Targeted Diazotransfer Reagents Enable Selective Modification of Proteins with Azides

Article abstract of DOI:10.1021/acs.bioconjchem.7b00110

In chemical biology, azides are used to chemically manipulate target structures in a bioorthogonal manner for a plethora of applications ranging from target identification to the synthesis of homogeneously modified protein conjugates. While a variety of methods have been established to introduce the azido group into recombinant proteins, a method that directly converts specific amino groups in endogenous proteins is lacking. Here, we report the first biotin-tethered diazotransfer reagent DtBio and demonstrate that it selectively modifies the model proteins streptavidin and avidin and the membrane protein BioY on cell surface. The reagent converts amines in the proximity of the binding pocket to azides and leaves the remaining amino groups in streptavidin untouched. Reagents of this novel class will find use in target identification as well as the selective functionalization and bioorthogonal protection of proteins.

Full text of DOI:10.1021/acs.bioconjchem.7b00110

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Communication
Targeted Diazotransfer Reagents Enable
Selective Modification of Proteins with Azides
Jonas Lohse, Lotteke Swier, Ruben Oudshoorn, Guillaume
Médard, Bernhard Kuster, Dirk Jan Slotboom, and Martin D. Witte
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00110 • Publication Date (Web): 29 Mar 2017
Downloaded from http://pubs.acs.org on April 4, 2017
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Targeted Diazotransfer Reagents Enable Selective Modification of Proteins
with Azides
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Jonas Lohse,† Lotteke J.Y.M. Swier,‡ Ruben C. Oudshoorn,‡ Guillaume Médard,§ Bernhard Kuster,§¶
Dirk-Jan Slotboom,‡ Martin D. Witte,†*
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†Chemical Biology II, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747
AG Groningen, The Netherlands
‡Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, 9747
AG Groningen, The Netherlands
§Chair for Proteomics and Bioanalytics, WZW, Technical University of Munich, 85354 Freising,
Germany
¶Center for Integrated Protein Science Munich (CIPSM), Munich, Germany
AUTHOR INFORMATION
Corresponding author
*E-mail: m.d.witte@rug.nl. Phone: +31-503635643
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Abstract
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In chemical biology azides are used to chemically manipulate target structures in a
bioorthogonal manner for a plethora of applications ranging from target identification to the
synthesis of homogeneously modified protein conjugates. While a variety of methods have
been established to introduce the azido group into recombinant proteins, a method that
directly converts specific amino groups in endogenous proteins is lacking. Here we report the
first biotin-tethered diazotransfer reagent DtBio and demonstrate that it selectively modifies
the model proteins streptavidin, avidin and the membrane protein BioY on cell surface. The
reagent converts amines in the proximity of the binding pocket to azides and leaves the
remaining amino groups in streptavidin untouched. Reagents of this novel class will find use
in target identification as well as the selective functionalisation and bioorthogonal protection
of proteins.
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Keywords:
Protein modification, chemical probes, ligand-tethered labelling, vitamin transporters
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The discovery that azides react in a truly orthogonal way in complex biological samples
opened up the possibility to selectively functionalize biomolecules in vitro and in vivo.1–3
Fluorophores, polyethylene glycol groups, carbohydrates, phosphates, proteins and small
molecule drugs have been conjugated to azide containing proteins using Staudinger ligation,
copper catalysed alkyne-azide cycloaddition (CuAAC) and strain promoted alkyne-azide
cycloaddition (SPAAC) reactions.4–6 The unique reactivity of the relatively small azido group
has been exploited for target identification.7–9 In contrast to affinity handles like biotin, azides
have a minimal effect on the biological activity, but they readily enable enrichment of the
target after being incorporated. Finally, masking essential amino groups of proteins and
peptides as azides provides a means to chemically control biological processes.10
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The aforementioned applications require the introduction of an azido group onto the protein
and over the past decade various methods have been reported that enable this. Both In situ
metabolic labelling1,9,11 and chemical modification of surface exposed amines of purified
proteins facilitate the global incorporation of azides (Figure 1).12 These methods are residue
specific, but not protein- and site-selective, which limits their applicability. Strategies with
increased site-selectivity have been developed to overcome this. Both co-translational
incorporation of non-canonical amino acids using techniques like stop codon suppression13,14
,16
and enzymatic modification of genetically engineered proteins15 have enabled the
incorporation of azides with pinpoint precision. By optimizing the reaction conditions, also
selective chemical labelling has been achieved. The N-terminal amino group of a protein can
be modified selectively at pH 8.5 using 1.75 equivalents of diazotransfer reagent 1 17
.
Co-translational incorporation of non-canonical amino acids and enzymatic modification
methods are also protein-specific and can therefore be employed in the context of more
complex surroundings, such as live cells and cell lysates. Disadvantage of both methods is
that they are restricted to genetically modified proteins. A suitable method to directly
convert an amino group in a target protein to an azide, in complex biological samples, is
lacking. Chemical modification with imidazole-1-sulfonyl azide
of the protein and it therefore forms an attractive starting point for the development of such
a method. However, diazotransfer reagent is not protein specific. Prior work on acylating
1 does not require engineering
1
and alkylating agents shows that targeting the reagent to the protein of interest by tethering
it to inhibitors or ligands addresses the issue of specificity.18–20 We therefore reasoned that
diazotransfer reagent
1 could be converted into specific chemical probes in a similar fashion.
Linking imidazole-1-sulfonyl azide to a ligand will direct the diazotransfer reagent to amino
groups proximal to the binding site of the ligand’s respective target and it therefore will
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increase both the protein specificity and the site-selectivity (Figure 1). We report here the
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viability of this approach and demonstrate that biotin tethered diazotransfer reagent
2
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(DtBio) selectively modifies biotin-binding proteins in complex mixtures. DtBio 2 can be used
to modify the previously challenging to target membrane protein BioY, the S-component of
9
the ECF BioY vitamin transporter from Lactococcus lactis, on the cell surface with a BODIPY
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reporter group 3 21
.
H
S
N
B
H
O
S
O
S
N
F
HN
N
N
N3
N
N3
N
F
H
NH
O
N
O
O
O
Dt 1
DtBio 2
BODIPY alkyne 3
O
S
N
O
O
S
N
N3
H2N
N
N
H2N
H2N
H2N
O
N
S
NH2
N
O
H2N
H2N
H2N
O
N3
H2N
Cu(II)
Cu(I)
N
H2N
N3
N3
NH2
H2N
NH2
N
N
= Ligand directing Dt-probe
= Bioorthogonal handle
= Conjugate/Reporter
Figure 1. Top: structures of the non-targeted (1) and targeted (2) diazotransfer reagents and the
BODIPY fluorophore (3) used as reporter group in the evaluation of the diazotransfer reaction.
Bottom: schematic representation of the targeted diazotransfer reagent concept. Ligands bearing
the diazotransfer group direct the reagent to the protein of interest. Upon binding an amino group
of the protein is transformed into an azide in the presence of Cu(II). The modified protein can be
further functionalized with bioorthogonal chemistry.
To assess if targeting diazotransfer reagents to proteins of interest is indeed feasible, we first
explored if tethering imidazole-1-sulfonyl azide 1 to
D-biotin results in the selective
functionalization of streptavidin. A lysine (K121) is located near the biotin-binding site of wild-type
core streptavidin.22 It has been shown that the ε-amine of this residue reacts with ligand-directed
reagents and it is therefore conceivable that it will also function as a diazotransfer acceptor, making
streptavidin a good model protein.23 To prepare the DtBio 2 required for these studies, we
synthesized p-nitrophenyl ester 4 from
D-biotin and coupled it to histamine (Scheme 1).
Subsequently histamine derivative 5 was transformed into DtBio 2. Initial attempts to react 5 with in
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situ prepared chlorosulfonyl azide, as was described for the synthesis of non-targeted imidazole-1-
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sulfonyl azide 1 24
were unsuccessful. The inherent instability of chlorosulfonyl azide impedes
,
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isolation and we therefore explored other sulfonyl azide transfer reagents to synthesize 2. Culhane
and Fokin showed that solid sulfonyl azide transfer reagents can be readily obtained by methylating
derivatives of 1 with methyl triflate.25,26 Gratifyingly, addition of one equivalent of sulfonyl azide
transfer reagent 6 to biotin-histamine 5 in DMF gave diazotransfer reagent 2.
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O
O
HN
H
HN
H
NH
H
a
b
NH
H
O
OH
S
S
O
O
NO2
4
O
O
HN
H
HN
c
NH
H
NH
H
H
H
N
H
N
O
S
S
S
N3
NH
N
O
N
O
N
O
5
DtBio 2
OTf
O
S
N3
N
N
O
6
Scheme 1. Synthesis of diazotransfer reagent DtBio 2 (a) EDC⋅HCl, p-nitrophenol, DMF, 24 h, rT, 72%,
(b) histamine, DMF, 16 h, rT, 83%, (c) sulfonyl azide transfer reagent 6, DMF, 2 h, 0 °C, 52%.
With DtBio
binding proteins by incubating a mixture of streptavidin (10
CuSO4 (1 mM) dissolved in PBS (pH 7.4) for 1 h with reagent
reacted with , we functionalized the introduced azido groups with BODIPY-alkyne
employing CuAAC.27 Upon fluorescence scanning of the SDS PAGE gel, two prominent bands
are detected that are absent when DtBio is excluded from the reaction mixture (Figure 2A
and Figure S1). The molecular weight of the respective bands match those of monomeric
(13 kDa) and tetrameric (52 kDa) core streptavidin (see Figure S1 for a gel image that reveals
2
in hand, we evaluated its ability to selectively transfer the diazo group to biotin-
M), ovalbumin (10 M) and
. To visualize the proteins that
ꢀ
ꢀ
2
2
3
2
,
tetramer labelling more clearly than shown in Figure 2A, where a 4–12% bis-tris gradient gel
was used, which is not optimal for visualization of the tetramer). Very little fluorescence is
detected at the molecular weight of ovalbumin (43 kDa), indicating that DtBio
with streptavidin. Treatments that undermine binding of DtBio to streptavidin, like heat-
denaturing and pre-incubating with -biotin, drastically reduce fluorescent labelling (Figure
2A) and further confirm that binding of DtBio to streptavidin is responsible for the observed
2 mainly reacts
2
D
2
selectivity. Besides increasing the selectivity, targeting the diazotransfer reagent also
enhances the labelling efficiency, as is apparent from the increased fluorescence intensity for
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samples treated with DtBio
(Figure 2A, lanes 6 and 7).
2
compared to those treated with non-targeted reagent 117
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By varying the assay conditions, we studied if the incubation time, the protein to probe ratio
and the amount of copper affected the labelling efficiency and selectivity. These experiments
revealed that an equimolar amount of probe gives the best signal-to-noise ratios, as judged
by comparing the labelling intensity of streptavidin and ovalbumin. Protein to probe ratios
above one-to-one result in increased non-specific modification of ovalbumin and stabilize the
streptavidin tetramer, while lower probe concentrations lead to less efficient labelling of
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streptavidin (Figure 2B). With an equimolar amount of probe
achieved within approximately 30 minutes. Adding DtBio together with sodium ascorbate,
ligand and BODIPY-alkyne to the protein mixture results in minimal labelling, which
suggests that diazotransfer is negligible during the copper catalysed click reaction (Figure 2C).
Finally, using dibenzocyclooctyne-TAMRA 728 as read-out to exclude that labelling by DtBio
2, saturation of labelling is
2
3
2
originates from traces of copper (II) used in the CuAAC visualisation step revealed that the
concentration of the copper catalyst can be lowered to 50 μM without a marked effect.
Decreasing the CuSO4 concentration further leads to a reduction in the fluorescence
intensity. Interestingly, DtBio 2 even reacts with streptavidin when CuSO4 was omitted during
the diazotransfer step, albeit with a decreased efficiency (Figure 2D). These results
corroborate those of copper independent protein modification with non-targeted reagent
1.17 Competition experiments and heat-inactivation confirm that this particular labelling of
tetrameric streptavidin in the complete absence of copper is activity dependent (Figure S5).
D
DtBio 2 (10 μM)
Dt 1 [mM]
+
-
-
-
-
+
+
-
+
-
-
0.01
-
-
1
-
A
B
C
-
time [h]
0
0
0.25 0.5
1
2
4
DtBio 2 [μM]
Biotin [mM]
-
-
0.01
1
1
5
10 25 50 100
kDa
75
Strp
Strp
4
Strp
4
OVA
D
CuSO4
10 50 100 500 1000
OVA
Strp
Strp
4
25
Strp
Strp
1
2
3
4
5
6
7
Figure 2. Biochemical evaluation of diazotransfer probe DtBio 2 with streptavidin (Strp) as target
protein. (A) A mixture of Strp and OVA labelled with indicated amount DtBio 2 (lane 1) or Dt 1 (lane
6,7) and clicked to BODIPY-alkyne 3 subsequent to diazotransfer is resolved on a bis-tris 4-12%
gradient gel and visualized by fluorescence scanning. Heat-inactivation with 1% SDS prior to
incubation (lane 3) and competition with D-biotin (lanes 4,5) were used as controls. Note: biotin and
derivatives thereof (such as DtBio 2) stabilize tetrameric Strp. (B) A mixture of streptavidin and OVA
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was incubated with indicated amount of DtBio 2 after which the modified proteins were visualized
as described above (resolved on a 15% Laemmli type SDS-PAGE gel). (C) Crop showing the labelling
intensity of monomeric streptavidin incubated with DtBio 2 (10 ꢀM) for the indicated time after
which the modified proteins were visualized as described above. (D) Crops showing the labelling
intensity of tetrameric and monomeric streptavidin incubated with DtBio 2 (10 ꢀM) in the presence
of the indicated amount of CuSO4 after which the modified proteins were visualized with DBCO-
TAMRA 7. For uncropped images and coomassie brilliant blue (CM) staining of the gels see Figures
S1-4.
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The biochemical assays on purified protein revealed that streptavidin gets labelled at low
concentrations of DtBio 2, only when the probe can bind to its target. Binding of 2 should position
the diazotransfer moiety in the proximity of the amino group of lysine K121 (Figure S6). Therefore, if
labelling is dependent on binding, DtBio 2 should exclusively modify this residue, while leaving the
N-terminal amine and lysines K80, K132 and K134, which are not in proximity of the reactive group,
untouched. To validate if this is indeed the case, we identified the modification sites by digesting
streptavidin that was priorly incubated with DtBio 2 with trypsin and analysing the tryptic peptides
with nano LC-MS/MS. We searched the raw MS data for modified peptides, taking into account that
converting the ε-amine of a lysine side chain into an azide obliterates the trypsin cleavage site at the
carboxy terminal end of the residue. The only modified residue identified was lysine K121 (all other
residues were identified only in their unmodified version, see SI excel file “Mass spectrometry”),
which demonstrates that the probe (1) binds to streptavidin and (2) only reacts with nearby amino
groups. These results indicate that in essence any biotin-binding protein should be amenable for
labelling with DtBio 2 as long as it contains a suitably positioned lysine group or N-terminus. Avidin
and its deglycosylated variant NeutrAvidin are structurally related to streptavidin and contain
a lysine residue (K111) oriented in a similar position in the extended loop between strands
seven and eight of the prominent β-barrel structure of the proteins.20 Indeed these proteins
are also modified by DtBio 2. Even though labelling is less efficient, presumably due to the
suboptimal linker length in the probe molecule, it demonstrates that the use of DtBio
limited to streptavidin (Figure S7).
2 is not
Having established that DtBio
2 labels purified biotin-binding proteins site-selectively in an
activity dependent manner, we extended its use to more biologically relevant settings. We
first determined the selectivity by reacting E. coli cell lysates containing spiked in streptavidin
with 10 μM of DtBio
2 (Figure 3A). Also under these conditions, specific labelling of
streptavidin with minimal background is observed. Again labelling could be abolished by
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heat-inactivating or pre-incubating with a competitor. Extending on this finding we tested
common hen egg white which contains 0.05% (weight/dry weight) native avidin. In order to
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probe the functionality of DtBio
solution of egg white with . Indeed the probe labels avidin at a protein dilution of 1:1000 in
HEPES buffer. Labelling could be abolished by heat-denaturing or pre-incubating with the
competitor biotin (Figure S8). These experiments prompted us to explore if DtBio could be
2 in avidin’s natural environment, we incubated a dilute
2
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2
employed on live cells to label BioY29, the biotin specific S-component of the energy-coupling
factor transporter protein complex ECF BioY, which transports biotin from the extracellular
space into the cytosol of the Gram-positive bacterium Lactococcus lactis. Analysis of the
crystal structure of BioY revealed the absence of primary amines in the proximity of the
biotin-binding site. For the purpose of labelling this membrane protein on the cell surface,
we introduced a lysine residue near the binding pocket by exchanging either asparagine 79,
located in a flexible loop between beta strands three and four, or arginine 93, which is buried
deeper inside the biotin binding pocket of the protein (N79K and R93K mutants,
respectively). To assure that these mutations do not affect biotin binding, we determined the
binding affinities of wild-type BioY and its mutants for biotin using isothermal titration
calorimetry. Both mutants bind biotin with affinities similar to the affinity of wild-type BioY,
and DtBio
2 binds to these mutants with ~5-fold and ~12-fold decreased affinities for R93K
and N79K, respectively. We used Lactococcus lactis strains expressing these variants of BioY
for the cell surface labelling experiments. After on-cell labelling and cell disruption, the lysate
was analysed by fluorescence scanning of the SDS-PAGE gel. N79K-BioY showed
concentration dependent labelling with DtBio
protein corresponds to BioY using higher probe concentrations. As expected, the mutant
could be visualized with BODIPY alkyne after diazotransfer of DtBio using CuAAC click
2 (Figure S9). We validated that the labelled
3
chemistry, while only background labelling was observed for wt-BioY and untreated samples.
HisTag purification of labelled N79K-BioY and a subsequent western blot directed against the
HisTag confirmed labelling of the target (Figure 3B)
.
A
B
D
-
Biotin
DtBio 2
-
-
-
+
+
-
-
+
+
+ -
+
+
+
kDa
75
kDa
25
spiked-in Strp
M
Strp
4
FL
BioY
N79K WT
N79K
25
15
BioY
BioY
CL
SL
Strp
FL
1
2
3
4
5
1 2 3 4 5 6
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Figure 3. Labelling of streptavidin in E.coli lysates (A). A mixture of Strp (0.5 μg) and E.coli lysate (2.0
mg/mL) was incubated with DtBio 2 (10 μM). The modified proteins are visualized by clicking them
to BODIPY-alkyne 3 subsequent to diazotransfer and resolving them on a 15% SDS-PAGE gel. (B) Cell
surface labelling of N79K-BioY and wt-BioY. Cells expressing either form of BioY were treated with
DtBio 2 (250 μM) in the presence of 1 mM CuSO4 for 1h, after which the modified proteins were
clicked to 3. Labelling of BioY was visualized with fluorescence scanning (FL) of the lysate subsequent
to cell disruption (top panel); detection of BioY subsequent to western blotting of the same gel using
HisProbe-HRP and chemiluminescence (CL) as read-out (middle); and silver staining of the same gel
post-transfer (lower panel) (lanes 1-3). To further confirm the labelling signal from N79K-BioY, the
same sample as in lane 2 (B) was used for Ni-NTA bead purification of N79K-BioY and the eluate is
compared to untreated purified N79K-BioY (lanes 5,6).
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In conclusion, we here report the first ligand tethered-diazotransfer reagent and show that this
reagent selectively modifies biotin-binding proteins in protein mixtures, cell lysates and on living
cells. The reagents rapidly label the target proteins in an activity-dependent manner. 50 ꢀM of
copper (II) sulfate is sufficient to efficiently catalyse the diazotransfer reaction, but labelling even
occurs in the absence of catalyst. Mass spectrometry analysis of tryptic digests of labelled
streptavidin revealed that targeting the reagent not only enhances the protein-specificity, but also
the site-selectivity. We showed that DtBio 2 can be used to label N79K BioY and further optimization
of the probe may lead to cell surface labelling methods that can be used to study uptake of biotin
by BioY. Finally, the reported method provides a starting point for the design of other
targeted diazotransfer reagents that modify different proteins in complex samples and may
find use in target identification strategies.
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ASSOCIATED CONTENT
Supporting information
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The supporting information is available free of charge on the ACS Publications website.
Labelling of streptavidin resolved on a 15% SDS-PAGE gel; Coomassie brilliant blue stains of
the gels in Figure 2; Uncropped images of the gels in Figure 2C and 2D; Docking pose of DtBio
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bound to streptavidin; MS2 spectrum of the modified peptide in streptavidin; Labelling of
streptavidin, neutravidin and avidin at saturating and subsaturating probe concentrations;
Labelling of avidin in egg white; Docking poses, binding affinities and labelling of BioY and
BioY mutants and ITC data; Procedures for the synthesis of DtBio
DtBio , generation of BioY mutants, purification of BioY and BioY mutants, determination of
binding affinities, cell surface labelling of BioY and MS analysis; H-NMR and 13C-NMR
spectra; LC-MS data of DtBio . Excel table “Mass spectrometry” including the MaxQuant
2, labelling of proteins with
2
1
2
search results for trypsin digested, diazotised streptavidin and spectrum for N-terminal
peptide.
AUTHOR INFORMATION
Corresponding author
*E-mail: m.d.witte@rug.nl. Phone: +31-503635643
ORCID
Jonas Lohse: 0000-0002-5904-2390
Guillaume Médard: 0000-0002-4782-4029
Martin D. Witte: 0000-0003-4660-2974
Notes
The authors declare no competing interests
ACKNOWLEDGMENTS
J. de Keyzer is acknowledged for technical assistance. T. Masini and Y. Unver are thanked for
generating the images of DtBio binding streptavidin and BioY, respectively. M.D.W. is
supported by The Netherlands Organisation for Scientific Research (NWO-VENI grant 722.012.003)
and a Marie Sklowdoska Curie Career Integration Grant (CIG PCIG14-GA-2013-631057).
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REFERENCES
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(1) Saxon, E., and Bertozzi, C. R. (2000) Cell Surface Engineering by a Modified Staudinger
Reaction. Science 287, 2007–2010.
(2) Prescher, J. A., Dube, D. H., and Bertozzi, C. R. (2004) Chemical remodelling of cell
surfaces in living animals. Nature 430, 873–877.
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60
(3) Bräse, S., Gil, C., Knepper, K., and Zimmermann, V. (2005) Organic Azides: An Exploding
Diversity of a Unique Class of Compounds. Angew. Chem. Int. Ed. 44, 5188–5240.
(4) Debets, M. F., van der Doelen, C. W. J., Rutjes, F. P. J. T., and van Delft, F. L. (2010) Azide:
A Unique Dipole for Metal-Free Bioorthogonal Ligations. ChemBioChem 11, 1168–1184.
(5) Thirumurugan, P., Matosiuk, D., and Jozwiak, K. (2013) Click Chemistry for Drug
Development and Diverse Chemical–Biology Applications. Chem. Rev. 113, 4905–4979.
(6) Agard, N. J., Baskin, J. M., Prescher, J. A., Lo, A., and Bertozzi, C. R. (2006) A Comparative
Study of Bioorthogonal Reactions with Azides. ACS Chem. Biol. 1, 644–648.
(7) Ovaa, H., van Swieten, P. F., Kessler, B. M., Leeuwenburgh, M. A., Fiebiger, E., van den
Nieuwendijk, A. M. C. H., Galardy, P. J., van der Marel, G. A., Ploegh, H. L., and Overkleeft, H.
S. (2003) Chemistry in Living Cells: Detection of Active Proteasomes by a Two-Step Labeling
Strategy. Angew. Chem. Int. Ed. 42, 3626–3629.
(8) Speers, A. E., Adam, G. C., and Cravatt, B. F. (2003) Activity-Based Protein Profiling in
Vivo Using a Copper(I)-Catalyzed Azide-Alkyne [3 + 2] Cycloaddition. J. Am. Chem. Soc. 125,
4686–4687.
(9) Grammel, M., and Hang, H. C. (2013) Chemical reporters for biological discovery. Nat.
Chem. Biol. 9, 475–484.
(10) Pawlak, J. B., Gential, G. P. P., Ruckwardt, T. J., Bremmers, J. S., Meeuwenoord, N. J.,
Ossendorp, F. A., Overkleeft, H. S., Filippov, D. V., and van Kasteren, S. I. (2015)
Bioorthogonal Deprotection on the Dendritic Cell Surface for Chemical Control of Antigen
Cross-Presentation. Angew. Chem. Int. Ed. 54, 5628–5631.
(11) Kiick, K. L., Saxon, E., Tirrell, D. A., and Bertozzi, C. R. (2002) Incorporation of azides into
recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl.
Acad. Sci. 99, 19–24.
(12) van Dongen, S. F. M., Teeuwen, R. L. M., Nallani, M., van Berkel, S. S., Cornelissen, J. J. L.
M., Nolte, R. J. M., and van Hest, J. C. M. (2009) Single-Step Azide Introduction in Proteins
via an Aqueous Diazo Transfer. Bioconjug. Chem. 20, 20–23.
(13) Chin, J. W., Santoro, S. W., Martin, A. B., King, D. S., Wang, L., and Schultz, P. G. (2002)
Addition of p -Azido-
Soc. 124, 9026–9027.
L -phenylalanine to the Genetic Code of Escherichia c oli. J. Am. Chem.
(14) Wang, Z. A., Kurra, Y., Wang, X., Zeng, Y., Lee, Y.-J., Sharma, V., Lin, H., Dai, S. Y., and Liu,
W. R. (2017) A Versatile Approach for Site-Specific Lysine Acylation in Proteins. Angew.
Chem. Int. Ed. 56, 1643–1647.
(15) Witte, M. D., Cragnolini, J. J., Dougan, S. K., Yoder, N. C., Popp, M. W., and Ploegh, H. L.
(2012) Preparation of unnatural N-to-N and C-to-C protein fusions. Proc. Natl. Acad. Sci. 109,
11993–11998.
(16) Fernández-Suárez, M., Baruah, H., Martínez-Hernández, L., Xie, K. T., Baskin, J. M.,
Bertozzi, C. R., and Ting, A. Y. (2007) Redirecting lipoic acid ligase for cell surface protein
labeling with small-molecule probes. Nat. Biotechnol. 25, 1483–1487.
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(17) Schoffelen, S., van Eldijk, M. B., Rooijakkers, B., Raijmakers, R., Heck, A. J. R., and van
Hest, J. C. M. (2011) Metal-free and pH-controlled introduction of azides in proteins. Chem.
Sci. 2, 701.
(18) Tsukiji, S., Miyagawa, M., Takaoka, Y., Tamura, T., and Hamachi, I. (2009) Ligand-
directed tosyl chemistry for protein labeling in vivo. Nat. Chem. Biol. 5, 341–343.
(19) Fujishima, S., Yasui, R., Miki, T., Ojida, A., and Hamachi, I. (2012) Ligand-Directed Acyl
Imidazole Chemistry for Labeling of Membrane-Bound Proteins on Live Cells. J. Am. Chem.
Soc. 134, 3961–3964.
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(20) Yamaguchi, T., Asanuma, M., Nakanishi, S., Saito, Y., Okazaki, M., Dodo, K., and
Sodeoka, M. (2014) Turn-ON fluorescent affinity labeling using a small bifunctional O-
nitrobenzoxadiazole unit. Chem Sci 5, 1021–1029.
(21) Anderson, L. N., Koech, P. K., Plymale, A. E., Landorf, E. V., Konopka, A., Collart, F. R.,
Lipton, M. S., Romine, M. F., and Wright, A. T. (2016) Live Cell Discovery of Microbial Vitamin
Transport and Enzyme-Cofactor Interactions. ACS Chem. Biol. 11, 345–354.
(22) Le Trong, I., Wang, Z., Hyre, D. E., Lybrand, T. P., Stayton, P. S., and Stenkamp, R. E.
(2011) Streptavidin and its biotin complex at atomic resolution. Acta Crystallogr. D Biol.
Crystallogr. 67, 813–821.
(23) Kunishima, M., Kato, D., Nakanishi, S., Kitamura, M., Yamada, K., Terao, K., and Asano,
T. (2014) Specific Labeling of Streptavidin for Better Understanding of Ligand Modification in
Modular Method for Affinity Labeling (MoAL). Chem. Pharm. Bull. (Tokyo) 62, 1146–1150.
(24) Goddard-Borger, E. D., and Stick, R. V. (2007) An Efficient, Inexpensive, and Shelf-Stable
Diazotransfer Reagent: Imidazole-1-sulfonyl Azide Hydrochloride. Org. Lett. 9, 3797–3800.
(25) Culhane, J. C., and Fokin, V. V. (2011) Synthesis and Reactivity of Sulfamoyl Azides and
1-Sulfamoyl-1,2,3-triazoles. Org. Lett. 13, 4578–4580.
(26) Sulfonyl azide transfer reagent 6 and diazotransfer reagents 1-2 may be shock sensitive
and should be handled using appropriate precautions.
(27) Hong, V., Presolski, S., Ma, C., and Finn, M. G. (2009) Analysis and Optimization of
Copper-Catalyzed Azide–Alkyne Cycloaddition for Bioconjugation. Angew. Chem. Int. Ed. 48,
9879–9883.
(28) Ning, X., Guo, J., Wolfert, M. A., and Boons, G.-J. (2008) Visualizing Metabolically
Labeled Glycoconjugates of Living Cells by Copper-Free and Fast Huisgen Cycloadditions.
Angew. Chem. Int. Ed. 47, 2253–2255.
(29) Berntsson, R. P.-A., ter Beek, J., Majsnerowska, M., Duurkens, R. H., Puri, P., Poolman,
B., and Slotboom, D.-J. (2012) Structural divergence of paralogous S components from ECF-
type ABC transporters. Proc. Natl. Acad. Sci. 109, 13990–13995.
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TOC GRAPHIC
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directing group diazotransfer
H
S
H
N
O
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HN
N3
N
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NH
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DtBio 2
N3
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site-selective modification
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Figure 1. Top: structures of the non-targeted (1) and targeted (2) diazotransfer reagents and the BODIPY
fluorophore (3) used as reporter group in the evaluation of the diazotransfer reaction. Bottom: schematic
representation of the targeted diazotransfer reagent concept. Ligands bearing the diazotransfer group direct
the reagent to the protein of interest. Upon binding an amino group of the protein is transformed into an
azide in the presence of Cu(II). The modified protein can be further functionalized with bioorthogonal
chemistry.
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Scheme 1. Synthesis of diazotransfer reagent DtBio 2 (a) EDC⋅HCl, p-nitrophenol, DMF, 24 h, rT, 72%, (b)
histamine, DMF, 16 h, rT, 83%, (c) sulfonyl azide transfer reagent 6, DMF, 2 h, 0 °C, 52%.
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Figure 2. Biochemical evaluation of diazotransfer probe DtBio 2 with streptavidin (Strp) as target protein.
(A) A mixture of Strp and OVA labelled with indicated amount DtBio 2 (lane 1) or Dt 1 (lane 6,7) and clicked
to BODIPY-alkyne 3 subsequent to diazotransfer is resolved on a bis-tris 4-12% gradient gel and visualized
by fluorescence scanning. Heat-inactivation with 1% SDS prior to incubation (lane 3) and competition with
D-biotin (lanes 4,5) were used as controls. Note: biotin and derivatives thereof (such as DtBio 2) stabilize
tetrameric Strp. (B) A mixture of streptavidin and OVA was incubated with indicated amount of DtBio 2 after
which the modified proteins were visualized as described above (resolved on a 15% Laemmli type SDS-
PAGE gel). (C) Crop showing the labelling intensity of monomeric streptavidin incubated with DtBio 2
(10 µM) for the indicated time after which the modified proteins were visualized as described above. (D)
Crops showing the labelling intensity of tetrameric and monomeric streptavidin incubated with DtBio 2
(10 µM) in the presence of the indicated amount of CuSO4 after which the modified proteins were visualized
with DBCO-TAMRA 7. For uncropped images and coomassie brilliant blue (CM) staining of the gels see
Figures S1-4.
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Figure 3. Labelling of streptavidin in E.coli lysates (A). A mixture of Strp (0.5 µg) and E.coli lysate (2.0
mg/mL) was incubated with DtBio 2 (10 µM). The modified proteins are visualized by clicking them to
BODIPY-alkyne 3 subsequent to diazotransfer and resolving them on a 15% SDS-PAGE gel. (B) Cell surface
labelling of N79K-BioY and wt-BioY. Cells expressing either form of BioY were treated with DtBio 2 (250 µM)
in the presence of 1 mM CuSO4 for 1h, after which the modified proteins were clicked to 3. Labelling of BioY
was visualized with fluorescence scanning (FL) of the lysate subsequent to cell disruption (top panel);
detection of BioY subsequent to western blotting of the same gel using HisProbe-HRP and
chemiluminescence (CL) as read-out (middle); and silver staining of the same gel post-transfer (lower
panel) (lanes 1-3). To further confirm the labelling signal from N79K-BioY, the same sample as in lane 2 (B)
was used for Ni-NTA bead purification of N79K-BioY and the eluate is compared to untreated purified N79K-
BioY (lanes 5,6).
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Table of contents: Biotin-tethered diazotransfer reagents enable site-selective modification of biotin-binding
proteins
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