N-Terminal Cysteine Bioconjugation with (2-Cyanamidophenyl)boronic Acids Enables the Direct Formation of Benzodiazaborines on Peptides

Benzodiazaborines (BDABs) have emerged as a valuable tool to produce stable and functional bioconjugates via a click-type transformation. However, the current available methods to install them on peptides lack bioorthogonality, limiting their applications. Here, we report a strategy to install BDABs directly on peptide chains using (2-cyanamidophenyl)boronic acids (2CyPBAs). The resulting BDAB is stabilized through the formation of a key intramolecular B–N bond. This technology was applied in the selective modification of N-terminal cysteine-containing functional peptides.


General Remarks
All reagents and solvents used were purchased from Fluorochem, Alfa Aesar, TCI or Sigma-Aldrich without further purifications. The solvents used in ESI-MS and LC-MS experiments were of spectroscopic quality. In case of air-sensitive reactions, solvents and triethylamine were obtained in anhydrous conditions by distillation under nitrogen. Particularly, anhydrous dichloromethane and tetrahydrofuran were obtained using a Pure Solv™ Micro 100 Liter solvent purification system with activated alumina column. Dipeptides were prepared by Ismael Compañón and Dr. Francisco Corzana from Departamento de Química, Centro de Investigación en Síntesis Química, Universidad de La Rioja. Cys-Bombesin, C-Ovalbumin, CysCys-Bombesin and GV-1001 peptides were purchased from GeneCust. Maleimide 25 was prepared according to a reported procedure. 1 All chemical procedures were performed in air at ambient temperature (~22 ºC) and pressure (1.0 atm) unless indicated otherwise. Reaction mixtures were analyzed by thin layer chromatography using Merck silica gel 60F254 aluminium plates and visualized by exposure to UV light or by dipping the plates in panisaldehyde or phosphomolybdic acid stains followed by heating. Column chromatography was performed with silica gel Geduran® Si 60 (0.040-0.063 mm) purchased from Merk. NMR spectra were recorded in a Bruker Fourier 300 and 400 (Bruker, Massachusetts, USA) using CDCl3 and D2O as deuterated solvents. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project Nº 022161c and ROTEIRO/0031/2013-PINFRA/22161/2016 (cofinanced by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). All coupling constants (J values) are expressed in Hertz (Hz) and chemical shifts (δ) in parts per million (ppm). Multiplicities are given as: s (singlet), br (broad) d (doublet), dd (double doublet), dt (double triplet), t (triplet), tt (triple triplet), q (quartet), quint (quintuplet) and m (multiplet).
Low resolution mass spectra were recorded in a LCQ Fleet Ion Trap Mass Spectrometer (Thermo Fisher Scientific, Germany) equipped with an electrospray interface. High resolution mass spectra were carried on in a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo ScientificTM Q ExactiveTM Plus). The Liquid chromatography-mass spectrometry (LC-MS) runs were performed using a Dionex Ultimate 3000 UHPLC+ system equipped with a Multiple-Wavelength detector and a imChem Surf C18 TriF 100 Å 3 μm 100 x 2,1 mm column connected to Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo ScientificTM Q ExactiveTM Plus). Semi-preparative RP HPLC was performed on a Dionex Ultimate 3000 system using a Phenomenex Luna® 10 μm S6 C18(2) 100 Å, LC Column 250 x 10 mm.

Kinetic Studies of Benzodiazaborine 2 Formation
The following reaction was performed under pseudo-first order conditions in a NMR tube: 10 equivalents of L-cysteine (30 mg, 0.248 mmol) were added to a 50 mM solution of cyanamide 1 (6.04 mg, 0.025 mmol) in deuterated ammonium acetate 20 mM at pH 7.0 (0.50 mL). The formation of BDAB 2 was monitored by 1 H-NMR spectroscopy over 26 h at room temperature. Figure S5. 1 H-NMR spectra of compound 2 formation as a function of time. Initial [1] = 50 mM, internal standard = deuterated ammonium acetate. Figure S6. 1 H-NMR kinetic study. Cyanamide 1 and diazaborine 2 concentrations versus time data were acquired from the integration of the respective 1 H-NMR signals relative to deuterated ammonium acetate, utilized as an internal standard. Figure S7. Linear fitting of cyanamide 1 kinetic data using the first order kinetic model. The plot of ln( [1]) (mM) versus time (h) was used to determine the pseudo-first order constant (kobs). 2CyPBA 1 concentration versus time data were acquired from the integration of the respective 1 H-NMR signals relative to deuterated ammonium acetate, utilized as an internal standard. S17 6. Benzodiazaborine 2 Stability Studies 6.1. Stability in PBS pH 7.4 Triplicated solutions of benzodiazaborine 2 (10 mM, 10 µL, 0.1 µmol) in PBS pH 7.4 (1.0 mL) were prepared and incubated at 25 °C for five days. 20 µL aliquots were taken over time for LC-MS analysis. The HPLC runs were carried out with a gradient of A (Milli Q water containing 0.1 % v/v Formic acid, FA) and B (acetonitrile containing 0.1 % v/v FA, Honeywell HPLC-grade). The mobile phase was t = 0-1 min, 5 % B; t = 10-11 min, 95.5 % B; t = 12 min, 5 % B; t = 15 min, stop at a flow rate of 0.2 mL / min. Diazaborine: RT 5.84 min, detection EIC. The peak areas of the EIC of 2 over time were converted into remaining concentration based on the following calibration curve (base peak m/z 266.0765, within 5 ppm range).

Stability in Presence of Glutathione
Triplicated solutions of benzodiazaborine 2 (10 mM, 10 µL, 0.1 µmol) in PBS pH 7.4 (1.0 mL) were prepared and incubated with 10 equivalents of glutathione (50 mM, 20 µL, 1.0 µmol) at 25 °C for three days. 20 µL aliquots were taken over time for LC-MS analysis. The HPLC runs were carried out with a gradient of A (Milli Q water containing 0.1 % v/v Formic acid, FA) and B (acetonitrile containing 0.1 % v/v FA, Honeywell HPLC-grade). The mobile phase was t = 0-1 min, 5 % B; t = 10-11 min, 95.5 % B; t = 12 min, 5 % B; t = 15 min, stop at a flow rate of 0.2 mL/min. Diazaborine: RT 5.84 min, detection EIC. The peak areas of the EIC of 2 over time were converted into remaining concentration based on the following calibration curve (base peak m/z 266.0765, within 5 ppm range).

ESI-MS Mechanistic Studies
The reactions between cyanamides 1, 3, 6 or 8 and cysteine models (L-cysteine and N-acetyl cysteine) were performed according to the following protocol: 1.0 equivalent of the cysteine model L-cysteine or Nacetyl cysteine (100 mM, 100 µL, 10.00 µmol) was added to a 2 mM solution of the respective cyanamide (100 mM, 100 µL, 10.00 µmol) in ammonium acetate solution 20 mM, pH 7.0 (500 µL). The reaction was stirred at 25 °C for 24 h and monitored by ESI-MS in Positive Mode. The reactions with cyanamides 1 and 3 were also monitored by LC-HRMS in Positive Mode. The HPLC runs were carried out with a gradient of A (Milli Q water containing 0.1 % v/v Formic acid, FA) and B (acetonitrile containing 0.1 % v/v FA, Honeywell HPLC-grade). The mobile phase was t = 0-1 min, 5 % B; t = 10-11 min, 95.5 % B; t = 12 min, 5 % B; t = 15 min, stop at a flow rate of 0.2 mL/min, detection EIC.       Figure S17. LC-MS chromatograms of Cys-Gly conjugate 10 and Cys-Gly dipeptide. A -Reaction mixture TIC after 48 h; B -Reaction detection at 210 nm, after 48 h; C -Reaction t0 -unreacted Cys-Gly EIC (base peak m/z 178.0645); D -Unreacted Cys-Gly EIC after 48 h with 8.0 eq of 1; E -Cys-Gly conjugate 10 EIC (base peak m/z 322.1140) after 48 h reaction. Cys-Gly conversion was calculated based on the EIC intensity (AUC) within δ 5 ppm range.

Computational Studies
All of the calculations were performed using the Gaussian09 program. 10 Computations were done using wb97xd functional 11 in conjunction with standard basis sets def2SVP and def2TZVP. 12 Geometry full optimizations were made at wb97xd/def2SVP level. Single point calculations using def2TZVP basis set were carried out over optimized geometries to obtain the energy values. Solvent effects (toluene) were considered using the PCM model. 13 The nature of stationary points was defined on the basis of calculations of normal vibrational frequencies (force constant Hessian matrix). The optimizations were carried out using the Berny analytical gradient optimization method. 14 Minimum energy pathways for the reactions studied were found by gradient descent of transition states in the forward and backward direction of the transition vector (IRC analysis). 15 Analytical second derivatives of the energy were calculated to classify the nature of every stationary point, to determine the harmonic vibrational frequencies, and to provide zero-point vibrational energy corrections. The thermal and entropic contributions to the free energies were also obtained from the vibrational frequency calculations, using the unscaled frequencies. Correction to free energy was made by substracing Strans contribution and considering a 1 M concentration. 16 NCI (non-covalent interactions) were computed using the methodology previously described. 17 Data were obtained with the NCIPLOT program. 18 A density cutoff of ρ=0.1 a.u. was applied and the pictures were created for an isosurface value of s=0.5 and colored in the [-0.03,0.03] a.u. sign(λ2)ρ range using VMD software. 19 Structural representations were generated using CYLView. 20

Models of addition
We studied the reaction of cysteine with substrates 1 and 6. There are two orientations (1 and 2 series) of the cyano group as illustrated in Figure S58. Consequently, four transition structures were calculated. For the comparison between NH (a series) and NMe (b series) derivatives the best orientation in each case was selected. Figure S60. Reaction between L-cysteine and compounds 1 (RE1a/RE2a) and 6 (RE1b/RE2b)