A helical chain being the most common secondary structure in biopolymers prefers one-handed (left-or right-handed) screw sense, when chiral moieties are incorporated into the main or side chain through the covalent bond. We here report that an achiral helical peptide prefers the one-handed helical screw sense by noncovalent interaction of its N-terminal amino group with a chiral carboxylic acid. Little is known about such phenomena in peptide systems typical of biopolymers, although it has been reported that synthetic helical polymers bearing carboxyl or amino groups in the repeating units induce the one-handed screw sense by addition of chiral small amines or acids to interact on their polymer side or main chains. 1 In our system, the acid-base interaction occurring in the N-terminal position of the peptide chain will lead to the predominance of one-handed screw sense of the entire peptide chain, namely through domino effect. For our purpose, the following N-deprotected nonapeptide 1 consisting of nonprotein amino acids [R-aminoisobutyric acid (Aib) and R, -dehydrophenylalanine (∆ Z Phe)] was synthesized. 2 Peptide 1 can be expected to generate two "enantiomeric" (leftand right-handed) helices, since Aib and ∆ Z Phe residues are achiral ones and strong inducers for forming a 3 10 -helix. 3 Actually, a helical conformation was evidenced by 1 H NMR spectroscopy on peptide 1 in CDCl 3 . In the NOESY experiment, marked crosspeaks were observed for the N i H-N i+1 H resonances in the segment of Aib(3) to Aib(9), 2 indicating that peptide 1 forms a 3 10 -or R-helix. 4 The solvent dependence on amide NH chemical shifts in CDCl 3 /(CD 3 ) 2 SO mixtures revealed that six NH resonances of ∆ Z Phe(4) to Aib(9) residues are shielded from solvent due to intramolecular hydrogen bonding, 2 of which the pattern corresponds to a 3 10 -helix. The helical conformation was also supported by the amide I absorption bands of its FT-IR spectrum in chloroform: 1660 and 1627 cm -1 , which can be assigned to saturated amino acid and ∆ Z Phe residues in a helical segment, respectively. 5 Furthermore, energy minimization of peptide 1 by the semiempirical molecular orbital method 6 gave a 3 10 -helical conformation ( Figure 1) characterized by 〈φ〉 ) (41.1°, 〈ψ〉 ) (37.6°, and 〈ω〉 ) 180.0°for average values of ∆ Z Phe(2) to ∆ ZPhe(8) residues, and the main-chain energy contour map was severely restricted into right-and left-handed helical regions (φ ) (60 to (40°, ψ ) (60 to (30°). 2 Therefore, peptide 1 having a strong helix-forming tendency forms a 3 10 -helical conformation in chloroform. 7 Peptide 1 could not show any CD signals due to the absence of chiral residues, thus taking both left-and right-handed helices with the same content in an equilibrium state (Figure 2, dotted line). However, intense split CD signals were induced around 282 nm assignable to ∆ Z Phe residues by the addition of enantiomerically pure Boc-L-Pro-OH (Boc ) t-butoxycarbonyl), as shown in Figure 2. The mirror image was obtained by the addition of Boc-D-Pro-OH, th...

Elastomeric vitrimer materials with tunable cross-link densities are prepared using cross-linking precursor polyesters with multiple COOH side groups in the presence of diepoxy cross-linkers and trans-esterification catalysts.

A diene-based cyclic polymer has been synthesized by the anionic polymerization of methyl sorbate (MS) by an N-heterocyclic carbene (NHC) in the presence of a bulky aluminum Lewis acid. We first polymerized methyl sorbate (MS) initiated by NHC in N,N-dimethylformamide (DMF) at 25 °C, poly(MS) with a number-average molecular weight (M) of 3.5 × 10 (M/M = 2.1) was obtained with a conversion of 93%. The structure was confirmed by H andC NMR and IR spectra, which revealed that the propagation proceeded via 1,2-addition as well as 1,4-addition. Although the polymerization did not occur in toluene in the absence of any additive, quantitative monomer consumption was observed in the presence of methylaluminum bis(2,6-di-tert-butyl-4-methylphenoxide) (MAD) to afford the poly(MS) with a 1,4-trans structure, 86% of threo diastereoselectivity, and a M of 23.0 × 10 with narrow molecular weight distribution (M/M = 1.1). From the matrix assisted laser desorption/ionization (MALDI-TOF) mass spectra of poly(MS) and the hydrogenated analogue, ring-closing occurred by nucleophilic attack of the anionic propagating center into the adjacent carbon of the α-terminal imidazolimium group to afford cyclic poly(MS). The cyclic formation in the present synthesis system was confirmed by DSC and viscosity measurements.

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Molecules essential for the induction of immunogenic cell death (ICD) are called damage-associated molecular patterns (DAMPs). The effects of oncolytic herpes simplex virus type 1 (HSV-1) on the production of DAMPs were examined in squamous cell carcinoma (SCC) cells. The cytopathic effects of HSV-1 RH2 were observed in mouse SCCVII cells infected at a high multiplicity of infection (MOI), and the amounts of viable cells were decreased. After being infected with RH2, ATP and high mobility group box 1 (HMGB1) were released extracellulary, while calreticulin (CRT) translocated to the cell membrane. A flow-cytometric analysis revealed an increase in the number of annexin-V and propidium iodide (PI)-stained cells; and the amount of cleaved poly (ADP-ribose) polymerase (PARP) was increased. The killing effect of RH2 was reduced by pan-caspase inhibitor z-VAD-fmk and the caspase-1 inhibitor z-YVAD-fmk, suggesting the involvement of apoptosis and pyroptosis. In C3H mice bearing synergic SCCVII tumors, the growth of tumors injected with the supernatant of RH2-infected cells was less than that of tumors injected with phosphate-buffered saline (PBS). These results indicate that oncolytic HSV-1 RH2 produces DAMPs from SCC cells to induce cell death. This may contribute to the enhancement of tumor immunity by oncolytic HSV-1.

Vacuolar H+-translocating inorganic pyrophosphatase is a single-protein enzyme and uses a simple substance as an energy donor. Functional domains of the enzyme were investigated by using antibodies specific to peptides corresponding to the putative substrate-binding site (DVGADLVGKVE) in the hydrophilic loop and the carboxyl terminal part. The antibody to the former peptide clearly reacted with the pyrophosphatases of different plant species, and strongly inhibited the hydrolytic activity of the purified enzymes and the proton pumping activity of membrane vesicles. These results indicate that the sequence functions as an actual substrate-binding site and is a common motif. The antibody to the carboxyl terminal part reacted only to the mung bean enzyme, suppressing its hydrolytic and proton pumping activities. The results suggest that the carboxyl terminus is exposed to the cytosol and is close to the catalytic site. H+-Pyrophosphatase hydrolyzed triphosphate and tetraphosphate at low rates. Phytic acid, myo-inositol hexaphosphate, inhibited the enzyme even in the presence of Mg2+. The concentration for 50% inhibition was 0.15 mM. The inhibition of H+-PPase by dicyclohexyldiimide was partly reversed by Mg2+. The catalytic site and the membrane topology of the enzyme are discussed.

Therapeutic proteins are indispensable in treating numerous human diseases. However, therapeutic proteins often suffer short serum half-life. In order to extend the serum half-life, a natural albumin ligand (a fatty acid) has been conjugated to small therapeutic peptides resulting in a prolonged serum half-life via binding to patients' serum albumin in vivo. However, fatty acid-conjugation has limited applicability due to lack of site-specificity resulting in the heterogeneity of conjugated proteins and a significant loss in pharmaceutical activity. In order to address these issues, we exploited the site-specific fatty acid-conjugation to a permissive site of a protein, using copper-catalyzed alkyne-azide cycloaddition, by linking a fatty acid derivative to p-ethynylphenylalanine incorporated into a protein using an engineered pair of yeast tRNA/aminoacyl tRNA synthetase. As a proof-of-concept, we show that single palmitic acid conjugated to superfolder green fluorescent protein (sfGFP) in a site-specific manner enhanced a protein's albumin-binding in vitro about 20 times and the serum half-life in vivo 5 times when compared to those of the unmodified sfGFP. Furthermore, the fatty acid conjugation did not cause a significant reduction in the fluorescence of sfGFP. Therefore, these results clearly indicate that the site-specific fatty acid-conjugation is a very promising strategy to prolong protein serum half-life in vivo without compromising its folded structure and activity.

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