Multinuclear Nuclear Magnetic Resonance Spectroscopy Is Used to Determine Rapidly and Accurately the Individual pKa Values of 2-Deoxystreptamine, Neamine, Neomycin, Paromomycin, and Streptomycin

Unambiguous assignments have been made for each individual pKa value of the amino group and guanidine substituents on 2-deoxystreptamine, neamine, neomycin, paromomycin, and streptomycin by pH-titration evaluation of their 1H, 13C, and 15N (by 1H-15N heteronuclear multiple-bond correlation (HMBC) spectra) NMR chemical shifts (δXs) as the reporter nuclei. These data require minor revisions of the literature data in terms of the assignment order for neomycin and paromomycin. In situ titrations and NMR spectroscopy are shown to be a powerful combination for rapidly (minutes) obtaining each distinct pKa value of the similar amine and guanidine functional groups, which decorate aminoglycoside antibiotics.


■ INTRODUCTION
Aminoglycosides are polyamine natural products consisting of amino sugars linked by glycosidic bonds, hence their class name. 1 Potentiometric titration and UV methods have been used to determine ionization constants (pK a ). However, in polyamines, no direct link can be drawn from an individual amino group to a specific pK a value by those techniques. 2 In this study, we determine the individual pK a values of four selected aminoglycoside alkaloids from Streptomyces and Micromonospora by detailed multinuclear nuclear magnetic resonance (NMR) spectroscopy, using 1 H, 13 C, and 15 N as the reporter nuclei. This combination, including 1 H-15 N HMBC spectroscopy, is a rapid and accurate method to gain quantitative data and thereby important molecular structural information.
The first aminoglycoside, streptomycin, was isolated from Streptomyces griseus in 1943 by Waksman. Indeed, Waksman and his research group also isolated the second aminoglycoside, neomycin, from Streptomyces fradiae in 1949 and, in the following years, another naturally occurring aminoglycoside, paromomycin, was discovered. 3−6 Aminoglycosides contain an aminocyclitol, a 1,3-diaminoinositol moiety; 2-deoxystreptamine, as found in neamine, neomycin, and paromomycin; or a streptidine ring as found in streptomycin, linked by glycosidic bonds to sugars, such as L-neosamine, D-neosamine, and Dribose as found in neamine, neomycin, and paromomycin; or to streptose and glucosamine as found in streptomycin. 7−9 Aminoglycosides are primarily used for the treatment of bacterial infection caused by Gram-negative (aerobic) or Gram-positive bacteria. 1,6,10,11 They act on the 16S fragment of ribosomal RNA (rRNA) located in the 30S subunit of the 70S bacterial ribosome, leading to cell death. 12−16 The amino functional group substituents around the different rings, including the similar, but slightly different two monosubstituted guanidines in streptomycin, are key to the biological activities of these natural product alkaloids. Knowing the pK a values of these polyamine alkaloids will afford a better understanding of their structure−activity relationships (SARs) especially the order in which these similar functional groups gain/lose protons, i.e., ammonium ions/free amine bases. Such data will potentially help in understanding the order of target rRNA binding of key basic functional groups.
It is clear that for polyamine molecules, when they are studied in aqueous solutions of different acidity, the 15 N nuclei are the primary pH/pD-dependent NMR detectors, i.e., socalled "NMR probes", while the 1 H and 13 C nuclei are only secondary probes. Hence, the 15 N NMR spectral data are the most important in this case, but unfortunately they are also much more difficult to obtain. This issue has been addressed previously using the methodology of 15 N observation, i.e., the determination of midpoints of 15 N NMR titration curves vs pH, and comparison of the pK a values found classically, by potentiometry or UV spectroscopic methods, 2 with those from the analysis of 15 N NMR data. After Coxon's work on the 15 N NMR spectroscopy of amino sugars, 17 Botto and Coxon reported on neomycin B and related aminoglycosides. 18 Paschal and Dorman reported on the nebramycin aminoglycosides 19 comparing the pK a values derived from 15 N and 13 C NMR spectroscopies, completing the assignment of the 15 N resonances of apramycin. 20 They showed that hydroxylation adjacent to one of the basic nitrogen atoms of apramycin changed the pK a values of all five amino functional groups in the molecule. 20 Serpersu and co-workers have reported solution NMR spectroscopic studies and conformational comparisons between isepamicin (from the kanamycin group of aminoglycosides), butirosin A, and ribostamycin, a neomycin group aminoglycoside. 21,22 They also made a detailed NMR study of neomycin B binding to the aminoglycoside phosphotransferase (3′)-IIIa. 23 Likewise, the order of the pK a values of tobramycin has been studied by 15 N NMR spectroscopy. 16,24 Studying whether aminoglycosides adopt similar conformations when bound to RNA and protein targets may have significant implications in the design of enzyme inhibitors and/or antibiotics. Therefore, we are studying new combinations of multinuclear NMR spectroscopic data as a rapid analytical method to deconvolute accurately the individual pK a data of these polyamines in order to know what chemical structure each molecule has at physiological pH. 16 ■ RESULTS 1. pK a Values of the Individual Amino Groups of 2-Deoxystreptamine. 2-Deoxystreptamine is the central moiety of neamine, neomycin, and paromomycin. It is a cyclohexane ring substituted with two primary amines with a plane of symmetry. As this 4,5-di-O-substituted moiety is central to our aminoglycosides of interest, it was chosen as a model compound on which to determine the accuracy and precision of measuring the pK a values of these polyamine alkaloids in situ using 1 H, 13 C, and 1 H-15 N HMBC NMR spectroscopies. The pK a values of the two individual nitrogen atoms of 2-deoxystreptamine, shown in Figure 1 and Table 1, were extracted from the inflection points of the nonlinear sigmoidal curves (using Prism 7). The error bars in these determinations about the mean of each chemical shift and of the pH meter values are from MS Excel, and the tiny spread in the calculated pK a values, typically ±0.05, is from Prism (n = 3). Using the NMR technique, these errors are, perhaps not surprisingly, small when titrating in situ and then measuring both the chemical shift and the local pD and then converting the latter into pH (pH = pD − 0.5). Here, we chose to follow the IUPAC Technical Report Guidelines of Popov et al., subtracting 0.5 from the measured pD value, 25 rather than subtracting 0.4, 26 or applying the equation pH = 0.929pD + 0.42 determined experimentally by Krezėl and Bal. 27 Typically, these errors are <0.1 ppm in 1 H, 13 C, and 15 N and likewise the differences in pH were <0.1, giving rise to measured pK a values of individual nitrogen atoms with an error typically <0.05 obtained with each of the three chosen nuclei. The effects of temperature on 1 H and 13 C NMR chemical shifts at 25, 35, and 50°C and of dilution were also shown to be minimal, if any, at the typical concentrations used in these NMR studies. After these initial studies (n = 3), all three of the NMR experiments were repeated (n = 2), and it was shown that this gave similarly reproducibly accurate data. Subsequent in situ NMR spectroscopic titrations of aminoglycosides in D 2 O were therefore performed at n = 1 providing accurate data.
Due to the symmetrical structure of 2-deoxystreptamine, the signals of H-1/3, C-1/3, and N-1/3 have the same chemical shifts (δ X ). Therefore, there are two inflections in the H-1/3, C-1/3, and N-1/3 sigmoidal curves. Each inflection point of each curve can be read as a pK a value. These two points are less easily seen in Figure 1A,B, but they are clearly seen using the expanded scale in Figure 1E,F from the reporter nuclei C-1/3 substituted with N-1/3 and from H-1/3 on those carbon atoms shown as NMR titration curves of H-1/3 and C-1/3.
In this work, the amino groups on 2-deoxystreptamine were numbered by comparing them to the 2-deoxystreptamine ring of neamine, neomycin, and paromomycin. The labeling of the symmetrical ring makes sense after a protonation event. In this context, the first pK a is easier to understand. The second less so, but it gives valuable information, which is relevant for the analysis of other molecules in the study. These amino group protons are highly fluctional on the NMR time scale. It is important to observe that the relative direction of travel of the chemical shifts is in the opposite directions from 1 H and 15 N to 13 C. 16 The two pK a values of 2-deoxystreptamine were determined to be 7.00 ± 0.05 and 9.26 ± 0.05.
2. pK a Values of the Individual Amino Groups of Neamine. Neamine has four primary amino group substituents on two rings, one of which is 2-deoxystreptamine, the other is a D-amino sugar on a D-glucose hexose template, Dneosamine. All the pK a determinations using 1 H, 13 C, and 1 H-15 N HMBC NMR spectroscopies were repeated twice. The average values of the chemical shifts of 1 H, 13 C, and 1 H-15 N HMBC of neamine, at different pHs, were plotted against the in situ measured pH values of the solution. The pK a values of each individual nitrogen atom of neamine, shown in Figure 2 and Table 2, were then extracted from the inflection points of the nonlinear sigmoidal curves.
After calculating, the average pK a values, using 1 H, 13 C, and 1 H-15 N HMBC NMR spectroscopic data, for each individual nitrogen atom on neamine are as follows: N-1 = 7.60, N-3 = 6.51, N-2′ = 7.11, and N-6′ = 8.31. The assignment order of the average ionization constants is N-6′ > N-1 > N-2′ > N-3. These pK a values are consistent in magnitude and in assignment order with those reported in the literature. 28 3. pK a Values of the Individual Amino Groups of Neomycin. Neomycin is the most basic aminoglycoside with its six primary amino group substituents on two amino sugar rings, D-neosamine and L-neosamine, and the central cyclohexane ring (a 4,5-O-disubstituted 2-deoxystreptamine). These three six-membered rings are themselves O-substituents pendant from a D-ribose furanose ring. Using the selected reporter nuclei of neomycin, whose 1 H, 13 C, and 1 H-15 N HMBC chemical shifts (ppm) are measured in 99.97% D 2 O at 25°C and pD 2.55 are shown in Figure 3A; we have followed the in situ titrations. The spectra obtained with neomycin are representative of the analytical data we have collected in each aminoglycoside titration experiment, e.g., from pD = 2.55 to 12.30 as a 1 H stack plot ( Figure 3B), the 1 H-15 N HMBC overlay at pD 5.25 (black) and pD 12.12 (red) ( Figure 3C), and 13 C-1 H heteronuclear single quantum correlation (HSQC) overlay at pD 6.12 (black) and pD 12.12 (red) ( Figure 3D). The average (n = 2) of the chemical shifts of 1 H, 13 C, and 1 H-15 N HMBC of neomycin at different pHs were plotted against the pH values of the solution. The pK a values of individual nitrogen atoms of neomycin ( Figure 3 and Table 3) were then extracted from the inflection points of the nonlinear   Table 4) were then extracted from the inflection points of the nonlinear sigmoidal curves.
The average pK a values, calculated using 1 H, 13 C, and 1 H-15 N HMBC NMR spectroscopic data, for each individual nitrogen atom on paromomycin are as follows: N-1 = 8.11, N-3 = 6.50, N-2′ = 8.06, N-2″' = 8.10, and N-6″' = 9.08, the new  pK a values of individual nitrogen atoms of neamine determined using 1 H NMR spectroscopy in D 2 O relative to the HDO peak at δ = 4.8 ppm, at 25°C. 28 b This work (n = 2). c The pK a value of N-6′ of neamine determined using 1 H NMR spectroscopy (in this work) is the average pK a of the values of N-6′ obtained using 1 H NMR spectroscopic data for H-6′a (8.33) and 6′b (8.35  5. pK a Values of the Individual Amino Groups of Streptomycin. Streptomycin has seven nitrogen atoms distributed within two guanidine groups on streptidine and secondary N-methylamine on an N-methyl-1-glucosamine ring. The streptose ring with its six-carbon atoms is a furanose, not a pyranose, and it is not a linear sugar with its methyl and aldehyde functional groups. The chemical shifts of 1 H, 13 Table 5) were extracted from the inflection points of the nonlinear sigmoidal curves.
The three average pK a values after calculating using 1 H, 13 C, and 1 H-15 N HMBC NMR spectroscopic data of the two guanidine groups (N-1 and N-3) and the secondary amine (Nmethyl, N-2′) on streptomycin are as follows: N-1 (guanidine) = 13.06, N-3 (guanidine) = 12.06, and N-2′ = 8. 16. The assignment order of the average (δ x ) ionization constants is as follows: N-1 > N-3 > N-2′. These pK a values are consistent in the assignment order with those reported in the literature. 32

■ DISCUSSION
The nitrogen atoms are flipping between protonated and unprotonated states, so they are sensitive to the pH of the environment and will naturally shift significantly in the NMR experiment. For carbon atoms, we are primarily detecting shifts on carbon atoms that are attached by one bond to a nitrogen atom that is either protonated or not. The NMR shifts seen are explained by inductive effects (the increased charge at the nitrogen atom causes a change in the carbon−nitrogen bond), but note that this is not simply the intuitive inductive effect, as the shift for 13 C goes in the "wrong" direction, as discussed previously. 16 We still see the shifts on titration in the proton chemical shifts that are two bonds away from the affected nitrogen atoms. In this case, the inductive effect matches our expectation and can be explained akin to swapping an electronegative atom for a proton in a simple alkane. Furthermore, the magnitude of the shift observed in titration also reflects the relative parts per million scale range ( 1 H = 20 ppm, 13 C = 200 ppm, and 15 N = 100 ppm).
The short total time taken for the experiment in this pK a by the in situ titration NMR spectroscopic method depends on the concentration. The accuracy and resolution depend upon having suitable signals present to monitor and follow in that titration. It is ideal to use well-resolved 1 H spectra right across the pH range under investigation, although it is possible to follow individual peaks as they move during the titration. If 13 C or 15 N data are needed, then samples will need to be of reasonable concentrations, more so for 15 N data, even though both HSQC and HMBC are 1 H-detected NMR spectroscopic experiments. While detecting 1 H alone is rapid down to concentrations of a few millimolar, this will not be true for 13 C or 15 N. However, for molecules of low molecular weight (perhaps under 500 Da), a few milligrams of the sample in a standard 5 mm NMR tube, typically in 0.4 mL of solvent, will give suitable 1 H-13 C HSQC and 1 H-15 N HMBC data to allow a pK a to be determined in only a few hours. At such concentrations, the 1 H measurement only takes minutes; indeed, titrating (adjusting) and then measuring the pH is the rate-limiting step that can still be achieved in minutes. Typically, the 1 H spectra are obtained in 1−2 min per data point (assuming either 8 or 16 scans per spectrum). For 1 H-13 C HSQC data, the standard experimental setup is ns = 2 with the number of increments as td = 256. This gives an acquisition time of ∼20 min per data point, although this can be reduced (e.g., ns = 1 and td = 128 would require ∼5 mins, but with reduced data quality). Similarly, the 1 H-15 N HMBC experiment uses ns = 8 and td = 128 requiring ∼40 min per data point. However, improvements in modern NMR spectrometers are routinely reducing the time needed for such experiments, with advances such as nonuniform sampling (NUS) and parallel acquisition (acquiring more than one data set simultaneously). Broadly, we have shown that the experimental pK a value derived from each of the three nuclei is similar, and thus if only 1 H can be used spectroscopically, that will suffice to provide accurate pK a values.
The chemical shifts corresponding to the H-1/3 of 2deoxystreptamine when measured at 0.630 M and 0.157 M, at low pD (∼2), did not shift with the change in concentration. Also, pK a data determined over a 1000-fold dilution using a sample of neomycin (218 and 0.200 mM) at four pH values were not significantly different when measured by 1 H NMR spectroscopy ( Figure 3E and Table 3). Likewise, there was no significant difference studying dissociation constants in D 2 O instead of pure H 2 O ( Figure 3F). Furthermore, NMR experiments at 25, 35, and 50°C measuring the proton and carbon chemical shifts gave constant, not temperature-  30 e This work (n = 2). f The pK a value of N-6′ of neomycin determined using 1 H NMR spectroscopy (in this work) is the average pK a of the values of N-6′ obtained using 1 H NMR spectroscopic data for 6′a (8.65) and 6′b (8.70), and the pK a value of N-6‴ of neomycin determined using 1 H NMR spectroscopy (in this work) is the average pK a of the values of N-6‴ obtained using 1 H NMR spectroscopic data for 6‴a (8.72) and 6‴b (8.75).

ACS Omega
http://pubs.acs.org/journal/acsodf Article dependent, responses. Therefore, we concluded that the pK a values of these aminoglycosides are not affected by changes in concentration or in the temperature at these typical NMR experiment ranges. The measurement of the individual ionization constants of polybasic compounds is known to be complex when the separate functional groups have similar constants. 33 These problems are particularly acute when measuring, e.g., in streptomycin, the basicity of two different monoalkyl guanidines, the most basic functional group in medicinal chemistry. This study is a continuation of our pK a investigations by multinuclear NMR spectroscopy on other aminoglycosides, tobramycin, kanamycin, amikacin, sisomicin, and netilmicin, recently published in this Journal. 16 The correct assignment of the pK a values of amines of an aminoglycoside is important for the characterization of the binding mode of those antibiotics to the RNA. pK a determination by the NMR spectroscopy method has also been studied in significant detail by the Hagele research group for other functional groups, including different organophosphorus compounds, aminophosphonates, and phosphinic acids, whilst not for aminoglycosides. 34−36 Using potentiometric titrations, the pK a values of neamine have been measured: pK a 1, pK a 2, and pK a 3 are 6.35 ± 0.2, 7.73 ± 0.15, and 8.62 ± 0.08, respectively, but care must be taken in determining thermodynamic properties. 37 Note that for the four amines, the potentiometric method could only resolve three values, presumably N-2′ is overlapping. The dependence of the solution structure of neamine on pH has also been determined by NMR and AMBER molecular dynamics analyses at pD 3.3, 6.5, and 7.4 in D 2 O at 25°C. In acid, it essentially showed only one conformer. 28 The pK a values determined by NMR titration experiments are as follows: pK a 1 = 6.44 ± 0.13 for N-3 of ring-II, pK a 2 = 7.23 ± 0.09 for N-2′ of ring-I, pK a 3 = 7.77 ± 0.19 for N-1 of ring-II, and pK a 4 = 8.08 ± 0.15 for N-6′ of ring-I. 28 In this work, using δ x , we determined the pK a values of neamine to be N-3 = 6.51, N-2′ = 7.11, N-1 = 7.60, and N-6′ = 8.31. Therefore, neamine exists as a mixture of tetra−/tri−/diprotonated species between pD 4.5 and 7.4, but it really is only a diprotonated species around physiological pDs. This may facilitate the binding of neaminelike aminoglycosides by favorable entropy of binding to the Asite of 16S rRNA, suggesting that novel aminoglycoside compounds carrying such a neamine-based pharmacophore  13 In a good example of the interplay between sterics and electronics, in the binding of streptomycin derivatives to nucleotides, the electrostatic forces are not always the dominant factor; the conformational fit is also important. 38 Westhof and co-workers have clearly established the importance of structural and physicochemical parameters, e.g., pK a , in aminoglycoside−rRNA interactions. 13,39 Not that every amino group must be (fully) charged, but that in such complexes the number of rings and hence the positions of  29 c This work (n = 1). d The pK a value of N-6‴ of paromomycin determined using 1 H NMR spectroscopy (in this work) is the average pK a of the values of N-6‴ obtained using 1 H NMR spectroscopic data for 6‴a (9.14) and 6‴b (9.16).

ACS Omega
http://pubs.acs.org/journal/acsodf Article positive charges promotes specific binding. Indeed, unlike in the example of streptomycin, 38 the strength of the interaction of neomycin with RNA is dominated by electrostatics, with the positively charged aminoglycoside displacing metal ions. 27 Asensio and co-workers have reported 30 that a complete characterization of the protonation equilibrium that accompanies the molecular recognition of neomycin B by a specific 23-mer RNA receptor has been achieved by employing a simple NMR analysis. There were dramatic alterations in the neomycin B amino functional group protonation state upon aptamer binding measured using uniformly enriched 15 Nneomycin. 30 Some values rose by ∼1 pK a unit, one only by 0.3, and the pK a of N6‴ remained unaltered. The formation of a neomycin−phosphotransferase enzyme complex was determined by isothermal titration calorimetry (ITC). The enthalpy of binding became more favorable (negative) at a higher pH where binding-linked protonation was attributable mostly to the amino groups. Multiple interactions may affect the affinity of the ligand for the enzyme and changes in pH will affect this. Therefore, it remains important to determine the thermodynamic parameters of aminoglycoside−target interactions under different experimental conditions before making attributions to specific sites and their effects on these global parameters. 28 Neomycin is the most basic aminoglycoside with its six primary amino groups. A primary alcohol replaces the N6′ primary amine (ammonium ion) in paromomycin whose X-ray crystal structure docked into the eubacterial ribosomal decoding A-site has been reported by Vicens and Westhof. 40 In this work, using δ X , the average pK a s of neomycin are shown to be N-3 = 6.86, N-2′ = 7.98, N-2‴ = 8.03, N-1 = 8.08, N-6′ = 8.65, and N-6‴ = 8.76. Likewise, the average pK a s of paromomycin are N-3 = 6.50, N-2′ = 8.06, N-2‴ = 8.10, N-1 = 8.11, and N-6‴ = 9.08. Significantly, in terms of the total number of positive charges, the number of aminoglycoside− RNA contacts is about the same in neomycin and paromomycin. 13 Other X-ray crystal structures of aminoglyco-side complexes and the decoding A-site oligonucleotides serve to emphasize the roles of the number of rings and the positive charges in the specific binding leading to miscoding. 13 Barbieri and Pilch have reported a complete thermodynamic characterization of the multiple protonation equilibria of paromomycin as its binding to different macromolecular targets is coupled to selective protonation of its five amino functional groups. 31 ITC studies conducted at a drug concentration of 45 mM revealed that the extent of paromomycin protonation showed that the binding of the drug to its pharmacologically relevant target, the 16S rRNA A-site, is consistent with the pK a values of the free base. With their co-workers, they concluded that it is important to study the protonation equilibria of the aminoglycoside amino groups as well as any concentration/ionic strength or temperature dependence, physicochemical analysis being a critical component of any drug design strategy. 29,31 The individual ionization constants, the pK a values of the individual amino groups of 2-deoxystreptamine, neamine, neomycin, paromomycin, and streptomycin, were therefore determined using chemical shift variation with 1 H, 13 C, and 1 H-15 N HMBC NMR spectroscopies (Table 6). This was experimentally verified to be a rapid, accurate, precise, and reproducible technique. 1 H NMR spectroscopy was the least time consuming (2 min for each data point), then 13 C (30 min for each data point), and then 1 H-15 N HMBC (∼45 min for each data point) NMR spectroscopy. This technique is both accurate (typically, errors of ±0.1 ppm for each nucleus gave rise to measured pK a values of ±0.05) and precise as followed from the small error bars and the results tallying with those obtained in other laboratories and by potentiometric methods. Also, experimentally determined was the reproducibility where the error bars were typically smaller than the (typical, small) symbols used in plotting the curves; again, this is shown to be accurate for each of the three nuclei. This allowed n = 3 to be reduced to n = 1 with no loss of accuracy across a range of concentrations (see Experimental Section, Figure 3E and Table   Table 6. Average pK a Values of Individual Nitrogen Atoms of the Indicated Aminoglycosides Using 1 H, 13 13 C, and 50.67 MHz for 15 N) spectrometer at 25°C (unless otherwise indicated). MestReNova and Bruker Topspin have been used for processing the spectra. 1 H and 13 C chemical shifts (δ x ) were observed and are reported in parts per million (ppm) relative to sodium trimethylsilylpropanoate (TMSP) at 0.00 ppm as an internal reference, and 1 H-15 N HMBC chemical shifts were measured relative to external nitromethane (CH 3 NO 2 in CDCl 3 (1:1, v/v)), recorded, and set at δ N 379.8 ppm, and the sr value (correction factor) is measured as −511.72 on our spectrometer. 42 The recording time differed for each isotope as follows: 2, 30, and ∼45 min per data point for 1 H, 13   The pH values were adjusted using 0.5 M NaOD and 0.5 M DCl. MestReNova and Bruker Topspin were used for the analysis of the recorded spectra. 1 H and 13 C NMR chemical shifts and 1 H-15 N HMBC spectra of each aminoglycoside were obtained in 99.97% D 2 O at 25°C starting, as set out above, in 0.4 mL in a 5 mm diameter NMR tube; then, data were collected at varying pH values diluted by titration with 0.5 M NaOD to a final volume of 0.6 mL. Both the change in solvent column height above the Bruker probe and the dilution makes essentially no difference to the quality of data collected. The transformed data were then plotted against the in situ pH values. The nonlinear sigmoidal curve and the inflection point of the sigmoidal curve were determined using GraphPad Prism 7 (version 2017), after the subtraction of 0.5 (following the IUPAC Technical Report Guidelines) in order to convert the measured pD values into pH values. 25 The individual pK a values of each basic functional group on each aminoglycoside are these inflection points.
The data obtained from these 1 H, 13 C, and 1 H-15 N HMBC NMR spectroscopic experiments were determined to be precisely reproducible by repetitions (n = 3) starting with the simple symmetrical diamine, 2-deoxystreptamine, as a model compound. Similarly, each of the different NMR experiments was then repeated for neomycin (n = 2) to calculate the errors in the measurement of the following: pH values, chemical shifts (δ), and pK a values. The majority of the error bars for the pH values and for the chemical shifts were of a similar size as the (small, typical) symbols used to plot the points on the nonlinear sigmoidal curves. Typically, pK a values were accurate to <±0.1 and sometimes even down to ±0.02. Therefore, having determined by experiment that the typical size of the errors is small, it was judged that n = 1 was sufficient for obtaining further NMR spectroscopic data for each of paromomycin and streptomycin.

Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
We thank the Government of the Kingdom of Saudi Arabia for fully funding this studentship to A.H.A. NMR equipment was provided by the Material and Chemical Characterization Facility at the University of Bath. We also acknowledge with gratitude the careful reading of a referee who alerted us to our omission 16 that the external nitromethane reference standard was recorded and set at δ N = 379.8 ppm, 42 and the measured sr correction value was −511.72 on our spectrometer.