Cyclic Peptide Mimetic of Damaged Collagen

Article abstract of DOI:10.1021/acs.biomac.0c00103

Collagen is the most abundant protein in humans and the major component of human skin. Collagen mimetic peptides (CMPs) can anneal to damaged collagen in vitro and in vivo. A duplex of CMPs was envisioned as a macromolecular mimic for damaged collagen. The duplex was synthesized on a solid support from the amino groups of a lysine residue and by using olefin metathesis to link the N termini. The resulting cyclic peptide, which is a monomer in solution, binds to CMPs to form a triple helix. Among these, CMPs that are engineered to avoid the formation of homotrimers but preorganized to adopt the conformation of a collagen strand exhibit enhanced association. Thus, this cyclic peptide enables the assessment of CMPs for utility in annealing to damaged collagen. Such CMPs have potential use in the diagnosis and treatment of fibrotic diseases and wounds.

Full text of DOI:10.1021/acs.biomac.0c00103

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Article
A Cyclic Peptide Mimetic of Damaged Collagen
Aubrey J. Ellison, I. Caglar Tanrikulu, Jesús M. Dones, and Ronald T. Raines
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.0c00103 • Publication Date (Web): 11 Mar 2020
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Biomacromolecules
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A Cyclic Peptide Mimetic of Damaged Collagen
Aubrey J. Ellison,† I. Caglar Tanrikulu,§,‡ Jesús M. Dones,†,‡ and Ronald T. Raines*,†,§,‡
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†Department of Chemistry and §Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706,
United States
‡Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
ABSTRACT: Collagen is the most abundant protein in
humans and the major component of human skin. Colla-
gen mimetic peptides (CMPs) can anneal to damaged col-
lagen in vitro and in vivo. A duplex of CMPs was envi-
sioned as a macromolecular mimic for damaged collagen.
The duplex was synthesized on a solid support from the
amino groups of a lysine residue and by using olefin me-
tathesis to link the N termini. The resulting cyclic peptide,
which is a monomer in solution, binds to CMPs to form a
triple helix. Among these, CMPs that are engineered to
avoid the formation of homotrimers but preorganized to
adopt the conformation of a collagen strand exhibit en-
hanced association. Thus, this cyclic peptide enables the
assessment of CMPs for utility in annealing to damaged
collagen. Such CMPs have potential use in the diagnosis
and treatment of fibrotic diseases and wounds.
Figure 1. Scheme showing the molecular mimicry of damaged col-
lagen by a cyclic “host” of two parallel collagen strands.
assembly of many possible complexes. To make a duplex
more amenable to rigorous analyses and more biomi-
metic, we sought to tether two parallel strands at both ter-
mini (Figure 1). This design not only minimizes the con-
formational entropy of the duplex, but also mimics natu-
ral collagen fibers that have a disrupted triple helix, as
might be found in damaged collagen. Here, we describe
the creation of a “host” and report on its interaction with
“guest” strands, that is, CMPs.
Keywords: fibrosis; host–guest; macrocycle; triple helix;
wound
INTRODUCTION
The collagen triple helix is the most abundant structure
adopted by a biopolymer within the human body. There,
collagen comprises 1/3 of the total protein, accounts for 3/4
of the dry weight of skin, and is the most prevalent com-
ponent of the extracellular matrix.1
EXPERIMENTAL METHODS
Collagen is damaged in fibrotic and other diseases
and in wounds. Collagen mimetic peptides (CMPs) can
anneal to damaged collagen.2 Such annealing could allow
for the delivery of diagnostic or therapeutic agents that
are conjugated to the CMP. Indeed, we have used such
CMP conjugates to anneal fluorescent dyes,3 a growth
factor,4 and even a sunscreen5 to natural collagen. Dam-
aged collagen in a human body is, however, a complex
target that confounds physicochemical analyses, compli-
cating the assessment of therapeutic potential for new
CMP designs.
We sought to develop a molecular mimic of damaged
collagen. We envisioned doing so with a collagen “du-
plex”, that is, two crosslinked CMPs. Several double-
stranded duplexes have been synthesized and used to
form collagen triple helices.6 In most of these precedents,
one or both ends of the peptides are free, allowing for the
General. Boc-flp-OH and Fmoc-flp-OH were from
OmegaChem (Lévis, Québec). (Here, “flp” refers to
(2S,4S)-4-fluoroproline.) Boc-Flp-OH, Fmoc-Hyp-OH,
and other amino acid derivatives, resins, Fmoc-OSu, and
HOBt were from Chem-Impex International (Wood Dale,
IL). (Here, “Flp” refers to (2S,4R)-4-fluoroproline and
“Hyp” refers to (2S,4R)-4-hydroxyproline.) DIC and 4-
methyl-piperidine were from Oakwood Chemical (Estill,
SC). Streptavidin-coated fluorescent blue particles were
product SVFP-106805 from Spherotech (Lake Forest,
IL). 6-Aminohexanoic acid and all other reagents were
from Sigma–Aldrich (St. Louis, MO) and were used with-
out further purification.
DMF was dried with a Glass Contour system from
Pure Process Technology (Nashua, NH). In addition,
DMF was passed through an associated isocyanate
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“scrubbing” column to remove any amines. Water was
purified with an Arium Pro system from Sartorius (Göt-
tingen, Germany).
+ H]+ calcd, 354.17; found, 354.17. 1H NMR (400 MHz,
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MeOD, δ): 7.78 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 7.5 Hz,
2H), 7.37 (t, J = 7.4 Hz, 2H), 7.33–7.26 (m, 2H), 4.33 (d,
J = 6.8 Hz, 2H), 4.18 (t, J = 6.9 Hz, 1H), 3.08 (t, J = 7.0
Hz, 2H), 2.27 (t, J = 7.4 Hz, 2H), 1.60 (p, J = 7.5 Hz, 2H),
1.49 (p, J = 7.1 Hz, 2H), 1.33 (p, J = 10.1, 6.0 Hz, 2H).
13C NMR (101 MHz, MeOD, δ): 157.49, 143.95, 141.20,
127.34, 126.71, 124.75, 119.50, 66.11, 47.13, 40.17,
33.71, 29.18, 25.97, 24.45.
Boc-Flp-OBn. Boc-Flp-OH (2.5 g, 10.7 mmol) was
dissolved in DMF. Solid Cs2CO3 (1.5 g, 10.7 mmol) was
added, and the reaction mixture was stirred for 10 min.
Benzyl bromide (1.27 mL, 10.7 mmol) was added drop-
wise, and the mixture was stirred for 16 h. The mixture
was then concentrated under reduced pressure. Crude
product was purified by chromatography on silica gel,
eluting with EtOAc (10% v/v) in hexanes to yield product
(2.26 g, 65%). HRMS–ESI (m/z): [M + H]+ calcd, 324.15;
found, 324.16. 1H NMR (400 MHz, CDCl3, δ): 7.35 (d, J
= 4.5 Hz, 5H), 5.32–5.19 (m, 1H), 5.19–5.14 (m, 1H),
5.14–5.04 (m, 1H), 4.58–4.38 (m, 1H), 3.99–3.75 (m,
1H), 3.61 (ddt, J = 36.1, 13.0, 3.9 Hz, 1H), 2.70–2.47 (m,
1H), 2.20–1.97 (m, 1H), 1.53–1.31 (m, 9H), which are
consistent with literature values for this known com-
pound.7
Boc-flp-Flp-OBn. Boc-Flp-OBn (2.26 g, 6.99 mmol)
was dissolved in 4 N HCl (8.0 mL), and the reaction mix-
ture was stirred for 30 min. The reaction mixture was then
concentrated under reduced pressure. The residue was
dissolved in DMF. DIEA (4.87 mL, 27.96 mmol) was
added dropwise. Solid PyBrOP (3.91 g, 8.39 mmol) and
Boc-flp-OH (1.79 g, 7.69 mmol) were added, and the re-
action mixture was stirred for 16 h. The residue was con-
centrated under reduced pressure, taken up in EtOAc, and
washed successively with 1.0 M HCl (2×), saturated
aqueous NaHCO3 (2×), and brine (2×). The organic layer
was dried over Na2SO4(s), filtered, and concentrated un-
der reduced pressure to yield crude product (3.96 g),
which was carried forward without further purification.
HRMS–ESI (m/z): [M + H]+ calcd, 439.20; found,
439.20. 1H NMR (400 MHz, CDCl3, δ): 7.35 (d, J = 4.0
Hz, 6H), 5.32–5.10 (m, 4H), 4.55 (dt, J = 20.6, 8.4 Hz,
1H), 4.06–3.77 (m, 4H), 3.66 (ddt, J = 36.6, 13.0, 3.5 Hz,
1H), 2.62 (dddt, J = 28.3, 16.6, 8.0, 1.8 Hz, 1H), 2.22–
1.99 (m, 2H), 1.85 (ddt, J = 71.3, 13.3, 6.7 Hz, 1H), 0.94
(d, J = 6.7 Hz, 3H), 0.85 (dd, J = 6.7, 2.3 Hz, 4H). 13C
NMR (101 MHz, CDCl3, δ): 172.16, 172.04, 155.00,
154.56, 135.47, 135.28, 128.65, 128.58, 128.52, 128.42,
128.33, 128.16, 92.65, 91.86, 90.86, 90.08, 77.25, 71.88,
71.83, 67.09, 67.02, 60.40, 57.72, 57.48, 53.58, 53.35,
53.15, 52.93, 37.77, 37.54, 36.71, 36.48, 28.36, 28.17,
27.98, 27.86, 19.03, 18.96, 18.92, 14.22.
The phrase “concentrated under reduced pressure”
refers to the removal of solvents and other volatile mate-
rials with a rotary evaporator at water-aspirator pressure
(<20 torr) while maintaining a water bath below 40 °C.
Residual solvent was removed from samples with a high
vacuum (<0.1 torr).
All procedures were performed in air at ambient tem-
perature (~22 °C) and pressure (1.0 atm) unless indicated
otherwise.
Instrumentation. Solid-phase peptide synthesis
was performed with a Liberty Blue Peptide Synthesizer
from CEM (Matthews, NC). Synthetic peptides were pu-
rified by HPLC with a Prominence instrument from Shi-
madzu (Kyoto, Japan) equipped with a VarioPrep 250/21
C18 column from Macherey–Nagel (Düren, Germany).
Molecular mass was determined by matrix-assisted laser
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desorption/ionization–time-of-flight
(MALDI–TOF)
mass spectrometry on an α-cyano-4-hydroxycinnamic
acid or sinapic acid matrix with a microflex LRF™ in-
strument from Bruker (Billerica, MA). Purity analyses
were performed with an Acquity UPLC® H-Class system
from Waters that was equipped with an Acquity photodi-
ode array detector, Acquity quaternary solvent manager,
Acquity sample manager with a flow-through needle, Ac-
quity UPLC® BEH C18 column (2.1 × 50 mm, 1.7-µm
particle size) and Empower 3 software. 1H and 13C NMR
spectra were acquired with an Avance III 400 spectrome-
ter from Bruker. Sedimentation equilibrium experiments
were performed with an XL-A analytical ultracentrifuge
and An-60 Ti rotor from Beckman Coulter (Brea, CA) at
the Biophysics Instrumentation Facility of the University
of Wisconsin–Madison (UW BIF). Beads were imaged
using a Eclipse Ti inverted confocal microscope from
Nikon (Melville, NY) at the Biochemistry Optical Core
of the University of Wisconsin−Madison. Flow cytome-
try was performed with an Accuri Flow Cytometer with
C-Sampler from BD (San Jose, CA) at the UW BIF. CD
data were acquired with a 420 CD spectrophotometer
from Aviv Biomedical (Lakewood, NJ) at the UW BIF.
Small-Molecule Synthesis. Fmoc-6-aminohexa-
noic acid. 6-Aminohexanoic acid (1.00 g, 7.62 mmol)
was dissolved in a saturated aqueous solution of NaHCO3
(50 mL). In a separate flask, Fmoc-OSu (2.82 g,
8.38 mmol) was dissolved in dioxane (50 mL). The two
solutions were combined, and the reaction mixture be-
came cloudy and was stirred for 16 h. The mixture was
then concentrated under reduced pressure. The residue
was dissolved in EtOAc and washed with aqueous 1.0 M
HCl and brine. The organic layer was dried over
Na2SO4(s), decanted, and concentrated under reduced
pressure. Crude product was purified by chromatography
on silica-gel, eluting with EtOAc (40% v/v) and acetic
acid (1% v/v) in hexanes to yield Fmoc-6-aminohexanoic
acid (2.56 g, 95%) as a white solid. HRMS–ESI (m/z): [M
Fmoc-Gly-flp-Flp-OBn. Crude Boc-flp-Flp-OBn
(3.96 g) was dissolved in 4 N HCl (8.0 mL), and the re-
action mixture was stirred for 30 min. The mixture was
then concentrated under reduced pressure. The residue
was dissolved in DCM. DIEA (3.65 mL, 20.97 mmol)
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Biomacromolecules
was added dropwise to the resulting solution. Solid Fmoc- acid was single-coupled at a 0.10-mmol scale. Additional
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Gly-OPfp (3.24 g, 6.99 mmol) was added, and the reac-
tion mixture was stirred for 16 h. The residue was con-
centrated under reduced pressure, taken up in EtOAc, and
washed successively with 1.0 M HCl (2×), saturated
aqueous NaHCO3 (2×), and brine (2×). The organic layer
was dried over Na2SO4(s), filtered, and concentrated un-
der reduced pressure. Crude product was purified by
chromatography on silica gel, eluting with dichloro-
methane followed by a methanol flush to yield product
(3.70 g, 83%). HRMS–ESI (m/z): [M + NH4]+ calcd,
635.23; found, 635.27. 1H NMR (400 MHz, CDCl3, δ):
7.75 (d, J = 7.5 Hz, 2H), 7.59 (dd, J = 7.5, 3.2 Hz, 2H),
7.43–7.26 (m, 9H), 5.86–5.48 (m, 1H), 5.44–5.02 (m,
4H), 4.85–4.67 (m, 2H), 4.45–4.02 (m, 4H), 4.03–3.60
(m, 3H), 2.62 (ddd, J = 20.3, 14.1, 8.1 Hz, 1H), 2.56–2.22
(m, 2H), 2.08 (dddd, J = 40.9, 18.3, 9.1, 4.8 Hz, 1H), 1.82
(s, 2H). 13C NMR (101 MHz, CDCl3, δ): 171.14, 168.75,
167.23, 156.27, 143.88, 141.26, 135.40, 128.58, 128.39,
128.29, 127.69, 127.10, 125.21, 119.95, 91.95, 77.25,
67.21, 57.96, 57.13, 47.08, 43.41, 35.31, 34.48, 34.26.
Fmoc-Gly-flp-Flp-OH. (3.70 g, 5.98 mmol) was dis-
solved in methanol (25 mL). The head space was purged
with N2(g). Pd/C (10% w/w, 0.64 g) was added, and the
flask was capped with a septum. H2(g) was added via a
balloon. The reaction was monitored by thin-layer chro-
matography and observed to be complete at 6 h. The re-
action mixture was filtered through diatomaceous earth
and concentrated under reduced pressure. Crude product
was purified by chromatography on silica gel, eluting
with 1% v/v acetic acid and 20% v/v methanol in EtOAc
to yield product (2.95 g, 93%). HRMS–ESI (m/z): [M –
amino acid and small-molecule carboxylic acid additions
were done at a 0.10-mmol scale and double-coupled.
MALDI (m/z): [M + H]+ calcd, 5757.05; found, 5757.07.
A 0.05-mmol scale synthesis afforded 7.6 mg (2.7%) of
host-o after purification.
Host-c. The closed host was synthesized by olefin
metathesis on the N-terminal 4-butenoic acids of the open
next on resin, following a procedure similar to that for the
stapling of peptide side chains.8 Peptide bound to resin
was added to a Schlenk flask. The resin was then dried for
at least 3 h on a high-vacuum manifold. Then, the flask
was purged rigorously with N2(g). While under N2(g), the
resin was pre-swelled in 2.5 mL of dry CH2Cl2 for at least
15 min prior to the addition 0.5 mL of a 2.5 mM solution
of the Grubbs G2 catalyst (C46H65Cl2N2PRu)9 in dry
CH2Cl2 using standard Schlenk techniques. The reaction
flask was equipped quickly with an oven-dried reflux
condenser, purged with N2(g), and heated in an oil bath at
40 °C for 36 h under N2(g). After 36 h, another 0.5-mL
aliquot of the Grubbs G2 catalyst solution was added, and
the flask was heated at 40 °C for another 36 h under N2(g).
Dry solvent was added over the course of the reaction to
maintain at least 3 mL of CH2Cl2. The reaction mixture
was then allowed to cool to room temperature and fil-
tered. The resin was washed with DCM to remove any
remaining catalyst. MALDI (m/z): [M + H]+ calcd,
5729.02; found, 5728.55. A 0.05-mmol scale synthesis
afforded 7.0 mg (2.4%) of host-c after purification.
Host-r. Following olefin metathesis, the resin was fil-
tered to remove catalyst and a small sample was taken to
confirm the generation of host-c by MALDI mass spec-
trometry. The resin was returned to a Schlenk flask and
suspended in DCE. Again following a literature prece-
dent,10 Umicore M2 (46.5 mg, 0.05 mmol) and Et3SiH
(0.80 mL, 9.4 mmol) were added to the flask. The vessel
was capped with a septum, and the reaction mixture was
heated to 60 °C for 72 h. The resin was washed with DCM
to remove any remaining catalyst. MALDI (m/z): [M +
H]+ calcd, 5731.03; found, 5731.67. A 0.05-mmol scale
synthesis afforded 9.8 mg (3.4%) of host-r after purifica-
tion.
Host-o–biotin and host-r–biotin. Biotin was conju-
gated to the Nε-amino group of a lysine residue installed
near the C-terminus of host-o and host-r. Specifically,
Fmoc-Lys(Boc)-OH was coupled to TGT S RAM resin
(0.22 mmol/g) followed by a (Gly-Ser)3 sequence synthe-
sized by the addition of Fmoc-protected amino acids. The
remainder of the host-o and host-r syntheses followed as
described above.
After cleavage from the resin, crude host-o (5.3 mg,
0.84 µmol) was dissolved in 500 µL of DMSO. A solution
of biotin–NHS ester (4.0 mg, 11.7 µmol) and DIEA (0.1
mL, 0.57 mmol) in 500 µL of DMSO was added, and the
mixture was allowed to react for 12 h. The host-o–biotin
conjugate was purified by HPLC. MALDI (m/z): [M +
H]+ calcd, 6543.38; found, 6541.63. A 0.05-mmol scale
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1
H]− calcd, 526.52; found, 526.18. H NMR (400 MHz,
MeOD, δ): 7.81 (d, J = 7.6 Hz, 2H), 7.74–7.64 (m, 2H),
7.48–7.28 (m, 4H), 5.49–5.03 (m, 3H), 4.72–4.20 (m,
4H), 4.20–3.43 (m, 5H), 2.85–2.27 (m, 3H), 2.26–2.06
(m, 1H). 13C NMR (101 MHz, CDCl3, δ): 177.16, 173.49,
172.00, 160.94, 147.71, 145.13, 132.21, 131.59, 131.14,
130.97, 130.78, 128.99, 123.79, 96.63, 96.29, 94.84,
94.48, 71.07, 68.30, 61.94, 61.35, 50.96, 46.99, 39.48,
39.26, 38.24, 38.03.
Peptide Synthesis. Peptides were prepared by au-
tomated solid-phase peptide synthesis. Fmoc-deprotec-
tion was achieved by treatment with 4-methyl-piperidine
(20% v/v) in DMF. Tripeptides, amino acids, and small-
molecule carboxylic acids (5 equiv) were activated by us-
ing DIC and HOBt. Peptides were cleaved from the resin
with 96.5:2.5:1.0 TFA/H2O/TIPSH (5 mL), precipitated
from diethyl ether at −80 °C, and isolated by centrifuga-
tion. Peptides were purified by preparative HPLC using a
gradient of 0–100% B over 50 min (A: H2O containing
0.1% v/v TFA; B: acetonitrile containing 0.1% v/v TFA).
The purity of each peptide was assessed to be >95% by
UPLC.
Host-o. The open host was synthesized by first doing
a 0.05-mmol coupling of Fmoc-Lys(Fmoc)-OH to TGT S
RAM resin (0.22 mmol/g). Next, Fmoc-6-aminohexanoic
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synthesis afforded 0.4 mg (0.52%) of host-o–biotin after
purification.
After cleavage from the resin, crude host-r (4.3 mg,
(5 equiv) was converted to an active ester by using DIC
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and HOBt. The peptide was then cleaved from the resin.
A solution of 5(6)-carboxyfluorescein (9.7 mg, 25.7
µmol), HATU (9.1 mg, 23.9 µmol), and DIEA (100 µL,
0.57 mmol) in 500 µL DMSO was allowed to react for 15
min. A solution of crude Ac-Lys-(Ser-Gly)3-(D-Pro-D-
Pro-Gly)7 (36.0 mg) dissolved in 500 µL of DMSO was
added to the solution and allowed to react for 12 h. The
peptide was then purified by HPLC. MALDI (m/z): [M +
Na]+ calcd, 2759.20; found, 2760.47. A 0.05-mmol syn-
thesis afforded 2.5 mg (1.8%) of Ac-Lys(fluorescein)-
(Ser-Gly)3-(D-Pro-D-Pro-Gly)7 after purification.
Biotin–fluorescein. Fmoc-Lys(Boc)-OH was coupled
first to TGT S RAM resin (0.22 mmol/g) followed by a
(Gly-Ser)3 sequence that was synthesized by the addition
of Fmoc-protected amino acids. The resulting decapep-
tide was treated on-resin with biotin–NHS ester (5 equiv).
The peptide was then cleaved from the resin and depro-
tected. A solution of 5(6)-carboxyfluorescein (9.5 mg,
25.2 µmol), HATU (8.7 mg, 22.8 µmol), and DIEA (100
µL, 0.57 mmol) in 500 µL DMSO were incubated for 15
min. This solution was added to a solution of crude biotin-
(Gly-Ser)3-Lys-NH2 (12.3 mg) in 500 µL of DMSO, and
the mixture was allowed to react for 12 h. The biotin–flu-
orescein conjugate was then purified by HPLC. MALDI
(m/z): [M + H]+ calcd, 1162.41; found, 1162.52. A 0.05-
mmol synthesis afforded 1.5 mg (2.5%) of biotin-(Gly-
Ser)3-Lys(fluorescein)-NH2 after purification.
Analytical Ultracentrifugation. A cyclic peptide
(100 µL of a 45 µM solution) and matching buffer
(110 µL) were placed in a cell with an Epon 12 mm dou-
ble-sector charcoal-filled centerpiece from Beckman
Coulter. Experiments were run at 4 °C for more than
seven days at speeds of 20,000, 26,000, 34,000, and
42,000 rpm, and gradients recorded at 235 nm were mon-
itored until superimposable 4 h apart. Equilibrium gradi-
ents at 4 °C were modeled as single and multiple non-
interacting species through nonlinear least-squares fits to
the gradient data, using a buffer density of 1.00037 g/mL
and partial specific volumes of 0.7471 and 0.7460 mL/g
calculated based on amino acid and functional group con-
tent for host-o and host-r, respectively.11 Non-sediment-
ing baseline-attenuance was applied during data analysis,
which was performed with programs written by D. R.
McCaslin (UW BIF) for IGOR PRO software from
WaveMetrics (Lake Oswego, OR).
Confocal Microscopy and Flow Cytometry. A
200-µL suspension of streptavidin-coated fluorescent
blue particles was added to 200 µL of a 58-µM solution
of biotinylated host-o or host-r in H2O, and the mixture
was agitated for 9 h. Beads were pelleted by centrifuga-
tion at 12,000 rpm for 2 min. The supernatant was re-
moved, and the beads were resuspended in 200 µL of H2O
and stored at 4 °C.
0.68 mmol) was dissolved in 500 µL of DMSO. A solu-
tion of biotin–NHS ester (6.7 g, 19.6 µmol) and DIEA
(0.1 mL, 0.57 mmol) in 500 µL of DMSO was added, and
the mixture was allowed to react for 12 h. The host-r–bi-
otin conjugate was purified by HPLC. MALDI (m/z): [M
+ H]+ calcd, 6517.37; found, 6519.48. A 0.05-mmol scale
synthesis afforded 0.2 mg (0.14%) of host-r–biotin after
purification.
(flp-Hyp-Gly)7. (flp-Hyp-Gly)7 was synthesized by
the addition (double-coupling) of Fmoc-protected amino
acids to preloaded Fmoc-Gly-Wang resin (0.65 mmol/g).
MALDI (m/z): [M + H]+ calcd, 2015.80; found, 2015.10.
A 0.05-mmol scale synthesis afforded 6.4 mg (6.4%) of
(flp-Hyp-Gly)7 after purification.
(flp-Flp-Gly)7. (flp-Flp-Gly)7 was synthesized by us-
ing Fmoc-Gly-flp-Flp-OH tripeptide and Fmoc-flp-OH
and Fmoc-Flp-OH monomers. Six segment condensa-
tions of tripeptide were followed by the addition of each
monomer, with each addition being double-coupled on
preloaded Fmoc-Gly-Wang resin (0.65 mmol/g). The
peptide was then cleaved from the resin and purified by
HPLC. MALDI (m/z): [M + H]+ calcd, 2028.77; found,
2028.75. A 0.05-mmol scale synthesis afforded 8.3 mg
(8.1%) of (flp-Flp-Gly)7 after purification.
(Pro-Pro-Gly)7. (Pro-Pro-Gly)7 was synthesized by
the addition of Fmoc-protected amino acids to preloaded
Fmoc-Gly-Wang resin (0.65 mmol/g). MALDI (m/z): [M
+ H]+ calcd, 1777.02; found, 1776.87. A 0.05-mmol scale
synthesis afforded 18.0 mg (20.2%) of (Pro-Pro-Gly)7 af-
ter purification.
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(Pro-Ile-Gly)7. (Pro-Ile-Gly)7 was synthesized by the
addition of Fmoc-protected amino acids to preloaded
Fmoc-Gly-Wang resin (0.65 mmol/g). MALDI (m/z): [M
+ H]+ calcd, 1890.31; found, 1890.30. A 0.05-mmol scale
synthesis afforded 9.6 mg (10.2%) of (Pro-Ile-Gly)7 after
purification.
Fluorescein–CMP.
Ac-Lys-(Ser-Gly)3-(Pro-Pro-
Gly)7 was synthesized by the addition of Fmoc-protected
amino acids to preloaded Fmoc-Gly-Wang resin (0.65
mmol/g) and then cleaved from the resin. 5(6)-Carbox-
yfluorescein (112.4 mg, 0.30 mmol), HATU (104.8 mg,
0.28 mmol), and DIEA (100 µL, 0.57 mmol) were incu-
bated for 15 min in 500 µL of DMSO. This solution was
added to a solution of crude Ac-Lys-(Ser-Gly)3-(Pro-Pro-
Gly)7 (74.2 mg) in 500 µL of DMSO, and the mixture was
allowed to react for 12 h. MALDI (m/z): [M + Na]+ calcd,
2759.20; found, 2760.47. A 0.05-mmol scale synthesis
afforded 3.5 mg (2.5%) of Ac-Lys(fluorescein)-(Ser-
Gly)3-(Pro-Pro-Gly)7 after purification.
Fluorescein–D-CMP. Ac-Lys-(Ser-Gly)3-(D-Pro-D-
Pro-Gly)7 was synthesized by amino acid addition on pre-
loaded Fmoc-Gly-Wang resin (0.65 mmol/g). Fmoc-
deprotection was achieved by treatment with 4-methyl-
piperidine (20% v/v) in DMF. The amino acid monomer
A 10-µL aliquot of suspended beads was treated with
Ac-Lys(fluorescein)-(Ser-Gly)3-(Pro-Pro-Gly)7 such that
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[ ]
θ
[ ]
θ
guest
× n
+
× n
res,guest
-
3
-
3
the final concentration of added peptide was 60 µM. Mix-
tures were allowed to anneal by heating the samples to
host
res,host
[ ]
θ
=
(1)
host+guest
n
+ n
res,host
res,guest
2
3
4
5
6
7
8
9
65 °C and cooling slowly to 4 °C at a rate of −12 °C per
hour. At 4 °C, beads were pelleted, the supernatant was
removed, and the beads were resuspended in water. This
process was repeated three times to wash the beads.
Finally, beads were resuspended in 300 µL of water. For
confocal images, beads (3 µL) were spotted on a
microscope slide and allowed to dry. For flow cytometry,
resuspended beads were imaged with 488-nm and 640-
nm lasers by using 530/30-nm and 675/25-nm filters, re-
spectively.
where [θ]host and [θ]guest are the respective mean residue
ellipticities for a host with nres,host residues and a guest
with nres,guest residues.
RESULTS
Design and Synthesis of a Host. We sought to
design a host in which two parallel CMPs are tethered to-
gether at both ends. Because lysine has two amino groups
that could serve to initiate synthesis, we began with lysine
immobilized via its carboxyl group. The collagen triple
helix forms with a one-residue stagger between its
strands, which the distance between the α and ε amino
groups of lysine helps to recreate. We reasoned that the
linkers on both ends must be flexible to allow for proper
folding. Accordingly, we condensed Fmoc-6-aminohexa-
noic acid spacer to both of the amino groups of lysine.
Then, we used standard solid-phase peptide synthesis
methods to add a (Pro-Pro-Gly)10 sequence to each amino
group.
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Circular Dichroism Spectroscopy. Solutions of
peptide were prepared in 50 mM HOAc. Many experi-
ments in this study involve mixtures of hosts and guest
peptides, and preparation of equimolar mixtures of the
components is of vital importance. To ensure equimolar
mixtures, relative concentrations were determined by in-
tegrating the absorbance of peptides at 218 nm during
UPLC, and concentrations of each peptide were calcu-
lated based on the absorbance of 180 µM (Pro-Pro-Gly)10
and the assumption that the extinction coefficient of all
Xaa-Yaa-Gly repeats were identical at 218 nm. Analyte
solutions were prepared such that the concentration of in-
dividual collagen strands was 180 µM. For example, so-
lutions contained 60 µM of a double-stranded host and
60 µM of a (Xaa-Yaa-Gly)7 peptide. To facilitate for-
mation of the most stable complex, analyte solutions were
heated to 65 °C, then cooled to 4 °C at a rate of −12 °C
per hour. Samples were left at 4 °C for at least 48 h before
data acquisition.
We considered flexibility as well in the design of the
N-terminal linker, which would close the macrocycle. 6-
Aminohexanoic acid spacers were attached at the N-ter-
mini of the two (Pro-Pro-Gly)10 peptides while still on the
solid support. Initial attempts to close the macrocycle en-
listed small molecules. Glutaric acid resulted in a mixture
of products containing one or two amides but no detecta-
ble macrocycle. Similarly, adding a cysteine to each end
and treating with dibromobimane12 provided a mixture of
products containing one or two thioethers but no macro-
cycle. A cyclic duplex that has been optimized to anneal
to endogenous single strands of collagen has been closed
by forming a disulfide bond,13 but that methodology was
unsuccessful in our hands. Finally, we sought to deploy
peptide “stapling”8,14 by condensing 3-butenoic acid to
the N termini (generating the open host) and using olefin
metathesis on-resin with Grubbs G2 catalyst to form a
macrocycle (the closed host). Mass spectrometry revealed
a decrease of 28 amu, consistent with the loss of ethylene
and successful tethering of the two ends (Figure S1). We
reduced the nascent alkene to endow greater flexibility
(the reduced host). The open, closed, and reduced hosts
CD spectra of peptides at 180 µM strand concentra-
tion were recorded at 4 °C with a 1-nm band-pass filter
and an averaging time of 3 s in a 0.1-cm path-length
quartz cuvette. For thermal denaturation experiments, the
CD signal was monitored at 226 nm as the sample was
heated at a rate of 12 °C/h in 3-°C steps. The value of Tm,
which is the temperature at the midpoint of the thermal
transition, was calculated by fitting the data to a Boltz-
mann sigmoidal curve with the program Prism from
GraphPad (La Jolla, CA).
CD spectra of equimolar non-interacting host and
guest mixtures, [θ]host+guest, were calculated based on the
spectra of individual components by using the equation:
Figure 2. Chemical structures of the open host (host-o), closed host (host-c), and closed, reduced host (host-r). The macrocyclic rings of
host-c and host-r contain 221 atoms.
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(host-o, host-c, and host-r, respectively) are depicted in
Figure 2.
Analysis of Host Self-Association with Ultra-
quenching was observed upon bead-labeling. The same
pattern was apparent for host-o and host-r coated beads
when mixed with the fluorescein–CMP conjugate. In con-
trast, treatment with fluorescein–CMP alone did not lead
to green fluorescence (data not shown). Host-coated
beads mixed with fluorescein–D-CMP, the enantiomeric
peptide incapable of forming a triple helix with either
host, showed a substantial reduction in bead-labeling
(Figure S3). This reduction is indicative of the specific
binding of fluorescein–CMP to hosts on the bead surface.
The qualitative results obtained with microscopy
were quantified by examining beads with flow cytometry,
where 10,000 events were evaluated for each sample in a
single run. Again, beads coated with both host-o and host-
r showed substantial labeling upon incubation with the
fluorescein–CMP conjugate (Figure 5). The fluorescence
with host-r was greater than that with host-o, which is not
closed on its N terminus. Moreover, the triple helix
formed by host-r with a CMP has greater kinetic stability
than does that with host-o, where agitation for 9 h dimin-
ished fluorescence.
Analysis of Host·CMP Complex Formation
with Circular Dichroism Spectroscopy. The colla-
gen triple helix generates a distinct circular dichroism
(CD) spectrum with maximum ellipticity near 226 nm.15
In our host·guest system, however, that diagnostic
method is complicated by two factors. First, the hosts
themselves have CD spectra like that of a triple helix (Fig-
ure 6A). Secondly, guest strands alone can form a homo-
trimeric helix as well as a host·guest complex, and those
two triple helices are likely to have indistinguishable CD
spectra. Nonetheless, interactions with a monomeric host
(e.g., host-r) could reveal trends, especially if analyses are
restricted to strands that do not form homotrimers. Ac-
cordingly, changes in CD signal upon mixing such
strands with a host could be attributed to the formation of
a host·guest triple helix.
centrifugation. To assess the aggregation state of host-
o and host-r in solution, we subjected their solutions to
analytical ultracentrifugation (AUC). Although sedimen-
tation-equilibrium data fitted to a single-species model
presents host-o as a dimer and host-r as a monomer in so-
lution, introduction of a multimer in the models improves
fits significantly by better representing the contributions
from high-molecular weight (MW) species. Monomer–
pentamer and monomer–trimer models best explain the
high-MW species observed in host-o and host-r gradients,
respectively (Figure S2). Monomer-multimer models for
host-o and host-r data collected at 34,000 rpm exemplifies
the abundance of each species across the gradients. The
presence of high-MW species in the host-o sample results
in an appreciable deviation from linearity (Figure 3A),
while the host-r gradient remains linear with only minor
deviation from sedimentation expected of a monomer
(Figure 3B). Thus, AUC indicates multimerization of
host-o, whereas host-r remains essentially in a mono-
meric state.
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Analysis of Host·CMP Complex Formation
with Fluorescence Spectroscopy
To reveal whether a triple helix forms between the hosts
and a CMP, we employed fluorescence spectroscopy.
Host-o and host-r were conjugated to biotin, and then
complexed to fluorescent beads that were coated with
streptavidin. A fluorescein probe was tethered to a CMP,
(Pro-Pro-Gly)7. Upon mixing, coinciding fluorescence
would indicate association and, presumably, triple-helix
formation (Figure 4A). Application of the positive control
biotin–fluorescein, (Ser-Gly)3 conjugated to both biotin
and fluorescein, reveals a green halo upon the red fluores-
cence of beads under confocal microscopy where the fo-
cal plane bisects the bead (Figure 4B). No fluorescent
Figure 3. Graphs of analytical ultracentrifugation data obtained at 34,000 rpm for host-o and host-r. (A) Data for host-o fitted to a monomer
+ pentamer mixture. (B) Data for host-r fitted to a monomer + trimer mixture.
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than each species can independently contribute. In com-
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parison, host-o·guest complexes do not yield similar lev-
els of cooperativity (Figure S4).
The results from temperature-denaturation experi-
ments are consistent with the spectral analysis above, and
also highlight the impact of macrocycle formation on host
structure. Although similar folded states can be imagined
for all hosts, macrocycle formation and flexibility limit
their conformations upon denaturation. This is clearly re-
flected in their denaturation profiles (Figure S5A).
Whereas a distinct transition is apparent for host-o (Tm =
45.6 °C), an extremely shallow transition is apparent for
the highly constrained host-c and increases after reduc-
tion to host-r. This trend is consistent with that observed
in CD spectra (Figure 6A). Furthermore, the thermal tran-
sitions for host-r·guest complexes become increasingly
recognizable when a more cooperative guest is selected
(Figure S5B), consistent with our analysis of host·guest
cooperativity (Figure 6C).
The peptides (flp-Hyp-Gly)7, (flp-Flp-Gly)7, and
(Pro-Pro-Gly)7 were used previously as invasive strands
to deliver cargo to damaged collagen.3-5 (Pro-Hyp-Gly)7
has also been employed for this purpose.19 Unlike the
other peptides, however, (Pro-Hyp-Gly)7 readily forms
homotrimers at room temperature, complicating its thera-
peutic use. As anticipated, application of (Pro-Hyp-Gly)7
to host-r does not enhance triple-helical content (Figure
S6).
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Figure 4. Binding of immobilized host-o and host-r to a fluorescent
CMP. (A) Scheme showing the experimental design. (B) Repre-
sentative confocal microscopy images. Streptavidin-coated fluores-
cent beads (red) were treated with host-o or host-r and then incu-
bated with fluorescein–CMP (green), which is Ac-Lys(fluores-
cein)-(Ser-Gly)3-(Pro-Pro-Gly)7. Also shown are images from a
negative control of untreated beads and a positive control of beads
treated with a biotin–fluorescein conjugate (green), which is biotin-
(Gly-Ser)3-Lys(fluorescein)-NH2. Scale bar: 10 µm.
The CMPs (flp-Hyp-Gly)7,3b,16 (flp-Flp-Gly)7,17
(Pro-Pro-Gly)7,17 and (Pro-Ile-Gly)718 cannot form stable
homotrimeric triple helices, exhibit low CD signal, and
are ideal for testing the utility of host-r as a mimic for
damaged collagen (Figure 6B). In contrast, a collagen sig-
nature is apparent in their mixtures with host-r (Fig-
ure 6C). To determine the extent of interaction between
host-r and each guest, we used data on individual species
to calculate CD spectra where the components of the mix-
ture do not interact. The calculated spectra explain the CD
signal observed for mixtures of host-r with (Pro-Pro-
Gly)7 and (Pro-Ile-Gly)7 (Figure 6C). In contrast, (flp-
Hyp-Gly)7 and (flp-Flp-Gly)7 interact cooperatively with
host-r, producing significantly higher triple-helix signal
Figure 5. Quantification of binding of immobilized host-o and
host-r to a CMP. Streptavidin-coated fluorescent beads were treated
with host-o–biotin or host-r–biotin, and then with fluorescein–
CMP, which is Ac-Lys(fluorescein)-(Ser-Gly)3-(Pro-Pro-Gly)7.
Beads were also treated with biotin–fluorescein conjugate (which
is biotin-(Gly-Ser)3-Lys(fluorescein)-NH2), host-o alone, or host-r
alone. Values represent the percent of the sample population with
fluorescein-labeling relative to that from treatment with biotin–flu-
orescein.
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Figure 6. Circular dichroism spectra of hosts (A), guest CMPs (B) and host·CMP complexes (C). Calculated spectra for non-interacting
mixtures of host-r and CMPs are shown (dashed gray lines) together with acquired spectra for host-r·CMP complexes (red lines). Spectra
were obtained in 50 mM HOAc at 4 °C.
DISCUSSION
deliver cargo to wound beds, which similarly display
damaged collagen on a 3-D surface.3a,4,5
A collagen duplex could be an effective mimic of dam-
aged collagen (Figure 1). To optimize a duplex for this
purpose, we synthesized two parallel strands of (Pro-Pro-
Gly)10 from the amino groups of an immobilized lysine
residue. The N-terminal closing of this acyclic duplex,
host-o, was achieved by olefin metathesis using the
Grubbs G2 catalyst. Reduction of the ensuing alkene af-
forded a cyclic duplex, host-r (Figure 2). This simple
modification appears to be critical, as it results in a dra-
matic increase in CD signal (Figure 6A).
Analytical ultracentrifugation revealed that host-r ex-
ists primarily as a monomer in solution (Figure 3B). Yet,
it exhibits triple-helical character by CD spectroscopy,
despite its topological inability to form a triple helix. This
finding is consistent with our recent discovery that two
singly crosslinked CMPs adopt a collagen-like structure
even in the absence of a third strand.20
Although host-r exhibits a lower triple-helix signal
than does host-o (Figure 6A), the benefits of macrocycle
formation outweigh any accompanying structural pen-
alty. Closing the macrocycle reduces multimerization
(Figure 3), improves the cooperativity of host·guest asso-
ciation (cf.: Figures 6C and S4), and enables better reten-
tion of a CMP on a host-coated bead surface (Figure 5).
Formation of a kinetically stable interaction with (Pro-
Pro-Gly)7 is especially interesting, as this CMP displays
little cooperativity with host-r. CD spectra calculated for
non-interacting species are likely overestimations due to
higher concentrations used to obtain data for individual
species. Perhaps more importantly, the bead-retention ex-
periment is unique in being a measurement done on a
solid support, which can enhance ligand association as
compared to solution-state measurements.21 Interestingly,
(Pro-Pro-Gly)7 has been used as an invasive strand to
Different CMPs were examined as “guests”—third
strands. Of those, (flp-Hyp-Gly)7 and (flp-Flp-Gly)7 are
superior at promoting the formation of a triple helix with
host-r (Figure 6C). Their ability to form a stable triple he-
lix can be attributed to the preorganization endowed by
their flp residues (which adopt a requisite Cγ-endo ring
pucker and ꢀ ~ −75° dihedral angle) and Hyp and Flp res-
idues (which adopt a requisite Cγ-exo ring pucker and
smaller ꢀ ~ −60° dihedral angle).1 Interestingly, the inter-
action of host-r with (flp-Hyp-Gly)7 produces a greater
rise in CD signal than that with (flp-Flp-Gly)7, even
though Flp-to-Hyp substitutions at the Yaa position of a
(Pro-Yaa-Gly)7 peptide lowers thermostability.22 This
finding is consistent with our recent discovery that (flp-
Hyp-Gly)7 is an optimal strand for annealing to damaged
collagen in vitro and ex vivo.3b Hence, we conclude that
host-r provides a reliable mimic of damaged collagen.
Although all CMPs tested are deficient in homotri-
mer formation, they are so due to different factors. The
peptides, (flp-Hyp-Gly)7 and (flp-Flp-Gly)7, feature resi-
dues with high triple-helical propensity, but cannot form
triple helices due to severe steric hindrance between Xaa
= flp and Yaa = Flp or Hyp residues on neighboring
strands.3b,7 In contrast, (Pro-Pro-Gly)7 and (Pro-Ile-Gly)7
lack the preorganization of their counterparts, and their
triple helices are not stable above 4 °C despite being un-
hindered by sterics. Thus, (Pro-Pro-Gly)7 and (Pro-Ile-
Gly)7 are not well-positioned for cooperative interactions
with host-r, which is corroborated by our findings (Figure
6C). Interestingly, we also observe similar uncooperative
behavior in mixtures of host-r and (Pro-Hyp-Gly)7, a pep-
tide that forms stable homotrimers up to 36 °C (Figure
S5).23 Together, our results point to engineered guest
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peptides, and especially to (flp-Hyp-Gly)7, as the optimal
CMPs for targeting collagen damage.
REFERENCES
1
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(1) Shoulders, M. D.; Raines, R. T. Collagen structure and
stability. Annu. Rev. Biochem. 2009, 78, 929-958.
CONCLUSIONS
(2) (a) Siebler, C.; Erdmann, R. S.; Wennemers, H. From
azidoproline to functionalizable collagen. Chimia 2013, 67, 891-
895; (b) Chattopadhyay, S.; Raines, R. T. Collagen-based
biomaterials for wound healing. Biopolymers 2014, 101, 821-833;
(c) Wahyudi, H.; Reynolds, A. A.; Li, Y.; Owen, S. C.; Yu, S. M.
Targeting collagen for diagnostic imaging and therapeutic delivery.
J. Controlled Release 2016, 240, 323-331; (d) Shekhter, A. B.;
Fayzullin, A. L.; Vukolova, M. N.; Rudenko, T. G.; Osipycheva,
V. D.; Litvitsky, P. F. Medical applications of collagen and
collagen-based materials. Curr. Med. Chem. 2019, 26, 506-516.
(3) (a) Chattopadhyay, S.; Murphy, C. J.; McAnulty, J. F.;
Raines, R. T. Peptides that anneal to natural collagen in vitro and
ex vivo. Org. Biomol. Chem. 2012, 10, 5892-5897; (b) Dones, J.
M.; Tanrikulu, I. C.; Chacko, J. V.; Schroeder, A. B.; Hoang, T. T.;
Gibson, A. L. F.; Eliceiri, K. W.; Raines, R. T. Optimization of
interstrand interactions enables burn detection with a collagen-
mimetic peptide. Org. Biomol. Chem. 2019, 17, 9906-9912; (c)
Song, J. Y.; Pineault, K. M.; Dones, J. M.; Raines, R. T.; Wellik,
D. M. Hox genes maintain critical roles in the adult skeleton. Proc.
Natl. Acad. Sci. USA, DOI: 10.1073/pnas.1913463117.
(4) Chattopadhyay, S.; Guthrie, K. M.; Teixeira, L.; Murphy, C.
J.; Dubielzig, R. R.; McAnulty, J. F.; Raines, R. T. Anchoring a
cytoactive factor in a wound bed promotes healing. J. Tissue Eng.
Regen. Med. 2014, 10, 1012-1020.
(5) Ellison, A. J.; Raines, R. T. A pendant peptide endows a
sunscreen with water-resistance. Org. Biomol. Chem. 2018, 16,
7139-7142.
(6) (a) Koide, T.; Homma, D. L.; Asada, S.; Kitagawa, K. Self-
complementary peptides for the formation of collagen-like triple
helical supramolecules. Bioorg. Med. Chem. Lett. 2005, 15, 5230-
5233; (b) Kotch, F. W.; Raines, R. T. Self-assembly of synthetic
collagen triple helices. Proc. Natl. Acad. Sci. USA 2006, 103, 3028-
3033; (c) Yamazaki, C. M.; Asada, S.; Kitagawa, K.; Koide, T.
Artificial collagen gels via self-assembly of de novo designed
peptides. Biopolymers 2008, 90, 816-823; (d) Yamazaki, C. M.;
Kadoya, Y.; Hozumi, K.; Okano-Kosugi, H.; Asada, S.; Kitagawa,
K.; Nomizu, M.; Koide, T. A collagen-mimetic triple helical
supramolecule that evokes integrin-dependent cell responses.
Biomaterials 2010, 31, 1925-1934; (e) Tanrikulu, I. C.; Raines, R.
T. Optimal interstrand bridges for collagen-like biomaterials. J.
Am. Chem. Soc. 2014, 136, 13490-13493; (f) Delsuc, N.;
Uchinomiya, S.; Ojida, A.; Hamachi, I. A host–guest system based
on collagen-like triple-helix hybridization. Chem. Commun. 2017,
53, 6856-6859.
Host-r is a macrocycle that contains two collagen-mi-
metic peptides and forms a collagen triple helix with a
third collagen-mimetic peptide. Its development provides
opportunities for new types of analyses. Historically, the
rigorous analysis of triple-helix formation has been com-
plicated by the process being termolecular. For example,
hysteresis often confounds analyses of the unfolding–re-
folding transition with three strands.24 As the basis of a
bimolecular rather than a termolecular system, host-r
could provide clarity as well as new insight.
In addition, we note that human type-I collagen,
which is the most abundant protein in the extracellular
matrix and connective tissue,1 is composed of two α1[I]
strands and one α2[I] strand. In collagen fibrils, a Gly-
Phe-Hyp-Gly-Glu-Arg sequence clusters integrins on the
surface of endothelial cells and promotes wound heal-
ing.25 Disruption of the triple helix in this region could be
especially deleterious to wound repair. Host-r variants
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composed
of
two
copies
of
the
GAOGPSGARGERGFOGERGVQGPOGPAGPR se-
quence of human α1[I] strands (where “O” refers to Hyp)
could lead to the discovery of CMPs that enable new ther-
apeutic interventions.13b
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bi-
omac.xxxxxxx
Figures S1–S6, NMR spectra, and UPLC traces (PDF)
AUTHOR INFORMATION
Corresponding Author
*Telephone: (617) 253-1470; E-mail: rtraines@mit.edu
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ORCID
I. Caglar Tanrikulu: 0000-0002-7165-0399
Ronald T. Raines: 0000-0001-7164-1719
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
We are grateful to Dr. Darrell R. McCaslin and Dr. Mar-
tha M. Vestling for facility support and contributive dis-
cussions, and to Prof. Clark R. Landis for use of his glove
box. A.J.E. was supported by Chemistry–Biology Inter-
face (CBI) Training Grant T32 GM008505 (NIH). This
work was supported by Grant R56 AR044276 (NIH).
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