Imino protons

Fig. 52. Suggested base-pairing scheme for metal derivatized M-DNA where the imino protons of N3-T and Nl-G are replaced by a divalent metal ion. Adapted from Ref. (177).

Protein-DNA complexes are usually formed by adding small portions of the protein solution to the DNA. The formation of the complex is best monitored by observing the changes of the DNA imino proton signals in a ID spectrum after each step. A reversed approach, i.e. adding the DNA to a protein solution, is not recommended because of the formation of higher order complexes by nonselective binding of protein to DNA. 

D or 3D HCCNH-TOCSY experiment that correlates the imino proton with H6/8, which can be independently assigned from other experiments. 

The well-resolved imino proton resonances from base-paired uracil and guanine resonances serve as powerful probes to characterize RNA-ligand interaction by NMR. The chemical shift of imino proton resonances is responsive to perturbations in the local chemical environment that result from RNA conformational... 

Figure 5.4. Titration of A-site oligonucleotide with paromomycin. Titration of the A-site oligonucleotide with paromomycin yields a specific 1 1 complex. Imino proton resonances for U1490 and G1491, which shift significantly upon binding of paromomycin, are labeled. The intermediate point in the titration demonstrates the appearance of new peaks from the bound form of the RNA.

Figure 5-51 (A) The low-field region of the one-dimensional H NMR spectrum of E. coli tRNAjVal at 27°C in H20. Resonances are identified by letters A - X. (B) NOESY spectrum of the same tRNA under similar conditions showing the imino-imino NOEs. In the lower right sector the connectivity traces of the acceptor helix and dihydrouridine helix are shown as solid and dotted lines, respectively. In the NOESY sample the two protons in peak EF are partially resolved whereas the two protons in peak T have coalesced. (C) NOESY spectrum of E. coli tRNA,Val at 32°C showing the imino and aromatic proton regions. AU-type imino protons have been connected horizontally by a dotted line to the cross-peak of their proximal C2-H or C8-H in the 7 to 9 ppm region, which has been labeled with the corresponding lower-case letter. From Hare et al.669 Courtesy of Brian Reid.

Since neither the 2V-methyl nor the 2V-benzyl derivative of D-mannose phenylhydrazone showed a color change or the presence of paramagnetic species following treatment with base, it was concluded that the colored radical anion from D-mannose phenylhydrazone must arise following the abstraction of the acidic imino proton by base. Thus, this must be a first reaction step before fission of the phenylhydrazine moiety can occur. 

Since the inosose phenylhydrazones could be acylated even in the presence of the strong base and isolated, it was concluded that the radical anions must arise following the abstraction of an acidic imino proton of the phenylhydrazone group by base. On interaction with oxygen the resulting anions (by an electron-transfer mechanism (29)) give... 

Observation of the imino-proton resonances - albeit somewhat shifted and broadening at low temperatures - of the central G C base pairs, indicates that hydrogen bonding (Watson-Crick type or related) is still possible after platination. 

Replacement of the proton at N1 of G by a metal ion has qualitatively similar consequences as substitution of the imino proton of T and U. The Pt(II) binding to this site increases the basicity of the purine ring, but it is not clear how the residual electron density is distributed. The N1 platinated guanine bases titrate with a pA a of 5 (176, 177), and from the high pD dependence of the H8 resonances in the H NMR spectra it is concluded that the most likely protonation site is N7 (cf. Fig. 16). In its protonated state, the nucleobase represents again a metal-stabilized rare tautomer. 

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