Orthogonal ring-closing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides

Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets.


Chemicals and instrumentation
Unless otherwise noted, chemicals were purchased from Sigma Aldrich, Merck, Okeanos, Roth or Alfa Aesar and were used without further purification. Protected Fmoc-amino acids and coupling reagents were purchased from Novabiochem and Iris Biotech GmbH. Building block 6 for hydrocarbon peptide stapling was purchased from Okeanos Tech. Co. LTD. All solvents were purchased from commercial suppliers and used without further purification. Analytical HPLC was performed using an Agilent 1100 Series with either a C18 HPLC column 3 µm (Macherey Nagel) or a C18 HPLC column 1.8 µm (Macherey Nagel). The system was run at a flow rate of 1.0 mL/min over 30 min using H 2 O (0.1% TFA) and MeCN (0.1% TFA) as solvents. Linear gradients were run over varying periods of time. The efficiency of nucleotide exchange was monitored by analytical HPLC using 50 mM KH 2 PO 4 (pH 7.0) and 10 mM TBABr and MeCN (0.1% TFA) as solvents. HPLC-MS analyses were performed with an Agilent 1100 Series connected to a Thermo LCQ Advantage mass spectrometer using a C18 HPLC column 3 µm (Macherey Nagel). The system was run at a flow rate of 1 mL/min over 15 min using H 2 O (0.1% formic acid) and MeCN (0.1% formic acid) as eluents. Semi preparative HPLC was carried out on a Agilent 1100 Series using a SP125/10 Nuclear C18 Gravity 5 µm column (Macherey Nagel) at a flow rate of 6 mL/min. Linear gradients using H 2 O (0.1% TFA) and MeCN (0.1% TFA) were run over varying periods of time. High resolution mass spectra were recorded on a QLT Orbitrap mass spectrometer coupled to an Acceka HPLC-System (HPLC column: Hypersyl GOLD, 50 mm x 1mm particle size 1.9 µm, ionization method: Electrospray Ionization). Automated Peptide synthesis was performed using a CEM-Discover microwave and a CEM-Liberty peptide synthesizer. Fluorescence polarization was measured with a Tecan Safire 2 . Absorbance measurements were performed on a Tecan infinite M200 and Thermo scientific Nanodrop 2000c. 1 H-and 13 C-NMR spectra were recorded on a Varian Mercury VX 500 or 400 spectrometer at room temperature. NMR spectra were calibrated to the solvent signals CDCl 3 (7.26 and 77.16) or DMSO (2.50 and 39.52). MicroScale Thermophoresis (MST) curves were measured on a NanoTemper Technologies Monolith NT.115.

Peptide synthesis General
Peptides were synthesized on solid-phase using the Fmoc-strategy and Rink Amide (MBHA) resin, Rink Amide NovaSyn TGR resin or ChemMatrix Rink Amide resin as solid support. Solvents and soluble reagents were removed by suction. Washings between coupling and deprotection were carried out in DMF and DCM using 1 mL solvent per 100 mg resin. Coupling efficiency was monitored by ESI-MS and/or HPLC analyses.

Amino acid coupling
Fmoc-Xaa-OH (4 eq.) was dissolved in freshly prepared solution of HCTU (3.9 eq., 0.5 M) with DIEA (8 eq.). Subsequently, this mixture was added to the resin and shaken for 30 min at room temperature. For coupling of the Alkyne building blocks (1 -4), the building block 6 and the subsequent amino acid: Fmoc-Xaa-OH (4 eq.) was dissolved in DMF in the presence of COMU (3.9 eq.), Oxyma (3.9 eq.) and DIEA (8 eq.), added to the resin and shaken for 1 h at room temperature. Except coupling of the Alkyne building blocks 1 -4 and the alkene building block 6, all couplings were performed as double couplings. All equivalents are calculated based on theoretical loading of the resin as provided by the vendor.

N-Acetylation
For preparation of N-acetylated peptides and whenever a quantitative yield even after recoupling treatments was not achieved, the free N-terminal amino group was acetylated using a solution of Ac 2 O/DIEA/DMF (1/1/8, v/v/v) for 2 x 10 min at room temperature.

Fluorescence labelling with FITC
Prior to fluorescence labelling with FITC a PEG-linker (Fmoc-O2Oc-OH) was coupled to the free N-terminus. A mixture of Fmoc-O2Oc-OH (5 eq.), COMU (4.9 eq.), Oxyma (4.9 eq.) and DIEA (10 eq.) in DMF was transferred to the resin and shaken at room temperature for 2 x 1 h. The resin was drained and washed with DMF (3x). The Fmoc group was removed as described above and the resin was treated with FITC (5 eq.) and DIEA (10 eq.) for 16 h at room temperature under exclusion of light. Afterwards, the resin was washed with DMF (3x), DCM (3x) and dried to constant weight in vacuo.

Ring closing alkyne metathesis
The dried resin was transferred under argon into a baked out Schlenk tube and swollen and shrunken alternating in dry diethyl ether and dry toluene (3x each). Afterwards 0.5 mL of a solution of the alkyne metathesis complex 5 (2 mg mL -1 ) in dry toluene was added and the reaction mixture was stirred at 40°C for 1.5 h. During the reaction time argon was bubbled through the reaction mixture to evaporate the 2butyne. After addition of 0.5 mL of fresh complex 5 solution the mixture was stirred at 40° C for 1.5 h. The resin was filtered off, washed with toluene (3x), DCM (3x) and dried to constant weight.

Ring closing olefin metathesis
The dried resin was swollen in DCE for 15 min. A solution of Grubbs 1 st generation catalyst (2 mg mL -1 ) in DCE was added to the resin and reacted for 2 h at room temperature. During the reaction time argon was bubbled through the reaction mixture to remove ethene. The procedure was repeated twice and the resin was washed with DCE (3x), DCM (3x), DMF (3x).

One pot ring closing alkyne and olefin metathesis
The dried resin was transferred under argon into a baked out Schlenk tube and swollen and shrunk alternating in dry diethyl ether and dry toluene (3x each). Afterwards 0.5 mL of a solution of the alkyne metathesis complex 5 (2 mg mL -1 ) and Grubbs 1 st generation catalyst (2 mg mL -1 ) in dry toluene was added and the reaction mixture stirred at 40° C for 1.5 h. During the reaction time argon was bubbled through the reaction mixture to evaporate the 2-butyne. After addition of 0.5 mL of fresh complex solution (alkyne complex 5 and Grubbs 1 st generation catalyst) the mixture was stirred at 40° C for 1.5 h. The resin was filtered off, washed with toluene (3x), DCM (3x) and dried to constant weight.

Dibromination of alkyne macrocycles
The dried resin was swollen in dry MeCN for 15 min and treated with a mixture of CuBr 2 in dry MeCN (2 mg mL -1 ) for 2 h. The reaction was performed in a Syringe reactor and the procedure was repeated twice. Afterwards, the resin was washed with MeCN (3x), DMF (3x), DCM (3x).

Cleavage from the resin
The dry resin was treated with a solution of TFA/EDT/TIS/H 2 O (94/1/2.5/2.5, v/v/v/v) 100 µL 10 mg -1 resin for 2 x 1 h and 1 x 5 min. The solvents were evaporated and the crude peptide was precipitated by the addition of cold diethyl ether. After centrifugation (10 min, 16.100 x g, 4°C) the supernatant was removed. The crude product was dissolved in H 2 O/MeCN (2/1, v/v) and lyophilized. The crude peptides were purified by semi-preparative HPLC.

Fmoc quantification
A defined amount of dry resin was transferred into an Eppendorf cap and treated with 0.5 mL deprotection solution for 15 min. The UV absorption of the supernatant was determined at 305 nm and the occupation density calculated using Beer-Lambert law (ԑ = 7800 cm -1 M -1 ).

Peptide quantification
The concentration of fluorescein labeled peptides was determined by UV absorption in 20 mM phosphate buffer (pH 8.5) at 496 nm (ԑ = 77.000 cm -1 M -1 ). The concentration of acetylated peptides was determined gravimetrically or via UV absorption at 280 nm.

Protein expression and purification
Expression and purification of Rab8a 6-176 was performed analog to full-length Rab8a according to established protocols. 1,2

Nucleotide exchange
Nucleotide exchange was performed according to previously established protocols. 2,3 Briefly, for nucleotide removal Mg 2+ was removed by addition of a 5-fold excess of EDTA and reacted for 1 h at room temperature. The protein solution was desalted using a PD-10 desalting column Sephadex G-25 DNA Grade (GE Healthcare) with elution buffer consisting of 20 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP. After removal of Mg 2+ the protein was diluted to 80 -100 µM before addition of ZnCl 2 (500 µM) and (NH 4 ) 2 SO 4 (200 mM). After addition of alkaline phosphatase (5 U mg -1 Rab protein) the mixture was incubated for 16 h at 4°C. For nucleotide exchange, the mixture contained a 5-fold excess of GppNHp during alkaline phosphatase incubation. Afterwards, the mixture was desalted using a PD-10 desalting column Sephadex G-25 DNA Grade (GE Healthcare) with elution buffer consisting of 25 mM HEPES (pH 7.5) 150 mM NaCl, 1 mM TCEP, 1 mM MgCl 2 and 1 µM GppNHp.

Microscale Thermophoresis (MST)
Rab8a 6-176 (GppNHp) was serially diluted in assay buffer and treated with 133 nM fluorescein-labeled peptide. After incubation for 4 h at room temperature the mixture was soaked into capillaries for microscale thermophoresis (MST) measurements. K d values were calculated after initial fluorescene analysis of the obtained MST curves using the software Monolith Affinity Analysis (NanoTemper Technologies).

Competition fluorescence polarization assay
Acetylated peptides were serially diluted in assay buffer (1:1) and were incubated with a mixture of 60 nm fluorescein-labeled peptide and Rab8a 6-176 (GppNHp) (15 µM or 100 µM depending on K d of the labeled peptide) for 1 h at room temperature. Fluorescence polarization was dertermined and half maximal inhibitory concentrations (IC 50 ) were calculated by nonlinear regression analysis of doseresponse curves using Prism 5.0 software (GraphPad). 5

Synthetic methods
Synthesis of the alkyne building blocks 1-4 was performed according to adapted protocols from Y. N. Belokon et al. 6 and G. H. Bird et al. 7 Schematic representation of the synthesis is summarized below.

Synthesis of Mo-complexes 5 and 33
Mo-complexes for RCAM 5 and 33 were prepared according to previously established procedures. 8

Synthesis of (S, R)-Ala-Ni(II)-BPB (36a,b)
Synthesis of the Ni-complexes 36a and 36b was carried out according to previously established protocols starting either from L-or D-Proline. 6

Synthesis of unprotected -methyl--alkinyl amino acids (38a-d)
To a solution of 37a-d in MeOH (40 mL), conc. hydrochloric acid (10 eq.) was added and the reaction mixture refluxed at 80 °C for 1 h. The reaction mixture was allowed to cool to room temperature and concentrated in vacuo. After addition of 20 mL water (20 mL) the aqueous layer was extracted with DCM (3 × 20 mL). The combined organic layers were washed with brine, dried over MgSO 4 and concentrated under reduced pressure. Recovered BPB was purified by precipitation as hydrochloric salt from acetone solution. 12 The aqueous layer was dried by lyophilization and the crude unprotected -methyl--alkinyl amino acid was used without any further purification.