DE10149568A1 - Detection of amines, especially aminoacids formed in sequencing of peptides by Edman degradation, by forming reaction product with halo-substituted phenylisothiocyanate - Google Patents

Abstract

The use of a halo-substituted phenylisothiocyanates (I) for: (a) derivatization and/or detection of primary and/or secondary amines (A); or (b) the analysis of polypeptides. The use of a halo-substituted phenylisothiocyanate of formula (I) for: (a) derivatization and/or detection of primary and/or secondary amines (A); or (b) the analysis of polypeptides. R1 - R5 = H, halo, 1-3C alkyl, or 1-3C mono- or poly-haloalkyl; and provided that at least one is 1-3C mono- or polyhaloalkyl, or halo. Independent claims are also included for: (1) new reaction products of halogen-substituted phenyl isothiocyanates (preferably, but not restricted to, (I)) with (A); and (2) a method for determining the sequence of polypeptides, including the stage of reacting the polypeptide with (I).

Description

The present invention relates to a method for determining the sequence of polypeptides.

The sequence analysis of polypeptides and proteins is for the modern biotechnology of of central importance. Although with increasing knowledge of the genome from different Organisms, including humans, the amino acid sequences of polypeptides and Proteins can be predicted based on the nucleic acid sequence that comes polypeptide sequence analysis is still of central importance insofar as starting from a sample present, the polypeptides contained therein are sequenced or must at least be sequenced in order to start from the sequenced N- Terminus of the polypeptide to design a probe that is then used to screen the Nucleic acids using the extensive nucleic acid database, be it in vivo, in vitro or in silico, the putative total nucleic acid sequence and thus also the Determine overall sequence of the protein.

In addition, there is still a need and need for polypeptides or proteins to be sequenced, in particular then, for example when searching for individual-specific deviations from the expected or particularly in a population common sequence. Generally, protein analysis is more recent in the so-called proteome analysis is increasingly used [1 Kahn, 1995]. Here, the Whole or parts, e.g. B. the nuclear fraction, the expressed proteins of an organism or a cell type at a certain growth phase, under the influence of Drugs or examined in morbidly changed condition. The proteins are with suitable methods, for example two-dimensional gel electrophoresis [2 Klose 1995], separated and the proteins in the gel made visible using suitable staining methods. Differences in the protein pattern can be seen, for example, between the growth phases or in the case of pathological changes. It applies z. B. to identify the proteins that a have different expression rates or due to pathological changes in the Cell can be modified. [3 Brockstedt 1998].

A first method for sequence analysis of polypeptides is the Sanger method in which 1-fluoro-2,4-dinitrobenzene is used to label N-terminal amino acids has been. After complete hydrolytic breakdown of the protein into individual amino acids was the labeled (terminal) amino acid using chromatographic techniques identified. This method was particularly disadvantageous for larger peptides connected, which resulted from the polypeptides via partial hydrolysis or enzymatic digestion into small fragments and then the N-terminal and The terminal amino acid of the fragments was determined and in addition the Amino acid composition had to be determined by means of amino acid analysis.

A process that is clearly associated with advantages over the Sanger process is the So-called Edman degradation, in which the respective Nterminal amino acid is split off one peptide chain after the other. [4 Edman 1950]. As part of the Edman degradation, phenyl isothiocyanate is grown under basic conditions coupled to the amino terminus of the polypeptide chain. A phenylthiocarbamyl is formed Polypeptide (PTC polypeptide). In the second step, the terminal is derivatized Amino acid selectively using anhydrous acid e.g. B. trifluoroacetic acid and thereby converted into the anilinothiazolinone derivative (ATZ derivative) of the amino acid. in the In the following the AS derivatized in this way is extracted and with the help of aqueous Acid in the more stable phenylthiohydantoin derivative (PTH derivative) of the amino acid isomerized. The PTH derivative of the amino acid is available in the today Automatic sequencers then automatically into high pressure liquid chromatography System injected. Here the amino acids are separated according to their retention time. The Detection is carried out by UV absorption. The process described is repeated cyclically, so that the primary structure of the polypeptide chain can be successively analyzed.

For the analysis and identification of the polypeptide, the turnover rates of the individual Substeps z. B. coupling, splitting or extraction of the so-called Edman degradation of vitally important. The highest possible should be for the overall process Sales rate, preferably over 90% and preferably over 95% achieved in order to be able to clearly identify a large number of mining stages. With less Incomplete breakdown leads to sales rates that in the subsequent Degradation steps the now cleaved amino acid by the background of the amino acids is overlaid from the previous steps, the so-called overlap, so that the Sequence is no longer clearly identifiable. An overview of protein sequencing can be found in [5, 6].

The process was carried out by Edman and Begg carried out manually in 1967 [7 Edman 1967]. Over the years made various technical improvements to the machines. single out are the introduction of dead volume-free valve systems [8 Wittmann-Liebold 1973], with which the individual reagents and solvents in the machine with air exclusion and without that liquid residues remain in the pipes. With the on-line Conversion [9 Wittmann-Liebold 1976] and on-line injection [9 Wittmann-Liebold 1985] was able to fully automate the entire chemical process and not just a part become. Hereby losses through manual handling could be minimized and also the oxygen-sensitive conversion can be carried out completely in the absence of air, which resulted in higher overall sales rates. With the development of the reactor cartridge and the related introduction of gas phase sequencing where some reagents gaseous doses [11 Hewick 1981], the by-products of the reaction were successful reduce and thus perform the analysis more sensitively. All technical improvements ultimately had the goal of increasing the sensitivity of the analysis. So were initially Micromole amounts of a purified protein required for successful analysis. Analyzes in the picomole range are now routinely carried out. The The actual chemical process remained almost unchanged over the years.

For the identification and characterization of proteins and peptides using Edman Protein sequencers such as the device are currently being degraded. "Procise cLc "from Applied Biosystems, Foster City, USA, or the device" Knauer 910 "from Company Knauer Wissenschaftlicher Gerätebau, Berlin. (Technical description of the devices can be found in the respective operating instructions [12, 13]).

The devices differ slightly in their technical design. Both are common Devices that contain the reagents and solvents in a bottle battery. about a valve system, these are sealed off in the reactor chamber and the Converter chamber dosed. Both devices identify the cleaved amino acid a liquid chromatograph with UV detection. The devices also differ minor in the use of individual reagents or solvents. The "Edman- Sequencing "or Edman degradation with the" Edman reagent "phenyl isothiocyanate as Standard method is not only these devices, but practically all currently on the Devices on the market. However, there are a variety of small ones Process variants. The devices are still designed to allow the user to make changes in the process according to your own ideas, such as other solvents can use. The terms wet phase sequencing (Knauer) or gas phase and pulsed liquid sequencing (Applied Biosystems) mean that individual reagents as Drops of liquid, as a fine spray or in gaseous form. Here, for the different reagents in the process also combined different dosage types become. Common to the process is that the protein is placed in a suitable reactor chamber Noncovalent membranes, e.g. B. is immobilized via hydrophobic interactions and the reagents are dosed into the reactor. The binding of the protein to the membrane is loosened or loosened by some reagents, so that an unfavorable process control leads to a washing out of the protein and thus the sensitivity is reduced or the continuation of the analysis becomes impossible. The manufacturers have with the individual Dosage types the chemical reaction to high turnover rates and low washing out optimized. In principle, all sequencing procedures with small changes to the Devices. You can also use liquid phase sequencing or Solid phase sequencing, in which the protein is covalently bound to carrier material, albeit with restrictions.

The sequence of protein sequencing in an automated machine is in general follows. The protein is taken up and taken up in a suitable solvent applied a filter. This filter consists of PVDF, for example (Polyvinylidene fluoride) (Sequelon membrane, Millipore, USA) or from pretreated Glass fiber (Applied Biosystems, USA). The protein does not bind covalently, e.g. B. about hydrophobic interactions so that it is immobilized on the filter or membrane is. The filter or membrane with the protein is placed in a reactor chamber. The reagents and solvents are then dosed. In principle, only one becomes Base (e.g. 6% trimethylamine in ethanol water (1: 1)) metered. Then 5% Phenyl isothiocyanate dosed in heptane and again trimethylamine. In the reactor chamber the coupling reaction takes place (approx. 20 min at 45 ° C). The excess reagents with an organic solvent (e.g. ethyl acetate, heptane, butyl chloride) washed out. Anhydrous acid (e.g. 100% trifluoroacetic acid) is introduced into the reactor chamber. dosed and the terminal amino acid is split off. The extraction of the cleaved derivatized amino acid is treated with an organic solvent (e.g. Butyl chloride, ethyl acetate). Here, the solvent passes through the reactor rinsed and caught in the converter in a small container like a vial. in the The protein shortened by one amino acid is available to the reactor for the next degradation step Available. An aqueous acid (e.g. 25% trifluoroacetic acid in Water) dosed. The isomerization reaction takes place at about 55 ° C for 30 min. The reagent is then dried and in a suitable solvent (e.g. 20% Acetonitrile in water) and resuspended via an injection valve with six Connections injected into the HPLC system. The converter is now ready for recording the amino acid split off in the meantime and the process is repeated cyclically. In the entire process is a multitude of other drying-mixing or Cleaning steps required for analysis. A full description can be found in the respective device documentation [12, 13].

As described in the original publication by Edman [4], is still used today Phenyl isothiocyanate used as a coupling reagent. However, in the Past varied attempts to replace this reagent with a higher one To achieve sensitivity of the analysis. The use of 4- N, N-dimethylaminoazobenzene 4'-isothiocyanate [14 Chang, 1978] a fluorescent Isothiocyanate, or the use of 311-PITC by Aebersold and co-workers for the sensitive mass spectrometric detection was developed [15 Hess 1995].

A disadvantage of the reagents presented so far for the substitution of phenyl isothiocyanate is for example, that they usually have a slow reaction speed compared to PITC with the amino terminus so that there is no complete conversion. The slow coupling rate is due to the steric hindrance of the reaction the larger isothiocyanate molecules or in the changed electronic properties of the Phenyl ring or the isothiocyanate used.

Another disadvantage of some of the compounds presented is that they do have a comparably fast coupling rate, but the cleavage reaction runs much slower, so that is why the required high Don't let sales rates of the overall process be realized. Some connections like to Example 4- (N-1-Dimethylaminonaphthalene-5-Sulfonylamino) Phenylisothiocyanate [16 Jin, 1986] have a high coupling rate and a high release rate, they have however, physico-chemical properties unfavorable for the overall process; so lie the fluorescent isothiocyanates at room temperature and normal pressure as a solid and must be kept in solution in a suitable solvent for the reaction. In the liquid phase sequencing that used to be used in the so-called Spinning Cup Reaction compartments or in manual procedures, this played Effect on reaction speed only a minor role. However, the technical conditions of these analyzers and the processes carried out therein, for example the comparatively high oxygen input during the process Limiting their sensitivity.

Are fluorescent isothiocyanates in the gas or used today Wet phase sequencers used, the following occurs during the coupling reaction Effect on: The coupling reagent, dissolved in a suitable organic solvent (e.g. Heptane) is metered into the reactor. This evaporates in the subsequent drying step Partial solvent. In the subsequent step, the base, for example 6% Trimethylamine in 50% ethanol and 50% water, dosed. The isothiocyanate precipitated largely on the filter with the result that the reaction rate with the protein is significantly restricted. Another disadvantage here is that this physical chemical properties also the removal of the excess added to the reaction Reagent is considerably more difficult (e.g. due to stationary charge of the used Coupling). In the process step of extracting the cleaved terminal Amino acid shows many of the proposed substitutes for PITC unfavorable properties. The extraction takes place through the solubility properties of the isothiocyanates used only incomplete and only a small proportion of the derivatized amino acid are detected, which is why the detection limit is based on the amount of protein used is reduced overall.

The present invention has for its object to provide a connection that instead of the phenyl isothiocyanate used in the prior art in the context of Edman degradation can be used and those described in the prior art Overcomes disadvantages. In particular, it is an object of the present invention to: to provide such connection, the at least several for a reliable Use of Edman mining, especially in its automated form, required Has properties, namely a high reaction rate with primary amines, a high cleavage rate, a low melting point, solubility in organic and inorganic solvents and a low detection limit for using this reagent derivatized amino acids with a suitable detection method.

Another object underlying the present invention is that a Reagent is provided, which is the state of the art in the context of the Edman Degradation used phenyl isothiocyanate replaced and the detection limit for the Overall process should further reduce compared to the prior art.

According to the invention, the object is achieved in a first aspect by using a halogen-substituted phenyl isothiocyanate of the formula


wherein R ₁ to R _{5 are} each independently selected from the group comprising hydrogen, halogen, alkyl and haloalkyl,
where halogen is selected from the group comprising fluorine, chlorine, bromine and iodine,
and alkyl is selected from the group consisting of methyl, ethyl, n-propyl and isopropyl;
provided that at least one of the radicals R ₁ to R _{5 is} a halogen and / or contains a halogen,
for the derivatization of amino acids, in particular of α-amino acids.

In one embodiment it is provided that the haloalkyl radical contains at least one Halogen is substituted.

In a further embodiment it is provided that the halogen-substituted Phenyl isothiocyanate is selected from the group consisting of 2,4-bis (trifluoromethyl) - Phenyl isothiocyanate, 2,5-bis (trifluoromethyl) phenyl isothiocyanate and 3,5- Bis (trifluoromethyl) phenyl isothiocyanate.

According to the invention, the object is achieved in a second aspect by the use of 3,5-bis (trifluoromethyl) phenyl isothiocyanate for the derivatization of amino acids, especially of α-amino acids.

In a second aspect, the problem is solved by an adduct of 3,5-bis (Trifluoromethyl) phenyl isothiocyanate and an amino acid.

In one embodiment of the first and second aspects of the present invention provided that the amino acid is an N-terminal amino acid of a polypeptide.

In a third aspect, the object is achieved according to the invention by using a halogen-substituted phenyl isothiocyanate of the formula


wherein R ₁ to R _{5 are} each independently selected from the group comprising hydrogen, halogen, alkyl and haloalkyl,
where halogen is selected from the group comprising fluorine, chlorine, bromine and iodine, and
Alkyl is selected from the group consisting of methyl, ethyl, n-propyl and isopropyl;
provided that at least one of the radicals R ₁ to R _{5 is} a halogen and / or contains a halogen,
for the analysis of polypeptides.

These halogen substituted phenyl isothiocyanates are also referred to herein as "halogen substituted phenyl isothiocyanates as described herein ", and referenced below Reference to the use of this term.

In one embodiment it is provided that the haloalkyl radical contains at least one Halogen is substituted.

In a further embodiment it is provided that the halogen-substituted Phenyl isothiocyanate is selected from the group consisting of 2,4-bis (trifluoromethyl) - Phenyl isothiocyanate, 2,5-bis (trifluoromethyl) phenyl isothiocyanate and 3,5- Bis (trifluoromethyl) phenyl isothiocyanate.

In one embodiment it is provided that 3,5-bis (trifluoromethyl) phenyl isothiocyanate is used to analyze polypeptides.

In a further embodiment it is provided that the analysis is the sequence analysis of the Is polypeptide.

In a still further embodiment it is provided that the analysis by means of Edman Dismantling takes place.

In a fourth aspect, the task is solved by a reaction product from a Halogen substituted phenyl isothiocyanate, especially a halogen substituted Phenyl isothiocyanate as described herein and an amino acid, preferably one α-amino acid.

In one embodiment it is provided that the amino acid is attached to at least one other Amino acid is bound, preferably to a polypeptide.

• - Bereitstellen des Polypeptids und
• - Umsetzen des Polypeptids mit einem Halogen-substituierten Phenylisothiocyanat, wie hierin beschrieben.

In a fifth aspect, the task is finally solved by a method for determining the sequence of polypeptides, comprising the following steps:

• - Providing the polypeptide and
• - Reacting the polypeptide with a halogen substituted phenyl isothiocyanate as described herein.

• - Abspalten der endständigen derivatisierten Aminosäure und
• - Detektierten der abgespaltenen endständigen derivatisierten Aminosäure.

In one embodiment it is provided that the inventive step further comprises at least one of the following steps:

• - Cleavage of the terminally derivatized amino acid and
• - Detected the cleaved terminal derivatized amino acid.

In a further embodiment it is provided that the steps of converting the Splitting and the detection can be repeated at least once.

In a still further embodiment it is provided that the implementation at a Temperature of about 20-95 ° C, preferably 20-85 ° C and preferably 40-45 ° C he follows.

In one embodiment it is further provided that the conversion under basic Conditions are carried out, preferably using a nitrogen base.

In a preferred embodiment it is provided that the reaction at a pH of 7 to 14, preferably at a pH of 8 to 10.

In a further embodiment it is provided that after the polypeptide has been reacted phenylthiocarbamoyl polypeptide obtained with phenyl isothiocyanate, preferably after Drying the phenylthiocarbamoyl polypeptide reacted with anhydrous acid while doing so the N-terminal amino acid of the polypeptide as a heterocyclic derivative in the form of a Anilinothiazolinone amino acid is split off.

In yet another embodiment, the anilinothiazolinone amino acid is from the Reaction batch is extracted and transferred directly to a detection system and identified.

Finally, in one embodiment it is provided that the anilinothiazolinone Amino acid extracted from the reaction mixture and into a phenylthiocarbamoyl Amino acid is transferred.

In a further embodiment it is provided that the anilinothiazolinone amino acid extracted from the reaction mixture and converted into a phenylhydantoin amino acid.

In one embodiment it is provided that, as a further step, verification and / or The amino acid derivative is identified.

In a still further embodiment it is provided that the detection and / or Identify by means of a method that is selected from the group consisting of Reverse phase HPLC, in particular microbore reverse phase HPLC, gas chromatography and mass spectrometry.

In one embodiment, the detection of the amino acid derivative by means of ECD Detector, UV detection, retention time, mass information and / or retention time and Mass information, or by means of negative chemical ionization.

In a further embodiment, the polypeptide is one with an N-terminal Amino acid reduced sequence with 3,5-bis (trifluoromethyl) phenyl isothiocyanate is implemented.

In a still further embodiment it is provided that at least one of the in the steps provided according to the invention are repeated.

The present invention is based on the surprising finding that with the herein described halogen-substituted phenyl isothiocyanate reagents and could be provided, which meet the specific requirements of a Phenyl isothiocyanate derivatives are sufficient as coupling reagents for Edman degradation and which allow an improvement in Edman degradation and in particular the detection limit significantly reduce for individual amino acids.

The halogen substituted phenyl isothiocyanates described herein have the following formula


wherein R ₁ to R _{5 are} each independently selected from the group comprising hydrogen, halogen, alkyl and haloalkyl, where halogen is independently selected from the group comprising fluorine, chlorine, bromine and iodine, alkyl is independently selected from the group comprising methyl, ethyl, n-propyl and isopropyl, in each case provided that at least one of the radicals R ₁ to R _{5 is} a halogen and / or contains a halogen. In the context of the present invention, it is provided that the halogen as such or the halogen in the haloalkyl is selected from the group of halogens mentioned above. Fluorine and chlorine are particularly preferred.

It is also within the scope of the present invention that the alkyl radical, be it the Alkyl radical as such or the alkyl radical of haloalkyl is selected from the group which Includes methyl, ethyl, n-propyl and isopropyl.

It is also within the scope of the present invention that the halogen directly to the Phenyl isothiocyanate, especially the phenyl ring, is bound. The haloalkyl includes preferably those alkyl radicals which are at least simple, but preferably two or are triple substituted. It is preferred that the double or triple substitution each used the same halogen. It is further preferred that two or three times Substitution takes place at the same carbon atom.

3,5-bis (trifluoromethyl) isothiocyanate is particularly preferred. The following statements made relate only to said 3,5-bis (trifluoromethyl) - Isothiocyanate. However, it is within the scope of the present invention that the in Connection with this connection specially made versions and described Advantages, embodiments, applications and advantages in general for the halogen substituted phenyl isothiocyanates as disclosed herein apply.

Combine the halogen substituted phenyl isothiocyanates described herein surprisingly, a multitude of those properties that can be seen from an "optimal" Edman reagent expected. To that extent, the use of those described herein Halogen-substituted phenyl isothiocyanates compared to those described in the prior art Edman's reagents surprisingly have clear advantages. The one here Halogen-substituted phenyl isothiocyanates described are described, for example in German Offenlegungsschrift 26 08 488. The use of the invention Halogen-substituted phenyl isothiocyanates described herein allow the implementation of Edman degradation to polypeptides or proteins in very low concentrations available. This enables a direct analysis of, for example, biological systems only weakly expressed polypeptides and proteins possible for the first time. Especially the analysis of poorly expressed polypeptides and proteins is common in modern biotechnology of particular interest, but also poses a problem in that the low concentrations in purification steps a selective purification of the polypeptides of interest is difficult to achieve and the specific losses here in particular to weigh heavily. In addition, it is reduced due to the lower limit of detection of polypeptide sequencing also possible, simplified polypeptide purification processes apply. This in turn results in further applications of polypeptide analysis using the Edman degradation, especially in the field of diagnostics and here again with high-throughput systems.

It is within the scope of the present invention that essentially the Edman degradation, such as he is described in the literature and known to the experts without further changes to the Experimental protocol except for the halogen substituted ones described herein Phenyl isothiocyanates instead of those otherwise used in the prior art Phenyl isothiocyanate derivatives can be carried out. This applies to both Preparation of the polypeptide, in particular its presentation in the reactor chamber used solvents and auxiliaries, the pH range, the Temperature range and after the derivatization of the amino acid, especially the N-terminal amino acid of a polypeptide with those described herein Halogen-substituted phenyl isothiocyanates in the subsequent reaction steps.

• 1. Bereitstellen eines zu sequenzierenden Polypeptids oder Proteins,
• 2. Umsetzen mit Kupplungsreagens, d. h. im vorliegenden Falle erfindungsgemäß unter Verwendung eines Halogen-substituierten Phenylisothiocyanates, wie hierin beschrieben,
• 3. Abspalten der endständig durch das Halogen-substituierte Phenylisothiocyanat derivatisierten Aminosäure,
• 4. Detektion der abgespaltenen endständigen derivatisierten Aminosäure, und
• 5. optional zyklisches Wiederholen der Schritte 2, 3 und 4.

Basically, Edman mining involves the following steps:

• 1. providing a polypeptide or protein to be sequenced,
• 2. Reaction with coupling reagent, ie in the present case according to the invention using a halogen-substituted phenyl isothiocyanate as described herein
• 3. cleaving off the amino acid derivatized by the halogen-substituted phenyl isothiocyanate,
• 4. Detection of the cleaved terminal derivatized amino acid, and
• 5. optional cyclical repetition of steps 2, 3 and 4.

Typically, after derivatization, the derivatized ones are extracted or separated Amino acid and then a measurement of the same. The said measurement can either be a direct measurement, or by conversion, for example by Isomerization in PTH and subsequent detection, or by conversion, for example by isomerization in PTH and additional derivatization and detection take place, or by conversion, for example by hydrolysis, in PTC and subsequent Detection take place, or by conversion, for example hydrolysis, in PTC and additional derivatization and detection.

In the reactions outlined above, the polypeptide is typically grown on a suitable carrier material bound covalently or non-covalently. suitable Carrier materials are glass, glass fibers, microporous glass balls, Plastic membranes (such as PVDF, polyethylene, Teflon and the like).

When using the halogen-substituted phenyl isothiocyanates according to the invention, As described herein, significant advantages also result from the low one Boiling point of these compounds. So it is possible that the clutch in the Excess, based on the amount of protein, added reagent after the reaction has taken place, also by an increase in temperature and an associated Evaporation of the reagent can be removed. Another benefit from the lesser Boiling point of the halogen-substituted phenyl isothiocyanates as described herein consists in the fact that the process control can be changed insofar as that after the Reaction of the isothiocyanate with the polypeptide chain the temperature in the reactor chamber can be increased. The volatile isothiocyanate evaporates, at least in part, whereby the subsequent washing steps, in which the reagent is additionally extracted, run more effectively. This form of carrying out the process shows that Edman sequencing through a lower amount of disruptive by-products improved analysis result.

The conversion of the amino acid, which is preferably carried out under basic conditions with one of the halogen-substituted phenyl isothiocyanates as described herein, is preferably carried out with the addition of water and / or an organic solvent. An example of such an organic solvent is ethanol. The one with the addition Base performed reaction can be a liquid or gaseous base include.

In the processes according to the invention, the halogen-substituted is typically used Phenyl isothiocyanate added in excess. Removal of the reagent follows preferably by extraction and / or evaporation, in principle also others Procedures can be applied as evaporation, however, due to the low Boiling point of the halogen-substituted phenyl isothiocyanates by means of removal To design temperature increases particularly efficiently. It is also under the present invention to perform a combination of extraction and evaporation.

If the reaction mixture is dried, this is typically done using an inert gas (Nitrogen or argon) is passed through the reaction vessel. Alternatively, too Drying is done by applying a vacuum. The subsequent washing step takes place preferably with organic solvents, such as ethyl acetate or Heptane. In general, solvents are preferred which do not dissolve the polypeptide or Wash. Another drying step then takes place, using or using of the measures described above.

In one embodiment it is provided that the anilinothiazolinone amino acid from the Reaction mixture extracted and transferred directly to a suitable detection system and is identified. The extraction takes place in that an organic solvent, such as for example, ethyl acetate and / or butyl chloride is passed through the reactor chamber. The cleaved amino acid dissolves, whereby the polypeptide shortened by one amino acid however, does not dissolve and remains in the reaction vessel. The extractant with the Amino acid is transferred to the detection system and the amino acid accordingly identified.

In a further embodiment it is provided that the anilinothiazolinone amino acid extracted from the reaction mixture and converted into a phenylthiohydantoin amino acid becomes. The extraction is carried out by an organic solvent, such as Ethyl acetate and / or butyl chloride is passed through the reactor chamber. The split off Amino acid dissolves, whereas the polypeptide shortened by one amino acid does not dissolves and remains in the reactor chamber. The extractant with the derivatized Amino acid is transferred to another reaction chamber, the so-called "converter". An aqueous acid, such as 25% trifluoroacetic acid, is metered in here, and the Isomerization reaction takes place.

In a further embodiment it is provided that the anilinothiazolinone amino acid extracted from the reaction mixture and into a phenylthiocarbamoyl amino acid (PTC-AS) is transferred. The extraction is done by using an organic solvent, such as ethyl acetate and / or butyl chloride is passed through the reaction vessel. The cleaved amino acid dissolves, whereas the polypeptide shortened by an amino acid does not dissolve and in the Reactor chamber remains. The extractant with the amino acid is in said another reaction chamber ("converter") transferred. Here, for example, water or an aqueous base is metered in and the hydrolysis reaction takes place. The PTC amino acid can subsequently identified directly or it is derivatized in a further step and then analyzed.

In a still further embodiment it is provided that the anilinothiazolinone Amino acid extracted from the reaction mixture and into a phenylthiohydantoin amino acid is transferred. The extraction is done by using an organic solvent, such as ethyl acetate and / or butyl chloride, is passed through the reaction vessel. The cleaved amino acid dissolves in contrast to the polypeptide shortened by an amino acid, which in the Reactor chamber remains. The extractant with the amino acid is converted into another Reactor chamber, the "converter" transferred. Here is an aqueous acid like for example 25% trifluoroacetic acid, added and the isomerization reaction takes place instead of. Then the phenylthiohydantoin amino acid in a further step derivatized. The derivatization takes place, as in the case of the PTC derivative Amino acid, e.g. B. in gas chromatography usual reagents, so that the double derivatized amino acid is accessible to a highly sensitive analysis method. there an easily ionizable compound can be coupled or formed, a fluorescent group can be coupled, or the boiling point of the compound through the Derivatization can be reduced.

A special advantage when using 3,5-bis (trifluoromethyl) - Phenyl isothiocyanate in the context of Edman degradation is in the detection or Identification step of those derivatized with 3,5-bis (trifluoromethyl) phenyl isothiocyanate To see amino acid. Although the methods described in the prior art, such as for example UV detection, [10, Wittmann-Liebold 1985] are basically used within the scope of the present invention, in particular more sensitive detection or detection methods possible. This is due to the fact that the invention 3,5-bis (trifluoromethyl) phenyl isothiocyanate has a high proportion of fluorine atoms in the Has molecule and this by suitable coupling to detectors, such as ECD (Electron Capture Detection) or by negative chemical ionization (NCI) or chemical ionization (CI) can be specifically detected. Instead of HPLC could also separate and characterize the amino acid derivatives obtained using gas chromatography with reference to standardized retention times respectively. The same applies in principle to those of those disclosed or described phenyl isothiocyanates, the fewer fluorine atoms, but still the have the above physicochemical properties.

It is within the scope of the present invention that the various amino acid derivatives be resolved using different separation principles and then, also using various, including physico-chemical, parameters be separated. In this respect, all possible combinations are fundamentally off Separation methods and detection methods possible, for example the use of Detectors such as ECD or NCI for separation using gas chromatography, gas-liquid Chromatography and liquid chromatography (reverse phase chromatography or Ion exchange chromatography). In the case of UV detection or ECD detection comes the essential information regarding the identity of the amino acid derivative from the Retention time.

In particular when using gas chromatography systems, it is necessary to: derivatized amino acids from Edman degradation (FM-TH-AS) to derivatize them to make the detection accessible. In general, it is with the invention Edman degradation possible, the resulting derivatized amino acids (in the configuration Anilinothiazolinon or Thiocarbamyl or Thiohydantoin) in a further step to derivatize that they have optimal properties for gas chromatographic analysis exhibit.

It is also within the scope of the present invention that the Edman degradation as automated method, as described herein in the introductory part, herein by Reference at this point should be taken up again and repeated, under Use of the halogen substituted phenyl isothiocyanates described herein, such as for example 3,5-bis (trifluoromethyl) phenyl isothiocyanate, as a "wet phase" process, is carried out as a "pulsed liquid" process or as a gas phase process.

With the "wet phase" process, the "pulsed liquid" process or the gas phase process Methods as can be used herein in the method according to the invention one or more reagents in the form of a small (preferably less than 10 ul, preferably less than 2 µl) in the form of a liquid drop ("wet phase"), in the form a spray ("pulsed liquid") or gaseous with a carrier gas (preferably Nitrogen or argon) metered into the reactor chamber in which the polypeptide is covalent or not covalently bound on a carrier material (preferably a glass fiber filter, one Membrane which preferably has hydrophobic properties, for example made of PVDF, or porous glass balls). The reagents are one of those herein described halogen-substituted phenyl isothiocyanates, (preferably dissolved in organic solvent), the base, preferably a nitrogen base, preferably a tertiary amine or a ring compound with a nitrogen atom contained in an organic Solvent or in water or a mixture of water and organic solvents can be dissolved, and the anhydrous acid, preferably trifluoroacetic acid. Here can any combination of the different dosing techniques for dosing the Reagents are used.

The invention is further illustrated below with the aid of the figures and examples, from which further features, embodiments and advantages of the invention result.

It shows

Fig. 1 shows the structural formula of 3,5-bis (trifluoromethyl) phenyl isothiocyanate,

Fig. 2A, the reaction of Ala with FM-PITC as a chromatogram,

FIG. 2B, the reaction of PITC with Ala and FM-PITC as a chromatogram,

Fig. 3A, the conversion of Arg with FM-PITC as a chromatogram,

Fig. 3B, the reaction of PITC with Arg and FM-PITC as a chromatogram,

Fig. 4A, the implementation of Asp with FM PITC as a chromatogram

FIG. 4B, the reaction of PITC with Asp and FM-PITC as a chromatogram,

Fig. 5A implementing Val with FM PITC as a chromatogram

Fig. 5B implementing Val with PITC and FM PITC as a chromatogram

Fig. 6A, the reaction of Ile with FM-PITC as a chromatogram,

Fig. 6B, the reaction of PITC with Ile and FM-PITC as a chromatogram,

Fig. 7 is a chromatogram of 3.5-bis (trifluoromethyl) phenyl isothiocyanate derivatized Arg, Asp, Ala, Val and Phe,

Fig. 8 is a chromatogram of derivatised with 3,5-bis (trifluoromethyl) phenyl isothiocyanate Ile, Pro, Met, Ser, and Lys,

As prepared in Example 1c), Fig. 9A shows a chromatogram of various alanine derivatives,

Fig. 9B is a chromatogram of various Ala-derivatives, prepared as in Example 1b),

FIG. 10A is a chromatogram of various Ala-derivatives, as prepared in example 1d), after five minutes,

FIG. 10B is a chromatogram of various Ala-derivatives, as prepared in example 1d), after ten minutes,

Fig. 10C is a chromatogram of various Ala-derivatives, as prepared in example 1d), after twenty minutes, and

FIG. 11A, the reaction of Lys with FM-PITC as a chromatogram, and

FIG. 11B, the implementation of Lys with PITC and FM-PITC as a chromatogram.

• - Lösung 1: Aminosäure-Lösung (c = 0,01 µmol/µl): freie Aminosäure gelöst in Pyridin/Wasser (1 : 1)
• - Lösung 2: PITC-Lösung: 5 Vol.-% Phenylisothiocyanat in reinem Acetonitril (ACN)
• - Lösung 3: 3,5-Bis(Trifluormethyl)-Phenylisothiocyanat (FM-PITC)-Lösung: 5 Vol.-% FM-PITC in reinem Acetonitril
• - Lösung 4: TMA-Lösung: 6 Vol.-% Trimethylamin in Ethanol
• - Lösung 5: TFA-Lösung: 40 Vol.-% Trifluoressigsäure

In the examples described below, various reagents and solutions were used, which are described in more detail below and are labeled in their composition. The implementation is not limited to the solvents or auxiliaries used, but can be carried out with conventional substitutes.

• - Solution 1: amino acid solution (c = 0.01 µmol / µl): free amino acid dissolved in pyridine / water (1: 1)
• - Solution 2: PITC solution: 5 vol.% Phenyl isothiocyanate in pure acetonitrile (ACN)
• - Solution 3: 3,5-bis (trifluoromethyl) phenyl isothiocyanate (FM-PITC) solution: 5 vol.% FM-PITC in pure acetonitrile
• - Solution 4: TMA solution: 6 vol .-% trimethylamine in ethanol
• - Solution 5: TFA solution: 40 vol .-% trifluoroacetic acid

example 1 Determining the clutch speed of FM-PITC experimental approaches a) Implementation of the free amino acid alanine with PITC

The procedure for the implementation of the free amino acid alanine with PITC was as follows:
Combine 40 µl solution 1, 40 µl solution 2 and 30 µl solution 4; mix; rinse with N ₂ ; Reaction at 55 ° C for 2 h; dry in a vacuum concentrator (Speed-Vac, Savant). Record for conversion to 50 µl ACN and add 50 µl solution 5; Hold reaction at 55 ° C for 30 min; dry in vacuum concentrator; take up in 1000 µl ACN / H ₂ O (1: 1); then suitable dilutions are made. The qualitative and quantitative determination is carried out by means of HPLC. The conditions for the HPLC measurements were identical in all experiments described here. An ABI 130a gradient system (Applied Biosystems, USA) as a pump and a UV detector type SPD 10A (Shimadzu, Japan) at a wavelength of 269 nm were used for the measurements. The data was recorded on the PC using an evaluation system from Knauer, Berlin. A column of the type YMC ODS AQ 150.2 mm, 3 μ 120A (YMC, Japan) was used for the chromatographic separation. The column was heated to 45 ° C. Buffer A 0.1% TFA in water, buffer B 0.08% TFA in acetonitrile, gradient% B 20% 0-8 min, injection at 8 min, 5B 20-60% 8-32.5 min,% was used B 60-80% 32.5-34 min.

b) reaction of the free amino acid alanine with FM PITC

The procedure for the implementation of the free amino acid alanine with FM-PITC was as follows:
Combine 40 µl solution 1, 40 µl solution 3 and 30 µl solution 4; mix; rinse with N ₂ ; Reaction at 55 ° C for 2 h; dry in vacuum concentrator.

For conversion: take up in 50 µl ACN + 50 µl solution 5; Reaction at 55 ° C for 30 min; dry in vacuum concentrator; take up in 1000 µl ACN / H ₂ O (1: 1); then suitable dilutions are made. The qualitative determination is carried out by means of HPLC as described under a).

A blank sample was used to verify by-products formed; this is under under the same conditions, a reaction with FM-PITC was carried out without an amino acid.

c) Kinetic analysis of the implementation of the free amino acid alanine with PITC

In order to investigate the kinetics of the reaction of the free amino acid alanine with PITC, the reaction was analyzed with a delayed addition of FM-PITC. The delayed addition of FM-PITC took place after 5, 10, 20, 30 and 60 minutes according to the following reaction scheme:
40 µl of solution 1 + 40 µl of solution 2 + 30 µl of solution 4 are placed in 5 reaction vessels; The reaction mixture was mixed, flushed with nitrogen and the reaction was carried out at 55 ° C. After 5, 10, 20, 30 and 60 min, 40 ul solution 3 are pipetted into the different reaction vessels. The reaction mixtures are kept at 55 ° C. for a total of 2 h. The procedure is continued as described under a) and b).

d) Kinetic analysis of the implementation of a free amino acid with FM PITC

In order to investigate the kinetics of the reaction of the free amino acid alanine with FM-PITC, the reaction was analyzed with a delayed addition of PITC to the reaction mixture described in b). The time-delayed addition of PITC took place after 5, 10, 20, 30 and 60 minutes in accordance with the following reaction scheme:
40 µl of solution 1 + 40 µl of solution 3 + 30 µl of solution 4 are placed in 5 reaction vessels; the reaction mixture was mixed, flushed with nitrogen and the reaction was carried out at 55 ° C. After 5, 10, 20, 30 and 60 min, 40 ul solution 2 are pipetted into the different reaction vessels. The reaction mixtures are kept at 55 ° C. for a total of 2 h. The procedure is continued as described under a) and b).

e) implementation of a free amino acid with simultaneous addition of FM PITC solution and PITC solution

For these investigations, solutions were prepared analogously to solution 1 of the following amino acids: isoleucine, alanine, valine, aspartic acid, lysine, arginine. The procedure for this reaction approach was as follows:
6 batches were carried out in parallel in 6 reaction vessels. Only one amino acid of the amino acids isoleucine, alanine, valine, aspartic acid, argenin and lysine was used in each reaction vessel. 40 .mu.l of the amino acid solution containing were combined with 40 .mu.l solution 3, 40 .mu.l solution 2 and 30 .mu.l solution 4. The respective reaction mixture was mixed, flushed with nitrogen and the reaction was then incubated at 55 ° C. for two hours. It was then dried in a vacuum concentrator (Speed-Vac). Here too, the procedure is as described under a) and b). The chromatograms 849, 852, 854, 856, 858, 860 shown in FIGS. 2B to 6B and 11B show the approach with simultaneous addition of the reagents. In comparison, the chromatograms 850, 851, 853, 855, 857, 859 shown in FIGS. 2A to 6A and 11A show the amino acids reacted only with FM-PITC, as described in b)).

Results

• a) Anhand der in a) beschriebenen Umsetzung einer freien Aminosäure, die als PTH- Aminosäure unter definierten Reaktionsbedingungen umgesetzt wurde, lassen sich die hierin verwendeten Reaktionsbedingungen überprüfen. Der gebildeten PTH-Aminosäure sowie Nebenprodukten lassen sich charakteristische Retentionszeiten zuordnen, beispielsweise durch Vergleich mit einem PTH-Aminosäure-Standard. Die Versuchsergebnisse bestätigen, dass die Reaktion sowohl qualitativ wie auch quantitativ möglich ist.
• b) Durch die in b) beschriebene Umsetzung einer freien Aminosäure mit dem erfindungsgemäßen 3,5-Bis(Trifluormethyl)-Phenylisothiocyanat (FM-PITC) unter den gleichen Reaktionsbedingungen wie bei Umsetzung der Aminosäure mit PITC konnte ebenfalls eine positive Reaktion in dem Sinne festgestellt werden, dass ein entsprechendes FM-PTH-Amionsäurederivat gebildet wurde und damit ein Nachweis bzw. eine Identifizierung einer Aminosäure unter Verwendung von 3,5-Bis(Trifluormethyl)- Phenylisothiocyanat. Der entsprechende Nachweis erfolgt mittels Chromatographie. Die chromatographische Messung der Blindprobe, die die entstandenen Nebenprodukte zeigt, bestätigen das vorstehende Ergebnis.
• c) Die in c) beschriebene kinetische Analyse der Umsetzung einer freien Aminosäure mit PITC und zeitlich verzögerter Zugabe von FM-PITC zeigt, dass überwiegend das PTH- Aminosäure-Derivat gebildet wird. Es werden bei der zeitlich verzögerten Zugabe von FM-PITC selbst nach 60 Minuten noch geringe Mengen von FM-PTH-Aminosäure 3,5- Bis(Trifluormethyl)-Phenylthiohydantoin-Aminosäure gebildet und sind detektierbar. (Chromatogramm 838).
• d) Darüber hinaus zeigt die anfängliche Umsetzung der freien Aminosäure mit FM-PITC, wie in d) beschrieben, dass schon nach wenigen Minuten der überwiegende Anteil an Aminosäure umgesetzt ist. Eine verzögerte Zugabe von PITC lässt erkennen, dass bereits nach fünf Minuten keine PTH-Aminosäure mehr detektierbar ist. (Chromatogramm 807).
• e) Die gleichzeitige Umsetzung der freien Aminosäuren mit FM-PITC und PITC, wie in Versuchsansatz e) beschrieben, zeigt, dass die Reaktion von FM-PITC mit einer freien Aminosäure schneller verläuft als mit PITC. Wie mittels chromatographischer Analyse des Reaktionsgeschehens gezeigt werden konnte, wurde im Vergleich zur PTH- Aminosäure eine deutlich größere Menge des FM-PTH-Aminosäure-Derivates detektiert. Hier bei konnte eine 3- bis 6fach höhere Konzentration festgestellt werden.

Based on the experimental approaches described above, the following can be stated:

• a) Using the conversion of a free amino acid described in a), which has been implemented as a PTH amino acid under defined reaction conditions, the reaction conditions used here can be checked. Characteristic retention times can be assigned to the PTH amino acid formed and by-products, for example by comparison with a PTH amino acid standard. The test results confirm that the reaction is possible both qualitatively and quantitatively.
• b) By reacting a free amino acid with the 3,5-bis (trifluoromethyl) phenyl isothiocyanate (FM-PITC) according to the invention described in b) under the same reaction conditions as when reacting the amino acid with PITC, a positive reaction in the sense was also found that a corresponding FM-PTH-amino acid derivative was formed and thus a detection or an identification of an amino acid using 3,5-bis (trifluoromethyl) phenyl isothiocyanate. The corresponding detection is carried out by means of chromatography. The chromatographic measurement of the blank sample, which shows the by-products formed, confirm the above result.
• c) The kinetic analysis of the reaction of a free amino acid with PITC and the delayed addition of FM-PITC described in c) shows that predominantly the PTH amino acid derivative is formed. With the delayed addition of FM-PITC, even after 60 minutes, small amounts of FM-PTH amino acid 3,5-bis (trifluoromethyl) phenylthiohydantoin amino acid are formed and are detectable. (Chromatogram 838).
• d) In addition, the initial conversion of the free amino acid with FM-PITC, as described in d), shows that the predominant amount of amino acid is converted after only a few minutes. A delayed addition of PITC shows that after five minutes no PTH amino acid can be detected. (Chromatogram 807).
• e) The simultaneous implementation of the free amino acids with FM-PITC and PITC, as described in experimental approach e), shows that the reaction of FM-PITC with a free amino acid proceeds faster than with PITC. As could be shown by means of chromatographic analysis of the reaction, a significantly larger amount of the FM-PTH amino acid derivative was detected compared to the PTH amino acid. A 3 to 6 times higher concentration was found here.

The result outlined above shows that the reaction of FM-PITC with the free Amino acid runs significantly faster than with PITC, is also important insofar as that is another step in automating Edman mining, in particular in the form of a high-throughput system, using 3.5 Bis (trifluoromethyl) phenyl isothiocyanate becomes possible.

Example 2 FM-PITC reactions with different amino acids

To demonstrate that the use of 3,5-bis (trifluoromethyl) - Phenyl isothiocyanate also with amino acids other than those described in Example 1 further amino acids with FM-PITC according to the following scheme implemented: The amino acids isoleucine, alanine, valine, aspartic acid, Argenin serine. Methionine proline, phenylalanine and lysine are used.

40 µl of the different amino acid solutions (c = 0.01 µmol / µl are pipetted into reaction vessels and 40 µl of solution 3 and 30 µl of solution 4 are added. This mixture is then mixed, flushed with nitrogen and the reaction at 55 ° C. incubated for one hour, the mixture is then dried and taken up for conversion into 50 μl of acetonitrile, 50 μl of solution 5 are then added to this mixture and the reaction is left to stand at 55 ° C. for 30 minutes, followed by drying again. The dried product is taken up in 1000 μl acetonitrile / H ₂ O (1: 1) with 0.1% TFA After the preparation of suitable dilutions, the FM-PTH-amino acid derivatives were determined qualitatively by means of HPLC under the conditions mentioned above. 7 and 8 show chromatograms FM-AS Standard 1 and FM-AS Standard 2 with 5 amino acids each from individual experiments superimposed in one image.

Results

The FM-PTH amino acid derivatives produced can be reproduced clearly from be distinguished from each other. The chromatograms show that every FM-PTH- Amino acid derivative is a characteristic peak with a defined retention time can be assigned.

Example 3 Manual Edman sequencing with FM-PITC using the example of different synthetic peptides

The manual sequencing was carried out based on [5]. Three synthetic peptides were used in three approaches for the investigation. peptide 1 has the sequence F-A-E-A-A-K-Y-A-S, peptide 2 has the sequence I-L-R-G-S-V-A-H-K, Peptide 3 has the sequence Y-T-D-P-N-T-F-S-S. The peptide was on a scale balanced and by adding the appropriate amount of 50% acetonitrile / water, 0.1% Trifluoroacetic acid diluted to a concentration of 50 µmol / µl. 40 µl of each Solution (n = 2 nmol) are pipetted into reaction vessels and vacuum dried.

Coupling reaction according to Edman degradation

20 .mu.l pyridine / water (1: 1) and 20 .mu.l 5vol .-% are added to the dried batch FM-PITC solution (in pure acetonitrile) given; the reaction takes place at 55 ° C for 40 min.

It is extracted twice with 80 μl of a mixture of heptane / ethyl acetate (2: 1), to remove excess reagent. The supernatant is discarded; the residue freeze-dried.

Spin-off according to Edman mining

5 μl of 100% trifluoroacetic acid are pipetted into the dried residue; the Reaction takes place at 55 ° C for 10 min .; then the reaction batch freeze-dried.

The residue is taken up in 30 ul water and twice with 40 ul each Butyl ester extracted. The extract with the cleaved amino acid derivative is in collected in a reaction vessel and dried in the Speed-Vac; that in the water leftover peptide is also freeze-dried and for the next Coupling reaction used.

Conversion according to Edman mining

The dried amino acid derivative is mixed with 10 ul 40% trifluoroacetic acid; offset; the reaction is carried out at 55 ° C for 30 min .; the reaction mixture is then by means of Speed-Vac dried.

The dried residue is dissolved in 20 ul 100% acetonitrile + 0.1% TFA and with another 20 μl of water are added, 2 μl of the solution are injected into the HPLC. The Determination takes place by means of HPLC under the running conditions mentioned above.

Result

Three degradation steps were carried out with the peptides. The (known) sequence The peptides of the synthetic peptides can be compared to those in Example 2 produced FM-PTH amino acid derivatives clearly verified. In the Chromatograms are superimposed on the three levels of degradation, which are amino acids characterized. The yield of the FM-PTH derivatives shows that the reaction and the Cleavage is almost complete. In the degradation steps n + 1 it is also possible no amino acid derivative from the preliminary degradation step, d. H. Dismantling step n, detect. A so-called overlap that is due to incomplete coupling or incomplete Separation would indicate here does not occur and thus confirms that of the invention underlying knowledge that the use of 3.5- Bis (trifluoromethyl) phenyl isothiocyanate allows optimization of Edman degradation.

Example 4 Edman sequencing with FM-PITC in the Knauer Sequencer Type 910, (Fa Knauer Wissenschaftlicher Gerätebau, Berlin) of synthetic peptides execution

From the above solution of peptide 2 with the sequence I-L-R-G-S-V-A-H-K (c = 50 µmol / µl) are pipetted onto a PVDF filter pretreated with Biobren. (Biobrene Plus, Applied Biosystems, USA, c = 10 µg / µl in 50% acetonitrile water, 3 µl on filter pipette and let dry. With the Knauer sequencer type 910 except for described modifications used the standard configuration [13]. With that, the Sequencer operated in the so-called wet phase mode (dosage of coupling reagent approx. 8 µl, Base approx. 4µl and acid approx. 3 µl as liquid drops in the reactor) as coupling reagent R1 is used instead of 5% PITC in heptane as a solution of 5% FM-PITC in heptane Solvent S3, with which the converted amino acid is redissolved and injected into the HPLC 50% acetonitrile / water is used. All other reagents and solvents are used as in the standard configuration. The current is for the dismantling Standard dismantling program used that belongs to [13] as an attachment. , , in the The program runs at the point where otherwise the automatic injection of the PTH Amino acid takes place in a detection system, the derivatized amino acid is collected and manually injected into an HPLC. The evaluation is carried out analogously to that in Example 3 described method.

The following HPLC system was used for detection purposes:
Pump ABI 130a gradient system, detector Shimadzu SPD 10A 269 nm, evaluation system Knauer HPLC-Box, Eurochrome software,
Column YMC ODS AQ 150.2 mm, 3µ 120A, oven 45 ° C, buffer A 0.1% TFA in water, buffer B 0.08% TFA in acetonitrile, gradient% B 0-8 min 20%, 8-32.5 min 20-60%, 32.5-34 min 60-80%.

The sequence of the peptide could be clearly identified in 6 stages of degradation the comparison of the sequence determined in Example 3) and the comparison with that in Example 2) synthesized derivatives emerges. It turns out that the reagent 3,5 Bis (trifluoromethyl) phenyl isothiocyanate for automatic execution in one Sequencing machines is ideal.

The literature references given here in square brackets are summarized below for the sake of clarity.
1. Kahn, K. (1995) From genome to proteome: looking at a cell's proteins. Science 270, 369-370.
2. Klose, J .; Kobalz, U. (1995) Two-dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome. Electrophoresis, 16, 1034-1059
3. Brockstedt, E., Rickers, A., Kostka, S., Laubersheimer, A., Dörken, B., Wittmann-Liebold, B., Bommert, K. and Otto, A. (1998) Identification of apoptosis- associated proteins in a human Burkitt Lymphoma cell line, cleavage of hnRNP A1 by caspase 3. J. Biol. Chem., 273, 28057-28064
4. Edman, P. (1950),. , ., Acta Chem Scand., 4, 283
5. Kamp, RM, Choli-Papadopoulou, T, Wittmann-Liebold, B .; (Eds.) 1997 Protein Structure Analysis, Springer Verlag Berlin,
6. Lottsspeich, F., Zorbas, H., (ed.) (1998) Bioanalytik, Spektrum Akad. Verlag,
7. Edman, P. and Begg, G. (1967) A protein sequenator. EurJBiochem 1, 80-97
8. Wittmann-Liebold, B patent specification
9. Wittmann-Liebold, B .; Graffunder, H. & Kohls, H. (1976) A device coupled to a modified sequenator for the automated conversion of anilinothiazolinones into PTH- amino acids. Analyte. Biochem. 75, 621-633;
10. Wittmann-Liebold, B .; Ashman, K. (1985) On-line detection of amino acid derivatives released by automatic Edman degradation of polypeptides. in Tschesche, H. (cd): Modern Methods in Protein Chemistry, Walter de Gruyter, Berlin, New York, 303-327
11. Hewick, R., M., Hunkapiller, MW, Hood, LE, Dreyer, WJ (1981) A gas-liquid solid phase peptide and protein sequenator. f Biol. Chem. 256, 7990-7997
12. Procise cLc manual, Applied Biosystems, USA
13. Handbook Knauer 910, Knaur Scientific Equipment, Berlin
14. Chang, JY, Brauer, D., Wittmann-Liebold, B. (1978) Micro-sequence analysis of peptides and proteins using 4-N, N-dimethylaminoazobenzene 4'-isothiocyanate / phenylisothiocyanate double coupling method. FEBS Lett., 93, 205-214
15. Hess, D., Nika, H., Chow, DT., Bures, EJ, Morrison, HD and Aebersold, R. (1995) Liquid chromatography-electrospray ionization mass spectrometry of 4- (3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins. Anal. Biochem, 224, 373-381
16. Jin, S.-W., Wittmann-Liebold, B. (1986): A New Sensitive Edman-Type Reagent: 4- (N-1-dimethylaminonaphthalene-5-sulfonylamino) phenyl isothiocyanate. Its Synthesis and Application for Micro-Sequencing of Polypeptides. FEBS Lett 198, 150
17. Wittmann-Liebold, B. (1986) Design of a multipurpose sequencer. In Shively, JE (ed): Methods in Protein Microcharacterization, Humana Press, Clifton, NJ, 249-277.

Those disclosed in the foregoing description, claims and drawings Features of the invention can be used both individually and in any combination Realization of the invention in its various embodiments may be essential.

Claims (28)

1. Use of a halogen-substituted phenyl isothiocyanate of the formula


wherein R ₁ to R _{5 are} each independently selected from the group comprising hydrogen, halogen, alkyl and haloalkyl,
where halogen is selected from the group comprising fluorine, chlorine, bromine and iodine, and
Alkyl is selected from the group consisting of methyl, ethyl, n-propyl and isopropyl;
provided that at least one of the radicals R ₁ to R _{5 is} a halogen and / or contains a halogen,
for the derivatization of amino acids, in particular of α-amino acids. 2. Use according to claim 1, characterized in that the haloalkyl radical with at least one halogen is substituted. 3. Use according to claim 1 or 2, characterized in that the Halogen-substituted phenyl isothiocyanate is selected from the group consisting of 2,4-bis (trifluoromethyl) - Phenyl isothiocyanate, 2,5-bis (trifluoromethyl) phenyl isothiocyanate and 3,5- Bis (trifluoromethyl) phenyl isothiocyanate. 4. Use of 3,5-bis (trifluoromethyl) phenyl isothiocyanate for the derivatization of Amino acids, especially α-amino acids. 5. Use according to one of claims 1 to 4, characterized in that the Amino acid is an N-terminal amino acid of a polypeptide. 6. Use of a halogen-substituted phenyl isothiocyanate of the formula


wherein R ₁ to R _{5 are} each independently selected from the group comprising hydrogen, halogen, alkyl and haloalkyl,
where halogen is selected from the group comprising fluorine, chlorine, bromine and iodine, and
Alkyl is selected from the group consisting of methyl, ethyl, n-propyl and isopropyl;
provided that at least one of the radicals R ₁ to R _{5 is} a halogen and / or contains a halogen,
for the analysis of polypeptides. 7. Use according to claim 6, characterized in that the haloalkyl radical with at least one halogen is substituted. 8. Use according to claim 6 or 7, characterized in that the Halogen-substituted phenyl isothiocyanate is selected from the group consisting of 2,4-bis (trifluoromethyl) - Phenyl isothiocyanate, 2,5-bis (trifluoromethyl) phenyl isothiocyanate and 3,5- Bis (trifluoromethyl) phenyl isothiocyanate. 9. Use of 3,5-bis (trifluoromethyl) phenyl isothiocyanate for the analysis of Polypeptides. 10. Use according to one of claims 6 to 9, characterized in that the Analysis is the sequence analysis of the polypeptide. 11. Use according to one of claims 6 to 11, characterized in that the Analysis carried out using Edman degradation. 12. Reaction product from a halogen-substituted phenyl isothiocyanate, in particular a phenyl isothiocyanate according to any one of claims 1 to 11, and an amino acid, preferably an α-amino acid. 13. Reaction product according to claim 12, characterized in that the amino acid at least one further amino acid is bound, preferably to a polypeptide. 14. A method for sequence determination of polypeptides comprising the following steps: - Providing the polypeptide and - Reacting the polypeptide with a halogen-substituted phenyl isothiocyanate according to one of claims 1 to 11. 15. The method according to claim 14, further comprising at least one of the following steps: - Cleavage of the terminally derivatized amino acid and - Detected the cleaved terminal derivatized amino acid. 16. The method according to claim 14 or 15, characterized in that the steps of Repositioning, splitting off and detection can be repeated at least once. 17. The method according to any one of claims 14 to 16, characterized in that the React at a temperature of about 20-95 ° C, preferably 20-85 ° C and preferably 40-45 ° C. 18. The method according to any one of claims 14 or 17, characterized in that the Implementation takes place under basic conditions, preferably using a Nitrogen base. 19. The method according to any one of claims 14 to 19, characterized in that the Reaction takes place at a pH of 7 to 14, preferably at a pH of 8 to 10. 20. The method according to any one of claims 8 to 10, characterized in that the after React the polypeptide with [insert bank 7 missing !!] phenyl isothiocyanate obtained Phenylthiocarbamoyl polypeptide, preferably after drying the phenylthiocarbamoyl Polypeptide reacted with anhydrous acid and thereby the N-terminal amino acid of Polypeptide as a heterocyclic derivative in the form of an anilinothiazolinone amino acid is split off. 21. The method according to claim 20, characterized in that the anilinothiazolinone Amino acid extracted from the reaction mixture and transferred directly to a detection system and is identified. 22. The method according to claim 20, characterized in that the anilinothiazolinone Amino acid extracted from the reaction mixture and into a phenylthiocarbamoyl Amino acid is transferred. 23. The method according to claim 20, characterized in that the anilinothiazolinone Amino acid extracted from the reaction mixture and into a phenylhydantoin amino acid is transferred. 24. The method according to any one of claims 14 to 23, characterized in that as a further Step detection and / or identification of the amino acid derivative is carried out. 25. The method according to claim 24, characterized in that the detection and / or Identify by means of a method that is selected from the group consisting of Reverse phase HPLC, in particular microbore reverse phase HPLC, gas chromatography and mass spectrometry. 26. The method according to any one of claims 14 to 25, characterized in that the Detection of the amino acid derivative using an ECD detector, UV detection, retention time, Mass information and / or retention time and mass information, or by means of negative chemical ionization takes place. 27. The method according to any one of claims 14 to 26, characterized in that the Polypeptide with a sequence reduced by one N-terminal amino acid with 3,5-bis- (Trifluoromethyl) phenyl isothiocyanate is reacted. 28. The method according to claim 27, characterized in that at least one of the in the Claims 14 to 27 provided steps are repeated.