Mutasynthetic supplementation of the AHBA blocked mutant strain of S. hygroscopicus, the geldanamycin producer, with 21 aromatic and heteroaromatic amino acids provided new nonquinoid geldanamycin derivatives. Large scale (5 L) fermentation provided four new derivatives in sufficient quantity for full structural characterisation. Among these, the first thiophene derivative of reblastatin showed strong antiproliferative activity towards several human cancer cell lines. Additionally, inhibitory effects on human heat shock protein Hsp90α and bacterial heat shock protein from H. pylori HpHtpG were observed, revealing strong displacement properties for labelled ATP and demonstrating that the ATP-binding site of Hsps is the target site for the new geldanamycin derivatives.

Thirteen new reblastatin derivatives, with alkynyl, amino and fluoro substituents on the aromatic ring, were prepared by a chemo-biosynthetic approach using an AHBA(-) mutant strain of Streptomyces hygroscopicus, the geldanamycin producer. The inhibitory potencies of these mutaproducts and of an extended library of natural products and derivatives were probed with purified heat shock proteins (Hsps), obtained from Leishmania braziliensis (LbHsp90) as well as from human sources (HsHsp90). We determined the activities of potential inhibitors by means of a displacement assay in which fluorescence-labelled ATP competes for the ATP binding sites of Hsps in the presence of the inhibitor in question. The results were compared with those of cell-based assays and, in selected cases, of isothermal titration calorimetry (ITC) measurements. In essence, reblastatin derivatives are also able to bind effectively to the ATP-binding site of LbHsp90, and for selected derivatives, moderate differences in binding to LbHsp90 and HsHsp90 were encountered. This work demonstrates that parasitic heat shock proteins can be developed as potential pharmaceutical targets.

The cover feature picture shows new reblastatin derivatives obtained by a chemo-biosemisynthetic approach with the corresponding aminobenzoic acid derivatives as starting building blocks. The fluoro and amino derivatives exerted high inhibitory activity on human and leishmania Hsp90, whereas the presence of an alkynyl substituent leads to selective activity towards Hsp90 from leishmania. The inhibitory activity was unraveled by using a novel protein microarray system with purified Hsp90 proteins and was further confirmed by cell-based assays and ITC measurements. More information can be found in the full paper by C. Zeilinger, A. Kirschning et al. on page 562 in Issue 6, 2018 (DOI: 10.1002/cbic.201700616).

Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA-blocked mutant with all possible monofluoro 3-aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies.

In diseases such as cancer, Alzheimer's disease or malaria, disease-related proteins take advantage of the heat shock protein (HSP) control system for their own activation or maturation. There is a quest to find inhibitors that specifically bind to the HSPs. Here, we report on a novel multiplexed assay system for inhibitor screening based on a protein microarray (MA) technique that was developed for routine applications with storable MAs. Purified HSPs are printed as full-length proteins on microarrays and used as a drug target for the screening of new inhibitors. Derivatives obtained by a combination of biological and chemical synthesis were tested as competitors of ATP with a suggested affinity for several HSP proteins which are hHSP from human, AtHSP83 (Arabidopsis thaliana) and HtpG from Helicobacter pylori. Some of these new derivatives exerted selectivity between human and bacterial heat shock proteins. Printed human HSP90 was used to test the binding of denatured proteins on the client binding site of human HSP90, since the full-length HSP maintains the capability to bind putative clients or cochaperones. Initial data revealed that the microarray application can be used to identify directly elevated heat-shock protein levels in cancer cell lysates. We suggest that microarray-based assaying of HSP levels can be used as a marker for determining stress levels.

Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family.

Covering 2005 to 2013. In this review recent progress in the development of heat shock proteins (Hsp90) in oncogenesis is illuminated. Particular emphasis is put on inhibitors such as geldanamycin and analogues that serve as a natural product show case. Hsp90 has emerged as an important target in cancer therapy and/or against pathogenic cells which elicit abnormal Hsp patterns. Competition for ATP by geldanamycin and related compounds abrogate the chaperone function of Hsp90. In this context, this account pursues three topics in detail: a) Hsp90 and its biochemistry, b) Hsp90 and its role in oncogenesis and c) strategies to create compound libraries of structurally complex inhibitors like geldanamycin on which SAR studies and the development of drugs that are currently in different stages of clinical testing rely.

Supplementing a culture of a mutant strain of Actinosynnema pretiosum that is unable to biosynthesize aminohydroxy benzoic acid (AHBA), with 3-azido-5-hydroxy-benzoic acid and 3-azido-5-amino-benzoic acid, unexpectedly yielded anilino ansamitocins instead of the expected azido derivatives. This is the first example of the bioreduction of organic azides. The unique nature of these results was demonstrated when 3-azido-5-amino-benzoic acid was fed to the corresponding AHBA blocked mutant of Streptomyces hygroscopicus, the geldanamycin producer. This mutasynthetic experiment yielded the fully processed azido derivative of geldanamycin.

Blocked mutants of Actinosynnema pretiosum, the producer of the highly cytotoxic antitumor agent ansamitocin, serve as powerful tools that allow synthetic chemists to generate natural product libraries. The power of this approach can be dramatically expanded when mutasynthesis is combined with chemical synthesis. This report provides illustrative examples of the application of this strategy to produce libraries based on the ansamycin antibiotics.

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.

AbstractReview: [semisynthesis, total synthesis, mutational biosynthesis or mutasynthesis of naural product libraries for use in medicinal chemistry.

Working hand in hand! The synthetic power of three mutant strains that produce ansamitocin and geldanamycin is combined with chemical synthesis, thus leading to 27 new ansamitocin derivatives. Structure–activity studies show that the N of the carbinolamide moiety is not important for cytotoxic activity but the α orientation of the OH group at C9 is key [see structures; chemical synthesis (red), mutasynthesis (blue), biosynthesis (black)]. Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Since its invention by Rinehart and Gottlieb, mutational biosynthesis (“mutasynthesis”) has become a useful tool in the portfolio of the synthetic natural product chemist for the preparation of complex natural product derivatives with pharmaceutical potential. Mutasynthesis requires the generation of mutants of a producer organism that are blocked in the formation of a biosynthetic building block of the end-product. Administration of mutasynthons to the blocked mutant results in new metabolites. A natural product suitable for mutasynthetic investigations is geldanamycin (1, Scheme 1), a potential antitumor drug that binds to the N-terminal ATP-binding domain of heat shock protein 90 (Hsp90) and inhibits its ATPdependent chaperone activities. Most geldanamycin derivatives reported to date are 17-aminated compounds and were obtained by semisynthesis. Recently, two groups have utilized blocked mutants of the microbial source of geldanamycin to prepare several new derivatives. Benzoquinone-containing Hsp90 inhibitors depend on reductive activation to the hydroquinone by the enzyme NAD(P)H/quinone oxidoreductase 1 (NQO1). As the activity of this enzyme in different patients is variable, derivatives that show binding to the ATP binding pocket of Hsp90 without the need for activation by NQO1 are highly desirable. Additionally, the quinone moiety of geldanamycin is held responsible for undesired side effects (for example, hepatotoxicity). The Michael addition of the thiol moiety of glutathione to the quinone is regarded as one source of problems. Related to geldanamycin 1 is reblastatin 2, which is saturated across C4–C5 and has a benzene chromophore instead of a benzoquinone or a hydroquinone moiety. Importantly, reblastatin shows lower cytotoxicity than geldanamycin but has a higher affinity for Hsp90. The genes required for the biosynthesis of 1 have been cloned, sequenced, and independently analyzed in several streptomycetes.The producing microorganism Streptomyces hygroscopicus var. geldanus NRRL 3602 creates geldanamycin through a biosynthetic machinery based on a polyketide synthase (PKS) and additional post-PKS enzymes. The biosynthesis of 1 is primed by the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA, 3), which originates from a shikimate-type biosynthetic pathway (Scheme 1). The PKS generates seco-progeldanamycin which is cyclised and released from the PKS by an amide synthase. The resulting progeldanamycin is then further modified by a set of tailoring enzymes, starting with the oxidation of C21 and C17, followed by O-methylation at C17, introduction of the carbamoyl moiety and finalized by dehydrogenation across C4–C5. The oxidation of the hydroquinone moiety to the quinone only takes place after oxidation at C21. Disruption of genes coding for AHBA formation leads to blocked mutants without affecting the modules of the polyketide biosynthetic genes (PKS 1). After our successful application of the mutasynthesis methodology for the generation of ansamitocin P-3 derivatives [a] S. Eichner, Prof. Dr. A. Kirschning Zentrum f r Biomolekulare Wirkstoffe (BMWZ), Leibniz Universit t Hannover Schneiderberg 1B, 30167 Hannover (Germany) Fax: (+ 49) 511-762-3011 E-mail : andreas.kirschning@oci.uni-hannover.de [b] Prof. Dr. H. G. Floss Department of Chemistry, University of Washington Seattle, Washington 98195-1700 (USA) [c] Dr. F. Sasse Abteilung Chemische Biologie, Helmholtz Zentrum f r Infektionsforschung (HZI) Inhoffenstrasse 7, 38124 Braunschweig (Germany) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900246. Scheme 1. Principal biosynthetic pathway of geldanamycin (1) (ACP = acyl carrier protein of last PKS-module) and structures of reblastatin (2) and 3amino-5-hydroxybenzoic acid (AHBA, 3).

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The microwave heated Suzuki–Miyaura cross-coupling reaction of boronic acids, boronic esters and organotrifluoroborates served as a model reaction in a singlemode equipment. The reaction conditions were optimized with respect to temperature and reaction time and were transferred to multimode equipment which is well suited for multiparallel synthesis in a larger scale. The source of the Pd species chosen included immobilized Pd complexes and Pd particles. In fact the increased time to reach the required reaction time in multimode chambers suitable for 48 parallel reactions has to be taken into account. The nature of the boronic acid has no impact on the efficiency of the catalytic process. However, heterogenized Pd species perform less well in multimode chambers with larger vial volumes, which we ascribe to diffusion phenomena.