Process For The Conjugation Of A Peptide Or Protein With A Reagent Comprising A Leaving Group Including A Portion Of PEG

Abstract

The invention relates to novel conjugating reagents capable of reaction with at least one nucleophile present in a peptide or protein, which contain at least one leaving group which is lost on reaction with said nucleophile, in which the leaving group includes a portion —(CH.sub.2CH.sub.2O).sub.n—, in which n is a number of six or more; and novel processes for the preparation of conjugates containing peptides or proteins made using such reagents.

Claims

1. A conjugating reagent capable of reaction with at least one nucleophile present in a peptide or protein, which contains at least one leaving group which is lost on reaction with said nucleophile, characterised in that said leaving group includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more.

2. A conjugating reagent as claimed in claim 1, in which n is from 6 to 9.

3. A conjugating reagent as claimed in claim 1, in which the portion —(CH.sub.2CH.sub.2O).sub.n— has a molecular weight of up to 5 kDa.

4. A reagent as claimed in claim 1 any one of the preceding claims, in which said leaving group is of the formula —SP, —OP, —SO.sub.2P, —OSO.sub.2P, —N.sup.+PR.sup.2R.sup.3, or is a group of formula —S—P—S—, —O—P—O—, —SO.sub.2—P—SO.sub.2—, —OSO.sub.2—P—OSO.sub.2—, or —N.sup.+R.sup.2R.sup.3—P—N.sup.+R.sup.2R.sup.3—, in which P is a group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— and each of R.sup.2 and R.sup.3 independently represents a hydrogen atom or a C.sub.1-4alkyl group or a group P.

5. A reagent as claimed in claim 4, in which said leaving group is of the formula —SO.sub.2P.

6. A reagent as claimed in claim 1, in which said leaving group includes —(CH.sub.2CH.sub.2O).sub.n—R.sup.1 where R.sup.1 is a capping group; or in which said —(CH.sub.2CH.sub.2O).sub.n— group has two points of attachment within the reagent.

7. A reagent as claimed in claim 1, in which said leaving group is —S—(CH.sub.2CH.sub.2O).sub.n—(CH.sub.2CH.sub.2)—S—, —O—(CH.sub.2CH.sub.2O).sub.n—(CH.sub.2CH.sub.2)—O—, —SO.sub.2—(CH.sub.2CH.sub.2O).sub.n—(CH.sub.2CH.sub.2)—SO.sub.2—, —OSO.sub.2—(CH.sub.2CH.sub.2O).sub.n—(CH.sub.2CH.sub.2)—OSO.sub.2—, —N.sup.+R.sup.2R.sup.3—(CH.sub.2CH.sub.2O).sub.n—(CH.sub.2CH.sub.2)—N.sup.+R.sup.2R.sup.3—, or one of the groups of formula ##STR00136## where R.sup.1 is a capping group and each of R.sup.2 and R.sup.3 independently represents a hydrogen atom or a C.sub.1-4alkyl group.

8. A reagent as claimed in claim 7, in which said leaving group is —SO.sub.2—(CH.sub.2CH.sub.2O).sub.n—(CH.sub.2CH.sub.2)—SO.sub.2—.

9. A reagent as claimed in claim 6, in which R.sup.1 represents a hydrogen atom, a C.sub.1-4 alkyl group, an optionally substituted aryl group, or a group of formula —CH.sub.2CH.sub.2CO.sub.2H or —CH.sub.2CH.sub.2NH.sub.2.

10. A reagent as claimed in claim 1, which carries a payload which is a diagnostic, therapeutic or labelling agent or a binding agent for a diagnostic, therapeutic or labelling agent.

11. A reagent as claimed in claim 1, which includes the functional grouping: ##STR00137## in which one L is a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more and the other L is a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more or is a leaving group of another structure; or which includes the functional grouping ##STR00138## in which one L is a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more.

12. A reagent as claimed in claim 1, which contains the functional grouping I, I′, II or III: ##STR00139## in which W represents an electron-withdrawing group; A represents a C.sub.1-5 alkylene or alkenylene chain; B represents a bond or a C.sub.1-4 alkylene or alkenylene chain; and either one L is a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more and the other L is a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more or is a leaving group of another structure, or both Ls together represent a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more; ##STR00140## in which W and A have the meanings given for the general formula I, m is 0 to 4, and L is a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more;
˜W-CR.sup.4R.sup.4′—CR.sup.4.L.L′  (II) in which W has the meaning given for the general formula I, and either (i) each R.sup.4 represents a hydrogen atom or a C.sub.1-4alkyl group, R.sup.4′ represents a hydrogen atom, and either L is a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more and L′ is a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more or is a leaving group of another structure, or L and L′ together represent a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more; or (ii) each R.sup.4 represents a hydrogen atom or a C.sub.1-4alkyl group, L represents a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more, and R.sup.4′ and L′ together represent a bond;
˜W-(CH═CH).sub.p—(CH.sub.2).sub.2-L   (III) in which W has the meaning given for the general formula I, p represents 0 or an integer of from 1 to 4, and L represents a leaving group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of six or more.

13. A reagent as claimed in claim 12 which has the general formula: ##STR00141## in which Q represents a linking group and D represents a payload which is a diagnostic, therapeutic or labelling agent or a binding agent for a diagnostic, therapeutic or labelling agent.

14. A reagent as claimed in claim 12, which contains the functional grouping: ##STR00142## in which Ar represents a phenyl group.

15. A reagent as claimed in claim 14, which has the general formula: ##STR00143## in which Q represents a linking group and D represents a payload which is a diagnostic, therapeutic or labelling agent or a binding agent for a diagnostic, therapeutic or labelling agent.

16. A reagent as claimed in either claim 13, in which D-Q includes a drug or a polymer or both a drug and a polymer.

17. A reagent as claimed in claim 16, in which the drug if present is a cytotoxic drug, and the polymer if present is polyethylene glycol.

18. A conjugating reagent capable of reaction with two nucleophiles present in a peptide or protein, which contains a leaving group which is lost on reaction with said nucleophiles, characterised in that said leaving group has two points of attachment within the conjugating reagent and includes a portion —(CH.sub.2CH.sub.2O).sub.n1— in which n1 is a number of two or more.

19. A conjugating reagent as claimed in claim 18, in which n1 is from 5 to 9.

20. A process for the conjugation of a peptide or protein, which comprises reacting said peptide or protein with a conjugating reagent as claimed in claim 1.

21. A process as claimed in claim 20, in which the protein is a receptor or ligand binding protein or an antibody or antibody fragment.

22. A protein or peptide conjugate prepared by a process as claimed in claim 20.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0143] FIG. 1 shows the results of Example 17.

[0144] FIG. 2 shows the results of Example 18.

[0145] FIG. 3 shows the results of Example 19.

[0146] FIG. 4 shows the results of Example 20.

[0147] FIGS. 5, 6 and 7 show the results of Example 21.

[0148] FIG. 8 shows the results of Example 22.

[0149] FIG. 9 shows the results of Example 25.

[0150] FIG. 10 shows the results of Example 49 obtained after 2 hours reaction time.

[0151] FIG. 11 shows the results of Example 49 obtained after 4 hours reaction time.

[0152] The following Examples illustrate the invention.

EXAMPLE 1

Synthesis of a Conjugation Reagent 2 Comprising 7 Repeat Unit Ethylene Glycol Leaving Groups

[0153] Step 1: Synthesis of Compound 1.

##STR00037##

[0154] To a stirred solution of 4-[2,2-bis[p-tolylsulfonyl)-methyl]acetyl]benzoic acid (1.50 g, Nature Protocols, 2006, 1(54), 2241-2252) in dimethylformamide (DMF, 70 mL) was added alpha-methoxy-omega-mercapto hepta(ethylene glycol) (3.20 g, Iris Biotech) and triethylamine (2.50 mL). The resulting reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. After 19 h, volatiles were removed in vacuo. The resulting residue was dissolved in water (2.4 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give 4-[2,2-bis[alpha-methoxy-omega-thio-hepta(ethylene glycol)]acetyl]-benzoic acid compound 1 as a thick clear colourless oil (1.77 g, 66%) m/z [M+H.sup.+]901.

[0155] Step 2: Synthesis of Reagent 2.

##STR00038##

[0156] To a stirred solution of 1 (1.32 g) in methanol:water (18 mL, 9:1 v/v) at room temperature was added Oxone® (2.70 g). After 2.5 h, the volatiles were removed in vacuo and water was azeotropically removed with acetonitrile (2×15 mL). The resulting residue was dissolved in dichloromethane (3×10 mL), filtered through a column of magnesium sulfate and washed with dichloromethane (2×7 mL). The eluent and washings were combined and the volatiles were removed in vacuo to give a thick clear pale yellow oil 1.29 g, 92%. A portion of the residue (700 mg) was dissolved in water:acetonitrile (1.50 mL, 3:1 v/v), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give 4-[2,2-bis[alpha-methoxy-omega-sulfonyl hepta(ethylene glycol)]acetyl]benzoic acid reagent 2 as a thick clear colourless oil (524 mg, 68%) m/z [M+H.sup.+]965.

EXAMPLE 2

Synthesis of a PEGylation Reagent 3 Comprising 7 Repeat Unit Ethylene Glycol Leaving Groups and a 10 kDa PEG

[0157] ##STR00039##

[0158] A stirred solution of reagent 2 (14 mg) in DMF (600 μL) was cooled to 0° C. under an argon atmosphere and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (5.5 mg) and N-methylmorpholine (NMM) (1.5 μL) were added. The solution was stirred for 0.5 h at 0° C. when a solution of 10 kDa H.sub.2N-PEG-OMe (132 mg) in dichloromethane (600 μL) was added. The resulting solution was stirred for 5 min at 0° C. before addition of further HATU (5.5 mg) and NMM (1.5 μL). The reaction solution was allowed to stir at 0° C. for 2 h before being warmed to room temperature. After 22 h, the volatiles were removed in vacuo. The resulting residue was dissolved in water and acetonitrile (v/v; 1/1, 2.0 mL), and purified by reverse phase C18-column chromatography cluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-mPEG(7u)sulfone-propanoyl-benzamide-PEG(10 kDa)OMe reagent 3 as a white powder (72 mg, 53%). .sup.1H NMR (600 MHz, MeOH-∂.sub.4) 3.35 (6H), 3.36 (3H), 3.40 (4H), 3.46-3.59 (10H), 3.56-3.70 (1065H, m, PEG), 3.71-3.77 (7H), 3.85-3.91 (4H), 4.76 (1H), 8.00 (2H), 8.18 (2H).

EXAMPLE 3

Synthesis of a Fluorescent Conjugation Reagent 4 Comprising 7 Repeat Unit Ethylene Glycol Leaving Groups and a Rhodamine B dye

[0159] ##STR00040##

[0160] Reagent 2 (38 mg) and Lissamine rhodamine B ethylenediamine (20 mg, Invitrogen) were dissolved in 1.5 mL of anhydrous DMF and cooled to 0° C. HATU (15 mg) was added and the solution was stirred for 2 min before addition of NMM (8.7 ). The reaction mixture was stirred for 2 h at 0° C. The crude reaction was partially concentrated under high vacuum for 3 h, then purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-mPEG(7u)sulfone-propanoyl-benzamide-rhodamine B reagent 4 as a purple solid (43 mg, 69%). m/z [M+H].sup.+ (1549, 80%), [M+2H].sup.2+ (783, 100%). .sup.1H NMR (600 MHz; MeOH-∂.sub.4) 1.45 (9H, s), 2.40-2.45 (8H, m), 3.40-3.46 (2H, m), 3.52-3.66 (m, PEG and CH.sub.2-Ts), 4.27 (1H, q), 8.01 (2H, d), 8.08 (1H, dd), 8.15 (2H, d), 8.66 (1H, d).

EXAMPLE 4

Synthesis of a Conjugation Reagent 5 Comprising 7 Repeat Unit Ethylene Glycol Leaving Groups and an Auristatin Cytotoxic Payload

[0161] ##STR00041##

[0162] To the TFA salt of val-cit-PAB-MMAE salt having the structure below:

##STR00042##

[0163] (25.0 mg) was added a solution of reagent 2 (15.6 mg) in DMF (1.5 mL) and stirred under an inert nitrogen atmosphere at room temperature for 5 min. The mixture was cooled to 0° C. and aliquots of HATU (6.1 mg) and NMM (1.8 μL) were added every 20 min for a total of 5 additions. After 1.5 h, the reaction mixture was warmed to room temperature. After 2 h, volatiles were removed in vacuo. The resulting residue was dissolved in water and acetonitrile (v/v; 1/1, 0.6 mL), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-mPEG(7u)sulfone-propanoyl-benzamide-val-cit-PAB-MMAE reagent 5 as a white powder (22.4 mg, 68%) m/z [M+2H.sup.2+] 1035.

EXAMPLE 5

Synthesis of a Conjugation Reagent 6 Comprising 7 Repeat Unit Ethylene Glycol Leaving Groups and an Auristatin Cytotoxic Payload

[0164] ##STR00043##

[0165] Step 1: Synthesis of Compound 7

##STR00044##

[0166] Stock solutions of HATU (394 mg) in DMF (4 ml) and NMM (114 μL) in DMF (4 mL) were prepared. To a DMF (10 mL) solution of reagent 2 (250 mg) was added H.sub.2N-dPEG-(24u)-(CO.sub.2.sup.tBu) (405 mg). The mixture was diluted with DMF (5 mL) and was stirred under an inert nitrogen atmosphere at room temperature for 5 min. The mixture was cooled to 0° C. and aliquots of HATU (1 mL) and NMM (1 mL) were added every 10 min for a total of 4 additions. After 40 min the reaction mixture was warmed to room temperature. After 2.5 h volatiles were removed in vacuo. The resulting residue was dissolved in water (2 mL), and the product was isolated by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v), the organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-PEG(24u)-.sup.tbutyl ester compound 7 as a thick clear pale yellow oil (422 mg, 76%) m/z [M−(tBu)+3H.sup.3+] 698.46 Da.

[0167] Step 2: Synthesis of Compound 8.

##STR00045##

[0168] Reagent 7 (382 mg) was dissolved in formic acid (5 mL). The reaction mixture was stirred under an inert nitrogen atmosphere at room temperature for 1 h. The formic acid was removed by lyophilisation. The resulting solid was dissolved in water (1 mL), and the product was isolated by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v), the organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-PEG(24u) acid compound 8 as a white solid (194 mg, 53%) m/z [M+3H.sup.3+] 698.33 Da.

[0169] Step 3: Synthesis of Reagent 6.

[0170] Reagent 8 (15 mg) was dissolved in DMF (0.2 mL) and val-cit-PAB-MMAE.TFA salt (11 mg) in DMF (0.2 mL) was added. The mixture was cooled to 0° C. and stirred under an inert atmosphere. HATU (2.7 mg) and NMM (0.7 mg) were added in succession and the reaction mixture was allowed to stir at 0° C. Additional amounts of HATU (2.7 mg) and NMM (0.4 mg) were added every 20 min for a total of 5 additions, and the reaction mixture was stirred at 0° C. After 1 h 40 min, the reaction was cooled to −20° C. for 16 h. The reaction solution was quenched with water (0.4 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-mPEG(7u)sulfone-propanoyl-benzamide-PEG(24u)-val-cit-PAB-MMAE reagent 6 as a thick clear colourless oil (7.9 mg, 34%) m/z [M+2H].sup.2+ 1599.98.

EXAMPLE 6

Synthesis of a Conjugation Reagent 9 Comprising 7 Repeat Unit Ethylene Glycol Leaving Groups and a Maytansinoid Cytotoxic Payload

[0171] ##STR00046##

[0172] To a stirred solution of the val-ala-PAB-AHX-DM1 having the structure below:

##STR00047##

[0173] (5.2 mg) in anhydrous DMF (400 μL) was added reagent 2 (6 mg) and stirred for 5 min at 0° C. HATU (2.62 mg) and NMM (0.44 mg) were added in succession and the reaction mixture was allowed to stir at 0° C. After 20 min, an additional amount of HATU (2.62 mg) and NMM (0.44 mg) was added and the reaction mixture was stirred at 0° C. After 2.5 h, the reaction was cooled to −20° C. for 16 h. The reaction solution was concentrated in vacuo and then purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-val-ala-PAB-AHX-DM1 reagent 9 as a clear colourless oil (6.5 mg, 74%) m/z [M+H.sup.+] 2030.

EXAMPLE 7

Synthesis of a Conjugation Reagent 10 Comprising 7 repeat Unit Ethylene Glycol Leaving Groups and a Maytansinoid Cytotoxic Payload

[0174] ##STR00048##

[0175] To a stirred solution of val-ala-PAB-AHX-DM1 (5.0 mg) in anhydrous DMF (400 μL) was added reagent 8 (13 mg) and stirred for 5 min at 0° C. HATU (2.62 mg) and NMM (0.44 mg) were added in succession and the reaction mixture was allowed to stir at 0° C. After 20 min, an additional amount of HATU (2.62 mg) and NMM (0.44 mg) was added and the reaction mixture was stirred at 0° C. After 2.5 h, the reaction was cooled to −20° C. for 16 h. The reaction solution was concentrated in vacuo and then purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrilc:0.05% trifluoroacctic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-PEG(24u)-val-ala-PAB-AHX-DM1 reagent 10 as thick yellow oil (5.6 mg, 41%) m/z [M−OH+H].sup.2+ 1571.5.

EXAMPLE 8

Synthesis of Conjugation Reagents 12, 13, 14 and 15 Comprising Either 2 Repeat Unit, 12 Repeat Unit, 1 kDa, 2 kDa or 5 kDa Polymeric Ethylene Glycol leaving Groups Respectively and a Maytansinoid Cytotoxic Payload

[0176] ##STR00049## ##STR00050##

[0177] Maytansanoid reagents bis-mPEG(12u)sulfone-propanoyl-benzamide-PEG(24u)-val-ala-PAB-AHX-DM1 12, bis-mPEG(1 kDa)sulfone-propanoyl-benzamide-PEG(24u)-val-ala-PAB-AHX-DM1 13, bis-mPEG(2 kDa)sulfone-propanoyl-benzamide-PEG(24u)-val-ala-PAB-AHX-DM1 14 and bis-mPEG(5 kDa)sulfone-propanoyl-benzamide-PEG(24u)-val-ala-PAB-AHX-DM1 15 were synthesised in an analogous way as that described for reagent 10 in Example 7, using the thiols 2-(2-methoxyethoxy)ethanethiol, alpha-methoxy-omega-mercapto dodeca(ethylene glycol), alpha-methoxy-omega-mercapto poly(ethylene glycol 1 kDa), alpha-methoxy-omega-mercapto poly(ethylene glycol 2 kDa) and alpha-methoxy-omega-mercapto poly(ethylene glycol 5 kDa) respectively instead of alpha-methoxy-omega-mercapto hepta(ethylene glycol) for the synthesis of compound 1 in Example 1.

EXAMPLE 9

Synthesis of Conjugation Reagents 17, 18, 19 and 20 Comprising Either 2 Repeat Unit, 12 Repeat Unit, 1 kDa, 2 kDa or 5 kDa Polymeric Ethylene Glycol Leaving Groups Respectively and an Auristatin Cytotoxic Payload

[0178] ##STR00051## ##STR00052##

[0179] Auristatin reagents bis-mPEG(12u)sulfone-propanoyl-benzamide-PEG(24u)-val-cit-PAB-MMAE 17, bis-mPEG(1 kDa)sulfone-propanoyl-benzamide-PEG(24u)-val-cit-PAB-MMAE 18, bis-mPEG(2 kDa)sulfone-propanoyl-benzamide-PEG(24u)-val-cit-PAB-MMAE 19 and bis-mPEG(5 kDa)sulfonc-propanoyl-benzamide-PEG(24u)-val-cit-PAB-MMAE 20 were synthesised in an analogous way as that described for reagent 6 in Example 5, using the thiols 2-(2-methoxyethoxy)ethanethiol, alpha-methoxy-omega-mercapto dodeca(ethylene glycol), alpha-methoxy-omega-mercapto poly(ethylene glycol 1 kDa), alpha-methoxy-omega-mercapto poly(ethylene glycol 2 kDa) and alpha-methoxy-omega-mercapto poly(ethylene glycol 5 kDa) respectively instead of alpha-methoxy-omega-mercapto hepta(ethylene glycol) for the synthesis of compound 1 in Example 1.

EXAMPLE 10

Synthesis of Conjugation Reagent 21 Comprising a Maytansinoid Cytotoxic Payload

[0180] ##STR00053##

[0181] Step 1: Synthesis of Compound 22.

##STR00054##

[0182] A solution of Fmoc-L-Glu-(OtBu)-OH (36 mg) in DMF (2 mL) was cooled to 0° C. under an argon atmosphere and (benzotriazol-1-yloxy)tris-(dimethylamino) phosphonium hexafluorophosphate (BOP) (41 mg) was added, followed by NH.sub.2-PEG(24u)-OMe (100 mg) and N,N-diisopropylethylamine (19 μL). The solution was allowed to warm to room temperature and after 22 h the volatiles were removed in vacuo. The resulting residue was dissolved in dichloromethane (1 mL) and purified by normal phase column chromatography eluting with dichloromethane:methanol (100:0 v/v to 80:20 v/v). The organic solvent was removed in vacuo to give Fmoc-L-Glu-[O.sup.tBu]-[PEG(24u)-OMe] as a colourless oil (84 mg, 67%). Piperidine (49 μL) was added to a solution of compound Fmoc-L-Glu-[O.sup.tBu]-[PEG(24u)-OMe](74 mg) in DMF (2 mL) under an argon atmosphere and the resulting solution stirred at room temperature for 22 h, after which the volatiles were removed in vacuo. The resulting residue was triturated with hexane (3×0.7 mL). The organic solvent was decanted each time and the resulting residue dried in vacuo to give the L-Glu-[OtBu]-[PEG(24u)-OMc] compound 22 as a white solid (61 mg, 97%). m/z [M+H].sup.+ (1097, 10%), [M+2H].sup.2+ (1035, 100%).

[0183] Step 2: Synthesis of Compound 23.

##STR00055##

[0184] A solution of compound 22 (26.6 mg) in DMF (550 μL) was cooled to 0° C. under an argon atmosphere to which HATU (10.5 mg) was added and the solution stirred for 0.5 h at 0° C. To this was added a solution of reagent 2 (32 mg) in DMF (550 μL). The resulting solution was stirred for 5 min at 0° C. before addition of NMM (2.9 μL) and HATU (10.5 mg). The reaction solution was allowed to stir at 0° C. for 2 h before being warmed to room temperature and stirred for a further 3.5 h. After this time the volatiles were removed in vacuo. The resulting residue was dissolved in water and acetonitrile (v/v; 1/1, 1.2 ml), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give bis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] as a colourless oil (30.5 mg, 55%). .sup.1H NMR (400 MHz, MeOH-∂.sub.4) 8.19 (2H, d), 8.04 (2H, d), 4.83-4.71 (1H, m), 4.58 (1H, dd,), 3.92-3.83 (6H, m), 3.78-3.56 (140H, m), 3.57-3.51 (6H, m), 3.40 (4H, dd), 3.36 (3H, s), 3.35 (6H, s), 2.41 (2H, t), 2.24-2.13 (1H, m), 2.10-1.98 (1H, m), 1.45 (9H, s); m/z [M+Na].sup.+ (2243, 50%), [M+H].sup.+ (2221, 40%), [M+Na+2H].sup.3+ (747, 100%). A solution of bis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] (30 mg) in dichloromethane (2 mL) under an argon atmosphere was cooled to 0° C. to which trifluoroacetic acid (500 μL) was added and the resulting solution stirred for 1.5 h. The reaction mixture was allowed to warm to room temperature and stirred for a further 1 h. After this time the volatiles were removed in vacuo. The resulting residue was dissolved in water and acetonitrile (v/v; 1/1, 0.6 mL), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-[OH]-[PEG(24u)-OMe] compound 23 as a colourless oil (20 mg, 68%). .sup.1H NMR (400 MHz, MeOH-∂.sub.4) 8.19 (2H, d), 8.04 (2H, d), 4.81-4.72 (1H, m), 4.59 (1H, dd), 3.92-3.84 (6H, m), 3.67-3.50 (146H, m), 3.40 (4H, dd), 3.36 (3H, s), 3.35 (6H, s), 2.48 (2H, t), 2.26-2.15 (1H, m), 2.15-2.03 (1H, m); m/z [M+H].sup.+ (2165, 55%), [M+2H].sup.2+ (1083, 60%), [M+2H+Na].sup.3+ (729, 100%).

[0185] Step 3: Synthesis of Reagent 21

[0186] A solution of compound 23 (15.0 mg) in DMF (600 μL) was cooled to 0° C. under an argon atmosphere. HATU (2.9 mg) was added and the solution stirred for 0.5 h at 0° C. To this was added a solution of val-ala-PAB-AHX-DM1 (9.2 mg) and NMM (0.84) in DMF (600 μL), which had been stirred at room temperature for 0.5 h. After 5 min, an additional amount of HATU (2.9 mg) and NMM (0.8 μL) was added and the reaction mixture stirred at 0° C. After 3 h, an additional amount of HATU (0.7 mg) was added and the reaction mixture stirred at 0° C. After a further 2 h, the reaction was stored at −20° C. for 16 h. The reaction solution was concentrated in vacuo and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-[val-ala-PAB-AHX-DM1]-[PEG(24u)-OMe] compound 21 as a thick clear colourless oil (14.3 mg, 64%). .sup.1H NMR (600 MHz, MeOH-∂.sub.4) (selected characteristic signals) 5.69 (1H, dd,), 6.59 (1H, dd), 6.68 (1H, s), 6.69 (1H, d), 7.10 (1H, s), 7.28 (2H, d), 7.57 (2H, d), 8.01 (2H, d), 8.16 (2H, d); m/z [M-AHX-DM1].sup.+ (2422, 40%).

EXAMPLE 11A

Synthesis of a Conjugation Reagent 24 Comprising 7 Repeat Unit Polymeric Leaving Groups and a Maytansinoid Cytotoxic Payload

[0187] ##STR00056##

[0188] A solution of compound 23 (12.4 mg) in DMF (500 μL) was cooled to 0° C. under an argon atmosphere. HATU (2.4 mg) was added and the solution stirred for 0.5 h at 0° C. To this was added a solution of val-cit-AHX-DM1 (made in an analogous way to compound 50 using AHX-DM1 instead of compound 47, 6.4 mg) and NMM (0.7 μL) in DMF (500 μL), which had been stirred at room temperature for 0.5 h. After 5 min, an additional amount of HATU (1.2 mg) and NMM (0.4 μL) was added and the reaction mixture stirred at room temperature. After 2 h, an additional amount of HATU (1.2 mg) and NMM (0.4 μL) was added and the reaction mixture stirred at room temperature. After a further 1 h, the reaction solution was concentrated in vacuo and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacctic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give bis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-[val-cit-AHX-DM1]-[PEG(24u)-OMe] 24 as a thick clear colourless oil (9.6 mg, 53%). m/z [M−H.sub.2O].sup.+(3148, 8%), [M−H.sub.2O].sup.2+ (1575, 40%), [M−H.sub.2O].sup.3+ (1050, 100%), 1036 [M−NHCO—H.sub.2O].sup.3+.

EXAMPLE 11B

Synthesis of a Conjugation Reagent 25 Comprising an Auristatin Cytotoxic Payload

[0189] ##STR00057##

[0190] Reagent 25 was synthesised in analogous way to reagent 24 of Example 11A from compound 23 and val-cit-PAB-MMAE TFA salt. Bis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-[val-cit-PAB-MMAE]-[PEG(24u)-OMe] 25 was isolated as a colourless oil. m/z [M+H].sup.+ (3270, 12%), [M+2H].sup.2+ (1636, 50%), [M+3H].sup.3+ (1091, 100%).

EXAMPLE 12

Synthesis of Conjugation Reagent 27 for Imaging and Chelating Applications Comprising 7 Repeat Unit Polymeric Ethylene Glycol Leaving Groups and Desferrioxamine

[0191] ##STR00058##

[0192] Step 1: Synthesis of Compound 28.

##STR00059##

[0193] To a solution of 21-(Boc-amino)-4,7,10,13,16,19-hexaoxaheneicosanoic acid (50 mg, Sigma-Aldrich) in DMF (2 mL) under an argon atmosphere at room temperature was added (benzotriazol-1-yloxy)tris-(dimethylamino) phosphonium hexafluorophosphate (BOP) (73 mg) and the solution was stirred for 0.5 h. To this was added a solution of desferrioxamine mesylate (87 mg, Sigma-Aldrich) and N,N-diisopropylethylamine (29 μL) in DMF (2 mL) which had been stirred at room temperature for 0.5 h. After 24 h the reaction solution was concentrated in vacuo and then purified directly by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 50:50 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the BocNH-PEG(6u)-desferrioxamine compound 28 as a white solid (50 mg, 46%) m/z [M+Na].sup.+ (1019, 50%), [M+H].sup.+ (997, 95%), [M+2H].sup.2+ (499, 100%); .sup.1H NMR (600 MHz; MeOH-δ.sub.4) 1.28-1.38 (7H, m), 1.43 (9H, s), 1.49-1.55 (6H, m), 1.60-1.67 (5H, m), 2.09 (3H, s), 2.41-2.47 (6H, m), 2.76 (4H, t), 3.14-3.19 (6H, m), 3.21 (2H, t), 3.50 (2H, t), 3.57-3.65 (27H, m), 3.70-3.73 (2H, t).

[0194] Step 2: Synthesis of Compound 29.

##STR00060##

[0195] A solution of compound 28 (49 mg) in dichloromethane (DCM) (3 mL) under an argon atmosphere was cooled to 0° C. and trifluoroacetic acid (TFA) (750 μL) was added. The solution was allowed to warm to room temperature. After 1 h, toluene (2 mL) was added to the reaction mixture and the volatiles were removed in vacuo. The resulting residue was dissolved in Buffer A (v/v): water:5% acetonitrile:0.1% formic acid (2 mL) and the aqueous solvent was removed by lyophilisation to remove any residual TFA and to give the NH.sub.2-PEG(6u)-desferrioxamine compound 29 as a white solid (44 mg, quantitative yield) which was used in the next step without any further purification: m/z [M+H].sup.+ (897, 100%), [M+2H].sup.2+ (449, 100%).

[0196] Step 3: Synthesis of Reagent 27.

[0197] A solution of reagent 2 (17 mg) in DMF (750 μL) was cooled to 0° C. under an argon atmosphere before HATU (8.0 mg) was added and the solution was stirred for 0.5 h at 0° C. To this was added a solution of compound 29 (23 mg) and NMM (2.1 μL) in DMF (750 μL) and the reaction mixture was stirred at 0° C. Additional amounts of reagent 2, HATU and NMM were added over 4 h. After a further 15 h, the reaction solution was concentrated in vacuo and then purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 50:50 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-PEG(6u)-desferrioxamine reagent 27 as a clear colourless oil (20.6 mg, 49%) m/z [M+H].sup.+ (1844, 95%), [M+2H].sup.2+ (922, 100%), [M+3H].sup.3+ (615, 100%).

EXAMPLE 13 (COMPARATIVE)

Synthesis of Conjugation Reagent 30 for Imaging and Chelating Applications Comprising Tosyl Leaving Groups and Desferrioxamine

[0198] ##STR00061##

[0199] To a solution of compound 29 (43 mg) in DMF (2.5 mL) was added NMM (6 μL) and the solution was stirred for 0.5 h at room temperature. To this was added a solution of the known 4-[2,2-bis[(p-tolylsulfonye-methyl]acetyl]benzoic acid (29 mg, Nature Protocols, 2006, 1(54), 2241-2252) in DMF (700 μL). Additional NMM was added to the reaction mixture after 1 h, 2 h, 19 h, 23 h and 25 h (15.6 μL in total). After 26 h the reaction solution was concentrated in vacuo and then purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the bis-tolylsulfone-propanoyl-benzamide PEG(6u)-desferrioxamine reagent 30 as a clear colourless oil (19 mg, 29%) m/z [M+H].sup.+ (1379, 98%), [M+2H].sup.2+ (690, 100%); .sup.1H NMR (600 MHz; MeOH-δ.sub.4) 1.28-1.36 (7H, m), 1.48-1.55 (6H, m), 1.59-1.65 (5H, m), 2.09 (3H, s), 2.40-2.46 (6H, m), 2.50 (6H, s), 2.74-2.78 (4H, m), 3.14-3.17 (5H, m), 3.55-3.61 (23H, m), 3.62-3.70 (12H, m), 3.75-3.80 (2H, m). 4.09-4.14 (1H, m), 7.44 (4H, d), 7.55 (2H, d), 7.62 (4H, d), 7.82 (2H, d).

EXAMPLE 14

Synthesis of a Conjugation Reagent 31 Comprising a hexaPEG-Disulfone Leaving Group and a Maytansinoid Cytotoxic Payload

[0200] ##STR00062##

[0201] Step 1: Synthesis of Compound 32.

##STR00063##

[0202] To a stirred solution of 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (542 mg, Nature Protocols, 2006, 1(54), 2241-2252) in DMF (40 mL) was added hexa(ethylene glycol) dithiol (400 μL) and Cs.sub.2CO.sub.3 (2.4 g). The resulting reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. After 40 h, volatiles were removed in vacuo. The resulting residue was dissolved in water (4 mL), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the hexaPEG-dithioether propanoyl-benzoic acid compound 32 as a white solid (180 mg, 33.1%) m/z [M+Na.sup.+] 525. .sup.1HNMR (600 MHz, CDCl.sub.3) 2.72-2.78 (4H, m, CH.sub.2-S), 2.95-3.04 (4H, m), 3.65-3.72(18H, m, PEG), 4.76 (1H, m), 8.11 (2H, d), 8.21 (2H, d).

[0203] Step 2: Synthesis of Compound 33.

##STR00064##

[0204] The hexaPEG-disulfone propanoyl-benzoic acid compound 33 was synthesised in an analogous way as that described for reagent 2 in Example 1, using compound 32 instead of compound 1.

[0205] Step 3: Synthesis of Compound 34.

##STR00065##

[0206] The hexaPEG-disulfone propanoyl-benzamide-PEG(24u)-.sup.tbutyl ester compound 34 was synthesised in an analogous way as that described for compound 7 in Example 5, using compound 33 instead of compound 2.

[0207] Step 4: Synthesis of Compound 35.

##STR00066##

[0208] The hexaPEG-disulfone propanoyl-benzamide-PEG(24u)-acid compound 35 was synthesised in an analogous way as that described for compound 8 in Example 5, using compound 34 instead of compound 7.

[0209] Step 5: Synthesis of Reagent 31.

[0210] The maytansanoid reagent hexaPEG-disulfone propanoyl-benzamide-PEG(24u)-val-ala-PAB-AHX-DM1 31 was synthesised in an analogous way as that described for reagent 10 in Example 7, using compound 35 instead of compound 8.

EXAMPLE 15 (COMPARATIVE)

Synthesis of a Conjugation Reagent 36 Comprising Tosyl Leaving Groups and a Rhodamine B Dye

[0211] ##STR00067##

[0212] Step 1: Synthesis of Compound 37.

##STR00068##

[0213] The 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzamide-PEG(12u)-acid compound 37 was synthesised in an analogous way as that described for reagent 8 in Example 5, using the known 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (Nature Protocols, 2006, 1(54), 2241-2252) instead of reagent 2.

[0214] Step 2: Synthesis of Reagent 36.

[0215] The 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzamide-PEG(12u)-rhodamine B reagent 36 was synthesised in an analogous way as that described for reagent 4 in Example 3, using compound 37 instead of reagent 2.

EXAMPLE 16

Synthesis of a Conjugation Reagent 38 Comprising a 7 Repeat Unit Polymeric Leaving Group, an Aryl Leaving Group and an Auristatin Cytotoxic Payload

[0216] ##STR00069##

[0217] Step 1: Synthesis of Compound 39.

##STR00070##

[0218] To a stirred solution of the known 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (200 mg, Nature Protocols, 2006, 1(54), 2241-2252) in dimethylformamide (DMF, 8 mL) was added alpha-methoxy-omega-mercapto hepta(ethylene glycol) (142 mg) and NMM (264 μL). The resulting reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. After 19 h, volatiles were removed in vacuo and the resulting residue was dissolved in acetonitrile (800 μL), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give mono-mPEG(7u)sulfone-propanoyl-benzamide-mono-(tosylmethyl) propanoyl)benzoic acid compound 39 as a thick clear colourless oil (132 mg, 47%) m/z [M+H.sup.+] 701.1.

[0219] Step 2: Synthesis of Reagent 40.

##STR00071##

[0220] To a stirred solution of compound 39 (116 mg) in methanol:water (5 mL, 9:1 v/v) at room temperature was added Oxone© (305 mg). After 75 min, the volatiles were removed in vacuo and water was azeotropically removed with acetonitrile (3×15 mL). The resulting residue was dissolved in dichloromethane (3×10 mL), filtered through a column of magnesium sulphate and washed with dichloromethane (2×7 mL). The eluent and washings were combined and the volatiles were removed in vacuo to give a thick clear pale yellow oil. The residue was dissolved in water:acetonitrile (800 μL, 1:4 v/v), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the 4-(3-((2-hepta(ethylene glycol) methoxyethyl) sulfonyl)-2-(tosylmethyl) propanoyl) benzoic acid compound 40 as a thick clear colourless oil (103 mg, 78%) m/z [M+H.sup.+] 733.2.

[0221] Step 3: Synthesis of Reagent 38

[0222] To val-cit-PAB-MMAE.TFA salt (25.0 mg) was added a solution of reagent 40 (11.8 mg) in DMF (1.5 mL) and stirred under an inert atmosphere at room temperature for 5 min. The mixture was cooled to 0° C. and aliquots of HATU (6.1 mg) and NMM (1.8 μL) were added over a period of 5 h for a total of 5 additions. After 1.5 h, the reaction mixture was warmed to room temperature. After 6 h, volatiles were removed in vacuo, and the resulting residue was dissolved in water and acetonitrile (v/v; 1/4, 0.6 mL), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give mono-mPEG(7u)sulfone-mono-(tosylmethyl propanoyl-benzamide-)-val-cit-PAB-MMAE reagent 38 as a bright white powder (13.0 mg, 44%) m/z [M+2H.sup.2+] 919.3.

EXAMPLE 17

PEGylation at a Reduced Fab Disulfide Bond using Reagent 3 and Comparison with the Known PEGylation Reagent 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzamide-PEG(10 kDa)-OMe 41 (Nature Protocols, 2006, 1(54), 2241-2252)

[0223] ##STR00072##

[0224] The interchain disulfide of a Fab (5.71 mg, 2.33 mg/mL in PBS produced by papain digestion of trastuzumab), was reduced by adding 50 μL of 0.5 M dithiothreitol (DTT) aqueous solution and incubating at 22° C. for 1 h. The reducing agent was then removed using a PD10 column equilibrated with 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5, and the reduced Fab diluted further to 1.24 mg/mL with the same buffer. Reagents 3 and 41 were dissolved independently in ultrapure water at 10 mg/mL and added to separate solutions of reduced Fab at 1.5 molar equivalents of PEG reagent to Fab. The PEGylation reactions were allowed to progress at 22° C., whereupon samples were taken for SDS-PAGE analysis after 2 and 4 h.

[0225] The % conversion to PEGylated Fab was estimated based on SDS-PAGE band density using an ImageQuant™ system (GE Healthcare). The results are shown in FIG. 1, which compares the extent of conjugation of PEGylation Reagent 3 (black bars) vs Reagent 41 (white bars) to the Fab. Reaction with reagent 3 gave 63% and 83% conversion to PEGylated Fab at 2 h and 4 h respectively. For reagent 41, conversions were 35% and 51% at 2 and 4 h respectively. These data show that reagent 3 reacts more quickly, producing a larger amount of PEGylated Fab in a shorter timeframe than reagent 41.

EXAMPLE 18

PEGylation at an Interferon-Alpha Disulfide Bond using Reagent 3 and Comparison with the Known PEGylation Reagent 41

[0226] Interferon α-2a (IFN) (4 mL, 0.93 mg/mL, in 20 mM Tris pH 8.0, ˜100 mM NaCl, protease inhibitors, 1 mM EDTA, 10% Glycerol) was buffer exchanged into phosphate buffered saline (PBS), pH 7.4 using Zeba™ spin 5 mL columns. IFN was reduced by treatment with DTT (25 mM) for 30 min at 22° C. Reductant was removed using a PD10 desalting column equilibrated with PBS at pH 7.4. Reagents 3 and 41 (1.5 eq. per IFN sulfide) were conjugated to IFN (0.7 mg/mL) at 4° C. Samples of the reaction mixtures were analysed by SDS-PAGE after 1 h, 2 h and 4 h incubation. The % conversion to PEGylated IFN was estimated based on SDS-PAGE band density using an ImageQuant™ system (GE Healthcare). The results are shown in FIG. 2, which compares the conjugation efficiency of fluorescent reagent 4 possessing polymeric ethylene glycol leaving groups and an analogous reagent 41 lacking polymeric leaving groups. Reagent 3 reacted faster and produced a greater amount of IFN-PEG(10 kDa)-OMe (band corresponding to approximately 35 kDa), compared to the equivalent timepoint for reagent 41.

EXAMPLE 19

Comparison of the Conjugation Efficiency of Fluorescent Reagent 4 Possessing Polymeric Ethylene Glycol Leaving Groups and an Analogous Reagent 36 Lacking Polymeric Leaving Groups

[0227] Using a similar conjugation method to that described in Example 17, reagents 4 and 36 were dissolved separately in 100% MeCN at 1.53 mg/mL and 1.67 mg/mL, respectively, prior to conjugation. To the Fab solution (1.24 mg/mL, 0.475 mL in PBS), reagent 4 or reagent 36 (18 μL) was added corresponding to 1.5 equivalents. The reaction was allowed to progress 22 h at 22° C. and samples were taken for SDS-PAGE analysis during the reaction. The % conversion to rhodamine-conjugated Fab was estimated based on SDS-PAGE band density using an ImageQuant™ system (GE Healthcare). The results are shown in FIG. 3, which compares the conversion to rhodamine conjugated Fab using reagent 4 (black bars) and reagent 36 (white bars), at 1.5 equivalents of reagent. Reagent 4 gave a higher conversion to conjugated Fab at each timepoint.

EXAMPLE 20

Comparison of the Antibody Conjugation of Cytotoxic Reagent 5 Possessing Polymeric Ethylene Glycol Leaving Groups with an Analogous Reagent 42 (4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzamide-val-cit-PAB-MMAE) Lacking Polymeric Leaving Groups

[0228] Reagent 42 was synthesised as described within WO2014064423.

[0229] Both reagents 5 and 42 were conjugated to the antibody trastuzumab using an analogous method to the Examples described in WO2014064423. Briefly, trastuzumab (5.2 mg/mL) was reduced with tris(2-carboxyethyl)phosphine (TCEP) at 40° C. for 1 h. Conjugation of the antibody with 1.5 molar equivalents of either reagent 5 or 42 per inter-chain disulfide bond was then performed by dissolving both reagent 5 and reagent 42 in DMF to a final concentration of 1.6 mM. The antibody solution was diluted to 4.21 mg/mL with 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5. Reagent 5 and 42 were added to separate aliquots of antibody. The final antibody concentration in the reaction was adjusted to 4 mg/mL with 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5. Each solution was mixed gently and incubated at 22° C. for 22 h. At 16 and 22 h an aliquot was taken, treated with N-acetyl-L-cysteine (20 eq. over reagent) for 1 h at 22° C. and then analysed by HIC. The % conversion of product with a drug to antibody ratio (DAR) of four was determined based on the % area of the absorbance peaks measured at 280 nm from the HIC chromatograms and results are shown in FIG. 4, which shows the comparison of conjugation of reagents 5 (black bars) and 42 (white bars) to an antibody. The results in FIG. 4 show that 6% of DAR 4 conjugate was produced with reagent 42 after 22 h, whereas reagent 5 gave 78% of DAR 4 conjugate after 22 h.

EXAMPLE 21

Comparison of the Antibody Conjugation of Cytotoxic Reagents 5, 6 and 10 all Possessing Polymeric Ethylene Glycol Leaving Groups with Analogous Reagents 42, 43 and 44 Lacking Polymeric Leaving Groups

[0230] Reagent 43 was synthesised as described within WO2014064423 and reagent 44 was synthesised as described within WO2014064424.

[0231] (a) Comparison of the antibody conjugation of cytotoxic reagent 43 (4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzamide-PEG(24u)-val-cit-PAB-MMAE) and reagent 5.

[0232] The reaction conditions used for conjugation of cytotoxic reagent 43 with antibody (5 mg scale) were the same as those described above for Example 20 with the following differences: The reagents 43 and 5 were prepared as 3.2 mM MeCN solutions. Aliquots of reaction solution were taken after 0.5, 1, 2, 4 and 22 h, quenched by treatment with N-acetyl-L-cysteine (1 h at 22° C.), and analysed by HIC. The results of these conjugation reactions are shown in FIG. 5, which is a time course analysis comparing the rate of conjugation (Average DAR) for reagent 5 and reagent 43 over 22 h. In FIG. 5, it can be seen that the rate of product formation was much faster for reagent 5 than for reagent 43. The DAR 4 species were purified from each reaction mixture using HIC and analysed by SDS-PAGE. From SDS-PAGE analysis, the % of half antibody species present for each sample were determined by ImageQuant™ analysis of the stained gel and the result is shown in Table 1, where the conjugate prepared from reagent 43 had almost twice the amount of half antibody species as the conjugate produced from reagent 5. This indicates the conjugate produced from reagent 5 had significantly more heavy to heavy interchain covalent bonding and better resembles the structure of the unconjugated antibody.

TABLE-US-00001 TABLE 1 % of half antibody species present by SDS- Sample PAGE analysis (H + L chain species) Trastuzumab-5 conjugate 27% Trastuzumab-43 conjugate 47%

[0233] (b) Comparison of the antibody conjugation of cytotoxic reagent 44 (4-[2,2-bis[(p-tolylsulfonyl)-methyl] acetyl] benzamide-PEG(24u)-val-ala-PAB-AHX-DM1) and reagent 10.

[0234] The reaction conditions used for conjugation of cytotoxic reagent 44 with antibody (3.8 mg scale) were the same as those described above for Example 20. An aliquot of reaction solution was taken after 16 and 22 h and quenched by treatment with N-acetyl-L-cysteine. Each aliquot was analysed by HIC and the % conversion to DAR 4 product determined. The result is shown in FIG. 6, which is a comparison of conjugation of reagents 10 (black bars) and 44 (white bars) to an antibody. It can be seen that reagent 10 gave more DAR 4 product after both 16 h and 22 h.

[0235] (c) Comparison of the antibody conjugation of cytotoxic reagent 43 (4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzamide-PEG(24u)-val-cit-PAB-MMAE) and reagent 6. The reaction conditions used for conjugation of cytotoxic reagent 6 with antibody (1 mg scale) were analogous to those described above for Example 20 with the following differences: Reagent 6 was prepared as a 3.2 mM MeCN solution and the MeCN concentration in the reaction was 5%. An aliquot of reaction solution was taken after 4 h, quenched by treatment with N-acetyl-L-cysteine, and analysed by analytical HIC and the % of DAR 4 product present determined. The results are shown in FIG. 7, which is a comparison of conjugation of reagents 6 (black bar) and 43 (white bar) to an antibody. It can be seen that reagent 6 (black bar) gave significantly more DAR 4 product after 4 h than reagent 43 (white bar).

EXAMPLE 22

Comparison of the Antibody Conjugation of Cytotoxic Reagent 12 Possessing Polymeric Ethylene Glycol Leaving Groups with an Analogous Reagent 44 without Polymeric Leaving Groups

[0236] The reaction conditions used for conjugation of cytotoxic reagent 12 with an antibody (1 mg scale) were the same as those described above for Example 20. An aliquot of reaction solution was taken after 4 h, quenched by treatment with N-acetyl-L-cysteine and analysed by HIC. The results arc shown in FIG. 8, which is a comparison of conjugation of reagents 44 (white bar) and 12 (black bar) to an antibody. In FIG. 8, it can be seen that reagent 12 gave a significantly higher yield of the DAR 4 product after 4 h compared with reagent 44.

EXAMPLE 23

Comparison of the Antibody Conjugation of Cytotoxic Reagents 14, 15 and 31 Possessing Polymeric Ethylene Glycol Leaving Groups with an Analogous Reagent 44 without Polymeric Leaving Groups

[0237] The reaction conditions used for conjugation of cytotoxic reagents 14, 15 and 31 with antibody (1 mg scale) were the same as those described above for Example 20. An aliquot of reaction solution was taken after 4 h and quenched by treatment with N-acetyl-L-cysteine. Each aliquot was analysed by HIC and the % of DAR 4 product present determined. Results for conjugation with reagents 14, 15, 31 and 44 are displayed in Table 2, where is can be seen that reagents 14, 15 and 31 gave over twice the amount of DAR 4 product compared to reagent 44 after 4 h.

TABLE-US-00002 TABLE 2 Comparison of conjugation of reagents 14, 15, 31 and 44 to an antibody after 4 h reaction. Reagent used for conjugation % DAR 4 product to an antibody after 4 h 44  9 14 24 15 23 31 28

EXAMPLE 24

Preparation of Antibody Drug Conjugates

[0238] Antibody drug conjugates were prepared from reagents 21, 24 and 25 by methods analogous to those described in WO2014064423 and WO2014064424. Briefly, antibody (trastuzumab or brentuximab) was reduced using tris(2-carboxyethyl)phosphine at 40° C. for 1 h. Conjugation of the antibody with 1.5 molar equivalents of reagent per inter-chain disulfide bond was then performed by dissolving reagents to a final concentration of 1.6 mM in either acetonitrile or DMF. The antibody solution was diluted to 4.21 mg/mL with 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5. Reagents were added to antibody and the final antibody concentration in the reaction was adjusted to 4 mg/mL with 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5. Each solution was mixed gently and incubated at 22° C. Antibody drug conjugate product was purified by hydrophobic interaction chromatography for each conjugate.

EXAMPLE 25

PEGylation of a Histidine-Tagged Interferon-Alpha using Reagent 3 at pH 6.6 and 7.9

[0239] The conjugation of interferon a-2a (IFN) possessing an 8-histidine tag with reagent 3 was carried out at 2.5 mg/mL at either pH 6.6 or pH 7.9, achieved by mixing sodium acetate pH 5.3 or sodium phosphate pH 8.0 with sodium phosphate pH 7.4 respectively. Reagent 3 at 1, 1.5 and 2 eq. per IFN was used. The resulting reaction mixtures were incubated for 22 h at 22° C. with an interim analysis at 4 h. The reaction mixtures were analysed by SDS-PAGE and the results are shown in FIG. 9. In FIG. 9, Lane M indicates Novex Protein Standards; lane 1 indicates IFN at pH 5.4; lane 2 indicates IFN at pH 8.0; lanes 3-5 indicate conjugated product from 1, 1.5 and 2 eq. reagent 3 respectively at 4 hat pH 6.6; lane 6 indicates conjugated product from 1.5 eq. reagent 3 at 4 h, pH 7.9; lanes 7-9 indicate conjugated product from 1, 1.5 and 2 eq. reagent 3 respectively at 22 h at pH 6.6; lane 10 indicates conjugated product from 1.5 eq. reagent 3 respectively at 22 hat pH 7.9.

EXAMPLE 26

Synthesis of a Disulfide Bridging Reagent 45

[0240] ##STR00073##

[0241] Step 1: Synthesis of Compound 46.

##STR00074##

[0242] To a stirred solution of aminohexanoic maytansine (AHX-DM1).TFA salt (29.4 mg) of formula:

##STR00075##

in dimethylformamide (DMF) (400 μL) was added a solution of 4-(N-Boc-amino)-1,6-heptanedioic acid bis-pentafluorophenyl ester (10.2 mg) in DMF (200 μL). The solution was cooled to 0° C. before addition of N,N-diisopropylethylamine (DIPEA) (13.5 μL). The solution was allowed to warm to room temperature and stirred for 18.5 h. The reaction solution was purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give 4-(N-boc-amino)-1,6-heptanediamide bis-AHX-DM1 compound 46 (assumed quantitative yield, 29.7 mg) as a white solid m/z [M+2H−2(H.sub.2O)—NHCO].sup.2+ 844 (100%), [M+H].sup.+ 1767.

[0243] Step 2: Synthesis of Cytotoxic Payload 47.

##STR00076##

[0244] Compound 46 (assumed quantitative yield, 29.7 mg) was dissolved in formic acid (700 μL) and the solution stirred at room temperature for 1.5 h. Volatiles were removed in vacuo and the residue converted to the trifluroacetic acid salt by dissolving in a buffer A:buffer B 50:50 v/v % mixture (1.5 mL, buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid). The solution was stirred at room temperature for 5 min before the solvent was removed by lyophilisation. The process was repeated to give 4-(amino)-1,6-heptanediamide bis-AHX-DM1 compound 47 as an off-white solid (18.0 mg, 60% over 2 steps) m/z [M+2H−2(H.sub.2O)—NHCO)].sup.2+ 794 (100%), [M+H].sup.+ 1667.

[0245] Step 3: Synthesis of Compound 48.

##STR00077##

[0246] Compound 48 was synthesised following the procedure described in patent (EP 0 624 377 A2) to give a white solid with spectroscopic data in agreement with that previously reported.

[0247] Step 4: Synthesis of Compound 49.

##STR00078##

[0248] Stock solutions of compound 48 (20.0 mg) in DMF (500 μL) and HATU (40.0 mg) in DMF (400 μL) were prepared. To a stirred solution of compound 47 (14.0 mg) in DMF (700 μL) was added aliquots of compound 48 stock solution (126.9 μL) and HATU stock solution (77.8 μL). The reaction solution was cooled to 0° C. before the addition of DIPEA (4.11 μL). The solution was stirred at 0° C. for 50 min before further aliquots of compound 48 stock solution (126.9 μL), HATU stock solution (77.8 μL) and DIPEA (4.11 μL) were added. The solution was stirred for 40 min at 0° C. The reaction solution was purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give 4-(Fmoc-val-cit-amido)-1,6-heptanediamide bis-AHX-DM1 compound 49 (assumed quantitative yield, 16.9 mg) as an off-white solid m/z [M+2H-2(H.sub.2O)].sup.2+ 1055 (100%).

[0249] Step 5: Synthesis of Compound 50.

##STR00079##

[0250] To a stirred solution of compound 49 (assumed quantitative yield, 16.9 mg) in DMF (500 μL) was added piperidine (3.04 μL). The reaction solution was stirred at room temperature for 1.5 h before purification by reverse phase C18-column chromatography eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give 4-(val-cit-amido)-1,6-heptanediamide bis-AHX-DM1 compound 50 as an off-white solid (8.8 mg, 55% over 2 steps) m/z [M+2H].sup.2+ 962 (100%).

[0251] Step 6: Synthesis of Disulfide Bridging Reagent 45 Comprising the Cytotoxic Payload 47.

[0252] Stock solutions of compound 8 (13.5 mg) in DMF (100 μL), HATU (10.0 mg) in DMF (200 μL) and NMM (5.83 μL) in DMF (94.2 μL) were prepared. To a stirred solution of 50 (1.8 mg) in DMF (80 μL) was added aliquots of compound 23 stock solution (16.5 μL) and HATU stock solution (20.2 μL). The reaction solution was cooled to 0° C. before adding an aliquot of NMM stock solution (5 μL). The solution was allowed to stir at 0° C. for 1 h before warming to room temperature. After 3.5 h, the reaction solution was purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give bis-mPEG(7u)sulfone-propanoyl-benzamide-val-cit-amido)-1,6-heptanediamide bis-AHX-DM1 reagent 45 as a solid (1.5 mg, 42%) m/z [M+4H−2(H.sub.2O)].sup.4+ 992 (100%), [M+3H−2(H.sub.2O)].sup.3+ 1321, [M+2H−2(H.sub.2O)].sup.2+ 1982.

EXAMPLE 27

Synthesis of a Disulfide Bridging Reagent 51 Comprising the Cytotoxic Payload 47.

[0253] ##STR00080##

[0254] Step 1: Synthesis of Reagent 51.

[0255] Stock solutions of HATU (10 mg) in DMF (200 μL) and NMM (5.83 μL) in DMF (94.2 μL) were prepared. Compound 23 (5.4 mg) was dissolved in a solution of compound 50 (3.6 mg) in DMF (254 μL) with stirring. To the stirred solution was added an aliquot of HATU stock solution (40 μL). The solution was cooled to 0° C. before an aliquot of NMM stock solution (10 μL) was added. After 50 min, further aliquots of HATU stock solution (6.67 μL) and NMM stock solution (1.67 μL) were added. The reaction solution was stirred at 0° C. for a further 20 minutes and purified directly by reverse phase C18-column chromatography eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give reagent 51 as an off-white solid (3.8 mg, 53%) m/z [M+4H−(H.sub.2O)—NHCO].sup.4+ 1003 (100%), [M+3H−2(H.sub.2O)—NHCO].sup.3+ 1331, [M+2H−2(H.sub.2O)].sup.2+ 2017.

EXAMPLE 28

Synthesis of a Disulfide Bridging Reagent 56 Comprising the Cytotoxic Payload 47

[0256] ##STR00081##

[0257] Step 1: Synthesis of Compound 57.

##STR00082##

[0258] A stock solution of hydroxybenzotriazole (HOBt, 6.6 mg) in DMF (200 μL) was prepared. To a stirred solution of cytotoxic payload 47 (10 mg) in DMF (500 μL) was added Fmoc-val-ala-PAB-PNP (3.7 mg) and an aliquot of HOBt stock solution (2 μL). The reaction solution was cooled to 0° C. before DIPEA (2.14 μL) was added. The reaction solution was then stirred at room temperature for 18 h before purification by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give Fmoc-val-ala-PAB-amido-1,6-heptanediamide bis-AHX-DM1 reagent 57.

[0259] Step 2: Synthesis of Compound 58.

##STR00083##

[0260] The bis-maytansinoid compound amine-val-ala-PAB-amido-1,6-heptanediamide bis-AHX-DM1 compound 58 was synthesised in an analogous way to that described for compound 50 using compound 57 instead of compound 49.

[0261] Step 3: Synthesis of Reagent 56.

[0262] The bis-maytansinoid reagent bis-mPEG(7u)sulfone-propanoyl-benzamide-Glu-[NH-PEG(24u)-OMe]-[val-ala-PAB-amido-1,6-heptanediamide bis-AHX-DM1] 56 was synthesised in an analogous way to that described for reagent 51, using compound 58 instead of compound 50.

EXAMPLE 29

Synthesis of a Disulfide Bridging Reagent 59 Comprising the Cytotoxic Payload AHX-DM1

[0263] ##STR00084##

[0264] To a stirred solution of val-cit-AHX-DM1 (5.0 mg) in anhydrous DMF (400 μL) was added reagent 8 (13 mg) and stirred for 5 min at 0° C. HATU (2.62 mg) and NMM (0.44 mg) were added in succession and the reaction mixture was allowed to stir at 0° C. After 20 min, an additional amount of HATU (2.62 mg) and NMM (0.44 mg) was added and the reaction mixture was stirred at 0° C. After 2.5 h, the reaction was cooled to −20° C. for 16 h. The reaction solution was concentrated in vacuo and then purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the bis-mPEG(7u)sulfone-propanoyl-benzamide-PEG(24u)-val-cit-AHX-DM1 reagent 59 as thick yellow oil (5.6 mg, 41%) m/z [M−OH+H].sup.2+ 1571.5.

EXAMPLE 30

Production of Antibody Drug Conjugate 60 using Disulfide Bridging Reagent 45; Antibody Drug Conjugate 61 using Disulfide Bridging Reagent 51 and Antibody Drug Conjugate 62 using Disulfide Bridging Reagent 56

[0265] Antibody drug conjugates were prepared by methods analogous to those described in WO2014064423 and WO2014064424. Briefly, antibody (trastuzumab or brentuximab) was reduced using tris(2-carboxyethyl)phosphine at 40° C. for 1 h. Conjugation of the antibody with 1.5 molar equivalents of reagent (i.e., 45, 51 or 56) per inter-chain disulfide bond was then performed by dissolving reagents to a final concentration of 1.6 mM in either acetonitrile or DMF. The antibody solution was diluted to 4.21 mg/mL with 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5. Reagents were added to antibody and the final antibody concentration in the reaction was adjusted to 4 mg/mL with 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5. Each solution was mixed gently and incubated at 22° C. Antibody drug conjugate product was purified by hydrophobic interaction chromatography for each conjugate to give products with a defined drug to antibody ratio (DAR). The % conversion to DAR4 product for reagents 45, 41 and 56 were 30%, 65% and 40%, respectively, as determined by HIC.

EXAMPLE 31

Synthesis of a PEG Conjugation Reagent 63 Comprising 7 Repeat Unit Ethylene Glycol Leaving Groups and Fluorinated Aryl Linker

[0266] ##STR00085##

[0267] Step 1: Synthesis of Compound 64:

##STR00086##

[0268] Formaldehyde (37% solution) (144 μL), piperidine (14.4 μL), and piperidine hydrochloride (134 mg) was added to a mixture of 4-acetyl-2-fluorobenzoic acid (200 mg) and 4-methylbenzene thiol (272 mg) in absolute ethanol (1.5 mL). The reaction mixture was heated at reflux for 2.5 h, during which time a further portion of formaldehyde (37% solution) (144 μL) was added (after 1.5 h). The reaction mixture was then cooled to room temperature and stirred overnight. Additional quantities of formaldehyde (37% solution) (288 μL) and piperidine (14.4 μL) were added and the reaction mixture was heated under reflux for a further 8 h. The reaction mixture was then cooled to room temperature and stirred overnight. Volatiles were removed in vacuo. The resulting residue was dissolved in water:acetonitrile (800 μL, 1:3 v/v) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-(methyl benzene sulfide propanoyl)-2-fluoro-benzoic acid compound 64 as pale orange crystals (196.0 mg, 39%) m/z [M+H].sup.+ 454.20 Da; .sup.1H NMR (400 MHz, CDCl.sub.3) 2.35 (6H, s, OMe), 3.20-3.30 (4H, m, CH.sub.2), 3.70-3.80 (1H, m, CH), 7.10 (4H, d, Ar—H), 7.15 (4H, d, Ar—H), 7.20 (1H, s, Ar—H), 7.35 (1H, d, Ar—H), 7.90 (1H, t, Ar—H).

[0269] Step 2: Synthesis of Compound 65:

##STR00087##

[0270] To a stirred solution of compound 64 (75 mg) in methanol:water (2.0 mL, 9:1 v/v) at room temperature was added Oxone® (304 mg, Sigma-Aldrich). After 2 h, the volatiles were removed in vacuo. The resulting residue was dissolved in dichloromethane (3×5.0 mL), filtered through a column of magnesium sulfate and washed with dichloromethane (2×5 mL). The eluent and washings were combined and the volatiles were removed in vacuo to give bis-(tosyl propanoyl)-2-fluoro-benzoic acid compound 65 as a white powder (84.0 mg, 98%) m/z [M−H].sup.+ 519.15 Da.

[0271] Step 3: Synthesis of Compound 66:

##STR00088##

[0272] To a solution of compound 65 (75 mg) in DMF (2.0 mL) was added Et.sub.3N (240 μL, Acros Organics) and MeO-PEG-(7u)-SH (154 mg, Iris Biotech). The reaction was stirred under an inert nitrogen atmosphere at room temperature. After 6 h, volatiles were removed in vacuo. The resulting residue was dissolved in water: acetonitrile (2.0 mL, 1:1 v/v) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-mPEG(7u)-sulfide-propanoyl-2-fluoro-benzoic acid compound 67 as colourless oil (101 mg, 78%) m/z [M−H].sup.+ 919.45 Da.

[0273] Step 4: Synthesis of Compound 67:

##STR00089##

[0274] To a stirred solution of compound 66 (80 mg) in methanol:water (2.0 mL, 9:1 v/v) at room temperature was added Oxone® (160 mg, Sigma-Aldrich). After 3 h, the volatiles were removed in vacuo. The resulting residue was dissolved in dichloromethane (3×4 mL), filtered through a column of magnesium sulfate and washed with dichloromethane (2×5 mL). The eluent and washings were combined and the volatiles were removed in vacuo. The resulting oil was dissolved in water and lyophilised to give bis-mPEG(7u)-sulfone-propanoyl-2-fluoro-benzoic acid compound 67 as a clear colourless oil (82 mg, 95%) m/z [M+H].sup.+ 983.26 Da.

[0275] Step 5: Synthesis of Compound 63:

[0276] Stock solutions of HATU (40.0 mg, Novabiochem) in DMF (200 μL) and NMM (11.8 μL, Acros Organics) in DMF (200 μL) were prepared. To a DMF (800 μL) solution of compound 67 (25.0 mg) was added H2N-PEG(24u)-OMe (23.4 mg, Iris Biotech). The resulting reaction mixture was stirred at room temperature under an inert nitrogen atmosphere. Aliquots of HATU (40.0 4) and NMM (40.0 μL) were added every 10 min for a total of 3 additions and the remaining aliquots of HATU (80.0 μL) and NMM (80.0 μL) were added at 30 min. After 60 min, volatiles were removed in vacuo. The resulting residue was dissolved in water (1.0 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-mPEG(7u)-sulfone-propanoyl-2-fluoro-benzamide-PEG(24u) reagent 63 as a clear colourless oil (17.5 mg, 40%) m/z [M+3H].sup.3+ 684.94 Da.

EXAMPLE 32

Synthesis of a PEG Conjugation Reagent 68 Comprising 7 Repeat Unit Ethylene Glycol Amino Leaving Groups

[0277] ##STR00090##

[0278] Step 1: Synthesis of Compound 69.

##STR00091##

[0279] Stock solutions of HATU (632 mg, Novabiochem) in DMF (1.6 mL) and NMM (183 μL, Acros Organics) in DMF (1.6 mL) were prepared. To a DMF (4.0 mL) solution of 4-(3-tosyl-2-(tosylmethyl)propanoyl)benzoic acid (199 mg, BioVectra) was added H2N-PEG(24u)-CO.sub.2.sup.tBu (400.0 mg, Iris Biotech). The resulting reaction mixture was stirred at room temperature under an inert nitrogen atmosphere. Aliquots of HATU (320 μL) and NMM (320 μL) were added every 10 min for a total of 5 additions. After 120 min, volatiles were removed in vacuo. The resulting residue was dissolved in water:acetonitrile (2.0 mL, 1:3 v/v) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-(tosyl propanoyl)-benzamide-PEG(24u)-.sup.tButyl ester compound 69 as a clear orange oil (184 mg, 33%) m/z [M+Na+H].sup.2+ 853.43 Da, [M−(tBu)+2H].sup.2+ 814.83 Da.

[0280] Step 2: Synthesis of Compound 68.

[0281] To a solution of 69 (32 mg) in DMF (500 μL) was added anhydrous sodium carbonate (24.1 mg, Fisher Scientific) and MeO-PEG-(7u)-NH.sub.2 (19 mg, Iris Biotech). The reaction was stirred at room temperature under an inert nitrogen atmosphere. After 18 h, volatiles were removed in vacuo. The resulting residue was dissolved in water (1.0 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-mPEG(7u)-amino-propanoyl-benzamide-PEG(24u)-.sup.tButyl ester reagent 68 as a clear colourless oil (15.0 mg, 38%) m/z [M+2H].sup.2+ 1026.09 Da, [M+3H].sup.3+ 665.45 Da.

EXAMPLE 33

Synthesis of a Conjugation Reagent 70 Comprising Maleimide Unit, 7 Repeat Unit Ethylene Glycol Leaving Groups

[0282] ##STR00092##

[0283] Step 1: Synthesis of Compound 71.

##STR00093##

[0284] 3,4-dibromofuran-2,5-dione (300 mg, Sigma Aldrich) was dissolved in acetic acid (4.0 mL, Fisher Scientific) and to this was added CO.sub.2.sup.tBu-PEG-(24u)-NH.sub.2 (705 mg, Iris Biotech). The reaction was stirred at 75° C. under an inert nitrogen atmosphere. After 3 h the reaction was stirred at room temperature and after 21 h, volatiles were removed in vacuo. The resulting residue was dissolved in water and acetonitrile (v/v; 1/1, 4.0 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give dibromo-maleimide-PEG(24u)-.sup.tButyl ester compound 71 as an off white opaque solid (427 mg, 51%) m/z [MH−(.sup.tBu)].sup.2+ 692.68 Da.

[0285] Step 2: Synthesis of Compound 70.

[0286] To a solution of 71 (250 mg) in DMF (1.5 mL) was added alpha-methoxy-omega-mercapto hepta(ethylene glycol) (186 mg, Iris Biotech) and triethylamine (145 μL). The resulting reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. Rapidly precipitated triethylamine hydrobromide was redissolved into the reaction mixture with DMF (1.0 mL) and after 30 min volatiles were removed in vacuo. The resulting residue was dissolved in formic acid (3 mL, Sigma Aldrich) and the reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. After 30 min trifluoroacetic acid (50 μL, Acros Organics) was added and after 60 min more trifluoroacetic acid (300 μL) was added. After 100 min, volatiles were removed in vacuo. The resulting residue was dissolved in water (1.5 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-mPEG(7u)-maleimide-PEG(24u)-acid reagent 70 as a waxy yellow solid (146 mg, 43%) m/z [MH.sub.3].sup.3+ 646.28 Da.

EXAMPLE 34

Synthesis of a Conjugation Reagent 72 Comprising 7 Repeat Unit Ethylene Glycol Leaving Group and an Auristatin Cytotoxic Payload

[0287] ##STR00094##

[0288] Step 1: Synthesis of Compound 73.

##STR00095##

[0289] To a stirred solution of 4-(3-tosylpropanoyl)benzoic acid (99 mg, Bioconjugate Chem. 2014, (25) 460-469) in dimethylformamide (DMF, 5.0 mL) was added alpha-methoxy-omega-mercapto hepta(ethylene glycol) (170 mg, Iris Biotech) and triethylamine (250 μL). The resulting reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. A further portion of triethylamine (120 μL) was added to the reaction mixture after 4 h. Volatiles were removed in vacuo after 95.0 h and the resulting residue was dissolved in water/acetonitrile (2 mL, 1:1 v/v) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give mPEG(7u)-sulfide-propanoyl-benzoic acid compound 73 as a white solid (200 mg, 126%, with residual solvent) m/z [M+H].sup.+ (533).

[0290] Step 2: Synthesis of Compound 74.

##STR00096##

[0291] To a stirred solution of 73 (200 mg) in methanol:water (5.5 mL, 9:1 v/v) at room temperature was added Oxone® (320 mg). After 4.5 h, the volatiles were removed in vacuo. The resulting residue was dissolved in dichloromethane (4×3 mL), filtered through a column of magnesium sulfate and washed with dichloromethane (2×3 mL). The eluent and washings were combined and the volatiles were removed in vacuo to give a thick clear oil. The residue was dissolved in water:acetonitrile (1.0 mL, 4:1 v/v), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give mPEG(7u)-sulfone-propanoyl-benzoic acid reagent 74 as a white solid (90 mg, 60%) m/z [M+H].sup.+ (565).

[0292] Step 3: Synthesis of Compound 72.

[0293] To a stirred solution of val-cit-PAB-MMAE (30 mg) in anhydrous DMF (500 μL) was added compound 73 (11 mg) and stirred for 5 min at 0° C. HATU (9.95 mg) and NMM (4.26 μL) were added in succession and the reaction mixture was allowed to stir at 0° C. After 0.5 h, and 2 h an additional amount of HATU and NMM was added and after a further 2.0 h the reaction solution was then concentrated in vacuo and then purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give the mPEG(7u)-sulfone-propanoyl-benzamide-val-cit-PAB-MMAE reagent 72 as off-white solid (15.0 mg, 46%) m/z [M+H].sup.+ (1671).

EXAMPLE 35

Synthesis of a Conjugation Reagent 75 Comprising 7 Repeat Unit Ethylene Glycol Leaving Groups and a Furan Linker

[0294] ##STR00097##

[0295] Step 1: Synthesis of Reagent 76

##STR00098##

[0296] To a suspension of N,N-dimethylmethyleneiminium iodide (407 mg) in DMF (3.0 mL) was added 5-acetylfuran-2-carboxylic acid (169 mg). The reaction mixture was heated to 125° C. and stirred under an inert atmosphere for 90 min. To a portion of the reaction mixture (1.0 mL) was added mPEG-(7u)-SH (325 mg) and Et.sub.3N (363 μL). The reaction mixture was stirred at room temperature under an inert atmosphere for 90 min. Volatiles were removed in vacuo. The resulting residue was dissolved in water (1.0 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-mPEG(7u)-sulfide-propanoyl-furan-2-carboxylic acid reagent 76 as pale yellow oil (119 mg, 36%). Yield calculation based on a ⅓.sup.rd portion of the initial reaction mixture used. .sup.1H NMR (400 MHz, DMSO) 2.60-2.70 (4H, m, CH.sub.2), 2.80-2.90 (4H, m, CH.sub.2), 3.24 (6H, s, OMe), 3.40-3.60 (m, PEG), 3.90-3.95 (1H, m, CH), 7.36 (1H, d, Ar—H), 7.69 (1H, d, Ar—H).

[0297] Step 2: Synthesis of Reagent 77

##STR00099##

[0298] To a solution of 76 (30.0 mg) in methanol:water (1.0 mL, 9:1 v/v) was added Oxone® (62.1 mg, Sigma-Aldrich). The reaction mixture was stirred at room temperature under an inert atmosphere for 120 min. The crude reaction mixture was concentrated in vacuo. The resulting residue was dissolved in dichloromethane (3×5.0 mL), filtered through a column of magnesium sulfate and washed with dichloromethane (2×5.0 mL). The eluent and washings were combined and the volatiles were removed in vacuo to give bis-mPEG(7u)-sulfone-propanoyl-furan-2-carboxylic acid reagent 77 as a cicar colourless oil (31.0 mg, 97%). m/z [M+H].sup.+ 955.28 Da.

[0299] Step 3: Synthesis of Reagent 75

[0300] Stock solutions of HATU (51.5 mg) in DMF (200 μL) and NMM (15.0 μL) in DMF (200 μL) were prepared. A DMF (500 μL) solution of bis-mPEG(7u)-sulfone-propanoyl-furan-2-carboxylic acid 77 (31.0 mg) was added to a DMF (500 μL) solution of H2N-PEG(24u)-OMe (29.5 mg, Iris Biotech). The resulting reaction mixture was stirred at room temperature under an inert nitrogen atmosphere. Aliquots of HATU (40 μL) and NMM (40 μL) were added at time 0 min, 10 min, 30 min and an aliquot of HATU (80 μL) and NMM (80 μL) was added at 90 min. After 120 min, volatiles were removed in vacuo. The resulting residue was dissolved in water:acetonitrile (1.0 mL, 1:1 v/v) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-mPEG(7u)-sulfone-propanoyl-furan-2 amide-PEG(24u)-OMe reagent 75 as clear colourless oil (9.6 mg, 18%). m/z [M+2H].sup.2+ 1012.76 Da.

EXAMPLE 36

Synthesis of a Conjugation Reagent 78 Comprising 7 Repeat Unit Ethylene Glycol Sulfide Leaving Groups

[0301] ##STR00100##

[0302] To a solution of 1-(4-hydroxyphenyl)-3-tosyl-2-(tosylmethyl)propan-1-one (400 mg) in DMF (8.0 mL) was added Et.sub.3N (708 μL, Acros Organics) and MeO-PEG-(7u)-SH (905 mg, Iris Biotech). The reaction was stirred under an inert nitrogen atmosphere at room temperature. After 48 h, volatiles were removed in vacuo. The resulting residue was dissolved in water: acetonitrile (2.0 mL, 1:1 v/v) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 79 as clear colourless oil (322 mg, 43%) m/z [M+H].sup.+ 873.49 Da.

EXAMPLE 37

Synthesis of a Conjugation Reagent 79 Comprising 7 Repeat Unit Ethylene Glycol Sulfone Leaving Groups

[0303] ##STR00101##

[0304] To a stirred solution of compound 78 (20.0 mg) in methanol:water (500 μL, 9:1 v/v) at room temperature was added Oxone® (42.2 mg. Sigma-Aldrich). After 3 h, the volatiles were removed in vacuo. The resulting residue was dissolved in water:acetonitrile (800 μL, 1:1 v/v) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 79 as clear colourless oil (19.5 mg, 91%) m/z [M+H].sup.+ 936.86 Da.

EXAMPLE 38

Synthesis of a Conjugation Reagent 80 Comprising 7 Repeat Unit Ethylene Glycol Sulfone Leaving Groups and a Phenol Ester Linker

[0305] ##STR00102##

[0306] Step 1: Synthesis of Compound 81:

##STR00103##

[0307] To a solution of mPEG(24u)-COOH (38.4 mg, Iris Biotech) in DMF (400 μL) was added HATU (26.1 mg) and DIPEA (12.0 μL). The reaction was stirred under an inert nitrogen atmosphere at room temperature for 5.0 min. To this mixture was added compound 79 (20.0 mg) dissolved in DMF (100 μL). The reaction was stirred under an inert nitrogen atmosphere at room temperature for a further 42 h. After 42 h, volatiles were removed in vacuo. The resulting residue was dissolved in water (800 μL) and purified by reverse phase C18-column chromatography cluting with buffer A (v/v): water: 0.05% trifluoroacctic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 81 as clear colourless oil (15.4 mg, 34%) m/z [M+2H-mPEG(7u) fragment].sup.2+ 824.34 Da.

[0308] Step 2: Synthesis of Compound 80:

[0309] To a stirred solution of compound 81 (14.0 mg) in methanol:water (500 μL, 9:1 v/v) at room temperature was added Oxone® (13.1 mg, Sigma-Aldrich). After 230 min, the volatiles were removed in vacuo. The resulting residue was dissolved in water (1.0 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 80 as clear colourless oil (12.7 mg, 88%) m/z [M+3H].sup.3+ 679.16 Da.

EXAMPLE 39

Synthesis of a Conjugation Reagent 82 Comprising 7 Repeat Unit Ethylene Glycol Sulfone Leaving Groups, Non-Aromatic Linker and an Auristatin Cytotoxic Payload

[0310] ##STR00104##

[0311] Step 1: Synthesis of Compound 83:

##STR00105##

[0312] To a stirred solution of 3-bromo-2-(bromomethyl) propionic acid (1.32 g, Alfa Aesar) in methanol (50.0 mL) at room temperature was added crushed sodium hydroxide pellets (428 mg, Fisher Scientific). The mixture was heated until the solids dissolved. The sodium salt of benzenesulfinic acid (1.76 g, Sigma Aldrich) was added and the reaction mixture was refluxed under an inert nitrogen atmosphere. After 4.0 h, the reaction mixture was cooled to room temperature and was stirred overnight under an inert nitrogen atmosphere. After 20 h, the volatiles were removed in vacuo. The resulting residue was dissolved in 0.5 M sodium hydroxide (30 mL) and was washed with diethyl ether (3×10.0 mL). The aqueous fraction was made acidic with 37% hydrochloric acid and gave a white precipitate. The precipitate was collected by Buchner filtration, the retentate was washed with distilled water (3×20.0 mL) and aqueous solvent was removed by lyophilisation to give compound 83 as a white powder (632 mg, 32%) m/z [M+H].sup.+ 369.08 Da; [M+Na].sup.+ 391.04 Da.

[0313] Step 2: Synthesis of Compound 84:

##STR00106##

[0314] To a stirred solution of compound 83 (111 mg) in dimethylformamide (2.0 mL) at room temperature was added alpha-methoxy-omega-mercapto hepta(ethylene glycol) (428 mg, Iris Biotech) and triethylamine (251 μL). The resulting reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. After 20 h, the crude reaction mixture was diluted with water (1.0 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 84 as clear colourless oil (154 mg, 64%) m/z [M-PEG-(7u)].sup.+ 473.05 Da.

[0315] Step 3: Synthesis of Compound 85:

##STR00107##

[0316] To a stirred solution of bis-mPEG(7u)-sulfide-propanoic acid (65.0 mg) in methanol:water (1.5 mL, 9:1 v/v) at room temperature was added Oxone® (151 mg, Sigma-Aldrich). After 3.0 h, the volatiles were removed in vacuo. The resulting residue was dissolved in water (800 μL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 85 as clear colourless oil (41.7 mg, 59%) m/z [M+H].sup.+ 860.91 Da.

[0317] Step 4: Synthesis of Compound 82:

[0318] Stock solutions of HATU (26.4 mg) in DMF (200 μL) and NMM (7.6 μL) in DMF (200 μL) were prepared. A DMF (200 μL) solution of compound 83 (12.0 mg) was added to a DMF (400 μL) solution of NH2-Val-Cit-PAB-MMAE (21.5 mg, Concortis). The resulting reaction mixture was stirred at room temperature under an inert nitrogen atmosphere. Aliquots of HATU (80 μL) and NMM (80 μL) were added at time 0 min and 40 min for a total of 2 additions. After 180 min, volatiles were removed in vacuo. The resulting residue was dissolved in water: acetonitrile (1.0 mL, 1:1 v/v) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give reagent 82 as a white powder (13.6 mg, 50%). m/z [M+2H].sup.2+ 983.45 Da.

EXAMPLE 40

Synthesis of a Conjugation Reagent 86 Comprising 7 Repeat Unit Ethylene Glycol Sulfone Leaving Groups and Non-Aromatic Linker

[0319] ##STR00108##

[0320] Step 1: Synthesis of Compound 87.

##STR00109##

[0321] To a stirred solution of compound 84 (15.0 mg) in toluene (250 μL) at room temperature was added mPEG-(23u)-OH (19.7 mg, Iris Biotech), triphenylphosphine (5.43 mg, Sigma Aldrich) and diisopropyl azodicarboxylate (4.1 μL, Sigma Aldrich). The resulting reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. After 30 min, additional triphenylphosphine (5.43 mg, Sigma Aldrich) and diisopropyl azodicarboxylate (4.10 Sigma Aldrich) were added. The reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. After 1.0 h, the volatiles were removed in vacuo. The resulting residue was dissolved in water (700 μL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 87 as clear colourless wax (18.5 mg, 54%) m/z [M-PEG-(7u)].sup.+ 750.19 Da.

[0322] Step 2: Synthesis of Compound 86.

[0323] To a stirred solution of compound 87 (14.5 mg) in methanol:water (1.0 mL, 9:1 v/v) at room temperature was added Oxone® (14.7 mg, Sigma-Aldrich). After 3.0 h, the reaction mixture was filtered through cotton wool (1.5 cm plug). The crude product was purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 86 as clear colourless wax (7.1 mg, 47%) m/z [M+3H].sup.3+ 629.91 Da.

EXAMPLE 41

Synthesis of a Bifunctional Conjugation Reagent 88 Comprising 7 Repeat Unit Ethylene Glycol Sulfone Leaving Groups and Poly Ethylene Glycol Spacer

[0324] ##STR00110##

[0325] Stock solutions of HATU (448 mg) in DMF (2.0 mL) and NMM (129 μL) in DMF (2.0 mL) were prepared. A DMF (500 μL) solution of amino-PEG-(11u)-amine (64.1 mg, Iris Biotech) was added to a DMF (2.0 mL) solution of 4-[2,2-bis[alpha-methoxy-omega-sulfonyl hepta(ethylene glycol)]acetyl]benzoic acid (250 mg). The resulting reaction mixture was stirred at room temperature under an inert nitrogen atmosphere. Aliquots of HATU (800 μL) and NMM (800 μL) were added at time 0 min and aliquots of HATU (200 μL) and NMM (200 μL) were added at time 50 min for a total of 2 additions. After 2.0 h, volatiles were removed in vacuo. The resulting residue was dissolved in water (1.0 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give reagent 88 as a clear colourless oil (200 mg, 69%). m/z [M+H].sup.2+ 1219.5 Da [M+2H].sup.3+ 813.35 Da, [M+3H].sup.4+ 610.28 Da.

EXAMPLE 42

Synthesis of a Conjugation Reagent 89 Comprising 7 Repeat Unit Ethylene Glycol Sulfone Leaving Groups and a Sulfonic Acid Side Chain

[0326] ##STR00111##

[0327] To a stirred solution of 3-(N-(3-sulfopropyl)-4-(3-tosyl-2-(tosylmethyl)propanoyl)benzamido) propanoic acid (Click Chemistry Tools LLC, 20.0 mg) in dimethylformamide (500 μL) at room temperature was added alpha-methoxy-omega-mercapto hepta(ethylene glycol) (30.2 mg, Iris Biotech) and triethylamine (23.6 μL). The resulting reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. After 3.5 h, the crude intermediate product was purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation. The resulting clear colourless residue was dissolved in methanol:water (750 μL, 9:1 v/v) and the solution was added to Oxone® (52.1 mg, Sigma-Aldrich). The resulting reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. After 4.5 h the crude product was purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (98:2 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 89 as a clear colourless oil (4.71 mg, 14%) m/z [M+H].sup.2+ 579.67 Da, [M+H.sub.2O].sup.2+ 588.17 Da.

EXAMPLE 43

PEGylation at a Reduced Fab Disulfide Bond Using Conjugation Reagents 63, 68 and 70

[0328] To the Fab of trastuzumab produced by papain digestion was added 1 M DTT stock solution (25 μL, final concentration 10 mM), the reduction mixture was briefly mixed using a vortex and incubated at 22° C. for 1 h. The reductant was then removed using a PD10 column (GE Healthcare) equilibrated with 20 mM sodium phosphate buffer pH 7.5, 150 mM NaCl, 20 mM EDTA. The reduced Fab was further diluted to 1.2 mg/mL with the same buffer. Reagents 63, 68 and 70 were dissolved independently in acetonitrile at 0.719 mM and added to separate solutions of reduced Fab (1.5 eq. reagent per Fab). The PEGylation reactions were allowed to progress at 22° C., whereupon samples were taken for HIC HPLC analysis after 22 h. HIC HPLC analysis of each reaction mixture showed that the % conversion to PEGylated Fab for reagents 63, 68 and 70 were 95%, 90% and 90% respectively.

EXAMPLE 44

Conjugation of Reagent 72, to Partially Reduced Trastuzumab

[0329] Trastuzumab (3.5 mg, 5.2 mg/mL) in 20 mM sodium phosphate, pH 7.5 (20 mM EDTA; 150 mM NaCl) was warmed to 40° C. for 15 min. To the trastuzumab, was added 2.08 mM TCEP (28 μL) and the resulting mixture was incubated at 40° C. for 1 h. After 1 h, the TCEP treated trastuzumab was cooled to 22° C. A portion of the trastuzumab solution (3.2 mg, 0.64 mL, 5.0 mg/mL) was diluted with 20 mM sodium phosphate, pH 7.5 (20 mM EDTA; 150 mM NaCl) (0.12 mL). Three vials were charged with the diluted mAb solution (1 mg, 238 μL). Solutions of reagent 72, (3.2 mM) were prepared in DMF. The reagent solutions (12.5 μL, 6.0 eq. per mAb) were added to separate vials, the reactions were mixed and then incubated at 22° C. At 22 h the reaction samples were quenched with N-acetyl-L-cysteine (20 eq. over reagent) prior to HIC analysis. HIC HPLC analysis showed that the average DAR of Trastuzumab conjugated with reagent 72 was 4.5.

EXAMPLE 45

Conjugation of Reagents 82 and 86 to Trastuzumab

[0330] Both reagents 82 and 86 were conjugated to the antibody trastuzumab using identical protocols. Briefly, trastuzumab (5.2 mg/mL) in buffer (20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 8.4) was reduced with TCEP (6 eq.) at 40° C. for 1 h. Conjugation of the antibody with 1.5 molar equivalents of either reagent 82 or 86 per inter-chain disulfide bond was then performed at 40° C. This was achieved by addition either reagent 82 or reagent 86 in acetonitrile (5% v/v acetonitrile in each reaction). The antibody solution was diluted to 4.0 mg/mL with 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 8.4. Each solution was the mixed gently and incubated at 40° C. for 22 h. At 22 h the reactions were treated with N-acetyl-L-cysteine (20 eq. over reagent) for 1 h at 22° C. The reactions were subsequently analysed by analytical HIC. The % conversion to product utilising reagents 82 and 86 were determined based on the % area of the absorbance peaks measured at 280 nm from the HIC chromatograms. For reagents 82 and 86, the % conversion to product values were 100% and 50% respectively.

EXAMPLE 46

PEGylation at a Reduced Fab Disulfide Bond Using Conjugation Reagents 75, 79 and 80 and Reagent 89

[0331] Reagents 75, 79, 80 and 89 were conjugated to Fab.sub.trast in a similar manner to that described for conjugation reagents 63, 68 and 70 within example 43. Following 22 h conjugation reaction, samples were analysed by SDS-PAGE under reducing and non-reducing conditions. The % conversion of PEGylated Fab.sub.trast with each of the reagents was estimated from the band density measurements using an ImageQuant™ LAS 4010 system (GE Healthcare) using gels stained with InstantBlue™. For reagents 75, 79, 80 and 89, the % conversion values were 90%, 20%, 75% and 40% respectively.

EXAMPLE 47

Synthesis of Conjugation Reagents 90 (Comparative) and 91 (Comparative) Comprising Either 3 or 5 Repeat Unit Polymeric Ethylene Glycol Leaving Groups Respectively and an Auristatin Cytotoxic Payload

[0332] ##STR00112##

[0333] Auristatin reagents bis-mPEG(3u)sulfone-propanoyl-benzamide-val-cit-PAB-MMAE 90 and bis-mPEG(5u)sulfone-propanoyl-benzamide-val-cit-PAB-MMAE 91 were synthesised in an analogous way as that described for reagent 5 in Example 4, using the thiols 2-[2-(2-methoxyethoxy)ethoxy]-ethanethiol and 2,5,8,11,14-pentaoxahexadecane-16-thiol respectively instead of alpha-methoxy-omega-mercapto hepta(ethylene glycol) for the synthesis of compound 1 in Example 1.

EXAMPLE 48

Synthesis of a PEGylation Reagent 92 Comprising a Single 7 Repeat Unit Ethylene Glycol Leaving Group and a 24 Unit PEG

[0334] ##STR00113##

[0335] The synthesis of mono-mPEG(7u)sulfone-(methyl-acryloyl)-benzamide-L-Glu-[OH]-[PEG(24u)-OMe] reagent 92 was carried out according to Nature Protocols (2006, 1, 2241-2252) using compound 23 in Example 10.

EXAMPLE 49

Comparison of the Antibody Conjugation of Cytotoxic Reagents 90 (Comparative), 91 (Comparative) and 5 Possessing Polymeric Ethylene Glycol Leaving Groups

[0336] The reaction conditions used for conjugation of cytotoxic reagents 90, 91 and 5 with trastuzumab (1 mg scale) were the same as those described above for Example 20, using 1.25% v/v MeCN as solvent in the conjugation reaction in place of DMF. Aliquots of reaction solution were taken after 2 and 4 h and each were quenched by treatment with N-acetyl-L-cysteine. Each aliquot was analysed by HIC and the % DAR product profile determined. Results for conjugation with reagents 90, 91 and 5 are displayed in Table 3 and FIGS. 10 (2 hours) and 11 (4 hours). It can be seen that the results obtained when using the reagent according to the present invention arc significantly improved compared to the results obtained using the comparison reagents, in that the homogeneity of the product is much higher, the product being predominantly the desired DAR4 conjugate.

TABLE-US-00003 TABLE 3 Comparison of conjugation of reagents 90, 91 and 5 to an antibody after 2 h and 4 h reaction. Reagent used % DAR % DAR for conjugation product after 2 h product after 4 h to an antibody 0 1 2 3 4 >4 0 1 2 3 4 >4 90 8.3 14.4 13.9 26.0 33.5 4.0 5.2 8.9 10.8 22.6 48.0 4.6 91 7.9 13.3 12.7 25.8 35.5 4.8 4.7 8.6 9.7 23.2 48.5 5.4 5 6.3 4.0 4.8 10.2 60.0 14.7 0.0 2.6 4.2 11.6 72.8 8.8

EXAMPLE 50

Pegylation at a Reduced Fab Disulfide Bond Using Reagent 92

[0337] Reagent 92 was conjugated to a reduced Fab using the same conditions as those described in Example 17. The reactions were subsequently analysed by SDS-PAGE and the major product, PEGylated Fab, was observed at 49 kDa against the molecular weight protein standards.

EXAMPLE 51

Synthesis of Conjugation Reagent 93 Comprising an Auristatin Cytotoxic Payload

[0338] ##STR00114##

[0339] Step 1: Synthesis of Compound 94.

##STR00115##

[0340] Boc-L-Glu (135 mg) and (benzotriazol-1-yloxy)tris-(dimethylamino) phosphonium hexafluorophosphate (BOP) (724 mg) were dissolved in anhydrous DMF (4 mL) and were stirred at 0° C. under a nitrogen atmosphere for 1.25 h. This solution was then added to a solution of H.sub.2N-PEG(12u)-Me (685 mg) and NMM (180 μL) in DMF (3 mL). The solution was then stirred under N.sub.2 for 4 h. The solution was then stirred 0-4° C. under a nitrogen atmosphere for 4.5 h. Further BOP (241 mg) and NMM (60 μL) were added, reaction mixture left for 24 h at 4° C. The volatiles were removed in vacuo and the resulting residue was purified by reverse phase C18-flash chromatography eluting with eluting with buffer A (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 65:35 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation. The material was repurified by normal phase flash chromatography eluting with ethyl acetate:methanol (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give Boc-Glu-[PEG(12u)-Me].sub.2 compound 94 as a colourless oil (450 mg). m/z [M+H].sup.+ (1331, 100%), [M+2H].sup.2+ (665, 100%).

[0341] Step 2: Synthesis of Compound 95.

##STR00116##

[0342] Compound 94 (450 mg) was dissolved in DCM (25 mL) to which was added TFA (2.5 mL). The solution stirred at room temperature for 5 h. After which the volatiles were removed in vacuo. The resulting residue was purified by reverse phase C18-flash chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 60:40 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give Glu-[HN-PEG(12u)-Me].sub.2 TFA compound 95 as a clear colourless gum (320 mg) m/z [M+Na].sup.1+ (1253.0, 10%) [M+H].sup.2+ (616.8, 100%).

[0343] Step 3: Synthesis of Compound 96

##STR00117##

[0344] To a stirred solution of Fmoc-L-Glu-(OtBu)-OH (36 mg) in anhydrous DMF (2 mL) was added HATU (37 mg). The reaction mixture was stirred at 0° C. under a nitrogen atmosphere for 1 h and then added to a solution of compound 95 (103.5 mg) and NMM (19 μL) in DMF (1 mL). Additional DMF (1 mL) was added. The stirred reaction was left to warm to room temperature over 5 h. The volatiles were removed in vacuo. The resulting pale yellow oil was purified by reverse phase C18-flash chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 50:50 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give Fmoc-L-Glu-(O.sup.tBu)-Glu-[HN-PEG(12u)-Me].sub.2 compound 96 (173 mg) as a white paste. m/z [M+1].sup.+ (1638, 100%) & [M+Na].sup.+ (1660, 57%).

[0345] Step 4: Synthesis of Compound 97

##STR00118##

[0346] To a stirred solution of compound 96 (173 mg) in anhydrous DMF (3.2 mL) was added piperidine (104 μL). The solution was stirred at room temperature under argon for 1.5 h. The volatiles were removed in vacuo and the residue triturated repeatedly with hexane. The product was dried in vacuo to give L-Glu-(O.sup.tBu)-L-Glu-[HN-PEG(12u)-Me].sub.2 compound 97 (152 mg) as a clear colourless oil. m/z [M+H].sup.1+ (1416.7, 85%), [M+2H].sup.2+ (708.5, 100%), [M+Na].sup.1+ (1438.7, 30%).

[0347] Step 5: Synthesis of Compound 98

##STR00119##

[0348] To a stirred solution of 4-[2,2-bis[alpha-methoxy-omega-sulfonyl hepta(ethylene glycol)]acetyl]benzoic acid (114 mg) in anhydrous DMF (3 mL) was added HATU (51 mg). Reaction mixture was stirred at 0° C. for 0.5 h then added to a solution of L-Glu(OtBu)-Glu-[HN-PEG(12u)-OMe].sub.2. (152 mg) in DMF (2 mL) and washed in with further DMF (1 mL), followed by NMM (15 μL). The reaction mixture was stirred at 0-15° C. for 3.5 h after which the volatiles were removed in vacuo. The resulting residue was purified by reverse phase C18-flash chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 55:45 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give Bis-mPEG(7u)sulfone-propanoyl-benzamide -L-Glu-(O.sup.tBu)-Glu-[HN-PEG(12u)-Me].sub.2 compound 98 (161 mg) as a clear colourless oil. m/z [M+H].sup.1+ (2366.7, 100%), [M+2H].sup.2+ (1184.0, 80%) [M+H.sub.2O].sup.3+ (795.5, 100%).

[0349] Step 6: Synthesis of Compound 99

##STR00120##

[0350] To the stirred solution of compound 98 (58 mg) in anhydrous DCM (6 mL) was added TFA (6 mL). Reaction mixture was stirred at room temperature for 2 h. after which the volatiles were removed in vacuo, dissolved in water (25 mL) and lyophilised to give Bis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-(OH)-Glu-[HN-PEG(12u)-OMe].sub.2 compound 99 (160.6 mg) as a clear colourless oil. m/z [M+H].sup.1+ (2306.8, 90%), [M+2H].sup.2+ (1153.0, 100%).

[0351] Step 7: Synthesis of Reagent 93

[0352] Reagent 93 was synthesised in analogous way to reagent 24 of Example 11A from compound 99 and val-cit-PAB-MMAE TFA salt. Bis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-(val-cit-PAB-MMAE)-Glu-[HN-PEG(12u)-Me].sub.2 93 was isolated as a colourless oil (69%). m/z [M+H].sup.1+ (3410.4, 90%), [M+2H].sup.2+ (1706.2, 60%), [M+3H].sup.3+ (1137.2, 85%), [M+4H].sup.4+ (852.8. 70%).

[0353] Conjugation of Brentuximab with conjugation reagent 93 was carried out as described within Example 21 for 16 h. Results from analytical HIC show that the conversion to DAR4 product was 67%.

EXAMPLE 52

Synthesis of Conjugation Reagent 100 Comprising an Auristatin Cytotoxic Payload

[0354] ##STR00121##

[0355] Reagent 100 was synthesised in analogous way to reagent 24 of Example 11A using compound 20B instead of compound 23 and val-cit-PAB-MMAE TFA salt instead of val-cit-AHX-DM1.

##STR00122##

[0356] Compound 20B was made in an analogous way to compound 23 in Example 10, using H.sub.2N-PEG(12u)-tri(m-dPEG(24u) instead of H.sub.2N-PEG(24u). Bis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-[val-cit-PAB-MMAE]-[PEG(12u)-tri(m-dPEG(24u))] 100 was isolated as a colourless oil. m/z [M+2H].sup.2+ (3166, 20%), [M+3H].sup.3+ (2111, 50%), [M+4H].sup.4+ (1583, 100%).

[0357] Conjugation of Brentuximab with conjugation reagent 100 was carried out as described within Example 21 for 16 h. Results from analytical HIC show that the conversion to DAR4 product was 65%.

EXAMPLE 53

Synthesis of Conjugation Reagent 101 Comprising two Auristatin Cytotoxic Payloads

[0358] ##STR00123##

[0359] Step 1: Synthesis of Compound 102.

##STR00124##

[0360] To a stirred solution of Fmoc-L-Glu-(O.sup.tBu)-OH (2 g) in anhydrous DMF (18 mL) was added HOBt (666 mg) and DIC (768 μL). The reaction mixture was stirred at 0° C. for 10 min and then 2.5 h at room temperature. H-L-Glu-(OtBu)-OH (1.19 g) and DIPEA (2.46 mL) were added and the reaction mixture was stirred for 18 h at room temperature. The reaction mixture was diluted with water (100 mL) and acidified to pH 2.0 by adding diluted HCl. The aqueous layer was extracted with EtOAc (3×100 mL), and the organic phases combined and washed with water (2×50 mL) and saturated brine solution (1×50 mL). The EtOAc layer was dried over Na.sub.2SO.sub.4 for 2 h and then concentrated on a rotary evaporator. The product was isolated by reverse phase C18-flash chromatography eluting with buffer A (v/v): water: 5% acetonitrile: 0.1% formic acid and buffer B (v/v): acetonitrile: 0.1% formic acid (100:0 v/v to 80:20 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound Fmoc-L-Glu-(OtBu)-L-Glu-(OtBu)-OH 102 (875 mg) as a white solid. m/z [M+H].sup.1+ (610.8, 85%), [M+Na].sup.1+ (633.1, 55%), [2M+Na].sup.+ (1243.2, 55%).

[0361] Step 2: Synthesis of Compound 103.

##STR00125##

[0362] To a stirred solution of Fmoc-L-Glu-(OtBu)-L-Glu-(OtBu)-OH (510 mg) and NH.sub.2-PEG(24u)-OMe (1 g) in anhydrous DMF (5 mL) was added and N,N-diisopropylethylamine (44 μL) and HATU (48 mg). The reaction mixture was stirred at 0° C. for 10 min and then 16 h at room temperature. The solution was concentrated in vacuo to 2 mL and the residue was purified by reverse phase C18-flash chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 83:17 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give Fmoc-Glu-(OtBu)-Glu-(OtBu)-PEG(24u)-OMe compound 103 644 mg) as a white paste. m/z [M+H].sup.1+ (1681.0, 40%), [M+Na].sup.1+ (1704.0, 30%) and [M+2H].sup.2+ (841.4, 55%).

[0363] Step 3: Synthesis of Compound 104.

##STR00126##

[0364] To a stirred solution of Fmoc-Glu-(OtBu)-G1u-(OtBu)-PEG(24u)-OMe (193 mg) in anhydrous DMF (900 μL) was added piperidine (34 μL) and the reaction mixture was stirred 1 h at room temperature. The solution was concentrated in vacuo to dryness and the residue triturated with Et.sub.2O (2×2.5 mL). The product was dried in vacuo to give H-L-Glu-(OtBu)-Glu-(OtBu)-PEG(24u)-OMe compound 104 (166 mg) as an off-white solid.

[0365] Step 4: Synthesis of Compound 105.

##STR00127##

[0366] Reagent 105 was synthesised in analogous way to compound 23 of Example 10 from compound 104 and 4-[2,2-bis[alpha-methoxy-omega-sulfonyl hepta(ethylene glycol)]acetyl]benzoic acid. Bis-mPEG(7u)sulfone-propanoyl-benzamide-Glu-(OtBu)-Glu-(OtBu)-PEG(24u)-OMe 105 was isolated as a colourless oil. m/z [M+H].sup.1+ (2407.2, 25%), [M+Na].sup.1+ (2429.4, 70%).

[0367] Step 5: Synthesis of Compound 106.

##STR00128##

[0368] Reagent 106 was synthesised in analogous way to reagent 23 of Example 10 from compound 105. Bis-mPEG(7u)sulfone-propanoyl-benzamide-Glu-(OH)-Glu-(OH)-PEG(24u)-OMe 106 was isolated as a colourless oil. m/z [M+H].sup.1+ (2294.2, 20%), [M+Na].sup.1+ (2317.4, 10%) and [M+2Na].sup.2+ (1217.4, 100%).

[0369] Step 6: Synthesis of Reagent 101.

[0370] To a stirred solution of compound 106 (28 mg), val-cit-PAB-MMAE TFA salt (31 mg) and HATU (14 mg) in anhydrous DMF (1.5 mL) was added N-methylmorpholine (7 μL) and the reaction mixture was stirred at 0° C. for 5 h. The solution was diluted with water (1 mL) and purified by reverse phase C18-flash chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.1% TFA and buffer B (v/v): acetonitrile:0.1% TFA (100:0 v/v to 60:40 v/v).

[0371] The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give bis-mPEG(7u)sulfone-propanoyl-benzamide-bis-[Glu-(val-cit-PAB-MMAE)]-PEG(24u)-OMe compound 101 (36 mg) as a white solid. m/z [M+2H].sup.2+ (2252.7, 20%), [M+3H].sup.3+ (1501.6.7, 40%) and [M+4H]4.sup.+ (1126.6, 100%).

[0372] Conjugation of Brentuximab with conjugation reagent 101 was carried out as described within Example 21 for 16 h. Results from analytical HIC show that the conversion to DAR4 product was 56%.

EXAMPLE 54

Synthesis of Conjugation Reagent 107 Comprising Two Auristatin Cytotoxic Payloads

[0373] ##STR00129##

[0374] Step 1: Synthesis of Compound 108.

##STR00130##

[0375] To a stirred solution of Boc-L-Glu(OH)—OH (51.6 mg) in anhydrous DMF (6 mL) was added BOP (277 mg). The solution was stirred at 0° C. for 20 min before Me0-PEG(24)-NH.sub.2 (500 mg) was added followed by NMM (69 μL). After 4 h, additional amounts of BOP (92 mg) and NMM (23 μL) were added. After a further 2.5 h, the reaction mixture was stored at −20° C. for 18 h before being concentrated in vacuo and purified by reverse phase column C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give a white solid (373 mg). Formic acid (6 mL) was added to the solid and the resulting mixture stirred under an inert atmosphere for 60 min before being concentrated in vacuo. The residue was dissolved in 95% water:5% acetonitrile:0.05% trifluoroacetic acid (-6 mL) and lyophilisation overnight to give TFA.H.sub.2N-Glu(PEG(24)-OMe)-PEG(24)-OMe, compound 108 as an off-white solid (330 mg). m/z [M+2H].sup.2+ (1144, 5%), [M+3H].sup.3+ (763, 35%), [M+4H].sup.+ (573, 100%).

[0376] Step 2: Synthesis of Compound 109.

##STR00131##

[0377] To a stirred solution of Fmoc-Glu(OtBu)-OH (2 g) in anhydrous DMF (18 mL) at 0° C. was added HOBt (666 mg) and DIC (768 μL). The reaction mixture was allowed to warm to RT and after 2 h, Glu(O.sup.tBu)-OH (1.19 g) and DIPEA (2.46 mL) were added. After stirring for 20 h, the reaction mixture was concentrated in vacuo and purified by reverse phase column C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v) The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give Fmoc-(L-Glu(O.sup.tBu)).sub.2-OH, compound 109, as a white solid (1.03 g). m/z [2M+H].sup.+ (1221, 15%), [M+H].sup.+ (611, 60%), [M−.sup.tBu+H].sup.+ (554, 65%), [M−2.sup.tBu+H].sup.+ (499, 100%)).

[0378] Step 4: Synthesis of Compound 110.

##STR00132##

[0379] To a stirred solution of compound 108 (330 mg) in anhydrous DMF (10 mL) was added compound 109 (100 mg). At 0° C., HATU (156 mg) and NMM (45 μL) were then added and the resulting solution stirred for 5 min before further addition of NMM (3 μL) and HATU (11 mg). The reaction solution was allowed to stir for another 20 min before being warmed to room temperature whereupon stirring was continued for a further 4 h. After this time, additional amounts of HATU (51 mg) and NMM (15 μL) were added. After a further 1.5 h, the mixture was stored at −20° C. for 18 h before being purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give Fmoc-(L-Glu(O.sup.tBu)).sub.2-L-Glu(PEG(24)-OMe)-PEG(24)-OMe, compound 110, as a white solid (193 mg). m/z [M+3H].sup.3+ (961, 20%), [M-.sup.tBu+4H].sup.4+ (707, 100%), [M−2.sup.tBu+4H].sup.4+ (693, 85%), [M+5H].sup.5+ (577, 75%).

[0380] Step 5: Synthesis of Compound 111.

##STR00133##

[0381] To a stirred solution of 111 (193 mg) in anhydrous DMF (1.5 mL) was added piperidine (20 μL). After 90 min, a further amount of piperidine (13 μL) was added and the reaction stirred for another 90 min before being stored at −20° C. for 18 h. The solvent was removed under high vacuum and the resulting residue triturated in hexane. The residue was further dried under high vacuum for 30 min before being dissolved in a 50:50 mixture of buffer A:buffer B (2 mL, buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacctic acid) and lyophilised overnight to give TFA.H2N-(L-Glu(OtBu)).sub.2-L-Glu(PEG(24)-OMe)-PEG(24)-OMe, compound 111, as a pale blue solid (186 mg). m/z [M+3H].sup.3+ (887, 20%), [M+4H].sup.4+ (666, 100%), [M+5H].sup.5+ (533, 30%).

[0382] Step 6: Synthesis of Compound 112.

##STR00134##

[0383] To a stirred solution of 4-[2,2-bis[alpha-methoxy-omega-sulfonyl hepta(ethylene glycol)]acetyl]benzoic acid (71 mg) in anhydrous DMF (1.5 mL) was added to HATU (28 mg). The mixture was cooled to 0° C. and stirred under an inert atmosphere for 30 min. A solution of 111 (186 mg) in anhydrous DMF (2.5 mL) was added, followed by HATU (22.9 mg) and NMM (14.7 μL), and the mixture allowed to warm to RT.

[0384] After 3 h, additional amounts of 4-[2,2-bis[alpha-methoxy-omega-sulfonyl hepta(ethylene glycol)]acetyl]benzoic acid (18 mg), HATU (50.8 mg) and NMM (15 μL) were added. After a further 1.5 h, further amounts of 4-[2,2-bis[alpha-methoxy-omega-sulfonyl hepta(ethylene glycol)]acetyl]benzoic acid (9 mg), HATU (51 mg) and NMM (15 μL) were added. The reaction mixture was stirred for a further 8 h and purified twice by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give 112, as a pale yellow oil (assumed quantitative). m/z [M+4H].sup.4+ (902, 60%), [M−.sup.tBu+4H].sup.4+ (888, 60%), [M−2.sup.tBu+4H].sup.4+ (874, 45%), [M−.sup.tBu+5H].sup.5+ (711, 100%).

[0385] Step 7: Synthesis of Compound 113.

##STR00135##

[0386] Formic acid (2 mL) was added to 112 under an inert atmosphere. The reaction mixture was stirred for 60 min before being concentrated in vacuo. The material was purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give 113, as a colourless oil (28.1 mg). m/z [M+3H].sup.3+ (1165, 5%), [M+4H].sup.4+ (874, 65%), [M+5H].sup.5+ (699, 100%).

[0387] Step 7: Synthesis of Reagent 107.

[0388] To a stirred solution of 113 (15 mg) in anhydrous DMF (270 μL) was added HATU (4 mg). The mixture was cooled to 0° C. and stirred under an inert atmosphere for 20 min. A solution of val-Cit-PAB-MMAE (12 mg) in anhydrous DMF (300 μL) was added, followed by HATU (2.5 mg) and NMM (2 μL), and the mixture allowed to warm to RT. After 4 h 20 min, additional amounts of HATU (3.3 mg) and NMM (0.9 μL) were added. The reaction mixture was stirred for a further 2 h before being stored at −20° C. for 18 h. Upon warming to RT, HATU (3.3 mg) and NMM (0.9 μL) were added to the stirred solution. After a 4.5 h, additional amounts of HATU (1.6 mg) and NMM (0.5 μL) were added and the reaction allowed to stir for a further 2.5 h before being stored at −20° C. for 18 h. The material was purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give 107, as a white solid (13.8 mg). m/z [M+4H].sup.4+ (1426, 5%), [M+5H].sup.5+ (1141, 70%), [M+6H].sup.6+ (951, 100%), [M+7H].sup.7+ (815, 20%).

[0389] Conjugation of Brentuximab with conjugation reagent 107 was carried out as described within Example 21 for 17 h. Results from analytical HIC show that the conversion to DAR4 product was 61%.