molecules

Review Synthesis of Nucleobase-Modified RNA Oligonucleotides by Post-Synthetic Approach

Karolina Bartosik , Katarzyna Debiec , Anna Czarnecka , Elzbieta Sochacka and Grazyna Leszczynska *

Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland; [email protected] (K.B.); [email protected] (K.D.); [email protected] (A.C.); [email protected] (E.S.) * Correspondence: [email protected]; Tel.: +48-42-6313150



Academic Editor: Katherine Seley-Radtke
 Received: 20 June 2020; Accepted: 20 July 2020; Published: 23 July 2020

Abstract: The chemical synthesis of modified oligoribonucleotides represents a powerful approach to study the structure, stability, and biological activity of RNAs. Selected RNA modifications have been proven to enhance the drug-like properties of RNA oligomers providing the oligonucleotide-based therapeutic agents in the antisense and siRNA technologies. The important sites of RNA modification/functionalization are the nucleobase residues. Standard phosphoramidite RNA chemistry allows the site-specific incorporation of a large number of functional groups to the nucleobase structure if the building blocks are synthetically obtainable and stable under the conditions of oligonucleotide chemistry and work-up. Otherwise, the chemically modified RNAs are produced by post-synthetic oligoribonucleotide functionalization. This review highlights the post-synthetic RNA modification approach as a convenient and valuable method to introduce a wide variety of nucleobase modifications, including recently discovered native hypermodified functional groups, fluorescent dyes, photoreactive groups, disulfide crosslinks, and nitroxide spin labels.

Keywords: phosphoramidite chemistry; RNA solid-phase synthesis; post-synthetic RNA modification; convertible nucleoside

1. Introduction Recently, a significant interest has been observed in the use of modified oligoribonucleotides in the fields of molecular biology, biochemistry, and medicine [1–3]. The representative sites of RNA modifications are the 50- and 30-ends of oligoribonucleotides, and the 20-positions of the ribose, phosphodiester residues, and nucleobase moieties. Notably, among 150 modified nucleosides identified in cellular RNA sequences, more than 95% of functional groups are installed in the nucleobase heterocycles (Figure1)[ 4,5]. Improved mass spectrometry approaches and highly sensitive chemical screens in cellular RNA isolation are still extending this list with new base-modifying units, e.g., cyclic N6-threonylcarbamoyladenosines (ct6A, ms2ct6A) [6,7], 2-methylthiomethylenethio-N6-isopentenyl adenosine (msms2i6A) [8], uniquely lipophilic 5-substituted 2-geranylthiouridines (mnm5ges2U, cmnm5ges2U) [9] and 2-methylthiocytidine (ms2C) [10]. RNA modifications affect the structure, stability, and biological activity of RNA biomolecules, particularly the translation process [11]. Experiments of the site-specific incorporation of nucleic base modifications have proved to be a particularly useful method for precisely assessing the role of various functional groups in the structure, function, and biosynthesis of RNA molecules [12–17].

Molecules 2020, 25, 3344; doi:10.3390/molecules25153344 www.mdpi.com/journal/molecules Molecules 2020, 25, 3344 2 of 37 Molecules 2020, 25, x FOR PEER REVIEW 2 of 37

FigureFigure 1. 1.Modified Modified positions positions distributed distributed in naturally existing existing RNA RNA nucleobases; nucleobases; Rb: Rb: ribose. ribose.

ValuableValuable data data on on RNA RNA structure structure andand cellularcellular activity can can be be also also extracted extracted from from biochemical, biochemical, biophysical,biophysical, and and structural structural studies studies involving involving unnatural unnatural base-modified base-modified RNARNA constructs.constructs. The The insertion insertion of artificialof artificial purine purine/pyrimidine/pyrimidine in the in place the ofplace naturally of naturally existing existing nucleosides nucleosides may change may change the local the hydrogen local bondinghydrogen system, bonding stacking system, interactions, stacking interactions, or puckering or puckering of ribose of residues, ribose residues, giving answersgiving answers as to which as modificationsto which modifications or interactions or are interactions essential for are the essential functional for RNAthe functional structure RNA [18–20 structure]. Important [18–20]. details onImportant RNA structure, details conformational on RNA structure, dynamics, conformational and RNA’s dynamics, homo- and and RNA’s heterotropic homo- and interactions heterotropic can be providedinteractions by oligoribonucleotides can be provided by oligoribonucleotides site-specifically labeled site-specifically with fluorescent labeled dyes,with fluorescent nitroxide radicals,dyes, nitroxide radicals, disulfide crosslinks, or photolabile groups [21–28]. disulfide crosslinks, or photolabile groups [21–28]. For years, chemically modified oligonucleotides have been extensively studied for the development For years, chemically modified oligonucleotides have been extensively studied for the development of new antisense and siRNA therapeutics [1,29–31]. Several nucleobase modifications were found to of new antisense and siRNA therapeutics [1,29–31]. Several nucleobase modifications were found improve the drug-like properties of oligonucleotides such as cellular uptake, stability in the cell, target to improvespecificity, the and drug-like binding affinity properties [31–33], of and oligonucleotides reduce undesirable such protein as cellular binding uptake, and immune stability stimulation in the cell, target[34,35]. specificity, Gratifyingly, and bindingthe incorporation affinity [of31 the–33 first-generation], and reduce undesirableinternucleotide protein linkage binding modifications and immune and stimulationsecond-generation [34,35]. sugar Gratifyingly, modifications the resulted incorporation in the current of the approval first-generation of two RNAi-based internucleotide therapeutics, linkage modificationsOnpattro (patisiran) and second-generation and Givlaari (givosiran) sugar [31]. modifications resulted in the current approval of two RNAi-basedGrowing therapeutics, interest in Onpattrothe modified (patisiran) RNA oligomers and Givlaari creates (givosiran) the need [31to ].develop chemistry that permitsGrowing the site-specific interest in theintroduction modified of RNA functionalizable oligomers groups creates into the RNA. need toThe develop most general chemistry way to that permitsaddress the the site-specific issue of site introduction selectivity is of through functionalizable the use of groups solid-phase into RNA. synthesis The phosphoramidite most general way to addresschemistry, the which issue of involves site selectivity two strategies: is through (1) the classical use of modified solid-phase monomer synthesis approach phosphoramidite via the chemistry,preparation which of a involves modified two phosphoramidite strategies: (1) classicalbuilding modifiedblock and monomer its subsequent approach incorporation via the preparation into the of aRNA modified chain; phosphoramidite and (2) a post-synthetic building RNA block modification and its subsequent approach incorporation based on selective into the chemicalRNA chain; andreaction(s) (2) a post-synthetic of precursor RNA unit(s) modification in the full-length approach oligonucleotide based on selective prepared chemical by the reaction(s) classical method. of precursor unit(s) inThis the review full-length for oligonucleotidethe first time summarizes prepared by the the methods classical method.of site-specific incorporation of nucleobase-modified units into RNA oligomers via the post-synthetic strategy. We focused on the This review for the first time summarizes the methods of site-specific incorporation of nucleobase functionalization due to the high abundance of nucleobase modifications in the nature nucleobase-modified units into RNA oligomers via the post-synthetic strategy. We focused on and the high convenience of the post-synthetic strategy to construct the nucleobase-labeled chemical the nucleobase functionalization due to the high abundance of nucleobase modifications in the nature or biophysical probes. The scope of post-synthetically reactive precursor oligonucleotides has been andlimited the high to RNA convenience oligomers of prepared the post-synthetic by the phosphoramidite strategy to construct chemistry. the To nucleobase-labeled facilitate the selection chemical of or biophysicalpost-synthetic probes. methods The best scope suited of to post-synthetically the assumed chemical–biological reactive precursor objectives, oligonucleotides a large number has of been limitedexperimental to RNA details oligomers have prepared been included. by the phosphoramidite chemistry. To facilitate the selection of post-synthetic methods best suited to the assumed chemical–biological objectives, a large number of experimental2. Solid-Phase details Synthesis have been of Modified included. RNA Oligomers via Phosphoramidite Chemistry

2. Solid-PhaseThe most Synthesis current method of Modified for the RNA incorporation Oligomers of via a modified Phosphoramidite nucleoside Chemistry into a site-specific position of oligoribonucleotides is the phosphoramidite chemistry, which was introduced by CaruthersThe most in current 1981 [36,37]. method The for preparation the incorporation of RNA of oligomers a modified by nucleoside this strategy into involves a site-specific a four-step position of oligoribonucleotidesreaction cycle including is the 5′-deblocking, phosphoramidite coupling, chemistry, capping, which and oxidation was introduced steps (Scheme by Caruthers 1). The in 1981synthesis [36,37]. Thetakes preparation the 3′→5′ of direction RNA oligomers and starts by thisfrom strategy the 5′-deprotection involves a four-step of fully reaction protected cycle includingribonucleoside 50-deblocking, attached coupling,to a solid support capping, (in andstandard, oxidation controlled-pore steps (Scheme glass,1 or). polystyrene The synthesis resins) takes thevia 30 the50 3′-hydroxyldirection and group. starts In the from second the 5 step,0-deprotection activation and of fully coupling, protected the nucleoside ribonucleoside deprived attached of the to 5′-protecting→ group reacts with a suitably protected phosphoramidite building block activated by the a solid support (in standard, controlled-pore glass, or polystyrene resins) via the 30-hydroxyl group. 5-substituted 1H-tetrazole or imidazole derivatives, yielding an unstable phosphite triester linkage. In the second step, activation and coupling, the nucleoside deprived of the 50-protecting group reacts withSince a suitably a small protectednumber of phosphoramidite unreacted 5′-hydroxyl building sites block remain activated active after by the coupling, 5-substituted in the third 1H-tetrazole step, capping, the acylating reagent is introduced to prevent them from reacting with the next monomeric or imidazole derivatives, yielding an unstable phosphite triester linkage. Since a small number of unit and elongating the missense strands. In the oxidation step, the support-linked dinucleoside unreacted 50-hydroxyl sites remain active after coupling, in the third step, capping, the acylating reagent is introduced to prevent them from reacting with the next monomeric unit and elongating Molecules 2020, 25, 3344 3 of 37

Molecules 2020, 25, x FOR PEER REVIEW 3 of 37 the missense strands. In the oxidation step, the support-linked dinucleoside phosphite triester P(III) is convertedphosphite totriester the more P(III) stable is converted phosphate to the triester more stable P(V) withphosphate an oxidation triester agent,P(V) with typically an oxidation iodine or tertagent,-butylhydroperoxide typically iodine or solutions. tert-butylhydroperoxide solutions.

SchemeScheme 1. General1. General scheme scheme of of RNA RNA solid-phase solid-phase synthetic synthetic cycle.cycle. R 1, RR2,2 R,R4: 4protecting: protecting groups groups for for 5′-OH, 50-OH, 20-OH,2′-OH, and and phosphate phosphate residue, residue, respectively; respectively; B(RB(R33): protected protected nucleobase; nucleobase; R5 R: 5capping: capping group. group.

TheThe removal removal of of the the 5′-protecting 50-protecting group group initiates initiates the the next next chain chain extension extension cycle. cycle. Repeating Repeating the the syntheticsynthetic cycle cycle provides provides a a full-length full-length oligonucleotideoligonucleotide (prepared (prepared in in the the trityl-on trityl-on or ortrityl-off trityl-o mode),ff mode), whichwhich can can be be released released from from the the solid solid support support andand deprotected.deprotected. ForFor successful successful solid-phase solid-phase RNA RNA synthesis, synthesis, the phosporamidite monomeric monomeric units units must must be be protected with a combination of orthogonal R1 transient and R2, R3, R4 permanent protecting groups. protected with a combination of orthogonal R1 transient and R2,R3,R4 permanent protecting groups.Several Several synthetic synthetic protocols, protocols, systems systems of protecting of protecting groups, groups, and deprotection and deprotection strategies strategies have been have demonstrated so far [38]. In practice, the building blocks are protected by employing one of the five been demonstrated so far [38]. In practice, the building blocks are protected by employing one of most common strategies: (1) 5’-O-DMTr-2’-O-TBDMS [39–41]; (2) 5’-O-DMTr-2’-O-TOM [42]; (3) 5’- the five most common strategies: (1) 5’-O-DMTr-2’-O-TBDMS [39–41]; (2) 5’-O-DMTr-2’-O-TOM [42]; O-DMTr-2’-O-Fpmp [43]; (4) 5’-O-DMTr-2’-O-TC [44], and (5) 5’-O-DOD(BzH)-2’-O-ACE [45,46] (3) 5’-O-DMTr-2’-O-Fpmp [43]; (4) 5’-O-DMTr-2’-O-TC [44], and (5) 5’-O-DOD(BzH)-2’-O-ACE [45,46] (Figure 2). (Figure2). Most of the synthetic approaches involve 30-O-β-cyanoethyldiisopropylphosphoramidites containing the transient, acid-labile 50-O-dimethoxytrityl group (DMTr) and permanent 20-protection such as silyl blockage (TBDMS or TOM, Figure2a,b), the acetal masking group (Fpmp, Figure2c) or carbothioate (TC, Figure2d). The deprotection of trityl-o ff RNA oligomers prepared via TBDMS/TOM and Fpmp chemistry starts from the removal of base-labile protecting groups from the phosphotriester backbone and exoamino functions of nucleobases (usually acyl or aminomethylene blockage (Figure2g). Typically, ammonolytic conditions are used such as aqueous (aq.) ammonia or aq. ammonia–methylamine solution (AMA). This alkaline step of deprotection runs simultaneously with the support cleavage. Optionally, the removal of β-cyanoethyl phosphate groups can be performed separately before the alkaline deprotection step using a weak base in an organic solvent (e.g., TEA/acetonitrile, 1:1 v/v or 10% diethylamine in acetonitrile). Separation of the released acrylonitrile by-product prevents the formation of 2-cyanoethyl-containing oligomer adducts observed when the strong basic conditions of RNA deprotection are used. Removal of the 20-protecting groups is performed as a final step of oligomer deprotection in the presence of fluoride agent (TBAF or Figure 2. The most common ribonucleoside phosphoramidite building blocks for solid-phase RNA TEA 3HF) for 20-silyl groups or under acidic conditions (AcOH) when 20-Fpmp acetal blockage is used. · synthesis; (a) 5′-O-DMTr–2′-O-TBDMS–3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite); (b) 5′- Molecules 2020, 25, x FOR PEER REVIEW 3 of 37

phosphite triester P(III) is converted to the more stable phosphate triester P(V) with an oxidation agent, typically iodine or tert-butylhydroperoxide solutions.

Scheme 1. General scheme of RNA solid-phase synthetic cycle. R1, R2, R4: protecting groups for 5′-OH, 2′-OH, and phosphate residue, respectively; B(R3): protected nucleobase; R5: capping group.

The removal of the 5′-protecting group initiates the next chain extension cycle. Repeating the synthetic cycle provides a full-length oligonucleotide (prepared in the trityl-on or trityl-off mode), which can be released from the solid support and deprotected. For successful solid-phase RNA synthesis, the phosporamidite monomeric units must be Moleculesprotected2020, 25with, 3344 a combination of orthogonal R1 transient and R2, R3, R4 permanent protecting groups.4 of 37 Several synthetic protocols, systems of protecting groups, and deprotection strategies have been demonstrated so far [38]. In practice, the building blocks are protected by employing one of the five Themost deprotection common strategies: of the TC-blocked (1) 5’-O-DMTr-2’- support-boundO-TBDMS oligonucleotide [39–41]; (2) 5’-O-DMTr-2’- is carriedO out-TOM on a[42] synthesizer; (3) 5’- columnO-DMTr-2’- in oneO step-Fpmp by [ filling43]; (4) the 5’- columnO-DMTr-2’- withO-TC neat [44], ethylenediamine. and (5) 5’-O-DOD(BzH)-2’- After TC removal,O-ACE the [45,46] “free” oligomer(Figure is 2). washed from the column using a small volume of water.

FigureFigure 2. 2. TheThe most common common ribonucleoside ribonucleoside phosphoramidite phosphoramidite building building blocks blocksfor solid-phase for solid-phase RNA

RNAsynthesis; synthesis; (a) 5′- (aO) 5-DMTr–2′-0-O-DMTr–2O-TBDMS–3′-0-O-TBDMS–3O-(2-cyanoethyl-0-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite);N,N-diisopropylphosphoramidite); (b) 5′- (b) 50-O-DMTr–20-O-TOM–30-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite); (c) 50-O-DMTr–20- O-Fpmp–30-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite); (d) 50-O-DMTr–20-O-TC-30-O-(2- cyanoethyl-N,N-diisopropylphosphoramidite); (e) 50-O-DOD–20-O-ACE–30-O-(methyl-N,N- diisopropyl) phosphoramidite; (f) 30-O-DMTr-20-O-TBDMS-50-O-(2-cyanoethyl-N,N-diisopropylphosphor-amidite) -reverse RNA phosphoramidite; (g) commonly used nucleobase protecting groups.

An alternative approach for the orthogonal protection of the 50- and 20-hydroxyl groups of monomeric units (prepared as methyl diisopropylphosphoramidites) is based on fluoro-labile 50-O-silyl protecting groups (DOD or BzH) and acid-labile 20-O-bis(2-acetoxyethoxy)methyl ortoester protection (ACE, Figure2e) [ 45,46]. The exocyclic amino functions of nucleobases are masked with base-labile acyl protecting groups. After 50-O-DOD/BzH removal (HF/TEA), the oligomer is subjected to a three-step deprotection protocol involving the release of phosphate groups by disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate (S2Na2), the removal of base-labile protecting groups, including acetyl groups from 20-O-ACE blockage using methylamine solution with simultaneous resin cleavage, and finally, the removal of acid-labile 20-O-bis(2-hydroxyethoxy)methyl orthoesters with AcOH/TEMED. In addition to the classical phosphoramidite approach proceeding in a 3 5 direction, interest has 0→ 0 also been focused on the RNA synthesis in the reverse direction (5 3 )[47,48]. This approach 0→ 0 utilizes 50-O-phosphoramidites (Figure2f) with a suitable N-protecting group (Bz for adenine, cytosine, Ac for cytosine, and iBu for guanine) and a 30-O-DMTr-20-O-TBDMS blockage of sugar residue. Activated 50-O-phosphoramidite is coupled with a nucleoside bearing a 50 succinate of ribonucleoside attached to a solid support leading (after oxidation) to the dimer formation with phophodiester linkage and a 30-O-DMTr-protecting group. The removal of DMTr opens the next synthetic cycle. Oligonucleotide deprotection is performed using the same standard TBDMS chemistry currently utilized in the conventional 3 5 synthesis. Although the reverse RNA synthesis is used to 0→ 0 a lesser extent than the conventional strategy, it offers a facile route to the assembly of 30-conjugated RNA constructs of long and medium lengths. Importantly, it was demonstrated that the use of 30-O-DMTr-50-phosphoramidites enhances the coupling efficiency, which surpassed 99% per step leading to high-purity RNA products [48]. These distinct advantages of 5 3 direction synthesis 0→ 0 made the reverse 30-O-DMTr-50-phosphoramidites commercially available. Molecules 2020, 25, 3344 5 of 37

After deprotection, synthetic oligomers (obtained in both conventional and reverse strategies) are purified using polyacrylamide gel electrophoresis (PAGE) or high-performance liquid chromatography (RP HPLC, IE HPLC) and identified by electrospray ionization mass spectrometry (ESI),Molecules matrix-assisted 2020, 25, x FOR PEER laser REVIEW desorption /ionization-time of flight mass spectrometry (MALDI-ToF), 5 of 37 and enzymatic digestion analysis. The solid-phase phosphoramidite method is limited to synthesizing RNA oligomers with lengthsThe solid-phase of 50–100 nucleotides phosphoramidite (longer methodRNAs molecules is limited can to synthesizing be synthesized RNA by oligomersthe enzymatic with ligation lengths of 50–100of chemically nucleotides modified (longer RNA RNAs fragment(s) molecules using can be T4 synthesized DNA or T4 byRNA the ligases enzymatic [49–51] ligation or DNA-zymes of chemically modified[52]). In RNA response fragment(s) to the using clinical T4 and DNA commercial or T4 RNA success ligases of [49 the–51 therapeutic] or DNA-zymes oligonucleotides, [52]). In response the to theclassical clinical phosphoramidite and commercial protocols success of were the therapeutictranslated for oligonucleotides, use in the large-scale the classical synthesis phosphoramidite [53]. Using protocolsautomated were translated computer-controlled for use in the synthesizers, large-scale synthesis the incorporation [53]. Using automated of several computer-controlled modifications synthesizers,(phosphorothioate the incorporation internucleotide of several linkage, modifications 2′-O-methoxyethyl, (phosphorothioate 2′-O-Me, 2′-F, internucleotidelocked-, morpholino- linkage, 20-Oand-methoxyethyl, Spiegelmer building 20-O-Me, blocks) 20-F, locked-, was scaled morpholino- up from gram- and Spiegelmer to kilogram-scale building production blocks) was under scaled upCurrent from gram- Good to Manufacturing kilogram-scale Practice production (cGMP). under Current Good Manufacturing Practice (cGMP).

3. Post-Synthetic3. Post-Synthetic Strategy Strategy for for Nucleobase Nucleobase RNARNA Modifications Modifications FirstFirst protocols protocols for for the the post-synthetic post-synthetic functionalizationfunctionalization of of nucleobases nucleobases were were elaborated elaborated for forDNA DNA oligomersoligomers about about three three decades decades ago ago [ 54[54–59].–59]. TheThe general concept concept of of post-synthetic post-synthetic RNA RNA modification modification approachapproach relies relies on on the the preparation preparation of of anan easilyeasily convertibleconvertible/reactive/reactive precursor precursor oligonucleotide oligonucleotide by bythe the chemicalchemical method, method, e.g., e.g., phosphoramidite phosphoramidite chemistry, chemistry, and its and subsequent its subsequent derivatization derivatization through through chemical chemical reaction(s) involving the reactive center(s) of precursor nucleoside(s). The representative reaction(s) involving the reactive center(s) of precursor nucleoside(s). The representative sites of sites of post-synthetic modifications are 5′- and 3′-ends of RNA oligomers, the 2′-position of sugar, post-synthetic modifications are 5 - and 3 -ends of RNA oligomers, the 2 -position of sugar, phosphate phosphate linkage, and nucleobase0 moieties0 (Figure 3). The derivatization0 of nucleobases attends the linkage, and nucleobase moieties (Figure3). The derivatization of nucleobases attends the C-4, C-5, C-4, C-5, and N-3 positions of uracil, the C-5 position of cytosine, the C-2 position of adenine, and the andC-8 N-3 and positions N-7 sites ofof uracil,guanosine the (Figure C-5 position 3). Some of of cytosine, the functional the C-2 groups position have of also adenine, been attached and the to C-8 andthe N-7 exocyclic sites of amino guanosine functions (Figure of C,3 A,). Someand G ofas thewell functional as the thiocarbonyl groups havegroup also of thiouridines been attached (Figure to the exocyclic3). amino functions of C, A, and G as well as the thiocarbonyl group of thiouridines (Figure3).

FigureFigure 3. Representative3. Representative sites sites of of post-synthetic post-synthetic modificationsmodifications present present in in sugar–phosphate sugar–phosphate backbone backbone andand nucleobase nucleobase residues residues with with the the highlighting highlighting of of uridine uridine and and thiouridine thiouridine ( a(a);); cytidine cytidine ( (bb);); adenosineadenosine (c); and(c guanosine); and guanosine (d). (d).

Post-syntheticPost-synthetic conversionsconversions of of RNA RNA oligomers oligomers can canbe performed be performed in the insolid the or solidliquid orphase liquid phase(Scheme (Scheme 2). 2). The The “in “in solid-phase” solid-phase” approach approach involves involves a a fully fully protected, protected, support-attached support-attached oligoribonucleotideoligoribonucleotide as as aa substratesubstrate for for the the post-synthetic post-synthetic reaction reaction (Scheme (Scheme 2A).2 AfterA). After conversion, conversion, the theoligomer oligomer is is released released from from the the resin resin and and deprotected. deprotected. Notably, Notably, the the use use of ofbasic basic conditions conditions for forthe the transformationtransformation process process can can promote promote the simultaneous the simultaneous removal removal of exoamino of exoamino and phosphate-protecting and phosphate- protecting groups as well as the support cleavage. In these cases, the post-synthetic reaction is carried groups as well as the support cleavage. In these cases, the post-synthetic reaction is carried out in out in a one-step conversion–deprotection process. Occasionally, the separate deprotection of the a one-step conversion–deprotection process. Occasionally, the separate deprotection of the phosphate phosphate groups is performed prior to post-synthetic conversion to avoid any side reactions of the groups is performed prior to post-synthetic conversion to avoid any side reactions of the oligomer oligomer with acrylonitrile released during removal of the β-cyanoethyl groups. with acrylonitrile released during removal of the β-cyanoethyl groups. Molecules 2020, 25, 3344 6 of 37 Molecules 2020, 25, x FOR PEER REVIEW 6 of 37

Scheme 2. Schematic representation of the most commonly used post-synthetic protocols of RNA Scheme 2. Schematic representation of the most commonly used post-synthetic protocols of RNA modification: (A) “In solid-phase” approach; (B) “In-solution” approach. modification: (A) “In solid-phase” approach; (B) “In-solution” approach. Alternatively, the post-synthetic RNA modification can be carried out in the liquid phase (SchemeAlternatively, 2B). This the “in-solution” post-synthetic approach RNA involves modification a fully can deprotected be carried and out released in the liquid precursor phase (Schemeoligoribonucleotide.2B). This “in-solution” After purification, approach the involves“free” oligomer a fully is deprotectedconverted to andthe target released product precursor and oligoribonucleotide.repurified to remove After the excess purification, of reagents. the “free” It is imperative oligomer isthat converted the presence to the of targetfree 2′-hydroxyl product and repurifiedgroups toexcludes remove the the use excess of highly of reagents. alkaline It is conversion imperative conditions that the presence that could of free promote 20-hydroxyl the strand groups excludescleavage the or use phosphate of highly migration. alkaline conversion conditions that could promote the strand cleavage or phosphateCorrect migration. incorporation of the final modified unit should be verified by careful analysis of the isolatedCorrect product incorporation by MALDI-ToF, of the ESI-MS, final modified and/or enzymatic unit should digestion. be verified by careful analysis of the isolatedThe product post-synthetic by MALDI-ToF, strategy ESI-MS, of RNA and / modificationor enzymatic has digestion. several important advantages in comparison to the classical modified monomer approach. Firstly, one single modified precursor The post-synthetic strategy of RNA modification has several important advantages in comparison oligoribonucleotide can provide several sequentially homologous, variously modified RNA to the classical modified monomer approach. Firstly, one single modified precursor oligoribonucleotide fragments [60–62]. Such a strategy significantly reduces the cost of synthesis compared to preparing can provide several sequentially homologous, variously modified RNA fragments [60–62]. each modified monomeric unit separately. Secondly, hypermodified monomeric units containing Such a strategy significantly reduces the cost of synthesis compared to preparing each modified highly reactive groups e.g., –COOH, -SO3H, -CHO can be replaced by structurally convenient, easy monomericconvertible unit precursor separately. phosphoramidites Secondly, hypermodified deprived of problematic monomeric functional units containing groups [62]. highly Finally, reactive the groupspost-synthetic e.g., –COOH, strategy -SO permits3H, -CHO introducing can be nucleosides/labels replaced by structurally that are sensitive convenient, to the easy conditions convertible of precursorsolid-phase phosphoramidites synthesis and/or deprived oligomer of deprotection problematic [63–65]. functional In some groups cases, [62]. the Finally, use of the post-synthetic post-synthetic strategyprotocol permits proved introducing to be the method nucleosides of choice/labels [63,64]. that are sensitive to the conditions of solid-phase synthesisThe and key/or to oligomer an effective deprotection post-synthetic [63–65 RNA]. In modification some cases, strategy the use of is post-synthetic the reactivity of protocol the provedprecursor to be nucleoside, the method which of choice should [63 ensure,64]. almost quantitative conversion without any perturbation of TheRNA key structure. to an eff Inective addition, post-synthetic the precursor RNA nucleoside modification should strategy offer an is easy the reactivity approach ofto theafford precursor the nucleoside,phosphoramidite which shouldbuilding ensure block almostand its quantitativeeffective incorporation conversion into without the RNA any chain. perturbation Therefore, of the RNA structure.choice of precursor In addition, compounds the precursor is limited nucleoside to nucleosides should that are off erfully an compatible easy approach with the to protocols afford the phosphoramiditeof RNA synthesis building and deprotection. block and its effective incorporation into the RNA chain. Therefore, the choiceThe of limited precursor stability compounds and hydrophilic is limited character to nucleosides of oligoribonucleotides that are fully compatible significantly with reduce the protocols the of RNAnumber synthesis of organic and reactions deprotection. that can be used in post-synthetic transformations of precursor RNA oligomers. In practice, the conditions of post-synthetic reactions are restricted to polar solvents, The limited stability and hydrophilic character of oligoribonucleotides significantly reduce moderate temperatures (less than 60 °C), and reaction times shorter than 24 h. Despite the above- the number of organic reactions that can be used in post-synthetic transformations of precursor mentioned restrictions, the conversions attending the heterocyclic bases represent the highest RNA oligomers. In practice, the conditions of post-synthetic reactions are restricted to polar chemical diversity demonstrated by several types of reactions, including nucleophilic aromatic solvents,substitution, moderate allylic temperatures substitution, (lesscarbon–carbon than 60 ◦C), bond-forming and reaction reaction times shorter via Sonogashira than 24 h. and Despite Stille the above-mentionedcouplings, cycloaddition restrictions, reactions, the conversions the derivatization attending of aliphatic the heterocyclic amino groups bases by represent the formation the highest of chemicalamide bonds, diversity and demonstratedfinally, the functionalization by several types of thiouridines of reactions, via includingdesulfuration, nucleophilic the formation aromatic of substitution,thioether bonds, allylic and substitution, disulfide bridges. carbon–carbon All of the bond-forming above-mentioned reaction post-synthetic via Sonogashira conversions and are Stille couplings,discussed cycloaddition in the following reactions, chapters. the derivatization of aliphatic amino groups by the formation of amide bonds, and finally, the functionalization of thiouridines via desulfuration, the formation of thioether bonds, and disulfide bridges. All of the above-mentioned post-synthetic conversions are discussed in the following chapters. Molecules 2020, 25, 3344 7 of 37

Molecules 2020, 25, x FOR PEER REVIEW 7 of 37 3.1. Nucleophilic Aromatic Substitution 3.1. Nucleophilic Aromatic Substitution Nucleophilic aromatic substitution (SNAr) is the most common post-synthetic reaction utilized in the synthesisNucleophilic of nucleobase-modified aromatic substitution oligoribonucleotides. (SNAr) is the most common It involves post-synthetic a convertible reaction RNA utilized oligomer equippedin the synthesis with a nucleosideof nucleobase-modified containing aoligoribonucleotides. good leaving group(s) It involves directly a convertible attached toRNA the oligomer heterobase equipped with a nucleoside containing a good leaving group(s) directly attached to the heterobase and easily displaced by nucleophile(s). The post-synthetic SNAr reaction enables synthesizing single- or multiple-modifiedand easily displaced target by nucleophile(s). oligonucleotides, The post-synthetic including double-derivatized SNAr reaction enables products synthesizing yielded single- by the or multiple-modified target oligonucleotides, including double-derivatized products yielded by the attachment of bifunctional nucleophile and designed for further derivatization with a chemical probe. attachment of bifunctional nucleophile and designed for further derivatization with a chemical probe. The literature provides several examples of convertible ribonucleosides subjected to post-synthetic The literature provides several examples of convertible ribonucleosides subjected to post-synthetic SNAr transformations (Figure4). Most of them contain aryl ether leaving groups ( 1–3, 6) directly SNAr transformations (Figure 4). Most of them contain aryl ether leaving groups (1–3, 6) directly attached to the C4 position of pyrimidine and the C6 position of purine heterobases. Others contain attached to the C4 position of pyrimidine and the C6 position of purine heterobases. Others contain a triazolyla triazolyl group group directly directly linked linked to to thethe C4C4 positionposition of the pyrimidine pyrimidine ring ring (5 ()5 or) or sulfonate sulfonate residue residue installedinstalled in the in the C6 position C6 position of purine of purine nucleoside nucleoside (7). The (7). Therepresentative representative leaving leaving groups groups are alsoare halidesalso attachedhalides to attached the C5 atomto the ofC5 uridine atom of ( 4uridine) and C2 (4) orand C6 C2 atoms or C6 of atoms purine of purine nucleosides nucleosides (8, 9). (8, 9).

FigureFigure 4. Convertible4. Convertible nucleosides nucleosides employed employed in the post-synthetic transformations transformations of ofprecursor precursor RNA RNA viavia nucleophilic nucleophilic aromatic aromatic substitution. substitution. TheThe leavingleaving groups are are indicated indicated in in the the frames. frames.

TheThe first first convertible convertible oligoribonucleotides oligoribonucleotides were were reported reported by Verdine by Verdine and and co-workers co-workers in 1995 in 1995 [66,67 ]. The[66,67]. post-synthetic The post-synthetic strategy attended strategy theattended exocyclic the aminoexocyclic groups amino of groups A, C, and of A, G C, nucleosides and G nucleosides and was an extensionand was of an earlier extension Verdine’s of earlier lab studiesVerdine’s on lab the studies convertible on the nucleoside convertible approach nucleoside in approach the derivatization in the of DNAderivatization oligomers of [ DNA54,55 ,57 oligomers,58,68]. Developed [54,55,57,58,68]. chemistry Developed permits chemistry the site-specific permits the incorporation site-specific of N-functionalizableincorporation of nucleobases N-functionalizable of A, C, and nucleobases G into RNA of and A, its C, further and G derivatization into RNA and to e.g., its crosslinked further nucleicderivatization acids (see to Scheme e.g., crosslinked 27A). The nucleic authors acids demonstrated (see Scheme 27A). the useThe ofauthors 4-O-(4-chlorophenyl)uridine demonstrated the use (1,of see 4- FigureO-(4-chlorophenyl)uridine4), 6- O-(4-chlorophenyl)inosine (1, see Figure 4), (6 ),6- andO-(4-chlorophenyl)inosine 2-fluoro-6-O-(4-nitrophenylethyl)inosine (6), and 2-fluoro-6-O- (9) (4-nitrophenylethyl)inosine (9) prepared as 5′-O-DMTr-2′-O-TBDMS-protected phosphoramidites to prepared as 50-O-DMTr-20-O-TBDMS-protected phosphoramidites to the synthesis of 11-nt precursor the synthesis of 11-nt precursor oligonucleotides 10–12 by standard 1 µmol phosphoramidite oligonucleotides 10–12 by standard 1 µmol phosphoramidite chemistry (Scheme3). For use in the chemistry (Scheme 3). For use in the automated RNA synthesis, the convertible Verdine’s automated RNA synthesis, the convertible Verdine’s phosphoramidites were applied as 0.1 M solutions phosphoramidites were applied as 0.1 M solutions in acetonitrile, while the coupling time was in acetonitrile,extended to while12 min. the Syntheses coupling proceeded time was with extended average to 12stepwise min. Synthesesyields of ca. proceeded 97%. The with following average stepwiseconversion–deprotection yields of ca. 97%. Thestep following of resin-bound conversion–deprotection oligonucleotides with step primary of resin-bound alkylamines oligonucleotides resulted in withthe primary substitution alkylamines of chlorophenyl resulted groups in the substitutionin aryl ethers of (oligomers chlorophenyl 10, 11 groups) as well in as aryl 2-fluoride ethers (oligomersleaving 6 10,groups 11) as well in O as6-protected 2-fluoride inosine leaving (oligomer groups in 12O) -protectedwith amines inosine providing (oligomer N4-alkylcytidine-(12) with amines14a– providingh), N6- 4 6 2 N -alkylcytidine-(alkyladenosine-(14a15a––hh),) N and-alkyladenosine-( N2-alkylguanosine-modified15a–h) and N RNAs-alkylguanosine-modified (16a–h). To eliminate RNAs the base- (16a–h). To eliminatepromoted theRNA base-promoted degradation, the RNA post-synthetic degradation, transformations the post-synthetic were transformations performed under were anhydrous performed underconditions anhydrous using conditions 7 M methanolic using 7 M ammonia, methanolic 8 M ammonia, solution 8 of M methylamine solution of methylamine in ethanol, orin ethanol, 2 M or 2methanolic M methanolic solutions solutions of other of other amines amines such such as as ethylenediamine, ethylenediamine, 1,4-diaminobutane, 1,4-diaminobutane, cystamine, cystamine, Molecules 2020, 25, 3344 8 of 37

Molecules 2020, 25, x FOR PEER REVIEW 8 of 37 ethanolamine, benzylamine, and 2-(methylthio)ethylamine. Reactions were performed at 42 ◦C for 12–18ethanolamine, h. After displacement, benzylamine, the and mixture 2-(methylthio)ethylamine. of oligomer and remaining Reactions amine were reagent performed was separatedat 42 °C for from 12–18 h. After displacement, the mixture of oligomer and remaining amine reagent was separated+ the support and then evaporated or eluted through a Dowex cation exchange column (NH4 form). from the support and then evaporated or eluted through a Dowex cation exchange column (NH4+ Subsequent desilylation involved TEA 3HF or 1 M TBAF/THF treatment. Target oligonucleotides were form). Subsequent desilylation involved· TEA·3HF or 1 M TBAF/THF treatment. Target purified by denaturing PAGE, affording oligomers 14–16 in the relative conversion yields above 65%. oligonucleotides were purified by denaturing PAGE, affording oligomers 14–16 in the relative Measurements of thermal stability of modified duplex RNA oligomers have revealed that in most conversion yields above 65%. Measurements of thermal stability of modified duplex RNA oligomers cases,have the revealed presence that of inN -alkyl most cases, modification the presence slightly of reducesN-alkyl modification duplex stability. slightly The reduces exceptions duplex were oligonucleotidesstability. The exceptionscarrying a tether were oligonucleotides with a “free” amine carrying group a (oligomers tether with14c a – “free”e, 15c – aminee and 16c group–e) for which(oligomers increased 14c duplex–e, 15c–e stability and 16c was–e) for observed. which increased duplex stability was observed.

SchemeScheme 3. 3.General General scheme scheme for for the the introduction introduction of N-alkyl-alkyl modifications modifications via via the the post-synthetic post-synthetic modificationmodification of support-linked of support-linked convertible convertible oligomers oligomers modified modified with with 4-O 4--(4-chlorophenyl)uridineO-(4-chlorophenyl)uridine (10 ), 6-O(-(4-chlorophenyl)inosine10), 6-O-(4-chlorophenyl)inosine (11), 2-fluoro-6- (11), 2-fluoro-6-O-(4-nitrophenylethyl)inosineO-(4-nitrophenylethyl)inosine (12), and ( 5-bromouridine12), and 5- bromouridine (13). Reagents and conditions: (1) 7 M NH3/MeOH (14a–16a), 8 M MeNH2/EtOH (14b– (13). Reagents and conditions: (1) 7 M NH3/MeOH (14a–16a), 8 M MeNH2/EtOH (14b–16b), 16b), 2 M methanolic solution of all other amines: H2NCH2CH2NH2 (14c–17c), H2N(CH2)4NH2 (14d– 2 M methanolic solution of all other amines: H2NCH2CH2NH2 (14c–17c), H2N(CH2)4NH2 16d), H2N(CH2)2SS(CH2)2NH2 (14e–16e), HOCH2CH2NH2 (14f–16f), PhCH2NH2 (14g–16g), (14d–16d), H2N(CH2)2SS(CH2)2NH2 (14e–16e), HOCH2CH2NH2 (14f–16f), PhCH2NH2 (14g–16g), H3CS(CH2)2NH2 (14h–16h); 45 °C, 18 h (12 h for 16a–h); (2) TEA3HF, 1 h, rt for 14a–h, 15a–h and 17c, H3CS(CH2)2NH2 (14h–16h); 45 ◦C, 18 h (12 h for 16a–h); (2) TEA 3HF, 1 h, rt for 14a–h, 15a–h and 17c, TBAF/THF, 24 h, rt for 16a–h. · TBAF/THF, 24 h, rt for 16a–h.

Currently,Currently, the the convertible convertible phosphoramidites phosphoramidites introduced introduced by by Verdine Verdine and and co-workers co-workers are are commercially available as 5′-O-DMTr-2′-O-TBDMS-protected building blocks. commercially available as 50-O-DMTr-20-O-TBDMS-protected building blocks. Based on the Verdine’s strategy, Babaylova and co-workers introduced an ethylenediamine Based on the Verdine’s strategy, Babaylova and co-workers introduced an ethylenediamine linker linker to the C5 position of uridine employing 5-bromouridine 4 (see Figure 4) as a convertible to the C5 position of uridine employing 5-bromouridine 4 (see Figure4) as a convertible nucleoside [ 69]. nucleoside [69]. The CPG-bound 5′-O-DMTr-2′(3′)-O-acetyl-C5-bromouridine was employed for the O O Thesynthesis CPG-bound of a 510-nt0- -DMTr-2 RNA oligomer0(30)- -acetyl-C5-bromouridine 13 (Scheme 3) at the 0.4 µmol was scale employed followed for theby treatment synthesis with of a 10-nt 2 RNAM solutionoligomer of13 ethylenediamine(Scheme3) at the in methanol 0.4 µmol (42 scale °C, 18 followed h). After by the treatment desilylation with process, 2 M solution amino- of ethylenediamineoligomer 17c was in methanolisolated by (42denaturating◦C, 18 h). PAGE After theelectrophoresis. desilylation The process, attachment amino-oligomer of a bifunctional17c was isolatedethylenediamine by denaturating nucleophile PAGE electrophoresis. in this fashion The provided attachment a convenient of a bifunctional substrate ethylenediamine for further nucleophilefunctionalization in this with fashion nitroxide provided radicals a (see convenient Scheme 21). substrate for further functionalization with nitroxideThe radicals. next exploration of Verdine’s ribonucleosides 1, 6, and 9 (see Figure 4) was demonstrated byThe the next Höbartner exploration group of to Verdine’s install the ribonucleosides nitroxide spin labels1, 6, and on 9 exocyclic(see Figure amino4) was groups demonstrated of RNA bynucleobases the Höbartner of C, group G, and to A install(Schemes the 4 nitroxideand 5) and spin evaluate labels the on potential exocyclic of aminoTEMPO- groups and proxyl- of RNA nucleobaseslabeled nucleobases of C, G, and for A probing (Schemes RNA4 and secondary5) and evaluate structures thepotential by PELDOR of TEMPO- experiments and proxyl-labeled [52,70], and nucleobasesconstruct, for among probing others, RNA riboswitches secondary of structures the correct by regulatory PELDOR experiments function [52]. [ 52 The,70 ], commercially and construct, amongavailable others, 5′-O riboswitches-DMTr-2′-O-TBDMS-protected of the correct regulatory Verdine’s function phosphoramidites [52]. The were commercially incorporated available into RNA by solid-phase synthesis using a polystyrene support in combination with 5′-O-DMTr-2′-O- 50-O-DMTr-20-O-TBDMS-protected Verdine’s phosphoramidites were incorporated into RNA by Molecules 2020, 25, 3344 9 of 37

Molecules 2020,, 25,, xx FORFOR PEERPEER REVIEWREVIEW 9 9 ofof 3737 solid-phase synthesis using a polystyrene support in combination with 50-O-DMTr-20-O-TOM-protected canonicalTOM-protected monomeric canonical units monomeric (products wereunits prepared(products aswere single prepared or double-modified as single or double-modified 12-, 13- or 20-nt oligomers)12-, 13- or [70 20-nt]. oligomers) [70].

SchemeScheme 4. 4.General General scheme scheme for for the the preparation preparation of spin-labeled RNA RNA oligomers oligomers 1919–21–21 withwith TEMPO TEMPO derivativederivative18a 18a. Reagents. Reagents and and conditions: conditions: (1)(1) 22 MM 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl (18a (18a) in) in methanol, 42 °C, 24 h; (2) 1 M TBAF/THF, 25 °C, 12 h. methanol,methanol, 42 42◦C, °C, 24 24 h; h; (2) (2) 1 1 M M TBAF TBAF/THF,/THF, 25 25◦ °C,C, 1212 h.

SchemeScheme 5. 5.Preparation Preparation of of oligomers oligomers 24a––bb andand 25a25a––bb containingcontaining TEMPO- TEMPO- oror proxyl-labeled proxyl-labeled cytidinecytidine/2′-/20-O-methylcytidineO-methylcytidine nucleotide. nucleotide. Reagents Reagents and and conditions: conditions: (1) (1) 2 M 2 M solution solution of of18a18a–b –inb in methanol,methanol, 42–55 42–55◦C, °C, 24–48 24–48 h; h; (2) (2) 8 8 M M aq. aq. MeNHMeNH2,, 37 37 °C,◦C, 2–5 2–5 h h or or 25% 25% aq. aq. NH NH3, 355, 55 °C,◦ C,2–5 2–5 h; (3) h; (3)1 M 1 M TBAFTBAF/THF,/THF, rt, rt, 8–16 8–16 h. h.

TheThe support-bound, support-bound, fullyfully protected precursors precursors 10 10andand 12 were12 were incubated incubated with 2 with M solution 2 M solution of 4- ofamino-2,2,6,6-tetramethylpiperidin-1-oxyl 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO-NH (TEMPO-NH2, 18a) in2 methanol, 18a) in at 42 methanol °C for 24 h at providing 42 ◦C for 24 hfully providing converted fully N4 converted-TEMPO-cytidine-N4-TEMPO-cytidine- and N2-TEMPO-guanosine-containing and N2-TEMPO-guanosine-containing RNAs, respectively. RNAs, 6 6 respectively.However, the However, same transformation the same transformation of O6-(4-chlorophenyl)inosine-oligomer of O6-(4-chlorophenyl)inosine-oligomer 11 to N6-TEMPO–11 to N6-TEMPO–adenosine–RNAadenosine–RNA turned out incomplete turned out even incomplete after a prolonged even after incubation a prolonged time and incubation temperature. time All and nucleobase substitutions were performed in one step with support cleavage and the deprotection of temperature.nucleobase substitutions All nucleobase were substitutions performed in were one step performed with support in one cleavage step with and support the deprotection cleavage of and base-labile protecting groups. After desilylation, the TEMPO-substituted RNA oligonucleotides 19– thebase-labile deprotection protecting of base-labile groups. protectingAfter desilylation, groups. the After TEMPO-substituted desilylation, the RNA TEMPO-substituted oligonucleotides 19 RNA– 21 were purified by anion-exchange HPLC or denaturing PAGE. The influence of TEMPO subsituents oligonucleotides 19–21 were purified by anion-exchange HPLC or denaturing PAGE. The influence on RNA conformation and the secondary structure of the hairpin and duplex structure was of TEMPO subsituents on RNA conformation and the secondary structure of the hairpin and investigated, demonstrating the potential of labeled oligomers as reporter probes in pulsed EPR duplex structure was investigated, demonstrating the potential of labeled oligomers as reporter spectroscopy. Although the resulting spin-labeled RNAs were obtained in good yields and high probesqualities, in pulsed further EPR work spectroscopy. was undertaken Although to improve the the resulting preparation spin-labeled of the precursor RNAs oligomers. were obtained To in goodthis purpose, yields Verdine’s and high 4- qualities,O-(p-chlorophenyl)uridine further work was building undertaken blocks protected to improve with the 2′-O preparation-TOM or 2′- of theO precursor-Me group oligomers. were used To in this the purpose, place of Verdine’scommercially 4-O -(p-chlorophenyl)uridine available 2′-O-TBDMS phosphoramidite building blocks Molecules 2020, 25, 3344 10 of 37

protected with 20-O-TOM or 20-O-Me group were used in the place of commercially available 20-OMolecules-TBDMS 2020, phosphoramidite 25, x FOR PEER REVIEW (Scheme 5)[ 52]. This change significantly improved the coupling 10 of 37 yields and shortened coupling times. In addition, 20-OMe modification was expected to increase the(Scheme stability 5) of [52]. nitroxide-labeled This change significantly oligomers. improved For post-synthetic the coupling transformations, yields and shortened fully coupling protected, polystyrene-linkedtimes. In addition, oligomers 2′-OMe modification22, 23 were incubatedwas expected with to aincrease 2 M methanolic the stability solution of nitroxide-labeled of TEMPO-NH 2 oligomers. For post-synthetic transformations, fully protected, polystyrene-linked oligomers 22, 23 (18a) or 3-amino-2,2,5,5-tetramethylpyrrolidin-1-oxyl (proxyl, 18b) at 42–55 ◦C for 24–48 h. Then, aq. were incubated with a 2 M methanolic solution of TEMPO-NH2 (18a) or 3-amino-2,2,5,5- MeNH2 or aq. NH3 were added to complete the cleavage of base-labile protecting groups (this step wastetramethylpyrrolidin-1-oxyl skipped during the preparation (proxyl, of18b short) at 42-55 TEMPO-labeled °C for 24–48 h. oligomers, Then, aq. MeNH which2 or were aq. NH deprotected3 were simultaneouslyadded to complete with displacement). the cleavage of Fully base-labile deprotected protecting TEMPO- groups (24a (this, 25a step) and was proxyl-labeled skipped during oligomers the preparation of short TEMPO-labeled oligomers, which were deprotected simultaneously with (24b, 25b) ranging from 9 to 27 nt were purified by PAGE or anion-exchange HPLC. For proxyl-labeled displacement). Fully deprotected TEMPO- (24a, 25a) and proxyl-labeled oligomers (24b, 25b) ranging RNAs 24b, 25b, the formation of an N4-methylcytidine-containing side product was observed, from 9 to 27 nt were purified by PAGE or anion-exchange HPLC. For proxyl-labeled RNAs 24b, 25b, resulting from the substitution of the 4-O-chlorophenyl group in an unreacted precursor oligomer the formation of an N4-methylcytidine-containing side product was observed, resulting from the withsubstitution methylamine. of the The 4-O-chlorophenyl nitroxide-labeled group RNAs in an carrying unreacted TEMPO precursor or proxyloligomer were with utilized methylamine. to obtain functionalThe nitroxide-labeled 53-nt and 118-nt RNAs riboswitch carrying RNAsTEMPO by or deoxyribozyme-catalyzed proxyl were utilized to obtain ligation functional as an alternative 53-nt and to protein-based118-nt riboswitch RNA RNAs ligation by [deoxyribozyme-catalyzed52]. ligation as an alternative to protein-based RNA ligationCommercially [52]. available 50-O-DMTr-20-O-TBDMS Verdine’s phosphoramidites were applied for the incorporationCommercially of available photolabile 5′-O 2-(2-nitrophenyl)propyl-DMTr-2′-O-TBDMS Verdine’s (NPP) phosphoramidites and 2-(2-nitrophenyl)ethyl were applied (NPE)for “cagingthe incorporation groups” (Scheme of photolabile6)[ 71]. 2-(2-nitrophenyl)propyl Solid-phase syntheses (NPP)of 15-nt and precursor 2-(2-nitrophenyl)ethyl oligomers 10 – (NPE)12 were performed“caging groups” according (Scheme to standard 6) [71]. coupling Solid-phase protocol. syntheses For displacementof 15-nt precursor reaction, oligomers fully protected10–12 were and support-boundperformed according oligomers to standard10–12 were coupling treated protocol. with For 2.5 Mdisplacement NPP-NH2 reaction,(26a) in fully methanol protected (1–2 and days, 2 25–42support-bound◦C) or NPE-NH oligomers2 (26b )10 in–12 acetonitrile were treated (2–5 with days, 2.5 42–60 M NPP-NH◦C). The (26a use) in of methanol sterically (1–2 hindered days, 25– amine 42 °C) or NPE-NH2 (26b) in acetonitrile (2–5 days, 42–60 °C). The use of sterically hindered amine 26b required an extra cleavage step with methylamine or aq. NH3 for complete base deprotection. 26b required an extra cleavage step with methylamine or aq. NH3 for complete base deprotection. In In the case of inosine-containing oligomer 11 the conversion with NPE–NH2 turned out not to work. Finally,the case oligomers of inosine-containing containing both oligomer NPP and 11 NPEthe conversion groups were with desilylated NPE–NH2 usingturned TEA out 3HFnot /toTEA work./NMP Finally, oligomers containing both NPP and NPE groups were desilylated using TEA3HF/TEA/NMP· mixture. An attempt to prepare 4-O-caged uridine- and 6-O-caged adenosine-containing oligomers by mixture. An attempt to prepare 4-O-caged uridine- and 6-O-caged adenosine-containing oligomers incubation of 10, 11 with NPP–OH nucleophilic reagent (alcohol analog of NPP–NH2) in MeCN for by incubation of 10, 11 with NPP–OH nucleophilic reagent (alcohol analog of NPP–NH2) in MeCN 7 days at 42 C (optionally in the presence of tertiary amine) proved to be ineffective. The presented for 7 days◦ at 42 °C (optionally in the presence of tertiary amine) proved to be ineffective. The methodologypresented methodology enabled simplifying enabled simplifying the synthesis the ofsynthesis caged RNAs of caged (compared RNAs (compared to the standard to the standard monomer approach),monomer since approach), the commercially since the commercially available Verdine’s available phosphoramidite Verdine’s phosphoramidite was used. The was expected used. The results of duplexexpected destabilization results of duplex caused destabilization by attached caused caging by groupsattached were caging achieved. groups were achieved.

SchemeScheme 6. 6.General General scheme scheme forfor post-synthetic incorporation incorporation of of2-(2-nitrophenyl)propyl 2-(2-nitrophenyl)propyl (NPP) (NPP) and 2- and 2-(2-nitrophenyl)ethyl(2-nitrophenyl)ethyl (NPE) (NPE) groups. groups. Reagents Reagents and and conditions: conditions: (1) 2.5 (1) M 2.5 solution M solution of 26a ofin 26aMeOH,in MeOH, 1–2

1–2days, days, rt rt or or 42 42 °C◦C or or 2.5 2.5 M M solution solution of of 26b26b inin MeCN, MeCN, 2–5 2–5 days, days, 42–60 42–60 °C;◦ C;(2) (2) 32% 32% aq. aq. NH NH3, rt,3, 40 rt, h 40 or h or 33%33% MeNH MeNH/MeOH,2/MeOH, rt, rt, overnight; overnight; (3) (3) TEATEA3HF3HF/TEA/NMP/TEA/NMP (4:3:6 (4:3:6 vv/v/v/v/v),), 2 2h, h, 42 42 °C.C. 2 · ◦ Molecules 2020, 25, 3344 11 of 37

MoleculesIn 2003, 2020, 25, Kierzek’s x FOR PEER REVIEW group employed 6-methylthiopurine riboside 7 (see Figure 11 of 374 ) and 2-methylthio-6-chloropurine riboside 8 as convertible nucleosides for post-synthetic In 2003, Kierzek’s group employed 6-methylthiopurine riboside 7 (see Figure 4) and 2- nucleophilic aromatic substitution with primary alkylamines, leading to N6-alkyladenosine- and methylthio-6-chloropurine riboside 8 as convertible nucleosides for post-synthetic nucleophilic 2-methylthio-N6-alkyladenosine-modified oligoribonucleotides (7-, 8- and 17-mers), respectively aromatic substitution with primary alkylamines, leading to N6-alkyladenosine- and 2-methylthio-N6- 6 6 2 6 (Schemesalkyladenosine-modified7 and8)[ 60]. Among oligoribonucleotides them, four naturally (7-, 8- occurring and 17-mers), tRNA respectively modifications (Schemes i A, m 7,A, 8) ms[60].i A, 2 6 andAmong ms m them,A were four inserted. naturally Both occurring convertible tRNA oligomers modifications30 and i6A,34 mwere6A, ms synthesized2i6A, and ms automatically2m6A were oninserted. 1 µmol scaleBoth convertible by the standard oligomers protocol 30 and of 34 phosphoramidite were synthesized chemistryautomatically on on solid 1 µmol support scale (CPG,by Fractosilthe standard 500 or protocol resins loaded of phosphoramidite with 50-O-DMTr-2 chemistry0-O-TBDMS-protected on solid support (CPG, convertible Fractosil nucleosides 500 or resins7, 8). Beforeloaded conversion, with 5′-O-DMTr-2′- the 6-methylthioO-TBDMS-protected group (6-SCH convertible3) of support-linked nucleosides 6-methylthiopurine-modified7, 8). Before conversion, the oligoribonucleotide6-methylthio group30 (6-SCH(Scheme3) of7 support-linked) was selectively 6-methylthiopurine-modified and quantitatively activated oligoribonucleotide by oxidation with30 a 20(Scheme mM solution 7) was of selectively monoperoxyphtalic and quantitatively acid (magnesium activated by salt oxidation form, 2.5 with h, rt) a 20yielding mM solution a mixture of of oligomersmonoperoxyphtalic31 and 32 decorated acid (magnesium with 6-methylsulfoxide salt form, 2.5 h, (6-S(O)CH rt) yielding3) a and mixture 6-methylsulfonyl of oligomers (6-S(O)31 and2 32CH 3) groups,decorated respectively. with 6-methylsulfoxide The oxidized oligomers (6-S(O)CH313), 32 and(still 6-methylsulfonyl attached to the (6-S(O) support)2CH were3) groups, reacted withrespectively. various primary The oxidized alkylamines. oligomers 31 To, 32 get (still oligomers attached to modified the support) with wereN 6reacted-methyladenosine with various or 6 primary alkylamines. To get oligomers modified with N6-methyladenosine or N6- N -isopentenyladenosine, 2 M MeNH2 in THF (12 h, rt) and isopentenylamine hydrochloride with isopentenyladenosine, 2 M MeNH2 in THF (12 h, rt) and isopentenylamine hydrochloride with TEA TEA in pyridine (12 h, 55 ◦C) were applied, respectively. Other amines, such as isoamylamine, in pyridine (12 h, 55 °C) were applied, respectively. Other amines, such as isoamylamine, neopentylamine, 1-methylpropylamine, 1-methylbutylamine, and propargylamine were used as neopentylamine, 1-methylpropylamine, 1-methylbutylamine, and propargylamine were used as a a solution in acetonitrile (1:9 v/v) and left for 12 h at rt. After conversion, the samples were evaporated, solution in acetonitrile (1:9 v/v) and left for 12 h at rt. After conversion, the samples were evaporated, and the aq. NH3/EtOH (3:1 v/v, 8 h, 55 ◦C) was added for the complete removal of the base-labile and the aq. NH3/EtOH (3:1 v/v, 8 h, 55 °C) was added for the complete removal of the base-labile protectingprotecting groups. groups. After After desilylation, desilylation, the fully deprotected oligomers oligomers were were purified purified on on silica silica gel gel 6 plates.plates. The The overall overall yields yields of ofN N-alkyladenosine-containing6-alkyladenosine-containing RNAs RNAs 33a33a–h– hwerewere in inthe the range range of 40–77% of 40–77% (13–34%(13–34% for for 17-mers). 17-mers). It wasIt was indicated indicated that that the the synthesis synthesis yields yields werewere notnot dependentdependent on the position of convertibleof convertible nucleoside nucleoside in the in RNA the sequence RNA sequence but on but the on steric the hindrancesteric hindrance and the and nucleophilic the nucleophilic character of thecharacter amino of group the amino in alkylamine group in alkylamine conversion conversion reagents. reagents.

SchemeScheme 7. 7.General General scheme scheme for the the synthesis synthesis of ofN6N-alkyladenosine-containing6-alkyladenosine-containing oligomers oligomers 33a–h 33aby the–h by thepost-synthetic post-synthetic modification modification of ofconvertible convertible oligomers oligomers bearing bearing 6-methylthiopurine 6-methylthiopurine riboside riboside 31 and31 32and. 32.Reagents Reagents and and conditions: conditions: (1) (1) 20 20 mM mM solution solution of of magnesium magnesium salt salt of of monoperoxyphtalic monoperoxyphtalic acid, acid, dioxane/water (9:1 v/v), 2.5 h, rt; (2) 32% aq. ammonia 16 h, 55 °C (33a); 2 M MeNH2 (800 equiv) in dioxane/water (9:1 v/v), 2.5 h, rt; (2) 32% aq. ammonia 16 h, 55 ◦C(33a); 2 M MeNH2 (800 equiv) in THF, 12 h, rt (33b); isopentenylamine hydrochloride (213 equiv), TEA (426 equiv), pyridine, 12 h, 55 THF, 12 h, rt (33b); isopentenylamine hydrochloride (213 equiv), TEA (426 equiv), pyridine, 12 h, 55 ◦C °C (33g); the other amines/MeCN (1:9 v/v; 500–1000 equiv of amine), 12 h, rt (33c–f,h); (3) aq. (33g); the other amines/MeCN (1:9 v/v; 500–1000 equiv of amine), 12 h, rt (33c–f,h); (3) aq. NH3/EtOH NH3/EtOH (3:1 v/v), 8 h, 55 °C; (4) 1 M TEAF in pyridine, 48 h, 55 °C. (3:1 v/v), 8 h, 55 ◦C; (4) 1 M TEAF in pyridine, 48 h, 55 ◦C.

ToTo prepare prepare the the 2-methylthio- 2-methylthio-NN6-alkyladenosine-modified-alkyladenosine-modified RNAs RNAs 35a35a–c (Scheme–c (Scheme 8), several8), several 6- 6-substitutedsubstituted purinepurine ribosidesribosides such as as dinitrothiophenyl-, dinitrothiophenyl-, pentafluorophenyl-, pentafluorophenyl-, and and chloro-substituted chloro-substituted 6 purinespurines were were tested tested as model as modelconvertible convertible nucleosides nucleosides [60]. Among [60]. them, Among 2-methylthio- them, 2-methylthio-N6-chloropurineN - ribosidechloropurine8 (see riboside Figure4 )8 exhibited (see Figure the 4) highest exhibited reactivity the highest with reactivity primary with amines. primary Therefore, amines. Therefore, the 5′-O-DMTr-2′-O-TBDMS-protected convertible phosphoramidite of 8 was introduced the 50-O-DMTr-20-O-TBDMS-protected convertible phosphoramidite of 8 was introduced into RNA into RNA sequences (7-, 8- and 17-mers) on the 1 µmol scale of the oligonucleotide synthesis. The sequences (7-, 8- and 17-mers) on the 1 µmol scale of the oligonucleotide synthesis. The trityl-off trityl-off support-linked precursor 34 (Scheme 8) was treated with alkylamines: 2 M MeNH2 in THF Molecules 2020, 25, 3344 12 of 37

Molecules 2020, 25, x FOR PEER REVIEW 12 of 37 support-linked precursor 34 (Scheme8) was treated with alkylamines: 2 M MeNH 2 in THF (2–5 h, 55 C) or isopentenylamine hydrochloride/TEA in pyridine (2–5 h, 55 C). For complete removal of ◦(2–5 h, 55 °C) or isopentenylamine hydrochloride/TEA in pyridine (2–5◦ h, 55 °C). For complete base-labile protecting groups, the samples were additionally treated with aq. NH3/EtOH (3:1 v/v, 8 h, removal of base-labile protecting groups, the samples were additionally treated with aq. NH3/EtOH 55 C). Displacement of the 6-chloro group with –NH involved the aq. ammonia and prolonged (3:1◦ v/v, 8 h, 55 °C). Displacement of the 6-chloro group2 with –NH2 involved the aq. ammonia and reactionprolonged time (16reaction h, 55 time◦C). After (16 h, desilylation, 55 °C). After the desilylation, fully deprotected the fully oligomers deprotected were oligomers purified on were silica 6 gelpurified plates. The on overall silica gel yields plates. of 2-methylthio- The overallN yields-alkyladenosine-modified of 2-methylthio-N6-alkyladenosine-modified oligoribonucleotides 35a –c rangeoligoribonucleotides from 20% to 65% 35a (16–24%–c range for from 17-mers). 20% to 65% (16–24% for 17-mers).

SchemeScheme 8. General8. General scheme scheme for for the the synthesis synthesis ofof 2-methyltio-2-methyltio-N66-alkyladenosine-containing-alkyladenosine-containing oligomers oligomers 35a35a–c by–c by the the post-synthetic post-synthetic modification modification ofof convertibleconvertible oligomers oligomers 3434 modifiedmodified with with 6-chloro-2-methyl 6-chloro-2-methyl

thiopurinethiopurine riboside. riboside. Reagents and and conditions: conditions: (1) 32% (1)32% aq. ammonia aq. ammonia 16 h, 55 16 °C h,(for 55 35a◦C); (for2 M MeNH35a); 22 M MeNH(10002 (1000equiv) equiv) in THF, in 2–5 THF, h, 2–555 °C h, 55(for◦ C35b (for); isopentenylamine35b); isopentenylamine hydrochloride hydrochloride (213 equiv), (213 TEA equiv), (426 TEA equiv), pyridine, 2–5 h, 55 °C (for 35c); (2) aq. NH3/EtOH (3:1 v/v), 8 h, 55 °C; (3) 1 M TEAF in pyridine, (426 equiv), pyridine, 2–5 h, 55 ◦C (for 35c); (2) aq. NH3/EtOH (3:1 v/v), 8 h, 55 ◦C; (3) 1 M TEAF in 48 h, 55 °C. pyridine, 48 h, 55 ◦C.

TheThe post-synthetic post-synthetic introduction introduction of of NN66-alkylated adenosines adenosines and and 2-methylthioadenosines 2-methylthioadenosines elaboratedelaborated by by Kierzek’s Kierzek’s group group seems seems toto bebe thethe method of of choice choice for for obtaining obtaining several several homologous homologous RNARNA oligomers, oligomers, especiallyespecially that that the the precursor precursor synthons synthons were were stable stable during during the synthesis the synthesis of 3′-O- of phosphoramidite/oligonucleotide and easily transformed to target N6-substituted6 adenosine-RNAs. 30-O-phosphoramidite/oligonucleotide and easily transformed to target N -substituted adenosine-RNAs. The post-synthetic SNAr reaction has shown to be attractive for the incorporation of 4-thiouridine The post-synthetic SNAr reaction has shown to be attractive for the incorporation of 4-thiouridine (s4U) into RNA oligomers. The preparation of s4U-RNAs by the classical approach requires a special (s4U) into RNA oligomers. The preparation of s4U-RNAs by the classical approach requires a special protection for 4-thiocarbonyl function in s4U-phosphoramidite to avoid the desulfuration by-product protection for 4-thiocarbonyl function in s4U-phosphoramidite to avoid the desulfuration by-product produced during the oxidation step [72,73]. This inconvenience has been overcome by employing the producedconvertible during nucleoside the oxidation approach. step Several [72,73]. examples This inconvenience of precursor has ribonucleosides been overcome were by reported employing for the convertibles4U-RNA nucleoside preparation, approach. including 4-(1,2,4-triazol-1-yl)-2-pyrimidon-1-yl- Several examples of precursor ribonucleosidesβ-D-ribofuranoside were ( reported5, see 4 forFigure s U-RNA 4) [74–76], preparation, 4-O-(2-nitrophenyl)uridine including 4-(1,2,4-triazol-1-yl)-2-pyrimidon-1-yl- (2, Figure 4) [76], and 4-O-(4-nitrophenyl)uridineβ-D-ribofuranoside (3, (5, seeFigure Figure 4) [77].4)[ 74–76], 4-O-(2-nitrophenyl)uridine (2, Figure4)[ 76], and 4-O-(4-nitrophenyl)uridine (3, FigureThe4)[ use77]. of 4-(1,2,4-triazol-1-yl)-2-pyrimidon-1-yl-β-D-ribofuranoside convertible nucleoside 5 wasThe demonstrated use of 4-(1,2,4-triazol-1-yl)-2-pyrimidon-1-yl- by Shah and Kumar [74,75] (Scheme β9A).-D-ribofuranoside The 5′-O-DMTr-2′- convertibleO-TBDMS-protected nucleoside 5 wasphosphoramidite demonstrated by of Shah 5 was and Kumar incorporated [74,75 ] into (Scheme 5- 9 orA). 7-nt The 5oligonucleotides0-O-DMTr-20-O -TBDMS-protected using standard phosphoramiditephosphoramidite of 5 chemistrywas incorporated with more into than 5- or 98% 7-nt oligonucleotides coupling efficiency. using The standard trityl-off, phosphoramidite CPG-linked chemistryprecursor with 36 more was than converted 98% coupling to s4U-RNA efficiency. by treatment The trityl-o withff, CPG-linked10% thioacetic precursor acid (CH363wasC(O)SH) converted in acetonitrile4 (12 h, rt). Then, 10% DBU in methanol (16 h, rt) was used to release the oligomer from the to s U-RNA by treatment with 10% thioacetic acid (CH3C(O)SH) in acetonitrile (12 h, rt). Then, 10% DBUsupport in methanol and to remove (16 h, the rt) base-labile was used protecting to release groups. the oligomer The 2′-O from-TBDMS the supportgroups were and cleaved to remove with the TBAF to give the final s4U-RNA 38. base-labile protecting groups. The 20-O-TBDMS groups were cleaved with TBAF to give the final s4U-RNA 38. Molecules 2020, 25, 3344 13 of 37 Molecules 2020, 25, x FOR PEER REVIEW 13 of 37

SchemeScheme 9. 9.( A(A)) GeneralGeneral scheme scheme for for the the synthesis synthesis of 4-thiouridine-containing of 4-thiouridine-containing oligomer oligomer 38 via the38 post-via the post-syntheticsynthetic reaction reaction of ofconvertible convertible oligomers oligomers 36 36andand 37 37withwith thioacetic thioacetic acid. acid. Reagents Reagents and and conditions: conditions: (1) 10%(1) 10% thioacetic thioacetic acid, acid, MeCN, MeCN, 12 12 h, h, rt; rt; (2) (2) 10% 10% DBU DBU/MeOH,/MeOH,16 16 h,h, 25–3025–30 ◦°C;C; (3) 1 M M TBAF/THF, TBAF/THF, 24 24 h, h, rt or neatrt or TEAneat TEA·3HF,3HF, 8 h, rt 8 orh, rt TEA or TEA·3HF/DMSO3HF/DMSO (1:1 (1:1v/v), v 24/v), h, 24 rt; h, ( Brt;) ( PreparationB) Preparation of 4-ofO 4--methyluridine-RNAO-methyluridine- · · 39.RNA Reagents 39. Reagents and conditions: and conditions: (1) 10% (1) DBU 10%/ MeOH,DBU/MeOH, 16 h, 3016 h,C; 30 (2) °C; TEA (2) TEA·3HF/DMSO3HF/DMSO (1:1 (1:1v/v ),v/ 24v), h,24 rt. ◦ · h, rt. Thioacetic acid reagent was also employed by Komatsu and co-workers to obtain s4U-derivative 38 byThioacetic the conversion acid reagent of 4- wasO-(4-nitrophenyl)uridine-containing also employed by Komatsu and co-workers oligomer to obtain37 sprepared4U-derivative using 50-O38-DMTr-2 by the 0 conversion-O-TBDMS of chemistry 4-O-(4-nitrophenyl)uridine-containing (Scheme9A) [ 77]. The conversion oligomer and 37base–deprotection prepared using 5′-O steps- wereDMTr-2′- performedO-TBDMS according chemistry to Shah’s (Scheme procedure 9A) [77]. [74] The in 94% conversion yield. After and desilylationbase–deprotection and purification steps were (RP andperformed IE HPLC), according the overall to Shah’s yield of procedure s4U-RNA [74] oligomer in 94% 38yield.was After determined desilylation as 10%. and purification The 4-nitrophenyl (RP 4 etherand leaving IE HPLC), group the overall was also yield eff ectivelyof s U-RNA displaced oligomer with 38 was methoxy determined substituent as 10%. by The the 4-nitrophenyl incubation of ether leaving group was also effectively displaced with methoxy substituent by the incubation of precursor oligomer 37 with 10% DBU/MeOH at 30 ◦C for 16 h (Scheme9B). Strong alkaline conditions precursor oligomer 37 with 10% DBU/MeOH at 30 °C for 16 h (Scheme 9B). Strong alkaline conditions permitted the simultaneous cleavage of base-labile protecting groups and resin. Further desilylation permitted the simultaneous cleavage of base-labile protecting groups and resin. Further desilylation (TEA 3HF/DMSO, 1:1 v/v) and HPLC-purification afforded 4-O-methyluridine-RNA 39 in an overall (TEA·3HF/DMSO,· 1:1 v/v) and HPLC-purification afforded 4-O-methyluridine-RNA 39 in an overall 10%10% yield. yield. 4 In 2004,In 2004, Avino Avino and co-workers and co-workers prepared prepared s U-RNAs s4U-RNAs by the by post-synthetic the post-synthetic conversion conversion of oligomers of containingoligomers 4-triazolyl-pyrimidine containing 4-triazolyl-pyrimidine riboside 40 and riboside 4-O-(2-nitrophenyl)uridine 40 and 4-O-(2-nitrophenyl)uridine41 with sodium 41 hydrogen with sulfidesodium NaSH hydrogen (Scheme sulfide 10A) [NaSH76]. To (Scheme avoid oligomer 10A) [76]. degradation To avoid oligomer under base degradation conversion under conditions, base theconversion 50-O-DMTr-2 conditions,0-O-Fpmp-protection the 5′-O-DMTr-2′- systemO-Fpmp-protection was applied for system monomeric was applied units. for The monomeric 6- and 10-nt RNAunits. oligomers The 6- and were 10-nt synthesized RNA oligomers on a 1 wereµmol synthesized scale using on CPG a 1 support.µmol scale The using trityl-on CPG support. support-linked The precursorstrityl-on 40support-linkedand 41 were precursors treated with 40 and a 0.2 41 M were NaSH treated solution with a in 0.2 DMF M NaSH (3 h, rt)solution to achieve in DMF complete (3 h, displacementrt) to achieve of complete the triazolyl displacement group. Then, of the concentratedtriazolyl group. ammonia Then, concentrated was added ammonia (4 h, 50 ◦wasC) toadded release the(4 oligomers h, 50 °C) to from release the supportthe oligomers and remove from the the support base-labile and remove protecting the base-labile groups. The protecting obtained groups. trityl-on The obtained trityl-on 2′-protected-RNAs were purified by RP HPLC and then treated with 10 mM 20-protected-RNAs were purified by RP HPLC and then treated with 10 mM aq. HCl (pH 2.5–3, 4 12 h,aq. rt), HCl yielding (pH 2.5–3, ca. 1012 ODh, rt), of yielding the final ca. s4 U-oligomer10 OD of the42 final. It wass U-oligomer found that 42. the It was triazolyl found derivatives that the (phosphoramiditetriazolyl derivatives building (phosphoramidite block and precursor building oligomer block and40) have precursor a limited oligomer shelf life 40) ( < have3 months), a limited while shelf life (<3 months), while the 4-O-(2-nitrophenyl)uridine derivatives appeared significantly more the 4-O-(2-nitrophenyl)uridine derivatives appeared significantly more stable (shelf life > 3 years). stable (shelf life > 3 years). Molecules 2020, 25, 3344 14 of 37 Molecules 2020, 25, x FOR PEER REVIEW 14 of 37

A NO2 B N N N N N O N N N N

N O N O N O

DMTr O O DMTr O O DMTr O O

O OFpmp O OFpmp O OTBDMS

40 41 43

(1) HNR1R2 (1) NaSH (2) base deprotection & resin cleavage (2) desilylation (3) Fpmp and DMTr removal (3) detritylation

R R S 1 N 2

NH N

N O N O R1, R2 a R1=H, R2=CH3 O O O O b R1=CH3, R2=CH3

O OH O OH 42 44a-b SchemeScheme 10. 10.(A ()A General) General scheme scheme forfor thethe synthesissynthesis of 4-thiouridine-containing oligomers oligomers 42 42viavia the the post-syntheticpost-synthetic reaction reaction of of convertible convertible oligomers oligomers 4040,, 41 with NaSH. NaSH. Reagents Reagents and and conditions: conditions: (1) (1)0.2 0.2M M NaSHNaSH/DMF,/DMF, 3 h, 3 rt;h, rt; (2) (2) conc. conc. NH NH3,3, 4 4 h, h, 50 50◦ °C;C; (3)(3) 10 mM aq. aq. HCl HCl (pH (pH 2.5–3), 2.5–3), overnight, overnight, rt. rt.(B) ( BGeneral) General schemescheme for for preparing preparing the theN N4-methylated4-methylated cytidine-RNAscytidine-RNAs 44a-b44a-b. .Reagents Reagents and and conditions: conditions: (1) (1)gaseous gaseous

methylamine,methylamine, 2 2 h, h, 65 65◦ °C,C, 11 barbar pressure (for (for 44a44a) or) or 40% 40% aq. aq. dimethylamine, dimethylamine, 2.5 h, 2.5 40 h,°C 40(for◦C 44b (for); (2)44b ); (2)NMP/TEA/TEA NMP/TEA/TEA3HF3HF (6:3:4 (6:3:4 v/vv//v/),v ),1.5 1.5 h, h,70 70°C; C;(3) (3)40% 40% aq. aq.AcOH, AcOH, 30 min, 30min, rt. rt. · ◦ TheThe convertible convertible approach approach based based onon thethe 4-triazolyl4-triazolyl pyrimidine pyrimidine nucleoside nucleoside 5, 5which, which was was initially initially introducedintroduced by by Shah Shah and and co-workers co-workers [ [74]74] for for s s44U-RNA preparation preparation (Scheme (Scheme 9),9), was was adopted adopted by by GuennewigGuennewig [78 [78]] and and Koch Koch [ 20 [20]] to to obtain obtain the the NN4-methyl-4-methyl- and N4,N,N4-dimethylcytidine-modified4-dimethylcytidine-modified oligonucleotidesoligonucleotides44a 44a––bb (Scheme(Scheme 10B).10B). Using Using the the “in “insolid-phase” solid-phase” approach, approach, the fully the protected fully protected CPG- CPG-linkedlinked precursor precursor oligomer oligomer 43 (prepared43 (prepared in trityl-on in trityl-on mode) mode) was treated was treated with withgaseous gaseous methylamine methylamine or aq. 40% dimethylamine at 40–65 °C for approximately 2 h. The displacement conditions enabled the or aq. 40% dimethylamine at 40–65 ◦C for approximately 2 h. The displacement conditions enabled effective substitution of the triazolyl group by alkyl and dialkyl amines, and the simultaneous the effective substitution of the triazolyl group by alkyl and dialkyl amines, and the simultaneous removal of base-labile protecting groups and support cleavage (optionally, alkaline deprotection was removal of base-labile protecting groups and support cleavage (optionally, alkaline deprotection was performed with aq. ammonia for 30 min at 40 °C when dimethylamine converting reagent was used performed[20]). After with desilylation, aq. ammonia the crude for RNAs 30 min were at purified 40 ◦C when by RP dimethylamineHPLC and detritylated. converting The RNAs reagent 44a– was usedb were [20]). againAfter purified desilylation by RP, the HPLC crude and RNAs analyzed were by purified ESI mass by RPspectrometry. HPLC and It detritylated. was found that The N RNAs4- 44amonomethylated–b were again purified cytidine-containing by RP HPLC oligomer and analyzed 44a does by not ESI affect mass the spectrometry. hybridization It affinity, was found while that 4 N -monomethylatedthe dimethylated cytidine cytidine-containing analog 44b significantly oligomer 44a reducesdoes notthe binding affect the affinity hybridization to target aRNAsffinity, and while thecan dimethylated be applied cytidinein negative analog miRNA44b significantlycontrol [78]. reducesIn addition, the bindingthe N4,N a4ffi-dimethylcytidine-modifiednity to target RNAs and can be appliedRNA was in used negative to identify miRNA critical control 23S rRNA [78]. Ininteractions addition, in the drug-dependentN4,N4-dimethylcytidine-modified ribosome stalling [20]. RNA was used to identify critical 23S rRNA interactions in drug-dependent ribosome stalling [20]. 3.2. Carbon–Carbon Bond-Forming Reaction via Sonogashira and Stille Couplings 3.2. Carbon–Carbon Bond-Forming Reaction via Sonogashira and Stille Couplings Palladium-catalyzed Sonogashira and Stille cross-coupling reactions between iodonucleobase- modifiedPalladium-catalyzed oligomers and terminal Sonogashira alkynes and or Stilleunsaturated cross-coupling stannanes, reactions respectively, between were iodonucleobase-employed for modifiedthe post-synthetic oligomers andfunctionalization terminal alkynes of RNA or unsaturatedby the formation stannanes, of the new respectively, C-C bond. were Contrary employed to the for thestandard post-synthetic protocol functionalization of the post-synthetic of RNA approach, by the which formation involves of thethe newfull-length C-C bond. oligonucleotide Contrary toas the standardsubstrate, protocol the Sonogashira of the post-synthetic Pd-catalyzed approach, cross-coupling which reactions involves are the performed full-length on oligonucleotide a column [79], as substrate,after the theincorporation Sonogashira of iodonucleoside Pd-catalyzed cross-couplingphophoramidite. reactions It means arethat performedthe synthesis on is ainterrupted column [79 ], after iodonucleoside insertion (without deprotection of the 5′-hydroxyl group), and the column is after the incorporation of iodonucleoside phophoramidite. It means that the synthesis is interrupted removed from the synthesizer. Then, the reagent mixture for cross-coupling (ethynyl reagent, CuI after iodonucleoside insertion (without deprotection of the 5 -hydroxyl group), and the column is and Pd catalyst) is loaded to the column, and after 2–3 h of coupling,0 the column is washed and removed from the synthesizer. Then, the reagent mixture for cross-coupling (ethynyl reagent, CuI reinstalled on the synthesizer. The solid-phase synthesis is continued to obtain the desired full-length and Pd catalyst) is loaded to the column, and after 2–3 h of coupling, the column is washed and oligonucleotide, which is followed by its deprotection and release from resin. This on-column method reinstalledhas been onfound the to synthesizer. present several The advantages solid-phase over synthesis solution is continuedderivatization to obtainsuch as the a low desired consumption full-length oligonucleotide, which is followed by its deprotection and release from resin. This on-column method has been found to present several advantages over solution derivatization such as a low consumption Molecules 2020, 25, 3344 15 of 37

Molecules 2020, 25, x FOR PEER REVIEW 15 of 37 of ethynyl reagent, efficient Pd cross-coupling reaction, and a simple washing step to separate reagents of ethynyl reagent, efficient Pd cross-coupling reaction, and a simple washing step to separate from the support-linked oligomer. reagents from the support-linked oligomer. TheThe post-synthetic post-synthetic Sonogashira Sonogashira coupling coupling reactions reactions were were extensively extensively exploited exploited in the in the Engels Engels group to constructgroup to construct the RNA the oligomers RNA oligomers with nitroxide with nitroxide spin labels spin [labels65,80 –[65,80–82]82] and pyrene and pyrene fluorophores fluorophores [83,84 ]. The[83,84]. authors The described authors describedthe attachment the attachment of spin label of spin 2,2,5,5-tetramethylpyrrolin-1-yloxyl-3-acetylene label 2,2,5,5-tetramethylpyrrolin-1-yloxyl-3- (TPA)acetylene to 5-iodouridine (TPA) to 5-iodouridine or 2-iodoadenosine or 2-iodoadenosine (both introduced (both introduced as a 50-O as-DMTr-2 a 5′-O-DMTr-2′-0-O-TBDMS-protectedO-TBDMS- phosphoramidites)protected phosphoramidites) by on-column by derivatization on-column derivatization using deoxygenated using solutions deoxygenated of Pd(0)(Ph solutions3P) 4 of, CuI in DMFPd(0)(Ph/TEA3P) [480, CuI], or in Pd(II)(PPh DMF/TEA3) 2 [80],Cl2, CuI or Pd(II)(PPh in DCM/TEA3)2Cl2, [ 65 CuI,81 in]. However, DCM/TEA the [65,81]. conditions However, typical the for 50-Oconditions-DMTr-3 0typical-O-TBDMS for 5′- phosphoramiditeO-DMTr-3′-O-TBDMS chemistry phosphoramidite turned out chemistry to be harmful turned for out TPA to be modification, harmful particularlyfor TPA modification, iodine in pyridine particularly/water iodine (used in in oxidation pyridine/water step) (usedand acidic in oxidation conditions step) of and detritylation. acidic To eliminateconditions thisof detritylation. problem, the To 50 -BzH-2eliminate0-O this-ACE-protected problem, the monomeric5′-BzH-2′-O-ACE-protected units were used monomeric (Scheme 11 ). units were used (Scheme 11). The combination of the mild tert-butylhydroperoxide oxidant with the The combination of the mild tert-butylhydroperoxide oxidant with the neutral fluoride for 50-O-BzH removalneutral significantly fluoride for 5′- improvedO-BzH removal the stability significantly of the improved nitroxide the spin stability label and of the increased nitroxide the spin effi labelciency fromand 4–10% increased for TBDMS the efficiency to 35–50% from 4–10% for ACE for chemistry TBDMS to [ 6535–50%,82]. Thefor ACE ACE-protected chemistry [65,82]. phosphoramidites The ACE- protected phosphoramidites of 5-iodouridine, 5-iodocytidine, and 2-iodoadenosine were of 5-iodouridine, 5-iodocytidine, and 2-iodoadenosine were incorporated into 12-, 15-, and 16-nt RNA incorporated into 12-, 15-, and 16-nt RNA oligomers, on a 0.2 µmol scale synthesis. The key reaction oligomers, on a 0.2 µmol scale synthesis. The key reaction of Sonogashira cross-coupling involved of Sonogashira cross-coupling involved iodo-RNAs 45–47 (Scheme 11), TPA 48, and a solution of iodo-RNAs 45–47 (Scheme 11), TPA 48, and a solution of Pd(II)(PPh3)2Cl2 and copper (I) iodide Pd(II)(PPh3)2Cl2 and copper (I) iodide in DCM/TEA (3.5:1.5 v/v) (the cross-coupling was performed v v in DCMtwice /toTEA obtain (3.5:1.5 quantitative/ ) (the yields). cross-coupling After 2.5 wash, the performed column was twice reinstalled to obtain on the quantitative synthesizer yields). to Afteraccomplish 2.5 h, the the column synthesis was of reinstalledthe RNA oligomer. on the synthesizerFully assembled to accomplish RNA was subjected the synthesis to deprotection of the RNA oligomer.and purified Fully by assembled anion-exchange RNA was HPLC. subjected to deprotection and purified by anion-exchange HPLC.

SchemeScheme 11. 11.General General scheme scheme of of a a Sonogashira Sonogashira couplingcoupling reaction between between iodonucleobase-modified iodonucleobase-modified oligomersoligomers45 –4547–47and and nitroxide nitroxide spin-labeled spin-labeled 2,2,5,5-tetramethylpyrrolin-1-yloxyl-3-acetylene (TPA) (TPA) 48. 48.

ReagentsReagents and and conditions: conditions: (1)(1) Pd(II)(PPh33)2)Cl2Cl2, 2CuI,, CuI, deoxygenated deoxygenated DCM/TEA DCM/TEA (3.5:1.5 (3.5:1.5 v/v), rt,v/ v2.5), rt,h; (3) 2.5 h; (3)Deprotection: Deprotection: (i) (i) 0.4 0.4 M M S2Na S2Na2, DMF/H2, DMF2O/H (98:22O (98:2 v/v), v rt,/v ), 30 rt, min; 30 min; (ii) 40% (ii) aq. 40% MeNH aq. MeNH2, rt, 122 , h; rt, (iii) 12 h; (iii)TEMED/CH TEMED/CH3COOH3COOH buffer buff (pHer (pH 3.8), 3.8), 60 °C, 60 ◦30C, min. 30 min.

TheThe combination combination of of ACE ACE phosphoramidite phosphoramidite chemistry chemistry and and Sonogashira Sonogashira coupling coupling was was applied applied forfor the preparationthe preparation of pyrene-modified of pyrene-modified RNA oligomers RNA oligomers53a–b and 53a54a–b (Scheme and 54a 12(Scheme)[83,84 ]. 12)Two [83,84]. fluorophores Two , 1-ethynylpyrenefluorophores, 1-ethynylpyrene52a and 1-(p-ethynyl-phenylethynyl)-pyrene 52a and 1-(p-ethynyl-phenylethynyl)-pyrene52b, were attached 52b, were by attached reaction by with 5-iodocytidine-reaction with 5-iodocytidine- or 2-iodoadenosine-containing or 2-iodoadenosine-containing oligomers 46 oligomers, 47 (prepared 46, 47 as (prepared 10- or 12-nt as 10- oligomers or 12- nt oligomers on a 0.2 µmol scale) and the solution of Pd(0)(PPh3)4, CuI in DCM/TEA by on-column on a 0.2 µmol scale) and the solution of Pd(0)(PPh3)4, CuI in DCM/TEA by on-column synthesis. synthesis. The coupling was performed twice for 2.5 h each to ensure quantitative reaction. Full- The coupling was performed twice for 2.5 h each to ensure quantitative reaction. Full-length oligomers length oligomers were deprotected using a standard order of reactions. The crude oligomers were were deprotected using a standard order of reactions. The crude oligomers were purified by IE HPLC, purified by IE HPLC, affording pure products 53a–b and 54a. affording pure products 53a–b and 54a. Molecules 2020, 25, 3344 16 of 37 Molecules 2020, 25, x FOR PEER REVIEW 16 of 37

SchemeScheme 12. Post-synthetic 12. Post-synthetic Sonogashira Sonogashira coupling coupling reactions reactions between between iodonucleobase-modified iodonucleobase-modified oligomers 46,oligomers47 and alkynyl 46, 47 pyrenes and alkynyl52a–b . pyrenes Reagents 52a–b and. conditions: Reagents and (1)Pd(0)(PPh conditions:3) 4, (1) CuI, Pd(0)(PPh DCM/TEA3)4, CuI,(3.5:1.5 v/v),DCM/TEA rt, 2.5 h; (3)(3.5:1.5 Deprotection: v/v), rt, 2.5 (i)h; 0.4(3) Deprotection: M S2Na2, DMF (i)/ H0.42 OM (98:2S2Na2v,/ vDMF/H), rt, 12 hO (ii) (98:2 40% v/v aq.), rt, MeNH 1 h (ii)2 40%, 55 ◦C, 10 min;aq. MeNH (iii) TEMED2, 55 °C,/ CH10 min;3COOH (iii) TEMED/CH buffer (pH3 3.8),COOH 60 ◦bufferC, 30 (pH min. 3.8), 60 °C, 30 min.

TheThe on-column on-column concept concept was was applied applied by theby groupthe group of Engels of Engels to the to post-synthetic the post-synthetic C–C bond-formingC–C bond- reactionforming via reaction palladium-mediated via palladium-mediated Stille cross-couplings Stille cross-couplings (Scheme (Scheme 13)[85 13)]. [85]. The The feasibility feasibility of Stilleof couplingStille coupling was tested was tested for 5-iodouridine- for 5-iodouridine- and and 2-iodoadenosine-RNA 2-iodoadenosine-RNA precursorsprecursors prepared prepared as as5- or 5- or 12-mers in three strategies: 2′-O-ACE (46, 47), 2′-O-TBDMS (55, 57), and 2′-O-TC (56) on 0.2 or 1 µmol 12-mers in three strategies: 20-O-ACE (46, 47), 20-O-TBDMS (55, 57), and 20-O-TC (56) on 0.2 scale. Iodo-oligomers were coupled with several tributylstannanes (RSnBu3, 58a–d) such as vinyl-, or 1 µmol scale. Iodo-oligomers were coupled with several tributylstannanes (RSnBu3, 58a–d) suchfuryl-, as vinyl-, thienyl-, furyl-, and benzothienyl(tributyl)-stannane. thienyl-, and benzothienyl(tributyl)-stannane. In contrast to Sonogashira In contrast post-synthetic to Sonogashira cross- coupling, the Stille reaction was performed on column after the completion of automated RNA post-synthetic cross-coupling, the Stille reaction was performed on column after the completion synthesis. The synthesizer column with fully assembled RNA was connected with the column- of automated RNA synthesis. The synthesizer column with fully assembled RNA was connected syringe system that loaded the solution of tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3, 15 with the column-syringe system that loaded the solution of tris(dibenzylideneacetone)dipalladium(0) equiv) and tri-2-furylphosphine (P(furyl)3, 30 equiv) in dry DMF and then a solution of alkylstannane (Pd(602(dba) equiv)3, 15 in equiv) DMF. andThe column tri-2-furylphosphine with reaction solution (P(furyl) was3, 30 placed equiv) in in the dry oven DMF at and60 °C then for a2–18 solution h. of alkylstannaneThen, the support-linked (60 equiv) modified in DMF. RNA The (still column closed with in the reaction synthesizer solution column) was placedwas washed, in the dried, oven at 60 ◦andC for subjected 2–18 h. to Then, deprotection. the support-linked The ACE-protected, modified polystyrene-bound RNA (still closed in oligomers the synthesizer were deprotected column) was washed,using dried,the standard and subjected protocol. to The deprotection. TBDMS-protected, The ACE-protected, CPG-linked oligomers polystyrene-bound were treated oligomers with conc were deprotectedNH3/EtOH using (3:1 v the/v, 35 standard °C, 18–24 protocol. h) to cleave The TBDMS-protected,the base-labile protecting CPG-linked groupsoligomers and resin, wereand then, treated withthey conc were NH desilylated.3/EtOH (3:1 Thev/ vTC, 35 chemistry◦C, 18–24 was h) totested cleave exclusively the base-labile for 5-iodouridine-RNA. protecting groups The and CPG- resin, andlinked/TC-protected then, they were desilylated. oligomer enclosed The TC in chemistrythe column was was testedtreatedexclusively with 20% Et2 forNH/MeCN 5-iodouridine-RNA. (3 min, rt) Theto CPG-linked deprotect the/TC-protected phosphate groups. oligomer The enclosed residual in blockage the column and wasCPG treatedresin were with cleaved 20% Et 2withNH /50%MeCN (3 min,solution rt) toof ethylenediamine deprotect the phosphate in dry toluene groups. (2 h, Thert), and residual finally, blockage fully deprotected and CPG RNA resin oligomer were cleaved was witheluted 50% from solution the column of ethylenediamine with 0.1 M TEAA in dry buffer. toluene All (2obtained h, rt), andoligomers finally, 59a fully–d and deprotected 60a–d were RNA oligomerpurified was by elutedIE HPLC. from In thea few column cases, withthe desired 0.1 M TEAAproducts bu offfer. Stille All cross-coupling obtained oligomers reactions59a were–d and 60aaccompanied–d were purified by reduced by IE HPLC. deiodinated In a few RNAs cases, or themethylated desired side products products. of Stille cross-coupling reactions were accompanied by reduced deiodinated RNAs or methylated side products. Molecules 2020, 25, 3344 17 of 37 Molecules 2020, 25, x FOR PEER REVIEW 17 of 37

SchemeScheme 13. 13.Post-synthetic Post-synthetic Stille Stille coupling coupling reaction between iodonucleobase-modified iodonucleobase-modified oligomers oligomers (46(,4746,4755, –5557–57) and) and tributylstannanes tributylstannanes RSnBu RSnBu33 (58a(58a––dd).). Reagents Reagents and and conditions: conditions: (1) Pd (1)2(dba) Pd32 (dba) (15 3 (15equiv), equiv), P(furyl) P(furyl)3 (303 equiv),(30 equiv), dry DMF, dry DMF,60 °C, 60 2–18◦ C, h; 2–18(2) Deprotection: h; (2) Deprotection: acid-labile 2′-O acid-labile-bis(2- 20-Oacetoxyethoxy)methyl-bis(2-acetoxyethoxy)methyl ortoester protection ortoester (ACE) protection strategy: (ACE) (i) 0.4 strategy: M S2Na2, (i)DMF/H 0.4 M2O S (98:22Na2 v,/v DMF), rt, /20H 2O (98:2min;v/v (ii)), rt, 40% 20 aq. min; MeNH (ii) 40%2, 55 aq. °C, MeNH10 min;2 (iii), 55 TEMED/CH◦C, 10 min;3 (iii)COOH TEMED buffer/CH (pH3COOH 3.8), 60 bu°C,ff 30er min; (pH TBDMS 3.8), 60 ◦C, 30 min;strategy: TBDMS (i) 37% strategy: aq. NH (i)3/EtOH 37% aq.(3:1 NH v/v),/ EtOH35 °C, (3:118–24v/ vh;), (ii) 35 NMP/TEA/TEAC, 18–24 h; (ii) NMP3HF (6:3:4/TEA /vTEA/v/v),3HF 60 °C, (6:3:4 3 ◦ · 90 min; TC strategy: (i) 20% Et2NH in MeCN, rt, 3 min; (ii) 50% NH2CH2CH2NH2 in dry toluene, rt, 2 v/v/v), 60 ◦C, 90 min; TC strategy: (i) 20% Et2NH in MeCN, rt, 3 min; (ii) 50% NH2CH2CH2NH2 in dry toluene,h; (iii) rt,0.1 2 M h; TEAA (iii) 0.1 buffer. M TEAA buffer.

3.3.3.3. Cycloaddition Cycloaddition Reactions Reactions TwoTwo types types of post-synthetic of post-synthetic cycloadditions cycloadditions were were employed employed in the in synthesis the synthesis of nucleobase-modified of nucleobase- oligoribonucleotides,modified oligoribonucleotides, Cu(I)-catalyzed Cu(I)-catalyzed cycloaddition cycloaddition of azides and of terminal azides alkynes and terminal (CuAAC alkynes strategy) and(CuAAC inverse strategy) electron and demand inverse Diels-Alder electron demand cycloaddition Diels-Alder (iEDDA). cycloaddition (iEDDA). TheThe CuAAC CuAAC reaction reaction (pioneered (pioneered byby RolfRolf Huisgen [86] [86 ]and and optimized optimized by by Sharpless Sharpless [87] [87 and] and MeldalMeldal [88 ]) [88]) has has been been exploited exploited for the for post-synthetic the post-synthetic incorporation incorporation of 1,4-disubstituted of 1,4-disubstituted 1,2,3-triazoles 1,2,3- triazoles to RNA oligomers by the reaction of ethynyl-modified oligoribonucleotide with an azido- to RNA oligomers by the reaction of ethynyl-modified oligoribonucleotide with an azido-containing containing reagent. The reverse substrate system is not preferred because azido-modified RNA reagent. The reverse substrate system is not preferred because azido-modified RNA oligomers oligomers interfere with the chemical conditions during automated RNA synthesis, particularly with interfere with the chemical conditions during automated RNA synthesis, particularly with P(III) P(III) form. The representative sites for direct attachment of the ethylene group are positions C7 in form.the 7-deazapurineThe representative and C5 sites in forthe direct pyrimidine attachment precursor of the nucleosides ethylene [50,89,90]. group are In positions some cases, C7 the in the 7-deazapurineterminal alkyne and group C5 in was the pyrimidineattached to the precursor nucleobases nucleosides via a linker [50, 89attached,90]. In to some the exoamino cases, the group terminal alkyneof guanosine group was or attachedN3 nitrogen to the of uridine nucleobases [35,91,92]. via a linker attached to the exoamino group of guanosine or N3 nitrogenIn 2010, ofthe uridine Beal’s group [35,91 applied,92]. a post-synthetic CuAAC strategy to introduce N2-modifed 2- aminopurineIn 2010, the ribonucleosides Beal’s group appliedinto an RNA a post-synthetic chain to design CuAAC RNA constructs strategy to for introduce siRNA technologyN2-modifed 2-aminopurine(Scheme 14) [35,91]. ribonucleosides A 2-propargylaminopurine into an RNA ribonucleoside chain to design was converted RNA constructs to 5′-O-DMTr,2′- for siRNAO- technologyTBDMS -cyanoethyl (Scheme 14 phosphoramidite)[35,91]. A and 2-propargylaminopurine incorporated into 12- and ribonucleoside21-nt RNA oligomers was by converted solid- tophase 50-O-DMTr,2 synthesis.0-O After-TBDMS deprotection,β-cyanoethyl alkynyl phosphoramiditeoligomer 61 was conjugated and incorporated with 2-azido-2-deoxy- into 12-D and- 21-ntmannose RNA (62a oligomers), N-azidoacetyl- by solid-phaseD-mannosamine synthesis. (62b), 3′-azidothymidine After deprotection, (AZT, alkynyl62c), or azide oligomer of N- 61 wasethylpiperidine conjugated with(62d). 2-azido-2-deoxy-D-mannoseClick products 63a–d were generated (62a), byN-azidoacetyl-D-mannosamine incubating an aqueous solution (of62b ), RNA oligomer 61 with the water-soluble tris-[1-(3-hydroxypropyl)-1H-[1,2,3]triazol-4- 30-azidothymidine (AZT, 62c), or azide of N-ethylpiperidine (62d). Click products 63a–d were generatedyl)methyl]amine by incubating Cu(I)-stabilizing an aqueous ligand solution(THPTA), ofCuSO RNA4, and oligomer sodium ascorbate61 with (system the water-soluble for an in situ generation of catalytically active Cu(I) species), and azide 62a–d (5 equiv) for 4 h at ambient tris-[1-(3-hydroxypropyl)-1H-[1,2,3]triazol-4-yl)methyl]amine Cu(I)-stabilizing ligand (THPTA), temperature. Complete conversions were observed for primary azides 62b,62d; however, the use of CuSO , and sodium ascorbate (system for an in situ generation of catalytically active Cu(I) species), secondary4 azides 62a,62c reduced conversion yields by 50%. Click products 63a–d were purified by and azide 62a–d (5 equiv) for 4 h at ambient temperature. Complete conversions were observed for denaturing PAGE and confirmed by ESI-MS. Azide 62b was also effectively employed for the post- 62b,62d 62a 62c primarysynthetic azides preparation ;of however, the di-click the 21-nt use of RNA secondary oligomer. azides Thermal, denaturationreduced conversion studies indicated yields by 50%.that Click oligomers products 63b–63ad showed–d were similar purified base by pairing denaturing stability PAGE and and specificity confirmed to adenosine-containing by ESI-MS. Azide 62b was also effectively employed for the post-synthetic preparation of the di-click 21-nt RNA oligomer. Thermal denaturation studies indicated that oligomers 63b–d showed similar base pairing stability and Molecules 2020, 25, 3344 18 of 37 Molecules 2020, 25, x FOR PEER REVIEW 18 of 37 Molecules 2020, 25, x FOR PEER REVIEW 18 of 37 specificitycounterparts. to adenosine-containing [35,91]. Click products counterparts. 63b and 63d [35, 91displayed]. Click productsreduced 63bbindingand 63daffinitydisplayed to off-target reduced bindingproteinscounterparts. affi (RNA-dependentnity [35,91]. to off-target Click proteinsproteinproducts kinase (RNA-dependent63b and and 63d adenosine displayed protein deaminases), reduced kinase binding and while adenosine affinity RNAi activityto deaminases), off-target was whilemaintainedproteins RNAi (RNA-dependent activity [35]. was maintained protein [kinase35]. and adenosine deaminases), while RNAi activity was maintained [35].

SchemeScheme 14. 14.Post-synthetic Post-synthetic Cu(I)-catalyzed Cu(I)-catalyzed cycloaddition of of azides azides and and terminal terminal alkynes alkynes (CuAAC) (CuAAC) reactionreactionScheme of of14.N N2 Post-synthetic-propargyl-2-aminopurine-RNA2-propargyl-2-aminopurine-RNA Cu(I)-catalyzed cycloaddition (61 (61) with) with azides of azides azides 62a-d62a and. Reagents–d terminal. Reagents and alkynes conditions: and (CuAAC) conditions: (i) 10 (i) 10mMreaction mM solution solution of N 2of-propargyl-2-aminopurine-RNA of62a62a andand 62b62b in inwater water or or40% 40% aq. (61 aq. DMSO) with DMSO azidessolution solution 62a-d of 62c of. Reagents62c or 0.5or 0.5M and Tris-HCl M Tris-HClconditions: solution solution (i) 10of of 62dmM solution (pH of 8.0), 62a and CuSO 62b4, in water sodium or 40% ascorbate, aq. DMSO tris-[1-(3-hydroxypropyl)-1solution of 62c or 0.5 M Tris-HClH-[1,2,3]triazol-4- solution of 62d (pH 8.0), CuSO4, sodium ascorbate, tris-[1-(3-hydroxypropyl)-1H-[1,2,3]triazol-4-yl)methyl]amine Cu(I)-stabilizingyl)methyl]amine62d (pH 8.0), ligand Cu(I)-stabilizing CuSO (THPTA),4, sodium rt, ligand 4 h. (THPTA), ascorbate, rt, 4 tris-[1-(3-hydroxypropyl)-1h. H-[1,2,3]triazol-4- yl)methyl]amine Cu(I)-stabilizing ligand (THPTA), rt, 4 h. FurtherFurther studies studies of the the Beal Beal group group focused focused on the on isertion the isertion of several of 7-substituted several 7-substituted 8-aza-7- 8-aza-7-deazaadenosinesdeazaadenosinesFurther studies into of 12-nt into the 12-nt siRNABeal groupsiRNA constructs focused constructs by on azide-alkyne by the azide-alkyne isertion cycloaddition of several cycloaddition 7-substituted (Scheme (Scheme 15) 8-aza-7- [89]. 15 )[ 7-89]. 7-Ethynyl-8-aza-7-deazaadenosine-containingEthynyl-8-aza-7-deazaadenosine-containingdeazaadenosines into 12-nt siRNA constructs oligomer by oligomer azide-alkyne 64 64was was cycloaddition synthesized synthesized (Scheme using using 15) standard [89]. standard 7- phosphoramiditephosphoramiditeEthynyl-8-aza-7-deazaadenosine-containing chemistry chemistry (1.0(1.0 mmol scale) scale) with oligomer with the the coupling64 coupling was time synthesized time prolonged prolonged to using 25 min to standard25 for min a 7- for ethynyl-modifiedphosphoramidite monomericchemistry (1.0 unit mmol (prepared scale) as with 5′-O -DMTr-2′-the couplingO-TBDMS, time prolonged NH-dmf toamidite 25 min with for TMS a 7- a 7-ethynyl-modified monomeric unit (prepared as 50-O-DMTr-20-O-TBDMS, NH-dmf amidite with blockageethynyl-modified on the ethynyl monomeric group unit to prevent(prepared partial as 5′- Ohydrolysis-DMTr-2′- duringO-TBDMS, the alkaline NH-dmf deprotection amidite with step). TMS TMS blockage on the ethynyl group to prevent partial hydrolysis during the alkaline deprotection step). Afterblockage deprotection, on the ethynyl precursor group oligomer to prevent 64 partialwas incubated hydrolysis with during aqueous the alkalinesolution deprotectionof THPTA ligand, step). After deprotection, precursor oligomer 64 was incubated with aqueous solution of THPTA ligand, CuSOAfter 4deprotection,, sodium ascorbate, precursor and oligomer azide 65a 64–f forwas 6.5 incubated h at rt or with 35 °C. aqueous Azides solutionwere dissolved of THPTA in aq. ligand, Tris- CuSO4, sodium ascorbate, and azide 65a–f for 6.5 h at rt or 35 ◦C. Azides were dissolved in aq. Tris-HCl HClCuSO buffer4, sodium with ascorbate,pH 8.0 (65a and), water azide ( 65b65a–df ),for or 6.5 DMSO h at rt(65e or ,35 65f °C.). Click Azides products were dissolved 66a–f were in aq.purified Tris- buffer with pH 8.0 (65a), water (65b–d), or DMSO (65e, 65f). Click products 66a–f were purified by byHCl denaturating buffer with pHPAGE 8.0 and(65a identified), water (65b by –MALDI-ToFd), or DMSO MS. (65e Study, 65f). ofClick the productsmodification 66a –impactf were onpurified RNA denaturatingpropertiesby denaturating PAGErevealed PAGE and a and identified substantial identified by destabilization by MALDI-ToF MALDI-ToF MS. ofMS. RNA Study Study duplexes ofof the modification modification modified impact with impact sterically on onRNA RNA propertiesdemandingproperties revealed revealed triazoles a substantial 66e a substantial and 66f destabilization. Oligomers destabilization ofwith RNA less of duplexes RNA hindrance duplexes modified (66a– d modified) with indicated sterically with only stericallydemanding a slight triazolesdecreasedemanding66e ofand duplex triazoles66f. stability Oligomers 66e and and 66f with a little. Oligomers less effect hindrance of withmodification (less66a– hindranced) indicatedon the base-pairing (66a only–d) a indicated slight specificity. decrease only a of slight duplex stabilitydecrease and of a duplex little e ffstabilityect of modification and a little effect on the of modification base-pairing on specificity. the base-pairing specificity.

Scheme 15. Post-synthetic CuAAC reaction of 7-ethynyl-8-aza-7-deazaadenosine-RNA (64) with SchemeazidesScheme 15. 65a 15.Post-synthetic–f . Post-syntheticReagents and CuAAC conditions: CuAAC reaction reaction (i) THPTA, of 7-ethynyl-8-aza-7-deazaadenosine-RNA of 7-ethynyl-8-aza-7-deazaadenosine-RNA CuSO4, sodium ascorbate, 0.05 M solution (64 ) ( with64 )of with azides65a

65ainazides–f .0.5 Reagents M 65a Tris-HCl–f. Reagents and (pH conditions: 8.0),and 0.5conditions: M (i) or THPTA, 0.15 (i) M THPTA, CuSOsolution4, CuSO sodium of 65b4, –sodiumd ascorbate, in water ascorbate, or 0.05 0.15 M 0.05M solution solution M solution of of65a 65e ofin– f65a 0.5in M Tris-HClDMSO,in 0.5 M (pH rt Tris-HCl (35 8.0), °C 0.5for (pH M65f or8.0),), 6.5 0.15 0.5 h. MM solutionor 0.15 M of solution65b–d inof water65b–d orin water 0.15 M or solution 0.15 M solution of 65e–f ofin 65e DMSO,–f in rt DMSO, rt (35 °C for 65f), 6.5 h. (35 ◦C for 65f), 6.5 h. Kellner and co-workers employed click chemistry to label unmodified RNAs with fluorescent dyesKellner Kellner(Scheme and and 16) co-workers co-workers[92]. For successful employed employed labeling, clickclick chemistry the unmodified to to label label RNAs unmodified unmodified 67 (prepared RNAs RNAs as with synthetic with fluorescent fluorescent RNA dyesdyes (Scheme (Scheme 16 16))[92 [92].]. For For successful successful labeling,labeling, the unmodified unmodified RNAs RNAs 6767 (prepared(prepared as assynthetic synthetic RNA RNA Molecules 2020, 25, 3344 19 of 37

Molecules 2020, 25, x FOR PEER REVIEW 19 of 37 Molecules 2020, 25, x FOR PEER REVIEW 19 of 37 oligomers or yeast tRNAPhe in vitro transcript) were incubated with bifunctional azido-bromo-coumarin oligomers or yeast tRNAPhe in vitro transcript) were incubated with bifunctional azido-bromo- 68 incoumarinoligomers denaturating 68 or in yeast denaturating conditions tRNAPhe conditions in (70% vitro aq. transcript) (70% DMSO, aq. DMSO, pH were 8.5, incubated pH 37 8.5,◦C, 37 3 with °C, h) to 3 bifunctional h) provide to provide products azido-bromo- products69 69with chemoselectivelywithcoumarin chemoselectively 68 in N3-alkylateddenaturating N3-alkylated conditions uridine. uridine. The (70% azidocoumarin-modified The aq. azidocoumarin-modifiedDMSO, pH 8.5, 37 °C, RNA 3 h) RNA to69 provide was69 was subjected subjectedproducts to 69to click conjugationclickwith chemoselectively conjugation with fluorescent with N3-alkylated fluorescent alkyne-containing alkyne-containinguridine. The AlexaFluor azidocoumarin-modified AlexaFluor 647 (70a 647) or (70a fluorescein RNA) or fluorescein69 was (70b subjected) in (70b bu)ff to iner of + 2+ pHbufferclick= 8, conjugationin of the pH dark, = 8, in with using the fluorescentdark, Cu using(generated alkyne-containingCu+ (generated in situ from in AlexaFluorsitu sodium from sodium ascorbate 647 (70a ascorbate) and or fluorescein Cu and) withCu2+ ()70b awith THPTA) ina ligandTHPTAbuffer (rt, of 2ligand pH h). Click= (rt,8, in 2 products theh). Clickdark, 71aproducts using–b wereCu 71a+ (generated analyzed–b were analyzed in by situ PAGE from by electrophoresis. PAGE sodium electrophoresis. ascorbate and Cu2+) with a THPTA ligand (rt, 2 h). Click products 71a–b were analyzed by PAGE electrophoresis.

SchemeScheme 16. 16.Chemoselective Chemoselective labeling labeling ofof RNARNA 6767 withwith azide-containing coumarine coumarine and and the the following following clickclickScheme reaction reaction 16. of Chemoselective69 ofwith 69 ethynyl-fluorescentwith ethynyl-fluorescent labeling of RNA dyes 67 70a dyeswith–b .70aazide-containing Reagents–b. Reagents and conditions: coumarine and conditions: (i) and 100 the mM (i) following 100 phosphate mM click reaction of 69 with ethynyl-fluorescent dyes 70a–b. Reagents and conditions: (i) 100 mM buffphosphateer (pH = 8.5),buffer 70% (pH=8.5), DMSO, 70% 37 DMSO,◦C, 3 h, 37 under °C, 3 lighth, under protection; light protection; (ii) PUS (ii) bu ffPUSer (100buffer mM (100 Tris-HCl, mM Tris-HCl,phosphate pH=8.0, buffer (pH=8.5),100 mM NH70%4OAc, DMSO, 1 mM 37 °C,MgCl 3 h,2), under2.5 mM light THPTA, protection; 0.5 mM (ii) CuSO PUS 4buffer, 5 mM (100 sodium mM pH = 8.0, 100 mM NH4OAc, 1 mM MgCl2), 2.5 mM THPTA, 0.5 mM CuSO4, 5 mM sodium ascorbate, Tris-HCl, pH=8.0, 100 mM NH4OAc, 1 mM MgCl2), 2.5 mM THPTA, 0.5 mM CuSO4, 5 mM sodium alkyne-fluorescentascorbate, alkyne-fluorescent dye at 50 µM dye concentration, at 50 µM concentration, rt, 2 h, under rt, 2 light h, under protection. light protection. ascorbate, alkyne-fluorescent dye at 50 µM concentration, rt, 2 h, under light protection. TheThe post-synthetic post-synthetic CuAAC CuAAC reaction reaction was wasemployed employed by Kerzhner by Kerzhner and co-workers and co-workersto introduce to introducethe nitroxide-spinThe thepost-synthetic nitroxide-spin labels CuAACinto RNA labels reaction oligomers into was RNA for employed EPR/PELDOR oligomers by Kerzhner for measurements EPR /andPELDOR co-workers [50] measurements (Scheme to introduce 17). The [50 ] 5′-theO nitroxide-spin-DMTr-5-ethynyl-2′-deoxyuridine labels into RNA oligomers phosphoramidite for EPR/PELDOR (additionally measurements protected[50] (Scheme with 17). The a (Scheme 17). The 50-O-DMTr-5-ethynyl-20-deoxyuridine phosphoramidite (additionally protected 5′-O-DMTr-5-ethynyl-2′-deoxyuridine phosphoramidite (additionally protected with a withtriisopropylsilyl a triisopropylsilyl group group on ethynyl on ethynyl moiety) moiety) was was effectively effectively introduced introduced to an to anRNA RNA oligomer oligomer by by triisopropylsilyl group on ethynyl moiety) was effectively introduced to an RNA oligomer by conventionalconventional solid-phase solid-phase synthesis. synthesis. Using Using thethe “in-solution” approach, approach, the the fully fully deprotected deprotected oligomer oligomer conventional solid-phase synthesis. Using the “in-solution” approach, the fully deprotected2+ oligomer 72 was conjugated with azide-functionalized nitroxide 73 with CuI instead of a Cu 2-ascorbate+ system 72 was conjugated with azide-functionalized nitroxide 73 with CuI instead of a Cu2+ -ascorbate system (due72 was to conjugatedthe reducible with nature azide-functionalized of nitroxide radicals) nitroxide with 73 the with excess CuI of instead THPTA of ain Cu DMSO-H-ascorbate2O solution system (due to the reducible nature of nitroxide radicals) with the excess of THPTA in DMSO-H O solution for(due 30 to min the at reducible 37 °C. The nature spin-labeled of nitroxide oligomer radicals) was withpurified the excessby HPLC of THPTA to afford in the DMSO-H product2O 742 solution in total for 30 min at 37 C. The spin-labeled oligomer was purified by HPLC to afford the product 74 in total 72%for 30 yield. min atThe 37◦ same°C. The oligomer spin-labeled spin labeling oligomer was was demonstrated purified by HPLC by Kerzhner to afford and the co-workers product 74 in in solidtotal 72% yield. The same oligomer spin labeling was demonstrated by Kerzhner and co-workers in solid phase72% yield. [90]. TheIn this same case, oligomer the total spin reaction labeling time was was demonstrated considerably by longerKerzhner (24 and h), andco-workers the conversion in solid phaseyieldphase [90 dropped ].[90]. In thisIn thisto case, 36%. case, the the total total reaction reaction time time was was considerably considerably longer longer (24 (24 h), h), and and the the conversion conversion yield droppedyield dropped to 36%. to 36%.

Scheme 17. Post-synthetic spin labeling of RNA oligomer 72 with the azide-functionalized nitroxide Scheme 17. Post-synthetic spin labeling of RNA oligomer 72 with the azide-functionalized nitroxide 73Scheme by click 17. chemistry. Post-synthetic Reagents spin andlabeling conditions: of RNA (i) oligomer CuI, THPTA, 72 with DMSO-H the azide-functionalized2O, 30 min, 37 °C. nitroxide 73 by73 by click click chemistry. chemistry. Reagents Reagents and and conditions: conditions: (i) CuI, THPTA, DMSO-H DMSO-H2O,2O, 30 30 min, min, 37 37°C.◦ C. Recently, Nakamoto and co-workers have demonstrated the use of CuAAC reaction for the Recently, Nakamoto and co-workers have demonstrated the use of CuAAC reaction for the incorporationRecently, of Nakamoto biotin to oligoribonucleotide and co-workers have modified demonstrated with a novel the use bifunctional of CuAAC nucleoside reaction foranalog the incorporationincorporation of of biotin biotin to to oligoribonucleotide oligoribonucleotide modifiedmodified with with a a novel novel bifunctional bifunctional nucleoside nucleoside analog analog Molecules 2020, 25, 3344 20 of 37

Molecules 2020, 25, x FOR PEER REVIEW 20 of 37 containing aryl trifluoromethyl diazirine and ethynyl moieties (Scheme 18)[93]. Precursor oligomer containing aryl trifluoromethyl diazirine and ethynyl moieties (Scheme 18) [93]. Precursor oligomer 75 was prepared using 5 -O-DMTr-2 -O-TBDMS building blocks and a standard protocol of 75 was prepared using 0 5′-O-DMTr-2′-0 O-TBDMS building blocks and a standard protocol of phosphoramiditephosphoramidite solid-phase solid-phase synthesis synthesis andand deprotection.deprotection. The The ethylene–RNA ethylene–RNA probe probe 75 75waswas treated treated 76 withwith biotynyl-TEG-azide biotynyl-TEG-azide 76 (80(80 equivalent) equivalent) in the in thepresence presence of sodium of sodium ascorbate, ascorbate, CuSO4 and CuSO tris[(1-4 and tris[(1-benzyl-1benzyl-1H-1,2,3-triazol-4-yl)methyl]amineH-1,2,3-triazol-4-yl)methyl]amine (TBTA) (TBTA) for 1 h at for rt. 1 The h at RP rt. HPLC The RP purified HPLC product purified 77 product was 77 wasidentified identified by MALDI-ToF by MALDI-ToF MS. MS.

SchemeScheme 18. 18.Post-synthetic Post-synthetic Cu-catalyzed Cu-catalyzed azide-alkyne azide-alkyne cycloaddition cycloaddition of of oligomer oligomer75 75with with biotynyl-TEG- biotynyl- azideTEG-azide76. Reagents 76. Reagents and conditions: and conditions: (i) sodium (i) ascorbate, sodium ascorbate, tris[(1-benzyl-1 tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]H-1,2,3-triazol-4- amineyl)methyl]amine (TBTA), CuSO (TBTA),4, DMSO, CuSO 0.24, MDMSO, phosphate 0.2 M phosphate buffer (pH buffer 7.0), rt,(pH 1 h.7.0), rt, 1 h.

BeyondBeyond the the CuAAC CuAAC cycloaddition, cycloaddition, the the inverse inverse electron electron demand demand Diels-Alder Diels-Alder reaction reaction (iEDDA) (iEDDA) was employedwas employed for the post-synthetic for the post-synthetic labeling of labeling norbornene-containing of norbornene-containing RNA oligomer RNA 78 oligomerwith disubstituted 78 with 1,2,4,5-tetrazine-fluorophoredisubstituted 1,2,4,5-tetrazine-fluorophore derivatives 79a derivatives–d (Scheme 79a 19–d)[ (Scheme94]. The 19) fast [94]. conjugation The fast conjugation between the ring-strainedbetween the olefin ring-strained group as olefin a dienophile group as and a dienophile highly electron-deficient and highly electron-deficient 1,2,4,5-tetrazine 1,2,4,5-tetrazine as a diene was discoveredas a diene by was the Foxdiscovered and Hilderbrand by the Fox groups and Hilderbrand in 2008 [95, 96groups]. To demonstratein 2008 [95,96]. the To use demonstrate of iEDDA strategy the in theuse post-synthetic of iEDDA strategy labeling in ofthe RNAs, post-synthetic the norbornene labeling modification of RNAs, the was norbornene introduced modification into three di wasfferent introduced into three different oligoribonucleotides (12-, 21-, and 24-mers) using 5-norbornene- oligoribonucleotides (12-, 21-, and 24-mers) using 5-norbornene-modified 50-O-DMTr-20-O-TBDMS modified 5′-O-DMTr-2′-O-TBDMS uridine phosphoramidite and standard conditions of RNA uridine phosphoramidite and standard conditions of RNA synthesis. Deprotected oligomers 78 synthesis. Deprotected oligomers 78 were conjugated with a 2- or 3-fold excess of tetrazine- were conjugated with a 2- or 3-fold excess of tetrazine-fluorophore derivatives 79a–d containing the fluorophore derivatives 79a–d containing the fluorophore Oregon Green 488 in aqueous solution (rt, fluorophore Oregon Green 488 in aqueous solution (rt, 1 h). Cycloaddition products were identified 1 h). Cycloaddition products were identified by PAGE and MS analysis. It was indicated that the byoverall PAGE and yields MS of analysis.cycloaddition It was reactions indicated were that comparable the overall regardless yields of of cycloaddition norbornene position reactions (the were comparablesingle- or double-stranded regardless of norbornene regions of RNAs). position However, (the single- they were or double-stranded dependent on the regions steric effects of RNAs). of However,the disubstituted they were tetrazine dependent derivatives on the steric withe theffects RNA of the oligomer disubstituted (reduced tetrazine yields were derivatives observed with for the RNAcycloadditions oligomer (reduced with tetrazinesyields were 79c observed,d). A bioorthogonalfor cycloadditions reaction with tetrazines of iEDDA79c between,d). A bioorthogonal tetrazine- reactionfluorophore of iEDDA 79a between and 21-nt tetrazine-fluorophore norbornene-modified79a and RNA 21-nt 78 norbornene-modified (prepared as a siRNA RNA duplex)78 (prepared was as aeffectively siRNA duplex) performed was in emammalianffectively performed cells. In contrast in mammalian to already known cells. In techniques, contrast tothe already fluorophore known techniques,can be attached the fluorophore to RNA after can the be target attached binding, to RNA which after minimizes the target the binding, distortion which of RNA minimizes transport the distortionand the offunctions RNA transport in the cells. and the functions in the cells. Molecules 2020, 25, 3344 21 of 37 Molecules 2020, 25, x FOR PEER REVIEW 21 of 37

SchemeScheme 19. 19.Inverse Inverse electron-demand electron-demand Diels–Alder Diels–Alder cycloaddition cycloaddition of of tetrazine-Oregon tetrazine-Oregon Green Green 488 488 conjugatesconjugates79a 79a–d–withd with norbornene-modified norbornene-modified RNARNA oligomer 7878.. Conditions: Conditions: (i) (i) water, water, rt, rt, 1 h. 1 h.

3.4.3.4. Derivatization Derivatization of of Amino-Modified Amino-Modified Oligoribonucleotides Oligoribonucleotides via via Formation Formation of of Amide Amide Linkage Linkage Amino-prefunctionalizedAmino-prefunctionalized oligoribonucleotides oligoribonucleotides provide provide a a very very important important class class of of substrates substrates forfor the post-syntheticthe post-synthetic modification modification/labeling/labeling of RNA of oligomers. RNA oligomers. Site-specifically Site-specifically introduced introduced amino-modified amino- unitsmodified are usually units are conjugated usually conjugated via amide via bond amide formation bond formation with active with active esters esters derived derived from from the the label andlabelN-hydroxysuccinimide and N-hydroxysuccinimide (NHS), (NHS), tetrafluorophenol tetrafluorophenol (TFP), (TFP),or N-hydroxybenzotriazole or N-hydroxybenzotriazole (HOBt). The(HOBt). active estersThe active react esters selectively react withselectively an aliphatic with an amino aliphatic function amino in function solution, in after solution, the deprotection after the deprotection of RNA oligomers, without any risk of side reactions with 2′–OH and aromatic amino of RNA oligomers, without any risk of side reactions with 20–OH and aromatic amino groups of groups of nucleobases. Based on this strategy, efficient methods for the incorporation of fluorescent nucleobases. Based on this strategy, efficient methods for the incorporation of fluorescent dyes, flavin dyes, flavin mononucleotide derivative, as well as nitroxide and diazirine labels into RNA oligomers mononucleotide derivative, as well as nitroxide and diazirine labels into RNA oligomers have been have been developed for their application in the structural and functional analysis of RNA molecules developed for their application in the structural and functional analysis of RNA molecules [69,97,98]. [69,97,98]. Hydantoin RNA modification has also been reported as an example of intramolecular Hydantoin RNA modification has also been reported as an example of intramolecular amide bond amide bond formation produced by cyclization within the N6-threonylcarbamoyl functional group 6 formationattached produced to an adenosine by cyclization precursor within [63,64]. the N -threonylcarbamoyl functional group attached to an adenosineIn 2011, precursor the group [63,64 of]. Muller employed amino-modified RNA oligomers 81a–c and 82a,c as substratesIn 2011, for the post-synthetic group of Muller amino employed groups conjugation amino-modified with fluorescent RNA oligomers dyes (Alexa488,81a–c and Cy582a,c and as substratesATTO647N) for post-synthetic and flavin mononucleotide amino groups (FAM) conjugation (Scheme with 20) fluorescent [97]. Primary dyes amino (Alexa488, groups Cy5 were and ATTO647N)attached to and the flavin C5 position mononucleotide of uridine (FAM)or C8 position (Scheme of 20 adenosine)[97]. Primary via a linker amino to groups increase were the attachedpost- to thesynthetic C5 position conversion of uridine yields between or C8 position amino nucleobases of adenosine and via bulky a linker size labels. to increase Two alkenyl- the post-synthetic and one conversionalkynylamino yields linkers between were amino utilized nucleobases that varied and in bulky terms size of length labels. and Two flexibility alkenyl- (Scheme and one alkynylamino20 a, b and linkersc). were utilized that varied in terms of length and flexibility (Scheme 20a–c). Molecules 2020, 25, 3344 22 of 37 Molecules 2020, 25, x FOR PEER REVIEW 22 of 37

SchemeScheme 20. 20.Post-synthetic Post-synthetic labeling labeling ofof amino-modifiedamino-modified oligomers oligomers 81a81a–c– candand 82a,c82a,c withwith active active esters esters (83,(83, 84) 84 of) Alexa488,of Alexa488, Cy5, Cy5, ATTO647N, ATTO647N, FMN, FMN,and and diazirine.diazirine. Reagents Reagents and and conditions: conditions: (i) (i)conjugation conjugation

withwith R1 –TFPR1–TFP and and R 2,3R2,3–NHS–NHS esters: esters: 81a81a–cc/82a,c/82a,c inin 0.1 0.1 M M borax borax buffer buffer at at pH pH 9.4 9.4 (for (for R1 R) 1or) or8.7 8.7 (for (for R2,3 R),2,3 ), dye in DMF, overnight, rt, under light protection; (ii) conjugation with R4–NHS (N- dye in DMF, overnight, rt, under light protection; (ii) conjugation with R4–NHS (N-hydroxysuccinimide) ester:hydroxysuccinimide) FMN–NHS ester wasester: generated FMN–NHS in ester situ usingwas generated TSTU (1.2 in equiv), situ using DIPEA TSTU (2.0 (1.2 equiv), equiv), DMF, DIPEA 1 h, rt; (2.0 equiv), DMF, 1 h, rt; (iii) conjugation with R5–NHS ester: 81a in siRNA buffer (Dharmacon), Na (iii) conjugation with R5–NHS ester: 81a in siRNA buffer (Dharmacon), Na HEPES pH 7.5, diazirine–NHS in DMSOHEPES (pH> 60 7.5, equiv), diazirine–NHS 2–4 h, rt. in DMSO (> 60 equiv), 2–4 h, rt.

ToTo prepare prepare the amino-modified the amino-modified oligomers oligomers81a– c81aand–c82a,c and the82a,c standard the standard phosphoramidite phosphoramidite chemistry chemistry was used with 5′-O-DMTr-2′-O-TBDMS-protected building blocks and CPG support (the was used with 50-O-DMTr-20-O-TBDMS-protected building blocks and CPG support (the aliphatic aliphatic amino group of the linkers was protected with trifluroacetyl). Oligomers were effectively amino group of the linkers was protected with trifluroacetyl). Oligomers were effectively deprotected deprotected (conc. NH3/MeNH2-EtOH, 1:1 v/v, 2 h, rt, then TEA3HF/DMF, 55 °C, 1.5 h) and purified (conc. NH /MeNH -EtOH, 1:1 v/v, 2 h, rt, then TEA 3HF/DMF, 55 C, 1.5 h) and purified by PAGE by PAGE3 electrophoresis.2 To attach the fluorescent dyes,· precursor oligomers◦ 81a–c and 82a,c were electrophoresis. To attach the fluorescent dyes, precursor oligomers 81a–c and 82a,c were dissolved in dissolved in borax buffer at an approximate pH 9; then, freshly prepared solution of active TFP ester borax83 (for buff Alexa488)er at an approximate or NHS ester pH 84 9;(for then, Cy5 freshly and ATTO647N) prepared solutionin DMF was of active added. TFP The ester labeling83 (for reactions Alexa488) or NHSwere estercarried84 out(for at Cy5 rt overnight and ATTO647N) in the dark. in DMF To conjugate was added. the The flavinmononucleotide labeling reactions were derivative, carried the out 5- at rt overnightaminoallyluridine-RNA in the dark. To conjugate 81a wasthe treated flavinmononucleotide with R4-NHS ester derivative, 84 generated the 5-aminoallyluridine-RNA in situ using TSTU 81acondensingwas treated reagent with R 4-NHS and DIPEA ester 84 ingenerated DMF. Oligomers in situ using containing TSTU condensing Alexa488, reagent Cy5, and DIPEA flavin in DMF.mononucleotide Oligomers containing derivative Alexa488, were purified Cy5, and using flavin mononucleotide denaturing PAGE derivative (yields were 15–54%), purified while using denaturingATTO647N-labeled PAGE (yields oligomers 15–54%), were while isolated ATTO647N-labeled by RP HPLC (yields oligomers 40–50%). were The isolatedstructures by of RP target HPLC (yieldsoligomers 40–50%). were The confirmed structures by ofMALDI-ToF target oligomers mass spectrometry. were confirmed by MALDI-ToF mass spectrometry. Recently, post-synthetic transformations of amino-modified oligoribonucleotides to photo-crosslinkable diazirine-derivatives have been employed to investigate the regulation of protein-RNARecently, interactions post-synthetic by 6-methyladenosine transformations of[98 ]. amino-modified For preparing theoligoribonucleotides diazirine-containing to photo- oligomer 87acrosslinkable(Scheme 20 diazirine-derivatives), (E)-5-(3-aminoprop-1-en-1-yl)uridine-RNA have been employed to investigate81a was the suspendedregulation of in protein-RNA siRNA buff er (Dharmacon,interactions Lafayette,by 6-methyladenosine Colorado, [98]. USA), For preparing then Na–HEPES the diazirine-containing (pH 7.5) and oligomer a solution 87a of (Scheme diazirine NHS20), ester (E)-5-(3-aminoprop-1-en-1-yl)uridine-RNA (>60 equiv) in DMSO were added. 81a Thewas suspended reaction was in siRNA vortexed buffer and (Dharmacon, incubated at ambientLafayette, temperature Colorado, USA), for 2–4 then hNa–HEPES in the dark. (pH 7.5) The and a mixture solution wasof diazirine purified NHS by ester RP (>60 HPLC equiv) and in DMSO were added. The reaction was vortexed and incubated at ambient temperature for 2–4 h in analyzed by mass spectrometry. The developed transformation worked also for 30-biotynylated 6-methyladenosine-modified amino RNA. Molecules 2020, 25, x FOR PEER REVIEW 23 of 37 Molecules 2020, 25, 3344 23 of 37

the dark. The mixture was purified by RP HPLC and analyzed by mass spectrometry. The developed transformationThe post-synthetic worked reaction also for of 3′-biotynylated amino oligomers 6-methyladenosine-modified with active esters was utilized amino for RNA. RNA site-specific spin labelingThe post-synthetic with nitroxide reaction radicals of for amino EPR measurements oligomers with (Scheme active 21esters) [69 was]. Amino-modified utilized for RNA guanosine site- orspecific uridine spin were labeling introduced with into nitroxide the terminal radicals position for EPR ofmeasurements 10-nt oligomers (Scheme via 21)linkers [69]. attached Amino- to modified guanosine or uridine were introduced into the terminal position of 10-nt oligomers via the N7 position of G (89) or C5 position of U (90). The precursor oligomers were prepared by linkers attached to the N7 position of G (89) or C5 position of U (90). The precursor oligomers were the complementary addressed modification method (oligomer 89)[99] or by the post-synthetic prepared by the complementary addressed modification method (oligomer 89) [99] or by the post- strategy (oligomer 90, see Scheme3). Both amino-modified RNAs were spin labeled with new synthetic strategy (oligomer 90, see Scheme 3). Both amino-modified RNAs were spin labeled with 2,5-bis(spirocyclohexane)-substitutednew 2,5-bis(spirocyclohexane)-substituted nitroxide nitroxide and conventional and conventional tetramethyl-substituted tetramethyl-substituted nitroxide 91b. The solution of NH -bearing oligomer 89,90 in HEPES-KOH (pH 9.0) was mixed with the nitroxide 91b. The solution2 of NH2-bearing oligomer 89,90 in HEPES-KOH (pH 9.0) was mixed with respectivethe respective derivative derivative of NHS of NHS ester ester nitroxide nitroxide91a 91a–b–dissolvedb dissolved in in DMSODMSO (rt, (rt, 2 2 h). h). The The final final products products 92a92a–b–andb and93a 93a–b–wereb were separated separated by by HPLC HPLC inin approximatelyapproximately 70% 70% of of conversion conversion yields. yields.

SchemeScheme 21. 21.Post-synthetic Post-synthetic labelinglabeling ofof amino-containingamino-containing precursor precursor oligomers oligomers 8989, 90, 90withwith nitroxide nitroxide derivativesderivatives91a,b 91a,b.. Reagents Reagents and conditions: conditions: (1) (1) precursor precursor oligomers oligomers 89, 9089 in, 9050 mMin 50 HEPES-KOH mM HEPES-KOH (pH (pH9.0), 9.0), 20 20 mM mM active active esters esters 91a,b91a,b in DMSO,in DMSO, rt, 2 rt,h. 2 h.

TwoTwo cyclic cyclic NN6-threonylcarbamoyladenosines6-threonylcarbamoyladenosines containing containing a hydantoin a hydantoin ring ring (ct6A (ct and6A 2- and 2-methylthio-analogmethylthio-analog msms22ct6A)A) have have been been recently recently discovered discovered at atposition position 37 37of ofanticodon anticodon loops loops in several in several prokaryoticprokaryotic and and eukaryotic eukaryotic tRNA tRNA sequences sequences [6 [6,7].,7]. To To determine determine the the e ffeffectect of of hydantoin hydantoin structurestructure onon the biologicalthe biological activity activity of tRNAs, of tRNAs, model 17-ntmodel RNA 17-nt oligomers RNA oligomers containing containing ct6A and ct6A ms and2ct6 Ams were2ct6A obtainedwere byobtained the post-synthetic by the post-synthetic methodology methodology based on the based cyclization on the cyclization of linear t6 Aof/ mslinear2t6A t6A/ms precursor2t6A precursor nucleosides (Schemenucleosides 22)[63 (Scheme,64]. The 22) post-synthetic [63,64]. The post-synthetic approach was approach the method was of the choice, method since of choice, the hydantoin since the ring 6 2 6 of cthydantoin6A/ms2ct 6ringA turned of ct A/ms out toct beA veryturned susceptible out to be very to hydrolysis susceptible under to hydrolysis conditions under of phosphoramidite conditions of phosphoramidite solid-phase RNA synthesis and deprotection. Oligomers 94a–b were synthesized solid-phase RNA synthesis and deprotection. Oligomers 94a–b were synthesized on CPG support at on CPG support at a 2.5 µmol scale using Ultra Mild 5′-O-DMTr-2′-O-TBDMS-NH-tac- a 2.5 µmol scale using Ultra Mild 5 -O-DMTr-2 -O-TBDMS-NH-tac-phosphoramidite building blocks phosphoramidite building blocks0 to retain 0 the carbamoyl linkage in t6A/ms2t6A intact. The to retain the carbamoyl linkage in t6A/ms2t6A intact. The L-threonine residue was protected with L-threonine residue was protected with TBDMS at the hydroxyl function and 2-(trimethylsilyl)ethyl TBDMS(TMSE) at at the the hydroxyl carboxyl function moiety in and both 2-(trimethylsilyl)ethyl t6A and ms2t6A phosphoroamidites. (TMSE) at the For carboxyl the preparation moiety in of both 6 2 6 t A94a and–b,ms botht precursorA phosphoroamidites. amidites were coupled For the twice preparation in extended of 94a time–b ,(15 both min precursor for t6A and amidites 25 min for were 6 2 6 coupledms2t6A) twice and anhydrous in extended conditions time (15 of min alkaline for t Adeprotection and 25 min were for msappliedt A) to and prevent anhydrous the TMSE conditions ester of alkalineamonolysis deprotection (fluorolabile were TMSE applied group was to preventcleaved during the TMSE desilylation, ester amonolysis simultaneously (fluorolabile with TBDMS TMSE group was cleaved during desilylation, simultaneously with TBDMS blockage). Fully deprotected t6A/ms2t6A-RNAs 94a–b were purified by IE HPLC and subjected to post-synthetic cyclization with Molecules 2020, 25, x FOR PEER REVIEW 24 of 37 MoleculesMolecules2020 2020, 25,, 25 3344, x FOR PEER REVIEW 24 of24 37 of 37 blockage). Fully deprotected t6A/ms2t6A-RNAs 94a–b were purified by IE HPLC and subjected to blockage). Fully deprotected t6A/ms2t6A-RNAs 94a–b were purified by IE HPLC and subjected to post-synthetic cyclization with aq. DMF solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide aq.post-synthetic DMF solution cyclization of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide with aq. DMF solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC HCl) and hydrochloride (EDC·HCl) and 1-hydroxy-1H-benzotriazole (HOBt) added in the 5- or 10-fold· molar hydrochlorideH (EDC·HCl) and 1-hydroxy-1H-benzotriazole (HOBt) added in the94a 5- or 10-fold94b molar 1-hydroxy-1excess to 94a-benzotriazole and 94b precursor (HOBt) oligomers, added in the respectively. 5- or 10-fold After molar the excess complete to cyclizationand (1precursor h, rt) excess to 94a and 94b precursor oligomers, respectively. After the complete cyclization (1 h, rt) oligomers,oligomers respectively. 95a–b were isolated After the by completecentrifugation cyclization through (1Amicon h, rt) oligomersin 80–90% conversion95a–b were yields. isolated The by oligomers 95a–b were isolated by centrifugation through Amicon in6 80–90%2 6 conversion yields. The centrifugationct6A/ms2ct6A-oligomers through Amicon 95a–b in were 80–90% characterized conversion by yields. MALDI-ToF The ct MSA/ms andct nucleosideA-oligomers composition95a–b were ct6A/ms2ct6A-oligomers 95a–b were characterized by MALDI-ToF MS and nucleoside composition characterizedanalysis. by MALDI-ToF MS and nucleoside composition analysis. analysis.

SchemeScheme 22. 22. Post-syntheticPost-synthetic cyclization cyclization of t6 ofA/ms t6A2t6/A-containingms2t6A-containing precursor precursor oligomers oligomers(94a,b) to ct (694aA- ,b) Scheme 22. Post-synthetic cyclization of t6A/ms2t6A-containing precursor oligomers (94a,b) to ct6A- toor ct 6msA-2ct or6A-modified ms2ct6A-modified RNA oligomers RNA (95a oligomers and 95b, respectively). (95a and 95b Reagents, respectively). and conditions: Reagents (i) 1-ethyl- and or ms2ct6A-modified RNA oligomers (95a and 95b, respectively). Reagents and conditions: (i) 1-ethyl- conditions:3-(3-dimethylaminopropyl)-carbodiimide (i) 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl) hydrochloride (5–10 equiv), (EDC HCl) 1-hydroxy-1 (5–10 equiv),H- 3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl) (5–10 equiv), · 1-hydroxy-1H- 1-hydroxy-1benzotriazoleH-benzotriazole (HOBt) (5-10 (HOBt)equiv), H (5-102O-DMF, equiv), 1 h, H rt.2O-DMF, 1 h, rt. benzotriazole (HOBt) (5-10 equiv), H2O-DMF, 1 h, rt. 3.5.3.5. Transformation Transformation of of Ester Ester Groups Groups 3.5. Transformation of Ester Groups Recently,Recently, a new a new 5-pivaloyloxymethyluridine 5-pivaloyloxymethyluridine (Pivom (Pivom5U) convertible5U) convertible nucleoside nucleoside has been has designed been Recently, a new 5-pivaloyloxymethyluridine (Pivom5U) convertible nucleoside has been fordesigned preparing for thepreparing 5-aminomethyluridine the 5-aminomethyluridine (R5U)-modified (R5U)-modified RNA RNA oligomers oligomers97a– 97ae (Scheme–e (Scheme 23 23))[62 ]. designed for preparing the 5-aminomethyluridine (R5U)-modified RNA oligomers 97a–e (Scheme 23) [62]. The studies were focused on the naturally existing 5-aminomethyluridine derivatives such as5 The[62]. studies The werestudies focused were focused on the naturallyon the naturally existing existing 5-aminomethyluridine 5-aminomethyluridine derivatives derivatives such such as nm as U, nm55U, mnm55U, inm5U,5 cmnm5U,τ and5 τm5U due to their crucial role in tuning the translation process mnmnm5U,U, mnm inm 5U,U, cmnminm5U, cmnmU, and5U, andm U τm due5U due to theirto their crucial crucial role role inin tuning the the translation translation process process (all modified uridines are positioned at the first anticodon letter of numerous tRNAs) and poor (all(all modified modified uridines uridines are are positioned positioned atat the the first first anticodon letter letter of of numerous numerous tRNAs) tRNAs) and and poor poor accessibility to oligomers containing these modifications [100,101]. accessibilityaccessibility to to oligomers oligomers containing containing these these modificationsmodifications [100,101]. [100,101].

Scheme 23. General scheme for post-synthetic transformation of 5-pivaloyloxymethyluridine SchemeScheme 23. 23.General General scheme scheme for for post-synthetic post-synthetic transformation of of 5-pivaloyloxymethyluridine 5-pivaloyloxymethyluridine (Pivom5U)-RNA 96 to R5U-modified oligoribonucleotides 97a–e. Reagents and conditions: (1) (Pivom(Pivom5U)-RNA5U)-RNA96 96 to to R R55U-modifiedU-modified oligoribonucleotides oligoribonucleotides 97a97a–e.– e Reagents. Reagents and conditions: and conditions: (1) TEA/MeCN (1:1 v/v), 20 min, rt; (2) 30% aq. NH3 (97a), 8 M MeNH2/EtOH (97b), 0.4 M (1)TEA/MeCN TEA/MeCN (1:1 (1:1 vv/v/v),), 20 20 min, min, rt; rt; (2) (2) 30% 30% aq. aq. NH NH3 (97a(97a), ), 8 8 M M MeNH MeNH2/EtOH/EtOH (97b (97b), 0.4), 0.4 M M NH2CH2CH=CMe2 in EtOH/H2O (4:1 v/v), TEA (97c), 1.5 3M NH2CH2CO2-NBu4+2/EtOH (97d), 1.5 M NH2CH2CH=CMe2 in EtOH/H2O (4:1 v/v), TEA (97c), 1.5 M NH2CH2CO2-NBu4+/EtOH+ (97d), 1.5 M NH CH CH=CMe- in EtOH+ /H O (4:1 v/v), TEA (97c), 1.5 M NH CH CO NBu /EtOH (97d), 1.5 M NH2 2CH2 2CH2SO3 2NBu4 /EtOH 2(97e), 1 h (for 97a–b) or 20 h (for 97c2 –e),2 60 °C;2− (3) TEA·3HF/NMP4 (1:1 - + NHNHCH2CHCH2CHSO2SO3 NBuNBu4 /EtOH+/EtOH (97e (97e), 1), h 1 (for h (for 97a97a–b) –orb )20 or h 20 (for h 97c (for–e97c), 60–e °C;), 60 (3) C;TEA·3HF/NMP (3) TEA 3HF (1:1/NMP v2/v), 242 h, rt.2 3− 4 ◦ · (1:1vv/v/v),), 24 24 h, h, rt. rt. It was found that a sterically hindered pivaloyloxyl group (-OPiv) attached to the C-5,1- It was found that a sterically hindered pivaloyloxyl group (-OPiv) attached to the C-5,1- pseudobenzylicIt was found carbon that of auridine sterically exclusively hindered promotes pivaloyloxyl the nucleophilic group substitution (-OPiv) attachedat this position to the pseudobenzylic carbon of uridine exclusively promotes the nucleophilic substitution at this position C-5,1-pseudobenzylicunder treatment with carbon ammonia, of uridine primary exclusively alkylamines, promotes or amino the acidsnucleophilic at elevated substitution temperature, at this under treatment with ammonia, primary alkylamines, or amino acids at elevated temperature, positionleading under to the treatment quantitative with transformation ammonia, primary of Pivom alkylamines,5U to 5-aminomethyluridines or amino acids at elevated[62,102]. temperature, leading to the quantitative transformation of Pivom5U to 5-aminomethyluridines [62,102]. leadingThe to the 5- quantitativeand 17-nt RNA transformation precursor oligomers of Pivom 965 Uwere to 5-aminomethyluridines synthesized by the standard [62,102 protocol]. of The 5- and 17-nt RNA precursor oligomers 96 were synthesized by the standard protocol of phosphoramiditeThe 5- and 17-nt chemistry RNA precursoron CPG support oligomers using 96a 5’-wereO-DMTr-2’- synthesizedO-TBDMS by thesystem standard of protection protocol phosphoramidite chemistry on CPG support using a 5’-O-DMTr-2’-O-TBDMS system of protection of(Pivom phosphoramidite5U–phosphoramidite chemistry were on coupled CPG support twice). The using post-synthetic a 5’-O-DMTr-2’- transformationO-TBDMS of Pivom system5U- of (Pivom5U–phosphoramidite were coupled twice). The post-synthetic transformation of Pivom5U- modified oligomers5 96 was conducted “in solid-phase” after the removal of β-cyanoethyl groups protectionmodified (Pivom oligomersU–phosphoramidite 96 was conducted “inwere solid-phase” coupled twice). after the The removal post-synthetic of β-cyanoethyl transformation groups of Pivom5U-modified oligomers 96 was conducted “in solid-phase” after the removal of β-cyanoethyl groups (TEA/MeCN, 1:1 v/v, 20 min, rt), simultaneously with the alkaline deprotection of nucleobases and resin cleavage. For this purpose, the CPG-linked Pivom5U-RNA 96 was incubated with Molecules 2020, 25, 3344 25 of 37

Molecules 2020, 25, x FOR PEER REVIEW 25 of 37 nitrogen(TEA/MeCN, nucleophiles 1:1 v/v, such20 min, as rt), ammonia, simultaneously primary with amines the alkaline (methylamine deprotection or of isopentenyloamine), nucleobases and or tetrabutylammoniumresin cleavage. For this salts purpose, of amino the acids CPG-linked in ethanolic Pivom or5 aqueousU-RNA 96 solutions was incubated at 60 ◦C. withTo get nitrogen oligomers 5 modifiednucleophiles with 5-aminomethyluridine such as ammonia, primary97a and 5-methylaminomethyluridine amines (methylamine or isopentenyloamine),97b, Pivom U-RNA or 96 wastetrabutylammonium incubated with aq. 30%saltsammonia of amino acids or 8M in ethanolicethanolic or solution aqueous of solutions MeNH2, at respectively 60 °C. To get for oligomers 1 h at 60 ◦C. To obtainmodified 5-isopentenylaminomethyluridine-RNA with 5-aminomethyluridine 97a and 5-methylaminomethyluridine (97c), precursor oligomer 96 97bwas, Pivom treated5U-RNA with 0.496 M solutionwas incubated of isopentenylammonium with aq. 30% ammonia tosylate or 8 (600 M ethanolic equiv) and solution TEA (6000of MeNH equiv)2, respectively in an EtOH–H for 21O h mixtureat 60 5 5 for°C. 20 hTo at obtain 60 ◦C. 5-isopentenylaminomethyluridine-RNA The preparation of cmnm U- and τm U-RNAs (97c), precursor required oligomer the use 96 of glycinewas treated and with taurine as alkylamine0.4 M solution conversion of isopentenylammonium reagents. To increase tosylate their (600 nucleophilic equiv) and TEA character (6000 asequiv) well in as an the EtOH–H solubility2O in organicmixture solvents, for 20 h both at 60 amino °C. The acids preparation were transformed of cmnm5U- to and the τm tetrabutylammonium5U-RNAs required the saltuse forms.of glycine Then, Pivomand 5taurineU-RNAs as alkylamine96 were treated conversion with reagents. 1.5 M ethanolic To increase solution their nucleophilic of amino acid character salts (3000as well equiv) as the for solubility in organic solvents, both amino acids were transformed to the tetrabutylammonium salt 20 h at 60 ◦C. After displacement reactions, the excess of salts was removed from oligomers 97c–e on 5 C18forms. cartridge. Then, Finally,Pivom U-RNAs all the oligomers 96 were treated were desilylatedwith 1.5 M ethanolic and isolated solution by IEof HPLCamino acid in 60–85% salts (3000 yields. equiv) for 20 h at 60 °C. After displacement reactions, the excess of salts was removed from oligomers The developed strategy was also applied for the preparation of 5-cyanomethyluridine-modified RNA 97c–e on C18 cartridge. Finally, all the oligomers were desilylated and isolated by IE HPLC in 60– (cnm5U-RNA) with potassium cyanide as a nucleophile; however, the conditions used for the complete 85% yields. The developed strategy was also applied for the preparation of 5-cyanomethyluridine- conversion of Pivom5U (20 h, 60 C) caused the partial degradation of RNA. modified RNA (cnm5U-RNA) with◦ potassium cyanide as a nucleophile; however, the conditions used 5 3.6.for Post-Synthetic the completeConversions conversion of of Pivom Sulfur-ContainingU (20 h, 60 °C) RNA caused Oligomers the partial degradation of RNA.

3.6.1.3.6. Post-Synthetic Post-Synthetic Conversions Conversion of ofSulfur-Containing 2-Thiouridine-Modified RNA Oligomers RNA Oligomers

3.6.1.A thiocarbonyl Post-Synthetic group Conversion in 2-thiouridines of 2-Thiouridine-Modified (s2Us) was found RNA to be Oligomers a reactive site for two post-synthetic RNA conversions. The first strategy was based on the oxidative desulfuration of s2U-containing A thiocarbonyl group in 2-thiouridines (s2Us) was found to be a reactive site for two post- 2 oligomerssynthetic leading RNA conversions. to the formation The firstof 4-pyrimidinone strategy was based nucleosides on the (HoxidativeU) [61 ]. desulfuration The second of approach s2U- 2 involvedcontaining the reactionoligomers of leading a nucleophilic to the formation substitution of 4-pyrimidinone between alkyl nucleosides halide and (H an2U) s U-oligomer[61]. The second where theapproach sulfur atom involved acts as the a nucleophile reaction of agent, a nucleophilic providing substitutionS-alkylated between analogs alkyl of 2-thiouridine halide and an [12 s].2U- 2 oligomerThe strong where tendency the sulfur of atom 2-thiouridine acts as a nucleophile (s U) to agent,desulfuration providing under S-alkylated oxidative analogs conditions of 2- wasthiouridine reported [12]. several times at the nucleoside and oligonucleotide levels [103–105]. The loss of a sulfurThe strong atom tendencyled to the of formation 2-thiouridine of (s uridine2U) to desulfuration and/or 4-pyrimidinone under oxidative nucleoside conditions (H was2U) in a ratioreported that several was dependenttimes at the onnucleoside the oxidizer and oligonucleotide (type, concentration), levels [103–105]. solvent, The pH,loss of and a sulfur type of 5-substituentatom led to attachedthe formation to the of nucleobase.uridine and/or The 4-pyrimidinone observation nucleoside that s2U-oligomers (H2U) in a wereratio that desulfured was underdependent conditions on the mimicking oxidizer (type, the oxidative concentration), stress solvent, in a cell pH, [104 and,105 type] enhanced of 5-substituent the need attached for a reliable to accessthe nucleobase. to H2Us-modifed The observation oligomers. that s2 TheU-oligomers attempt were to incorporatedesulfured under an H conditions2U phosphoramidite mimicking the into 2 theoxidative RNA chain stress turnedin a cell [104,105] out to beenhanced not straightforward the need for a reliable by theaccess classical to H Us-modifed approach oligomers. because of The attempt to incorporate an H2U phosphoramidite into the RNA chain turned out to be not H2U-RNA instability under alkaline conditions [104]. In 2015, Chwialkowska and co-workers straightforward by the classical approach because of H2U-RNA instability under alkaline conditions published a selective post-synthetic transformation of oligomers modified with 2-thiouridine [104]. In 2015, Chwialkowska and co-workers published a selective post-synthetic transformation of and 5-substituted 2-thiouridines (R5s2U-RNA, 98a–f) exclusively to the 4-pyrimidinone-containing oligomers modified with 2-thiouridine and 5-substituted 2-thiouridines (R5s2U-RNA, 98a–f) derivatives (R5H2U-RNA, 99a–f, Scheme 24)[61]. Six types of R5s2U modifications were selected to exclusively to the 4-pyrimidinone-containing derivatives (R5H2U-RNA, 99a–f, Scheme 24) [61]. Six 2 2 showtypes the of generality R5s2U modifications of the method: were selected 2-thiouridine to show (stheU, generality R2 = H), of 2 0the-O-methyl-2-thiouridine method: 2-thiouridine (s (s2U,Um, 5 2 R1 R=2 OMe= H),,R 2′-2O=-methyl-2-thiouridineH), 5-methyl-2-thiouridine (s2Um, (m R1 s= U,OMe, R2 = R-CH2 = H),3), 5-metoxycarbonylmethyl-2-thiouridine,5-methyl-2-thiouridine (m5s2U, R2 = - 5 2 5 2 mcmCHs3),U, 5-metoxycarbonylmethyl-2-thiouridine, R2 = CH2COOMe), 5-methylaminomethyl-2-thiouridine mcm5s2U, R2 = CH2COOMe), (mnm 5-methylaminomethyl-2-s U, R2 = CH2NHCH3), 5 2 andthiouridine 5-taurinomethyl-2-thiouridine (mnm5s2U, R2 = CH2 (NHCHτm s U,3), R and2 = 5-taurinomethyl-2-thiouridineCH2NHCH2CH2SO3H). Notably, (τm5s2 allU, selected R2 = 2-thiouridinesCH2NHCH2CH are2SO naturally3H). Notably, existing all selected ribonucleosides 2-thiouridines present are atnaturally the first existing (“wobble”) ribonucleosides position of numerouspresent at tRNA the first biomolecules. (“wobble”) position of numerous tRNA biomolecules.

SchemeScheme 24. 24.Post-synthetic Post-synthetic desulfuration desulfuration ofof 2-thiouridine-modified 2-thiouridine-modified oligomers oligomers 98a98a–f–. f Reagents. Reagents and and ® conditions:conditions: (i) (i) 2 mM2 mM aq. aq. KHSO KHSO44 (Oxone(Oxone®),), rt, rt, 5-30 5–30 min. min. Molecules 2020, 25, 3344 26 of 37

Molecules 2020, 25, x FOR PEER REVIEW 26 of 37 Precursor oligomers 98a–f were synthesized by phosphoramidite chemistry using a 50-O-DMTr,2Precursor0- O oligomers-TBDMS 98a protection–f were synthesized system for by the phosphoramidite preparation of chemistry monomeric using units a 5′-O and- a specialDMTr,2′- protocolO-TBDMS of protection P(III) P(V) system oxidation for the preparation and work-up of monomeric to retain theunits s 2and-thiocarbonyl a special protocol function → intactof P(III) [100,→101P(V),106 oxidation,107]. Synthetic and work-up R5s2U-modified to retain the precursor s2-thiocarbonyl oligomers function were intact of varied [100,101,106,107]. length (from 11 to 23Synthetic nts) with R5s 2-thiouridines2U-modified precursor located both oligomers in single were and of double-stranded varied length (from regions. 11 to Fully 23 nts) deprotected with 2- andthiouridines purified oligomers located both98a in–f singlewere subjected and double-stranded to oxidative regions. desulfuration Fully deprotected by incubation and purified in 2 mM oligomers 98a–f were subjected to oxidative® desulfuration by incubation in 2 mM aqueous solution aqueous solution of KHSO4 (Oxone , 10-fold molar excess) at 25 ◦C for 5–30 min. The reactions of KHSO4 (Oxone®, 10-fold molar excess) at 25 °C for 5–30 min. The reactions were terminated by were terminated by loading on a C18 Sep-Pack cartridge and a standard desalting process. It was loading on a C18 Sep-Pack cartridge and a standard desalting process. It was found that more than found that more than 30 min exposition of the R5H2U–RNA products to the oxidizing agent leads to 30 min exposition of the R5H2U–RNA products to the oxidizing agent leads to further unidentified further unidentified oxidative lesions. The products 99a–f (obtained in conversion yields 87–95%) oxidative lesions. The products 99a–f (obtained in conversion yields 87–95%) were analyzed by wereMALDI-ToF analyzed bymass MALDI-ToF spectroscopy, mass indicating spectroscopy, the selectivity indicating of the reaction selectivity toward of the the reaction 2-thiocarbonyl toward the 2-thiocarbonylfunction. function. InIn 2012, 2012, highly highly lipophilic lipophilicS -geranylatedS-geranylated derivatives derivatives of of 5-methylaminomethyl-2-thiouridine 5-methylaminomethyl-2-thiouridine 5 2 5 2 (mnm(mnmges5gesU)2U) and and 5-carboxymethylaminomethyl-2-thiouridine5-carboxymethylaminomethyl-2-thiouridine (cmnm (cmnm5ges2U)ges wereU) wereidentified identified at the at thefirst first anticodon anticodon position position in bacterial in bacterial tRNAs tRNAs bearing bearing Lys, Glu, Lys, and Glu, Gln [9]. and To Gln understand [9]. To understandthe biological the biologicalrole of a roleunique of aS unique-geranylS modification,-geranyl modification, the model 2-geranylthiouridine-RNA the model 2-geranylthiouridine-RNA oligomer 103 (ges oligomer2U- 103RNA,(ges2 homologueU-RNA, homologue of anticodon of arm anticodon domain arm of E. domain coli tRNA ofLysE.) was coli tRNAsynthesizedLys) was and synthesized characterized and characterized(Scheme 25) (Scheme [12]. Three 25)[ chemical12]. Three routes chemical were routeselaborated were for elaborated preparing for the preparing ges2U-RNA the oligomer, ges2U-RNA oligomer,including including two post-synthetic two post-synthetic strategies strategies based on basedthe selective on the geranylation selective geranylation of CPG-linked of CPG-linked (Scheme (Scheme25A) or25 A) released/deprotected or released/deprotected 2-thiouridine-modified 2-thiouridine-modified precursor precursor oligomers oligomers (Scheme (Scheme 25B). The 25 B). synthesis of the s2U-RNA2 oligomer 100 was performed routinely, using a 5′-O-DMTr-2′-O-TBDMS The synthesis of the s U-RNA oligomer 100 was performed routinely, using a 50-O-DMTr-20-O-TBDMS protectionprotection system system and andtert tert--butylhydroperoxidebutylhydroperoxide as an an oxidizing oxidizing reagent. reagent. In Inthe the first first “in “in solid-phase” solid-phase” method (Scheme 25A), detritylated, support-linked s2U-RNA 100 was deprived of β-cyanoethyl method (Scheme 25A), detritylated, support-linked s2U-RNA 100 was deprived of β-cyanoethyl groups groups from phosphate residues (TEA/MeCN, rt, 30 min) and then alkylated with > 200-fold molar from phosphate residues (TEA/MeCN, rt, 30 min) and then alkylated with > 200-fold molar excess of excess of geranyl bromide and an equimolar amount of triethylamine in ethanol (3 h, rt). The geranyl bromide and an equimolar amount of triethylamine in ethanol (3 h, rt). The converted product converted product 101 was subjected to alkaline deprotection and support cleavage under mild 101 was subjected to alkaline deprotection and support cleavage under mild alkaline conditions (8 M alkaline conditions (8 M NH3/EtOH, rt, 8 h) to avoid substitution of the thioalkyl group with a NHnitrogen3/EtOH, nucleophile, rt, 8 h) to avoid leading substitution to the formation of the thioalkyl of isocytidine group withderivatives a nitrogen [108]. nucleophile, After desilylation, leading to 2 theges formation2U-oligomer of isocytidine 103 was purified derivatives by anion-exchange [108]. After desilylation,HPLC, affording ges aU-oligomer geranylated 103productwas in purified a total by anion-exchangeof 40% yield. HPLC, affording a geranylated product in a total of 40% yield.

SchemeScheme 25. 25.( A(A)) Post-synthetic Post-synthetic geranylation geranylation of s s2U-oligomer 100100 inin solid solid phase. phase. Reagents Reagents and and conditions:conditions: (1) (1) TEA TEA/MeCN/MeCN (1:1(1:1 v/v), rt, rt, 20 20 min; min; (2) (2) geBr geBr (213 (213 equiv), equiv), TEA TEA (213 (213equiv), equiv), EtOH, EtOH, rt, 3 h; rt, (3) 3 h; 2 (3)8 8 M M NH NH3/EtOH,3/EtOH, rt, rt, 8 h; 8 h;(4) (4)TEA TEA3HF/NMP3HF/NMP (1:1 (1:1v/v), vrt,/v ),24 rt, h; 24(B) h; Post-synthetic (B) Post-synthetic geranylation geranylation of s U- of 2 · s U-oligomeroligomer 102102 in in solution. solution. Reagents Reagents and and conditions: conditions: (1) conc. (1) conc. NH3/EtOH NH3/ EtOH (3:1 v/v (3:1), 40v / °C,v), 40 16 ◦ h;C, (2) 16 h; (2)TEA 3HF/NMP3HF/NMP (1:1 (1:1 v/vv/),v), rt, rt, 24 24 h; h;(3) (3) geBr geBr (213 (213 equiv), equiv), TEA TEA (213 (213 equiv), equiv), EtOH/H EtOH2O/ H(1:1O v (1:1/v), rt,v/ v3), h. rt, 3 h. · 2 InIn the the second second approach, approach, post-synthetic post-synthetic s s22→ges22 transformationtransformation was was performed performed in in solution solution → (Scheme(Scheme 25 B)25B) by by the the treatment treatment of of fully fully deprotecteddeprotected/released/released s s22U-RNAU-RNA oligomer oligomer 102102 withwith EtOH-water EtOH-water solutionsolution of of geBr geBr and and TEA. TEA. The The mixture mixture waswas shakenshaken vigorously for for 3 3h hat at rt. rt. Then, Then, the the oligomer oligomer was was Molecules 2020, 25, 3344 27 of 37

Molecules 2020, 25, x FOR PEER REVIEW 27 of 37 filtered through a NAP-G25 column and purified by IE HPLC to furnish pure ges2U-RNA 103 at 2 a totalfiltered of 67% through yield. a NAP-G25 The structure column of gesand2 U-RNApurified productby IE HPLC103 towas furnish confirmed pure ges byU-RNA MALDI-ToF 103 at mass a total of 67% yield. The structure of ges2U-RNA product 103 was confirmed by MALDI-ToF mass spectrometry and nucleoside composition analysis. spectrometry and nucleoside composition analysis. 3.6.2. Post-Synthetic Conversion of 4-Thiouridine-Prefunctionalized RNA Oligomers 3.6.2. Post-Synthetic Conversion of 4-Thiouridine-Prefunctionalized RNA Oligomers Due to the more nucleophilic character of the sulfur atom in s4U than other functional groups in Due to the more nucleophilic character of the sulfur atom in s4U than other functional groups in nucleic acids, the selective S-labeling was reported for both the entire s4U-tRNA molecules [109,110] nucleic acids, the selective S-labeling was reported for both the entire s4U-tRNA molecules [109,110] and s4U/s4dU-modified oligonucleotides [97,111–113]. The post-synthetic functionalization of s4U was and s4U/s4dU-modified oligonucleotides [97,111–113]. The post-synthetic functionalization of s4U appliedwas applied predominantly predominantly for the site-directedfor the site-directed spin labeling spin labeling of oligomers of oligomers to extract to extract the long-range the long-range distances in RNAdistances or protein–RNA in RNA or protein–RNA complexes complexes by EPR and by NMREPR and techniques NMR techniques [97,112– [97,112–114].114]. Two general Two general methods 4 of smethodsU-RNA of functionalization s4U-RNA functionalization with thiol-specific with thiol-specific reagents reagents were reported, were reported, namely namely by S-alkylation by S- (Schemealkylation 26A) (Scheme and mixed 26A) disulfide and mixed formation disulfide (Schemeformation 26 (SchemeB). 26B).

SchemeScheme 26. 26.(A )( Post-syntheticA) Post-synthetic s4U-RNA s4U-RNA nitroxide nitroxide labeling labeling with iodoacetamidewith iodoacetamide spin label spin105 label. Reagents 105. andReagents conditions: and conditions: (i) 100 mM (i) phosphate 100 mM phosphate buffer (pH buffer 8.0), (pH 100 8.0), equiv 100 ofequiv105 ofin 105 H2O in/EtOH H2O/EtOH (7:3 v (7:3/v), rt, overnight,v/v), rt, in theovernight, dark; ( B) in Post-synthetic the dark; s (4BU-RNA) Post-synthetic nitroxide labeling s4U-RNA with nitroxide methanesulfonylthioalkyl labeling with reagentsmethanesulfonylthioalkyl107a–c. Reagents and reagents conditions: 107a–c. (i) Reagents 100 mM and phosphate conditions: bu (i)ffer 100 (pH mM 8.0), phosphate dithiothreitol buffer (pH (DTT), rt, 308.0), min-2 dithiothreitol h; (ii) 10 (DTT), mM phosphate rt, 30 min-2 bu h;ff er(ii) (pH 10 mM 6.8), phosphate or a mixture buffer of 90(pH mM 6.8), phosphate or a mixture bu offfer 90 (pH mM 8.0) andphosphate DMF (9:1 bufferv/v), rt,(pH 12 8.0) h. and DMF (9:1 v/v), rt, 12 h.

s4U-precursors4U-precursor oligomers oligomers104 104were were preparedprepared by the standard standard phosphoramidite phosphoramidite chemistry chemistry using using 5’-O5’--DMTr-2’-O-DMTr-2’-O-TBDMS-protectedO-TBDMS-protected monomeric monomeric unitsunits andand β-cyanoethyl-cyanoethyl protection protection for for s4-thiocarbonyl s4-thiocarbonyl groupgroup to retainto retain the the thio-function thio-function intact intact during during thethe P(III)P(III) → P(V) oxidation oxidation step. step. → 4 In theIn the studies studies of of post-synthetic post-synthetic appending appending of nitroxide labels labels to to the the thiocarbonyl thiocarbonyl group group of ofs U s 4U residue,residue, the the iodo-containing iodo-containing proxyl proxyl spin spin labellabel waswas employed (Scheme (Scheme 26A) 26A) [112]. [112 ].Fully Fully deprotected deprotected s4U-RNAs 104 (30- and 31-nt) were dissolved in 100 mM phosphate buffer (pH 8.0), treated with s4U-RNAs 104 (30- and 31-nt) were dissolved in 100 mM phosphate buffer (pH 8.0), treated with solution of 3-(2-iodoacetamido)proxyl 105 in H2O/EtOH (7:3 v/v), and incubated overnight at rt in the solution of 3-(2-iodoacetamido)proxyl 105 in H O/EtOH (7:3 v/v), and incubated overnight at rt in the dark. S-Alkylated oligomers 106 were isolated 2(the NAP-10 column and ethanol precipitation) in 80% dark. S-Alkylated oligomers 106 were isolated (the NAP-10 column and ethanol precipitation) in 80% yields on average. Similar procedures of s4U-labeling were employed by others [49,97,114] to gain 4 yieldsmodels on average.for the structural Similar and procedures functional of analysis s U-labeling of RNA. were employed by others [49,97,114] to gain modelsThe for thefast structuralattachment and of functionalnitroxide radical analysis to the of RNA. s4-thiocarbonyl function of s4U-RNA oligomers 4 4 wasThe achieved fast attachment by applying of nitroxide the methanethiosulfonate radical to the s -thiocarbonyl spin label function reagents of s107aU-RNA–c (Scheme oligomers 26B) was achieved[97,113,115,116]. by applying “Free” the methanethiosulfonate s4U-precursor RNAs 104 spin (23- label and reagents 57-nt) 107awere– incubatedc (Scheme with26B) [dithiothreitol97,113,115,116 ]. “Free”(DTT) s4U-precursor reducing agent RNAs in 100104 mM(23- phosphate and 57-nt) buffer were incubated(pH 8.0) for with 30 min-2 dithiothreitol h at rt. After (DTT) the reducing removal agentof in 100DTT mM excess, phosphate the oligomers buffer (pH were 8.0) for treated 30 min-2 with h at appropriate rt. After the 1-oxyl-2,2,5,5-tetramethylpyrroline- removal of DTT excess, the oligomers weremethanethiosulfonates treated with appropriate 107a–c 1-oxyl-2,2,5,5-tetramethylpyrroline- in phosphate buffer overnight at rt to afford methanethiosulfonates products 108a–c. Reagents107a–c in phosphate107a–c were buffer found overnight to be at effective rt to aff ord for products the thioalkyl108a – transferc. Reagents to the107a sulfur–c were of 4-thiouridine found to be e withffective forformation the thioalkyl of the transfer disulfide to thetethered sulfur group. of 4-thiouridine However, the with labile formation nature of of the the disulfide disulfide bond tethered requires group. However,the storage the of labile labeled nature RNAs of at the -20 disulfide °C to minimize bond requiresthe nitroxide the storageradical detachment. of labeled RNAs at 20 C to − ◦ minimize the nitroxide radical detachment.

Molecules 2020, 25, 3344 28 of 37

Molecules 2020, 25, x FOR PEER REVIEW 28 of 37

3.6.3.3.6.3. Post-Synthetic Post-Synthetic Formation Formation of of Disulfide Disulfide CrosslinksCrosslinks TheThe site-specific site-specific disulfide disulfide crosslinking crosslinking of RNA of RNA represents represents a powerful a powerful tool for tool studying for studying the structure, the folding,structure, and functionfolding, and of RNA function by locking of RNA RNA by locking into a single RNA foldedinto a single and homogeneous folded and homogeneous structure. First, disulfidestructure. crosslinks First, were disulfide found crosslinks in cellular were tRNAs found between in neighboring cellular tRNAs 4-thiouridines between neighboring [117]. A synthetic 4- procedurethiouridines for the[117]. site-specific A synthetic insertion procedure of for the the intrahelical site-specific disulfide insertion crosslink of the intrahelical into RNA disulfide oligomers wascrosslink based on into the RNA oxidation oligomers of thioalkyl-modified was based on the nucleobases oxidation of positioned thioalkyl-modified at the 30- and nucleobases 50-ends of oligomers,positioned which at the after 3′- and forming 5′-ends the of duplex, oligomers, were which able toafter crosslink forming with the theduplex, opposite were strandable to [crosslink66,118,119 ]. Thewith ethane-thiol the opposite tethers strand were [66,118,119]. attached toThe the ethane-thiol exocyclic aminotethers groupwere attached of cytosines to the (Scheme exocyclic 27 aminoA) or to thegroup N3 atom of cytosines of uracils (Scheme (Scheme 27A)27B), or which to the were N3 both atom introduced of uracils (Schemeto oligomers 27B), as whichS-protected were both mixed disulfidesintroduced109 toand oligomers112, respectively. as S-protected The mixed synthesis disulfides of S-protected 109 and 112N4-(2-thioethyl)cytidines-modified, respectively. The synthesis of 4 oligomersS-protected (8-nt N model-(2-thioethyl)cytidines-modified of hairpin) utilized the Verdine oligomers convertible (8-nt model nucleoside of hairpin) 1 utilized (see Figure the Verdine4) and the displacementconvertible reactionnucleoside with 1 (see cystamine Figure 4) reagent and the (details displacement are given reaction in Scheme with cystamine3)[ 66], whereas reagentS (details-tert-butyl 3 3 are given in Scheme 3) [66], whereas S-tert-butyl N -(2-thioethyl)uridines were transformed to 5′-O- N -(2-thioethyl)uridines were transformed to 50-O-DMTr-20-O-TBDMS-protected phosphoramidite DMTr-2′-O-TBDMS-protected phosphoramidite as well as nucleoside-modified CPG resin, after as well as nucleoside-modified CPG resin, after which they were introduced to RNA (12- or 72-nts which they were introduced to RNA (12- or 72-nts models of hairpin or yeast tRNAPhe, respectively) models of hairpin or yeast tRNAPhe, respectively) by standard phosphoramidite chemistry [118,119]. by standard phosphoramidite chemistry [118,119]. The mixed disulfides 109, 112 were reduced with TheDTT mixed under disulfides aqueous 109conditions, 112 were (2–12 reduced h, rt-55 with°C) to DTT obtain under the free aqueous thiols conditions110, 113. After (2–12 the h,removal rt-55 ◦ C) to obtainof DTT the excess free (precipitation, thiols 110, 113 Qiagen. After cartridge, the removal or dialysis), of DTT excessdithiol-containing (precipitation, oligomers Qiagen 110 cartridge,, 113 or dialysis),were redissolved dithiol-containing in 25 mM ammonium oligomers 110acetate, 113 [66]were or redissolved100 mM phosphate in 25 mM buffer ammonium (pH 8.0) [118,119] acetate [ 66] or 100and mMexposed phosphate to air for bu ff5–20er (pH h at 8.0) rt to [118 form,119 the] and disulfide exposed bonds. to air Denaturing for 5–20 h atPAGE rt to formisolation the disulfideof the bonds.target Denaturing RNA products PAGE 111 isolation, 114 revealed of the nearly target quantitative RNA products formation111, 114 of revealeddisulfide nearlycrosslinks. quantitative Since formationthe formation of disulfide of disulfide crosslinks. crosslinks Since is themild, formation highly selective, of disulfide and crosslinksdoes not alter is mild, the RNA highly structure selective, andor does folding, not alterit was the found RNA to structure be a valuable or folding, method it was for found the structural to be a valuable and biochemical method forevaluation the structural of andRNA biochemical molecules. evaluation of RNA molecules.

SchemeScheme 27. 27.(A ()A General) General scheme scheme for for post-syntheticpost-synthetic disulfide disulfide crosslinking crosslinking formation formation between between cytidines; cytidines; reagentsreagents and and conditions: conditions: (i)(i) DTTDTT in H H22O,O, 2 2h, h, 55 55 °C;◦ C;(ii) (ii)25 mM 25 mM NH4 NHOAc,4OAc, air oxidation, air oxidation, 20 h, rt; 20 (B h,) rt; (B)General General scheme scheme for for post-synthetic post-synthetic disulfide disulfide crosslinking crosslinking formation formation between between uridines; uridines; reagents reagents and and

conditions:conditions: (i) DTT(i) DTT in 100in 100 mM mM phosphate phosphate bu ffbufferer (pH (pH 8.0), 8.0), 12 h,12 rth, or rt 40or◦ 40C; °C; (ii) 100(ii) 100 mM mM phosphate phosphate buff er (pHbuffer 8.0), air(pH oxidation, 8.0), air oxidation, 5–12 h, rt.5–12 h, rt.

TheThe thiol-adenine-containing thiol-adenine-containing oligomer oligomer homologoushomologous with the the branched branched region region of ofpre-mRNA pre-mRNA was was post-syntheticallypost-synthetically reacted reacted with with benzophenone benzophenone reagent reagent to indicate to indicate photo-crosslinks photo-crosslinks with with proteins proteins under splicingunder conditions splicing conditions [120]. A two-step [120]. A strategy two-step was strategy chosen was to chosen introduce to introduce the benzophenone the benzophenone reagent into pre-mRNAreagent into substrates pre-mRNA (Scheme substrates 28). Firstly, (Scheme a reactive28). Firstly, thiol a reactive functionality thiol functionality was introduced was intointroduced synthetic 10-ntinto RNA synthetic oligomer 10-nt115 RNAusing oligomer Verdine 115 convertibleusing Verdine inosine convertible nucleoside inosine6 (seenucleoside Figure 46), (see which Figure permits 4), which permits the installation of cystein disulfide tether at the N6 position of adenosine (see Scheme the installation of cystein disulfide tether at the N6 position of adenosine (see Scheme3). After the 3). After the incorporation of synthetic 115 oligomer into full-length pre-mRNA molecules via the incorporation of synthetic 115 oligomer into full-length pre-mRNA molecules via the ligation procedure, the disulfide was reduced to thiol-RNA 117 using DTT. The RNA construct 117 was subjected to MoleculesMolecules2020 2020, 25,, 25 3344, x FOR PEER REVIEW 29 of29 37 of 37

ligation procedure, the disulfide was reduced to thiol-RNA 117 using DTT. The RNA construct 117 derivatizationwas subjected with to highly derivatization effective thiol-specificwith highly effective benzophenone thiol-specific maleimide benzophenone photoreagent maleimide in 50% aq. DMFphotoreagent for 1 h at rt. in 50% aq. DMF for 1 h at rt.

SchemeScheme 28. Post-synthetic 28. Post-synthetic preparation preparation of mRNA–benzophenone of mRNA–benzophenone conjugate conjugate118 via118 N6-(2-thioethyl) via N6-(2- o adenosine-precursorthioethyl)adenosine-precursor117. Reagents 117. Reagents and conditions: and conditions: (i) 5 mM (i) DTT5 mM in DTT 20 mMin 20 NaHCOmM NaHCO3, 1 h,3, 1 30 h, C; (ii)30 20 o mMC; (ii) benzophenone 20 mM benzophenone maleimide maleimide in 50% aq.in 50% DMF, aq. 1 DMF, h, rt; (iii)1 h, irradiation rt; (iii) irradiation with a 302with nm a 302 lamp. nm lamp. The obtained benzophenone-pre-mRNA 118 was incubated in HeLa nuclear extract under standard splicingThe conditions obtained and benzophenone-pre-mRNA crosslinked with proteins 118 by was irradiation incubated with in a HeLa 302 nm nuclear lamp. extract under standard splicing conditions and crosslinked with proteins by irradiation with a 302 nm lamp. 4. Conclusions 4. Conclusions Every year, new strategies are discovered to synthesize modified oligonucleotides. Nevertheless, there isEvery still a year, growing new strategies demand are for discovered the development to synthesize of new modified approaches, oligonucleotides. particularly Nevertheless, toward RNA oligomers,there is still the preparationa growing demand of which for is morethe development challenging of than new their approaches, DNA counterparts. particularly Unlike toward enzymatic RNA procedures,oligomers, the the chemical preparation synthesis of which of RNA is oligomersmore challenging offers the than possibility their DNA of site-specific counterparts. incorporation Unlike enzymatic procedures, the chemical synthesis of RNA oligomers offers the possibility of site-specific of modified nucleosides, making RNA synthesis an important tool for many interdisciplinary incorporation of modified nucleosides, making RNA synthesis an important tool for many studies. The hereby presented concept of post-synthetic oligonucleotide modification represents interdisciplinary studies. The hereby presented concept of post-synthetic oligonucleotide an important alternative for standard phosphoramidite chemistry, particularly when target modifying modification represents an important alternative for standard phosphoramidite chemistry, groups/labels are unstable or reactive under conditions of solid-phase synthesis. The great advantage particularly when target modifying groups/labels are unstable or reactive under conditions of solid- of post-syntheticphase synthesis. reactions The great is thatadvantage one building of post-synthetic block with areactions reactive is group that one can reactbuilding with block a wide with variety a of reagents,reactive group providing can react several with variously a wide varietymodified of oligomers reagents, providing of homologous several sequences. variously modifiedIn addition, reportedoligomers precursor of homologous oligomers sequences. can be easilyIn addition, adapted reported to incorporate precursor newoligomers modifications can be easily using adapted already well-optimizedto incorporate post-synthetic new modifications reactions. using Somealready of well-optimized the post-synthetic post-synthetic transformations reactions. are Some bioorthogonal of the andpost-synthetic have been successfullytransformations employed are bioorthogonal to study and and have monitor been successfully RNA nucleic employed acids into study their and native environmentmonitor RNA [121 nucleic–123]. acids in their native environment [121–123].

AuthorAuthor Contributions: Contributions:Writing—G.L., Writing—G.L., K.B. K.B. andand K.D.;K.D.; Contribution to to the the manuscript manuscript writing writing A.C.; A.C.; Supervision Supervision E.S.E.S. and and G.L. G.L. All All authors authors have have read read and and agreed agreed toto thethe publishedpublished version version of of the the manuscript. manuscript. Funding:Funding:This This work work was was founded founded by by the the NationalNational ScienceScience Center, Poland, Poland, UMO-2017/25/B/ST5/00971 UMO-2017/25/B/ST5/00971 to toE.S. E.S. ConflictsConflicts of Interest:of Interest:The The authors authors declare declare no no conflict conflict ofof interest.interest.

AbbreviationsAbbreviations

AcAc acetyl acetyl ACEACE bis(2-acetoxyethoxy)methyl bis(2-acetoxyethoxy)methyl AMAAMA ammonia-methylamineammonia-methylamine solution solution AZTAZT 30-azidothymidine 3′-azidothymidine BzBz benzoyl benzoyl BzHBzH benzhydryloxybis-(trimethylsiloxy)silyl benzhydryloxybis-(trimethylsiloxy)silyl cmnmcmnm5ges25gesU2U 5-carboxymethylaminomethyl-2-geranylthiouridine5-carboxymethylaminomethyl-2-geranylthiouridine cmnmcmnm5U5U 5-carboxymethylaminomethyluridine 5-carboxymethylaminomethyluridine cnm5U 5-cyanomethyluridine Molecules 2020, 25, 3344 30 of 37

CPG controlled pore glass ct6A cyclic N6-threonylcarbamoyladenosine dbf dibutylaminomethylene DBU 1,8-diazabicyclo [5.4. 0]undec-7-ene DCM dichloromethane DIPEA N,N-diisopropylethylamine dmf dimethylaminomethylene DMTr 4,40-dimethoxytrityl DOD bis(trimethylsiloxy)cyclododecyloxysilyl DTT dithiothreitol EDC HCl 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride · EPR electron paramagnetic resonance ESI-MS electrospray ionization mass spectrometry FMN flavin mononucleotide Fpmp 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl geBr geranyl bromide ges2U 2-geranylthiouridine H2U 4-pyrimidinone nucleoside HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid HOBt N-hydroxybenzotriazole i6A N6-isopentenyladenosine iBu isobutyryl IE HPLC ion-exchange high performance liquid chromatography iEDDA inverse electron demand Diels-Alder cycloaddition inm5U 5-(isopentenylaminomethyl)uridine m5s2U 5-methyl-2-thiouridine m6A N6-methyladenosine MALDI-ToF-MS matrix-assisted laser desorption/ionization-time of flight mass spectrometry mcm5s2U 5-metoxycarbonylmethyl-2-thiouridine, mnm5ges2U 5-methylaminomethyl-2-geranylthiouridine mnm5s2U 5-methylaminomethyl-2-thiouridine mnm5U 5-methylaminomethyluridine ms2C 2-methylthiocytidine ms2ct6A 2-methylthio cyclic N6-threonylcarbamoyladenosine ms2i6A 2-methylthio-N6-isopentenyladenosine ms2m6A 2-methylthio-N6-methyladenosine ms2t6A 2-methylthio-N6-threonylcarbamoyladenosine msms2i6A 2-methylthiomethylenethio-N6-isopentenyladenosine NHS N-hydroxysuccinimide nm5U 5-aminomethyluridine NMP N-methyl-2-pyrrolidone NPE 2-(2-nitrophenyl)ethyl NPP 2-(2-nitrophenyl)propyl OPiv pivaloyloxyl P(furyl)3 tri-2-furylphosphine Pac phenoxyacetyl PAGE polyacrylamide gel electrophoresis Pd2(dba)3 tris(dibenzylideneacetone)dipalladium(0) PELDOR pulsed electron-electron double resonance Pivom5U 5-pivaloyloxymethyluridine R5H2U 5-substituted 4-pyrimidinone nucleoside R5s2U 5-substituted 2-thiouridine R5U 5-substituted uridine Rb ribose RP HPLC reverse-phase high performance liquid chromatography rt room temperature Molecules 2020, 25, 3344 31 of 37 s2U 2-thiouridine 2 s Um 20-O-methyl-2-thiouridine 4 s dU 4-thio-20-deoxyuridine s4U 4-thiouridine SNAr nucleophilic aromatic substitution t6A N6-threonylcarbamoyladenosine Tac 4-(tert-butylphenoxy)acetyl TBAF tetrabutylammonium fluoride TBDMS tert-butyldimethylsilyl TBTA tris[(1-benzy-1H-1,2,3-triazol-4-yl)methyl]amine TC 1,1-dioxo-1l6-thiomorpholine-4-carbothioate TEA triethylamine TEA 3HF triethylamine trihydrofluoride × TEAA triethylammonium acetate TEAF triethylammonium fluoride TEG triethylene glycol TEMED N,N,N0,N0-tetramethylethylenediamine TEMPO 2,2,6,6-tetramethylpiperidin-1-oxyl TEMPO-NH2 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl TFP tetrafluorophenol THPTA tris-[1-(3-hydroxypropyl)-1H-[1,2,3]triazol-4-yl)methyl]amine TMS trimethylsilyl TMSE 2-(trimethylsilyl)ethyl TOM triisopropylsilyloxymethyl TPA 2,2,5,5-tetramethylpyrrolin-1-yloxyl-3-acetylene Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride TSTU N,N,N0,N0-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate τm5U 5-taurinomethyluridine τm5s2U 5-taurinomethyl-2-thiouridine

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