Quantitative F NMR method validation and application to the quantitative analysis of a fluoro-polyphosphates mixture

Quantitative ¹⁹F NMR (QNMR) was developed and employed to determine the amounts of four kinds of fluoropolyphosphates (FPPs) in a mixture containing sodium monofluoro-phosphate (MFP), sodium monofluoro-dipolyphosphate (MFDPP), sodium monofluoro-tripolyphosphate (MFTPP) and sodium difluoro-tripolyphosphate (DFTPP). The amounts of these ingredients cannot be measured by high-performance liquid chromatography (HPLC) because no high-purity standard samples are available. The main parameter “delay time (d1) between two scans” of affecting the response of NMR signal was determined by measuring longitudinal relaxation time (T1) to be 25 s. By using NaF as an internal reference to measure the amount of MFP solution with known concentration and then to compare the deviation between experiment value and real value, a simple and effective approach for checking out the validity of ¹⁹F QNMR method was carried out. Six experiments with different mole ratios of NaF/MFP over 200 times were repeated and the relative standard deviation (R.S.D.) of results is less than 2.0%, the limit of detection of ¹⁹F QNMR can reach to 1.0 mmol/L. NaF was used as an internal reference for the quantitative analysis of FPPs mixture. The amount of each FPP in FPPs mixture obtained by ¹⁹F QNMR was calculated and the R.S.D.s of results were less than 4.81%.     

F NMR applications in chemical biology

Although fluorine plays only a minor role in biology, its physicochemical properties have proven incredibly useful in the design of analogues of biologically important molecules. Analysis of organofluorine compounds by ¹⁹F NMR was largely confined to synthetic chemists but this technique is finding increasing applications in biological systems. The fluorine atom with its relative small size and 100% natural isotope abundance represents an attractive option for biological NMR studies. In this paper we review the recent literature highlighting the exploitation of ¹⁹F NMR in a range of research areas at the interface of chemistry and biology.     

The preparation and analysis of fluoro-polyphosphates by F, P, 2D F-P HETCOR and 2D P-P COSY NMR

A complex mixture of fluoro-polyphosphates (FPPs) and polyphosphates was prepared by heating a mixture of NaF and sodium tripolyphosphate (STPP) at 600 °C in nitrogen atmosphere. Two-dimensional ³¹P-¹⁹F heteronuclear correlation spectroscopy (HETCOR) NMR was developed in identifying the atomic connection between F and P in the mixed FPPs. ¹⁹F, ³¹P and ³¹P-³¹P correlation spectroscopy (COSY) NMR methods were employed to identify the components of the mixture and measure the chain length of each FPP ingredient. NMR results clearly demonstrated that the mixture contains four kinds of fluoro-phosphates with different chain length of polyphosphate, which are monofluoro-phosphate (MFP), monofluoro-dipolyphosphate (MFDPP), monofluoro-tripolyphosphate (MFTPP) and difluoro-tripolyphosphate (DFTPP). Other phosphates and polyphosphates also were found in the mixture.     

Solid phase β-lactams synthesis using the Staudinger reaction, monitored by F NMR spectroscopy

We report the use of ¹⁹F NMR as a simple means to monitor reactions on a solid phase. Multi-step sequences including protection, coupling, deprotection, condensation, cycloaddition and cleavage steps are described in the case of multicomponent reactions involving fluorinated α-aminoesters, aldehydes and acid chlorides.     

Strategies for organic impurity quantification by H NMR spectroscopy: Constrained total-line-shape fitting

A constrained total-line-shape (CTLS) fitting strategy for organic impurity analysis from ¹H NMR spectra was developed and assessed by studying two examples. In general, total-line-shape fitting allows integration of overlapping lines without suffering from baseline artifacts as much as traditional integration methods. It is shown here that the constrained total-line-shape fitting, where the spectral structures of the multiplets to be fitted are taken into account in form of constraints, allows quantification of seriously overlapping lines and when the signals are close to the root of major signals. Also, a method for removal of ¹³C satellite signals is described. The results indicate that our approach significantly improves the usefulness of qNMR in impurity analysis and that impurity levels of 0.1 mol%, which in some cases means down to 0.01 wt%, can be easily determined with relative standard error smaller than 10%.     

Identification and quantification of the main organic components of vinegars by high resolution 1H NMR spectroscopy

A detailed analysis of the proton high-field NMR spectra of vinegars (in particular of Italian balsamic vinegars) is reported. A large number of organic substances belonging to different classes, such as carbohydrates, alcohols, organic acids, volatile compounds and amino acids, were assigned. The possibility of quantification of the substances identified in the whole vinegar sample, without extraction or pre-concentration steps, was also tested. The data validity was demonstrated in terms of precision, accuracy, repeatability and inter-day reproducibility. The effects of the most critical experimental parameters (sample concentration, water suppression and relaxation time) on the analysis response were also discussed. ¹H NMR results were compared with those obtained by traditional techniques (GC-MS, titrations), and good correlations were obtained. The results showed that ¹H NMR with water suppression allows a rapid, simultaneous determination of carbohydrates (glucose and fructose), organic acids (acetic, formic, lactic, malic, citric, succinic and tartaric acids), alcohols and polyols (ethanol, acetoin, 2,3-butanediol, hydroxymethylfurfural), and volatile substances (ethyl acetate) in vinegar samples. On the contrary, the amino acid determination without sample pre-concentration was critical. The ¹H NMR method proposed was applied to different samples of vinegars, allowing, in particular, the discrimination of vinegars and balsamic vinegars.     

Assessment of historic Tilia codrata wood by solid-state C CPMAS NMR spectroscopy

Historic lime (Tilia cordata Mill.) wood samples, differing by their provenance, conservation status and period have been investigated by solid-state carbon-13 cross polarization magic angle sample spinning nuclear magnetic resonance (¹³C CPMAS NMR) spectroscopy. Structural and chemical modifications were assessed by comparing the historic samples with a reference wood sample. The conventional NMR measurements followed by the ¹³C resonance integral intensities of the wood samples have been carried out in order to acquire information of the chemical changes due to the natural ageing process taking place over the years. The main results concern the decrease of the carbohydrates moiety, especially the decrease of the hemicelluloses and amorphous cellulose signals, while the signals for aliphatic and methoxyl carbons from lignin present and increase of the intensity up to 120 years then start to decrease. At the same time a slight widening of the amorphous carbohydrate signals was observed, which may evidence the occurring of some chemical rearrangements, with the formation of new chemical species. These lead in the ¹³C NMR spectra to the line broadening of the signals induced by their chemical shifts dispersion.     

Chiral selectors for enantioresolution and quantitation of the antidepressant drug fluoxetine in pharmaceutical formulations by (19)F NMR spectroscopic method

¹⁹F NMR spectroscopy was applied to the quantitative determination of fluoxetine enantiomers using different chiral recognition agents in pharmaceutical formulations. Several parameters affecting the enantioresolution including the type and concentration of chiral selector, concentration of fluoxetine and temperature were studied. The chiral selectors investigated are the cyclic oligosaccharides α-, β- and γ-cyclodextrin and a diamino derivative of methylated α-cyclodextrin (DAM-α-CD), linear polysaccharides (maltodextrin with dextrose equivalents of 4.0-7.0, 13.0-17.0 and 16.5-19.5) and the macrocyclic antibiotic vancomycin. Among the chiral selectors used, DAM-α-CD turned out to give the best resolution of the ¹⁹F NMR signals of (R)- and (S)-fluoxetine. The calibration curve was linear for (R)- and (S)-fluoxetine over the range 0.10-1.35 mg mL⁻¹, the detection limits (S/N = 3) being 5.9 and 7.5 μg mL⁻¹ for the pure solutions of (R)- and (S)-fluoxetine, respectively. The recovery studies performed on pharmaceutical samples ranged from about 90 to 110% with relative standard deviations of <8%. The results showed that the proposed method is rapid, precise and accurate. Applying statistical Student's t-test revealed insignificant difference between the real and measured contents at the 95% confidence level.     

Mercury(II) cyanide complexes with alkyldiamines: solid-state/solution NMR,computational, and antimicrobial studies

Mercury cyanide complexes of alkyldiamines (1–6), [Hg(L)(CN)₂] (where L?=?en (1,2-diaminoethane), pn (1,3-diaminopropane), N-Me-en, N, N′-Me₂-en, N, N′-Et₂-en, and N, N′-ipr₂-en), have been synthesized and characterized by elemental analysis, IR, ¹³C, and ¹⁵N solution NMR in DMSO-d₆, as well as ¹³C, ¹⁵N, and ¹⁹⁹Hg solid-state NMR spectroscopy. Complexes 1 and 2 have been studied computationally, built and optimized by GAUSSIAN03 using DFT at B3LYP level with LanL2DZ basis set. Binding modes of en and bn (where bn?=?1,4-diaminobutane) toward Hg(CN)₂ are completely different. Complexes with en and pn show chelating binding to Hg(II), while bn behaves as a bridging ligand to form a polymeric structure, [Hg(CN)₂-bn]_∞ [B.A. Al-Maythalony, M. Fettouhi, M.I.M. Wazeer, A.A. Isab. Inorg. Chem. Commun., 12, 540 (2009).]. The solution ¹³C NMR of the complexes demonstrates a slight shift of the ?C≡N (0.9 to 2?ppm) and ?C–NH₂ (0.25 to 6?ppm) carbon resonances, while the other resonances are relatively unaffected. ¹⁵N labeling studies have shown involvement of alkyldiamine ligands in coordination to the metal. The principal components of the ¹³C, ¹⁵N, and ¹⁹⁹Hg shielding tensors have been determined from solid-state NMR data. Antimicrobial activity studies show that the complexes exhibit higher antibacterial activities toward various microorganisms than Hg(CN)₂.     

NMR spectra of 1,3,5-triazines with fluorinated substituents

The NMR spectra of a series of 1,3,5-triazines (1) in which one or more of the substituent groups are fluorinated are described, and the results are discussed in terms of bonding and geometry of the molecules. Remote effects of unsymmetrical substitution are observed in the fluorine-19 and carbon-13 spectra, in some molecules indicating restricted rotation about the bond joining an amino group to a ring carbon.     

In vitro enzymatic oxidation of a fluorine-tagged sulfido substrate analogue: a 19F NMR investigation

1H-decoupled 19F NMR has been used to monitor the highly regioselective oxidation of a fluorine-tagged thia-fatty acid derivative by castor stearoyl-ACP delta9 desaturase. The major enzymatic product, after reductive work-up, was identified as 9-fluoro-1-nonanol. This compound could be easily distinguished from substrate and a 9-sulfoxy by-product on the basis of its 19F NMR chemical shift and spiking experiments using authentic standards. Structural assignment of the cleavage product was confirmed by GC-MS analysis of the enzymatic products.     

Analysis of F and C NMR spectra of tetrafluorophthalic anhydride and its derivatives

¹⁹F and ¹³C NMR spectra of perfluorinated compounds (i.e., tetrafluorophthalic anhydride, its hydroxyl- and amino-derivatives, N-pentafluorophenyltetrafluorophthalimide, and hexafluoroindan-1,3-dione) were analysed. Different signals in NMR spectra were assigned based on the analysis of spin-spin coupling constants. All assignments made were further confirmed by density functional theory (DFT) calculations of ¹³C chemical shifts and J_C,F coupling constants.     

Isotopic finger-printing of active pharmaceutical ingredients by 13C NMR and polarization transfer techniques as a tool to fight against counterfeiting

The robustness of adiabatic polarization transfer methods has been evaluated for determining the carbon isotopic finger-printing of active pharmaceutical ingredients. The short time stabilities of the adiabatic DEPT and INEPT sequences are very close to that observed with the one pulse sequence, but the DEPT long time stability is not sufficient for isotopic measurements at natural abundance or low enrichment. Using the INEPT sequence for ¹³C isotopic measurements induces a dramatic reduction in the experimental time without deterioration in short time or long time stability. It appears, therefore, to be a method of choice for obtaining the isotopic finger-print of different ibuprofen samples in a minimum time. The results obtained on 13 commercial ibuprofen samples from different origins show that this strategy can be used effectively to determine ¹³C distribution within a given molecule and to compare accurately differences in the isotopic distribution between different samples of the given molecule. The present methodology is proposed as a suitable tool to fight against counterfeiting.     

Solid-state NMR studies of pharmaceutical solids in polymer matrices

Biodegradable drug-delivery systems can be formulated to release drug for hours to years and have been used for the controlled release of medications in animals and humans. An important consideration in developing a drug-delivery matrix is knowledge of the long-term stability of the form of the drug and matrix after formulation and any changes that might occur to the drug throughout the delivery process. Solid-state NMR spectroscopy is an effective technique for studying the state of both the drug and the matrix. Two systems that have been studied using solid-state NMR spectroscopy are presented. The first system studied involved bupivacaine, a local anesthetic compound, which was incorporated into microspheres composed of tristearin and encapsulated using a solid protein matrix. Solid-state ¹³C NMR spectroscopy was used to investigate the solid forms of bupivacaine in their bulk form or as incorporated into the tristearin/protein matrix. Bupivacaine free base and bupivacaine-HCl have very different solid-state NMR spectra, indicating that the molecules of these compounds pack in different crystal forms. In the tristearin matrix, the drug form could be determined at levels as low as 1:100 (w/w), and the form of bupivacaine was identified upon loading into the tristearin/protein matrix. In the second case, the possibility of using solid-state ¹³C NMR spectroscopy to characterize biomolecules lyophilized within polymer matrices is evaluated by studying uniformly ¹³C-labeled asparagine (Asn) in 1:250 (w/w) formulations with poly(vinyl pyrrolidone) (PVP) and poly(vinyl alcohol) (PVA). This work shows the capability of solid-state NMR spectroscopy to study interactions between the amino acid and the polymer matrix for synthetic peptides and peptidomimetics containing selective ¹³C labeling at the Asn residue.     

Hydrolysis of Volatile Ammonium Oxofluorotitanate According to 19F, 17O, and 49Ti NMR Data

Hydrolysis of NH₄TiO_xF_5-2x (x0.4) (I) was investigated by ¹⁹F, ¹⁷O, and ⁴⁹Ti NMR. The interaction of complex I with water is accompanied by the formation of [TiF₆]^2- and multinuclear titanium forms. The composition of the main forms resulting from hydrolysis of I has been established. The bonding of titanium octahedra into dimers and other oligomers occurs by formation of hydroxyl bridges, considerably lowering the pH of the solution. Close analogy has been found between hydrolysis of the title complex and that of titanium tetrafluoride.     

Local fluorine environments in nanoscopic magnesium hydr(oxide) fluorides studied by F MAS NMR

¹⁹F MAS NMR signals of a variety of X-ray amorphous magnesium hydroxide (oxide) fluorides (MgF_x(OH(R))_2−x (0 < x ≤ 2)) prepared using different synthesis routes are compared and assigned. Relations between their ¹⁹F signal positions and local coordinations are elaborated on the basis of the chemical behaviour and data available for the few crystalline reference compounds known so far. Supported by the superposition model, ¹⁹F signals appearing between −150 ppm and −200 ppm were subdivided into three groups due to local [FMg₆], [FMg₅] and [FMg₄], and [FMg₃] environments, respectively.     

Local environments and dynamics of hydrogen atoms in protonated forms of ion-exchangeable layered perovskites estimated by solid-state H NMR

The local environments and dynamics of hydrogen atoms in five samples of protonated forms of ion-exchangeable layered perovskites, Dion-Jacobson-type H[LaNb₂O₇] and H[LaTa₂O₇], Ruddlesden-Popper-type H₂[SrTa₂O₇] and H₂[La₂Ti₃O₁₀], and H_1.8[(Sr_0.8Bi_0.2)Ta₂O₇] derived from an Aurivillius phase, Bi₂Sr₂Ta₂O₉, have been investigated by solid-state ¹H nuclear magnetic resonance spectroscopy (NMR). Solid-state ¹H NMR with a magic-angle spinning technique conducted at room temperature reveals that the mean electron densities around the ¹H nuclei in these protonated forms are relatively low, and that they decrease in the following order: H_1.8[(Sr_0.8Bi_0.2)Ta₂O₇]>H[LaNb₂O₇]>H₂[SrTa₂O₇]>H[LaTa₂O₇]>H₂[La₂Ti₃O₁₀]. The temperature-dependent solid-state ¹H broad-line NMR spectra measured at 140-400 K reveal a decrease in the signal width for all of these five samples upon heating due to motional narrowing. The NMR spectra of H[LaNb₂O₇] and H[LaTa₂O₇] are different from the other three protonated forms due to the weaker dipole-dipole interactions at low temperatures and lower mobility of the hydrogen atoms at high temperatures.     

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by 19F NMR

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of ¹⁹F NMR methods to characterize complex exchange processes with multiple excited states.     

Solid-state Na and C NMR characterization of Na3C60

Two separate samples of Na₃C₆₀ were prepared by direct reaction of C₆₀ with sodium metal vapor, and subjected to different annealing times of 10 days and 16 days. Solid-state ¹³C and ²³Na NMR, along with elemental analysis, powder X-ray diffraction (XRD) and Raman spectroscopy, were used to characterize both samples. The Raman spectra of both materials have a single peak at 1447 cm⁻¹ which correspond to the A_g peak of C₆₀³⁻, consistent with the stoichiometry of Na_xC₆₀ with x=3. The powder XRD patterns are also virtually identical for both samples. However, solid-state ²³Na and ¹³C NMR spectra of the two samples are significantly different, suggesting a relationship between annealing times and the final structure of the alkali fulleride. Variable-temperature ²³Na magic-angle spinning (MAS) NMR experiments reveal the existence of two or three distinct sodium species and reversible temperature-dependent diffusion of sodium ions between octahedral and tetrahedral interstitial sites. ¹³C MAS NMR experiments are used to identify resonances corresponding to free C₆₀ and fulleride species, implying that the samples are segregated-phase materials composed of C₆₀ and non-stoichiometric Na₃C₆₀. Variable-temperature ¹³C MAS NMR experiments reveal temperature-dependent motion of the fullerides.     

F NMR spectroscopy as useful tool for determining the structure in solution of coordination compounds of MF5 (M = Nb, Ta)

The salts [S(NMe₂)₃][MF₆] (M = Nb, 2a; M = Ta, 2b) and [S(NMe₂)₃][M₂F₁₁] (M = Nb, 2c; M = Ta, 2d) have been prepared by reacting MF₅ (M = Nb, 1a; M = Ta, 1b) with [S(NMe₂)₃][SiMe₃F₂] (TASF reagent) in the appropriate molar ratio. The solid state structure of 2b has been ascertained by X-ray diffraction. The 1:1 molar ratio reactions of 1a with a variety of organic compounds (L) give the neutral adducts NbF₅L [L = Me₂CO, 3a; L = MeCHO, 3b; L = Ph₂CO, 3c; L = tetrahydrofuran (thf), 3d; L = MeOH, 3e; L = EtOH, 3f; L = HOCH₂CH₂OMe, 3g; L = Ph₃PO, 3h; L = NCMe, 3i] in good yields. The complexes MF₅L [M = Nb, L = HCONMe₂, 3j; M = Nb, L = (NMe₂)₂CO, 3k; M = Ta, L = (NMe₂)₂CO, 3l; M = Nb, L = OC(Me)CHCMe₂, 3m] have been detected in solution in admixture with other unidentified products, upon 2:1 molar reaction of 1 with the appropriate reagent L. The ionic complexes [NbF₄(tht)₂][NbF₆], 4a, and [NbF₄(tht)₂][Nb₂F₁₁], 4b, have been obtained by combination of tetrahydrothiophene (tht) and 1a, in 1:1 and 2:3 molar ratios, respectively. The treatment of 1 with a two-fold excess of L leads to the species [MF₄L₄][MF₆] [M = Nb, L = HCONMe₂, 5a; M = Ta, L = HCONMe₂, 5b; M = Nb, L = thf, 5c; M = Ta, L = thf, 5d; M = Nb, L = OEt₂, 5e]. The new complexes have been fully characterised by NMR spectroscopy. Moreover, the revised ¹⁹F NMR features of the known compounds MF₅L [M = Ta, L = Me₂CO, 3n; M = Ta, L = Ph₂CO, 3o; M = Ta, L = MePhCO, 3p; M = Ta, L = thf, 3q; M = Nb, L = CH₃CO₂H, 3r; M = Nb, L = CH₂ClCO₂H, 3s; M = Ta, L = CH₂ClCO₂H, 3t], TaF₄(acac), TaF₄(Me-acac) and [TaF(Me-acac)₃][TaF₆] (Me-acac = methylacetylacetonato anion) are reported.