Strategies for the Syntheses of Pactamycin and Jogyamycin.

Pactamycin and jogyamycin are aminocyclopentitol natural products, where each core carbon bears a stereodefined alcohol or amine moiety. Their structural complexity, coupled with the diversity of functional groups coexisting in a condensed space, make them fascinating synthetic targets in their own right. Pactamycin and its derivatives bind to the 30S ribosomal subunit and display activity against parasites responsible for drug-resistant malaria and African sleeping sickness; however, efforts to develop their therapeutic potential have been hampered by their cellular toxicity. Interestingly, bioengineered analogues display differences in selectivity and toxicity towards mammalian cells, spurring efforts to develop flexible strategies to thoroughly probe structure-activity relationships (SAR), particularly in analogues lacking the C7 hydroxyl group of pactamycin. This review compares and contrasts approaches towards pactamycin and jogyamycin, including two successful total syntheses of the former. The implications of each route for preparing analogues to inform SAR and lead to compounds with increased selectivity for binding malarial over human ribosomes are briefly discussed.

Stereocontrolled Synthesis of the Aminocyclopentitol Core of Jogyamycin via an Ichikawa Rearrangement Reaction.

Jogyamycin is a member of the aminocyclopentitol class of natural products that exhibits significant antiprotozoal activities against diseases that include African sleeping sickness and malaria. Herein, we report a route to the core of this natural product via an underutilized Ichikawa rearrangement as a key step. This route efficiently forms the cyclopentane ring from simple and easily accessible starting materials and rapidly installs the C1/C4/C5 polar functional groups. In addition, this strategy shows excellent potential for the preparation of analogues of jogyamycin to study how structural changes impact the selectivity in binding to the ribosome.

Total Synthesis of Pactalactam, an Imidazolidinone-Type Pactamycin Analogue.

The first total synthesis of pactalactam was accomplished using substrate-controlled stereoselective aziridination and regioselective aziridine ring-opening to construct three continuous amino groups on an octasubstituted cyclopentane core. The cyclopentane framework was obtained by ring-closing metathesis and aldol coupling using a l-threonine-derived oxazoline compound. Cyclic urea formation, m-acetylphenyl group introduction by Chan-Lam coupling, and primary alcohol-selective acylation yielded the reported pactalactam structure. The presence of pactalactam in the fermentation broth of pactamycin-producing bacteria was also confirmed.

Asymmetric Synthesis and Biological Activities of Pactamycin-Inspired Aminocyclopentitols.

Pactamycin is a structurally unique aminocyclitol antibiotic with broad-spectrum cell growth inhibitory activity. To explore the bountiful activity of the aminocyclitol core of pactamycin, an efficient, modular, and asymmetric synthesis of aminocyclopentitols resembling the pactamycin pharmacophore has been developed employing a SmI2-mediated imino-pinacol coupling strategy. Two of the compounds exhibited antitumor activity against A375 melanoma cells.

NAD+ -Dependent Dehydrogenase PctP and Pyridoxal 5'-Phosphate Dependent Aminotransferase PctC Catalyze the First Postglycosylation Modification of the Sugar Intermediate in Pactamycin Biosynthesis.

The unique five-membered aminocyclitol core of the antitumor antibiotic pactamycin originates from d-glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Herein, we examined the timing of introduction of an amino group into certain sugar-derived intermediates by using recombinant enzymes that were encoded in the pactamycin biosynthesis gene cluster. We found that the NAD+ -dependent alcohol dehydrogenase PctP and pyridoxal 5'-phosphate dependent aminotransferase PctC converted N-acetyl-d-glucosaminyl-3-aminoacetophonone into 3'-amino-3'-deoxy-N-acetyl-d-glucosaminyl-3-aminoacetophenone. Further, N-acetyl-d-glucosaminyl-3-aminophenyl-ß-oxopropanoic acid ethyl ester was converted into the corresponding 3'-amino derivative. However, PctP did not oxidize most of the tested d-glucose derivatives, including UDP-GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.

A Highly Promiscuous ß-Ketoacyl-ACP Synthase (KAS) III-like Protein Is Involved in Pactamycin Biosynthesis.

ß-Ketoacyl-acyl carrier protein (ß-Ketoacyl-ACP) synthase (KAS) III catalyzes the first step in fatty acid biosynthesis, involving a Claisen condensation of the acetyl-CoA starter unit with the first extender unit, malonyl-ACP, to form acetoacetyl-ACP. KAS III-like proteins have also been reported to catalyze acyltransferase reactions using coenzyme A esters or discrete ACP-bound substrates. Here, we report the in vivo and in vitro characterizations of a KAS III-like protein (PtmR), which directly transfers a 6-methylsalicylyl moiety from an iterative type I polyketide synthase to an aminocyclopentitol unit in pactamycin biosynthesis. PtmR is highly promiscuous, recognizing a wide array of S-acyl-N-acetylcysteamines as substrates to produce a suite of pactamycin derivatives with diverse alkyl and aromatic features. The results suggest that KAS III-like proteins may be used as versatile tools for modifications of complex natural products.

Analysis of ribosomal inter-subunit sites as targets for complementary oligonucleotides.

The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined. Based on these properties we selected rRNA targets for hybridization with complementary 2'-O-methyl oligoribonucleotides (2'-OMe RNAs). Further, the inhibition efficiencies of the designed ribosome-interfering 2'-OMe RNAs were tested using a ß-galactosidase assay in a translation system based on the E. coli extract. Several of the oligonucleotides displayed IC50 values below 1 µM, which were in a similar range as those determined for known ribosome inhibitors, tetracycline and pactamycin. The calculated opening and van der Waals stacking energies of the rRNA targets correlated best with the inhibitory efficiencies of 2'-OMe RNAs. Moreover, the binding affinities of several oligonucleotides to both 70S ribosomes and isolated 30S and 50S subunits were measured using a double-filter retention assay. Further, we applied heat-shock chemical transformation to introduce 2'-OMe RNAs to E. coli cells and verify inhibition of bacterial growth. We observed high correlation between IC50 in the cell-free extract and bacterial growth inhibition. Overall, the results suggest that the computational analysis of potential rRNA targets within the conformationally dynamic regions of inter-subunit bridges can help design efficient antisense oligomers to probe the ribosome function.

Interrogating the Tailoring Steps of Pactamycin Biosynthesis and Accessing New Pactamycin Analogues.

Pactamycin is a bacteria-derived aminocyclitol antibiotic with a wide-range of biological activity. Its chemical structure and potent biological activities have made it an interesting lead compound for drug discovery and development. Despite its unusual chemical structure, many aspects of its formation in nature remain elusive. Using a combination of genetic inactivation and metabolic analysis, we investigated the tailoring processes of pactamycin biosynthesis in Streptomyces pactum. The results provide insights into the sequence of events during the tailoring steps of pactamycin biosynthesis and explain the unusual production of various pactamycin analogues by S. pactum mutants. We also identified two new pactamycin analogues that have better selectivity indexes than pactamycin against malarial parasites.

Diastereoselective Synthesis of the Aminocyclitol Core of Jogyamycin via an Allene Aziridination Strategy.

Oxidative allene amination provides rapid access to densely functionalized amine-containing stereotriads through highly reactive bicyclic methyleneaziridine intermediates. This strategy has been demonstrated as a viable approach for the construction of the densely functionalized aminocyclitol core of jogyamycin, a natural product with potent antiprotozoal activity. Importantly, the flexibility of oxidative allene amination will enable the syntheses of modified aminocyclitol analogues of the jogyamycin core.

Mechanism-Based Trapping of the Quinonoid Intermediate by Using the K276R Mutant of PLP-Dependent 3-Aminobenzoate Synthase PctV in the Biosynthesis of Pactamycin.

Mutational analysis of the pyridoxal 5'-phosphate (PLP)-dependent enzyme PctV was carried out to elucidate the multi-step reaction mechanism for the formation of 3-aminobenzoate (3-ABA) from 3-dehydroshikimate (3-DSA). Introduction of mutation K276R led to the accumulation of a quinonoid intermediate with an absorption maximum at 580 nm after the reaction of pyridoxamine 5'-phosphate (PMP) with 3-DSA. The chemical structure of this intermediate was supported by X-ray crystallographic analysis of the complex formed between the K276R mutant and the quinonoid intermediate. These results clearly show that a quinonoid intermediate is involved in the formation of 3-ABA. They also indicate that Lys276 (in the active site of PctV) plays multiple roles, including acid/base catalysis during the dehydration reaction of the quinonoid intermediate.

Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine.

Malaria inflicts an enormous burden on global human health. The emergence of parasite resistance to front-line drugs has prompted a renewed focus on the repositioning of clinically approved drugs as potential anti-malarial therapies. Antibiotics that inhibit protein translation are promising candidates for repositioning. We have solved the cryo-EM structure of the cytoplasmic ribosome from the human malaria parasite, Plasmodium falciparum, in complex with emetine at 3.2 Å resolution. Emetine is an anti-protozoan drug used in the treatment of ameobiasis that also displays potent anti-malarial activity. Emetine interacts with the E-site of the ribosomal small subunit and shares a similar binding site with the antibiotic pactamycin, thereby delivering its therapeutic effect by blocking mRNA/tRNA translocation. As the first cryo-EM structure that visualizes an antibiotic bound to any ribosome at atomic resolution, this establishes cryo-EM as a powerful tool for screening and guiding the design of drugs that target parasite translation machinery.

A single PLP-dependent enzyme PctV catalyzes the transformation of 3-dehydroshikimate into 3-aminobenzoate in the biosynthesis of pactamycin.

Natural amino donation: A PLP-dependent aminotransferase PctV, encoded in the pactamycin biosynthetic gene cluster, was found to catalyze the formation of 3-aminobenzoate from 3-dehydroshikimate with L-glutamate as the amino donor. The PctV reaction comprises a transamination and two dehydration reactions. This is the first report of a simple 3-ABA synthase in nature.

Crystal structure of a bioactive pactamycin analog bound to the 30S ribosomal subunit.

Biosynthetically and chemically derived analogs of the antibiotic pactamycin and de-6-methylsalicylyl (MSA)-pactamycin have attracted recent interest as potential antiprotozoal and antitumor drugs. Here, we report a 3.1-Å crystal structure of de-6-MSA-pactamycin bound to its target site on the Thermus thermophilus 30S ribosomal subunit. Although de-6-MSA-pactamycin lacks the MSA moiety, it shares the same binding site as pactamycin and induces a displacement of nucleic acid template bound at the E-site of the 30S. The structure highlights unique interactions between this pactamycin analog and the ribosome, which paves the way for therapeutic development of related compounds.

Diastereoselective synthesis of vicinal tertiary diols.

A strategy for the synthesis of differentiated vicinal tertiary diols is described. The key step is a high-yielding, diastereoselective LaCl3·2LiCl-mediated addition of a Grignard or organolithium reagent to ketone 2a. The reaction is believed to proceed via a 1,3-chelated intermediate. One of the adducts has been transformed into a functionalized cyclopentenone resembling the core structure of pactamycin.

Enantioselective synthesis of pactamycin, a complex antitumor antibiotic.

Medicinal application of many complex natural products is precluded by the impracticality of their chemical synthesis. Pactamycin, the most structurally intricate aminocyclopentitol antibiotic, displays potent antiproliferative properties across multiple phylogenetic domains, but it is highly cytotoxic. A limited number of analogs produced by genetic engineering technologies show reduced cytotoxicity against mammalian cells, renewing promise for therapeutic applications. For decades, an efficient synthesis of pactamycin amenable to analog derivatizations has eluded researchers. Here, we present a short asymmetric total synthesis of pactamycin. An enantioselective Mannich reaction and symmetry-breaking reduction sequence was designed to enable assembly of the entire carbon core skeleton in under five steps and control critical three-dimensional (stereochemical) functional group relationships. This modular route totals 15 steps and is immediately amenable for structural analog synthesis.

Mutasynthesis of fluorinated pactamycin analogues and their antimalarial activity.

A mutasynthetic strategy has been used to generate fluorinated TM-025 and TM-026, two biosynthetically engineered pactamycin analogues produced by Streptomyces pactum ATCC 27456. The fluorinated compounds maintain excellent activity and selectivity toward chloroquine-sensitive and multidrug-resistant strains of malarial parasites as the parent compounds. The results also provide insights into the biosynthesis of 3-aminobenzoic acid in S. pactum.

Total synthesis of pactamycin and pactamycate: a detailed account.

This article describes synthetic studies that culminated in the first total synthesis of pactamycin and pactamycate and, in parallel, the two known congeners, de-6-MSA-pactamycin and de-6-MSA-pactamycate, lacking the 6-methylsalicylyl moiety. Starting with L-threonine as a chiron, a series of stereocontrolled condensations led to a key cyclopentenone harboring a spirocyclic oxazoline. A series of systematic functionalizations led initially to the incorrect cyclopentanone epoxide, which was "inverted" under solvolytic conditions. Installation of the remaining groups and manipulation of the oxazoline eventually led to pactamycin, pactamycate, and their desalicylyl analogues.

Access to the pactamycin core via an epoxide opening cascade.

A synthetic strategy to establish five contiguous stereocenters, in a stereocontrolled manner, on the core structure of pactamycin is described. This sequence exploits the use of a Lewis acid mediated epoxide opening cascade to set the relative configuration of the C4-C5 diol while reversing the configuration at C7. This sequence provides the oxygenated core of pactamycin in just 11 steps.

Diastereocontrolled construction of pactamycin's complex ureido triol functional array.

An advanced intermediate in a projected synthesis of pactamycin has been prepared. Early installation of the C1-dimethylurea functionality allows for its participation in a diastereoselective, chelation-controlled addition of organometal nucleophiles to the C5 prochiral ketone. Four of the molecule's six stereocenters are set with a ketone functional handle provided for subsequent manipulation.