A bifunctional amino acid to study protein-protein interactions

Article abstract of DOI:10.1039/d0ra09110c

Protein-protein interactions (PPIs) play crucial roles in regulating essentially all cellular processes. Photo-cross-linking represents a powerful method to study PPIs. To fulfil the requirements for the exploration of different PPIs, there is a continuous demand on the development of novel photo-reactive amino acids with diverse structural properties and functionalities. Reported herein is the development of a bifunctional amino acid termed dzANA, which contains a diazirine, for photo-cross-linking, and a terminal alkyne group, for bioorthogonal tagging. Using known PPIs between histone posttranslational modifications (PTMs) and their binding partners as models, we demonstrate that the dzANA-harbouring peptide-based photoaffinity probes could efficiently and selectively capture the weak and transient PPIs mediated by histone modifications. Our study indicates the potential of dzANA to identify and characterize unknown PPIs. This journal is

Full text of DOI:10.1039/d0ra09110c

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PAPER
A bifunctional amino acid to study protein–protein
interactions†
Cite this: RSC Adv., 2020, 10, 42076
*
Tangpo Yang,‡ Xin Li
and Xiang David Li
Protein–protein interactions (PPIs) play crucial roles in regulating essentially all cellular processes. Photo-
cross-linking represents a powerful method to study PPIs. To fulfil the requirements for the exploration
of different PPIs, there is a continuous demand on the development of novel photo-reactive amino acids
with diverse structural properties and functionalities. Reported herein is the development of
a bifunctional amino acid termed dzANA, which contains a diazirine, for photo-cross-linking, and
a terminal alkyne group, for bioorthogonal tagging. Using known PPIs between histone posttranslational
modifications (PTMs) and their binding partners as models, we demonstrate that the dzANA-harbouring
peptide-based photoaffinity probes could efficiently and selectively capture the weak and transient PPIs
mediated by histone modifications. Our study indicates the potential of dzANA to identify and
characterize unknown PPIs.
Received 25th October 2020
Accepted 12th November 2020
DOI: 10.1039/d0ra09110c
rsc.li/rsc-advances
reactivity.7–10 These two features of diazirine ensure high cross-
linking specicity and efficiency, as it can be installed close
Introduction
enough to the binding sites without inducing undesired steric
hindrance, and thereby selectively and rapidly label the PPIs of
interest upon photo-activation.
Almost all biological processes, including DNA replication and
gene expression, cellular transportation and secretion, signal
transduction, and metabolism, rely on the exquisite regulation
of interactions among their key protein components.1,2 Given
the important roles played by protein–protein interactions
(PPIs) in normal physiology, aberrant regulation of PPIs may
lead to human diseases, such as Alzheimer's disease and
cancer. Identication and characterization of PPIs are there-
fore critical for not only fundamental understanding of
complex cellular processes but also the development new
therapeutic strategies to treat human diseases. A variety of
methods, including affinity purication, yeast two-hybrid
screening, among others, have been developed to study
PPIs.3 However, results obtained from these methods may
suffer from a high false-positive rate. In addition, it remains
a challenge to identify and characterize weak or transient PPIs,
including those mediated by posttranslational modications
(PTMs).
We and others have reported the development of various
diazirine-based photo-reactive amino acids (Fig. 1).11–24 These
unnatural amino acids are readily incorporated into peptides
and proteins to offer versatile photoaffinity probes for PPIs
mapping. To facilitate the detection and identication of the
targeted PPIs, the peptide- or protein-based probes are usually
decorated with epitope sequences or bioorthogonal chemical
handles. Recognition of an epitope by antibodies may vary in
affinity and selectivity case-by-case, leading to loss of target
proteins or false-positive results. In contrast, the bioorthogonal
handles, for example, alkyne and azide, are inert to different
biomolecules and are covalently conjugated to uorescent dyes
(e.g., rhodamine) or affinity tags (e.g., biotin), enabling the
subsequent visualization or isolation of the proteins labelled by
photoaffinity probes with high reliability. The introduction of
both photo-reactive and bioorthogonal functionalities into one
probe at the same time, however, can sometimes be trouble-
some or even technically challenging.25,26
Here, we report the development of dzANA (Fig. 1),
a bifunctional amino acid containing both a diazirine moiety
and a terminal alkyne group. Using a collection of PPIs
mediated by histone posttranslational modications (PTMs)
as models, we showed that the peptide-based chemical
probes armed with dzANA could robustly capture the known
binding partners, suggesting the potential of this novel
bifunctional amino acid to identify and characterize
unknown PPIs.
Photo-cross-linking represents a powerful method to study
PPIs, which converts the noncovalent interactions between
a protein of interest and its binding partners into covalent
linkages, facilitating the following identication and charac-
terization processes.4–6 Among all the frequently used photo-
reactive groups, diazirine is known for its small size and high
Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong,
China. E-mail: xiangli@hku.hk
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra09110c
‡ Current address: Department of Cellular and Molecular Pharmacology,
University of California, San Francisco, California 94158, United States.
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purchased from Sigma-Aldrich and used without further puri-
cation. In-solution reactions were monitored by thin-layer
chromatography (TLC) silica gel 60 F254 from Merck. Plates
were visualized by UV light or 1% KMnO4. Flash column chro-
matography was carried out using silica gel purchased from
Grace.
Instrumentation
1H NMR (300 MHz or 400 MHz), 13C NMR (75 MHz, 100 MHz)
were conducted on a Bruker spectrometer at 25 ꢀC and were
calibrated using residual undeuterated solvent as an internal
reference. Chemical shis were reported in ppm and coupling
constants (J) were quoted to the nearest 0.1 Hz. High resolution
mass spectrometry (HRMS) was recorded using a Bruker maXis
II High Resolution QTOF. Peptides were analyzed by LC-MS with
an Agilent 1260 Innity HPLC system connected to a Thermo
Finnigan LCQ DecaXP MS detector. Peptides were further
puried by a preparative HPLC system with Waters 2535
Quaternary Gradient Module, Waters 515 HPLC pump, Waters
SFO System Fluidics Organizer and Waters 2767 Sample
Manager.
Photo-cross-linking were performed with ENF-260C/FE
hand-hang UV lamp (Spectroline). In-gel uorescence scan-
ning was performed using a Typhoon 9410 variable mode
imager from GE Healthcare Life Sciences (excitation 532 nm,
emission 580 nm). All images were processed by ImageJ so-
ware (National Institutes of Health), and contrast was adjusted
appropriately.
Fig. 1 Structures of reported photo-reactive unnatural amino acids
and dzANA developed in this study.
Molecular cloning, protein expression and purication
Plasmids construction, protein expression and purication
were performed as previously described:12,24 GST-SPIN1 (full-
length), GST-ING2 (208–270), BHC80 (486–543), Sirt3 (102–
399), BRD4 (44–168), Sirt5 (34–302), GST-HP1 (23–74).
Photo-cross-linking
Indicated probes in the presence or absence of different
concentrations of corresponding competitors were incubated
with recombinant proteins (60 ng mLꢁ1) in binding buffer
(50 mM HEPES, 150 mM NaCl, 2 mM MgCl2, 0.1% Tween-20,
20% glycerol, pH 7.5) at 4 ꢀC for 10 min. Then the samples
were exposed to 365 nm UV irradiation for 20 min in 96-well
(recombinant proteins, 75 mL per well) on ice.
Scheme 1 Synthetic route for free and Fmoc-protected dzANA. TEA,
triethylamine; Ts, p-tosyl; DCM, dichloromethane; Bu, butyl; TMS,
trimethylsilyl; THF, tetrahydrofuran; Me, methyl; Boc, tert-butylox-
ycarbonyl; HOSA: hydroxylamine-O-sulfonic acid; TFA, trifluoroacetic
acid; Fmoc-OSu, 9-fluorenylmethyl N-succinimidyl carbonate.
Cu(I)-catalyzed azide–alkyne cycloaddition/click chemistry
Aer photo-cross-linking, 100 mM rhodamine-N3 (10 mM stock
in DMSO) was added, followed by 1 mM TCEP (freshly prepared
50 mM stock in H2O) and 100 mM TBTA (10 mM stock in DMSO).
Finally, the reaction was initiated by the addition of 1 mM
CuSO4 (freshly prepared 50 mM stock in H2O). The reactions
were incubated at room temperature for 1 h with regular vor-
texing. Aer quenching by adding 5 volumes of ice-cold acetone,
the samples were placed at ꢁ20 ꢀC overnight to precipitate
proteins.
Experimental
Reagents
Fmoc-protected amino acids were purchased from GL Biochem.
Fmoc-N-3-(trimethyl)-L-lysine chloride (Fmoc-Lys(Me3)-OH)
were purchased from Chem-Impex International (Wood Dale,
IL). All the other chemical reagents and solvents were
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the rhodamine-labelled BSA were performed as previously
describe.
Synthesis
The synthesis of dzANA and its derivatives was followed the
synthetic route listed in Scheme 1.
Preparation of compound 2. To a mixture of 3-butyn-1-ol
(3.88 g, 55.36 mmol), TEA (15.43 mL, 2 eq.) and DCM (100
mL) was added a solution of p-tosyl chloride (11.61 g, 1.1 eq.) in
anhydrous DCM over 30 min at 0 ꢀC. The solution was then
stirred at room temperature for 3 h and then poured into
100 mL of ice/H2O mixture. The aqueous layer was separated
and extracted with DCM. The combined organic layers were
washed with sat. NH4Cl and brine, respectively; dried over
Na2SO4 and concentrated. Compound 2 was obtained by ash
column chromatography as a colourless liquid (11.8 g, 95%
yield). 1H NMR (300 MHz, CDCl3) d 7.81 (d, J ¼ 8.1 Hz, 2H), 7.36
(d, J ¼ 8.0 Hz, 2H), 4.11 (t, J ¼ 7.1 Hz, 2H), 2.56 (td, J ¼ 7.0 Hz, J
¼ 2.5 Hz, 2H), 2.46 (s, 3H), 1.97 (t, J ¼ 2.6 Hz, 1H); 13C NMR (100
MHz, CDCl3) d 145.06, 132.69, 129.90, 127.88, 78.48, 70.80,
67.52, 21.56, 19.36.
Fig. 2 (a) Structures of probe 1, probe 2, H3K4me3 and H3K4me0
peptides. (b) Selective labelling of SPIN1 and ING2 by probe 1, and
BHC80 by probe 2. For all the photo-cross-linking experiments,
protein concentration used was 60 mg mLꢁ1. After UV irradiation (365
nm) for 20 min on ice, the probe-labelled proteins were conjugated to
Rho-N3 and visualized by in-gel fluorescence scanning.
Preparation of compound 3. To a solution of tosylate 2 (5.0 g,
22.29 mmol) in dry THF was slowly added n-butyl lithium (2.4 M
in THF, 11.15 mL, 1.2 eq.) at ꢁ78 ꢀC. Then the solution was
stirred at ꢁ78 ꢀC for 1.5 h. To this dark brownish solution was
slowly added TMSCl (3.96 mL, 1.4 eq.). The mixture was stirred
at ꢁ78 ꢀC for 1 h and then was allowed to warm to room
temperature over 3 h. The solution was poured into ice/H2O
mixture. The residue was extracted with ethyl acetate. The
combined organic extracts were washed with sat. NH4Cl and
brine, respectively; dried over Na2SO4 and concentrated.
Compound 3 was obtained by ash column chromatography as
a colourless liquid (5.13 g, 99% yield). 1H NMR (300 MHz,
CDCl3) d 7.81 (d, J ¼ 8.3 Hz, 2H), 7.36 (d, J ¼ 8.1 Hz, 2H), 4.08 (t, J
¼ 7.3 Hz, 2H), 2.60 (t, J ¼ 7.3 Hz, 2H), 2.46 (s, 3H), 0.12 (s, 9H);
13C NMR (150 MHz, CDCl3) d 144.96, 132.92, 129.92, 127.97,
100.33, 87.49, 67.57, 21.69, 20.74, ꢁ0.10.
In-gel uorescence scanning
Aer precipitation, proteins were spin down at 6000g for 5 min
at 4 ꢀC. The supernatant was discarded, and the pellet was
washed with ice-cold methanol twice and air-dried for 10 min.
The proteins were resuspended in 1ꢂ LDS loading buffer
(Invitrogen) with 50 mM DTT and heated at 80 ꢀC for 8 min
before resolving by SDS-PAGE. The labelled proteins were
visualized by scanning on a Typhoon 9410 model.
Preparation of compound 4. To a solution of tosylate 3
(5.13 g, 17.30 mmol) in acetone (30 mL) was added lithium
bromide (3.0 g, 2 eq.). The suspension was stirred at room
temperature overnight. The resulting suspension was dissolved
in water and the aqueous solution was extracted with ethyl
acetate. The combined organic layers were washed with brine
and dried over Na2SO4. The solution was concentrated and
compound 4 was obtained by ash column chromatography as
a colourless liquid (2.45 g, 69% yield). 1H NMR (400 MHz,
CDCl3) d 3.42 (t, J ¼ 7.5 Hz, 2H), 2.77 (t, J ¼ 7.5 Hz, 2H), 0.15 (s,
9H); 13C NMR (100 MHz, CDCl3) d 103.32, 87.13, 29.30, 24.42,
0.08.
Preparation of compound 5. The Grignard reagent was
prepared according to the literature reported by Jarvo et al.41 To
a ame-dried 50 mL two necks round ask equipped with a stir
bar and condenser was added with magnesium turnings
(352 mg, 2.2 eq.). Then anhydrous THF (10 mL) was added via
Peptide synthesis
All peptides used in this research were synthesized on Rink-
Amide MBHA resin followed standard Fmoc-based solid-phase
peptide synthesis protocol. Aer the coupling of all amino
acids, the removal of protecting groups and cleavage of peptides
from the resin were done by incubating the resin with cleavage
cocktail containing 95% triuoroacetic acid (TFA), 2.5% triiso-
propylsilane, 1.5% water and 1% thioanisole for 2 h. Peptides
were puried by preparative HPLC with an XBridge Prep
OBDTM C18 column (30 mm ꢂ 250 mm, 10 mm, Waters).
Mobile phase used were water with 0.1% TFA (buffer A) and 90%
acetonitrile (ACN) in water with 0.1% TFA (buffer B).
Cross-linking yield determination
The chemical conjugation of BSA to rhodamine and the deter- syringe followed by iodine under argon.
A solution of
mination of photo-cross-linking yield of selected proteins using compound 4 (2.7 g, 2 eq.) in THF (5 mL) was slowly added.
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with ethyl acetate. The combined organic layers were washed
with brine and dried over Na2SO4. The solution was concen-
trated and compound 6 was obtained by ash column chro-
matography purication to give a white solid (900 mg, 98%). 1H
NMR (400 MHz, MeOD) d 4.07 (dd, J ¼ 9.1, 5.0 Hz, 1H), 2.68 (t, J
¼ 7.2 Hz, 2H), 2.64–2.53 (m, 2H), 2.40 (ddd, J ¼ 9.5, 6.6, 2.7 Hz,
2H), 2.21 (t, J ¼ 2.7 Hz, 1H), 2.17–2.02 (m, 1H), 1.92–1.74 (m,
1H), 1.44 (s, 9H).
Preparation of compound 7. Compound 6 (0.76 g, 2.68
mmol) was dissolved in about 30 mL of liquid ammonia. The
solution was stirred at ꢁ40 ꢀC to ꢁ30 ꢀC for 5 h. Then the
ꢀ
reaction ask was cooled to ꢁ50 C and a solution of hydrox-
ylamine-O-sulfonic acid (0.31 g, 1.3 eq.) in anhydrous methanol
was dropped into the mixture over 30 min. The reaction was
then allowed to stir at ꢁ40 ꢀC to ꢁ30 ꢀC for 10 h and the
ammonia was evaporated. The suspension was ltered through
Celite® to remove the precipitant. The lter cake was washed
with several portions of anhydrous methanol. The combined
washings were concentrated until no smell of ammonia could
be detected. The oil was re-dissolved in 10 mL of anhydrous
ꢀ
methanol and cooled to 0 C. TEA (0.75 mL, 2 eq.) was added
followed by adding iodine solution in MeOH until a brown
colour persisted. The reaction was stirred at room temperature
for 1 h, then concentrated and the residue was re-dissolved in
ethyl acetate. The organic solution was washed by water and
brine, respectively; dried over Na2SO4. The solution was
concentrated and compound 7 was obtained by ash column
Fig. 3 (a) Structures of probe 3 to probe 7, (b) selective labelling of
Sirt3, HP1, BRD4, and Sirt5 by corresponding probes. Photo-cross-
linking experiments followed the same conditions as listed in Fig. 2.
1
chromatography as a colourless liquid (0.43 g, 54% yield). H
NMR (400 MHz, MeOD) d 5.08 (s, 1H), 4.06 (d, J ¼ 8.2 Hz, 1H),
2.25 (t, J ¼ 2.7 Hz, 1H), 2.02 (td, J ¼ 7.5, 2.7 Hz, 2H), 1.71–1.57
(m, 3H), 1.57–1.46 (m, 3H), 1.44 (s, 9H); 13C NMR (150 MHz, d6-
DMSO) d 173.70, 155.58, 83.14, 78.06, 71.75, 52.74, 31.42, 28.18,
28.06, 27.93, 24.93, 12.67; IR (CHCl3): 3300, 2980, 2125,
Meanwhile, the reaction ask was heated with heat gun to
gently reux until the brown colour disappeared. The addition
of compound 4 lasted for 30 min. Then the reaction was stirred
at room temperature for 2 h. The mixture was cooled to ꢁ40 ꢀC
and a solution of Boc protected L-pyroglutamate methyl ester
(1.6 g, 6.58 mmol) in anhydrous THF (10 mL) was added to the
1715 cmꢁ1
.
Preparation of compound 8. Compound 7 (300 mg, 1.02
mmol) was dissolved in 4 M HCl/THF (1 : 1, 2 mL in total). The
mixture was stirred at room temperature for 3 h. Then solvent
was removed under reduced pressure, the resulting white solid
was used for next step without any further purication. The
solid was dissolved in water (5 mL) and dioxane (10 mL) fol-
lowed by adding NaHCO3 (213 mg, 2.5 eq.). To the mixture was
added a solution of Fmoc-OSu (0.42 g, 1.2 eq.) in dioxane (2 mL)
over 15 min. The solution was stirred for 24 h at room
temperature. All the organic solvents were removed, and the
residue was dissolved in 5 mL of water. The solution was acid-
ied to pH 3–4 using 1 M HCl. The aqueous solution was
extracted with ethyl acetate. The combined organic layers were
dried over Na2SO4. The solution was concentrated and
compound 8 was obtained by ash column chromatography as
a white solid (200 mg, 47% yield). 1H NMR (400 MHz, d6-DMSO)
d 7.89 (d, J ¼ 7.5 Hz, 2H), 7.71 (d, J ¼ 7.4 Hz, 2H), 7.59 (d, J ¼
8.2 Hz, 1H), 7.42 (t, J ¼ 7.5 Hz, 2H), 7.33 (t, J ¼ 7.4 Hz, 2H), 4.33–
4.20 (m, 3H), 3.88 (s, 1H), 2.81 (s, 1H), 2.05–1.94 (m, 2H), 1.71–
1.28 (m, 6H); 13C NMR (100 MHz, d6-DMSO) d 173.41, 156.11,
143.84, 143.77, 140.74, 127.64, 127.06, 125.23, 120.12, 71.74,
65.57, 53.17, 46.66, 31.41, 28.74, 28.06, 25.11, 12.71; LRMS (EI,
ꢀ
mixture over 20 min. The reaction was kept stirring at ꢁ40 C
for 2 h. Then saturated NH4Cl was added to quench the reac-
tion. The aqueous solution was extracted with ethyl acetate and
the combined extracts were washed with brine and dried over
Na2SO4. Solvent was removed in vacuo and compound 5 was
obtained by ash column chromatography purication as col-
ourless oil (2.2 g, 91% yield). 1H NMR (400 MHz, CDCl3) d 5.07
(d, J ¼ 6.56 Hz, 1H), 4.28 (br, 1H), 3.74 (s, 3H), 2.66 (t, J ¼
7.04 Hz, 2H), 2.61–2.46 (m, 4H), 2.19–2.13 (m, 1H), 1.94–1.85
(m, 1H), 1.44 (s, 9H), 0.13 (s, 9H); 13C NMR (100 MHz, CDCl3)
d 207.43, 172.86, 155.51, 105.61, 85.20, 80.09, 52.90, 52.46,
41.72, 38.55, 28.36, 26.61, 14.45, 0.12; IR (CHCl3) 3367, 2959,
2178, 1732 cmꢁ1; HRMS (EI, 20 eV) for C14H23NO5Si (M ꢁ t-Bu)+:
313.1340; found: 313.1328.
Preparation of compound 6. To a solution of compound 5
(1.20 g, 3.25 mmol) in THF (15 mL) with ice bath was added 1 M
LiOH (2 eq.) over 20 min. Aer the addition of LiOH, the reac-
tion was allowed to come back to room temperature and stirred
for 1 h. The mixture was then cooled to 0 ꢀC and acidied to pH
3–4 using 0.1 M HCl. The resulting suspension was extracted
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We next sought out to test the ability of dzANA in capturing
PPIs. To this end, the histone PTM-mediated PPIs were chosen
as the models. Histones, the scaffold proteins for the package of
DNA in eukaryotic cell nuclei, are extensively decorated by
diverse PTMs.27 By recognizing the histone PTMs, many effector
proteins bind to histones to regulate DNA-templated biological
processes, such as gene expression.28–30 Because of the dynamic
nature of histone PTMs, the PPIs mediated by these modica-
tions are normally weak or even transient, rendering them
stringent models to evaluate the performances of chemical
probes to capture PPIs.
To develop dzANA-based photoaffinity probes for the study
of histone PTMs, we rst focused on a well-studied trimethy-
lation mark at histone H3 Lys 4 (H3K4me3). Our design of probe
1 (Fig. 2a) was based on the H3K4me3 peptide (residues 1–15),
in which the dzANA was incorporated at the original Ala 7 site,
a position that is near the K4me3 binding pocket to ensure the
cross-linking efficiency and specicity, but is not too close to
interfere with the PPI of interest. Two known binding partners
of H3K4me3, SPIN1 (ref. 31) and ING2,32,33 were used as positive
controls to validate the capability of probe 1 in the identication
of H3K4me3-mediated PPIs. The two proteins were rst incu-
bated with probe 1. Aer UV-induced photo-cross-linking, the
probe 1-labelled proteins were conjugated to rhodamine-azide
(Rho-N3) via ‘click chemistry’ and resolved by SDS-PAGE.
Subsequent in-gel uorescence scanning showed that both
SPIN1 and ING2 could be labelled by probe 1, but not by
a control probe 2 lacking the trimethylation mark (Fig. 2b).
Interestingly, experiments using BHC80,34 an unmodied
histone H3 tail binder, led to a completely reversed cross-
linking pattern that the trimethylated probe 1 failed to
capture the protein while the unmodied probe 2 induced
robust labelling (Fig. 2b). This observation matched with the
previous discovery that the K4me3 mark could disrupt the
BHC80–H3 tail interaction. It was worth noting that the probe 1-
induced labelling toward SPIN1 and ING2, and the probe 2-
induced labelling toward BHC80, could be competed off by the
addition of their corresponding native peptide ligands (Fig. 2b),
further demonstrating that the cross-linking was modication-
Fig. 4 Quantification of photo-cross-linking yield of selected pho-
toaffinity probes toward their corresponding binding partners. Photo-
cross-linking experiments followed the same conditions as listed in
Fig. 2.
20 eV) m/z 389.2 (M+ ꢁ N2, 0.14), 178.1 (100); HRMS (EI, 20 eV)
for C24H23NO4 (M+ ꢁ N2): calcd 389.1622, found 389.1630.
Results
To synthesize dzANA, a seven-step synthetic route was designed dependent instead of non-specic labelling.
(Scheme 1). Briey, 3-butyn-1-ol 1 was treated with p-tosyl
Encouraged by above results, we further designed and
chloride in the presence of triethylamine to give compound 2, synthesized a series of dzANA-based probes (probe 3 to probe 6,
whose terminal alkyne was further protected by trimethylsilyl Fig. 3a) to expand our tests to PPIs mediated by different
group to yield 3. Nucleophilic substitution with LiBr converted histone PTMs at varied positions. As shown in Fig. 3b, all of
the tosylate into organobromide 4, which was used for the these probes could robustly and selectively label their known
preparation of corresponding Grignard reagent. The following binding proteins, which were the crotonylation mark at H3K4
ring-opening reaction of the Boc protected L-pyroglutamate (probe 3) toward Sirt3,35 the trimethylation mark at H3K9 (probe
methyl ester by the in situ generated organomagnesium species 4) toward HP1,36,37 the acetylation mark at H3K9 (probe 5)
afforded the intermediate 5 with the aminononynoic acid toward BRD4,38 and the malonylation mark at H3K9 (probe 6)
skeleton. Simultaneous removal of the trimethylsilyl group and toward Sirt5,39,40 while the corresponding control probe 2 and
the methyl ester under an alkaline environment provided the probe 7 without the modications showed no cross-linking
ketone 6. Subsequent diazirine synthesis using the ketone as signals.
a precursor led to the formation of 7, which was further
To obtain a more quantitative assessment on how efficient
deprotected by triuoroacetic acid to give the desired dzANA. To the dzANA-harbouring probes captured their target proteins, we
facilitate the solid-phase peptide synthesis, the Fmoc-protected repeated the photo-cross-linking experiments of ING2, SPIN1,
dzANA (8) was also prepared. The overall yield of the whole BHC80, and Sirt3 using the corresponding probes. To each of
synthetic route was 15%.
the resulting samples, we parallelly loaded a series of different
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i.e., mapping protein–protein binding regions. To build a pho-
toaffinity probe for the identication of PPIs, a common prac-
tice is to incorporate two unnatural amino acids, one with
photo-reactivity and another with bioorthogonality. Under
most circumstances, such photoaffinity probes are incompetent
to mapping protein–protein binding regions. This is because
that the proteins labelled by the photoaffinity probes should be
digested into short peptides for mass spectrometry-based
protein identication. During this process, the peptide-based
photoaffinity probes will also be digested. As a result, the bio-
orthogonal handles that are subsequently conjugated to affinity
tags for protein purication will be separated from the photo-
reactive groups that cross-linked to the regions mediated the
PPIs, which will eventually lead to the loss of information on the
binding regions. Incorporation of dzANA into peptide probes at
the positions close to key residues for binding would allow
efficient identication of both the PPIs involved and mapping of
the binding sites (Fig. 5a).
Most of the studies on PPIs have been carried out by using
puried recombinant proteins or cell extracts in vitro. The PPIs
are known to be dynamic and regulated by a series of cellular
mechanisms in living cells, which are very different from the
situation in vitro. The identication of PPIs in living cells should
provide more direct information for unravelling their interac-
tion networks. However, simultaneous incorporation of both
a photo-reactive and a bioorthogonal unnatural amino acids
into proteins co-translationally has been technically chal-
lenging. The newly developed bifunctional dzANA holds the
potential to be incorporated into proteins by engineering the
cellular translational machinery. It may facilitate the in situ
xing of PPIs in living cells and the isolation of the captured
proteins for further investigations (Fig. 5b).
Fig. 5 Potential applications of dzANA in (a) mapping PPIs binding
sites, and (b) study PPIs in living cells.
Conclusions
In summary, we have developed a novel bifunctional amino
acid, dzANA, carrying both a diazirine moiety and a terminal
alkyne handle, which is readily incorporated into peptides
through standard Fmoc-based solid-phase peptide synthesis
strategy. We showed that photoaffinity probes harbouring
dzANA could effectively and specically capture the weak and
transient PPIs mediated by histone PTMs, suggesting the
potential of this amino acid as a useful tool to study unknown
PPIs. By integrating both photo-reactive and bioorthogonal
functionalities into one amino acid, dzANA is expected to
amount of bovine serum albumin (BSA) pre-labelled by rhoda-
mine in SDS-PAGE as standards.24 Based on the in-gel uores-
cence scanning results (Fig. 4), all the four tested proteins
showed satisfactory cross-linking yields, ranging from 13% to
50%.
Discussion
To full the requirements for the explorations of PPIs in various facilitate the mapping of PPI binding sites and the study of PPIs
contexts and for obtaining different information about the PPIs, in living cells. Such explorations are our important next steps
there is a continuous demand on the development of novel and will be reported in due course.
photo-reactive amino acids with diverse structural properties
and functionalities. Compared with unnatural amino acids with
only photo-reactivity, dzANA is expected to pose advantages in
Conflicts of interest
two different applications, including the mapping of protein– There are no conicts to declare.
protein binding site and identication and characterization of
PPIs in living cells.
Acknowledgements
A comprehensive understanding of PPIs requires not only
the identication of the binding partners but also the identi- This study is supported by National Natural Science Foundation
cation of the areas where two proteins interact with each other, of China (91753130 to X. D. L.) and Excellent Young Scientists
This journal is © The Royal Society of Chemistry 2020
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Fund of China (Hong Kong and Macau) (21922708 to X. D. L.). 13 X. Xie, X. M. Li, F. Qin, J. Lin, G. Zhang, J. Zhao, X. Bao,
The authors acknowledge the support from the Hong Kong
Research Grants Council (RGC) Collaborative Research Fund
(CRF C7029-15G and C7017-18G to X. D. L.), the Areas of
R. Zhu, H. Song, X. D. Li and P. R. Chen, Genetically
Encoded Photoaffinity Histone Marks, J. Am. Chem. Soc.,
2017, 139(19), 6522–6525.
Excellence Scheme (AoE/P-705/16 to X. D. L.), the General 14 E. M. Tippmann, W. Liu, D. Summerer, A. V. Mack and
Research Fund (GRF 17121120, 17126618, and 17125917 to X.
D. L.), and the RGC Postdoctoral Fellowship (Scheme 2020/21 to
X. L.).
P. G. Schultz,
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genetically encoded diazirine
15 B. Van der Meijden and J. A. Robinson, Synthesis and
application of photoproline - a photoactivatable derivative
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