Enhanced Permeability and Binding Activity of Isobutylene‐Grafted Peptides

Abstract We present a new peptide‐macrocyclization strategy with an isobutylene graft. The reaction is mild and proceeds rapidly and efficiently both for linear and cyclic peptides. The resulting isobutylene‐grafted peptides possess improved passive membrane permeability due to the shielding of the polar backbone of the amides, as demonstrated by NMR spectroscopy and molecular dynamics simulations. The isobutylene‐stapled structures are fully stable in human plasma and in the presence of glutathione. This strategy can be applied to bioactive cyclic peptides such as somatostatin. Importantly, we found that structural preorganization forced by the isobutylene graft leads to a significant improvement in binding. The combined advantages of directness, selectivity, and smallness could allow application to peptide macrocyclization based on this attachment of the isobutylene graft.


Enhanced permeability and binding activity of isobutylene-grafted peptides Table of Contents
General methods S1 Synthesis and characterization of peptides S6 Circular Dichroism spectroscopy S15 Parallel Artificial Membrane Permeability Assay (PAMPA) S15 Tryptophan fluorescence spectroscopy S16 Stability test of Stapled Somatostatin S16 ROESY spectra of peptides IV and IV' S17 MD simulations data S17 References S19

General methods
Chromatography. Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel 60 F 254 plates, using UV at 254 nm or staining with ninhydrin for visualization. Column chromatography was performed with Material Harvest silica gel 60. Reverse-phase column chromatography was conducted with Varian Bond Elut ® C18. The HPLC was conducted on Agilent 1100 Series fitted with G1322A degasser, G1311A pump, G1313A autosampler and G1315 DAD, with YMC-Pack Pro C18 column 120 Å S-5 µm 10 mm x 250 mm (product no. AS12S05-2510WT) for preparative scale. The eluent was solvent B, water with 0.1% trifluoroacetic acid (TFA), and C, acetonitrile with 0.1% trifluoroacetic acid, unless otherwise noted and gradients specific to the compound. spectrometer, which was routinely calibrated with (1S)-(+)-10-camphorsulfonic acid. Spectra were recorded at 298K with a 0.1 cm quartz cell over the wavelength range 250-189 nm at 50 nm·min -1 , with a bandwidth of 1.0 nm, the response time of 1 s, resolution step width of 1 nm and sensitivity of 20-50 Mdeg. Each spectrum represents the average of 5 scans.
Parallel Artificial Membrane Permeability Assay (PAMPA). The PAMPA EvolutionTM instrument was used to determine permeability. In PAMPA, a sandwich is formed such that each composite well is divided into two chambers, separated by a 125 μm thick microfilter disc (0.45 μm pores), coated with Pion GIT-0 phospholipid mixture. The effective permeability, Pe, of each compound was measured at the customer-specified pHs in the donor compartment using low-binding, low UV Prisma buffer. The drug-free acceptor compartment was filled with acceptor sink buffer containing a scavenger at the start of the test. The proprietary scavenger mimics serum proteins and blood circulation, thus creating sink conditions.

S3
In the default protocol the aqueous solutions of studied compounds are prepared by diluting and thoroughly mixing 3 µL of DMSO stock in 600 µL of Prisma HT buffer. Final concentration of organic solvent (DMSO) in aqueous buffer is ≤ 0.5% (v/v).
The reference solution is identical to the donor at time zero, so that any surface adsorption effects from the plastic is compensated. The PAMPA sandwich was assembled and allowed to incubate for ~15 hours. The solutions in the donor compartment were un-stirred within duration of the experiment. Thus, the thickness of the aqueous boundary layer expected to be about 1000 μm. The sandwich was then separated, and both the donor and receiver compartments were assayed for the amount of drug present by comparison with the UV spectrum obtained from reference standards. Mass balance was used to determine the amount of material remaining in the membrane filter and on the plastic (%R).
Ketoprofen, Verapmil and Propanolol were used as reference compounds.

Buffers preparation
pH of Prisma HT buffer was adjusted to the requested values using 1.0 M solution of NaOH. Actual pH the buffers was 7.40±0.05.

Stock solutions preparation
Sample powders pre-weighed in glass vials were brought to the room temperature at the day of the experiment. The samples were diluted with an organic solvent (DMSO) to prepare stock solutions at concentration ~10 mM. The stock solutions were further diluted in buffer at 7.40 producing the aqueous sample solutions at concentrations ~50 µM. The amount of DMSO in the resulting solution was <0.5% (v/v). The solutions were filtered prior assaying the samples.

Molecular dynamics (MD) simulations with time averaged restraints (MD-tar).
The simulations on peptides IV and IV' were carried out with AMBER 16 package [S1] implemented with ff14SB [S2] and GAFF [S3] force fields. The parameters and charges for the unnatural amino acids were generated with the antechamber module of AMBER, using GAFF force field and AM1-BCC method for charges. [S4] Prior to MD-tar productive simulations, we performed an equilibration protocol consisting of an initial minimization of the water box of 5000 steps, followed by a 2500-step minimization of the whole system. Then, the TIP3P water [S5] box was heated at constant volume until 298 K, using a time constant for the heat bath coupling of 1 ps.
The equilibration finished with 200 ps of MD simulation without restraints, at a constant pressure of 1 bar and turning on the Langevin temperature scaling with a collision frequency of 1 ps. Nonbonded interactions were cut-off at 8.0 Å and updated every 25 steps. Periodic boundary conditions and the Particle Mesh Ewald method [S6] were turned on in every step of the equilibration protocol to evaluate the long-range electrostatic forces, using a grid spacing of approximately 1 Å. The ROESY-derived distances were imposed as time-averaged constraint, applying an r −6 averaging. The equilibrium distance range was set to r exp − 0.2 Å ≤ r exp ≤ 0.2 Å.
Trajectories were run at 298 K, with a decay constant of 2000 ps and a time step of 1 fs. The force constants rk 2 and rk 3 used in each case were 10 kcal·mol −1 ·Å −2 . The overall simulation length was 20 ns. The coordinates were saved each 1 ps, obtaining MD trajectories of 20000 S4 frames each. A convergence within the equilibrium distance range was obtained in the simulations.

Unrestrained (MD simulations on somatostatin and stapled somatostatin. Each peptide was
immersed in a water box with a 10 Å buffer of TIP3P water molecules. A two-stage geometry optimization approach was performed. The first stage minimizes only the positions of solvent molecules and the second stage is an unrestrained minimization of all the atoms in the simulation cell. The systems were then gently heated by incrementing the temperature from 0 to 300 K under a constant pressure of 1 atm and periodic boundary conditions. Harmonic restraints of 30 kcal·mol -1 were applied to the solute, and the Andersen temperature-coupling scheme was used to control and equalize the temperature.
The time step was kept at 1 fs during the heating stages, allowing potential inhomogeneities to selfadjust. Long-range electrostatic effects were modelled using the particle-mesh-Ewald method. An 8 Å cut-off was applied to Lennard-Jones and electrostatic interactions. Each system was equilibrated for 2 ns with a 2 fs time step at a constant volume and temperature of 300 K. Production trajectories were then run for additional 500 ns under the same simulation conditions.

Polar surface area (PSA) for glycopeptides IV and IV'. This parameter was obtained by MD-tar
simulations with CPPTRAJ software, [S7] included in AMBER16, using the 'surf' flag and the mask :1-10@O,N.

S5
Peptides II-IV were synthesized following a similar protocol, employing a stepwise micro-wave assisted solid-phase peptide synthesis on a Liberty Blue synthesizer. In these cases, the peptides were purified by HPLC using a Phenomenex Luna C18(2) column (10 μ, 250 mm × 21.2 mm) and a dual absorbance detector, with a flow rate of 20 mL/min.

Stapled peptides
The linear peptide (0.02 mmol) was dissolved in 10 mL of DMF and K 2 CO 3 (0.10 mmol) and tris(2carboxyethyl)phosphine (TCEP, 0.02 mmol) were then added. The solution was stirred for 1 h at rt. 3bromo-2-bromomethyl-1-propene (0.025 mmol) was then added and stirred for additional 12 h. The crude peptide was purified by reversed-phase HPLC to obtain the corresponding stapled derivative. In all cases the yield was ≥ 75%. Finally, they were purified by HPLC.