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WO1997035844A1 - Technique de preparation de thiohydantoines d'acides amines - Google Patents

Technique de preparation de thiohydantoines d'acides amines Download PDF

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Publication number
WO1997035844A1
WO1997035844A1 PCT/AU1997/000200 AU9700200W WO9735844A1 WO 1997035844 A1 WO1997035844 A1 WO 1997035844A1 AU 9700200 W AU9700200 W AU 9700200W WO 9735844 A1 WO9735844 A1 WO 9735844A1
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WIPO (PCT)
Prior art keywords
acid
isothiocyanate
peptide
amino acid
thiocyanate
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Application number
PCT/AU1997/000200
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English (en)
Inventor
Adam Inglis
Albert Peng Sheng Tseng
Olga V. Antonova
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Garvan Institute Of Medical Research
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Publication date
Application filed by Garvan Institute Of Medical Research filed Critical Garvan Institute Of Medical Research
Priority to AU21440/97A priority Critical patent/AU2144097A/en
Publication of WO1997035844A1 publication Critical patent/WO1997035844A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/86—Oxygen and sulfur atoms, e.g. thiohydantoin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/534—Production of labelled immunochemicals with radioactive label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides
    • G01N33/6821—Sequencing of polypeptides involving C-terminal degradation

Definitions

  • 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.
  • 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.
  • the thiocyanate is ammonium thiocyanate.
  • thiocyanic acid (HSCN) is used.
  • the isothiocyanate is selected from the group consisting of guanidine isothiocyanate, trimethylsilyl-isothiocyanate, phosphoryl- isothiocyanate (e.g. diphenyl phosphorylisothiocyanatidate) and benzoyl- isothiocyanate.
  • 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.
  • an organic fluoroacid such as trifluoroacetic acid or heptafluorobutyric acid, methanesulphonic acid, sulphuric acid, phosphoric acid and hydrochloric acid.
  • 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.
  • a peptide used in this specification will cover dipeptides up to large peptides including proteins of any size.
  • 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.
  • the peptide is reacted with tritiated hydrochloric acid (HCl) and tritiated acetic acid.
  • HCl tritiated hydrochloric acid
  • 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.
  • 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.
  • TFA trifluoroacetic acid
  • 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.
  • 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
  • Figure 8 shows the incorporation of tritium into the peptide FLSYKA with the reactivity profile aligned with the HPLC chromatogram.
  • 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.
  • 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.
  • 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.
  • 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.
  • the radioactivity of the amino acid thiohydantoins can be increased further by cleaving in the presence of tritium.
  • 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
  • vapour phase chemistry 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.
  • 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.
  • Fig. 6 indicates that the commercial isothiocyanate reagent (DPP- ITC) reacts less effectively than ammonium thiocyanate.
  • DPP- ITC isothiocyanate reagent
  • 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.
  • the cleavage reagent like the cyclization reagents, is both acidic and volatile.
  • the cleavage reagent is both acidic and volatile.
  • several degradation cycles may be made without compromising the reaction chemistry because of build up of salts or reactive by-products.
  • 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.
  • Vapour phase reactions highlight the reactivity nature of HSCN.
  • the reaction also went smoothly.
  • 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.
  • 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.
  • the cleavage reagent like the cyclization reagents, is both acidic and volatile.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ -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.

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Abstract

Technique de préparation de thiohydantoïnes d'acides aminés non marquées ou radio-marquées, soit seules soit en tant que résidu C-terminal d'un peptide. La technique consiste à faire réagir, en présence d'un acide fort, l'acide aminé ou le peptide avec de l'anhydride acétique, de l'acide acétique ou de l'acide acétique tritié et un thiocyanate ou un isothiocyanate, la réaction se produisant soit en phase liquide soit en phase vapeur.
PCT/AU1997/000200 1996-03-27 1997-03-27 Technique de preparation de thiohydantoines d'acides amines WO1997035844A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU21440/97A AU2144097A (en) 1996-03-27 1997-03-27 Method for preparing amino acid thiohydantoins

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPN8963A AUPN896396A0 (en) 1996-03-27 1996-03-27 Method for preparing amino acid thiohydantoins
AUPN8963 1996-03-27

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WO1997035844A1 true WO1997035844A1 (fr) 1997-10-02

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0217634A2 (fr) * 1985-09-26 1987-04-08 Beckman Research Institute of the City of Hope Séquençage de peptides
AU3741293A (en) * 1992-03-25 1993-10-21 Garvan Institute Of Medical Research Method for preparation of amino acid thiohydantoins
US5521097A (en) * 1991-08-28 1996-05-28 Seiko Instruments Inc. Method of determining amino acid sequence of protein or peptide from carboxy-terminal

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0217634A2 (fr) * 1985-09-26 1987-04-08 Beckman Research Institute of the City of Hope Séquençage de peptides
US5521097A (en) * 1991-08-28 1996-05-28 Seiko Instruments Inc. Method of determining amino acid sequence of protein or peptide from carboxy-terminal
AU3741293A (en) * 1992-03-25 1993-10-21 Garvan Institute Of Medical Research Method for preparation of amino acid thiohydantoins

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
ANALYTICAL BIOCHEMISTRY, Vol. 224, No. 2, (1995), BAILEY J.M. et al., "Automated Carboxy- Terminal Sequence Analysis of Polypeptides Containing C-Terminal Proline", pages 588-596. *
BIOCHEMISTRY, Vol. 21, (1982), MEUTH J.L. et al., "Stepwise Sequence Determination from the Carboxyl Terminus of Peptides", pages 3750-3757. *
BIOCHEMISTRY, Vol. 29, (1990), BAILEY J.M. and SHIVELY J.E., "Carboxy Terminal Sequencing : Formation and Hydrolysis of C-Terminal Peptidylthiohydantoins", pages 3145-3156. *
DERWENT ABSTRACT, Accession No. 87-246019/35, Class S03; & JP,A,62 167 480 (SHIMADZU SEISAKUSHO KK), 23 March 1987. *
JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS, Vol. 25, (1992), INGLIS, ADAM S. et al., "Formation of Proline Thiohydantoin With Ammonium Imocyanate : Progress Towards a Viable C-terminal Amino Acid Sequencing Procedure", pages 163-171. *
METHODS IN PROTEIN SEQUENCE ANALYSIS [Proc. Int. Conf.] 9th (1993), Meeting Date 1992, EDITORS IMAHORI K. and SAKIYAMA, F. Authors, INGLIS A.S. and DE LUCA C., "A New Chemical Approach to C-Terminal Microsequence Analysis Via the Thiohydantoin", pages 71-78. *
MOLECULAR BIOLOGY, BIOCHEMISTRY AND BIOPHYSICS, 8, (1975), Protein Sequence Determination: A Sourcebook of Methods and Techniques. 2nd Rev. and Enl. Ed., MATSUO H. and NAVITA K., "Improved Tritium - Labelling for Quantitative-C Terminal Analysis", pages 104-113. *
RADIOPHARMACEUTICALS AND LABELLED COMPOUNDS. PROCEEDINGS OF THE SYMPOSIUM ON NEW DEVELOPMENTS IN RADIOPHARMACEUTICALS AND LABELLED COMPOUNDS (1973), Meeting Date 1973, Vol. 2, MORGAT J.L. and FROMAGEOT P., "Preparation of Tritium-Labelled Peptidic Hormones of High Specific Radioactivity", pages 109-19. *
THE JOURNAL OF BIOCHEMISTRY, Vol. 74, No. 1, (1973), 179-181, YAGI K. et al., "C-Terminal Amino Acid of D-Amino Acid Oxidase". *

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