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Multi-Functional Chemical Agents, and the Method for Protein Modification

a chemical agent and multi-functional technology, applied in the field of biotechnology, can solve the problems of a huge limitation of engineered proteins, the difficulty of developing synthetic parallels of natural systems, and the difficulty of precise labeling of native proteins,

Active Publication Date: 2021-06-03
DEPT OF BIOTECHNOLOGY MINIST OF SCI & TECH GOVERNMENT OF INDIA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent aims to develop chemical agents and a method for selectively modifying the protein backbone of native or un-engineered proteins, as well as other functional biological molecules such as antibodies. The modifications can be regulated by the chemical agents, allowing for the attachment of various tags such as fluorescent labels, drugs, and enzymes. The technology enables the creation of protein-based reporters, cyclization, protein-protein conjugation, enzyme-protein conjugation, and enzyme-antibody conjugation, among others.

Problems solved by technology

The ability in developing a synthetic parallel of the natural system is challenging as the site-specific modification of proteins requires controlled condition which is limited by the chemistry available.
The task of precise labeling of native proteins is still challenging and would require multiple approaches to meet the desired targets.
The site-directed mutagenesis route works well but operates through an engineered protein which is a huge limitation.
However, it is bogged down by the requirement of a specific affinity tag as a pre-requisite, whereas the chemoenzymatic methods have very limited scope of application.
Needless to mention, that this technique can't be used with native proteins.
The engineered monoclonal antibody approach is not practical; hence, both the approved drugs are synthesized using chemical methods.
However, in absence of an enabling technology, both the approved drugs are sold as heterogeneous mixtures [brentuxinab vedotin (Seattle Genetics; anti-CD30mAb, PAB linker, MMAE drug) and adotrastuzumab emtansine (Genentech; anti-HER2 mAb, SMCC linker, maytansine drug)].
This is further complicated by the presence of several copies of each amino acid.
It is a grand challenge to distinguish one residue from multiple copies of an amino acid present in protein for a site-selective modification.
The limitation of this method is that it works well for cases where a ligand is known for binding selectively to the site of interest.

Method used

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  • Multi-Functional Chemical Agents, and the Method for Protein Modification
  • Multi-Functional Chemical Agents, and the Method for Protein Modification
  • Multi-Functional Chemical Agents, and the Method for Protein Modification

Examples

Experimental program
Comparison scheme
Effect test

example 1

General Procedures

[0120]The reagents, proteins, and enzymes were purchased from Sigma-Aldrich, Alfa Aeser and Merck Novabiochem. The organic solvents used were reagent grade. Aqueous buffers were prepared freshly using Millipore Grade I water (Resistivity>5 MΩ cm, Conductivity<0.2 μS / cm, TOC<30 ppb). Mettler Toledo (FE20) pH meter was used to adjust the final pH. The reaction mixture for the small molecules was stirred (Heidolph, 500-600 rpm). Proteins were either vortexed or incubated in incubator-shaker Thermo Scientific MaxQ 8000 (350 rpm, 25-37° C.). Cellulose membrane (MWCO, 6-8 kD) from Spectrum labs was used for dialysis. Amicon® Ultra-0.5 mL 3-kDa MWCO Centrifugal Filters from Merck Millipore was used to remove small molecules from protein mixture, desalting and buffer exchange. Organic solvents were removed by BUCHI rotavapor R-210 / 215 whereas aqueous samples were lyophilized by CHRiST ALPHA 2-4 LD plus lyophilizer. Circular Dichroism (CD) measurements were recorded on JASC...

example 2

Procedures for Synthesis of Multifunctional Chemical Agents and Characterization Data

[0124]Following reagents represent Class 2 (A). For the synthesis of the reagents of other classes, condensation of respective Fn1 and Fn2 using similar protocol and linkers are followed.

example 2a

Synthesis of 2-hydroxy-4-(oxiran-2-ylmethoxy)benzaldehyde (24)

[0125]

[0126]In 5 ml round bottom flask, 2,4-dihydroxybenzaldehyde (138 mg, 1 mmol) and K2CO3 (138 mg, 1 mmol) were dissolved in dry DMF (1 ml). To this solution, epibromohydrin (86 μl, 1 mmol) was added and stirred at room temperature. After 8 h, reaction mixture was carried out for ethylacetate:n-hexane (30:70) and water work up. The collected organic layers were dried with anh. sodium sulphate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography using ethyl acetate:n-hexane (3:97) to give 24 (68 mg, 35% yield). 1H NMR (400 MHz, CDCl3) δ 11.45 (s, 1H), 9.73 (s, 1H), 7.44 (d, J=8.7 Hz, 1H), 6.57 (dd, J=8.7, 2.3 Hz, 1H), 6.43 (d, J=2.2 Hz, 1H), 4.31 (dd, J=11.1, 2.9 Hz, 1H), 3.97 (dd, J=11.1, 5.9 Hz, 1H), 3.41-3.32 (m, 1H), 2.93 (t, J=4.5 Hz, 1H), 2.76 (dd, J=4.8, 2.6 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 194.6, 165.6, 164.6, 135.5, 115.7, 108.8, 101.5, 69.2, 49.8, 44.7. HRMS (ESI) [MH...

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Abstract

A multifunctional chemical agents comprising functional agents Fn1, Fn2 and linkers, for the linchpin directed (LDM), protein directed (PDPM) modifications of proteins, and Fn1 accelerated kinetic labeling by Fn2.

Description

FIELD OF INVENTION[0001]The invention is in the field of biotechnology with specific reference to site selective modification of proteins.BACKGROUND OF THE INVENTION[0002]The diversity of structure and function of proteins emerges from their co-translational and post-translational modification. In efforts to mimic this process of nature, synthetic modification of protein has emerged as a wonder tool. It offers a broad range of applications for proteins being used from probes to therapeutics. The ability in developing a synthetic parallel of the natural system is challenging as the site-specific modification of proteins requires controlled condition which is limited by the chemistry available. Further, the need for molding the operating parameters and reactions for modification to a near biologically similar condition becomes relevant so as not to disrupt the protein architecture or function. Over many decades, a number of methodologies have emerged for modifying both the natural and...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K1/107C07K1/02C07K14/805
CPCC07K1/1075C07K14/805C07K1/02C07K1/006C07K1/13C07K1/22
Inventor RAI, VISHALADUSUMALLI, SRINIVASA RAO
Owner DEPT OF BIOTECHNOLOGY MINIST OF SCI & TECH GOVERNMENT OF INDIA