CH622788A5 - Process for obtaining carrier-bound gibberellins - Google Patents

Description

The invention relates to a process for the production of carrier-fixed gibberellins, the chemical linking of the low-molecular phytohormone with high-molecular, mostly insoluble carriers, e.g. Polysaccharides, is to be understood.

Such materials consisting of carrier and ligand have recently become increasingly important due to their biological activity. This applies above all to affinity chromatography, in which the specific interaction between ligand and ligand-affine substances is used to separate the latter. It is known that the fixation of animal hormones to the carrier led to the discovery of hormone-specific receptor proteins and thus basic steps in the mode of action of those hormones could be elucidated. Corresponding results with regard to the mechanism of action and metabolism can also be expected when using carrier-fixed phytohormones. However, carrier-fixed gibberellins are not yet known.

1) General information about the beam fixation can be found at:

Axén, R., J. Porath u. S. Ernbeck - Nature 214, 1302-1304 (1967)

Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides.

Axén, R. u. P. Vretblad - Acty Chem. Scand. 25, 2711-2716

(1971)

Binding of proteins to polysaccharides by means of cyanogen halides. Cyanogen bromide - treated Sephadex.

Baum, G. - Enzyme Engineering, Vol. 2 (eds, E.K. Pye and L.B. Wingard, Jr.) - Plenum Press, New York - London 1974, pp. 63-64

Derivatives of controlied pore glass for affinity chromatography.

Cuatrecasas, P. - J. Biol. Chem. 245, 3059-3065 (1970) Protein purification by affinity chromatography. Derivatives of agarose and polyacrylamide beads.

Cuatrecasas, P. u. J. Parikh - Biochemistry 11, 2291-2299

(1972)

Adsorbents for affinity chromatography. Use of N-hydroxysuc-cinimide esters of agarose.

Turkovâ, J., O. Hubalkovâ, M. Krivakova u. J. (5oupek -

Biochim. Biophys, Acta 322.1-9 (1973)

Affinity chromatography on hydroxyalkyl methacrylate gels.

2) Information on carrier fixation and the use of adsorbents in affinity chromatography can be found at: Cuatrecasas, P. - Advan. Enzymol. Relat. Areas Mol. Biol. 36, 29-89 (1972)

Affinity chromatography of macromolecules.

Weetall, H.H. - Separation and Purification Methods 2,199-

229 (1973) - Affinity Chromatography.

Methods in Enzymology, vol. 34 (eds. W. B. Jacoby et al.

M. Wilchek), Academic Press, New York 1974 - Affinity

Techniques.

3) Information about receptor proteins of animal hormones can be found at:

Cuatrecasas, P. - Proc. Nat. Acad. Be. USA 69, 1277-1281, (1972)

Affinity chromatography and purification of the insulin reeep-tor of liver cell membranes.

Dufau, M.L., D.W. Ryan, A.J. Baukai and K.J. Catt - J. Biol. Chem. 250, 4822-4824 (1975) - Gonadotropin reeep-tors.

Solubilization and purification by affinity chromatography. Ludens, J.H., J.R. De Vries u. D. D. Fanestil - J. Biol.

Chem. 247.7533-7538 (1972)

Criteria for affinity chromatography of steroid binding macromolecules.

Sica, V., J. Parikh, E. Nola, A.G. Puca u. P. Cuatrecasas - J. Biol. Chem. 248, 6543-6558 (1973)

Affinity chromatography and the purification of estrogen receptors.

4) Information on the production and use of carrier-fixed phytohormones can be found at:

Venis, M.A. - Proc. Nat. Acad. Be. USA 68, 1824-1827 (1971)

Stimulation of RNA transcription from pea and com DNA by protein retaines on Sepharose coupled to 2,4-di-chlorophen-oxyacetic acid.

Venis, M.A. Biochem. J. 127, 29 P (1972)

Promotion of deoxyribonucleic acid - dependent ribonucleic acid synthesis by protein isolated on an plant hormone affinity column.

The invention is therefore based on the object of developing a method for obtaining gibberellins fixed to the carrier.

The process according to the invention is characterized in that gibberellins in the form of their anhydrides or their derivatives containing amino groups are reacted with a carrier to form an acid amide group. A high molecular weight polysaccharide is preferably used as the carrier.

The acid amide grouping can be done in different ways

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produce. As found, one possibility is that an anhydride of a gibberellin with a carrier containing primary amino groups, e.g. an aminoalkylaga rose. The goal is also achieved if a gibberellin derivative with a primary amino group is reacted directly with an activated carrier; this activation can e.g. by means of cyanogen bromide or by using a polysaccharide containing imidocarbonate structures. Finally, the reaction of a gibberellin derivative with a primary amino group with carboxyl groups attached to the support (e.g. 10 a polysaccharide) and activated with N-hydroxysuccinimide leads to gibberellins fixed to the support. In the latter two processes, the gibberellin derivative can be e.g. a gibberellinaminoalkylamide and agarose or carboxyl groups activated by cyanogen bromide, which are bound to agarose via an aminoalkyl group and activated with N-hydroxysuccinimide.

By varying the alkyl chain lengths [- (CH2) n—], the gibberellin molecules can be linked to the high-molecular polysaccharide matrix of the agarose via side arms of different lengths, which is of great importance for affinity-chromatographic purposes.

The method according to the invention makes it possible for the first time to obtain gibberellins which are fixed on the carrier. They are of technical importance for the targeted production of plant matter; Because this makes previously unrealizable studies of the mechanism of action of the gibberellins that intervene in crucial differentiation and development processes in plants. 30th

The process is to be explained below using a few exemplary embodiments with reaction schemes.

example 1

Conversion of gibberellic anhydride 35

with aminopentyl agarose 1.4 ml aminopentyl agarose (formula 1; n = 5) were in

622 788

4 ml of 0.1 m sodium borate buffer pH 8.5 suspended and reacted with 25.8 mg of gibberellic anhydride (formula 2) at 4 ° C. for 30 hours. The reaction product obtained (formula 3; n = 5) was washed successively with 0.1 n sodium hydrogencarbonate and water. The quantitative determination showed a content of 2.46 xM gibberellic acid per ml agarose gel.

Example 2

Reaction by cyanogen bromide-activated agarose with gibberellic acid 4-amino-n-butylamide 15 ml agarose gel (formula 4) were activated with 3 g cyanogen bromide and in 15 ml 0.1 n sodium hydrogen carbonate with 162.7 mg gibberellic acid 4-amino-n -butylamide (formula 5; n = 4) reacted at 4 ° C for 20 hours. After the excess amide had been washed out, the gel was reacted with 10 ml of a 1N ethanolamine solution in 0.1N sodium hydrogen carbonate. This was followed by washing with 0.1N sodium hydrogen carbonate and water. Analysis of the reaction product (formula 3; n = 4) revealed a content of 10.9 (ÎM gibberellic acid per ml agarose gel.

Example 3

Reaction of the N-hydroxysuccinimide ester of carboxyethyl agarose with gibberellic acid 4-amino-n-butylamide 1 ml agarose gel (formula 6; m = 2), which contains carboxyl groups activated by N-hydroxysuccinimide in the spacer part, was in 3 ml 0.1 M sodium acetate buffer pH 6.3 with 20 mg gibberellic acid 4-amino-n-butylamide (formula 5; n = 4)

implemented over 15 hours at 4 ° C. After washing the reaction product (formula 7; m = 2, n = 4) with 0.1 m sodium acetate buffer pH 6.3 and water, a content of 0.56 [xM gibberellic acid per ml agarose gel was determined.

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1 sheet of drawings

Claims (10)

622788 1. A process for the production of carrier-fixed gibberellins, characterized in that gibberellins in the form of their anhydrides or their derivatives containing amino groups are reacted with a carrier to form an acid amide group. 2. The method according to claim 1, characterized in that a high molecular weight polysaccharide is used as the carrier. 2nd PATENT CLAIMS 3. The method according to claim 1 or 2, characterized in that reacting an anhydride of a gibberellin with a carrier which contains primary amino groups. 4. The method according to claim 1 or 2, characterized in that a gibberellin derivative having a primary amino group is reacted directly with an activated carrier. 5. The method according to claim 1 or 2, characterized in that reacting a gibberellin derivative having a primary amino group with carboxyl groups bound to the support and activated with N-hydroxysuc-cinimide. 6. The method according to claim 3, characterized in that gibberellic anhydride is reacted with an amino-alkyl-agarose. 7. The method according to claim 4, characterized in that reacting a gibberellin derivative with a primary amino group with a polysaccharide activated by cyanogen bromide or with a polysaccharide containing imidocarbonate structures. 8. The method according to claim 7, characterized in that reacting a gibberellinaminoalkylamide containing a primary amino group with agarose activated by cyanogen bromide. 9. The method according to claim 5, characterized in that reacting a gibberellin derivative having a primary amino group with carboxyl groups bound to a polysaccharide and activated by N-hydroxysuccinimide. 10. The method according to claim 9, characterized in that a Gibberellinaminoalkylamid, which contains a primary amino group, with carboxyl groups which are bonded to an agarose via an aminoalkyl grouping and activated with N-hydroxysuccinimide.