Design and synthesis of a monofluoro-substituted aromatic amino acid as a conformationally restricted 19F NMR label for membrane-bound peptides

Article abstract of DOI:10.1002/ejoc.201301737

A monofluoro-substituted amino acid was designed to serve as a conformationally restricted label for solid-state ¹⁹F NMR distance measurements in membrane-bound peptides. The aromatic cis and trans isomers of 1-amino-3-(4-fluorophenyl)cyclobuta

Full text of DOI:10.1002/ejoc.201301737

FULL PAPER
DOI: 10.1002/ejoc.201301737
Design and Synthesis of a Monofluoro-Substituted Aromatic Amino Acid as a
19
Conformationally Restricted F NMR Label for Membrane-Bound Peptides
Anton N. Tkachenko,[a] Pavel K. Mykhailiuk,*[a,b] Dmytro S. Radchenko,[a,c] Oleg Babii,[d]
Sergii Afonin,*[e] Anne S. Ulrich,*[d,e] and Igor V. Komarov[c]
Keywords: Amino acids / Proteins / Membrane proteins / Protein modifications / Fluorine / Solid-state NMR spectroscopy
A monofluoro-substituted amino acid was designed to serve
as a conformationally restricted label for solid-state 19F NMR
distance measurements in membrane-bound peptides. The
aromatic cis and trans isomers of 1-amino-3-(4-fluorophenyl)-
cyclobutanecarboxylic acid were synthesized in five steps
from diethyl 2-(4-fluorophenyl)propanedioate. They were in-
corporated into the antimicrobial peptide gramicidin S to re-
rasubstituted amino acid cannot racemize, it showed full
compatibility with solid-phase peptide synthesis protocols.
According to circular dichroism analysis and molecular mod-
eling, the 19F-labeled analogues of the known helix-inducing
amino acid (1-aminocyclobutane-1-carboxylic acid) do not
disrupt the peptide conformation when substituted for Phe,
neither in a β-turn nor in an α-helix.
place a native D-phenylalanine residue. Because the Cα-tet-
ing the following strict criteria: (1) the side chain of the
amino acid has to be conformationally constrained to place
the 19F reporter group in a geometrically well-defined posi-
tion relative to the peptide backbone; (2) the amino acid
should be compatible with the standard procedures of pept-
ide synthesis and, clearly, (3) has to be similar to the natural
amino acid which it replaces in terms of steric size, polarity,
ionizability, conformational propensities, etc. These criteria
exclude most of the known fluorinated amino acids from
the list of potential 19F labels.[3]
Introduction
19F NMR spectroscopy is a powerful technique for the
structure analysis of peptides and proteins. The high gyro-
magnetic ratio, the absence of a natural abundance back-
ground, the unique sensitivity of 19F NMR chemical shifts
to local environment, and especially access to long-range
distances of up to 18 Å, make the 19F nucleus an attractive
NMR reporter.[1] Solid-state 19F NMR experiments, in par-
ticular, benefit from these features, because selective 19F la-
bels can be used to determine the conformation, alignment,
and dynamic behavior of membrane-bound peptides with a
unique sensitivity and distance range that is unprecedented
by any other NMR label.[2] For this approach, carefully de-
signed 19F-substituted amino acids are incorporated into
peptides in place of the natural amino acid residues, meet-
Recently, several 19F-labeled amino acids, usually bearing
[4]
a CF3 or a CF2 group[5] have been designed, synthesized
and incorporated into peptides for NMR analysis. The
main reasons for having developed such multi-fluorine-sub-
stituted NMR labels are the ease with which orientational
parameters can be extracted from the dipolar 19F–19F cou-
plings, and advantages in spectral referencing.[2d,2n] To date,
only a single conformationally constrained monofluoro-
substituted amino acid has been shown to be suitable for
19F NMR structure analysis. Namely, 4-F-phenylglycine
(compound 1) was used as a selective label to replace vari-
ous hydrophobic residues in several membrane-active pept-
ides.[2n,6] Unfortunately, this amino acid racemizes exten-
sively during the Fmoc (9-fluorenylmethoxycarbonyl) solid-
phase peptide synthesis (SPPS),[6b] such that two peptide
epimers are produced, which could be separated chromato-
graphically. This drawback, together with the timely ap-
pearance of a nonracemizable alternative,[4a–4c] was the
prime reason for discontinuing its use as an NMR label.
The monofluoro-substituted amino acids, however, are
useful not only for measuring peptide orientational param-
eters,[6] but they are actually superior over CF3/CF2 groups
for determining inter-fluorine distances from discrete 19F–
[a] Enamine Ltd.,
Oleksandra Matrosova 23, 01103 Kyiv, Ukraine
E-mail: Pavel.Mykhailiuk@gmail.com
Pavel.Mykhailiuk@mail.enamine.net
http://www.enamine.net/
[b] Faculty of Chemistry, Taras Shevchenko National University of
Kyiv,
Volodymyrska 64, 01601 Kyiv, Ukraine
http://www.chem.univ.kiev.ua/
[c] Institute of High Technologies, Taras Shevchenko National
University of Kyiv,
Volodymyrska 60, 01601 Kyiv, Ukraine
[d] Institute of Organic Chemistry and CFN, Karlsruhe Institute
of Technology (KIT),
Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany
[e] Institute of Biological Interfaces (IBG-2), KIT,
P. O. B. 3640, 76021 Karlsruhe, Germany
E-mail: sergiy.afonin@kit.edu
anne.ulrich@kit.edu
http://www.ibg.kit.edu/nmr/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301737.
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19F NMR Label for Membrane-Bound Peptides
19F spin pairs.[6a,6g] In such NMR experiments, CF3/CF2 acid 3 might partially decompose during peptide synthesis.
groups display strong intra-group dipolar interactions that More important for our choice in favor of 4 was the fact
obscure the much weaker inter-group interactions.[2j,2p] that the native peptide backbone conformation is disturbed
Therefore, we set out to prepare novel monofluoro-substi- by cyclopropane-derived amino acid residues more than by
tuted amino acids suitable for 19F NMR studies that cannot many other α,α-disubstituted analogues.[8d,8e] Another ad-
racemize during peptide synthesis. Here, we report on the vantage of 4 is that this molecule is not chiral, which will
rational design and synthesis of an amino acid derivative simplify the synthesis by avoiding asymmetric steps or te-
that is fully compatible with SPPS and may be used as a dious resolution of enantiomers. Given that amino acid 4 is
selective 19F label to replace aromatic amino acid residues quaternary, racemization cannot occur at the α-carbon
(phenylalanine, Phe, and tyrosine, Tyr) in peptides. The re- atom, which was the main drawback of the known label 1.
sulting Cα-tetrasubstituted amino acids are structural ana-
logues of 1-aminocyclobutane-1-carboxylic acid. Hence,
they are expected to exert the same the conformational
preferences as this known β-turn and α-helix-inducer.[7]
Results and Discussion
Design
The principles of designing the target structure are sum-
marized in Scheme 1. The F-substituent should be placed
at the para-position of the phenyl ring (Scheme 1, step a);
in this case, rotation around the opposite C–C bond will
not change the position of the 19F-reporter group relatively
to the peptide backbone in the labeled peptides. To restrict
any other conformational mobility of the side chain, and to
prevent racemization during peptide synthesis, conforma-
tionally rigid cyclopropane or restricted cyclobutane frag-
ments could be introduced in the intermediate template 2
(Scheme 1, step b), giving two possible F-label candidates,
the amino acids 3 and 4. The latter might be regarded as
superior for several reasons. First, the cyclopropane ring is
known to be susceptible to nucleophilic attack when acti-
Scheme 1. Design of the conformationally restrained 19F NMR la-
bel 4, to be used as a replacement for the aromatic proteinogenic
amino acids in peptides; (a) attachment of the monofluoro reporter
group; (b) restriction of the side chain mobility.
Amino Acid Synthesis
Synthesis of the target compound 4 started with the
vated by electronegative substituents.[8a–8c] Thus, amino fluoro-substituted diester 5 (Scheme 2).[9] Reduction of 5
Scheme 2. Synthesis of amino acids 4a·HCl and 4b·HCl. Reagents and conditions: (a) LiAlH4, Et2O, reflux; (b) Et3N, MsCl, CH2Cl2;
(c) CH2(COOEt)2, NaH, dioxane, reflux; (d) KOH, EtOH, reflux; (e) SOCl2, CH2Cl2, reflux; (f) NaN3, H2O, acetone, 0 °C; (g) toluene,
90 °C, then tBuOH; (h) 20% aq. HCl, reflux.
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with LiAlH4 in diethyl ether afforded alcohol 6 according 9b, separately, to reflux in diluted aqueous HCl solution
to a reported protocol.[9] Mesylation of 6[10] followed by gave the target amino acids 4a·HCl and 4b·HCl.
double alkylation of diethyl malonate gave cyclobutane 7 in
good yield. Monohydrolysis of 7 with 1 equiv. KOH in eth-
anol afforded acid 8 as an inseparable mixture of two iso-
mers (3:2). A Curtius reaction was then performed, and the
diastereomeric products were separated by column
chromatography to afford the pure compounds 9a (51%)
and 9b (33%). The stereochemical configuration of the ob-
tained isomers was determined by X-ray diffraction analysis
of the crystalline ester 9a (Figure 1). Finally, heating 9a and
Conformational Preferences
Having obtained two stereoisomers of the new mono-
fluoro-substituted 19F NMR label, we performed molecular
modeling calculations to gain an impression of how these
restricted side chains would fit into the structure of a typi-
cal peptide. For a representative backbone conformation,
an ideal α-helix was constructed from ten alanine or ten
leucine residues using the program Discovery Studio,[12] and
the novel amino acid residues 4a or 4b were placed at the
fifth position of each decamer, using the structural param-
eters obtained in the X-ray study.[11] Figure 2 (A) illustrates
the obtained results. In an ideal, all-l α-helix, all the χ1
rotamers are allowed for a LPhe residue,[13] and steric
clashes only occur when a bulky side chain occupies posi-
tion i-3. Such putative clashes can only occur in a limited
sector of the rotameric space, which is actually opposite to
the one that can be occupied by 4a/4b side chains. There-
fore, the new 19F labels should be well-compatible with any
α-helical environment. In the context of a β-turn, the intrin-
sic flexibility of any (l- or d-)Phe side chain is even greater
than in an α-helix, and should thus also be fully compatible
with the new amino acid (Figure 2, B). We have selected the
antimicrobial peptide gramicidin S {[cyclo(Val-Orn-Leu-
Figure 1. X-ray diffraction analysis of 9a.[11] The anisotropic dis-
placement ellipsoids of atoms are shown at 30% probability levels
(see the Supporting Information), with fluorine in green, nitrogen
in blue, and oxygen in red.
Figure 2. Visualization of the expected impact of the sterically restrained side chains of 4a and 4b residues on the neighboring amino
acid residues in an ideal α-helical peptide (A), and within a β-turn of gramicidin S (B). Natural LPhe (A) and DPhe (B) are displayed on
the very left, with their preferred torsion angles around which the free rotation is allowed (indicated as χ1 and χ2). In the middle column
of the panels, the monofluoro-substituted 19F NMR labels are displayed in their sterically constrained configuration, as determined from
X-ray crystallography. The molecular models on the very right show an overlay of all possible χ1 rotamers (18 in total, spaced by 20°) of
LPhe (A) and DPhe (B) together with the sterically confined positions of 4a (dark red) and 4b (dark blue). In the molecular models,
carbon atoms are grey, nitrogen are blue, oxygen are red, fluorine are green; hydrogen bond patterns are marked by green dotted lines.
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19F NMR Label for Membrane-Bound Peptides
DPhe-Pro)2]; GS}[14] as a test system to incorporate the new worth mentioning that neither isomerization nor decompo-
NMR label, because it contains two DPhe-Pro units that sition of amino acid 4a was observed during this synthesis,
are an obvious choice for a DPhe/4 substitution. The calcu- and the linear peptide 11 was obtained as a single stereoiso-
lations suggested that both 4a and 4b residues should be mer. In contrast, under identical conditions, amino acid 1
readily accommodated in the molecule of gramicidin S, had previously racemized.[5b,6b,16]
without perturbing the native backbone conformation. No-
The GS analogue 12 was obtained by cyclization of 11,
tably, in both backbone environments of an α-helix and a performed in dichloromethane under high dilution condi-
β-turn, the orientational constraints that could be obtained tions, using PyBOP/HOBt/DIPEA to activate the carbox-
from the new 19F NMR reporters (i.e., their C–F vectors) ylic acid group (Scheme 3). The NBoc protecting groups on
are almost equivalent for 4a and 4b, therefore, any of the ornithine residues were removed by trifluoroacetic acid. Pu-
cis- and trans-isomers of the novel amino acid could be used rification of the crude product by reverse-phase high-per-
as a label. Given the negligible steric clashes of the new formance liquid chromatography (RPLC) afforded the pure
amino acid residues in the cyclic structure of GS, we pro- peptide cyclo[Val-Orn-Leu-4a-Pro-Val-Orn-Leu-DPhe-Pro]
ceeded to synthesize the peptide analogue labeled with (ar- (12).
bitrarily selected) 4a.
Conformational Analysis
Peptide Synthesis
To evaluate the impact of 4a on the structure of GS ex-
Amino acid 4a was incorporated into GS by replacing perimentally, a circular dichroism (CD) study was per-
one of the two d-phenylalanine residues. First, amino acid formed. Solutions of RPLC-purified natural GS and 12
4a was converted into the Fmoc-protected form 10a by were studied. Similar to GS, 12 was only poorly soluble in
treatment with FmocCl/(CH3)3SiCl in dichlorometh- phosphate buffer (PB; 10 mm, pH 7.4). In the membrane-
ane.[4f,4g,15] The building block 10a was used to manually modeling environments, namely in PB with excess detergent
synthesize the linear decapeptide 11 on a 2-chlorotrityl resin micelles (20 mm SDS, detergent/peptide of Ͼ300:1 mol/
preloaded with d-phenylalanine.[16] It was found that mol), both peptides provided the CD spectra with almost
1.5 equiv. of 10a, which we used to save material, was suf- identical appearance (Figure 3). All the CD spectral fea-
ficient to be completely incorporated into the polypeptide tures known for GS[5b,6a,15b] were the same in the spectra
sequence. A mixture of 1-hydroxybenzotriazole (HOBt), of 12. These results proved the equivalency of the secondary
(benzotriazole-1-yl)oxy-tris-pyrrolidino-phosphonium structures of the studied peptides in the tested environ-
hexafluorophosphate (PyBOP), and N-ethyldiisopropyl- ments. The replacement of DPhe by 4 is therefore compati-
amine (DIPEA) was used to activate the carboxylic acid ble with the backbone conformation of GS, as was pre-
group of each amino acid during the coupling step. The dicted by the calculations described above. The Cα-tetra-
linear peptide was cleaved from the resin by using a hexa- substituted amino acids developed here are direct structural
fluoroisopropanol/dichloromethane cocktail under condi- derivatives of the well-known β-turn and α-helix-inducer 1-
tions that left the ornithine Boc-protection intact.[17] It is aminocyclobutane-1-carboxylic acid. Since the derivati-
Scheme 3. Synthesis of the GS analogue 12. Reagents and conditions: (a) (CH3)3SiCl, DIPEA, FmocCl, CH2Cl2, 0–25 °C; (b) SPPS, 2-
chlorotrityl resin; (c) i. PyBop, HOBt, DIPEA, CH2Cl2, high dilution; ii. trifluoroacetic acid, thioanisole, water.
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using silica gel (230–400 mesh) as the stationary phase. 1H, 19F, and
13C NMR spectra were recorded at 9.4 and 11.7 Tesla. Chemical
shifts are reported in ppm downfield of tetramethylsilane (TMS,
zation is made at the Cγ position, the steric vicinity around
Cα should remain similar to that in the parent amino acid.
Hence, 4 is expected to have very similar conformational
preferences.[7] The new amino acids may thus be especially
useful for incorporating a mono-fluoro label into α-helical
and β-turn structured backbones, without risking destabili-
zation of the backbone. Along the same line of arguments,
the same secondary structure propensities are expected for
the other Cγ-substituted amino acids, which would explain,
1
for H, 13C) or CFCl3 (19F). Mass spectrometry analyses were per-
formed with a liquid chromatography–mass spectrometry instru-
ment (Agilent 1100) with chemical ionization, a gas chromatog-
raphy–mass spectrometry instrument with electron impact ioniza-
tion, or with a Bruker Autoflex III MALDI (matrix-assisted laser
desorption/ionization). MALDI samples were co-crystallized with
a matrix of α-cyano-4-hydroxycinnamic acid from acidic H2O/
for example, their good compatibility with α-helical back- MeCN/MeOH solutions on a stainless steel target.
bones.[6f,6g]
2-(4-Fluorophenyl)propane-1,3-diol (6): Anhydrous Et2O (700 mL)
was placed into a large (2 L) three-necked round-bottomed flask
equipped with mechanical stirrer, a condenser, and a drop funnel.
LiAlH4 (9.6 g, 0.25 mol) was added carefully while stirring. To this
suspension, 5[9] (26.0 g, 0.10 mol, oil) was added dropwise with vig-
orous stirring. The obtained mixture was heated to reflux for 2 h,
and then cooled to room temperature. Water (9 mL) was slowly
added (2–3 drops per minute) followed by 10% aq. NaOH (9 mL)
and water again (32 mL). The white precipitate was filtered off,
the filter cake was washed with diethyl ether (200 mL), dried with
MgSO4, and the solvents were evaporated in vacuo. Vacuum distil-
lation (110 °C at 0.5 Torr) gave diol 6 (13.4 g, 69 mmol, 69% yield)
as a colorless viscous oil. The spectral characteristics were identical
to those reported previously.[9]
Diethyl 3-(4-Fluorophenyl)cyclobutane-1,1-dicarboxylate (7): Diol 6
(13.4 g, 79 mmol) was dissolved in dichloromethane (250 mL), then
triethylamine (33 mL, 237 mmol) was added and the obtained mix-
ture was cooled to –30 °C. MsCl (14.6 mL, 189 mmol) was added
Figure 3. Circular dichroism spectra of native GS and the 19F-lab-
eled analogue (12) in the presence of detergent micelles (20 mm
SDS).
dropwise, taking care to keep the temperature below –20 °C. Sub-
sequently, the mixture was warmed to room temperature, and then
washed with water (100 mL), 10% aq. citric acid (100 mL), satu-
rated aq. NaHCO3 (100 mL), and brine (100 mL). The obtained
solution was dried with Na2SO4, followed by vacuum drying to
give the corresponding di-mesylate (26.0 g, 79 mmol, 100% yield)
as a viscous reddish oil, which crystallized upon storage. The prod-
uct (26.0 g) was placed into a 2 L three-necked round-bottomed
flask, equipped with a mechanical stirrer and a Dimroth condenser,
followed by addition of anhydrous dioxane (300 mL) and diethyl
malonate (13 mL, 88 mmol). The formed solution was heated to
reflux, and NaH (6.4 g, 0.16 mol, 60% in mineral oil) was added
in very small portions over 3 h. The reaction mixture was then
heated to reflux for additional 18 h. After cooling to room tem-
perature, the mixture was filtered through a silica gel pad and vac-
uum dried. The residue was partitioned between hexane (200 mL)
and water (200 mL) and the aqueous layer was additionally ex-
tracted with hexane (2ϫ150 mL). The combined organic fractions
were washed with brine, dried with Na2SO4, and vacuum dried to
afford an orange oil (18.6 g, 63 mmol, 79%). The product was used
Conclusions
We have synthesized the novel monofluoro-substituted
conformationally restricted amino acids 4a and 4b. These
amino acids were designed to be used as solid-state 19F
NMR labels to measure long-range 19F–19F distances in
membrane-bound peptides. The compatibility of 4a with
standard SPPS protocols was demonstrated by incorporat-
ing it into the antimicrobial peptide gramicidin S, by replac-
ing d-phenylalanine in one of the β-turns. In contrast to
the previously used monofluoro-substituted label 1, the new
amino acid did not racemize during peptide synthesis.
Computational analysis and circular dichroism showed that
the backbone structure of the 19F-labeled peptide was not
perturbed by the conformationally restricted side chain,
which is well-compatible with a β-turn. For α-helical pept-
ides, we also predict that amino acid 4 will be a suitable
NMR label that can be used to determine orientational
constraints and to conduct long-range distance measure-
ments.
1
in the next step without purification. H NMR (500 MHz, CDCl3,
TMS): δ = 1.29 (t, JH,H = 7.0 Hz, 3 H, CH2CH3), 1.33 (t, JH,H
7.0 Hz, 3 H, CH2CH3), 2.68 [t, JH,H = 11.0 Hz, 2 H, C(CHH)2C],
2.95 [t, JH,H = 10.5 Hz, 2 H, C(CHH)2C], 3.61 (quint, JH,H
=
=
9.5 Hz, 1 H, ArCH), 4.24 (q, JH,H = 7.0 Hz, 2 H, CH2CH3), 4.30
(q, JH,H = 7.0 Hz, 2 H, CH2CH3), 7.02 (t, JH,H = 8.5 Hz, 2 H, m-
ArH], 7.21 (br. t, 2 H, o-ArH) ppm.
1-(Ethoxycarbonyl)-3-(4-fluorophenyl)cyclobutanecarboxylic Acid
(8): Diester 7 (8.2 g, 28 mmol) was dissolved in ethanol (80 mL)
and the solution was heated to reflux. A solution of KOH (1.5 g,
27 mmol) in ethanol (70 mL) was added dropwise over 1 h under
vigorous stirring. The mixture was heated to reflux for an ad-
ditional 15 min, and then cooled to room temperature. The white
Experimental Section
General: Solvents were purified according to standard procedures.
Analytical thin-layer chromatography (TLC) was performed using
Polychrom SI F254 plates. Column chromatography was performed
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19F NMR Label for Membrane-Bound Peptides
precipitate bis-potassium salt of the corresponding dicarboxylic
acid (0.9 g, 3 mmol) was filtered off and discarded, and the filtrate
was dried under reduced pressure. The residue was dissolved in
water (80 mL), and the solution was extracted with diethyl ether
(2ϫ 50 mL). The organic phase contained the starting diester 7
(1.7 g, 6 mmol). The aqueous layer was acidified by 6 n aqueous
HCl solution until pH ca. 1, and the product was extracted with
dichloromethane (3ϫ 50 mL). The combined extracts were washed
with brine (50 mL), dried with Na2SO4, and filtered. Evaporation
of the solvent gave the crude monoester 8 (5.6 g) as a reddish oil.
This material was dissolved in dichloromethane (100 mL), and an
aq. solution of KMnO4 (6%, 150 mL) was added. The hetero-
geneous mixture was vigorously stirred for 16 h, then diluted sulfu-
ric acid (20%, 50 mL) and saturated aq. NaHSO3 solution (70 mL)
was added until two colorless layers formed. The organic layer was
separated, dried with Na2SO4, and vacuum dried to give the desired
compound 8 (4.3 g, 16 mmol, 58% yield) as a 3:2 mixture of the
2 H, C(CHH)2C], 3.71 (quint, JH,H = 9.0 Hz, 1 H, ArCH), 4.19 (q,
JH,H = 7.2 Hz, 2 H, CH2CH3) 5.33 (br. s, 1 H, NH), 6.98 (t, JH,H
= 8.8 Hz, 2 H, m-ArH], 7.28 (m, 2 H, o-ArH) ppm. 13C NMR
(126 MHz, CDCl3, TMS):
δ = 14.2 (s, CH2CH3), 28.3 [s,
C(CH3)3], 33.3 (s, ArCH), 38.6 [s, C(CH2)2C], 55.6 (s, CNHBoc),
2
61.4 (s, CH2CH3), 80.1 [s, C(CH3)3], 115.1 (d, JC,F = 21 Hz, m-
ArC), 128.1 (s, 3JC,F = 9 Hz, o-ArC), 139.9 (s), 155.4 (s, NHCOO),
1
161.5 (d, JC,F = 244 Hz, FC), 173.2 (s, COOEt) ppm. 19F NMR
(376 MHz, CDCl3, CFCl3): δ = –117.26 (s) ppm. C18H24FNO4
(337.39): calcd. C 64.08, H 7.17, N 4.15; found C 64.38, H 6.99, N
3.87.
cis-1-Amino-3-(4-fluorophenyl)cyclobutanecarboxylic Acid Hydro-
chloride (4a·HCl): Compound 9a (2.10 g, 6.2 mmol) was suspended
in 20% aq. HCl (20 mL) and the mixture was heated to reflux for
2 h. After cooling to room temperature, the solution was vacuum
dried. The solid residue was triturated with diethyl ether (80 mL)
and the precipitate was filtered and dried in air to give 4a·HCl
(1.25 g, 6.0 mmol, 96% yield), m.p.Ͼ220 °C (decomp.). 1H NMR
(500 MHz, D2O, TMS): δ = 2.49 [t, JH,H = 11.0 Hz, 2 H, (CHH)2],
2.95 [t, JH,H = 10.0 Hz, 2 H, (CHH)2], 3.78 [quint, JH,H = 9.5 Hz, 1
H, ArCH], 7.03 [app. t, JH,H = 9.0 Hz, 2 H, m-ArH], 7.24 (m, 2 H,
o-ArH) ppm. 13C NMR (126 MHz, D2O, TMS): δ = 31.5 (s,
ArCH), 37.4 [s, (CH2)2], 53.4 (s, CCOOH), 115.3 [d, 2JC,F = 21 Hz,
1
cis- and trans-diastereomers, appearing as a yellow viscous oil. H
NMR (500 MHz, CDCl3, TMS): δ = 1.26–1.38 (m, 3 H, CH2CH3),
2.65–2.83 [m, 2 H, C(CHH)2C], 2.93–3.07 [m, 2 H, C(CHH)2C],
3.60–3.75 (m, 1 H, ArCH), 4.21–4.37 (m, 2 H, CH2CH3), 7.02 (m,
2 H, m-ArH), 7.22 (m, 2 H, o-ArH), 8.82 (br, 1 H, COOH) ppm.
Ethyl 1-[(tert-Butoxycarbonyl)amino]-3-(4-fluorophenyl)cyclobutane-
carboxylates (9a/9b): A solution of 8 (4.0 g, 15 mmol) in dichloro-
methane (40 mL) and SOCl2 (3.3 mL, 45 mmol) was heated to re-
flux for 1 h until gas evolution ceased. The reaction mixture was
vacuum-dried and re-dissolved/re-dried three times from CCl4
(30 mL) to remove traces of SOCl2. The final residue was dissolved
in acetone (20 mL) and the obtained solution was added dropwise
to a stirring ice-cooled solution of NaN3 (4.9 g, 75 mmol) in water
(16 mL). The obtained mixture was stirred for 1 h at 0–5 °C and
then poured into crushed ice (ca. 80 g). The mixture was extracted
with diethyl ether (3ϫ 60 mL) and the combined organic fractions
were dried with MgSO4, concentrated by vacuum-drying to ca.
20 mL, and added dropwise to preheated toluene (120 mL, 90 °C)
under vigorous stirring. After 3 h, the evolution of gas stopped,
tBuOH (30 mL) was added and the mixture was heated to reflux
for 18 h. After cooling to room temperature, the solution was vac-
uum dried. The residue was purified by column chromatography
(hexane/ethyl acetate, 7:1) to give 9a (2.6 g, 7.7 mmol, 51% yield)
and 9b (1.7 g, 5.0 mmol, 33% yield), both as white crystalline sol-
ids.
3
4
m-ArC], 128.1 [d, JC,F = 9 Hz, o-ArC], 139.2 [d, JC,F = 4 Hz],
161.4 [d, JC,F = 241 Hz, FC], 174.1 (s, COOH) ppm. 19F NMR
1
(376·MHz, D2O, CFCl3): δ = –115.00 [tt, JH,F = 10.0, 5.0 Hz] ppm.
C11H13ClFNO2 (245.68): calcd. C 53.78, H 5.33, N 5.70; found C
53.59, H 5.31, N 5.84.
trans-1-Amino-3-(4-fluorophenyl)cyclobutanecarboxylic Acid Hydro-
chloride (4b·HCl): Compound 9b (1.30 g, 3.8 mmol) was suspended
in 20% aq. HCl (20 mL) and the mixture was heated to reflux for
2 h. After cooling to room temperature, the solution was vacuum
dried. The solid residue was triturated with diethyl ether (60 mL)
and the precipitate was filtered and dried in air to give 4b·HCl
(0.74 g, 3.5 mmol, 92% yield), m.p. Ͼ 220 °C (decomp.). 1H NMR
(500 MHz, D2O, TMS): δ = 2.70 [m, 2 H, (CHH)2], 2.89 [m, 2 H,
(CHH)2], 3.83 (quint, JH,H = 9.5 Hz, 1 H, ArCH), 7.04 (app. t,
JH,H = 9.0 Hz, 2 H, m-ArH), 7.24 (m, 2 H, o-ArH) ppm. 13C NMR
(126 MHz; D2O; TMS): δ = 31.3 (s, ArCH), 36.5 [s, (CH2)2], 55.3
2
3
(s, CCOOH), 115.3 (d, JC,F = 21 Hz, m-ArC), 128.3 (d, JC,F
=
4
1
9 Hz, o-ArC), 139.0 (d, JC,F = 3 Hz), 161.5 (d, JC,F = 241 Hz,
FC), 173.6 (s, COOH) ppm. 19F NMR (376 MHz, D2O, CFCl3): δ
= –114.88 (tt, JH,F = 10.0, 5.0 Hz) ppm. C11H13ClFNO2 (245.68):
calcd. C 53.78, H 5.33, N 5.70; found C 53.65, H 5.02, N 5.32.
Compound 9a: M.p. 78–79 °C. Rf = 0.36. 1H NMR (400 MHz,
CDCl3, TMS): δ = 1.32 (t, JH,H = 7.2 Hz, 3 H, CH2CH3), 1.43 [s,
9 H, C(CH3)3], 2.15–2.59 [br. m, 2 H, C(CHH)2C], 3.00 [m, 2 H,
C(CHH)2C], 3.61 (quint, JH,H = 8.8 Hz, 1 H, ArCH), 4.27 (q, JH,H
cis-1-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-3-(4-fluoro-
phenyl)cyclobutanecarboxylic Acid (10a): Compound 4a·HCl
(1.20 g, 5.8 mmol) was suspended in anhydrous dichloromethane
(30 mL). The solution was cooled in an ice bath and chlorotrimeth-
ylsilane (2.2 mL, 17.7 mmol) and DIPEA (4.5 mL, 28.7 mmol)
were added while stirring (the solution became clear after the ad-
dition). The mixture was stirred for 30 min, then FmocCl (1.5 g,
5.93 mmol) was added. After stirring for 3 h at room temperature,
the solvent was removed under reduced pressure and the residue
was partitioned between 5% aq. NaHCO3 (50 mL) and diethyl
ether (50 mL), and the organic fraction was discarded. The aque-
ous layer was acidified with 1 n HCl to pH ca. 1 and extracted
with ethyl acetate (3ϫ 50 mL). The combined organic layers were
washed with water (50 mL) and brine (50 mL), dried with Na2SO4,
and filtered. Evaporation of the solvent gave crude 10a (2.20 g)
which was purified by crystallization (hexane/ethyl acetate, 8:1) to
give the pure product (1.62 g, 3.7 mmol, 65% yield). 1H NMR
(600 MHz, [D6]DMSO, TMS): δ = 2.25 [t, JH,H = 11.0 Hz, 2 H,
= 7.2 Hz, 2 H, CH2CH3), 5.30 (br. s, 1 H, NH), 6.98 (t, JH,H
8.4 Hz, 2 H, m-ArH], 7.23 (br. s, 2 H, o-ArH) ppm. 13C NMR
(126 MHz, CDCl3, TMS): 14.2 (s, CH2CH3), 28.3 [s,
=
δ
=
C(CH3)3], 32.5 (s, ArCH), 39.8 [s, C(CH2)2C], 54.5 (s, CNHBoc),
2
61.5 (s, CH2CH3), 79.9 [s, C(CH3)3], 115.1 [d, JC,F = 21 Hz, m-
3
ArC], 128.1 [s, JC,F = 8 Hz, o-ArC], 140.3 (s), 154.7 (s, NHCOO),
1
161.5 (s, JC,F = 245 Hz, FC), 174.1 (s, COOEt) ppm. 19F NMR
(376 MHz, CDCl3, CFCl3): δ = –117.51 (s) ppm. C18H24FNO4
(337.39): calcd. C 64.08, H 7.17, N 4.15; found C 64.32, H 7.18, N
4.02.
Crystals suitable for X-ray analysis were obtained by slow evapora-
tion of the solvent from a diluted solution of 9a in hexane at room
temperature.
Compound 9b: M.p. 111–112 °C; Rf = 0.29. 1H NMR (400 MHz,
CDCl3, TMS): δ = 1.27 (t, JH,H = 7.2 Hz, 3 H, CH2CH3), 1.45 [s,
9 H, C(CH3)3], 2.57 [br. s, 2 H, C(CHH)2C], 2.75 [t, JH,H = 11.0 Hz,
Eur. J. Org. Chem. 2014, 3584–3591
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3589

P. K. Mykhailiuk, S. Afonin, A. S. Ulrich et al.
FULL PAPER
C(CHH)2C], 2.92 [t, JH,H = 10.5 Hz, 2 H, C(CHH)2C], 3.51 (quint, membrane-mimicking solutions to a final peptide concentration of
JH,H = 9.0 Hz, 1 H, ArCH), 4.24 [t, JH,H = 6.0 Hz, 1 H, 75 μg/mL (for GS: 65 μm; for 12: 63 μm). The temperature was
(C6H4)2CHCH2], 4.30 [d, JH,H = 6.5 Hz, 2 H, (C6H4)2CHCH2],
7.15 [t, JH,H = 8.5 Hz, 2 H, fluorinyl-H], 7.30–7.35 (m, 4 H), 7.43
[t, JH,H = 8.0 Hz, 2 H, fluorinyl-H], 7.73 (d, JH,H = 8.0 Hz, 2 H),
7.91 (d, JH,H = 7.0 Hz, 2 H), 8.05 (s, 1 H, NH), 12.62 (br, 1 H,
COOH) ppm. 13C NMR (151 MHz, [D6]DMSO, TMS): δ = 32.3
(s, ArCH), 47.2 [s, (C6H4)2CHCH2], 53.8 (s, CCOOH), 65.8 [s,
maintained at 20 °C.
Supporting Information (see footnote on the first page of this arti-
cle): Copies of NMR spectra of 4, 7–10, and 12; characterization
of peptide 12, RPLC of GS and crystallographic analysis of 9a.
2
(C6H4)2CHCH2], 115.5 (d, JC,F = 21 Hz, m-ArC), 120.6 (s), 125.7
Acknowledgments
3
(s), 127.5 (s), 128.1 (s), 128.8 (d, JC,F = 8 Hz, o-ArC), 141.2 (s),
1
141.5 (s), 144.3 (s), 155.3 (s, NHCOO), 161.2 (d, JC,F = 242 Hz,
The authors are grateful to Prof. Andrey A. Tolmachev and En-
amine Ltd. for financial support of the work, and to Prof. Oleg V.
Shishkin for crystallographic analysis and Dr. Marina Berditsch for
providing the natural gramicidin S. One of us (A. N. T.) acknowl-
edges a Deutscher Akademischer Austauschdienst (DAAD) grant.
FC), 175.6 (s, COOH) ppm. 19F NMR (376 MHz, [D6]DMSO,
CFCl3): δ = –116.98 (s) ppm. C26H22FNO4 (431.46): calcd. C 72.38,
H 5.14, N 3.25; found C 72.63, H 4.81, N 3.34.
Peptide Synthesis
Cyclo[Val-Orn-Leu-10a-Pro-Val-Orn-Leu-DPhe-Pro] (12): d-Phen-
ylalanine pre-loaded 2-chlorotrityl resin (50 mg, 1 equiv.) with 100–
200 mesh was used with a loading of 0.73 mmol/g. The coupling
reactions of the amino acids were performed by using the following
combination: Fmoc-amino acid (4 equiv.) + 6Cl-HOBt (4 equiv.),
HCTU (3.9 equiv.), DIPEA (8 equiv.). Fmoc-Leu (52 mg), Fmoc-
(Boc)Orn (66 mg), Fmoc-Val (50 mg), Fmoc-Pro (50 mg). 6Cl-
HOBt (25 mg), HCTU (59 mg), DIPEA (51 μL). The coupling re-
action of building block 10a (24 mg, 1.5 equiv.) was performed with
6Cl-HOBt (10 mg, 1.6 equiv.), HCTU (22 mg, 1.5 equiv.) and
DIPEA (19 μL, 3 equiv.). Fmoc-amino acids and the reagents were
pre-mixed in N-methyl-2-pyrrolidone (1 mL) for 2 min before add-
ing to the resin. The coupling time was 2 h, except for building
block 10a (3 h). The Fmoc-protecting group was cleaved by treating
the resin with 22% piperidine in dimethylformamide for 30 min.
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After completion of the synthesis, the resin was washed with
dichloromethane and dried under vacuum for 24 h. The linear
peptide was cleaved from the resin by mixing with 1:3 hexa-
fluoroisopropanol/dichloromethane (2ϫ25 min). All volatiles were
blown off under argon. The residue was taken up in an acetonitrile/
water mixture and frozen. Lyophilization afforded the crude linear
peptide 11. Compound 11 was dissolved in dichloromethane
(400 mL), a solution of PyBOP (57 mg, 3 equiv.) and HOBt (15 mg,
3 equiv.) in dimethylformamide (1 mL) followed by DIPEA (38 μL,
6 equiv.) were added. The reaction mixture was stirred for 7 h, then
another portion of the coupling reagents was added. After 13 h,
the solvent was evaporated under reduced pressure, and the residue
was dissolved in acetonitrile/water and lyophilized. A deprotection
mixture (10 mL) – trifluoroacetic acid, triisopropylsilane and water
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cold diethyl ether (15 mL) and centrifuged. The white precipitate
12 was lyophilized from acetonitrile/water and purified using RPLC
conditions identical to GS purification, i.e., with an acetonitrile/
water gradient (with 5 mm HCl)[6b] to obtain the desired cyclic
peptide (3.2 mg, overall yield 7.4%). MALDI-TOF: m/z calcd. [M
+ H]+ 1185; found 1186.
Circular Dichroism (CD) Spectroscopy: CD spectra were measured
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free background, which was later subtracted. Lyophilized peptide
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Received: November 19, 2013
Published Online: April 24, 2014
Eur. J. Org. Chem. 2014, 3584–3591
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3591