Facile t-boc and FMOC Amino Acid Chiral Separations by HPLC

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Facile t-bc and FMC Amino Acid Chiral Separations by PLC J. T. Lee, T. E. Beesley Advanced Separation Technologies 37 Leslie Court, P.. Box 297 Whippany, NJ 07981 USA Poster PLC 2002

Abstract Macrocyclic glycopeptide-based chiral stationary phases (CSPs) have become more popular due to their multimodal capability, broad selectivity and ruggedness. wing to their unique chiral ionic character, the enantiomer resolution of a wide variety of acids, bases and amphoteric racemates has been an easier task. Recently, with the advances of genomics/proteomics a huge demand for amino acids and peptides analysis has evolved. Specifically, the chiral analysis of N-blocked amino acids, i.e. t- BC and FMC, has become essential. There are three types of macrocyclic stationary phases suitable for this type of analysis. The unique phenomenon of complementary separations among these chiral stationary phases (vancomycin, teicoplanin, ristocetin A) renders them very effective tools for separating a wide variety of amino acids and N-blocked amino acids. A large number of t-bc and FMC amino acids have been tested, and baseline resolution is easily achieved for every racemate tested. In some cases, the selectivity/resolution can be greater than 5. The method development protocol and optimization methods are very straightforward in two mobile phase types, the polar organic phase and the reversed phase mode. The polar organic and reversed phase systems with the use of volatile buffers like ammonium trifluoroacetate and/or ammonium acetate are very compatible with LC/MS and LC/MS/MS platforms. The chiral recognition mechanisms involved in this type of chromatography will be proposed along with the rationale for optimization.

Proposed Structures of Glycopeptide CSPs 3 C N N 2 N N A Cl N N B Cl C 3 N 2 C 3 C N N C 2 N C 3 N N N CC 3 A B N C N C 3 D N C 3 N 2 Vancomycin Ristocetin A

Proposed Structures of Glycopeptide CSPs NR C 2 NCC 3 N C C 2 Cl N B N A N C Cl N N D N 2 C 2 Teicoplanin

N-FMC (9-Fluorenylmethyl Chlorophormate) Amino Acids Column: CIRBITIC T in Reversed Phase Mode Peak 1: 6.77 Peak 2: 11.57 Peak 1: 5.55 Peak 2: 6.73 Peak 1: 7.87 Peak 2: 10.30 Alanine Aspartic Acid Glutamic Acid Mobile Phase: 40/60: Me/0.1% TEAA, p=4.1

N-FMC (9-Fluorenylmethyl Chloroformate) Amino Acids Column: CIRBITIC T in Reversed Phase Mode Peak 1: 6.60 Peak 2: 13.33 Peak 1: 5.98 Peak 2: 12.49 Peak 1: 5.87 Peak 2: 12.88 Norleucine Norvaline Methionine Mobile Phase: 40/60: Me/0.1% TEAA, p=4.1

N-FMC (9-Fluorenethylmethyl Chloroformate) Amino Acids Column: CIRBITIC R in Polar rganic Mode Peak 1: 2.95 Peak 2: 4.40 Peak 1: 8.11 Peak 2: 13.66 Peak 1: 4.05 Peak 2: 5.16 Alanine Glutamine Isoleucine Mobile Phase: 100/0.1%: Me/N4TFA (LC/MS Compatible)

N-FMC (9-Fluorenethylmethyl Chloroformate) Amino Acids Column: CIRBITIC R in Polar rganic Mode Peak 1: 4.20 Peak 2: 5.61 Peak 1: 4.51 Peak 2: 7.17 Peak 1: 5.4 Peak 2: 9.90 Norleucine Norvaline Methionine Mobile Phase: 100/0.1%: Me/N4TFA (LC/MS Compatible)

N-FMC (9-Fluorenylmethyl Chloroformate) Amino Acids Column: CIRBITIC R in Reversed Phase Mode Peak 1: 7.66 Peak 2: 9.95 Peak 1: 5.79 Peak 2: 9.87 Peak 1: 6.22 Peak 2: 8.78 Asparagine Glutamine Serine Mobile Phase: 30/70: Me/20mM N4Ac (LC/MS Compatible)

N-FMC (9-Fluorenethylmethyl Chloroformate) Amino Acids Column: CIRBITIC R in Reversed Phase Mode Peak 1: 4.13 Peak 2: 7.44 Peak 1: 3.80 Peak 2: 7.68 Peak 1: 3.35 Peak 2: 6.07 Alanine Methionine Phenylalanine Mobile Phase: 50/50: Me/20mM N4Ac (LC/MS Compatible)

p Effect Column: CIRBITIC T FMC Methionine FMC Peak 1: 6.65 Peak 2: 13.14 FMC Peak 1: 4.57 Peak 2: 12.38 FMC + FMC-L-Methionine Peak 1: 3.68 Peak 2: 9.67 p 4.1 p 5.0 p 5.5 Mobile phase: 40/60: Me/20mM N4Ac

p Effect Column:CIRBITIC T FMC Aspartic acid Peak 1: 4.41 Peak 2: 5.38 Peak 1: 3.14 Peak 2: 3.60 FMC FMC p 4.1 p 5.0 Mobile Phase: 40/60: Me/20mM N4Ac

CIRBITIC T (Reversed Phase Mode) LC LC/MS Compatible Mobile Phase Example: FMC Alanine Mobile Phase: Me/Buffer*, 40/60 Peak 1: 6.77 Peak 2: 11.57 Peak 1: 6.56 Peak 2: 9.41 *Buffer: 0.1% TEAA, p=4.1 *Buffer: 20mM N4Ac, p=4.1

CIRBITIC T (Reversed Phase Mode) LC LC/MS Compatible Mobile Phase Example: FMC Leucine Me/ Buffer*, 40/60 Peak 1: 6.10 Peak 2: 10.58 Peak 1: 6.97 Peak 2: 11.88 *Buffer: 0.1% TEAA, p=4.1 *Buffer: 20mM N4Ac, p=4.1

CIRBITIC T vs R FMC Aspartic acid FMC Peak 1: 3.14 Peak 2: 3.60 Peak 1: 7.36 Peak 2: 9.62 Mobile phase: 40/60: Me/20mM N4Ac, p=5.0

N-t-BC Amino Acids Column: CIRBITIC R Peak 1: 6.26 Peak 2: 8.74 Peak 1: 7.79 Peak 2: 8.32 Peak 1: 6.13 Peak 2: 7.55 Alanine Asparagine Glutamine Mobile Phase: 20/80: Me/0.1 % TEAA, p=4.1

N-t-BC Amino Acids Column:CIRBITIC R Peak 1: 8.01 Peak 2: 9.24 Peak 1: 5.64 Peak 2: 6.42 Peak 1: 7.33 Peak 2: 8.43 Isoleucine Serine Valine Mobile Phase: 20/80: Me/0.1% TEAA, p=4.1

N-t-BC Amino Acids Column: CIRBITIC R Peak 1: 5.43 Peak 2: 6.32 Peak 1: 6.06 Peak 2: 13.24 Peak 1: 4.84 Peak 2: 10.12 istidine Phenylalanine Tryptophan Mobile Phase: 20/80: Me/0.1% TEAA, p=6.0

N-t-BC Amino Acids Column: CIRBITIC T Peak 1: 4.36 Peak 2: 5.10 Peak 1: 8.66 Peak 2: 10.08 Peak 1: 4.23 Peak 2: 4.69 Alanine Phenylalanine Glutamine Mobile Phase: 10/90: Me/0.1% TEAA, p=4.1

N-t-BC Amino Acids Column: CIRBITIC T Peak 1: 4.43 Peak 2: 8.62 Peak 1: 4.87 Peak 2: 10.94 Methionine Phenylglycine Mobile Phase: 20/80: Me/0.1% TEAA, p=4.1

Conclusions 1. f the three macrocyclic glycopeptides (CSPs) investigated, CIRBITIC T (Teicoplanin) and CIRBITIC R (Ristocetin A) are the best choices for FMC and t-bc amino acids. 2. For FMC amino acids, both reversed phase and polar organic mode work pretty well in most cases on CIRBITIC R while reversed phase is the best choice for CIRBITIC T. 3. For t-bc amino acids, reversed phase mode is the viable choice for both CIRBITIC T and R. 4. Both reversed phase and polar organic phase systems can be converted to LC/MS platforms. 5. It appears that carboxylate group of the analytes is the key interaction site with chiral amino group of the CSPs.

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