Dirigent Proteins Guide Asymmetric Heterocoupling for the Synthesis of Complex Natural Product Analogues

Article abstract of DOI:10.1021/jacs.0c13164

Phenylpropanoids are a class of abundant building blocks found in plants and derived from phenylalanine and tyrosine. Phenylpropanoid polymerization leads to the second most abundant biopolymer lignin while stereo- and site-selective coupling generates an array of lignan natural products with potent biological activity, including the topoisomerase inhibitor and chemotherapeutic etoposide. A key step in etoposide biosynthesis involves a plant dirigent protein that promotes selective dimerization of coniferyl alcohol, a common phenylpropanoid, to form (+)-pinoresinol, a critical C2 symmetric pathway intermediate. Despite the power of this coupling reaction for the elegant and rapid assembly of the etoposide scaffold, dirigent proteins have not been utilized to generate other complex lignan natural products. Here, we demonstrate that dirigent proteins from Podophyllum hexandrum in combination with a laccase guide the heterocoupling of natural and synthetic coniferyl alcohol analogues for the enantioselective synthesis of pinoresinol analogues. This route for complexity generation is remarkably direct and efficient: three new bonds and four stereocenters are produced from two different achiral monomers in a single step. We anticipate our results will enable biocatalytic routes to difficult-to-access non-natural lignan analogues and etoposide derivatives. Furthermore, these dirigent protein and laccase-promoted reactions of coniferyl alcohol analogues represent new regio- and enantioselective oxidative heterocouplings for which no other chemical methods have been reported.

Full text of DOI:10.1021/jacs.0c13164

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Dirigent Proteins Guide Asymmetric Heterocoupling for the
Synthesis of Complex Natural Product Analogues
Stacie S. Kim and Elizabeth S. Sattely*
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ABSTRACT: Phenylpropanoids are a class of abundant building
blocks found in plants and derived from phenylalanine and
tyrosine. Phenylpropanoid polymerization leads to the second most
abundant biopolymer lignin while stereo- and site-selective
coupling generates an array of lignan natural products with potent
biological activity, including the topoisomerase inhibitor and
chemotherapeutic etoposide. A key step in etoposide biosynthesis
involves a plant dirigent protein that promotes selective
dimerization of coniferyl alcohol, a common phenylpropanoid, to
form (+)-pinoresinol, a critical C2 symmetric pathway intermedi-
ate. Despite the power of this coupling reaction for the elegant and
rapid assembly of the etoposide scaffold, dirigent proteins have not
been utilized to generate other complex lignan natural products.
Here, we demonstrate that dirigent proteins from Podophyllum hexandrum in combination with a laccase guide the heterocoupling of
natural and synthetic coniferyl alcohol analogues for the enantioselective synthesis of pinoresinol analogues. This route for
complexity generation is remarkably direct and efficient: three new bonds and four stereocenters are produced from two different
achiral monomers in a single step. We anticipate our results will enable biocatalytic routes to difficult-to-access non-natural lignan
analogues and etoposide derivatives. Furthermore, these dirigent protein and laccase-promoted reactions of coniferyl alcohol
analogues represent new regio- and enantioselective oxidative heterocouplings for which no other chemical methods have been
reported.
We hypothesized that an analogue of etoposide bearing E-ring
substituents less prone to oxidation might limit unwanted
hepatic metabolism.
INTRODUCTION
■
Ubiquitous phenylpropanoid monomers in plants (termed
monolignols) are the starting point for the biosynthesis of two
very different classes of molecules: polymeric lignin, the
structural component of the lignified plant cell wall, and
lignans, bioactive dimeric natural products that typically
contain multiple chiral centers and aryl ethers (see Scheme
1A for common modes of coupling). While nonselective
polymerization of monolignol radical species results in the
synthesis of lignin,1 regio- and enantioselective coupling routes
are thought to lead to diverse optically active dimeric lignans
that accumulate in the tissue of both edible and medicinal
plants.2−4 One important example is etoposide, an anticancer
drug that is semisynthetically produced from a lignan natural
product, (−)-podophyllotoxin, found in Podophyllum spp.
(Scheme 1A).
Etoposide is currently manufactured using a semisynthetic
approach that requires sourcing a related lignan, (−)-podo-
phyllotoxin, from the medicinal plant Podophyllum spp.
Recently, we established a complete biosynthetic pathway for
4′-demethyl (−)-epipodophyllotoxin, or etoposide aglycone
(EA) through heterologous expression of previously
known11−16 and newly identified17 Podophyllum hexandrum
(Ph) genes in Nicotiana benthamiana (Nb). Given the modular
nature of EA biosynthesis, we considered that analogous
couplings of alternative monolignol building blocks could
enable efficient modification of the etoposide E ring for the
formation of analogues.
Etoposide and related drugs (etopophos and teniposide) act
as reversible topoisomerase inhibitors by intercalating at the
interface of the topoisomerase IIβ and DNA cleavage complex
and stabilizing the double stranded break, thereby inducing
apoptosis of cancer cells.5 However, one liability of etoposide
is the propensity of the drug to undergo demethylation by
human liver enzyme CYP3A4 to generate a quinone with an
altered activity profile (Scheme 1A, highlighted in green).6−10
Received: December 21, 2020
Published: March 29, 2021
https://doi.org/10.1021/jacs.0c13164
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© 2021 American Chemical Society
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Scheme 1. Proposed Enantioselective Production of Hetero-(+)-Pinoresinol Analogues for Biosynthesis of Etoposide
a
Analogues
a
A. Laccase oxidizes coniferyl alcohol and generates CA radical species that undergo three unique modes of coupling.13,18,19 (Note on the coupling
product naming convention: coupling products of the same monomer combination are collectively referred by their assigned compound number,
and specific coupling modes are denoted by lowercase letters: a, 8-O-4′; b, 8-8′; and c, 8-5′.) In presence of PhDIR, oxidized radical species of
coniferyl alcohol form predominantly 8-8′ coupling products enantioselectively. The 8-8′ coupling product (+)-pinoresinol can be subsequently
processed by enzymes involved in the etoposide aglycone (EA) biosynthetic pathway. The methoxy group of etoposide highlighted in green is
prone to oxidation by human CYP3A4 and therefore is a target for modification using an engineered biosynthesis route. B. For each coupling mode
(8-8′ coupling mode shown here; see Figure S1 for all other possible products), there are three possible types of coupling products when a mixture
of two monomers is used: one heterocoupling and two distinct homocoupling products. We propose that the use of coniferyl alcohol analogues for
(+)-pinoresinol analogue synthesis will lead to etoposide variants with modifications at the E ring.
The EA pathway involves convergent coupling of two
coniferyl alcohol (CA) monomers for rapid complexity
generation (route shown in Scheme 1A shows the native
biosynthetic pathway that begins with dimerization of two CA
monomers).13,18,19 This key step involves oxidation of CA
monomers to their corresponding radical species by a
peroxidase or a laccase.19,20 Dirigent proteins (DIRs),
described from Podophyllum and other plant species, then
direct the resulting radicals toward a remarkable stereoselective
C2 symmetric dimerization.13,21,22 DIRs are not known to
affect the rate of monolignol oxidation, nor are they thought to
impact the net rate of dimerization. However, DIRs do change
the relative ratio of different coupling products. In the absence
of DIRs, monolignol radicals undergo spontaneous and
nonselective couplings. This mechanism is supported empiri-
cally such that stereoselectivity is observed only in the presence
of both an oxidizing agent (e.g., enzymatic or inorganic) and a
DIR.13,23 In contrast, multiple racemic coupling products are
formed if only an oxidizing agent is present (for examples of
other coupling modes, see Scheme 1A).
Comparison of crystal structures of enantiocomplementary
dirigent proteins (a (+)-pinoresinol-forming DIR from Pisum
sativum [PsDRR206]24 and a (−)-pinoresinol-forming DIR
from Arabidopsis thaliana [AtDIR6]25) suggests that the active
sites exhibit contrasting conformations, likely serving as
“molds” for the differential binding of two radical substrates
for the si-si or re-re coupling, respectively. The ability of
dirigent proteins to mediate a highly regio- and enantiose-
lective coupling reaction to generate (+)-pinoresinol raises the
intriguing possibility that these proteins could couple other
monolignols, including those not typically found in nature. To
date, (+)-pinoresinol-forming DIRs have exhibited high
substrate specificity toward CA;13 however, their ability to
guide heterocoupling reactions (beneficial for the desired
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etoposide analogue generation described here) has not been
explored. Stereoselectivity determined at this PhDIR-mediated
(+)-pinoresinol-forming step that funnels the supply of CA
into the EA pathway is crucial, because several of the
remaining pathway enzymes are known to be enantiospe-
cific.15,26 This proposed engineered route could yield valuable
non-natural and heterocoupled lignan analogues that would
enable specific modifications of the E ring of etoposide.
To determine the inherent substrate flexibility of DIRs and
expand the utility of this direct complexity-generating reaction,
we investigated whether DIRs could mediate stereoselective
heterocoupling of various analogues of the natural substrate,
coniferyl alcohol (Scheme 1B). Here, we show that PhDIR is
capable of promoting the coupling of synthetic and natural
monolignols to form non-natural heterocoupled lignans. The
resulting non-natural heterocoupling products retain the
necessary stereochemistry to be further processed by the rest
of the EA pathway enzymes for etoposide analogue biosyn-
thesis. We anticipate the complex lignan scaffolds we produced
will have direct utility in a biosynthetic route to etoposide
analogues that are less prone to E-ring metabolism and
formation of the reactive etoposide quinone (Scheme 1B,
highlighted in blue).
RESULTS AND DISCUSSION
■
CA Analogue Design and Preparation. In choosing
substrate designs to test for dirigent protein mediated coupling,
we considered previous structure−activity relationships that
had been established for the parent drug etoposide. For
example, it has been shown that replacement of both the 3′-
and 5′-methoxy groups by hydrogens in the E ring results in
only a moderately decreased topoisomerase inhibition, and
replacement of only the 5′-methoxy group by a hydrogen
results in comparable topoisomerase inhibition as the parent
compound.27,28 In contrast, methylation of the 4′-hydroxy
group resulted in the loss of drug activity, consistent with
recent evidence from the ternary crystal structure of
topoisomerase IIβ, DNA, and etoposide that suggests an
important favorable interaction between the functional group
and topoisomerase residue D479.5 These data, in addition to
the crystal structure of the DNA cleavage complex, suggested
that activity would not be compromised by replacement of the
E ring methoxy substituents with functional groups at the 3′
and 5′ positions anticipated to prevent the unwanted
metabolism. With this hypothesis in mind, analogues of 1
(3-11) were prepared, containing phenolic substituents with
varying degrees of electronegativity (Figure 1). Compounds 3
and 4 are naturally occurring monolignols produced by plants
that differ by the number of methoxy substituents ortho to the
phenol. Monomers 5, 6, and 7 were designed to explore
significantly altered electronic states of the aromatic ring,
relative to coniferyl alcohol, with the strongly electron-
withdrawing trifluoromethoxy, fluorine, and bromine groups
replacing the methoxy group, respectively. Additionally, the
bromine group of 7 could serve as a synthetic handle for
functionalization of the coupling product to diversify the
product profile post-coupling. Compounds 8 and 9 replace the
methoxy group with mildly electron-donating methyl groups.
Both 10 and 11 retain the alkyl aryl ethers at the 3′ position.
For 10, the electronic state of the phenolic ring is expected to
be altered by the presence of the 5′-fluorine substitution
compared to 1. Monomer 11 probes the effects of a bulkier
alkoxy group substitution.
Figure 1. Relative rates of monolignol oxidation and coupling product
formation catalyzed by TvLac. Each monomer is present as an
equimolar mixture with 1. 1, 3, and 4 are naturally occurring
monolignols; all others are synthetic. For substrate oxidation, data
points indicate the mean of peak integrations for the corresponding
monomer species (m/z of [M-H2O+H]+) detected by LC-MS; EIC
integrations are normalized to the peak integration at the initial
reaction time points per species. For coupling product formation, data
points indicate the mean of peak integrations for the corresponding
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electron-donating aromatic substituents, exhibited noticeably
slower oxidation rates in comparison to 1. Compound 8, with
one methyl substituent, showed slower oxidation compared to
1, while compound 9 with two methyl groups showed a more
closely matched oxidation trend as 1. More importantly, the
rates of TvLac oxidation of 10 and 11 are sufficiently matched
with that of 1, and both of these monomers would be
particularly useful for generating E-ring modified etoposide
variants. As anticipated, in the corresponding coupling
reactions, appreciable levels of the desired heterocoupling
products accumulate over the course of the assay (Figure 1,
coupling products [heterocoupling products in green]; see
Figure S1 for enumeration of all possible coupling products).
In contrast, slower-to-oxidize electron-deficient monolignols
(relative to 1) do not couple as efficiently with 1 to generate
heterocoupling products. Taken together, a trend emerges
where the relative rates of TvLac oxidation for the two
monomers present in a mixture determine the identity of the
major product formed. In this model, the collision of oxidized
monolignols dictates identity of the coupling product, a
process that is controlled by the relative concentration of these
monolignol radical species. These data confirm that efficient
formation of the desired heterocoupling products requires a
close match in monomer oxidation rates.
Expression and Extraction of Functional PhDIR. To
test if dirigent proteins could influence the regio- and
enantioselectivity of these heterocoupling reactions, we first
needed sufficient supply of PhDIR for in vitro assays. PhDIR
was expressed in N. benthamiana leaves using Agrobacterium-
mediated transient expression as described previously.17 While
DIRs have previously been successfully expressed in heterol-
ogous hosts including Pichia pastoris and plant cell
cultures,30−32 for plant leaf expression, the apoplastic fluid
wash method (APW) has been employed as a secretion and
extraction method for heterologous protein expression in N.
benthamiana, without the need for further processing of the
extracted proteins.25,33,34 Since many annotated dirigent
proteins, including PhDIR, are computationally predicted (by
Figure 1. continued
regioisomeric products detected by LC-MS (m/z of [M-H2O+H]+, a
common ion to all regioisomeric products a−c); EIC integrations are
normalized to the maximum product EIC integration observed over
the entire time course; relative ratios of products are unchanged from
the raw data. CA-CA (2) denotes the natural dimer, CA-X the
heterocoupling product, and X-X the non-CA dimer, where X denotes
the non-CA analogue. Error bars show standard deviations of data
from triplicate reactions. Consumption of coupling products (e.g., 2
past ∼5 min) is likely due to nonselective TvLac activity resulting in
oligomerization.
Oxidation Rates of Monolignol Analogues. Dirigent
proteins are thought to harness reactive monolignol radicals
and direct selective dimerization after an initial oxidation
event. In these transformations, several oxidizing reagents,
inorganic or enzymatic, have been used to catalyze the initial
oxidation of coniferyl alcohol.13,29 We anticipated that efficient
heterocoupling between 1 and a different monolignol
analogue, necessary for the E-ring modified etoposide
analogue, would only occur if the oxidation rates of each
were closely matched. To probe this, we first examined relative
rates of oxidation of our panel of monomers in vitro, using a
fungal laccase from Trametes versicolor (TvLac) that is known
to catalyze single electron oxidation of naturally occurring
monolignols (1, 3, and 4). Time-course of each substrate
oxidation in the presence of 1 was investigated using liquid
chromatography−mass spectrometry (LC-MS) to track
substrate consumption during the reaction. For naturally
occurring monolignols, 4 exhibited the fastest oxidation rate
with TvLac, followed by 1, and 3 with the slowest rate of
oxidation (Figure 1, mixed substrates). As previously
reported,29 the decreasing trend of oxidative rates with
decreasing number of methoxy substituents attested to the
importance of electron-donating nature of the methoxy group
for single-electron oxidation mechanism of laccases. In line
with these observations, compounds 5, 6, and 7, which lack the
Table 1. Reaction Yields of 8-5′ and 8-8′ Coupling Products and Enantiomeric Excess of (+)-Pinoresinol Analogues
a
b
c
b
c
d
e
monomers
coupling
CA-CA (2)
oxidant plant extract
8-5′, b(μM)
[%]
8-8′, c(μM)
[%]
8-8′ regioisomeric content (%)
% ee
1
TvLac
TvLac
TvLac
TvLac
TvLac
TvLac
TvLac
EV
PhDIR
EV
PhDIR
EV
PhDIR
EV
PhDIR
EV
PhDIR
EV
PhDIR
EV
250 47
57 10
86 11
[50]
[11]
[17]
[11]
[12]
[8]
122 26
446 29
[24]
[89]
[7]
[17]
[3]
33
89
28
60
19
21
15
94
2
88
2
91
0
31
1
94
−2
82
0
89
−1
47
1 + 8
CA-MCA (17)
MCA-MCA (26)
CA-FMCA (19)
FMCA-FMCA (28)
CA-ECA (20)
33
83
15
11
5
3
5
2
5
55
62 14
41
136 19
2
3
[2]
[5]
1 + 10
1 + 11
[27]
[2]
24
10
N/A
N/A
2
145
49
4
8
[29]
[10]
[26]
[<2]
[18]
[<2]
[4]
f
f
f
N/A
N/A
f
129
<10g
3
182 12
54 11
168 11
147 23
[36]
[11]
[34]
[29]
<5
62
<6
12
88 16
<10g
ECA-ECA (29)
PhDIR
20 10
a
b
Plant extracts obtained with APW from leaves expressing empty vector (EV) or PhDIR. Reaction yield was estimated from a standard curve using
EIC peak integration as analyzed by LC-MS on C18 chromatography. For 17 and 26, standard curves of 2b and 2c were used to estimate yields.
c
Error values are standard deviations of triplicates. 100% theoretical yield corresponds to 500 μM product generated from coupling of 500 μM each
d
substrate (1 mM total substrate concentration). 8-8′ regioisomeric content was calculated by the following formula: [8-8′, b]/([8-8′, b]+[8-5′,
e
c])*100%. ee: enantiomeric excess of (+)-pinoresinol or analogue over (−)-pinoresinol or analogue, calculated using the EIC peak integration
f
ratios on chiral chromatography, averaged from triplicate reactions. Not applicable; 8-5′ coupling product was not observed with the fluorine
g
substituent. Below the limit of linearity of 10 μM.
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SignalP) to contain N-terminal signal peptides that direct the
proteins to the apoplast, the extracellular space in plants,35,36
we applied a similar apoplastic extraction strategy to the N.
benthamiana leaves expressing PhDIR without adding an
extrinsic signal peptide and carried out activity assays of the
protein without further purification. To control for different
environmental factors such as salt concentration and presence
of native N. benthamiana laccases and peroxidases, the APW
protocol was performed in parallel using plants transformed
with an empty vector (EV), and the resulting extracts were
used as negative controls. To validate dirigent protein
expression, 1 was subjected to oxidation by TvLac in the
presence of plant extract from leaves expressing PhDIR. As
expected, on the basis of previously characterized activity of a
(+)-pinoresinol-forming DIR,13 the regioisomeric profile was
nearly inverted compared to that in the absence of PhDIR,
with the (+)-pinoresinol (2b) regioisomeric content increasing
from 33% (EV control) to 89% (PhDIR extract) (Figure S2a
and Table 1; reaction scheme shown in Scheme 1A).
Altered Regioselective Product Distributions Medi-
ated by PhDIR. With functional PhDIR in hand, we next
tested its capacity to direct the outcome of oxidative
heterocoupling of CA and each of the alternative monomers
shown in Figure 1. In our initial analysis of the reaction
outcomes, we observed that the addition of PhDIR to reactions
catalyzed by TvLac did not alter the ratio of all homo- and
heterocoupling products compared to TvLac alone (see Figure
2: unfilled bars in the graphs indicating all products from
homocoupling, e.g., 2 and 21-29, or heterocoupling, e.g., 12-
20). This observation is in accordance with previous literature
findings that dirigent proteins do not possess catalytic activity;
accordingly, the rate of formation of the three coupling
product types is dictated only by the TvLac oxidation step.
We next quantified the impact of the PhDIR addition on the
distribution of regioisomers formed for each coupling type.
This analysis revealed that the addition of PhDIR significantly
influences the regioselectivity of the heterocoupling reaction,
shifting the major product to the 8-8′ regioisomer for
heterocoupling of all monolignol analogues with 1 (Figures 2
and S2, filled, slash-patterned columns for products 12-20; and
Table 1). More specifically, the desired 8-8′ heterocoupling
product 19b comprised 94% of the quantified CA-FMCA
heterocoupling products 19b-c (6.2-fold increase compared to
the negative control, i.e., reactions with TvLac and EV extract,
at 15%). Similarly, in reactions with PhDIR, 20b constitutes
62% of the quantified CA-ECA heterocoupling products 20b-c
(∼12-fold increase from <5% in control reaction). In addition,
couplings of 1 with 3-9 each resulted in a similar trend of
regioselectivity increase in heterocoupling reaction (Figures
2c,d and S2b−f for products 12-18), revealing remarkable
flexibility in the ability of PhDIR to couple 1 to both natural
and non-natural monolignols. These data show promise that
the E ring modification of etoposide could be accomplished
through a biosynthetic route from production of (+)-pinor-
esinol analogues. Moreover, in the context of plant
metabolism, it is notable that 1, 3, and 4 are common and
abundant metabolites found throughout the plant kingdom.
Our in vitro results raise the possibility that naturally occurring
optically active heterocoupled lignans, e.g., (+)-demethoxypi-
noresinol (CA-pCA 8-8′ heterocoupling product, 12b) from
Gladiolus segetum37 and (+)-medioresinol (CA-SA 8-8′
heterocoupling product, 13b) from Eucommia ulmoides,38
originate with pinoresinol-like couplings catalyzed by an
Figure 2. End-point coupling product distributions in the presence
and absence ofPhDIR. Couplings of equimolar (a) 1 and 10, (b) 1
and 11, (c) 1 and 6, and (d) 1 and 8. Total content of the three
coupling product types for each reaction was estimated by summing
up peak integrations of the corresponding ion species (m/z of [M-
H2O+H]+) detected by LC-MS, and each coupling product
composition (type and coupling mode) was normalized to the total
integrations. Values reflect only relative product concentrations to
compare reactions. Colors indicate reactions carried out with empty
vector control (black, on the left) or PhDIR (blue, on the right)
extracts. Filled, slash-patterned columns indicate levels of 8-8′
coupling products. Bar heights indicate the mean of triplicates, and
error bars show standard deviations.
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Figure 3. PhDIR-mediated regio- and stereoselective heterocoupling of coniferyl alcohol and selected analogues. EICs as analyzed by LC-MS with
C18 and chiral chromatography show separation of regioisomers and enantiomers, respectively, of end-point TvLac reaction products in the
absence and presence of PhDIR extracts (black and blue lines, respectively) from an equimolar substrate mixture. Authentic standards, purchased
[(+)-2b] or purified from preparatory-scale reactions with and without PhDIR (all other standards), are shown in gray. (For 17 and 20, the c/c′
suffix denotes the 5-8′/8-5′ regioisomeric coupling products.) Control reactions were supplemented with an equal volume of plant extracts
transformed with an empty vector (EV). C18 traces are to scale, and chiral traces are normalized per reaction. Chromatographic peaks
corresponding to the (+)-pinoresinol analogues (structures shown above) are denoted with red dotted lines, and their regioisomers (C18) and
enantiomers (chiral) are denoted with black dotted lines. Relative stereochemistry assignments are supported by the identical 1H NMR spectra of
purified racemic 8-8′ coupling products isolated from the TvLac-only control reactions and enantioenriched 8-8′ coupling products from TvLac-
PhDIR reactions (Figure S11). Regioisomeric products are distinguishable based on MS/MS fragmentation pattern (Figure S12).
oxidase and mediated by dirigent proteins. However, the
biosynthetic origins of these molecules have not been
described to date.
coupling; one hypothesis is that the pocket preferentially binds
an initial CA monolignol but is tolerant of CA analogues as the
second monomer.
Unlike the high regioselectivity observed for PhDIR-
mediated heterocoupling reaction, homocoupling of CA
analogues was not as selective. Regioselectivity observed for
homocouplings of 10 and 11 only moderately increased
compared to the striking heterocoupling regioselectivity
increase (e.g., ∼2-fold increase for 29b vs ∼12-fold increase
for 20b). In the coupling reaction of 1 and 6, the addition of
PhDIR did not increase the apparent percent composition of 8-
8′ homocoupled product FCA-FCA, 24b (Figure S6). More
generally, we did not observe any increase in regioselectivity in
the formation of non-natural dimers derived from 3-9 (Figures
S3−S9). In line with these results, dirigent proteins have
previously been shown to not impact the oxidative
dimerization of 3 and 4.13 It is possible that the larger increase
in regioselectivity observed in heterocoupling reaction
compared to non-CA dimerization (when it occurs) can be
explained by substrate specificity in the binding pocket of
PhDIR. Although speculative, PhDIR binding pocket is
thought to take two units of monolignol radicals to facilitate
Asymmetric Heterocoupling Guided by PhDIR.
Encouraged by the increased regioselectivity observed in the
PhDIR-mediated heterocoupling of CA with all monolignol
analogues, we next investigated enantiomeric ratios of the
heterocoupled 8-8′ products formed in our assays. In the
dimerization of 1, the native substrate of PhDIR, the major
product was confirmed to be (+)-pinoresinol with an
enantiomeric excess (ee) >85% compared to <5% ee in
control experiments without PhDIR (Figure 3 and Table 1).
Notably, for the heterocoupling products formed at the highest
levels, e.g., 19b and 20b, we determined that the 8-8′
heterocoupling products formed selectively in the presence of
PhDIR were also produced in high enantioselectivity with 94%
ee and 89% ee, respectively. In addition, heterocoupling
products formed (albeit at lower levels) in the coupling
reactions of 1 with each of 3-9 also proceeded with high
enantioselectivity (71−93% ee) in the presence of PhDIR
(Table 2 and Figures S3−S9). These results reveal the
remarkable flexibility of dirigent proteins for guiding the
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we compared the activities of various oxidants: TvLac,
horseradish peroxidase (HRP), and ammonium persulfate
(APS). When we subjected an equimolar mixture of 1 and 6 to
a free coupling reaction in the presence of each of these
oxidants, we observed that the relative oxidation rate
differences observed between 1 and 6 were similar regardless
of the oxidant used (Figures 4a), where 6 was much slower to
oxidize compared to 1. We also examined the effect of varying
pH on the reaction. Because the pKa of the phenols vary
depending on the substituent identity, we hypothesized that
the pH of TvLac oxidation reactions could impact the relative
rate of monomer oxidations. While TvLac activity was
comparable at either pH 4 and the enzyme’s optimal pH of
5.5, relative oxidation rates did not differ at different pH
conditions (Figure S13).
Table 2. Selective Heterocoupling Mediated by PhDIR
As an alternative approach to improve heterocoupling
product yields, we tested whether superstoichiometric amounts
of 6 compared to 1 would result in higher accumulation of
heterocoupled products 15 (and homocoupling products 24)
(Figure 4b,c and S14). While CA homocoupling products (2)
remained as the major product, this strategy was effective for
increasing the formation of 15 from the coupling of 1 and 6.
Altered stoichiometry was even more beneficial in the coupling
of 1 and the methyl analogue 8 (Figure 4d) and enabled the
product isolation described below. However, these yield
improvements are maximal at a 1:3 ratio of 1 and 8; at higher
ratios, enantioselectivity begins to drop when DIR is included
in the reaction (Figure S15). Additionally, increasing the
amount of PhDIR in the assay improved regioselectivity while
the absolute yield of 17b remained constant.
Preparatory-Scale Biosynthesis of Synthetic Lignan
Analogues Using PhDIR Plant Extracts. Careful optimiza-
tion of in vitro TvLac-PhDIR reactions allowed isolation and
characterization of desired non-natural (+)-pinoresinol ana-
logues (Table 3). Time-courses of TvLac-EV and TvLac-
PhDIR reactions were taken to gain more insight into this
coupled enzyme-protein reaction prior to product isolation and
to avoid further apparent TvLac-catalyzed polymerization of
the desired coupling products (Figure S16). In assays
containing PhDIR, accumulation of the desired products was
highest at a point coinciding with full consumption of
monolignol substrates. These reaction conditions were
employed for preparatory-scale in vitro coupling reactions of
1 with 10 or 11. For coupling of 1 and 8, a molar ratio of 1:3
(8 in excess) was used, while the amount of PhDIR extract was
kept consistent as other coupling reactions. The 18% isolated
yield (29% reaction yield, Table 1) of the CA-FMCA 8-8′
heterocoupling product 19b (Table 3) exceeds the yields of
racemic ( )-pinoresinols (natural coupling product; at most
20%) obtained from coupling reactions promoted by either
laccase alone or a synthetic oxidant, as previously reported in
literature.35,39 This is an exciting proof of concept for the
robustness and flexibility of using plant chemistry for the direct
enantioselective synthesis of natural product analogues not
readily obtained through chemical methods.
a
b
See Table 1 footnote. See Table 1 footnote; reaction yield was
c
estimated using the standard curve of 2b. See Table 1 footnote.
enantioselective formation not only of C2 symmetric dimers
but also for 8-8′ heterocoupling products bearing four
contiguous stereocenters.
Relative to the 8-8′ heterocoupling products, we observed
lower regio- and enantioselectivity for the formation of the
corresponding non-natural 8-8′ homocoupling products in the
presence of PhDIR (Table 1 and Figure S10). Of these
products, the highest ee was obtained for 28b (82% ee),
suggesting that PhDIR is capable of accepting the monolignols
that closely resemble 1 in structure (e.g., 10). In line with this
hypothesis, monomer 11, bearing a bulky ethoxy substituent,
was converted to its 8-8′ dimer (29b) with only modest
selectivity in the presence of PhDIR (47% ee), and
homocoupling of 8, with a methyl group substituent, showed
only a modest increase in enantioselectivity in the presence of
PhDIR (31% ee for 26b); in these reactions, the 8-5′
regioisomer was still the major product. Moreover, in the
coupling of 1 with 3-7 and 9, no enantioselectivity increase was
observed for the homocouplings of the monolignol analogues
(Figures S3−S9), further suggesting that the promiscuity of
PhDIR is specific to the asymmetric heterocoupling mecha-
nism.
CONCLUSIONS
■
Optimization of Oxidation Reaction for Heterocou-
pling. For the formation of some of the desired pinoresinol
analogues (e.g., 15b and 17b), yield was limited by the slow
oxidation of the monolignol analogue relative to 1. To address
this, different reaction parameters were considered. In an effort
to better couple the oxidation rates of monolignol substrates,
Taken together, our results show that plant-derived dirigent
protein can be used to promote the regio- and stereoselective
coupling of non-natural monolignols and CA to make
functionalized variants of (+)-pinoresinol, a precursor to
clinically used chemotherapeutics. The transformation involves
coupling of achiral precursors and formation of three new
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Figure 4. Optimization of heterocoupling product formation. (a) Relative oxidation rates and product formation in a coupling reaction of 1 and 6,
with TvLac, HRP, and APS as oxidants. For data normalization, see Figure 1 caption. (b) Coupling product formation in a TvLac oxidation of 0.5
mM 1 and 0, 0.5, 1.5, or 4.5 mM 6 (1:0, 1:1, 1:3, and 1:9 ratios, respectively). Data points indicate the mean of EIC peak integration (m/z of [M-
H2O+H]+) normalized to the maximum product level per coupling product type. (c and d) End-point product distributions of (part c) 1 and 6 at a
molar ratio of 1:9 (500 μM and 4.5 mM, respectively) and (part d) 1 and 8 at a molar ratio of 1:3 (500 μM and 1.5 mM, respectively). See Figure 2
caption for data normalization.
Table 3. Isolated Yields of the (+)-Pinoresinol Analogues
a
b
product
isolated yield (mg)
PhDIR extract used (ml)
yield (%)
ee (%)
8-8′ CA-MCA, 17b
8-8′ CA-FMCA, 19b
8-8′ CA-ECA, 20b
8-8′ FMCA-FMCA, 28b
8-8′ ECA-ECA, 29b
0.2
1.2
0.7
1.2
0.5
3
3.5
4
3.5
2.9
4
18
9
19
9
73
93
93
82
61
a
100% theoretical yield corresponds to 500 μM product generated from 500 μM limiting monomer (1) concentration (1:1 molar ratio of 1 and 10
b
or 11, and 1:3 molar ratio of 1 and 8). ee: enantiomeric excess of (+)-pinoresinol analogue over (−)-pinoresinol analogue, calculated using the
EIC peak integration ratios on chiral chromatography.
bonds and four stereocenters in a single step. Importantly,
there is no analogous synthetic method for this direct coupling
and complexity-generating reaction. We anticipate that the
expanded substrate versatility of DIRs described here,
combined with future protein engineering and optimization
efforts to overcome the limitations of the initial oxidation step,
will enable the production of etoposide analogues with altered
substituents that still function as topoisomerase inhibitors but
are resistant to degradation by human liver cytochrome P450s.
While in vitro biochemical characterization of dirigent
proteins has begun to shed light on their role in selective
coupling of monolignols, our observation that heterocoupling
products can be formed suggests the potential for previously
unrealized activities of dirigent proteins in plants, including
those characterized to date that mediate (+)-pinoresinol or
(−)-pinoresinol-forming reactions.13,30,35,40,41 In addition, a
related protein from cotton, the (+)-gossypol-forming dirigent
protein, promotes stereoselective aryl−aryl bond forma-
tion.34,42 Notably, low concentrations of both (+)-gossypol
and gossypol-6-methyl ether (which can be viewed as a
heterocoupling product of hemigossypol and hemigossypol-6-
methyl ether) have been found in the native plants, suggesting
the possibility of other dirigent-protein-mediated heterocou-
pling reactions occurring in nature.43
The typical plant genome, e.g., Arabidopsis thaliana, contains
on the order of 20−50 putative DIR homologues, the vast
majority of which have not been characterized, and it is unclear
whether they are associated with natural product biosyn-
thesis.44,45 However, the diversity of known optically active
lignans found in plants suggests additional members of this
class of proteins could also promote alternative stereoselective
coupling reactions.46,47 In our study, we demonstrate an
expanded substrate versatility of DIRs promoting stereo-
selective heterocoupling of natural and non-natural monolignol
analogues. Furthermore, we showcase synthesis and isolation
of optically active coupling products derived from a broad
substrate scope using crude plant extracts containing functional
DIRs. These results show promise for combining conventional
synthetic chemistry and biosynthesis engineering techniques to
access synthetic analogues of complex natural products that
can be optimized for use in the clinic.
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were analyzed on the LC-MS on the positive ion mode at the same
time. Time points were recorded every 1.5−1.6 min (from the
timestamps of the data files), which accounts for the duration of data
collection as well as sample injection performed by the instrument.
In-Vitro LC-MS End-Point Analysis with C18 and Chiral
Chromatography. For assays involving PhDIR apoplastic plant
extract, the protocols by Gasper et al. were followed with
modifications.25 Enzyme assays were prepared by diluting the plant
extract of leaves expressing either PhDIR or EV (transformed with
agrobacteria at OD600 nm = 0.1) 5-fold in 0.1 M MES buffer containing
0.3 M NaCl and 0.333 mU/μL TvLac at pH 5.5. Substrate stock
solutions were prepared from 100 mM solutions in DMSO for 1 and
8-11 and in MeOH for 1 and 3-7. (In our hands, use of methanol
stock solutions resulted in increased production of coupling products
derived from 3-7 (CA-X and X-X) in comparison to that when
DMSO stock solutions were used.) Reactions were initiated by
addition of 1.875 mM equimolar substrate solutions in 1.875%
DMSO or MeOH for a final total substrate concentration of 1 mM at
30 °C. After 20 min for assays containing 4 and 40 min for the rest,
the coupling reactions were quenched by addition of an equal volume
of acetonitrile. The reactions were centrifuged at 15000 rpm for 10
min, and the supernatants were filtered for LC-MS anaylsis on the
positive ion mode.
EXPERIMENTAL SECTION
■
Transient Expression of P. hexandrum Genes by Agro-
Infiltration. Agrobacterium tumafaciens (GV3101, gentamicinR;
agrobacteria or agro) transformants with P. hexandrum genes in
pEAQ-HT (kanamycinR) vectors were prepared as described
previously.17 Agrobacteria (gentR and kanR) grown on LB plates
supplemented with gentamicin and kanamycin were resuspended in
LB media and centrifuged at 8000 x g for 5 min. Supernatant was
discarded, and the cell pellets were resuspended in 500 μL of 10 mM
MES buffer at pH 5.6 with 10 mM MgCl2 and 150 μM
acetosyringone for 1−2 h induction at room temperature. The
induced cell suspension was further diluted with induction media to
the desired inoculum level measured by optical density measurement
at 600 nm. The agrobacterial suspension was infiltrated on the
underside of the N. benthamiana leaves with a needleless 1-ml syringe.
Plants were 5−6 weeks old at the time of infiltration, grown under a
16-h light cycle. Biological replicates consisted of leaves from different
N. benthamiana plants from the same batch, unless otherwise noted.
Apoplastic Fluid Wash Protocol (APW). Plant extracts were
obtained from N. benthamiana leaves by APW as described
previously.25,48 Briefly, PhDIR-harboring or empty pEAQ-HT vector
was transiently expressed in N. benthamiana by agro-infiltration
(OD600 nm = 0.1−0.3) for 5 days. Leaves were vacuum-infiltrated with
0.1 M MES and 0.3 M NaCl at pH 5.5 through three cycles of pulling
vacuum to 150 Torr and slow release to atmospheric pressure. The
infiltrated leaves were layered on top of a piece of parafilm and
wrapped around a pipet tip and placed inside a syringe in place of a
plunger. The syringe assembly was placed in a conical tube and was
centrifuged at 1500 x g for 15 min at 4 °C. The extracts collected at
the bottom of the conical tubes were transferred to clean tubes and
centrifuged at 14000 x g for 10 min at 4 °C to clear undesired cellular
debris. The supernatants were ultrafiltrated (Amicon Ultra-0.5 mL 10-
kD) at 14000 x g for 10 min at 4 °C, and the retentate was aliquoted,
flash-frozen in liquid nitrogen, and stored at −80 °C until use at 5-fold
dilution.
Time-Course of TvLac Oxidation on LC-MS. Enzyme assays
contained a final concentration of 1 mM equimolar mixture (1%
MeOH in water) and 0.1 mU/μL TvLac in 1.5 mM MES buffer at pH
5.5. When TvLac was substituted with HRP or APS (Figure 4a), 5 pg/
μL HRP and 10 mM H2O2, or 1 mM APS was used. When the molar
ratio of the mixed substrates was varied (Figure 4b−d, S14 and S15),
the amount of the limiting monomer (often 1) was kept constant at
500 μM with the final total substrate concentration ranging 0.5−5
mM. (E.g., for a molar ratio of 1:3, the final concentrations of the
limiting and excess monomers were 500 μM and 1.5 mM,
respectively, with the final total substrate concentration of 2 mM.)
For the varied pH study (Figure S13), citrate buffer was used for pH 2
and 4, HEPES buffer at pH 7, and Tris buffer at pH 8.5. (Because
there is no chromatography in this analysis, to minimize salt
contamination in the mass spectrometer, it was important to use
organic buffer reagents and ammonium hydroxide or sulfuric acid for
adjusting pH.) For standards (Figure S17), bovine serum albumin was
supplemented instead of TvLac at 152 ng/μL. Reactions were
initiated by addition of 2 mM substrate stock solutions (equi-volume
dilution to a final concentration of 1 mM). Substrates were added to
the reaction at ∼1 min prior to the first injection of each sample. Real-
time reaction samples were analyzed on the LC-MS on the positive
ion mode through direct injection. Three samples were analyzed
within the same worklist at a time, and reaction replicates were
randomized over different worklists. Time points were recorded every
1.5−1.6 min, which accounts for the time of data collection as well as
sample injection performed by the instrument.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c13164.
■
sı
General methods and materials, synthetic procedures,
supplementary figures, supplementary table, NMR
spectra, and supplementary references (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Elizabeth S. Sattely − Department of Chemical Engineering
and Howard Hughes Medical Institute, Stanford University,
Stanford, California 94305, United States; orcid.org/
0000-0002-7352-859X; Email: sattely@stanford.edu
Author
Stacie S. Kim − Department of Chemical Engineering,
Stanford University, Stanford, California 94305, United
States; orcid.org/0000-0003-4755-0598
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c13164
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Warren Lau for help and guidance in
the initial development of this project; Mark Smith (Medicinal
Chemistry Knowledge Center, ChEM-H) for help with
designing medicinally relevant monolignol analogues; Russ Li
for preparation of p-coumaryl alcohol; Curt Fischer and Yuqin
Dai (Metabolomics Knowledge Center, ChEM-H) for helpful
discussions and 6545 qToF usage; Stephen Lynch (Stanford
NMR facility director) for consultation on NMR techniques;
Kevin Smith for help with NMR analysis; Alex Engel for critical
reading of the manuscript; and Kevin Smith, Ryan Nett,
Time-Course of In Vitro TvLac-EV vs TvLac-PhDIR Assays on
LC-MS. Enzyme assays contained a final concentration of 1 mM
equimolar substrate mixture, 1% MeOH, 0.333 mU/μL TvLac, and
20% v/v PhDIR or EV plant extract (transformed with agrobacteria at
OD600 nm = 0.3) in 20 mM MES buffer at pH 5.5. Reactions were
initiated by addition of substrate stock solutions at 1.875 mM
(1.875% DMSO in water). Substrates were added to the reaction at 1
min prior to the first injection of each sample. Reaction replicates
Ricardo De La Pena, and Bailey Schultz for helpful discussions
̃
and feedback. This work was supported by the National
Science Foundation Graduate Research Fellowship under
Grant No. DGE-1656518, NIH R01GM121527, and a AAAS
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ABBREVIATIONS
■
LC-MS, liquid chromatography−mass spectrometry; EIC,
extracted ion chromatogram; HPLC, high-performance liquid
chromatography; Lac, laccase; EV, empty vector (control);
DIR, dirigent protein; CYP, cytochrome P450
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