LK-3, an amphipathic
dimeric peptide linked by two disulfide bonds,
and related isomeric bundles were synthesized, and their cell penetrating
abilities were investigated. The measurements using size exclusion
chromatography and dynamic light scattering show that LK-3 and its
isomers form cell penetrating oligomers. Calculations, performed for
various types of peptide isomers, elucidate a strong correlation between
the amphipathic character of dimers and cell penetration ability.
The results suggest that the amphipathicities of LK-3 and related
bundle dimers are responsible for their oligomerization propensities
which in turn determine their cell penetrating abilities. The observations
made in this study provide detailed information about the mechanism
of cell uptake of LK-3 and suggest a plausible insight of the early
stage of nanoparticle formation of the cell penetrating amphipathic
peptides.
Candida antarctica lipase B (CAL-B) exhibits remarkable enantioselectivity for various chiral sec-alcohols, and the enantioselectivity is structurally well-understood. Two substituents at the chiral center of a sec-alcohol separately bind two pockets, namely, large and medium binding pockets. It has been believed that the medium pocket is too small to accommodate a large substituent (larger than an ethyl group), and thus, bulky secalcohols bearing two large substituents have been regarded as a poor substrate for CAL-B. However, we found that CAL-B can catalyze the transesterification of N-Boc-protected rac-2-amino-1-phenylethanol (1a) enantioselectively with a moderate reaction rate. X-ray crystallography and computer modeling revealed that the rotation of the Leu278 side chain creates a space to accept the N-Boc-aminomethylene group of 1a. Moreover, a sec-alcohol substrate with less than one hydrogen atom at the γ-position from the hydroxyl group is required to achieve a moderate reaction rate. On the basis of this observation, we diversified bulky N-Boc-protected rac-2-amino-1-arylethanols for the transesterifications with high enantioselectivities (E > 200).
Most lipases resolve secondary alcohols in accordance with the "Kazlauskas rule" to give the R enantiomers. In a similar manner to other lipases, Candida rugosa lipase (CRL) exhibits R enantioselectivity towards heptan-2-ol, although the enantiomeric ratio (E) is low (E=1.6). However, unexpected enantioselectivity (i.e., S enantioselectivity, E=58) of CRL towards 4-(tert-butoxycarbonylamino)butan-2-ol, which has a similar chain length to heptan-2-ol, has been observed. To develop a deeper understanding of the molecular basis for this unusual enantioselectivity, we have conducted a series of molecular modeling and substrate engineering experiments. The results of these computational and experimental analyses indicated that a hydrogen bond between the Ser450 residue and the nitrogen atom of the carbamate group is critical to stabilize the transition state of the S enantiomer.
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