Complete Enantiomeric Separation of Phenylthiocarbamoylated Amino Acids on a Tandem Column of Reversed and Chiral Stationary Phases

Abstract

The enantiomeric separation of phenylthiocarbamoyl derivatives of amino acids (PTC-AAs) was studied on a series of reversed phase HPLC columns coupled to the chiral phase HPLC columns. First, the five chiral phases (native, 0.2, 3.3, 7.5 and 16.9 phenylcarbamoylated/β-cyclodextrins, Ph/CD) were newly prepared by modification of β-cyclodextrin with phenyl isocyanate and were examined for the enantiomeric separation of PTC-AAs. Among them, the 3.3Ph/CD phase gave the best enantiomeric separation (α ≥ 1.04). However, the separation of the individual PTC-AAs was not sufficient. Next, these separations were investigated on various reversed phase HPLC columns, and octyl silica was selected in terms of the suitability of the mobile phase adopted for the enantiomeric separation mentioned above. The effects of the column temperature, the ion-pairing reagent, and the final content of methanol were also studied on the tandem column of octyl silica and the 3.3Ph/CD phase. Under the best conditions (100 mM ammonium acetate (pH 6.5) containing 1 mM butanesulfonate with 0-40% methanol as the mobile phase), all the individual PTC-AAs were well separated within 150 min. The applicability of the method was demonstrated by the sequence/configuration analysis of a peptide containing a d-amino acid ([d-Thr(2)]leucine enkephalin-Thr).

Establishment of a Two-Dimensional HPLC-MS/MS Method Combined with DCl/D₂O Hydrolysis for the Determination of Trace Amounts of D-Amino Acid Residues in Proteins

“…An enantioselective sequencing method combining Edman degradation and enantioselective analysis of the produced PTH amino acids has also been reported [18]. For the screening of diastereomeric peptides containing D-amino acids, the use of an LC-MS/MS method is also effective [17].…”

“…Normally, the proteins were enzymatically digested, and the peptides containing the target D-amino acid residues are determined as diastereomers [16][17]. Edman degradation combined with enantioselective analysis of the produced PTH amino acids has also been reported [18]. However, these methods are complicated and applicable to limited protein/peptide samples having specific target sequences.…”

Interpretation on Band‐Broadening in Chromatography with Spatial Peak Profiles Obtained Using Whole‐Column Detection

“…Sometimes, peaks with larger retention times exhibit narrower widths than those with a smaller retention time in non-linear chromatography. [11][12][13][14] This can be explained by the fact that the higher solute migration speeds occur during the latter part of the elution at which the greater solvent strengths in LC or the higher temperatures in GC are encountered. Thus, the peak widths obtained on the chromatogram do not reflect how they may look on the spatial coordinate in the column.…”

Detection of DBD-Carbamoyl Amino Acids in Amino Acid Sequence and Configuration Determination of Peptides with Fluorogenic Edman Reagent 7-[(N,N-Dimethylamino)sulfonyl]-2,1,3- benzoxadiazol-4-yl Isothiocyanate

“…DBD-NCS was synthesized as previously described (13 (16). Pyridine and TFA were of amino acid sequencing grade (Wako Pure Chemicals, Japan).…”

“…The mobile phase for the ␤-CD series chiral columns were usually carried out by water with organic modifiers (16,28). Although most of the DBD-CA amino acid enantiomers could also be resolved on native ␤-CD or phenylcarbamoylated ␤-CD (Ph-CD) or ES-1/4 Ph-CD columns (16) with neutral mobile phases such as ammonium acetate/MeOH (data not shown), the peaks were broad and one or two DBD-CA amino acid enantiomers could not be separated under these conditions.…”