The synthesis of EPR differentiable spinlabels and their coupling to uridine

Article abstract of DOI:10.1080/15257770701490522

For EPR measurements of RNA, DNA, or proteins, the occurrence of the paramagnetic species is necessary. The aim of this work is to improve the synthesis of two different EPR spinlabels 2,2,6,6-tetra methyl-3,4-dehydro- piperidin-N-oxyl-4-acetylene (TEMPA) 6 and 15N-labeled TEMPA 6* and their coupling to uridine. The yield of the synthesis of TEMPA could be increased to 40% and the second nitroxide 2,2,6,6-tetramethyl-3,4-dehydro-piperidin-15N-oxyl- 4-acetylene 6* could be synthesized with a yield of 11%. Copyright Taylor & Francis Group, LLC.

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The Synthesis Of Epr Differentiable
Spinlabels And Their Coupling To Uridine
Olga Frolow a , Bela E. Bode b & Joachim W. Engels a
a Johann W. Goethe Universität Frankfurt am Main, Institut für
Organische Chemie und Chemische Biologie (OCCB), Frankfurt am
Main, Germany
b Institut für Physikalische und Theoretische Chemie, Frankfurt am
Main, Germany
Published online: 10 Dec 2007.
To cite this article: Olga Frolow , Bela E. Bode & Joachim W. Engels (2007): The Synthesis Of Epr
Differentiable Spinlabels And Their Coupling To Uridine, Nucleosides, Nucleotides and Nucleic Acids,
26:6-7, 655-659
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Nucleosides, Nucleotides, and Nucleic Acids, 26:655–659, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701490522
THE SYNTHESIS OF EPR DIFFERENTIABLE SPINLABELS
AND THEIR COUPLING TO URIDINE
Olga Frolow
Johann W. Goethe Universita¨t Frankfurt am Main, Institut fu¨r
2
Organische Chemie und Chemische Biologie (OCCB), Frankfurt am Main, Germany
Bela E. Bode
Institut fu¨r Physikalische und Theoretische Chemie, Frankfurt am
2
Main, Germany
Joachim W. Engels
Johann .W. Goethe Universita¨t Frankfurt am Main, Institut fu¨r
2
Organische Chemie und Chemische Biologie (OCCB), Frankfurt am Main, Germany
For EPR measurements of RNA, DNA, or proteins, the occurrence of the paramagnetic species is
2
necessary. The aim of this work is to improve the synthesis of two different EPR spinlabels 2,2,6,6-
tetramethyl-3,4-dehydro-piperidin-N-oxyl-4-acetylene (TEMPA) 6 and 15 N-labeled TEMPA 6* and
their coupling to uridine. The yield of the synthesis of TEMPA could be increased to 40% and the
second nitroxide 2,2,6,6-tetramethyl-3,4-dehydro-piperidin-15 N-oxyl-4-acetylene 6* could be synthe-
sized with a yield of 11%.
Keywords RNA structure; sonogashira cross-coupling; side-specific label; nitroxide;
EPR
INTRODUCTION
The structure determination of macromolecules, like RNA, DNA, or
proteins, is not straightforward but needs a variety of analytic methods. Be-
sides X-ray, NMR, and FRET, electron paramagnetic resonance (EPR) can
be applied to elucidate the structure of such systems. Indeed, EPR spec-
troscopy already proved to be a powerful technique to characterize the local
surrounding of paramagnetic centers in proteins.[1] DNA,[2] and RNA.[3]
One requirement to determine the three dimensional structure is the
availability of three in EPR distinguishable spinlabels. In most cases the rad-
ical 2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene (TPA) could be used to
obtain paramagnetic species. The use of this spinlabel or similar compounds
The authors thank SFB 579 for financial support and Nelly Piton for synthetic advice.
Address correspondence to Joachim W. Engels, Johann .W. Goethe Universitaet Frankfurt am Main,
Institut fuer Organische Chemie und Chemische Biologie (OCCB), Max-von-Laue Str. 7, Frankfurt am
Main, D-60438, Germany. E-mail: joachim.engels@chemie.uni-frankfurt.de
655

656
O. Frolow et al.
SCHEME 1 Total synthesis of spinlabel TEMPA 6 and 6∗.
is described for proteins, DNA, and RNA. Gannett[4] published a synthesis of
the spinlabel 6 2,2,6,6-tetramethyl-3,4-dehydro-piperidin-N -oxyl-4-acetylene
(TEMPA) in 2001 and showed its application for DNA labeling. In our group
the coupling of nitroxide to RNA could be accomplished and a new spinla-
bel 6* had to be synthesized.
Synthesis
To be able to synthesize the 15N -labeled TEMPA (Scheme 1), the
synthesis of both radicals has to start with a ring closure reaction.[5]
Reaction of phorone 1 with ammonia in water yields the six-membered
ring 2 with 98% for 14N - and 91% for 15N -TEMPA. The oxidation of
the nitrogen was performed following the standard procedure with hy-
drogen peroxide and led to a very good yield of 96% for compound
3 (70% for 3*). For economical reasons and higher reactivity, we used
trimethylsilyl instead of triisopropylsilyl-protected acetylene. Product 4
could be obtained as a greenish yellow solid in good yield. The reagents
for the elimination step to compounds 5 and 5* could also be changed
from thionylchloride in pyridine to methanesulfonylchloride (MsCl) in
triethylamine. The use of these conditions facilitates the purification of
4-acetylene-(trimethylsilyl)-2,2,6,6-tetramethyl-piperid-3,4-en-N -oxyl
(5).
The silyl-group was cleaved with TBAF in 88% yield.
The synthesis of the 15N -labeled compound was performed by the same
strategy. Although the structures of both spinlabels are identical, their re-
activity is quite different. Thus the reaction time and the temperature of
the reaction had to be varied for each step. During the synthesis all com-

Synthesis of EPR Differentiable Spinlabels
657
TABLE 1 EPR parameters for the three spinlabels
Spinlabel
g-factor
Aiso [mT]
TPA
2.006
2.0055
2.0055
1.4
1.5
2.1
14N -TEMPA 6
15N -TEMPA 6*
pounds could be crystallized and analyzed (elemental analysis, 1H-NMR and
13C-NMR).
cw-EPR
The continuous wave-EPR (cw-EPR) measurements were carried out with
ESP300 cw X-Band spectrometer of the Bruker company at the frequency of
9.4267 GHz at 25 K. Both samples had the concentration of 10−4 mol/L in
toluene and were deoxygenated with argon.
Due to a different geometry the five (TPA) and six-membered rings
(TEMPA) possess distinct g-factors (Table 1). The 15N -labeling (S = 1/2
instead of S = 1 for 14N ) affects the hyperfine-coupling (Aiso) of the nitrox-
ide 6*, which scales with the gyromagnetic ratio γ 15N / γ 15N = 1.4. The
effect is reflected in the EPR-spectra (2 transitions instead of 3), shown in
Figure 1.
FIGURE 1 cw-EPR spectrum of spinlabels 6 and 6∗.

658
O. Frolow et al.
SCHEME 2 Sonogashira cross-coupling.
Cross-Coupling
For pulsed EPR distance measurements in RNA the synthesized spin-
labels will be coupled to the nucleotide base through a Sonogashira
cross-coupling during solid-phase synthesis after a method already devel-
oped in our laboratory for TPA.[3]
To optimize the conditions for solid phase, the reaction between both
synthesized spinlabels 6 and 6* and 2ꢁ,3ꢁ,5ꢁ-tri-O-acetyl-5-iodo-uridine 7 was
tested in solution (Scheme 2) and the kinetics thereof were followed by
HPLC (Figure 2).
FIGURE 2 Comparison of two HPLC-spectra (sp=spinlabel; UI =5-Iodo-uridine; Usp =spinlabled
uridine).

Synthesis of EPR Differentiable Spinlabels
659
TABLE 2 Different test conditions for the cross-coupling with uridine
Spinlabel
Solvent
DCM
DCM
THF
DMF
DCM
Piperidine
Pd
CuI
Yield
1
2
3
4
5
6
TPA
Pd(II)
Pd(II)
Pd(II)
Pd(II)
Pd(0)
Pd(0)
x
x
x
x
x
/
96%
90%
97%
28%
69%
76%#
TEMPA
TEMPA
TEMPA
TEMPA
TEMPA
#
The hydroxy groups of the sugar are deprotected.
Gradient: 0 min. 10% AcCN + 90% of 0.1% tri-ethylammonium-acetate.
0.1–20 min. 100% AcCN + 0% of 0.1% TEAA.
to 30 min. 100% AcCN + 0% of 0.1% TEAA.
30.1 min. 10% AcCN + 90% of 0.1% TEAA.
The reaction with the spinlabel TPA (Table 2) was taken as reference.
Bis(triphenylphosphine)-palladium-dichloride and tetrakis(triphenylphos-
phine)palladium were used as Pd(II) and Pd(0) respectively . Reactions 1
to 5 are performed with triethylamine as a base.
Yield was calculated by HPLC after a reaction time of 90 min. at room
temperature. Reaction 3 in THF with Pd(II) looks like the best condition
for the coupling with spinlabel 6.
CONCLUSIONS
After the coupling tests in solution the transfer to the solid phase will
be performed and the stability of the labeled RNA will be checked via Tm-
measurements and CD-spectra.
To optimize the PELDOR parameters for the new nitroxides we are syn-
thesizing different linker systems. On the basis of these results, we will in-
corporate the three different spin labels into RNA-duplexes to validate the
method and then apply it to the determination of more complex 3D-RNA
structures.
REFERENCES
1. Hubbell, W.; Cafiso, D.S.; Altenbach C. Identifying conformational change with side-directed spin
labeling. Nature Struct. Biol. 2000, 7(9), 735
2. Schiemann, O.; Piton, N.; Mu, Y.; Stock, G.; Engels, J.W.; Prisner, T.F. A PELDOR-based nanometer
distance ruler for oligonucleotides. J. Am. Chem. Soc. 2004, 126, 5722.
3. Piton, N.; Schiemann, O.; Mu, Y.; Stock, G.; Prisner, T.F.; Engels, J.W. Synthesis of spin-labeled RNAs
for long range distance measurements by PELDOR. Nucl. Nucl. Nucleic Acids. 2005, 24(5-7), 771.
4. Gannett, P.M.; Darian, E.; Powell, J.H.; Johnson, E.M. A short procedure for synthesis of 4-ethynyl-
2,2,6,6-tetramethyl-3,4-dehydroxy-piperidine-1-oxyl nitroxide. Synthetic Communications. 2001, 31(14),
2137.
5. Kelemen, P.; Lugtenburg, J.; Klumperman, B. 15N NMR spectroskopy of labeled alkoxyamines. 15N-
labeled model compounds for nitroxide-trapping studies on free-radical (co)polymerization. J. Org.
Chem. 2003, 68, 7322.