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EP0697112A1 - Sequen age c-terminal de peptides pouvant renfermer de la proline - Google Patents

Sequen age c-terminal de peptides pouvant renfermer de la proline

Info

Publication number
EP0697112A1
EP0697112A1 EP94910704A EP94910704A EP0697112A1 EP 0697112 A1 EP0697112 A1 EP 0697112A1 EP 94910704 A EP94910704 A EP 94910704A EP 94910704 A EP94910704 A EP 94910704A EP 0697112 A1 EP0697112 A1 EP 0697112A1
Authority
EP
European Patent Office
Prior art keywords
terminal
reaction
thiohydantoin
peptide
carboxylate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94910704A
Other languages
German (de)
English (en)
Other versions
EP0697112A4 (fr
Inventor
Jerome M. Bailey
John E. Shively
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
City of Hope
Original Assignee
City of Hope
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/094,024 external-priority patent/US5432092A/en
Application filed by City of Hope filed Critical City of Hope
Publication of EP0697112A1 publication Critical patent/EP0697112A1/fr
Publication of EP0697112A4 publication Critical patent/EP0697112A4/fr
Withdrawn legal-status Critical Current

Links

Definitions

  • This invention relates to a method for the degradation from the C-terminus of peptides which may include a proline residue.
  • Application Serial No. 08/094,024 illustrates sequential use of diphenyl phosphoroisothio ⁇ cyanatidate and a heterocyclic amine for C-terminal peptide degradation.
  • the peptide which is preferably bound to a solid phase is converted to a carboxylate salt by triethylamine or similar base.
  • the carboxylate is reacted with diphenyl phosphoroisothiocyanatidate.
  • a heterocyclic amine such as pyridine is added.
  • This invention provides a method for the C-terminal degradation of peptides which may include a proline residue.
  • the method of the invention entails (1) formation of a carboxylate on the C-terminal amino acid of the peptide to be sequenced, (2) reaction of the carboxylated peptide with diphenyl phosphoroisothiocyanatidate and a heterocyclic amine to produce a thiohydantoin derivative, (3) protonating the thiohydantoin derivative, and (4) cleaving the protonated thiohydantoin derivative to produce a shortened peptide and a thiohydantoin derivative of the C-terminal amino acid of the peptide to be sequenced.
  • polypeptides to be sequenced are preferably either non-covalently applied to the porous tetrafluoroethylene (Zitex) or covalently attached to carboxylated polyethylene (PE-COOH) . See application Serial No. 07/576,943 and patent 5,180,807.
  • Figure 1 is a schematic of one C-terminal sequencer useful in the practice of the invention.
  • Figure 2 illustrates the practice of the invention to sequence YGGFL covalently coupled to carboxylic acid modified polyethylene (PE-COOH) .
  • R4 is gas phase pyridine.
  • Figure 3 illustrates the practice of the invention to sequence YGGFL covalently coupled to PE-COOH.
  • R4 is a solution of tetrazole in dimethylformamide.
  • FIGS 4A to 4F illustrate the practice of the invention to sequence YGGFL covalently coupled to PE-COOH.
  • R4 is a solution of tetrazole in acetonitrile.
  • Figure 5 illustrates the practice of the invention to sequence LAP covalently coupled to PE-COOH.
  • Figure 6 illustrates the practice of the invention to sequence AGSE covalently coupled to PE-COOH.
  • Figure 7 illustrates the practice of the invention to sequence Superoxide Dismutase non-covalently coupled to polytetrafluoroethylene (Zitex) .
  • Figure 8 illustrates the practice of the invention to sequence Ribonuclease A non-covalently coupled to polytetrafluoroethylene (Zitex) .
  • Figure 9 illustrates the practice of the invention to sequence hemoglobin a chain non-covalently coupled to polytetrafluoroethylene (Zitex) .
  • the invention provides a preferably sequential four-step method for degradation of a peptide which may include proline.
  • a carboxylate is formed at the C-terminus of the peptide to be sequenced by reaction with an organic or inorganic base.
  • the specific base utilized is not critical.
  • the carboxylation is preferably carried out by reaction of the peptide to be sequenced with a solution of the selected base in an appropriate solvent.
  • Tertiary trial yl amines are preferred.
  • Primary and secondary alkyl amines may also be utilized.
  • Alkali metal bases such as sodium or potassium hydroxide are effective and may be utilized in an aqueous solution. Sodium trimethylsilanolate in methyl alcohol solution is appropriate.
  • alkyl amines such as triethylamine are preferably present in a concentration of about 5% by volume.
  • the preferred carboxylation reagent for use in the practice of this invention is a solution containing 40% to 60% by volume of triethylamine in an anhydrous methanol. The carboxylation reaction is appropriately conducted at a temperature from 30° to 70°C.
  • the peptide carboxylate formed in step 1 is converted to a thiohydantoin by reaction with diphenyl phosphoroisothiocyanatidate and with an aromatic heterocyclic ring containing nitrogen. Sequential reaction, first with diphenyl phosphoroisothiocyanatidate and then with an aromatic heterocyclic ring containing nitrogen, permits sequencing through Asp and Glu.
  • the diphenyl phosphoroisothiocyanatidate and amine reagents are utilized in organic solvents such as acetonitrile, dimethylformamide, ethyl acetate, benzene and toluene.
  • the concentration of the diphenyl phosphoroisothiocyanatidate in the solvent is preferably from 2% to 70% by volume.
  • the reactions whether simultaneous or sequential are conducted at a temperature from 15°C. to 90°C, preferably 50°C. to 70°C. '
  • the amine reagent rapidly promotes removal of the phosphoryl moiety from the phosphoroisothio ⁇ cyanatidate reaction product.
  • the invention includes the use of any aromatic heterocyclic compound in which nitrogen is present in the ring.
  • Amines useful in the invention include, but are not limited to, pyridine, derivatized pyridines such as dimethylaminopyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrole, pyrazole, imidazole, triazole, or tetrazole.
  • Pyridine is preferred and may be used either per se, e.g., in the gas phase, or in an organic solvent medium such as acetonitrile or dimethylformamide at any concentration in excess of 0.1% by volume.
  • Protonation of the thiohydantoin product of Step 2 may be accomplished with any of a number of acids. Trifluoromethanesulfonic acid and trifluoroacetic acid are preferred. Acids found to be useful include hydrochloric, acetic and formic. The protonation reaction is appropriately conducted at a temperature of from about 30°C. to 90°C, preferably 50°C.
  • a unique feature of this invention is the efficiency with which the protonated thiohydantoin derivative is cleaved to provide a shortened peptide and a thiohydantoin derivative of the C-terminal amino acid.
  • Cleavage is best achieved by reaction with the sodium trimethylsilanolate.
  • the sodium trimethylsilanolate is utilized as a 0.01M to 1.0M, preferably 0.1M solution in an alcohol.
  • a preferred solvent contains equal parts of methanol and t-butanol. See PCT application US90/02723.
  • the trimethylsilanolate ion such as those having the monovalent cations IT 1" , Li + , Rb + , and Cs + may be utilized.
  • the trimethyl group may be replaced with other alkyl groups or with phenyl groups.
  • the preferred cleavage reagent is gas phase water (water vapor) at a temperature of 30°C. to
  • the thiohydantoin derivative of the C-terminal amino acid residue is analyzed by reverse phase HPLC.
  • the free carboxylate is regenerated, for example, by a second treatment with sodium trimethylsilanolate.
  • Each activated PE-COOH strip was inserted into a continuous flow reactor (CFR) (Shively et al., 1987) containing a solution of leucine enkephalin (YGGFL) in 50% aqueous DMF overnight at 22°C.
  • CFR continuous flow reactor
  • YGGFL leucine enkephalin
  • the microbore tubing on one end of the CFR was sealed by heating and then pinched closed with pliers.
  • the support was rinsed with coupling solvent and acetonitrile, and then dried in a vacuum centrifuge.
  • reagent and solvent bottles associated with the instrument depicted in Figure 1 are shown.
  • Four reagent bottles, R2-R5, and five solvent bottles, S1-S5, are utilized in the practice of the invention illustrated by the ensuing examples.
  • Reagents from bottles R2-R4 and solvents from bottles S1-S4 are delivered to the continuous flow reactor (CFR) .
  • Reagent from bottle R5 and solvent from bottle S5 are delivered to the conversion flask (CF) .
  • the CF serves to convert the ATZ derivative of the cleaved amino acid into a PTH (phenylthiohydantoin) just before analysis by HPLC.
  • the CF serves as a place to hold the cleaved thiohydantoin amino acid just prior to injection into the HPLC.
  • Table I The composition of the reagents and solvents is set forth in Table I.
  • a gentle pressure (1.5 atms) of argon is applied to each bottle.
  • Argon was chosen because of its chemical inertness. Other suitable inert gases could be helium and nitrogen.
  • pressure regulators P1-P5 .
  • PI is for S1-S4, P2 is for S5, S6, R5, S6, P3 is for R2 and R3, P4 is for Rl and R4, and P5 is for blow out functions and argon delivery functions (drying, etc.).
  • a solenoid actuated valve on P4 is opened in order to let the argon pass through the valve to the bottle (Rl) . Since each bottle is sealed, the argon pressure pushes the solvent through the line at the bottom to the valve block (in this case Q2) .
  • a solenoid actuated valve on Q2 and a valve on SW1 (for venting) is opened to allow the solvent flow into the valve block, Q2 and on into the CFR. Once the CFR is full, the flow is stopped by closing the valves and the reaction is allowed to continue for the desired length of time.
  • Flask (45°C) (sec) pressurize S4 3 deliver S4 60 pressurize SI 3 deliver SI 60 pressurize S4 3 deliver S4 60 pressurize SI 3 deliver SI 60 blow out SI 60 pressurize S3 3 deliver S3 30 blow out S3 45 pressurize R3 3 deliver R3 4
  • R3 reaction 120 blow out R3 20 pressurize R3 3 deliver R3 4
  • the first time is pressure, the second delivery, the third reaction time, and the fourth blowout.
  • the steps in the initial program described in Table II are performed only once for a particular sample and are only performed at the beginning of a sequencing experiment.
  • the "pressurize S4" step means that the S4 bottle is allowed to pressurize with argon for 30 seconds.
  • S4 reagent in this case trifluoromethanesulfonic acid
  • Table III describes the sequence of events which will derivatize the C-terminal amino acid to a thiohydantoin and specifically cleave it to leave a shortened polypeptide ready for continued sequencing.
  • the sequence of four events which, as illustrated, entails treatment of the polypeptide sample with diphenyl phosphoroisothiocyanatidate (R2) , rinsing with ethyl acetate/acetonitrile (S3) , treatment with gas phase pyridine (R4) , and rinsing with ethyl acetate/acetonitrile (S3) , is repeated three times in order to complete derivatization of the C-terminal amino acid.
  • the polypeptide C-terminal amino acid is derivatized to a thiohydantoin, except in the case of proline.
  • the sample is then extensively washed with ethyl acetate/acetonitrile (S3) in order to remove any remaining isothiocyanate reagent and pyridine present in the CFR or in various lines that add UV absorbing impurities to the HPLC chromatogram of the related thiohydantoin amino acid.
  • S3 ethyl acetate/acetonitrile
  • S4 gas phase trifluoromethanesulfonic acid
  • This treatment is then followed by reaction with vapor phase water (SI) to specifically hydrolyze the newly formed thiohydantoin proline.
  • Methanol (S2) is then delivered to the CFR in order to dissolve any thiohydantoin proline formed and carry it to the CF where it is then dried.
  • the acid/water/methanol treatment has no effect on the other 19 commonly occurring amino acids.
  • C-terminal thiohydantoins other than proline are not cleaved by the acid/water treatment and still must be cleaved by treatment with sodium trimethylsilanolate (R3) .
  • This example describes the sequencing of YGGFL (5.6 nmoles) covalently coupled to carboxylic acid modified polyethylene, for four cycles utilizing a computer automated C-terminal sequencer as depicted by Figure 1 and the program set forth above in which the R4 reagent is pyridine delivered in the gas phase, with the exception that the SI and S4 reaction steps are not included.
  • Figure 2 shows the chromatograms resulting from cycles 1-4.
  • the derivatized C-terminal amino acid is identified by retention time on a c-18 reverse phase column.
  • the separation of the thiohydantoin amino acids was performed on a 2.1 x 250 mm Reliasil C-18 column at 35°C. with a flow rate of 0.15 ml/min.
  • Solvent A is 0.1% trifluoroacetic acid in water.
  • Solvent B is 80% acetonitrile, 10% water, and 10% methanol. Gradient elution is performed as follows: 0% B for 2 min., 0-4% B for 35 min. , and 35-50% B for 10 min. Absorbance is monitored at 265 nm.
  • Example 1 is repeated with the exception that R4 is a solution of 0.1 grams tetrazole in 30 milliliters of dimethylformamide. The results are depicted by Figure 3.
  • Example 1 is repeated with the exception that R4 is a solution of 0.1 grams tetrazole in 30 milliliters of acetonitrile. The results are depicted by Figures 4A, 4B, 4C, 4D, 4E and 4F.
  • EXAMPLE 4 This example describes the sequencing of the tripeptide LAP (15 nmoles) , covalently coupled to carboxylic acid modified polyethylene, for four cycles utilizing a computer automated C-terminal sequencer as depicted by Figure 1 and the program set forth above. HPLC separation of the amino acid thiohydantoins is performed as described in Example 1. The R4 reagent is pyridine delivered in the gas phase. The results are depicted by Figure 5.
  • EXAMPLE 5 This example describes the sequencing of the tetrapeptide AGSE (9 nmoles) , covalently coupled to carboxylic acid modified polyethylene, for four cycles utilizing a computer automated C-terminal sequencer as depicted by Figure 1 and the program set forth above with the exception that the SI and S4 reaction steps are not included.
  • HPLC separation of the amino acid thiohydantoins is performed as described in Example 1.
  • the R4 reagent is pyridine delivered in the gas phase. The results are depicted by Figure 6.
  • EXAMPLE 6 This example describes the sequencing of the protein Superoxide Dismutase (400 pmoles) , non-covalently applied to a Zitex strip (1mm x 10mm) , for four cycles utilizing a computer automated C-terminal sequencer as depicted by Figure 1 and the program set forth above with the exception that the SI and S4 reaction steps are not included.
  • HPLC separation of the amino acid thiohydantoins is performed as described in Example 1.
  • the R4 reagent is pyridine delivered in the gas phase. The results are depicted by Figure 7.
  • This example describes the sequencing of the protein Ribonuclease A (4.5 nmoles), non-covalently applied to a Zitex strip (1mm x 10 mm) , for four cycles utilizing a computer automated C-terminal sequencer as depicted by Figure 1 and the program set forth above with the exception that the SI and S4 reaction steps are not included.
  • HPLC separation of the amino acid thiohydantoins is performed as described in Example 1. The results are depicted by Figure 8.
  • This example describes the sequencing of the protein Hemoglobin chain (4.1 nmoles), non-covalently applied to a Zitex strip (1 mm x 10 mm) , for four cycles utilizing a computer automated C-terminal sequencer as depicted by Figure 1 and the program set forth above with the exception that the SI and S4 reaction steps are not included.
  • HPLC separation of the amino acid thiohydantoins is performed as described in Example 1. The results are depicted by Figure 9.
  • This example involves a solution phase experiment in which C-terminal Asp did not derivatize to a thiohydantoin with simultaneous reaction of diphenyl phosphoroisothiocyanatidate and pyridine.
  • Pro-Phe-Asp 60 nmol N-protected with an acetyl group was reacted with diphenyl phosphoroisothiocyanatidate (0.06 mol) and pyridine (0.12 mmol) in acetonitrile for 40 minutes at 50°C.
  • the total reaction volume was 0.1 ml.
  • the peptide solution was evaporated to dryness by vacuum centrifugation.

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  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

On décrit une méthode de séquençage de protéines en partant du carboxy terminal, qui fait appel à du diphényle phosphoroisothiocyanatidate et à une amine hétérocyclique, pour produire un dérivé thiohydantoïne de l'acide aminé C-terminal. Le dérivé est facilement scindé. La méthode est utile pour un séquençage portant sur l'ensemble des 20 acides aminés naturels.
EP94910704A 1993-07-26 1994-02-15 Sequen age c-terminal de peptides pouvant renfermer de la proline Withdrawn EP0697112A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US08/094,024 US5432092A (en) 1991-12-03 1992-02-07 C-terminal peptide sequencing using diphenyl phosphoroisothiocyanatidate and pyridine
PCT/US1994/001742 WO1995022060A1 (fr) 1993-07-26 1994-02-15 Sequençage c-terminal de peptides pouvant renfermer de la proline

Publications (2)

Publication Number Publication Date
EP0697112A1 true EP0697112A1 (fr) 1996-02-21
EP0697112A4 EP0697112A4 (fr) 1998-07-29

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EP94910704A Withdrawn EP0697112A4 (fr) 1993-07-26 1994-02-15 Sequen age c-terminal de peptides pouvant renfermer de la proline

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Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5254475A (en) * 1990-08-13 1993-10-19 City Of Hope Sequential C-terminal degradation of peptides and proteins
US5185266A (en) * 1991-10-15 1993-02-09 Applied Biosystems, Inc. Cleavage method for acyl thiohydantoins and use of the method in c-terminal peptide sequencing

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EP0697112A4 (fr) 1998-07-29

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