Cross-Correlation

are the

What I Always Wondered About Coherence Selection, but was Afraid to Ask.
In treatments of spin coherence during multiple-pulse experiments it is often stated (see, for example, Ernst et al [l] p. 293) that quadrature detection selects the operator I , so that we should focus on phase changes of this operator.I have always been bugged by this statement because: 1. Quadrature detection is a purely engineering device.It is likely that many NMR instruments will not have quad detectors in the near future."Digital quad detection" is really a fancy way to describe one step of a high frequency Ff of a non-quad signal performed inside a DSP. 2. I is non-Hermitian, and its "expectation value" is trivially related to that ofl+ (they are complex conjugates of each other).
The answer to these questions is that the argument is both wrong and unnecessary, so it should not be invoked without proper qualification.I am quibbling to complain about this, but by avoiding a swindle and thinking correctly we might learn something interesting, or avoid errors.
This convention seldom has any bad consequence since the phase changes in (I_) are in direct correspondence to the phase shift of the high-frequency observable (Ix) in the fixed frame.Further, the originators of this convention did a good deed by focusing on one bunch of matrix elements, thereby promoting a universal language.Finally, not every book repeats this argument (for example Freeman) though many do.
This statement comes from writing that the quad detector gives a signal S = (Ix)-i( Iy) (equation A), and that I_= Ix -ily (equation B).Therefore, obviously S = (I_) (see Ernst et al, equation 6.3.2).This is in error, in a slightly interesting way.The error is that the "i" = H factors in these equations are not identical.Rewrite equation A as S = (Ix) -j(Iy) where j2 = -1, but ij is not equal to -1.The quantity j was introduced (in fact by me in J. Chem.Phys.54,  1418 (1971), as "i") as a convenient way to organize data inside the computer, and has no deep physical significance.The relation between i andj may be a "hypercomplex" one similar to the relation between the hierarchies of hypercomplexity in 2D and higher NMR, as described in Ernst et al. p. 307-308, and implemented by States and Haberkorn.The argument would still be a swindle applied to quad coil detection [2] where both (Ix) and (Iy) are detected in the fixed frame.As everyone knows, hypercomplex numbers are related to quaternions (invented by Hamilton) and I tried to educate myself about these, to see if they could be useful other than as a description.I did not get any useful insight If there are careful NMR papers on this topic I would appreciate learning of them.Incidentally, electrical engineers now routinely use quad detection, but I have found only one book that explicitly treats the quad outputs as real and imaginary.I think they lose a lot by not doing so, but maybe they know that they will get confused.Sara Kunz and I discussed this point in the January JMR (hope you liked the cover!).When you need problem-solving power in an NMR probe, Varian has your solution.

/J4
Varian probe design for NMR performance excellence at all field strengths and for all magnet types is achieved through precise balancing of sensitivity and RF homogeneity.This balanced design, combined with state-of-the-art components such as GaAs FET preamplifiers, give Varian probes unparalled experimental performance.Exacting handcraftsmanship, use of susceptibility-corrected RF coils, and RF coil shape ensure unmatched performance in lineshape, resolution, and water suppression.
The Penta probe is an exciting new addition to the distinguished Varian family of NMR products, designed and built to the most demanding specifications in the industry.
All Varian probes produce guaranteed levels of performance in even the toughest NMR applications.Don't settle for less in probe design and performance.Contact the Varian office nearest you for more information on the new Penta probe.Product and company information is updated regularly at our web site: www.varian.com.

varian @
Varian Probe Design for Maximum

NMR Performance
Innovative designs that achieve the highest sensitivity in multiple pulse n-D applications are a direct result of the careful optimization and balance of many performance factors:

Probe Design Factor
• Lineshape • RF homogeneity Long long ago as a postdoc at Saclay (France), I spent a very long time building a high resolution spectrometer from scratch, knowing that if I got the probe materials wrong, all the lines would be broad and the experiment useless.Not surprisingly, I found >200 Hz linewidths, even after redesigning the probe a couple of times.I was using a Pound spectrometer as the rf unit, and eventually it dawned on me that the problem was radiation damping (1), greatly enhanced by the positive feedback (regeneration) in the Pound box (2).Going to a conventional rf transmitter solved the problem.It turned out that this phenomenon had been anticipated by Losche in East Germany, but I had overlooked this work until Jack Powles pointed it out.This cautionary tale served • to sensitize me once and for all to the insidious dangers of radiation damping in high resolution NMR.Now, some forty years later, this same demon is beginning to cause more widespread troubles in high resolution NMR when we use aqueous solutions in high polarizing fields.Consequently, several authors have proposed methods to suppress radiation damping by negative feedback (3), Q-switching (4) or the application of bipolar field gradients (5).
We have tried a different approach, compensating the radiation damping field (a torque about the -x axis) with a DANTE sequence of tiny rotations about the +x axis that just balance the effect of the radiation damping field.As the transverse NMR signal decays with time, the flip angle of the DANTE pulses is reduced at a matching rate.One complex data point is acquired in each interpulse interval.Once the DANTE frequency has been tuned to the water resonance, the inherent spectrometer stability ensures that it stays at resonance and maintains the correct phase.The water signal then decays quite slowly and the rest of the spectrum is unaffected.

1
H dipolar interactions of peptide backbone amides has been quantitatively measured, and this cross-correlation was demonstrated to be directly proportional to the generalized order parameter S 2 (1).However, in principle, the strength of the relaxation interference depends on the angles between the unique axes of the CSA and dipolar tensors, so neither CSA values nor values of the spectral density J ( ro) are determined from such transverse cross-correlation experiments.Measurement of the longitudinal cross-correlation rate can be readily carried out and, in combination with the transverse cross-correlation rate, can be useful for elucidating dynamics.11zese /actors, along with computer driven automatic testing and e11viro11me11tal stress screening of t/ze final product, ensure t/zat t/ze perfon11a11c,?, quality and reliability meet AMT's exacting standards.

Sample Preparation and Handling in the SubMicro World
Our contributions the past several issues have addressed the performance and capabilities offered with a prototype I. 7 mm SubMicro Inverse-Detection Gradient or SMIDG™ NMR probe built for us by Nalorac.Performance notwithstanding, it is still necessary to get the sample into the tube and then into the spectrometer before what the probe contributes begins to matter.If sample preparation and the subsequent handling of the sample are deficient in technique, the difficulty in working with submicromole samples is proportionately increased.For these reasons, we thought it might be worthwhile to address sample preparation and handling in this contribution.

1
Much of the work our group at Pharmacia & Upjohn does is related to the isolation and characterization of impurities contained in candidate drugs and the study of the products of degradation after candidate drug molecules are subjected to various stress challenges, photooxidation, etc.To support this work, standard analytical and preparative HPLC methods are employed to isol~te the sample(s) intended for study.At this point, pooled fractions from a preparative chromatographic system often contain contaminants that produce "chemical noise" in an NMR spectrum.To reduce the level of chemical noise-causing contaminants in our samples, pooled fractions containing the analyte of interest are subjected to cleanup via a chromatographic trapping procedure.The first step in the procedure is to reduce the percentage of organic mobile phase modifier (acetonitrile, methanol, etc.) in the pooled fraction by rotary evaporation or dilution with water.The resulting primarily aqueous solution is then pumped onto a 10 x 250 mm K.romasil Cl8 column which has been charged with water.This step traps the analyte on the head of the K.romasil column and irreversibly retains the majority of hydrophobic contaminants (mobile phase stationary phase, oil and grease from glassware, etc).Ionic mobile phase additives (trifluoroacetic acid, ammonium formate, etc) remaining from chromatographic isolation are then washed from the trapping column with 10 column volumes of water.The analyte is then eluted from the trapping column with 100% acetonitrile.Finally, the resulting analyte solution obtained from the trapping column is transferred to a specially cleaned (washed 3X with HPLC grade hexane) 10 ml conical vial and lyophilized.The dried samples are transferred, under vacuum, if necessary, to an argon atmosphere glove box where the NMR sample is prepared.The approximate sample recovery for this method of sample preparation is -70%.For submicromole quantities of material, all sample preparation is done in the glovebox to avoid the introduction of water since a significant percentrage of the samples are prepared in d6-DMSO.Preparation on the bench outside of the glovebox is undesirable.Using a I µmole sample of ibuprofen (1), for example, when the sample is prepared on the bench, the largest peak in the spectrum is the water that the sample picks up during preparation (see Figure I).Note that the water peak is approximately the same size as the methyl doublet furthest upfield when all peaks are plotted on-scale.

USA
To illustrate the type of sample that can be obtained using the procedure just briefly outlined, a 50 µg sample of linezolidrn (2, PNU-100766) was subjected to preparative HPLC isolation and chemical noise cleanup.A 50 µg sample was injected onto an reversed phase, isocratic, preparative HPLC system employing an acetonitrile, water, and trifluoroacetic acid mobile phase.The linezolid peak was collected and the resulting fraction processed through the chemical noise cleanup procedure described above.Assming 70% recovery following chemical noise cleanup, the final, isolated sample was estimated to contain -35 µg or about 0.1 µmole of sample.The sample was prepared on the benchtop, giving rise to the intense water resonance at 3.3 ppm, which is the strongest resonance in the spectrum.For weaker samples, this level of water contamination would be totally unacceptable as noted in the text.
The isolated sample of linezolid was concentrated in the bottom of a conical bottom vial and then passed into the glove box for sample preparation.An aliquot of 28 µl of 99.992% d6-DMSO (Cambridge Isotope Laboratories) was added to the sample vial using a flexible teflon needle (Hamilton #90624) small enough to be threaded to the bottom of the l.7 mm NMR tubes attached to a Hamilton 50 µl gas-tight syringe.Mixing and complete dissolution were accomplished by slowly drawing the sample in and out of the teflon needle several times before drawing it into the needle for the final time.At that point, the teflon needle was inserted into the l.7 mm NMR tube to the bottom of the tube.As the needle was then withdrawn the plunger of the syringe was slowly depressed to inject the sample into the tube.Considerable hangup of the solvent on the walls of the tube due to capillarity is possible so care should be taken during this step in the process.After the sample has been completely introduced to the tube, the tube should be shaken down to be certain that all solution hanging on the walls of the tube is down.We have found sample columns in the 1. 7 mm tubes in the range of 20-22 mm (24-26.4µl) to be well suited for ease of shimming.Sample volume in the tube can be calculated at 1.2 µI/mm of column height.This volume of solution can be easily transferred using the method just described if the initial sample is prepared with 28 µl of solvent.• The 1 H reference proton spectrum of the 50 µg sample of 2 subjected to the chromatographic workup and handling just described is shown plotted above a GHSQC spectrum of the antibiotic in Figure 2. The GHSQC spectrum was acquired as 2048 x 64 States-TPPI hypercomplex files in slightly <5 h with 128 transients accumulated/ti increment.The data were processed by linear prediction to 192 files in Ft followed by zero-filing to 256 points prior to transformation.Gaussian multiplication was used used prior to Fourier transformation with the weighting function optimized to the data.States-TPPI hypercomplex files with 32 steady state transients and 64 transients accumulated/t1 increment.Total acquisiton time was slightly <5 h.Data were processed using linear prediction to 192 files in F1 and zero-filling to 256 points.Gaussian multiplication optimized for the data in both frequency domains was applied prior to Fourier transfonnation • As will be noted from the proton reference spectrum shown in Figure 2, the sample preparation just described affords a sample for NMR analysis that is free of any chromatogaphic artifacts and that is also reasonably dry.All of the protonated carbon responses are easily observed in the short <5 h data acquisition.In comparison, a sample of this size studied in a 3 mm probe would typically require at least an overnight acquisition to provide comparable data.1-3Long-range GHMBC data were accessible on the -0.1 µmole sample of 2 in an overnight acquisition.In contrast, long-range data on a sample of this size in a 3 mm gradient inverse micro probe would generally be either out ofreach or would require a full weekend of data acquisition.The lowest level 3 mm long-range data acquired in conjunction with a structure elucidation problem we are aware of was in the elucidation of the structure of crytolepicarboline, which was done on -0.25 µmole (398 mw).4 Work

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recently reported from these laboratories has demonstrated that it is feasible to acquire long-range GHMBC data on samples as small as 0.04 µmole over a weekend.4 Regarding the water peak in the proton reference spectrum plotted above the contour plot in Figure 2, note that it is only slightly larger than the N-acetyl methyl singlet.In contrast, for the lµmole ibuprofen sample preapred on the benchtop, the water was there the size of the methyl doublet, roughly five times the intensity of the corresponding water peak when prepared in the glove box as in Figure 2 (when the molar ratio of the two samples are considered).Furthermore, it is likely that the water in the spectrum in Figure 2 is residual to the chromatographic preparation of the sample.In contrast, the water in the spectrum in Figure I was absorbed by the solvent during preparation of the sample from a reference standard using serial dilution techniques.If the sample in Figure 1 had been chromatographically prepared, it is probable that the water resonance would have been correspondingly more intense.
In conclusion, SMIDG NMR probe technology now allows the spectroscopic characterization of samples that can be readily obtained from single semi-preparative HPLC isolation followed by treatment to reduce chemical noise in the sample arising from chromatographic artifacts, etc.This capability can be expected to greatly enhance the ability to characterize impurities and degradants of pharmaceuticals, their metabolites, combinatorial chemisitry products when the chemistry has led to unanticipated reaction products, and minor natural products.
Many of these types of problems have in the past either been out of reach of all but proton NMR experiments, or have required extensive chromatographic effort with sample pooling to obtain sufficient quantities for even direct correlation inverse-detected heteronuclear experiments.
Position Available Rick Beger has just accepted a position with the FDA and will be joining them in the very near future.data base as well as the internal data base so that the accuracy of calculations is increased.
• Search according to user-defined fields (lab book number, project name, chemical name, operator, etc.).
• Search multiple user data bases at a time • Manual or automatic numbering of atoms in molecules prior to prediction.Same atomic numbering schemes for all nuclei.
• Calculates spin-spin interactions, emulates off-resonance, DEPT, J-modulation and much morel • Recognizes spectral differences between the following types of isomeric structures: cis-trans isomers of alkenes; cis-trans and endo-exo isomers of cyclic structures; syn-anti isomers of amides, oximes, hydrazones and nitrosoamines.
• Calculates the spin-spin interaction of carbon nuclei with magnetic nuclei of other elements, proceeding from the natural ratio of magnetic isotopes.

Spectral Representation
• Emulates the operation of an actual spectrometer, allowing the user to choose among different modes of obtaining spectra (operating frequency, off-resonance, splitting, DEPT, J-modulation, CH3, CH2, CH and C-subspectra).
• Easily attributes spectral peaks and chemical shifts to nuclei and vice versa.

Directly-coupled CE-NMR and CEC-NMR in Drug Metabolism
Direct coupling of HPLC with NMR spectroscopy is now a routine commercially-available technique which has proved useful in many areas of analytical chemistry particularly for drug metabolite identification and the information content of such studies has been extended by the further hyphenation with mass spectrometry.There have been a number of extensions to the approach involving other types of separation and Jonathan Sweedler has reported developments in capillary electrophoresis coupling to NMR (CE-NMR).This methodology and that of capillary electrochromatography (CEC-NMR) has now been evaluated using an extract of human urine containing the metabolites of acetaminophen.This work has been a collaboration with Ian Wilson at Zeneca Pharmaceuticals, and Professors Ernst Bayer, Klaus Albert and their research teams at the University of Tiibingen, Germany.The metabolism of acetaminophen (4hydroxyacetanilide) has been studied extensively using NMR spectroscopy and it is known that the two major metabolites are the glucuronide and sulfate conjugates of the phenolic hydroxyl group.More recently the metabolism of acetaminophen has been used by us as a model system to investigate the usefulness of both HPLC-NMR and HPLC-NMR-MS.
1.5 ml of human urine was collected 3 hours after a normal therapeutic dose of acetaminophen and was extracted by passing it down a C18 solid phase extraction cartridge previously conditioned with methanol and HCl and the fraction containing the metabolites was washed off with 100% methanol and evaporated to dryness.The experimental arrangement for the CE-NMR and CEC-NMR experiments has been reported by the Tubingen group (Anal.Chem., in press).The continuous-flow CE-NMR experiment gave the result shown in Figure l(a) which is viewed as a contour plot with CE separation time on the vertical axis and the NMR chemical shift on the horizontal axis.The peaks spread throughout the figure arise from formate from the buffer at 88.4, residual water in the D 2 0 buffer at 84.7 and a small amount of glycine which remained from earlier use of a glycine-containing buffer at 83.5.In addition at separation times of 49 min and 73 min sets of peaks related to acetaminophen can be observed.The first eluting can be assigned to acetaminophen glucuronide from the known aromatic proton and N-acetyl methyl chemical shifts taken together with the diagnostic shifts of the glucuronic acid moiety.The second set of acetaminophen-related resonances show aromatic proton peaks consistent with the sulfate conjugate of acetaminophen.Finally, a third component is detected in the continuous-flow CE-NMR experiment and the NMR • spectrum of this component is consistent with the endogenous compound hippurate.This shows the expected aromatic proton resonances together with a singlet from the glycyl methylene group.
Directly-coupled CEC-NMR spectroscopy was also achieved using the same sample.The result is shown in Figure l(b).The CEC-NMR result is similar to that from the CE-NMR experiment with the glucuronide conjugate eluting first .at 36 min, followed by the sulfate at 40 min and then hippurate at 50 min.Individual rows from the continuous-flow CEC-NMR experiment are shown in Figure 2 with the assignments as marked.
For CE-NMR, based on the observed signal-noise ratio seen in the NMR spectrum and given the known volume of the CE-NMR detection cell, it is estimated that approximately 10 ng of each metabolite was detected with a quality of result which would have allowed unambiguous identification of the molecules.Although CE-NMR remains a

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technically difficult procedure it has potential for identification of small quantities of analytes.The CEC-NMR spectra show better signal-noise ratio and this is a combination of both greater loading and active NMR detection volume.TecMag's upgrade kits contained everything needed for the transition.Each kit included a National Instruments PCI 96-bit interface board, PCI to NuBus adapter boards, and new headers for boards inside our Aries and two Libras.The upgrades for two of the systems were $650 each, including TecMag's 20% academic institution discount.For our oldest Libra with the NBE board, the upgrade kit was $950.This kit included an extra adapter board that ran from the clone to the Libra's NBE board.Both of the adapter boards for this system connect to the same board in the clone, so no additional expansion slots are tied up.Each system required a board that ran from the clone to the spectrometer's MACINTl board.
Three headers needed to be replaced on two boards for the Aries and the newest Libra.Four headers needed to be replaced on three boards for the old Libra; the extra header was for the NBE board.One of the Libra's had old wire-wrapped boards.Replacing the headers for that system only required careful use of tweezers to pull out the old ones.No soldering was involved.The other Libra and Aries had printed circuit boards, which have soldered connections to the headers.Each of the pins on the headers are fragile and closely spaced, so removing the old connections and soldering the new ones with your typical lab soldering iron would be very difficult and not worth the risk of damaging the expensive headers that probably paid for some TecMag vacations.Our electronics shop had some gear that they use for circuit boards, so they replaced the headers for two of the spectrometers.
PowerMac versions of the latest MacNMR software were available for free by downloading from the TecMag website.A few drivers also had to be replaced; these too were also available on their website.
It's certainly not as exciting as laser-polarized xenon, but any contribution I can make to insuring competitiveness in the computer market place is worth it.
Please credit this contribution to the Raychem account. Sincerely,

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UMEA UNIVERSITY The aim of this approach is to find a suitable way of handling an independent matrix of complex spectra (X) and its relation to a dependent matrix of properties ( Y, i.e. reactivities, toxicities, or other activities).Small systematic information in X, related to Y, is commonly hidden in other dominant variation in X (instrwnental and or sample instabilities, temperature effects, different measuring conditions etc.) .If these unwanted systematic variation is of similar magnitude as the the variation sought for, then normal PLS regression can handle this using FID's or frequency spectra in X.However, if the looked-for variation is minor then orthogonal scatter correction ( OS C) on the FID 's turned out to be quite useful i.e.OSC removes variation in X which is_ not related ( orthogonal) to Y.
A typical set of CP/MAS NMR spectra of dissolving pulps could be illustrative.Using PLS modelling on 17 FID's without OSC resulted in 5 components totally explaining 85 .5 % of the quality variables in Y.However, three of the components described most of the variation in X (98.6 %) but almost no variation in Y (1.5 %).Performing OSC on the FID's as a preprossesing method improved the predictive ability of the procedure significantly.Only 3 components were significant explaining 99.2 % in Y (cross-validated value, see below).The resulting loading spectrum after FT ( showing only those carbon signals related to the Y changes), showed amazing improvement relative to the experiment using unpreprocessed data.----------------- Now, the fundamentally superior precision and stability of digital signal processing is available from a precedent-setting series of NMR spectrometers.With its digital advantage, the Bruker AVANCE'" series sets revolutionary standards for performance, long-term reliability and ease of use, whether for routine applications or the most demanding research.

Best regards 2d
The modular architecture of the Bruker AVANCE design makes extensive use of digital signal processing technology, incorporating high performance RISCbased processors into the lock, filters, timing control unit, gradient generation, and many other key areas of the system.The result is increased sensitivity, higher dynamic range, cleaner spectra, flat baselines and unprecedented stability.
The AVANCE Series of high performance spectrometers.
The comprehensive AVANCE 'family of NMR spectrometers was developed in direct response to the increasing demands of the NMR community for greater performance and stability in a highly automated, easy to use instrument.Within the AVANCE series of DPX, DAX, OMX and DSX systems there is a virtual continuum of configuration options from 200 to 750 MHz, including solids, liquids and imaging .Whatever the environment or application, there is an appropriate AVANCE model to choose from.Your Bruker representative will be happy to recommend a configuration that is optimum for your needs -today and tomorrow.The Influence of Adsorbed Benzene on Na-sites in NaY Zeolite Investigated by 2D Triple-quantum 23 Na MAS NMR Spectroscopy Dear Prof. Shapiro:

For complete details or to arrange a demonstration please contact your nearest
The effect of benzene adsorption on 23 Na resonance and the quadrupolar interaction in NaY zeolites is studied by two-dimensional triple-quantum MAS 23 Na NMR spectroscopy.
The pulse program is based on the two-pulse sequence proposed by Frydman et al. [1], and adds a z-filter pulse [2] to reduce the errors from the phase-cycling.The 2D-spectrum of dry NaY zeolite shows two kinds of 23 Na signal, the gaussian-like down-field peak along SQ-axis may be correlated to SI-Na and the high-field peak with quadrupole-splitting to SII-Na [3,4].In Fig. 1, with increasing benzene-loading, the SII-Na signal changes from a quadrupole-splitting lineshape to a gaussian-like lineshape and displaces to a new position in the 2D-spectra.For SI-Na, the calculated isotropic chemical • shifts (ocs) are -7.3,-7.1, -6.5, -6.1 and -6.1 ppm referred to solid NaCl, for the NaY zeolites with loadings of•O, 0.3, 0.8, 2.0 and 5.0 C6D6 per supercage.This reflects a small effect of down-field shift of SI-Na in the vicinity of adsorbed benzene molecules on SII or the 12R window site [5].Also, the calculated second-order-quadrupolar-effect (SOQE) parameters are 1.3, 1.2, 1.2, 1.3 and 1.2 MHz for the same loadings.In addition, for benzene-adsorbed SII-Na, the calculated ocs parameters are -21.9,-22.5 and -23.3 ppm for the loadings of 0.8, 2.0 and 5.0 C6D6 per supercage, and the calculated SOQE parameters are 2.3, 1.9 and 1.7 MHz for the same loadings.The ring current effect of benzene may give rise to the up-field trend of the ocs parameter for SIT-Na adsorbed with benzene.Nevertheless, adsorption of benzene may reduce the electric field gradient at SII-Na as well and results in a decrease in magnitude of SOQE for SII-Na.H NMR spectroscopy of paramagnetic heme proteins has provided a wealth of information on the electronic and molecular structure of the heme prosthetic group and the nearby amino acids. 1 In comparison, much less information is available about the 13 C resonances arising from carbons in the prosthetic group.This stems from the inherently low sensitivity associated with the observation of 13 C nuclei, and from the lack of a general process for isotopic labeling of the heme.
Although many of the problems associated with the poor sensitivity encountered with 13 C NMR experiments of paramagnetic heme proteins have been overcome with indirect-detected experiments such as HMQC, 2 the assignment of quaternary carbons in paramagnetic hemes continues to be challenging.Hetero-correlated experiments based on the relatively large value of 1 JcH (HMQC) typically meet the condition T 2 • 1 < 1 Icw Consequently, development of coherence and antiphase cancellation are less problematic in HMQC than in experiments based on 3 JHH, such as COSY.In fact, HMQC has been successfully applied to a number of heme proteins to obtain 1 H and 13 C assignments corresponding to heme C~ units. 2 • 3 In contrast, the application of hetero-correlated experiments to the detection of long range 1 H-13 C correlations in fast relaxing systems has been less successful.2• 4 This is largely due to the fact that the condition T/ > 2 JcH is typically encountered in paramagnetic hemes.Consequently, the assignment of quaternary carbons, which are typically obtained with HMBC experiments in diamagnetic molecules, are not readily obtained in fast relaxing paramagnetic heme proteins.
We have recently reported a biosynthetic approach for the isotopic labeling of heme which may be applicable to most heme proteins with removable hemes. 5In this letter we wish to communicate that the assignment of quaternary carbons in a paramagnetic active site has been carried out with a 13 C-13 C double quantum coherence experiment, INADEQUATE, 6 and a protein containing doubly-labeled heme, taking advantage of the relatively large value of 1 Jcc• The heme in mitochondrial cytochrome b 5 was labeled as shown in Fig. 1, utilizing 5-13 Co-aminolevulinic acid as a source of 13 C-label.The INADEQUATE spectrum obtained from 13 Clabeled-heme cytochrome b 5 is shown in Fig. 2. We believe that this is the first demonstration of a 13 C-13 C double quantum coherence experiment applied to a paramagnetic heme incorporated into a protein.Furthermore, it is clear that all the expected type I and type III correlations Please credit this contribution to Lydia Chang's account.
Yours sincerely I don't know if Ray Freeman likes the films of Federico Fellini, but I could not help associating this one-of-a-kind book with those films, especially Amarcord.There is a whole cavalcade of basic knowledge, well-developed conclusions, little tricks and secrets, all presented with lots of artistry, love of the subject, and superior elegance.This association was further supported by the way how Professor Freeman chose the title for his new book, comparing the dance of nuclear spins choreographed by pulse sequences to that of ballet dancers.There is one point, however, where this association breaks down; "Spin Choreography" is much more than looking to the past.We find not only those basic ideas and results started at the dawn of high resolution NMR spectroscopy, but also lots of brand new applications, as revolutionary as very broad band decoupling using adiabatic pulses, for example.At the same time, this book is edited in a highly systematic fashion, carries a lot of educational value, and can be useful material for any university course.
The book consists of twelve chapters, and an Appendix -explaining an intentionally modest number of acronyms (with original literature references) -and a five-page Index.Each chapter starts with a content summary, and is completed with carefully selected references.These references embrace practically the whole history of high resolution NMR, making direct connections between the late forties/early fifties and the upcoming end of the century.The first three chapters provide the basic tools for proceeding with the rest efficiently (Energy Levels; Vector model, and Product Operator Formalism).
In the following nine chapters one will find some condensed matter -quite an encyclopedia of most of the fundamental NMR phenomena and examples of their use.Discussion of spin echoes is followed by that of soft radio frequency pulses, touching on some aspects of multidimensional spectroscopy.Those readers (most of us, I guess), who would miss the enlightening illustrations well-known from "A Handbook of NMR" and the many lectures of Prof. Freeman, will be compensated by titles (and attitude), such as "Separating the wheat from the chaff' (Chapter 6).This chapter is about selection of information of interest against artifacts or unwanted magnetization of any kind, using phase cycling, gradients, various filters and data processing approaches.Broadband decoupling, making heteronuclear experiments feasible at very high fields, is the subject of the next chapter.The discussion starts from basic theory, surveys various approaches popular at times (many still being used), concluding with the use of adiabatic pulse techniques.Two-dimensional spectroscopy in the next chapter covers the basic homo-and heteronuclear correlation techniques, including multiple-quantum and J-correlations, with a brief look at higher dimensionality and applications for isotope-labeled substances.The nuclear Overhauser effect is discussed in an independent chapter (Chapter 9)."In defense of noise" is the title of the next chapter, where various sources of noise, its suppression, treatment of noisy data in data processing, and, as the title hints, use of noise (in stochastic excitation and noise decoupling, for example) are discussed.It is rare to see such a thoughtful summary of this aspect of signal processing and data treatment.The last two chapters deal with water, including suppression techniques and radiation damping, and various ways of measuring J-coupling constants, in both time and frequency domain.
Each chapter focuses on a more-or-less stand-alone subject, which leads to some overlap and repetition, but each time in a different context.The text is well supported with lots of graphical illustrations and example spectra (real or simulated), and all processes are described with the aid of product operators.This book can be recommended to anyone who uses NMR and wants to learn more about the subject.It makes an excellent source for university courses, •and serves as a reference book, too.And, above all, it is pleasant reading, even in the evening when the music is on -let the spins dance!--------------------------l As a result of the shift towards high throughput screening and combinatorial analytical methods, we at Advanced Chemistry Development are pursuing any possibility to support this exciting field .In collaboration with Varian NMR Instruments, specifically Evan Williams and Paul Keifer, we have enhanced our NMR software offerings to support Combinatorial NMR applications.ACD/Combi-NMR presently allows the user to access NMR data directly from the spectrometer and process using a Group Macro processing feature.We have used this to process the data obtained from a 96-well plate but have tested the bulk processing on over 200 FIDs.An alternative of course is to obtain the appropriate set of phasefiles from the spectrometer.Following processing the spectra automatically populate a database identical to those generated in our ACD/NMR Manager product.
The Combi/NMR module can be used to allow spectral, subspectral or multi-subspectral searching with the "hits" displayed on a 96-well plate format using color coding to show responses to the search.An example is shown below where the multi-subspectral search was performed on the spectrum immediately below and matches, spectra containing the highlighted subspectra, were located within the 96-well plate as shown at right.Green highlighted buttons indicate the hits with the lower screen indicating one of these hits and the upper screen showing the pattern for matching.ACD/Combi NMR therefore allows the user to remove the data from the spectrometer and perform necessary data handling at the desk.Following this data handling the Combinatorial plate data sets can be stored as individual databases or merged into a larger overall database containing up to 500,000 spectra.Each entry in the database can be associated with up to 16,000 textual user data fields that are fully searchable through the database interface.These of course could include Notebook #'s, Chemist's Name, Solvent details, Synthesis ID# or whatever. .Iii Iii Iii Iii Iii Iii Iii Iii Following acquisition of a Cambi data set the user has the opportunity to add suggested structures and associate them with particular spectra in the dataset.Presently this can be done directly through our integrated ChemSketch structure drawing package (which can read ChemDraw, ISIS.skc, molfiles or many other formats).Using the ACD H1 NMR prediction algorithms we generate predicted spectra for each of the suggested structures and display them on screen for direct visual comparison with the experimental spectra.This comparison could obviously be performed just by manually screening for matches but we perform a statistical analysis based on the differences in shifts between the experimental and predicted spectra and produce a Cambi-result factor which varies between 0 and 1, 1 being a perfect match.The obtained values are then displayed using color coding to display ranges for the match factors.Databasing these spectra including structures allows future searches of groups of such databases by structure or substructure as well as the user definable textual data.
Our future improvement plans for • this development project include quantitation capabilities, ignoring exchangeable protons as an option during the spectral matching procedure and utilizing user databases containing structures and assignments pertinent to the chemistry of the user or laboratory in question.We look forward to our continuing collaborations with Varian as well as suggestions from your readers regarding possibly useful features that could be incorporated into ACD/Combi NMR.

Best wishes Barry!
Tony Williams tony@acdlabs.com

Are Computers Male or Female?
Five reasons to believe computers are male: 1.They have a lot of data, but are still clueless.2. They are supposed to help you solve problems, but half the time they ARE the problem.3.As soon as you commit to one you realize that, if you had waited a little longer, you could have obtained a better model.• 4 .In order to get their attention, you have to turn them on.5. Big power surges knock them out for the rest of the night .

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Five reasons to believe computers are female: 1 .No one but the Creator understands their internal logic.2. The native language they use to communicate with others is incomprehensible to everyone else. 3 .The message "Bad command or file name" is about as informative as, "If you don't know why I'm mad at you, then I'm certainly not going to tell you. 114.Even your smallest mistakes are stored in long-term memory for later retrieval.5.As soon as you make a commitment to one, you find yourself spending half your paycheck on accessories for it.

2 1
Figure I. GHSQC spectrum of 1 µmole (206 µg) of ibuprofen dissolved in 25 µI 99.996% d 6 -DMSO.The data were recorded as 2048 x 128 States-TPPIhypercomplex files with 1 transient accumulated/t 1 increment to give a total acquisition time of 6 min.All direct responses are visible, including the weak response for the methine of the sec-butyl group.The proton reference spectrum was acquired as a single transient following the application of a 90° pulse.The sample was prepared on the benchtop, giving rise to the intense water resonance at 3.3 ppm, which is the strongest resonance in the spectrum.For weaker samples, this level of water contamination would be totally unacceptable as noted in the text.

Figure 2 .
Figure 2. GHSQC spectrum of a 50 µg sample of the oxazolidinone antibiotic linzeolid (PNU-100766) subjected to the chromatographic workup described above.Sample recovery was estimated at 70%, affording a sample in the 1. 7 mm SMIDG NMR tube, assuming complete transfer, of -35 µg or -0.1 µmole (mw 349).The data were acquired using a Varian INOVA 600 spectrometer equipped with a Nalorac Z•SPEC SMIDG™-600-1.7 probe.The IH reference spectrum plotted above the contour plot was plotted with all peaks on scale.The largest peak in the spectrum arises from residual water at -3.3 ppm.The spectrum was acquired as 2048 x 64 States-TPPI hypercomplex files with 32 steady state transients and 64 transients accumulated/t1 increment.Total acquisiton time was slightly <5 h.Data were processed using linear prediction to 192 files in F1 and zero-filling to 256 points.Gaussian multiplication optimized for the data in both frequency domains was applied prior to Fourier transfonnation •
Thus, there is a post-doctoral position available to study the structures, metal ion binding and dynamicsof aptamer, telomere and damaged DNAs in NIH/ ACS funded studies.We have a Varian Unityplus 400 with 5 & 8 mm pfg probes and a Varian !nova 500, three channel spectrometer with pfg 5 & 8 mm probes.The group has extensive computational support including IBM and SUN workstations.Please send cv and three letters of recommendation to me at: Philip H. Bolton, Chemistry Department, Wesleyan University, Middletown, CT 06459 pbolton@wesleyan.edun •-•-•o ..... ., ......... O•l.f.' Ol•" ~•.., .ttn:.or ~~-1 H prediction window with zoom-in, and tables of chemical shifts and coupling constants Features of ACD/ Predictor • Contains the History Window which displays all the structures used for spectral calculations during the current program session.• Allows you to load/ save all the structures to/ from a file on a disk, print them, and import a new list of structures from an SDFfile (up to 99 per session).• Calculates spectra based on internal DAT files with tens of thousands of experimental chemical shifts (see table opposite) • For ACD / HNMR Predictor, 3D molecular structure minimization and Karplus relationships are used to predict proton-proton coupling constants • The new Calculation Protocol window with histogram display allows direct examination, as a histogram plot, on a nucleus by nucleus basis, of which structures within the database were used for the predictions.In this way all database structures utilized for the prediction of a single molecule can be screened one nuclear center at a time.• Increased accuracy through a proprietary self-training system.For a new class of compounds, you can create and/ or update your own user data base, which is completely searchable, with experimental chemical shifts.The next time you open your data base and make calculations for any new compound, the program can use information from both your opened

PLS Modeling ofFID Data Preprocessed by Orthogonal Scatter Correction.
Bruker representative.

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