FgaPT2, a biocatalytic tool for alkyl-diversification of indole natural products

Article abstract of DOI:10.1039/c9md00177h

Aromatic prenyltransferases from natural product biosynthetic pathways display relaxed specificity for their aromatic substrates. While a growing body of evidence suggests aromatic prenyltransferases to be more tolerant towards their alkyl-donor substrates, most studies aimed at probing their donor-substrate specificity are limited to only a small set of alkyl pyrophosphate donors, restricting their broader utility as biocatalysts for synthetic applications. Here, we assess the donor substrate specificity of an l-tryptophan C4-prenyltransferase, also known as C4-dimethylallyltryptophan synthase, FgaPT2 from Aspergillus fumigatus, using an array of 34 synthetic unnatural alkyl-pyrophosphate analogues, and demonstrate FgaPT2 can catalyze the transfer of 25 of the 34 non-native alkyl groups from their corresponding synthetic alkyl-pyrophosphate analogues at N1, C3, C4 and C5 position of tryptophan in a normal and reverse manner. The kinetic studies and regio-chemical analysis of the alkyl-l-tryptophan products suggest that the alkyl-donor transfer by FgaPT2 is a function of the stability of the carbocation and the steric factors in the active site of the enzyme. Further, to demonstrate the biocatalytic utility of FgaPT2, this study also highlights the FgaPT2-catalyzed synthesis of a small set of alkyl-diversified indolocarbazole analogues. These results reveal FgaPT2 to be more tolerant to diverse non-native alkyl-donor substrates beyond their known acceptor substrate promiscuity and set the stage for its development as a novel biocatalytic tool for the differential alkylation of natural products for drug discovery and other synthetic applications.

Full text of DOI:10.1039/c9md00177h

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RESEARCH ARTICLE
FgaPT2, a biocatalytic tool for alkyl-diversification
of indole natural products†
Cite this: DOI: 10.1039/c9md00177h
Chandrasekhar Bandari,‡ Erin M. Scull,‡ Tejaswi Bavineni, Susan L. Nimmo,
*
Eric D. Gardner, Ryan C. Bensen, Anthony W. Burgett and Shanteri Singh
Aromatic prenyltransferases from natural product biosynthetic pathways display relaxed specificity for their
aromatic substrates. While a growing body of evidence suggests aromatic prenyltransferases to be more
tolerant towards their alkyl-donor substrates, most studies aimed at probing their donor-substrate specific-
ity are limited to only a small set of alkyl pyrophosphate donors, restricting their broader utility as bio-
catalysts for synthetic applications. Here, we assess the donor substrate specificity of an L-tryptophan C4-
prenyltransferase, also known as C4-dimethylallyltryptophan synthase, FgaPT2 from Aspergillus fumigatus,
using an array of 34 synthetic unnatural alkyl-pyrophosphate analogues, and demonstrate FgaPT2 can cat-
alyze the transfer of 25 of the 34 non-native alkyl groups from their corresponding synthetic alkyl-
pyrophosphate analogues at N1, C3, C4 and C5 position of tryptophan in a normal and reverse manner.
The kinetic studies and regio-chemical analysis of the alkyl-L-tryptophan products suggest that the alkyl-
donor transfer by FgaPT2 is a function of the stability of the carbocation and the steric factors in the active
site of the enzyme. Further, to demonstrate the biocatalytic utility of FgaPT2, this study also highlights the
FgaPT2-catalyzed synthesis of a small set of alkyl-diversified indolocarbazole analogues. These results re-
veal FgaPT2 to be more tolerant to diverse non-native alkyl-donor substrates beyond their known acceptor
substrate promiscuity and set the stage for its development as a novel biocatalytic tool for the differential
alkylation of natural products for drug discovery and other synthetic applications.
Received 25th March 2019,
Accepted 5th June 2019
DOI: 10.1039/c9md00177h
rsc.li/medchemcomm
great promise as useful biocatalysts for the production and di-
versification of bioactive NPs due to their broad substrate
Introduction
Prenylation is one of the important late-stage modifications
found in nature,1–5 which can enhance the lipophilicity of
compounds and alter the binding specificity of the biological
scope and remarkable regiospecificity.
Dimethylallyltryptophan synthases (DMATSs) are a sub-
class of aromatic PTs, responsible for the regioselective
prenylation of tryptophan moieties using dimethylallyl pyro-
phosphate (DMAPP) as the prenyl donor. DMATS are known
to have relaxed acceptor substrate specificities2,17 that use
DMAPP as the preferred alkyl donor. DMATS capable of selec-
tively alkylating all seven positions of the indole ring have
been biochemically characterized.2,17–19 Further, prenylation
can take place in a normal or reverse fashion, referring to C1′
or C3′ attachment of DMAPP, respectively.17,19 Currently, crys-
tal structures of several aromatic PTs are available in the Pro-
tein Data Bank (PDB), which have enabled a better under-
standing of substrate recognition and catalytic mechanisms.
All these structures have revealed aromatic PTs to have a
common structural motif known as the “ABBA PT” fold,
made up of a 10-stranded antiparallel β-barrel surrounded by
α-helices. This β-barrel contains a hydrophobic reaction
chamber capable of binding both donor and acceptor sub-
strates to carry out the prenyl transfer reaction.20–29
targets.6,7 Aromatic prenyltransferases are
a subset of
prenyltransferases (PTs) involved in the biosynthesis of natu-
ral products (NPs), which catalyze the transfer of a C5, C10, or
C15 isoprenoid chain from the corresponding prenyl pyro-
phosphate donors to an electron-rich aromatic acceptor such
as an indole or phenolic molecule. Prenylated NPs display a
diverse spectrum of biological activities, including anti-in-
flammatory,8,9 antibacterial,10–12 anticancer,13,14 and anti-
viral15,16 (Fig. 1). Due to the high structural complexity and
chemical reactivity of many NPs, purely synthetic methods for
analogous alkylation reactions are unavailable due to poor
regio- and chemo-specificity. Therefore, aromatic PTs show
Department of Chemistry and Biochemistry, University of Oklahoma, Stephenson
Life Sciences Research Center, 101 Stephenson Parkway, Norman, Oklahoma
73019, USA. E-mail: shanteri.singh@ou.edu
† Electronic supplementary information (ESI) available: These include HPLC
chromatograms, HRMS, NMR table, and NMR spectra for all new compounds.
See DOI: 10.1039/c9md00177h
FgaPT2 from Aspergillus fumigatus is a C4-DMATS that cat-
alyzes the normal prenylation of L-tryptophan (L-Trp) using
DMAPP. This prenylation is the first committed step in the
‡ These authors contributed equally.
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electron density at C3, support the possibility of initial re-
verse prenylation at C3, followed by a Cope-rearrangement to
produce the final C4 prenylated product of L-Trp.33 Later re-
ports using C4 substituted L-Trp substrates that resulted in
five different regio-isomers of dimethylallyl L-Trp (N1, C3, C5,
C6, and C7) strengthened the argument of direct prenylation
at C4 of L-Trp by FgaPT2.34
Several studies have revealed that FgaPT2 has a broad and
diverse acceptor substrate scope, including substituted tryp-
tophan, L-Trp containing cyclic dipeptides, isomers of
L-tyrosine, indolocarbazoles, and phenolic molecules.35–40
A
Fig. 1 Representative biologically active prenylated natural products
in which the prenyl moieties are derived from DMAPP (blue) or GPP
(red) through PT-catalyzed prenylation reaction.
few alternative alkyl donors have also been reported with
FgaPT2 including the C10 geranyl pyrophosphate (GPP),
2-butenyl pyrophosphate, 2-pentenyl pyrophosphate, and
benzyl pyrophosphate.41,42 These studies revealed that GPP
was poorly accepted by FgaPT2,30 while 2-butenyl pyrophos-
phate produced a mixture of C4 and C5 alkylated products,
and the bulkier 2-pentenyl and benzyl pyrophosphates pro-
duced C5 alkylated tryptophan products (Fig. 2B).41,42 Our re-
cent study of the tyrosine-O-prenyltransferase, SirD, demon-
strated its ability to accept 16 of the 21 diverse unnatural
allylic donors (1–21 in Fig. 3A).43 This prompted our investi-
gation of the alkyl-donor substrate scope of FgaPT2 to assess
its potential use as a biocatalytic tool for differential alkyl-
ation of indole-containing NPs.
Herein, we report the alkyl donor substrate scope of
FgaPT2 using our library of 34 synthetic allylic and benzylic
alkyl donors with varying carbon chain lengths and substitu-
ents (Fig. 3A). Our systematic evaluation of alkyl pyrophos-
phate (alkyl-PP) donors revealed FgaPT2 can utilize 25 out of
the 34 diverse alkyl-PP analogues tested. The subsequent
structural characterization of alkyl-diversified L-Trp analogues
revealed that FgaPT2 predominantly catalyzes the normal al-
kylation of C4 and C5, and less frequently, the normal or re-
verse prenylation of C3 and N1 of L-Trp. Furthermore, we
were able to demonstrate the potential utility of FgaPT2 as a
chemoenzymatic late stage diversification tool by producing a
library of alkylated indolocarbazole analogues. These ana-
logues were assayed for cytotoxic activity against the human
colon cancer cell line HCT-116.
biosynthetic pathway of the ergot alkaloid, fumigaclavine C
(Fig. 2A).30 The reaction proceeds through the generation of
an allylic carbocation via pyrophosphate dissociation,
followed by electrophilic aromatic substitution at C4 of L-Trp
to generate an arenium intermediate.31 The subsequent de-
protonation at C4 restores the aromaticity, generating
4-dimethylallyltryptophan (4-DMAT).31,32 The crystal structure
of FgaPT2 in complex with L-Trp and the unreactive substrate
analogue, dimethylallyl S-thiolopyrophosphate (DMASPP),
shows the Michaelis complex, where the plane of the di-
methylallyl group is stacked against the plane of the indole
ring, with C4 of L-Trp at a distance of 3.8 Å from C1′ of
DMASPP, and C3 of L-Trp at a distance of 3.5 Å from C3′ of
DMASPP.20 This structure also revealed that Lys174 is posi-
tioned properly to function as a base in the final re-
aromatization step.20 Subsequent mutational studies of
FgaPT2 revealed that the K174A mutant produced a C3 re-
verse prenylated product.33 This data, along with the high
Results and discussion
Synthesis of alkyl-PP analogues
In the present study, 34 unnatural alkyl-PP analogues (1–34
in Fig. 3A) were evaluated for their potential as alkyl donors
for the FgaPT2 catalyzed alkylation with L-Trp. These alkyl-PP
analogues include 21 previously reported allylic analogues
(1–21),43
a benzyl analogue, 22, and 12 new benzyl-
substituted alkyl-PP analogues (23–34). The syntheses of all
analogues were carried out using previously reported
methods.44–46 All synthetic alkyl-PP analogues were purified
and confirmed by high-resolution electrospray ionization
(ESI) mass spectroscopy (HRMS) (see ESI† Table S2) and 1H
and 31P NMR spectroscopy. Of the 34 alkyl-PP analogues, the
natural alkyl donor, dimethylallyl (1), 2-butenyl (7) and benzyl
Fig. 2 (A) Reaction catalyzed by FgaPT2 in the biosynthetic pathway
of fumigaclavine C.30 (B) Previously known FgaPT2 catalyzed alkylation
reaction using unnatural alkyl-pyrophosphate donors.41,42 The regio-
chemistry of the alkyl group transfer on L-tryptophan has been labelled
in red.
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in the linear direction of the designed alkyl-PP analogues was
kept <10, to be consistent with the ability of FgaPT2 cata-
lyzed reaction, which barely accepted a C10 GPP as a
substrate.30
In vitro activity assays with alkyl-PP analogues
In order to assess the ability of FgaPT2 to accept diverse un-
natural alkyl-PP analogues, the recombinant FgaPT2 was
overproduced in Escherichia coli BL21(DE3) cells transformed
with the codon-optimized synthetic FgaPT2 gene in a pET28a
vector. The resulting FgaPT2 with N-terminal His6-fusion pro-
tein was purified to homogeneity via Ni-NTA affinity chroma-
tography as described previously,28 yielding ∼40 mg L−1 pure
N-terminal His6-fusion protein. Standard uniform assay con-
ditions (1.2 mM alkyl-PP analogue, 1 mM L-Trp, 5 μM
FgaPT2, 25 mM Tris, 5 mM MgCl2, 50 mM KCl, pH 7.5, 16 h
at 35 °C) were adopted to facilitate the initial assessment of
the donor substrate specificity of FgaPT2 with synthetic alkyl-
PP analogues. Negative controls were included to ensure that
the product formation was enzymatic. Production of alkyl-L-
Trp derivatives was determined by the difference in the reten-
tion time of L-Trp and alkyl-L-Trp products using a reverse
phase high-pressure liquid chromatography (RP-HPLC) end-
point assay with subsequent confirmation by HRMS coupled
with liquid chromatography (LC) analysis for all positive reac-
tions (see ESI† Table S3) to afford complete analysis of the
desired product.
The initial assessment of FgaPT2 catalyzed alkylation of
L-Trp with 34 synthetic alkyl-PP analogues based upon RP-
HPLC of analytical scale reaction is illustrated in Fig. 3B,
wherein 25 of the 34 synthetic analogues (Fig. 3A) in FgaPT2
catalyzed reaction produced the corresponding alkyl-L-Trp. All
products were subsequently confirmed to be mono-alkylated
products from HRMS coupled with liquid chromatography
(LC) analysis (see ESI† Table S3). Of the 25 positive reactions,
12 led to appreciable (>50%) conversion, an additional seven
led to moderate (20–50%) conversion, while six offered de-
tectable product formation (<5%) under the conditions de-
scribed (Fig. 3B, see ESI,† Fig. S2). Analysis of HPLC and
HRMS results suggested more than one product for eight
(Fig. 3B, 4, 6–9, 17, 19, 20) of the 19 analytical scale reactions
with product yield >20% (Fig. 3B, 1–9, 17–20, 22, 24, 25,
27–29). Notably, the 3-carbon allyl pyrophosphate 10 was ac-
cepted as a substrate although with a very low conversion
rate of 1%, resulting in the formation of allyl-L-Trp. However,
11 with a methyl group substituted at the β-carbon of allyl py-
rophosphate was not accepted by FgaPT2. No product forma-
tion was observed for 12 that has a carbonyl group at
β-carbon. The bulky cinnamyl analogue (13) and its deriva-
tives (14–16, and 21) produced only a trace amount of the
alkylated product with a low yield (<2%). In addition, while
o-methoxy (24), m-methoxy (25) and p-halogen (27–29)
substituted benzyl analogues were accepted by FgaPT2, intro-
duction of bulky groups at the para-position, including
p-methoxy (23), p-methyl (26), p-nitro (30) and di- and tri-
Fig. 3 (A) Library of synthetic alkyl-PP analogues assessed in FgaPT2-
catalyzed reactions with L-Trp. (B) The percent conversion of alkyl-PP
analogues to the corresponding alkyl-L-Trp catalyzed by FgaPT2 based
upon RP-HPLC. The standard deviation for the total product conver-
sion was obtained from two experimental replicates (see ESI† Table S7
for the exact values used to generate this figure). Reactions were
performed with 1.2 mM alkyl-PP analogue, 1 mM L-Trp, 5 μM FgaPT2,
25 mM Tris, 5 mM MgCl2, and 50 mM KCl at pH 7.5 for 16 h at 35 °C.
No product formation was observed in the absence of FgaPT2 or the
alkyl-PP analogue. The blue, green, yellow, and black colors in the bar
diagram denote C4, C5, C3, and N1 alkyl-L-Trp regio-isomers as con-
firmed by NMR, whereas the red color represents the alkyl-L-Trp iso-
mer with an undetermined regio-chemistry.
(22) were previously reported to be substrates for FgaPT2 cat-
alyzed reaction with L-Trp (Fig. 2B)41,42 and were included
here for comparison purposes. Several factors were consid-
ered while designing these alkyl-PP analogues (Fig. 3A) in-
cluding the effect of the carbon chain length (10 < 7 < 1 < 2
< 3 < 6 < 13) to explore the steric factors in the active site
and the electronic nature of the substituents (1, versus 8 and
9) to understand their potential utility in transferring diverse
functional groups. We also included conjugated diene ana-
logues (17–21) to grasp the contribution of carbocation reso-
nance stabilization, in addition to introducing methyl groups
at different positions (7, 1, 2, 10, 11, 18–20) to explore the
methyl group effect. The benzylic pyrophosphate analogues
(22–34) were tested to explore the inductive and resonance
contribution of substituents. The length of the carbon chain
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substituted benzyl analogues (31–34), did not serve as sub-
strates for FgaPT2 catalyzed reaction with L-Trp due to the po-
tential steric hindrance in the active site of the enzyme.
These results suggest the FgaPT2 catalyzed reaction with
L-Trp is sterically limited by bulky substitution on the alkyl-
donor.
22 > 4; 22 > 27 > 28 > 29. Furthermore, a 9-fold higher kcat
value of 22 compared to 4 suggests that the resonance stabili-
zation of the carbocation by the benzylic group contributes to
the rate of the reaction. However, the kcat values of the cyclo-
hexyl (4) and benzyl analogues (22) were nearly 310- and 36-
fold lower than the kcat value of 1. When an electron with-
drawing group such as chloro (8) or bromo (9) replaced one
of the methyl groups of 1, the efficiency of the enzyme was
lowered nearly 150–250-fold compared to the natural alkyl
donor, 1, which explains the importance of the methyl group
in stabilizing the carbocation. Surprisingly, the dienyl ana-
logues (17–20) displayed relatively higher rates with just ∼2-
fold lower kcat values compared to the natural alkyl donor, 1,
implying that the extended resonance stabilization of the
carbocation favors the FgaPT2 catalyzed reaction. However,
the KM values of the dienyl analogues were 5–10-fold higher
than the KM value of the natural alkyl donor, 1 and the trend
of KM values correlated with the increasing length of the car-
bon chain (C5–C7), in the order 17 < 19 < 20.
In order to understand the structural basis for the alkyl
group specificity, we analyzed the alkyl-binding site within
the crystal structure of FgaPT2 (PDB: 3I4X),20 where the pyro-
phosphate moiety of DMASPP is anchored in the active site
by hydrogen bonding interaction between the oxygen atom of
phosphate groups and the amine side chains of arginine and
lysine residues (Arg100, Arg257, Arg404, Lys187 and Lys259)
(Fig. 4). The alkyl group is encaged within the hydrophobic
cavity formed by the side chains of Tyr189, Tyr191, Leu245,
Tyr261, Met328, Tyr345, Tyr398, and Tyr413 residues (Fig. 4).
The carbocation formed during the reaction is stabilized via
π-interactions with 2 aromatic rings: substrate tryptophan on
one side and Tyr345 on the other side.20 A closer analysis of
the active site revealed that the the methyl groups of DMASPP
are within 3.6 Å and 4.4 Å from the side chain of Met328 and
Kinetic studies and structural analysis
Michaelis–Menten kinetic parameters for all 19 alkyl-PP ana-
logues with product conversion >20% (Table 1) and for L-Trp
with representative alkyl-PP analogues (see ESI† Table S1)
were studied. The kinetic parameters, rates (kcat
) and
Michaelis constants (KM) were determined from a nonlinear
regression fit of initial velocities versus the concentration of
the alkyl-PP analogue by GraphPad Prism 5.04. Interestingly,
the KM value of L-Trp for FgaPT2 with various alkyl-PP donors
did not change drastically with the nature of the alkyl donors
and remained within the range of 0.20 0.08 mM (see ESI†
Table S1). Consistent with our previous observations with
tyrosine-O-prenyltransferase SirD,43 the alkyl-PP analogues
that contained groups to enhance the stability of the
carbocation via increased conjugation and resonance stabili-
zation correlated with the increased turnover of the product.
For example, the kcat values of the reaction correlated with
conjugation by a methyl group in the order 1 > 2 > 3 > 7
(Table 1). In addition, the KM values increased with the in-
creased length of the carbon chain in the order 1 < 2 ≅ 3 ≅
5 < 6 and 17 < 18 ≅ 19 ≅ 20. Removing one methyl group of
1 decreased the efficiency (kcat/KM) of 7 88-fold compared to
1. In addition, changing one or both methyl groups of 1 to
an ethyl group (2 and 3) lowered the enzyme efficiency 17–20
times compared to 1. Similarly, the kcat values of the reaction
correlated with resonance stabilization in the order 17 > 7;
Table 1 Kinetic parameters for FgaPT2 with 1.2 mM L-Trp and varied concentrations of alkyl-PP analogues in 25 mM Tris, 5 mM MgCl2, and 50 mM
KCl, at pH 7.5 and 35 °C
Alkyl-PP analogue
Kcat (min−1
)
KM (mM)
kcat/KM (mM−1 min−1
)
Relative catalytic efficiencya
1
2
3
4
5
6
7
8
50
9.9 0.4
6.5 0.1
0.160 0.006
5.04 0.18
3.2 0.4
0.57 0.02
1.3 0.1
0.47 0.01
26
28
20.2 0.9
16.9
1.36 0.02
1.93 0.05
1
0.14 0.01
0.48 0.06
0.37 0.02
0.16 0.02
0.31 0.03
1.4 0.2
0.14 0.01
0.53 0.08
0.35 0.03
0.66 0.06
1.1 0.1
349 28
100
21
17
3
1
5.88
5.04
0.28
4.48
0.64
1.14
0.70
0.38
10.92
7.14
4.34
3.39
1.74
0.95
0.064
0.19
0.13
0.12
1.0 0.1
16
2
2.2 0.4
3.9 0.4
2.5 0.4
1.3 0.1
9
17
18
19
20
22
24
25
27
28
29
1
1
39
26
16
12
4
3
2
2
1.3 0.1
2
1.39 0.2
0.22 0.01
0.57 0.04
0.73 0.06
0.4 0.02
0.43 0.03
0.32 0.03
6.2 0.3
3.4 0.2
0.23 0.02
0.69 0.04
0.46 0.03
0.42 0.04
0.170 0.005
0.276 0.004
0.196 0.004
0.134 0.003
a
Relative catalytic efficiency is defined as the percent kcat/KM when compared to 1.
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Despite the fact that the present analysis suggests the ste-
ric factor to be a major contributing aspect to the low or no
turn-over of alkyl donors containing long carbon chains or
bulky aromatic groups, a more definite answer requires in-
depth mutational studies or 3D structural studies of FgaPT2
in the presence of diverse unnatural analogues. Remarkably,
all amino acids involved in the shielding of the dimethylallyl
carbocation are conserved among the DMATS enzymes.
Therefore, the conclusions for the alkyl-PP substrate specific-
ity drawn from the present study of FgaPT2 are likely to be
valid for other DMATS orthologs as well. Overall, while the
active site of wild-type FgaPT2 is optimized for the efficient
transfer of the dimethylallyl group, future structure-based en-
gineering is expected to produce enzyme variants with en-
hanced efficiency and altered substrate scope.
Fig. 4 The active site of the FgaPT2 structure (PDB: 3I4X)20 displaying
distances between DMASPP and the active site residues, where
pyrophosphate interactions and alkyl group distances within the active
site are colored cyan and yellow, respectively.
Regio-chemistry of the alkyl-tryptophan products
Tyr398, respectively (Fig. 4). Therefore, considering the C–C
bond length is about ∼1.5 Å, any additional increase in size
of the alkyl group beyond the length of DMAPP will have po-
tential steric clash with Met328 and/or Tyr398. This explains
the higher KM values seen in the case of alkyl-PP analogues
with a longer carbon chain, especially the dienyl analogues
(17, 18, 19, and 20) which have a good catalytic ability and in-
creased KM values. Similarly, the C2 and C3 of DMASPP are
in the range of 3.7–4.5 Å from the side chain of Tyr345 and
Tyr261 (Fig. 4). Therefore, the C2-methyl substitution in 19
and 20 will have additional steric constraints from the side
chains of Tyr345 and Tyr261 and potentially explains the
lowered catalytic efficiency.
To assess the regio-specificity of FgaPT2 catalyzed alkyl trans-
fer, the 19 analytical scale reactions with an alkyl-tryptophan
yield >20% (reaction involving 1–9, 17–20, 22, 24, 25, 27–29,
Fig. 3B) were scaled up. Subsequently, all alkyl-L-Trp ana-
logues (35–55 in Fig. 5) were purified via RP-HPLC, and 1H
and 2D NMR experiments were carried out in deuterated
DMSO or methanol. While eight of the 19 alkyl-transfer reac-
tions involving alkyl donors 4, 6–9, 17, 19 and 20 yielded two
or more mono-alkylated products based on RP-HPLC and
HRMS analysis, regio-specific alkyl-group attachment of
Among the benzylic analogues, the mono-substituted ana-
logues (24, 25, 27, 28, and 29) displayed similar KM values,
which were 2–3-fold higher than the KM value of the benzyl an-
alogue, 22. The 10-fold higher kcat value of o-methoxy (24) com-
pared to m-methoxy (25) suggests that benzylic cation stabiliza-
tion by an o-methoxy group increases the efficiency of the
FgaPT2 catalyzed alkylation reaction. Among the para-
substituted halogens, the kcat values followed the order benzyl
(22) > p-fluorobenzyl (27) > p-chloro (28) ≅ p-bromo (29),
suggesting a combination of steric factors and possible stabili-
zation of the carbocation of the 27 carbocation via back dona-
tion of the non-bonded electron pairs of the fluorine atom to
the 27 carbocation through effective 2p–2p orbital interaction.
The observed kcat values of para-substituted halogens were ∼5–
10-fold lower than the kcat value of 22, which is consistent with
the inductive effect of the halogens. Surprisingly, while the
para-substituted halogens were accepted by FgaPT2, p-methoxy
and p-methyl were not substrates for FgaPT2 catalyzed reaction
even with their ability to contribute to the stabilization of the
benzylic cation, likely due to the steric factors in the active site
of the enzyme. These results emphasize the fact that the
FgaPT2 catalyzed Friedel–Craft alkylation reaction proceeds via
a stabilized carbocation,41 and the steric factor and the orienta-
tion of the alkyl group in the active site have a major role in
the ability of FgaPT2 to utilize alkyl donors.
Fig.
5 NMR characterized structures of isolated alkyl-tryptophan
products obtained from FgaPT2 reaction with L-Trp and alkyl-PP ana-
logues. The red colored bonds represent the COSY correlation be-
tween the protons across the bond and arrows represent HMBC corre-
lations of representative compounds, respectively (see the ESI† for
NMR Table S5). * represents known compounds.
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minor products of reactions involving 6, 8, 9, 17, 19, and 20
could not be attained due to insufficient intensity of NMR
signals. The regiospecificity of 21 alkyl-tryptophan analogues
1
was determined using H and 2D NMR analysis. The detailed
NMR analysis of 21 alkyl-tryptophan analogues revealed that
FgaPT2 is able to catalyze alkyl transfer at four different posi-
tions (N1, C3, C4, and C5) of tryptophan using unnatural al-
kyl donors, resulting in normal alkylation at C4 and C5 and
normal or reverseC5′ alkylation at C3 and N1 of tryptophan
(Fig. 5). Among the 21 alkyl-tryptophan analogues character-
ized by NMR (see ESI† Table S5), the alkyl group of seven
were at C4 (derived from 1–5, 7, and 24), 11 at C5 (derived
from 4, 6–9, 18, 22, 25, 27–29), one at C3 (derived from 17)
forming hexahydro-pyrroloindoline product, and two were at
the N1 (derived from 19 and 20) position of tryptophan, re-
spectively (see ESI† Fig. S1 and S4).
Fig.
6
The active site of the FgaPT2 structure (PDB: 3I4X)20 in
complex with L-Trp and DMASPP displaying distances between C1′ of
DMASPP and C4 and C5 of L-Trp, and C3′ of DMASPP and C3 of L-Trp.
Intriguingly, apart from the strict C4 alkylation of trypto-
phan by FgaPT2 using the natural alkyl donor 1, the unnatu-
ral alkyl donors, 2, 3 and 5, which contained an ethyl group
(2 and 3) in place of one or both methyl group/s of 1, or the
similarly sized conformationally restricted cyclopentyl ana-
logue (5) selectively added the alkyl group at the C4 position
of tryptophan. Alkyl-PP analogues (4 and 7) with a smaller al-
kyl chain in the linear direction compared to 1 resulted in a
mixture of C4 and C5 regio-isomers, and alkyl-PP analogues
with a methyl group of 1 replaced with a halogen (8 and 9) or
a larger alkyl chain (6 and 18) predominantly produced the
C5 alkylated product. While most of the unnatural benzylic
analogues (22, 25, 27, 28, and 29) produced selectively C5 al-
kylation of tryptophan, the o-methoxy benzyl analogue (24)
produced a single C4-alkylated tryptophan. Careful analysis
of the C4 alkylating analogues (1, 2, 3, 5, and 24) suggests
that C4 substitution is preferred when the carbon chain
length is less than 6 while C3′ is also disubstituted to facili-
tate stacking interaction with the indole ring. Therefore, the
active site steric factors in combination with improper stack-
ing interaction of the alkyl group with the indole ring con-
ceivably displaces the carbocation resulting in direct addition
of the alkyl group to C5 of tryptophan, as seen for 11 of the
C5-alkyl-Trp products. The addition of alkyl groups at the C5
position of tryptophan by most of the alkyl-PP analogues is
consistent with the orientation of DMASPP with respect to
the indole ring in the active site of the FgaPT2 crystal struc-
ture,20 where C5 of tryptophan is just 3.5 Å from the C1′ of
the alkyl group compared to a distance of 3.8 Å between C4
of tryptophan and the C1′ of the alkyl group (Fig. 6).
rearrangement. Importantly, due to the conjugated double
bonds in analogues 17, 19 and 20, there are three possible reso-
nance structures for the carbocation (Fig. 7, A-, B- and C-form),
with the divinyl B-form being the most stabilized resonance
form. As seen from the Michaelis complex within the crystal
structure of FgaPT2, the C3 of tryptophan is just 3.5 Å from C3′
of DMASPP (Fig. 6), and thus, it is likely that the stable ‘B-form’
Among the dienyl analogues, except for 18, which alkylated
the C5 position due to possible steric hindrance in the active
site, the rest of the dienyl analogues (17, 19, and 20) produced
C3, N1-normal, and N1-reverseC5′ alkylation. While in all three
cases alkylation can be explained as direct addition of the alkyl
group to the respective position (via carbocations A- or C-form
for 17 (Fig. 7A), A-form for 19 (Fig. 7B) and C-form for 20
(Fig. 7C), respectively), an alternative explanation is that these
products could have arisen from the attachment of C3′ of the al-
kyl group (B-form in Fig. 7) at C3 of tryptophan followed by a
Fig. 7 Proposed mechanism for FgaPT2 catalyzed alkyl-transfer reac-
tion of tryptophan with (A) 17, (B) 19 and (C) 20.
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as the representative candidate, which was previously demon-
strated to be taken by FgaPT2 with the native alkyl-donor, 1.38
The synthesis of 56 was carried out using previously reported
methods.47 Standard assay conditions (1.2 mM alkyl-PP ana-
logue, 1 mM 7-hydroxy-K252c, 5 μM FgaPT2, 25 mM Tris, 5
mM MgCl2, 50 mM KCl, pH 7.5, 16 h at 35 °C) were adopted
to facilitate the assessment of the donor substrate specificity
of FgaPT2 using synthetic alkyl-PP analogues (Fig. 3A) with
56. From this pilot study, six of the 34 synthetic alkyl-PP ana-
logues led to the production of the corresponding alkylated
indolocarbazoles in moderate yields (≥15%, Fig. 8 and ESI†
Fig. S3). The product yield decreased with the size of the alkyl
chain, in the order 1 > 2 > 3 and 5 > 6, respectively. Surpris-
ingly, the dienyl analogues with comparable carbon chain
lengths as alkenyl analogues did not serve as substrates for
FgaPT2 catalyzed reaction with 56. Subsequently, all six ana-
lytical scale reactions were scaled-up and purified via RP-
HPLC for the investigation of regio-chemical alkyl substitu-
tion on 56 (Fig. 8, ESI† Tables S4 and S6). The HRMS analysis
of FgaPT2 catalyzed reaction with 56 using 1, 2, 3, 4, 5 or 6 as
alkyl donors provided the mass corresponding to the mono-
alkylated product of 56 (see ESI† Table S4). Based on the de-
Fig.
8
FgaPT2-catalyzed
alkyl-diversification
of
7-hydroxy-
indolocarbazole (56). NMR characterized structures of pure isolated
alkyl-7-hydroxy-indolocarbazole products (57–62) of the enzymatic re-
action of 56 with alkyl-PP analogues. The percent conversion in the
standard analytical scale reaction for each compound is enclosed in
parentheses (see the ESI† for NMR, Table S6).
tailed analysis of H, COSY, H-13C HSQC, and H-13C HMBC
spectra, all six compounds confirmed the C3-normal alkyl-
ation of 56 (see ESI† Table S6). Thus, FgaPT2 catalyzes regio-
specific C3-alkylation of 56 in the presence of natural and un-
natural alkyl-donors. The C3 position of 56 structurally corre-
sponds to the C5 position of tryptophan. These results, along
with the C5-alkylation of L-Trp using most of the unnatural
alkyl-PP donors, imply that the preferred position for FgaPT2
catalyzed alkyl transfer is the C5 position of the indole ring.
Finally, the cytotoxic activity of compounds 56–62 was
studied in human colon carcinoma (HCT-116) cells. While
the C3-alkyl substituted analogues of 56 displayed 3–6-fold
diminished cytotoxicity in HCT116 (Table 2), generation of
these analogues demonstrated the biocatalytic utility of
FgaPT2 in differential alkylation of indole-containing natural
products. Notably, this is the first report on DMATS-catalyzed
differential alkylation of a natural product, and it clearly sets
the stage for the utility of FgaPT2 for differential alkylation of
other natural products.
1
1
1
adds at the reactive C3 position of tryptophan due to the
carbocation stability and its proximity to C3 of tryptophan. Sub-
sequently, the C3 reverse alkylated iminium intermediate un-
dergoes a [1,3] alkyl shift in the presence of 17 to yield a normal
or reverseC5′ C3 alkylation product of 17 (Fig. 7A), both of which
are structurally indistinguishable. Similarly, the C3 reverse
alkylated iminium intermediate undergoes an aza-Cope
rearrangement in the presence of 19 and 20 to yield an N1 nor-
mal and N1 reverseC5′ alkylation product with 19 and 20, respec-
tively (Fig. 7B and C). Especially in the case of 19 and 20, the di-
rect addition to N1 of the indole ring (using A-form for 19 and
C-form for 20) seems unlikely as the indole nitrogen is very
poorly nucleophilic when compared to the C3 position. Thus,
these product studies involving C4, C5, C3, and N1 alkyl-
tryptophan analogues suggest that the the regioselectivity of tryp-
tophan alkylation by FgaPT2 depends primarily on the nature of
the alkyl donor. Engineering studies of FgaPT2 along with struc-
tural characterization using a combination of acceptor and non-
native donor analogues are underway to understand the molecu-
lar description responsible for FgaPT2 regioselectivity.
Conclusions
In conclusion, this work has revealed that FgaPT2 is a highly
permissive enzyme and accepts diverse allylic and benzylic
unnatural alkyl-donor substrates. While FgaPT2 can transfer
alkyl groups at C4, C5, N1 and C3 positions of L-Trp, the C5
position is the preferred alkylation position for FgaPT2 with
Alkyl-diversification of indolocarbazole, 7-hydroxy-K252c (56)
To subsequently assess the practical utility of FgaPT2 for
alkyl-diversification of NPs, indolocarbazole 56 was selected
Table 2 Table of anticancer activity of 56–62 against HCT-116 cells. The averaged IC50 values show the standard deviation for the indolocarbazole
compounds from two experimental replicates
Compound number
56
57
58
59
60
61
62
IC50 (μM)
11
1
62 12
38
1
35
1
61 23
58
6
31
1
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1
unnatural alkyl donors. Further, our studies involving the
synthesis of differently alkylated indolocarbazole highlight
the utility of FgaPT2 for broader applications such as alkyl-
diversification of indole-containing natural products for drug
discovery and other synthetic applications. Future enzyme
mutational studies along with substrate engineering is
expected to drive the development of more efficient and se-
lective FgaPT2-based biocatalysts.
NH4OH : H2O 7 : 2 : 1 v/v): Rf = 0.45; H NMR (400 MHz, deute-
rium oxide) δ 5.99 (td, J = 6.2, 1.4 Hz, 1H), 4.53–4.40 (m, 2H),
2.29 (q, J = 1.3 Hz, 3H). 31P NMR (162 MHz, deuterium oxide)
δ −7.28 (d, J = 21.9 Hz), −10.61 (d, J = 22.0 Hz). HR-ESI-MS
(m/z): [M − H]− calcd for C4H8BrO7P2−, 308.8934, observed,
308.8932.
Benzyl pyrophosphate (22)
22 was obtained as a white solid (78%) from benzylbromide
using TBAPP. TLC (iPrOH : NH4OH : H2O 7 : 2 : 1 v/v): Rf = 0.4;
1H NMR (400 MHz, deuterium oxide) δ 7.42–7.36 (m, 2H),
7.35–7.23 (m, 3H), 4.88 (d, J = 6.6 Hz, 2H); 31P NMR (162
MHz, deuterium oxide) δ −8.10 (d, J = 21.5 Hz), −10.80 (d, J =
21.5 Hz); HR-ESI-MS (m/z): [M − H]− calcd for C7H9O7P2−,
266.9824; observed, 266.9828.
Experimental
General materials
Unless otherwise stated, all chemicals and reagents were pur-
chased from Sigma-Aldrich (St. Louis, MO, USA), Acros (New
Jersey, USA), Alfa-Aesar (Ward Hill, MA, USA) or TCI
(Portland, OR, USA) and were of reagent grade or better. The
PD-10 column and Ni-NTA superflow columns were pur-
chased from GE Healthcare (Piscataway, NJ). The HCT-116
cell line was purchased from ATTC (ATCC® CCL-247™).
p-Methoxybenzyl pyrophosphate (23)
23 was obtained as a white solid (82%) from p-methoxybenzyl
bromide using TBAPP. TLC (iPrOH : NH4OH : H2O 7 : 2 : 1 v/v):
General methods
1
Rf = 0.39; H NMR (400 MHz, deuterium oxide) δ 7.31 (d, J =
All reactions were conducted in oven- or flame-dried glass-
ware under a nitrogen atmosphere with anhydrous solvents,
unless otherwise noted. Reactions were monitored by thin-
layer chromatography (TLC) (EMD Millipore Corp, Billerica,
MA, USA) and visualization was accomplished with UV light
(254 nm) followed by staining with phosphomolybdic acid so-
lution or anisaldehyde solution and heating. Flash column
chromatography was performed using ACS grade solvents
and silica gel (SiliCycle Inc, P60, particle size 40–63 μm).
HPLC was conducted using an Agilent 1220 system equipped
with a DAD detector.
8.5 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 4.78 (d, J = 6.3 Hz, 2H),
3.70 (s, 3H); 31P NMR (162 MHz, deuterium oxide) δ −8.54 (d,
J = 21.1 Hz), −10.89 (d, J = 21.1 Hz); HR-ESI-MS (m/z): [M −
H]− calcd for C8H11O8P2−, 296.9929; observed, 296.9939.
o-Methoxybenzyl pyrophosphate (24)
24 was obtained as a white solid (32%) from o-methoxybenzyl
alcohol using TEAP. TLC (iPrOH: NH4OH : H2O 7 : 2 : 1 v/v): Rf
= 0.39; 1H NMR (400 MHz, deuterium oxide) δ 7.32 (dd, J =
7.5, 1.7 Hz, 1H), 7.21 (td, J = 7.9, 1.7 Hz, 1H), 6.95–6.74 (m,
2H), 4.83 (d, J = 6.8 Hz, 2H), 3.69 (s, 3H); 31P NMR (162 MHz,
deuterium oxide) δ −8.45 (d, J = 21.4 Hz), −10.76 (d, J = 21.2
General procedure of synthesis of alkyl-PP analogues
Hz); HR-ESI-MS (m/z): [M
296.9929; observed, 296.9927.
−
H]− calcd for C8H11O8P2−,
The procedures for the synthesis of alkyl-PP analogues 1–4,
6–8, and 10–21 and phosphorylation using bis-
IJtriethylammonium)
phosphate
(TEAP)
and
tris-
m-Methoxybenzyl pyrophosphate (25)
IJtetrabutylammonium) pyrophosphate (TBAPP) have been
reported previously.43
25 was obtained as
a
white solid (30%) from
m-methoxybenzyl using TEAP. TLC (iPrOH : NH4OH : H2O 7 : 2 :
1
2-Cyclopentylideneethyl pyrophosphate (5)
1 v/v): Rf = 0.39; H NMR (400 MHz, deuterium oxide) δ 7.25
(t, J = 8.0 Hz, 1H), 6.99 (d, J = 2.6 Hz, 2H), 6.84 (dd, J = 8.2,
2.5 Hz, 1H), 4.85 (d, J = 6.6 Hz, 2H); 31P NMR (162 MHz, deu-
terium oxide) δ −7.37 (d, J = 22.5 Hz), −10.75 (d, J = 21.7 Hz);
HR-ESI-MS (m/z): [M − H]− calcd for C8H11O8P2−, 296.9929; ob-
served, 296.9926.
The title product was obtained as an ivory solid (33%) from
2-cyclopentylideneethan-1-ol48 using TEAP. TLC (iPrOH :
NH4OH : H2O 7 : 2 : 1 v/v): Rf = 0.35; H NMR (400 MHz, deute-
rium oxide) δ 5.56 (tt, J = 7.3, 2.4 Hz, 1H), 4.45 (t, J = 7.0 Hz,
2H), 2.40–2.20 (m, 4H), 1.65 (dq, J = 20.8, 6.7 Hz, 4H). 31P
NMR (162 MHz, deuterium oxide) δ −7.73 (d, J = 22.0 Hz),
−10.51 (d, J = 21.9 Hz). HR-ESI-MS (m/z): [M − H]− calcd for
C7H13O7P2−, 271.0142; observed, 271.0148.
1
p-Methylbenzyl pyrophosphate (26)
26 was obtained as a white solid (32%) from p-methylbenzyl
alcohol using TEAP. TLC (iPrOH: NH4OH : H2O 7 : 2 : 1 v/v): Rf
(Z)-3-Bromobut-2-en-1-yl pyrophosphate (9)
1
= 0.39; H NMR (400 MHz, deuterium oxide) δ 7.22 (d, J = 7.9
The title product was obtained as a white solid (74%) from
crude (Z)-3-bromo-1-chlorobut-2-ene (prepared from (Z)-3-
bromo-but-2-en-1-ol49 using the procedure described for (Z)-3-
chloro-1-chlorobut-2-ene50) using TBAPP. TLC (iPrOH :
Hz, 2H), 7.10 (d, J = 7.8 Hz, 2H), 4.77 (d, J = 6.3 Hz, 2H), 2.16
(s, 3H); 31P NMR (162 MHz, deuterium oxide) δ −6.95 (d, J =
22.6 Hz), −10.81 (d, J = 21.8 Hz); HR-ESI-MS (m/z): [M − H]−
calcd for C8H11O7P2−, 280.9980; observed, 280.9978.
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p-Fluorobenzyl pyrophosphate (27)
Hz); HR-ESI-MS (m/z): [M
327.0035; observed, 327.0027.
−
H]− calcd for C9H13O9P2−,
27 was obtained as a white solid (29%) from p-fluorobenzyl
alcohol using TEAP. TLC (iPrOH : NH4OH : H2O 7 : 2 : 1 v/v): Rf
= 0.4; 1H NMR (400 MHz, deuterium oxide) δ 7.37 (dd, J =
8.2, 5.7 Hz, 2H), 7.02 (t, J = 8.7 Hz, 2H), 4.85 (dd, J = 17.1, 6.8
Hz, 2H); 31P NMR (162 MHz, deuterium oxide) δ −7.64 (d, J =
22.3 Hz), −10.85 (d, J = 21.6 Hz); HR-ESI-MS (m/z): [M − H]−
calcd for C7H8FO7P2−, 284.9729; observed, 284.9728.
3,5-Difluorobenzyl pyrophosphate (33)
33 was obtained as
a white solid (28%) from 3,5-
difluorobenzyl alcohol using TEAP. TLC (iPrOH : NH4OH : H2O
1
7 : 2 : 1 v/v): Rf = 0.37; H NMR (400 MHz, deuterium oxide) δ
6.94 (d, J = 7.5 Hz, 2H), 6.75 (t, J = 9.8 Hz, 1H), 4.84 (d, J = 7.3
Hz, 2H). 31P NMR (162 MHz, deuterium oxide) δ −6.99 (d, J =
21.9 Hz), −10.82 (dd, J = 18.2, 10.6 Hz); HR-ESI-MS (m/z): [M −
H]− calcd for C7H7F2O7P2−, 302.9635; observed, 302.9629.
p-Chlorobenzyl pyrophosphate (28)
28 was obtained as a white solid (35%) from p-chlorobenzyl
alcohol using TEAP. TLC (iPrOH : NH4OH : H2O 7 : 2 : 1 v/v): Rf
1
3,4,5-Trimethoxybenzyl pyrophosphate (34)
= 0.41; H NMR (400 MHz, deuterium oxide) δ 7.40–7.06 (m,
4H), 4.79 (d, J = 6.8 Hz, 2H); 31P NMR (162 MHz, deuterium
oxide) δ −7.77 (d, J = 21.7 Hz), −10.90 (d, J = 21.9 Hz); HR-ESI-
MS (m/z): [M − H]− calcd for C7H8ClO7P2−, 300.9434; observed,
300.9428.
34 was obtained as
a white solid (31%) from 3,4,5-
trimethoxybenzyl alcohol using TEAP. TLC (iPrOH : NH4OH:
1
H2O 7 : 2 : 1 v/v): Rf = 0.35; H NMR (400 MHz, deuterium ox-
ide) δ 6.76 (d, J = 2.0 Hz, 2H), 4.80 (dd, J = 6.4, 1.7 Hz, 2H),
3.75 (s, 6H), 3.64 (d, J = 1.9 Hz, 3H). 31P NMR (162 MHz, deu-
terium oxide) δ −7.38 (d, J = 21.6 Hz), −10.88 (d, J = 21.9 Hz);
HR-ESI-MS (m/z): [M − H]− calcd for C10H15O10P2−, 357.0140;
observed, 357.0135.
p-Bromobenzyl pyrophosphate (29)
29 was obtained as a white solid (72%) from p-bromobenzyl
bromide using TBAPP. TLC (iPrOH : NH4OH : H2O 7 : 2 : 1 v/v):
1
Rf = 0.41; H NMR (400 MHz, deuterium oxide) δ 7.40 (d, J =
Determination of structures
8.4 Hz, 2H), 7.20 (d, J = 8.5 Hz, 2H), 4.77 (d, J = 6.9 Hz, 2H);
31P NMR (162 MHz, deuterium oxide) δ −10.38 (d, J = 21.4
Hz), −11.25 (d, J = 20.7 Hz); HR-ESI-MS (m/z): [M − H]− calcd
for C7H8BrO7P2−, 344.8929; observed, 344.8927.
High-resolution mass spectrometry (HRMS) and liquid chro-
matography mass spectrometry (LCMS) were conducted on
an Agilent 6545-QTOF W/1290 HPLC mass spectrometer at
the University of Oklahoma Department of Chemistry and
Biochemistry. NMR spectra were obtained on Varian VNMRS
400 or 500 MHz instruments at the NMR facility of the De-
partment of Chemistry and Biochemistry of the University of
Oklahoma using 99.9% DMSO-d6 with 0.05% v/v TMS or
99.9% D2O or 99.8% MeOH-d4 and 99.9% acetone-d6 (Cam-
bridge Isotope Laboratories, MA, USA). 1H, 13C, and 31P
chemical shifts were referenced to internal solvent reso-
nances. Multiplicities are indicated by s (singlet), d (doublet),
t (triplet), q (quartet), quin (quintet), m (multiplet), and br
(broad). Chemical shifts are reported in parts per million
(ppm) and coupling constants J are given in Hz. All NMR
spectra were recorded at ambient temperature and processed
using MestReNova software.
p-Nitrobenzyl pyrophosphate (30)
30 was obtained as a brown solid (28%) from p-nitrobenzyl al-
cohol using TEAP. TLC (iPrOH: NH4OH : H2O 7 : 2 : 1 v/v): Rf =
1
0.37; H NMR (400 MHz, deuterium oxide) δ 8.05 (d, J = 8.5
Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 4.91 (d, J = 7.4 Hz, 2H); 31P
NMR (162 MHz, deuterium oxide) δ −7.29 (d, J = 21.9 Hz),
−10.78 (d, J = 21.7 Hz); HR-ESI-MS (m/z): [M − H]− calcd for
C7H8NO9P2−, 311.9674; observed, 311.9677.
2,4-Dimethoxybenzyl pyrophosphate (31)
31 was obtained as
a white solid (30%) from 2,4-
dimethoxybenzyl alcohol using TEAP. TLC (iPrOH: NH4OH :
1
H2O 7 : 2 : 1 v/v): Rf = 0.35; H NMR (400 MHz, deuterium ox-
ide) δ 7.28 (d, J = 9.0 Hz, 1H), 6.50 (dd, J = 4.5, 2.1 Hz, 2H),
4.79 (d, J = 6.3 Hz, 2H), 3.71 (s, 3H), 3.70 (s, 3H); 31P NMR
(162 MHz, deuterium oxide) δ −6.58 (d, J = 21.9 Hz), −10.58
(d, J = 21.7 Hz); HR-ESI-MS (m/z): [M − H]− calcd for
C9H13O9P2−, 327.0035; observed, 327.0048.
In vitro FgaPT2 assay
The recombinant FgaPT2 was overproduced in Escherichia
coli BL21(DE3) cells transformed with the codon-optimized
synthetic FgaPT2 gene in a pET28a vector. The resulting
FgaPT2 with N-terminal His6-fusion protein was purified to
homogeneity via Ni-NTA affinity chromatography as de-
scribed previously.28 In vitro FgaPT2 reactions were
conducted in a volume of 20 μl with 1.2 mM alkyl-PP ana-
logue (1–34), 1 mM L-Trp or 7-hydroxy-K252c and 5 μM puri-
fied FgaPT2 in 25 mM Tris buffer pH 7.5, 5 mM MgCl2, and
50 mM KCl, incubated at 35 °C for 16 h. Reactions were
quenched by adding an equal volume of methanol followed
by centrifugation (10 000 × g for 15 min) to remove the
3,4-Dimethoxybenzyl pyrophosphate (32)
32 was obtained as
a white solid (35%) from 3,4-
dimethoxybenzyl alcohol using TEAP. TLC (iPrOH: NH4OH :
1
H2O 7 : 2 : 1 v/v): Rf = 0.35; H NMR (400 MHz, deuterium ox-
ide) δ 7.06 (d, J = 1.8 Hz, 1H), 6.98–6.87 (m, 2H), 4.79 (d, J =
6.2 Hz, 2H), 3.74 (s, 3H), 3.72 (s, 3H); 31P NMR (162 MHz,
deuterium oxide) δ −7.20 (d, J = 21.7 Hz), −10.83 (d, J = 21.8
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precipitated protein, and product formation for each reaction
was subsequently analyzed by RP-HPLC using method A or
method C (Fig. S2 and S3†). Negative controls included reac-
tion in the absence of an enzyme or alkyl-PP analogue. For
each reaction, the percent yield was based upon the integra-
tion of species at 254 nm and calculated by dividing the inte-
grated area of the product by the sum of the integrated areas
of the product and/or and the remaining substrate. All puta-
tive products were subsequently confirmed by HRMS in posi-
tive (+) and/or negative (−) mode (see general methods) (Ta-
ble S3†). Kinetic parameters were assessed using 1.2 mM
L-Trp and variable alkyl-PP analogues (0.1–6 mM). Assays
were conducted in triplicate and all rates were confirmed to
be linear. The kinetic curves were fit to Michaelis–Menton ki-
netics using Prism 5.04 (GraphPad Software, Inc. La Jolla, CA
92037 USA). The reactions with >20% product yield were
performed in 2–5 mL volume under standard conditions,
products were purified by semi-prep HPLC using method B
or method D and the putative new products were confirmed
by NMR (Tables S5 and S6†).
HPLC method D
Semi-preparative RP HPLC was conducted using a Gemini-NX
C-18 (5 μm, 10 mm × 250 mm) column (Phenomenex, Tor-
rance, California, USA) to purify the 7-hydroxy-K252c ana-
logues [gradient of 20% B to 60% B over 10 min, 60% B to
100% B over 10 min, 100% B over 3 min, 100% B to 20% B
over 0.1 min, 20% B for 7 min (A = ddH2O with 0.1% TFA; B
= acetonitrile); flow rate = 2 mL min−1; A254].
The biological activity of alkyl-7-hydroxy-K252c analogues
HCT-116 cells were seeded out in 96-well plates containing 75
μl of McCoy's media at a cell number of 2.0e3 per well. Fol-
lowing a 24 h incubation, 25 μl of medium and 20 μl of
CellTiter-Blue (Promega) were added per well to one 96-well
plate and incubated at 37 °C for 90 min. Fluorescence was
detected (544 nm excitation; 590 nm emission) on a plate
reader (GloMax Explorer, Promega) to establish the baseline
viability at the time of treating. Compounds were serially di-
luted in the medium at 4X concentration and delivered to the
cells in a volume of 25 μl. After a 48 h incubation, CellTiter-
Blue was added for 90 min, and fluorescence was measured
as previously described. Growth relative to untreated cells
was calculated and expressed as IC50 values on GraphPad
Prism software utilizing the four-parameter-dose–response
curve. Experiments were run in duplicate (n = 2) utilizing
Taxol as an internal control.
HPLC method A
To monitor FgaPT2 reactions of L-tryptophan, analytical
reverse-phase (RP) HPLC was conducted employing a Gemini-
NX C-18 (5 μm, 4.6 mm × 250 mm) column (Phenomenex,
Torrance, California, USA) [gradient of 10% B to 25% B over
10 min, 25% B to 100% B over 15 min, 100% B for 3 min,
100% B to 10% B over 0.1 min, 10% B for 7 min (A = ddH2O
with 0.1% TFA; B = acetonitrile); flow rate = 1 mL min−1;
Conflicts of interest
There are no conflicts to declare.
A254]. The reaction was monitored by means of the retention
time difference between the starting material and product.
Acknowledgements
This work was supported by University of Oklahoma start-up
funding and by the National Institutes of Health grant
AI141950. We gratefully acknowledge the University of Okla-
homa, Department of Chemistry and Biochemistry NMR and
Mass Spectrometry facility for analytical support. Thanks to
J. S. Thorson for graciously providing the FgaPT2 expression
strain.
HPLC method B
Semi-preparative RP HPLC was conducted using a Gemini-NX
C-18 (5 μm, 10 mm × 250 mm) column (Phenomenex, Tor-
rance, California, USA) to purify the L-Trp analogues [gradient
of 10% B to 25% B over 10 min, 25% B to 100% B over 15
min, 100% B for 3 min, 100% B to 10% B over 0.1 min, 10%
B for 7 min (A = ddH2O with 0.1% TFA; B = acetonitrile); flow
rate = 2 mL min−1; A254].
Notes and references
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