A fluorogenic probe for recognizing 5-hydroxylysine inspired by serine/threonine ligation

Article abstract of DOI:10.1039/c3cc47465h

The 5-lysyl-hydroxylation has been found among protein post-translational modifications. In this report, we have devised a fluorescence turn-on probe which allows for selectively recognizing 5-hydroxylysine-containing peptides. the Partner Organisations 2014.

Full text of DOI:10.1039/c3cc47465h

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A fluorogenic probe for recognizing 5-hydroxylysine
inspired by serine/threonine ligation†
Cite this: Chem. Commun., 2014,
0, 5298
5
Chun Ling Tung,‡ Hiu Yung Lam,‡ Jianchao Xu and Xuechen Li*
Received 30th September 2013,
Accepted 22nd October 2013
DOI: 10.1039/c3cc47465h
www.rsc.org/chemcomm
The 5-lysyl-hydroxylation has been found among protein post-
translational modifications. In this report, we have devised a fluores-
cence turn-on probe which allows for selectively recognizing
5-hydroxylysine-containing peptides.
Protein posttranslational modifications (PTMs) account for the
Fig. 1 5-Hydroxylysine in proteins.
increased structural and functional diversity of proteins in higher
1
organisms. They play key roles in tuning protein conformations, factor 65 kDa subunit), CROP (cisplatin resistance-associated
2
localization and binding properties. In addition to the well- overexpressed protein), and LUC7L2 (splicing regulatory protein
1
3
studied PTMs including protein glycosylation, lipidation, and LUC7-like 2). It is very likely that the lysyl-hydroxylation is also
phosphorylation, other types of protein posttranslational modi- a type of protein posttranslational modification that is abundant
fications have been identified and shown to play important roles in animal proteins. The biological significance of posttransla-
3,4
in many biological pathways. Protein posttranslational modifi- tional 5-lysyl-hydroxylation requires extensive in-depth studies.
cations pose a challenge for the investigation using traditional On the other hand, it remains essential to investigate whether
biochemical and biological approaches, as their biosynthesis is there are other proteins whose lysine residues are hydroxylated.
not genetically controlled. Recently, chemical biology has pro- Thus, it will be of great value to develop a profiling strategy to
vided an array of important and complementary tools to discover globally identify 5-hydroxylysine-containing proteins.
new types of protein posttranslational modifications and study
As there are many side-chain functional groups present in pro-
their biological roles. Various chemical approaches have been teins, searching for a chemical reagent mediating an orthogonal
developed to detect and elucidate many protein posttranslational reaction to selectively react with 5-hydroxylysine would be very difficult
modifications, including protein glycosylation, lipidation, phos- and challenging. The key issue and challenge is the chemoselectivity.
5–9
phorylation, acetylation and AMPylation.
Noting that 5-hydroxylysine contains a 1,2-hydroxylamino group,
The hydroxylation of the lysine residue within the proteins plays a distinct from side-chain functional groups of other naturally occurring
crucial role in stabilizing intra- and intermolecular crosslinks of amino acids, we directed our thinking to serine/threonine ligation
1
0
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collagen proteins (Fig. 1). Hydroxylysines also exist in ‘‘noncollagen’’ (STL), recently developed by us. A side-chain unprotected peptide
proteins, such as the C1q subcomponent of a complement, acetyl- salicylaldehyde (SAL) ester reacts with another peptide with an
cholinesterase, mannose binding proteins, type I and II macrophage N-terminal serine or threonine residue to afford an N,O-benzylidene
11,12
scavenger receptors, bovine conglutinin and adiponectin.
Lysyl acetal linked product and subsequently acidolysis releases the ligated
hydroxylase is an oxygenase enzyme that catalyzes the hydroxylation peptide with a natural peptidic bond (Scheme 1). Studies have shown
of lysine to hydroxylysine. More recently, Jmjd6 has been shown to that this reaction is chemoselective, without being interfered with by
14
catalyze lysyl-5-hydroxylation of many proteins associated with other side-chain functional groups.
RNA metabolism, processing and splicing, including U2AF65
Towards the development of a fluorogenic probe to recognize
splicing factor U2 small nuclear ribonucleoprotein auxiliary 5-hydroxylysine, we noted that the umbelliferone (i.e. 1) is a
(
1
5
fluorescent moiety, while the ester protected form (i.e. 2) and
Department of Chemistry, The University of Hong Kong, Hong Kong, P. R. China.
16
ortho-aldehyde containing form (i.e. 3) do not exhibit fluores-
E-mail: xuechenl@hku.hk; Fax: +86 852 28571586
cence (Fig. 2). Hence, we hypothesized that the ortho-aldehyde
umbelliferone ester (i.e. 4) containing a salicylaldehyde function-
ality and a fluorescent coumarin group would react with a
†
Electronic supplementary information (ESI) available: Details of the experi-
mental procedure. See DOI: 10.1039/c3cc47465h
‡
CLT and HYL contributed equally.
5
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Scheme 3 (a) MeOH, LiBF , trimethylorthoformate, 4 h, 70%; (b) trimethyl-
2 2 2
acetic acid, EDCI, DMAP, CH Cl , 97%; (c) TFA–H O, 70%; (d) pyridine
acetate buffer (mole : mole, 1 : 1).
pivalic ester 10, hoping that the reaction would be slowed down so
that the dynamic change of the reaction could be monitored. The
preparation of pivalic ester 10 is shown in Scheme 3. The ortho-
aldehyde 4-methylumbelliferone 7 was prepared according to the
literature. Next, the aldehyde group was first protected with
dimethyl acetal to give 8 before esterification with pivalic acid was
performed. Upon acidolysis, compound 10 was obtained.
Scheme 1 Serine/Threonine Ligation (STL).
17
To the end, compound 10 and serine methyl ester were mixed in the
reaction solvent (pyridine acetate buffer) at a concentration of 5 mM at
room temperature. To measure the fluorescence changes, a 25 ml
aliquot reaction solution at a different time point during the reaction
process was taken and diluted to 62 mM with the same solvent mixture
and the fluorescence intensity was recorded at lex = 320 nm.
As seen in Fig. 2, the reaction mixture indeed showed a strong
fluorescence with the maximum lem value observed at 425 nm.
The fluorescence intensity increased over time and reached the
maximum at 2 h. These results clearly indicated that compound
Fig. 2 Fluorescence spectra (at a concentration of 62 mM) of 10 upon
reaction with serine methyl ester (lex = 320 nm).
10 could serve as a fluorescence turn-on probe to react with the
1,2-hydroxylamino group.
Having established the validity of this approach, we next sought
to expand upon the general concept of selectively recognizing
-hydroxylysine. Thus, we synthesized an array of peptides, includ-
ing the one containing a 5-hydroxylysine residue (12) and the control
peptides with random sequences (13–15) (Table 1), and then studied
their reactions with compound 10.
LC-MS analysis of the reaction progression revealed that the
reaction of peptide 12 (1.0 equiv.) and probe 10 (1.2 equiv.) at a
concentration of 5 mM proceeded smoothly to completion in
1
,2-hydroxylamino group in a similar way to the salicylaldehyde
ester-induced serine/threonine ligation to give a ligated product
i.e. 6) (Scheme 2), after which the free hydroxyl group of the
5
(
umbelliferone would be liberated and the electron withdrawing
aldehyde group would be converted into an N,O-acetal group;
thus fluorescence could be likely regenerated via a fluorescence
turn-on mechanism.
To test the feasibility of our thinking, we started to perform a
model study. Since the ligation is generally very rapid, we chose
2
hours, which correlated well with the fluorescent analysis (Fig. 3).
To further confirm that compound 16 was formed, we treated
the reaction product under the acidic conditions (TFA/H O). As
2
determined by LC-MS, the putative compound 16 was converted
into the desired compound 17 (Scheme 4).
Table 1 5-Hydroxylysine-containing peptide and model peptides
Entry
Sequence
1
2
3
4
Ac-A-P-K(5-OH)-V-T-S-L-OH (12)
H-A-P-K-V-T-S-L-OH (13)
H-A-G-L-H-A-OH (14)
H-R-E-N-Q-D-W-Y-F-P-M-H-T-OH (15)
Scheme 2 Strategy of a fluorogenic probe to recognize a 1,2-hydroxyl-
amino group.
The peptide was synthesized by the standard SPPS.
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Fig. 3 Fluorescence spectra of 5-hydroxylysine-containing peptide 12 reacting
with probe 10. Dilution 100Â with (a) pyridine acetate buffer; (b) water.
Fig. 5 Fluorescence spectra of peptides 12–15 at different concentra-
tions reacting with probe 10. Dilution 100Â with water. [10] = 5 mM.
its fluorescence turn-on properties. The current limitation
residing in the reaction medium (i.e. pyridine acetate buffer)
would restrict its application scope. Our future efforts will be
oriented to the improvement of probe 10 enabling detection of
5-hydroxylysine-containing peptides/proteins under physio-
logical conditions.
We thank the General Research Fund (HKU703811P) of the
Research Grants Council and Seed Funding from the University
of Hong Kong (201210159054) for the financial support.
Notes and references
Scheme 4 5-Hydroxylysine peptide 12 reacting with 10.
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changes for the control peptides (13–15) upon addition of
probe 10. The peptides did not cause any significant changes
in the fluorescence emission intensity (Fig. 4).
We further studied the detection limits of the analytes, by
measuring the fluorescence intensity of peptides 12–15 at
concentrations from 0.01 mM to 5 mM. The result indicates
that the probe can detect a hydroxylysine-containing peptide at
a concentration below 1 mM (Fig. 5).
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In summary, we have devised and synthesized an
umbelliferone-based fluorogenic probe, which has allowed us
8 Peptides with N-terminal serine or threonine are expected to react
as well.
1
8
to selectively detect 5-hydroxylysine-containing peptides via
5300 | Chem. Commun., 2014, 50, 5298--5300
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