Achieving High Affinity and Selectivity for Asymmetric Dimethylarginine by Putting a Lid on a Box

Article abstract of DOI:10.1002/anie.201814645

The methylation states of Lys and Arg represent a particularly challenging set of targets to distinguish selectively in water using synthetic receptors. To date, trimethyllysine (Kme3) is the only post translational modification (PTM) of the eight possible methylation states of Lys and Arg that can be recognized selectively. Here, we report the first synthetic receptor capable of selectively recognizing asymmetric dimethylarginine (Rme2a). This was achieved by using a biased dynamic combinatorial chemistry (DCC) library to generate a receptor mimicking the 5-sided box-like shape of Rme2 reader proteins, a feature that has been hypothesized to impart selectivity. Additionally, we synthesized a thioether-linked analogue of the resulting receptor to provide a novel scaffold with maintained selectivity but greater stability. This work introduces strategies that can be applied towards achieving selectivity based on subtle differences in hydrophilic guests in aqueous solutions.

Full text of DOI:10.1002/anie.201814645

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Accepted Article
Title: Achieving High Affinity and Selectivity for Asymmetric
Dimethylarginine by Putting a Lid on a Box
Authors: Alexandria Mullins, Nicholas Pinkin, Joshua Hardin, and
Marcey L. Waters
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201814645
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10.1002/anie.201814645
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Achieving High Affinity and Selectivity for Asymmetric
Dimethylarginine by Putting a Lid on a Box
Alexandria G. Mullins, Nicholas K. Pinkin, Joshua A. Hardin, Marcey L. Waters*[a]
Abstract: The methylation states of Lys and Arg represent a
particularly challenging set of targets to distinguish selectively in water
using synthetic receptors. To date, Kme3 is the only post translational
modification (PTM) of the eight possible methylation states of Lys and
Arg that can be recognized selectively. Here we report the first
synthetic receptor capable of selectively recognizing Rme2a. This
was achieved using a biased DCC library to generate a receptor
mimicking the 5-sided box-like shape of Rme2 reader proteins, a
feature that has been hypothesized to impart selectivity. Additionally,
we have synthesized a thioether-linked analogue of the resulting
receptor to provide a novel scaffold with maintained selectivity, but
greater stability. This work introduces strategies that can be applied
towards achieving selectivity based on subtle differences in
hydrophilic guests in aqueous solutions.
mimicking the 5-sided box-like shape of Rme2 reader proteins
(Fig. 1b) through a unique receptor design with a two-flapped lid
(Fig. 2d).
Supramolecular chemistry is increasingly being applied to
problems in chemical biology. However, an ongoing fundamental
challenge that limits its application to biological problems is
achieving selective molecular recognition of hydrophilic guests in
water.1–4 Methylated lysine (Kme) and arginine (Rme) (Fig. 1a)
are protein post-translational modifications (PTMs) that represent
a noteworthy example of difficult targets to distinguish in water.
These PTMs are of significant interest due to their role in
mediating gene expression and their dysregulation in numerous
diseases5,6. Much work has gone into developing synthetic
receptors that bind PTMs selectively in aqueous solution, with
applications in sensing, tagging, enrichment, and enzymatic
assays.7–12 However, achieving selectivity for one methylation
state of Lys or Arg has been a persisting challenge, as all eight
possible methylation states can participate in electrostatic
interactions, cation-π interactions, and H-bonding, with the
exception of trimethyllysine (Kme3) which cannot H-bond. Indeed,
the lower desolvation cost of Kme3 may be why it has been the
easiest to bind with high affinity (< 1 µM) and selectivity (> 10-fold
over Kme2).13 Attempts at selectively binding lower methylation
states of lysine14 or dimethyl arginine (Rme2)15 have been met
with limited success (see Fig. 2b,c). Herein, we describe the first
selective receptor for asymmetric dimethylarginine (Rme2a),
which is associated with a number of diseases, including cancer,
neurogenic, metabolic, and muscular disorders.5 This receptor,
N2G2, binds more tightly than all reported reader proteins for
Rme2.16 Both high affinity and selectivity were achieved by
Figure 1. (a) Rme2a, Rme2s, and Kme3, highlighting regions capable or
hydrogen-bonding (red) or cation- interactions (blue). (b) A crystal structure of
Kme3 (orange) and an NMR structure of Rme2a (green) in their respective
binding pockets.
Previously our group has utilized dynamic combinatorial
chemistry (DCC) 17 to develop novel receptors for methylated Lys
and Arg using disulfide exchange, and identified a number of A2X
receptors, where X is a varied monomer imposing selectivity (Fig.
2a,c). Switching X from a phenyl to a naphthyl monomer resulted
in the first synthetic receptor for Rme2a, A2D.15 While A2D
remains the tightest binding receptor for Rme2a,18 it does not
exhibit any selectivity over Kme3, binding both equally in the low
micromolar range.15 Inspection of the binding pockets of reported
synthetic receptors for methylated Lys and Arg as compared to
those of their respective reader proteins suggested a feature that
may explain the lack of selectivity: A2D has a spherical binding
pocket, similar to Kme3 reader proteins, but reader proteins for
Rme2 have a conserved 5-sided box with an open face for Rme2
to thread in (Fig. 1b, S1).16 The Rme2 reader proteins provide
excellent selectivity over Kme3, a feat that has been unachievable
using synthetic receptors to date.15,18 It has been proposed that
the narrower cleft formed by the top and bottom aromatic faces of
what has been described as a “rectangle cuboid” pocket provides
selectivity over Kme3, although this has not been experimentally
demonstrated.19 Additionally, we envisioned that the sides and
[a]
Department of Chemistry, CB 3290, University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599, United States
E-mail: mlwaters@email.unc.edu
Supporting information for this article is given via a link at the end of
the document.
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back of the box could further control selectivity for Rme2a over
Rme2s by constraining the size of the binding pocket and
providing additional CH- interactions. This may disfavor Rme2s,
which has H-bond donors on all three N atoms, resulting in more
disperse hydrophilic regions (Fig 1a).
Figure 2. Gas-phase models of previously reported receptors (a) A2N,13 (b)
CB7,18 (c) A2D,15 and the proposed box-like receptor (d) N2G2 shown bound to
Kme3 (orange) or Rme2a (green). The reported affinities were measured with
free amino acids for CB7 and with histone peptides for A2N and A2D.
To evaluate these hypotheses, we screened libraries
consisting of different combinations of three previously reported
building blocks E, G, and N (Fig. 3a).13,20 Compound G was
included in all libraries, as it has been reported to form rectangular
receptors with E,20 and the extended width of G was expected to
be necessary to accommodate methylated Arg. Monomers E and
N both introduce a -2 charge and contain a para-substituted
dithiobenzene, predicted to give the correct height to a receptor
for Rme2 but not Kme3. However, N provides a second benzene
ring that can act as a “lid”, providing the fifth face of the receptor,
as is observed in Rme2 reader proteins. Moreover, N has
previously been shown to impart significant selectivity for binding
an NMe over an NH group in a receptor for methylated Lys by
providing two CH- interactions and creating a deeper aromatic
pocket.13 Thus, three libraries consisting of E+G, E+N+G, and
N+G, were investigated to determine which features, if any, are
important for Rme2a selectivity. Amplification of a species in a
dynamic combinatorial library (DCL) generally correlates with
binding, although statistical preferences must be kept in mind.17
These libraries were screened against tetrameric peptides, Ac-
XGGY-NH2, containing a variable residue X (=R, Rme2a, Rme2s,
and Kme3), a Gly-Gly spacer, and a Tyr tag for concentration
determination (Figure 3b-d). Additional libraries templated with
the lower methylation states of Arg and Lys are reported in the SI
and demonstrate no selective amplification.
Figure 3. (a) Monomers E, G, and N (b-d) HPLC traces of a libraries at 254 nm
after equilibrating in pH 8.5 50 mM sodium borate buffer; [peptide] = 1.35 mM;
(b) [G] = 0.67 mM, [E] = 0.67 mM; (c) [E] = 0.34 mM, [G] = 0.67 mM, [N] = 0.34
mM; (d) [G] = 0.67 mM, [N] = 0.67 mM. (Shifted peaks due to analysis on a
different day)*. (e) Proposed box-like receptors.
Interestingly, despite the fact that all three libraries have the
potential to form the same rectangular scaffold, the amplified
species varied significantly between PTMs and libraries. In the
E+G library, the rectangular E2G2 macrocycle was not amplified
by any PTMs, suggesting that a box-like shape alone is not
sufficient for Arg recognition (Figure 3b and e). Indeed, Kme3
resulted in the only amplified species, EG3, which has a larger
pocket than E2G2. Since E2G2 is statistically preferred over EG3,
lack of E2G2 amplification is consistent with preferential binding.
Introducing one equivalent of monomer
N provided
amplification of two isomers of ENG2 in the presence of both
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Rme2a and Rme2s (Figure 3c and e). Thus, these results support
selectivity for Rme2 over Kme3, presumably because of a narrow
cleft that cannot accommodate Kme3 (see Fig. S10). However,
ENG2 appears to lack selectivity between Rme2s and Rme2a.
Monomer E introduces additional carboxylates on both edges of
the pocket that can H-bond with the NH groups of both Rme2a
and Rme2s, which may allow for binding both Rme2a and Rme2s.
In contrast to the E+N+G library, the N+G library resulted in
a single amplified peak, corresponding to N2G2, in the presence
of Rme2a only. Note that this peak was also weakly amplified in
the E+N+G library with Rme2a but not Rme2s. While the other
three isomers of N2G2 were observed, none were amplified (see
Fig. S14). Replacing monomer E with another unit of monomer N
to give N2G2 allows for CH- interactions with Rme2a, regardless
of whether both N units are parallel or antiparallel (Fig. 3e and
S11). In contrast, Rme2s would have to thread an NH into the
aromatic cage in order to bind, which would have a higher
desolvation cost. Thus, the deeper aromatic pocket may give rise
to the selectivity between Rme2a and Rme2s. As with ENG2, we
expect that the cleft between the two naphthalenes is too narrow
for Kme3, with a width of 7.8-7.9 Å, which compares well to the
distances seen in Rme2 reader proteins of 6.7-7.2 Å.19 The lack
of amplification of any rectangular species by Kme3 in any of the
libraries further supports the hypothesis that a narrower cleft
excludes Kme3 binding.
Because of its apparent selectivity, we isolated N2G2 by RP-
HPLC for additional characterization. We undertook tandem mass
spectrometry experiments to determine the connectivity of
monomers N and G in the macrocycle, specifically looking for
whether our modeled alternating connectivity of monomers was
accurate.21 We reasoned that the disulfide bonds of an NG, NN,
and GG species should all fragment and ionize equivalently and
therefore we could determine their connectivity by the population
of fragmented species observed. The lack of NN or GG species
supported the modeled constitutional isomer of N2G2 with
alternating N and G monomers necessary for forming the
predicted box (Fig. S18).
similar dihedral angle as disulfides and would be stable in the
presence of thiols such as glutathione, so we hypothesized an
analogue of the DCC-identified receptor would maintain
selectivity over Kme3 with a box-like geometry while providing a
more stable linkage to access a larger breadth of future
applications. The binding of thioether cyclophanes has not
previously been explored as a class of synthetic receptors for
biomolecules.
Table 1. Binding Data N2G2 with for Rme and Kme Peptides.
Peptide
Kd (µM) [a]
1.2±0.3
13±1
Selectivity
H3R8me2a
H3K9me3
H3R8me2s
H3R8me1
H3R8K9
-
11
11
17
19
14±2
21±3
23±2
[a] Conditions: 25° C, 10 mM Na2HBO3/NaH2BO3, pH 8.5. Average of two runs.
A variant of monomer N in which the thiols were replaced
with bromomethyl groups was synthesized22 to provide a site for
nucleophilic substitution with the thiol of monomer G (Scheme S1).
This synthetic route provided the four possible isomers of
thioether-linked N2G2 (thN2G2) in
a statistical distribution.
Preliminary binding studies revealed that the fourth isomer
(thN2G2-4) demonstrated modestly tighter binding to Rme2a than
the others. Therefore this isomer was used for continued studies.
It is worth noting that while thN2G2-4 and the isomer of N2G2 that
was amplified both displayed the longest retention times, we were
unable to confirm that they are the same isomer (vide infra).
ITC binding studies of thN2G2-4 revealed weaker binding
affinity for Rme2a (Kd = 25±3 µM) compared to the disulfide-linked
N2G2, although still tighter than any reported Rme2a reader
proteins, which have Kd’s ranging from 42 to >1000 µM (Table
S1).23 Selectivity over Kme3 and the other methylation states of
arginine was maintained (Table S2). The weaker binding may be
the result of several factors, including a smaller binding pocket
due to shorter C-S bonds, which could make binding less
favorable, or could allow for greater edge-face interactions
between the naphthyl groups in the unbound state, which would
need to be disrupted to bind Rme2a. Additionally, while the
disulfides in N2G2 are included as a result of disulfide exchange,
they may contribute favorably to binding via dispersion
interactions, as sulfur is quite polarizable.
To further probe the recognition of Rme2a, the change in
the 1H NMR spectrum of the tetrameric peptide Ac-Rme2aGGY-
NH2 in the bound state with both N2G2 and thN2G2-4 was
investigated under saturating conditions. While N2G2 appeared to
aggregate under these conditions, thN2G2-4 was well behaved. In
the presence of N2G2 and thN2G2-4 the methyl groups on Rme2a
were upfield shifted by 1.9 ppm and 1.7 ppm, respectively,
indicating significant CH- interactions, consistent with binding
within the aromatic pocket. The alkyl chain of Rme2a, in contrast,
is only minimally shifted, suggesting that binding is primarily at the
guanidinium group. Furthermore, the peptide backbone and other
Isothermal calorimetry (ITC) was used to characterize the
binding of N2G2 to a short model peptide sequence representative
of
the
histone
H3
tail
(H3R8meXK9meZ
=
Ac-
YGGQTARmexKmezSTG-NH2, where X, Z = 0; X = 0, Z = 3; X =
1, Z = 0; or X = 2; Z = 0) (Table 1). Binding affinities indicate that
N2G2 is indeed selective, exhibiting >10-fold selectivity over both
Rme2s and Kme3, consistent with amplification results.
Additionally, the receptor is approximately 20-fold selective over
Rme and unmethylated Arg. The binding constant for N2G2 to
H3R8me2a (Kd = 1.2±0.3 µM) is tighter than all known Rme2
reader proteins (5 to >1000 µM, see Table S1). and demonstrates
4-fold greater affinity than the previously reported receptor A2D to
the same peptide (Kd = 5.1±0.6 µM) as well as improved
selectivity over Rme2s (7-fold for A2D).15 Thus, creating a
narrower cleft increased selectivity over Kme3 more than 10-fold
and improved the absolute binding affinity.
Furthermore,
inclusion of the aromatic “lids” appears to be required for
selectivity over Rme2s.
We further explored whether we could use N2G2 as a
template to create a receptor that would be more stable in the
reducing conditions of biological assays, by investigating a
thioether analogue of N2G2 (thN2G2). Thioether bonds maintain a
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10.1002/anie.201814645
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sidechains are not significantly shifted (Fig. S38). When
compared to the reported NMR binding data for A2D (Table S3),
it is notable that the methyl groups experience almost a 2-fold
greater ∆δ for thN2G2-4, but less shifting for all of the protons on
the carbon chain. Thus, thN2G2-4 is a well behaved selective
receptor for Rme2a with protein-like binding affinity and linkages
that are stable to a reducing environment.
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for Rme2a, N2G2, which is tighter than any of the known Rme2
reader proteins.23 Selectivity was achieved by using principles of
molecular recognition gleaned from native reader proteins,
including mimicking the cuboid shape which provides steric
occlusion of Kme3 and formation of a deeper aromatic pocket
through the incorporation of aromatic “lids”, which disfavors
binding of Rme2s. Selective molecular recognition of hydrophilic
guests in water is an ongoing challenge, and this approach
allowed us to select for a more hydrophilic guest, Rme2a, over a
less hydrophilic guest, Kme3. In a general sense, this work
advances the strategies that can be applied to achieve selectivity
for subtle differences in hydrophilic guests in aqueous solution.
Additionally, we have demonstrated that thioether-linked
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receptors provide
a novel scaffold capable of selectively
recognizing PTMs. The potential to develop receptors selected
from DCC studies into analogues that are stable to the reducing
environment of a cell is particularly appealing. While some degree
of binding affinity was lost relative to the disulfide-linked receptor,
binding was nonetheless tighter than any reported native reader
protein for Rme2a23 and selectivity over Kme3 and unmethylated
arginine was observed. Further functionalization of this receptor
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This material is based upon work supported by the National
Science Foundation (CHE-1608333).
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Keywords: dimethylarginine • histone post-translational
modifications • molecular recognition in water • epigenetics •
synthetic receptors
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Entry for the Table of Contents
Layout 2:
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Author(s), Corresponding Author(s)*
Page No. – Page No.
Title
Tailor-made: By introducing “lids” on a box-like receptor, a perfect fit for
asymmetric dimethylarginine is achieved, to the exclusion of symmetric
dimethylarginine and trimethyllysine.
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