Process for preparing peptides and N-carbamoyl-protected...

Details Process for preparing peptides and N-carbamoyl-protected... Process for preparing peptides and N-carbamoyl-protected...

1998-04-03

2001-06-26

Prats, Francisco (Department: 1631)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Enzymatic production of a protein or polypeptide

C435S129000, C435S136000, C435S171000, C435S071100, C435S071200, C435S195000, C435S227000, C530S300000, C530S302000, C530S331000, C530S330000, C530S329000, C530S328000, C530S335000, C530S337000, C530S343000, C530S345000, C564S255000, C560S345000

Reexamination Certificate

active

06251625

ABSTRACT:

The invention relates to a process for the enzymatic preparation of protected di- or oligopeptides and removal of the protective group used by splitting off.
Synthetic short-chain peptides are increasingly used in pharmacology and in parenteral feeding. Kyotorphin (L-Tyr-L-Arg), which promotes the release of enkephalins, that is to say endogenous substances which have an analgesic and tranquillizing action in the brain (Hughes, 1975), may be mentioned as an example of a pharmacologically active dipeptide.
The enzymatic preparation of di- or oligopeptides is already known and generally makes use of protective group technology. The use of a formyl protective group is thus described in U.S. Pat. No. 571,037, that of an acetyl protective group in JP 62074296, that of a benzyl protective group in U.S. Pat. No. 4,935,355 or that of a phenacetyl protective group in Tetrahedron 1992, 48, 1115. These variants have the disadvantage that the protective groups in some cases are not inexpensive (benzyl, phenacetyl), can be split off again only with difficulty (acetyl, formyl) or can be removed (benzyl) only under quite specific conditions (hydrogenolysis).
The object of the invention was therefore to develop a process for the synthesis of peptides which is particularly simple and inexpensive, allows simple and gentle removal of the protective group by splitting off, and allows simple working up and separating-off of the enzyme.
This object of the invention for the preparation of a peptide of the general formula I
wherein
R
1
and R
2
independently of one another denote hydrogen, (C
1
-C
6
) alkyl, which can optionally be interrupted or substituted by heteroatoms, such as N, O or S, one [sic] or several times, it being possible for the heteroatoms in their turn to be substituted by hydrogen, (C
1
-C
4
) alkyl or benzyl or to be bonded to the alkyl group via a double bond, phenyl or benzyl, both of which can optionally be substituted by halogen or hydroxyl once or several times, heteroaralkyl, such as 3-indolylmethyl, 2-, 3- or 4-pyridylmethyl,
R
3
denotes (C
1
-C
4
) alkoxy, NH
2
, hydroxyl, NR
1
R
2
, benzyloxy, which can optionally be substituted by halogen, nitro, NH
2
, (C
1
-C
4
) alkyl, (C
1
-C
4
) alkoxy once or several times, or one or more units of the type II
is achieved by a process in which compounds of the type III or a salt form thereof,
wherein R
1
, R
2
and R
3
have the abovementioned meaning and
R
4
denotes hydrogen, (C
1
-C
4
) alkyl, phenyl, which can optionally be substituted by halogen, (C
1
-C
4
) alkyl, (C
1
-C
4
) alkoxy, nitro, CN, CF
3
, (C
1
-C
6
) alkoxycarbonyl, COOH or —NR
1
R
2
once or several times, aralkyl, such as benzyl, which can be substituted in its turn by halogen, (C
1
-C
4
) alkyl or (C
1
-C
4
) alkoxy, naphthyl, heteroaralkyl, such as 2-, 3- or 4-thienyl, 2-, 3- or 4-pyridyl or 2-quinolyl
are reacted with a carbamoylase, optionally in the presence of a solvent, or the carbamoyl protective group is split off chemically, optionally in the presence of a solvent and optionally in the presence of an acid.
It has furthermore been found that carbamoyl-protected peptides of the general structure III,
wherein
R
1
and R
2
independently of one another denote hydrogen, (C
1
-C
6
) alkyl, which can optionally be interrupted or substituted by heteroatoms, such as N, O or S, one [sic] or several times, it being possible for the heteroatoms in their turn to be substituted by hydrogen, (C
1
-C
4
) alkyl or benzyl or to be bonded to the alkyl group via a double bond, phenyl or benzyl, both of which can optionally be substituted by halogen or hydroxyl once or several times, heteroaralkyl, such as 3-indolylmethyl, 2-, 3- or 4-pyridylmethyl,
R
3
denotes (C
1
-C
4
) alkoxy, NH
2
, hydroxyl, NR
1
R
2
, benzyloxy, which can optionally be substituted by halogen, nitro, NH
2
, (C
1
-C
4
) alkyl, (C
1
-C
4
) alkoxy once or several times,
R
4
denotes hydrogen, (C
1
-C
4
) alkyl, phenyl, which can optionally be substituted by halogen, (C
1
-C
4
) alkyl, (C
1
-C
4
) alkoxy, nitro, CN, CF
3
, (C
1
-C
6
) alkoxycarbonyl, COOH or —NR
1
R
2
once or several times, aralkyl, such as benzyl, which car be substituted in its turn by halogen, (C
1
-C
4
) alkyl or (C
1
-C
4
) alkoxy, naphthyl, heteroaralkyl, such as 2-, 3- or 4-thienyl, 2-, 3- or 4-pyridyl or 2-quinolyl
are obtained by a process in which a compound of the type IV or a salt form of IV, in which R
1
and R
4
have the abovementioned meaning,
is reacted with a compound of the type V, or of [sic] an acid addition salt thereof, wherein R
2
and R
3
have the abovementioned meaning,
optionally in the presence of a solvent and optionally in the presence of a base.
The term “alkyl groups” is to be understood as meaning both “straight-chain” and “branched” alkyl groups.
The term “straight-chain alkyl group” is to be understood as meaning, for example, radicals such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the term “branched alkyl group” is to be understood as meaning radicals such as, for example, isopropyl or tert.-butyl.
The term halogen represents fluorine, chlorine, bromine or iodine. The term “alkoxy group” represents radicals such as, for example, methoxy, ethoxy, propoxy, butoxy, isopropoxy, isobutoxy or pentoxy.
Dipeptides, such as, for example, aspartam, can preferably be prepared by this process.
Another advantage of the process is that the peptide coupling can be carried out in a highly dense suspension, it being possible for the substrate and product to be present in partial [sic] solid form.
The novel process for the synthesis of peptides using, as the intermediate product, the carbamoyl-protected peptides described comprises three reaction steps.
A) Preparation of the N-carbamoyl-amino acid or of the N-carbamoyl-amino acid derivative
B) Linking of the peptide bond between the carbamoyl-protected electrophile and the nucleophile
C) Removal of the carbamoyl protective group by splitting off
The preparation of the N-carbamoyl-amino acid or of the N-carbamoyl-amino acid derivative can be carried out in a manner known per se from the literature. Preferably, the reaction of the amino acid or of the amino acid derivative with an isocyanate of the general structure VI,
R
1
—NCO  VI
wherein R
1
has the meaning given above, in a two-phase system of H
2
O/organic solvent, it being possible for the organic solvent to be, for example, toluene, chlorobenzene or tert.butyl methyl ether, is carried out. The reaction is carried out at temperatures between 0° C. and 120° C., preferably between 20° C. and 70° C. If appropriate, an inorganic base, such as, for example, NaOH, KOH, K
2
CO
3
, Na
2
CO
3
, potassium bicarbonate or sodium bicarbonate, or an organic base, such as, for example, triethylamine or tributylamine, can be added to the reaction mixture. The phenyl isocyanate of the structure III is advantageously used in a slight excess.
Reactions to give the N-carbamoyl-protected amino acids or amino acid derivatives with a cyanate of the structure VII,
M
⊖⊖
OCN  VII
wherein M
⊕
represents Na
⊕
, K
⊕
, Ag
⊕
, Pb
⊕
(OCN) or ammonium ions, such as, for example, tetraethylammonium, tetrabutylammonium or benzyltriethylammonium, are particularly preferred.
The reaction is carried out in a manner known per se in aqueous solution at temperatures between 0° C.-120° C., preferably between 60° C.-100° C., and if appropriate with the addition of an inorganic base, such as, for example, NaOH, KOH, K
2
CO
3
or Na
2
CO
3
. It has proved favourable if the reaction is carried out in the presence of slight excesses of 1.01-2 equivalents, preferably 1.1-1.2 equivalents, of cyanate.
The linking of the peptide bond is carried out in a manner known per se with the aid of a hydrolase (Jakubke, Kuhl and Könnecke; Angew. Chem. 1985, vol. 97, p.p. 79-87). It is irrelevant for the reaction conditions here whether the peptide linking proceeds under kinetic or thermodynamic control. The process according to the inventio

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