Peptide Nucleic Acid Monomer And A Preparation Method Thereof
Abstract:
The disclosure in various embodiments is related to a peptide nucleic acid monomer (VI) and an improved process for preparation of PNA (VI) using nosyl- group containing starting material. The process includes reacting amino acid with a nosyl group. The nosyl protected amino acid is alkylated with 2-(Boc-amino)ethyl bromide to obtain an intermediate. The nosyl group of the obtained intermediate is removed to form a PNA backbone. The PNA backbone upon addition of a modified nucleobase results in the formation of a PNA monomer (VI). The obtained PNA monomer exhibits either chiral or achiral geometric properties.
Fig.VI
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Notices, Deadlines & Correspondence
Amrita Vishwa Vidyapeetham, Amritanagar, Ettimadai,
Coimbatore, Tamil Nadu, India, Pin Code-641 112.
Inventors
1. Dr. Prasanna RAMANI
Dhanvanthri Lab, Department of Sciences, Amrita Vishwa Vidyapeetham, Amrita University, Amrita Nagar, Coimbatore, Tamil Nadu, India, Pin Code-641 112.
Specification
PEPTIDE NUCLEIC ACID MONOMER AND A PREPARATION METHOD THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] None.
FIELD OF THE INVENTION
[0002] This invention relates generally to nucleic acids and in particular to peptide
nucleic acid (PNA) monomers for use in building peptide nucleic acid oligomers and method of preparation thereof.
DESCRIPTION OF THE RELATED ART
[0003] Peptide nucleic acid (PNA) is a potential bio-molecular tool with broad range of
applications. PNAs are considered to be the most important backbone modifications of DNA, which was developed by Nielsen and co-workers, where the entire sugar-phosphate backbone is replaced by a homomorphous, chiral/achiral and uncharged peptide backbone based on N-(2-aminoethyl) glycine units (aegPNA). The nucleobases are attached to the backbone through a carbomethylene linker. The inter-nucleobases distance in PNA is preserved, allowing its binding to the target DNA/RNA sequences with high sequence specificity and affinity. PNA is considered to be an exact DNA mimic in terms of base-pair recognition. But, PNAhas poor solubility compared to both DNA and RNA. Low cellular permeability limits its applications for antigene or antisense therapies (via infra). PNAs lacks in probe to recognize the PNA-DNA hybridization event.
[0004] U.S patent 5859191 to Miller et al., discloses a method for the site-specific
modification of peptide alpha amines at specific N-alpha positions. The method of modification consists of treating a free terminal alpha amine with an aryl sulfonamide
activating agent, to form an activated amide which is deprotonated with a base and modified by the addition of a substituent group.
[0005] US patent 5714331 to Buchardt et al., discloses a method to enhance binding
affinity, sequence specificity and solubility of PNAs.US patent 7022851 to Kim et al., discloses the preparation of monomers for the preparation of PNA (peptide nucleic acid) oligomers and provides method for the synthesis of both predefined sequence PNA oligomers and random sequence PNA oligomers.
[0006] “Synthesis of achiral and chiral peptide nucleic acid (PNA) monomers using
“Mitsunobu reaction” Falkiewicz et al., Tetrahedron, 2001 discusses the synthesis of N-, C-protected pseudodipeptides with a reduced peptide bond and a peptide nucleic acid (PNA) monomers, based on the Mitsunobu reaction of N-Boc-β-amino alcohols with N-o-nitrobenzenesulfonyl-protected (oNBS-protected) amino acid esters.
[0007] “A convenient synthesis of chiral peptide nucleic acid (PNA) monomers”
Kosynkina et al., Tetrahedron Letters, 1994 discusses the preparation of chiral peptide nucleic acid monomers containing amino acid chains from BOC-protected amino acids.
[0008] The present disclosure describes a process, which is simple and cheap, for the
preparation of chiral or achiral PNA monomers.
SUMMARY OF THE INVENTION
[0009] The disclosure relates toa process for synthesizing PNA monomers which forms
the building blocks of PNA oligomers.
[0010] In various embodiments process for preparation of a peptide nucleic acid (PNA)
monomer, includes providing a nosyl-group containing starting material of formula (II)
where, R1 is -H, -CH3, -CH(CH3)2 , -CH2CH(CH3)2, -CH2OH, -CH2C6H5OH and R3 is o-nosyl or p-nosyl. The nosyl-group containing starting material of formula (II) is alkylated with 2-(Boc-amino) ethyl bromide to form an intermediate of formula (IV).
where, R2 in (IV) is an amine-protecting group. The nosyl group is removed to obtain a PNA backbone of formula (V).
A nucleobase is added to (V) to obtain a PNA monomer.
[0011] In various embodiments a nosyl-group containing starting material (II) is obtained by a reaction between an amino acid containing a first reaction mixture with nosyl chloride. In
AMRT-008300IN -4-
one embodiment the first reaction mixture includes dichloro methane and triethyl amine or diisopropyl ethylamine. In one embodiment the reaction is carried out at a temperature between 0-35°C. In some embodiments the nosyl product is obtained by stirring the mixture for 20- 30 h.
[0012] In various embodiments alkylating includes reacting the nosylated starting material (II) and with a second reaction mixture. Second reaction mixture includes cesium carbonate or 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU), or triethylamine or pyridine and dimethylformamide as solvent. In some embodiments, the alkylation is performed at a temperature between 25-40°C. In some embodiments the alkylation is performed for a period of 10-24 h. In one embodiment the intermediate (IV) is further purified by column chromatography.
[0013] In various embodiments, removal of the nosyl group includes deprotecting the nosyl group having a third reaction mixture under nitrogen atmosphere at 0°C. The reaction mixture is stirred at a temperature between 20-30°C for a period of 15-20 h. In some embodiments the third reaction mixture includes a DMF medium having potassium carbonate and thiophenol.
[0014] In various embodiments, the starting material is an amino acid selected from the group which includes of alanine, glycine, and valine. In some embodiments the obtained PNA monomer is chiral or achiral.
[0015] In various embodiments, to obtain a PNA monomer a nuclobase is added by a condensation reaction between a modified nucleobase and a compound of formula (V). The nucleobase used is a modified nucleobase which is thymine-1- acetic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The invention has other advantages and features which will be more readily
apparent from the following detailed description of the invention and the appended claims, when taken in conjunction with the accompanying drawings, in which:
[0017] FIG. 1 illustrates a method of preparing a peptide nucleic acid (PNA) monomer.
[0018] FIG. 2A illustrates starting materials, intermediates and products obtained during
the process.
[0019] FIG. 2B illustrates nosyl protection of the amino acid.
[0020] FIG. 2C illustrates alkylation reaction of the nosyl protected amino acid.
[0021] FIG. 2D illustrates removal of the noysl group.
[0022] FIG. 2E illustrates addition of a nucleobase to form a PNA monomer.
[0023] FIG. 2F illustrates a complete reaction to form a PNA monomer from an amino
acid starting material.
DETAILED DESCRIPTION
[0024] While the invention has been disclosed with reference to certain embodiments, it
will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.
[0025] Throughout the specification and claims, the following terms take the meanings
explicitly associated herein unless the context clearly dictates otherwise. The meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on." Referring to the drawings, like numbers indicate like parts throughout the views. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.
[0026] The word “exemplary” is used herein to mean “serving as an example, instance,
or illustration.” Any implementation described herein as “exemplary” is not necessarily to be construed as advantageous over other implementations. The ingredients are indicated to be in weight % throughout this specification.
[0027] In various embodiments a process 100 for preparing a peptide nucleic acid
(PNA) monomer is disclosed. The PNA monomer is of formula (VI) as shown in FIG. 2A.
[0029] In one embodiment of the invention the process 100 for preparing a peptide
nucleic acid (PNA) monomer is illustrated in four steps 101-104 of FIG. 1. An amino acid (I) is protected with a nosyl group in a nosylation step 101 to form a nosyl protected amino acid (II). The nosyl protected amino acid (II) reacts with a 2-(Boc-amino)ethyl bromide (III) to form an intermediate (IV) as shown in an alkylation reaction step 102. In the next step 103, removal of the nosyl protection group in (IV) results in formation of a PNA backbone (V). In the final step 104, a nucleobase molecule is reacted with the previously obtained PNA backbone (V) to form the PNA monomer (VI) as shown in a condensation reaction 104.
R\ R\'
[0030] In various embodiments R1 of (I) is H or amino acid side chain such as -CH3, -
CH(CH3)2, -CH2CH(CH3)2, -CH2-OH, -CH2-