WO2003031984A2 - Method for analysing the sequence of polypeptides - Google Patents

Abstract

The invention relates to the use of a halogen-substituted phenylisothiocyanate of formula (I): in which R1 to R5 respectively are selected independently of one another from the group containing hydrogen, halogen, alkyl and haloalkyl, whereby the halogen is selected from the group containing fluorine, chlorine, bromium and iodine and the alkyl is selected from the group containing methyl, ethyl, n-propyl and isopropyl; with the proviso that at least one of the groups R1 to R5 is a halogen and/or contains a halogen. Said halogen-substituted phenylisothiocyanate is used to obtain derivatives of and/or to identify primary or secondary amines.

Description

 Methods for sequence analysis of polypeptides

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

The sequence analysis of polypeptides and proteins is of central importance for modern biotechnology. Although with increasing knowledge of the genome of various organisms, including humans, the amino acid sequences of polypeptides and proteins can be predicted on the basis of the nucleic acid sequence, polypeptide sequence analysis is still of central importance in so far as that from a present sample the ones contained therein Polypeptides must be sequenced or at least sequenced in order to design a probe based on the N-terminus of the polypeptide which is then used for screening the nucleic acids using the extensive nucleic acid data base, be it in vivo, in vitro or in silico is used to determine the putative total nucleic acid sequence and thus also the total sequence of the protein.

In addition, there is still the need and the need to sequence polypeptides or proteins, in particular then, for example when looking for individual-specific deviations from the expected sequence or one which is particularly widespread in a population. In general, protein analysis has recently been increasingly used in so-called proteome analysis [1 Kahn, 1995]. The whole or parts, e.g. examines the core fraction, the expressed proteins of an organism or a cell type at a certain growth phase, under the influence of medication or in a pathologically altered state. The proteins are separated using known methods, for example two-dimensional gel electrophoresis [2 Klose 1995], and the proteins are made visible in the gel 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 e.g. identify the proteins that have a different expression rate or are modified by pathological changes in the cell [3 Brockstedt 1998].

A first method for sequence analysis of polypeptides is the Sanger method, in which l-fluoro-2,4-dinitrobenzene was used to label N-terminal amino acids. After the complete hydrolytic decomposition of the protein into individual amino acids, the labeled (terminal) amino acid was identified using chromatographic techniques. This method was associated with disadvantages, in particular in the case of larger peptides, which resulted from the fact that the polypeptides were broken down into small fragments via partial hydrolysis or enzymatic digestion, and then the N-terminal and C-terminal amino acid of the fragments were determined, and the amino acid composition was also determined by means of amino acid analysis had to become.

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

The conversion rates of the individual sub-steps, for example coupling, cleavage or extraction of the so-called Edman degradation, are of crucial importance for the analysis and identification of the polypeptide. For the overall process, the highest possible turnover rate, preferably of over 90% and preferably of over 95%, should be achieved in order to be able to clearly identify a large number of degradation stages. At lower conversion rates, the incomplete degradation means that in the subsequent degradation steps, the amino acid that is now split off is overlaid by the background of the amino acids from the previous steps, the so-called overlap, so that the sequence can no longer be clearly identified. An overview of protein sequencing can be found in [5,6]. The process was carried out manually until Edman and Begg introduced automatic sequencing devices in 1967 [7 Edman 1967]. Various technical improvements have been made to the machines over the years. Particularly noteworthy are the introduction of dead volume-free valve systems [8 Wittmann-Liebold 1973], with which the individual reagents and solvents are dosed in the machine in an air-tight manner and without liquid residues remaining in the lines. With the on-line conversion [9 Wittmann-Liebold 1976] and the on-line injection [9 Wittmann-Liebold 1985] the entire chemical process and not just a part of it could be fully automated. As a result, losses due to manual handling could be minimized and the oxygen-sensitive conversion could also be carried out entirely without air, which led to higher overall sales rates. With the development of the reactor cartridge and the associated introduction of gas phase sequencing, in which some reagents are dosed in gaseous form [11 Hewick 1981], it was possible to reduce the by-products of the reaction and thus make the analysis more sensitive. Ultimately, all technical improvements were aimed at increasing the sensitivity of the analysis. In the beginning, micromole amounts of a purified protein were required for a successful analysis. Analyzes in the range of a few picomoles are now routinely carried out. The actual chemical process has remained almost unchanged over the years.

For the identification and characterization of proteins and peptides by means of Edman degradation, protein sequencers are currently being used, such as, for example, the “Procise cLc” device from Applied Biosystems, Foster City, USA, or the “Knauer 910” device from Knauer Wissenschaftlicher Gerätebau, Berlin, used. (Technical descriptions of the devices can be found in the respective operating instructions [12, 13]).

The devices differ slightly in their technical design. Both devices have in common that the reagents and solvents are contained in a bottle battery. These are dosed into the reactor chamber and the converter chamber with the exclusion of air via a valve system. Both devices identify the cleaved amino acid with a liquid chromatograph with UV detection. The devices also differ slightly in the use of individual reagents or solvents. The use of the “Edman sequencing” method or Edman degradation with the “Edman reagent” phenyl isothiocyanate as the standard method is not only common to these devices, but to practically all devices currently on the market. However, there are a variety of small ones Process variants. The devices are also designed so that the user can make changes in the process according to their own ideas, such as using other solvents. The terms wet phase sequencing (Knauer) or gas phase and pulsed liquid sequencing (applied biosystems) mean that individual reagents are dosed as drops of liquid, as a fine spray or in gaseous form. Different dosage types can also be combined for the different reagents in the process. What is common to the processes is that the protein is immobilized in a reactor chamber on suitable membranes noncovalently, for example via hydrophobic interactions, and the reagents are metered into the reactor. The binding of the protein to the membrane is loosened or loosened by some reagents, so that an unfavorable process leads to a washing out of the protein and thus the sensitivity is reduced or the analysis cannot be continued. With the individual dosage types, the manufacturers have optimized the chemical reaction to high turnover rates and low washing out. In principle, all sequencing procedures can be carried out with small changes to the devices. Liquid phase sequencing or solid phase sequencing, in which the protein is covalently bound to carrier material, can also be carried out, albeit with restrictions.

The sequence of protein sequencing in a machine is generally as follows. The protein is taken up in a suitable solvent and applied to a filter. This filter consists, for example, of PVDF (polyvinylidene fluoride) (Sequelon membrane, Millipore, USA) or of pretreated glass fiber (Applied Biosystems, USA). The protein does not bind covalently, for example via hydrophobic interactions, so that it is immobilized on the filter or membrane. The filter or membrane with the protein is placed in a reactor chamber. The reagents and solvents are then dosed. In principle, a base (eg 6% trimethylamine in ethanol water (1: 1)) is metered in first. Then 5% phenyl isothiocyanate in heptane and again trimethylamine. The coupling reaction takes place in the reactor chamber (approx. 20 min at 45 ° C). The excess reagents are washed out with an organic solvent (e.g. ethyl acetate, heptane, butyl chloride). Anhydrous acid (for example 100% trifluoroacetic acid) is metered into the reactor chamber and the terminal amino acid is split off. The extracted derivatized amino acid is extracted with an organic solvent (eg butyl chloride, ethyl acetate). Here, the solvent is flushed through the reactor and in the converter in a small container such as caught in a vial. The amino acid-shortened protein is available in the reactor for the next degradation step. An aqueous acid (eg 25% trifluoroacetic acid in water) is metered into the converter. The isomerization reaction takes place at about 55 ° C for 30 min. The reagent is then dried and taken up in a suitable solvent (for example 20% acetonitrile in water) and injected into the HPLC system via an injection valve with six connections. The converter is now ready for the absorption of the amino acid which has been split off in the meantime and the process is repeated cyclically. A multitude of further drying, mixing or cleaning steps are required for the analysis in the entire process. A complete description can be found in the respective device documentation [12, 13].

As described in the original publication by Edman [4], phenyl isothiocyanate is still used as a coupling reagent. In the past, however, there have been numerous attempts to replace this reagent in order to achieve a higher sensitivity of the analysis. For example, the use of 4-N, N-dimethylaminoazobenzene 4'-isothiocyanate [14 Chang, 1978], a fluorescent isothiocyanate, or the use of 311-PITC, developed by Aebersold and co-workers for sensitive mass spectrometric detection was [15 Hess 1995].

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

Another disadvantage of some of the compounds presented is that although they have a comparatively high coupling rate, the splitting reaction proceeds much more slowly, so that the high conversion rates required for the overall process cannot be achieved for this reason. Some compounds such as 4- (Nl-dimethylaminonaphthalene-5-sulfonylamino) phenylisothiocyanates [16 Jin, 1986] have a high coupling rate and a high cleavage rate, but they have physico-chemical properties which are unfavorable for the overall process; So the fluorescent isothiocyanates are at room temperature and normal pressure as a solid and must be used for Reaction can be kept in solution in a suitable solvent. In the previously common liquid phase sequencing, which was carried out in the so-called spinning cup reaction compartments or in manual processes, this effect on the reaction rate only played a minor role. However, the technical circumstances of these analysis devices and the processes carried out therein, for example the comparatively high oxygen input during the process, limited their sensitivity.

If fluorescent isothiocyanates are used in the gas or wet phase sequencing devices used today, the following effect occurs during the coupling reaction: The coupling reagent, dissolved in suitable organic solvents (e.g. heptane), is metered into the reactor. In the subsequent drying step, the solvent partially evaporates. In the subsequent step, the base, for example 6% trimethylamine in 50% ethanol and 50%) water, is metered in. The isothiocyanate precipitates largely on the filter with the result that the reaction rate with the protein is considerably restricted. Another disadvantage here is that these physico-chemical properties also make it considerably more difficult to remove the excess reagent added to the reaction (e.g. due to the stationary charge of the coupling reagent used). In the process step of extracting the cleaved terminal amino acid, many of the proposed substitutes for PITC show unfavorable properties. The extraction is only incomplete due to the solubility properties of the isothiocyanates used and only a small proportion of the derivatized amino acid can be detected, which is why the detection limit is reduced overall based on the amount of protein used.

The present invention has for its object to provide a compound which can be used instead of the phenyl isothiocyanate used in the prior art in the context of Edman degradation and overcomes the disadvantages described in the prior art. In particular, it is an object of the present invention to provide such a compound which has at least several of the properties required for reliable use of Edman degradation, in particular in its automated form, namely a high reaction rate with primary amines, a high rate of elimination, one low melting point, solubility in organic and inorganic solvents and a low detection limit for amino acids derivatized with this reagent using a suitable detection method. Another object on which the present invention is based is that a reagent is provided which replaces the phenyl isothiocyanate used in the prior art in the context of Edman degradation and is intended to further reduce the detection limit for the overall process 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

(I)

wherein Rj 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] to R _{5 is} a halogen and / or contains a halogen,

for derivatization and / or detection of primary and / or secondary amines. In one embodiment it is provided that the haloalkyl radical is substituted with at least one halogen.

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 includes.

The object is achieved in a second aspect by the use of 3,5-bis (trifluoromethyl) phenyl isothiocyanate for the derivatization and / or for the detection of primary and / or secondary amines.

In a second aspect, the object is achieved by an adduct of 3,5-bis (trifluoromethyl) phenyl isothiocyanate and an amino acid.

In embodiments according to the first and second aspects of the present invention it is provided that the primary amine is an amino acid, preferably an α-amino acid. Alternatively, the primary and / or secondary amine is a biogenic amine or a chemical compound which contains an amino group as a functional group. It is fundamentally within the scope of the present invention that the amino acid, like the primary or secondary amine, can be in the L or D form.

In one embodiment of the first and second aspects of the present invention it is 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

(I)

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] to R is a halogen and / or contains a halogen,

for the analysis of polypeptides.

These halogen-substituted phenyhsothiocyanates are also referred to herein as "halogen-substituted phenyhsothiocyanates, as described herein" and are referred to using this term.

In one embodiment it is provided that the haloalkyl radical is substituted with at least one halogen.

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) - Includes phenyl isothiocyanate.

In one embodiment it is provided that 3,5-bis (trifluoromethyl) phenyl isothiocyanate is used for the analysis of polypeptides.

In a further embodiment it is provided that the analysis is the sequence analysis of the polypeptide. In a still further embodiment it is provided that the analysis is carried out by means of Edman degradation.

In a further embodiment it is provided that the derivatized amine is detected and / or identified as a further step, as is provided in the context of the method according to the invention. Derivatized amine as used herein is the primary or secondary amine which is reacted with the halogen substituted phenyl isothiocyanate as described herein, or the reaction product thereof.

In a further embodiment it is provided that the detection and / or identification is carried out by means of a method which is selected from the group comprising reverse phase HPLC, gas chromatography and mass spectrometry. Microbore reverse phase HPLC is particularly preferred.

In yet another embodiment it is provided that the derivatized amine is detected by means of OCD detection and / or UV detection and retention time. It is also provided that the detection is carried out using mass information and / or retention time. Finally, it is also provided that the detection is carried out by means of mass information and / or retention time and mass information, the mass information being obtained by ionizing the molecules by means of negative chemical ionization.

In ^'Λveiteren embodiments it is provided that the detection means of ECD-detection and / or UV detection and retention time is carried out. Alternatively, it can be provided that the detection is carried out by means of mass spectrometry alone, or by means of separation and mass spectrometry. In the event that mass spectrometry is used, the ionization required is preferably carried out by negative chemical ionization.

In a fourth aspect, the object is achieved by a reaction product of a halogen-substituted phenyl isothiocyanate, in particular a halogen-substituted phenyl isothiocyanate, as described herein, and a primary and / or secondary amine, preferably an amino acid and preferably an α-amino acid.

In one embodiment it is provided that the amino acid is bound to at least one further amino acid, preferably to a polypeptide. In a fifth aspect, the object is finally achieved 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.

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

Cleavage of the terminal derivatized amino acid and detection of the cleaved terminal derivatized amino acid.

In a further embodiment it is provided that the steps of converting, splitting off and detecting are repeated at least once.

In a still further embodiment it is provided that the reaction takes place at a temperature of approximately 20-95 ° C., preferably 20-85 ° C. and preferably 40-45 ° C.

In one embodiment it is further provided that the reaction takes place under basic conditions, preferably using a nitrogen base.

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

In a further embodiment it is provided that the phenylthiocarbamoyl polypeptide obtained after reacting the polypeptide with phenylisothiocyanate, preferably after drying the phenylthiocarbamoyl polypeptide, is reacted with anhydrous acid and the N-terminal amino acid of the polypeptide as a heterocyclic derivative in the form of an anilinothiazolinone becomes. In yet another embodiment, the anilinothiazolinone amino acid is extracted from the reaction mixture and transferred directly to a detection system and identified.

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

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

In one embodiment it is provided that the amino acid derivative is detected and / or identified as a further step.

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

In one embodiment, the amino acid derivative is detected by means of an 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 reacted with 3,5-bis (trifluoromethyl) phenyl isothiocyanate with a sequence reduced by one N-terminal amino acid.

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

The present invention is based on the surprising finding that with the halogen-substituted phenyl isothiocyanates described herein, it was possible to determine and provide reagents which meet the specific requirements of a phenyl isothiocyanate derivative as a coupling reagent for Edman degradation and which allow an improvement in Edman degradation and in particular significantly reduce the detection limit for individual amino acids.

The halogen-substituted phenysothiocyanates described herein have the following formula

(I)

in which R] to R _{5 are} each independently selected from the group comprising hydrogen, halogen, alkyl and haloalkyl.

In preferred embodiments, 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 RI to R5 is a halogen and / or contains a halogen. In the context of the present invention, it is provided in a preferred embodiment 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 comprising methyl, ethyl, n-propyl and isopropyl.

It is also within the scope of the present invention that the halogen is bonded directly to the phenyl isothiocyanate, in particular the phenyl ring. The haloalkyl preferably comprises those alkyl radicals which are at least monosubstituted, but preferably are substituted twice or three times. It is preferred that the double or triple substitution in each case uses the same halogen. It is further preferred that the double or triple substitution takes place on the same carbon atom.

The use of 3,5-bis (trifluoromethyl) isothiocyanate is particularly preferred. The statements made below relate only to said 3,5-bis (trifluoromethyl) isothiocyanate. However, it is within the scope of the present invention that the particulars made and described advantages, embodiments, applications and advantages in connection with this compound apply in general to the halogen-substituted phenylthothiocyanates as disclosed herein.

The halogen-substituted phenyhsothiocyanates described herein surprisingly combine a large number of those properties which are expected from an “optimal” Edman reagent. To this extent, the use of the halogen-substituted phenyhsothiocyanates described here is surprisingly clear compared to the Edman reagents described in the prior art The halogen-substituted phenyhsothiocyanates described here are described, for example, in German Offenlegungsschrift 26 08 488. The use according to the invention of the halogen-substituted phenyhsothiocyanates described herein enables Edman degradation to be carried out on polypeptides or proteins which are present in very low concentrations. This makes it possible for the first time to directly analyze, for example, poorly expressed polypeptides and proteins in biological systems, especially the analysis of poorly expressed polypeptides and proteins one is of particular interest in modern biotechnology, but is also a problem insofar as the low concentrations in the purification steps make it difficult to selectively purify the polypeptides of interest and the specific losses here weigh particularly heavily. In addition, the reduced detection limit in the context of polypeptide sequencing also makes it possible to use simplified polypeptide purification procedures. This in turn results in further applications of polypeptide analysis using Edman degradation, in particular in the field of diagnostics and here again in systems with a high throughput, i.e. H. so-called high-throughput systems.

It is within the scope of the present invention that essentially the Edman degradation, as described in the literature and known to those skilled in the art, without further changes to the test protocol with the exception of the halogen-substituted ones described herein Phenyhsothiocyanate can be carried out instead of the Phenyhsothiocyanatderivate otherwise used in the prior art. This applies both to the preparation of the polypeptide, in particular its presentation in the reactor chamber, the solvents and auxiliaries used, the pH value range, the temperature range and the implementation of the after the derivatization of the amino acid, in particular the N-terminal amino acid of a polypeptide the halogen-substituted phenyl isothiocyanates described herein in the subsequent reaction steps.

Basically, Edman mining involves the following steps:

1. providing a polypeptide or protein to be sequenced,

2. React with coupling reagent, d. H. 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 the cleavage, the derivatized amino acid is extracted or separated and subsequently measured. Said measurement can either be a direct measurement, or be carried out 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, 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 described above, the polypeptide is typically covalently or non-covalently bound to a suitable carrier material. 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 phenylsothiocyanates according to the invention, as described herein, significant advantages also result from the low boiling point of these compounds. It is thus possible that the reagent added in the coupling in excess, based on the amount of protein, after the reaction has taken place, can also be removed by increasing the temperature and associated evaporation of the reagent. Another advantage of the lower boiling point of the halogen-substituted phenylthothiocyanates, as described herein, is that the process control can be modified insofar as the temperature in the reactor chamber can be increased after the isothiocyanate has reacted with the polypeptide chain. The volatile isothiocyanate evaporates, at least in part, making the subsequent washing steps, in which the reagent is additionally extracted, more effective. Due to this form of process control, the Edman sequencing according to the invention has an improved analytical result due to a smaller amount of disruptive by-products.

The reaction of the amino acid, which is preferably carried out under basic conditions, with one of the halogen-substituted phenylsothiocyanates, as described here, is preferably carried out with the addition of water and / or an organic solvent. An example of such an organic solvent is ethanol. The reaction carried out with the addition of a base can comprise a liquid or gaseous base. Suitable bases are, for example, those selected from the group referred to as nitrogen bases, that is, for example, the tertiary amines, for example trimethylamine or triethylamine, bases from the group of the morpholines, for example 4-methylmorpholine, bases from the group, the pyrrolidones, Includes pyridines and pyrimidines. Bases which do not contain nitrogen can also be used for the reaction. It is also within the scope of the present invention to use a mixture of two or more bases.

In the processes according to the invention, the halogen-substituted phenyl isothiocyanate is typically added in excess. Removal of the reagent is preferably carried out by extraction and / or evaporation, although processes other than evaporation can in principle also be used, but the removal by means of an increase in temperature is the result of the low boiling point of the halogen-substituted phenysothiocyanates to design particularly efficiently. It is also within the scope of the present invention to carry out a combination of extraction and evaporation.

If the reaction mixture is dried, this is typically done by passing an inert gas (nitrogen or argon) through the reaction vessel. Alternatively, drying can also be done by applying a vacuum. The subsequent washing step is preferably carried out using organic solvents, such as, for example, ethyl acetate or heptane. Solvents that do not dissolve or wash out the polypeptide are generally preferred. A further drying step then takes place using or using the measures described above.

In one embodiment it is provided that the anilinothiazolinone amino acid is extracted from the reaction mixture and transferred directly to a suitable detection system and identified. The extraction is carried out by passing an organic solvent, such as ethyl acetate and / or butyl chloride, through the reactor chamber. The amino acid split off dissolves, but the polypeptide shortened by one amino acid 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 is identified accordingly.

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

In a further embodiment it is provided that the anilinothiazolinone amino acid is extracted from the reaction mixture and converted into a phenylthiocarbamoyl amino acid (PTC-AS). The extraction is carried out by passing an organic solvent, such as ethyl acetate and / or butyl chloride, through the reaction vessel. The split off amino acid dissolves whereas the amino acid truncated polypeptide does not dissolve and remains in the reactor chamber. The extractant with the amino acid is transferred to said further reaction chamber (“converter”). Here, for example, water or an aqueous base is metered in and the hydrolysis reaction takes place. The PTC amino acid can then be identified directly or it is derivatized in a further step and then analyzed.

In yet another embodiment, it is provided that the anilinothiazolinone amino acid is extracted from the reaction mixture and converted into a phenylthiohydantoin amino acid. The extraction is carried out by passing an organic solvent, such as ethyl acetate and / or butyl chloride, through the reaction vessel. The split off amino acid dissolves in contrast to the polypeptide shortened by one amino acid, which remains in the reactor chamber. The extractant with the amino acid is transferred to another reactor chamber, the “converter”. Here, an aqueous acid, such as 25% trifluoroacetic acid, is added and the isomerization reaction takes place. The phenylthiohydantoin amino acid is then derivatized in a further step As in the case of the PTC derivative of the amino acid, derivatization is carried out using reagents that are customary in gas chromatography, for example, so that the doubly derivatized amino acid is accessible to a highly sensitive analysis method, in which case an easily ionizable compound can be coupled or formed, a fluorescent one Group are coupled, or the boiling point of the compound can be reduced by the derivatization.

A particular advantage when using 3,5 - bis (trifluoromethyl) phenyl isothiocyanate in the context of Edman degradation is the detection or identification step of the amino acid derivatized with 3,5 - bis (trifluoromethyl) phenyl isothiocyanate. Although the methods described in the prior art, such as UV detection, [10, Wittmann-Liebold 1985] can in principle be used, more sensitive detection or detection methods are possible within the scope of the present invention. This is due to the fact that the 3,5-bis (trifluoromethyl) phenyl isothiocyanate according to the invention has a high proportion of fluorine atoms in the 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, a separation and characterization of the amino acid derivatives obtained by means of gas chromatography with reference to standardized could Retention times take place. The same also applies in principle to those of the phenysothiocyanates disclosed or described herein which have fewer fluorine atoms but still have the above physico-chemical properties.

It is within the scope of the present invention that the different amino acid derivatives are dissolved using different separation principles and then, also using different, among other things, physico-chemical parameters. In this respect, all possible combinations of separation methods and detection methods are fundamentally possible, for example the use of detectors such as ECD or NCI in the case of separation by means of gas chromatography, gas-liquid chromatography and

Liquid chromatography (reverse phase chromatography or ion exchange

Chromatography). In the case of UV detection or ECD detection, the essential information regarding the identity of the amino acid derivative comes from the retention time.

In particular when using gas chromatography systems, it is necessary to derivatize the derivatized amino acids from Edman degradation (FM-TH-AS) in order to make them accessible for detection. In general, it is possible in the Edman degradation according to the invention to derivatize the resulting derivatized amino acids (in the configuration anilinothiazolinone or thiocarbamyl or thiohydantoin) in a further step in such a way that they have optimal properties for gas chromatographic analysis.

It is further within the scope of the present invention that the Edman degradation is described as an automated process as described in the introductory part, which is to be considered as repeated and repeated here by reference at this point, using the halogen-substituted phenyl isothiocyanates described herein, such as for example 3,5 - bis (trifluoromethyl) phenyl isothiocyanate, as a "wet phase" process, as a "pulsed liquid" process or as a gas phase process.

In the “wet phase” method, the “pulsed liquid” method or the gas phase method, as can be used here in the method according to the invention, one or more reagents are in the form of a small (preferably less than 10 μl, preferably less than 2 μl) ) in the form of a liquid drop ("wet phase"), in the form of a spray ("pulsed liquid") or in gaseous form with a carrier gas (preferably nitrogen or argon) metered into the reactor chamber in which the polypeptide is covalently or non-covalently bound on a carrier material (preferably a glass fiber filter, a membrane which preferably has hydrophobic properties, for example made of PVDF, or porous glass balls). The reagents here are one of the halogen-substituted phenysothiocyanates described here (preferably dissolved in organic solvent), the base, preferably a nitrogen base, preferably a tertiary amine, such as trimethylamine or triethylamine, or a ring compound with a nitrogen atom, in an organic solvent or can be dissolved in water or a mixture of water and organic solvents, and the anhydrous acid, preferably trifluoroacetic acid. Any combination of the different metering techniques can be used for metering the reagents.

As can be seen from the above illustration, the halogen-substituted phenyl isothiocyanate disclosed herein can react with a primary amine of a peptide, in particular from the terminal amino acid of the peptide and, after cleavage of the said terminal amino acid, can be detected selectively. It is within the scope of the present invention that the reaction with the halogen-substituted phenyl isothiocyanate described herein is not limited to the reaction with peptides. Rather, a reaction generally takes place with primary or secondary amines. In the broadest sense, this reaction is therefore a derivatization reaction of primary and / or secondary amines. With the derivatization of primary and secondary amines according to the invention, it is possible to specifically analyze said primary and secondary amines from or in a mixture of substances, in particular in a mixture of these classes of substances. With the derivatization, the polarity of those containing the primary or secondary amine group _. Substance reduced so that it is or becomes more accessible for gas chromatographic analysis. Due to the electronegative properties of the derivatization reagent, ie the halogen-substituted phenylisothiocyanate described herein, due to the halogen atoms, the reagent is particularly suitable for detection with electron capture detectors or for detection by means of negative chemical ionization and wet spectrometric detection. With this selective detection, the ratio of signal to noise is increased many times over, so that a much more sensitive analysis of the target substances, ie of primary and secondary amines, is made possible. Using the halogen-substituted phenyl isothiocyanate described here, the detection of primary and secondary amines is to be carried out down to the attomole range or in even smaller amounts, and thus ultimately possible to identify the primary or secondary amine containing compound. With the use according to the invention of the halogen-substituted phenyhsothiocyanates described herein, primary and secondary amines are now accessible for direct gas chromatographic measurement.

The derivatization or the method for the detection of primary and / or secondary amines is carried out according to the following steps:

a) providing a sample,

b) optionally carrying out a purification step with the aim of removing certain components of the sample or increasing the proportion of primary and / or secondary amines to be derivatized or detected,

c) provision of a suitable solvent,

d) optionally adding a nitrogen ase,

e) adding the isothiocyanate to the reaction batch and allowing the sample or reaction batch to react,

f) optionally removing the excess reagent and / or the auxiliary substances, and

g) Analysis of the reaction mixture thus obtained.

In principle, the sample to be analyzed can be any sample. Accordingly, the sample can come from the technical or analytical area. The analytical area includes in particular the areas of biology, biochemistry and medicine as well as forensics as well as that of process technology. The samples are preferably of biological origin and in particular blood, urine, stool, lymph fluid or cerebrospinal fluid. However, the sample can also be an air sample, a soil sample, or a water sample. Finally, the sample can also be a solid substance, such as hair or a solid sample in general. The amine contained in the sample, in particular primary or secondary amine, or presumably contained therein primary and / or secondary amine, can preferably be such an amine as is used in starting materials for the production of industrial products, such as solvents, for example. Textile and flotation aids, invert soaps, surfactants, bactericides, corrosion inhibitors, anti-foaming agents and additives are included. However, amines as they can be detected or detected according to the invention also represent constituents or reactive groups of pharmaceuticals, including drugs. Amines as they can be detected or detected according to the invention also represent, for example, various metabolic products. Of particular interest in In connection with the present invention are primary and secondary amines which are biogenic amines.

Biogenic amines are primary mono- and diamines and, in the broader sense, certain derivatives thereof. Such biogenic amines are typically formed in the organism by decarboxylation of amino acids. In the context of the present invention, biogenic amines can be detected using the halogen-substituted phenyhsothiocyanates described herein and thus allow conclusions to be drawn about the state of an organism, in particular the presence of desired or undesired biogenic amines, which in turn allow conclusions to be drawn about diseases or disease states. It is also possible to monitor the success of the therapy or to determine the pharmacokinetics of active substances containing primary or secondary amines. Using the halogen-substituted phenylisothiocyanates disclosed herein and the corresponding methods for derivatizing or detecting primary and / or secondary amines, the following biogenic amines can be detected particularly preferably or the samples containing them can be analyzed using the method according to the invention. Tyramine, dopamine, noradrenaline and adrenaline, which are neurotransmitters, hormones or tissue hormones and which are the decarboxylation product of the amino acid tyrosine. Tryptamine, serotonin and melatonin, which are neurotransmitters, hormones and tissue hormones and which are the decarboxylation product of tryptophan. Histamine, which is a tissue hormone and is involved in a variety of reactions, including allergic reactions, and is the decarboxylation product of the amino acid histidine. Cysteamine, which is an essential component of co-enzyme A and the decarboxylation product of cysteine. Betaalanin, which is also a building block of coenzyme A and the decarboxylation product of the amino acid Is asparagine. Gamma aminobutyric acid, which is an important neurotransmitter and the decarboxylation product of glutamic acid. Propanolamine, which has an essential function as a cobalamin and is the decarboxylation product of threonine. Ethanolamine, which is an essential component of the phosphatides, which in turn are involved in the construction of the cytoplasmic membrane, and the decarboxylation product of serine. Cadaverine, which is a bacterial breakdown product of lysine. Spermidine, spermine and putrescine, which are involved in the regulation of DNA and RNA synthesis and cell proliferation and are the decarboxylation product of the amino acid omithin. Agmatine, which is a bacterial degradation product and the decarboxylation product of arginine. ß-Alanine, which arises from L-aspartic acid and is important as a precursor for pantothenic acid. Aminoacteon, which arises from L-2-aminoacetoacetic acid and is a precursor for vitamin B12. 5- Amino-4-oxovaleric acid, which is formed from succinylglycine and is a precursor for porphyrins. Cadaverine, which is made from L-lysine and is a precursor for alkaloids. Dopamine, which is derived from L-dopa and is a neurotransmitter, precursor for the catecholamines, L-noradrenaline and L-adrenaline, and is also a proto-alkaloid. Serotonin, which arises from 5-hydroxy-L-tryptophan and is a neurotransmitter and precursor of the hormone melatonin. Tryptamine, which arises from L-tryptophan and causes contraction of the smooth muscles and has a growth-promoting effect on plants. Tyramine, which arises from L-tyrosine and causes the contraction of the smooth muscles.

In addition, there are a large number of other biogenic amines for which the use according to the invention or the method according to the invention is suitable. If there is a causal connection between the primary or secondary amine to be detected according to the invention and a disease, the corresponding method can be used as a diagnostic method for the respective disease.

The purification steps optionally carried out in accordance with step b) have the aim of removing any constituents of the sample which interfere with the reaction or of increasing the concentration of the primary and / or secondary amines to be detected or derivatized in a preliminary step. For example, phase separation can take place in the event that the sample is a dispersion or contains larger solids. The purification is carried out according to principles which are known to the person skilled in the art, extraction being possible, for example, with the aim of incorporating the primary and secondary amines into a corresponding solvent phase. In step c), the sample is then taken up in a suitable solvent, in which the reaction with the halogen-substituted phenylisothiocyanate according to the invention should ultimately take place. In principle, a large number of solvents are suitable, the suitability of the particular solvent being essentially determined by the amine to be specifically detected. As an example, reference is made here to the solvents mentioned in connection with the method according to the invention for the sequencing of proteins or peptides, which are in principle also suitable for this application. A suitable solvent can be, for example, a mixture of 50% pyridine, 6% trimethylamine and 44% water. The isothiocyanate is preferably present in excess.

With the optional addition of a nitrogen base according to step d), those bases can in principle be used, as are also used in connection with the method according to the invention for the sequence analysis of polypeptides. Nitrogen bases such as trimethylamine, triethylamine or pyridine are particularly preferred.

With the addition of the isothiocyanate to the optionally prepared sample, the actual reaction takes place, which is carried out under normal conditions. Usual conditions refer to z. B. 50 ° C to 55 ° C for 30 to 40 min, preferably 50 ° C for 40 min.

After step e), in particular after the reaction mixture has been reacted, it can optionally be provided that an isomerization reaction of the halogen-substituted phenylisothiocyanate used takes place. This reaction generally increases the sensitivity of detection. Reaction conditions which lead to isomerization of the halogen-substituted phenylisothiocyanate reacted with the amine are known to those skilled in the art and are also described, for example, in connection with the method according to the invention for the sequence analysis of polypeptides. For example, the isomerization can be carried out with 25% trifluoroacetic acid, preferably after the reaction mixture has dried.

The analysis according to step g) will in particular be carried out using the derivatized amine, the respective amine being dried before being taken up in a solvent suitable for the respectively selected analytical method. When using a Gas chromatography system is preferred, for example, a redissolution of the dried amine in 70% acetone or in pure acetone. In the gas chromatographic system, the respective amine can be identified as a derivative by comparing the retention times or by means of mass spectrometric determination. The actual detection can take place both in the context of the method according to the invention for determining the sequence of polypeptides and in the derivatization and / or detection of primary and / or secondary amines according to the invention also by means of UV or fluorescence detection. The derivatization ensures that when an electron capture detector (ECD detector) is used, the molecule contains at least one functional group that has strongly electronegative properties. The electron capture detector is a specific detector that has high response with electronegative elements or electronegative functional groups such as fluorine (-F), chlorine (-C1) or bromine (-Br) or nitro groups (-NO ₂ ) , ie a high signal response, so that these molecules are selectively detected.

The detection can also take place in a mass spectrometer, preferably in a mass spectrometer which is equipped with an ion source for the so-called negative chemical ionization. In this process, which is described in detail in the literature, preference is given to ionizing molecules which contain electronegative functional groups such as, for example, fluorine (-F), chlorine (-C1) or bromine (-Br) substitutes or nitro groups (-NO ₂ ). The derivatized substances can thus be selectively detected with an exceptionally high sensitivity

As used herein, the term primary and / or secondary amine means any chemical compound that contains, as a functional group, at least one primary and / or secondary amine or amino group. It is within the scope of the definition that the compound can also have one or more other functional groups.

The reaction conditions described in connection with the method according to the invention for determining the sequence of polypeptides basically also apply to the use of the halogen-substituted phenyl isothiocyanate, as described herein, for the derivatization and / or for the detection of primary and / or secondary amines. 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

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

2A shows the implementation of Ala with FM-PITC as a chromatogram,

2 B shows the implementation of Ala with PITC and FM-PITC as a chromatogram,

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

3 B shows the conversion of Arg with PITC and FM-PITC as a chromatogram,

4 A shows the implementation of Asp with FM-PITC as a chromatogram,

4 B shows the implementation of Asp with PITC and FM-PITC as a chromatogram,

5 A shows the implementation of Val with FM-PITC as a chromatogram,

5 B shows the implementation of Val with PITC and FM-PITC as a chromatogram,

6 A shows the implementation of Ile with FM-PITC as a chromatogram,

6 B shows the implementation of Ile with PITC and FM-PITC as a chromatogram,

7 shows a chromatogram of Arg, Asp, Ala, Val and Phe derivatized with 3,5-bis (trifluoromethyl) phenyl isothiocyanate, FIG. 8 shows a chromatogram of Ile, Pro, Met derivatized with 3,5-bis (trifluoromethyl) phenyl isothiocyanate. Ser and Lys, FIG. 9A shows a chromatogram of various alanine derivatives as produced in Example lc), FIG. 9B shows a chromatogram of various Ala derivatives as produced in Example lb),

10A shows a chromatogram of various Ala derivatives as produced in Example ld) after five minutes, FIG. 10B shows a chromatogram of various Ala derivatives as produced in Example Id) after ten minutes, FIG. 10C shows a chromatogram of different Ala Derivatives as prepared in Example ld) after twenty minutes, and FIG. 11 A shows the reaction of Lys with FM-PITC as a chromatogram, FIG. 11 B shows the reaction of Lys with PITC and FM-PITC as a chromatogram, FIG. 12 a / b the detection of FM thiohydantoin valine by means of electron impact ionization in the

Selected ion mode, Fig. 13 a / b Detection of FM thiohydantoin valine by means of negative chemical ionization in the selected ion mode, 14 detection of FM thiohydantoin valine by means of electron capture detection,

Fig. 15 a / b detection of FM thiohydantoin isoleucine using negative chemical

Ionization in scan mode Fig. 16 a / b detection of FM thiohydantoin glutamine by means of negative chemical

Ionization in scan mode, Fig. 17 a / b step 1 of the sequence analysis of β-lactoglobulin and detection of FM

Thiohydantoin leucine by means of negative chemical ionization, Fig. 18 a / b step 2 of the sequence analysis of β-lactoglobulin, in particular detection of

FM-thiohydantoin isoleucine by means of negative chemical ionization, Fig. 19 a / b step 3 sequence analysis of β-lactoglobulin, in particular detection of FM-thiohydantoin-valine by means of negative chemical ionization, Fig. 20 a / b detection and identification of the reaction product of an amine and FM-PITC by means of negative chemical ionization, and FIG. 21 shows the result of the sequencing of a synthetic peptide with the sequence FA-

E-A-A-K-Y-A-S, showing the first three dismantling steps.

In the examples described below, various reagents and solutions were used, which are described in more detail below and 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:

- Solution 2: PITC solution: 5 vol.% Phenyl isothiocyanate in pure acetonitrile (ACN)

- Solution 3: 3.5 - bis (trifluoromethyl) - phenyl isothiocyanate (FM-PITC) solution: 5% by volume 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 ul solution 1, 40 ul solution 2 and 30 ul solution 4; mix; rinse with N ₂ ; Reaction at 55 ° C for 2h; dry in a vacuum concentrator (Speed-Vac, Savant). Record for conversion to 50μl ACN and add 50μl solution 5; Hold reaction at 55 ° C for 30 min; dry in vacuum concentrator; take up in 1OOOμl ACN / H ₂ O (1: 1); Suitable dilutions are then 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 269nm were used for the measurements. The data was recorded on the PC using an evaluation system from Knauer, Berlin. A column type YMC ODS AQ 150 * 2mm, 3μ 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% 0-8min, injection at 8min, 5B 20-60% 8-32.5min,% B 60 was used -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 ul solution 1, 40 ul solution 3 and 30 ul solution 4; mix; rinse with N ₂ ; Reaction at 55 ° C for 2h; dry in vacuum concentrator.

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

A blank sample was used to verify the formation of by-products; For this purpose, a reaction with FM-PITC without an amino acid is carried out under the same conditions. c) Kinetic analysis of the conversion 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 each, 40 μl of 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 as described under a) and b).

d) Kinetic analysis of the conversion 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). PITC was added after 5, 10, 20, 30 and 60 minutes according to the following reaction scheme:

40 μl of solution 1 + 40 μl of solution 3 + 30 μl of solution 4 are initially introduced into 5 reaction vessels; The reaction mixture was mixed, flushed with nitrogen and the reaction was carried out at 55.degree. After 5, 10, 20, 30 and 60 min, 40 μl of 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 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 μl of the amino acid solution containing 40 μl of solution 3, 40 μl of solution 2 and 30 μl of solution 4 were combined. 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 (SpeedVac). 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:

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 the reaction of a free amino acid described in b) with the 3,5-bis (trifluoromethyl) phenyl isothiocyanate (FM-PITC) according to the invention same reaction conditions as when reacting the amino acid with PITC, a positive reaction was also found in the sense that a corresponding FM-PTH-amino acid derivative was formed and thus a detection or 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 just 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 test approach e), shows that the reaction of FM-PITC with a free

Amino acid runs 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. Here a 3 to 6 times higher concentration could be determined.

The result described above, that the reaction of FM-PITC with the free amino acid proceeds significantly faster than with PITC, is also important insofar as it is a further step in the automation of Edman degradation, especially in the form of high Throughput system, using 3,5-bis (trifluoromethyl) phenyl isothiocyanate is possible. Example 2: Reactions of FM-PITC with different amino acids:

In order to demonstrate that the use of 3,5-bis (trifluoromethyl) phenyl isothiocyanate according to the invention also takes place with amino acids other than those described in Example 1, further amino acids were reacted with FM-PITC according to the following scheme: Here the amino acids isoleucine, alanine , Valine, aspartic acid, argenin serine. Methionine proline, phenylalanine and lysine are used.

40 μl each 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. for 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 for 30 minutes at 55 ° C. The mixture is then dried again in 1000 μl of 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, FIGS Chromatograms FM-AS Standard 1 and FM-AS Standard 2, each with 5 amino acids from individual experiments, superimposed in one image.

Results:

The FM-PTH amino acid derivatives can be reproducibly distinguished from one another. The chromatograms show that each FM-PTH amino acid derivative can be assigned a characteristic peak with a defined retention time.

Example 3: Manual Edman sequencing with FM-PITC using the example of various 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 FAEAAKYAS, peptide 2 has the sequence ILRGSVAHK, peptide 3 has the sequence YTDPNTFSS. The peptides were weighed out on a balance and diluted to a concentration of 50 pmol / μl by adding the appropriate amount of 50% acetonitrile / water, 0.1% trifluoroacetic acid. 40μl of the respective solution (n = 2 nmol) are pipetted into reaction vessels and vacuum dried.

Coupling reaction according to Edman degradation:

20 μl of pyridine / water (1: 1) and 20 μl of 5% by volume FM-PITC solution (in pure acetonitrile) are added to the dried mixture; 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) in order 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 .; the reaction mixture is then freeze-dried.

The residue is taken up in 30 ul water and extracted twice with 40 ul butyl ester. The extract with the cleaved amino acid derivative is collected in a reaction vessel and dried in the Speed-Vac; the residual in the water peptide is used the next coupling reaction also freeze-dried and for ^'.

Conversion according to Edman degradation:

The dried amino acid derivative is mixed with 10 ul 40% trifluoroacetic acid; the reaction takes place at 55 ° C for 30 min .; the reaction mixture is then dried using SpeedVac. The dried residue is dissolved in 20 ul 100% acetonitrile + 0.1% TFA and mixed with another 20 ul water, 2 ul of the solution are injected into the HPLC. The determination is carried out by means of HPLC under the running conditions mentioned above.

Result:

Three degradation steps were carried out with the peptides. The (known) sequence of the peptides of the »synthetic peptides can be clearly verified by comparison with the FM-PTH amino acid derivatives prepared in Example 2. In the chromatograms shown in FIG. 21, the three stages of degradation are superimposed and the amino acids are identified. The yield of the FM-PTH derivatives shows that the reaction and the cleavage proceed almost completely. Also, in the degradation steps n + 1, virtually no amino acid derivative from the preliminary degradation step, i.e. H. Detection step n, detect. A so-called overlap, which would indicate incomplete coupling or incomplete cleavage, does not occur here and thus confirms the knowledge on which the invention is based that the use according to the invention of 3,5-bis (trifluoromethyl) phenyl isothiocyanate permits optimization of the Edman degradation ,

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

Execution:

From the above-mentioned solution of peptide 2 with the sequence ILRGSVAHK (c = 50pmol / μl) are pipetted onto a PVDF filter pretreated with biobren. (Biobrene Plus, Applied Biosystems, USA, c = 10μg / μl in 50% acetonitrile water, pipette 3μl onto filter and let dry. With the Knauer Sequencer Type 910 the standard configuration was used except for the modifications described [13] Sequencer operated in the so-called wet phase mode (dosage of coupling reagent approx. 8μl, base approx. 4μl and acid approx. 3μl as drops of liquid in the reactor) Instead of 5% PITC in heptane, a solution of 5% FM-PITC in Heptane is used, as 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 standard degradation program is used for the degradation used that is described as an attachment to [13]. In the course of the program, the derivatized amino acid is collected at the point at which the PTH amino acid is otherwise automatically injected into a detection system and injected manually into an HPLC. The evaluation is carried out analogously to the method described in Example 3.

The following HPLC system was used for detection purposes:

Pump ABI 130a gradient system, detector Shimadzu SPD 10A 269nm, evaluation system Knauer HPLC-Box, Eurochrome software, column YMC ODS AQ 150 * 2mm, 3μ 120A, oven 45 ° C, buffer A 0.1% TFA in water, buffer B 0.08 % TFA in acetonitrile, gradient% B 0-8min 20%, 8- 32.5min 20-60%, 32.5-34 min 60-80%

The sequence of the peptide could be clearly identified in 6 stages of degradation, which is evident from the comparison of the sequence determined in Example 3) and the comparison with the derivatives synthesized in Example 2). It turns out that the reagent 3,5 bis (trifluoromethyl) phenyl isothiocyanate is outstandingly suitable for the automatic execution in a sequence machine.

Example 5: Reaction conditions for the detection of primary and / or secondary amines

In addition to the above reaction conditions, the following reaction conditions for the detection or derivatization of primary and / or secondary amines can also be detected according to the disclosure given herein.

The primary and / or secondary amine to be detected or the sample suspected of containing it is typically dissolved in a solvent consisting of 50% pyridine, 6% trimethylamine and 44% water. The derivatization reagent, for example 3,5-bis (trifluoromethyl) phenyl isothiocyanate, is added to the sample as a 5% solution in acetonitrile. The reaction conditions are 50 to 55 ° C and the times 30 to 40 min. 50 ° C. for 40 min is preferred. The isomerization reaction is carried out with 25% trifluoroacetic acid, and the reaction product obtained is dried and dissolved in 70% acetone. Example 6: Detection of the implementation of the free amino acid with FM-PITC by means of gas chromatography:

This example shows, among other things, the use according to the invention of the reagent described for the detection of amines, since the class of amino acids represents a subclass from the group of amines.

experimental approaches

Implementation of the free amino acids with FM-PITC:

For the implementation of the free amino acids valine, isoleucine and glutamine with FM-PITC, the procedure was analogous to example 1 as follows:

Combine 40 ul solution 1, 40 ul solution 3 and 30 ul solution 4; mix; rinse with N ₂ ; Reaction at 55 ° C for 2h; dry in vacuum concentrator.

To carry out the isomerization reaction, also called conversion, proceed as follows: take the sample in 50μl ACN and add 50μl solution 5; _reaction at 55 ° C for 30min; dry in vacuum concentrator; take up in lOOOμl acetone; then suitable dilutions are made. The qualitative and quantitative determination is carried out using gas chromatography. For this purpose, an Agilent 6890N gas chromatograph equipped with an electron capture detector, an Agilent 6890N gas chromatograph equipped with an Agilent 5973 MSD mass-selective detector in the configuration with electron impact ionization (EI) and in the configuration with negative chemical ionization (NCI) were used. Helium was used as the carrier gas and methane was used as the reagent gas. The gas chromatographs were equipped with a temperature programmable inlet system. The temperature program was as follows: Initial 50 ° C for 0.02 min, heating at 720 ° C / min to 270 ° C, keeping the temperature for 1 min. The inlet was operated in split mode. The split ratio was varied depending on the concentration at hand. Usual split ratios were between 1 to 1 and 1 to 200. Usually 1 μl was injected. An HP 5 MS was used as the column. The temperature gradient was programmed from 50 ° C to 280 ° C. Results:

FIG. 12a shows a section of the total ion current of a measurement in the EI in the selected ion monitoring mode (German: selection ion monitoring mode). Selected ion monitoring mode means that the quadrupole mass filter is preset for ions with a specific mass to charge ratio. These preselected ions can pass through and are detected. Ions with a different mass to charge ratio cannot pass through the mass filter and are detected. A peak at 18.85 min can be clearly identified. The associated averaged spectrum FIG. 12b identifies the FM thiohydantoin valine derivative with the mass 370 and the specific fragment 328 (cleavage of the propyl side group). 13a shows the same sample measured with negative chemical ionization as a total ion current. 13a shows the associated mass spectrum. The chromatographic peak has the same retention time and masses 370 and 328 clearly identify the substance. Fig. 14 shows the same sample in the • electron capture detector. The amino acid was also clearly identified here.

15a shows the analysis of FM thiohydantoin isoleucine in an NCI measurement in scan mode (German: scan mode). Scan mode means that the quadrupole mass filter analyzes the individual masses one after the other in a preset range, i.e. takes up a complete mass spectrum of the preselected area for a short period of time. The derivatized amino acid can be clearly identified as a chromatographic peak in this example at 11:00 min. 15b shows the associated mass spectrum. The derivatized amino acid is clearly identified by its mass 384 and fragment 356 (elimination of carbon monoxide). r

Figure 16a shows the analysis of FM thiohydantoin glutamine. The amino acid elutes in this chromatographic separation at 10.80 min and is identified by its mass 399 and fragment 371 (FIG. 16b). Example 7: Execution of an automatic protein sequencing of β-lactoglobulin in the Knauer sequencer with FM-PITC as coupling reagent and detection of the cleaved, derivatized amino acids after gas chromatographic separation and mass spectrometric detection with negative chemical ionization:

experimental approach

The automatic protein sequencing was carried out analogously, as described in Example 4. Β-lactoglobulin was used as the protein sample.

The cleaved and converted amino acids were redissolved in acetone, transferred to suitable vessels and injected into the gas chromatography system. Figs. 17a, 17b, 18a, 18b, 19a and 19b each show an example of the total ion current in the scan mode of the first three degradation steps of the sequence. The amino acids leucine (Fig. 17a, b), isoleucine (Fig. 18a, b) and valine (Fig. 19a, b) were identified. This corresponds to the amino terminal sequence of β-lactoglobulin described in the literature. The respective amino acids were identified by comparing the retention times with the derivatives produced analogously to Examples 3 and 6 and the respective specific masses and fragment masses.

Example 8: Detection and identification of amines by derivatization with 3,5-bis (trifluoromethyl) phenyl isothiocyanate using gas chromatography

Separation and mass spectrometric detection with negative chemical ionization:

experimental approach

A solution containing 5% FM-PITC 5% trimethylamine in acetonitrile was added to a sample that is present in a solution of 50% ethanol 50% water and which contains other substances besides dimethylamine. The reaction was carried out at 55 ° C. for 40 min and the sample was then dried, redissolved in 70% acetone and injected into the gas chromatography system. 20a shows the total ion current of the reaction of Dimethylamine with 3,5-bis (trifluoromethyl) phenyl isothiocyanate. The reaction product has a retention time of 9.08 minutes and is clearly identified via mass 317, as shown in FIG. 20b.

The references cited 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 AI by caspase 3. J. Biol. Chem., 273, 28057-28064

4. Edman, P. (1950), ..., Acta Chem Scand., 4, 283

5. Kamp, R.M., 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. Eur J Biochem 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 aeids. Analyte. Biochem. 75, 621-633;

10 Wittmann-Liebold, B .; Ashman, K. (1985) On-line detection of amino aeid derivatives released by automatic Edman degradation of polypeptides. in Tschesche, H. (ed): Modern Methods in Protein Chemistry, Walter de Gruyter, Berlin, New York, 303-327

11 Hewick, R., M., Hunkapiller, M.W., Hood, L.E., Dreyer, W.j. (1981) A gas-liquid solid phase peptide and protein sequenator. J. Biol. Chem. 256, 7990-7997

12. Procise cLc manual, Applied Biosystems, USA 13. Handbook Knauer 910, Knaur Scientific Equipment, Berlin

14. Chang, J.Y., Brauer, D., Wittmann-Liebold, B. (1978) Micro-sequence analysis of peptides and proteins using 4-N, N-dimethylaminoazobenzene 4'-isothiocyanate / phenyhsothiocyanate double coupling method. FEBSLett., 93, 205-214.

15. Hess, D., Nika, H., Chow, D.T., Bures, E.J., Morrison, H.D. 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-l-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, J.E. (ed): Methods in Protein Microcharacterization, Humana Press, Clifton, NJ, 249-277

The features of the invention disclosed in the preceding description, the claims and the drawings can be essential both individually and in any combination for realizing the invention in its various embodiments.

Claims

Expectations

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

(I)

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 derivatization and / or detection of primary and / or secondary amines.

2. Use according to Anspmch 1, characterized in that the haloalkyl radical is substituted with at least one halogen.

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 derivatization and / or for the detection of primary and / or secondary amines.

5. Use according to one of claims 1 to 4, characterized in that the primary amine is an amino acid, preferably an α-amino acid.

6. Use according to one of claims 1 to 4, characterized in that the primary and / or secondary amine is a biogenic amine.

7. Use according to one of claims 1 to 4, characterized in that the amino acid is an N-terminal amino acid of a polypeptide.

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

(I)

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.

9. Use according to Anspmch 8, characterized in that the haloalkyl radical is substituted with at least one halogen.

10. Use according to Anspmch 8 or 9, 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.

11. Use of 3,5-bis (trifluoromethyl) phenyl isothiocyanate for the analysis of polypeptides.

12. Use according to one of claims 8 to 11, characterized in that the analysis is the sequence analysis of the polypeptide.

13. Use according to one of claims 8 to 12, characterized in that the analysis is carried out by means of Edman degradation.

14. Use according to one of claims 1 to 13, characterized in that detection and / or identification of the derivatized amine is carried out as a further step.

15. Use according to Anspmch 14, characterized in that the detection and / or identification is carried out by means of a method which is selected from the G ppe, which comprises reverse phase HPLC, in particular microbore reverse phase HPLC, gas chromatography and mass spectrometry.

16. Use according to one of claims 1 to 15, characterized in that the detection of the derivatized amine by means of ECD detection and / or UV detection and retention time, and / or mass information and / or retention time and mass information, and / or mass information and / or retention time and mass information, the ionization of the molecules taking place by means of negative chemical ionization.

17. Reaction product from a halogen-substituted phenyl isothiocyanate, in particular a phenyl isothiocyanate according to one of claims 1 to 13, and a primary and / or secondary amine, preferably an amino acid and preferably an α-amino acid.

18. Reaction product according to Anspmch 17, characterized in that the amino acid is bound to at least one further amino acid, preferably to a polypeptide.

19. 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 any one of claims 1 to 13.

20. The method according to claim 19, further comprising at least one of the following steps:

Cleavage of the terminal derivatized amino acid and detection of the cleaved terminal derivatized amino acid.

21. The method according to claim 19 or 20, characterized in that the steps of converting, splitting off and detecting are repeated at least once.

22. The method according to any one of claims 19 to 21, characterized in that the reaction takes place at a temperature of about 20-95 ° C, preferably 20-85 ° C and preferably 40-45 ° C.

23. The method according to any one of claims 19 or 22, characterized in that the reaction takes place under basic conditions, preferably using a nitrogen base.

24. The method according to any one of claims 19 to 23, characterized in that the reaction takes place at a pH of 7 to 14, preferably at a pH of 8 to 10.

25. The method according to any one of claims 19 to 24, characterized in that the phenylthiocarbamoyl polypeptide obtained after reacting the polypeptide with phenyl isothiocyanate, preferably after drying the phenylthiocarbamoyl polypeptide, reacted with anhydrous acid and thereby the N-terminal amino acid of the polypeptide as a heterocyclic Derivative in the form of an anilinothiazolinone amino acid is split off.

26. The method according to Anspmch 25, characterized in that the anilinothiazolinone amino acid is extracted from the reaction mixture and transferred directly to a detection system and identified.

27. The method according to Anspmch 25, characterized in that the anilinothiazolinone amino acid is extracted from the reaction mixture and converted into a phenylthiocarbamoyl amino acid.

28. The method according to Anspmch 25, characterized in that the anilinothiazolinone amino acid is extracted from the reaction mixture and converted into a phenylhydantoin amino acid.

29. The method according to any one of claims 19 to 28, characterized in that the amino acid derivative is detected and / or identified as a further step.

30. The method according to Anspmch 29, characterized in that the detection and / or identification takes place by means of a method which is selected from the group comprising reverse phase HPLC, in particular microbore reverse phase HPLC, gas chromatography and mass spectrometry.

31. The method according to any one of claims 19 to 30, characterized in that the detection of the amino acid derivative by means of ECD detection and / or UV detection and retention time, and / or mass information and / or retention time and mass information, and / or mass information and / or retention time and mass information, the molecules being ionized by means of negative chemical ionization.

32. The method according to any one of claims 19 to 31, characterized in that the polypeptide is reacted with a sequence reduced by one N-terminal amino acid with 3,5-bis (trifluoromethyl) phenyl isothiocyanate.

33. The method according to claim 32, characterized in that at least one of the steps provided in claims 19 to 32 is repeated.