Fluorous aryldiazirine photoaffinity labeling reagents

Article abstract of DOI:10.1021/ol901955y

Two fluorous versions of trifluoromethyldiazirine derivatives have been designed and synthesized. The new photoaffinity labeling reagents have reactivity similar to that of their aryltrifluoromethyldiazirine parent when activated in MeOH, while the reacti

Full text of DOI:10.1021/ol901955y

ORGANIC
LETTERS
2009
Vol. 11, No. 21
4882-4885
Fluorous Aryldiazirine Photoaffinity
Labeling Reagents
Zhiquan Song and Qisheng Zhang*
DiVision of Medicinal Chemistry and Natural Products, The UniVersity of North
Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
qszhang@unc.edu
Received August 23, 2009
ABSTRACT
Two fluorous versions of trifluoromethyldiazirine derivatives have been designed and synthesized. The new photoaffinity labeling reagents
have reactivity similar to that of their aryltrifluoromethyldiazirine parent when activated in MeOH, while the reaction products can be efficiently
separated over fluorous silica gel. The alcohol group in the two reagents is further converted to activated carboxylic acid and amine, which
enable coupling both reagents with small molecules and macromolecules under mild conditions.
Photoaffinity labeling1-5 has been extensively used to
identify the ligand binding site of various enzymes and
receptors. A photoaffinity label is designed by attaching a
photoactive group to a substrate, a ligand, or an inhibitor.
After the probe is bound reversibly to the active site, the
complex is photoirradiated to generate reactive species that
chemically derivatize the target through a rapid and irrevers-
ible covalent bond with a binding site residue. Traditionally,
a photoaffinity probe consists of a substrate specific for its
cognate receptor, a photoreactive group for cross-linking, and
a tag to identify the cross-linked products. Azide, diazirine,
and benzophenone are the most commonly used photoreac-
tive groups, while the tag is usually a radiolabel. The cross-
linked proteins are then identified on the basis of the tag
and analyzed by liquid chromatography/tandem mass spec-
trometry (LC/MS/MS) after trypsin digestion. However,
living organisms often exhibit large dynamic ranges in
protein expression levels, ranging from an estimated value
of 104 in yeast to 109-1012 in plasma.6 Without selective
enrichment of subsets of proteins,7-9 the photoaffinity
labeling will likely fail to identify the direct target whose
expression level is low. In addition, synthesis of a radiola-
beled compound with high specific activities is sometimes
challenging. Recently, the alkyne functional group has been
introduced as a tag in a photoaffinity probe.10 The cross-
linked complexes are separated from the proteome through
a Cu(I)-catalyzed chemical reaction11 with immobilized azide
on the solid phase, fluorescence dye, or biotin. However,
this approach is highly dependent on the efficiency of the
coupling reaction, which varies from protein to protein and,
therefore, may fail to capture the desired protein target(s).
Moreover, proteins that are not cross-linked may also be
pulled out due to protein-protein interactions. Developing
new strategies for photocross-linking is therefore of signifi-
cance and an urgent need.
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10.1021/ol901955y CCC: $40.75
Published on Web 10/06/2009
 2009 American Chemical Society

Small molecules containing perfluorinated groups (fluorous
molecules) can be separated from nonfluorous species
through selective retention on fluorinated silica gel.12 This
property has been used for the recycling and reuse of
catalysts,13,14 removal of reaction intermediates,15-17 and
fluorous mixture synthesis of libraries of compounds.18-21
Recently, fluorinated peptides6 and oligonucleotides22 were
also efficiently separated from nonfluorinated counterparts
through fluorous solid-phase extraction (FSPE). We envision
that a fluorous photoreactive group, when coupled to
bioactive small molecules and activated in a biological
system, would cross-link with the small molecules’ targets
and enrich them for characterization based on the fluorous
tag.
Scheme 1. Synthesis of Fluorous Aryldiazirines
Among the known photoreactive groups, aryltrifluorom-
ethyldiazirine (Figure 1) has been widely used in biological
sponding iodide 4b and isopropylmagnesium chloride1,23 and
then reacted with 3 to afford the fluorous ketone 5b in 51%
yield. In this process, it is important to generate the
perfluoroalkylmagnesium chloride at -78 °C to minimize
its potential decomposition. The ketone 5b was then con-
verted to the diazirine via a standard literature protocol:2 (i)
formation of the tosyloxime 6b in 50% yield through reaction
with hydroxylamine followed by the treatment with tolu-
enesulfonyl chloride (TsCl) and (ii) ammonolysis of 6b to
furnish the diaziridine, which was subsequently oxidized to
the corresponding diazirine with iodine. Removal of the TBS
protective group with tetrabutylammonium fluoride (TBAF)
then provided the desired diazirine 1b in 62% yield. The
diazrine 1c with the C6F13 substituent was synthesized in an
analogous fashion. The UV-vis spectra of both 1b and 1c
display an absorption at about 350 nm, which is consistent
with the formation of diazirine.1 For comparison, diazirine
1a was also synthesized. This was accomplished through
reduction of the commercially available 4-(3-(trifluorometh-
yl)-3H-diazirin-3-yl)benzoic acid 7 with NaBH4 in the
presence of BF3 in 57% yield24,25 (Scheme 1B).
Figure 1. Chemical structures of fluorous aryldiazirines.
systems due to its relatively small size, high stability under
acidic or basic conditions in the dark, the long wavelength
(365 nm) used for activation, and high reactivity of the
corresponding carbene. We thus chose to develop fluorous
aryltrifluoromethyldiazirine analogues as novel photoaffinity
labeling reagents. In our design, the trifluoromethyl (CF3)
group is replaced with a longer perfluoroalkyl chain to render
the resulting compounds fluorous. For further derivatization
and coupling to bioactive molecules, a p-hydroxymethyl
group is also added to the designed photolabeling reagents
(Figure 1).
The synthesis of the fluorous aryl diazirines 1b and 1c
starts with the commercially available methyl 4-(hydroxym-
ethyl)benzoate 2 (Scheme 1A). The benzyl alcohol in 2 was
protected as the tert-butyldimethyl silyl (TBS) ether 3 by
reaction with TBSCl in 94% yield. The perfluoropropyl-
magnesium chloride was generated in situ from the corre-
Photoactivation of aryltrifluoromethyldiazirine in MeOH is
known to generate the corresponding methyl ether adduct in
(13) Zhang, Q.; Luo, Z.; Curran, D. P. J. Org. Chem. 2000, 65, 8866–
8873
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67, 1781–1785
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(23) Omote, M. E., Y.; Tarui, A.; Sato, K.; Ando, A. Tetrahedron:
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Asymmetry 2007, 18, 2768–2772.
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G. D.; Arevalo, E.; Olsen, R. W.; Raines, D. E.; Forman, S. A.; Cohen,
J. B.; Miller, K. W. J. Med. Chem. 2006, 49, 4818–4825.
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3947–3950
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(18) Zhang, Q. S.; Lu, H. J.; Richard, C.; Curran, D. P. J. Am. Chem.
Soc. 2004, 126, 36–37
(19) Zhang, W.; Lu, Y. M.; Chen, C. H. T.; Zeng, L.; Kassel, D. B.
J. Comb. Chem. 2006, 8, 687–695
(20) Fukui, Y.; Bruckner, A. M.; Shin, Y.; Balachandran, R.; Day, B. W.;
Curran, D. P. Org. Lett. 2006, 8, 301–304
(21) Zhang, W.; Luo, Z.; Chen, C. H.; Curran, D. P. J. Am. Chem. Soc.
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Hennig, H.; Beck-Sickinger, A. G.; Welzel, P. Angew. Chem., Int. Ed. 2002,
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Org. Lett., Vol. 11, No. 21, 2009
4883

high yield1,26,27 (Figure 2) and was thus used as the model
reaction to compare the reactivity of 1b and 1c with that of
1a. A solution of 1a, 1b, or 1c (0.7 mM for each) in MeOH
and 1c could be used for enrichment of the cross-linked
products, the reaction mixtures in Figure 2 were loaded onto
a column packed with fluorous silica gel (Figure 3). Each
Figure 3. Separation of the photoirradiated reaction mixture by
fluorous silica gel: (A) HPLC chromatograms of fractions eluted
by 50% MeOH in H2O (fraction 1); (B) HPLC chromatograms of
fractions eluted by 100% MeOH (fraction 2). HPLC conditions:
80% MeOH in H2O at 1 mL/min on a Thermo Betasil C18 column
(150 × 4.6 mm).
column was first eluted with 50% MeOH in water (fraction
1) and then 100% MeOH (fraction 2). All fractions were
then concentrated and analyzed by reversed-phase HPLC.
As shown in Figure 3, the MeOH adduct with the CF3
substituent was mainly in the first fraction (>95%). In
contrast, the adducts with the C3F7 and C6F13 substituents
were not detectable in the first fraction but were completely
recovered in the second fraction. These data suggest that both
1b and 1c could be used to separate the cross-linked products
from nonfluorous components in the reaction mixtures.
The hydroxyl group in the fluorous diazirines 1b and 1c can
be easily converted to other functional groups for attachment
to a variety of molecules under mild conditions. As examples,
Figure 2. HPLC chromatograms of 1 in MeOH upon photoirra-
diation (A) before irradiation; (B) after irradiation. The MeOH
solution of 1 was irradiated at 365 nm for 5 min followed by 312
nm for 5 min. The red lines denote chromatograms detected at 350
nm, while the black lines are those at 217 nm. HPLC conditions:
80% MeOH in H2O at 1 mL/min on a Thermo Betasil C18 column
(150 × 4.6 mm).
was irradiated in parallel at 365 nm (8 W) for 5 min. Under
these conditions, each reaction mixture showed two major
1
products formed in approximately 1:1 ratio. The H NMR
spectra indicated that one product is the MeOH adduct 13.
We speculated that the other product is the diazo isomer of
1 and therefore irradiated the reaction mixtures for an
additional 5 min at 312 nm (8 W). Indeed, this two-step
irradiation protocol resulted in the formation of only one
major product as judged by the HPLC chromatograms. As
shown in Figure 2, the product in each reaction mixture has
a different retention time than its corresponding starting
material as indicated by their absorption at both 217 nm
(black lines) and 350 nm (red lines). NMR spectroscopic
and mass spectrometric data further confirmed that 13 was
formed in the reaction. In addition, we carried out the
photoirradiation experiments in i-PrOH and n-BuOH. All
three diazirines 1a-c were completely consumed in the two-
step irradiation protocol. These data collectively suggest that
new fluorous aryldiazirines 1b and 1c undergo photolysis in
a manner similar to that of their parent photoaffinity-labeling
reagent 1a.
Scheme 2
.
Transformations of Fluorous Aryldiazirines
One key feature of fluorous compounds is their selective
retention on fluorous silica gel compared to nonfluorous
compounds. To test whether the new fluorous diazirines 1b
4884
Org. Lett., Vol. 11, No. 21, 2009

we developed synthetic routes that transformed the hydroxyl
group in 1c to activated carboxylic acid and amino groups
(Scheme 2). Oxidation of 1c with basic potassium permanganate
provided the corresponding carboxylic acid 9 in 85% yield,
which was activated as ester 10 in 78% yield by reaction with
N-hydroxysuccinimide (NHS) in the presence of dehydrating
agent dicyclohexylcarbodiimide (DCC). To synthesize the
amino derivative 12, compound 1c was first mesylated with
methanesulfonyl chloride (MsCl), and the resulting intermediate
was treated with NaN3 to generate 11 in 90% yield. Under
Staudinger’s conditions,28 the azido group in 11 was reduced
to the free amine to provide 12 in 77% yield. Because NHS
esters and amines are widely used for coupling to different
molecules under mild conditions,29 these analogues should
enable the easy application of 1b and 1c to various biological
systems. Furthermore, these transformations, performed under
different reaction conditions, highlight the stability of the
diazirine moiety in both 1b and 1c.
Both reagents have similar photoreactivity as the parent
compound 1a, and the resulting photoadducts are selectively
retained on fluorous reversed-phase silica gel. Furthermore,
both fluorous probes can be easily converted into activated
carboxylic acids and amines for facile coupling to different
molecules under mild conditions. We are currently applying
the fluorous diazirines to different biological systems and
will report the results in due course.
Acknowledgment. We thank Dr. David Lawrence (Uni-
versity of North Carolina) for critical reading of the
manuscript. This work was supported by the startup fund
from the University of North Carolina at Chapel Hill.
Supporting Information Available: Characterization of
new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
In summary, we have developed two novel fluorous
aryldiazirines (1b and 1c) as photoaffinity labeling reagents.
OL901955Y
Org. Lett., Vol. 11, No. 21, 2009
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