Dabitc Derivatives of Amino Acids

DABITC reacts with the NH2-terminal end of an amino acid in basic media to give a 4-dimethylamino azobenzene thiohydantoin (DABTH) amino acid via a DABTC derivative, in a manner similar to the Ed-man method, where a PTH amino acid is obtained by the reaction of phenylisothiocyanate (PITC). The presence of excess free amino acid does not, in any case, interfere with the analysis.

Two-dimensional TLC on polyamide sheets by ascending solvent flow is used to identify all DABTH amino acids except DABTH-Ile/Leu. No phase equilibrium is necessary; H2O-acetic acid (2 : 1, v/v) is used for the first dimension and toulene-w-hexane-acetic acid (2:1:1, v/v) is used for the second. For discrimination between DABTH-Ile/Leu, one-dimensional separation on polyamide using formic acid-ethanol (10 : 9, v/v) or one-dimensional separation on silica gel (Merck) using chloroform-ethanol (100 : 3, v/v) is carried out. The successful identification of DABTH amino acids relies on skilful running of the small polyamide sheet and interpretation of the pattern of spots.

Detection of DABITC derivatives of amino acids The use of DABITC reagent during amino acid sequencing of proteins has distinct advantages over the use of dansyl chloride; for example, the colour difference between DABITC, DABTC derivatives and DABTH-amino acids greatly facilitates direct visualization and identification. DABTH amino acids are coloured compounds having absorption maxima at 520 nm in acid media (s = 47 000). Thus, using the visible region, the quantitation and

Table 9 RF values for Dns amino acids in various solvent systems on polyamide sheets

Dnsaminoacid RFin solvent systems

Table 9 RF values for Dns amino acids in various solvent systems on polyamide sheets

Dnsaminoacid RFin solvent systems

A

B

C

D

E

F

G

H

I

J

1.

Ala

0.53

0.48

0.49

0.69

0.69

0.57

0.81

0.68

0.43

0.76

2.

Arg

0.05

0.03

0.03

0.91

0.39

0.09

0.76

0.22

0.01

0.06

3.

Asp

0.08

0.07

0.10

0.69

0.38

0.10

0.88

0.37

0.12

0.19

4.

Cys

0.03

0.03

0.04

0.19

0.43

0.22

0.78

0.09

0.03

0.06

5.

Glu

0.15

0.10

0.15

0.66

0.88

0.02

0.88

0.34

0.05

0.30

6.

Gly

0.32

0.21

0.32

0.69

0.63

0.48

0.80

0.48

0.28

0.69

7.

His

0.07

0.05

0.13

0.96

0.76

0.32

0.84

0.36

0.06

0.18

8.

Ile

0.77

0.54

0.65

0.40

0.57

0.71

0.78

0.76

0.60

0.84

9.

Leu

0.70

0.49

0.59

0.34

0.57

0.71

0.78

0.75

0.54

0.80

10.

Lys (mono)

0.35

0.21

0.38

0.22

0.09

0.63

0.72

0.58

0.09

0.79

11.

Lys (di)

0.53

0.37

0.48

0.78

0.69

0.35

0.82

0.40

0.39

0.76

12.

Met

0.52

0.36

0.51

0.43

0.59

0.68

0.80

0.62

0.55

0.81

13.

Phe

0.57

0.38

0.53

0.31

0.43

0.68

0.77

0.62

0.51

0.81

14.

Pro

0.85

0.66

0.71

0.55

0.74

0.46

0.84

0.75

0.69

0.90

15.

Ser

0.12

0.07

0.16

0.81

0.71

0.49

0.82

0.42

0.10

0.44

16.

Thr

0.15

0.10

0.26

0.81

0.74

0.57

0.82

0.56

0.16

0.56

17.

Tyr

0.63

0.47

0.61

0.00

0.00

0.84

0.73

0.65

0.58

0.91

18.

Val

0.72

0.56

0.61

0.47

0.67

0.71

0.81

0.80

0.61

0.88

19.

Dns-OH

0.00

0.01

0.00

0.51

0.54

0.16

0.74

0.00

0.04

0.04

20.

Dns-NH2

0.51

0.38

0.47

0.71

0.17

0.96

0.49

0.60

0.40

0.91

Solvent systems: A, benzene-acetic acid (9:1, v/v): B, toluene-acetic acid (9:1, v/v); C, toluene-ethanol-acetic acid (17:1 : 2, v/v): D, water-formic acid (200:3, v/v); E, water-ethanol-ammonium hydroxide (17:2:1, v/v); F, ethyl acetate-ethanol-ammonium hydroxide (20:5:1); G, water-ethanol-ammonium hydroxide (14:15:1, v/v); H, n-heptane-nbutanol-acetic acid (3:3:1, v/v); I, chlorobenzene-acetic acid (9:1, v/v); J, ethyl acetate-methanol-acetic acid (20 : 1 : 1).

Table 10 Various solvent systems for TLC of dansyl amino acids

Solvent systems

Ratio

1.

HCOOH

1.5%

Benzene-acetic acid

9: 1

2.

Formic acid

1.5%

Benzene-acetic acid

4.5:1

3.

H2O-pyridine-HCOOH

93:35:3.5

Benzene-acetic acid

4.5:1

4.

NH4Cl # NH3 # ethanol

80g # 22 mL # 10 mL

Benzene-pyridine-HOAc

75 : 2 : 6

5.

H2O-propanol-formic acid

100 : 5 : 2

Benzene-acetic acid

9:1

6.

Ethyl acetate-MeOH-HOAc

20 : 1 : 1

Benzene-HOAc-BuOH

90 : 10 : 5

7.

Formic acid

1.5%

Benzene-acetic acid

9:2

8.

Benzene-anhydrous HOAc, followed by

9:1

EtOAc-MeOH-anhydrous HOAc in the same direction

10:1

Formic acid

1.5%

9.

H2O-pyridine-HCOOH

93:35:3.5

Benzene-acetic acid

4.5:1

10.

Formic acid

3%

Benzene-acetic acid

9:1

11.

Me-acetate-iso-PrOH-NH3

9:7:4

CHCl3-MeOH-HOAc

15:5:1

CHCl3-EtOAc-MeOH-HOAc

45 : 75 : 22.5 : 1

Pet ether-t-BuOH-HOAc

5:2:2

12.

CHCl3-MeOH

9:1

13.

CCl4-2-methoxyethanol

17:3

14.

Benzene-pyridine-acetic acid

80 : 20 : 2

Solvents at serial no. 1-8 : two-dimensional TLC on polyamide layers. Solvents at serial no. 9-14 : one-dimensional TLC on silica gel layers.

Table 11 hRF Valuesof 10dansylaminoacidson silica gel thin Table 13 hRF Values of DNP amino acids on silica gel thin layers (Sl. no. = serial number)

layers

Sl. Dansyl amino acid no.

Solvent system

S1 S2

1. Dansyl-L-alanine

2. Dansyl-L-isoleucine

3. Dansyl-L-leucine

4. Dansyl-L-methionine

5. Dansyl-L-proline

6. N-O-dansyl-L-tyrosine

7. A/-a-dansyl-L-tryptophan

8. Dansyl-L-phenylalanine

9. Dansyl-L-valine 10. Dansyl-L-norvaline

62 61 60 50 27

80 92 85 85 49

83 85 80 89 65

86 64 62 55 31

60 84 72 30 39

55 73 40 60 18

51 53 46 40 21

77 76 74 52 40

72 88 65 48 35

75 81 68 45 37

Sl. Dansyl amino acid no.

Solvent system

Sl. Dansyl amino acid no.

Solvent system

Table 12 hRF Values of 10 dansylamino acids on silica gel thin layers

Ai

A2

A3

A4

A5

1.

N-a-dansyl-L-asparagine

56

75

53

30

35

2.

Dansyl-L-aspartic acid

66

72

60

64

30

3.

a-Dansyl-L-arginine

7

12

3

2

3

4.

N-N-didansyl-L-cystine

84

83

68

85

18

5.

Dansyl-L-cysteic acid

82

80

25

15

11

6.

Dansyl-L-glutamic acid

80

90

84

74

55

7.

Dansyl-L-glutamine

62

77

63

41

40

8.

N-dansyl-L-lysine

16

20

10

6

8

9.

N-dansyl-L-serine

72

85

72

58

32

10.

Dansyl-L-threonine

76

88

76

68

45

N-DNP-l-amino acid

Solvent system

S1, n-heptane-BuOH-HOAc (20:8:3, v/v); S2, dichloro-methane-MeOH-propionic acid (30: 1 : 0.5, v/v); S3, chloro-form-HOAc-ethylacetate (24 : 5 : 4, v/v); S4, chloroform-MeOH-ethyl acetate (23 : 8 : 2, v/v); S5, chloroform-propionicacid-ethyl acetate (23 : 6 : 4, v/v); RF values are average of five determinations.

identification of these derivatives become more convenient and sensitive (10 pmol on a polyamide plate). Exposure to HCl vapours turns all yellow spots to red or blue on polyamide sheets.

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