A click chemistry constructed affinity system for 2-oxoglutaric acid receptors and binding proteins

Article abstract of DOI:10.1039/c4ob01005a

An ingenious and specific affinity resin designed to capture the 2-oxoglutaric acid (2-OG) binding proteins was constructed by appending a 2-OG tag to the solid resin via a Cu-catalyzed Huisgen click reaction. The so-obtained affinity resin was able to recognize, retain and separate the established 2-OG binding protein NtcA in both the pure form and crude cellular extract, thus constituting a valuable means of searching for novel 2-OG receptors with a view to exploring the signalling pathways of 2-OG, a key Krebs cycle intermediate with unprecedented signalling functions. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob01005a

Organic &
Biomolecular Chemistry
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A “click” chemistry constructed affinity system for
2-oxoglutaric acid receptors and binding proteins†
Cite this: Org. Biomol. Chem., 2014,
12, 6470
Yang Wang,a,b Zeinab Assaf,a,c Xinjun Liu,b Fabio Ziarelli,d Amel Latifi,c
Otmane Lamrabet,c Gilles Quéléver,a Fanqi Qu,b Cheng-Cai Zhangc and Ling Peng*a
An ingenious and specific affinity resin designed to capture the 2-oxoglutaric acid (2-OG) binding proteins
was constructed by appending a 2-OG tag to the solid resin via a Cu-catalyzed Huisgen “click” reaction.
The so-obtained affinity resin was able to recognize, retain and separate the established 2-OG binding
protein NtcA in both the pure form and crude cellular extract, thus constituting a valuable means of
searching for novel 2-OG receptors with a view to exploring the signalling pathways of 2-OG, a key Krebs
cycle intermediate with unprecedented signalling functions.
Received 15th May 2014,
Accepted 30th June 2014
DOI: 10.1039/c4ob01005a
www.rsc.org/obc
the balance in carbon and nitrogen metabolism,6–8 epigenetic
regulation mediated by 2-OG dependent oxygenases,9 gene-
Introduction
2-Oxoglutaric acid (2-OG) (Scheme 1) is a strategically impor- ration of the “onco-metabolite” 2-hydroxyglutarate via misregu-
tant intermediate of the Krebs cycle since it constitutes the lated metabolism in various cancers, etc.10 The signalling
precursor and the carbon skeleton for nitrogen assimilation, pathways of 2-OG are rather complex and remain largely un-
leading to the synthesis of various biomolecules and the pro- explored because of the difficulties faced in identifying the
duction of cellular energy. In addition to its canonical roles, corresponding 2-OG receptors or 2-OG binding proteins. The
2-OG has recently regained considerable attention as a key sig- prerequisite therefore in achieving the ultimate goal of better
nalling molecule1–5 in different organisms as it plays impor- understanding the signalling role of 2-OG is finding a simple
tant roles in various signalling pathways such as regulation of and effective way of searching and identifying potential 2-OG
receptors. To this end, affinity chromatography appears to be
the method of choice by virtue of its high specificity, easy
manipulation and simple scale-up.11 In this method, a compe-
tent tag bearing 2-OG mimics shall be immobilized onto the
solid support via an appropriate spacer arm, and the resulting
affinity resin (Fig. 1) is expected to capture the 2-OG binding
proteins via specific interactions between the 2-OG binding
site of the proteins and the 2-OG mimic attached on the resin.
An elution step using 2-OG containing eluent would then
Scheme 1 Structure of 2-OG, the 2-OG analogue DMPA and the
affinity resin 1 with DMPA as a 2-OG tag on the solid support.
aAix-Marseille Université and CNRS, Centre Interdisciplinaire de Nanoscience de
Marseille, UMR 7325, 13288 Marseille, France. E-mail: ling.peng@univ-amu.fr
bCollege of Chemistry and Molecular Sciences, Wuhan University, P. R. China
cAix-Marseille Université and CNRS, Laboratoire de Chimie Bactérienne, UMR 7283,
Marseille, France
dAix-Marseille Université and CNRS, Spectropôle, FR 1739 Marseille, France
†Electronic supplementary information (ESI) available: Schemes S1–S3,
Fig. S1–S3, synthesis of 2, 3 and 6, 1H and 13C NMR spectra of compounds 2–5,
IR and 13C HRMAS NMR spectra of resins 1, 6, 7. See DOI: 10.1039/c4ob01005a
Fig. 1 The principle of using 2-OG affinity chromatography to capture
the potential 2-OG receptors.
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deliver the 2-OG binding proteins enabling their subsequent
purification and identification.
The foremost requirement to guarantee a successful affinity
chromatographic study is establishing a competent affinity
resin with high affinity and specificity, where the apt tag to
sense the 2-OG binding proteins is of paramount importance.
However, the native 2-OG does not appear to be the tag of
choice as the α-keto carboxylic acid entity within 2-OG is rather
labile, and ready to undergo different transformations in bio-
logical media containing various enzymes. This is certainly the
biggest concern during our consideration to devise an appro-
priate affinity column to capture the 2-OG binding proteins in
the crude cell extracts or biological media where many
enzymes or proteins may react with the α-keto carboxylic acid
entity. Furthermore, 2-OG is a small molecule, and any struc-
tural modification with relatively large functional moieties will
inevitably modify binding characteristics towards 2-OG
receptors.4,12–15 Taking all these into consideration, we opted
for the nonmetabolisable 2-OG analogue, 2,4-dimethylene-
pentanedioic acid (DMPA), as the 2-OG tag to construct the
affinity resin 1 (Scheme 1) for its structural resemblance to,
and resulting ability to mimic, 2-OG in signaling.14 In
addition, it is practical for chemical modification and conju-
gation of DMPA onto the solid support. For linking the 2-OG
tag onto the solid resin, we chose the hydrophilic ethyleneoxy
spacer in order to provide a hydrophilic environment allowing
the 2-OG tag to search for and bind to the 2-OG binding
proteins. To anchor the 2-OG tag onto the resin, we wished to
harness the Cu-catalyzed Huisgen reaction,16 a well acknow-
ledged “click” reaction for affording clean products efficiently
and rapidly in high yield under mild conditions, hence allow-
ing a facile and economic solid support synthesis.
Based on this rationale, we report here the synthesis,
characterization and biological validation of the so-devised
affinity resin 1 that can selectively and specifically recognize,
retain and separate the 2-OG binding protein NtcA, a known
2-OG receptor involved in regulating the carbon/nitrogen
balance and heterocyst differentiation in diazotrophic
cyanobacteria.4,12–15,17 To our knowledge, this is the first
example of an affinity resin using a 2-OG mimic tag validated
with a known 2-OG receptor.18 This affinity system hence
holds great promise in searching for 2-OG receptors or 2-OG
binding proteins involved in signal transduction and meta-
bolic regulation.
Scheme 2 Preparation of the affinity resin 1.
Results and discussion
ponding azides 5, which were further conjugated to the alkyne-
bearing resin 6 via a Cu-catalyzed Huisgen cycloaddition to
yield the resin 7. Gratifyingly, the Cu-catalyzed “click” reaction
proved to be very powerful and rewarding as it proceeded
smoothly, and was accomplished rapidly within an hour
(Fig. S1†). As shown in the IR spectra (Fig. 2A), the peak at
3289 cm−1 corresponding to the vibration of the C–H bond in
the terminal alkynyl –CuC–H moiety of 6 disappeared follow-
ing the “click” reaction, and a very strong signal was generated
The construction of the affinity resin 1 was achieved according
to the strategy presented in Scheme 2. The precursor of the
2-OG tag featuring the ethyleneoxy spacer (5 in Scheme 2) was
obtained via an Horner–Wadsworth–Emmons (HWE) reac-
tion20 by condensing the corresponding aldehyde 2 with the
phosphonate 3 in the presence of sodium hydride, affording 4
with an excellent yield of 85% as a mixture of E/Z isomers (E/Z
= 3/1).21 Subsequent treatment with NaN3 delivered the corres-
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ponding carboxylate functions was highlighted by the signal of
the carbonyl group shifting from 1707 cm−1 (characteristic of
the ester groups in 7) to 1658 cm−1 (characteristic of the car-
boxylate functions in 1). This was further confirmed with the
results obtained by 13C CPMAS NMR. As clearly depicted in
Fig. 2B, the signal concerning the ester carbonyl groups in 7
wore off at 166 ppm, and the new broad peak corresponding to
the carboxylate carbonyl groups in 1 emerged at 175 ppm.
Moreover, the signals relating to the ethyl moieties in the ester
groups of 7 disappeared at 14 and 60 ppm, further stressing
the successful hydrolysis of 7 to afford the resin 1.
To verify that the above-obtained affinity resin 1 could
indeed recognize and bind 2-OG binding proteins, we exam-
ined its interaction with NtcA, an established 2-OG receptor
regulating nitrogen metabolism and responsible for heterocyst
formation in cyanobacteria.4,17 Bovine serum albumin (BSA)
was used as a negative control of non-2-OG binding protein.
No retention of BSA on resin 1 was observed (data not shown),
whereas 1 could effectively retain NtcA on the column, as indi-
cated by the revelation of NtcA in the solution before and after
passing through the affinity column composed of 1 (Fig. 3A,
lanes 2, 3, 4 and 5). The retained NtcA on the affinity column
was then effectively eluted out using a solution containing
2-OG (Fig. 3A, lane 6), authenticating that the retention of
NtcA on the resin 1 is specific via the explicit interaction
between NtcA and the 2-OG tag featured on 1.
Fig. 2 IR and 13C CPMAS NMR spectra of the intermediate products
6 and 7, and the final affinity resin 1.
around 1707 cm−1, relating to the carbonyl group of the ester
functions of 7. The successful conversion of 6 to 7 was further
affirmed by 13C cross polarization magic angle spinning
(CPMAS) NMR, an excellent non-destructive method for the
in situ analysis of the solid phase reaction.22–24 As illustrated
in Fig. 2B, the disappearance of the signals associated with the
alkynyl moiety at 70–80 ppm in 6 was accompanied by the
emergence of the signals at 14 and 60 ppm corresponding to
the ethyl groups within the ester functions of 7. Collectively,
these results indicate a rapid and complete conversion of 6 to
deliver 7 via “click” chemistry. Subsequent alkaline hydrolysis
of 7 furnished the final resin 1, which was successfully charac-
terized by both IR and CPMAS NMR. As we can see in Fig. 2A,
the hydrolysis of the ester groups in 7 to give the corres-
Fig. 3 Protein revelation of resin 1 capturing and eluting NtcA from (A)
a pure NtcA solution and (B) E. coli cell lysate. Lane 1: protein molecular
weight marker; lane 2: NtcA or E. coli cell lysate as a reference; lane 3:
the supernatant from the incubation buffer; lanes 4 and 5: elution with
PBS; lane 6: elution with 2-OG solution (0.5 M) in PBS buffer; lane 7:
elution with PBS; lane 8: elution with NaCl solution; lane 9: elution with
PBS. The pH of PBS and 2-OG elution was controlled at pH = 7.5.
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We further tested the robustness of the affinity resin 1 in 31P NMR spectrum was recorded at 121 MHz on a Varian
identifying and capturing the 2-OG receptor NtcA from cell Mercury-VX300 spectrometer. Chemical shifts were reported
lysate. To this end, we assessed the ability of resin 1 to fish out relatively to 85% H3PO4 as the external standard. The solid-
the NtcA protein from the total cellular extract of an E. coli state 13C cross polarization magic angle spinning (CPMAS)
strain which over-expresses NtcA. We found that 1 could NMR spectra were recorded on a Bruker Avance-400 MHz NMR
indeed selectively and specifically anchor NtcA, and then spectrometer. HRMS were recorded using Waters Micromass
release it when using 2-OG elution (Fig. 3B). Altogether, these GCT Premier and QStar Elite Mass spectrometers. IR spectra
results demonstrate the robust specificity of the affinity resin 1 were recorded using a Bruker Alfa IR spectrometer. The micro-
and its potential use in detecting and searching for 2-OG wave reaction was performed in a CEM Discover SP microwave
receptors.
reactor.
Synthesis of 4. To a mixture of 85% NaH (229 mg,
8.11 mmol) in freshly distilled DME (10.0 mL) was added a
solution of 3 (2.73 g, 8.11 mmol) in freshly distilled DME
(10.0 mL) over a period of 10 min. The reaction mixture was
Conclusions
In summary, we have successfully established the affinity resin stirred at room temperature for 3 h until the disappearance of
1 for specific recognition, capture and release of the 2-OG powdered NaH. Then, a solution of 2 (1.63 g, 5.40 mmol) in
receptor NtcA via affinity chromatography. This resin features freshly distilled DME (10.0 mL) was added to the reaction
an affinity tag bearing a 2-OG mimic, which was appended to mixture over a period of 10 min at −50 °C. The resulting reac-
the solid support resin via a hydrophilic linker. Cu-catalyzed tion mixture was allowed to warm up to room temperature and
“click” chemistry was implemented to conjugate the tag to the stirred for 2 h before quenching by addition of a saturated
resin, and proved to be very efficient and rewarding. The so- NH4Cl (20.0 mL), and then followed by extraction with ethyl
obtained 2-OG resin could not only retain the 2-OG receptor acetate (3 × 20.0 mL). The combined organic layers were dried
NtcA in its pure form, but also identify and capture NtcA from over anhydrous MgSO4, filtered and concentrated under
the cell extract containing a mixture of various macro- reduced pressure. The crude residue was purified by flash
molecules, before allowing its release from the column with an chromatography using a gradient of petroleum ether–ethyl
eluent containing 2-OG. Altogether, these results illustrate the acetate (6 : 1–4 : 1, v/v), yielding 4 as a colorless oil (2.22 g,
1
power and value of this resin in specifically recognizing, 85%, E/Z ≈ 3/1). H NMR (300 MHz, CDCl3) δ 7.80 (d, J = 8.10
sufficiently binding and effectively releasing the 2-OG receptor Hz, 2H, PhCH3), 7.35 (d, J = 8.40 Hz, 2H, –PhCH3), 6.98 (t, J =
NtcA. Consequently, this resin constitutes a novel useful 5.70 Hz, 1H, E: CvCHCH2), 6.23 (s, 1H, Z: CvCHH), 6.19–6.14
means of searching for new 2-OG receptors with a view to (m, 2H, E: CvCHH, Z: CvCHCH2), 5.55 (s, 1H, Z: CvCHH),
investigate the signaling pathways of 2-OG and complete our 5.49 (s, 1H, E: CvCHH), 4.46 (d,
J = 4.80 Hz, 1H,
understanding of its signaling roles. We are actively pursuing Z: CvCHCH2), 4.23–4.14 (m, 8H, CvCHCH2, CH2OTs,
in this direction.
OCH2CH3), 3.70 (t, J = 4.80 Hz, CH2CH2OTs), 3.61–3.56 (m, 4H,
OCH2), 3.31 (s, 2H, E: CH2), 3.29 (s, 2H, Z: CH2), 2.45 (s, 3H,
–PhCH3), 1.32–1.25 (m, 6H, –CH2CH3); 13C NMR (150 MHz,
CDCl3) δ 166.9, 166.8, 145.2, 144.0, 141.0, 138.6, 137.5, 133.2,
130.6, 130.2, 129.3, 128.2, 126.7, 125.6, 70.9–68.2, 61.2, 60.9,
35.3, 29.3, 21.9, 14.5; HRMS cacld for C23H32O9SNa+ 507.1659,
Experimental section
General methods
All the reactions were carried out under argon. Monotosylated found 507.1659.
triethylene glycol was synthesized according to the literature.25
Synthesis of 5. To a solution of 4 (2.18 g, 4.50 mmol) in
The detailed synthesis of compounds 2 and 3 and resin 6 is CH3CN (50.0 mL) was added NaN3 (1.47 g, 22.5 mmol). The
described in the ESI.† Anhydrous 1,2-dimethoxyethane (DME) reaction mixture was refluxed for 10 h, and then was allowed
was distilled in the presence of sodium-benzophenone. An- to cool down to room temperature and filtered. The so
hydrous CH2Cl2 and DMSO were prepared by distillation in the obtained organic phase was concentrated under reduced
presence of CaH2. 85% NaH was prepared by washing 60% pressure, and the crude residue was purified by flash
NaH in mineral oil with petroleum ether and drying in a chromatography using a gradient of petroleum ether–ethyl
vacuum. The Merrifield resin (loading capacity: 3.1 mmol g−1
)
acetate (6 : 1–4 : 1, v/v), yielding pale yellow oil 5 as a mixture of
was purchased from Iris Biotech (Germany). All the other E/Z isomers (863 mg, 54%, E/Z ≈ 3/1). 1H NMR (300 MHz,
reagents were purchased from Sigma-Aldrich (China) or Acros CDCl3) δ 7.00 (t, J = 5.90 Hz, 1H, E: CvCHCH2), 6.23 (s, 1H,
Organics (China) without any further purification. Silica gel Z: CvCHH), 6.19 (s, 2H, E: CvCHH, Z: CvCHCH2), 5.54 (s,
(200–300 mesh) used for flash chromatography was purchased 1H, Z: CvCHH), 5.48 (s, 1H, E: CvCHH), 4.49 (d, J = 4.80 Hz,
from Qing Dao Hai Yang Chemical Industry Co. (China). 1H 1H, Z: CvCHCH2), 4.25–4.14 (m, 6H, CvCHCH2, OCH2CH3),
NMR and 13C NMR spectra were recorded at 300 MHz and 75 3.70–3.63 (m, 6H, –OCH2), 3.41 (t, J = 4.95 Hz, CH2N3), 3.32 (s,
or 150 MHz respectively on Varian Mercury-VX300 and VX600 2H, E: CH2), 3.29 (s, 2H, Z: CH2), 1.32–1.24 (m, 6H, –CH2CH3);
spectrometers. Chemical shifts are reported in parts per 13C NMR (150 MHz, CDCl3) δ 166.6, 166.5, 143.8, 140.8,
million (ppm) with TMS as an internal reference. The 138.3, 137.2, 130.3, 129.0, 126.4, 125.2, 70.6–69.8, 68.0, 60.8,
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60.6, 50.6, 35.0, 29.0, 14.2; IR (cm−1) ν 2106.1 (–N3); HRMS cia, Sweden). Proteins were concentrated on Vivaspin columns
cacld for C16H26N3O6+ 356.1816, found 356.1815.
and used for subsequent analyses. Proteins were fractionated
Synthesis of 7. To a mixture of 6 (150 mg, 0.465 mmol) in by SDS-PAGE (12.5% gel) and stained using the SeeBand pro-
3.00 mL THF and N,N-diisopropylethylamine (2 : 1, v/v) were cedure (Euromedex, France).
added
5
(196 mg, 0.552 mmol) and CuI (5.00 mg,
Preparation of the E. coli cell lysate. A BL21DE3 culture
0.0263 mmol). The reaction mixture was stirred at 35 °C for expressing the ntcA-6his recombinant gene was prepared as
24 h, and then allowed to cool down to room temperature and explained above. After induction of the synthesis of the recom-
filtered. The obtained resin was washed successively with THF, binant protein, the obtained pellet was resuspended in 2 mL of
water, saturated EDTA solution, water, MeOH, CH2Cl2 and PBS. 20 µL of the protease inhibitor (Roche, France) was added.
diethyl ether (30.0 mL for each) and dried in a vacuum, yield- After disrupting the cell by ultrasonication (3 × 15 min), the
ing 160 mg desired resin 7 as a pale green powder. IR (cm−1
)
lysate was centrifuged for 10 min at 12 300 rpm. The super-
ν
1707 (CvO); 13C CPMAS NMR
(OCH2CH3), 14.0 (OCH2CH3).
δ
166.0 (CvO), 60.6 natant was loaded onto the resin 1 for affinity chromatography.
Affinity chromatography. Affinity resin 1 (20 mg) was pre-
Synthesis of 1. To a mixture of 7 (50.0 mg, 0.155 mmol) in equilibrated by washing with PBS (4 × 1 mL) and 0.5 M EDTA
1.50 mL THF was added 1 M LiOH (1.50 mL). The reaction solution (6 × 1 mL), and then again with PBS (4 × 1 mL). After
mixture was stirred at 35 °C for 24 h, and then allowed to cool pre-equilibration, NtcA (10 µg/45 µL) or E. coli cell lysate (200 µL)
down to room temperature and filtered to obtain the resin. was added to resin 1, followed by incubation and agitation
The obtained resin was washed successively with THF, water, (gentle agitation every 5 min) at room temperature for 3 h.
saturated NaCl solution, MeOH, CH2Cl2 and diethyl ether Resin 1 was then washed twice with PBS (45 µL). The elution
(30.0 mL for each) and dried in a vacuum, yielding 40 mg resin was realized with 2-OG (45 µL, 0.5 M). Repeated incubation and
1 as a yellow powder. IR (cm−1): ν 1707 (CvO); 13C CPMAS agitation (gentle agitation every 5 min) were performed at room
NMR δ 175.9 (CvO).
temperature for 3 h; resin 1 was washed again with PBS (45 µL),
13C CPMAS NMR. The solid-state 13C CPMAS NMR spectra regenerated with NaCl (45 µL, 1 M), and washed for the last
were obtained on a Bruker Avance-400 MHz NMR spectrometer time with PBS (1 mL). The regenerated resin was stored at
operating at a 13C resonance frequency of 106 MHz using a −20 °C. The presence of the protein at each step of the
commercial Bruker double-bearing probe. About 30 mg of chromatography was assessed by SDS-PAGE as explained above.
each sample were placed in zirconium dioxide rotors of 4 mm
outer diameter and spun at a magic angle spinning rate of 10
kHz. The cross polarization (CP) technique22 was applied with
Acknowledgements
a ramped 1H-pulse starting at 100% power and decreasing
until 50% during the contact time (2 ms) in order to circum- We are grateful for the financial support from the “ProKrebs”
vent Hartmann–Hahn mismatches.23 To improve the resolu- project of the ANR program of “Physique, Chimie et du
tion, a dipolar decoupling TPPM15 pulse sequence was Vivant”, Région PACA, CNRS, Wuhan University and Aix-Mar-
applied during the acquisition time. To obtain a good signal- seille Université. Y.W. and X.L. are supported by the overseas
to-noise ratio in the 13C CPMAS experiment 6000 scans were Ph.D. fellowship from China Scholarship Council. We thank
accumulated using a delay of 2.5 s. The 13C chemical shifts Dr Emily Witty for English correction.
were referenced to tetramethylsilane and calibrated with the
glycine carbonyl signal, set at 176.5 ppm.
Production and purification of the NtcA recombinant
protein. A DNA fragment corresponding to the entire coding
Notes and references
region of ntcA (alr4392) was amplified by performing PCR
using the ntcA forward primer 5′-C -ATGATCGTGA
CACAAGATAA3′ (NdeI site underlined) and the ntcA reverse
primer 5′-G -AGTGAACTGT CTGCTGAGAG T3′ (SacI site
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̲A̲T̲A̲T̲G̲
̲A̲G̲C̲T̲C̲
underlined). The PCR product was cloned into the pET28
vector (Novagen, USA). A clone confirmed by DNA sequencing
was transformed into the BLDE3 E. coli strain (Novagen, USA).
The recombinant clones obtained were grown in LB rich
medium supplemented with Kanamycin (50 µg mL−1) and
glucose (0.2%) with an OD at 600 nm of 0.3–0.4. The culture
was then washed twice with LB medium to eliminate the
glucose. The expression of the recombinant protein was then
induced by adding 1 mM isopropyl-β-D-thiogalactoside (IPTG)
for 4 h. The recombinant proteins were purified using Hitrap
columns as recommended by Pharmacia. Imidazole was
removed from purified proteins using PD10 columns (Pharma-
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