Interdependent Sequence Selectivity and Diastereoselectivity in the Alkylation of DNA by Decarbamoylmitomycin C

Abstract

Mitomycin C (MC), an antitumor drug, and decarbamoylmitomycin C (DMC), a derivative of MC, alkylate DNA and form deoxyguanosine monoadducts and interstrand crosslinks (ICLs). Interestingly, in mammalian culture cells, MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-β). The molecular basis for the stereochemical configuration exhibited by DMC has been investigated using biomimetic synthesis. Here, we present the results of our studies on the monoalkylation of DNA by DMC. We show that the formation of 1"-β-deoxyguanosine adducts requires bifunctional reductive activation of DMC, and that monofunctional activation only produces 1"-α-adducts. The stereochemistry of the deoxyguanosine adducts formed is also dependent on the regioselectivity of DNA alkylation and on the overall DNA CG content. Additionally, we found that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by mitomycins: At 0 °C, both deoxyadenosine (dA) and deoxyguanosine (dG) alkylation occur whereas at 37 °C, mitomycins alkylate dG preferentially. The new reaction protocols developed in our laboratory to investigate DMC-DNA alkylation raise the possibility that oligonucleotides containing DMC 1"-β-deoxyguanosine adducts at a specific site may be synthesized by a biomimetic approach.

Synthesis of Oligonucleotides containing the cis‐Interstrand Crosslink Produced by Mitomycins in their Reaction with DNA

“…In our previous work, we found that the structure of the nucleobases flanking the target sequence influences both alkylation yields and the ratio of diastereomeric monoadducts . In order to fine tune conditions that favor the formation of 1“‐β adducts, we further investigated this effect by comparing the frequency of 2 b and 2 a in 5′‐AGCT; 5′‐TGCA and 5′‐AGCA sequence contexts.…”

“…In order to fine tune conditions that favor the formation of 1“‐β adducts, we further investigated this effect by comparing the frequency of 2 b and 2 a in 5′‐AGCT; 5′‐TGCA and 5′‐AGCA sequence contexts. Duplex oligonucleotides were reacted with fully reduced DMC according to a protocol described previously . Alkylated oligonucleotides were digested by nucleases and phosphatases to the individual nucleosides and the adducted nucleosides ( 2 a and 2 b ) were identified by their UV spectra, their retention time and co‐elution with synthetically prepared standards to confirm their identity .…”

“…Duplex oligonucleotides were reacted with fully reduced DMC according to a protocol described previously . Alkylated oligonucleotides were digested by nucleases and phosphatases to the individual nucleosides and the adducted nucleosides ( 2 a and 2 b ) were identified by their UV spectra, their retention time and co‐elution with synthetically prepared standards to confirm their identity . A method to calculate the percent yields (frequency) for adducts 2 a and 2 b is described in the supporting information and in previous work .…”

“…Alkylated oligonucleotides were digested by nucleases and phosphatases to the individual nucleosides and the adducted nucleosides ( 2 a and 2 b ) were identified by their UV spectra, their retention time and co‐elution with synthetically prepared standards to confirm their identity . A method to calculate the percent yields (frequency) for adducts 2 a and 2 b is described in the supporting information and in previous work . Results (Table ) showed that the yield of β‐monoalkylation ( 2 b ) in duplex DNA is enhanced when adenine is located at the 3′ position of the GpC step.…”

“…Until now, there was no method to access isomeric 1“‐β monoadducts or β‐ICLs produced by mitomycins ( 2 b , 3 b , Figure ). We recently showed that the formation of 1”‐β adducts requires an alternative “bifunctional” reductive activation whereby mitomycins are fully reduced preceding DNA alkylation (Scheme B) . Under these conditions, both α and β nucleophilic additions are possible.…”