CS245626B1 - A method for cleaving tert-butoxycarbonyl protecting groups in peptide synthesis on carriers - Google Patents

Abstract

Method for cleaving tert-butyloxycarbonyl protecting groups in the synthesis of peptides on supports. In this method, a peptide with a protected terminal amino group is treated with a solution of 1 and 51 wt. hydrogen bromide in a mixture of ethylene chloride and acetic acid, which contains 50 to 90 vol. % methylene chloride. The method can be used in peptide chemistry.

Description

The invention relates to the cleavage of tert-butyloxycarbonyl (Boc) protecting groups in the synthesis of peptides on supports.

To protect alpha-amino groups of amino acids in solid phase peptide synthesis / Merrifield R. B .: J. Am. Chem. Soc. 85, 2149 (1963), the tert-butyloxycarbonyl moiety is the most commonly used today (Carpino L. A., J. Am, Chem. Soc. 79, 4427 (1957), which is cleavable by acidic reagents at room temperature.

The most common cleaving agent is a 30-80% solution of trifluoroacetic acid in methylene chloride / Gutte B., Merrifield R. B., J. Biol. Chem. 246, 1922 (1971). Trifluoroacetic acid is an expensive chemical that is difficult to recover from solid phase filtrates.

In addition to trifluoroacetic acid, the use of a solution of hydrogen chloride in acetic acid, formic acid or dioxane and some other reagents is also described (Barany G., Merrifield R. B. Solid Phase Peptide Synthesis, p. 102, in The Peptides, Vol. 2, Gross B., Meienhofer J. ed., Academic Press, New York, 1980]. Hydrochloride solutions are readily available and inexpensive, but cannot be stored for long periods. The other agents described are either difficult to access or do not operate universally.

The object of the invention, which alleviates the above-mentioned disadvantages, is a method of cleaving tert-butyloxycarbonyl protecting groups with a solution of hydrogen halide in acetic acid in the synthesis of peptides on supports, said group protecting the terminal amino group of the peptides. % of a solution of 1 to 5 wt. % hydrogen bromide in a mixture of methylene chloride and acetic acid containing 50 to 90% by volume of methylene chloride.

Mehtylene chloride allows the carrier to swell, acetic acid increases the solubility of hydrogen bromide. The solution is cheap and can be stored for up to 1 year. Table 1 shows the time required for complete cleavage of the tert-butyloxycarbonyl group from the Boc-Gly on the support, depending on the concentration of hydrogen bromide in the CH ₂ Cl ₂ -CH ₃ COOH (5: 1 v / v) mixture. The table shows that a 3% solution is sufficient for complete cleavage of the Boc group.

Table 1% HBr in solution Time to cleave the Boc-group from Boc-Gly-P / min /

20 May

2

1

P - carrier

Hydrogen bromide in methylene chloride and acetic acid is an easily available reagent. It is obtained by diluting 35% hydrogen bromide in acetic acid, a solution commonly used in peptide chemistry, and adding methylene chloride.

They form stable solutions. The advantage in its use is a shortened reaction time compared to cleavage with trifluoroacetic acid solutions. Although the use of said reagent with hydrogen bromide may also effect cleavage of the peptides alone from the carrier, due to the advantage of a reduced reaction time, this effect will not occur.

The described method of cleavage of tert-butyloxycarbonyl protecting groups can be used in conventional peptide syntheses, as exemplified below:

Amide benzylthiopropionyl-beta-D-tyrosyl-phenylalanyl-alpha-aspartyl-butyryl-S-benzylcysteinyl-prolyl-N ^G -tosyl-D-arginyl-glycine

245636

The sequential synthesis of said peptides was carried out on a chloromethylated polystyrene resin sulfurized with 14 divinylbenzene (chlorine content 2.9%), esterified with Boo-Gly (0.3 mmol Gly / g carrier).

Solid phase synthesis was performed on an automatic peptide synthesizer and 4 g of esterified carrier (1.2 mmol Gly) was used. The cleavage of the Βοσ-groups after each amino acid attachment to the peptide chain was carried out by treatment with 34 HBr in CH 3 Cl 2 - CH 3 COOH (5: 1 v / v) for 3 minutes, condensing each amino acid with symmetric anhydride protected amino acids (3 eq, 30 to 60 min).

Upon completion of the synthesis, the supported peptide was ammonolyzed to give 1.04 g (64%) of the product, mp 144-152 ° C. An analytical sample was překrystalyzován twice from methanol - water, mp 154-156 ° C, [oí.J ³⁰ = -10.3 DEG / c 0.4, dimethyl formamide /.

It was chromatographically uniform in the n-butanol-tert-butanol-acetic acid-water system (2: 2: 1: 1 v / v). Amino Acid Composition: Tyr 0.92, Phe 0.98, Abu 0.99, Asp 0.95,

Cys / S-Bzl / 1.02, Pro 1.07, Arg 0.99, Gly 1.06. For ^C 66 ^H 83 ^N 13 ° 13 ^N 3 · ^{1.5 H} 2 ° / ^{1 39O | /} calculated! 57.04 4 C, 6.24 4 N ·; found: 56.88 4 C, 5.97 4 H, 12.84 4 N.

Claims (1)

A process for the cleavage of tert-butyloxycarbonyl protecting groups in the synthesis of peptides on carriers with a solution of hydrogen halide in acetic acid, said group protecting the terminal amino group of the peptides, characterized in that the peptide is treated for 2 to 10 min with 1 to 54 wt. % hydrogen bromide in a mixture of methylene chloride and acetic acid containing 50 to 90% by volume of methylene chloride.