Identification of asm19 as an acyltransferase attaching the biologically essential ester side chain of ansamitocins using N-desmethyl-4,5-desepoxymaytansinol,not maytansinol,as its substrate

The potent antitumor activity of the ansamitocins, polyketides isolated from Actinosynnema pretiosum, is absolutely dependent on a short acyl group esterified to the C-3 oxygen of the macrolactam ring. Asm19, a gene in the ansamitocin biosynthetic gene cluster with homology to macrolide O-acyltransferase genes, is thought to encode the enzyme catalyzing this esterification. A mutant carrying an inactivated asm19 no longer produced ansamitocins but accumulated N-desmethyl-4,5-desepoxymaytansinol, rather than maytansinol, indicating that the acylation is not the terminal step of the biosynthetic sequence. Bioconversion experiments and in vitro studies with recombinant Asm19, expressed in Escherichia coli, showed that the enzyme is very specific toward its alcohol substrate, converting N-desmethyl-4,5-desepoxymaytansinol (but not maytansinol) into ansamitocins, but rather promiscuous toward its acyl substrate, utilizing acetyl-, propionyl-, butyryl-, isobutyryl-, as well as isovaleryl-CoA.     

Dual carbamoylations on the polyketide and glycosyl moiety by asm21 result in extended ansamitocin biosynthesis

Carbamoylation is one of the post-PKS modifications in ansamitocin biosynthesis. A novel ansamitocinoside with carbamoyl substitution at the C-4 hydroxyl group of the N-β-D-glucosyl moiety was identified from the ansamitocin producer, Actinosynnema pretiosum. Through biotransformation, the carbamoyltransferase gene asm21 was suggested to be responsible for the carbamoylation of the glucosyl moiety. Three new derivatives without the backbone carbamoyl group were isolated from an asm21 mutant and characterized by NMR spectroscopy. Among them, 18-O-methyl-19-chloroproansamitocin was the major product and the preferred substrate for macrolactam C-7 carbamoylation by Asm21. However, Asm21 exhibited higher catalytic efficiency toward the glucosyl moiety. Furthermore, the dual carbamoylations and N-glycosylation were precisely demonstrated in vivo. This work represents the first biochemical characterization of an O-carbamoyltransferase performing dual actions on both a polyketide backbone and a glycosyl moiety during ansamitocin biosynthesis.     

The post-polyketide synthase modification steps in the biosynthesis of the antitumor agent ansamitocin by Actinosynnema pretiosum

The functions of six genes in the ansamitocin biosynthetic gene cluster of Actinosynnema pretiosum have been investigated by gene inactivation and chemical analysis of the mutants. They encode a halogenase (asm12), a carbamoyltransferase (asm21), a 20-O-methyltransferase (asm7), a 3-O-acyltransferase (asm19), an epoxidase (asm11), and an N-methyltransferase (asm10), respectively, and are responsible for the six post-PKS modification steps in ansamitocin formation. Several of the enzymes have relaxed substrate specificities, resulting in multiple parallel pathways in a metabolic grid, albeit with a preferred sequence of reactions as listed above.     

Functional expression of genes involved in the biosynthesis of the novel polyketide chain extension unit, methoxymalonyl-acyl carrier protein, and engineered biosynthesis of 2-desmethyl-2-methoxy-6-deoxyerythronolide B

A subcluster of five genes, asm13-17, from the ansamitocin biosynthetic gene cluster of Actinosynnema pretiosum was coexpressed in Streptomyces lividans with the genes encoding the 6-deoxyerythronolide B (6-DEB) synthase from Saccharopolyspora erythraea, in which the methylmalonate-specifying AT6 domain had been replaced by the methoxymalonate-specifying AT8 domain from the FK520 cluster of Streptomyces hygroscopicus. The engineered strain produced the predicted product, 2-desmethyl-2-methoxy-DEB, instead of 6-DEB and 2-desmethyl-6-DEB, which were formed in the absence of the asm13-17 cassette, indicating that asm13-17 are sufficient for synthesis of this unusual chain extension unit. Deletion of asm17, encoding a methyltransferase, from the cassette gave 6-DEB instead of its hydroxy analogue, indicating that methylation of the extender unit is required for its incorporation.     

Broad substrate specificity of the amide synthase in S. hygroscopicus--new 20-membered macrolactones derived from geldanamycin

The amide synthase of the geldanamycin producer, Streptomyces hygroscopicus, shows a broader chemoselectivity than the corresponding amide synthase present in Actinosynnema pretiosum, the producer of the highly cytotoxic ansamycin antibiotics, the ansamitocins. This was demonstrated when blocked mutants of both strains incapable of biosynthesizing 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase starter unit of both natural products, were supplemented with 3-amino-5-hydroxymethylbenzoic acid instead. Unlike the ansamitocin producer A. pretiosum, S. hygroscopicus processed this modified starter unit not only to the expected 19-membered macrolactams but also to ring enlarged 20-membered macrolactones. The former mutaproducts revealed the sequence of transformations catalyzed by the post-PKS tailoring enzymes in geldanamycin biosynthesis. The unprecedented formation of the macrolactones together with molecular modeling studies shed light on the mode of action of the amide synthase responsible for macrocyclization. Obviously, the 3-hydroxymethyl substituent shows similar reactivity and accessibility toward C-1 of the seco-acid as the arylamino group, while phenolic hydroxyl groups lack this propensity to act as nucleophiles in the macrocyclization. The promiscuity of the amide synthase of S. hygroscopicus was further demonstrated by successful feeding of four other m-hydroxymethylbenzoic acids, leading to formation of the expected 20-membered macrocycles. Good to moderate antiproliferative activities were encountered for three of the five new geldanamycin derivatives, which matched well with a competition assay for Hsp90α.     

Identification of a set of genes involved in the formation of the substrate for the incorporation of the unusual "glycolate" chain extension unit in ansamitocin biosynthesis

The unusual "glycolate" extender unit at C-9/C-10 of ansamitocin is not derived from 2-hydroxymalonyl-CoA or 2-methoxymalonyl-CoA, as demonstrated by feeding experiments with the corresponding 1-13C-labeled N-acetylcysteamine thioesters but is formed from an acyl carrier protein (ACP)-bound substrate, possibly 2-methoxymalonyl-ACP, elaborated by enzymes encoded by a subcluster of five genes, asm12-17, from the ansamitocin bisosynthetic gene cluster.     

Chemoenzymatic approaches toward dechloroansamitocin P-3

[reaction: see text] The enantioselective total synthesis of proansamitocin, a key biosynthetic intermediate of the highly potent antitumor agent ansamitocin P-3, is described which bears a diene-ene RCM as the key macrocyclization step. Feeding of proansamitocin to an AHBA block mutant Actinosynnema pretiosum (HGF073) yielded ansamitocin P-3 as well as dechloroansamitocin P-3, the latter also being formed upon fermentation in the presence of 3-amino-5-methoxybenzoic acid.     

Asymmetric Total Synthesis of Fluvirucinine A(1)

Diastereoselective vinyl addition to an amide carbonyl group and amide enolate induced aza-Claisen rearrangement are the key steps in the first asymmetric total synthesis of fluvirucinine A(1) (1), the aglycon of fluvirucin A(1). Fluvirucins are a class of macrolactam antibiotics produced by actinomycete strains that show promising biological properties.     

Total syntheses of the thiopeptides amythiamicin C and D

The thiopeptides amythiamicin C and D were synthesized by employing amide bond formation, a Stille cross-coupling reaction, and two Negishi cross-coupling reactions as key transformations. The central 2,3,6-trisubstituted pyridine ring of the target compounds was introduced as a 2,6-dibromo-3-iodopyridine, which was selectively metalated at the 3-position and connected to the complete Southern fragment of the amythiamicins by a Negishi cross-coupling. For the synthesis of amythiamicin C, this step was followed by a Negishi cross-coupling at C-6 of the pyridine core. Subsequent attachment of the Eastern fragment was achieved by amide bond formation and macrolactam ring closure by a Stille cross-coupling at C-2. The Eastern bithiazole fragment of the amythiamins was constructed also by regioselective metalation and cross-coupling reactions. The pivotal step involved the diastereoselective addition of 4-bromothiazole-2-magnesium bromide to a chiral sulfinyl imine. For the synthesis of amythiamicin D, the order of cross-coupling at C-6, amide bond formation, and cross-coupling at C-2 was changed. The amide bond formation to the Eastern fragment was performed first and it was subsequently attempted to close the macrolactam by an intramolecular regioselective Stille cross-coupling at C-2. Despite the low regioselectivity of this reaction it paved the way to the immediate completion of the amythiamicin D synthesis when followed by a Negishi cross-coupling at C-6 with 2-zincated methyl thiazole-5-carboxylate.     

Metabolism studies of the anti-tumor agent maytansine and its analog ansamitocin P-3 using liquid chromatography/tandem mass spectrometry

Maytansine, a potent clinically evaluated plant-derived anti-tumor drug, and its microbial counterpart, ansamitocin P-3, showed a substantially higher cytoxicity than many other anti-tumor drugs. Owing to a shortage of material and lack of sufficiently sensitive analytical methods at the time, no metabolism studies were apparently carried out in conjunction with the initial preclinical and clinical studies on maytansine, but some products of decomposition during the period of storage of the formulated drug were reported. In the current study, the in vitro metabolism of maytansine and ansamitocin P-3 was studied after incubation with rat and human liver microsomes in the presence of NADPH, and with rat and human plasma and whole blood, using liquid chromatography/multi-stage mass spectrometry. Unchanged ansamitocin P-3 and 11 metabolites and unchanged maytansine and seven metabolites were profiled and the structures of some metabolites were tentatively assigned based on their multi-stage electrospray ion-trap mass fragmentation data and in some cases accurate mass measurement. The major pathway of ansamitocin P-3 metabolism in human liver microsomes appears to be demethylation at C-10. Oxidation and sequential oxidation/demethylation also occurred, although to a lesser extent. However, the major pathway of maytansine metabolism in human liver microsomes is N-demethylation of the methylamide of the ester moiety. Several minor pathways including O/N-demethylation, oxidation and hydrolysis of the ester bond were also observed. There were no differences in maytansine metabolism between rat and human liver microsomes; however, the rate of metabolism of ansamitocin P-3 was different in rat and human liver microsomes. About 20% of ansamitocin P-3 was converted to its metabolites in rat liver microsomes and about 70% in human liver microsomes under the same conditions. Additionally, 10-O-demethylated ansamitocin P-3 was also detected in the urine after i.v. bolus administration of ansamitocin P-3 to Sprague-Dawley male rats. No metabolites were detected following incubation of maytansine and ansamitocin P-3 with human and rat whole blood and plasma.     

Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes

HSAF was isolated from Lysobacter enzymogenes , a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here we describe the HSAF biosynthetic gene cluster, which contains only a single-module polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/NRPS are six genes that encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activates L-ornithine and that the four-domain NRPS is able to catalyze the formation of a tetramic acid-containing product from acyl-S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms.     

Concise total synthesis of the thiazolyl peptide antibiotic GE2270 A

The potent antibiotic thiazolylpeptide GE2270 A was synthesized starting from N-tert-butyloxycarbonyl protected valine in a longest linear sequence of 20 steps and with an overall yield of 4.8 %. Key strategy was the assembly of the 2,3,6-trisubstituted pyridine core by consecutive cross-coupling reactions starting from 2,6-dibromo-3-iodopyridine. The complete Southern fragment was installed by Negishi cross-coupling of 3-zincated 2,6-dibromopyridine at the terminal 2-iodothiazole of a trithiazole (87 %). The substituent at C-6 representing the Northern part of the molecule was introduced in form of the truncated tert-butyl 2-bromothiazole-4-carboxylate after metalation to a zinc reagent by another Negishi cross-coupling (48 %). Decisive step of the whole sequence was the macrocyclization to a 29-membered macrolactam, which was conducted as an intramolecular Stille cross-coupling occurring at C-2 of the pyridine core and providing the desired product in 75 % yield. The required stannane was obtained by amide bond formation (87 %) between a complex dithiazole fragment representing the Eastern part of GE2270 A and a 3,6-disubstituted 2-bromopyridine. Final steps included attachment of a serine-proline amide dipeptide to the Northern part of the molecule (65 %), formation of the oxazoline ring and silyl ether deprotection (55 % overall).     

Preparation of Thermocleavable Conjugates Based on Ansamitocin and Superparamagnetic Nanostructured Particles by a Chemobiosynthetic Approach

A combination of mutasynthesis, precursor‐directed biosynthesis and semisynthesis provides access to new ansamitocin derivatives including new nanostructured particle–drug conjugates. These conjugates are based on the toxin ansamitocin and superparamagnetic iron oxide–silica core shell particles. New ansamitocin derivatives that are functionalized either with alkynyl‐ or azido groups in the ester side chain at C‐3 are attached to nanostructured iron oxide core–silica shell particles. Upon exposure to an oscillating electromagnetic field these conjugates heat up and the ansamitocin derivatives are released by a retro‐Diels–Alder reaction. For example, one ansamitocin derivative exerts strong antiproliferative activity against various cancer cell lines in the lower nanomolar range while the corresponding nanostructured particle‐drug conjugate is not toxic. Therefore, these new conjugates can serve as dormant toxins that can be employed simultaneously in hyperthermia and chemotherapy when external inductive heating is applied.     

Hydrolysis behavior of prednisolone 21-hemisuccinate/beta-cyclodextrin amide conjugate: involvement of intramolecular catalysis of amide group in drug release

Prednisolone 21-hemisuccinate/beta-cyclodextrin (beta-CyD) amide conjugate was prepared by binding prednisolone 21-hemisuccinate covalently to the amino group of mono(6-deoxy-6-amino)-beta-CyD through amide linkage. Prednisolone 21-hemisuccinate was intramolecularly transformed to prednisolone 17-hemisuccinate, and the parent drug, prednisolone, was slowly released from the 21-hemisuccinate with a half life of 69 h in pH 7.0 at 37 degrees C; the drug release at 25 degrees C was less than 10% for 48 h. In sharp contrast, the hydrolysis of prednisolone 21-hemisuccinate/beta-CyD amide conjugate was significantly faster (half life of 6.50 min at 25 degrees C) and gave prednisolone and mono(6-deoxy-6-succimino)-beta-CyD as products. The hydrolysis of the beta-CyD amide conjugate was subject to a specific-base catalysis in the alkaline region. The rapid hydrolysis of the conjugate can be ascribed to the involvement of an intramolecular nucleophilic catalysis of the amide group in the reaction. The succinic acid, bound to a drug through ester linkage at one carboxylic group and bound to a pro-moiety through amide linkage at another carboxylic group, may be useful as a spacer for construction of the immediate release type prodrugs of CyDs.     

Preparative isolation and purification of anti‐tumor agent ansamitocin P‐3 from fermentation broth of Actinosynnema pretiosum using high‐performance counter‐current chromatography

Ansamitocin P‐3 is a potent anti‐tumor maytansinoid found in Actinosynnema pretiosum. However, due to the complexity of the fermentation broth of Actinomycete, how to effectively separate ansamitocin P‐3 is still a challenge. In this study, both analytical and preparative high‐performance counter‐current chromatography were successfully used to separate and purify ansamitocin P‐3 from fermentation broth. A total of 28.8 mg ansamitocin P‐3 with purity of 98.4% was separated from 160 mg crude sample of fermentation broth in less than 80 min with the two‐phase solvent system of hexane–ethyl acetate–methanol–water (0.6:1:0.6:1, v/v/v/v). The purity and structural identification were determined by HPLC, ¹H NMR, ¹³C NMR and mass spectroscopy.     

On the biosynthetic origin of methoxymalonyl-acyl carrier protein, the substrate for incorporation of "glycolate" units into ansamitocin and soraphen A

Feeding experiments with isotope-labeled precursors rule out hydroxypyruvate and TCA cycle intermediates as the metabolic source of methoxymalonyl-ACP, the substrate for incorporation of "glycolate" units into ansamitocin P-3, soraphen A, and other antibiotics. They point to 1,3-bisphosphoglycerate as the source of the methoxymalonyl moiety and show that its C-1 gives rise to the thioester carbonyl group (and hence C-1 of the "glycolate" unit), and its C-3 becomes the free carboxyl group of methoxymalonyl-ACP, which is lost in the subsequent Claisen condensation on the type I modular polyketide synthases (PKS). d-[1,2-(13)C(2)]Glycerate is also incorporated specifically into the "glycolate" units of soraphen A, but not of ansamitocin P-3, suggesting differences in the ability of the producing organisms to activate glycerate. A biosynthetic pathway from 1,3-bisphosphoglycerate to methoxymalonyl-ACP is proposed. Two new syntheses of R- and S-[1,2-(13)C(2)]glycerol were developed as part of this work.     

Xanthomonins I–III: A New Class of Lasso Peptides with a Seven‐Residue Macrolactam Ring

Lasso peptides belong to the class of ribosomally synthesized and post‐translationally modified peptides. Their common distinguishing feature is an N‐terminal macrolactam ring that is threaded by the C‐terminal tail. This lasso fold is maintained through steric interactions. The isolation and characterization of xanthomonins I–III, the first lasso peptides featuring macrolactam rings consisting of only seven amino acids, is now presented. The crystal structure of xanthomonin I and the NMR structure of xanthomonin II were also determined. A total of 25 variants of xanthomonin II were generated to probe different aspects of the biosynthesis, stability, and fold maintenance. These mutational studies reveal the limits such a small ring imposes on the threading and show that every plug amino acid larger than serine is able to maintain a heat‐stable lasso fold in the xanthomonin II scaffold.     

Combined muta- and semisynthesis: a powerful synthetic hybrid approach to access target specific antitumor agents based on ansamitocin P3

Access of four new tumor specific folic acid/ansamitocin conjugates is reported that relies on a synthetic strategy based on the combination of mutasynthesis and semisynthesis. Two bromo‐ansamitocin derivatives were prepared by mutasynthesis or by a modified fermentation protocol, respectively, that served as starting point for the semisynthetic introduction of an allyl amine linker under Stille conditions. A sequence of standard coupling steps introduced the pteroic acid/glutamic acid/cysteine unit to the modified ansamitocins. All new derivatives, including those that are expected to be generated after internalization of the folic acid/ansamitocin conjugates into the cancer cell and reductive cleavage of the disulfide linkage showed good to strong antiproliferative activity (IC₅₀ <10 nM ) for different cancer cell lines. Finally, the four conjugates were exposed to two cancer cell lines [cervix carcinoma, KB‐3‐1 (FR+) and lung carcinoma, A‐459 (FR?)], the latter devoid of the membrane‐bound folic acid receptor (FR?). All four conjugates showed strong antiproliferative activity for the FR+ cancer cell line but were inactive against the FR? cell line. The synthetic strategy pursued is based on the combination of mutasynthesis and semisynthesis and proved to be powerful for accessing new ansamitocin derivatives that are difficult to prepare by total synthesis.     

Deciphering indolocarbazole and enediyne aminodideoxypentose biosynthesis through comparative genomics: insights from the AT2433 biosynthetic locus

AT2433, an indolocarbazole antitumor antibiotic, is structurally distinguished by its aminodideoxypentose-containing disaccharide and asymmetrically halogenated N-methylated aglycon. Cloning and sequence analysis of AT2433 gene cluster and comparison of this locus with that encoding for rebeccamycin and the gene cluster encoding calicheamicin present an opportunity to study the aminodideoxypentose biosynthesis via comparative genomics. The locus was confirmed via in vitro biochemical characterization of two methyltransferases--one common to AT2433 and rebeccamycin, the other unique to AT2433--as well as via heterologous expression and in vivo bioconversion experiments using the AT2433 N-glycosyltransferase. Preliminary studies of substrate tolerance for these three enzymes reveal the potential to expand upon the enzymatic diversification of indolocarbazoles. Moreover, this work sets the stage for future studies regarding the origins of the indolocarbazole maleimide nitrogen and indolocarbazole asymmetry.     

Enthalpies of interaction of N-acetyl amides of sarcosine and N-methyl-L-alanine dissolved in N,N-dimethylformamide at 25°C

Enthalpies of dilution of N-acetyl amides of sarcosine and N-methyl-L-alanine dissolved in N,N-dimethylformamide have been measured calorimetrically at 25°C. The enthalpic pairwise interaction coefficients calculated there from are negative, indicating a energetically favorable interaction. The results were used to make a comparison with other peptides with regard to the methylation of amide groups. Substituting a primary amide hydrogen by a methyl group gives a smaller positive change of the pairwise interaction coefficient than substituting a secondary amide hydrogen.