15N nuclear magnetic resonance studies of [1-15N]nicotinamide adenine dinucleotides

The synthesis of [1-¹⁵N]nicotinamide adenine dinucleotide is described. Chemical shift data from ¹⁵N NMR studies are presented for the pyridine ring nitrogen of labeled NAD and related compounds. The results indicate a ¹⁵N label in the N-1 position to be highly sensitive to the redox-state of the pyridine moiety, with an upfield shift of over 100 ppm observed upon reduction of NAD⁺ to NADH. The feasibility of conducting ¹⁵N NMR studies of pyridine nucleotide binding to dehydrogenases is discussed.     

NMR studies of coupled low- and high-barrier hydrogen bonds in pyridoxal-5'-phosphate model systems in polar solution

The 1H and 15N NMR spectra of several 15N-labeled pyridoxal-5'-phosphate model systems have been measured at low temperature in various aprotic and protic solvents of different polarity, i.e., dichloromethane-d2, acetonitrile-d3, tetrahydrofuran-d8, freon mixture CDF3/CDClF2, and methanol. In particular, the 15N-labeled 5'-triisopropyl-silyl ether of N-(pyridoxylidene)-tolylamine (1a), N-(pyridoxylidene)-methylamine (2a), and the Schiff base with 15N-2-methylaspartic acid (3a) and their complexes with proton donors such as triphenylmethanol, phenol, and carboxylic acids of increasing strength were studied. With the use of hydrogen bond correlation techniques, the 1H/15N chemical shift and scalar coupling data could be associated with the geometries of the intermolecular O1H1N1 (pyridine nitrogen) and the intramolecular O2H2N2 (Schiff base) hydrogen bonds. Whereas O1H1N1 is characterized by a series of asymmetric low-barrier hydrogen bonds, the proton in O2H2N2 faces a barrier for proton transfer of medium height. When the substituent on the Schiff base nitrogen is an aromatic ring, the shift of the proton in O1H1N1 from oxygen to nitrogen has little effect on the position of the proton in the O2H2N2 hydrogen bond. By contrast, when the substituent on the Schiff base nitrogen is a methyl group, a proton shift from O to N in O1H1N1 drives the tautomeric equilibrium in O2H2N2 from the neutral O2-H2...N2 to the zwitterionic O2-...H2-N(2+) form. This coupling is lost in aqueous solution where the intramolecular O2H2N2 hydrogen bond is broken by solute-solvent interactions. However, in methanol, which mimics hydrogen bonds to the Schiff base in the enzyme active site, the coupling is preserved. Therefore, the reactivity of Schiff base intermediates in pyridoxal-5'-phosphate enzymes can likely be tuned to the requirements of the reaction being catalyzed by differential protonation of the pyridine nitrogen.     

15N nuclear magnetic resonance spectroscopy. Natural-abundance 15N spectra of aliphatic oximes

¹⁵N chemical shifts of the Z and E isomers of twenty-two ketoximes and fourteen aldoximes have been determined at the natural-abundance level of ¹⁵N, using Fourier transform methods. The influences of π delocalization, methyl substituents and solute concentration on the oxime nitrogen shielding have been determined. The ¹⁵N shifts for oximes of several cycloalkanones have been measured and the influence of ring size on the chemical shifts is discussed.     

15N nuclear magnetic resonance spectroscopy. Natural abundance 15N NMR of monosubstituted indoles

¹⁵N chemical shifts of twenty-four substituted indoles have been determined in natural abundance (in organic solvents) using Fourier transform NMR. The overall chemical shift range is 27 ppm, with groups in the 2-, 3- and 5-ring positions showing the largest substituent effects. Substituents capable of resonance interaction with the indole nitrogen give shifts in the expected directions but they cannot be correlated with known substituent parameters. Compounds measured in DMSO give 0·2 to 10·2 ppm downfield shifts with respect to the same compound measured in CDCl₃. ¹³C NMR data for previously unreported compounds are also reported.     

Tetrazoloazines. 15N nuclear magnetic resonance and infrared absorption spectroscopy

The reaction of 4-chloro-2-phenylquinazoline with K¹⁵NN₂ has been studied by ¹⁵N-NMR. spectroscopy. ¹⁵N-chemical shifts in 5-phenyl-1 (3)-[¹⁵N]-tetrazolo[1,5-c]quinazoline and -Nα(Nγ)-[¹⁵N]-4-azido-2-phenylquinazoline are reported. The characteristic IR. absorption frequencies of the tetrazole group have been determined in a series of annelated ¹⁵N-labelled compounds. From these studies and the chemistry of the labelled tetrazoles, it is concluded that all haloazines examined react with KN₃ by the direct nucleophilic substitution mechanism. An addition of nucleophile-ring opening-ring closure (ANRORC) mechanism was not observed. The synthesis of several ¹⁵N-labelled tetrazoloazines is described.     

Studies of protein hydration in aqueous solution by high-resolution nuclear magnetic resonance spectroscopy

Individual hydration water molecules in aqueous protein solutions have been observed using experimental schemes for homonuclear two-dimensional and heteronuclear three-dimensional NMR experiments in H₂O solution, which do not require suppression of the solvent line by presaturation. In these experiments, the location of the hydration waters is determined from their nuclear Overhauser effects (NOE s) with individual hydrogen atoms of distinct amino acid residues. In the basic pancreatic trypsin inhibitor (BPTI ), four internal water molecules that had been reported in three different crystal forms were also found to be in the same locations in the solution structure, with lifetimes with respect to exchange of the water protons in excess of 0.3 ns. Additional NOE s with polypeptide protons located on the protein surface may involve either hydration water molecules or hydroxyl protons of amino acid side chains. Their total number is small compared to the number of NOE s expected from the hydration water molecules identified in the crystal structures of BPTI .     

13C, 15N and 29Si nuclear magnetic resonance studies of some aminosilanes and aminodisilanes

¹³C, ¹⁵N (at natural abundance) and ²⁹Si NMR data (chemical shifts and coupling constants) are reported for aminosilanes R₂R′SiNHR¹ (1), bis(silyl)amines Me₂R′SiNHSiMe₃ (2), 1,2-bis(amino)-ethanes (3), bis(amino)silanes RR′Si(NHR¹)₂ (4), 1,2-bis(amino)tetramethyldisilanes (5) and 1,1,2,2-tetrakis(amino)dimethyldisilanes (6). The δ¹⁵N values depend more on the nature of the substituents R¹(H, alkyl, aryl) at the nitrogen atom (in the same way as for other amines) than on different substituents at the silicon atom. A linear correlation between ¹J(²⁹Si¹⁵N) and ¹J(²⁹Si¹³C) is proposed for silanes in which the SiN unit is replaced by the SiCH unit. This correlation comprises all ¹J(²⁹Si¹⁵N) values for aminosilanes R_4-nSi(N)n (n = 1–4) and—most likely—also for aminodisilanes, and it predicts ¹J(²⁹Si¹⁵N)>0 if the corresponding value |¹J(²⁹Si¹³C)|>25 Hz. For the first time a two-bond coupling across Si, ²J(²⁹Si ¹⁵N) = 6.9 Hz, has been observed for 6a. In the case of 6b (R¹ = ^sBu) all resonances for the diastereomers are resolved in the ¹⁵N and ²⁹Si NMR spectra in contrast to the ¹H and ¹³C NMR spectra.     

Natural abundance nitrogen-15 nuclear magnetic resonance spectral studies on selected donors

The natural abundance 15N-NMR chemical shifts of selected aliphatic amines, 2-substituted pyridine type compounds, bialicyclic tertiary amines have been measured as a function of the nature of the solvent. In the case of cyclic aliphatic amines, like piperidine, morpholine, piperazine, thiomorpholine, the nitrogen is more shielded in concentrated solution compared to that in dilute solution whereas in the hydrogen bonding and protonating solvents there is a prominent deshielding. 2-Substituted pyridines studied can be further divided into four sub groups. The site of hydrogen bonding and protonation in 2-amino, 2-hydroxy and 2-mercapto pyridines have been conclusively proved from the 15N-NMR chemical shifts and the well-known tautomeric forms of the above compounds. Similarly in the case of 2-(2-thienyl)pyridine and 2-(3-thienyl)pyridine, the site of donation has been proved as the nitrogen of the pyridine ring in both the compounds. In a similar manner, the site of hydrogen bonding and protonation in two individual compounds 2-anilinopyridine and 2-(2-pyridyl)benzimidazole have also been established. Among the bialicyclic amines, 1,2-diazabicyclo[2.2.2]octane (DABCO) behaved differently from the other two compounds. In both 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), it was possible to show that N1-nitrogen in both the compounds is the site of donation. The effect of the second donor site on the 15N-NMR chemical shift, the site of donation in the selected compounds and some typical compounds reported in literature have been presented and discussed.     

Proton adsorption and acid-base properties of Tunisian illites in aqueous solution

Suspensions of illite clay minerals samples of three different origins (American illite from Montana, It(Mo), Tunisian glauconite from Gafsa, It(Ga), and Tunisian illite — chlorite mixed layer from El Hamma, It(Ha)) were prepared. The Fourier Transform Infrared (FTIR) spectroscopy, X-ray Diffractometry (XRD), Electron Scanning Microscopy (SEM) and surface area measurements were used to characterize the three illite samples having different extent of isomorphic substitution. The layers have a permanent negative charge due to isomorphic substitutions and pH dependent charges on the surface hydroxyls on the edges. Surface speciation of these samples was investigated using continuous potentiometric titration and mass titration curves between pH 4 and 11 at 0.001 M, 0.01 M, and 0.1 M NaCl solutions at ambient temperature. The two methods revealed point of zero charge (PZC) of the amphoteric edges sites approximately similar to the purified samples, in the range ～7.5–8.5, ～8.2–8.7, and ～ 9.0–9.3 for It(Mo), It(Ha) and It(Ga), respectively. The PZCs of freshly prepared dispersions are higher than those reported in the literature indicating basic character of these samples (pH of equilibrium suspensions in distilled water were ～7.9–9). In the present study, the focus was on the surface charge characteristics. A simple SCM model approach is presented to explain the illite H⁺ adsorption data. Surface weak acidic sites and surface ionization constants were calculated from titration data using regression methods. Sites with pKa ₁ ^int values of 6–6.7; 5.4–5.8; 6.1–6.6 and sites at pKa ₂ ^int values of 9.2–9.9; 10.2–10.4; 9.3–10 for It(Mo), It(Ga) and It(Ha), respectively, were assigned to amphoteric Al-OH and/or Fe-OH and Si-OH groups on the edges of illite samples.     

Nitrogen-15 nuclear magnetic resonance spectra of ketenimines

Nitrogen-15 NMR spectra of 11 ketenimines have been taken both at the natural-abundance level of ¹⁵N and with the aid of ¹⁵N-labeling. The nitrogen chemical shifts are substantially different from those of neutral imines and are upfield, more like those of protonated imines. The results are in accord with significant delocalization of the nitrogen lone-pair. Furthermore, there is a rough parallelism between the ¹³C shifts of the terminal carbons and the ¹⁵N shifts.     

Reconstitution and biochemical characterization of a new pyridoxal-5'-phosphate biosynthetic pathway

The substrates for Bacillus subtilis PLP synthase (YaaD and YaaE) are identified, and the first reconstitution of PLP biosynthesis using this pathway is described. Three partial reactions catalyzed by YaaD are also identified.     

Progress in nuclear magnetic resonance studies of surfactant systems

Nuclear magnetic resonance (NMR), as a powerful technology, is widely used to characterize the physicochemical properties of surfactants in solution. As a sensitive technique to molecular environment, NMR is beyond the reach of other spectral methods in surfactant systems. Recent years, intensive investigations of surfactants by NMR were reported but not well summarized; therefore, we highlight these significant progresses, which may shed light on the challenges to understand their behavior and mechanisms in surfactant systems. The theory of various NMR methods was introduced, including chemical shifts, diffusion, relaxation, 2D nuclear Overhauser effect spectroscopy and rotating frame nuclear Overhauser effect spectroscopy. The behavior, interaction, and mechanisms among surfactants and other molecules from NMR technologies were discussed. Challenges to understand the behavior and mechanisms in surfactant systems and instrumentation limits are addressed as perspectives.     

Nuclear magnetic resonance relaxation studies of polyacrylamide solution

 The cohesive interaction among polymer chains in a polyacrylamide (PAAm)–D₂O solution has been studied by NMR relaxation. The NMR relaxation times of PAAm in the good solvent D₂O were measured at different temperatures. The results show that the solution system has a high local viscosity and that its relaxation characteristic is soft-solid-like. The temperature dependence of the relaxation behavior of the solution is obviously different from that of ordinary polymer solutions. The difference lies in the relaxation behavior of the methylene protons in the main chain of PAAm, as shown by analyzing the relaxation process with single exponential and biexponential decays. As the temperature increases, the solvation is weakened, leading polymer chains to form curling coils, thus hindering the movement of the methylene protons among the main chains. It can be expected from the existence of 80% fast-relaxing protons that there are a zhigh number of entanglements among the polymer chains in PAAm solution. The information about entanglements among the polymer chains can be deduced from the biexponential dependence of the spin–spin relaxation on the concentration of the polymer solutions. Received: 14 April 1999/Accepted in revised form: 12 October 1999     

Salt-induced micellization of a triblock copolymer in aqueous solution: a 1H nuclear magnetic resonance spectroscopy study

An aqueous micellar solution of a PEO-PPO-PEO triblock copolymer, pluronic F88 (EO103PO39EO103), in the presence of salt (KCl) has been investigated by 1H NMR spectroscopy. The hydrogen-bonding structure in water is directly changed by the strong polarization effect of added salt, which indirectly weakens the interaction of polymer molecules with water. Both EO and PO blocks are dehydrated by the addition of salt in a similar way, whereas the solubility of the PO blocks may be affected in a more pronounced way, which results in the decrease of the critical micellization temperature (CMT). It is found that the addition of salt favors a more compact micellar core, where the water content is decreased and an effective PO-PO interaction is increased. Increasing the salt concentration would result in a decrease in the number of gauche conformers in the PPO chain, which may be the deeper reason for the decreasing solubility of PPO segments in aqueous salt solution. The temperature region over which the micellization occurs is broad, indicating that micelles and unimers coexist over an extended temperature range, whereas this transition region is significantly narrowed by the addition of salt. The addition of salt offers a good substitute way of changing the temperature to induce micellization. The critical micellization salt concentration (CMSC) is determined to be 1.0 mol l-1 for KCl in 2.5% aqueous pluronic F88 solution at 25 degrees C, and the transition region in which both free and associated copolymer molecules coexist is defined to range from 1 to 2 mol L-1.