Practical Synthesis of Cap‐4 RNA

Abstract Eukaryotic mRNAs possess 5′ caps that are determinants for their function. A structural characteristic of 5′ caps is methylation, with this feature already present in early eukaryotes such as Trypanosoma. While the common cap‐0 (m7GpppN) shows a rather simple methylation pattern, the Trypanosoma cap‐4 displays seven distinguished additional methylations within the first four nucleotides. The study of essential biological functions mediated by these unique structural features of the cap‐4 and thereby of the metabolism of an important class of human pathogenic parasites is hindered by the lack of reliable preparation methods. Herein we describe the synthesis of custom‐made nucleoside phosphoramidite building blocks for m6 2Am and m3Um, their incorporation into short RNAs, the efficient construction of the 5′‐to‐5′ triphosphate bridge to guanosine by using a solid‐phase approach, the selective enzymatic methylation at position N7 of the inverted guanosine, and enzymatic ligation to generate trypanosomatid mRNAs of up to 40 nucleotides in length. This study introduces a reliable synthetic strategy to the much‐needed cap‐4 RNA probes for integrated structural biology studies, using a combination of chemical and enzymatic steps.


Introduction
Posttranscriptional processing adds anotherl ayer of information to RNA.T his affects mRNA splicing, [1] nuclear export, [2] initiation of translation, [3] and stability. [4,5] So-called 5' caps, with the characteristic andu nique feature of an N7-methyl guanosine connected over a5 '-to-5' triphosphate bridge to the first nucleotide of nascent transcripts are hallmarks of eukaryotic mRNA processing. [6,7] For highere ukaryotes it is known that 5' caps are involved in all the above-mentionedp rocesses. For example, m 7 Gc aps are responsible for the recruitment of an uclear cap-binding complex, which plays ak ey role fort he splicing process and is involved in the packaging process of RNA into ribonucleoprotein particles. This is an essential step for export out of the nucleus. [8,9] Moreover,t he 5' cap binds to the eukaryotict ranslation initiation factor 4E (eIF4E), which is ak ey component for the translational machinery and which itself is directedt ot he 5' end of the mRNA. [10] Furthermore, the 5' cap acts as ap hysical hindrance to 5'!3' exoribonucleases, so that decappinge nzymes are necessary to start the controlled decomposition of mRNA. [11] This increases the lifetimeo fm RNA [12] and enables an additional level of regulation. [13] Our growing understanding of the functionality of 5' caps and the consequences of disrupting cap-dependentb iochemical processes that result in severe medical disorders make 5' caps attractive for the development of novel pharmaceutic and therapeutic approaches. [14] Potential applications forc ap analogues are foreseeable in antiviral therapy, cancer treatment (including eIF4E targeting and mRNA-based immunotherapy), spinal muscular atrophy treatment, and treatment of geneticd iseases by mRNA-based protein replacement therapy. [15] Essential for the success of RNA-based therapeutics is the development of methods to introduce mRNA into cells and the improvement of stabilitya nd translation efficiency of the therapeutic RNA itself. [15,16] The high potentialo fm RNA-capbased approaches has been demonstrated in the first application of 5'-capped RNAs in preclinical studies. [15] Significant structural diversity is encountered for mRNA 5' caps. The most commonc aps in eukaryotes are cap-0 (m 7 GpppN), whichi st ypically for lower eukaryotes, cap-1 (m 7 GpppNm) and cap-2 (m 7 GpppNmNm), which are typically for higher eukaryotes. [11] They differ in the complexity of their methylation pattern and are named after how many of the first nucleotideshave a2 '-O-methyl group. Recent studies have shown that 2'-O-methylations play ak ey role in cellular discrimination of endogenousf rom pathogenic RNA. [11] Although variousm odificationso ft hese caps have been discovered, [11] surprisingly,t he most complex methylation pattern known in naturew as found in early eukaryotes, [17] more precisely,w ithin the Trypanosomatidae family from the Trypanosomatida order and the Kinetoplastida class. [18][19][20][21][22] The relevant cap-4 shows seven additional methylations in the first four nucleotides relative to cap-0 ( Figure 1). [17] Eukaryotic mRNAs possess 5' caps that are determinants for their function. As tructural characteristico f5 ' caps is methylation, with this feature already present in early eukaryotes such as Trypanosoma. While the commonc ap-0 (m 7 GpppN) showsa rather simple methylation pattern,t he Trypanosoma cap-4 displays seven distinguished additional methylations within the first four nucleotides. The study of essential biological functions mediatedb yt hese unique structuralf eatures of the cap-4 andt hereby of the metabolism of an important class of human pathogenic parasites is hindered by the lack of reliable preparation methods. Herein we describe the synthesis of custom-made nucleoside phosphoramidite building blocks for m 6 2 Am and m 3 Um, their incorporation into short RNAs, the efficient constructiono fthe 5'-to-5' triphosphate bridge to guanosine by using as olid-phase approach, the selective enzymatic methylation at positionN 7o ft he inverted guanosine, and enzymatic ligation to generate trypanosomatidm RNAs of up to 40 nucleotides in length. This study introduces ar eliable synthetic strategy to the much-needed cap-4 RNA probes for integrated structuralb iology studies, using ac ombination of chemicaland enzymatic steps.
The study of essential biological functions mediated by these unique structural features of cap-4 and thereby of the metabolism of an importantc lass of human pathogenicp arasites is hindered by the lack of reliable preparation methods. To the best of our knowledge,o nly the chemical synthesis of the terminal four-nucleotide fragment of cap-4 has been published thus far.T his was accomplished by obtaining the tetranucleotide fragment (5'-p-(m 6 2 Am)(Am)(Cm)(m 3 Um)) first by the phosphoramidite solid-phase method. The 5'-phosphorylated tetranucleotide was then chemically coupled with m 7 GDP to yield the cap-4 structure. [23] In this study,w es et out to develop ap ractical synthetic route toward Trypanosoma cruzi trans-spliced leader (SL) RNA of as pecific 39-nucleotide (nt) sequencet hat harborst he unique hypermethylated cap-4 structure. [24][25][26][27] We obtained this challenging target by a combination of chemical and enzymatic methods, and provide ap ractical protocol for cap-4 RNAs of anys equence of up to 40 nt to the research community.

Synthesis of m 3 Um and m 6 Am building blocks
The functionalization of commercially available2 '-O-methyluridine 1 into the desired m 3 Um phosphoramidite 3 involved three reactions, which are summarized in Scheme 1. Our route began with alkylation of N3 under basic conditions using iodomethane. After concentration to ad ry solid, the crude product was directly transformed into the dimethoxytritylated compound 2 by using 4,4'-dimethoxytriphenylmethyl chloride (DMTCl) in pyridine. Finally,p hosphitylation was executed with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (CEP-Cl) in the presenceo fN,N-diisopropylethylamine (DIPEA). Starting from nucleoside 1,o ur route providesp hosphoramidite 3 with 45 %o verall yield in three transformations involving two chromatographic purifications;i nt otal, 0.85 go fc ompound 3 was prepared over the course of this study.
Scheme2.Five-step synthesis of m 6 Am phosphoramidite for RNA solidphase synthesis. mine. Compound 7 was tritylated to give compound 8,a nd finally phosphitylated with CEP-Cli nt he presence of DIPEA. Startingf rom nucleoside 4,o ur route provides phosphoramidite 9 in 46 %o verall yield over five steps involving five chromatographic purifications;i nt otal, 0.75 go fc ompound 9 was prepared during the course of this study.

Synthesis of cap-4 RNA
With the hypermethylated nucleoside phosphoramidites in hand, we continued with asynthetic strategy that was originally introduced by Debart, Decroly,a nd co-workers. [29b] This approachr elies on RNA assembly and introductiono ft he cap on solid support, enabling the removal of excessr eagents by simple washing whichmakes the synthesis straightforward and convenient. However,i nsteado fr elying on RNA solid-phase synthesis with the base-labile 2'-O-pivaloyloxymethyl( PivOM)protectedn ucleoside phosphoramidites, we optimized our protocolf or the implementation of standard 2'-O-tert-butyldimethylsilyl (TBDMS) and/or 2'-O-[(triisopropylsilyl)oxy]methyl (TOM) phosphoramidites ( Figure 2). Attachmento ft he GDP was derived from earlier protocols for the synthesis of 5'-triphosphate DNA and RNA on solid support, [30] by converting the free 5'-OH and otherwise protected oligonucleotide into its 5'-H-phosphonate derivative.This derivative was then activated as the phosphoroimidazolide by amidative oxidation. Then, coupling of guanosine diphosphate (GDP) as tributylammonium salt on the RNA bound to the solid support was followed by deprotection of acyl groups and releasef rom the solid support using methylaminei nw ater/methanol (AMA). Subsequently,s ilyl deprotection was achieved with tetrabutylammonium fluoride in THF, resulting in the desired Gppp-RNA in solution.
Finally,t he obtained cap-4 RNA was readily ligatedu sing T4 DNA ligase and DNA or 2'-O-methyl RNA splints. Figure 4i llustrates at ypicale nzymatic ligationo fa3 9-nt T. cruzi cap-4 spliced leader RNA, using a1 .2-fold excesso ft he unmodified RNA fragment and 1.2-fold excesso ft he splint ( Figure 4). Ligation yields were about 65 %, and the product was purified by anion-exchange chromatography.M ass spectrometric analysis using LC-electrospray ionization confirmed the integrity of the cap-4 RNA product.

Conclusion
We developed ap ractical route towardh ypermethylated cap-4 RNAs. This included the three-and five-step syntheses of m 3 Um and m 6 2 Am phosphoramidites. Assembly of the G-5'-ppp-5'-RNA was entirely conducted on solid phase along the lines of ap reviously established protocol, however,w ith the difference of using 2'-O-silyl-protectedb uildingb locks, which required adaption of the RNA deprotection steps. The final N7 methylation was performed enzymatically using Ecm1 methyltransferase. We exemplified the chemoenzymatic approach by the synthesis of a3 9-nt T. cruzi trans-spliced leader RNA that awaits applicationsi ns tructural biologys tudies. Very recently, initial insightb yX -ray crystallography into how cap-4 is recognized by a T. cruzi eIF4E5 translation factor homologue has become available using as hort m 7 G-ppp-(m 6 2 Am)(Am)(Cm)-(m 3 Um) synthetic oligonucleotide. [33] With the access to larger cap-4 RNAs presentedh erein, structure elucidation of ribosomal complexes bound to cap-4 RNAs appearsa ccomplishable in the near future.

Experimental Section
77 mmol) was dissolved in dry dimethyl sulfoxide (2 mL). Sodium hydride (18.6 mg, 0.77 mmol) was added to the reaction mixture and stirred at room temperature under argon atmosphere until bubbling stopped. Then, methyl iodide (0.05 mL, 0.77 mmol) was added dropwise. The solution was stirred for another 5h.A ll volatiles were evaporated under high vacuum. The residue was co-evaporated once with methanol and twice with pyridine before it was dissolved in anhydrous pyridine (1 mL). Dimethoxytrityl chloride (0.32 g, 0.93 mmol) and 4-dimethylaminopyridine (14.2 mg, 0.12 mmol) were added. The reaction solution was stirred under argon at room temperature overnight before the reaction was quenched by adding methanol. All volatiles were evaporated, the residue was diluted in methylene chloride and was washed three times with as olution of 5% citric acid, once with as aturated sodium bicarbonate solution, and once with as aturated sodium chloride solution. The product was isolated after column chromatography (100:0.1 to 100:0.5 methylene chloride/methanol). Yield:0 .24 g( 54 %) of 2 as white foam. 1     15 mmol;p repared as described below following ref. [28]) which was previously dried over phosphorpentoxide under high vacuum for 2h.This solid was diluted in dry pyridine (8 mL) and stirred under argon atmosphere in the dark at 85 8Cf or 48 h. Then all volatiles were removed under vacuum and the residue was co-evaporated twice with toluene. The residue was filtrated and washed with methylene chloride. The solid N,Ndimethylformamide-azine dihydrochloride can be recycled. The filtrate was washed with as olution of 5% citric acid, as aturated solution of sodium bicarbonate and as aturated solution of sodium chloride. The organic fractions were united and the volatiles were removed under vacuum. The residue was then purified by column chromatography (silica, 0.5-1.  The residue was diluted in dry pyridine (8 mL). 4,4'-O-Dimethoxytrityl chloride (0.32 g, 0.92 mmol) was dried for 2h on high vacuum before it was added to the reaction solution, and the mixture was stirred overnight at room temperature together with molecular sieves under argon atmosphere. The reaction was quenched with methanol (0.5 mL) before all volatiles were removed under reduced pressure. The residue was co-evaporated three times with toluene. Afterward the product was purified by column chromatography (silica, 0-3 %m ethanol in methylene chloride). Another column chromatography (12-25 %a cetone in toluene) can be necessary to further improve purity.Y ield:0 .382 g( 81 %) of 8 as white foam.  dihydrochloride: [28] Thionyl chloride (1.4 mL, 19.3 mmol) was added dropwise to dry DMF (7 mL) under cooling with ice water.T his transparent, slightly yellow solution was stirred for 20 hu nder argon. As olution of hydrazine monohydrate (0.24 mL, 1equiv,5 .0 mmol) in dry DMF (7 mL) was added in drops and under cooling. This slightly yellow suspension was stirred for another 24 hu nder argon atmosphere. Commercially available modified nucleoside building blocks:2 '-Omethyl adenosine and 2'-O-methyl cytidine (ChemGenes Corporation), were incorporated using modified synthesis cycles with longer coupling times (6 min).
RNA phosphitylation, hydrolysis, oxidative amidation was performed in analogy to ref.
[29a],w hile Gpp attachment was performed in analogy to ref. [29b].C onstruction of the triphosphatebridged inverted guanosine to the RNA on the solid phase in-volved four steps, all of them performed under argon atmosphere. RNA phosphitylation was carried out by applying 0.5 mL of phosphitylation solution (2 mL diphenylphosphite, 8mLa nhydrous pyridine) over 300 s. This treatment was repeated twice. After washing the beads with acetonitrile, hydrolysis was performed using 0.5 mL of hydrolysis solution (1.0 mL of 1 m aqueous triethylammonium bicarbonate, 5mLw ater and 4mLa cetonitrile) over ap eriod of 5min. This treatment was repeated four times. The solid support was then rinsed with 20 mL of acetonitrile before being dried under vacuum for several hours. Oxidative amidation was done by incubation with 0.5 mL of as olution containing 600 mg imidazole, 2mL N,O-bis(trimethylsilyl)acetamide, 4mLa nhydrous acetonitrile, 4mLb romotrichloromethane and 0.4 mL trimethylamine for 30 min. This treatment was repeated four times. The solid support was intensively rinsed with dry acetonitrile. Finally,Gpp attachment was achieved by application of 0.5 mL of coupling solution (0.28 m guanosine 5'-diphosphate di-tributylammonium salt [preparation see above] in dry DMF,5 00 mm zinc chloride) over 8h at room temperature. This treatment was repeated three times.
Gppp-capped RNA deprotection: The solid supports with the Gppp-capped and otherwise fully protected oligos were rinsed with DBU in acetonitrile (6 mL;1m), then washed with acetonitrile and dried. Cleavage from the support and base deprotection was effected by treating the solid support with a1 :1 mixture of 40 % aqueous methylamine and 30 %a queous ammonia (AMA) in a screw-cap vial for 2h at 40 8C. The resulting suspensions were filtered and the filtrates dried. Removal of 2'-O silyl protecting groups was carried out with of 1 m tetrabutylammonium fluoride trihydrate in THF (1 mL) for 6h at 40 8C. The reaction was quenched by the addition of 1 m triethylammonium acetate solution (pH 7.4, 1mL) and then concentrated to approximately 1mL. This viscous solution was desalted by size exclusion chromatography on aH iPrep 26/10 Sephadex G25 column (GE Healthcare). The crude products were evaporated and re-dissolved in water (1 mL). Quality assessment of the crude Gppp-capped RNAs via anion-exchange HPLC on aD ionex DNAPac PA-100 column (4 250 mm); conditions:f low 1mLmin À1 ;e luent A: 25 mm Tris hydrochloride, 6 m urea, pH 8.0, eluent B: 500 mm sodium perchlorate, 25 mm Tris hydrochloride, 6 m urea, pH 8.0;g radient:0 -60 %Bin 45 minutes; temperature:4 0 8C, UV detection at 260 nm. The Gppp-capped RNAs were isolated by anion-exchange HPLC on aD ionex DNAPac PA-100 column (4 250 mm);C onditions:f low 1mLmin À1 ;e luents: see above;t emperature 40 8C; UV detection at 260 nm. The product fractions were diluted with an equal amount of triethylammonium bicarbonate buffer (100 mm,p H7.4) and loaded onto equilibrated C18 Sep-Pak (Waters Corporation). The cartridge was washed with water and the RNA was eluted with acetonitrile/water (1:1). All volatiles were evaporated and the residue re-dissolved in water (1 mL). Yields were determined UV photometrically.The quality of the product was analyzed via anion-exchange HPLC as described above and by reversed-phase LC-ESI-MS.
Enzymatic N7 methylation of Gppp-RNA: The enzymatic transformation was conducted in analogy to refs. [31] and [32].I ns hort, lyophilized Gppp-RNA 10 (4 nmol) was dissolved in buffer (8.0 mL, 1.5 m NaCl, 200 mm Na 2 HPO 4 ,p H7.4) followed by the addition of an aqueous solution of S-adenosylmethionine (2.0 mL, 12 nmol), and the addition of water to obtain at otal volume of 68.8 mL. To the mixed solution, enzymes were added successively (1.6 mLo f 50 mm MTAN, 1.6 mLo f5 0mm LuxS, 8 mLo f5 0mm Ecm1 (Figures S1 and S2 in the Supporting Information)). The mixture was incubated for 45 min at 37 8C. The solution was extracted twice with an equal volume of chloroform/isoamyl alcohol solution (24:1, v/v). The or-ganic layers were rewashed twice with an equal volume of water. The aqueous layers were combined and lyophilized, to eliminate remaining organic solvents. Analysis of the methylation reaction and purification of the product was performed by anion-exchange chromatography (conditions see above). The integrity of the product was confirmed by LC-ESI mass spectrometry (conditions see below).