Detection method for amino acid derivative

A method for detecting amino acid derivatives in which 2-anilino-5-thiazolinone derivatives of amino acids are reacted with an amino compound of fluorescein or rhodamine derivative amino compounds to form phenylthiocarbamyl amino acids derivatives. The phenylithiocarbamyl amino acids derivatives are detected with a high level of sensitivity by a fluorescence spectrophotometer.

BACKGROUND OF THE INVENTION 
1. Field of the invention 
This invention relates to a method for detecting amino acid derivatives for 
application to sequence determination from the amino(N) terminal of a 
protein, more particularly to a method for detecting amino acid derivative 
with high sensitivity by using an amino compound containing a fluorescent 
amino compound. 
2. Description of the prior art 
For the detection of amino acids in the final step of the 
phenylisothiocyanate method according to Edman, P. Acta. Chem. Scand. 10 
761 (1956), which is an N-terminal sequence determination method, it has 
been a usual practice as shown in FIG. 2 that thiazolinone derivatives are 
treated with an acid to form phenylthiohydantoin (PTH) derivatives and 
these derivatives are determined spectrophotometrically. 
Although the prior art method for spectrophotometrically detecting PTH 
derivatives is simple and convenient as detection means, it cannot fully 
cope with a recent trend toward more highly sensitive analysis of a 
protein with a smaller amount of specimen. 
For the high-sensitivity detection of amino acids, methods of using .sup.32 
S PITC labelling or .sup.135 I PITC labelling are shown in the following 
publication, PITC standing for phenylisothiocyanate: 
W. G. Lauer, Fundamental Techniques in Virology, Eds. K. Habel and N.P. 
Salgman p. 379 (1961) Academic Press N.Y. 
C. J. Burrell, P. D. Cooper, J. M. Swann, Aust J. Chem. 28 2289, (1975). 
In the above methods, radioactive isotope derivatives take part in the main 
reaction in the Edman degradation method. If high radioactivity is used 
for the purpose of realizing high sensitivity, not only radioactive 
disintegration increases, which adversely affects a yield itself in the 
Edman amino acid sequence determination, but also the contamination of the 
environment occurs. 
SUMMARY OF THE INVENTION 
Therefore, it is an object of the present invention to provide a novel 
method for detecting amino acids which can be applied to an amino acid 
sequence determination from the amino terminal of a protein with higher 
sensitivity and no contamination of the environment. 
This and other objects of the invention are accomplished by a novel method 
comprising the steps of reacting a 2-anilino-5-thiazolinone derivative of 
an amino acid with an amino compound of fluorescein or of a rhodamine 
derivative to form a phenylthiocarbamyl amino acid derivative and 
detecting the phenylthiocarbamyl amino acid derivative.

DETAILED DESCRIPTION OF THE INVENTION 
FIG. 1 shows principle flow diagram of the present invention. The method 
according to the present invention comprises the steps of reacting a 
2-anilino-5-thiazolinone derivative with a fluorescent amino compound such 
as an aminofluorescein or a rhodamine derivative amino compound, to form a 
PTC amino acid derivative and detecting the amino acid derivative with 
high sensitivity by making use of a fluorophotometer. In FIG. 1, each of 
R.sub.1, R.sub.2 and R.sub.3 is an amino acid side chain and X denotes 
fluorescein or a rhodamine derivative. 
The present invention will now be described in more detail with reference 
to the following examples. 
EXAMPLE 1 
This example illustrates a basic embodiment of the method according to the 
present invention in which a 2-anilino-5-thiazolinone derivative of an 
amino acid (ATZ) is reacted with 4-aminofluorescein to form a 
phenylthiocarbamyl amino acid derivative and the reaction product is 
detected with very high sensitivity. 
In this Example, a dipeptide (Ala-Gly) is used instead of a protein for 
simplification. 
i) Synthesis and formation of ATZ-Ala (see Molecular Biology, Biochemistry 
and Biophysics, Vol. 8, "Protein Sequence Determination" edited by Saul B. 
Neadleman, Springer-Verlag, p. 253) 
The reaction proceeds according to the following formulae [IV] and [V]: 
##STR1## 
Ala-Gly was dissolved in a 50% aqueous pyridine solution, and 2M NaOH was 
added thereto to adjust the pH value to 8.6. Subsequently, PITC was added 
thereto. Since the pH value lowered with the addition of PITC, 2M NaOH was 
added to keep the pH value at 8.6. After the variation in the pH value was 
hardly observed, the solution was heated at 40.degree. C. for 1 hr. After 
the completion of the reaction, the reaction mixture was washed with 
benzene. Benzene dissolved in the water phase was purged with a nitrogen 
gas, and the pH was adjusted to 2 by the addition of 1M HCl, thereby 
preparing PTC-Ala Gly in the form of a white precipitate. 
##STR2## 
PTC-Ala-Gly thus prepared was dissolved in TFA, and the solution was heated 
at 50.degree. C. for 5 min. After the completion of the reaction, the 
reaction mixture was evaporated to dryness. Butyl chloride was added to 
the residue to thoroughly dissolve the product therein, and the resulting 
solution was passed through a cellulose column. The by-product and Gly 
were adsorbed on the column. The effluent was collected and evaporated to 
dryness, thereby preparing ATZ-Ala in the form of a white crystal. 
ii) Coupling reaction 
##STR3## 
phenylthiocarbamyl amino acid aminoflouroescein As shown in the 
above-described reaction formula [VI], the amino group of 
4-aminofluorescein attacks the carbonyl group of ATZ amino acid. This 
reaction is the reverse of a reaction for cleaving a peptide bond between 
PITC and the peptide. The reaction was conducted at room temperature for 3 
min. in a solvent comprising pyridine (10%) and demethylformamide. 
iii) Detection of phenylthiocarbamyl amino acid The reaction mixture was 
applied to high-pressure liquid chromatography (HPLC), and the reaction 
product was detected with ultraviolet absorption spectra (at 269 nm). The 
results are shown in FIG. 3. 
FIG. 3(i) is an HPLC chromatogram of a thiazolinone derivative, while FIG. 
3(ii) is an HPLC chromatogram after the reaction of the thiazolinone 
derivative for 3 min. In FIGS. 3(i) and 3(ii), (a) is a peak of a 
thiazolinone derivative of alanine and (b) is a peak of a 
phenylthiocarbamyl derivative of alanine. As can be seen from the 
drawings, the coupling reaction between the thiazolinone derivative of 
alanine and 4-aminofluorescein was completed in 3 min at room temperature, 
and the thiazolinone derivative was detected in the form of a 
thiocarbamylalanine derivative. 
(Conditions of HPLC) 
column: 
SERVA Cat. No. 42318 
250 mm.times.4.6 mm 
SERVACHROM Packing 
solvent system: 
______________________________________ 
buffer solution 
organic solvent 
______________________________________ 
0.015M CH.sub.3 CN--MeOH 
NaOAc + AcOH (4:1) 
pH 5 
1 5 
______________________________________ 
effluent flow rate: 1.5 ml/min 
detection wave length 269 nm 
EXAMPLE 2 
This Example shows the results of an experiment wherein a 
2-anilino-5-thiazolinone derivative of an amino acid was reacted with 
4-aminofluorescein to form a phenylthiocarbamyl amino acid derivative and 
the maximum sensitivity with which the amino acid derivative can be 
detected was investigated. 
The phenylthiocarbamyl leucine derivative prepared by the same way from a 
dipeptide (Leu-Gly) was dissolved in methanol, and 1 femto (10.sup.-15) 
mol, 10 femto mol and 100 femto mole (10 .mu.1 ) thereof were applied to 
HPLC and detected with a fluorophotometer (an excitation wavelength of 500 
nm and a fluorescence wavelength of 516 nm) respectively. The results are 
shown in FIG. 4. 
The comparison of sensitivity of detection according to the present 
invention with that of the conventional phenylthiohydantoin amino acid 
detection method (PTH method) gave the following results, i.e., revealed 
that the sensitivity of detection according to the present invention was 
much higher than that according to the prior art method. 
______________________________________ 
detection method 
sensitivity of detection 
______________________________________ 
PTH method less than 10 pico mol 
present invention 
1 femto mol 
______________________________________ 
(Condition of HPLC) 
column: 
SHISEIDO CAPSEL C18 AG Type 
150 mm.times.4.6 mm 
solvent system: 
A: 10 mM Na.sub.2 HPO.sub.4 - NaH.sub.2 PO.sub.4 ; pH 8.0 
B: MeOH 
gradient elution 
flow rate: 1 ml/min 
temperature: 40 .degree. C. 
detection: 
excitation wavelength: 486 nm 
fluorescence wavelength: 513 nm 
EXAMPLE 3 
This Example illustrates an embodiment in which a mixture of 
phenylthiocarbamyl amino acid derivatives of amino acids constituting a 
protein was separated into components and detected by HPLC (the condition 
of HPLC were the same as those of Example 2). 
A mixture of phenylthiocarbamyl amino acid derivatives synthesized by 
making use of dipeptides containing predetermined amino acids according to 
the procedures described in the Example 1 was analyzed by HPLC under 
conditions shown in Example 2. The results are shown in FIG. 5. As can be 
seen from the drawing, the phenylthiocarbamyl derivatives of 20 amino 
acids which are components constituting a protein were excellently 
separated. 
An important feature of the present invention resides in that a fluorescent 
amino compound is reacted with thiazolinone derivative of an amino acid to 
form a phenylthiocarbamyl derivative of the amino compound and the amino 
acid derivative is detected without using any radioisotope. 
In the above Examples, embodiments in which 4-aminofluorescein was used 
were described. However, it is apparent that other fluorescent amino 
compounds can be used, and the present invention is not limited to the 
above Examples only. As is apparent from the foregoing description, the 
method for detecting an amino acid derivative with high sensitivity is 
very useful from the viewpoint of industry.