DD211553A1 - PROCESS FOR THE PREPARATION OF 4-AZIDO-CINEMA-AEURESUCCINIMIDYLESTER - Google Patents

Abstract

The invention relates to a process for the preparation of 4-azido-cinnamic acid succinimidyl ester of the formula which can be used as a photoactivatable heterobifunctional crosslinker for a wide variety of molecular biological problems. The object of the invention is to provide a heterobifunctional crosslinker which reacts easily and selectively with biologically active molecules such as proteins and nucleic acids with cross-linking. The novel process for the preparation of 4-azido-cinnamic acid succinimidyl ester is characterized in that 4-azido-cinnamic acid is reacted with N-hydroxysuccinimide in the presence of a suitable acylation catalyst such as dicyclohexylcarbodiimide in an aprotic solvent at 0-25 ° C.

Description

Dr. C. Lindig

H. Scheer

Dr. P. Westermann

"Process for the preparation of 4-azido-zirconic acid succinimidyl ester"

Field of application of the invention

The invention relates to a process for the preparation of the 4-azido-cinnamic acid succinimidyl ester of the formula

Il -CH = CH-C-O-N

which can be used as photoactivatable heterobifunctional crosslinker for a variety of molecular biological problems.

The use of cross-linkers for systematic investigations of the structural and functional properties of various complex molecular biology systems has undergone significant intensification in recent years. · Chemical crosslinking reagents of defined extension and specific geometry have been used as probes to determine the quaternary structure of protein and ribonucleoprotein complexes and have been used for elucidation The spatial arrangement and contact surfaces of the individual proteins in multi-enzyme complexes, in membrane preparations or other molecular associates such as ribosomes and virus particles. "The application has been extended to the labeling of surface receptors for macromolecular ligands. By coupling photoactivatable, heterobifunctional reagents to macromolecular ligands

._ Λ Α Λ #. Γ »rt Λ -4

photoreactive derivatives of ligands were obtained via the non-photosensitive, chemically reactive group, which were crosslinked after binding to the receptor by photoactivation with the same.

Recent aspects of crosslinking technology have arisen in the field of various Enzyra immunoassays in medical diagnostics by coupling enzymes to antibodies as well as producing tumor-specific potential therapeutic agents by conjugating selectively cytotoxic proteins with raonoclonal antibodies against tumor-associated antigens,

Characteristic of the Known Technical Solutions Due to the increasing importance for molecular biological research, the synthesis and application of a number of heterobifunctional reagents as crosslinkers with partly very specific goals has been described in recent years (for reviews see »K» PETERS and FM RICHARDS, Ann Rev. Biochera., 46, 523 (1977) and M. Das and CF Fox, Ann. Rev. Biophys · Bioeng., 8, 165 (1979)). N-succinimidyl 6- (4'-azido-2 ¹ -nitrophenylamino) hexanoate, which is unsuitable for various purposes because of its long and flexible spacer group, is known, however. The synthesis of the 4-azido-zisntsäuresuccinisnidylesters and its application as a heterobifunctional crosslinker there is no information so far ·

Object of the invention

The object of the invention is to provide a heterobifunctional crosslinker which reacts easily and selectively with biologically active molecules such as proteins and nucleic acids with cross-linking. The requirements to be met by such a crosslinker are as follows:

1.) The two functional groups should be in the.

Distinguish reactivities to the extent that a controlled sequential implementation for each group is possible;

2.) between the functional groups, a mean optimal distance of about 10 R should be programmed by a suitable inflexible spacer as possible; 3.) the reaction should be carried out under as gentle conditions as possible so that the original biological activities of the biomacromolecules to be crosslinked are largely retained after conjugation;

4.) excess reagent must be easily removable after reaction.

The object of the invention is to decouple a production method for a crosslinker with the properties mentioned.

Explanation of the essence of the invention

The above-mentioned requirements for the crosslinker to be developed according to the invention are met by the hitherto unknown compound ^ -azido-cinnamic acid succinimidyl ester. For the ester, the abbreviation SAC (derived from jSucciniraidyl-4-azido-innatamate) is used below. The succinimidooxycarbonyl group quantitatively accepts free primary and secondary aliphatic amino groups in both organic and aqueous media at room temperature within a few minutes ·· The azidophenyl group, as a photoactivatable group, has two properties: it is completely inert chemically, thus allowing the removal of excess reagent after modification of the first component. Only after photolysis by irradiation with long-wave UV light does the azidophenyl group react to form a reactive phenylnitrene. This reacts nonspecifically and

therefore does not require a special functional group in the binding area of the second component for the crosslinking of the modified component.

The linking of the two functional groups by a vinyl group results in the desired little variable geometry with a distance of the functional groups between 8.8 and 9.2 Angstrom.

The preparation process of SAC according to the invention is characterized in that 4-azido-cinnamic acid _# must be present in a particularly pure form for this purpose, with N-hydroxysuccinimide in the presence of a suitable acylation catalyst such as dicyclohexylcarbodiimide in a suitable aprotic solvent such as tetrahydrofuran, dimethylformamide , Acetonitrile or dichloromethane, preferably tetrahydrofuran, at temperatures between 0 and 25 ⁰ C, preferably at 0 ⁰ C ,, and the formed SAC after removal of Dicyclohexylharnstoffs by known methods by recrystallization from a suitable solvent, preferably acetone, purified. All operations are performed under dim red light.

The process enables the production of SAC in good yields and high purity.

The SAC prepared according to the invention is capable of crosslinking with each other and with ribosomal RNA after reaction with ribosalar proteins when the azido group is activated by UV light in the range 300-400 nm. The inventive method will be explained in more detail below.

exemplary

Carefully purified 4-azido-cinnamic acid and 0.608 g (5.28 mmol) of N-hydroxysucciniride are dissolved in 20 ml of absolute tetrahydrofuran, with exclusion of moisture (argon), of 1 0 g (5.28 mmol) by recrystallization from ethyl acetate.

suspended. After cooling the suspension to ⁰ ° C., 1.2 g (5.81 mmol) of dicyclohexylcarbodiimide in 5 ml of absolute tetrahydrofuran are added with stirring. After 15 minutes, the batch is removed from the ice bath and stirred until complete reaction at room temperature. Then, the precipitated dicyclohexylurea is separated by filtration and the filtrate brought to dryness on a rotary vacuum evaporator. Recrystallization of the residue from acetone-heptane gives 1.25 g (83% of theory) of mp SAC 141 -. 143 ⁰ C. IR Spektrumj 1595 (C = C phenyl), 1623 (C = C double bond conj ♦) ^ 1733 (C = O conc.-unsaturated ester) and 2120 cm -1 (N = N stretching vibration). Mass spectrum (Hochai solves): 286 = M ⁺ , 258 = M - H ₂ , 172 = N ₃ -C ₅ H ₄ - CH = CH-CO ''' *

Claims (1)

invention claim Process for the preparation of 4-azido-cinnamic acid succinimidyl ester (SAC) O
O characterized in that 4-azido-cinnamic acid with N-hydroxysucciniraid in the presence of a suitable acylation catalyst such as dicyclohexylcarbodximide in a suitable aprotic solvent such as tetrahydrofuran, dimethylformamide, acetonitrile or dichloromethane, preferably tetrahydrofuran, at temperatures between 0 and 25 ⁰ C, preferably at 0 ° C> and after formation of the dicyclohexylurea the SAC formed is purified by recrystallization from a suitable solvent, preferably acetone, according to methods known per se.