Applications of heteronuclear NMR spectroscopy in biological and medicinal inorganic chemistry

Abstract

There is a wide range of potential applications of inorganic compounds, and metal coordination complexes in particular, in medicine but progress is hampered by a lack of methods to study their speciation. The biological activity of metal complexes is determined by the metal itself, its oxidation state, the types and number of coordinated ligands and their strength of binding, the geometry of the complex, redox potential and ligand exchange rates. For organic drugs a variety of readily observed spin I = 1/2 nuclei can be used (¹H, ¹³C, ¹⁵N, ¹⁹F, ³¹P), but only a few metals fall into this category. Most are quadrupolar nuclei giving rise to broad lines with low detection sensitivity (for biological systems). However we show that, in some cases, heteronuclear NMR studies can provide new insights into the biological and medicinal chemistry of a range of elements and these data will stimulate further advances in this area.

Keywords: ADP, adenosine diphosphate; AES, atomic emission spectroscopy; AMP, adenosine monophosphate; ATP, adenosine triphosphate; BNCT, boron neutron capture therapy; BPG, 2,3-bisphosphoglycerate; BSA, bovine serum albumin; BSH, sodium borocaptate; Bioinorganic chemistry; Biological systems; DNA, deoxyribonucleic acid; EDTA-N4, ethylenediaminetetraacetamide; EFG, electric field gradient; GMP, guanosine monophosphate; HMQC, heteronuclear multiple quantum correlation; Heteronuclear NMR spectroscopy; Im, imidazole; In, indazole; MQF, multiple quantum filtered; MRI, magnetic resonance imaging; Medicinal inorganic chemistry; Metallopharmaceuticals; NOE, nuclear Overhauser effect; PET, positron emission tomography; Quadrupolar nuclei; RBC, red blood cell; RNA, ribonucleic acid; SDS, sodium dodecyl sulfate; rRNA, ribosomal ribonucleic acid; tRNA, transfer ribonucleic acid.

Fig. 1

Some of the areas of medicinal inorganic chemistry (adapted from Ref. [160]).

Fig. 2

⁷Li NMR spectrum of a blood sample treated with Li₂CO₃ recorded in 30 min using a home-built solenoid coil at 7 T field strength. The chemical shift of lithium accumulated in RBCs has been set to 0 ppm. The RBC and plasma ⁷Li resonances are separated by 13 ppm by the addition of 20 mg of [Tm(DOTP)]⁵⁻ as shift reagent (adapted from Ref. [29]).

Fig. 3

(a) Variable-temperature ²³Na NMR spectra of 0.80 M Na₂(5′-GMP) obtained on a Bruker Avance-500 spectrometer (B₀ = 11.75 T) operating at 132.26 MHz for ²³Na. (b) ²³Na NMR spectra of d(TG₄T) obtained on a Bruker Avance-600 spectrometer (B₀ = 14.1 T). An inversion-recovery sequence was used to partially suppress the large signal from the free Na⁺ ions. A recovery delay of 13 ms (close to the null point) was used. Typically, 250k scans were accumulated with a recycle time of 50 ms. The d(TG₄T) DNA sample was prepared at 8 mM strand concentration in 10 mM sodium phosphate buffer (pH 7.1) and 100 mM NaCl. All ²³Na chemical shifts are referenced to Na⁺(aq) at 0.0 ppm (adapted from Ref. [36]).

Fig. 4

Variable-temperature ³⁹K NMR spectra of 0.53 M Na₂(5′-GMP) in presence of 0.10 M K⁺ obtained on a Bruker Avance-500 spectrometer (B₀ = 11.75 T) operating at 23.33 MHz for ³⁹K nucleus. Typically, 500k scans were accumulated with a recycle time of 10 ms. All ³⁹K chemical shifts are referenced to K⁺(aq) at 0.0 ppm (adapted from Ref. [36]).

Fig. 5

³⁹K NMR spectrum of human RBCs resuspended in the NMR buffer at 37 °C. K_i⁺ and K_o⁺ represent the intra- and extracellular ³⁹K peaks, respectively. Hematocrit was 61.2%. One division in the spectrum is equal to 50 ppm (adapted from Ref. [42]).

Fig. 6

⁸⁷Rb NMR spectra (referenced to internal Rb⁺(aq) at 0.0 ppm) of perfused rat heart during Rb⁺ load and washout. The hearts were perfused with phosphate-free Krebs–Henseleit buffer. The perfusate was equilibrated with 95% O₂/5% CO₂, with the pH maintained at 7.4. The buffer used for Rb⁺ loading contained 0.94 mM Rb⁺ and 3.76 mM K⁺ (adapted from Ref. [45]).

Fig. 7

¹³³Cs NMR spectra of human erythrocytes suspended in a buffer containing 140 mM NaCl and 10 mM CsCl. The origin of the chemical shift scale is arbitrary (adapted from Ref. [49]).

Fig. 8

⁴³Ca NMR spectra of the titration of 0.33 mM equine apolysozyme with ⁴³Ca²⁺ at pH 6.0. 200k scans were collected in each experiment: (a) 0.23 equiv. of ⁴³Ca²⁺; (b) 0.46 equiv. of ⁴³Ca²⁺; (c) 0.69 equiv. of ⁴³Ca²⁺; (d) 0.92 equiv. of ⁴³Ca²⁺; (e) 1.15 equiv. of ⁴³Ca²⁺ (adapted from Ref. [68]).

Fig. 9

Schematic illustration of the BSH anion. There is a net negative charge of 2 in the boron-hydride cage. δ(¹¹B) of free BSH are referenced to saturated H₃BO₃ at 0.0 ppm (adapted from Ref. [73]).

Fig. 10

The ¹¹B (a) single quantum and (b) MQF (τ = 1.4 ms) NMR spectra of 100 mM borate solution with 4 mM ferricytochrome-c at 5 °C, 11.4 T, and pH 9.7 (adapted from Ref. [75]).

Fig. 11

Backbone structure of human serum transferrin (coordinates supplied by Zuccola [245]); only the C-lobe site is occupied by iron in this structure.

Fig. 12

²⁷Al NMR spectra (referenced to external 1.0 M [Al(NO₃)₃] in D₂O at 0.0 ppm) of ovotransferrin (oTf, 1.13 mM, pH 7.5), serotransferrin (sTr, 1.09 mM, pH 7.3), and lactoferrin (lTf, 0.73 mM, pH 7.5) in the presence of 20 mM Na₂¹³CO₃ and 2.0 equiv. of Al³⁺ (75% H₂O/25% D₂O, 150 mM KCl, 25 °C) at four magnetic fields: 7.0 T (ν_o = 78.2 MHz), 9.4 T (ν_o = 104.3 MHz), 11.7 T (ν_o = 130.3 MHz), and 14.1 T (ν_o = 156.4 MHz) (adapted from Ref. [82]).

Fig. 13

²⁷Al NMR spectra (referenced to external 1.0 M [Al(NO₃)₃] in D₂O at 0.0 ppm) of (a) 1.20 mM ovotransferrin in the presence of 5 mM Na₂¹³C₂O₄ and 2.0 equiv. of Al³⁺ (75% H₂O/25% D₂O, 150 mM KCl, pH 7.4, 25 °C), and (b) 1.13 mM ovotransferrin in the presence of 20 mM Na₂¹³CO₃ and 2.0 equiv. of Al³⁺ (75% H₂O/25% D₂O, 150 mM KCI, pH 7.6, 25 °C) at a magnetic field strength of 11.7 T (130.3 MHz) (adapted from Ref. [81a]).

Fig. 14

²⁰⁷Pb (104.435 MHz) NMR spectra of the ²⁰⁷Pb²⁺ forms of bacterially expressed mammalian calmodulin (1.47 mM, 4.0 equiv. ²⁰⁷Pb²⁺, 100 mM KCl, pH 7.1, 85k scans) and its C-terminal domain fragment TR₂C (1.11 mM, 2.0 equiv. ²⁰⁷Pb²⁺, 100 mM KCl, pH 7.0, 80k scans) (adapted from Ref. [114]).

Fig. 15

2D [¹H,²⁰⁷Pb] HMQC NMR spectrum of [²⁰⁷Pb(EDTA-N₄)]²⁺ (Pb-ethylenediaminetetraacetamide), 99.9% D₂O, pH 6.1, 25 °C). Coupling is observed from ²⁰⁷Pb to ¹H through three bonds. The lead chemical shift was referenced to external 1 M [Pb(NO₃)₂] in 99.9% D₂O at pH 3.3 (adapted from Ref. [112]).

Fig. 16

¹³C and ⁴⁵Sc NMR spectra (referenced to external neat TMS and 1.0 M ScCl₃ in D₂O, respectively, at 0.0 ppm) of 1.05 mM ovotransferrin in the presence of 10 mM Na₂¹³CO₃ and 1.9 equiv. of Sc³⁺ (75% H₂O/25% D₂O, 150 mM KCI, pH 7.6, 25 °C) (adapted from Ref. [82c]).

Fig. 17

Variable pH ⁵¹V NMR spectra (referenced to external neat [VOCl₃] at 0.0 ppm) of a 0.15 M NaCl aqueous solution of (a) [V^IVO(maltolato)₂] (10 mM) and (b) NH₄[V^VO₂(maltolato)₂] (10 mM) at 25 °C (adapted from Ref. [136]).

Fig. 18

⁵⁷Fe NMR spectra (referenced to external [Fe(CO)₅] at 0.0 ppm) of ⁵⁷Fe-cytochrome-c (⁵⁷Fe-Cyt-c, 3 mM in 50 mM phosphate buffer, pH 7, 298 K), ⁵⁷Fe-carbonmonoxymyoglobin (⁵⁷Fe-MbCO, 15 mM in 50 mM phosphate buffer, pH 7.1, 296 K), and ferrocene (Cp₂Fe, 0.8 M in C₆H₆) (adapted from Refs. [158c,158d]).

Fig. 19

Structure of vitamin B₁₂.

Fig. 20

⁵⁹Co NMR spectra of natural B₁₂ derivatives in solution (externally referenced to 1 M aqueous [Co(NH₃)₆]Cl₃, and subsequently converted to ppm downfield from 1 M aqueous K₃[Co(CN)₆] for the sake of literature consistency). Superimposed on the experimental traces are best Lorentzian fits characterized by the isotropic shift δ and full-width-at-half-height (FWHH) values indicated on the right (adapted from Ref. [174]).

Fig. 21

2D [¹H,¹⁰⁹Ag] HMQC spectrum (referenced to external 1 M AgNO₃ at 0.0 ppm) of yeast ¹⁰⁹Ag-metallothionein (6 mM in 18 mM phosphate buffer in D₂O, pD 6.5, 298 K). The spectrum shows the connectivities of all seven silver atoms with the cysteine H_β protons of the yeast Ag-metallothionein (adapted from Ref. [190]).

Fig. 22

Observed ¹¹³Cd chemical shifts for structurally characterized ¹¹³Cd-substituted metalloproteins: mononuclear (top) and polynuclear (bottom). All shifts are reported relative to external 0.1 M [Cd(ClO₄)₂]. The chemical shift position is correlated to ligand composition, where S represents sulfur from cysteine, S* represents sulfur from methionine, O represents oxygen from carboxylate or water, and N represents nitrogen from histidine (adapted from Ref. [201a]).

Fig. 23

¹¹³Cd NMR spectra of mammalian MTs (mouse MT-1, human MT-3, rabbit MT-2) acquired at the indicated frequencies. Schematic structures of the two metal clusters are reported (adapted from Ref. [201a]).

Fig. 24

Three-dimensional coronal ¹⁷O MRI (Fourier series window) images of natural abundance H₂¹⁷O in the rat brain acquired at 9.4 T. Three adjacent coronal ¹⁷O images (0.1 ml voxel size; total acquisition time of 11 s) and the corresponding proton anatomical images are presented herein (adapted from Ref. [220a]).

Fig. 25

In vivo³³S NMR spectrum (referred to the ³³S signal of 1 M Na₂SO₄ in H₂O in a coaxial cell) of an L. lithophaga homogenate. The peak at −6.8 ppm is assigned to the amino acid taurine (adapted from Ref. [223]).

Fig. 26

¹⁹F NMR spectrum at 282 MHz with proton decoupling of a urine sample from a patient treated orally with capecitabine at a dose of 2900 mg/day administered twice daily at 12 h interval. Urine fraction 0–12 h collected after the first dose of 1450 mg and 14-fold concentrated, pH of the sample: 5.5. The chemical shifts are expressed relative to the resonance peak of trifluoroacetic acid (5% (w/v) aqueous solution) used as external reference (REF). Assignments: F⁻, fluoride ion; CAP, capecitabine; 5′dFCR, 5′-deoxy-5-fluorocytidine; 5′dFUR, 5′-deoxy-5-fluorouridine; FC, 5-fluorocytosine; FU, 5-fluorouracil; ?, unknown; FUPA, α-fluoro-β-ureidopropionic acid; FBAL, α-fluoro-β-alanine; FHPA, 2-fluoro-3-hydroxypropanoic acid; OHFC, 6-hydroxy-5-fluorocytosine; FUH₂, 5,6-dihydro-5-fluorouracil; FAC, fluoroacetic acid. J, ¹J(¹³C–¹⁹F) coupling constant (adapted from Ref. [16h]).

Fig. 27

Catabolic pathway of capecitabine. All the compounds (except CFBAL) are represented in neutral form. CAP, capecitabine; 5′dFCR, 5′-deoxy-5-fluorocytidine; 5′dFUR, 5′-deoxy-5-fluorouridine; FU, 5-fluorouracil; FUH₂, 5,6-dihydro-5-fluorouracil; FUPA, α-fluoro-β-ureidopropionic acid; FBAL, α-fluoro-β-alanine; CFBAL, N-carboxy-α-fluoro-β-alanine; F⁻, fluoride ion; FHPA, 2-fluoro-3-hydroxypropanoic acid; FAC, fluoroacetic acid; FC, 5-fluorocytosine; OHFC, 6-hydroxy-5-fluorocytosine.