Aldol sensor-inspired fluorescent probes for measuring protein citrullination

Article abstract of DOI:10.1039/c9ob02737h

Protein citrullination is an important posttranslational modification on an arginine residue. However, high quality fluorescent probes for measuring the citrullination level and capturing citrullinated proteins are quite limited. Inspired by the similarit

Full text of DOI:10.1039/c9ob02737h

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: H. Chen, H. Zhao,
L. Xiang, H. Wu, Y. Liang, X. Huang and J. Zhang, Org. Biomol. Chem., 2020, DOI: 10.1039/C9OB02737H.
Volume 15
Number 47
21 December 2017
Pages 9945-10124
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
Accepted Manuscripts are published online shortly after acceptance,
rsc.li/obc
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
ISSN 1477-0520
PAPER
I . J. Dmochowski et al.
Oligonucleotide modifi cations enhance probe stability for
shall the Royal Society of Chemistry be held responsible for any errors
single cell transcriptome in vivo analysis (TIVA)
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
rsc.li/obc

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Page 1 of 5
OrPgleanaisce&dBoionmotoaledcjuulsatr mChaermgiinstsry
View Article Online
DOI: 10.1039/C9OB02737H
COMMUNICATION
Aldol sensors-inspired fluorescent probes for measuring protein
citrullination
Huihong Chen, Hailong Zhao, Lingling Xiang, Haiting Wu, Yunshi Liang, Xin-An Huang,* Jing Zhang*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Protein citrullination is one important posttranslational substrate-based
assay,
fluorescence
polarization,
modification on arginine residue. However, high quality fluorescent supramolecular host sensor,6 reactive fluorescent turn-on
probes for measuring citrullination level and capturing citrullinated probes. Among these methods, acid-promoted condensation
proteins are quite limited. Inspired by the similarity between acid- reaction between ureido group and 1,2-dicarbonyl group
promoted citrulline-labeling reaction and Aldol reaction, here we (represented by phenylglyoxal derivatives and their hydrates)
present “turn-on” and “turn-off” fluorescent probes for measuring constitutes the basis for chemically characterizing protein
citrulline levels based on the scaffold of Aldol sensors. Further citrullination level and capturing citrullinated proteins (Scheme
application of the modified probe showed great potential to 1A, second step). This reaction, which is generally believed to
simultaneously monitor and capture citrullinated peptides.
go through an “addition-cyclization” process, has long been
adopted to screen PAD inhibitors with COLDER assay still being
widely used nowadays.7 Meanwhile, phenylglyoxal-
functionalized beads and phenylglyoxal-biotin conjugate
Over the past few decades, it is increasingly recognized that
posttranslational modifications (PTMs) of protein are
widespread and they are crucial for cell state determination.1
Among the amino acid residues subjected to PTMs, arginine is
one of the major players. With a unique pendant guanidinium
group enriched in hydrogen-bonding capability, arginine
residue is currently known to subject to four major PTMs:
(
Biotin-PEG-GBA) were synthesized to selectively capture and
enrich citrullinated peptides (Scheme 1B).8 And based on this,
Thompson et al. developed rhodamine−phenylglyoxal
Rh−PG) conjugate. After reacting this probe with cell lysates or
a
(
purified protein under acidic condition, the citrullinated
proteins can be visualized by gel separation and fluorescent
imaging, resulting in comparable outcomes as antibody-based
method.9 Recently, Nakagawa group developed a series of
fluorescent turn-on probes built on glyoxal-fluorescein hybrids.
These probes are represented by compound 4MEBz-FluME in
Scheme 1B. The fluorescence of these probes can be turned off
via an intramolecular donor-excited photoinduced electron
transfer (d-PeT) mechanism. Upon reacting with citrulline, the
electron-deficiency of glyoxal group was diminished and the
fluorescence was resumed.10
Even with these advances, there is still a great shortage of
chemical probes suitable for performing multiple functions such
as detecting and capturing protein citrullination to meet the
diverse biochemical study needs, especially probes with low
fluorescence background. In view of this, herein we explored a
novel type of bifunctional glyoxal-based citrulline-reactive
fluorescent probes. This is inspired by the previously reported
Aldol sensors which could be fluorescently switched on/off to
monitor the activity of Aldol enzymes (Scheme 2A).11 These
sensors are a set of aromatic aldehydes with or without
conjugated triple or double bond. Depending on the existence
of the conjugation pendant, the aldehyde/aldol pair can be
either fluorescently turned on or turned off. When there is no
methylation,
citrullination,
ADP-ribosylation,
and
phosphorylation.2 Protein citrullination, which is catalyzed by
five peptidyl arginine deiminase (PADs), converts positively
charged guanidino-containing arginine residue to neutral
ureido-containing citrulline residue (Scheme 1A, first step).3
Recent studies suggest that dysregulation of protein
citrullination is highly involved in tumorigenesis, and also in
inflammatory diseases especially rheumatoid arthritis (RA).4
Therefore, citrullination level measurement and citrullinated
protein capture are of great value for elucidating the biological
function of protein citrullination.
The conversion of arginine residue to citrulline residue
results in minimal disturbance on molecular weight and
molecular size. However, dramatic change in hydrophobicity,
electrostatic interaction and chemical reactivity are noticed in
this process. Based on these differences, several strategies for
measuring protein citrullination level were developed5
including color development reagent (COLDER) assay, anti-
citrulline antibody, ammonia release assay, fluorescent
Artemisinin Research Center, Guangzhou University of Chinese Medicine, 12
Jichang Road, Guangzhou 510405, China.
Email: jingzhang@gzucm.edu.cn; xinanhuang@gzucm.edu.cn
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
π-conjugated bond (exemplified by compound
A in Scheme 2A),
Please do not adjust margins

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021PAlsepaosseePdtyoLtnd.ot adjust margins
Organic & Biomolecular Chemistry
Page 2 of 5
COMMUNICATION
Journal Name
the fluorescence is turned off after Aldol reaction because the
aromatic aldehyde system was disrupted. In contrast, when
View Article Online
DOI: 10.1039/C9OB02737H
there is conjugated bond (exemplified by compound
B in
Scheme 2A), the electron-deficient formyl group quenches the
fluorescence of aromatic system resulting in low fluorescence
background, and the fluorescence of probe can be recovered
when the formyl group was disrupted after Aldol reaction.
Scheme 2. Aldol sensors-inspired fluorescent probes for measuring and capturing
protein citrullination.
The syntheses of compounds Napdial and Napdialyne are
outlined in Figure 1. Briefly, Napdial can be prepared by a one-
step SeO2-oxidation starting from acetonaphthone analogue.
For probe Napdialyne, the key intermediate
by a Pd-catalyzed Sonogashira coupling reaction between
terminal alkyne compound and 2-bromo-6-
methoxynaphthalene.11c However, subsequent oxidation of
compound using classic SeO2-oxidation led to no reaction,
3 can be accessed
2
3
probably due to the electron-withdrawing effect of alkynyl
substitute. Instead, a combination of I2/DMSO yielded the
desired glyoxal product with satisfactory yield.12 For compound
Napdial, 1H NMR and 13C NMR spectra showed that it exists as
ca. 1:1 ratio of hydrate state and glyoxal state. For compound
Napdialyne, NMR spectra suggested that it is a hydrate. The
reason of incomplete hydration of Napdial is not clear at this
moment. For both compounds, mass spectrum displayed
glyoxal forms rather than hydrate form, probably due to water
loss during ion fragmentation.
With Napdial and Napdialyne in hand, we next tested their
reactivity with free L-citrulline. As expected, both probes
reacted smoothly with L-citrulline under acidic condition
(TFA/H2O 1:1), and the adducts (Napdial-Cit and Napdialyne-Cit)
can be successfully separated as pure compounds and their
structures were confirmed.
Scheme 1. (A) PAD-catalyzed protein citrullination and chemoselective reaction between
citrulline and phenylglyoxal under acidic condition; (B) Previously reported phenylglyoxal
analogues for protein citrullination measurement.
In this work, we reasoned that the reaction between glyoxal
group and citrulline residue could result in diminishing of the
electro-deficiency on glyoxal group, and this is similar to the
outcome of Aldol reaction. Based on this, we firstly built two
probes (namely Napdial and Napdialyne) consisting of a glyoxal
group and a fluorescence turn on/off group (Scheme 2B).
Further, we modified the methoxyl group on Napdialyne to
introduce a N3 handle (namely Napdial-N3) for a second function
such as capturing citrullinated peptide. Since these probes are
predominantly existed in glyoxal hydrate form, the hydrate
structure rather than aldehyde structure was adopted in this
report.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Page 3 of 5
Organic & Biomolecular Chemistry
Please do not adjust margins
Journal Name
COMMUNICATION
Napdialyne (50 µM) was incubated witVhiew AdrtiicflfeeOrnelinnet
DOI: 10.1039/C9OB02737H
concentrations of L-citrulline in the presence of 20%
trichloroacetic acid, then the reaction mixture was diluted ten
times before fluorescence measurement. The whole procedure
is relatively simple, and extra separation or neutralization step
was not needed. For probe Napdial, it exhibited a linear
decrease in the range of 0-600 µM L-citrulline reaction
concentration at emission wavelength of 450 nm (Fig. 3B-C),
which is consistent with its mechanism as a “turn-off” probe
(Fig. 3A). For “turn-on” probe Napdialyne, a linear increase was
observed at L-citrulline reaction concentration from 0 to 250
µM (Fig. 4B-C), suggesting that this probe is more sensitive to
detect low concentration of citrulline. Further, the reaction
condition and dilution solvents were investigated (Fig. 4C and
Fig. S1), and results showed that fluorescence intensity was
briefly linearly increased over time at 50 oC reaction
temperature and the optimal dilution solvent was DMSO.
Meanwhile, we also measured the selectivity profile of
Napdialyne. As shown in Fig. S2, Napdialyne displayed low
fluorescence intensity towards arginine, lysine or buffers
(HEPES, Tris), which is consistent with the previous report that
the glyoxal compounds selectively react with citrulline residue
under acidic condition.
Figure 1 Synthetic routine to probes Napdial and Napdialyne, and their reactions with L-
citrulline.
Having prepared both probes and their citrulline adducts, a
proof-of-concept study was conducted by comparing their
fluorescence spectra (Fig. 2). As expected, probe Napdial
showed a fluorescence upon UV irradiation at 320 nm with a
maximum emission wavelength at 470 nm, while its purified
adduct Napdial-Cit showed no fluorescence in this range (Fig.
2A). On the contrary, probe Napdialyne displayed no
fluorescence, while its purified adduct Napdialyne-Cit displayed
a strong fluorescence with a maximum emission wavelength at
375 nm (Fig. 2B). The “turn-on” and “turn-off” fluorescence
properties of these two citrulline adducts are very similar to the
products of Aldol sensors, confirming our hypothesis that the
glyoxal moiety is critical for modulating the fluorescence of
citrulline probe just as that of formyl group for Aldol sensors.
Meanwhile, the ratio of Napdialyne-Cit/Napdialyne
fluorescence intensities was 340-fold at maximum emission
wavelength. This extra low fluorescent background indicated
that the fluorescence of probe Napdialyne can be sufficiently
quenched intramolecularly, which is especially useful for
measuring low concentration of citrullinated product when high
concentration of probe is present. The quantum yields of
Napdialyne and Napdialyne-Cit were listed in Table S1.
Figure 3 (A) Schematic illustration of the reaction between probe Napdial and L-citrulline;
(B-C) Fluorescence spectra (B) and dose-dependent relationship of fluorescence intensity
(C) on L-citrulline concentration. Reaction condition: Napdial (100 µM), L-citrulline (10
mM to 0 mM, 2-fold dilution), 20% trichloroacetic acid in ddH2O, 50 oC, 3 h. The reaction
mixture was diluted 10-times with MeOH before measurement. λex = 324 nm, λem = 450
nm, n = 2. L-Cit: L-citrulline.
Figure 2 Fluorescence emission spectra comparison of Napdial, Napdialyne and their
citrulline adducts at 10 µM concentrations, λex = 320 nm. For Napdial and Napdial-Cit in
panel A, solvent: CH3OH; For Napdialyne and Napdialyne-Cit in panel B, solvent: DMSO.
Next, the fluorescence response of probes to citrulline
concentration was assessed. Briefly, probe Napdial (100 µM) or
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins