Discovery and Reconstitution of the Cycloclavine Biosynthetic Pathway - Enzymatic Formation of a Cyclopropyl Group

Dorota Jakubczyk, Lorenzo Caputi, Anaëlle Hatsch, Curt A. F. Nielsen, Melanie Diefenbacher, Jens Klein, Andrea Molt, Hartwig Schröder, Johnathan Z. Cheng, Michael Naesby, and Sarah E. O’Connor

ACIE, 2015, 54, 5117-5121

CO2H N NH2 H O O 8 enzymes + P P N O O O H O O L-tryptophan DMAPP NH cycloclavine

Steph McCabe Wipf Group- Current Literature 27th June 2015

Steph McCabe@ Wipf Group Slide 1 of 12 6/27/15 N H Ergot Alkaloids

NH • Ergot alkaloids are produced by fungi of the Clavicipitaceae and Trichocomaceae families, which infect the seeds of grass or grains

• Consumption of ergot contaminated grains leads to ergotism (St. Anthony’s fire); § ergotismus convulsivus characterized by hallucinations and paranoia § ergotismus gangrenous characterized by gangrene of the feet, legs, hands and arms

• Ergot alkaloids are characterized by the presence of a tetracyclic Ergoline core and can be further classified into Lysergic acid or Clavine subclasses based on the oxidation state of the substituent at C8

8 NH D H

HO2C 8 Me 8 NH C NH D H D H A B 2 C C N H 1 Ergot on wheat stalks A B 2 Ergoline Core A B 2 N N H 1 H 1

Lysergic acid subclass Clavine subclass

O O N N NH O Et2N OH H O N O NH N N H H H H

NH NH NH NH

ergotamine Lysergic acid diethylamide (LSD) festuclavine cycloclavine

Wallwey, C. and Li, S.,Nat. Prod. Rep., 2011, 28, 496 Jakubczyk, D., Cheng, J. Z. and O’Connor, S., Nat. Prod. Rep., 2014, 31, 1328 Steph McCabe@ Wipf Group Slide 2 of 12 6/27/15 N H Biological Activity

• The broad spectrum of bioactivity exhibited by ergot alkaloids is related to their ability to act as an agonist or antagonist toward neuroreceptors for dopamine, serotonin and adrenaline.

8 6 NH H 9 10 5 4 H11 16 3 12 2 13 1 15 NH 14 Ergoline

6 6 NH2 NHMe 6 NH2 5 HO 5 5 10 10 4 11 11 11 HO 16 3 12 16 12 16 12 2 13 13 13 1 15 15 15 N HO 14 HO 14 14 H OH OH serotonin (5-HT) dopamine (DA) epinephrine (adrenalin)

Jakubczyk, D., Cheng, J. Z. and O’Connor, S., Nat. Prod. Rep., 2014, 31, 1328

Steph McCabe@ Wipf Group Slide 3 of 12 6/27/15 N H Examples of FDA Approved Drugs with an Ergoline Core

NH Br OH N OH NH NH O O O N H O N N H H

N NH R N Ergometrine R = Me R = H Me Nicergoline Use: induces contractions, Methysergide Methylergometrine Use: treatment of senile prevents post partum Use migraine treatment Use: prevents post partum dementia haemorrhage MOA: antagonist of haemorrhage, migraine treatment MOA: partial agonist/antagonist of MOA: alpha-1A adrenergic Mechanism of Action (MOA): serotonin 5-HT2B receptors receptor antagonist acts on alpha-adrenergic serotonergic, dopaminergic and serotonergic receptors and alpha adrenergic receptors

N O NH O N N NH O N O S N H OH O N H O N H H H H Br NH NH NH

Pergolide Cabergoline Bromocriptine Use: treatment of Parkinson’s Disease Use: treatment of Use: treatment of pituitary tumors, MOA: agonist of dopaminergic receptors hyperprolactinaemia Parkinson's disease, (*withdrawn from the US market in 2007 and Parkinson's disease hyperprolactinaemia, due to increased risk of cardiac MOA: agonist of MOA: agonist valvulopathy) dopamine D2 receptors. of dopamine D2 receptors DrugBank 4.0: Nucleic Acids Res., 2014, 42(1),1091-7. Steph McCabe@ Wipf Group Slide 4 of 12 6/27/15 N H Cycloclavine

N H

Cycloclavine • First isolated in 1969 from the seeds of the African morning glory (Ipomea hildebrandtii) by Hoffman and co-workers

• Subsequently isolated Aspergillus japonicus, a species of in filamentous fungus

• Absolute configuration assigned from X-ray crystallographic analysis of the methobromide salt

• No significant biological activities reported thus far Ipomea hildebrandtii

• 3 total syntheses, including an earlier synthesis from our group in 14 linear steps and 1.2% overall yield

steps Boc O N 1. IMDAF OH HN 2. LAH N N HO H H CO2Me (±)-cycloclavine Hofmann et. al.,Tetrahedron, 1969, 25, 5879 Furuta, T., et al., Agric. Biol. Chem., 1982, 46, 1921 Incze, M., et al., Tetrahedron, 2008, 64, 2924 Petronijevic, F. R. and P. Wipf JACS, 2011, 133, 7704 Jabre, N. D., et al. ,Tetradhedron Lett,, 2014, 56, 197

Steph McCabe@ Wipf Group Slide 5 of 12 6/27/15

N Biosynthetic Pathway Elucidation: H Reconstitution of the Ergot Alkaloid Synthesis (EAS) Genes NH

• All ergot alkaloids are derived from common a biosynthetic intermediate - chanoclavine-I. The mechanisms of most downstream biosynthetic transformations are unknown

• Recently a 16.8 kbp biosynthetic cluster, containing 8 genes in the genome of cycloclavine producer Aspergillus japonicus was identified (O'Connor et al)

• 7 genes are homologous to genes previously implicated in the biosynthesis of festuclavine or agroclavine.

• Role of easH unknown

equipped with a 1.7mm cryogenic TCI probe. The structure of cycloclavine 6 was solved by H 1 13 1 1 1 13 lysergic acid N means H-NMR, C-NMR, H, H-ROESY, and H, C-HMBC.subclass H OH O

HN SI-2 Supporting Figures agroclavine H H EasD/ FgaDH H H N N H Figure S1 Organization of the AspergillusH japonicus cycloclavine 6 gene cluster NAD NADH HN HN H N ?? N chanoclavine-I chanoclavine-I aldehyde clavine subclass H H

NAD NADH HN HN festuclavine cycloclavine

Steph McCabe@ Wipf Group Slide 6 of 12 6/27/15

13 N Biosynthetic Pathway Elucidation: H Reconstitution of the Ergot Alkaloid Synthesis (EAS) Genes NH

• This paper aimed to validate whether this cluster was responsible for cycloclavine biosynthesis by reconstitution of the eight genes in S. cerevisiae

• Chanoclavine-I was synthesized by heterologous expression of dmaW, easF, easE and easC in a S. cerevisiae strain

CO2H CO2H DmaW/ EasF/ NH2 prenyl transferase NH2 methyl transferase

N DMAPP PPi N SAM S-homo H H cysteine L-tryptophan DMAT

HN H H EasE/ oxidase N EasC/ catalase H CO2H

N HN H Chanoclavine-I N-Me-DMAT

Microbial Cell Factories, 2014, 13, 95 Nat. Prod. Rep., 2011, 28, 496

Steph McCabe@ Wipf Group Slide 7 of 12 6/27/15 Angewandte. Communications

N Biosynthetic Pathway Elucidation: H Reconstitution of the Ergot Alkaloid Synthesis (EAS) Genes NH

• In Vivo transformation of the chanoclavine-I producing strain with easD, easA, and easG produced festuclavine

• Addition of easH produced cycloclavine as the major product along with festuclavine

• In vitro incubation of chanoclavine-I aldehyde with easA, eas G easH produced cycloclavine

• Cycloclavine was reconstituted in S. cerevisiae with a final concentration of 529 mg. L-1

OH O

easD/ eas A/ H H H H Reductase N oxidase N H H NAD NADH NADH NAD HN HN chanoclavine-I chanoclavine-I aldehyde

H eas G/ N Reductase H O NADH NAD HN festuclavine H H N H N H N H HN H easH/ eas G/ incubation of chanoclavine-I aldehyde dioxygenase Reductase with easA, eas G and easH HN HN cycloclavine festuclavineFigure 1. Ergot-alkaloid biosynthetic pathway. a) Biosynthesis of festuclavine (4), agroclavine (5), and cycloclavine (6) from l-tryptophan and NADH NAD optimised ratio > 7:1 dimethylallyl pyrophosphate (DMAPP). b) LC–MS chromatograms showing that EasA, EasG, and EasH are required to generate cycloclavine (6) from chanoclavine-I aldehyde (3). i–iv) Authentic standards of chanoclavine-I aldehyde (3; i), agroclavine (5; ii), festuclavine (4; iii), and cycloclavine (6, iv). v,vi) Reaction products from the incubation of Aj_EasA, Aj_EasG, and Aj_EasH with chanoclavine-I aldehyde (3) and cofactors: Steph McCabe@ Wipf Group Slide 8 of 12 v) by-product, festuclavine (4); vi) predominant enzymatic6/27/15 product, cycloclavine (6).

both cycloclavine (6) and festuclavine (4) have been isolated derivatized ergot alkaloids have not been subject to such from A. japonicus,[3a] we hypothesize that this gene cluster efforts. To examine whether we could produce the complex produces a mixture of these two compounds in the native ergot alkaloid cycloclavine (6) in high yields in S. cerevisiae, host, though how this ratio is impacted by environmental we integrated multiple copies of cycloclavine-pathway genes conditions is unknown. To assess whether increased amounts into the genome of the commonly used yeast strain S288C. of EasH would impact the festuclavine/cycloclavine ratio, we The best strain had an additional copy of easG, two additional constructed a strain carrying the entire eight-gene cluster copies of dmaW and easD, and three additional copies of easC supplemented with additional copies of easH from plasmid and easH. Moreover, additional copies of the host genes pdi1 vectors. We observed a clear gene-dose-dependent increase in (protein disulfide isomerase) and fad1 (FAD synthetase) the ratio of cycloclavine (6) to festuclavine (4) as the copy were also included to assist in the production of the disulfide- number of easH increased (see Figure S4). and flavin-containing enzyme EasE (see Table S1 in the Complex ergot alkaloids constitute a rich source of Supporting Information). This strain resulted in the produc- biologically active compounds, and a robust production tion of cycloclavine (6) with a final concentration of 1 platform for these molecules will improve accessibility and 529 mgLÀ in the growth medium when fermentation was prospects for commercial application. Whereas chanoclavine- carried out for 160 h in a 1 L fermenter and a fed-batch I(2) has been successfully reconstituted in S. cerevisiae as well regime was used with restricted feeding starting after 40 h as another heterologous host, Aspergillus nidulans,[5, 11] more (Figure 2). Additionally, the strain produced festuclavine (4)

5118 www.angewandte.org 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2015, 54, 5117 –5121 N H Role of EasH in the Biosynthesis of Cycloclavine

Figure S20 Detection• Authors of intermediate propose in in vitro cyclo EasHclavine biosynthetic generates reactions the cyclopropyl moiety through an oxidative mechanism by intercepting one of the Conditions are as described in the methods for in vitro endpoint assays unless otherwise specified. (a) EasA (A.intermediates japonicus) + EasH (A. japonicus generated) + co-factors during = intermediate the 10 (Mconversion+= of chanoclavine-I aldehyde into cycloclavine 237); (b, c) EasA (A. japonicus) + EasH (A. japonicus) + (after 2 h) EasG (A. japonicus) yielded cycloclavine 6 + festuclavine 4. Upon incubation of chanoclavine-I aldehyde 3 with Aj_EasA and Aj_EasH (with co-factors), + intermediate with• M+ Hypothesis= 237 has been observed. was Addition supported of Aj_EasG generates by the cycloclavine detection of intermediate (M = 237) upon incubation of chanoclavine-A aldehyde with 6 which supports mechanismAj_EasA proposed and in figure Aj_EasH. 3 (main text). Confirmed by trapping studies.

Figure S22 “Trapping” of iminium intermediate 8 by selective reduction reaction with

NaBD3CN yielded deuterium labelled festuclavine 11. LC-MS chromatograms of: (a) Deuterated festuclavine 11. (b) Festuclavine 4 standard. O O

H H H easA / H H N N reductase N spontaneous H H H

HN HN HN M+ = 237

D H easH/ N H H N eas G/ N N NaBD CN N dioxygenase 3 H reductase H H H H MeOH, H2O rt, 3h HN HN HN NADH NAD HN HN

Steph McCabe@ Wipf Group Slide 9 of 12 6/27/15

32 N H Common Biosynthetic Cyclopropanations

Electrophilic attack of a carbenium ion on a double bond Internal nucleophilic substitution (SNi) (most substrates are isoprenoid) H2O PLP Ad N PLP-Enz NH2 S N PLP-Enz

Co2H Co2H H

iPr iPr Reaction of an intermediate cation with an existing !-methyl group Transition Metal-Mediated Radical Cyclisation (common in steroids) H O H OH II R1 (iv)Fe (iii)Fe Fe R1 R3 R3

R2 R4 R2 R4 OH Enz-B O H (iv)Fe (iii)Fe H2N H HO2C HO2C H H HOOC Enz-PLP N Enz-PLP N IIFe

HO HO

SAM mediated cyclopropanation Ad

H3C S (SAM) H

CO2H H2N

Ad (SAM) H3C S H R

O O O

7 7 7 OH OH OH Wessjohann, L. A. and Brandt, W., Chem. Rev., 2003, 103, 1625 Steph McCabe@ Wipf Group Slide 10 of 12 6/27/15 N H Possible Mechanisms of Cyclopropanation

• 1) Hydroxylation/ Halogenation, loss of leaving group & cyclisation

• 2) Abstraction of a benzylic H by NAD+, followed by cyclisation

• 3) Abstraction of a benzylic H by an iron cofactor followed by radical cyclisation

• Closest known easH homologues have demonstrated hydroxylase activity

R N R=OH, Cl H

1)easH HN

H NAD N N N eas G 2)easH H H H H N H HN NADH NAD HN HN HN 3)easH

IVFe O H N N H H

HN HN

Steph McCabe@ Wipf Group Slide 11 of 12 6/27/15 N H Conclusions

CO2H N NH2 H O O 8 enzymes + P P N O O O H O O L-tryptophan DMAPP NH cycloclavine

• Cycloclavine was produced in high yields (529 mg. L-1) through yeast fermentation

• First example of successful reconstitution of a complex, downstream ergot alkaloid • Identiﬁcation of easH as the gene responsible for generating the cyclopropyl moiety through an oxidative mechanism

Steph McCabe@ Wipf Group Slide 12 of 12 6/27/15