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Synthesis of S-adenosyl-L-methionine analogs and their use for sequence-specific transalkylation of DNA by methyltransferases

Nature Protocols volume 1, pages 1879–1886 (2006)Cite this article

Abstract

Here we describe a one-step synthetic procedure for the preparation of S-adenosyl-L-methionine (AdoMet) analogs with extended carbon chains replacing the methyl group. These AdoMet analogs function as efficient cofactors for DNA methyltransferases (MTases), and we provide a protocol for sequence-specific transfer of extended side chains from these AdoMet analogs to DNA by DNA MTases. Direct chemoselective allylation or propargylation of S-adenosyl-L-homocysteine (AdoHcy) at sulfur is achieved under the acidic conditions needed to protect other nucleophilic positions in AdoHcy. The unsaturated bonds in β position to the sulfonium center of the resulting AdoMet analogs are designed to stabilize the transition state formed upon DNA MTase-catalyzed nucleophilic attack at the carbon next to the sulfonium center and lead to efficient transfer of the extended side chains to DNA. Using these protocols, sequence-specific functionalized DNA can be obtained within one to two weeks.

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Figure 1
Figure 2: One-step chemical syntheses of doubly activated cofactor analogs 3 and 4 starting from AdoHcy 2.
Figure 3: Sequence-specific modifications of the hemimethylated duplex ODN I·II by the DNA adenine-N6 MTase M.TaqI with the cofactor analogs 3 and 4 (4 used as a 7:3 mixture of diastereoisomers).
Figure 4: Reversed-phase HPLC analysis of enzymatically fragmented duplex ODN IR·II obtained after modification with cofactor analog 3 (trace a; R, CH2CH=CH2) and cofactor analog 4 (used as a 7:3 mixture of diastereoisomers; trace b; R, CH2C≡CCH3) in the presence of M.TaqI.
Figure 5: Principle of the DNA protection assay used to analyze and quantify the enzymatic activities of M.TaqI with the doubly activated cofactor analogs 3 and 4 and the natural cofactor 1.
Figure 6: Gel showing phage λ DNA modified by M.TaqI with the doubly activated cofactor analogs 3 and 4 (4 used as a 7:3 mixture of diastereoisomers) or the natural cofactor AdoMet 1 and investigated by DNA protection.

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Acknowledgements

The authors are grateful to K. Glensk for preparing M.TaqI. C.D. thanks the Deutsche Forschungsgemeinschaft for a stipend within the Graduiertenkolleg 440. G.L. thanks M. Krenevièienë for recording NMR spectra for cofactor analog 4. The M.TaqI expression vector pAGL15-M13 was kindly provided by J. Benner and C. Baxa, New England Biolabs. This work was supported by grants from the VolkswagenStiftung, the Howard Hughes Medical Institute and the Lithuanian Science and Study Foundation.

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Authors and Affiliations

  1. Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, Aachen, D-52056, Germany

    Christian Dalhoff & Elmar Weinhold

  2. Laboratory of Biological DNA Modification, Institute of Biotechnology, V. Graičično 8, Vilnius, LT-02241, Lithuania

    Gražvydas Lukinavičius & Saulius Klimašauskas

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  1. Christian Dalhoff
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Correspondence to Elmar Weinhold.

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We have filed a patent application on the doubly activated AdoMet analogs and made a license agreement with a company. Thus, we are eligible for possible future personal royalties.

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Dalhoff, C., Lukinavičius, G., Klimašauskas, S. et al. Synthesis of S-adenosyl-L-methionine analogs and their use for sequence-specific transalkylation of DNA by methyltransferases. Nat Protoc 1, 1879–1886 (2006). https://doi.org/10.1038/nprot.2006.253

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