US20050247001A1

US20050247001A1 - Building block forming a c-c or a c-hetero atom bond uponreaction

Info

Publication number: US20050247001A1
Application number: US10/507,829
Authority: US
Inventors: Alex Gouliaev; Henrik Pedersen; Kim Jensen; Mikkel Lundorf; Christian Sams; Jakob Felding; Michael Godskesen
Original assignee: Nuevolution AS
Current assignee: Nuevolution AS
Priority date: 2002-03-15
Filing date: 2003-03-14
Publication date: 2005-11-10
Also published as: WO2003078446A2; EP1487850A2; AU2003218629A1; WO2003078446A3

Abstract

A building block having the dual capabilities of transferring genetic information and functional entity precursor to a recipient reactive group is disclosed. The building block may be used in the generation of a single complex or libraries of different complexes, wherein the complex comprises an encoded molecule linked to an encoding element. Libraries of complexes are useful in the quest for pharmaceutically active compounds.

Description

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a building block comprising a complementing element and a precursor for a functional entity. The building block is designed to transfer the functional entity precursor with an adjustable efficiency to a recipient reactive group upon recognition between the complementing element and an encoding element associated with the reactive group. The invention also relates to a method for transferring a functional entity precursor to recipient a reactive group.

BACKGROUND

The transfer of a chemical entity from one mono-, di- or oligonucleotide to another has been considered in the prior art. Thus, N. M. Chung et al. (Biochim. Biophys. Acta, 1971, 228,536-543) used a poly(U) template to catalyse the transfer of an acetyl group from 3′-O-acetyladenosine to the 5′-OH of adenosine. The reverse transfer, i.e. the transfer of the acetyl group from a 5′-O-acetyladenosine to a 3′-OH group of another adenosine, was also demonstrated.
Walder et al. Proc. Natl. Acad. Sci. USA, 1979, 76, 51-55 suggest a synthetic procedure for peptide synthesis. The synthesis involves the transfer of nascent immobilized polypeptide attached to an oligonucleotide strand to a precursor amino acid attached to an oligonucleotide. The transfer comprises the chemical attack of the amino group of the amino acid precursor on the substitution labile peptidyl ester, which in turn results in an acyl transfer. It is suggested to attach the amino acid precursor to the 5′0 end of an oligonucleotide with a thiol ester linkage.
The transfer of a peptide from one oligonucleotide to another using a template is disclosed in Bruick RK et al. Chemistry & Biology, 1996, 3:49-56. The carboxy terminal of the peptide is initially converted to a thioester group and subsequently transformed to an activated thioester upon incubation with Ellman's reagent. The activated thioester is reacted with a first oligo, which is 5′-thiol-terminated, resulting in the formation of a thio-ester linked intermediate. The first oligonucleotide and a second oligonucleotide having a 3′0 amino group is aligned on a template such that the thioester group and the amino group are positioned in close proximity and a transfer is effected resulting in a coupling of the peptide to the second oligonucleotide through an amide bond.

SUMMARY OF THE INVENTION

The present invention relates to a building block of the general formula:
Complementing Element-Linker-Carrier-C—F-connecting group-Functional entity precursor
capable of transferring a Functional entity precursor to a recipient reactive group, wherein

- Complementing Element is a group identifying the Functional entity precursor,
- Linker is a chemical moiety comprising a spacer and a S—C-connecting group, wherein the spacer is a valence bond or a group distancing the Functional entity precursor to be transferred from the complementing element and the S—C-connecting group connects the spacer with the Carrier
- Carrier is arylene, heteroarylene, C₁-C₆alkylene, C₁-C₆alkenylene, C₁-C₆alkynylene, or —(CF₂)_m— substituted with 0-3 R¹wherein m is an integer between 1 and 10;

R¹are independently selected from —H, —OR², —NR² ₂, —Halogen, —NO₂, —CN, —C(Halogen)₃, —C(O)R², —C(O)NHR², C(O)NR² ₂, —NC(O)R², —S(O)₂NHR², —S(O)₂NR² ₂, —S(O)₂R², —P(O)₂—R², —P(O)—R², —S(O)—R², P(O)—OR², —S(O)—OR², —N⁺R² ₃, wherein R²is H, C₁-C6 alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, or aryl,

- C—F-connecting group is chosen from the group consisting of —SO₂—O—, —O—SO₂—O—, —C(O)—O—, —S⁺(R³RRrr)—, —C—U—C(V)—O—, —P⁺(W)₂—O—, —P(W)—O— where U is —C(R²)₂, —NR²— or —O—; V is ═O or ═NR²and W is —OR²or —N(R²)₂

Functional entity precursor is —C(H)(R³)—R⁴or functional entity precursor is heteroaryl or aryl optionally substituted with one or more substituents belonging to the group comprising R³and R⁴.
Wherein R³and R⁴independently is H, alkyl, alkenyl, alkynyl, alkadienyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, optionally substituted with one or more substituents selected from the group consisting of SnR⁵R⁶R⁷, Sn(OR⁵)R⁶R⁷, Sn(OR⁵)(OR⁶)R⁷, BR⁵R⁶, B(OR⁵)R⁶, B(OR⁵)(OR⁶), halogen, CN, CNO, C(halogen)₃, OR⁵, OC(═O)R⁵, OC(═O)OR⁵, OC(═O)NR⁵R⁶, SR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, N₃, NR⁵R⁶, N⁺R⁵R⁶R⁷, NR⁵OR⁶, NR⁵NR⁶R⁷, NR⁵C(═O)R⁶NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, NC, P(═O)(OR⁵)OR⁶, P⁺R⁵R⁶R⁷, C(═O)R⁵, C(═NR⁵)R⁶, C(═NOR⁵)R⁶, C(═NNR⁵R⁶), C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶, C(═O)NR⁵NR⁶R⁷, C(═NR⁵)NR⁶R⁷, C(═NOR⁵)NR⁶R⁷or R⁸, wherein,
R⁵, R⁶, and R⁷independently is H, alkyl, alkenyl, alkynyl, alkadienyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, optionally substituted with one or more substituents selected from the group consisting of halogen, CN, CNO, C(halogen)₃, ═O, OR⁸, OC(═O)R⁸, OC(═O)OR⁸, OC(═O)NR⁸R⁹, SR⁸, S(═O)R⁸, S(═O)₂R⁸, S(═O)₂NR⁸R⁹, NO₂, N₃, NR⁸R⁹, N⁺R⁸R⁹R¹⁰, NR⁵OR⁸, NR⁵NR⁶R⁷, NR⁸C(═O)R⁹, NR⁸C(═O)OR⁹, NR⁸C(═O)NR⁹R¹⁰, NC, P(═O)(OR⁸)OR⁹, P⁺R⁵R⁶R⁷, C(═O)R⁸, C(═NR⁸)R⁹, C(═NOR⁸)R⁹, C(═NNR⁸R⁹), C(═O)OR⁸, C(═O)NR⁸R⁹, C(═O)NR⁸OR⁹C(═NR⁵)NR⁶R⁷, C(═NOR⁵)NR⁶R⁷or C(═O)NR⁸NR⁹R¹⁰, wherein R⁵and R³may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring, wherein,
R⁸, R⁹, and R¹⁰independently is H, alkyl, alkenyl, alkynyl, alkadienyl, cycloalkyl, 20 cycloheteroalkyl, aryl or heteroaryl and wherein R⁸and R⁹may together form a 3-8 membered heterocyclic ring or R⁸and R¹⁰may together form a 3-8 membered heterocyclic ring or R⁹and R¹⁰may together form a 3-8 membered heterocyclic ring.
In the present description and claims, the direction of connections between the various components of a building block should be read left to right. For example an S—C-connecting group —C(═O)—NH— is connected to a Spacer through the carbon atom on the left and to a Carrier through the nitrogen atom on the right hand side.
The term “C₃-C₇cycloheteroalkyl” as used herein refers to a radical of totally saturated heterocycle like a cyclic hydrocarbon containing one or more heteroatoms selected from nitrogen, oxygen, phosphor, boron and sulphur independently in the cycle such as pyrrolidine (1-pyrrolidine; 2-pyrrolidine; 3-pyrrolidine; 4-pyrrolidine; 5-pyrrolidine); pyrazolidine (1-pyrazolidine; 2-pyrazolidine; 3-pyrazolidine; 4-pyrazolidine; 5-pyrazolidine); imidazolidine (1-imidazolidine; 2-imidazolidine; 3-imidazolidine; 4 imidazolidine; 5-imidazolidine); thiazolidine (2-thiazolidine; 3-thiazolidine; 4-thiazolidine; 5-thiazolidine); piperidine (1-piperidine; 2-piperidine; 3-piperidine; 4-piperidine; 5-piperidine; 6-piperidine); piperazine (1-piperazine; 2-piperazine; 3-piperazine; 4-piperazine; 5-piperazine; 6-piperazine); morpholine (2-morpholine; 3-morpholine; 4-morpholine; 5-morpholine; 6-morpholine); thiomorpholine (2-thiomorpholine; 3-thiomorpholine; 4-thiomorpholine; 5-thiomorpholine; 6-thiomorpholine); 1,2-oxathiolane (3-(1,2-oxathiolane); 4-(1,2-oxathiolane); 5-(1,2-oxathiolane); 1,3-dioxolane (2-(1,3-dioxolane); 4-(1,3-dioxolane); 5(1,3dioxolane); tetrahydropyrane; (2-tetrahydropyrane; 3-tetrahydropyrane; 4-tetrahydropyrane; 5-tetrahydropyrane; 6-tetrahydropyrane); hexahydropyridazine (1-(hexahydropyridazine); 2-(hexahydropyndazine); 3-(hexahydropyridazine); 4-(hexahydropyridazine); 5-(hexahydropyridazine); 6-(hexahydropyridazine)), [1,3,2]dioxaborolane, [1,3,6,2]dioxazaborocane
The term “aryl” as used herein includes carbocyclic aromatic ring systems of 5-7 carbon atoms. Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems as well as up to four fused fused aromatic- or partially hydrogenated rings, each ring comprising 5-7 carbon atoms.
The term “heteroaryl” as used herein includes heterocyclic unsaturated ring systems containing, in addition to 2-18 carbon atoms, one or more heteroatoms selected from nitrogen, oxygen and sulphur such as furyl, thienyl, pyrrolyl, heteroaryl is also intended to include the partially hydrogenated derivatives of the heterocyclic systems enumerated below.
The terms “aryl” and “heteroaryl” as used herein refers to an aryl which can be optionally substituted or a heteroaryl which can be optionally substituted and includes phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N-hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3-anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl), indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-1-yl, 1,2,3-triazol-2- yl 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6pyrimidinyl), pyrazinyl, pyridazinyl (3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6 quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6-benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl (2-(2,3-dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro-benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro-benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyI), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f]azepin-1-yl, 5H-dibenz[b,f]azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11-dihydro-5H-dibenz[b,f]azepine (10,11-dihydro-5H-dibenz[b,f]azepine-1-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,11 -dihydro-5H-dibenz[b,f]azepine-4-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-5-yl).
The Functional Entity carries elements used to interact with host molecules and optionally reactive elements allowing further elaboration of an encoded molecule of a library. Interaction with host molecules like enzymes, receptors and polymers is typically mediated through van der waal's interactions, polar- and ionic interactions and pi-stacking effects. Substituents mediating said effects may be masked by methods known to an individual skilled in the art (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis; 3rd ed.; John Wiley & Sons: New York, 1999.) to avoid undesired interactions or reactions during the preparation of the individual building blocks and during library synthesis. Analogously, reactive elements may be masked by suitably selected protection groups. It is appreciated by one skilled in the art that by suitable protection, a functional entity may carry a wide range of substitutents.
The Functional Entity Precursor is a masked. Functional Entity that is incorporated into an encoded molecule. After incorporation, reactive elements of the Functional Entity may be revealed by un-masking allowing further synthetic operations. Finally, elements mediating recognition of host molecules may be un-masked.
In a certain aspect of the invention, Functional entity precursor is —C(H)(R¹¹)—R¹¹′ or functional entity precursor is heteroaryl or aryl substituted with 0-3 R¹¹, 0-3 R¹³and 0-3 R¹⁵, wherein
R¹¹and R¹¹′ are independently H, or selected among the group consisting of a C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₈alkynyl, C₄-C₈alkadienyl, C₃-C₇cycloalkyl, C₃-C₇cyclo-heteroalkyl, aryl, and heteroaryl, said group being substituted with 0-3 R¹², 0-3 R¹³and 0-3 R¹⁵,
or R¹¹and R¹¹′0 are C₁-C₃alkylene-NR¹² ₂, C₁-C₃alkylene-NR¹²C(O)R¹⁶, C₁-C₃alkylene-NR¹²C(O)OR¹⁶, C₁-C₂alkylene-O—NR¹² ₂, C₁-C₂alkylene-O—NR¹²C(O)R¹⁶, C₁-C₂alkylene-O—NR¹²C(O)OR¹⁶substituted with 0-3 R¹⁵,

- where R¹²is H or selected independently among the group consisting of C₁C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl, heteroaryl, said group being substituted with 0-3 R¹³and 0-3 R¹⁵,
- R¹³is selected independently from —N₃, —CNO, —C(NOH)NH₂, —NHOH, —NHNHR¹⁷, —C(O)R¹⁷, —SnR¹⁷ ₃, —B(OR¹⁷)₂, —P(O)(OR¹⁷)₂or the group consisting of C₂-C₆alkenyl, C₂-C₆alkynyl, C₄-C₈alkadienyl said group being substituted with 0-2 R¹⁴,
- where R¹⁴is independently selected from —NO₂, —C(O)OR¹⁷, —COR¹⁷, —CN, —OSiR¹⁷ ₃, —OR¹⁷and —NR¹⁷ ₂;
- R¹⁵is ═O, —F, —Cl, —Br, —I, —CN, —NO₂, —OR¹⁷, —NR¹⁷ ₂, —NR¹⁷—C(O)R¹⁶, —NR¹⁷—C(O)OR¹⁶, —SR¹⁷, —S(O)R¹⁷, —S(O)₂R¹⁷, —COOR¹⁷, —C(O)NR¹⁷ ₂and —S(O)₂NR¹⁷ ₂,
- R¹⁶is H, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl, aryl or C₁-C₆alkylene-aryl substituted with 0-3 substituents independently selected from —F, —Cl, —NO₂, —R², —R², —SiR² ₃;

R¹⁷is selected independently from H, C₁-C₆alkyl, C₃-C₇cycloalkyl, aryl, C₁-C₆alkylene-aryl,

G is H or C₁-C₆alkyl and n is 1,2,3 or 4.
The function of the carrier is to ensure the transferability of the functional entity precursor. To adjust the transferability a skilled chemist can design suitable substitutions of the carrier by evaluation of initial attempts. The transferability may be adjusted in response to the chemical composition of the functional entity precursor, to the nature of the complementing element, to the conditions under which the transfer and recognition is performed, etc.
In a preferred embodiment, the carrier is selected from the group consisting of arylene, heteroarylene or —(CF₂)_m— substituted with 0-3 R¹wherein m is an integer between 1 and 10, and C—F-connecting group is —SO₂—O—. Due to the high reactivity of such compounds a broad range of recipient reactive groups may be employed in the construction of carbon-carbon bonds or carbon-hetero atom bonds.
In another preferred embodiment of the invention, the carrier is —(CF₂)_m— wherein m is an integer between 1 and 10, the C—F-connecting group is —SO₂—O—; and the functional entity precursor is aryl or heteroaryl substituted with 0-3 R¹¹, 0-3 R¹³and 0-3 R¹⁵.
The C—F-connecting group determines in concert with the carrier the transferability of the functional entity precursor. In a preferred embodiment, the C—F-connecting group is —S⁺(R¹¹)—,
In another preferred embodiment, the C—F-connecting group is chosen from the group consisting of —SO₂—O—, and —S⁺(R¹⁷)—; wherein R¹⁷is selected independently from H, C₁-C₆alkyl, C₃-C₇cycloalkyl, aryl, C₁-C₆alkylene-aryl.
In the presence of a catalyst comprising transition metals such as Pd, Ni or Cu, an aromatic moiety may be transferred from the C—F-connecting group to a recipient reactive group. Further, the transfer may be initiated by adding the catalyst, independently of the annealing of encoding - and complementing elements.
The S—C-connecting group provide a means for connecting the Spacer and the Carrier. As such it is primarily of synthetic convenience and does not influence the function of a building block.
The spacer serves to distance the functional entity precursor to be transferred from the bulky complementing element. Thus, when present, the identity of the spacer is not crucial for the function of the building block. It may be desired to have a spacer which can be cleaved by light. In this case, the spacer is provided with e.g. the group
In the event an increased hydrophilicity is desired the spacer may be provided with a polyethylene glycol part of the general formula:
In a preferred embodiment, the complementing element serves the function of transferring genetic information e.g. by recognising a coding element. The recognition implies that the two parts are capable of interacting in order to assemble a complementing element—coding element complex. In the biotechnological field a variety of interacting molecular parts are known which can be used according to the invention. Examples include, but are not restricted to protein-protein interactions, protein-polysaccharide interactions, RNA-protein interactions, DNA-DNA interactions, DNA-RNA interactions, RNA-RNA interactions, biotin-streptavidin interactions, enzyme-ligand interactions, antibody-ligand interaction, protein-ligand interaction, etc.
The interaction between the complementing element and coding element may result in a strong or a weak bonding. If a covalent bond is formed between the parties of the affinity pair the binding between the parts can be regarded as strong, whereas the establishment of hydrogen bondings, interactions between hydrophobic domains, and metal chelation in general results in weaker bonding. In general relatively weak bonding is preferred. In a preferred aspect of the invention, the complementing element is capable of reversible interacting with the coding element so as to provide for an attachment or detachment of the parts in accordance with the changing conditions of the media.
In a preferred aspect of the invention, the interaction is based on nucleotides, i.e. the complementing element is a nucleic acid. Preferably, the complementing element is a sequence of nucleotides and the coding element is a sequence of nucleotides capable of hybridising to the complementing element. The sequence of nucleotides carries a series of nucleobases on a backbone. The nucleobases may be any chemical entity able to be specifically recognized by a complementing entity. The nucleobases are usually selected from the natural nucleobases (adenine, guanine, uracil, thymine, and cytosine) but also the other nucleobases obeying the Watson-Crick hydrogen-bonding rules may be used, such as the synthetic nucleobases disclosed in U.S. Pat. No. 6,037,120. Examples of natural and non-natural nucleobases able to perform a specific pairing are shown in FIG. 2. The backbone of the sequence of nucleotides may be any backbone able to aggregate the nucleobases is a sequence. Examples of backbones are shown in FIG. 4. In some aspects of the invention the addition of non-specific nucleobases to the complementing element is advantegeous, FIG. 3
The coding element can be an oligonucleotide having nucleobases which complements and is specifically recognised by the complementing element, i.e. in the event the complementing element contains cytosine, the coding element part contains guanine and visa versa, and in the event the complementing element contains thymine or uracil the coding element contains adenine.
The complementing element may be a single nucleobase. In the generation of a library, this will allow for the incorporation of four different functional entities into the template-directed molecule. However, to obtain a higher diversity a complementing element preferably comprises at least two and more preferred at least three nucleotides. Theoretically, this will provide for 42 and 43, respectively, different functional entities uniquely identified by the complementing element. The complementing element will usually not comprise more than 100 nucleotides. It is preferred to have complementing elements with a sequence of 3 to 30 nucleotides.
The building blocks of the present invention can be used in a method for transferring a functional entity precursor to a recipient reactive group, said method comprising the steps of

- providing one or more building blocks as described above and
- contacting the one or more building blocks with a corresponding encoding element associated with a recipient reactive group under conditions which allow for a recognition between the one or more complementing elements and the encoding elements, said contacting being performed prior to, simultaneously with, or subsequent to a transfer of the functional entity precursor to the recipient reactive group.

The encoding element may comprise one, two, three or more codons, i.e. sequences that may be specifically recognised by a complementing element. Each of the codons may be separated by a suitable spacer group. Preferably, all or at least a majority of the codons of the template are arranged in sequence and each of the codons are separated from a neighbouring codon by a spacer group. Generally, it is preferred to have more than two codons on the template to allow for the synthesis of more complex encoded molecules. In a preferred aspect of the invention the number of codons of the encoding element is 2 to 100. Still more preferred are encoding elements comprising 3 to 10 codons. In another aspect, a codon comprises 1 to 50 nucleotides and the complementing element comprises a sequence of nucleotides complementary to one or more of the encoding sequences.
The recipient reactive group may be associated with the encoding element in any appropriate way. Thus, the reactive group may be associated covalently or noncovalently to the encoding element. In one embodiment the recipient reactive group is linked covalently to the encoding element through a suitable linker which may be separately cleavable to release the reaction product. In another embodiment, the reactive group is coupled to a complementing element, which is capable of recognising a sequence of nucleotides on the encoding element, whereby the recipient reactive group becomes attached to the encoding element by hybridisation. Also, the recipient reactive group may be part of a chemical scaffold, i.e. a chemical entity having one or more reactive groups available for receiving a functional entity precursor from a building block.
The recipient reactive group may be any group able to participate in cleaving the bond between the carrier and the functional entity precursor to release the functional entity precursor. Typically, the recipient reactive group is a nucleophilic atom such as S, N, 0, C or P. Scheme 1a shows the transfer of an alkyl group and scheme 1b shows the transfer of an vinyl group.
Alternatively, the recipient reactive group is a organometallic compound as shown in scheme 2.
According to a preferred aspect of the invention the building blocks are used for the formation of a library of compounds. The complementing element of the building block is used to identify the functional entity. Due to the enhanced proximity between reactive groups when the complementing entity and the encoding element are contacted, the functional entity precursor together with the identity programmed in the complementing element is transferred to the encoding element associated with recipient reactive group. Thus, it is preferred that the sequence of the complementing element is unique in the sense that the same sequence is not used for another functional entity. The unique identification of the functional entity enable the possibility of decoding the encoding element in order to determine the synthetic history of the molecule formed. In the event two or more functional entities have been transferred to a scaffold, not only the identity of the transferred functional entities can be determined. Also the sequence of reaction and the type of reaction involved can be determined by decoding the encoding element. Thus, according to a preferred embodiment of the invention, each different member of a library comprises a complementing element having a unique sequence of nucleotides, which identifies the functional entity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Two setups for Functional Entity Transfer
FIG. 2. Examples of specific base pairing
FIG. 3. Example of non-specific base-pairing
FIG. 4. Backbone examples
FIG. 5 Three examples of building blocks

DETAILED DESCRIPTION OF THE INVENTION

A building block of the present invention is characterized by its ability to transfer its functional entity precursor to a recipient reactive group. This is done by forming a new covalent bond between the recipient reactive group and cleaving the bond between the carrier moiety and the functional entity precursor of the building block.
Two setups for generalized functional entity precursor transfer from a building block are depicted in FIG. 1. In the first example, one complementing element of a building block recognizes a coding element carrying another functional entity precursor, hence bringing the functional entities in close proximity. This results in a reaction between functional entity precursor 1 and 2 forming a covalent bond between these concurrent with the cleavage of the bond between functional entity precursor 2 and its linker. In the second example, a template brings together two building blocks resulting in functional entity precursor transfer from one building block to the other.
FIG. 5 illustrates three specific compounds according to the invention. For illustrative purposes the individual features used in the claims are indicated. The upper compound is an example of a building block wherein the linker is backbone attached at the 3′-position. The first part of the linker, i.e. the spacer, is an aliphatic chain ending in a nitrogen atom. The nitrogen atom bridges to the S—C-connecting group, which is an N-acylated arylmethyleamine. The carrier attached to the left hand side carbonyl group of the S—C-connecting group is a benzene ring holding the C—F Connecting group in the para position. The C—F Connecting group is a positively charged sulfur atom which is attached to the Functional Entity Precursor, in this case a benzyl group. When the building block is presented to a nucleophilic recipient reactive group, such an amine or a thiol, Functional Entity Precursor is transferred to benzylate the recipient reactive group.
The middle compound illustrates a 5′0 attachment of a linker. The linker is linked through a phosphate group and extends into a three membered aliphatic chain. Through another phosphate group and a PEG linker the complementing element is linked via an amide bond to the Carrier. When the building block is presented to a nucleophile the Functional Entity Precursor is transferred resulting in an alkylation of the nucleophile.
The lower compound illustrates a nucleobase attachment of the linker. The linker attaches to the 5 position of a pyrimidine type nucleobase and extents through an α-β unsaturated N-methylated amide to the S—C-connecting group, which is a 4-amino methyl benzoic acid derivative. The functional entity precursor can be transferred to a nucleophilic recipient reactive group e.g. an amine or a thiol forming an allylic amine or thiol.
According to the invention, the functional entity precursor is of the formula —C(H)(R³)—R⁴or functional entity precursor is heteroaryl or aryl optionally substituted with one or more substituents belonging to the group comprising R³and R⁴. In a further preferred embodiment,
R³and R⁴independently is H, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₄-C₈alkadienyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl, optionally substituted with one or more substituents selected from the group consisting of
SnR⁵R⁶, R⁷, Sn(OR⁵)R⁶R⁷, Sn(OR⁵)(OR⁶)R⁷, BR⁵R⁶, B(OR⁵)R⁶, B(OR⁵)(OR⁶), halogen, CN, CNO, C(halogen)₃, ═O, OR⁵, OC(═O)R⁵, OC(═O)OR⁵, OC(═O)NR⁵R⁶, SR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, N₃, NR⁵R⁶, N⁺R⁵R⁶R⁷, NR⁵OR⁶, NR⁵NR⁶R⁷, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, NC, P(═O)(OR⁵)OR⁶, P⁺R⁵R⁶R⁷, C(═O)R⁵, C(═NR⁵)R⁶, C(═NOR⁵)R⁶, C(═NNR⁵R⁶), C(═O)OR, C(═O)NR⁵R⁶, C(═O)NR⁵OR₆, C(═O)NR⁵NR⁶R⁷, C(═NR⁵)NR⁶R⁷, C(═NOR⁵)NR⁶R⁷or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₄-C₈alkadienyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in another prefered embodiment,
R³and R⁴independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl, optionally substituted with one or more substituents selected from the group consisting of halogen, CN, C(halogen)₃, ═O, OR⁵, OC(═O)R⁵, OC(═O)OR⁵, OC(═O)NR⁵R⁶, SR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵OR⁶, NR5NR⁶R⁷, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, P(═O)(OR5)OR⁶, C(═O)R⁵, C(═NR⁵)R⁶, C(═NOR⁵)R⁶, C(═NNR⁵R⁶), C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶, C(═O)NR⁵NR⁶R⁷, C(═NR⁵)NR⁶R⁷, C(═NOR⁵)NR⁶R⁷or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl, optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, OC(═O)R⁵, OC(═O)OR⁵, OC(═O)NR⁵R⁶, SR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵R⁶, NR⁵NR⁶R⁷, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, P(═O)(OR⁵)OR⁶, C(═O)R⁶, C(═NR⁵)R⁶, C(═NOR⁵)R⁶, C(═NNR⁵R⁶), C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶, C(═O)NR⁵NR⁶R⁷, C(═NR⁵)NR⁶R⁷, C(═NOR⁵)NR⁶R⁷or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl, optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, phenyl, naphtyl, thienyl, furyl, pyridyl, quinolinyl or isoquinolinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl and wherein R⁶and R5 may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl or morpholinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 38 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, phenyl, naphtyl, thienyl, furyl, pyridyl, quinolinyl or isoquinolinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁵, wherein,
R⁵, R⁶, R⁷and R³independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, phenyl or naphtyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R6 and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, thienyl, furyl, pyridyl, quinolinyl or isoquinolinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, R⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁵independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R^6,NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl or morpholinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R^6,R⁷and R⁸independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, phenyl, naphtyl, thienyl, furyl, pyridyl, quinolinyl or isoquinolinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵s, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NRBR⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, phenyl or naphtyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, thienyl, furyl, pyridyl, quinolinyl or isoquinolinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁶, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl and wherein R⁵and R⁶may together form a 38 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, methyl, ethyl, propyl or butyl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl or morpholinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NRR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, methyl, ethyl, propyl or butyl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, phenyl, naphtyl, thienyl, furyl, pyridyl, quinolinyl or isoquinolinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, methyl, ethyl, propyl or butyl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, phenyl or naphtyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃,═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, methyl, ethyl, propyl or butyl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is H, thienyl, furyl, pyridyl, quinolinyl or isoquinolinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁶C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, methyl, ethyl, propyl or butyl and wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring,
in still another prefered embodiment,
R³and R⁴independently is methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁶R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁶)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl,
in still another prefered embodiment,
R³and R⁴independently is aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl or morpholinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein, R⁵, R⁶, R⁷and R⁸independently is H, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl,
in still another prefered embodiment,
R³and R⁴independently is phenyl, naphtyl, thienyl, furyl, pyridyl, quinolinyl or isoquinolinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl,
in still another prefered embodiment,
R³and R⁴independently is phenyl or naphtyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁸, R⁷and R⁸independently is H, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl,
in still another prefered embodiment,
R³and R⁴independently is thienyl, furyl, pyridyl, quinolinyl or isoquinolinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl,
in still another prefered embodiment,
R³and R⁴independently is methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl,
in still another prefered embodiment,
R³and R⁴independently is aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl or morpholinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl,
in still another prefered embodiment,
R³and R⁴independently is phenyl, naphtyl, thienyl, furyl, pyridyl, quinolinyl or isoquinolinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl,
in still another prefered embodiment,
R³and R⁴independently is phenyl or naphtyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl,
in still another prefered embodiment,
R³and R⁴independently is thienyl, furyl, pyridyl, quinolinyl or isoquinolinyl optionally substituted with one or more substituents selected from the group consisting of F, Cl, CN, CF₃, ═O, OR⁵, S(═O)R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, NR⁵R⁶, NR⁵C(═O)R⁵, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, C(═O)R⁵, C(═NOR⁵)R⁶, C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶or R⁸, wherein,
R⁵, R⁶, R⁷and R⁸independently is H, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl,
in still another prefered embodiment,
R³and R⁴independently is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl or heteroaryl
in still another prefered embodiment,
R³and R⁴independently is H,
in still another prefered embodiment,
R³and R⁴independently is C₁-C₆alkyl, C₃-C₇cycloalkyl or C₃-C₇cycloheteroalkyl,
in still another prefered embodiment,
R³and R⁴independently is methyl, ethyl, propyl or butyl
in still another prefered embodiment
R³and R⁴independently is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl
in still another prefered embodiment
R³and R⁴independently is aziridinyl, pyrrolidinyl, piperidinyl or morpholinyl
in still another prefered embodiment,
R³and R⁴independently is aryl or heteroaryl
in still another prefered embodiment,
R³and R⁴independently is phenyl or naphthyl
in still another prefered embodiment,
R³and R⁴independently is thienyl, furyl, pyridyl, quinolinyl or isoquinolyl
Experimental Section
General Procedure 1: Preparation of Carrier-Functional Entity Reagents:
The 4-halobenzoic acid (25 mmol) is added to a ice cooled solution of chloro sulfonic acid (140 mmol). The mixture is slowly heated to reflux and left at reflux for 2-3 hours. The mixture is added to 100 mL ice and the precipitate collected by filtration. The filtrate is washed with water (2×50 mL) and the dried in vacuo affording the corresponding sulfonoyl chloride in 60-80% yield. The 3-chlorosulfonyl-4-halo-benzoic acid derivate (5 mmol) is dissolved in EtOH (5 mL) and added to a ice cooled mixture of NaOEt (10 mL, 2M). The mixture is stirred o/n at rt. Acetic acid (40 mmol) is added and the mixture is evaporated in vacuo. Water (10 mL) is added and pH adjusted to pH=2 (using 1M HCl). The product is extracted with DCM (2×25 mL), dried over Na₂SO₄and evaporated in vacuo affording the desired products.

EXAMPLE 1

General Procedure (1)

3-Ethoxysulfonyl4-fluorobenzoic acid
¹H-NMR (DMSO-d₆): δ 8.49 (d, 1H), 7.85 (dd, 1H), 7.5 (d, 1H), 4.32 (q, 2H), 1.32 (t, 3H)

EXAMPLE 2

General Procedure (1)

4-chloro-3-Ethoxysulfonylbenzoic acid
¹H-NMR (DMSO-d₆): δ 8.49 (d, 1H), 7.85 (dd, 1H), 7.5 (d, 1H), 4.32 (q, 2H), 1.32 (t, 3H)

EXAMPLE 3

4-Methylsulfanyl benzoic acid (0.5 g, 2.97 mmol, commercially available from Aldrich, cat no. 145521) was added to methyl p-toluene solfunate (0.61 g, 3.27 mmol). The mixture was heated to 140° C. for 1 hour in a sealed vessel. After cooling to rt the mixture was trituated with diethyl ether. Filtration and drying in vacuo yielded 844 mg (80%) of the desired product (>95% pure by ¹H nmr).
¹H nmr (DMSO-d6): 8.20-8.10 (m, 4H), 7.45 (d, 2H), 7.08 (d, 2H), 3.29 (s, 6H), 2.30 (s, 3H).
General Procedure 2: Solid Phase Preparation of Carrier-Functional Entity Reagents for Alkylation Building Blocks:
Ps=Polystyrene resin. Alternatively other acid labile linkers may be employed.
Step 1:
A polystyrene resin with a wang linker (4-hydroxymethylphenol linker)(50 mg˜50 umol), a bi-functional carrier (200 umol, 4 equiv) in a solvent such as THF, DCM, DCE, DMF, NMP or a mixture thereof (500 uL) and a base such as TEA, DIEA, pyridine (400 umol, 8 equiv), optionally in the presence of DMAP (100 umol), are allowed to react at temperatures between −20° C. and 60° C., preferably between 0° C. and 25° C., for 1-24 h, preferably 14 h. The resin is washed with the solvent composition used during the reaction (5×1 mL) and used in the following step.
Step 2:
A functional entity precursor carrying a hydroxy group in the position of the intended attachment to the C—F-connecting group (200 umol, 4 equiv) in a solvent such as THF, DCM, DCE, DMF, NMP or a mixture thereof (500 uL) and a base such as TEA, DIEA, pyridine (400 umol, 8 equiv), optionally in the presence of DMAP, are added to the resin bound carrier isolated in step 1 and allowed to react at temperatures between 0° C. and 100° C., preferably between 25° C. and 80° C., for 248 h, preferably. 4-16 h. The resin is washed with the solvent composition used during the reaction (5×1 mL).
Step 3:
The desired Carrier-Functional entity reagent is cleaved from the resin obtained in step 2 by treatment with an acid like TFA, HF or HCl in a solvent such as THF, DCM, DCE or a mixture thereof (1 mL) at temperatures between −20° C. and 60° C., preferably between 0° C. and 25° C., for 14 h, preferably 1-2 h. Upon filtration, the resin is washed with the solvent composition used during cleavage (2×1 mL) and the combined filtrates are evaporated in vacuo. The isolated product may be purified by chromatography.

Assembly of Building Blocks

The Carrier-Functional entity reagent may be bound to the Spacer by several different reactions as illustrated below.

Formation of an Amide Bond Between a Carboxylic Acid of the Carrier and an Amine Group of a Spacer

General Procedure 3: Preparation of Building Blocks by Loading a Carrier-Functional Entity Reagent onto a Nucleotide Derivative Comprising an Amino Group:
15 uL of a 150 mM building block solution of FE¹-Carrier-COOH is mixed with 15 μL of a 150 mM solution of EDC and 15 μL of a 150 mM solution of N-hydroxy-succinimide (NHS) using solvents like DMF, DMSO, water, acetonitril, THF, DCM, methanol, ethanol or a mixture thereof. The mixture is left for 15 min at 25° C. 45 μL of an aminooligo (10 nmol) in 100 mM buffer at a pH between 5 and 10, preferably 6.0-7.5 is added and the reaction mixture is left for 2 hours at 25° C. Excess building block and organic by-products were removed by extraction with EtOAc (400 μL). Remaining EtOAc is evaporated in vacuo using a speedvac. The building block is purified following elution through a BiORad micro-spin chromatography column, and analyzed by electron spray mass spectrometry (ES-MS).

EXAMPLE 4

General Procedure ( )

Where Oligo is 5′0 XCG ATG GAT GCT CCA GGT CGC 3′, X=5′ amino C₆(Glen catalogue# 10-1906-90), Expected molecular weight: 6313.22 MS (calc.)=6543,43; MS (found)=6513,68*
* Observed molecular weight of the cleaved sulfonic ester: 6513.68 Expected molecular weight of the cleaved ester. 6514.37 The quantitative loss of the ethyl group Is probably due to the presence of pipeddine during the recording of the LCMS data.
General Procedure 4: Loading of a Carrier Coupled Functional Entity onto an Amino Ontgo:
25 μl 100 mM carrier coupled functional entity dissolved in DMF (dimethyl formamide) was mixed with 25 μl 100 mM EDC (1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride) in DMF for 30 minutes at 25° C. The mixture was added to 50 μl amino oligo in H₂O with 100 mM HEPES (2-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-ethanesulfonic acid) pH 7.5 and the reaction was allowed to proceed for 20 minutes at 25° C. Unreacted carrier coupled functional entity was removed by extraction with 500 μl EtOAc (ethyl acetate), and the oligo was purified by gel filtration through a microspin column equilibrated with 100 mM MES (2-(N-morpholino) ethanesulfonic acid) pH 6.0.
Oligonucleotide used:
Oligo A: 5′-YACGATGGATGCTCCAGGTCGC
Y=Amino modifier C6 (Glen#10-1906)

EXAMPLE 5

General Procedure 4

Carrier—Functional Entity: (4-Carboxy-phenyl)-dimethyl-sulfonium
Mass: 6789.21 (observed using ES-MS), 6790.65 (calculated)
General Procedure 5: Preparation of Arylation Building Blocks:

Funtional Entity-OH is a phenol, n is an integer between 3 and 6.
Step 1
To a solution of the bis-sulfonylchloride (Ward, R. B.; J. Org. Chem.; 30; 1965; 3009-3011; Qiu, Weiming; Burton, Donald J.; J. Fluorine Chem.; 60; 1; 1993; 93100)(3 umol) in DMF, DMSO, acetonitril, THF or a mixture thereof (150 uL) is a phenolic functional entity in excess (1.05-1.8 mmol) in DMF, DMSO, acetonitril, THF or a mixture thereof (150 uL) added slowly at temperatures between −20° C. and 100° C. preferably at 0-50° C. in the presence of a base such as TEA, DIEA, pyridine, Na-HCO₃or K₂CO₃.
Step2
The reaction mixture from step 1 is added to a solution of an aminooligo (10 nmol) in 100 mM buffer at a pH between 5 and 10, preferably 6.0-7.5 optionally in the presence of NHS. The reaction mixture is left for 2 hours at 25° C. Excess building block and organic by-products were removed by extraction with EtOAc (400 μL). Remaining EtOAc is evaporated in vacuo using a speedvac. The building aminooligo is purified following elution through a BiORad micro-spin chromatography column, and analyzed by electron spray mass spectrometry (ES-MS).

Use of Building Blocks

General Procedure 6: Alkylation of Oligonucleotide Derivatives Containing a Nucleophilic Recipient Group Using a Building Block of the Invention:
An oligonucleotide building block carrying functional entity FE¹is combined at 2 μM final concentration with one equivalent of a complementary building block displaying a nucleophilic recipient group. Reaction proceeds at temperatures between 0° C. and 100° C. preferably between 15° C.-50° C. for 148 hours, preferably 10-20 hours in DMF, DMSO, water, acetonitril, THF, DCM, methanol, ethanol or a mixture thereof, pH buffered to 4-10, preferably 6-8. Organic by-products are removed by extraction with EtOAc, followed by evaporation of residual organic solvent for 10 min in vacuo. Pd catalyst is removed and oligonucleotides are isolated by eluting sample through a BiORad micro-spin chromatography column. Coupling efficiency is quantified by ES-MS analysis.
General Procedure 7: Transfer of Functional Entity from a Carrier Oligo to Recipient Reactive Group.
A carrier coupled functional entity oligo (Example 1)(250 pmol) was added to a scaffold oligo B (200 pmol) in 50 μl 100 mM MES, pH 6. The mixture was incubated overnight at 25° C. Subsequently, the mixture was purified by gel filtration using a microspin column equilibrated with H₂O and transfer of the functional entity was verified by electron spray mass spectrometry (ES-MS). Transfer efficiency is expressed in percent and were calculated by dividing the abundance of scaffold oligo carrying transferred functional entities to total abundance of scaffold oligos (with and without transferred functional entities).

EXAMPLE 6

General Procedure 7

Mass (“X”): 6583.97 (observed), 6583.31 (calculated). Abundance: 65.79 (arbitrary units)
Mass (“Y”): 6599.73 (observed), 6597.34 (calculated). Abundance: 29.23 (arbitrary units)
Mass (“Z”): 6789.36 (observed), 6790.65 (calculated)
Transfer efficiency calculated as: 29.23/ (29.23+65.79) ═0.3076˜31%
General Procedure 8: Arylation of Oligonucleotide Derivatives Containing a Nucleophilic Recipient Group Using a Building Block of the Invention:
An oligonucleotide building block carrying functional entity FE¹is combined at 2 μM final concentration with one equivalent of a complementary building block displaying a nucleophilic recipient group. In the presence of a Pd catalyst, the reaction proceeds at temperatures between 0° C. and 100° C. preferably between 15° C.-50° C. for 1-48 hours, preferably 10-20 hours in DMF, DMSO, water, acetonitrile, THF, DCM, methanol, ethanol or a mixture thereof, pH buffered to 4-10, preferably 6-8. Organic by-products are removed by extraction with EtOAc, followed by evaporation of residual organic solvent for 10 min in vacuo. Pd catalyst is removed and oligonucleotides are isolated by eluting sample through a BiORad micro-spin chromatography column. Coupling efficiency is quantified by ES-MS analysis.
General Procedure 9: General Route to the Formation of Alkylating/Vinylating Monomer Building Blocks with a Thio-Succinimid S—C-Connecting Group and Use of These:
R¹and R²may be used to tune the reactivity of the sulphate to allow appropriate reactivity. Chloro and nitro substitution will increase reactivity. Alkyl groups will decrease reactivity. Ortho substituents to the sulphate will due to steric reasons direct incoming nucleophiles to attack the R-group selectively and avoid attack on sulphur. E.g.
3-Aminophenol (6) is treated with maleic anhydride, followed by treatment with an acid e.g. H₂SO₄or P₂O₅and heat to yield the maleimide (7). The ring closure to the maleimide may also be achieved when an acid stable O-protection group is used by treatment with or Ac₂O with or without heating, followed by O-deprotection. Alternatively reflux in Ac₂O, followed by O-deacetylation in hot water/dioxane to yield (7).
Further treatment of (7) with SO₂Cl₂with or without triethylamine or potassium carbonate in dichloromethane or a higher boiling solvent will yield the intermediate (8), which may be isolated or directly further transformed into the aryl alkyl sulphate by the quench with the appropriate alcohol, in this case MeOH, whereby (9) will be formed. The organic building block (9) may be connected to an oligo nucleotide, as follows.
A thiol carrying oligonucleotide in buffer 50 mM MOPS or hepes or phosphate pH 7.5 is treated with a 1-100 mM solution and preferably 7.5 mM solution of the organic building block (9) in DMSO or alternatively DMF, such that the DMSO/DMF concentration is 5-50%, and preferably 10%. The mixture is left for 1-16 h and preferably 24 h at 25° C. To give the alkylating in this case methylating monomer building block (10).
The reaction of the alkylating monomer building block (10) with an amine carrying monomer building block may be conducted as follows:
The coding oligonucleotide (1 nmol) is mixed with a thio oligonucleotide loaded with a building block (1 nmol)(10) and an amino-oligonucleotide (1 nmol) in hepes-buffer (20 μL of a 100 mM hepes and 1 M NaCl solution, pH=7.5) and water (39 uL). The oligonucleotides are annealed to the template by heating to 50° C. and cooled (2° C./second) to 30° C. The mixture is then left o/n at a fluctuating temperature (10° C. for 1 second then 35° C. for 1 second), to yield the template bound methylamine (11).
A vinylating monomer building block may be prepared and used similarily as described above for an alkylating monomer building block. Although instead of reacting the chlorosulphonate (8 above) with an alcohol, the intermediate chlorosulphate is isolated and treated with an enolate or O-trialkylsilylenolate with or without the presence of fluoride. E.g.
Formation of the vinylating monomer building block (13):
The thiol carrying oligonucleotide in buffer 50 mM MOPS or hepes or phosphate pH 7.5 is treated with a 1-100 mM solution and preferably 7.5 mM solution of the organic building block (12) in DMSO or alternatively DMF, such that the DMSO/DMF concentration is 5-50%, and preferably 10%. The mixture is left for 1-16 h and preferably 2-4 h at 25° C. To give the vinylating monomer building block (13).
The sulfonylenolate (13) may be used to react with amine carrying monomer building block to give an enamine (14a and/or 14b) or e.g. react with an carbanion to yield (15a and/or 15b). E.g.
The reaction of the vinylating monomer building block (13) and an amine or nitroalkyl carrying monomer building block may be conducted as follows:

The coding oligonucleotide (1 nmol) is mixed with a oligonucleotide building block (1 nmol)(13) and an amino-oligonucleotide (1 nmol) or nitroalkyl-oligonucleotide (1 nmol) in 0.1 M TAPS, phosphate or hepes-buffer and 300 mM NaCl solution, pH=7.5-8.5 and preferably pH=8.5. The oligonucleotides are annealed to the template by heating to 50° C. and cooled (2° C./second) to 30° C. The mixture is then left o/n at a fluctuating temperature (10° C. for 1 second then 35° C. for 1 second), to yield template bound (14a/b or 15a/b).



DCC	N,N′-Dicyclohexylcarbodiimide
DhbtOH
	3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
DIC	Diisopropylcarbodiimide
DIEA	Diethylisopropylamin
DMAP	4-Dimethylaminopyridine
DNA	Deoxyribosenucleic Acid
EDC	1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide.HCl
HATU	2-(1H-7-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
	hexafluorophosphate
HBTU	2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
	hexafluoro-phosphate
HOAt	N-Hydroxy-7-azabenzotriazole
HOBt	N-Hydroxybenzotriazole
LNA	Locked Nucleic Acid
NHS	N-hydroxysuccinimid
OTf	Trifluoromethylsulfonate
OTs	Toluenesulfonate
PNA	Peptide Nucleic Acid
PyBoP	Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
	hexafluoro-phosphate
PyBroP	Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate
RNA	Ribonucleic acid
TBTU	2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetra-
	fluoroborate
TEA	Triethylamine
RP-HPLC	Reverse Phase High Performance Liquid Chromatography
TBDMS-Cl	Tert-Butyldimethylsilylchloride
5-Iodo-dU	5-iodo-deoxyriboseuracil
TLC	Thin layer chromatography
(Boc)₂O	Boc anhydride, di-tert-butyl dicarbonate
TBAF	Tetrabutylammonium fluoride
SPDP	Succinimidyl-propyl-2-dithiopyridyl

Claims

1. A building block of the general formula

Complementing Element-Linker-Carrier-C—F-connecting group—Functional entity precursor

capable of transferring a Functional entity precursor to a recipient reactive group, wherein

Complementing Element is a group identifying the Functional entity precursor,

Linker is a chemical moiety comprising a spacer and a S—C-connecting group, wherein the spacer is a valence bond or a group distancing the Functional entity precursor to be transferred from the complementing element and the S—C-connecting group connects the spacer with the Carrier,

Carrier is arylene, heteroarylene, C₁-C₆alkylene, C₁-C₆alkenylene, C₁-C₆alkynylene, or —(CF₂)_m— substituted with 0-3 R¹wherein m is an integer between 1 and 10;

R¹are independently selected from the group consisting of —H, —OR², —NR² ₂, -Halogen, —NO₂, —CN, —C(Halogen)₃, —C(O)R², —C(O)NHR², C(O)NR² ₂, —NC(O)R², —S(O)₂NHR², —S(O)₂NR², —S(O)₂R², —P(O)₂—R², —P(O)—R², —S(O)—R², P(O)—OR², —S(O)—OR², and —N⁺R² ₃, wherein R²is H, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, or aryl,

C—F-connecting group is selected from the group consisting of —SO₂—O—, —O—SO₂—O—, —C(O)—O—, —S⁺(R³)—, —C—U—C(V)—O—, —P⁺(W)₂—O—, and —P(W)—O—, where U is —C(R²)₂—, —NR²— or —O—; V is ═O or ═NR²and W is —OR²or —N(R²)₂,

Functional entity precursor is —C(H)(R³)—R⁴or functional entity precursor is heteroaryl or aryl optionally substituted with one or more substituents belonging to the group comprising R³and R⁴,

Wherein R³and R⁴independently is H, alkyl, alkenyl, alkynyl, alkadienyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, optionally substituted with one or more substituents selected from the group consisting of SnR⁵R⁶R⁷, Sn(OR⁵)R⁶R⁷, Sn(OR⁵)(OR⁶)R⁷, BR⁵R⁶, B(OR⁵)R⁶, B(OR⁵)(OR⁶), halogen, CN, CNO, C(halogen) ₃, OR⁵, OC(═O) R⁵, OC(═O)OR⁵, OC (═O) NR⁵R⁶, SR⁵, S(═O) R⁵, S(═O)₂R⁵, S(═O)₂NR⁵R⁶, NO₂, N₃, NR⁵R⁶, N⁺R⁵R⁶R⁷, NR⁵OR⁶, NR⁵N R⁶R⁷, NR⁵C(═O)R⁶, NR⁵C(═O)OR⁶, NR⁵C(═O)NR⁶R⁷, NC, P(═O)(OR⁵)OR⁶, P⁺R⁵R⁶R⁷, C(═O) R⁵, C(═NR⁵)R⁶, C(═NOR⁵)R⁶, C(═NNR⁶), C(═O)OR⁵, C(═O)NR⁵R⁶, C(═O)NR⁵OR⁶, C(═O)NR⁵NR⁶R⁷, C(═NR⁵)NR⁶R⁷, C(═NOR⁵)NR⁶R⁷and R⁸, wherein,

R⁵, R⁶, and R⁷independently is H, alkyl, alkenyl, alkynyl, alkadienyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, optionally substituted with one or more substituents selected from the group consisting of halogen, CN, CNO, C(halogen)₃, ═O, OR⁸, OC(═O)R⁸, OC(═O)OR⁸, OC(═O)NR⁸R⁹, SR⁸, S(═O)R⁸, S(═O)₂R⁸, S(═O)₂NR⁸R⁹, NO₂, N₃, NR⁸R⁹, N⁺R⁸R⁹R¹⁰, NR⁵OR⁶, NR⁵NR⁶R⁷, NR⁸C(═O)R⁹, NR⁸C(═O)OR⁹, NR⁸C(═O)NR⁹R¹⁰, NC, P(═O)(OR⁸)OR⁹, P⁺R⁵R⁶R⁷, C(═O)R⁸, C(═NR⁸)R⁹, C(═NOR⁸)R⁹, C(═NNR⁸R⁹), C(═O)OR⁸, C(═O)NR⁸R⁹, C(═O)NR⁸OR⁹C(═NR⁵)NR⁶R⁷, C(═NOR⁵)NR⁶R⁷or C(═O)NR⁸NR⁹R¹⁰, wherein R⁵and R⁶may together form a 3-8 membered heterocyclic ring or R⁵and R⁷may together form a 3-8 membered heterocyclic ring or R⁶and R⁷may together form a 3-8 membered heterocyclic ring, wherein,

R⁸, R⁹, and R¹⁰independently is H, alkyl, alkenyl, alkynyl, alkadienyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl and wherein R⁸and R⁹may together form a 3-8 membered heterocyclic ring or R⁸and R¹⁰may together form a 3-8 membered heterocyclic ring or R⁹and R¹⁰may together form a 3-8 membered heterocyclic ring.

2. A compound according to claim 1 wherein,

Functional entity precursor is —C(H)(R¹¹)—R¹¹′ or functional entity precursor is heteroaryl or aryl substituted with 0-3 R¹¹, 0-3 R¹³and 0-3 R¹⁵, wherein

R¹¹and R¹¹′0 are independently H, or selected from the group consisting of a C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₄-C₈alkadienyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl, and heteroaryl, said group being substituted with 0-3 R¹², 0-3 R¹³and 0-3 R¹⁵,

or R¹¹and R¹¹′ are C₁-C₃alkylene-NR¹² ₂, C₁-C₃alkylene-NR¹²C(O)R¹⁶, C₁-C₃alkylene-NR¹²C(O)OR¹⁶, C₁-C₂alkylene-O—NR¹² ², C₁-C₂alkylene-O—N R¹²C(O)R¹⁶, or C₁-C₂alkylene-O—NR¹²C(O)OR¹⁶substituted with 0-3 R¹⁵,

where R¹²is H or selected independently from the group consisting of C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl, and heteroaryl, said group being substituted with 0-3 R¹³and 0-3 R¹⁵,

R¹³is selected independently from the group consisting of —N₃, —CNO, —C(NOH)NH₂, —NHOH, —NHNHR¹⁷, —C(O)R¹⁷, —SnR¹⁷ ₃, —B(OR¹⁷)₂, and —P(O)(OR¹⁷)₂or the group consisting of C₂-C₆alkenyl, C₂-C₆alkynyl, and C₄-C₈alkadienyl, said group being substituted with 0-2 R¹⁴,

where R¹⁴is independently selected from the group consisting of —NO₂, —C(O)OR¹⁷, —COR¹⁷, —CN, —OSiR¹⁷ ₃, —OR¹⁷and —NR¹⁷ ₂;

R¹⁵is ═O, —F, —Cl, —Br, —I, —CN, —NO₂, —OR¹⁷, —NR¹⁷ ₂, —NR¹⁷—C(O)R¹⁶, —NR¹⁷—C(O)OR¹⁷, —SR¹⁷, —S(O)R , —S(O)₂R¹⁷, —COOR¹⁷, —C(O)NR¹⁷ ₂or —S(O)₂NR¹⁷ ₂,

R¹⁶is H, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl, aryl or C₁-C₆alkylene-aryl substituted with 0-3 substituents independently selected from —F, —Cl, —NO₂, —R², —OR , —SiR² ₃;

R¹⁷is selected independently from the group consisting of H, C₁-C₆alkyl, C₃-C₇cycloalkyl, aryl, C₁-C₆alkylene-aryl,

G is H or C₁-C₆alkyl and n is 1,2,3 or 4.

3. A compound according to claim 2 wherein,

or R¹¹and R¹¹′0 are C₁-C₃alkylene-NR¹² ₂, C₁-C₃alkylene-NR¹²C(O)R¹⁶, C₁-C₃alkylene-NR¹²C(O)OR¹⁶, C₁-C₂alkylene-O—NR¹² ₂, C₁-C₂alkylene-O—NR¹²C(O)R¹⁶, C₁-C₂alkylene-O—NR¹²C(O)OR¹⁶substituted with 0-3 R¹⁵,

where R¹²is H or selected from the group consisting of C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl, and heteroaryl, said group being substituted with 0-3 R¹³and 0-3 R¹⁵,

R¹³is selected from the group consisting of —N₃, —CNO, —C(NOH)NH₂, —NHOH, —NHNHR¹⁷, —C(O)R¹⁷, —SnR¹⁷ ₃, —B(OR¹⁷)₂, and —P(O)(OR¹⁷)₂or the group consisting of C₂-C₆alkenyl, C₂-C₆alkynyl, and C₄-C₈alkadienyl, said group being substituted with 0-2 R¹⁴,

where R¹⁴is selected from the group consisting of —NO₂, —C(O)OR¹⁷, —COR¹⁷, —CN, —OSiR¹⁷ ₃, —OR¹⁷and —NR¹⁷ ₂;

R¹⁵is ═O, —F, —Cl, —Br, —I, —CN, —NO₂, —OR¹⁷, —NR¹⁷ ₂, —NR¹⁷—C(O)R¹⁶, —NR¹⁷—C(O)OR¹⁶, —SR¹⁷, —S(O)R¹⁷, —S(O)₂R¹⁷, —COOR¹⁷, —C(O)NR¹⁷ ₂or —S(O)₂NR¹⁷ ₂,

R¹⁶is H, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl, aryl or C₁-C₆alkylene-aryl substituted with 0-3 substituents independently selected from the group consisting of —F, —Cl, —NO₂, —R², —OR², and —SiR² ₃; wherein R¹⁷is selected independently from the group consisting of H, C₁-C₆alkyl, C₃-C₇cycloalkyl, aryl, and C₁-C₆alkylene-aryl.

4. A compound according to claim 1 wherein, Functional entity precursor is —C(H)(R¹¹)—R¹¹′0 wherein R¹¹and R¹¹′ are C₁-C₃alkylene-NR¹² ₂, C₁-C₃alkylene-NR¹²C(O)R¹⁶, C₁-C₃alkylene-NR¹²C(O)OR¹⁶, C₁-C₂alkylene-O—NR¹² ₂, C₁-C₂alkylene-O—NR¹²C(O)R¹⁶, or C₁-C₂alkylene-O—NR¹²C(O)OR¹⁶substituted with 0-3 R¹⁵.

5. A compound according to claim 1 wherein, Functional entity precursor is —C(H)(R¹¹)—R¹¹′ wherein R¹¹and R¹¹′ are independently H, or selected from the group consisting of a C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₄-C₈alkadienyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl, and heteroaryl, said group being substituted with 0-3 R¹², 0-3 R¹³and 0-3 R¹⁵.

6. A compound according to claim 2 wherein, Functional entity precursor is —C(H)(R¹¹)—R¹¹′ wherein

R¹¹and R¹¹′ are independently H, or selected from the group consisting of a C₁-C₆alkyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl, and heteroaryl, said group being substituted with 0-3 R¹²and 0-3 R¹⁵,

where R¹²is H or selected from the group consisting of C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₇cycloalkyl, C₃-C₇cycloheteroalkyl, aryl, and heteroaryl,

R¹⁷is selected from the group consisting of H, C₁-C₆alkyl, C₃-C₇cycloalkyl, and C₁-C₆alkylene-aryl.

7. A compound according to claim 1 wherein, Functional entity precursor is heteroaryl or aryl substituted with 0-3 R¹¹, 0-3 R¹³and 0-3 R¹⁵.

8. A compound according to claim 2 wherein C—F-connecting group is selected from the group consisting of —SO₂—O—, —O—SO₂—O—, —C(O)—O—, —S⁺(R¹¹)—, —C—U—C(V)—O—, —P⁺(W)₂—O—, and —P(W)—O— where U is —C(R²)₂—, —NR²— or —O—; V is ═O or ═NR²and W is —OR²or —N(R²)₂.

9. A compound according to claim 2 wherein C—F-connecting group is —S⁺(R¹¹)—.

10. A compound according to claim 1 wherein C—F-connecting group is selected from the group consisting of —SO₂—O—, —O—SO₂—O—, —C(O)—O—, —S⁺(R¹⁷)—, —C—U—C(V)—O—, —P⁺(W)₂—O—, and —P(W)—O— where U is —C(R²)₂—, —NR²— or —O—; V is ═O or ═NR²and W is —OR²or —N(R²)₂, wherein R¹⁷is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, aryl, or C₁-C₆alkylene-aryl.

11. A compound according to wherein C—F-connecting group is chosen from the group consisting of —SO₂—O—, and —S⁺(R¹⁷)—; wherein R¹⁷is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, aryl, or C₁-C₆alkylene-aryl.

12. A compound according to claim 1 wherein, Spacer is a valence bond, C₁-C₆alkylene-A—, C₂-C₆alkenylene-A—, C₂-C₆alkynylene-A—, or

said spacer optionally being connected through A to a linker selected from

—(CH₂)_n—B—,

and —(CH₂)_n—S—S—(CH₂)_m—B—

where A is a valence bond, —C(O)NR¹⁷—, —NR¹⁷—, —O—, —S—, or —C(O)—O—; B is a valence bond, —O—, —S—, —NR¹⁷— or —C(O)NR¹⁷— and connects to S—C-connecting group; and n and m independently are integers ranging from 1 to 10; and R¹⁷is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, aryl, or C₁-C₆alkylene-aryl.

13. A compound according to claim 1 wherein, Spacer is a valence bond, C₁-C₆alkylene-A—, C₂-C₆alkenylene-A—, C₂-C₆alkynylene-A—, or

said spacer optionally being connected through A to a linker selected from —(CH₂)_n—B—,

and

where A is a valence bond, —C(O)NR¹⁷—, —NR¹⁷—, —S—, or —C(O)—O—; B is —O—, —S—, —NR¹⁷—, or —C(O)NR¹⁷— and connects to S—C-connecting group; and n and m independently are integers ranging from 1 to 6; and R¹⁷is H, C₁-C₆alkyl, C₃-C₇cycloalkyl, aryl, or C₁-C₆alkylene-aryl.

14. A compound according to claim 1 wherein, S—C-connecting group is a valence bond, —NH—C(═O)—,

15. A compound according to claim 2 wherein, the carrier is selected from the group consisting of arylene, heteroarylene and —(CF₂)_m— substituted with 0-3 R¹wherein m is an integer between 1 and 10, and C—F-connecting group is —SO₂—O—, and the functional entity precursor is —C(H)(R¹¹)—R¹¹′.

16. A compound according to claim 1 wherein, the carrier is —(CF₂)_m— wherein m is an integer between 1 and 10, the C—F-connecting group is —SO₂—O—; and the functional entity precursor is aryl or heteroaryl substituted with 0-3 R¹¹, 0-3 R¹³and 0-3 R¹⁵.

17. A compound according to claim 1 wherein Complementing element is a nucleic acid.

18. A compound according to claim 1 where Complementing element is a sequence of nucleotides selected from the group consisting of DNA, RNA, LNA PNA, and morpholino derivatives.

19. A library of compounds according to claim 1, wherein each different member of the library comprises a complementing element having a unique sequence of nucleotides, which identifies the functional entity.

20. A method for transferring a functional entity precursor to a recipient reactive group, comprising the steps of

providing one or more building blocks according to claim 1,

contacting the one or more building blocks with a corresponding encoding element associated with a recipient reactive group under conditions which allow for a recognition between the one or more complementing elements and the encoding elements, said contacting being performed prior to, simultaneously with, or subsequent to a transfer of the functional entity precursor to the recipient reactive group.

21. The method according to claim 20, wherein the encoding element comprises one or more encoding sequences comprised of 1 to 50 nucleotides and the one or more complementing elements comprises a sequence of nucleotides complementary to one or more of the encoding sequences.

22. The method of claim 20, wherein the recipient reactive group is a nucleophilic S- or N-atom, which may be part of a chemical scaffold, and the activating catalyst is contains palladium.