DD226014A1 - PROCESS FOR PREPARING THE W-TERT. BUTYLERS OF AMINODIC ACETONES - Google Patents

Abstract

The invention relates to a process for the preparation of selectively protected in the side chain a-Aminodicarbonsaeurederivaten by enzyme-catalyzed hydrolysis of the corresponding a-, v-di-tert. Butylesterderivate. Such selectively protected derivatives of aspartic acid and glutamic acid are used in the synthesis of peptides and thus in the pharmaceutical industry.

Description

Process for the preparation of CJ.-tert. Butyl ester of aminodicarboxylic acids

Area of application of the invention

The invention relates to a process for the preparation of selectively ü-protected aspartic acid and Glutaminsäurederivaten by enzyraatischer hydrolysis of corresponding oC -, OJ -Diesterderivate. Such selectively protected derivatives of aspartic acid and glutamic acid are used in the synthesis of peptides and thus in the pharmaceutical industry.

Characteristics of the known technical solutions The synthesis of aspartic acid or Glu ta ^ ^ insavrehsl term peptides requires the selective protection of the side chains functional relation to the necessarily free for peptide coupling oL carboxyl. The benzyl ester or the tert-3-butyl ester are used as protective groups. While the preparation of U) -benzyl esters of Asp and Glu can be achieved by chemical methods in good yields in a one-step reaction, (Houben-Weyl, Methods of Organic Chemistry Vol. 15/1 Georg Thieme Verlag, Stuttgart 1974, p. 645 ff .) is the preparation of corresponding (C tert butyl considerably more complicated in practice two variants are used (Houben-Weyl, methods of organic Chemistry Vol 15/1 Georg Thieme Verlag, Stuttgart 1974, 3. 649 ff....):

1.) starting from N-protected amino acid (Z-Asp or Z-Glu) is prepared via the anhydride oC-methyl ester, which by means of isobutene in the side chain in the tert. Butyl ester is transferred. Subsequent alkaline hydrolysis of dL- methyl ester and hydro-

Holytic cleavage of the amino protecting group leads to the desired .beta.-value. butyl

2. N-Benzyloxycarbonyl-asparagine or -glutamine is converted into the methyl ester. Subsequently, the cleavage of the side chain amide takes place. By reaction with isobutene, the side chain function in the tert. Butyl ester transferred. The subsequent cleavage of the N - and C ⁰ ^ -protective groups corresponding to 1. leads to the desired W -tert. Butyl derivative.

According to Sirr (EP-P3 006 856) can be ^ -tert. Butyl esters of aspartic acid and glutamic acid by direct conversion with isobutene and subsequent purification produce »The different rates of esterification at the cL - and the W -Carboxylgruppe be exploited. In this process, the CO monoester formed is always contaminated with cL monoester in accordance with the ratio of the formation rates of oL and iO monoesters. For further use of the O -tert. However, butyl monoesters in peptide synthesis must be run with cL- tert. Butyl monoesters can be excluded »It is therefore a costly quantitative separation of the physico-chemical properties very similar cL and CJ -Monoester required.

An analogous process for the preparation of iJ-tert. Butyl ester of glutamic acid by direct conversion of glutamic acid with tert-butyl acetate was already in 1963 by Taschner et al. (Taschner, E. et al., Ann. Ghem. 663, (1963) ISS). In peptidchsmischen practice is to ensure the isomeric freedom of the LO -tert. 3utylesters the precedence over the temporary protection of the C ^ -Carboxylgruppe priority given to the more complex, but safe, to ensure the appropriate purity of the peptides to be synthesized * In addition to the above chemical methods are no enzymatic method for the preparation of (J-tert Butylesterderivaten known.

Object of the invention

It is the object of the invention, LO -tert. Butyl esters of aspartic acid and glutamic acid and their N-acyl derivatives using fewer steps and to the exclusion of the possibility of contamination with the corresponding egg -tert. Produce butyl ester or its N-acyl derivative in good yields,

Explanation of the essence of the invention

The object of the invention is to develop a method in which cL -, U? Di-tert -sutyl ester derivatives are selectively converted to the U) monoester derivatives.

The object has been achieved by the fact that the """""""""T" in isobutene / sulfuric acid easily and in high yields producible cL -, LO- di-tert-butyl ester derivatives of general formula I, where R = H, Acyl and η = 1 or 2, are selectively converted into the Lo monoester derivatives under the action of enzymes., The selectivity of the hydrolysis is ensured by the selectivity of the enzymes »

R-NH-CH-COOC (CH-)

ι O

COOC (CH ₃ ) ₃

As a result of the enzyraatic hydrolysis, the corresponding W-term. Butylestsr obtained, which may be contaminated with incomplete hydrolysis only with diester. However, this, as well as the enzyme, can be separated off in a simple manner. For hydrolysis, enzymes or enzyme-containing mixtures with sufficient esterase activity with respect to substrates of general formula I can be used in this connection. The use of readily available proteolytic enzymes, such as cL- chymotrypsin or pronase F, has proven particularly useful

only impurities are avoided that complicate product isolation. For example, the use of defatted pork and bovine pancreas (pancreatin) also provides good results. In addition, can be at similar procedure, the O-Senzylester of aspartic acid or glutamic acid or · create their N-acyl derivatives by enzymatic hydrolysis of the corresponding OC "lO -Dibenzylesterderivate.

The invention will be explained in more detail with reference to embodiments.

Embodiments Example 1;

100 mg of H-Glu (OBu ^t ) -OBu ^t , dissolved in 5 ml of 0.05 M TRIS / HCl 0.005 M CaCl ₂ , pH 7.5, are mixed with 20 mg of oC-chymotrypsin. The mixture is stirred at 37 ⁰ C · The quantitative analysis of the thin-layer chromatographic examination (QTLC) of the batch S ^ days ~ (on KG 60 - Glass pre-coated plates Merck after development with ninhydrine or Senzidin Fa.) Showed a 91% formation of H- GIu (OBu) -OH. To isolate the desired monoester, the concentrated mixture is chromatographed on a column filled with silica gel 60 (2 × 10 cm) in chloroform with increasing ethanol content. 3 ml fractions were collected. From the fractions. 53-61, 3.2 mg H-Glu (OBut) -OBu and from fractions 85-102 80.1 mg H-Glu (OBu) -OH were obtained by concentration iV.

Example 2:

10 mg H-Glu (03u ^t ) -Obu ^t , dissolved in 1 ml of 0.05 M TRIS / HCl 0.005 M CaCl-, pH 7.5, are mixed with 5 mg pancreatin.

After stirring for 3 days at 37 ⁰ C the QTLC showed 56%, after 5 days 68% and after 10 days 74% formed H-Glu (OBu *) -

Isolation as in Example 1.

Example 3;

100 mg of H-Glu (OBu ^Z) -OBu ¹¹ was dissolved in 5 ml of 0.05 M TRIS / HCl 0.005 M CaCl-t pH 7.5, are mixed with 20 mg pronase P and the reaction stirred at 37 ⁰ C. After 20 h, another 10 mg of Pronase P are added and the mixture is stirred for a further 1 h. The QTLC gives a 93% formation of H-Glu (OBu ¹ ") -OH»

Example 4

43 mg of H-ASp (OBu ¹¹ ) -OBu ¹¹ , dissolved in 5 ml of 0.05 M TRIS / HCl 0.005 M CaCl ₂ , pH 7.5, are mixed with Pronase P 12 mg. After stirring for 14 h at 37 ⁰ C a 9O50ige formation of H-Asp could (0BU ^t) -0H detected (QTLC).

Example 5

120 mg of Z-Glu (OBu *) - OBu * are suspended in 5 ml of 0.05 M TRIS / HCl 0.005 M CaCl ₂ , pH 7.5. 15 mg pronase P are added and the mixture was stirred at 37 ⁰ C · After 22 h and 44 h are respectively an additional 15 mg pronase P added. After a reaction time of 66 h, 40 % of Z-Glu (OBu 1 -GH were formed (QTCL) The product isolation was carried out as described in Example 1.

Example 6

24 g of H-Asp (03u ^t ) -Obu ^t , dissolved in 0.5 ml of 0.05 M TRIS / HCL 0.05 M CaCl ₂ , pH 7.5, are mixed with 5 mg pancreatin and the approach at 37 ⁰ C stirred. After 7 h, 75 % H-Asp (OBu ¹ ") OH had been formed (QTLC).

Example 7:

5 mg of Z-Asp (OBzl) -QBzI dissolved in 0.05 ml of N, N-dimethylacetamide are mixed with 2 rag o6-chymotrypsin in 0.15 ml of 0.1 M TRIS / HCl, pH 8. After stirring for 16 h at 37 ⁰ C 53% Z-Asp (OBzl) -OH were formed (QTLC). The isolation was carried out analogously to Example 1

Example 8:

As in Example 7, only in place of o (- chymotrypsin uses pronase P. After 16 h, 95% M Z-Asp (OBzI) -OH was formed (QTLC).

Example 9

Like Example 7, only pancreatin is used instead of oL- chymotrypsin. After 16h, 74% Z-Asp (CBzI) -CH was detected (QTLC).

Claims (4)

invention claim 1. A process for the preparation of the ü-tert. 3-butyl ester of amino-dicarboxylic acids corresponding to the general formula R-NH-CH-COOH Vn
COOC (CH ₃ ) ₃ where R = H, acyl η = 1, 2 , characterized in that the corresponding cC- IO- Oi-tert-butyl esters are converted into the desired tert-butyl- butanol esters using enzymes. 2. The method according to item 1, characterized in that proteases are used as enzymes. 3 · Method according to item 1, characterized in that oL-chymotrypsin is used as enzyme, 4. The method according to item 1, characterized in that is used as the enzyme Pronase P ·