Patent Application
20240190924
Originally developed by Peter Nielsen and coworkers in the early 1990's, peptide nucleic acids (PNAs) have emerged as a promising class of nucleic acid mimics for biological and biomedical applications because of their tight and specific binding, and resistance to enzymatic degradation by proteases and nucleases. Building on this initial molecular scaffold, it has been shown that PNAs, which do not adopt a well-defined conformation, can be preorganized into either a left-handed (LH) or right-handed (RH) helical motif by installing an appropriate stereogenic center at the gamma backbone (
The recognition orthogonality and the translational capability of the (LHγPNA)/(PNA)/(RHγPNA) system has generated considerable interest in its applications in molecular computing and cell therapy. With respect to the latter, the utilization of relatively short, orthogonal LHγPNAs for programming cell-cell interaction is attractive, for instance, in directing chimeric antigen receptor T (CAR-T) cells to engage cancer cells, because of the relative ease of production, the close proximity of the two cell types required for effective cancer cell killing, and their inertness to hybridization with DNA or RNA upon internalization into the cell's nucleus and cytoplasm. However, with current design, the charge-neutral backbone imposes a major challenge for their handling and molecular manipulation. Due their poor water solubility, these molecules and the corresponding complexes are prone to aggregation and association with other macromolecules and surfaces in a nonspecific manner, making them inferior for molecular self-assembly. Likewise, when covalently attached to cells or other materials, they have a propensity to collapse onto the surface, and in the former case, are buried in the membrane, rendering them ineffective for hybridization with their complementary partners that are in solution or covalently attached to other extracellular components or material's surfaces.
There is a need for LHγPNA and RHγPNA monomers and oligomers containing strategically placed or fully-modified groups, such as phosphate, guanidine, and dihydroxypropyl gamma-side chain, as well as methods for easy modification of groups attached to the gamma side chain.
According to an aspect or embodiment, a method of making a peptide nucleic acid monomer is provided, comprising:
phosphorylating compound 1, where n is 1, 2, 3, or 4, R1 is an amine-protecting group:
with trichlorophosphorus (PCl3), phosphoramidous acid, N,N-bis(1-methylethyl)-, bis(phenylmethyl) ester, or 4,3-Benzodioxaphosphepin, 3-chloro-15-dihydro-, 3-oxide), R2—OH, and R3—OH, to produce compound 2, where R2 and R3 are, independently, H, benzyl
t-butyl, propionitrilyl
or 4-nitrophenylethylenyl
reducing compound 2 to produce compound 3:
reacting compound 3 with Dess-Martin periodinane, followed by treating with NH2CH2C(O)OCH3 and DCM, or reacting compound 3 with DMSO:TEA followed by treating with NH2CH2C(O)OCH3, to produce compound 4:
and
conjugating compound 4 with a nucleobase by reacting compound 4 with RCH2C(O)OH, where R is a nucleobase in which α-amines of R are protected with an amine-protecting group to produce compound 5:
According to another aspect or embodiment, a method of making a peptide nucleic acid monomer is provided, comprising:
reducing compound 6:
where R4 is an amine-protecting group, and one of R5 and R6 is H, and the other of R5 and R6 is:
where m is 1, 2, 3, or 4, to produce compound 7:
reacting compound 7 with Dess-Martin periodinane, followed by treating with NH2CH2C(O)OCH3 (methyl glycinate), or reacting compound 7 with DMSO:TEA followed by treating with NH2CH2C(O)OCH3, to produce compound 8:
conjugating compound 8 with a nucleobase by reacting compound 8 with RCH2C(O)OH, where R is a nucleobase in which α-amines of R are protected with an amine-protecting group to produce compound 9:
and
removing the terminal methyl group of 9 to produce compound 10:
According to another aspect or embodiment, a compound having the structure:
or a salt thereof is provided, along with enantiopure compositions comprising a stereoisomer of that compound, as well as a racemic mixture of stereoisomers of that compound in any relative proportion. A peptide nucleic acid comprising a residue (one or more residues) of the monomer also is provided in aspects or embodiments.
According to another aspect or embodiment, a solid-phase peptide nucleic acid synthesis method is provided comprising sequentially extending a sequence of peptide nucleic acid monomers from a substrate to produce a peptide nucleic acid comprising the sequence of peptide nucleic acid monomers and wherein the sequence of peptide nucleic acid monomers comprising at least one residue of a γPNA monomer having the structure:
where R4 is an amine-protecting group, and one of R5 and R6 is H, and the other of R5 and R6 is
where m is 1, 2, 3, or 4,
deprotecting the peptide nucleic acid to remove the allyloxycarbonyl group from R5 and R6, e.g., using Pd(PPh3)4 (palladium-tetrakis(triphenylphosphine)) or PhSiH3 (phenylsilane), and linking the unprotected amine of R5 or R6 with an amine-reactive compound to modify the peptide nucleic acid; and
cleaving the peptide nucleic acid from the substrate
According to another aspect or embodiment, a peptide nucleic acid monomer, having the structure:
where one of R5 and R6 is H, and the other of R5 and R6 is:
where m is 1, 2, 3, or 4. IN another aspect or embodiment, a γPNA is provided, comprising a monomer residue of that peptide nucleic acid monomer.
According to another aspect or embodiment, a method of making a peptide nucleic acid monomer is provided, comprising:
adding an amine protecting group to the terminal amine and adding a 4,4′-dimethoxytrityl (DMT) group to the primary hydroxyl group of compound 11:
where R11 is —(CH2)n—, and n is 1-4,
to produce compound 12, where R12 is an amine-protecting group;
reducing compound 12 and reacting compound 12 with Dess-Martin periodinane followed by methyl glycinate to produce compound 13:
conjugating compound 13 with a nucleobase by reacting compound 13 with RCH2C(O)OH, where R is a nucleobase in which α-amines of R are protected with an amine-protecting group and removing the methyl group from the terminal carboxymethyl group to produce compound 14:
phosphorylating the DMT-protected oxygen with:
dichloroacetic acid,
tetrazole, and I2, to produce compound 15:
where R13 and R14 are, independently, H, benzyl, t-butyl, propionitrilyl, or 4-nitrophenylethylenyl.
According to another aspect or embodiment, a solid-phase peptide nucleic acid synthesis method is provided. comprising sequentially extending a sequence of peptide nucleic acid monomers from a substrate to produce a peptide nucleic acid comprising the sequence of peptide nucleic acid monomers and wherein the sequence of peptide nucleic acid monomers comprising at least one residue of a γPNA monomer having the structure:
where R11 is —(CH2)n—, and n is 1-4 and R12 is an amine-protecting group; and
phosphorylating the DMT-protected oxygen with:
dichloroacetic acid,
tetrazole, and I2; and
cleaving the peptide nucleic acid from the substrate.
The terms Fig., Figs., Figure, and Figures are used interchangeably in the specification to refer to the corresponding figures in the drawings.
The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values. As used herein “a” and “an” refer to one or more.
As used herein, the term “comprising” is open-ended and may be synonymous with “including”, “containing”, or “characterized by”. The term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting of” excludes any element, step, or ingredient not specified in the claim. As used herein, embodiments “comprising” one or more stated elements or steps also include, but are not limited to embodiments “consisting essentially of” and “consisting of” these stated elements or steps.
A polymer, such as a PNA, e.g., a γPNA, “comprises” or is “derived from” a stated monomer if that monomer is incorporated into the polymer. Thus, the incorporated monomer that the polymer comprises is not the same as the monomer prior to incorporation into a polymer, in that at the very least, certain linking groups are incorporated into the polymer backbone or certain groups are removed in the polymerization process, such as the loss of a water molecule in forming a peptide (amide) bond characteristic of the reaction of an amino group with a carboxyl group during PNA synthesis. A polymer is said to comprise a specific type of linkage if that linkage is present in the polymer. An incorporated monomer is a “residue”. A typical monomer for a nucleic acid or nucleic acid analog is referred to as a nucleotide or a nucleotide residue when incorporated into a polymer.
A “moiety” (pl. “moieties”) is a part of a chemical compound, and comprises groups, such as functional groups. As such, a nucleobase moiety is a nucleobase that is modified by attachment to another compound moiety, such as a polymer monomer, e.g. the nucleic acid or nucleic acid analog monomers described herein, or a polymer, such as a nucleic acid or nucleic acid analog as described herein. In chemical structures provided herein, wavy lines indicate the location of a bond linking the depicted moiety or group to a remainder of a described compound or molecule.
“Alkyl” refers to straight, branched chain, or cyclic hydrocarbon groups including from 1 to about 20 carbon atoms, for example and without limitation C1-3, C1-6, C1-10 groups, for example and without limitation, straight, branched chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and the like. An alkyl group can be, for example, a C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted. Non-limiting examples of straight alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Branched alkyl groups comprises any straight alkyl group substituted with any number of alkyl groups. Non-limiting examples of branched alkyl groups include isopropyl, isobutyl, sec-butyl, and t-butyl. Non-limiting examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptlyl, and cyclooctyl groups. Cyclic alkyl groups also comprise fused-, bridged-, and spiro-bicycles and higher fused-, bridged-, and spiro-systems. A cyclic alkyl group can be substituted with any number of straight, branched, or cyclic alkyl groups. “Alkylene” and “substituted alkylene” refer to divalent alkyl and divalent substituted alkyl, respectively, including, without limitation, ethylene (—CH2—CH2—).
“Aryl,” alone or in combination refers to an aromatic ring system such as phenyl, benxyl, or naphthyl. “Aryl” also includes aromatic ring systems that are optionally fused with a cycloalkyl ring. A “substituted aryl” is an aryl that is independently substituted with one or more substituents attached at any available atom to produce a stable compound. Common substituents include, but are not limited to halide atoms, such as Cl, Br, and F. “Optionally substituted aryl” refers to aryl or substituted aryl. “Arylene” denotes divalent aryl, and “substituted arylene” refers to divalent substituted aryl. “Optionally substituted arylene” refers to arylene or substituted arylene. As used herein, the term “polycyclic aryl group” and related terms, such as “polycyclic aromatic group” means a group composed of at least two fused aromatic rings. “Heteroaryl” or “hetero-substituted aryl” refers to an aryl group substituted with one or more heteroatoms, such as N, O, P, and/or S. Arylalkyl refers to moieties comprising alkyl and aryl constituents.
“Carboxyl” or “carboxylic” refers to group having the indicated number of carbon atoms and terminating in a —C(O)OH group, thus having the structure -R—C(O)OH, where R is a divalent organic group that includes linear, branched, or cyclic hydrocarbons. Non-limiting examples of these include: C1-8 carboxylic groups, such as ethanoic, propanoic, 2-methylpropanoic, butanoic, 2,2-dimethylpropanoic, pentanoic, etc.
A conformationally preorganized nucleic acid analog is a nucleic acid analog that has a backbone (a preorganized backbone) that forms only either a right-handed helix or a left-handed helix, depending on the structure of the nucleic acid backbone. As shown herein, an example of a conformationally preorganized nucleic acid analog is γPNA, which has a chiral center at the γ carbon, and, depending on, and due to, the chirality of the groups at the γ carbon, forms a right-handed helix or a left-handed helix.
In the context of the present disclosure, a PNA monomer, e.g., a γPNA monomer, refers to a monomer comprising at least one nucleobase and a backbone element (backbone moiety), which in a peptide nucleic acid is N-(2-aminoethyl)-glycine, which in γPNA has a chiral center at the gamma carbon. PNA monomers also comprise reactive amine and carboxyl groups that permit polymerization under specific conditions, as in SPPS. For chemical synthesis of PNAs, the nucleobases and backbone monomers may contain modified groups, such as blocked amines, as are known in the art. A “PNA monomer residue” refers to a single PNA monomer that is incorporated into a PNA oligomer, with adjacent PNA monomers being linked by an amide bond. A “genetic recognition reagent”, in context of the present disclosure, refers generically to a peptide nucleic acid that comprises a sequence of nucleobases that is able to hybridize to a complementary nucleic acid or nucleic acid analog sequence on a nucleic acid by cooperative base pairing, e.g., Watson-Crick base pairing or Watson-Crick-like base pairing.
In reference to
Referring to
According to one aspect or embodiment, a PNA monomer synthesis method is provided that may be conducted as a one-pot reaction to install a phosphate group on amine-protected serine or homoserine, e.g., Fmoc-serine or Fmoc-homoserine (see, e.g., compound 2,
A benzyl (Bn) protecting group may be used to protect the phosphate due to its stability toward both acid and base conditions employed in the synthesis, and that it can be simultaneously removed in a final cleavage step of solid-phase peptide synthesis (SPPS). A single, rather than double, Bn-protection may be used to achieve efficient monomer coupling, while providing complete protection for a phosphate group.
For the schemes shown in
In chemical synthesis schemes and methods described herein, the chemical structures of expected intermediate compounds are provided as illustrative of a major, intended product of a stated reaction, and certain illustrative reaction conditions, solvents, co-factors, catalysts, initiators, and other reactants are described. Choices of solvents, and other compounds and compositions in any give reaction mixture, as well as their concentration in the reaction mixture, and physical reaction conditions may be varied and optimized by one of ordinary skill in the art based on the present disclosure and the desired reaction outcome.
More generally, according to aspects or embodiments of the present disclosure, and as exemplified by the scheme of
where n is 1, 2, 3, or 4, and R1 is an amine-protecting group, e.g., with trichlorophosphorus (PCl3), phosphoramidous acid, N,N-bis(1-methylethyl)-, bis(phenylmethyl) ester (e.g., CAS Number: 108549-23-1), or 4,3-Benzodioxaphosphepin, 3-chloro-1,5-dihydro-, 3-oxide (e.g., CAS Number: 49785-01-5), R2—OH, and R3—OH, to produce compound 2:
where R2 and R3 are, independently, H, benzyl
t-butyl, propionitrilyl
or 4-nitrophenylethylenyl
Compount 2 may be then reduced to produce compound 3:
Compound 3 may be then reacted with Dess-Martin periodinane, followed by treating with NH2CH2C(O)OCH3 and DCM, to produce compound 4. Alternatively compound 3 may be reacted with DMSO:TEA followed by treating with NH2CH2C(O)OCH3, to produce compound 4:
Compound 4 may then be conjugated with a nucleobase by reacting compound 4 with RCH2C(O)OH, where R is a nucleobase in which α-amines of R are protected with an amine-protecting group, such as Boc, to produce compound 5:
Conjugation of compound 4 may be performed using HBTU. Conjugation of a nucleobase to a PNA backbone, e.g. reacting a secondary amide of a PNA precursor with RCH2C(O)OH, where R is a nucleobase, according to any aspect or embodiment described herein, also may be performed in the presence of alternatives to HBTU, such as, for example and without limitation: HATU (e.g., 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), HCTU (e.g., O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), TATU (e.g., O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate), TBTU (e.g., 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate), BOP (e.g., benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), PyBOP (e.g., Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), PyAOP (e.g., tripyrrolidin-1-yl(triazolo[4,5-b]pyridin-3-yloxy)phosphanium; hexafluorophosphate), PyBrOP (e.g., bromo(tripyrrolidin-1-yl)phosphanium; hexafluorophosphate), BOP-Cl (e.g., Phosphoric acid bis(2-oxooxazolidide) chloride), DCC (e.g., N,N′-dicyclohexylcarbodiimide), DIC (e.g., N,N′-Diisopropylcarbodiimide), or EDC HCl (e.g., N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride).
Compound 1 may be:
resulting in a product, compound 5 of:
respectively.
R1 may be Fmoc. The nucleobase R may be any nucleobase, either natural (adenine, thymine, guanine, cytosine, or uracil), or non-natural, e.g. as depicted in
Protecting groups (e.g., for protecting amines during synthesis of compounds described herein) are broadly-known in the art and include, for example and without limitation: 9-fluorenylmethyloxy carbonyl (Fmoc), t-butyloxycarbonyl (Boc), tert-butyl (tBu), benzhydryloxycarbonyl (Bhoc), benzyloxycarbonyl (Cbz), O-nitroveratryloxycarbonyl (Nvoc), benzyl (Bn), allyloxycarbonyl (alloc), trityl (Trt), dimethoxytrityl (DMT), I-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl (Dde), diathiasuccinoyl (Dts), benzothiazole-2-sulfonyl (Bts) and monomethoxytrityl (MMT) groups.
PNA oligomers, e.g. comprising from 2 to 25 PNA monomer residues, as described herein, may be synthesized using solid-phase peptide synthesis (SPPS) methods, as are broadly-known, and using manual or automated equipment, including microwave-assisted methods and devices. SPPS involves the successive addition of protected amino acid derivatives, such as PNA monomers, to a growing peptide chain immobilized on a solid phase, including deprotection and washing steps to remove unreacted groups and also side products. While the SPPS methods of making PNA oligomers described herein rely on specialized reagents, such as novel monomers as described herein, the overall deprotection, conjugation, capping, and washing steps, and choice of protecting groups, may be accomplished using standard methodologies, modified as described using unique reagents and steps. Fmoc SPPS is very common, and uses Fmoc to protect terminal amines, removal of which (deprotection) permits addition of a single Fmoc-protected PNA monomer. While Fmoc SPPS is depicted throughout this document, other SPPS methods are contemplated, with appropriate choice of protective groups.
PNA oligomers, including conformationally organized RHγPNA and/or LHγPNA oligomers, have two or more PNA monomer residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive PNA residues. Individual monomer residue of the PNA oligomer may be linked to (covalently attached to) a nucleobase, as depicted herein. Each individual monomer of a PNA oligomer may be linked to the same or a different nucleobase to form a defined sequence of nucleobases that may be complementary to, or bind to a nucleic acid or a nucleic acid analog, including other PNAs, as is broadly-known in the nucleic acid and peptide nucleic acid fields (see, e.g., International Patent Publication Nos. WO 2014/169206, WO 2018/058091, WO 2019/126638, and WO 2019/236979, the disclosure of each of which is incorporated herein by reference, also providing useful PNA oligomers). In a PNA oligomer, the sequence of nucleobases, and the sequence of PNA monomers, as described herein is produced by the stepwise addition of individual PNA monomers by SPSS protocols.
According to one aspect or embodiment, a PNA monomer synthesis method is provided that may be conducted as a one-pot reaction to install a protective allyloxycarbonyl group (—C(O)OCH2CHCH2 or Alloc) at the gamma carbon (see, e.g., compounds 10a-d and 10′a-d,
where R9 and R10 are, independently, H, benzyl, t-butyl, propionitrilyl, or 4-nitrophenylethylenyl;
to add phosphate, guanidine, or dihydroxypropyl functionality, respectively.
According to aspects or embodiments of the present disclosure, a method of making a peptide nucleic acid monomer, as exemplified in
where R4 is an amine-protecting group, and one of R5 and R6 is H, and the other of R5 and R6 is:
where m is 1, 2, 3, or 4 (methylenyl, dimethylenyl, trimethylenyl, or tetramethylenyl), to produce compound 7:
It is noted that the location of the H atom for R5 and R6 will determine chirality of the resultant monomer. Next compound 7 may be reacted with Dess-Martin periodinane, followed by treating with NH2CH2C(O)OCH3 (methyl glycinate), or reacted with DMSO:TEA followed by treating with methyl glycinate, to produce compound 8:
Compound 8 may then be conjugated to a nucleobase at the secondary amine by linking with RCH2C(O)OH, where R is a nucleobase in which α-amines of R are protected with an amine-protecting group to produce compound 9:
The terminal methyl group of 9 is then removed to produce compound 10:
According to one aspect or embodiment, a PNA monomer synthesis method is provided that may be conducted as a one-pot reaction to install a phosphate at the gamma carbon (see, e.g., compounds 15a-d and 15′a-d,
In one aspect or embodiment, a method of making a peptide nucleic acid monomer is provided. The method comprises, first, adding an amine protecting group to the terminal amine and adding a 4,4′-dimethoxytrityl (DMT) group to the primary hydroxyl group of compound 11:
where R11 is —(CH2)n—, and n is 1-4, to produce compound 12;
where R12 is an amine-protecting group, such as Fmoc. Compound 12 may then be reduced, and reacted with Dess-Martin periodinane and methyl glycinate to produce compound 13:
Compound 13 may next be conjugated with a nucleobase by reacting compound 13 with RCH2C(O)OH, where R is a nucleobase in which α-amines of R are protected with an amine-protecting group, and the methyl group may be removed from the terminal carboxymethyl group, to produce compound 14:
The DMT-protected oxygen of compound 14 may then be phosphorylated with dichloroacetic acid,
tetrazole, and I2, to produce compound 15:
where R13 and R14 may be, independently, H, benzyl, t-butyl, propionitrilyl, or 4-nitrophenylethylenyl.
It is noted that in all phosphorylated γPNA monomers described herein, one or more of the oxygen atoms of the phosphate group may be connected to benzyl, t-butyl, propionitrilyl, or 4-nitrophenylethylenyl groups. These groups may serve to protect the phosphate group and may be removed during cleavage of the PNA oligopeptide from the solid support during SPSS incorporation of the PNA monomer.
PNA monomers synthesized as described above, for example as shown in
In the case of the process exemplified in
where R9 and R10 are, independently, H, benzyl, t-butyl, propionitrilyl, or 4-nitrophenylethylenyl;
glyceric acid; dyes, such as cyanine, FAM, FITC, Rhodamine dyes; and R15—C(O)OH (e.g., organic acids or carboxylic acids), where R15 may be an alky or aryl group, including alkyl-aryl, or substituted alkyl, aryl, or alkyl-aryl groups.
In the case of the process exemplified in
The methods described herein may be used to produce a genetic recognition reagent, that binds specifically to fully or partially complementary nucleic acid or PNA strands. The genetic recognition reagent comprises a plurality of nucleobase moieties, each attached to a PNA backbone monomer residue, and forming a part of the larger genetic recognition reagent comprising at least two PNA monomer residues, and therefore at least two nucleobases (nucleobase moieties). Depending upon choice of nucleobases in the sequence, the genetic recognition reagents described herein can bind a single nucleic acid or PNA strand, or invade or otherwise hybridize to two strands of fully-complementary, partially-complementary or non-complementary double-stranded nucleic acids. As used herein, a monovalent nucleobase binds one nucleobase on a single nucleic acid strand, while a divalent nucleobase binds to two nucleobases, one on a first nucleic acid strand, and another on a second nucleic acid strand. Any choice of divalent and/or monovalent nucleobases may be selected for incorporation into a PNA oligomer as described herein.
Complementary refers to the ability of polynucleotides (nucleic acids) to hybridize (bind) to one another, forming inter-strand base pairs. Base pairs are formed by hydrogen bonding between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair (hybridize or bind) in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. When using RNA as opposed to DNA, uracil rather than thymine is the base that is complementary to adenosine. Two sequences comprising complementary sequences can hybridize if they form duplexes under specified conditions, such as in water, saline (e.g., normal saline, or 0.9% w/v saline) or phosphate-buffered saline), or under other stringency conditions, such as, for example and without limitation, 0.1×SSC (saline sodium citrate) to 10×SSC, where 1×SSC is 0.15M NaCl and 0.015M sodium citrate in water. Hybridization of complementary sequences is dictated, e.g., by salt concentration and temperature, with the melting temperature (Tm) lowering with increased mismatches and increased stringency. Perfectly matched sequences are said to be “fully complementary”, though one sequence (e.g., a target sequence in an mRNA) may be longer than the other, as in the case of the small recognition reagents described herein in relation to the much longer target sequences on which they concatenate, such as mRNAs containing repeat expansions. Two complementary strands of nucleic acid bind in an antiparallel orientation, with one strand in a 5′ to 3′ orientation, and the other in a 3′ to 5′ orientation. PNA permits both parallel and antiparallel orientation, though for γPNA antiparallel binding is preferred.
Examples of applications for the oligomers described herein is in the treatment of genetic diseases with repeat expansion of small sequences, such as those listed in Table 1.
200-1,700
500-4,500
Based on Table 1, the nucleobase sequence of the PNA oligomers described herein would bind to the repeat sequences shown in that table, either to a single strand, or to two strands in the case of divalent nucleobases for genetic recognition reagents. The PNA oligomer may bind to a single repeat or contain more than one iterations of the repeated sequence. It should be noted that, depending on their sequence, not all repeated sequences will form a hairpin structure under normal conditions, but can be induced into a triplex “hairpin” structure by a genetic recognition reagent comprising divalent nucleobases. The repeated nature of the sequences dictate that for a three-base repeat, three different frameshifts may be useful for each sequence, and for a four-base repeat, four different frameshifts may be useful. In use, PNA oligopeptides as described herein may be compounded or formulated into a pharmaceutical composition, including one or more pharmaceutically-acceptable excipients.
The following are illustrative examples of the preparation of γPNA monomers and oligomers as described herein.
The overall scheme for this reaction is essentially as depicted in
Compound 3: To a cold solution of compound 2 in anhydrous DME was added NMM followed by IBCF under argon atmosphere. After stirring the reaction mixture at the same temperature for 10 minutes, the mixture was filtered, and the residue was washed with DME. The collected filtrate was treated with an aqueous NaBH4 solution at −10 C. The reaction mixture was poured into water. The precipitate was collected by vacuum filtration and washed with water to afford compound 3 as a white solid, which was used in the next step without further purification.
Compound 4: 6 mmol of pyridine in anhydrous CH2Cl2 was added to an argon-purged flask containing Dess-Martin periodinane (1 mmol) in anhydrous CH2Cl2 to generate a clear solution. This stock solution was used in the oxidation step within 20 min. To a solution of compound 3 (1 eq) was added (1.9 eq.) of freshly prepared periodinane stock solution in one portion. After 1 h, the clear solution was diluted with 5% MeOH:CH2Cl2 and was quenched by adding 1:1 saturated aqueous NaHCO3/sodium bisulfite and stirring the resulting mixture for 5 min. The mixture was washed with saturated aqueous NaHCO3 and brine, and the organic layer was dried (Na2SO4), filtered, and concentrated. The resulting oily residue was used in the next step without further purification.
To an aldehyde solution in anhydrous DCM was added glycine hydrochloride followed by DIPEA. After 1 h at room temperature, NaB(OAc)3H was added to the reaction mixture and left for overnight stirring. The reaction mixture was diluted with 10% MeOH:DCM: 2% aq. HCl solution and the solution was extracted with 10% MeOH:DCM. The combined organic layer was dried (Na2SO4) and removed. The resultant crude was purified using silica gel column chromatography to obtain compound 4.
Compound 5: The mixture of nucleobase acid, HBTU, DIPEA, and compound 4 was stirred at room temperature for 10-15 hours. After consuming one of the starting materials, the reaction mixture was concentrated under a vacuum. The resultant residue was dissolved in water and extracted with 10% MeOH:DCM (2×). The combined organic layer was dried and removed. The obtained residue was purified by using silica gel column chromatography.
1N NaOH was added to the solution of methyl ester monomer and CaCl2.XH2O in IPA:H2O mixture. After 3 h at room temperature, the reaction mixture was acidified with acetic acid and concentrated. The remaining residue was treated with MeOH and water. The obtained precipitate was collected by vacuum filtration and washed with water.
The overall scheme for this reaction is essentially as depicted in
Compound 7: To a cold solution of compound 6 in anhydrous DME was added NMM followed by IBCF under argon atmosphere. After stirring the reaction mixture at the same temperature for 10 minutes, the mixture was filtered, and the residue was washed with DME. The collected filtrate was treated with an aqueous NaBH4 solution at −5° C. The reaction mixture was poured into water. The obtained precipitate was collected by vacuum filtration and washed with water to afford compound 7 as a white solid, which was used in the next step without further purification.
Compound 8: To a cold solution of compound 7 (1 eq) was added Dess-Martin periodinane (1.15 eq.) in portion-wise and warmed to room temperature. After 1 h, the solution was diluted with ether and quenched by adding 1:1 saturated aqueous NaHCO3/sodium thiosulfate and stirring the resulting mixture for 5 minutes. The mixture was washed with saturated aqueous NaHCO3 and brine, and the organic layer was dried (Na2SO4), filtered, and concentrated. The resulting residue was used in the next step without further purification.
To an aldehyde solution (1eq) in anhydrous DCM was added glycine hydrochloride (1.5eq) followed by DIPEA (2.5 eq). After 1 h at room temperature, NaB(OAc)3H (2 eq) was added to the reaction mixture and left for overnight stirring. The reaction mixture was diluted with DCM solution, and the resulting solution was washed with 10% sodium bicarbonate solution. The combined organic layer was dried (Na2SO4) and removed. The resultant crude was purified using silica gel column chromatography to obtain compound 8.
Compound 9: To the mixture of nucleobase acid (1 eq), HBTU (1 eq), DIPEA (1.1 eq) in anhydrous DMF was added compound 8 (1 eq) at room temperature and stirred for an additional 10-15 hours. After consuming one of the starting materials, the reaction mixture was concentrated under a vacuum. The resultant residue was dissolved in water and extracted with ethyl acetate (2×). The combined organic layer was dried and removed. The obtained residue was purified by using silica gel column chromatography.
1N NaOH (1.5 eq) was added to the solution of Methyl ester monomer (1 eq) and CaCl2.XH2O (20 eq) in IPA:H2O mixture. After 3 h at room temperature, the reaction mixture was acidified with acetic acid and concentrated. The remaining residue was treated with MeOH and water. The obtained precipitate was collected by vacuum filtration and washed with water and purified by silica gel column chromatography to obtain pure monomer series 9.
The overall scheme for this reaction is essentially as depicted in
Compound 13: (i) To a cold solution of compound 2 in anhydrous DME was added NMM followed by IBCF under argon atmosphere. After stirring the reaction mixture at the same temperature for 10 minutes, the mixture was filtered, and the residue was washed with DME. The collected filtrate was treated with an aqueous NaBH4 solution at −10 C. The reaction mixture was poured into water. The precipitate was collected by vacuum filtration and washed with water to afford compound 12 as a white solid, which was used in the next step without further purification.
(ii): 6 mmol of pyridine in anhydrous CH2Cl2 was added to an argon-purged flask containing Dess-Martin periodinane (1 mmol) in anhydrous CH2Cl2 to generate a clear solution. This stock solution was used in the oxidation step within 20 min. To a solution of step 1 compound (1 eq) was added (1.9 eq.) of freshly prepared periodinane stock solution in one portion. After 1 h, the clear solution was diluted with Ether and was quenched by adding 1:1 saturated aqueous NaHCO3/sodium bisulfite and stirring the resulting mixture for 5 min. The mixture was washed with saturated aqueous NaHCO3 and brine, and the organic layer was dried (Na2SO4), filtered, and concentrated. The resulting oily residue was used in the next step without further purification.
(iii) To an aldehyde solution in anhydrous DCM was added glycine hydrochloride followed by DIPEA. After 1 h at room temperature, NaB(OAc)3H was added to the reaction mixture and left for overnight stirring. The reaction mixture was diluted with DCM and the solution was extracted with DCM. The combined organic layer was dried (Na2SO4) and removed. The resultant crude was purified using silica gel column chromatography to obtain compound 13.
Compound 14: The mixture of nucleobase acid, HBTU, DIPEA, and compound 13 was stirred at room temperature for 10-15 hours. After consuming one of the starting materials, the reaction mixture was concentrated under a vacuum. The resultant residue was dissolved in water and extracted with Ethyl acetate (2×). The combined organic layer was dried and removed. The obtained residue was purified by using silica gel column chromatography.
1N NaOH was added to the solution of methyl ester monomer and CaCl2.XH2O in IPA:H2O mixture. After 3 h at room temperature, the reaction mixture was acidified with acetic acid and concentrated. The remaining residue was treated with MeOH and water. The obtained precipitate was collected by vacuum filtration and washed with water and purified by silica gel column chromatography.
Six sets of PNA oligomers were prepared as shown in
In further detail, Fmoc based solid-phase synthesis was used to to prepare all PNA oligomers depicted in
For Example 5, all assays were conducted using standard protocols. Selected PNA monomers, and the oligomers of
From the HPLC, the conversion of five alloc to five amines in the P4 series was very efficient. The polarity of alloc PNA was least followed by glyceric acid oligomer P4c. The polarity of P4a, P4b, and P4 (NH2) was almost identical on HPLC.
In further detail, to validate that the post-modification of PNA oligomers will not influence the PNA-PNA or PNA-RNA thermal stability; we measured the UV-melting profiles. The thermal stability of P2:R1 was higher than P1:R1 due to the preorganized helical structure of P2. The UV-melting profiles of singly modified oligomer (P3 series) with a complementary RNA R1 showed a similar melting transition as the P2-R1 duplex. The presence of either modification on the PNA did not disturb the thermal stability of the duplex. Moreover, the five alternate alterations with different charges in parent PNA P2 (P4 series) also presented similar melting temperatures. No electrostatic interactions between gamma epsilon guanidine (P4b) or repulsion between gamma epsilon phosphate (P4a) and phosphate (R1) groups were noted.
Referring to
To determine the mobility and orthogonal binding of prepared negatively charged PNA, EMSA comparing the orthogonal binding of Right-handed (P4a) and Left-handed (P5a) with complementary RNA R1 at physiologically relevant conditions was performed.
The present invention has been described with reference to certain exemplary embodiments, dispersible compositions and uses thereof. However, it will be recognized by those of ordinary skill in the art that various substitutions, modifications or combinations of any of the exemplary embodiments may be made without departing from the spirit and scope of the invention. Thus, the invention is not limited by the description of the exemplary embodiments, but rather by the appended claims as originally filed.
This application is the United States national phase of International Application No. PCT/US22/18300, filed Mar. 1, 2022, and claims priority to U.S. Provisional Application, No. 63/154,840, filed Mar. 1, 2021, the disclosures of which are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/18300 | 3/1/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63154840 | Mar 2021 | US |
