WO2004050231A2

WO2004050231A2 - (bio) organic oligomers for the preparation of macromolecules

Info

Publication number: WO2004050231A2
Application number: PCT/DK2003/000821
Authority: WO
Inventors: Kurt Vesterager Gothelf; Raymond S. Brown; Anne Thomsen; Morten Nielsen
Original assignee: Aarhus Universitet
Priority date: 2002-11-29
Filing date: 2003-11-28
Publication date: 2004-06-17
Also published as: AU2003283214A8; AU2003283214A1; WO2004050231A3

Abstract

A macromolecular architecture suitable for use in molecular electronics. And in the manufacture of conductors and semi-conductors has been synthesised using linear and branched oligomers of organic molecules. The incorporation of a bi-or tri-functional organic compound in an oligonucleotide chain and the application of these for formation of covalently linked organic and metal-organic oligomers has led to a novel molecular architecture. Furthermore, the iterative serial synthesis of linear and branched organic oligomers by automated methods such as DNA-synthesis or peptide synthesis, using bi- or tri-functional organic monomers is described herein. The novel compounds may be used to position and arrange nanoscale substrates such as biomolecules, biological structures, colloids, supramolecular structures forming covalently linked assemblies for use as conducting wires and components in electronic devices.

Description

MACROMOLECULAR ARCHITECTURES

FIELD OF THE INVENTION

The present invention relates to new methods for the synthesis of linear and branched oligomers of organic molecules. More specifically one part of the invention is directed to the incorporation of a bi- or tri-functional organic compound in an oligonucleotide chain and the application of these for formation of covalently linked organic and metal-organic oligomers. Another aspect of the invention is directed to the iterative serial synthesis of linear and branched organic oligomers by automated methods such as DNA-synthesis or peptide synthesis, using bi- or tri-functional organic monomers. The novel compounds embodied in the invention may be used to position and arrange nanoscale substrates such as biomolecules, biological structures, organic compounds, colloids, supramolecular structures and mixtures thereof. In particular the compounds and covalently linked assemblies of the compounds may be used as conducting wires and components in electronic devices.

BACKGROUND OF THE INVENTION

DNA-directed assembly of organic compounds and materials

The major obstacle for the realization of electronics based on single molecule components and the construction of nanomachines is the lack of ability to arrange and connect the single components. Top-down methods such as e.g. photo-lithography or scanning probe microscopy are either not suitable for single molecule manipulations or the methods cannot connect single molecules.

Bottom-up methods using- molecular self-organisation are another strategy for constructing nanostructures. The development in supramolecular chemistry has shown that complex geometric arrangements of components can be obtained by self-assembly (Marquis er a/., Angew. Chem. Int. End. 2002, 41, 2761 and references cited therein), however, for a more exact engineering of a desired nanostructure and the positioning of such, DNA is the most promising material, due to its unique base recognition capabilities (Storhoff and Mirkin, Chem. Rev. 1999, 99, 1849-1862).

It is one aspect of the present invention to apply organo-nucleotide hybrids for the modular construction of linear and two-dimensional organic oligomers. This part of the invention includes the parallel DNA-directed covalent coupling of several or many of the hybridized organo-nucleotide hybrids. The number of reports on organic reactions controlled by attached DNA-chains is limited to few papers. In these reports it has only been possible to direct the formation of covalent bonds between two, different or identical compounds using a DNA template. In the first 5 example Saito and co-workers reported a photoreversible DNA-template dependent coupling of oligonucleotides through 5-vinyl-deoxyuridine (Fujimoto ef al. J. Am. Chem. Soc. 2000, 122, 5646-5647). Czlapinski and Sheppard demonstrated that two salicylaldehyde derivatives, functionalized with a spacer element and an oligonucleotide chain in the 5-position of salicylaldehyde, could specifically assemble to a single metal-

10 salen complex by ethylenediamine and Mn(OAc)₂ (or Ni(II)⁺²), when the reaction was templated by a third DNA or RNA chain. (Czlapinski and Sheppard, T. L.; J. Am. Chem. Soc. 2001, 123, 8618-8619). In two papers Liu and co-workers have demonstrated that a variety of organic reactions could be directed by attachment of the two reacting compounds to complementary oligonucleotides. (Gartner ef al. Angew. Chem. Int. Ed.

15 2002, 41, 1796; Gartner et al. Angew. Chem. Int. Ed. 2002, 41, 1796). They showed that reactions such as nucleophilic addition, nucleophilic substitution, amide formation, reductive amination, Henry reaction, nitro-Michael addition, Wittig reaction, 1,3-dipolar cycloaddition and Heck reaction could be controlled by the DNA-templates and the reactions selectively proceeded only when attached to complementary sequences.

20 However, the methods described above for DNA-directed synthesis cannot be applied for the reaction between three or more organo-nucleotide components.

In a different approach assembly several organo-DNA components via DNA hybridization of the two DNA strands of the organic compound with complementary hybrids has been

25 attempted. Shi and Bergstrom applied a rigid tetrahedral linker, which was functionalized at each terminus with the same self-complementary oligonucleotide. This type of compound assembled by hybridization into a series of discrete cyclic supramolecular structures (Shi and Bergstrom Angew. Chem. Int. Ed. 1997, 36, 111-113). In a more recent example Bunz and co-workers applied linear oligophenylacetylenes or linear

30 organometallic complexes having a DMTr group at one terminus and a phosphoramidite group at the other terminus (Waybright ef a/., 3. Am. Chem. Soc. 2001, 123, 1828-1833). These compounds were incorporated into a DNA strand by automated DNA-synthesis. In this manner the organic compound is in principle functionalized with a DNA strand in each terminus. Several of these compounds were assembled into programmed linear (not

35 geometrical linear) supramolecular structures by pairing of complementary DNA-strands. However, both of the methods described above cannot be applied for the formation of covalent bonds between the organic residues and thus the stability and geometric control of the assemblies obtained is limited. Organic Oligomers by automated synthesis

Iterative synthesis is a useful method for the synthesis of organic oligomers. Especially the fully automated solidphase synthesis of peptides (Merrifield Adv. Enzymoi. 1969, 32,221-) and oligonucleotides (Beaucage and Iyer Tetrahedron 1992, 48, 2223-) are extremely important techniques for making small peptides and short DNA fragments and a variety of peptide and DNA-synthesizers are commercially available. The above described automated oligomer syntheses are based on the formation of two fundamental functional groups: amide for the peptide synthesizers and phosphate esters for the DNA synthesizers. These reactions have been highly optimized to allow for the synthesis of oligomers consisting of up to more than 100 units (amino acids or nucleotides). In addition to the synthesis of peptides based on naturally occurring amino acids peptide synthesizers can also be applied to produce oligomers of other amino acids including PNA (Peptide Nucleic Acids).

Iterative synthesis of other types of organic oligomers based on the formation other functional groups have also been developed (Feuerbacher and Vδgtle Top. Curr. Chem. 1998, 197, 1-18). One of the methods related to the present invention is the iterative synthesis of phenylacetylene dendrimers (Xu and Moore Angew. Chem. 1993, 105, 1394- 1397) and linear oligophenylacetylenes (Zhang et al. J. Am. Chem.1992, 114, 2273-2274, Tour Chem. Rev. 1996, 96, 537-553). These syntheses are based on the repeated Sonogashira coupling between terminal acetylenes and haloarenes. In this manner Tour and coworkes synthesized a geometrically linear conducting wires having a length of 128 A. (Schumm et al. Angew. Chem. Int. Eng. Ed. 1994, 33, 1360-1363, Jones ef al. J. Org. Chem. 1997, 62, 1388-1410). One limitation of the above described procedure is that only oligomers consisting of n² (n < 5, i.e. 2,4,8,16) phenylacetylene units can be formed. It is one aspect of the present invention to provide a fundamentally new method also for the iterative synthesis of linear and branched organic oligomers, which can for example be used as rigid organic wires.

Molecular electronics and Nanotechnology Molecules studied as molecular wires have mainly been conjugated organic molecules and carbon nanotubes. Among the most popular systems studied are oligo(phenylacetylene) organic wires. This type of wires have the advantage that they possess a rigid linear highly conjugated structure in which the delocalized network of π-symmetry orbitals provides a pathway through which the movement of electrons is easy (Reed and Tour Sci. Am. 2000, June, p68-75). Tour and coworkes demonstrated that a 128 A long molecular wire of this type could be synthesized by solution or solidphase synthesis (Jones II et al., J. Org. Chem. 1997, 62, 1388-1410). More complex derivatives such as oligo(phenylacetylene) having substuents that alter the electronic proberties or oligo(phenylacetylene) chains incorporating e.g. a porphyrin unit have been prepared (Dirk et al, Tetrahedron 2001, 57, 5109-5121; Tour et al, Chem. Eur. J. 2001, 7, 5118-5134). A number of potential molecular components have also been synthesized, including switches, rectifiers, memory devices and recently also transistors and in several cases these individual components have been tested (Joachim et al, . Nature 2000, 408, 541-548; Park et al, Nature 2002, 417, 722-725; Liang et al, Nature 2002, 417, 725-729, WO 02/35580 A2, WO 02/09117 Al, WO 01/27972 A2). The most frequent method for testing of the electronic properties of single molecular components is either by trapping a molecule at each electrode in a break junction or by scanning probe techniques on self-assembled monolayers containing the component (Reed et al. Science 1997, 278, 252-254; Mayor et al, Angew. Chem. Int. Ed. 2002, 41, 1183; Fan et al. J. Am. Chem. Soc. 2002, 124, 5550-5560). Despite the availability of both wires and components the major obstacle for the realization of molecular electronic circuits remains to be how the molecular components are connected to each other. One aspect of the present invention is to provide a solution to this problem. The application of carbon nanotubes is an alternative approach to molecular electronics. Carbon nanotubes are easier to visualize and to some extent easier to handle due to their increased size and they have a number of unique electronic properties (Baughman et al. Nature 2002, 297, 787 and references cited therein). However, the lacking ability to connect single carbon nanotubes into circuits is also for these system the major unsolved problem in the realization of molecular electronics.

SUMMARY OF THE INVENTION

The present invention provides methods for the preparation of a macromolecular architecture, cf. claims 1, 22 and 45. The invention furthermore provides compounds useful in these methods, cf. claims 43 and 72, as well as macromolecular architectures obtainable from said methods, cf. claim 76, and various uses of the macromolecular architectures, cf. claims 80-83.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Schematic representation of the LANA (Linear Activated Nucleotide Analog) and its conversion by automated oligonucleotide synthesis into the LANAO (Linear Activated Nucleotide Analog in Oligonucleotide). The protective group (PG¹) can be a dimethoxytrityl group or other protective groups compatible with nucleotide synthesis. The Phos group is an phosphorous moiety e.g. a phosphoramidite that can be activated to react with a hydroxygroup. The organic compound contains functional groups F¹ and F² in each terminal. The functional groups can be activated to make a covalent coupling between to a first and a second organic compound. The functional groups may be different or preferably identical (F¹ = F² = F).

Figure 2. Schematic representation of the TANA (Tripoidal Activated Nucleotide Analog) and its conversion by automated oligonucleotide synthesis into the TANAO (Tripoidal Activated Nucleotode Analog in Oligonucleotide). The protective group (PG¹) can be a dimethoxytrityl group or other protective groups compatible with nucleotide synthesis. The Phos group is an phosphorous moiety eg. a phosphoramidite that can be activated to react with a hydroxygroup. The protective group PG² can be a Fmoc group of any other protective group compatible with automated oligonucleotide synthesis and orthogonal to PG¹. The organic compound contains functional groups F¹ and F² in each terminal. The functional groups can be activated to make a covalent coupling between to a first and a second organic compound.

Figure 3. Schematic presentation of the oligonucleotide directed coupling of L-ANAO compounds into CALTO (Covalently assembled L-ANAO and/or TANAO Oligonucleotide functionalized) and CALT (Covalently assembled LANA and/or TANA) compounds. Step 1: The l-ANAOs are mixed under conditions at which complementary DNA-strands i.e. the pairs o2 and o2', and o3 and o3' hybridize and assemble the L-ANAOs. Step 2: The functional groups are activated for reaction and a covalent bond between the three

L-ANAOs under formation of a CALTO compound. Step 3: The oligonucleotide strands are cleaved from the CALTO compound to give a CALT compound.

Figure 4. Example of the oligonucleotide directed coupling of two TANAO compounds into CALTO and CALT compounds. Step 1: The TANAOs are mixed under conditions at which complementary DNA-strands 06 and 06' hybridize and assemble the TANAOs. Step 2: The functional groups are activated for reaction and a covalent bond between the two TANAOs under formation of a CALTO compound. Step 3: The oligonucleotide strands are cleaved from the CALTO compound to give a CALT compound.

Figure 5. Example of the oligonucleotide directed coupling of three LANAO compounds and two TANAO compounds into CALTO and CALT compounds. Step 1 : The compounds are mixed under conditions at which complementary DNA-strands hybridize. Step 2: The functional groups are activated for reaction and a covalent bond between the five compounds under formation of a CALTO compound. Step 3: The oligonucleotide strands are cleaved from the CALTO compound to give a CALT compound.

Figure 6a. Example of the oligonucleotide-directed coupling applying L-ANAO and TANAO compounds for which the backbone is based on phenylacetylene units and the functional groups are a salicylaldehyde derivatives. Step 1: The three compounds are brought together by hybridization of the oligonucleotide chains. Step 2: Addition of ethylene diamine and a metal salt such as Mn(OAc)₂ makes the functional groups having nearby hybridized oligonucleotide chains react under formation of two metal-salen complexes. Step 3: The oligonucleotide strands are cleaved from the CALTO compound by chemical or biological methods to give a CALT compound.

Figure 6b. Examplified method for the assembly of LANAO (or TANAO) compounds having no complimentary oligonucleotide strands by the use of a oligonucleotide template. Step 1: The nucleotide template is added and undergoes hybridization with the two LANAOs. Step 2: The salicylaldehyde moieties of the termini having hybridized oligonucleotides undergo formation of the metal-salen complex in the presence of a 1,2-diamine and a suitable metal salt.

Figure 7. Schematic illustration of the indirect iterative synthesis of DCALT (Double

Covalently Linked LANA and TANA) and CALT compounds based on the methods applied for iterative oligonucleotide synthesis. Step 1 : the first LANA compound is linked to the solid support via a spacer. Step 2: A number of n LANA compounds are linked to each other in n cycles in iterative oligonucleotide synthesis. Step 3 the oligo-LANA compound is cleaved from the solid support. Step 4 The functional groups are activated for the formation of a second covalent bond between the single LANA compounds in oligo-LANA to give DCALT. (The order of step 3 and 4 may be reversed). Step 5: The first covalent bond removed by chemical cleavage of the phosphate moieties or the linkers to give the CALT compound.

Figure 8. Schematic illustration of the indirect iterative synthesis of DCALT (Double Covalently Linked LANA and TANA) and CALT compounds involving the application of a TANA. Step 1 and oligo-LANA compound containing one TANA moiety is synthesized by standard oligonucleotide synthesis. Step 2: The terminal OH group of the oligomer is blocked with a protective group PG³. Step 3: The protective group PG² of the third branch of TANA is removed, Step 4: The iterative synthesis is continued from the liberated OH group by introducing further LANA (and possibly also TANA) compounds. Step 5: the oligo- LANA/TANA compound is cleaved from the solid support and the functional groups are activated for the formation of a second covalent bond between the single LANA and TANA compounds to give DCALT. (The order of cleavage and coupling may be reversed). Step 6: The first covalent bond removed by chemical cleavage of the phosphate moieties or the linkers to give the CALT compound.

Figure 9. An example of the indirect iterative synthesis of a salen linked CALT compound. Step 1. The three components are linked by phosphate derivative by iterative synthesis and the oligomer is cleaved from the solid support. Step 2: By addition of a 1,2-diamine and a suitable metal salt the salen moieties are formed making a second covalent link between the modules. Step 3 the first covalent bond is cleaved to give the CALT compound.

Figure 10. A list of compounds for iterative (oligonucleotide or peptide) synthesis embodied in the invention. For iterative synthesis using the oligonucleotide approach PG is a protected hydroxygroup for which PG¹ is a base labile protective group such as a trityl derivative and A² is a phosphorous derivative such as a phosphoramidite. PG² is a protective group which is orthogonal to PG¹. For application in automated peptide synthesis PG is a protected amine and A² is a carboxylic acid or any derivative thereof. PG² is a protective group which is orthogonal to PG¹.

Figure 11. Schematic illustration of the indirect iterative synthesis of DCALT and CALT compounds based on the methods applied for iterative peptide synthesis. Step 1: the first

LPA (Linear Peptide Analog) compound is linked to the solid support via a spacer. Step 2: A number of n LPA compounds are linked to each other on n cycles in an iterative synthesis.

Step 3: the oligo-LANA compound is cleaved from the solid support. Step 4: The functional groups are activated for the formation of a second covalent bond between the single LPA compounds in oligo-LPA to give DCALT. (The order of step 3 and 4 may be reversed). Step

5: The first covalent bond removed by chemical cleavage of the phosphate moieties or the linkers to give the CALT compound.

Figure 12. Schematic illustration of the indirect iterative synthesis of DCALT and CALT compounds involving the application of a TPA (Tripoidal Peptide Analog). Step 1 : An oligo- LPA compound containing one TPA moiety is synthesized by standard iterative peptide synthesis. Step 2: The terminal NH₂ group of the oligomer is blocked with a protective groups PG². Step 3: The protective group PG¹ of the third branch of TPA is removed. Step 4: The iterative synthesis is continued from the liberated NH₂ group by introducing further LPA (and possibly also TPA) compounds. Step 5: the oligo-LPA/TPA compound is cleaved from the solid support and the functional groups are activated for the formation of a second covalent bond between the single LPA and TPA compounds to give DCALT. (The order of cleavage and coupling may be reversed). Step 6: The first covalent bond removed by chemical cleavage of the phosphate moieties or the linkers to give the CALT compound.

Figure 13. Illustration of the formation of the second covalent bond after coupling of the modules by iterative synthesis (by the peptide or oligonucleotide methods). In this example the second covalent bond is formed in a Sonogashira coupling using acetylene as the linker leading to the formation of a rigid phenylactylene-based DCALT compound. Figure 14. A series of reactions which can be applied for the formation of rigid linear bonds between aryl and/or arylacetylene groups.

Figure 15. Synthesis of prototypes 1 and 2 of the linear and tripoidal compounds, respectively.

Figure 16. Synthesis of the protected salicyl terminus 8 containing an ester linker.

Figure 17. Synthesis of the protected salicyl termini 10a and 10b containing amide linkers.

Figure 18. Final steps in the synthesis of the LANA 13 containing an ester linker.

Figure 19. Final steps in the synthesis of the LANA 16 containing an amide linker. Figure 20. Synthesis of the symmetric TANA compound 20.

Figure 21. Synthesis of the unsymmetric TANA compound 23.

Figure 22. Oligonucleotide sequences on L-ANAO¹, L-ANAO², L-ANAO³ and TANAO¹.

Figure 23. a) Programmed assembly of LANAO compounds, a) PAGE under denaturing conditions of the coupling reactions involving LANAO¹, LANAO² and L-ANAO³. Column 1: L-ANAO¹ and L-ANAO², Column 2: LANAO² and LANAO³, Column 3: LANAO¹ and LANAO³, Column 4: L-ANAO¹, L-ANAO² and L-ANAO³, Column 5: L-ANAO¹ and LANAO² no Mn(OAc)₂, Column 6: L-ANAO¹ and L-ANAO² no ethylenediamine, Column 7: L-ANAO¹ and L-ANAO² no ethylenediamine and no Mn(OAc)₂, Column 8: DNA marker.

Figure 24. PAGE under denaturing conditions of the coupling reactions involving TANAO¹, L-ANAO¹ and L-ANAO². Column 1: TANAO¹ (no coupling reaction) Column 2: TANAO¹ and LANAO³, Column 3: TANAO¹ and 2 equivalents of L-ANAO¹, Column 4: TANAO¹, LANAO¹ and L-ANAO³

Figure 25. Proposed synthesis of the LPA (Linear Peptide Analog) 28.

Figure 26. Proposed synthesis of the TPA (Tripoidal Peptide Analog) 31.

Figure 27. Proposed synthesis of the LANA 34. Figure 28. Proposed synthesis of the TANA 37. The conversion of 36 into 37 is performed as the conversion of 17a into 23 (see Figures 20 and 21).

Figure 29. Exemplified iterative solid phase synthesis of an Oligo-LPA compound followed by formation of a ethynyl bond between the single modules in the oligomer to give a DCALT compound.

Figure 30. Examples of attachment of macromolecular architectures to electrodes El, E2, E3 and E4.

Figure 31. Right-hand side: a set of 100*4 electrodes that can be addressed individually, having 4-armed macromolecular architectures bridged between each of the sets of 4 electrodes. Left-hand side: enlargement of a single macromolecular architecture trapped in between 4 electrodes.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods for the preparation of macromolecular architectures that can be useful within various fields of nano-technology. According to the novel methods, simple compounds can - as building blocks - be used to create macromolecular architectures of predetermined (pre-programmed) structure. The methods offer tools for the preparation of particularly designed structures.

Method involving hybridisation of oligonucleotides

In a first method for the preparation of a macromolecular architecture, the specific hybridisation of oligonucleotides assembles individual compounds so as to form the macromolecular architecture in a predetermined manner whereafter covalent links are introduced in order to "freeze" said structure. The oligonucleotides may thereafter be removed from the structure.

In particular, the first method comprises the following steps:

(a) providing at least three organic compounds, including a first compound, a second compound and a third compound, each compound comprising at least two structural domains, including a first structural domain and a second structural domain, each structural domain comprising in proximity (i) one or more functional groups and (ii) an oligonucleotide chain, the oligonucleotide chain of the first structural domain of the first compound being at least partly complementary to the oligonucleotide chain of the second structural domain of the second compound, and the oligonucleotide chain of the second structural domain of the first compound being at least partly complementary to the oligonucleotide chain of the first structural domain of the third compound; and

(b) hybridising the oligonucleotide chain of the first structural domain of said first compound to the at least partly complementary oligonucleotide chain of the second structural domain of said second compound, and hybridising the oligonucleotide chain of the second structural domain of said first compound to the at least partly complementary oligonucleotide chain of the first structural domain of said third compound, thereby establishing base-paired hybrids, whereby the functional group(s) of the first structural domain of said first compound is brought in proximity to the functional group(s) of the second structural domain of said second compound, and the functional group(s) of the second structural domain of said first compound is brought in proximity to the functional group(s) of the first structural domain of said third compound; and

(c) establishing a covalent link between the first structural domain of said first compound and the second structural domain of said second compound, and establishing a covalent link between the second structural domain of said first compound and the first structural domain of said third compound; and

(d) optionally partly or completely cleaving the oligonucleotide chain from the first structural domain of the first compound and from the second structural domain of the second compound, and partly or completely cleaving the oligonucleotide chain from the second structural domain of the first compound and from the first structural domain of the third compound.

re Step (a)

Initially, at least three organic compounds, including a first compound, a second compound and a third compound, are provided. It should be understood that due to the complexity and size of the macromolecular architecture, a larger number of organic compounds may be used, such as for example up to several hundred or up to thousand, compounds, e.g. 10-1000 compounds. It should also be understood that the first compound, the second compound, etc. may be structurally similar, and that the denomination "first compound", "second compound", etc. are used to differentiate between particular molecules and to explain the mode of design and hybridisation. However, as will be understood from the following, one major advantage of the present method is that a variety of compounds may be used and that the hybridisation "match" between the oligonucleotides of the compound can be used to determine the structuring of compounds and thereby the structure of the macromolecular architecture.

Each of the at least three organic compounds comprises at least two structural domains, namely a first structural domain and a second structural domain. Each of the domains comprises a domain core, one or more functional groups, and an oligonucleotide chain, where the oligonucleotide chain may be linked to the domain core via a spacer. The at least two domain cores may be linked directly together or may be linked together via a scaffold. In the latter instance, the scaffold may carry further domains. Thus, in one embodiment, at least one of the first compound, the second compound, and the third compound comprises at least three structural domains, including a first structural domain, a second structural domain and a third structural domain. In a more particular embodiment, at least one of the first compound, the second compound, and the third compound comprises at least four structural domains, including a first structural domain, a second structural domain, a third structural domain, and a fourth structural domain.

In the embodiment where all compound only have two domains, the resulting macromolecular architecture is said to be "linear" meaning that the assembly of compounds results in an extending chain or wire, i.e. extending in "one dimension". Such a construction may then be "linear" in the geometrical sense, or may be curved, coiled, etc.

The purpose of introducing organic compounds comprising more than two structural domains is to make branches in the macromolecular architecture. Providing organic compounds with more than two structural domains makes it possible to build macromolecular architectures in more than one dimension. In a particular example, the presence of a larger number of organic compounds comprising two structural domains and one organic compound comprising three structural domains, makes the organic compound with three structural domains a potential branching point in said particular macromolecular architecture. Thus, said macromolecular architecture might potentially extend in two dimensions or potentially also in three dimensions. In the embodiment where compounds having three structural domains are used predominantly or exclusively, it is possible to create a framework of hexagonal structures.

Thus in one embodiment, the use of a larger number of organic compounds exclusively comprising two structural domains, including a first structural domain and a second structural domain, result in a one-dimensional macromolecular architecture.

In a further embodiment the use of a larger number of organic compounds having two structural domains, and the use of a larger number of organic compounds having three structural domains, result in a two-dimensional architecture. This two-dimensional architecture might have one, two or multiple branching points. The number of organic compounds having three structural domains in relation to the number of organic compounds having two structural domains determine how many branching points are provided.

In a further embodiment, the use of a larger number of organic compounds having two structural domains, three structural domains and four structural domains may result in a three-dimensional architecture. This three-dimensional architecture possibly has multiple branching points. The number of organic compounds having three and four structural domains in relation to organic compounds having two structural domains determine how many branching points are provided.

Thus, this first method allows a particularly designed combination of organic compounds comprising two, three or four structural domains. Also, the number of and ratio between said organic compounds comprising two, three or four structural domains may be used to determine the overall form of the macromolecular architecture. Thus, the huge flexibility in the number, the ratio between and the combination of said organic compounds comprising two, three or four structural domains gives a possibility to build macromolecular architectures in a predetermined geometric manner.

When turning to the individual domains, it is presently believed that the domain core thereof advantageously consists of a rigid structure such as a carbocyclic or heterocyclic structure. In some presently preferred embodiments, the domain core of the structural domains is an aryl group or heteroaryl group, e.g. an aryl group, such as a phenyl group or naphthyl group, in particular a phenyl group. Such a domain core may, as stated above, be linked directly to other domains or may be linked to the scaffold.

In one embodiment, the domain core (e.g. a phenyl group) carries one functional group and one oligonucleotide chain, where the oligonucleotide chain preferably is linked to the domain core via a spacer. Alternatively, the domain core carries two functional groups in addition to said oligonucleotide chain. In a further alternative, the domain core has three functional groups in addition to said oligonucleotide chain.

In the embodiment where the domain core is a phenyl group, the position of said functional groups on the phenyl ring of course influences the overall structure of the macromolecular architecture. For the embodiments where the domain core is a phenyl ring, the positions of the oligonucleotide chain and the functional groups can be defined relative to position for attachment of the domain core to another domain core or the scaffold.

In one embodiment the phenyl ring has one functional group. If the domain core has just one functional group in addition to the oligonucleotide chain, said functional group may be attached to said phenyl ring in the para-position and the oligonucleotide chain may be attached in the ortho- or meta-position. In another embodiment, the functional group may be attached to the phenyl ring in meta-position and the oligonucleotide chain may be attached in the second meta-position or in the para-position. In a yet further embodiment, the functional group may be attached to the phenyl ring in ortho-position and the oligonucleotide chain may be attached in the one of the ortho-positions or in the para- position. Any second functional group is preferably positioned in the ortho-position relative to the first functional group.

The scaffold defines the spatial orientation of the two or more structural domains of the compounds, and will therefore influence on the structure of the macromolecular architecture. It is envisaged that the scaffold in one embodiment may present two structural domains in a more or less linear manner thereby facilitating a geometrically linear structure, which may be useful as electrically conductive wires, etc.

With the aim of reducing the flexibility of the macromolecular architecture, it is believed that scaffolds comprising moieties selected from aryl groups, heteroaryl groups, ethylene groups, acetylene groups, etc. are particularly relevant. It is presently believed that aryl groups, heteroaryl groups and acetylene groups, such as phenyl groups and acetylene groups, are particularly relevant.

Illustrative examples of scaffolds are acetylene, acetylene-phenyl-acetylene, acetylene- phenyl-acetylene-phenyl-acetylene (see Formulae 1 and 3), 1,3,5-triacetylenebenzene (see Formulae 2 and 4), tetraacetylenemethane, diacetylene, biarylene such as biphenylene, triarylene such as triphenylene.

In one embodiment, when taken together the domain cores and the scaffold of at least the first compound comprises an arylacetylene motif, such as a phenylacetylene motif. In another embodiment, when taken together the domain cores and the scaffold of at least the first compound comprises a arylethynylaryl motif, such as a phenylacetylenephenyl motif. As mentioned above, each of the structural domains comprises an oligonucleotide chain optionally linked to the domain core via a spacer. Although the oligonucleotide may be linked directly to the domain core, it is envisaged that the oligonucleotide preferably is linked via a spacer of 1-50 atoms, such as 2-50 atoms, typically 2-40 atoms, such as 2-30 atoms or 2 to 20 atoms, in particular 3-10 atoms.

The oligonucleotide chains have some special key functions. For the first the pairing of the chains brings the functional groups in proximity, enabling the functional groups to react and form a covalent link. The formation of this covalent link is critical for the stability and rigidity of the macromolecular architecture. The covalent link ensures that the architecture is rigid and tightly bound. Another key idea of using oligonucleotide chains is the means of nature's own machinery for recognition. Choosing appropriate oligonucleotide chains enable the chains to recognise at least partly complementary chains and thereafter self- hybridise. This gives a possibility to create a macromolecular architecture in a predetermined manner.

When used herein, the term "oligonucleotide" and similar expressions are intended to cover any sequence of nucleotides, nucleotide analogues, nucleotide derivatives, and nucleotide mimetics, and hybrids thereof. Examples hereof are DNA, RNA, LNA, PNA, an hybrids thereof etc. The important feature of the selected "oligonucleotide" is the ability to form hybrids with another oligonucleotide.

In the present method it is provided that an oligonucleotide chain of one structural domain is at least partly complementary to an oligonucleotide chain of another structural domain for the oligonucleotide chains to hybridise. The meaning of "at least partly" refers to the situation wherein one or more mismatches occur between two oligonucleotide chains, or where one oligonucleotide is slightly longer than the otherwise "complementary" oligonucleotide.

In the present method it is sufficient that the oligonucleotide chains is only partly complementary for the hybrid to be established. The maximum number of mismatches between two oligonucleotide chains should at the highest be 20%. Mismatches also include the situation where a nucleotide unit lacks the nucleobase. Preferably, however the oligonucleotide chains should be complementary.

The each oligonucleotide typically has a length of 4-30 nucleotides. For oligonucleotides shorter than 4, it is believed that the hybridisation would be too weak, and oligonucleotides of more than 30 nucleotides of length are not believed to provide further advantages. In a presently preferred embodiment the oligonucleotide chain comprises 6-20 nucleotides, in particular 8-16 nucleotides.

Overall, the melting temperature (T_m) of the hybridised oligonucleotide chains is of relevance for practical reasons. Thus it is presently believed that the T_m of the hybridised oligonucleotides should be at least 15°C for the annealing between the oligonucleotide chains to take place. It is presently believed that the T_m advantageously should be at least 20°C, in particular at least 25°C.

In one particular embodiment the oligonucleotide chain is connected to the domain core via a spacer. The spacer typically has a length of 1-50 atoms, such as 2-50 atoms, more typically 2-40 atoms, such as 2-30 atoms or 2-20 atoms, preferably 2-10 atoms.

The spacer may be considered as a saturated or partly unsaturated straight or branched hydrocarbon chain optionally terminated or interrupted by one or more atoms or groups selected from -O-, -S-, -S-S-, -S0₂-, -P(0,S)-, -P(S)₂-, -P(0)₂-, -P(0,NR^a)-, -PO(CH₃)-, - PO(NHR^a)-, -N(R^a)- wherein R^a is selected from hydrogen, Cι-₄-alkyl and amine protecting groups, >C=0, >C=S, arylene (such as phenylene), and heteroarylene. Thus, the spacer may comprise one or more amide, ester, thioester, amino, disulfide, ether, phosphate, and thioether, etc. functionalities, and optionally aromatic or mono/polyunsaturated groups. The spacer may furthermore be substituted as defined above for "alkyl" and "aryl"/"heteroaryl". More specific examples are alkylenes, alkenylenes, polyoxyethylene such as polyethylene glycol, oligoamides (e.g. peptides) such as poly-β-alanine, polyglycine, and polylysine. Even more specific examples are -(CH₂)₂-₂₀-, ^_((CH₂)₀-ιo- CH=CH(CH₂)o-ιo)-, -((CH₂)o-ιoO(CH₂)o-₁₀)-, -((CH₂)₀-₁₀S(CH₂)₀-₁₀)-, -((CH₂)₀-₁₀NR^a(CH₂)₀-₁₀)-, and -COO(CH₂)_!-₂₀, and branched and substituted variant thereof.

In a particular preferred embodiment the oligonucleotide chain is connected to the spacer via a phosphate diester, alternatively a thiophosphate ester, alternatively a phosphate diester.

As mentioned above, the oligonucleotide chain is attached the domain core preferably via a spacer. The oligonucleotide chain is in one presently preferred embodiment attached to the spacer via a terminal nucleotide in said oligonucleotide chain, i.e. either via the 3'-end or via the 5'-end. In another embodiment the oligonucleotide chain is attached via any other position in the chain, i.e. either via the sugar moiety, via the nucleobase moiety or via the phosphate moiety. Preferably, however, the oligonucleotide is linked via a terminal nucleotide. In the present method it is suggested that the oligonucleotide chain (via the spacer) and the functional group(s) are in close proximity to each other. This implies that the hybridisation of oligonucleotides of two structural domains brings the functional groups of said domain together to form a covalent link. The spatial proximity between the oligonucleotide chain and the functional groups in each structural domain makes the virtual concentration of contacting functional groups higher and thus increases the possibility of a reaction between said functional groups. The likelihood of establishing a covalent link between said functional groups are higher as the functional groups are brought in close proximity due to the reduced spatial distance between the oligonucleotide chain and said functional groups.

The term "proximity" typically means that the distance between the functional group(s) of two domains where the oligonucleotides thereof are hybridised is less than 100A such as less than 5θA, preferably less than 2θA.

The functional groups of the structural domains of the compounds are present for the purpose of rendering it possible to introduce covalent links between structural domains of two compound.

Illustrative examples of functional groups are hydroxy groups, aldehyde groups, ketone groups. Such functional groups may either be present in the compounds in step (a) as the "free groups" or may be present in a protected form. Protective groups are known to the person skilled in the art, see e.g. Greene, T. W. and Wuts, P. G. M., "Protective Groups in Organic Synthesis", 2^nd ed., John Wiley, N.Y. (1991), and MJ. Gait, Oligonucleotide Synthesis, IRL Press, 1984). Illustrative examples of protected forms of aldehydes and ketones are acetals/ketals of the general type CH(OC_!-₄-alkyl)₂ and CH(OC₁. -alkylene-0), and thioacetals/thioketals of the general type CH(SC_!-₄-alkylene)₂, CH(S-C₁- -alkylene-S), CH(S-C₁_₄-alkylene-0)₂.

The presently preferred aldehyde group is formyl (-CHO). The presently preferred ketone group is C₁-₄-C(=0)-.

Presently preferred functional groups are hydroxy groups, aldehyde groups, carboxylic acid groups, ketone groups.

This being said, it is believed that a certain degree of correspondence should exist between the functional group(s) of the first structural domain of the first compound and the second structural domain of the second compound. E.g. in one embodiment, at least the first structural domain of the first compound and the second structural domain of the second compound each comprise two functional groups. In a further embodiment, at least one functional group of the first structural domain of the first compound is identical to a functional group of the second structural domain of the second compound. In a more particular embodiment, at least two functional groups of the first structural domain of the first compound are identical to two functional groups of the second structural domain of the second compound.

In one embodiment at least one functional group of the first structural domain of the first compound is an aldehyde or a protected form thereof, and at least one functional group of the second structural domain of the second compound is an aldehyde or a protected form thereof.

In another embodiment, at least one functional group of the first structural domain of the first compound is a ketone or a protected form thereof, and at least one functional group of the second structural domain of the second compound is a ketone or a protected form thereof.

In a more particular embodiment, at least two functional groups of the first structural domain of the first compound are identical to two functional groups of the second structural domain of the second compound.

In one embodiment, two functional groups in a structural domain are identical to two functional groups in another structural domain. These functional groups are preferably aldehyde or a protected form thereof and as a second functional group hydroxide. As an example, the two functional groups of the first structural domain of the first compound are an aldehyde (or a protected form thereof) and hydroxide, respectively, and the two functional groups of the second structural domain of the second compound are an aldehyde (or a protected form thereof) and hydroxide, respectively. Particularly, the first structural domain of the first compound is a salicylaldehyde or a protected form thereof, and the second structural domain of the second compound is a salicylaldehyde or a protected form thereof.

In a still further embodiment, at least one functional group in a structural domain is identical to a functional group in another structural domain. In a particular embodiment this functional group is an aldehyde or a protected form thereof, or a ketone or a protected form thereof.

Alternatively, the functional groups may be selected from iodo, bromo, chloro, triflato, ethynyl, -C≡CSiMe₃, -C=C-SnBu₃, -C≡C-SnMe₃, -C≡C-Si'Pr₃, -B(OH)₂, -B(OEt)₂, - -B(OCH₂CH₂0), -B(OC(CH₃)₂C(CH₃)₂0), -SnBu₃, -SnMe₃, -C(0)Me, -CHO, -COOH, NH₂, CH₂- NH₂, as well as groups illustrated infra.

The compound may be prepared as described further below. re Step (b)

In the subsequent step, the matching oligonucleotides are allowed to hybridise. The three or more organic compounds are typically provided in solution so as to facilitate suitable hybridisation. The solution is typically aqueous containing a buffer and low concentrations of salts (50-200 mM), but the hybridisation can also take place in pure water or in organic solvents as will be known to the person skilled in the art of oligonucleotides.

As mentioned in a previous section the hybridisation of oligonucleotide chains between two structural domains brings the functional groups of the domains in proximity of each other thereby increasing the possibility for the formation of a covalent link.

re Step (c)

It should be understood that the length of the optional spacer introduces a certain degree of flexibility that may be beneficial in view of the reaction mechanism involved in formation of the covalent link.

One advantage of the present method is that covalent links can be established in parallel between multiple sets of structural domains. Once the oligonucleotide chains have hybridised to assemble the macromolecular architecture, the covalent links may preferably be established between the individual domains in a single step. This is typically preferred over establishing the covalent links stepwise, i.e. establishing a covalent link after just two oligonucleotide chains have hybridised.

The formation of the covalent link may take place directly, i.e. the functional groups of corresponding domains may be selected so that they readily react with each other. Alternatively, the functional groups of corresponding domains may react after suitable deprotection.

The establishment of a covalent link may however also in a presently preferred embodiment be accompanied by a coupling reactant. The coupling reactant may be used to activate the functional groups, e.g. acid groups, hydroxy groups, etc. or the coupling reactant may be used to link corresponding functional groups. As an example of the latter, coupling reactants like diamines and derivative thereof may be used in combination with aldehyde or ketone groups (or protected form of any of these). The diamine can, e.g., be a 1,2-diamine or a derivative thereof, an ethylene diamine or a derivative thereof or a diaminoarene or a derivative thereof.

The establishment leads to a covalent link including two imine bonds (see e.g. Figure 6a). When starting from salicylaldehyde or the ketone variants thereof, the resulting structure is known as a salen complex. It is also envisaged that the imine bond may be reduced so as to form an amine bond. In a suitable embodiment, two aldehydes such as salicylaldehyde, undergo reductive amination using a diamine such as ethylenediamine. Typically, reductive amination is performed using sodium cyanoborohydride in an acidic medium, such as at pH 3 to 6.

Still further, the establishment of a covalent link may be conducted in the presence of a metal or metal salts. Illustrative examples of salts are Mn, Ni, Co, Fe, Al, and U.

Illustrative examples of metal salts are Mn(OAc)₂, Mn(CI)₃, NiCI₂, Ni(OAc)₂, CoCl₃, FeCI₃, AICI₃, U0₂(N0₃), AI((N0₃)₃ . The resulting structure is known as a metal salen complex.

re Step (d)

In a final step, the oligonucleotides may be cleaved from the structural domains. This may be accomplished by biological or chemical means. The cleavage may be performed directly while the oligonucleotides are still hybridised, or a de-hybridisation may be performed before cleavage as the case may be. Also, cleavage may or may not involve cleavage of the spacer.

Biological cleavage may be performed by DNAases, RNAases or other enzymes capable of cleaving nucleotides, phosphate groups, etc. or any groups of the spacer.

Chemical cleavage may be cleavage of an amide bond (e.g. hydrolysis), a thiophosphate bond, a carbon-carbon bond, an ester bond (e.g. hydrolysis) or cleavage of a disulphide bond by thiols or other reactants, by tris(2-carboxyethyl)phosphine (TCEP) (see Burmeϊster Getz, E., Xiao, M., Chakrabarty, T., Cooke, R. & Selvin, P.R.; A comparison between the sulfhydryl reductants Tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal. Biochem. 273, 73-80 (1999). or by elimination of a proton or silyl group in the beta-position to the phosphate diester group by using base or fluoride. In most instances, the cleavage will leave a part being a hydroxy group, a phosphate group, a thiol group, an alkene grup, one or two or a few nucleotides or any chemical functionality obtained by cleavage of the spacer, as the case may be.

The macromolecular architecture typically has a molecular weight of at least 5000 Da. However, in many instances, the molecular weight may be as high as at least 20,000 Da or even at least 50,000 Da.

When turning to possible compounds for use as starting materials in the first method of the invention, the following illustrative examples of the formulae A-D can be given

A1

C1

wherein n is an integer of 0-2;

R¹, R² and R⁸ are independently selected from aldehyde groups, ketone groups, carboxylic acid groups, and protected forms thereof; each R³ is independently selected from hydrogen, optionally substituted C^o-alkyl, optionally substituted Cι-₂₀-alkoxy, optionally substituted aryl, optionally substituted aryloxy, a polyether of the formula 0(CH₂CH₂)_pOCH₂CH₃ wherein p is 1-10, and a disulfide of the formula Q_t-io-alkyl-SS-Q_t-io-alkyl;

R⁴, R⁵ and R⁹ are independently selected from hydrogen and phenol protecting groups; and R⁶, R⁷ and R¹⁰ are independently selected from iodo, bromo, chloro, triflato, ethynyl,

-C--CSiMe₃, -C≡C-SnBu₃, -C≡C-SnMe₃, -C≡C-Si'Prs, -B(OH)₂, -B(OEt)₂, -B(OCH₂CH₂0),

-B(OC(CH₃)₂C(CH₃)₂0), -SnBu₃, -SnMe₃, -C(0)Me, -CHO, and COOH; spacer¹, spacer² and spacer³ are independently selected from a single bond and spacers having a length of 1-50 atoms, and X¹, X² and X³ are independently selected from oligonucleotides of 4-30 nucleotides. A particular subgroups of compounds are those selected from

wherein n is an integer of 0-2;

R³ is independently selected from hydrogen, optionally substituted C_α-₂o-alkyl, optionally substituted C^o-alkoxy, optionally substituted aryl, optionally substituted aryloxy, a polyether of the formula 0(CH₂CH₂)_pOCH₂CH₃ wherein p is 1-10, and a disulfide of the formula Q-jo-alkyl-SS-C_t-io-alkyl; and the structural domains T¹, T² and T³ are selected from

wherein X is selected from oligonucleotides of 4-30 nucleotides and wherein m is an integer of 0-10.

In one embodiment R¹, R² and R⁸ are selected from aldehyde groups and protected forms of the aldehyde group such as acetals of the general type CH(OC₁.₄-alkyl)₂ and CH(OC_!-₄- alkylene-O), thioacetals of the general type CH(SC₁.₄-alkylene)₂, CH(S-C_!-₄-alkylene-S), CH(S-C₁.₄-alkylene-0)₂, ketone groups such as -C(=0)-C₁.₄-alkyl (e.g. -C(=0)-Me, -C(=0)-Et) or -C(=0)-aryl (e.g. -C(=0)-Ph) and protected forms thereof; R⁴ is selected from hydrogen, tetrahydropyranyl, methoxymethyl, benzyloxymethyl, methoxyethoxyethyl, methylthiomethyl, 2-(trimethylsi!yl)ethoxymethyl, allyl, formyl, acetyl, pivaloyl, benzoyl, and methyl-, t-butyl-, vinyl- and benzyl carbonates. The above compounds are believed to be novel in themselves and therefore represent a special aspect of the present invention.

This first method of the invention is further illustrated by the following with reference to the Figures.

Accordingly, one embodiment takes advantage of a new class of organic molecules referred to as Linear Activated Nucleotide Analogues, hereafter referred to as LANA (see Figure 1). The LANA compounds contain two functional groups F^- and F² that are preferably identical (in that case they are just assigned F). They can be activated for a reaction with a similar or complementary functionality to connect the two groups. More specifically the functional groups could be an aldehyde or a ketone that can react in the presence of a diamine such as ethylenediamine or derivatives thereof (F¹ = F² = F). In particular, the functional group could be a salicylaldehyde derivative that can react in the presence of ethylene diamine or derivatives thereof to form salen compounds. This coupling reaction can be performed in the presence of salts of metals such as Mn(OAc)₂, MnCI₃, U0₂(N0₃)₂, AICI₃, AI((N0₃)₃, NiCI₂, Ni(OAc)₂, FeCI₃, CoCI₃ or others to form metal salen complexes. The LANA compound is furthermore camouflaged like an activated nucleotide analogue for automated DNA-synthesis by using a protecting group (PG¹) such as dimethoxytrityl group (or other acid labile protecting groups) to one of the terminal hydroxyl groups of the LANA compound and to attach a phosphate derivative (Phos) such as 2-cyanoethyl diisopropylphosphoramidite (or other phosphorous derivatives for automated DNA- synthesis) to the other terminal hydroxygroup. This can be incorporated at any sequence position, into a single stranded oligonucleotide strand via phosphate diesters by automated DNA-synthesis. One could consider the LANA molecule to be incorporated in the middle of an oligonucleotide with a chain length of 9-61 nucleotides, such as 13-41 nucleotides (assuming that the LANA counts as one "nucleotide"). Such an "oligonucleotide" can be synthesised by standard protocols for automated DNA-synthesis preferably using the phosphoamidite protocol. The DNA-functionalized LANA compounds obtained in this manner are hereafter referred to as L-ANAO. The L-ANAO is cleaved from the resin, deprotected and purified by standard protocols.

Alternatively, the covalent link or coupling to the LANA compound may be via the use of a reductive amination reaction.

Another embodiment of the invention takes advantage of Tripodal Activated Nucleotide Analogues, hereafter referred to as TANA (see Figure 2). The three termini of TANA are functionalised by functional groups F that can be activated for reaction with a similar functionality to form a covalent bonding with two such groups intermolecular (Figure 2). More specifically, the functional group could be an aldehyde or a ketone that can react in the presence of a diamine such as ethylenediamine or derivatives thereof. In particular the functional group could be a salicylaldehyde derivative that can react in the presence of ethylene diamine or derivatives thereof to form salen compounds. This reaction can be performed in the presence of salts of metals such as Mn(OAc)₂, MnCI₃, U0₂(N0₃)₂, AICI₃, AI((N0₃)₃, NiCI₂, Ni(OAc)₂, FeCI₃, CoCI₃ or others to form metal salen complexes. The TANA compound is furthermore camouflaged like an activated nucleotide analog for automated DNA-synthesis by using a protecting group (PG¹) such as dimethoxytrityl group (or other acid labile protecting groups) to one of the terminal hydroxygroups of the TANA compound and to attach a phosphate derivative (Phos) such as 2-cyanoethyl diisopropylphosphoramidite (or other phosphorous derivatives for automated DNA- synthesis) to the other terminal hydroxygroup. The TANA compound also embodies the possibility of synthesis of a branched nucleotide in which TANA represents the branching point. This is obtained by incorporating a protecting group PG², for the third terminal hydroxyl group. PG² could for example be a Fmoc protecting group or other protecting groups that are compatible with automated DNA-synthesis (in particular by the phosphoamidite method), and orthogonal to the other protecting groups applied. In this manner the TANA compound, can be functionalised with three oligonucleotide chains in the three terminals of any desired length between 4-30 nucleotides each, preferably 6-20 nucleotides each. The derivatised TANA compound containing 3 oligonucleotide chains is hereafter referred to as TANAO. The TANAO compound obtained by automated synthesis is cleaved from the resin and deprotected and purified by standard methods.

In a further embodiment, one or more of the functional groups of TANA and TANAO compounds are different functional groups.

As an example, Figure 3 (upper part) illustrates the mixing in solution and hybridisation of complimentary DNA oligonucleotides of 3 LANAO's into double stranded DNA hybrids. Figure 4 (upper part) illustrates the mixing in solution and hybridisation of 2 TANAO compounds into double stranded DNA hybrids. Figure 5 illustrates the mixing in solution and hybridisation of a mixture of 3 L-ANAO compounds with 2 TANAO compounds to give double stranded DNA hybrids.

Following the routes in Figures 3-5, the functional groups of the L-ANAOs and TANAOs undergo reaction under formation of at least one covalent link between the structural domains when adding a coupling reagent such as e.g. ethylene diamine and derivatives thereof or 1,2-diaminoarenes for imine formation with a functional group such as a ketone or an aldehyde in particular a salicylaldehyde derivative. The same reaction may also be used for a reductive amination adding a coupling reagent such as e.g. ethylene diamine and derivatives thereof for amine formation in the presence of a reducing agent, such as sodium cyanoborohydride, and a functional group such as a ketone or an aldehyde in particular an salicylaldehyde derivative. Addition of coupling reagents can proceed in the presence of a metal such as Mn(OAc)₂, MnCI₃, U0₂(N0₃)₂, AICI₃, AI((N0₃)₃, NiCI₂, Ni(OAc)₂, FeCI₃, CoCI₃ or other metal salts.

The formation of at least one covalent bond between the hybridised structures can also be applied a catalyst or reagent for reactions such as eg. the Sonogashia reaction, Ulman reaction, Stille coupling, Suzuki coupling and others (see Figure 14). The reactions leading to formation of covalent bonds can proceed to take place only between two functional groups F, for which the oligonucleotide chains attached to the same terminals in the LANAO or TANAO are hybridised. Functional groups F, for which the oligonucleotide at the same terminal of the LANAO or TANAO is single stranded, do not undergo coupling to or reaction with another functional group at F at a LANAO or TANAO as illustrated in step 2 in Figures 3-5.

This provides the intriguing possibility of, in a first step, to prepare precursors for macromolecular architectures, where the precursors are prepared from e.g. 2-10 compounds and where the precursors have free single stranded oligonucleotides and ("inactive") hybridised oligonucleotides. In a subsequent step, further build-up of the macromolecular architecture may be conducted as illustrated herein using the precursors as the first compound, the second compound, etc.

Macromolecular architectures obtained by assembly of at least 3, preferably more, LANAOs or TANAOs, by covalent bonds are hereafter referred to as CALTO (Covalently Assembled LANAOs and/or TANAOs, Oligonuctide functionalized). Compounds obtained by assembly of at least 3, preferably more, LANAOs or TANAOs, by covalent bonds from which the oligonucleotide chains have been cleaved by biological or chemical methods, are hereafter referred to as CALT (Covalently Assembled LANAO and/or TANAO, (without Oligonucleotides)).

Another aspect of the invention relates to the cleavage of the nucleotide chains attached to CALTO compounds as exemplified in Figures 3-5. That may be cleavage of exclusively single stranded chains, double stranded chains or in particular both single and double stranded oligonucleotide chains. This cleavage may proceed by using biological methods such as DNases or RNases or other enzymes capable of cleaving nucleotides, phosphate groups of any functionality in the organic part of molecule that links the nucleotide to the organic compound. The cleavage may also be performed by chemical methods that cleaves either the oligonucleotide chain or in particular any functionality in the organic part of molecule that links the nucleotide to the organic compound. Chemical cleavage of the spacer may be by hydrolysis of an amide bond or an ester bond, or by cleavage of an disulfide bond by thiols or other reductants, by tris(2-carboxyethyl)phosphine (TCEP), or by elimination of an proton or silyl group in the beta-position to the phosphate diester group by using base or fluoride. By one or more of these cleavage methods, the oligonucleotide chains may be removed to give CALT compounds (step 3, Figures 3-5). The residue R in the CALT compounds obtained after cleavage of the oligonucleotide may be a hydroxygroup, a phosphate group, one or two nucleotides or any chemical functionality obtained by cleavage of the spacer; in particular thiol, alkene, hydroxygroup, carboxylate (or acid derivative), carboxamide or sulfonate.

In particular the assembly of LANAO and TANAO by DNA hybridisation and subsequent formation of covalent bonds as described above for subsequent formation of covalent bonds relates to such LANAO and TANAO as illustrated in Figure 6. In LANAO¹ and LANAO² the substituent R' can be hydrogen or an alkyl group of any length, linear or branched , or an Oalkyl or an alkyl chain terminated by a functional group such as NH₂, OH, COOH, SH, S0₃H or any salts (e.g. Na, K) or protected derivatives thereof. The "Spacer" in these LANAO and TANAO compounds consists of an organic motive that bridges the organic molecule to the oligonucleotide chain and can be a wide variety of organic groups. In particular the spacer can be an amide functionality of the type -C(0)NR*(CH₂)_nO- (n = 2- 20) in which the terminal oxygen is linked to an phosphate group of the oligo nucleotide chain and R* is H or any alkylgroup (e.g. Me, Et, Pr), alkylarene (e.g. Bn) or alkenylgroup (e.g. allyl); or the spacer can be an ester of the type -C(0)0(CH₂)_nO- (n = 2-20) in which the terminal oxygen is linked to an phosphate group of the oligo nucleotide chain.

The method of the invention typically involves the subsequent parallel formation of 1 or more covalent bonds between hybridised assemblies of L-ANAOs or TANAOs in any number and combination of the two types of compounds, by a chemical or enzymatic reaction. This relates for example to the formation of imine bonds between two aldehyde or keto groups by the addition of an diamine. In particular this relates to the formation of salen compounds, in functional groups adjacent to hybridized oliginucleotides, by the reaction of a salicylaldehyde derivative incorporated in the LANAO's and TANAO shown in Figure 6, with an 1,2-diamine, for example ethylenediamine. Preferably the salen formation takes place in the presence of a metal salt such as Mn(OAc)₂, MnCI₃, U0₂(N0₃), AICI₃, AI((N0₃)₃, NiCI₂, Ni(OAc)₂, FeCI₃, CoCI₃ or other metal salts. The parallel formation of a salen moieties between hybridised assemblies of LANAO's and TANAO's is exemplified in Figure 6a, step 2, using ethylene diamine as the coupling reagent.

A key feature of the formation of covalent bonds between assemblies of LANAOs or TANAOs in any number and combination, which is also embodied in the invention, is that the formation of covalent bonds can proceed only when the functional group of the LANAO or TANAO is located at a structural domain of the compound for which the nearest attached oligonucleotide chain is hybridised with another LANAO or TANAO compound. This in particular relates to reaction of LANAO's or TANAO's such as illustrated in Figure 6, which have salicylaldehyde-type structural domains to which is attached a spacer that connects to the oligonucleotide chain. At the suitable conditions (suitable conditions relates to: buffer; temperature; concentrations of LANAO_nTANAO_m hybrid, ethylenediamine and metalsalt; reaction time (see the examples)) the reaction leading to formation of salens or metalsalens can take place only for those salicylaldehyde domains for which the nearest attached oligonucleotide chain is hybridised with an oligonucleotide chain on another LANAO or TANAO compound. Thus, when the oligonucleotides bring the functional groups into proximity the functional groups will react with each other under the given appropriate conditions.

The invention provides a variety of CALTO assemblies obtained by oligonucleotide directed assembly and in particular CALTO assemblies connected via salen or metal salen complexes as illustrated in Figure 6a. The invention also allows for the connection of any CALTO compounds via the single stranded oligonucleotide chains by hybridisation with another CALTO (or single or more LANAO or TANAO compounds) and subsequent formation of covalent bonds to form a new and larger CALTO compound. In a subsequent step (step (d)) the nucleotide chains attached to CALTO compounds are cleaved. That may be cleavage of exclusively single stranded chains, double stranded chains or in particular both single and double stranded oligonucleotide chains. This cleavage may proceed by using biological methods or chemical method as described above. By one or more of these cleavage methods, the oligonucleotide chains may be removed to give CALT compounds (step 3, Figure 6). The residue R" in the CALT compounds obtained after cleavage of the oligonucleotide may be a hydroxy group, a phosphate group, one or two or a few nucleotides or any chemical functionality obtained by cleavage of the spacer; in particular thiol groups, alkene groups, hydroxy group, carboxylate (or acid derivatives), carboxamide or sulfonate.

Method involving templates

In a second and alternative method for the preparation of a macromolecular architecture, the specific hybridisation of oligonucleotides assembles individual compounds so as to form the macromolecular architecture in a predetermined manner whereafter covalent links are introduced in order to stabilise and "freeze" said structure.

The main difference between the first method and the present second method, is the introduction of a third oligonucleotide chain, which is involved in the hybridisation and assembling of individual compounds. This third oligonucleotide chain functions as a template to the oligonucleotide chains in the individual compounds. After formation of a covalent link, the oligonucleotide chains may thereafter be removed from the structure just as the third oligonucleotide chain is removed.

In particular, the second method comprises the following steps:

(a) providing at least three organic compounds, including a first compound, a second compound and a third compound, and at least one oligonucleotide template, each compound comprising at least two structural domains, including a first structural domain and a second structural domain, each structural domain comprising in proximity (i) one or more functional groups and (ii) an oligonucleotide chain, the oligonucleotide chain of the first structural domain of the first compound being non- complementary to the oligonucleotide chain of the second structural domain of the second compound, and/or the oligonucleotide chain of the second structural domain of the first compound being non-complementary to the oligonucleotide chain of the first structural domain of the third compound, furthermore the oligonucleotide chain of the first structural domain of said first compound and the oligonucleotide chain of said second structural domain of said second compound being at least partly complementary to the at least one oligonucleotide template, and/or the oligonucleotide chain of the second structural domain of said first compound and the oligonucleotide chain of the first structural domain of said third compound being at least partly complementary to the at least one oligonucleotide template, said oligonucleotide template; and

(b) hybridising the oligonucleotide chain of the first structural domain of said first compound and the oligonucleotide chain of the second structural domain of the second compound with the at least one oligonucleotide template, and/or hybridising the oligonucleotide chain of the second structural domain of said first compound and the oligonucleotide chain of the first structural domain of the third compound with the at least one oligonucleotide template, thereby establishing one or two base-paired hybrid(s), whereby the functional group(s) of the first structural domain of the first compound is brought in proximity to the functional group(s) of the second structural domain of the second compound, and/or the functional group(s) of the second structural domain of the first compound is brought in proximity to the functional group(s) of the first structural domain of the third compound; and

(c) establishing a covalent link between the first structural domain of said first compound and the second structural domain of said second compound by conducting a reaction involving the functional group(s) of said first structural domain of said first compound and said second structural domain of said second compound, and/or establishing a covalent link between the second structural domain of said first compound and the first structural domain of said third compound by conducting a reaction involving the functional group(s) of said second structural domain of said first compound and said first structural domain of said third compound; and

re Step (a)

The individual compounds in the present method are similar to the compounds provided in the first method. This similarity includes the structural domains in each compound and the domain cores herein, the scaffold, the presence of functional group(s) in each structural domain and the attachment of an oligonucleotide chain to each domain core in the structural domain.

Differing from the first method though, is the appearance of at least one oligonucleotide template and the non-complementarity between the oligonucleotide chains of two structural domains of separate compounds.

In the method, four oligonucleotide chains on four separate domain cores are provided. The oligonucleotide chain of the first structural domain of the first compound being non- complementary to the oligonucleotide chain of the second structural domain of the second compound, and/or the oligonucleotide chain of the second structural domain of the first compound being non-complementary to the oligonucleotide chain of the first structural domain of the third compound.

The wording "and/or" suggests that it is adequate that just the oligonucleotide chain in the first structural domain of the first compound is non-complementary to the oligonucleotide chain in the second structural domain of the second compound. The oligonucleotide chain in the second structural domain of the first compound may optionally be non- complementary to the oligonucleotide chain in the first structural domain of the third compound.

For the proper understanding the oligonucleotide chain in the first structural domain of a compound and the oligonucleotide chain in the second structural domain of an adjacent compound is called "oligonucleotide pairs" in the following sections. The optional non-complementarity implies to the use of a larger number of compounds in the macromolecular architecture as well. It is adequate that just two "oligonucleotide pairs" between two structural domains are non-complementary, leaving the "oligonucleotide pairs" between other structural domains complementary. Alternatively, all the "oligonucleotide pairs" between two structural domains are non-complementary, or a part of the "oligonucleotide pairs" between two structural domains are non- complementary.

The introduction of non-complementary oligonucleotide chains is necessary when a oligonucleotide template is provided. In the present method, the oligonucleotide chain of the first structural domain of the first compound and the oligonucleotide chain of the second structural domain of the second compound is at least partly complementary to the at least one oligonucleotide template, and/or the oligonucleotide chain of the second structural domain of the first compound and the oligonucleotide chain of the first structural domain of the third compound is at least partly complementary to the at least one oligonucleotide template as well. Preferably, however, one oligonucleotide template is provided for each oligonucleotide pair.

The oligonucleotide template is used to assemble individual compounds by means of hybridisation with the "oligonucleotide pairs". This hybridisation only takes place when the third oligonucleotide chain is at least partly complementary with two oligonucleotide chains comprising the "oligonucleotide pair".

The oligonucleotide template typically has a length of 8-60 base pairs, preferably between 16-40 base pairs. The oligonucleotide template should be at least partly complementarity to each of the oligonucleotides of the oligonucleotide pair. At least partly complementary is to be understood as described above for the first method of the invention.

Otherwise, the provisions for providing the compound and the oligonucleotide template(s) follows the provision given for the first method of the invention.

re Steps (b)-(d)

The provisions for hybridisation, formation of a covalent link and for cleavage of the oligonucleotide chains follows the provisions given for the first method of the invention. It is envisaged that the first method of the invention and the second method of the invention may be combined in a manner where some oligonucleotide chains of structural domains are hybridised to other nucleotide chains of structural domains according to the first method, whereas still other oligonucleotide chains of structural domains are hybridised to a template according to the second meth'od.

In such an embodiment, the hybridisation with a oligonucleotide template only occurs between a fraction of the oligonucleotide pairs, whereas the other oligonucleotide pairs are non-complementary to the oligonucleotide template by mutually complementary. Thus, in this embodiment the oligonucleotide template is preferably used as a "helper" for assembling fractions of the macromolecular architecture held together by complementary oligonucleotide pairs.

In another embodiment the a number of oligonucleotide templates are used to assemble the whole macromolecular architecture, meaning that all the oligonucleotide pairs are non- complementary but complementary to the oligonucleotide template. It is also envisaged that one or only a few oligonucleotide templates can be use, in which instance each oligonucleotide template has a considerable length so a to hybridise to a number of oligonucleotide pairs.

This second method of the invention is further illustrated by the following with reference to the Figures.

The invention also embodies the hybridisation of two non-complementary oligonucleotide chains a and b on two different LANAO and/or TANAO compounds with a oligonucleotide template that has sequenses which are complementary to both a and b. The template thus brings the two functional groups of the LANAO and/TANAO compounds in close proximity as illustrated for a salicylaldehyde functional group in Figure 6b. In this example, the two non-complementary nucleotide chains o2 and o3' are hybridised with an oligonucleotide template containing the complementary sequenses of both o2 and o3'. The spacing between the sequenses o2 and o3' in the template is typically 0-10 nucleotides or a spacer similar to the one defined above.

The formation of covalent bonds between assemblies of LANAO's or TANAO's in any number and combination can proceed only either i) when the functional group of the LANAO or TANAO is located at a termini of the compound for which the nearest attached oligonucleotide chain is hybridized with another LANAO or TANAO compound or ii) when the nearest attached oligonucleotide chain is hybridized with a template that brings two non-complementary oligonucleotide chains together on two different LANAO and/or TANAO compounds. These two approaches to DNA hybridization and subsequent formation of covalent bonds may proceed in parallel when using several (>2) LANAO and/or TANAO compounds.

The compounds defined as particularly useful as starting materials in the first method of the invention are also believed to be particularly useful in the second method of the invention.

Method involving iterative synthesis In a third method, the specific stepwise unmasking and coupling of organic compounds to a growing macromolecular architecture attached to a solid support, assembles individual compounds so as to form the macromolecular architecture in a predetermined manner whereafter covalent links are introduced in order to "freeze" said structure. The method opens for the possibility to automate the construction of the macromolecular architecture and thereby offers an advantageous tool for making complicated structures from simple organic compounds.

In particular, the method comprises the following steps:

(a) providing a solid support having reactive groups, and at least two organic compounds, including a first compound and a second compound, each compound comprising a first structural domain and a second structural domain, said first structural domains of said compounds including in proximity one or more functional groups and a reactive group, and said second structural domain of said compounds including in proximity one or more functional groups and a masked reactive group,

(b) immobilising said first compound to said solid support by attaching the first structural domain of said first compound to the solid support, this attachment involving the reactive group in said first structural domain of said first compound, (c) unmasking the masked reactive group in the second structural domain of said first compound, thereby exposing said second structural domain of said first compound for a reaction with the first structural domain of the second compound,

(d) coupling the second structural domain of said first compound to the first structural domain of said second compound, this coupling involving the unmasked reactive group in said second structural domain of said first compound and the reactive group in said first domain of said second compound, thereby bringing the functional group(s) of the second structural domain of the first compound in proximity to the functional group(s) of the first structural domain of the second compound,

(e) optionally repeating the procedure of unmasking (c) and coupling (d) using further compounds equivalent to the first compound and/or the second compound provided in (a), and

(fl) detaching the obtained product from the solid support and subsequently establishing a further covalent link at least between the second structural domain of said first compound and the first structural domain of said second compound, or

(f2) establishing a covalent link at least between the second structural domain of said first compound and the first structural domain of said second compound, and subsequently detaching the obtained product from the solid support.

As will be immediately recognised, the method takes advantage of a procedure similar to that used in the preparation of oligonucleotides and peptides. Thus, equipment and other means known to be applicable in the synthesis (in particular automated synthesis) of oligonucleotides and peptides may be adapted to the method of the present invention.

re step (a)

Initially, a solid support is provided. The solid support material is used to immobilised the compounds so as to facilitate assembly and synthesis of the macromolecular architecture. In view of the above, it will be understood that solid supports used in connection with oligonucleotide synthesis and peptide synthesis can be used. Such solid supports should carry groups that can undergo reaction with the reactive group of the first domain of the first compound (see below, in particular step (d)). Initially, two organic compounds, including a first compound and a second compound, are provided. Furthermore each compound comprises a first structural domain and a second structural domain. The first structural domain includes a reactive group and the second structural domain includes a masked reactive group.

The individual compounds in the present (third) method are somewhat similar to the compounds used in first method of the invention and the second method of the invention. This similarity includes the domain core comprising the structural domains in each compound, the scaffold, and the presence of functional group(s) in each structural domain. The difference between the compounds used in the third method is the absence of an oligonucleotide chain in each structural domain. Instead the structural domains either comprise a reactive group or a masked reactive groups, see further below. However, the definitions and preferences mentioned further above with respect to these compounds, including the functional groups, the domain core, the spacers and the scaffold, etc. also applies here, mutatis mutantis. Thus, the compounds used in the third method of the invention may also include a third structural domain, and even a fourth structural domain.

In one embodiment the functional groups are preferably aldehyde or a protected form thereof and as a second functional group hydroxide. As an example, the functional groups of the first structural domain of the first compound are an aldehyde (or a protected form thereof) and hydroxide, respectively, and the functional groups of the second structural domain of the second compound are an aldehyde (or a protected form thereof) and hydroxide, respectively. Particularly, the first structural domain of the first compound is a salicylaldehyde or a protected form thereof, and the second structural domain of the second compound is a salicylaldehyde or a protected form thereof. The establishment of a covalent link may however also in a presently preferred embodiment be accompanied by a coupling reactant. The coupling reactant may be used to activate the functional groups, e.g. acid groups, hydroxy groups, etc. or the coupling reactant may be used to link corresponding functional groups. As an example of the latter, coupling reactants like diamines and derivative thereof may be used in combination with aldehyde or ketone groups (or protected form of any of these). The diamine can be, for instance, a 1,2-diamine or a derivative thereof, an ethylene diamine or a derivative thereof or a diaminoarene or a derivative thereof.

The establishment leads to a covalent link including two imine bonds. When starting from salicylaldehyde or the ketone variants thereof, the resulting structure is known as a salen complex. It is also envisaged that the imine bond may be reduced so as to form an amine bond. In a suitable embodiment, two aldehydes such as salicylaldehyde, undergo reductive amination using a diamine such as ethylenediamine. Typically, reductive amination is performed using sodium cyanoborohydride in an acidic medium, such as at pH 3 to 6. Still further, the establishment of a covalent link may be conducted in the presence of a metal or metal salts. Illustrative examples of salts are Mn, Ni, Co, Fe, Al, and U. Illustrative examples of metal salts are Mn(OAc)₂, Mn(CI)₃, NiCI₂, Ni(OAc)₂, CoCI₃, FeCI₃, AICI₃, U0₂(N0₃), AI((N0₃)₃ . The resulting structure is known as a metal salen complex.

Besides the functional groups specifically suggested for the structural domains of the compounds of the first method of the invention, a further embodiment involves compounds where the structural domains include one functional group which is particular adapted to undergo the Sonogashira reaction, the Ulman reaction, the Stille coupling, the Suzuki coupling and others C-C coupling reactions (see Figure 14). A particularly interesting embodiment of the invention involves compounds where the structural domains include functional groups suitable for reductive amination, such as the use of salicylaldehyde with a diamine.

Examples of groups for such reactions that in typical instances are metal catalysed (in particular noble metal (e.g. palladium) catalysed), are those selected from iodo, bromo, chloro, triflato, ethynyl, -C≡CSiMe₃, -C=C-SnBu₃, -CsC-SnMe₃, -C≡C-Si^^, -B(OH)₂, -B(OEt)₂, -B(OCH₂CH₂0), -B(OC(CH₃)₂C(CH₃)₂0), -SnBu₃, -SnMe₃, -C(0)Me, COOH, and -CHO.

Alternative to the oligonucleotide chains, the compounds for use in this method comprises above a "reactive group" and a "masked reactive group". The term "masked" means that the respective reactive group is intended to be preserved under the conditions prevailing, but that the reactive group can be liberated under suitable conditions. Particularly, the masked reactive group is not intended to react with reactive or functional groups of other domains.

In one embodiment, the group for masking the masked reactive group is any group compatible with automated DNA synthesis or automated peptide synthesis. Thus, the type of masked reactive groups is typically selected with due regard to the protocol for synthesis.

If more than two structural domains are present in the selected compounds, each compound will normally have one reactive group and two or more masked reactive groups. Thus, the first structural domain always comprises a reactive group, whereas the "second", the "third" and the "fourth" structural domain typically each comprises a "masked reactive group".

Such "masked reactive groups" of different domains within the same molecular may be different so that they, if desired, can be unmasked independently. If three structural domains are provided in a particular embodiment, the "masked reactive groups" on two of such structural domains are different from each other. If a "fourth" structural domain is provided, making a whole of four structural domains, the "masked reactive groups" on three such structural domains are different from each other. It is thought that this difference between the "masked reactive groups" allows the site-specific coupling of organic compounds and thus the predetermination of the macromolecular architecture.

Alternatively, the masked groups are similar or identical whereby the further reaction (step (d)) proceeds via two or more unmasked reactive groups in parallel.

In a presently preferred embodiment the "masked reactive group" is independently selected from hydroxy (-OH), Prot-O-, carboxylic acid (-COOH), Prot-C(=0)-, amino (- NHR^a), Prot-NR^a-, thiolo (-SH), and Prot-S-, where R^a is hydrogen or C^-alkyl, and wherein Prot is a protecting group for -OH, -COOH, -NHR^a and -SH, respectively. Specific examples of "masking groups" for used in oligonucleotide synthesis are acid labile groups, such as dimethoxytrityl group, Fmoc or other groups.

As mentioned above there is only one reactive group in one compound. The reactive group provided in the method may, e.g., be any group selected hydroxy (-OH), Act-O-, carboxylic acid (-COOH), Act-C(=0)-, amino (-NHR^a), Act-NR^a, thiolo (-SH), and Act-S-, where R^a is hydrogen or C_α-₄-alkyl, and wherein Act is an activation group for -OH, -COOH, -NHR^a and -SH, respectively.

In one preferred embodiment the reactive group is COOH or an active ester thereof. In another embodiment, the reactive group is selected from substituted O- phosphoramidite, optionally substituted O-phosphortriester, optionally substituted O- phosphordiester, optionally substituted H-phosphonate, and optionally substituted O- phosphonate. Alternatively, the reactive group is selected from hydroxy, -P(N'Pr₂)OCH₂CH₂CN), -COOH and -COOtBu.

In a presently preferred embodiment the reactive group is any group compatible with automated DNA synthesis and automated peptide-synthesis. re Step (b)

The idea behind the third method for the preparation of macromolecular architectures is that the solid support should function as a fundament for the coupling of compounds. The solid support functions as an immobiliser of the first compound, whereby the masked reactive group(s) of the first compound can be unmasked to be able to react with the second compound, etc.

re Step (c-d)

Subsequent to coupling of the first compound to the solid support, a second compound can, after unmasking of the masked reactive group(s) of the first compound, be coupled to this immobilised first compound, a third compound can, after unmasking of the masked reactive group(s) of the first compound, be coupled to this second compound, and so forth.

The coupling between two structural domains leads to the formation of a covalent link. The coupling of compounds involves the reactive group, which is present in one of the structural domains of each compound, as previous mentioned. If there is more than two structural domains in a compound, the other structural domains are masked with a "masked reactive group". At the very beginning of the synthesis the reactive group in the first compound reacts, and this leads to immobilisation to the solid support. The other domain(s) of this first compound is/are "masked" with the "masked reactive group". By "unmasking" a "masked reactive group" the "unmasked reactive group" may react with another compound comprising a reactive group. In this way "unmasking" of one structural domain exposes the domain for further reactions and so on, the repetition enables the growth of a macromolecular architecture (see also step (e)).

Suitable methods for unmasking the masked reactive groups will be apparent for the person skilled in the art.

A key feature in the method is the possibility to keep control of the "unmasking" of specific "masked reactive groups" of the domains in the compound. If a compound comprises three structural domains, two structural domains initially are "masked" with "masked reactive groups". With the "unmasking" of one specific structural domain, this structural domain is exposed for a reaction with a reactive group of another compound. The growth of a chain of compounds may thus proceed in this particular direction. A compound comprising three structural domains thus may function as a branching point in the particular macromolecular architecture thereby allowing for "growth" of the macromolecular architecture in two directions. Thus, in the embodiment where all compounds only have two domains, the resulting macromolecular architecture is said to be "linear" meaning that the assembly of compounds results in an extending chain or wire, i.e. extending in "one dimension". Such a construction may then be "linear" in the geometrical sense, or may be curved, coiled, etc.

The purpose of introducing organic compounds comprising more than two structural domains is to make branches in the macromolecular architecture. Providing organic compounds with more than two structural domains makes it possible to build macromolecular architectures in more than one dimension. In a particular example, the presence of a larger number of organic compounds comprising two structural domains and one organic compound comprising three structural domains, makes the organic compound with three structural domains a potential branching point in said particular macromolecular architecture. Thus, said macromolecular architecture might potentially extend in two dimensions or in three dimensions.

The covalent link formed in the reaction between the reactive group in one compound and an "unmasked reactive group" in a second compound is preferably either an amide bond (a peptide bond) or a phospodiester bond.

In a presently preferred embodiment the macromolecular architecture obtained in the coupling between the second structural domain of the first compound and the first structural domain of the second compound is performed by automated DNA synthesis.

In a further presently preferred embodiment the macromolecular architecture obtained in the coupling between the second structural domain of the first compound and the first structural domain of the second compound is performed by automated peptide synthesis.

The control of the "masked reactive groups" is the keystone in the method. The compounds used in the method are in this way predetermined (pre-programmed). This pre-programming of compounds offers tools for the preparation of particularly designed macromolecular architectures. It is thought that this difference between the "masked reactive groups" allows the site-specific coupling of organic compounds and thus the predetermination of the macromolecular architecture.

re Step (e)

In one presently preferred embodiment the coupling between two structural domains of two respective compounds is made by iterative synthesis, i.e. steps (c)-(d) are repeated as often as necessary. In some interesting embodiments, the steps (c) and (d) are repeated at least 5 times, such as at least 10 times or at least 25 times, or even at least 50 times.

re Step (f)

In the further step of the method of the invention, a further covalent link is formed between the functional groups of the two structural that are brought in proximity as a consequence of the coupling reactions, cf. step (d).

The covalent links may be established while the product is attached to the solid support (step (f2)). Alternatively the product are removed form the solid support and the covalent links are established afterwards (step (fl)).

A great advantage of the present method is that the covalent links between functional groups of structural domains may be established in one step. The covalent links are preferably made established in parallel. The meaning of this is that the covalent links are established in a single step either while the "product" is attached to the solid support (step (f2)) or after the product has been removed from the solid support (step (fl)). The assembling of the macromolecular architecture has in both variants thus been done prior to the establishing of covalent links.

In a special embodiment, the covalent bonds prepared in step (d) may subsequently be cleaved so as to remove constraints in the structure. In this embodiment, the macromolecular architecture may exclusively be linked together by the covalent bonds synthesised in step (fl) or step (f2).

The method includes the stepwise coupling of compounds, as mentioned in the previous sections. Another main advantage of the method is the possibility to functionalize the structural domains with oligonucleotide chains. This allows a combination of the first methods of the invention and the second method of the invention with the present (third) method of the invention. The meaning of this should be understood in the way that fractions of the macromolecular architecture are made in as in the third method and that these fractions are put together using either the first method or the second method. On the other hand, fractions of the macromolecular architecture may be made using the first method or the second method and these fractions being put together using the third method.

In the first instance, it is believed that step (b) should be preceded by a step wherein an oligonucleotide chain is synthesised and that step (e) should be succeeded by a step where an oligonucleotide is synthesisted. In this manner, a structure corresponding to the starting materials of the first method and the second method may be prepared.

When turning to possible compounds for use in the above method, the following illustrative examples of the formulae E-H can be given.

E1

G1

wherein n is an integer of 0-2; R¹, R² and R⁸ are independently selected from aldehyde groups and optionally protected aldehyde groups such as acetals; each R³ is independently selected from hydrogen, optionally substituted C_t-₂₀-alkyl, optionally substituted

a polyether of the formula 0(CH₂CH₂)_pOCH₂CH₃ wherein p is 1-10; optionally substituted alkylaryl; a disulfide of the formula Ci-io-alkyl-SS-Q-io- alkyl; and

R⁴, R⁵ and R⁹ are independently selected from hydrogen and phenol protecting groups; and R⁶, R⁷ and R¹⁰ are independently selected from iodo, bromo, chloro, triflato, ethynyl, -C≡CSiMe₃, -C≡C-SnBu₃, -C≡C-SnMe₃, -C≡C-Si'Prs, -B(OH)₂, -B(OEt)₂, -B(OCH₂CH₂0), -B(OC(CH₃)₂C(CH₃)₂0), -SnBu₃, -SnMe₃, -C(0)Me, and -CHO; spacer¹, spacer² and spacer³ are independently selected from a single bond and spacers having a length of 1-20 atoms,

Q¹ is selected hydroxy (-OH), Act-O-, carboxylic acid (-COOH), Act-C(=0)-, amino (-NHR^a), Act-NR^a, thiolo (-SH), and Act-S-, where R^a is hydrogen or C^-alkyl, and wherein Act is an activation group for -OH, -COOH, -NHR^a and -SH, respectively; Q² and Q³ are independently selected from hydroxy (-OH), Prot-O-, carboxylic acid

(-COOH), Prot-C(=0)-, amino (-NHR^a), Prot-NR^a-, thiolo (-SH), and Prot-S-, where R^a is hydrogen or Cι-₄-alkyl, and wherein Prot is a protecting group for -OH, -COOH, -NHR^a and -SH, respectively. In a particular embodiment, the compound is of formula E wherein n is 1, R¹ and R² are acetal-protected formyl, R³ is hydrogen or a Cι-₄-alkyl group, R⁴ and R⁵ independently are selected from benzoyl and acetyl hydroxy protecting groups, spacer¹ and spacer² independently are selected from C(=O)NR^a-(CH₂)₂-ι₀, wherein R^a is hydrogen or C^-alkyl, and Q² is selected from OH, (O-(dimethoxytrityl), and NHFmoc, and Q is selected from OH, 0-P(N'Pr₂)OCH₂CH₂CN), COOH and COOtBu.

In another particular embodiment, the compound is of formula F wherein R¹, R² and R⁸ are acetal-protected formyl, R⁴, R⁵ and R⁹ independently are selected from benzoyl and acetyl hydroxy protecting groups, spacer¹, spacer² and spacer³ independently are selected from C(=0)NR^a-(CH₂)₂-ιo, wherein R^a is hydrogen or Cι-₄-alkyl, and Q² selected from OH, (O- (dimethoxytrityl), and NHFmoc, Q¹ is selected from OH, 0-P(NⁱPr₂)OCH₂CH₂CN), COOH and COOtBu, and Q³ is selected from OH, O-Fmoc, O-carboxymethyl, O-TBDMS, TIPS, NHFmoc and SH.

In a further interesting embodiment, the organic compound is selected from

wherein n is an integer of 0-2; R³ is independently selected from hydrogen, optionally substituted

optionally substituted Q-₂₀-alkoxy, optionally substituted aryl, optionally substituted aryloxy, a polyether of the formula 0(CH₂CH₂)_pOCH₂CH₃ wherein p is 1-10, and a disulfide of the formula

and the structural domains T is selected from

"(CH₂)_m

f-BuO

wherein m is an integer of 0-5, and the structural domains T² and T³ are independently selected from

in m is an integer of 0-5 and x is an integer of 2-10.

These compounds are believed to be novel in themselves and therefore represent a special aspect of the present invention.

The method of the invention is further illustrated by the following with reference to the Figures.

Another main aspect of the invention embodies the covalent connection of two or more LANA and/or TANA compounds to each other by a serial method, in particular by the method applied for automated DNA synthesis. Accordingly, one embodiment of the application of only LANA compounds as exemplified in Figure 7. First the LANA compound is attached to the solid support via a standard linker to the solid support or an oligonucleotide chain and a linker. The functional group FG¹ may be an OH group or a phosphorous derivative such as e.g. a phosphoamidite group. The protecting group PG¹ may be an DMTr, MMTr or Tr group. By automated synthesis, several LANA compounds can now be coupled via a phosphorous derivative by automated synthesis (n = 1-200). In step 3 the compound is detached from the resin to yield a linear poly-LANA compound in which the single units are linked by phosphorous derivative preferably phosphate diesters. The terminal functionalities R¹ and/or R² may hydroxy groups, phosphate or oligonucleotide chains.

A further embodiment of the application of the poly-LANA compound obtained as illustrated in Figure 7, for formation of new covalent bonds between the single LANA units in the poly- LANA compound to form a double covalently linked linear assembly of LANA/TANA compounds hereafter referred to as DCALT. An important aspect of this is that the (non- phosphorous) covalent bonds are only formed between two LANA (or TANA) compounds that have been linked via automated synthesis by a phosphate diester or other phosphate derivatised bonds. In particular the non-phosphorous bonds formed can be (i) a salen or metal salen functionality obtained from two salicylaldehyde functional groups by the addition of an 1,2-diamine and preferably also a metal (ii) an aryl-aryl bond formed by an Ulman coupling between two arylhalides (halide = CI, Br, I) (iii) and aryl-acetylene-aryl linkage obtained by a Sonogashia coupling between an aryl acetylene and an arylhalide (iv) an aryl-acetylene-aryl linkage obtained by a Sonogashia coupling between two arylhalides and acetylene, (v) aryl-acetylene-aryl obtained by a Stille coupling between an arylacetylene(trialkyl)tin compound and an arylhalide, (vi) an aryl-acetylene-aryl obtained by a Stille coupling between two arylhalides and l,2-bis(trialkylstannyl)acetylene (vii) an aryl-acetylene-aryl linkage obtained by a Suzuki coupling between an arylacetyleneboron(III) compound and an arylhalide or (viii) and aryl-diacetylene-aryl linkage obtained by an Glaser or Eglinton reactions between two phenyl acetylene compounds.

In still another embodiment, the phosphate linkage between these DCALT compounds may be removed by chemical or biological methods to give CALT compounds as illustrated in step 3 Figure 7.

Still another embodiment aspect of this invention is the serial automated covalent assembly of mixtures of several LANA compounds (1+n+m+p, n = 0-200, m = 0-100, p=0-100) with one or more TANA compounds. This is exemplified for the synthesis of an oligo(LANA-TANA) compound containing one TANA in Figure 8. The TANA compound is functionalized as previously mentioned. The branching is obtained by blocking the chain with a protecting group PG³ (e.g. acetyl) (step 2, Figure 8) and by activating the other branch by removing PG² (PG²: for example and Fmoc group) and continuing the chain in step 4 from there. In another aspect each of the ends can be functionalized with oligonucleotide chains in the automated synthesis. Embodied in this aspect of the invention is also the formation of a second set of covalent bonds between the single units in the oligo(LANA-TANA) compound in a fashion similar as described above for the poly-LANA compounds to give DCALT compounds as exemplified in Figure 8.

In particular this reaction relates to the application of LANA and/or TANA compounds such as illustrated in Figure 9 in automated synthesis of Oligo-LANA/TANA compounds. This is exemplified by the combination of 2 LANA compounds and 1 TANA compound in Figure 9.

The invention embodies any application of LANA compounds in automated oligonucleotide synthesis as a nucleotide analog and any application of TANA compounds in automated oligonucleotide synthesis of linear or branched oligo's. For example synthesis of an oligonucleotide chain based on any combination of the oxyribo and/or deoxyribonucleotides A,C,G,T and U with one or more LANA compounds, in particular with LANA compound such as 1 (Figure 6).

The invention also embodies the application of the general 6 types of LANA compounds and one general type of TANA compounds listed in Figure 10. In these compounds the PG¹ can be a DMTr, MMTr or Tr group and the Phos group is any phosphate derivative applied in automated DNA-synthesis in particular 2-cyanoethyl diisopropyl-phosphoramidite. For the TANA-compounds PG³ can be a base labile protecting group such as Fmoc or a silyl- protective groups or any other protective group that is orthogonal to DMTr and cyanoethyl protective groups. The invention also relates to the application of the LANA and TANA compounds listed in Figure 10 in automated synthesis to form oligo-LANA compounds and/or oligo-LANA/TANA compounds. In particular this applies to the subsequent formation of non-phosphorous covalent linear bonds such as: aryl-aryl bond formed by an Ulman coupling between two arylhalides (halide = CI, Br, I); aryl-acetylene-aryl linkage obtained by a Sonogashia coupling between an aryl acetylene and an arylhalide; aryl-acetylene-aryl obtained by a Stille coupling between an arylacetylene(trialkyl)tin compound and an arylhalide or an aryl-diacetylene-aryl linkage obtained by an Glaser coupling between two phenyl acetylene compounds.

The invention also embodies the application in iterative solid phase peptide synthesis of organic compounds that a) contain at least one phenylacetylene unit and b) are also functionalized with an amino group or a protected derivative thereof and a carboxylic acid or a protected derivative thereof (see Figures 10-13). This includes the covalent connection of two or more compounds equivalent to LANA and/or TANA compounds by using automated peptide synthesis (Linear Peptide Analogs referred to as: LPA and Trigonal Peptide Analog referred to as TPA). In this approach the LPA and TPA compounds have the same functional groups F, but instead of protected hydroxy groups the LPA compounds have one spacer functionalized with an NH₂, NHFmoc or NHBoc group and the other termini with a COOH group or any protected derivatives. For the TPA compound the third termini is a NH₂ or COOH group that is protected with a protecting group that is orthogonal to standard peptide synthesis. The oligo-LPA/TPA compounds are thus interconnected by amide bonds. It should generally be understood that most of the many known methodologies known within the field of peptide synthesis will be applicable in combination with the present method.

The functional groups of the oligo-LPA/TPA compounds can by reaction in fashion similar as described for oligo-LANA/TANA compounds also lead to CALT-compounds. The application of LPA and TPA compounds also relate to the incorporation of these in PNA chains. Thus, DCALT and CALt compounds may be prepared by this approach. It should be understood that DCALT, and similarly CALT, also covers Double covalently assembled LPA and/or TPA compounds.

The macromolecular architectures

Following the above, the present invention also provides the novel macromolecular architectures resulting from the above methods.

In one specific embodiment, the invention provides a macromolecular architecture that comprises at least one arylacetylenearyl motif and at least one metal salen complex of the formula

wherein M is a metal selected from Mn, Ni, Co, Fe, Al, and U. Such an architecture may be prepared according to the methods described herein, but it is envisaged that other method may be applicable now or in the future. In some very interesting embodiments, the molecular weight of the macromolecular architecture is at least 5000 Da. However, in many instances, the molecular weight may be as high as at least 20,000 Da or even at least 50,000 Da.

In further interesting embodiments, the macromolecular architecture is electrically conducting.

The macromolecular architecture may be characterised by PAGE (polyacrylamide gel electrophoresis) for nucleotide containing macromolecular architectures, MALDI-TOF MS (Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry) and ES-MS (electro spray mass spectroscopy).

In the present context, the term "d-^-alky!" means a linear, cyclic or branched hydrocarbon group having 1 to 20 carbon atoms, such as methyl, ethyl, propyl, /so-propyl, cyclopropyl, butyl, tert-butyl, /so-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, decyl, undecyl, dodecyl, etc. Analogously, the term "Cj-_Kj-alkyl" means a linear, cyclic or branched hydrocarbon group having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, /so-propyl, pentyl, cyclopentyl, hexyl, cyclohexyl, decyl, and the term "Cι-₄-alkyl" is intended to cover linear, cyclic or branched hydrocarbon groups having 1 to 4 carbon atoms, e.g. methyl, ethyl, propyl, /so-propyl, cyclopropyl, butyl, /^'so-butyl, tert-butyl, cyclobutyl. Typical examples of "Cι-₄-alkyl" are methyl, ethyl, propyl, /^'so-propyl, butyl, tert-butyl, and /^'so-butyl.

In the present context, i.e. in connection with the terms "alkyl", the term "optionally substituted" means that the group in question may be substituted one or several times, preferably 1-3 times, with group(s) selected from hydroxy, -e-alkoxy {i.e. C_t-₆-alkyl-oxy), C₂-₆-alkenyloxy, carboxy, oxo (forming a keto or aldehyde functionality), C^- alkoxycarbonyl, d-β-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C₁-₆-alkyl)amino; carbamoyl, mono- and di(C₁-₆-alkyl)aminocarbonyl, amino-Ci-₆-alkyl- aminocarbonyl, mono- and di(C₁-_s-alkyl)amino-C₁-₆-alkyl-aminocarbonyl, Cj-₆-alkylcarbony- lamino, cyano, guanidino, carbamido, Cx-β-alkanoyloxy, sulphono, Ci-₆-alkyIsulphonyloxy, nitro, sulphanyl, d-₆-alkylthio, halogen, where any aryl and heteroaryl may be substituted as specifically describe below for "optionally substituted aryl and heteroaryl".

Preferably, the substituents are selected from hydroxy, Cι-₅-aikoxy, carboxy, C_α-₆- alkoxycarbonyl, Ci-₆-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, amino, mono- and di(Cι-₆-alkyl)amino, carbamoyl, mono- and di(C₁.₆-alkyl)aminocarbonyl, amino-Ci-₆-alkyl-aminocarbonyl, mono- and di^-β-alky amino-Ci-e-alkyl-aminocarbonyl, Ci-₆-alkylcarbonylamino, cyano, carbamido, halogen, where aryl and heteroaryl may be substituted 1-5 times, preferably 1-3 times, with C^-alkyl, -₄-alkoxy, nitro, cyano, amino or halogen. Especially preferred examples are hydroxy, -₆-alkoxy, carboxy, aryl, heteroaryl, amino, mono- and di(Cι-₆-alkyl)amino, and halogen, where aryl and heteroaryl may be substituted 1-3 times with Cι-₄-alkyl, Q-₄-alkoxy, nitro, cyano, amino or halogen. In the present context the term "aryl" means a fully or partially aromatic carbocyclic ring or ring system, such as phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl, among which phenyl is a preferred example.

The term "heteroaryl" means a fully or partially aromatic carbocyclic ring or ring system where one or more of the carbon atoms have been replaced with heteroatoms, e.g. nitrogen (=N- or -NH), sulphur, and/or oxygen atoms. Examples of such heteroaryl groups are oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, coumaryl, furyl, quinolyl, benzothiazolyl, benzotriazolyl, benzodiazolyl, benzooxozolyl, phthalazinyl, phthalanyl, triazolyl, tetrazolyl, isoquinolyl, acridinyl, carbazolyl, dibenzazepinyl, indolyl, benzopyrazolyl, phenoxazonyl.

In the present context, i.e. in connection with the terms "aryl" and "heteroaryl", the term "optionally substituted" means that the group in question may be substituted one or several times, preferably 1-5 times, in particular 1-3 times) with group(s) selected from hydroxy (which when present in an enol system may be represented in the tautomeric keto form), -₆-alkyl, Cι-₆-alkoxy, oxo (which may be represented in the tautomeric enol form), carboxy, Cι-₆-alkoxycarbonyl, Cα-₆-alkylcarbonyl, formyl, aryl, aryloxy, aryloxy- carbonyl, arylcarbonyl, heteroaryl, amino, mono- and di(Cι-₆-alkyl)amino; carbamoyl, mono- and di(C₁-₆-alkyl)aminocarbonyl, amino- -₆-alkyl-aminocarbonyl, mono- and d Ci-e-alky amino-Ci-s-alkyl-aminocarbonyl, Cι-₆-alkylcarbonylamino, cyano, guanidino, carbamido, Q-₆-alkanoyloxy, sulphono, Cι-₆-alkylsulphonyloxy, nitro, sulphanyl, dihalogen- Cα-₄-alkyl,

halogen, where aryl and heteroaryl representing substituents may be substituted 1-3 times with d-₄-alkyl, -₄-alkoxy, nitro, cyano, amino or halogen. Preferred examples are hydroxy, Ci-₆-alkyl, Q-e-alkoxy, carboxy, Cι-₆-alkoxy- carbonyl, Cι-₆-alkylcarbonyl, aryl, amino, mono- and di(C₁.₆-alkyl)amino, and halogen, wherein aryl may be substituted 1-3 times with C_t-₄-alkyl, C^-alkoxy, nitro, cyano, amino or halogen.

"Halogen" includes fluoro, chloro, bromo, and iodo.

"Act" is an activation group for -OH, -COOH, -NHR^a and -SH, respectively. Activation groups for -OH are, e.g., selected from optionally substituted O- phosphoramidite, optionally substituted O-phosphortriester, optionally substituted O- phosphordiester, optionally substituted H-phosphonate, and optionally substituted O- phosphonate.

The term "phosphoramidite" means a group of the formula -P(OR )-N(R^y)_2/ wherein R^x designates an optionally substituted alkyl group, e.g. methyl, 2-cyanoethyl, or benzyl, and each of R^y designate optionally substituted alkyl groups, e.g. ethyl or isopropyl, or the group -N(R^y)₂ forms a morpholino group (-N(CH₂CH₂)₂0). R^x preferably designates 2- cyanoethyl and the two R^y are preferably identical and designate isopropyl. Thus, an especially relevant phosphoramidite is N,N-diisopropyl-0-(2-cyanoethyl)phosphoramidite.

Typical examples of activation groups for -OH are -P0₂ ^"0-(2-chlorophenyl), -P0₃ ^2", and -P(NⁱPr₂)OCH₂CH₂CN.

Activation groups for carboxylic acids are of the types known to the person skilled in the art, namely active esters, acid anhydrides, and acid halides. For activation of the carboxylic acids are used standard reagents for generating acid chlorides or reagents such as DCC, BOP, CDI, EEDQ, HBTU, PyBOP, MSNT, PyBroP and WSC-HCI (EDC) in the presence or absence of additives such as HOBt and DMAP (for abbreviations see: Novabiochem Catalog 2002/2003, p316-320).

"Prot" is a protecting group for -OH, -COOH, -NHR^a and -SH, respectively. Protection (and deprotection) can be performed by methods known to the person skilled in the art (see, e.g., Greene, T. W. and Wuts, P. G. M., "Protective Groups in Organic Synthesis", 2^nd ed., John Wiley, N.Y. (1991), and M.J. Gait, Oligonucleotide Synthesis, IRL Press, 1984).

Illustrative examples of hydroxy protection groups are optionally substituted trityl, such as 4,4'-dimethoxytrityl (DMTr), 4-monomethoxytrityl (MMTr), and trityl (Tr), optionally substituted 9-(9-phenyl)xanthenyl (pixyl), optionally substituted ethoxycarbonyloxy, p- phenylazophenyloxycarbonyloxy, tetraahydropyranyl (thp), 9-fluorenylmethoxycarbonyl (Fmoc), methoxytetrahydropyranyl (mthp), silyloxy such as trimethylsilyl (TMS), triisopropylsilyl (TIPS), terf-butyldimethylsilyl (TBDMS), triethylsilyl, and phenyldimethyl- silyl, benzyloxycarbonyl or substituted benzyloxycarbonyl ethers such as 2-bromo benzyloxycarbonyl, terf-butylethers, alkyl ethers such as methyl ether, acetals (including two hydroxy groups), acyloxy such as acetyl or halogen substituted acetyls, e.g. chloroacetyl or fluoroacetyl, isobutyryl, pivaloyl, benzoyl and substituted benzoyls, methoxymethyl (MOM), benzyl ethers or substituted benzyl ethers such as 2,6- dichlorobenzyl (2,6-Cl₂Bzl). Further suitable hydroxy protection groups for phosphoramidite oligonucleotide synthesis are described in Agrawal, ed. "Protocols for Oligonucleotide Conjugates"; Methods in Molecular Biology, vol. 26, Humana Press, Totowa, NJ (1994) and Protocols for Oligonucleotides and Analogs, vol 20, (Sudhir Agrawal, ed.), Humana Press, 1993, Totowa, NJ.

The term "phenol protecting groups" is intended to cover essentially the same groups as "hydroxy protecting groups". Illustrative examples of phenol protecting groups include ethers such as tetrahydropyranyl, methoxymethyl, benzyloxymethyl, methoxyethoxyethyl, methylthiomethyl, 2-(Trimethylsilyl)ethoxymethyl, and allyl, as well as esters such as formyl, acetyl, pivaloyl, and benzoyl, and carbonates such as methyl carbonate, t-butyl carbonate, vinyl carbonate and benzyl carbonate.

Illustrative examples of carboxy protection groups are allyl esters, methyl esters, ethyl esters, 2-cyanoethylesters, trimethylsilylethylesters, benzyl esters (Obzl), 2-adamantyl esters (O-2-Ada), cyclohexyl esters (OcHex), 1,3-oxazolines, oxazoler, 1,3-oxazolidines, amides or hydrazides.

Illustrative examples of amino protection groups are Fmoc (fluorenylmethoxycarbonyl), BOC (tert-butyloxycarbonyl), trifluoroacetyl, allyloxycarbonyl (alloc, AOC), benzyl- oxycarbonyl (Z, Cbz), substituted benzyloxycarbonyls such as 2-chloro benzyloxycarbonyl ((2-CIZ), monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), phthaloyl, and 9-(9- phenyl)xanthenyl (pixyl). Further suitable amino protection groups for phosphoramidite oligonucleotide synthesis are also described in Agrawal (see above).

Illustrative examples of thiolo protecting groups are trityl (Tr), acetamidomethyl (acm), trimethylacetamidomethyl (Tacm), 2,4,6-trimethoxybenzyl (Tmob), tert-butylsulfenyl (StBu), 9-fluorenylmethyl (Fm), 3-nitro-2-pyridinesulfenyl (Npys), and 4-methylbenzyl (Meb). Further suitable mercapto protection groups for phosphoramidite oligonucleotide synthesis are also described in Agrawal (see above).

Typical protection groups for -OH and -SH are selected from optionally substituted trityl, such as trimethoxytrityl, dimethoxytrityl (DMTr), monomethoxytrityl (MMTr), and trityl, and 9-(9-phenyl)xanthenyl (pixyl), tetrahydropyranyl (thp), TBDMS and TIPS; typical protection groups for -NH(R^a) are selected from trityl, such as dimethoxytrityl (DMTr), monomethoxytrityl (MMTr), and trityl, and 9-(9-phenyl)xanthenyl (pixyl), optionally substituted, tetrahydropyranyl (thp), t-BOC, Fmoc, and carboxybenzyl.

It should be understood that the protecting groups used herein may be selected so that when the compounds are incorporated in an architecture according to the invention, it will be possible to perform either a simultaneous deprotection or a sequential deprotection of the functional groups.

Applications The macromolecular architectures synthesised and assembled according to the methods described herein have applications within the field of nanotechnology and especially in the field of molecular electronics.

The linear and branched covalently linked macromolecular architectures are conjugated or partly conjugated compounds that can act as conductors or semiconductors following the principles outlined in Reed, M. A.; Tour, J. M., Scientific American, 2000, June, 68-75). Thus, the macromolecular architectures of the invention can be applied in electronic circuitry as a molecular circuit or as extensions of conventional circuits and devices.

The dimensions of the macromolecular architectures embodied in this invention may be up to 1 μm x 1 μm, such as 100 nm xlOO nm, when placed on a planar surface and larger for assemblies of macromolecular architectures. The termini of the macromolecular architectures may be functionalised with organic or biological moieties that can attach some or all of the termini of the macromolecular architectures to an appropriate material such as e.g. metal electrodes.

The linear and in particular branched macromolecular architectures may self-assemble to bridge two or more electrodes on a surface.

The invention embodies the attachment of two or more of the termini of one or more macromolecular architectures to electrodes for application of the macromolecular architectures in electronic devices. Such an electronic device may incorporate one or more macromolecular architectures that may operate in parallel or in a serial fashion.

The invention also embodies the incorporated in the macromolecular architectures or bridging two one more of the macromolecular of the invention of other organic or metalorganic compounds that have electronic properties such as rectifiers, transistors, capacitors, switches and optoelectronic, storage or memory devices. Such derivatised macromolecular architectures can be applied as nano-electronic molecular circuits.

In particular the attachment of the macromolecular architectures to electrodes may be performed in the following manner: a) Each of the termini of the macromolecular architecture that are to be attached to electrodes placed on a non-conductive surface can be functionalized with a small organic molecule that enables attachment of the individual termini to individual electrodes. For attachment to gold or platinum electrodes such small organic molecule should contain at least one of the following functional groups: thiol, sulfide, disulfide or diselenide

For attachment to silver or cupper electrodes such small organic molecule should contain at least one of the following functional groups such as thiol, sulfide, disulfide, diselenide or a carboxylic acid.

b) Each of the individual electrodes to which an individual terminus of the macromolecular architecture shall attach are functionalized with individual oligonucleotides, and each of the individual termini of the macromolecular architecture are functionalized with an oligonucleotide that is complementary with the individual oligonucleotide at the electrode to which it should bind. This will allow for the tunneling of electrons to and from the electrode, possibly via the double stranded oligonucleotide, to the macromolecular architecture.

According to the methods described above, several macromolecular architectures will bind to each electrode, but due to the geometrical arrangement of the electrodes which are designed by lithography to fit the geometrical shape of the macromolecular architecture, only one or few (<50) macromolecular architectures will attach to all of the electrodes (2- 10).

Examples of the attachment of 2-, 3- and 4-armed macromolecular architectures to electrodes according to this principle by self-assembly from a solution is shown in Figure 30.

The connectivity of individual macromolecular architectures to from 2 to 10 electrodes allows for the specific and controlled input and output of electrons via specific termini of the macromolecular architecture which can therefore be applied as an electronic component or circuit of its own according to the specific design and nature of the macromolecular architecture.

Several macromolecular architectures can in this manner be arranged on a surface by self assembly in a framework of several electrode groups made by lithographic techniques in which the single electrodes can be addressed electronically. This is exemplified in Figure 31. The macromolecular architectures embodied in this invention may also be applied as a skeleton in nanoscaled assemblies of structures such as biomolecules, biological structures, organic compounds, metalorganic compounds, colloids, carbon nanotubes, supramolecular structures and mixtures thereof. In particular the macromolecular architectures can be applied to position isolated or mixtures of peptides, oligonucleotides, dendrimers, polymers, photoactive molecules, single walled carbon nanotubes and colloidal particles with each other and/or on surfaces.

In the enclosed Examples, the inventors have fully demonstrated that the macromolecular architectures can be applied as rigid parts of nano-mechanical devices and in nano-robots; the macromolecular architectures can be used to transport a photogenerated charge over distances of 5-1000 nanometers, such as 5-500 nanometers, such as 5-100 nanometers; the macromolecular architectures can be applied for transport of current to nanomechanical devices; the macromolecular architectures can be applied to assemble carbon nanotubes; the macromolecular architectures can be applied to arrange photoactive metal such as e.g. erbium and yttrium for application nano-fotonics; and the macromolecular architectures can be applied in the detection of gene sequences.

EXAMPLES

PART A

ORGANIC SYNTHESIS

General Conditions: Standard Schlenk and vacuum line techniques were employed using argon as the inert atmosphere for all manipulations of air- or moisture-sensitive compounds. Yields refer to isolated chromatographically and spectroscopically homogeneous materials, unless otherwise stated. Commercially available starting materials were used without further purification. Solvents were dried according to standard procedures. Purification of the products was carried out by flash chromatography (FC) using Merck silica gel 60 (230-400 mesh). ^X and ¹³C NMR spectra were recorded at 400 and 100 MHz, respectively, using CDCI₃ as the solvent and were reported in ppm downfield from TMS (δ = 0) for ^XH NMR and relative to the central CDCI₃ resonance (δ = 77.00) for ¹³C NMR. Mass spectra and high resolution mass spectra were obtained on an LC-TOF spectrometer (Micromass). Triethylamine was distilled from CaH₂ under argon prior to use. Pyridine was dried over molecular sieves. 2-Hydroxy-5-iodobenzoic acid (Aldrich), Λ/_/Λ/-diisopropylcyanoethyl- chlorophosphoramidite (Aldrich), DMTrCI (Lancaster), Bis(triphenylphosphine)palladium(II) chloride (Aldrich) and 5-iodoanthranilic acid (Aldrich) were purchased from commercial 5 sources. 1,4-Diethynylbenzene (Takahashi et al. Synthesis 1980, 627; Yu et al. J. Org. Chem. 1999, 64, 2070), 1,3,5-triethynylbenzene (Weber et al. J. Chem. Soc. Perkin Trans 2, 1988, 1251; Yu et al. J. Org. Chem. 1999, 64, 2070), 2,5-diiodobenzoic acid (25), 2,5- dididodecyloxy-l,4-diethynylbenzene (24) and Λ/-methyl-3-aminopropan-l-ol (Deslongchamps et al. Can. J. Chem. 1979, 57, 3262; Koepke et al. J. Org. Chem. 1979, 10 44, 2718) were synthesized according to literature procedures.

l,4-Bis[(5-fert-butyl-3-formyl-4-hydroxyphenyl)ethynyl]benzene (1) (Figure 15)

Bis(triphenylphosphine)palladium(II)chloride (29.3 mg, 0.042 mmol) and copperiodide (15.9 mg, 0.084 mmol) were stirred in a Schlenk flask under vacuum for 30 min. 5-Iodo- 3-tert-butylsalicylaldehyde (356 mg, 1.17 mmol), 1,4-diethynylbenzene (52.7 mg, 0.42

20 mmol) and NEt₃ (15 mL) were added and the reaction mixture was stirred under argon at 60 °C for 18 h. The reaction mixture was poured into 10% aqueous NH CI (15 mL) and extracted with CH₂CI₂ (3 x 15 mL). The combined organic fractions were washed with water, dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (CH₂CI₂/pentane, 1 : 1) to yield 1 as yellow crystals (77 mg,

25 39%) : ^αH NMR (CDCI₃, 400 MHz) δ 1.36 (s, 18H), 7.43 (s, 4H), 7.53 (d, J = 1.6 Hz, 2H), 7.59 (d, J = 1.6 Hz, 2H), 9.79 (s, 2H), 11,87 (s, 2H); ¹³C NMR (CDCI₃, 100 MHz) δ 28.1, 34.0, 87.0, 89.3, 113.0, 119.5, 121.9, 130.4, 134.1, 136.0, 138.0, 160.4, 195.6.

l,3,5-Tris[(5-tert-butyl-3-formyl-4-hydroxyphenyl)ethynyl]benzene (2) (Figure 30 15)

Bis(triphenylphosphine)palladium(II)chloride (288 mg, 0.41 mmol) and copperiodide (156 mg, 0.82 mmol) were stirred in a Schlenk flask under vacuum for 30 min. 5-Iodo-3-tert- butylsalicylaldehyde (2.60g, 8.55 mmol), 1,3,5-triethynylbenzene (306 mg, 2.04 mmol) and NEt₃ (30 mL) were added and the reaction mixture was stirred under argon at 50 °C for 21 h. The reaction mixture was poured into 10% aqueous NH₄CI (40 mL) and extracted with CH₂CI₂ (3 x 20 mL). The combined organic fractions were washed with water, dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (CHzClz/pentane, 1: 1 → 3:2) to yield 2 as white crystals (841 mg, 61%): ^XH NMR (CDCI₃, 400 MHz) δ 1.44 (s, 27H), 7.61 (s, 3H), 7.64 (s, 3H), 7.66 (s, 3H), 9.87 (s, 3H), 11,97 (s, 3H); ¹³C NMR (CDCI3, 100 MHz) δ 29.3, 35.2, 87.0, 89.9, 114.0, 120.7, 124.2, 134.0, 135.5, 137.3, 139.3, 161.8, 196.9.

3-Formyl-2-hydroxy-5-iodo-benzoic acid (4) (Figure 16)

A round bottomed flask equipped with a tight screw-cap lid was charged with 2-hydroxy-5- iodobenzoic acid (3.0 g, 11.3 mmol) under argon. Hexamethylenetetramine (7.0 g, 49.9 mmol) and trifluoroacetic acid (30 mL) were added and the suspension was stirred overnight at 90 °C in the closed flask. The reaction mixture was poured into aqueous HCl (1 M, 60 mL) and stirred for 5 h. The precipitate was filtered off, washed thoroughly with aqueous HCl (1 M) and water and dried under vacuum. Due to the high polarity of this compound column chromatography was not possible, and the crude material was used directly in the following reaction. 3-([l,3]Dioxan-2-yl)-2-hydroxy-5-ϊodobenzoic acid (5) (Figure 16)

5

To a round bottomed flask charged with freshly prepared 4 (3.0 g, 10.3 mmol) suspended in toluene (30 mL) was added 1,3-propanedioI (5.0 mL, 69 mmol) and H₂S0₄ (100 μL, 1.9 mmol). The reaction mixture was refluxed for 16 h, allowed to cool to rt and neutralized with saturated aqueous NaHC0₃. Water (20 mL) was added and the mixture was extracted with CH_CCI₂ (5 x 20 mL). The combined organic fractions were washed with water (20 mL), dried over MgS0 and concentrated. The crude product was recrystallized from toluene and pentane to yield 5 as white crystals (1.556 g, ~35% over two steps): mp: 88-89 °C; ¹H NMR (CDCi₃, 400 MHz) δ 1.39 (d, J = 12.8, 1H), 1.91-2.06 (m, 1H), 3.89 (dd, J = 12.0, 2H), 4.08 (dd, J = 11.2 Hz, J = 4.4 Hz, 2H), 5.65 (s, 1H), 7.84 (d, J = 2.0 Hz, 1H), 8.02 (d, J = 2.0 Hz, 1H); ¹³C NMR (CDCI₃, 100 MHz) δ 26.0, 67.5, 80.9, 95.3, 116.0, 129.9, 139.1, 141.6, 158.6, 171.5; HRMS calcd 372.9549 ([M + Na]⁺), found 372.9553.

3-Bromo-l-(dimethoxytrityloxy)propane (6)

Br ^^ ODMTr

To a Schlenk flask charged with 3-bromo-l-propanol (256 μL, 2.95 mmol), DMTrCI (1,00 g, 2.95 mmol) and DMAP (36 mg, 0.30 mmol) in CH₂CI₂ (10 mL), was added NEt₃ (412 μL, 2.95 mmol) dropwise while stirring. The reaction mixture was stirred at rt for 4 h and poured into water (20 mL). The organic layer was separated and the aqueous phase was extracted with CH₂CI₂ (2 x 10 mL). The combined organic fractions were washed with water (10 mL), dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (pentane/Et₂0, 9: 1) to yield 6 as slightly reddish crystals (1,078 g, 83%): ^αH NMR (CDCI₃, 400 MHz) δ 2.04 (qui, J = 6.2 Hz, 2H), 3.13 (t, J = 6.2 Hz, 2H), 3.50 (t, J = 6.8 Hz, 2H), 3.72 (s, 6H), 6.76 (d, J = 6.4 Hz, 4H), 7.14-7.25 (m, 7H), 7.35 (d, J = 6.4 Hz, 2H); ¹³C NMR (CDCI₃, 100 MHz) δ 31.2, 33.7, 55.6, 61.2, 92.3, 113.3, 127.9, 129.4, 130.3, 136.5, 145.3, 158.6; HRMS calcd 463.0885 ([M + Na]⁺), found 463.0894.

3-(Dimethoxytrityloxy)prop-l-yl 3-([l,3]dioxan-2-yl)-2-hydroxy-5-iodo- benzoate (7) (Figure 16)

To a Schlenk flask charged with 5 (450 mg, 1.29 mmol) and 6 (600 mg, 1.36 mmol) in DMF (2 mL), NEt₃ (190 μL, 1.36 mmol) was added dropwise. The reaction mixture was warmed to 40 °C, stirred for 16 h and poured into water (10 mL). The DMF-water mixture was extracted with Et₂0 (3 x 10 mL) and the combined organic fractions were washed with water (10 mL), dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (CH₂CI₂/MeOH, 98: 2) to yield 7 as a colorless oil (580 mg, 63%): ^XH NMR (CDCI₃, 400 MHz) δ 1.45 (d, J = 13.6 Hz, 1H), 2.03 (qui, J = 6.0 Hz, 2H), 2.22 (m, 1H), 3.24 (t, J = 5.6 Hz, 2H) 3.75 (s, 6H), 4.01 (t, J = 12.0 Hz, 2H), 4.25 (dd, J = 11.2 Hz, J = 4.8 Hz, 2H), 4.48 (t, J = 6.4 Hz, 2H), 5.84 (s, 1H), 6.79 (d, J = 6.79, 4H), 7.18 (t, J = 9.2 Hz, 2H), 7.24-7.32 (m, 5H), 7.41 (d, J = 7.6 Hz, 2H), 7.94 (d, J = 2.4 Hz, 1H), 8.06 (d, J = 2.4 Hz, 1H), 11.21 (s, 1H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.9, 29.3, 55.3, 59.3, 63.5, 67.8, 80.4, 86.2, 95.8, 113.3, 114.7, 127.1, 128.2, 128.4, 129.0, 130.2, 136.4, 138.8, 142.0, 142.2, 145.1, 158.6, 169.2; HRMS calcd 733.1274 ([M + Na]⁺), found 733.1277.

3-(Dimethoxytrityloxy)prop-l-yl 2-benzoyloxy-3-([l,3]dioxan-2-yl)-5-ϊodo- benzoate (8) (Figure 16)

8

To a Schlenk flask charged with 7 (100 mg, 0.14 mmol) dissolved in THF was added NaOH (1 M, 210 μL, 0.21 mmol) followed by dropwise addition of benzoylchloride (25 μL, 0.21 mmol). The reaction mixture was stirred at rt for 3 min. It was then neutralized with aqueous NH₄OH (20 mL) and extracted with Et₂0 (3 x 15 mL). The combined organic phases were dried over MgS0 and concentrated. The crude product was purified by flash chromatography on silica gel (CH₂CI₂/MeOH, 99: 1) to yield 8 as a colorless oil (93 mg, 81%): ^αH NMR (CDCI₃, 400 MHz) δ 1.34 (d, J = 14.0 Hz, IH), 1.74 (qui, J = 5.7 Hz, 2H),

2.15 (m, IH), 3.05 (t, J = 6.0 Hz, 2H), 3.73-3.83 (m, 8H), 4.15 (dd, J = 10. 4 Hz, J = 3.6

Hz, 2H), 4.30 (m, 2H), 5.57 (s, IH), 6.78 (dd, J = 6.8 Hz, J = 2.0 Hz, 2H), 6.83 (dd, J =

6.8 Hz, J = 2.0 Hz, 2H), 7.17 (dd, J = 6.8 Hz, J = 2.4 Hz, 2H), 7.23-7.29 (m, 5H), 7.36

(d, J = 7.2 Hz, IH), 7.50 (t, J = 7.6 Hz, 2H), 7.56 (d, J = 7.6 Hz, IH), 7.64 (t, J = 7.6 Hz, IH) 8.16 (d, J = 8.4 Hz, 2H), 8.22 (d, J = 2.4 Hz, IH), 8.25 (d, J = 2.4 Hz, IH); ¹³C NMR

(CDCI₃, 100 MHz) δ 25.6, 29.2, 55.6, 59.5, 63.3, 67.7, 86.0, 90.0, 96.6, 113.3, 128.0,

128.2, 128.4, 129.3, 129.4, 130.1, 130.2, 130.2, 130.4, 130.5, 134.7, 136.5, 141.0,

141.1, 145.2, 158.6, 163.2, 164.6; HRMS calcd 837.1537 ([M + Na]⁺), found 837.1552.

l-Benzoyloxy-6-[l,3]dioxan-2-yl-2-(/V-(3-hydroxyprop-l-yl)aminocarbonyl-4- iodobenzene (9a) (Figure 17)

9a

To a Schlenk flask charged with 5 (513 mg, 1.47mmol) dissolved in CH₂CI₂ (20 mL) was added benzoylchloride (170 μL, 1.47 mmol) and the mixture was cooled to 0 °C. Pyridine (237 μL, 2.93 mmol) was slowly added to the reaction mixture, which was allowed to warm up to rt and stirred for 3 h under argon. 3-Amino-l-propanol (183 μL, 2.39 mmol) was then added before the mixture again was cooled to 0 °C. EDC (458 mg, 2.39 mmol) was added to the reaction mixture, which was allowed to warm up to rt and stirred for 4 h under argon. The reaction mixture was poured into water (20 mL) and extracted with CH₂CI₂ (3 x 20 mL). The combined organic fractions were washed with water, dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (EtOAc/pentane, 1 : 1 → EtOAc/pentane, 7:3) to yield 9a as white crystals (374 mg, 50%): *H NMR (CDCI₃, 400 MHz) δ 1.23 (d, J = 13.6 Hz, IH), 1.43 (qui, J = 5.8 Hz, 2H), 1.95-2.10 (m, IH), 2.62 (t, J = 6.2 Hz, IH), 3.35 (dt, J = 6.1 Hz, 2H), 3.43 (dt, J = 5.7 Hz, 2H), 3.66 (dd, J = 11.0 Hz, 2H), 4.02 (dd, J = 4.8 Hz, J = 11.2 Hz, 2H), 5.46 (s, IH), 6.44-6.54 (m, IH), 7.48 (t, J = 7.6 Hz, 2H), 7.62 (t, J = 7.4 Hz, IH), 7.93 (d, J = 2.4 Hz, IH), 8.03 (d, J = 2.4 Hz, IH), 8.13 (d, J = 8.2 Hz, 2H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.5, 32.3, 36.9, 59.4, 67.6, 90.9, 97.0, 128.7, 129.1, 130.4, 132.4, 133.9, 134.5, 138.7, 138.9, 145.6, 165.3, 165.7; HRMS calcd 534.0390 ([M + Na]⁺), found 534.0400.

l-Benzoyloxy-6-[l,3]dioxan-2-yl-2-[/V-(3-hydroxyprop-l-yl)->V-methyl- aminocarbonyi]-4-iodo-benzene (9b) (Figure 17)

9b

To a Schlenk flask charged with 5 (150 mg, 0.43mmol) dissolved in CH₂CI₂ (6 mL) was added benzoylchloride (48 μL, 0.45 mmol) and the mixture was cooled to 0 °C. Pyridine (71 μL, 0.88 mmol) was slowly added to the reaction mixture, which was allowed to warm up to rt and stirred for 3 h under argon. Λ/-Methyl-3-amino-l-propanol (62 mg, 0.70 mmol) was then added before the mixture again was cooled to 0 °C. EDC (124 mg, 0.65 mmol) was added to the reaction mixture, which was allowed to warm up to rt and stirred for 4 h under argon. The reaction mixture was poured into water (15 mL) and extracted with CH₂CI₂ (3 x 15 mL). The combined organic fractions were washed with water, dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (EtOAc/pentane, 1: 1 → EtOAc/pentane, 7:3) to yield 9a as white crystals (121 mg, 54%): Unless otherwise mentioned the signals are from two diastereomers *H NMR (CDCI₃, 400 MHz) δ 1.22-1.28 (m, 2 x IH), 1.54-1.62 (m, 2 x 2H), 1.98-2.10 (m, 2 x IH), 2.85 (s, 3H, 2. diastereomer), 2.88 (s, 3H, 1. diastereomer), 3.20-3.26 (m, 2 x 2H), 3.32-3.38 (m, 2 x IH), 3.40-3.54 (m, 2 x 2H), 3.63-3.73 (m, 2 x 2H), 3.98-4.08 (m, 2 x 2H), 5.47 (s, IH, 2. diastereomer), 5.50 (s, IH, 1. diastereomer), 7.42-7.48 (m, 2 x 2H), 7.56-7.62 (m, 2 x 2H), 8.01-8.02 (m, 2 x IH), 8.07-8.11 (m, 2 x 2H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.6, 29.3 (1. diastereomer), 30.5 (2. diastereomer), 32.5 (2. diastereomer), 37.2 (1. diastereomer), 43.7 (1. diastereomer), 48.1 (2. diastereomer), 58.0 (1. diastereomer), 59.5 (2. diastereomer), 67.6, 90.3, 96.9, 125.8 (2. diastereomer), 128.8 (1. diastereomer), 129.0, 130.4, 133.0, 134.3, 134.4, 136.7, 137.7, 145.4, 164.6, 167.5; HRMS calcd 548.0546 ([M + Na]⁺), found 548.0564. l-Benzoyloxy-2-(VV-(3-dimethoxytrϊtyloxyprop-l-yl)-aminocarbonyl)-6- [l,3]dioxan-2-yl-4-iodo-benzene (10a) (Figure 17)

10a

To a Schlenk flask charged with 9a (2.43 g, 4.76 mmol) dissolved in CH₂CI₂ (40 mL) was added DMTrCI (2.42 g, 7.14 mmol) and DMAP (87 mg, 0.71 mmol). Finally NEt₃ (1.06 mL, 7.61 mmol) was added and the reaction mixture was stirred under argon at rt for 4 h. The

10 reaction mixture was poured into water (35 mL) and extracted with CH₂CI₂ (3 x 35 mL). The combined organic fractions were washed with water, dried over MgS0 and concentrated. The crude product was purified by flash chromatography on silica gel (EtOAc/pentane/NEt₃, 40: 59: 1) to yield 10a as yellow crystals (3.36 g, 90%): *H NMR (CDCI₃, 400 MHz) δ 1.23 (d, J = 13.0 Hz, IH), 1.55 (qui, J = 6.1 Hz, 2H), 1.96-2.10 (m,

15 IH), 2.99 (t, J = 5.6 Hz, 2H), 3.29 (dt, J = 6.4 Hz, 2H), 3.66 (dd, J = 11.4 Hz, 2H), 3.70 (s, 6H), 4.02 (dd, J = 5.0 Hz, J = 11.0 Hz, 2H), 5.46 (s, IH), 6.37 (t, J = 5.4 Hz, IH), 6.70 (d, J = 8.8 Hz, 4H), 7.12-7.19 (m, 7H), 7.28 (d, J = 8.4 Hz, 2H), 7.43 (t, J = 7.8 Hz, 2H), 7.59 (t, J = 7.4 Hz, IH), 7.83 (d, J = 2.0 Hz, IH), 8.02 (d, J = 2.4 Hz, IH), 8.04 (d, J = 8.2 Hz, 2H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.6, 29,5, 38.4, 55.5, 61.8, 67.6, 86.4, 90.8,

20 97.0, 113.3, 127.0, 128.1, 128.3, 128.9, 129.0, 130.1, 130.5, 132.9, 133.9, 134.2, 136.3, 138.5, 138.6, 144.9, 145.6, 158.6, 164.5, 164.8; HRMS calcd 836.1696 ([M + Na]⁺), found 836.1794.

l-Benzoyloxy-2-(/V-(3-dimethyloxytrityloxyprop-l-yl)-/V-methyl-aminocarbonyl)- 25 6-[l,3]dioxan-2-yl-4-iodo-benzene (10b) (Figure 17)

To a Schlenk flask charged with 9b (120 mg, 0.23 mmol) dissolved in CH₂CI₂ (5 mL) was added DMTrCI (116 mg, 0.34 mmol) and DMAP (4 mg, 0.034 mmol). Finally NEt₃ (51 μL, 0.36 mmol) was added and the reaction mixture was stirred under argon at rt for 4 h. The reaction mixture was poured into water (10 mL) and extracted with CH₂CI₂ (3 x 10 mL). 5 The combined organic fractions were washed with water, dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (EtOAc/pentane/NEt₃, 35:64: 1) to yield 10b as yellow crystals (163 mg, 87%): Unless otherwise mentioned the signals are from two diastereomers ¹H NMR (CDCI₃, 400 MHz) δ 1.16-1.25 (m, 2 x IH), 1.60 (qui, J = 6.9 Hz, 2H, 1. diastereomer), 1.66-1.78 (m, 2H, 2.

10 diastereomer), 1.94-2.09 (m, 2 x IH), 2.78 (s, 3H, 2. diastereomer), 2.80 (s, 3H, 1. diastereomer), 2.88-2.97 (m, 2 x 2H), 3.32-3.41 (m, 2 x 2H), 3.62-3.72 (m, 2 x 8H), 3.96-4.05 (m, 2 x 2H), 5.46 (s, IH, 2. diastereomer), 5.47 (s, IH, 1. diastereomer), 6.70-6.75 (m, 2 x 4H), 7.09-7.22 (m, 2 x 7H), 7.25-7.31 (m, 2 x 2H), 7.37-7.43 (m, 2 x 2H), 7.47 (d, J = 2.0 Hz, IH, 1. diastereomer), 7.50-7.56 (m, IH (2. diastereomer) and 2

15 x IH), 7.97 (d, J = 1.6 Hz, IH, 1. diastereomer), 7.99 (d, J = 1.8 Hz, IH, 2. diastereomer), 8.02-8.07 (m, 2 x 2H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.7, 27.6 (1. diastereomer), 29.3 (2. diastereomer), 32.8 (2. diastereomer), 37.7 (1. diastereomer), 45.3 (1. diastereomer), 48.9 (2. diastereomer), 55.5, 60.9 (2. diastereomer), 61.0 (1. diastereomer), 67.6, 86.0 (1. diastereomer), 86.3 (2. diastereomer), 90,3 (1.

20 diastereomer), 90.4 (2. diastereomer), 96.9 (1. diastereomer), 97.0 (2. diastereomer),

113.3 (1. diastereomer), 113.4 (2. diastereomer), 127.0, 128.0 (1. diastereomer), 128.1 (2. diastereomer), 128.3 (2. diastereomer), 128.4 (1. diastereomer), 128.9, 129.0, 130.2, 130.5, 133.5 (2. diastereomer), 133.6 (1. diastereomer), 134.1, 134.3, 136.5, 136.6 (1. diastereomer), 136.7 (2. diastereomer), 137.3 (1. diastereomer), 137.4 (2. diastereomer),

25 145.1 (2. diastereomer), 145.2 (1. diastereomer), 145.3, 158.6, 164.3 (2. diastereomer),

164.4 (1. diastereomer), 165.8 (1. diastereomer), 166.3 (2. diastereomer); HRMS calcd 850.1853 ([M + Na]⁺), found 850.1817.

l,4-Bis[(4-benzoyloxy-5-(3-dimethoxytrityloxyprop-l-yloxy)carbonyl-3-([l,3]- 30 dioxan-2-yl)-phenyi)ethynyl]benzene (11) and l-[(4-benzoyloxy-5-(3- dimethoxytrityloxyprop-l-yloxy)carbonyl-3-([l,3]-dioxan-2-yl)-phenyl)ethynyl]- 4-[(4-benzoyloxy-5-(3-hydroxyprop-l-yloxy)carbonyl-3-(l,3-dioxan-2-yl)- phenyl)ethynyl]benzene (12) (Figure 18)

35

11 12

To a Schlenk flask charged with 8 (93 mg, O.llmmol) were added 1,4-diethynylbenzene (6.9 mg, 0.055 mmol), Bis(triphenylphosphine)-palladium(IL)chloride (4.0 mg, 0.006 5 mmol) and copperiodide (1.1 mg, 0.006 mmol) and the mixture was stirred under vacuum for 30 min. NEt₃ (5 mL) was added and the reaction mixture was stirred under argon at 53 °C for 19 h. The reaction mixture was poured into 10% aqueous NH₄CI (10 mL) and extracted with CH₂CI₂ (3 x 10 mL). The combined organic fractions were washed with water, dried over MgS0₄ and concentrated. The crude product was purified by flash

10 chromatography on silica gel (Et₂0/pentane, 9: 1) to yield 11 as a yellow oil (23 mg, 28%) and 12 as a yellow oil (34 mg, 51%). 11: *H NMR (CDCI₃, 400 MHz) δ 1.29 (d, J = 12.4 Hz, 2H), 1.72 (qui, J = 5.8 Hz, 4H), 2.04-2.17 (m, 2H), 3.02 (t, J = 5.8 Hz, 4H), 3.67 (s, 12H), 3.76 (dd, J = 12.2 Hz, 4H), 4.12 (dd, J = 11.2 Hz, J = 4.4 Hz, 4H), 4.27 (t, 6.4 Hz, 4H), 5.57 (s, 2H), 6.72 (d, J = 8.8 Hz, 8H), 7.10 (t, J = 7.4 Hz, 2H), 7.16-7.23 (m, 12H),

15 7.31 (d, J = 7.2, 4H), 7.42-7.48 (m, 8H), 7.58 (t, J = 7.4, 2H), 8.02 (d, J = 2.4 Hz, 2H), 8.07 (d, J = 2.4 Hz, 2H), 8.13 (d, J = 7.6 Hz, 4H); ¹³C NMR (CDCI₃, 100 MHz) δ 29.2, 30.0, 55.4, 59.5, 63.2, 67.7, 86.0, 90.0, 90.3, 97.1, 113.2, 121.4, 123.2, 124.6, 126.9, 128.0, 128.4, 128.9, 129.4, 130.2, 130.5, 131.9, 133.3, 133.9, 135.1, 135.5, 136.5, 145.2, 148.4, 158.6, 163.9, 164.7; HRMS calcd 1521.5399 ([M + Na]⁺), found 1521.5505. 12: ^JH

20 NMR (CDCI₃, 400 MHz) δ 1.35 (d, J = 13.6 Hz, 2H), 1.72-1.83 (m, 4H), 2.10-2.24 (m, 2H), 3.09 (t, J = 5.8 Hz, 2H), 3.61 (dt, J = 5.6 Hz, 2H), 3.74 (s, 6H), 3.83 (dd, J = 12.8 Hz, 4H), 4.18 (dd, J = 4.2, J = 11.8, 4H), 4.33 (t, J = 6.2 Hz, 4H), 5.64 (s, IH), 5.65 (s, IH), 6.79 (d, J = 8.8 Hz, 4H), 7.17 (t, J = 6.8 Hz, IH), 7.22-7.34 (m, 6H), 7.38 (d, J = 3.6 Hz, 2H), 7.48-7.59 (m, 8H), 7.62-7.71 (m, 2H), 8.09 (d, J = 2.4 Hz, IH), 8.10 (d, J =

25 2.4 Hz, IH), 8.13 (d, J = 2.4 Hz, IH), 8.20 (d, J = 7.8 Hz, 2H), 8.22 (d, J = 2.4 Hz, IH), 8.25 (d, J = 7.8 Hz, 2H); ¹³C NMR (CDCI₃, 100 Hz) δ 29.9, 30.5, 31.7, 55.4, 59.1, 59.5, 62.4, 63.2, 66.1, 67.7, 86.0, 89.9, 90.0, 90.2, 90.4, 97.1, 113.2, 121.4, 121.6, 123.1, 123.2, 124.6, 126.9, 128.0, 128.3, 128.9, 129.0, 129.4, 130.2, 130.5, 131.9, 133.3, 133.9, 134.1, 135.0, 135.2, 135.5, 136.5, 145.2, 148.2, 148.4, 158.5, 163.9, 164.5,

30 164.7, 164.8; HRMS calcd 1219.4092 ([M + Na]⁺), found 1219.4094. l-[(4-Benzoyloxy-5-(3-dimethoxytrityloxyprop-l-yloxy)carbonyl-3-([l,3]- dioxan-2-yl)-phenyl)ethynyl]-4-[(4-benzoyloxy-5-(3-(2-cyanoethoxy-Λ/,iV- diisopropylamino-phosphanyloxy)prop-l-yloxy)carbonyl-3-(l,3-dioxan-2-yl)- phenyl)ethynyl]benzene (13) (Figure 18)

To a Schlenk flask charged with 12 (67 mg, 0.056 mmol) dissolved in THF (5 mL) was

10 added diisopropylethylamine (49 μL, 0.28 mmol) and the solution was cooled to 0 °C. N,N- diisopropylcyanoethylchlorophosphoramidite (44 μL, 0.20 mmol) was added dropwise while stirring. The reaction mixture was stirred under argon at rt for 16 h. The reaction was quenched by addition of MeOH (0.5 ml) and the solvent was removed by vacuum distillation. The resulting oil was dissolved in EtOAc and washed with 5.5% aqueous

15 NaHC0₃ (2 x 10 mL), washed with water (10 mL), dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (EtOAc/pentane/NEt₃, 4:5: 1 → EtOAc/NEt₃, 9: 1) to yield 13 as a yellow oil (41 mg, 52%): *H NMR (CDCI₃, 400 MHz) δ 1.14-1.31 (m, 14H), 1.74 (qui, J = 7 Hz, 4H), 2.02-2.18 (m, 2H), 2.51 (t, J = 6.4 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 3.02 (t, J = 5.6 Hz, 2H), 3.44-3.55 (m, 4H), 3.67 (s,

20 6H), 3.76 (dd, J = 11.8 Hz, 4H), 4.10 (dd, J = 4.4 Hz, J = 11.2 Hz, 4H), 4.19-4.29 (m, 4H), 5.566 (s, IH), 5.572 (s, IH), 6.71 (d, J = 8.8 Hz, 4H), 7.09 (t, J = 7.4 Hz, IH), 7.15- 7.22 (m, 6H), 7.31 (d, J = 7.6 Hz, 2H), 7.40-7.52 (m, 8H), 7.54-7.62 (m, 2H), 8.02 (s, 2H), 8.06 (d, J = 2 Hz, IH), 8.13 (d, J = 7.6 Hz, 2H), 8.15 (d, J = 2.4 Hz, IH), 8.18 (d, J = 7.4 Hz, 2H); ¹³C NMR (CDCI₃, 100 MHz) δ 19.3, 23.6, 24.5, 27.9, 29.1, 42.0, 54.1, 57.4,

25 58.2, 58.9, 61.3, 61.9, 66.5, 84.8, 88.7, 88.8, 88.97, 89.00, 95.9, 112.0, 113.1, 120.1, 120.2, 121.9, 122.0, 123.3, 125.6, 126.7, 127.1, 127.6, 127.7, 128,2, 128.9, 129.2, 130.6, 132.0, 132.1, 132.6, 132.7, 133.8, 133.9, 134.2, 135.3, 144.0, 147.1, 147.2,157.3, 162.6, 163.4; HRMS calcd 1419.5171 ([M + Na]⁺), found 1419.5170. l,4-Bis[(4-benzoyloxy-5-()V-(3-dϊmethoxytrityloxyprop-l-yl)-aminocarbonyl)-3- ([l,3]-dioxan-2-yl)-phenyl)ethynyl]benzene (14a) (Figure 19)

^14a

Bis(triphenylphosphine)palladium(II)chloride (111 mg, 0.16 mmol) and copperiodide (30 mg, 0.16 mmol) were stirred in a Schlenk flask under vacuum for 30 min. 10a (1.35 g, 1.66 mmol), 1,4-diethynylbenzene (100 mg, 0.79 mmol) and THF (18 mL) were added. Finally NEt₃ (7 mL) was added and the reaction mixture was stirred under argon at 53 °C for 16 h. The reaction mixture was poured into saturated aqueous NaCI ( 25 mL) and extracted with Et₂0 (3 x 25 mL). The combined organic fractions were washed with water, dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (EtOAc/pentane, 1: 1 → EtOAc/pentane, 7:3) to give 14a as yellow foam (753 mg, 64%): ²H NMR (CDCI₃, 400 MHz) δ 1.24 (d, J = 13.6 Hz, 2H), 1.59 (qui, J = 6.0 Hz, 4H), 1.96-2.10 (m, 2H), 3.02 (t, J = 5.4 Hz, 4H), 3.32 (dt, J = 6.0 Hz, 4H), 3.67 (s, 12H), 3.69 (dd, J = 11.6 Hz, 4H), 4.04 (dd, J = 4.6 Hz, J = 11.4 Hz, 4H), 5.52 (s, 2H), 6.42 (t, J = 5.6 Hz, 2H), 6.70 (d, J = 8.8, 8H), 7.08-7.21 (m, 14H), 7.30 (d, J = 7.2 Hz, 4H), 7.41 (s, 4H), 7.43 (t, J = 8.0 Hz, 4H), 7.58 (t, J = 7.2 Hz, 2H), 7.66 (d, J = 2.0 Hz, 2H), 7.89 (d, J = 2 Hz, 2H), 8.08 (d, J = 7.8 Hz, 4H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.7, 29.5, 38.5, 55.4, 61.9, 67.6, 86.5, 90.1, 90.3, 97.5, 113.3, 121.7, 123.2, 127.0, 128.1, 128.4, 129.0, 129.1, 130.1, 130.5, 131.4, 131.9, 132.6, 132.7, 132.8, 134.1, 136.4, 145.0, 145.7, 158.6, 164.9, 165.4; HRMS calcd 1519.5719 ([M + Na]⁺), found 1519.8694.

l,4-Bis[(4-benzoyloxy-5-( -(3-(dimethoxytrϊtyloxyprop-l-yl)- v-methyl- aminocarbonyl)-3-([l,3]-dioxan-2-yl)-phenyl)ethynyl]benzene (14b) (Figure 19)

14b

Bis(triphenylphosphine)palladium(II)chloride (14 mg, 0.020 mmol) and copperiodide (4 mg, 0.020 mmol) were stirred in a Schlenk flask under vacuum for 30 min. 10b (176 mg, 5 0.21 mmol), 1,4-diethynylbenzene (13 mg, 0.10 mmol) and THF (6 mL) were added. Finally NEt₃ (4 mL) was added and the reaction mixture was stirred under argon at 53 °C for 16 h. The reaction mixture was poured into saturated aqueous NaCI (10 mL) and extracted with Et₂0 (3 x 10 mL). The combined organic fractions were washed with water, dried over MgS0 and concentrated. The crude product was purified by flash

10 chromatography on silica gel (EtOAc/pentane, 1: 1 → EtOAc/pentane, 7:3 ) to yield 14b as yellow foam (99 mg, 64%): Unless otherwise mentioned the signals are from two diastereomers *H NMR (CDCI₃, 400 MHz) δ 1.28-1.36 (m, 2 x 2H), 1.74 (q, J = 7.0 Hz, 4H, 1. diastereomer), 1.80-1.89 (m, 4H, 2. diastereomer), 2.02-2.20 (m, 2 x 2H), 2.90 (s, 6H, 2. diastereomer), 2.93 (s, 6H, 1. diastereomer), 3.00-3.06 (m, 2 x 4H), 3.46-3.52

15 (m, 2 x 4H), 3.72-3.82 (m, 2 x 16H), 4.08-4.16 (m, 2 x 4H), 5.61 (s, 2H, 2. diastereomer), 5.63 (s, 2H, 1. diastereomer), 6.78-6.83 (m, 2 x 8H), 7.16-7.32 (m, 2 x 14H), 7.34-7.43 (m, 2 x 6H), 7.46-7.54 (m, 2 x 8H), 7.60-7.67 (m, 2 x 2H), 7.92-7.96 (m, 2 x 2H), 8.14-8.19 (m, 2 x 4H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.7, 27.6 (1. diastereomer), 29.3 (2. diastereomer), 32.8 (2. diastereomer), 37.8 (1. diastereomer),

20 45.3 (1. diastereomer), 49.0 (2. diastereomer), 55.4, 60.9 (2. diastereomer), 61.0 (1. diastereomer), 67.6 (2. diastereomer), 67.7 (1. diastereomer), 86.1 (1. diastereomer), 86.3 (2. diastereomer), 90.1, 90.3, 97.4 (1. diastereomer), 97.5 (2. diastereomer), 113.3, 121.4 (1. diastereomer), 121.5 (2. diastereomer), 123.2, 126.9, 128.0, 128.3 (2. diastereomer), 128.4 (1. diastereomer), 128.9, 129.1, 130.1 (2. diastereomer), 130.2 (1.

25 diastereomer), 130.5, 130.8 (2. diastereomer), 131.0 (1. diastereomer), 131.6, 131.9, 132.7, 132.8, 134.0, 136.5 (2. diastereomer), 136.6 (1. diastereomer), 145.2 (1. diastereomer), 145.26 (2. diastereomer), 145.31, 158.6, 164.5 (2. diastereomer), 164.6 (1. diastereomer), 166.7 (1. diastereomer), 167.2 (2. diastereomer).

30 l-[(4-Benzoyloxy-5-(W-(3-dimethoxytrityloxyprop-l-yl)-aminocarbonyl)-3- ([l_/3]-dioxan-2-yl)-phenyl)ethynyl]-4-[(4-benzoyloxy-5-(Λf-(3-hydroxyprop-l- yI)aminocarbonyl)-3-(l,3-dioxan-2-yl)phenyl)ethynyl]benzene (15) (Figure 19)

5

To a Schlenk flask charged with 14a (103 mg, 0.069 mmol) dissolved in CH₂CI₂ (5 mL) was added AcOH (5 mL) and reaction mixture was stirred at rt for 55 min. The reaction mixture was poured into 5.5 % aqueous NaHC0₃ (132 mL) to obtain a pH value at 7 and then extracted with CH₂CI₂ (3 x 20 mL). The combined organic fractions were washed with

10 water, dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (EtOAc/pentane, 8: 2) to yield 15 as yellow foam (48 mg, 60%): H NMR (CDCI₃, 400 MHz) δ 1.32 (d, J = 13.2 Hz, 2H), 1.53 (qui, J = 6.0 Hz, 2H), 1.66 (qui, J = 6.0 Hz, 2H), 2.04-2.18 (m, 2H), 2.83 (t, J = 6.2 Hz, IH), 3.10 (t, J = 5.4 Hz, 2H), 3.39 (dt, J = 6.0 Hz, 2H), 3.44 ( dt, J = 6.3 Hz, 2H), 3.52 (dt, J = 5.6 Hz, 2H),

15 3.75 (s, 6H), 3.76 ( dd, J = 12.4 Hz, 4H), 4.11 (dd, J = 4.4 Hz, J = 11.6 Hz, 4H), 5.58 (s, 2H), 6.50 (t, J = 5.4 Hz, IH), 6.59 (t, J = 6.4 Hz, IH), 6.76 (d, J = 8.8 Hz, 4H), 7.16-7.28 (m, 7H), 7.36 (d, J = 7.2 Hz, 2H), 7.49 (s, 4H), 7.50 (t, J = 8 Hz, 2H), 7.56 (t, J = 7.8 Hz, 2H), 7.66 (t, J = 7.6 Hz, IH), 7.69 (t, J = 7.6 Hz, IH), 7.73 (d, J = 2.0, IH), 7.85 (d, J = 2.4 Hz, IH), 7.96 (d, J = 2.4 Hz, 2H), 8.15 (d, J = 7.6 Hz, 2H), 8.22 (d, J = 7.6 Hz, 2H);

20 ¹³C NMR (CDCI₃, 100 MHz) δ 25.6, 25.7, 29.5, 32.3, 36.8, 38.5, 55.4, 59,3, 61.9, 67.6, 86.5, 89.9, 90.2, 90.3, 90.5, 97.5, 113.3, 121.7, 122.0, 123.1, 123.3, 127.0, 128.1, 128.4, 128.8, 128.99, 129.05, 129.1, 130.1, 130.46, 130.49, 131.0, 131.4, 131.9, 132.6, 132.7, 132.8, 132,9, 133.1, 134.1, 134.4, 136.4, 145.0, 145.6, 145.7, 158.6, 164.9, 165.4, 166.6; HRMS calcd 1217.4412 ([M + Na]⁺), found 1217.5266.

25 l-[(4-Benzoyloxy-5-(W-(3-dimethoxytrityloxyprop-l-yl)aminocarbonyl)-3-([l_/3]- dioxan-2-yl)-phenyl)ethynyl]-4-[(4-benzoyloxy-5-(Λt-3-(2-cyanoethoxy-7v,/V- diisopropylamino-phosphanyloxy)prop-l-yl)aminocarbonyl)-3-(l,3-dioxan-2- yl)phenyl)ethynyl]benzene (16) (LANA) (Figure 19)

30

To a Schlenk flask charged with 15 (277 mg, 0.23 mmol) dissolved in THF (15 mL) was added diisopropylethylamine (320 μL, 1.39 mmol) and the solution was cooled to 0 °C. 5 /V_/W-diisopropylcyanoethylchlorophosphoramidite (181 μL, 0.97 mmol) was added dropwise while stirring. The reaction mixture was stirred under argon at rt for 16 h. The reaction was quenched by addition of MeOH (1 ml) and the solvent was removed by vacuum distillation. The resulting oil was dissolved in EtOAc (15 mL) and washed with 5.5% aqueous NaHC0₃ (15 mL), dried over MgS0₄ and concentrated. The crude product was

10 purified by flash chromatography on silica gel (DCM/pentane/NEt₃, 2:7: 1 →

EtOAc/pentane/NEt₃, 4:5: 1) to yield 16 as a yellow oil (107 mg, 33%): *H NMR (CDCI₃, 400 Hz) δ 1.07 (d, J = 7.2 Hz, 6H), 1.10 (d, J = 7.2 Hz, 6H), 1.25 (d, J = 13.6 Hz, 2H), 1.54-1.66 (m, 4H), 1.98-2.12 (m, 2H), 2.47 (t, J = 6.6 Hz, 2H), 3.03 (t, J = 5.8 Hz, 2H), 3.28-3.39 (m, 4H), 3.43-3.55 (m, 4H), 3.56-3.75 (m, 12H), 4.01-4.08 (m, 4H), 5.52 (s,

15 2H), 6.41 (t, J = 5.6 Hz, IH), 6.51 (t, J = 5.8 Hz, IH), 6.71 (d, J = 8.8 Hz, 4H), 7.09-7.21 (m, 7H), 7.30 (d, J = 7.2 Hz, 2H), 7.42 (s, 4H), 7.44 (t, J = 8.0 Hz, 2H), 7.48 (t, J = 8.4 Hz, 2H), 7.59 (t, J = 7.6 Hz, IH), 7.61 (t, J = 7.6 Hz, IH), 7.66 (d, J = 2 Hz, IH), 7.76 (d, J = 2 Hz, IH), 7.89 (s, 2H), 8.08 (d, J = 7.8 Hz, 2H), 8.16 (d, J = 8.0 Hz, 2H); ¹³C NMR (CDCI₃, 100 MHz) δ 19.3, 23.6, 24.4, 28.2, 29.4, 36.5, 37.2, 41.9, 54.2, 57.2, 57.4, 60.6,

20 66.4, 85.2, 88.8, 88.9, 89.0, 89.1, 96.2, 112.1, 116.8, 120.4, 120.5, 121.9, 122.0, 125.8, 126.8, 127.1, 127.7, 127.8, 128.8, 129.2, 129.3, 130.1, 130.6, 131.3, 131.40, 131.44, 131.5, 131.6, 132.9, 133.0, 135.1, 143.7, 144.4, 144.5, 157.4, 163.6, 163.8, 164.1, 164.2; HRMS calcd 1417.5490 ([M + Na]⁺), found 1417.1564.

25 l,3,5-Tris[(4-benzoyloxy-5-( V-3-dimethoxytrityloxyprop-l-yl)aminocarbonyl)-3- ([l,3]-dioxan-2-yl)-phenyl)ethynyl]benzene (17a) (Figure 20)

17a

Bis(triphenylphospine)palladium(II)chloride (148 mg, 0.211 mmol) and copperiodide (40 mg, 0.211 mmol) were stirred in a Schlenck flask under vacuum for 30 min. 10a (3.44 g, 4.23 mmol), triethynylbenzene (159 mg, 1.06 mmol) and THF (90 mL) were added. Finally NEt₃ (22.5 mL) was added and the reaction mixture was stirred under argon at 52 °C for 16 h. The reaction mixture was diluted with CH₂CI₂ (100 mL) and then washed with water (3 x 100 mL) and brine (100 mL). The organic layer was dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (CHzCIJEtOAc, 90: 10) to yield unreacted 10a (1.06 g, 1.30 mmol) and 17a as white foam (866 mg, 37%): *H NMR (CDCI₃, 400 MHz) δ 1.23 (d, J = 12.8 Hz, 3H), 1.55 (qui, J = 6.4 Hz, 6H), 2.02 (m, 3H), 2.99 (t, J = 6.4 Hz, 6H), 3.29 (qui, J = 6.0 Hz, 6H), 3.67 (s, 18H), 3.7 (t, J = 12.0 Hz, 6H), 4.04 (dd, J = 11.2 Hz, J = 5.2 Hz, 6H), 5.53 (s, 3H), 6.42 (t, J = 5.2 Hz, 3H), 6.70 (d, J = 8.8 Hz, 12H), 7.12-7.19 (m, 21H), 7.30 (d, J = 8.4 Hz, 6H), 7.44 (t, J = 7.6 Hz, 6H), 7.55 (s, 3H), 7.59 (t, J = 7.6 Hz, 3H), 7.67 (d, J = 2.2 Hz, 3H), 7.91 (d, J = 2.2 Hz, 3H), 8.09 (d, J = 8.4 Hz, 6H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.7, 29.5, 38.5, 55.4, 61.9, 67.6, 86.5, 88.8, 89.5, 113.4, 121.4, 127.0, 128.1, 128.4, 129.0, 129.1, 130.1, 130.5, 131.5, 132.7, 132.8, 132.9, 134.1, 134.6, 136.4, 145.0, 145.9, 158.6, 164.9, 165.4. l,3,5-Tris[(4-benzoyloxy-5-( v-(3-dimethoxytrityloxyprop-l-yl)-/V-methyl- aminocarbonyl)-3-([l,3]-dioxan-2-yl)-phenyl)ethynyl]benzene (17b) (Figure 20)

Bis(triphenylphosphine)paliadium(II)chloride (7.7 mg, 0.011 mmol) and copperiodide (2.1 mg, 0.011 mmol) were stirred in a Schlenck flask under vacuum for 30 min. 10b (174 mg, 0.21 mmol), triethynylbenzene (8.0 mg, 0.053 mmol) and THF (8 mL) were added. Finally

10 NEt₃ (0.4 mL) was added and the reaction mixture was stirred under argon at 52 °C for 16 h. The reaction mixture was diluted with CH₂CI₂ (10 mL) and then washed with water (3 x 10 mL) and brine (10 mL). The organic layer was dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel (EtOAc/pentane/NEt₃, 60:40: 1) to yield unreacted 10b (46.2 mg, 0.056 mmol) and 17b as white foam (62.3

15 mg, 46%): ^XH NMR (CDCI₃, 400 MHz) δ 1.24 (d, J = 12.6 Hz, 3H), 1.57 (qui, J = 5.9 Hz, 6H), 2.04 (m, 3H), 2.82 (s, 1. diastereomer), 2.86 (s, 2. diastereomer, tot 1. and 2. diastereomer 9H), 2.95 (q, J = 6.0 Hz, 6H), 3.41 (s, br, 6H), 3.64-3.71 (m, 24H), 4.04 (dd, J = 11.2 Hz, J = 4.8 Hz, 6H), 5.54 (s, 1. diastereomer), 5.55 (s, 2. diastereomer, tot. 1. and 2. diastereomer, 3H), 6.72 (d, J = 8.8 Hz, 12H), 7.12-7.19 (m, 21H), 7.27-7.33

20 (m, 9H), 7.40-7.46 (m, 9H), 7.54 (m, 3H), 7.87 (m, 1. and 2. diastereomer, 3H), 8.09 (m, 1. and 2. diastereomer, 6H);

¹³C NMR (CDCI₃, 100 MHz) δ 25.4, 27.3, 29.1, 32.5, 37.4, 44.9, 48,6, 55.1, 55.7, 60.6, 60.7, 67.2, 67.3, 85.7, 85.9, 88.1, 88.4, 89.1, 89.5, 97.0, 97.1, 112.9, 113.0, 120.6, 120.8, 123.6, 123.7, 126.6, 127.7, 128.0, 128.4, 128.5, 128.6, 128.7, 128.8, 129.8,

25 130.2, 130.5, 130.6. 130.7, 131.5, 131.6, 131.7, 131.8, 131.9, 132.0, 132.5, 133.7, 134.3, 135.0, 136.2, 144.8, 145.0, 158.3, 164.1, 164.2, 166.3, 166.8. l,3-Bis[(4-benzoyloxy-5-(/V-3-dimethoxytrityloxyprop-l-yl)aminocarbonyl)-3- ([l,3]-dioxan-2-yl)-phenyl)ethynyl]-5-[(4-benzoyloxy-5-(/V-(3-hydroxyprop-l- yl)aminocarbonyl)-3-([l,3]-dioxan-2-yl)-phenyl)ethynyl] benzene (18) and 1,3- Bϊs[(4-benzoyloxy-5-( v-(3-hydroxyprop-l-yl)aminocarbonyl)-3-([l,3]-dioxan-2- yl)-phenyl)ethynyl]-5-[(4-benzoyloxy-5-(iV-3-dimethoxytrityloxyprop-l- yl)aminocarbonyl)-3-([l,3]-dioxan-2-yl)-phenyl)ethynyl]benzene (19) (Figure 20)

18 19

10

To a round bottomed flask charged with 17a (485 mg, 0.22 mmol) dissolved in CH₂CI₂ (15 mL) was added dropwise glacial AcOH (15mL). The reaction mixture was stirred for 8 minutes, quickly diluted with CH₂CI₂ (50 mL) and neutralized with saturated NaHC0₃. The organic layer was washed with water (3 x 80 mL), dried over MgS0 and concentrated. The

15 crude hydrolyzed mixture was purified by flash chromatography on silica gel

(CH₂CIJEtOAc, 85: 15 → 60:40 → 0: 100) to yield unhydroiized 17a (248 mg, 0112 mmol), 18 (118 mg, 28%) and 19 (27 mg, 7.6%). 18: *H NMR (CDCI₃, 400 MHz) δ 1.23 (d, J = 12.8 Hz, 6H), 1.48 (qui, J = 6.4 Hz, 2H), 1.60 (qui, J = 6.4 Hz, 4H), 2.02 (m, 3H), 2.99 (t, J = 6.4 Hz, 4H), 3.29-3.42 (m, 6H), 3.47 (q, J = 5.1 Hz, 2H), 3.67 (s, 12H), 3.70 (t, J =

20 12.0 Hz, 6H), 4.04 (dd, J = 11.2 Hz, J = 5.2 Hz, 6H), 5.53 (s, 3H), 6.42 (t, J = 5.2 Hz, 2H), 6.51 (t, J = 6.2 Hz, IH), 6.70 (d, J = 8.8 Hz, 8H), 7.12-7.19 (m, 14H), 7.30 (d, J = 8.4 Hz, 4H), 7.42-7.51(m, 6H), 7.55- 7.62 (m, 6H), 7.67 (d, J = 2.2 Hz, 2H), 7.78 (d, J = 2.2, IH), 7.91 (d, J = 2.2 Hz, 3H), 8.09 (d, J = 8.4 Hz, 4H), 8.16 (d, J = 8.0, 2H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.3, 25.4, 29.2, 32.1, 36.5, 83.2, 55.2, 59.1, 61.6, 67.3, 86.2,

25 88.6, 88.8, 89.0, 89.2, 97.1, 97.2, 113.1, 121.1, 121.4, 123.6, 123.7, 126.7,

127.8,.128.1, 128.6, 128.7, 128.8, 129.8, 130.2, 130.8, 131.2, 132.3, 132.4, 132.5, 132.6, 132.7, 133.0, 133.8, 134.1,134.3, 136.1, 144.7, 145.5, 145.6, 158.4, 164.6, 165.1, 166.4; HRMS calcd xx.xxxx ([M + Na]⁺), found xx.xxxx. 19: *H NMR (CDCI₃, 400 MHz) δ 1.23 (d, J = 12.8 Hz, 6H), 1.48 (qui, J = 6.4 Hz, 4H), 1.60 (qui, J = 6.4 Hz, 2H), 2.02 (m, 3H), 2.99 (t, J = 6.4 Hz, 2H), 3.29-3.40 (m, 6H), 3.47 (s, br, 4H), 3.67 (s, 6H), 3.70 (t, J = 12.0 Hz, 6H), 4.04 (dd, J = 11.2 Hz, J = 5.2 Hz, 6H), 5.53 (s, 3H), 6.46 (t, J = 5 5.2 Hz, IH), 6.58 (t, J = 6.2 Hz, 2H), 6.70 (d, J = 8.8 Hz, 8H), 7.12-7.19 (m, 7H), 7.30 (d, J = 8.4 Hz, 2H), 7.42-7.51(m, 6H), 7.55-7.62 (m, 6H), 7.67 (d, J = 2.2 Hz, IH), 7.78 (d, J = 2.2, 2H), 7.91 (d, J = 2.2 Hz, 3H), 8.09 (d, J = 8.4 Hz, 2H), 8.16 (d, J = 8.0, 4H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.4, 25.8, 28.8, 29.2, 32.0, 36.5, 38.2, 55.1, 59.0, 61.6, 67.3, 67.5, 86.1, 88.6, 88.7, 89.0, 89.2, 97.1, 113.0, 113.1, 120.9, 121.1, 121.2, 123.6, 10 123.7, 123.8, 123.9, 126.7, 126.9, 127.8, 127.9, 128.0, 128.4, 128.6, 128.7, 128.8,

129.5, 129.8, 130.2, 130.6, 131.0, 132.3, 132.4, 132.5, 132.5, 132.9, 133.2, 133.8, 134.0, 134.3, 135.0, 135.0, 135.9, 136.1, 144.7, 145.5, 145.6, 158.3, 158.3, 158.4,

164.6, 165.0. 165.2, 166.3;

15 l,3-Bis[(4-benzoyloxy-5-(/V-(3-dimethoxytrityloxyprop-l-yl)-aminocarbonyl)-3- ([l,3]-dioxan-2-yl)-phenyl)ethynyl]-5-[(4-benzoyloxy-5-(W-(3-(2-cyanoethoxy- /V,Λ -diisopropylamino-phosphanyloxy)prop-l-yl)-aminocarbonyl)-3-([l,3]- dioxan-2-yl)-phenyl)ethynyl]benzene (20)

(Figure 20) 20

20 The alcohol 18 (42.7 mg, 0.022 mmol) was a stirred under vacuum in a Schlenk flask for 45 min. CH₂C!₂ (3 mL) and diisopropylethylamine (15.6 μL, 0.09 mmol) were added and the solution was quickly cooled to 0 °C. /V_/tV-diisopropylcyanoethylchlorophosphoramidite (10 μL, 0.045 mmol) was added dropwise and the reacting mixture was stirred at rt for 30 min under argon. The reacting mixture was diluted with CH₂CI₂ (5 mL) and washed with saturated NaHC0₃ (4 x 4 mL) and brine (4 x 4 mL). The organic layer was dried over MgS0₄ and concentrated. The crude product was purified by precipitation in pentane to yield 20 as yellow crystals (41.6 mg, 89%): *H NMR (CDC1₃, 400 MHz) δ 1.08 (m, 12H), 1.23 (d, J = 12.8 Hz, 6H), 1.55-1.75 (m, 6H), 2.02 (m, 3H), 2.48 (t, J = 7 Hz, 2H), 2.99 (t, J = 6.4 Hz, 4H), 3.27-3.42 (m, 8H), 3.45 (m, 2H), 3.67 (b,s, 14H), 3.70 (t, J = 12.0 Hz, 6H), 4.04 (dd, J = 11.2 Hz, J = 5.2 Hz, 6H), 5.53 (s, 3H), 6.42 (t, J - 5.2 Hz, 2H), 6.51 (t, J = 6.2 Hz, IH), 6.70 (d, J = 8.8 Hz, 8H), 7.12-7.19 (m, 14H), 7.30 (d, J = 8.4 Hz, 4H), 7.42-7.51 (m, 6H), 7.55-7.62 (m, 6H), 7.67 (d, J = 2.2 Hz, 2H), 7.78 (d, J = 2.2, IH), 7.91 (d, J = 2.2 Hz, 3H), 8.09 (d, J = 8.4 Hz, 4H), 8.16 (d, J = 8.0, 2H); ³¹P NMR δ 148.90.

l,3-Bis[(4-benzoyloxy-5-(/V-3-dimethoxytrityloxyprop-l-yl)aminocarbonyl)-3- ([l,3]-dioxan-2-yl)-phenyl)ethynyl]-5-[(4-benzoyloxy-5-( V-3-(9- fluoroenylmethoxycarbonyloxy)prop-l-yl)aminocarbonyl)-3-([l,3]-dioxan-2-yl)- phenyl)ethynyl]benzene (21) (Figure 21)

21 To a Schlenk flask charged with 9-fluorenylmethyl chloroformate (64.2 mg, 0.25mmol) and Λ/_//V-dimethyl-4-aminopyridine (0.76 mg, 0.0062 mmol) was added dropwise a solution of 18 (118 mg, 0.062 mmol) in distilled pyridine (4mL). The reaction mixture was stirred at rt for 16h under argon, then diluted with CH₂CI₂ (10 mL) and washed with water (3 x 10 5 mL). The organic layer was dried over MgS0 and concentrated. The crude was purified by flash chromatography on silica gel (CH₂CI₂/EtOAc, 85: 15) to yield 21 as white foam (98 mg, 74%): ^XH NMR (CDCI₃, 400 MHz) δ 1.23 (d, J = 12.8 Hz, 6H), 1.60 (qui, J = 6.4 Hz, 4H), 1.74 (q, J = 6.4, 2H), 2.02 (m, 3H), 2.99 (t, J = 6.4 Hz, 4H), 3.33 (m, 6H), 3.67 (s, 12H), 3.70 (t, J = 12.0 Hz, 6H), 4.04 (m, 8H), 4.15 (t, J = 7.7, IH), 4.30 (d, J = 7.7, 2H),

10 5.53 (s, 3H), 6.42 (m, 2H), 6.70 (d, J = 8.8 Hz, 8H), 7.12-7.29 (m, 16H), 7.30 (m, 6H), 7.42-7.47 (m, 6H), 7.50-7.62 (m, 8H), 7.67 (d, J = 2.2 Hz, 2H), 7.69 (d, J = 7.7, 2H), 7.78 (d, J = 2.2, IH), 7.91 (d, J = 2.2 Hz, 3H), 8.09 (d, J = 8.4 Hz, 4H), 8.16 (d, J = 8.0, 2H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.3, 25.4, 28.5, 29.2, 36.6, 38.2, 46.7, 55.2, 61.6, 65.5, 67.3, 69.8, 86.2, 88.6, 89.0, 89.2, 97.1, 97.2, 113.1, 120.0, 121.2, 121.4, 123.6,

15 123.7, 125.1, 126.7, 127.1, 127.8, 128.1, 128.6, 128.7, 128.8, 129.8, 130.9, 131.2, 132.3, 132.3, 132.5, 132.6, 133.0, 133.8, 134.0, 134.3, 136.1, 141.2, 143.3, 144.7, 145.6, 155.1, 158.3, 164.6, 164.9, 165.1, 165.4.

l-[(4-Benzoyloxy-5-(yV-3-dimethoxytrityloxyprop-l-yl)aminocarbonyl)-3-([l,3]- 20 dioxan-2-yl)-phenyl)ethynyl]-3-[(4-benzoyloxy-5-(/V-(3-hydroxyprop-l- yl)aminocarbonyl)-3-([l,3]-dioxan-2-yl)-phenyl)ethynyl]-5-[(4-benzoyloxy-5- (W-(3-(9-fluoroenylmethoxycarbonyloxy)prop-l-yl)aminocarbonyl)-3-([l,3]- dioxan-2-yl)-phenyl)ethynyl]benzene (22) (Figure 21)

22

To a round bottomed flask charged with 21 (114 mg, 0.054 mmol) dissolved in CH₂CI₂ (5 mL) was added dropwise glacial AcOH (5mL). The reaction mixture was stirred for 10 min, 5 quickly diluted with CH₂CI₂ (50 mL) and neutralized with saturated NaHC0₃. The organic layer was washed with water (3 x 80 mL), dried over MgS0₄ and concentrated. The crude hydrolyzed mixture was purified by flash chromatography (CH₂CI₂/EtOAc, 85: 15 → 60:40 → 0: 100) to yield unhydrolized 21 (60 mg, 0.028 mmol) and 22 (24 mg, 25%): ^X NMR (CDCI₃, 400 MHz) δ 1.23 (d, J = 12.8 Hz, 6H), 1.46 (qui, J = 6.4 Hz, 2H), 1.60 (qui, J =

10 6.4 Hz, 2H), 1.74 (qui, J = 6.4, 2H), 2.02 (m, 3H), 2.99 (t, J = 6.4 Hz, 2H), 3.29-3.43 (m, 6H), 3.47 (q, J = 5.1, 2H), 3.67 (s, 12H), 3.70 (t, J = 12.0 Hz, 6H), 4.04 (dd, J = 11.2 Hz, J = 5.2 Hz, 6H), 4.15 (t, J = 7.7, IH), 4.30 (d, J = 7.7, 2H), 5.53 (s, 3H), 6.45-6.51 (m, 2H), 6.68 (t, J = 6.2, IH), 6.70 (d, J = 8.8 Hz, 4H), 7.12-7.29 (m, 9H), 7.30 (m, 4H), 7.42-7.62 (m, 14H), 7.67 (d, J = 2.2 Hz, IH), 7.69 (d, J = 7.7, 2H), 7.78 (d, J = 2.2, IH),

15 7,80 (d, J = 2.2, IH), 7.91 (d, J = 2.2 Hz, 3H), 8.09 (d, J = 8.4 Hz, 2H), 8.16 (d, J = 8.0, 4H); ¹³C NMR (CDCI₃, 100 MHz) δ 25.3, 25.4, 28.5, 29.2, 32.1, 36.5, 36.6, 38.2, 46.7, 55.1, 59.0, 61.6, 65.5, 67.3, 69.7, 86.2, 88.6, 88.7, 88.8, 88.9, 89.0, 89.2, 97.1, 97.2,

113.0, 120.0, 121.1, 121.3, 121.4, 123.6, 123.7, 125.1, 126.7, 127.1, 127.8, 128.0, 128.5, 128.6, 128.7, 128.8, 129.8, 130.2, 130.7, 130.8, 132.3, 132.4, 132.5, 132.5,

20 132.6, 132.9, 133.0, 133.8, 134.0, 134.1, 134.3, 136.1, 141.2, 143.2, 144.7, 145.5,

155.1, 158.3, 164.6, 165.1, 165.1, 165.4, 166.2. l-[(4-Benzoyloxy-5-(/V-3-(dimethoxytrϊtyloxyprop-l-yl)-aminocarbonyl)-3- ([l,3]-dioxan-2-yl)-phenyl)ethynyl]-3-[(4-benzoyloxy-5-(Vv-(3-(9- 5 fluoroenylmethoxycarbonyl)prop-l-yl)aminocarbonyl)-3-([l,3]-dioxan-2-yl)- phenyl)ethynyl]-5-[(4-benzoyloxy-5-(/V-(3-(2-cyanoethoxy-/V,iV- diisopropylamino-phosphanyloxy)prop-l-yl)aminocarbonyl)-3-([l,3]-dioxan-2- yl)-phenyl)ethynyl]benzene (23) (Figure 21)

10 23

The alcohol 23 (25.5 mg, 0.014 mmol) was a stirred under vacuum in a Schlenk flask for 45 min. CH₂CI₂ (1.5 mL) and diisopropylethylamine (9.75 μL, 0.056 mmol) were added and the solution was quickly cooled to 0 °C. Λ/,Λ/-diisopropylcyanoethylchlorophosphoramidite (6.25 μL, 0.028 mmol) was added dropwise and the reacting mixture was stirred at rt for

15 30 min under argon. The reacting mixture was diluted with CH₂CI₂ (3 mL), washed with saturated NaHC0₃ (2 x 4 mL) and brine (2 x 4 mL). The organic layer was dried over MgS0 and concentrated. The crude product was purified by precipitation in pentane to yield 22 as yellow crystals (19.8 mg, 70%): ^ NMR (CDCI₃, 400 MHz) δ 1.14 (m, 12H), 1.23 (d, J = 12.8 Hz, 6H), 1.46 (qui, J = 6.4 Hz, 2H), 1.60 (qui, J = 6.4 Hz, 2H), 1.74

20 (qui, J = 6.4, 2H), 2.02 (m, 3H), 2.58 (t, 2H), 2.99 (t, J = 6.4 Hz, 2H), 3.29-3.47 (m, 8H), 3.45 (m, 2H), 3.67 (s, 13H), 3.70 (t, J = 12.0 Hz, 6H), 4.04 (m, 8H), 4.15 (t, J = 7.7, IH), 4.30 (d, J = 7.7, 2H), 5.53 (s, 3H), 6.45-6.49 (m, 2H), 6.68 (t, J = 6.2, IH), 6.70 (d, J = 8.8 Hz, 4H), 7.12-7.29 (m, 9H), 7.30 (m, 4H), 7.42-7.62 (m, 14H), 7.67- 7.72 (m, 3H), 7.78 (d, J = 2.2, IH), 7,80 (d, J = 2.2, IH), 7.91 (d, J = 2.2 Hz, 3H), 8.09

25 (d, J = 8.4 Hz, 2H), 8.16 (d, J = 8.0, 4H); ³¹P NMR δ 148.89. BIOCHEMISTRY

Standard laboratory denaturing PAGE

A 14.5 per cent polyacrylamide gel (30: 1.6 acrylamide: bisacrylamide) contained 8M urea, 90 mM Tris-borate buffer (pH 8.3) and 2.5 mM EDTA was used. DNA samples (1.0 mg) were in 8M urea, 40 mM EDTA (pH 7.0, NaOH) and boiled (without marker dyes) for 1 min before loading. After electrophoresis at 150 volts the gel was soaked in 50 per cent ethanol for 10 min. Then water for 5 min and then in ethidium bromide 1 mg/L for 10 min and finally in water for 15 min. DNA was visible under UV light.

A modified denaturing PAGE system

A 7.5 per cent polyacrylamide gel (30: 1.6 acrylamide:bisacrylamide) contained 8 M urea and 50 mM Tricine buffer (pH 8.1, NaOH) was used. DNA samples (0.5 mg) were loaded in 6 M urea (without marker dyes). After electrophoresis at 100 volts the gel was soaked in 50 per cent ethanol for 10 min. Then water for 5 min and then in ethidium bromide 1 mg/L for 10 min and finally in water for 15 min. DNA was visible under UV light.

Oligonucleotide synthesis

The LANA compounds 13 or 16 were incorporated into different DNA oligonucleotides on an automated DNA synthesizing machine at the 0.2 mmol scale using standard procedures for the phosphoramidite method (the TBPA group was used as the labile base protecting groups for nucleotides and was removed at the end of the synthesis by treatment with 30% aqueous NH₃ for 25 min at 50 °C). The modified DNA oligonucleotide with the DMTr group on the 5-hydroxy group was purified by RP HPLC with a Oligo R3 column before the DMTr group was removed with 70 % aqueous AcOH at rt for 20 min. Finally the sample was freeze dried. The symmetric TANA compound 20 was incorporated into an oligonucleotide chain in the same manner as the LANA compound. The unsymmetric TANA compound 23 was incorporated in a branched oligonucleotide chain in the method descibed for oligonucleotide synthesis using an asymmetric doubler (Glen Research). After finishing the synthesis of the second arm the DMTr group was removed and the free OH blocked using Ac₂0. The synthesis of the oligonucleotide on the third arm was initiated by removing the Fmoc group using 0.1% DBU for 30 min.. The product was purified as described above. DNA synthesis was conducted at DNA Technology A/S, Aarhus, Denmark. LANAO and TANAO (Figure 22)

To remove the dioxane protecting group from the modified DNA oligonucleotide each sample was dissolved in 132 mL of water, 66 mL of 1.5 M NaOAc buffer (pH 4.0) and 2 mL of 100 mM EDTA (pH 7.0, NaOH) and incubated for 2 h at 37 °C. After the incubation 125 mL 1 M CAPS buffer (pH 10.4, NaOH) was added to the solution to obtain a pH value of 7. Then 975 mL of absolute ethanol was added to the solution, which was placed at 20 °C overnight so that LANAO could precipitate. The sample was centrifuged at 10 0000 Xg for 15 min and the supernatant was poured away. 1.3 ml of 75 per cent ethanol was added to the yellow pelleted deprotected modified DNA oligonucleotide to dissolve salts and buffers and then centrifuged again. The supernatant was poured away. This wash step was repeated two more times. The LANAO was dried briefly in a vacuum centrifuge and then redissolved in 100 mL of 10 mM EPPS buffer (pH 8.0, NaOH) and frozen at 20 °C. Standard laboratory denaturing polyacryamide gel electrophoresis (PAGE) was performed to examine the purity of the sample and to estimate its concentration.

Formation of CALTO¹, CALTO² and CALTO³ (Figure 23)

A mixture of LANAO¹ and LANAO² (or LANAO³ and LANAO⁴) each at 10 mM in 100 mL buffer consisting of 0.5 mM MnOAc₂ and 1.0 mM ethylenediamine in 50 mM EPPS-NaOH pH 8.0 . This solution was incubated for 2 h at 35 °C and then cooled to rt. A modified denaturing PAGE was performed to analyse CALTO¹ (or CALTO²). CALTO³: mixture of LANAO¹, LANAO² and LANAO³ each at 10 mM in 100 mL buffer consisting of 50 mM TAPS (pH 8.0, NaOH) and 100 mM KCI was heated at 95 °C for 2.5 min and then cooled to 5 °C at a rate of 2 °C per min. 1 mL 100 mM Mn(OAc)₂ and 1 mL 25 mM ethylenediamine were added so that there final concentrations were 1 mM and 250 mM respectively. This solution was incubated for 2 h at 35 °C and then cooled to rt. A modified denaturing PAGE was performed to analyse CALTO³.

Formation of CALTO⁴ (Figure 24) A mixture of LANAO¹, LANAO², LANAO³ and TANAO¹ each at 10 mM in 100 mL buffer consisting of 50 mM TAPS (pH 8.0, NaOH) and 100 mM KCI was heated at 95 °C for 2.5 min and then cooled to 5 °C at a rate of 2 °C per min. 1 mL 100 mM Mn(OAc)2 and 1 mL 25 mM ethylenediamine were added so that there final concentrations were 1 mM and 250 mM respectively. This solution was incubated for 2 h at 35 °C and then cooled to rt. A modified denaturing PAGE was performed to analyse CALTO⁴. Use of other metal salts to prepare CALTO

Several other metal salts can be used in place of Mn(OAc)2 to prepare CALTO from a mixture of LANAOs. These include U0₂(OAc)₂, Co(N0₃)₂, FeCI₃, AICI₃ and LuCl₃.

PART B

Procedures enabling the synthesis of 28, 31, 34 and 37 and iterative oligomer synthesis

Bis-l,4-[(3-carboxy-4-iodophenyl)ethynyl]-di-2,5-(dodecyloxy)benzene (26)

26

Synthesized according to a procedure for a analogous regioselective coupling. (Balavoine, F.; Madec, D.; Mioskowski, C. Tetrahedron Lett. 1999, 40, 8351)

Typical experimental procedure Compound 25 (1 equiv.) dissolved in THF is added dropwise to a suspension of 24 (2.4 equiv.), (PPh₃)₂PdCI₂ (0.1 equiv) and Cul (0.1 equiv) in a mixture of THF and triethylamine.

The reaction is stirred overnight at rt and the quenched with saturated NH₄CI and finally extracted with EtOAc. The combined organic extracts are evaporated in vacuo and the residue purified by Si0₂ chromatography.

l-[(3-Carboxy-4-iodophenyl)ethynyl]-4-[4-(2-(t-butyloxycarbonylaminoethyl)-l- aminocarbonyl)-4-iodophenyl)ethynyl]-di-2,5-(dodecyloxy)benzene (27)

Synthetized from 26 by a peptide coupling of one equivalent of 26 with mono-Λ/-t-BOC- ethylenediamine. The crude product will contain a mixture of 26, 27 and the diamide.

Typical experimental procedure

A mixture of 27 (1 equiv) and DMAP (0.05 equiv) in CH₂CI₂ is cooled to 0 °C and EDC (1 equiv) is added. The mono-Λ/-r-BOC-3-ethylenediamine dissolved in CHCI₂ is added dropwise and the reaction mixture is allowed to warm up to rt and stirred for 4 h under argon. The reaction mixture is poured into 0.1 N HCl and extracted with CH₂CI₂ (3 x 20 mL). The combined organic fractions are washed with water, dried over MgS0₄ and concentrated. The crude product is purified by flash chromatography on silica gel.

l-[(3-Carboxy-4-iodophenyl)ethynyl]-4-[4-(2-(9-fluorenylmethoxy- carbonyl)aminoethyl)-l-aminocarbonyl)-4-iodophenyl)ethynyl]-di-2,5- (dodecyloxy)benzene (28)

Obtained by standard acidic BOC removal in trifluoroacetic acid/methylene chloride from 27 and subsequent protection by FmocCl in pyridine/methylene chloride. Tris-l,3,5-[3-(methoxycarbonyl-4-iodophenyl)ethynyl] benzene (29)

Synthesized from 24 and 1,3,5-triethynylbenzene in a Sonogashira coupling in analogy to the synthesis of 25. Due to the polarity of the crude product it is converted into the corresponding trimethyl ester before purification to give 29.

Typical experimental procedure 1,3,5-Triethynylbenzene (1 equiv) dissolved in THF is added dropwise to a suspension of 24 (3.5 equiv.), (PPh₃)₂PdCI₂ (0.1 equiv) and Cul (0.1 equiv) in a mixture of THF and triethylamine. The reaction is stirred overnight at rt. The precipitate is filtered off and acidified with 1 N HCl. EtOAc is added to the suspension and the aqueous is extracted 3 times with EtOAc. After removal of the solvent in vacuo the residue is dissolved in MeOH containing H₂SO₄ (0.2 equiv) and refluxed overnight. The solution is neutralized using 1 N NaOH and extracted with CH₂CI₂. The combined organic extracts are dried over MgS0₄. After evaporation of the solvent in vacuo, the residue is purified by Si0₂ chromatography.

l-[(3-Carboxy-4-iodophenyl)ethynyl]-3,5-bis-[3-(2-(f- butyloxycarbonylaminoethyl)-l-aminocarbonyl)-4-iodophenyl)ethynyl]benzene (30)

Compound 29 is heated with 2 equivalents of mono-/V-Boc-ethylenediamine for 2 h to give a mixture 29, monoamide, diamide and triamide. The desired diamide is obtained after chromatography and selective hydrolysis of the remaining methyl ester refluxing the crude product in a solution of LiOH in H₂0/THF/MeOH. The compound is obtained after acidification of the solution to pH 2-3 with HCl followed by extraction with EtOAc.

l-[(3-Carboxy-4-iodophenyl)ethynyl]-3-[3-(2-(t-butyloxycarbonylaminoethyl)-l- amϊnocarbonyl)-4-iodophenyl)ethynyl]-5-[3-(2-(9-fluorenylmethoxy- carbonyl)aminoethyl)-l-aminocarbonyl)-4-iodophenyl)ethynyl] benzene (31)

Obtained by acidic removal of one BOC protecting group. Controlling the reaction time will lead to the desired monoamine, wich will after purification be protected with an Fmoc- group using standard condition to give 31.

l,4-Bis[(3-(3-dimethoxytrityloxyprop-l-yloxy)carbonyl-4-iodophenyl)ethynyl]- di-2,5-(dodecyloxy)benzene (32)

32

Synthesized by reaction of 25 with 3-bromo-l-(dimethoxytrityIoxy)propane (6) in analogy with the reaction described for 7. Typical experimental procedure

To a Schlenk flask charged with 31 (1 mmol) and 6 (2.4 mmol) in DMF (5 mL), NEt₃ (2.4 mmol) is added dropwise. The reaction mixture is warmed to 40 °C, stirred for 16 h and poured into water (10 mL). The DMF-water mixture is extracted with Et₂0 (3 x 10 mL) and the combined organic fractions are washed with water (10 mL), dried over MgS0₄ and concentrated. The crude product is purified by flash chromatography on silica gel to give 32.

l-[(3-(3-Dimethoxytrityloxyprop-l-yloxy)carbonyl-4-iodophenyl)ethynyl]-4-[(3- (3-hydroxyprop-l-yloxy)carbonyl-4-iodophenyl)ethynyl-di-2,5- (dodecyloxy)benzene (33)

Typical experimental procedure

To a Schlenk flask charged with 32 (0.5 mmol) dissolved in CH₂CI₂ (20 mL) is added AcOH (20 mL) and reaction mixture is stirred at rt for 30 min. The reaction mixture is poured into 5.5 % aqueous NaHC0₃ (132 mL) to obtain a pH value at 7 and then extracted with CH₂CI₂ (3 x 20 mL). The combined organic fractions are washed with water, dried over MgS0₄ and concentrated. The crude product is purified by flash chromatography on silica gel.

l-[(3-(/V-(3-Dimethoxytrityloxyprop-l-yloxy)carbonyl)-4-iodophenyl)ethynyl]-4- [(3-(/V-3-(2-cyanoethoxy- v,iV-diisopropylamino-phosphanyloxy)prop-l- yloxy)carbonyl)-4-iodophenyl)ethynyl] di-2,5-(dodecyloxy)benzene (34)

Typical experimental procedure

To a Schlenk flask charged with 33 (0.2 mmol) dissolved in THF (15 mL) was added diisopropylethylamine (1.3 mmol) and the solution was cooled to 0 °C. N,N- diisopropylcyanoethylchlorophosphoramidite (0.9 mmol) was added dropwise while stirring. The reaction mixture was stirred under argon at rt for 16 h. The reaction was quenched by addition of MeOH (1 ml) and the solvent was removed by vacuum distillation. The resulting oil was dissolved in EtOAc (15 mL) and washed with 5.5% aqueous NaHC0₃ (15 mL), dried over MgS0₄ and concentrated. The crude product was purified by flash chromatography on silica gel.

Tris-l,3,5-[3-carboxy-4-iodophenyl)ethynyl]benzene (35)

Compound 35 is obtained after selective hydrolysis of the three methyl esters in 29 by refluxing the compound in LiOH in H₂0/THF/MeOH. The compound is obtained after acidification of the solution to pH 2-3 with HCl followed by extraction with EtOAc.

l,3,5-Tris[(3-(3-dimethoxytrityloxyprop-l-yloxy)carbonyl-4 iodophenyl)ethynyl]benzene (36)

Synthesized by reaction of 35 with 3-Bromo-l-(dimethoxytrityloxy)propane (6) in analogy with the reaction described for 32.

Typical experimental procedure

To a Schlenk flask charged with 31 (1 mmol) and 6 (4 mmol) in DMF (10 mL), NEt₃ (4 mmol) is added dropwise. The reaction mixture is warmed to 40 °C, stirred for 16 h and poured into water (10 mL). The DMF-water mixture is extracted with Et_zO (3 x 10 mL) and the combined organic fractions are washed with water (10 mL), dried over MgS0₄ and concentrated. The crude product is purified by flash chromatography on silica gel to give 36. l-[(3-(3-Dimethoxytrityloxyprop-l-oxycarbonyl)-4-iodophenyl)ethynyl]-3-[(3- (3-(9-fluoroenylmethoxycarbonyl)prop-l-yloxycarbonyl)-4-iodophenyI)ethynyl]- 5-[(3-(3-(2-cyanoethoxy-/V,/V-diisopropylamino-phosphanyloxy)prop-l- yloxycarbonyl)-4-iodophenyl)ethynyl] benzene (37)

Compound 37 is synthesized from 36 in several steps in complete analogy to the experimental procedures described for the conversion of compound 17a into 20.

ITERATIVE SYNTHESIS

Oligonucleotide analog synthesis

The LANA and TANA compounds 13, 16, 20, 23, 34 and 37 are all functionalized as nucleotide analogs and can be applied in iterative nucleotide synthesis as described in figure 7 and figure 8. General conditions applied in automated oligonucleotide synthesis based on the phosphoamidite method can be applied (see: Brown T. and Dorcas, J. S., (1995) "Modern machine-aided methods of oligonucleotide synthesis. In Oligonucleotides and Analogues a Practical Approach. Ed. F. Eckstien, IRL Press Oxford UK). Prolonged reaction times may be used in the coupling reactions to ensure high yields. The unsymmetric TANA compounds 23 and 37 can be incorporated in a branched oligonucleotide analog in the method described for oligonucleotide synthesis using an asymmetric doubler (Glen Research). After finishing the synthesis of the second arm the DMTr group is removed and the free OH blocked using Ac₂0. The synthesis of the oligonucleotide on the third arm is initiated by removing the Fmoc group using 0.1% DBU for 30 min.

The oligomers are cleaved from the resin using standard methods. (Formation of the 5 second covalent bond may be accomplished before cleavage from the resin.)

Peptide analog synthesis

The LPA and TPA compounds 28 and 31 are suitable for application in iterative peptide synthesis based on the Fmoc strategy and can be applied as described in figure 11, figure

10 12 and figure 29. Standard methods are used for the peptide synthesis. (Atherton E. and Sheppard R. C, Fmoc Solid phase peptide synthesis - a practical approach, IRL Press, New York, 2000.) Since the carboxylic acid on an aromatic residue decreased reactivity for activation and reaction with amines may be observed. This is circumvented by using prolonged reaction times and strong activators such as e.g. HBTU. Standard solid supports

15 and linkers for peptide synthesis are used and the peptide analogs are attached to the resin and cleaved from the resin due to standard procedures (Gloor A. P.; Hoare S. M.; Lawless K.; Steinauer R.A.; White P.; Yong C. W., Synthesis notes, Novabiochem). The unsymmetric TPA compound 31 can be incorporated in a branched peptide analog. After finishing the synthesis of the second arm the Fmoc group is removed and the free -NH₂

20 blocked using Ac₂0. The synthesis of the oligonucleotide on the third arm is initiated by removing the rBoc group of the third arm of the TPA by using 30% TFA in CH₂CI₂.

Formation of DCALT compounds.

The oligomers obtained by oligonucleotide or peptide synthesis as described above may be

25 subjected to the parallel formation of a second covalent bond.

Metal salen formation (Typical experimental procedure): (Figure 9) The oligomer containing salicylaldehyde termini (after liberation of the protected aldehyde at pH 4) is dissolved in alcohol containing 1 mM Mn(OAc) and 250 mM ethylenediamine. The mixture is stirred for 2 h at rt. The DCALT compounds is precipitated by addition of water and

30 isolated by filtration.

By a Sonogashira Coupling (Typical experimental procedure): (Figure 29) The oligomer containing iodoaryl termini (1 mmol of Art) is dissolved in DMF (5 mL) containing Pd(PPh₃)₂CI₂ (0.035 mmol) and Cul (0.11 mmol). Acetylene is bubbled through the 35 solution which is heated to 60 °C for 12h. Water (1 mL) is added and the oligomer is isolated by filtration.

Cleavable linker Introduction of a cleavable linker²² into the module further extends the design possiblities by enabling the total or selective removal of the DNA from assembled nanostructures. LMs were synthesized with disulfide links (ssLM) between the organic backbone and the adjoining oligonucleotides. These ssLM modules couple with the same efficiency as LM modules. Surprisingly the manganese salen-coupled organic backbone was found to be chemically unstable to reducing agents. However, aluminium salen-coupled components are unaffected by treatment with tris(2-carboxyethyl)phosphine (TCEP). Mild conditions (1 mM TCEP, 1 h, RT) completely reduce disulphide-linked oligonucleotides in contrast to a higher concentration of dithiotreitol (50 mM) needed. As expected when aluminium salen- coupled ssLM dimers are treated with TCEP, the DNA products released are two 15-mer oligonucleotides and a 15-bp duplex. Following reduction, the newly exposed backbone sulphydryls can be used for attachment to a surface or a nanodevice or the site-specific introduction of additional functional groups.

Coupling with Al³⁺ and disulphide reduction. 10 μl of 5 μM LM or S-SLM modules in 500 mM KCI, 10 mM TAPS (pH 8.4) were heated in a thermocycler at 55°C for 5 min and cooled slowly to 5°C (at 5°C per min). The coupling reaction was performed by addition of 1.0 mM (EDA), 0.25 mM AI(N0₃)₃ and incubation for 3 h at 35°C. Disulphide reduction was by addition of 0.2 μl of 50 mM TCEP (pH 7.0) for 1 h at RT. Analysis of the reaction products was by electrophoresis at 50 V in 7.5% polyacrylamide gels (30: 1.6) in 50 mM TAPS (pH 8.4) with and without 8 M urea. Samples were loaded in 15% (v/v) glycerol or 7.5 M urea without heating or addition of dyes.

Claims

1. A method for the preparation of a macromolecular architecture, said method comprising the following steps: (a) providing at least three organic compounds, including a first compound, a second compound and a third compound, each compound comprising at least two structural domains, including a first structural domain and a second structural domain, each structural domain comprising in proximity (i) one or more functional groups and (ii) an oligonucleotide chain, the oligonucleotide chain of the first structural domain of the first compound being at least partly complementary to the oligonucleotide chain of the second structural domain of the second compound, and the oligonucleotide chain of the second structural domain of the first compound being at least partly complementary to the oligonucleotide chain of the first structural domain of the third compound; and

(d) optionally partly or completely cleaving the oligonucleotide chain from the first structural domain of the first compound and the oligonucleotide chain from the second structural domain of the second compound, and partly or completely cleaving the oligonucleotide chain from the second structural domain of the first compound and oligonucleotide chain from the first structural domain of the third compound.

2. The method according to claim 1, wherein any of the first compound, the second compound and the third compound comprises a third structural domain.

3. The method according to claim 2, wherein any of the first compound, the second compound and the third compound comprises a fourth structural domain.

4. The method according to any of the preceding claims, wherein at least the first compound comprises an arylacetylene motif.

5. The method according to claim 4, wherein the arylacetylene motif is a phenylacetylene motif.

6. The method according to claim 4, wherein at least the first compound comprises a arylethynylaryl motif.

7. The method according to claim 6, wherein the arylethynylaryl motif is a phenylethynylphenyl motif.

8. The method according to any of the preceding claims, wherein at least the first structural domain of the first compound and the second structural domain of the second compound comprise two functional groups.

9. The method according to any of the preceding claims, wherein at least one functional group of the first structural domain of the first compound is identical to a functional group of the second structural domain of the second compound.

10. The method according to claim 9, wherein at least one functional group of the first structural domain of the first compound is an aldehyde or a protected form thereof, and at least one functional group of the second structural domain of the second compound is an aldehyde or a protected form thereof.

11. The method according to claim 9, wherein at least one functional group of the first structural domain of the first compound is a ketone or a protected form thereof, and at least one functional group of the second structural domain of the second compound is a ketone or a protected form thereof.

12. The method according to any of the preceding claims, wherein at least two functional groups of the first structural domain of the first compound are identical to two functional groups of the second structural domain of the second compound.

13. The method according to claim 12, wherein the first structural domain of the first compound is a salicylaldehyde or a protected form thereof, and the second structural domain of the second compound is a salicylaldehyde or a protected form thereof.

5

14. The method according to any of the preceding claims, wherein the oligonucleotide is linked via a spacer having a length of 2-50 atoms, typically 2-40 atoms, such as 2-30 atoms or 2 to 20 atoms.

10 15. The method according to any of the preceding claims, wherein at least the oligonucleotide chain of the first structural domain of the first compound is connected to the domain core of said first structural domain of said first compound via a phosphate diester, and the oligonucleotide chain of the second structural domain of the second compound is connected to the domain core of said second structural domain of said second

15 compound via a phosphate diester.

16. The method according to any of the preceding claims wherein the covalent link is established in step (c) by a reaction conducted at least between the functional group(s) of the first structural domain of the first compound and the functional group(s) of the second

20 structural domain of the second compound in the presence of a coupling reactant or a catalyst.

17. The method according to claim 16, wherein the coupling reactant is a diamine or a derivative thereof.

25

18. The method according to any of the preceding claims, wherein the coupling reaction in step (c) is performed in the presence of a metal or metal salts.

19. The method according to any of the preceding claims, wherein the covalent link is a 30 part of a salen complex.

20. The method according to to any of the preceding claims, wherein the covalent link is part of a metal salen complex or formed by reductive amination.

35 21. The method according to any of the preceding claims, wherein the covalent links established in step (c) are established in parallel.

22. A method for the preparation of a macromolecular architecture, said method comprising the following steps: (a) providing at least one oligonucleotide template, and at least three organic compounds, including a first compound, a second compound and a third compound, each compound comprising at least two structural domains, including a first structural domain and a second structural domain, each structural domain comprising in proximity (i) one or more functional groups and (ii) an oligonucleotide chain, the oligonucleotide chain of the first structural domain of the first compound being non- complementary to the oligonucleotide chain of the second structural domain of the second compound, and/or the oligonucleotide chain of the second structural domain of the first compound being non-complementary to the oligonucleotide chain of the first structural domain of the third compound, furthermore the oligonucleotide chain of the first structural domain of said first compound and the oligonucleotide chain of said second structural domain of said second compound being at least partly complementary to one of the at least one oligonucleotide template(s), and/or the oligonucleotide chain of the second structural domain of said first compound and the oligonucleotide chain of the first structural domain of said third compound being at least partly complementary to one of the at least one oligonucleotide template(s), said oligonucleotide template; and

(b) hybridising the oligonucleotide chain of the first structural domain of said first compound and the oligonucleotide chain of the second structural domain of the second compound with one of the at least one oligonucleotide template(s), and/or hybridising the oligonucleotide chain of the second structural domain of said first compound and the oligonucleotide chain of the first structural domain of the third compound with one of the at least one oligonucleotide template(s), thereby establishing base-paired hybrid(s), whereby the functional group(s) of the first structural domain of the first compound is brought in proximity to the functional group(s) of the second structural domain of the second compound, and/or the functional group(s) of the second structural domain of the first compound is brought in proximity to the functional group(s) of the first structural domain of the third compound; and

(c) establishing a covalent link between the first structural domain of said first compound and the second structural domain of said second compound by conducting a reaction involving the functional group(s) of said first structural domain of said first compound and the functional group(s) of said second structural domain of said second compound, and/or establishing a covalent link between the second structural domain of said first compound and the first structural domain of said third compound by conducting a reaction involving the functional group(s) of said second structural domain of said first compound and the functional group(s) of said first structural domain of said third compound; and

(d) optionally partly or completely cleaving the oligonucleotide chain from the first 5 structural domain of the first compound and the oligonucleotide chain from the second structural domain of the second compound, and partly or completely cleaving the oligonucleotide chain from the second structural domain of the first compound and the oligonucleotide chain from the first structural domain of the third compound.

10 23. The method according to claim 22, wherein any of the first compound, the second compound and the third compound comprises a third structural domain.

24. The method according to claim 23, wherein any of the first compound, the second compound and the third compound comprises a fourth structural domain.

15

25. The method according to any of the claims 22-24, wherein at least the first compound comprises an arylacetylene motif.

26. The method according to claim 25, wherein the arylacetylene motif is a 20 phenylacetylene motif.

27. The method according to claim 25, wherein at least the first compound comprises a arylethynylaryl motif.

25 28. The method according to claim 27, wherein the arylethynylaryl motif is a phenylethynylaryl motif.

29. The method according to any of the claims 22-28, wherein at least the first structural domain of the first compound and the second structural domain of the second compound

30 comprise two functional groups.

30. The method according to any of the claims 22-29, wherein at least one functional group of the first structural domain of the first compound is identical to a functional group of the second structural domain of the second compound.

35

31. The method according to claim 30, wherein at least one functional group of the first structural domain of the first compound is an aldehyde or a protected form thereof, and at least one functional group of the second structural domain of the second compound is an aldehyde or a protected form thereof.

32. The method according to claim 30, wherein at least one functional group of the first structural domain of the first compound is a ketone or a protected form thereof, and at least one functional group of the second structural domain of the second compound is a

5 ketone or a protected form thereof.

33. The method according to any of the claims 22-32, wherein at least two functional groups of the first structural domain of the first compound are identical to two functional groups of the second structural domain of the second compound.

10

34. The method according to claim 33, wherein the first structural domain of the first compound is a salicylaldehyde or a protected form thereof, and the second structural domain of the second compound is a salicylaldehyde or a protected form thereof.

15 35. The method according to any of the claims 22-34, wherein the oligonucleotide is linked via a spacer having a length of 2-50 atoms, typically 2-40 atoms, such as 2-30 atoms or 2 to 20 atoms.

36. The method according to any of the claims 22-35, wherein at least the oligonucleotide 20 chain of the first structural domain of the first compound is connected to the domain core of said first structural domain of said first compound via a phosphate diester, and the oligonucleotide chain of the second structural domain of the second compound is connected to the domain core of said second structural domain of said second compound via a phosphate diester. 25

37. The method according to any of the claims 22-36, wherein the covalent link is established in step (c) by a reaction conducted at least between the functional group(s) of the first structural domain of the first compound and the functional group(s) of the second structural domain of the second compound in the presence of a coupling reactant or a

30 catalyst.

38. The method according to claim 37, wherein the coupling reactant is a diamine or a derivative thereof.

35 39. The method according to any of the claims 22-38, wherein the coupling reaction in step (c) is performed in the presence of a metal or metal salts or wherein the coupling reaction is a reductive amination in the presence of metal salts.

40. The method according to any of the claims 22-39, wherein the covalent link is a part of a salen complex.

41. The method according to claim 40, wherein the covalent link is part of a metal salen 5 complex.

42. The method according to any of the claims 22-41, wherein the covalent links established in step (c) are established in parallel.

10 43. An organic compound selected from any of formula A-D:

A1

15

C1

wherein n is an integer of 0-2;

R¹, R² and R⁸ are independently selected from aldehyde groups, ketone groups, carboxylic acid groups, and protected forms thereof;

each R³ is independently selected from hydrogen, optionally substituted C_t-₂₀-alkyl, optionally substituted Cι-₂₀-alkoxy, optionally substituted aryl, optionally substituted aryloxy, a polyether of the formula 0(CH₂CH₂)_pOCH₂CH₃ wherein p is 1-10, and a disulfide of the formula Q-io-alkyl-SS-Ci-io-alkyl;

R⁴, R⁵ and R⁹ are independently selected from hydrogen and phenol protecting groups; and R⁶, R⁷ and R¹⁰ are independently selected from iodo, bromo, chloro, triflato, ethynyl, -C≡CSiMe₃, -C≡C-SnBu₃, -C≡C-SnMe₃, -OC-Si'Pra, -B(OH)₂, -B(OEt)₂, -B(OCH₂CH₂0), -B(OC(CH₃)₂C(CH₃)₂0), -SnBu₃, -SnMe₃, -C(0)Me, -COOH, -CHO;

spacer¹, spacer² and spacer³ are independently selected from a single bond and spacers having a length of 1-50 atoms, and

X¹, X² and X³ are independently selected from oligonucleotides of 4-30 nucleotides.

44. The organic compound according to claim 43, which is selected from

wherein n is an integer of 0-2;

R³ is independently selected from hydrogen, optionally substituted C_t-₂₀-alkyl, optionally substituted

optionally substituted aryl, optionally substituted aryloxy, a polyether of the formula 0(CH₂CH₂)_pOCH₂CH₃ wherein p is 1-10, and a disulfide of the formula Ci-io-alkyl-SS-Q-iQ-alkyl; and

the structural domains T¹, T² and T³ are selected from

45. A method for the preparation of a macromolecular architecture, said method comprising the following steps:

5 (d) coupling the second structural domain of said first compound to the first structural domain of said second compound, this coupling involving the unmasked reactive group in said second structural domain of said first compound and the reactive group in said first domain of said second compound, thereby bringing the functional group(s) of the second structural domain of the first compound in proximity to the functional group(s) 10 of the first structural domain of the second compound,

(e) optionally repeating the procedure of unmasking (c) and coupling (d) using further compounds equivalent to the first compound and/or the second compound provided in (a), and 15

20 (f2) establishing a covalent link at least between the second structural domain of said first compound and the first structural domain of said second compound, and subsequently detaching the obtained product from the solid support.

46. The method according to claim 45, wherein any of the first compound and the second 25 compound comprises a third structural domain.

47. The method according to claim 46, wherein any of the first compound and the second compound comprises a fourth structural domain.

30 48. The method according to any of the claims 45-47, wherein at least the first compound comprises an arylacetylene motif.

49. The method according to claim 48, wherein the arylacetylene motif is a phenylacetylene motif.

35

50. The method according to claim 48, wherein at least the first compound comprises a arylethynylaryl motif.

51. The method according to claim 50, wherein the arylethynylaryl motif is a phenylethynylphenyl motif.

52. The method according to any of the claims 45-51, wherein at least the first structural 5 domain of the first compound and the second structural domain of the second compound comprise two functional groups.

53. The method according to any of the claims 45-52, wherein at least one functional group of the first structural domain of the first compound is identical to a functional group

10 of the second structural domain of the second compound.

54. The method according to claim 53, wherein at least one functional group of the first structural domain of the first compound is an aldehyde or a protected form thereof, and at least one functional group of the second structural domain of the second compound is an

15 aldehyde or a protected form thereof.

55. The method according to claim 53, wherein at least one functional group of the first structural domain of the first compound is a ketone or a protected form thereof, and at least one functional group of the second structural domain of the second compound is a

20 ketone or a protected form thereof.

56. The method according to any of the claims 45-55, wherein at least two functional groups of the first structural domain of the first compound are identical to two functional groups of the second structural domain of the second compound.

25

57. The method according to claim 56, wherein the first structural domain of the first compound is a salicylaldehyde or a protected form thereof, and the second structural domain of the second compound is a salicylaldehyde or a protected form thereof.

30 58. The method according to any of the claims 45-51, wherein one functional group of either the first structural domain of the first compound or the second structural domain of the second compound is selected from iodo and bromo, and one functional group of the other of the first structural domain of the first compound and the second structural domain of the second compound is selected from iodo, bromo and acetylene.

35

59. The method according to any of the claims 45-58, wherein the covalent link is established in step (fl) or (f2) by a reaction conducted at least between the functional group(s) of the first structural domain of the first compound and the functional group(s) of the second structural domain of the second compound in the presence of a coupling reactant or a catalyst.

60. The method according to claim 59, wherein the coupling reactant is a diamine or a 5 derivative thereof.

61. The method according to any of the claims 45-60, wherein the coupling reaction in step (c) is performed in the presence of a metal or metal salts.

10 62. The method according to any of the claims 60-61, wherein the covalent link is a part of a salen complex.

63. The method according to claim 62, wherein the covalent link is part of a metal salen complex.

15

64. The method according to any of the claims 45-63, wherein the covalent links established in step (fl) or (f2) are established in parallel.

65. The method according to any of the claims 45-64, wherein the coupling between the 20 second structural domain of the first compound and the first structural domain of the second compound is conducted by automated DNA synthesis.

66. The method according to any of the claims 45-64, wherein the coupling between the second structural domain of the first compound and the first structural domain of the

25 second compound is conducted by automated peptide synthesis.

67. The method according to any of the claims 45-66, wherein the coupling between two domains of two respective compounds is made by iterative synthesis.

30 68. The method according to any of the claims 45-67, where steps (c) and (d) are repeated at least 5 times.

69. The method according to any of the claims 45-68, wherein the coupling between two structural domains of two respective compounds results in the formation of a

35 phosphodiester.

70. The method according to any of the claims 45-68, wherein the coupling between two structural domains of two respective compounds results in the formation of an amide.

71. The method according to any of the claims 45-70, wherein the coupling between two structural domains of two respective compounds results in the formation of a phosphodiester.

72. An organic compound comprising the following structural formulas E-H:

E1

Q¹ — Spacer¹ R³ Spacer²-Q²

G1

wherein n is an integer of 0-2;

R¹, R² and R⁸ are independently selected from aldehyde groups, ketone groups and protected forms thereof;

each R³ is independently selected from hydrogen, optionally substituted C_α-₂₀-alkyl, optionally substituted Q-₂₀-alkoxy, a polyether of the formula 0(CH₂CH₂)_pOCH₂CH₃ wherein p is 1-10; optionally substituted alkylaryl; a disulfide of the formula Cj-jo-alkyl-SS- Ci-io-alkyl; and

R⁴, R⁵ and R⁹ are independently selected from hydrogen and phenol protecting groups; and

R⁶, R⁷ and R¹⁰ are independently selected from iodo, bromo, chloro, triflato, ethynyl, -C≡CSiMe₃, -C≡C-SnBu₃, -C≡C-SnMe₃, -C≡C-Si'Prs, -B(OH)₂, -B(OEt)₂, -B(OCH₂CH₂0), -B(OC(CH₃)₂C(CH₃)₂0), -SnBu₃, -SnMe₃, -C(0)Me, and -CHO;

spacer¹, spacer² and spacer³ are independently selected from a single bond and spacers having a length of 1-50 atoms such as 1-20 atoms, Q¹ is selected hydroxy (-OH), Act-O-, carboxylic acid (-COOH), Act-C(=0)-, amino (-NHR^a), Act-NR^a, thiolo (-SH), and Act-S-, where R^a is hydrogen or C^-alkyl, and wherein Act is an activation group for -OH, -COOH, -NHR^a and -SH, respectively;

5 Q² and Q³ are independently selected from hydroxy (-OH), Prot-O-, carboxylic acid (-COOH), Prot-C(=0)-, amino (-NHR^a), Prot-NR^a-, thiolo (-SH), and Prot-S-, where R^a is hydrogen or Cι_₄-alkyl, and wherein Prot is a protecting group for -OH, -COOH, -NHR^a and -SH, respectively.

10 73. The organic compound according to claim 72, which is of formula E wherein n is 1, R¹ and R² are acetal-protected formyl, R³ is hydrogen or a C^-alkyl group, R⁴ and R⁵ independently are selected from benzoyl and acetyl hydroxy protecting groups, spacer¹ and spacer² independently are selected from C(=O)NR^a-(CH₂)₂-₁₀, wherein R^a is hydrogen or Q_t-₄-alkyl, and Q² is selected from OH, (O-(dimethoxytrityl), and NHFmoc, and Q is

15 selected from OH, 0-P(N'Pr₂)OCH₂CH₂CN), COOH and COOtBu.

74. The organic compound according to claim 72, which is of formula F wherein R¹, R² and R⁸ are acetal-protected formyl, R⁴, R⁵ and R⁹ independently are selected from benzoyl and acetyl hydroxy protecting groups, spacer¹, spacer² and spacer³ independently 20 are selected from C(=O)NR^a-(CH₂)₂-₁₀, wherein R^a is hydrogen or C^-alkyl, and Q² selected from OH, (O-(dimethoxytrityl), and NHFmoc, Q¹ is selected from OH, O- P(N'Pr₂)OCH₂CH₂CN), COOH and COOtBu, and Q³ is selected from OH, O-Fmoc, O- carboxymethyl, O-TBDMS, TIPS, NHFmoc and SH.

25 75. The organic compound according to claim 72, which is selected from

30

wherein n is an integer of 0-2; R is independently selected from hydrogen, optionally substituted Cι-₂₀-alkyl, optionally substituted .^-alkoxy, optionally substituted aryl, optionally substituted aryloxy, a polyether of the formula 0(CH₂CH₂)_pOCH₂CH₃ wherein p is 1-10, and a disulfide of the formula Q-jo-alkyl-SS-Ci-io-alkyl; and

the structural domains T¹ is selected from

wherein m is an integer of 0-5 and x is an integer of 2-10.

76. A macromolecular architecture, said architecture comprising at least one arylacetylenearyl motif and at least one metal salen complex of the formula

wherein M is a metal selected from Mn, Ni, Co, Fe, Al, and U.

10 77. The macromolecular architecture according to claim 76 that has a molecular weight of at least 5000 Da.

78. The macromolecular architecture according to any of claims 76-77, which is electrically conducting.

15

79. The macromolecular architecture according to any of claims 76-78, which is prepared according to the methods defined in any of claims 1-21, or 45-71.

80. The use of a supramolecular architecture as defined herein for the manufacture of a 20 device in molecular electronics.

81. The use of a supramolecular architecture as defined herein for the manufacture of conductors.

25 82. The use of a supramolecular architecture as defined herein for the manufacture of semi-conductors.

83. The use of a supramolecular architecture as defined herein for the manufacture of a device in molecular electronics. 30