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. 2021 Oct 13;27(57):14263-14272.
doi: 10.1002/chem.202102338. Epub 2021 Sep 8.

Synthesis of Oligonucleotides Containing Trans Mitomycin C DNA Adducts at N6 of Adenine and N2 of Guanine

Owen Zacarias  1 , Ana G Petrovic  2 , Rinat Abzalimov  3   4 , Padmanava Pradhan  5 , Elise Champeil  1   4
Affiliations

Synthesis of Oligonucleotides Containing Trans Mitomycin C DNA Adducts at N6 of Adenine and N2 of Guanine

Owen Zacarias et al. Chemistry. .

Abstract

Mitomycin C, (MC), an antitumor drug, is a DNA alkylating agent currently used in the clinics. Inert in its native form, MC is reduced to reactive mitosenes, which undergo nucleophilic attack by guanine or adenine bases in DNA to form monoadducts as well as interstrand crosslinks (ICLs). Although ICLs are considered the most cytotoxic lesions, the role of each individual adduct in the drug's cytotoxicity is still not fully understood. Synthetic routes have been developed to access modified oligonucleotides containing dG MC-monoadducts and dG-MC-dG ICL at a single position of their base sequences to investigate the biological effects of these adducts. However, until now, oligonucleotides containing monoadducts formed by MC at the adenine base had not been available, thus preventing the examination of the role played by these lesions in the toxicity of MC. Here, we present a route to access these substrates. Structural proof of the adducted oligonucleotides were provided by enzymatic digestion to nucleosides and high-resolution mass spectral analysis. Additionally, parent oligonucleotides containing a dG monoadduct and a dG-MC-dG ICL were also produced. The stability and physical properties of all substrates were compared via CD spectroscopy and UV melting temperature studies. Finally, virtual models were created to explore the conformational space and structural features of these MC-DNA complexes.

Keywords: alkylation; conformational studies; deoxyadenosine adducts; interstrand crosslinks; site-specific mitomycin C.

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Figures

Figure 1.
Figure 1.
Monoadducts and ICLs generated by Mitomycin C in its reaction with DNA. (A): Monofunctional pathway (B): Bifunctional pathway.
Figure 2.
Figure 2.
A) HPLC Chromatogram of the products from the reaction between MC and duplex (8).(9) under monofunctional activation. Blue line: Absorption at 260 nm, red line: Absorption at 320 nm. B) Alkylation mechanism. C) Oligonucleotides produced.
Figure 3.
Figure 3.
HPLC chromatograms of enzymatic digests from oligonucleotides 10, 11 and 12. Co-injection with authentic standards 1a and 4a.
Figure 4.
Figure 4.
Synthesis of trans crosslink 13.
Figure 5.
Figure 5.
Fragmentation pattern and mitosene containing fragments detected by MS/MS fragmentation in collision activated mode. (11) : [an-B] with n=1,2,3,5,8–14,16,17, 20,22,24 and wn with n=1–7,24. (12) : [an-B] with n=1–8, 10–14,16,17,20, 24 and wn with n=1–7,24. (13) : top strand [an-B] with n=1,4,5,7,8, 10–12 and wn with n=1,3,4–6, 8,10–12; bottom strand: [an-B] with n=1–6, 12 and wn with n=1–3,5,6,9,11.
Figure 6.
Figure 6.
HPLC chromatogram (A260) of products from the reactions between 8 and 9 (A), 8 and 15 (B), 8 and 16 (C), 8 and 17 (D). HPLC analysis performed at 45°C for (A); (B) and (D) and at 30°C for (C).
Figure 7.
Figure 7.
UV-Vis spectra of 10, 11, 12 and trans-ICL 13.
Figure 8.
Figure 8.
CD spectra of double stranded 10 (dG-Adduct; A260: 0.43 OD), double stranded 11 (dA-adduct; A260: 0.45 OD), double stranded 12 (dA and dG Bis-Adduct; A260: 0.80 OD), 13 (crosslinked; A260: 0.46 OD) and control duplex (DS-std; A260: 0.51 OD).
Figure 9.
Figure 9.
Melting curves for the trans-ICL (13, red), the control duplex (DS-std, black), the dG monoadduct (10, green), the dA monoadduct (11, blue) and the dA and dG-bisadduct (12, purple).
Figure 10.
Figure 10.
Representative structures associated with the lowest energy cluster for each of the model systems, with bulk model of the mitosene displayed in green.
Figure 11.
Figure 11.
Magnified region surrounding the mitosene for representative structures (lowest energy clusters) of model systems containing the adduct(s).
Scheme 1.
Scheme 1.
Reductive activation pathways of Mitomycin C.
See this image and copyright information in PMC

References

    1. Hata T, Hoshi T, Kanamori K, Matsumae A, Sano Y, Shima T, Sugawara R, J. Antibiot 1956, 9, 141–146. - PubMed
    1. Bradner WT, Cancer Treat. Rev 2001, 27, 35–50. - PubMed
    1. Verweij J, Pinedo H, in Cancer Chemotherapy and Biological Modifiers: Annual 11 (Eds.: Pinedo HM, Chabner BA, Longo DL), Elsevier Science Publishers B.V., Amsterdam, 1990, p. 67.
    1. Chabner BA, Amrein PC, Druker BJ, Michaelson MD, Mitsiades CS, Goss PE, Ryan DP, Ramachandra S, Richardson PJ, Supko JG, Wilson WH, “Antineoplastic Agents” in Goodman & Gilman’s The Pharmacological Basis of Therapeutics (Eds.: Brunton LL, Lazo JS, Parker KL), McGraw-Hill Publishers, New York, 2005, pp. 1315–1403.
    1. Paz MM, Pritsos CA, “The Molecular Toxicology of Mitomycin C” in Advances Molecular Toxicology Vol. 6 (Ed.: Fishbein JC), Elsevier Science Publishers B.V., Amsterdam, 2012, pp. 244–286.

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