DE10312625A1 - Method for preparing functional peptide nucleic acids, useful e.g. in gene therapy and as detection probes, using protected synthon that allows subsequent reaction with functional carboxylic acid - Google Patents

Abstract

Method for producing a functional PNA (peptide nucleic acid) oligomer (A) in which PNA monomer units, containing protected adenine, guanine, cytosine or thymidine, are reacted with Fmoc-omega -amino acid-Boc-PNA-OH (X) (Fmoc = fluorenylmethoxycarbonyl; Boc = tert-butoxycarbonyl). Method for producing a functional PNA (peptide nucleic acid) oligomer (A) in which PNA monomer units, containing protected adenine, guanine, cytosine or thymidine, are reacted with Fmoc-omega -amino acid-Boc-PNA-OH (X) (Fmoc = fluorenylmethoxycarbonyl; Boc = tert-butoxycarbonyl), and after synthesis of the PNA oligomer, a functional molecule having a free carboxy group is incorporated, followed by removal of the protecting group. Independent claims are also included for the following: (1) compounds of formula Boc-NH-CH2CH2-N[CO-(CH2)n-NHFmoc]-CH2COOH (I); (2) method for preparing (I); (3) compounds of formulae (II) and (III); and (4) method for preparing (III) by reacting Fmoc-omega -amino acid with pentafluorophenol. [Image] n : 1-15; each B : A, T, G or C; each R : Fmoc or functional carboxylic acid residue (Q); R1>hydrogen or Q; a-h : 0-10; x1 to x3, y1, y2 and z1 to z5 each : 0 or more. Provided that: (1) y1 and y2 are not both zero, (2) not all x and all z are simultaneously zero, and (3) when all x = zero, R1> = Q.

Description

The present invention relates to a new process for the production of a functional peptide nucleic acid monomer, a functional peptide nucleic acid oligomer made by this method and its intermediates. In particular, the invention relates a manufacturing process that involves the introduction of one type or two or several types of a functional molecule post-synthetic according to the introduction a precursor PIVA monomer unit in a PNA oligomer.

Nucleic acids consist of DNA and RNA, which control the genetic information of living things. In contrast, peptide nucleic acids (PNA) refer to modified nucleic acids in which the sugar-phosphate framework of a nucleic acid has been converted into an N- (2-aminoethyl) glycine framework ( 1 ). Although the sugar-phosphate frameworks of DNA / RNA charge negatively under neutral conditions, which leads to electrostatic repulsion between the complementary strands, the basic framework of the PNA is inherently not charged, which is why there is no electrostatic repulsion , Compared to traditional nucleic acids, the PNA have a higher ability to form double strands and have a pronounced ability to recognize base sequences. Since the PNA are also extremely stable with regard to nucleases and proteases in the organism and are not degraded by them, investigations were carried out into their usability for gene therapy as an antisense molecule.

A result of using PNA with the technique that is common Using the DNA as a medium is that it has become possible is, the disadvantages of DNA, which so far could not be overcome remove. So e.g. can PNA in the "DNA microarray technology" for rapid and large volume systematic analysis of genetic information as well as being the first Recently developed "molecular signals" for use as Probes for the detection of a base sequence with the emission of fluorescent Light specifically recognized can be used. There with both Applications as a medium DNA-free enzyme resistance is used, accurate sampling is required when using these technologies. Precise compliance with this requirement is the key to Achieving a higher Accuracy when working with these techniques.

On the other hand, PNA is completely enzyme resistant is, can by replacing DNA with PNA in DNA microarray technology and overcome the technical disadvantages mentioned above with molecular signals be suggesting further benefits from using these technologies gives hope.

Although there are many other areas on which the PNA advances to further advances in addition to DNA microarray technology and the molecular signals could, it is necessary in these areas to construct new PNA monomers, so that the PNA can function effectively, through implementation an effective introduction functional molecules into the PNA monomers.

Since the usual methods of solid phase peptide synthesis are used for the processes of PNA oligomer synthesis, the classification of the PNA monomer units according to the PNA basic structure results in two types, namely PNA monomers of the Fmoc type and those of the tBoc type ( 2 ).

worin X Guanin, Thymin, Cytosin oder Adenin bedeutet.Methods for synthesizing Fmoc-type PNA monomer units have already been established. Since their oligomer synthesis can be carried out using a conventional automated DNA synthesizer, the synthesis can be carried out on a small scale in the following way:

where X is guanine, thymine, cytosine or adenine.

verwendet, was zu wirksameren Syntheseverfahren führte:

PNA monomer units of the tBoc type were originally like

used, which led to more effective synthetic methods:

However, since the Fmoc type mentioned above was developed that made handling easier, the frequency decreases the use of the tBoc type.

When introducing a functional molecule that different from the four types of nucleic acid bases guanine, thymine, cytosine and adenine, e.g. when introducing a photofunctional molecule, however, it happens frequently before that when the to be introduced functional molecule is unstable under alkaline conditions, a tBoc type of PNA backbone, which is not used under alkaline conditions, very suitable is. The inventors in the present application already have one Patent application for a "procedure for the preparation of t-butoxycarbonylaminoethylamine and amino acid derivatives "(JA-PA No. 2000-268 638).

In addition, from the state of the Technique five examples for Syntheses of monomer units of photofunctional oligo-PNA are known. Although the above path is followed in all of these syntheses, their yields have either not been described or are extremely low (Peter E. Nielsen, Gerald Haaiman, Anne B. Eldrup PCT Int. Appl. (1998) WO 985 295 A1 19 981 126, T.A. Tran, R.-H. Mattern, B.A. Morgan (1999) J. Pept. Res. 53, 134-145, Jesper Lohse et al. (1997) Bioconjugate Chem., 8, 503-509, Hans-Georg Batz, Henrik Frydenlund Hansen et al., PCT Int. Appl. (1998) WO 9 837 232 A2 19 980 827, Bruce Armitage, Troels Koch et al. (1998) Nucleic Acid Res., 26, 715-720). In addition, since the structures of the compounds used can Have property that they are comparatively alkaline resistant are expected to be produced at a high yield can, whereby a method is used which corresponds to the method mentioned similar to the prior art is, namely the subsequent synthesis route A, if under alkaline Conditions an inconsistent Chromophore linked becomes.

However, since it often happens that photofunctional molecules or other functional molecules expensive methods for the synthesis of more suitable functional are PNA desired namely procedures for a particularly quick introduction of these functional molecules (1) for the effective introduction functional molecules into a PNA framework in the construction of functional PNA monomer units (2) for synthetic routes considering of the price-performance ratio and (3) for the adaptation with regard to the use of genetic diagnostic Drugs.

Given the problems mentioned above The inventors now found a new method for producing functional PNA monomers, the in the almost quantitative synthesis of a photofunctional PNA monomer 4 using a t-butoxycarbonylaminoethylamine derivative 6 for the PNA backbone and in Condensation with an active ester form 5, which is the pentafluorophenyl group 1 contains as indicated in path B below.

In addition, another procedure was used for the Synthesis of functional PNA monomers using a benzyloxycarbonyl-ω-amino acid derivative instead of the above t-butoxycarbonylaminoethylamine derivative 6 for the PNA backbone (path C) found. Patent applications have already been filed for these processes filed.

After all, there have been procedures for the industrial synthesis of functional PNA established in the synthesis functional PNA monomers according to method according to route B or C exist with subsequent polymerization of these monomers. In particular, it has now become possible size Amounts of functional PNA for use as PNA probes using existing ones Process for synthesizing functional PNA industrially.

On the other hand, there were also procedures to produce functional PNA improved to the value for money too improve and an extremely quick introduction functional molecules to enable. It is known e.g. a process in which functional molecules in PNA oligomers Postsynthetic with the help of the following precursor PNA monomer unit introduced be, this method differs from that described above Use of functional PNA monomer units differs (Oliver Seitz: Tetrahedron Letters 1999, 40, 4161-4164).

This procedure is based on the introduction the precursor PNA monomer unit into a PNA oligomer the functional PNA by additional introduction of a functional molecule synthesized.

However, this method has the disadvantage that the types of functional molecules that are introduced can, are limited.

As indicated below, the in Commercially available Succinimide esters are not introduced as a photofunctional molecule. It is necessary to first introduce a linker like Fmoc-Gly in order to this photofunctional molecule introduce to be able which makes the above connection difficult to use.

They have also been in the past DNA, RNA and PNA oligomers as fluorescent probes for introduction to Cells used, which of course must be able to To pass cell membrane. However, since the surface of the cell membrane is negative it’s extremely difficult to use DNA / RNA oligomers, that are inherently negatively charged.

On the other hand, although PNA oligomers are electrically neutral, the results obtained showed that a Passage through the cell membrane is problematic. When introducing PIVA oligomers in a cell must go through this introduction Pretreatment of the membrane surface be relieved or they must with the help of a transfection reagent.

In the case of the introduction of PNA oligomers under Application of the mentioned Treatment exists even though the function of the probe is demonstrated can, however, no guarantee that the behavior naturally shown by the organism emerges exactly. Moreover this only applies to a cell while for numerous other cells of an organism their use is practically impossible.

Because of the current situation is the development of a fluorescent PNA probe that crosses the cell membrane able to penetrate, desirable.

It must be stated that fluorescent PNA probes that penetrate the cell membrane capital, already exist. Examples of these are (1) fluorescent PNA probe containing an oligopeptide that penetrate the cell membrane is linked to PNA, and (2) a fluorescent PNA probe, where a phospholipid that is able to penetrate the cell membrane linked to PNA is. However, it is expected that the proportion of these probes that differ from the PNA by enzymes such as proteases within the cells are broken down after permeation of the cell membrane, whereby these remain inside the cell. As this becomes a surplus leads to PNA probes, the incapable were to capture the target, increasing their ability to penetrate the membrane is lost and it is difficult to use them in the subsequent washing stages To remove from the cell, this means that the natural gene expression system the cell can no longer be expressed exactly.

An object of the present invention is thus the provision of a new method with cheap Price-performance ratio for the synthesis of functional PNA, which allows functional molecules to be very quickly introduced can be of compounds used and of new functional PNA.

Through intensive studies of the mentioned above The inventors surprisingly have problems found that by optimizing the structure of the precursor PNA monomer units the problems of the prior art can be eliminated and that functional PNA is synthesized within a very wide range can be thereby realizing the present invention.

An object of the present invention is in particular to provide a method for producing a functional PNA oligomer, in which a PNA monomer unit with adenine, guanine, cytosine or thymine, protected by a protective group, with Fmoc-ω-amino acid ^Boc PNA-OH implemented and after synthesis of the PNA oligomer, a functional molecule with a free carboxylic acid is introduced into the PNA oligomer, after which the protective group is removed again.

The invention further relates to the method described above, in which Fmoc-ω-amino acid- ^Boc PNA-OH is produced by reacting Fmoc-ω-amino acid pentafluorophenyl ester with ^Boc PNA-OH.

The invention further relates to that Process described above, in which Fmoc-ω-amino acid pentafluorophenyl ester by Implementation of Fmoc-ω-amino acid and Pentafluorophenol is produced.

The invention further relates to that The method described above, in which, after the introduction of a functional molecule, another functional molecule introduced becomes.

The invention further relates to that The method described above in which the functional mullet introduced is selected under a photofunctional molecule, a membrane permeable functional molecule, an organ-selective functional molecule, a bactericidal functional molecule molecule and a molecular recognizer functional molecule.

The invention further relates to that Process described above in which the introduced functional molecule is a photofunctional molecule and contains a membrane permeable functional molecule.

The invention further relates to that Process described above, in which the photofunctional molecule FITC, ROX, TAMRA or Dabcyl is functional and the membrane permeable molecule is a water soluble amino acid.

The invention further relates to that Process described above, in which the adenine, guanine, cytosine or thymine protective Group is a group Z

The invention further relates to that The method described above in which the synthesis of the PNA oligomer the condensation and extension in a PNA chain using a solid phase support for the tBoc process includes.

The invention further relates to this The method described above, in which the solid phase carrier for the MBHA tBoc method is.

The invention further relates to this Process described above, in which the introduction of a functional molecule with free carboxylic acid by dehydrogenation condensation with a primary amino group which by selective removal of the protecting group from the Fmoc group is obtained by piperidine treatment.

worin n eine ganze Zahl von 1 bis 15 bedeutet.The invention further relates to the process described above, in which the Fmoc-ω-amino acid ^Boc PNA-OH is a compound of the following general formula I:

where n is an integer from 1 to 15.

• a) Umsetzung der Fmoc-ω-Aminosäure mit Pentafluorphenol auf einer Stufe, auf der Fmoc-ω-aminosäurepentafluorphenylester erzeugt wird,
• b) Einführung der Fmoc-ω-Aminosäure in ^BocPNA-OH durch Umsetzung des Fmoc-ω-Aminosäurepentafluorphenylesters mit ^BocPNA-OH auf einer Stufe, auf der Fmoc-ω-aminosäure-^BocPNA-OH erzeugt wird;
• c) Erzeugung des PNA-Oligomers durch Umsetzung einer PNA-Monomereinheit mit Fmoc-ω-Aminosäure-^BocPNA-OH auf einer Stufe, auf der aus Fmoc-ω-aminosäure-^BocPNA-OH PNA-Oligomer erzeugt wird und
• d) Durchführung der Einführung eines funktionellen Moleküls in das PNA-Oligomer durch Dehydrierungskondensation einer primären Aminogruppe, die durch selektive Entfernung einer Schutzgruppe von einer Fmoc-Gruppe durch Piperidinbehandlung auf einer Stufe erhalten wird, auf welcher aus dem PNA-Oligomer ein funktionelles PNA-Oligomer erzeugt wird.

The invention further relates to the method described above, in which the method comprises one or more of the following steps a) to d):

• a) reaction of the Fmoc-ω-amino acid with pentafluorophenol at a stage at which Fmoc-ω-amino acid pentafluorophenyl ester is produced,
• b) introduction of the Fmoc-ω-amino acid in ^Boc PNA-OH by reacting the Fmoc-ω-amino acid pentafluorophenyl ester with ^Boc PNA-OH at a stage at which Fmoc-ω-amino acid ^Boc PNA-OH is generated;
• c) Generation of the PNA oligomer by reacting a PNA monomer unit with Fmoc-ω-amino acid- ^Boc PNA-OH at a stage at which Fmoc-ω-amino acid- ^Boc is used to produce PNA-OH PNA-oligomer and
• d) Carrying out the introduction of a functional molecule into the PNA oligomer by dehydrogenation condensation of a primary amino group, which is obtained by selective removal of a protective group from an Fmoc group by piperidine treatment at a stage at which a functional PNA oligomer is formed from the PNA oligomer is produced.

worin n eine ganze Zahl von 1 bis 15 bedeutet.The invention also relates to a compound of the following general formula I:

where n is an integer from 1 to 15.

worin n eine ganze Zahl von 1 bis 15 bedeutet, das die Einführung der Fmoc-ω-Aminosäure durch Umsetzung der Fmoc-ω-Aminosäure mit Pentafluorphenol und Umsetzung des Reaktionsproduktes mit ^BocPNA-OH umfasst.The invention also relates to a process for producing a compound of the general formula I:

where n is an integer from 1 to 15, which comprises introducing the Fmoc-ω-amino acid by reacting the Fmoc-ω-amino acid with pentafluorophenol and reacting the reaction product with ^Boc PNA-OH.

worin die Symbole B jeweils unabhängig voneinander dieselbe oder unterschiedliche Bedeutung haben und Adenin, Guanin, Cytosin oder Thymin bedeuten und die Symbole R jeweils unabhängig voneinander dieselbe oder unterschiedliche Bedeutung haben und eine Fmoc-Gruppe oder ein funktionelles Carbonsäurederivat, R¹ ein Wasserstoffatom oder ein funktionelles Carbonsäurederivat, a bis h ganze Zahlen von 0 bis 10, X₁ bis X₃, Y₁, Y₂ und Z₁ bis Z₅ jeweils ganze Zahlen von 0 oder mehr bedeuten, X₁ + X₂ + X₃ ≥ 0, Y₁ + Y₂ > 0 und Z₁ + Z₂ + Z₃ + Z₄ + Z₅ ≥ 0, mit der Maßgabe, dass X₁ + X₂ + X₃ und Z₁ + Z₂ + Z₃ + Z₄ + Z₅ nicht gleichzeitig 0 bedeuten, und für den Fall, dass X₁ + X₂ + X₃ = 0, R¹ ein funktionelles Carbonsäurederivat bedeutet.The invention also relates to a compound of general formula II

wherein the symbols B each independently have the same or different meaning and adenine, guanine, cytosine or thymine and the symbols R each independently have the same or different meaning and an Fmoc group or a functional carboxylic acid derivative, R ^{1 is} a hydrogen atom or a functional one Carboxylic acid derivative, a to h are integers from 0 to 10, X ₁ to X ₃ , Y ₁ , Y ₂ and Z ₁ to Z ₅ each represent integers from 0 or more, X ₁ + X ₂ + X ₃ ≥ 0, Y ₁ + Y ₂ > 0 and Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ ≥ 0, with the proviso that X ₁ + X ₂ + X ₃ and Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ does not simultaneously mean 0, and in the event that X ₁ + X ₂ + X ₃ = 0, R ¹ denotes a functional carboxylic acid derivative.

The present invention also relates to the compound described above, in which Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ = 0 and R ^{1 represents} a hydrogen atom.

The invention also relates to Compound described above, in which R is a carboxylic acid derivative of Includes methyl red.

The invention also relates to the compound described above, in which X ₁ + X ₂ + X ₃ = 9 and Y ₁ + Y ₂ = 1.

The invention further relates to the compound described above, in which X ₁ = 3, X ₂ = 6 and Y ₁ = 1.

The invention further relates to the compound described above, in which R or R ^{1 represents} a cell membrane-permeable functional molecular derivative.

The invention further relates to the compound described above, in which R ^{1 is} a functional carboxylic acid derivative.

The invention further relates to the compound described above, in which X ₁ = Z ₁ = 1.

The invention further relates to the compound described above, in which Y ₁ ≥ 2 and Z ₂ = 1.

The invention further relates to the Connection described above, where a ≤ 6, b ≤ 4 and f ≤ 6.

The invention further relates to the compound described above, in which R ^{1 is} a photofunctional carboxylic acid derivative.

worin n eine ganze Zahl von 1 bis 15 bedeutet.The invention also relates to a compound of the following general formula III described above:

where n is an integer from 1 to 15.

worin n eine ganze Zahl von 1 bis 15 bedeutet, bei dem die Fmoc-ω-aminosäure mit Pentafluorphenol umgesetzt wird.The invention also relates to a process for producing a compound of the general formula III:

where n is an integer from 1 to 15, in which the Fmoc-ω-amino acid is reacted with pentafluorophenol.

The present invention ensures an almost quantitative synthesis of photofunctional PNA oligomers by introducing a precursor PNA monomer unit in which Fmoc-ω-amino acid is introduced into a PNA backbone, namely Fmoc-ω-amino acid- ^Boc PNA-OH in a PNA oligomer with subsequent post-synthetic introduction of a functional molecule.

According to the above characteristics it is in the manufacturing method according to the invention not necessary for the one to be introduced functional molecule commercial Bernsteinsäureimidester to use. A carboxyl group is used instead having connection, which can be introduced easily and quantitatively can. The manufacturing method according to the invention thus shows a very favorable price-performance ratio.

In addition, by separating the resin after introduction the precursor PNA monomer units into the functional PNA oligomer different functional molecules introduced into every resin become. The method according to the invention allows therefore an extremely quick one Synthesis of the functional PNA oligomer.

worin die Symbole B jeweils unabhängig voneinander dieselbe oder unterschiedliche Bedeutung haben und Adenin, Guanin, Cytosin oder Thymin bedeuten und die Symbole R jeweils unabhängig voneinander dieselbe oder unterschiedliche Bedeutung haben und eine Fmoc-Gruppe oder ein funktionelles Carbonsäurederivat, R¹ ein Wasserstoffatom oder ein funktionel les Carbonsäurederivat, a bis h ganze Zahlen von 0 bis 10, X₁ bis X₃, Y₁, Y₂ und Z₁ bis Z₅ jeweils ganze Zahlen von 0 oder mehr bedeuten, X₁ + X₂ + X₃ ≥ 0, Y₁ + Y₂ > 0 und Z₁ + Z₂ + Z₃ + Z₄ + Z₅ ≥ 0, mit der Maßgabe, dass X₁ + X₂ + X₃ und Z₁ + Z₂ + Z₃ + Z₄ + Z₅ nicht gleichzeitig 0 bedeuten, und für den Fall, dass X₁ + X₂ + X₃ = 0, R¹ ein funktionelles Carbonsäurederivat, worin Z₁ + Z₂ + Z₃ + Z₄ + Z₅ = 0, und R¹ ein Wasserstoffatom bedeuten.An example of a functional PNA oligomer that can be effectively synthesized by the process according to the invention is the compound of the general formula II:

wherein the symbols B each independently have the same or different meaning and adenine, guanine, cytosine or thymine and the symbols R each independently have the same or different meaning and an Fmoc group or a functional carboxylic acid derivative, R ^{1 is} a hydrogen atom or a functional one les carboxylic acid derivative, a to h are integers from 0 to 10, X ₁ to X ₃ , Y ₁ , Y ₂ and Z ₁ to Z ₅ each represent integers from 0 or more, X ₁ + X ₂ + X ₃ ≥ 0, Y ₁ + Y ₂ > 0 and Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ ≥ 0, with the proviso that X ₁ + X ₂ + X ₃ and Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ do not simultaneously mean 0, and in the event that X ₁ + X ₂ + X ₃ = 0, R ^{1 is} a functional carboxylic acid derivative, in which Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ = 0, and R ^{1 represents} a hydrogen atom.

According to the invention, the same or different functional molecules arbitrarily on a variety selected Places are introduced into the compound of formula II. The piperidine treatment and the post-synthetic introduction of a functional molecule can at the same time after the introduction of a PNA oligomer using the above-mentioned precursor MNA monomer units carried out be, this method with a view to rapid construction of the system, which has the function of cell membrane permeation PNA oligomers improved, is essential. The method according to the invention is far superior in this regard.

An example of a compound produced in this way is a compound in which Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ > 0, R is a cell membrane-permeable molecular derivative and R ^{1 is} a functional carboxylic acid derivative in the represent formula II above.

This probe can basically be divided into three domains, namely a fluorescence-labeled domain, a domain with the function of cell membrane permeation and a molecule-recognizing domain, each of these domains being connected to one another by linker sites (the section is represented by the suffixes from Z ₁ to Z ₅ ).

Both commercial products as well as new ones Fluorescence-labeled PNA monomer units, for which the inventors already a PCT application has been submitted to the present invention was come for the fluorescence-labeled compound in question.

The molecular recognition site is synthesized using a commercially available PNA unit. This is characterized by the use of a new PNA unit of the general formula I for which a patent application has been filed in Japan for the domain for the function of membrane permeation. The This new PNA unit of the general formula I is a precursor unit which was developed for the post-synthetic introduction of functional molecules and is characterized in that it enables the simultaneous introduction of molecules with the same function as mentioned above after the introduction of a large number of these new PNA units in a row.

According to the invention numerous functional molecules without being limited to photofunctional molecules, light and extremely effective introduced in PNA become.

Examples of such functional molecules are Naphthalimide, flavin, dabcyl, biotin, FAM, rhodamine, TAMRA, ROX, HABA, pyrene and coumarin-type photofunctional monomer units, Membrane-permeable functional molecules, organ-selective functional ones molecules bactericidal functional molecules and molecule-recognizing functional molecules.

The term "functional" is not limited to the photo functionality here, but rather means all Types of functions that are recently connected by a particular one Modification to be conferred, membrane permeability, organ selectivity, bactericidal Function and molecular recognition function includes.

According to the invention, the term “functional PNA” does not mean just the direct link of the PNA monomers through a 2- (N-aminoethyl) glycine skeleton, but also those that have a hydrocarbon chain etc. in the form of a included linkers in between.

Other aspects and advantages of Invention will be apparent from the following description with the attached Drawings that exemplify the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following are the characteristics of the method according to the invention explained in more detail.

The synthetic route of the oligo-PNA according to the invention is given below:

First, as indicated below, the Fmoc-ω-amino acid is optionally reacted with pentafluorophenol (PfpOH), after which the Fmoc-ω-amino acid pentafluorophenyl ester (Fmoc-ω-amino acid OPfp) is used to synthesize the Fmoc-ω-amino acid ^Boc PNA-OH becomes:

To obtain a solution of the Fmoc-ω-amino acid OPfp which is used in the subsequent steps, for example, preferably either an organic solvent such as DMF or a water-soluble one ches solvent containing acetone and water can be used. The use of a water-soluble solvent offers advantages in terms of aftertreatment, such as cleaning.

worin n eine ganze Zahl von 0 bis 15 bedeutet, wird durch Umsetzung von Fmoc-ω-Aminosäure mit PfpOH in einer DMF-Lösung unter Zugabe von DCC erhalten.The above Fmoc-ω-amino acid OPfp, such as that of Formula III below

where n is an integer from 0 to 15 is obtained by reacting Fmoc-ω-amino acid with PfpOH in a DMF solution with the addition of DCC.

A DMF solution of ^Boc PNA-OH and diisopropylethylamine is then added in order to arrive at the Fmoc-ω-amino acid ^Boc PNA-OH.

Since the Fmoc-ω-amino acid ^Boc PNA-OH acts as a precursor of the PNA monomer unit, it can be referred to as a precursor PNA monomer unit.

Although an integer from 1 to 15 for n in formula I expediently selected can be a higher one Value in terms of steric rejection reduction (hindrance) while to be preferred to hybrid formation.

Then, as indicated below, synthesized the oligomer Ia using the precursor PNA monomer unit.

In particular, a PNA monomer unit, which has adenine, guanine, cytosine or thymine and with a group Z is protected as an N-benzyloxycarbonyl group with the precursor PNA monomer unit implemented, after which the PNA chain successively condenses and with the help of a solid phase support for the tBoc process extended becomes.

Although it is necessary, the tBoc group before the condensation of the PNA chain, there is still no restrictions with regard to the method that is suitable for this, i.e. that the usual Procedures can be used. For the subsequent condensation becomes an ordinary condensing agent like HATU, HBTU or BOP.

Also, although there is none special restrictions with regard to the solid support if he is for the tBoc process comes into question, MBHA particularly preferred. After that is, as indicated below, by the Fmoc group by piperidine treatment for conversion into an amino group and for the production of Ib die Protective group selectively split off.

In addition, as indicated below, becomes a functional molecule with a free carboxylic acid group for the above amino group of Ib dehydrated and condensed to Ic.

Although in view of the above Carboxylic acid group no particular restrictions there is an aliphatic carboxylic acid group in terms of reactivity an aromatic carboxylic acid group preferable. An aliphatic carboxylic acid group is therefore due their higher production efficiency preferable.

The spin-off also takes place the protecting group of the Fmoc group by piperidine treatment, preferably for a long period of time. The duration of this treatment is in particular 20 to 40 minutes and very particularly 30 minutes.

Regarding the condensing agent there are no particular restrictions and similar like the one mentioned above PNA chain condensation becomes a typical condensing agent like HATU, HBTU or BOP.

It should be noted that the introduction of functional molecules immediately after the condensation of the Fmoc-ω-amino acid ^Boc PNA-OH (method 1) or after the successive condensation of all PNA monomer units including the Fmoc-ω-amino acid ^Boc PNA-OH done (method 2).

Finally, as stated below, the target PAN oligomer Id through simultaneous execution separation from the carrier resin and by splitting off the Z groups.

There are no particular restrictions in terms of the condition for separation and deprotection, provided that they are carried out after the protective group has been split off from the Fmoc group. So e.g. separation and deprotection are preferred under ordinary Conditions like TFA / TFMSA / p-cresol / thioanisole = 60/25/10/10 performed.

As described above, the method according to the invention in contrast to the methods of the prior art used for synthesis of the functional monomers requires the synthesis of an active ester make the functional molecules can be used immediately. In addition, after synthesis Different functional molecules from Ia are introduced, which is a rapid and enables parallel synthesis of different types of PNA probes, which was difficult according to the prior art.

worin die Symbole B jeweils unabhängig voneinander dieselbe oder unterschiedliche Bedeutung haben und Adenin, Guanin, Cytosin oder Thymin bedeuten und die Symbole R jeweils unabhängig voneinander dieselbe oder unterschiedliche Bedeutung haben und eine Fmoc-Gruppe oder ein funktionelles Carbonsäurederivat, R¹ ein Wasserstoffatom oder ein funktionelles Carbonsäurederivat, a bis h ganze Zahlen von 0 bis 10, X₁ bis X₃, Y₁, Y₂ und Z₁ bis Z₅ jeweils ganze Zahlen von 0 oder mehr bedeuten, X₁ + X₂ + X₃ ≥ 0, Y₁ + Y₂ > 0 und Z₁ + Z₂ + Z₃ + Z₄ + Z₅ ≥ 0, mit der Maßgabe, dass X₁ + X₂ + X₃ und Z₁ + Z₂ + Z₃ + Z₄ + Z₅ nicht gleichzeitig 0 bedeuten, und für den Fall, dass X₁ + X₂ + X₃ = 0, R¹ ein funktionelles Carbonsäurederivat bedeutet.In the process according to the invention, which comprises a reaction between Fmoc-ω-amino acid ^Boc PNA-OH and a molecule with a PNA chain, compounds such as those of the following general formula II are preferably synthesized:

wherein the symbols B each independently have the same or different meaning and adenine, guanine, cytosine or thymine and the symbols R each independently have the same or different meaning and an Fmoc group or a functional carboxylic acid derivative, R ^{1 is} a hydrogen atom or a functional one Carboxylic acid derivative, a to h are integers from 0 to 10, X ₁ to X ₃ , Y ₁ , Y ₂ and Z ₁ to Z ₅ each represent integers from 0 or more, X ₁ + X ₂ + X ₃ ≥ 0, Y ₁ + Y ₂ > 0 and Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ ≥ 0, with the proviso that X ₁ + X ₂ + X ₃ and Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ does not simultaneously mean 0, and in the event that X ₁ + X ₂ + X ₃ = 0, R ¹ denotes a functional carboxylic acid derivative.

In the process of the invention, compounds of formula II are particularly preferably synthesized as compounds in which R is a carboxylic acid derivative of methyl red, X ₁ + X ₂ + X ₃ = 9, Y ₁ + Y ₂ = 1, Y ₁ = 3 and X ₂ = 6 or Y ₁ = 1 etc.

In addition, in the compounds of the above-mentioned general formula II, as examples of compounds in which a plurality of functional molecules are introduced, compounds are preferably synthesized in which R and R ^{1 are} cell membrane-permeable functional molecules. Such compounds are usually compounds in which R is a derivative of a cell membrane permeable functional molecule, etc., while R ^{1 is} a functional carabonic acid derivative of a photofunctional molecule, etc., and compounds in which the functional molecules are present at a variety of sites terminal sections is introduced and these functional molecules are given a variety of functions. Such connections can be shown schematically as indicated below.

Such compounds are compounds in which X ₁ = Z ₁ = Z ₂ = 1 and Y ₁ ≥ 2 in the aforementioned formula II.

These connections are in the future to be preferred to the ease of synthesis, the synthesis costs, etc.

Although there are no particular restrictions with regard to the above compounds, provided that a, b and f each represent an integer from 0 to 10, there are also in the case of connections in which e.g. a ≤ 6, b ≤ 4 and f ≤ 6, none Problems with synthesis or practicality.

The introduction of linker sites prevented if necessary, the interference between individual functional Sites and base sequence recognition sites while improving of reliability the molecular function. The expressions PNA, PNA monomer and PNA oligomer in the present application mean that they contain link sites at the ends and / or inside.

In addition to the linker sites above can f to h in general formula I can be selected as sites preventing mutual inference between the sites or domains serve.

Examples of groups that link sites form, are unbranched or branched hydrocarbons and their ether forms. In view of the ease of insertion and the cost point will be unbranched hydrocarbon groups and especially those with 1 to 6 carbon atoms are particularly preferred. Also are preferred ether forms in view of their universal usability.

Connections where the above Variety of functional molecules introduced are preferred, e.g. according to Koch T., Hansen H.F., Andersen P., Larsen T., Batz H.G., Otteson K. and Orum H .: Peoptide Res. 1997, 49, 80-88.

Base sequence recognition sites can be sequenced in oligomers by solid phase synthesis conventional PNA monomers can be converted. Commercially available Boc-7-aminoheptane or Boc-6-aminocaproic acid etc. can be used for linker sites.

The introduction of a photofunctional molecule as the only functional molecule allows the fluorescent label. It can but also compounds are synthesized that have other functions exhibit. Although the most diverse fluorescence emission wavelengths under Use of standard commercial products Fluorescent labeling compounds of the active ester type, such as by FITC, ROX, TAMRA and Dabcyl as fluorescent label sites chosen can be are the fluorescent labeling compounds that are introduced not limited to that.

An example of further, in the compounds of the invention insertable Functions is the membrane permeation function. These menopausal permeation function sites can on similar Manner using a compound of the general mentioned above Formula I introduced become. examples for functional molecules, which is the membrane permeability are arginine. It can but also preferred lysine, serine and other water-soluble amino acids be used.

Furthermore, it is also possible under Using the Fmoc amino acid unit a variety of amino acids introduce. examples for this synthesis is given in Examples 20 and 21. The above both connections are only model connections for one Fluorescence PNA probe with membrane permeation function. The present Invention is not limited to these two compounds.

The probes mentioned are thereby characterized that they are based solely on the PNA type and complete are enzyme resistant. In particular, the above probes include Membrane permeation function mainly those that have a covalent Binding of the PNA and a peptide chain or a phospholipid with membrane permeation function include. These probes have a better membrane permeation function on as soon as they enter a cell and it is expected that the peptide chain or the phospholipid is broken down by enzymes becomes. Their disadvantage is that a probe that is Degradation due to non-detection of the target is subjected to the washing stage is not complete can be removed.

Because the probe constructed here itself is not degraded enzymatically within the cells, a probe, which the target has not recognized is completely removed at the washing stage be what an accurate determination of the amount of the gene expressed allows.

It must be pointed out that in addition to the compounds with these functional properties organ-selective functional molecules like lactose and Tris-X, bactericidal functional molecules like tanatin and cecropin as well as molecule-recognizing functional molecules how viologen are also introduced according to the invention without restriction can and that such compounds practically in large quantities and at low Costs can be used.

EXAMPLES

The invention is exemplified below illustrated, the invention being not limited to these examples.

example 1

Synthesis of Fmoc-Gly- ^Boc PNA-OH (1)

A solution of Fmoc-Gly-OH (891 mg, 3.0 mmol) and PfpOH (754 mg, 4.5 mmol) in DMF (12 ml) was with DCC (845 mg, 4.5 mmol) at 0 ° C during 30 min and then at room temperature for 15 hours. The reaction mixture was then filtered to remove the DC urea, after which the filtrate was evaporated in vacuo to give the crude Fmoc-Gly-OPfp. A solution of Fmoc-Gly-OPfp and ^Boc PNA-OH (436 mg, 2.0 mmol) in DMF (16 ml) was added with diisopropylethylamine (445 µl, 2.6 mmol), after which the reaction mixture was at room temperature for 15 hours was stirred. The reaction mixture was then evaporated in vacuo and the residue was subjected to flush chromatography (0-50% MeOH / CH ₂ Cl ₂ ) to give Fmoc-Gly- ^Boc PNA-OH (121 mg, 12%). 1H NMR (DMSO · d ₆ ) δ 7.88 (d, J = 7.0 Hz, 2 H), 7.72 (d, J = 7.0 Hz, 2 H), 7.62 (brt) and 7.56 (brt) (1 H), 7.41 (t, J = 7.0 Hz, 2 H), 7.33 (t, J = 7.0 Hz, 2 H), 7.18 (m , 2 H), 6.85 (brt) and 6.79 (brt) (1 H), 4.35 - 4.15 (m, 3 H), 4.05 - 3.85 (m, 3 H) , 3.77 (m, 1H), 3.40-3.25 (m, 2H), 3.10 (m) and 3.03 (s) (2H), 1.37 (brs, 9H) ); ¹³ C NMR (DMSOd ₆ ) δ 172.2 (d), 169.10 (d), 156.34 (d), 155.58 (d), 143.83, 140.66, 127.58, 127.04, 125.24, 120.04, 77.77 (d), 65.71, 47.34 (d), 46.72, 46.65 (d), 29.23 (d), 28, 14 (d); FABMS m / z 498 [(M + H) ⁺ ].

Example 2

Synthesis of Fmoc-C ₇ -OPfp

DCC (392.0 mg, 1.9 mmol) was ice-cooled with a DMF solution (2.5 ml) of Fmoc-C ₇ -OH (381.9 mg, 1.0 mmol) and PfpOH (349.7 mg , 1.9 mmol) was added, after which the reaction mixture was stirred at 0 ° C. for 30 minutes and then at room temperature overnight. The reaction mixture was then filtered to remove the TLC urea, after which the filtrate was concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (CH ₂ Cl ₂ ). After concentration, the residue was recrystallized from hexanes, whereby Fmoc-C ₇ -OPfp was obtained as a white powder (537.5 mg, 98%). ¹ H NMR (CDCl ₃ ) x 7.76 (d, J = 7.6 Hz 2 H), 7.59 (d, J = 7.6 Hz, 2 H), 7.40 (t, J = 7.4 Hz, 2 H), 7.31 (t, J = 7.4 Hz, 2 H), 4.70 - 4.73 (brt, 1 H), 4.47 - 4.40 (brd, 2 H), 4.22 (t, J = 6.42 Hz, 1 H), 3.20 (q, J = 5.94 Hz, 2 H), 2.66 (t, J = 7.38 Hz , 2 H), 1.80 - 1.75 (m, 2 H), 1.55 - 1.50 (m, 2 H), 1.45 - 1.34 (m, 6 H); ¹³ C-NMR (CDCl ₃ ) x 169.44, 156.43, 143.98, 141.96 (m), 141.29, 140.23, 138.67 (m), 136.99 (m), 127.60, 126.96, 124.97, 119.91, 66.49, 55.73, 47.29, 41.34 (d), 34.89, 33.22, 29.85, 28.70 , 26.42, 25.43, 24.60; HRMS (FAB ⁺ ) calculated for C ₂₉ H ₂₇ F ₅ NO ₄ [(M + H) ⁺ ] 547.5131, found 548.1861.

Example 3

Synthesis of Fmoc-Gly- ^Boc PNA-OH (2)

A solution of NaHCO ₃ (67.2 mg, 0.8 mmol), H ₂ O (1.0 ml) and acetone (6.0 ml) was added to Fmoc-Gly-OPfp (240.9 mg 0.52 mmol) and ^Boc PNA-OH (87.3 mg, 0.4 mmol) was added, after which the reaction mixture was stirred at room temperature for 6 hours. The reaction mixture was then cooled to 0 ° C and adjusted to pH 3.0 using cooled 1N aqueous HCl. The solution, to which 1% aqueous citric acid was added, was then extracted with EtOAc, the combined organic layers washed with brine, dried over MgSO ₄ and then evaporated in vacuo. The residue was subjected to flush chromatography (1-5% MeOH / CH ₂ Cl ₂ ) to give the crude Fmoc-Gly- ^Boc PNA-OH (157.3 mg x 80%) as an amorphous white powder.

Example 4

Synthesis of Fmoc-β-Ala- ^Boc PNA-OH (1)

A solution of Fmoc-β-Ala-OH (311 mg, 1.0 mmol) and PfpOH (334 mg, 1.75 mmol) in DMF (2.5 ml) was treated with DCC (288 mg, 1.4 mmol) at 0 ° C for 30 min and then at room temperature for 15 hours. The reaction mixture was then filtered to remove the DC urea, the filtrate was evaporated in vacuo and the residue was subjected to flush chromatography (CH ₂ Cl ₂ ). The crude Fmoc-β-Ala-OPfp was recrystallized with hexane and CH ₂ Cl ₂ to give the crude Fmoc-β-Ala-OPfp (429 mg, 90%) as a white powder. A solution of Fmoc-β-Ala-OPfp (100 mg, 0.21 mmol) and ^Boc PNA-OH (41 mg, 0, 19 mmol) in DMF (2 ml) was treated with diisopropylethylamine (36 μl, 0.21 mmol ) was added, after which the reaction mixture was stirred at room temperature for 15 hours. The reaction mixture was then evaporated in vacuo and the residue was subjected to flush chromatography (0-10% MeOH / CH ₂ Cl ₂ ) to give Fmoc-Gly- ^Boc PNA-OH (41 mg, 42%). ¹ H NMR (DMSOd ₆ ) δ 7.88 (d, J = 7.4 Hz, 2 H), 7.68 (d, J = 7.4 Hz, 2 H), 7.41 (t, J = 7.3 Hz, 2H), 7.33 (t, J = 7.3 Hz, 2 H), 7.18 (m, 2 H), 6.83 (brt) and 6.72 (brt) (2 H), 4.3 - 4.2 (m, 4 H), 4.05 - 3.9 (m, 3 H), 3.33 (brt) and 3.29 (brt) (2H), 3.19 (m, 2 H), 3.07 (brq) and 3.02 (brq) (2 H), 1.36 (brs, 9 H), ¹³ C NMR (DMSOd ₆ ) δ 171, 20 (d), 170.85 (d), 155.93, 155.56, 143.87, 140.69, 127.55, 127.01, 125.09, 120.05, 77.73 (d), 65 , 30 (d), 59.69, 47.35 (d), 46.68, 46.49 (d), 37.99 (d), 36.72 (d), 2814 (d).

Example 5

Synthesis of Fmoc-β-Ala- ^Boc PNA-OH (2)

To a solution of NaHCO ₃ (92.4 mg, 1.1 mmol), H ₂ O (1.15 ml) and acetone (1.25 ml) were added Fmoc-β-OPfp (476.0 mg 1.0 mmol) and ^Boc PNA-OH (87.3 mg, 0.55 mmol) was added, after which the reaction mixture was stirred at room temperature for 6 hours. The reaction mixture was then cooled to 0 ° C and adjusted to pH 3.0 using cooled 1N aqueous HCl. The solution, to which 1% aqueous citric acid was added, was then extracted with EtOAc, after which the combined organic layers were washed with brine, dried over MgSO ₄ and then concentrated in vacuo were steamed. The residue was subjected to flush chromatography (1-5% MeOH / CH ₂ Cl ₂ ), evaporated in vacuo, dissolved in CH ₂ Cl ₂ and evaporated again in vacuo to give the crude Fmoc-β- ^Boc PNA-OH ( 225.9 mg x 80%) as an amorphous white powder.

Example 6

Synthesis of Fmoc-GABA- ^Boc PNA-OH (1)

A solution of Fmoc-GABA-OPfp (100 mg, 0.20 mmol) and ^Boc PNA-OH (40 mg, 0.18 mmol) in DMF (2 ml) was treated with diisopropylethylamine (34 μl, 0.20 mmol) after which the reaction mixture was stirred at room temperature for 15 hours.

The reaction mixture was then evaporated in vacuo and the residue was subjected to flush chromatography (0-20% MeOH / CH ₂ Cl ₂ ) to give Fmoc-GABA- ^Boc PNA-OH (43 mg, 45%). ¹ H NMR (DMSO · d ₆ ) δ 7.88 (d, J = 7.4 Hz, 2 H), 7.68 (d, J = 7.4, 2 H), 7.41 (t, J = 7.4 Hz, 2 H), 7, 33 (t, J = 7, 4 Hz, 2 H), 7, 29 (m, 1 H), 6, 82 (brt) and 6.71 (brt) (1 H), 4.3 - 4.2 (m, 4 H), 4.05 - 3.9 (m, 3 H), 3.35 - 3.25 (m, 2 H), 3.1 - 2.95 (m, 4H), 1.36 (brs, 9H); ¹³ C NMR (DMSO • d ₆ ) δ 172.2 (d), 171.5 (d), 156.03, 155.60 (d), 143.89, 140.68, 127.54, 127.00 , 125.50, 120.04, 77.70 (d), 65.19, 54.84, 47.89 (d), 46.97 (d), 46.72, 38.20 (d), 29 , 23 (d), 28.14 (d), 24.98 (d).

Example 7

Synthesis of Fmoc-GABA OPfp

DCC (248 mg, 1.2 mmol) was added while cooling with ice a DMF solution (2.5 ml) of Fmoc-GABA-OH (325 mg, 1.0 mmol) and PfpOH (221 mg, 1.2 mmol) after which the reaction mixture was stirred at 0 ° C for 30 minutes and then at room temperature for 15 minutes. The reaction mixture was then filtered off, after which the filtrate was concentrated under reduced pressure. The residue was then subjected to flush chromatography (CH ₂ Cl ₂ ) to give Fmoc-GABA-OPfp as a white powder (463 mg, 94%). ¹ H NMR (CDCDl ₃ ) δ 7.77 (d, J = 7.5 Hz, 2 H) 7.59 (d, J = 7.5 Hz, 2 H), 7.40 (t, J = 7 , 5 Hz, 2 H), 7, 31 (t, J = 7.5 Hz, 2 H), 4.85 (brs, 1 H), 4.45 (d, J = 6.3 Hz, 2 H) ), 4.21 (t, J = 6.3 Hz, 2 H), 3.32 (d, J = 6.5 Hz, 2 H), 2.71 (t, J = 6.5 Hz, 2H ), 1.98 (t, J = 6.5 Hz, 2H); ¹³ C NMRCDCl ₃ δ 169.02, 156.49, 143.83, 141.87 (m), 141.28, 140.23 (m), 138.61 (m) 136.94 (m) , 127.62, 127.46, 124.89, 119.88, 66.52, 47.25, 39.92, 30.42, 25.03.

Example 8

Synthesis of Fmoc-GABA- ^Boc PNA-OH (2)

A solution of NaHCO ₃ (67.2 mg, 0.8 mmol), H ₂ O (1.0 ml) and acetone (5.0 ml) was added to Fmoc-GABA-OPfp (255.5 mg, 0.52 mmol ) and ^Boc PNA-OH (87.3 mg, 0.4 mmol) were added, after which the reaction mixture was stirred at room temperature for 8 hours. The reaction mixture was then cooled to 0 ° C and adjusted to pH 3.0 using cooled 1N aqueous HCl. The solution, to which 1% aqueous citric acid was added, was then extracted with EtOAc, the combined organic layers washed with brine, dried over MgSO ₄ and then evaporated in vacuo. The residue was subjected to flush chromatography (1-5% MeOH / CH ₂ Cl ₂ ), evaporated in vacuo, dissolved in CH ₂ Cl ₂ and again evaporated in vacuo to give the crude Fmoc-GABA- ^Boc PNA-OH ( 175.9 mg · 84%) as an amorphous white powder.

Example 9

Synthesis of Fmoc-C ₄ - ^Boc PNA-OH

A solution of NaHCO ₃ (67.2 mg, 0.8 mmol), H ₂ O (1.0 ml) and acetone (4.0 ml) was added to Fmoc-C ₄ -OPfp (323.5 mg, 0.64 mmol) and ^Boc PNA-OH (87.3 mg, 0.4 mmol) were added, after which the reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was then cooled to 0 ° C and adjusted to pH 3.0 using cooled 1N aqueous HCl. The solution, to which 1% aqueous citric acid was added, was then extracted with EtOAc, the combined organic layers washed with brine, dried over MgSO ₄ and then evaporated in vacuo. The residue was subjected to flush chromatography (1-5% MeOH / CH ₂ Cl ₂ ), evaporated in vacuo, dissolved in CH ₂ Cl ₂ and again evaporated in vacuo to give the crude Fmoc-C ₄ - ^Boc PNA-OH (190.7 mg x 88%) as an amorphous white powder. ¹ H NMR (CDCl ₃ ) x 7.76 (d, J = 6.7, 2 H), 6.96 (mi) and 6.66 (ma) (brd, J = 6.7 Hz, 2 H) ), 7.41 - 7.37 (m, 2 H), 7, 32.7, 28 (m, 2 H), 7, 14 (ma) and 6.68 (mi) (m, 1 H), 5.54 (ma) and 5.43 (mi) (brt, 1 H) 4.45 (mi) and 4.37 (ma) (m, 2 H), 424 - 4.21 (m, 1 H) , 4.08 - 3.95 (m, 2 H) 3.54 - 3.48 (m, 2 H), 3.29 - 3.11 (m, 4 H), 2.43 - 2.25 ( m, 2H), 1.70-1.29 (m, 13H); ¹³ C NMR (CDCl ₃ ) x 174.39, 173.07, 171.95, 157.51, 156.79 (d), 156.11, 144.06 (d), 141.16, 127.46 (d), 126.90 (d), 119.77 (d), 81.42, 79.67, 66.39 (d), 53.35, 49.46 (d), 49.17, 48, 60, 47.15 (d), 40.89, 40.32 (d), 38.68, 31.87, 31.41, 29.59, 29.11 (d), 28.28, 21.77 (d); HRMS (FAB ⁺ ) calcd for C ₂₉ H ₃₇ N ₃ O ₇ [(M + H) ⁺ ] 539, 2632, found. 540.2707.

Example 10

Synthesis of Fmoc-C ₅ - ^boc PNA-OH

A solution of NaHCO ₃ (67.2 mg, 0.8 mmol), H ₂ O (1.0 ml) and acetone (7.5 ml) was added to Fmoc-C ₅ -OPfp (311.0 mg, 0.6 mmol) and ^Boc PNA-OH (87.3 mg, 0.4 mmol) were added, after which the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was then cooled to 0 ° C and adjusted to pH 3.0 using cooled 1N aqueous HCl. The solution, to which 1% aqueous citric acid was added, was then extracted with EtOAc, the combined organic layers washed with brine, dried over MgSO ₄ and then evaporated in vacuo. The residue was subjected to flush chromatography (1-5% MeOH / CH ₂ Cl ₂ ), evaporated in vacuo, dissolved in CH ₂ Cl ₂ and evaporated again in vacuo to give the crude Fmoc-C ₅ - ^Boc PNA-OH (198.0 mg, 90%) as an amorphous white powder. ¹ H NMR (DMSO-d ₆ ) x 7.88 (d, J = 7.4 Hz, 2 H), 7.68 (d, J = 7.2 Hz, 2 H), 7.41 (t , J = 7.4 Hz, 2 H), 7.32 (t, J = 7.4 Hz, 2 H), 7.22 (brt, 1 H), 6.81 (ma) and 6.67 ( mi) (brt, 1 H), 4.33 (mi) and 4, 29 (ma) (brd, 2 H), 4.20 (t, J = 7, 1 Hz, 1 H), 4, 08 ( mi) and 3, 90 (ma) (brs, 2 H), 3.09 - 2.94 (m, 4 H), 2.30 (ma) and 2.14 (mi) (brt, 2 H), 1.51-1.45 (m, 2H), 1.41-1.31 (brs, 11H), 1.29-1.21 (m, 8H); ¹³ C NMR (CDCl ₃ ) x 175.1 (d), 172.27 (d), 157.20 (t), 156.63, 144.43 (d), 141.69, 128.07, 127 , 45, 125.40 (d), 120.35, 81.71, 80.00, 67.36 (d), 50.42, 49.81 (d), 48.90 (d), 47.82 (d), 41.77, 41.16, 40.64, 39.19, 33.12, 32.75, 29.45 (d), 28.81, 26.59, 24.97, 24.70 ; HRMS (FAB ⁺ ), calc. For C ₃₀ H ₃₉ N ₃ O ₇ [(M + H) ⁺ ] 553.2788, found. 554.2873.

Example 11

Synthesis of Fmoc-C ₆ - ^Boc PNA-OH

A solution of NaHCO ₃ (67.2 mg, 0.8 mmol), H ₂ O (1.0 ml) and acetone (6.0 ml) was added to Fmoc-C ₆ -OPfp (331.9 mg, 0.6 mmol) and ^Boc PNA-OH (87.3 mg, 0.4 mmol) were added, after which the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was then cooled to 0 ° C and adjusted to pH 3.0 using cooled 1N aqueous HCl. The solution, to which 1% aqueous citric acid was added, was then extracted with EtOAc, the combined organic layers washed with brine, dried over MgSO ₄ and then evaporated in vacuo. The residue was subjected to flush chromatography (1-5% MeOH / CH ₂ Cl ₂ ), evaporated in vacuo, dissolved in CH ₂ Cl ₂ and evaporated again in vacuo to give the crude Fmoc-C ₆ - ^Boc PNA-OH (197.0 mg, 87%) as an amorphous white powder. ¹ H NMR (DMDO-d ₆ ) x 7.88 (d, J = 7.7 Hz, 2 H), 7.68 (ma) and 7.63 (mi) (brd, J = 7.4 Hz , 2 H), 7.40 (t, J = 7.4 Hz, 2 H), 7, 32 (t, J = 7, 4 Hz, 2 H), 7, 22 (brt, 1 H), 6 , 79 (ma) and 6.66 (mi) (brt, 1 H), 4.39 (mi) and 4.29 (ma) (brd, 2 H), 4.20 (brt, J = 6.7 Hz, 1 H), 4.08 (mi) and 3.91 (ma) (brs, 2 H), 3.10 - 2.97 (m, 4 H), 2.31 (ma) and 2, 15 (mi) (brt, 2H), 1.50-1.47 (m, 2H), 1.41-1.36 (m, 11H), 1.28-1.24 (brd, 6H) ); ¹³ C NMR (CDCl ₃ ) x 175.23 (d), 142.41 (d), 157.11 (d), 156.60, 144.34 (d), 141.69, 128.07, 127 , 45, 125.40 (d), 120.35, 81.68, 80.00 (d), 67.72, 67.50 (d), 53.87, 50.77, 50.14 (d) , 48.90, 47.82 (d), 41.29 (d), 41.36, 40.69, 39.18, 33.19, 32.96, 30.00, 29.11, 28.81 , 26.75 (d), 25.27 (d), 24.80 (d); HRMS (FAB ⁺ ) calcd for C ₂₉ H ₄₁ N ₃ O ₇ [(M + H) ⁺ ] 567.2945, found. 568.3027.

Example 12

Synthesis of Fmoc-C ₇ - ^Boc PNA-OH

A solution of NaHCO ₃ (67.2 mg, 0.8 mmol), H ₂ O (1.0 ml) and acetone (7.0 ml) was added to Fmoc-C ₇ -OPfp (328.5 mg, 0.6 mmol) and ^Boc PNA-OH (87.3 mg, 0.4 mmol) were added, after which the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was then cooled to 0 ° C and adjusted to pH 3.0 using cooled 1N aqueous HCl. The solution, to which 1% aqueous citric acid was added, was then extracted with EtOAc, after which the combined organic layers were washed with brine, dried over MgSO ₄ and then in vacuo were evaporated. The residue was subjected to flush chromatography (1-5% MeOH / CH ₂ Cl ₂ ) to give the crude Fmoc-C ₇ - ^Boc PNA-OH (196.1 mg, 84%) as an amorphous white powder. ¹ H NMR (DMDO-d ₆ ) x 7.88 (d, J = 7.7 Hz, 2 H), 7.68 (ma) and 7.63 (mi) (brd, J = 7.4 Hz , 2 H), 7.40 (t, J = 7.4 Hz, 2 H), 7.32 (t, J = 7.4 Hz, 2 H), 7.22 (brt, 1 H), 6 , 79 (ma) and 6.79 (mi) (brt, 1 H), 4.39 (mi) and 4.29 (ma) (brd, J = 6.9 Hz, 2 H), 4.05 ( t, J = 6.7 Hz, 1H), 4.08 (mi) and 3.91 (ma) (brs, 2H), 3.12-2.95 (m, 4H), 2.31 (mi) and 2.15 (ma) (brt, 2 H), 1.50 - 1.47 (m, 2 H), 1.42 - 1.34 (m, 11 H), 1.25 (brd , 2 H); ¹³ C NMR (CDCl ₃ ) x 174.72, 172.19, 156.52, 156.05, 143.78 (d), 141.14, 127.51, 126.89, 124.86 (d) , 119.79, 79.43 (d), 66.80 (d), 53.33, 50.19, 49.20, 48.50, 47.14 (d), 41.18 (d), 38 , 60, 32.28 (d), 29.60, 28.83, 28.26, 26.27 (d), 24.68 (d), 21.77 (d); HRMS (FAB ⁺ ) calc. For C ₃₂ H ₄₃ N ₃ O ₇ [(M + H) ⁺ ] 581.3101, found. 582.3171.

Example 13

Synthesis of Fmoc-C ₁₀ - ^Boc PNA-OH

To a solution of NaHCO ₃ (67.2 mg, 0.8 mmol), H ₂ O (1.0 ml) and acetone (7.0 ml) was added Fmoc-C ₁₀ -OPfp (353, 7 mg, 0.6 mmol) and ^Boc PNA-OH (87.3 mg, 0.4 mmol) were added, after which the reaction mixture was stirred at room temperature for 24 hours.

The reaction mixture was then cooled to 0 ° C and adjusted to pH 3.0 using cooled 1N aqueous HCl. The solution, to which 1% aqueous citric acid was added, was then extracted with EtOAc, the combined organic layers washed with brine, dried over MgSO ₄ and then evaporated in vacuo. The residue was subjected to flush chromatography (1-5% MeOH / CH ₂ Cl ₂ ), evaporated in vacuo, dissolved in CH ₂ Cl ₂ and evaporated again in vacuo to give the crude Fmoc-C ₁₀ - ^Boc PNA-OH (218.5 mg, 88%) as an amorphous white powder. ¹ H NMR (CDCl ₃ ) * 9.60 (brs, 1 H), 7.73 (d, J = 7, 6 Hz, 2 H), 7, 58 (d, J = 6, 8 Hz, 2 H), 7.37 (t, J = 6, 8 Hz, 2 H), 7, 29 (t, J = 7, 2 Hz, 2 H), 5.52 (ma) and 5.35 (mi) (brd, 1 H), 5.00 (s, 1 H), 4.45 (mi) and 4.40 (ma) (brd, J = 6.4 Hz, 2 H), 4.23 - 4, 22 (m, 1 H), 4.09 (mi) and 4.04 (ma) (brs, 2 H), 3.57 - 3.46 (m, 2 H), 3.29 - 3.03 ( m, 4H), 1.66-1.58 (brs, 2H), 1.52-1.37 (m, 11H), 1.33-1.20 (brs, 12H); ¹³ C NMR (CDCl ₃ ) x 174.5 (d), 172.43 (d), 171.64 (d), 157.83 (d), 156.98, 156.03, 143.78 (d ), 141.17, 127.52, 126.90, 124.86 (d), 119.70 (d), 81.08, 79.43 (d), 67.26, 66.43, 50.14 , 49.29, 48.145 (d), 47.17 (t), 41.47, 40.99, 40.16, 38.63, 32.90, 32.43 (d), 29.55 (d) 29.22 (m), 28.28, 26.56 (d), 24.98, 24.75; HRMS (FAB ⁺ ) calc. For C ₃₅ H ₄₉ N ₃ O ₇ [(M + H) ⁺ ] 623.3571, found. 624.3643.

Example 14

Synthesis of Fmoc-C ₁₁ -OPfp

DCC (309.5 mg, 3.0 mmol) was ice-cooled with a DMF solution (2.5 ml) of Fmoc-C ₁₁ -OH (437.5 mg, 2.0 mmol) and PfpOH (276.6 mg , 3.0 mmol) was added, after which the reaction mixture was stirred for 30 minutes at 0 ° C. and then at room temperature for 18 hours. The reaction mixture was then filtered to remove the DC urea, after which the filtrate was concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (CH ₂ Cl ₂ ). After concentration, the residue was recrystallized from hexanes, whereby Fmoc-C ₁₁ -OPfp was obtained as a white powder (575.6 mg, 96%). ¹ H NMR (CDCl ₃ ) δ 7.79 (d, J = 7.6 Hz, 2 H), 7.63 (d, J = 7.2 Hz, 2 H), 7.43 (t, J = 7.6 Hz, 2 H), 7.34 (t, J = 7.2 Hz, 2 H), 4.86 (brt, 1 H), 4.47 (mi) and 4.44 (ma) (brd, 2 H), 4.25 (t, 1 H), 3.22 (q, J = 6.1 Hz, 2 H), 2.68 (t, J = 7.2 Hz, 2 H) , 1.80 (m, 2H), 1.56-1.52 (m, 2H), 1.47-1.42 (m, 2H), 1.39-1.30 (m, 12th H); HRMS (FAB ⁺ ) calc. For C ₃₃ H ₃₄ F ₅ NO ₄ [(M + H) ⁺ ] 603.6194, found. 604.2490.

Example 15

Synthesis of Fmoc-C ₁₁ - ^Boc PNA-OH

To a solution of NaHCO ₃ (67.2 mg, 0.8 mmol), H ₂ O (1.0 ml) and acetone (10 ml) was added Fmoc-C ₁₁ -OPfp (362.2 mg, 0.6 mmol) and ^Boc PNA-OH (87.3 mg, 0.4 mmol) was added, after which the reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was then cooled to 0 ° C and adjusted to pH 3.0 using cooled 1N aqueous HCl. The solution, to which 1% aqueous citric acid was added, was then extracted with EtOAc, the combined organic layers washed with brine, dried over MgSO ₄ and then evaporated in vacuo. The residue was subjected to flush chromatography (1-5% MeOH / CH ₂ Cl ₂ ), evaporated in vacuo, dissolved in CH ₂ Cl ₂ and evaporated again in vacuo to give the crude Fmoc-C ₁₁ - ^Boc PNA-OH (227, 6 mg, 89%) as an amorphous white powder. ¹ H NMR (CDCl ₃ ) 9.62 (brs, 1 H), 7.74 (d, J = 7.6 Hz, 2 H), 7.57 (d, J = 7.5 Hz, 2 H), 7.37 (t, J = 7.1 Hz, 2 H) , 7.28 (t, J = 6.8 Hz, 2 H), 5.53 (ma) and 5.35 (mi) (brs, H), 5.00 (brt, 1 H), 4.43 (mi) and 4.37 (ma) (brd, J = 6.4 Hz, 2 H), 4.22 - 4.19 (m, 1 H), 3.23 - 3.08 (m, 4 H) ), 2.36 (ma) and 2.21 (mi) (brt, J = 7.0 Hz, 2 H), 1.69 - 1.58 (brs, 2 H), 1.52 - 1.40 (m, 11H), 1.29-1.25 (brd, 14H); ¹³ C NMR (CDCl ₃ ) x 175.45 (d), 172.428 (d), 157.91 (d), 157.01, 156.57, 144.33 (d), 141.72, 128.05 , 127.43, 125.40 (d), 120.35, 81.63, 80.00 (d), 67.77, 66.97, 50.67, 49.84, 49.20 (d), 47.72 (t), 42.10, 41.53, 40.70, 39.18, 33.51, 33.07, 30.03, 29.83 (m), 28.82, 27.00 ( d), 25.44, 25, 35; HRMS (FAB ⁺ ) calcd for C ₃₆ H ₅₁ N ₃ O ₇ [(M + H) ⁺ ] 637.3727, found. 638.3794.

Example 16

Synthesis of PNA oligomer 1 (H ₂ NGATp ·· -GACGC-CONH ₂ )

(Method 1)

According to the solid-phase tBoc method described by Koch et al. (Koch T., Hansen HF, Andersen P., Larsen T., Batz HG, Otteson K., ⌀rum HJ, Peptide Res. 1997, 49, 80-88) were first successive condensation reactions with the solid phase carrier MBHA (50 mg ) with different types of PNA monomer units with a PNA precursor monomer unit Fmoc-Gly- ^Boc PNA-OH (each 20 μmol) as well as HBTU (7.6 mg, 20 μmol) and DIEA (7.0 μl, 20 μmol) performed as a condensing agent. After the condensation of the Fmoc-Gly- ^Boc PNA-OH, the protective group was removed from the Fmoc group by piperidine treatment (20% piperidine in DMF, room temperature, 30 min), after which the photoactive carboxylic acid derivative p-methyl red (10.8 mg, 40 μmol) was condensed on the amino group obtained using HBTU (15.2 mg, 40 μmol) and DIEA (7.0 μl, 40 μmol) as the condensing agent, as a result of which the photoactive molecule was incorporated at the target site. After the successive condensation reactions using thymine, adenine and guanine PNA monomer units had been completed, the cleavage from the solid phase support and the cleavage of the protective group from the Z group were carried out using the cleavage reagent TFA / TFMSA / p-cresol / thioanisole ( 60/25/10/10), after which the target PNA oligomer 1 was aftertreated. UV λmax (H ₂ O) 303, 548 (nm).

Example 17

Synthesis of PNA oligomer 1 (H ₂ NGATp ·· -GACGC-CONH ₂ )

(Method 2)

According to the solid-phase tBoc method described by Koch et al. (Koch T., Hansen HF, Andersen P., Larsen T, Batz HG, Otteson K., ⌀rum HJ, Peptide Res. 1997, 49, 80-88) were first successive condensation reactions with the solid phase carrier MBHA (50 mg) with different types of PNA monomer units with a PNA precursor monomer unit Fmoc-Gly- ^Boc PNA-OH (each 20 μmol) and HBTU (7.6 mg, 20 μmol) as a condensing agent. After the successive condensation of all PNA units, the protective group was removed from the Fmoc group by piperidine treatment (20% piperidine in DMF, room temperature, 30 min), after which the photoactive carboxylic acid derivative p-methyl red (10.8 mg, 40 μmol ) was condensed on the amino group obtained using HBTU (15.2 mg, 40 μmol) and DIEA (13.9 μl, 40 μmol) as the condensing agent, as a result of which the photoactive molecule was incorporated at the target site. Finally, the cleavage from the solid phase support and the cleavage of the protective group of the Z group were carried out using the cleavage reagent TFA / TFMSA / p-cresol / thioanisole (60/25/10/10) at the same time, after which the target PNA oligomer 2 was aftertreated , UV λmax (H ₂ O) 302, 550 (nm).

Example 18

Synthesis of PNA Oligomer 2 (H ₂ NGATm · -GACGC-CONH ₂ )

(Method 2)

According to the solid-phase tBoc method described by Koch et al. (Koch T., Hansen HF, Andersen P., Larsen T., Batz HG, Otteson K., ⌀rum HJ, Peptide Res. 1997, 49, 80-88) were first successive condensation reactions with the solid phase carrier MBHA (50 mg ) with different types of PNA monomer units with a PNA precursor monomer unit Fmoc-Gly- ^Boc PNA-OH (each 20 μmol) as well as HBTU (7.6 mg, 20 μmol) and DIEA (7.0 μl, 40 μmol) as a condensing agent leads. After the successive condensation of all PNA units, the protective group was removed from the Fmoc group by piperidine treatment (20% piperidine in DMF, room temperature, 30 min), after which the photoactive carboxylic acid derivative m-methyl red (10.8 mg, 40 μmol) was added the obtained amino group was condensed with HBTU (15.2 mg, 40 μmol) and DIEA (13.9 μl, 40 μmol) as a condensing agent, whereby the photoactive molecule was incorporated at the target site. Finally, the cleavage from the solid phase support and the cleavage of the protective group of the Z group were carried out using the cleavage reagent TFA / TFMSA / p-cresol / thioanisole (60/25/10/10) at the same time, after which the target PNA oligomer 2 was aftertreated , UV λmax (H ₂ O) 308, 570 (nm).

Example 19

Synthesis of PNA oligomer 3 (H ₂ NGATo ·· -GACGC-CONH ₂ )

(Method 2)

According to the solid-phase tBoc method described by Koch et al. (Koch T., Hansen HF, Andersen P., Larsen T, Batz HG, Otteson K., ⌀rum HJ, Peptide Res. 1997, 49, 80-88) were first successive condensation reactions with the solid phase carrier MBHA (50 mg) with different types of PNA monomer units with a PNA precursor monomer unit Fmoc-Gly- ^Boc PNA-OH (each 20 μmol) as well as HBTU (7.6 mg, 20 μmol) and DIEA (7.0 μl, 40 μmol) as Condensing agent carried out. After the successive condensation of all PNA units, the protective group was removed from the Fmoc group by piperidine treatment (20% piperidine in DMF, room temperature, 30 min), after which the photoactive carboxylic acid derivative o-methyl red (10.8 mg, 40 μmol) was added the obtained amino group was condensed with HBTU (15.2 mg, 40 μmol) and DIEA (13.9 μl, 40 μmol) as a condensing agent, whereby the photoactive molecule was incorporated at the target site. Finally, the cleavage from the solid phase support and the cleavage of the protective group of the Z group were carried out using the cleavage reagent TFA / TFMSA / p-cresol / thioanisole (60/25/10/10) at the same time, after which the target PNA oligomer was obtained 3 treated. UV λmax (H ₂ O) 302, 561 (nm).

Example 20 Synthesis of the fluorescence PNA probe 1 incorporating the membrane permeability function

The connection 31 was after the Method 2 described above synthesized.

Corresponding to the one described above Solid phase tBoc method according to Koch et al. (Koch T., Hansen H.F., Andersen P., Larsen T., Batz H.G., Otteson K., ⌀rum H.J., Peptide Res. 1997, 49, 80-88) was first a condensation reaction with the solid phase support MBHA (50 mg) with the thymine PNA monomer unit (7.7 mg, 20 μmol) and HBTU (7.6 mg, 20 μmol) and DIEA (3.5 μl, 20 μmol) performed as a condensing agent. This process was repeated two more times (construction the base sequence recognition domain).

The ω-amino acid linker Boc-7-aminoheptanoic acid (5.2 mg, 20 μmol), the PNA precursor monomer unit Fmoc-C ₅ - ^Boc PNA-OH (10.0 mg, 20 μmol) and then again Boc- 7-aminoheptanoic acid was successively condensed with HBTU (7.6 mg, 20 μmol) and DIEA (3.5 μl, 20 μmol) (construction of the linker site and the functional domain having membrane permeability).

After the successive condensation all The Fmoc group became a protective group by piperidine treatment (20% piperidine in DMF, room temperature, 3 min). The functional Carboxylic acid derivative Fmoc-Arg (Mts) -OH (23.1 mg, 40 mmol) was then treated with HBTU (15.2 mg, 40 mmol) and DIEA (7.0 μl, 40 mmol) as a condensing agent, which makes the functional molecule introduced at the destination (Post-synthetic introduction membrane permeability).

After splitting off the protecting group from the Boc group by TFA treatment (95% TFA / 5% m-cresol) was treated with FITC (9.3 mg, 25 mmol) in the presence of DIEA (17.4 μl, 100 mmol) by shaking at Room temperature during Fluorescent labeling of the PNA oligomer was carried out for 12 hours.

Finally, after the protective group had been split off from the rest of the Fmoc group using piperidine (20% piperidine in DMF, room temperature, 3 min), the solid phase support was split off using the cleavage reagent TFA / TFMSA / p-cresol / thioanisole (60/25/10 / 10 /) was carried out, after which aftertreatment was carried out to obtain the target fluorescent PNA probe 1. MALDI-TOF MS. calcd. 2096.26 (m + H ⁺ ), found 2,096.36.

Example 21 Synthesis of the fluorescence PNA probe 2 incorporating the membrane permeability function

The implementation was the same Similar to compound 31 until the post-synthetic uptake of the functional molecule.

Corresponding to the one described above Solid phase tBoc method according to Koch et al. (Koch T., Hansen H.F., Andersen P., Larsen T., Batz H.G., Otteson K., ⌀rum H.J., Peptide Res. 1997, 49, 80-88) was first a condensation reaction with the solid phase support MBHA (50 mg) with the thymine PNA monomer unit (7.7 mg, 20 μmol) and HBTU (7.6 mg, 20 μmol) and DIEA (3.5 μl, 20 μmol) performed as a condensing agent. This process was repeated two more times (construction the base sequence recognition domain).

The ω-amino acid linker Boc-7-aminoheptanoic acid (5.2 mg, 20 μmol), the PNA precursor monomer unit Fmoc-C ₅ - ^Boc PNA-OH (10.0 mg, 20 μmol) and then again Boc- 7-aminoheptanoic acid was successively condensed with HBTU (7.6 mg, 20 μmol) and DIEA (3.5 μl, 20 μmol) (construction of the linker site and the functional domain having membrane permeability).

After the successive condensation all Units became the protective group of the Fmoc group through piperidine treatment (20% piperidine in DMF, room temperature, 3 min). The functional Carboxylic acid derivative Fmoc-Arg (Mts) -OH (23.1 mg, 40 mmol) was then treated with HBTU (15.2 mg, 40 mmol) and DIEA (7.0 μl, 40 mmol) as a condensing agent, which makes the functional molecule introduced at the destination (Post-synthetic introduction membrane permeability).

After re-treatment with piperidine (50% piperidine in DMF, room temperature, 3 min) to split off the Protecting group from the Fmoc group was Fmoc-Arg (Mts) -OH (23.1 mg, 40 μmol) with HBTU (15.2 mg, 40 μmol) and DIEA (7.0 μl, 40 μmol) condensed as a condensing agent (additional inclusion of the membrane permeability function).

After splitting off the protecting group from the Boc group by TFA treatment (95% TFA / 5% m-cresol) was treated with FITC (9.3 mg, 25 mmol) in the presence of DIEA (17.4 μl, 100 mmol) by shaking at Room temperature during Fluorescent labeling of the PNA oligomer was carried out for 12 hours.

Finally, after the protective group had been split off from the rest of the Fmoc group using piperidine (20% piperidine in DMF, room temperature, 3 min), the solid phase support was split off using the cleavage reagent TFA / TFMSA / p-cresol / thioanisole (60/25/10 / 10 /), after which post-treatment was carried out to obtain the target fluorescent PNA probe 1. MALDI-TOF MS. Calc. 2252.44 (m + H ⁺ ), found 2252.33.

IMPORTANCE THE INVENTION

According to the invention, many and different functional molecules into the PNA without restriction on photofunctional molecules easily introduced and many and different functional molecules can be effective and easily inserted into the PNA what enables the construction of a number of PNA those in gene therapy and for other purposes can be used.

The present examples and designs thus illustrate, but do not limit, the invention the invention is not limited to the details described here, but can be in the frame and equivalent to the attached claims be modified.

SHORT DESCRIPTION THE DRAWINGS

The invention, together with the objects and advantages, can be best understood from the present description of the preferred embodiments together with the accompanying drawings become. Show:

1 - The differences in structure and state of charge between DNA and PNA and

2 - the structures of two types of PNA monomer units.

Claims (28)

Process for producing a functional PNA oligomer, in which a PNA monomer unit with adenine, guanine, cytosine or thymine, protected by a protective group, is reacted with Fmoc-ω-amino acid- ^Boc PNA-OH and, after synthesis of the PNA oligomer, is a functional one Molecule with a free carboxylic acid is introduced into the PNA oligomer, after which the protective group is removed again. A method according to claim 1, in which Fmoc-ω-amino acid- ^Boc -PNA-OH is produced by reacting Fmoc-ω-amino acid pentafluorophenyl ester with ^Boc PNA-OH. The method of claim 2, wherein the Fmoc-ω-amino acid pentafluorophenyl ester by converting Fmoc-ω-amino acid and Pentafluorophenol is produced. Method according to one of claims 1 to 3, in which after the introduction of a functional molecule another functional molecule introduced becomes. Method according to one of claims 1 to 4, wherein the functional introduced Mülekül is selected under a photofunctional molecule, a membrane permeable functional molecule, an organ-selective functional molecule, a bactericidal functional molecule molecule and a molecule-recognizing functional Molecule. Method according to one of claims 4 or 5, wherein the functional introduced molecule under a photofunctional molecule and a membrane permeable functional molecule selected becomes. The method of claim 6, wherein the photofunctional molecule FITC, ROX, TAMRA or Dabcyl and the membrane is meable functional molecule a water soluble amino acid is. Method according to one of claims 1 to 7, wherein the Adenine, guanine, cytosine or thymine protecting group a group Z is. Method according to one of claims 1 to 8, wherein the synthesis of the PNA oligomer condensation and extension in a PNA chain using a solid phase support for the tBoc process. Method according to one of claims 1 to 9, wherein the solid phase carrier for the tBoc method MBHA is. Method according to one of claims 1 to 10, wherein the introduction of a functional molecule with free carboxylic acid by dehydrogenation condensation with a primary amino group which by selective removal of the protecting group from the Fmoc group is obtained by piperidine treatment. Process according to one of claims 1 to 11, in which the Fmoc-ω-amino acid ^Boc PNA-OH is a compound of the following general formula I:

where n is an integer from 1 to 15. A process according to claim 3, wherein the process comprises one or more of the following steps a) to d): a) reaction of the Fmoc-ω-amino acid with pentafluorophenol at a step on which Fmoc-ω-amino acid pentafluorophenyl ester is produced, b) introduction the Fmoc-ω-amino acid in ^Boc PNA-OH by converting the Fmoc-ω-amino acid pentafluor phenyl esters with ^Boc PNA-OH on a stage at which Fmoc-ω-amino acid ^Boc PNA-OH is generated; c) Generation of the PNA oligomer by reaction of a PNA monomer unit with Fmoc-ω-amino acid- ^Boc PNA-OH at a stage at which Fmoc-ω-amino acid- ^Boc is used to produce PNA-OH PNA-oligomer and d) implementation the introduction of a functional molecule into the PNA oligomer by dehydrogenation condensation of a primary amino group, which is obtained by selective removal of a protective group from an Fmoc group by piperidine treatment at a stage at which a functional PNA oligomer is produced from the PNA oligomer. Compound of the following general formula I:

where n is an integer from 1 to 15. Process for producing a compound of general formula I:

where n is an integer from 1 to 15, which comprises introducing the Fmoc-ω-amino acid by reacting the Fmoc-ω-amino acid with pentafluorophenol and reacting the reaction product with ^Boc PNA-OH. Compound of the general formula II

wherein the symbols B each independently have the same or different meaning and adenine, guanine, cytosine or thymine and the symbols R each independently have the same or different meaning and an Fmoc group or a functional carboxylic acid derivative, R ^{1 is} a hydrogen atom or a functional one Carboxylic acid derivative, a to h are integers from 0 to 10, X ₁ to X ₃ , Y ₁ , Y ₂ and Z ₁ to Z ₅ each represent integers from 0 or more, X ₁ + X ₂ + X ₃ ≥ 0, Y ₁ + Y ₂ > 0 and Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ ≥ 0, with the proviso that X ₁ + X ₂ + X ₃ and Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ does not simultaneously mean O, and in the event that X ₁ + X ₂ + X ₃ = 0, R ¹ denotes a functional carboxylic acid derivative. A compound according to claim 16, wherein Z ₁ + Z ₂ + Z ₃ + Z ₄ + Z ₅ = 0 and R ^{1 represents} a hydrogen atom. A compound according to claim 17, wherein R is a carboxylic acid derivative of methyl red. A compound according to claim 18, wherein X ₁ + X ₂ + X ₃ = 9 and Y ₁ + Y ₂ = 1. A compound according to claim 19, wherein X ₁ = 3, X ₂ = 6 and Y ₁ = 1. A compound according to claim 16, wherein R or R ^{1 is} a cell membrane permeable functional molecular derivative. A compound according to claim 21, wherein R ^{1 is} a functional carboxylic acid derivative. A compound according to claim 21 or 22, wherein X ₁ = Z ₁ = 1. A compound according to any one of claims 21 to 23, in which Y ₁ ≥ 2 and Z ₂ = 1. A compound according to any one of claims 21 to 24, in which a ≤ 6, b ≤ 4 and f ≤ 6. A compound according to any one of claims 21 to 25, wherein R1 is one photofunctional carboxylic acid derivative represents. Compound of the following general formula III:

where n is an integer from 1 to 15. Process for producing a compound of general formula III:

where n is an integer from 1 to 15, in which the Fmoc-ω-amino acid is reacted with pentafluorophenol.