Recent advance of photo-crosslinker
using tetrazole

Literature seminar #2
M1 Shinpei Takamaru
2023/06/22 (Thu)Contents
Ø IntroductionØStandard method to identify target proteinØConventional photo-crosslinker

Ø MainØTetrazole as photo-crosslinker
Ø2-acyl-5-carboxytetrazole (ACT)ØPPI mapping by using ACT in living cells

Ø Summary2Introduction
drug /
proteinprotein

What ? ( identification )How ? ( interaction mechanism )identifying binding protein or binding site
→ understand the mechanism of action→ improve the drug
3

Standard method for identifying target protein
Ø①

Co-IP

Major two methods
to identify thetarget protein① Co-IP② crosslinking

https://www.lifeasible.com/custom-solutions/plant/analytical-services/gene-function-analysis/coimmunoprecipitation-co-ip-assay/

4reacts with lysine rapidly, planting the reagent at specific localizations on protein. When the pendant aryl
sulfonyl fluoride is cast proximal to the target residues,
the increased effective concentration enables aryl sulfonyl fluoride to react with multiple weakly nucleophilicamino acid residues. This proximity-enhanced reaction
decreases the nonspecific crosslinks and exhibits abetter structural compatibility than the traditional bissuccinimide ester crosslinker. In addition, NHSF isinteracting protein whenever they are in acells and accumulate the crosslinked pro
identification with high sensitivity. The lategenetic encodability, and the spontaneouscrosslinking ability make GECX uniquely
applications in vivo.

Standard method for identifying target Aprotein
series of latent bioreactive Uaas have b
Figure 3

Ø②and genetically encoded into proteins t

crosslinking

Major two methods
to identify thetarget protein
Liu, J., Yang, B., & Wang, L. Current Opinion in Chemical Biology 2023, 74, 102285.

① Co-IP② crosslinking

enrichment
tag
Current Opinion in Chemical Biology

Crosslinking mechanism of small molecule crosslinker containing one weakly reactive group whose reactivity is enhanced by pro
www.sciencedirect.com

pharmacophore

photo-reacting
Current Opinion in Chemical Biology
group
5

re
m
sp

correct distance to be bridged by the cross-linker (Scheme 2).
The cross-link can then be created between two lysine residues
in proximity on the same peptide (intrapeptide cross-link) or
between two lysines separated from each other in the amino acidsequence or even located in diﬀerent proteins (interpeptide
cross-link). From a structural standpoint, interpeptide crosslinks are the most interesting ones and serve as the basis for

M
irr
th
of
in
Ra
re
ph
pr
as
co
ch
im

Photo-crosslinker and Chemical crosslinker
Ø Photo-crosslinker

Scheme 2. Reaction of Amine-Reactive NHS Esters,
Exempliﬁed for BS3 (Upper Panel). Reaction Products withProteins Include Intrapeptide Cross-links (Type 1; Left),Ø ChemicalInterpeptide
Cross-linksacrosslinker(Type 2; Middle), and “Dead-End”
Cross-links or “Mono-links” (Type 0; Right)

3.

Burton, N. R., Kim, P., & Backus, K. M.
Organic and Biomolecular Chemistry 2021, 19(36), 7792-7809.

To
alt
be
m
ly
ex
lin
ph
to
ar
lig
sti
wa
bi

Piersimoni,L., et al.one
Chem.
Rev. 2022,
122, 7500-7531.
Cross-links can becreated withinprotein(intraprotein,
red orblue only) or between diﬀerent proteins (interprotein, red and blue
6
mixed).

a7506

Conventional photo-crosslinker

Burton, N. R., Kim, P., & Backus, K. M. Organic and Biomolecular Chemistry 2021, 19(36), 7792-7809.

7

Conventional photo-crosslinker

Organic & Biomolecular Chemistry

View Article Online

Reviewtural motifs naturally present in many natural products andbioactive molecules. When photoactivated with 350 nm light,
pyrones yield a reactive ketene intermediate while pyrimidones
aﬀord either a bicyclic urea or isocyanate intermediate, all ofwhich are susceptible to nucleophilic crosslinking with adjacent amino acids.75
Continuing in the theme of nature-inspired photoactivatable handles, another functional group found in many natural
products is the tetrazole moiety (Fig. 1B). The photolability
and subsequent nucleophilic capture of the diaryltetrazole(DAT) was first reported by Rolf Huisgen in 1961.76 Nearly forty
years later, the tetrazole functionality was debuted as abioorthogonal group for bioconjugation, functioning via irreversible loss of nitrogen to form a reactive nitrile imine species
that is then trapped by an alkene through a 1,3-dipolar cycloaddition.77 This reactive intermediate could also be intercepted with carboxylate functionalities and as such was proposed as a photoreactive group for PAL experiments.36,78 A
similar handle, the 2-aryl-5-carboxytetrazole (ACT; Fig. 1B), wasdeveloped in parallel and Burton,
was reported
higherN. R., Kim,toP.,have& Backus,
K. M.crossOrganic
Biomolecular
Chemistry
2021, 19(36),matched
7792–7809.
linkingyieldandwhen
comparedwith structurally
benzo798
phenone and diazirine probes. In addition to DAT and ACT,
other photoreactive groups identified within the past five years
include o-nitrobenzyl alcohols (o-NBA; Fig. 1B), as lysine
specific photoactivatable groups, and o-(hydroxymethyl)
phenols (o-HMP) as tryptophan specific groups.80,81 Upon

Problem

ownloaded by The University of Tokyo on 6/8/2023 2:40:09 PM.

Ø extremely short half-lives→ very low target capturing yields
Ø the nitrone, carbene, anddiradical intermediates→ react non-selectively with any
proximal C–H/X–H bonds(X = N, O, S)→ high backgroundNon-radical intermediate photo-crosslinker

Battenberg, O. A., Nodwell, M. B., & Sieber, S. A.
Journal of Organic Chemistry 2011, 76(15), 6075–6087.

Current Opinion in Chemical Biology

Liu, J., Yang, B., & Wang, L. Current Opinion in Chemical Biology 2023, 74, 102285.

schematic view of photoaffinity labeling. PG: photoactivatable crosslinking group; CG: crosslinking group. (b) A schematic view of protein crossg with photoactivatable small molecule crosslinkers. The crosslinker can have one or two PGs. (c-e) Photoreactive mechanisms for tetrazole (c), 2enzyl alcohol (d), and photocaged quinone methide (e).

9Contents
Ø IntroductionØStandard method to identify target proteinØConventional photo-crosslinker

Ø MainØTetrazole as photo-crosslinker
Ø2-acyl-5-carboxytetrazole (ACT)ØPPI mapping by using ACT in living cells

Ø Summary
10

plinaryu.edu.cn
emistry

ocedurescoupling reaction, especially under the physiologically relevant
conditions, was warranted in order to achieve its full potential
for chemical biology research. Nallani’s group and Zhao’s groupindependently reported photo-induced reactions of tetrazoles
with a number of endogenous amino acids,6 but neither publication
has considered the reactivity of carboxylic acids. Given the highTetrazole photo-click chemistry
ChemComm

6. Downloaded by The University of Tokyo on 6/18/2023 9:16:51 AM.

g is acontrolme andinkingneithern, they
activity
resultsrgetingrazolesarkablento thecleanlyaffordry’’ hasantagesdducts,eactionhand-

Fig. 1 Photo-inducible reactions of tetrazoles. (a) Photo-induced tetrazole–alkene addition. (b) Photo-induced reactions of tetrazole 1a and carboxylic
acids.

Fig. 3 The competition reaction of carboxylic acid addition and 1,3-dipolar
cycloaddition of tetrazole 1b.

ü nitrile imine intermediate B
could react with a carboxylic acidand a hydroxamic acid (entries 2–14). The complete conversionof 1b was observed in all reactions while most additives did notinterfere with the photo-induced reaction. Nonetheless, it isnoteworthy that in the presence of Tyr, Lys or Cys amino acids
the yield of 9 decreased to a different degree (entries 10–12),
Zhao, S., et al. Chem. Commun. 2016, 52, 4702-4705.
presumably due to the reactions between the nitrile11imine
Li, Z., et al. Angew. Chem. Int. Ed. 2016, 55, 2002-2006.
intermediate and phenol, amino or thiol groups.6,13
The initial bioconjugation was attempted between the myoglobin
protein and tetrazole 1b. Analysis using electrospray ionization
mass spectrometry (ESI-MS) indicated the formation of a new
species into which the reactive intermediate was incorporated

Fig. 4indicatin
Da = 17 8
sites (blderivedIn an atetrazo
tryptic
presenand E4(Fig. 4predomcurrenand eaWeaminoacids. andWe1,3-dipolarinvestigated
the reactionof tetrazole 1b
carboxylic
acid addition
cycloadditionof tetramodel for various carboxylic acids, the reaction rates were genous
8 andquantitatedtheof product 9 byzole Boc-Asp-OtBu
1b in ethyl acetate(organic
solvent) andan yields
acetonitrile/
determined on the basis of HPLC analysis (see the ESI† for with
PBS bufferanalysis(1 : 1) mixture,
respectively.
TheS9,
resultssuggesteddetails, Fig. S1–S7). The apparent second-order rate constants HPLC-MS(Table2 and Fig.
ESI†).
In the absence ofwere derived (Table 1 and Table S2, ESI†) based on the assumption that the in situ generated reactive nitrile imine reacted selectively
any additive, the reaction was determined to give 9 in 68% yieldthat both the photolysis of diaryltetrazoles and the intramolecular with carboxylic acids over alkenes in ethyl acetate, but acted1). inThereactionwas(Fig.
thencontrarily
the buffer
mixture3). repeated in the presence of
O - N acyl transfer are very rapid processes.10,11 For unsub- (entry
Beforeutilizing thetetrazoles tomodify native
proteins,
a an alkyne
stituted diphenyl-tetrazole 1a, the apparent second-order rate various
additives,
includingprotected
aminoacids,!1 !1constant was estimated to be 2.8 M s , while the yield of the robustness screen of this photo-inducible reaction was desirable
to assess, rapidly and discretely, its tolerance to other functionalgroups in the aqueous solution, especially those within the endoTable 1 Kinetic study of photo-induced
reactions of tetrazoles
with
!1 !1
b
a
genous amino acids.12 We investigated the reaction of tetrazole 1b
X
Y
kCOOH [M s ]Yield (%)
propionic acidwith Boc-Asp-OtBu 8 andquantitatedthe yields of product 9 bydesiredproduct
HH
2.8
45
HPLC-MS analysis (Table 2 and Fig. S9, ESI†). In the absence of
p-OMe
H
5.5
56
any additive, the reaction was determined to give 9 in 68% yield
H
p-OMe
7.4
87
(entry 1). The reaction was then repeated in the presence of
H
0.28
5.9
p-CO2Mevarious additives, including protected amino acids, an alkyneePublished on 02 March 2016. Doc acidaOptimized di-aryl-tetrazoleH
p-CO2Me
p-OMe

Tetrazole

p-CO2Me
p-OMe
p-CO2Me
X

0.05
0.88
0.21

Y

2.8
19
4.7
!1 !1

kCOOH [Ms ]Yieldb (%)

on mixture1aof tetrazoleacid (1 mM)in
H (100 mM)
H and propionic
2.8
45
CN (1 : 1) 1b
within a p-OMe
quartz test
tube
wasirradiated
using
a
H
5.5
56
1c (302 nm,
H 6 W) at p-OMe
7.4
87
ld UV lamp
room temperature.
The product
H
0.28
5.9
1d
p-CO2Mes were analyzed
by reversed-phase
HPLC-MSand quantified
by
0.05
2.8
1e
H
p-CO2Meng the integrated
peaks
to2Me
that ofa standard;
S2
p-OMe
0.88see the ESI (Table
19
1f
p-CO
b
0.21of irradiation.4.7
p-OMe yieldp-CO
after
5 min
S1–S7) for1gdetails. HPLC
2Mehydrolysis
Fig. cycloaddition
2 The effect of pH
on the product distribution of reaction of
product
product
propionic
acid with tetrazole
1b. (a) Structures of the three observed
products
in PBS/MeCN (1 : 1) solution. (b) Reactions carried out at pH
Fig. 2 The effect of pH on the product distribution of reaction of propionic acid with tetrazole 1b. (a) Structures of
the threeobserved products
PBS/MeCN(1:1)
solution.
(b) Reactions
carried out atwere
pH values
ranging from 2.5using
tovaluesranging
fromin2.5
to 10.
The
yieldsof products
quantified
10. The yields of products were quantified using HPLC-MS. The data shown are an average of three replicate
HPLC-MS.
experiments. The data shown are an average of three replicate experiments.

aReaction mixture of tetrazole (100 mM) and propionic acid (1 mM) in
PBS/MeCN (1 : 1) within a quartz test tube was irradiated using aühandheld
tetrazoles
with
group
UV lamp(302only
nm, 6electronic-donatingW) at room temperature. The
product
mixtures were analyzed by reversed-phase HPLC-MS and quantified bygive Society
highproductyield
nal is © ThecomparingRoyalof Chemistry
the integrated
peaks
to2016that of a standard; see the ESI (Table S2band Fig. S1–S7) for details. HPLC yield after 5 min of irradiation.

Fig. 2 The effect of pH on the product distribution of reaction of
propionic acid with tetrazole 1b. (a) Structures of the three observed
products in PBS/MeCN (1 : 1) solution. (b) Reactions carried out at pH
values ranging from 2.5 to 10.Chem.
The yields
of products were
quantifiedusing
Commun.,2016,
52, 4702--4705
HPLC-MS. The data shown are an average of three replicate experiments.

ü the highest yield of the desired product 4b wasobserved under neutral or slightly acidic| 4703
conditions (pH = 7.1 or 5.5)
Zhao, S., et al. Chem. Commun. 2016, 52, 4702-4705. (Fig. modified) 12

This journal is © The Royal Society of Chemistry 2016a Reaction mixture of tetrazole (100 mM) and propionic acid (1 mM) in PBS/MeCN (1:1) within a quartz test tube was irradiated usinga handheld UV lamp (302 nm, 6 W) at room temperature. The product mixtures were analyzed by reversed-phase HPLC-MS andquantified by comparing the integrated peaks to that of a standard; see the ESI (Table S2 and Fig. S1–S7) for details. b HPLC yield after
5 min of irradiation.

Chem. Commun., 2016, 52, 4702--4705 | 4703

Published on 02 March 2016. Downloaded by The Univ

noteworthy that in the presence of Tyr, Lys or Cys amino acids
the yield of 9 decreased to a different degree (entries 10–12),
presumably due to the reactions between the nitrile imine
intermediate and phenol, amino or thiol groups.6,13
The initial bioconjugation was attempted between the myoglobin
protein and tetrazole 1b. Analysis using electrospray ionization
mass spectrometry (ESI-MS) indicated the formation of a new
species into which the reactive intermediate was incorporated
with equal stoichiometry (Fig. 4a). The conversion was about 12%.

Amino Acid Selectivity
Table 2

Photo-induced reaction of tetrazole 1b with acid 8atryptic digestion and tandem mass spectrometry confirmed thepresence of modified-carboxyl groups at positions E18, E27, E37
and E41 by the appearance of expected y and b fragment ions(Fig. 4b and Tables S3–S7, ESI†). These active residues, aspredominant sites of modifications identified in database undercurrent conditions, were exposed on the surface of myoglobin
and easily accessible to the reaction.
We subsequently investigated the possibility of using thetetrazole as a new photo-crosslinking functionality in the fieldof photo-affinity labelling, which could preferentially react withcarboxylic acid residues. As a proof-of-concept study, we designed
and de novo synthesized the active site-directed small molecule
probe 11 based on SAHA (10), a widely used inhibitor for histone
deacetylases (HDACs) (Fig. 5a and Scheme S1, ESI†).15 The designof this probe was inspired by the phenyl azide and benzophenone
derivatives of SAHA reported by Richon’s group and Cravatt’sgroup, respectively.16 In parallel, we also prepared a pseudoprobe (12) that lacked the strong Zn2+ chelating group (hydroxamicacid) and hence should bind HDACs much more weakly.15 Both 11
and 12 contain a terminal alkyne group as the ‘clickable’ moiety toallow conjugation of a desired tag after photoreaction.
We first tested probe 11 for the labelling of HDACs in HepG2cell lysates (Fig. 5b). The results confirmed that both HDAC1
and HDAC2 could be labelled by probe 11 (lanes 1–3), and thesignal intensity was competed away by SAHA (lanes 4 and 5). In
comparison, pseudo-probe 12 labelled neither HDAC1 nor
HDAC2 (lane 6). The labelling event was dependent on the
UV irradiation (lane 7), while there was hardly any labelling in
the denatured cell lysates (lane 8). The tetrazole-SAHA 11 should becovalently linked
2 to HDAC1 and HDAC2 upon UV irradiation
because the detected HDACs survived under the harsh denaturingcondition that is able to disrupt any noncovalent protein–protein/inhibitor interaction. Moreover, successful labelling was realizedby photolysis of probe 11 in living
was Chem.
also competed
Li,cells,
Z., et whichal. Angew.
Int. Ed. 2016, 55, 2002-2006.
away by excess SAHA (Fig. 5c and Fig. S10, ESI†). To pinpoint
the potential site(s) that reacted with the tetrazole moiety of 11,
Zhao, S., et al. Chem. Commun. 2016, 52, 4702-4705. (Fig. modified) 13

ü The complete conversion of 1b was observed in
all reactions

ü most additives did not interfere with the photoinduced reaction.

Entry

Additive

Yieldb (%)

1
2
3
4
5
6
7
8
9
10
11
12
13
14

None
Boc-Asn-ONpc
Boc-Gln-ONp
Boc-Ser-OMe
Boc-Thr-OMe
H-Arg-OMe
H-His-OMe
Boc-Trp-OSud
Boc-Met-OSu
Boc-Tyr-OMe
H-Lys-OMe
H-Cys-OEt
n-C4H9CCH
CH3CONHOH

68
74
74
64
69
61
66
66
78
40
40
33
64
65

a

Reaction mixture of tetrazole 1b (100 mM), 8 (2 mM) and additive(2 mM) in PBS/MeCN (1 : 1) within a quartz test tube was irradiatedusing a handheld UV lamp (302 nm, 6 W) for 2 h. b HPLC yield of 9 after 2 h.
c
Np = p-nitrophenyl. d Su = succinimide.

4704 | Chem. Commun., 2016, 52, 4702--4705

ü in the presence of Tyr, Lys or Cys, the yield of 9
decreased to a different degree (entries 10-12)→ presumably due to the reactions between the
nitrile imine intermediate and phenol, amino or
thiol groups.
→ Asp, Glu, Cys, Lys selectivity
ü Interestingly, of all functional groups tested,
CO H appeared most reactive at physiologicalpH, better than other common nucleophiles
including thiols, amines, and alcohols.

This journal is © The Royal Society of Chemistry 2016

-dipolar

ersion
id not, it iso acids
10–12),
imine

oglobinzationa new

Photo-crosslinking of myoglobin
View Article Online

Communication

https://www.uniprot.org/uniprotkb/P02144/entry#sequences

Fig. 4 Photo-labelling of myoglobin using tetrazole 1b. (a) ESI-MS analysis indicating the formation of the anticipatedFig.
4 1bPhoto-labelling
tetrazole
1b.
(a)modification
ESI-MSsites
analysistetrazoleadduct (expected Da = 17of
813).myoglobin(b) Residues E18, using
E27, E37 andE41 identified
as the(blue),
the locations ofthe
which formation
were indicated byof
the arrows.
View derived from atetrazole
crystal structureof myoglobin
ID: 3WI8).
indicating
the anticipated1badduct(PDB(expected
Da = 17 813). (b) Residues E18,Zhao,
E27,S.,
E37
E41 identified
as the52,
modification
et andal. Chem.
Commun. 2016,
4702-4705.
sites (blue), the locations of which were indicated by the arrows. View
derived from a crystal structure of myoglobin (PDB ID: 3WI8).14

ü Analysis using ESI-MS indicated the formation of a new species (+1 modification)
ü Subsequent tryptic digestion and tandem mass spectrometry
In an analogous experiment conducted in the absence of the→ confirmed the presence of modified-carboxyl groups at positions E18, E27, E37
tetrazole, no modification of the protein was observed. SubsequentandE41and tandem mass spectrometry confirmed thetryptic
digestion
presence of modified-carboxyl groups at positions E18, E27, E37
and E41 by the appearance of expected y and b fragment ions(Fig. 4b and Tables S3–S7, ESI†). These active residues, aspredominant sites of modifications identified in database undercurrent conditions, were exposed on the surface of myoglobin
and easily accessible to the reaction.

14

SAHA and HDAC
Ø As a proof-of-concept study, small molecule inhibitor SAHA waschosenü SAHA (suberoylanilide hydroxamic acid)inhibits HDAC
→ inhibits cell proliferation and exhibits
anti-tumor effects→ effective in the treatment of acute
myeloid leukemia
https://ruo.mbl.co.jp/bio/product/epigenome/pickup/deacetylase.html

15

SAHA-probe can detect HDAC1 and HDAC2

Communication

With successful profiling of HDACs inØusing
Proof-of-concept
study
the photo-activatable probe, we

between tetrazolesüreaction
Tetrazole-SAHA
probe and(11)carboxyli
appliedto protein labeling both in vitr
was designed

widespread utility of this coupling reactio

ü Pseudo-probe (12) lacked
cross-linking technologies
and other
the strong Zn2+ chelating
on-going and will be reported in duegroup (hydroxamic acid)

This work was supported by geneütheTetrazole-SAHA
probe
National ‘‘Young
1000(11)
Talents Pl
can modify
and EngineerinH
Chemistry
andHDAC1
Molecular
Peking-Tsinghua
Center for Life Sci
HDAC2
Science Foundation of China (Grant Noü Tetrazole-SAHA covalently
linked to HDAC1 and HDAC2

Notes and references

1 For recent reviews, see: (a) Y. Takaok
Angew. Chem., Int. Ed., 2013, 52, 4088–
G. J. L. Bernardes, Chem. Rev., 2015, 115
N. Chen, Acta Chim. Sin., 2015, 73, 657
2 C. Brieke, F. Rohrbach, A. Gottschalk
Angew. Chem., Int. Ed., 2012, 51, 8446–

Zhao, S., et al. Chem. Commun. 2016, 52, 4702-4705. 16

2 March 2016. Downloaded by The University of Tokyo on 6/18/2023 9:16:51 AM.

This work was support
the National ‘‘Young 1000
Chemistry and Molecular
Peking-Tsinghua Center f
Science Foundation of Chin
Successful labeling was realized in living cells
Fig. S11. HPLC-MS analysis of SAHA after the treatment of the MS sample
preparation

Notes and refereØ MS/MS spectrum
Table S8. MS/MSand b/y ion
assignments
by Mascot(inspectrum
vitro; HDAC1
and
SAHA-probe11)confirming 203E of
1 byFor
recentreviews, see: (aHDAC1 was labeledprobe
11
Angew. Chem., Int. Ed., 20
YGEYFPGTGDLR
Res.
mass
b ion
b++
y ion G. y++m/z
J. L.z Bernardes,
Chem. R
Y
163.1
164.1
82.5
2
895.9129
N. Chen,Acta Chim. Sin., 2
G
57.0
221.1 111.0
1629.8815.4 3597.2752
2 C. Brieke, F. Rohrbach, AE
101.0
768.3 384.7
1572.7786.9 4447.9564
Angew.
Chem.,
Int. Ed., 201Y
163.1
931.4 466.2
1025.5513.3
3 (a)431.7
S. A. Fleming, TetrahedroF
147.1
1078.5 539.7
862.4
P
97.1
1175.5 588.3
715.4 and358.2A. J. Wilson, Chem. Soc.
G
128.1
1232.5 616.7
618.3 Rev.,
309.7 2011, 111, 4405–4417.
T
101.0
1333.6 667.3
561.3
4 (a)281.2Y. Wang, C. I. Vera an
G
128.1
1390.6 695.8
460.3 (b)230.6
R. K. V. Lim and Q.
115.0
1505.6 753.8
403.2
202.1
D
(c) Z. Yu, Y. Pan, Z. Wang, J
113.1
1618.7 809.9
288.2
144.6
L
2012,
51, 10600–10604; (d)
R
156.1
175.1
88.1
2014, 50, 9595.
Fig. 5 Photo-affinity labelling of HDACs in cell lysates and living cells.
5 H. Meier
H. Heimgartn
ü(a)successful
labeling
wasprobes.
realizedbyü fromMS/MS
Spectrum,
E203 andclose
Structures of
SAHA and(b) Western
blotting
analysisof biotin6 (a) Y. Zhang, W. Liu andphotolysis
11 in(IA)livingcells
to SAHA
was modified
enriched
activeof(A)probe
or inactive
HepG2
cell lysates treatedwith probesin(b) W. Siti, A. K. Khan, H.-P
41
the absence (!) or presence (+) of competitor SAHA, with orwithout
17
Zhao, S., et al. Chem. Commun.
2016, 52,
4702-4705.Chem.,
Org.
Biomol.
2015,
irradiation using a handheld UV lamp (10 min). (c) Western blotting of
7 S. D. Varfolomeev, I. V. U
HDACs enriched from in situ HepG2 cells treated with tetrazole-SAHA
2002, 67, 1099–1108.
8 Z. Li, L. Qian, L. Li, J. C.
probe 11 and competitor SAHA.
S. Q. Yao, Angew. Chem., IØ HDACs enriched from in situ HepG2 cell
treated with tetrazole-SAHA probe 11Znd alkenes developed by Lin’s group is particularly remarkable
whereas the tetrazoles or alkenes need to be first introduced into therotein of interest.4 The photolysis of tetrazole A rapidly and cleanly
enerates nitrile imine dipole B that reacts with an alkene to afford
yrazoline cycloadduct C (Fig. 1a).4 This ‘‘photoclick chemistry’’ has
ince been applied in a wide range of studies, due to the advantages
ncluding the generation of strongly fluorescent cycloadducts,
he small size of the bioorthogonal alkene tag, superior reaction
inetics and the mild photoactivation procedure using a handeld UV lamp.

rch 2016. Downloaded by The University of Tokyo on 6/18/2023 9:16:51 AM.

Short summary

ü photo-induced diaryltetrazoleacid coupling reaction as a
Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary
biochemical conjugation strategy
Studies, Peking University, Beijing 100871, China. E-mail: tuopingluo@pku.edu.cn
Beijing National Laboratory for Molecular Science (BNLMS), College of Chemistry
diaryltetrazole
can Chemistry
react andwith
and Molecular üEngineering,
Key Laboratory of Bioorganic
Molecular Engineeringof Ministry ofamino
Education, College
of ChemistryandGlu,selective
acids
(Asp,
Molecular Engineering, Peking University, Beijing 100871, China
Fig. 1 Photo-inducible reactions of tetrazoles. (a) Photo-induced tetrazole–Cys, Lys)alkene addition. (b) Photo-induced reactions of tetrazole 1a and carboxylic
Electronic supplementary
information (ESI) available: Experimental procedures
acids.
nd characterization data. See DOI: 10.1039/c5cc10445a
ü successfully profiling HDACs incell lysates and living cells

702 | Chem. Commun., 2016, 52, 4702--4705

ü this coupling reaction could be
extensively applied to proteinlabeling (in vitro & in vivo)

This journal is © The Royal Society of Chemistry 2016

Fig. 5 Photo-affinity labelling of HDACs in cell lysates
Zhao, S., et al. Chem. Commun. 2016, 52, 4702-4705. 18(a) Structuresof SAHA and probes. (b) Western blotting aenriched active (A) or inactive (IA) HepG2 cell lysates treate
the absence (!) or presence (+) of competitor SAHA,
irradiation using a handheld UV lamp (10 min). (c) Wes
HDACs enriched from in situ HepG2 cells treated with

Contents
Ø IntroductionØStandard method to identify target proteinØConventional photo-crosslinker

Ø MainØTetrazole as photo-crosslinker
Ø2-acyl-5-carboxytetrazole (ACT)ØPPI mapping by using ACT in living cells

Ø Summary
19Advance of photo-crosslinker using tetrazole
・ACT (2-aryl-5-carboxytetrazole): non-radical intermediate

Herner, A., et al. Journal of the American Chemical Society 2016, 138(44), 14609–14615.

20

carboxylic acid presumably reacts with the nitrile imine generated
by irradiation of tetrazole 1a to form an adduct intermediate 2,
which is followed by an intramolecular O - N acyl transfer togive 3.5 The reinvestigation of this photo-induced tetrazole-acidcoupling reaction, especially under the physiologically relevant
conditions, was warranted in order to achieve its full potential
for chemical biology research. Nallani’s group and Zhao’s groupindependently reported photo-induced reactions of tetrazoles
with a number of endogenous amino acids,6 but neither publication
has considered the reactivity of carboxylic acids. Given the highactions involving proteinogenic amino
ely valuable transformations, with theeins and enable the studies of activitychemically induced cross-linking is aation method given the precise control
ieved in terms of wavelength, time andphoto-induced covalent cross-linking
hly reactive species and hence neither
y would be expected.3 In addition, theycell damage caused by the high reactivity
diates as well as non-reproducible results
ncy, the category of proteins and targetinghoto-induced reaction of diaryltetrazolesy Lin’s group is particularly remarkablekenes need to be first introduced into thehotolysis of tetrazole A rapidly and cleanlyole B that reacts with an alkene to afford
Fig. 1a).4 This ‘‘photoclick chemistry’’ hasde range of studies, due to the advantages
Herner, A., et al. Journal of the American Chemical Society 2016, 138(44), 14609–14615.
n of strongly fluorescent cycloadducts,
iteon BTK
proteinand theproposed ligand-directed cross-linking mechanism. (a) Sequence of therthogonal
alkenetag, superior
reaction
otoactivation
procedureusing a1a-modiﬁed
handS/MS
spectrumfor probe
tripeptide fragment, EMR, is shown with the fragment ions

Reaction mechanismdel of probe 1a bound to BTK (PDB code: 3K54) showing a proximal Glu-488 residue located on aring. (c) Proposed mechanism of the ligand-dependent nucleophilic addition to the carboxy-nitrile
Sciences, Academy
for Advanced
Interdisciplinary
neratethe speciﬁc
photoadduct.

ng 100871, China. E-mail: tuopingluo@pku.edu.cn
Molecular Science (BNLMS), College of Chemistry
Laboratory of Bioorganic Chemistry andry of Education, College of Chemistry andUniversity, Beijing 100871, China
rmation (ESI) available: Experimental procedures
DOI: 10.1039/c5cc10445a

g yield, we incubatedate Dasatinib or JQ-1
UV irradiation, andtifyingly, ACT-based
oss-linking with theiror 1a (Figure 3a) andof probe 1a to the kinase active site brings the C5 of the ACT
ca. 6.9
fromthecarboxylate
Glu-488;tetrazole–
indeed, it is theFig.Å
1 awayPhoto-inducible
reactionsof tetrazoles.
(a)of
Photo-induced
Zhao, S., etal. Chemical
Communications
2016, 52(25), 4702–4705.
addition. (b) Photo-induced
tetrazole
1a and carboxylic
only alkene
nucleophilicside chainreactions
withinof 9.0
Å fromthe electrophilic
acids.
site. Certainly, because the ACT is completely solvent exposed
and highly mobile, these distances may vary as the ACT orients
itself dynamically relative to the BTK protein. We propose that21

Dasatinib and JQ-1
Ø Dasatinib

Ø JQ-1

Krebs, S., et al. Journal of Nuclear Medicine 2020, 61(11), 1580-1587.

Mu, J., et al. Journal of Molecular and Cellular Cardiology 2020, 149,1–14.
22

Dasatinib
Journalandof theJQ-1
AmericanconjugatedChemical Society with PAL.

Society

Articleenrichment tag

tetrazole

diazirinebenzophenone

Herner, A., et al. Journal of the American Chemical Society 2016, 138(44), 14609–14615. (Fig. modified) 23

Journal of the American Chemical Society

ArticleBinding affinity assay

Figure 1. Dasatinib and JQ-1-derived photoaﬃnity probes containing 2-aryl-5-carboxy-tetrazole (ACT), diazirine (DA), or benzophenone (BP)photoaﬃnity label (PAL) and their biological activities. (a) Structures of the photoaﬃnity probes. (b) Kinome proﬁling of Dasatinib-derived
photoaﬃnity probes. A panel of 82 protein kinases were surveyed in this assay, and inhibition constants, Ki, are given in micromolar. See Table S1 in
the Supporting Information for Ki values. The nonreceptor tyrosine kinase targets are indicated by blue arrows. NV, no value. (c) Plots of theinhibition of BRD-2, -3, and -4 by JQ-1-derived photoaﬃnity probes. See Table S2 in the Supporting Information for Ki values.

Ø synthesized probes did not strongly inhibit ligand-POI affinity→ BTK, BRD4 were used
Ø For JQ-1, inhibition efficiency was improved

24

Herner, A., The
et al. Journal
the American
2016, 138(44),14609–14615.
and oﬀ-targets of Dasatinib and JQ-1−two drugs proﬁled
linkerof length
wasChemicalvaried Society
to adjustthe distanceof the11,12extensively in the literature.
Compared with DA and BP,
reactive intermediate from the target binding site (Figure 1a).
ACT gave higher yields in the ligand-directed photo-crossAn alkyne tag was placed on the photoaﬃnity labels to enablelinking reactions with the recombinant target proteins. In
the click chemistry-mediated detection and enrichment of theaddition, the ACT-based probes facilitated the in situ target
targets from cell lysates.21 To determine how the attachment ofEvaluating the efficiency and selectivity of photoaffinity-labeling
Journal of the American Chemical SocietyØ For BTK, all probes showed irradiation and ligand-dependent labelingØ For BRD4, irradiation and ligand-dependent labeling other than 6a/6b
Journal of the American Chemical Society→ exhibited strong background labeling

Articleal of the American Chemical Society

ArticleFigure 1. Dasatinib and JQ-1-derived photoaﬃnity probes containing 2-aryl-5-carbox
photoaﬃnity label (PAL) and their biological activities. (a) Structures of the photo
photoaﬃnity probes. A panel of 82 protein kinases were surveyed in this assay, and inhi
the Supporting Information for Ki values. The nonreceptor tyrosine kinase targets arinhibition of BRD-2, -3, and -4 by JQ-1-derived photoaﬃnity probes. See Table S2 inand oﬀ-targets of Dasatinib and JQ-1−two drugs proﬁled

11,12
Figure 1. Dasatinib and JQ-1-derived photoaﬃnity probes containingextensively
2-aryl-5-carboxy-tetrazole(ACT),
diazirine(DA), withor benzophenone
in the literature.
Compared
DA and BP,(BP)photoaﬃnity label (PAL) and their biological activities. (a) Structures of the photoaﬃnity probes. (b) Kinome proﬁling of Dasatinib-derived
ACT
gave
higheryieldsintheligand-directed
photo-crossphotoaﬃnity probes. A panel of 82 protein kinases were surveyed in this assay, and inhibition constants, Ki, are given in micromolar. See Table S1 inlinkingtargets
reactionswith therecombinant
are indicatedby bluearrows. NV, target
no value.proteins.
(c) Plots Inof thethe Supporting Information for Ki values. The nonreceptor tyrosine kinase
theinACT-based
probesfacilitatedin situ target
values.
inhibition of BRD-2, -3, and -4 by JQ-1-derived photoaﬃnity probes. addition,
See Table S2
the Supporting
Information
for Ki theidentiﬁcation in a manner similar to the DA-based ones.
and oﬀ-targets
and JQ-1−twodrugs proﬁled
linker2016,
length138(44),was varied
to adjust the distance
25 of theHerner,ofA.,Dasatinib
et al. 11,12
Journalof the American
ChemicalThe
Society14609–14615.
RESULTSAND DISCUSSION
extensively
intheliterature.
Compared
with
DA
and
BP,
reactiveintermediatefromthetargetbindingsite(Figure 1a).
2. Evaluating the eﬃciency and selectivity of photoaﬃnity-labeling of recombinant
proteins by the small-molecule probes. (a)Evaluating
the on the photoaﬃnity labels
ACT gave higher yields in the ligand-directed photo-cross- Design,
An alkyne
tag was placed
enableSynthesis,
and Biological Evaluation oftothe
beling eﬃciency using in-gel ﬂuorescence (top panels). For reaction setup,linking
0.5 reactions
μg of BTK
(ﬁnal
concentration≈0.1In μM),
smallwith therecombinanttarget proteins.
the0.2
clickμM
chemistry-mediateddetection
and acylenrichmentof thePhotoaﬃnity
Probes.
Whereas
the
tosyl
and
imidazolyl
21
addition,
ACT-based probes facilitatedthe in situ
To determine
how theattachment oftargets
cell lysates.employedgroupswere
successfullyin labelingendogenous
le probe, 10 μM Dasatinib (for competition only) in 50 μL of PBS were used.
For thephotoirradiation,a hand-held
UVtargetlamp
witha fromwavelength
identiﬁcation in a manner similar to the DA-based ones.
label
aﬀectstheideal
inhibitory
speciﬁcity,
targetsphotoaﬃnity
in living
cells,
they
are notaﬃnityactivity
labels andfor general
nm for ACT (5 min) and BP (20 min) or 365 nm for DA (10 min) was used. (b) Evaluating the BRD4 labeling eﬃciency
usingin-gelproﬁling
was carriedout forabout
Dasatinib-derived
targetkinome
identiﬁcation
becauseof concerns
their stabilityprobes
inANDμM
DISCUSSION
13 and(Figure1b
in vitro
binding
ence (top panels). For reaction setup, 0.4 μg of BRD4 (ﬁnal concentration ≈0.4RESULTSμM), 0.2
small-molecule probe, 5 μgcellularof BSA,
and
10TableμMS1), while
milieu.
We
hypothesized
thattheACTshouldserveassay
as anwasperformed forlabel
JQ-1-derived(Figure considerations:1c and Table S2).
Design, Synthesis, and Biological Evaluation of the ideal photoaﬃnity
based onprobes
the following■■

or competition only) in 50 μL of PBS were used. The equal loading of proteins was veriﬁed by SYPRO Ruby staining of the same gels

The link
reactive
An alkynthe clicktargets fr
photoaﬃ
kinome(Figure 1
performe
We founinhibitor
modestshorter llargest reably dueFigure 2. Evaluating the eﬃciency and selectivity of photoaﬃnity-labeling of recombinant proteins by the small-molecule probes. (a) Evaluating the
BTK labeling eﬃciency using in-gel ﬂuorescence (top panels). For reaction setup, 0.5 μg of BTK (ﬁnal concentration ≈0.1 μM), 0.2 μM smallmolecule probe, 10 μM Dasatinib (for competition only) in 50 μL of PBS were used. For photoirradiation, a hand-held UV lamp with a wavelengthof 302 nm for ACT (5 min) and BP (20 min) or 365 nm for DA (10 min) was used. (b) Evaluating the BRD4 labeling eﬃciency using in-gel
ﬂuorescence (top panels). For reaction setup, 0.4 μg of BRD4 (ﬁnal concentration ≈0.4 μM), 0.2 μM small-molecule probe, 5 μg of BSA, and 10 μM
JQ-1 (for competition only) in 50 μL of PBS were used. The equal loading of proteins was veriﬁed by SYPRO Ruby staining of the same gels(bottom panels). See Supporting Information for procedures of click chemistry with TAMRA-azide and polyacrylamide gel electrophoresis.

ACT showed higher photo-crosslinking yields

Figure 3. Quantifying the cross-linking eﬃciency of the photoaﬃnity labels with recombinant target proteins by LC-MS. (a−c) Deconvoluted
masses of the product mixture after incubating 2.5 μM human BTK387−659 with 25 μM Dasatinib probe 1a, 2a, or 3a in 100 μL of PBS for 15 min
followed by photoirradiation with a hand-held UV lamp for 5 min (302 nm for ACT and BP, 365 nm for DA) on ice. (d−f) Deconvoluted masses ofthe product mixture after incubating 2.5 μM BRD444−168 with 5 μM JQ-1 probe 4a, 5a, or 6a in 100 μL of PBS followed by photoirradiation with ahand-held UV lamp for 5 min (302 nm for ACT and BP, 365 nm for DA) on ice. The cross-linking yield was calculated using the following equation: 26
A., etal. Journal
the American
Society2016,
138(44),14609–14615.
Iphotoadductrepresent
theof ioncounts ofChemical
the target
proteinandthe photoadduct,
yield% = Iphotoadduct/(Itarget protein + Iphotoadduct), where Itarget protein and Herner,
respectively, and marked at the upper-right of the spectra.

ü ACT-based probes 1a and 4a showed robust photo-crosslinking with their targets,
while DA-based probes 2a and 5a gave crosslinked products in much lower yields
rhodamine-azide.22 For BTK, all probes showed irradiation andligand-dependent labeling, with 1a and 3a giving the strongest
ﬂuorescence (Figure 2a and Figure S2), suggesting other(Figure 2b) as well as labeling of BSA which was added to thereaction mixture to prevent nonspeciﬁc binding of BRD4 to theplastic surface (Figure S4). In the K562 cell lysate spiked with

Quenched product yield was almost the samea)b)

5 min 302-nm
photoirradiation

11.09 (1a)12.65 (2a)0 min

11.09 (1a)0 min

11.21 (quenched product)

5 min 365-nm
photoirradiation

12.70 (2a)12.55
(quenchedproduct)

Figure S6. HPLC and LC-MS analyses of the photoactivation of (a) 1a and (b) 2a (100 μM) in PBS. Red trace = absorbance at 254 nm; blue trace = absorbance a 365 nm. The percent conversion
was calculated to be 38% for 1a and 44% for 2a based on absorbance at 365 nm.

LC-MSü quenched product’s yields from 1a and 2a were almost the same
Herner, A., et al. Journal of the American Chemical Society 2016, 138(44), 14609–14615. 27[M+H+][M+H+]

Determination of the cross-linking site on BTK protein

Journalof Society
the American
Chemical Societyn ChemicalØ MS/MS
spectrumØ binding model

Articleü Glu-488 is the only nucleophilic side chain
within 9.0
Å frommechanism.
the electrophilic
Figure ü
4. Determinationof thewas
cross-linking
site onprotein and the proposed ligand-directedcross-linking(a) Sequencesiteof theonly Glu-488
detected
asBTKlabeledfull-length His6-(TEV)-G-hBTK387−659. The MS/MS spectrum for probe 1a-modiﬁed tripeptide fragment, EMR, is shown with the fragment ions
(BTK
has 25(b)Glu,
14 Asp)annotated on
the structure.
A dockingmodel of probe 1a bound toHerner,
BTK A.,(PDB
showing Chemicala proximal
Glu-488
residue 14609–14615.
located on a 28
et al.code:
Journal3K54)of the American
Society
2016, 138(44),loop 6.9 Å away from the C5 of the tetrazole ring. (c) Proposed mechanism of the ligand-dependent nucleophilic addition to the carboxy-nitrile
imine followed by the O → N acyl shift to generate the speciﬁc photoadduct.

studies. To quantify the photo-cross-linking yield, we incubatedrecombinant protein targets with appropriate Dasatinib or JQ-1

of probe 1a to the kinase active site brings the C5 of the ACT
ca. 6.9 Å away from the carboxylate of Glu-488; indeed, it is theArtices developed by Lin’s group is particularly remarkablee tetrazoles or alkenes need to be first introduced into theinterest.4 The photolysis of tetrazole A rapidly and cleanly
ocietynitrile imine dipole B that reacts with an alkene to afford
cycloadduct C (Fig. 1a).4 This ‘‘photoclick chemistry’’ hasapplied in a wide range of studies, due to the advantages
the generation of strongly fluorescent cycloadducts,
size of the bioorthogonal alkene tag, superior reaction
reactionproceduremechanismnd the mildØphotoactivation
using a handamp.

ArticleProposed mechanism

hua Center for Life Sciences, Academy for Advanced Interdisciplinary
ing University, Beijing 100871, China. E-mail: tuopingluo@pku.edu.cn
onal Laboratory for Molecular Science (BNLMS), College of Chemistry
ar Engineering, Key Laboratory of Bioorganic Chemistry andngineering of Ministry of Education, College of Chemistry andngineering, Peking University, Beijing 100871, China
supplementary information (ESI) available: Experimental procedures
rization data. See DOI: 10.1039/c5cc10445a

em. Commun., 2016, 52, 4702--4705

Fig. 1 Photo-inducible
reactionsof tetrazoles.
(a) Photo-induced
tetrazole–
Zhao, S., etal. Chemical
Communications
2016, 52(25), 4702–4705.
alkene addition. (b) Photo-induced reactions of tetrazole 1a and carboxylic
acids.

This journal is © The Royal Society of Chemistry 2016

Herner, A., et al. Journal of the American Chemical Society 2016, 138(44), 14609–14615.

site on BTK protein and the proposed ligand-directed cross-linking mechanism. (a) Sequence of the
MS/MS spectrum for probe 1a-modiﬁed tripeptide fragment, EMR, is shown with the fragment ionsdel of probe 1a bound to BTK (PDB code: 3K54) showing a proximal Glu-488 residue located on aring. (c) Proposed mechanism of the ligand-dependent nucleophilic addition to the carboxy-nitrile

29

criteria: (1) at least two unique peptides were identiﬁed in thecan be potentially overcome by increasing theMS, and (2) the area under the curve (AUC)−a measurement
between ACT and the ligand to allow the survey ofof MS signal intensity and reliability−for the parent drugsurrounding the ligand-binding pocket.
pretreated sample is not detectable. Using these criteria, six
In summary, we have developed a new photoaﬃ
kinases were identiﬁed by probe 1a, ﬁve of which also appearedaryl-5-carboxytetrazole (ACT), for eﬃcient ininACT-enabledprobe 2a-treated cells, indicating
ACT works
similarly toDAcapture and subsequent identiﬁcation. The atin situtargetidentification
ChemicalSociety was generally w(Figure 5a, Table S3). However, probe 1a failed to identify Abl Journal
ACTof theto American
two drugmolecules
without signiﬁcantly altering the binding aﬃnity an
Compared with DA and BP, ACT provide
mechanism of target capture through which the phcarboxy-nitrile imine intermediate reacts with
nucleophile near the target active site.
Journal of the American Chemical Society
ArticleAs a
displayed the cleanest and most eﬃcient cross-link
recombinant target proteins in vitro among theaﬃnity labels tested. In the in situ target identiﬁcwith two previously proﬁled drugs, Dasatinib andsuccessfully captured the desired targets in both ceﬃciency comparable to DA. While aniline was use
group in the present study, a wide range of hetero
explored in the future with a goal to identify
enhanced solubility and photo-cross-linking reacti

FigureVennof the identiﬁed proteinby (a)
ü 5.
both
ACTdiagrams(2-acyl-5-carboxytetrazole)
andtargets
diazirine
are efficient in the target identification
Dasatinib-derived photoaﬃnity probes 1a and 2a; and (b) JQ-1EXPERIMENTAL SECTION(K562 cell)
derived photoaﬃnity probes 4a and 5a.
In-Gel Fluorescence Analysis of BTK Labelingü ACT did not improve identification regardless of higher crosslinking
Probes inyield
Vitro.than
One diazirine
microliter of 0.5 mM Dasatinib
Figure1.
Dasatinib
andJQ-1-derived
containing
2-aryl-5-c
protein,but
presumably
a lack
proximal
nucleophilicside identification.
both ACTdue
andtoDAare ofefficientin thein situ target
competition experiments)photoaﬃnity
or DMSOprobes(without
Dasatinib
photoaﬃnity label (PAL) and their biological activities. (a) Structures of thechains near the kinase active site. For JQ-1 targets, probes 4a photoaﬃnity
was added toA panel
0.5 μgBTKkinases
in 50were
μLsurveyedof PBS.
Afterof 82ofproteinin thisassay,incanHerner, A., et al. Journal of the American Chemical Society 2016, probes.
138(44), 14609–14615.
(Fig. modified)30
The nonreceptor tyrosine
targ
Informationfor 10Ki values.
for 15 min,
1 μL ofμM photoaﬃnity
probe kinase
in DMSand 5a successfully captured the bromodomain proteins BRD- the Supporting
inhibition
BRD-2, -3, and-4 by JQ-1-derived
See Table
Afterofadditional
incubation
at r.t. photoaﬃnity
for 30 min,probes.
the mixture
2, -3, and -4 with minimum oﬀ-targets (Figure 5b, Table S4),a hand-held
302 andnm JQ-1−two
UV lamp,drugs
ca. 2−3cm from
oﬀ-targetsof Dasatinib
proﬁledThesuggesting both ACT and DA are eﬃcient in the in situ target and with
11,12
Figure 1. Dasatinib and JQ-1-derived photoaﬃnity probes containingextensively
2-aryl-5-carboxy-tetrazole(ACT),
diazirine(DA),orbenzophenone(BP)sample.
A
premixed
click
reaction
cocktail
(6
μL,
1:3:1
in
the
literature.
Compared
with
DA
and
BP,
reac
identiﬁcation. Comparison
oflabelour(PAL)dataotheractivities.
literaturephotoaﬃnity
and with
their biological(a) Structures of the photoaﬃnity probes. (b) Kinome proﬁling of Dasatinib-derived
t
ACT
gave
higheryieldsintheligand-directed
photo-crossAn
TBTA in 1:4 DMSO- BuOH/
CuSO
4 in water/1.7
photoaﬃnity probes. A panel of 82 protein kinases were surveyed in this assay,
and inhibition
constants, KmM
i, are given in micromolar. See Table S1 in■reported MS-based target identiﬁcation studies revealed that

Abl and Dasatinib interaction
Abl

Dasatinib
Dasatinib

D325

E329

ü D325 and E329 are present near Dasatinib → probably crosslinking was occurred
Vajpai, N., et al. THE JOURNAL OF BIOLOGICAL CHEMISTRY 2008, 283(26), 18292–18302.
(PDB 2GQG) https://www.rcsb.org/structure/2gqg (Fig. modified)31

Short summaryü Tetrazole has selectivity to amino acids (Asp, Glu, Cys, Lys)
ü ACT (2-acyl-5-carboxytetrazole) can serve as an effective
photoaffinity label for target identification both in vitro and in livingcellsü unique photo-crosslinking mechanism (non-radical)→ lead to reduced background reactions with nonspecific targets→ facile mapping of the ligand-binding site
ü Compared to DA and BP, ACT showed higher cross-linking yields withthe desired targets in vitroü achieve efficient in situ target capture and subsequent identification
Herner, A., et al. Journal of the American Chemical Society 2016, 138(44), 14609–14615. 32

Contents
Ø IntroductionØStandard method to identify target proteinØConventional photo-crosslinker

Ø MainØTetrazole as photo-crosslinker
Ø2-acyl-5-carboxytetrazole (ACT)ØPPI mapping by using ACT in living cells

Ø Summary33

T

a powerful chemical strategy involves the use of geneticallyChart 1), and the identiﬁcation of a new ACTK-spec
rradiated, which
could be attributed to higher
encoded photo-cross-linkers that permanently link transientpyrrolysyl-tRNA synthetase for site-speciﬁc incorporation
ncentrationof the GST
mutant complexes(Figure 2cwith
anda burst of light.
protein−protein
interaction
ACTK into proteins in Escherichia coli and in mammalian c). Because
can charge
othergenetically
ACTKs encoded photoBasedmPyTKRS
on the structures,the reported
One of the ACTK analogs, mPyTK, exhibited robust and ssite-selectively,
wecontain
expressed
SjGST
mutantsphenyl azide suchcross-linkers
one of thethree moieties:
selective photo-cross-linking of a GST dimer in bacteria. I
1
2
benzophenone
such
as
pBpa,
and
diazirinesuch
as
as pAzF,
FTK, TTKandPhTK,
respectively,atpositionACT incorporation
to cells as unnaturalamino
comparison study, mPyTK
showedacids
signiﬁcantly higher cr
3,4
5−8
mericanChemicalSocietyCommunicatio
AbK
and
their
derivatives(Chart1).
Althoughthese
ed their photo-cross-linking eﬃciency in E. coli
linking eﬃciency than AbK when both are incorporated at
geneticallyencodedonly
photo-cross-linkers
Westernblot analysis,
mPyTK, FTK haveand proven to besame location of GST. Moreover, the mPyTK-encoded adap
incross-linked
Chart 1, thedimer
key intermediate,
ethyl order
2-aryl-ofprotein, Grb2, showed a stimulus and position-dependhewith the eﬃciency
II
Chart
1.
Genetically
EncodedPhoto-Cross-Linkers
oxylate,obtained
Cu -catalyzed
capture of its transient interaction partner, epidermal gro
K> FTKwas(Figure2d),throughpresumably
due to thefactor receptor (EGFR), in mammalian cells.
with
theto
nethyldensity2H-tetrazole-5-carboxylate
at N-methyl-pyrrole ring, which
helps
Because pyrrolysyl-tRNA synthetase (PylRS) and its varia
motogenerated
salt14 (Schemes
S1−S4imine
in Supporting
carboxy-nitrile
and increase
have shown tremendous versatility in charging various ly
ological(5),media.
PhTK
the Kakehi tetrazole synthesis
derivatives into proteins site-selectively in bacteria, yeast
Tgive
photoreacts
withproximal
nucleophilic
residues
the ethyl 2-phenyl-2H-tetrazole-5mammalian cells,13 we decided to append ACT motif onto th
15 to determine which nucleophilic residues
soughtamino group via simple acylation reaction. For the synthesi
diate(Scheme S5 in Supporting Informae GST monomer
might react
the photohydrolysis
and coupling
withwith
Fmoc-lysine·
oxy-nitrile
imine
intermediate.
Toaﬀorded
this end,theweReceived: March 15, 2017
moval of theprotectinggroup
the GST-E52mPyTK and surveyed the chemical
Published: April 19, 2017
1−5 (Chart 1). To identify PylRS mutants
urrounding mPyTK. Four nucleophilic residues
e mPyTK (1), an
MmPylRSlibrary,
in locatedwhich© 2017 American
Chemical Society
6078
DOI: 10.1021/jacs.7b0
139 andØK141)
wereidentiﬁed
thatare
GeneticallyencodedACT(2-aryl-5J. Am. Chem. Soc. 2017, 139, 6078−
nding
the N-methylpyrrole-lysineside(Figurechainmthe electrophilic
nitrileimine
carbonstructureofcarboxytetrazole)
Methanosarcina
mazeiparticipates
PylRS in
ine
which
one
of these four residues
16(Y306,
L309,
C348
and
Y384;to Figure
Ø we
give
robustandsite-selectivegMP
reaction,mutatedtheseresidues
alaninephotod, wassubjected
to successive
roundsof thethephoto-cross-linking
activityof
the resulting
crosslinking
reactivity
ound the Glu92 → Ala mutation completely
Tian, 3.
Y., etal. Journal of thethe
AmericanChemicalSociety 2017, 139(17), 6078–6081.
FigureIdentifying
mPyTK
photo-cross-linking
site in GST. (a) A
ovalent dimer formation whereas other mutations
Tian, Y., & Lin, Q. Chemical Communications 2018, 54(35), 4449–4452. 34
close-up view of the nucleophilic residues from the opposite GST(Figure 3b). This result is consistent with themonomer (colored in gray) surrounding mPyTK in GST monomern reactivity as E92 is closest to mPyTK with a(colored in yellow). The side chains of proximal resides (E92, M133,
ance between the carboxylate oxygen and theC139 and K141) are rendered in tube model. (b) Coomassie blue
rbon of 2.8 Å (Figure 3a). Similar results were
stained SDS-PAGE gel showing UV-dependent cross-linking of theCommunication

GST photo-crosslinking by mPyTK
tetrazole

diazirine

ü GST dimer formation for the E52mPyTK mutant
with ∼53% yield at 5 min, but not for WT→ ACT moiety is responsible for dimer crosslinking

ü AbK exhibited very weak reactivity as the dimer
band was detected only by Western blot
→ higher yield by using tetrazole
Tian, Y., et al. Journal of the American Chemical Society 2017, 139(17), 6078–6081. 35

Figure S5. Comparison of the photo-cross-linking efficiencies of mPyTK and Ab
Coomassie blue stained SDS-PAGE gel after GST-E52AbK or -E52mPyTK muta
photoirradiated for 0, 1, 5 or 15 min. 365 nm UV was used for GST-E52Abk while 302

Based on Western blot analysis, only mPyTK, FTK andshowed the cross-linked dimer with the eﬃciency order of
K > TTK > FTK (Figure 2d), presumably due to thest electron density at N-methyl-pyrrole ring, which helps toze the photogenerated carboxy-nitrile imine and increase
Identifying
the mPyTK photo-crosslinking
site in GST
time in biologicalmedia.
Communication
ause ACT photoreacts with proximal nucleophilic residues
oteins, we sought to determine which nucleophilic residues
e opposite GST monomer might react with the photoated carboxy-nitrile imine intermediate. To this end, we
model of the GST-E52mPyTK and surveyed the chemical
onment surrounding mPyTK. Four nucleophilic residues
M133, C139 and K141) were identiﬁed that are located
3.0 Å from the electrophilic nitrile imine carbon (Figureo determine which one of these four residues participates
oss-linking reaction, we mutated these residues to alanine
xamined the photo-cross-linking activity of the resulting
nts. We found the Glu92 → Ala mutation completely
Figure 3. Identifying the mPyTK photo-cross-linking site in GST. (a) A
hed the covalent dimer formation whereas other mutations
close-up view of the nucleophilic residues from the opposite GST
o eﬀect (Figure 3b). This result is consistent with theü modelmonomer(coloredof
in thegray)GST-E52mPyTK
surrounding mPyTK in GST monomer
mity-driven reactivity as E92 is closest to mPyTK with a(colored →in yellow).
The side chains ofresidues
proximal resides
(E92,
M133,
Four
nucleophilic
(E92,
M133,
ated distance between the carboxylate oxygen and theC139 and K141) are rendered in tube model. (b) Coomassie blue
C139 andgelK141)
identified
imine carbon of 2.8 Å (Figure 3a). Similar results were
stained SDS-PAGE
showingwere
UV-dependent
cross-linking of the
GST-mPyTK
alaninemutants. completely
Asterisk indicatesabolishedan impurity derived
ned when the alanine scan was conducted with the GSTü E92A
mutation
thefrom
Ni-NTA
aﬃnitypuriﬁcation.
The
proteins
werephotoirradiatedTK mutant (Figure S7 in SI). We propose a photo-crosscovalent
dimer
formationwith a hand-held
302 nm
UV lampon ice for 15 min before SDS-PAGE.
g mechanism in which the E92 carboxylate undergoes(c) Proposedmechanism
for mPyTK-mediatedphoto-cross-linking ofü othermutations
had no effectophilic addition to the photogenerated carboxy-nitrile
GST dimer. The two cleavage pathways are marked with blue and red
followed by 1,4-acyl shift (Figure 3c). The rearranged
Tian, Y., et al. Journal of the American Chemical Society 2017, 139(17), 6078–6081. 36
dashlines on the cross-linked structure (see Figure S8 in SI for details).
linked structure was supported by tandem mass spectromata in which the two fragment ions derived from the two
te cleavage pathways were positively identiﬁed (Figure S8
ligand20 revealed that 9 residues surrounding the ligand, A91,). For comparison, we performed photo-cross-linking
D104, V105, Q106, F108, K109, L111, W121 and N143, could

lure, Fangtian Huang and Royston Carter*
EGFR Figureand 1Grb2

wth factor
SH2) domain
in Grb2n-activatedn of EGFRs
EGF–
emporaling cells, we
modified
nce energy
ing EGFRd Grb2 fusednt energy
occur if CFP
quires directhese
F resulted inmpartments
ase in FRET

EGF

Membrane
Grb2

NH2
SH3
SH2
SH3

KinaseKinaseKinase
NH2
SH3

Y1068
Y1086

YFP
f

Y1068PY1086P

SH2
SH3

f
CFP
Current Biology

470nm

FRET
436nm

436nm

Grb2

YFP
f

f
10-50Å
CFP

535nm

A., et al. Current
Biology 2000,
10, 1395-1398.
Hypothetical model of EGFR–Grb2 Sorkin,interaction
measuredby FRET.
In
our FRET registration system, the excitation of CFP at 436 nm will
result in emission at 470 nm if no YFP is in close proximity. If the
distance between CFP and YFP is less than 50 Å, however, due toformation of the EGFR–CFP/Grb2–YFP complex, then the energy will37

Photo-crosslinking in mammalian cells
Ø crystal structure ofØ Photo-crosslinking with EGFR
Journalthedomain
American Chemical Society in HEK293T cells
Grb2 of
SH2

■

UV

kDa
250
170
100
70

55

35
25
15

AUTHOR INFORMATION- - +Corresponding
Author*qinglin@buﬀalo.edu
Grb2―EGFR complex

ORCID

EGFR

Jun Qu: 0000-0002-1346-6809
Wenshe R. Liu: 0000-0002-7078-6534
Qing Lin: 0000-0002-9196-5718
Present Address#

Sichuan University College of Che

Notes

Figure S10. Photo-cross-linking with EGFR by Grb2-D104mPyTK. The lysates of HEK293T
Figure have
4. Photo-cross-linkingof photo-crosslink
EGFR by the mPyTK-encodedGrb2
ü 9 residues
a potential to
with
EGFR
cells expressing
either
EGFR or EGFR/Grb2-D104mPyTK were analyzed directly by SDS-PAGE
The antibody.
authors declare no competing ﬁ
followed by western blot using an anti-EGFR
mutantsinmammaliancells.
(a)A
close-upview
ofGrb2-SH2domainü D104mPyTK mutant gave the highest photo-cross-linking yield followed by V105mPyTK,
in complex with phosphotyrosine-containing heptapeptide (PDB code:
ACKNOWLEDGMENTS
Q106mPyTK
and N143mPyTK
1TZE), highlighting
the proximalmutants
residues surrounding pY (rendered incyan tube
model)
that wereselected for theresidue
mPyTK mutagenesis.
(b)
Weinteraction
gratefully acknowledge
ü may reactwiththe same
nucleophilicon EGFR acrosstheinterface the NIH■

Comparison of the eﬃciency of the mPyTK-containing
W.R.L.)
for38ﬁnancial sup
Tian, Y., et al. JournalGrb2-SH2of the American Chemical161158
Society 2017,to139(17),6078–6081.
domain mutants for photo-cross-linking with EGFR in HEK293T cells.
Koland at University of Iowa for pro
Cells were exposed to 302 nm UV light for 5 min before lysis. The Grb2EGFP plasmid, and Carlo Ramilcross-linked proteins in cell lysates were immunoprecipitated with theassistance.
anti-FLAG antibody-immobilized agarose beads. The samples wereComplex
Grb2―EGFR
Complex

Photo-crosslinking in mammalian cells

Grb2

Grb2

B

A
Grb2
WT
D104mPyTK
B
UV
- EGF
- + + - + +stimulation
0
1
5
EGF - + - + - + 15- 30
+
time
(min)

EGF
stimulation 0 1
time
(min)

5 15 30
Grb2―EGFR
ComplexGrb2―EGFR
Complex

Grb2

Grb2

Grb2

B

EGF
Figure S11. Photo-cross-linking with EGFR by Grb2-D104mPyTK is UV, EGF stimulation, andstimulation 0 1 5 15 30 stimulation time dependent. (A) Comparison of Grb2-D104mPyTK with wt-Grb2 in photo-crosstime
Figure S11. Photo-cross-linking
with EGFR by Grb2-D104mPyTK
is UV,inEGF
stimulation,linking with EGFR
the absence
and andpresence of UV irradiation and/or EGF stimulation. (B)(min)
stimulation time dependent. (A) Comparison of Grb2-D104mPyTK
with
wt-Grb2
inphoto-crossGrb2―EGFR
Effect of EGF stimulation time on EGFR photo-capture yield as detected in western blot using an
Complexlinking with EGFR in the absence and presence of UV
irradiation
and/or EGF stimulation. (B)anti-myc
antibody.

ü the photo-crosslinking of EGFR is EGF stimulation and photoirradiation-dependent
ü highest photo-cross-linking yield was when cells were stimulated with EGF for 15 min→ Grb2−EGFR interaction is transient and dynamic

Effect of EGF stimulation time on EGFR photo-capture yield as detected in western blot using ananti-myc antibody.
Tian, Y., et al. Journal of the American Chemical Society 2017, 139(17), 6078–6081. (Fig. modified)

39

Contents
Ø IntroductionØStandard method to identify target proteinØConventional photo-crosslinker

Ø MainØTetrazole as photo-crosslinker
Ø2-acyl-5-carboxytetrazole (ACT)ØPPI mapping by using ACT in living cells

Ø Summary40

Summary✗ conventional photo-crosslinkers (benzophenone, diazirine)• very low target capturing yields• react non-selectively (any proximal X-H bonds) → difficult for identifying
target by MS• high backgroundü Tetrazole has selectivity to amino acids (Asp, Glu, Cys, Lys)
ü unique photo-crosslinking mechanism (non-radical)→ lead to reduced background reactions with nonspecific targets→ facile mapping of the ligand-binding site41

whereas the tetrazoles or alkenes need to be first introduced into therotein of interest.4 The photolysis of tetrazole A rapidly and cleanly
enerates nitrile imine dipole B that reacts with an alkene to afford
yrazoline cycloadduct C (Fig. 1a).4 This ‘‘photoclick chemistry’’ has
ince been applied in a wide range of studies, due to the advantages
ncluding the generation of strongly fluorescent cycloadducts,
he small size of the bioorthogonal alkene tag, superior reaction
inetics and the mild photoactivation procedure using a handeld UV lamp.

Summaryü diaryltetrazole or ACT (2-aryl-5carboxytetrazole)
Peking-Tsinghua Center
for Life Sciences, Academy for successfully
Advanced Interdisciplinary
Studies, Peking University, Beijing 100871, China. E-mail: tuopingluo@pku.edu.cn
profiling proteins in vitro andBeijing National Laboratory for Molecular Science (BNLMS), College of Chemistry
and Molecular Engineering,
Laboratory of Bioorganic Chemistry andlivingKeycells

Molecular Engineering of Ministry of Education, College of Chemistry andMolecular Engineering, Peking University, Beijing 100871, China
Electronic supplementary information (ESI) available: Experimental proceduresnd characterization data. See DOI: 10.1039/c5cc10445a

Fig. 1 Photo-inducible reactions of tetrazoles. (a) Photo-induced tetrazole–alkene addition. (b) Photo-induced reactions of tetrazole 1a and carboxylic
acids.

ü Although more improvement is needed, these coupling reactions could
be extensively applied to low-background protein labelingüCommun.,Other2016,
crosslinking
alsoof Chemistry 2016702 | Chem.
52, 4702--4705 methods without radical intermediate
This journal is © Theare
Royal Society
emerging→ allow the identification of elusive transient and dynamic protein-protein
interactions in vitro, in cell lysates, and in living cells
42Thank you
for your kind attention!