Amino Acids and Their Derivatives

A broad variety of racemic amino acids has already been resolved by chiral TLC. Table 1 summarizes the analytical separations achieved in this field on 20 cm x 20 cm Chiralplates (Cat. No. 811058, Ma-cherey-Nagel; thickness 0.25 mm). The separations can be easily transferred to the 10 cm x 10 cm HPTLC-CHIR plates with concentrating zone since they are precoated with the same chiral selector.

With eluents A, B and C, 2 |L of a 1% solution of the racemates in methanol or methanol-water and with eluent D, 2 | L of a 0.5% solution of the ra-cemates in 0.1 molL"1 hydrochloric acid-methanol 1 : 1 were applied to the plates. Migration time increases from 0.5 h (eluent A) to 1.5 h (eluent C). Detection was performed by dipping the plates for 3 s in a 0.3% ninhydrin solution in acetone and then heating at 110°C for c. 5 min. Red spots appear on a white background.

The amount of solute applied to the plates (10-20 |g) is an order of magnitude greater than that generally employed in TLC; the use of HPTLC-CHIR layers improves the sensitivity of the method.

Thus far, 84 proteinogenic and nonproteinogenic amino acids have been separated without derivatiz-ation using mostly methanol-water-acetonitrile (50 : 50 : 200, v/v/v) as eluent. Usually the d enan-tiomer is the more retained. Racemic serine shows low resolution, while threonine and basic amino acids have not been resolved as yet.

The separations of enantiomeric amino acids reported in Table 2 using a variety of chiral selectors are very interesting since they also include the unresolved compounds mentioned above. Round, compact spots are generally obtained on silica gel plates eluted with 2-0-[(R)-2-hydroxypropyl]-P-cyclodex-trin solutions as deduced from Rs values which are equal to or higher than the a value for all the amino acids with the only exception of dl-citrulline (Rs = 0.94). Visualization is performed by spraying the plates with a salicyladehyde solution (1.5 g) in 100 mL toluene and then heating at 50°C for 10 min (yellow spots).

Table 3 gives the performance of cellulose plates, which are very effective in resolving racemates of aromatic and basic amino acids. Home-made layers of microcrystalline cellulose powder (commercially available from Merck, and Fluka) can be obtained with optimal homogeneity by spreading an aqueous suspension with about 25% chiral material. The plates are dried at room temperature and do not require activation before use. Polar mixtures (i.e. ethanol-pyridine-water) are the best eluents since they separate enantiomers as efficiently as an aqueous solvent (i.e. 0.1 mol L"1 NaCl) but give rise to more compact spots.

Chiralplates were very effective in resolving ra-cemates of N-alkyl, N-carbamyl and N-formyl amino acids and of several dipeptides (Table 4). It is worth noting that the dipeptide with the C-terminal l-con-figuration always has a higher retention than the one with the C-terminal d-configuration. Some racemic dipeptides were also resolved on microcrystalline cellulose with pyridine-water (2 : 1 or 4 : 1) and on SIL C18-50/UV254 plates using BSA as chiral mobile phase additive.

Derivatization of amino acids may be used to improve chiral recognition, detectability and sensitivity of the method and to label amino acids residues of peptides and proteins, especially the N-terminal amino acid. Dimethylaminonaphthalenesulfonyl (dansyl) amino acids form when primary and secondary amino acids react with dansyl chloride, generating strongly fluorescent compounds. The best chromatographic conditions for their separation are reported in Table 5. The most complete study, performed on KC18 F plates (Whatman) using ^-CD as chiral agent, concerns the racemates of 26

Table 1 Enantiomeric separation of proteinogenic and nonproteinogenic amino acids on Chiralplatesa

Racemate

hRF1b

hRF2

ac

Eluentd

Ala

69(D)

73

1.22

D

Ser

73(D)

76

1.17

D

Abu

48

52

1.17

A

Val

54(D)

62

1.39

A

Nva

49(D)

56

1.32

A

Leu

53(D)

63

1.51

C

Ile

47(D)

58

1.55

A

Nle

53(D)

62

1.44

A

allo-Ile

51(D)

61

1.50

A

t-Leucine

40(D)

51

1.56

A

Met

54(D)

59

1.23

A

Eth

52(D)

59

1.32

A

Pro

41(D)

47

1.27

A

cis-Hype

41(L)

59

2.08

A

allo-Hyp

41(L)

59

2.08

A

Pipecolic acid

51

58

1.32

D

Phe

49(D)

59

1.49

A

Homophenylalanine

49(D)

58

1.43

A

Tyr

58(D)

66

1.40

A

Dopa

47(L)

58

1.55

B

Trp

51(D)

61

1.50

A

Asp

50(D)

55

1.22

A

Glu

54(D)

59

1.22

A

Gln

41(L)

55

1.76

A

Thyroxine

38(D)

49

1.56

A

PhenylGly

57(D)

67

1.53

A

CyclopentylGly

43

50

1.32

A

(l-Methylcyclopropyl)-Gly

49

57

1.38

A

(2-Thienyl)-Gly

55

66

1.58

A

3-Cyclopentyl-Ala

46

56

1.49

A

3-(1-Naphthyl)-Ala

49(D)

56

1.32

A

3-(2-Naphthyl)-Ala

44(D)

59

1.82

A

Met(O2)f

62(D)

66

1.18

A

Eth(O2)

55

59

1.18

A

Seleno-Met

53(D)

61

1.38

A

S-Methylthio-Cys

47(D)

55

1.38

A

S-Methylthio-HomoCys

44

52

1.38

A

S-(2-Chlorobenzyl)-Cys

45

58

1.68

A

S-(3-Thiobutyl)-Cys

53

64

1.58

A

S-(2-Thiopropyl)-Cys

53

64

1.58

A

O-Benzyl-Ser

54(D)

65

1.58

A

O-Benzyl-Tyr

48(D)

64

1.92

A

3,3-Dimethyl-Nva

40(D)

56

1.91

A

4-Methyl-Trp

50

58

1.38

A

5-Methyl-Trp

52

63

1.57

A

6-Methyl-Trp

52

64

1.64

A

7-Methyl-Trp

51

64

1.70

A

4-Methoxy-Phe

52

64

1.64

A

5-Methoxy-Trp

55

66

1.58

A

3-Chloro-Ala

57

64

1.34

A

4-Amino-Phe

33

47

1.80

A

4-Bromo-Phe

44

58

1.75

A

4-Chloro-Phe

46

59

1.68

A

2-Fluoro-Phe

55

61

1.28

A

4-Iodo-Phe

45(D)

61

1.91

A

4-Nitro-Phe

52

61

1.44

A

3-Fluoro-Tyr

64

71

1.37

A

5-Bromo-Trp

46

58

1.61

A

a-Methyl-Ser

56(L)

67

1.59

B

a-Methyl-Abu

50

60

1.50

A

a-Methyl-Val

51

56

1.22

A

Table 1 Continued

Racemate

hRb

hRF2

ac

Eluentd

a-Methyl-Leu

48

59

1.55

A

a-Methyl-Met

56(D)

64

1.39

A

a-Methyl-Phe

53(L)

66

1.72

A

a-Methyl-Tyr

63(D)

70

1.37

A

a-Methyl-Dopa

46(L)

66

2.27

B

a-Methyl-Trp

54

65

1.58

A

a-Methyl-Asp

52(D)

56

1.17

A

a-Methyl-Glu

58(L)

62

1.18

A

a-Ethyl-Ala

55

61

1.28

A

a-Propyl-Ala

55

63

1.39

A

a-Butyl-Ala

51

63

1.63

A

a-Difluoromethyl-Phe

63

70

1.37

A

a-Propenyl-Phe

57

63

1.28

A

2'-Methyl-Phe

43(D)

54

1.55

A

ß-Methyl-Phe

36(R, R)

56(S, S)

2.26

A

2'-Methyl-^-methyl-Phe

48(S, R)

55(R, S)

1.32

A

2'-6'-Dimethyl-Phe

38(D)

52

1.76

A

ß-Methyl-p-nitro-Phe

43(R, R)

62(S, S)

2.16

A

52(S, R)

60(R, S)

1.38

A

2',5'-Dimethyl-4-methoxy-Phe

45(D)

57

1.61

A

ß-Hydroxy-Phe

49(R, R)

63(S, S)

1.77

A

ß-Methyl-Tyr

52(R, R)

67(S, S)

1.87

A

55(S, R)

67(R, S)

1.66

A

2'-Methyl-Tyr

54(D)

62

1.39

A

2',5'-Dimethyl-Tyr

56(D)

67

1.59

A

aMigration distance 13 cm; chamber saturation. bhRF = RF x 100.

dA = methanol-water-acetonitrile 50 : 50 : 200 (v/v/v); B = methanol-water-acetonitrile 50 : 50 : 30 (v/v/v); C = methanol-water 10 : 80 (v/v); D = acetone-methanol-water 10 : 2 : 2 (v/v/v). eHyp = hydroxyproline. fMet(O2) = methionine sulfone.

aMigration distance 13 cm; chamber saturation. bhRF = RF x 100.

dA = methanol-water-acetonitrile 50 : 50 : 200 (v/v/v); B = methanol-water-acetonitrile 50 : 50 : 30 (v/v/v); C = methanol-water 10 : 80 (v/v); D = acetone-methanol-water 10 : 2 : 2 (v/v/v). eHyp = hydroxyproline. fMet(O2) = methionine sulfone.

common and uncommon dansyl (Dns) amino acids. Similar results can be obtained on 10 cm x 10 cm SIL C18-50/UV254 layers with the same eluents but with lower migration times. Some enantiomeric Dns-amino acids such as Lys, Met, Nva, Pro and aromatic compounds show low selectivity coefficients with CD. Therefore, it can be useful to resolve these ra-cemates on RP-18W/UV254 plates with eluents containing BSA since very high a values have been achieved.

Other N- and C-terminal substituents studied by chiral TLC include 2,4-dinitrophenyl (DNP), 3,5-dinitro-2-pyridyl (DNPy), 3,5-dinitrobenzoyl (DNB), o-nitrophenylsulfenyl (o-NPS), 9-fluorenyl-methoxycarbonyl (FMOC), methylthiohydanthoin (MTH), phenylthiohydanthoin (PTH), i-butoxycar-bonyl (i-BOC), carbobenzoxy (CBZ), phthalyl, acetyl, p-nitroanilide (pNA) and ^-naphthylamide (0NA) (Table 6).

The maximum ARF for the enantiomers of FMOC amino acids was obtained at different concentrations of 2-propanol. It is worth noting that this is the first time optical isomers have been separated with eluents containing BSA in the presence of very high levels (12-36%) of organic modifier. The resulting spots have the shape of a reversed triangle. FMOC-dl-Asn and FMOC-dl-Gln are not resolved. The order of retention of the d and l forms of the different compounds is variable. The d forms of FMOC-Pro, FMOC-Trp and FMOC-Met are more retained than the l forms, whereas the opposite is noted for the other amino acids. This behaviour is also shown from DNP-amino acids and other N-derivatives.

Most DNP, DNPy and DNB-dl-amino acids are resolved on RP-18W/UV254 plates with 0.1 molLacetate buffer solutions containing 2% iso-propanol and different BSA concentrations (2-6%) but few of them show chiral separation with phosphate buffer (0.05 molL — potassium dihydrogen phosphate + 0.05 mol L_1 disodium hydrogen phosphate), an eluent of higher pH (6.86) than that previously used. Enantiomeric DNPy-Ala, DNPy-Nva and DNP-Eth(O2) are completely separated at low

Table 2 Resolution of racemic amino acids by chiral TLC

Racemate

hRF1a

hRF2

ab

Separation technique

Ala

18(D)

53

5.13

Slurry of silica gel (Merck) and (-)-brucine brought to pH 7.1

Ser

12(D)

50

7.33

with 0.1 mol L~1 NaOH and spread on 20 cm x 20 cm plates.

Thr

16(D)

29

2.15

Eluent: butanol-aceticacid-water 3 : 1 : 4 (v/v/v). Migration dis

Ile

16(D)

35

2.82

tance, 10 cm; development time 0.5 h.

Met

18(D)

29

1.86

Visualization: ninhydrin.

Phe

27(D)

40

1.80

Tyr

22(D)

29

1.45

Trp

17(D)

31

2.19

Trp

59(D)

72

1.77

SIL C18-50/UV254 plates (Cat. No. 711308, MN) 10 cm x 10 cm,

Trp-NH2c

31(L)

40

1.48

thickness 0.20 mm.

4-Methyl-Trp

42

65

2.56

Eluent: 0.05 mol L~1 NaHCO3 # 0.05 mol L~1 Na2CO3 contain

5-Methyl-Trp

37

61

2.53

ing 6% BSA and 6% isopropanol (pH 9.8); for the resolution

6-Methyl-Trp

66

78

1.82

of 7-methylTrp, 5-methoxyTrp and Kynurenine, 0.05 mol L~1

7-Methyl-Trp

41

50

1.43

sodium tetraborate was used. Migration distance, 8 cm;

5-Methoxy-Trp

42

49

1.32

development time 1 h 50 min.

4-Fluoro-Trp

51

66

1.86

Visualization: p-dimethylaminobenzaldehyde.

5-Fluoro-Trp

43

63

2.23

6-Fluoro-Trp

42

54

1.62

Kynurenine

69(D)

80

1.80

3-(1-Naphthyl)-Ala

34(D)

40

1.29

Val

62(D)

68

1.30

DC plastikfolien, Kieselgel 60 F254 (Merck), 20 cm x20cm,

Gln

59(D)

66

1.35

thickness 0.2 mm.

Arg

50(D)

59

1.44

Eluent: acetonitrile-water 1:2.5 for Arg, His and Lys and 1.5 : 2

Cit

65(D)

69

1.20

for the others; the water containing 6.5 • 10 ~3 mol L~1 2-O-[(R)-

His

46(D)

55

1.43

2-hydroxypropyl]-^-CD.

Lys

49(D)

60

1.56

Migration distance, 18 cm at 19 ° C.

cTryptophanamide.

cTryptophanamide.

temperature (10°C) and pH values (0.5molL_1 acetic acid) where their retention by the layer is sufficient. The unresolved racemates include DNPy-Ser, DNP-Asp, DNP-Glu and DNPy-Trp. The first three amino acids are markedly polar and are only slightly retained by silanized silica gel plates, even when eluted with acidic solution; this may be the reason for their not being resolved. In general, planar chromatography clearly separates the enantiomers of N-derivatized hydrophobic amino acids.

The complete resolution of DNPy-DL-Trp is obtained on layers of SIL C18-50/UV254 with ^-CD as chiral agent.

The optical isomers of PTH-amino acids are sensitive to light and readily racemize. Racemization of these optical active derivatives is observed on silanized silica gel plates with acidic eluents. MCTA plates may be useful since they are able to separate enantiomeric MTH-Phe, MTH-Tyr, MTH-Pro and PTH-Pro with neutral aqueous-alcoholic eluents.

Among C-terminal substituents, the enantiomeric ^NA derivatives of amino acids were well separated on silanized silica gel plates with ^-CD as mobile phase modifier while pNA derivatives show discordant results. In fact, dl-Leu-pNA is fully resolved but dl-Ala-pNA failed since the latter optical antipodes do not form inclusion complexes of sufficient stability with ^-CD. In addition, BSA seems efficient in the enantioseparation of pNA derivatives.

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