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WO1990004183A1 - Method for preparation of thiohydantoins and for protein sequence analysis - Google Patents

Method for preparation of thiohydantoins and for protein sequence analysis Download PDF

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WO1990004183A1
WO1990004183A1 PCT/AU1989/000433 AU8900433W WO9004183A1 WO 1990004183 A1 WO1990004183 A1 WO 1990004183A1 AU 8900433 W AU8900433 W AU 8900433W WO 9004183 A1 WO9004183 A1 WO 9004183A1
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peptide
amino acid
protein
thiohydantoin
terminal
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PCT/AU1989/000433
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French (fr)
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Adam Sinclair Inglis
Franca Casagranda
John Francis Kelly Wilshire
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Commonwealth Scientific And Industrial Research Organisation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • 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
    • 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

  • This invention relates to a method for the preparation of amino acid thiohydantoins and for the determination of the amino acid sequence of a protein or peptide from the C-terminal end thereof.
  • Such decomposition not only lowers the yield of amino acid thiohydantoin (IV) produced by the cleavage reaction, but also causes the formation of by-products which are likely to interfere with the unequivocal identification of the thiohydantoin (IV) released in each cycle, a step which is crucial for the success of the sequencing method.
  • this method also, there is provided a process for the preparation of amino acid thiohydantoins utilising this Schlack-Kumpf procedure.
  • a method for the C-terminal degradation of a protein or peptide which comprises the steps of:
  • the shortened protein or peptide produced in the cleavage reaction is suitable for a further degradation cycle.
  • the cleavage reaction has been attempted using either acids of varying strengths or mild organic bases such as acetohydroxamic acid or triethylamine. These approaches were apparently based on the belief that mild conditions were mandatory, particularly when basic reagents were being used. However, it has now been found that cleavage with a strong inorganic base at a suitable concentration (such as aqueous 0.5M potassium or other alkali metal hydroxide) in the presence of a water-miscible organic solvent (such as methanol), and optionally an antioxidant (such as dithioerythritol or dithiothreitol), gives excellent results provided that the reaction time is short.
  • a strong inorganic base at a suitable concentration (such as aqueous 0.5M potassium or other alkali metal hydroxide) in the presence of a water-miscible organic solvent (such as methanol), and optionally an antioxidant (such as dithioerythritol or dithiothreitol), gives
  • the C-terminal amino acid carboxyl group is activated prior to said coupling reaction.
  • Activation with acetic anhydride and acetic acid is preferred, however other known activation procedures such as that described by Kubo et.al. (1971) and variations thereof, may also be used for the activation step.
  • an aqueous solution of the strong inorganic base is used, such as a solution having a concentration of >0.2M, and preferred solutions comprise alkali metal hydroxides such as sodium or potassium hydroxide of >0.2M in aqueous methanol.
  • Preferred cleavage conditions in accordance with the present invention include the use of 0.5 potassium hydroxide in 33% aqueous methanol in the presence of dithioerythritol as antioxidant, however it will be appreciated that other strong inorganic base/organic solvent/antioxidant combinations may be used without departing from the broad ambit of the invention.
  • a method for the determination of the C- terminal amino acid of a protein or peptide which comprises the steps of C-terminal degradation of the protein or peptide by the method broadly described above, followed by identification of the C-terminal amino acid thiohydantoin formed by the cleavage reaction.
  • the method of this aspect of the invention enables the successful sequencing of the original protein or peptide from the C-terminal end by subjecting the shortened protein or peptide to one or more further degradation cycles in accordance with this invention, followed by identification of the (or each) C-terminal amino acid thiohydantoin as it is formed by the cleavage reaction. While this aspect of the invention is exemplified in the present specification with a procedure suitable for manual sequencing, it will be apparent to those skilled in the art that the simple operations involved can readily be automated using currently available technology, for example that used for automatic N-terminal amino acid sequencing, with appropriate programming of the controlling systems.
  • the determination of the amino acid sequence of the protein or peptide is carried out with the protein or peptide covalently attached by its N-terminal end to a solid support.
  • the solid support may, for example, consist of activated glass, glassfibre or other appropriate polymeric material.
  • the proteins or peptides may also be sequenced after they have been strongly adsorbed (i.e. non-covalently bound) to the solid support.
  • appropriate reaction conditions will need to be employed, for example reagents that could cause dissolution of the sample should just wet out the support, then be dried off before introducing further solvents or other reagents.
  • R x represents the residue of an amino acid, which comprises the step of coupling the carboxyl group of a compound of the general formula (V):
  • R x is as defined above and R 2 represents H or R 3 -CO- in which R 3 represents a lower alkyl (for example, methyl) group, an amino acid residue or a peptide residue, with thiocyanic acid or a thiocyanate to form a substituted thiohydantoin derivative of the general formula (Ila):
  • R x may represent the residue of any of the 20 common amino acids, namely alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine ( ), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) or valine (V).
  • the method of this aspect of the invention is of particular utility in that it enables the preparation of several amino acid thiohydantoins which previously could not be prepared readily, for example the thiohydantoins of serine, arginine, threonine and proline.
  • the amino acid thiohydantoins can be readily prepared in accordance with the present invention by a microscale in situ procedure, for example from the corresponding acyl amino acid such as the N-acetyl amino acid or the corresponding propionyl or benzoyl amino acid.
  • Acetylserine (100 nmoles) was added to a mixture of acetic anhydride/acetic acid (4:1, 120 ⁇ l) in a screw cap Eppendorf vial and the resultant mixture allowed to react for 5 minutes at 80°C.
  • Thiocyanic acid (HSCN) ( ⁇ 1.8M, 30 ⁇ l) was then added and the mixture heated at 80°C for 30 minutes and then dried under vacuum.
  • the residue was treated at room temperature with base (0.2M KOH in 33% methanol/water) containing dithioerythrit ⁇ l, the mixture stirred, and the cleavage reagent removed immediately and analysed for the corresponding thiohydantoin by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • HPLC provides a rapid and sensitive method for the identification and quantitation of amino acid thiohydantoins, and the following Table sets out retention times for a number of the products prepared in accordance with this Example (elution from a HP 1090 HPLC using a Water Pico Tag column of a flow rate of 1.0 ml/min and a column temperature of 40°C).
  • Figure 1 shows a typical HPLC profile for the thiohydantoins of the amino acids: aspartic acid (AT-asp), asparagine (AT-asn), glutamine (AT-gln), glutamic acid (AT-glu), glycine (AT-gly), alanine (AT-ala), lysine (AT-lys), serine (AT-ser), threonine (AT-thr), tyrosine (AT-tyr), valine (AT-val), proline (AT-pro), methionine (AT-met), isoleucine (AT-ile), leucine (AT-leu), phenylalanine (AT-phe) and tryptophan (AT-trp).
  • aspartic acid AT-asp
  • asparagine AT-asn
  • glutamine AT-gln
  • glutamic acid AT-glu
  • glycine AT-gly
  • alanine AT-ala
  • Peptides were covalently bound to activated glass beads (usually in 60-80% yield) by the method described by euth et.al. (1982).
  • Beads (1.5-4.0mg) containing approximately 20 nmol peptide were subjected to a modified sequencing procedure which is based on that of Meuth et.al., (1982), i.e. using activation with acetic acid and acetic anhydride and with thiocyanic acid as reagent.
  • Cleavage was achieved with 0.5M KOH in 33% aqueous methanol in the presence of dithioerythritol, and the cleavage solution analysed by HPLC for thiohydantoin content (see Experimental Protocol).

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Abstract

A method for the preparation of amino acid thiohydantoins and for sequencing a protein or peptide comprises c-terminal degradation of a protein or peptide by i) coupling the c-terminal carboxyl group with thiocyanic acid or a thiocyanate to form a substituted thiohydantoin derivative and ii) cleaving the substituted thiohydantoin derivative with a strong inorganic base in the presence of a water-miscible organic solvent, to give a shortened protein or peptide and a c-terminal amino acid thiohydantoin which can be identified for sequencing purposes.

Description

METHOD FOR PREPARATION OF THIOHYDANTOINS AND FOR PROTEIN SEQUENCE ANALYSIS
This invention relates to a method for the preparation of amino acid thiohydantoins and for the determination of the amino acid sequence of a protein or peptide from the C-terminal end thereof.
Application of the thiocyanate degradation procedure (Schlac and Ku pf 1926) .to a peptide or protein (I) converts the C-terminal amino acid into a substituted thiohydantoin derivative (II) which can be subsequently cleaved to give a shortened (by one amino acid) peptide or protein (III), together with the C- terminal amino acid thiohydantoin (IV):
(i) (ϋ)
—CONHCHRCOOH - -CON—CHR —COOH + HN—CHR
(I) (III)
Figure imgf000003_0001
H H (ID (IV)
There is a strong similarity between this Schlack- Kumpf thiocyanate degradation procedure and the Edman degradation used for N-terminal sequencing, in that both procedures give rise to thiohydantoin derivatives - phenylthiohydantoins (PTHs) in the case of the Edman degradation. Although potentially useful for many years, the Edman procedure only became routine with (i) the advent of highly sensitive detection techniques, e.g. high performance liquid chromatography (HPLC), and (ii) the use of protective agents, e.g. thiols, to counteract phenylthiohydantoin instability.
Although several attempts have been made to exploit the Schlack-Kumpf procedure in the sequencing of a protein or peptide, an acceptable sequencing method has not previously been developed. It will be noted that the reaction scheme as set out above consists of two steps, namely (i) coupling and (ii) cleavage. The objective of successful sequencing lies in maximising the reaction yields in both steps, whilst at the same time making sure that the C-terminal amino acid thiohydantoin (IV), the species which corresponds to the original C-terminal amino acid, is protected from decomposition (since the thiohydantoin ring is fairly unstable). Such decomposition not only lowers the yield of amino acid thiohydantoin (IV) produced by the cleavage reaction, but also causes the formation of by-products which are likely to interfere with the unequivocal identification of the thiohydantoin (IV) released in each cycle, a step which is crucial for the success of the sequencing method.
It is an object of the present invention to provide a method utilising the Schlack-Kumpf procedure as generally described above by which the sequence of the protein or peptide can be successfully determined from the C-terminal end. In this method also, there is provided a process for the preparation of amino acid thiohydantoins utilising this Schlack-Kumpf procedure.
In accordance with a first aspect of the present invention, there is provided a method for the C-terminal degradation of a protein or peptide, which comprises the steps of:
(i) coupling the carboxyl group of the C-terminal amino acid residue of said protein or peptide with thiocyanic acid or a thiocyanate to form a substituted thiohydantoin derivative, and (ii) cleaving said substituted thiohydantoin derivative with a strong inorganic base in the presence of a water-miscible organic solvent and optionally an antioxidant to form a shortened protein or peptide and the C-terminal amino acid thiohydantoin.
The shortened protein or peptide produced in the cleavage reaction is suitable for a further degradation cycle.
For amino acid determinations, the cleavage reaction has been attempted using either acids of varying strengths or mild organic bases such as acetohydroxamic acid or triethylamine. These approaches were apparently based on the belief that mild conditions were mandatory, particularly when basic reagents were being used. However, it has now been found that cleavage with a strong inorganic base at a suitable concentration (such as aqueous 0.5M potassium or other alkali metal hydroxide) in the presence of a water-miscible organic solvent (such as methanol), and optionally an antioxidant (such as dithioerythritol or dithiothreitol), gives excellent results provided that the reaction time is short. Using these cleavage reagents, the cleavage reaction can be conveniently performed at room temperature, but both lower and higher temperatures (for example, from 0°C to 70°C) may be used provided the time of reaction is varied accordingly.
Preferably, the C-terminal amino acid carboxyl group is activated prior to said coupling reaction. Activation with acetic anhydride and acetic acid is preferred, however other known activation procedures such as that described by Kubo et.al. (1971) and variations thereof, may also be used for the activation step.
The presence of an antioxidant is preferred during the cleavage reaction since in the absence of an antioxidant inferior results are obtained.
Preferably, an aqueous solution of the strong inorganic base is used, such as a solution having a concentration of >0.2M, and preferred solutions comprise alkali metal hydroxides such as sodium or potassium hydroxide of >0.2M in aqueous methanol.
Preferred cleavage conditions in accordance with the present invention include the use of 0.5 potassium hydroxide in 33% aqueous methanol in the presence of dithioerythritol as antioxidant, however it will be appreciated that other strong inorganic base/organic solvent/antioxidant combinations may be used without departing from the broad ambit of the invention.
In a second aspect of the present invention, there is provided a method for the determination of the C- terminal amino acid of a protein or peptide, which comprises the steps of C-terminal degradation of the protein or peptide by the method broadly described above, followed by identification of the C-terminal amino acid thiohydantoin formed by the cleavage reaction.
The method of this aspect of the invention enables the successful sequencing of the original protein or peptide from the C-terminal end by subjecting the shortened protein or peptide to one or more further degradation cycles in accordance with this invention, followed by identification of the (or each) C-terminal amino acid thiohydantoin as it is formed by the cleavage reaction. While this aspect of the invention is exemplified in the present specification with a procedure suitable for manual sequencing, it will be apparent to those skilled in the art that the simple operations involved can readily be automated using currently available technology, for example that used for automatic N-terminal amino acid sequencing, with appropriate programming of the controlling systems.
Preferably, in accordance with the present invention, the determination of the amino acid sequence of the protein or peptide is carried out with the protein or peptide covalently attached by its N-terminal end to a solid support. The solid support may, for example, consist of activated glass, glassfibre or other appropriate polymeric material. However, as an alternative the proteins or peptides may also be sequenced after they have been strongly adsorbed (i.e. non-covalently bound) to the solid support. Where the protein or peptide is adsorbed to the solid support, appropriate reaction conditions will need to be employed, for example reagents that could cause dissolution of the sample should just wet out the support, then be dried off before introducing further solvents or other reagents.
In accordance with yet another aspect of the present invention, there is provided a method for the preparation of an amino acid thiohydantoin of the general formula (IVa):
HN — CHR! I I IVa
Figure imgf000007_0001
H wherein Rx represents the residue of an amino acid, which comprises the step of coupling the carboxyl group of a compound of the general formula (V):
R2NHCHR1C00H V
wherein Rx is as defined above and R2 represents H or R3-CO- in which R3 represents a lower alkyl (for example, methyl) group, an amino acid residue or a peptide residue, with thiocyanic acid or a thiocyanate to form a substituted thiohydantoin derivative of the general formula (Ila):
R, — CON CHRX
Figure imgf000008_0001
H
and then cleaving said substituted thiohydantoin derivative with a strong inorganic base in the presence of a water-miscible organic solvent, and optionally an antioxidant.
Preferred reagents and conditions for this method are described above.
It will of course be appreciated that Rx (and R2 where appropriate) may represent the residue of any of the 20 common amino acids, namely alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine ( ), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) or valine (V). The method of this aspect of the invention is of particular utility in that it enables the preparation of several amino acid thiohydantoins which previously could not be prepared readily, for example the thiohydantoins of serine, arginine, threonine and proline. The amino acid thiohydantoins can be readily prepared in accordance with the present invention by a microscale in situ procedure, for example from the corresponding acyl amino acid such as the N-acetyl amino acid or the corresponding propionyl or benzoyl amino acid.
The following Examples demonstrate the preparation of amino acid thiohydantoins in accordance with this invention, and application of the method of this invention to
(a) the identification of the C-terminal amino acid of peptides and proteins in solution, and
(b) the sequencing of several peptides covalently attached to glass beads.
EXAMPLE 1 Preparation of Amino Acid Thiohydantoins.
Acetylserine (100 nmoles) was added to a mixture of acetic anhydride/acetic acid (4:1, 120μl) in a screw cap Eppendorf vial and the resultant mixture allowed to react for 5 minutes at 80°C. Thiocyanic acid (HSCN) (~1.8M, 30μl) was then added and the mixture heated at 80°C for 30 minutes and then dried under vacuum. The residue was treated at room temperature with base (0.2M KOH in 33% methanol/water) containing dithioerythritσl, the mixture stirred, and the cleavage reagent removed immediately and analysed for the corresponding thiohydantoin by high performance liquid chromatography (HPLC). Using this procedure, histidine, lysine, serine, arginine, threonine and proline have been converted into their thiohydantoins, each of which showed a single peak when analysed by HPLC. HPLC provides a rapid and sensitive method for the identification and quantitation of amino acid thiohydantoins, and the following Table sets out retention times for a number of the products prepared in accordance with this Example (elution from a HP 1090 HPLC using a Water Pico Tag column of a flow rate of 1.0 ml/min and a column temperature of 40°C).
Amino Acid Retention
Thiohydantoin Time (mins)
Lysine 3.14
Serine 3.35
Threonine 3.88
Proline 4.92
Figure 1 shows a typical HPLC profile for the thiohydantoins of the amino acids: aspartic acid (AT-asp), asparagine (AT-asn), glutamine (AT-gln), glutamic acid (AT-glu), glycine (AT-gly), alanine (AT-ala), lysine (AT-lys), serine (AT-ser), threonine (AT-thr), tyrosine (AT-tyr), valine (AT-val), proline (AT-pro), methionine (AT-met), isoleucine (AT-ile), leucine (AT-leu), phenylalanine (AT-phe) and tryptophan (AT-trp).
EXAMPLE 2 C-Terminal Amino Acid Determinations.
(i) Experimental Protocol. The protein or peptide ("20-50 nmoles) was dissolved in a mixture of acetic anhydride/acetic acid (4:1, 120μl) and allowed to react for 5 minutes at 80βC. The mixture was then treated with the thiocyanic acid solution (~1.8M, 30μl), heated at 80°C for 30 minutes and then vacuum dried. The residue was treated at room temperature with a solution (50μl) of 0.5M KOH in 33% methanol/water containing dithioerythritol (at least 10 mg per 100 ml cleavage solution), stirred and the cleavage solution immediately removed and analysed for the 2-thiohydantoin by HPLC.
(ii) Discussion and Results.
Thiocyanic acid ("1.8M solution in acetone) (Dwulet and Gurd 1977) was used as the coupling reagent. Ribonuclease, lysozyme or AlaGlyPhe (50 nmol) was dissolved in a mixture of acetic anhydride, acetic acid and thiocyanic acid (200-fold excess). This mixture was heated and then vacuum-dried, and the residue treated at room temperature with 0.5M KOH in 33% aqueous methanol in the presence of dithioerythritol. The mixture was then analysed by HPLC: the thiohydantoins of Phe, Val and Leu respectively were readily detected by the solution method (see Experimental Protocol) (see Fig.2). Using the same method, a C-terminal amino acid determination of myoglobin has been performed and its C-terminal amino acid, namely glycine, identified.
EXAMPLE 3 Solid-Phase C-Terminal Sequencing. (i) Experimental Protocol.
Glass beads ("2-10 mg) bound with peptide or protein ("15-30 nmoles) were placed in a 1.5ml screw-cap Eppendorf vial and acetic anhydride/acetic acid (4:1, 120μl) solution was added. The mixture was then stirred (vortexed) and heated at 80°C for 5 minutes in a water bath. Thiocyanic acid ("1.8M, 30μl) was then added, and the mixture stirred and heated for a further 30 minutes at 80°C. The vial was removed from the water bath, and the supernatant liquid removed carefully by pipette and discarded. The glass beads were then washed with 200μl aliquots of DMF (x2) followed by 70% acetonitrile/water (x3). To the beads, 0.5M KOH in 33% methanol/water solution (50μl) containing dithioerythritol (see Example 2) was added and the mixture intermittently stirred. After 3 minutes at room temperature, the cleavage solution was removed by pipette and analysed by HPLC for thiohydantoin content. The glass beads were immediately washed (200μl aliquots with 70% acetonitrile containing acetic acid ("5%), 70% acetonitrile (x2), acetonitrile (x2) and finally rinsed with the above activating solution (acetic anhydride/acetic acid) (x2) before the next cycle was commenced.
(ii) Discussion and Results.
Peptides were covalently bound to activated glass beads (usually in 60-80% yield) by the method described by euth et.al. (1982). Beads (1.5-4.0mg) containing approximately 20 nmol peptide were subjected to a modified sequencing procedure which is based on that of Meuth et.al., (1982), i.e. using activation with acetic acid and acetic anhydride and with thiocyanic acid as reagent. Cleavage was achieved with 0.5M KOH in 33% aqueous methanol in the presence of dithioerythritol, and the cleavage solution analysed by HPLC for thiohydantoin content (see Experimental Protocol).
With the substances examined so far, activation and coupling at 80°C, followed by cleavage with base as described above at room temperature have given both extensive and unequivocal sequence information. In addition, experiments have established that there is a variation in the reactivities of different amino acids at different temperatures and it may be preferable to use temperatures as low as 30°C for some, as high as 85°C for others, in some sequencing applications. The washing procedure is different to any described previously, and the results show that it effectively removes side products and excess reagents. In principle, other combinations of polar and non-polar solvents should be able to remove these. The solvents should, of course, ensure also that the peptide or protein is freed from acid before the cleavage step. Usually the recommended procedure is satisfactory, but sometimes addition of a base (that will not cleave the thiohydantoin) can be advantageous for proteins. Removal of both base and water are essential after the cleavage to ensure a good coupling step at the following cycle. However, the novelty and improvement effected with the present method is illustrated in the use of the methanolic KOH solution, which gives rapid cleavage, low background and high repetitive yields from cycle to cycle.
Using the procedure described, it has been possible to sequence for the first time through an aspartyl residue, the three successive C-terminal residues (AlaAspVal) of the peptide shown being determined unequivocally (see Fig.3). In addition, leucine-enkephalin has been completely sequenced (see Fig.4(a)), and the synthetic decapeptide (YLAIYVMAFV) sequenced through to the penultimate residue (see Fig.4(b)).
REFERENCES:
1. Dwulet, F.E., Gurd, F.R.N. (1979). Int. J. Pep. Prot. Res. 13:122-129.
2. Meuth, J.L., Harris, D.E., Dwulet, F.E., Crowl- Powers, M.L., Gurd, F.R.N. (1982). Biochem. 21:3750-3757.
3. Schlack, P., Kumpf, W. (1926). Hoppe-Seyler's Z Physiol. Chem. 154:125-170.
4. Kubo, H., Nakajima, T., Tamura, Z. (1971). Chem: & Pharm. Bull. 19:210-211.

Claims

CLAIMS :
1. A method for the C-terminal degradation of a protein or peptide, which comprises the steps of: (i) coupling the carboxyl group of the C-terminal amino acid residue of said protein or peptide with thiocyanic acid or a thiocyanate to form a substituted thiohydantoin derivative, and (ii) cleaving said substituted thiohydantoin derivative with a strong inorganic base in the presence of a water-miscible organic solvent to form a shortened protein or peptide and the C-terminal amino acid thiohydantoin.
2. A method according to claim 1, wherein said carboxyl group is activated prior to said coupling reaction.
3. A method according to claim 2, wherein said activation comprises reaction with acetic anhydride and acetic acid.
4. A method according to claim 1, wherein said cleavage reaction is carried out in the presence of an antioxidant.
5. A method according to claim 4, wherein said antioxidant is dithioerythritol or dithiothreitol.
6. A method according to claim 1, wherein said strong inorganic base is an alkali metal hydroxide of a concentration of >0.2M.
7. A method according to claim 6, wherein said strong inorganic base is 0.5M potassium hydroxide.
8. A method according to claim 1, wherein said water- miscible organic solvent is methanol.
9. A method according to claim 1, wherein said cleavage reaction is carried out using 0.5M potassium hydroxide in 33% aqueous methanol in the presence of dithioerythritol as antioxidant.
10. A method for the determination of the C-terminal amino acid of a protein or peptide, which comprises the steps of:
(i) C-terminal degradation of said protein or peptide by the method according to any of claims 1 to 9; and
(ii) identification of the C-terminal amino acid thiohydantoin formed by the cleavage reaction.
11. A method according to claim 10, wherein the shortened protein or peptide formed by the cleavage reaction is subjected to one or more further degradation cycles, with each such cycle being followed by identification of the C-terminal amino acid thiohydantoin formed by the cleavage reaction.
12. A method according to claim 10 or claim 11, wherein said protein or peptide is attached by its N- terminal end to a solid support prior to said C-terminal degradation.
13. A method according to claim 12, wherein said attachment is by covalent bonding or adsorption.
14. A method according to claim 12, wherein said solid support is activated glass, glass fibre or a polymeric material.
15. A method according to claim 14, wherein said solid support comprises glass beads and said protein or peptide is covalently bound thereto.
16. A method for the preparation of an amino acid thiohydantoin of the general formula (IVa):
HN — CHR,,
IVa
Figure imgf000017_0001
H
wherein Rα represents the residue of an amino acid, which comprises the step of coupling the carboxyl group of a compound of the general formula (V):
RjjNHCHRiCOOH
wherein R is as defined above and R2 represents H or R3-CO- in which R3 represents a lower alkyl group, an amino acid residue or a peptide residue, with thiocyanic acid or a thiocyanate to form a substituted thiohydantoin derivative of the general formula (Ila):
R2 — CON CHRX
Figure imgf000017_0002
H
and then cleaving said substituted thiohydantoin derivative with a strong inorganic base in the presence of a water-miscible organic solvent.
17. A method according to claim 16, wherein said carboxyl group is activated prior to said coupling reaction.
18. A method according to claim 17, wherein said activation comprises reaction with acetic anhydride and acetic acid.
19. A method according to claim 16, wherein said cleavage reaction is carried out in the presence of an antioxidant.
20. A method according to claim 19, wherein said antioxidant is dithioerythritol or dithiothreitol.
21. A method according to claim 16, wherein said strong inorganic base is an alkali metal hydroxide of a concentration >0.2M.
22. A method according to claim 21, wherein said strong inorganic base is 0.5M potassium hydroxide.
23. A method according to claim 16, wherein said water-miscible organic solvent is methanol.
24. A method according to claim 16, wherein said cleavage reaction is carried out using 0.5M potassium hydroxide in 33% aqueous methanol in the presence of dithioerythritol as antioxidant.
PCT/AU1989/000433 1988-10-07 1989-10-06 Method for preparation of thiohydantoins and for protein sequence analysis WO1990004183A1 (en)

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WO1996007916A1 (en) * 1994-09-08 1996-03-14 The Perkin-Elmer Corporation Acetic anhydride activation for c-terminal protein sequencing
JP2006521377A (en) * 2003-03-27 2006-09-21 ランケナー インスティテュート フォー メディカル リサーチ New IDO inhibitors and methods of use
AU2004315596B2 (en) * 2003-08-29 2011-11-24 President And Fellows Of Harvard College Inhibitors of cellular necrosis
US9499521B2 (en) 2014-12-11 2016-11-22 President And Fellows Of Harvard College Inhibitors of cellular necrosis and related methods
US9586880B2 (en) 2008-12-23 2017-03-07 President And Fellows Of Harvard College Small molecule inhibitors of necroptosis
US9725452B2 (en) 2013-03-15 2017-08-08 Presidents And Fellows Of Harvard College Substituted indoles and pyrroles as RIP kinase inhibitors

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Cited By (10)

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Publication number Priority date Publication date Assignee Title
WO1996007916A1 (en) * 1994-09-08 1996-03-14 The Perkin-Elmer Corporation Acetic anhydride activation for c-terminal protein sequencing
JP2006521377A (en) * 2003-03-27 2006-09-21 ランケナー インスティテュート フォー メディカル リサーチ New IDO inhibitors and methods of use
AU2004315596B2 (en) * 2003-08-29 2011-11-24 President And Fellows Of Harvard College Inhibitors of cellular necrosis
US8143300B2 (en) 2003-08-29 2012-03-27 President And Fellows Of Harvard College Inhibitors of cellular necrosis
US8741942B2 (en) 2003-08-29 2014-06-03 President And Fellows Of Harvard College Inhibitors of cellular necrosis
US9586880B2 (en) 2008-12-23 2017-03-07 President And Fellows Of Harvard College Small molecule inhibitors of necroptosis
US9725452B2 (en) 2013-03-15 2017-08-08 Presidents And Fellows Of Harvard College Substituted indoles and pyrroles as RIP kinase inhibitors
US9499521B2 (en) 2014-12-11 2016-11-22 President And Fellows Of Harvard College Inhibitors of cellular necrosis and related methods
US9944628B2 (en) 2014-12-11 2018-04-17 President And Fellows Of Harvard College Inhibitors of cellular necrosis and related methods
US10508102B2 (en) 2014-12-11 2019-12-17 President And Fellows Of Harvard College Inhibitors of cellular necrosis and related methods

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