Synthesis and Studies of Bulky Cycloalkyl α,ß-Dehydroamino Acids that Enhance Proteolytic Stability.

Three new bulky cycloalkyl α,ß-dehydroamino acids (ΔAAs) have been designed and synthesized. Each residue enhances the rigidity of model peptides and their stability to proteolysis, with larger ring sizes exhibiting greater effects. Peptides containing bulky cycloalkyl ΔAAs are inert to conjugate addition by a nucleophilic thiol. The results suggest that these residues will be effective tools for improving the proteolytic stability of bioactive peptides.

Synthesis and evaluation of potent yaku'amide A analogs.

Two full-length analogs of the anticancer peptide yaku'amide A (1a) and four partial structures have been synthesized. These analogs were identified by computational studies in which the three E- and Z-ΔIle residues of the natural product were replaced by the more accessible dehydroamino acids ΔVal and ΔEnv. Of the eight possible analogs, modeling showed that the targeted structures 2a and 2b most closely resembled the three-dimensional structure of 1a. Synthesis of 2a and 2b followed a convergent route that was streamlined by the absence of ΔIle in the targets. Screening of the compounds against various cancer cell lines revealed that 2a and 2b mimic the potent anticancer activity of 1a, thereby validating the computational studies.

Multi-CRAFTI: Relative Collision Cross Sections from Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Line Width Measurements.

Determination of collision cross sections (CCS) using the cross-sectional areas by the Fourier transform ion cyclotron resonance (CRAFTI) technique is limited by the requirement that accurate pressures in the trapping cell of the mass spectrometer must be known. Experiments must also be performed in the energetic hard-sphere regime such that ions decohere after single collisions with neutrals; this limits application to ions that are not much more massive than the neutrals. To mitigate these problems, we have resonantly excited two (or more) ions of different m/z to the same center-of-mass kinetic energy in a single experiment, subjecting them to identical neutral pressures. We term this approach "multi-CRAFTI". This facilitates measurement of relative CCS without requiring knowledge of the pressure and enables determination of absolute CCS using internal standards. Experiments with tetraalkylammonium ions yield CCS in reasonable agreement with the one-ion-at-a-time CRAFTI approach and with ion mobility spectrometry (IMS) when differences in collision energetics are taken into account (multi-CRAFTI generally yields smaller CCS than does IMS due to the higher collision energies employed in multi-CRAFTI). Comparison of multi-CRAFTI and IMS results with CCS calculated from structures computed at the M06-2X/6-31+G* level of theory using projection approximation or trajectory method values, respectively, indicates that the computed structures have CCS increasingly smaller than the experimental CCS as m/z increases, implying the computational model overestimates interactions between the alkyl arms. For ions that undergo similar collisional decoherence processes, relative CCS reach constant values at lower collision energies than do absolute CCS values, suggesting a means of increasing the accessible upper m/z limit by employing multi-CRAFTI.

Towards a streamlined synthesis of peptides containing α,ß-dehydroamino acids.

Investigation of a strategy to streamline the synthesis of peptides containing α,ß-dehydroamino acids (ΔAAs) is reported. The key step involves generating the alkene moiety via elimination of a suitable precursor after it has been inserted into a peptide chain. This process obviates the need to prepare ΔAA-containing azlactone dipeptides to facilitate coupling of these residues. Z-dehydroaminobutyric acid (Z-ΔAbu) could be constructed most efficiently via EDC/CuCl-mediated dehydration of Thr. Formation of Z-ΔPhe by this or other dehydration methods was unsuccessful. Production of the bulky ΔVal residue could be accomplished by DAST-promoted dehydrations of ß-OHVal or by DBU-triggered eliminations of sulfonium ions derived from penicillamine derivatives. However, competitive formation of an oxazoline byproduct remains problematic.

Convergent Total Synthesis of Yaku'amide†A.

Total synthesis of the anticancer peptide natural product yaku'amide A is reported. Its ß-tert-hydroxy amino acids were prepared by regioselective aminohydroxylation involving a chiral mesyloxycarbamate reagent. Stereospecific construction of the E- and Z-ΔIle residues was accomplished through a one-pot reaction featuring anti dehydration, azide reduction, and O→N acyl transfer. Alkene isomerization was negligible during this process. These methods enabled a highly convergent and efficient synthetic route to the natural product.

Impact of Dehydroamino Acids on the Structure and Stability of Incipient 310-Helical Peptides.

A comparative study of the impact of small, medium-sized, and bulky α,ß-dehydroamino acids (ΔAAs) on the structure and stability of Balaram's incipient 310-helical peptide (1) is reported. Replacement of the N-terminal Aib residue of 1 with a ΔAA afforded peptides 2a-c that maintained the 310-helical shape of 1. In contrast, installation of a ΔAA in place of Aib-3 yielded peptides 3a-c that preferred a ß-sheet-like conformation. The impact of the ΔAA on peptide structure was independent of size, with small (ΔAla), medium-sized (Z-ΔAbu), and bulky (ΔVal) ΔAAs exerting similar effects. The proteolytic stabilities of 1 and its analogs were determined by incubation with Pronase. Z-ΔAbu and ΔVal increased the resistance of peptides to proteolysis when incorporated at the 3-position and had negligible impact on stability when placed at the 1-position, whereas ΔAla-containing peptides degraded rapidly regardless of position. Exposure of peptides 2a-c and 3a-c to the reactive thiol cysteamine revealed that ΔAla-containing peptides underwent conjugate addition at room temperature, while Z-ΔAbu- and ΔVal-containing peptides were inert even at elevated temperatures. These results suggest that both bulky and more accessible medium-sized ΔAAs should be valuable tools for bestowing rigidity and proteolytic stability on bioactive peptides.