Conformationally rigid trifluoromethyl-substituted α-amino acid designed for peptide structure analysis by solid-state 19F NMR spectroscopy

Article abstract of DOI:10.1002/anie.200600346

(Figure Presented) Designing a sturdy label: The fluorinated amino acid L-CF₃-Bpg (see picture), which was specifically designed as a ¹⁹F NMR label for structure analysis of membrane-bound peptides, does not display the drawbacks of

Full text of DOI:10.1002/anie.200600346

Angewandte
Chemie
them by 19F NMRspectroscopy. The advantages of observing
this isotope arise from its high sensitivity, the absence of
biological background signals, its wide range of chemical
shifts, and strong dipolar interactions.[1,2] An FAA is usually
incorporated into a peptide by solid-phase peptide synthesis
(SPPS), though some other methods have also been devel-
oped.[1,3] In most NMRapplications the FAAs simply serve to
probe qualitative changes in the local environment.[1] Only
recently, FAAs have been incorporated into membrane-
bound peptides to determine explicit structural parameters in
terms of orientational constraints and/or interatomic distan-
[4,5]
ces by solid-state 19F NMRspectrosopy.
These solid-state
NMRstudies are particularly informative about membrane-
active peptides, as they can reveal their conformation,
alignment, and dynamic behavior in a lipid bilayer under
quasi-native conditions.[4] However, only a limited number of
FAAs are applicable to this approach, as they must meet
certain structural requirements.[5] All of the compounds (1–6)
used in the past face certain drawbacks in terms of either their
structural interpretation and/or their chemical incorporation
into peptides.
Labeled Amino Acids
DOI: 10.1002/anie.200600346
Conformationally Rigid Trifluoromethyl-
Substituted a-Amino Acid Designed for Peptide
19
Structure Analysis by Solid-State F NM R
Here we report the design, synthesis, and characterization
of a novel FAA that avoids the problems of 1–6 and is ideally
Spectroscopy
Pavel K. Mikhailiuk, Sergii Afonin,
Alexander N. Chernega, Eduard B. Rusanov,
Maxim O. Platonov, Galina G. Dubinina,
Marina Berditsch, Anne S. Ulrich,* and
Igor V. Komarov*
The incorporation of specifically fluorine-labeled l-a-amino
acids (FAAs) into polypeptides is a prerequisite for analyzing
[*] Dr. M. Berditsch, Prof. A. S. Ulrich
Universität Karlsruhe (TH)
Institut für Organische Chemie
Fritz-Haber-Weg 6, 76133 Karlsruhe (Germany)
Fax : (+49)721-608-48-23
suited as a label for structure analysis of membrane-bound
peptides by solid-state 19F NMRspectroscopy. The principles
of its design are based on the known advantages and
disadvantages of the previously used FAAs,[4,5] given that
the following criteria have to be satisfied. First, the 19F
reporter group has to be rigidly attached to the peptide
backbone in a well-defined position with respect to the
molecular framework. Any conformational flexibility of the
side chain around c1 and c2 would make the structural
interpretation of the NMRparameters ambiguous, as is the
case for 1 and 2.[4d,5,6] In 3 and 4 the position of the 19F reporter
group is well-defined, but analysis of the chemical shift
anisotropy of the single 19F substituent in 3 must still take into
account the value of c1.[4c,e,5,7] Fast rotation of the CF3 group in
4 renders all 19F interactions axially symmetric and colinear
E-mail: anne.ulrich@ibg.fzk.de
P. K. Mikhailiuk, Dr. M. O. Platonov, Dr. G. G. Dubinina,
Prof. I. V. Komarov
Department of Chemistry
Kyiv National Taras Shevchenko University
Volodymyrska 64, 01033 Kyiv (Ukraine)
Fax : (+38)044-225-12-73
and
Enamine Ltd.
Matrosova 23, Kiev 01103 (Ukraine)
E-mail: ik214@yahoo.com
Homepage: http://www.enamine.net
Dr. A. N. Chernega, Dr. E. B. Rusanov
Institute of Organic Chemistry of NAS of Ukraine
Murmanska 5, 02094 Kyiv (Ukraine)
ꢀ
with the Ca Cb bond, which makes it ideal for orientational
Dr. S. Afonin
analysis.[4a,b,5] While the chemical shift of a monofluorinated
FAA is difficult to reference,[2,5,8] this problem does not arise
when the absolute dipolar splitting of a CF3 group is
analyzed.[4a,b,5]
Forschungszentrum Karlsruhe
Institut für biologische Grenzflächen
Hermann-von-Helmholtz-Platz 1
76344 Eggenstein-Leopoldshafen (Germany)
It is also important that the FAA should not disturb the
structure and function of a peptide. Finally, the FAA should
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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5659

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be chemically stable and sufficiently reactive, and should
compound 10,[12] which was treated directly with tBuLi to
generate the carbanion and then with methylformate to
produce aldehyde 11. A mixture of compound 11 and the
corresponding hemiacetal 12 was used in the Strecker syn-
thesis with (R)-a-phenylglycinol as the chiral auxiliary.[13]
Products 13 and 14 were separated chromatographically.
The X-ray crystal structure of 14[14] confirmed the absolute
configuration at the newly formed chiral center to be S
(Figure 1).
maintain its l configuration during peptide synthesis. Even
under optimized conditions of Fmoc-SPPS the previously
used FAAs were found to racemize (3, 4), to eliminate HF (5),
or to be highly unreactive (6).[5,7]
In search of a rigid l-amino acid that would satisfy all of
the above-mentioned criteria, we focused on the bicyclo-
[1.1.1]pentane system. Placing CF3 and aminocarboxylate
moieties at the bridgehead positions of this skeleton yields the
19F-labeled amino acid 7 (3-(trifluoromethyl)bicyclopent-
ꢀ
[1.1.1]-1-ylglycine, CF3-Bpg) as the target. The Ca Cb and
ꢀ
C CF3 bonds in 7 are colinear; the side
chain can rotate only around these
bonds. In contrast to 3 and 4, in 7 the
CF3 group and the a-carbon atom are
separated by a saturated bicyclic cage.
This should make 7 less prone to racemization, since the
transmission of electronic effects across the bicyclo-
[1.1.1]pentane cage is far less pronounced than through an
aromatic ring.[9] Neither degradation by the loss of HF (as in
5) nor an unusually low reactivity of the aminocarboxylate
moiety as a result of steric hindrance (as in 6), are expected
for 7 under SPPS conditions.
The synthesis of 7 commences from 8 and proceeds via
[1.1.1]propellane 9[10,11] as the key intermediate (Scheme 1).
Addition of CF3I at the “inverted” carbon atoms of 9 led to
Figure 1. Molecular structure of 14.
Monitoring the asymmetric Strecker reaction by HPLC
we found that the initial 14/13 ratio was 1:1. However,
equilibration of purified 13 in MeOH led to isomerization,
giving a 14/13 ratio of 4:1. Repetition of the isomerization–
separation procedure provided 14 in 80% yield. The desired l
ꢀ
amino acid 7 was obtained from 14 by cleavage of the PhCH
CH2OH bond and hydrolysis of the intermediate Schiff base.
Compound 7 was optically pure (> 95% ee), as evidenced by
1
the H NMRspectrum of the corresponding methyl ester in
the presence of a chiral lanthanide shift reagent.
To examine the suitability of 7 as a 19F-labeled amino acid
for the analysis of peptides by NMRspectroscopy ( 19F NMR
label), we first compared its calculated properties[15] with
those of the nonpolar proteinogenic amino acids and the
aforementioned 19F-labeled compounds. The shape and steric
volume of 7 resemble closely the corresponding values of Leu,
Ile, Met, Trp, and Phe. The similarity to Pro, Val, and Ala is
poorer. The nonaromatic character of 7 constitutes its main
difference to Trp and Phe. If one considers the values of
octanol/water partition coefficients, a substitution of Leu, Ile,
or Met by 7 would cause minimal change in the mean
hydrophobicity, which is an important prerequisite for the
proper folding of a polypeptide.[16] According to all param-
eters it is apparent that 7 is closer to the natural nonpolar
amino acids than any of the previously used phenylglycine
derivatives (3, 4).
To address the suitability of 7 experimentally, the new
FAA was incorporated in the 21-mer sequence of the
Scheme 1. Reagents and conditions: a) MeLi, pentane, ꢀ788C, 0.5 h;
b) CF3I, pentane, RT, 20 h; c) tBuLi, Et2O, ꢀ788C, 1 h; d) CH3OCHO,
Et2O, ꢀ788C!RT, 3 h; e) (R)-a-phenylglycinol, CH2Cl2, RT, 2 h;
f) (CH3)3SiCN, RT, 10 h; g) chromatographic separation; h) MeOH,
reflux, 3 h; i) Pb(OAc)4, CH2Cl2, 08C, 5 min; j) 6m HCl, reflux, 2 h;
k) chromatography on Dowex-50.
antimicrobial peptide PGLa in the very same way as 3 and
[4a,b,h]
4 had been previously used as 19F NMRlabels.
The
substituted positions were Ile9 (15), Ala10 (16), Ile13 (17),
and Ala14 (18). Previously, the FAAs 3 and 4 had racemized
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completely under the SPPS conditions used to produce
PGLa.[4a,b,5,7] Now, all of the synthetic peptides containing 7
were obtained as single diastereomers, as shown by HPLC/
MS. It is known that PGLa assembles as an a helix when
bound to a lipid bilayer and permeabilizes bacterial mem-
branes.[4a,b] The biological activity of 15–18 was tested by
bacterial-growth inhibition assays, which can respond strongly
to single-point mutations (see the Supporting Information).
This proved that none of the labeled analogues display any
functional deviations from the wild-type peptide, suggesting
that the new building block 7 did not induce any structural
perturbations. Structure analysis of the labeled peptides 15–
18 by circular dichroism proved that no distortion was
induced by the new unit in any of the positions examined
(see Figure 2).
can be used to study their alignment, structure, and dynamics
by means of solid-state 19F NMRspectroscopy.
Received: January 26, 2006
Revised: May 15, 2006
Published online: July 25, 2006
Keywords: amino acids · fluorine · membrane-active peptides ·
.
NMR spectroscopy
[1] M. A. Danielson, J. J. Falke, Annu. Rev. Biophys. Biomol. Struct.
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Glaser, C. Sachse, P. Tremouilhac, M. Berditchevskaia in NMR
Spectroscopy of Biological Solids (Ed.: A. Ramamoorthy), CRC,
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[6] M. Cotten, C. Tian, D. D. Busath, R. B. Shirts, T. A. Cross,
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Figure 2. Circular dichroism spectra of the PGLa wild-type peptide and
its l-CF3-Bpg-labeled analogues 15–18 in the presence of sodium
dodecyl sulfate micelles (5 mmol), normalized to the peptide concen-
tration.
[8] a) R. W. Glaser, A. S. Ulrich, J. Magn. Reson. 2003, 164, 104;
b) R. Ulrich, R. W. Glaser, A. S. Ulrich, J. Magn. Reson. 2003,
164, 115.
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Org. Chem. 2005, 70, 1029.
[10] R. Pellicciari, M. Raimondo, M. Marinozzi, B. Natalini, G.
Costantino, C. Thomsen, J. Med. Chem. 1996, 39, 2874.
[11] K. B. Wiberg, S. T. Waddell, J. Am. Chem. Soc. 1990, 112, 2194.
[12] J. L. Adcock, A. A. Gack, J. Org. Chem. 1992, 57, 6206.
[13] T. K. Chakraborty, G. V. Reddy, K. A. Hussain, Tetrahedron
Lett. 1991, 32, 7597.
Finally, we reconstituted the labeled peptides into
mechanically oriented phospholipid bilayers for a compre-
hensive solid-state 19F NMRanalysis. In brief, 15–18 behaved
the same way as the PGLa analogues that had been previously
labeled with 3 and 4.[4a,b,5,7] Namely, 1) they bind to and
uniformly align in 1,2-dimyristoyl-sn-glycero-3-phosphocho-
line (DMPC) bilayers; 2) they rotate around the bilayer
normal; 3) they realign in
a concentration-dependent
[14] CCDC-293724 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif.
[15] The physical properties of the amino acids (see the Supporting
Information) were calculated by QikProp (www.schrodinger.
com).
manner; and 4) for a given concentration the labeled posi-
tions exhibit the expected dipolar coupling and sign compat-
ible with the known a-helical conformation. Therefore, we
conclude that CF3-Bpg satisfies all chemical and conforma-
tional requirements as a good labeling unit for peptide
structure analysis by solid-state 19F NMRspectroscopy.
[16] C. Branden, J. Tooze, Introduction to Protein Structure, Garland
Publishing, New York, 1999.
[17] Compound 7 is available at Enamine Ltd (http://www.enamine.
net).
In summary, we have synthesized the novel conforma-
tionally rigid fluorinated l a-amino acid 7 (CF3-Bpg). The
overall yield of the synthesis is 35%, which makes it attractive
for an intermediate-scale production of 7.[17] This amino acid
was designed as a label for membrane-bound peptides and
Angew. Chem. Int. Ed. 2006, 45, 5659 –5661
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5661