Synthesis of enantiomerically pure (2S,3S)-5,5,5-trifluoroisoleucine and (2R,3S)-5,5,5-trifluoro-allo-isoleucine

Article abstract of DOI:10.3762/bjoc.9.236

A practical route for the stereoselective synthesis of (2S,3S)-5,5,5- trifluoroisoleucine (L-5-F3Ile) and (2R,3S)-5,5,5-trifluoro-alloisoleucine (D-5-F3-allo-Ile) was developed. The hydrophobicity of L-5-F3Ile was examined and it was incorporated into a m

Full text of DOI:10.3762/bjoc.9.236

Synthesis of enantiomerically pure
(2S,3S)-5,5,5-trifluoroisoleucine and
(2R,3S)-5,5,5-trifluoro-allo-isoleucine
Holger Erdbrink‡, Elisabeth K. Nyakatura‡, Susanne Huhmann,
Ulla I. M. Gerling, Dieter Lentz§, Beate Koksch* and Constantin Czekelius*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 2009–2014.
Department of Chemistry and Biochemistry, Freie Universität Berlin,
Takustr. 3, 14195 Berlin, Germany
doi:10.3762/bjoc.9.236
Received: 19 June 2013
Email:
Accepted: 11 September 2013
Published: 02 October 2013
Beate Koksch* - beate.koksch@fu-berlin.de; Constantin Czekelius* -
cczekeli@chemie.fu-berlin.de
This article is part of the Thematic Series "Organo-fluorine chemistry III".
Guest Editor: D. O'Hagan
* Corresponding author ‡ Equal contributors
§ Responsible for X-ray analysis.
Keywords:
© 2013 Erdbrink et al; licensee Beilstein-Institut.
License and terms: see end of document.
amino acids; CD-spectroscopy; fluorine; helix propensity;
organo-fluorine; trifluoroisoleucine
Abstract
A practical route for the stereoselective synthesis of (2S,3S)-5,5,5-trifluoroisoleucine (L-5-F3Ile) and (2R,3S)-5,5,5-trifluoro-allo-
isoleucine (D-5-F3-allo-Ile) was developed. The hydrophobicity of L-5-F3Ile was examined and it was incorporated into a model
peptide via solid phase peptide synthesis to determine its α-helix propensity. The α-helix propensity of 5-F3Ile is significantly lower
than Ile, but surprisingly high when compared with 4’-F3Ile.
Introduction
Due to the unique physicochemical properties of fluorine, Numerous studies have focused on the incorporation of fluori-
namely its small size, extremely low polarizability and the nated aliphatic amino acids in helical folds [3-7], even though
strongest inductive effect among all chemical elements [1], their intrinsic tendency to adopt this secondary structure (i.e.
fluorine substitution has become a powerful tool for modu- their α-helix propensity) was shown to be considerably reduced
lating the properties of pharmaceuticals and biologically active when compared to their canonical analogues [8,9]. Thus, if
compounds. In this respect, the incorporation of amino acids enhanced thermal stabilities of helical assemblies containing ali-
with fluorinated side chains has been established as an efficient phatic fluorinated building blocks have been observed, this
strategy to alter distinct properties of peptides and proteins, stability was mainly attributed to a higher hydrophobicity or the
such as hydrophobicity, acidity/basicity, and conformation [2]. formation of a fluorous core [2,10]. Tirrell et al. found that the
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Beilstein J. Org. Chem. 2013, 9, 2009–2014.
biological function of the helical GCN4 transcription factor can analogues with retained α-helix propensity is of general interest
be retained, when utilizing racemic mixtures of 5,5,5-trifluoro- for site-specific modification of coiled-coil positions in parallel
isoleucine (5-F3Ile) as isoleucine surrogates for protein syn- packing arrangements, especially when solid phase peptide syn-
thesis in Escherichia coli [11,12]. Moreover, studying the thesis is employed.
impact of global Ile substitution with 5-F3Ile in comparison to
the substitution of Val by 4,4,4-trifluorovaline (4-F3Val) Herein, we report a flexible approach to enantiomerically pure
through protein expression, they showed that the replacement of (2S,3S)-5,5,5-trifluoroisoleucine (L-5-F3Ile) and (2R,3S)-5,5,5-
the δ-CH3 group of Ile by CF3 resulted in an approximately trifluoro-allo-isoleucine (D-5-F3-allo-Ile). Since the relation-
eight-fold higher thermal stability of the respective GCN4 ship of side chain hydrophobicity and α-helix propensity is of
analogue than the replacement of the γ-CH3 group of Val by crucial importance for the overall stability of helical assemblies,
CF3 [12]. This finding was explained by the substantial loss of these two properties were examined for L-5-F3Ile, as one of the
side-chain entropy of Val due to steric restriction between the possible fluorinated analogues of proteinogenic isoleucine.
significantly larger γ-CF3 group and the helix. In agreement
Results and Discussion
with these findings, we previously showed that the replacement
of a CH3 group by a CF3 substituent in close proximity to the Amino acid synthesis
α-carbon of aliphatic amino acids dramatically reduces their We have recently described a new method for the conjugate tri-
α-helix propensity [13].
fluoromethylation of α,β-unsaturated acyloxazolidinones [13].
Using this approach, all four diastereoisomers of N-Boc-
Since isolated α-helices are only marginally stable in solution, protected 4,4,4-trifluorovaline as well as (2S,3S)-4,4,4-trifluoro-
they are often stabilized in proteins by being wound around isoleucine were prepared from the corresponding products in
each other in a superhelix, a so-called coiled-coil arrangement, enantiomerically pure form via diastereoselective auxiliary-
where hydrophobic side chain interactions are maximized induced amination. It was found that both diastereoisomers of
within a helical interface. Coiled-coil structures are based on a trifluorovaline and trifluoroisoleucine show extremely low
(pseudo-) repetitive sequence (abcdefg)n, the so-called heptad α-helix propensities compared to their non-fluorinated
repeat, in which hydrophobic side chains are primarily located analogues valine (Val) and isoleucine (Ile), which we attribute
at the a- and d-positions on one side of the helix, while most of to steric clashes of the larger γ-CF3 group with the helix back-
the other positions are hydrophilic or charged [2,14]. In a bone [13]. We wondered whether the replacement of the δ-CH3
parallel helix alignment of coiled-coils, two fundamentally group of Ile by CF3 might retain α-helix propensity. In light of
different packing geometries occur at hydrophobic core posi- the fact that 5,5,5-trifluoroisoleucine has mostly been synthe-
tions a and d. The α−β bond vector of the amino acid side chain sized and incorporated into peptides as diastereomeric mixtures
can either point out of the hydrophobic core, termed parallel so far [11,12,16], we decided to extended our previous ap-
packing, or into the core and thus directly towards the neigh- proach towards the synthesis of (2S,3S)-5,5,5-trifluoroiso-
boring helix (perpendicular packing). As a consequence, leucine. For this, we envisioned acyloxazolidinone 5 as an inter-
hydrophobic β-branched amino acids (Ile or Val) are the most mediate for the synthesis of enantiomerically pure L-5,5,5-tri-
stabilizing amino acids in parallel packing arrangements, fluoroisoleucine [13,17]. For the synthesis of this building block
because they project the hydrocarbon side chain from the we started from enantiomerically pure alcohol 1, which was
β-carbon atom directly into the helical interface, whereas prepared using a modified protocol reported by Wang and
perpendicular packing precludes β-branched residues from Resnick from commercially available 4,4,4-trifluorobutanoic
occupying these sites and Leu is favored [14,15]. Therefore, the acid by diastereoselective enolate alkylation followed by reduc-
preparation of enantiomerically pure fluorinated isoleucine tion with lithium borohydride (Scheme 1) [18].
Scheme 1: Synthesis of optically active N-acyloxazolidinone 5 from 4,4,4-trifluorobutanoic acid. Conditions: (a) TsCl, DMAP (cat.), pyridine, 0 °C to rt,
12 h, 79%; (b) NaCN, NaI (cat.), DMSO, 60 °C, 2.5 h, 85%; (c) HCl (conc.), reflux, 2.5 h, 80%; (d) NEt3, PivCl, THF, −78 °C to 0 °C, 90 min, then
n-BuLi, (S)-4-benzyloxazolidin-2-one, THF, −78 °C to rt, overnight, 84%.
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Beilstein J. Org. Chem. 2013, 9, 2009–2014.
Fluorinated alcohol 1 was transformed into the corresponding By plotting their side chain van der Waals volume versus their
tosylate ester 2 and subsequently reacted with sodium cyanide retention time from an RP-HPLC experiment, we previously
to afford nitrile 3. Acid hydrolysis of 3 provided the enan- investigated the relationship between size and hydrophobicity
tiomerically pure carboxylic acid (R)-4 [19], which was then of various fluorinated and non-fluorinated amino acids [26]. In
coupled to the oxazolidinone via the mixed anhydride this experiment the non-polar phase of a reversed-phase column
(Scheme 1). With N-acyloxazolidinone 5 in hand, the optically serves as a mimic of a biological membrane or the kind of
active fluorinated α-amino acids (L-5,5,5-trifluoroisoleucine 7 hydrophobic interactions, that would be present in hydrophobic
and D-5,5,5-trifluoro-allo-isoleucine 10) were synthesized by cores of proteins and in ligand–receptor binding [27]. We
stereoselective, auxiliary-induced amination following pro- extended these initial studies by 2-aminoheptanoic acid (Aha)
cedures reported by Evans and coworkers (Scheme 2) [17,20- as an amino acid with an unbranched aliphatic side chain. The
24]. For this, acyloxazolidinone 5 was transformed into the van der Waals volumes of the amino acid side chains were
α-azido derivative which upon reduction and auxiliary removal calculated according to Zhao et al. [28].
gave the N-Boc protected L-amino acid 7 (L-5-F3Ile). Likewise,
the corresponding D-amino acid 10 (D-5-F3-allo-Ile) was Aha correlates very well with its smaller non-fluorinated
prepared by α-bromination followed by nucleophilic azide dis- analogues and their retention time increases non-linearly with
placement under stereochemical inversion (Scheme 2).
increasing side chain volume (Figure 1). As expected, the
enlargement of the aliphatic side chain results in an increase in
The absolute configuration of the amino acids was confirmed hydrophobicity.
by crystal structure determination of intermediate 6, based on
the known auxiliary configuration (see Supporting Information In agreement with previous studies that focused on other fluori-
File 1).
nated amino acids [26], also the retention times of 5-F3Ile and
4’-F3Ile do not fit into the correlation between side chain
volume and retention time (Figure 1). Although similar in size,
Hydrophobicity of L-5-F3-Ile
We investigated the relationship between side chain volume and the two fluorinated stereoisomers of Ile are less hydrophobic
hydrophobicity of L-5-F3Ile. Since size and hydrophobicity are than Aha. F6Leu is similar to Aha in hydrophobicity, while ex-
known to be essential factors for secondary structure formation, hibiting a much larger volume. In a free energy perturbation
we compared 5-F3Ile with previously studied (2S,3S)-4’-F3Ile study, the hydration energy of F6Leu was shown to be
[13], as well as with (S)-5,5,5,5’,5’,5’-hexafluoroleucine 1.1 kcal/mol higher than that of leucine [29]. This, together with
(F6Leu), which was prepared according to procedures reported our previous and new findings, suggests that there are two
by Keese and coworkers [25].
factors determining the overall hydrophobicity of fluorinated
Scheme 2: Synthesis of enantiomerically pure (2S,3S)-5-F3Ile and (2R,3S)-5-F3-allo-Ile. TMGA = 1,1,3,3-tetramethylguanidinium azide, Trisyl-N3 =
2,4,6-triisopropylbenzene-sulfonyl azide.
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Beilstein J. Org. Chem. 2013, 9, 2009–2014.
Table 1: Ellipticity [Θ] at 222 nm was taken from normalized CD data.
Fraction helix [fhelix] and helix propensities [ω] were calculated from
[Θ222 nm] applying a modified Lifson–Roig theory [31-33].
peptide
[Θ222 nm]
fhelix
ω
K-Ile
–13813 ± 156 0.40 ± 0.01 0.52 ± 0.05
–10776 ± 216 0.31 ± 0.01 0.26 ± 0.03
K-5-F3Ile
K-4’-F3Ile [13] –3602 ± 130
0.10 ± 0.01
0
model peptide, whereas the absence of these minima in the
K-4’-F3Ile spectrum demonstrates a complete loss of helicity in
the corresponding peptide (Figure 2). The drastic decrease in
helix propensity upon fluorination has been previously attrib-
uted to a possible burial of fluorocarbon side chains in the
unfolded state of the model peptide [8]. The exposure of these
side chains in the helical state would lead to unfavourable helix
formation energetics, due to the hydrophobic nature of fluoro-
carbon side chains. Moreover, due to its branching on the
β-carbon atom, the side chain atoms of Ile come in close prox-
Figure 1: Retention times of Fmoc amino acids plotted against the van
der Waals volume of their side chains. Non-fluorinated amino acids are
depicted as black triangles; their correlation is shown as a black line.
Fluorinated amino acids are represented by green triangles.
amino acids [26]. On one hand, substitutions of hydrogen by imity to the peptide backbone [34], leading to a reduced
fluorine increase the solvent accessible surface area and thus α-helicity in comparison to its unbranched analogue
lead to an increase in hydration energy. On the other hand, the ([ω]Leu= 1.06 [8]). This effect is amplified for K-4’-F3Ile which
C–F bond is more polarized than the C–H bond, and electro- carries the voluminous CF3 group on its β-carbon atom. The
static interactions of the fluorinated group with the solvent are close proximity of this sterically demanding group to the
energetically more favored. As a consequence, fluoroalkyl side peptide backbone seems to prevent the formation of α-helical
chains possess two seemingly contrary physicochemical prop- structures, resulting in a helix propensity of zero [13]. Here, we
erties, hydrophobicity and polarity, and the combination of both show that if isoleucine’s δ-methyl group is substituted with
leaves fluorinated amino acids to be less hydrophobic than their CF3, the α-helix propensity of this amino acid is partially
surface area would suggest.
retained.
α-Helix propensity of L-5-F3Ile
In general, fluorination of amino acids leads to a dramatic
decrease in helix propensity [8,9,13]. As the extreme of this
effect, we previously reported the complete loss of helix
propensity when the β-methyl group in β-branched hydrophobic
amino acids is replaced by a CF3-substituent [13]. We now
investigated the α-helix propensity of 5-F3Ile according to
methods established by Cheng et al., who showed that when an
amino acid of interest is incorporated into an α-helical polyala-
nine model peptide (KX), its α-helix propensity can be calcu-
lated from circular dichroism (CD) spectroscopy [8,9]. There-
fore, 5-F3Ile was converted into its Fmoc analogue and subse-
quently used in solid-phase synthesis of K-5-F3Ile applying
standard Fmoc-based chemistry (see Supporting Information
File 1) [30]. The α-helix propensity [ω] was calculated from CD
data (Table 1).
Figure 2: CD spectra of K-5-F3Ile and K-Ile peptides (KX:
Ac-YGGKAAAAKAXAAKAAAAK-NH2). The K-4’-F3Ile spectrum [13] is
shown for comparison. Spectra were recorded at pH 7 in 1 mM phos-
phate, borate, and citrate buffer with 1 M NaCl at 0 °C. Depicted
spectra are normalized and represent the mean of three independent
measurements at three different concentrations (80 µM, 50 µM, and
30 µM).
Although the helix propensity of 5-F3Ile is half of that for Ile,
two distinct minima at 208 nm and 222 nm in the corres-
ponding CD spectrum clearly indicate a helical structure of the
2012

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We synthesized two diastereoisomers of 5,5,5-trifluoroiso-
leucine ((2S,3S)-5-F3Ile and (2R,3S)-5-F3-allo-Ile) in enan-
tiomerically pure form. The hydrophobicity of (2S,3S)-5-F3Ile
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Supporting Information File 1
Experimental procedures, characterization data, copies of
all 1H, 13C, and 19F NMR spectra of all new compounds.
[http://www.beilstein-journals.org/bjoc/content/
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Acknowledgements
This work has been generously supported by the DFG in the
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