Iminoboronates are efficient intermediates for selective, rapid and reversible N-terminal cysteine functionalisation

Formyl benzeno boronic acids rapidly and selectively react with N-terminal cysteines to yield a reversible boronated thiazolidine that may be used in the interactive orthogonal modification of peptides.


Isolation of the products
L-Cysteine (40.4 mg, 0.333 mmol) was dissolved in 0.5 mL of water and then added to a solution of 2formylbenzeneboronic acid (50.0 mg, 0.333 mmol) in 2 mL of water previously prepared. The reaction occurred without stirring, at room temperature for 30 minutes. A precipitate was formed, decanted, and washed 3x with water. The crystals were dried under vacuum to give 48.9 mg of pure product. The mother liquor and washing water were combined and evaporated to give 28.8 mg of a residue, that 1 H-NMR analysis show that is the same product as that obtained in the crystals. Therefore, the total mass of 1 was 77.7 mg (0.333 mmol, 99% yield).  + , 217.9, 194.7. Anal. Calcd for C 10 H 10 BNO 3 S: C, 51.10; H,4.29;B,4.60;N,5.96;O,20.42;S,13.64. Found: C,51.44;H,4.28;N,6. L-Cysteine (23.3mg, 0.192 mmol) was dissolved in 0.5 mL of water and added to a solution of 3-fluoro-2formylbenzeneboronic acid (32.4 mg, 0.192 mmol) in 1 mL of water previously prepared. The reaction occurred without stirring, at room temperature for 30 min. A precipitate was formed, decanted, and washed 3x with water. The crystals were dried under vacuum to give 41.5 mg of pure product. The mother liquor and washing water were combined and evaporated to give 3.5 mg of a residue, that 1 H-  0.192 mmol) was dissolved in 0.5 mL of water:acetonitrile (1:1) and added to a solution of (3-formylthiophen-2-yl)boronic acid (30.0 mg, 0.192 mmol) in 2 mL of water:acetonitrile (1:1) previously prepared. The reaction occurred without stirring, at room temperature for 24 hours. The solvent was evaporated to a final volume of about 1 mL, a precipitate was formed, decanted and washed 3x with water. The precipitate was dried under vacuum to give 34.2 mg (0.142 mmol, 74%) of 3 as a 4:1 mixture of diastereoisomers. The mother liquor and filtrates were collected and evaporated until dryness to give 12.9 mg of a residue, that 1 H-NMR analysis shows that this fraction was impure. Therefore, we ignored this fraction. L-Cysteine (37.0 mg, 0.305 mmol) was dissolved in 0.5 mL of water and added to a solution of (2acetylphenyl)boronic acid (50.1 mg, 0.306 mmol) in 4.1 mL of water and 0.6 mL of acetonitrile previously prepared. The reaction occurred without stirring, at room temperature for 24 hours. A precipitate was formed, decanted, and washed 3x with water. The crystals were dried under vacuum to give 27.9 mg (0.112 mmol, 37%) of 4. The mother liquor and washing water were combined and evaporated to give 12.9 mg of a residue, that 1 H-NMR analysis show that this fraction was impure. Therefore, we ignored this fraction. In a round bottom flask, under inert atmosphere, 4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) (88 mg, 0.44 mmol) was dissolved in Acetonitrile (9 mL). After a solution of TEA (246 µL, 1.763 mmol) and 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethan-1-aminium 2,2,2-trifluoroacetate (224 mg, 0.881 mmol) [4] in Acetonitrile (4.5 mL), was added and the solution was stirred at room temperature for 1 h. The reaction mixture was concentrated and the residue was purified by column chromatography (silica gel, hexane:EtOAc (3:1 -1:1)) to afford 28 (10.1 mg, 0.033 mmol, 7.5 % yield). 7  Reaction buffer: was prepared by mixing Ammonium acetate solution pH 7.4 with dimethylformamide (DMF) in 10:1 ratio (vol:vol).

-UV-vis spectra
Reaction buffer (2.99 mL) was pipetted into the cuvette, followed by benzaldehyde (60 mM solution in DMF,10 µL, 0.60 µmol). The solution was mixed and a spectrum was measured. The reaction was then initiated by addition of L-cysteine (60 mM solution in DMF:H 2 O 1:1,15 µL, 0.90 µmol), the cuvettes were capped, shaken and the and spectra was measured. S1 is a known compound and was obtained according to the corresponding procedure as the reported mixture of diastereoisomers. [5] Wavelength nm. product S1 (0.2 mM) benzaldehyde (0.2 mM) reaction time 90 min Figure S 1 -Absorvance spectra for cysteine, benzaldehyde, S1 and at 90 min reaction time in reaction buffer.
[ Reaction buffer (2.99 mL) was pipetted into the cuvette, followed by 2FBBA (60 mM solution in DMF,10 µL, 0.60 µmol). The solution was mixed and a spectrum was measured. The reaction was then initiated by addition of L-cysteine (60 mM solution in DMF:H 2 O 1:1,15 µL, 0.90 µmol), the cuvettes were capped, shaken and the and spectra was measured.

Determination of rate constant
Reactions were performed under pseudo-first-order conditions in quartz cuvettes (50 mm path length; total volume ~3 mL) with solutions pre-equilibrated at room temperature (23 °C). Reaction buffer (3 ml) was pipetted into the cuvette, followed by 2-formylphenylboronic acid (15 µL, 3.00 µmol) stock solution.
The solution was mixed. The reaction was then initiated by addition of L-cysteine (in H 2 O) (10, 15, 20, 25 9 or 30 mM) (15 µL). The cuvettes were capped, shaken and the absorbance at 288 nm was measured repeatedly at 1 second intervals over 150 seconds.
We measure the reaction adding the following concentrations of L-cysteine (  To a solution of 2-formylphenylboronic acid (5.0 mg, 0.033 mmol) in a mixture of Acetate buffer 50mM (0.25 ml):D 2 O (0.25 ml) and DMF (0.025 ml) at pH = 7.4 was added L-cysteine (3.4 mg, 0.028 mmol). After the formation of the product at (about 90% conversion at 2min), new spectra were taken at 24h, 4 and 7days. No degradation of the product is observed. Compound 1 (2 mg, 8.51 µmol) was suspended in a mixture of Acetate buffer 50mM (0.25 ml):D 2 O (0.250 ml) and DMF (0.025 ml) at pH=4.5. And 1 H-NMR was measured at: 30 min, 24 h, 4 and 7 days. No degradation of 1 (with a characteristic peak at δ 6.31 (s, 1H)) is observed by the appearance of new products in the NMR, however the product did start to precipitate overtime, and in the 7 th day, almost no product was in solution. Compound 1 (2.0 mg, 8.5 µmol) was suspended in a mixture of Acetate buffer 50mM (0.25 ml):D 2 O (0.250 ml) and DMF (0.025 ml) at pH=9.0. And 1 H-NMR was measured at: 30 min, 24 h, 4 days and 7 days. Product 1 (with a characteristic peak at δ 6.31 (s, 1H)) disappear and the characteristic peak of 2FBBA start to emerge after only 30 min.

Computational Studies
All calculations were performed using the GAUSSIAN 09 software package, [6] and the M06-2X functional, without symmetry constraints. That is a hybrid meta-GGA functional developed by Truhlar and Zhao, [7] and it was shown to perform very well for main-group kinetics, providing a good description of long range effects such as van der Waals interactions or π-π stacking. [8,9] The optimized geometries were obtained with a standard 6-31G+(d,p) [10] basis set, accounting for solvent effects (water) by means of the Polarisable Continuum Model (PCM) initially devised by Tomasi and coworkers [11] with radii and nonelectrostatic terms of the SMD solvation model, developed by Truhler et al. [12] . Transition state optimizations were performed with the Synchronous Transit-Guided Quasi-Newton Method (STQN) developed by Schlegel et al, [13] following extensive searches of the Potential Energy Surface. Electronic energies were converted to free energy at 298.15 K and 1 atm by using zero point energy and thermal energy corrections based on structural and vibration frequency data calculated at the same level.

DFT calculated mechanism
The first part of the reaction corresponds to the formation of an imine intermediate resulting from the condensation of the amine group in Cys and the aldehyde carbonyl in the boronic acid. The model used for the calculations includes an explicit water molecule and the free energy profile obtained is represented in Figure S2 while a schematic illustration of the path is presented in Scheme S1.  The reaction starts with the formation of a N-C bond between the Cys amine group and the carbonyl Catom of 2FBBA, from A to B, in a process with a barrier of 8 kcal/mol and a slightly positive free energy balance (ΔG = 2 kcal/mol). In the next step, from B to C, there is protonation of the O-atom from the original carbonyl group while the carboxylic group of the amino acid is deprotonated. Thus, in C there is an ammonium ion (N + ) and a carboxylate group (COO -). In this step the neighbour water molecule acts as proton shuttle, resulting in a barrierless and exergonic process (ΔG = -7 kcal/mol). From C to C', there is a re-orientation of the water molecule and, then, in the following step (from C' to D) the N-atom of the ammonium ion in C' is deprotonated regenerating both the amine and the carboxylic groups, in D. This occurs with a small barrier (ΔG ‡ = 4 kcal/mol) and is an almost thermoneutral process. (ΔG = 1 kcal/mol). After a re-orientation of the water molecule and the corresponding change in the H-bonds network (from D to D'), the reaction proceeds with protonation of the OH group involving the O-atom from the original aldehyde. The proton involved is originated the carboxylic group (COOH) and results in the loss of one water molecule, from D' to E. This step has the highest barrier so far (ΔG ‡ = 10 kcal/mol) and is slightly exergonic (ΔG = -2 kcal/mol). In E there is an iminium ion and a carboxylate group, and two nearby water molecules: the one originally included in the model plus another, formed in the last step. A change in the H-bonds configuration transforms E into E' and, from here, the imine is produced in the last step of the mechanism (from E' to F) with deprotonation of the iminium ion and reprotonation of the carboxylate group. The proton transfer process is assisted by one neighbour water molecule and also by one of the OH groups attached to the boron atom, resulting in a barrier of ΔG ‡ = 7 kcal/mol and a free energy balance of ΔG = 1 kcal/mol. The overall mechanism for the formation of the imine intermediate is rather facile with a barrier of 11 kcal/mol (measured between C/C' and TS D'E ) and presents a favourable overall free energy balance of ΔG = -4 kcal/mol, as expected for a well known reversible reaction: formation of an imine from an amine and an aldehyde. 35 The second part of the mechanism goes from the imine to the final product, 1, and the corresponding path is represented in Figure S3 (free energy profile) and Scheme S2 (schematic representation).

36
The second part of the reaction mechanism starts with F', an imine similar to F but with a different conformation of the B(OH) 2 group. In the following step, from F' to G there is formation of a N-B bond and, thus, in G there is a new boron chelate 5-member ring (C 3 NB), an iminium group (C=N + ) and a tetravalent, formally negative, B-atom. This step as a negligible barrier of 3 kcal/mol and is slightly endergonic (ΔG = 2 kcal/mol). From G to G', there is a re-orientation of the -CH(COOH)CH 2 SH branch, attached to the N-atom, in a process that brings the S-atom and the C-atom of the iminium ion in the relative position necessary for the formation of the corresponding S-C bond, that will occur in the following step. Here, from G' to H, that bond is formed and, at the same time, there is transfer of a proton from the thiol to one of the OH groups bonded to the B-atom. Overall, in H, a second 5-member ring is formed, fused with the first one by the C-N bond, the nitrogen becomes an amine (tertiary), and there is one water molecule coordinated to the boron atom. The proton transfer from SH to OH is assisted by a neighbour water molecule, revealing once more the importance of the solvent molecules as shuttles in the proton exchange steps of the mechanism. Nevertheless, the barrier associated with this step (ΔG ‡ = 20 kcal/mol) is the highest of the path and the free energy balance indicates an endergonic process (ΔG = 7 kcal/mol). The subsequent step, from H to I, corresponds to dissociation of the water molecule from the boron coordination in an easy process with a barrier of only 2 kcal/mol and a favourable balance of ΔG = -6 kcal/mol. A re-orientation of the three water molecules and of the COOH group brings I to I'. In the following step, from I' to J, there attack of the carboxylic group on the trivalent B-atom with simultaneous proton loss. Thus, in J, there is a third 5-member ring (BNC 2 O) fused with the other two, forming another B-chelating ring. Also, in J, the proton lost by the carboxylic group is protonating a water molecule, forming an hydronium ion (H 3 O + ) stabilized by H-bonds to the two surrounding water molecules. The barrier for this step is 6 kcal/mol and the process is exergonic by 9 kcal/mol. Proton exchange between the free hydronium ion and the amine N-atom forms the final product, K, that correspond to 1 with three water molecules. Of these molecules, one was included in the initial model while the other two are reaction by-products resulting from the two condensation processes along the path: formation of the imine from the initial reagents, and coordination of the carboxylic group to the boron atom. The final process, from J to K, is very favourable, from the thermodynamic point a view, with a free energy balance of ΔG = -12 kcal/mol, reflecting the basicity of the amine N-atom. This stability of the final product provides the reaction the driving force, being due in good part to multiple B-coordination and the corresponding chelate effect existing in the product.
The reaction is clearly favourable, from the thermodynamic point of view, with a balance of ΔG = -19 kcal/mol, corroborating the observed stability of the product. Moreover, the calculated overall energy barrier is ΔG ‡ = 21 kcal/mol (measured from C/C' to TS G'H ) being slightly high for the experimental observations of a fast room temperature reaction. That difference is most probably due to the simplicity of model employed, with a reduced number of explicit water molecules in a reaction were proton transfer process are of major importance. Importantly, the B-atom as a two-fold role in the reaction mechanism. On one hand, it provides activation of the imine group by means of N-B coordination, promoting the formation of the S-C bond and, on the other, it provides stabilization of the final product through multiple boron-coordination and the corresponding chelate effect.