Philip J. Sidebottom

Structure, Biosynthetic Origin, and Engineered Biosynthesis of Calcium-Dependent Antibiotics from Streptomyces coelicolor

The calcium-dependent antibiotic (CDA), from Streptomyces coelicolor, is an acidic lipopeptide comprising an N-terminal 2,3-epoxyhexanoyl fatty acid side chain and several nonproteinogenic amino acid residues. S. coelicolor grown on solid media was shown to produce several previously uncharacterized peptides with C-terminal Z-dehydrotryptophan residues. The CDA biosynthetic gene cluster contains open reading frames encoding nonribosomal peptide synthetases, fatty acid synthases, and enzymes involved in precursor supply and tailoring of the nascent peptide. On the basis of protein sequence similarity and chemical reasoning, the biosynthesis of CDA is rationalized. Deletion of SCO3229 (hmaS), a putative 4-hydroxymandelic acid synthase-encoding gene, abolishes CDA production. The exogenous supply of 4-hydroxymandelate, 4-hydroxyphenylglyoxylate, or 4-hydroxyphenylglycine re-establishes CDA production by the DeltahmaS mutant. Feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues.

Engineered urdamycin glycosyltransferases are broadened and altered in substrate specificity.

Combinatorial biosynthesis is a promising technique used to provide modified natural products for drug development. To enzymatically bridge the gap between what is possible in aglycon biosynthesis and sugar derivatization, glycosyltransferases are the tools of choice. To overcome limitations set by their intrinsic specificities, we have genetically engineered the protein regions governing nucleotide sugar and acceptor substrate specificities of two urdamycin deoxysugar glycosyltransferases, UrdGT1b and UrdGT1c. Targeted amino acid exchanges reduced the number of amino acids potentially dictating substrate specificity to ten. Subsequently, a gene library was created such that only codons of these ten amino acids from both parental genes were independently combined. Library members displayed parental and/or a novel specificity, with the latter being responsible for the biosynthesis of urdamycin P that carries a branched saccharide side chain hitherto unknown for urdamycins.

NMR quantification using an artificial signal.

We have developed QUANTAS (QUANTification by Artificial Signal), which is a software‐based protocol for concentration measurement by NMR. QUANTAS is an absolute intensity external standard method for quantification by NMR that compensates for various experimental parameters. It is applicable to all nuclei and modern spectrometers. QUANTAS is demonstrated here for 1H and 19F NMR, enabling heteronuclear integrals to be compared. It can be applied using fixed probe tuning, matching and pulse length, for samples with the same effective loading on the probe coil as the appropriate reference spectrum. Otherwise, an optimised tuning and matching approach is adopted for every sample together with explicit PULCON (PUlse Length‐based CONcentration measurements) absolute intensity corrections. Copyright

Biosynthesis of the Putative Siderophore Erythrochelin Requires Unprecedented Crosstalk between Separate Nonribosomal Peptide Gene Clusters

The genome of the erythromycin-producing bacterium Saccharopolyspora erythraea contains many orphan secondary metabolite gene clusters including two (nrps3 and nrps5) predicted to govern biosynthesis of nonribosomal peptide-based siderophores. We report here the production by S. erythraea, even under iron-sufficient conditions, of a 2,5-diketopiperazine siderophore candidate we have named erythrochelin. Deletion of the nonribosomal peptide synthetase (NRPS) gene ercD within the nrps5 cluster abolished erythrochelin production. The tetrapeptide backbone of erythrochelin (alpha-N-acetyl-delta-N-acetyl-delta-N-hydroxyornithine-serine-delta-N-hydroxyornithine-delta-N-acetyl-delta-N-hydroxyornithine) suggests an orthodox colinear model for erythrochelin assembly. Curiously, the delta-N-acetyltransferase required for erythrochelin biosynthesis is encoded within a remote NRPS-cluster (nrps1) whose own NRPS contains an inactivating mutation. Disruption of the nrps1 gene mcd abolished erythrochelin biosynthesis, which could then be restored by addition of synthetic L-delta-N-acetyl-delta-N-hydroxyornithine, confirming an unprecedented example of functional crosstalk between nrps clusters.

Biotransformation of alosetron: Mechanism of hydantoin formation

The formation of the hydantoin biotransformation product (3) of Alosetron (1) has been shown to proceed via migration of the tricyclic lactam moiety and occurs with complete retention of both hydrogens in the C-7′ methylene group.

A new approach to the optimisation of non-uniform sampling schedules for use in the rapid acquisition of 2D NMR spectra of small molecules.

Non-uniform sampling allows the routine, rapid acquisition of 2D NMR data. When the number of points in the NUS schedule is low, the quality of the data obtained is very dependent of the schedule used. A simple proceedure for finding optimium schedules has been developed and is demonstrated for the multiplicity edited HSQC experiment.

The squalestatins: cleavage of the bicyclic core via the novel 6,8-dioxabicyclo[3.2.1]octane ring system

Squalestatin S1 1 has been converted into its 4,7-bis(2-methoxyethoxymethyl)ether 4,5-dimethyl ester 11 and thence to its 3-(tert-butoxycarbonyl)amino derivative 12via a Schmidt degradation. Acid-catalysed hydrolysis of 12 brought about a molecular rearrangement of the 2,8-dioxa- to the novel 6,8-dioxa-bicyclo[3.2.1]octane ring system 3. Oxidation of 3 followed by methanolysis gave the novel spiroketal 17. Treatment of 3 with trimethyl phosphonoacetate gave the acyclic derivative 18.

Synthesis of the bicyclic core structure of squalestatin 1

The butenolides 2 and 3 have been converted into the dioxabicyclo[3.2.1] loctane derivative 15, a late-stage precursor to squalestatin 1 16.

Antony J. Williams, Gary E. Martin and David Rovnyak (Eds): Modern NMR Approaches to the Structure Elucidation of Natural Products, Volume 1: Instrumentation and Software

references. The book is well produced and in many places the clarity is enhanced by the effective use of colour. As is the nature of such multi-author works there is overlap between some of the chapters and the style and quality vary between them. The gestation period for this book has been 4 years. As a result, some chapters, presumably those whose authors submitted their material on time, are not quite as up to date as others. In any multi-topic book each reader will take something different from it. Here are some of my highlights and impressions. In the hidden gem category is the section covering some of the sample preparation issues encountered when only small amounts of analyte are available. This is tucked into the chapter on microprobes. The advances in and power of using NMR features for dereplication as outlined in Chapter 8 also caught my eye. The chapter on computer-assisted structure elucidation seemed at times like an extended advertisement for one particular commercial package. That said, it is clear that much has been achieved in this area. I am left with the impression of a tool that, after appropriate training in its use, will provide help to experts rather than being a replacement for them. Finally, there is a chapter that from its title appears to be in the wrong book. What is a chapter on atomic force microscopy doing in an NMR book? However, had I skipped this chapter I would have missed one of the best, concise accounts of the structure elucidation process using NMR that I have come across. This culminates by highlighting the difficulties NMR has in solving the structures of molecules where the ratio of protons to heavy atoms is low. It then rightly makes the point that in such cases you may need to consider using other methods. With this in mind it would have been nice to see some coverage of the crystalline sponge approach to X-ray crystallography pioneered by Makoto Fujita’s group. Of course this might be the surprise held back for volume 2—we will just have to wait and see. It is tempting to assume from the title that this book will only contain material of interest to those engaged in the structure elucidation of natural products. In my opinion such an assumption would be a mistake. Those working in other fields where small molecule structure elucidation or the analysis of organic mixtures is necessary, should also find something in this book to interest them. The chapters on magnets and probes are relevant to most NMR spectroscopists. The stated aim of the editors is “to expose the reader to stateof-the-art technologies in hardware, software and methods”. They expect “that many of these will become more commonplace in the near future” and “forewarned is forearmed”. Thus, for most readers they hope to provide a look into the future. To do it, in this, the first of a planned two volume series, they have put together 13 chapters written by experts in their fields. These are split more or less evenly between hardware and software and in most cases contain an extensive list of literature

Production of novel derivatives of a gastrin antagonist (GW1) using biotransformation

Publisher Summary This chapter describes production of novel derivatives of a gastrin antagonist (GW1) using biotransformation. The idea of using micro-organisms to carry out biotransformations has proved to be a powerful aid to drug metabolism. The use of micro-organisms for metabolite production offers various advantages over more traditional approaches, such as (1) the use of radiochemicals can often be avoided, (2) micro-organisms are readily amenable to scale up supporting the production of gram quantities of known or putative mammalian metabolites, and (3) the demand for animal studies is reduced which is important both from an economic and a humanitarian standpoint. Sixty-five organisms are screened for possible biotransformation of GW1. Twenty showed significant (> 60%) substrate utilization. Further analysis shows that four organisms (all Streptomycetes) are worthy of scale-up based on both the diversity and the levels of compounds produced. Isolated yields of compound are typically 0.2–4 mg from each organism processed.