Nature loves nitrogen heterocycles

Accepted Manuscript Nature Loves Nitrogen Heterocycles Christopher T. Walsh PII: DOI: Reference:

S0040-4039(14)01939-X http://dx.doi.org/10.1016/j.tetlet.2014.11.046 TETL 45430

To appear in:

Tetrahedron Letters

Received Date: Revised Date: Accepted Date:

24 October 2014 8 November 2014 12 November 2014

Please cite this article as: Walsh, C.T., Nature Loves Nitrogen Heterocycles, Tetrahedron Letters (2014), doi: http:// dx.doi.org/10.1016/j.tetlet.2014.11.046

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Nature Loves Nitrogen Heterocycles

Christopher T. Walsh ChEM-H Institute, Stanford University Stanford, CA [email protected] 781-956-6192

Keywords: nitrogen heterocycles biosynthesis amino acid morphing enzymatic transformations

Abstract: Nitrogen-containing heterocycles are central to to the chemical reactions that occur in all organisms. The metabolic tranformation of amino acids into five, six, and seven-member heterocycles reveals the chemical logic and enzymatic machinery for shunting primary metabolites into bioactive heterocyclic nitrogen scaffolds.

Dedicated to Harry Wasserman: I first met Harry Wasserman when I was a second year assistant professor in the MIT Chemistry Department at MIT. We had breakfast together at the Nassau Inn at Princeton in 1974 and I was immediately captivated by his enthusiasm and intensity and his open invitation to hear me discuss my plans to uncover novel enzyme-mediated chemistry in biological systems. Subsequently when Harry’s son Steven enrolled as a biology graduate student at MIT, I was one of

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two supervisors (David Botstein was the other) for his dissertation research. Harry was always interested but properly distant about Steven’s progress during that five year interval: it was all good and Steven for years has been a Professor at UCSD where he, like his father at Yale, has been recognized as an outstanding teacher. Among his many chemical interests Harry Wasserman was interested in natural products and amino-acid based heterocycles that were constituents of many such scaffolds. Over the past decade in my research group we became focused on enzymes that morph amino acid building blocks into scaffolds with a variety of heterocyclic frameworks in order to decipher the chemical logic and molecular machinery for their construction. Heterocycles in Natural Products: Nitrogen heterocycles are the building blocks of life. They are the key constituents of both DNA and RNA and the proteinsynthesizing ribosomes are essentially RNA machines. Different nitrogen heterocycles are predominant functional groups in many coenzymes that mediate primary metabolic transformations: from the monocyclic pyridine aldehyde in the the coenzyme form of vitamin B6 and the pyridine carboxamide in the nicotinamide coenzymes and the methyl thiazolium reaction center in vitamin B1. For the bicyclic pterin ring of folate coenzymes and the tricyclic isoalloaxine ring of vitamin B2 coenzyme forms, the nitrogen heterocycles are key reaction centers. We have noted elsewhere (1) how medicinal chemists , in a riff on Nature’s molecules, have artfully constructed heterocyclic ring moieities in a variety of contemporary drugs, including the pyrazine ring in bortezomib, the pyrazole in crizotinib, the triazole in anastrazole as well as the aminothiazole in cephalosporins and the aza-indole scaffold in PLX4032. While both the biosynthesis and reactivity patterns of the coenzymes noted above have been well studied over past decades (2), knowledge of routes and mechanisms of assembly of many nitrogen heterocycles in natural product classes have only been deciphered in recent years, often enabled by genome sequencing in microbial producers and the study of encoded biosynthetic enzymes. We summarize below

2

some recent efforts from our group and others to decipher previously unknown routes to natural product heterocyclic scaffolds. Multiple biosynthetic routes to β -lactams: scheme 1 here As one example it is now apparent that the famous β-lactam four membered ring can be fashioned by four distinct enzymatic routes (scheme 1). Isopenicillin N synthase forms the four ring and then the five ring of isopenicillin N (1) as O2 is reduced by four electrons to two water molecules (3; 4), while clavulanate (2) and natural carbapenems (3) form via an intramolecular attack of the amine on a carboxylic-phosphoric anhydride (5, 6). In nocardicins (4) a different logic entails as a serine residue is converted to the lactam (7). Finally, although the mechanism for generation of the hydroxy-β-lactam (5) in the wildfire toxin tabtoxin is not fully resolved, it is clear that it represents a diversion of intermediates from the lysine biosynthetic pathway in the producing pseudomonad strains (8). Pyrrole group formation and transfer: At the level of a biological five-membered ring nitrogen heterocycle, the pyrrole ring occupies a central place and its biosynthesis from aminolevulinate and conversion to tetrapyrrole macrocycles in porphyrin and corrin macrocycles had been well studied (9, 10). Also, the pyrrole ring is the business end of the bicyclic indole side chain of tryptophan. Trp biosynthesis is well understood (2), as are the reactions of its indole ring as a nucleophile in enzymatic prenylations that can functionalize all nonbridgehead carbons and the nitrogen (11) in the assembly of hundreds of indole terpene scaffolds. scheme 2 here Parallel logic is in play (scheme 2) to generate the pyrroles in pyrrolnitrin (6), pyoluteorin (7), and in chlorobiocin (8), where the amino acid proline is the precursor (12). We demonstrated that proline is activated and tethered as thioesters bound covalently to carrier protein domains and then oxidized enzymatically by

3

four electrons to the pyrrolyl thioesters. These activated pyrrole moieties could then be chlorinated or methylated and used for condensative transfers to nucleophilic cosubstrates. In the assembly of the tripyrrolic prodigiosin scaffold (9), the first pyrrole is assembled by the above route from a prolyl thioester and then elongated with a decarboxylative thioester condensation of a malonyl nucleophilic substrate and condensation with a serine unit to generate the HBM intermediate (13). The third pyrrole is assembled by yet a distinct route (14). Indolocarbazoles with pyrrolic intermediates: Of special medicinal interest are the natural products staurosporine (10)and rebeccamycin (11), the former as a potent but promiscuous protein kinase inhibitor (15) ), the latter as a potent inhibitor of mammalian DNA topoisomerase 1 (16). The core framework is a hexacyclic indolocarbazole , where the pyrrole moiety is in distinct oxidation states (scheme 3). We have investigated the biosynthetic pathway involving four enzymestwo flavoproteins and two hemeproteins to carry out 10-14 electron oxidations of the starting tryptophans (17-19). Oxidative dimerization of the imine derived from oxidation of tryptophan yields chromopyrrolic acid (CPA)(12) as intermediate ( and progenitor to other indolocarbazole structures). Four electron oxidation of CPA yields the aglycone of staurosporin or rebeccamycin before N-glycosyltransferases complete the pathway (20). scheme 3 here Oxazoles and Thiazoles: Oxazoles and thiazoles and their precursor oxazoline and thiazoline rings with two hetero atoms embedded in the five-membered ring are useful in several biological (as well as medicinal) contexts (scheme 4AB). In the oxazoline and thiazoline oxidation states the nitrogen atoms are basic and act as high affinity ligands for Fe (III) in microbial siderophores, essential for bacterial and fungal acquisition of iron (21). scheme 4AB here

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On oxidation to the heteroaromatic oxazole and thiazole the basicity is lost but these are stable rigid structures. When strung together and macrocyclized as in patellamides (13) and telomastatin (14) (figure 4A), they can function as high affinity specific ligands for protein targets (22; 23). We have shown that the oxazoline and thiazoline-containing siderophores such as the plague bacterium’s yersiniabactin (15) and pre-acinetobactin (figure 4B) (16) are built on nonribosomal peptide synthetase assembly lines (24; 25), where serine, threonine, and cysteine residues are cyclodehydrated. An orthogonal route for conversion of X-Ser and X-Cys peptide bonds into oxazoles and thiazoles occurs in posttranslational cyclodehydrations and oxidations of nascent proteins (26; 27). After heterocyclization the nascent peptides are proteolytically carved out of the precursor protein by macrocyclization. Mutant forms of the precursor proteins allow structural diversity in the final products as shown for patellamides (28).

Pyridines from protein precursors: Thiopeptide antibiotics such as thiocillins (17) and thiostreptons (18) (sceheme 5A) approach the apotheosis of posttranslational modification of protein precursors (with up to 20 modification steps) (29; 30). Not only are there oxazoles and thiazoles in a 26 atom macrocycle, but there is also a central pyrdine/dihydropyridine embedded in the core and bearing three thiazolyl substituents such that the core scaffold is the tetracyclic 2,3,6thiazolyl- (dihydro)-pyridine (31) . We have shown that the pyridine is formed in the last step, from condensation of two dehydroalanyl residues (in turn derived from two serine residues by dehydration) (32) (scheme 5B). Pyridine formation constitutes the macrocyclization step and represents a remarkable morphing of the original acyclic protein precursor into a rigidified scaffold that binds tightly to bacterial ribosomes (33). scheme 5AB here Diketopiperazines and epidithiodiketopiperazines: Nonribosomal peptide synthetase assembly logic on dipeptiyl units is also at the core of many additional

5

natural products. Internal capture of the Trp-Pro-thioester-enzyme intermediate releases the diketopiperazine (DKP) brevianamide-F (19) that is the substrate for subsequent prenylations and oxidations to yield, inter alia, the fumitremorgins (20) (34) (scheme 6). Analogously the celebrated fungal toxin gliotoxin (21)is assembled from a comparable Phe-Ser-dipeptide assembly line (35). Most notable in this and related epidithiopiperazine frameworks is the installation of sulfur atoms at the Cα positions of the dipeptides during biosynthesis. Oxidation to the disulfide creates the characteristic epidisulfide bridge across the DKP that is the hallmark of this toxin family and thiol-disulfide interchange chemistry with host molecules and proteins is a likely source of toxicity (36). scheme 6 here Anthranilate and Tryptophan: short pathways to fungal alkaloid scaffold complexity: During tryptophan biosynthesis in microbes (Trp is an essential dietary amino acid in higher eukaryotes), anthranilate (ortho-aminobenzaote) is a key metabolic precursor. Anthranilate (Ant) and tryptophan are related as grandparent to daughter molecules (scheme 7) (37). These can be paired by aspergilli and penicillium molds to some remarkably complex heterocyclic frameworks in nonribosomal peptide synthetase (NRPS) pathways and associated tailoring enzymes of only two to four enzymatic steps (Walsh et al, 2013). A variety of tryptophan-derived natural products have the indole ring converted to the tricyclic pyrroloindole moiety. This is seen in both aszonalenin and ardeemin and can occur in later metabolites in the fumiquinaziline pathway (1). In the cases examined, the reaction of the C3 of the indole ring as nucleophile towards an electrophilic prenyl group of an electrophilic iron-oxo species, sets up the indoline for capture by a neighboring nitrogen to yield the tricyclic pyrroloindole. A simple two module NRPS assembly line generates an Ant-Trp-S-enzyme intermediate where the thioester is captured intramolecularly by the Ant-NH2 group to release a six-seven fused bicyclic benzodiazepinedione product (22) (scheme 9). This can be prenylated enzymatically at C3 of the indole ring and the resulting

6

indoline captured intramolecularly to produce the fused pentacyclic framework of aszonalenin (23) (38). Only two enzymes required. Scheme 7 here Analogously, a three module NRPS from Aspergillus fumigatus activates Ant, Trp, Ala and the tripeptidyl-S-enzyme intermediate suffers intermolecular capture again by the Ant-NH2. Cyclodehydration gives tricyclic fumiquinazoline F (24) as the only product (39). In a related aspergillus, the trimodular NRPS assembly line make an isomeric Ant-D-Ala-Trp-thioester intermediate which cyclizes during release to a regioisomeric quinazolinedione (25). A dedicated tailoring enzyme again prenylates C3 of the indole and now intramolecular closure generates the hexacyclic scaffold of ardeemin(26) (40), a potent blocking ligand for multidrug resistant export pumps in cancer cells. The hexacyclic framework is again constructed with only two biological catalysts in the pathway, almost incredible efficiency in complexity generation from primary metabolites. Pyrazines and Dimethylbenzimidazole: Progress has also been achieved in defining the biological routes to additional heterocycles, including ones with N-O and N-N linkages. One such is the six membered piperazic acid with a direct N-N bond, found in a variety of peptide-derived antibiotics, including piperazimycins (27) )(41). It appears that N5-hydroxy-ornithine is an intermediate scheme 8 here but how that is further derivatized to achieve elimination/cyclization is not yet understood (42) . As a final example of Nature’s ability to produce nitrogen heterocycles in efficient and still mechanistically mysterious routes, the protein BluB converts the three ring flavin mononucleotide (28) to the two ring dimethylbenzimidazole(DMB) (29) (43). DMB is then used as the bottom axial ligand for the Cobalt(III) in the corrin ring of vitamin B12. We have termed BluB an oxygen-consuming “flavin destructase” (scheme 9).

7

scheme 9 here References

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23 Shin-ya, K.; Wierzba, K.; Matsuo, K.; Ohtani, T.; Yamada, Y.; Furihata, K.; Hayakawa, Y.; Seto, H. J Am Chem Soc 2001, 123, 1262. 24 Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & biology 2002, 9, 333. 25 Wuest, W. M.; Sattely, E. S.; Walsh, C. T. J Am Chem Soc 2009, 131, 5056. 26 Li, Y. M.; Milne, J. C.; Madison, L. L.; Kolter, R.; Walsh, C. T. Science 1996, 274, 1188. 27 Arnison, P. G.; Bibb, M. J.; Bierbaum, G.; Bowers, A. A.; Bugni, T. S.; Bulaj, G.; Camarero, J. A.; Campopiano, D. J.; Challis, G. L.; Clardy, J.; Cotter, P. D.; Craik, D. J.; Dawson, M.; Dittmann, E.; Donadio, S.; Dorrestein, P. C.; Entian, K. D.; Fischbach, M. A.; Garavelli, J. S.; Goransson, U.; Gruber, C. W.; Haft, D. H.; Hemscheidt, T. K.; Hertweck, C.; Hill, C.; Horswill, A. R.; Jaspars, M.; Kelly, W. L.; Klinman, J. P.; Kuipers, O. P.; Link, A. J.; Liu, W.; Marahiel, M. A.; Mitchell, D. A.; Moll, G. N.; Moore, B. S.; Muller, R.; Nair, S. K.; Nes, I. F.; Norris, G. E.; Olivera, B. M.; Onaka, H.; Patchett, M. L.; Piel, J.; Reaney, M. J.; Rebuffat, S.; Ross, R. P.; Sahl, H. G.; Schmidt, E. W.; Selsted, M. E.; Severinov, K.; Shen, B.; Sivonen, K.; Smith, L.; Stein, T.; Sussmuth, R. D.; Tagg, J. R.; Tang, G. L.; Truman, A. W.; Vederas, J. C.; Walsh, C. T.; Walton, J. D.; Wenzel, S. C.; Willey, J. M.; van der Donk, W. A. Natural product reports 2013, 30, 108. 28 Donia, M. S.; Hathaway, B. J.; Sudek, S.; Haygood, M. G.; Rosovitz, M. J.; Ravel, J.; Schmidt, E. W. Nature chemical biology 2006, 2, 729. 29 Walsh, C. T.; Acker, M. G.; Bowers, A. A. The Journal of biological chemistry 2010, 285, 27525. 30 Liao, R.; Duan, L.; Lei, C.; Pan, H.; Ding, Y.; Zhang, Q.; Chen, D.; Shen, B.; Yu, Y.; Liu, W. Chemistry & biology 2009, 16, 141. 31 Hughes, R. A.; Moody, C. J. Angewandte Chemie 2007, 46, 7930. 32 Bowers, A. A.; Walsh, C. T.; Acker, M. G. J Am Chem Soc 2010, 132, 12182. 33 Harms, J. M.; Wilson, D. N.; Schluenzen, F.; Connell, S. R.; Stachelhaus, T.; Zaborowska, Z.; Spahn, C. M.; Fucini, P. Molecular cell 2008, 30, 26. 34 Grundmann, A.; Li, S. M. Microbiology 2005, 151, 2199. 35 Balibar, C. J.; Walsh, C. T. Biochemistry 2006, 45, 15029. 36 Srinivasan, U.; Bala, A.; Jao, S. C.; Starke, D. W.; Jordan, T. W.; Mieyal, J. J. Biochemistry 2006, 45, 8978. 37 Walsh, C. T.; Haynes, S. W.; Ames, B. D. Natural product reports 2012, 29, 37. 38 Yin, W. B.; Grundmann, A.; Cheng, J.; Li, S. M. The Journal of biological chemistry 2009, 284, 100. 39 Ames, B. D.; Walsh, C. T. Biochemistry 2010, 49, 3351. 40 Haynes, S. W.; Gao, X.; Tang, Y.; Walsh, C. T. ACS chemical biology 2013, 8, 741. 41 Oelke, A. J.; France, D. J.; Hofmann, T.; Wuitschik, G.; Ley, S. V. Natural product reports 2011, 28, 1445. 42 Neumann, C. S.; Jiang, W.; Heemstra, J. R., Jr.; Gontang, E. A.; Kolter, R.; Walsh, C. T. Chembiochem : a European journal of chemical biology 2012, 13, 972. 43 Taga, M. E.; Larsen, N. A.; Howard-Jones, A. R.; Walsh, C. T.; Walker, G. C. Nature 2007, 446, 449.

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H N

H2N HO

O

O

SH

O2

NH

O

H OH O H N Fe S

II

IV

COO

Fe O

HO

2 H2O

OH

O

S

COO

III OH Fe S

N O

COO

R

PPi

COO

AMP

OOC

N H

COO

NH2

COO S

O

R

R

O2 O

H

N

COO

COO

COO

N

N

N

ATP

NH2

isopenicillin N COOH

O

2

O

AMP + PPi

N

NH2 N H

OOC

NH2

COO

Carbapenem

3S,5S-carbapenam carboxylate

H N

O

N

O

O

HN

OH Fe

O

lactam ring formed first H20 released Fe=O intermediate

O

II

N

O

ATP

O

S N

H O

H2O

O

O

1

O R

H N

H 2N

O2

O

N OOC

OH

O2 NH2

O N

O

NH3 COO

Carboxyethyl Arginine NH2

HO

O

H N O

OH

4

O

OH

N COOH

Nocardicin G

O

H N

H2N

COOH

5

O

N COO

Clavulanate 3

OH

Cl NO2

N H

OH

pyrrolnitrin

N H

O

pyoluteorin

6

7

OH

OH

O O

O

O

O

O O

Cl

OH

Cl

OH

chlorobioicn

O NH

8 O NH NH

N

Prodigiosin 9

Cl

H N

N H3C

O

N

N H

Cl

H

NH

COOH

N H

OH

Cl

StaD

OH

11

H N

HOOC

NH

StaO

O

Rebeccamycin

10

N H

N

O

Staurosporine

NH2

O

HO

O

COOH

H N

O

O

N H

N H

Chromopyrrolic acid 12

H N

COOH

StaP StaC

N H

O

N H

Staurosporine aglycone

-Gly-Ser-

Oxazoline O

H N

N H

O

HO N H

O H

H N

O

Oxazole

O

O

O

N

N H

O

N H

dehydrogenase

H2O

N O

B

O

H N

N H

O

HO N H

S H

H N

O

O N H

S

O

N

N H

S

H2O

N S

B

-Gly-Cys-

O O

O

N

Thiazole

Thiazoline

N H

NH

N HN

N

N

S

O

S O

O

O

N N

N

N

N

O O

Patellamide C 13

O N

N O

N

O

O H

S

Telomestatin 14

Preacinetobactin N

16

HO N H+ OH N O

S

S N

N H

OH

HO

S HO

N

NH

O

OH O

H2O

Yersiniabactin

N NH

15 OH HO

O

O N H

Acinetobactin

N O

O

S

S

H N

N H

N

O OH

S

N O HO

S

MeO HN

O N N

HN

NH N O

S

17

O

S

O OH

HO

S

O

HN HO

N S

N S

18

OH

O O

HN N

O

NH

N

O O

NH NH

O

NH

N O N NH

N S

O

HN O

N N

NH2

HN

O

S

HN O OH

OH

O

N NH2

O O

N

S N H

N H

O H O

N H

O HOHO

N H O

N H

N

N H

Brevianamide F

N

N

O

Fumitremorgin B

Trypostatin B

20

19

OH

O NH2

N H

O

S O

SH O NH

HN

OH O

H O

N OH

OHS

O

N

S

S

N OH

Dihydro gliotoxin

OH

N O

Gliotoxin 21

OH

OH

HO COOH N H

NH2

Anthranilate parent

NH

NH2 N H

Indole glycerol-3-P daughter

O N H NH2

COOH OPO3H

grandaughter metabolite

H N

O

N H

N

O

NH

S O

Tryptophan

HN

O

NH

O

N H

N N H

O

O

Aszonalenin

Benzodiazepinedione

23

22

NH

O N H NH2

H N O

O

O N

S

Ant-D-Trp-L-Ala-S-enz

N

O

N

NH

N

Fumiquinazoline F 24

O

O

NH

O

O

N

NH

N

N

NH

NH

25

Ardeemin

26

HO

NH N

HN

HN

O O N

O

O O

O O

OH NH

N HN

Cl

Piperazimycin A 27

OPO3H OPO3H HO N

OH N

OH

HO

OH

O N

O

NH

N

N H

O

28

29

H N H N

N

O

H N

N O

O

N R

N

S O

O HN

N H

S N

N S