A trifluoromethylphenyl diazirine-based SecinH3 photoaffinity probe

Article abstract of DOI:10.1039/c2cc16477a

The synthesis of a trifluoromethylphenyl diazirine photoaffinity probe of the cytohesin inhibitor SecinH3 is described. The probe exhibits improved labelling efficiency over a benzophenone-based probe and thus is more suitable for photoaffinity labelling

Full text of DOI:10.1039/c2cc16477a

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A trifluoromethylphenyl diazirine-based SecinH3 photoaffinity probew
Barbara Albertoni, Jeffrey S. Hannam, Damian Ackermann, Anton Schmitz and
Michael Famulok*
Received 18th October 2011, Accepted 28th November 2011
DOI: 10.1039/c2cc16477a
The synthesis of a trifluoromethylphenyl diazirine photoaffinity
probe of the cytohesin inhibitor SecinH3 is described. The probe
exhibits improved labelling efficiency over a benzophenone-based
probe and thus is more suitable for photoaffinity labelling in
complex biological samples.
proteins with a coiled-coil amino terminal domain, a central Sec7
domain bearing the GEF function, and a pleckstrin homology
(PH) domain. The 1,2,4-triazole derivative SecinH3 1 (Fig. 1)
binds the Sec7 domain of cytohesin-1, -2 (ARNO), and -3 and
inhibits the GDP/GTP exchange.14,15 Application of SecinH3
in human cells, flies and mice revealed the involvement of
cytohesins in insulin signalling14,16 and recently their role as
cytoplasmic ErbB receptor activators.17 Moreover, SecinH3 is
increasingly being used as an indirect Arf6-inhibitor, owing to
the requirement of cytohesins for Arf6 activation and the
absence of any direct, specific Arf6 inhibitor.18–24
Photoaffinity probes are powerful tools for investigating inter-
actions between small organic molecules and proteins. When
combined with mass spectrometry, the identification of both
the interacting proteins and their respective interaction sites
becomes possible.1–4 These probes contain three crucial elements:
(i) an affinity unit, i.e. the ligand which interacts with the target
protein, (ii) a photoreactive moiety, which upon irradiation
covalently binds the probe to the protein, and (iii) a reporter
group, for detection and enrichment of the labelled protein. An
ideal photoactivatable group should be small, stable in the
absence of light, and highly and indiscriminately reactive upon
irradiation at wavelengths which do not damage the biological
sample.5 Commonly used photoactivatable groups include
aryl azides, benzophenones, and 3-aryldiazirines.6 Though
the advantages of 3-trifluoromethyl-3-phenyl-diazirine (TPD)7
over the other photoactivatable groups were recognised from their
inception, its use in photoaffinity labelling was limited in the past
by synthetic restrictions and limited availability of TPD derivatives
with convenient reactive groups.8,9 The recent establishment
of various diazirine compatible reactions8,10 and the increasing
commercial availability of TPD derivatives now allow their
more widespread use. The exceptional reactivity at relatively
long wavelengths (E360 nm), the superior properties, and its
structure make TPD an ideal photoactivatable group for the
modification of our small organic cytohesin inhibitor SecinH3.
In particular the lack of formation of slowly reacting isoforms,
as observed for benzophenones and aryl azides,5,11,12 is an
important advantage of TPD for experiments aimed at binding
site identification.13
Using the benzophenone derivatised probe Bio-SecinPP 2,
we have previously demonstrated specific binding of SecinH3
to the Sec7 domain of the members of the cytohesin family.25
However, the labelling efficiency was insufficient for allowing
the determination of the binding site by mass spectrometry of
LC-separated protein fragments or affinity based protein profiling
in whole proteomes.
Here we report the design and synthesis of SecinH3-TPD 3
(Fig. 1), a SecinH3 probe for the determination of the SecinH3
binding site on cytohesins, the analysis of specificity in complex
mixtures and as a lead structure for analyses in living cells, tissues,
or organisms. Additionally, we compare the results of the
SecinH3-TPD probe with those of an analogous benzophenone
containing compound. For SecinH3-TPD we expected an
improved labelling efficiency, which we confirmed by this work.
Structure–activity-relationship (SAR) studies on SecinH3
showed that removal of the thiophenol moiety in R1 led to loss
of inhibition of GEF activity. On the other hand, modification of
this group was tolerated to some extent. Similarly, the replace-
ment of the methoxy group for other ethoxy substituents affects
the biological activity of the compound only to a minor extent.25
These results indicate the suitability of the terminal thiophenyl
group as a site for substitution with the TPD moiety by amine
coupling reaction with commercially available 4-(1-azi-2,2,2-tri-
fluoroethyl)-benzoic acid (TPD-COOH), and of the 3-methoxy
residue as a site for reporter group attachment.25
Cytohesins are a class of small guanine nucleotide exchange
factors (GEFs) for the Arf (adenosine diphosphate ribosylation
factor) family of G-proteins. They are E47 kDa multidomain
To allow the enrichment of labelled protein in proteome-wide
profiling experiments, we employed desthiobiotin coupled to the
SecinH3-probe via an aminoethoxyethyl spacer (Fig. 1). Desthio-
biotin, an analogue of biotin, binds less tightly to streptavidin.
Desthiobiotin-containing probes can therefore be eluted from
streptavidin by washing with biotin-containing buffers, allowing
for straightforward recovery of proteins labelled with desthiobiotin
after enrichment by streptavidin chromatography.26
LIMES Institute, Chemical Biology & Medicinal Chemistry Unit,
University of Bonn, 53121 Bonn, Germany.
E-mail: m.famulok@uni-bonn.de; Fax: +49-228-735388
w Electronic supplementary information (ESI) available: Synthetic
procedures and characterization of compounds. See DOI: 10.1039/
c2cc16477a
c
1272 Chem. Commun., 2012, 48, 1272–1274
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Fig. 1 Structures of the cytohesin inhibitor SecinH3 and the photoreactive probes SecinH3-TPD and dt-SecinPP. Blue: reporter group
(desthiobiotin or biotin). Red: photoactive group (benzophenone or 3-trifluoromethyl-3-phenyl-diazirine, TPD).
The synthesis of SecinH3-TPD 3 is shown in Scheme 1.
Treatment of piperonoyl chloride with potassium thiocyanate
to obtain the acylthioisocyanate in situ, followed by addition
of the Boc-protected diglycol gave thiocarbamate 5. The 1,2,
4-substituted triazole ring 6 was obtained by cyclisation of 5
with 4-nitrophenylhydrazine.24 Deprotection of 6 with formic
acid led to amine 7, which was then coupled to desthiobiotin
by a standard peptide coupling reaction. The nitro-triazole 8 was
subsequently reduced employing a standard palladium catalyzed
hydrogenation to afford 9. The benzoic acid TPD-COOH was
converted to the corresponding benzoyl chloride before reaction
with 9 giving the requisite final product SecinH3-TPD 3. All steps
led to the desired product in good purity and satisfactory yield.
The ability of SecinH3-TPD to label cytohesins in an
irradiation-dependent way was tested on the purified Sec7
domain of cytohesin-2, called ARNO-Sec7. SecinH3-TPD in
5-fold excess was incubated on ice with a His6-tagged ARNO-Sec7
construct (0.5 mM) for 10 min in the dark, before irradiation
with UV light at 365 nm. The proteins were separated by 10%
SDS-PAGE and transferred on nitrocellulose membranes by
Western blotting. Labelled proteins were detected with a
NeutrAvidin DyLight 800 fluorescent conjugate, while the total
amount of protein was detected with a hexahistidine-specific
antibody (His-tag in Fig. 2A). A strong biotin signal was observed
only in the irradiated samples, demonstrating that SecinH3-
TPD covalently reacts with ARNO-Sec7 upon UV activation
(S in Fig. 2A).
As a control of specificity we used the purified PAZ domain
of Argonaute 1 and measured the efficiency of its labelling by
Secin-TPD (P in Fig. 2A). A slight degree of labelling could be
detected, but when the same experiment was performed in the
presence of ARNO as a specific competitor, no residual
labelling of PAZ was obtained (S + P in Fig. 2A), indicating
that SecinH3-TPD labels cytohesins in a specific fashion.
To verify that the SecinH3-TPD probe has the same binding
site on ARNO as SecinH3 we performed a competition
experiment by adding an excess of unmodified SecinH3 to
the labelling reaction. Indeed, when ARNODPBR (an ARNO
construct lacking the last 14 amino acids required for its
autoinhibition)27 was incubated with equimolar concentration
of SecinH3-TPD (1 mM) and excess of SecinH3 (14 mM), the
amount of labelled protein was strongly reduced (Fig. 2B),
indicating that the binding sites of SecinH3-TPD and SecinH3
on cytohesins overlap.
Next, we directly compared the efficiencies of the TPD and
benzophenone derivatives of SecinH3 in labelling ARNODPBR.
To ensure optimal comparability, the desthiobiotinylated
probe dt-SecinPP 4 (Fig. 1) was used instead of biotinylated 2.25
Scheme 1 Synthesis of the photoreactive probe SecinH3-TPD: (i) potassium thiocyanate, acetone, 55 1C, then Boc-2-(2-aminoethoxy)ethanol, 55 1C;
(ii) 4-nitrophenylhydrazine, EtOH, 80 1C, 8 h; (iii) formic acid, 2.5 h; (iv) D-desthiobiotin, HBTU, N,N-diisopropylethylamine, DMF, 1 h; (v) 10% Pd/C,
EtOH/THF, 9 bar H2, 2 h; (vi) TPD-COOH, oxalyl chloride, THF, DMF (catalytic), 2.5 h; (vii) N,N-diisopropylethylamine, CH2Cl2, 2 h.
c
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Chem. Commun., 2012, 48, 1272–1274 1273

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This work was supported by grants from the Deutsche
Forschungsgemeinschaft (SFB 645), and the European Science
Foundation (Synapta). B. A. thanks the Roche Research
Foundation and the GRK804 for scholarships.
Notes and references
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