Azide Phosphoramidite in Direct Synthesis of Azide-ModifiedOligonucleotidesMaksim A. Fomich,† Maksim V. Kvach,† Maksim J. Navakouski,‡ Christoph Weise,§

Alexander V. Baranovsky,∥ Vladimir A. Korshun,⊥,# and Vadim V. Shmanai*,†

†Institute of Physical Organic Chemistry, National Academy of Sciences of Belarus, Surganova 13, 220072 Minsk, Belarus‡Primetech ALC, Surganova 13, 220072 Minsk, Belarus§Institute of Chemistry and Biochemistry, Freie Universitat Berlin, Thielallee 63, 14195 Berlin, Germany∥Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevicha 5/2, 220141 Minsk, Belarus⊥Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia#Gause Institute of New Antibiotics, Bol’shaya Pirogovskaya 11, 119021 Moscow, Russia

*S Supporting Information

ABSTRACT: Azide and phosphoramidite functions were found tobe compatible within one molecule and stable for months insolution kept frozen at −20 °C. An azide-carrying phosphoramiditewas used for direct introduction of multiple azide modifications intosynthetic oligonucleotides. A series of azide-containing oligonucleo-tides were modified further using click reactions with alkynes.

In recent years, alkyne- and azide-modified oligonucleotideshave been widely used for the synthesis of a variety of

bioconjugates via azide−alkyne click chemistry.1 While terminalalkynes1 and in several cases even cyclooctynes2 can be directlyintroduced into oligonucleotides using phosphoramiditereagents, the approach is believed to be not applicable forobtaining of azide-modified oligonucleotides.3 Attempts toisolate3a an azide-containing phosphoramidite or to use it inoligonucleotide synthesis3b failed, presumably due to aStaudinger reaction of the azide with P(III) in thephosphoramidite function. Indeed, both H-phosphonate3a,4

and phosphotriester5 protocols using P(V) monomers areemployed for the direct synthesis of azide-modified oligonu-cleotides. Other methods of azide incorporation intooligonucleotides6 include postsynthetic modifications (acyla-tion of amine derivatives,2e,h,7 nucleophilic substitution on alkylhalide or alkyl sulfonate modified oligonucleotides,8 conversionof an amine to an azide with a diazo transfer reagent,9 clickreaction of alkyne-modified oligonucleotides with polyfunc-tional azides10) and some enzymatic approaches (incorporationof azide-modified triphosphates, etc.).11

With the introduction of strain-promoted azide−alkynecycloaddition (SPAAC) the use of azide oligonucleotides hasbecome an attractive option, as the azido function can becoupled with both terminal alkynes in a Cu(I)-catalyzedreaction (CuAAC) and strained cycloalkynes in a noncatalyticmanner. The synthesis of azide oligonucleotides, however, issophisticated to some extent, and the demand for astraightforward and convenient procedure for the incorporationof azide functionality into oligonucleotides is very high. If azideoligonucleotides were readily available, this would positively

impact the development of click-chemistry-based approaches innucleic acid chemistry.There are several communications reporting the compati-

bility of azide with P(III) functions, phosphoramidites andphosphites, within synthetic intermediates in one-pot prepara-tions of both nucleoside3a,12 and other13 azide derivativescontaining P(V). Recently, an important observation wasreported4b that a Staudinger reaction does not disturb theazide group in the support-bound growing nucleotide chainupon further assembly using phosphoramidites. This allowedthe use of azide-containing solid supports to produce 3′-azide-modified oligonucleotides in automated phosphoramiditesynthesis.14

These results encouraged us to investigate more thoroughlythe conditions for coexistence of azide and phosphoramiditefunctions in one molecule and to evaluate the utility of such areagent in automated oligonucleotide synthesis.We chose (3R,5S)-5-hydroxymethyl-3-hydroxypyrrolidine

(trans-4-hydroxy-L-prolinol)2a,15 1 as the structural basis forreagents allowing incorporation modifications into oligonucleo-tides in terminal positions or replacing any nucleoside withinthe sequence (Scheme 1). Acylation of 1 with the succinimideester of 4-azidobutanoic acid gave corresponding diol 2. Theprimary hydroxyl was then protected with 4,4′-dimethoxytritylchloride leading to the precursor 3, which was transformed tophosphoramidite 4 by N,N-diisopropylamino-2-cyanoethoxy-chlorophosphine. Alternatively, diol 2 was converted to thecontrolled pore glass support 8 by transient pivaloylation of

Received: July 22, 2014

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primary hydroxyl, Dmt protection of secondary OH-group of 5,removal of pivaloyl residue, and coupling with carboxyl CPG.Phosphitylation of compound 3 with N,N-diisopropylamino-

2-cyanoethoxychlorophosphine in the presence of DIEAproceeded smoothly and was completed in 5 min as monitoredby TLC. Subsequent workup with sodium bicarbonate anddrying over sodium sulfate resulted in the final solution ofcompound 4, which was stored for further NMR studies andautomated synthesis of modified oligonucleotides without anyadditional treatment.For the purpose of NMR study the synthesis of

phosphoramidite 4 from 3 was slightly modified and performedin CDCl3 instead of CH2Cl2. The resulting solution of 4 inCDCl3 was aliquoted and stored at room temperature or at−20 °C.The structure of 4 was confirmed by analysis of 1H, 13C,

COSY, HSQC, HMBC, NOESY, and 31P NMR spectraimmediately after synthesis. 1H and 31P NMR spectra ofaliquoted samples were registered also in 1, 4, 12, and 50 weeks.The initial purity of phosphoramidite 4 was determined as aratio of integral lines of P(III) to combined P(III) + P(V)species in 31P NMR and was approximately 94% (Figure 1A).Surprisingly, the phosphoramidite did not undergo noticeabledestruction upon storage in solution at −20 °C for 50 weeks(90% residual content, Figure 1B). At room temperature itdecomposed much faster (18% of 4 remained after 1 week,Figure 1C). All our attempts to obtain phosphoramidite 4 inthe solid state by evaporation of the solvent or freeze-drying(lyophilization) destroyed it almost completely (maximum 8%of 4 was determined while analyzing the solid product, Figure1D). These results demonstrate that phosphoramidite 4 can besuccessfully obtained and stored at −20 °C in solution;however, it decomposes fast at higher temperatures andimmediately upon concentration.Then reagents 4 and 8 were applied for automated

incorporation of the azide residue at the 5′-terminus of anoligonucleotide by two methods (Scheme 2).

The first method (Scheme 2, method A) is based ontraditional DNA synthesis in the 3′ → 5′ direction withstandard phosphoramidites. In this case, the azide residue wasincorporated in the 5′-position at the last coupling step usingphosphoramidite 4, which was prepared and stored as asolution in CH2Cl2 according to the procedure describedabove. The concentration of phosphoramidite 4 wasdetermined as 0.15 M by trityl assay (see SupportingInformation) and used as such in DNA-synthesis. The couplingtime of the azide phosphoramidite was increased to 8 min. Theadvantage of method A is the use of the standardphosphoramidites and CPGs for oligonucleotide assembly.The only disadvantage is that the azide phosphoramidite shouldbe stored at −20 °C in solution or alternatively be freshlysynthesized before use.We prepared the same oligonucleotide using the second

route (Scheme 2, method B) starting from azide-modified CPG8 by condensation of reversed phosphoramidites dABz, dCAc,dGdmf, dT in the 5′ → 3′ direction.16 Azide solid supports14

were never before applied to the synthesis of 5′-azide-modifiedoligonucleotides. At first glance, the stability of azide CPGduring oligonucleotide synthesis and upon storage is anadvantage of method B. However, reversed phosphoramiditesare much more expensive vs standard reagents,17 and 5′ → 3′synthesis is used for nonroutine purposes.Coupling of azide phosphoramidite 4 at the 5′-terminus

resulted in the oligonucleotide containing the Dmt-group. Twooptional isolation and purification methods can be applied tothe oligonucleotide ON1 (Table 1) synthesized according toScheme 2A. The Dmt-group can be automatically removed at

Scheme 1. Preparation of Azide Phosphoramidite and AzideSolid Supporta

aKey: Su, succinimidyl; Dmt, dimethoxytrityl; CEP, P(OCH2-CH2CN)Ni-Pr2; DIEA, N,N-diisopropylethylamine; DIC, diisopropyl-carbodiimide; CPG, controlled pore glass.

Figure 1. 31P NMR spectra of azide phosphoramidite 4: after synthesis(A); after 50 weeks of storage at −20 °C (B); after 1 week at 20 °C(C); after evaporation (D).

Scheme 2. Incorporation of Azide Residue at 5′-Terminus ofOligonucleotide

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the last detritylation step (“Dmt-Off”) or left after the finalsynthesis cycle (“Dmt-On”). We applied both methods forpurification of ON1. After cleavage from the solid support andammonia deprotection, “Dmt-Off” oligonucleotide ON1 waspurified by polyacrylamide gel electrophoresis (PAGE), and the“Dmt-On” oligonucleotide was purified by RP-HPLC and thenmanually detritylated. The identity of synthesized oligonucleo-tides was verified by MALDI-MS, and the data were in goodaccordance with calculated values.18

The coupling efficiency of phosphoramidite 4 was highenough to allow preparation of multilabeled azide oligonucleo-tides ON2 and ON5. By three sequential condensations ofphosphoramidite 4, ON2 containing three azide residues wasobtained. After the last coupling step the Dmt-group was left,the “Dmt-On” oligonucleotide ON2 was cleaved from the solidsupport in concentrated ammonia, purified by RP-HPLC, andmanually detritylated. Oligonucleotide ON5 contained threenonsequential azide groups in the internal positions of thechain and fluorescein label (FAM) at the 5′-terminus to provideeasy visualization of click derivatives.To confirm the presence and availability of azide functions in

synthesized oligonucleotides, ON1 and ON2 were modifiedwith Cy5-alkyne and FAM-alkyne in the presence of Cu(I) toafford oligonucleotides ON3 and ON4.18

We envisioned that azide phosphoramidite 4 is suitable forthe preparation of more complex oligonucleotides which couldbe useful for further preparation of self-assembling DNAstructures. As a proof of principle, we explored the possibility ofmultiple click modification of ON5 with alkyne-modifiedoligonucleotide T10 (Scheme 3). ON5 was reacted with 6equiv of T10 alkyne and precipitated in acetone. The reactiongives the desired trimodified ON6 as a major product (Figure2), which can be easily isolated by PAGE.18

To conclude, azide phosphoramidite 4 and azide solidsupport 8 were synthesized and showed good performance inthe preparation of azide-modified oligonucleotides suitable forfurther click modifications. Azide support was applied for thefirst time for the preparation of 5′-azide-modified oligonucleo-tides. Reagent 4 is suitable for the introduction of multipleazide modifications into oligonucleotides. Although azidephosphoramidite was not isolated in the solid state, its shelflife in solution is at least one year at −20 °C without substantialloss of coupling efficiency in oligonucleotide synthesis. Thisfinding paves the way for the preparation of a great variety ofazide-carrying phosphoramidite reagents and their use inautomated solid phase synthesis of azide-modified oligonucleo-tides for various applications, including DNA nanotechnology.

Table 1. Sequences of Synthesized Oligonucleotidesa

no. sequence, 5′→3′ON1 X-CACGACGTTGTAAAACGACON2 X-X-X-CACGACGTTGTAAAACGACON3 Cy5x-CACGACGTTGTAAAACGACON4 FAMx-FAMx-FAMx-CACGACGTTGTAAAACGACON5 FAM-CACGA-X-CATTG-X-TAAAA-X-CCACCON6 FAM-CACGA-(T10)

x-CATTG-(T10)x-TAAAA-(T10)

x-CCACCaX is a modification from azide phosphoramidite 4; FAMx and Cy5x

are fluorophores introduced via X by CuAAC reaction withcorresponding alkyne derivatives;18 FAM was introduced withfluorescein phosphoramidite.19

Scheme 3. Triple Click Modification of Oligonucleotide ON5 with T10 Alkyne

Figure 2. PAGE analysis of the reaction between ON5 and T10: (A)the starting ON5; (B) crude reaction mixture: ON6 and byproducts;L, loading line.

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■ ASSOCIATED CONTENT*S Supporting Information

Experimental procedures and NMR spectra of all newcomponds, HPLC traces, and MALDI mass spectra ofoligonucleotides. This material is available free of charge viathe Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author

*E-mail: shmanai@ifoch.bas-net.by.Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the National Academy of Sciencesof Belarus (3.1.03 “Convergence”). We thank Primetech ALC(Minsk, Belarus) for the synthesis of modified oligonucleotidesand Dr. Olga Sharko (Institute of Physical Organic Chemistry,Minsk, Belarus) for the thorough reading of the manuscript andhelpful discussions. For mass spectrometry (C.W.), we wouldlike to acknowledge the assistance of the Core FacilityBioSupraMol supported by the DFG. V.A.K. was supportedby the Molecular and Cellular Biology Program of the RussianAcademy of Sciences and Russian Foundation for BasicResearch (Project 13-04-01317).

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