WO1997035844A1

WO1997035844A1 - Method for preparing amino acid thiohydantoins

Info

Publication number: WO1997035844A1
Application number: PCT/AU1997/000200
Authority: WO
Inventors: Adam Inglis; Albert Peng Sheng Tseng; Olga V. Antonova
Original assignee: Garvan Institute Of Medical Research
Priority date: 1996-03-27
Filing date: 1997-03-27
Publication date: 1997-10-02
Also published as: AUPN896396A0

Abstract

Method of preparing unlabelled or radio-labelled amino acid thiohydantoins, either in isolation or as the C-terminal residue of a peptide comprising reacting the amino acid or the peptide in the presence of a strong acid with acetic anhydride, acetic acid or tritiated acetic acid and a thiocyanate or an isothiocyanate, the reaction carried out either in liquid or vapour phase.

Description

Method for Preparing Amino Acid Thiohydantoins

Technical Field

The present invention relates to a method of preparing radiolabelled or unlabelled amino acid thiohydantoins either singly or as the C-terminal residue of a peptide. This labelling should facilitate identification of the C- terminal amino acids of peptides and proteins and will also enable detection of the C-terminal peptides in enzyme and other digests of proteins. Background Art

Chemical methods for C-terminal sequencing have advanced to the stage where automated commercial instruments are becoming available, albeit without the performance that is characteristic of automatic N-terminal amino acid sequencers. One of the major shortcomings has been the analysis for the proline residue, which has been reported not to react in the procedures described for thiohydantoin formation via oxazolinones at the C- terminus.

In Australian Patent No 661138 (the disclosure of which is incorporated herein by reference) the present inventors set out an efficient method of forming amino acid thiohydantoins including proline thiohydantoin. This method resulted from the finding that acidic conditions were required for both the cyclization and the cleavage to overcome the specific problems associated with proline thiohydantoin formation and its subsequent cleavage from a peptide. The present inventors have now shown that this chemistry lends itself to the incorporation of tritium into the amino acid thiohydantoin and so provides a useful radio-label on the C-terminal amino acids of proteins and peptides. Furthermore, the present inventors have found surprisingly that a rearrangement of the experimental protocol will allow vapour phase reaction which results in a much cleaner reaction and the absence of by-products. Disclosure of Invention In a first aspect, the present invention consists in a method of preparing radio-labelled amino acid thiohydantoins either in isolation or as the C-terminal residue of a peptide comprising reacting the amino acid or the peptide in the presence of a strong acid with acetic anhydride in tritiated acetic acid and a thiocyanate or an isothiocyanate. In a preferred embodiment of the first aspect of the present invention the thiocyanate is ammonium thiocyanate. In a further preferred embodiment of the first aspect of the present invention thiocyanic acid (HSCN) is used.

In a still further preferred embodiment of the first aspect of the present invention the isothiocyanate is selected from the group consisting of guanidine isothiocyanate, trimethylsilyl-isothiocyanate, phosphoryl- isothiocyanate (e.g. diphenyl phosphorylisothiocyanatidate) and benzoyl- isothiocyanate.

In another preferred embodiment, the strong acid is selected from the group consisting of an organic fluoroacid, such as trifluoroacetic acid or heptafluorobutyric acid, methanesulphonic acid, sulphuric acid, phosphoric acid and hydrochloric acid.

As set out in Australian Patent No 661138, the method developed by the present inventors involves acid cleavage of the C-terminal amino acid thiohydantoin. This cleavage step also lends itself to modification to enable radiolabelling of the amino acid thiohydantoin.

It will be appreciated that the scope of the term "a peptide" used in this specification will cover dipeptides up to large peptides including proteins of any size.

In a second aspect, the present invention consists in a method of cleaving and tritiating a C-terminal amino acid thiohydantoin from a peptide comprising reacting the peptide with a strong acid in the presence of tritium.

In a preferred embodiment the peptide is reacted with tritiated hydrochloric acid (HCl) and tritiated acetic acid.

As will be readily appreciated by those skilled in the art, an increase in the level of labelling can be achieved by combining the first and second aspects of the present invention.

In a third aspect, the present invention consists in an improved method of preparing amino acid thiohydantoins either in isolation or as a peptidyl isothiocyanate or as the C-terminal residue of a peptide comprising reacting the amino acid or peptide in the presence of a strong acid with an acylating agent and a thiocyanate or an isothiocyanate, the improvement comprising conducting the reaction in a vapour phase.

The preferred reagents for this reaction are the same as those described in the inventors' previous work. The present inventors have found surprisingly that a rearrangement of the experimental protocol will allow vapour phase reaction which results in a much cleaner reaction and the substantial absence of unwanted by¬ products. For example, the sample is placed on an insoluble support, which is in turn held above the reaction mixture in the sample vial. In this manner, it is possible to separate or isolate the sample from the large excess of reagents, the thiocyanate salt in particular. It will be appreciated, however, that other means may be used to carry out the reaction in a vapour phase. The rationale for the improved reactivity in the inventors' patented procedure was that the acidity provided by the trifluoroacetic acid (TFA) in the mixture catalysed the cyclization reaction. Effective vapour phase reaction establishes that TFA also liberates reactive (volatile) thiocyanic acid from the (non-volatile) ammonium thiocyanate salt.

Because of the absence of salts and their by-products in the reaction mixture, and the volatile nature of the reagents on the insoluble support, the method is now infinitely better for cycling without the need for solvent extractions to reduce the background generated from the solution by¬ products. One major application of this method is to C-terminal "ladder sequencing"; that is, instead of identifications being made after individual degradation cycles, several cycles are made and the resulting mixture is placed on a mass spectrometer where the peptides generated at each cycle generate a ladder of masses that correspond to the loss of particular amino acids at each cycle.

In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described by reference to the following examples and Figures. Brief Description of Drawings

Fig. 1 gives equations for cyclization to peptidyl thiohydantoin (I) and cleavage to amino acid thiohydantoin (II) and expected sites of tritiation;

Fig. 2 shows tritium incorporation in amino acid thiohydantoins where acetylalanine and acetylmethionine were treated with a proline reaction mixture containing tritiated acetic acid. The thiohydantoins were radioactive (AcA and AcM) and on cleavage with tritiated acid, the reactivity remained in their respective thiohydantoins (A and M);

Fig. 3 shows coupling (A) and cleavage (B) results for acetylproline. HPLC separation of the products indicates that no shift in either the time of elution or radioactivity occurred on cleavage; Fig. 4 shows that the sensitivity of detection is increased approximately 30% on cleavage of acetyl Met-TH with tritiated HCl; Fig. 5 shows a comparative result with the liquid system for preparation of the thiohydantoins of acetylmethionine and acetylamine, and the vapour phase method. Both vials had the same amounts of reagents but the background for the vapour phase is greatly reduced;

Fig. 6 shows yields of products obtained for reaction of acetylproline with ammonium thiocyanate (A) and an isothiocyanate (DPP-ITC) (B) under vapour phase conditions; Figure 7 shows HPLC traces for the formation of proline thiohydantoin from acetylproline using acetic anhydride (A) and acetic anhydride/acetic acid (B) in the proline reaction mixture. Both solutions gave a clean reaction product eluting at 10.1 min, panel B showing an increase in yield was obtained on addition of acetic acid to the solution; and Figure 8 shows the incorporation of tritium into the peptide FLSYKA with the reactivity profile aligned with the HPLC chromatogram. Modes for Carrvinfi Out the Invention EXPERIMENTAL Cyclization Thiohydantoins were made using a scaled down version of the proced ire previously used for proline thiohydantoin (AU 661138). The acetyl amino acid or peptide (ca. 2.5 nm) was reacted in a 1.5 ml polypropylene tube with a screw cap fitted with an O-ring seal, the solution comprising ammonium thiocyanate (11 mg), acetic anhydride (100 ul), tritiated acetic acid (25 ul) and trifluoroacetic acid (4 ul). (Here the acetic acid was tritiated and prepared by addition of tritiated water (1.7 mCi, 95 ul) to acetic acid anhydride (500 ul) and leaving the mixture over night at room temperature). Cleavage The amino acid thiohydantoin was cleaved under anhydrous conditions using HCl in acetic acid. This was prepared in situ by the addition of water to acetyl chloride. The tritiated reagent was made by adding tritiated water (1.7 mCi, 10 ul)to acetyl chloride (40 ul). Chromatography A 1090 Hewlett-Packard HPLC with a 100 x 2.2 mm Hypersil column was used for chromatography of the reaction. Gradient elution was used with buffer A being 0.1% TFA and buffer B being 90% acetonitrile in 0.1% TFA. Aliquots (50 ul) to be analysed for radioactivity from the separations were diluted with 5 ml of Packard LCS-cocktail and counted for 5 minute periods in a Beckton Instruments scintillation counter. RESULTS

The rationale used by Matsuo et al [Biochem. Biophys. Res. Commun. 22:69-74 1966] for tritiation of the C-terminal amino acids of proteins was that treatment with acetic anhydride-pyridine-tritiated water would produce an oxazolinone at the carboxy terminus. Formation of this unstable cyclic derivative requires removal of hydrogen atoms from the C-terminal residue which would be partially replaced by tritium on hydrolytic opening of the ring in the presence of tritiated water. They were unable to explain the chemistry for aspartic acid and proline which was intractable yet did react under more forcing reaction conditions. These two amino acids also gave reaction problems for many years with various procedures that produced thiohydantoins, all based on reaction with either thiocyanic acid, thiocyanate salts or organic isothiocyanates. The discovery by the present inventors that proline thiohydantoin could be prepared from reaction of C-terminal proline with ammonium thiocyanate in the presence of trifluoracetic acid, acetic acid and acetic anhydride also implies that the reaction pathway does not require formation of an intermediate oxazolinone as has been previously proposed for thiohydantoin production. The present inventors suggested that an isothiocyanate intermediate could be formed in a manner akin to the acid- catalysed formation of carboxylic acid esters and that the presence of the strong acid would aid its cyclization to the thiohydantoin. In this process the loss of hydrogen from the nitrogen of the peptide bond must occur concomitantly with addition of hydrogen to the nitrogen of the isothiocyanate (see Fig. 1). The successful incorporation of tritium into model amino acids and peptides in the present experiments by substituting acetic acid with tritiated acetic acid in the standard protocol for thiohydantoin preparation is consistent with this supposition.

Fig. 2 the radioactive traces for HPLC of the products of reaction of acetylmethionine and acetylalanine with the tritiation procedure. They coincide with each of the amino acid thiohydantoin peaks. Treatment of the thiohydantoins with HCl in acetic acid caused a shift in the elution times of the alanine and methionine peaks and their radioactivity as expected for cleavage of the acetyl groups from the molecule. Earlier work by the present inventors established that the acetyl group of proline is cleaved during the cyclization (A) and so the elution times of the peaks of radioactivity and UV absorption are unchanged for the amino acid after treatment with HCl (B) in Fig. 3.

The radioactivity of the amino acid thiohydantoins can be increased further by cleaving in the presence of tritium. As described earlier, the HCl cleavage reagent was prepared in situ for these experiments by adding an equivalent amount of water to acetyl chloride. The former was replaced with tritiated water for these experiments. Fig. 4 shows that the sensitivity is increased by cleavage of acetyl Met-TH with tritiated HCl. Elimination of major background products without solvent extraction

As another aspect of the present invention, the possibility of using vapour phase chemistry on a sample loaded onto an insoluble support as a means of decreasing the level of background products was investigated.

Although by-products can of course be tolerated when they are well removed from products of interest on chroma tograms, low background is usually required for scaling down analytical methods. Since the extra acidity provided by the addition of trifluoracetic acid to the thiocyanate mixture in the present methods causes liberation of reactive thiocyanic acid from the ammonium thiocyanate salt, and both thiocyanic acid and trifluoracetic acid are volatile, reaction should occur with their vapours. To test this, the sample was placed on a glass fibre disc supported above the reaction mixture in the sample vial. The disc was first wet with acetic anhydride/acetic acid (4:1) in these experiments to aid reactivity and maintain the concentration of these less volatile reagents in the mixture on the disc. Reaction was effective and Fig. 5 shows a comparative result with the liquid system for preparation of the thiohydantoins of acetylmethionine and acetylalanine.

The vapour phase system possesses several advantages over the liquid phase procedure. Firstly, the amount and number of by-products is greatly diminished which makes identification of the released amino acid thiohydantoin more certain, as Fig. 5 shows. As expected, the salt peak at the beginning of the chromatography trace has virtually disappeared which is not trivial because the acidic amino acid thiohydantoins elute close to the front and salt peaks with current HPLC procedures. More importantly, there are no significant interfering peaks where other thiohydantoins would be expected to be eluted. When peaks obtained from the latter are also radioactive (after tritiation as above) assignments of C-terminal residues should be very soundly based. While the background obtained in the first commercial C-terminal sequencer is much better than that shown for the liquid phase system in Fig. 5, it does have major peaks, especially one near leucine. Fig. 6 indicates that the commercial isothiocyanate reagent (DPP- ITC) reacts less effectively than ammonium thiocyanate. A major concern with the liquid phase by-products is that they might also react with the thiohydantoin, either modifying it or destroying it, thereby rendering it unreactive to further chemistry and cycles. This would be consistent with the low yields and low repetitive yields obtained in the past with the thiohydantoin chemistry.

While the reaction proceeds without acetic acid in the mixture, it has a synergistic effect, which is illustrated in Fig. 7. Because of the cleanliness of the reaction and the volatility of the reagents, there is no need for solvent extractions prior to the cleavage reaction. Apart from cost economies, the possibility of wash out of the sample is minimised and the choice of the solid support is wider in such a system. TFA treated glass fibre disc (Applied Biosystems) was used in this work.

Advantages also accrue because the cleavage reagent, like the cyclization reagents, is both acidic and volatile. Hence, by drying the sample support after each reaction, several degradation cycles may be made without compromising the reaction chemistry because of build up of salts or reactive by-products. While the present inventors were able to show that two or more cycles may be made also by only drying using the liquid system, the background would have interfered with the identification of some amino acid thiohydantoins because of an overlapping peak with the proline derivative. The vapour phase procedure could be more easily adapted to automatic microsequencing than the liquid system in that the reagents and products do not present the same problems of removal from the sample support. This would apply especially to peptides which might normally be washed off unless covalently bound to the support. DISCUSSION

Vapour phase reactions highlight the reactivity nature of HSCN. When only the vapours of the proline reagent mixture were used to derivatise the acetyl amino acid, the reaction also went smoothly. In this case, the relative concentrations of reactants would be expected to be much different because of the low vapour pressures of acetic anhydride and acetic acid relative to HSCN and TFA. Importantly, in this mode of the nucleophilic attack on the carbonyl carbon would also be expected to proceed via the volatile free acid. It is the first example actually confirming the free acid, rather than the thiocyanate ion, provides the more active nucleophilic species.

The vapour phase system possesses several advantages over the liquid phase procedure. Firstly, the amount and number of by-products is greatly diminished which makes identification of the released amino acid thiohydantoin more certain, as Fig. 5 shows. As expected, the salt peak at the beginning of the chromatography trace has virtually disappeared but, more importantly, there are no significant interfering peaks where thiohydantoins would be expected to be eluted. When peaks obtained from the latter are also found to be radioactive (after tritiation as described above) assignments of C-terminal residues should be very soundly based. While the background obtained in the first commercial C-terminal sequencer is much better than that shown for the liquid phase system in Fig. 5 , it does have major peaks, especially one near leucine. Furthermore, the use of salts and reagents of relatively low volatility in that system would appear to preclude the type of reaction that has been employed here.

Secondly, because of the cleanliness of the reaction and the volatility of the reagents, there is no need for solvent extractions prior to the cleavage reaction. Apart from cost economies, the possibility of wash out of the sample is minimised and the choice of the solid support is wider in such a system. The present inventors used a TFA treated glass fibre disc (Applied Biosystems) in this work.

Advantages also accrue because the cleavage reagent, like the cyclization reagents, is both acidic and volatile. Hence, by simply drying the sample support after each reaction, several degradation cycles may be made without compromising the reaction chemistry because of build up of salts or reactive by-products. In their earlier work, the present inventors were able to show that two or more cycles could be made successfully also by just drying after liquid phase reaction [Inglis and De Luca Methods in Protein Sequence Analysis Eds. Imahori and Sakyama Plenum Press 71-78 1993 ], but the background obviously would have interfered with the identification of some amino acid thiohydantoins, and one background peak actually overlapped with the proline derivative in which we were interested.

Advances in the chemistry and instrumentation over the past 8 years have confirmed that the C-terminal thiohydantoin methodologies have potential to rival the N-terminal sequencing in terms of sensitivity. In principle, there is excessive hardware on the N-terminal sequencers commensurate with the needs of the C-terminal instrument because the former require an additional conversion reaction after the cleavage reaction, whereas the latter does not. This conversion of the thiazolinone of the cleaved amino acid to the more stable thiohydantoin requires a second reaction flask on the instrument. However, instrumentation presently available can be utilised advantageously for the present vapour phase methodology, especially as the current N-terminal sequencers allow reagent flows in either direction through the reaction cartridge which give a useful increased flexibility to their operations. These instruments have been designed for analyses of extremely small amounts of substances with correspondingly small reagent volumes. However, there is also an attendant need to remove by-products thoroughly to prevent them from interfering with the identification of derivatives. The results shown here obtained without any solvent clean-up-reflect the great advantage of this vapour phase approach for high sensitivity work.

Finally, the vapour phase procedure could be more easily adapted to automatic microsequencing than the liquid system in that the reagent products do not present the same problems of removal from the sample support; this would apply especially to peptides which might be washed off unless covalently bound to the support.

The discovery by the present inventors that a proline thiohydantoin could be prepared from reaction of C-terminal proline with ammonium thiocyanate, in the presence of trifluoroacetic acid, acetic acid and acetic anhydride, also implies that the reaction pathway does not require formation of an intermediate oxazolinone as has been previously proposed for thiohydantoin production. The present inventors suggested that an isothiocyanate intermediate could be formed in a manner akin to the acid- catalysed formation of carboxylic acid esters and that the presence of the strong acid would aid its cyclization to the thiohydantoin.

For the cyclization step, addition of hydrogen to the nitrogen of the isothiocyanate (see Fig. 1(1)) must occur concomitantly with the loss of hydrogen from the nitrogen of the peptide bond. Since the hydrogen can originate from the acidic reaction mixture, tritium atoms in it should also compete with hydrogen atoms for attachment to the isothiocyanate moiety. Similarly, during cleavage (Fig. 1(H)) it would be expected that a second tritium could be added to the thiohydantoin derivative.

By replacing acetic acid with tritiated acetic acid in the ammonium thiocyanate reaction mixture for thiohydantoin formation, tritium can be incorporated into the thiohydantoin formed at the C-terminus of a peptide.

This label could facilitate the identification of the C-terminal peptide in enzymic or chemical digests of proteins. Incorporation of radioactivity in a peptide is demonstrated in Fig. 8 using the peptide Phe-Leu-Ser-Tyr-Lys- Ala (FLSYKA). A single peak would not be expected in a peptide of this formula because the N-terminal amino and the amino ε-group of the lysine residue may only be partially acetylated. Cleavage of the amino acid thiohydantoin with tritiated acid is also convenient and increases the amount of radioactivity introduced during cyclization. Tritium labelling should also be a useful adjunct to C-terminal amino acid sequencing where the radioactivity of the released tritiated amino acid tliiohydantoins can be used to confirm assignments based on absorption of the derivatives at 269 nm, particularly when either background, scarcity of sample or the presence of an unusual amino acid causes problems of identification.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A method of preparing radio-labelled amino acid thiohydantoins either in isolation or as the C-terminal residue of a peptide comprising reacting the amino acid or the peptide in the presence of a strong acid with acetic anhydride in tritiated acetic acid and a thiocyanate or an isothiocyanate.

2. A method according to claim 1 wherein the thiocyanate is ammonium thiocyanate.

3. A method according to claim 1 wherein the thiocyanate is thiocyanic acid.

4. A method according to claim 1 wherein the isothiocyanate is selected from the group consisting of guanidine isothiocyanate, trimethylsilyl- isothiocyanate, phosphoryl-isothiocyanate, diphenyl phosphorylisothiocyanatidate, and benzoyl-isothiocyanate.

5. A method according to any one of claims 1 to 4 wherein the strong acid is selected from the group consisting of an organic fluoroacid, methanesulphonic acid, sulphuric acid, phosphoric acid and hydrochloric acid.

6. A method according to claim 5 wherein the organic fluoroacid is selected from trifluoroacetic acid and heptafluorobutyric acid.

7. A method of cleaving and tritiating a C-terminal amino acid thiohydantoin from a peptide comprising reacting the peptide with a strong acid in the presence of tritium.

8. A method according to claim 7 wherein the peptide is reacted with tritiated hydrochloric acid and tritiated acetic acid.

9. An improved method of preparing radio-labelled amino acid thiohydantoins either in isolation or as the C-terminal residue of a peptide comprising preparing the radiolabelled amino acid thiohydantoins according to the method of any one of claims 1 to 8, the improvement comprising conducting the reaction in a vapour phase.

10. An improved method of preparing amino acid thiohydantoins either in isolation or as a peptidyl isothiocyanate or as the C-terminal residue of a peptide comprising reacting the amino acid or peptide in the presence of a strong acid with an acylating agent and a thiocyanate or an isothiocyanate, the improvement comprising conducting the reaction in a vapour phase.

11. A method according to claim 10 wherein the acylating agent is acetic anhydride in acetic acid.

12. A method according to claim 10 wherein the acylating agent is acetyl chloride.

13. A method according to any one of claims 10 to 12 wherein the thiocyanate is ammonium thiocyanate.

14. A method according to any one of claims 10 to 12 wherein the thiocyanate is thiocyanic acid.

15. A method according to any one of claims 10 to 12 wherein the isothiocyanate is selected from the group consisting of guanidine isothiocyanate, trimethylsilyl-isothiocyanate, phosphoryl-isothiocyanate, diphenyl phosphorylisothiocyanatidate, and benzoyl-isothiocyanate.

16. A method according to any one of claims 10 to 15 wherein the strong acid is selected from the group consisting of an organic fluoroacid, methanesulphonic acid, sulphuric acid, phosphoric acid and hydrochloric acid.

17. A method according to claim 16 wherein the organic fluoroacid is selected from trifluoroacetic acid and heptafluorobutyric acid.

18. A method according to claim 10 in which the amino acid or peptide is reacted in the presence of trifluoroacetic acid with ammonium thiocyanate, acetic anhydride in acetic acid.