The 5‐Methylisocytosine β‐D‐Ribopyranosyl (4′ → 2′)‐Oligonucleotide

In the context of Eschenmoser's work on pyranosyl‐RNA (‘p‐RNA’), we investigated the synthesis and base‐pairing properties of the 5‐methylisocytidine derivative. The previously determined clear‐cut restrictions of base‐pairing modes of p‐RNA had led to the expectation that a 5‐methylisocytosine β‐D ‐ribopyranosyl (= D ‐pr(^MeisoC)) based (4′ → 2′)‐oligonucleotide would pair inter alia with D ‐pr(isoG) and L ‐pr(G) based oligonucleotides (D ‐pr and L ‐pr = pyranose form of D ‐ and L ‐ribose, resp.). Remarkably, we could not observe pairing with the D ‐pr(isoG) oligonucleotide but only with the L ‐pr(G) oligonucleotide. Our interpretation concludes that this – at first hand surprising – observation is caused by a change in the nucleosidic torsion angle specific for isoC.     

Efficient Automated Solid‐Phase Synthesis of DNA and RNA 5′‐Triphosphates

A fast, high‐yielding and reliable method for the synthesis of DNA‐ and RNA 5′‐triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5‐chloro‐saligenyl‐N,N‐diisopropylphosphoramidite was coupled to the 5′‐end. Oxidation of the formed 5′‐phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5′‐cycloSal‐oligonucleotides. Reaction of the support‐bonded 5′‐cycloSal‐oligonucleotide with pyrophosphate yielded the corresponding 5′‐triphosphates. The 5′‐triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer.     

The MOE Modification of RNA: Origins and Widescale Impact on the Oligonucleotide Therapeutics Field

In an article published by Helvetica Chimica Acta in 1995, chemist P. Martin describes the synthesis of 2′-O-alkylated ribonucleosides for use in therapeutic antisense oligonucleotides (ASOs). This work was motivated by the need for a modified ribose structure that was compatible with solid-phase synthesis protocols and that, when incorporated into an oligonucleotide, would render it resistant to nucleases without attenuating its ability to hybridize to a complementary RNA target. Martin described a robust route to 2′-O-alkylribonucleosides in which the ribose 2′-OH group is substituted with 2′-ethylene glycol derivatives. Oligonucleotides containing these modifications displayed überraschende Eigenschaften – ‘surprising properties’ – notably, higher affinity and specificity for RNA substrates and greater stability to nucleases relative to their unmodified counterparts. Today, the 2′-ethylene glycol modification is universally known in the field as the 2′-O-methoxyethyl (MOE) modification. The chemistry features in four ASO drugs and many others in clinical trials. Here, we 1) summarize the synthesis of the MOE-modified ribose; 2) outline the properties of MOE-modified oligonucleotides as reported in Martin’s article; 3) highlight the first approved MOE-modified ASO drugs, mipomersen and nusinersen; and 4) survey MOE-modified ASOs in clinical development. In the outlook, we put these developments into context and consider future possibilities for the MOE modification.     

Synthesis and Base Pairing Properties of 1′,5′‐Anhydro‐L‐Hexitol Nucleic Acids (L‐HNA)

Oligonucleotides composed of 1′,5′‐anhydro‐arabino‐hexitol nucleosides belonging to the L series (L ‐HNA) were prepared and preliminarily studied as a novel potential base‐pairing system. Synthesis of enantiopure L ‐hexitol nucleotide monomers equipped with a 2′‐(N⁶‐benzoyladenin‐9‐yl) or a 2′‐(thymin‐1‐yl) moiety was carried out by a de novo approach based on a domino reaction as key step. The L oligonucleotide analogues were evaluated in duplex formation with natural complements as well as with unnatural sugar‐modified oligonucleotides. In many cases stable homo‐ and heterochiral associations were found. Besides T_m measurements, detection of heterochiral complexes was unambiguously confirmed by LC‐MS studies. Interestingly, circular dichroism measurements of the most stable duplexes suggested that L ‐HNA form left‐handed helices with both D and L oligonucleotides.     

Oligodeoxynucleotides Containing 2′‐Deoxy‐1‐methyladenosine and Dimroth Rearrangement

2′‐Deoxy‐1‐methyladenosine was incorporated into synthetic oligonucleotides by phosphoramidite chemistry. Chloroacetyl protecting group and controlled anhydrous deprotection conditions were used to avoid Dimroth rearrangement. Hybridization studies of intramolecular duplexes showed that introduction of a modified residue into the loop region of the oligonucleotide hairpin increases the melting temperature. It was shown that modified oligonucleotides may be easily transformed into oligonucleotides containing 2′‐deoxy‐N⁶‐methyladenosine.     

Functionalization of 2″‐C‐(Piperazinomethyl)‐2′,3′‐BcNA (Bicyclic Nucleic Acids) with Pyren‐1‐ylcarbonyl Units

Herein, we describe the incorporation of 2″‐C‐(piperazinomethyl)‐2′,3′‐BcNA (Bicyclic Nucleic Acids) into oligonucleotides via phosphoramidite chemistry and their subsequent solid‐phase functionalization with pyren‐1‐ylcarbonyl units after oligonucleotide synthesis. Thermal denaturation measurements showed that one modification led to increased thermal stability of the resulting duplex, and that two modifications could be incorporated in close proximity without decreasing the duplex stability (compared to the duplex stability of unmodified RNA). Fluorescence studies of the modified duplexes revealed that the structure and intensity of the fluorescence spectra were largely sequence‐dependent. Furthermore, molecular‐modeling studies showed that the pyrene moieties are placed in the major groove, and that the configuration at C(2″) is important for the thermal stability of the duplex.     

Synthesis of Photolabile 5′‐O‐Phosphoramidites for the Photolithographic Production of Microarrays of Inversely Oriented Oligonucleotides

The photolabile 3′‐O‐{[2‐(2‐nitrophenyl)propoxy]carbonyl}‐protected 5′‐phosphoramidites ( 16 – 18 ) were synthesized (see Scheme) for an alternative mode of light‐directed production of oligonucleotide arrays. Because of the characteristics of these monomeric building blocks, photolithographic in situ DNA synthesis occurred in 5′→3′ direction, in agreement with the orientation of enzymatic synthesis. Synthesis yields were as good as those of conventional reactions. The resulting oligonucleotides are attached to the surface via their 5′‐termini, while the 3′‐hydroxy groups are available as substrates for enzymatic reactions such as primer extension upon hybridization of a DNA template (see Fig. 2). The production of such oligonucleotide chips adds new procedural avenues to the growing number of applications of DNA microarrays.     

Synthesis,Characterization, and Repair of a Flexible O6‐2′‐Deoxyguanosine‐alkylene‐O6‐2′‐deoxyguanosine Intrastrand Cross‐Link

Oligonucleotides tethered by an alkylene linkage between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines, which lack a phosphodiester linkage between these residues, have been synthesized as a model system of intrastrand cross‐linked (IaCL) DNA. UV thermal denaturation studies of duplexes formed between these butylene‐ and heptylene‐linked oligonucleotides with their complementary DNA sequences revealed about 20 °C reduction in stability relative to the unmodified duplex. Circular dichroism spectra of the model IaCL duplexes displayed a signature characteristic of B‐form DNA, suggesting minimal global perturbations are induced by the lesion. The model IaCL containing duplexes were investigated as substrates of O⁶‐alkylguanine DNA alkyltransferase (AGT) proteins from human and E. coli (Ada‐C and OGT). Human AGT was found to repair both model IaCL duplexes with greater efficiency towards the heptylene versus butylene analog adding to our knowledge of substrates this protein can repair.     

Oligonucleotides Containing Novel 4′‐C‐ or 3′‐C‐(Aminoalkyl)‐Branched Thymidines

The synthesis of four novel 3′‐C‐branched and 4′‐C‐branched nucleosides and their transformation into the corresponding 3′‐O‐phosphoramidite building blocks for automated oligonucleotide synthesis is reported. The 4′‐C‐branched key intermediate 11 was synthesized by a convergent strategy and converted to its 2′‐O‐methyl and 2′‐deoxy‐2′‐fluoro derivatives, leading to the preparation of novel oligonucleotide analogues containing 4′‐C‐(aminomethyl)‐2′‐O‐methyl monomer X and 4′‐C‐(aminomethyl)‐2′‐deoxy‐2′‐fluoro monomer Y (Schemes 2 and 3). In general, increased binding affinity towards complementary single‐stranded DNA and RNA was obtained with these analogues compared to the unmodified references (Table 1). The presence of monomer X or monomer Y in a 2′‐O‐methyl‐RNA oligonucleotide had a negative effect on the binding affinity of the 2′‐O‐methyl‐RNA oligonucleotide towards DNA and RNA. Starting from the 3′‐C‐allyl derivative 28 , 3′‐C‐(3‐aminopropyl)‐protected nucleosides and 3′‐O‐phosphoramidite derivatives were synthesized, leading to novel oligonucleotide analogues containing 3′‐C‐(3‐aminopropyl)thymidine monomer Z or the corresponding 3′‐C‐(3‐aminopropyl)‐2′‐O,5‐dimethyluridine monomer W (Schemes 4 and 5). Incorporation of the 2′‐deoxy monomer Z induced no significant changes in the binding affinity towards DNA but decreased binding affinity towards RNA, while the 2′‐O‐methyl monomer Z induced decreased binding affinity towards DNA as well as RNA complements (Table 2).     

Diffusion of Oligonucleotides from within Iron‐Cross‐Linked,Polyelectrolyte‐Modified Alginate Beads: A Model System for Drug Release

An analytical model to describe diffusion of oligonucleotides from stable hydrogel beads is developed and experimentally verified. The synthesized alginate beads are Fe³⁺‐cross‐linked and polyelectrolyte‐doped for uniformity and stability at physiological pH. Data on diffusion of oligonucleotides from inside the beads provide physical insights into the volume nature of the immobilization of a fraction of oligonucleotides due to polyelectrolyte cross‐linking, that is, the absence of a surface‐layer barrier in this case. Furthermore, the results suggest a new simple approach to measuring the diffusion coefficient of mobile oligonucleotide molecules inside hydrogels. The considered alginate beads provide a model for a well‐defined component in drug‐release systems and for the oligonucleotide‐release transduction steps in drug‐delivering and biocomputing applications. This is illustrated by destabilizing the beads with citrate, which induces full oligonucleotide release with nondiffusional kinetics.     

Nucleotides,Part LXIX,Synthesis of Phosphoramidite Building Blocks of Isoxanthopterin N8‐(2′‐Deoxy‐β‐D‐ribonucleosides): New Fluorescence Markers for Oligonucleotide Synthesis

The chemical synthesis of isoxanthopterin and 6‐phenylisoxanthopterin N⁸‐(2′‐deoxy‐β‐D ‐ribofuranosyl nucleosides) is described as well as their conversion into suitably protected 3′‐phosphoramidite building blocks to be used as marker molecules for DNA synthesis. Applying the npe/npeoc (=2‐(4‐nitrophenyl)ethyl/[2‐(4‐nitrophenyl)ethoxy]carbonyl) strategy, we used the new building blocks in the preparation of oligonucleotides by an automated solid‐support approach. The hybridization properties of a series of labelled oligomers were studied by UV‐melting techniques. It was found that the newly synthesized markers only slightly interfered with the abilities of the labelled oligomers to form stable duplexes with complementary oligonucleotides.     

5‐Aza‐7‐deazaguanine DNA: Recognition and Strand Orientation of Oligonucleotides Incorporating Anomeric Imidazo[1,2‐a]‐1,3,5‐triazine Nucleosides

The base‐pairing properties of oligonucleotides containing the anomeric 5‐aza‐7‐deazaguanine 2′‐deoxyribonucleosides 1 and 5 are described. The oligonucleotides were prepared by solid‐phase synthesis, employing phosphoramidite or phosphonate chemistry. Stable `purine'⋅purine duplexes with antiparallel (aps) chain orientation are formed, when the α‐D ‐anomer 5 alternates with the β‐D ‐anomeric 2′‐deoxyguanosine ( 2 ) within the same oligonucleotide chain. Parallel (ps) oligonucleotide duplexes are observed, when the β‐D anomer 1 alternates with 2 . A renewed reversal of the chain orientation (ps→aps) occurs when compound 1 pairs with 2′‐deoxyisoguanosine ( 6 ). In all cases, it is unnecessary to change the orientation within a single strand when α‐D units alternate with their β‐D counterparts. Heterochiral base pairs of 5 (α‐D ) with 2′‐deoxyisoguanosine (β‐D ) are well accommodated in duplexes with random base composition and parallel chain orientation. Base pairs of 5 (α‐D ) with 2′‐deoxyguanosine (β‐D ) destabilize duplexes with antiparallel chains.     

2′‐Ethynyl‐DNA: Synthesis and Pairing Properties

2‐Ethynyl‐DNA was developed as a potential DNA‐selective oligonucleotide analog. The synthesis of 2′‐arabino‐ethynyl‐modified nucleosides was achieved starting from properly protected 2′‐ketonucleosides by addition of lithium (trimethylsilyl)acetylide followed by reduction of the tertiary alcohol. After a series of protecting‐group manipulations, phosphoramidite building blocks suitable for solid‐phase synthesis were obtained. The synthesis of oligonucleotides from these building blocks was successful when a fast deprotection scheme was used. The pairing properties of 2′‐arabino‐ethynyl‐modified oligonucleotides can be summarized as follows: 1) The 2′‐arabino‐ethynyl modification of pyrimidine nucleosides leads to a strong destabilization in duplexes with DNA as well as with RNA. The likely reason is that the ethynyl group sterically influences the torsional preferences around the glycosidic bond leading to a conformation not suitable for duplex formation. 2) If the modification is introduced in purine nucleosides, no such influence is observed. The pairing properties are not or only slightly changed, and, in some cases (deoxyadenosine homo‐polymers), the desired stabilization of the pairing with a DNA complementary strand and destabilization with an RNA complement is observed. 3) In oligonucleotides of alternating deoxycytidine‐deoxyguanosine sequence, the incorporation of 2′‐arabino‐ethynyl deoxyguanosine surprisingly leads to the formation of a left‐handed double helix, irrespective of salt concentration. The rationalization for this behavior is that the ethynyl group locks such duplexes in a left‐handed conformation through steric blockade.     

Oligodeoxynucleotide Duplexes Containing (5′S)‐5′‐C‐Alkyl‐Modified 2′‐Deoxynucleosides: Can an Alkyl Zipper across the DNA Minor‐Groove Enhance Duplex Stability?

A series of oligonucleotides containing (5′S)‐5′‐C‐butyl‐ and (5′S)‐5′‐C‐isopentyl‐substituted 2′‐deoxyribonucleosides were designed, prepared, and characterized with the intention to explore alkyl‐zipper formation between opposing alkyl chains across the minor groove of oligonucleotide duplexes as a means to modulate DNA‐duplex stability. From four possible arrangements of the alkyl groups that differ in the density of packing of the alkyl chains across the minor groove, three (duplex types I – III , Fig. 2) could experimentally be realized and their duplex‐forming properties analyzed by UV‐melting curves, CD spectroscopy, and isothermal titration calorimetry (ITC), as well as by molecular modeling. The results show that all arrangements of alkyl residues within the minor groove of DNA are thermally destabilizing by 1.5–3°/modification in T_m. We found that, within the proposed duplexes with more loosely packed alkyl groups (type‐ III duplexes), accommodation of alkyl residues without extended distorsion of the helical parameters of B‐DNA is possible but does not lead to higher thermodynamic stability. The more densely packed and more unevenly distributed arrangement (type‐ II duplexes) seems to suffer from ecliptic positioning of opposite alkyl groups, which might account for a systematic negative contribution to stability due to steric interactions. The decreased stability in the type‐ III duplexes described here may be due either to missing hydrophobic interactions of the alkyl groups (not bulky enough to make close contacts), or to an overcompensation of favorable alkyl‐zipper formation presumably by loss of structured H₂O in the minor groove.     

(2′‐O‐Methyl‐RNA)‐3′‐PNA Chimeras: A New Class of Mixed Backbone Oligonucleotide Analogues with High Binding Affinity to RNA

The automated on‐line synthesis of DNA‐3′‐PNA chimeras 1 – 4 and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras 5 – 8 is described, in which the 3′‐terminal part of the oligonucleotide is linked to the N‐terminal part of the PNA via N‐(ω‐hydroxyalkyl)‐N‐[(thymin‐1‐yl)acetyl]glycine units (alkyl=Et, Ph, Bu, and pentyl). By means of UV thermal denaturation, the binding affinities of all chimeras were directly compared by determining their T_m values in the duplex with complementary DNA and RNA. All investigated DNA‐3′‐PNA chimeras and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras form more‐stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. Interestingly, a N‐(3‐hydroxypropyl)glycine linker resulted in the highest binding affinity for DNA‐3′‐PNA chimeras, whereas the (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras showed optimal binding with the homologous N‐(4‐hydroxybutyl)glycine linker. The duplexes of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras and RNA were significantly more stable than those containing the corresponding DNA‐3′‐PNA chimeras. Surprisingly, we found that the charged (2′‐O‐methyl‐RNA)‐3′‐PNA chimera with a N‐(4‐hydroxybutyl)glycine‐based unit at the junction to the PNA part shows the same binding affinity to RNA as uncharged PNA. Potential applications of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras include their use as antisense agents acting by a RNase‐independent mechanism of action, a prerequisite for antisense‐oligonucleotide‐mediated correction of aberrant splicing of pre‐mRNA.     

Oligonucleotides Containing 7‐Deaza‐2′‐deoxyinosine as Universal Nucleoside: Synthesis of 7‐Halogenated and 7‐Alkynylated Derivatives,Ambiguous Base Pairing,and Dye Functionalization by the Alkyne–Azide ‘Click’ Reaction

Oligonucleotides containing 7‐deaza‐2′‐deoxyinosine derivatives bearing 7‐halogen substituents or 7‐alkynyl groups were prepared. For this, the phosphoramidites 2b – 2g containing 7‐substituted 7‐deaza‐2′‐deoxyinosine analogues 1b – 1g were synthesized (Scheme 2). Hybridization experiments with modified oligonucleotides demonstrate that all 2′‐deoxyinosine derivatives show ambiguous base pairing, as 2′‐deoxyinosine does. The duplex stability decreases in the order C_d>A_d>T_d>G_d when 2b – 2g pair with these canonical nucleosides (Table 6). The self‐complementary duplexes 5′‐d(F⁷c⁷I‐C)₆, d(Br⁷c⁷I‐C)₆, and d(I⁷c⁷I‐C)₆ are more stable than the parent duplex d(c⁷I‐C)₆ (Table 7). An oligonucleotide containing the octa‐1,7‐diyn‐1‐yl derivative 1g , i.e., 27 , was functionalized with the nonfluorescent 3‐azido‐7‐hydroxycoumarin ( 28 ) by the Huisgen–Sharpless–Meldal cycloaddition ‘click’ reaction to afford the highly fluorescent oligonucleotide conjugate 29 (Scheme 3). Consequently, oligonucleotides incorporating the derivative 1g bearing a terminal C?C bond show a number of favorable properties: i) it is possible to activate them by labeling with reporter molecules employing the ‘click’ chemistry. ii) Space demanding residues introduced in the 7‐position of the 7‐deazapurine base does not interfere with duplex structure and stability (Table 8). iii) The ambiguous pairing character of the nucleobase makes them universal probes for numerous applications in oligonucleotide chemistry, molecular biology, and nanobiotechnology.     

Electron Attachment to Hydrated Oligonucleotide Dimers: Guanylyl‐3′,5′‐Cytidine and Cytidylyl‐3′,5′‐Guanosine

The dinucleoside phosphate deoxycytidylyl‐3′,5′‐deoxyguanosine (dCpdG) and deoxyguanylyl‐3′,5′‐deoxycytidine (dGpdC) systems are among the largest to be studied by reliable theoretical methods. Exploring electron attachment to these subunits of DNA single strands provides significant progress toward definitive predictions of the electron affinities of DNA single strands. The adiabatic electron affinities of the oligonucleotides are found to be sequence dependent. Deoxycytidine (dC) on the 5′ end, dCpdG, has larger adiabatic electron affinity (AEA, 0.90 eV) than dC on the 3′ end of the oligomer (dGpdC, 0.66 eV). The geometric features, molecular orbital analyses, and charge distribution studies for the radical anions of the cytidine‐containing oligonucleotides demonstrate that the excess electron in these anionic systems is dominantly located on the cytosine nucleobase moiety. The π‐stacking interaction between nucleobases G and C seems unlikely to improve the electron‐capturing ability of the oligonucleotide dimers. The influence of the neighboring base on the electron‐capturing ability of cytosine should be attributed to the intensified proton accepting–donating interaction between the bases. The present investigation demonstrates that the vertical detachment energies (VDEs) of the radical anions of the oligonucleotides dGpdC and dCpdG are significantly larger than those of the corresponding nucleotides. Consequently, reactions with low activation barriers, such as those for O? C σ bond and N‐glycosidic bond breakage, might be expected for the radical anions of the guanosine–cytosine mixed oligonucleotides.     

Floxuridine Homomeric Oligonucleotides “Hitchhike” with Albumin In Situ for Cancer Chemotherapy

Automated attachment of chemotherapeutic drugs to oligonucleotides through phosphoramidite chemistry and DNA synthesis has emerged as a powerful technology in constructing structure‐defined and payload‐tunable oligonucleotide–drug conjugates. In practice, however, in vivo delivery of these oligonucleotides remains a challenge. Inspired by the systemic transport of hydrophobic payloads by serum albumin in nature, we report the development of a lipid‐conjugated floxuridine homomeric oligonucleotide (LFU20) that “hitchhikes” with endogenous serum albumin for cancer chemotherapy. Upon intravenous injection, LFU20 immediately inserts into the hydrophobic cave of albumin to form an LFU20/albumin complex, which accumulates in the tumor by the enhanced permeability and retention (EPR) effect and internalizes into the lysosomes of cancer cells. After degradation, cytotoxic floxuridine monophosphate is released to inhibit cell proliferation.     

Synthesis and Pairing Properties of 3′‐Deoxyribopyranose (4′→2′)‐Oligonucleotides (‘p‐DNA')

The preparation and the pairing properties of the new 3′‐deoxyribopyranose (4′→2′)‐oligonucleotide (=p‐DNA) pairing system, based on 3′‐deoxy‐β‐D ‐ribopyranose nucleosides is presented. D ‐Xylose was efficiently converted to the prefunctionalized 3‐deoxyribopyranose derivative 4‐O‐[(tert‐butyl)dimethylsilyl]‐3‐deoxy‐D ‐ribopyranose 1,2‐diacetate 8 (obtained as a 4 : 1 mixture of α‐ and β‐D ‐anomers; Scheme 1). From this sugar building block, the corresponding, appropriately protected thymine, guanine, 5‐methylcytosine, and purine‐2,6‐diamine nucleoside phosphoramidites 29 – 32 were prepared in a minimal number of steps (Schemes 2–4). These building blocks were assembled on a DNA synthesizer, and the corresponding p‐DNA oligonucleotides were obtained in good yields after a one‐step deprotection under standard conditions, followed by HPLC purification (Scheme 5 and Table 1). Qualitatively, p‐DNA shows the same pairing behavior as p‐RNA, forming antiparallel, exclusively Watson‐Crick‐paired duplexes that are much stronger than corresponding DNA duplexes. Duplex stabilities within the three related (i.e., based on ribopyranose nucleosides) oligonucleotide systems p‐RNA, p‐DNA, and 3′‐O‐Me‐p‐RNA were compared with each other (Table 2). Intrinsically, p‐RNA forms the strongest duplexes, followed by p‐DNA, and 3′‐O‐Me‐p‐RNA. However, by introducing the nucleobases purine‐2,6‐diamine (D) and 5‐methylcytosine (M) instead of adenine and cytosine, a substantial increase in stability of corresponding p‐DNA duplexes was observed.