AU682343B2 - C-terminal sequencing of peptides which may include proline - Google Patents

Description

WO 95/22060 PCTI/US94/01742 -1- C-TERMINAL SEQUENCING OF PEPTIDES WHICH MAY INCLUDE PROLINE This application is a continuation-in-pa application Serial No. 08/094,024 1' 26 July 1993 which is a continuation- art of application Serial No. 07/801,94 ed 3 December 1991, now patent .7.

FIELD OF THE INVENTION This invention relates to a method for the degradation from the C-terminus of peptides which may include a proline residue.

BACKGROUND OF THE INVENTION Kenner, et al. J. Chem. Soc. 673-678 (1953) describes a C-terminal degradation experiment which required 110 hours to quantitatively form a thiohydantoin amino acid. Bailey patent 5,180,807 describes an improvement in which diphenyl phosphoroisothiocyanatidate is used concurrently with a heterocyclic amine, such as pyridine.

U&X- -1c P;&a;Ey Dr-ria.e. n ,..r..\illustrates sequential use of diphenyl phosphoroisothiocyanatidate and a heterocyclic amine for C-terminal peptide degradation. In a first step, the peptide which is preferably bornd to a solid phase is converted to a carboxylate salt by triet ;lamine or similar base. In a second step, the carboxylate is reacted with diphenyl phosphoroisothiocyanatidate.

In a third step, a heterocyclic amine such as pyridine is added.

-1a- SUMMARY OF THE INVENTION This invention provides a method for degrading a peptide which comprises: forming a thiohydantoin derivative of the C-terminal amino acid of the peptide; protonating the thiohydantoin derivative of the C-terminal amino acid; and cleaving the protonated thiohydantoin derivative of the C-terminal amino acid from the peptide.

The polypeptides to be sequenced are preferably either non-covalently applied to the porous tetrafluoroethylene (Zitex) or covalently attached to carboxylated polyethylene (PE-COOH). See U.S. Patents No. 5,059,540 and 5,180,807.

a •g* o I I SUMMARY OF THE INVENTION This invention provides a method for degrading a peptide which inclu forming a carboxylate on the C-terminal amino acid of the eptide; converting the carboxylated C-terminal amin acid into a thiohydantoin derivative of the C-terminal amino acid; protonating the thiohydantoin rivative of the C-terminal amino acid; and cleaving the proto ed thiohydantoin derivative of the C-terminal amino acid from the peptid e The poly ptides to be sequenced are preferably either non-covalently applied to the po us tetrafluoroethylene (Zitex) or covalently attached to carboxylated polye ylene (PE-COOH). See application Serial No. 07/576,943 and patent ,180,807.

DESCRIPTION OF THE FIGURES Figure 1 is a schematic of one C-terminal sequencer useful in the practice of the 20 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 illustrated the practice of the invention to sequence YGGFL covalently coupled to PE-COOH. R4 is a solution of tetrazole in dimethylformamide.

Figures 4A to 4F illustrate the practice of the invention to sequence YGGFL a covalently coupled to PE-COOH. R4 is a solution of tetrazole in acetonitrile.

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3 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 Dismuta3e 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).

Throughout the description and claims of This specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

S

DETAILED DESCRIPTION OF THE INVENTION The invention provides a preferably sequential four-step method for degradation of a peptide which "may include proline.

STEP 1. CARBOXYLATION OF THE PEPTIDE 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 trialkyl 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.

I

I I WO 95/22060 PCT/US94/01742 -4- When utilized in aqueous solution, alkyl amines such as triethylamine are preferably present in a concentration of about 5% by volume. When such amines are utilized in an organic solvent solution, a concentration of about 40% to 60% by volume is appropriate. The selection of an appropriate concentration of base in the solvent utilized is within the skill of the art. 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 0

C.

STEP 2. FORMATION OF THE THIOHYDANTOIN DERIVATIVE OF THE PEPTIDE TO BE SEQUENCED 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 0 C. to 0

C.'

The amine reagent rapidly promotes removal of the phosphoryl moiety from the phosphoroisothiocyanatidate reaction product. The invention includes -il- ill ~I ii I WO 95/22060 PCT/US94/01742 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, 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.

STEP 3. PROTONATION OF THE THIOHYDANTOIN 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 preferably STEP 4. CLEAVAGE OF THE C-TERMINAL THIOHYDANTOIN 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.OM, preferably 0.1M solution in an alcohol. A preferred solvent contains equal parts of methanol and t-butanol. See PCT application US90/02723.

Other salts of the trimethylsilanolate ion, such as those having the monovalent cations K Li+, Rb+, and Cs may be utilized. The trimethyl group may be replaced with other alkyl groups or with phenyl groups.

I Is*li WO 95/22060 PCT/US94/01742 -6- When the C-terminal derivative is thiohydantoin proline, the preferred cleavage reagent is gas phase water (water vapor) at a temperature of 30°C. to preferably 50"C. 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.

COVALENT COUPLING OF PEPTIDES TO CARBOXYLIC ACID MODIFIED POLYETHYLENE FILM Samples of PE-COOH film were treated with an IN solution of HC1 for 1-2 minutes at room temperature, in order to convert the surface carboxylic acid groups to the free acid form, prior to the covalent attachment of peptide samples.

The strips of PE-COOH film (1x12.5 mm) were then activated by an excess of DCC (dicyclohexylcarbodiimide) in anhydrous DMF (1 g/l ml) for 1 hour at room temperature. At the end of the activation reaction, the excess reagent was removed by rinsing the strips with anhydrous DMF.

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. The microbore tubing on one end of the CFR was sealed by heating and then pinched closed with pliers. After the coupling reaction, the support was rinsed with coupling solvent and acetonitrile, and then dried in a vacuum centrifuge.

DESCRIPTION OF FIGURE 1 SEQUENCER AND PROGRAM Below is described the instrument and detailed description of the program used for the C-terminal sequencing of C-terminal proline containing j Iq P-I WO 95/22060 PCT/US94/01742 -7polypeptides. This program can be used for the other 19 amino acids as well. It is therefore applicable to all of the common amino acids found in proteins.

The overall design of the sequencer sh by Figure 1 is similar in some respects to t gas phase sequencer described by Calaycay et al., Anal.Biochem.

192:23-31 (1991).

The reagent and solvent bottles associated with the instrument depicted in Figure 1 are shown. Four reagent bottles, R2-R5, and five solvent bottles, 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 anr solvent from bottle S5 are delivered to the conver ion flask In N-terminal sequencing, the CF serves to convert the ATZ derivative of the ;leaved amino acid into a PTH (phenylthiohydantoin) just before analysis by HPLC.

In C-terminal sequencing, the CF serves as a place to hold the cleaved thiohydantoin amino acid just prior to injection into the HPLC. The composition of the reagents and solvents is set forth in Table I.

I WO 95/22060 PCTIUS94/01742 -8- TABLE I COMPOSITION OF REAGENTS AND SOLVENTS FOR PROLINE PEPTIDES R1 R2 Diphenyl phosphoroisothiocyanatidate in acetonitrile (3.0 M) R3 0.10 M sodium trimethylsilanolate in methanol, 50% t-butyl alcohol R4 Pyridine (delivered in the gas phase) 2.0% trifluoroacetic acid in water 51 Water (delivered in the gas phase) S2 Methanol S3 25% acetonitrile, 75% ethyl acetate S4 Trifluoromethanesulfonic acid (delivered in the gas phase) Methanol 6 To deliver the reagents and solvents to the CFR, 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. There are a total of five pressure regulators (P1-P5). P1 is for S1-S4, P2 is for S5, S6, R5, S6, P3 is for R2 and R3, P4 is for R1 and R4, and P5 is for blow out functions and argon delivery functions (drying, etc.). When it is time to deliver a reagent or example Ri), a solenoid actuated valve on P4 opened in order to let the argon pass through valve to the bottle (RI).

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). There is a solenoid actuated valve on Q2, and a valve on SW1 II L -M

L

WO 95/22060 PCTIUS94/01742 -9- (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. After the reaction, the Angar valve (B01) and SW1 (to waste) is opened to allow argon to pass through the valve blocks Ql and Q2. This pushes the reagent or solvent in the CFR out to waste or to the CF, depending on which solenoid is actuated on the three-way switching valve just after the CFR. The program for sequencing therefore consists of only opening and closing solenoid actuated valves at various times.

The program for C-terminal sequencing utilizing the sequencer depicted in Figure 1 is set forth in Tables II and III.

TABLE II C-TERMINAL SEQUENCER INITIAL PROGRAM FOR PROLINE PEPTIDES Continuous Flow Conversion Duration Reactor (55 0 C) Flask (45"C) (sec) pressurize S4 3 deliver S4 pressurize Sl 3 deliver Sl pressurize S4 3 deliver S4 pressurize Sl 3 deliver S1 blow out S1 pressurize S3 3 deliver S3 blow out S3 pressurize R3 3 deliver R3 4 R3 reaction 120 blow out R3 pressurize R3 3 deliver R3 4 R3 reaction 120 blow out R3 s- I- -q u WO 95/22060 PCT/US94/01742 TABLE III C-TERMINAL SEQUENCER PROGRAM SUMMARY FOR PROLINE PEPTIDES Continuous Flow Conversion Durationa/ Reactor (55 0 C) Flask (45"C) (sec) R2 reaction S3 rinse R4 reaction S3 rinse R2 reaction S3 rinse R4 reaction S3 rinse R2 reaction S3 rinse R4 reaction S3 rinse S3 rinse S3 rinse 53 rinse S4 reaction Sl reaction S4 reaction S4 reaction S2 rinse S2 to CF S3 rinse S2 rinse S3 rinse S2 rinse Raise Temp. to R3 reaction R3 to CF Temp. back to pause R3 pressurize pause R3 to CF Dry 3, 2, 120, 3,60,---,30 3, 2, 120, 3,60,---,30 3, 2, 120, 3,60,---,30 3, 1, 3, 3, 3, 3, 30, 3, 60, 3, 60, 3, 60, 3, 2, 180 Dry in CF pressurize delivery to loop Loop to CF pressurize delivery to loop Loop to CF CF vent CF to HPLC pause pause CF to waste pressurize S5 deliver S5 Empty CF 600 3 4 8 3 4 8 3 3, 2, 3 120 a/ The first time is pressure, the second delivery, the third reaction time, and the fourth blowout.

4 1- I WO 95/22060 PCT/US94/01742 -11- 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.

The second step, "Deliver S4", the valve on P1 which corresponds to S4 is still open to maintain pressure on S4, but the solenoid on the reagent block (Q2) for S4 is also opened, permitting S4 vapor to flow into the CFR. Additionally, the solenoid on the three-way switching valve (SW1) is opened in order to permit equalization of pressure in the closed system and to allow any overflow to go to a waste bottle.

This flow is maintained for sixty seconds. At the end of sixty seconds, all of the solenoid actuated valves are closed and the S4 reagent, in this case trifluoromethanesulfonic acid, is then blown out of the CFR to the waste bottle by actuated valve B01 and the waste valve (SW1). This permits argon to push the contents of the CFR to waste. The same procedure, by actuation of the appropriate solenoids, is repeated for the remaining steps in the program.

The purpose of this program is to convert the C-terminal carboxylic acid group to a carboxylate.

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 rinsing with ethyl acetate/acetonitrile (S3), treatment with gas phase pyridine and rinsing with ethyl acetate/acetonitrile is repeated three times in order to complete derivatization of the C-terminal amino acid.

12 At this stage, 90% or greater of 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. The sample is then treated with gas phase trifluoromethanesulfonic acid (S4) in order to protonate the thiohydantoin ring formed in the case when the C-terminal amino acid is proline. This treatment is then followed by reaction with vapor phase water (S1) 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 me 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 15 treatment with sodium trimethylsilanolate (R3).

Cleavage is accomplished when sodium trimethylsilanolate in methanol and t-butanol (R3) is brought into the CFR, allowed to react for 180 seconds, Then the contents of the CFR are pushed into the CF and combined with the methanol extract described above. Once in the CF, the alcoholic solution ooo 20 containing the thiohydantoin is dried by blowing a stream of argon on it for 600 seconds. This is accomplished by opening valves (SW2 and SW3) under the CF o o as well as the valve which vents the CF.

The following examples are intended to illustrate particular embodiments and not limit the scope of the invention.

•0 i

I,

WO 95/22060 PCT/US94/01742 -13- EXAMPLE 1 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 S1 and S4 reaction steps are not included.

The product of each cycle is subjected to HPLC.

Figure 2 shows the chromatograms resulting from cycles 1-4. In each case, 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, water, and 10% methanol. Gradient elution is performed as follows: 0% B for 2 min., 0-4% B for min., and 35-50% B for 10 min. Absorbance is monitored at 265 nm.

EXAMPLE 2 Example 1 is repeated with the exception that R4 is a solution of 0.1 grams tetrazole in milliliters of dimethylformamide. The results are depicted by Figure 3.

EXAMPLE 3 Example 1 is repeated with the exception that R4 is a solution of 0.1 grams tetrazole in milliliters of acetonitrile. The results are depicted by Figures 4A, 4B, 4C, 4D, 4E and 4F.

r -I III WO 95/22060 PCT/US94/01742 -14- 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 EXAMPLE 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 Sl 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 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 S1 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.

WO 95/22060 PCT/US94/01742 EXAMPLE 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 S1 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.

EXAMPLE 8 This example describes the sequencing of the protein Hemoglobin a chain (4.1 nmoles), non-covalently applied to a Zitex strip (1 mm x 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 S1 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.

EXAMPLE 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 The total reaction volume was 0.1 ml. At the end of the reaction period, the peptide solution was evaporated to dryness by vacuum centrifugation. The i-- WO 95/22060 PCT/US94/01742 -16peptide products were re-dissolved in 0.1 ml of 0.1% trifluoroacetic acid in water and analyzed by reverse phase HPLC as described, in Bailey et al., Biochem. 29:3145-3156 (1990). A single peptide product was found. The mass of this peptide, obtained by FAB/MS, was found to be equivalent to the starting peptide, N-acetyl-Pro-Phe-Asp (MH+=420).

Claims (29)

1. A method for degrading a peptide which comprises: forming a thiohydantoin derivative of the C-terminal amino acid of the peptide; protonating the thiohydantoin derivative of the C-terminal amino acid; and cleaving the protonated thiohydantoin derivative of the C-terminal amino acid from the peptide.

2. A method according to claim 1, wherein the thiohydantoin derivative is formed by a method comprising the steps of: forming a carboxylate on the C-terminal amino acid of the peptide; and converting the carboxylated C-terminal amino acid into a 15 thiohydantoin derivative of the C-terminal amino acid.

3. A method according to claims 2, wherein the carboxylate is formed by reacting the peptide with a base.

4. A method according to claim 3, wherein the base is a tertiary trialkylamine. 20

5. A method according to claim 3, wherein the base is triethylamine.

6. A method according to claims 2, wherein the carboxylated peptide is converted into a thiohydantoin derivative by reacting the peptide with a phosphoroisothiocyanatidate derivative and an aromatic heterocyclic ring :containing nitrogen.

7. A method according to claim 6, wherein the phosphoroisothiocyanatidate derivative is diphenyl phosphoroisothiocyanatidate.

8. A method according to claim 6, wherein the aromatic heterocyclic ring containing nitrogen is selected from the group consisting of pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyrrole, pyrazole, imidazole, triazole, and tetrazole, or derivatives thereof.

9. A method according to claim 6 or 8, wherein the aromatic heterocyclic ring containing nitrogen is pyridine or a derivative thereof.

VT:C:Winword\ViotetIStuartnodeI 789.doc -18- A method according to claims 1, wherein the thiohydantoin derivative is protonated using an acid.

11. A method according to claim 10, wherein the acid is selected from the group consisting of hydrochloric acid, acetic acid, formic acid, trifluoromethanesulfonic acid and trifluoroacetic acid.

12. A method according to claim 11, wherein the acid is trifluoromethanesulfonic acid or trifluoroacetic acid.

13. A method according to claims 10, 11 or 12, wherein protonation occurs at a temperature of between about 30 0 C to about 900C.

14. A method according to claim 2, wherein the thiohydantoin derivative is cleaved using a trialkylsilanolate salt or a triarylsilanolate salt. A method according to claim 14, wherein the hydantoin derivative is cleaved using sodium trimethylsilanolate.

15

16. A method according to claim 1, wherein the C-terminal amino acid is proline.

17. A method according to claims 16, wherein the hydantoin derivative is cleaved using water vapor at a temperature between about 30°C to about 70 0 C.

18. A method according to claim 16, wherein the temperature of the water S 20 vapor is about 500C.

19. A method according to claims 6, 7, 8 or 9, wherein the aromatic heterocyclic ring containing nitrogen is present in molar excess with respect to the phosphoroisothiocyanatidate derivative.

20. A method according to claims 6, 7 8, 9 or 19, wherein the aromatic heterocyclic ring containing nitrogen and the phosphoroisothiocyanatidate derivative are in an inert polar solvent.

21. A method according to claim 20, wherein the inert polar solvent is acetonitrile or dimethylformamide.

22. A method according to claim 1, wherein the peptide is attached to a solid support. I- -19-

23. A method for degrading a peptide in which proline is the C-terminal amino acid which comprises: forming a thiohydantoin derivative of the proline residue; protonating the thiohydantoin derivative of proline; and cleaving the protonated thiohydantoin derivative of proline from the peptide.

24. A method according to claim 23, wherein the thiohydantoin derivative is formed by a method comprising the steps of: forming a carboxylate on the proline residue; and converting the carboxylated proline residue into a thiohydantoin derivative of the proline.

A method according to claim 24, wherein the carboxylate is formed by reacting the peptide with a base. 15

26. A method according to claim 24, wherein the carboxylated peptide is converted into a thiohydantoin derivative by reacting the peptide with a phosphoroisothiocyanatidate derivative and an aromatic heterocyclic ring .containing nitrogen.

27. A method according to claim 24, wherein the thiohydantoin derivative is 20 protonated using an acid.

28. A method according to claim 24, wherein wherein the hydantoin derivative is c',aved using water vapor at a temperature between about 30°C to about 700C.

29. A method according to claim 1 substantially as hereinbefore described with reference to any one of the examples. Dated: 14 February 1997 PHILLIPS ORMONDE FITZPATR!CK Attorneys for: CITY OF HOPE s 7' Q I'^:C:WinwordcViolet\Stuartknode1789.doc II- Ill