VEGF neutralizing prodrugs for the treatment of ocular conditions

Abstract

The present invention relates to a pharmaceutical composition comprising one or more pharmaceutically acceptable excipient(s) and a VEGF neutralizing prodrug, which comprises a VEGF neutralizing biologically active moiety, for use in a method for the treatment of one or more ocular conditions.

Claims

1. A method for the treatment of one or more ocular conditions, the method comprising: intraocularly administering, by injecting into the aqueous humor, the vitreous body, or the lens of an eye, a pharmaceutical composition comprising: one or more pharmaceutically acceptable excipient(s); and a VEGF neutralizing carrier-linked prodrug comprising a VEGF neutralizing biologically active moiety covalently and reversibly bound to a reversible prodrug linker moiety, wherein the reversible prodrug linker moiety is attached to a hydrogel carrier moiety, either directly or via a spacer moiety; wherein the step of intraocularly administering comprises two intraocular administrations of the prodrug; and wherein a time period between the two intraocular administrations of the VEGF neutralizing prodrug is at least 10 weeks.

2. The method of claim 1; wherein the carrier of the carrier-linked prodrug is a PEG-based hydrogel.

3. The method of claim 1; wherein the prodrug comprised in the pharmaceutical composition has a concentration of 5 to 200 mg prodrug/ml pharmaceutical composition.

4. The method of claim 1; wherein the composition comprises 8 to 80 weight percent of VEGF neutralizing biologically active moiety based on the total weight of the prodrug.

5. The method of claim 1; wherein the injection is carried out with an injection volume ranging from 10 to 200 l per injection.

6. The method of claim 1; wherein the VEGF neutralizing prodrug comprises in bound form at least one biologically active moiety selected from the group of drugs consisting of: antisense RNA, antisense DNA, ribozymes, and RNAi molecules targeting a VEGF nucleic acid; anti-VEGF aptamers, anti-VEGF antibodies, anti-VEGF antibody fragments, DARPins, and soluble VEGF receptor decoys that prevent binding of a VEGF to its cognate receptor; antisense, ribozymes, and RNAi molecules targeting a cognate VEGF receptor (VEGFR) nucleic acid; anti-VEGFR aptamers and anti-VEGFR antibodies that bind to a cognate VEGFR receptor; anti-VEGFR antibody fragments that bind to a cognate VEGFR receptor; and VEGFR tyrosine kinase inhibitors.

7. The method of claim 1; wherein the VEGF neutralizing prodrug comprises in bound form at least one biologically active moiety selected from the group consisting of ranibizumab, bevacizumab, pegaptanib, aflibercept, MP0112, KH902, ESBA1008, AL 39324, ALG-1001, and bevasiranib.

8. The method of claim 1; wherein the VEGF neutralizing prodrug comprises in bound form ranibizumab.

9. The method of claim 1; wherein the VEGF neutralizing prodrug comprises a moiety of formula (F-i): ##STR00148## wherein: D is ranibizumab; the dashed line indicates attachment to the carrier or to the optional spacer moiety; X.sup.1 is C or S(O); X.sup.2 is C(R.sup.7, R.sup.7a) or C(R.sup.7, R.sup.7a)C(R.sup.8, R.sup.8a); R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, R.sup.7, R.sup.7a, R.sup.8, and R.sup.8a are independently selected from the group consisting of H and C.sub.1-4 alkyl; optionally, one or more of the pair(s) R.sup.1a/R.sup.4a, R.sup.1a/R.sup.5a, R.sup.4a/R.sup.5a, R.sup.4a/R.sup.5a, R.sup.7a/R.sup.8a form a chemical bond; optionally, one or more of the pair(s) R.sup.1/R.sup.1a, R.sup.2/R.sup.2a, R.sup.4/R.sup.4a, R.sup.5/R.sup.5a, R.sup.7/R.sup.7a, R.sup.8/R.sup.8a are joined together with the atom to which they are attached to form a C.sub.3-7 cycloalkyl or a 4-membered to 7-membered heterocyclyl; optionally, one or more of the pair(s) R.sup.1/R.sup.4, R.sup.1/R.sup.5, R.sup.1/R.sup.6, R.sup.4/R.sup.5, R.sup.7/R.sup.8, R.sup.2/R.sup.3 are joined together with the atoms to which they are attached to form a ring A; optionally, R.sup.3/R.sup.3a are joined together with the nitrogen atom to which they are attached to form a 4-membered to 7-membered heterocycle; A is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 4-membered to 7-membered heterocyclyl, and 8-membered to 11-membered heterobicyclyl; and optionally, the moiety of formula (F-i) is further substituted, provided that the hydrogel marked with the asterisk in formula (F-i) is not replaced by a substituent, and that R.sup.3 and R.sup.3a are independently of each other H or are connected to N through an SP.sup.3-hybridized carbon atom.

10. The method of claim 9; wherein X.sup.1 is C.

11. The method of claim 9; wherein the VEGF neutralizing prodrug comprises a moiety of formula (f-ii): ##STR00149## wherein: the dashed line indicates attachment to the carrier; R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, X.sup.2, and D are used as defined in claim 9; R.sup.10 is selected from H and C.sub.1-6 alkyl; and SP.sup.9 is a further spacer moiety, which together with the moiety NR.sup.10C(O) forms the optional spacer moiety; and wherein the moiety of formula (F-ii) is optionally further substituted, provided that the hydrogel marked with the asterisk in formula (F-ii) is not replaced by a substituent and that R.sup.3 and R.sup.3a are independently of each other H or are connected to N through an SP.sup.3-hybridized carbon atom.

12. The method of claim 9; wherein R.sup.1 and R.sup.1a are both H.

13. The method of claim 9; wherein the VEGF neutralizing prodrug comprises a moiety of formula (F-iiia) or (F-iiib): ##STR00150## ##STR00151## wherein: the dashed line indicates attachment to the carrier; R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, R.sup.7, R.sup.7a, R.sup.8, R.sup.8a, X.sup.2, and D are used as defined in claim 9; and R.sup.10 is selected from H and C.sub.1-6 alkyl; and SP.sup.9 is a further spacer moiety, which together with the moiety NR.sup.10C(O) forms the optional spacer moiety; and wherein the moiety of formula (F-iiia) or (F-iiib) is optionally further substituted, provided that the hydrogen marked with the asterisk in formula (F-iiia) and (F-iiib) is not replaced by a substituent, and that R.sup.3 and R.sup.3a are independently of each other H or are connected to N through an SP.sup.3-hybridized carbon atom.

14. The method of claim 9; wherein the VEGF neutralizing prodrug comprises a moiety of formula (F-iva) or (F-ivb): ##STR00152## ##STR00153## wherein: the dashed line indicates attachment to the carrier; R.sup.3 and R.sup.3a are used as defined in claim 9; R.sup.10b is C.sub.1-6 alkyl; and SP.sup.0 is a further spacer moiety, which together with the moiety NHC(O) or NR.sup.10C(O) forms the optional spacer moiety; and wherein the moiety of formula (F-iva) or (F-ivb) is optionally further substituted, provided that the hydrogen marked with the asterisk is not replaced by a substituent and that R.sup.3 and R.sup.3a are independently of each other H or are connected to N through an SP.sup.3-hybridized carbon atom.

15. The method of claim 9; wherein: R.sup.3 is H; and R.sup.3a is methyl.

16. The method of claim 9; wherein R.sup.3 and R.sup.3a of are both H.

17. The method of claim 1; wherein the ocular condition is a disease characterized by ocular neovascularization.

18. The method of claim 17; wherein the disease characterized by ocular neovascularization is an ocular disease selected from the group consisting of optic disc neovascularization, iris neovascularization, retinal neovascularization, choroidal neovascularization, corneal neovascularization, vitreal neovascularization, glaucoma, pannus, pterygium, macular edema, macular degeneration, age-related macular degeneration, diabetic retinopathy, diabetic retinal ischemia, diabetic macular edema, vascular retinopathy, retinal degeneration, retrolental fibroplasias, retinoblastoma, retinopathy of prematurity of macular degeneration, corneal graft neovascularization, central retinal vein occlusion, pathological myopia, ocular tumors, uveitis, inflammatory diseases of the eye, and proliferative vitreoretinopathy.

19. The method of claim 1; wherein the pharmaceutical composition further comprises at least one drug in its free form selected from the group consisting of: antisense RNA, antisense DNA, ribozymes, and RNAi molecules targeting a VEGF nucleic acid; anti-VEGF aptamers, anti-VEGF antibodies, anti-VEGF antibody fragments, DARPins, anticalins, lipocalins, and soluble VEGF receptor decoys that prevent binding of a VEGF to its cognate receptor; antisense, ribozymes, and RNAi molecules targeting a cognate VEGF receptor (VEGFR) nucleic acid; anti-VEGFR aptamers and anti-VEGFR antibodies that bind to a cognate VEGFR receptor; anti-VEGFR antibody fragments that bind to a cognate VEGFR receptor; and VEGFR tyrosine kinase inhibitors.

20. The method of claim 1; wherein the pharmaceutical composition further comprises one or more additional prodrug(s), which one or more additional prodrug(s) comprise(s) in bound form at least one biologically active moiety selected from the group consisting of: antisense RNA, antisense DNA, ribozymes, and RNAi molecules targeting a VEGF nucleic acid; anti-VEGF aptamers, anti-VEGF antibodies, anti-VEGF antibody fragments, DARPins, anticalins, lipocalins, and soluble VEGF receptor decoys that prevent binding of a VEGF to its cognate receptor; antisense, ribozymes, and RNAi molecules targeting a cognate VEGF receptor (VEGFR) nucleic acid; anti-VEGFR aptamers and anti-VEGFR antibodies that bind to a cognate VEGFR receptor; anti-VEGFR antibody fragments that bind to a cognate VEGFR receptor; and VEGFR tyrosine kinase inhibitors.

21. The method of claim 1; wherein the carrier of the carrier-linked prodrug is a hyaluronic-acid-based hydrogel.

Description

EXAMPLES

(1) Materials and Methods

(2) Lucentis and Ranibizumab are used synonymously throughout the following examples.

(3) Materials:

(4) Amino 4-arm PEG5000 was obtained from JenKem Technology, Beijing, P. R. China. Cithrol DPHS was obtained from Croda International Pic, Cowick Hall, United Kingdom.

(5) cis-1,4-cyclohexanedicaboxylic acid was obtained from TCI EUROPE N.V., Boerenveldseweg 6-Haven 1063, 2070 Zwijndrecht, Belgium.

(6) Isopropylmalonic acid was obtained from ABCR GmbH & Co. KG, 76187 Karlsruhe, Germany.

(7) N-(3-maleimidopropyl)-39-amino-4,7,10,13,16,19,22,25,28,31,34,37-dodecaoxa-nonatriacontanoic acid pentafluorophenyl ester (Mal-PEG12-PFE) was obtained from Biomatrik Inc., Jiaxing, P. R. China.

(8) Oxyma pure, HATU, HOAt, HOBt, PyBOP, TBTU, COMU, Fmoc-L-Asp(OBzl)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-His(OTrt)-OH, Fmoc-Ado-OH and Rink amide resin were purchased from Merck Biosciences GmbH, Schwalbach/Ts, Germany.

(9) Boc-Lys(Boc)-OSu was purchased from Senn chemicals AG, Dielsdorf, Switzerland. Fmoc-N-Me-L-Asp(OtBu)-OH was purchased from Bachem, Bubendorf, Switzerland. Fmoc-N-Me-L-Asp(OBzl)-OH was purchased from Peptides International, Louisville, Ky., USA. 1,9-bis-Boc-1,5,9-triazanonan was purchased from PolyPeptide Laboratories A/S, Hillerd, Denmark.

(10) (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl 4-nitrophenyl carbonate was purchased from Chemzon Scientific Inc., Lachine, QC, Canada.

(11) -[3-(o-pyridyldisulfido)propanoylamido]--succinimidyl ester dodeca(ethylene glycol) (OPSS-PEG.sub.12-NHS) was purchased from Iris Biotech GmbH, Marktredwitz, Germany.

(12) All other chemicals were from Sigma-ALDRICH Chemie GmbH, Taufkirchen, Germany.

(13) Methods:

(14) Reactions were performed with dry solvents (DCM, THF, ACN, DMF, dioxane, MeOH, toluene) stored over molecular sieve purchased from Sigma-ALDRICH Chemie GmbH, Taufkirchen, Germany. Generally, reactions were stirred at room temperature and monitored by HPLC/MS or TLC.

(15) RP-HPLC was done on a 10020 mm or 10040 mm C18 ReproSil-Pur 300 ODS-3 5 column (Dr. Maisch, Ammerbuch, Germany) or XBridge BEH300 C18 OBD Prep 10 m 30150 mm or 5 m 10150 mm (Waters, Eschborn, Germany) connected to a Waters 600 or 2535 HPLC System and Waters 2487 or 2489 Absorbance detector, respectively. Linear gradients of solution A (0.1% TFA in H.sub.2O) and solution B (0.1% TFA in acetonitrile) were used. HPLC fractions containing product were combined and lyophilized.

(16) Flash chromatography purifications were performed on an Isolera One system from Biotage AB, Sweden, using Biotage KP-Sil silica cartridges and n-heptane, ethyl acetate, and methanol as eluents. Products were detected at 254 nm. For products showing no absorbance above 240 nm fractions were screened by LC/MS.

(17) Analytical ultra-performance LC (UPLC) was performed on a Waters Acquity system equipped with a Waters BEH300 C18 column (2.150 mm, 1.7 m particle size) coupled to a LTQ Orbitrap Discovery mass spectrometer from Thermo Scientific.

(18) HPLC-Electrospray ionization mass spectrometry (HPLC-ESI-MS) was performed on a Waters Acquity UPLC with an Acquity PDA detector coupled to a Thermo LTQ Orbitrap Discovery high resolution/high accuracy mass spectrometer or Waters Micromass ZQ both equipped with a Waters ACQUITY UPLC BEH300 C18 RP column (2.150 mm, 300 , 1.7 m, flow: 0.25 mL/min; solvent A: UP-H.sub.2O+0.04% TFA, solvent B: UP-Acetonitrile+0.05% TFA.

(19) MS spectra of PEG products showed a series of (CH.sub.2CH.sub.2O) moieties due to polydispersity of PEG staring materials. For easier interpretation only one single representative m/z signal is given in the examples.

(20) Buffer exchange was performed on a HiTrap or HiPrep column (GE Healthcare) connected to an Aekta Purifier 100 system.

(21) Cationic ion exchange chromatography was performed either on a Source 15 S 6 mL column connected to an Aekta Purifier 100 system using 20 mM MES, pH 5.7 and 20 mM MES, 500 mM NaCl, pH 5.7 as mobile phase A and B, respectively.

Example 1

(22) Synthesis of Backbone Reagent 1a and 1g

(23) ##STR00104##

(24) Backbone reagent 1a was synthesized as described in example 1 of WO 2011/012715 A1 except for the use of Boc-DLys(Boc)-OH instead of Boc-LLys(Boc)-OH.

(25) MS: m/z 888.50=[M+10H.sup.+].sup.10+ (calculated=888.54)

(26) ##STR00105##

(27) Backbone reagent 1g was synthesized from amino 4-arm PEG5000 1b according to the following scheme:

(28) ##STR00106##

(29) For synthesis of compound 1b, amino 4-arm PEG5000 (MW ca. 5350 g/mol, 10.7 g, 2.00 mmol, HCl salt) and bis(pentafluorophenyl)carbonate (4.73 g, 12.0 mmol) were dissolved in 43 mL of DCM (anhydrous) and DIPEA (3.10 g, 24.0 mmol, 4.18 mL) was added at room temperature. After 10 min, 1,9-bis-boc-1,5,9-triazanonane (5.30 g, 16.0 mmol) was added and the mixture was stirred for 15 min. Then additional 1,9-bis-boc-1,5,9-triazanonane (0.33 g, 1.0 mmol) was added. After complete dissolution, the reaction mixture was filtered and the solvent was evaporated at room temperature.

(30) The residue was dissolved in 40 mL iPrOH and diluted with 320 mL MTBE. The product was precipitated over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 200 mL of cooled MTBE (0 C.). The product was dried in vacuo over night.

(31) Yield 11.1 g (83%) white solid 1b.

(32) MS: m/z 1112.86=[M+6H].sup.6+ (calculated=1113.04).

(33) For synthesis of compound 1c, the boc-protected compound 1b (11.1 g, 1.66 mmol) was dissolved in 40 mL of 3 M HCl in MeOH and stirred for 20 min at 45 C., then for 10 min at 55 C. For precipitation, 10 mL MeOH and 200 mL of MTBE were added and the mixture was stored for 16 h at 20 C. The precipitate was collected by filtration through a glass filter Por. 3 and washed with 200 mL of cooled MTBE (0 C.). The product was dried in vacuo over night.

(34) Yield 9.14 g (89%) white powder 1c (HCl salt).

(35) MS: m/z 979.45=[M+6H].sup.6+ (calculated=979.55).

(36) For synthesis of compound 1d, compound 1c (9.06 g, 1.47 mmol, HCl salt) and bis(pentafluorophenyl)carbonate (6.95 g, 17.6 mmol) were dissolved in 50 mL of DCM (anhydrous) and DIPEA (4.56 g, 35.3 mmol, 6.15 mL) was added at room temperature. After 10 min, 1,9-bis-boc-1,5,9-triazanonane (7.80 g, 23.5 mmol) was added and the mixture was stirred for 15 min. Then additional 1,9-bis-boc-1,5,9-triazanonane (0.49 g, 1.5 mmol) was added. After complete dissolution, the solvent was evaporated at room temperature.

(37) The residue was dissolved in 35 mL iPrOH at 40 C. and diluted with 200 mL MTBE. The product was precipitated over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 200 mL of cooled MTBE (0 C.). The product was dried in vacuo over night to give 1d as a white solid.

(38) Yield 11.6 g (90%) white solid 1d.

(39) MS: m/z 1248.08=[M+7H].sup.7+ (calculated=1248.27).

(40) For synthesis of compound 1e, the boc-protected compound 1d (11.4 g, 1.31 mmol) was dissolved in 40 mL of 3 M HCl in MeOH and stirred for 20 min at 45 C., then for 10 min at 55 C. For precipitation, 10 mL MeOH and 200 mL of MTBE were added and the mixture was stored for 16 h at 20 C. The precipitate was collected by filtration through a glass filter Por. 3 and washed with 200 mL of cooled MTBE (0 C.). The product was dried in vacuo over night to give white powder 1e.

(41) Yield 7.60 g (75%) white powder 1e (HCl salt).

(42) MS: m/z 891.96=[M+8H].sup.8+ (calculated=892.13).

(43) For synthesis of compound 1f, compound 1e (7.56 g, 0.980 mmol, HCl salt) and bis(pentafluorophenyl)carbonate (9.27 g, 23.0 mmol) were dissolved in 250 mL of DCM (anhydrous) and DIPEA (6.08 g, 47.0 mmol, 8.19 mL) was added at 35 C. After 10 min, 1,9-bis-boc-1,5,9-triazanonane (5.30 g, 16.0 mmol) was added and the mixture was stirred for 15 min. Then additional 1,9-bis-boc-1,5,9-triazanonane (0.33 g, 1.0 mmol) was added. After complete dissolution, the solvent was evaporated at room temperature.

(44) The residue was dissolved in 250 mL iPrOH at 60 C. and diluted with 1350 mL MTBE. The product was precipitated over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 400 mL of cooled MTBE (0 C.). The product was dried in vacuo over night to give 1f as a glassy solid.

(45) Yield 11.1 g (83%) glassy solid 1f.

(46) MS: m/z 1312.01=[M+10H].sup.10+ (calculated=1312.21).

(47) For synthesis of backbone reagent 1g, the boc-protected compound 1f (7.84 g, 0.610 mmol) was dissolved in 16 mL of MeOH at 37 C. and 55 mL of a precooled solution of 4 M HCl (4 C.) in dioxane was added at room temperature. The mixture was stirred without cooling for 20 min. After 20 min 110 mL of 3M HCl in MeOH was added. The solution was partitioned in 24 Falcon tubes (50 mL) and precipitated by adding 40 mL cold MTBE (20 C.) to each Falcon tube. After centrifugation at 3214 rcf for 1 min, the supernatant was decanted and the glassy solid was dissolved in 5 mL MeOH per Falcon tube and precipitated by adding 40 mL cold MTBE (20 C.) to each Falcon tube again. The supernatant was discarded and the remaining solid was dried in vacuo over night.

(48) Yield 5.74 g (87%) white glassy solid 1g (HCl salt).

(49) MS: m/z 965.46=[M+10H].sup.10+ (calculated=965.45).

Example 2

(50) Synthesis of Crosslinker Reagents 2d, 2g, 2k, and 2o

(51) Crosslinker reagent 2e was prepared from azelaic acid monobenzyl ester and PEG10000 according to the following scheme:

(52) ##STR00107##

(53) For the synthesis of azelaic acid monobenzyl ester 2a, a mixture of azelaic acid (37.6 g, 200 mmol), benzyl alcohol (21.6 g, 200 mmol), p-toluenesulfonic acid (0.80 g, 4.2 mmol), and 240 mL toluene was refluxed for 7 h in a Dean-Stark apparatus. After cooling down, the solvent was evaporated and 300 mL sat. aqueous NaHCO.sub.3 solution were added. This mixture was extracted with 3200 mL MTBE. The combined organic phases were dried over Na.sub.2SO.sub.4 and the solvent was evaporated. The product was purified on 2340 g silica using ethyl acetate/heptane (10:90.fwdarw.25:75) as eluent. The eluent was evaporated and the residue was dried in vacuo over night.

(54) Yield 25.8 g (46%) colorless oil 2a.

(55) MS: m/z 279.16=[M+H].sup.+ (calculated=279.16).

(56) For synthesis of compound 2b, azelaic acid monobenzyl ester 2a (3.90 g, 14.0 mmol) and PEG 10000 (40.0 g, 4.00 mmol) were dissolved in 64 mL dichloromethane and cooled with an ice bath. A solution of DCC (2.89 g, 14.0 mmol) and DMAP (0.024 g, 0.020 mmol) in 32 mL dichloromethane was added. The ice bath was removed and mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 C. and the solid was filtered off. The solvent was evaporated in vacuo.

(57) The residue was dissolved in 65 mL dichloromethane and diluted with 308 mL MTBE at room temperature. The mixture was stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 250 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(58) Yield 40.8 g (97%) white powder 2b.

(59) MS: m/z 835.50=[M+14H].sup.14+ (calculated=835.56).

(60) For synthesis of compound 2c, compound 2b (40.6 g, 3.86 mmol) was dissolved in methyl acetate (250 mL) and 203 mg of palladium on charcoal was added. Under a hydrogen atmosphere of ambient pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo over night.

(61) Yield 37.2 g (93%) glassy solid 2c.

(62) MS: m/z 882.53=[M+13H].sup.13+ (calculated=882.51).

(63) For synthesis of compound 2d, compound 2c (32.0 g, 3.10 mmol) and TSTU (3.73 g, 12.4 mmol) were dissolved in 150 mL dichloromethane at room temperature. Then DIPEA (1.60 g, 12.4 mmol) was added and the mixture was stirred for 1 h. The resulting suspension was filtered and the filtrate was diluted with 170 mL dichloromethane, washed with 140 mL of a solution of 750 g water/197 g NaCl/3 g NaOH. The organic phase was dried over MgSO.sub.4 and the solvent was evaporated in vacuo.

(64) The residue was dissolved in 200 mL toluene, diluted with 180 mL MTBE at room temperature and stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 100 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(65) Yield 28.8 g (88%) white powder 2d.

(66) MS: m/z 795.47=[M+15H].sup.15+ (calculated=795.54).

(67) Crosslinker reagent 2g was prepared from azelaic acid monobenzyl ester and PEG6000 according to the following scheme:

(68) ##STR00108##

(69) For synthesis of compound 2e, azelaic acid monobenzyl ester 2a (6.50 g, 23.3 mmol) and PEG 6000 (40.0 g, 6.67 mmol) were dissolved in 140 mL dichloromethane and cooled with an ice bath. A solution of DCC (4.81 g, 23.3 mmol) and DMAP (0.040 g, 0.33 mmol) in 40 mL dichloromethane was added. The ice bath was removed and mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 C. and the solid was filtered off. The solvent was evaporated in vacuo.

(70) The residue was dissolved in 70 mL dichloromethane and diluted with 300 mL MTBE at room temperature. The mixture was stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 500 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(71) Yield 41.2 g (95%) white powder 2e.

(72) MS: m/z 833.75=[M+8H].sup.8+ (calculated=833.74).

(73) For synthesis of compound 2f, compound 2e (41.2 g, 6.32 mmol) was dissolved in methyl acetate (238 mL) and ethanol (40 mL), then 400 mg of palladium on charcoal was added. Under a hydrogen atmosphere of ambient pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo over night.

(74) Yield 38.4 g (96%) glassy solid 2f.

(75) MS: m/z 750.46=[M+9H].sup.9+ (calculated=750.56).

(76) For synthesis of compound 2g, compound 2f (38.2 g, 6.02 mmol) and TSTU (7.25 g, mmol) were dissolved in 130 mL dichloromethane at room temperature. Then DIPEA (3.11 g, 24.1 mmol) was added and the mixture was stirred for 1 h. The resulting suspension was filtered, the filtrate was diluted with 100 mL dichloromethane and washed with 200 mL of a solution of 750 g water/197 g NaCl/3 g NaOH. The organic phase was dried over MgSO.sub.4 and the solvent was evaporated in vacuo.

(77) The residue was dissolved in 210 mL toluene, diluted with 430 mL MTBE at room temperature and stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 450 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(78) Yield 35.8 g (91%) white powder 2g.

(79) MS: m/z 857.51=[M+8H].sup.8+ (calculated=857.51).

(80) Crosslinker reagent 2k was prepared from isopropylmalonic acid monobenzyl ester and PEG10000 according to the following scheme:

(81) ##STR00109##

(82) For the synthesis of isopropylmalonic acid monobenzyl ester rac-2h, isopropylmalonic acid (35.0 g, 239 mmol), benzyl alcohol (23.3 g, 216 mmol) and DMAP (1.46 g, 12.0 mmol) were dissolved in 100 mL acetonitrile. Mixture was cooled to 0 C. with an ice bath. A solution of DCC (49.4 g, 239 mmol) in 150 mL acetonitrile was added within 15 min at 0 C. The ice bath was removed and the reaction mixture was stirred over night at room temperature, then the solid was filtered off. The filtrate was evaporated at 40 C. in vacuo and the residue was dissolved in 300 mL MTBE. This solution was extracted with 2300 mL sat. aqueous NaHCO.sub.3 solution, then the combined aqueous phases were acidified to pH=1-3 using 6 N hydrochloric acid. The resulting emulsion was extracted with 2300 mL MTBE and the solvent was evaporated. The combined organic phases were washed with 200 mL sat. aqueous NaCl and dried over MgSO.sub.4. The product was purified on 340 g silica using ethyl acetate/heptane (10:90.fwdarw.20:80) as eluent. The eluent was evaporated and the residue was dried in vacuo over night.

(83) Yield 9.62 g (17%) colorless oil rac-2h.

(84) MS: m/z 237.11=[M+H].sup.+ (calculated=237.11).

(85) For synthesis of compound 2i, isopropylmalonic acid monobenzyl ester rac-2h (945 mg, 4.00 mmol) and PEG 10000 (10.0 g, 4.00 mmol) were dissolved in 20 mL dichloromethane and cooled with an ice bath. A solution of DCC (825 mg, 4.00 mmol) and DMAP (6 mg, 0.05 mmol) in 10 mL dichloromethane was added. The ice bath was removed and mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 C. and the solid was filtered off. The solvent was evaporated in vacuo.

(86) The residue was dissolved in 20 mL dichloromethane and diluted with 150 mL MTBE at room temperature. The mixture was stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 500 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(87) Yield 9.63 g (92%) white powder 2i.

(88) MS: m/z 742.50=[M+16H].sup.16+ (calculated=742.51).

(89) For synthesis of compound 2j, compound 2i (3.38 g, 0.323 mmol) was dissolved in methyl acetate (100 mL) and 105 mg of palladium on charcoal was added. Under a hydrogen atmosphere of ambient pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo over night.

(90) Yield 3.25 g (98%) glassy solid 2j.

(91) MS: m/z 731.25=[M+16H].sup.16+ (calculated=731.25).

(92) For synthesis of compound 2k, compound 2j (3.10 g, 0.302 mmol) and TSTU (0.364 g, 1.21 mmol) were dissolved in 15 mL dichloromethane at room temperature. Then DIPEA (0.156 g, 1.21 mmol) was added and the mixture was stirred for 45 min. The resulting suspension was filtered and the filtrate was washed with 210 mL of a 0.5 M phosphate buffer pH=6.5. The organic phase was dried over MgSO.sub.4 and the solvent was evaporated in vacuo. The residue was dissolved in 20 mL toluene, diluted with 10 mL MTBE at room temperature and stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 250 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(93) Yield 2.66 g (84%) white powder 2k.

(94) MS: m/z 743.37=[M+16H].sup.16+ (calculated=743.38).

(95) Crosslinker reagent rac-2o was prepared from cis-1,4-cyclohexanedicarboxylic acid and PEG10000 according to the following scheme:

(96) ##STR00110##

(97) For the synthesis of cis-1,4-cyclohexanedicarboxylic acid monobenzyl ester rac-21, cis-1,4-cyclohexanedicarboxylic acid (20.0 g, 116 mmol), benzyl alcohol (11.3 g, 105 mmol) and DMAP (710 mg, 5.81 mmol) were dissolved in 200 mL THF. Mixture was cooled to 0 C. with an ice bath. A solution of DCC (49.4 g, 239 mmol) in 100 mL THF was added within 15 min at 0 C. The ice bath was removed and the reaction mixture was stirred over night at room temperature, then the solid was filtered off. The filtrate was evaporated at 40 C. and the residue was dissolved in 300 mL MTBE. This solution was extracted with 2300 mL sat. aqueous NaHCO.sub.3 solution, then the combined aqueous phases were acidified to pH=1-3 using 6 N hydrochloric acid. The resulting emulsion was extracted with 2300 mL MTBE and the solvent was evaporated. The combined organic phases were washed with 200 mL sat. aqueous NaCl and dried over MgSO.sub.4. The product was purified on 340 g silica using ethyl acetate/heptane (10:90.fwdarw.20:80) as eluent. The eluent was evaporated and the colorless oily residue crystallized during drying in vacuo over night.

(98) Yield 4.82 g (16%) colorless crystals rac-21.

(99) MS: m/z 263.13=[M+H].sup.+ (calculated=263.13).

(100) For synthesis of compound 2m, cis-1,4-cyclohexanedicarboxylic acid monobenzyl ester rac-21 (2.10 g, 8.00 mmol) and PEG 10000 (20.0 g, 10.0 mmol) were dissolved in 50 mL dichloromethane and cooled with an ice bath. A solution of DCC (1.65 g, 8.00 mmol) and DMAP (0.012 g, 0.10 mmol) in 25 mL dichloromethane was added. The ice bath was removed and mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 C. and the solid was filtered off. The solvent was evaporated in vacuo.

(101) The residue was dissolved in 55 mL dichloromethane and diluted with 300 mL MTBE at room temperature. The mixture was stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 250 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(102) Yield 18.2 g (87%) white powder 2m.

(103) MS: m/z 745.76=[M+16H].sup.16+ (calculated=745.77).

(104) For synthesis of compound 2n, compound 2m (9.00 g, 0.857 mmol) was dissolved in methyl acetate (100 mL) and 157 mg of palladium on charcoal was added. Under a hydrogen atmosphere of ambient pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo over night.

(105) Yield 8.83 g (100%) glassy solid 2n.

(106) MS: m/z 734.50=[M+16H].sup.16+ (calculated=734.50).

(107) For synthesis of compound 2o, compound 2n (8.92 g, 0.864 mmol) and TSTU (1.04 g, 3.64 mmol) were dissolved in 35 mL dichloromethane at room temperature. Then DIPEA (0.447 g, 3.46 mmol) was added and the mixture was stirred for 45 min. The resulting suspension was filtered and the filtrate was washed with 210 mL of a 0.5 M phosphate buffer pH=6.5. The organic phase was dried over MgSO.sub.4 and the solvent was evaporated in vacuo.

(108) The residue was dissolved in 50 mL toluene, diluted with 25 mL MTBE at room temperature and stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 400 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(109) Yield 7.62 g (84%) white powder 2o.

(110) MS: m/z 702.60=[M+16H].sup.16+ (calculated=702.59).

Example 3

(111) Preparation of Hydrogel Beads 3a, 3b, 3c, and 3d Containing Free Amino Groups

(112) In a cylindrical 250 mL reactor with bottom outlet, diameter 60 mm, equipped with baffles, an emulsion of 218 mg Cithrol DPHS in 100 mL undecane was stirred with an isojet stirrer, diameter 50 mm at 580 rpm, at ambient temperature. A solution of 250 mg 1a and 2205 mg 2d in 22.1 g DMSO was added and stirred for 10 min at RT to form a suspension. 1.1 mL TMEDA were added to effect polymerization. The mixture was stirred for 16 h. 1.7 mL of acetic acid were added and then after 10 min 100 mL of a 15 wt % solution of sodium chloride in water was added. After 10 min, the stirrer was stopped and phases were allowed to separate. After 2 h the aqueous phase containing the hydrogel was drained.

(113) For bead size fractionation, the water-hydrogel suspension was diluted with 40 mL ethanol and wet-sieved on 125, 100, 75, 63, 50, 40, and 32 m steel sieves using a Retsch AS200 control sieving machine for 15 min. Sieving amplitude was 1.5 mm, water flow 300 mL/min. Bead fractions that were retained on the 63 and 75 m sieves were pooled and washed 3 times with 0.1% AcOH, 10 times with ethanol and dried for 16 h at 0.1 mbar to give 670 mg of 3a as a white powder.

(114) Amino group content of the hydrogel was determined to be 0.145 mmol/g by conjugation of a Fmoc-amino acid to the free amino groups on the hydrogel and subsequent Fmoc-determination.

(115) 3b was prepared as described for 3a except for the use of 350 mg 1a, 2548 mg 2g, 26.1 g DMSO, 257 mg Cithrol DPHS, 1.5 mL TMEDA, and 2.4 mL acetic acid, yielding 550 mg 3b as a white powder, free amino groups 0.120 mmol/g.

(116) 3c was prepared as described for 3a except for the use of 250 mg 1a, 3019 mg rac-2k, 32.7 g DMSO, 290 mg Cithrol DPHS, 1.1 mL ml TMEDA, and 1.7 mL acetic acid, yielding 770 mg 3c as a white powder, free amino groups 0.126 mmol/g.

(117) 3d was prepared as described for 3a except for the use of 250 mg 1a, 2258 mg rac-2o, 22.6 g DMSO, 222 mg Cithrol DPHS, 1.1 mL ml TMEDA, and 1.7 mL acetic acid, yielding 186 mg 3d as a white powder, free amino groups 0.153 mmol/g.

Example 4

(118) Synthesis of Linker Reagent 4c

(119) Linker reagent 4c was synthesized according to the following scheme:

(120) ##STR00111##
Synthesis of 4a:

(121) Fmoc-L-Asp(OtBu)-OH (1.00 g, 2.43 mmol) was dissolved with DCC (0.70 g, 3.33 mmol) in DCM (25 mL). Oxyma pure (0.51 g, 3.58 mmol) and collidine (0.50 mL, 3.58 mmol) were added in one portion and a solution of N-Boc-ethylenediamine (0.41 g, 2.56 mmol) in DCM (15 mL) was added slowly. After stirring the mixture for 90 min at RT the formed precipitate was filtered off and the filtrate washed with aqueous HCl (0.1 M, 50 mL). The aqueous layer was extracted with DCM (220 mL) and the combined organic fractions were washed with sat. aqueous NaHCO.sub.3 (325 mL) and brine (150 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude solid was purified by flash chromatography. The intermediate N-boc-N(N-fmoc-4-tert.-butyl-L-aspartoyl)-ethylenediamine was obtained as white solid (0.98 g, 1.77 mmol, 73%).

(122) MS: m/z 554.29=[M+H].sup.+, (calculated=554.29).

(123) N-boc-N(N-fmoc-4-tert.-butyl-L-aspartoyl)-ethylenediamine (0.98 g, 1.77 mmol) was dissolved in THF (15 mL), DBU (0.31 mL) was added and the solution was stirred for 12 min at RT. The reaction was quenched with AcOH (0.5 ml), concentrated in vacuo and the residue purified by flash chromatography to give 4a (0.61 g, 1.77 mmol, 73% over 2 steps) as white solid.

(124) MS: m/z 332.38=[M+H].sup.+, (calculated=332.22).

(125) Synthesis of 4b:

(126) 6-Acetylthiohexanoic acid (0.37 g, 1.95 mmol) was dissolved in DCM (19.5 mL) and Oxyma pure (0.35 g, 2.48 mmol) and DCC (0.40 g, 1.95 mmol) added in one portion. The solution was stirred for 30 min at RT, filtered, and the filtrate added to a solution of 4a (0.61 g, 1.77 mmol) in DCM (10.5 mL). DIPEA (0.46 mL, 2.66 mmol) was added to the solution and the reaction stirred for 2 h at RT. The solution was washed with aqueous H.sub.2SO.sub.4 (0.1 M, 230 mL), sat. aqueous NaHCO.sub.3 (220 mL) and brine (120 mL). The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude material was purified by flash chromatography to give N-boc-N(N-6-acetylthiohexyl-4-tert.-butyl-L-aspartoyl)-ethylenediamine (0.65 g, 1.30 mmol, 73% over 2 steps) as white solid.

(127) MS: m/z 504.27=[M+H].sup.+, (calculated=504.28).

(128) N-boc-N(N-6-Acetylthiohexyl-4-tert.-butyl-L-aspartoyl)-ethylenediamine (0.60 g, 1.18 mmol) was dissolved in TFA (5 mL) and TES (0.13 mL) and water (0.13 ml) were added. The mixture was stirred for 30 min at RT. TFA was removed in a stream of N.sub.2, and crude 4b dissolved in H.sub.2O/ACN 1:1 and purified by RP-HPLC.

(129) Yield: 0.39 g, 0.85 mmol (TFA salt), 72%.

(130) MS: m/z 348.25=[M+H].sup.+, (calculated=348.16).

(131) Synthesis of 4c:

(132) 4b (TFA salt, 0.38 g, 0.80 mmol) was dissolved in DMF (5 mL) and (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl 4-nitrophenyl carbonate (0.26 g, 0.88 mmol) and DIPEA (0.28 mL, 1.6 mmol) were added. The resulting suspension was diluted with DCM (5 mL) and stirred for 3 h at RT. More DIPEA (0.28 mL 1.6 mmol) was added and stirring continued for 2 h. DCM was concentrated in vacuo, the residue diluted with H.sub.2O/ACN 3:1 and purified by RP-HPLC to give N-(5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl-oxo carbonyl-N(N-6-acetylthio hexyl-L-aspartyl)-ethylenediamine (0.31 g, 0.62 mmol, 77%) as colorless oil.

(133) MS: m/z 504.16=[M+H].sup.+, (calculated=504.17).

(134) N-(5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl oxocarbonyl-N(N-6-acetylthiohexyl-L-aspartyl)-ethylenediamine (150 mg, 0.30 mmol) was dissolved in DCM (17.5 mL) and NHS (41 mg, 0.36 mmol), DCC (74 mg, 0.36 mmol) and DMAP (4 mg, 0.03 mmol) were added in one portion. The reaction was stirred for 1 h at RT and the resulting suspension filtered. The precipitate was washed with a small amount of DCM and the combined filtrates concentrated in vacuo. 4c was purified by RP-HPLC to give a colorless oil (144 mg, 0.24 mmol, 80%).

(135) MS: m/z 601.18=[M+H].sup.+, (calculated=601.18).

Example 5

(136) Preparation of Maleimide Functionalized Hydrogel Beads 5a

(137) 259.3 mg of dry hydrogel beads 3a was incubated for 15 min in 10 mL 1% n-propylamine in NMP and subsequently washed two times with 1% n-propylamine in NMP and two times with 2% DIPEA in NMP. 171 mg of maleimide-NH-PEG.sub.12-PFE was dissolved in 1 mL NMP and added to the washed hydrogel beads 3a. The hydrogel suspension was incubated for 2 h at room temperature. Resulting maleimide functionalized hydrogel beads 5a were washed five times each with NMP followed by 20 mM succinate, 1 mM Na.sub.2EDTA, 0.01% Tween20, pH 3.0, followed by water, followed by 0.1% acetic acid, 0.01% Tween20.

(138) Maleimide functionalized hydrogel beads 5b, 5c, and 5d were prepared accordingly using 3b, 3c, and 3d.

Example 6

(139) Synthesis of Transient Lucentis-Linker-Hydrogel Prodrug 6c

(140) 4.6 mg Lucentis (depicted in the scheme below as Lucentis-NH.sub.2) (460 L of 10 mg/mL Lucentis in 10 mM histidine, 10 wt % ,-trehalose, 0.01% Tween20, pH 5.5) was buffer exchanged to 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, pH 7.4 and the concentration of Lucentis was adjusted to 16.4 mg/mL. 6 mg of Linker reagent 4c was dissolved in 100 L DMSO to yield a concentration of 100 mM. 1 molar equivalent of linker reagent 4c relative to the amount of Lucentis was added to the Lucentis solution. The reaction mixture was mixed carefully and incubated for 5 min at room temperature. Subsequently, 2 additional molar equivalents of linker reagent 4c were added to the Lucentis solution in 1 molar equivalent steps and after addition of each equivalent the reaction mixture was incubated for 5 min at room temperature yielding a mixture of unmodified Lucentis and the protected Lucentis-linker monoconjugate 6a.

(141) The pH of the reaction mixture was adjusted to pH 6.5 by addition of 1 M sodium citrate, pH 5.0 and Na.sub.2EDTA was added to a final concentration of 5 mM. To remove the protecting groups of 6a 0.5 M NH.sub.2OH (dissolved in 10 mM sodium citrate, 140 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 6.5) was added to a final concentration of 45 mM and the deprotection reaction was incubated at room temperature for 4 h yielding the Lucentis-linker monoconjugate 6b. The mixture of Lucentis and Lucentis-linker monoconjugate 6b was buffer exchanged to 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, 5 mM Na.sub.2EDTA, 0.01% Tween 20, pH 6.5 and the overall concentration of the two Lucentis species was adjusted to 11.8 mg/mL. The content of Lucentis-linker monoconjugate 6b in the mixture was 20% as determined by ESI-MS.

(142) 4 mg of the Lucentis/Lucentis-linker monoconjugate 6b mixture in 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, 5 mM Na.sub.2EDTA, 0.01% Tween 20, pH 6.5 were added to 1 mg of maleimide functionalized hydrogel beads 5a and incubated overnight at room temperature yielding transient Lucentis-linker-hydrogel prodrug 6c.

(143) ##STR00112##

Example 7

(144) In Vitro Release Kinetics Determination of In Vitro Half-Life

(145) Lucentis-linker-hydrogel prodrug 6c (containing approximately 1 mg Lucentis) was washed five times with 60 mM sodium phosphate, 3 mM Na.sub.2EDTA, 0.01% Tween20, pH 7.4 and finally suspended in 1 mL of the aforementioned buffer. The suspension was incubated at 37 C. The buffer of the suspension was exchanged after different time intervals and analyzed by HPLC-SEC at 220 nm. Peaks corresponding to liberated Lucentis were integrated and the total of liberated Lucentis was plotted against total incubation time. Curve fitting software was applied to determine first-order cleavage rates.

Example 8

(146) Synthesis of Linker Reagent 8e

(147) Linker reagent 8e was synthesized according to the following scheme:

(148) ##STR00113##

(149) To a solution of N-Boc-ethylenediamine (2.08 g, 12.98 mmol) and NaCNBH.sub.3 (775 mg, 12.33 mmol) in MeOH (20 mL, anhydrous) a solution of 2,4,6-trimethoxybenzaldehyde (2.29 g, 11.68 mmol) in 40 mL anhydrous MeOH/DCM (1:1 v/v) was added over 2 h via syringe pump. The mixture was stirred for 90 min, acidified with 0.4 M HCl (60 mL) and stirred further 15 min. The reaction mixture was extracted with ethyl acetate (5). The combined organic phases were washed with saturated NaHCO.sub.3 and brine and dried over Na.sub.2SO.sub.4. Solvents were removed in vacuo and the residue was dried in high vacuum (<0.1 mbar). Crude N-Boc-N-Tmob-ethylenediamine 8a was used in the next reaction step without further purification. Yield: 3.70 g (10.87 mmol, 84%) of a colorless solid. MS: m/z 341.21=[M+H].sup.+, (calculated=341.21).

(150) A solution of 8a (1.7 g, 4.99 mmol) in DCM (40 ml, anhydrous, mol. sieve) was added to a solution of DCC (1.34 g, 6.50 mmol), Oxyma pure (995 mg, 7.00 mmol), Fmoc-L-Asp(OBn)-OH (2.22 g, 4.98 mmol) and 2,4,6-collidine (1.24 mL, 9.53 mmol) in DCM (40 ml, anhydrous, mol. sieve). The reaction mixture was stirred for 3 h at RT. The precipitate was filtered off and the filtrate was washed with 0.1 M HCl, sat. NaHCO.sub.3 and brine. Organic phase was dried over Na.sub.2SO.sub.4 and solvents were removed in vacuo. The crude material was purified by flash chromatography to give 8b (3.19 g, 4.15 mmol, 83%) as off white solid. MS: m/z 768.35=[M+H].sup.+, (calculated=768.35).

(151) To a solution of 8b (8.59 g, 11.19 mmol) in THF (98 mL) DBU (2 mL) was added. The solution was stirred for 12 min at RT, and the solvent was concentrated in vacuo. Flash chromatography afforded 4.89 g 8c (8.96 mmol, 80%). MS: m/z 546.28=[M+H].sup.+, (calculated=546.28).

(152) 6-Tritylmercaptohexanoic acid (2.04 g, 5.22 mmol) was dissolved in DCM (20 mL, anhydrous, mol. sieve) and DCC (1.08 g, 5.22 mmol) and Oxyma pure (945 mg, 6.65 mmol) were added. After 30 min, 8c (2.59 g, 4.75 mmol) and DIPEA (1.24 mL, 7.12 mmol) were added. The reaction mixture was stirred for 22 h at RT. The mixture was extracted with 1 N H.sub.2SO.sub.4 (2), sat. NaHCO.sub.3 (2) and brine. The organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuo and 8d was purified by flash chromatography. Yield: 4.10 g (4.47 mmol, 94%). MS: m/z 940.12=[M+Na].sup.+, (calculated=940.43).

(153) To a solution of 8d (4.10 g, 4.47 mmol) in i-PrOH (60 mL), water (20 mL) and LiOH (322 mg, 13.41 mmol) was added and the reaction mixture was stirred for 1 h at RT. Toluene (300 mL) was added and the organic phase was with 0.1 N HCl and with brine. The organic phase was dried over Na.sub.2SO.sub.4, filtrated and concentrated in vacuo. 8e was purified by flash chromatography. Yield: 3.53 g (4.26 mmol, 95%). MS: m/z 827.93=[M+H].sup.+, (calculated=828.39).

Example 9

(154) Synthesis of Linker Reagent 9c

(155) Linker reagent 9c was synthesized according to the following scheme:

(156) ##STR00114##

(157) 8e (300 mg, 0.36 mmol) was dissolved in HFIP/water/TES (39:1:1 v/v/v, 4.1 mL). Under stirring TFA (0.35 mL) was added and the reaction stirred for 75 min. The solvents were evaporated in a stream of argon. The residue was partitioned between water (20 mL) and DCM (40 mL). The water phase was collected, the DCM phase washed with water (5 mL) and both water phases combined. The pH of the resulting solution was adjusted with pH 7.4 sodium phosphate buffer (0.5 M, 5 mL) and a solution of 2,2-dithiodipyridine in 1 mL was added and the resulting suspension stirred for 30 min. The mixture was lyophilized and the residue suspended in ACN/water (7:3 v/v, 9 mL) and filtered. The filtrate was purified by RP-HPLC to give 9a. Yield: 90 mg, 0.17 mmol (TFA salt), 47%, MS: m/z 415.25=[M+H].sup.+, (calculated=415.15).

(158) 9a (90 mg, 0.17 mmol) was dissolved in DCM (1.2 mL) and (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl 4-nitrophenyl carbonate (138 mg, 0.47 mmol) and 2,4,6-collidine (129 L, 0.98 mmol) was added with stirring. DMF (1 mL) was added to facilitate dissolution of the formed precipitate. After 2 h and 8 h DIPEA (19 L, 0.11 mmol, each) was added and the reaction stirred for 48 h. The reaction was quenched with AcOH (38 L) and DCM evaporated in a stream of nitrogen. The residue was diluted with ACN/water/TFA (1:1:0.002 v/v/v) and purified by RP-HPLC to give 9b. Yield: 64 mg, 0.11 mmol, 65%, MS: m/z 571.09=[M+H].sup.+, (calculated=571.15).

(159) 9b (32 mg, 56 mol) was dissolved in DCM. With stirring NHS (8 mg, 67 mol), DCC (14 mg, 67 mol) and DMAP (0.7 mg, 6 mmol) were added. The reaction was stirred and after 1.5 h and 3.5 h DCC was added (3.5 mg, 11 mol and 2.3 mg, 7 mol, respectively). The solvent was evaporated in a stream of argon and the residue suspended in water/ACN/TFA (1:9:0.01 v/v/v, 3 mL) and filtered. The filtrate was purified by RP-HPLC to give 9c (28 mg, 42 mol, 75%). MS: m/z 668.17=[M+H].sup.+, (calculated=668.17).

Example 10

(160) Synthesis of Linker Reagent 10f

(161) Linker reagent 10f was synthesized according to the following scheme:

(162) ##STR00115## ##STR00116##

(163) Fmoc-N-Me-L-Asp(OtBu)-OH (1 g, 2.35 mmol) was dissolved in DCM (35 mL) and DCC (0.68 g, 3.29 mmol), Oxyma pure (0.5 g, 3.53 mmol) and 2,4,6-collidine (0.49 mL, 3.76 mmol) were added. N-Boc-N-methyl-ethylenediamine was dissolved in DCM (15 mL) and added slowly by syringe to the reaction mixture. The reaction was stirred for 16 h, the precipitate filtered off and the filtrate washed with 0.1 M HCl (50 mL). The water layer was reextracted twice with DCM (20 mL). The organic layers were combined and washed with sat. sodium bicarbonate solution (150 mL, 225 mL) and brine (150 mL). The organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuo and 10a was purified by flash chromatography. Yield: 1.36 g (2.33 mmol, 99%). MS: m/z 582.32=[M+H].sup.+, (calculated=582.32).

(164) 10a (1.36 g, 2.33 mmol) was dissolved in THF (20 mL) and DBU (0.4 mL) was added and the reaction stirred for 12 min. AcOH (0.5 mL) was added and the mixture concentrated in vacuo and 10b was purified by flash chromatography. Yield: 0.82 g (2.28 mmol, 98%). MS: m/z 360.25=[M+H].sup.+, (calculated=360.25).

(165) 6-Acetylmercaptohexanoic acid (0.49 g, 2.58 mmol) was dissolved in DCM (25 mL) and DCC (0.53 g, 2.58 mmol), Oxyma pure (0.47 g, 3.19 mmol) were added. The reaction was stirred for 45 min and filtered using a syringe with frit into a solution of 10b (0.82 g, 2.28 mmol) in DCM (12 mL). DIPEA (0.61 mL, 3.42 mmol) was added and the reaction stirred. Due to insufficient conversion Oxyma pure (0.2 g, 1.4 mmol) and DIPEA (0.25 mL, 1.4 mmol) were added and the reaction stirred for 16 h. Due to insufficient conversion 6-acetylmercaptohexanoic acid (0.49 g, 2.58 mmol), DCC (0.53 g, 2.58 mmol) and Oxyma pure (0.47 g, 3.19 mmol) were stirred for 30 min and filtered into the reaction. DIPEA (0.6 mL, 3.42 mmol) was added and the reaction stirred for 2.5 h. The reaction mixture was washed with 0.1 M H.sub.2SO.sub.4 (20 mL), saturated sodium bicarbonate solution (220 mL) and brine (20 mL). The organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuo and 10c was purified by flash chromatography. Yield: 0.74 g (1.39 mmol, 60%). MS: m/z 532.30=[M+H].sup.+, (calculated=532.31).

(166) 10c (0.74 g, 1.39 mmol) was dissolved in TFA/TES/water (95:2.5:2.5 v/v/v, 5.25 mL) and stirred for 30 min. The mixture was concentrated in vacuo and purified by RP-HPLC to give 10d (0.38 g, 0.78 mmol, 56%) MS: m/z 376.19=[M+H].sup.+, (calculated=376.19). 10d (0.38 g, 0.78 mmol) was dissolved in DCM (7 mL) and (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl 4-nitrophenyl carbonate (0.35 g, 1.17 mmol) and 2,4,6-collidine (0.45 mL, 3.51 mmol) were added and the reaction stirred. After 1 h 2,4,6-collidine (0.2 mL, 1.56 mmol) was added and the reaction stirred for 20 h. The reaction was concentrated in vacuo and purified by RP-HPLC to give 10e (0.26 g, 0.49 mmol, 63%) MS: m/z 532.20=[M+H].sup.+, (calculated=532.20).

(167) 10e (0.26 g, 0.49 mmol) was dissolved in DCM (3.6 mL) and DCC (0.12 g, 0.59 mmol), NHS (68 mg, 0.59 mmol) and DMAP (6 mg, 0.05 mmol) were added. The reaction was stirred for 1 h. The resulting suspension was filtered and the precipitate washed with DCM (2 mL). The filtrate was purified by flash chromatography to give 10f as white foam. Yield: 0.27 g (0.43 mmol, 87%). MS: m/z 629.21=[M+H].sup.+, (calculated=629.21).

Example 11

(168) Synthesis of Linker Reagent 11d

(169) Linker reagent 11d was synthesized according to the following scheme:

(170) ##STR00117##

(171) 2-Chlorotritylchloride resin (1.4 mmol/g, 357 mg, 0.5 mmol) was weighted into a 10 mL syringe with frit. The resin was swollen twice with 2 mL DCM. N-Fmoc-N-methyl-L-aspartate(OtBu)-OH (532 mg, 1.25 mmol and DIPEA (305 L, 1.75 mmol) were dissolved in DCM (3 mL) and drawn into the syringe. The syringe was agitated for 1 h. MeOH (0.5 mL) was drawn into the syringe and the syringe agitated for 30 min. The resin was washed 5 times with DCM (4 mL) and 5 times with DMF (4 mL). The resin was agitated 3 times for 5 min with DMF:DBU:piperidine (96:2:2 v/v/v, 4 mL). The resin was washed 5 times with DMF (4 mL). 6-tritylmercaptohexanoic acid (488 mg, 1.25 mmol) and HATU (475 mg, 1.25 mmol) were dissolved in DMF (3 mL) and DIPEA (436 L, 2.5 mmol) added. After 1 min preincubation the solution was drawn into the syringe and the syringe agitated for 1 h. The resin was washed 5 times with DMF (4 mL), 5 times with DCM (4 mL) and twice with MeOH (4 mL) and dried in vacuo. A solution of HFIP/TES/acetic acid (90/5/5 v/v/v, 3 mL each) were drawn into the syringe and the syringe agitated twice for 30 min. The solvent of the collected filtrates was evaporated in a stream of nitrogen. The residue was suspended in ACN/water (1:1 v/v, 5 mL) and filtered off. 2,2-dithiodipyridine (220 mg, 1 mmol) in ACN (0.5 mL) was added to the filtrate. pH of the reaction was adjusted to 7 with pH 7.4 sodium phosphate buffer (0.5 M, 1.2 mL) and stirred for 15 min. The product was directly purified by RP-HPLC to give 11a (117 mg, 0.27 mmol, 53%) MS: m/z 443.30=[M+H].sup.+, (calculated=443.17).

(172) 11a (117 mg, 0.27 mmol) was dissolved in DCM (2.4 mL). PyBOP (166 mg, 0.32 mmol), DIPEA (185 L, 1.06 mmol) and N-Boc-N-methylethylenediamine (57 L, 0.32 mmol) were added and the reaction stirred for 45 min. Acetic acid (185 L) was added and the solvents removed in a stream of nitrogen. The residue was dissolved with ACN/water/TFA (2:1:0.003 v/v/v) and purified by RP-HPLC to give 11b (135 mg, 0.23 mmol, 85%) MS: m/z 599.27=[M+H].sup.+, (calculated=599.29).

(173) 11b (135 mg, 0.23 mmol) was dissolved in TFA/TES/water (95:2.5:2.5 v/v/v, 5 mL). After 15 min the solvents were evaporated in a stream of nitrogen. The residue was diluted with ACN/water 1:1 and lyophilized. The residue was dissolved in DCM (1.5 mL), and (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl 4-nitrophenyl carbonate (80 mg, 0.27 mmol) was added. With stirring DIPEA (78 L, 0.46 mmol) was slowly added until the reaction stayed light yellow colored. More DIPEA (39 L, 0.23 mmol) was added and the reaction stirred for 1 h. After addition of DIPEA (20 L, 0.12 mmol) the reaction was stirred for 30 min. Acetic acid (140 L) were added and the solvents removed in a stream of nitrogen. The residue was diluted with ACN/water/TFA (1:1:0.002 v/v/v, 3 mL) and purified by RP-HPLC to give 11c (69 mg, 0.12 mmol, 51%). MS: m/z 599.19=[M+H].sup.+, (calculated=599.19).

(174) 11c (69 mg, 0.12 mmol) was dissolved in DCM and NHS (16 mg, 0.14 mmol), DCC (29 mg, 0.14 mmol) and DMAP (1.4 mg, 0.012 mmol) were added and the reaction stirred for 75 min. The solvent was evaporated in a stream of nitrogen and the residue was suspended in ACN/water/TFA (1:1:0.002 v/v/v, 3.5 mL) and filtered. The filtrate was purified by RP-HPLC to give 11d (60 mg, 0.09 mmol, 75%). MS: m/z 696.20=[M+H].sup.+, (calculated=696.20).

Example 12

(175) Synthesis of Linker Reagent 12e

(176) Linker reagent 12e was synthesized according to the following scheme:

(177) ##STR00118##

(178) To a solution of N-Boc-N-methylethylenediamine (2 g, 11.48 mmol) and NaCNBH.sub.3 (819 mg, 12.63 mmol) in MeOH (20 mL) was added 2,4,6-trimethoxybenzaldehyde (2.08 mg, 10.61 mmol) portion wise. The mixture was stirred at RT for 90 min, acidified with 3 M HCl (4 mL) and stirred further 15 min. The reaction mixture was added to saturated NaHCO.sub.3 solution (200 mL) and extracted 5 with CH.sub.2Cl.sub.2. The combined organic phases were dried over Na.sub.2SO.sub.4 and the solvents were evaporated in vacuo. The resulting N-Boc-N-methyl-N-tmob-ethylenediamine (12a) was completely dried in high vacuum and used in the next reaction step without further purification. Yield: 3.76 g (11.48 mmol, 89% purity, 12a: double Tmob protected product=8:1). MS: m/z 355.22=[M+H].sup.+, (calculated=355.23).

(179) To a solution of 12a (2 g, 5.65 mmol) in CH.sub.2Cl.sub.2 (24 ml) COMU (4.84 g, 11.3 mmol), N-Fmoc-N-methyl-L-Asp(OBn)-OH (2.08 g, 4.52 mmol) and 2,4,6-collidine (2.65 mL, 20.34 mmol) were added. The reaction mixture was stirred for 3 h at RT, diluted with CH.sub.2Cl.sub.2 (250 mL) and washed 3 with 0.1 M H.sub.2SO.sub.4 (100 mL) and 3 with brine (100 mL). The aqueous phases were re-extracted with CH.sub.2Cl.sub.2 (100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtrated and the residue concentrated to a volume of 24 mL. 12b was purified using flash chromatography. Yield: 5.31 g (148%, 6.66 mmol) MS: m/z 796.38=[M+H].sup.+, (calculated=796.38).

(180) To a solution of 12b [5.31 g, max. 4.51 mmol ref. to N-Fmoc-N-Me-L-Asp(OBn)-OH] in THF (60 mL) DBU (1.8 mL, 3% v/v) was added. The solution was stirred for 12 min at RT, diluted with CH.sub.2Cl.sub.2 (400 mL) and washed 3 with 0.1 M H.sub.2SO.sub.4 (150 mL) and 3 with brine (150 mL). The aqueous phases were re extracted with CH.sub.2Cl.sub.2 (100 ml). The combined organic phases were dried over Na.sub.2SO.sub.4 and filtrated. 12c was isolated upon evaporation of the solvent and used in the next reaction without further purification. MS: m/z 574.31=[M+H].sup.+, (calculated=574.31).

(181) 12c (5.31 g, 4.51 mmol, crude) was dissolved in acetonitrile (26 mL) and COMU (3.87 g, 9.04 mmol), 6-tritylmercaptohexanoic acid (2.12 g, 5.42 mmol) and 2,4,6-collidine (2.35 mL, 18.08 mmol) were added. The reaction mixture was stirred for 4 h at RT, diluted with CH.sub.2Cl.sub.2 (400 mL) and washed 3 with 0.1 M H.sub.2SO.sub.4 (100 mL) and 3 with brine (100 mL). The aqueous phases were re extracted with CH.sub.2Cl.sub.2 (100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtrated and 12d was isolated upon evaporation of the solvent. Product 7i was purified using flash chromatography. Yield: 2.63 g (62%, 94% purity) MS: m/z 856.41=[M+H].sup.+, (calculated=856.42).

(182) To a solution of 12d (2.63 g, 2.78 mmol) in i-PrOH (33 mL) and H.sub.2O (11 mL) was added LiOH (267 mg, 11.12 mmol) and the reaction mixture was stirred for 70 min at RT. The mixture was diluted with CH.sub.2Cl.sub.2 (200 mL) and washed 3 with 0.1 M H.sub.2SO.sub.4 (50 mL) and 3 with brine (50 mL). The aqueous phases were re-extracted with CH.sub.2Cl.sub.2 (100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtrated and 12e was isolated upon evaporation of the solvent. 12e was purified using flash chromatography. Yield: 2.1 g (88%) MS: m/z 878.4=[M+Na].sup.+, (calculated=878.40).

Example 13

(183) Synthesis of Linker Reagent 13g

(184) Linker reagent 13g was synthesized according to the following scheme:

(185) ##STR00119## ##STR00120##

(186) To a solution of N,N-dimethylethylenediamine (441 mg, 5 mmol) and NaCNBH.sub.3 (439 mg, 7 mmol) in MeOH (21 mL) was added a solution of 2,4,6-trimethoxybenzaldehyde (1.34 g, 6.85 mmol) in DCM and MeOH (17 mL each) with a syringe. The mixture was stirred at RT for 60 min, acidified with 0.5 M HCl (50 mL) and stirred further 15 min. The reaction mixture was brought to pH>12 by adding 1 M NaOH and extracted 4 times with ethyl acetate (1100 mL, 350 mL). The combined organic phases were dried over MgSO.sub.4 and the solvents were evaporated in vacuo. The resulting N,N-dimethyl-N-tmob-ethylenediamine (13a) was completely dried in high vacuum and used in the next reaction step without further purification. Yield: 1.63 g (contains double Tmob protected product). MS: m/z 269.19=[M+H].sup.+, (calculated=269.19).

(187) 13a (268 mg, ca 0.83 mmol) was dissolved in THF (5 mL) and 2,3-pyridinedicarboxylic anhydride (149 mg, 1 mmol), DIPEA (362 L, 2.08 mmol) and DMF (1 mL) were added. The solution was stirred for 15 min, quenched with AcOH (362 L) and the THF removed in a stream of nitrogen. The residue was diluted with ACN/water/TFA (1:1:0.002 v/v/v, 4 mL) and purified by RP-HPLC to give 13b (244 mg, 0.46 mmol, 55%). MS: m/z 418.20=[M+H].sup.+, (calculated=418.20).

(188) 12e (500 mg, 0.58 mmol) was dissolved in TFA/TES/DTT/water (85:5:5:5 v/v/v/v, 10 mL) and stirred for 4 h. The solvents were removed in a stream of nitrogen and the residue suspended in ACN/water and filtered. The filtrate was purified by RP-HPLC to give 13c (71 mg, 0.21 mmol, 36%). MS: m/z 334.43=[M+H].sup.+, (calculated=334.18).

(189) 13c (71 mg, 0.21 mmol) was dissolved in ACN/water (1:1, 1 mL) and 2,2-dithiodipyridine (93 mg, 0.42 mmol) in ACN/water (1:1, 1 mL, suspension) and pH 7.4 sodium phosphate buffer (0.5 M, 1 mL) were added. The resulting solution was stirred for 15 min and directly purified by RP-HPLC to give 13d (64 mg, 0.15 mmol, 68%). MS: m/z 443.30=[M+H].sup.+, (calculated=443.18).

(190) 13b (91 mg, 0.22 mmol) was dissolved in DMF (1 mL) and PyBOP (113 mg, 0.22 mmol) was added. DIPEA (50 L, 0.29 mmol) was added and the solution stirred for 15 min. 13d (64 mg, 0.145 mmol) was dissolved in DCM (1.5 mL) and added to the DMF solution. The reaction was stirred for 100 min and AcOH (50 L) was added. DCM was removed in a stream of nitrogen and the residue dissolved in ACN/water/TFA and purified by RP-HPLC to give 13e (26 mg, 31 mol, 21%). MS: m/z 842.14=[M+H].sup.+, (calculated=842.36).

(191) 13e (26 mg, 31 mol) was dissolved in HFIP/TES/water (39:1:1 v/v/v, 1 mL) and TFA (83 L) was added with stirring. After 90 min the solvents were evaporated in vacuo and the residue purified by RP-HPLC to give 13f (11 mg, 17 mol, 54%). MS: m/z 662.28=[M+H].sup.+, (calculated=662.28).

(192) 13f (11 mg, 17 mol) was dissolved in DCM (1.5 mL), DCC (4.2 mg, 20 mol), NHS (2.3 mg, 20 mol) and DMAP (0.2 mg, 1 mol) were added and the suspension stirred. After 1 h and 2 h the aforementioned amount of DCC and NHS were added again. After 3 h the solvent was evaporated in a stream of nitrogen. The residue was suspended in ACN/water/TFA (1:1:0.002, 3 mL) and filtered. The filtrate was purified by RP-HPLC to give 13g (14 mg, 16 mol (TFA salt), 94%). MS: m/z 759.30=[M+H].sup.+, (calculated=759.30).

Example 14

(193) Synthesis of Linker Reagent 14f

(194) Linker reagent 14f was synthesized according to the following scheme:

(195) ##STR00121##

(196) A solution of p-monomethoxytrityl chloride (1.54 g, 5 mmol) in DCM (10 mL) was slowly added with stirring to a solution of 3-(methylamino)propylamine (4.4 g, 50 mmol) in DCM (10 mL). After 2 h diethyl ether (166 mL) was added. Brine (100 mL) was mixed with NaOH (4 M, 80 L) and the reaction washed with this mixture (333 mL). The organic layer was washed with brine (30 mL), dried with MgSO.sub.4 and concentrated in vacuo. Yield (14a): 1.79 g (4.95 mmol, 99%).

(197) 14a (0.36 g, 1 mmol) was dissolved in THF (7 mL) and DIPEA (0.44 ml, 2.5 mmol) and a solution of quinolinic anhydride (0.18 g, 1.2 mmol) in THF (3 mL) were added with stirring. After 30 min a solution of 13a (0.54 g, 2 mmol) in DMF (2 mL) and PyBOP (0.78 g, 1.5 mmol) were added and the reaction stirred for 1 h. The reaction was diluted with ethyl acetate (50 mL) and washed with NaOH (1 M, 20 mL). The aqueous phase was reextracted with ethyl acetate (220 mL) and the collected organic phases combined and concentrated in vacuo and purified using flash chromatography to give 14b (0.4 g, 0.53 mmol, 53%) MS: m/z 760.41=[M+H].sup.+, (calculated=760.41).

(198) 14b (0.4 g, 0.53 mmol) was dissolved in ACN (3 mL) and HCl (0.4 M, 3 mL) was added and the solution stirred for 4 h. The reaction was quenched with NaOH (1 M, 15 mL) and extracted with DCM (530 mL). The combined organic phase was dried over MgSO.sub.4, concentrated in vacuo and 14c was purified by flash chromatography. Yield: 0.32 g (0.42 mmol, 81%, MMT salt) MS: m/z 488.31=[M+H].sup.+, (calculated=488.29).

(199) 11a (49 mg, 0.11 mmol) and 14c (114 mg, 0.15 mmol) were dissolved in DCM (1.4 mL) and PyBOP (62 mg, 0.12 mmol) and DIPEA (38 L, 0.22 mmol) were added with stirring. After 2 h AcOH (40 L) was added and the solvents evaporated in vacuo. The residue was dissolved in ACN/water/TFA (1:1:0.002 v/v/v, 5 mL) and purified by RP-HPLC to give 14d (104 mg, 0.1 mmol TFA salt, 92%). MS: m/z 912.19=[M+H].sup.+, (calculated=912.44).

(200) 14d (104 mg, 0.1 mmol) was dissolved in HFIP/TES/water (39:1:1 v/v/v, 3 mL) and TFA (0.25 mL) was added with stirring. After 2 h TFA (0.25 mL) was added with stirring and the reaction stirred further for 20 h. The mixture was concentrated in vacuo and the residue dissolved in ACN/water/TFA (1:1:0.002 v/v/v, 3 mL) and purified by RP-HPLC to give 14e (28 mg, 35 mol TFA salt, 35%). MS: m/z 676.13=[M+H].sup.+, (calculated=676.30).

(201) 14e (25 mg, 32 mol) was dissolved in DCM (2 mL) and DCC (10 mg, 48 mol), NHS (5.5 mg, 48 mol) and DMAP (0.4 mg, 3.2 mol) were added and the suspension stirred. After 1.5 h the aforementioned amount of DCC and NHS were added again. After 3 h the solvent was evaporated in a stream of nitrogen. The residue was suspended in ACN/water/TFA (1:1:0.002 v/v/v, 4 mL) and filtered. The filtrate was purified by RP-HPLC to give 14f (28 mg, 31.6 mol TFA salt, 99%). MS: m/z 773.31=[M+H].sup.+, (calculated=773.31).

Example 15

(202) Synthesis of Linker Reagent 15f

(203) Linker reagent 15f was synthesized according to the following scheme:

(204) ##STR00122##

(205) To a solution of 11a (0.29 g, 0.65 mmol) and PyBOP (0.34 g, 0.65 mmol) in ACN (5 mL) DIPEA (0.57 mL, 3.27 mmol) was added. This mixture was stirred at room temperature for one minute, before N,N-dimethylaminopropane-1,3-diamine (0.12 mL, 0.98 mmol) was added. After 30 min AcOH (0.7 mL) was added to quench the reaction. This mixture was diluted with water and purified by RP-HPLC. The obtained intermediate was dissolved in TFA (3 mL) and the mixture was stirred for 30 min before TFA was removed by a stream of N.sub.2. The residue was dried in vacuo over night to give 15a (0.42 g, 0.61 mmol (2TFA salt), 93%) as colorless oil, which was used without any further purification. MS: m/z=471.21=[M+H].sup.+, (calculated 471.21).

(206) To a solution of 15a (0.23 mg, 0.33 mmol) in DCM (5 mL) DMAP (8 mg, 65 mol), NHS (75 mg, 0.65 mmol) and DCC (135 mg, 0.65 mmol) were added. After stirring for 3 h the reaction was quenched by addition of AcOH (10 L). The solvent was removed by a stream of N.sub.2 and the residue was suspended in H.sub.2O/ACN/TFA (1:1:0.002 v/v/v) and filtered. The filtrate was purified by RP-HPLC to give 15b (83 mg, 0.10 mmol (2TFA salt), 32%) as a colorless oil. MS: m/z=568.23=[M+H].sup.+, (calculated 568.23).

Example 16

(207) Synthesis of Purification Tag 16e

(208) Purification tag 16e was synthesized according to the following scheme:

(209) ##STR00123## ##STR00124##

(210) To a suspension of 6,6-dithiodinicotinic acid (0.62 g, 2 mmol) in ACN (20 mL) were added PyBOP (2.08 g, 4 mmol) and DIPEA (1.29 g, 1.74 mL, 10 mmol) and the mixture was stirred for 1 min. The obtained brown solution was added to a solution of 1,9-bis-Boc-1,5,9-triazanonane (1.99 g, 6 mmol) in a mixture of ACN (20 mL) and DMF (5 mL) and stirred for 2 h. The reaction mixture was diluted with EtOAc (150 mL) and the organic layer was washed with aq. HCl (10 mM, 5100 mL), saturated NaHCO.sub.3 solution (3100 mL) and brine (100 mL), subsequently. After drying over MgSO.sub.4 and filtration, the solvent was removed in vacuo and the crude residue was purified by flash chromatography to give 16a (1.92 g, max. 2 mmol) as a light yellow foam. The product contains a small, non-separable amount of tripyrrolidine phosphoramide, which is removed in the next step. MS: m/z=935.47=[M+H].sup.+, (calculated 935.47).

(211) 16a (1.92 g, max. 2 mmol) was dissolved in TFA (10 mL) and the solution was stirred for 10 min. The reaction mixture was added dropwise to ice-cold diethyl ether (160 mL) to precipitate the product. The resulting suspension was centrifuged at 7000G and 2 C. for 3 min. The supernatant was discarded and the precipitate was dissolved in methanol (10 mL). This solution was added dropwise to ice-cold diethyl ether (160 mL) and the formed suspension was centrifuged at 7000G and 2 C. for 3 min. After discarding the supernatant the precipitation procedure was accomplished two more times like described above. The remaining oily precipitate was dried in vacuo to give 16b (1.77 g, 1.45 mmol (6TFA salt), 73%) as a light brown, very hygroscopic powder. MS: m/z=535.26=[M+H].sup.+, (calculated 535.26).

(212) To a solution of 16b (3.30 g, 2.7 mmol) in DMF (90 mL) were added DIPEA (5.4 mL, 31 mmol) and Boc-L-Lys(Boc)-OSu (5.62 g, 12.7 mmol). The mixture was stirred for 14 h before it was diluted with ethyl acetate (600 mL). The organic layer was washed with aq. HCl (10 mM, 5300 mL), sat. NaHCO.sub.3 solution (3300 mL) and brine (300 mL) and was dried over MgSO.sub.4. After filtration the solvent was removed in vacuo and the crude residue was purified by flash chromatography to give 16c (5.52 g, max. 2.7 mmol) as light yellow foam with 90% purity. MS: m/z=924.54=[M+2H].sup.2+, (calculated 924.53).

(213) 16c (5.52 g, max. 2.7 mmol) was dissolved in TFA (20 mL). After stirring for 15 min the product was precipitated by adding the reaction mixture dropwise to ice-cold diethyl ether (160 mL). The resulting suspension was centrifuged at 7000G and 2 C. for 3 min. The supernatant was discarded and the precipitate was dissolved in methanol (10 mL). This solution was added dropwise to ice-cold diethyl ether (160 mL) and the formed suspension was centrifuged at 7000G and 2 C. for 3 min. After discarding the supernatant the precipitation procedure was accomplished two more times like described above. The remaining oily precipitate was dried in vacuo to give 16d (4.96 g, 2.27 mmol (10TFA salt), 84%) as a light brown, hygroscopic powder. MS: m/z=1046.64=[M+H].sup.+, (calculated 1046.64).

(214) To a solution of 16d (1.53 g, 0.7 mmol) in dry DMF (20 mL) was added a solution of N,N-dimethylglycine (1.16 g, 11.2 mmol), PyBOP (5.83 g, 11.2 mmol) and DIPEA (3.23 g, 4.36 mL, 25 mmol) in DMF (35 mL) and stirred for 1 h. The mixture was then concentrated in vacuo to an approximate volume of 10 mL. To this residue water was added to a total volume of 100 mL and the solution was acidified to pH 1-2 by adding TFA. The turbid mixture was centrifuged at 5000G and 2 C. for 3 minutes. The oily precipitate was discarded and the supernatant was purified by RP-HPLC to give 16e (1.05 g, 0.37 mmol (10TFA salt), 53%) as a colorless oil. MS: m/z=864.54=[M+2H].sup.2+, (calculated 864.54).

Example 17

(215) Synthesis of Linker Reagent 17g

(216) Linker reagent 17g was synthesized according to the following scheme:

(217) ##STR00125## ##STR00126##

(218) 2-Chlorotritylchloride resin (1.4 mmol/g, 1.43 g, 2 mmol) was weighted into a 20 ml syringe with frit. The resin was swollen twice with 10 mL DCM. N-Fmoc-N-methyl-L-Asp(OtBu)-OH (1.06 g, 2.5 mmol) was dissolved in DCM (6 mL) and drawn into the syringe. DIPEA (436 L, 2.5 mmol) was dissolved in DCM (1 mL) and drawn into the syringe. The syringe was agitated for 5 min. DIPEA (654 L, 3.75 mmol) was dissolved in DCM (1 mL) and drawn into the syringe. The syringe was agitated for 1 h. MeOH (2 mL) was drawn into the syringe and the syringe agitated for 30 min. The resin was washed 5 times with DMF (10 mL). The resin was agitated 3 times for 5 min with DMF:DBU:piperidine (96:2:2 v/v/v 7 mL). The resin was washed 5 times with DMF (5 mL). 6-Tritylmercaptohexanoic acid (1.95 g, 5 mmol) and PyBOP (2.6 g, 5 mmol) were dissolved in DMF (6 mL) and DIPEA (3.5 mL, 20 mmol) added. After 1 min preincubation the solution was drawn into the syringe and the syringe agitated for 3 h. The resin was washed 5 times with DMF (7 mL), 5 times with DCM (7 mL). A solution of HFIP/DCM ( v/v, 8 mL each) were drawn into the syringe and the syringe agitated 3 times for 30 min. The collected filtrates were concentrated in vacuo. Crude 17a (0.84 g, 1.45 mmol, 73%) was used without further purification in the next step. MS: m/z 598.18=[M+Na].sup.+, (calculated=598.26).

(219) 17a (1.67 g, 2.9 mmol) was dissolved in DCM (20 mL) and N-Boc-N-methylethylenediamine (0.62 mL, 3.48 mmol) and PyBOP (1.81 g, 3.48 mmol) were added. DIPEA (2.02 mL, 11.6 mmol) was added and the reaction stirred for 1 h. AcOH (2 mL) was added, the mixture diluted with DCM (40 mL) and washed with water (220 mL). The organic layer was dried over MgSO.sub.4 and concentrated in vacuo and the crude residue was purified by flash chromatography to give 17b (1.74 g, 2.38 mmol, 82%). MS: m/z=754.19=[M+Na].sup.+, (calculated 754.39).

(220) 17b (1.74 g, 2.38 mmol) and triphenylmethanol (0.62 g, 2.38 mmol) were dissolved in DCM (7.2 mL) and TFA (7.2 mL) was added with stirring. The reaction was stirred for 90 min and the solvents were removed in a stream of nitrogen over 45 min. The residue was co-evaporated with DCM. The residue was suspended in ACN/water/TFA (2:1:0.003 v/v/v, 14 mL) and filtered. The filtrate was purified by RP-HPLC to give 17c (0.9 g, 1.3 mmol TFA salt, 55%). MS: m/z 576.20=[M+H].sup.+, (calculated=576.29).

(221) 17c (0.9 g, 1.3 mmol) was dissolved in DCM (20 mL) and (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl 4-nitrophenyl carbonate (0.46 g, 1.56 mmol) was added. DIPEA (0.45 mL, 2.6 mmol) was slowly added and the reaction stirred for 30 min. DIPEA (0.11 mL, 0.65 mmol) was added and the reaction stirred for 30 min. Again, DIPEA (0.11 mL, 0.65 mmol) was added and the reaction stirred for 60 min. AcOH (0.68 mL) was added and the mixture concentrated in vacuo and the crude residue was purified by flash chromatography to give 17d (1.04 g, max. 1.3 mmol). MS: m/z=754.28=[M+Na].sup.+, (calculated 754.28).

(222) 17d (1.04 g, max. 1.3 mmol) was dissolved in HFIP/TES/water (39:1:1 v/v/v, 8.2 mL) and TFA (0.66 mL) was added. After stirring for 15 min the reaction was concentrated in vacuo, the residue suspended in ACN/water/TFA (1:1:0.002 v/v/v 12 mL) and filtered. The filtrate was purified by RP-HPLC to give 17e (0.32 g, 0.65 mmol, 50%). MS: m/z 490.19=[M+H].sup.+, (calculated=490.19).

(223) 17e (181 mg, 0.37 mmol) was dissolved in ACN/water/TFA (1:1:0.002 v/v/v, 3 mL). 16e (1.05 g, 0.37 mmol (10TFA salt) was dissolved in ACN/water (1:1 v/v, 20 mL). Both solutions were combined and pH 7.4 sodium phosphate (0.5 M, 4 mL) was added and the mixture stirred for 30 min. The pH of the solution was adjusted to ca. pH 2 by addition of ACN/water/TFA (1:1:0.22 v/v/v) and ACN was removed in vacuo. The residue was purified by RP-HPLC to give 17f (0.47 g, 0.24 mmol 5TFA salt, 65%). MS: m/z 676.86=[M+2H].sup.2+, (calculated=676.86).

(224) 17f (0.18 g, 94 mol) was dissolved in ACN (6 mL) and NHS (92 mg, 0.8 mmol) and DCC (166 mg, 0.8 mmol) were added and the reaction stirred for 1 h. The solvent was removed in vacuo and the residue suspended in ACN/water/TFA (0.15:0.85:0.001 v/v/v, 6 mL) and filtered. The filtrate was purified by RP-HPLC to give 17g (129 mg, 64 mol 5TFA salt, 68%). MS: m/z 725.37=[M+H].sup.+, (calculated=725.37).

Example 18

(225) Synthesis of Linker Reagent 18i

(226) Linker reagent 18i was synthesized according to the following scheme:

(227) ##STR00127## ##STR00128##

(228) N-Boc-ethylenediamine (0.77 g, 4.8 mmol) was dissolved in DCM (15 mL) and 6-tritylmercaptohexanoic acid (2.25 g, 5.76 mmol) and PyBOP (3.0 g 5.76 mmol) were added with stirring. DIPEA (2.52 ml, 14.4 mmol) was added and the reaction stirred for 1 h. The reaction was diluted with diethyl ether (150 mL) and washed with slightly basic brine (330 mL, prepared from 100 mL brine and 3 mL 0.1 M aq. NaOH). The organic phase was washed once more with brine (30 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo and purified using flash chromatography to give 18a as a white foam. Yield: 2.55 g (4.79 mmol, 99%) MS:

(229) m/z 555.24=[M+Na].sup.+, (calculated=555.27).

(230) 18a (2.55 g, 4.79 mmol) was dissolved in THF (26 mL) and transferred into an oven-dried argon filled round-bottom flask. Borane-THF complex in THF (1 M, 17.7 mL, 17.71 mmol) was added and the reaction stirred for 15 h. MeOH (5.4 mL) was added slowly and N,N-dimethyl ethylenediamine (3.11 mL, 28.8 mmol) was added and the reaction refluxed for 2.5 h. After cooling the reaction was diluted with ethyl acetate and washed with sat. sodium bicarbonate solution (2125 mL) and brine (1125 mL). The organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuo to give 18b which was used without further purification in the next step. Yield: 2.38 g (4.59 mmol, 96%) MS: m/z 519.27=[M+H].sup.+, (calculated=519.31).

(231) 18b (1.19 g 2.29 mmol) was dissolved in DCM and (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl 4-nitrophenyl carbonate (1.02 g, 3.44 mmol) and 2,4,6-collidine (1.36 mL, 10.32 mmol) were added and the reaction stirred for 23 h. The reaction was concentrated in vacuo and purified using flash chromatography to give 18c. Yield: 1.19 g (1.77 mmol, 79%) MS: m/z 697.18=[M+Na].sup.+, (calculated=697.29).

(232) 18c (0.5 g, 0.74 mmol) was dissolved in DCM (2.5 mL) and triphenylmethanol (0.19 g, 0.74 mmol) and TFA (2.5 mL) were added. The reaction was stirred for 40 min, concentrated in a stream of argon and dried in vacuo (<0.1 mbar). The residue was dissolved in ACN/water (7:3 v/v, 10 mL) and purified by RP-HPLC to give 18d (0.50 g, 0.84 mmol, 114%). MS: m/z 575.33=[M+H].sup.+, (calculated=575.26).

(233) 2-Chlorotritylchloride resin (1.22 mmol/g, 0.87 g, 1 mmol) was weighted into a 10 ml syringe with frit. The resin was swollen with 5 mL DCM and washed with DCM (54 mL). N-Fmoc-L-Asp(OtBu)-OH (1.1 g, 2.7 mmol) was dissolved in DCM (5 mL) and DIPEA (0.66 mL, 3.78 mmol) was added and the solution drawn into the syringe. The syringe was agitated for 1 h. MeOH (0.5 mL) was drawn into the syringe and the syringe agitated for 15 min. The resin was washed 5 times with DCM (4 mL) and 5 times with DMF (5 mL). The resin was agitated 3 times for 5 min with DMF:DBU:piperidine (96:2:2 v/v/v 4 mL). The resin was washed 5 times with DMF (4 mL). Acetic anhydride (0.51 mL, 5.4 mmol) and DIPEA (1.9 mL, 10.8 mmol) were dissolved in DMF (6 mL) and the solution was drawn into the syringe and the syringe agitated for 15 min. The resin was washed 5 times with DMF (4 mL), 5 times with DCM (4 mL). A solution of HFIP/DCM ( v/v, 5 mL each) were drawn into the syringe and the syringe agitated 3 times for 10 min. The collected filtrates were concentrated in vacuo. Crude 18e (0.29 g, 1.23 mmol, 114%) was used without further purification in the next step. MS: m/z 254.38=[M+Na].sup.+, (calculated=254.12).

(234) 18e (65 mg, 0.28 mmol) was dissolved in DCM (3 mL) and PyBOP (0.18 g, 0.34 mmol) and DIPEA (0.15 mL, 0.84 mmol) were added. 18d (0.18 g, 0.31 mmol) was dissolved in DCM (3 mL) and added to the reaction. The reaction was stirred for 1 h and concentrated in vacuo. The residue was purified by RP-HPLC to give 18f (97 mg, 0.12 mmol, 44%). MS: m/z 810.02=[M+Na].sup.+, (calculated=810.34).

(235) 18f (97 mg, 0.12 mmol) was dissolved in TFA/TES/water (92:2.5:2.5 v/v/v, 3 mL) and the reaction stirred vigorously for 3 h while a stream of argon was passed through the solution. After 3 h the solvents were removed in a stream of argon and the residue was purified by RP-HPLC to give 18g (16 mg, 33 mol, 27%). MS: m/z 490.21=[M+H].sup.+, (calculated=490.19).

(236) 18g (16 mg, 33 mol) was dissolved in ACN/water (1:1 v/v, 3 mL) and a solution of 2,2-dithiodipyridine (14 mg, 65 mol) in ACN (0.1 mL) was added followed by pH 7.4 sodium phosphate buffer (0.5 M, 0.1 mL). The reaction was stirred for 4.5 h and the product directly purified by RP-HPLC to give 18h (14 mg, 23 mol, 71%). MS: m/z 598.98=[M+H].sup.+, (calculated=599.19).

(237) 18h (14 mg, 23 mol) was dissolved in DCM (3 mL) and NHS (3 mg, 28 mol), DCC (6.5 mg, 30 mol) and DMAP (0.3 mg, 2 mol) were added. The reaction was stirred for 1.5 h and the solvent removed in a stream of argon. The residue was suspended in ACN/water/TFA (9:1:0.01 v/v/v, 3 mL) and filtered. The filtrate was purified by RP-HPLC to give 18i (17 mg, 23 mol, 100%). MS: m/z 696.20=[M+H].sup.+, (calculated=696.20).

Example 19

(238) Synthesis of Crosslinker Reagents 19c, 19g

(239) Crosslinker reagent 19c was prepared from suberic acid monobenzyl ester 2a and PEG6000 accordingly to the following scheme:

(240) ##STR00129##

(241) For synthesis of compound 19a, azelaic acid monobenzyl ester 2a (6.50 g, 23.3 mmol) and PEG 6000 (40.0 g, 6.67 mmol) were dissolved in 140 mL dichloromethane and cooled with an ice bath. A solution of DCC (4.81 g, 23.3 mmol) and DMAP (0.040 g, 0.33 mmol) in 40 mL dichloromethane was added. The ice bath was removed and mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 C. and the solid was filtered off. The solvent was evaporated in vacuo.

(242) The residue was dissolved in 70 mL dichloromethane and diluted with 300 mL MTBE at room temperature. The mixture was stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 500 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(243) Yield 41.2 g (95%) white powder 19a.

(244) MS: m/z 833.75=[M+8H].sup.8+ (calculated=833.74).

(245) For mass spectra of polydisperse PEG containing compounds, one single mass peak was selected.

(246) For synthesis of compound 19b, compound 19a (41.2 g, 6.32 mmol) was dissolved in methyl acetate (238 mL) and ethanol (40 mL), then 400 mg of palladium on charcoal was added. Under a hydrogen atmosphere of ambient pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo over night.

(247) Yield 38.4 g (96%) glassy solid 19b.

(248) MS: m/z 750.46=[M+H].sup.9+ (calculated=750.56).

(249) For mass spectra of polydisperse PEG containing compounds, one single mass peak was selected.

(250) For synthesis of compound 19c, compound 19b (38.2 g, 6.02 mmol) and TSTU (7.25 g, mmol) were dissolved in 130 mL dichloromethane at room temperature. Then DIPEA (3.11 g, 24.1 mmol) was added and the mixture was stirred for 1 h. The resulting suspension was filtered, the filtrate was diluted with 100 mL dichloromethane and washed with 200 mL of a solution of 750 g water/197 g NaCl/3 g NaOH. The organic phase was dried over MgSO.sub.4 and the solvent was evaporated in vacuo.

(251) The residue was dissolved in 210 mL toluene, diluted with 430 mL MTBE at room temperature and stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 450 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(252) Yield 35.8 g (91%) white powder 19c.

(253) MS: m/z 857.51=[M+8H].sup.8+ (calculated=857.51).

(254) For mass spectra of polydisperse PEG containing compounds, one single mass peak was selected.

(255) Crosslinker reagent 19g was prepared from isopropylmalonic acid monobenzyl ester and PEG8000 according to the following scheme:

(256) ##STR00130##

(257) For the synthesis of isopropylmalonic acid monobenzyl ester rac-19d, isopropylmalonic acid (35.0 g, 239 mmol), benzyl alcohol (23.3 g, 216 mmol) and DMAP (1.46 g, 12.0 mmol) were dissolved in 100 mL acetonitrile. Mixture was cooled to 0 C. with an ice bath. A solution of DCC (49.4 g, 239 mmol) in 150 mL acetonitrile was added within 15 min at 0 C. The ice bath was removed and the reaction mixture was stirred over night at room temperature, then the solid was filtered off. The filtrate was evaporated at 40 C. in vacuo and the residue was dissolved in 300 mL MTBE. This solution was extracted with 2300 mL sat. aqueous NaHCO.sub.3 solution, then the combined aqueous phases were acidified to pH=1-3 using 6 N hydrochloric acid. The resulting emulsion was extracted with 2300 mL MTBE and the solvent was evaporated. The combined organic phases were washed with 200 mL sat. aqueous NaCl and dried over MgSO.sub.4. The product was purified on 340 g silica using ethyl acetate/heptane (10:90.fwdarw.20:80) as eluent. The eluent was evaporated and the residue was dried in vacuo over night.

(258) Yield 9.62 g (17%) colorless oil rac-19d.

(259) MS: m/z 237.11=[M+H].sup.+ (calculated=237.11).

(260) For synthesis of compound rac-19e, isopropylmalonic acid monobenzyl ester rac-19d (2.25 g, 9.50 mmol) and PEG 8000 (19.0 g, 2.38 mmol) were dissolved in 100 mL dichloromethane and cooled with an ice bath. A solution of DCC (1.96 g, 9.50 mmol) and DMAP (14 mg, 0.12 mmol) in 10 mL dichloromethane was added. The ice bath was removed and mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 C. and the solid was filtered off. The solvent was evaporated in vacuo.

(261) The residue was dissolved in 40 mL dichloromethane and diluted with 270 mL MTBE at room temperature. The mixture was stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 500 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(262) Yield 18.5 g (92%) white powder rac-19e.

(263) MS: m/z 737.43=[M+13H].sup.13+ (calculated=737.42).

(264) For mass spectra of polydisperse PEG containing compounds, one single mass peak was selected.

(265) For synthesis of compound rac-19f, compound rac-19e (18.4 g, 2.18 mmol) was dissolved in methyl acetate (160 mL) and 254 mg of palladium on charcoal was added. Under a hydrogen atmosphere of ambient pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo over night.

(266) Yield 17.7 g (98%) glassy solid rac-19f.

(267) MS: m/z 723.51=[M+13H].sup.13+ (calculated=723.55).

(268) For mass spectra of polydisperse PEG containing compounds, one single mass peak was selected.

(269) For synthesis of compound rac-19g, compound rac-19f (13.6 g, 1.65 mmol) and TSTU (1.96 g, 6.60 mmol) were dissolved in 60 mL dichloromethane at room temperature. Then DIPEA (852 mg, 6.60 mmol) was added and the mixture was stirred for 45 min. The resulting suspension was filtered, the filtrate was diluted with 70 mL ethyl acetate and washed with 70 mL of a 0.5 M phosphate buffer pH=6.5. The organic phase was dried over MgSO.sub.4 and the solvent was evaporated in vacuo. The residue was dissolved in 80 mL toluene, the remaining solid was filtered off and washed with 20 mL of toluene. The combined toluene fractions were diluted with 35 mL MTBE at room temperature and stored over night at 20 C. The precipitate was collected by filtration through a glass filter Por. 3, and washed with 600 mL of cooled MTBE (20 C.). The product was dried in vacuo over night.

(270) Yield 12.1 g (87%) white powder rac-19g.

(271) MS: m/z 738.51=[M+13H].sup.13+ (calculated=738.49).

(272) For mass spectra of polydisperse PEG containing compounds, one single mass peak was selected.

Example 20

(273) Preparation of Hydrogel Beads 20a Containing Free Amino Groups

(274) 20a was prepared as described for 3c except applying a stirrer speed of 560 rpm, the use of 398 mg 1b, 2690 mg 19c, 27.8 g DMSO, 274 mg Cithrol DPHS, 1.8 mL TMEDA, 2.7 mL acetic acid, yielding 0.22 g on the 50 m sieve, 0.33 g on the 63 m sieve, and 0.52 g on the 75 m sieve of 20a as a white powder, free amino groups 0.152 mmol/g.

(275) Preparation of Hydrogel Beads 20b Containing Free Amino Groups.

(276) 20b was prepared as described for 3c except applying a stirrer speed of 580 rpm, the use of 250 mg 1b, 2168 mg rac-19g, 21.8 g DMSO, 215 mg Cithrol DPHS, 1.1 mL TMEDA, 1.7 ml, acetic acid, yielding 0.09 g on the 50 m sieve, 0.17 g on the 63 m sieve, and 0.54 g on the 75 m sieve of 20b as a white powder, free amino groups 0.154 mmol/g.

Example 21

(277) Preparation of a Maleimide Containing Histidine-Tag 21

(278) ##STR00131##

(279) 0.78 g of RINK amide MBHA resin (100-200 mesh, 0.64 mmol/g amines) were transferred into a 20 mL syringe equipped with a frit and swollen in 10 mL DCM. The solvent was expelled and the resin was washed three times with NMP, the solvent was discarded each time. The resin was treated for 15 minutes and 5 min, respectively, with 5 mL 20% piperidine in DMF to remove the Fmoc protection group, the solvent was discarded each time. The resin was washed ten times with NMP. 775 mg Fmoc-His(Trt)-OH and 475 mg HATU were dissolved in 3 mL 0.5M HOAT in DMF and 850 L DIPEA were added. The solution was drawn into the syringe and the suspension was allowed to incubate under gentle shaking for 3 h at ambient temperature. The solvent was discarded and the resin was washed five times with NMP. The Fmoc-protecting group was removed as described above. The next five couplings were each performed using a coupling solution of 620 mg Fmoc-His(Trt)-OH, 77 mg HOBt, 160 mg TBTU and 850 L DIPEA in 3 mL NMP. The reaction was each time allowed to go for 1 hour at ambient temperature under gentle shaking followed by washing with NMP and Fmoc-deprotection. Afterwards, the resin was treated with a solution of 385 mg Fmoc-Ado-OH, 475 mg HATU, 850 L DIPEA dissolved in 3 mL 0.5M HOAt in DMF for 1 h at ambient temperature followed by washing with NMP and Fmoc deprotection. The resin was treated with 169 mg maleimido propionic acid, 475 mg HATU, 850 L DIPEA in 3 mL 0.5 M HOAt in DMF for eight hours at ambient temperature under gentle shaking. The solvent was discarded and the resin washed five times with NMP and ten times with DCM. The solvent was discarded each time. Finally, the resin was treated with each time 5 mL of a solution of 95% TFA/2.5% TIPS/2.5% water for two times 30 min and two times one hour. The solution was expelled into separate 50 mL Falcon tubes and the TFA evaporated under a continuous nitrogen stream. To the residue, 45 mL of ice cold ether were added and the Falcon tube was centrifugated and the supernatant discarded. The precipitate was taken up in 50% ACN/water and purified via prep. HPLC and lyophilized to give 21.

(280) Yield 0.15 g (26%).

(281) MS: m/z 1136.49=[M+H].sup.+ (calculated=1136.49).

Example 22

(282) Preparation of PEGylated Hydrogel Beads in DMSO 22

(283) Dry hydrogel beads as e.g. described in 3c were transferred into a syringe equipped with a frit and NMP (5 mL/100 mg hydrogel beads) was added. The hydrogel was allowed to swell for 10 min at ambient temperature under gentle shaking. The solvent was expelled and the hydrogel washed ten times with each time DMSO, followed by ten washes with each time 1% TMEDA/DMSO (5 mL/100 mg hydrogel beads), the solvent was each time discarded. 0.2 eq (based on amine content of the hydrogel beads) PEG-NHS were dissolved in a solution containing 2 eq (based on amine content of the hydrogel beads) TMEDA in DMSO at 37 C. for 15 min. The solution was drawn into the syringe and the resulting hydrogel suspension was allowed to incubate under gentle shaking. The solvent was expelled and the hydrogel was washed five times with DMSO (5 mL/100 mg hydrogel beads), followed by five washing cycles with 0.1% acetic acid/0.01% Tween 20 (5 mL/100 mg hydrogel beads). Fresh 0.1% acetic acid/0.01% Tween 20 was pulled into the syringe to give a suspension of 10 mg/mL hydrogel based on initial weight. Amine content of the hydrogel beads was determined as described above. Based on equation (2) this refers to the degree of PEGylation.

(284) Example 22a was prepared according to the procedure described above using 50 mg of hydrogel 20a and 60.8 mg 40 kDA PEG-NHS to give PEGylated hydrogel beads with an amine content of 0.141 mmol/g.

(285) Example 22b was prepared according to the procedure described above using 51.3 mg of hydrogel 20b and 59.5 mg 40 kDA PEG-NHS to give PEGylated hydrogel beads with an amine content of 0.118 mmol/g.

Example 23

(286) Preparation of Maleimide Functionalized Hydrogel Beads 23

(287) PEGylated hydrogel beads as a suspension of 10 mg/mL based on initial weight of hydrogel beads prior to PEGylation were transferred into a syringe equipped with a frit. The solvent was expelled and the hydrogel washed ten times with water (5 mL/100 mg hydrogel beads), the solvent was discarded each time. The hydrogel beads were then washed ten times with NMP and five times with 2% DIPEA in NMP. 5 eq of Mal-PEG.sub.6-Pfp (based on amine content of the hydrogel beads) were dissolved in NMP (1 mL/50 mg reagent) and added to the washed hydrogel beads. The hydrogel suspension was incubated for 2 h at room temperature. Resulting maleimide functionalized hydrogel beads were washed five times each with NMP and afterwards with 0.1% acetic acid/0.01% Tween20.

(288) Maleimide content of hydrogel beads was determined by conjugation of a Fmoc-cysteine to the maleimide groups on the hydrogel and subsequent Fmoc-determination as described by Gude, M., J. Ryf, et al. (2002) Letters in Peptide Science 9(4): 203-206.

(289) Example 23a was prepared according to the procedure described above using 35 mg of 22a (based on initial dry weight of 20a) and 13 mg Mal-PEG.sub.6-Pfp.

(290) Example 23b was prepared according to the procedure described above using 35 mg of 22b (based on initial dry weight of 20b) and 17 mg Mal-PEG.sub.6-Pfp.

Example 24

(291) Synthesis of Transient Lucentis-Linker-Hydrogel Prodrug

(292) 80 mg Lucentis (depicted in the scheme below as Lucentis-NH.sub.2) (2000 L of 40 mg/mL Lucentis in 10 mM histidine, 10 wt % ,-trehalose, 0.01% Tween20, pH 5.5) was buffer exchanged to 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, pH 7.4 and the concentration of Lucentis was adjusted to 13.5 mg/mL. 6 mg of Linker reagent 4c was dissolved in 100 L DMSO to yield a concentration of 100 mM. 1 molar equivalent of linker reagent 4c relative to the amount of Lucentis was added to the Lucentis solution. The reaction mixture was mixed carefully and incubated for 5 min at room temperature. Subsequently, 1.3 additional molar equivalents of linker reagent 4c were added to the Lucentis solution yielding a mixture of unmodified Lucentis and the protected Lucentis-linker monoconjugate 24a.

(293) The pH of the reaction mixture was adjusted to pH 6.5 by addition of 1 M sodium citrate, pH 5.0 and Na.sub.2EDTA was added to a final concentration of 5 mM. To remove the protecting groups of 24a 0.5 M NH.sub.2OH (dissolved in 10 mM sodium citrate, 140 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 6.5) was added to a final concentration of 45 mM and the deprotection reaction was incubated at room temperature for 4 h yielding the Lucentis-linker monoconjugate 24b. The mixture of Lucentis and Lucentis-linker monoconjugate 24b was buffer exchanged to 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, 5 mM Na.sub.2EDTA, 0.01% Tween 20, pH 6.5 and the overall concentration of the two Lucentis species was adjusted to 12.1 mg/mL.

(294) ##STR00132##

Example 25

(295) Synthesis of Transient Lucentis-Linker-Hydrogel Prodrug 25a

(296) 20 mg of the Lucentis/Lucentis-linker monoconjugate 24b mixture in 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, 5 mM Na.sub.2EDTA, 0.01% Tween 20, pH 6.5 were added to 2.5 mg of maleimide functionalized hydrogel beads 5a and incubated overnight at room temperature yielding transient Lucentis-linker-hydrogel prodrug 25a.

(297) Example 25b was prepared according to the procedure described for 25a using 2.5 mg (based on dry weight of 20a) 23a.

(298) Example 25c was prepared according to the procedure described for 25b using 2.5 mg (based on dry weight of 20b) 23b.

Example 26

(299) Blocked Hydrogel Beads 26

(300) Hydrogel beads were synthesized according to the procedure described in example 3c and functionalized with maleimide groups according to the procedure described in example 5a. Afterwards, 4 mL of the hydrogel suspension at 10 mg/mL were transferred into a 20 mL syringe equipped with a frit. The solvent was expelled and the hydrogel washed 10 times with 5 mL 10 mM histidine/10 wt % ,-trehalose/0.01% Tween20/pH 5.5. The solvent was expelled and 5 mL 1 mM -mercaptoethanol in 10 mM histidine/10 wt % ,-trehalose/0.01% Tween20/pH 5.5 were drawn into the syringe. The resulting suspension was allowed to incubate at ambient temperature under gentle shaking for 5 min. The solvent was discarded and the hydrogel treated 9 additional times with 5 mL 1 mM -mercaptoethanol in 10 mM histidine/10 wt % ,-trehalose/0.01% Tween20/pH 5.5. The solvent was each time discarded. The hydrogel beads were then washed 10 times with each time 5 mL 10 mM histidine/10 wt % ,-trehalose/0.01% Tween20/pH 5.5, the solvent was discarded each time. The hydrogel beads were then washed ten times with each time 5 mL PBS-T/pH 7.4, the solvent was discarded each time. Finally, fresh PBS-T/pH 7.4 was drawn into the syringe and the suspension transferred into a Falcon tube to give 26.

Example 27

(301) Antibody Binding to Lucentis Hydrogel Beads 27

(302) 20 L of hydrogel suspensions 25a-c (35 volume-%) in PBS-T buffer were mixed with 400 L first antibody solution in PBS-T with 1% BSA (Sigma, A3059) and incubated for 1 h at 200 rpm in 1.5 mL Eppendorf tubes. For Lucentis hydrogel beads a 1:100 dilution of antibody ab771 (Anti-human IgG Fab fragment antibody [4A11] (ab771)Abcam, Cambridge, UK) was used. Hydrogel beads were sedimented through a centrifugation step at 100 g for 1 min in a tabletop centrifuge. The supernatant was removed by pipetting and care was taken not to remove any hydrogel beads. Washing of the beads was accomplished via two rounds of washing steps, which included addition of 1 mL PBS-T buffer, centrifugation at 100 g for 1 min and careful removal of the supernatant by pipetting. 400 L of the secondary antibody in PBS-T with 1% BSA (Sigma, A3059) were added to the beads and incubated for 1 h at 200 rpm. For Lucentis hydrogel beads a 1:50 dilution of antibody ab97041 (Goat Anti-Mouse IgG H&L (Phycoerythrin) preadsorbed (ab97041)Abcam, Cambridge, UK) was used. The supernatant was removed by pipetting and care was taken not to remove any hydrogel beads. Washing of the beads was accomplished via four rounds of washing steps, which included addition of 1 mL PBS-T buffer, centrifugation at 100 g for 1 min and careful removal of the supernatant by pipetting. The washed beads were resuspended in 200 L PBS-T and transferred completely into black 96-well plates (black, non-binding, Art. no. 655900, Greiner bio-one GmbH, 72636 Frickenhausen, Germany). The fluorescence intensity was determined with a Tecan Infinite M200 fluorescence plate reader (Excitation 495 nm, Emission 575 nm, Number of flashes 25, Integration time 20 s, Multiple reads per well 55 (Border 250 m), Optimal gain).

(303) Data Analysis

(304) Determination of antibody binding to PEG-modified Lucentis hydrogel beads was achieved in comparison with a standard curve of unmodified Lucentis hydrogel beads 25a. The unmodified Lucentis hydrogel beads were mixed with placebo hydrogel beads 26 in different ratios. The plot (percentage of unmodified Lucentis hydrogel beads versus fluorescence intensity) was fitted in a linear fashion. The percentage of antibody binding to PEGylated Lucentis hydrogel beads was back-calculated according to the obtained calibration curve.

(305) Results

(306) TABLE-US-00002 Residual antibody binding [% DevMean - Lucentis in relation to unmodified residual antibody Hydrogel Lucentis hydrogel] binding 25b 6.2 1.9 25c 5.3 0.2 Definitions: DevMean = Deviation from mean = | X|, = mean value

Example 28

(307) In Vitro Release KineticsDetermination of In Vitro Half-Life 28a

(308) 50.3 mg of dense Lucentis-linker-hydrogel prodrug suspension 25a (containing approximately 1.68 mg Lucentis) was washed five times with 60 mM sodium phosphate, 3 mM Na.sub.2EDTA, 0.01% Tween20, pH 7.4 and finally suspended in 1 mL of the aforementioned buffer. The suspension was incubated at 37 C. The buffer of the suspension was exchanged after different time intervals and analyzed by HPLC-SEC at 220 nm. Peaks corresponding to liberated Lucentis were integrated and the total of liberated Lucentis was plotted against total incubation time. Curve fitting software was applied to determine first-order cleavage rates.

(309) Release Kinetic of 28a:

(310) TABLE-US-00003 t [d] Lucentis release [g] Lucentis release [%] 0 0 0 4.85 141.0 8.4 12.9 262.2 15.6

(311) Example 28b was prepared according to the procedure described in 28a but with 35.0 mg of dense Lucentis-linker-hydrogel prodrug suspension 25b (containing approximately 1.14 mg Lucentis).

(312) Release Kinetic of 28b:

(313) TABLE-US-00004 t [d] Lucentis release [g] Lucentis release [%] 0 0 0 4.85 96.9 8.5 12.9 172.7 15.1

(314) Example 28c was prepared according to the procedure described in 28a but with 25.3 mg of dense Lucentis-linker-hydrogel prodrug suspension 25c (containing approximately 0.73 mg Lucentis).

(315) Release Kinetic of 28c:

(316) TABLE-US-00005 t [d] Lucentis release [g] Lucentis release [%] 0 0 0 4.85 51.8 7.3 12.9 96.5 13.5

Example 29

(317) Preparation of Thiol Functionalized Hydrogel Beads 29

(318) 100 mg of dry hydrogel beads as described in 3c with an amine content of 0.097 mmol/g were transferred into a 20 mL syringe equipped with a frit. The hydrogel was swollen in NMP and the solvent was discarded. The hydrogel was washed with five times with each time 5 mL NMP, the solvent was discarded each time. The hydrogel beads were washed five times with each time 5 mL 2% DIPEA in NMP, the solvent was discarded each time. The hydrogel beads were washed five times with 5 mL DMSO, the solvent was discarded each time. 17.7 mg OPSS-PEG.sub.12-NHS were dissolved in 1 mL DMSO and drawn into the syringe. The suspension was allowed to incubate for 2 hours under gentle shaking at ambient temperature. The solvent was expelled and the hydrogel beads washed five times with each time 5 mL DMSO and five times each time 5 mL 15 mM succinate/100 mM NaCl/5 mM EDTA/pH 4.0, the solvent was each time discarded. 10 mL 15 mM succinate/100 mM NaCl/5 mM EDTA/pH 4.0 buffer were drawn into the syringe and the resulting suspension was transferred into a Falcon Tube. 10 L Tween20 were added. 1 mL of this hydrogel suspension was transferred into a 5 mL syringe equipped with a frit and the solvent was expelled. The hydrogel beads were incubated with 1 mL of a 50 mM TCEP solution in water for 10 min at ambient temperature under gentle shaking. The solvent was expelled and the hydrogel beads were washed five times with each time 1 mL of a 50 mM TCEP solution in water, the solvent was discarded each time. The hydrogel beads were washed ten times with 1 mL 15 mM succinate/100 mM NaCl/5 mM EDTA/0.01% Tween20/pH 4.0, the solvent was each time discarded. 1 mL 15 mM succinate/100 mM NaCl/5 mM EDTA/0.01% Tween20/pH 4.0 was drawn into the syringe to give 29 as a hydrogel suspension of 10 mg/mL based on initial dry weight of the hydrogel beads.

Example 30

(319) Preparation of a Thiol Functionalized Histidine Tag 30

(320) ##STR00133##

(321) 0.2 g of Rink amide MBHA resin (100-200 mesh, 0.64 mmol/g amines) were transferred into a 5 ml, syringe equipped with a frit and swollen in 2 mL DMF for 10 min. The solvent was expelled and the resin was washed five times with 5 mL DMF, the solvent was discarded each time. The resin was treated for two times 15 minutes with 5 mL 20% piperidine in DMF, the solvent was discarded each time to remove the Fmoc protecting group. The resin was washed ten times with 2 mL DMF. 198 mg Fmoc-His(Trt)-OH and 146 mg HATU were dissolved in 1 mL 0.5M HOAT in DMF and 223 L DIPEA were added. The solution was drawn into the syringe and the suspension was allowed to incubate under gentle shaking for 1.5 hours at ambient temperature. The solvent was discarded and the resin was treated again for 1.5 hours with a solution of 198 mg Fmoc-His(Trt)-OH, 146 mg HATU and 223 L DIPEA in 1 mL 0.5M HOAT in DMF. The solvent was discarded and the resin was washed ten times with 2 mL DMF each time, the solvent was each time discarded. The Fmoc-protecting group was removed as described above. The next five couplings were each performed using a coupling solution of 198 mg Fmoc-His(Trt)-OH, 146 mg HATU and 223 L DIPEA in 1 mL 0.5M HOAt in DMF. The reaction was each time allowed to go for 1.5 hours at ambient temperature under gentle shaking followed by washing with DMF and Fmoc-deprotection. Afterwards, the resin was treated with a solution of 123 mg Fmoc-Ado-OH, 122 mg HATU, 223 L DIPEA dissolved in 1 mL 0.5M HOAt in DMF for 1.5 hours at ambient temperature followed by washing with DMF and Fmoc deprotection. The resin was treated with 112 mg 3-(tritylthio-)propionic acid, 122 mg HATU and 223 L DIPEA in 1 mL 0.5 M HOAt in DMF for one hour at ambient temperature under gentle shaking. The solvent was discarded and the resin washed ten times with DMF and ten times with DCM. The solvent was discarded each time. The resin was treated with each time 3 mL of a solution of 95% TFA/2.5% TIPS/2.5% water for two times 30 min and once for one hour. The solution was expelled into separate 50 mL Falcon tubes and the TFA evaporated under a continuous nitrogen stream. To the residue, 45 mL of ice cold ether were added and the Falcon tube was centrifugated and the supernatant discarded. The precipitate was taken up in 50% ACN/water and purified via prep. HPLC and lyophilized to give 30.

(322) Yield 8.1 mg (6%).

(323) MS: m/z 1073.46=[M+H].sup.+ (calculated=1073.46).

Example 31

(324) Preparation of 2-(2-pyridyldisulfanyl)ethanol 31

(325) ##STR00134##

(326) 250 mg aldrithiol-2 were dissolved in 10 mL 50 mM phosphate/ACN pH 7.4 and 95 L 2-mercaptoethanol were added. The solution was allowed to stir for 5 min at ambient temperature. Then, additional 50 mg of aldrithiol-2 were added. The solution was stirred for 16 h at ambient temperature. The crude product was purified via prep. HPLC and lyophilized to give 31.

(327) Yield 140.6 mg (55%).

(328) MS: m/z 188.02=[M+H].sup.+ (calculated=188.02).

Example 32

(329) Blocking of Thiol Functionalized Hydrogel 32

(330) 4 mg (based on dry weight of the initial hydrogel) of thiol functionalized hydrogel beads 29 were transferred as a suspension of 10 mg/mL in 15 mM succinate/100 mM NaCl/5 mM EDTA/0.01% Tween20/pH 4.0 into a 5 mL syringe equipped with a frit. The solvent was expelled and 322 L of a 5 mM solution of 31 in 15 mM succinate/100 mM NaCl/5 mM EDTA/0.01% Tween20/pH 4.0 was drawn into the syringe. The suspension was allowed to incubate at ambient temperature under gentle shaking for 16 h. The solvent was expelled and the hydrogel beads were washed ten times with 2 mL 15 mM succinate/100 mM NaCl/5 mM EDTA/0.01% Tween20/pH 4.0, the solvent was discarded each time.

Example 33

(331) Synthesis of Transient Lucentis-Linker-Hydrogel Prodrug 33d

(332) 360 mg Lucentis (depicted in the scheme below as Lucentis-NH.sub.2) (9 mL of 40 mg/mL Lucentis in 10 mM histidine, 10 wt % ,-trehalose, 0.01% Tween20, pH 5.5) was buffer exchanged to 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, pH 7.4 and the concentration of Lucentis was adjusted to 20 mg/mL. Linker reagent 11d was dissolved in DMSO to yield a concentration of 100 mM. 5 molar equivalents of linker reagent 11d relative to the amount of Lucentis were added to the Lucentis solution in 2, 2, and 1 molar equivalent steps. The reaction mixture was mixed carefully after each linker reagent addition and incubated for 5 min at room temperature yielding a mixture of unmodified Lucentis and the protected Lucentis-linker monoconjugate 33a.

(333) The pH of a quarter of the reaction mixture was adjusted to pH 6.5 by addition of 1 M sodium citrate, pH 5.0 and Na.sub.2EDTA was added to a final concentration of 5 mM. To remove the (5-methyl-2-oxo-1,3-dioxol-yl)-methyl oxocarbonyl protecting group of 33a 0.5 M NH.sub.2OH (dissolved in 10 mM sodium citrate, 140 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 6.5) was added to a final concentration of 45 mM and the deprotection reaction was incubated at room temperature for 135 min yielding the Lucentis-linker monoconjugate 33b. The mixture of Lucentis and Lucentis-linker monoconjugate 33b was buffer exchanged to 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 and subsequently concentrated to a concentration of 15 mg/mL. The protein solution was cooled to 4 C. and 1 molar equivalent of 25 mM DTT in 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 with respect to the overall Lucentis content was added to remove the Pys protecting group yielding the Lucentis-linker monoconjugate 33c. The mixture of unmodified Lucentis and the Lucentis-linker monoconjugate 33c was buffer exchanged to 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 and subsequently concentrated to a concentration of 28.7 mg/mL. The content of Lucentis-linker monoconjugate 33c in the mixture was 15% as determined by Ellman's assay.

(334) 79.7 mg of the Lucentis/Lucentis-linker monoconjugate 33c mixture in 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 was added to 5.1 mg of maleimide functionalized hydrogel beads 5c, the pH was adjusted to pH 5 by addition of 0.5 M succinic acid, pH 6.0 and incubated overnight at room temperature yielding transient Lucentis-linker-hydrogel prodrug 33d. Excess maleimides were blocked by eight incubation steps with 1 molar equivalent (with respect to maleimide content of maleimide functionalized hydrogel beads 5c) 1 mM 2-mercaptoethanol in 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 for 15 min at room temperature. In vitro release kinetic analysis to determine in vitro half-life of 33d was performed according to Example 7.

(335) ##STR00135## ##STR00136##

Example 34

(336) Synthesis of Transient Lucentis-Linker-Histidine-Tag Conjugate 34d

(337) 120 mg Lucentis (depicted in the scheme below as Lucentis-NH.sub.2) (3 mL of 40 mg/mL Lucentis in 10 mM histidine, 10 wt % ,-trehalose, 0.01% Tween20, pH 5.5) was buffer exchanged to 60 mM sodium phosphate, 100 mM sodium chloride, pH 7.4 and the concentration of Lucentis was adjusted to 20 mg/mL. Linker reagent 14f (only 1 regioisomer is depicted in the scheme below) was dissolved in DMSO to yield a concentration of 100 mM. 2 molar equivalents of linker reagent 14f relative to the amount of Lucentis were added to the Lucentis solution. The reaction mixture was mixed carefully and incubated for 5 min at room temperature. Subsequently, 1 additional molar equivalent of linker reagent 14f was added. Additional incubation for 5 min at room temperature yielded a mixture of unmodified Lucentis and the protected Lucentis-linker monoconjugate 34a. Incubation at room temperature for 4 h led to a quantitative conversion of Lucentis-linker monoconjugate 34a to Lucentis-linker monoconjugate 34b.

(338) The mixture of Lucentis and Lucentis-linker monoconjugate 34b was buffer exchanged to 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 and the protein concentration was adjusted to 11.8 mg/mL. The protein solution was cooled to 4 C. and 1 molar equivalent of 25 mM DTT in 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 with respect to the overall Lucentis content was added to remove the Pys protecting group yielding the Lucentis-linker monoconjugate 34c. The mixture of unmodified Lucentis and the Lucentis-linker monoconjugate 34c was buffer exchanged to 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 and subsequently concentrated to a concentration of 17.2 mg/mL.

(339) To 106.5 mg of the Lucentis/Lucentis-linker monoconjugate 34c mixture in 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 l molar equivalent with respect to the overall Lucentis content of maleimide containing histidine-tag 21 was added and the pH was adjusted to pH 5 by addition of 0.5 M succinic acid, pH 6.0. Incubation at room temperature for 4.5 h yielded the Lucentis-linker-histidine-tag conjugate 34d, which was purified by cation exchange chromatography from Lucentis and the excess maleimide containing histidine-tag 21.

(340) ##STR00137## ##STR00138##

Example 35

(341) In Vitro Release KineticsDetermination of In Vitro Half-Life of Transient Histidine-Tag Conjugates

(342) Cation exchange chromatography purified Lucentis-linker-histidine-tag conjugate 34d was buffer exchanged to 60 mM sodium phosphate, 3 mM Na.sub.2EDTA, 0.01% Tween20, pH 7.4 and the concentration was adjusted to 1 mg/mL. After incubation at 37 C. for different time intervals 200 g protein sample was analyzed by cation exchange chromatography. Amount of released Lucentis was determined by comparison of the peak areas of released Lucentis and Lucentis-linker-histidine-tag conjugate 34d and plotted against incubation time. Curve fitting software was applied to determine first-order cleavage rates.

Example 36

(343) Synthesis and Purification of Transient Tagged Lucentis-Linker Monoconjugate 36b

(344) 400 mg Lucentis (depicted in the scheme below as Lucentis-NH.sub.2) (10 mL of 40 mg/mL Lucentis in 10 mM histidine, 10 wt % ,-trehalose, 0.01% Tween20, pH 5.5) was buffer buffer exchanged to 60 mM sodium phosphate, 100 mM sodium chloride, pH 7.4 and the concentration of Lucentis was adjusted to 20.8 mg/mL. Linker reagent 17g was dissolved in DMSO to yield a concentration of 100 mM. 4.5 molar equivalents of linker reagent 17g relative to the amount of Lucentis were added to the Lucentis solution. The reaction mixture was mixed carefully and incubated for 5 min at room temperature yielding a mixture of unmodified Lucentis and the protected, tagged Lucentis-linker monoconjugate 36a.

(345) The mixture of Lucentis and protected, tagged Lucentis-linker monoconjugate 36a was buffer exchanged to 60 mM sodium phosphate, 100 mM sodium chloride, pH 6.5. To remove the (5-methyl-2-oxo-1,3-dioxol-yl)-methyl oxocarbonyl protecting group of 36a 0.5 M NH.sub.2OH (dissolved in 10 mM sodium citrate, 140 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 6.5) was added to a final concentration of 45 mM and the deprotection reaction was incubated at room temperature for 2.5 h yielding the tagged Lucentis-linker monoconjugate 36b which was separated from unmodified Lucentis by cation exchange chromatography.

(346) ##STR00139##

Example 37

(347) Synthesis of Transient Lucentis-Linker-Hydrogel Prodrug 37a Using Thiol/Activated Disulfide Conjugation Chemistry

(348) 61.5 mg of the Lucentis/Lucentis-linker monoconjugate 33b mixture (c=41 mg/mL) in 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 was added to 6.6 mg of thiol functionalized hydrogel beads 29. The hydrogel loading reaction was incubated for 60 h at 4 C. followed by 16 h at room temperature yielding transient Lucentis-linker-hydrogel prodrug 37a.

(349) ##STR00140##

Example 38

(350) Synthesis of Transient Lucentis-Linker-Hydrogel Prodrug 38a Using Thiol/Maleimide Conjugation Chemistry

(351) 10 mg Lucentis-linker monoconjugate 34c at a concentration of 20 mg/mL is added to 5 mg maleimide functionalized hydrogel 5c and the reaction mixture is incubated at pH 5 and room temperature overnight yielding the transient Lucentis-linker-hydrogel prodrug 38a. Excess maleimides are blocked by eight incubation steps with 1 molar equivalent (with respect to maleimide content of maleimide functionalized hydrogel beads 5c) 1 mM 2-mercaptoethanol in 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 for 15 min at room temperature.

(352) ##STR00141##

Example 39

(353) Synthesis of Transient Lucentis-Linker-Hydrogel Prodrug 39a Using Thiol/Activated Disulfide Conjugation Chemistry

(354) 50 mg of the Lucentis/Lucentis-linker monoconjugate 34b mixture at an overall Lucentis concentration of 40 mg/mL in 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 is added to 5 mg of thiol functionalized hydrogel beads 29 and the reaction mixture is incubated at room temperature overnight yielding the transient Lucentis-linker-hydrogel prodrug 39a. Excess thiol groups on the hydrogel are blocked by incubation with 5 molar equivalents (with respect to thiol content of thiol functionalized hydrogel beads 29) 1 mM solution of 31 in 15 mM succinic acid, 100 mM sodium chloride, 5 mM Na.sub.2EDTA, pH 4.0 for 16 h at room temperature.

(355) ##STR00142##

Example 40

(356) Synthesis of Transient Lucentis-Linker-Histidine-Tag Conjugate 40a

(357) Transient Lucentis-linker-histidine-tag conjugate 40a was prepared according to Example 33 using linker reagent 9c. Instead of maleimide functionalized hydrogel 5c 1 molar equivalent with respect to overall Lucentis content of maleimide functionalized histidine-tag 21 was used.

(358) ##STR00143##

Example 41

(359) Synthesis of Transient Lucentis-Linker-Histidine-Tag Conjugate 41a

(360) Transient Lucentis-linker-histidine-tag conjugate 41a was prepared according to Example 34 using linker reagent 13g.

(361) ##STR00144##

Example 42

(362) Synthesis of Transient Lucentis-Linker-Hydrogel Prodrug 42a

(363) Transient Lucentis-linker-hydrogel prodrug 42a was prepared according to Example 33 using linker reagent 15b. Due to the absence of the (5-methyl-2-oxo-1,3-dioxol-yl)-methyl oxocarbonyl protecting group no hydroxylamine assisted deprotection step was performed.

(364) ##STR00145##

Example 43

(365) Synthesis of Transient Lucentis-Linker-Hydrogel Prodrug 43a

(366) Transient Lucentis-linker-hydrogel prodrug 43a was prepared according to Example 33 using linker reagent 18i.

(367) ##STR00146##

(368) TABLE-US-00006 TABLE 1 Half-lives of synthesized Lucentis-linker-hydrogel prodrugs and Lucentis-linker-histidine tag conjugates. Prodrug/Conjugate Half-life at pH 7.4 and 37 C./d 6c 37 40a 37 33d 6 41a 5 34d 30 42a 28 43a 17

Example 44

(369) Synthesis of Transient Lucentis-Linker-Hydrogel Prodrug 44a

(370) 1147 mg of the Lucentis/Lucentis-linker monoconjugate 6b mixture in 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, 5 mM Na.sub.2EDTA, 0.01% Tween 20, pH 6.5 were added to 153 mg of maleimide functionalized hydrogel beads 5c and incubated overnight at room temperature yielding transient Lucentis-linker-hydrogel prodrug 44a.

(371) ##STR00147##

Example 45

(372) Evaluation of Binding Affinity of Lucentis Released from Lucentis-Linker-Hydrogel Prodrug 44a

(373) Active VEGF binding concentration of the Lucentis released from Lucentis-linker-hydrogel prodrug 44a was measured on a Biacore surface plasmon resonance system (Biacore T200, Pharmacia, Piscataway, N.J.). VEGF was covalently immobilized onto the carboxymethylated dextran sensor chip (CM5) using the amine coupling kit (GE Healthcare). The binding of Lucentis to VEGF was determined by monitoring the change in the resonance units before and after injection for 180 s. The active VEGF binding concentration was determined using a standard calibration curve prepared from serial dilution of reference material from 5 g/ml to 0.156 g/ml. The ratio of this active binding concentration by the total protein concentration determined by Bradford assay or UV-Vis absorbance gives the percent binding.

(374) For Lucentis-linker-hydrogel prodrug 44a it was shown that Lucentis released after 28 days and after more than 120 days showed 8010% binding.

Example 46

(375) Ranibizumab Measurements and Analysis

(376) For both groups, a qualified ligand-binding assay was designed to measure the concentrations of ranibizumab in rabbit vitreous matrix. For the Lucentis-linker-hydrogel prodrug 44a qualification, the hydrogel was determined to not interfere with ranibizumab quantitation at all concentrations tested. The assay used recombinant human VEGF-A to capture ranibizumab in rabbit vitreous samples. Bound ranibizumab was detected using an anti-human F(ab)2 conjugated to horseradish peroxidase (HRP), and a peroxidase substrate (TMB) was used for color development. The drug level was quantitated using absorbance spectrophotometry using a microplate reader. The concentration of ranibizumab in the study samples was calculated from the standard curve, and the minimum quantifiable concentration (MQC) in rabbit vitreous matrix was 1.5 ng/mL. The vitreous concentrations were plotted and analyzed using MATLAB software.

(377) TABLE-US-00007 TABLE 2 Ranibizumab concentrations (ng/mL) measured in vitreous humor after a 0.5 mg/eye intravitreal dose of Ranibizumab Time Individual point Animals Mean Std Dev of (day) Animal Eye.sup.a ng/mL (ng/mL) Mean 0.25 A1001 L 192186 201106 45418 0.25 A1001 R 231717 0.25 A1002 L 239944 0.25 A1002 R 140579 2 B1003 L 151619 153967 24199 2 B1003 R 124811 2 B1004 L 155490 2 B1004 R 183949 7 C1005 L 37730 58209 17247 7 C1005 R 50149 7 C1006 L 73097 7 C1006 R 71859 11 D1007 L 17493 17855 4869 11 D1007 R 13029 11 D1008 L 16311 11 D1008 R 24587 14 E1009 L 14788 11332 2983 14 E1009 R 9484 14 E1010 L 12768 14 E1010 R 8288 .sup.aL = left eye, R = right eye

(378) TABLE-US-00008 TABLE 3 Ranibizumab concentrations (ng/mL) measured in the vitreous humor following a 1.7 mg/eye intravitreal dose of Lucentis-linker-hydrogel prodrug 44a. Time Individual point Animals Mean Std Dev of (day) Animal Eyes.sup.a ng/mL (ng/mL) Mean 0.25 A2001 L 8097 10449 2771 0.25 A2001 R 14261 0.25 A2002 L 8744 0.25 A2002 R 10696 2 B2003 L 41525 37010 5387 2 B2003 R 34128 2 B2004 L 41534 2 B2004 R 30854 7 C2005 L 61395 64741 4887 7 C2005 R 68766 7 C2006 L 59709 7 C2006 R 69094 11 C2007 L 52090 44180 7745 11 C2007 R 34221 11 C2008 L 48043 11 C2008 R 42367 14 E2009 L 46066 46112 8243 14 E2009 R 35003 14 E2010 L 48671 14 E2010 R 54706 21 F2011 L NO ND ND SAMPLE.sup.b 21 F2011 R LTR 21 F2012 L LTR 21 F2012 R LTR 24 G2013 L 15259 12901 7308 24 G2013 R 2514 24 G2014 L 19609 24 G2014 R 14224 28 H2015 L LTR ND 28 H2015 R LTR ND 28 H2016 L 23589 28 H2016 R 23284 .sup.aL = left eye, R = right eye .sup.bno sample in tube LTR = lower than reportable ND = not determined

Abbreviations

(379) aq. aqueous Asp aspartate Boc tert-butyloxycarbonyl CIEC cationic ion exchange chromatography COMU (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate DBU 1,8-diazabicyclo(5.4.0)undec-7-ene DCC dicyclohexylcarbodiimide DCM dichloromethane DIPEA diisopropylethylamine DMAP dimethylaminopyridine DMF dimethylformamide DMSO dimethylsulfoxide DTT dithiothreitol EDTA ethylenediaminetetraacetic acid Fmoc fluorenylmethyloxycarbonyl HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate HFIP hexafluoroisopropanol HPLC high performance liquid chromatography iPrOH isopropanol Lys lysine max. maximal Maleimide-NH-PEG12-PFE N-(3-maleimidopropyl)-39-amino-4,7,10,13,16,19,22,25,28,31,34,37-dodecaoxa-nonatriacontanoic acid pentafluorophenyl ester Me methyl MeOAc methyl acetate MeOH methanol MES 2-(N-morpholino)ethanesulfonic acid MMT 4-methoxytriphenylmethyl MS mass spectrometry MTBE methyl-tert-butyl ether NHS N-hydroxysuccinimide Oxyma Pure ethyl 2-cyano-2-(hydroxyimino)acetate PEG polyethyleneglycol Pys 2-pyridinesulfenyl PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate RP-HPLC reversed phase high performance liquid chromatography RT room temperature sat. saturated Su N-hydroxysuccinimidyl tBu and t-Bu tert.-butyl TAN 1,5,9-triazanonane TES triethylsilane TFA trifluoroacetic acid THF tetrahydrofurane TLC thin layer chromatography TMEDA N,N,N,N-tetramethylethylene diamine Tmob 2,4,6-trimethoxybenzyl Trt trityl TSTU O(N-succinimidyl)-N,N,N,N-tetramethyluronium tetrafluoroborate