necessary, but has been shown in some Cases as economically sensible. Different immobilization techniques can be used for the realization of 3 to 4 consecutive syntheses with only one enzyme batch can be successfully applied (Kennedy, J.F., Cabral, J.M.S. 1987, "Enzyme Immobilization ", Biotechnology, Vol. 7a, pp 347 to 404, VCH Weinheim, Buchholz, K., Kasche, V., "Biocatalysts and Enzyme Technology", 1977, VCH Weinheim).

When using an immobilized enzyme several times, the synthesis yield a total of up to 2.5 times higher than when using free enzyme (The yield per mg of immobilized enzyme is compared with the yield per mg of free enzyme).

Depending on the enzyme used, the pH of the reaction medium was Acetate or phosphate buffer set to a value in the range of 5.5 to 7.8.

The tough reaction mixture generally does not allow mechanical through mixture. The shear forces caused by stirring or kneading are also called possible source of danger for the enzyme.

The course of the reaction was determined using HPLC (High Performance Liquid Chromatography) and TLC (Thin Layer Chromatography). The product was cleaned by HPLC with upstream extraction steps. Purification by columns chromatography is also possible. However, the separation selectivity is here Lower compared to HPLC.

Embodiments

The enzymatic linkage of three serine derivatives with galactose is described. In the example, the amino acids, lactose and target compounds are abbreviated according to the internationally applicable rules. The following abbreviations are also used:

Aloc - allylloxycarbonyl
Boc - tert-butyloxycarbonyl
Z - benzyloxycarbonyl
OMe - methyl ester.

Amounts for the synthesis of Aloc- (Galβ1) Ser-OMe (Example 1):
Lactose 1 mmol
Aloc-Ser-OMe 2 mmol
E. coli 200 U beta-galactosidase
Acetone (pA) 150 µl
K-phosphate buffer (0.1 M, pH = 7.0.1 mmol MgCl ₂ and Immol dithiothreitol) 850 µl.

Batch amounts for the synthesis of Boc- (Galβ1) Ser-OMe (Example 2):
Lactose 1 mmol
Boc-Ser-OMe 3 mmol
E. coli 200 U beta-galactosidase
2-butanone (pA) 100 µl
K-phosphate buffer (0.1 M, pH = 7.0.1 mmol MgCl ₂ and Immol dithiothreitol) 850 µl.

Amounts for the synthesis of Z- (Galβ1) Ser-OMe (Example 3):
Lactose 1 mmol
Z-Ser-OMe 3 mmol
E. coli 200 U beta-galactosidase
2-butanone (pA) 150 µl
K-phosphate buffer (0.1 M, pH = 7.0.1 mmol MgCl ₂ and Immol dithiothreitol) 850 µl.

The stated amounts of lactose, serine derivative and most of the aqueous phosphate buffer are combined in succession in a sealable glass vessel (approx. 3 cm ³ reaction volume). The enzyme is then added as a suspension, each with 20 ul K-phosphate buffer. After adding the organic solvent, the reaction mixture is mixed with a spatula and the reaction vessel is closed. The synthesis takes place in a thermomixer at 37 ° C and a shaking frequency of 1000 rpm. Depending on the synthesis, the reaction time is 15 to 20 h.

After the reaction has ended, the reaction mixture is taken up in 10 ml of methanol and filtered. The filtrate is evaporated on a rotary evaporator at 40 ° C and 30 mbar. The residue is taken up in 10 ml of water. The unreacted serine derivative is shaken out of the aqueous solution with 20 ml of methylene chloride in a funnel and separated. The remaining aqueous phase is again concentrated on a rotary evaporator (40 ° C., 30 mbar) and then purified using preparative HPLC.

HPLC conditions:
Column: 250 × 10 mm, NH2, 10 µm
Eluent: acetonitrile / water = 80/20
Flow: 6 ml / min
Detection: UV / VIS and light scatter detector.

The product was identified and characterized by NMR and MS under Use of literature data.

Table 1

Yields of synthesis

By purifying the products with preparative HPLC, a loss of up to 40% occur. Generally about 60 to 80% of the actual isolate the resulting product. The main cause of this loss is a pH Lowering caused hydrolysis of the glycosidic bond, which in the Concentration of the reaction mixtures on the rotary evaporator with simultaneous Presence of traces of acid can occur.

Claims (7)

1. Process for the enzymatic synthesis of glycoamino acid derivatives, in particular galactosylamino acid derivatives, of the form Y- (Galβ1) Ser-OX [NY-3-O-β-D-galactopyronosyl-L-serine-X-ester]
wherein
Y = Aloc (allyloxycarbonyl-), Boc (tert-butoxycarbonyl-) or Z (benzyloxycarbonyl-) and
X = methyl, ethyl or -H,
with lactose as galactosyl donor and serine derivatives of the type Y-Ser-OX as galactosyl acceptors in the presence of β-galactosidases of different origin, characterized in that the reaction in an aqueous-organic reaction medium with 50 to 95% by weight of water and 50 up to 5 wt .-% organic solvent is carried out. 2. Process for the enzymatic synthesis of glycoamino acid derivatives, in particular their galactosylamino acid derivatives, according to claim 1, characterized in that at least 2 moles, preferably 2 to 4 moles, of serine per mole of lactose Derivatives are used.   3. Process for the enzymatic synthesis of glycoamino acid derivatives, in particular der Galactosylaminosäurederivaten, according to claim 1 and 2, characterized characterized in that initially a mixture of lactose and serine derivatives, Enzymes and aqueous buffer solutions are prepared, then the organic solution added medium and finally the reaction itself is carried out. 4. Process for the enzymatic synthesis of glycoamino acid derivatives one or more of claims 1 to 3, characterized in that for the aqueous-organic reaction system as organic solvent acetone, Acetonitrile, ethyl acetate, 2-butanone or cyclohexane can be used. 5. Process for the enzymatic synthesis of glycoamino acid derivatives one or more of claims 1 to 4, characterized in that the Composition of the aqueous-organic system 75 to 90 wt .-% water and 25 to 10% by weight of organic solvent. 6. Process for the enzymatic synthesis of glycoamino acid derivatives one or more of claims 1 to 5, characterized in that the Reaction between 35 and 45 ° C is carried out. 7. Process for the enzymatic synthesis of glycoamino acid derivatives one or more of claims 1 to 6, characterized in that the Immobilized, preferably cross-linked or microencapsulated, be used.