Synthesis of a complete set of L-difluorophenylalanines, L-(F2)Phe, as molecular explorers for the CH/π interaction between peptide ligand and receptor

Article abstract of DOI:10.1016/S0040-4039(99)02191-7

A complete set of difluorophenylalanines in the L-configuration [L- (F₂)Phe] (namely, L-(2,3-F₂)Phe, L-(2,4-F₂)Phe, L-(2,5-F₂)Phe, L-(2,6- F₂)Phe, L-(3,4-F₂)Phe, L-(3,5-F₂)Phe) w

Full text of DOI:10.1016/S0040-4039(99)02191-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 923–927
Synthesis of a complete set of L-difluorophenylalanines,
L-(F2)Phe, as molecular explorers for the CH/π interaction
between peptide ligand and receptor
Tsugumi Fujita, Takeru Nose, Ayami Matsushima, Kazushi Okada, Daisuke Asai,
Yasuko Yamauchi, Naoto Shirasu, Takeshi Honda, Daiki Shigehiro and
Yasuyuki Shimohigashi ∗
Laboratory of Structure–Function Biochemistry, Department of Molecular Chemistry, Graduate School of Sciences,
Kyushu University, Fukuoka 812-8581, Japan
Received 8 October 1999; accepted 19 November 1999
Abstract
A complete set of difluorophenylalanines in the L-configuration [L-(F2)Phe] (namely, L-(2,3-F2)Phe, L-(2,4-
F2)Phe, L-(2,5-F2)Phe, L-(2,6-F2)Phe, L-(3,4-F2)Phe, L-(3,5-F2)Phe) was prepared and incorporated into the
thrombin receptor-tethered ligand peptide SFLLRNP to identify the phenyl hydrogens of the Phe-2 residue involved
in the CH/π receptor interaction. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: amino acids; amino acid derivatives; fluorine; fluorine compounds; molecular recognition; peptides; polypeptides.
1. Introduction
Phenylalanine (Phe), an aromatic amino acid, is often crucially important in biologically active pep-
tides to elicit intrinsic activity, and on that occasion, the side-chain phenyl group plays an essential role
in the ligand–receptor interaction.1 The molecular mechanism of this so-called hydrophobic interaction,
however, has never been elucidated in detail. When Phe interacts with the alkyl side chains of amino acids
Leu, Ile, Val, or Ala, the Phe–phenyl π system would function as a hydrogen acceptor. This interaction is
denoted as a CH/π interaction, the concept of which has recently been established by Nishio et al.2 When
Phe interacts with aromatic amino acids such as Phe, Tyr, Trp, and His, Phe–phenyl should be involved in
two different types of π–π interactions, i.e., the face-to-face π–π stacking interaction and the edge-to-
face CH/π interaction. To better understand the molecular mechanism of peptide interactions involving
the Phe residue, differentiation of these Phe–phenyl π interactions is requisite. We have postulated that
∗
Corresponding author.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(99)02191-7
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these π–π interactions can apparently be distinguished by replacing fluorophenylalanines for the Phe
residue.
Fluorine can substitute the benzene hydrogens (CHs) without changing the atomic size, and thus
it is highly likely that multiple fluorine replacements of benzene hydrogens could clarify whether
essential hydrogens exist on Phe–phenyl or whether the π system is required for the interaction with
the receptor. For fluorophenylalanines (Fn)Phe, there are 19 isomers in total (three of (F1)Phe, six
of (F2)Phe, six of (F3)Phe, three of (F4)Phe, and one of (F5)Phe), and only several of them such as
mono- and pentafluorophenylalanines have been prepared in optically pure states. Most of the di-, and
tetrafluorinated derivatives were synthesized as racemates, and almost no data have been reported for
trifluorinated derivatives.3 In the present study, we synthesized a complete set of optically pure L- and D-
difluorinated phenylalanines, difluorophenylalanines [(F2)Phe] (namely, (2,3-F2)Phe, (2,4-F2)Phe, (2,5-
F2)Phe, (2,6-F2)Phe, (3,4-F2)Phe and (3,5-F2)Phe) (Fig. 1). Furthermore, we incorporated all six isomers
of L-(F2)Phe, respectively, into a thrombin receptor-tethered ligand peptide S/Phe/LLRNP to clarify the
specific edge-to-face CH/π interaction.4
Fig. 1. The side chain structure of difluorophenylalanines [(F2)Phe]
2. Experiments and results
The synthesis and optical resolution of difluorophenylalanines were carried out by the method shown
in Fig. 2.5 Six different difluorobenzyl bromides 1 (120 mmol) (JRD Fluorochemicals Ltd, Surrey, UK)
were coupled with diethyl acetamidomalonate (100 mmol), respectively, in abs. EtOH which dissolved
Na (120 mmol) or NaOEt (120 mmol) by refluxing for 2 h. After filtration and evaporation, the residue
was treated with cold water to collect the precipitate, which was recrystallized from EtOH–water to
afford diethyl [difluorophenylmethyl]acetamidomalonate 2 (av. yield, 90%) (Table 1). Diethyl ester 2 (70
mmol) was then subjected to simultaneous saponification in MeOH (200 ml)/2 M NaOH (280 mmol) at
40°C for 3 h. The resulting malonic acid derivatives 3 were liberated by 6 M HCl and extracted with
ethyl acetate. Unlike non-fluorinated derivatives in the literature,5 3 appeared to be quite unstable, and
thus the residue of evaporation was directly refluxed in para-xylene for 2 h. This afforded decarboxylated
acetyl-DL-difluorophenylalanines (Ac-DL-(F2)Phe, 4) in very high yield (av. yield, 92%) (Table 1).
Ac-DL-(F2)Phe was digested with Aspergillus genus acylase (Tokyo Chemical Industry) to resolve into
Ac-D-(F2)Phe and L-(F2)Phe. To a solution (pH 8.0) of Ac-DL-(F2)Phe (50 mmol) in 0.2 M NaOH (275
ml) was added acylase (400 mg) in 0.2 M AcONa, and the solution was incubated at 38°C for 48 h. After
removal of the enzyme by Norit, Ac-D-(F2)Phe was extracted with EtOAc from the aqueous solution
(pH 3.0) and purified by recrystallization (av. yield, 91%). The liberated free amino acid L-(F2)Phe was
collected and purified by applying to a column (3.8×22 cm) of Dowex 50X8 (H+ form) eluted with 2
M NH4OH (av. yield after recrystallization, 92%). Optical purity of L-(F2)Phe was confirmed by high-

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Fig. 2. Synthetic route of difluorophenylalanines [(F2)Phe]
Table 1
The melting point of compounds related to difluorophenylalanines
performance liquid chromatography (HPLC) using a chiral column SUMICHIRAL OA 5000 (0.46×15
cm, 5 mm; Sumika Chemical Analysis Service, Osaka). The analytical data are shown in Tables 1 and 2.
In due course, we achieved, for the first time, the synthesis of all six isomers of optically pure L- and
D-difluorophenylalanines (F2)Phe. Prudechenko6 reported the synthesis of five DL-(F2)Phe racemates,
except for DL-(2,3-F2)Phe, by the azlactone method, in which difluorinated benzaldehydes were coupled
with benzoylglycine. Although all six isomers of DL-(F2)Phe racemates are now commercially available
(JRD), none of the optically pure stereoisomers of L- or D-(F2)Phe are available. For peptide synthesis,
however, amino-protected derivatives of optically active amino acids usually in the L-configuration are
required, and among the L-(F2)Phe isomers only Boc and Fmoc derivatives of L-(3,4-F2)Phe and L-(3,5-
F2)Phe are available from several commercial sources. Thus, in this study, we prepared Boc derivatives
of all of the L-(F2)Phe isomers with di-tert-butyldicarbonate (av. yield, 94%). These Boc-L-(F2)Phe
isomers were used for the syntheses of thrombin receptor-tethered ligand peptide SFLLRNP (denoted
by one-letter amino acid codes), in which L-(F2)Phe isomers were incorporated into position 2 to replace
Phe(=F) essential for the receptor activation.
Peptides were synthesized by the manual solid-phase synthesis method. Coupling reactions (0.1
mmol) were carried out with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) in the presence of 1-hydroxybenzotriazole (HOBt) for 30 min. After HF treatment, the peptides
were purified by Sephadex G-15 followed by preparative reversed-phase HPLC (Cica–Merck, LiChros-

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Table 2
Optical properties of difluorophenylalanines and their derivatives
pher RP-18 (e) (5 µ): 25×250 mm). The total yield of peptides from the resin was av. 43%. The purity
was verified by analytical HPLC and amino acid analyses. All the peptides synthesized were evaluated
in the assay for human platelet aggregation by the standard turbidimetric procedure using a hema tracer.
It was found that any replacement of the phenyl hydrogens at the ortho and meta positions diminishes
the activity considerably (6–35% activity of parent SFLLRNP). This finding, together with previous
assay results,3 clarified an important aspect of the structure, which led to the conclusion that Phe-
2-phenyl of S/Phe/LLRNP is in the edge-to-face CH/π interaction with the receptor aromatic group,
utilizing the benzene ring-edge along with hydrogens at positions 5 and 6 (Fig. 3). Furthermore, the high
activity (140–150%) of L-(2,4-F2)Phe- and L-(3,4-F2)Phe-containing S/Phe/LLRNP analogs confirmed
the previous result that the fluorine atom at position 4 increases biological activity.3,7 This fluorine
appears to increase the acidity of the hydrogen at adjacent position 5, resulting in a reinforcement of
the CH/π interaction and consequently in enhancement of biological activity. Collectively, benzene
hydrogen/fluorine replacement was found to enable an effective structural examination of the Phe
residue, i.e., to identify the hydrogens in the CH/π interaction, and to strengthen the CH/π interaction.
Fluorophenylalanines would substantiate the structural examination to elucidate the functional roles of
the Phe residue in biologically active peptides.
Fig. 3. Putative edge-to-face CH/π interaction between the peptide ligand (3,4-F2)Phe–phenyl and the receptor Phe–phenyl
groups

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Li, G.; Haskell-Luevano, C.; Shenderovich, M. Biopolymers 1997, 43, 219–266.
2. Nishio, M.; Umezawa, Y.; Hirota, M.; Takeuchi, Y. Tetrahedron 1995, 51, 8665–8701.
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