EP1427710A2 - Method for the production of cna - Google Patents

Abstract

The invention relates to a novel method for producing CNA oligomers and artificial supramolecular CNA-p-RNA pairing systems. The invention also relates to the use of the same, especially in biotechnological assays.

Description

Process for the preparation of CNA

description

The invention relates to a process for the production of CNA oligomers and polymers, as well as a process for the production of the CNA monomer building blocks required therefor, and the use of the CNA oligomers for the formation of artificial supramolecular pairing systems, in particular in biotechnological assays.

In recent years, many new procedures based on biochip technology have been established in the field of clinical diagnostics or in the field of pharmaceutical target and drug screening. So far, interest has mainly focused on DNA chips, e.g. B. to detect the presence of specific nucleic acid sequences in a sample by means of hybridization.

Based on this, the idea was obvious to use molecular pairing systems for the thermodynamically controllable generation of supramolecular structures for biotechnological assays, such as those used for. B. are described in WO99 / 15893. For this purpose, interesting chemical substances, such as. B. peptides or proteins fixed to nucleic acid derivatives with specific sequences. The desired supramolecular systems can then be generated intermediately by pairing with nucleic acid fragments with corresponding sequences. If fragments with a known sequence are attached to a surface as a receptor at a defined location, chemical substances attached to a fragment with a complementary sequence can even be addressed in a spatially resolved manner.

Naturally occurring nucleic acids, such as DNA and RNA, are, however, only suitable as addressable "carriers" in molecular self-organizing systems if approximately physiological conditions are adhered to due to the insufficient chemical stability of naturally occurring nucleic acids, e.g. B. against nucleases or basic or acidic conditions, on the other hand, their mating behavior by process-related factors, such as. B. influences the salt content and the temperature of the medium, which greatly limits their use in biotechnological assays.

For this reason, it has long been sought for artificial molecular pairing systems that are suitable for the construction of supramolecular architectures and at the same time tolerate a wide variety of assay conditions.

A pairing system that has been known for some time is p-RNA oligomers (Helv. Chim. Acta 1993, 76, 2161-2183, Helv. Chim. Acta 1995, 78, 1621-1635, Helv. Chim. Acta 1996, 79, 2316-2345 , Helv. Chim. Acta, 1997, 80, 1901-1951, Helv. Chim. Acta, 2000, 83, No. 6, 1079-1107). Because of their modified sugar-phosphate backbone, their specific interaction with biologically active Substances such as enzymes, DNA or RNA strands excluded, the sensitivity of the hybridization of complementary p-RNA oligomers z. B. compared to the salt concentration in a sample medium, however, remains a problem even when using p-RNAs.

Because of their uncharged peptide backbone, cyclohexyl or heterocyclohexyl nucleo-amide oligomers (CNA oligomers), as described in WO99 / 15509, represent a suitable alternative to the charged phosphate-containing pairing system. In particular, their use in assays in which it is not possible to work in a physiological medium, the use of such CNA pairing systems seems suitable. A disadvantage, however, is the strongly hydrophobic nature of the CNAs, which makes them usable in polar solvents, such as. B. water is restricted.

In addition, the production of CNA oligomers, as described in WO99 / 15509, has hitherto been difficult. In particular, the complex, multi-stage synthesis route, primarily due to a complex protective group strategy, and the reaction procedure under drastic conditions lead to low yields on CNA oligomers. In addition to the low yields, the CNA oligomers have so far not been able to be produced on a larger scale, which has made their use in biotechnological assays difficult to date. Furthermore, the production of the monomer units required for the synthesis of CNA oligomers is complex and possible with only average yields (WO99 / 15509), so that the availability of these starting materials also makes the technical use of CNA oligomer products difficult.

The task was now to provide a method for CNA oligomerization with which larger amounts of CNA oligomers are synthetically accessible and with which high yields of CNA oligomer can be achieved

The object can be achieved by a process for the preparation of CNA oligomers, which comprises the following process steps: a) activation of the carboxyl function of a CNA monomer block, b) subsequent esterification of the CNA monomer block with free hydroxyl groups of a support under basic conditions and c) subsequent structure of the Oligomers are solved via repetitive cycles, starting from the cleavage of the protective group on the amino group of the cyclohexyl ring of the CNA monomer and subsequent attachment of a further activated CNA monomer, using CNA monomers of the formula (I),

NHBoc formula (I)

wherein A, D and F can independently represent a -CR ³ R ⁴ -, -NR ⁵ -, -O- or -S- group and E for a -CR ⁶ - group, where R ³ , R ⁴ , R ⁵ or R ⁶ independently of one another for a hydrogen atom or a C 1 -C ₂ alkyl group and where B is a nucleobase, preferably selected from the group adenine, guanine, cytosine, thymine, uracil, isoguanine, isocytosine, xanthine or hypoxanthine, whose primary amino groups can be in unprotected or Boc-protected form. The groups A, D and F preferably represent a carbon atom and the radicals R ⁴ -R ^{6 represent} hydrogen atoms. Heterocyclic compounds of the formula (I) preferably have an oxygen or a sulfur atom or one or two at positions A, D and F nitrogen atoms.

Surprisingly, in contrast to the conventional DNA or RNA oligomerization processes, the CNA oligomerization described here can be carried out using only Boc-protected as well as already base-unprotected CNA monomers. As a result, the final process step for deprotection, usually by treatment of the pMBz-protected oligomers with the addition of 2M NaOH at 55 ° C. for several hours, can be avoided (WO99 / 15509). The high yield losses resulting from fragmentation under the harsh pMBz deprotection conditions can thus be avoided. In contrast, the present production method for CNA oligomers according to the invention proceeds with approximately 100% yield. Since each coupling step proceeds quantitatively, there are no intermediate free cyclohexylamino groups at the end of a synthesis cycle, which also makes masking such wear sequences unnecessary. This also enables the synthesis of longer CNA oligomers in larger quantities.

In a preferred embodiment, the synthesis of the CNA oligomers from the corresponding monomer units on a solid phase, e.g. B. a Tentagel resin (Tentagel S-HMB, Rapp polymers), carried out based on the Merrifield peptide synthesis (Merrifield, J. Amer. Chem. Soc. 1963, 85, 2149). For this, one with a linker, such as. B. a hydroxymethylbenzoyl linker (HMB linker), derivatized solid phase and the loading of the carrier by activating the carboxyl function of one of the monomer units, for. B. with HATU (N - [(dimethylamino) (3H-1, 2,3-triazolo (4,5b) pyridin-3-yloxy) methylene] - N-methylmethanaminium hexafluophosphate), DIC, TBTU or HBTU and subsequent connection to the free hydroxyl function of the polymer carrier under basic conditions. The subsequent build-up of the oligomer now takes place via repetitive cycles, which consist of the elimination of a temporary Boc protective group, the amine function of the monomer unit covalently bonded to the support on the cyclohexyl ring and subsequent attachment of a monomer activated in solution. The preferred coupling times are between 3 to 6 hours. A synthesis scheme is shown in Table 1 as an example.

Table 1: Scheme of a synthesis cycle for the production of CNA oligomers

As already mentioned, one advantage of this synthesis lies in the monomer units that can be used to construct a CNA oligomer. It is therefore advantageous to use the monomer units on the nucleobases only in a Boc-protected or even completely unprotected manner. In the case of the guanine building block, the 2-amino function of the nucleobase Boc is preferably used in a protected manner. The cytosine building block is preferably used on the nucleobase N ⁴ -Boc-protected, the adenine building block is preferably used on the nucleobase both N ⁶ -Boc-protected and unprotected, and the thymine building block is preferably used on the nucleobase N ³ - Boc protected as well as unprotected used for oligomerization. In all cases in which the monomer component is Boc-protected, the Boc protective group is removed again directly during the Boc deprotection step (see Table 1, process step 1), so that after each coupling step, all Nucleobases are unprotected. If the bases of the CNA monomer building blocks are already Boc-unprotected, they can also be used directly to build up an oligomer.

Accordingly, preference is given to using CNA monomers from the group 3-tert-butoxycarbonyl-1 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] thymine (Formula (II)), 1 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethylcyclohex-1-yl] thymine (Formula (III)), N ⁶ -ter. - Butoxycarbonyl-9 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethylcyclohex-1-yl] -adenine (formula (IV)), 9 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] adenine (formula (V)), N ⁴ -tert.-butoxycarbonyl-1 - [(1 R, 2R, 4R) -2 -tert.-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -cytosine (formula (VI)), 1 - [(1 R, 2R, 4R) -2-tert.-butoxycarbonylamino-4-carboxymethyl-cyclohex- 1-yl] -cytosine (formula (VII), N ² -tert.-butoxycarbonyl-9 - [(1 R, 2R, 4R) -2-tert.-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] - guanine (formula (VIII)), 9 - [(1R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] guanine (formula (IX)) are selected.

(V) (VI) (VII)

(VIII) (IX)

In principle, oligomerization is possible without taking into account the stereoselectivity of the CNA monomer building blocks. B. racemic mixtures of a CNA monomer or enantiomerically pure CNA monomer units can be used in any order as a starting material for the oligomerization. However, enantiomerically pure or at least enantiomerically enriched CNA oligomers are preferably used to produce CNA oligomers, since z. B. CNA-CNA duplexes or CNA-p-RNA heteroduplicates succeed only with oligomers which have been built up from either (R) or (S) -CNA monomer units. The stereoselective preparation of such CNA oligomers is achieved simply by using enantiomeric (R) or (S) -CNA monomers as a starting material for the oligomerization.

In order to increase the solubility of the CNA oligomers in polar solvents, in particular with respect to aqueous media, hydrophilic groups can be introduced via suitable linkers. In addition, hydrophilic groups at the N- or C-terminal end of the CNA oligomers allow an active transport of these molecules by applying electrical fields, such as. B. in U.S. Patent 5,605,662 and in P.N. Gilles et al. [Nature Biotechnology, 17, 365-370 (1990)].

As a linker for the introduction of N-terminal phosphate groups z. B. hydroxycarboxylic acid derivatives, such as butyric acid proven (WO99 / 15509). The synthesis of phosphorylated CNA oligomers can be immensely simplified and the yield increased by using an already phosphorylated hydroxycarboxylic acid derivative in an improved synthesis variant. So can e.g. B. by using phosphorylated butyric acid under the standard coupling conditions used, this grouping can be introduced directly in high yields.

Another alternative method for increasing the hydrophilicity of the CNA oligomers is to attach terminal lysine or oligolysine residues to the C- and / or N-terminal end of the CNA oligomers.

Analogously, simple CNA-peptide or CNA-protein conjugates can also be produced, which lead to better solubility in polar media. However, such conjugates are primarily of interest as functional components in biotechnological assays. In particular, conjugates containing antibodies whose functional domains, peptidic antigens, receptors, structural proteins, glycoproteins or enzymes are of interest for the construction of biochip surfaces.

Furthermore, the connection of many markers, such as. B. of fluorescent dyes, radioactively labeled amino acids or biotin, at the terminal ends of the CNA oligomers.

The present invention further provides an advantageous process for the preparation of CNA monomers which are particularly suitable for oligomerization and which can be used directly for carrying out the described oligomerization. In addition to the direct usability of the CNA monomer products for oligomerization, a further advantage of the synthesis according to the invention is their high yield, based on an improved protecting group strategy.

In the process according to the invention for the production of CNA monomers, an iodine lactam of the formula (XIII)

in which A, D and F can independently represent a -CR ³ R ⁴ group and E a -CR ⁶ group, where R ³ , R ⁴ or R ^{6 have} the meaning given above, the iodine lactam preferably 8 -Iodine-2-azabicyclo [3.3.1] nonan-3-one is coupled to a nucleobase in the presence of a base. As bases come e.g. B. organic bases, such as DBU, DBN, or carbonates, such as alkali and alkaline earth carbonates in question, preferred bases are NaH, OBu and K ₂ CO ₃ . NaH is particularly preferably used as the base in a molar ratio of 1: 1 to the lactam.

Thymine, preferably its lithium salt, adenine, N ⁶ -benzoyl-adenine, N ⁶ -dimethylaminoethylene-adenine, cytosine, N ⁴ -benzoyl-cytosine or 2-amino-6-chloro-purine are suitable as nucleobases. The secondary amine group of the lactam ring is then BOC-protected. The further primary and secondary amine groups of the base can also be BOC-protected. A targeted prior protection of the amino functions of the base part of the monomer with other protective groups is surprisingly not necessary. As a result, the necessary separation of these protective groups is also eliminated.

After the introduction of the BOC protective group on the N ^{2 of} the lactam, the ring is cleaved with a lithium salt, preferably with lithium hydroxide monohydrate, to give the desired CNA monomer. To synthesize the guanine building block, the chlorine substituent of the purine must be converted into a carbonyl group before the lactam cleavage.

The CNA monomers thus produced can be used directly in the CNA oligomer synthesis without further derivatization. So are another subject In the present invention, CNA monomers whose 2-amino function of the cyclohexane ring are simply Boc-protected and whose nitrogen atoms in the base part are not protected or are partially Boc-protected. CNA monomers from the group are particularly preferred

3-tert.butoxycarbonyl-1 - [(1 R, 2R, 4R) -2-tert.butoxycarbonylamino-4-carboxymethylcyclohex-1-yl] thymine, 1 - [(1 R, 2R, 4R) -2 -tert.Butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -thymine, 9 - [(1 R, 2R, 4R) -2-tert.butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -adenine, N ^6- tert.butoxycarbonyl-9 - [(1 R, 2R, 4R) -2- tert.butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -adenine, N ⁴ - tert.butoxycarbonyl-1 - [(1 R , 2R, 4R) -2-tert.butoxycarbonylamino-4-carboxymethylcyclohex-1-yl] cytosine, 1 - [(1 R, 2R, 4R) -2-tert.butoxycarbonylamino-4-carboxymethylcyclohex-1 -yl] -cytosine, N ² -tert.Butoxycarbonyl-9 - [(1 R, 2R, 4R) -2- tert.butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -guanine, 9 - [(1R, 2R, 4R) -2- tert.butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] guanine.

With the method for CNA oligomerization according to the invention, CNA oligomers can be provided in a sufficient amount, for example. B. in biotechnological assays as a pairing system.

Surprisingly, it can be shown that CNA oligomers with p-RNA oligomers with complementary sequences pair very specifically with one another and form supramolecular heteroduplexes whose chemical properties and structure are hardly influenced by biological samples. The single strands of the heteroduplex do not pair with DNA, RNA or PNA. Complementary CNA and p-RNA form a ladder structure in the heteroduplex which is more stable than that of naturally occurring nucleic acids and whose specific pairing is less dependent on external factors such as e.g. B. depends on the solvent used. This results in a very good thermodynamic control of the hybridization and dehybridization in a wide variety of media, in particular the control can be carried out independently of naturally occurring association processes. Due to the high stability of the intermolecular binding of the CNA oligomers with their more complete p-RNA oligomers, even relatively short sequences can still pair with each other in a sequence-specific manner. It also helps that stability of CNA oligomers with their complementary p-RNA oligomers is greatly reduced by base mismatches. Furthermore, p-RNA and CNA pair exclusively in Watson-Crick mode, which increases their selectivity. Another advantage of CNA oligomers is their usability in media with a low salt concentration, since even then a thermodynamically stable selective pairing with complementary p-RNA oligomers is achieved. In addition, the CNA oligomers stabilize a CNA-p-RNA heteroduplex at elevated temperatures and in basic or acidic media.

Due to the artificial oligomer backbone and the linear ladder structure, CNA-p-RNA heteroduplex molecules are not degraded enzymatically and the association of double-stranded nucleic acids binding biological molecules, such as. B. histones, transcription factors, repressors, ribosomes etc. is minimized.

Thus, depending on the assay conditions, adapted pairing systems from CNA-CNA, p-RNA-p-RNA homoduplices or CNA-p-RNA heteroduplices can now be used.

Suitable cyclohexyl or heterocyclohexyl nucleo-amide oligomers (CNA oligomers), as are suitable for the formation of CNA-p-RNA heteroduplexes, contain a peptidic cyclohexylamide backbone, as exemplified in structural formula (X).

Structural formula (X)

In structural formula (X), B represents any nucleobase, such as. B. adenine, guanine, cytosine, thymine, uracil, isoguanine, isocytosine, xanthine or hypoxanthine. The nucleobases are bonded to the cyclohexyl radical in the 1 ^' position. The cyclohexyl or heterocyclohexyl residues are linked to each other via 2 ^' -4 ^Λ amide bonds to form a CNA oligomer. In principle, a stereochemically regular CNA oligomer structure is advantageous for pairing with p-RNA oligomers, with (S) -CNA oligomers with (L) -p-RNA and (R) -CNA oligomers with (D) -p- Hybridize RNA.

A large number of monomeric cyclohexyl or heterocyclohexyl nucleo-amine building blocks (CNA monomers) which can be used to build up corresponding oligomers (structural formula (XI)) and processes for their preparation are described in WO99 / 15509. R1

Structural formula (XI)

In structural formula (XI), B represents a nucleobase and R1 represents an NH ₂ group. R2 represents a CH ₂ -COOH group. A, D and F can independently of one another stand for a -CR ³ R ⁴ -, -NR ⁵ -, -O- or -S- group and E for a -CR ⁶ - group, where R ³ , R ⁴ , R ⁵ or R ^{6 can} independently represent a hydrogen atom or a d -CC ₂ alkyl group. CNA monomers in which the radicals R ³ and R ^{4 are} hydrogen atoms are particularly preferred.

For the formation of herteroduplicates according to the invention, p-RNA oligomers are suitable, as described, for. B. in (Helv. Chim. Acta 1993, 76, 2161-2183, Helv. Chim. Acta 1995, 78, 1621-1635, Helv. Chim. Acta 1996, 79, 2316-2345, Helv. Chim. Acta, 1997 , 80, 1901-1951). The p-RNA oligomers can also be modified so that p-RNA conjugates are formed which can contain peptides, proteins or markers known to the person skilled in the art. Linkers as described in DE 19741 738 A1 are preferably used in the preparation of such conjugates.

The CNA oligomers described can hybridize with p-RNA oligomers to form heteroduplices, as shown, for example, in structural formula (XII), where B and B ^' each represent bases which are complementary to one another.

Structural formula (XII)

The CNA-p-RNA heteroduplices have relatively high melting points, i.e. the pairing is relatively stable. So even short oligomers such. B. 5-mers form stable heteroduplexes in aqueous solution. However, heteruplexes of 7- to 12-mers are preferred. Of course, nothing stands in the way of using longer oligomers for heteroduplex formation.

In addition, it can be shown that the CNA-p-RNA heteroduplex formation is quite high even when using short CNA and p-RNA oligomers under low salt conditions. Working under low salt conditions is particularly important for biotechnological processes, since the salt concentration affects the conformation or the activity of biological substances such as e.g. B. strongly influenced by DNA or proteins. The addition of cations in order to saturate the negative charges of the duplex can be minimized by the variation of CNA-CNA and CNA-p-RNA parental systems. EXAMPLES

General preliminary remarks on the exemplary embodiments:

All pure solvents used (Flukä) are kept over a molecular sieve and used without further purification.

Oligomerization by means of solid phase synthesis is carried out using a "TentaGel S HMB" resin (rapp polymers) with a capacity of 0.23 mmol / g. The reactions are carried out in a Chirana syringe (5 ml volume for a quantity of over 100 mg resin or 2 ml volume for a resin quantity below 100 mg) provided with a fritted filter insert.

The HPLC tests are carried out with a Beckman System Gold ^ M

Chromatography system with programmable solvent module "126" and one

Diode array UVΛ / is detector module "168" performed.

The RP-HPLC is performed on an analytical scale using a LiChrospher

C-18 Hibar column (5 μm, 4 x 250 mm, Merck) at a flow rate of 1 ml / min and on a semi-preparative scale using a LiChrospher C-18 column (10 μm, 4 x 250 mm, Merck) with a flow of 1 ml / min.

The following solvents are used for elution: Eluent A: water, 0.1%

TFA (v / v); Eluent B: MeCN, 0.1% TFA (v / v).

The following solvent gradients are made from eluent for the analytical HPLC runs

A and eluent B selected:

Method A: Continuous increase in the solvent content of Eluent B from 10% to 60% within 30 minutes;

Method B: Continuous increase in the solvent content of Eluent B from 10% to 40% within 30 minutes;

Method C: Continuous increase in the solvent content of Eluent B from 10% to

30% within 40 min.

The following solvent gradient is made from eluent for the semi-preparative HPLC runs

A and eluent B selected: Method D: Continuous increase in the proportion of eluent B from 10% to 40% within 30 minutes.

The UV data specified in the exemplary embodiments were obtained using a Jasco V-530 UV / Vis spectrophotometer, the CD spectra were recorded using a Jasco J710 spectropolarimeter, and the melting curves (Tm curves) were measured by UV measurement using a Perkin Elmer Lamda 2 UVΛ / is spectrophotometer, equipped with a temperable quartz cuvette holder and a fitted micro-thermocouple (Keithley Instruments DAS-801-AT-Bus measurement card (Quick-Basic 4.0 driver)) that can be inserted into the measuring cuvette.

The Tm curves are measured at a temperature gradient between 5 ° and 90 ° C with a heating or cooling rate of 1 ° C / 40s. For this purpose, the samples are dissolved in degassed Tris-HCl buffer (5mM; pH 7.0) and added to the quartz cuvettes (10x10mm, 1x10mm, Hellma; d = 10 or d = 1). The measurement data is evaluated with the MicroCal Origin software using polynomial regression (600 points, 9th order).

The mass spectra were recorded with an electrospray ionization mass spectrometer (ESI-MS) (Finnigan LCQ ion trap MS (Finnigan, USA)) with a mixture of MeCN / water (1: 1) containing 0.1% TFA as solvent.

Synthesis of the monomer building blocks

(/) -Thymine building block Scheme 1

4

(a) NaH, Li salt of thymine, DMF, 0 ° C / RT - (b) (Boc) ₂ 0, TEA, D AP, CH ₂ CI ₂ - (c) LiOH, THF / H ₂ 0 / CH ₃ 0H, 0 ° C / RT - (d) Li0 ₂ H, THF / H ₂ 0 / CH ₃ OH, OT / RT

example 1

1 - [(1R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] thymine 2

1.26 g (10 mmol) of thymine are placed in 50 ml of THF at 0 ° C. and 4 ml (10 mmol) of 2.5M butyllithium in hexane are carefully added. After the solution has been stirred for 10 minutes, the THF is distilled off in vacuo and the residue (monolithium salt of thymine) is taken up in 10 ml of DMF.

A second flask is charged with 0.4 g (10 mmol) NaH (60%) and 2.65 g (10 mmol) (1 R, 5R, 8R) -8-iodo-2-azabicylclo at 0 ° C [ 3.3.1] nonan-3-one 1 added. After about 30 minutes, the H ₂ evolution is complete and the above lithium salt solution can be added. Then it is overnight (approx. 16 hours) Room temperature stirred. The solvent is distilled off in vacuo

Residue dissolved in water and placed on an XAD-16 column; the inorganic constituents are first separated off with water (including unreacted thymine) and then the organic product is dissolved from the column with methanol. Evaporation of the methanol fractions gives a residue which is stirred in methylene chloride for further purification and then suction filtered. (1.33 g)

A further amount of product can be isolated from the mother liquor by chromatography (EtOAc / CH ₃ OH = 5: 1). (0.65 g)

1.98 g (75%)

Mp> 300 ° C (decomp.)

R _F = 0.55 (acetone: CH ₂ CI ₂ : CH ₃ OH: EtOAc: H ₂ O: AcOH = 9: 6: 2: 2: 2: 1)

HPLC:> 98% [Cyclobond I, CH ₃ CN / (H ₂ O + 0.01 N TBAS) = 80:20]

[α] _D ²² = + 55.5 ° (c = 0.5; CH ₃ OH)

¹ H NMR (400 MHz, d ₆ -DMSO) 1.52-1.65 (m, 3H); 1.80-1.88 (m, 4H); 1.96-2.12 (m,

3H); 2.16-2.23 (m, 1H); 2.40-2.48 (m, 1H); 3.61 u. 4.17 (2m, 2H, CH- [NH] and CH-N);

7.52 (d, 1H, = CH); 7.94 (d, 1H, NH-CO); 11, 27 (s, 1H, CO-NH-CO)

Example 2

1 - [(1R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] thymine 2

1.26 g (10 mmol) of thymine, 2.65 g (10 mmol) (1 R, 5R, 8R) -8-iodo-2-azabicylclo [3.3.1] nonan-3-one 1 and 3.5 g (25 mmol) K ₂ CO ₃ in 50 ml DMSO are stirred at room temperature for 48 hours. For working up, the DMSO is first distilled off in vacuo and the residue is boiled out with carbon tetrachloride. The remaining residue is dissolved in water and placed on an XAD-16 column; the inorganic constituents are first separated off with water (including unreacted thymine) and then the organic constituents are removed with methanol Product detached from the column. Evaporation of the methanol fractions gives a residue which is chromatographed for further purification. (SiO ₂ :

EtOAc / EtOH = 5: 1)

1.0 g (38%)

For physical data, see example 1

Example 3

3-tert.butoxycarbonyl-1 - [(1 R, 5R, 8R) -3-oxo-2 tert.butoxycarbonyl-2-azabicylo [3.3.1] -nonan-8-yl] -thymine 3

3

2.1 g (8 mmol) 1 - [(1 R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] -thymine 2, 1, 38 ml

(10 mmol) triethylamine, 4.36 g (20 mmol) di-tert-butyl pyrocarbonate and 50 mg DMAP are refluxed for approx. 4 hours in 50 ml methylene chloride and left to stand overnight at room temperature. (DC control: EtOAc) If the

50 ml of water are added and the mixture is stirred vigorously for about 10 minutes. The organic phase is then separated off, and the aqueous phase is extracted with

Methylene chloride, dries the combined organic phases and evaporates it

Solvent. The residue is purified by column chromatography. (SiO ₂ ,

EtOAc)

2.85 g (77%)

Mp 116 ° C

R _F = 0.41 (EtOAc)

HPLC:> 98% [LiChrospher RP 18, CH ₃ CN / (H ₂ O + 0.01 N TBAS) = 75:25]

[α] _D ²² = + 5.75 ° (c = 0.38; CH ₃ OH)

¹ H-NMR (400 mHz, d ₆ -DMSO) 1, 34 u. 1, 43 (2s, 18 H, 2 tert Bu); 1, 34-1, 36 (m,

1H); 1.67-1.83 (m, 6H); 1.90-2.08 (m, 2H); 2.13-2.26 (m, 2H); 2.57-2.69 (m, 1H); 4.22 u. 4.30 (2m, 2H, CH- [NH] and CH-N); 7.56 (d, 1H, = CH); Example 4

3-tert.butoxycarbonyl-1 - [(1 R, 2R, 4R) -2-tert.butoxycarbonylamino-4-carboxymethyl-cyclohex-1-y l] -thymine 4

OH

4

1.5 g (36 mmol) of lithium hydroxide monohydrate are dissolved in 25 ml of water and 3.1 ml (36 mmol) of H ₂ O ₂ (35%) are added at 0.degree. Then 5.6 g (12 mmol) of 3-tert-butoxycarbonyl-1 - [(1 R, 5R, 8R) -3-oxo-2 part. butoxycarbonyl-2-azabicylo [3.3.1] - nonan-8-yl] -thymine 3 dissolved in 165 ml THF added dropwise at 0 ° C; 50 ml of methanol are then added and the mixture is stirred for about 18 hours at room temperature. If peroxides are still present in the solution, they are destroyed with a little Na ₂ SO ₃ solution; THF / CH ₃ OH is then distilled off in vacuo, water and methylene chloride are added to the remaining aqueous phase, and the mixture is adjusted to pH 3 with dilute HCl, the organic phase is separated off and the aqueous phase is extracted with methylene chloride. After drying and distilling off the solvent, a residue is obtained which is purified by chromatography. (SiO ₂ : EtOAc) 4.2 g (73%)

RF = 0.71 (EtOAc / CH ₃ OH = 5: 1))

HPLC: 96% [Cyclobond I, CH ₃ CN / (H ₂ O + 0.01 N TBAS) = 92.5: 7.5] [] _D ²² = + 24.8 ° (c = 0.56; CH ₃ OH)

¹ H NMR (400 MHz, CDCI ₃ ) 1.05-1.25 (m, 2H); 1, 32 u. 1.59 (2s, 18H, 2 tert.Bu); 1.57-1.68 (m, 1H); 1.80-2.08 (m, 3H); 1, 91 (s, 3H, CH ₃₎ 2.10-2.23 (m, 1H); 2.22-2.37 (m, 2H); 3.86 u. 4.33 (2m, 2H, CH- [NH] and CH-N); 4.73 (d, 1H, HN-CO); 7.15 (s, 1H, = CH); approx.10.0 (br.s, 1H, CO ₂ H) Example 5

1 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethylcyclohex-1-yl] thymine 5

9.3 g (20 mmol) 3-tert-butoxycarbonyl-1 - [(1 R, 5R, 8R) -3-oxo-2 tert.butoxycarbonyl-2-azabicylo [3.3.1] -nonan-8-yl] -thymine 3 are placed in 450 ml of THF and a solution of 4.2 g (100 mmol) of lithium hydroxide monohydrate in at 30 ° C. within 30 minutes

100 ml of water added dropwise. Then 70 ml of methanol are added and so long

-10 ° C stirred until there is no more starting product. (approx. 5 hours; DC

Control: EtOAc) The mixture is then stirred for 12 hours at room temperature and for 15-20 hours at 45-55 ° C. (TLC control: CH ₂ CI ₂ / CH3OH = 5: 1) The solvent

THF / CH ₃ OH is distilled off under vacuum, the aqueous residue with dilute

HCL set to PH3 and extracted with methylene chloride. The organic phase is dried, evaporated and the residue is purified by chromatography. (SiO2:

CH ₂ CI ₂ / CH ₃ OH = 5: 1)

3.5 g (46%)

Mp 245 ° C (dec.)

R _F = 0.36 (CH ₂ CI ₂ / CH ₃ OH = 5: 1)

HPLC: 98.7% [LiChrospher RP 18, CH ₃ CN, (H ₂ O + 0.01 N TBAS) as gradient (from

90% to 60% in 30 min.), Flow 1 ml / min.]

[α] _D ²² = + 22.6 ° (c = 0.57; CH ₃ OH)

¹ H NMR (400 MHz, d ₆ -DMSO) 1, 3-1, 32 (m, 2H); 1, 26 (s, 9H, tert.Bu); 1, 63-1, 94 (m,

5H); 1, 73 (s, 3H, CH _3); 2.06-2.21 (m, 2H); 3.75 (m, 1H, CH- [NH]); 4.18 (m, 1H, CH-

N); 6.74 (d, 1H, NH-boc); 7.55 (s, 1H, -CH =); 11.06 (s, 1H, CO-NH-CO); 12.05 (s,

1 H, (/?) - Adenine building block Scheme 2

6 13

(a NaH, adenine, DMF, 0 ° C / RT - (b) (Boc) ₂ 0, TEA, DMAP, CH ₂ CI ₂ - (c) LiOH, THF / H ₂ 0 / CH ₃ OH, -10 ° C / RT - (d) NaH, N -dimethylaminomethylene-adenine, DMF, 0 ° C / RT - (e) NaH, N ⁶ -benzoyl-adenine, DMF, 0 / RT - (f) LiOH, CH ₃ OH / H ₂ 0, -15 ° C then RT

Example 6

N ⁶ -Benzoyl-9 - [(1 R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] -adenine 6

0.4 g (10 mmol) of NaH (60%) are placed in 50 ml of DMF at 0 ° C. and 2.65 g

(10 mmol) (1 R, 5R, 8R) -8-iodo-2-azabicylclo [3.3.1] nonan-3-one 1 was added. After about 30 minutes, the evolution of H _{2 has} ended and 2.4 g (10 mmol) of N ⁶ -benzoyl-adenine can be added. Then remove the cooling and stir

Mix at room temperature overnight (approx. 15 hours). The DMF is then distilled off under vacuum, the residue is taken up in about 100 ml of water and the

Reaction product extracted several times with methylene chloride. The organic phase is washed with saturated sodium chloride solution, dried and evaporated. The solid residue is taken up in isopropanol and residual solid

Aspirated product. [0.4 g (17%) N ⁶ -benzoyl-adenine] The filtrate is evaporated and chromatographed. (SiO ₂ : First EtOAc / CH ₃ OH = 5: 1 then CH ₂ CI ₂ / CH ₃ OH =

5: 1)

2.85 g (75.8%)

Mp> 300 ° C

R _F = 0.57 (CH ₂ CI ₂ / CH ₃ OH = 5: 1)

[] _D ²² = -35.1 ° (c = 1, 1; CH ₃ OH)

HPLC: 94% [Cyclobond I, CH ₃ CN / (H ₂ O + 0.01 N TBAS) = 90:10]

¹ H NMR (400 MHz, d ₆ -DMSO) 1.52-1.74 (m, 3H); 2.05-2.34 (m, 5H); 2.48-2.56 (m,

1 H); 4.25 u. 4.62 (2m, 2H, CH- [NH] and CH-N); 7.52-8.07 (m, 5H, Ph); 8.11 (d, 1H,

HN [CH]); 8.68 u. 8.75 (2s, 2H, 2-CH =); 11, 15 (s, 1 H, NH-CO) Example 7

N ⁶ - (Dimethylaminomethylene) -9 - [(1 R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] -adenine 8

0.8 g (20 mmol) of NaH (60%) are placed in 100 ml of DMF at 0 ° C. and 5.3 g

(10 mmol) (1 R, 5R, 8R) -8-iodo-2-azabicylclo [3.3.1] nonan-3-one 1 was added. After about 30 minutes, the H ₂ evolution is complete and 3.8 g (20 mmol) of N ⁶ -

(Dimethylaminomethylene) adenine can be added. Then the cooling is removed and the mixture is stirred at room temperature for about 48 hours

The DMF is then distilled off in vacuo, the residue is taken up in water and applied to an XAD 16 column to remove inorganic constituents; after washing with water, the product is detached from the column with methanol.

After the solvent has been evaporated off, the residue is purified by chromatography. (SiO ₂ : CH ₂ CI ₂ / CH ₃ OH = 5: 1)

5 g (76.5%)

R _F = 0.24 (CH ₂ CI ₂ / CH ₃ OH = 10: 1)

[] _D ²² = -81 ° (c = 0.4; CH ₃ OH)

HPLC:> 98% [Cyclobond I, CH ₃ CN / (H ₂ O + 0.01 N TBAS) = 80:20]

¹ H NMR (400 MHz, d ₆ -DMSO) 1, 45-1, 74 (m, 3H); 2.02-2.29 (m, 5H); 2.46-2.55 (m,

1 H); 3.12 u. 3.18 (2s, 6H, 2 CH _3); 4.22 u. 4.51 (2m, 2H, CH- [NH] and CH-N); 8.05 (d,

1 H, HN- [CH]); 8.42 u. 8.44 (2s, 2H, 2-CH =); 8.92 (s, 1H, N = CH-N) Example 8

9 - [(1R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] adenine 7

0.8 g (20 mmol) of NaH (60%) are placed in 50 ml of DMF at 0 ° C. and 5.3 g (20 mmol) (1 R, 5R, 8R) -8-iodo-2-azabicylclo [ 3.3.1] nonan-3-one 1 added. After about

The H ₂ evolution is complete for 30 minutes and 2.3 g (20 mmol) of adenine can be added. The cooling is then removed and the mixture is stirred in

Room temperature overnight (approx. 15 hours). The DMF is then distilled off under vacuum, the residue is dissolved in about 100 ml of water and removed to remove

By-products extracted several times with ethyl acetate; the aqueous phase is boiled with about 5 g of NaHCO ₃ and cooled and placed on an XAD 16 column; With

Water washes the inorganic salts from the column first and then the reaction product with methanol. After evaporating the

Methanol phases, the residue is boiled up once in ethyl acetate, cooled and suction filtered.

4.4 g (80%)

Mp> 300 ° C

R _F = 0.6 (CH ₂ CI ₂ / CH ₃ OH = 5: 1)

[α] _D ²² = -43 ° (c = 0.4; CH ₃ OH)

HPLC: 98% [Cyclobond I, CH ₃ CN / (H ₂ O + 0.01 N TBAS) = 70:30]

¹ H NMR (400 MHz, d ₆ -DMSO) 1, 47-1, 73 (m, 3H); 2.04-2.28 (m, 5H); 2.47-2.56 (m,

1 H); 4.22 u. 4.48 (2m, 2H, CH- [NH] and CH-N); 7.30 (s, 2H, NH _2); 8.13 (d, 1H, HN-

CO); 8.16 u. 8.37 (2s, 2H, 2-CH =) Example 9

9 - [(1R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] adenine 7

1.35 g (10 mmol) adenine, 2.65 g (10 mmol) (1 R, 5R, 8R) -8-iodo-2-azabicylclo [3.3.1] nonan-3-one 1 and 2.1 g (15 mmol) of potassium carbonate are stirred in 50 ml of dimethyl sulfoxide for 2 days at room temperature. The dimethyl sulfoxide is then largely distilled off under good vacuum at moderate temperature, the residue is taken up in water and added to an XAD 16 column. The inorganic constituents can be washed out with water together with unreacted adenine; the pre-purified reaction product is then washed off the column with methanol. The residue from the methanol phase is purified by chromatography. (SiO2: CH ₂ CI ₂ / CH ₃ OH = 5: 1) 2.0 g (73%) Physical data see example 8

Example 10

N ⁶ -Benzoyl-N ⁶ -tert.butoxycarbonyl-9 - [(1 R, 5R, 8R) -3-oxo-2-tert.butoxycarbonyl-

2-azabicylo [3.3.1] nonan-8-yl] adenine 13

13

1.88 g (5 mmol) N ⁶ -benzoyl-9 - [(1 R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] - adenine 6, 3.27 g (15 mmol) of di-tert-butyl pyrocarbonate and 100 mg of DMAP in 50 ml of acetonitrile are stirred for 24 hours at room temperature. After the solvent has been evaporated off, the residue is chromatographed. (SiO2: EtOAc)

2.14 g (74.2%)

R _F = 0.77 (EtOAc / CH ₃ OH = 5: 1)

¹ H-NMR (400 mHz, d ₆ -DMSO) 1, 25 u. 1.52 (2s, 18H, 2tert.Bu); 1.61-1.92 (m, 3H);

2.02-2.15 (m, 2H); 2.20-2.28 (m, 1H); 2.31-2.45 (m, 2H); 2.76-2.88 (m, 1H); 4.89 u.

5.15 (2m, 2H, CH- [NH] and CH-N); 7.52-7.81 (m, 5H, Ph); 8.87 u. 8.88 (2s, 2H, 2- CH =)

Example 11

N ⁶ -Di- (tert.butoxycarbonyl) -9 - [(1 R, 5R, 8R) -3-oxo-2-tert.butoxycarbonyl-2-azabicylo [3.3.1] nonan-8-yl] -adenine 11

11

10 g (36.7 mmol) 9 - [(1 R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] -adenine 7 in

350 ml of methylene chloride are mixed with 5.08 ml (36.7 mmol) of TEA, 32 g (147 mmol)

Pyrocarbonate di-tert-butyl ester and 200 mg DMAP combined at room temperature and then stirred under reflux for about 4 hours. (TLC control: CH ₂ CI ₂ / CH ₃ OH = 5: 1)

After the solvent has been distilled off, the residue is passed through

Column chromatography cleaned. (SiO ₂ : EtOAc)

16.3 g (77.5%)

R _F = 0.58 (EtOAc)

[ _α ] _D ²² = .49.5 ° (c = 1, 1; CH ₃ OH)

HPLC: 98% [Cyclobond I, CH ₃ CN]

¹ H-NMR (400 mHz, CDCI ₃ ) 1, 48 u. 1.61 (2s, 27H, 3tert.Bu); 1.76-1.95 (m, 3H); 2,15-

2.45 (m, 4H); 2.50-2.57 (m, 1H); 2.80-2.90 (m, 1H); 4.93 u. 5.25 (2m, 2H, CH- [NH] u.

CH-N); 8.32 u. 8.87 (2s, 2H, 2-CH =) Example 12

N ⁶ - (Dimethylaminomethylene) -9 - [(1 R, 5R, 8R) -3-oxo-2-tert.butoxycarbonyl-2-azabicylo [3.3.1] nonan-8-yl] -adenine 9

5 g (13.4 mmol) of N ⁶ - (dimethylaminomethylene) -9 - [(1 R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] -adenine 8 are added in 250 ml methylene chloride with 1.86 ml

TEA, 5.85 g (26.8 mmol) di-tert-butyl pyrocarbonate and 20 mg DMAP approx. 60

Stirred at room temperature. After the solvent has been evaporated off, the residue is chromatographed. (SiO ₂ : EtOAc / CH ₃ OH = 5: 1)

5 g (87.5%)

R _F = 0.23 (EtOAc / CH ₃ OH = 5: 1))

¹ H NMR (400 MHz, CDCI ₃ ) 1, 6 (s, 9H, tert.Bu); 1.75-2.56 (m, 8H); 2.78-2.89 (m, 1H);

3.22 u. 3.27 (2s, 6H, 2CH _3); 4.87 u. 5.30 (2m, 2H, CH- [NH] and CH-N); 8.12 u. 8.54

(2s, 2H, 2-CH =); 8.95 (s, 1H, N = CH-N)

Example 13

N ⁶ -tert.Butoxycarbonyl-9 - [(1 R, 2R, 4R) -2-tert.butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -adenine 12

12

2.14 g (3.7 mmol) N ⁶ -benzoyl-N ⁶ -tert.butoxycarbonyl-9 - [(1 R, 5R, 8R) -3-oxo-2-tert.butoxycarbonyl-2-azabicylo [3.3. 1] nonan-8-yl] -adenine 13 are placed in 80 ml THF. 0.9 g (21.5 mmol) of LiOH monohydrate dissolved in 20 ml of water are added dropwise at 0 ° C. within 30 minutes. Then add 13 ml of methanol and stir approx.

16 hours at room temperature. After distilling off the organic

Solvent in vacuo, add a little water to the residue, adjust to p 3 with dilute HCl, suck off the reaction product and wash it with

Water and acetone.

1.0 g (55.5%)

Mp> 300 ° C

R _F = 0.6 (CH ₂ CI ₂ / CH ₃ OH = 5: 1)

[] _D ²² = + 3.9 ° (c = 51; CH ₂ CI ₂ / CH ₃ OH = 1: 1)

HPLC: 98% [Cyclobond I, CH ₃ CN / (H ₂ O + 0.01 N TBAS) = 95: 5]

¹ H-NMR (400 mHz, d ₆ -DMSO) 1, 04 u. 1, 47 (2s, 18H, 2tert.Bu); 1.08-1.33 (m, 2H);

1.78-2.03 (m, 4H); 2.14-2.29 (m, 3H); 4.01 u. 4.31 (2m, 2H, CH- [NH] and CH-N); 6.85

(d, 1H, NH- [CH]); 8.4 u. 8.54 (2s, 2H, -CH =) 9.9 (s, 1H, NH-C); 12.13 (s, 1 H, CO ₂ H) Example 14

N ⁶ -tert.Butoxycarbonyl-9 - [(1 R, 2R, 4R) -2-tert.butoxycarbonylamino-4-carboxymethyl-cyclohex-1 -yl] -adenine 12

boc

12

11.24 g (19.6 mmol) N ⁶ -di (tert-butoxycarbonyl) -9 - [(1 R, 5R, 8R) -3-oxo-2-tert-butoxycarbonyl-2-azabicylo [3.3.1 ] nonan-8-yl] -adenine 11 are placed in 390 ml THF. 4.11 g (98 mmol) of LiOH monohydrate dissolved in 100 ml of water are added dropwise at -10 ° C. in the course of 30 minutes. Then 65 ml of methanol are added and the mixture is stirred for 2 hours at -10 ° C. and then for about 16 hours at room temperature. After the organic solvents have been distilled off in vacuo, a little water is added to the residue, the mixture is adjusted to p 3 with dilute HCl, the reaction product is filtered off with suction and washed with water and acetone. 6.6 g (68.5%) Physical data see example 13

Example 15

N ⁶ -tert.Butoxycarbonyl-9 - [(1 R, 2R, 4R) -2-tert.butoxycarbonylamino-4-carboxymethyl-cyclohex-1 -yl] -adenine 12

12

5.3 g (9.25 mmol) N ⁶ -Di- (tert.butoxycarbonyl) -9 - [(1 R, 5R, 8R) -3-oxo-2-tert.butoxycarbonyl-2-azabicylo [3.3.1 ] nonan-8-yl] -adenine 11 are placed in 180 ml of methanol. At -15 ° C., 89 g (45 mmol) of LiOH monohydrate, dissolved in 100 ml of water, are added dropwise within 30 minutes. The mixture is stirred at -15 ° C. until the starting product has disappeared and then at room temperature for about 16 hours. After distilling off the organic solvent in vacuo, the aqueous residue is one with dilute HCl to p _H 3, and the reaction product extracted with methylene chloride. The product is purified by chromatography. (SiO ₂ : CH ₂ CI ₂ / CH ₃ OH = 5: 1) 3.8 g (84%)

0.53 g (16%) N ⁶ - (tert.butoxycarbonyl) -9 - [(1 R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] adenine physical For data see example 13 Example 16

9 - [(1R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethylcyclohex-1-yl] adenine 10

10

5.0 g (12 mmol) N ⁶ - (dimethylaminomethylene) -9 - [(1 R, 5R, 8R) -3-oxo-2-tert.butoxycarbonyl-2-azabicylo [3.3.1] nonan-8-yl ] -adenine 9 are placed in 240 ml of methanol. At -15 ° C., 2.52 g (60 mmol) of LiOH monohydrate, dissolved in 60 ml of water, are added dropwise within 30 minutes. The mixture is stirred at -15 ° C. until the starting product has disappeared and then at room temperature for about 16 hours. After the organic solvents have been distilled off in vacuo, the aqueous residue is adjusted to p 3 with dilute HCl, and the reaction product is extracted with methylene chloride. The product is purified by chromatography. (SiO ₂ : acetone / CH ₂ CI ₂ / CH ₃ OH / EtOAc / H ₂ O / AcOH = 9: 6: 2: 2: 2: 1) 2.4 g (51.3%) mp 221 ° C (Dec) RF = 0.31 (CH ₂ CI ₂ / CH ₃ OH = 5: 1)

HPLC:> 98% [LiChrospher RP 18, CH ₃ CN / (H ₂ O + 0.01 N TBAS) = 15:85] [α] _D ²² = + 19.5 ° (c = 0.51; CH ₃ OH)

¹ H NMR (400 MHz, d ₆ -DMSO) 1.09 (s, 9H, tert.Bu); 1.05-1.31 (m, 2H); 1.76-2.01 (m, 4H); 2.08-2.26 (m, 3H); 3.98 u. 4.22 (2m, 2H, CH- [NH] and CH-N); 6.79 (d, 1H, NH-CO); 7.07 (s, 2H, NH _2); 8.07 u. 8.11 (2s, 2H, = CH); 12.2 (br.s, 1 H, CO ₂ H)

As a by-product, 1.3 g (40%) of 9 - [(1 R, 5R, 8R) -3-oxo-2-azabicylo [3.3.1] nonan-8-yl] -adenine 7 are isolated. (f?) - Cytosine building block Scheme 3

17 18

(a) Cytosine, NaH, DMF, O / RT - (b) (Boc) ₂ 0, TEA, DMAP, CH ₂ CI ₂ - (c) LiOH, CH ₃ 0H / H ₂ 0, -15 ° C / RT - (d) NaH, N ⁴ - benzoyl cytosine, DMF, 0 ° C / RT - (e) LiOH, THF / H ₂ 0 / CH ₃ OH, -10 ° C / RT

Example 17

N ⁴ -Benzoyl-1 - [(1R, 5R, 8R) -3oxo-2-azabicyclo [3.3.1] nonan-8-yl] cytosine 17

17

0.4 g (10 mmol) of NaH (60%) are placed in 50 ml of DMF at 0 ° C. and 2.65 g (10 mmol) (1 R, 5R, 8R) -8-iodo-2-azabicylclo [ 3.3.1] nonan-3-one 1 added. After about 30 minutes, the H ₂ evolution is complete and 2.15 g (10 mmol) of N ⁴ -Benzoyl- cytosine can be added. The cooling is then removed and the mixture is stirred at room temperature overnight (approx. 18 hours). The DMF is distilled off under vacuum, the residue is taken up in methylene chloride and washed twice with saturated sodium chloride solution. The organic phase is dried and evaporated. The solid residue is purified by chromatography. (SiO ₂ :

EtOAc / CH ₃ OH = 5: 1)

2.55 g (72%)

Mp> 300 ° C

R _F = 0.52 (CH ₂ CI ₂ / CH ₃ OH = 5: 1)

[α] _D ²² = + 88.7 ° (c = 0.8; CH ₃ OH)

HPLC:> 97% [LiChrospher RP 18, CH ₃ CN / (H ₂ O + 0.01 N TBAS = 30:70, flow 1 ml / min.]

¹ H NMR (400 MHz, d ₆ -DMSO) 1.49-1.69 (m, 3H); 1.85-1.98 (m, 1H); 2.01-2.25 (m,

4H); 2.44-2.53 (m, 1H); 3.75 u. 4.38 (2m, 2H, CH- [NH] and CH-N); 7.32-8.3 (m, 8H,

Ph, N-CH = CH, NH- [CH]); 11, 23 (s, 1H, NH-CO)

Example 18

1 - [(1R, 5R, 8R) -3oxo-2-azabicyclo [3.3.1] nonan-8-yl] cytosine 14

14

0.96 g (24 mmol) of NaH (60%) are placed in 75 ml of DMF and 2.22 g (20 mmol) of cytosine are added; then stir until the H ₂ evolution has ended. Then 5.3 g (20 mmol) (1 R, 5R, 8R) -8-iodo-2-azabicylclo [3.3.1] nonan-3-one 1 are added and the mixture is stirred at room temperature for a further 15 hours. The DMF is distilled off under vacuum and the residue is dissolved in 2.7 ml (20 mmol) of KH ₂ PO ₄ in 100 ml of water added. The aqueous phase is first washed twice with ethyl acetate, then placed on an XAD 16 column. The column is washed well with water and then with methanol. The product is obtained from the methanol fraction, which is stirred with ethyl acetate, suction filtered and dried.

3.42 g (69%)

Mp> 300 ° C

R _F = 0.26 (acetone / CH ₂ CI ₂ / CH ₃ OH / EtOAc / H ₂ O / AcOH = 9: 6: 2: 2: 2: 1)

[α] _D ²² = + 103 ° (c = 0.4; CH ₃ OH)

HPLC: 98% [Cyclobond I, CH ₃ CN / (H ₂ O + 0.01N TBAS) = 75:25]

¹ H NMR (400 MHz, d ₆ -DMSO) 1, 45-1, 63 (m, 3H); 1, 7-1, 83 (m, 1H); 1.9-2.1 (m, 3H);

2.12-2.2 (m, 1H); 2.39-2.48 (m, 1H); 3.65 u. 4.21 (2m, 2H, CH- [NH] and CH-N); 5.71 u.

7.71 (2d, 2H, N-CH = CH); 6.80-7.15 ("m", 2H, NH ₂ ); 7.92 (d, 1H, NH- [CH])

Example 19

1 - [(1R, 5R, 8R) -3oxo-2-azabicyclo [3.3.1] nonan-8-yl] cytosine 14

14

1.11 g (10 mmol) of cytosine, 2.65 g (10 mmol) (1 R, 5R, 8R) -8-iodo-2-azabicylclo [3.3.1] nonan-3-one 1 and 2.1 g (15 mmol) K ₂ CO ₃ in 50 ml DMSO are stirred for 2 days at room temperature. The DMSO is largely distilled off under good vacuum, the residue is taken up in water and placed on an XAD 16 column; this is first washed well with water, then the reaction product can be dissolved with methanol. The final purification is carried out by chromatography. (SiO2: acetone / CH ₂ CI ₂ / CH ₃ OH / EtOAc / H ₂ O / AcOH = 9: 6: 2: 2: 2: 1) 1 g (40%)

For physical data see example 18

Example 20

N ⁴ -Benzoyl-N ⁴ - (tert.butoxycarbonyl) -1 - [(1R, 5R, 8R) -3oxo-2-tert.butoxycarbonyl-2-azabicyclo [3.3.1] nonan-8-yl] cytosine 18

18

3.5 g (10 mmol) of N ⁴ -benzoyl-1 - [(1R, 5R, 8R) -3oxo-2-azabicyclo [3.3.1] nonan-8-yl] cytosine 176.55 are dissolved in 100 ml of acetonitrile g (30 mmol) of tert-butyl pyrocarbonate and 250 mg of DMAP were stirred for 24 hours at room temperature. The

The reaction mixture is evaporated under vacuum and the residue is purified by chromatography. (SiO ₂ : EtOAc)

3.86 g (70%)

Schmp.181 ° C

R _F = 0.61 (EtOAc)

[α] _D ²² = + 15.3 ° (c = 1; CH ₃ OH)

HPLC: 98% [Cyclobond I, CH ₃ CN / (H ₂ O + 0.01N TBAS) = 98: 2]

¹ H NMR (400 MHz, d ₆ -DMSO) 1.27 and 1.41 (2s, 18H, 2tert.Bu); 1.45-1.54 (m, 1H);

1.72-1.82 (m, 1H); 1.83-1.97 (m, 3H); 1.99-2.12 (m, 1H); 2.2-2.36 (m, 2H); 2.65-2.75

(m, 1H); 4.37 and 4.41 (2m, 2H, CH- [NH] and CH-N); 6.97 and 8.31 (2d, 2H, N-CH = CH);

7.55-7.88 (m, 5H, Ph) Example 21

N ⁴ -di (tert.butoxycarbonyl) -1 - [(1R, 5R, 8R) -3oxo-2-tert.butoxycarbonyl-2-azabicyclo [3.3.1] nonan-8-yl] cytosine 15

15

9.0 g (36.2 mmol) of 1 - [(1 R, 5R, 8R) -3oxo-2-azabicyclo [3.3.1] nonan-8-yl] cytosine 14.5 ml ( 36.5 mmol) TEA, 31.6 g (145 mmol)

Pyrocarbonate di-tert.butyl ester and 200 mg DMAP are under approx. 4 hours

Reflux cooked. (TLC control: acetone / CH ₂ CI ₂ / CH ₃ OH / EtOAc / H ₂ O / AcOH =

9: 6: 2: 2: 2: 1) After evaporation, the residue is stirred with approx. 50 ml EtOAc, cooled and suction filtered. (9.8 g) A further 4.1 g can be isolated from the mother liquor by chromatography. (SiO ₂ : EtOAc)

13.9 g (70%)

Mp 208 ° C

R _F = 0.62 (EtOAc)

[α] _D ²² = +16.1 ° (c = 0.99; CH ₂ CI ₂ )

HPLC:> 98% [LiChrospher RP 18, CH ₃ CN / (H ₂ O + 0.01 N TBAS = 60:40, flow 1 ml / min.]

¹ H-NMR (400 mHz, CDCI ₃ ) 1, 53 u. 1.56 (2s, 27H, 3tert.Bu); 1.53-1.56 (m, 1H); 1, 78-

1.88 (m, 1H); 1.96-2.2 (m, 4H); 2.31-2.48 (m, 2H); 2.68-2.79 (m, 1H); 4,35u. 4.63

(2m, 2H, CH- [NH] and CH-N); 7.03 u. 7.72 (2d, 2H, N-CH = CH) Example 22

N ⁴ -tert.Butoxycarbonyl-1 - [(1 R, 2R, 4R) -2-tert.butoxycarbonylamino-4-carboxymethyl-cyclohex-1 -yl] -cytosine 16

16

5 g (9 mmol) of N ⁴ -benzoyl-N - (tert.butoxycarbonyl) -1 - [(1 R, 5R, 8R) -3oxo-2-tert.butoxycarbonyl-2-azabicyclo [3.3.1] nonan-8 -yl] -cytosine are placed in 180 ml of THF. At -10 ° C. 1.9 g (45 mmol) of LiOH monohydrate are dissolved in within 30 minutes

45 ml of water added dropwise. After the addition of 30 ml of methanol, 2 hours at -

10 ° C and about 18 hours at room temperature. The organic solvents are distilled off in vacuo, the aqueous phase is adjusted to pπ3 with dilute HCl and extracted with methylene chloride. This is dried, evaporated and the residue is purified by chromatography. (SiO ₂ : CH ₂ CI ₂ / CH ₃ OH = 95: 5)

1.8 g (43%)

Mp> 300 ° C

R _F = 0.7 (CH ₂ CI ₂ / CH ₃ OH = 5: 1))

[] _D ²² = + 29.6 ° (c = 0.54 CH ₃ OH)

HPLC: 98% [Cyclobond I, CH ₃ CN / (H ₂ O + 0.01N TBAS) = 90:10]

¹ H-NMR (400 mHz, d ₆ -DMSO) 1.05 u. 1, 25 (2s, 18H, 2tert.Bu); 1, 0-1, 14 (m, 1H); 1, 4-

1.78 (m, 6H); 1.9-2.05 (m, 2H); 3.6 u. 4.22 (2m, 2H, CH- [NH] and CH-N); 6.62 (d, 1H,

NH- [CH]); 6.7 u. 7.89 (2s, 2H, N-CH = CH) 9.5-12.5 (2H, NH-C, CO ₂ H) (R) -Guanine building block Scheme 7

22 21

(a) 6-chloro-2-amino-puπn, NaH, DMF, 0 ° C / RT - (b) (Boc) ₂ 0, TEA, DMAP, CH ₂ CI ₂ - (c) (CH ₃ ) ₃ N , 3-HO-Propιonιtrιl, DBU, CH ₂ CI ₂ , 0 ° C then RT then LiOH, CH ₃ 0H / H ₂ 0, -15 ° C / RT - (d) (CH ₃ ) ₃ N, 3-HO -Propιonιtrιl, DBU, CH ₂ CI ₂ , 0 ° C then RT - (e) LiOH, THF / H ₂ 0 / CH ₃ OH, 0 ° C / RT

Example 23

2-Amlno-6-chloro-9 - [(1R, 5R, 8R) -3oxo-2-azabicyclo [3.3.1] nonan-8-yl] -purine 19

19 2 g (50 mmol) NaH (60%) are placed in 150 ml DMF. 13.25 g (50 mmol) (1 R, 5R, 8R) -8-iodo-2-azabicylclo [3.3.1] nonan-3-one in portions at 0 ° C. in a short time

1 added; after about 30 minutes at 0 ° C. the H ₂ evolution is complete and 8.5 g

(50 mmol) 2-amino-6-chloropurine are added. Then the

Reaction mixture stirred for 24 hours at room temperature. After the DMF has been distilled off under vacuum, the solid residue is suction-filtered with about 70 ml of water and washed with acetone: 7.8 g of pure reaction product; from the

Filtrate can again 3.9 g of product by chromatography (SiO: CH ₂ CI ₂ / CH ₃ OH

= 9: 1) can be isolated.

11.7 g (76%)

Mp 275 ° C

R _F = 0.55 (CH ₂ CI ₂ / CH ₃ OH = 5: 1)

HPLC: 98% [Cyclobond I, CH ₃ CN / (H ₂ O + 0.01 N TBAS) = 92.5: 7.5]

[] _D ²² = -10.8 ° (c = 1; CH ₃ OH)

¹ H NMR (400 MHz, d ₆ -DMSO) 1, 57-1, 73 (m, 3H); 1.92-2.28 (m, 5H); 2.47-2.56 (m,

1 H); 4.24 (m, 1H, HC- [NH]); 4.39 (m, 1H, CH-N); 6.82 (s, 2H, NH _2); 7.92 (d, 1H, NH-

CO); 8.36 (s, 1H, -CH =)

Example 24

2-Amino-6-chloro-9 - [(1R, 5R, 8R) -3oxo-2-azabicyclo [3.3.1] nonan-8-yl] -purine 19

2.65 g (10 mmol) (1 R, 5R, 8R) -8-iodo-2-azabicylclo [3.3.1] nonan-3-one 1, 1.7 g (10 mmol) 2-amino-6- chlor-purine and 2.1 g (15 mmol) of K ₂ CO ₃ are stirred in 50 ml of DMSO for 24 hours at room temperature. After the DMSO has been distilled off under vacuum, the residue is pasted in 50 ml of water, filtered off with suction and extracted with acetone washed; 1.8 g of pure product are obtained, and a further 0.3 g is isolated from the filtrate by chromatography (SiO ₂ : CH ₂ CI ₂ / CH ₃ OH = 9: 1).

2.1g (69%)

For physical data, see Example 28

Example 25

2- [di- (tert.butylcarbonyl) amino] -6-chloro-9 - [(1 R, 5R, 8R) -3oxo-2-tert.butoxycarbonyl-2-azabicyclo [3.3.1] nonane-8- yl] -purin 20

20

12.73 g (41.5 mmol) of 2-amino-6-chloro-9 - [(1 R, 5R,

8R) -3oxo-2-azabicyclo [3.3.1] nonan-8-yl] -purine 19, 5.75 ml (41, 5 mmol) TEA, 31, 7 g

(145 mmol) pyrocarbonate di-tert.butyl ester and 150 mg DMAP for approx. 12 hours under

Reflux cooked. (TLC control: (CH ₂ CI ₂ / CH ₃ OH = 5: 1) This is worked up

The solvent is distilled off and the residue is purified by chromatography. (SiO ₂ :

EtOAc)

18.6 g (74%)

R _F = 0.74 (EtOAc)

HPLC:> 98% [LiChrospher RP 18, CH ₃ CN / (H ₂ O + 0.01 N TBAS) = 90:10]

[α] _D ²² = -62 ° (c = 1, 1; CH ₃ OH)

¹ H NMR (400 mHz, d ₆ -DMSO) 1, 36 u. 1, 48 (2s, 27H, 3tert.Bu); 1.60-1.85 (m, 3H);

2.02-2.13 (m, 2H); 2.17-2.27 (m, 1H); 2.35-2.49 (m, 2H); 2.77-2.89 (m, 1H); 4.85 (m,

1 H, HC- [NH]); 5.21 (m, 1H, CH-N); 8.99 (s, 1H, -CH =) Example 26

N ² -tert.Butoxycarbonyl-9 - [(1 R, 2R, 4R) -2-tert.butoxycarbonylamino-4-carboxymethyl-cyclohex-1 -yl] -guanine 22

22

4.1 g (6.75 mmol) of 2- [di- (tert-butylcarbonyl) amino] -6-chloro-9 - [(1 R, 5R, 8R) -3oxo-2-azabicyclo [3.3.1] nonan-8-yl] -purine 20 are placed in 30 ml of methylene chloride at 0 ° C. Then 2.3 g (32.5 mmol) of 3-hydroxypropionitrile, 3.84 g (65 mmol) of trimethylamine and 1.5 g (9.25 mmol) of DBU are added and the mixture is stirred for 16 hours at room temperature. After distilling off all volatile constituents under vacuum, the residue is acidified with aqueous KH ₂ PO solution and extracted several times with methylene chloride. After drying and evaporation, a solid residue (4.26 g) is obtained from the organic phase, which is dissolved in 145 ml of methanol and at -15 ° C. within 30 minutes with a solution of 1.52 g (36.2 mmol) of LiOH monohydrate in 36 ml of water. Then the mixture is stirred at -10 ° C. for 2 hours and at room temperature for 20 hours. For workup, the methanol is distilled off under vacuum, the aqueous residue diluted with a little water, then with dilute hydrochloric acid to p _H 3 and extracted with methylene chloride. The reaction product is purified by chromatography. (SiO2: acetone / CH ₂ CI ₂ / CH ₃ OH / EtOAc / H ₂ O / AcOH = 9: 6: 2: 2: 2: 1) 2.87 g (84%) mp. > 300 ° C (dec.)

RF = 0.79 (acetone / CH ₂ CI ₂ / CH ₃ OH / EtOAc / H ₂ O / AcOH = 9: 6: 2: 2: 2: 1) RF = 0.37 (CH ₂ CI ₂ / CH ₃ OH = 5: 1)

HPLC:> 98% [Cyclobond I, CH ₃ CN / (H ₂ O + 0.01 N TBAS) = 90:10] [α] _D ²² = + 3.41 ° (c = 1; CH ₃ OH) ¹ H-NMR (400 mHz, d ₆ -DMSO) 1, 18 u. 1, 48 (2s, 18H, 2tert.Bu); 1, 1-1.22 (m, 1H); 1.74-1.84 (m, 1H); 1.85-1.98 (m, 4H); 2.10-2.25 (m, 3H); 3.87-4.13 (m, 2H, CH- [NH], CH-N); 6.78 (d, 1H, NH-C [H]); 7.79 (s, 1H, N-CH = N); 11, 02, 11, 28 u. 12.1 (3s, 3H, NH, NH, CO ₂ H)

Example 27

N ² -di (tert-butylcarbonyl) -9 - [(1 R, 5R, 8R) -3oxo-2-tert-butoxycarbonylamino-2-azabicyclo- [3.3.1] nonan-8-yl] guanine 21

21

37.8 g (62.3 mmol) of 2- [di- (tert.butylcarbonyl) amino] -6-chloro-9 - [(1 R, 5R, 8R) -3oxo-2-tert.butoxycarbonyl-2- azabicyclo [3.3.1] nonan-8-yl] purine 20 are placed in 190 ml of methylene chloride. At 0 ° C., 21.4 ml (313 mmol) of 3-hydroxypropionitrile, 62.3 ml (approx. 660 mmol) of trimethylamine and 9.13 ml (59 mmol) of DBU are added and the mixture is then stirred for 15 hours at room temperature. Then all volatile constituents are distilled off in vacuo, the residue is mixed with 300 ml of aqueous KH ₂ PO solution and extracted with methylene chloride. After drying and evaporation, the organic phase gives a solid residue, which is treated with diisopropyl ether and suction filtered (about 27.2 g). The purification is carried out by chromatography. (SiO ₂ : EtOAc / CH ₃ OH = 10: 1) 25.5 g (69.5%) RF = 0.28 (EtOAc / CH ₃ OH = 5: 1) Example 28:

Solid phase synthesis of (?,?, R) -enatiomers or (S, S, S) -enantiomers of CNA oligomers.

28.1 Solid phase synthesis of the CNA oligomer (?) - CNA [H (AATAT) OH] 28.1.1 Loading of the support with C-terminal monomers 5 or 10. Tentagel S HMB resin (100mg; 28μmol; capacity = 0.28mmol / g) is placed in a 2 ml syringe and swollen with anhydrous DMF (1.5 ml) for 5 min at room temperature and then the solvent is removed. The monomer 5 (32 mg, 84 μmol) is suspended in anhydrous DMF (200 μl) and 200 μl of a solution of HATU (32 mg, 84 μmol) in anhydrous DMF and DIPEA (36 mg, 280 μmol) are added. After shaking gently for five minutes, a pale yellow solution is obtained. After adding the solution to the resin, the resulting suspension is shaken at room temperature for 8 hours. The reaction solution is then removed and the resin DMF (6 x 1.5 ml) and CH 2 Cl 2 (6 x 1.5 ml) washed and dried in vacuo.

The reaction cycle described is repeated using the same stoichiometric amounts of reactants, the solid phase being incubated with the monomers 5 or 10 overnight at room temperature. After the reaction has ended, the excess reactants are removed. The resin adduct obtained is washed with DMF (6 x 1.5 ml), CH 2 Cl 2 (6 x 1.5 ml) and diethyl ether (3 x 1.5 ml) and then dried thoroughly in vacuo.

After a sample of the monomers attached to the resin support has been split off, HPLC analysis (method A) provides a loading of 0.11-0.14 mmol / g. For the quantitative determination of the loading of the resin with the respective monomers, a defined amount (approx. 3-4 mg) of the resin (mHarz) is treated with 2N NaOH (150 μl) for 30 min at room temperature. The resin is then filtered off and washed twice with the same amount of 2N NaOH (150 μl). The filtrates are combined and made up to a volume of 1 ml with 2N NaOH. For the spectrometric examination of the filtrate, a 100 μl sample of this solution is taken and diluted to 1 ml with 2N NaOH. The absorbance of the filtrate diluted in this way is at 273 nm, in the case of the presence of CNA-thymine derivatives -1 -1 (^ 273nm ⁼ 1500 lτnol ^« cm), respectively at 260nm, in the presence of

-1 -1 CNA-adenine derivatives (E26O nm ⁼ 15400 l'mol -cm), determined. The capacity

X [mmol / g] of the resin can be calculated from this using the values obtained using the following formula:

X = V x E / (ε x d x m resin)

To cap free hydroxyl groups, the resin is suspended in CH2CI2 (700 μl) and with Ac2θ (206.7 mg; 2.0 mmol) and DIPEA (256.7 mg; 2.0 mmol) for 15 min if the adenine monomer is present, or 1 h if a thymine is present -Monomers, treated at room temperature. Excess reactants are then removed and the resin is washed with CH2Cl2 (10 × 1.5 ml) and then dried thoroughly.

28.1.2 Preactivation of Monomers 5 and 10

HATU (42 μmo) l (Perseptive Biosystems) is added to a solution of the monomers 5 or 10 (42 μmol) in anhydrous DMF (150 μl). The mixture is then shaken for 1 min. After five minutes of incubation of the resulting suspension with DIEA (10.9 mg, 84 μmol) at room temperature, a pale yellow solution is obtained. The monomer activated in this way can now be used for the solid phase reaction (oligomerization).

28.1.3 Checking the coupling efficiency

The efficiency of the coupling reaction is checked by RP-HPLC (method C). For this purpose, a sample of the resin (approx. 1 mg) is taken from the reaction mixture and treated with 2N NaOH / MeOH (90 μl, 1: 1) for 5-10 min. The sample solution is then neutralized with 2N HCl (90 μl) and analyzed by RP-HPLC.

28.1.4 Synthesis protocol (see Table 1):

100 mg of the resin (14 μmol; capacity: 0.14 mmol / g), loaded with the monomers 5 or 10, are placed in a syringe (2 ml). The N-terminal Boc protecting group is split off with TFA-CH ₂ CI ₂ 1: 1 (1.5 ml), process step 1) for 5 min at room temperature. The reaction solution is removed and fresh deprotection reagent [TFA-CH2CI2 1: 1 (1.5 ml)] is added and the mixture is incubated at RT for a further 30 min. The resin is washed with CH2CI2 (2 x 1.5 ml), neutralized with 1M DIEA in DMF (4 x 1.5 ml), then washed again with DMF (6 x 1.5 ml) and with CH2CI2 (6 x 1.5 ml) (process steps 2 to 4, Table 1). The Boc-deprotected resin is dried in a vacuum and then treated with anhydrous DMF (1.5 ml) for 5 min. After removal of the DMF, the solution of activated monomer 5 or 10 (42 μmol) (s 1.1.2) is added. The suspension is shaken for 4 hours (process step 5, Table 1). After coupling is complete, the supernatant is filtered off and the resin is washed with DMF (6 x 1.5 ml) and with CH2CI2 (6 x 1.5 ml)

(Process step 6, table 1). A sample of the resin-bound coupling product is taken for HPLC analysis (see method C above). Since the coupling is quantitative, a subsequent capping of free terminal amino functions is not necessary.

28.1.5 Cleavage of the N-terminally Substituted Oligomers from the Carrier After completion of the oligomerization, the product is cleaved from the carrier by treating the resin-bound oligomer with 2 ml of a 2M aqueous NaOH solution in MeOH (1: 1) at room temperature for two hours. After neutralizing the solution with 2M HCl, the oligomer obtained can be purified by RP-HPLC (Method D). The product-containing fractions are combined and concentrated by means of lyophilization.

ESI-MS: (f?) - CNA [H (AATAT) OH] (1360.7 [M + H] ⁺ , M _C alc- = 1360.7 RP-HPLC (Method C): (?) - CNA [H (AATAT) OH] (R _t = 15.0 min). Tm value = 27.4 °.

All CNA oligomers synthesized by this method are listed in Table 2 below. Table 2: Characterization of the CNAs using RP-HPLC and ESI-MS

(R) - (X) n and (S) - (X) _n stand for (1 'R, 2'R, 4'R) -CNA- [H (X) _n OH], (1' S, 2'S , 4'S) - CNA- [H (X) _n OH], X: are the monomer units T, C, G and A, phba: (HO) 2P (= O) -O- (CH2) 3-C (= O) radical, Lys: lysine residue, Ac: acetyl residue. b) Method B: Merck LiChrospher, 5 μm, 250 x 4.0 mm; Elution with water (0.1% TFA), and a linear gradient of MeCN [(0.1% TFA), 10-40% within 30 min]. c) Method C: Merck LiChrospher, 5 μm, 250 x 4.0 mm; Elution with water (0.1% TFA), and a linear gradient of MeCN [(0.1% TFA), 10-30% within 40 min]. d) The observed molecular weight was obtained from the single positively charged molecular ion of the respective CNA oligomer.

28.1.6 CNA / p-RNA hybridization

The oligonucleotide strands of the CNA and p-RNA were dissolved in 5 mM Tris HCl buffer in such a way that a CNA concentration of 5 μM and an equimolar p-RNA concentration of 5 μM was present. The solution was then examined in the temperature range from 35 ° C. to -8 ° C. (feed speed 80 s / ° C.) at a wavelength of 265 nm by absorption spectroscopy. The absorption values were recorded with a Perkin Elmer device (Lambda 2). FIG. 1A shows that even the short 5-mer heteroduplex (FIG. 1B) pairs in a stable manner. The melting point is 14 ° C. In addition, this example shows that mating also occurs under low salt conditions.

Example 29:

Improve the solubility of CNAs by introducing a

Butyrophosphatgruppe.

29.1 Addition of di (p-methoxyphenyl) phenylmethyl-protected 4-hydroxybutanoic acid to the N-terminus of the resin-bound pentamer (R) - CNA [H (AATAT) OH].

A solution of 4- (4,4-dimethoxytrityloxy) butanoic acid (57mg, 140 μmol) [14] and HATU (53 mg, 140 μmol) in DMF (400 μl) is added before the addition of DIEA (27 mg, 210 μmol) Stirred for 10 min, after a further 2 min the solution is added to 100 mg of the Tentagel S HMB resin carrier (0.14 mmol / g, 0.014 mmol) loaded with the pentamer residue (f?) - CNA [H (AATAT) OH]. The resin is then analogous to Process steps 1-4 (Table 1) treated. Finally, the supernatant is filtered off, the modified resin DMF (4 x 4 ml) and CH2Cl2 (2 x 4 ml) are washed.

29.2 Detritylation

For detritylation, the modified resin is treated with a 6% solution of dichloroacetic acid in CH2CI2 five times for two minutes each, washed with CH2CI2 (4 x 4 ml) and with MeCN (4 x 4 ml) and finally overnight under high vacuum over P2O5 dried.

29.3 Phosphorylation and Oxidation:

The dried modified resin is swollen for 10 min with 2 ml of a 0.5M solution of pyridinium hydrochloride in anhydrous MeCN. The reaction vessel is then placed under an Ar atmosphere and a solution of 0.5M pyridinium hydrochloride in MeCN (780 μl) and MeCN (145 μl) is added. A solution of bis- (2-cyanoethyl) -Λ /, Λ / -diisopropylamino phosphoramidite in anhydrous MeCN (75 μl, 300 mM) is then pipetted into the modified resin and shaken for 45 min. After the supernatant has been removed, fresh phosphorylation reagent is added to the resin once more and treated for 45 min. The reaction cycle described is carried out four times in total. The resin is then freed from the supernatant and washed with MeCN (6 × 1 ml) and then treated 3 × with 1.5 ml of an iodine-containing oxidation solution consisting of 5 mmol iodine, 223 mmol collidine in MeCN / H2θ (64:36) for 2 min. After complete oxidation, the phosphorylated oligomer is washed 5 times with 1.5 ml of a 0.5M solution of pyridinium hydrochloride in anhydrous MeCN and 5 times with 1.5 ml of MeCN.

29.4 Elimination of Cyanoethyl Groups

The loaded resin is treated for 15 h at room temperature with 1.5 ml of a 2.7M solution of DBU in MeCN and then washed with MeCN (6 x 1.5 ml) and with CH2Cl2 (6 x 1.5 ml). 29.5 Cleavage of the N-terminal substituted oligomer from the support

After the synthesis is complete, the phosphorylated product is left for two hours

Treatment at room temperature with 2 ml of a 2M aqueous NaOH solution in

Cleave MeOH (1: 1) from the support. After neutralization with 2M HCl, the phosphorylated oligomer crude product is purified by RP-HPLC (Method D). The oligomer fractions obtained are combined and concentrated by means of lyophilization.

ESI-MS: (K) -CNA [phba (AATAT) OH] (1527.0 [M + H] ⁺ , 764.5 [M + 2H] ²⁺ ; M _C alc- =

1526.6 RP-HPLC (Method C): () -CNA [phba (AATAT) OH] (R _t = 22.9 min). Tm value

= 33.6 °.

Example 30:

Improve the solubility of CNAs by directly introducing 4-

[[Bis (phenylmethoxy) phosphinyl] oxy] butanoic acid

30.1 Synthesis of 4-hydroxybutanoic acid methyl ester

Butyrolactone (12 g, 140 mmol), MeOH (130 ml) and conc. Sulfuric acid (8 drops) are refluxed together for 8 h. The mixture is allowed to cool to RT and NaHCO3 (1 g) is added with ice / salt cooling and the mixture is stirred for a further 10 min. The solid residue is filtered off and the solvent is removed on the RV. The colorless liquid obtained is purified by chromatography on silica gel (14 × 5.5 cm, Et2θ / CH2Cl2 3: 2). The pure ester fractions are combined and 1.5 g (12.7 mmol, 9%) of the ester result after removal of the solvent.

30.2 Synthesis 4 - [[bis (phenylmethoxy) phosphinyl] oxy] butanoic acid methyl ester

6.9 g (19.7 mmol) of tribenzyl phosphite are abs. CH2CI2 (70 ml) dissolved and at

0 ° C portions of iodine (4.59 g, 18 mmol) were added. The iodine dissolves within 30 minutes, so that a clear colorless solution results. HBU methyl ester (1.94 g, 16.4 mmol) are abs. CH2CI2 (150 ml) dissolved and with an ice / salt mixture on -

Cooled 10 ° C and added pyridine (5.3 ml = 5.18 g, 65.7 mmol). Now the solution of phosphorylation reagent is added dropwise within 30 min and the mixture is stirred for a further 10 min. After adding 20% citric acid (3 x 80 ml) shaken out and dried twice with water (80 ml) over Na2S04. Chromatography on silica gel (11.5 × 5.5 cm, 400 ml CH2Cl2, each 200 ml CH2Cl2 / MeOH 20: 1 and 15: 1 then with 10: 1 until the product elutes) provides the desired product (4.75 g , 12.1 mmol, 74%).

30.3 Synthesis of 4 - [[bis (phenylmethoxy) phosphinyl] oxy] butanoic acid 4 - [[bis (phenylmethoxy) phosphinyl] oxy] butanoic acid methyl ester (2 g, 5.3 mmol) is dissolved in MeOH (150 ml) with 1 M NaOH (15 ml) was added and the mixture was stirred at RT for 6 h. The reaction volume is concentrated to half at RT on the RV, mixed with 1 M NaOH (60 ml) and the solution extracted three times with Et2θ. The watery

Phase is isolated with conc. HCl acidified (pH 1-2) and extracted three times with ethyl acetate. The combined organic phases are dried over Na2SO4 and the reslutant milky suspension is purified on silica gel (8 × 5.5 cm, CH2Cl2 / MeOH 8: 1). 1.2 g of the desired product (3.3 mmol, 62%) are obtained.

30.4 Synthesis of (R) -CNA [phba (ATATT) OH]

65 mg (6 μmol) of the carrier coated with (R) -CNA [H (ATATT) OH] oligomer are placed in a syringe (2 ml). The N-terminal Boc protective group is removed by exposure to TFA-CH2CI2 1: 1 (1.5 ml) for 5 min. Fresh deprotection reagent is added and the Boc deprotection reaction is carried out for a further 30 min at RT. The reaction solution is removed, the support is washed with CH2CI2 (2x1.5 ml), neutralized with 1 M DIPEA in DMF (4x1.5 ml) and washed with DMF (6x1.5 ml) and CH2CI2 (6x1.5 ml) (operations 2-4). The Boc-deprotected carrier is then abs for 5 min. Swell DMF (1.5 ml) and filter off the solvent. 4 - [[Bis (phenylmethoxy) phosphinyl] oxy] butanoic acid (6.6 mg, 18 μmol) are abs. DMF (250 μl) dissolved, HATU (6.9 mg, 18 μmol) added and DIPEA (6.3 μl, 36 μmol) pipetted in. After about 2 minutes, a clear, slightly yellow colored solution is obtained, which is added to the Boc-deprotected solution. The coupling is carried out at RT for 4 h. After splitting off the benzyl protecting group with TFA-H2θ-triethylsilane (2 ml; 95: 2.5: 2.5), the phosphorylated pentamer results in almost quantitative yield. Example 31:

C-terminal peptide conjugation of a CNA oligonucleotide in solution.

31.1 Iodoacetylation of (S) -CNA [Ac (AATAT) -Lys-OH] on the ε-amino function of lysine

690 nmol (S) -CNA [Ac (AATAT) -Lys-OH are dissolved in 800μl 0.1 M sodium hydrogen carbonate / DMF (1: 2) and with 150 equivalents of N-succinimidyl iodoacetate (102 μmol, 28.9 mg) with exclusion of light Shaken for 2 hours. The

Product (S) -CNA [Ac (AATAT) -Lys (N ^ε -lodacetyl) -OH] is purified directly using preparative RP-HPLC and desalted using an RP-C18 cartridge. For poorly soluble oligonucleotides, DMSO with 3 equivalents of pyridine, based on the oligonucleotide used, can be used as the solvent mixture.

31.2 Conjugation of (S) -CNA [Ac (AATAT) -Lys (Nε-iodoacetyl) -OH] with H-Cys-Ser-Lys-Val-Gly-OH (conjugate 1).

106 nmol (S) -CNA [Ac (AATAT) -Lys (N ^ε -lodacetyl) -OH] are dissolved in 20μl DMF and then with a solution of H-Cys-Ser-Lys-Val-Gly-OH (256 nmol , 162 μg) in 20 μl DMF / EDTA-Borax-HCl buffer (pH = 7.5). The mixture is shaken for 2 hours with the exclusion of light until the oligonucleotide has reacted completely. The CNA conjugate (conjugate 1) is then purified directly using RP-HPLC and desalted using an RP-C18 cartridge.

Example 32:

N-terminal peptide conjugation of resin-bound CNA oligonucleotides

(Conjugate 2)

C-terminally bound to resin (S) -CNA [Lys (TTTTT) OH] (200 nmol, based on the oligonucleotide, loading 0.11 mmol / g, 2 mg resin) are mixed with 300 μl of 0.1M sodium hydrogen carbonate / DMF (1: 2) solution and 150 equivalents of iodoacetic acid N-succinimidyl ester (30 μmol, 8.5 mg) was added and shaken for 2 hours with the exclusion of light. The resin beads are then filtered and washed with DMF. The resulting iodoacetyl derivative (S) -CNA [(N ^ε -lodacetyl) -Lys (TIII ι) - resin] is then mixed with 400 nmol of H-Cys-Ala-Ala-Ala-Gly-NH ₂ in 200 μl DMF / EDTA -Borax-HCI buffer (pH = 7.5) (2: 1) implemented. After washing with DMF and dichloromethane, the product can be split off from the resin with 2M NaOH. After neutralization of the NaOH solution with 1 M HCl, the crude product is purified directly using RP-HPLC and desalted using an RP-C18 cartridge.

Example 33:

Fluorescent labeling of a CNA oligonucleotide in solution

A solution of 50 μl DMF and 780 nmol DIPEA is added to a solution of 130 nmol (R) -CNA [Ac- (CTGAA) -Lys] in 50 μl DMF. The combined solution is then reacted with a Cy5 labeling kit (approx. 150 nmol) (Amersham Pharmacia Biotech, Prod.Nr .: PA15001). The reaction mixture is shaken for 24 hours with the exclusion of light until the oligonucleotide is completely converted. The labeled oligonucleotide can be purified directly using RP-HPLC and desalted using an RP-C18 cartridge. The compound obtained is shown together with the associated mass spectrum in FIGS. 2A and B.

Claims

201nr03.wo claims:

1. A process for the preparation of CNA oligomers comprising the following process steps: a) activation of the carboxyl function of a CNA monomer block, b) subsequent esterification of this monomer block with a free hydroxyl function of the support under basic conditions and c) subsequent construction of the oligomer over repetitive cycles starting from the splitting off of the protective group on the amino group of the cyclohxyl ring of the CNA monomer and subsequent attachment of a further activated CNA monomer, CNA monomers of the formula (I) being used,

NHBoc formula (I)

wherein A, D and F can independently represent a -CR ³ R ⁴ -, -NR ⁵ -, -O- or -S- group and E for a -CR ⁶ - group, where R ³ , R ⁴ , R ⁵ or R ^{6 can} independently represent a hydrogen atom or a C 1 -C ₂ alkyl group and in which B represents a nucleobase, the primary amino groups of which can be present in unprotected or Boc-protected form.

A method according to claim 1, characterized in that the nucleobase B is selected from the group adenine, guanine, cytosine, thymine, uracil, isoguanine, isocytosine, xanthine or hypoxanthine.

3. The method according to any one of claims 1 or 2, characterized in that CNA monomers are used which are selected from the group 3-tert-butoxycarbonyl-1 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino- 4-carboxymethyl-cyclohex-1-yl] thymine (formula (II)), 1 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] thymine (Formula (III)), N ^6- tert-butoxycarbonyl-9 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -adenine (Formula (IV )), 9 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -adenine (formula (V)), N ⁴ - tert-butoxycarbonyl-1 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] cytosine (formula (VI)), 1 - [(1 R, 2R, 4R) -2 -tert- butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -cytosine (formula (VII), N ² -tert.-butoxycarbonyl-9 - [(1 R, 2R, 4R) -2-tert.-butoxycarbonylamino- 4-carboxymethyl-cyclohex-1-yl] guanine (formula (VIII)), 9 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1 -yl] guanine (formula (IX)) are selected.

4. The method according to any one of claims 1 to 3, characterized in that HATU, DIC, TBTU or HBTU is used to activate the carbonyl function of a CNA monomer block.

5. The method according to any one of claims 1 to 4, characterized in that the CNA oligomer or the CNA monomer unit is additionally coupled with hydrophilic groups, markers or biologically active substances.

6. The method according to claim 5, characterized in that phosphorylated butyric acid is used as the linker for coupling.

7. A process for the preparation of CNA monomers for CNA oligomer synthesis comprising the following process steps:

a) coupling of an iodine lactam of the formula (XIII)

(XIII)

in which A, D and F can independently represent a -CR ³ R ⁴ group and E represents a -CR ⁶ -, where R ³ , R ⁴ or R ^{6 have} the meaning given above, in the presence of a base on a Nucleobase from the group thymine, adenine, N ⁶ -benzoyl-adenine, N ⁶ -dimethylaminoethylene-adenine, cytosine, N ⁴ -benzoyl-cytosine or 2-amino-6-chloro-purine, b) subsequent protection of the nitrogen of the lactam ring with a Boc protective group and c) nucleophilic ring cleavage to the desired CNA monomer, the chlorine substituent being converted into a keto group if the 2-amino-6-chloro-purine is used.

8. The method according to claim 7, characterized in that 8-iodo-2-azabicyclo [3.3.1] nonan-3-one is used as iodine lactam.

9. The method according to claim 7 or 8, characterized in that the nucleophilic ring cleavage to the CNA monomer is carried out with LiOH or LiOOH.

10. The method according to claim 7 to 9, characterized in that the introduction of the Boc protective groups is carried out by adding di-tert-butyl pyrocarbonate in the presence of triethylamine and DMAP.

11. The method according to any one of claims 7 to 10 for the production of the thymine monomer, characterized in that a lithium salt of the thymine is used as starting material.

12. The method according to any one of claims 7 to 10 for the production of the guanine monomer, characterized in that the conversion of the chlorosubstitute is carried out in a keto group in the presence of (CH ₃ ) ₃ N, 3-OH-propionitrile and DBU.

13. CNA monomers for direct oligomerization with the structure 3-tert-butoxycarbonyl-1 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -thymine ( Formula (II)), 1 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethylcyclohex-1-yl] thymine (formula (III)), N ⁶ -tert.-butoxycarbonyl -9 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -adenine (formula (IV)), 9 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] -adenine (formula (V)), N ⁴ tert-butoxycarbonyl-1 - [(1 R, 2R, 4R) -2-tert.- butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] cytosine (formula (VI)), 1 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] - cytosine (formula (VII), N ² -tert.-butoxycarbonyl-9 - [(1 R, 2R, 4R) -2-tert.-butoxycarbonylamino-4-carboxymethyl-cyclohex-1-yl] guanine (formula (VIII )), 9 - [(1 R, 2R, 4R) -2-tert-butoxycarbonylamino-4-carboxymethylcyclohex-1-yl] guanine (formula (IX)) are selected.

14. Heteroduplice containing a CNA oligomer and a complementary p-RNA oligomer.

15. Heteroduplice according to claim 14 containing CNA oligomers with a 2 ^, -4 ^' - cyclohexyl polyamide backbone.

16. Heteroduplice according to claim 14 or 15, characterized in that the CNA oligomers are composed of monomers of the structural formula (XI), R2 / ,.

I D,

B

R1 structural formula (II)

in which B stands for a nucleobase and R1 for an NH ₂ group and R2 for a CH ₂ -COOH group and A, D and F independently of one another a - CR ³ R ⁴ -, -NR ⁵ -, -O- or - S group and E can be a -CR ⁶ group, where R ³ , R ⁴ , R ⁵ or R ^{6 can} independently represent a hydrogen atom or a C 1 -C ₂ -alkyl group.

17. Heteroduplice according to one of claims 14 to 16 with the structural formula (XII)

Structural formula (XII) where B and B ^'represent complementary nucleobase pairs.

18. Heteroduplice according to one of claims 14 to 17, characterized in that the CNA oligomer or the p-RNA oligomer is modified.

19. Heteroduplice according to claim 18, characterized in that the CNA oligomer and / or the p-RNA oligomer is modified with a peptide, a protein, a radioactive marker or a dye and / or the CNA oligomer with an N-terminal Phosphate group is modified.

20. Heteroduplice according to one of claims 18 or 19, characterized in that the modifications are terminal.

21. Heteroduplice according to one of claims 18 to 20, characterized in that N-terminal hydroxycarboxylic acid residues or terminal lysine residues are present as linkers on the CNA oligomer.

22. Modified CNA oligomers characterized in that they are covalently linked to a fluorescent dye.

23. Use of CNA-p-RNA heteroduplice in biotechnological assays.

24. Use of modified CNA oligomers according to claim 22 in biotechnological assays.