Structural Diversification of Hapalindole and Fischerindole Natural Products via Cascade Biocatalysis

Article abstract of DOI:10.1021/acscatal.0c05656

Hapalindoles and related compounds (ambiguines, fischerindoles, welwitindolinones) are a diverse class of indole alkaloid natural products. They are typically isolated from the Stigonematales order of cyanobacteria and possess a broad scope of biological activities. Recently the biosynthetic pathway for assembly of these metabolites has been elucidated. In order to generate the core ring system, l-tryptophan is converted into the cis-indole isonitrile subunit before being prenylated with geranyl pyrophosphate at the C-3 position. A class of cyclases (Stig) catalyzes a three-step process, including a Cope rearrangement, 6-exo-trig cyclization, and electrophilic aromatic substitution, to create a polycyclic core. The formation of the initial alkaloid is followed by diverse late-stage tailoring reactions mediated by additional biosynthetic enzymes to give rise to a wide array of structural variations observed in this compound class. Herein, we demonstrate the versatility and utility of the Fam prenyltransferase and Stig cyclases toward the core structural diversification of this family of indole alkaloids. Through the synthesis of cis-indole isonitrile subunit derivatives, and with the aid of protein engineering and computational analysis, we have employed cascade biocatalysis to generate a range of derivatives and gained insights into the basis for substrate flexibility in this system.

Full text of DOI:10.1021/acscatal.0c05656

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Research Article
Structural Diversification of Hapalindole and Fischerindole Natural
Products via Cascade Biocatalysis
Robert M. Hohlman, Sean A. Newmister, Jacob N. Sanders, Yogan Khatri, Shasha Li, Nikki R. Keramati,
Andrew N. Lowell, K. N. Houk, and David H. Sherman*
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ABSTRACT: Hapalindoles and related compounds (ambiguines, fischerindoles, welwitindolinones) are a diverse class of indole
alkaloid natural products. They are typically isolated from the Stigonematales order of cyanobacteria and possess a broad scope of
biological activities. Recently the biosynthetic pathway for assembly of these metabolites has been elucidated. In order to generate
the core ring system, L-tryptophan is converted into the cis-indole isonitrile subunit before being prenylated with geranyl
pyrophosphate at the C-3 position. A class of cyclases (Stig) catalyzes a three-step process, including a Cope rearrangement, 6-exo-
trig cyclization, and electrophilic aromatic substitution, to create a polycyclic core. The formation of the initial alkaloid is followed by
diverse late-stage tailoring reactions mediated by additional biosynthetic enzymes to give rise to a wide array of structural variations
observed in this compound class. Herein, we demonstrate the versatility and utility of the Fam prenyltransferase and Stig cyclases
toward the core structural diversification of this family of indole alkaloids. Through the synthesis of cis-indole isonitrile subunit
derivatives, and with the aid of protein engineering and computational analysis, we have employed cascade biocatalysis to generate a
range of derivatives and gained insights into the basis for substrate flexibility in this system.
KEYWORDS: Stig cyclase, Fam prenyltransferase, hapalindole, fischerindole, diversification, biocatalysis
While each hapalindole subgroup has a characteristic ring
connectivity and stereochemistry, current evidence indicates
that they are all derived from a cis-indole isonitrile core
combined with a geranyl monoterpene subunit. Through
biosynthetic analysis, the mechanism for assembly of the
hapalindole core was recently elucidated. L-Tryptophan is
converted to cis-indole isonitrile precursor 1 by a three-enzyme
cascade8a followed by geranylation at the C-3 position.8f In the
presence of a Stig cyclase(s), this 3-geranyl cis-indole isonitrile
(3-GC) intermediate 2 undergoes a Cope rearrangement, 6-
exo-trig cyclization, and a terminal electrophilic aromatic
substitution (EAS) at either the C-2 or C-4 indole position
INTRODUCTION
■
Hapalindoles are a large family of indole alkaloids that have
been isolated from the cyanobacterial order Stigonematales.1
Along with their related compounds, fischerindoles, ambi-
guines, and welwitindolinones, there have been at least 81
members isolated from over 18 cyanobacterial strains.2 They
have also been shown to have antimicrobial,3 antimycotic,3e,4
anticancer,5 and immunomodulatory activity.6 Hapalindole/
fischerindole metabolites possess three distinguishing features:
a polycyclic ring system, diverse stereochemical variations, and
the late-stage introduction of a range of functional groups such
as additional rings, halogens, hydroxyls, isothiocyanate, and
others. Due to these unique structural features and diverse
biological activities, a number of total syntheses have been
devoted to this family of indole alkaloids.7 However, these
efforts have been hindered due to the highly functionalized
ring system and variant stereochemical patterns. Recently,
work by several groups has explored the biogenesis of these
metabolites,8 which has uncovered new prenyltransferases,
cyclases, halogenases, and other unique biosynthetic enzymes.
Received: December 23, 2020
Revised: March 21, 2021
Published: April 5, 2021
https://doi.org/10.1021/acscatal.0c05656
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Figure 1. (A) Proposed biosynthesis of hapalindole type molecules. L-Tryptophan is converted into the cis-indole isonitrile core (1) (in blue) via a
three-enzyme cascade followed by geranylation (in red) at the C-3 position to afford the 3-GC intermediate (2). In the presence of the cyclase(s), 2
undergoes a Cope rearrangement, 6-exo-trig cyclization, and electrophilic aromatic substitution (EAS) at the C-4 or C-2 position to afford the core
hapalindole or fischerindole structure, respectively. (B) The approach described herein includes the chemical synthesis of unnatural cis-indole
derivatives to show that the geranyltransferase (FamD2) and Stig cyclases can produce a range of unnatural 12-epi-hapalindole U (3), 12-epi-
fischerindole U (4), and 12-epi-hapalindole C (5) compounds.
to afford the fischerindole or hapalindole core, respectively
(Figure 1A).8f Subsequent late-stage tailoring of these
molecules provides further access to the ambiguines and
welwitindolinones.
demonstrate herein that key Stig cyclases can be employed as
biocatalytic tools to produce numerous unnatural hapalindole
and fischerindole compounds while also providing further
mechanistic insights into the unusual cyclization cascade
(Figure 1).
Recently, cyanobacterial-derived Stig cyclases have attracted
interest for their ability to catalyze a multistep core-forming
cyclization cascade. Previous work has shown that the Stig
cyclases exist in a dimeric state that may involve higher order
oligomeric complexes to catalyze cyclization, including a
terminal C−H functionalization reaction.8h,p Depending on
the components of this oligomeric complex, different regio-
and stereochemical outcomes have been observed.8f,g,i,s,t
Structural and mutagenesis studies have revealed the key
residues responsible for core hapalindole and fischerindole
formation alongside computational analysis that has examined
the cyclase-mediated Cope rearrangement and terminal EAS.8h
Although gram-scale total syntheses of the hapalindoles have
been achieved on a few select metabolites,7o,t further
diversification is necessary for drug lead exploration. The
Stig cyclases show potential as novel biocatalytic tools for
developing unnatural hapalindole and fischerindole metabo-
lites. Recently, we showed that fluorinated unnatural
fischerindole and hapalindole derivatives could be produced
using a microscale in vitro transcription/translation system
with genes encoding prenyltransferase and Stig cyclase
proteins.9 To validate and explore this approach further, we
RESULTS AND DISCUSSION
■
Substrate Synthesis. To pursue our analysis, the chemical
synthesis of unnatural cis-indole isonitrile derivatives was
initiated. We synthesized these molecules via a Horner−
Wadsworth−Emmons (HWE) olefination reaction from
carboxyaldehyde indole derivatives on the basis of the ability
to produce unnatural derivatives in a facile manner (Table 1).
While the HWE reaction is known to stereochemically favor
trans-(E) product formation, we found that performing the
reaction at 0 °C yields a 50:50 ratio of the cis-(Z) and trans
isomers. Using normal-phase chromatography, these isomers
are readily separated to obtain the desired Z-isomer product.
The majority of these derivatives were designed to have
functional group modifications at the indole C-5 and C-6
positions, which included halogens (10−16), methoxy groups
(8, 9), benzyl groups (6), and a cyano (7) functionality. Two
of the isomers (17 and 18) were modified at the C-7 position
of the indole ring by replacement with a nitrogen atom. For
derivatives 16 and 18, the carboxyaldehyde indole precursor
was commercially unavailable. Thus, we obtained the indole
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Table 1. cis-Indole Isonitrile Derivatives Generated in This
Study and Isolated Percent Yield of the Desired Z Isomer
Table 2. TTN Values for Geranylation Reaction with
a
FamD2
compound
TTN value
1
6
7
1495
1615
N/A
b
8
9
1481
1902
947
1547
1625
803
755
1174
710
10
11
12
13
14
15
16
17
18
19
compound
substituent
yield of Z isomer (%)
6
7
8
9
R1 = OBzn, R2 = H, X = C,Y = NC
R1 = CN, R2 = H, X = C, Y = NC
R1 = OMe, R2 = H, X = C, Y = NC
R1 = H, R2 = OMe, X = C, Y = NC
R1 = Cl, R2 = H, X = C, Y = NC
R1 = H, R2 = Cl, X = C, Y = NC
R1 = F, R2 = H, X = C, Y = NC
R1 = H, R2 = F, X = C, Y = NC
R1 = Br, R2 = H, X = C, Y = NC
R1 = H, R2 = Br, X = C, Y = NC
R1 = I, R2 = H, X = C, Y = NC
R1 = H,R2 = H,X = N,Y = NC
R1 = F, R2 = H, X = N, Y = NC
R1 = H, R2 = H, X = C, Y = CN
11
22
16
5
10
11
12
13
14
15
16
17
18
19
11
16
18
23
18
23
18
13
18
16
1281
1366
1454
a
Assay conditions: 1 μM FamD2, 2 mM substrate, 1.5 mM GPP, 5
mM MgCl2, 50 mM glycine (pH 10.0), 100 μL, 37 °C, 200 rpm, 1 h.
b
Compound 7 showed no conversion in the assay.
fluorinated cis-indole isonitrile derivatives 12 and 13 to
generate the new hapalindole compounds 22 and 23,
respectively.9 We sought to analyze and confirm its scope
beyond fluorinated derivatives. Initially, few additional
substrates showed conversion to the cyclized product.
Lowering the pH and concentration of FamD2 did not
increase the cyclase activity. As Ca2+ has been shown to be an
important cyclase cofactor, we supplemented the reaction
mixture to test its effect on FamC1 activity.8g−i,s Upon the
addition of 5 mM calcium chloride, we observed increased
production of cyclized products in 8 of the 14 unnatural
substrates. On the basis of HPLC analysis, we estimated that
the conversion values ranged from 10% to >99%. Scale-up
efforts of 2 mg of each substrate enabled the structural
characterization of these compounds. We confirmed that, in all
but one case, the corresponding 12-epi-hapaplindole U
derivative was produced. In contrast, FamC1 catalyzed the
formation of tricyclic 12-epi-hapalindole C derivative 31 from
azaindole compound 17 (Table 3). Tricyclic hapalindoles are
normally a minor product observed in enzymatic reactions
from previously studied Stig cyclases. We initially reasoned that
the unnatural electronegative N-7 of the azaindole inhibited
the terminal EAS reaction at the C-4 position. However, C-4
tetracycle formation was observed with the C-5-fluoro
derivative 12, which suggests that the selectivity may be
guided by a skeletal variation of the N-7 position instead of the
electronics of the indole ring.
HpiC1 Cyclase. While FamC1 demonstrated the ability to
convert 8 of 14 substrates, we sought a cyclase with greater
flexibility. We next examined HpiC1 from Fischerella sp. ATCC
43239, which shares an 84% sequence homology with FamC1.
Complexes consisting of only HpiC1 have been shown to
produce 12-epi-hapalindole U and trace levels of 12-epi-
hapalindole C.8g,h A sequence comparison revealed only three
active site variations between HpiC1 and FamC1.8h Analytical
reactions showed an increased scope, as 10 of the 14 substrates
were converted to hapalindole products with conversion values
ranging from 30 to >99%. As with FamC1, the majority of the
compounds were confirmed to be the corresponding 12-epi-
hapalindole U derivatives. Similarly to reactions with FamC1,
precursor and added the carboxyaldehyde functionality via a
Vilsmeier−Haack reaction that offered 20 and 21 (Exper-
imental Section), allowing us to obtain derivatives 16 and 18.
FamD2 Prenyltransferase. With a suite of cis-indole
isonitrile derivatives in hand, we assessed the flexibility of
FamD2 (also known as AmbP1), the prenyltransferase from
Fischerella ambigua UTEX 1903,8a,f for the first key trans-
formation of 1. Through initial analytical reactions, we
observed geranylation of 13 of the 14 substrates, with only
the 5-cyano derivative 7 failing to be geranylated. This result
was rationalized on the basis of previous structural studies of
FamD2 that revealed conformational flexibility in the active
site in the presence of a Mg2+ cofactor.8n In the case of 7, we
hypothesized that either steric hindrance or unfavorable
electronics played a role in the lack of C-3 geranylation.
Because substrate 16 (containing a large 5-iodo group) was
able to be geranylated, we believed that steric hindrance was
less likely to be a factor. Upon examination of the crystal
structure of FamD2,8n we hypothesized that the highly polar 5-
cyano functionality is interacting with a specific residue or the
backbone of FamD2, disrupting the derivative from a favorable
position for geranylation. Because the 3-GC intermediate has
been shown to undergo a 1,2-shift,8f we sought a different
method to analyze the efficiency of the geranylation step.
Using an HPLC assay, total turnover numbers (TTN) were
calculated over a 1 h reaction period at pH 10 to achieve
maximum enzyme efficiency. Further cyclization reactions were
conducted at pH 7 or 7.8, as the cyclase activity is greater
under more acidic conditions.8h,s It is possible that FamD2
turnover is attenuated at a more neutral pH, but this was not
tested. At pH 10, TTN values ranged from 710 to 1902, which
highlights the versatility and efficiency of FamD2 as a
biocatalyst over a range of unnatural substrates (Table 2).
FamC1 Cyclase. We next investigated the FamC1 cyclase
from the Fischerella ambigua UTEX 1903 fam biosynthetic
gene cluster. The homodimeric form of FamC1 produces 12-
epi-hapalindole U8f and was previously shown to accept
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Table 3. Structures of 12-epi-Hapalindole U and 12-epi-Hapalindole C Derivatives Produced by FamC1 and HpiC1 from
a
Unnatural cis-Indole Isonitrile or cis-Indole Nitrile Substrates
a
Percent conversions, isolated yield values, and tetracyclic:tricyclic ratio (ratio estimated by NMR and/or HPLC) are shown below each derivative.
N/A denotes that the derivative was either not produced by FamC1 or was not screened further in this study due to the enhanced versatility of
HpiC1. HPLC conversion values were determined after 4 h in 100 μL reactions. Isolated yield values were determined from overnight reactions.
we observed the production of tricyclic 12-epi-hapalindole C
derivatives 31 and 32 from cis-indole isonitrile substrates 17
and 18, respectively (Table 3).
exhibits low percent conversions for 14 and 15 and completely
fails to convert 16, we hypothesize that the two mutations
present in HpiC1 (Phe138 and Leu147) reduce steric
hindrance in the area of the active site that interacts with the
C-5 and C-6 substituents of the indole, resulting in higher
conversion efficiencies.
We hypothesized that the increased scope and reaction
efficiency of HpiC1 could be attributed to the amino acid
differences in the active site,8h resulting in less steric hindrance
to accommodate larger substituents. We investigated the three
amino acid differences in the active site between HpiC1 and
FamC1 using site-directed mutagenesis to assess their role in
the substrate scope, which are Val51, Phe138, and Leu147 in
HpiC1. The HpiC1_V51I mutant revealed no changes in the
substrate scope. Although single mutants (HpiC1_F138L and
HpiC1_L147F) failed to show noticeable changes in scope, the
percent conversions of substrates 8, 14, and 16 were reduced
(Table S36 in the Supporting Information). The attenuated
substrate scope of the double mutant (HpiC1_F138L_L147F)
closely matched that of WT FamC1 (Figures S12−S22 in the
Supporting Information). This mutagenesis study revealed that
two of the three active site residue differences (138 and 147)
were responsible for the change in substrate scope and
conversion values between the two cyclases. Given that FamC1
FimC5 Cyclase. Following an analysis of two hapalindole-
producing cyclases, we explored FimC5, a fischerindole
producing cyclase derived from Fischerella muscicola UTEX
1829. This cyclase produces 12-epi-fischerindole U8g and was
used in our previous study to generate the two fluorinated
derivatives 33 and 37.9 Analytical reactions showed the
production of cyclized products for 8 of the 14 substrates
tested with estimated conversion values ranging from 20% to
>99%. The majority of the substrates were characterized as the
corresponding 12-epi-fischerindole U derivatives. With sub-
strate 17, FimC5 catalyzed the formation of a 50:50 mixture of
two products: the previously observed tricyclic derivative 31
and the new tetracyclic derivative 36. The structural assign-
ment of 36 was also confirmed using X-ray crystallography
(Figure 2). In the case of derivative 18, we only observed
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Figure 2. X-ray crystal structure of 36. The NMR-assigned structure has been added to highlight the structural similarity. Nitrogen atoms have been
highlighted in blue with same numbers to orient the reader.
Table 4. Structures of 12-epi-Fischerindole U Derivatives Produced by FimC5 from Unnatural cis-Indole Isonitrile or cis-
a
Indole Nitrile Substrates
a
Percent conversions, isolated yields, and tetracyclic:tricyclic ratio (ratio estimated by NMR and/or HPLC) are shown below each derivative.
HPLC conversion values were determined after 4 h in 100 μL reactions. Isolated yield values were determined from overnight reactions.
production of the tricyclic derivative 32 (Table 4), with the C-
5 fluorine appearing to limit reactivity at the C-2 position of
the indole ring.
A previous analysis of FimC5 indicated that two active site
amino acid residues (Phe101 and Ser138) play a key role in
selectivity toward the indole C-2 or C-4 position for the
terminal EAS.8g,h We decided to explore this further through
site-directed mutagenesis with unnatural 3-GC derivatives.
Three mutants of HpiC1 were produced to match the same
residues identified in the FimC5 active site (Y101F, F138S,
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Figure 3. Quantum mechanical density functional theory computations comparing the energetics of tetracyclic hapalindole formation and
tetracyclic fischerindole formation starting from the cationic intermediates derived from 2, 17, and 18. The tetracyclic fischerindole formation (left
side) is only influenced by inductive effects, while the tetracyclic hapalindole formation (right side) is also influenced by resonance effects.
and Y101F_F138S). Our results provide further support that
these residues are critical for the selectivity of the terminal
reaction. For the Y101F and F138S mutants, we observed the
coproduction of fischerindole and hapalindole products.
However, upon screening the HpiC1 double mutant, we
observed almost complete conversion to fischerindole
metabolites. The F138S mutation also aided in uncovering
the substrate selectivity differences between HpiC1 and
FimC5, as product formation was either inhibited or
completely abolished in select substrates. Exceptions to these
results include compounds 13 and 17, which displayed a 50:50
ratio of products for all mutants, and compound 18, which led
to formation of the tricyclic hapalindole 32 for all HpiC1
mutants tested (Table S37 and Figures S1−S11 in the
Supporting Information).
Discovery and Isolation of Nitrile-Containing Com-
pounds 30 and 40. Hapalindole-type indole alkaloids are
noted for containing a rare isonitrile (−NC) moiety, although
nitrile (−CN)-containing fischerindole and ambiguine mole-
cules have been isolated previously.1,3c,10 Derivation of the
nitrile functionality from an isonitrile rearrangement has been
suggested previously,10 and we decided to examine this
hypothesis by screening the Stig cyclases using cis-indole
nitrile derivative 19, which was accepted by the prenyltransfer-
ase FamD2 at a TTN value comparable to that of the native
isonitrile compound (Table 2). A cyclized product from wild-
type HpiC1 was not initially observed with 19; however, the
generation of two new products from each of the three
HpiC1→FimC5 mutant cyclases in varying ratios was
observed. Scale-up and structural characterization of products
derived from the HpiC1 Y101F mutant led to identification of
the 12-epi-hapalindole U nitrile derivative 30 (we also
observed small amounts of the corresponding tricyclic
hapalindole derivative). Wild-type HpiC1 and FamC1 were
then rescreened under the optimized conditions (Experimental
Section), resulting in the production of 30 for both enzymes.
To our knowledge, these are the first nitrile-containing
hapalindole molecules reported. With this result in hand, we
returned to wild type FimC5 cyclase and, under the same
optimized conditions, retested substrate 19. We observed the
production of the nitrile-containing 12-epi-fischerindole U
derivative 40 that was inadvertently overlooked in our initial
analysis. Thus, our results demonstrated that the FamC1,
HpiC1, and FimC5 cyclases have the ability to catalyze
cyclization of the isonitrile- and nitrile-containing indole
subunits. However, the nitrile substrate appeared to have
lower conversion and isolated yield values (Tables 3 and 4) in
comparison to the native isonitrile substrate, suggesting that
the nitrile moiety affects turnover from the 3-GC intermediate
to the terminal tetracyclic product. Regardless, this result
supports that the nitrile functionality may come from early
modifications of the intermediates, but the source remains
unknown.
Computational Analysis of the Reactivity of Sub-
strates 17 and 18. To investigate why substrates 17 and 18
form tricyclic hapalindole compounds 31 and 32 with FamC1/
HpiC1, respectively, while tetracyclic fischerindole 36 is
formed with FimC5, we performed quantum mechanical
density functional theory computations (Figure 3). For
substrates 17 and 18, as well as the parent indole intermediate
2, we began with the tricyclic cationic intermediates T and
considered two possible electrophilic aromatic substitutions:
(1) reaction at C-4 to form the tetracyclic hapalindole scaffolds
H and (2) reaction at C-2 to form the tetracyclic fischerindole
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scaffolds F. As shown in Figure 3, formation of hapalindole
scaffolds is favored for the parent indole but disfavored for the
azaindoles. The difference can be understood by a consid-
eration of resonance structures that stabilize the hapalindole
scaffold. While the conversion of indole cation 2-T into 2-H is
exergonic by 1.9 kcal/mol and has a low free energy barrier of
7.4 kcal/mol, the conversion of azaindole cation 17-T into 17-
H is endergonic by 10.0 kcal/mol and has a higher barrier of
14.7 kcal/mol. In particular, of the four resonance structures in
addition to the iminium structure, the resonance structure
shown places a positive charge on the electronegative nitrogen,
resulting in less stabilization in comparison with the parent
indole derivative, where this positive charge is on a carbon.
The conversion of fluorinated azaindole cation 18-T into 18-H
is only slightly more favorable due to near-cancellation of the
resonance and inductive effects of F; this reaction is
endergonic by 9.8 kcal/mol and has a barrier of 14.0 kcal/
mol. Given the higher barriers to tetracyclic hapalindole
formation for 17 and 18, it is not surprising that FamC1 and
HpiC1 fail to catalyze this reaction; instead, deprotonation of
cations 17-T and 18-T yield tricyclic compounds 31 and 32,
respectively. Unlike the case for tetracyclic hapalindole
formation, the conversion of azaindoles into tetracyclic
fischerindole scaffolds does not exhibit differential resonance
effects in comparison to the parent indole because the
azaindole nitrogen and fluorine atoms are not on positively
charged positions in any of the four additional resonance
structures that stabilize each tetracyclic fischerindole scaffold.
Instead, tetracyclic fischerindole formation is influenced by
smaller inductive effects. Thus, the conversion of indole 2-T
into 2-F is exergonic by 10.1 kcal/mol and the conversion of
azaindole 17-T into 17-F (with an additional inductively
withdrawing nitrogen atom) is exergonic by only 8.5 kcal/mol.
The conversion of fluorinated azaindole 18-T into 18-F, which
contains inductively withdrawing nitrogen and fluorine atoms,
is exergonic by only 6.5 kcal/mol. The free energy barriers,
which are 1.7, 1.9, and 2.9 kcal/mol, respectively, follow the
same trend in which addition of inductively withdrawing atoms
raises the transition-state energy. However, because these
inductive effects are smaller than resonance effects, FimC5 can
efficiently catalyze the conversion of 17 into 36. Although it is
important to note that these reactions take place in an enzyme
active site, these computations addressing the innate reactivity
of azaindoles reveal that tetracyclic hapalindole formation has a
substantially higher free energy barrier in comparison to
tetracyclic fischerindole formation. Accordingly, it is not
surprising that FimC5 catalyzes tetracyclic fischerindole
formation while FamC1 and HpiC1 form tricyclic products
rather than tetracyclic hapalindoles. Evidently, the enzyme
active sites do not overcome the innate difference in ease of
tetracyclic product formation.
values observed in the analytical-scale reactions. We believe
that, through the filtration, workup, and purification process, a
significant amount of material was lost and optimizing isolation
methods may be necessary to address this issue. At the 5 mg
scale with HpiC1, we observed that the tricyclic minor product
was generated in a higher ratio in comparison to those for the 2
mg or analytical reactions. While these two compounds are
separable, further scalability represents an objective for protein
engineering to maximize tetracycle formation. However,
FamC1 did show lower production of the tricyclic product
but appears to be less efficient and flexible in comparison to
HpiC1.
CONCLUSION
■
Through this work, we have shown the biocatalytic potential of
core biosynthetic enzymes to produce unnatural hapalindole
and fischerindole derivatives. The three cyclases investigated
for this work, FamC1, HpiC1, and FimC5, along with select
mutants and FamD2 geranyltransferase have demonstrated
their ability to accept numerous unnatural cis-indole isonitrile
(and nitrile) derivatives. These new compounds are poised for
an investigation of biological activity by direct means or for
additional semisynthetic or biocatalytic modifications. While
HpiC1 appears to be the most versatile Stig cyclase, there are
numerous additional homologues and heteromeric combina-
tions that can be screened to further probe the full scope of
Stig cyclase biocatalytic versatility. This work further supports
the growing opportunity to employ natural product bio-
synthetic enzymes for the assembly of complex, bioactive small
molecules and as a complement to synthetic chemistry
approaches.
EXPERIMENTAL SECTION
■
General Considerations. All NMR spectra were acquired
on a Varian 400 and 600 MHz and Bruker 800 MHz
spectrometers. Proton and carbon signals are reported in parts
per million (δ) using residual solvent signals as an internal
standard. Analytical HPLC analysis was performed on a
Shimadzu 2010 EV APCI spectrometer equipped with an
LUNA C18 250 × 4.6 mm column, with a mobile phase
gradient of 70−100% acetonitrile in water over 16 min at 40
°C, and was monitored by UV absorption at 280 nm. LC-MS
analysis was performed on a Agilent Infinity II TOF instrument
using an XBridge C18 2.1 × 150 mm column, with a mobile
phase gradient of 70−100% acetonitrile in water over 12 min.
Preparative-scale HPLC was performed on a Shimadzu 20-AT
instrument equipped with an LUNA C18 250 × 10 mm
column for 2 mg reactions and an LUNA C8 250 × 21 mm
column for 5 mg reactions, with a mobile phase gradient of
50−100% or 60−100% acetonitrile in water over 60 min.
Optical rotations were obtained using a Jasco P2000
polarimeter at 25 °C.
Analysis of Stig Cyclase Biocatalytic Ability. In this
work, we explored the biocatalytic versatility of select Stig
cyclases by assessing their substrate scope and ability to
generate new derivatives. We analyzed HpiC1, select FamC1,
and FimC5 reactions at both 2 and 5 mg scales to assess how
scalability may affect the overall yield, which ranged from 10 to
60% (Tables 3 and 4) for both reactions. The isolated yields of
certain substrates (22 and 26 for HpiC1 and 39 for FimC5)
were coincident with, or exceeded, the levels of the native
substrate, suggesting that certain substituents enhance cyclase
reactivity. In the majority of cases though, the isolated yields
appeared to be significantly lower than the percent conversion
Escherichia coli strain BL21(DE3) was used for protein
expression. Plasmid pET28H8T8g was used for cloning and
expression of N-truncated FamC1 and FimC5. Plasmid
pET28a was used for cloning and expression of FamD2,
HpiC1, and HpiC1 mutants. Isopropyl β-D-thiogalactopyrano-
side (IPTG) was used to induce expression; DNase and
lysozyme were purchased form Sigma-Aldrich. Ni-NTA
agarose from Invitrogen was used to purify His-tag proteins.
All chemicals were purchased from Sigma-Aldrich, ACROS,
and Combi-Blocks. Multiplicities are abbreviated as follows:
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singlet (s), doublet (d), triplet (t), quartet (q), doublet of
doublets (dd), triplet of doublets (td), doublet of doublets of
doublets (ddd), triplet of doublets of doublets (tdd), and
multiplet (m). Chemical abbreviations are as follows: ethyl
acetate (EtOAc), dichloromethane (DCM), tetrahydrofuran
(THF), potassium bis(trimethylsilyl)amide (KHMDS), acetic
acid (AcOH), sodium sulfate (Na2SO4), diethyl ether (Et2O),
phosphorus oxychloride (POCl3), sodium hydroxide (NaOH),
acetonitrile (CH3CN), magnesium chloride (MgCl2), calcium
chloride (CaCl2), and sodium chloride (NaCl).
analysis for the starting material (Tables S3−S15 in the
Supporting Information). For the structure analysis and
isolated yield values of the enzymatic products, the reactions
were scaled up to 2 and 5 mg of starting material (10 and 25
mL, respectively) and the reaction mixtures incubated at 37 °C
overnight or until HPLC showed consumption of the starting
material. The products were extracted with EtOAc and purified
by preparative HPLC as described in General Considerations.
All products were obtained as white solids. The purified
compounds were concentrated, dissolved in C6D6, and
analyzed using Varian 600 MHz and Bruker 800 MHz NMR
spectrometers.
Chemical Synthesis of cis-Indole Isonitrile Deriva-
tives. All derivatives were prepared using methods previously
described.8f,9 Briefly, in a 50 mL two-neck round-bottom flask
purged with nitrogen at −78 °C (dry ice/acetone), diethyl
(isocyanomethyl)phosphonate (0.37 mL, 2.26 mmol) (diethyl
(cyanomethyl)phosphonate was used for the production of
19) was diluted with THF (5 mL). KHMDS (1 M THF, 2.60
mL, 2.60 mmol) was added dropwise, and the reaction mixture
was stirred at −78 °C for 15 min. In a separate 4 mL vial, an
indole-3-carboxaldehyde derivative (1.13 mmol) was dissolved
in THF (5 mL), and the resulting solution was added dropwise
to the KHMDS solution at −78 °C. The resulting mixture was
stirred at 0 °C (cryocool) overnight or until TLC showed
consumption of the starting material. The resulting solution
was quenched by the addition of AcOH (0.15 mL, 2.6 mmol)
and concentrated. The resulting residue was diluted with
EtOAc (20 mL), washed with 1 M aqueous potassium
phosphate buffer (20 mL, pH 7) and brine, dried with
Na2SO4, and concentrated to a residue. The residue was
dissolved in EtOAc and purified by flash chromatography (24−
100% pentane/Et2O, SiO2) to afford the title compound as
reported below. Yields and spectral data are reported below.
(Z)-5-(Benzyloxy)-3-(2-isocyanovinyl)-1H-indole (6). Blue
solid, 17 mg, 11%. 1H NMR (400 MHz, acetone-d6): δ 5.17 (s,
2H), 5.90 (d, J = 8.9 Hz, 1H), 6.90−6.99 (m, 2H), 7.32 (t, J =
7.3 Hz, 1H), 7.37−7.47 (m, 4H), 7.51 (d, J = 7.5 Hz, 2H),
8.14 (d, J = 2.7 Hz, 1H), 10.80 (s, 1H). 13C NMR (151 MHz,
acetone): δ 169.52, 154.15, 137.99, 130.84, 128.32, 127.70,
127.58, 127.55, 127.32, 124.44, 113.46, 112.60, 109.49, 101.38,
70.17.
Protein Expression and Purification. The expression
and purification of proteins were performed as described.8g,9
Briefly, a single BL21(DE3) colony was inoculated in LB
medium containing 50 μg/mL of kanamycin and grown
overnight at 37 °C with shaking at 200 rpm. The main culture
(1 L) was inoculated at a dilution of 1:100 in a 2.8 L Fernbach
flask containing TB medium and the same concentration of
antibiotic. The cells were grown (37 °C, 200 rpm) to an
optical density (A600 nm) of 1.0. The culture flasks were chilled
in ice, the contents were induced with IPTG (0.2 mM), and
the flasks were further incubated (18 °C, 200 rpm) for 16 h.
The cells were harvested (5000 rpm, 4 °C, 15 min), flash-
frozen, and stored at −80 °C until purification. The cell pellets
were resuspended at 4 °C in the lysis buffer (10 mM HEPES,
50 mM NaCl, 0.2 mM TCEP, 10% glycerol, 20 mM
imidazole), containing 0.5 mg/mL of lysozyme, 1 mM
PMSF, and 1 mL of 2 mg/mL DNase. The mixture was
stirred for 30 min and sonicated on ice for a total time of 120 s
using 10 s pulses followed by a 50 s pause. The cellular debris
was removed by centrifugation (65000g, 4 °C, 35 min). The
clarified lysate was loaded onto Ni-NTA agarose column
equilibrated with lysis buffer. The column was washed with
two column volumes of wash buffer (10 mM HEPES, 300 mM
NaCl, 0.2 mM TCEP, 10% glycerol, 20 mM imidazole) and
the His-tagged protein was eluted with elution buffer (10 mM
HEPES, 50 mM NaCl, 0.2 mM TCEP, 10% glycerol, 300 mM
imidazole). The fractions were pooled and dialyzed overnight
or by using a PD10-desalting column (GE Healthcare) with
storage buffer (10 mM HEPES, 50 mM NaCl, 0.2 mM TCEP,
10% glycerol). The purified protein was analyzed by SDS-
PAGE gel for purity, measured by Nanodrop using a calculated
molar extinction coefficient for concentration, and flash-frozen
in liquid nitrogen for storage at −80 °C.
Analytical, TTN, and Scale-Up Enzymatic Reactions.
cis-Indole isonitrile derivatives were synthesized as described
below. For the initial assays, a 50 μL reaction mixture
containing 10 μM FamD2, 15 μM cyclase, 1 mM substrate, 1
mM GPP, 5 mM MgCl2, 50 mM Tris pH 7.8 buffer, and 5 mM
CaCl2 was incubated at 37 °C for 4 h. The reaction was
quenched twice with a 3× volume of EtOAc. The organic
layers were combined, dried, and redissolved in 100 μL of
acetonitrile for LCMS and HPLC analysis. HPLC conversion
values were determined by the area under the curve of the
residual starting material and newly formed product. For later
assays, a 100 μL reaction mixture containing 5 μM FamD2, 20
μM cyclase, 1 mM substrate, 1.5 mM GPP, 5 mM MgCl2, 50
mM of Tris pH 7.0 buffer and 7.5 mM CaCl2 was incubated,
quenched, and analyzed as previously described. For FamD2
TTN assays, a 100 μL reaction mixture containing 1 μM
FamD2, 2 mM substrate, 1.5 mM GPP, 5 mM MgCl2, and 50
mM Glycine pH 10.0 buffer was incubated at 37 °C for 1 h.
The reaction was quenched and analyzed as described
previously. TTN values were determined by standard curve
(Z)-3-(2-Isocyanovinyl)-1H-indole-5-carbonitrile (7). Tan
solid, 25 mg, 22%. 1H NMR (400 MHz, acetone-d6): δ 6.06 (d,
J = 8.9 Hz, 1H), 7.06 (dt, J = 9.2, 4.7 Hz, 1H), 7.53 (dd, J =
8.5, 1.6 Hz, 1H), 7.71 (dd, J = 8.4, 0.8 Hz, 1H), 8.24−8.29 (m,
1H), 8.32 (s, 1H), 11.38 (s, 1H). 13C NMR (101 MHz,
acetone): δ 171.15, 138.20, 129.86, 127.72, 126.16, 124.72,
124.10, 120.80, 114.08, 111.07, 104.41
(Z)-3-(2-Isocyanovinyl)-5-methoxy-1H-indole (8). Red
1
solid, 36 mg, 16%. H NMR (400 MHz, acetone-d6): δ 3.84
(s, 3H), 5.89 (d, J = 8.8 Hz, 1H), 6.87 (dd, J = 8.8, 2.4 Hz,
1H), 6.97 (d, J = 6.0 Hz, 1H), 7.30 (d, J = 2.4 Hz, 1H), 7.41
(d, J = 8.7 Hz, 1H), 8.07−8.15 (m, 1H), 10.77 (s, 1H). 13C
NMR (151 MHz, dmso): δ 169.82, 154.74, 130.64, 127.65,
127.49, 125.45, 124.09, 113.16, 113.00, 109.24, 100.54, 55.83.
(Z)-3-(2-Isocyanovinyl)-6-methoxy-1H-indole (9). Tan
1
solid, 5 mg, 5%. H NMR (599 MHz, acetone-d6): δ 3.82 (s,
4H), 5.91 (d, J = 8.8 Hz, 1H), 6.82 (dd, J = 8.7, 2.3 Hz, 1H),
6.93 (dt, J = 9.8, 4.8 Hz, 1H), 7.04 (d, J = 2.3 Hz, 1H), 7.64 (d,
J = 8.7 Hz, 1H), 8.03 (d, J = 2.3 Hz, 1H), 10.67 (s, 1H). 13C
NMR (151 MHz, dmso): δ 169.84, 156.73, 136.44, 125.96,
125.32, 124.46, 123.68, 119.32, 110.83, 109.34, 95.19, 55.67.
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(Z)-5-Chloro-3-(2-isocyanovinyl)-1H-indole (10). Tan
solid, 20 mg, 11%. H NMR (400 MHz, acetone-d6): δ 5.98
(d, J = 8.9 Hz, 1H), 6.93−7.06 (m, 0H), 7.22 (dd, J = 8.6, 2.0
Hz, 1H), 7.55 (d, J = 8.6 Hz, 1H), 7.82 (d, J = 2.0 Hz, 1H),
8.22 (d, J = 2.0 Hz, 1H), 11.04 (s, 1H).
Chemical Synthesis of Indole-3-carboxaldehyde De-
rivatives. All derivatives were prepared using the same
method. Briefly, in a 25 mL round-bottom flask purged with
nitrogen at 0 °C (ice−water bath), POCl3 (1.38 mL, 14.7
mmol) was stirred in dry DMF (4 mL) for 20 min. In a
separate 4 mL vial, the reactant indole compound (2.94 mmol)
was dissolved in dry DMF (4 mL) and added to the POCl3
solution at 0 °C. The reaction mixture was slowly brought to
room temperature and stirred for 1 h or until TLC showed
consumption of the starting material. The reaction mixture was
cooled to 0 °C and quenched with ice−water and 1 M NaOH
(5 mL each). The reaction mixture was stirred at room
temperature for 1 h or until TLC showed consumption of the
intermediate. The resulting solution was extracted with EtOAc
(2 × 10 mL), and the extract was washed with brine, dried
with Na2SO4, and concentrated to a residue. The residue was
dissolved in EtOAc and purified by flash chromatography (16−
100% hexanes/EtOAc, SiO2) to afford the title compound as
reported below. Yields and spectral data are reported below.
5-Iodo-1H-indole-3-carbaldehyde (20). Off-white solid,
1
(Z)-6-Chloro-3-(2-isocyanovinyl)-1H-indole (11). Yellow
1
solid, 36 mg, 16%. H NMR (599 MHz, acetone-d6): δ 5.96
(d, J = 8.9 Hz, 1H), 6.94 (dt, J = 9.5, 4.9 Hz, 1H), 7.16 (dd, J =
8.5, 1.9 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H), 7.75 (d, J = 8.5 Hz,
1H), 8.17−8.20 (m, 1H), 11.02 (s, 1H). 13C NMR (151 MHz,
acetone): δ 170.89, 137.02, 128.93, 128.64, 128.47, 126.68,
124.71, 121.72, 120.25, 112.65, 110.61.
(Z)-5-Fluoro-3-(2-isocyanovinyl)-1H-indole (12). Tan
1
solid, 36.7 mg, 18%. H NMR (400 MHz, acetone-d6): δ
5.95 (d, J = 8.9 Hz, 1H), 6.94 (p, J = 4.8 Hz, 1H), 7.03 (td, J =
9.1, 2.5 Hz, 1H), 7.52 (td, J = 9.9, 3.5 Hz, 2H), 8.23 (s, 1H),
10.97 (s, 1H).
(Z)-6-Fluoro-3-(2-isocyanovinyl)-1H-indole (13). Tan
1
solid, 48 mg, 23%. H NMR (400 MHz, acetone-d6): δ 5.97
(d, J = 8.9 Hz, 1H), 6.93−7.01 (m, 2H), 7.27 (dd, J = 9.7, 2.3
Hz, 1H), 7.77 (dd, J = 8.7, 5.2 Hz, 1H), 8.17 (s, 1H), 10.93 (s,
1H).
1
259 mg, 46%. H NMR (400 MHz, acetone-d6): δ 7.42 (dd,
J = 8.5, 0.6 Hz, 1H), 7.58 (dd, J = 8.6, 1.8 Hz, 1H), 8.22 (s,
1H), 8.56−8.66 (m, 1H), 10.01 (s, 1H).
(Z)-5-Bromo-3-(2-isocyanovinyl)-1H-indole (14). Red
1
solid, 51 mg, 18%. H NMR (400 MHz, acetone-d6): δ 5.96
5-Fluoro-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (21).
1
(d, J = 8.9 Hz, 1H), 6.97 (dt, J = 9.2, 4.7 Hz, 1H), 7.33 (dd, J =
8.6, 1.9 Hz, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.95 (d, J = 1.8 Hz,
1H), 8.20 (d, J = 2.2 Hz, 1H), 11.08 (s, 1H). 13C NMR (101
MHz, acetone): δ 170.77, 135.27, 129.69, 128.97, 128.80,
126.21, 124.63, 121.61, 114.62, 114.27, 110.11.
White solid, 88 mg, 18%. H NMR (400 MHz, acetone-d6):
δ 8.20 (dd, J = 8.8, 2.8 Hz, 1H), 8.31 (dd, J = 2.8, 1.8 Hz, 1H),
8.48 (s, 1H), 10.01 (s, 1H), 11.69 (s, 1H).
Chemical Synthesis of Geranyl Diphosphate Triam-
monium. Geranyl diphosphate was synthesized as described
previously.11 In a 10 mL round-bottom flask purged with
nitrogen, tris(tetrabutylammonium) hydrogen pyrophosphate
(1.0 g, 1.04 mmol) was dissolved in CH3CN (1.0 mL).
Geranyl chloride (0.09 mL, 0.475 mmol) was added, and the
reaction mixture was stirred at room temperature for 2 h.
Dowex 50WX8 Resin Preparation. Dowex 50WX8 resin
(20 g, hydrogen form) was washed with half-saturated aqueous
ammonium chloride (5 × 50 mL) and water (5 × 50 mL) until
the pH of the supernatant equaled 5. The slurry was rinsed
twice with ion exchange buffer (2% isopropyl alcohol in 25
mM aqueous ammonium bicarbonate), loaded into a flash
column, and equilibrated with ion exchange buffer.
(Z)-6-Bromo-3-(2-isocyanovinyl)-1H-indole (15). Red
1
solid, 63 mg, 23%. H NMR (400 MHz, acetone-d6): δ 5.98
(d, J = 8.9 Hz, 1H), 6.85−7.00 (m, 1H), 7.29 (dd, J = 8.5, 1.8
Hz, 1H), 7.67−7.76 (m, 2H), 8.18 (d, J = 1.9 Hz, 1H), 10.99
(s, 1H). 13C NMR (151 MHz, acetone): δ 169.98, 136.50,
127.61, 126.03, 123.70, 123.38, 119.69, 115.57, 114.74, 109.70,
104.77.
(Z)-5-Iodo-3-(2-isocyanovinyl)-1H-indole (16). Red solid,
1
60 mg, 18%. H NMR (400 MHz, acetone-d6): δ 5.98 (d, J =
8.9 Hz, 1H), 7.00 (d, J = 9.0 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H),
7.51 (dd, J = 8.6, 1.7 Hz, 1H), 8.16 (d, J = 3.0 Hz, 2H), 11.03
(s, 1H). 13C NMR (151 MHz, acetone): δ 169.93, 152.01,
134.82, 130.90, 129.54, 127.63, 127.06, 123.71, 114.15, 108.91,
83.50.
Purification. The reaction mixture was concentrated to
afford an orange residue that was diluted with ion exchange
buffer. The crude mixture was chromatographed with two
column volumes of ion exchange buffer (75 mL). The fractions
were combined, concentrated by rotary evaporation, flash-
frozen, and lyophilized for 2 days. The resulting white powder
was diluted with 0.1 M ammonium bicarbonate (4 mL) and
50% isopropyl alcohol/CH3CN (10 mL), vortexed for 30 s and
centrifuged (2000 rpm, rt, 5 min). The organic layer was
extracted, the residual 0.5 mL of yellow liquid was diluted with
50% isopropyl alcohol/CH3CN and the dilution/vortex/
centrifugation process was repeated twice. The combined
organic layers were concentrated to afford a while solid. The
white solid was taken up in 50% isopropyl alcohol/25%
CH3CN/25% 0.1 M aqueous ammonium bicarbonate and
chromatographed with cellulose. The resulting fractions were
combined and lyophilized, affording the title compound as a
white powder (138 mg, 80.3%).
(Z)-3-(2-Isocyanovinyl)-1H-pyrrolo[2,3-b]pyridine (17).
1
White solid, 26 mg, 13%. H NMR (599 MHz, acetone-d6):
δ 1.70−1.84 (m, 0H), 3.56−3.66 (m, 0H), 6.01 (d, J = 8.9 Hz,
1H), 6.97 (dt, J = 9.8, 5.1 Hz, 1H), 7.20 (dd, J = 7.9, 4.7 Hz,
1H), 8.20 (dd, J = 7.9, 1.6 Hz, 1H), 8.26 (s, 1H), 8.35 (dd, J =
4.6, 1.6 Hz, 1H), 11.38 (s, 1H). 13C NMR (151 MHz,
acetone): δ 170.07, 148.22, 144.26, 127.05, 126.63, 123.64,
118.94, 116.61, 108.38, 105.03.
(Z)-5-Fluoro-3-(2-isocyanovinyl)-1H-pyrrolo[2,3-b]-
1
pyridine (18). White solid, 38 mg, 18%. H NMR (400 MHz,
acetone-d6): δ 6.04 (d, J = 8.9 Hz, 1H), 6.95 (dt, J = 9.5, 5.0
Hz, 1H), 8.04 (dd, J = 9.3, 2.7 Hz, 1H), 8.26 (t, J = 2.2 Hz,
1H), 8.33 (d, J = 2.1 Hz, 1H), 11.44 (s, 1H). 13C NMR (151
MHz, acetone): δ 170.20, 156.91, 155.31, 144.88, 132.67,
132.48, 129.23, 123.38, 119.29, 119.25, 112.50, 112.35, 108.61.
(Z)-3-(1H-indol-3-yl)acrylonitrile (19). Yellow solid, 30 mg,
1
16%. H NMR (400 MHz, acetone-d6): δ 5.37 (dd, J = 11.8,
1H NMR (400 MHz, D2O/ND4OD): δ 1.92 (d, J = 1.3 Hz,
3H), 1.98 (s, 3H), 2.01 (d, J = 1.3 Hz, 3H), 2.39 (d, J = 6.5
Hz, 2H), 2.41−2.49 (m, 2H), 5.45−5.53 (m, 1H), 5.74 (dt, J =
6.1, 3.9 Hz, 1H). 13C NMR (151 MHz, D2O/ND4OD): δ
1.1 Hz, 1H), 7.14−7.31 (m, 2H), 7.50−7.60 (m, 1H), 7.67 (d,
J = 11.7 Hz, 1H), 7.77−7.86 (m, 1H), 8.37 (s, 1H), 11.02 (s,
1H).
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Research Article
142.46, 133.67, 124.40, 120.46, 62.61, 39.10, 25.92, 25.16,
17.26, 15.89. 31P NMR (162 MHz, D2O/ND4OD): δ −9.93
(d, J = 21.6 Hz), −6.08 (d, J = 21.6 Hz).
Angeles, California 90095, United States; orcid.org/
0000-0002-2196-4234
Yogan Khatri − Life Sciences Institute, University of Michigan,
Ann Arbor, Michigan 48109-2216, United States;
orcid.org/0000-0002-2432-7679
Shasha Li − Life Sciences Institute and Department of
Medicinal Chemistry, University of Michigan, Ann Arbor,
Michigan 48109-2216, United States
Nikki R. Keramati − Life Sciences Institute, University of
Michigan, Ann Arbor, Michigan 48109-2216, United States
Andrew N. Lowell − Life Sciences Institute, University of
Michigan, Ann Arbor, Michigan 48109-2216, United States;
orcid.org/0000-0001-5357-5279
Structure Determination. Colorless plates of 36 were
grown from a diethyl ether/hexanes solution of the compound
at 4 °C. A crystal of dimensions 0.06 × 0.04 × 0.02 mm was
mounted on a Rigaku AFC10K Saturn 944+ CCD-based X-ray
diffractometer equipped with a low-temperature device and a
Micromax-007HF Cu-target microfocus rotating anode (λ =
1.54187 A) operated at 1.2 kW power (40 kV, 30 mA). The X-
ray intensities were measured at 85(1) K with the detector
placed at a distance 42.00 mm from the crystal. A total of 2028
images were collected with an oscillation width of 1.0° in ω.
The exposure times were 5 s for the low-angle images and 45 s
for the high angle images. Rigaku d*trek images were exported
to CrysAlisPro for processing and corrected for absorption.
The integration of the data yielded a total of 61153 reflections
to a maximum 2θ value of 139.83°, 7384 of which were
independent and 5502 were greater than 2σ(I). The final cell
constants (Table S36) were based on the xyz centroids of 8462
reflections above 10σ(I). An analysis of the data showed
negligible decay during the data collection. The structure was
solved and refined with the Bruker SHELXTL (version 2018/
3) software package, using the space group P212121 with Z = 4
for the formula C44H56N6O. All non-hydrogen atoms were
refined anisotropically with the hydrogen atoms placed in a
combination of idealized and refined positions. Full-matrix
least-squares refinement based on F2 converged at R1 = 0.0885
and wR2 = 0.2349 (for I > 2σ(I)) and R1 = 0.1102 and wR2 =
0.2582 for all data. Additional details are presented in Tables
S38−S44 and are given in the Supporting Information.
K. N. Houk − Department of Chemistry and Biochemistry,
University of California, Los Angeles, Los Angeles, California
90095, United States; orcid.org/0000-0002-8387-5261
Complete contact information is available at:
https://pubs.acs.org/10.1021/acscatal.0c05656
Author Contributions
R.M.H., A.N.L., and D.H.S. designed the research. R.M.H. and
N.R.K. performed all experiments and characterized all
compounds. S.A.N. and Y.K. aided in the expression and
purification of all proteins. J.N.S. performed quantum
mechanical computations, and J.N.S. and K.N.H. wrote the
computational section of the manuscript. S.L. cloned the
expression plasmids for FamD2, FamC1, HpiC1, and FimC5.
A.N.L. and R.M.H. synthesized unnatural cis-indole isonitrile
substrates. R.M.H., S.A.N., and D.H.S. contributed to the
preparation of the manuscript.
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscatal.0c05656.
ACKNOWLEDGMENTS
■
We are grateful to the National Science Foundation under the
CCI Center for Selective C−H Functionalization (CHE-
1700982), National Institutes of Health (R35 GM118101),
and the Hans W. Vahlteich Professorship (to D.H.S.) and ACS
MEDI Pre-Doctoral Fellowship (to R.M.H) for financial
support. J.N.S. acknowledges support from the National
Institute of General Medical Sciences of the National Institutes
of Health (F32 GM122218). The authors thank Dr. Jeffery
Kampf (University of Michigan Department of Chemistry) for
X-ray crystallographic work and Dr. Pavel Nagorny for access
to their polarimeter. Funding from NSF grant CHE-0840456
helped defray costs of small molecule X-ray instrumentation.
Computational resources were provided by the UCLA Institute
for Digital Research and Education (IDRE) and by the San
Diego Supercomputing Center (SDSC) through XSEDE
(ACI-1548562).
Full amino acid sequences, mutagenic primers, TTN
tables, compound characterization tables, mutagenesis
percent conversion tables, mutagenesis HPLC traces,
NMR spectra, MS traces, crystallographic data and
parameters, and computational methods (PDF)
Crystallographic data for 36 (CIF)
AUTHOR INFORMATION
■
Corresponding Author
David H. Sherman − Life Sciences Institute, Department of
Medicinal Chemistry, and Department of Microbiology &
Immunology, Department of Chemistry, University of
Michigan, Ann Arbor, Michigan 48109-2216, United States;
orcid.org/0000-0001-8334-3647; Email: davidhs@
umich.edu
REFERENCES
■
(1) Walton, K.; Berry, J. P. Indole Alkaloids of the Stigonematales
(Cyanophyta): Chemical Diversity, Biosynthesis and Biological
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Authors
Robert M. Hohlman − Life Sciences Institute and Department
of Medicinal Chemistry, University of Michigan, Ann Arbor,
Michigan 48109-2216, United States; orcid.org/0000-
0002-0361-1611
Sean A. Newmister − Life Sciences Institute, University of
Michigan, Ann Arbor, Michigan 48109-2216, United States
Jacob N. Sanders − Department of Chemistry and
Biochemistry, University of California, Los Angeles, Los
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