3-Trifluoromethyl-3-aryldiazirine photolabels with enhanced ambient light stability

Article abstract of DOI:10.1039/c5cc09518b

Ambient light stable 3-trifluoromethyl-3-aryldiazirine photolabels are developed via stabilization of the strained three membered diazirine ring by replacing the phenyl ring with electron withdrawing heterocyclic rings. Photolabeling studies reveal that these ambient light stable photolabels are equally efficient in photolabeling target proteins as the traditional 3-trifluoromethyl-3-phenyldiazirine and found to significantly increase the aqueous solubility of the photoaffinity labels.

Full text of DOI:10.1039/c5cc09518b

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3-Trifluoromethyl-3-Aryldiazirine Photolabels with Enhanced
Ambient Light Stability
Arun Babu Kumar,a Jeremiah D. Tipton,b,‡ and Roman Manetscha,§,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
10
Ambient light stable 3-trifluoromethyl-3-aryldiazirine photolabels respectable amounts of specific labeling products.1,
In
are developed via stabilization of the strained three membered particular, the 3-trifluoromethyl-3-phenyl-diazirine introduced
diazirine ring by replacing the phenyl ring with electron by Brunner is the most widely used photolabel producing a
withdrawing heterocyclic rings. Photolabeling studies reveal that singlet carbene as the reactive intermediate, which inserts into
these ambient light stable photolabels are equally efficient in carbon-, nitrogen-, oxygen- or sulfur-containing bonds at
photolabeling target proteins as the traditional 3-trifluoromethyl- almost diffusion-controlled reaction rates.4,
11
Despite the
3-phenyldiazirine and found to significantly increase the aqueous promise of photoactivatable 3H-diazirines as photolabeling
solubility of the photoaffinity labels.
agents, the cumbersome synthesis, the instability to ambient
light conditions, and the limited aqueous solubility represent
major drawbacks for PAL. Herein we report for the first time
on the design, synthesis and evaluation of 3-pyridyl- and 3-
Photoaffinity labeling (PAL) is an established approach to
investigate interactions between a ligand and a biological
receptor, utilizing a photoactivatable probe to form a covalent
bond between the ligand and the biological receptor upon UV
light irradiation of the ligand-receptor complex.1, 2 Many types
of photoactivatable groups have been developed over the past
40 years, of which 3H-diazirines, arylazides, and
pyrimidyl-substituted 3-trifluoromethyl-diazirines
1 and 2 (Fig.
1) as photoaffinity labels displaying favorable ambient light
stability without compromising the photoactivated insertion
reactivity.
Ambient light conditions commonly promote the
benzophenones emerged as the widely used photoprobes.3, 4 spontaneous decomposition of the 3H-diazirines due to ring
strain energy of the three-membered diazirine ring. Based on
previous reports, we speculated to minimize the ambient light
mediated 3H-diazirine's decay through electron withdrawing
groups while retaining the ability to rapidly react with UV light
3H-Diazirines, arylazides, and benzophenones photoreact into
highly reactive intermediates such as carbenes, nitrenes, and
biradicals respectively, which covalently crosslink to
biological receptor.5
a
(λext
= 320 - 400 nm) to the corresponding carbene
Ideally,
a photoactivatable probe should be readily
intermediate.12 As the trifluoromethyl substituent of 3-
trifluoromethyl-3-phenyl-diazirine is a very strong electron
withdrawing group,13 as well as previous studies have shown
that aromatic diazirines photochemically produce higher ratios
synthesized, chemically stable and susceptible to smooth
photolysis at long wavelengths (λext ≥ 300 nm) to exclude
photooxidative or other photochemical damage of the
biological target. Comparative studies with ion channels,6
glucose transporter proteins,7 yeast RNA Polymerase III,8 and
peptide thymopentin9 identified 3H-diazirines to be best suited
for PAL, while arylazides and benzophenones failed at yielding
of carbene over the rearranged diazo byproduct compared to
aliphatic diazirines,1,
14, 15
we decided to replace the phenyl
group with an electron withdrawing pyridine or pyrimidine
ring. Furthermore, we speculated that the use of a pyridyl- or
pyrimidyl-substituent will slightly increase the insertion
reactivity of the photoactivated intermediate as pyridyl
carbenes have been shown to be more reactive than
corresponding phenyl analogues.16 With this understanding,
we prepared pyridine and pyrimidine photolabels
starting from the alcohols
1
and
2
4
and 917 (Scheme 1A). For
N
N
N
N
N
N
N
CF3
N
CF3
CF3
HO
HO
HO
N
3
1
2
Fig. 1. Design of ambient light stable pyridine and pyrimidine derived 3-
trifluoromethyl-3-aryldiazirines and
1
2
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comparative studies, we also prepared the conventional
Journal Name
Table 1. Comparison of ambient light stability of modified trifluoromethylaryl
diazirines
1
and
2
versus the conventional trifluoromethylphenyl diazirine
3. A
Brunner-type 3-trifluoromethyl-3-phenyl-diazirine
previously reported.14, 18
3
as
solution of trifluoromethylaryl diazirines
1
,
2
or 3 in d4-methanol was exposed to
light from two linear fluorescent lamps (28 W each) at room temperature and
the photodecomposition of the diazirines were followed by 19F NMR. The picture
shots of the 19F NMR shows that upon exposure to ambient light the
conventional trifluoromethylphenyl diazirine decomposes faster as indicated by
the decomposition product peak (pointed out by arrow), while the pyridinyl
derivative is much more resistant to the photodecomposition and the
pyrimidinyl diazirine is virtually intact.
The photoactivation and the photostability of diazirines 1 -
were investigated first by 19F NMR utilizing deuterated
3
methanol to scavenge the carbene intermediates.19, 20 Previous
19F NMR studies have demonstrated that photoactivation of
diazirine
3 lead predominantly to the corresponding singlet
carbene, which rapidly forms the methanol insertion product
19
(Scheme 1B).14,
In parallel, a side reaction occurs by
into a linear diazo compound, which
rearranging diazirine
3
under continuous light exposure slowly decomposes to the
reactive carbene leading ultimately to the methanol insertion
product.
Similarly, a 19F NMR experimental set-up was chosen to
monitor the photoactivation of diazirines
1-
3. The exchange of
the phenyl ring in the 3-trifluoromethyl-3-phenyl diazirine
3
by
the electron withdrawing pyridine or pyrimidine ring did not
affect the ratio between the carbene and the linear diazo
intermediate (Supporting Information Fig. S1). Across control
compound
3 and the modified photolabels 1 and 2, the linear
diazo intermediate peaked at about 30% and decreased as
irradiation continued. These results indicated that the
photolabeling efficiency, with respect to the ratio of carbene
to linear diazo ratio, would not be affected by switching the
phenyl moiety with a pyridine or a pyrimidine.
diazirine photolabel
increase the light stability of diazirine photolabels
Solutions of the conventional diazirine and modified
photolabels and in deuterated methanol were exposed to
3
to determine the effectiveness to
We subjected the modified 3-trifluoromethyl-3-
1
and
2.
aryldiazirine photolabels
1 and 2 to an ambient light stability
3
test along with the conventional 3-trifluoromethyl-3-phenyl
1
2
ambient light conditions using linear fluorescent lamps, and
the rate of decomposition was observed over a period of one
i) n-BuLi, ether
ii) methyltrifluoro-
acetate, ether
4 h, 75-76%
O
X
CF3
Br
Br
A
TBS-Cl or TBDPS-Cl,
imidazole, DCM, RT,
16 h, 90-91%
month. As shown by the 19F NMR, 3-phenyl diazirine
3 had
X
N
X
N
N
already undergone significant photodecomposition after 7
days of light exposure (Table 1). In contrast, during the same
OH
OR
OR
5
10
: X = CH, R = TBS
: X = N, R = TBDPS
6
11
4
9
: X = CH, R = TBS
: X = N, R = TBDPS
: X = CH
: X = N
period of ambient light exposure, pyridine photolabel
negligibly photodecomposed whereas pyrimidyl photolabel
1
2
i) NH2OH.HCl,
AcONa, ethanol,
reflux, 16 h
ii) reaction
conditions a
or b,
42 to 61 %
over 2 steps
was virtually unaffected. Exposing the probes 1-3 to light for a
period of one month continued this stability trend. As
determined by 19F NMR, only 27% of the conventional
i) Ag2O, ether, 2 to
16 h
ii) TBAF, THF, RT,
2 h, 76 to 85% over 2
steps
N
NH
HN
TsON
CF3
N
X
liquid NH3,
ether, 16 h,
68 to 94%
CF3
CF3
photolabel
exposure (Supporting Information Table S1), whereas 79% of
the pyridine photolabel and 90 % of the pyrimidine
photolabel remained unaffected. Similar stability trends
were observed when photolabels were exposed to light
from an incandescent bulb (Supporting Information Table S2).
No appreciable decomposition of diazirines was detected
in a control experiment when compounds were kept in the
dark at room temperature for a period of one month
(Supporting Information Table S3), suggesting probes and
to be equally stable compared to the conventional
phenyldiazirine . Furthermore, the near-UV/Vis absorption
spectra show that the maximum absorption λmax was 350 nm
for , whereas the absorption coefficient ε was decreased by
approximately 15% for pyiridine and 30% for pyrimidine
3 remained intact after one month of ambient light
N
X
N
X
N
1
OH
OR
OR
2
7: X = CH, R = TBS
12
8: X = CH, R = TBS
13
1: X = CH
: X = N
: X = N, R = TBDPS
: X = N, R = TBDPS
2
1-3
Reaction conditions: a) X = CH, pyridine, DMAP, Ts2O, DCM, 0oC to RT, 61%
b) X = N, DIEA, DMAP, TsCl, DCM, -50oC to 0oC, 42%
1-3
B
N
N
X
CF3
1-3
hν
hν
Y
N
D
1
2
CF3
D3CO
X
CF3
N
X
CF3
Y
CD3OD
OH
δ ≅ -59 ppm
linear diazo
hν
3
X
Y
Y
OH
OH
OH
δ ≅ -67 ppm
diazirine
1-3
h
ν
δ ≅ -78 ppm
methanol-d 4 adduct
carbene
1
2
X and Y = N or CH
(Supporting Information Table S4). Importantly, these results
proved our hypothesis of increasing the ambient light stability
Scheme
aryldiazirines
1
(A) Synthesis of pyridine and pyrimidine 3-trifluoromethyl-3-
and (B) Photoactivation reaction of diazirine photoprobes
1
2
2 | J. Name., 2012, 00, 1-3
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N
N
A
OH
by stabilizing the diazirine ring with electron withdrawing
substitutions to be correct.
OH
O
NH
NHAc
F3C
X
HO
HO
O
Y
Photolabeling experiments are commonly conducted in
aqueous buffer solutions, and it was predicted that pyridine
14
O
: X = Y = CH
15: X = CH, Y = N
16: X = Y = N
O
O
X
O
N
HN
N
NH
and pyrimidine probes
solubility than the conventional 3-trifluoromethyl-3-phenyl
diazirine To demonstrate the aqueous solubility
enhancements of pyridine and pyrimidine photoprobes,
compounds were derivatized with N-acetyl tryptophan or
4-quinolinol to yield corresponding esters 14 16 or ethers 17
19 (Fig. 2A). The aqueous solubility of compounds 14 19 were
1 and 2 will possess a better aqueous
Y
N
N
N
CF3
H
N
F3C
X
S
O
3.
O
Y
20
: X = Y = CH
17
: X = Y = CH
18: X = CH, Y = N
19: X = Y = N
N
21: X = CH, Y = N
22: X = Y = N
1-3
-
-
-
experimentally determined at pH = 7.4 and 5.0 using a
previously reported HPLC-based assay (Supporting Information
Table S4).21 As expected, in comparison to conventional 3-
trifluoromethyl-3-phenyl-diazirines 14 and 17, the pyridyl
probes 15 and 18 were approximately 30-250-fold more
soluble, whereas pyrimidinyl probes 16 and 19 were 100-7,500
times more soluble.
To investigate whether pyridyl- and pyrimidyl-substituted
diazirines
1
and
2
are suitable for photoaffinity labeling of
22 were
protein targets, mannose photoaffinity probes 20
-
designed to label concanavalin A (Con A) (Fig. 2A). Previously, a
specifically designed diazirine-derived mannose photoaffinity
probe was reported to covalently crosslink to Con A at the
saccharide binding sites.22 Diazirine probes 20
-22 were
designed with a biotin moiety to facilitate western blot
Fig. 2 (A) Compounds 14-19 synthesized for solubility studies and photoaffinity
probes 20, 21 and 22 designed to evaluate photolabeling activities (B) Coomassie
visualization and post-labeling enrichment (synthesis of 20
, 21
stained gel of photolabeled Con A and control samples (C) Western blot analysis
to detect biotinylation of Con through photolabeling. The western blot
indicates that Con is photolabeled with the pyridinyl and pyrimidinyl
photolabels 21 and 22 as effectively as the conventional phenyl derived
incubated with Con A and subjected to photoaffinity labeling photolabel 20. (D) Photolabeling of Con A with 21 and 22 in the presence of the
native ligand mannose at different concentrations. The western blot analysis
suggests that the extent of photolabeling of Con A with photoprobes 21 or 22
correlates indirectly to the concentration of competing mannose ligand.
and 22 detailed in Supporting Information Scheme S3).
A
A
In the first labeling experiment, probes 20-22 were
by exposure to UV light (> 320 nm). In another set of
experiment, probes 20 22 were incubated with Con A without
-
being exposed to UV light to investigate if there is any labelling
in the absence of photoactivation. As a control, Con A in
incubation buffer in absence of any photoaffinity probe was
also subjected to photoactivation under UV light. The control,
which contained Con A without any photoaffinity probe, was
visualized with coomassie stain but did not make any visible
spot in the anti-biotin peroxidase antibody western blot (Fig.
2B and 2C). In contrast, the samples containing Con A and label
observed that the crosslinking of 20-22 was significantly
suppressed (Fig. 2D) depending on the concentration of
mannose ligand. These results suggested that the
photolabeling of 20-22 occurred at the mannose-specific
binding sites of the target protein Con A and not in an
unspecific way on the protein surface.
Finally, in order to determine the photolabeled site of Con
20, 21 or 22 that were photoactivated with UV light were
A, samples photolabeled with 20-22 were subjected to trypsin
visible with both coomassie stain and anti-biotin peroxidase
antibody western blot. This data indicates that the biotin
containing photoaffinity labels derived from the modified
photoaffinity labels 21 and 22 and the conventional 3-
trifluoromethyl-3-phenyldiazirine photoaffinity label 20
successfully labeled the protein target upon photoactivation.
However, samples containing Con A along with photoaffinity
digestion and subsequent analysis by liquid chromatography
coupled to a linear ion trap-Orbitrap spectrometry. The labeled
peptides with a neutral mass of 1826.9023, 1827.8971 and
1828.8940 Da, for the samples photolabeled with 20, 21 and
22 respectively, were found (Fig. 3A). Upon subtracting the
mass of denitrogenated photolabels from the mass of labeled
peptides, all the resultant masses corresponded to the mass of
the peptide sequence Val91 - Lys101 (VGLSASTGLYK) with high
mass accuracy (< 3 ppm) (Supporting Information Table S5).
Further fragmentation of the labeled peptide in the Orbitrap
mass spectrometer confirmed the identity and sequence of the
labeled peptide (Supporting Information Table S6 and Fig. S3).
It is noteworthy that Hamachi and co-workers previously
reported the same peptide sequence (Val91 - Lys101) to be
labeled, in their efforts to photolabel Con A with their 3-
trifluoromethyl-3-phenyldiazirine derived photoaffinity label.22
labels 20, 21 or 22 that were not photoactivated with UV light
were detected only with coomassie stain and not in the
western blot analysis, proving that Con A is tagged only upon
photoactivation of the photoaffinity probes. These data
suggest that the modified labels
proteins upon photoactivation as efficiently as the
conventional diazirine photolabel
1 and 2 are capable of tagging
3.
Since mannose is the natural ligand of Con A, addition of
mannose prior to the photolabeling should inhibit the binding
of the photoaffinity labels 20-22 to Con A and impede
thephotoaffinity labeling. In presence of mannose ligand, we
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Fig. 3 (A) Mass spectra of peptides labeled with photolabels 20-22. The MS/MS
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peptide is Val91 - Lys101 (VGLSASTGLYK) residue. (B) Virtual docking of the
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3-phenyldiazirine by a pyridine or pyrimidine ring in 3-position.
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pyridine and pyrimidine photolabels also showed significant
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Acknowledgements
This research was funded by the National Institutes of Health
(R01 GM097118). We thank Dr. S. Mahajan and Dr. D.
Ramamoorthy for their guidance with the western blot and
virtual docking studies respectively.
Notes and references
4 | J. Name., 2012, 00, 1-3
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