Purpose: The amino functional group substituents around the different rings of aminoglycoside antibiotics are key to the biological activities of these clinically important natural product alkaloids. The ionisation constant (pKa) is the pH at which functional groups are 50% ionized. The pKa values of any medication play a significant role in the physicochemical data as they are relevant to drug activity. This study is on determining individual pKa values by detailed Nuclear Magnetic Resonance (NMR) spectroscopy of selected aminoglycoside alkaloids from Streptomyces and Micromonospora. In order to determine the individual pKa values, and such resolution is not available by potentiometric methods, different NMR reporter nuclei have been employed. Studying the pKa values of these alkaloids will afford a better understanding of the order in which these similar functional groups gain/lose protons, data of immediate relevance to their structure-activity relationships (SAR). Such data will potentially help in understanding the order of target mRNA binding of key basic functional groups. The aim is to measure pKa values of individual amines on aminoglycosides by using new combinations of 1H, 13C, and 15N NMR spectroscopic data.
Methods: Aminoglycoside analyte solutions (0.73-0.15 M aminoglycoside in 99.97% D2O) were prepared at a ~10 mg/mL concentration. NMR spectra including 1H, 13C, HSQC, HMBC, NOESY, and 15N HMBC were recorded on Bruker Avance III 400 and 500 MHz spectrometers. Trimethylsilylpropanoic acid (TMSP) was used as a reference for 1H and 13C NMR spectroscopy. 15N chemical shifts were measured relative to external CH3NO2 set at -511.72 ppm. The pH values were adjusted using 0.5 M NaOD/DCl in D2O. MestReNova was used for analysis of the recorded spectra. The nonlinear sigmoidal curve and the inflection point of the sigmoidal curve were determined using GraphPad Prism 7 (Version 2017), after subtraction of 0.5 to convert the measured pD values into pH values.
Results: The pKa values of 1-NH2 and 3-NH2 of 2-deoxystreptamine are 9.14 and 7.05. The order of ionisation constants for neamine is: N-6' (8.32) > N-1 (7.60) > N-2' (7.20) > N-3 (6.50), for neomycin is:
N-6''' (8.78) ≈ N-6' (8.70) > N-1 (8.05) ≈ N-2' (8.15) ≈ N-2''' (8.00) > N-3 (6.40), for tobramycin is:
N-6' (9.10) > N-1 (7.50) ≈ N-2' (7.75) ≈ N-3'' (7.68) > N-3 (6.70), and for sisomicin is: N-6' (9.30) > N-3'' (8.50) > N-2' (8.04) > N-1 (7.42) > N-3.
Conclusion: 1H, 13C, and 15N NMR spectroscopy is a powerful tool for the measurement of individual pKa values. Moreover, because of its sensitivity, 1H NMR spectroscopy is less time consuming (2 min for each sample) than 13C (30 min for each sample) and 15N HMBC (45 min for each sample) NMR spectroscopy. Unambiguous assignments have been made for each individual amine substituent on these clinically important aminoglycoside antibiotics.
We thank the Government of the Kingdom of Saudi Arabia for fully funding this studentship.