AU4159396A

AU4159396A - Synthesis of peptide nucleic acids (pnas) and analogues via submonomer approach

Info

Publication number: AU4159396A
Application number: AU41593/96A
Authority: AU
Inventors: Thomas Horn; Lutz S Richter; Ronald N Zuckermann
Original assignee: Chiron Corp
Current assignee: Novartis Vaccines and Diagnostics Inc
Priority date: 1994-11-14
Filing date: 1995-11-13
Publication date: 1996-06-06
Also published as: CA2203565A1; EP0792283A1; WO1996015143A1; JPH10509947A

Description

Synthesis of Peptide Nucleic Acids (PNAs) and Analogues Via Submonomer Approach

Inventors: Lutz Ritcher, Ronald Zuckerman, and Thomas Horn

Description

Technical Field

The present invention relates generally to chemical synthesis technologies. More particularly, the present invention relates to the synthesis of N-substituted oligomers and particularly to Peptide Nucleic Acids (PNAs) and analogues to form poly-PNAs.

Background of the Invention

Several types of amide polymers with nucleobase substituents, also known as poly-PNAs, have been described in Zuckermann, The Chemical Synthesis of Peptidomimetic Libraries, in Current Opinion in Structural Biology, Volume 3, 580-586 (1993). The described amide polymers included (a) nylon-based oligonucleotide analogues with a polyamide backbone; (b) polyamide oligonucleotide analogues with an alternating serine-glycine backbone; and (c) polyamide oligonucleotide analogues with an N-aminoethylglycine backbone. The PNA containing the N-aminoethylglycine backbone was shown to hybridize to complementary oligonucleotides obeying the Watson-Crick hydrogen bonding rules. See Egholm et al., Nature 365:566-568 (1993). A PNA containing oligomer was also effective in direct analysis of single base mutation by polymerase chain reaction (PCR). This method described in Orum et al., Nuc Acids Res 2K23V 5332-5336 (1993), took advantage of the higher thermal stability of poly- PNA binding to nucleic acids to detect single base mutations.

Disclosure of the Invention

One aspect of the invention relates to a method of synthesizing PNA monomers using three submonomers, referred herein to as acylating agents, displacing agents, and nucleobase agents. The method to produce a monomer comprises:

(a) providing a submonomer acylating agent of the following structure: O

II L— a— C— Y comprising a leaving group, L, capable of nucleophilic displacement, a backbone R., and a carbonyl group;

(b) protecting said carbonyl group of said acylating agent with a substituent, P„ which can be, for example, a solid support;

(C) providing a submonomer displacing agent of the following formula:

Ammo Am.no Qroυp Oraup comprising a first and second amino group, wherein said second amino group is protected;

(d) reacting the displacing agent with the acylating agent to displace the leaving group with the first amino group to obtain a secondary amine of the following structure:

R H O

I I II

Pd— N-Rd—N— Ra— C — Ps

(e) providing a submonomer nucleobase agent of the following structure:

Rn I

Linker I

C=O I Y comprising a reactive carbocanyl group; rø condensing said nucleobase agent with said secondary amine to obtain a tertiary amide with the following structure.

The invention further relates to a method of synthesizing PNA polymers by repeating the method of synthesizing the monomer. Before the method is repeated, the second amino group is deprotected and then acylated with an acylating agent. When the steps (b) - (f) are repeated, the submonomers can be substituted with any desired submonomer to obtain polymers of varying composition.

Another method for synthesizing a PNA monomer is a four step cycle comprising two acylating agents, one displacing agent, and an altered nucleobase, e.g. a persilylated nucleobase. The method comprises:

(a) providing a submonomer first acylating agent of the following structure:

O

II L— Ra— C— Y comprising a leaving group, L, capable of nucleophilic displacement and a carbonyl group;

(c) providing a submonomer displacing agent of the following formula:

d l Amino An lino Group Gi -oup comprising a first and second amino group, wherein said second amino group is protected; (d) reacting the displacing agent with the acylating agent to displace the leaving group with the first amino group to obtain a secondary amine of the following structure:

R H O

I I II

Pd-N-Rd-N— Ra— C — Ps (e) providing a second acylating agent of the following structure:

O

(f) reacting said second acylating agent with said secondary amine to obtain a tertiary amide by condensation with the following structure:

(g) providing an altered nucleobase capable of displacing the leaving group; (h) reacting the altered nucleobase to form the monomer under displacement of the leaving group.

Another object of the invention is a PNA monomer compound comprising a non- natural nucleobase of the following structure:

Yet another object of the invention is a PNA polymer of comprising at least one non-natural nucleobase of the following structure:

Modes of Carrying Out The Invention A. Definitions The term "oligomer" includes polymers such as poly-PNAs, produced by the process of the invention, including homopolymers, copolymers, and interpolymers of any length. More specifically, oligomers may be comprised of a single repeating monomer, two alternating monomer units, two or more monomer units randomly and/or deliberately spaced relative to each other. Regardless of the type of poly-PNAs produced, the poly-PNAs of the invention are produced by the same general procedure which includes repeating a three-step cycle (described below in detail) wherein a new monomer unit is added in each cycle until an oligomer of desired length is obtained. The oligomer is preferably 1-100 monomers, more preferably 2-50, or 2-20, and most preferably 2-10 monomers.

An submonomer "acylating agent" refers to an acylating reagent used in the method of the invention. Such acylating agents, also referred to as "acyl submonomers," comprise a reactive carbonyl or carbonyl equivalent connected by a backbone, R_, to a leaving group. The reactive carbonyl or carbonyl equivalent reacts with the second amino group of the submonomer displacing agent or amine bound substrate. The backbone, R, with the backbone of the displacing agent, Ra, together will be of sufficient length and flexibility to permit the nucleobases or binding agents of the oligomer to hybridize with target polynucleotides, or to bind to the desired protein, such as polymerase or DNA binding protein. The leaving group is such that it may be displaced by nucleophilic displacement by an amine. Suitable acyl submonomers include, without limitation, haloacetic acid, 3-halo-propionic acid, 2- halopropionic acid, 2-haloethyl isocyanate, 2-halomethyl-benzoic acid, 4-halomethyl-2- methoxybenzoic acid, 5-bromomethyl-pyridine-2-carboxylic acid, halocrotonic, 2-haloethyl- haloformate, halomethyl-benzoic acid, and the like.

A "leaving group" refers to a moeity capable of nucleophilic displacement by an amine, e.g., R-NH₂. Any leaving group can be used here provided it is readily removed by nucleophilic displacement. Non-limiting examples of leaving groups useful in the invention include halo, such as bromo, chloro, iodo, O-tosyl, O-triflyl, O-mesyl, and the like.

Carbonyl or carbonyl equivalent" includes, without limitation, carboxylic acids, esters, anhydrides, acyl halides, and isocyantes (in the synthesis of polycarbamates of the invention), which include a carbonyl group or are readily converted to include a carbonyl group. Esters used will generally be "reactive" forms. A carbonyl group will generally be represented

O ll_ by A thioester is also an example of a carbonyl equivalent. Both the acyl and nucleobase submonomers contain a carbonyl or carbonyl equivalent for acylation. These acylation agents may be more potent in the presence of additional agents such as carbodϋmide. A "displacing agent" or "displacing submonomer" refer to a compound containing a first amino group connected by a backbone, Ra, to a second amino group. The first amino group capable of (1) effecting a nucleophilic displacement of the leaving group in an acyl submonomer and (2) being acylated by a nucleobase submonomer. The second amino group is protected to prevent the second group from reacting during acylation by a nucleobase submonomer. For displacing agents that are asymmetrical without the protecting group, the protection also prevents the second amino group from reacting during nucleophilic displacement. The backbone, R , with the backbone of the displacing agent, R«, together will be of sufficient length and flexibility to permit the nucleobases or binding agents of the oligomer of the instant invention to bind with a desired protein, such as a polymerase or DNA binding protein, or hybridize with a desired target polynucleotides. Examples of displacing submonomers include without limitation 1 -(protected amino)-2-amino-ethane, amino, (protected amino) methyl benzene; and 1 -(protected, methyl anιino)-2-amino-ethane A submonomer "nucleobase agent" also referred to as a "nucleobase submonomer" is a compound comprising a nucleobase or binding agent with at least one reactive carbonyl or carbonyl group equivalent. Optionally, a linker can be included in the submonomer between the nucleobase and carbonyl group. In a second acylation reaction, the nucleobase submonomer reacts with the first amino group of the displacing agent to incorporate the nucleobase into the monomer or oligomer.

The term "nucleobase," includes adenine, cytosine, guanine, thymine, and uracil, and refers to any substituent that can bond non-covalently with polynucleotides, such as DNA or RNA, or with other components, such as proteins, that naturally occurring polynucleotides interact with. Such non-covalent bonds include, for example, hydrogen bonding or hydrophobic interactions. These substituents exhibit one or more properties that are similar to naturally occurring nucleobases. These characteristics include an ability to participate in base pairing, structural similarity, etc.

PNA to polynucleotide interaction can be assayed by standard polynucleotide hybridization procedures. Examples of such assays range from annealing experiments to Southern or Northern blots to PCR assays. Detection assays of PNA interaction with other components, such as proteins, will vary and will be chosen for convenience. For example, a PNA that inhibits polymerase activity can be easily measured by standard polymerase assay. Alternatively, if the PNA is to bind irreversibly to the polymerase, other analytical techniques, such as gel electrophoresis, can be also be used.

Examples of nucleobases include "naturally occurring" ones, adenine, cytosine, guanine, thymine, and uracil. Generally, nucleobases are substantially planar heterocyclic compounds comprising one to three rings. Also included are not only the blown purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include alkylated purines or pyrimidines, acylated purines or pyrimidines, and other heterocycles. Other groups included in the term nucleobase are intercalating agents, which are usually flat aromatic rings. Such intercalating agents include without limitation: acridine, anthracene, indole, quinoline, isoquinoline, quinones, dihydroquinones, anthracyclines, tetracyclines, anthracyclinone, etc. Examples of non-naturally occurring nucleobases include isocytosine and isoguanine, formulas shown below:

Other examples of non-naturally occurring nucleobases are described in Benner et al, U.S. Pat. No. 5,432,272, herein incorporated by reference.

"Binding agents" are radicals that will modify the target molecule, which short of destroying the target molecule, cannot be reversed. Binding agents, unlike nucleobases, may not exhibit any characteristics similar to those of naturally occurring nucleobases. DNA interactive agents are one example of binding agents. These agents can modify DNA by covalent bonding, breaking the DNA, or by causing crosslinking of the DNA. Examples of DNA interactive agents include, but are not limited to, protected and unprotected derivatives of nitrogenous mustards, cis-plantinum, and enediyne-derivatives. A linker of the nucleobase submonomer can be included between the nucleobase and the carbonyl group. The linker may be wanted to help position the nucleobase in the desired conformation, which permits it to interact with, for example, the target nucleobase or protein. The linker can be of any composition to provide the desired length and flexibility. Examples of linkers include hydrocarbons, hydrocarbyl and hydrocarbylene substituents with none, one, or two hydrogens removed respectively. These substituents may include, for example, nitrogen, sulfur, and oxygen atoms, as well as hydrogen and carbon. Generally, the structure of the linker is as follows.

wherein Ri, Rϋ, Ra;, and R is a bond to the adjacent carbon or a radical which does not substantially sterically inhibit synthesis or monomer/oligomer activity; preferably, the radicals is hydrogen or an alkyl from one to four carbons;

X is O, N, S, P(O)χ, or a bond; m = 0-8; and n = 1-8.

Preferred R' groups are saturated hydrocarbons between 0-4 carbons; even more preferably, 1-2 carbons.

An "altered nucleobase" is a nucleobase which has been altered to activate, so that the compound react with an acyl submonomer to displace the leaving group. By displacing the leaving group, the altered nucleobase can be incorporated into the monomer/oligomer.

The protected amino group bound to a substrate utilized to initiate solid phase monomer or oligomer synthesis or the second amino group of the displacing agent can be either a primary or secondary amine. Typically, these substituents are designated R, R-, or R₂ in the structures of the present application. Two factors for choosing the composition of R are (1) the efficiency level of monomer/oligomer synthesis; and (2) the efficiency level of the monomer/oligomer activity, e.g., polynucleotide hybridization. Generally, R can be of any composition. However, R will not substantially sterically hinder synthesis or monomer/oligomer activity or function. For example, for DNA hybridization, R is not so bulky that it substantially impedes hydrogen bonding for the desired degree of hybridization. Further considerations for choosing R include its tendency to sterically inhibit synthesis.

Examples of suitable R include hydrocarbon, hydrocarbyl and hydrocarbylene substituents with none, one, or two hydrogens removed, respectively. These substituents may include, for example, nitrogen, sulfur, and oxygen atoms, as well as hydrogen and carbon. Further, these substituents may be saturated or unsaturated, aliphatic, alicyclic or aromatic. When rings are included the structure, usually one, two or three rings are included. The rings may be fused or bridged or spiro-fused. Preferably, R is a radical containing 12 or fewer carbon atoms, 4 or fewer nitrogen atoms, 4 or fewer oxygen atoms, and 2 or fewer sulfur atoms. More preferably, R is a radical is alkyl containing 6 or fewer carbon atoms; most preferably, R is hydrogen.

The spacer between the nucleobases is comprised of the following structure:

O R

I I — Ra— C-N-Rd — where R, and R are the backbones of the acylating and displacing agent. The spacer between the nucleobase is preferably of a length and flexibility to permit, for example, hydrogen bond base pairing between the nucleobases of the invention of the desired target polynucleotide. The dimensions of the backbones can be chosen to conform to the known measurements of double stranded DNA. According to X-ray crystallography data, the double stranded DNA helix is approximately 2 nm thick and completes a 360° turn every 10 nucleotides. The nucleotides are stacked 0.34 nm apart in the double helix. Though PNA polymers can be constructed to these dimensions, hybridization assays, such as Southern blots, can be used to determine if the PNA polymers can effectively anneal with native polynucleotides. In addition, PNA monomers and oligomers can be, for example, inhibitors of enzymes and polynucleotide regulatory proteins. The backbones can be chosen to enhance such inhibitor qualities by testing various constructs in protein/PNA binding assays. The backbones of the acylating and displacing agent, R. and R_d, can include without limitation hydrocarbon radicals such as alkyl, aryl, and arylalkyl moieties. These groups may be saturated or unsaturated, aliphatic, alicyclic or aromatic. Structures comprising ring structures, usually include one, two or three rings, which may be fused, or bridged or spiro- fused. Typically, R. and Rd are hydrocarbon backbones such as ethylene.

The following is an example of a class of acylating and displacing agents which together can create oligomers with sufficient length and flexibility to bind to natural polynucleotides. Preferred acylating agents of this example include those of the following formula:

Preferred displacing agents to be utilized with the acylating agents of this example are of either two following formula:

Suitable A*, Di, and ∑ include hydrocarbon, hydrocarbyl and hydrocarbylene substituents with none, one, or two hydrogens removed, respectively. These substituents may include, for example, nitrogen, sulfur, and oxygen atoms, as well as hydrogen and carbon. Further, these substituents may be saturated or unsaturated, aliphatic, alicyclic or aromatic. When rings are included the structure, usually one, two or three rings are included. The rings may be fused or bridged or spiro-fused. Preferably, R is a radical containing 12 or fewer carbon atoms, 4 or fewer nitrogen atoms, 4 or fewer oxygen atoms, and 2 or fewer sulfur atoms. More preferably, R is a radical is alkyl containing 6 or fewer carbon atoms; most preferably, R is hydrogen.

A radical designated R-, is a terminal group that can be at either terminus of the monomers or oligomers of the instant invention. This radical is not critical to the invention and can be chosen at convenience from a variety of substituents. For example, R. can be a protecting group, or an amino acid; DNA interacting agent; or even a cholesterol substituent to confer hydrophobicity. Because this R. is a radical at the terminus of the monomer or oligomer, Rt generally will not substantially interfere with the hybridizing or inhibiting properties of the monomer/oligomer.

The "activity" of a monomer or oligomer of the instant invention will be, for example, the ability of the monomer/oligomer to hybridize with naturally occurring polynucleotides or proteins. Alternatively, a monomer/oligomer will be considered active if can act as an inhibitor, e.g., a polymerase inhibitor.

A group is "protected" when any group is reversibly altered to prevent it from participating in an undesired reaction or bonding, usually in a synthesis reaction. When desired, the group can be deprotected to resume it original configuration. Groups can be protected with protecting groups to prevent, for example, reaction or bonding of carboxylic acids, thiols, and the like. Such groups and their preparation and introduction are conventional in the art and include salts, esters and the like.

A "substrate" or "solid support" is a conventional solid support material used in peptide synthesis. In addition, a substrate can act as a protecting group. Non-limiting examples of such substrates or supports include a variety of support resins and connectors to the support resins such as those which are photocleavable, DKP-forming linkers (DKP is diketopiperazine; see, e.g., WO90/09395 incorporated herein by reference), TFA-cleavable, HF-cleavable, fluoride ion cleavable, reductively cleavable and base-labile linkers. Examples or solid support material include controlled pore glass, polystyrene co-polymers, polyacrylamide, silica, polyethyleneglycol, polyethylene and polypropylene grafts.

B. General Method

Both PNA monomers and oligomers can be synthesized utilizing the three step cycle described herein. Each N-substituted PNA monomer can be synthesized directly on a solid substrate (support) from three reactants: acylating agent, displacing agent, and the nucleobase agent. Step 1 : Acylation

⁰ ?ι

L— Ra— C— Y HN"~« L— Ra— ? C-N

Carbonyl Qroup αroup

Step 2: Nucleophilic Displacement

Step 3: Acylation

The backbone of the monomer is assembled in the first two steps of the synthesis cycle. The first reaction is an acylation step where the carbonyl group of the acylating agent reacts with an amine. The acylating agent comprises a carbonyl group; a backbone, R.; and a leaving group, L.

The second step is a nucleophilic displacement of the leaving group by the first amino group of the displacing agent. The displacing agent comprises a first and a second amino group and a backbone, Rd. The first amino group is a primary amine, and the second step produces a secondary amine.

The third step is another acylation in which the nucleobase agent reacts with the first amino group of the displacing agent to produce a tertiary amide. The nucleobase agent comprises of a carbonyl group; an optional linker; and a nucleobase, R-,

Monomers and polymers can be synthesized utilizing either solution or solid phase methods. To initiate either solution or solid phase synthesis, a protected primary or secondary amine is provided. The amine is protected to ensure that synthesis proceeds in the desired direction. Preferably, monomers and polymers are synthesized on a substrate or solid support. Thus, the initial amine is attached to a solid support is utilized to initiate synthesis with an acylating agent. The amine may be a primary or secondary amine. In addition to the substrate, alkyl, aryl, and arylalkyl groups are examples of substituents of preferred secondary amines. Substrates to be utilized are those conventional solid support material used in peptide synthesis. Examples of solid support material include controlled pore glass, polystyrene co¬ polymers, polyacrylamide, silica, polyethylene and polypropylene grafts. Optionally, a linker can be placed between the support and the amino group, poly-lysine, for example. A variety of support bound resins including a cleavable linkers can be used, for example, p- methylbenzhydrylamine-resin-HCl or benzhydrylamine-resin-HCl; Bachem, Torrance, California, USA.

Preferably, synthesis of oligomers or monomers begins with a primary amine bound to a support and an acylating agent. A preferred substrate comprising a primary amine is, fluorenylmethoxy-carbonyl-4-methoxy-4'-(y-caA^ linked to alanyl-aminomethyl resin, the resin comprising chloromethylated polystyrene, 1% divinylbenzene, 1% DVB cross-linked. Acylating submonomers have been generally defined above. The following are preferred compounds to be used as acylating agents for the present invention, halo-crotonic, 2-halo-propionic acid, 2-halo-ethyl-haloformate, ω-haloalkyl carboxylic acid, halo-methyl benzoic acid, haloalkyl sulfonic acid, and 2-haloalkyl sulfonyl chloride.

The optimum ratio of these reactants will vary depending on the specific amine and acylating submonomer used. If, for example, bromoacetic acid and polystyrene beads are utilized, the acylating agent will be in excess of the substrate bound amine by about 1.01 to 10 fold; more preferably, in about 3 to about 10 fold excess; even more preferably, in about 10 fold excess.

In addition to the ratio of the reactants, the concentration will also vary depending on the conditions and quantities to be used. Typically, if bromoacetic acid is used, it will be at a concentration of between about 0.1 M to about 5 M; more typically, about 0.5 M. An activation agent can be included to facilitate acylation. Examples of activating agents include, without limitation, alkylphosphonic anhydrides, such as propane phosphonic anhydride; carbodiimides, such as dϋsopropylcarbodϋmide (DIC) and dicyclohexylcarbodiimide (DCC); uronium salts, such as HBTU, TBTU, HATU, TNTU, TSTU; phophonium salts, such as BOP, PyBOP, PyClOP,PyBrOP, PyNOP, PyFOP; carbonyldϋmidazole (CDI) and derivatives, such as dimethylated CDI; EEDQ; HOBt, and N- hydroxysuccinimides. The concentration of such activating agents will vary depending on the acylating submonomer and the amine to be reacted. When DIC is utilized with bromoacetic acid and polystyrene beads, the concentration of the activating agent will be approximately the same as the concentration of bromoacetic acid.

Solvents for solid phase synthesis are preferably dipolar and non-protic or non- polar. Examples of such solvents, include without limitation, dimethylforamide (DMF), dimetiiylsulfoxide (DMSO), dichloromethane, dichloroethane, and N-methyl morpholine. When bromoacetic acid is reacted with an amine bound substrate, DMF is preferred.

In one embodiment of the invention, this acylation reaction is performed at room temperature and atmospheric pressure. Though the reaction may not be driven to completion, the occurrence of unwanted side reactions is acceptable. Higher temperatures, such as 40°C - 50°C can be utilized to drive the reaction to completion. However, at these temperatures, the occurrence of unwanted side reactions can increase to affect the overall yield.

The time allowed for the components to react can vary depending on the temperature, pressure, reactants, and other components used. The time can vary from 5 minutes to 24 hours. As an example, when bromoacetic acid, a primary amine bound to a substrate, and DIC, as the activating agent, are the reactants, the time given is about 30 minutes at room temperature and atmospheric pressure.

To begin the reaction, the substrate bound amine is deprotected if necessary.

Next, the resin is washed thoroughly with the solvent alone. After the resin is drained, the appropriate amount of solvent containing the acylating submonomer and activating agent is added. Preferably, the solution and resin are agitated during the reaction by bubbling, for example, argon or nitrogen. Once the reaction time is completed, the solution is drained and the resin is washed again with the solvent alone.

For the next step, the displacing agent is added to displace the leaving group, now bound to the support. Displacing submonomers have been generally defined above. The following are preferred compounds to be used as displacing agents for the present invention, 1 -

(protected amino)-2-amino-ethane; R and S, l-amino-2-(protected amino)-propane; R and S, 1- (protected amino)-2-amino-propane; R and S, protected amino, amino ethyl benzene; R and S, amino, (protected amino) ethyl benzene; 1 -(protected, methyl, aιnino)-2-amino-ethane; N- (protected), N-aryl, 1,2 diamino ethane; 1 -(protected _ιmino)-3-amino- propane; R and S, 1- (protected ammo)-3-amino-2-methyl-propane; R and S, 1 -(protected an no)-3-amino-2-aryl- propane; R and S, 1 -(protected ammo)-3-amino-2-(hydroxy, methyl)-propane; R and S, 1- (protected anιmo)-3-amino-2-fluoro-propane; R and S, 1 -(protected, aryl, ammo)-3-amino- ethane; R and S, 1 -(protected ammo)-3-amino-2-carboxy-propane; R and S, 1 -(protected ammo)-3-amino-2-aminomethyl-propane; R and S, 1 -(protected ammo)-3-amino-2-thiomethyl- propane; R and S, l-(protected an_mo)-3-amino-2-trifluoromethyl-propane; R and S, 1- (protected amino)-3-amino-2-cyclopropylpropane, cis- and trans- 1 -(protected amino)-2- aminocyclopropane; and cis- and trans- 1 -(protected am o)-2-aminomethylcyclopropane; 1- ammo-2-(protected aminomethyl)cyclopropane.

Examples of protecting groups that can be utilized to protect the amino group are, without limitation, p-methoxybenzyloxycarbonyl; 2(3,4 dimethoxyphenyl) prop-2- pyloxycarbonyl; 2-nitro-4,5-dimethoxybenzyloxycarbonyl; t-butoxycarbonyl (tBOC); vinyloxycarbonyl; and allyloxycarbonyl.

Solvents for solid phase synthesis are preferably dipolar and non-protic or non- polar. Examples of such solvents, include without limitation, dimethylforamide (DMF), dimethylsulfoxide (DMSO), dichloromethane, dichloroethane, and N-methyl morpholine. When 4-(methoxybenzyloxy)carbonyl-ethylenediamine is utilized as the displacing agent for solid phase synthesis, DMSO is the preferred solvent.

The optimum ratio of these reactants will vary depending on the specific displacing agent used. One factor to consider is that one equivalent of acid is generated from the nucleophilic displacement. The resulting acid can decrease the yield of the reaction by protonating the displacing agent. This problem can be circumvented by adding a base that does not disrupt the reaction. If, for example, 4-(methoxybenzyloxy)carbonyl-ethylenediamine is utilized as the displacing agent, the agent will be in excess of the substrate by about 1.01 to 40 fold; more preferably, in about 3 to about 20 fold excess; even more preferably, in about 10 fold excess. In addition to the ratio of the reactants, the concentration will also vary depending on the conditions and quantities to be used. Typically, if 4- (met oxybenzyloxy)cari)onyl-ethylenediamine is used, it will be at a concentration of between about 0.1 M to about 10 M; more typically, about 0.5 M.

In one embodiment of the invention, the displacement reaction is performed at room temperature and atmospheric pressure. Though the reaction may not be driven to completion, the occurrence of unwanted side reactions is acceptable. Higher tempertures, such as 40°C-50°C, can be utilized to decrease the reaction time.

At room temperature and atmospheric pressure, the reaction time for the displacement step is somewhat longer thaN the acylation times. For example, if monoprotected ethylenediamine is utilized as the displacing submonomer, the reactant time is preferably between about 20 minutes and about 12 hours; more preferably, about 1 hour to 4 hours; even more preferably, the reaction time is about two hours.

The final step of the cycle, is another acylation reaction. To prepare this step, the beads are drained and washed with the buffer of the displacement step and then washed wit the buffer of the final step. Nucleobase submonomers have been generally defined above. The following ar preferred compounds to be used as nucleobase agents for the present invention. 1- carboxymethylthymidine; 1-caιfcoxymethyluracil; l-c-irboxymethyl-5-hydroxymethyluracil; 1- carboxymethyl-5-fluorouracil; l-carboxymethyI-5-bromouracil; 1-carboxymethylcytosine; 9- carboxymethyladenine; l-carboxymethy-2-ammopyrido[4,5-c]imidazole; 8-azido-9- carboxymethyladenine; l-carboxymethylguanine; l-hydro-6-oxo-9-carboxymethylpurine; 2,4- dioxo-9-caΛoxymethylpurine; l-methyl-6-imino-9-carboxymethylpurine; 3-methyl-6-imino-9- carboxymethyl-3,6-dihydropurine; 2-amino-6-methoxy-9-carboxymethylpurine; 1- carboxymethyl-5-methylcytosine; l-cart>oxymethyl-3-methylthymine; l-carboxymethyl-6- methylthymine; l-carboxymethyl-6-methylthiopurine; l-carboxymethyl-2-deoxo-2- thionothymine; l-carboxymethyI-6-methylpurine; l-carboxymethyl-6-methyluracil; 6- methylammo-9-cari oxymethypurine; 6-methoxy-9-carboxymethylpurine; l-carboxymethyl-5- trifluoromethyluracil; l-carboxvmethyl-4-amino-6-oxo-l,2,5-triazine; l-carboxymethyl-4,6- dioxo-5-hydro- 1,2, 5-triazine; 1 -carboxymethyl-3,5-dihydro-4, 6-dioxo- 1 ,2, 5-triazine; 1 - c»ri)θxvmethyl-5-hydroxvmethylcytosine; l-carboxymethyI-2-oxo-4-methylpyrimidine; 6- methylamino-9-carboxypurine; 2-clιloro-4-amino-9-carboxymethylpurine; 2-amino-4-oxo-9- carboxymethylpurme; 2,4-diar no-9-caιtoxymethylpurine; 2-oxo-4-amino-5-iodo-pyrimidine; 2,4-dioxo-5-iodopyrimidine. In addition, it may be advatageous to introduce protecting groups for the functional groups of the nucleobase. These protecting groups are standard in the art and have been reviewed in recently in Beaucage et al., Tetrahedron 48(12): 2223-2311 (1992). The optimum ratio of the nucleobase submonomer and the substrate bound amine vary depending on the specific amine and acylating submonomer used. If, for example, thymidine nucleobase agent, the submonomer will be in excess of the substrate bound amine by about 1.01 to 20 fold; more preferably, in about 3 to about 10 fold excess; even more preferably, in about 10 fold excess.

In addition to the ratio of the reactants, the concentration of them will also vary depending on the conditions and quantities to be used. Typically, if a thymidine nucleobase agent is used, for example, the submonomer will be at a concentration of between about 0.1 M to about 10 M; more typically, between about 0.2 M and about 0.5 M.

Like the first step of the cycle, an activation agent can be included to facilitate acylation. The activation agents to be used for the first step can also be utilized for this acylation step.

Solvents for solid phase synthesis are preferably dipolar and non-protic. Examples of such solvents, include without limitation, dimethylsulfoxide (DMSO), dichloromethane, dichloroethane, and N-methyl morpholine. For this step, a 1:1 mixture of DMSO and N-methyl-morpholine is preferred. In one embodiment of the invention, this acylation reaction is performed at room temperature and atmospheric pressure. Though the reaction may not be driven to completion, the occurrence of unwanted side reactions is acceptable. Higher temperatures, such as 40°C - 50°C can be utilized to drive the reaction to completion. However, at these temperatures, the occurrence of unwanted side reactions can increase to affect the overall yield. The time allowed for the components to react can vary depending on the temperature, pressure, reactants, and other components used. The time can vary from 5 minutes to 24 hours. Preferably, the time given for the reaction is about 30 minutes at room temperature and atmospheric pressure.

The desired nucleobase also can be incorporated into the monomer/oligomer in two steps rather than one acylating step. For example, the first amino group of the displacing agent can be reacted with a second acyl submonomer, where R, is the linker of desired length and flexibility to place the nucleobase into the wanted conformation. Next, an altered nucleobase can be provided to displace the leaving group of the second acylating agent to incorporate the nucleobase into the monomer/oligomer. An example of an altered nucleobase is a persilylated nucleobase. The acylating reaction is performed under similar conditions as the nucleobase agent addition.

The optimum ratio of these reactants will vary depending on the specific altered nucleobase agent used. If, for example, persilylated nucleobases are utilized, the agent will be in excess of the substrate by about 1.01 to 10 fold; more preferably, in about 3 to about 10 fold excess; even more preferably, in about 10 fold excess.

In addition to the ratio of the reactants, the concentration will also vary depending on the conditions and quantities to be used. Typically, if persilylated nucleobase is used, it will be at a concentration of between about 0.1 M to about 10 M; more typically, about 0.5 M. Solvents for solid phase synthesis are preferably dipolar and non-protic or non- polar. Examples of such solvents, include without limitation, dimethylforamide (DMF), dimethylsulfoxide (DMSO), dichloromethane, dichloroethane, and N-methyl morpholine. When persilylated nucleobases are utilized for solid phase synthesis, DMF is the preferred solvent. The displacement reaction is preferably performed at atmospheric pressures. Typically, this reaction will take place at elevated temperatures between 20°C and 150°C; more typically, between 50°C and 11 °C; even more typically, between 60°C and 100°C; most typically, at about 80°C.

The time allowed for the components to react can vary depending on the temperature, pressure, reactants, and other components used. The time can vary from 5 minutes to 24 hours. Preferably, the time given for the reaction is about 30 minutes to six hours; more preferably for 3 hours at 60°C-100°C and atmospheric pressure.

Synthesis of the monomer is complete after the nucleobase is incorporated into the monomer. To repeat the cycle to construct oligomers, the second amino group of the displacing agent bound to the substrate is deprotected to permit acylation with the next acyl submonomer. The same acyl and displacing submonomers need not be used throughout the oligomer. The backbones of each monomer in the oligomer can vary if desired. Further, synthesis of monomers and oligomers can be automated utilizing peptide synthesizers, such as

Biosyn, Millipore, or Applied Biosystems.

The PNAs of the instant invention can be utilized for diagnostic and therapeutic purposes. For example, PNA oligomers can be used as probes in standard polynucleotide detection and analytical procedures, such as Southerns and Northerns. As shown in the

Example below, when the PNAs of the instant invention hybridize with DNA, these PNADNA hybrids remain intact at higher temperatures than naturally occurring DNADNA hybrids. Thus, the instant PNAs can be employed in a PCR assay to detect mutations. Such an assay is described in Orum et al., Nuc Acids Res 2K23V 5332-5336 (1993). Further, PNAs of the instant invention can be applied in anti-sense therapy. For example, a PNA can be designed to bind to an undesired cancer gene. By binding to the gene, the PNA can prevent transcription and/or translation of the gene to prevent cancer activation.

PNAs can also be used to bind materials other than polynucleotides. For example, PNAs can be designed to bind irreversibly to viral polymerase, like HTV, to prevent viral replication.

C. Examples

The examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.

Example 1 : Synthesis of PNA with an N-aminoethylglycine Backbone

The oligomer was synthesized by incorporating monomers utilizing a four-step cycle. First, a resin-bound amine is acylated with a haloacetic acid submonomer acylating agent.

Next, the halo leaving group is displaced with a monoprotected ethylenediamine submonomer displacing agent. The unprotected amino group is acylated with a nucleobase derivative, a submonomer nucleobase agent. Finally, the protected amino group is deprotected to permit the next round of monomer incorporation.

All the reaction steps for this experiment were performed at room temperature and atmospheric pressure. Synthesis began by deprotecting the amine bound to a substrate.

The resin used was fluorenylmethoxycarbonyl-4-methoxy-4'-(γ-carboxypropyloxy)-benzhydryl- amine linked to alanyl-aminomethyl resin, the resin comprising chloromethylated polystyrene,

1% divinylbenzene, 1% DVB cross-linked; manufactured by Bachem California, Torrance, California, USA For a final yield of 50 μmoles, the experiment began with 0.5 mmoles of amine, calculated from the substitution level of the resin.

The resin was washed three times with 1 mL of dimethylformamide (DMF). The deprotection of the 9-fluorenylmethoxycarbonyl (Fmoc) group was performed with 20% (v/v) piperidine, in DMF. After one prewash with this reagent, the resin was bubbled with argon for 30 minutes. To remove the reagent, the resin was drained and washed first with 1 mL DMF five times.

After deprotection, the resin was washed several times with DMF, by adding the solvent to the resin, agitating the mixture with argon, and drained. Next, for the first step of the cycle, a ten fold excess of the acyl submonomer, bromoacetic acid, and activating agent, carbodϋmide, in DMF was added to the substrate bound amine. To the resin, 0.5 M bromoacetic acid, 0.5 M carbodϋmide in DMF was added to the resin, and the mixture was agitated for approximately 30 minutes by bubbling argon through the mixture. The solution was drained from the resin, and the resin was washed several times with DMF. Before beginning the displacement step, the resin was washed several times with

DMSO. Next, a ten fold excess of displacing submonomer, 4-(methoxybenzyloxy) carbonyl- ethylenediamine, was added to displace the leaving group, now bound to the resin. For this experiment, 0.5 M 4-(methoxybenzyloxy)carbonyl-ethylenediamine in DMSO was added to the resin, and the mixture was agitated for approximately two hours by bubbling argon through the mixture. The solution was drained from the resin, and the resin was washed several times with DMSO.

Before the third step of the cycle, the resin was washed several times with a l l mixture of DMSO and N-methyl morpholine. To acylate the first amino group of the displacing group, now bound to the resin, a ten fold excess of the nucleobase submonomer, 5- carboxymethyl-thymine, was added. The activating agent, PyBroP, was added in powder form to the resin. Next, 1.0 mL of a 0.5 M solution of 5-carboxymethyl-thymine in DMSO/N-methyl morpholine (1:1) was added to the resin and the activating agent. When the solvent was added the activating agent was at a final concentration of 0.5 M and at 10 fold excess of the resin. The resin and solution was agitated for approximately 30 minutes by bubbling argon. The solution was drained from the resin, and the resin was washed several times with the 1 : 1 mixture of DMSO and N-methyl morpholine. Finally, the second amino group of the displacing agent was deprotected. The resin was washed three times with 1 mL of dichloromethane. The deprotection of the Moz group was performed with 1% (v/v) trifluoroacetic acid (TFA), 2.5% (v/v) thioamisole and 2.5% (v/v) ethanedithiol in dichloromethane. After one prewash with this cleavage cocktail, the mixture was bubbled with the resin for 10 minutes. To neutralize the cocktail, the resin was drained and washed first with 1 mL dichloromethane three times. Next, the resin was washed with 10% (v/v) triethylamine in dichloromethane. The solution was then bubbled with a solution of 10% (v/v) triethylamine in dichloromethane for 10 minutes. After deprotection, the resin was washed several times with DMF. The three step cycle was repeated to construct a PNA octamer of oligothymine.

Annealing experiments were performed utilizing this oligomer and decamer of oligoadenine comprising a naturally occurring DNA phosphodiester backbone.

The oligomers dissolved in a buffer of 50 mM 3-N-morpholine-2hydroxy- propane sulfonic acid (MOPS), pH 7.0, 140 mM NaCl, and 10 mM MgCl₂. The mixture was heated to 90°C, then permitted to cool slowly for 30 minutes to allow the oligomers to anneal. Next, the absorbance of the mixture was measured at a wavelength of 260 nm as the mixture was heated. The absorbance of single stranded polynucleotides is greater than double stranded. From the data, a curve of absorbance versus temperature was plotted. The melting temperature (T_m) was determined from the curve. The melting temperature of the hybrid is when half of the hydrogen bonds have been broken. The T_m is the temperature when absorbance of mixture is half the theoretical maximum limit. The T_m for the PNA:DNA mixture was approximately 58°C.

A control experiment was performed utilizing a oligo-thymine and a oligo¬ adenine decamers. The data showed that the T_m was below 20°C, as expected from the literature data (T„ = 17.76°C) for a thymine adenine DNA decamer. See the Handbook of Chemistry and Molecular Biology, 3rd ed., Nucleic Acids, Vol. I, G.D. Fasman (ed ), CRC Press, Boca-Raton 1975, page 584.

Claims

WHAT IS CLAIMED:

1. A method of synthesizing a monomer comprising a nucleobase substituent comprising:

(a) providing a substrate, P., with primary or secondary amine bound thereto:

»

(b) acylating said amine bound to a substrate with a submonomer acylating agent of the structure.

L— Ra— V C— Y wherein L is a leaving group capable of nucleophilic displacement, to provide an acylated amine bound to a substrate having positioned thereon a leaving group for nucleophilic displacement of the structure.

O

L—Ra—C—N ϊ^*— linker— Ps

(c) reacting said acylated amine with a submonomer displacing agent comprising first and second amino groups of the structure:

wherein said first amino group is capable of nucleophilic displacement of said leaving group and wherein said second amino group is protected to prevent acylation of said second amino group from unwanted by the submonomer nucleobase agent, to provide a secondary amine having positioned thereon a protected second amino group of the structure: R₂ H O Ri

Pd— N-Rd—N — Ra— C— N— hnker— p_s

>

(d) acylating said secondary amine with a submonomer nucleobase agent of the structure:

Rn

I Linker

I C=O

I Y

wherein R„ is a nucleobase protected to prevent unwanted side- reactions of the functional groups of said nucleobase, to provide a tertiary amide of the structure.

2. The method of Claim 1, further comprising

(d) deprotecting said protected second amino group to provide a a primary or secondary amine bound to a substrate of the structure:

The method of Claim 2, wherein said leaving group is halo.

4. A method of synthesizing an oligomer comprising a nucleobase substituent comprising:

(a) providing a substrate, P„ with primary or secondary amine bound thereto:

L— Ra— ? C— Y wherein L is a leaving group capable of nucleophilic displacement, to provide an acylated amine bound to a substrate having positioned thereon a leaving group for nucleophilic displacement of the structure:

O R-

II I ' .

L—Ra—C—N— linker— Ps

(c) reacting said acylated amine with a submonomer displacing agent comprising first and second amino groups of the structure.

wherein said first amino group is capable of nucleophilic displacement of said leaving group and wherein said second amino group is protected to prevent acylation of said second amino group from unwanted by the submonomer nucleobase agent, to provide a secondary amine having positioned thereon a protected second amino group of the structure.

Pd— N T-— Rd— V N — Ra— ? C — N ϊ '— „ inker— p_s

(d) acylating said secondary amine with a submonomer nucleobase agent of the structure.

Rn

I

Linker I

C=O I Y wherein Ra is a nucleobase protected to prevent unwanted side- reactions of the functional groups of said nucleobase, to provide a tertiary amide of the structure::

(e) deprotecting said protected second amino group to provide a a primary or secondary amine bound to a substrate of the structure (f) repeating steps (b) through (e).

5. The method of Claim 4, wherein each submonomer acylating agent is haloacetic acid.

6. The method of Claim 5, wherein each submonomer displacing agent is a protected ethylenediamine.

7. The method of Claim 6, wherein said submonomer nucleobase agent is a mixture of carboxymethylthymine, carboxymethyluracil, carboxymethyladenine, carboxymethylcytosine, carboxymethylguanine, and carboxymethylinosine.

8. A compound comprising monomers of the following structure:

wherein R is a radical which does not substantially sterically hinder synthesis or activity and contains: 12 or fewer carbon atoms,

4 or fewer nitrogen atoms, 4 or fewer oxygen atoms, and 2 or fewer sulfur atoms; R„ is a nucleobase or a binding agent; the linker is a divalent linking radical capable of positioning R„ in the desired conformation;

Rti and R_ are terminal groups; and wherein at said oligomer comprises least two different R„.

9. The compound of Claim 8, wherein R_<* and R, are the same from monomer to monomer.

10. The compound of Claim 9, wherein R' is hydrogen.

11. The compound of Claim 10, wherein said linker is:

wherein i, Ra, Rjjj, and R_JV is a bond to the adjacent carbon or a radical which does not substantially sterically inhibit synthesis or activity;

Xis O. N, S, P(O)_X) or a bond; m = 0-8; and n = l-8.

12. The compound of Claim 11, wherein Rj, R-j, Rai, and R are hydrogen or alkyl comprising one to four carbon atoms.

13. The compound of Claim 12, wherein n = 1 to 4, R;, Ra are hydrogen, X is a bond and m = 0.

14. The compound of Claim 13, wherein Ra is ethyl and R, is methyl.

15. The compound of Claim 14, wherein R„ is selected from the group consisting of thyminyl, uracilyl, adeninyl, guaninyl, cytosinyl, inosinyl.

16. A compound having the following structure:

wherein

Re is a nucleobase or binding agent; the linker is a radical capable of positioning said R. in the desired conformation; Ri and R₂ are radicals which do not substantially sterically hinder synthesis or activity and contains: 12 or fewer carbon atoms,

4 or fewer nitrogen atoms, 4 or fewer oxygen atoms, and 2 or fewer sulfur atoms; Rt is a terminal group; and

R, is not methyl when and Rd is ethyl.

17. The compound of Claim 16, wherein said linker is:

wherein

RJ, Rjj, Rjϋ, and R-y is a bond to the adjacent carbon or a radical which does not substantially sterically inhibit synthesis or activity; X is O, N, S, P(O)„ or a bond; m ^~ 0-8; and n = l-8.

18. The compound of Claim 17, wherein Rj, Ra, Ra., and R* are hydrogen or alkyl comprising one to four carbon atoms.

19. The compound of Claim 18, wherein n = 1 to 4, Rj, Ra are hydrogen, X is a bond and m - 0.