Photolabile protecting groups for nitroxide spin labels

Article abstract of DOI:10.1002/ejoc.201301692

Nitroxide spin labels can be protected against critical conditions of DNA/RNA or peptide synthesis by reduction and alkylation with light-sensitive groups such as nitrobenzyl- or aminocoumarins. High chemical stability qualifies tetraethylisoindoline 5 an

Full text of DOI:10.1002/ejoc.201301692

FULL PAPER
DOI: 10.1002/ejoc.201301692
Photolabile Protecting Groups for Nitroxide Spin Labels
Ibrahim Seven,[a][‡] Timo Weinrich,[a][‡] Markus Gränz,[b] Christian Grünewald,[a]
[a]
[b]
[b]
[a]
´
Silke Brüß, Ivan Krstic, Thomas F. Prisner, Alexander Heckel, and
Michael W. Göbel*[a]
Keywords: Tetraethylisoindoline / EPR spectroscopy / Photochemistry / Phosphoramidites / TEMPO / Radicals / DNA
Nitroxide spin labels can be protected against critical condi-
tions of DNA/RNA or peptide synthesis by reduction and alk-
ylation with light-sensitive groups such as nitrobenzyl- or
aminocoumarins. High chemical stability qualifies tetraethyl-
isoindoline 5 and, with some restrictions, (2,2,6,6-tetrameth-
ylpiperidin-1-yl)oxy (TEMPO) precursor 4 as building blocks
for spin-labeled biopolymers. The yield of recovered
nitroxides (80–95%) is sufficient for PELDOR experiments. A
protected TEMPO phosphoramidite was successfully used to
synthesize a short spin-labeled DNA with high yield and pu-
rity.
ligation involve thioesters and free thiol groups. Although
thiol-free methods for RNA ligation based on deoxy-
ribozymes exist,[5] it appeared to us that protecting groups
for nitroxides might significantly improve the synthesis of
complex spin labels and of complex spin-labeled biopoly-
mers.
Introduction
Electron paramagnetic resonance (EPR) is well estab-
lished as a method to investigate local dynamics in biomac-
romolecules.[1] In addition to that, advanced spectroscopic
techniques such as PELDOR can also determine distances
and angles between spins.[2] To develop the full range of
possible applications, preparative techniques for the site-
specific incorporation of spin labels also have to be im-
proved.[1,3] Post-synthetic attachment of persistent nitroxide
labels is quite popular, because even large target molecules
may be modified with a minimum of preparative effort in
sufficient yield. The precise determination of distances and
angles by EPR, however, requires linking nitroxides to the
target molecule in a rigid manner,[4] because local dynamics
of linker structures may blur the information expected from
the experiment. Best results in this respect are generally ob-
tained when spin labels are introduced during chain as-
sembly of the biopolymer. A clear drawback of this strategy
is nitroxide decomposition owing to side reactions during
the synthesis of oligonucleotide or peptide chains. To build
up large target structures, ligation steps to assemble several
fragments are also required.[5] Enzymatic ligation of oligo-
nucleotides can be very efficient. Thiol components present
in the typical buffers, however, cause permanent damage of
nitroxide labels.[5] Similarly, important protocols for peptide
Results and Discussion
Temporary protection of nitroxides has been achieved
previously by reduction and acylation,[6] silylation,[6a] or
alkylation[7] of the resulting hydroxylamine. Unfortunately,
acyl groups are removed by nucleophiles used in oligonucle-
otide synthesis such as ammonia and methylamine. The re-
cently introduced R2N-O-CH3 group requires meta-chloro-
peroxybenzoic acid for demethylation, conditions hardly
compatible with oligonucleotides.[7] We therefore tested
[a] Institut für Organische Chemie und Chemische Biologie,
Goethe Universität Frankfurt,
Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany
E-mail: M.Goebel@chemie.uni-frankfurt.de
http://web.uni-frankfurt.de/fb14/goebel/
[b] Institut für Physikalische und Theoretische Chemie, Goethe
Universität Frankfurt,
Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany
[‡] These authors contributed equally to this work.
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301692.
Scheme 1. Nitroxide spin labels (A) are temporarily transformed
into C by reduction (B) and alkylation with light-sensitive residues.
C must be stable under conditions of oligonucleotide and peptide
synthesis. After photochemical deprotection, nitroxide A is formed
under air.
Eur. J. Org. Chem. 2014, 4037–4043
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4037

M. W. Göbel et al.
FULL PAPER
light-sensitive alkyl groups (Scheme 1).[8] Their removal
from RNA and peptides does not require further reagents
and, when done properly, is known not to affect the sam-
ple.[9] Hydroxylamines, once deprotected under air, form the
corresponding nitroxides spontaneously.[6,10]
Compound 1 was obtained by hydrogenation of commer-
cial
(4-acetamido-2,2,6,6-tetramethylpiperidin-1-yl)oxy
(TEMPO) and alkylation with 2-nitrobenzyl bromide (Fig-
ure 1). The latter step requires exclusion of oxygen to avoid
reoxidation of the hydroxylamine intermediate (B Ǟ A in
Scheme 1). In a similar way, compounds rac-2–5 were pre-
pared from nitroxides 6 and 7.[11] Good to excellent yields
were found with exception of 5 (42%). Steric hindrance
slowed down alkylation in this case and caused high levels
of oxidation back to 7.
Figure 2. EPR spectra of compound 5 after irradiation at 365 nm
for 9 min in CH3CN (black) and CH3CN/H2O, 2:1 (red).
(Top) Baseline-corrected EPR signal at room temperature, rescaled
to adapt unequal concentrations (0.272 mm in CH3CN, 0.319 mm
in CH3CN/H2O). (Bottom) Double integral of upper spectra in
percentage of recovered amount of nitroxides.
Table 1. Yield of nitroxide spin labels after photolysis of com-
pounds 1–5 and air oxidation. Volume fraction of water: 33%. All
analyses were run at least in triplicate (errorϮ10%).
Solvent
1
rac-2
rac-3
4
5
CH3CN[a]
H2O/CH3CN[a]
CH3CN[b]
95%
50%
80%
65%
80%
65%
80%
80%
92%
88%
95%
90%
95%
95%
Figure 1. Structures of nitroxides and their protected derivatives.
H2O/CH3CN[b]
[a] Quantification by cw EPR at X-band frequency. [b] Quantifica-
tion by HPLC.
The recovery of nitroxide radicals from the protected
precursors was tested first by EPR (Figure 2). Solutions
(0.2–0.3 mm; 0.06–0.09 μmol of precursor) in acetonitrile or
66% aqueous CH3CN were irradiated at 405 nm (coumarin
4, Ն60 min) or 365 nm (all others, 9 min) while being ex-
posed to air. After deprotection and air oxidation, the pres-
ence of nitroxides was reviewed and quantified by EPR
(Table 1). Although the accuracy of the individual experi-
ments is limited, consistent data could be obtained by re-
peating them many times (Table 1). It is important to check
for complete deprotection in the case of 4 (TLC or HPLC).
Although good recovery of spin labels was seen with aceto-
nitrile as solvent, the presence of water reduced some yields
significantly. Notable exceptions are compounds 4 and 5.
When studied by HPLC, photochemical deprotection of 4
and 5 showed encouraging yields of radicals 6 and 7 from
88 to 95% in aqueous CH3CN. No more than 12% of re-
threitol (DTT). 2,3-Dimethylbenzoic acid as an internal
standard allowed quantification of recovered compounds 4
and 5 by HPLC. Only minor losses of protected hydroxyl-
amines were found under most conditions tested (Table 2).
Table 2. Resistance of protected spin labels against typical condi-
tions used in peptide and oligonucleotide chemistry. Recovery as
determined by HPLC.
[a]
[a]
Compound TFA[a]
I2
NH3
HF·Et3N[a] DTT[a]
4
5
97%[b]
92–100%
100%
100%
100%
98%
100%
98%
100%
97%[c]
[a] For details see Supporting Information. [b] 7 % TFA, 2 min.
[c] 33% TFA, 4 h.
By starting from tert-butyloxycarbonyl (Boc) protected
duced amine was found after deprotection of 4 (6% in dry 4-amino-TEMPO 8,[12] coumarin derivative 9 was obtained
CH3CN) and 5% (3%) after photolysis of 5, respectively.
by hydrogenation and alkylation as described above (31%;
To test the compatibility of compounds 4 and 5 with con- Figure 3). Removal of the Boc group with trimethylsilyl
ditions used in solid-phase syntheses of peptides or nucleic iodide then yielded 79% of compound 10. O-Sulfonylation
acids, the protected spin labels were challenged by 32% of 3Ј-O-acetyl-5Ј-O-DMT-2Ј-deoxyuridine[14] with 2,4,6-tri-
aqueous NH3, iodine in aqueous acetonitrile, trifluoroacetic isopropylbenzenesulfonyl chloride[13] activated the nu-
acid (TFA), triethylamine trihydrofluoride, and dithio- cleoside for displacement with amine 10 to give 22% of
4038
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 4037–4043

Photolabile Protecting Groups for Nitroxide Spin Labels
product 11. After deacetylation (96%), nucleoside 12 was isolated without difficulty in 50–60% yield. Photochemical
converted into phosphoramidite 13 by standard methods deprotection (365 nm) of 15 to produce nitroxide DNA 16
(71%). For comparison, phosphoramidite 14 with an un- critically depends on the pH of the buffer (Table 3). Al-
protected spin label was also synthesized according to pub- though amine 17 is the dominant product at pH = 4.6, at
lished procedures.[15]
pH = 7.4 50% of 17 and 50% of 16 could be observed by
HPLC. The identity of both oligonucleotides was con-
firmed by mass spectrometry. Protonation makes nitrogen
a better leaving group and may therefore favor N–O bond
cleavage in the photochemical step. In agreement with that
idea, deprotection at pH = 9 led to the best results: 85%
of nitroxide 16 and only 15% of amine 17 were obtained.
According to EPR, the degree of spin labeling in the crude
deprotection mixture was 95%; 80–90% of nitroxide 16
were found already at pH = 7.4 when acetonitrile was pres-
ent during irradiation (volume fraction of water: 33%). In
an attempt towards spin-labeled RNA we also used
Höbartner’s method[3b] based on convertible nucleosides:
the nitroxide is introduced after chain assembly of the
RNA, thus avoiding critical conditions. After optimization,
high degrees of labeling could be achieved. However, con-
sistent with earlier observations,[5] enzymatic ligation
caused significant decomposition of nitroxides (see the Sup-
porting Information). In contrast, incubation of protected
DNA 15 with dithiothreitol at concentrations typical for
ligation protocols did not induce any visible changes.
Table 3. Photochemical deprotection of oligonucleotide 15. Ratio
of nitroxide 16 versus amine 17 formation as a function of pH.
Each sample was irradiated for 30 min (365 nm).
Solvent
pH
8.0
4.6
7.4
9.0
9.5
H2O[a]
H2O[b]
20:80
10%
50:50
40%
75:25
65%
85:15
80%
85:15
95%
[a] Quantification by HPLC. [b] Percentage of spin labeling in the
crude irradiated product as determined by continuous wave (cw)
EPR at X-band frequency; phosphate buffers for pH = 4.6–8.0;
carbonate buffers for pH = 9.0 and 9.5.
Figure 3. Synthesis of spin-labeled phosphoramidites and DNA oli-
gonucleotides.
Conclusions
Both phosphoramidites were incorporated into the
11mer DNA sequences 15 and 16 on a commercial synthe-
The formation of nitroxide spin labels from hydroxyl-
sizer by using the unmodified standard protocol for DNA amines by spontaneous air oxidation is a very mild and ef-
(see the Supporting Information). According to trityl as- ficient procedure that will hardly interfere with sensitive
says, both amidites 13 and 14 coupled with good yields. functional groups present in oligonucleotides or peptides.
However, with unprotected TEMPO a complex mixture was The removal of photolabile groups from hydroxylamines,
obtained from which pure 16 could not be isolated easily. however, is a critical step that may cause considerable losses
When the whole group of peaks around 16 was separated of spin labels. Although the recovery yields obtained from
by HPLC, the labeling degree according to EPR was only compounds 1–rac-3 in pure acetonitrile are good, we are
5%. Most early work with nitroxide-containing phos- reluctant to apply these molecules in the synthesis of com-
phoramidites[16] neither reported yields of purified oligonu- plex samples. Future work will focus on compounds 4 and
cleotides nor special conditions to minimize degradation. 5. High chemical stability and promising yields of nitroxides
Some authors, however, have tried to quantify nitroxide qualifies them as building blocks for spin-labeled oligonu-
losses in phosphoramidite chemistry owing to the presence cleotides. As a proof of concept, a nucleoside derivative of
of acids and iodine[17] or suggested optimized methods for 4 was used to synthesize DNA oligonucleotide 16. The ro-
chain assembly.[3a,3d,4] The poor result with unprotected bustness of precursor DNA 15, the yield and purity of 16
amidite 14, therefore, may be improved to some extent, but both compare favorably to samples prepared from unpro-
major losses are likely unavoidable. In contrast, HPLC- tected phosphoramidite 14. For peptide syntheses, com-
purified DNA 15 that contains the nitroxide precursor was pound 5 is preferable owing to its superior acid stability. In
Eur. J. Org. Chem. 2014, 4037–4043
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4039

M. W. Göbel et al.
FULL PAPER
(dd, J = 12.4, 3.6 Hz, 2 H, CH2), 1.17 (s, 7 H, NH and 2 CH3),
1.15 (t, J = 12.0 Hz, 2 H, CH2), 1.04 (s, 6 H, 2 CH3) ppm. 13C
NMR (62.9 MHz, CDCl3): δ = 165.4, 134.9, 130.9, 128.1, 127.2,
50.4, 44.2, 42.3, 34.6, 28.7 ppm. MS (ESI): m/z (%) = 261.5 (100)
[M + H+]. HRMS (MALDI): calcd. for C16H25N2O [M + H+]
261.1961; found 261.1965.
unprotected form, even robust nitroxide 7 decomposes to a
large extent under most conditions shown in Table 2 (see
Table S3 in the Supporting Information). It is noteworthy
that the coumarin protecting group of 4 can be removed by
two-photon excitation. This property may offer the poten-
tial to generate spin labels with high levels of spatial and
temporal resolution. The small amounts of sample used in
such experiments will allow short deprotection times in the
range of seconds.
N-(2,2,6,6-Tetramethylpiperidin-4-yloxyl)benzamide (6): A solution
of
N-(2,2,6,6-tetramethylpiperidin-4-yl)benzamide
(3.07 g,
11.8 mmol, 1.00 equiv.) in MeOH (50 mL) was basified with aque-
ous NaOH (5 m; pH = 10–12), and Na2WO4 (0.97 g, 2.95 mmol,
0.25 equiv.) was added. The reaction mixture was cooled to 0 °C,
H2O2 (30%; 9.04 mL, 89 mmol, 7.5 equiv.) was added slowly, and
the mixture was stirred at ambient temperature for 43 h. Sub-
sequently, the mixture was diluted with CH2Cl2, and K2CO3 (3 g)
was added. The phases were separated, and the aqueous layer was
extracted with CH2Cl2. The combined organic layers were dried
with MgSO4 and concentrated under reduced pressure. Purification
by silica gel chromatography (CH2Cl2/MeOH, 19:1) yielded 2 as an
orange foam (2.80 g, 87%). Rf = 0.15 (cyclohexane/EtOAc, 3:1).
MS (ESI): m/z (%) = 277.8 (100) [M + e– + 2 H+]. HRMS
(MALDI): calcd. for C16H25N2O2 [M + e– + 2 H+] 277.1910; found
277.1910 (MALDI spectra of nitroxides are dominated by the peak
of the reduced and protonated hydroxylamine).
Experimental Section
General Procedure for the Conversion of Nitroxide Radicals into
Alkylated Hydroxylamines: A solution of the corresponding ni-
troxide (500 mg) in dry tetrahydrofuran (THF; 30 mL) was stirred
with palladium (10% on charcoal) under hydrogen (balloon) at am-
bient temperature. The reaction flask was evacuated and purged
with argon. Et3N (3 equiv.) and the corresponding alkyl bromide
(1.5 equiv.) in dry THF (5 mL) were added, and the mixture al-
lowed to react at the given temperature for the time indicated be-
low. The reaction mixture was then filtered through Celite and con-
centrated at reduced pressure. The resulting residue was purified by
silica gel chromatography.
(؎)-N-{1-[1-(2-Nitrophenyl)ethoxy]-2,2,6,6-tetramethylpiperidin-4-
yl}benzamide (rac-2): According to the general procedure, nitroxide
6 (500 mg, 1.81 mmol) was hydrogenated with Pd/C (200 mg 10%)
for 3.5 h. Alkylation with Et3N (1.41 mL, 10.0 mmol) and 1-(1-
bromoethyl)-2-nitrobenzene (0.63 g, 2.74 mmol) at ambient tem-
perature for 42 h and subsequent chromatography (cyclohexane/
EtOAc, 3:1) yielded rac-2 as light yellow solid (0.65 g, 84%). M.p.
149 °C. Rf = 0.21 (cyclohexane/EtOAc, 3:1). λmax = 225, 273 nm
(MeOH). 1H NMR (300 MHz, [D6]DMSO): δ = 8.12 (d, J =
7.7 Hz, 1 H, CONH), 7.87–7.74 (m, 5 H, Ar-H), 7.55–7.40 (m, 4
H, Ar-H), 5.18 (q, J = 6.6 Hz, 1 H, CHCH3), 4.21–4.08 (m, 1 H,
CHNH), 1.75–1.69 (m, 1 H, CH2), 1.61–1.38 (m, 3 H, CH2), 1.56
(d, J = 6.6 Hz, 3 H, CHCH3), 1.27 (s, 3 H, CH3), 1.22 (s, 3 H,
CH3), 1.03 (s, 3 H, CH3), 0.47 (s, 3 H, CH3) ppm. 13C NMR
(75.4 MHz, [D6]DMSO): δ = 165.6, 147.9, 139.2, 134.6, 133.3,
131.0, 128.6, 128.4, 128.1, 127.2, 123.4, 78.4, 59.8, 59.4, 45.1, 45.0,
33.8, 32.8, 22.7, 20.6, 20.5 ppm. HRMS (MALDI): calcd. for
C24H32N3O4 [M + H+] 426.2387; found 426.2392.
N-[2,2,6,6-Tetramethyl-1-(2-nitrobenzyloxy)piperidin-4-yl]acetamide
(1): According to the general procedure, 4-acetamido-TEMPO
(500 mg, 2.34 mmol) was hydrogenated with Pd/C (60 mg 10%) for
30 min. Alkylation with Et3N (1.70 mL, 12.1 mmol) and 2-ni-
trobenzyl bromide (750 mg, 3.47 mmol) at reflux temperatures for
2 h and subsequent chromatography (EtOAc) yielded 1 as a color-
less solid (549 mg, 67%). M.p. 188 °C from ethyl acetate. Rf = 0.41
1
(EtOAc). λmax = 259 nm (MeOH). H NMR (400 MHz, CDCl3): δ
= 8.06 (dd, J = 8.0, J = 1.0 Hz, 1 H, Ar-H), 7.87 (d, J = 7.6 Hz, 1
H, Ar-H), 7.66 (t, J = 8.0 Hz, 1 H, Ar-H), 7.43 (t, J = 8.0 Hz, 1
H, Ar-H), 5.25 (d, J = 7.2 Hz, 1 H, NH), 5.20 (s, 2 H, CH2), 4.23–
4.12 (m, 1 H, CH), 1.96 (s, 3 H, acetyl-H), 1.84–1.80 (m, 2 H,
CH2), 1.37 (t, J = 12.4 Hz, 2 H, CH2), 1.28 (s, 6 H, 2 CH3) 1.19
(s, 6 H, 2 CH3) ppm. 13C NMR (62.9 MHz, CDCl3): δ = 169.3,
146.8, 134.8, 133.6, 128.3, 127.5, 124.5, 75.1, 60.3, 45.7, 41.0, 32.8,
23.5, 21.1 ppm. MS (ESI): m/z (%) = 350.3 (100) [M + H+]. HRMS
(MALDI): calcd. for C18H28N3O4 [M + H+] 350.20743; found
350.20774. C18H27N3O4 (349.42): calcd. C 61.87, H 7.79, N 12.03;
found C 61.57, H 7.60, N 12.07.
(؎)-N-{1-[1-(4,5-Dimethoxy-2-nitrophenyl)ethoxy]-2,2,6,6-tetra-
methylpiperidin-4-yl}benzamide (rac-3): According to the general
procedure, nitroxide 6 (500 mg, 1.81 mmol) was hydrogenated with
Pd/C (200 mg 10 %) for 2.5 h. Alkylation with Et3N (1.41 mL,
10.0 mmol) and 1-(1-bromoethyl)-4,5-dimethoxy-2-nitrobenzene
(0.79 g) at ambient temperature for 76 h and subsequent
chromatography (cyclohexane/EtOAc, 3:1) yielded rac-3 as a light
yellow foam (0.87 g, 98%). Rf = 0.16 (cyclohexane/EtOAc, 3:1).
λmax = 224, 300, 341 nm (MeOH). 1H NMR (300 MHz, [D6]-
DMSO): δ = 8.12 (d, J = 7.7 Hz, 1 H, CONH), 7.81–7.78 (m, 2 H,
Ar-H), 7.50–7.40 (m, 4 H, Ar-H), 7.18 (s, 1 H, Ar-H), 5.32 (q, J =
6.5 Hz, 1 H, CHCH3), 4.20–4.09 (m, 1 H, CHNH), 3.92 (s, 3 H,
OCH3), 3.85 (s, 3 H, OCH3), 1.76–1.70 (m, 1 H, CH2), 1.63–1.38
(m, 3 H, CH2), 1.56 (d, J = 6.0 Hz, 3 H, CHCH3), 1.29 (s, 3 H,
CH3), 1.24 (s, 3 H, CH3), 1.08 (s, 3 H, CH3), 0.52 (s, 3 H, CH3)
ppm. 13C NMR (75.4 MHz, [D6]DMSO): δ = 165.6, 153.0, 147.4,
139.7, 135.2, 134.6, 131.0, 128.2, 127.2, 109.6, 106.8, 78.9, 59.7,
59.4, 56.1, 56.0, 45.2, 45.0, 33.8, 32.9, 22.8, 20.6, 20.5 ppm. HRMS
(MALDI): calcd. for C26H36N3O6 [M + H+] 486.2598; found
N-(2,2,6,6-Tetramethylpiperidin-4-yl)benzamide: Benzoyl chloride
(0.78 mL, 6.77 mmol, 1.05 equiv.) was added dropwise to a solution
of 4-amino-2,2,6,6-tetramethylpiperidine (1.00 g, 6.40 mmol,
1.00 equiv.) and pyridine (0.65 mL, 8.05 mmol, 1.30 equiv.) in THF
(30 mL). The reaction mixture was stirred at ambient temperature
for 3.5 h, and water (30 mL) was added. The solution was brought
to pH ≈ 12 with aqueous NaOH solution (5 m) and extracted with
CH2Cl2. The organic layer was dried with MgSO4 and concentrated
under reduced pressure. The resulting residue was co-evaporated
with toluene and suspended in water. The aqueous layer was acidi-
fied with concd. HCl solution (pH ≈ 2) and extracted with CH2Cl2.
Subsequently, the aqueous layer was basified with aqueous NaOH
(5 m; pH ≈ 12). The resulting precipitate was filtered off and washed
with cold water. The residue was co-evaporated with toluene to give
the title compound as a colorless solid (1.17 g, 70%). M.p. 122 °C.
Rf = 0.31 (br.) (CH2Cl2/MeOH, 9:1). 1H NMR (300 MHz, [D6]-
DMSO): δ = 8.13 (d, J = 7.9 Hz, 1 H, CONH), 7.84–7.81 (m, 2 H,
Ar-H), 7.53–7.41 (m, 3 H, Ar-H), 4.34–4.22 (m, 1 H, CHNH), 1.68 486.2595.
4040
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 4037–4043

Photolabile Protecting Groups for Nitroxide Spin Labels
N-(1-{[7-(Diethylamino)-2-oxo-2H-chromen-4-yl]methoxy}-2,2,6,6- HRMS (MALDI): calcd. for C28H44N3O5K [M + K+] 540.28343;
tetramethylpiperidin-4-yl)benzamide (4): According to the general
procedure, nitroxide 6 (500 mg, 1.81 mmol) was hydrogenated with
Pd/C (200 mg 10 %) for 2.5 h. Alkylation with Et3N (1.41 mL,
10.0 mmol) and 4-(bromomethyl)-7-(diethylamino)-2H-chromen-2-
one (0.84 g, 2.71 mmol) at ambient temperature for 72 h and subse-
quent chromatography (1st: CH2Cl2/cyclohexane/EtOAc, 10:5:1;
2nd: cyclohexane/EtOAc, 4:1) yielded 4 as a yellow solid (0.74 g,
81 %). M.p. decomp. Rf = 0.12 (CH2Cl2/cyclohexane/EtOAc,
10:5:1). λmax = 237, 379 nm (MeOH). 1H NMR (300 MHz, [D6]-
DMSO): δ = 8.20 (d, J = 8.2 Hz, 1 H, CONH), 7.83 (d, J = 7.2 Hz,
2 H, Ar-H), 7.52–7.42 (m, 4 H, 3 Ar-H, 5-H), 6.68 (dd, J = 8.2, J
= 2.5 Hz, 1 H, 6-H), 6.54 (d, J = 2.5 Hz, 1 H, 8-H), 6.08 (s, 1 H,
3-H), 4.98 (s, 2 H, OCH2), 4.32–4.18 (m, 1 H, CHNH), 3.43 (q, J
= 6.7 Hz, 4 H, 2 NCH2), 1.78–1.73 (m, 2 H, CH2), 1.63–1.55 (m,
2 H, CH2), 1.24 and 1.22 (2s, 12 H, 4 CH3), 1.12 (t, J = 6.7 Hz, 6
H, 2 NCH2CH3) ppm. 13C NMR (75.4 MHz, [D6]DMSO): δ =
165.7, 160.8, 155.7, 152.3, 150.3, 134.7, 131.0, 128.1, 127.2, 125.4,
108.6, 105.4, 104.4, 96.8, 74.23, 59.8, 48.6, 44.6, 43.9, 32.4, 20.6,
12.3 ppm. HRMS (MALDI): calcd. for C30H40N3O4 [M + H+]
506.3013; found 506.3005.
found 540.28460.
4-[(4-Amino-2,2,6,6-tetramethylpiperidin-1-yloxy)methyl]-7-(diethyl-
amino)-2H-chromen-2-one (10): A suspension of 9 (1.26 g,
2.51 mmol, 1.00 equiv.) in acetonitrile (40 mL) was cooled to 0 °C,
and trimethylsilyl iodide (0.89 mL, 6.27 mmol, 2.5 equiv.) was
added in portions (100 μL/10 min). Subsequently, a concd.
NaHCO3 solution (50 mL) was added, and the reaction mixture
was stirred for 30 min. After addition of CH2Cl2/MeOH (9:1), the
organic layer was separated, and the aqueous layer was extracted
with CH2Cl2/MeOH (9:1). The combined organic layers were dried
with MgSO4, and the solvent was evaporated under reduced pres-
sure. Purification by silica gel chromatography (CH2Cl2/MeOH,
9:1) gave 10 as a light yellow solid (0.79 g, 79 %). Rf = 0.32
1
(CH2Cl2/MeOH, 9:1). H NMR (500 MHz, CDCl3): δ = 7.41 (d, J
= 9.0 Hz, 1 H, 5-H), 6.67 (dd, J = 9.0, 2.2 Hz, 1 H, 6-H), 6.53 (d,
J = 2.3 Hz, 8-H), 6.06 (s, 1 H, 3-H), 5.90 (br. s, 2 H, NH2), 4.96
(s, 2 H, OCH2), 3.42 (q, J = 6.9 Hz, 4 H, CH2), 3.25 (t, J = 12.2 Hz,
1 H, CHNH2), 1.75 (d, J = 9.8 Hz, 2 H, CHH), 1.42 (t, J = 12.2 Hz,
2 H, CHH), 1.19 (s, 6 H, CH3), 1.17 (s, 6 H, CH3), 1.11 (t, J =
7.0 Hz, 6 H, CH2CH3) ppm. 13C NMR (125.8 MHz, CDCl3): δ =
160.8, 155.7, 152.2, 150.3, 125.4, 108.6, 105.3, 104.3, 96.8, 74.2,
59.5, 44.9, 44.0, 41.8, 32.3, 20.5, 12.3 ppm. MS (ESI): m/z = 403.0
[M + H+]. HRMS (MALDI): calcd. for C23H36N3O3 [M + H+]
402.27512; found 402.27524.
1,1,3,3-Tetraethyl-2-(2-nitrobenzyloxy)isoindoline (5): According to
the general procedure, nitroxide 7[11] (500 mg, 2.03 mmol) was hy-
drogenated with Pd/C (70 mg 10%) for 30 min. Alkylation with
Et3N (0.85 mL, 6.05 mmol) and 2-nitrobenzyl bromide (0.65 g,
3.01 mmol) at reflux temperatures for 2 h and subsequent
chromatography (CH2Cl2/n-hexane, 1:1) yielded 5 as a brightly yel-
low solid (327 mg, 42%). M.p. 81 °C (ethanol). Rf = 0.51 (CH2Cl2/
n-hexane, 1:2). λmax = 259, 264, 271 nm (MeOH). 1H NMR
(400 MHz, CDCl3): δ = 8.07 (dd, J = 8.2, J = 1.0 Hz, 1 H, Ar-H),
7.85 (d, J = 7.6 Hz, 1 H, Ar-H), 7.67 (dt, J = 7.6, J = 1.2 Hz, 1 H,
Ar-H), 7.46 (t, J = 8.0 Hz, 1 H, Ar-H), 7.25–7.21 (m, 2 H, Ar-H),
7.07–7.03 (m, 2 H, Ar-H), 5.29 (s, 2 H, CH2), 2.10–1.79 (m, 8 H,
4 CH2CH3), 0.95–0.78 (m, 12 H, 4 CH2CH3) ppm. 13C NMR
(100.6 MHz, CDCl3): δ = 147.3, 142.1, 134.5, 133.5, 129.4, 127.9,
126.3, 124.5, 123.4, 73.6, 73.3, 30.4, 29.3, 9.4, 9.0 ppm. MS (ESI):
m/z (%) = 383.5 (100) [M + H+]. HRMS (MALDI): calcd. for
C23H31N2O3 [M + H+] 383.23292; found 383.23249. C23H30N2O3
(382.50): calcd. C 72.22, H 7.91, N 7.32; found C 72.13, H 7.86, N
7.16.
3Ј-O-Acetyl-N-(1-{[7-(diethylamino)-2-oxo-2H-chromen-4-yl]-
methoxy}-2,2,6,6-tetramethylpiperidin-4-yl)-5Ј-O-DMT-2Ј-deoxy-
cytidine (11): To a solution of 3Ј-O-acetyl-5Ј-O-DMT-2Ј-deoxyurid-
ine[14] (0.693 g, 1.21 mmol, 1.00 equiv.) in CH2Cl2 (40 mL) Et3N
(1.53 mL, 10.89 mmol, 9.00 equiv.) and 4-(dimethylamino)pyridine
(0.02 g, 0.18 mmol, 0.15 equiv.) were added. The reaction mixture
was cooled to 0 °C, treated with 2,4,6-triisopropylbenzene-1-sulfu-
ryl chloride (0.48 g, 1.59 mmol, 1.32 equiv.) and stirred at 0 °C for
10 min. The mixture was warmed to ambient temperature and
stirred for 2 h. The reaction was quenched with concd. NaHCO3
solution. The organic layer was separated, and the aqueous layer
was extracted with CH2Cl2. The combined organic layers were
dried with MgSO4, and the solvent was removed under reduced
pressure. The residue was dissolved in CH2Cl2, and Et3N (1.53 mL,
10.89 mmol, 9.00 equiv.) and amine 10 (0.73 g, 1.81 mmol,
tert-Butyl-1-{[7-(diethylamino)-2-oxo-2H-chromen-4-yl]methoxy}- 1.50 equiv.) were added. The reaction mixture was stirred at ambi-
2,2,6,6-tetramethylpiperidin-4-ylcarbamate (9): A solution of 4- ent temperature for 19 h, concentrated in vacuo and purified by
[(tert-butyloxycarbonyl)amino]-2,2,6,6-tetramethylpiperidin-1- silica gel chromatography (EtOAc/Et3N, 100:1) to give nucleoside
oxyl[12] 8 (1.73 g, 6.38 mmol, 1.00 equiv.) in THF (45 mL) was
treated with Pd/C (200 mg 10%) and stirred under hydrogen (bal-
loon) at ambient temperature for 1.5 h. The flask was evacuated
11 as a light yellow solid (0.25 g, 22%). Rf = 0.18 (EtOAc). 1H
NMR (500 MHz, [D6]DMSO): δ = 7.63 (d, J = 7.6 Hz, 1 H, NH),
7.58 (d, J = 7.6 Hz, 1 H, 6-H), 7.43 (d, J = 9.1 Hz, 1 H, Ar-H),
and flushed with argon. Et3N (2.69 mL, 19.14 mmol, 3.00 equiv.) 7.37 (m, 2 H, Ar-H), 7.32 (t, J = 7.7 Hz, 2 H, Ar-H), 7.25–7.23 (m,
and 4-(bromomethyl)-7-(diethylamino)-2H-chromen-2-one (2.97 g,
9.57 mmol, 1.50 equiv.) were added slowly. After stirring at ambient
temperature for 67 h, the reaction mixture was filtered through sil-
ica gel and concentrated in vacuo. Purification by silica gel
chromatography (CH2Cl2/EtOAc, 10:1) gave 9 as a light yellow so-
lid (0.98 g, 31 %). Rf = 0.44 (CH2Cl2/EtOAc, 10:1). 1H NMR
(500 MHz, CDCl3): δ = 7.23 (d, J = 9.0 Hz, 1 H, 5-H), 6.54 (dd, J
= 9.0, 2.5 Hz, 1 H, 6-H), 6.50 (d, J = 2.5 Hz, 8-H), 6.28 (s, 1 H, 3-
H) 4.94 (s, 2 H, OCH2), 4.27 (br. s, 1 H, NHCH), 3.83 (br. s, 1 H,
5 H, Ar-H), 6.89 (dd, J = 8.9, 1.6 Hz, 4 H, Ar-H), 6.68 (dd, J =
9.2, 2.4 Hz, 1 H, 6-H), 6.53 (d, J = 2.4 Hz, 1 H, 8-H), 6.18 (t, J =
6.3 Hz, 1 H, H1Ј), 6.08 (s, 1 H, 3-H), 5.57 (d, J = 7.6 Hz, 1 H, 5-
H), 5.23–5.21 (m, 1 H, H3Ј), 4.97 (s, 2 H, CH2ON), 4.28–4.20 (m,
1 H, NHCH), 4.07 (q, J = 3.4 Hz, 1 H, H4Ј), 3.74 (s, 6 H, OCH3),
3.42 (q, J = 6.9 Hz, 4 H, CH2CH3), 3.33–3.30 (m, 1 H, H5Ј), 3.22
(dd, J = 10.3, 3.1 Hz, 1 H, H5ЈЈ), 2.34–2.24 (m, 2 H, H2Ј, H2ЈЈ),
2.04 (s, 3 H, COCH3), 1.80 (d, J = 15.0 Hz, 2 H, CHHCH), 1.38
(t, J = 12.3 Hz, 2 H, CHHCH), 1.22 (s, 6 H, CH3), 1.20 (s, 6 H,
CHNH), 3.40 (q, J = 7.0 Hz, 4 H, CH2), 1.85 (d, J = 11.5 Hz, 2 CH3), 1.11 (t, J = 7.0 Hz, 6 H, CH2CH3) ppm. 13C NMR
H, CHH), 1.45 (s, 6 H, tBu), 1.33 (t, J = 12.5 Hz, 2 H, CHH), 1.26 (125.8 MHz, [D6]DMSO): δ = 170.0, 162.6, 160.8, 158.1, 155.8,
(s, 6 H, CH3), 1.21 (s, 6 H, CH3), 1.20 (t, J = 7.0 Hz, 6 H, CH2CH3) 154.8, 152.3, 150.3, 144.6, 139.5, 135.3, 135.2, 129.7, 127.9, 127.7,
ppm. 13C NMR (125.8 MHz, CDCl3): δ = 162.5, 156.3, 155.3,
151.9, 150.5, 124.6, 108.5, 106.5, 106.0, 98.0, 74.6, 60.6, 46.1, 44.9,
42.2, 32.9, 28.6, 21.1, 12.6 ppm. MS (ESI): m/z = 503.5 [M + H+].
126.8, 125.5, 113.3, 108.7, 105.4, 104.4, 96.9, 94.9, 86.0, 84.9, 82.8,
74.31, 74.25, 63.5, 59.8, 55.1, 44.6, 44.0, 40.7, 37.2, 32.4, 20.8,
20.7, 12.3 ppm. MS (ESI): m/z (%) = 957.7 [M + H+]. HRMS
Eur. J. Org. Chem. 2014, 4037–4043
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4041

M. W. Göbel et al.
FULL PAPER
(MALDI): calcd. for C55H65N5O10Na [M + Na+] 978.46236; found
978.46477.
DMSO): δ = 147.6, 147.1 ppm. MS (ESI): m/z (%) = 1116.0 [M
+ H+]. HRMS (MALDI): calcd. for C62H80N7O10Na [M + Na+]
1136.55965; found 1136.56008.
N-(1-{[7-(Diethylamino)-2-oxo-2H-chromen-4-yl]methoxy}-2,2,6,6-
tetramethylpiperidin-4-yl)-5Ј-O-DMT-2Ј-deoxycytidine (12): To a
solution of nucleoside 11 (0.25 g, 0.26 mmol, 1.00 equiv.) in MeOH
(30 mL) NaHCO3 (0.33 g, 3.91 mmol, 15.00 equiv.) was added. The
suspension was stirred at ambient temperature for 6 h, a mixture
of CH2Cl2/MeOH/Et3N (95:5:1) was added, and the solution was
filtered through neutral silica gel. After evaporation under reduced
pressure, the title compound is obtained as a yellow foam (0.23 g,
Photochemical Removal of Protecting Groups: 300 μL of a 0.2–
0.3 mm solution of the alkylated hydroxylamines was illuminated
in a round glass cuvette (Carl Roth, 50 mmϫ10 mm diameter). For
compounds 1, rac-2, rac-3, 5 and 15 a custom-built setup contain-
ing three light-emitting diodes was used (Nichia NCCU033,
365 nm, each with 100 mW optical output power). The irradiation
of compound 4 was performed in a custom-built setup containing
96%). Rf = 0.24 (CH2Cl2/MeOH, 19:1). 1H NMR (500 MHz, [D6]- a single LED (Roithner H2A1-H405; 405 nm, 105 mW optical out-
DMSO): δ = 7.61 (d, J = 7.5 Hz, 1 H, NH), 7.58 (d, J = 8.0 Hz,
6-H), 7.43 (d, J = 9.0 Hz, Ar-H), 7.38 (d, J = 9.5 Hz, 2 H, Ar-H),
7.32 (t, J = 7.7 Hz, 2 H, Ar-H), 7.26–7.24 (m, 5 H, Ar-H), 6.90
(dd, J = 8.9, 1.7 Hz, 4 H, Ar-H), 6.68 (dd, J = 9.1, 2.4 Hz, 1 H, 6-
H), 6.53 (d, J = 2.4 Hz, 1 H, 8-H), 6.16 (t, J = 6.3 Hz, 1 H, H1Ј),
6.08 (s, 1 H, 3-H), 5.53 (d, J = 7.5 Hz, 1 H, 5-H), 5.30 (d, J =
4.7 Hz, 1 H, H3Ј), 4.97 (s, 2 H, CH2ON), 4.36 (d, J = 4.1 Hz, 1 H,
3Ј-OH), 4.27–4.20 (m, 2 H, H4Ј, NHCH), 3.74 (s, 6 H, OCH3),
3.42 (q, J = 6.9 Hz, 4 H, CH2CH3), 3.21–3.16 (m, 2 H, H5Ј, H5ЈЈ),
2.20–2.15 (m, 1 H, H2Ј), 2.05–2.00 (m, 1 H, H2ЈЈ), 1.79 (d, J =
13.3 Hz, 2 H, CHHCH), 1.38 (t, J = 12.3 Hz, 2 H, CHHCH), 1.22
(s, 6 H, CH3), 1.20 (s, 6 H, CH3), 1.11 (t, J = 7.0 Hz, 6 H, CH2CH3)
ppm. 13C NMR (125.8 MHz, [D6]DMSO): δ = 162.6, 160.8, 158.1,
155.8, 154.9, 152.3, 150.3, 144.8, 139.6, 135.5, 135.3, 129.7, 127.9,
127.7, 126.8, 125.5, 113.2, 108.7, 105.4, 104.4, 96.9, 94.5, 85.7, 85.2,
84.7, 74.3, 70.0, 63.4, 62.0, 59.8, 55.1, 44.6, 44.0, 40.7, 40.5, 32.4,
20.7, 12.3 ppm. MS (ESI): m/z (%) = 915.6 [M + H+]. HRMS
(MALDI): calcd. for C53H63N5O9Na [M + Na+] 936.45180; found
936.45396.
put power).
Recovery of Spin Labels, Detection by EPR: The cw EPR measure-
ments were performed at X-band frequency (9.54 GHz) with a
Bruker E500 spectrometer equipped with a TE102 cavity. Experi-
mental parameters: 100 kHz modulation frequency, 0.1 mT modu-
lation amplitude, 0.2 mW microwave power, 40.96 ms time con-
stant, 40.96 ms conversion time, 1024 points, 7 mT sweep width, 20
scans. Directly after irradiation, 20 μL aliquots were placed in
quartz EPR tubes of 1 mm inner diameter. The EPR signal was
recorded as the first derivative of the absorption signal. Double
integral intensities were calculated after baseline correction. Ni-
troxide spin concentrations were determined by analyzing these in-
tensities with those of calibrated test samples.
Supporting Information (see footnote on the first page of this arti-
cle): Additional preparative details and characterization data; sta-
bility tests for compounds 4, 5, and nitroxide 7; HPLC conditions
to determine nitroxide recovery from precursors 4 and 5; UV/Vis
spectra of compounds 1–5; synthesis, analysis, and photochemical
deprotection of DNA 15; partial loss of spin labels during enzy-
Deoxycytidine Phosphoramidite with Protected Spin Label 13: To a
solution of nucleoside 12 (0.44 g, 0.48 mmol, 1.00 equiv.) in CH2Cl2
(30 mL) Et3N (0.40 mL, 2.88 mmol, 6.00 equiv.) and N,N-(diiso-
propylamino)(cyanoethyl)phosphonamidic chloride (0.34 g,
1.44 mmol, 3.00 equiv.) were added. The reaction mixture was
stirred at ambient temperature for 21 h. Subsequently concd.
NaHCO3 solution (40 mL) was added and stirred at ambient tem-
perature for 5 min. The organic layer was separated, and the aque-
ous layer was extracted with CH2Cl2. The combined organic layers
were dried with MgSO4, and the solvent was removed under re-
duced pressure. Purification by silica gel chromatography (EtOAc/
Et3N, 100:1) gave the title compound as a yellow foam (0.38 g,
71 %). Rf = 0.19, 0.39 (EtOAc; 2 diastereomers). 1H NMR
(500 MHz, [D6]DMSO): δ = 7.66–7.59 (m, 2 H, NH, 6-H), 7.43 (d,
J = 9.0 Hz, Ar-H), 7.39–7.36 (m, 2 H, Ar-H), 7.31 (m, 2 H, Ar-H),
7.27–7.23 (m, 5 H, Ar-H), 6.90–6.86 (m, 4 H, Ar-H), 6.68 (dd, J =
9.2, 2.4 Hz, 1 H, 6-H), 6.53 (d, J = 2.4 Hz, 1 H, 8-H), 6.19–6.14
(m, 1 H, H1Ј), 6.08 (s, 1 H, 3-H), 5.56 and 5.55 (2d, J = 7.0 Hz, 1
H, 5-H), 4.97 (s, 2 H, CH2ON), 4.53–4.46 (m, 1 H, NHCH), 4.29–
4.20 (m, 1 H, H3Ј), 4.00–3.95 (m, 1 H, H4Ј), 3.74 (s, 3 H, OCH3),
3.73 (s, 3 H, OCH3), 3.69–3.47 [m, 4 H, POCH2, NCH(CH3)2],
3.42 (q, J = 6.8 Hz, 4 H, CH2CH3), 3.29–3.22 (m, 2 H, H5Ј, H5ЈЈ),
2.76 (t, J = 7.5 Hz, 1 H, CHHCN), 2.65 (t, J = 7.5 Hz, 1 H,
CHHCN), 2.36–2.29 (m, 1 H, H2Ј), 2.24–2.17 (m, 1 H, H2ЈЈ), 1.79
(d, J = 12.5 Hz, 2 H, CHHCH), 1.38 (t, J = 12.3 Hz, 2 H,
CHHCH), 1.22–1.17 (m, 15 H, CH3), 1.13–1.08 (m, 15 H, CH3,
CH2CH3) ppm (2 diastereomers due to chiral P). 13C NMR
(125.8 MHz, [D6]DMSO): δ = 162.6, 160.8, 158.1, 155.8, 154.8,
152.3, 150.3, 144.7, 139.9, 135.3, 135.2, 131.6, 129.7, 127.9, 127.7,
127.6, 126.8, 125.5, 119.0, 118.8, 118.6, 113.2, 108.7, 105.4, 104.4,
96.9, 94.8, 94.7, 85.9, 85.8, 84.9, 74.3, 63.0, 62.7, 59.8, 58.4, 58.3,
58.2, 58.1, 55.1, 44.6, 44.5, 44.0, 42.6, 42.5, 40.7, 32.4, 24.3 22.6,
20.7, 19.8, 19.4, 14.1, 12.3 ppm. 31P NMR (202.5 MHz, [D6]-
matic ligation; H and 13C NMR spectra of all key intermediates
1
and final products.
Acknowledgments
Financial support by the Deutsche Forschungsgemeinschaft (col-
laborative research center 902) is gratefully acknowledged.
[1] a) J. F. W. Keana, Chem. Rev. 1978, 78, 37–64; b) H. Dugas,
Acc. Chem. Res. 1977, 10, 47–54; c) T. Gnewuch, G. Sosnovsky,
Chem. Rev. 1986, 86, 203–238; d) I. Sengupta, P. S. Nadaud,
C. P. Jaroniec, Acc. Chem. Res. 2013, 46, 2117–2126; e) C. Al-
tenbach, A. K. Kusnetzow, O. P. Ernst, K. P. Hofmann, W. L.
Hubbell, Proc. Natl. Acad. Sci. USA 2008, 105, 7439–7444; f)
E. Bordignon, H.-J. Steinhoff, in ESR spectroscopy in mem-
brane biophysics (Eds.: M. A. Hemminga, L. J. Berliner),
Springer Science, New York, 2007, pp. 129–164.
[2] a) G. Jeschke, Annu. Rev. Phys. Chem. 2012, 63, 419–446; b) O.
Schiemann, T. F. Prisner, Quart. Rev. Biophys. 2007, 40, 1–53.
[3] a) O. Schiemann, N. Piton, J. Plackmeyer, B. E. Bode, T. F.
Prisner, J. W. Engels, Nat. Protoc. 2007, 2, 904–923; b) G. Sic-
oli, F. Wachowius, M. Bennati, C. Höbartner, Angew. Chem.
Int. Ed. 2010, 49, 6443–6447; Angew. Chem. 2010, 122, 6588–
6592; c) S. A. Shelke, S. T. Sigurdsson, Eur. J. Org. Chem. 2012,
2291–2301; d) M. Azarkh, V. Singh, O. Okle, I. T. Seemann,
D. R. Dietrich, J. S. Hartig, M. Drescher, Nat. Protoc. 2013, 8,
131–147.
[4] N. Barhate, P. Cekan, A. P. Massey, S. T. Sigurdsson, Angew.
Chem. Int. Ed. 2007, 46, 2655–2658; Angew. Chem. 2007, 119,
2709–2712.
[5] L. Büttner, J. Seikowski, K. Wawrzyniak, A. L. Ochmann, C.
Höbartner, Bioorg. Med. Chem. 2013, 21, 6171–6180.
[6] a) F. W. Keana, G. S. Heo, G. T. Gaughan, J. Org. Chem. 1985,
50, 2346–2351; b) D. R. Alessi, J. E. T. Corrie, J. Feeney, I. P.
4042
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 4037–4043

Photolabile Protecting Groups for Nitroxide Spin Labels
Trayer, D. R. Trentham, J. Chem. Soc. Perkin Trans. 1 1991,
2243–2247; c) A. T. Yordanov, K. Yamada, M. C. Krishna, A.
Russo, J. Yoo, S. English, J. B. Mitchell, M. W. Brechbiel, J.
Med. Chem. 2002, 45, 2283–2288; d) K. E. Fairfull-Smith, F.
Brackmann, S. E. Bottle, Eur. J. Org. Chem. 2009, 49, 1902–
1915.
[11] K. S. Chan, K. W. Mak, M. K. Tse, S. K. Yeung, B. Z. Li, Y. W.
Chan, J. Organomet. Chem. 2008, 693, 399–407.
[12] A. Glaser, S. Haremza, S. Schambony, PCT Int. Appl. 2010,
WO 2010/023115 A1.
[13] F. Seela, W. Herdering, A. Kehne, Helv. Chim. Acta 1987, 70,
1649–1660.
[7] B. A. Chalmers, J. C. Morris, K. E. Fairfull-Smith, R. S.
Grainger, S. E. Bottle, Chem. Commun. 2013, 49, 10382–10384.
[14] A. Heckel, M. C. R. Buff, M.-S. L. Raddatz, J. Müller, B.
Pötzsch, G. Mayer, Angew. Chem. Int. Ed. 2006, 45, 6748–6750;
Angew. Chem. 2006, 118, 6900–6902.
[15] a) C. Giordano, F. Fratini, D. Attanasio, L. Cellai, Synthesis
2001, 565–572; b) P. Cekan, S. T. Sigurdsson, J. Am. Chem.
Soc. 2009, 131, 18054–18056.
ˇ
[8] P. Klán, T. Solomek, C. G. Bochet, A. Blanc, R. Givens, M.
Rubina, V. Popik, A. Kostikov, J. Wirz, Chem. Rev. 2013, 113,
119–191.
[9] C. Brieke, F. Rohrbach, A. Gottschalk, G. Mayer, A. Heckel,
Angew. Chem. Int. Ed. 2012, 51, 8446–8476; Angew. Chem. [16] A. Spaltenstein, B. H. Robinson, P. B. Hopkins, J. Am. Chem.
2012, 124, 8572–8604.
Soc. 1988, 110, 1299–1301.
[10] a) V. D. Sholle, V. A. Golubev, É. G. Rozantsev, Bull. Acad. Sci.
USSR Div. Chem. Sci. (Engl. Transl.) 1972, 21, 1163–1165; b)
K. Hideg, J. Csekö, H. O. Hankovszky, P. Sohár, Can. J. Chem.
1986, 64, 1482–1490.
[17] R. A. Persichetti, R. R. Sinden, J.-L. Duh, A. M. Bobst, Synth.
Commun. 1991, 21, 1013–1019.
Received: November 11, 2013
Published Online: May 27, 2014
Eur. J. Org. Chem. 2014, 4037–4043
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4043