Macathiohydantoin L, a Novel Thiohydantoin Bearing a Thioxohexahydroimidazo [1,5-a] Pyridine Moiety from Maca (Lepidium meyenii Walp.).

Five new thiohydantoin derivatives (1-5) were isolated from the rhizomes of Lepidium meyenii Walp. NMR (1H and 13C NMR, 1H-1H COSY, HSQC, and HMBC), HRESIMS, and ECD were employed for the structure elucidation of new compounds. Significantly, the structure of compound 1 was the first example of thiohydantoins with thioxohexahydroimidazo [1,5-a] pyridine moiety. Additionally, compounds 2 and 3 possess rare disulfide bonds. Except for compound 4, all isolates were assessed for neuroprotective activities in corticosterone (CORT)-stimulated PC12 cell damage. Among them, compound (-)-3 exhibited moderate neuroprotective activity (cell viability: 68.63%, 20 µM) compared to the positive control desipramine (DIM) (cell viability: 88.49%, 10 µM).

Bioactive constituents study of Pugionium cornutum L. Gaertn on intestinal motility.

Thirty-one compounds were obtained and identified from the dried roots of Mongolian medicinal and edible plant, Pugionium cornutum L. Gaertn. Eight of them were new ones, and named as pugcornols A (1), B (2), C (3), and D (4), pugcornosides A (5), B (6), C (7), and D (8), respectively. Among them, 1-4 were rare naturally occurring thiohydantoin derivatives. Meanwhile, all the isolates were determined for their activities on isolated mice jejunum contraction, and ten of them increased height of the spontaneous contractions. Moreover, we partly clarified the mechanism of 5-(methylsulfinyl)-pentanenitrile (9), a characteristic compound in P. cornutum by using different kinds of inhibitor.

A method for preparation of amino acid thiohydantoins from free amino acids activated by acetyl chloride for development of protein C-terminal sequencing.

A novel and efficient method to prepare amino acid thiohydantoins, which are required as reference standards for development of C-terminal protein sequencing, is reported. Amino acid thiohydantoins were prepared using a straightforward method involving reaction of 20 free amino acids with acetyl chloride as activating reagent and trimethylsilyl isothiocyanate (TMS-ITC) as derivatizing reagent. The products were characterized by HPLC, uv spectra, amino acid analysis, MS, and NMR. Different reaction conditions were investigated and the chemical mechanism of the formation of amino acid thiohydantoins was illustrated.

HPLC purification and separation of 5,5'-substituted-2,4-imidazolidinedithiones.

Several 5,5'-substituted-2,4-imidazolidinedithiones, synthesized from aldehydes or ketones, have been purified by HPLC using poly(styrene-divinylbenzene) packings. Purified 5,5'-substituted-2,4-imidazolidinedithiones have been identified in column effluent by UV absorbance and corroborated by mass spectrometry. Several silica-based, polymeric, and poly(styrene-divinylbenzene)-based packings were evaluated as matrices for resolution of a mixture of purified 5,5'-substituted-2,4-imidazolidinedithiones.

Microsequence analysis of peptides and proteins: trimethylsilylisothiocyanate as a reagent for COOH-terminal sequence analysis.

A reinvestigation of the isothiocyanate-based chemistry for cyclic degradations of peptides and proteins revealed that the reagent trimethylsilylisothiocyanate (TMS-ITC) gives superior results in terms of coupling efficiency and lack of complicating side reactions. Acetic anhydride (10 min at various temperatures) was used to activate the carboxyl terminus, and 6 N HCl (30 min at room temperature) was used for cleavage as originally described by G.R. Stark (Biochemistry 8, 4735, 1968). Reaction conditions for efficient coupling were explored using subtractive chemistry on bradykinin, a nonapeptide, and separation of the reaction products by reverse-phase high-performance liquid chromatography. The products were analyzed by fast atom bombardment-mass spectrometry and shown to be the N-acetylated starting material and the N-acetylated des-Arg9 derivative of bradykinin. The pseudo-first-order rate constants measured at 50, 70, and 90 degrees C were 5.6 X 10(-5), 5.1 X 10(-4), and 8.6 X 10(-4) s-1, respectively. In order to obtain complete couplings within 30-40 min at 50 degrees C, the effect of pyridine catalysis was studied. The addition of 0.225 M pyridine resulted in roughly doubling the rates at 50 and 70 degrees C. In the case of bradykinin, the reaction with TMS-ITC in the presence of the pyridine catalyst at 50 degrees C was complete in 15 min. In order to apply this methodology to the analysis of proteins, the thiohydantoin derivatives of amino acids were synthesized and separated by reverse-phase HPLC. The derivatives were also characterized by mass spectrometry. The above reaction conditions were tested on 3 nmol of sperm whale apomyoglobin for three cycles of degradation. The sample was first coupled to p-phenylene diisothiocyanate-derivatized aminopropyl glass with a 90% yield. The approximate initial yield of glycine at cycle one was 30%. The first three cycles corresponded exactly to the predicted carboxy-terminal sequence of myoglobin. These results demonstrate the feasibility of a new Stark reagent for automated carboxy-terminal chemistry.

A modified system for thiazolinone conversion to thiohydantoin derivatives and their separation by high-pressure liquid chromatography.

An improved and very simple procedure for thiazolinone conversion to thiohydantoin derivatives and their separation by reverse-phase high-pressure liquid chromatography is described. Trifluoroacetic acid (10%) in ethyl acetate has been employed as a conversion reagent to circumvent the deamidation of acid amides and methylation of acidic amino acids, with a concomitant increase in the detection limits of these residues. Additionally, a very simple procedure has been developed for the separation of phenylthiohydantoin (PTH) derivatives of amino acids. The system takes advantage of the computer-controlled precise mixing of the solvents A and B to achieve accurate pH and thus avoid the necessity of pH adjustment of a buffer. The procedure is simple and highly reproducible, and separates all the 20 known PTH amino acids. The efficiency of the method has been examined on synthetic and natural proteins/peptides, in manual and autoconversion systems, over a period of more than 18 months.

Separation of dimethylaminoazobenzenethiohydantoin amino acids by high-performance liquid chromatography at low picomole concentrations.

The separation of all common dimethylaminoazobenzenethiohydantoin (DABTH) amino acids derived from modified Edman sequencing can be achieved by using high-performance liquid chromatography. All derivatives, including DABTH-Ile and DABTH-Leu, can be readily separated in a solvent mixture of sodium acetate buffer and 1% ethylene dichloride in acetonitrile. The high absorbance of the DABTH amino acids at 436 nm makes possible the quantitative determination of these derivatives at picomole concentrations in a relatively short time (30-40 min).

Separation of phenylthiohydantoin-amino acids by high-pressure liquid chromatography.

A method for the separation of all phenylthiohydantoin (PTH)-amino acids except PTH-arginine and PTH-histidine by high-pressure liquid chromatography on a silica column is described. Elution is performed with a concave solvent gradient from hexane-methanol-propanol (3980:9:11) to methanol-propanol (9:11). A complete run is achieved in 40 min with a pressure drop of 1000 p.s.i. over the 250 mm X 2.1 mm column. Eluted peaks of 2-5 nmole are easily detected by their ultraviolet absorption at 254 nm. This method is superior to existing gas-liquid and thin-layer chromatographic techniques since all PTH-amino acids except PTH-arginine and PTH-histidine may be both separated and quantitated in a single run of 40 min. The use of the technique in conjunction with an automated peptide sequence analyser is illustrated.