Delivering Structural Information on the Polar Face of Membrane-Active Peptides:19F-NMR Labels with a Cationic Side Chain

Article abstract of DOI:10.1002/anie.201607161

Conformationally constrained non-racemizing trifluoromethyl-substituted lysine isosteres [(E)- and (Z)-TCBLys] with charged side chains are presented as a new type of¹⁹F-NMR labels for peptide studies. Design of the labels, their synthesis, incorporation into peptides and experimental demonstration of their application for solid state NMR studies of membrane-active peptides are described. A series of fluorine-labeled analogues of the helical amphipathic antimicrobial peptide PGLa(Nle) was obtained, in which different lysine residues in the original peptide sequence were replaced, one at a time, by either (E)- or (Z)-TCBLys. Antimicrobial activities of the synthesized analogues were practically the same as those of the parent peptide. The structural and orientational parameters of the helical PGLa(Nle) peptide in model bilayers, as determined using the novel labels confirmed and refined the previously known structure. (E)- and (Z)-TCBLys, as a set of cationic¹⁹F-NMR labels, were shown to deliver structural information about the charged face of amphipathic peptides by solid state¹⁹F-NMR, previously inaccessible by this method.

Full text of DOI:10.1002/anie.201607161

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International Edition: DOI: 10.1002/anie.201607161
German Edition: DOI: 10.1002/ange.201607161
NMR Spectroscopy
Delivering Structural Information on the Polar Face of Membrane-
19
Active Peptides: F-NMR Labels with a Cationic Side Chain
Oleg M. Michurin, Sergii Afonin,* Marina Berditsch, Constantin G. Daniliuc, Anne S. Ulrich,
Igor V. Komarov,* and Dmytro S. Radchenko*
Abstract: Conformationally constrained non-racemizing tri-
fluoromethyl-substituted lysine isosteres [(E)- and (Z)-
TCBLys] with charged side chains are presented as a new
type of 19F-NMR labels for peptide studies. Design of the labels,
their synthesis, incorporation into peptides and experimental
demonstration of their application for solid state NMR studies
of membrane-active peptides are described. A series of
fluorine-labeled analogues of the helical amphipathic antimi-
crobial peptide PGLa(Nle) was obtained, in which different
lysine residues in the original peptide sequence were replaced,
one at a time, by either (E)- or (Z)-TCBLys. Antimicrobial
activities of the synthesized analogues were practically the
same as those of the parent peptide. The structural and
orientational parameters of the helical PGLa(Nle) peptide in
model bilayers, as determined using the novel labels confirmed
and refined the previously known structure. (E)- and (Z)-
TCBLys, as a set of cationic 19F-NMR labels, were shown to
deliver structural information about the charged face of
amphipathic peptides by solid state 19F-NMR, previously
inaccessible by this method.
transport,[3] amyloidogenesis,[4] among others.[5] The structure,
dynamics and alignment of MAPs in lipid bilayers are
important parameters for unraveling the molecular mecha-
nisms of their biological actions.
Only very few of the current techniques are suitable for
structural studies of MAPs in their membrane-bound state
under ambient conditions, because of the inherent complexity
of the membrane systems. This complexity and conforma-
tional plasticity of MAPs make it nearly impossible to
perform X-ray diffraction or electron microscopy studies.
The biophysical methods which use oriented membranes
(fluorescence-,[6] infrared-,[7] and oriented circular dichroism
spectroscopy,[8] colorimetry,[9] interface-sensitive X-ray or
neutron scattering[10] and quartz crystal microbalance[11]
)
provide only low-resolution structural information or suffer
from technical problems (e.g. radiation damage or membrane
dehydration).
As the most appropriate methodology, solid state NMR
spectroscopy (ssNMR) of the peptides labeled by 2H, 15N, 13C,
19F isotopes is used for decades to study MAPs at near-atomic
resolution.[12a] In particular, 19F-ssNMR method has been
successfully used to obtain structural information for more
than a dozen MAPs.[12b–e]
M
embrane-active peptides (MAPs) realize their functions
via interactions with biological membranes and play crucial
roles in many fundamental biological processes, including
innate immune response,[1] membrane fusion,[2] intracellular
Unlike labeling by 2H, 15N or 13C, introduction of
a fluorine-substituted amino acid in place of natural residues,
shown to be useful for qualitative structural assessments,[13]
might significantly change structure and properties of the
labeled peptides.[14] While it might be beneficial for the design
of peptides with improved characteristics for practical appli-
cation, it is completely unacceptable for peptide studies by
19F-ssNMR, where an ideal label should be non-perturbing
and conformationally rigid in order to provide correct
structural constraints by NMR.[12b–d]
[*] O. M. Michurin, Dr. D. S. Radchenko
Enamine Ltd.
vul. Chervonotkatska 78, 02094 Kyiv (Ukraine)
E-mail: Dmitry.Radchenko@mail.enamine.net
Prof. I. V. Komarov
Institute of High Technologies
Taras Shevchenko National University of Kyiv
vul. Volodymyrska 60, 01601 Kyiv (Ukraine)
E-mail: ik214@yahoo.com
Currently known 19F-labels (1–6) are shown in Figure 1;
they were used to substitute non-polar natural amino
acids.[12b–d,15] The only exception is the amino acid 7[16] which
has a polar side chain. To date, no fluorine-substituted amino
acids with charged side chains have ever been described as
labels for 19F-ssNMR studies of MAPs. One of the reasons for
this is the challenging design: a fluorine-containing substitu-
ent will perturb the polar charged side chains much more than
will modify the hydrophobic non-polar aliphatic residues.
Charged amino acid residues are known to be fundamen-
tally important for the structure and functions of numerous
MAPs. For example, interaction of anionic lipid head groups
with the positively charged side chains is a prerequisite for the
MAPs to bind to natural membranes.[17] Another example is
the ladder-like pattern of positively and negatively charged
amino acid residues forming multiple salt bridges, which has
Dr. S. Afonin, Prof. A. S. Ulrich
Institute of Biological Interfaces (IBG-2)
Karlsruhe Institute of Technology (KIT)
POB 3640, 76021 Karlsruhe (Germany)
E-mail: Sergiy.Afonin@kit.edu
Dr. M. Berditsch, Prof. A. S. Ulrich, Dr. D. S. Radchenko
Institute of Organic Chemistry (IOC), KIT
Fritz-Haber-Weg 6, 76131 Karlsruhe (Germany)
Dr. C. G. Daniliuc
Institute of Organic Chemistry
Westfalische Wilhelms-Universitꢀt Mꢁnster
Corrensstrasse 40, 48149 Mꢁnster (Germany)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201607161.
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lysine analogues, TCBLys) which were selected for the
synthesis.
The synthesis (Scheme 2) was based on the construction
of 1,3-functionalized cyclobutane derivative 14 using a [2+2]
cycloaddition reaction between allene and CF3-substituted
acrylonitrile 13.[21] In our hands, the transformations leading
Figure 1. Known CF3-substituted labels for 19F-ssNMR and the natural
amino acid residues that they could substitute in MAPs (in brackets,
Aib=a-aminoisobutyric acid).
been recently described as a “charge zipper” motif in several
MAPs.[18]
Based on 19F-ssNMR analysis, it was recently shown that
a lack of 19F-labels on the charged side of an amphipathic
peptide can lead to ambiguity in determining its membrane-
bound orientation.[19] The polar/charged face of peptides, as of
now, has to be considered the side of MAPs largely unex-
plored by the 19F-ssNMR. Here, we describe the first
representatives of charged 19F-labels, i.e., cationic lysine
analogues suitable for labeling MAPs.
In our design of cationic 19F-labels, lysine (8) served as
a template; we modified the molecule with the intention of
introducing minimal perturbations to the amino acid structure
and properties (Scheme 1), as required for the 19F-ssNMR
Scheme 2. Synthesis of (Z)-TCBLys (10a), (E)-TCBLys (10b) and their
orthogonally N-protected derivatives (20a,b) for Fmoc-SPPS. a) KCN,
H2SO4, <108C, 15 min; b) AcCl, reflux, 5 h; c) pyrolysis, 5508C, 4 h;
d) allene, PhH, hydroquinone, 2008C, 24 h; e) KOH, EtOH–H2O,
reflux, 12 h; f) LiAlH4, Et2O, RT, 12 h; g) CH3SO2Cl, Et3N, CH2Cl2,
À108C, 30 min; h) KCN, DMF, 1008C, 12 h; i) LiAlH4, Et2O, RT, 12 h;
j) Boc2O, RT, 2 h; k) NaIO4, RuCl3, CH3CN–H2O, RT, 30 min; l) NH3,
Ti(O-i-Pr)4, TMSCN, i-PrOH, RT, 1 h, then Ac2O, Et3N, THF, RT, 12 h,
chromatographic separation; m) 6n HCl, reflux, 12 h, ion-exchange
chromatography; n) Boc2O, TMSCl, DIPEA, CH2Cl2, À158C, 18 h;
o) FmocCl, RT, 5 h.
Scheme 1. Design of lysine analogue TCBLys (both diastereomers are
shown) as a label for 19F-ssNMR. a) Introduction of the CF3-group;
b) fixation of c1 and c2.
to the cyclobutane ring formation were scalable, despite the
high temperature required for the pyrolysis of 12. The
subsequent hydrolysis of the nitrile group in 14 followed by
LiAlH4 reduction produced the alcohol 15. It was trans-
formed to nitrile 16 via the corresponding mesylate. Reduc-
tion of the nitrile group in 16 with LiAlH4 furnished a volatile
primary amine, which, in its crude form, reacted with di-tert-
butyl pyrocarbonate (Boc2O) to give the Boc-protected
derivative 17. Oxidative cleavage of the double bond in 17
by NaIO4–RuCl3 afforded the Boc-N-protected amino ketone
18. A modified Strecker reaction using Ti(Oi-Pr)4 as water
scavenger and mild Lewis acid[22] was employed to obtain
studies of MAPs. The g-position on the lysine side chain
appeared to be superior to the other sites for attaching the
electron-withdrawing CF3-substituent (structure 9). The fluo-
rine-containing group in this position resides the farthest from
all the other functional groups and has the least influence on
their chemical properties. This is especially important for
preserving the high basicity of the e-amino group, which
critically determines the charged character of the amino acid
in peptides at ambient pH. A comparison of the pKa for
butan-1-amine (10.7) and 4,4,4-trifluorobutan-1-amine
(9.7)[20] justifies this design strategy. The conformational
restriction of the fragment connecting the aminocarboxylate
moiety to the NMR-reporter CF3 group can be achieved
a
mixture of diastereomeric acylated amino nitriles
(19a:19b = 3:1) after treating the intermediate amino nitrile
with acetic anhydride. The diastereomers were separated by
column chromatography, and the major isomer was identified
as (Z)- by NMR and X-ray analysis (see the Supporting
À
À
by incorporating a CH2 linker between the a- and g-
positions,[15e,16] leading to the cyclobutane-derived amino
acids 10a,b (trifluoromethyl- and cyclobutane-containing
2
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Information). Refluxing each aminonitrile (19a,b) in 6n HCl
achieved full hydrolysis and provided the amino acids
10a,10b.
To be suitable for Fmoc (Fmoc = 9-fluorenylmethoxy-
carbonyl) solid state peptide synthesis (Fmoc-SPPS), the
amino groups in the synthesized lysine analogues 10a,b
should be orthogonally protected. Selective Ne protection
could be accomplished using 1.3 equiv. Boc2O at low temper-
ature (À158C); only a trace amount of the Na-Boc-derivative
was formed. A subsequent one-pot reaction with FmocCl led
to the smooth formation of compounds 20a and 20b. This
approach yielded regioselective orthogonal protection with-
out the need to isolate the intermediate compounds.
Next, we tested compatibility of 20a,b with Fmoc-SPPS by
synthesizing Lys/TCBLys-substituted analogues of PGLa-
(Nle), a Met/Nle (Nle = norleucine) mutant of the antimicro-
bial peptide PGLa. This peptide is a typical representative of
a-helical amphipathic peptides whose thorough 19F-ssNMR
analysis has been recently performed using non-polar 19F-
label 3.[23] The lysine residues at positions 12, 15, and 19 were
substituted, one at a time, with either (E)- or (Z)-TCBLys to
yield a total of 6 labeled analogues (Figure 2). Despite being
Figure 3. Properties of the Lys/TCBLys mutants of PGLa(Nle). a) CD
spectra in aqueous (left: PB) and membrane-mimicking (right:
micelles of lyso-MPC in PB, peptide/detergent=1/100 molmolÀ1) envi-
ronments. All peptides are at 0.05 mgmLÀ1 concentration. b) MIC
(mgmLÀ1) against E. coli and S. aureus; c) Representative 19F-ssNMR
spectra from labeled peptides in oriented DMPC multibilayers as
a function of temperature; peptide/lipid=1/50 molmolÀ1; the bilayer
normal was aligned at 08 to the direction of the static magnetic field.
MPC) micelles. Therefore, we concluded that residues of the
TCBLys diastereomers resemble lysine residues regarding
their structural impact on the polypeptide backbone.
Figure 2. Amino acid sequence, helical wheel and amphipathicity of
the a-helical PGLa(Nle); shown by arrows are the positions labeled
with the new 19F-label 10a and 10b (TCBLys) and previously addressed
with 3 (CF3-Bpg); mutations are indicated in brackets (N*=Nle).
Determination of the minimum inhibitory concentrations
(MICs) of the Lys/TCBLys mutants served as a sensitive assay
to probe for functional perturbations imposed by the labeling.
Gratifyingly, we found that all mutant peptides were anti-
microbially active. As the MIC values of the labeled peptides
were close to that of the parent peptide (Figure 3b), it is safe
to conclude that the labeling does not affect significantly the
key function of PGLa(Nle).
PGLa(Nle) was previously shown to adopt an amphi-
pathic a-helical structure in membranes, and re-orient with
respect to the bilayer normal when the lipids change their
phase state.[20] Using identical conditions, we measured
the 19F-ssNMR spectra of all TCBLys-substituted peptides
in oriented 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC) bilayers. We observed three distinct orientational
states of the labeled peptides at different temperatures
(Figure 3c), in full agreement with the previous study.
The earlier 19F-ssNMR structure analysis of PGLa(Nle)[23]
(Figure 4a) had been performed with only four structural
constrains, which is the minimal number required to find an
unambiguous solution for the three orientational parameters
a,a-disubstituted, 20a and 20b showed sufficient reactivity,
similar to that of Fmoc-Lys(Boc)-OH, and were thus found to
be fully compatible with Fmoc-SPPS.
One of the main goals of developing TCBLys was to
obtain a 19F-label with structure and properties similar to
lysine. As a first criterion to assess the similarity, we evaluated
the basicity of the e-amino group of the 19F-label (see the
Supporting Information). The pKa values in the (E)- and (Z)-
TCBLys residues (as determined using E-19 and Z-19
peptides) were 9.3 Æ 0.1 and 9.7 Æ 0.1, respectively. These
values are close to the corresponding pKa value of the lysine
side chain (10.3). Hence, the impact of the CF3-group on the
basicity of the (E)- and (Z)- residues is small.
The structural impact of the Lys/TCBLys-substitution was
further evaluated by circular dichroism (CD) spectroscopy
(Figure 3a). All six tested peptides adopted random coil
conformations in aqueous phosphate buffer (PB) while being
structured as a-helices in the presence of lyso-lipid (lyso-
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ethyl)-3(trifluoromethyl)cyclobutane carboxylic acids for 19F-
ssNMR structural studies of MAPs. These 19F-labels are the
first of their kind with a charged (cationic) side chain, thus
suitable to probe the polar face of amphiphilic peptides. Both
diastereomers of the novel TCBLys-label were demonstrated
to have close structural and functional similarity to lysine. The
two diastereomers could be used as independent labels in
a single position to obtain two independent orientational
constraints. The simultaneous use of hydrophobic as well as
charged 19F-labels was demonstrated to considerably increase
the precision of the peptide orientational analysis, and will
thus extend the applicability of 19F-ssNMR to previously
inaccessible systems.
Acknowledgements
D.S.R. acknowledges financial support from the Alexander
von Humboldt (AvH) Foundation for a postdoctoral fellow-
ship. I.V.K. and O.M.M. thank the AvH for a Georg Forster
Research Award. This work was partially supported by GRK
2039 from the German Research Society (DFG) and by the
Helmholtz Association program “BIFTM”.
Keywords: amino acids · fluorine · lysine · peptides ·
solid state 19F-NMR spectroscopy
[1] M. Pasupuleti, A. Schmidtchen, M. Malmsten, Crit. Rev.
Biotechnol. 2012, 32, 143 – 171.
[2] S. Martens, H. T. McMahon, Nat. Rev. Mol. Cell Biol. 2008, 9,
543 – 556.
[3] M. Pooga, ꢀ. Langel, Methods Mol. Biol. 2015, 1324, 3 – 28.
[4] M. Landreh, J. Johansson, H. Jçrnvall, J. Mol. Biol. 2014, 426,
2159 – 2166.
Figure 4. 19F-ssNMR structure analysis of PGLa(Nle) in fluid DMPC
bilayers. The best-fit dipolar wave plots (left panels) and corresponding
error plots (right panels: RMSD t–1) are calculated from experimental
19F-ssNMR constraints based on a) four CF3-Bpg-labels, b) six TCBLys-
labels, and c) four CF3-Bpg plus six TCBLys-labels. RMSD=root mean
square deviation.
[5] Handbook of Biologically Active Peptides (Ed.: A. Kastin),
Academic Press, London, 2013.
[6] J. R. Lakowicz, Principles of Fluorescence Spectroscopy,
Springer, Berlin, 2006.
[7] X. Chen, J. Wang, A. P. Boughton, C. B. Kristalyn, Z. Chen, J.
Am. Chem. Soc. 2007, 129, 1420 – 1427.
[8] Y. Wu, H. W. Huang, G. A. Olah, Biophys. J. 1990, 57, 797 – 806.
[9] T. Sheynis, J. Sykora, A. Benda, S. Kolusheva, M. Hof, R. Jelinek,
Eur. J. Biochem. 2003, 270, 4478 – 4487.
[10] T. Salditt, C. Li, A. Spaar, Biochim. Biophys. Acta Biomembr.
2006, 1758, 1483 – 1498.
[11] A. Mechler, S. Praporski, K. Atmuri, M. Boland, F. Separovic,
L. L. Martin, Biophys. J. 2007, 93, 3907 – 3916.
[12] a) M. Fillion, M. Auger, Biophys. Rev. 2015, 7, 311 – 320; b) S. L.
Grage, S. Afonin, A. S. Ulrich, Methods Mol. Biol. 2010, 618,
183 – 207; c) V. S. Kubyshkin, I. V. Komarov, S. Afonin, P. K.
Mykhailiuk, S. L. Grage, A. S. Ulrich in Fluorine in Pharma-
ceutical and Medicinal Chemistry: From Biophysical Aspects to
Clinical Applications (Eds.: V. Gouverneur, K. Mꢁller), Imperial
College Press, London, 2012, pp. 91 – 139; d) K. Koch, S. Afonin,
M. Ieronimo, M. Berditsch, A. S. Ulrich, Top. Curr. Chem. 2012,
306, 89 – 118; e) A. N. Smith, M. A. Caporini, G. E. Fanucci, J. R.
Long, Angew. Chem. Int. Ed. 2015, 54, 1542 – 1546; Angew.
Chem. 2015, 127, 1562 – 1566.
(t, 1, Smol) that define the peptide alignment in the membrane.
In this work, we have now sampled new orientational
constraints from the three lysine positions in PGLa(Nle) on
the previously inaccessible charged face of the helix. Two
diastereomers of the 19F-label doubled the number of
constraints from one position. The use of two stereoisomers
had already been demonstrated to be effective in determining
the membrane orientation of an alamethicin with another 19F-
label 2.[15d] In the case of PGLa(Nle), six independent TCBLys
constraints yielded a structural solution (Figure 4b) that is
fully consistent with the previous analysis. Moreover, using
the entire set of 19F-ssNMR data for PGLa(Nle) labeled with
3, 10a, and 10b, we could further refine the orientation of this
peptide in lipid bilayers. As seen from Figure 4c, when four
CF3-Bpg and six TCBLys labels were used together, the best-
fit solution is much more precise, in terms of both the tilt
angle t and the azimuthal rotation angle 1.
In conclusion, we have designed and synthesized con-
formationally restricted (E)- and (Z)-1-amino-3-(2-amino-
[13] Y. Suzuki, B. C. Buer, H. M. Al-Hashimi, E. N. G. Marsh,
Biochemistry 2011, 50, 5979 – 5987.
4
www.angewandte.org
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These are not the final page numbers!

Angewandte
Communications
Chemie
[14] a) H. Meng, K. Kumar, J. Am. Chem. Soc. 2007, 129, 15615 –
15622; b) A. Niemz, D. A. Tirrell, J. Am. Chem. Soc. 2001, 123,
7407 – 7413; c) E. N. G. Marsh, Acc. Chem. Res. 2014, 47, 2878 –
2886; d) S. Huhmann, E. K. Nyakatura, H. Erdbrink, U. I. M.
Gerling, C. Czekelius, B. Koksch, J. Fluorine Chem. 2015, 175,
32 – 35.
[16] A. N. Tkachenko, P. K. Mykhailiuk, S. Afonin, D. S. Radchenko,
V. S. Kubyshkin, A. S. Ulrich, I. V. Komarov, Angew. Chem. Int.
Ed. 2013, 52, 1486 – 1489; Angew. Chem. 2013, 125, 1526 – 1529.
[17] V. Teixeira, M. J. Feio, M. Bastos, Prog. Lipid Res. 2012, 51, 149 –
177.
[18] T. H. Walther, A. S. Ulrich, Curr. Opin. Struct. Biol. 2014, 27,
63 – 68.
[15] a) R. W. Glaser, C. Sachse, U. H. N. Dꢁrr, P. Wadhwani, A. S.
Ulrich, J. Magn. Reson. 2004, 168, 153 – 163; b) P. K. Mikhailiuk,
S. Afonin, A. N. Chernega, E. B. Rusanov, M. O. Platonov, G. G.
Dubinina, M. Berditsch, A. S. Ulrich, I. V. Komarov, Angew.
Chem. Int. Ed. 2006, 45, 5659 – 5661; Angew. Chem. 2006, 118,
5787 – 5789; c) P. K. Mykhailiuk, S. Afonin, G. V. Palamarchuk,
O. V. Shishkin, A. S. Ulrich, I. V. Komarov, Angew. Chem. Int.
Ed. 2008, 47, 5765 – 5767; Angew. Chem. 2008, 120, 5849 – 5851;
d) D. Maisch, P. Wadhwani, S. Afonin, C. Bçttcher, B. Koksch,
A. S. Ulrich, J. Am. Chem. Soc. 2009, 131, 15596 – 15597; e) A. N.
Tkachenko, D. S. Radchenko, P. K. Mykhailiuk, S. Afonin, A. S.
Ulrich, I. V. Komarov, Angew. Chem. Int. Ed. 2013, 52, 6504 –
6507; Angew. Chem. 2013, 125, 6632 – 6635; f) V. Kubyshkin, S.
Afonin, S. Kara, N. Budisa, P. K. Mykhailiuk, A. S. Ulrich, Org.
Biomol. Chem. 2015, 13, 3171 – 3181.
[19] P. Wadhwani, E. Strandberg, J. van den Berg, C. Mink, J. Bꢁrck,
R. A. M. Ciriello, A. S. Ulrich, Biochim. Biophys. Acta Bio-
membr. 2014, 1838, 940 – 949.
[20] W. K. Hagmann, J. Med. Chem. 2008, 51, 4359 – 4369.
[21] H. K. Hall, E. P. Blanchard, E. L. Martin, Macromolecules 1971,
4, 142 – 146.
[22] A. V. Chernykh, D. S. Radchenko, O. O. Grygorenko, D. M.
Volochnyuk, S. V. Shishkina, O. V. Shishkin, I. V. Komarov, RSC
Adv. 2014, 4, 10894 – 10902.
[23] D. S. Radchenko, S. Kattge, S. Kara, A. S. Ulrich, S. Afonin,
Biochim. Biophys. Acta Biomembr. 2016, 1858, 2019 – 2027.
Received: July 30, 2016
Revised: September 14, 2016
Published online: && &&, &&&&
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Communications
NMR Spectroscopy
Illuminating the “dark side”: A cyclobu-
tane-based a-amino acid was designed as
the first 19F-NMR label carrying a charged
side chain. Two diastereomers of the
amino acid were used to obtain solid
state NMR constraints for the previously
inaccessible cationic face of a membrane-
embedded peptide PGLa(Nle). The label
showed similarity to lysine allowing its
use in NMR studies of lysine-rich pep-
tides in membranes.
O. M. Michurin, S. Afonin,* M. Berditsch,
C. G. Daniliuc, A. S. Ulrich,
I. V. Komarov,*
D. S. Radchenko*
&&&& — &&&&
Delivering Structural Information on the
Polar Face of Membrane-Active Peptides:
19F-NMR Labels with a Cationic Side
Chain
6
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