Modified DNA oligomers possess interesting possibilities for avariety of biomedical and synthetic applications. However, the onlycurrent methods for creating brominated strands involve costly,often commercial synthesis. Thus, a novel synthetic route forproducing these modified oligonucleotides in a more cost-effectivemanner, involving the bromination of natural DNA oligomers viamild brominating reagent sodium monobromoisocyanurate(SMBI), is here developed. Upon successful bromination, thestrands may be functionalized for various organic reactions,including Suzuki coupling reactions and click reactions after azidosubstitution of bromine. Conditions for said reactions withbrominated oligonucleotide strands were analyzed and refined.
Why brominate natural DNA?Bromine’s ability to act as a stableleaving group means that the elementplays an integral role in many organicsubstitution reactions. Accordingly, abrominated DNA strand has the capacityto be modified further via thesesubstitution reactions, facilitating theaddition of various functional groupsand thereby priming the strand forparticipation in many well-establishedpractical applications in the worlds ofmedicine and research science.
BROMINATION REACTIONSA
B
C
Exact Mass: 2473.39
PURPOSE
Progress Toward Bromination & Functionalization of Natural DNASamantha Horwitz1, Emin B Atuk2, Janarthanan Jayawickramarajah31Kent State University, Dept. of Chemistry & Biochemistry, 2-3Tulane University, Dept. of Chemical & Biomolecular Engineering
The strands that were reacted with sodiumazide were further explored in a clickreaction featuring said strands combinedwith coumarin and an azide linker,synthesized via the pathway depicted in A.The characteristic peak in fluorescenceintensity around 500 nm of reacted DNAfractions after HPLC purification (B) as wellas the shift in retention time of the strandafter the reaction (D) serve to validate thereaction’s success.
AcknowledgementsThank you to Berk Atuk, Dr. Jayawickramarajah, and all members of the Jayawickramarajah lab for providing help and motivation for this work.
We thank the National Science Foundation for financial support through grantsDMR-1460637 and DMR-1852274
Optimization Conditions for Electrophilic Aromatic Bromination of DNA:
• Temperature• Salt concentration• pH (acidic)• Solid-Phase Reaction• Strand Specificity
FUNCTIONALIZATION REACTIONS
BROMINATION
FUTURE DIRECTIONS
FLUOROGENIC CU(I)-CATALYZED AZIDE-ALKYNE CLICK CHEMISTRY
A
C
D
Most electrophilic aromaticbromination reactions are carried outby NBS, a reagent that necessitatesharsh conditions that would destroyDNA to function. SMBI, depicted inA, provides milder conditions forbromination. SMBI has generallybeen used only to brominate singlenucleosides via the reaction depictedin B. However, recent MALDI results(C) from a previously run reactionwithin this lab between SMBI inDMF and ssDNA sequenceTGGGGTT indicate a masscorresponding to successfulbromination of the reaction strand.
After obtaining positive results using theTGGGGTT strand, other strands werestudied under various conditions toascertain whether these preliminaryresults were reproducible. Strands usedwere in both free, aqueous form as wellas bound to reaction beads in solidphase. Although few conclusive resultsprecipitated, reaction conditionscomprised of acidic solution, moderatelyhigh temperature, and reaction strandsattached to said reaction beads had themost promising HPLC traces uponpurification, evidenced by theperceivable shift between the retentiontime of unmodified versus reactedstrands.
HPLC Trace of SH15 vs Unmodified (TTAGGG)3
B
While the bromination reaction trialscontinued, functionalizationreactions—wherein commerciallysynthesized, brominated DNA strands(TTTTTA[Br]TTTTT and (TTA[Br]GGG)3)were obtained and utilized in reactionschemes as displayed right—occurredsimultaneously. Our aim was to applypractical functional modifications, suchas the Suzuki coupling reaction (1),sodium azide addition (2) and clickchemistry reactions (3), to the strands.HPLC traces of purifications followingthe reactions show definitive shifts inretention times between brominated,unmodified strands and modifiedstrands.
MALDI of Azido Substituted TTTTTA(Br)TTTTT
3375.841
NMR Spectrum of Coumarin with Azide Linker (Product from A)
Much remains to be investigated and refined in terms of the brominationand functionalization of natural DNA as proposed by this project. Namely,a similar fluorophore labelling reaction as depicted above is possible forproducts of the Suzuki cross-coupling reaction with attachment of afluorescent boronic acid derivative. Similarly, with respect to brominationof the strands themselves, the use of external catalysts to enhanceelectrophilicity of Br+ should be examined. Ultimately, consistentlysuccessful bromination of DNA strands would provide a cost-effectiveroute for the development of such biomedical applications asradiosensitizers for tumor treatment as well as poly adenylation inhibitorsfor treatment of various cancers and other malignancies.
• Maity, J. and Stromberg, R. An Efficient and Facile Methodology for Bromination of Pyrimidine and Purine Nucleosides with Sodium Monobromoisocyanurate (SMBI). Molecules 2013, 18, 12740-12750.
• Shaugnessy, K.H. Palladium-Catalyzed Modification of Unprotected Nucleosides, Nucleotides, and Oligonucleotides. Molecules 2015, 20, 9419-9454.
• Zayas et al. Strain Promoted Click Chemistry of 2- or 8-Azidopurine and 5-Azidopyrimidine Nucleosides. Bioconjugate Chemistry 2015, 26, 1519-15532.
