Highly fluorinated amino acids have been used to stabilize helical proteins for potential application in various protein-based biotechnologies. To gain further insight into the effect of these highly fluorinated amino acids on helix formation exclusively, we measured the helix propensity of three highly fluorinated amino acids: (S)-5,5,5,5',5',5'-hexafluoroleucine (Hfl), (S)-2-amino-4,4,4-trifluorobutyric acid (Atb), and (S)-pentafluorophenylalanine (Pff). We have developed a short chemoenzymatic synthesis of Hfl with extremely high enantioselectivity (>99%). To measure the helix propensity (w) of the amino acids, alanine-based peptides were synthesized, purified, and investigated by circular dichroism spectroscopy (CD). On the basis of the CD data, the helix propensity of hydrocarbon amino acids can decrease up to 24-fold (1.72 kcal.mol-1.residue-1) upon fluorination. This difference in helix propensity has previously been overlooked in estimating the magnitude of the fluoro-stabilization effect (which has been estimated to be 0.32-0.83 kcal.mol-1.residue-1 for Hfl), resulting in a gross underestimation. Therefore, the full potential of the fluoro-stabilization effect should provide even more stable proteins than the fluoro-stabilized proteins to date.
Chan-Lam coupling is one of the most popular and easy methods to perform arylation of amines (N-arylations). This cross-coupling is generally performed by reacting aryl boronate derivatives with a variety of substrates involving nitrogen containing functional groups such as amines, amides, ureas, hydrazine, carbamates. This article summarizes the synthetic applications of this reaction and the efforts of scientists to develop novel and efficient methodologies for this reaction.
Ion-pairing interactions are important for protein stabilization. Despite the apparent electrostatic nature of these interactions, natural positively charged amino acids Lys and Arg have multiple methylenes linking the charged functionality to the backbone. Interestingly, the amino acids Lys and Orn have positively charged side chains that differ by only one methylene. However, only Lys is encoded and incorporated into proteins. To investigate the effect of side chain length of Lys on ion-pairing interactions, a series of 12 monomeric alpha-helical peptides containing potential Glu-Xaa (i, i+3), (i, i+4) and (i, i+5) (Xaa = Lys, Orn, Dab, Dap) interactions were studied by circular dichroism (CD) spectroscopy at pH 7 and 2. At pH 7, no Glu-Xaa (i, i+5) interaction was observed, regardless of the Xaa side chain length. Furthermore, only Lys was capable of supporting Glu-Xaa (i, i+3) interactions, whereas any Xaa side chain length supported Glu-Xaa (i, i+4) interactions. Side chain conformational analysis by molecular mechanics calculations showed that the side chain length of Lys enables the Glu-Xaa (i, i+3) interaction with lower energy conformations compared to residues with side chain lengths shorter than that of Lys. Furthermore, these calculated low energy conformers were consistent with conformations of intra-helical Glu-Lys salt bridges in a non-redundant protein structure database. Importantly, the CD spectra for peptides with Glu-Lys interactions did not alter significantly upon changing the pH because of a greater contribution to these interactions by forces other than electrostatics. Incorporating side chains just one methylene shorter (Orn) resulted in significant pH dependence or lack of interaction, suggesting that nature has chosen Lys to form durable interactions with negatively charged functional groups.
On-chip droplet splitting is one of the fundamental droplet-based microfluidic unit operations to control droplet volume after production and increase operational capability, flexibility, and throughput. Various droplet splitting methods have been proposed, and among them the acoustic droplet splitting method is promising because of its label-free operation without any physical or thermal damage to droplets. Previous acoustic droplet splitting methods faced several limitations: first, they employed a cross-type acoustofluidic device that precluded multichannel droplet splitting; second, they required irreversible bonding between a piezoelectric substrate and a microfluidic chip, such that the fluidic chip was not replaceable. Here, we present a parallel-type acoustofluidic device with a disposable microfluidic chip to address the limitations of previous acoustic droplet splitting devices. In the proposed device, an acoustic field is applied in the direction opposite to the flow direction to achieve multichannel droplet splitting and steering. A disposable polydimethylsiloxane microfluidic chip is employed in the developed device, thereby removing the need for permanent bonding and improving the flexibility of the droplet microfluidic device. We experimentally demonstrated on-demand acoustic droplet bi-splitting and steering with precise control over the droplet splitting ratio, and we investigated the underlying physical mechanisms of droplet splitting and steering based on Laplace pressure and ray acoustics analyses, respectively. We also demonstrated droplet tri-splitting to prove the feasibility of multichannel droplet splitting. The proposed on-demand acoustic droplet splitting device enables on-chip droplet volume control in various droplet-based microfluidic applications.
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