Conformationally unambiguous spin labeling for distance measurements

Article abstract of DOI:10.1002/chem.200902162

An iDEER about molecular structure: Measurements of interspin distances through double electron-electron resonance (DEER) experiments allow information to be gained about the structure of molecules (see figure). The less variability in the orientation of the N-O axis of nitroxyl spin labels with respect to the labeled molecule, the smaller the distribution of the interspin distance and thus the better the conclusion.

Full text of DOI:10.1002/chem.200902162

DOI: 10.1002/chem.200902162
Conformationally Unambiguous Spin Labeling for Distance Measurements
Muhammad Sajid,[a] Gunnar Jeschke,[b] Michael Wiebcke,[c] and Adelheid Godt*[a]
Electron paramagnetic resonance (EPR) spectroscopy on
site-directed spin-labeled molecules is a powerful tool to in-
vestigate structures of oligomers and polymers. One key
technique is the double electron–electron resonance
(DEER) experiment that provides information on the dis-
tance between paramagnetic centers.[1,2] The most reliable
structural information will be gained if this distance is unaf-
fected by conformational flexibility of the spin label. 3-Ethy-
condensed with an imide moiety. The halo and ethynyl sub-
ꢁ
stituents permit attachment through C C cross coupling, a
method broadly applied for spin labeling.[3,6] Spin-density
transfer onto the N-bound phenylene moiety is negligible
(see the Supporting Information), an aspect of special signif-
icance when the labeling sites are connected through a poly-
conjugated bridge. Herein, the synthesis (see the Supporting
Information), structure, and application of spin labels 1 and
their diamagnetic precursors 2 are described together with a
comparative DEER study to evaluate the quality of the new
spin labels as conformationally unambiguous spin labels
(CUSL).[7]
ACHTUNGTRENNUNGnyl-1-oxyl-2,2,5,5-tetramethylpyrroline is essentially a rigid
spin label and has been used for studies on the flexibility of
shape-persistent nanoscopic molecules including oligonucle-
otides.[3] However, because the C C triple bond and the N
ꢀ
ꢁ
ꢁ
O bond are not co-linear, the free rotation around C C
X-ray structure analysis on single crystals of spin label 1c
(Figure 2) shows an almost planar isoindolinedicarboximide
moiety. The angles O23-N21-N9 (177.67(3)8) and N21-N9-
single bonds results in conformational isomers that differ in
the distances between the paramagnetic centers. For oligo-
nucleotides, ribbon-shaped spin labels with integrated quino-
lone or cytosine moieties for base pairing are an elegant so-
lution.[4] Herein, we introduce the spin labels 1 (Figure 1), in
which the chemically very robust isoindoline nitroxide[5] is
Figure 2. Structure of spin label 1c in the crystal.
C6 (177.14(3)8) deviate only by about three degrees from
1808. Consequently, the two bonds O23-N21 and N9-C6 are
essentially co-linear. Thus, spin labels 1 should allow confor-
Figure 1. Conformationally unambiguous spin labels (CUSLs) 1 and their
diamagnetic precursors 2.
ꢁ
mationally unambiguous labeling through C C cross cou-
pling despite free rotation around single bonds in the spin-
label moiety.
[a] M. Sajid, Prof. Dr. A. Godt
Department of Chemistry, Bielefeld University
Universitꢀtsstr. 25, 33615 Bielefeld (Germany)
Fax : (+49)0521-1066417
The quality of the new spin label as a CUSL was probed
through DEER measurements on oligo(para-phenyleneethy-
nylene) 4 (oligoPPE, Scheme 1) and comparison of the data
with those from oligoPPE 5 (Figure 3), an oligoPPE carrying
a standard, conformationally ambiguous label.[8] We deliber-
ately chose a very short oligoPPE to minimize the contribu-
tion of the flexibility of the oligoPPE backbone to the distri-
bution of the distance between the two unpaired electrons.
OligoPPE 4 was synthesized through coupling of spin-label
E-mail: godt@uni-bielefeld.de
[b] Prof. Dr. G. Jeschke
Lab. Phys. Chem., ETH Zꢁrich, 8093 Zꢁrich (Switzerland)
[c] Dr. M. Wiebcke
Institute of Inorganic Chemistry, Leibniz University Hannover
Callinstr. 9, 30167 Hannover (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902162.
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Chem. Eur. J. 2009, 15, 12960 – 12962

COMMUNICATION
ly, the oscillation persists much longer for 4, the oligoPPE
with the CUSL, than for 5 (note the different time axes). As
a measure for the relative width of the distance distribution,
we used the ratio between the amplitude D2 of the second
oscillation and the amplitude D5 of the fifth oscillation. We
found a smaller ratio D2/D5 =2.75ꢂ0.15 for 4 than for 5
(3.34ꢂ0.20) corresponding to decay of the oscillation for 4
that is significantly slower than for 5, even when considering
the different signal-to-noise ratio. Hence, the CUSL pro-
vides a significantly narrower distance distribution.
Scheme 1.
To check whether the absolute width of the distribution is
reduced with the CUSL, the data shown in Figure 3b were
subjected to Tikhonov regularization with the same opti-
p
mum regularization parameter a= 10ꢃ10ꢁ3. To compen-
sate for the different label lengths, the resulting distance dis-
tributions were superimposed by a relative shift along the
distance axis (Figure 3c). Indeed, the distribution is slightly
but significantly narrower when using the CUSL. This nar-
rowing is most visible in the wing of the distribution towards
short distances (arrow in Figure 3c), and owing to the same
regularization parameter being used, cannot be an artifact
of data analysis.
The only moderate narrowing of the distribution is due to
the flexibility of the oligoPPE backbone, which causes a dis-
tribution of the angle between the backbone directions at
the two ends. This angular distribution, in turn, causes a
leveraged broadening of the distance distribution by the
label and the larger the leverage is, the longer the label.
To test whether spin labels 1 can be used for site-specific
labeling of DNA or RNA, 5-iodo-2’-deoxyuridine was cou-
pled with spin label 1d by using [PdACHTNUTRGNE(UNG PPh3)4] and CuI as the
catalysts under stringent exclusion of oxygen (Scheme 2).
Figure 3. a) Structure of spin-labeled oligoPPE 5. b) Background-correct-
ed time-domain data for oligoPPE 4 (i, top time axis) and oligoPPE 5 (ii,
bottom time axis) obtained at 50 K in perdeuterated o-terphenyl. The
dotted lines denote definition of the amplitude D2 of the second and D5
of the fifth oscillation in each trace. c) Label-to-label distance distribu-
tions for 4 (solid line, top distance axis) and 5 (dashed line, bottom dis-
tance axis). The arrow denotes the wing of the distribution in which
broadening due to conformational ambiguity is most easily seen.
Scheme 2.
Before workup, the reaction mixture was stirred in air, be-
cause we had learned from coupling of spin label 1a with
triisopropylsilylethyne that some of the nitroxyl 1c was re-
duced to the corresponding hydroxylamine, which was rap-
idly reoxidized upon exposure of the reaction mixture to air.
Reversible reduction of nitroxyls to hydroxylamine by CuI
has been described.[9] The oxidation of the hydroxylamine
seems to be catalyzed by in-situ formed CuII: isolated hy-
droxylamine dissolved in THF is oxidized very slowly in air
but is instantaneously oxidized after addition of CuCl2. The
smooth coupling of the spin label 1d with a nucleoside sug-
gests that the spin labels 1 can be used for post-synthetic la-
precursor 2d with diiodobenzene 3 and subsequent N oxida-
tion with peracid (Scheme 1), which demonstrates one of
the options of how to attach the CUSL 1.
Owing to the different lengths between the point of back-
ꢁ
bone attachment and the midpoint of the N O bond for the
two labels, the comparison of data from 4 and 5 requires
some caution. First, we compared orientation-averaged
background-corrected time-domain data (Figure 3b). Clear-
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A. Godt et al.
[3] a) A. Marko, D. Margraf, H. Yu, Y. Mu, G. Stock, T. Prisner, J.
Chem. Phys. 2009, 130, 064102; b) D. Margraf, B. E. Bode, A.
Marko, O. Schiemann, T. F. Prisner, Mol. Phys. 2007, 105, 2153–
2160; c) B. E. Bode, D. Margraf, J. Plackmeyer, G. Dꢁrner, T. F. Pris-
ner, O. Schiemann, J. Am. Chem. Soc. 2007, 129, 6736–6745; d) S.
Obeid, M. Yulikov, G. Jeschke, A. Marx, Angew. Chem. 2008, 120,
6886–6890; Angew. Chem. Int. Ed. 2008, 47, 6782–6785; e) A. Spal-
tenstein, B. H. Robinson, P. B. Hopkins, J. Am. Chem. Soc. 1988,
110, 1299–1301; f) O. Schiemann, N. Piton, J. Plackmeyer, B. E.
Bode, T. F. Prisner, J. W. Engels, Nat. Protoc. 2007, 2, 904–923; g) N.
Piton, Y. Mu, G. Stock, T. F. Prisner, O. Schiemann, J. W. Engels,
Nucleic Acids Res. 2007, 35, 3128–3143.
[4] a) T. R. Miller, S. C. Alley, A. W. Reese, M. S. Solomon, W. V.
McCallister, C. Mailer, B. H. Robinson, P. B. Hopkins, J. Am. Chem.
Soc. 1995, 117, 9377–9378; b) P. Cekan, A. L. Smith, N. Barhate,
B. H. Robinson, S. Th. Sigurdsson, Nucleic Acids Res. 2008, 36,
5946–5954; c) O. Schiemann, P. Cekan, D. Margraf, T. F. Prisner, S.
Th. Sigurdsson, Angew. Chem. 2009, 121, 3342–3345; Angew. Chem.
Int. Ed. 2009, 48, 3292–3295.
beling of appropriately functionalized oligonucleotides.
Therefore, spin label 1d is a potential substitute for the cur-
rently used spin labels 3-ethynyl-1-oxyl-2,2,5,5-tetramethyl-
pyrroline[3d–g] and 1-oxyl-2,2,6,6-tetramethylazacyclohex-3-
ene.[10] Besides being a CUSL, it offers the advantage of
being very robust toward redox reactions.[5b]
In summary, spin labels 1 and their diamagnetic precur-
sors 2 can be attached through cross-coupling to the com-
pounds of interest, for example, artificial rod-like com-
pounds such as oligoPPE or biomolecules such as DNA.
These spin labels provide access to data that are unbiased
by conformational flexibility within the spin label, which is
clearly documented by DEER experiments with spin-la-
beled oligoPPE 4.
[5] a) D. G. Gillies, L. H. Sutcliffe, X. Wu, J. Chem. Soc. Faraday Trans.
1994, 90, 2345–2349; b) J. P. Blinco, J. L. Hodgson, B. J. Morrow,
J. R. Walker, G. D. Will, M. L. Coote, S. E. Bottle, J. Org. Chem.
2008, 73, 6763–6771.
[6] a) D. J. Keddie, K. E. Fairfull-Smith, S. E. Bottle, Org. Biomol.
Chem. 2008, 6, 3135–3143; b) K. E. Fairfull-Smith, S. E. Bottle, Eur.
J. Org. Chem. 2008, 5391–5400.
Acknowledgements
Financial support by DFG (JE 246/4-2, GO 555/4-3) is gratefully ac-
knowledged. M.S. thanks the Higher Education Commission of Pakistan
for a fellowship.
[7] The use of spin labels 1 for three-spin systems has been described,
see: G. Jeschke, M. Sajid, M. Schulte, A. Godt, Phys. Chem. Chem.
Phys. 2009, 11, 6580–6591.
ꢁ
Keywords: C C coupling
resonance · EPR spectroscopy · nitroxyls · spin labeling
·
double electron–electron
[8] a) A. Godt, C. Franzen, S. Veit, V. Enkelmann, M. Pannier, G.
Jeschke, J. Org. Chem. 2000, 65, 7575–7582; b) A. Godt, M. Schulte,
H. Zimmermann, G. Jeschke, Angew. Chem. 2006, 118, 7722–7726;
Angew. Chem. Int. Ed. 2006, 45, 7560–7564.
[9] M. A. Schwartz, J. W. Parce, H. M. McConnell, J. Am. Chem. Soc.
1979, 101, 3592–3595.
[10] P. M. Gannett, E. Darian, J. Powell, E. M. Johnson II, C. Mundoma,
N. L. Greenbaum, C. M. Ramsey, N. S. Dalal, D. E. Budil, Nucleic
Acids Res. 2002, 30, 5328–5337.
[1] a) A. D. Milov, A. B. Ponomarev, Y. D. Tsvetkov, Chem. Phys. Lett.
1984, 110, 67–72; b) R. E. Martin, M. Pannier, F. Diederich, V.
Gramlich, M. Hubrich, H. W. Spiess, Angew. Chem. 1998, 110,
2993–2998; Angew. Chem. Int. Ed. 1998, 37, 2833–2837; c) M. Pan-
nier, S. Veit, A. Godt, G. Jeschke, H. W. Spiess, J. Magn. Reson.
2000, 142, 331–340; d) G. Jeschke, A. Bender, H. Paulsen, H. Zim-
mermann, A. Godt, J. Magn. Reson. 2004, 169, 1–12.
[2] a) Y. D. Tsvetkov, A. D. Milov, A. G. Maryasov, Usp. Khim. 2008,
77, 515–550; Russ. Chem. Rev. 2008, 77, 487–520; b) G. Z. Sowa,
P. Z. Qin, Prog. Nucleic Acid Res. Mol. Biol. 2008, 82, 147–197;
c) O. Schiemann, T. F. Prisner, Q. Rev. Biophys. 2007, 40, 1–53.
Received: August 3, 2009
Published online: October 21, 2009
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Chem. Eur. J. 2009, 15, 12960 – 12962