Design and synthesis of an all-in-one 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl functional group for photo-affinity labeling

Article abstract of DOI:10.1016/j.bmc.2009.06.048

A novel radioisotope-free photo-affinity probe containing the 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl functional group was designed and synthesized. This very compact functionality is envisaged to allow photochemically-induced coupling of a compound

Full text of DOI:10.1016/j.bmc.2009.06.048

Bioorganic & Medicinal Chemistry 17 (2009) 5388–5395
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of an all-in-one 3-(1,1-difluoroprop-2-ynyl)-3H-
diazirin-3-yl functional group for photo-affinity labeling
*
Nag S. Kumar, Robert N. Young
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel radioisotope-free photo-affinity probe containing the 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-
yl functional group was designed and synthesized. This very compact functionality is envisaged to allow
photochemically-induced coupling of a compound to its target followed by click reaction coupling with
an azido-biotin reagent in order to facilitate purification of the labeled target. In a proof-of-concept study
we have shown that 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl functional group could be photolyzed
to efficiently furnish the methanol adduct 23 and that the generated highly unstable carbene does not
react with the neighboring acetylene moiety. A subsequent click reaction with the azido-biotin derivative
25 proceeded smoothly to give triazole 26. This chemical probe should thus be of unique value for facil-
itating identification of the molecular structure of the target of a bioactive compound.
Received 2 May 2009
Revised 19 June 2009
Accepted 22 June 2009
Available online 27 June 2009
Keywords:
Photo-affinity labeling
Click chemistry
3-(1,1-Difluoroprop-2-ynyl)-3H-diazirin-3-
yl group
Ó 2009 Elsevier Ltd. All rights reserved.
Leukotriene
5-Lipoxygenase
1. Introduction
ecules. In addition, radioactive tagging methods do not offer a
ready handle for target molecule enrichment and isolation. To ad-
Photo-affinity labeling (PAL) is an efficient and reliable tool
used to identify, isolate and characterize novel biological mole-
cules and potential drug targets, particularly when the target mol-
ecule is unknown.1–10 In PAL, a ligand incorporates a photoreactive
group in its structure which can be irradiated when it complexes
with its target. Irradiation gives rise to a highly reactive intermedi-
ate, which in turn forms a covalent bond between the ligand and
the target protein. Even though many types of photosensitive
groups have been used such as azides, diazirine, and benzophenon-
es that are suitable for PAL, aromatic diazirines have enjoyed wide-
spread application and are considered to be the most effective PAL
functional group because they generate carbenes, which react with
many functional groups including C–H bonds11,12 and they can be
activated at a long wavelength (k >300 nm), thus, preventing dam-
age or competing activation of the examined biological system.
In order to identify and facilitate the isolation of the photo-la-
beled product, a reporter group such as a radioactive label, a biotin
tag or a fluorescent tag is often incorporated into the PAL ligand.7
Radioactive labeling is often used for tracing the tagged molecules
because of detection sensitivity and because radioactive labels do
not need to employ sterically demanding tracer groups that may
perturb interactions of the biological target and the labeled re-
agent. However, the hazardous nature of the radioactive ligand
inhibits use and complicates the direct analysis of the tagged mol-
dress these disadvantages, biotin tagging has been developed
which takes advantage of the high affinity between biotin for avi-
din to allow affinity purification of the probe-target adduct. How-
ever, the large and highly polar biotin unit often has unfavorable
effects on the bioactivity of the probe.13 To address this issue, a
number of ‘fishing’ techniques have been devised wherein ligands
are made which incorporate either an alkyl azide or terminal al-
kyne instead of biotin. Once the PAL ligand–target complex is
photo-reacted, the azide or the alkyne group can be used as a tag
for the consecutive attachment of a detectable group (e.g., biotin)
using either azido-targeting or alkyne-targeting bioconjugation
reactions such as Cu(I) catalyzed 1,3-dipolar cycloaddition (click
chemistry).14–20
Recently, Hosoya and co-workers have prepared a compact ‘all-
in-one’ (3-azidodifluoromethyl-3H-diazirin-3-yl)benzene deriva-
tive 1 to be used for radioisotope-free PAL.21 Unfortunately, azi-
do-containing compounds are potentially explosive, and in our
hands a derivative (3) of compound 1 exploded when it was either
treated with palladium, Cu(I), or Cu(II) catalyst (Fig. 1).22 In a sep-
arate attempt to make the 3-azidodifluoromethyl-3H-diazirin-3-yl
group, the azido-O-tosyl-oxime 4 decomposed to give the unde-
sired nitrile 5 when it was treated with ammonia under reaction
conditions similar to those reported by Hosoya and co-workers. Gi-
ven that 3-azidodifluoromethyl-3H-diazirin-3-yl group appeared
to be unstable and more importantly, explosive in nature under
our reaction conditions, we envisioned that a compound bearing
an acetylene moiety instead of an azide should be more suitable.
* Corresponding author. Tel.: +1 778 782 3351; fax: +1 778 782 3765.
E-mail address: roberty@sfu.ca (R.N. Young).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.048

N. S. Kumar, R. N. Young / Bioorg. Med. Chem. 17 (2009) 5388–5395
5389
N N
F
N
N
N3
F
F
F
R3SiO
R
1
2
N N
CF3
N N
F
O
O
N
N
F
S
S
S
S
O
O
6
7
N N
F
Pd2dba3 / dppf
or CuI or CuO
N3
Exploded
F
I
3
OTs
N
HN NH
N3
liq. NH3
O
O
N
N
S
N
F
F
S
S
S
O
O
4
5
Figure 1. Decomposition of the azido group.
This is indeed the case and we report herein the first synthesis of
ynyl)-3H-diazirin-3-yl group to any aryl halide. The key intermedi-
ate 14, corresponding to A, was prepared from commercially avail-
able 2-(methylthio)thiazole (8) (Scheme 2). The most acidic H-5
proton was abstracted with LDA and upon treatment with tetrahy-
dropyran-4-one (9) gave alcohol 11 which was subsequently meth-
ylated to give ether 12. The methyl group on the sulfide was
removed using a mild, one-pot, three step procedure via Pummerer
rearrangement of the corresponding sulfoxide to give thiol 13. Pal-
ladium catalyzed cross-coupling35 of thiol 13 with 1,4-diiodoben-
zene (10) gave the target intermediate 14.
We next turned our attention to the coupling of compound
14 with methyl 2,2-difluoro-4-(triisopropylsilyl)-3-butynoate
15,36 corresponding to B (Scheme 3). Initially, compound 14
was treated with nBuLi followed by addition of ester 15 at
ꢀ78 °C, however, there were competing side reactions which
gave undesired byproducts 17 and 18 as judged by high-resolu-
tion mass spectrometry and TLC. The ratio of the desired product
16, increased when 14 and 15 were premixed in THF followed
by addition of nBuLi at ꢀ78 °C, yet there was still considerable
amount of byproduct 18. However, when the same reaction
was performed at ꢀ110 °C, byproduct 18 was not detected by
either MS or TLC.
the photo-affinity probe containing
a 3-(1,1-difluoroprop-2-
ynyl)-3H-diazirin-3-yl group (2).
In order to illustrate the potential use of the ‘all-in-one’ 3-(1,1-
difluoroprop-2-ynyl)-3H-diazirin-3yl group we report the synthe-
sis of a derivative (7) of a known 5-lipoxygenase (5-LO) inhibitor
623 and we also describe a sequence of carbene-trapping followed
by click chemistry using an azido-biotin reagent 25.
Leukotrienes (LTs) are lipid mediators implicated in numerous
diseases, including inflammation, atherosclerosis, and respiratory
diseases such as asthma24–32 and 5-LO plays a key role in the biosyn-
thesis of LTs from arachidonic acid. Therefore, blocking LT biosyn-
thesis could lead to the development of novel therapies for such
diseases. There are some indications of multiple protein complexes
involved in LT biosynthesis in cells33 and compounds such as 6 have
shown anomalous LT inhibitory activity in cells34 and may interact
with other undefined protein targets. Hence, an affinity ligand such
as compound 7 bearing the 3-(1,1-difluoroprop-2-ynyl)-3H-diazi-
rin-3-yl group could be used to ‘fish out’ 5-LO or any other protein
this molecule might interact with within the LT biosynthesizing
cells. The results of these studies will be published elsewhere.
To synthesize the target compound 7, ketone 16 was first re-
acted with hydroxylamine hydrochloride in the presence of pyri-
dine to give the corresponding oxime 19 (Scheme 4). The oxime
was tosylated with TsCl and upon treatment with ammonia gave
diaziridine 21. Subsequent oxidation of compound 21 using Swern
oxidation gave diazirine 22. Finally the TIPS protecting group was
removed using TBAF to give the desired ‘all-in-one’ diazirine PAL
probe 7.
2. Results and discussion
Retrosynthetic analysis indicated that target compound 7 could
be synthesized by coupling aryl halide A with ester B (Scheme 1).
The key intermediate A could, in turn, be prepared from commer-
cially available 2-(methylthio)thiazole (8), tetrahydropyran-4-one
(9), and 1,4-diiodobenzene (10). This route was chosen in order
to provide flexibility of adding the key 3-(1,1-difluoroprop-2-

5390
N. S. Kumar, R. N. Young / Bioorg. Med. Chem. 17 (2009) 5388–5395
N N
X
O
N
O
P
O
N
+
F
F
S
S
MeO
S
S
O
O
F
F
B
A
7
X = Halide
P = Protecting group
O
I
N
+
+
S
O
S
I
9
10
8
Scheme 1. Retrosynthetic analysis of compound 7.
NaH / MeI
N
S
O
O
i) LDA / THF -78°C
ii)
N
N
DMF
S
S
0°C - rt
96%
S
S
S
OH
11
O
O
O
8
9
12
57%
1) i. mCPBA / CHCl3
ii. Ca(OH)2
2) TFAA 40°C
3) i. Et3N / MeOH
ii. NH4Cl
10
Pd2dba3 / dppf
I
O
Et3N / NMP
O
N
N
80°C
37% (from
S
S
SH
S
O
O
12
)
14
13
Scheme 2. Synthesis of the key intermediate 14.
O
F
O
N
F
O
F
S
S
O
O
F
nBuLi
THF
+
14
TIPS
16
TIPS
15
O
O
N
N
S
S
S
O
O
17
18
Scheme 3. Coupling reaction of compounds 14 and 15.
A solution of compound 7 in methanol was irradiated with a
365 nm centered wavelength UV light (UVP, UVGL-25, 4 W) to
cleanly give the methanol adduct 23, presumably by insertion
of the photo-generated carbene species 24 into methanol
(Scheme 5). This adduct was characterized by a methoxy signal
(d = 3.46 ppm) and a methine hydrogen (d = 4.47 ppm) which ap-
peared as a triplet due to 2 fluorine coupling. The formation of
the methanol adduct 23 confirms that the neighboring acetylene
does not react with the highly reactive carbene species 24. Final-
ly, the photolyzed adduct 23 was treated with an azido-biotin

N. S. Kumar, R. N. Young / Bioorg. Med. Chem. 17 (2009) 5388–5395
5391
OH
F
O
N
F
NH2OH.HCl
Pyr. / EtOH
O
O
N
N
F
F
60 °C
S
S
S
S
O
O
14
70% (from
)
TIPS
TIPS
OTs
16
19
TsCl / Et3N
DMAP / DCM
0 °C
94%
N
HN NH
F
F
F
O
Liq. NH3 / Et2O
82%
N
O
N
F
S
S
S
S
O
O
TIPS
TIPS
21
20
1) (COCl)2 / DMSO / CH2Cl2
- 78 °C
2) Et3N
97%
N N
N N
F
F
TBAF
THF
O
O
N
N
F
F
S
S
S
S
O
61%
O
TIPS
7
22
Scheme 4. Synthesis of ‘all-in-one’ diazirine PAL probe 7.
N N
F
F
ν
h
(365 nm)
O
O
N
N
F
F
MeOH, rt
S
S
S
S
O
O
7
24
insertion into
MeOH
O
F
O
N
F
S
S
O
23
Scheme 5. Photoreaction of all-in-one diazirine PAL probe 7 with MeOH.
derivative 2537 in the presence of CuSO4, sodium ascorbate, and
TBTA (triazole ligand) to promote click coupling between the
azide and alkyne (Scheme 6). Consequently, the triazole 26
was isolated in pure form in moderately good yield. We also re-
peated the reaction where compound 7 was converted in one
pot to triazole 26 without isolating intermediate 23 and its
two step yield was 46%. Compounds 7, 23, and 26 are currently
being tested against the 5-LO enzyme. Due to the smaller
appendage we expect compounds 7 and 23 to be more potent
than 26.
In summary, we have prepared the first ‘all-in-one’ radioisotope
free PAL containing the 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-
yl functional group. We have shown that this photo probe could be
photolyzed in methanol with 365 nm wavelength UV light to give
the methanol adduct and that the highly unstable carbene thus
generated does not react with the neighboring acetylene moiety.
The methanol adduct reacted smoothly with the azido-biotin
derivative 25 by click chemistry. The screening of compounds 7,
23, and 26 against 5-LO and the photolabeling studies in cells with
compound 7 will be reported in due course.

5392
N. S. Kumar, R. N. Young / Bioorg. Med. Chem. 17 (2009) 5388–5395
O
CuSO4
Na Ascorbate
HN
NH
+
23
TBTA
H
N
N3
CH3OH / H2O
rt, 1 h
S
O
63%
25
N
S
O
S
O
O
F2C
O
S
N
N
H
N
N
NH
HN
O
26
Scheme 6. Click reaction of the methanol adduct 23 with the biotin azide 25.
0
0
0
0
Due to its very compact structure and selective reactivity, com-
pounds incorporating the 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-
3-yl group should be useful alternatives wherever aryl-3-(trifluo-
romethyl)-3H-diazirines are now used.
2.67 (3H, s, SCH3), 2.14 (2H, ddd, J2eq ,3ax = 4.4 Hz, J2ax ,3ax = 11.2 Hz,
J3ax ,3eq = 14.6 Hz, H-3ax0), 2.06 (1H, s, OH), 1.88 (2H, m, H-3eq0); 13C
NMR (CDCl3): d 166.3 (C-2), 147.1 (C-5), 137.3 (C-4), 68.4 (C-40), 63.6
(C-20), 39.7 (C-30), 16.6 (SCH3). HRMS calcd for (C9H13NO2S2 + H)+
232.0465, found 232.0461.
0
0
3. Experiment
3.3. 5-(4-Methoxytetrahydropyran-4-yl)-2-(methylthio)thiazole
(12)
3.1. General methods
1H and 13C NMR spectra were recorded with a Bruker TCI cryo-
probe at frequencies of 600 and 150 MHz, respectively. All assign-
ments were confirmed with the aid of two-dimensional 1H, 1H
(COSY), and 1H, 13C (HSQC) experiments. Processing of the spectra
was performed with MestRec software. The high-resolution mass
spectra were recorded in positive ion-mode with an ESI ion source
on an Agilent Time-of-Flight LC/MS mass spectrometer. Analytical
thin-layer chromatography (TLC) was performed on aluminum
plates pre-coated with Silica Gel 60F-254 as the adsorbent. The
developed plates were air-dried, exposed to UV light and/or dipped
in KMnO4 solution and heated. Column chromatography was per-
formed with Silica Gel 60 (230–400 mesh). All procedures with
diazirine functionality were carried out in flask/column covered
by aluminum foil.
NaH (0.31 g, 60% in oil, 7.75 mmol) was added to a stirred solu-
tion of compound 11 (1.11 g, 4.81 mmol) in DMF (20 mL) at 0 °C un-
der N2. After stirring for 10 min, MeI (0.36 mL, 5.78 mmol) was
added and the mixture was stirred at rt for 2 h. The reaction mixture
was partitioned between EtOAc (200 mL) and H2O (100 mL). The or-
ganic phase was washed with brine (100 mL), dried over anhydrous
Na2SO4, and concentrated. The crude product was purified by flash
chromatography (EtOAc–hexanes, 1:2) to give 12 as a white solid
(1.14 g, 96%). Mp 63–65 °C; 1H NMR CDCl3): d 7.45 (1H, s, H-4),
0
0
0
0
0
0
3.83 (2H, ddd, J2ax ,3eq = 3.6 Hz, J2ax ,3ax = 7.9 Hz, J2ax ,2eq = 11.5 Hz,
H-2ax0), 3.76 (2H, ddd, J2eq ,3ax = J2eq ,3eq = 3.9 Hz, J2ax ,2eq = 11.5 Hz,
H-2eq0), 3.10 (3H, s, OCH3), 2.71 (3H, s, SCH3), 2.11–2.02 (4H, m, H-
3ax0, H-3eq0); 13C NMR (CDCl3): d 167.4 (C-2), 143.5 (C-5), 139.8
(C-4), 72.9 (C-40), 63.7 (C-20), 50.1 (OCH3), 36.7 (C-30), 16.8 (SCH3).
HRMS calcd for (C10H15NO2S2 + H)+ 246.0622, found 246.0617.
0
0
0
0
0
0
3.2. 5-(4-Hydroxytetrahydropyran-4-yl)-2-(methylthio)thiazole
(11)
3.4. 2-(4-Iodophenylthio)-5-(4-methoxytetrahydropyran-4-
yl)thiazole (14)
A solution of 2-(methylthio)thiazole 8 (5.0 g, 38.1 mmol) in THF
(15 mL) was added to a stirred solution of freshly prepared LDA
(nBuLi (22.0 mL, 2.5 M in hexane, 55.0 mmol) and diisopropylamine
(7.8 mL, 55.3 mmol) in THF (10 mL)) at ꢀ78 °C under N2. After stir-
ring for 20 min, tetrahydropyran-4-one 9 (5 mL, 54.1 mmol) was
added and the mixture was stirred at ꢀ78 °C for 2 h. The reaction
mixture was partitioned between EtOAc (300 mL) and H2O
(100 mL). The organic phase was washed with brine (100 mL), dried
over anhydrous Na2SO4, and concentrated. The crude product was
recrystallized with Et2O and hexanes to give 11 as a white solid
(5.1 g, 57%). Mp 114–115 °C; 1H NMR (CDCl3): d 7.46 (1H, s, H-4),
To a stirred solution of 12 (2.00 g, 8.16 mmol) in CHCl3 (60 mL) at
0 °C was added mCPBA (1.86 g, 77%, 8.27 mmol) and the mixture
was stirred for 90 min. Ca(OH)2 (1.00 g, 13.48 mmol) was added
and the mixture was stirred at rt for 20 min, filtered, and concen-
trated to give essentially pure sulfoxide. The residue was dissolved
in TFAA (25 mL) and refluxed for 30 min and concentrated. The mix-
ture was diluted with MeOH–Et3N (1:1, 50 mL) and evaporated to
dryness. The residue was dissolved in CHCl3 (300 mL) and washed
with satd NH4Cl (100 mL), dried over anhydrous Na2SO4, filtered,
and concentrated to provide thiol 13. In a separate flask, 1,4-diiodo-
benzene (10) (4.00 g, 12.12 mmol), Pd2dba3 (300 mg, 0.33 mmol)
and dppf (740 mg, 1.34 mmol) was dissolved in NMP (40 mL) fol-
3.89 (2H, ddd, J2ax ,3eq = 2.2 Hz, J2ax ,3ax = J2ax ,2eq = 11.3 Hz, H-2ax0),
0
0
0
0
0
0
3.80 (2H, ddd, J2eq ,3ax = J2eq ,3eq = 4.0 Hz, J2ax ,2eq = 11.3 Hz, H-2eq0),
0
0
0
0
0
0

N. S. Kumar, R. N. Young / Bioorg. Med. Chem. 17 (2009) 5388–5395
5393
lowed by the addition of Et3N (2.0 mL, 16.15 mmol). The mixture
was purged with N2 for 15 min and then a solution of thiol 13
(ꢁ8.16 mmol) in NMP (10 mL) was added. The reaction mixture
was heated at 80 °C for 24 h and then partitioned between EtOAc
(300 mL) and brine (100 mL). The organic phase was washed with
brine (100 mL), dried over anhydrous Na2SO4, filtered, and concen-
trated. The crude product was purified by flash chromatography
(EtOAc–hexanes, 1:5?2:5) to give 14 as a white solid (1.32 g,
rable isomers. According to 1H NMR and 13C NMR spectra, the puri-
fied 20 contains a small amount of byproduct 17.
Major isomer: 1H NMR (CDCl3): d 7.86 (2H, d, J2
= 8.3 Hz, H-
000,3000
2000), 7.59 (2H, d, J2 ,3 = 8.5 Hz, H-200), 7.57 (1H, s, H-4), 7.37 (2H, d,
00 00
J2 ,3 = 8.5 Hz, H-300), 7.34 (2H, d, J2
= 8.1 Hz, H-3000), 3.82–3.71
00 00
000,3000
(4H, m, H-20), 3.09 (3H, s, OCH3), 2.47 (3H, s, CH3), 2.08–1.97 (4H,
m, H-30), 1.07–1.03 (3H, m, SiCH), 1.04 (18H, d, JCH;CH ¼ 5:4 Hz,
3
SiCHCH3); 13C NMR (CDCl3):
d 161.8 (C-2), 158.1 (1C, t,
37%). Mp 93–94 °C; 1H NMR (CDCl3): d 7.74 (2H, d, J2 ,3 = 8.5 Hz,
JC,F = 30.7 Hz, C-500), 147.4 (C-5), 145.7 (C-1000), 140.8 (C-4), 134.1
(C-1”), 131.9 (C-4000), 131.2 (C-200), 130.1 (C-400), 129.8 (C-300),
129.7 (C-3000), 129.1 (C-2000), 107.8 (1C, t, JC,F = 236.9 Hz, C-600),
96.0 (1C, t, JC,F = 4.6 Hz, C-800), 95.2 (1C, t, JC,F = 36.5 Hz, C-700),
72.8 (C-40), 63.4 (C-20), 50.0 (OCH3), 36.4 (C-30), 21.8 (CH3), 18.4
(SiCH), 10.8 (SiCHCH3). HRMS calcd for (C35H44F2N2O5S3Si + H)+
735.2227, found 735.2239.
00 00
H-200), 7.48 (1H, s, H-4), 7.33 (2H, d, J2 ,3 = 8.5 Hz, H-300), 3.78 (2H,
00 00
ddd, J2ax ,3eq = 3.4 Hz, J2ax ,3ax = 9.8 Hz, J2ax ,2eq = 11.5 Hz, H-2ax0),
0
0
0
0
0
0
3.72 (2H, ddd, J2eq ,3ax = J2eq ,3eq = 4.1, J2ax ,2eq = 11.5 Hz, H-2eq0),
3.05 (3H, s, OCH3), 2.03–1.96 (4H, m, H-30); 13C NMR (CDCl3): d
165.2 (C-2), 145.7 (C-5), 140.4 (C-4), 138.9 (C-200), 135.2 (C-300),
131.3 (C-100), 95.9 (C-400), 72.7 (C-40), 63.3 (C-20), 49.9 (OCH3), 36.4
(C-30). HRMS calcd for (C15H16INO2S2 + H)+ 433.9745, found
433.9740.
0
0
0
0
0
0
3.7. 2-(4-(3-(1,1-Difluoro-3-triisopropylsilyl-prop-2-ynyl)diaz-
iridin-3-yl)phenylthio)-5-(4-methoxytetrahydropyran-4-yl)-
thiazole (21)
3.5. 2-(4-(2,2-Difluoro-1-hydroxyimino-4-triisopropylsilyl-but-
3-ynyl)phenylthio)-5-(4-methoxytetrahydropyran-4-yl)thiazole
(19)
Liquid ammonia (ꢁ2 mL) was condensed into a stirred solution
of 20 (310 mg, 0.42 mmol) in Et2O (5 mL) at ꢀ78 °C under N2 in a
pressure tube. The pressure tube was sealed and the mixture was
stirred at rt for 16 h. After removing excess ammonia, the mixture
was diluted with Et2O (100 mL) and washed with H2O (50 mL),
brine (50 mL), dried over anhydrous Na2SO4, filtered, and concen-
trated. The crude product was purified by flash chromatography
(EtOAc–hexanes, 1:2) to give 21 as colorless syrup (200 mg, 82%).
nBuLi (0.41 mL, 2.5 M in hexanes, 1.03 mmol) was added to a
stirred solution of 14 (300 mg, 0.69 mmol) and methyl 2,2-di-
fluoro-4-(triisopropylsilyl)-3-butynoate (15) (300 mg, 1.03 mmol)
in THF (20 mL) at ꢀ110 °C under Ar. The mixture was stirred at
ꢀ110 °C?ꢀ78 °C for 30 min and then the reaction was quenched
with 1 M aqueous HCl (20 mL). The reaction mixture was extracted
with Et2O (2 ꢂ 100 mL) and the combined organic layer was
washed with H2O (50 mL), brine (50 mL), dried over anhydrous
Na2SO4, filtered, and concentrated. The residue was dissolved in
pyridine–EtOH (2:1, 15 mL), followed by addition of hydroxyl-
amine hydrochloride (100 mg, 1.46 mmol). The mixture was stir-
red at 60 °C for 4 h and then partitioned between Et2O (100 mL)
and 1.0 M aqueous HCl (50 mL). The organic phase was washed
with H2O (50 mL), brine (50 mL), dried over anhydrous Na2SO4, fil-
tered, and concentrated. The crude product was purified by flash
chromatography (EtOAc–hexanes, 1:3?1:2) to give 19 as colorless
syrup (280 mg, 70% for the two steps) as 1:5 mixture of E- and Z-
isomers. According to 1H NMR and 13C NMR spectra, the purified
19 contains a small amount of byproduct 17.
1H NMR (CDCl3): d 7.68 (2H, d, J2 ,3 = 8.3 Hz, H-300), 7.61 (2H, d,
00 00
J2 ,3 = 8.4 Hz, H-200), 7.51 (1H, s, H-4), 3.78 (2H, ddd,
J
00 00
2ax0,3eq0 = 3.4 Hz, J2ax ,3ax = 9.9 Hz, J2ax ,2eq = 11.5 Hz, H-2ax0), 3.73
0
0
0
0
(2H, ddd, J2eq ,3ax = J2eq ,3eq = 4.1, J2ax ,2eq = 11.5 Hz, H-2eq0), 3.05
(3H, s, OCH3), 2.89 (1H, d, JNHa,NHb = 8.6 Hz, NH), 2.23 (1H, d,
JNHa,NHb = 8.7 Hz, NH), 2.04–1.97 (4H, m, H-30), 1.10–1.05 (3H, m,
0
0
0
0
0
0
SiCH), 1.06 (18H, d, JCH;CH ¼ 5:4 Hz, SiCHCH3); 13C NMR (CDCl3):
3
d 164.5 (C-2), 146.1 (C-5), 140.4 (C-4), 134.4 (C-100), 133.7 (C-400),
132.9 (C-200), 130.1 (C-300), 112.1 (1C, t, JC,F = 237.1 Hz, C-600), 96.6
(1C, t, JC,F = 38.1 Hz, C-700), 94.6 (1C, t, JC,F = 4.9 Hz, C-800), 72.7
(C-40), 63.3 (C-20), 60.2 (1C, t, JC,F = 32.3 Hz, C-500), 49.9 (OCH3),
36.4 (C-30) 18.4 (SiCH), 10.8 (SiCHCH3). HRMS calcd for
(C28H39F2N3O2S2Si + H)+ 580.2299, found 580.2291.
Majorisomer: 1H NMR (CDCl3): d 9.35 (1H, br s, NOH), 7.63 (2H, d,
00 00
J2 ,3 = 8.5 Hz, H-300), 7.55 (1H, s, H-4), 7.52 (2H, d, J2
= 8.4 Hz, H-
00,300
200), 3.85–3.71 (4H, m, H-20), 3.07 (3H, s, OCH3), 2.05–1.97 (4H, m,
3.8. 2-(4-(3-(1,1-Difluoro-3-triisopropylsilyl-prop-2-ynyl)diazirin-
3-yl)phenylthio)-5-(4-methoxytetrahydropyran-4-yl)thiazole (22)
H-30), 1.10–1.02 (3H, m, SiCH), 1.03 (18H, d, JCH;CH ¼ 5:0 Hz,
3
SiCHCH3); 13C NMR (CDCl3): d 164.1 (C-2), 151.2 (1C, t, JC,F = 30.2 Hz,
C-500), 146.4 (C-5), 140.4 (C-4), 134.0 (C-400), 132.5 (C-300), 130.2 (C-
200), 128.4 (C-100), 109.1 (1C, t, JC,F = 233.1 Hz, C-600), 96.5 (1C, t,
JC,F = 37.4 Hz, C-700), 94.4 (1C, t, JC,F = 4.6 Hz, C-800), 72.8 (C-40), 63.4
(C-20), 50.0 (OCH3), 36.3 (C-30) 18.4 (SiCH), 10.9 (SiCHCH3). HRMS
calcd for (C28H38F2N2O3S2Si + H)+ 581.2139, found 581.2132.
Oxalyl chloride (60
tion of dry DMSO (60
lL, 0.69 mmol) was added to a stirred solu-
L, 0.84 mmol) in CH2Cl2 (15 mL) at ꢀ78 °C un-
l
der N2. After 5 min, a solution of diaziridine 21 (300 mg, 0.52 mmol)
in CH2Cl2 (3 mL) was added and the mixture was stirred at ꢀ78 °C for
15 min. Et3N (0.36 lL, 2.65 mmol) was added and the mixture was
allowed to warm to rt over 90 min. The reaction was quenched with
H2O (5 mL) and the organic layer was dried over anhydrous Na2SO4,
filtered, and concentrated. The crude product was purified by flash
chromatography (EtOAc–hexanes, 1:3) to give 22 as colorless syrup
3.6. 2-(4-(2,2-Difluoro-1-(4-methylphenylsulfonyloxy)imino-4-
triisopropylsilyl-but-3-ynyl)phenylthio)-5-(4-methoxytetra-
hydropyran-4-yl)thiazole (20)
(290 mg, 97%). 1H NMR (CDCl3): d 7.57 (2H, d, J2 ,3 = 8.4 Hz, H-200),
00 00
7.50 (1H, s, H-4), 7.30 (2H, d, J2 ,3 = 8.4 Hz, H-300), 3.78 (2H, ddd, J
00 00
p-Toluenesulfonyl chloride (110 mg, 0.58 mmol), 4-(dimethyl-
amino)pyridine (4 mg, 0.03 mmol) and Et3N (80 lL, 0.57 mmol)
2ax0,3eq0 = 3.3 Hz, J2ax ,3ax = 9.9 Hz, J2ax ,2eq = 11.5 Hz, H-2ax0), 3.73
0
0
0
0
(2H, ddd, J2eq ,3ax = J2eq ,3eq = 4.0, J2ax ,2eq = 11.5 Hz, H-2eq0), 3.05
(3H, s, OCH3), 2.04–1.96 (4H, m, H-30), 1.09–1.02 (3H, m, SiCH),
0
0
0
0
0
0
were added to a stirred solution of 19 in CH2Cl2 (8 mL) at 0 °C un-
der N2. After stirring for 90 min at 0 °C, the mixture was poured
into H2O (20 mL) and extracted with CH2Cl2 (2 ꢂ 50 mL). The com-
bined organic extracts were washed with H2O (50 mL), dried over
anhydrous Na2SO4, filtered, and concentrated. The crude product
was purified by flash chromatography (EtOAc–hexanes, 1:2) to
give 20 as colorless syrup (320 mg, 94%) as 1:4 mixture of insepa-
1.03 (18H, d, JCH;CH ¼ 5:4 Hz, SiCHCH3); 13C NMR (CDCl3): d 164.4
3
(C-2), 146.2 (C-5), 140.4 (C-4), 133.2 (C-100), 132.9 (C-200), 132.3 (C-
400), 128.2 (C-300), 110.2 (1C, t, JC,F = 235.5 Hz, C-600), 97.5 (1C, t,
JC,F = 5.0 Hz, C-800), 95.6 (1C, t, JC,F = 38.5 Hz, C-700), 72.7 (C-40), 63.3
(C-20), 49.9 (OCH3), 36.4 (C-30), 31.4 (1C, t, JC,F = 36.2 Hz, C-500), 18.3

5394
N. S. Kumar, R. N. Young / Bioorg. Med. Chem. 17 (2009) 5388–5395
(SiCH), 10.7 (SiCHCH3). HRMS calcd for (C28H37F2N3O2S2Si + H)+
578.2268, found 578.2276.
m, 2 ꢂ H-3000, H-5000), 1.33–1.23 (2H, m, H-4000); 13C NMR (DMSO):
d 172.5 (CONH), 163.4 (C-2), 162.6 ((HN)2CO), 145.4 (C-5), 141.2
(1C, dd, JC,Fa = JC,Fb = 26.8 Hz, triazole-C-4), 135.5 (C-100), 132.6 (C-
300), 131.4 (C-400), 130.2 (C-200), 124.9 (triazole-C-5), 116.9 (1C, dd,
JC,Fa = 39.8 Hz, JC,Fb = 44.9 Hz, C-600), 82.0 (1C, dd, JC,Fa = 24.6 Hz,
JC,Fb = 31.9 Hz, C-500), 72.4 (C-40), 62.6 (C-20), 60.9 (SCH2CH), 59.1
(SCHCH), 57.5 (OCH3), 55.3 (SCH), 49.3 (OCH3), 49.1 (CH2triazole),
39.8 (SCH2), 38.5 (CH2CH2triazole), 35.8 (C-30), 35.0 (NHCOCH2),
28.0 (C-4000), 27.9 (C-5000), 25.0 (C-3000). HRMS calcd for
(C32H41F2N7O5S3 + H)+ 738.2377, found 738.2395.
3.9. 2-(4-(3-(1,1-Difluoroprop-2-ynyl)diazirin-3-yl)phenylthio)-
5-(4-methoxytetrahydropyran-4-yl)thiazole (7)
TBAF (0.7 mL, 1 M in THF, 0.7 mmol) was added to a stirred solu-
tion of 22 (260 mg, 0.45 mmol) in THF (15 mL) at rt under N2. After
stirring for 40 min, the mixture was concentrated and the residue
was purified by flash chromatography (EtOAc–hexanes, 1:2) to give
7 as pale yellow syrup (115 mg, 61%). 1H NMR (CDCl3): d 7.58 (2H, d,
J2 ,3 = 8.6 Hz, H-200), 7.51 (1H, s, H-4), 7.28 (2H, d, J2 ,3 = 8.5 Hz,
3.12. One pot synthesis of triazole 26 from compound 7
00 00
00 00
H-300), 3.78 (2H, ddd, J2ax ,3eq = 3.4 Hz, J2ax ,3ax = 9.8 Hz, J2ax ,2eq
=
=
0
0
0
0
0
0
11.4 Hz, H-2ax0), 3.73 (2H, ddd, J2eq ,3ax = J2eq ,3eq = 4.2, J2ax ,2eq
A solution of compound 7 (5 mg, 11.8 lmol) in MeOH (1 mL)
was irradiated with 365 nm wavelength UV light (UVP, UVGL-25,
4 W) at rt for 90 min, followed by the addition of biotin azide
0
0
0
0
0
0
11.5 Hz, H-2eq0), 3.06 (3H, s, OCH3), 3.01 (1H, t, J8 ,F = 5.1 Hz, H-800),
2.04–1.97 (4H, m, H-30); 13C NMR (CDCl3): d 164.0 (C-2), 146.3 (C-
5), 140.5 (C-4), 133.6 (C-100), 132.8 (C-200), 131.6 (C-400), 128.2 (C-
300), 110.3 (1C, t, JC,F = 236.4 Hz, C-600), 80.6 (1C, t, JC,F = 6.4 Hz, C-
800), 73.6 (1C, t, JC,F = 40.4 Hz, C-700), 72.8 (C-40), 63.3 (C-20), 49.9
(OCH3), 36.4 (C-30), 31.1 (1C, t, JC,F = 35.4 Hz, C-500). HRMS calcd for
(C19H17F2N3O2S2 + H)+ 422.0808, found 422.0803.
00
(25) (3.5 mg, 10.7
mol), CuSO4 (100 mM aqueous solution, 10
dium ascorbate (100 mM aqueous solution, 30
l
mol), TBTA (50 mM MeOH solution, 20
L, 1 mol), and so-
L, 3 mol) at rt un-
lL,
1
l
l
l
l
l
der Ar. The mixture was stirred at rt for 1 h and concentrated. The
crude product was purified by preparative TLC (CH2Cl2–MeOH;
10:1) to give compound 26 as pale yellow syrup (4 mg, 46%).
3.10. 2-(4-(2,2-Difluoro-1-methoxy-but-3-ynyl)phenylthio)-5-
(4-methoxytetrahydropyran-4-yl)thiazole (23)
Acknowledgments
A solution of compound 7 (10 mg, 23.7
l
mol) in MeOH (1.5 mL)
We thank the British Columbia Government Leading Edge
Endowment Fund, Merck Frosst and Simon Fraser University for
financial support.
was irradiated with 365 nm wavelength UV light (UVP, UVGL-25,
4 W) at rt while monitoring by TLC. The reaction proceeded
smoothly and all 7 was consumed by 90 min. The reaction mixture
was concentrated and the residue was purified by flash chromatog-
raphy (hexanes–EtOAc, 2:1) to give compound 23 as pale yellow
Supplementary data
syrup (7 mg, 69%). 1H NMR (CDCl3): d 7.62 (2H, d, J2
= 8.3 Hz,
00,300
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.06.048.
H-200), 7.51 (1H, s, H-4), 7.49 (2H, d, J2 ,3 = 8.2 Hz, H-300), 4.47
00 00
(1H, t, J5 ,F = 8.6 Hz, H-500), 3.78 (2H, ddd, J2ax ,3eq = 3.3 Hz, J2ax ,3ax
=
=
00
0
0
0
0
10.0 Hz, J2ax ,2eq = 11.4 Hz, H-2ax0), 3.73 (2H, ddd, J2eq ,3ax
0
0
0
0
References and notes
J2eq ,3eq = 4.1, J2ax ,2eq = 11.4 Hz, H-2eq0), 3.46 (3H, s, OCH3), 3.06
0
0
0
0
(3H, s, OCH3), 2.81 (1H, t, J8 ,F = 4.2 Hz, H-800), 2.05-1.97 (4H, m,
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H-30); 13C NMR (CDCl3): d 165.1 (C-2), 145.8 (C-5), 140.5 (C-4),
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yl)thiazole (26)
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To a stirred solution of 23 (5 mg, 11.7
was added biotin azide (25) (3.5 mg, 10.7
MeOH solution, 20 L, 1 mol), CuSO4 (100 mM aqueous solution,
mol), and sodium ascorbate (100 mM aqueous solution,
mol) at rt under Ar. The mixture was stirred at rt for
1 h and concentrated. The crude product was purified by prepara-
tive TLC (CH2Cl2–MeOH ; 10:1) to give compound 26 as pale yellow
syrup (5 mg, 63%). 1H NMR (DMSO): d 8.40 (1H, s, triazole), 7.98
l
mol) in MeOH (0.5 mL)
lmol), TBTA (50 mM
l
l
10
30
l
l
L, 1
L, 3
l
l
(1H, t, JNH;CH ¼ 5:7 Hz, CONH), 7.73 (1H, s, H-4), 7.64 (2H, d,
2
J2 ,3 = 8.3 Hz, H-300), 7.47 (2H, d, J2 ,3 = 8.2 Hz, H-200), 6.41 (1H, s,
00 00
00 00
00
00
NH), 6.35 (1H, s, NH), 5.09 (1H, dd, J5 ,Fa = 7.1 Hz, J5 ,Fb = 16.1 Hz,
H-500), 4.46 (2H, t, JCH
¼ 6:0 Hz, CH2triazole), 4.29 (1H, m,
2;CH2
SCHCH), 4.11 (1H, m, SCH2CH), 3.61–3.58 (4H, m, H-20), 3.51 (2H,
dt, JCH
¼ JNH;CH ¼ 6:0 Hz, CH2CH2triazole), 3.24 (3H, s, OCH3),
2;CH2
2
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(1H, d, Ja,b = 12.4 Hz, SCH2), 2.02 (2H, t, J
¼ 7:0 Hz, NHCOCH2),
CH2;CH2
1.97–1.90 (4H, m, H-30), 1.62–1.56 (1H, m, H-5000), 1.49–1.41 (3H,
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