HU193104B - Process for preparing n up alpha -/9-fluorenyl-methyl-oxy-carbonyl/-aminoacids and pentafluoro-phenyl esters thereof - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to N- (9-fluorenylmethyloxycarbonyl) amino acids of formula I and pentafluorophenyl esters thereof, wherein R 1 is an α-aminocarboxylic acid which is commonly used in peptide chemistry, optionally containing a protecting group. and R2 is hydrogen, or R1 and R2 taken together to form a 1,2-ethylene or 1,3-propylene moiety, such that (9-fluorenylmethyl) of formula (11) is selected from the group consisting of: (pentafluorophenyl) carbonate in a mixture of water and a water-miscible organic solvent in the presence of an acid scavenger with a c-aminocarboxylic acid of formula (III) wherein R1 and R2 are as defined above, and optionally, for the preparation of pentafluorophenyl esters. the N- (9-fluorenylmethyloxycarbonyl) amino acid of formula I, wherein R1 and R2 are as defined above, and pentafluorophenol dicyclohexylcarbodiimide. in an organic solvent medium. F F R 2 R 1 I HN — CH — COOH -1-

Description

The present invention relates to a novel process for the preparation of the N-tG-fluorenylmethyloxycarbonyl amino acids of the formula (I) and their pentafluorophenyl esters, wherein

R ¹ represents one, relating to the 2-position carbon chain containing protective groups customary in peptide α-amino carboxylic acid and optionally

R ² is hydrogen or R ¹ and R ² together are 1,2-ethylene or 1,3-propylene.

In recent years, the previously known [J.Am.Chem.Soc. 92, 5748 (1970); J.Org. Chem., 37, 3404 (1972)]

The use of a 9-fluorenylmethyloxycarbonyl group, hereafter referred to as the Fmoc group, is widely used to protect the amino group both in solution and in solid phase peptide synthesis [Int.J.Peptide Protein Slit. 13, 35 (1979) and 18, 237 (1981); Experientia 38: 773-82 (1982)]. Later, it was demonstrated in methodological studies [J. Org. Chem., 44, 1622. (1979); Int.J.Peptide Protein Rés. 15

59 (1980)], in the coupling reactions in solution, the Fmoc group is partially cleaved due to the basicity of the amine component to be acylated, resulting in double acylated products. This side reaction can be avoided with an acidic additive (e.g., 1-hydroxybenzotriazole) [J. Org. Chem. 45, 72 (1980)] or highly reactive pentafluorophenyl esters [J. Org. Chem. 35, 3563 (1970); Justus Liebig’s Ann. Chem. 1973, 1421; Tetrahedron Lett. 1974, 1785] which significantly increase the coupling reaction rate compared to the Fmoc group cleavage. The demonstration of the benefits of using pentafluorophenyl ester of Fmoc amino acids in peptide synthesis in solution can be found in Synthesis 1983, p.

CJjab has been shown [Can.J.Chem. 60, 976 (1982); Synthesis 1983, 671; Peptides, Structure and Function (editors of Hruby and Rich), Pierce Chem.Co., Rockford, 1984, p.79], that in the preparation of Fmoc-amino acids,

In the reaction of 9-fluorenylmethyloxycarbonyl chloride (Fmoc-Cl) with the amino acids, not only the desired Fmoc amino acids but also Fmoc dipeptides are formed in an amount of 3-20%, presumably because of the relatively slow reaction rate. Fmoc amino acids react with the starting amino acids to form anhydride mixed with the reagent. Fmoc amino acids are very difficult to purify from Fmoc dipeptides with similar properties.

To avoid side reactions, succinimidyl, phthalimidyl, benzthiazolyl-1, and a number of substituted phenyl-9-fluorenylmethyl carbonates were introduced, of which 9-fluorenylmethylsuccinimidyl carbonate (Fmoc-OSu) was found to be the most preferred reagent. Enter Fmoc Group [Peptides, Structure and Function (editors of Hruby and Rich), Pierce 2

Chem.Co., Rockford, 1984, p.79]. Although the data in the publication include 9-fluorenylmethylpentafluorophenyl carbonate (hereinafter Fmoc-OPfp), it is not characterized, as is with 68% Fmoc-Ala-OH.

Surprisingly, it has been found that the Fmoc-amino acids of formula (I) and their pentafluorophenyl esters can be prepared in pure form, in good yield and in readily recoverable form, by reacting Fmoc-OPfp of formula (II) with water and in an aqueous solvent-miscible organic solvent in the presence of an acid acceptor with an α-aminocarboxylic acid of formula (III) wherein R ¹ and R ² are as defined above and optionally forming the pentafluorophen amino acid, wherein R ¹ and R ² are as defined above, and pentafluorophenol in the presence of dicyclohexylcarbodiimide in an organic solvent medium.

Preferably, Fmoc-OPfp is prepared from Fmoc-Cl and pentafluorophenol in ether solution by addition of triethylamine. The precipitated triethylammonium chloride was removed by washing with water and the reagent was isolated by recrystallization from n-hexane after removal of the solvent. Fmoc-OPfp is a white, crystalline non-hygroscopic material that can be stored well at room temperature, as opposed to Fmoc-Cl, which is only stable at low temperatures, and water-sensitive Fmoc-OSu.

Reagents such as Fmoc-Cl and Fmoc-OSu are usually used in excess in reactions to protect or activate amino acids. In the process of this invention, equimolar amounts of reagents are sufficient to protect or activate the amino acids. In addition, it is also possible to react with the relatively more expensive Fmoc-OPfp reagent in amounts less than the equivalent (up to 0.9 equivalents) for large amounts of available amino acids. Again, the process of the present invention provides a very good yield of 75-90%. A further advantage is that the materials obtained in the first step do not need to be prepared in pure form for the preparation of the pentafluorophenyl esters.

The water-miscible organic solvent used in the process of the present invention is C 3 -C 5 aliphatic ketones, tetrahydrofuran, dioxane, dimethylformamide or dimethylsulfoxide. A preferred representative of these is acetone.

Inorganic and organic compounds with weak basic properties can be used as acid binders. Examples include

Tertiary amines having from 1 to 4 carbon atoms, tertiary bases containing a ring nitrogen atom, alkali metal bicarbonates and alkali metal carbonates. Preferably, sodium bicarbonate is used.

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The organic solvents used in the dicyclohexylcarbodiimide reaction include ether-type solvents such as tetrahydrofuran, dioxane, halogenated hydrocarbons such as chloroform, dichloromethane, alkylated acid amides such as dimethylformamide or C 1-4 alkyl esters of C 1-4 alkanoic acids such as ethyl. .

Further details of the present invention are illustrated by the following non-limiting examples.

Abbreviations used to designate amino acids and their derivatives follow the suggestion of IUPAC-1UB [Biochem. J. 2t9, 345 (1984)].

Melting point determinations were made by dr. They were made in a Tottoli apparatus (Büchi, Switzerland).

The specific optical rotation was measured using a Perkin Elmer 141 numeric photoelectric polarimeter.

The purity of the products was occasionally compared to control compounds on a precast silica gel adsorbent (DC-Fertigplatten, Merck). The Fmoc-amino acids were chromatographed in ethyl acetate: (pyridine: acetic acid: water = 20: 6 ?, 11) = = 9: 1; Chromatograms are first visualized under UV light at 256 nm and then, after chlorination, visualized with potassium iodide-tholidine reagent.

Example 1 (9-Fluorenylmethyl) - (pentafluorophenyl) carbonate (II)

A solution of 93.5 g (0.4 mol) of 9-fluorenylmethyloxycarbonyl chloride and 73.6 g (0.4 mol) of pentafluorophenol in 1.0 L of anhydrous ether was cooled to 0 ° C and stirred. 56.0 ml (0.4 mol) of triethylamine were added dropwise thereto. The reaction mixture was stirred for 2 hours at 0 ° C and washed three times with 200 ml of water. The organic layer was dried over anhydrous sodium sulfate and clarified with charcoal. The ether was evaporated in vacuo and the crystalline residue was recrystallized from 500 ml of n-hexane. 139.7 g (86%) of Fmoc-OPfp are obtained. M.p. 83-84 ° C; Rf = 0.80 (chromatographed in ethyl acetate).

Example 2

N * - (carbonyl-9-fluorenylmethyl-oxy) -L-alanine

To a suspension of L-alanine (0.29 g, 3.3 mmol) and sodium bicarbonate (0.59 g, 7.0 mmol) in water (10 mL) and acetone (15 mL) was added 1.22 g (3) with stirring at room temperature. 0 mmol) of FmocOPfp. The reaction mixture was stirred overnight. The next day, the disappearance of the reagent was monitored by TLC, acidified to pH 2 with 18% hydrochloric acid and extracted with ethyl acetate (3 x 20 mL). The organic layer was washed with 1N hydrochloric acid (20 mL) and water (20 mL), dried over anhydrous sodium sulfate and concentrated in vacuo to 3 mL. To the residue is added n-hexane until turbid and the mixture is refrigerated. The next day, the precipitated crystalline material was filtered off and washed with n-hexane. 0.42 g (93.3%) of Fmoc-Ala-OH is obtained; 141-142 ° C; [<x] D = _18,5 ⁵ ° (c = 0; dimethylformamide);

R / = 0.5.

Proceeding as described above, the Fmoc amino acids of the formula (1), which may have additional protecting groups as shown in Table 1, are prepared.

Example 3 hr- (9-Fluorenylmethyloxycarbonyl) -L-alanine (pentafluorophenyl) ester

Fmoc-Ala-OH was prepared in the amounts described in Example 2. The ethyl acetate solution of Fmoc-Ala-OH and pentafluorophenol obtained during the main reaction mixture was cooled and dicyclohexylcarbodiimide (0.64 g, 3.1 mmol) was added with stirring. . After stirring for 2 hours in an ice bath, the precipitated dicyclohexylurea was filtered off and the filtrate was concentrated in vacuo to 3 ml. To the residue is added n-hexane until turbid and the mixture is crystallized in a refrigerator. 1.08 g (73.9%) of Fmoc-Ala-OPfp is obtained. 173-175 ° C; [α] D = -22.3 ° (c = 1, chloroform); Rf = 0.80.

Using the same procedure as described above, the N * - (9-fluorenylmethyloxycarbonyl) amino pentafluorophenyl esters listed in Table 2 and optionally other protecting groups are prepared.

Generation of Table 1 Fmoc Amino Acids with Fmoc-OPfp

amino acid (origin derivative) Fmoc amino acid Yield,% Melting point C below 93.3 141-142 Gly • 84.2 171-172 Leu 94.4 151-152 He 83.0 141-142 With 90.0 143-144 Pro 82.0 114-115 Phe 86.2 181-182 Trp 89.1 163-165 Met 84.2 127-130 Cys (Bzl) 99.0 126-128 Lys (Tos) 91.8 134-136 Z-Lys 88.7 86-88 Lys (Z) 81, 7 113-114 Asp (0 * Bu) 78.2 147-148 Glu (O ^f Bu) 91.0 47-51 Pip 63.7 150-151 Ser 77.8 90-94 Thr 90.0 amorphous Tyr 70.2 98-107 Asn 82.5 184-135 Gin 91.8 206-208

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Table 2: Preparation of pentafluorophenyl ester of Fmoc amino acids

Fmoc-amino amino acids occurs Fmoc-pentafluoro phenyl ester Yield,% Melting point, ° C Gly 72 158-160 below 74 173-175 Leu 78 113-114 He 70 94-96 With 77 121-123 Pro 89 125-127 Phe 90 152-154 Trp 85 184-185 Met 68 101-103 Cys (Bzl) 59 132-134 Lys (Z) 96 105-106 Z-Lys 90 127-128 Asp (0 ^l Bu) 63 97-100 Glu (0 ^t Bu) 95 119-120 Ser 83 124-126 Thr 81 126-128 Tyr 70 152-156 Lys (Tos) 85 113-118 Lys (Boc) 68 87-90

PATENT CLAIMS

Claims (6)

A process for the preparation of N * - (9-fluorophenylmethyloxycarbonyl) amino acids of the formula I and their pentafluorophenyl esters - wherein R 'represents an α-aminocarboxylic acid customary in peptide chemistry, optionally having a protecting group attached to the 2-position and R ² is hydrogen, or R ^J and R ² together represent a 1,2-ethylene or 1,3-propylene group - preparation, characterized in that (II) (9-fluorenylmethyl) formula - (pentafluoro phenyl) carbonate water and a water-immiscible organic solvent of an acid-binding agent in the presence of a-amino carboxylic acid of a general formula (III) - wherein R ¹ and R ² je10 Lentes above - is reacted, and then, if desired, the pentafluorophenyl esters for the preparation of the resulting N * - (9-fluorenylmethyloxycarbonyl) amino acid of formula (I) wherein R ¹ and R ² are The above - and pentafluorophenol are reacted in the presence of dicyclohexifcarbodiimide in an organic solvent medium. 2. The process of claim 1 wherein water and A mixture of water-miscible organic solvent and water-acetone (1: 1.5) was used. 3. A process according to any one of the preceding claims wherein the acid scavenger is sodium bicarbonate. Process according to any one of the preceding claims, characterized in that the reaction is carried out at room temperature. 30. Process according to any one of the preceding claims, characterized in that ethyl acetic acid is used as the organic solvent in the dicyclohexylcarbodiimide esterification reaction. 6. The process of claim 5, wherein the dicyclohexylcarbodiimide esterification reaction is carried out at a temperature of -5 ° C to + 5 ° C. 1 page drawing