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Graphical abstract, Highlights • Studies on proline hydroxylase selectivity reveals new products. • Proline hydroxylases can produce dihydroxylated 5-, 6-, and 7-membered ring products. • Proline hydroxylases can accept bicyclic substrates. • Bicyclic products arise via bifurcation: two C-H bonds are accessible to the reactive oxidising species. • The results have implications for other oxygenases, including those catalysing protein modifications. • The results highlight the potential for amino acid hydroxylases in biocatalysis., Studies on the substrate selectivity of recombinant ferrous-iron- and 2-oxoglutarate-dependent proline hydroxylases (PHs) reveal that they can catalyse the production of dihydroxylated 5-, 6-, and 7-membered ring products, and can accept bicyclic substrates. Ring-substituted substrate analogues (such hydroxylated and fluorinated prolines) are accepted in some cases. The results highlight the considerable, as yet largely untapped, potential for amino acid hydroxylases and other 2OG oxygenases in biocatalysis.

Whereas trimers and tetramers of β- C - d -arabinofuranosyl carbohydrate amino acids adopt well defined conformations based around a repeating β-turn like structure, stabilised by (i, i-2) inter-residue hydrogen bonds, there is no indication of any secondary structure in the tetramer of the epimeric α- C - d -arabinofuranosyl carbohydrate amino acid, where the C-2 and C-5 substituents of the tetrahydrofuran ring are trans to each other.

Publisher Summary This chapter presents the introduction of two-dimensional (2D) methods, specifically in the context of determining homonuclear correlations. This is so that the 2D concept is introduced with a realistically useful experiment in mind, although it also becomes apparent that the general principles involved are common to any 2D experiment. Following this, a variety of correlation techniques are presented for identifying scalar (J) couplings among homonuclear spins, which for the most part means protons although methods for correlating spins of low abundance nuclides such as carbon are also discussed. Thes chapter introduces the operation and use of pulsed field gradients. The techniques themselves begin with basic correlation spectroscopy (COSY), which maps nuclei sharing a mutual scalar coupling within a molecule, making the proton–proton COSY one of the workhorse techniques in organic structure elucidation. In the chapter, some variants of the basic COSY experiment that display a number of beneficial characteristics are presented. The chapter discusses techniques such as total correlation spectroscopy (TOCSY), which provides an alternative to the COSY approach for establishing correlations within a molecule. This provides efficient transfer of information along a network of coupled spins, a feature that can be extremely powerful in the analysis of more complex spectra. The final method—incredible natural-abundance double-quantum transfer experiment (INADEQUATE)—establishes correlations between like spins of low natural abundance.

This chapter brings together a collection of the most commonly used NMR techniques for the identification of a chemical structure and applies these to the structure elucidation and spectrum assignment of a single molecule. The descriptions highlight how these techniques may be combined in a systematic manner to define the structure and stereochemistry of an illustrative compound. A range of 1D and 2D spectra are reproduced and their interpretation described in detail.

This chapter initially provides a comprehensive introduction to how 2D nuclear magnetic resonance (NMR) spectroscopy functions, extending the idea presented previously for 1D techniques into an additional second dimension. It considers how 2D data sets are generated and describes the practicalities associated with their use. This includes aspects such as quadrature detection, 2D lineshapes, possible artefacts, and details of data acquisition and processing. The concept of coherence and coherence transfer between nuclei is then introduced since this lies at the heart of many NMR correlation methods. The chapter then descries how pulsed field gradients operate and how these are used within modern NMR techniques for signal selection and signal suppression. Finally, the notion of ultrafast single-scan 2D NMR is introduced.

Publisher Summary This chapter describes the most widely used one-dimensional (1D) techniques, ranging from the optimization of the single-pulse experiment to the multiplicity editing of heteronuclear spectra and the concept of polarization transfer, another feature central to pulse nuclear magnetic resonance (NMR) methods. This includes the universally employed methods for the editing of carbon spectra according to the number of attached protons. Specific requirements for the observation of certain quadrupolar nuclei that posses extremely broad resonances are also considered. The approach to any structural or mechanistic problem will invariably start with the acquisition of 1D spectrum. As these provide the foundations for further work, it is important that these be executed correctly and full use be made of the data they provide before more extensive and potentially time-consuming experiments are undertaken. There are two extreme cases to be considered for the single-pulse experiment—in particular, whether one is striving for optimum sensitivity for a given period of data accumulation or whether one requires accurate quantitative data from the sample. The experimental conditions for meeting these criteria can be quite different, so each shall be considered separately.

This chapter considers NMR methods that are specifically tailored to qualitatively and quantitatively investigate the interaction of small molecules with protein receptors, and as such find use in protein–ligand screening. The basic phenomenon of the ligand binding equilibrium is first introduced, and then various methods to investigate this are described. The methods focus primarily in ligand-observed techniques, and cover the most common approaches, including direct ligand observation with relaxation editing, saturation transfer difference (STD), water-LOGSY (ligand observation with gradient spectroscopy), and exchange-transferred nuclear Overhauser effects (NOEs). The concept of competition experiments for detecting high-affinity ligands is introduced along with the notion of competitive ligand screening via a reporter or spy molecule. Finally, methods that employ observation of the isotopically labelled protein receptor are also introduced as an alternative to ligand-observed techniques.

Publisher Summary This chapter principally considers a collection of experimental methods that find widespread use in modern high-resolution nuclear magnetic resonance (NMR). They are sequence segments that are used within the techniques already encountered to enhance their information content or to overcome a range of experimental limitations; in addition to the familiar simple rf pulses, they are the components used to construct modern NMR experiments. Depending on context, they may be considered as essential to the correct execution of the desired experiment or viewed as an optional extra to enhance performance. For example, the broadband decoupling of protons during carbon acquisition routinely makes use of the so-called “composite-pulse decoupling” (CPD) schemes to achieve efficient removal of all proton couplings and is nowadays considered essential. Pulsed field gradients (PFGs) could equally well have been included in this chapter as in many cases they serve as an alternative means of signal selection to traditional phase-cycling procedures. The chapter concludes with a discussion on some newer experimental methods that have not yet found widespread use but have the potential to become more widely employed in the future.

Publisher Summary This chapter describes the practical aspects of performing nuclear magnetic resonance (NMR) spectroscopy. The chapter discusses the operating principles of the spectrometer and the handling of NMR data. It explains how to prepare the sample and the spectrometer before attempting experiments, how to calibrate the instrument, and how to monitor and measure its performance. NMR spectrometers are expensive instruments, representing one of the largest financial investments a chemical laboratory is likely to make, and to get the best results from these, they must be operated and maintained in the appropriate manner. The chapter explores some of the fundamental experimental aspects of relevance to high-resolution, solution state NMR spectroscopy, from instrumental procedures through to the preparation of samples for analysis.

Publisher Summary This chapter introduces the basic model for the description of nuclear magnetic resonance (NMR) methods and describes how this provides a simple picture of the behavior of chemical shifts and spin–spin couplings during pulse experiments. This model is then used to visualize nuclear spin relaxation, a feature of central importance for the optimum execution of all NMR experiments (indeed, it seems early attempts to observe NMR failed most probably because of a lack of understanding at the time of the relaxation behavior of the chosen samples). Methods for measuring relaxation rates also introduce multipulse NMR sequences. To understand the modern NMR techniques together the information and their potential applications, it is necessary to develop an understanding of some elementary principles. When considering the effects of scalar coupling on a resonance, it is convenient to remove the effects of chemical shift altogether by choosing the reference frequency of the rotating frame to be the chemical shift of the multiplet of interest.

Publisher Summary This chapter concerns with a fundamentally different form of interaction among nuclear spins through-space magnetic interactions (dipolar couplings) that give rise to the nuclear Overhauser effect (NOE). This brings about changes in resonance intensities and is intimately related to nuclear spin relaxation. The importance of the NOE in modern structure elucidation can hardly be overstated. It is unique in its ability to provide the chemist with information on 3D molecular geometry. Such information can be obtained because the NOE depends on, internuclear separations such that only those spins that are close in space are able to demonstrate this effect. However, the practical implementation of both 1D and 2D NOE experiments is described, including the widely used pulsed field gradient 1D NOE methods. Rotating-frame NOE (ROE) techniques are also described, which find greatest utility in the study of larger molecules for which the NOE can be poorly suited, having application in studies of host–guest complexes, oligomeric structures, and supramolecular systems.

Publisher Summary This chapter considers methods for separating chemical shifts and coupling constants in spectra that are based on two-dimensional (2D) methods. Unlike the techniques encountered that exploit scalar ( J ) couplings to correlate the chemical shifts of interacting spins, ‘J-resolved’ experiments aim to separate (or resolve) chemical shifts from scalar couplings, thus allowing the chemist to examine one parameter without complications arising from the other. For example, the analysis of crowded proton spectra is often complicated by the overlap of neighboring multiplets, making the extraction of coupling constants or the accurate measurement of chemical shifts difficult or even impossible. In practice, J-resolved methods are subject to a number of technical difficulties that may limit their effectiveness in this respect, and the methods have found far less use in routine structural work than the shift correlation experiments. The methods are based on 2D spectroscopy in which the shift and coupling parameters are resolved by presenting chemical shifts in f 2 and only spin couplings in f 1 that may be heteronuclear or homonuclear couplings, depending on the details of the experiment. These techniques were the most widely studied methods in the early development of 2D nuclear magnetic resonance (NMR) spectroscopy and may be understood with reference to the vector model, being based on simple spin-echoes.

Publisher Summary This chapter focuses on the use of high-resolution nuclear magnetic resonance (NMR) methods for studying diffusion in physically homogeneous (isotropic) solutions. Such self diffusion arises from the random translational (Brownian) motion of molecules (or their complexes) driven by thermal energy of the system and may be characterized quantitatively by the so-called self-diffusion coefficient. The study of molecular diffusion in solution potentially offers insights into a range of physical molecular properties including molecular size, shape, aggregation, encapsulation, complexation and hydrogen bonding, and NMR-based measurements have been applied to many areas of chemistry for over four decades. Diffusion measurements have become routinely accessible with conventional high-resolution NMR spectrometers through the introduction of actively shielded pulsed field gradient probe heads. All modern NMR-based diffusion measurements rely on the application of field gradients to encode the physical location of a molecule or complex in solution and so characterize its diffusion along the direction of the applied field gradient, typically the z axis of conventional gradient probe heads. The chapter extends into the study of self-diffusion, an area now routinely amenable to investigation on spectrometers equipped with standard pulsed field gradient hardware.