Design and synthesis of a tag-free chemical probe for photoaffinity labeling

Article abstract of DOI:10.1002/ejoc.200700188

The novel aromatic diazirine-containing benzoic acid 22 was prepared via the diazirine 11 as the key intermediate. After formylation of the aryl ring and cleavage of the methyl ether function of aldehyde 12, the phenolic hydroxy group was converted into the ether 21 terminating in an alkyne function. Oxidation of the aldehyde to the carboxylic acid provided the chemical probe 22 designed for tag-free photoaffinity labeling. In a proof-of-concept study it could be shown that irradiation of the simple ester 23 indeed yields the methanol insertion product 24. A subsequent click reaction with benzyl azide 20 led to the triazole 25. A more complicated example was realized with the esterification of bafilomycin A₁ (27) with acid 22. Wiley-VCH Verlag GmbH & Co. KGaA, 2007.

Full text of DOI:10.1002/ejoc.200700188

FULL PAPER
DOI: 10.1002/ejoc.200700188
Design and Synthesis of a Tag-Free Chemical Probe for Photoaffinity Labeling
Timo Mayer[a] and Martin E. Maier*[a]
Keywords: Photoaffinity labeling / Click chemistry / Triazoles / 1,3-Dipoles / Diazirines / Nitrogen heterocycles
The novel aromatic diazirine-containing benzoic acid 22 was
prepared via the diazirine 11 as the key intermediate. After
formylation of the aryl ring and cleavage of the methyl ether
function of aldehyde 12, the phenolic hydroxy group was
converted into the ether 21 terminating in an alkyne func-
tion. Oxidation of the aldehyde to the carboxylic acid pro-
vided the chemical probe 22 designed for tag-free photoaf-
finity labeling. In a proof-of-concept study it could be shown
that irradiation of the simple ester 23 indeed yields the meth-
anol insertion product 24. A subsequent click reaction with
benzyl azide 20 led to the triazole 25. A more complicated
example was realized with the esterification of bafilomycin
A1 (27) with acid 22.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
gate substantially less active than the parent ligand. In or-
der to circumvent these shortcomings compact bifunctional
probes carrying a small functional group for selective modi-
fication after photoaffinity labeling (post-PAL modifica-
tion, PPALM) have recently been described (cf. 3, 4, and
5). These are formed by catalyzed azide–alkyne [3+2] cyclo-
addition[13–16] or Staudinger–Bertozzi ligation reac-
tions.[17,18] In fact, Suzuki and co-workers showed that the
azidoalkyl group of an ether derivative of diazirine 3 largely
survives the conditions used for photoactivation of the 3-
(trifluoromethyl)-3H-diazirin-3-yl group.[19] They also
showed that the sequence of photoreaction followed by cy-
cloaddition with benzyl azide worked in vitro on a deriva-
tive of 4.[20]
Introduction
A timeless challenge in biology and drug discovery in-
volves finding a matching pair between one entity and a
large number of possible partners. Thus, libraries of small
molecules have been screened for binding to or inhibition
of an enzyme.[1,2] Also the target of an active compound,
frequently a natural product, needs to be found. Depending
on the nature of the ligand, activity-based profiling[3] or
affinity chromatography can be used. However, in some
cases like low-abundance proteins, recourse to photoaffinity
labeling (PAL) has to be made.[4] Besides target fishing,
PAL can be used to elucidate the structural and functional
properties of biological systems. In PAL a ligand is con-
nected to a photoreactive group. Irradiation of the bound
ligand–label conjugate generates a reactive intermediate
that hooks onto the protein of interest by forming a cova-
lent bond. Among various photophores, aryl(trifluorometh-
yl)diazirines[5] have found widespread applications. They
are readily photolyzed to reactive carbenes at around
360 nm.[6] Furthermore, they can be kept small enough that
the biological activity of the modified ligand does not suffer
too much. In order to identify the PAL products after bind-
ing and photolysis, a reporter group should be contained
within the photophore or somewhere else in the ligand.[7]
Historically, radioisotopes have been employed in this con-
text (cf. compounds 1,[8,9] Figure 1). As an alternative to
radioisotopes, photoaffinity biotinylation can be used (cf.
compound 2,[10–12] Figure 1). However, the polar and big
biotin-anchored tag often renders the ligand-affinity conju-
Figure 1. Some important examples of photoaffinity probes based
on 3-(trifluoromethyl)-3H-diazirines and azides.
[a] Institut für Organische Chemie, Universität Tübingen,
Auf der Morgenstelle 18, 72076 Tübingen, Germany
Fax: +49-7071-295137
In connection with the task of localizing the binding
partner of a natural product a tag-free 4-[3-(trifluorometh-
yl)-3H-diazirinyl]benzoic acid was sought. The idea was to
E-mail: martin.e.maier@uni-tuebingen.de
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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T. Mayer, M. E. Maier
FULL PAPER
shorten the side-chain of benzoic acid 2 and terminate it
with an azide or alkyne. This should allow selective fishing
of a labeled protein by click chemistry. In this paper we
detail the synthesis of the bifunctional benzoic acid 22 and
illustrate the sequence of carbene trapping followed by click
chemistry on a simple model system.
Results and Discussion
Starting from 3-bromoanisole (6) the key intermediate 3-
(3-methoxyphenyl)diazirine 11 was prepared using the clas-
sical sequence[21] for introduction of the 3-(trifluoromethyl)-
diazirin-3-yl group (Scheme 1). Compared with the litera-
ture methods some steps were slightly modified. Thus, met-
alation of bromide 6 with n-butyllithium followed by acyl-
ation of the aryllithium intermediate with methyl trifluo-
roacetate gave ketone 7 in excellent yield. Ketone 7 was con-
verted into the oxime 8 using hydroxylamine hydrochloride
in the presence of pyridine. Treatment of the oxime 8 (E/Z
mixture) with tosyl chloride led to the p-tolylsulfonyloxime
9. All these steps proceeded in almost quantitative yields.
Formation of the diaziridine 10 using ammonia followed by
oxidation of 10 with silver oxide[21a,21b] furnished diazirine
11. The most convenient way to introduce the formyl group
involves Friedel–Crafts alkylation of anisole 11 with dichlo-
romethyl methyl ether in the presence of TiCl4 followed by
aqueous work-up.[22] These conditions gave the desired al-
dehyde 12 (41%) along with the regioisomer 13 (9%). The
two aldehydes could be separated by flash chromatography.
In order to prepare for the attachment of the side-chain,
the aryl methyl ether was cleaved using boron tribromide in
dichloromethane which led to hydroxybenzaldehyde 14.
It was then planned to form an ether bond between phen-
ol 14 and a suitable alkynol derivative. As shown in
Scheme 2 the tosylate 16 was prepared in two steps by
monoalkylation of ethanediol with propargyl bromide[23]
followed by tosylation of the alkynol 15. In order to demon-
strate a click reaction of the alkyne functionality, we chose
the simple benzyl azide 20. This compound was easily ac-
cessible by routine steps from 3-methoxybenzaldehyde (17).
Thus, reduction of the aldehyde to the benzyl alcohol 18
was followed by bromination using PBr3 leading to benzyl
bromide 19. A final nucleophilic substitution of the bro-
mide with sodium azide in DMSO provided the azide 20.
The methoxy group would later facilitate the identification
of the click product by NMR spectroscopy.
Scheme 1. Synthesis of the 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]
benzaldehyde 14.
Alkylation of the 2-hydroxybenzaldehyde 14 with the tos-
ylate 16 was performed at a slightly elevated temperature in
the presence of potassium carbonate and catalytic amounts
of tetrabutylammonium iodide in DMF (Scheme 3). These
conditions gave the ether 21 in 75% yield. The etherifi-
cation was also possible under Mitsunobu conditions.[24]
However, the yield was lower and separation of the byprod-
Scheme 2. Preparation of the alkyne 16 and benzyl azide 20.
ucts turned out to be problematic. The final oxidation of of sodium chlorite with sulfamic acid to be suitable.[25] This
the aldehyde 21 to the acid 22 was rather problematic. Typi- way the benzoic acid 22 could be obtained in 94% yield.
cal reagents for related oxidation reactions such as perman- In large-scale reactions, simple extraction of the reaction
ganate did not work.[21] Finally, we found the combination mixture followed by concentration of the organic layer pro-
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A Tag-Free Chemical Probe for Photoaffinity Labeling
vided the pure acid 22 as an amorphous solid without any
further purification. As a model the acid 22 was esterified
with 2-propanol using the condensing agent 1-ethyl-3-(di-
methylaminopropyl)carbodiimide (EDC) providing ester 23
in reasonable yield.
Scheme 3. Synthesis of the photoaffinity probe 22 by alkylation of
phenol 14 and oxidation of the benzaldehyde 21.
Scheme 4. Photoreaction of the alkynyl-functionalized phenyl(tri-
fluoromethyl)diazirine 23 in methanol followed by a click reaction
with benzyl azide 20.
By irradiating a solution of diazirine 23 in methanol with
a UV lamp (8 W, 366 nm), the corresponding carbene was
generated, as evidenced by the isolation of the methanol
adduct 24 (Scheme 4). This adduct is characterized by the
methoxy signal (δ = 3.41 ppm) and the methine hydrogen
(δ = 4.48 ppm). Owing to the CF3 group the latter appears
as a quartet (2J = 6.6 Hz). Although the yield of adduct 24
was moderate, its formation shows that the side-chain does
not react with the intermediate carbene. According to LC-
MS analysis, irradiation on a larger scale produces the re-
duced derivative (2,2,2-trifluoroethyl substituent) as a side-
product. In a subsequent click reaction between alkyne 24
and the azide 20 the triazole 25 was formed. Remarkably,
the order of events could also be reversed. Thus, the diazir-
ine function seems to be compatible with the conditions of
the click reaction, as indicated by the successful formation
of triazole 26. This shows that the alkyne function might
be used as a handle for conjugation with other small groups
prior to irradiation. Subjection of diazirine 26 to irradiation
in methanol gave compound 25 as well.
cordingly, reaction of bafilomycin A1 (27) with acid 22
(3 equiv.) in the presence of the condensing agent EDC and
DMAP provided the ester 28 in good yield (Scheme 5).
Even though an excess of acid 22 was used, besides ester 28
some unreacted 27 was recovered. In the NMR spectrum
the signal of the methine 21-H resonates at δ = 5.11–
5.20 ppm as opposed to 3.53 ppm in 27. The carbon atom
C-21 appears at δ = 75.6 ppm ([D6]acetone) as opposed to
70.4 ppm.
In order to illustrate the potential use of acid 22 we
treated it with the natural product bafilomycin A1 (27). This
macrolactone, isolated from Streptomyces griseus, is a well-
known inhibitor of V-ATPase.[26,27] In this regard it is
closely related to concanamycin A.[28] As has been de-
scribed before, the hydroxy group at C-21 can be selectively
acylated, alkylated, and oxidized without affecting the V-
ATPase activity too much.[29] On the other hand, modifica-
Scheme 5. Esterification of bafilomycin A1 (27) at 21-OH with acid
tions to 7-OH cause a significant reduction in activity. Ac- 22 yielding ester 28.
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T. Mayer, M. E. Maier
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We also demonstrated a click reaction with the ester 28. bafilomycin A1 derivatives 28 and 33 are currently being
It seemed that the azido-biotin derivative 32 would be a tested for inhibition of V-ATPase. Owing to the smaller ap-
suitable reagent (Scheme 6).[30] In order to prepare this rea- pendage one would expect a higher activity for 28 over 33.
gent, 2-bromoethylamine was converted into azidoethyl-
amine (29) by treating it with sodium azide.[31] In a separate
operation biotin (30) was activated as its N-hydroxysuc- Conclusions
cinimide derivative 31[32] in a condensation reaction per-
In this paper we have described the synthesis of the novel
formed in the presence of the reagent EDC. Thereafter,
bifunctional probe 22 featuring the 3-(trifluoromethyl)-3H-
treatment of N-hydroxysuccinimidobiotin (31) with azido-
diazirin-3-yl group as well as an alkynyl side-chain. In a
ethylamine (29) in DMF in the presence of triethylamine
model study we have shown that the carbene insertion prod-
furnished the azide[30] 32 in good yield. When the azido-
uct can be evidenced by a CuI-catalyzed Huisgen alkyne–
biotin derivative 32 was treated with the bafilomycin A1
derivative 28 under typical click conditions (copper sulfate,
azide 1,3-dipolar cycloaddition reaction. Esterification of
bafilomycin A1 (27) with the acid 22 gave the ester 28. In a
sodium ascorbate as reducing agent) in a mixture of tBuOH
click reaction with the biotinyl azide 32 the triazole 33 was
and H2O the ligation product 33 could be isolated in a
obtained. With the availability of biotinyl azides such as 32
moderate yield as a colorless amorphous solid. The two
or fluorescence dyes containing an azide group,[13] applica-
tions of 22 in chemical biology present itself. Studies along
these lines are currently being pursued.
Experimental Section
General: 1H and 13C NMR: Bruker Avance 400 spectrometer,
Bruker AMX 600 spectrometer; spectra were recorded at 295 K in
CDCl3, [D6]acetone, or [D6]DMSO; chemical shifts are calibrated
to the residual proton and carbon resonances of the solvent: CDCl3
(δH = 7.25, δC = 77.0 ppm), [D6]acetone (δH = 2.04, δC = 29.8 ppm),
[D6]DMSO (δH = 2.49, δC = 39.5 ppm). Melting points: Büchi
Melting Point B-540, uncorrected. HRMS (FT-ICR): Bruker Dal-
tonic APEX 2 with electron-spray ionization (ESI). The minimal
resolution of this machine is 1 ppm (∆m/mϫ106). Flash
chromatography: J. T. Baker silica gel, 43–60 µm. Thin-layer
chromatography: Macherey–Nagel Polygram Sil G/UV254 plates.
Analytical LC-MS: HP 1100 Series connected with an ESI-MS Ag-
ilent G1946C detector, positive mode with a fragmentor voltage
of 40 eV; column: Nucleosil 100–5, C-18 HD, 5 mm, 70ϫ3 mm
Macherey–Nagel; eluent: NaCl solution (5 m)/acetonitrile; gradi-
ent: 0–10–15–17–20 min with 20–80–80–99–99% acetonitrile; flow:
0.5 mLmin–1. All solvents used in the reactions were distilled be-
fore use. Dry tetrahydrofuran was distilled from sodium and benzo-
phenone, whereas dry CH2Cl2, dimethylformamide, pyridine, and
DMSO were distilled from CaH2. Petroleum ether with a boiling
range of 40–60 °C was used. Reactions were generally run under
argon. All commercially available compounds were used as received
unless stated otherwise. Reactions with diazirine-containing com-
pounds were protected from light. Bafilomycin A1 was a gift from
Dr. Carlo Farina, Brane Discovery Srl, via Zambeletti 25, 20021
Baranzate (MI), Italy.
2,2,2-Trifluoro-1-(3-methoxyphenyl)ethanone
(7):[21b,33]
nBuLi
(2.5  in hexane, 28 mL, 1.5 equiv.) was slowly added to a solution
of 1-bromo-3-methoxybenzene (8.9 g, 6.0 mL, 47.6 mmol) in dry
THF (100 mL) at –78 °C. The reaction mixture was stirred at this
temperature for 2 h before a solution of methyl trifluoroacetate
(8.1 g, 6.3 mL, 63.3 mmol, 1.3 equiv.) in dry THF (40 mL) was
added over a period of 30 min. After the mixture had been stirred
for an additional 2 h, a solution of conc. HCl (10.5 mL) in meth-
anol (25 mL) was added. The resulting solution was warmed up to
0 °C, diluted with Et2O (60 mL), washed with water (2ϫ10 mL),
dried with MgSO4, and filtered. The solvent was evaporated in
vacuo and the crude product purified by kugelrohr distillation,
60 °C/10–1 mbar (ref.[33] 84–85 °C/12 Torr) to give the title com-
Scheme 6. Synthesis of the biotin azide 32 and its click reaction
with the alkyne 28.
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A Tag-Free Chemical Probe for Photoaffinity Labeling
pound (9.38 g, 46.0 mmol, 96%) as a colorless liquid. Rf = 0.21
(hexane/EtOAc, 5:1). H NMR (400 MHz, CDCl3): δ = 3.86 (s, 3
H, CH3), 7.24 (dd, J = 8.1, 2.0 Hz, 1 H, 4-H), 7.44 (t, J = 8.0 Hz, (CH3), 58.0 (q, JCF = 35.9 Hz, CCF3), 113.6 (C-2), 115.8 (C-4),
4-H), 7.14 (s, 1 H, 2-H), 7.19 (d, J = 7.6 Hz, 1 H, 6-H), 7.32 (t, J
1
= 8.0 Hz, 1 H, 5-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 55.3
2
1 H, 5-H), 7.55 (s, 1 H, 2-H), 7.65 (d, J = 7.6 Hz, 1 H, 6-H) ppm.
120.3 (C-6), 123.5 (q, 1JCF = 278.8 Hz, CF3), 129.9 (C-5), 133.0 (C-
1), 159.7 (C-3) ppm.
13C NMR (100 MHz, CDCl3): δ = 55.5 (CH3), 113.9 (d, J = 1.5 Hz,
1
C-2), 116.6 (q, JCF = 291.3 Hz, CF3), 122.3 (C-4), 122.7 (q, J =
3-(3-Methoxyphenyl)-3-(trifluoromethyl)-3H-diazirine (11): A solu-
tion of sodium hydroxide (2.4 g, 60.0 mmol) in water (30 mL) was
added slowly to a boiling solution of silver(I) nitrate (10.2 g,
60.0 mmol) in water (60 mL). The precipitate was filtered and
washed with water (100 mL), acetone (100 mL), and Et2O (100 mL)
to give Ag2O as a brown solid. The freshly prepared Ag2O (9.5 g,
41.0 mmol, 3.8 equiv.) was added to a solution of diaziridine 10
(2.38 g, 10.9 mmol) in dry Et2O (100 mL) and stirred at room tem-
perature for 3.5 h. The dispersion was filtered, the filter cake rinsed
with Et2O, and the organic layer dried with MgSO4. Filtration and
evaporation of the solvent gave the pure diazirine 11 (2.17 g,
10.04 mmol, 92%) as a colorless liquid. Rf = 0.84 (CHCl3/EtOAc,
2
3.0 Hz, C-6), 130.1 (C-5), 131.0 (C-1), 160.0 (C-3), 180.4 (q, JCF
= 35.1 Hz, CCF3) ppm.
2,2,2-Trifluoro-1-(3-methoxyphenyl)ethanone Oxime (8):[33] A solu-
tion of 2,2,2-trifluoro-1-(3-methoxyphenyl)ethanone (7) (9.38 g,
46.0 mmol) and hydroxylamine hydrochloride (4.2 g, 60.4 mmol,
1.3 equiv.) in pyridine (100 mL) and dry ethanol (50 mL) was re-
fluxed at an oil-bath temperature of 85 °C for 14 h. The solvent
was removed under reduced pressure and the resulting residue par-
titioned between Et2O (80 mL) and water (60 mL). The organic
layer was washed with 1  HCl (3ϫ10 mL) and water (1ϫ10 mL),
dried with MgSO4, and filtered. Evaporation of the solvent af-
forded the pure oxime (9.8 g, 44.72 mmol, 97%) as a colorless li-
1
20:1). H NMR (400 MHz, CDCl3): δ = 3.80 (s, 3 H, CH3), 6.69
1
(s, 1 H, 2-H), 6.77 (d, J = 7.6 Hz, 1 H, 4-H), 6.91–6.97 (m, 1 H, 6-
quid (E/Z mixture, 6:4 ratio). Rf = 0.63 (CHCl3/EtOAc, 10:1). H
H), 7.30 (t, J = 8.1 Hz, 1 H, 5-H) ppm. 13C NMR (100 MHz,
NMR (400 MHz, CDCl3): δ = 3.83 (s, 3 H, CH3), 6.98–7.11 (m, 3
H, 2-H, 4-H, 6-H), 7.33 (t, J = 8.3 Hz, 0.4 H, 5-H), 7.40 (t, J =
7.8 Hz, 0.6 H, 5-H), 9.19 (s, 0.6 H, NOH), 9.34 (s, 0.4 H,
NOH) ppm. 13C NMR (100 MHz, CDCl3): δ = 55.36 (CH3), 55.38
(CH3) 113.9 (C-2), 114.2 (C-2), 116.1 (C-4), 116.3 (C-4), 118.2 (q,
2
CDCl3): δ = 28.4 (q, JCF = 40.3 Hz, CCF3), 55.3 (CH3), 112.2 (d,
1
J = 1.5 Hz, C-2), 115.2 (C-4), 118.7 (C-6), 122.1 (q, JCF
=
274.4 Hz, CF3), 130.0 (C-5), 130.5 (C-1), 159.8 (C-3) ppm.
2-Methoxy-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzaldehyde
(12) and 4-Methoxy-2-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzal-
dehyde (13):[22a] TiCl4 (460 mg, 2.4 mmol, 1.5 equiv.) at 0 °C, fol-
lowed by dichloromethyl methyl ether (276 mg, 2.4 mmol,
1.5 equiv.) was slowly added to a stirred solution of diazirine 11
(350 mg, 1.62 mmol) in dry CH2Cl2 (15 mL). The reaction mixture
was warmed up to room temperature and stirred at this tempera-
ture for 1 h before it was cooled again to 0 °C and quenched with
water (10 mL). The organic phase was diluted with CH2Cl2 (15 mL)
and washed with water (2 ϫ5 mL), saturated NaHCO3 solution
(2ϫ5 mL), and water (2ϫ5 mL). The organic layer was dried with
MgSO4, filtered, and concentrated in vacuo. The resulting crude
product was purified by flash chromatography (hexane/EtOAc, 5:1)
to afford aldehyde 12 (161 mg, 0.66 mmol, 41%) and the isomeric
aldehyde 13 (34 mg, 0.14 mmol, 9%) as yellow oils.
1
1JCF = 282.5 Hz, CF3), 120.5 (q, JCF = 275.2 Hz, CF3), 120.7 (C-
6), 120.8 (C-6), 127.0 (C-1) 129.65 (C-5), 129.73 (C-5), 131.1 (C-1),
2
147.5 (q, 2JCF = 32.9 Hz, CCF3), 147.7 (q, JCF = 30.7 Hz, CCF3),
159.40 (C-3), 159.54 (C-3) ppm.
2,2,2-Trifluoro-1-(3-methoxyphenyl)-N-{[(4-methylphenyl)sulfonyl]-
oxy}ethanimine (9): Toluenesulfonyl chloride (4.3 g, 22.5 mmol,
1.5 equiv.) was added to a solution of 2,2,2-trifluoro-1-(3-meth-
oxyphenyl)ethanone oxime (8) (3.3 g, 15.0 mmol) in pyridine
(50 mL) and the resulting solution was refluxed for 2 h at an oil-
bath temperature of 125 °C. After cooling, the solvent was evapo-
rated and the residue partitioned between Et2O (250 mL) and water
(60 mL). The organic layer was washed with 1  HCl (3ϫ15 mL)
and water (1ϫ15 mL), dried with MgSO4, and filtered. Evapora-
tion of the solvent afforded the pure title compound (5.5 g,
14.7 mmol, 98%) as a colorless solid, m.p. 111 °C. Rf = 0.17 (tolu-
ene). 1H NMR (400 MHz, CDCl3): δ = 2.47 (s, 3 H, CH3), 3.81 (s,
3 H, OCH3), 6.87 (s, 1 H, 2-H), 6.93 (d, J = 7.6 Hz, 1 H, 4-H),
7.04 (dd, J = 8.5, 1.9 Hz, 1 H, 6-H), 7.37 (t, J = 7.3 Hz, 3 H, 5-H,
3Ј-H, 5Ј-H), 7.87 (d, J = 8.3 Hz, 2 H, 2Ј-H, 6Ј-H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 21.7 (CH3), 55.4 (OCH3), 113.9 (C-2),
117.1 (C-4), 119.5 (q, 1JCF = 277.4 Hz, CF3), 120.4 (C-6), 125.6 (C-
5), 129.2 (C-3Ј, C-5Ј), 129.9 (C-2Ј, C-6Ј), 130.0 (aryl C), 131.1 (aryl
C), 146.1 (C-4Ј), 153.9 (q, 2JCF = 33.6 Hz, CCF3), 159.5 (C-3) ppm.
1
Aldehyde 12: Rf = 0.40 (hexane/EtOAc, 5:1). H NMR (400 MHz,
CDCl3): δ = 3.92 (s, 3 H, CH3), 6.68 (s, 1 H, 3-H), 6.83 (d, J =
8.1 Hz, 1 H, 5-H), 7.82 (d, J = 8.1 Hz, 1 H, 6-H), 10.42 (s, 1 H,
2
CHO) ppm. 13C NMR (100 MHz, CDCl3): δ = 28.6 (q, JCF
=
40.3 Hz, CCF3), 55.8 (CH3), 109.5 (C-3), 118.6 (C-5), 121.8 (q, 1JCF
= 274.4 Hz, CF3), 125.4 (C-1), 129.0 (C-6), 136.6 (C-4), 161.5 (C-
2), 188.7 (CHO) ppm.
1
Aldehyde 13: Rf = 0.28 (hexane/EtOAc, 5:1). H NMR (400 MHz,
CDCl3): δ = 3.88 (s, 3 H, CH3), 7.05 (dd, J = 8.7, 2.2 Hz, 1 H, 5-
H), 7.16 (s, 1 H, 3-H), 7.89 (d, J = 8.6 Hz, 1 H, 6-H), 10.38 (s, 1
3-(3-Methoxyphenyl)-3-(trifluoromethyl)diaziridine (10):[21b,33]
A
2
H, CHO) ppm. 13C NMR (100 MHz, CDCl3): δ = 27.2 (q, JCF
=
solution of 2,2,2-trifluoro-1-(3-methoxyphenyl)-N-{[(4-meth-
ylphenyl)sulfonyl]oxy}ethanimine (9) (5.5 g, 14.73 mmol) in dry
CH2Cl2 (65 mL) was added to liquid ammonia (50 mL) at –78 °C
over a period of 1 h. The solution was stirred at –78 °C for 12 h
and then warmed up to room temperature overnight which led to
evaporation of the excess ammonia and some of the CH2Cl2. The
colorless solid was removed by filtration and the filtrate was
washed with water (3ϫ5 mL). The organic layer was dried with
MgSO4, filtered, and concentrated in vacuo. The resulting crude
product was purified by flash chromatography (CHCl3/EtOAc,
20:1) to give the diaziridine 10 (2.39 g, 11.0 mmol, 74 %) as a
slightly yellow liquid. Rf = 0.30 (CHCl3/EtOAc, 20:1). 1H NMR
(400 MHz, CDCl3): δ = 2.24 (d, J = 8.6 Hz, 1 H, NH), 2.78 (d, J
= 8.3 Hz, 1 H, NH), 3.81 (s, 3 H, CH3), 6.97 (d, J = 8.3 Hz, 1 H,
42.5 Hz, CCF3), 55.8 (CH3), 116.2 (aryl C), 116.8 (aryl C), 121.6
(q, JCF = 275.2 Hz, CF3), 129.1 (aryl C), 130.8 (aryl C), 133.8
(aryl C), 164.6 (C-4), 188.2 (CHO) ppm.
1
2-Hydroxy-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzaldehyde
(14): Tribromoborane (1  in CH2Cl2, 5.5 mL, 5.5 mmol, 1.1
equiv.) was added slowly to a solution of 2-methoxy-4-[3-(trifluoro-
methyl)-3H-diazirin-3-yl]benzaldehyde (12) (1.2 g, 4.91 mmol) in
dry CH2Cl2 (15 mL) at –20 °C. The reaction was warmed up to
0 °C, stirred at this temperature for 1 h, and then quenched with
water (5 mL). The solution was diluted with CH2Cl2 (60 mL), the
layers were separated, and the organic layer washed with saturated
NaCl solution (3ϫ10 mL), dried with MgSO4, filtered, and con-
centrated in vacuo. The crude product was purified by flash
Eur. J. Org. Chem. 2007, 4711–4720
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4715

T. Mayer, M. E. Maier
FULL PAPER
chromatography (hexane/CH2Cl2, 2:1) to give the hydroxy aldehyde
oxyphenyl)methanol (18) (1.0 g, 7.24 mmol) in dry CH2Cl2 (25 mL)
at 0 °C. The mixture was stirred at room temperature for 90 min
14 (870 mg, 3.78 mmol, 77%) as a colorless solid, m.p. 44 °C. Rf =
0.32 (hexane/CH2Cl2, 2:1). 1H NMR (400 MHz, CDCl3): δ = 6.75– before it was treated with cold water (5 mL). The layers were sepa-
6.80 (m, 2 H, 3-H, 5-H), 7.59 (d, J = 7.8 Hz, 1 H, 6-H), 9.91 (s, 1
rated and the water phase extracted with CH2Cl2 (3ϫ5 mL). The
H, CHO), 11.04 (s, 1 H, OH) ppm. 13C NMR (100 MHz, CDCl3): combined organic layers were washed with water (2ϫ10 mL), a
2
δ = 28.4 (q, JCF = 41.0 Hz, CCF3), 115.8 (C-3), 117.3 (d, J =
saturated NaHCO3 solution (1ϫ10 mL), a saturated NaCl solu-
tion (1ϫ10 mL), dried with MgSO4, filtered, and concentrated in
vacuo to afford the pure bromide 19 (1.1 g, 5.47 mmol, 76%) as a
1
1.5 Hz, C-5), 120.8 (C-1), 121.6 (q, JCF = 275.2 Hz, CF3), 133.9
(C-6), 138.0 (C-4), 161.3 (C-2), 195.9 (CHO) ppm. HRMS (ESI):
calcd. for C9H5F3N2O2 [M + H]+ 229.02304; found 229.02302.
colorless liquid. Rf = 0.25 (petroleum ether). H NMR (400 MHz,
1
CDCl3): δ = 3.81 (s, 3 H, OCH3), 4.46 (s, 2 H, CH2), 6.84 (dd, J =
8.2, 2.2 Hz, 1 H, 4-H), 6.93 (s, 1 H, 2-H), 6.97 (d, J = 7.6 Hz, 1 H,
6-H), 7.25 (t, J = 7.8 Hz, 1 H, 5-H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 33.5 (CH2), 55.3 (CH3), 114.2 (C-4), 114.4 (C-2), 121.3
(C-6), 129.8 (C-5), 139.1 (C-1), 159.7 (C-3) ppm.
2-(Prop-2-ynyloxy)ethanol (15): Finely pulverized NaOH (2.16 g,
54 mmol, 1.2 equiv.) was added to a vigorously stirred solution of
3-bromoprop-1-yne (5.34 g, 44.9 mmol) and ethylene glycol (5.57 g,
89.8 mmol, 2 equiv.) at 0 °C. The resulting suspension was refluxed
at 45 °C for 4 h, filtered, and the filtrate extracted with CHCl3
(3ϫ25 mL). The combined organic layers were washed with water
(3 ϫ 10 mL), dried with MgSO4, filtered, and concentrated in
vacuo. The crude product was purified by flash chromatography
(hexane/EtOAc, 2:1) to afford alcohol 15 (1.62 g, 16.18 mmol,
30%) as a colorless liquid. Rf = 0.18 (CHCl3). 1H NMR (400 MHz,
CDCl3): δ = 2.43 (t, J = 2.3 Hz, 1 H, CϵCH), 2.71 (s, 1 H, OH),
3.56–3.61 (m, 2 H, CH2), 3.68–3.72 (m, 2 H, CH2), 4.15 (d, J =
2.3 Hz, 2 H, CH2CϵC) ppm. 13C NMR (100 MHz, CDCl3): δ =
58.2 (CH2CϵC), 61.4 (CH2OH), 71.1 (CH2CH2OH), 74.7
(CϵCH), 79.4 (CϵCH) ppm.
2-(Prop-2-ynyloxy)ethyl Toluenesulfonate (16): Solid p-toluenesul-
fonyl chloride (1.14 g, 6 mmol, 1.1 equiv.) was added to a stirred
solution of 2-(prop-2-ynyloxy)ethanol (15) (546 mg, 5.46 mmol) in
pyridine (10 mL) at 0 °C. After 17 h saturated NaHCO3 solution
(20 mL) was added and the reaction mixture was stirred at the same
temperature for another 10 min before it was poured into a satu-
rated NaHCO3 solution (20 mL) at room temperature. The re-
sulting mixture was extracted with Et2O (3ϫ20 mL). The com-
bined organic layers were washed with 1  HCl (3ϫ20 mL), water
(1 ϫ 20 mL), dried with MgSO4, filtered, and concentrated in
vacuo. The crude product was purified by flash chromatography
(hexane/EtOAc, 3:1) to give the tosylate 16 (1.13 g, 4.45 mmol,
82 %) as a colorless liquid. Rf = 0.62 (hexane/EtOAc, 3:2). 1H
NMR (400 MHz, CDCl3): δ = 2.41 (t, J = 2.4 Hz, 1 H, CϵCH),
2.42 (s, 3 H, CH3), 3.68–3.72 (m, 2 H, pTsOCH2CH2), 4.09 (d, J
= 2.3 Hz, 2 H, CH2CϵC), 4.14–4.18 (m, 2 H, pTsOCH2), 7.33 (d,
J = 8.1 Hz, 2 H, 2-H, 6-H), 7.79 (d, J = 8.3 Hz, 2 H, 3-H, 5-
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 21.6 (CH3), 58.3
(CH2CϵC), 67.1 (pTsOCH2), 68.8 (pTsOCH2CH2), 75.0 (CϵCH),
78.9 (CϵCH), 128.0 (C-2, C-6), 129.8 (C-3, C-5), 132.9 (C-1), 144.9
(C-4) ppm.
1-(Azidomethyl)-3-methoxybenzene (20): A solution of sodium az-
ide (0.5 , 190.3 mg, 2.93 mmol, 1.1 equiv.) in DMSO (5.86 mL),
which was prepared by stirring the sodium azide in DMSO at room
temperature for 24 h, was added to 1-(bromomethyl)-3-methoxy-
benzene (19) (535 mg, 2.66 mmol). The mixture was stirred for 1 h
at room temperature before it was quenched with water (10 mL).
The mixture was extracted with Et2O (3ϫ10 mL). The combined
organic layers were washed with water (2ϫ10 mL) and a saturated
NaCl solution (1ϫ10 mL), dried with MgSO4, filtered, and con-
centrated in vacuo. The pure azide 20 (372 mg, 2.28 mmol, 86%)
1
was obtained as a colorless liquid. Rf = 0.16 (petroleum ether). H
NMR (400 MHz, CDCl3): δ = 3.82 (s, 3 H, CH3), 4.30 (s, 2 H,
CH2), 6.84–6.92 (m, 3 H, 2-H, 4-H, 6-H), 7.29 (t, J = 7.8 Hz, 1 H,
5-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 54.7 (CH2), 55.2
(CH3), 113.6 (aryl C), 113.8 (aryl C), 120.4 (C-6), 129.8 (C-5), 136.8
(C-1), 159.9 (C-3) ppm.
2-[2-(Prop-2-ynyloxy)ethoxy]-4-[3-(trifluoromethyl)-3H-diazirin-3-
yl]benzaldehyde (21): Potassium carbonate (710 mg, 5.14 mmol,
1.7 equiv.) was added to a cooled solution (0 °C) of 2-hydroxybenz-
aldehyde 14 (695 mg, 3.02 mmol) in dry DMF (60 mL) followed by
stirring of the mixture for 30 min. The cooling bath was removed,
then the tosylate 16 (870 mg, 3.42 mmol, 1.1 equiv.) dissolved in
DMF (10 mL) was added, followed by Bu4NI (120 mg, 0.3 mmol,
0.1 equiv.). The mixture was stirred at 55 °C for 16 h. It was then
diluted with EtOAc (100 mL), washed with 1  HCl (1ϫ40 mL)
and a saturated NaCl solution (1ϫ40 mL), dried with MgSO4, fil-
tered, and concentrated in vacuo. The crude product was purified
by flash chromatography (petroleum ether/Et2O, 6:1) to give the
slightly yellow aldehyde 21 (706 mg, 2.2 mmol, 75%), m.p. 40 °C.
1
Rf = 0.45 (toluene/EtOAc, 25:1). H NMR (400 MHz, CDCl3): δ
= 2.47 (t, J = 2.2 Hz, 1 H, CϵCH), 3.94–3.96 (m, 2 H, Ar-
OCH2CH2), 4.24–4.27 (m, 4 H, ArOCH2, CH2CϵC), 6.73 (s, 1 H,
3-H), 6.83 (d, J = 8.3 Hz, 1 H, 5-H), 7.83 (d, J = 8.1 Hz, 1 H, 6-
H), 10.47 (s, 1 H, CHO) ppm. 13C NMR (100 MHz, CDCl3): δ =
(3-Methoxyphenyl)methanol (18): Sodium borohydride (777 mg,
20.6 mmol, 0.5 equiv.) was added to a solution of 3-methoxybenzal-
dehyde (17) (5.6 g, 41.1 mmol) in THF (120 mL) and H2O (4 mL)
and the mixture stirred at room temperature for 20 min. Then,
water (120 mL) was added and the solution stirred for another
5 min. The mixture was extracted with CH2Cl2 (3ϫ75 mL). The
combined organic layers were dried with MgSO4, filtered, and con-
centrated in vacuo. This way the benzylic alcohol 18 (5.58 g,
40.39 mmol, 98%) was obtained as a colorless liquid, sufficiently
pure for the subsequent step. Rf = 0.18 (CHCl3). 1H NMR
(400 MHz, CDCl3): δ = 1.96 (t, J = 5.2 Hz, 1 H, OH), 3.80 (s, 3
H, OCH3), 4.65 (d, J = 5.5 Hz, 2 H, CH2), 6.80–6.85 (m, 1 H, 4-
H), 6.90–6.94 (m, 2 H, 2-H, 6-H), 7.26 (t, J = 8.1 Hz, 1 H, 5-
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 55.2 (CH3), 65.2 (CH2),
112.2 (C-2), 113.2 (C-4), 119.1 (C-6), 129.5 (C-5), 142.5 (C-1), 159.8
(C-3) ppm.
2
28.5 (q, JCF = 41.0 Hz, CCF3), 58.6 (CH2CϵC), 67.7 (Ar-
OCH2CH2), 68.3 (ArOCH2), 75.1 (CϵCH), 79.0 (CϵCH), 110.8
1
(C-3), 118.9 (C-5), 121.7 (q, JCF = 275.2 Hz, CF3), 125.6 (C-1),
128.9 (C-6), 136.6 (C-4), 160.9 (C-2), 188.8 (CHO) ppm. HRMS
(ESI): calcd. for C14H11F3N2O3 [M + Na]+ 335.0614; found
335.0612.
2-[2-(Prop-2-ynyloxy)ethoxy]-4-[3-(trifluoromethyl)-3H-diazirin-3-
yl]benzoic Acid (22): Sulfamic acid (400 mg, 4.13 mmol, 1.85 equiv.)
was added to a solution of aldehyde 21 (695 mg, 2.23 mmol) in
THF (20 mL) and water (10 mL). The mixture was stirred for 5 min
at room temperature before sodium chlorite (365 mg, 4.04 mmol,
1.81 equiv.) dissolved in water (4 mL) was added. The reaction mix-
ture was stirred for 3.5 h at room temperature and then extracted
with CH2Cl2 (3ϫ20 mL). The combined organic layers were dried
1-(Bromomethyl)-3-methoxybenzene (19): Phosphorous tribromide
(705 mg, 2.6 mmol, 0.36 equiv.) was added to a solution of (3-meth-
4716
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 4711–4720

A Tag-Free Chemical Probe for Photoaffinity Labeling
with MgSO4, filtered, and concentrated in vacuo to afford the pure
benzoic acid 22 (685 mg, 2.09 mmol, 94%) as a colorless solid, m.p.
70 °C. Purification by flash chromatography was accompanied by
some loss of product. Rf = 0.16 (CHCl3). 1H NMR (400 MHz,
and H2O (0.75 mL) at room temperature under nitrogen. Then, a
0.01  aqueous solution of sodium ascorbate (0.37 mg, 0.19 mL,
1.84 µmol) was added followed by a 0.005  aqueous solution of
copper(II) sulfate pentahydrate (0.05 mg, 0.04 mL, 0.18 µmol). Af-
CDCl3): δ = 2.48 (t, J = 2.3 Hz, 1 H, CϵCH), 3.95–3.99 (m, 2 H, ter being stirred for 20 h, water (2 mL) was added and the aqueous
ArOCH2CH2), 4.26 (d, J = 2.3 Hz, 2 H, CH2CϵC), 4.37–4.41 (m,
2 H, ArOCH2), 6.78 (s, 1 H, 3-H), 6.92 (d, J = 8.3 Hz, 1 H, 5-H),
8.16 (d, J = 8.3 Hz, 1 H, 6-H), 10.58 (br. s, 1 H, CO2H) ppm. 13C
NMR (100 MHz, CDCl3): δ = 28.3 (q, 2JCF = 41.0 Hz, CCF3), 58.5
phase extracted with CH2Cl2 (3ϫ5 mL). The combined organic
layers were washed with water, dried with MgSO4, filtered, and
concentrated in vacuo. The residue was purified by flash
chromatography (petroleum ether/EtOAc, 1:1) to give the triazole
(CH2CϵC), 66.7 (ArOCH2CH2), 69.4 (ArOCH2), 75.6 (CϵCH), 25 (5.7 mg, 10.6 µmol, 58%) as a colorless oil.
78.5 (CϵCH), 111.3 (C-3), 119.6 (C-1), 119.9 (d, J = 1.5 Hz, C-5),
b) From diazirine 26: A stirred 0.75 m solution of diazirine 26
1
121.7 (q, JCF = 275.2 Hz), 134.3 (C-6), 135.9 (C-4), 157.4 (C-2),
(2 mg, 3.7 µmol) in MeOH (5 mL) was irradiated at 366 nm with a
UV lamp (8 W, Waldmann, type 600352) at a distance of 1 cm for
1 h in a Duran Schlenk tube under nitrogen. The reaction mixture
164.5 (CO2H) ppm. HRMS (ESI): calcd. for C14H11F3N2O4 [M +
Na]+ 351.0563; found 351.0560.
Isopropyl 2-[2-(Prop-2-ynyloxy)ethoxy]-4-[3-(trifluoromethyl)-3H- was concentrated at reduced pressure and the resulting crude prod-
diazirin-3-yl]benzoate (23): To a solution of benzoic acid 22 uct was purified by flash chromatography (petroleum ether/EtOAc,
(100.0 mg, 0.3 mmol) in dry CH2Cl2 (4 mL) was added DMAP 1:1) to afford insertion product 25 (1.14 mg, 2.1 µmol, 57%) as a
(73.3 mg, 0.6 mmol, 2 equiv.), followed by propan-2-ol (0.05 mL, colorless oil. This material was identical in all respects (TLC, LC-
1
0.6 mmol, 2 equiv.), and EDC (0.06 mL, 51.2 mg, 0.33 mmol, MS and H NMR) to the triazole prepared by the other route.
1.1 equiv.). The resulting solution was stirred at room temperature
Rf = 0.25 (petroleum ether/EtOAc, 1:1). 1H NMR (400 MHz,
for 16 h and then diluted with CH2Cl2 (35 mL). The mixture was
CDCl3): δ = 1.30 [d, J = 6.1 Hz, 6 H, CH(CH3)2], 3.40 (s, 3 H,
washed with water (3 ϫ5 mL), dried with MgSO4, filtered, and
OCH3), 3.76 (s, 3 H, ArOCH3), 3.89–3.94 (m, 2 H, ArOCH2CH2),
concentrated in vacuo. The crude product was purified by flash
4.17–4.23 (m, 2 H, ArOCH2), 4.47 (q, J = 6.5 Hz, 1 H, CHCF3),
chromatography (petroleum ether/Et2O, 5:1) to give the ester 23
4.75 (s, 2 H, OCH2-triazole), 5.12–5.23 [m, 1 H, CH(CH3)2], 5.48
(61.0 mg, 0.17 mmol, 55%) as a colorless viscous oil. Rf = 0.32
(s, 2 H, NCH2), 6.80 (s, 1 H, 2Ј-H), 6.83–6.88 (m, 2 H, 4Ј-H, 6Ј-
(petroleum ether/Et2O, 5:1). 1H NMR (400 MHz, CDCl3): δ = 1.34
H), 7.01 (d, J = 8.1 Hz, 1 H, 5-H), 7.03 (s, 1 H, 3-H), 7.26 (t, J =
[d, J = 6.3 Hz, 6 H, CH(CH3)2], 2.44 (t, J = 2.3 Hz, 1 H, CϵCH),
8.0 Hz, 1 H, 5Ј-H), 7.64 (s, 1 H, 5ЈЈ-H), 7.74 (d, J = 7.8 Hz, 1 H,
3.89–3.94 (m, 2 H, ArOCH2CH2), 4.16–4.21 (m, 2 H, ArOCH2),
6-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 21.9 [CH(CH3)2],
4.27 (d, J = 2.3 Hz, 2 H, CH2CϵC), 5.16–5.28 [m, 1 H, CH(CH3)
54.1 (NCH2), 55.3 (ArOCH3), 58.4 (OCH3), 64.9 (OCH2-triazole),
2], 6.70 (s, 1 H, 3-H), 6.80 (d, J = 8.1 Hz, 1 H, 5-H), 7.74 (d, J =
2
68.4 [CH(CH3)2], 68.6 (OCH2CH2O), 81.0 (q, JCF = 31.0 Hz,
8.1 Hz, 1 H, 6-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 21.9
CCF3), 112.8 (C-3), 113.7 (C-2Ј), 114.1 (C-4Ј), 120.3 (C-6Ј), 120.5
[CH(CH3)2], 28.4 (q, 2JCF = 41.0 Hz, CCF3), 58.6 (CH2CϵC), 67.9
1
(C-5), 122.7 (C-1, C-5ЈЈ), 123.4 (q, JCF = 282.0 Hz, CF3), 130.1
(ArOCH2CH2), 68.72 [CH(CH3)2], 68.74 (ArOCH2), 74.8
(C-5Ј), 131.4 (C-6), 136.0 (C-1Ј), 137.5 (C-4), 145.5 (C-4ЈЈ), 158.2
1
(CϵCH), 79.4 (CϵCH), 111.5 (C-3), 118.6 (C-5), 121.8 (q, JCF
=
(C-2), 160.1 (C-3Ј), 165.4 (CO2iPr) ppm. HRMS (ESI): calcd. for
C26H30F3N3O6 [M + H]+ 538.21593; found 538.21562; ∆(rel.) =
0.58 ppm.
275.2 Hz, CF3), 123.0 (C-1), 131.8 (C-6), 133.8 (C-4), 158.0 (C-2),
165.0 (CO2iPr) ppm. HRMS (ESI): calcd. for C17H17F3N2O4 [M +
Na]+ 393.1033; found 393.1034.
Isopropyl 2-(2-{[1-(3-Methoxybenzyl)-1H-1,2,3-triazol-4-yl]meth-
oxy}ethoxy)-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoate (26):
The alkyne 23 (24.0 mg, 64.8 µmol) and the azide 20 (12.7 mg,
77.8 µmol, 1.2 equiv.) were suspended in tBuOH (1 mL) and H2O
(0.2 mL) at room temperature under nitrogen. Then, a 0.01  aque-
ous solution of sodium ascorbate (1.3 mg, 0.66 mL, 6.48 µmol) was
added, followed by a 0.005  aqueous solution of copper(II) sulfate
pentahydrate (0.16 mg, 0.13 mL, 0.65 µmol). After being stirred for
20 h, water (3 mL) was added and the aqueous phase was extracted
with CH2Cl2 (3 ϫ 10 mL). The combined organic layers were
washed with water, dried with MgSO4, filtered, and concentrated
in vacuo. The crude product was purified by flash chromatography
(petroleum ether/EtOAc, 1:1) to give the triazole 26 (12.4 mg,
23.2 µmol, 36%) as a colorless oil. Rf = 0.34 (petroleum ether/
EtOAc, 1:1). 1H NMR (400 MHz, CDCl3): δ = 1.29 [d, J = 6.1 Hz,
6 H, CH(CH3)2], 3.76 (s, 3 H, OCH3), 3.88–3.93 (m, 2 H, Ar-
OCH2CH2), 4.13–4.19 (m, 2 H, ArOCH2), 4.74 (s, 2 H, OCH2-
triazole), 5.11–5.21 [m, 1 H, CH(CH3)2], 5.48 (s, 2 H, NCH2), 6.66
(s, 1 H, 3-H), 6.79 (d, J = 7.1 Hz, 2 H, 5-H, 2Ј-H), 6.83–6.89 (m,
2 H, 4Ј-H, 6Ј-H), 7.26 (t, J = 7.8 Hz, 1 H, 5Ј-H), 7.61 (s, 1 H, 5ЈЈ-
H), 7.73 (d, J = 8.1 Hz, 1 H, 6-H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 21.8 [CH(CH3)2], 28.3 (q, 2JCF = 41.0 Hz, CCF3), 54.1
Isopropyl 2-[2-(Prop-2-ynyloxy)ethoxy]-4-(2,2,2-trifluoro-1-meth-
oxyethyl)benzoate (24): A stirred 1 m solution of diazirine 23
(5.5 mg, 14.9 mmol) in MeOH (15 mL) was irradiated at 366 nm
with a UV lamp (8 W, Waldmann, type 600352) at a distance of
1 cm for 1 h in a Duran Schlenk tube under nitrogen. Thereafter,
the reaction mixture was concentrated at reduced pressure and the
resulting crude product was purified by flash chromatography (pe-
troleum ether/EtOAc, 8:1) to afford the insertion product 24
(2.1 mg, 5.61 mmol, 38%) as a colorless oil. Rf = 0.16 (petroleum
1
ether/EtOAc, 8:1). H NMR (400 MHz, CDCl3): δ = 1.35 [d, J =
6.3 Hz, 6 H, CH(CH3)2], 2.44 (t, J = 2.2 Hz, 1 H, CϵCH), 3.41 (s,
3 H, OCH3), 3.91–3.95 (m, 2 H, ArOCH2CH2), 4.21–4.25 (m, 2 H,
ArOCH2), 4.28 (d, J = 2.3 Hz, 2 H, CH2CϵC), 4.48 (q, J = 6.6 Hz,
1 H, CHCF3), 5.19–5.30 [m, 1 H, CH(CH3)2], 7.02 (d, J = 8.1 Hz,
1 H, 5-H), 7.05 (s, 1 H, 3-H), 7.75 (d, J = 8.1 Hz, 1 H, 6-H) ppm.
13C NMR (100 MHz, CDCl3): δ = 21.9 [CH(CH3)2], 58.4 (OCH3),
58.7 (CH2CϵC), 68.0 (ArOCH2CH2), 68.5 [CH(CH3)2], 68.6 (Ar-
OCH2), 74.7 (CϵCH), 79.5 (CϵCH), 81.0 (2JCF = 31.5 Hz, CCF3),
112.9 (C-3), 120.6 (C-5), 123.0 (C-1), 123.4 (1JCF = 281.8 Hz, CF3),
131.4 (C-6), 137.4 (C-4), 158.1 (C-2), 165.6 (CO2iPr) ppm. HRMS
(ESI): calcd. for C18H21F3O5 [M + Na]+ 397.1233; found 397.1236.
Isopropyl 2-(2-{[1-(3-Methoxybenzyl)-1H-1,2,3-triazol-4-yl]- (NCH2), 55.3 (ArOCH3), 64.9 (OCH2-triazole), 68.5 (Ar-
methoxy}ethoxy)-4-(2,2,2-trifluoro-1-methoxyethyl)benzoate (25): a)
From alkyne 24: The alkyne 24 (6.9 mg, 18.4 µmol) and the azide
20 (3.6 mg, 22.1 µmol, 1.2 equiv.) were suspended in tBuOH (1 mL)
OCH2CH2), 68.6 [CH(CH3)2], 68.7 (ArOCH2), 111.4 (C-3), 113.7
1
(C-2Ј), 114.1 (C-4Ј), 118.5 (C-5), 120.3 (C-6Ј), 121.8 (q, JCF
=
275.2 Hz, CF3), 122.7 (C-5ЈЈ), 122.8 (C-1), 130.1 (C-5Ј), 131.8 (C-
Eur. J. Org. Chem. 2007, 4711–4720
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4717

T. Mayer, M. E. Maier
FULL PAPER
6), 133.8 (C-4), 136.0 (C-1Ј), 145.4 (C-4ЈЈ), 158.1 (C-2), 160.1 (C-
3Ј), 164.9 (COO) ppm. HRMS (ESI): calcd. for C25H26F3N5O5 [M
+ H]+ 534.19585; found 534.19543; ∆(rel.) = 0.79 ppm.
41.0 Hz, CCF3), 37.9 (C-6), 38.3 (C-16), 39.0 (C-22), 40.6 (C-20),
41.6 (C-8), 42.2 (C-9), 42.9 (C-18), 55.6 (14-OMe), 58.8
(CH2CϵC), 60.1 (2-OMe), 68.7 (ArOCH2CH2), 69.6 (ArOCH2),
71.5 (C-17), 75.6 (C-21), 76.1 (CϵCH), 76.6 (C-23), 77.3 (C-15),
80.3 (C-7), 80.6 (CϵCH), 83.3 (C-14), 99.8 (C-19), 112.3 (Ar-3),
NMR Spectroscopic Data for Bafilomycin A1 (27): 1H NMR
(400 MHz, [D6]acetone): δ = 0.76 (d, J = 6.8 Hz, 3 H, Me-33), 0.86
(d, J = 6.8 Hz, 3 H, Me-30), 0.91 (m, 9 H, Me-25, Me-28, Me-32),
0.98 (d, J = 7.1 Hz, 3 H, Me-31), 1.03 (d, J = 7.1 Hz, 3 H, Me-27),
1.08–1.13 (m, 2 H, 20-H), 1.22–1.30 (m, 1 H, 22-H), 1.77 (q, J =
7.0 Hz, 1 H, 18-H), 1.83–1.90 (m, 2 H, 8-H, 24-H), 1.92 (s, 3 H,
Me-29), 1.97 (d, J = 0.8 Hz, 3 H, Me-26), 1.99–2.03 (m, 9-H), 2.11–
2.20 (m, 2 H, 16-H, 20-H), 2.49–2.58 (m, 1 H, 6-H), 3.22 (s, 3 H,
14-OMe), 3.27–3.31 (m, 1 H, 7-H), 3.44 (dd, J = 10.4, 2.0 Hz, 1 H,
23-H), 3.53 (ddd, J = 15.4, 10.3, 5.4 Hz, 1 H, 21-H), 3.62 (s, 3 H,
2-OMe), 4.05 (t, J = 8.8 Hz, 1 H, 14-H), 4.11 (d, J = 5.6 Hz, 1 H,
7-OH), 4.17 (ddd, J = 10.7, 4.2, 1.5 Hz, 1 H, 17-H), 4.71 (d, J =
4.3 Hz, 1 H, 17-OH), 5.0 (dd, J = 8.3, 1.0 Hz, 1 H, 15-H), 5.13 (dd,
J = 14.9, 9.1 Hz, 1 H, 13-H), 5.24 (d, J = 2.0 Hz, 1 H, 19-OH),
5.78 (d, J = 10.6 Hz, 1 H, 11-H), 5.95 (d, J = 8.8 Hz, 1 H, 5-H),
1
119.4 (Ar-5), 122.9 (q, JCF = 273.7 Hz, CF3), 124.7 (Ar-1), 125.1
(C-11), 126.9 (C-13), 132.4 (Ar-6), 132.7 (C-4), 133.7 (Ar-4), 134.2
(C-3), 134.4 (C-12), 141.9 (C-2), 144.8 (C-10), 145.7 (C-5), 158.7
(Ar-2), 166.0 (ArCO2), 167.7 (C-1) ppm. HRMS (ESI): calcd. for
C49H67F3N2O12 [M + Na]+ 955.45383; found 955.45387; ∆(rel.) =
0.04 ppm.
2-Azidoethylamine (29): 2-Bromoethylamine hydrobromide
(500 mg, 2.44 mmol) was added to a solution of sodium azide
(475.9 mg, 7.32 mmol, 3 equiv.) in H2O (2 mL). The stirred solution
was heated to 75 °C for 21 h before it was cooled to 0 °C. Et2O
(2 mL) was added followed by solid KOH (800 mg). The organic
phase was separated and the aqueous layer extracted with Et2O
(3ϫ10 mL). The combined organic layers were dried with MgSO4,
filtered, and the solvent removed carefully by rotary evaporation
(35 °C, 750 mbar) to afford the azide 29 (171 mg, 1.99 mmol, 82%)
as a colorless liquid. Rf = 0.39 (EtOAc/MeOH, 3:1). 1H NMR
(400 MHz, CDCl3): δ = 1.27 (s, 2 H, NH2), 2.80–2.84 (m, 2 H,
CH2N3), 3.30 (t, J = 5.7 Hz, 2 H, CH2NH2) ppm. 13C NMR
(100 MHz, CDCl3): δ = 41.2 (CH2NH2), 54.6 (CH2N3) ppm.
6.66 (dd, J = 10.9, 4.0 Hz, 1 H, 12-H), 6.70 (s, 1 H, 3-H) ppm. 13
C
NMR (100 MHz, [D6]acetone): δ = 7.3 (Me-31), 10.3 (Me-30), 12.6
(Me-32), 14.1 (Me-26), 14.7 (Me-33), 17.7 (Me-27), 20.3 (Me-29),
21.7 (Me-25), 22.2 (Me-28), 28.7 (C-24), 37.9 (C-6), 38.2 (C-16),
41.6 (C-8), 41.9 (C-22), 42.2 (C-9), 43.0 (C-18), 44.5 (C-20), 55.6
(14-OMe), 60.1 (2-OMe), 70.4 (C-21), 71.5 (C-17), 76.9 (C-23), 77.4
(C-15), 80.3 (C-7), 83.3 (C-14), 99.7 (C-19), 125.2 (C-11), 126.9 (C-
13), 132.7 (C-4), 134.1 (C-3), 134.4 (C-12), 141.9 (C-2), 144.8 (C-
10), 145.7 (C-5), 167.7 (C-1) ppm.
1-{[5-(2-Oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-
pentanoyl]oxy}pyrrolidine-2,5-dione (31): N-Hydroxysuccinimide
(56.5 mg, 0.49 mmol, 1.2 equiv.) followed by EDC (86.9 µL,
0.49 mmol, 1.2 equiv.) was added to a solution of -biotin
(100.0 mg, 0.41 mmol) in DMF (10 mL). The solution was stirred
for 21 h at room temperature. The solvent was evaporated in vacuo
and the resulting residue dissolved in CH2Cl2 (200 mL). The or-
ganic phase was washed with NaHCO3 solution (2ϫ10 mL) and a
saturated NaCl solution (1ϫ10 mL), dried with MgSO4, and fil-
tered. The solvent was removed under vacuo to give biotin-NHS
31 (71.0 mg, 0.21 mmol, 51 %) as a colorless solid. 1H NMR
(400 MHz, [D6]DMSO): δ = 1.35–1.54 (m, 3 H, 4Ј-H, 5Ј-H), 1.58–
1.68 (m, 3 H, 3Ј-H, 5Ј-H), 2.57 (d, J = 11.4 Hz, 1 H, SCH2), 2.66
(t, J = 7.3 Hz, 2 H, 2Ј-H), 2.78–2.85 [m, 5 H, CH2CH2 (succinyl),
SCH2], 3.06–3.12 (m, 1 H, SCH), 4.11–4.16 (m, 1 H, 3a-H), 4.27–
4.32 (m, 1 H, 6a-H), 6.36 (s, 1 H, 1-NH), 6.42 (s, 1 H, 3-NH) ppm.
13C NMR (100 MHz, [D6]DMSO): δ = 24.3 (C-3Ј), 25.4 [CH2CH2
(succinyl)], 27.6 (C-5Ј), 27.8 (C-4Ј), 30.0 (C-2Ј), 39.9 (SCH2), 55.2
(SCH), 59.2 (C-6a), 61.0 (C-3a), 162.7 [(HN)2CO], 168.9 (CO2),
170.3 [N(CO)2] ppm.
21-O-{2-[2-(Prop-2-ynyloxy)ethoxy]-4-[3-(trifluoromethyl)-3H-di-
aziren-3-yl]benzoyl}bafilomycin A1 (28): Benzoic acid 22 (10.5 mg,
32.1 µmol, 2 equiv.) followed by DMAP (4.1 mg, 33.7 µmol,
2.1 equiv.), and EDC (6.0 µL, 33.7 µmol, 2.1 equiv.) were added to
a stirred solution of bafilomycin A1 (10.0 mg, 16.1 µmol) in CH2Cl2
(2 mL) . After stirring for 24 h at room temperature, additional
acid 22 (5.3 mg, 16.1 µmol, 1 equiv.), DMAP (2.3 mg, 19.3 µmol,
1 equiv.), and EDC (3.4 µL, 19.3 µmol, 1.2 equiv.) were added and
stirring was continued for a further 24 h. The solvent was evapo-
rated in vacuo and the resulting crude product purified by flash
chromatography (hexane/EtOAc, 3:2) to afford ester 28 (11.9 mg,
12.7 µmol, 79%) as a colorless solid as well as unreacted bafilomy-
cin A1 (2.0 mg, 3.2 µmol). Rf = 0.56 (hexane/EtOAc, 3:2). 1H NMR
(600 MHz, [D6]acetone): δ = 0.81 (d, J = 6.9 Hz, 3 H, Me-33), 0.87
(d, J = 6.9 Hz, 3 H, Me-30), 0.91 (d, J = 6.5 Hz, 6 H, Me-25, Me-
32), 0.96 (d, J = 6.9 Hz, 3 H, Me-28), 1.01 (d, J = 7.1 Hz, 3 H,
Me-31), 1.04 (d, J = 6.9 Hz, 3 H, Me-27), 1.35–1.40 (m, 1 H, 20-
H), 1.68 (ddq, J = 16.9, 10.5, 6.5 Hz, 1 H, 22-H), 1.84–1.89 (m, 2
H, 8-H, 18-H), 1.92 (s, 3 H, Me-29), 1.93–1.96 (m, 1 H, 24-H), 1.97 N-(2-Azidoethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-
(d, J = 1.2 Hz, 3 H, Me-26), 2.01 (d, J = 10.8 Hz, 1 H, 9-H), 2.16
(ddq, J = 17.6, 6.9, 0.6 Hz, 1 H, 16-H), 2.40 (dd, J = 11.8, 4.9 Hz,
1 H, 20-H), 2.51–2.57 (m, 1 H, 6-H), 2.94 (t, J = 2.4 Hz, 1 H,
pentanamide (32): Et3N (0.03 mL, 0.20 mmol, 1 equiv.) was added
to a solution of 2-azidoethylamine (29) (24.1 mg, 0.28 mmol,
1.4 equiv.) in DMF (3 mL), followed by the addition of biotin-NHS
CϵCH), 3.23 (s, 3 H, 14-OMe), 3.28–3.31 (m, 1 H, 7-H), 3.63 (s, 31 (70.0 mg, 0.20 mmol) in DMF (2 mL). The resulting solution
3 H, 2-OMe), 3.63–3.65 (m, 1 H, 23-H), 3.88–3.90 (m, 2 H, Ar-
was stirred at room temperature for 24 h. The solvent was evapo-
OCH2CH2), 4.05 (app. t, J = 8.9 Hz, 1 H, 14-H), 4.11 (d, J =
rated in vacuo and the crude product purified by flash chromatog-
5.5 Hz, 1 H, 7-OH), 4.19 (ddd, J = 10.7, 4.3, 1.6 Hz, 1 H, 17-H), raphy (acetone/MeOH, 10:1) to give the desired azido-biotin 32
4.25 (d, J = 2.4 Hz, 2 H, CH2CϵCH), 4.27–4.30 (m, 2 H, Ar-
OCH2), 4.78 (d, J = 3.7 Hz, 1 H, 17-OH), 4.97 (dd, J = 8.6, 1.2 Hz,
1 H, 15-H), 5.11–5.20 (m, 2 H, 13-H, 21-H), 5.45 (d, J = 1.8 Hz, 1
H, 19-OH), 5.79 (d, J = 10.8 Hz, 1 H, 11-H), 5.95 (d, J = 8.8 Hz,
(47.7 mg, 0.15 mmol, 75%) as a colorless solid. Rf = 0.22 (acetone/
MeOH, 10:1). 1H NMR (400 MHz, [D6]DMSO): δ = 1.20–1.38 (m,
2 H, 4Ј-H), 1.39–1.55 (m, 3 H, 3Ј-H, 5Ј-H), 1.55–1.65 (m, 1 H, 5Ј-
H), 2.06 (t, J = 7.3 Hz, 2 H, 2Ј-H), 2.56 (d, J = 12.9 Hz, 1 H,
1 H, 5-H), 6.66 (dd, J = 14.9, 10.8 Hz, 1 H, 12-H), 6.70 (d, J = SCH2), 2.80 (dd, J = 12.4, 5.1 Hz, 1 H, SCH2), 3.05–3.11 (m, 1 H,
0.6 Hz, 1 H, 3-H), 6.90 (d, J = 0.6 Hz, 1 H, 3-ArH), 7.01 (dd, J =
8.1, 0.8 Hz, 1 H, 5-ArH), 7.78 (d, J = 8.1 Hz, 1 H, 6-ArH) ppm.
13C NMR (100 MHz, [D6]acetone): δ = 7.4 (Me-31), 10.3 (Me-30),
SCH), 3.19–3.24 (m, 2 H, CH2CH2N3), 3.31 (d, J = 7.6 Hz, 2 H,
CH2N3), 4.08–4.14 (m, 1 H, 3a-H), 4.26–4.32 (m, 1 H, 6a-H), 6.35
(s, 1 H, 1-NH), 6.42 (s, 1 H, 3-NH), 8.03 (t, J = 5.3 Hz, 1 H,
12.8 (Me-32), 14.1 (Me-26), 14.6 (Me-33), 17.7 (Me-27), 20.3 (Me- CONH) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 25.2 (C-3Ј),
2
29), 21.6 (Me-25), 22.2 (Me-28), 28.7 (C-24), 29.1 (q, JCF
=
28.0 (C-5Ј), 28.2 (C-4Ј), 35.1 (C-2Ј), 38.1 (CH2CH2N3), 39.9
4718
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 4711–4720

A Tag-Free Chemical Probe for Photoaffinity Labeling
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(SCH2), 50.0 (CH2N3), 55.4 (SCH), 59.2 (C-6a), 61.0 (C-3a), 162.7
[(HN)2CO], 172.4 (CONH) ppm. HRMS (ESI): calcd. for
C12H20N6O2S [M + H]+ 313.14412; found 313.14419; ∆(rel.) =
0.22 ppm.
[4]
21-O-{2-(2-{[1-(2-{[5-(2-Oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl)pentanoyl]amino}ethyl)-1H-1,2,3-triazol-4-yl]methoxy}ethoxy)-4-
[3-(trifluoromethyl)-3H-diaziren-3-yl]benzoyl}bafilomycin A1 (33):
Azido-biotin 32 (2.0 mg, 6.4 µmol, 1.2 equiv.) was added to a
stirred solution of bafilomycin A1 derivative 28 (5.0 mg, 5.1 µmol)
in H2O (1 mL) and tBuOH (1 mL) at room temperature. An aque-
ous sodium ascorbate solution (0.01 , 55 µL, 0.5 µmol) was added
followed by an aqueous solution of copper(II) sulfate pentahydrate
(0.005 , 10 µL, 0.05 µmol). The reaction mixture was stirred for
24 h at 35 °C before further sodium ascorbate solution (55 µL,
0.5 µmol) and copper(II) sulfate pentahydrate solution (10 µL,
0.05 µmol) were added. After another 24 h of stirring the solvent
was removed under vacuum and the crude product purified by flash
chromatography (CH2Cl2/MeOH, 10:1) to afford the ligation prod-
uct 33 (2.0 mg, 1.6 µmol, 31%) as a colorless solid. Owing to the
small amount a 13C spectrum could not be acquired. According to
the 1H NMR spectrum, the purified 33 contains a small amount
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[10]
1
of unreacted azide 32. Rf = 0.18 (CH2Cl2/MeOH, 10:1). H NMR
[11]
(600 MHz, [D6]acetone): δ = 0.81 (d, J = 7.0 Hz, 3 H, Me-33), 0.87
(d, J = 6.6 Hz, 3 H, Me-30), 0.91 (m, 6 H, Me-25, Me-32), 0.96 (d,
J = 6.7 Hz, 3 H, Me-28), 0.99 (d, J = 7.0 Hz, 3 H, Me-31), 1.04
(d, J = 7.0 Hz, 3 H, Me-27), 1.35–1.48 (m, 3 H, 20-H, 4Ј-H), 1.54–
1.72 (m, 4 H, 22-H, 3Ј-H, 5Ј-H), 1.72–1.80 (m, 1 H, 5Ј-H), 1.84–
1.89 (m, 2 H, 8-H, 18-H), 1.92 (s, 3 H, Me-29), 1.93–1.96 (m, 1 H,
24-H), 1.98 (m, 3 H, Me-26), 2.00–2.02 (m, 1 H, 9-H), 2.13–2.16
(m, 1 H, 16-H), 2.19 (t, J = 7.2 Hz, 2 H, NHCOCH2), 2.40 (dd, J
= 11.8, 4.4 Hz, 1 H, 20-H), 2.51–2.57 (m, 1 H, 6-H), 2.67–2.71 (m,
2 H, SCH2), 2.90–2.94 (m, 3 H), 3.17–3.22 (m, 1 H, SCH), 3.23
(s, 3 H, 14-OMe), 3.28–3.31 (m, 1 H, 7-H), 3.35–3.42 (m, 4 H,
CH2CH2triazole), 3.63 (s, 3 H, 2-OMe), 3.65–3.68 (m, 1 H, 23-H),
3.88–3.91 (m, 2 H, ArOCH2CH2), 4.03–4.11 (m, 2 H, 7-OH, 14-
H), 4.16–4.20 (m, 1 H, 17-H), 4.26–4.29 (m, 2 H, ArOCH2), 4.30–
4.34 (m, 1 H, 3a-H), 4.48–4.52 (m, 1 H, 6a-H), 4.68 (s, 2 H, OCH2-
triazole), 4.77 (d, J = 4.2 Hz, 1 H, 17-OH), 4.98 (m, 1 H, 15-H),
5.17 (m, 2 H, 13-H, 21-H), 5.50 (s, 1 H, 19-OH), 5.72 (s, 1 H, NH),
5.80 (d, J = 10.4 Hz, 1 H, 11-H), 5.92 (s, 1 H, NH), 5.94–5.97 (m,
1 H, 5-H), 6.66 (dd, J = 14.9, 11.0 Hz, 1 H, 12-H), 6.71 (s, 1 H, 3-
H), 6.89 (s, 1 H, 3-ArH), 7.02 (d, J = 8.9 Hz, 1 H, 5-ArH), 7.29–
7.37 (m, 1 H, triazole), 7.81 (d, J = 8.1 Hz, 1 H, 6-ArH), 7.96 (s,
1 H, CONH) ppm. HRMS (ESI): calcd. for C61H87F3N8O14S [M
+ Na]+ 1267.59068; found 1267.58951; ∆(rel.) = 0.90 ppm.
[12]
[13]
[14]
[15]
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Supporting Information (see also the footnote on the first page of
this article): Copies of the 1H and 13C NMR spectra of the de-
scribed compounds.
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Acknowledgments
[20]
[21]
Financial support by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie is gratefully acknowledged.
We also thank Paul Schuler (Institute of Organic Chemistry) for
measuring capillary NMR spectra of the bafilomycin derivatives.
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Eur. J. Org. Chem. 2007, 4711–4720
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4719

T. Mayer, M. E. Maier
FULL PAPER
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Received: March 2, 2007
Published Online: July 23, 2007
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