2D spectroscopic techniques

The IR-2D spectroscopic technique applied in Section IV.C (30,42) utilized the frequency domain after selectively bleaching individual one-excitonic states using a narrowband intense pump pulse, a broadband probe pulse... 

With the backbone assigned, it is necessary to continue with the assignment of the side-chain protons. Commonly, another 2D spectroscopic technique, TOCSY (total correlation spectroscopy), is employed in addition to COSY. This... 

Chemists, biochemists, biotechnologists, and physicists now routinely use NMR spectroscopy as a powerful research tool. The effective application of ID and 2D NMR expteriments depends largely on the skill and innovation of the user. This book is intended to provide practical knowledge to research workers in the use of NMR spectroscopic techniques to elucidate the structure of organic molecules. Every attempt has been made to prevent the book from becoming too technical, and the underlying principles behind many of the experiments have been described nonmathematically. 

Preparative and analytical HPLC were carried out in an ODS column using gradient elution. The gradient was composed of methanol, water and formic acid. The chemical structures of the new pigments were elucidated by UV-VIS, 2D NMR and LC-MS. MS conditions were capillary 3 kV, cone 30 and 60 V, extractor 7 V, sources block temperature 120°C, desolvation temperature 150°C [257],The chromatographic profile of the SEC fraction containing the new pigments is shown in Fig. 2.116. The chemical structures of the new derivatives identified by various spectroscopic techniques are shown in Fig. 2.117. 

The volumes in the series of Spectroscopic Techniques An Interactive Course are delivered with special versions of ID WIN-NMR and 2D WIN-NMR. They are a supplement for this course to be installed on a stand-alone PC and to be used exclusively for processing the experimental data supplied in the NMR data base. They cannot be used to process the users personal NMR data. The full version of ID WIN-NMR and 2D WIN-NMR software must be installed for this purpose and a special copy protection dongle (a WIBU key for the single user mode, or a Net-HASP key for the multi-user/network mode) must be used. Note also that for 2D WIN-NMR a standard 16-bit and a more powerful 32-bit version exist. Please refer to the description in the corresponding Bruker manuals [2.1, 2.2]. 

Diffusion-ordered spectroscopy (DOSY)45 is a NMR spectroscopic technique that separates the NMR signals of different compounds according to their diffusion coefficient (D, their rate of diffusion in a particular medium). A series of spin echo spectra is measured with different pulsed field gradient strengths, and the signal decays are fitted to give diffusion coefficients for each compound present. In 2D DOSY this... 

We are beginning to develop a detailed understanding of these methods (18,21,30,33,34,37-40,42,44,47-49), many of which are described in this book. We have recently demonstrated a series of novel nonlinear all-IR spectroscopic techniques (IR-pump-IR-probe, IR-three-pulse photon echoes, IR-dynamic hole burning, IR-2D spectroscopy), all of them utilizing intense femtosecond IR pulses, with the intention to develop new multidimensional spectroscopic tools to study the structure and the dynamics of proteins (30,31,41,42,50-53). We shall summarize in this contribution our work, its underlying principles, and its applications. 

We have presented two types of nonlinear IR spectroscopic techniques sensitive to the structure and dynamics of peptides and proteins. While the 2D-IR spectra described in this section have been interpreted in terms of the static structure of the peptide, the first approach (i.e., the stimulated photon echo experiments of test molecules bound to enzymes) is less direct in that it measures the influence of the fluctuating surroundings (i.e., the peptide) on the vibrational frequency of a test molecule, rather than the fluctuations of the peptide backbone itself. Ultimately, one would like to combine both concepts and measure spectral diffusion processes of the amide I band directly. Since it is the geometry of the peptide groups with respect to each other that is responsible for the formation of the amide I excitation band, its spectral diffusion is directly related to structural fluctuations of the peptide backbone itself. A first step to measuring the structural dynamics of the peptide backbone is to measure stimulated photon echoes experiments on the amide I band (51). 

The complete assignment of individual resonances for a protein can, in principle, be achieved by using multidimensional NMR spectroscopic techniques. For simplicity, the following three 2D NMR H techniques will be discussed. They are COSY (Section 13.3), NOESY (Section 13.4), and TOCSY. These techniques allow for the identification of resonances for nuclei that are connected through bonds, those that are in close proximity in space, and those that are within a given spin system, respectively. 

The observed temperature dependence of the absorption cross section of 3-pentanone and the corresponding fluorescence intensity offers the possibility for a new type of temperature measurements.This technique gives access to 2D-temperature distributions between 300 and 1000 K relevant for precombustion conditions that could hardly be assessed with other laser spectroscopic techniques developed for combustion thermometry. By calculating temperatures from the ratio of simultaneously acquired intensity distributions, the measurement is independent on local tracer concentrations. Measurements in inhomogeneously mixed environments are therefore feasible. 

In concluding this review it must be noted that there are many other techniques that are being utilized to increase our understanding about the structure of synthetically important carbanions. A partial listing of these techniques would include the theoretical approaches taken by Schleyer, Streitweiser, Houk and others and classical spectroscopic techniques. There exist also a number of useful NMR techniques in addition to the 2D-HOESY method previously mentioned. These NMR techniques include analysis of chemical shifts, Li- N spin-spin splitting, Li quadrupolar coupling o and rapid injection which has proven useful as a technique for structural investigations of aliphatic carba-nions. Last, but certainly not least, the excellent thermochemical measurements recently reported by Arnett and coworkers serve to correlate the solid state structural studies with solution species. A... 

The structure of a given biflavonoid is typically elucidated using intensive ID and 2D NMR (COSY, NOESY).[10, 11] Circular dichroism (CD) is used to determine the absolute configuration in molecules that possess stereocenters. [12] Occasionally, additional spectroscopic techniques are used (MS, IR, UV and single crystal X-ray). 

Phytochemical studies on Narcissus obesus have resulted in the isolation of a new alkaloid named obesine (420) (see Fig. 24), accompanied by several known Amaryllidaceae alkaloids (74). The stereochemistry and structural determination of the alkaloid 420 have been carried out by spectroscopic analyses and by application of 2D NMR techniques. Although the H-6)8 proton (5 4.38) is masked by the H-3 proton (5 4.30-4.40), the H-6a proton (54.02) was assigned at higher field on the basis of the nuclear Overhauser effect (NOE) with H-12 endo (5 3.10) observed in the 2D NMR experiment. On the other hand, the a disposition of H-3 was confirmed by the NOE between H-3 (5 4.30-4.40) and H-12 exo (5 3.01). In the C-NMR spectrum of 420 a characteristic signal due to the C-11 carbon was observed at 82.7 (singlet) ppm. Also, a comparison of the H and NMR spectra of obesine (420) with those of the related alkaloid 3-epi-marconine (303) (50) was performed. 

An intriguing cytotoxic and highly methylated tripeptide, milnamide A (57), was recently isolated by Crews and coworkers from the orange sponge Auletta cf. constricta, collected in Papua New Guinea (55). Cytotoxicity observed in the crude extract was partitioned into a dichloromethane layer which was further purified to yield milnamide A and an unrelated cyclic peptide. The structure is based on spectroscopic data, especially 2D NMR techniques the stereochemistry has not been established. A related tripeptide from the South African sponge Hemiasterella minor (also reported without stereochemistry) lacks only the C(l) methylene, and thus the p-carboline ring system (56). 

If the teaching versions of ID WIN-NMR and 2D WIN-NMR have not been installed already as part of Spectroscopic Techniques An interactive Course they must be installed as outlined below. The CD-ROM also contains the NMR-SIM, ID WIN-NMR and 2D WIN-NMR manuals as pdf files plus a copy of Adobe Acrobat Reader required to read pdf files. Finally the CD-ROM contains a file resulLpdf containing additional information and results appertaining to the various Check its in this book. 

In all the books in the series Spectroscopic Techniques An Interactive Course the emphasis is on the interactive method of learning and this volume follows the same approach. The remaining chapters in this book all have a similar format, a short written introduction and number of Check its for the reader to complete. Before each Check it is a short introduction which may include the discussion of new concepts or the advantage or disadvantage of a particular pulse sequence etc. The Check its are then used to illustrate the points being discussed either by displaying the processed data in ID WIN-NMR or 2D WIN-NMR or in the case of the Bloch simulator in a spherical or other display modes. 

In keeping with the overall philosophy of Spectroscopic Techniques An Interactive Course the reader is encouraged by a series of Check its to become familiar with the software tools NMR-SIM, ID WIN-NMR and 2D WIN-NMR and to try their own simulations. To assist the reader the character format used is the same as used in the other books in this series ... 

Table VI summarizes the alkaloids encountered in the Sri Lankan flora, together with the classes to which they belong and the plant and its part from which each alkaloid was isolated. A total of 43 plant species, including 13 endemic species, yielded alkaloids (Table IV). From these 197 alkaloids were isolated, and 51 were found to be new (Table VI). The structures of most of the new alkaloids were elucidated by modern spectroscopic techniques, including 2D NMR methods. Except for 8-hydroxyquinoline-4-carbaldehyde (1), artabotrine (50), 8-methoxyouregidione (51), cyathocaline (53), koenoline (64), and monogagaine (181), the new bases have not been evaluated for their biological activity.

The structural elucidation of naturally occurring oligostilbenes depends on a modern spectroscopic evidence, such as MS, NMR, IR and UV. All kinds of 2D-NMR techniques (including COSY, HMQC, HMBC, NOESY) play important roles in the structural elucidation of oligostilbenes. We will mainly summarize their 1H NMR and l3C NMR characteristics in this section. 

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