Stereoretentive post-translational protein editing

Chemical post-translational methods allow convergent side-chain editing of proteins without needing to resort to genetic intervention. Current approaches that allow the creation of constitutionally native side chains via C–C bond formation, using off-protein carbon-centered C· radicals added to unnatural amino acid radical acceptor (SOMOphile, singly occupied molecular orbital (SOMO)) “tags” such as dehydroalanine, are benign and wide-ranging. However, they also typically create epimeric mixtures of d/l-residues. Here, we describe a light-mediated desulfurative method that, through the creation and reaction of stereoretained on-proteinl-alanyl Cβ· radicals, allows Cβ–Hγ, Cβ–Oγ, Cβ–Seγ, Cβ–Bγ, and Cβ–Cγ bond formation to flexibly generate site-selectively edited proteins with full retention of native stereochemistry under mild conditions from a natural amino acid precursor. This methodology shows great potential to explore protein side-chain diversity and function and in the construction of useful bioconjugates

Synthetic Studies Toward the Skyllamycins: Total Synthesis and Generation of Simplified Analogues

Herein, we report our synthetic studies toward the skyllamycins, a highly modified class of nonribosomal peptide natural products which contain a number of interesting structural features, including the extremely rare α-OH-glycine residue. Before embarking on the synthesis of the natural products, we prepared four structurally simpler analogues. Access to both the analogues and the natural products first required the synthesis of a number of nonproteinogenic amino acids, including three β-OH amino acids that were accessed from the convenient chiral precursor Garner’s aldehyde. Following the preparation of the suitably protected nonproteinogenic amino acids, the skyllamycin analogues were assembled using a solid-phase synthetic route followed by a final stage solution-phase cyclization reaction. To access the natural products (skyllamycins A–C) the synthetic route used for the analogues was modified. Specifically, linear peptide precursors containing a C-terminal amide were synthesized via solid-phase peptide synthesis. After cleavage from the resin the N-terminal serine residue was oxidatively cleaved to a glyoxyamide moiety. The target natural products, skyllamycins A–C, were successfully prepared via a final step cyclization with concomitant formation of the unusual α-OH-glycine residue. Purification and spectroscopic comparison to the authentic isolated material confirmed the identity of the synthetic natural products.AR

Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis.

Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues were also shown to be nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis

Total Synthesis of Natural Products with Antimicrobial Activity

Natural products are an essential source of many modern medicines. Examples of important natural products include the antibiotic penicillin and the antimalarial quinine. One significant class of bioactive natural products are non-ribosomal peptides (NRPs) and two prototypical members of this class are the extremely important antibiotics, penicillin and vancomycin. Currently, bacterial resistance to antibiotics is one of the most pressing global health issues. The need for new antibiotics with novel mechanisms of action is paramount. This thesis describes the total synthesis of the recently isolated antimicrobial NRPs teixobactin and skyllamycins A-C. Chapter two of this thesis describes the first total synthesis of teixobactin, a novel cyclic NRP antibiotic isolated in 2015. This was carried out via a solid-phase peptide synthesis (SPPS) strategy with a late stage cyclisation reaction. The synthetic natural product possessed potent activity against a number of clinically relevant Gram-positive bacterial pathogens. Chapters three and four describe investigations towards the total synthesis of skyllamycins A-C, a family of structurally complex cyclic NRPs. These natural products inhibit the growth of bacterial biofilms, a mechanism by which bacteria evade antibiotics. The most unusual feature of these natural products is the presence of an α-OH-glycine (Gly) moiety, which to date has only been found in one other linear peptide natural product. Chapter three details the synthesis of the non-proteinogenic amino acids present in the natural products and their incorporation into the synthesis of four skyllamycin analogues that omit the unusual α-OH-Gly residue. These analogues were analysed for their biofilm growth inhibition activity. Chapter four describes the completion of the first total synthesis of skyllamycins A-C. This was achieved through a SPPS strategy followed by a late stage cyclisation and concomitant formation of the unusual α-OH-Gly residue in one step

FIELD-SCALE VERIFICATION OF NITROUS OXIDE EMISSION REDUCTION WITH DCD IN DAIRY-GRAZED PASTURE USING MEASUREMENTS AND MODELLING

Nitrous oxide (N 2 O) from agricultural soils is a major source of greenhouse gas emissions in New Zealand. N 2 O is produced by the microbial break-down of animal excreta and fertiliser N applied to agricultural soils. Nitrification inhibitors are seen in New Zealand as a potential technology to reduce N 2 O emissions from agricultural soils. A review of lysimeter and field studies using the nitrification inhibitor dicyandiamide (DCD) reported an average reduction in N 2 O emission of 67 ± 6% from animal urine Accordingly, the objectives of this study were: 1) to test whether the same level of N 2 O reduction is achieved under grazed conditions where excretal-N is non-uniformly deposited, and 2) to apply the process-based NZ-DNDC model to simulate the effect of DCD on emission reductions. Two circular 1260-m 2 treatment plots at Massey University Dairy Farm 4 were grazed simultaneously for 5 h, by 20 cattle on each plot. The following day, DCD was applied in 800 L water to one of the plots at 10 kg ha -1 . N 2 O emissions were measured periodically for 20 days following a grazing event, using equal arrays of 20 soil chambers in either plot, and soil and environmental variables were monitored. The cumulative N 2 O emissions over the 20 day period were 220 ± 90 g N 2 O-N ha -1 and 110 ± 20 g N 2 O-N ha -1 (based on the arithmetic mean and standard error of the chambers) for the untreated and DCD-treated plots respectively. This suggests a reduction in N 2 O emission from DCD application of ~50 ± 40% from a single grazing event. However, this result should be treated with caution, because the possibility of sampling error due to the chamber distribution cannot be excluded. NZ-DNDC simulated N 2 O emissions of 185 g N 2 O-N ha -1 and 73 g N 2 O-N ha -1 for the untreated and DCD-treated areas respectively, corresponding to a reduction in N 2 O emissions from DCD application of 60%. This level of reduction is consistent with that found in experiments with individual urine patches. A sensitivity analysis was conducted on the NZ-DNDC model to find the variability in the predicted N 2 O emissions that would result from uncertainty in the input parameters. Varying the parameters' initial soil NO 3 -and NH 4 + , soil organic carbon and bulk density within plausible ranges resulted in N 2 O emissions from the no-DCD area ranging from 120 to 259 g N 2 O-N ha -1 . This range is in good agreement with the measured emissions.

Synthesis of β-Thiol Phenylalanine for Applications in One-Pot Ligation- Desulfurization Chemistry

The efficient synthesis of a β-thiol phenylalanine derivative is described starting from Garner’s aldehyde. The utility of this amino acid in peptide ligation–desulfurization chemistry is described, including the trifluoroethanethiol (TFET)-promoted one-pot assembly of the 62 residue peptide hormone augurin

Total Synthesis of Fellutamide B and Deoxy-Fellutamides B, C, and D

The total syntheses of the marine-derived lipopeptide natural product fellutamide B and deoxy-fellutamides B, C, and D are reported. These compounds were accessed through a novel solid-phase synthetic strategy using Weinreb amide-derived resin. As part of the synthesis, a new enantioselective route to (3R)-hydroxy lauric acid was developed utilizing a Brown allylation reaction followed by an oxidative cleavage-oxidation sequence as the key steps. The activity of these natural products, and natural product analogues was also assessed against Mycobacterium tuberculosis in vitro

Total Synthesis of Fellutamide B and Deoxy-Fellutamides B, C, and D

The total syntheses of the marine-derived lipopeptide natural product fellutamide B and deoxy-fellutamides B, C, and D are reported. These compounds were accessed through a novel solid-phase synthetic strategy using Weinreb amide-derived resin. As part of the synthesis, a new enantioselective route to (3R)-hydroxy lauric acid was developed utilizing a Brown allylation reaction followed by an oxidative cleavage-oxidation sequence as the key steps. The activity of these natural products, and natural product analogues was also assessed against Mycobacterium tuberculosis in vitro