# Roles of D-Amino Acids on the Bioactivity of Host Defense Peptides
## 5 Mechanisms of Bioactivities
### Influence of Secondary Structure on Bioactivity  
Currently, the vast majority of studies dedicated to the mechanism of d-AA incorporation into HDPs had been performed on linear peptides with helical propensities and on peptides targeting either bacteria or, to a limited extent, cancer. [PERSON] et al. [96] had investigated the role of helicity and hydrophobicity on the bioactivity of anticancer peptides by showing that d-AA can be used to improve peptide specificity toward cancer cells, particularly by substituting them on the polar face of the helix to modulate their cytotoxicity while its placement on the non-polar face could still maintain the anticancer activity. Similarly, [PERSON] et al. [97] had also extended their investigation to studythe role of helicity and hydrophobicity in \(\alpha\)-helical AMP by showing that d-AA substitution at the non-polar face led to stronger antimicrobial activity and lower toxicity owing to the stronger preference toward bacterial cells. Particularly, it was found that high helicity led to stronger antimicrobial activity whereas disruption of the helicity led to decrease in the hydrophobicity and lower hemolytic activity. Both studies revealed that the incorporation of d-AA into the studied peptides affected the helicity and consequently influencing the hydrophobicity and its resulting bioactivity. Thus far, there have been virtually no studies on the detailed insights into the mechanism of d-AA-containing peptides with other types of structures and targets in spite of the fact that these peptides have already been synthesized and their activities tested, such as in the case of d-AA-containing peptides having \(\beta\)-hairpin [48] or \(\beta\)-sheet structures [9]. Two available studies had provided a rudimentary look at the potential challenges of understanding the mechanism of d-AA-containing HDPs with structures other than linear [98; 99], namely the cyclic polymxin B (i.e., a natural AMP with a ring structure attached to a largely linear tail). It was found via fluorescence quenching and colorimetry that the ring structure crucially affected both binding to the membrane surface as well as depth of insertion into the membrane. The diastereomer analogues differed from the native all-l form in only the chirality of the ring structure. However, when compared to the all-l peptides the diastereomers binds strongly to the lipid head groups and did not insert deeply into the membrane, which is consistent with the observations for linear, helical peptides. However, instead of preserving antimicrobial activity, the incorporation of d-AAs nearly inactivated the activity of cyclic diastereomer. No explanation was provided, but this result calls into question whether conclusions drawn from d-AA modification of linear peptides can also be extrapolated to the cyclic form. Another work that provided some insight into the mechanism of non-helical diastereomers was carried out by [PERSON] et al. [48], who synthesized an 18-residue diastereomer composed primarily of alternating Val and Lys. The diastereomer exhibited membrane lytic activity against a number of cancer cell lines and had low cytotoxicity against normal mammalian cells such as human umbilical vein endothelial cell (HUVEC). Instrumental analysis and liposomal fluorescence dye leakage assay revealed that the diastereomer was unstructured in solution but adopted a \(\beta\)-hairpin structure upon contact with the negatively-charged membrane. The peptide was unstructured in solution because the absence of compensatory interactions and the high density of positively-charged Lys residues resulted in intrastrand charge repulsion and prevented the N- and C-terminal strands from folding into \(\beta\)-hairpins. However, once bound to the negatively-charged membranes, the positively-charged Lys were neutralized owing to formation of ion pairs thereby allowing the terminal strands to collapse and form the hairpin structure.