Binding of the 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs to tRNA(phe.)

Abstract

Background: Three new analogs of berberine with aryl/ arylalkyl amino carbonyl methyl substituent at the 9-position of the isoquinoline chromophore along with berberrubine were studied for their binding to tRNA(phe) by wide variety of biophysical techniques like spectrophotometry, spectrofluorimetry, circular dichroism, thermal melting, viscosity and isothermal titration calorimetry.

Methodology/ principal findings: Scatchard binding isotherms revealed that the cooperative binding mode of berberine was propagated in the analogs also. Thermal melting studies showed that all the 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs stabilized the tRNA(phe) more in comparison to berberine. Circular dichroism studies showed that these analogs perturbed the structure of tRNA(phe) more in comparison to berberine. Ferrocyanide quenching studies and viscosity results proved the intercalative binding mode of these analogs into the helical organization of tRNA(phe). The binding was entropy driven for the analogs in sharp contrast to the enthalpy driven binding of berberine. The introduction of the aryl/arylalkyl amino carbonyl methyl substituent at the 9-position thus switched the enthalpy driven binding of berberine to entropy dominated binding. Salt and temperature dependent calorimetric studies established the involvement of multiple weak noncovalent interactions in the binding process.

Conclusions/ significance: The results showed that 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs exhibited almost ten folds higher binding affinity to tRNA(phe) compared to berberine whereas the binding of berberrubine was dramatically reduced by about twenty fold in comparison to berberine. The spacer length of the substitution at the 9-position of the isoquinoline chromophore appears to be critical in modulating the binding affinities towards tRNA(phe).

Figure 1. Chemical structures.

(A) cloverleaf structure of tRNA, (B) berberine, (C) berberrubine and (D) analogs B2, B3, B4.

Figure 2. Absorption and fluorescence spectral studies of the analogs with tRNA.

(A) Absorption spectral changes of analog B4 (curve 1) with increasing concentration of tRNA (curves 2–8), (B) fluorescence spectral changes of analog B4 (curve 1) with increasing concentration of tRNA (curves 2–8) (C) Scatchard plot for analog B4-tRNA complexation from absorbance and (D) Scatchard plot for analog B4-tRNA complexation from fluorescence.

Figure 3. Job plot for the binding of the analogs with tRNA.

(A) analog B3 and (B) analog B4.

Figure 4. Stern-Volmer plots for the quenching of fluorescence by increasing concentration of [Fe(CN)₆]⁴⁻.

(A) analog B3 and (B) analog B4. Symbols are in the absence (▪) and in the presence (•) of tRNA.

Figure 5. Circular dichroism spectra of the alkaloid-tRNA complexes.

tRNA (50 µM) treated with increasing concentrations of analogs (A) B2 and (B) B4.

Figure 6. Representative ITC profiles for the complexation of the berberine analogs to tRNA.

The profiles shown represent the sequential titration of successive aliquots of analogs (A) B2 and (B) B4 to tRNA (curve at the bottom), along with the dilution profiles (curves on the top offset for clarity). The top panel represents the raw data and the bottom panel shows the integrated heat data after correction of the heat of dilution. The symbols (▪) represent the data points that were fitted to a one-site model and the solid lines represent the best-fit data.

Figure 7. Plots of variation of salt dependent thermodynamic parameters.

(A) Plot of log K_a versus log [Na⁺] for the binding of analogs B2 (▪), B3 (•) and B4 (▴) to tRNA and (B) partitioned polyelectrolytic (ΔG⁰_pe) (shaded) and nonpolyelectrolytic (ΔG⁰_t) (black) contributions to the Gibbs energy of the B4-tRNA complexation at different [Na⁺] concentrations.

Figure 8. Plots of variation of temperature dependent thermodynamic parameters.

(A) Plot of variation of enthalpy of binding (ΔH⁰) with temperature for binding of analog B2 (▪), B3 (•) and B4 (▴) to tRNA. (B) Plot of variation of ΔG⁰ (open symbols) and ΔH⁰ (closed symbols) versus TΔS⁰ for the binding of analogs B2 (▪), B3 (•) and B4 (▴) to tRNA, respectively.