Z-inducing chimera composition

Abstract

C-5 propyl-G2-tagged pyrimidine and C-8 propyl-G2-tagged purine nucleoside. These nucleosides are useful as precursors for modified DNA and / or RNA strands. The resulting strands may have non-canonical confirmations (NCCs).

Description

Z-INDUCING CHIMERA COMPOSITIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and is a non -provisional of, U. S. Patent Application 63 / 719,300 (filed November 12, 2024), the entirety of which is incorporated herein by reference.STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number R35GM139336 awarded by the National Institute of General Medical Sciences (NIGMS). The government has certain rights in the invention.REFERENCE TO A SEQUENCE LISTING

[0003] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference. The computer readable tile is named Seqeuence.xml and was created on October 23, 2025 (22 kb).BACKGROUND OF THE INVENTION

[0004] Deoxyribonucleic acid (DNA) is canonically a right-handed helix (B-DNA). In addition to B-DNA, other non-canonical confirmations (NCCs) are known, such as G-quadruplex formation, i-motifs, R-loops, triplex and cruciform structures, supercoiling, bubbles, H-DNA, A-DNA, Z-DNA, and BZ-DNA junction structures. Growing evidence suggests these NCCs play a major role in regulating cellular activities. For example, repetitive sequences with a high propensity to form NCCs have been identified in promoter regions, and replication origins suggesting their pivotal role in biological processes While the occurrence of NCC is transient, driven by unfavorable folding, these conformations still seem to play a decisive role in the regulation of DNA replication and gene expression, altering DNA-protein interactions, transcription and translation, and processing of pre-mRNA.

[0005] The existence of Z-DNA conformation in the promoter sites is implicated in cancer, autoimmune diseases and neurological diseases, such as Alzheimer’s disease. Z-DNA binding proteins, the class of proteins that recognize the left-handed Z-form of DNA and RNA, include important enzymes. For example, Adenosine Deaminase Acting on RNA 1 (AD ARI), is involved in RNA editing by converting adenosine to inosine, and it plays a key role in cancer and autoimmune disorders.

[0006] Despite repeated observations of the presence of Z-DNA structures and their implications in altering cellular processes, the use of Z-DNA in both cellular and animal models has been challenging due, at least in part, to the instability of Z-DNA structures and the transient appearance of Z-DNA in cellular events. A variety of methodologies have been used to induce NCCs of DNA but these typically rely on controlling the sequence of modified nucleotides. Improved compositions and methods of generating NCCs of DNA in a sequence-independent manner are therefore desired.

[0007] The discussion above is merely provided for general background information and is not intended to be used as an aid in determining the scope of the claimed subject matter.SUMMARY

[0008] This disclosure provides C-5 propyl -G2-tagged pyrimidine and C-8 propyl-G2-tagged purine nucleosides. These nucleosides are useful as precursors for modified DNA and / or RNA strands. The resulting strands may have non-canonical confirmations (NCCs).

[0009] In a first embodiment, a composition of matter is provided. The composition of matter comprising an organic moiety (G2); a propyl linker; a nitrogenous base that is: a purine base with a C8 position that links the purine base to the organic moiety (G2) with the propyl linker, or a pyrimidine base with a C5 position that links the pyrimidine base to the organic moiety (G2) with the propyl linker: and a ribose that is directedly connected to the nitrogenous base at a Cl’ position of the ribose.

[0010] In a second embodiment, a composition of matter is provided. The composition of matter comprising a compound with a structure of:wherein O1is an alcohol or a protected alcohol, G1is a nitrogenous base having a structure selected from:wherein G2is an indole, a benzoimidazole or a methoxyphenyl and N1is a primary amine or a protected primary amine.

[0011] This brief description of the invention is intended only to provide a brief overview of subject matter disclosed herein according to one or more illustrative embodiments, and does not serve as a guide to interpreting the claims or to define or limit the scope of the invention, which is defined only by the appended claims. This brief description is provided to introduce an illustrative selection of concepts in a simplifiedform that are further described below in the detailed description. This brief description is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. The claimed subject matter is not limited to implementations that solve any or all disadvantages noted in the background.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] So that the manner in which the features of the invention can be understood, a detailed description of the invention may be had by reference to certain embodiments, some of which are illustrated in the accompanying drawings. It is to be noted, however, that the drawings illustrate only certain embodiments of this invention and are therefore not to be considered limiting of its scope, for the scope of the invention encompasses other equally effective embodiments. The drawings are not necessarily to scale, emphasis generally being placed upon illustrating the features of certain embodiments of the invention. In the drawings, like numerals are used to indicate like parts throughout the various views. Thus, for further understanding of the invention, reference can be made to the following detailed description, read in connection with the drawings in which:

[0014] FIG. 1 depicts a generic structure for some embodiments of disclosed nucleotide precursors.

[0015] FIG. 2A, FIG. 2B and FIG. 3 depict sequence alignment of examples of DNA duplexes that incorporate modified nucleotides made from the disclosed nucleotide precursors, EcoRI restriction site is shaded gray. Xmal, Smal restriction site is in italics.

[0016] FIGs. 4A to 4W illustrate CD spectra of the DNA duplexes showing formation of NCCs.

[0017] FIGs. 5A to 5H are graphs of normalized fluorescence showing promiscuity of DNA duplexes towards DNase I (FIG. 5 A, FIG. 5B), EcoRI (FIG. 5C, FIG. 5D), Xmal (FIG. 5E and FIG. 5F), and Smal (FIG. 5G and FIG. 5H).DETAILED DESCRIPTION OF THE INVENTION

[0018] This disclosure provides nucleotide precursors that produce modified nucleotides that induce non-canonical confirmations (NCCs) of DNA duplexes. The nucleotide precursors have a general structure of Formula A. Also see FIG. 1

[0019] The nucleotide precursors of Formula A include a phosphoramidite moiety. Phosphoramidite moi eties are used in a conventional chemical synthetic process that sequentially adds one nucleotide at a time to a growing DNA or RNA chain on a solid support. Accordingly, the nucleotide precursors of Formula A are useful for forming DNA and RNA. In one embodiment, a composition of matter is provided that comprises a nucleic acid (either DNA or RNA, either single strand or duplexed) that comprises at least one nucleotide formed from the disclosed precursors.

[0020] Referring again to Formula A, O1is an alcohol (-OH) or a protected alcohol ( OP). In those embodiments where O1is a protected alcohol, the protecting group (P) is not particularly limited. For example, in one embodiment, O1is a 4,4’-dimethoxytrityl (DMTr) protected alcohol (DMTrO).

[0021] G1is a nitrogenous base that is a modified purine or pyrimidine having a structure selected from Formula B-l, Formula B-2, Formula B-3 or Formula B-4:wherein G2is an organic moiety and N1is a primary amine (-NH2) or a protected primary amine (e.g. -NHP or -NP). The modified purine or pyrimidine is connected to the organic moiety G2by a propyl linker (— CH2CH2CH2— ).

[0022] In those embodiments wherein N1is a protected primary' amine, the protecting group is not particularly limited. For example, N!may be selected from

[0023] In one embodiment, N1is selected such that the nitrogenous base (G1) has a structure selected fromo

[0024] G2is an organic moiety. In one embodiment, the organic moiety (G2) is a benzene, a pyridine, a pyrimidine, a pyrazine, a pyrazine. Each of these organic moieties may be substituted or unsubstituted. In another embodiment, G2has a structure of Formul a G2-1.R3Rt ^R2H4" H2Formula G2-1wherein H1, H2, H\ H4and H5are independently selected from C and N and R1, R2, RJ, R4and R3are independently selected from II, an alkyl, a halogenated alkyl, an aryl, a primary amine, a secondary amine (NHR6, wherein R6is an alkyl or an aryl), a tertiary amine (NR, R8wherein R', RSare independently selected alkyl or aryl), an alcohol, a ether (OR9wherein R9is an alkyl or aryl), a halogen (F, Cl, Br, I), a thiol, a nitro, anitrile, an amide (C(=O)NR1UR11wherein R10and R11are independently selected from H, alkyl and aryl), a carboxylic acid, an ester (C(=O)R12wherein R12is an alkyd or aryl), a thioether (SRi3wherein R13is an alkyl or aryl), a sulfoxide (S(=O)R14wherein R14is an alkyl or aryl), a sulfone (S(=O)(=O)R13wherein R15is an alkyl or aryl), a sulfonamide (S(=O)(=O)NR16R' ' wherein R16and R17are independently selected alkyl or aryl), a sulfoximine (SRls(::::O)(::::NR19) wherein R18and R19are independently selected alkyl or aryl). In one embodiment, H1, H2, H3, H4and H5are selected such that one is N and four are C. In another embodiment, H1, II2, H3, H4and H' are selected such that two are N and three are C.

[0025] As used herein, “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 10 carbon atoms (“Cl-10 alkyl”). In some embodiments, an alkyl group has I to 9 carbon atoms (“Cl -9 alkyl”). In some embodiments, an alkyl group has I to 8 carbon atoms (“Cl -8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“Cl-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“Cl -6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“Cl -5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“Cl -4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“Cl -3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“Cl-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Cl alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”). Examples of Cl -6 alkyl groups include, without limitation, methyl (Cl), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents.

[0026] As used herein, “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n ■ 2 aromatic ring system (e.g., having 6, 10, or 14 n electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“CIO aryl”; e.g., naphthyl such as 1 -naphthyl and 2-naphthyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the and ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C6-14 aryl. In certain embodiments, the aryl group is a substituted C6-14 aryl.

[0027] In one embodiment, the organic moiety (G2) is a pyrrole, a furan, a thiophene, an imidazole, a thaizole, an oxazole, a triazole or a pyrazole. Each of these organic moieties may be substituted or unsubstituted. In another embodiment, G2has a structure of Formula G2-2:Formula G2-2wherein H6, H', H8and H9are independently selected from C, O, S, N and NH and H10is selected from C and N, provided at least one of H6, H7, H8, H9and Hf0is selected from O, S, N and NH. R20, R21, R22, R23are independently selected from H, an alkyl, a halogenated alkyl, an aryl, a primary amine, a secondary amine (NHR6, wherein R6is analkyl or an aryl), a tertiary amine (NR7, R8wherein R', R8are independently selected alkyl or aryl), an alcohol, a ether (OR9wherein R9is an alkyl or aryl), a halogen (F, Cl, Br, I), a thiol, a nitro, a nitrile, an amide (C(=O)NR10R1!wherein Ri0and Rnare independently selected from H, alkyl and aryl), a carboxylic acid, an ester (C(=O)R12wherein Ri2is an alkyl or aryl), a thioether (SR13wherein R13is an alkyl or aryl), a sulfoxide (S(=O)R14wherein R14is an alkyl or aryl), a sulfone (S(:::O)(:::O)R13wherein R13is an alkyl or aryl), a sulfonamide (S(=O)(=O)NRl6R! 7wherein Rloand R17are independently selected alkyl or aryl), a sulfoximine (SR18(:::O)(=NR19) wherein R18and R19are independently selected alkyl or aryl). In one embodiment, Hi0is N and H6, H7, H8and H9are C. In another embodiment, H10is N, one of H6, H7, H8and H9is N and three of H6, H', H8and H9are C. In another embodiment, H10is C, one of Hb, H7, H8and H9is O and three of H6, H7, H8and H9are C. In another embodiment, H10is C, one of H6, H', H8and H9is S and three of H6, H', H8and H9are C, In another embodiment, Hi0is C, two of H6, H7, H8and H9are N and two of H°, H7, H8and H9are C. In another embodiment, H10is C, three of H6, H7, FI8and H9are N and one of H6, H7, H8and H9is C. In another embodiment, H10is C, two of Hb, H7, H8and H9are S and two of H6, H7, H8and H9are C. In another embodiment, H10is C, one of H6, H7, H8and H9is O, one of H°, H7, H8and H9is N and two of H6, H7, H8and H9are C In another embodiment, H10is C, one of H6, H7, H8and H9is S, one of H6, H7, H8and H9is N and two of H6, H7, H8and H9are C.

[0028] In one embodiment, the organic moiety (G2) is an indole, a benzimidazole, a benzothiophene, an isoindole or an indolizine Each of these organic moieties may be substituted or unsubstituted. In another embodiment, G2has a structure of Formula G2-3 A, Formula G2-3B, Formula G2-3C or Formula G2-3D:Formula G2-3A Formula G2-3Bp28Formula G2-3C Formula G2-3Dwherein H”, H12, H13, H14and H15are independently selected from C, S, N and NH provided at least one of H11, H12, H13, H14and H15is selected from S, N and NH. R24, R25, R26, R27, R28, R29and R30are independently selected from H, an alkyl, a halogenated alkyl, an aryl, a primary amine, a secondary amine (NHR6, wherein R6is an alkyl or an aryl), a tertiary amine (NR7, R8wherein R7, R8are independently selected alkyl or aryl), an alcohol, a ether (OR9wherein R9is an alkyl or aryl), a halogen (F, Ci, Br, I), a thiol, a nitro, a nitrile, an amide (C(=O)NR10R11wherein Rwand R11are independently selected from H, alkyl and and), a carboxylic acid, an ester (C(=O)R12wherein R12is an alkyl or aryl), a thioether (SR13wherein R13is an alkyl or aryl), a sulfoxide (S(=O)R14wherein R14is an alkyl or aryl), a sulfone (S(=O)(=O)R1:>wherein Rl3is an alkyl or aryl), a sulfonamide (S(;::O)(:;:O)NRiDR17wherein R16and R1' are independently selected alkyl oraryl), a sulfoximine (SR18(=O)(=NR19) wherein R18and R19are independently selected alkyl or aryl). In one embodiment, one of H12and H13is N, one of H12and H13is C, and H11, H14and Hi5are C. In another embodiment, H12and H1Jare C, one of H11, H14and H15is N and two of Hn, H14and H15are C. In another embodiment, H12and H13are C, one of Hu, H14and H15is S and two of Hu, H14and H15are C.

[0029] In one embodiment, the organic moiety (G2) is a quinoline, an isoquinoline or a quinazoline. Each of these organic moieties may be substituted or un substituted In another embodiment, G has a structure of Formula G2-4A or Formula G2-4B:Formula G2-4A Formula G2-4B

[0030] wherein FIlb, H1', II18, H19, H20, II21, II22and H23are independently selected from C and N provided at least one of H16, H17, H18, H19, H20, H21, H22and H23is N. R31, R32, R3J, R34, R33, R36, R3 / and R38are independently selected from H, an alkyl, a halogenated alkyl, an aryl, a primary amine, a secondary amine (NHR6, wherein R6is an alkyl or an aryl), a tertiary amine (NR7, R8wherein R', R8are independently selected alkyl or aryl), an alcohol, a ether (OR9wherein R9is an alkyl or aryl), a halogen (F, Cl, Br, I), a thiol, a nitro, a nitrile, an amide (C(=O)NR10R11wherein R10and Rnare independently selected from H, alkyl and aryl), a carboxylic acid, an ester (C(=O)R12wherein Ri2is an alkyl or aryl), a thioether (SR1wherein R13is an alkyl or aryl), a sulfoxide (S(=O )R14wherein R14is an alkyl or aryl), a sulfone (S(::O)(;;O)R15wherein R15is an alkyl or aryl), a sulfonamide (S(=O)(=O)NRlbR17wherein R16and R17are independently selected alkylor and), a sulfoximine (SR18(=O)(=NR19) wherein R18and Rlyare independently selected alkyl or aryl). In one embodiment, one of H16, H17, H18, H19, H20, H21, H22and H23is N and seven of Hlb, H1', H18, H!y, H20, H21, H22and H23are C. In another embodiment, two of H16, H17, H18, H19, H20, H21, H22and H23is N and six of H16, H17, H18, H19, H20, H21, H22and H23are C. In one embodiment, G2is an indole selected from 2 -benzoimidazole, 2-indole and 3-indole Examples of organic moieties (G2) include:, where X1is CH or NH.

[0031] In another embodiment, G2is a methoxyphenyl selected from 2 -methoxy phenyl, 3 -methoxy phenyl and 4-methoxy phenyl:

[0032] The ring numbering of purine and pyrimidine nucleotides and ribose is conventional, and the relevant numbers are shown below. Select substituents (e.g. O1, N1) have been omitted from the image below for clarity of illustration, A ribose is shown below but the numbering system also applies to deoxyribose. As used in this specification, the term “ribose” includes ribose, deoxyribose, 2’-OMe-ribose and 2’-F-ribose. G2is connected to the C8 position of a purine by a propyl linker. G2is connected to the C5 position of a pyrimidine by a propyl linker. A ribose or deoxyribose is, in some embodiments, directly connected to N9 of the purine or N1 of a pyrimidine. In some embodiments, the purine or pyrimidine is directly connected to a ribose or deoxyribose at C 1’ of the ribose or deoxyribose and N9 or N 1 of the purine or py rimidine, respectively, thereby forming a nucleoside. In some embodiments, the ribose or deoxyribose includes a phosphoramidite at the C3’ position. In one embodiment, the phosphoramidite is a 2-cyanoethyl-A, Ar-diisopropyl-phosphoramidite that is connected to the oxygen of the C3’ position. The C5’ position of the ribose or deoxyribose is directly connected to O’.oG!o( - O -r1 PO \.f \) / o

[0033] Systematic biochemical evaluation re z:vealed that strategic placement of the resulting nucleotides fine-tuned the susceptibility of DNA duplexes to nucleases and restriction enzymes. This offers a new approach to modulate sequence recognition and stability and demonstrates the utility of the disclosed modified nucleotide precursors. Table 1 provides non-limiting examples of nucleotide precursors.Table 1Compound Structure1APrecursor for uracil with a 3-propyl indole at C5 0^■'NHDMTrO.HA(T AFj> — CNPrecursor for cytosine with a 3-propyl-indole at C5H, A ON-ANHVtrxz x— -\ / / \\N" N \ DMTrO^A-CNPrecursor for guanine with a 3-propyl-indole at C8HN |\ _■__< / ’■ I l4B DMTrOxComparativeExample°Y°— CNY TPrecusor for adenine with a 3-(prop-2-yn-l-yl)-indole at C8

[0034] Conventional solid-phase synthesis was used to produce single-strand DNAs as shown in Table 2A and Table 2B (comparative examples) that used conventional nucleotides (upper case) and / or modified nucleotides (lower case).Table 2A: 3 -propyl -indole modified DNAName Sequence SEQ ID NO.WT TTGAATTCCCGGGTCCAAA SEQ ID NO: 1Z1 TTgAATTCCCGGGTCCAAA SEQ ID NO: 2Z4 TTgaauuCCCGGGTCCAAA SEQ ID NO: 4Z5 TTGAATuCCCGGGTCCAAA SEQ ID NO: 6Z6 TTGAAuuCCCGGGTCCAAA SEQ ID NO: 8cWT G ACC CGGG A ATTC TT SEQ ID NO. 9cZ3 GACCCGGGAAuucTT SEQ ID NO: 10cZ4 GACCCGGGAauucTT SEQ ID NO: 11cZ5 GA C C C GGGaauucTT SEQ ID NO: 12u = Uracil with a 3-propyl-indole at C5c = Cytosine with a 3-propyl-indole at C5g = Guanine with a 3-propyl-indole at C8a = Adenine with a 3-propyl-indole at C8Table 2B (comparative examples): 3-propargyl-indole modified DNAName Sequence SEQ ID NO.Zll TTgAATTCCCGGGTCCAAA SEQ ID NO: 3Z44 TTgaauuCCCGGGTCCAAA SEQ ID NO: 5Z55 TTGAATuCCCGGGTCCAAA SEQ ID NO: 7u = Uracil with a 3-(prop-2-yn-l-yl)-indole at C5c = Cytosine with a 3-(prop-2-yn- l-yl)-indole at C5g = Guanine with a 3 -(prop-2 -yn-l-yl)-indole at C8a = Adenine with a 3 -( prop-2 -yn-l-yl)-indole at C8

[0035] The DNA of Table 2A was used to form duplexes 1-20 (see FIG. 2 A and FIG. 2B, F is a 5’-(6-FAM)-label and Q is a 3’-(Dabcyl)-label). Likewise, the DNA of Table 2B was used to form duplexes 21-23 (see FIG. 3). Standard solid-state chemistry was used on an ABI 394 DNA synthesizer to synthesize the duplexes using coupling times extended to 600 seconds to couple 1A, IB, 2A, 2B, 3A and 4B. No reduction in coupling efficiency was observed for propyl-linked indole-modified nucleoside phosphoramidites 1A, 2A, 3A or 4A. Based on DMT detection, the overall yield of Zl-Z6 and cZ3-cZ5 oligos was approximately 80%-85%, which was comparable to the yield of wild-type (WT) sequences. This indicates modification at C-5 and C-8 using flexible propyl (Pr) linkage does not impact coupling efficiency. However, a decline in the coupling efficiency of propargyl-linked indole modified nucleoside phosphoramidites (IB, 2B, 3B, and 4B, comparative examples), reducing the overall yield to 15%-20% for Z11-Z55 oligonucleotides, respectively.

[0036] Circular dichroism (CD) was used to evaluate the confirmation of duplexes 1- 23. Melting temperature was used to evaluate their stability. See Table 3. Notably, the propargyl linker of duplexes 21-23 did not produce NCCs.Table 3: Analysis of duplexesMelting TempDuplex (°C) Observed CD spectral feature 1 (control) 51 B2 44 Modified B3 39 Modified B4 33 BZ 5 44 BZ 6 41 Modified B7 37 Modified B8 31 Modified B9 43 B10 37 BZ11 36 BZ 12 30 Z 13 39 B 14 35 Modified B15 36 Z 16 32 BZ 17 33 Modified B18 38 Modified B19 39 Modified B20 43 B 21 (comparative example) 42 B22 (comparative example) 34 B23 (comparative example) 33 B

[0037] CD spectra of the duplexes are depicted in FIGs. 4A to 4W. CD spectra were recorded at 25 °C using a 10 mm pathlength quartz cuvette in the range of 220 nm to 330 nm with a scanning speed of 20 nm per min, 1 nm bandwidth, 8s response time, and three accumulations. Modified B-DNA: B-DNA structure that exhibits deviations in molar ellipticity at 280 / 245 nm, as detected by CD spectroscopy, indicating alterations to the canonical B-form while retaining overall B-DNA characteristics.

[0038] Unusual spectral transitions were observed across all duplexes with increasing levels of propyl-linked indole modification, characterized by varying degrees of decreased or increased molar ellipticity near 280 / 245 nm. This is indictive of the formation of unusual B-DNA conformations, potentially comprising modified B-DNA forms. For example, as the number propyl-linked indole modifications increased, the conformation of duplexes 2-4 progressively shifted from the B-form to a BZ-like conformation). This trend continued in duplexes 5-8. Duplex 5 adopted a BZ conformation, while duplexes 6-8 adopted modified B-form and progressively lower melting temperatures, correlating with the number of propyl-linked indole modifications on the complementary'' strand. Duplexes 9-12 showed a structural transition from B-form in the least propyl-linked indole modified duplex 9 to BZ in duplexes 10-11 and ultimately to Z-form in duplex 12, indicating that the introduced uridine and adenine propyl-linked indole modifications on the complementary strand had influenced bothduplex stability and conformation. Duplexes 13—16 followed a similar consistent trend, wherein the minimally modified duplex adopted a canonical B-form and exhibited the highest thermal stability. However, progressive incorporation of propyl-linked indole modifications on the complementary strand induced a conformational shift from distorted B-form to BZ and ultimately Z-DNA, correlating with a stepwise reduction in melting temperature. These findings further validate that increased propyl-linked indole modification density drives conformational transitions and destabilizes duplex thermal stability.

[0039] Two propyl-linked indole modified uracil that are adjacent are present in duplexes 13-16, and those duplexes both exhibit the lowest melting temperatures and adopt more distorted conformations. Given that the duplexes contain a palindromic d(AAUU) sequence in the EcoRi site, the propyl-linked indole modifications introduced at the dU and dA positions may have derived conformational inversion even in the absence of high salt, likely owing to the steric effects of the bulky indole moiety forcing this conformational adaptation. Duplexes 17-20, which contained the highest density of propyl-linked indole modified bases on the template strand, exhibited an inverse trend whereby the least-modified duplex (17) showed the lowest melting temperature, whereas the most extensively propyl-linked indole modified duplex (20) displayed the least destabilizing effect. With increasing number of propyl-linked indole modifications introduced on the complementary' strand, duplex conformation progressively shifted from a distorted B-form to a canonical B-form, indicating increased structural stabilization with greater modification density on both strands. This suggests that the successive incorporation of propyl-linked indole modified dAs contributed to the enhanced duplex stability. Additional propyl-linked indole dG modifications did not lead to substantial reductions in melting temperature. These findings may indicate that propyl-linked indole dU and propyl-linked indole dA have a greater impact on conformational transitions. Overall, the melting temperature i s most strongly impacted when the 3’-d(CUUAA)-5’ sequence is modified with propyl-linked indole.

[0040] Duplexes 21-23, containing propargyl-linked indole modifications, adopted a canonical B-form. Increasing number of propargyl-linked indole modifications on the template strand resulted in a progressive decrease in melting temperature.

[0041] Enzymatic Experiments: Because most NCC of nucleic acids and modified nucleic acids are resistant to DNase I, the sensitivity of the duplexes towards DNase I cleavage was assessed. DNase I is an endonuclease that nonspecifically, but preferentially, cleaves double-stranded B-DNA at 3' hydroxyl and 5' phosphoryl nucleotides using a single-strand nicking mechanism. Despite the known high promiscuity of DNA duplexes towards nuclease activity mediated by DNase I, sensitivity of the modified duplexes towards DNase I was observed to vary based on the position of the propyl-linked indole modification. For example, the number of propyl-linked indole modifications on the complementary strand of duplexes 1-4 was strategically increased one base at a time. Results showed a progressive reduction in DNase I sensitivity with increasing propyl-linked indole modification density. Among all duplexes, except duplex 17, the least propyl-linked indole-modified duplex exhibited the highest susceptibility to DNase I, but enzymatic sensitivity significantly decreased as additional propyl-linked indole modifications were introduced, either on the template strand or the complementary strand.

[0042] Significant nuclease resistance was observed in duplexes 4, 8, 12, 16, and 17-20. Among duplexes adopting distorted B-form conformations, DNase I sensitivity varied depending on the position and number of propyl-linked indole modifications. For instance, duplexes 6-8, with stepwise introduction of propyl-linked indole modifications on the complementary strand, demonstrated progressively increased resistance to DNase I. Notably, when the template strand was modified with propyl-linked indole, all corresponding duplexes exhibited substantial resistance to enzymatic cleavage, irrespective of modification pattern. The duplexes containing Z6, except for duplex 13, significantly deviated in melting points with greater deviations from the B-DNA structure, showed a lower promiscuity towards DNase I. While DNase I exhibits peakendonuclease activity at 37°C, it is also capable of cleaving DNA at a lower rate, maintaining its activity at room temperature, albeit with reduced efficiency.

[0043] Since the melting temperatures of duplexes 15 and 12 are below 37°C, their resistance to DNase I cleavage was investigated to determine if this resulted from partial melting into single-stranded structures or the adoption of Z conformation. When DNase I activity was assessed at 25 °C, no change in DNase I resistance was observed for duplexes 12 and 15 compared to that of the wild-type duplex under the same conditions.

[0044] Promiscuity of the duplex towards restriction-modification (R-M) enzymes was also assessed. R-M enzymes show both nuclease and methylation activities that resemble primitive immune systems that destroy foreign DNA in bacteria. R-M enzymes are programmed to protect host chromosomes by recognizing and cutting foreign DNA from invading viruses. EcoRI is known to first scan DNA duplexes in one dimension to find its cleavage site and then cleave between G and A in a d(GAATTC) palindrome. A 2-D NMR analysis of dodecamer structure with an EcoRI site showed that structural anomalies, such as kinks or increased flexibility of a duplex, facilitate scission.Modified bases were strategically introduced at the EcoRI recognition site, ranging from one to five modifications on both the template and complementary strands. Similar to DNase I, when PrLI modifications were introduced on the complementary strand, at the EcoRI recognition site, duplexes 1-4 exhibited a progressive decrease in EcoRI sensitivity with increasing modification density EcoRI did not, however, digest all duplexes containing PrLI modified restriction site except for duplex 2. This resistance is expected, as the modifications were introduced directly at the restriction site. Similarly, resistivity of the duplex was increased when the template strand was modified with 1B-4B. This line of evidence suggests that the PrLI modification induced steric effects by the indole moiety may abolish the interaction between the duplex and EcoRI. To assess whether the reduced melting temperatures contributed to EcoRI resistance in duplexes 12 and 15, cleavage efficiency at 25 °C was evaluated by examining site-specific digestion. No significant differences in cleavage patterns were observed.

[0045] A modification to DNA was introduced in close proximity to a specific DNA sequence recognized by a restriction enzyme and evaluated the effect on scission. The susceptibility of the duplexes to Smal and Xmal cleavage also appears to be influenced by the position of PrLI modifications, even when they are not directly located within the recognition or cleavage sites. For example, duplexes 17-20, which contain the highest density of PrLI modifications on both the template and complementary strands, exhibited the lowest susceptibility to Smal and Xmal. In contrast, all other duplexes displayed varying degrees of enzyme tolerance, depending on the number and position of the introduced modifications. For instance, duplex 12 exhibited the lowest susceptibility to Xmal / Smal cleavage. To evaluate whether this resistance was temperature-dependent, enzymatic activity at 25 °C and assessed and observed no significant differences in cleavage pattern. Smal and Xmal are isoschizomers, i.e., restriction enzymes that recognize the same DNA sequence, such as CCCGGG, but may not cut at the same location. Xmal cleaves between the external cytosines, while Smal cleaves between CG in the CCCGGG to introduce blunt end scission. Both endonucleases have been shown to form a stable, specific complex with DNA. However, the landing of endonuclease on the duplex leads to the bending of DNA such that Smal bends the DNA towards the major groove, similar to EcoRI, while Xmal bends the DNA towards the minor groove. This resistance occurs despite the modifications being positioned adjacent to the restriction site, suggesting that downstream modifications may influence cleavage of upstream restriction sites. A profound loss of sensitivity towards Xmal and Smal was observed for duplexes modified with IB, 2B, 3B, and 4B showing that resistivity gradually increased with the increasing number of propargyl-linked indole modifications. These results suggests that rigidity of the propargyl linker impacted the enzyme recognition site more strongly, even though the modification was not positioned directly on the restriction site. All duplexes 21, 22 and 23, containing propargyl linked indole, were resistant to DNase I, EcoRI, and Xmal, despite the degree or site of modification. However, the promiscuity of these duplexes towards Smal slightly deviates from thistrend For example, duplex 23 shows lower resistance to Smal compared to duplexes 21 and 22.

[0046] For each assay, the 10 pmol duplex samples were prepared by incubating 10 pmol of fluorophore-labelled oligonucleotide sequence with 25 pmol of quencher-labelled complementary oligonucleotide sequence (sample volume: 10 µL). After addition of respective endonuclease, the reaction samples were incubated for the specified amount of time in a TC 9639 Thermal Cycler (Benchmark). For DNase I digestion assays, the duplex samples were treated with 1.4 units of DNase I (Thermo Scientific, REF# EN0521, 0.56 u / pL). The reactions were incubated for 15 minutes at 37 °C For EcoRl digestion assays, the duplex samples were treated with 70 units of EcoRI- HF (NEB, R3101M, 28 u / pL). The reactions were incubated overnight in a thermocycler at 37°C. For Xmal digestion assays, the duplex samples were treated with 7 units of Xmal (NEB, R0180S, 2.8 u / pL). The reactions were incubated overnight at 37°C. For Smal digestion assays, the duplex samples were treated with 7 units of Smal (Thermo Scientific, REF# ER0665, 2.8 u / pL) at 25°C. To assess enzymatic digestions in all endonuclease digestion assays, the treated duplex samples were diluted with 185 µL of 1X PBS (Duplex Cf= 50 nM) post-incubation, and fluorescence measurement was performed at 20 °C. The emission wavelength range utilized for measuring treated duplex samples was, λem=500 to 600 nm, and the excitation wavelength was λex=488 nm, with a 2.5nm slit width. An unmodified, wild-type duplex used as the control, and was measured alongside each modified duplex, to ensure consistency in conditions between each experiment.

[0047] Data Analysis for Bar Diagrams: FRET data analysis for endonuclease assays included baseline correction and normalization. During the baseline correction step, background fluorescence intensity at λmax514 nm (at 20°C) from the untreated sample of each duplex was subtracted that of the corresponding enzyme-treated sample. The corrected fluorescence values were then normalized to the wild-type (WT) duplex using the following equation:Normalized Fluorescence Intensity = - - - - - - r x 100y(WTd} - (WTe)wherein Feis fluorescence intensity of modified duplex with enzyme, Fdis fluorescence intensity of modified duplex without enzyme, WTeis fluorescence intensity of wild type duplex with enzyme and WTdis fluorescence intensity of wild type duplex without enzyme. The average of the normalized values from three replicates was calculated for each duplex and used to generate bar diagrams providing a visual representation of each duplex that showed relative susceptibility to endonuclease activity compared to that of WT. The resulting graphs are depicted in FIGs. 5 A to 5H which provide a comparative analysis of the promiscuity of duplexes 1-23 towards DNase I (A-B), EcoRI (C-D), Xmai (E-F), and Smal (G-H). Enzymatic assays were performed using FRET assays with a fluorophore-labeled template strand and a quencher-labeled complementary strand. Each duplex was incubated at 37°C with corresponding enzyme, and enzymatic activity was analyzed using FRET at 20°C using a fluorescence spectrometer. Fluorescence intensity measured was normalized against the control wild-type duplex 1.

[0048] Materials and methods: All chemicals and solvents were purchased from Sigma-Aldrich, Fisher Scientific, Oakwood Chemicals and Ambeed Chemicals and used without further purification. Reactions were monitored by thin-layer chromatography (TLC) using Merck precoated silica plates (Silica Gel 60 F254, 0.25 mm). TLC plates were visualized under ultraviolet light at 254 nm and by charring using ceric ammonium molybdate and p-anisaldehyde solutions. Chromatography was performed on a Teledyne ISCO CombiFlash Rf 200i using disposable silica cartridges. ¹H,13C and31P NMR spectra were acquired on an OXFORD NMR AS500 (1H at 500.0 MHz, 13C at 126.0 MHz, and 31P at 202.0 MHz). Residual solvent peaks were used as internal references and expressed in parts per million (ppm). Spin multiplicities were represented as s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), ddd (doublet of doublet of doublet), dt (doublet of triplet), m (multiplet) and brs (broad singlet), whilecoupling constants (J) are given in Hertz. Mass spectra were recorded by either the Proteomics Facility at the Advanced Science Research Centre, CUNY or Novatia, LLC.

[0049] Solid-phase synthesis of DNA sequences and purification: All DNA reagents for DNA synthesis were purchased from Glen Research. Wild type (WT) and complementary wild type (cWT) DNA sequences were purchased from Integrated DNA Technologies. All modified DNA sequences were synthesized by following standard solid-phase DNA synthesis protocols on an AB 1394 DNA synthesizer (Biolytics) using a 0.2 pmol scale. Synthesis was performed in standard mode using 2-cyanoethyl-N, N-diisopropylphosphoramidites. A 0.1 M solution of each phosphoramidite and 0.25 M 5-Ethylthio-l-H-Tetrazole, as an activator, were used for DNA synthesis. Iodine solution (0.02 M Iodine in THF / Py / Water) was used for oxidizing phosphoramidites. The reaction volume and duration for coupling natural phosphoramidites were 220 pL and 90 seconds, respectively, while for modified phosphoramidites, the reaction volume was kept at 220 pL, while the coupling time was extended to 600 seconds. The DNA deprotection was carried out by incubating DNA on CPG with 30% aq. NH4OH (37°C), 24 h). DNA sequences were purified using high-performance liquid chromatography (Agilent) equipped with a C-18 reverse-phase column (Phenomenex). Chromatography used 0.1 M Triethylammonium Acetate (TEAA) and acetonitrile as mobile phase, and a linear gradient from 5% to 100% acetonitrile over 65 minutes was used to elute oligonucleotides. A UV-vis spectrophotometer (Agilent) was employed to quantify the purified DNA to obtain the stock concentration.

[0050] CD Spectral analysis: Circular dichroism (CD) measurements were conducted using a Jasco-1500 Circular Dichroism spectrometer. The data were collected using a quartz cuvette with a 10 mm pathlength, in the wavelength range of 220 nm to 330 nm, with a data pitch of 2 nm, an integration time of 8 seconds, a spectral bandwidth of 1 nm, and a scanning speed of 20 nm / min. Three accumulations were averaged for each spectrum. A blank measurement was first taken with 400 µL of PBS buffer 200 pmol of DNA samples in 400 pL of PBS buffer were analyzed. Blank spectra weresubtracted from the sample spectra, which were subsequently smoothed and zeroed at 320 nm.

[0051] Melting Point Measurements: To analyze the thermal stability of the duplexes, 50 nM of each duplex were prepared by incubating 10 pmol of fluorescently labelled template oligonucleotide sequence with 25 nmol of dabcyl labelled complementary oligonucleotide sequence in 200 pL of PBS (GIBCO™ IX PBS, pH::=7.4). Each duplex sample was placed into a reduced volume quartz cuvette (Horiba, path length=3mm), and fluorescence intensity was measured using the FluoroMax Plus spectrofluorometer (Horiba) with a TCI temperature controller (Quantum Northwest) and an EXT -440 liquid cooling system (Koolance). The emission wavelength utilized in the thermal stability assays correlated with fluoresceine (2em=514 nm), with the excitation wavelength at Acz=488 nm, and slit width of 2.5 nm. Measurements were taken at 1 °C) intervals (tolerance=±0.5°C), across a temperature range of 5 °C)to 70 °C).

[0052] The precursor nucleoside phosphoramidites were synthesized via classical Sonogashira cross-coupling reaction, followed by hydrogenation, protection of hydroxyl and amine groups, and subsequent conversion to phosphoramidites.

[0053] A variety of organic moiety (G2) y be appended to the nitrogenous base using conventional synthetic chemistry. For example, a halogenated nitrogenous base may be coupled to 3-(prop-2-yn-l-yl)-G2moiety.

[0054] One of ordinary skill in the art, after benefitting from reading this disclosure, would be able to generate a variety of nucleosides. The following are non-limiting examples.

[0055] Synthesis of precursors 1 and IB

[0056] Synthesis of 5-(3-(l / 7-indol-3-yl)prop-l -yn-l-y])-l -((2R,4S,5R)-4-hydroxy-5 (hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(17 / ,3 / / )-dione ( )■

[0057] To a solution of 5-iododeoxyuridine (5) (3.0 g, 8.50 mmol) and 3-(prop-2-yn-l-yl)-l / / -indole (1.7 g, 11.0 mmol) were added to a mixture of Pd(PPli3)4 (0.98 g, 0.85 mmol) and Cui (0.48 g, 2.50 mmol) in anhydrous DMF (30 mL) under a Ar atmosphere in a flask equipped with a gas inlet tube and a magnetic stirrer. Then pump / purge cycles were applied with the addition of Ar gas Et₃N (5.90 mL, 42.0 mmol) was added and then the mixture was heated at 55 °C for 2 h. The progress of the reaction was followed using TLC. After completion of the reaction, evaporated under reduced pressure and the crude product was purified by flash chromatography (0-10% MeOH in CH2CI2). The desired compound 6 (2.3 g, 6.0 mmol, 71%) was obtained as a white solid.

[0058] Synthesis of 5-(3-(l / / -indol-3-yl)propyl)-l-((2R,4S,5R)-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3 / / )-dione (7):

[0059] To a solution of compound 6 (2.0 g, 5.2 mmol) in methanol (40 mL), 10% Pd / C (0.56 g, 0.52 mmol) was added under stirring. The reaction mixture was then stirred under hydrogen atmosphere at 50 °C for 12 h. After completion of the reaction, the reaction mixture was filtered through celite to remove the catalyst. The filtrate was evaporated under reduced pressure to yield the pure compound 7 (1.5 g, 3.9 mmol, 74 %) as a white solid.

[0060] Synthesis of 5-(3-(lJT-indol-3-yl)propyl)-l-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyr)methoxy)methyl)-4-hydroxytetrahydrofiiran-2-yl)pyrimidine-2,4(17 / ,37 )-dione (8):

[0061] To a solution of compound 7 (1.0 g, 2.6 mmol ) in dry pyridine (15 mL), DMAP (32 mg, 0.26 mmol) was added. To this mixture, a solution of 4, ’dim ethoxy trityl chloride (1 1 g, 3.1 mmol) in 5 mL, of anhydrous pyridine was added in four equal portions over the time of 1 h. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the solvent was evaporated under reduced pressure. The resulting crude mixture was purified by flash chromatography (0- 2% MeOH in CH2CI2 with 0.2% of EtjN) yielding the desired compound S (0,90 g, 1,31 mmol, 50%) as a yellow foam.

[0062] Synthesis of (2R,3S,5R)-5-(5-(3-(l / 7-indol-3-yl)propyl)-2,4-dioxo-3,4-dihydropyrimidin- 1 (277)-yl )-2-((bi s(4 -methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphorami dite (1 ):

[0063] To a solution of compound 8 (0.6 g, 0.87 mmol) in anhydrous CH₂Cl₂, N, N-diisopropylethylamine (0.75 mL, 4.36 mmol) was added under stirring. The reaction mixture was cooled to 0 °C, and 2-cyanoethyl- / V, / V-diisopropylchlorophosphoramidite (0.3 mL, 1.40 mmol) was added under an argon atmosphere. The mixture was allowed to warm to room temperature and stirred for 1.0 h. The reaction mixture was then diluted with anhydrous CH2CI2 (30 mL) and washed successively with 5% NaHCCh (30 mL) and brine solution (30 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography using (10-60% EtOAc in hexane containing 0.5% EtsN) as the eluent. The desired compound 1 A was obtained as a 1:1,3 mixture of two diastereomers in the form of a white foam (0.52 g, 0.6 mmol, 68% yield).

[0064] Synthesis of 5-(3-(l / 7-indol-3-yl)prop-l-yn-l-yl)-l-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl (methoxy )methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimi dine-2,4(17 / ,377)-dione (9):OH

[0065] To a solution of compound 6 (1.0 g, 2,6 mmol) in dry pyridine (15 mL), DMAP (32 mg, 0.26 mmol) was added under stirring. To this mixture, a solution of 4,4’-dimethoxytrityl chloride (1.1 g, 3.1 mmol) in 5 mL of anhydrous pyridine was added in four equal portions over the time of 1 h. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the solvent was evaporated under reduced pressure. The resulting crude mixture was purified by flash chromatography (0- 2% MeOH in CH2CI2, with 0.2% of EtsN) yielding the desired compound 9 (0.95 g, 1.39 mmol, 53%) as a yellowish foam.

[0066] Synthesis of (2R,3S,5R)-5-(5-(3-(l / / -indol-3-yl)prop-l-yn-l-yl)-2,4-dioxo- 3,4-dihydropyrimidin-l(2J )-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cy anoethyl) diisopropylphosphoramidite (1 B):

[0067] To a solution of compound 9 (0.5 g, 0.73 mmol) in anhydrous CH2CI2, N, N- diisopropylethylamine (0.63 mL, 3.66 mmol) was added under stirring. The reaction mixture was cooled to 0 °C, and 2-cyanoethyl-AC'V-diisopropylchlorophosphoramidite (0.25 mL, 1.2 mmol) was added under an argon atmosphere. The mixture was allowed to warm to room temperature and stirred for 1.0 h. The reaction mixture was then diluted with anhydrous CH2CI2 (30 mL) and washed successively with 5% NaHCCL (30 mL) and brine solution (30 mL). The organic phase was dried over anhydrous Na2SCL and concentrated under reduced pressure The crude product was purified by column chromatography using (10-50% EtOAc in hexane containing 0.5% ELN) as the eluent. The desired compound IB was obtained as a 1:1.2 mixture of two diastereomers in the form of a white foam (037 g, 0.43 mmol, 58% yield).

[0068] Synthesis of precursors 2A and 2B

[0069] Synthesis of 5-(3-(lJ7-indol-3-yl)prop-l-yn-] -yl)-4-amino-l-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(l / / )-one (11):

[0070] To a solution of 5-iodo-2'-deoxycytidine (10) (2.0 g, 5.66 mmol) and 3-(prop-2-yn-l-yl)-177-indole1(1.14 g, 7.36 mmol) were added to a mixture of Pd(PPh3)4 (0.66 g, 0.57 mmol) and Cui (0.32 g, 1.70 mmol) in anhydrous DMF (30 mL) under a Ar atmosphere in a flask equipped with a gas inlet tube and a magnetic stirrer. Then pump / purge cycles were applied with the addition of Ar gas Et₃N (3.95 mL, 28.3 mmol)was added and then the mixture was heated at 55 °C for 1.5 h. The progress of the reaction was followed using TLC. After completion of the reaction, evaporated under reduced pressure and the crude product was purified by flash chromatography (0-10% MeOH in CH2CI2). The desired compound 11 (1.5 g, 3.9 mmol, 70%) was obtained as a white solid.

[0071] Synthesis of 5-(3-(177-indol-3-yl)propyl)-4-amino-l-((2R,4S,5R)-4-hydroxy-5 (hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(l / / )-one (12):12

[0072] To a solution of compound 11 (1.3 g, 3.4 mmol) in methanol (40 mL), 10% Pd / C (0.36 g, 0.34 mmol) was added under stirring. The reaction mixture was then stirred under a hydrogen atmosphere at 50 °C for 12 h. The progress of the reaction was followed using TLC. After completion of the reaction, the reaction mixture was filtered through celite to remove the catalyst. The filtrate was evaporated under reduced pressure to yield the pure compound 12 (1.1 g, 2.9 mmol, 84 %) as a white solid.

[0073] Synthesis of 5-(3-(l / f-indol-3-yl)propyr)-4-amino-l-((2R,4S,5R)-5-((bis(4 methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidin-2(l / / )-one (13):

[0074] To a solution of compound 12 (1.0 g, 2.6 mmol) in dry pyridine (15 mL), DMAP (32 mg, 0.26 mmol) was added. To this mixture, a solution of 4,4’-dimethoxytrityl chloride (1.1 g, 3 1 mmol) in 5 mL of anhydrous pyridine was added in four equal portions over the time of 1 h. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the solvent was evaporated under reduced pressure. The resulting crude mixture wus purified by flash chromatography using (0-2% MeOH in CH2CI2, with 0.2% of Et₃N) yielding the desired compound 13 (0.97 g, 1.41 mmol, 54%) as a yellowish foam.

[0075] Synthesis ofN-(5~(3-(l / / -indol-3-yl)propyl)-l-((2R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyrimidin-4-yl)acetamide (14):

[0076] To a solution of compound 13 (0.85g, 1.24 mmol) in dry DMF (12 mL), acetic anhydride (0.13 mL, 1.36 mmol) was added dropwise over 15 minutes at 0 °Cunder an argon atmosphere The reaction mixture was then stirred at room temperature for 8 h. Progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The crude product was purified by flash chromatography using (0-2% MeOH in CH2CI2, with 0.2% of Et., N) yielding the desired compound 14 (0.55 g, 0.76 mmol, 61%) as a pale-yellow solid.

[0077] Synthesis of (2R,3S,5R)-5-(5-(3-(177-indol-3-yl)propyl)-4-acetamido-2-oxopyri midin- l(2 / 7)-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl (2-cy anoethyl) diisopropylphosphoramidite (2A):

[0078] To a solution of compound 14 (0.4 g, 055 mmol) in anhydrous CH2CI2, N, N-diisopropylethylamine (0.47 mL, 274 mmol) was added under stirring. The reaction mixture was cooled to 0 °C, and 2-cyanoethyl-A7, A diisopropylchlorophosphoramidite (0.19 mL, 0.88 mmol) was added under an argon atmosphere. The mixture was allowed to warm to room temperature and stirred for 1.5 h. The reaction mixture was then diluted with anhydrous CH2Cl2 (20 mL) and washed successively with 5% NaHCCE (20 mL) and brine solution (20 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography using (10-70% EtOAc in hexane containing 0,5% EtsN) as the eluent. The desired compound 2A was obtained as a 1: 1.4 mixture of two diastereomers in the form of a white foam (0.28 g, 0.31 mmol, 56% yield).

[0079] Synthesis of 5-(3-(lJ / -indol-3-yl)prop-l-yn-l-yl)-4-amino-l-((2R,4S,5R)-5- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidin-2(l / / )-one (15):

[0080] To a solution of compound 11 (1.1 g, 2.9 mmol) in dry pyridine (10 mb), DMAP (35 mg, 0.29 mmol) was added under stirring. To this mixture, a solution of 4,4’-dimethoxytrityl chloride (1.2 g, 3.5 mmol) in 5 mL of anhydrous pyridine was added in four equal portions over the time of 1 h. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the solvent was evaporated under reduced pressure. The resulting crude mixture was purified by flash chromatography using (0~2% MeOH in CH2CI2, with 0.2% of EtsN) as the eluent, yielding the desired compound 15 (1.1 g, 1.6 mmol, 56%) as a white solid.

[0081] Synthesis of N-(5-(3-(lH-indol-3-yl)prop-l-yn-l-yl)-l -((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyrimidin-4-yl)acetamide (16):

[0082] To a solution of compound 15 (1.0 g, 1.5 mmol) in dry DMF (15 mL), acetic anhydride (0.15 mL, 1.6 mmol) was added dropwise over 15 minutes at 0 °C under an argon atmosphere. The reaction mixture was then stirred at room temperature for 10 h. Progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The crude product was purified by flash chromatography (0-2% MeOH in CH2CI2, with 0.2% of EtsN) as the eluent, yielding the desired compound 16 (0.6 g, 0.83 mmol, 57%) as a pale-yellow solid.

[0083] Synthesis of (2R,3S,5R)-5-(5-(3-(l / 7-indol-3-yl)prop-l-yn-l-yr)-4-acetamido-2-oxopyrimidin-l(2 / / )-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (2B):

[0084] To a solution of compound 16 (0.3 g, 0.41 mmol) in anhydrous CH2CI2, N,N-diisopropylethylamine (0.36 mL, 2.1 mmol) was added under stirring. The reaction mixture was cooled to 0 °C, and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.14 mL, 0.66 mmol) was added under an argon atmosphere. The mixture was allowed to warm to room temperature and stirred for 1.5 h. The reaction mixture was then diluted with anhydrous CH2CI2 (20 mL) and washed successively with 5% NaHCO3 (20 mL) and brine solution (20 mL). The organic phase was dried over anhydrous Na2SC>4 and concentrated under reduced pressure The crude product was purified by column chromatography using (10-60% EtOAc in hexane containing 0.5% EhN) as the eluent. The desired compound 2B was obtained as a 1:1.1 mixture of two diastereomers in the form of a white foam (0.24 g, 0.26 mmol, 64% yield).

[0085] Synthesis of precursors 3A and 3B

[0086] Synthesis of 8-(3-(lH-indol-3-yl)prop-l-yn-] -yl)-2-amino-9-((2R,4S,5R)-4-hydroxy-5-hydroxymethyl)tetrahydrofuran-2-yl)- 1,9-dihydro-6H-purin-6-one (18):18

[0087] To a solution of 8-Bromo-2'-deoxyguanosine (17) (2.0 g, 5.78 mmol) and 3-(prop-2-yn-1-yl)-1H-indole1(1.17 g, 7.51 mmol) were added to a mixture of Pd(PPli3)4 (0.67 g, 0.58 mmol) and Cui (0.33 g, 1.73 mmol) in anhydrous DMF (40 mL) under a Ar atmosphere in a flask equipped with a gas inlet tube and a magnetic stirrer. Thenpump / purge cycles were applied with the addition of Ar gas Et N (4.03 mL, 28.9 mmol) was added and then the mixture was heated at 60 °C for 3 h. The progress of the reaction was followed using TLC. After completion of the reaction, evaporated under reduced pressure and the crude product was purified by flash chromatography (0-15% MeOH in CH2CI2). The desired compound 18 (1.4 g, 3.3 mmol, 58%) was obtained as a brown solid.

[0088] Synthesis of 8-(3-(lJTf-indol-3-yl)propyl)-2-amino-9-((2R,4S,5R)-4-hydroxy- 5-(hydroxymethyl)tetrahydrofuran-2-yl)- 1,9-dihydro-6H-purin-6-one (19):

[0089] To a solution of compound 18 (1.3 g, 3.1 mmol) in methanol (60 mL), 10% Pd / C (0.33 g, 0.31 mmol) was added under stirring. The reaction mixture was then stirred under a hydrogen atmosphere at 50 °C for 12 h. The progress of the reaction was followed using TLC. After completion of the reaction, the reaction mixture was filtered through celite to remove the catalyst. The filtrate was evaporated under reduced pressure to yield the pure compound 19 (1.0 g, 2.4 mmol, 76 %) as a pale-yellow solid,

[0090] Synthesis of (E)-N'-(8-(3-(l / / -indol-3-yl)propyl)-9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-177-purin-2-yl)-N, N-dirnethylformimi damide (20)

[0091] To a solution of compound 19 (0.80 g, 1.88 mmol) in anhydrous DMF (10 mL) under argon atmosphere, dimethylformamide dimethylacetal (5.0 mL, 37.7 mmol) was added under stirring. The reaction mixture was continued to stir at room temperature for 1 h. The progress of the reaction was followed using TLC. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by flash chromatography using (0-10% MeOH in DCM) as eluent. The pure product 20 (0.66 g, 1.4 mmol, 73%) was obtained as a white solid.[00921 Synthesis of (E)-N'-(8-(3-(l / / -indol-3-yl)propyl)-9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl )m ethoxy )methyl)-4-hydroxytetrahydrofuran-2-yl)-6-oxo-6, 9-diliydro-l / / -purin-2-yl)-N, N-dimethylformimidamide (21):

[0093] To a solution of compound 20 (0.5 g, 1.04 mmol) in dry pyridine (10 mL), DMAP (13 mg, 0.10 mmol) was added. To this mixture, a solution of 4,4’-dimethoxytrityl chloride (0.42 g, 1.25 mmol) in 3 mL of anhydrous pyridine was added in four equal portions over the time of 1 h. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the solvent was evaporated under reduced pressure. The resulting crude mixture was purified by flash chromatography (0- 5% MeOH in CH2CI2, with 0.2% of Et₃N) as the eluent, yielding the desired compound 21 (0.43 g, 0.55 mmol, 53%) as a pale-yellow solid.

[0094] Synthesis of (2R,3S,5R)-5-(8-(3-(U / -indol-3-yl)propyl)-2-(((E)- (dimethylamino)methylene)amino)-6-oxo-L6-dihydro-9H-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl)dii sopropylphosphorami di te (3A):

[0095] To a solution of compound 21 (0.35 g, 0.45 mmol) in anhydrous CH2CI2, N,N-diisopropylethylamine (0.39 mL, 2.24 mmol) was added under stirring. The reaction mixture was cooled to 0 °C, and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.15 mL, 0.72 mmol) was added under an argon atmosphere. The mixture was allowed to warm to room temperature and stirred for 1.5 h. The reaction mixture was then diluted with anhydrous CH2Cl2 (20 mL) and washed successively with 5% NaHCO3 (20 mL) andbrine solution (20 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting crude mixture was purified by flash chromatography (10-60% acetone in hexane containing 0.5% EtsN) as the eluent. The desired compound 3A was obtained as a 1:1.1 mixture of two diastereomers in the form of a pale-yellow foam (0.24 g, 0.22 mmol, 55% yield).

[0096] Synthesis of (E)-N'-(8-(3-(l / 7-indol-3-yl)prop-l-yn-l-yl)-9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-l / -purin-2-yl)-N, N-dimethylfoirnimidamide (22):

[0097] To a solution of compound 18 (0.7 g, 1.66 mmol) in anhydrous DMF (8 mL) under argon atmosphere, dimethylformamide dimethylacetal (4.4 mL, 33.3 mmol) was added under stirring. The reaction mixture was stirred at room temperature for 1 h. The progress of the reaction was followed using TLC. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by flash chromatography using (0-10% MeOH in DCM as the eluent. The pure compound 22 (0.52 g, 1.1 mmol, 66%) was obtained as a pale-yellow solid.

[0098] Synthesis of (E)-N'-(8-(3-(lJ7-indol-3-yl)prop-l-yn-l -yl)-9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-l / / -purin-2-yl)-N, N-dimethylformimidamide (23):

[0099] To a solution of compound 22 (0.4 g, 0.84 mmol) in dry pyridine (8 mL), DMAP (10 mg, 0.1 mmol) was added under stirring. To this mixture, a solution of 4,4’-dimethoxytrityl chloride (0.34 g, 1.01 mmol) in 4 mL of anhydrous pyridine was added in four equal portions over the time of 1 h. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the solvent was evaporated under reduced pressure. The resulting crude residue was purified by flash chromatography (0-5% MeOH in CH2CI2, with 0.2% of EtsN) as an eluent, yielding the desired compound 23 (0.38 g, 0.49 mmol, 58%) as a pale-yellow form.

[0100] Synthesis of (2R,3S,5R)-5-(8-(3-(l / / -indol-3-yl)prop-l-yn-l-yl)-2-(((E)-(dimethylamino)methylene)amino)-6-oxo-l,6-dihydro-9 / 7-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl)dii sopropylphosphorami di te (3 B):

[0101] To a solution of compound 23 (0.3 g, 0.39 mmol) in anhydrous CH2Cl2, N, N-diisopropylethylamine (0.34 mL, 1.93 mmol) was added under stirring. The reaction mixture was cooled to 0 °C, and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.13 mL, 0.62 mmol) was added under an argon atmosphere. The mixture was allowed to warm to room temperature and stirred for 1.5 h. The reaction mixture was then diluted with anhydrous CH2Cl2 (20 mL) and washed successively with 5% NaHCO3 (20 mL) and brine solution (20 mL). The organic phase was dried over anhydrous Na SCh and concentrated under reduced pressure. The crude residue was purified by column chromatography using (10—50% acetone in hexane containing 0.5% Et₃N) as the eluent. The desired compound 3B was obtained as a 1:1.2 mixture of two diastereomers in the form of a pale-yellow foam (0.26 g, 0.27 mmol, 69% yield),

[0102] Synthesis of precursors 4A and 4B

[0103] Synthesis of (2R,3S,5R)-5-(8-(3-(lJ¥-indol-3-yl)prop-l-yn-l-yl)-6-amino- 9 / / -purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol (25):

[0104] To a solution of 8-Bromo-2’-deoxyadenosine (24) (2.0 g, 6.1 mmol) and 3-(prop-2-yn-l-yl)-l / / -indole (1.2 g, 7.90 mmol) were added to a mixture of Pd(PPhj)4 (0.7 g, 0.61 mmol) and Cui (0.35 g, 1.8 mmol) in anhydrous DMF (30 mL) under a Ar atmosphere in a flask equipped with a gas inlet tube and a magnetic stirrer. Then pump / purge cycles were applied with the addition of Ar gas Et₃N (3.4 mL, 24.0 mmol) was added and then the mixture was heated at 55 °C for 2 h. The progress of the reaction was followed using TLC. After completion of the reaction, evaporated under reduced pressure and the crude product was purified by flash chromatography (0-10% MeOH in CH2Cl2). The desired compound 25 (1.4 g, 3.5 mmol, 57%) was obtained as a brown solid.

[0105] Synthesis of (2R,3S,5R)-5-(8-(3-(1 -indol-3-yl)propyl)-6-amino-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3 -ol (26):

[0106] To a solution of compound 25 (1.3 g, 3.2 mmol) in methanol (40 mL), 10% Pd / C (0.34 g, 0.32 mmol) was added under stirring. The reaction mixture was then stirred under a hydrogen atmosphere at 50 °C for 12 h. After completion of the reaction, the reaction mixture was filtered through celite to remove the catalyst. The filtrate was evaporated under reduced pressure to yield the pure compound 26 (1.0 g, 2.4 mmol, 76 %) as a white solid.

[0107] Synthesis of (E)-N'-(8-(3-(lH-indol-3-yl)propyl)-9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)-N, N-dimethylformimidamide (27):

[0108] To a solution of compound 26 (0.8 g, 1.96 mmol) in anhydrous DMF (16 mL) under argon atmosphere, dimethylformamide di methyl acetal (3.12 mL, 23.5 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (0-10% MeOH in CH2CI2 with 0.2% of Et₃N). The pure product 27 (0.62 g, 1.34 mmol, 68%) was obtained as a pale-yellow solid.

[0109] Synthesis of (E)-N'-(8-(3-(ll / -indol-3-yl)propyl)-9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-9A'-purin-6- yl)-N, N-dimethylformimidamide (28):5

[0110] To a solution of compound 27 (0.6 g, 1.29 mmol) in dry pyridine (12 mL), DMAP (16 mg, 0.129 mmol) was added. To this mixture, a solution of 4,4’-dimethoxytrityl chloride (0.52 g, 1.55 mmol) in 4 mL of anhydrous pyridine was added in four equal portions over the time of 1 h. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the solvent was evaporated under reduced pressure. The resulting crude mixture was purified by flash chromatography (0- 3% MeOH in CH2CI2 with 0.2% of Et₃N) yielding the desired compound 28 (0.54 g, 0.71 mmol, 55%) as a white solid.

[0111] Synthesis of (2R,3S,5R)-5-(8-(3-(l / Z-indol-3-yl)propyl)-6-(((E)- (dimethylamino)methylene)amino)-9 / / -purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (4A):

[0112] To a solution of compound 28 (0.35 g, 0.46 mmol) in anhydrous CH₂Cl₂, N,N-diisopropylethylamine (0.4 mL, 2,28 mmol) was added under stirring. The reaction mixture was cooled to 0 °C, and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.16 mL, 0.73 mmol) was added under an argon atmosphere. The mixture was allowed to warm to room temperature and stirred for 1 5 h. The reaction mixture was then diluted with anhydrous CH2CI2 (20 mL) and washed successively with 5% NaHCO₃ (20 mL) and brine solution (20 mL). The organic phase was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography using (10-50% acetone in hexane containing 0.5% EtsN) as the eluent. The desired compound 4A was obtained as a 1:1.1 mixture of two diastereomers in the form of a white foam (0.28 g, 0.29 mmol, 63% yield).

[0113] Synthesis of (E)-N'-(8-(3-(lH-indol-3-yl)prop-l-yn-l-yl)-9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yr)-9 / 7-purin-6-yl)-N, N-dimethylformimidamide (29):

[0114] To a solution of compound 25 (0.8 g, 1.98 mmol) in anhydrous DMF (10 mL) under argon atmosphere, dimethylformamide dimethylacetal (5.26 mL, 39.6 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by flash chromatography using (0-10% MeOH in DCM) as the eluent. The desired product 29 (0.56 g, 1.22 mmol, 62%) was obtained as a yellow solid.

[0115] Synthesis of (E)-N'-(8-(3-(17 / -indol-3-yl)prop-l-yn-l-yl)-9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-97 / -purin-6-yl)-N, N-dimethylformimidamide (30):

[0116] To a solution of compound 29 (0.45 g, 0.98 mmol) in dry pyridine (12 mL), DMAP (12 mg, 0.1 mmol) was added. To this mixture, a solution of 4,4’ -dimethoxytrityl chloride (0.4 g, 1.17 mmol) in 8 mL of anhydrous pyridine was added in four equal portions over the course of 1 h. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the solvent was evaporated under reduced pressure. The resulting crude mixture was purified by flash chromatography (0-3% MeOH in CH₂Cl₂ with 0.2% Et₃N) yielding the desired compound 30 (0.4 g, 0.53 mmol, 54%) as a pale-yellow solid.

[0117] Synthesis of (2R,3S,5R)-5-(8-(3-(lH-indol-3-yl)prop-l-yn-l -yl)-6-(((E)-(dimethylamino)methylene)amino)-9 / 7-purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cy anoethyl) diisopropylphosphoramidite (4B):

[0118] To a solution of compound 30 (0.3 g, 0.39 mmol) in anhydrous CH2Cl2, N, N-diisopropylethylamine (0.34 mL, 1.97 mmol) was added under stirring. The reaction mixture was cooled to 0 °C, and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0,13 mL, 0,63 mmol) was added under an argon atmosphere. The mixture was allowed to warm to room temperature and stirred for 1 5 h. The reaction mixture was then diluted with anhydrous CH2CI2 (20 mL) and washed successively with 5% NaHCO3 (20 mL) and brine solution (20 mL). The organic phase was dried over anhydrous Na? SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography using (10-40% acetone in hexane containing 0.5% EtsN). The desired compound 4B was obtained as a 1:1.2 mixture of two diastereomers in the form of a paleyellow foam (0.23 g, 0.24 mmol, 61% yield).

[0119] Synthesis of precursor 5A

[0120] Synthesis of 1 -((27?,45,57?)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-(3-(4-methoxyphenyl)prop-l -yn-1 -yl)pyrimidine-2, 4(1 / 7, 377)-dione (31):

[0121] To a stirred solution of 5 -iododeoxy uridine (5) (3.03 g, 8.55 mmol) in anhydrous N,N-dimethylformamide (DMF) (30 mL) were added copper(I) iodide (0.49 g, 2.57 mmol) and DIPEA (4.42 g, 34.2 mmol) under an inert atmosphere. After stirring for 5 minutes at room temperature, tetrakis(triphenylphosphine)palladium(0) [Pd(PPh₃)₄] (1.0 g, 0.86 mmol) was added, and the reaction mixture was stirred at room temperature for 4 hours. The progress of the reaction was monitored by thin-layer chromatography (TLC). The mixture was extracted with di chloromethane (2 x 50 mL), washed with water (25 mL) and brine (25 mL). The organic phase was dried over anhydrous Na₂SO₄ and then concentrated under reduced pressure. The crude product was purified by flash column chromatography using a gradient of 0-10% methanol in dichloromethane to afford compound 31 (2.29 g, 6.16 mmol, 72.0%) as a white solid.

[0122] Synthesis of 1 -((22?,45’,5A)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-(3-(4- ethoxyphenyl)propyl)pyrimidine-2,4(l / / ,3Z / )-dione (32):31

[0123] Compound 31 (1.0 g, 2.69 mmol) was dissolved in methanol (15 mL) in a round-bottom flask. Pd / C (0.29 g, 0.27 mmol 10 mol%) was added, and the reaction mixture was subjected to three vacuum / H₂ purge cycles before being placed under a hydrogen atmosphere (balloon). The mixture w7as stirred at room temperature for 24 hours. After completion, the reaction mixture was filtered through Celite to remove the catalyst, and the solvent was evaporated under reduced pressure to afford pure product 32 (0.96 g, 2.55 mmol, 95%) as a white solid.

[0124] Synthesis of l-((27?,45’,57?)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-(3-(4- methoxyphenyl)propyl)pyrimidine-2, 4(1 / 7, 3H)-dione (33):

[0125] In a round bottom flask under argon atmosphere, compound 32 (0.85 g, 2.26 mmol) was dissolved in anhydrous pyridine (10 mL) followed by the addition of 4- (dimethylamino)pyridine (24.0 mg, 0.2 mmol). Solution of 4,4’-dimethoxytrityl chloride (DMTrCl) (0.92 g, 2.71 mmol) in 10 mL of anhydrous pyridine was added in 3 portions over 15 minutes and the reaction mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography using a gradient of 0-20% acetone in dichloromethane to afford compound 33 (1.27 g, 1.87 mmol, 83%) as awhile solid.

[0126] Synthesis of (2 / ,35)5 / ?)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-(3-(4-methoxyphenyl)propyl)-2,4-dioxo-3,4-dihydropyrimidin-I(2 / / )-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (5A):

[0127] Under an argon atmosphere, compound 33 (0.8 g, 1.18 mmol) was dissolved in anhydrous di chi orom ethane (CH2CI2) (10 ml) in a round-bottom flask. N, JN- Diisopropylethylamine (1.01 ml, 5.89 mmol) was added, and the reaction mixture was cooled to 0 °C. 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.56 g, 2.36 mmol) was then added dropwise. The mixture was allowed to warm to room temperature and stirred for 3 hour. After completion, the reaction was diluted with anhydrous CH2CI2, washed sequentially with 5% aqueous NaHCO3 and brine, then dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography using a 0-40% gradient of acetone in hexane to yield compound 5A (0.89 g, 1.01 mmol, 86%) as a mixture of two diastereomers in a form of a white foam.

[0128] Synthesis of precursor 6A

[0129] Synthesis of 4-amino-l-((2A,45,5 / ?)-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-5-(3-(4-methoxyphenyl)prop-l-yn-l-yl)pyrimidin-2(17 / )-one (35):

[0130] To a stirred solution of 5-iodo-2'-deoxycytidine (10) (2.0 g, 5.66 mmol) in anhydrous N,N-dimethylformamide (30 mL) were added copper(I) iodide (324.0 mg, 1.70 mmol) and DIPEA (2.93 g, 22.64 mmol) under an inert atmosphere. After stirring for 5 minutes at room temperature, tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4] (0.65 g, 0.56 mmol) was added, and the reaction mixture was heated to 40 °C and stirred for 2 hours. The progress of the reaction was monitored by thin-layer chromatography (TLC). The mixture was extracted with di chloro ethane (2 x 50 mL), washed with water (25 mL) and brine (25 mL). The organic phase was dried over anhydrous Na2SO4 and then concentrated under reduced pressure. The crude product was purified by flash column chromatography using a gradient of 0-10% methanol in di chloromethane to afford compound 35 (1.85 g, 4.98 mmol, 88%) as a white solid.

[0131] Synthesis of 4-amino-l-((2A,41S’,5A)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-(3-(4-methoxyphenyl)propyl)pyrimidin-2(l / / )-one (36):

[0132] Compound 35 (1.50 g, 4.04 mmol) was dissolved in dry methanol (30 mL) in a round-bottom flask. Pd / C (10 mol%) (86.2 mg, 0.81 mmol) was added, and the reaction mixture was subjected to three vacuum / H₂ purge cycles before being placed under a hydrogen atmosphere (balloon). The mixture was stirred at room temperature for 24 hours. After completion, the reaction mixture was filtered through a Celite to remove the catalyst, and the solvent was evaporated under reduced pressure to afford pure product 36 (1.27 g, 3.39 mmol, 84%) as a white solid.

[0133] Synthesis of 4-amino- 1 -((27^,43’, 5 )-5 -((bi s(4- methoxyphenyl)(phenvl [methoxy )methyl)-4-hydroxytetrahydrofuran-2-yl)-5-(3-(4- methoxyphenyl)propyl)pyrimidin-2(l / / )-one (37):

[0134] In a round bottom flask under argon atmosphere, compound 36 (1.20 g, 3.2 mmol) was dissolved in anhydrous pyridine (15 mL) followed by the addition of 4- (dimethylamino)pyridine (78.0 mg, 0.64 mmol). Solution of 4,4’-dimethoxytrityl chloride(DMTrCl) (1.30 g, 3.84 mmol) in 10 mL of anhydrous pyridine was added in portions over 15 minutes and the reaction mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography using a gradient of 0 -10% methanol in di chloromethane to afford compound 37 (1.26 g, 1.86 mmol, 58%) as a white solid.

[0135] Synthesis ofN-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-(3-(4-methoxyphenyl)propyl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide (38):

[0136] To a solution of compound 37 (1.20 g, 1.77 mmol) in anhydrous N, N- dimethylformamide (DMF) (10 mL), acetic anhydride (217.0 mg, 2.12 mmol) was added dropwise over 15 minutes at 0 °C under an argon atmosphere The reaction mixture was then stirred at room temperature for 24 hours. The progress of the reaction was monitored by thin-layer chromatography (TLC). The mixture was extracted with dichloromethane (2 x 25 mL), washed with water (25 mL) and brine (25 mL). The organic phase was dried over anhydrous Na2SO4 and then concentrated under reduced pressure. The crude product was purified by flash column chromatography using a gradient of 0-20% acetone in dichloromethane to afford compound 38 (1.03 g, 1.43 mmol, 81%) as a white solid.

[0137] Synthesis of (2R,3S,5R)-5-(4-acetamido-5-(3-(4-methoxyphenyl)propyl)-2-oxopyrimidin-1(2H)-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphorami dite (6A):

[0138] Under an argon atmosphere, compound 38 (1.0 g, 1.39 mmol) was dissolved in anhydrous dichloromethane (10 mL) in a round-bottom flask. N,N-Diisopropylethylamine (0.90 g, 6.95 mmol) was added, and the reaction mixture was cooled to 0 °C. 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (495.0 mg, 2.09 mmol) was then added dropwise. The mixture was allowed to warm to room temperature and stirred for 2 hour. After completion, the reaction was diluted with anhydrous dichloromethane (50 mL), washed sequentially with 5% aqueous NaHCO3 (30 mL) and brine (20 mL), then dried over Na2SO4. The solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography using a 0-40% gradient of acetone in hexane to yield compound 6A (1.06 g, 1.15 mmol, 83%) as a mixture of two diastereomers in a form of a white foam.

[0139] Synthesis of precursor 7 A

[0140] Synthesis of 2-amino-9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-8-(3-(4-methoxyphenyl)prop-1-yn-1-yl)-1,9-dihydro-6H-purin-6-one (41):17 41

[0141] To a stirred solution of 8-Bromo-2'-deoxyguanosine (17) (2.00 g, 5.78 mmol) in anhydrous N,N-dimethylformamide (DMF, 20 mL) were added copper(I) iodide (0.33 g, 1.73 mmol) and DIPEA (2.99 g, 23.13 mmol) under an inert atmosphere. After stirring for 5 minutes at room temperature, tetrakis(triphenylphosphine)palladium(0) (0.67 g, 0.58 mmol) was added, and the reaction mixture was stirred at 60 °C for 3 hours The progress of the reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the reaction mixture was evaporated under reduced pressure, and the crude product was purified by flash chromatography (0–15% MeOH in CH2Cl2). The desired compound 41 (1.49 g, 3.64 mmol, 63%) was obtained as a brown solid.

[0142] Synthesis of2-amino-9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-8-(3-(4-methoxyphenyl)propyl)-1,9-dihydro-6H-purin-6-one (42):

[0143] Compound 41 (1.0 g, 2.41 mmol) was dissolved in methanol (20 mL) in a round-bottom flask. PtO2 (109,4 mg, 0.48 mmol) was added, and the reaction mixture was subjected to three vacuum / H2 purge cycles before being placed under a hydrogen atmosphere (balloon). The mixture was stirred at room temperature for 12 hours. After completion, the reaction mixture was filtered through Celite to remove the catalyst, and the solvent was evaporated under reduced pressure to afford pure product 42 (0.76 g, 1.83 mmol, 76%) as a solid.

[0144] Synthesis of (E)-N'-(9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-8-(3-(4-methoxyphenyl)propyl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide (43):

[0145] To a stirred solution of compound 42 (0.70 g, 1.68 mmol) was dissolved in anhydrous DMF (10 mL) under argon atmosphere, dimethylformamide dimethylacetal (DMF-DMA) (2.80 g, 23.52 mmol) was added and the reaction was stirred at room temperature for 3 hrs. The progress of the reaction was monitored by TLC. After completion of the reaction, the solvent was evaporated under reduced pressure and the crude product thus obtained was purified by combi-flash chromatography (0-15% MeOH in CH2CI2) The desired compound 43 (404.66 mg, 0.86 mmol, 51%) was obtained as a white solid.

[0146] Synthesis of (E)-N'-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-8-(3-(4-methoxyphenyl)propyl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide (44):

[0147] In a round bottom flask under argon atmosphere, compound 43 (350.0 mg, 0.74 mmol) was dissolved in anhydrous pyridine (10 mL) followed by the addition of 4-(dimethylamino)pyridine (18.3 mg, 0.15 mmol). Solution of 4,4’-dimethoxytrityl chloride (DMTrCl) (301.6 mg, 0.89 mmol) in 5 mL of anhydrous pyridine was added in portions over 15 minutes and the reaction mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure, and the crude product was purified by flash column chromatography using a gradient of 0 -20% acetone in di chloromethane to afford compound 44 (232.0 mg, 0.30 mmol, 40%) as a white solid.

[0148] Synthesis of (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(((E)-(dimethylamino)methylene)amino)-8-(3-(4-methoxyphenyl)propyl)-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (7A):

[0149] Under an argon atmosphere, compound 44 (0.20 g, 0.26 mmol) was dissolved in anhydrous dichloromethane (10 mL) in a round-bottom flask. N,N-Diisopropylethylamine (168.0 mg, 1.30 mmol) was added, and the reaction mixture was cooled to 0 °C. 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (92.30 mg, 0.39 mmol) was then added dropwise. The mixture was allowed to warm to room temperature and stirred for 2 hour. After completion, the reaction was diluted with anhydrous di chloromethane (50 mL), washed sequentially with 5% aqueous NaHCO3 (30 mL) and brine (20 mL), then dried over Na2SO4. The solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography using a 0-50% gradient of acetone in hexane to yield compound 7A (201.0 mg, 0.21 mmol, 81%) as a mixture of two diastereomers in a form of a yellow foam.

[0150] Synthesis of precursor 8 A

[0151] Synthesis of (2R,3S,5R)-5-(6-amino-8-(3-(4-methoxyphenyl)prop-1-yn-1-yl)-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol (47):47

[0152] To a stirred solution of 8-Bromo-2'-deoxyadenosine (24) (2.00 g, 6.06 mmol) in anhydrous N, N-dimethyIformamide (DMF) (20 mL) were added copper(I) iodide (346.6 mg, 1.82 mmol) and DIPEA (3.13 g, 24.2 mmol) under an inert atmosphere. After stirring for 5 minutes at room temperature, tetrakis(triphenylphosphine)palladium(0) (705.0 mg, 0.61 mmol) was added, and the reaction mixture was stirred at 55 °C for 2 hours The progress of the reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the reaction mixture was evaporated under reduced pressure and the crude product was purified by flash chromatography (0–15% MeOH in CH2Cl2). The desired compound 47 (1.68 g, 4.24 mmol, 70%) was obtained as a yellow solid.

[0153] Synthesis of (2R,3S,5R)-5-(6-amino-8-(3-(4-methoxyphenyl)propyl)-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol (48):4748

[0154] Compound 47 (1.60 g, 4.05 mmol) was dissolved in methanol (20 ml..) in a round-bottom flask. PtCl₂ (184.0 mg, 0.81 mmol) was added, and the reaction mixture was subjected to three vacuum / H₂ purge cycles before being placed under a hydrogen atmosphere (balloon). The mixture was stirred at room temperature for 12 hours. After completion, the reaction mixture was filtered through Celite to remove the catalyst, and the solvent was evaporated under reduced pressure to afford pure product 48 (1.28 g, 3.2 mmol, 79%) as a solid.

[0155] Synthesis of (E)-N'-(9-((27?,4S,57?)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-8-(3-(4- ethoxyphenyl)propyl)-97 -purin-6-yl)- jVyV-dimethylformimidamide (49):

[0156] To a stirred solution of compound 48 (1,20 g, 3.00 mmol) was dissolved in anhydrous DMF (10 mL) under argon atmosphere, dimethylformamide dimethylacetal (DMF-DM A) (5.00 g, 42.00 mmol) was added and the reaction was stirred at room temperature for 3 hrs. The progress of the reaction was monitored by TLC. After completion of the reaction, the solvent was evaporated under reduced pressure and the crude product thus obtained was purified by combi-flash chromatography (0- 15% MeOH in CH₂Cl₂). The desired compound 49 (0.71 g, 1.56 mmol, 52%) was obtained as a white solid.

[0157] Synthesis of (E)-N'-(9-((2A5,45,5 / ?)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-8-(3-(4- methoxyphenyl)propyl)-9 / 7-purin-6-yl)- / V, A-diraethylformimidamide (50):7:

[0158] In a round bottom flask under argon atmosphere, compound 49 (600.0 mg, 1.32 mmol) was dissolved in anhydrous pyridine (10 mL) followed by the addition of 4-(dimethylamino)pyridine (32.3 mg, 0.26 mmol). Solution of 4,4’-dimethoxytrityl chloride (DMTrCl) (536.7 mg, 1.58 mmol) in 5 mL of anhydrous pyridine was added in portions over 15 minutes and the reaction mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure, and the crude product was purified by flash column chromatography using a gradient of 0-20% acetone in di chloromethane to afford compound 50 (0.40 g, 0.53 mmol, 40%) as a white solid.

[0159] Synthesis of (27?,35,57?)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(((E)-(dimethylamino)methylene)amino)-8-(3-(4-methoxyphenyl)propyl)-9 / / -purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) ethyl(isopropyl)phosphoramidite (8A):

[0160] Under an argon atmosphere, compound 50 (300.00 g, 0.40 mmol) was dissolved in anhydrous di chloromethane (10 mL) in a round-bottom flask. N. N-Diisopropylethylamine (258.5 mg, 2.00 mmol) was added, and the reaction mixture was cooled to 0 °C. 2-Cyanoethyl-AUV-diisopropylchlorophosphoramidite (142.00 mg, 0.60 mmol) was then added dropwise. The mixture was allowed to warm to room temperature and stirred for 1 hour. After completion, the reaction was diluted with anhydrous dichloromethane (50 mL), washed sequentially with 5% aqueous NaHCO3 (30 mL) and brine (20 mL), then dried over Na₂SO₄. The solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography using a 0-50% gradient of acetone in hexane to yield compound 8A (230,0 mg, 0.24 mmol, 60%) as a mixture of two diastereomers in a form of a yellow foam.

[0161] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do notdiffer from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

Claims

What is claimed is:

1. A composition of matter comprisingan organic moiety (G2);a propyl linker:a nitrogenous base that is:a purine base with a C8 position that links the purine base to the organic moiety (G2) with the propyl linker, ora pyrimidine base with a C5 position that links the pyrimidine base to the organic moiety (G2) with the propyl linker; anda ribose that is directedly connected to the nitrogenous base at a Cl’ position of the ribose.

2. The composition of matter as recited in claim 1, wherein the nitrogenous base is purine, wherein the purine has a N9 position that directly connects to the CT position of the ribose.

3. The composition of matter as recited in claim 2, further comprising a phosphoramidite connected to a C3’ position of the ribose.

4. The composition of matter as recited in claim 3, wherein the ribose is a deoxyribose.

5. The composition of matter as recited in claim 3, wherein the phosphoramidite is a 2-cyanoethyl-A / , A'-diisopropylchlorophosphoramidite.

6. The composition of matter as recited in claim 3, wherein the organic moiety (G2) is an indole, a substituted indole, a benzoimidazole, a substituted benzoimidazole, a benzothiophene, a substituted benzothiophene, an isoindole, a substituted isoindole, an indolizine or a substituted indolizine.

7. The composition of matter as recited in claim 3, wherein the organic moiety (G2) is an indole.

8. The composition of matter as recited in claim 3, wherein the organic moiety (G2) is 3-indole.

9. The composition of matter as recited in claim 3, wherein the organic moiety (G2) is a benzene, a substituted benzene, a pyridine, a substituted pyridine, a pyrimidine, a substituted pyrimidine, a pyrazine, a substituted pyrazine, a pyrazine or a substituted pyrazine,10. The composition of matter as recited in claim 3, wherein the organic moiety (G2) is a pyrrole, a substituted pyrrole, a furan, a substituted furan, a thiophene, a substituted thiophene, an imidazole, a substituted imidazole, a thaizole, a substituted thiazole, an oxazole, a substituted oxazole, a triazole a substituted triazole, a pyrazole or a substituted pyrazole.

11. The composition of matter as recited in claim 3, wherein the organic moiety (G2) is a quinoline, a substituted quinoline, an isoquinoline, a substituted isoquinoline, a quinazoline or a substituted quinazoline.

12. The composition of matter as recited in claim 1, wherein the nitrogenous base is pyrimidine, wherein the pyrimidine has a N1 position that directly connects to the Cl’ position of the ribose.

13. The composition of matter as recited in claim 12, further comprising a phosphoramidite connected to a C3’ position of the ribose.

14. The composition of matter as recited in claim 13, wherein the phosphoramidite is a 2-cyanoethyl-? / , Ar-diisopropylchlorophosphoramidite.

15. The composition of matter as recited in claim 14, wherein the organic moiety (G2) is an indole, a benzoimidazole or a methoxy phenyl.

16. The composition of matter as recited in claim 12, wherein the organic moiety (G2) is an indole, a benzoimidazole or a methoxy phenyl.

17. A nucleic acid comprising a plurality of nitrogenous bases, at least one of which is the composition of matter as recited in claim 1.

18. A nucleic acid comprising a plurality of nitrogenous bases, at least one of which is the composition of matter as recited in claim 1, wherein the ribose is a deoxyribose.

19. A composition of matter comprising a compound with a structure ofwherein O' is an alcohol or a protected alcohol, G1is a nitrogenous base having a structure selected from:wherein G2is an indole, a benzoimidazole or a methoxyphenyl and N1is a primary amine or a protected primary amine.

20. The composition of matter as recited in claim 19, wherein G2is 3-indole.

Images