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  • The HRTOF MS of showed

    2021-09-24

    The HRTOF-MS of showed a quasi-molecular ion peak at / 525.3044 [M+Na], corresponding to a quasi-molecular formula of CHNONa (Calcd for CHNONa: 525.3053). IR absorptions at 1680 and 1522cm suggested the presence of amide functions. Based on an analysis of 1D and 2D NMR spectra, the presence of one phenylalanine (Phe) and two isoleucines (Ile) was easily deduced. The structure of the remaining amino apexbt chemical residue was determined from 2D NMR spectral data (): the COSY spectrum indicated the connectivities H-2″/H-3″/H-6″ and H-4″/H-5″. The chemical shifts of C-4″ ( 67.4) and H-4″ ( 3.88) showed that C-4″ was an oxymethine carbon. The HMBC correlations between H-2″/C-1″ and H-6′′/C-4′′confirmed the connections of C-1″/C-2″ and C-3′′/C-4′′. Thus, this amino acid residue was confirmed to be 4-hydroxyisoleucine (4-OH Ile). The HMBC correlations H-2″/C-4″, H-5″/C-3″ and H-6″/C-2″ support the 4-OH Ile fragment. We encountered a problem in determining the amino acid sequences; the four NH signals of appear as an overlapped broad hump at 7.40–7.90 (in DMF-), which made it impossible to use them for sequencing by HMBC spectroscopy. Therefore, N-methylation of was performed. Compound was treated with CHI and NaH in DMF at 0°C to give pentamethylated compound (30% after HPLC separation). The strong -Me signals of showed intense HMBC cross-peaks and the following HMBC correlations clarified the amino acid sequences of : NCH-2/C-2 and 1′′′, NCH-2′/C-1 and 2′, NCH-2′′/C-1′ and C-2′′ and NCH-2′′′/C-1′′ and 2′′′ (). The configurations of Phe and Ile were determined to be -Phe and -Ile by hydrolysis (6M HCl) followed by the advanced Marfey’s method. At the same time, the stereochemistry at the α-position of 4-OH Ile was also elucidated: HPLC of the - and -FDLA (1-fluoro-2,4-dinitrophenyl-5-leucineamide) derivatives of 4-OH Ile in the hydrolysates gave peaks at 6.69 and 10.48min, respectively, which established that the α-position of 4-OH Ile had an -configuration. When the hydrolysis mixture was purified using a strong cation exchange (SCX) resin and SCC, cyclo-4-hydroxy isoleucine () was obtained in 18% yield (). The coupling constants between H-2/H-3 and H-3/H-4 are 11.4 and 9.6Hz, respectively, which indicates the anti/anti-configuration of H-2/H-3/H-4. Thus, the absolute stereochemistry of was determined to be 2,3,4. (2,3,4)-Cyclo-4-hydroxyisoleucine was reported as a component of funebrine (), which was isolated from the plant . The coupling pattern of was identical to the data in the literature. Thus, the structure of was determined to be as shown in . The structures of compounds – were determined essentially in the same manner as described for and by comparison of their NMR data with those of (). To investigate the structure–activity relationship, we synthesized H-Ile-Phe-OH (linear tetrapeptide, ), cyclo-(Ile-Phe) (cyclic pentapeptide, ) and cyclo-(Ile-Phe) (cyclic hexapeptide, ) (). Compounds and were prepared via cyclization of the linear peptides, and , respectively. The glycine-uptake inhibitory activities and the GlyT1/GlyT2 selectivities of the isolated and synthesized peptides were measured () as previously reported. T98G cells (2×10cells/well) were plated in a 96-well plate and incubated at 37°C in a humidified incubator. After 24h, the cells were washed and incubated at room temperature with 250nM [H]-glycine and test sample. After 15min, the cells were washed and lysed with 0.5M NaOH, a scintillation cocktail was added, and the cell lysates were counted using a scintillation counter. The GlyT1/GlyT2 selectivities were also measured in the same way using COS7 cells transfected with rat GlyT1 or GlyT2. Of the tested samples, showed the most potent inhibitory activity with high GlyT1 selectivity. The linear tetrapeptide () showed weak glycine-uptake inhibitory activity in T98G. The cyclic pentapeptide () showed a moderate inhibitory activity, while cyclic hexapeptide () showed only weak activity. Apicidin (), a cyclic tetrapeptide that was reported as a histone deacetylase (HDAC) inhibitor, showed moderate inhibitory activity, but no selectivity was observed against GlyT subtypes. These results indicate that the cyclic tetrapeptide structure is important for strong inhibitory activity against GlyT, and GlyT1/GlyT2 selectivity is affected by the amino acid components.