Fluorocyclopropane-Containing Proline Analogue: Synthesis and Conformation of an Item in the Peptide Chemistʼs Toolbox

Over the years, numerous modifications to the structure of proline have been made in order to tune its effects on bioactive compounds. Notably, the introduction of a cyclopropane ring or a fluorine atom has produced interesting results. Herein, we describe the synthesis of a proline containing fluorocyclopropane. This modified amino acid was inserted into a tripeptide, whose conformation was studied by nuclear magnetic resonance and density functional theory calculations.


■ INTRODUCTION
As a subunit of peptides, amino acids have attracted a great deal of interest from the scientific community. Indeed, combinations of the 21 natural amino acids are responsible for the wide variety of peptides that preform vital functions in all living matter. Among these amino acids, proline plays an important role in the peptide conformation. Indeed, in contrast to the other amino acids, proline forms a tertiary amide moiety, which is unable to act as a hydrogen bond donor with the other amino acids. This feature along with the rigidity of the five-membered ring increases the ratio of the cis conformer, which induces turns into the peptide structure, thus triggering interesting biological properties. 1 Therefore, numerous studies have focused on the modification of the structure of the proline backbone to modulate these effects. Notably, the introduction of a cyclopropane ring increases the rigidity of the resulting modified proline and impacts its bioactivity. For example, 2,3-methanoproline is an inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase, which contributes to the biosynthesis of ethylene by plants. 2 In addition, the cis-3,4-methanoproline inhibits the growth of Escherichia coli and Salmonella typhimurium, 3 where this scaffold was integrated into the structure of an inhibitor of STAT3, a translation factor responsible for the growth of cancer cells. 4 Furthermore, a nitrile-containing 4,5-methanoproline constitutes a part of the structure of saxagliptin, a medicine used in the treatment of type 2 diabetes (Figure 1). 5 Another strategy to modify the structure of proline relies on the introduction of fluorine atom(s) at the carbon 3-and/or 4position. 6 Initially, this scaffold was used to gain insights into the post-translational modifications of the proline leading to 4hydroxyproline in collagen. 7 Owing to the so-called gauche effect of fluorine, the absolute configuration at carbon-4 has a significant impact on the conformation of 4-fluoroproline. 8 This motif was used to study the conformation, stability, and folding of several proteins 9 and was used as the core of a thrombin inhibitor. 10 Notably, the introduction of this motif into the structure of neurotensin displayed interesting biological effects. 11 ■ RESULTS AND DISCUSSION Stemming from our interest in the synthesis and applications of fluorocyclopropanes, 12 we were eager to combine the features of cyclopropane and the fluorine atoms to access a new fluorocyclopropane-containing proline analogue ( Figure 2). Indeed, this original scaffold might offer interesting conformation modifications or properties by combining the particular properties of both the fluorine atom and cyclopropane.
We postulated that this modified proline could be obtained via two different strategies: (1) the intermolecular cyclopropanation, followed by intramolecular amide bond formation ( Figure 2, strategy a) or (2) the intramolecular cyclopropanation of a diazoacetamide ( Figure 2, strategy b).
The intermolecular cyclopropanation reaction followed by intramolecular amide bond formation (Scheme 1) was first investigated. Pleasingly, the cyclopropanation of fluoroalkene 1a with ethyl diazoacetate 2a in the presence of 2 mol % Rh 2 (OPiv) 4 afforded (±)-trans-3a and (±)-cis-3a in 87% yield and a modest 3:1 trans/cis mixture after selective monodeprotection of the biscarbamate moiety. 13 The saponification of (±)-trans-3a followed by the deprotection of the second carbamate group afforded the hydrochloride salt of the amino acid (±)-trans-4a. Unfortunately, the use of acidic or basic conditions was both unproductive to build up the fivemembered ring. Similarly, the use of a coupling agent [e.g., T 3 P, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, etc.] to access the amide bond was unsuccessful to construct the fluorocyclopropane-containing proline analogue. This low reactivity might be explained either by the strain induced by the adjacent cyclopropane ring and/or by the lower nucleophilicity of the amine residue, resulting from the presence of the fluorine atom at the vicinal position.
Alternatively, we turned our attention to the second approach based on the intramolecular cyclopropanation (Scheme 2). N-Benzyl allylamine 1b, 14 N-(4-nitrobenzyl) allylamine 1c, and N-(4-chlorobenzyl) allylamine 1d were reacted with bromoacetyl bromide to afford the corresponding amides 2b−d. From 2b, the substitution of bromide with sodium azide and the subsequent Staudinger reaction gave access to amine 4b. From 2c−d, the substitution of bromide with NBoc 2 followed by the removal of the carbamate functions delivered the expected ammonium chloride of 4c−d. The latter 4b−d compounds were converted into the corresponding diazo compounds 5b−d after the reaction with sodium nitrite. Diazo 5b underwent an intramolecular cyclopropanation reaction in the presence of Rh 2 (OPiv) 4 (0.5 mol %) to give (±)-6b in 55% isolated yield.
Having developed this intramolecular cyclopropanation reaction, the enantioselective version was investigated using chiral Rh and Ru catalysts (Scheme 3). The first screening of chiral Rh catalysts revealed that the amide-based Rh catalysts were the only complexes that furnished the target compound 6b. After a survey of the catalysts and the screening of the reaction conditions, it was found that Rh 2 ((S)-MEPY) 4 afforded an interesting enantiomeric excess (ee) (62%) and a decent 53% isolated yield (Scheme 3, entry 4). Rh 2 ((S)-BPTCP) 4 , 15 which has been proven to be a highly efficient Rh catalyst for cyclopropanation, particularly in fluorinated series, delivered the expected cyclopropane from 5c in 92% isolated yield and 70% ee when the reaction was carried out in CHCl 3 at −35°C (Scheme 3, entry 13). We next examined the use of Ru-Pheox, which is also very efficient in many cyclopropanation reactions. 16 When Ru-Pheox 3 (Scheme 3, entry 9) was used in the intramolecular cyclopropanation of 5b at 0°C, the expected target was obtained with the best ee (77%) 17 and in very good isolated yield (80%). Unfortunately, all attempts to improve either the yield or the ee were unsuccessful.
Hence, to tackle this limitation, the racemic mixture was resolved using preparative supercritical fluid chromatography (SFC) on a chiral stationary phase. This preparative separation was carried out on a 450 mg scale of the racemate. Compound (−)-6b was used to complete the synthesis of the fluorocyclopropane-containing proline analogue. Homologation of lactam (−)-6b was performed by reductive cyanation, leading to the formation of amino-nitrile (−)-cis-7b in high yield (70%) with good diastereoselectivity (4:1). The final hydrolysis of nitrile (−)-cis-7b under acidic conditions, followed by the deprotection of the amine, afforded the free amino acid (−)-cis-8b in 76% yield (Scheme 4).
To further study the effect of this fluorocyclopropanecontaining proline on the conformation of peptides and more precisely on the cis/trans equilibrium of proline, (−)-cis-8b was then introduced into a tripeptide structure (Scheme 5). To this end, the amine moiety was protected with a Boc group, and the C-coupling was performed with alanine in the presence of (2- (1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) to afford dipeptide (−)-10 in 65% yield. Xray crystallographic analysis of this compound confirmed the relative and absolute configuration of all stereogenic centers.
Finally, deprotection of the amine using HCl followed by Ncoupling with a second alanine with HBTU led to the formation of tripeptide (−)-11 in 37% isolated yield.
The conformation of this tripeptide (−)-11 was studied using nuclear magnetic resonance (NMR) and density functional theory (DFT) calculations.
NMR Spectroscopy and DFT Calculations. The 1D 19 F spectrum of tripeptide (−)-11 in CDCl 3 at 298 K showed two signals at −200.94 and −204.05 ppm, indicating the presence of two species with proportions of 75 and 25%, respectively ( Figure  S64). This was supported by the observation of two sets of N−H amide signals at 5.01/5.29 and 6.86/8.06 ppm in the 1D 1 H Scheme 2. Strategy B: Synthesis of Diazoacetamide Followed by Intramolecular Cyclopropanation Scheme 3. Catalytic Asymmetric Cyclopropanation Reaction to Access Enantioenriched 6b−d ACS Omega http://pubs.acs.org/journal/acsodf Article spectrum, assigned to NH15 and NH5, respectively ( Figure  S66), leading to a repeated pattern of cross-peaks in the 2D NMR 1 H− 1 H correlation spectroscopy (COSY) spectrum ( Figure S70a). Moreover, additional cross-peaks, between each signal pair of NH15 and NH5 presumably due to the chemical exchange, were clearly observed in the 2D 1 H− 1 H nuclear Overhauser effect spectroscopy (NOESY) NMR spectrum ( Figure S70b). These observations suggested that the conformation of these two tripeptide (−)-11 species is sensitive to the "Ala−Pro" bond cis−trans isomerism. 18 This hypothesis was confirmed by the presence of a NOE cross-peak between H7 and H13, and intense NOE cross-peaks between H13 and H10 for the minor cis conformer and the major trans conformer (Scheme 6 and Figure S71). All proton, fluorine, and carbon resonance assignments of the trans (Table S1) and cis ( Examination of the short-and medium-range NOEs for the two isomers, in combination with the scalar coupling constants and the slow H/D exchange of the NH5 amide proton (Table  S3), suggested a turn centered on the proline residue.
According to the experimental observations (NOE and H/D exchange), interstrand hydrogen bonds C 12 O···H 5 N for the trans conformer and C 16 O···H 5 N for the cis conformer were tested as initial conformations in the DFT structure calculations using the Gaussian software. 19 Analysis of the (Φ, Ψ) angles (Table S4) and the potential NOEs in the calculated structures ( Figure 3 and Table S3) revealed that the sequence of compound (−)-11 consists of an inverse γ turn centered on the Ala-FPro-Ala segment for the trans isomer and a type β VIa1 turn centered on Boc-Ala-FPro-Ala segment for the cis isomers. 20 In summary, a fluorocyclopropane ring-fused proline analogue was synthesized via carbene transfer reactions in the presence of a transition-metal chiral catalyst. The isomers were optically resolved and then converted to the corresponding tripeptides in high yield and then analyzed by spectral and computational chemical analyses. The resulting access to proline derivatives and elucidation of the structural and conformational details will be helpful for the design of a new medicinal entry for drug discovery.

■ EXPERIMENTAL SECTION
General Techniques. Air-and/or water-sensitive reactions were conducted in oven-dried glassware in argon. All solvents were distilled from appropriate drying agents prior to use.  ACS Omega http://pubs.acs.org/journal/acsodf Article All reagents were used as received from commercial suppliers unless otherwise stated. High-resolution mass spectroscopy (HRMS) spectra were recorded on a Waters LCT Premier, infrared (IR) spectra were recorded on a PerkinElmer Spectrum 100, and optical rotations were recorded on a PerkinElmer Polarimeter 341. NMR experiments were carried out by using Bruker DXC 300 and Bruker AVIII 500 or 600 MHz spectrometers (Bruker, Wissembourg, France) equipped with a 10 A gradient amplifier giving a maximum gradient of 50 G cm −1 and a 5 mm TXO { 1 H, 13 C, 19 F} probe including shielded zgradients for 500 MHz or a CPTXI { 1 H, 15 N, 13 C} cryoprobe for 600 MHz. Measuring frequencies were 500 MHz or 600 MHz for 1 H, 125 MHz for 13 C, and 470 MHz for 19 F. 1 H and 13 C NMR chemical shifts are reported in parts per million using the residual peak of CDCl 3 (7.28 and 77.0 ppm) as the internal standard. 19 F NMR spectra were referenced (δ = 0.0 ppm) to calculated the 19 F frequency in the CFCl 3 reference compound. 21 1D NMR Measurements. The proton one-dimensional experiments were performed with standard Bruker "zg" parameters using 16 scans. One-dimensional 13 C{ 1 H} NMR spectra with broadband proton decoupling in order to remove the 13 C− 1 H scalar coupling were recorded with standard Bruker "zgpg" parameters. One-dimensional 19 F{ 1 H} NMR spectra with broadband proton decoupling in order to remove the 19 F− 1 H scalar coupling were recorded with standard Bruker "zgig" parameters. For 1 H NMR experiments, the acquisition time was 4.54 s and the relaxation delay (D1) was 2 s; for 13 C{ 1 H} NMR experiments, the acquisition time was 1.05 s and the relaxation delay was 5 s; for 19 F{ 1 H} NMR spectra, the acquisition time was 14 s and the relaxation delay was 5 s. 2D NMR Measurements. 1 H− 1 H COSY. The spectrum was acquired with 2048 data points in f2 and with 256 increments in f1 using 24 scans for each free induction delay (FID). The recycling delay was 2 s; the acquisition mode was DQD; and one-time zero filling in f1 and pure sine bell window function in f1 and f2 dimensions were applied before the Fourier transformation. 1 H− 13 C HMQC. The spectrum was acquired with 2048 data points in f2 and with 256 increments in f1 using 16 scans for each FID. The recycling delay was 2 s; the acquisition mode was DQD; and one-time zero filling in f1 and pure sine bell window functions were applied to f2 and f1 dimensions before the Fourier transformation. 1 H− 13 C HMBC. The spectrum was acquired with 2048 data points in f2 and with 256 increments in f1 using 32 scans for each FID. The delay for evolution of longrange couplings was 0.062 s and the recycling delay was 2 s; the acquisition mode was DQD; one-time zero filling in f1 and pure sine bell window functions were applied to f2 and f1 dimensions before the Fourier transformation. 1 H− 1 H NOESY. The spectrum was acquired with 2048 data points in f2 and with 256 increments in f1 using 24 scans for each FID. The recycling delay was 3 s; pure-phase line shapes were obtained by using time proportional phase incrementation (TPPI) phase cycling, where the mixing time of 0.5 s was used; and one-time zero filling in f1 and pure sine bell window functions were applied to f2 and f1 dimensions before Fourier transformation. 1 H− 19 F HOESY. The spectrum was acquired with 2048 data points in f2 and with 256 increments in f1 using 32 scans for each FID. The recycling delay was 3 s; pure-phase line shapes were obtained by using TPPI (states-TPPI) phase cycling; where the mixing time of 1 s was used; and one-time zero filling in f1 and pure sine bell window functions were applied to f2 and f1 dimensions before the Fourier transformation. DFT calculations. Starting from the tripeptide (−)-11 structure, we carried out the geometry optimization of the two possible trans and cis isomers using the DFT at the B3LYP/cc-PVTZ level of theory, followed by a frequency calculation at the same level of theory using the Gaussian software09. 19 General Procedure for the Synthesis of N-Benzyl Allylamines 1c and 1d. In a sealed flask, the appropriate benzyl amine (2 equiv, 63.5 mmol) was added to a solution of K 2 CO 3 (2 equiv, 63.5 mmol, 8.8 g) in acetonitrile (25 mL), and then, 3-chloro-2-fluoroprop-1-ene (1 equiv, 31.7 mmol, 3.0 g) was added at 0°C. The mixture was heated to 40°C for 18 h. Water was added to the reaction mixture and extracted three times with CH 2 Cl 2 . The combined organic layers were dried over MgSO 4 , filtered, and concentrated. The crude product was purified by column chromatography (SiO 2 , petroleum ether/ EtOAc) to afford the desired compound 1.