New peptide architectures through C–H activation stapling between tryptophan–phenylalanine/tyrosine residues

Natural peptides show high degrees of specificity in their biological action. However, their therapeutical profile is severely limited by their conformational freedom and metabolic instability. Stapled peptides constitute a solution to these problems and access to these structures lies on a limited number of reactions involving the use of non-natural amino acids. Here, we describe a synthetic strategy for the preparation of unique constrained peptides featuring a covalent bond between tryptophan and phenylalanine or tyrosine residues. The preparation of such peptides is achieved in solution and on solid phase directly from the corresponding sequences having an iodo-aryl amino acid through an intramolecular palladium-catalysed C–H activation process. Moreover, complex topologies arise from the internal stapling of cyclopeptides and double intramolecular arylations within a linear peptide. Finally, as a proof of principle, we report the application to this new stapling method to relevant biologically active compounds.

Peptide NMR spectra comparison between compounds 2g and 1g. a, NMR H region of peptide 2g and its linear precursor 1g. b, Plot of the 13 C chemical shift differences ( 13 C ∆ cyclic-linear ) between stapled peptide 2g and its linear counterpart 1g. c, Summary of NOE connectivities and temperature coefficients of the NH amide protons (∆ /∆T) of peptide 1g (bottom left) and 2g (bottom right). The thickness of the bars reflects the intensity of the NOEs, i.e. weak ( ), medium ( ) and strong ( ). I-F: miodophenylalanine. Figure 6 Peptide NMR spectra comparison between compounds 2h and 1h. a, NMR H region of peptide 2h and its linear precursor 1h. b, Plot of the 13 C chemical shift differences ( 13 C ∆ cycliclinear ) between stapled peptide 2h and its linear counterpart 1h. c, Summary of NOE connectivities and temperature coefficients of the NH amide protons (∆ /∆T) of peptide 1h (bottom left) and 2h (bottom right). The thickness of the bars reflects the intensity of the NOEs, i.e. weak ( ), medium ( ) and strong ( ). I-F: m-iodophenylalanine. Figure 7 Peptide NMR spectra comparison between compounds 2j and 1j. a, NMR H region of peptide 2j and its linear precursor 1j. b, Plot of the 13 C chemical shift differences ( 13 C ∆ cyclic-linear ) between stapled peptide 2j and its linear counterpart 1j. c, Summary of NOE connectivities and temperature coefficients of the NH amide protons (∆ /∆T) of peptide 1j (bottom left) and 2j (bottom right). The thickness of the bars reflects the intensity of the NOEs, i.e. weak ( ), medium ( ) and strong ( ). I-F: m-iodophenylalanine.

General experimental information
Reactions were monitored by HPLC-MS at 220 nm using a HPLC Waters Alliance HT comprising a pump (Edwards RV12) with degasser, an autosampler and a diode array detector. Flow from the column was split to a MS spectrometer. The MS detector was configured with an eletrospray ionization source (micromass ZQ4000) and nitrogen was used as the nebulizer gas. Data acquisition was performed with MassLynx software. For compounds 2a-2c, 2l, 2m, 1j-BODIPY, 5, 6, and 16, yields are estimated from the integration of the peak areas in the HPLC-MS crude. Other yields are for the isolated pure compound. All microwave reactions were carried out in 10 mL sealed glass tubes in a focused mono-mode microwave oven ("Discover" by CEM Corporation) featured with a surface sensor for internal temperature determination. Cooling was provided by compressed air ventilating the microwave chamber during the reaction. When stated, the final crude was purified via flash column chromatography Combi Flash ISCO RF provided with dual UV detection.
NMR spectra of peptides in DMSO-d 6 were acquired with either a Bruker DMX-500 MHz spectrometer or Bruker Avance III 600 MHz and Bruker Avance 800 MHz spectrometers equipped with TCI cryoprobes. The spectra were referenced relative to the residual DMSO signal ( 1 H, 2.49 ppm; 13 C, 39.5 ppm). 1 H resonances were unequivocally assigned by two-dimensional NMR experiments (COSY, TOCSY and NOESY and/or ROESY). Then, the 13 C resonances were straightforwardly assigned on the basis of the cross-correlations observed in the 1 H-13 C HSQC spectra.
Mixing times for TOCSY spectra were 70 ms, for NOESY spectra 300-450 ms and for ROESY experiments were 200 ms. The temperature coefficients for the amide protons of each peptide were determined via 1 H spectra in the range 298-313 K with a step size of 5 K. Chemical shifts (δ) are reported in ppm. Multiplicities are referred by the following abbreviations: s = singlet, d = doublet, t = triplet, dd = double doublet, dt = double triplet, q = quartet, p = pentuplet and m = multiplet. HRMS (ESI positive) were obtained with a LTQ-FT Ultra (Thermo Scientific) mass Spectrometer. IR spectra were obtained on a Thermo Nicolet NEXUS. CD Spectroscopy. Circular dichroism (CD) measurements were performed using a Jasco J-815 spectrophotometer. The spectra were recorded from 260 to 170 nm using a 1.0 mm path-length quartz cuvette at 2 nm bandwidth, 50 nm/min scan speed, 0.5 s response time, 0.2 nm data pitch and three accumulations. The background signal of the buffer alone was subtracted for each spectrum. CD spectra were converted from raw ellipticity (θ, mdeg) to mean molar ellipticity per residue ([θ], deg cm 2 dmol −1 ).
All the samples were dissolved in a buffer of 25 mM Na 2 HPO 4 (pH 7) at both 100 and 200 μM final peptide concentration. Additionally, new determinations were made in 10% of 2,2,2-trifluoroethanol (TFE) to increase the propensity to form secondary structures. To ensure no interference of peptide aromatic moiety on the spectra profiles, the previously reported 3-(2-Phenyl-1H-indol-3-yl)propanoic acid 1 was also analysed at identical conditions as for the tested compounds.
General procedure for SPPS. 2 All peptides were manually synthesized in polystyrene syringes fitted with a polyethylene porous disc using Fmoc-based SPPS. Solvents and soluble reagents were removed by suction. The Fmoc group was removed with piperidine-DMF (1:4) (1×1 min, 2×5 min).
Peptide synthesis transformations and washes were performed at r.t.
Resin loading (only for 2-Chlorotrityl resin). Fmoc-XX-OH (1.0 eq.) was attached to the resin (1.0 eq.) with DIEA (3.0 eq.) in DCM at r.t for 10 min and then DIEA (7.0 eq.) for 40 min. The remaining trityl groups were capped adding 0.8 μL MeOH/mg resin for 10 minutes. After that, the resin was filtered and washed with DCM (4 x 1 min), DMF (4 x 1 min). The loading of the resin was determined by titration of the Fmoc group. 2 Peptide elongation. After the Fmoc group was eliminated, the resin was washed with DMF (4 x 1 min), DCM (3 x 1 min), DMF (4 x 1 min). The completion of the coupling was monitored with the ninhydrin (free primary amine) or chloranil (free secondary amine) tests. 3 Then, the resin was filtered and washed with DCM (4 x 1 min) and DMF (4 x 1 min) and the Fmoc group was eliminated.
Final cleavage. The resin bound peptide was treated with the corresponding TFA cleavage cocktail.
Then, the resin was washed with DCM and the combined eluates were evaporated under vacuum.
Then, the residue was washed with Et 2 O, dissolved in ACN:H 2 O and lyophilized furnishing the corresponding peptide.