Synthesis of trifluoromethyl-substituted proline analogues as19F NMR labels for peptides in the polyproline II conformation

Article abstract of DOI:10.1002/anie.200801022

(Chemical Presented) As good as a Pro: Another CF₃-substituted amino acid, CF₃MePro (see structure), has been added to the arsenal of ¹⁹F NMR labels; it is particularly suited for the study of proline-rich peptides. This amino acid was carefully designed and chosen from other synthesized isomers, according to strict selection criteria, as the most stable, nonracemizable, conformationally restricted, and compatible with solid-phase peptide-synthesis protocols.

Full text of DOI:10.1002/anie.200801022

Angewandte
Chemie
DOI: 10.1002/anie.200801022
Amino Acids
Synthesis of Trifluoromethyl-Substituted Proline Analogues as
19F NMR Labels for Peptides in the Polyproline II Conformation**
Pavel K. Mykhailiuk, Sergii Afonin, Gennady V. Palamarchuk, Oleg V. Shishkin,
Anne S. Ulrich,* and Igor V. Komarov*
The location of proline (Pro) in proteins is peculiar: it is most
frequently found in loops, turns, and flanking positions of
stable secondary-structure elements.[1] Such positioning is a
consequence of the structural properties of Pro: first, the lack
of a second hydrogen atom on the a-amino group prevents
hydrogen bonding within a polypeptide, and second, the cyclic
nature of the side chain reduces the conformational freedom
and typically restricts the torsion angle, f, to (À63 Æ 15)8.[1a]
Polyprolines, (Pro)n, in aqueous solvents exist in a “poly-l-
proline II” (PPII) conformation;[2] this conformation is also
readily adopted by various proline-rich peptides (PRPs) and
protein segments, which are important as structural or
recognition motifs.[3–5]
Major obstacles in identifying and studying the native
PPII conformation are 1) the lack of intramolecular hydrogen
bonds, 2) the close similarity to the random coil (RC)
conformation when observed by conventional spectroscopic
methods, and 3) the frequent coexistence with other extended
conformations.[3c,5d] One of the very few ways to address such
ambiguities experimentally is to use solid-state NMR spec-
troscopy on selectively isotope-labeled peptides (by using 15N,
2
13C, H, or 19F isotopes). 19F NMR spectroscopy in particular
benefits from the high sensitivity of the 19F nucleus and does
not suffer from any natural-abundance background either.[6]
This approach is particularly well suited to study the structure,
alignment, and dynamics of peptides embedded in lipid
membranes.[7] It requires the selective incorporation of a
suitable 19F-substituted amino acid into the peptide backbone
to serve as an NMR fluorine label (FL). Such FLs should
1) be conformationally rigid to place the 19F reporter group
(preferably CF3) in a well-defined position, 2) be compatible
with standard solid-phase peptide-synthesis protocols, 3) be
chemically stable in a polypeptide under the conditions of
study, and 4) not perturb the native structure/function of the
peptide.
19F-labeled amino acids with nonpolar side chains (1, 2)
have already been successfully used as FLs.[7,8] Unfortunately,
the unique properties of Pro rule out the use of 1 or 2 as a
replacement for this residue. At first glance, the known CF3-
substituted proline analogues (3–5) may be considered as FLs.
Though, in 3 and 4,[9] criterion (1) from above is not fulfilled.
Amino acid 5[10] has no such deficiency because the CF3 group
is directly attached to the Ca carbon atom. However, the CF3
moiety could severely alter the steric and electronic environ-
ment of the peptide backbone and could reduce the chemical
reactivity of the amino acid.[11]
[*] P. K. Mykhailiuk, Prof. A. S. Ulrich
Institut für Organische Chemie
Karlsruhe Institute of Technology (KIT)
Fritz-Haber-Weg 6, 76133 Karlsruhe (Germany)
Fax: (+49)721-608-48-23
Homepage: http://hikwww8.fzk.de:8080/imb/bionmr/
Dr. S. Afonin, Prof. A. S. Ulrich
Institut für Biologische Grenzflächen
Karlsruhe Institute of Technology (KIT)
POB 3640, 76021 Karlsruhe (Germany)
E-mail: anne.ulrich@ibg.fzk.de
P. K. Mykhailiuk, Prof. I. V. Komarov
Organic Chemistry Department
Kyiv National Taras Shevchenko University
Volodymyrska 64, 01033 Kyiv (Ukraine)
E-mail: ik214@yahoo.com
Homepage: http://418Lab.chem.univ.kiev.ua/
P. K. Mykhailiuk, Prof. I. V. Komarov
Enamine Ltd.
Matrosova 23, 01103 Kyiv (Ukraine)
Homepage: http://www.enamine.net/
Our present research was motivated by the structural and
functional importance of proline in proteins and by the wish
to expand the repertoire of available FLs. We have designed
and synthesized the isomeric CF3-substituted 3,4- and 4,5-
methanoproline analogues 6 and 7 and compared them in
order to select the best FL candidate to be used in place of
Pro. Metal-catalyzed trifluoromethyl cyclopropanation of
G. V. Palamarchuk, Prof. O. V. Shishkin
STC, Institute for Single Crystals
National Academy of Science of Ukraine
Lenina ave. 60, 61001 Kharkiv (Ukraine)
[**] Financial support from the Alexander von Humboldt Foundation is
gratefully acknowledged (Institute Partnership Grant 3 Fokoop
DEU/1054096).
=
C C bonds, by using CF3CHN2, was recently shown to be
practically beneficial.[12] We utilized this approach in the
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200801022.
construction of 6 and 7.
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Communications
The synthesis of 6 started from optically pure 3,4-
dehydroproline derivative 8.[13] Extensive experimentation
was needed to find the optimal reaction conditions with
suppression of electrophilic attack of the carbene at the OtBu
group (Scheme 1). The latter reaction prevailed if CuCl was
Figure 1. Molecular structure of 12b.
Scheme 1. Reagents and conditions: a) excess CF3CHN2, CuOTf, RT;
b) column chromatography on silica gel Merck 60; c) excess CF3CHN2,
CuCl, RT; d) HBr (48%), reflux, 6 h; e) chromatography on Dowex-50.
Tf: trifluoromethanesulfonyl, Boc: tert-butoxycarbonyl.
Information), and the assignment was also confirmed for 7b
by X-ray analysis of the precursor 12b (Figure 1).
In principle, each isomer of 6 and 7 could now be used as
an FL. They all possess a CF3 group, this group is sufficiently
distant from the backbone to prevent perturbation, and the
used as a catalyst; the only isolated product in that case was 9. side chains are conformationally more restricted than in 3 and
By increasing the reaction time and applying CuOTf, we 4. However, amino acids 6a,b are not preferable, because of
managed to isolate two diastereomeric products, 10a,b, in a the poor total yield of their synthesis. In the case of 7b and 7c,
modest yield (15%). The deprotected amino acids 6 were the cyclopropane ring was found to be unstable at low pH
obtained after 10 was heated to reflux in 48% HBr, and the values. By contrast, 7a is stable at both high and low pH
products were purified by ion-exchange column chroma- values, and it was therefore selected as a promising FL to
tography.
The synthesis of 7 turned out to be more fruitful. The
replace Pro in peptides.
In order to address the suitability of 7a as an FL and to
reaction of 11 with 2,2,2-trifluoromethyldiazomethane in the examine its influence on the PPII conformation, the proline-
presence of catalytic amounts of CuCl produced trifluoro- rich cell-penetrating peptide SAP (14, (VRLPPP)3) was
methyl-substituted proline analogues 12a–c in an overall selected.[15] This peptide is known to self-assemble above
yield of 66% (Scheme 2). The molar ratio of 12a/12b/12c was 50 mm in aqueous solutions, and it is believed to exist as a
1.0:0.9:0.7. In contrast to the corresponding reaction with 8, mixture of the PPII and RC conformations below this
the formation of side products 13[14] was observed only with concentration. The peptide VRLPPPVRLP 7a PVRLPPP
prolonged reaction times. The three isomers of 7 were (15), containing a Pro/7a substitution, was synthesized.
À
À
obtained by standard deprotection methods.
Neither degradation nor low reactivity of 7a was observed.
The relative configurations of 6a,b and 7a–c were No racemization was detected either.
determined by NMR spectroscopy (see the Supporting
We have also synthesized the SAP analogue VRLPPPVR-
2-PPPVRLPPP (16), labeled with the previously reported FL
[8c,d] in the position of Leu9. Comparison of the CD spectra of
2
peptides 14–16 at different concentrations (Figure 2) revealed
that FL 2 did not affect the SAP structure, whereas FL 7a
unexpectedly stabilized the PPII conformation of SAP. It can
be seen that 14 and 16 exhibit spectral changes up to 50 mm,
indicating oligomerization beyond this point, while 15 oligo-
merized at a concentration that was twice as high. Moreover,
for all concentrations of 15, a positive band was observed at
223 nm, the hallmark of the PPII conformation. The same
result is seen in the temperature series of 15 (Figure 2D). The
positive band at 223 nm persisted even at 508C, in contrast to
the results with native SAP.[15]
In summary, 7a was demonstrated to be a promising label
for solid-state 19F NMR analysis of PRPs in the light of its
chemical stability, compatibility with solid-phase peptide
synthesis, and propensity to maintain, and even to stabilize,
Scheme 2. Reagents and conditions: a) CF3CHN2, CuCl, RT; b) column
chromatography on silica gel Merck 60; c) excess CF3CHN2, CuCl, RT;
d) NaOH, MeOH, RT; e) TFA/CH2Cl2, RT. TFA: trifluoroacetic acid
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Angewandte
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Keywords: amino acids · conformation analysis · fluorine ·
.
NMR spectroscopy · peptides
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Figure 2. CD spectra of A) wild-type SAP peptide 14, B) CF3MePro-
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the PPII conformation; this stabilization is probably a
consequence of the conformational properties of 7a.
Received: March 3, 2008
Published online: June 24, 2008
Angew. Chem. Int. Ed. 2008, 47, 5765 –5767
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5767