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Macromolecular conjugates for visualization and separation of proteins and cells

a technology of macromolecules and conjugates, applied in the field of synthetically prepared macromolecules, can solve the problems of loss of the ability to bind a given antigen, high production cost of antibodies, and easy degradation of antibody molecules, so as to avoid steric hindrance of binding, the effect of inexpensive preparation of polymeric conjugates

Inactive Publication Date: 2018-02-22
INST OF ORGANIC CHEM & BIOCHEMISTRY OF THE ACAD OF SCI OF THE CZECH REPUBLIC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a new method for creating polymeric conjugates that can be used to target enzymes and inhibit their activity. These conjugates are made using a strong interaction between a protein called biotin and a protein called avidin, which is attached to a resin. The conjugate can be easily separated from other molecules and used in various imaging techniques. The use of these conjugates has advantages over traditional antibodies, including lower cost and better stability. The polymer backbone can also be modified as needed, allowing for different fluorescent compounds or inhibitors to be attached. Overall, this method provides a more efficient and specific way to target enzymes and study their activity.

Problems solved by technology

First, the production of antibodies is very expensive.
Like other proteins, antibody molecules are susceptible to degradation: generally they must be stored at low temperatures, and if necessary, frozen in aliquots.
Their repeated thawing often leads to loss of their ability to bind a given antigen.
Another disadvantage is their own creation and method of preparation, since the immunization of an animal may not always lead to successful production of antibodies—they often may not be produced at all, or can be non-specific to the antigen.
Another disadvantage is the fact that for a close group of enzymes (ie. homologs, either paralogs or orthologs), it is often impossible to use the same antibody (recognizing native proteins) due to differences in amino acid residues on their surface.

Method used

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  • Macromolecular conjugates for visualization and separation of proteins and cells
  • Macromolecular conjugates for visualization and separation of proteins and cells
  • Macromolecular conjugates for visualization and separation of proteins and cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

on of GCPII Inhibitor with a Linker (Compound A)

[0053]

Di-tert-butyl 2-(3-(6-((4-bromobenzyl)amino)-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)pentanedioate; Compound A2: 300 mg (0.615 mmol, 1 eq) di-tert-butyl 2-(3-(6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)pentanedioate (Compound A1, prepared according to [10]) and 120 mg (0,646 mmol, 1.05 eq) of 4-bromobenzaldehyde was dissolved in 5 ml methanol in a round-bottom flask. 50 μl of glacial acetic acid was added and, after rapid mixing, 120 mg (1.85 mmol, 3.0 eq) of sodium cyanoborohydride in one portion. After 12 hours, the reaction was stopped by adding 10 ml of water. After 10 minutes, the reaction mixture was further diluted with 50 ml of water and was extracted three times with ethyl acetate (3×25 ml). The organic phase was dried and evaporated and the raw product was purified by chromatography on silica gel (eluent: EtOAc+1% ammonia saturated in water, TLC analysis, Rf=0.55). The weight of the obtained pure product was 395 mg (...

example 2

of Inhibitor of Carbonic Anhydrase IX (Compound B)

[0056]Compound B was prepared according to the scheme below:

methyl 4-(4-((tert-butoxycarbonyl)amino)butoxy)benzoate, Compound B1: 161 mg (1 eq, 1.06 mmol) of methyl 4-hydroxybenzoate, 300 mg (1.5 eq, 1.59 mmol) of tert-butyl (4-hydroxybutyl) carbamate and 400 mg (1.5 eq, 1.59 mmol) of triphenylphosphine was mixed in 10 ml of THF. 312 μl (1.5 eq, 1.59 mmol) of DIAD was then added all at once to the solution and the reaction was stirred overnight. The reaction mixture was then evaporated and the raw product was purified by column chromatography (He:EtOAc 4:1, RF=0.25). The weight of the obtained white powder was 260 mg, representing a 75% yield.

[0057]Note: the methyl 4-hydroxybenzoate had the same RF as the product, so 1.5 eq was used with other compounds.

[0058]MS (ESI+): counted for C17H25O5N [MNa]+ 346.17. Found 346.2. 1H NMR (400 MHz; CDCl3) δ 7.95 (d; J=8.9 Hz; 2H); 6.87 (d; J=8.9 Hz; 2H); 4.71 (s; 1H); 3.99 (t; J=6.2 Hz; 2H); 3.85...

example 3

on of HIV-1 Protease Inhibitor with a Linker (Compound C)

[0062]Compound C, based on a commercially available HIV protease inhibitor drug ritonavir (RTV), was synthesized according to the below depicted scheme:

[0063]Isolation of ritonavir (RTV) from commercially available capsules: RTV is suspended in capsules in an oily mixture of rather non-polar compounds. 50 tablets (100 mg RTV each) were cut open and the oily substance was squeezed out into a round-bottom shaped 2 l flask. 200 ml of hexan was added along with 500 ml of diethyl ether. The resulting suspension was triturated and sonicated for 3 hours until all oil turned into a white precipitate. This precipitate was filtered and again triturated / sonicated in pure diethyl ether, after which the pure RTV was filtered. 3.6 g of RTV was obtained (isolation yield 72%). The purity of RTV was determined by HPLC and was well above 99% (analytical HPLC RT=23.7 min).

Partial hydrolysis of ritonavir (RTV), thiazol-5-ylmethyl ((2S,3S,5S)-5-am...

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Abstract

Macromolecular water-soluble conjugates based on synthetic copolymers to which at least one affinity tag, at least one imaging probe, and at least one targeting ligand are bound via covalent bonds. The macromolecular conjugate may be used in identification, visualization, quantification or isolation of proteins and / or cells. The targeting ligand may be attached to the synthetic copolymer via a flexible linker.

Description

FIELD OF ART[0001]The invention describes synthetically prepared macromolecules having properties of monoclonal antibodies, said macromolecules being capable of replacing the use of antibodies in scientific research, in diagnostics, in biochemical investigations and for the preparation of targeted drugs. These synthetic macromolecules, targeted and binding specifically to certain proteins, are suitable for the visualization, identification and isolation of biomolecules and / or cells in biochemistry, molecular biology and medicine and as targeting ligands in the pharmacy and diagnostics.BACKGROUND ART[0002]The discovery and the subsequent use of monoclonal antibodies allowing detection and specific binding of biologically important molecules caused a revolution in biochemistry and molecular biology as well as in the diagnosis and treatment of numerous serious diseases. In science, this discovery led to the development of many important techniques today considered routine, such as West...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/534A61K47/58A61K49/00C08F220/58G01N33/533
CPCG01N33/534A61K47/58A61K49/0054C08F220/58G01N33/533C08F2500/01C07K5/00C07K17/08C08F220/18G01N33/60G01N33/531G01N33/532
Inventor SACHA, PAVELKONVALINKA, JANSCHIMER, JIRIKNEDLIK, TOMASNAVRATIL, VACLAVTYKVART, JANSEDLAK, FRANTISEKMAJER, PAVELCIGLER, PETRSUBR, VLADIMIRULBRICH, KARELSTROHALM, JIRI
Owner INST OF ORGANIC CHEM & BIOCHEMISTRY OF THE ACAD OF SCI OF THE CZECH REPUBLIC
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