Use of Crystallography and Molecular Modeling for the Inhibition of the Botulinum Neurotoxin A Protease

Article abstract of DOI:10.1021/acsmedchemlett.1c00325

Botulinum neurotoxins (BoNTs) are extremely toxic and have been deemed a Tier 1 potential bioterrorism agent. The most potent and persistent of the BoNTs is the "A"serotype, with strategies to counter its etiology focused on designing small-molecule inhibitors of its light chain (LC), a zinc-dependent metalloprotease. The successful structure-based drug design of inhibitors has been confounded as the LC is highly flexible with significant morphological changes occurring upon inhibitor binding. To achieve greater success, previous and new cocrystal structures were evaluated from the standpoint of inhibitor enantioselectivity and their effect on active-site morphology. Based upon these structural insights, we designed inhibitors that were predicted to take advantage of π-πstacking interactions present in a cryptic hydrophobic subpocket. Structure-activity relationships were defined, and X-ray crystal structures and docking models were examined to rationalize the observed potency differences between inhibitors.

Full text of DOI:10.1021/acsmedchemlett.1c00325

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Letter
Use of Crystallography and Molecular Modeling for the Inhibition of
the Botulinum Neurotoxin A Protease
Lewis D. Turner, Alexander L. Nielsen, Lucy Lin, Antonio J. Campedelli, Nicholas R. Silvaggi,
Jason S. Chen, Amanda E. Wakefield, Karen N. Allen, and Kim D. Janda*
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ABSTRACT: Botulinum neurotoxins (BoNTs) are extremely
toxic and have been deemed a Tier 1 potential bioterrorism
agent. The most potent and persistent of the BoNTs is the “A”
serotype, with strategies to counter its etiology focused on
designing small-molecule inhibitors of its light chain (LC), a
zinc-dependent metalloprotease. The successful structure-based
drug design of inhibitors has been confounded as the LC is highly
flexible with significant morphological changes occurring upon
inhibitor binding. To achieve greater success, previous and new
cocrystal structures were evaluated from the standpoint of inhibitor
enantioselectivity and their effect on active-site morphology. Based
upon these structural insights, we designed inhibitors that were
predicted to take advantage of π−π stacking interactions present in
a cryptic hydrophobic subpocket. Structure−activity relationships were defined, and X-ray crystal structures and docking models
were examined to rationalize the observed potency differences between inhibitors.
KEYWORDS: BoNT/A, structure-based drug design, enantioselectivity, hydroxamate, structure−activity relationships, π−π stacking
effects such as spread from the injection site, iatrogenic
botulism, and respiratory compromise.13
otulinum neurotoxins (BoNTs) possess incredible
Bpotency and are among the most toxic agents known to
humankind. Seven serotypes (A−G) have been identified,1
with serotype A responsible for the majority (52%) of botulism
cases within the United States between 1975−2009.2 Its
remarkable toxicity (intravenous LD50 = 1−2 ng/kg)3 and
persistence within human tissue (t1/2 = months to years),4
coupled with its ease of production and dissemination, has led
to categorization by the Centers for Disease Control and
Prevention (CDC) as a Tier 1 potential bioterrorism agent.5
BoNTs are produced by a species of diverse family of
anaerobic bacteria known as Clostridium botulinum and also
pose a risk of foodborne botulism from incorrect handling of
food products, particularly canned goods.6,7
The remarkable potency of BoNT/A has been utilized in a
multitude of therapeutic and cosmetic applications in which
aberrant muscular spasticity is implicated, with several Food
and Drug Administration (FDA)-approved formulations
available for various and limited indications.8 Debilitating
disorders such as cerebral palsy,9 chronic migraines,10 and
sialorrhea11 and cosmetic imperfections such as facial
wrinkles12 have been successfully treated using BoNT/A
formulations. The prolific use of BoNT/A for these therapeutic
applications comes with associated risks: misuse, overdose, or
poor injection technique of BoNT/A has established side
The effects associated with BoNT/A intoxication arise from
its ability to prevent exocytotic vesicle fusion at neuronal
termini, thus preventing neurotransmitter release and disrupt-
ing normal muscular function, which results in the flaccid
paralysis that is characteristic of botulism.14 The 150 kDa
BoNT/A holotoxin consists of a heavy chain (HC, 100 kDa)
and a light chain (LC, 50 kDa) that are connected by a single
disulfide bond. Facilitation by the HC binding to cell surface
receptor sites, the LC, a zinc-dependent metalloprotease, is
translocated to the cytosol.15 Once internalized, the LC
selectively cleaves synaptosomal-associated protein 25 (SNAP-
25), a crucial component of the soluble N-ethylmaleimide
sensitive factor (NSF) attachment protein receptor (SNARE)
complex that is responsible for vesicular fusion to the
synaptosomal membrane.16 Current FDA-approved postintox-
ication treatments for BoNT/A are limited to antibody-based
antitoxins which have a short therapeutic window (12−24 h
Received: June 7, 2021
Accepted: July 20, 2021
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postexposure), owing to their inability to enter the neuronal
compartment.17 A recent advance in this field has outlined the
use of a chimeric toxin-based delivery method which utilizes
the intrinsic membrane-penetrating ability of the BoNT/X
transmembrane domain to facilitate the internalization of a
linked BoNT/A-specific antibody. This strategy was successful
in sequestering LC activity in vitro and in vivo.18 Although
promising, there remains a large deficit of postintoxication
treatments for BoNT/A. An alternative strategy is the
development of small-molecule inhibitors.
Currently, several examples of small-molecule inhibitors for
the BoNT/A LC have been identified and primarily function
through active-site zinc ion chelation.19,20 Some of these
inhibitors (Figure 1A) have been cocrystallized within the LC,
Figure 2. (A) DCHA and previous branched derivatives, values for 3
and 4 are IC50. (B) Alternative view of DCHA bound within the
active site of the LC. Structural water molecules are outlined in red
spheres, with nearby polar contacts indicated by yellow dashed lines.
(C) Cocrystal structure of 3 bound within the active site of the LC
(PDB 7N18) determined at a resolution of 2.1 Å. Both R and S
enantiomers are shown. The hydroxyethyl group has displaced one of
the water molecules observed in the structure of the complex with
DCHA. Nearby polar contacts are indicated by yellow dashed lines.
network with amino acid residues His223, Glu351, and Arg363
(Figure 2B). Compounds 3 and 4 were designed with two
purposes in mind: (1) extend additional chemical composition
into the polar space of the active site, and (2) investigate the
effect of chiral inhibitors upon BoNT/A LC inhibition.26
Specifically, the hydroxyethyl appendage was hypothesized to
displace one of the ordered water molecules within the enzyme
active site. Moreover, previous BoNT/A LC inhibition data
revealed a ∼4- and 21-fold preference for the R over the S
enantiomer of 3 and 4, respectively.26 To investigate this
selectivity, we have determined the cocrystal structure of
racemic 3 bound within the BoNT/A LC (Figure 2C).
Although the inhibitor could be unambiguously placed
within the active site, electron density at the stereogenic center
of 3 was poorly defined (Figure S1). As we crystallized with the
racemic mixture, and that both enantiomers had comparable
potencies, we concluded that each enantiomer had similar
occupancies within the LC crystal lattice and an average of the
two structures was observed. Despite this, electron density for
the phenyl ring, the hydroxamate, and the hydroxyethyl
appendage was well-defined, with the hydroxyethyl OH group
displacing one water molecule (Figure 2C) to form a H-bond
network with residues His223, Glu351, and Arg363. Notably,
the inclusion of the 2-chloro substituent in 4 greatly improved
enantiomeric selectivity;26 evaluation of the binding poses of
both DCHA and 3 show the phenyl ring making a π−π
stacking interaction with Phe194. Based on these insights, we
posit that the 2-chloro substituent of 4 acts as a “molecular
brake” and reduces the free rotation of the phenyl ring, causing
a greater chiral effect at the stereocenter.
Figure 1. (A) Previous hydroxamate-based inhibitors of the BoNT/A
LC. (B) Overlay of X-ray crystal structures of DCHA (purple, PDB
2IMA)24 and 1 (cyan, PDB 4HEV)22 bound within the active site of
the LC. The 370 loop adopts an Phe369-in−Asp370-out con-
formation. (C) X-ray crystal structure of 2 (green, PDB 2IMB)24
bound within the active site of the LC. The 370 loop adopts an
Phe369-out−Asp370-in conformation. Hydrogen bonds and cation−π
interactions are outlined in yellow and cyan dashed lines, respectively.
and distinct morphological changes in the S1′ subsite have
been observed (Figure 1B,C). Lipophilic inhibitors 2,4-
dichlorocinnamic hydroxamic acid (DCHA) and adaman-
tane-based inhibitor 1 possess almost identical binding poses
and occupy the S1′ subsite, a small hydrophobic cleft
comprising of residues Phe194, Ile161, Asp370, and Phe369.
The phenyl ring of DCHA forms an offset π−π stack with
Phe194, with the 370 loop adopting a Phe369-in−Asp370-out
conformation with Phe369, forming a partial lid above the
inhibitors.21,22 Conversely, hydrophilic inhibitor L-arginine-
based hydroxamate 2 causes a conformational inward flip of
Asp370, forming H-bond interactions with the guanidinium
moiety of 2.23,24 Moreover, Phe194 has also rotated to form a
cation−π interaction with 2. Additional examples of small-
molecule hydroxamate inhibitors have also outlined a high
degree of flexibility within the S1′ region of the BoNT/A LC.25
The advancement of structural information for the BoNT/A
LC has enabled structure-guided design of enantioselective
chiral inhibitors, as outlined by Stowe et al. (Figure 2A).26
Analysis of the active-site binding pose of DCHA identified
two proximal water molecules involved in an H-bonding
Recently, a central focus of the development of BoNT/A LC
inhibitors has been the irreversible inhibition of the enzyme.
Success in this area has been found through a bifunctional
strategy, wherein coordination to the active-site zinc ion allows
proximal positioning of a pendent electrophilic warhead for the
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simultaneous irreversible modification of a noncatalytic
cysteine.27,28 Previously synthesized 5 possessed an electro-
philic methanethiosulfonate warhead and the same branched
pharmacophore as 3 and 4 and exhibited modest irreversible
inhibition against the BoNT/A LC with a kinact/Ki value of 19.4
M−1·s−1 (Figure 3A).27 As proof-of-concept for this covalent
Compound 2 is the smallest ligand, but had a comparable
pocket size to that of 6. Upon visual inspection of the binding
poses of DCHA and 2, Pro69 exhibited a large shift toward
solvent, resulting in a larger binding pocket for 2 (Figure S2).
We envisioned that we could design inhibitors based upon
the branched pharmacophore of 6 to take advantage of this
unexplored, cryptic, hydrophobic subpocket. Specifically, we
postulated that we could create a tripartite π−π stacking
interaction with Phe163/194 and a phenyl ring by replacement
of the alkyl chain in 6. To bolster this hypothesis, we
conducted docking studies of the benzylamine-based derivative
7 using Glide (Figure 4A).
Figure 4. (A) Docking pose of 7 bound to the BoNT/A LC. (B) 2D
representation of the predicted binding mode of 7 in the BoNT/A LC
active site. Coordination of the hydroxamate and π−π stacking
interactions are outlined in red and blue, respectively.
Figure 3. (A) Previous branched hydroxamate-based inhibitors of the
BoNT/A LC. (B) X-ray crystal structure of 6 bound in the active site
of the LC (PDB 6XCF).27 Accommodation of the alkyl moiety in the
hydrophobic subpocket results in disruption of the face-to-face π−π
stack between the 2,4-dichlorocinnamic phenyl ring and Phe194. (C)
Alternative view of DCHA (purple) bound with the LC. The 2,4-
dichlorocinnamic phenyl ring and Phe194 form an offset face-to-face
π−π stack.
As posited, the benzylamine ring adopted a pose which
occupied the hydrophobic subpocket with both Phe163 and
194 forming offset π−π stacking interactions. We further
strengthened our docking strategy by showing that the
predicted docking pose of 6 had excellent overlap with the
crystallized binding mode of 6, with the alkyl chain occupying
the cryptic hydrophobic subpocket (Figure S3). To investigate
this hypothesis, we designed a series of aniline- and
benzylamine-based derivatives (Figure 5). The design included
potential hydrophobic/hydrogen-bonding contacts derived
through either methyl/ethyl groups as well as OH and NH2
moieties that were strategically placed on the aromatic ring.
approach, previously synthesized 6 was included as a
noncovalent control and was crystallized in complex with the
BoNT/A LC (Figure 3B).27 This structure revealed a new
cryptic hydrophobic subpocket adjacent to the main S1′
binding pocket that was absent in the crystal structure of
DCHA bound to the BoNT/A LC (Figure 3C). The π−π
stacking interaction observed between the 2,4-dichlorophenyl
ring in DCHA and the side chain of Phe194 is not present
upon binding of 6. The induced fit caused by the alkyl moiety
results in a large shift and rotation of the side chain of Phe194,
and together with Phe163, form the edges of the new
hydrophobic subpocket effectively “sandwiching” the alkyl
chain.
Formation of this cryptic hydrophobic subpocket has not
previously been observed; to quantify the size of this additional
binding pocket and the main S1′ pocket, we used Dpocket, a
module within the Fpocket suite, to calculate pocket volumes
(Table S1) for DCHA, 1−3, and 6.29,30 Dpocket defines the
pocket using atoms within 4 Å of the ligand, thus it is expected
that larger ligands give rise to larger binding pockets. With the
exception of 2, the results clearly reflected this expectation,
with 6 filling additional subpockets within the active site
compared to other ligands that only occupied the S1′ pocket.
Figure 5. Aniline- and benzylamine-based target library.
C
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a
Scheme 1. General Route to Benzylamine- and Aniline-Based Final Compounds
a
31 and 32 represent multiple intermediates. Reagents and conditions: (A) RNH2, CHCl3, or CH2Cl2, rt; (B) RNH2, HATU or EDC, DIPEA,
DMF, rt; (C) PPTS, EtOH, 65 °C or 10% TFA−CH2Cl2, rt.
Table 1. IC50 Values for 7−28 When Evaluated against the FRET-Based SNAPtide Assay; See SI-6.1 for Dose Response Curves
a
Average of at least three experimental replicates.
Synthesis of target compounds 7−28 (Scheme 1) began
with ring opening of previously synthesized cyclic anhydride
2927 with O-tetrahydropyran-protected hydroxylamine to
afford the carboxylic acid intermediate 30 in a moderate
yield. Acid 30 was subjected to amide coupling conditions with
various benzylamines to afford protected hydroxamates (31)
that were then deprotected using pyridinium p-toluenesulfo-
nate (PPTS) to yield the final target compounds. Notably,
amide coupling conditions for 30 using less-reactive/ortho-
substituted aniline reagents resulted in no formation of the
desired product. Fortunately, a change in order of steps (route
2) reacting aniline reagents with 29 directly to give carboxylic
acids (32) was successful. Furthermore, optimization of
workup procedures allowed both the amide coupling and
deprotection step to be combined, allowing rapid access to
final compounds 7−28. Further details on the synthetic routes
employed can be found in SI-4.0.
Having established solid synthetic routes, hydroxamates 7−
28 were evaluated for BoNT/A LC inhibition (Table 1) using
a previously described27 fluorescence resonance energy transfer
(FRET)-based assay that uses a truncated form of the BoNT/
A LC (amino acids 1−425) and a SNAPtide substrate.31,32
Compounds were preincubated for 30 min at various
concentrations with 10 nM BoNT/A LC, followed by addition
of 4 μM substrate, and reaction progress fluorescently
monitored at λex/em = 490/523 nm.
The initial target hydroxamate 7 had an IC50 value of 4.0
μM, thus showing no real improvement when compared to the
alkyl-containing counterpart 6 (IC50 = 5.7 μM). This suggested
that the hypothesized π−π stacking interaction with residues
Phe163 and 194 did not provide significant binding
stabilization or was unlikely to be occurring. Addition of a
methyl group in the ortho (8) and meta (9) position produced
a ∼2-fold increase in potency, whereas the para-substituted
D
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compound 10 exhibited minimal effect on the potency. Of
note, addition of an ethyl group (11) in the para position
resulted in a ∼2-fold loss in potency when compared to
unsubstituted benzylamine 7. With the docked structure of 7
(Figure 4A) in mind, this suggested that 11 may be too large,
potentially clashing with Phe192 deep within the hydrophobic
subpocket.
Hydroxamates 12−17 were designed to explore hydrogen-
bonding potential within the hydrophobic subpocket. Sub-
stitution at either the ortho, meta, or para position with polar
OH and NH2 groups for benzylamine-based inhibitors resulted
in a large decrease in potency. This was readily interpreted as
repulsive interactions between the hydrophobic residues that
line the subpocket and the appended polar groups, and that H-
bonding contacts may be untenable. Unsubstituted aniline 18
exhibited a submicromolar IC50 value of 0.8 μM, a ∼7- and 5-
fold increase over 6 and 7, respectively. This indicated
optimum positioning and alignment of the aniline ring for
the predicted π−π stacking interaction with Phe163/194. To
further explore these differences, we conducted docking of 18
and compared the docked pose to that of 7 (Figure 6).
exhibited a loss of potency when compared to unsubstituted
aniline 18. As might be posited from the docking models
(Figure 6), 7 was expected to extend further into the
hydrophobic subpocket than 18; hence, the polar groups
would be better tolerated when placed toward the entrance of
the hydrophobic subpocket. This premise was bolstered by the
superior potency of ortho-substituted compounds 19, 23, and
26 when compared to their meta-substituted (20, 24, and 27)
and para-substituted (21, 25, and 28) analogues. Ethyl-
containing aniline 22 also exhibited a loss in potency but was
still superior to its benzylamine counterpart 11. The reduced
extendibility of the aniline compounds may allow for larger
groups in the para position to be better accommodated deep
within the hydrophobic subpocket. Compound 23 exhibited an
IC50 of 0.3 μM and was the most potent compound evaluated.
The improved potency of this compound when compared to
that of 24 and 25 indicated that there may be a H-bond
forming between the phenol and residues near the entrance of
the hydrophobic subpocket. However, this potency increase
was not observed for dianiline 26, which also possessed the
capacity to H-bond. Therefore, it was unclear what the precise
reasoning was for the superior potency of 23. In summary,
aniline-based molecules 18−28 exhibited superior inhibitory
potency when compared to that of benzylamine-based
structures 7−17, which we propose is facilitated through
π−π stacking interactions with the aniline phenyl ring and
Phe163/194.
To further investigate the effect of chirality upon inhibitor
binding, we employed chiral supercritical fluid chromatography
(SFC) to separate enantiomers of the three best inhibitors 18,
19, and 23. This approach allowed us rapid access to
enantiopure material without undertaking the cumbersome
asymmetric synthetic approach outlined previously;26 details
pertaining to SFC are outlined in SI-4.4. Relative stereo-
chemistry of enantiomers was assigned through their specific
rotation of plane-polarized light and their BoNT/A LC
inhibition was assessed using the FRET-based assay (Table 2).
Differences in inhibitory potency was observed, with the (+)
configuration being slightly preferred. These hydroxamates did
not exhibit the same magnitude of enantiomer selectivity that
was observed for 3 and 4.26 The lack of the hydroxyethyl
moiety and subsequent loss of the H-bond network with amino
acids His223, Glu351, and Arg363 (Figure 2C) for inhibitors
18, 19, and 23 suggested that this interaction was crucial for
the development of exquisitely enantioselective inhibitors of
BoNT/A LC using this pharmacophore. The fluid morphology
Figure 6. (A) Docking poses of 7 (blue) and 18 (yellow) bound
within the BoNT/A LC. (B) 2D representation of the predicted π-
sandwich interaction between Phe163 and 194 and the aniline ring of
18. The π−π stacking interactions are outlined in blue.
The aniline phenyl ring in 18 was predicted to form a double
offset π−π interaction with Phe163 and 194 (Figure S4) and
exhibited a more planar orientation with respect to the
phenylalanine ring systems than what was observed for 7. The
additional methylene group present in 7 was foreseen to
disrupt the planarity of the π−π stacking interactions and
could account for the lower potency observed for the
benzylamine-based compounds. Addition of a methyl group
in the ortho position (19) improved the potency, whereas the
meta-substituted (20) and para-substituted (21) derivatives
Table 2. IC50 Values for Enantiopure Compounds 18, 19, and 23
a
Average of at least three independent experiments.
E
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of the BoNT/A LC active site, coupled with the fact that both
groups flanking the stereocenter are aromatic, suggests that
both ring systems may have the capacity to occupy both the
main S1′ pocket and the hydrophobic subpocket.
Amanda E. Wakefield − Department of Biomedical
Engineering and Department of Chemistry, Boston University,
Boston, Massachusetts 02215, United States; orcid.org/
0000-0001-7962-2686
In summary, we have designed, synthesized, and biochemi-
cally evaluated a comprehensive series of chiral benzylamine-
and aniline-based inhibitors of the BoNT/A LC. With the aid
of cocrystal structures and docking, molecules were designed
to take advantage of a π−π stacking interaction to improve
inhibitor potency. Aniline-based hydroxamates possessed
superior potency compared to that of the benzylamine-based
hydroxamates, which was attributed to a more planar
orientation of the π−π stacking interaction, as predicted by
docking. Moreover, the cocrystal structure of 3 has revealed
that displacement of an active-site water molecule by the
hydroxyethyl moiety is crucial to enantiomer selectivity. We
envision that both occupation of the hydrophobic subpocket
by aniline functionalities and the crucial H-bond network
interaction obtained from the hydroxyethyl moiety can be
combined into one entity that will possess not only improved
potency but also greater enantioselectivity; work on such
compounds is currently being conducted.
Karen N. Allen − Department of Chemistry, Boston University,
Boston, Massachusetts 02215, United States; orcid.org/
0000-0001-7296-0551
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00325
Author Contributions
L.D.T. conceptualized the study; L.D.T., A.L.N., and A.J.C.
carried out chemical synthesis and characterization; L.D.T.,
L.L., and A.J.C. carried out biochemical assays and data
analysis; N.R.S. carried out crystallization studies and refine-
ment; A.L.N. and A.E.W. performed computational modeling
and calculations; J.S.C. carried out and overlooked SFC;
L.D.T. wrote the original draft of the manuscript, and all
authors revised the final version; K.N.A. and K.D.J. acquired
funding and supervised the study.
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00325.
■
ACKNOWLEDGMENTS
sı
■
The authors thank James Conley for his assistance regarding
crystallography, Dr. Joseph Barbieri for providing truncated
BoNT/A LC, and Emily Sturgell and Brittany Sanchez for their
contribution to SFC. This work was supported by the National
Institutes of Health Grant No. R01 AI153298, the Fulbright
Scholar Program (A.L.N.), the Natural Sciences and Engineer-
ing Research Council of Canada PGSD3-502274 (L.L.), and
the Skaggs Institute for Chemical Biology (L.L. and K.D.J.).
This is Scripps Research manuscript #30105.
Supporting figures, tables, biological materials and
methods, chemistry experimental details, IC50 curves,
compound HPLC chromatograms, and NMR spectra
(PDF)
Accession Codes
X-ray diffraction data, coordinates, and structure factors for the
X-ray crystal structure of 3 bound within the BoNT/A LC is
deposited on the PDB under accession code 7N18.
ABBREVIATIONS
■
BoNT/A, botulinum neurotoxin A; CDC, Center for Disease
Control and Prevention; DCHA, 2,4-dichlorocinnamic hy-
droxamic acid; DIPEA, diisopropylethylamine; DMF, dime-
thylformamide; EDC, 1-ethyl-3-(3-(dimethylamino)propyl)-
carbodiimide; FDA, Food and Drug Administration; FRET,
fluorescence resonance energy transfer; HATU, 1-[bis-
(dimethlyamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate; HC, heavy chain;
LC, light chain; NSF, N-ethylmaleimide sensitive factor; PDB,
protein data bank; PPTS, pyridinium p-toluenesulfonate; SFC,
supercritical fluid chromatography; SI, Supporting Informa-
tion; SNARE, NSF attachment protein factor; SNAP-25,
synaptosomal-associated protein-25; TFA, trifluoroacetic acid
AUTHOR INFORMATION
Corresponding Author
Kim D. Janda − Department of Chemistry, Scripps Research,
La Jolla, California 92037, United States; orcid.org/
0000-0001-6759-4227; Email: kdjanda@scripps.edu
■
Authors
Lewis D. Turner − Department of Chemistry, Scripps
Research, La Jolla, California 92037, United States;
orcid.org/0000-0003-0660-8247
Alexander L. Nielsen − Department of Chemistry, Scripps
Research, La Jolla, California 92037, United States;
orcid.org/0000-0003-1195-0143
Lucy Lin − Department of Chemistry, Scripps Research, La
Jolla, California 92037, United States; orcid.org/0000-
0001-9546-6919
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