Double-headed nucleosides: Synthesis and applications

Double-headed nucleoside monomers have immense applications for studying secondary nucleic acid structures. They are also well-known as antimicrobial agents. This review article accounts for the synthetic methodologies and the biological applications of double-headed nucleosides.

Double-headed nucleosides are synthetically derived nucleoside scaffolds that are known to impact significantly secondary structures in nucleic acids [29]. Some oligonucleotides containing a particular double-headed nucleotide monomer have been found to form a three-way junction structure with a hairpin loop and two flanking sequences [30,31]. Moreover, these nucleotides have been found to orient the additional nucleobase towards the core of the duplex to participate in Watson-Crick base pairing [32][33][34]. The incorporation of the double-headed nucleosides into oligonucleotides followed by their duplex formation studies against complimentary oligonucleotide strands had described a very selective zipper-interaction [35], whereas a relative stabilization was observed due to stacking of these additional nucleobases across the minor groove [31]. The biological activity of the acyclic double-headed nucleosides was assessed through in vitro studies on Gram-positive bacteria Staphylococcus aureus, Listeria inovanii and Gram-negative bacteria Klebsiella pneumoniae, Salmonella sp., and Escherichia coli [20]. Triazolyl double-headed nucleosides showed efficacy against eosinophil-derived neurotoxin, which is an eosinophil secretion protein and a member of the Ribonuclease A (RNase A) superfamily [36]. Double-headed nucleosides were also found to be active against orthopox viruses, vaccinia virus, and cowpox virus under in vitro conditions [11], whereas few double-headed nucleoside analogues showed a moderate cytostatic activity against human cervix carcinoma HeLa cells [37].
It is pertinent to mention that Sharma et al. [29] have reviewed the double-headed nucleotides in the recent past with a focus on their effects in nucleic acid duplexes and other secondary structures. Herein, we focused on the synthetic protocols used for accessing a variety of double-headed nucleoside monomers. Thus, this review is the comprehensive compilation of the synthetic protocols available for the production of double-headed nucleoside monomers and their applications. For better overview the review has been structured based on the types of the sugar moiety of the nucleoside and the position of the attachment of the additional base, either directly or through a linker on the sugar.

Review Furanosyl double-headed nucleosides
Based on literature reports most of the double-headed nucleosides comprised a pentofuranosyl sugar moiety. Various synthetic methodologies have been developed for the introduction of the additional nucleobase/heterocyclic system directly or via a linker at the C-2′/C-3′/C-4′/C-5′ position of the pentofuranosyl moieties. We have categorized the double-headed furanosylnucleosides depending on the position of the attachment of the additional nucleobase/heterocyclic system at the particular carbon of the pentofuranosyl moiety of the nucleoside ( Figure 1).

1,2-Furanosyl double-headed nucleosides
Herein, all nucleosides comprising furanosyl ring structures are included, with the first nucleobase attached to the C-1′ position and the second nucleobase introduced at the C-2′ position either with or without a linker (Figure 1).
The synthesized double-headed nucleosides 4a,b were dimethoxytritylated (DMTr), phosphitylated, and incorporated into DNA oligonucleotides using the standard automated phosphoramidite method. The UV-based melting temperature (T m ) of hybrids of the modified oligonucleotides with complementary DNA strands were studied. The analysis of the melting temperature of the duplex and extensive molecular dynamics studies revealed that the synthesized double-headed nucleotides behave as functional dinucleotide mimics and hybridize with complementary targets neatly with their Watson-Crick faces compatible with natural DNA [39]. Scheme 1: Synthesis of 2′-(pyrimidin-1-yl)methyl-or 2′-(purin-9-yl)methyl-substituted double-headed nucleosides 4a-f of arabinofuranosyluracil.
Nielsen and co-workers [42] additionally synthesized 2′-(Nbenzoylcytosin-1-yl)methylarabinofuranosyl-N-benzoylcytosine (7) from uridine using a similar methodology. Thus, the nucleophilic epoxide ring opening in spironucleoside 2 with uracil in DMF in a N 1 -regioselective manner afforded the TIPDS-protected double-headed nucleoside 5 having two uracil bases (the additional uracil being attached through a methylene linker to the 2′-position of arabinouracil). Subsequently, the two uracil bases of the TIPDS-protected double-headed nucleoside 5 were converted to N-benzoylated cytosines in a three-step onepot procedure in 55% yield. For this conversion, the carbonyl group at the 4-position of uracil was first activated by tosylation, which was followed by conversion to the amine upon reaction with ammonia and protection of the newly introduced amino group with benzoyl chloride to afford the double-headed nucleoside 6. The removal of the silyl protecting group with NEt 3 ·3HF in THF yielded 2′-(N-benzoylcytosin-1-yl)methylarabinofuranosyl-N-benzoylcytosine (7, Scheme 2) [42].
The double-headed nucleoside 7 was dimethoxytritylated and phosphitylated following the standard procedure and incorporated into oligonucleotides to study its effects on duplex stability. The single incorporation in oligonucleotides and study of the melting temperature (T m ) of its duplex hybridized with a complementary DNA strand revealed an increase in T m by 4 °C with respect to the normal duplex. This indicated the participation of both nucleobases of the double-headed nucleotides in Watson-Crick base pairing. The same group also showed that a multiple incorporation of the double-headed nucleotide is also tolerated, but the double-headed nucleotides with the present design were not suitable as triplex-forming oligonucleotides [42].
The double-headed nucleoside 11 was dimethoxytritylated and phosphitylated following the standard procedures and incorporated once into a 13-mer oligodeoxynucleotide and an LNAmodified oligodeoxynucleotide sequence, and four-times in the middle of a 12-mer oligodeoxynucleotide sequence in order to study the effect of the additional nucleobase in duplexes, bulged duplexes, and in three way junctions [35]. The designed doubleheaded nucleoside was found to be reasonably well tolerated in duplexes and stabilized three-way junctions. Significant conformational changes in these secondary structures have also been induced [35].
Both double-headed nucleosides 14 and 15, when incorporated into oligonucleotides were found to stabilize three-way junction in both DNA-DNA and DNA-RNA duplexes and when introduced into a (+1)-zipper motif, cross strand interactions were observed in a DNA-DNA duplex [43].
TIPDS protection of uridine (16), followed by the treatment of the product with acetic anhydride/acetic acid in DMSO produced the protected nucleoside 17 [45,46] (Scheme 5). Next, the fully protected nucleoside 17 was subjected to chlorination using thionyl chloride in dichloromethane, followed by the treatment of the product with N 3 -benzoylthymine under basic conditions (K 2 CO 3 in DMF) to produce the nucleoside 18. The removal of the tert-butyldimethylsilyl group from nucleoside 18, followed by dimethoxytritylation at the primary hydroxy and phosphitylation at the secondary hydroxy group afforded the double-headed nucleoside monomer 19 (Scheme 5) [45].
The synthesized double-headed nucleoside 19 was introduced in oligonucleotides and its impact on the secondary nucleic acid structure was studied. It was revealed that the double-headed nucleoside 19 was well accommodated in a hybrid DNA:RNA duplex and stabilized bulged duplex and three way junctions [45]. The potential of the double-headed nucleoside 19 in secondary nucleic acid structures was compared with the earlier reported monomer 11 and found to be inferior to double-headed nucleoside 11 due to the 3′-endo conformation which placed the 2′-substituent towards the minor groove rather than to the duplex core [35].
Vilarrasa and co-workers [47] synthesized 2′-uracil-1-yl and 2′-thymin-1-yl derivatives of 2′-deoxythymidine starting from uridine (16). The synthesis started with the TIPDS protection of 16 followed by introduction of an azide group in the C-2′ position of the molecule to afford nucleoside 22. The treatment of azide 22 with pyrrolidine in acetonitrile followed by hydrogenation afforded aminonucleoside 23, which was used as a key intermediate for the synthesis of the double-headed nucleosides 24 and 25 (Scheme 6) [47].
The same group [47] also synthesized the C-2′ isomeric nucleosides 28 and 29, i.e., with inverted configuration at C-2′ as compared to nucleosides 24 and 25 (Scheme 7). The synthesis of these two nucleosides was carried out through the formation of the anhydro nucleoside 26 and its transformation into the aminonucleoside 27. The key intermediate nucleoside 27 was then treated with 3-ethoxypropenoyl isocyanate or 3-methoxy-  2-methylpropenoyl isocyanate in a mixture of benzene and DMF, followed by acidification with sulfuric acid affording the nucleosides 28 and 29, respectively in high yields (Scheme 7) [47].
The double-headed nucleoside 33 was dimethoxytritylated, phosphitylated, and incorporated into duplex and its ability to recognize complementary base pairs was monitored by UV melting curve analysis [33]. Hybridization data revealed that the synthesized double-headed nucleotide recognized itself either through formation of Watson-Crick base pairs with two com-plementary adenosines or through the formation of T:T (thymine:thymine) base pairs that resulted in the formation of two novel nucleic acid motifs. The novel nucleic acid motifs could be incorporated either single or multiple times in dsDNA duplexes without altering its stability. It was revealed by molecular dynamics (MD) simulations that the DNA sugar-phosphate backbone accommodated modified nucleotide by stretching or curling up as required and all the four base pairs based upon the structure of the synthesized double-headed nucleotide could be accommodated in the similar way as the T:A (thymine:adenine) base pair in the motif. The nucleic acid motifs may also be used in designing nanoscale DNA structures where a specific duplex twist is required [33].
The double-headed nucleoside 37 was dimethoxytritylated, phosphitylated, and incorporated into 11-to 13-mer oligonucleotides using the standard automated phosphoramidite method. The UV-based melting temperature (T m ) of hybrids of the modified oligonucleotides with complementary DNA strands were studied. The analysis of the melting temperature of the resulting duplex revealed that the synthesized doubleheaded nucleotide behaved as a compressed dinucleotide and combination of all natural nucleobases on compressed scaffold can form Watson-Crick base pairs with complementary bases [34].
The double-headed nucleoside 41 was phosphitylated and then incorporated into oligonucleotides and was found to form highly stable DNA duplexes and three way junctions. There was a four-fold increase in the intensity of the pyrene excimer signal observed when an oligonucleotide containing two incorporations of the double-headed nucleoside 41 hybridized with an RNA target whereas the pyrene-pyrene excimer band almost vanished when the oligonucleotide was hybridized with a DNA target. The double-headed nucleoside 41 has potential in DNA invader probes as well as in RNA targeting and detection [23].

1,3-Furanosyl double-headed nucleosides
In this section, all double-headed nucleosides with furanosyl ring structures are collected. The first nucleobase is attached at the anomeric position of the furanosyl ring structure and the second nucleobase is connected to the C-3′ position with or without a linker ( Figure 1).
The double-headed nucleosides 46a-c and 50a-e were evaluated for their inhibitory potency towards RNase A and eosinophil-derived neurotoxin (EDN). Among all the nucleosides, the double-headed nucleoside 50c showed a stronger preference for EDN than for ribonuclease A whereas all other derivatives were found to be more specific for ribonuclease A [36].
Vilarrasa and co-workers [47] also synthesized the doubleheaded nucleosides 63 and 64 with downwards orientation of the additional nucleosides at the C-3′ position. The synthesis was carried out via formation of anhydride 61. Azidation, followed by reduction of the corresponding nucleoside with tin chloride produced nucleoside 62 which was treated as a key intermediate for the production of the double-headed nucleosides 63 and 64. Reaction of nucleoside 62 with 3-ethoxypropenoyl isocyanate or 3-methoxy-2-methylpropenoyl isocyanate in a solution mixture of benzene and DMF, followed by acidification with sulfuric acid produced nucleosides 63 and 64, respectively (Scheme 14) [47].

1,4-Furanosyl double-headed nucleosides
A literature search revealed two different categories of 1,4-furanosyl double-headed nucleosides. In the first category, the first nucleobase was a natural (attached at C-1′ position) and the second nucleobase was an aromatic moiety, which was at-tached at the C-4′ position without any linker ( Figure 1). Whereas the second category of nucleosides contained first natural nucleobase at the C-1′ position and a second natural nucleobase attached at the C-4′ position with a methylene linker. The nucleosides of the second type may also contain a hydroxymethyl group at the C-4′ position.
The tert-butyldimethylsilyl-protected (TBDMS) nucleoside 76 was first hydrolyzed using NaOH, which was followed by TBDMS deprotection using tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuran (THF) to afford the double-headed nucleoside 77. The TBDMS-protected nucleoside 73 was first hydrolyzed using NaOH followed by the reaction with TBDMSCl and benzoyl chloride to get the N 6 -benzoyl-3',5'-O-diTBDMS-protected nucleoside 74. Removal of the silylprotecting groups in the double-headed nucleoside 74 with TBAF in THF resulted in the formation of the desired doubledheaded nucleoside 75 (Scheme 17) [54].
The double-headed nucleosides 75 and 77 were 4-methoxytritylated and phosphitylated following the standard procedures and incorporated into oligonucleotides. Extrahelical A-T base interactions were observed when these double-headed nucleoside monomers were placed in opposite strands of the duplex with separation of one regular base pair from each other [54].
Lazrek et al. [51] synthesized C-5′-modified double-headed nucleosides 96a-g, where a 1,2,3-triazolo ring acted as the linker between the nucleobase and the sugar moiety. First, seven N 9 /N 1 -propargylpurine/pyrimidine nucleobases 13b, 45 and 53a-e were synthesized by treating the nucleobases with propargyl bromide in the presence of K 2 CO 3 . Nucleoside 94 was synthesized from thymidine (93) which was first tritylated at the C-5′ primary hydroxy position followed by acetylation at the C-3′ secondary hydroxy group [61]. Next, detritylation and tosylation of the protected nucleoside 94 followed by treatment with lithium azide in DMF and saturated methanolic ammonia solution afforded nucleoside 95. Refluxing of nucleoside 95 with 13b, 45 and 53a-e in toluene produced the desired nucleosides 96a-g (Scheme 21) [51].
Shaikh et al. [14] reported the synthesis of double-headed nucleosides where an aromatic moiety or a nucleobase is attached at the C-5′ position of the nucleoside. The synthetic methodology started with the 5′-epoxide 97, which was synthesized from 3′-O-(tert-butyldimethylsilyl)thymidine in three steps, where the oxidation of the C-5′-hydroxy group followed by a Wittig reaction with methylenetriphenylphosphorane (Ph 3 P=CH 2 ) produced the 5′-methylene derivative [62]. Finally, oxidation with meta-chloroperbenzoic acid (mCPBA) afforded the nucleoside 97. Treatment of the nucleoside 97 with Grignard reagent PhMgBr in THF produced nucleoside 98, whose secondary hydroxy group was protected by reaction with pixyl chloride to afford the nucleoside 99. The removal of the tertbutyldimethylsilyl protecting group under standard conditions afforded the double-headed nucleoside 100 (Scheme 22) [14].
Opening of the epoxide ring in nucleoside 97 with sodium azide in DMF produced nucleoside 101, whose secondary hydroxy group was protected by reaction with pixyl chloride to afford nucleoside 102. The azido nucleoside 102 was a key intermediate, which was used for the synthesis of a variety of 1,2,3-triazolyl-linked double-headed nucleosides. Thus the treatment of azido nucleoside 102 with phenylacetylene in the presence of sodium ascorbate and copper sulfate in a solvent mixture of t-BuOH, water and pyridine, followed by the removal of the tert-butyldimethylsilyl protecting group gave nucleoside 103 (Scheme 22) [14]. Under similar reaction conditions, the treatment of nucleoside 102 with N 1 -benzoyl-5-ethynyluracil followed by desilylation produced the double-headed nucleoside 104, whereas the reaction of the azido nucleoside 102 with trimethylsilylacetylene (TMS-acetylene) followed by desilylation produced the nucleoside 105 (Scheme 23) [14].
Subsequently, the double-headed nucleoside 107 was incorporated into oligonucleotides [31,33,35,63,64] and when the duplex was generated with complementary DNA and RNA sequences, the additional nucleobase was positioned in the minor groove of the duplex. However, the presence of the additional nucleobase resulted in a thermal destabilization of the duplex as compared to unmodified duplexes. The introduction of two These nucleoside monomers were converted into phosphoramidites and then incorporated into oligonucleotide sequences, followed by thermal hybridization studies that indicated that the 5′-(S)-C-position is ideal for placing an additional nucleobase in the minor groove and interstrand stacking effects decreased with an increase in the length of the linker [31,65].
The incorporation of the double-headed nucleoside monomer 114 into oligonucleotides failed to stabilize three-way junctions [43] which is contrary to the double-headed nucleoside 11 which stabilized three-way junction very efficiently [35].
The nucleoside monomer 118 was phosphitylated and then incorporated into oligodeoxynucleotides but stabilization in the

Bicyclic double-headed nucleosides
In this section we have included the double-headed nucleoside monomers, which have a locked nucleic acid type conformation and the additional nucleobase is attached at one of the carbon or nitrogen atoms constituting the bridge (Figure 1). All examples discussed herewith are constituted by furanosyl carbohydrate moiety.
The double-headed nucleoside monomers 125a-c were incorporated into oligodeoxyribonucleotides via phosphoramidite derivatization of the C-3′ hydroxy group present in the moiety. The oligonucleotides thus synthesized were found to stabilize the duplex formed with complementary DNA [69].
The synthesized double-headed nucleosides were phosphitylated and incorporated into oligonucleotides and the melting temperatures were evaluated against unmodified DNA strands. Oligonucleotides with fourteen consecutive incorporations of different double-headed nucleosides were synthesized and the DNA duplexes showed increased stability owing to increased stacking interactions among the nucleobases of the opposite strands [72]. Molecular dynamics simulations demonstrated the exposure of Watson-Crick/Hoogsteen faces of additional nucleobases for their recognition in the major groove.
Both double-headed nucleoside monomers 143 and 146 were phosphoramidated at the C-3′ hydroxy group and incorporated into oligonucleotides. The synthesized oligonucleotides were found to decrease the thermal stability of the duplexes. However, their potential in triplex forming oligonucleotides was also studied which concluded the formation of most stable triplexes with single incorporations of additional pyrimidine nucleobases connected via a propylene linker [71].
Hrdlicka and co-workers [24] also synthesized 5-C-triazolylfunctionalized double-headed nucleosides 154a,b starting from 5-C-ethynyl-functionalized LNA uridine 152. The LNA uridine 152 was reacted with 1-azidopyrene (153a) and 1-azidomethylpyrene (153b) separately under copper-catalyzed alkyne azide cycloaddition (CuAAC) reaction conditions to yield the The synthesized double-headed nucleosides 151a-d and 154a,b were phosphitylated, incorporated into oligonucleotides and characterized with respect to thermal denaturation, enzymatic stability, and fluorescence properties. The incorporation of the double-headed nucleosides 151a-d and 154a,b into oligo-nucleotides failed to form thermostable duplexes with complementary DNA and RNA strands but exhibited a potential resistance towards 3′-exonuclease. The synthesized double-headed nucleosides 151c,d and 154a,b when incorporated into oligonucleotides enabled fluorescent discrimination of targets with single nucleotide polymorphisms (SNPs) [24].
The double-headed nucleosides 157a-c were introduced into nonamer oligonucleotides by phosphoramidite chemistry [15]. a single incorporation of double-headed nucleosides 157a-c into oligonucleotides resulted in the formation of unstable duplexes with complementary DNA and RNA strands whereas four consecutive incorporations led to increased duplex stability due to an efficient stacking of heteroaromatic triazoles as revealed by CD spectroscopy and molecular dynamics simulations [15,22]. The double-headed nucleoside 157a was further used for the synthesis of 5-(phenyltriazol)-2′-deoxyuridine-modified 2′-O-methyl mixmer antisense oligonucleotides (AOs). The obtained AOs were investigated for their potential to induce exon skipping in DMD (Duchenne muscular dystrophy) transcript using H2K mdx mouse myotubes. It was found that exon-23 skipping potential of oligonucleotide containing 5-(phenyltriazole)-2′-deoxyuridine (157a) building blocks placed distantly was slightly better than oligonucleotides containing the 5-(phenyltriazole)-2′-deoxyuridine (157a) building blocks placed consecutively [80].
Nielsen and co-workers [26]  The synthesized double-headed nucleosides 159 and 163 were reacted with 2-cyanoethyl-N,N-diisopropyl-phosphoramidochloridite in the presence of DIPEA (N,N-diisopropylethylamine) to afford phosphoramidites 160 and 164 which were then incorporated into oligodeoxynucleotides using automated solid phase synthesis. The synthesized oligonucleotides were removed from the solid support by treatment with concentrated aqueous ammonia which resulted in the formation of incorporated monomers 161 and 165 by simultaneous removal of tertbutyldimethylsilyl and amidine protecting groups, respectively (Scheme 41 and Scheme 42) [26].
The incorporation of the double-headed nucleosides 159 and 163 into oligonucleotides resulted in the formation of thermally stable DNA:RNA duplexes due to an efficient π-π stacking between two or more phenyltriazoles in the major groove. The more stable duplex was obtained when oligonucleotide containing monomer 165 was hybridized with the complementary RNA strand due to the best stacking shown by sulfonamide-substituted phenyltriazoles in the major groove [26,27]. Single incorporations of 5-C-triazolylbenzenesulfonamide-substituted monomer 165 at four positions within the gap region of RNase H gapmer antisense oligonucleotides (ASOs) reduced wild-type and mutant huntingtin mRNA in human patient fibroblasts. A structural model of the catalytic domain of human RNase H bound to ASO:RNA heteroduplexes was created which was utilized for explaining the activity and selectivity observations in cells and in the biochemical assays [81].
Sharma and co-workers [27]  The incorporation of the double-headed nucleoside 167 into oligonucleotides resulted in the formation of an equally stable DNA:RNA duplex as in the case of double-headed nucleoside 163 irrespective of the positional orientation of the sulfonamide group due to an efficient π-π stacking between two or more phenyltriazoles in the major groove [27]. On the other hand, the incorporation of the double-headed nucleosides 171 and 175 into oligonucleotides resulted in the formation of less stable DNA:RNA duplexes because of the poor stacking by the alkynyl group as compared to triazolyl groups in double-headed nucleosides 163 and 167 [27]. The double-headed nucleotide 173 was fully accepted by KOD (kodakaraensis), Phusion, and Klenow DNA polymerases as substrate which resulted in the formation of fully extended DNA. KOD DNA polymerase was found to be the best enzyme to produce DNA containing the double-headed nucleotide 173 in good yield and Phusion DNA polymerase amplified the template containing double-headed nucleotide 173 efficiently by PCR (polymerase chain reaction) [82].
The incorporation of the double-headed nucleosides 181 and 183 multiple times into oligonucleotides resulted in the formation of stable DNA:RNA duplexes due to the perfect stacking of the aromatic moieties in the major groove of the duplex [22]. The double-headed nucleoside 183 containing a phenylpyrazole moiety exhibited better π-π stacking interactions in the major groove with itself and with an adjacent double-headed nucleoside (157a) incorporated as compared to the doubleheaded nucleoside 181 containing a flexible phenylfuran moiety. There was not any change in the geometry of the duplexes observed upon introduction of double-headed nucleosides 181 and 183 as revealed by CD spectroscopy and molecular modeling. The synthesized oligonucleotides containing consecutive triazole-functionalized double-headed nucleosides 183 and 157a were found to form highly stable duplexes due to a large aromatic overlap of their substituents at the 5-position due to which they can be utilized as a simple tool in high affinity RNA targeting oligonucleotides [22].

Acyclic double-headed nucleosides
In this section, double-headed nucleosides are included that have an acyclic carbohydrate moiety and the heterocyclic moieties/nucleobases are terminally attached at the sugar moiety ( Figure 1).
These double-headed nucleosides when incorporated into oligonucleotides destabilized both DNA and RNA duplexes. However, nucleosides with 2′(S)-configuration were found to destabilize duplexes and bulged motifs to a lesser extent than the other stereoisomers [89].
The synthesized double-headed nucleosides 218b-d may exhibit potential biological activities due to the resistance of the C-glycosidic moiety towards hydrolytic or enzymatic cleavage [90] and the enhanced hydrophilicity which results in an increased transportation to biological systems [16].
The acyclic double-headed nucleosides 232a and 233-235 were screened for their in vitro antibacterial activity against the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus and for their antifungal activity against Candida albicans using the agar diffusion method [103]. Among the tested compound, derivative 235 showed fair activity against E. coli and C. albicans but was inactive against S. aureus whereas compound 234 showed activity only against S. aureus [19].
The double-headed C-nucleosides 240-242 were tested in vitro against Gram-positive bacteria Staphylococcus aureus, Listeria inovanii and Gram-negative bacteria Klebsiella pneumoniae, Salmonella sp., and Escherichia coli. All the double-headed nucleosides except derivative 242 showed moderate antibacterial activity in comparison with the known antibiotic combination amoxicillin/clavulanic acid (AMC) [20]. The structural and electronic properties of the double-headed nucleosides were explored theoretically by performing semiempirical molecular orbital, ab initio Hartree-Fock (HF), and density functional theory (DFT) calculations and their geometries were optimized at the level of Austin Model 1 (AM1) [104].
Compound 247 was treated with adenine in the presence of sodium hydride in DMF at 105 °C to incorporate two adenine moieties affording compound 248. The benzoylation of compound 248, followed by treatment with methanolic ammonia at low temperature produced the corresponding N-benzoylated adenine derivative 249 [111]. The cleavage of the diacetal in compound 249 was achieved with 75% TFA/water resulting in compound 250, which was considered as the acyclic doubleheaded nucleoside without any protection of the primary hydroxy groups (Scheme 65) [111].
In a similar reaction sequence, compound 251 was treated with N 3 -benzoylthymine to afford compound 252, which was treated with 75% TFA-water for deprotection of the hydroxy groups to afford the final monomer 253 (Scheme 66) [111].
The synthesized acyclo nucleosides 250 and 253 were phosphitylated and incorporated into oligonucleotides to evaluate the effects on duplex stability. It was observed that the hybridization properties of the oligonucleotides with one acyclic achiral nucleoside, i.e., 250 or 253 when incorporated in the middle of a 12-mer or 13-mer decreased with complementary DNA or RNA [111]. Four pyrimidine nucleobases 254a-d were treated with methyl iodide in the presence of sodium hydroxide to get methylthio derivatives 255a-d, which were treated with 2,2bis(bromomethyl)-1,3-diacetoxypropane (256) in the presence of NaH in DMF to afford the mono-headed acyclic nucleosides 257a-d [112]. The second nucleobase was introduced in compounds 257a-d by repeating the reaction with the desired nucleobase under otherwise identical conditions (NaH/DMF) giving the acyclic double-headed pyrimidine nucleosides 258a-d. Finally, the treatment of compounds 258a-d with NaOMe in methanol produced the unprotected nucleosides 259a-d (Scheme 67) [112].
The double-headed nucleoside 261 was obtained by a two-step reaction sequence starting from compound 256, which was first reacted with theophylline in DMF, to give the acyclic doubleheaded purine nucleoside 260 followed by treatment with NaOMe in methanol to get the unprotected product 261. In the sequence, all reactions were carried out under microwave irradiation conditions (Scheme 68) [112]. The branched chain tetraseco-nucleosides 259a-d and 261 were synthesized because acyclic nucleosides of tetraseco-type were found to possess interesting antiviral activities [21,[113][114][115].

Conclusion
Among the variety of modified nucleosides, double-headed nucleoside monomers are an important class of compounds, which have shown their importance in nucleoside chemistry. Here, we have focused on the available methodologies for the synthesis of several double-headed nucleosides. For a systematic discussion, we have classified them into three different categories, i.e., double-headed nucleosides with additional head/ nucleobase on the sugar moieties, nucleobase moieties and on acyclic carbohydrate moieties. We have subdivided the category of monocyclic furanosyl double-headed nucleosides into 1,2-furanosyl-, 1,3-furanosyl-, 1,4-furanosyl-, and 1,5-furanosyl double-headed nucleosides depending on the position of the aglycon moiety in the furanosyl ring and systematically described their synthetic methodologies. Next, we elaborated the procedures for the synthesis of bicyclic furanosyl doubleheaded nucleosides, followed by procedures for the development of base to base double-headed nucleosides. The chemical strategies for the synthesis of pyranosyl double-headed nucleosides and acyclic double-headed nucleosides were also described. Along with the methodologies for the development of double-headed nucleoside monomers, the synthetic approach for their incorporation into the oligonucleotides was also elaborated in this review. Biological applications of the synthesized nucleosides were also described.

Future Direction
Double-headed nucleosides are important structural scaffolds that modulate nucleic acid structures. Rationally designed nucleosides can tune interstrand and intrastrand interactions that are exhibited in nucleic acids. As a consequence, these synthetic scaffolds can be exploited rationally in biomolecular designs and medicinal chemistry. These modified double-headed nucleosides could be incorporated into oligonucleotides to explore their potential as antisense nucleosides. Similarly, as some of these nucleosides have shown their potential as antimicrobial agents, they could be explored extensively for their biological activity. This review will help researchers to get an insight into the available procedures for the synthesis of doubleheaded nucleosides and briefly explores their role in modulating nucleic acid structures and in medicinal chemistry. The researchers working in the field of modified nucleosides will be encouraged further to take up challenges for the synthesis of currently unexplored double-headed nucleosides with extensive configurations, connectivity through different linkers, and exploration of different purine and pyrimidine moieties as nucleobases.