Chemically modifying peptides

Abstract

A method for chemically modifying a peptide, derivative or analogue thereof is described. The method comprises contacting a peptide, derivative or analogue thereof with a fluoro-heteroaromatic compound to activate the peptide, derivative or analogue thereof. The activated peptide, derivative or analogue thereof is then contacted with a nucleophile or base to create a chemically modified peptide, derivative or analogue thereof.

Claims

1. A method for chemically modifying a peptide, derivative or analogue thereof, the method comprising: (i) contacting a peptide, derivative or analogue thereof comprising at least one nucleophilic side chain with a fluoro-heteroaromatic compound to activate the peptide, derivative or analogue thereof due to the formation of a leaving group on the peptide, derivative or analogue thereof, wherein the leaving group comprises the heteroaromatic compound, which is covalently bonded to the nucleophilic side chain, and at least a portion of the nucleophilic side chain; and (ii) contacting the activated peptide, derivative or analogue thereof with a nucleophile, wherein the nucleophile displaces the leaving group and creates a covalent bond between the peptide, derivative or analogue thereof and the nucleophile to create a chemically modified peptide, derivative or analogue thereof, wherein the derivative or analogue is: a) a peptide where one or more of the amino acids residues of the peptide are replaced by residues with similar side chains or peptide backbone properties; b) a peptide where terminal groups thereof are protected by N- and C-terminal protecting groups with similar properties to acetyl or amide groups; c) a peptoid; d) a retropeptoid; e) a peptide-peptoid hybrid; or f) a peptide where at least one of the amino acids residues of the peptide is a D-amino acid.

2. The method according to claim 1, wherein the fluoro-heteroaromatic compound contains at least one nitrogen atom in its aromatic ring.

3. The method according to claim 1, wherein the fluoro-heteroaromatic compound comprises at least one hydrogen atom, wherein each hydrogen atom is covalently bonded to a carbon atom in the aromatic ring, or the fluoro-heteroaromatic compound comprises a perfluoroaromatic compound, or the fluoro-heteroaromatic compound comprises a chloro-fluoro-heteroaromatic compound.

4. The method according to claim 1, wherein the nucleophilic side chain reacts in an SNAr type reaction with the fluoro-heteroaromatic compound to displace a fluorine atom and create a covalent bond between the nucleophilic side chain and the heteroaromatic compound.

5. The method according to claim 1, wherein step (i) of the method comprises dissolving a peptide, derivative or analogue thereof in a solvent, and adding a base thereto before the fluoro-heteroaromatic compound is added to the dissolved peptide to create a reaction solution.

6. The method according to claim 5, wherein the molar ratio of the peptide, derivative or analogue thereof to the fluoro-heteroaromatic compound in step (i) is between 1:1 and 1:100, or between 1:5 and 1:50, or between 1:10 and 1:40, or between 1:20 and 1:30 and/or the molar ratio of the activated peptide, derivative or analogue thereof to the nucleophile or base in step (ii) is between 1:1 and 1:100, or between 1:5 and 1:10, or between 1:10 and 1:40, or between 1:20 and 1:30.

7. The method according to claim 1, wherein the nucleophile: a) comprises an organic molecule possessing nucleophilic functionality; and/or b) includes at least one group possessing nucleophilic functionality which is selected from a thiol group, a hydroxyl group, a primary amine group, a secondary amine group and a selenol group; and/or c) is selected from the group consisting of: a thiol containing sugar, a thiol containing nucleoside, a thiol containing alkyl chain, a thiol containing PEGylating agent, a thiol containing fluorescent tag, a thiol containing antibody, a hydroxyl containing sugar, a hydroxyl containing nucleoside, a hydroxyl containing alkyl chain, a hydroxyl containing PEGylating agent, a hydroxyl containing fluorescent tag, a hydroxyl containing antibody, an amine containing sugar, an amine containing nucleoside, an amine containing alkyl chain, an amine containing PEGylating agent, an amine containing fluorescent tag, an amine containing antibody, a selenol group, a selenol containing sugar, a selenol containing nucleoside, a selenol containing alkyl chain, a selenol containing PEGylating agent, a selenol containing fluorescent tag and a selenol containing antibody; and/or d) is glutathione.

8. The method according to claim 1, wherein the chemical modification comprises conjugation of a chemical entity onto the peptide.

9. The method according to claim 1, wherein at least one of the nucleophilic side chains comprises an amine group.

10. The method according to claim 1, wherein at least one of the nucleophilic side chains comprises a thiol group.

11. The method according to claim 1, wherein at least one of the nucleophilic side chains comprises an alcohol group.

12. The method according to claim 1, wherein at least one of the nucleophilic side chains comprises a selenol group.

13. A chemically modified peptide, derivative or analogue thereof obtained by the method according to claim 1.

14. The chemically modified peptide, derivative or analogue thereof according to claim 13, wherein the chemically modified peptide, derivative or analogue thereof is functionalised or tagged with a selected chemical entity.

15. The method according to claim 2, wherein the fluoro-heteroaromatic compound contains one, two or three nitrogen atoms in the aromatic ring.

16. The method according to claim 1, wherein the fluoro-heteroaromatic compound contains at least one fluorine atom, where each fluorine atom is covalently bonded to a carbon atom in the aromatic ring.

17. The method according to claim 5, wherein the base is N,N-diisopropylethylamine (DIPEA).

18. The method according to claim 5, wherein the solvent is 2,2,2-trifluoroethanol (TFE).

19. The method according to claim 8, wherein the chemical entity is selected from the group consisting of a sugar, a thiosugar, a nucleoside, an alkyl chain, PEG, a fluorescent tag and an antibody.

20. The method according to claim 9, wherein the amine group is provided on an amino acid residue within the peptide, derivative or analogue thereof.

21. The method according to claim 9, wherein the amine group is provided on a lysine residue in the peptide, derivative or analogue thereof.

22. The method according to claim 10, wherein the thiol group is provided on a cysteine residue or modified cysteine residue in the peptide, derivative or analogue thereof.

23. The method according to claim 11, wherein the alcohol group comprises a phenol group.

24. The method according to claim 11, wherein the alcohol group is provided on a serine or threonine residue within the peptide, derivative or analogue thereof.

25. The method according to claim 12, wherein the selenol group is provided on a selenocysteine residue within the peptide, derivative or analogue thereof.

26. The chemically modified peptide, derivative or analogue thereof according to claim 14, wherein the chemical entity is selected from the group consisting of a sugar, a thiosugar, a nucleoside, an alkyl chain, PEG, a fluorescent tag and an antibody.

Description

(1) For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:

(2) FIG. 1 is a schematic diagram showing the common method used in the prior art for chemical modification of a protein or peptide, where a chemical entity is conjugated to a protein or peptide using a linker;

(3) FIG. 2 is a schematic diagram showing the method developed for chemical modification of a protein or peptide according to the present invention;

(4) FIG. 3 shows one approach for chemical modification of a protein or peptide used in the prior art, where a disulfide bond is present in the final product;

(5) FIG. 4 shows an alternative approach for chemical modification of a protein or peptide used in the prior art, where the alpha-chirality of the amino acids involved is racemised;

(6) FIG. 5 shows a linker used in the prior art for homogeneous antibody-drug conjugation;

(7) FIG. 6 shows a molecule developed in the prior art for use as a linker;

(8) FIG. 7 shows a protein which has been chemically modified, using a linker, according to the teachings of the prior art;

(9) FIG. 8 shows how a protein can be chemically modified, using maleimide as a linker, according to the teachings of the prior art;

(10) FIG. 9 shows how a protein can be chemically modified, using bromomaleimide as a linker, according to the teachings of the prior art;

(11) FIG. 10 shows various embodiments of suitable fluoro-heteroaromatic compounds which can be reacted with a peptide, derivative or analogue thereof to create an activated peptide, derivative or analogue thereof according to the invention;

(12) FIG. 11 shows the reaction of Peptide 2 with thiophenolate;

(13) FIG. 12 is the liquid chromatography-mass spectrometry (LCMS) spectrum for the crude reaction mixture resulting from the reaction shown in FIG. 12;

(14) FIG. 13 is the mass spectrometry (MS) data from the peak at 2.508 minutes in the LCMS spectrum of FIG. 13;

(15) FIG. 14 shows the reaction of a fluoro-pyridine activated peptide with thioacetate;

(16) FIG. 15 is the LCMS spectrum for the crude reaction mixture resulting from the reaction shown in FIG. 17;

(17) FIG. 16 is the mass spectrometry (MS) data from the peak at 2.071 minutes in the LCMS spectrum of FIG. 18;

(18) FIG. 17 is the mass spectrometry (MS) data from the peak at 2.834 minutes in the LCMS spectrum of FIG. 18;

(19) FIG. 18 is a schematic showing the method developed for accessing dehydroalanine containing peptides according to the present invention; and

(20) FIG. 19 is a schematic showing the method developed for accessing multiply chemically tagged peptides according to the present invention.

EXAMPLES

(21) As mentioned previously, it is desirable to be able to chemically modify peptides, analogues and derivatives thereof. It is often desirable to chemically modify a peptide, analogue or derivative thereof by attaching a chemical entity to the said peptide, analogue or derivative thereof. Much work has been done in developing this process in the prior art.

(22) One method developed in the prior art involves linking the peptide, analogue or derivative thereof to the chemical entity by means of a linker. A schematic showing how this can be achieved is shown in FIG. 1 and examples using linkers developed by ThioLogics are shown in FIGS. 5, 6, 7, 8 and 9. As discussed above, linkers that are used often produce more than one product (poor control of regio- and stereo-isomer formation) which presents a significant challenge in terms of purification and characterization.

(23) Alternatively, the Davis group at Oxford have devised an approach that results in a disulfide linkage being present in the final product, as shown in FIG. 3, or involves the formation of a dehydroalanine intermediate, as shown in FIG. 4. Drawbacks with both of these approaches are discussed above.

(24) The applicant has developed a new technology platform that allows chemical moieties to be attached to a peptide or protein in a manner that does not involve a linker. FIG. 2 is a schematic illustrating this embodiment of the present invention.

(25) An alternative embodiment of the present invention allows the formation of dehydroalanine intermediates by a novel method. A schematic illustrating this embodiment is shown in FIG. 18.

(26) The applicant has also developed a new technology platform that allows multiple chemical moieties to be attached to a peptide or protein. FIG. 19 is a schematic illustrating this embodiment of the present invention.

(27) Materials and Methods

(28) Five different peptides, referred to as peptides 1 to 4, were prepared where: Peptide 1 has the structure AcNH-Y-C-G-G-G-C-A-L-CONH.sub.2; Peptide 2 has the structure AcNH-A-C-Y-G-S-I-L-A-R-T-CONH.sub.2; Peptide 3 has the structure AcNH-F-C-G-G-G-C-A-L-CONH.sub.2; and Peptide 4 has the structure AcNH-F-S-G-G-G-S-A-L-CONH.sub.2;

(29) Peptides 1-4 was prepared using automated Fmoc-SPPS methods on a Liberty 1 peptide synthesizer (CEM) with microwave-assisted couplings (single coupling per amino acid; 10 min, 75 C. (50 C. for Fmoc-cys(trt)-OH coupling). Solid phase synthesis was conducted using Rink amide resin (0.7 mol/g loading) on a 0.1 mol scale, employing PyBOP and DIPEA as activator and base, respectively. Following on-resin synthesis of the appropriate sequence, N-terminal capping was achieved using Ac.sub.2O/DMF (20%, 215 min) with shaking at room temperature. Finally, peptides were cleaved from the resin as the C-terminal amide by treatment of beads with a cleavage cocktail containing 90% TFA, 5% TIPS and 5% water with shaking at room temperature for 4 h. After removal of volatiles in vacuo, the product was triturated and washed using Et.sub.2O.

(30) The structures of the fluoro-heteroaromatics and fluoro-aromatic used are shown in FIG. 15.

(31) The peptides were reacted according to procedures A and B as described below:

(32) Procedure A

(33) Solid peptide (approx. 2 mg, 2.5 mol) was dissolved in DMF (0.5 mL) in a 1.5 mL plastic Eppendorf tube, to which DIPEA (50 mM in DMF, 0.5 mL) was added. The fluoro-heteroaromatic or fluoroaromatic was then added in 25 equivalents and the tube was shaken at room temperature for 4.5 h. After removal of volatiles under vacuum, each reaction mixture was re-dissolved in a 1:1 mixture of H.sub.2O and MeCN (1 mL) and analyzed by LCMS (ESI+).

(34) Procedure B

(35) Solid peptide (approx. 2 mg, 2.5 mol) was dissolved in TFE (0.5 mL) in a 1.5 mL plastic Eppendorf tube, to which DIPEA (50 mM in TFE, 0.5 mL) was added. The fluoro-heteroaromatic or fluoroaromatic was added in 25 equivalents and the tube was shaken at room temperature for 4.5 h. After removal of volatiles under vacuum, each reaction mixture was re-dissolved in a 1:1 mixture of H.sub.2O and MeCN (1 mL) and analyzed by LCMS (ESI+) and .sup.19F NMR (100 L D.sub.2O added).

(36) Procedure C

(37) DIPEA (20 L) was added to a solution of peptide (0.3 mg) in MeCN (0.5 mL) and water (0.5 mL) in a 1.5 mL plastic Eppendorf tube. A sulphur nucleophile was added in 5 equivalents and the tube was shaken at room temperature for 4 h and then analyzed by LCMS (ESI+).

(38) The inventor has found that when the reagents are reacted according to procedure B the fluoro-heteroaromatic selectively reacts with cysteine residues instead of the tyrosine residues.

(39) LC-MS Conditions:

(40) Peptides and peptoids were characterised by LC-MS, ESI-LC MeCN (TQD mass spectrometer and an Acquity UPLC from Waters) using an Acquity UPLC BEH C8 1.7 m (2.1 mm50 mm) column and (C18 as of Jun. 2, 2015 3 pm) with a flow rate of 0.6 ml min.sup.1, a linear gradient of 5-95% of solvent B over 3.8 min (A=0.1% formic acid in H.sub.2O, B=0.1% formic acid in MeCN) and injection volume of 1 l.

(41) QToF (mass spectrometer and an Acquity UPLC from Waters) using an Acquity UPLC BEH C8 1.7 m (2.1 mm50 mm) column with a flow rate of 0.6 ml min.sup.1, a linear gradient of 0-99% of solvent B over 5 min (A=0.1% formic acid in H.sub.2O, B=0.1% formic acid in MeCN) and injection volume of 3 l.

(42) Peptides and peptoids identities were also confirmed by MALDI-TOF mass spectra analysis (Autoflex II ToF/ToF mass spectrometer Bruker Daltonik GmBH) operating in positive ion mode using an -cyano-4-hydroxycinnamic acid (CHCA or CHHA) matrix. Data processing was done with MestReNova Version 10.0.

(43) TQD

(44) ESI-LC MeCN (TQD): Acquity UPLC BEH C8 1.7 m (2.1 mm50 mm) (C18 as of Jun. 2, 2015 3 pm) Mobile phase: water containing formic acid (0.1% v/v):Acetonitrile Flow rate 0.6 ml min.sup.1 Injection volume: 1 l Gradient:

(45) TABLE-US-00001 Time (min) % A % B 0 95 5 0.2 95 5 4 5 95 4.5 5 95 5 95 5 Data processing: MestReNova 10.0
QToF Accurate mass: Acquity UPLC BEH C18 1.7 m (2.1 mm100 mm) Mobile phase: water containing formic acid (0.1% v/v):Acetonitrile Flow rate: 0.6 ml min.sup.1 Injection volume: 3 l Gradient:

(46) TABLE-US-00002 Time (min) % A % B 0 100 0 5 1 99 6 1 99 6.1 100 0 7 100 0 Data processing: MestReNova 10.0
MALDI Autoflex II ToF/ToF mass spectrometer Bruker Daltonik GmBH 337 nm nitrogen laser Sample preparation 1 mg/ml, 1 l spotted on matrix Operating in positive ion mode using an -cyano-4-hydroxycinnamic acid (CHCA or HCCA) matrix Data acquisition: reflecton mode of analysis Data processing: MestReNova 10.0

EXAMPLE 1

Reacting Peptides 1 to 4 with Fluoro-Heteroaromatic or Fluoroaromatic Compounds to Obtain Activated Peptides

(47) Peptides 1 to 4 were further reacted using either procedure A or procedure B to create a stock of modified peptides, as set out in table 1.

(48) TABLE-US-00003 TABLE 1 Reaction of peptides 1 to 4 according to procedure A or B LCMS Fluoro- spectrum and Peptide heteroaromatic chromatogram Product 1* One major peak in the LCMS chromatogram with a retention time of 2.617 minutes, the spectrum for the peark shows an [M + H].sup.+ at 1081.629 m/z. 2 One major peak in the LCMS chromatogram with a retention time of 3.242 minutes, the spectrum for this peak shows an [M + H].sup.+ peak at 1603 m/z. 2 A peak in the LCMS chromatogram with retention times of 3.650 minutes. The spectrum for this peaks show an [M + H].sup.+ peak at 1802 m/z.. 2 One major peak in the LCMS chromatogram with a retention time of 2.683 minutes, the spectrum for this peak shows an [M + H].sup.+ peak at 1357 m/z. 2 One major peak in the LCMS chromatogram with a retenetion time of 4.067 minutes, the spectrum for this peak shows an [M + H].sup.+ peak at 1740 m/z. 0 2 One major peak in the LCMS chromatogram with a retention time of 3.458 minutes, the spectrum for this peak shows an [M + H].sup.+ peak at 1691 m/z. 2 Two major peaks in the LCMS chromatogram with retention times of 2.750 minutes and 3.833 minutes. The spectrum for these peaks show an [M + 2MeCN + H].sup.+ peak at 1288 m/z and an [M + H]+ peak at 1686 m/z. 3 One major peak in the LCMS chromatogram with a retention time of 2.292 minutes, the spectrum for this peak shows an [M + H].sup.+ peak at 880 m/z. 3 One major peak in the LCMS chromatogram with a retention time of 2.942 minutes, the spectrum for this peak shows an [M + H].sup.+ peak at 994 m/z. 3 Two major peaks in the LCMS chromatogram with retention times of 3.650 minutes and 3.883 minutes. The spectrum for these peaks show an [M + H].sup.+ peak at 1238 m/z and an [M + H].sup.+ peak at 982 m/z. 0 3 Major peak in the LCMS chromatogram with a retention time of 3.083 minutes. The spectrum for this peak shows an [M + H].sup.+ peak at 1030 m/z. 3 Two major peaks in the LCMS chromatogram with retention times of 4.058 minutes and 4.300 minutes. The spectrum for these peaks show an [M + H].sup.+ peak at 1181 m/z and an [M + H].sup.+ peak at 1189 m/z. 3 Two major peaks in the LCMS chromatogram with retention times of 3.558 minutes and 2.583 minutes. The spectrum for these peaks showed [M + H].sup.+ peaks at 1132 m/z, 929 m/z. 34 One major peak in the LCMS chromatogram with a retention time of 3.275 minutes, the spectrum for this peak shows [M + H].sup.+ peaks at 1169 m/z and 1155 m/z. 3 Two major peaks in the LCMS chromatogram with retention times of 3.458 minutes and 2.758 minutes. The spectrum for these peaks show an [M + H].sup.+ peak at 1100 m/z and an [M + H]+ peak at 914 m/z. 0 3 One major peak in the LCMS chromatogram with retention times of 3.458 minutes. and 2.958 minutes. The spectrum for this peak show an [M + H].sup.+ peak at 1066 m/z. 3 Two major peaks in the LCMS chromatogram with retention times of 3.208 minutes and 2.417 minutes. The spectrum for these peaks show [M + H].sup.+ peaks at 1064 m/z, 896 m/z and 879 m/z. 4 Two major peaks in the LCMS chromatogram with retention times of 2.125 minutes and 2.197 minutes. The spectrum for these peaks show [M + H].sup.+ peaks at 868 m/z, 1000 m/z and 848 m/z. 4 Two major peaks in the LCMS chromatogram with retention times of 3.650 minutes and 2.708 minutes. The spectrum for these peaks show [M + H].sup.+ peaks at 1206 m/z and 951 m/z. 4 One major peak in the LCMS chromatogram with a retention time of 3.558 minutes, the spectrum for this peak shows an [M + H].sup.+ peak at 1100 m/z. 0 4 One major peak in the LCMS chromatogram with a retention time of 3.275 minutes, the spectrum for this peak shows an [M + H].sup.+ peak at 1066 m/z. 4 One major peak in the LCMS chromatogram with retention times of 3.467 minutes. The spectrum for this peak show an [M + H].sup.+ peak at 1140 m/z. 4 Two major peaks in the LCMS chromatogram with retention times of 2.133 minutes and 3.025 minutes. The spectrum for these peaks show [M + H].sup.+ peaks at 884 m/z and 1032 m/z. *The reaction for this entry was carried out using procedure B. All other reactions were performed using procedure A.

(49) It will be readily appreciated that while a selection of fluoro-heteroaromatic compounds were used in this instance many more fluoro-heteroaromatic compounds could be used to create a peptide with a suitable leaving group. FIG. 10 shows the structures of some suitable fluoro-heteroaromatic compounds. Alternatively, it will be appreciated that further fluoro-heteroaromatic compound could be used, such as fused six-membered rings.

EXAMPLE 2

Reaction of Peptide 6 with Thiophenolate

(50) Peptide 6 was reacted with thiophenolate according to the following reaction procedure:

(51) To a solution of Peptide 6 (2 mg, 1.8 mol) in MeCN (0.5 mL) and water (0.5 mL) in a 1.5 mL plastic Eppendorf tube, was added DIPEA (20 L). Sodium thiophenolate was added in 5 equivalents and the tube was shaken at room temperature for 4 h and then analyzed by LCMS (ESI+).

(52) An LCMS spectrum of the crude reaction mixture is shown in FIG. 12, and MS data from the peak at 2.508 minutes is shown in FIG. 13. Analysis of the crude reaction mixture suggested that the thio-fluoroheteroaryl group was substituted by the thiophenolate. The peak at 1008.796 m/z in FIG. 13 corresponds to an [M+H].sup.+ peak for a single thiophenolate substituted product.

(53) FIG. 11 shows the reaction which occurred.

(54) Accordingly, one embodiment of the present invention is a method for attaching a chemical moiety to a peptide or protein in a manner that does not involve a linker.

EXAMPLE 3

Reaction of Peptide 6 with Thioacetate

(55) Peptide 6 was reacted with potassium thioacetate according to the following reaction procedure:

(56) To a solution of Peptide 6 (2 mg, 1.8 mol) in MeCN (0.5 mL) and water (0.5 mL) in a 1.5 mL plastic Eppendorf tube, was added DIPEA (20 L). Potassium thioacetate was added in 5 equivalents and the tube was shaken at room temperature for 4 h and then analyzed by LCMS (ESI+).

(57) An LCMS spectrum of the crude reaction mixture is shown in FIG. 15, and MS data from the peaks at 2.071 minutes and 2.834 minutes is shown in FIGS. 16 and 17 respectively. Analysis of the crude reaction mixture suggested that the thio-fluoroheteroaryl group was eliminated to afford the dehydroalanine. The peak at 715.567 m/z in FIG. 17 corresponds to an [M+H].sup.+ peak for the mono-eliminated product and the peak at 898.458 m/z in FIG. 20 corresponds to an [M+H].sup.+ peak for the di-eliminated product.

(58) FIG. 14 shows the reaction which occurred.

(59) Accordingly, an alternative embodiment of the present invention is a new method enabling the creation of dehydroalanine containing peptides. The general approach for this is shown in FIG. 18. Dehydroalanine containing peptides can be used as reactive intermediates in the formation of bioconjugates, as demonstrated by the work carried out by the Davis group. For instance, dehydroalanine containing peptides are well known to act as substrates for the addition of various sulphur nucleophiles.

EXAMPLE 4

Further Reaction of Activated Peptides with Sulphur Nucleophiles

(60) The activated peptides obtained by reacting peptide 2 with various fluoroheteroaromatic compounds in Example 1 were further reacted with various sulphur nucleophiles according to Procedure C.

(61) TABLE-US-00004 TABLE 2 Reaction of Peptides 6, 8, 9, 10, 11a and 11b with sulphur nucleophiles according to procedure C Sulphur Peptide Nucleophile Product 6 8 0 9 9 10 11a and 11b 11a and 11b 0

(62) When peptide 6 was exposed to sodium thiophenolate, the sulphur nucleophile displaced two heteroaromatic groups on peptide 6 to give product 201. This results in original peptide 2 having been tagged, without using a linker, at a cysteine residue and a serine residue.

(63) Similarly, when peptides 8, 10 and 11a were reacted with sodium thiophenolate peptide 8 reacted to give product 202, peptide lo reacted to give product 206, and peptide 11a reacted to give product 207, all of which have undergone a displacement reaction. This results in original peptide 2 having been tagged, without using a linker, at a cysteine residue.

(64) For the aforementioned reactions the peptides underwent displacement reactions but the heteroaromatic group attached on the tyrosine resides were unreacted. Accordingly, the inventors have shown that it is possible to selectively tag molecules at chosen residues (i.e. cysteine or serine) in the presence of an activated tyrosine residue. Additionally, the inventors have shown that sometimes instead of displacing a heteroaromatic group the sulphur nucleophile instead displaces one or more of the halogens on the aromatic ring. Due to the presence of multiple halogens on the ring, it is possible to add multiple tags to a heteroaromatic group. An example of this is where peptide 11a reacted with sodium thiophenolate to give the product 207. An example of displacing only one halogen can be seen in the reaction between peptide 9 and the nucleophile 1-Thio--D-glucose tetraacetate which affords the product 205.

EXAMPLE 5

Further Reaction of Activated Peptides with Sulphur Nucleophiles

(65) The activated peptides obtained by reacting peptide 3 with various fluoroheteroaromatic compounds in Example 1 were further reacted with various sulphur nucleophiles according to Procedure C.

(66) TABLE-US-00005 TABLE 3 Reaction of Peptides 12 and 14b with sulphur nucleophiles according to procedure C Sulphur Peptide(s) Nucleophile Product 12 14b

(67) When peptide 12 was exposed to 1-thio--D-glucose tetraacetate, the sulphur nucleophile displaced one of the fluorine atoms on the fluoro-heteroaromatic bridge to give product 301. Similarly when peptide 14b was exposed to 1-thio--D-glucose tetraacetate, the sulphur nucleophile displaced on of the fluorine atoms on the fluoro-heteroaromatic bridge to give product 302. This demonstrates that it is possible to tag a fluoro-heteroaromatic containing cyclic peptides without causing degradation of the cyclic peptide. This approach offers a novel route to prepare tagged cyclic peptides.

(68) It should be noted that when peptide 3 was modified using a hexafluorobenzene , to give peptide 19, the inventors found that this modified peptide did not react with the sulphur nucleophile to give a tagged cyclic product. This highlights the advantage over the prior art afforded through the application of a perfluoro-heteroaromatic reagent to activate the peptides.

EXAMPLE 6

Further Reaction of Activated Peptides with Sulphur Nucleophiles

(69) The activated peptides obtained by reacting peptide 4 with various fluoroheteroaromatic compounds in Example 1 were further reacted with various sulphur nucleophiles according to Procedure C.

(70) TABLE-US-00006 TABLE 4 Reaction of Peptides 22a, 22b, 23a and 27b with sulphur nucleophiles according to procedure C Sulphur Peptide Nucleophile Product 22a and 22b 23a 0 23a 27b

(71) When peptide 22b was exposed to 1-thio--D-glucose tetraacetate, the sulphur nucleophile displaced one heteroaromatic group on peptide 22b to give product 402. This results in original peptide 4 having been tagged, without using a linker, at a serine residue.

(72) When peptide 23a was exposed to 1-thio--D-glucose tetraacetate, the sulphur nucleophile displaced one heteroaromatic group on peptide 23a to give product 405. This results in original peptide 4 having been tagged, without using a linker, at a serine residue.

(73) Additionally peptide 22ba reacted with 1-thio--D-glucose tetraacetate to give the product 403, where the sulphur nucleophile displaced two halogens on each aromatic ring, thereby adding multiple tags to the heteroaromatic group. Further examples of this include where peptide 23a reacted with sodium thiophenolate to give the multiply tagged product 404 and where peptide 27b reacted with sodium thiophenolate to give the multiply tagged product 406.

SUMMARY

(74) Advantages of the invention include the possibility of chemically modifying activated peptides under mild conditions in a traceless fashion. Traceless chemical modification is not currently available through other published methodologies most of which require the use of a linker moiety being present in the final product. The methodology requires peptide activation via reaction with a halogenated heteroaromatic followed by nucleophilic displacement of the halogenated heteroaromatic. Selectivity between serine/cysteine activated residues and tyrosine activated residues has been demonstrated. Nucleophilic displacement occurs directly without the need to install a dehydro-alanine type motif which in other published methodologies leads to the loss of stereo-chemical integrity.

(75) However, if the installation of a dehydro-alanine type motif is desired, the activated peptide can be reacted under mild conditions to install the motif into the peptide backbone.

(76) Activation of a peptide through a halogenated heteroaromatic has also been shown to offer a route to attach multiple chemical moieties to a linear or cyclic peptide. The aforementioned reaction has been shown not to be possible if carried out with a published halogenated aromatic (e.g. hexafluorobenzene) activated peptide.