Synthesis of new spin labels for Cu-free click conjugation

Article abstract of DOI:10.1016/j.tetlet.2011.03.077

New pyrroline nitroxides attached to a terminal acetylenic sulfone, a dibenzocyclooctyne or a cyclooctyne carboxylic acid were synthesized and tested in Cu-free click reactions to conjugate these new spin labels with 4-azido-TEMPO, azidophenylalanine and

Full text of DOI:10.1016/j.tetlet.2011.03.077

Tetrahedron Letters 52 (2011) 2747–2749
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of new spin labels for Cu-free click conjugation
a
b
c
b
a,
⇑
}
Tamás Kálai , Mark R. Fleissner , József Jeko , Wayne L. Hubbell , Kálmán Hideg
a Department of Organic and Medicinal Chemistry, University of Pécs, H-7602 Pécs, PO Box 99, Hungary
b Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA 90095-7008, USA
c Department of Chemistry, College of Nyíregyháza, H-4440 Nyíregyháza, Sóstói st. 31/B, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
New pyrroline nitroxides attached to a terminal acetylenic sulfone, a dibenzocyclooctyne or a cyclooc-
tyne carboxylic acid were synthesized and tested in Cu-free click reactions to conjugate these new spin
labels with 4-azido-TEMPO, azidophenylalanine and an azidophenylalanine-containing protein.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 13 December 2010
Revised 9 March 2011
Accepted 18 March 2011
Available online 8 April 2011
Keywords:
Amino acids
Acetylenes
Click chemistry
Free radicals
Spin labeling
The incorporation of nitroxides into various biological and non-
biological structures followed by EPR analysis has emerged as an
important technology. The spin labeling method, introduced by
McConnell and co-worker,1 is an effective tool to explore structure,
dynamics and interactions of complex biomolecules such as pro-
teins, nucleic acids, and polysaccharides. Spin labels can be site-
specifically introduced into proteins by covalent chemical bond
formation with sulfhydryl-specific reagents, such as the reaction
of methanethiosulfonates with cysteine,2 or by genetic incorpora-
tion.3 However, this particular labeling approach is not useful
when native, reactive thiols are present in the protein of interest.
To circumvent this limitation, it has recently become possible to
investigate biomolecules in vivo or in vitro by using bioorthogonal
chemical reactions (i.e., reactions based on functional groups not
found in biology). Such reactions must have fast rates under phys-
iological conditions and be inert to the myriad of functionalities
present in biomolecules.4 To that end, an orthogonal spin labeling
strategy based on the genetically encoded amino acid p-acetyl-
mentioned oxo-hydroxylamine conjugation, such as Staudinger
ligations,7 Diels–Alder reactions with alkenes, and azide–alkyne
reactions, which are 1,3-dipolar cycloadditions of organic azides
with alkynes.8,9 In the presence of Cu(I), azide–alkyne coupling
has been established as one of the most versatile means for the
covalent assembly of complex molecules10 and tagging fluoro-
phores to biomolecules.11
Although bioorthogonal techniques are quite widespread in
fluorescence labeling,12 their applications in spin labeling are cur-
rently limited. Our laboratory reported a series of paramagnetic
monofunctional and bifunctional azides, and monofunctional acet-
ylenes for modifying biomolecules in Cu(I) catalyzed reactions.13
However, it would be most useful to develop nitroxide reagents
that can react with azides or acetylenes under physiological condi-
tions in the absence of cytotoxic Cu(I) ions., that is, Cu-free click
conjugation. In this report, we consider spin labels with an acety-
lene function that undergo facile reaction with azides in the ab-
sence of Cu(I). Because the chemical properties of stable free
nitroxide radicals limit the range of reactions and procedures
applicable, it was obvious that the ‘clickable’ (acetylene) part could
not be formed on the nitroxide ring, but that the nitroxide should
be attached to an appropriate acetylene moiety. We have explored
two different strategies to promote the [3+2] cycloaddition under
mild conditions: (1) use of an alkyne activated with an electron-
withdrawing group,14,15 and (2) use of a strained alkyne.16 As far
as we know, this is the first report of the synthesis and model reac-
tions for azide specific, biocompatible spin label reagents.
L-phenylalanine was recently introduced, in which a ketoxime-
linked spin label was generated by selective modification of the
ketone group with a hydroxylamine nitroxide.5
In addition to p-acetyl-
genetically encode over seventy unnatural amino acids,6 including
many with bioorthogonal functional groups (e.g., p-azido- -phen-
L-phenylalanine, it is now possible to
L
ylalanine). These unnatural amino acids allow several other
bioorthogonal reactions for attaching spin labels beyond the afore-
Treatment of compound 117 with chlorosulfonic acid at 0 °C in
an aromatic electrophilic substitution reaction18 gave sulfonyl
⇑
Corresponding author. Tel.: +36 72 536 221; fax: +36 72 536 219.
E-mail address: kalman.hideg@aok.pte.hu (K. Hideg).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.03.077

2748
T. Kálai et al. / Tetrahedron Letters 52 (2011) 2747–2749
chloride 2 in 73% yield. Reaction of compound 2 with HCl gas in
ethyl acetate containing an equivalent amount of ethanol gave
the hydroxylamine salt, which was treated with 3 equiv of alumi-
num chloride and 3 equiv of bis(trimethylsilyl)acetylene in dichlo-
romethane19 to give compound 3 (HO-4429) in 15% yield. Reaction
of compound 3 with 4-azido-TEMPO (4)20 in CH2Cl2 at room tem-
perature yielded biradical 5 as a mixture of the 1,4-disubstituted
triazole as the main isomer and a non-separable minor (1,2-disub-
stituted) isomer,14 suggesting that compound 3 is a candidate for
azido-specific spin labeling (Scheme 1).
Br
a
+
N
O
7
O
HO
O
6
8
N
O
+
O
b
O
N
EtO
NH
Because ethynyl sulfones are powerful Michael acceptors19 that
can react with many nucleophiles, we explored using strained
acetylenes, namely cyclooctynes, for generating azide specific nitr-
oxide reagents in order to improve selectivity. Currently, several
cyclooctyne rings have been reported for copper-free click chemis-
try.21 For modification with a nitroxide, we chose the dibenzocyc-
looctyne, 11,12-didehydro-5,6-dihydrodibenzo[a,e]cycloocten-5-
ol (6),22 which was alkylated with the allylic bromide23 7 in the
presence of NaH in a THF/DMF (10:7) mixture. The resulting ether
8 (HO-4389) showed limited water solubility, but underwent a
[3+2] cycloaddition reaction in CH2Cl2 at ambient temperature
O
O
O
EtO
N
NH
N
N
3
9
O
N
with N-protected
D
,L
-4-azidophenylalanine ester (9)24 to give an
10
O
inseparable mixture of regioisomers of compound 10 (Scheme 2).
Another possibility was the application of 1-fluorocyclooct-2-
ynecarboxylic acid (11),25 an easily available cyclooctyne moiety,
to react with a paramagnetic amine. Treatment of 11 in THF with
1,10-carbonyldiimidazole (CDI) gave an imidazolide that was not
isolated, but used directly for acylating 1226 to give amide 13
(HO-4451) in 39% yield. Reaction of compound 13 in CH2Cl2 with
the protected azidophenylalanine 9 gave a 1:1 mixture of triazole
regioisomers of compound 14, suggesting its applicability for mod-
ification of azides (Scheme 3).
Scheme 2. Reagents and conditions: (a) THF, NaH (1.0 equiv) 30 min, 0 °C, then 7
(1.0 equiv), DMF, 40 °C, 1 h, rt 12 h, 43%; (b) 9 (1.0 equiv), CH2Cl2, rt, 1 h, 45%
(isolated).
H
N
O
NH2
CO2H
a
F
F
+
N
O
N
O
The utility of compound 13 for modifying azide-containing pro-
teins was demonstrated by reacting it with a T4 lysozyme mutant
11
13
12
b
O
SO2Cl
EtO
O
N
N
N
H
O
F
NH
O
a
N
N
N
O
1
O
N
2
O
S
O
14
b
Scheme 3. Reagents and conditions: (a) CDI (1.1 equiv), THF, reflux 10 min, then 12
(1.0 equiv), reflux, 30 min, 39%; (b) 13 (1.0 equiv), 9 (1.0 equiv), CH2Cl2, rt, 1 h, 38%
(isolated).
N3
O
+
N
N
O
4
O
containing p-azido-L-phenylalanine at site 109 (T4L 109p-AzF). The
c
resulting X-band EPR spectrum of the triazole-linked nitroxide side
chain (Fig. 1) shows restricted nitroxide motion, as evidenced by
the reduced intensities of the low- and high-field resonance lines
relative to the central line, indicating that compound 13 is linked
to the protein.
3
O
S
N
N
In conclusion, we have synthesized new, azido-specific,
biocompatible nitroxide reagents suitable for generating spin labels
via Cu-free click chemistry. All of the new labels were tested in mod-
el reactions, and their triazole adducts were isolated for mass spec-
trometric identification. The utility of compound 13 was
O
N
N
N
O
O
demonstrated by modifying a p-azido-L-phenylalanine-containing
5
protein for the assembly of spin label-protein conjugates. This
orthogonal azide and cyclooctyne click reaction spin labeling meth-
odology proved superior to modification of ketones with a nitroxide
hydroxylamine5 in that the reaction is faster and takes place at neu-
tralpH. It is anticipatedthathighly specificspin label reagentscan be
Scheme 1. Reagents and conditions: (a) HSO3Cl, excess, 0 °C?rt, 2 h, then work-up,
PbO2 (0.2 equiv), O2, 10 min, 73%; (b) HCl gas, excess, EtOH (1.0 equiv), EtOAc, then
evaporation, AlCl3 (3 equiv), Me3SiC„CSiMe3 (3 equiv), CH2Cl2, 0 °C? rt, 24 h, then
work-up with H2O, NaNO2 (1 equiv), 15%; (c) 4 (1 equiv), CH2Cl2, rt, 12 h, 23%
(isolated).

T. Kálai et al. / Tetrahedron Letters 52 (2011) 2747–2749
2749
Supplementary data
N
Protein
N
N
H
N
O
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.03.077.
F
References and notes
N
O
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Figure 1. Room temperature EPR spectrum of a T4L 109p-AzF modified with
compound 13 in a 30% sucrose solution (sucrose was added to the protein solution
only to increase the solution viscosity, which aids in spectral analysis). Expression
of the T4L 109p-AzF mutant was performed as described by Fleissner et al.5 using
}
the pEVOL-pAzF plasmid27 and 2 mM p-azido-
L-phenylalanine. Sweep width is
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100 Gauss.
}
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}
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prepared using this methodology, and the synthesis and biophysical
studies of other azido-specific spin label reagents28 for click chemis-
try is currently an ongoing area of research in our laboratories.
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Acknowledgements
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27. Young, T. S.; Ahmad, I.; Yin, J. A.; Schultz, P. G. J. Mol. Biol. 2010, 395, 361.
28. The water solubilities of the acetylenic nitroxide reagents are limited.
However, only very low concentrations of nitroxide reagent are necessary for
spin labeling reactions.
This work was supported by grants from the Hungarian Na-
tional Research Fund (OTKA K 81123), the National Institutes of
Health (EY05216), and the Jules Stein Professorship. The authors
thank Mária Balog and Evan Brooks for technical assistance, and
Krisztina Kish for elemental analysis.