New Insights into the Binding Site and Affinity of the Interaction between Biotin and PAMAMs-NH₂ via NMR Studies

Abstract

Biotin–dendrimer conjugates (such as biotin-PAMAMs-NH2) are important macromolecules in the field of host–guest chemistry and widely used systems for delivery. The similar chemical structures of the inner and outer layers of PAMAM-NH2 make it difficult to illuminate the interaction and the binding affinity of biotin-PAMAMs-NH2. By utilizing NMR techniques including 1H NMR titration, CSSF-TOCSY, STDD methods, and 2D DOSY analysis, we demonstrate a method to sort out these interactions. The methylene protons of the inner and outer layers of PAMAM-NH2 are successfully identified and accurately positioned so that the carboxylic acid groups of biotins are having ionic interactions with the outermost amine groups of PAMAM-NH2. The inner PAMAM-NH2 is protonated when reaching the isoelectric point of PAMAM-NH2, increasing the hydrodynamic radius. On the basis of the NMR experiments, a model is proposed, where the carboxylic acid groups and heterocyclic skeleton of biotin arched over the outer layers of PAMAM-NH2 like a bridge. Furthermore, using STDD epitope mapping, the binding affinity between biotin and PAMAM-NH2 was quantified. The diffusion behavior of biotin-G5 PAMAM-NH2 complex is more complicated than that of biotin-G3 PAMAM-NH2 complex due to steric hindrance. The results provide a theoretical basis for understanding these complicated drug delivery systems.

Effect of Ionization Degree of Poly(amidoamine) Dendrimer and 5-Fluorouracil on the Efficiency of Complex Formation—A Theoretical and Experimental Approach

“…As can be seen, the spectrum of dendrimer exhibited six broad bands centered at 2.37, 2.57, 2.68, 2.76, 3.19, and 3.24 ppm. The chemical shift attribution was made based on the literature [37]. Upon the addition of 5FU, discernible proton chemical signals of G4PAMAM were strongly influenced by the presence of the drug.…”

Interactions between PAMAM−NH2 and 6‐Mercaptopurine: Qualitative and Quantitative NMR studies

“…The change of all PAMAMÀ NH 2 protons is probably because the pH in the solution is lower than the isoelectric point and the inner layer of PAMAMÀ NH 2 is protonated by the hydrogen ion. [15] STD and STDD analysis STD NMR can be used to study low affinity binding between host and guest. [20a, 26] Saturation transfer identifies specific binding from guest to host at the molecular level.…”

“…Because STD NMR detects only the bound ligand fraction, the presence of excess ligand is beneficial as this maximizes the number of ligand-receptor interactions and results in increased sensitivity in the NMR experiment. [15,26] To study the binding of 6-MP to PAMAMÀ NH 2 a molar ratio of 80 : 1 was chosen for the STD analysis. The obtained STD spectrum is presented in Figure 5.…”

“…Dendrimers can be functionalized with a large number of chemical versatile surface groups, but the interior remains relative nonpolar with nanoscale cavities. [11] It has been reported that PAMAMÀ NH 2 is used as carrier for different anticancer drugs such as 5-fluorouracil, [12] cisplatin, [13] doxorubicin, [14] methotrexate, [4b] biotin, [15] phenobarbital, [16] sulfamethoxazole [17] and capecitabine. [18] Although it has been reported that PAMAM is a drug carrier capable to encapsulate 6-MP and hence improve the water solubility, [3] the detailed information about the molecular interaction mechanisms and the binding site of 6-MP with PAMAMÀ NH 2 are still not clear.…”

“…[12] Recently, we explored the interaction mechanism of biotin and PAMAMÀ NH 2 by NMR, this work identified new structural difference between the inner and outer layers in dendrimers at atomic-level. [15] Herein, we report the interaction between PAMAMÀ NH 2 and 6-MP by using various 1D and 2D NMR techniques including 1 H and 13 C chemical shift titrations, diffusion coefficient determinations, nuclear Overhauser effect spectroscopy ( 1 H-1 H NOESY), relaxation time measurements, Saturation Transfer Difference (STD) spectroscopy and Saturation Transfer Double Difference (STDD). The combination of these techniques enabled the qualitative and quantitative study of the interaction between 6-MP and PAMAMÀ NH 2 .…”