Adam Allerhand

Natural Abundance Carbon‐13 Partially Relaxed Fourier Transform Nuclear Magnetic Resonance Spectra of Complex Molecules

Proton‐decoupled partially relaxed Fourier transform (PRFT) NMR of carbon‐13 in natural abundance was used to determine spin–lattice relaxation times (T1) of individual carbons in solutions of cholesteryl chloride, sucrose, and adenosine 5′‐monophosphate (AMP) at 15.08 MHz and 42°C. With the exception of a few side‐chain groups, all protonated carbons have T1 values of less than 1 sec. Some side‐chain carbons on cholesteryl chloride show evidence of internal reorientation and have relaxation times of up to 2 sec. Nonprotonated carbons have T1 values in the range 2–8 sec. These relaxation times are sufficiently short to make ordinary Fourier transform NMR a very sensitive technique in the study of complex molecules without the need for spin‐echo refocusing schemes. Integrated intensities and nuclear Overhauser enhancements prove that, except for two of the three nonprotonated carbons in AMP, all 13C nuclei in these compounds relax mainly through 13C–1H dipolar interactions. Measured T1 values of protonated...

Theory of Nuclear Overhauser Enhancement and 13C–1H Dipolar Relaxation in Proton‐Decoupled Carbon‐13 NMR Spectra of Macromolecules

Expressions are presented for the nuclear Overhauser enhancement (NOE), the spin‐lattice relaxation time (T1), and the spin‐spin relaxation time (T2) of a 13C nucleus relaxing by a dipolar interaction with one proton under conditions of complete proton decoupling, and without assuming that the extreme narrowing limit applies. Specific equations are derived for a C–H group in a rigid molecule rotating isotropically and also for a C–H group with one degree of internal motion attached to a molecule undergoing isotropic rotational reorientation. Numerical results are presented for T1, T2, and the NOE of a C–H group in a rigid molecule (undergoing purely dipolar relaxation) as a function of the rotational correlation time (τR) and the resonance frequency (ωC). T1 goes through a minimum when τRωC ≈ 0.8. The NOE varies from the expected value of 2.988 in the extreme narrowing limit to 1.153 when 1/τR is much smaller than the resonance frequency. The numerical results indicate that the signal‐to‐noise ratio in pr...

Fluorine NMR Spectra and Conformational Isomerization of 1,1‐Difluorocyclohexane

The 19F high‐resolution spectrum of 1,1‐difluorocyclohexane has been observed at temperatures from −93° to +40°C. The rate of conformational isomerization was determined by a complete line‐shape analysis method for temperatures from −71° to +6°C. Activation parameters ΔH‡ and ΔGcc‡ in kilocalories/mole and ΔScc‡ in entropy units were 9.0, 9.8, −3.3 and 8.6, 9.8, −5.0 for 1:1 vol/vol solutions in CS2 and CFCl3, respectively, at the coalescence temperature of 228°K. In the low‐temperature spectra of the CS2 solution, the resonance of the equatorial fluorine is shifted 15.64 ppm downfield from the axial and JFF is 235.3 cps; also J (Hax–Fax) the axial‐axial vicinal H–F coupling constant is 34.3 cps and J (Heq–Fax) is 11.5 cps. At temperatures above −10°C, neglect of the H–F coupling in the line‐shape analysis produces systematic errors in the apparent exchange rate.

Spin‐Echo NMR Studies of Chemical Exchange. III. Conformational Isomerization of Cyclohexane and d11‐Cyclohexane

Equations derived previously for NMR chemical‐exchange effects have been used in proton spin‐echo studies at 26.85 Mc/sec of the chair—chair isomerization of cyclohexane and d11‐cyclohexane in 1:1 v/v solutions in CS2. The d11‐cyclohexane is particularly suited for such studies because of its very long natural relaxation time T20, and exchange rates of 0.53 to 3.8×104 sec−1 were determined for it in the temperature range between −98° and +25°C. The heteronuclear coupling in d11‐cyclohexane does not affect the spin‐echo results. However, in cyclohexane the homonuclear coupling and the shorter T20 restricted the temperature range of accurate rate determinations to −60° and −25°C, respectively. The exchange rates are the same within experimental error for both compounds, as are ΔH‡, ΔFcb‡, and ΔScb‡ which were found to be 9.1 and 10.3 kcal/mole and −5.8 eu. The latter are compared with the results of several previous studies, and it is demonstrated that, in most if not all of the studies, there are systemati...

Studies of individual carbon sites of hemoglobins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy.

Proton-decoupled natural abundance 13C NMR spectra of carbon monoxide hemoglobins were recorded at 15.18 MHz by the Fourier transform method, under conditions of spectrometer sensitivity sufficient for detection of individual carbon resonances. The aromatic region of each spectrum contains broad bands of methine carbon resonances, and some relatively narrow peaks arising from nonprotonated carbons. Resonances of heme carbons were detected in spectra of carbon monoxide hemoglobins, but not in spectra of ferrihemoglobin (as a result of paramagnetic effects). Spectra of carbon monoxide hemoglobins from various species yielded only a few well resolved individual carbon resonances, most notably those of Cgamma of tryptophan residues. A comparison of the spectra of human adult, human fetal, chicken AII, and bovine fetal hemoglobins yielded specific assignments for all resonances of Cgamma of tryptophan residues. In the cases of human fetal, chicken AII, and bovine fetal hemoglobins, each tryptophan yielded a completely resolved individual carbon resonance. The chemical shift difference between the resonances of Cgamma of Trp-130beta and Cgamma of Trp-37beta is about 6 ppm. The chemical shift difference between Trp A12[14]alpha and Trp A12[15]beta is 1 ppm or less. A comparison of the chemical shifts of analogous tryptophan residues of the four carbon monoxide hemoglobins suggests very similar conformations in solution.

Natural abundance carbon-13 nuclear magnetic resonance spectra of the canine sciatic nerve.

The proton-decoupled natural abundance carbon-13 nuclear magnetic resonance spectrum of the canine sciatic nerve is virtually identical to that of canine adipose tissue and markedly similar to that of liquid triolein. No resonances assignable to cholesterol, glycolipids, or sphingolipids are detectable in the sciatic nerve spectrum despite their abundance in the myelin sheath of this nerve. However, many such resonances are observed in lipid extracts of the nerve. Chronmatographic analysis of specimens of canine and rabbit sciatic nerve has revealed that these contain sufficient triglyceride to account quantitatively for the observed spectrum. Proton nuclear magnetic resonance and spin-labeling results for preparations containing myelin, especially those derived from the peripheral nerve, should be critically examined for experimental artifacts reflecting the triglyceride content.

Spin‐Echo NMR Studies of Chemical Exchange. IV. Intramolecular Exchange of a Coupled AB System

A mathematical and experimental study of the spin‐echo amplitudes in a Carr—Purcell train of echoes is reported for a coupled AB system undergoing intramolecular exchange. The density‐matrix approach was used to derive a set of four coupled recursion relations for the amplitudes of successive echoes. It seems unlikely that the set of equations can be reduced to a simple analytical function applicable to the general case, so a program was written to investigate the properties of the system numerically with a high‐speed computer. Such an analysis predicts that when the exchange rate 1/2τ is small compared with the coupling constant A in rad sec−1, the amplitude of the echo train is modulated. The modulation frequency depends on A, 1/2τ, and on the chemical shift δω and the rf pulse separation tcp. The modulation diminishes as the exchange rate increases, and it disappears when 1/2τ≳3A. In the latter case, the train of echoes decays exponentially with an apparent decay constant 1/T2 which is described by the...

Ultra-high-resolution NMR. IV. A simple technique for measurements of sample temperature gradients

La methode de mesure des gradients de temperature est basee sur le fait que deux resonances quelconques qui ont differentes dependances du deplacement chimique vis-a-vis de la temperature presentent un elargissement de raie spectrale different du aux gradients de deplacement chimique

Ultra-high-resolution NMR. III. Performance of WALTZ-16 proton decoupling

In a recent report (1) we used the nonprotonated carbon of toluene to demonstrate the feasibility of ultra-high-resolution NMR. We achieved an instrumental broadening (Win) of as little as 6.6 mHz (1). However, a nonprotonated carbon does not place a severe requirement on proton decoupling efficiency. In this Communication we show that WALTZ16 proton decoupling, developed by Freeman and co-workers (2, 3), is an extraordinarily effective low-power proton decoupling method for ultra-high-resolution NMR. It should be noted that the experimental tests of WALTZ-16 performance reported by Shaka et al. (Fig. 3 of Ref. (2)) were done under conditions of 0.25 Hz line broadening and are therefore not necessarily indicative that WALTZ-16 is suitable for ultra-high-resolution NMR. We need a low-power decoupling method that yields 5 mHz or less residual broadening of r3C resonances for the whole typical range of ‘H chemical-shift offsets relative to the ‘H carrier frequency. Before the development of highly efficient low-power proton-decoupling methods in recent years it would have been impossible to obtain ultra-high-resolution 13C NMR spectra, for two reasons. First, even with high-power (b10 W) ‘H irradiation, older proton-decoupling schemes such as random-noise modulation (4) and square-wave modulation (5) left large residual unresolved 13C-‘H splittings. Second, we have found that even if the residual broadening had been absent, temperature gradients in the sample resulting from uneven sample heating by the high decoupling power would have caused enough of a chemical-shift gradient in the sample to nullify ultra-high resolution. Detailed measurements of temperature gradients as a function of gas flow rate, decoupling power, and other variables will be presented in a separate report. To determine the limits imposed by ‘H decoupling on the linewidths of 13C resonances under ultra-high-resolution conditions, we examine here the effect of offset of the ‘H carrier frequency on the linewidth of the methyl resonance of acetone (Fig. 1), recorded with WALTZ16 decoupling. The experiments were done on a slightly modified (I) Nicolet NT-200 NMR spectrometer at a 13C resonance frequency of 50.3 MHz, with a standard 12 mm probe and a sample volume of 4.2 ml. Standard singlepulse 13C excitation was used. A hard-wired WALTZ16 d&al control box, purchased from Bio-Magnetic Instruments of West Lafayette, Indiana, was used to create the required 180” phase shifts in the ‘H decoupler circuit of the Nicolet NT-200 NMR spectrometer. In our sample (see caption of Fig. 1) lJ& = 126.85 Hz and 4JCH = 1.50

Nuclear Magnetic Relaxation Study of the Anisotropy of Rotational Reorientation in Liquid Boron Trichloride

Boron‐11 and chlorine‐35 nuclear magnetic relaxation measurements were used to determine the anisotropy of rotational reorientation in liquid BCl3. If the classical diffusion model is used, then the ratio of the rotational diffusion constants parallel and perpendicular to the C3 axis in 11B35Cl3 is 0.75 + 0.25, independent of temperature in the range − 90 to 30°C. The error estimate includes the effect of uncertainties in the 11B and 35Cl quadrupole coupling constants for the liquid. It is also shown that the determination of the anisotropy of rotational motion in liquids from relaxation times dominated by a quadrupolar mechanism is very sensitive to errors in the nuclear quadrupole coupling constants. This problem is least serious when the motion is nearly isotropic, as in BCl3.