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Chemical mechanisms involved during the biosynthesis of tropolones
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Chemical mechanisms involved during the biosynthesis of tropolones Russell J Cox1,2 and Ahmed Al-Fahad1 Tropolones are seven-membered aromatic rings which feature in the core of several important bioactive natural products including colchicine and stipitatic acid. Studies of their biosynthesis over nearly 70 years have revealed four parallel routes from polyketide, terpene, alkaloid and shikimate precursors, but the key steps all involve ring expansion of an alkylated 6-membered ring. Recent studies in fungi have revealed details of the individual chemical steps at the molecular level, but detailed molecular biosynthetic pathways in other organisms remain obscure. Addresses 1 School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, UK 2 Institute for Organic Chemistry, Liebniz University of Hannover, Schneiderberg 1B, 30167 Hannover, Germany Corresponding author: Cox, Russell J ([email protected], [email protected]) and
Current Opinion in Chemical Biology 2013, 17:532–536 This review comes from a themed issue on Mechanisms Edited by Hung-wen Liu and Tadhg Begley For a complete overview see the Issue and the Editorial Available online 16th July 2013 1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2013.06.029
Tropolone natural products Natural products containing the tropolone structural motif are relatively rare, but they are distributed in plants, bacteria and fungi and frequently possess useful bioactivities. The earliest examples colchicine (1, Scheme 2) and stipitatic acid (2, Scheme 4) were discovered well before structural and theoretical methods were advanced enough to determine their chemical structures. However in a seminal contribution to both natural products chemistry and organic chemistry more generally, Dewar realised that 1 [1] and 2 [2] were tropolones which consisted of a sevenmembered aromatic ring and published his findings in 1945. Following this realisation a flurry of publications described new naturally occurring tropolones, synthetic methods for their construction and speculations and initial experiments to determine their biosynthetic origin. Investigations of tropolone biosynthesis have continued, on and off, for roughly the past 70 years, often reflecting advances in methodology such as the use of radioisotopes and then stable isotopes as in vivo tracers and more latterly the use of molecular biology and in vitro enzymology to reveal Current Opinion in Chemical Biology 2013, 17:532–536
individual mechanistic steps. While the natural products themselves have been reviewed recently [3], this review will focus on what is known of the biosynthetic steps and chemical mechanisms. Biosynthesis in bacteria
Tropolones are rarely found in bacteria. However the biosynthesis of the sulfur containing tropolones thiotropocin 3 in Pseudomonas sp. [4] and its tautomer tropodithietic acid 4 in Phaeobacter sp. [5,6] have been investigated using isotopic labelling and volatiles from these species have been shown to contain tropone 5 and tropolone 6 itself (Scheme 1). v-Cyclohexyl fatty acids 7 from Alicyclobacillus sp. have also been investigated and it appears likely that these are produced by sequential reductions of a tropolone species [7]. In all three cases it has been shown that phenylacetic acid 8 is the precursor of the seven member ring, which itself is derived either directly, or indirectly v*ia phenylalanine, from the shikimate pathway. Two mechanisms have been suggested to account for the observed labelling patterns from isotopically labelled precursors. In the first mechanism (pathway A, Scheme 1) it is proposed that oxidative hydroxylation of phenylacetic acid, analogous to the known degradation pathway in Escherichia coli, forms the triol 9 which is the substrate for ring expansion in a mechanism analogous to that observed during fungal tropolone biosynthesis. An alternative mechanism (pathway B, Scheme 1) has been proposed which involves the intermediacy of a hydroxycyclopropane 10 which could also convert to a tropolone. While some proposed intermediates have been observed the detailed enzymatic and chemical steps remain to be elucidated. Recently Bugg and Xin have investigated the E. coli nonheme-iron-dependent dioxygenase enzyme MhpB (Scheme 1) which is normally involved in the oxidative degradation of catechols [8]. Interestingly this enzyme can accept de-aromatised substrates bearing a CH2OH substituent and catalyse a pinacol-type rearrangement to form tropolone products (Scheme 1). While this reaction does not appear to be naturally relevant in the bacterium E. coli it is interesting to note that this type of mechanism could account for the production of thiotropocin in bacteria and the biosynthesis of fungal tropolones (section ‘Stipitatic acid biosynthesis’). Biosynthesis in plants
Two of the most well-known tropolone natural products are derived from plants. Colchicine 1 (Scheme 2) is www.sciencedirect.com
Tropolone biosynthesis Cox and Al-Fahad
533
Scheme 1
OH O
HR HS
X
A
OH
S
OH
OH
S S
S
OH
OH
O
O
8
O
[O] B
O
O
O
9
OH
O
OH
3
O
4
[O]
HS
O HS
OH O
O
O [O] OH
CO2H 5
6
O HS
[ OH]
O
E. coli
CO2H
CO2H
HO
OH OH
OH OH
HS
n
10
7
HS
O
MhpB Fe2+
R
R
Current Opinion in Chemical Biology
Proposed biosynthetic pathways to seven-membered ring compounds from bacteria. Coloured atoms are for illustrative purposes only.
obtained from the autumn crocus Colchicum autumnale and extracts of this plant have been known to alleviate the symptoms of various diseases including rheumatism and gout for hundreds, and possibly thousands, of years [9,10]. Pure colchicine was obtained in the early 1800s and it has
remained of biological and medical significance ever since [11]. b-Thujaplicin 11, also known as hinokitiol, is a C10 tropolone obtained from the wood of various trees such as Cupressus lusitanica (Mexican white cedar) and Thuja plicata (western red cedar) and which shows antibacterial
Scheme 2
13
MeN
HS
MeO
12
HR
HS
MeO
OH
MeO OMe
[O]
MeO HR
O
MeO NMe H
MeO MeO MeO
O
MeO MeO
H
O MeO
HR
OMe
N-formyl demecolcine 14
N
Me O
HS HR O
MeO
X
12
O OMe
O-methylandrocymbin 13
X HR HS O HR
NHAc MeO
12
MeO
(S)-autumnaline 12
MeO
HR NMe H
MeO
MeO
OMe
13
MeO
NMe H
12
HO
O
HR
13
Colchicine 1
HR
O
MeO
NMe H
MeO MeO HR
HS O OMe Current Opinion in Chemical Biology
Later steps during the biosynthesis of colchicine 1 and proposed 2-electron rearrangement mechanism following hydroxylation at C-13 of O-methylandrocymbin 13. Coloured atoms are for illustrative purposes only. www.sciencedirect.com
Current Opinion in Chemical Biology 2013, 17:532–536
534 Mechanisms
Scheme 3
OPP
1'
2'
2'
2' 3'
1'
1' 3'
10'
3'
1' 2'
O HO 3'
11
Current Opinion in Chemical Biology
Proposed Biosynthetic route to b-thujaplicin 11 in C. lusitanica. Red and green atoms are for illustrative purposes only.
and antifungal properties. Although numerous other tropolones are known in plants, significant studies of plant tropolone biosynthesis have focussed on 1 and 11. Colchicine biosynthesis
Colchicine 1 has undergone one of the most detailed isotopic labelling investigations of any secondary metabolite. In a series of four back-to-back papers published in 1998 (but describing work initiated during the 1960s and executed over 30 years) Battersby and coauthors conclusively demonstrate the biosynthetic route from (S)autumnaline 12 v*ia O-methylandrocymbin 13 and N-formyldemecolcine 14 (Scheme 2) [12,13,14,15]. The key transformation to form the tropolone comes relatively late in the pathway when O-methylandrocymbin 13 is the substrate for oxidative rearrangement. Battersby showed that the oxidation selectively removes 12HS and 13-HS, cleaving the C–C-bond between carbons 12 and 13. This discovery was achieved using selectively 3 H-labelled autumnaline 12 in a series of feeding experiments with growing Colchicum autumnale plants followed by isolation of labelled 1. The rearrangement is clearly related to those occurring during b-thujaplicin 11 and stipitatic acid 2 biosynthesis in which a carbon is oxidised and incorporated into the ring. However it differs in cleaving an ‘external’ C–C bond during the rearrangement. No further mechanistic details have been obtained and it remains to be seen whether the oxidation and rearrangement steps are coupled as they are during stipitatic acid biosynthesis, or whether the oxidative and rearrangement steps are catalysed by separate enzymes. Battersby himself suggested a single electron rearrangement mechanism as a plausible route to 1. However, knowledge of the mechanism involved during the ring expansion to form fungal tropolones also allows a 2electron mechanism (Scheme 2) to be proposed which is fully consistent with the geometry of the proposed intermediate if 13-HS is abstracted to form an intermediate 13-oxygenated species. The geometry of the reacting bonds in such a species is perfectly aligned to allow the ring expansion and concomitant cleavage of the 12,13bond. Current Opinion in Chemical Biology 2013, 17:532–536
b-Thujaplicin biosynthesis
Investigations of b-thujaplicin 11 biosynthesis have been complicated because of the difficulties of working with tree-derived material. However the terpenoid origin of 11 was confirmed in a feeding experiment using [10-14C]geraniol [16] and a callus culture of Cupressus lusitanica (Scheme 3). This result was bolstered by feeding [1-13C]glucose, [2-13C]-glucose and [U-13C]-glucose to the same culture and examining the isolated b-thujaplicin using 13 C-INADEQUATE NMR. The results were consistent with construction of the tropolone ring v*ia a limonane skeleton, followed by a ring-expansion which incorporates the ring-methyl group into the ring (Scheme 3). Although no further mechanistic or pathway details have been thus far elucidated, the similarities with the construction of tropolones in fungi (section ‘Biosynthesis in fungi’) are obvious. Biosynthesis in fungi Stipitatic acid biosynthesis
One of the first natural products recognised to be a tropolone was stipitatic acid 2 (Scheme 4). This compound was first isolated by Raistrick in the 1930s [17]], but elucidation of its structure had to await the insight of Dewar in 1945. The high-titre and reliable production of 2 by Talaromyces stipitatus (previously known as Penicillium stipitatum) prompted the use of radioactive and stable isotopes in feeding experiments in this organism. The distribution of incorporated label in 2 obtained after feeding [1-14C]-D-glucose suggested that the pathway did not proceed v*ia aromatic amino acids or shikimate [18]. Further feeding studies using [1-14C] and [2-14C]acetate, as well as [1,3-14C2]-malonate pointed to a polyketide origin for 2 [19]. It was then proposed that 3methylorcinaldehyde 15 and 3-methylorsellinic acid 16 were likely to be precursors for stipitatic acid biosynthesis, after an efficient conversion of a radioactive aldehyde 15 more readily than the acid 16 into 2 [20]. Later studies investigated the oxidative ring expansion mechanism, which was proposed to occur either v*ia a pinacoltype rearrangement or v*ia an oxidative ring opening and recyclization to a benzenoid intermediate [21]. Growing T. stipitatus under 18O2 supported the former mechanism www.sciencedirect.com
Tropolone biosynthesis Cox and Al-Fahad
535
Scheme 4
R
O
H
OH
OH
H
OH
O
TropB
OH
O
O
O
O
OH
O OH
TropD
OH
OH
O 18
15 R = H 16 R = OH
O
OH
OH
O
OH
O
OH
stipitaldehyde 19 via non-released intermediate
TropC
OH
O [O]
OH O stipitatonic acid 17 -CO2
TropC
O His
OH
O
H
His NH
H O
N IV O
Fe
O H H
H
O
OH Asp
O
H O
N N H
His
His NH
H O
N III O
Fe
O H H
OH Asp
H
O
N
O H
N H
His
O
H O
OH
HO
NH Asp N II O
Fe
O O H+
H
O
HO
N N H
His
O
stipitatic acid 2
Current Opinion in Chemical Biology
Early steps of stipitatic acid biosynthetic pathway and the proposed mechanism of tropolone nucleus formation catalysed by TropB and TropC. Coloured atoms are for illustrative purposes only.
as no observed incorporation of 18O at the 6-OH of stipitatic acid was detected. This suggested the involvement of a monooxygenase during the formation of the tropolone ring [22]. No further details about the ring expansion could be gained from isotopic feeding experiments, but more information about the enzymes which catalyse late stages of stipitatic acid biosynthesis were obtained from in vitro assays using a T. stipitatus cell-free extract to decarboxylate stipitatonic acid 17 [23]. In addition, a non-reducing polyketide synthase (nr-PKS, known as methylorcinaldehyde synthase or MOS), which produces the proposed tropolone precursor 3-methylorcinaldehyde 15, was discovered in the fungus Acremonium strictum which is itself a troplone producer [24]. After the availability of the genome sequence of T. stipitatus in 2007, it was possible to locate a putative gene cluster encoding stipitatic acid biosynthesis. A BLAST search for a putative MOS revealed a candidate gene cluster containing eleven open reading frames, including those encoding a nr-PKS, a mono-oxygenase and a decarboxylase [25]. Gene knockouts and heterologous expression approaches were then applied to this cluster which was confirmed to be responsible for stipitatic acid 2 production. To-date, four genes (tropA-D) have been deleted and found to be crucial for the biosynthesis of 2. The knockout of the nr-PKS, encoded by tropA, abolished tropolone formation. Additionally, tropA was heterologously expressed in Aspergillus oryzae which then produced the aldehyde 15. The deletion of tropB (encoding a FAD-dependent monooxygenase) resulted in the accumulation of 15, whereas a knockout of tropC (encoding a non-heme iron dioxygenase) gave rise to a new www.sciencedirect.com
dienone intermediate 18. The dienone 18 was determined to be a direct product of TropB v*ia in vitro enzyme assay. The tropC gene was also successfully expressed in Escherichia coli and incubation of TropC protein with 18 yielded a new tropolone, named stipitaldehyde 19. The oxidative mechanism of TropC was proposed to occur in two steps (Scheme 4). The first step is likely to be the formation of an iron-oxo species hydroxylating the 3-methyl of 18 which is then followed by a catalytic ring expansion rearrangement. After the formation of the seven-membered ring, initiation of the synthesis of the maleic anhydride moiety of stipitatonic acid 17 is catalysed by TropD which is a cytochrome P450 monooxygenase. Inactivation of the tropD gene accumulated 19. Analyses of the genomes of many other filamentous fungi show that gene clusters homologous to the trop cluster are widespread and probably responsible for the synthesis of many tropolones and related compounds such as the azaphilones. Outlook
The biosynthesis of tropolones appears to have evolved in parallel in bacteria, plants and fungi. In bacteria the biosynthetic pathways known to-date have a shikimate origin; in plants origins from alkaloids and terpenes are known; while fungi use a polyketide route. In all cases, however, the seven-membered ring appears to be derived by an oxidative ring expansion of a 6-membered precursor, albeit with different structural features in each case. Only in the case of fungi have isolated enzymes been explored, and even here the exact chemical mechanisms have not been determined unambiguously. There is thus significant scope for detailed investigations of tropolone Current Opinion in Chemical Biology 2013, 17:532–536
536 Mechanisms
biosynthesis, particularly in plants and bacteria where there is not yet knowledge about the precise chemical intermediates or the enzymes which catalyse each step.
Acknowledgements Research into fungal tropolone biosynthesis in fungi has been supported by EPSRC (EP/F066104/1), the University of Bristol and Al Baha University (A. A-F.).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1. Dewar MJS: Structure of colchicine. Nature 1945, 155: 141-142. This paper describes the first correct structure elucidations of tropolones. The realisation that aromaticity was not restricted to 6-membered rings revolutionised ideas about structure and bonding in organic chemistry and led to the development of new theoretical and computational methods in organic chemistry. 2. Dewar MJS: Structure of stipitatic acid. Nature 1945, 155:50-51. See annotation to Ref. [1]. 3.
Bentley R: A fresh look at natural tropolonoids. Nat Prod Rep 2008, 25:118-138.
4.
Cane DE, Wu Z, Vanepp JE: Thiotropocin biosynthesis, shikimate origin of a sulfur-containing tropolone derivative. J Am Chem Soc 1992, 114:8479-8483.
5.
Thiel V et al.: Identification and biosynthesis of tropone derivatives and sulfur volatiles produced by bacteria of the marine Roseobacter clade. Org Biomol Chem 2010, 8:234-246.
6.
Geng H, Bruhn JB, Nielsen KF, Gram L, Belas R: Genetic dissection of tropodithietic acid biosynthesis by marine roseobacters. Appl Environ Microbiol 2008, 74:1535-1545.
7.
Moore BS, Walker K, Tornus I, Handa S, Poralla K, Floss HG: Biosynthetic studies of v-cycloheptyl fatty acids in Alicyclobacillus cycloheptanicus. Formation of cycloheptanecarboxylic acid from phenylacetic acid. J Org Chem 1997, 62:2173-2185.
8.
Xin M, Bugg TDH: Biomimetic formation of 2-tropolones by dioxygenasecatalysed ring expansion of substituted 2,4cyclohexadienones. ChemBioChem 2010, 11:272-276. This paper demonstrated the formation of tropolones by a ring expansion mechanisms catalysed by both a non-heme iron containing enzyme, and an inorganic iron complex.
9.
Graham W, Roberts JB: Intravenous colchicine in the management of gouty arthritis. Ann Rheum Dis 1953, 12:16-19.
10. Hartung EF: History of the use of colchicum and related medicaments in gout. Ann Rheum Dis 1954, 13:190-200. 11. Massarotti A, Coluccia A, Silvestri R, Sorba G, Brancale A: The tubulin colchicine domain: a molecular modeling perspective. Chemmedchem 2012, 7:33-42. 12. McDonald E, Robert R, Woodhouse RN et al.: Biosynthesis. Part 27. Colchicine: studies of the phenolic oxidative coupling and ring-expansion processes based on incorporation of multiply
Current Opinion in Chemical Biology 2013, 17:532–536
labelled 1-phenethylisoquinolines. J Chem Soc Perkin Trans 1 1998:2979-2987. This paper describes the comprehensive and exhaustive determination of the intermediates involved during the biosynthesis of colchicine. It is instructive not just in elucidating the pathway, but also in the variety and creativity of the experiments and the very high level of technical skill and ingenuity. 13. Barker AC, Julian DR, Ramage R et al.: Biosynthesis. Part 28. Colchicine: definition of intermediates between Omethylandrocymbine and colchicine and studies on speciosine. J Chem Soc Perkin Trans 1 1998:2989-2994. See annotation to Ref. [12]. 14. Woodhouse RN, McDonald E, Ramage R et al.: Biosynthesis. Part 29. Colchicine: studies on the ring expansion step focusing on the fate of the hydrogens at C-3 of autumnaline. J Chem Soc Perkin Trans 1 1998:2995-3001. See annotation to Ref. [12]. 15. Sheldrake PW, Suckling KE, Woodhouse RN et al.: Biosynthesis. Part 30. Colchicine: studies on the ring expansion step focusing on the fate of the hydrogens at C-4 of autumnaline. J Chem Soc Perkin Trans 1 1998:3003-3009. See annotation to Ref. [12]. 16. Fujita K, Yamaguchi K, Itose T, Sakai RK: Biosynthetic pathway of beta-thujaplicin in the Cupressus lusitanica cell culture. J Plant Physiol 2000, 156:462-467. 17. Birkinshaw JH, Chambers AR, Raistrick H: Studies in the biochemistry of micro-organisms 70. Stipitatic acid, C8H6O5, a metabolic product of Penicillium stipitatum Thom. Biochem J 1942, 36:242-251. A masterpiece of understated writing which set the scene for the huge interest in tropolones during the 1940s, 1950s and 1960s. 18. Bentley R: Aromatic synthesis in molds: formation of the tropolone, stipitatic acid. Biochim Biophys Acta 1958, 29:666-667. 19. Bentley R: Biosynthesis of tropolones in Penicillium stipitatum 3. Tracer studies on formation of stipitatonic and stipitatic acids. J Biol Chem 1963, 238:1895-1902. 20. Bryant R, Light R: Stipitatonic acid biosynthesis — incorporation of [formyl-C-14]-3-methylorcylaldehyde and [C14]stipitaldehydic acid, a new tropolone metabolite. Biochemistry 1974, 13:1516-1522. 21. Ferretti LD, Richards JH: The biogenesis of the mold tropolones. Proc Natl Acad Sci U S A 1960, 46:1438-1444. 22. O’Sullivan M, Schwab J: Verification of the mechanism of oxidative ring expansion in the biosynthesis of stipitatic acid by Talaromyces stipitatus. Bioorg Chem 1995, 23:131-143. 23. Bentley R, Thiessen CP: Biosynthesis of tropolones in Penicillium stipitatum V. Preparation and properties of stipitatonic acid decarboxylase. J Biol Chem 1963, 238:3811-3816. 24. Bailey AM et al.: Characterisation of 3-methylorcinaldehyde synthase (MOS) in Acremonium strictum: first observation of a reductive release mechanism during polyketide biosynthesis. Chem Commun 2007, 39:4053-4055. 25. Davison J, Al Fahad A, Cai M et al.: Genetic, molecular, and biochemical basis of fungal tropolone biosynthesis. Proc Natl Acad Sci U S A 2012, 109:7642-7647. This paper describes in detail the genes, enzymes and intermediates involved in tropolone biosynthesis in fungi, involving combined molecular biological, enzymological and chemical investigations.
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Chemical mechanisms involved during the biosynthesis of tropolones Russell J Cox1,2 and Ahmed Al-Fahad1 Tropolones are seven-membered aromatic rings which feature in the core of several important bioactive natural products including colchicine and stipitatic acid. Studies of their biosynthesis over nearly 70 years have revealed four parallel routes from polyketide, terpene, alkaloid and shikimate precursors, but the key steps all involve ring expansion of an alkylated 6-membered ring. Recent studies in fungi have revealed details of the individual chemical steps at the molecular level, but detailed molecular biosynthetic pathways in other organisms remain obscure. Addresses 1 School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, UK 2 Institute for Organic Chemistry, Liebniz University of Hannover, Schneiderberg 1B, 30167 Hannover, Germany Corresponding author: Cox, Russell J ([email protected], [email protected]) and
Current Opinion in Chemical Biology 2013, 17:532–536 This review comes from a themed issue on Mechanisms Edited by Hung-wen Liu and Tadhg Begley For a complete overview see the Issue and the Editorial Available online 16th July 2013 1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2013.06.029
Tropolone natural products Natural products containing the tropolone structural motif are relatively rare, but they are distributed in plants, bacteria and fungi and frequently possess useful bioactivities. The earliest examples colchicine (1, Scheme 2) and stipitatic acid (2, Scheme 4) were discovered well before structural and theoretical methods were advanced enough to determine their chemical structures. However in a seminal contribution to both natural products chemistry and organic chemistry more generally, Dewar realised that 1 [1] and 2 [2] were tropolones which consisted of a sevenmembered aromatic ring and published his findings in 1945. Following this realisation a flurry of publications described new naturally occurring tropolones, synthetic methods for their construction and speculations and initial experiments to determine their biosynthetic origin. Investigations of tropolone biosynthesis have continued, on and off, for roughly the past 70 years, often reflecting advances in methodology such as the use of radioisotopes and then stable isotopes as in vivo tracers and more latterly the use of molecular biology and in vitro enzymology to reveal Current Opinion in Chemical Biology 2013, 17:532–536
individual mechanistic steps. While the natural products themselves have been reviewed recently [3], this review will focus on what is known of the biosynthetic steps and chemical mechanisms. Biosynthesis in bacteria
Tropolones are rarely found in bacteria. However the biosynthesis of the sulfur containing tropolones thiotropocin 3 in Pseudomonas sp. [4] and its tautomer tropodithietic acid 4 in Phaeobacter sp. [5,6] have been investigated using isotopic labelling and volatiles from these species have been shown to contain tropone 5 and tropolone 6 itself (Scheme 1). v-Cyclohexyl fatty acids 7 from Alicyclobacillus sp. have also been investigated and it appears likely that these are produced by sequential reductions of a tropolone species [7]. In all three cases it has been shown that phenylacetic acid 8 is the precursor of the seven member ring, which itself is derived either directly, or indirectly v*ia phenylalanine, from the shikimate pathway. Two mechanisms have been suggested to account for the observed labelling patterns from isotopically labelled precursors. In the first mechanism (pathway A, Scheme 1) it is proposed that oxidative hydroxylation of phenylacetic acid, analogous to the known degradation pathway in Escherichia coli, forms the triol 9 which is the substrate for ring expansion in a mechanism analogous to that observed during fungal tropolone biosynthesis. An alternative mechanism (pathway B, Scheme 1) has been proposed which involves the intermediacy of a hydroxycyclopropane 10 which could also convert to a tropolone. While some proposed intermediates have been observed the detailed enzymatic and chemical steps remain to be elucidated. Recently Bugg and Xin have investigated the E. coli nonheme-iron-dependent dioxygenase enzyme MhpB (Scheme 1) which is normally involved in the oxidative degradation of catechols [8]. Interestingly this enzyme can accept de-aromatised substrates bearing a CH2OH substituent and catalyse a pinacol-type rearrangement to form tropolone products (Scheme 1). While this reaction does not appear to be naturally relevant in the bacterium E. coli it is interesting to note that this type of mechanism could account for the production of thiotropocin in bacteria and the biosynthesis of fungal tropolones (section ‘Stipitatic acid biosynthesis’). Biosynthesis in plants
Two of the most well-known tropolone natural products are derived from plants. Colchicine 1 (Scheme 2) is www.sciencedirect.com
Tropolone biosynthesis Cox and Al-Fahad
533
Scheme 1
OH O
HR HS
X
A
OH
S
OH
OH
S S
S
OH
OH
O
O
8
O
[O] B
O
O
O
9
OH
O
OH
3
O
4
[O]
HS
O HS
OH O
O
O [O] OH
CO2H 5
6
O HS
[ OH]
O
E. coli
CO2H
CO2H
HO
OH OH
OH OH
HS
n
10
7
HS
O
MhpB Fe2+
R
R
Current Opinion in Chemical Biology
Proposed biosynthetic pathways to seven-membered ring compounds from bacteria. Coloured atoms are for illustrative purposes only.
obtained from the autumn crocus Colchicum autumnale and extracts of this plant have been known to alleviate the symptoms of various diseases including rheumatism and gout for hundreds, and possibly thousands, of years [9,10]. Pure colchicine was obtained in the early 1800s and it has
remained of biological and medical significance ever since [11]. b-Thujaplicin 11, also known as hinokitiol, is a C10 tropolone obtained from the wood of various trees such as Cupressus lusitanica (Mexican white cedar) and Thuja plicata (western red cedar) and which shows antibacterial
Scheme 2
13
MeN
HS
MeO
12
HR
HS
MeO
OH
MeO OMe
[O]
MeO HR
O
MeO NMe H
MeO MeO MeO
O
MeO MeO
H
O MeO
HR
OMe
N-formyl demecolcine 14
N
Me O
HS HR O
MeO
X
12
O OMe
O-methylandrocymbin 13
X HR HS O HR
NHAc MeO
12
MeO
(S)-autumnaline 12
MeO
HR NMe H
MeO
MeO
OMe
13
MeO
NMe H
12
HO
O
HR
13
Colchicine 1
HR
O
MeO
NMe H
MeO MeO HR
HS O OMe Current Opinion in Chemical Biology
Later steps during the biosynthesis of colchicine 1 and proposed 2-electron rearrangement mechanism following hydroxylation at C-13 of O-methylandrocymbin 13. Coloured atoms are for illustrative purposes only. www.sciencedirect.com
Current Opinion in Chemical Biology 2013, 17:532–536
534 Mechanisms
Scheme 3
OPP
1'
2'
2'
2' 3'
1'
1' 3'
10'
3'
1' 2'
O HO 3'
11
Current Opinion in Chemical Biology
Proposed Biosynthetic route to b-thujaplicin 11 in C. lusitanica. Red and green atoms are for illustrative purposes only.
and antifungal properties. Although numerous other tropolones are known in plants, significant studies of plant tropolone biosynthesis have focussed on 1 and 11. Colchicine biosynthesis
Colchicine 1 has undergone one of the most detailed isotopic labelling investigations of any secondary metabolite. In a series of four back-to-back papers published in 1998 (but describing work initiated during the 1960s and executed over 30 years) Battersby and coauthors conclusively demonstrate the biosynthetic route from (S)autumnaline 12 v*ia O-methylandrocymbin 13 and N-formyldemecolcine 14 (Scheme 2) [12,13,14,15]. The key transformation to form the tropolone comes relatively late in the pathway when O-methylandrocymbin 13 is the substrate for oxidative rearrangement. Battersby showed that the oxidation selectively removes 12HS and 13-HS, cleaving the C–C-bond between carbons 12 and 13. This discovery was achieved using selectively 3 H-labelled autumnaline 12 in a series of feeding experiments with growing Colchicum autumnale plants followed by isolation of labelled 1. The rearrangement is clearly related to those occurring during b-thujaplicin 11 and stipitatic acid 2 biosynthesis in which a carbon is oxidised and incorporated into the ring. However it differs in cleaving an ‘external’ C–C bond during the rearrangement. No further mechanistic details have been obtained and it remains to be seen whether the oxidation and rearrangement steps are coupled as they are during stipitatic acid biosynthesis, or whether the oxidative and rearrangement steps are catalysed by separate enzymes. Battersby himself suggested a single electron rearrangement mechanism as a plausible route to 1. However, knowledge of the mechanism involved during the ring expansion to form fungal tropolones also allows a 2electron mechanism (Scheme 2) to be proposed which is fully consistent with the geometry of the proposed intermediate if 13-HS is abstracted to form an intermediate 13-oxygenated species. The geometry of the reacting bonds in such a species is perfectly aligned to allow the ring expansion and concomitant cleavage of the 12,13bond. Current Opinion in Chemical Biology 2013, 17:532–536
b-Thujaplicin biosynthesis
Investigations of b-thujaplicin 11 biosynthesis have been complicated because of the difficulties of working with tree-derived material. However the terpenoid origin of 11 was confirmed in a feeding experiment using [10-14C]geraniol [16] and a callus culture of Cupressus lusitanica (Scheme 3). This result was bolstered by feeding [1-13C]glucose, [2-13C]-glucose and [U-13C]-glucose to the same culture and examining the isolated b-thujaplicin using 13 C-INADEQUATE NMR. The results were consistent with construction of the tropolone ring v*ia a limonane skeleton, followed by a ring-expansion which incorporates the ring-methyl group into the ring (Scheme 3). Although no further mechanistic or pathway details have been thus far elucidated, the similarities with the construction of tropolones in fungi (section ‘Biosynthesis in fungi’) are obvious. Biosynthesis in fungi Stipitatic acid biosynthesis
One of the first natural products recognised to be a tropolone was stipitatic acid 2 (Scheme 4). This compound was first isolated by Raistrick in the 1930s [17]], but elucidation of its structure had to await the insight of Dewar in 1945. The high-titre and reliable production of 2 by Talaromyces stipitatus (previously known as Penicillium stipitatum) prompted the use of radioactive and stable isotopes in feeding experiments in this organism. The distribution of incorporated label in 2 obtained after feeding [1-14C]-D-glucose suggested that the pathway did not proceed v*ia aromatic amino acids or shikimate [18]. Further feeding studies using [1-14C] and [2-14C]acetate, as well as [1,3-14C2]-malonate pointed to a polyketide origin for 2 [19]. It was then proposed that 3methylorcinaldehyde 15 and 3-methylorsellinic acid 16 were likely to be precursors for stipitatic acid biosynthesis, after an efficient conversion of a radioactive aldehyde 15 more readily than the acid 16 into 2 [20]. Later studies investigated the oxidative ring expansion mechanism, which was proposed to occur either v*ia a pinacoltype rearrangement or v*ia an oxidative ring opening and recyclization to a benzenoid intermediate [21]. Growing T. stipitatus under 18O2 supported the former mechanism www.sciencedirect.com
Tropolone biosynthesis Cox and Al-Fahad
535
Scheme 4
R
O
H
OH
OH
H
OH
O
TropB
OH
O
O
O
O
OH
O OH
TropD
OH
OH
O 18
15 R = H 16 R = OH
O
OH
OH
O
OH
O
OH
stipitaldehyde 19 via non-released intermediate
TropC
OH
O [O]
OH O stipitatonic acid 17 -CO2
TropC
O His
OH
O
H
His NH
H O
N IV O
Fe
O H H
H
O
OH Asp
O
H O
N N H
His
His NH
H O
N III O
Fe
O H H
OH Asp
H
O
N
O H
N H
His
O
H O
OH
HO
NH Asp N II O
Fe
O O H+
H
O
HO
N N H
His
O
stipitatic acid 2
Current Opinion in Chemical Biology
Early steps of stipitatic acid biosynthetic pathway and the proposed mechanism of tropolone nucleus formation catalysed by TropB and TropC. Coloured atoms are for illustrative purposes only.
as no observed incorporation of 18O at the 6-OH of stipitatic acid was detected. This suggested the involvement of a monooxygenase during the formation of the tropolone ring [22]. No further details about the ring expansion could be gained from isotopic feeding experiments, but more information about the enzymes which catalyse late stages of stipitatic acid biosynthesis were obtained from in vitro assays using a T. stipitatus cell-free extract to decarboxylate stipitatonic acid 17 [23]. In addition, a non-reducing polyketide synthase (nr-PKS, known as methylorcinaldehyde synthase or MOS), which produces the proposed tropolone precursor 3-methylorcinaldehyde 15, was discovered in the fungus Acremonium strictum which is itself a troplone producer [24]. After the availability of the genome sequence of T. stipitatus in 2007, it was possible to locate a putative gene cluster encoding stipitatic acid biosynthesis. A BLAST search for a putative MOS revealed a candidate gene cluster containing eleven open reading frames, including those encoding a nr-PKS, a mono-oxygenase and a decarboxylase [25]. Gene knockouts and heterologous expression approaches were then applied to this cluster which was confirmed to be responsible for stipitatic acid 2 production. To-date, four genes (tropA-D) have been deleted and found to be crucial for the biosynthesis of 2. The knockout of the nr-PKS, encoded by tropA, abolished tropolone formation. Additionally, tropA was heterologously expressed in Aspergillus oryzae which then produced the aldehyde 15. The deletion of tropB (encoding a FAD-dependent monooxygenase) resulted in the accumulation of 15, whereas a knockout of tropC (encoding a non-heme iron dioxygenase) gave rise to a new www.sciencedirect.com
dienone intermediate 18. The dienone 18 was determined to be a direct product of TropB v*ia in vitro enzyme assay. The tropC gene was also successfully expressed in Escherichia coli and incubation of TropC protein with 18 yielded a new tropolone, named stipitaldehyde 19. The oxidative mechanism of TropC was proposed to occur in two steps (Scheme 4). The first step is likely to be the formation of an iron-oxo species hydroxylating the 3-methyl of 18 which is then followed by a catalytic ring expansion rearrangement. After the formation of the seven-membered ring, initiation of the synthesis of the maleic anhydride moiety of stipitatonic acid 17 is catalysed by TropD which is a cytochrome P450 monooxygenase. Inactivation of the tropD gene accumulated 19. Analyses of the genomes of many other filamentous fungi show that gene clusters homologous to the trop cluster are widespread and probably responsible for the synthesis of many tropolones and related compounds such as the azaphilones. Outlook
The biosynthesis of tropolones appears to have evolved in parallel in bacteria, plants and fungi. In bacteria the biosynthetic pathways known to-date have a shikimate origin; in plants origins from alkaloids and terpenes are known; while fungi use a polyketide route. In all cases, however, the seven-membered ring appears to be derived by an oxidative ring expansion of a 6-membered precursor, albeit with different structural features in each case. Only in the case of fungi have isolated enzymes been explored, and even here the exact chemical mechanisms have not been determined unambiguously. There is thus significant scope for detailed investigations of tropolone Current Opinion in Chemical Biology 2013, 17:532–536
536 Mechanisms
biosynthesis, particularly in plants and bacteria where there is not yet knowledge about the precise chemical intermediates or the enzymes which catalyse each step.
Acknowledgements Research into fungal tropolone biosynthesis in fungi has been supported by EPSRC (EP/F066104/1), the University of Bristol and Al Baha University (A. A-F.).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1. Dewar MJS: Structure of colchicine. Nature 1945, 155: 141-142. This paper describes the first correct structure elucidations of tropolones. The realisation that aromaticity was not restricted to 6-membered rings revolutionised ideas about structure and bonding in organic chemistry and led to the development of new theoretical and computational methods in organic chemistry. 2. Dewar MJS: Structure of stipitatic acid. Nature 1945, 155:50-51. See annotation to Ref. [1]. 3.
Bentley R: A fresh look at natural tropolonoids. Nat Prod Rep 2008, 25:118-138.
4.
Cane DE, Wu Z, Vanepp JE: Thiotropocin biosynthesis, shikimate origin of a sulfur-containing tropolone derivative. J Am Chem Soc 1992, 114:8479-8483.
5.
Thiel V et al.: Identification and biosynthesis of tropone derivatives and sulfur volatiles produced by bacteria of the marine Roseobacter clade. Org Biomol Chem 2010, 8:234-246.
6.
Geng H, Bruhn JB, Nielsen KF, Gram L, Belas R: Genetic dissection of tropodithietic acid biosynthesis by marine roseobacters. Appl Environ Microbiol 2008, 74:1535-1545.
7.
Moore BS, Walker K, Tornus I, Handa S, Poralla K, Floss HG: Biosynthetic studies of v-cycloheptyl fatty acids in Alicyclobacillus cycloheptanicus. Formation of cycloheptanecarboxylic acid from phenylacetic acid. J Org Chem 1997, 62:2173-2185.
8.
Xin M, Bugg TDH: Biomimetic formation of 2-tropolones by dioxygenasecatalysed ring expansion of substituted 2,4cyclohexadienones. ChemBioChem 2010, 11:272-276. This paper demonstrated the formation of tropolones by a ring expansion mechanisms catalysed by both a non-heme iron containing enzyme, and an inorganic iron complex.
9.
Graham W, Roberts JB: Intravenous colchicine in the management of gouty arthritis. Ann Rheum Dis 1953, 12:16-19.
10. Hartung EF: History of the use of colchicum and related medicaments in gout. Ann Rheum Dis 1954, 13:190-200. 11. Massarotti A, Coluccia A, Silvestri R, Sorba G, Brancale A: The tubulin colchicine domain: a molecular modeling perspective. Chemmedchem 2012, 7:33-42. 12. McDonald E, Robert R, Woodhouse RN et al.: Biosynthesis. Part 27. Colchicine: studies of the phenolic oxidative coupling and ring-expansion processes based on incorporation of multiply
Current Opinion in Chemical Biology 2013, 17:532–536
labelled 1-phenethylisoquinolines. J Chem Soc Perkin Trans 1 1998:2979-2987. This paper describes the comprehensive and exhaustive determination of the intermediates involved during the biosynthesis of colchicine. It is instructive not just in elucidating the pathway, but also in the variety and creativity of the experiments and the very high level of technical skill and ingenuity. 13. Barker AC, Julian DR, Ramage R et al.: Biosynthesis. Part 28. Colchicine: definition of intermediates between Omethylandrocymbine and colchicine and studies on speciosine. J Chem Soc Perkin Trans 1 1998:2989-2994. See annotation to Ref. [12]. 14. Woodhouse RN, McDonald E, Ramage R et al.: Biosynthesis. Part 29. Colchicine: studies on the ring expansion step focusing on the fate of the hydrogens at C-3 of autumnaline. J Chem Soc Perkin Trans 1 1998:2995-3001. See annotation to Ref. [12]. 15. Sheldrake PW, Suckling KE, Woodhouse RN et al.: Biosynthesis. Part 30. Colchicine: studies on the ring expansion step focusing on the fate of the hydrogens at C-4 of autumnaline. J Chem Soc Perkin Trans 1 1998:3003-3009. See annotation to Ref. [12]. 16. Fujita K, Yamaguchi K, Itose T, Sakai RK: Biosynthetic pathway of beta-thujaplicin in the Cupressus lusitanica cell culture. J Plant Physiol 2000, 156:462-467. 17. Birkinshaw JH, Chambers AR, Raistrick H: Studies in the biochemistry of micro-organisms 70. Stipitatic acid, C8H6O5, a metabolic product of Penicillium stipitatum Thom. Biochem J 1942, 36:242-251. A masterpiece of understated writing which set the scene for the huge interest in tropolones during the 1940s, 1950s and 1960s. 18. Bentley R: Aromatic synthesis in molds: formation of the tropolone, stipitatic acid. Biochim Biophys Acta 1958, 29:666-667. 19. Bentley R: Biosynthesis of tropolones in Penicillium stipitatum 3. Tracer studies on formation of stipitatonic and stipitatic acids. J Biol Chem 1963, 238:1895-1902. 20. Bryant R, Light R: Stipitatonic acid biosynthesis — incorporation of [formyl-C-14]-3-methylorcylaldehyde and [C14]stipitaldehydic acid, a new tropolone metabolite. Biochemistry 1974, 13:1516-1522. 21. Ferretti LD, Richards JH: The biogenesis of the mold tropolones. Proc Natl Acad Sci U S A 1960, 46:1438-1444. 22. O’Sullivan M, Schwab J: Verification of the mechanism of oxidative ring expansion in the biosynthesis of stipitatic acid by Talaromyces stipitatus. Bioorg Chem 1995, 23:131-143. 23. Bentley R, Thiessen CP: Biosynthesis of tropolones in Penicillium stipitatum V. Preparation and properties of stipitatonic acid decarboxylase. J Biol Chem 1963, 238:3811-3816. 24. Bailey AM et al.: Characterisation of 3-methylorcinaldehyde synthase (MOS) in Acremonium strictum: first observation of a reductive release mechanism during polyketide biosynthesis. Chem Commun 2007, 39:4053-4055. 25. Davison J, Al Fahad A, Cai M et al.: Genetic, molecular, and biochemical basis of fungal tropolone biosynthesis. Proc Natl Acad Sci U S A 2012, 109:7642-7647. This paper describes in detail the genes, enzymes and intermediates involved in tropolone biosynthesis in fungi, involving combined molecular biological, enzymological and chemical investigations.
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