Chemo‐Enzymatic Synthesis of Position‐Specifically Modified RNA for Biophysical Studies including Light Control and NMR Spectroscopy

The investigation of non‐coding RNAs requires RNAs containing modifications at every possible position within the oligonucleotide. Here, we present the chemo‐enzymatic RNA synthesis containing photoactivatable or ¹³C,¹⁵N‐labelled nucleosides. All four ribonucleotides containing ortho‐nitrophenylethyl (NPE) photocages, photoswitchable azobenzene C‐nucleotides and ¹³C,¹⁵N‐labelled nucleotides were incorporated position‐specifically in high yields. We applied this approach for the synthesis of light‐inducible 2′dG‐sensing riboswitch variants and detected ligand‐induced structural reorganization upon irradiation by NMR spectroscopy. This chemo‐enzymatic method opens the possibility to incorporate a wide range of modifications at any desired position of RNAs of any lengths beyond the limits of solid‐phase synthesis.     

Light regulation of aptamer activity: an anti-thrombin aptamer with caged thymidine nucleobases

"Caged" derivatives of a 15 nucleotide ssDNA anti-thrombin aptamer have been synthesized in which thymidine nucleotides are modified with photolabile protecting groups. One caged thymidine in a key location is enough to completely mask the aptamer's function in respect to their affinity for thrombin and their inhibition of the blood clotting cascade. With light (366 nm) the caging group can be removed, yielding the unmodified active aptamer.     

Prediction of the three-dimensional structure of the enzymatic domain of t-PA

Summary Tissue plasminogen activator (t-PA), an enzyme of the fibrinolytic system, is responsible for lysis of fibrin via activation of plasminogen, and therefore for degradation of blood clots. There are currently no X-ray crystal structure data of the t-PA molecule available either in whole or in part. We therefore predicted the three-dimensional structure of the protease domain by means of computer-graphical methods.The model obtained forms a basis for understanding the binding of plasminogen to the active site of t-PA. In addition, the interactions of various inhibitors with t-PA were studied by modeling them into the active site. The model also yields an explanation for the observed amidolytic activity of t-PA in the single chain form.     

Synthesis via pummerer intermediates. II.. Disproportionation reactions of 3-(methylsulfinyl)chromones and 3-(methyl-sulfinyl)quinolinones with hydrochloric acid

Sulfoxides ( 1 and 10 ) gave oxidation-reduction products when treated with 5N hydrochloric acid. 8-Methoxy-3-(methylsulfinyl)-4H-benzopyran-4-one (1) gave 8-methoxy-3-(methylthio)-4H-1-benisopyran-4-one ( 4 ) and 8-methoxy-3-(methylsulfonyl)-4H-1-benzopyran-4-one ( 5 ), whereas 1-melhyl-3-(methylsulfinyl)-4(1H)quinolinone ( 10 ) gave 1-methyl-3-(methylthio)-4(1H)-quinolinone ( 12 ) and 1-methyl-4(1H)-quinolinone ( 13 ).     

Synthesis via pummerer intermediates. III. Rearrangements of 3-(methylsulfinyl)quinolinones, 3-(methylsulfinyl)cinnolinones, 3-(methylsulfinyl)chromanones and 3-(methylsulfinyl)chromones with acetic anhydride and with thionyl chloride

The reaction of 1-methyl-3-(methylsulfinyl)-4(1H)quinolinone ( 1 ) with acetic anhydride and thionyl chloride gave 3-[[(acetyloxy)methyl]thio]]-1-methyl-4(1H)quinolinone ( 2 ) and 3-[(chloromethyl)thio]-1-methyl-4(1H)quinolinone ( 3 ) respectively. 3-(Methylsulfinyl)-4(1H)cinnolinone ( 4 ) gave the corresponding products when treated under similar conditions. Treatment of 8-methoxy-3-(methylsulfinyl)-4H-1-benzopyran-4-one ( 11 ) with acetic anhydride and thionyl chloride gave bis addition vinyl Pummerer products 2,3-bis(acetyloxy)-2,3-dihydro-8-methoxy-3-(methylthio)-4H-1-benzopyran-4-one ( 12 ) and 2,3-dichloro-2,3-dihydro-8-methoxy-3-(methylthio)-4H-1-benzopyran-4-one ( 13 ), respectively.     

Inactivation of Competitive Decay Channels Leads to Enhanced Coumarin Photochemistry

In the development of photolabile protecting groups, it is of high interest to selectively modify photochemical properties with structural changes as simple as possible. In this work, knowledge of fluorophore optimization was adopted and used to design new coumarin- based photocages. Photolysis efficiency was selectively modulated by inactivating competitive decay channels, such as twisted intramolecular charge transfer (TICT) or hydrogen-bonding, and the photolytic release of the neurotransmitter serotonin was demonstrated. Structural modifications inspired by the fluorophore ATTO 390 led to a significant increase in the uncaging cross section that can be further improved by the simple addition of a double bond. Ultrafast transient absorption spectroscopy gave insights into the underlying solvent-dependent photophysical dynamics. The chromophores presented here are excellently suited as new photocages in the visible wavelength range due to their simple synthesis and their superior photochemical properties.     

Green-Light Activatable BODIPY and Coumarin 5’-Caps for Oligonucleotide Photocaging

We synthesized two green-light activatable 5’-caps for oligonucleotides based on the BODIPY and coumarin scaffold. Both bear an alkyne functionality allowing their use in numerous biological applications. They were successfully incorporated in oligonucleotides via solid-phase synthesis. Copper-catalyzed alkyne-azide cycloaddition (CuAAC) using a bisazide photo-tether gave cyclic oligonucleotides that could be relinearized by activation with green light and were shown to exhibit high stability against exonucleases. Chemical ligation as another example for bioconjugation yielded oligonucleotides with an internal strand break site. Irradiation at 530 nm or 565 nm resulted in complete photolysis of both caging groups.