A unifying paradigm for naphthoquinone-based meroterpenoid (bio)synthesis

Article abstract of DOI:10.1038/nchem.2829

Bacterial meroterpenoids constitute an important class of natural products with diverse biological properties and therapeutic potential. The biosynthetic logic for their production is unknown and defies explanation via classical biochemical paradigms. A l

Full text of DOI:10.1038/nchem.2829

ARTICLES
PUBLISHED ONLINE: 31 JULY 2017 | DOI: 10.1038/NCHEM.2829
A unifying paradigm for naphthoquinone-based
meroterpenoid (bio)synthesis
1
†
1†
2†
2
2
*
Zachary D. Miles , Stefan Diethelm , Henry P. Pepper , David M. Huang , Jonathan H. George
and Bradley S. Moore1,3
*
Bacterial meroterpenoids constitute an important class of natural products with diverse biological properties and
therapeutic potential. The biosynthetic logic for their production is unknown and defies explanation via classical
biochemical paradigms. A large subgroup of naphthoquinone-based meroterpenoids exhibits a substitution pattern of the
polyketide-derived aromatic core that seemingly contradicts the established reactivity pattern of polyketide phenol
nucleophiles and terpene diphosphate electrophiles. We report the discovery of a hitherto unprecedented enzyme-
promoted α-hydroxyketone rearrangement catalysed by vanadium-dependent haloperoxidases to account for these
discrepancies in the merochlorin and napyradiomycin class of meroterpenoid antibiotics, and we demonstrate that the
α-hydroxyketone rearrangement is potentially a conserved biosynthetic reaction in this molecular class. The biosynthetic
α-hydroxyketone rearrangement was applied in a concise total synthesis of naphthomevalin, a prominent member of the
napyradiomycin meroterpenes, and sheds further light on the mechanism of this unifying enzymatic transformation.
eroterpenoids are natural products of mixed biosynthetic To explain prenylation at the non-nucleophilic C3 carbon of the
1
–6
2,3,6,7
origin that exhibit antimicrobial , anticancer
and THN core, the involvement of ‘promiscuous’ PTases has been pos-
8,9
25–27
M
antioxidant activities . They are formed in nature by the tulated
. To the best of our knowledge, no literature precedence
regioselective addition of an electron-rich, polyketide-derived aro- exists for prenylation at such a non-nucleophilic aromatic carbon by
matic nucleophile to an electrophilic terpene diphosphate, a reac- a PTase, and therefore the lack of a satisfying explanation called for
tion catalysed by the ABBA prenyltransferase (PTase) class of further investigation.
10,11
enzymes . Despite the impressive body of knowledge that per-
Herein we report the discovery of a halogenation-induced
12
tains to terpenoid biosynthesis , surprisingly little is known about α-hydroxyketone rearrangement to account for the enigmatic sub-
the biosynthesis of these hybrid compounds. Moreover, meroterpe- stitution pattern of the prevalent class II meroterpenoid natural pro-
noids are ubiquitous across all domains of life and are a prevalent ducts (Fig. 1, bottom). This unifying transformation occurs in the
source of natural product structural diversity in fungi . Among bac- biosynthesis of both the merochlorin and napyradiomycin classes
teria, biosynthetic pathways to secondary metabolites that incorpor- of compounds through the action of the vanadium-dependent
13
ate terpene fragments are often difficult to unravel with established chloroperoxidase (VCPO) Mcl24 (merochlorin) and a homologous
terpene biosynthetic concepts1
4–16
.
enzyme NapH3 (napyradiomycin) that does not harbour the same
Recently, a group of hybrid isoprenoid–polyketide natural halogenation ability, yet catalyses a mirrored α-hydroxyketone
17
products, which include naphthomevalin (1) , napyradiomycin rearrangement. In addition, our investigations identified a PTase,
A1 (2)1 , neomarinone (3) , naphterpin (4) and merochlorins NapT8 (napyradiomycin), responsible for the upstream biosynthetic
8,19
20
8,21
1,22
A, B and D (5–7) , have attracted the attention of the biosyn- reaction in the napyradiomycin biosynthetic pathway, which
thetic community because of their unique molecular architectures includes prenylation of a halogenated meroterpenoid precursor
Fig. 1). As demonstrated previously by C-labelling studies20,21,23, and further underlines the novelty of biosynthetic transformations
1
3
(
these naphthoquinone-based meroterpenoids derive from a tetra- within bacterial meroterpenoid biosynthetic pathways.
hydroxynaphthalene (THN) (8) precursor formed through the In a joint effort that involves synthetic chemistry and biochem-
action of a single polyketide synthase using the substrate istry, we set out to exploit the unexpected discovery of this novel
malonyl-coenzyme A (ref. 24). Single or multiple prenylation enzymatic rearrangement to develop a biosynthesis-inspired
events, in addition to further embellishment by a multitude of pre- synthetic entry into the naphthomevalin class of meroterpenoids.
dominantly uncharacterized modifying enzymes, adorn the THN As such, the presented synthesis is designed to follow a blueprint
precursor and form the complex final products. Curiously, the of enzymatic reactions, and is therefore in the strictest sense biomi-
most-abundant subgroup of THN-derived meroterpenes (Fig. 1b, metic. Moreover, our synthetic endeavour provides additional
class II) exhibit a C3-prenylation pattern, which contradicts the insight into the enzymatic transformation and lends credence to
expected C2/C4-nucleophilic reactivity of the parent THN mol- this rearrangement. We demonstrate that the amalgamation of bio-
ecule, as the C3 position is not nucleophilic and not poised for chemical investigations into an enzymatic reaction and its cognate
such an alkylation reaction. As such, they clearly distinguish them- synthetic application harbours great potential to address complex
selves from the C2- or C4-prenylated meroterpenes, which include questions and advance the fields of both biosynthetic and
2
8,29
neomarinone (3) and merochlorins A (5) and B (6) (Fig. 1a, class I). synthetic chemistry
.
1
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093, USA.
2
3
Department of Chemistry, The University of Adelaide, Adelaide, South Australia 5005, Australia. Skaggs School of Pharmacy and Pharmaceutical
†
Sciences, University of California San Diego, La Jolla, California 92093, USA. These authors contributed equally to this work.
*
e-mail: bsmoore@ucsd.edu; jonathan.george@adelaide.edu.au
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2829
ARTICLES
a
b
Class I
Class II
HO
O
HO
O
O
H
Cl
Cl
Cl
O
O
3
3
2
HO
OH
HO
O
O
HO
OH
HO
O
Cl
O
HO
OH
4
O
4
Neomarinone (3)
2
3
Naphthomevalin (1)
Napyradiomycin A1 (2)
HO
HO
O
HO
O
O
HO
O
HO
OH
Cl
Cl
O
THN (8)
3
3
H
4
HO
HO
OH
HO
O
Merochlorin A (5)
H
O
Merochlorin B (6)
Merochlorin D (7)
Naphterpin (4)
HO
OH
2
HO
O
HO
O
X
α-hydroxyketone
rearrangement
X
R
Cl
O
Cl
OH
3
HO
OH
R
HO
HO
4
R
OH
O
R = Isoprenoid appendage
X = Cl or alkyl
Figure 1 | Bacterial THN-derived meroterpenes. a,b, THN-derived meroterpenoids can be divided into two classes based on C2 or C4 prenylation
class I (a)) or C3 prenylation (class II (b)). The majority of naphthoquinone-based terpenoids are categorized as class II. Naphthomevalin (1) and
(
napyradiomycin A1 (2) share features of both class I and II, with terpene units at both C2 and C3. All of the compounds could originate from a C4-prenylated
molecule, with an α-hydroxyketone rearrangement of the prenyl appendage from C4 to C3 accounting for the creation of class II (bottom).
Results
12 by water gives α-hydroxyketone 13, which undergoes a second
Characterization of an additional Mcl24 product. The merochlorins chlorination at C2 to give the geminal dichloride 14 (path b). The
1
8
are a group of recently isolated bacterial meroterpenoid antibiotics addition of water is supported by assays conducted in OH2,
that include both the C4-prenylated members merochlorin A (5) wherein product 10 showed the requisite m/z shift of +2 of the
and B (6), as well as the C3-substituted analogues merochlorin C primary ionization peak (Supplementary Fig. 3). The subsequent
1
(
not shown) and D (7) . Produced by a single biosynthetic gene α-hydroxyketone rearrangement of 14 produces the C3-substituted
cluster, this system offers a unique opportunity to study the THN derivative 10, which shares the same carbon skeleton as mer-
chemistry that leads to the cryptic C3 prenylation observed in ochlorin D (7). To the best of our knowledge, this is the first report
merochlorin D (7). We recently reported that the VCPO Mcl24 of a halogenation-mediated α-hydroxyketone rearrangement in nature,
mediates
followed by intramolecular cyclization(s) to convert pre-merochlorin biosynthesis . It is particularly remarkable that a single enzyme,
9) into the stereochemically complex natural products 5 and 6 in a Mcl24, encoded from a 41-open reading frame gene cluster, is
a
chlorination-induced oxidative dearomatization and resembles a suggested α-hydroxyketone rearrangement in aurachin
3
4
(
30,31
single enzymatic reaction (Fig. 2) . However, the formation of the responsible for generating a significant portion of the structural
C3-prenylated merochlorin analogues remained elusive. Remarkably, complexity and diversity of all of the merochlorin natural products.
the only PTase in the mcl gene cluster (Mcl23) showed selectivity
30
for the prenylation of THN (8) at C4 to produce 9 exclusively . Chemical chlorination of pre-merochlorin (9) and analogue 17.
Therefore, the possible promiscuity during the prenylation event We previously established a chemical chlorination protocol using
could be excluded in the biosynthesis of the merochlorins.
a combination of N-chlorosuccinimide (NCS) and i-Pr NH to
2
3
1
During our investigations aimed towards a detailed characteriz- mimic the Mcl24 chemistry . Under these conditions, pre-
ation of Mcl24, we observed that a minor, previously uncharacterized, merochlorin (9) was converted into deschloromerochlorin A (15)
product of the reported reaction increased in abundance under basic and deschloromerochlorin B (16) (Fig. 3). However, we did not
reaction conditions (Fig. 2a and Supplementary Fig. 1). A detailed observe the formation of a C3-prenylated product resulting from
structural analysis indicated that the newly formed product 10 was an α-hydroxyketone rearrangement under these conditions. We
a C3-prenylated THN derivative that exhibited dichlorination at C2 were curious to probe the influence that a change in the electronic
(Fig. 2b). In addition, a circular dichroism (CD) spectrum was properties of the substrate might have on the rearrangement
obtained of 10, and the pronounced features are indicative of an chemistry. Such a study might be of interest, as some of the
enantiopure chiral molecule, which demonstrates the enantiospecifi- proposed intermediates en route to a rearranged α-hydroxyketone
city of Mcl24 (Supplementary Fig. 2). This finding suggests that 10 is product possess polar character and would hence be influenced by
formed through an α-hydroxyketone rearrangement, which accounts such electronic changes in the substrate. Consequently, we subjected
for the alkyl migration from C4 to C3 (Fig. 2c). We propose a mech- the less electron-rich analogue 17 to our chemical chlorination
anism wherein Mcl24 first chlorinates 9 at C2 and the C3 phenol to protocol. Interestingly, besides the expected merochlorin A analogue
give intermediate 11. Subsequent oxidative dearomatization to form a 18, two additional products, 19 and 20, were isolated.
benzylic carbocation intermediate 12, followed by intramolecular
We propose that the initial dichlorination of 17 gives intermedi-
3
1
cyclization gives rise to 5 and 6, as reported previously (path a) . ate 21, which undergoes oxidative dearomatization to give benzylic
A similar benzylic carbocation intermediate was also proposed in carbocation 22, in analogy to the chemistry performed by Mcl24
previous total syntheses of both merochlorin A and B (refs 32,33). (Fig. 2c). Cyclization of 22 affords the merochlorin A analogue
Alternatively, trapping of the intermediate benzylic carbocation 18. Alternatively, trapping of the carbocation intermediate 22 by
2
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2829
ARTICLES
a
1
0
b
HO
OH
HO
O
O
Cl
Mcl24
H2O2
Cl
2
HO
OH
HO
OH
4
5
15
*
16
Pre-merochlorin (9)
× Cl+
10
6
α-hydroxyketone
rearrangement
2
c
HO
O
pH 8.0
HO
OH
2
Cl
Cl
Cl
O
Cl
HO
HO
O
O
H
11
14
Oxidative
dearomatization
Cl–
Cl+
–
HO
OH
HO
OH
Cl
Cl
H2O
Path b
2
+
HO
O
HO
O
pH 6.5
OH
1
2
13
No H O
2
2
Path a
No enzyme
Merochlorin A (5)
or B (6)
16
17
18
19
20
21
22
23
24
Time (min)
Figure 2 | Mcl24-mediated reaction of pre-merochlorin (9). a, Reversed-phase HPLC (254 nm) of the standard reaction of Mcl24 with 9 at pH 8.0, pH 6.5,
excluding H and excluding enzyme. When assayed at a lower pH, the major product observed was merochlorin A (5). When assayed at a higher pH, the
2 2
O
product distribution changed substantially, with 10 as the major product. Consistent with the reaction requirements of VHPO enzymes, the removal of
hydrogen peroxide (or enzyme) resulted in no turnover. All of the products other than 10, including isochloro-merochlorin B (asterisk) and the uncharacterized
31
degradation product of 9 at ∼16.5 min, were reported in a previous study . b, Production of the C3-prenylated THN analogue 10 through an α-hydroxyketone
rearrangement of 9. c, The proposed mechanism involves a chlorination-induced enzymatic α-hydroxyketone rearrangement.
chloride at C4, followed by chlorination of the resultant chloroenol which was converted into the methyl ketone 26 via Friedel–Crafts
3
8
at C2, would deliver product 19. Finally, the addition of water at acylation with Ac O followed by demethylation using AlCl .
2
3
C4 of carbocation 22 followed by C2 chlorination would give α- Protection of 26 as a bis-methoxymethyl (MOM) ether followed
hydroxyketone 23, which does not undergo an α-hydroxyketone by base-induced aromatization via a Dieckmann-type cyclization
rearrangement such as that observed in the enzymatic reaction. then gave the protected THN derivative 27. Selective C4 geranyla-
Instead, intramolecular hemiacetalization to form 24, followed by tion was achieved through a palladium-mediated allylation using
3
0,39
fragmentation, gives rise to compound 20. It seems probable that ethyl geranyl carbonate (28) as the electrophile . The resulting
the presence of excess base influences the reaction outcome by pro- naphthol 29 was subsequently subjected to Pb(OAc) -mediated oxi-
4
3
2,40
moting hemiacetalization at the expense of the desired α-hydroxy- dative dearomatization
followed by in situ dichlorination of the
ketone rearrangement. Furthermore, the different solvent intermediate enol at C2 using NCS to give dichlorodiketone 30.
properties of the enzymatic versus the chemical chlorination can Selective removal of one chlorine substituent from C2 of 30 was
influence the reaction outcome, and either prevent or promote the achieved using a lithium diisopropylamide (LDA)-mediated
rearrangement chemistry by the impact on the stability of polar reduction to give a monochloride that is protected from further
4
1
intermediates on the reaction path. Our failure to mimic the dechlorination by enolization . Subsequent cleavage of the acetate
natural α-hydroxyketone rearrangement in these initial experiments protecting group delivered enol 31, which was then prenylated at
raised questions about the thermodynamic requirements for the C2 to give α-hydroxyketone 32 as a single diastereomer because
enzymatic 1,2-alkyl shift and motivated further synthetic studies.
of the steric hindrance of one face of the enolate derived from 31
by the bulky geranyl substituent. Removal of the MOM protecting
Biomimetic total synthesis of (±)-naphthomeꢀalin. The novel groups from 32 under mild acidic conditions then gave α-hydroxy-
enzymatic α-hydroxyketone rearrangement discovered in the ketone 33, and thus set the stage for the key biomimetic α-hydroxy-
merochlorin system offered an opportunity to develop a general ketone rearrangement. Initial attempts to rearrange 33 using acidic
synthetic entry into the class II meroterpenes (Fig. 1b). A strategy or basic conditions failed to give C3-geranylated naphthomevalin
that takes advantage of a biomimetic α-hydroxyketone rearrangement (1). However, simply heating 33 in toluene at 110 °C for 16 hours
seemed particularly attractive, as it would exploit the predisposed efficiently produced (±)-naphthomevalin (1) in quantitative yield.
C4-nucleophilic reactivity of THN to build up a precursor molecule All of the spectroscopic data for 1 matched the reported data of
efficiently, which would then undergo alkyl migration to arrive at the the natural product (Supplementary Information). The relative con-
required C3-substitution pattern. Moreover, despite their promising figuration of 1, with a trans relationship between the C2-prenyl
antibacterial activity, only three total syntheses of naphthomevalin/ and C3-geranyl substituents, is supported by its facile conversion
3
5–37
napyradiomycin meroterpenoids have been reported to date
.
into the epoxide natural product A80915G under mild basic
1
7
As outlined in Fig. 4, our biomimetic synthesis of naphthomeva- conditions (Supplementary Information). Further experiments
lin (1) commenced with methyl (3,5-dimethoxyphenyl)acetate (25), showed that the conversion of 33 into 1 occurred at a significantly
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2829
ARTICLES
a
HO
O
HO
O
H
H
O
X = OH
ref. 31)
O
(
+
HO
HO
X
OH
Deschloro-merochlorin B (16)
NCS/i-Pr2NH
Deschloro-merochlorin A (15)
X
OH
CH2Cl2
O
Cl
Cl
O
Cl
O
Cl
X = H
O
+
O
+
O
O
X = OH: pre-merochlorin (9)
X = H: analogue (17)
Cl
20
Fragmentation
Cl
18
19
+
2
x Cl X = H
−
1
. Cl at C4
+
Cyclization
OH
2
. Cl at C2
b
−
OH
Cl
O
O
Cl
Cl
Hemiacetal
formation
Cl
1
. H2O at C4
−
Cl
2
Cl
O
−
Cl
+
2. Cl at C2
O
Cl
Oxidative
dearomatization
+
O
4
O
O
OH
21
22
23
24
Figure 3 | Chemical chlorination of pre-merochlorin (9) or analogue 17. Pre-merochlorin (9) and its less electron-rich analogue 17 were subjected to our
previously established NCS/i-Pr NH chemical chlorination conditions. a, An alternative outcome was observed for the NCS/i-Pr NH-mediated oxidative
2
2
dearomatization of 9 and its analogue 17 (Supplementary Table 2 gives the product yields). Whereas 9 converted into the two deschloro-merochlorin
analogues 15 and 16, chlorination of substrate 17 produced a mixture of products 18–20. Interestingly, no products that result from an α-hydroxyketone
rearrangement were observed under these conditions. b, A possible mechanistic rationale for the formation of products 19 and 20 involves the initial
formation of carbocation 22 through oxidation and chloride loss. Intermediate 22 can subsequently be trapped by chloride to provide product 19, or
alternatively undergo water addition followed by a C–C bond cleavage to arrive at compound 20. NCS, N-chlorosuccinimide.
lower temperature (60 °C) when water was used as the solvent, rearrangement has been proposed for a similar α-hydroxyketone
3
4,43
and thus represented an unusual example of an ‘on-water’ catalysed rearrangement recently observed in aurachin biosynthesis
α-hydroxyketone rearrangement .
.
42
Our synthesis of naphthomevalin (1) is biomimetic as it mirrors Characterization of an α-hydroxyketone rearrangement in
several steps of the proposed biosynthesis, namely C4 geranylation, napyradiomycin biosynthesis. After unravelling the origin of C3
oxidative dearomatization, C2 chlorination, C2 prenylation and prenylation in merochlorin biosynthesis coupled with the
α-hydroxyketone rearrangement. Thus, in conjunction with the syn- successful application of the key α-hydroxyketone rearrangement
thesis of 1, we were able to prepare additional proposed biosynthetic in the total synthesis of naphthomevalin (1), it remained unclear
intermediates free of protecting groups, which were instrumental in if this rearrangement can, indeed, serve as a general rule for the
later enzymatic studies (vide infra).
C3-prenylation pattern in all naphthoquinone-derived meroterpenoids.
With a synthesis of naphthomevalin and various analogues in We therefore set out to explore whether an analogous α-hydroxyketone
hand, we envisioned further characterization of the thermal rearrangement is involved in the biosynthesis of naphthomevalin
α-hydroxyketone rearrangement from a theoretical perspective. As (1) using the proposed biosynthetic intermediates generated in
shown in Fig. 5, three α-hydroxyketone derivatives (33, 34 and our biomimetic synthesis as substrates for enzymatic studies.
3
5) were subjected to the originally established reaction conditions
In 2007, we characterized the naphthomevalin-related napyra-
4
4
(110 °C in toluene) for the 1,2-alkyl shift. For each reaction, we also diomycin biosynthetic gene cluster and identified genes that
calculated kinetic and thermodynamic parameters using quantum encoded two PTases and three vanadium-dependent haloperoxi-
chemical methods (Supplementary Information gives the full dases (VHPOs). The VCPO NapH1 was subsequently shown to
details). The calculations support a concerted 1,2-suprafacial-shift catalyse the stereoselective chlorination–cyclization of the dimethyl-
4
5
mechanism for the α-hydroxyketone rearrangement of both 33 allyl group of a methylated analogue of naphthomevalin (1) .
and the dichlorinated derivative 35 (which closely resembles the Therefore, we set out to investigate the function of the putative
1
0,11
putative intermediate 14 in the Mcl24 reaction of 9) with an internal ABBA aromatic PTases
encoded by napT8 and napT9. We
proton transfer. The rearrangement of 33 and 35 is thermodynami- surmised that these genes encode enzymes responsible for the
cally favoured, in part by the formation of a more-conjugated, sequential addition of the dimethylallyl and geranyl moieties to
planar structure in products 1 and 36. In contrast, monochlorinated THN at the electron-rich C2 and C4 positions (and not, as originally
derivative 34 was unreactive, as it possesses a near planar bicyclic suggested, at the electron-deficient C3), which results in an inter-
structure, which indicates full conjugation of the π system. mediate primed for an α-hydroxyketone rearrangement. Indeed,
Clearly, geminal disubstitution at C2 is necessary for the migration NapT8 was cloned, heterologously expressed, purified and found
to occur. An alternative two-step mechanism that involves a retro- to catalyse the addition of dimethylallyl pyrophosphate (DMAPP)
[2,3]-Wittig rearrangement followed by an aromatic Claisen to the C2 position of 34 to generate 33 (Fig. 6a). Furthermore,
4
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2829
ARTICLES
EtO2CO
OH
28
MOMO
OH
MeO
HO
O
1. MOMCl, i-Pr2NEt MOMO
CH2Cl2
2. NaH, DMF
1
2
. Ac2O, HClO4
. AlCl3, CH2Cl2
Pd(PPh3)4, Et3B
THF, 50 °C
2
CO2Me
2
CO Me
6
2
0% over
steps
74% over
2 steps
29%
MOMO
OH
MeO
HO
MOMO
OH
4
4
(+46% recovered 27)
Geranylation at C4
25
26
27
2
9
Pb(OAc)4, CHCl3
Oxidative dearomatization,
−40 °C; then NCS
chlorination at C2
53%
HO
OH
Cl
MOMO
OH
MOMO
O
Cl
1. LDA, THF, −78 °C
Cl
O
Cl
HCl, MeOH, RT
9%
2. KOH, MeOH, 60 °C
HO
O
MOMO
MOMO
O
5
53% over 2 steps
dechlorination
OH
OH
OAc
3
4
31
30
Br
NaH, DMF, 0 °C
5%
Proposed biosynthetic
intermediate
5
Prenylation at C2
PhMe, 110 °C, 16 h
or
H O, 60 °C, 16 h
2
MOMO
O
HO
O
HO
O
Cl
O
Cl
or
Cl
HCl, MeOH, RT
59%
MeOH-H O, 60 °C, 40 h
2
MOMO
HO
O
HO
OH
quant.
α-hydroxyketone
rearrangement
OH
OH
O
Naphthomevalin (±)-1
Proposed biosynthetic
intermediate
32
33
Figure 4 | Biomimetic total synthesis of (±)-naphthomevalin (1) via a thermal α-hydroxyketone rearrangement. This 11-step total synthesis of
±)-naphthomevalin was designed to incorporate several steps of its proposed biosynthesis, which include the C4 geranylation of a THN derivative followed
(
later by an α-hydroxyketone rearrangement to shift the geranyl group to C3. In addition to the natural product, two putative biosynthetic intermediates were
synthesized for use in enzymatic studies.
NapT8 was only active when the isoprenoid DMAPP was utilized under substrate-saturating conditions were measured (Supplementary
and activity was stimulated by the presence of exogenous MgCl2 Fig. 11). Under these conditions, NapH3 increased the rate nearly
–
1
(
Supplementary Fig. 7). To the best of our knowledge, this rep- tenfold (0.374 ± 0.021 µM min for the NapH3-catalysed reaction
–1
resents the first example of a PTase that catalyses the prenylation compared with 0.041 ± 0.002 µM min for the non-enzymatic reac-
of a halogenated carbon centre. tion) when assayed at a 10 µM enzyme concentration. The turnover
−
1
We next turned our attention to the identification of an enzyme number (k ) for NapH3 is 0.038 ± 0.002 min , and remained
cat
that could potentially catalyse an analogous α-hydroxyketone constant when assayed with either synthetic or enantiopure
rearrangement of 33 to give naphthomevalin (1). We immediately (NapT8-produced) 33. To ascertain that naphthomevalin (1) is,
focused on the orphan napyradiomycin VHPO homologues indeed, a biosynthetic intermediate, we further subjected racemic
encoded by napH3 and napH4 that share about a 57% pairwise 1 to NapH1 to yield the natural product napyradiomycin A1 (2)
amino acid sequence identity with the previously characterized (Fig. 6a). To gain insight into the stereochemistry of the enzymatic
NapH1 (ref. 46). Although NapH4 proved recalcitrant to all of the reactions of NapT8 and NapH3, CD spectra were measured for
attempts at recombinant expression, we were able to clone, hetero- the enzymatically produced 33 (Supplementary Fig. 12) and 1
logously express and purify NapH3. Recombinant NapH3 was sub- (Supplementary Fig. 13). The CD spectrum of 33 formed from
jected to activity assays with putative synthetic pre-napyradiomycin racemic 34 and NapT8 demonstrated the presence of an enantio-
substrates (Supplementary Figs 8 and 9), along with the enzymatic pure chiral molecule, that is, a kinetic resolution had taken place.
product of the NapT8 reaction (33). Of all of the substrates The CD spectrum of 1 generated by NapH3 catalysis on racemic 33
evaluated, NapH3 exhibited an efficient capacity to mediate the also indicated an enantiopure chiral molecule. Furthermore,
C4-to-C3 α-hydroxyketone rearrangement of the geranyl moiety unreacted 33 re-isolated from the NapH3 reaction has the opposite
only on incubation with both synthetic and NapT8-produced 33, CD spectrum to that of 33 generated from prenylation of 34 with
to form naphthomevalin (1) (Fig. 6a and Supplementary Fig. 8). NapT8. These observations prove conclusively that the prenylation
Incubation of 33 with the VHPOs NapH1 and Mcl24, on the other and α-hydroxyketone steps are under strict enzymatic stereocontrol,
hand, did not catalyse the formation of naphthomevalin (1) dictated by NapT8 and NapH3, respectively.
(
Supplementary Fig. 10), although we could measure some slow,
non-enzymatic conversion, as similarly observed in the synthetic halogenation activity of VHPOs , H O or exogenous Na VO
4
In contrast to the standard assay conditions required for the
4
7
2
2
3
conversion and NapT8 assays. To compare the non-enzymatic rate were not required for NapH3 activity. Furthermore, this enzyme
with that of the NapH3-catalysed reaction, the initial velocities did not show chlorination or bromination activity when assayed
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2829
ARTICLES
a
HO
O
O
Cl
α-hydroxyketone
rearrangement
PhMe, 110 °C
HO
OH
quant.
Naphthomevalin (±)-1
ΔG°soln = −5.4 kcal mol-1
33
‡
-1
ΔG soln = +37.1 kcal mol
b
HO
O
Cl
Cl
α-hydroxyketone
rearrangement
PhMe, 110 °C
HO
OH
68%
O
3
6
35
‡
-1
ΔG°soln = −8.6 kcal mol-1
ΔG soln = +36.9 kcal mol
c
HO
OH
Cl
No reaction
PhMe, 110 °C
HO
OH
O
34
37
ΔG°soln = +11.4 kcal mol
‡
-1
-1
ΔG soln = +35.7 kcal mol
Figure 5 | Theoretical and experimental studies on the thermally induced α-hydroxyketone rearrangement. a–c, Attempted 1,2-shift of three
‡
°
α-hydroxyketone substrates (33 (a), 35 (b) and 34 (c)), with ΔG soln and ΔG soln denoting computed free energy changes from the reactant to the transition
state and from the reactant to the product, respectively. Structures with and without the ‘‡’ symbol are the computed transition state and reactant
geometries, respectively. These studies show that geminal disubstitution at C2 is required for the α-hydroxyketone rearrangement to be thermodynamically
favoured. soln, solution.
with monochlorodimedone (MCD), a commonly used substrate to novel α-hydroxyketone rearrangement. Yet the requirement for
4
8,49
assess the capacity of VHPOs to form hypohalous acid . The disubstitution of C2 remains in both parallel enzymatic transform-
observation that NapH3 catalyses the α-hydroxyketone rearrange- ations, which is in line with our previous synthetic and theoretical
ment on an already chlorinated/dearomatized intermediate further observations. Studies are ongoing to identify the origin of the
supports the lack of halogenation ability. A multiple-sequence align- initial chlorination event in napyradiomycin biosynthesis.
ment of the VHPO homologues from both the merochlorin and
napyradiomycin gene clusters revealed a conserved serine residue Discussion and conclusions
that is present as a phenylalanine residue (F378) only in NapH3 The merochlorin and napyradiomycin families of natural products
(Supplementary Fig. 14). This residue was found to be critical for have proved valuable model biosynthetic pathways to address the
the turnover in NapH1, wherein the mutation to histidine abolished long-standing question of how a counterintuitive substitution
45
the native activity . To test if this single mutation would be suffi- pattern is generated in the largest subclass of bacterial meroterpenoids.
cient to confer halogenation ability, the NapH3 F378S variant was We identified two enzymes with a highly conserved primary sequence,
expressed, purified and assayed using the standard MCD assay; capable of mediating an α-hydroxyketone rearrangement that leads
however, no activity was observed (data not shown).
to a non-standard prenyl substitution of the parent THN core.
When taken together, the general biosynthetic transformation for Remarkably, these enzymes invoke similar, yet in one instance dis-
both pathways first employs chlorination-mediated oxidative dearo- jointed, chemistry. In the biosynthesis of the merochlorins, the
matization, followed by subsequent α-hydroxyketone rearrangement VHPO Mcl24 acts as a multitasking halogenating enzyme that
through a presumed acid/base catalysis. There are very few examples can catalyse both the oxidative dearomatization of a simple precur-
of a rearrangement of a long-chain carbon moiety in natural product sor molecule and the movement of a prenyl appendage. In contrast,
biosynthesis; it has only been observed as a reaction that results in a its homologue in the napyradiomycin biosynthetic pathway,
2
9
shunt product in hapalindole/ambiguine biosynthesis and it NapH3, requires a C2-chlorinated substrate that is predisposed for
is suggested in the biosynthesis of aurachin3 . In merochlorin the rearrangement. Despite the close relationship between the two
biosynthesis, both reactions are catalysed by a single enzyme, VHPO homologues, NapH3 appears to have lost its capability as a
Mcl24. The increased production of 10 when the pH is increased haloperoxidase and has evolved into an exclusive and selective catalyst
from 6.5 to 8.0 implies a specific catalytic base residue with a pKa for the requisite rearrangement chemistry. Furthermore, we have ident-
of ∼7, such as a histidine residue. In napyradiomycin biosynthesis, ified the PTase NapT8, which is capable of prenylating a halogenated
NapH3 catalyses the ‘second half’ of the Mcl24 reaction and pre- carbon centre on the THN core, and thereby primes the substrate for
sumably participates in a solely acid/base catalysis to promote this the α-hydroxyketone rearrangement catalysed by NapH3.
4,43
6
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.2829
ARTICLES
a
Data aꢀailability. All data and any associated accession codes and
references are available in the online version of this paper.
34
1
33
2
Received 12 October 2016; accepted 15 June 2017;
published online 31 July 2017
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Additional information
Supplementary information and chemical compound information are available in the
online version of the paper. Reprints and permissions information is available online at
www.nature.com/reprints. Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. Correspondence and
requests for materials should be addressed to J.H.G. and B.S.M.
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Competing financial interests
The authors declare no competing financial interests.
8
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