WO1998058256A1 - Peptido-oligonucleotides (pon) et leurs banques combinatoires - Google Patents

Peptido-oligonucleotides (pon) et leurs banques combinatoires Download PDF

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WO1998058256A1
WO1998058256A1 PCT/US1998/012580 US9812580W WO9858256A1 WO 1998058256 A1 WO1998058256 A1 WO 1998058256A1 US 9812580 W US9812580 W US 9812580W WO 9858256 A1 WO9858256 A1 WO 9858256A1
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pon
composition
substrate
group
pons
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PCT/US1998/012580
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Ryszard Cole
Weiguo Liu
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The University Of North Carolina At Chapel Hill
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C07K14/003—Peptide-nucleic acids (PNAs)

Definitions

  • the present invention provides a novel embodiment or libraries enclosing large numbers of nucleotide like substances referred to as Peptido Oligonucleotides (PONs), and a powerful technique that efficiently select individual PONs against specific DNA or RNA targets in celMines for antisense therapeutics.
  • PONs Peptido Oligonucleotides
  • the peptido oligonucleotides (PONs) in this invention consists of natural and unnatural L- or D-amino acids, purine and pyrimidine derived nucleobases, and a four-carbon-chain connecting the nucleobases and the amino acids toeether through amide linkages to form a peptide backbone.
  • These three types of building blocks are arranged to allow a three-bond distance between the nucleobases and the backbone, and a six- bond distance between each nucleobase attached on the backbone.
  • the arrangement provides the new PONs with optimum affinity to the complementary sequences of natural DNA or RNA molecules and by doing so render ⁇ ese analogs desirable features as potential antisense therapeutics.
  • this new construction of peptido oligonucleotides allows easy incorporation of various of functionalities in the molecule for a given sequence. By simply varying the connecting amino acids during standard peptide synthesis, one has the opportunity of generating very large numbers of antisense PONs that have different chemical and physical properties but are all complementary to a single target sequence.
  • a library consisting of 20 10 PONs can be generated in theory by randomly choosing the connecting amino acids only from the proteogenic amino acid pool.
  • the so obtained PON library is screened in a proper cell line bearing the target nucleic acid sequence, and only those PONs that efficiently penetrated the cell membranes, survived cellular degradation, and bonded strongly and selectively to the target sequences are selected.
  • the connecting amino acid sequences of selected PONs are determined and sufficient quantities of the compounds are synthesized for further advanced testing. This new technology significantly improves the odds of developing clinically useful antisense therapeutics.
  • the invention generally relates to the creation and application of a large body of synthetic organic compounds that are capable of recognizing and binding to nucleic acids in a sequence- specific manner. Specifically, the invention provides a novel methodology for generation of very large numbers of defined mixtures of nucleotide like substances, i.e. peptido oligonucleotide (PON) combinatorial libraries, and the screening of the same for antisense agents that are effective in vivo against specific DNA or RNA target sequences.
  • PON peptido oligonucleotide
  • the peptido oligonucleotide of this invention involves a hybrid of peptides and nucleotides with amino acids and ⁇ ucleoside analogs alternately inter-connected through amide linkages to form oligomers that resemble nucleic acids.
  • the geometry and topology of the base portions of the oligomer are preserved to function just like a nucleic acid in recognizing and base pairing with complementary sequences.
  • the invention also relates to the novel synthetic processes for preparation of optically active amino acid nucleosides as building blocks and the construction and screening of peptido oligonucleotide combinatorial libraries.
  • ODNs Antisense oligodeoxyribonucleotides
  • ODNs Antisense oligodeoxyribonucleotides
  • These synthetic oligonucleotides can bind specifically by Watson-Crick base pairing to complementary DNA or RNA sequences and thus inhibit gene expression either by direct intervention of translation or transcription, or via activation of RNase H.
  • the synthetic phosphorothioates and methylphosphonate ODNs are more resistant to nucleases but still have the problem of not efficiently penetrating the cell membranes.
  • these two classes of ODNs are both produced as mixtures of diastereomers, and this could be the cause of some of the non- sequence-specific side effects observed (see Kibler-Herzog, L., Zon, G., Uznanski, B., Whittier, G., Wilson, W. D., Nucleic Acid Res. 1991, 19, 2979-2986; Lesnikowski, Z., J., Jaworska, M., Stec, W. J., Nucleic Acid Res. 1990, 18, 2109-21 15).
  • a common disadvantage of the above antisense oligonucleotides is that for a given sequence of a target nucleic acid, only one complementary antisense oligo from each of the above category can be prepared. These oligos are then tested individually for antisense activities and sometimes modified with peripheral attachment of functionalities. Such a process is time-consuming and has been deemed inefficient in selecting effective antisense therapeutics.
  • the complexity of interactions between antisense agents and biological systems should be considered when designing new antisense oligonucleotides. There are clearly many other factors besides Watson-Crick base pairing that decide the effectiveness of an antisense agent in inhibiting gene expression.
  • an antisense oligonucleotide For an antisense oligonucleotide to be effective as an therapeutic agent, it must meet the criteria of ( 1) can be synthesized easily and in bulk; (2) being stable in vivo; (3) being able to enter the target cell; (4) can be retained by the target cell; (5) being able to interact with cellular targets: and (6) not interact in a non-sequence-specific manner with other macromolecules. None of the currently available ODN analogs meet all of these criteria (Stein, C. A. & Cheng, Y.-C, Science , 1993. 261 , 1004- 1012).
  • a practical approach for selecting such an antisense oligonucleotide would be that for a given target sequence of a gene, a large number of oligonucleotides with the same antisense sequence but different structures and chemical/physical properties are synthesized simultaneously and then tested for the desired biological activities as a group in order to identify a proper candidate for further development.
  • a lead compound is generated first for a given therapeutic target. This initial drug candidate may have some desired biological activities along with undesired side effects or toxicity. Based on the structure of this lead compound, an array of derivatives and analogs are prepared and tested. From them, the final drug with the highest biological activity and lowest side effects is identified.
  • the conventional practice in developing antisense drugs by generating one antisense molecule for each target gene sequence is replaced with the generation of a large group of antisense molecules for each target gene sequence.
  • the antisense sequence of the oligonucleotides is conserved as the pharmacophore as in the lead compound, while the rest of the structure such as the types of backbone, the various side chains on the backbone, and the functional groups on the bases are altered to generate a great variety of analogs and derivatives from which the best antisense molecule for the purpose is selected.
  • the technology described in this invention has a clear advantage in the rapid development of very large numbers of new, potent antisense agents which have the required properties of stability, affinity, permeation, and ultimately, favorable pharmacokinetics. That is indeed the objective of this invention.
  • the primary objective of the present invention is to provide a new class of polymeric molecules capable of forming duplex or triplex structures with nucleic acids in a sequence specific manner.
  • a further objective of the invention is to formulate and construct analogs that function like Peptide Nucleic Acids in the existing art but overcome some of the disadvantages associated with PNA such as solubility and cellular uptake.
  • the present invention provides a novel embodiment or libraries of large numbers of nucleotide like substances referred to as Peptido Oligonucleotides (PONs), and a powerful technique that efficiently select individual PONs against specific DNA or RNA targets in cell lines for antisense therapeutics.
  • PONs Peptido Oligonucleotides
  • the peptido oligonucleotides (PONs) in this invention consists of natural and unnatural L- or D-amino acids, purine and pyrimidine derived nucleobases, and a four-carbon-chain connecting the nucleobases and the amino acids together through amide linkages to form a peptide backbone.
  • a library consisting of 20 10 PONs can be generated in theory by randomly choosing the connecting amino acids only from the proteogenic amino acid pool.
  • the so obtained PON library is screened in a proper cell line bearing the target nucleic acid sequence, and only those PONs that efficiently penetrated the cell membranes, survived cellular degradation, and bonded strongly and selectively to the target sequences are selected.
  • the connecting amino acid sequences of selected PONs are determined and sufficient quantities of the compounds are synthesized for further advanced testing. This new technology will significantly improve the odds of developing clinically useful antisense therapeutics.
  • At least a portion of the PONs of the invention has the stereochemically defined composition in the form of S-(pX-AA) n -Y
  • S is a hydrogen or a linker or a modifying group or a peptide.
  • Y is a hydrogen or a modifying group or an amino acid or a peptide.
  • AA is one of any natural and unnatural amino acids excluding pX.
  • pX is an optically active amino acid nucleoside having the structure of
  • composition involves defined chiral centers bearing either (R ) or (S) configurations.
  • the compounds of the invention generally are prepared by solid phase peptide synthesis techniques.
  • a novel enzyme catalyzed enantioselective hydrolysis reaction was applied to prepare both (R ) and (S) enantiomers of the pX through resolution of the racemic mixtures synthesized by established methods.
  • Fig. 1 is the synthetic scheme for preparation of ⁇ -bromo- ⁇ -aminobutyric acid derivatives.
  • Fig. 2 is the synthetic scheme for preparation PON monomer pT , here p is ⁇ -aminobutyric acid, and T is thymine.
  • Fig. 3 is the synthetic scheme for preparation PON monomer pC, where p is ⁇ -aminobutyric acid, and C is cytosine.
  • Fig. 4 is the synthetic scheme for preparation PON monomers pG and pA, where p is ⁇ - aminobutyric acid, G is Guanine and A is adenine.
  • Fig. 5 is the reaction scheme for enzymatic resolution of racemic ethyl ⁇ -t- butoxycarbonylamino- ⁇ -( l-(2,4-dihydroxy-5-methylpyrimidyl)butyrate ((dl)-Boc-pT-OEt) to obtain both (S)-, and (R)-Boc-pT.
  • Fig. 6 is the chrial HPLC monitoring of the resolution process.
  • the 4 chromatograms are (dl)- Boc-pT-OEt, its reaction mixture with papain, (S) and (R) Boc-pT, and isolated (S)-Boc-pT.
  • Fig. 7 is the synthetic scheme for preparation of deoxyt_hymidine-2'-amino-5'-carboxylic acid.
  • Fig. 8 is me synthetic scheme for preparation of dideoxycytodine-2'-amino-5'-carboxylic acid.
  • Fig. 9 is a demonstration of reactions catalyzed by uridine phosphorylase and purine nucleoside phosphorylase.
  • Fig, 1 1. is a demonstrattion of properly protected amino acid nucleosides for peptide synthesis.
  • Fig. 12. is the synthetic scheme for preparation of properly protected dideoxycytodine-2'- amino-5' -carboxylic acid.
  • Fig. 13 is the chemical structure of the PON (lys-(pT-gly) I0 -gly-NH_).
  • Fig. 14 is a computer model of a double helex formed between a PON (ala-pX) n and a complementary single stranded DNA (dX) n .
  • Fig. 15 is an example of constructing primary PON libraries applying one-bead-one-peptide approach.
  • Fig. 16 is an example of constructing secondary PON libraries by coupling mixtures of the connecting amino acids.
  • oligonucleotides refers to polymeric molecules having repeated units formed in a specific sequence from naturally occurring bases and sugars joined together through phosphodiester bonds. These molecules include fragments of DNA, RNA, and their derivatives.
  • oligonucleotide analogs refers to those compounds that function like oligonucleotides but have modified or completely re-designed structures.
  • peptide nucleic acid PNA relates to a special group of oligonucleotide analogs having a peptide backbone with side chains having nucleobases that are capable of engaging in hydrogen bonding with an oligonucleotide having a complementary sequence.
  • the peptido oligonucleotides (PONs) of the present invention refers to a new class of peptide nucleic acids through novel assembly of subunits consisting of natural and unnatural amino acids, natural and modified nucleobases, and a bridging molecule that effectively link the nucleobases with the amino acids.
  • the bridging molecule in this invention itself is an amino acid in nature. It is attached to nucleobases through displacement of an co-leaving group in a N-protected ⁇ -amino butyric acid by one of the nucleophilic nitrogen's on the nucleobase.
  • interconnecting amino acids in this invention relates to a series of natural and unnatural amino acids including D-, and L-amino acids, ⁇ , ⁇ -disubstituted amino acids, and amino acids with secondary amino groups or amino groups inco ⁇ orated in a ring system.
  • the term "combinatorial library” refers to an embodiment of usually a large number of different substances generated systematically and simultaneously through ordered, well controlled synthetic steps in combination with a random distribution of a number of defined building components.
  • the basic building units for the peptido oligonucleotides (PONs) of this invention include an array of ⁇ -aminobutyric acid derived amino-acid-nucleosides, and are referred to as
  • PON monomers designated as pX, while X equals to A (adenine), T (thymine), C (cytosine),
  • p represents the ⁇ -aminobutyric acid portion of the nucleoside serving as a spacer and a linker with free or protected amino carboxylic acid bifunctionalities.
  • a general representation of pX is illustrated in the following structure: HOOCCHNH 2 CH 2 CH 2 -X wherein, X is a nucleoside base or its modified derivatives
  • the general approach for preparing PON monomers is to attach the properly protected amino acid directly to various protected or unprotected nucleobases and then manipulate the adducts to release the desired functionalities.
  • we also disclose a novel chemo-enzymatic process for preparation of optically active 2-amino-4-acyI-butyric acid analogs (acyl pyrimidine and purine nucleobases) as PON monomers or building blocks.
  • the bromoacid is treated with hydrogen chloride gas in anhydrous ethanol to produce the ethyl ester which is then protected at the amino group with Boc by reacting with di-tert-butyldicarbonate in aqueous sodium carbonate to yield ethyl ⁇ -amino- ⁇ -bromo-N-t-butoxycarbonylbutyrate.
  • the protected ⁇ -bromo-aminoester is then attached to various nucleobases by displacement of the bromo group of the amino ester with the nitrogen's in the nucleobases (Fig.2-4).
  • the resulting amino ester nucleoside is hydrolyzed by sodium hydroxide or lithium hydroxide to generate the free acid.
  • This synthetic sequence involves very strong acid and high temperature conditions which could lead to rac ⁇ mization at the ⁇ -carbon if an optically active amino acid is involved.
  • optically active POX monomers such as the 2-aminobutyric acid derivative is. at present, intended for both enantiomers.
  • optically active POX monomers such as the 2-aminobutyric acid derivative
  • racemic final product we could obtain both L-, and D-amino acid nucleosides for construction of a variety of homogenous PON stereomers.
  • Libraries of PON stereomers can be screened for binding affinity with complementary nucleic acids and the optimum conformation and stereochemistry of the PON can be selected.
  • a part of the present invention refers to a process in which two enantiomers in a racemic mixture of amino acid nucleoside are separated physically after treating the mixture with an enantioselective reagent.
  • This reagent reacts preferentially to one of the two enantiomers in the racemic mixture yielding a product with different chemical structure from the un-reacted enantiomer thereby generating the differences for the separation of the two.
  • a general representation of the racemic amino nucleosides is illustrated in the following structure
  • X is a nucleoside base or its modified derivatives
  • R is H; alkyl or substituted alkyl; alkenyl or substituted alkynyl; alkaryl or substituted alkaryl; aralkyl or substituted arakyl; cyclic or heterocyclic ring systems.
  • R is H; alkyl or substituted alkyl; alkenyl or substituted alkynyl; alkaryl or substituted alkaryl; aralkyl or substituted arakyl; alcyclic; cyclic and heterocyclic ring systems.
  • Y is CH, or CH H, or O or S .
  • Z is a protecting group including Fmoc, Boc, Cbz, Pht, etc.
  • the enantioselective reagents include optically active molecules bearing acid or base functionalities that serve the purpose of general acid or base catalyses. Representing members of these reagents include hydrolytic enzymes such as papain, trypsin, subtlisine, chymotrypsin, acylases, esterases, lypases, and other proteases.
  • the reaction involves enantioselective hydrolysis of either the carboxylic ester R 2 or the nitrogen protecting group Z.
  • the resulting optically active carboxylic acid or the free amine are both significantly more water soluble than the un-reacted starting material and are thus easily separated.
  • the hexane extracts are combined, dried, and concentrated to dryness to obtain the optically pure ethyl ⁇ - (R)-amino- ⁇ -( l-(2,4-dihydroxy-5-methylpyrimidyl)-N-t-butoxycarbonylbutyrate which is then hydrolyzed chemically with NaOH to give ⁇ -(R)-amino- ⁇ -( 1 -(2.4-dihydroxy-5- methylpyrimidyl)-N-t-butoxycarbonylbutyric acid .
  • the aqueous layer is concentrated under vacuum to a small volume and then 100% ethanol is added to precipitate the enzyme protein and inorganic salt.
  • racemic ⁇ -amino- ⁇ -(l-(2,4-dihydroxy-5- methylpyrimidyl)-N-t-butoxycarbonylbutyric acid is treated with papain in a mixture of ethanol/hexane/buffer (1/1/3) at room temperature for 24 hours. After about 50% acid to ester conversion, the reaction mixture is filtered to remove the enzyme, diluted with water, and extracted repeatedly with hexane.
  • optically active amino acid nucleosides are used as the basic building blocks - PON monomers (pXs), for the construction of a vast variety of PONs and PON libraries.
  • nucleoside analogs bearing aminocarboxylic acid functionalities can also serve as building blocks for construction of PONs.
  • PON monomers can be prepared ether by attaching amino acids to the nucleobases or by direct derivatization of deoxyribonucleosides.
  • amino acid bifunctionality into dexoyribonucleosides.
  • the commercially available 2',3'- - dideoxy-3'-azido-.hymidine (AZT) (Fig.7) is used as one of the starting materials.
  • AZT is first oxidized to the 5'-carboxy-derivative by an appropriate oxidizing agent such as chromic acid, potassium permagnate, and ruthenium trichloride. Since direct hydrogenadon of the acid resulted to its decomposition, the methyl ester derivative is prepared and reduced by hydrogenadon on pd/C to give the amino ester which is then hydrolyzed by sodium ethoxide in ethanol/water to yield the deoxythymidine amino acid.
  • an appropriate oxidizing agent such as chromic acid, potassium permagnate, and ruthenium trichloride. Since direct hydrogenadon of the acid resulted to its decomposition, the methyl ester derivative is prepared and reduced by hydrogenadon on pd/C to give the amino ester which is then hydrolyzed by sodium ethoxide in ethanol/water to yield the deoxythymidine amino acid.
  • the 5'-carboxylic ester was hydrolyzed to the free acid which was re-esterified followed by Pd/C .catalyzed hydrogenadon and base hydrolysis to yield the desired deoxycytodine amino acid.
  • the corresponding amino acids of the purine deoxyribo ⁇ ucleosides are prepared through a novel process based on base exchange reactions catalyzed by a group of enzymes termed Nucleoside Phosphoralases (Fig.9).
  • Uridine phosphorylase catalyzes the equilibrium reactions of uridine and other pyrimidine nucleoside with inorganic phosphate to give the free pyrimidine nucleobase and ribose-1 -phosphate.
  • Purine nucleoside phosphorylase catalyzes the same equilibrium reactions between a purine-nucleoside and its free base plus ribose-1- phosphate, in which the equilibrium is heavily tilted toward nucleoside formation. Since ribose-1 -phosphate is the common intermediate in both enzyme reactions, when the two reactions are combined the ribose-1 -phosphate generated from the first reaction is immediately utilized in the second reaction to react with purine nucleobases in forming purine nucleoside.
  • the mixture are stirred at 30 °C until more than 80% of starting material is converted to the corresponding purine nucleoside, and then filtered to recover the enzyme.
  • the filtrate is first extracted with chloroform to remove the nucleobases, and then with ethyl acetate or butanol to obtain the product.
  • a purine analog such as adenine or guanine catalyzed by the combined nucleoside phosphorylases system as above
  • the corresponding purine nucleoside amino acid are prepared in reasonable yields (Fig.10).
  • the ability of the PON to form a duplex with single stranded DNA or RNA was studied by molecular modeling (Fig.14).
  • a standard double-stranded DNA was called on from the data base.
  • the phosphorodiester linkages on one strand of the DNA was replaced with the (S)- alanine- (S)- ⁇ -aminobutyric acid linkage as in the PON molecule.
  • the ⁇ -carbons and ⁇ - nitrogen's in both alanine and ⁇ -aminobutyric acid were kept in the same planes as their neighboring carbonyl groups. Rotating the two planes along Y-axis yields several conformations of lower energy than that of the original phosphodiester backbone.
  • 0 -gly-NH 2 /(dA) 12 as a comple t ely complementary PON-DNA duplex was recorded as 74 °C, very close to the T m value (73 °C) for the corresponding PNA-DNA duplex but significantly higher than that (25 °C) of (dT),,/(dA) I2 as a standard DNA-DNA duplex. Melting temperatures (TJ for PON-DNA duplex with one and two mismatches are 59 °C and 49 °C respectively.
  • the peptido oligonucleotide (PON) combinatorial libraries of the present invention are generated by connecting PON monomers alternating with natural or synthetic amino acids as spacers.
  • the PON monomers and the spacer amino acids are linked in such a way that the nucleobase sequence of the resulting PONs are well defined while the backbone that connects these nucleobases are altered in a combinatorial manor to generate large numbers of antisense molecules complementary to a given target sequence.
  • the resulting peptido oligonucleotides produced by this process will possess different chemical, physical, and biological properties.
  • a typical PON consists at least a Dortion of a simrjle repeat of pX and AA as core PON in the form of -(AA-pX-) ⁇ -, where n is 0 or as above and AA represents natural and synthetic amino acids with a general structure of
  • Rl is H; alkyl or substituted alkyl; alkenyl or substituted alkynyl; alkaryl or substituted alkaryl; aralkyl or substituted arakyl; alcyclic; cyclic and heterocyclic ring systems.
  • R2 is H; alkyl or substituted alkyl; alkenyl or substituted alkynyl; alkaryl or substituted alkaryl; aralkyl or substituted arakyl; acyclic; cyclic and heterocyclic ring systems.
  • Z is H or alkyl or alkenyl or a protecting group including Fmoc, Boc, Cbz, Pht, etc.
  • S is a bond or an atom or a group of atoms
  • S, Rl, R2 and Z can be interconnected in one or more ring systems.
  • pXs and AAs are coupled together through standard reactions for peptide bond formation including both solution and solid phase peptide synthesis.
  • the PONs are assembled on solid phase resins following Merrifield method or its modified versions.
  • the sequence of the PONs is predetermined based on the sequence of the target gene fragment. These target sequences are usually selected according to their sensitivities to antisense inhibitions and the functions of their protein products.
  • a target RNA sequence of AATTTCCGGG for example, dictate the types of pX and their order of introduction as pTpTpApApApApGpGpCpCpC for the corresponding PON library.
  • Each pX is separated by an amino acid (AA) as the spacer in the actual PON molecules for proper base pairing with the target.
  • AA amino acid
  • the general structure of core PONs for this target library is therefore pT-AA-pT-AA-pA-AA-pA-AA-pA-AA-pG-AA-pG-AA-pC-AA-pC-AA, where each and every individual AA can be any amino acids in any combinations. If AAs are only draw from a pool of the 20 gene encoded natural amino acids, the library will in theory contain 20 10 members of PONs with the same pX sequence but different AA combinations.
  • the size of a certain PON library for a specific target sequence is defined by the maximum number of PONs it covers, which, in turn depends on the length of the target sequence and the variety of AAs inco ⁇ orated. It is generally expressed as x n , where n is the number of bases in the target sequence or in the PONs and x is the number of amino acids among which the AAs are selected. This expression only applies to libraries of core PON structures.
  • the actual size of a PON library can be larger than x n when extra AAs or a strings of AAs are attached to either C or N or both terminals of the core PONs.
  • a PON library for a target sequence can be further divided into sublibraries where the pX sequence is the same but the connecting AAs of the PONs are subdivided according to their chemical, physical and biological properties.
  • a library with PONs containing partially or wholly D-amino acids as the connecdng AAs is regarded as a D-AA sublibrary; while the one with PONs containing pXs bearing a D or R chiral center is referred as a D-pX sublibrary. If the PONs in a library contain both D-AAs and D-pXs, then the library is called a D-PON library assuming the unspecified PON libraries as L-PON sublibraries.
  • a PON library with PONs containing, as the connecting AAs, predominantly lipophilic amino acids, or hydrophilic amino acids, or anionic amino acids, or cationic amino ' acids, or ⁇ , ⁇ - disubstituted amino acids is referred to as a lipophilic sublibrary, or a hydrophilic sublibrary; or an anionic sublibrary, or a cationic sublibrary, or a disubstituted sublibrary.
  • a sublibrary can enclose sub-sublibraries according to the further differences of fuctionalities inco ⁇ orated into the PONs.
  • a cationic PON sublibrary can contain sub-sublibraries with PONs inco ⁇ orating predominantly lysine, or arginine, or other positively charged amino acids as me connecting AAs.
  • Each sub-sub library can be divided further and further into branched smaller libraries based on more and more detailed differentiation's of the fuctionalities.
  • a target nucleic acid sequence, or an antisense PON against the sequence typically contains 10 to 20 nucleobases. If the PON library against this sequence include all possible amino acids as the connecting AAs. it would be too large to be practically constructed due to the demand for huge amounts of the total mixture in order to include every possible PONs in a detectable quantity.
  • Currently available screening and analytical techniques require a minimum of about 100 picomoles of a PON present in the mixture to be effectively selected.
  • the average molecular weight for a PON with 10 nucleobases is approximately 3000. If a specific 10 nucleobase PON library include all 20 natural amino acids independently as connecting AAs, the total number of individual PONs in the library will be 20 10 . For each member of the PON in the library to be present in a quantity above 100 picomoles, the mass of the total mixture will have to be larger than 3.1 metric tons. Such a library is obviously impractical to construct and screen. It is therefore necessary to construct smaller sublibraries to probe certain general features of antisense PONs at the beginning of the research. Once some of these general features are understood, further branched sublibraries are constructed for another round of investigations until the desired PONs are selected.
  • SAR structure activity relationships
  • the ability of a PON to recognize and bind to complementary sequences of nucleic acids is primarily influenced by the space between the neighboring nucleobases in the PON molecule and by the distance from the nucleobase to the backbone.
  • Stereochemistry is another first tier variable that affect binding.
  • electronic charge is an important factor since the target nucleic acids are highly charged molecules. Contributions from these first tier variables to the binding affinity of PONs are generally independent of other structural changes confined within the scope of their definitions. Therefore, the very first sets of PON sublibraries are constructed and screened primarily for their binding affinities to complementary DNA or RNA sequences.
  • PON libraries consisting ⁇ -aminobutyric acid-based nucleosides as pXs
  • other amino acid-nucleosides such as ( ⁇ )-, and ( ⁇ )-2'-amino-5 ' -carboxyl- deoxyribonucleosides, and D-, and L-threonine-based nucleosides are also included as alternative PON monomers.
  • PONs consisting of these nucleosides could be ether invaribale or variable.
  • Invariable PONs contain a single type of p as pX throughout a PON molecule resulting an even construction in terms of distances between each nucleobases and from the nucleobase to the backbone.
  • variable PONs inco ⁇ orate more than one type of p within each PON chain so that the property and geometry of each pX in the PON molecule could be different.
  • PON sub-libraries bearing primary structure features are screened, usually in vitro, for their affinities and specificity's of binding to target DNA or RNA sequences (Fig.15).
  • a set of second tier PON sub-libraries are generated by varying the connecting AAs in the PON molecule with fixed primary structure features (Fig.16). Since the changes of AAs in a PON molecule does not significantly affect the distances between each nucleobases and the distance from the nucleobase to the backbone, their effect on the binding affinities of the PON to the complementary DNA or RNA sequences is secondary to the changes of pXs, except the electronic charge. Positively or negatively charged functional groups on the backbone will likely impose certain effects on the binding affinity of the resulting PONs due to the ionic or electrostatic interactions between the PON and the nucleic acid target.
  • an antisense PON that can be readily delivered in vivo to the target nucleic acid and binds to it with strong affinity and high specificity could be selected in relatively a short period of time, providing a powerful tool for generadon of effective antisense therapeutics in the treatment of gene related diseases.
  • Homogeneous PONs refer to those peptido oligonucleotides having a uniformed linker molecule as p. such as 2-aminobutyric acid, and a single amino acid as the connecting AA. These PONs are prepared for representative sequences such as (gly-pT) 10 , and are usually constructed on resin beads, released and isolated as free peptides, and tested individually for binding to complementary target nucleic acid sequences such as (dA) I0 . Measurement of melting temperatures of the resulting duplex or triplex of PON-nucleic acid hybrids are the most commonly used methods for determining their binding affinity.
  • the ability of a PON to recognize and bind to a complementary nucleic acid sequence can also be tested by gel retardation experiments in which complementary and non-complementary sequences of single strand nucleic acids are exposed to the testing PON before subjecting to agarose gel electrophoresis.
  • the RNA strands that are not complementary to the PON sequence migrates normally on th ⁇ . gel, but the ones that are complementary to the PON will move much slower on the gel due to the formation of PON- RNA hybrids.
  • Heterogeneous PONs have two or more amino acids serving as the connecting AAs and sometimes include different combinations of p build into pXs. These compounds are, in most cases, synthesized as defined mixtures either in a solution or a solid phase following standard combinatorial library generation methods.
  • the solid phase PONs are constructed on synthetic resins according to established methods for preparation of peptide combinatorial libraries.
  • the nucleobase X is selected among A, T, C, G, and U depending on the sequence of the target nucleic acid, while p is the ⁇ -aminobutyric acid linker or other alternative spacer with aminocarboxylic acid bi- functionalities.
  • the pX can be selected from at least the following three types of amino acid nucleosides and their stereoisomers: 3'-amino-5'-carboxythymidine (A), 3-oxy-(l-thymidyl)-threonine (B), and 4- (l-thymidyl)-2-aminobutyric acid (C).
  • A 3'-amino-5'-carboxythymidine
  • B 3-oxy-(l-thymidyl)-threonine
  • C 4- (l-thymidyl)-2-aminobutyric acid
  • the AA-resins are evenly divided into three groups and coupled respectively with properly protected A, B, and C. After deprotection, the resins are combined, well mixed and coupled with a specific Boc-AA.
  • the resins are, again, evenly divided into three groups and coupled with A, B, and C respectively. This process is repeated until the resulting peptide chain contains 10 pX units.
  • the library would have contained in theory a total of 59049 different types of PON molecules all having 10 thymine units complementary to (dA) I0 with one or more of defined amino acid as the connecting AA.
  • Similar (pT) !0 libraries can be generated with a single 4-(l-thymidyl)-2-aminobutyric acid as pX, while a variety of positively or negatively charged amino acid are inco ⁇ orated into the PON chain as connecting AAs in a combinatorial fashion.
  • PON secondary libraries are sub-grouped according to the properties of the connecting AAs.
  • Cationic PONs contain mostly lysine, arginine, etc as connecting AAs while anionic PONs generally consist of aspartic acid, glutamic acid, and their derivatives.
  • PONs with serine, threonine, cystine, histidine, tyrosine, etc as major components tend to be more hydrophilic while those inco ⁇ orating mostly valine, phenylalanine, tryptophan, proline, and 2,2- disubstituted amino acids as connecting AAs are more likely to be lipophilic.
  • Each of those PON sub-libraries are screened for their ability to penetrate cell membranes in order to establish certain relationships between membrane penetration and the PON's chemical and physical properties such as lipophilicity and electronic charge. Based on the knowledge obtained from the experiments, new sub-libraries with mixed functionalities are constructed and screened until a proper combination of functionalities and sequences of connecting AAs is found best for the resulting PON to penetrate cell membranes and to bind to target sequences.
  • Screening of secondary PON libraries are generally performed in cell-lines carrying the gene of target sequence. Libraries are first labeled with ratio active amino acids at the N terminal of each PON molecule, and then released from resins. The soluble free PON mixtures are incubated with target cells at an appropriate temperature for a varying lengths of time and then aliquots of cells are taken at specific time, laid on the surface of 500 ⁇ l of pre -chilled silicone oil, and centrifuged for 30 seconds in a Eppendorf centrifuge at ambient temperature.
  • the bottom of the tube which contains the cell pellet, is removed using dog toenail clippers, briefly inverted on absorbent paper to drain, and then transferred to a scintillation vial and counted for determining the apparent cell uptake of the PONs in the specific sub-library. After comparing apparent cell uptake of various PON sub-libraries, those having the most promising cell uptake are selected for further investigation.
  • Cells selected from above experiments are treated with detergents or physical forces such as-sonication and pressure to break the membranes.
  • the total nucleic acids including DNAs and RNAs are isolated and digested with endonucleases.
  • the antisense PONs that successfully penetrated the cell's membranes, reached to the active site, and bound specifically to the target would have formed doublex or triplex of PON-nucleic acid hybrids. These hybrids are resistant to nuclease digestion's and will remain as doublex and triplex of the same length as the starting PONs after the nuclease treatment.
  • the PON-nucleic, acid hybrids are then separated by electrophoresis on agarose gels.
  • Racemic homoserine (10 g, 84 mmole) was stirred in a 500 mL pressure tube with 30% (HBr/HOAc, w/w) hydrogen bromide in acetic acid (100 mL, 50 mmole HBr). The tube was sealed and then heated slowly in a water bath to 78 oC with good stirring. After holding at the temperature for 5 hours, the mixture was cooled down to room temperature, mixed with diethvl ether (100 mL), and filtered.
  • the product can also be obtained from homoserine ⁇ -lactone by following the same procedure.
  • ⁇ -amino- ⁇ -bromobutyric acid hydrogenbromide (7.4 g, 28.1 mmole) was dissolved in 100 mL of absolute ethanol. The solution was cooled to 5 oC in an ice bath, and slowly ' bubbled with gaseous hydrogen chloride (HCI) continuously for 8 hours.
  • HCI gaseous hydrogen chloride
  • the starting ester ( 1.5 g, 4.2 mmole) was dissolved in 50 mL of a mixture of water and acetonitrile (80 : 20) with 1,500 units of commercial protease papain (Sigma, 50 mg). The mixture was stirred at room temperature for 4 hours and the progress of the reaction was monitored by chiral HPLC. When about 50% of one enantiomer ester is converted to the corresponding acid, the mixture was extracted repeatedly with diethyl ether (4 x 30 mL).
  • the ether extracts were combined, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain 0.8 g (53%) of optically active ethyl ⁇ (R)-t- butoxycarbonylamino- ⁇ -( l-(2,4-dihydroxy-5-methylpyrimidyl)butyrate.
  • the aqueous phase was dried by liopholizadon.
  • the residue was reslurried in absolute ethanol (50 mL), heated to 60 oC, then cooled to 5 oC and filtered. The filtrate was concentrated to small volume (10 mL) for crystallization.
  • a mixture of ethanol (4 mL) and hexane (mL) was added to a final ratio of ethanol hexane/buffer (1/1/3). The mixture was stirred vigorously at room temperature for 24 hours and the progress to the reaction was monitored bv HPLC.
  • the reaction mixture was filtered to remove insoluble proteins, diluted with water, and extracted repeatedly with hexane.
  • the hexane extracts was combined, washed with water, dried, and concentrated to dryness to obtain the optically pure active ⁇ -(S)-amino- ⁇ -(l-(2.4-dihydroxy-5-methylpyrimidyl)-N-t- butoxycarbonylbutyrate.
  • the aqueous layer is concentrated under vacuum to near dryness followed by crystallization to give optically pure ⁇ -(R)-amino- ⁇ -( 1 -(2,4-dihydroxy-5- methylpyrimidyl)-N-t-butoxycarbonylbutyric acid.
  • the protocols chosen for construction of the PONs in this invention was based on standard Boc chemistry of solid phase peptide synthesis.
  • 0.1 mM of Boc-Gly-MBHA (p-methylbenzhydrylamine ) resin was used as starting material on an automated peptide synthesizer. After deprotection of the Boc-group with trifluoroacetic acid
  • This peptido oligonucleotide is >93% pure by RP-HPLC:
  • Solvent A 0.1 % (W/V) TFA/H.0
  • Example 12 Synthesis of resin-bind PON combinatorial libraries based on one-bead-one-peptide strategy.
  • a 10-nucleobase library was constructed with (S)- ⁇ -t-butoxycarbonylamino- ⁇ -(l-(2,4- dihydroxy-5-methylpyrimidyl)butyric for all pXs, and different combinations of glycine, alanine, phenylalanine, lysine, and aspartic acid as connecting AAs.
  • Standard solid phase peptide synthetic procedures are followed as in example 1 1.
  • Boc-glycine MB HA resins were deprotected, and coupled with (S)- ⁇ -t-butoxycarbonylamino- ⁇ -(l-(2,4-dihydroxy-5- methylpyrimidyl)butyric (pX). After deprotection of the Boc group on the pX, the resins were equally divided into four proportions and each was coupled respectively with properly protected Boc- alanine, phenylalanine, lysine(Cl-Z), and aspartic acid (OBzl).
  • Example 13 Synthesis of soluble PON combinatorial libraries for in vivo screening.
  • the solution of the PON library is incubated with target cells at a proper temperature for a varying lengths of time and then aliquots of cells are taken at specific time, laid on the surface of 500 .
  • ⁇ l of pre -chilled silicone oil and centrifuged for 30 seconds in a Eppendorf centrifuge at ambient temperature.
  • the bottom of the tube, which contains the cell pellet is removed using dog toenail clippers, briefly inverted on absorbent paper to drain, and then transferred to a scintillation vial and counted for determining the apparent cell uptake of the PONs in the specific sub-library.
  • Example 14 Recognition of deoxyadenasine 12-mer (dA) l2 by PON (lys-(pT-gly) l0 -gly-NH,).
  • UV absorbance An increase in UV absorbance is observed during the thermal denaturation as the ordered, native structure of a nucleic acid base-pair stacking is disrupted.
  • hypochromicity the change in UV absorbance is a measure of base-pairing and base-stacking between two complementary strands.
  • the resulting UV absorbance profile as a function of temperature is known as a melting curve with the midpoint of the curve defining the melting temperature, T m , at which 50% of the double strand is dissociated into its two single strands.
  • T m melting temperature
  • the measurement of UV absorbance melting curves provides qualitative and quantitative structural information about the nucleic acid bound to its complementary strand.
  • the T m is dependent upon the concentration of the oligonucloetide the properties to the solvent (buffer: pH, ionic strength, ect.).
  • Binding studies were carried out by hybridizing the PON described above to its complementary oligonucleotide "dA 12 ", followed by thermal denaturation and measurement of the UV absorbance as a function of temperature.
  • Synthetic oligodeoxynucleotide (ODN) dT P and its complementary oligodeoxynucleotide dA 12 were used as reference nucleic acids.
  • the samples were prepared in 50mM phoshate (Na 2 HP0 4 )(pH 7.4) and 140 mM NaCl buffer at 5 mM dA 12 , 10 mM dT 12 and 10 mM PON T I0 ..
  • the T ra of the PON:ODN was significantly higher than that of the control ODN dimer dA 12 : dT 12 . This indicates very strong interaction between the two strands. Even with single and double mismatch ODNs (dA ⁇ : dT, , dA 10 : dT 2 ) the T m was still higher than the control ODN dimer. This adds support to the binding interaction of the PON to the ODN and that this interaction is due to base-pair interaction rather than a non-specific interaction.
  • dNu deoxyoligonucleotide
  • pNu Peptido-Oligonucleotide
  • pT 10 PON T,
  • peptido oligonucleotide PON
  • its libraries are the introduction of an element of plurality into the oligonucleotides by altemately connecting an amino acid and a nucleoside through peptide synthesis.
  • the resulting peptide oligonucleotides are not only highly analogous to nucleic acids in terms of recognizing and base-pairing with complementary sequences, and possessing the peptide backbone that resist nuclease degradation, but also have the flexibility of carrying various combinations of functionalities within the molecule without changing the nucleotide sequence or increasing the length of the peptide chain.
  • PONs peptide oligonucleotides
  • the potential utility of the peptide oligonucleotides (PONs) of this invention is far reaching.
  • the ability of antisense oligonucleotides to recognize and bind to specific sequences in a DNA or RNA molecule is the foundation for their wide spread applications.
  • Extensive research and development work has been carried out in using antisense oligonucleotides for treatment or diagnoses of gene related diseases, especially cancer, AIDS, and other genetic disorders.
  • the PONs of this invention can be further explored in other applications outside traditional antisense arena. These PONs are designed in a way that allows to easily inco ⁇ orate various functional groups in the backbone of the oligomers.
  • oligomers recognize and bind to specific gene sequences
  • the functional groups on their backbones can serve as catalytic arms that reach across to the complementary strain and perform certain chemical reactions ⁇
  • This combination of sequence specificity with catalytic activity in one synthetic oligonucleotide provides a perfect tool to design artificial enzymes for gene surgery.
  • DNAs and RNAs could potentially be cleaved, ligated, alkylated, oxidized, reduced, halogenated, etc. on any base at any desired sequence catalyzed by these specifically designed cataly ⁇ c PONs.
  • These synthetic catalytic PONs could also be used as probes for mechanistic studies of a vast variety of DNA or RNA processing enzymes such as nucleases, ligases, RNAases, and polymerases.
  • DNA or RNA processing enzymes such as nucleases, ligases, RNAases, and polymerases.
  • One example of the application of these catalytic PONs is the design and synthesis of artificial sequence-specific endonucleases. Restriction endonucleases are extremely important tools in molecular biology and biochemistry. They are widely used in gene isolation, DNA sequencing, and recombinant DNA technology. However, these enzymes recognize relatively small size DNA sequences, (usually 4-6 bp) and thus generate too many fragments from a large DNA substrate.
  • PNA protein nucleic acid
  • Lam, Kit S.; Lebl, Michal. Peptide library screening based on the one-bead one-peptide concept. Biol. Chem., Proc. Chin. Pept. Symp., 3rd, 1995, 273-277.
  • Lam, Kit S.; Lebl, Michal. Synthesis and screening of a "one -bead-one-compound” combinatorial peptide library.”, Methods Mol. Cell. Biol., 1996, 6, 46-56.

Abstract

L'invention concerne des banques de substances ressemblant aux nucléotides, appelées peptio-oligonucléotides (PON). Les PON de l'invention se composent d'acides aminés D et L naturels et non naturels, de nucléobases dérivées de purine ou de pyrimidine et d'une chaîne à quatre atomes de carbone, reliant entre eux les nucléobases et les acides aminés par des liaisons amides pour former un squelette peptidique.
PCT/US1998/012580 1997-06-16 1998-06-15 Peptido-oligonucleotides (pon) et leurs banques combinatoires WO1998058256A1 (fr)

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US8932992B2 (en) 2001-06-20 2015-01-13 Nuevolution A/S Templated molecules and methods for using such molecules
US9096951B2 (en) 2003-02-21 2015-08-04 Nuevolution A/S Method for producing second-generation library
US9109248B2 (en) 2002-10-30 2015-08-18 Nuevolution A/S Method for the synthesis of a bifunctional complex
US9121110B2 (en) 2002-12-19 2015-09-01 Nuevolution A/S Quasirandom structure and function guided synthesis methods
US9574189B2 (en) 2005-12-01 2017-02-21 Nuevolution A/S Enzymatic encoding methods for efficient synthesis of large libraries
US10731151B2 (en) 2002-03-15 2020-08-04 Nuevolution A/S Method for synthesising templated molecules
US10730906B2 (en) 2002-08-01 2020-08-04 Nuevolutions A/S Multi-step synthesis of templated molecules
US11118215B2 (en) 2003-09-18 2021-09-14 Nuevolution A/S Method for obtaining structural information concerning an encoded molecule and method for selecting compounds
US11225655B2 (en) 2010-04-16 2022-01-18 Nuevolution A/S Bi-functional complexes and methods for making and using such complexes
CN114163471A (zh) * 2020-09-11 2022-03-11 南京华狮新材料有限公司 一种长链高丝氨酸衍生物的制备方法
US11965209B2 (en) 2003-09-18 2024-04-23 Nuevolution A/S Method for obtaining structural information concerning an encoded molecule and method for selecting compounds

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Cited By (16)

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Publication number Priority date Publication date Assignee Title
US8932992B2 (en) 2001-06-20 2015-01-13 Nuevolution A/S Templated molecules and methods for using such molecules
US10669538B2 (en) 2001-06-20 2020-06-02 Nuevolution A/S Templated molecules and methods for using such molecules
US10731151B2 (en) 2002-03-15 2020-08-04 Nuevolution A/S Method for synthesising templated molecules
US10730906B2 (en) 2002-08-01 2020-08-04 Nuevolutions A/S Multi-step synthesis of templated molecules
US9284600B2 (en) 2002-10-30 2016-03-15 Neuvolution A/S Method for the synthesis of a bifunctional complex
US10077440B2 (en) 2002-10-30 2018-09-18 Nuevolution A/S Method for the synthesis of a bifunctional complex
US9109248B2 (en) 2002-10-30 2015-08-18 Nuevolution A/S Method for the synthesis of a bifunctional complex
US11001835B2 (en) 2002-10-30 2021-05-11 Nuevolution A/S Method for the synthesis of a bifunctional complex
US9121110B2 (en) 2002-12-19 2015-09-01 Nuevolution A/S Quasirandom structure and function guided synthesis methods
US9096951B2 (en) 2003-02-21 2015-08-04 Nuevolution A/S Method for producing second-generation library
US11118215B2 (en) 2003-09-18 2021-09-14 Nuevolution A/S Method for obtaining structural information concerning an encoded molecule and method for selecting compounds
US11965209B2 (en) 2003-09-18 2024-04-23 Nuevolution A/S Method for obtaining structural information concerning an encoded molecule and method for selecting compounds
US9574189B2 (en) 2005-12-01 2017-02-21 Nuevolution A/S Enzymatic encoding methods for efficient synthesis of large libraries
US11702652B2 (en) 2005-12-01 2023-07-18 Nuevolution A/S Enzymatic encoding methods for efficient synthesis of large libraries
US11225655B2 (en) 2010-04-16 2022-01-18 Nuevolution A/S Bi-functional complexes and methods for making and using such complexes
CN114163471A (zh) * 2020-09-11 2022-03-11 南京华狮新材料有限公司 一种长链高丝氨酸衍生物的制备方法

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