Conjugation Method for Carrier-Linked Prodrugs

Abstract

The present invention relates to reagents comprising a substituted acyl borate or a substituted hydroxylamine, to a method of synthesizing a carrier-linked prodrug using said reagents and to carrier-linked prodrugs obtainable by said method.

Claims

1. A reagent of formula (I)
D-(L.sup.1-L.sup.2-A).sub.a   (I), wherein -D is a biologically active moiety; each -L.sup.1- is independently a reversible prodrug linker; each -L.sup.2- is independently a chemical bond or a spacer; each -A is —X.sup.0 or —Y.sup.0; a is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 or 8; —X.sup.0 is a substituted acyl borate; and —Y.sup.0 is a substituted hydroxylamine.

2. The reagent of claim 1, wherein -D is selected from the group consisting of small molecule biologically active moieties, oligonucleotide moieties, peptide nucleic acid moieties, peptide moieties and protein moieties.

3. The reagent of claim 1 or 2, wherein -L.sup.1- is of formula (a-i): ##STR00070## wherein the dashed line indicates the attachment to a primary or secondary amine of -D by forming an amide bond; —X— is —C(R.sup.4R.sup.4a)—; —N(R.sup.4)—; —O—; —C(R.sup.4R.sup.4a)—C(R.sup.5R.sup.5a)—; —C(R.sup.5R.sup.5a)—C(R.sup.4R.sup.4a)—; —C(R.sup.4R.sup.4a)—N(R.sup.6)—; —N(R.sup.6)—C(R.sup.4R.sup.4a)—; —C(R.sup.4R.sup.4a)—O—; —O—C(R.sup.4R.sup.4a)—; or —C(R.sup.7R.sup.7a)—; >X.sup.1═ is C; or S(O); —X.sup.2— is —C(R.sup.8R.sup.8a)— or —C(R.sup.8R.sup.8a)—C(R.sup.9R.sup.9a)—; ═X.sup.3 is ═O; ═S; or ═N—CN; —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.6, —R.sup.8, —R.sup.8a, —R.sup.9, —R.sup.9a are independently selected from the group consisting of —H and C.sub.1-6 alkyl; —R.sup.3, —R.sup.3a are independently selected from the group consisting of —H and C.sub.1-6 alkyl, provided that in case one of —R.sup.3, —R.sup.3a or both are other than —H they are connected to N to which they are attached through a sp.sup.3-hybridized carbon atom; —R.sup.7 is —N(R.sup.10R.sup.10a); or —NR.sup.10—(C═O)—R.sup.11; —R.sup.7a, —R.sup.10, —R.sup.10a, —R.sup.11 are independently of each other —H; or C.sub.1-6 alkyl; optionally, one or more of the pairs —R.sup.1a/—R.sup.4a, —R.sup.1a/—R.sup.5a, —R.sup.1a/—R.sup.7a, —R.sup.4a/—R.sup.5a, —R.sup.8a/—R.sup.9a form a chemical bond; optionally, one or more of the pairs —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.8/—R.sup.8a, —R.sup.9/—R.sup.9a are joined together with the atom to which they are attached to form a C.sub.3-10 cycloalkyl; or 3- to 10-membered heterocyclyl; optionally, one or more of the pairs —R.sup.1/—R.sup.4, —R.sup.1/—R.sup.5, —R.sup.1/—R.sup.6, —R.sup.1/—R.sup.7a, —R.sup.4/—R.sup.5, —R.sup.4/—R.sup.6, —R.sup.8/—R.sup.9, —R.sup.2/—R.sup.3 are joined together with the atoms to which they are attached to form a ring A.sup.0; optionally, R.sup.3/R.sup.3a are joined together with the nitrogen atom to which they are attached to form a 3- to 10-membered heterocycle; A.sup.0 is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl; C.sub.3-10 cycloalkyl; 3- to 10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl; and wherein -L.sup.1- is substituted with 1, 2, 3, 4, 5, 6, 7 or 8 moieties -L.sup.2-A and wherein -L.sup.1- is optionally further substituted, provided that the hydrogen marked with the asterisk in formula (a-i) is not replaced by -L.sup.2-A or a substituent.

4. A reagent of formula (II))
Z-(L.sup.2′-B.sup.0).sub.b   (II), wherein —Z is a carrier; -L.sup.2′- is a chemical bond or a spacer; —B.sup.0 is selected from the group consisting of —X.sup.0 and —Y.sup.0; b is an integer of at least 1; —X.sup.0 is a substituted acyl borate; and —Y.sup.0 is a substituted hydroxylamine.

5. The reagent of claim 4, wherein —Z is a water-soluble carrier.

6. The reagent of claim 4 or 5, wherein —Z comprises a C.sub.8-24 alkyl moiety or a polymeric moiety.

7. The reagent of any one of claims 4 to 6, wherein —Z comprises a PEG-based polymer.

8. The reagent of any one of claims 4 to 6, wherein —Z comprises a hyaluronic acid-based polymer.

9. The reagent of any one of claims 4 to 6, wherein —Z comprises a random coil protein moiety.

10. The reagent of claim 9, wherein —Z comprises a PG moiety.

11. The reagent of any one of claims 5 to 10, wherein b is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12

12. The reagent of any one of claims 1 to 11, wherein -L.sup.2- and -L.sup.2′- are selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.y1)—, —S(O).sub.2N(R.sup.y1)—, —S(O)N(R.sup.y1)—, —S(O).sub.2—, —S(O)—, —N(R.sup.y1)S(O).sub.2N(R.sup.y1a)—, —S—, —N(R.sup.y1)—, —OC(OR.sup.y1)(R.sup.y1a)—, —N(R.sup.y1)C(O)N(R.sup.y1a)—, —OC(O)N(R.sup.y1)—, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T-, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one or more —R.sup.y2, which are the same or different and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.y3)—, —S(O).sub.2N(R.sup.y3)—, —S(O)N(R.sup.y3)—, —S(O).sub.2—, —S(O)—, —N(R.sup.y3)S(O).sub.2N(R.sup.y3a)—, —S—, —N(R.sup.y3)—, —OC(OR.sup.y3)(R.sup.y3a)—, —N(R.sup.y3)C(O)N(R.sup.y3a)—, and —OC(O)N(R.sup.y3)—; —R.sup.y1 and —R.sup.y1a are independently of each other selected from the group consisting of —H, -T, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one or more —R.sup.y2, which are the same or different, and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.y4)—, —S(O).sub.2N(R.sup.y4)—, —S(O)N(R.sup.y4)—, —S(O).sub.2—, —S(O)—, —N(R.sup.y4)S(O).sub.2N(R.sup.y4a)—, —S—, N(R.sup.y4), —OC(OR.sup.y4)(R.sup.y4a)—, N(R.sup.y4)C(O)N(R.sup.y4a)— and —OC(O)N(R.sup.y4)—; each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; wherein each T is independently optionally substituted with one or more —R.sup.y2, which are the same or different; each —R.sup.y2 is independently selected from the group consisting of halogen, —CN, oxo(═O), —COOR.sup.y5, —OR.sup.y5, —C(O)R.sup.y5, —C(O)N(R.sup.y5R.sup.y5a), —S(O).sub.2N(R.sup.y5R.sup.y5a), —S(O)N(R.sup.y5R.sup.y5a), —S(O).sub.2R.sup.y5, —S(O)R.sup.y5, —N(R.sup.y5)S(O).sub.2N(R.sup.y5aR.sup.y5b), —SR.sup.y5, —N(R.sup.y5R.sup.y5a), —NO.sub.2, —OC(O)R.sup.y5, —N(R.sup.y5)C(O)R.sup.y5a, —N(R.sup.y5)S(O).sub.2R.sup.y5a, —N(R.sup.y5)S(O)R.sup.y5a, —N(R.sup.y5)C(O)OR.sup.y5a, —N(R.sup.y5)C(O)N(R.sup.y5aR.sup.y5b), —OC(O)N(R.sup.y5R.sup.y5a), and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and each —R.sup.y3, —R.sup.y3a, —R.sup.y4, —R.sup.y4a, —R.sup.y5, —R.sup.y5a and —R.sup.y5b is independently selected from the group consisting of —H, and C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

13. The reagent of any one of claims 1 to 12, wherein -L.sup.2- and -L.sup.2′- are selected from the group consisting of C.sub.1-10 alkyl, phenyl, naphthyl, azulenyl, indenyl, indanyl, C.sub.3-10 cycloalkyl, 3- to 10- membered heterocyclyl and -A and —B.sup.0 are —X.sup.0.

14. The reagent of claim 13, wherein -L.sup.2- and -L.sup.2′- are selected from C.sub.1-10 alkyl and phenyl.

15. The reagent of any one of claims 1 to 12, wherein -L.sup.2- and -L.sup.2′- are C.sub.1-10 alkyl and -A and —B.sup.0 are —Y.sup.0.

16. The reagent of claim 15, wherein -L.sup.2- and -L.sup.2′- are C.sub.6 alkyl.

17. The reagent of any one of claims 1 to 14, wherein —X.sup.0 is of formula (III) ##STR00071## wherein the dashed line indicates attachment to -L.sup.2- or -L.sup.2′-, respectively; —X.sup.1, —X.sup.2 and —X.sup.3 are independently of each other selected from the group consisting of —F, —OR, —N.sup.+R.sub.3, —N.sup.+R.sub.2OR, —N.sup.+R.sub.2SR and —N.sup.+R.sub.2NR.sub.2; each —R is independently selected from the group consisting of —H, -T.sup.0, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T.sup.0, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one or more —R.sup.x1, which are the same or different and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T.sup.0-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.x2)—, —S(O).sub.2N(R.sup.x2)—, S(O)N(R.sup.x2), —S(O).sub.2—, —S(O)—, —N(R.sup.x2)S(O).sub.2N(R.sup.x2a)—, —S—, —N(R.sup.x2)—, —OC(OR.sup.x2)(R.sup.x2a), —N(R.sup.x2)C(O)N(R.sup.x2a)—, and —OC(O)N(R.sup.x2)—; each T.sup.0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 30-membered heteropolycyclyl; wherein each T.sup.0 is independently optionally substituted with one or more —R.sup.x1, which are the same or different; each —R.sup.x1 is independently selected from the group consisting of halogen, -T.sup.0, —CN, oxo (═O), —COOR.sup.x3, —OR.sup.x3, —C(O)R.sup.x3, —C(O)N(R.sup.x3R.sup.x3a), —S(O).sub.2N(R.sup.x3R.sup.x3a), —S(O)N(R.sup.x3R.sup.x3a), —S(O).sub.2R.sup.x3, —S(O)R.sup.x3, —N(R.sup.x3)S(O).sub.2N(R.sup.x3aR.sup.x3b), —SR.sup.x3, —N(R.sup.x3R.sup.x3a), —NO.sub.2, —OC(O)R.sup.x3, —N(R.sup.x3)C(O)R.sup.x3a, —N(R.sup.x3)S(O).sub.2R.sup.x3a, —N(R.sup.x3)S(O)R.sup.x3a, —N(R.sup.x3)C(O)OR.sup.x3a, —N(R.sup.x3)C(O)N(R.sup.x3aR.sup.x3b), —OC(O)N(R.sup.x3R.sup.x3a), and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; each —R.sup.x2, —R.sup.x2a, —R.sup.x3, —R.sup.x3a, —R.sup.x3b is independently selected from the group consisting of —H and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; optionally, two or three of —X.sup.1, —X.sup.2 and —X.sup.3 are joined together with the atom to which they are attached to form a ring A.sup.0; A.sup.0 is selected from the group consisting of 3- to 10-membered heterocyclyl and 8- to 30-membered heteropolycyclyl.

18. The reagent of claim 17, wherein —X.sup.1, —X.sup.2 and —X.sup.3 are —F.

19. The reagent of any one of claim 1 to 12, 15 or 16, wherein each —Y.sup.0 is independently of formula (IV) ##STR00072## wherein the dashed line indicates attachment to -L.sup.2- or -L.sup.2′-, respectively; —R.sup.a1 is selected from the group comprising —COOR.sup.x1, —OR.sup.x1, —C(O)R.sup.x1, —C(O)N(R.sup.x1R.sup.x1a), —S(O).sub.2N(R.sup.x1R.sup.x1a), —S(O)N(R.sup.x1R.sup.x1a), —S(O).sub.2R.sup.x1, —S(O)R.sup.x1, —S(O).sub.2OR.sup.x1, —N(R.sup.x1R.sup.x1a), -T.sup.0, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl; wherein -T.sup.0, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl are optionally substituted with one or more —R.sup.x2, which are the same or different and wherein C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T.sup.0-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.x3)—, —S(O).sub.2N(R.sup.x3)—, —S(O)N(R.sup.x3)—, —S(O).sub.2—, —S(O)—, —N(R.sup.x3)S(O).sub.2N(R.sup.x3a)—, —S—, —N(R.sup.x3)—, —OC(OR.sup.x3)(R.sup.x3a)—, —N(R.sup.x3)C(O)N(R.sup.x3a)—, and —OC(O)N(R.sup.x3)—; —R.sup.x1, —R.sup.x1a, —R.sup.x1b are independently of each other selected from the group consisting of —H, -T.sup.0, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl; wherein -T.sup.0, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl are optionally substituted with one or more —R.sup.x2, which are the same or different and wherein C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T.sup.0-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.x3)—, —S(O).sub.2N(R.sup.x3)—, —S(O)N(R.sup.x3)—; —S(O).sub.2—, —S(O)—, —N(R.sup.x3)S(O).sub.2N(R.sup.x3a)—, —S—, —N(R.sup.x3)—, —OC(OR.sup.x3)(R.sup.x3a)—, —N(R.sup.x3)C(O)N(R.sup.x3a)—, and —OC(O)N(R.sup.x3)—; each T.sup.0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T.sup.0 is independently optionally substituted with one or more —R.sup.x2, which are the same or different; each —R.sup.x2 is independently selected from the group consisting of halogen, -T.sup.0, —CN, oxo(═O), —COOR.sup.x4, —OR.sup.x4, —C(O)R.sup.x4, —C(O)N(R.sup.x4R.sup.x4a), —S(O).sub.2N(R.sup.x4R.sup.x4a), —S(O)N(R.sup.x4R.sup.x4a), —S(O).sub.2R.sup.x4, —S(O)R.sup.x4, —N(R.sup.x4)S(O).sub.2N(R.sup.x4aR.sup.x4b), —SR.sup.x4, —N(R.sup.x4R.sup.x4a), —NO.sub.2, —OC(O)R.sup.x4, —N(R.sup.x4)C(O)R.sup.x4a, —N(R.sup.x4)S(O).sub.2R.sup.x4a, —N(R.sup.x4)S(O)R.sup.x4a, —N(R.sup.x4)C(O)OR.sup.x4a, —N(R.sup.x4)C(O)N(R.sup.x4aR.sup.x4b), —OC(O)N(R.sup.x4R.sup.x4a), and C.sub.1-4 alkyl; wherein C.sub.1-4 alkyl is optionally substituted with one or more halogen, which are the same or different; each —R.sup.x3, —R.sup.x3a, —R.sup.x4, —R.sup.x4a, —R.sup.x4b is independently selected from the group consisting of —H and C.sub.1-4 alkyl; wherein C.sub.1-4 alkyl is optionally substituted with one or more halogen, which are the same or different.

20. The reagent of claim 19, wherein —R.sup.a1 of formula (IV) is selected from the group consisting of methyl, ethyl, propyl, ##STR00073## wherein —R.sup.c1 and —R.sup.c1a are independently of each other selected from the group consisting of —H, -T.sup.0 and C.sub.1-6 alkyl; —R.sup.d is selected from the group consisting of C.sub.1-6 alkyl and —NO.sub.2, —CN, —C(O)OR.sup.e,, —S(O).sub.2OR.sup.e; each -T.sup.0 is independently of each other selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each -T.sup.0 is independently optionally substituted with one or more —R.sup.e, which are the same or different; each —R.sup.e is independently selected from the group consisting of C.sub.1-6 alkyl; n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; m is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; p is selected from 0, 1, 2, 3, 4 and 5;

21. The reagent of any one of claim 19 or 20, wherein —R.sup.a1 ##STR00074##

22. A method of synthesizing a carrier-linked prodrug, the method comprising the step of reacting a reagent of formula (I) of any one of claims 1 to 3 or 12 to 21 under aqueous conditions with a reagent of formula (II) of any one of claims 4 to 21, with one of -A and —B.sup.0 being —X.sup.0 and the other one being —Y.sup.0, and forming an amide bond between moiety -A and a moiety —B.sup.0.

23. The method of claim 22, wherein the method is performed at a pH ranging from and including 1 to 8.

24. The method of claim 22 or 23, wherein the method is performed at a pH ranging from and including 3 to 5.

25. The method of any one of claims 22 to 24, wherein the method is conducted in an aqueous buffer, which aqueous buffer may comprise additives.

26. The method of any one of claims 22 to 25, wherein the reaction is conducted in a solvent mixture selected from the group consisting of THF/H.sub.2O, CH.sub.3CN/H.sub.2O, tBuOH/H.sub.2O and DMSO/tBuOH/H.sub.2O.

27. The method of any one of claims 22 to 26, wherein the method of the present invention is performed for 1 minute to 5 hours.

28. A carrier-linked prodrug obtainable by the method of any one of claims 22 to 27.

Description

EXAMPLES

[0648] Materials and Methods

[0649] Materials:

[0650] The protein moiety “PA” is obtained as described in WO 2011/144756 A1, wherein the sequence was a 20-mer of SEQ ID NO:1 as disclosed therein.

[0651] Hyaluronic acid (HA, 90-130 kDa lab grade, batch 214-9272) was purchased from Contipro Biotech, Czech Republic.

[0652] Sunbright® GL2-200PA, was purchased from NOF Europe N.V., Grobbendonk, Belgium.

[0653] Dipentafluorophenylcarbonate was obtained from IRIS Biotech GmbH, Germany.

[0654] COMU, EDC.HCl, NHS, TBTU and PyBOP were purchased from Novabiochem, Germany.

[0655] Fmoc-N-Me-Asp(OBn)-OH was obtained from Peptide International Inc., Louisville, Ky., USA. All other protected amino acids were obtained from Bachem, Switzerland.

[0656] HMPB-ChemMatrix® resin, all other chemicals and solvents were purchased from Sigma-ALDRICH Chemie GmbH, Taufkirchen, Germany.

[0657] Polypeptides Fmoc-GP(80)-OH, Ac-GP(80)-OH and Fmoc-Lys(Cbz)-GP(80)-OH were synthesized by standard Fmoc synthesis strategy using a Biotage® Initiator+Alstra™ peptide synthesizer with microwave (Biotage AB, Sweden) on HMPB-ChemMatrix® resin preloaded with glycine. Besides standard Fmoc amino acids Fmoc-Gly-Pro-OH was used to introduce proline into the sequence. The sequence of GP(80) is SEQ ID NO:2: (GGPGGPGPGGPGGPGPGGPG).sub.4

[0658] Methods:

[0659] Reactions were performed with anhydrous solvents (DCM, THF, ACN, DMF, MeOH, NMP). Generally, reactions were stirred at room temperature and monitored by HPLC/MS or TLC.

[0660] RP-HPLC was performed on a XBridge BEH300 C18 OBD Prep 10 μm 30×150 mm or 5 μm 10×150 mm (Waters, Eschborn, Germany) connected to a Waters 600 or 2535 HPLC System and Waters 2487 or 2489 Absorbance detector, respectively. If not indicated otherwise, linear gradients of solution A (0.1% TFA in H.sub.2O (v/v)) and solution B (0.1% TFA in acetonitrile (v/v)) were used. HPLC fractions containing product were combined and lyophilized.

[0661] 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, dichloromethane, acetonitrile and methanol as eluents. Products were detected at 254 nm. For products showing no absorbance above 240 nm fractions were screened by TLC or LC/MS.

[0662] 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 or an Agilent technologies 1290 Infinity II system with a G4212A diode array and a G6120B single quad MS system. A Waters ACQUITY UPLC BEH300 C18 RP column (2.1×50 mm, 300 Å, 1.7 μm, flow: 0.25 mL/min; solvent A: water+0.04% TFA (v/v), solvent B: acetonitrile+0.05% TFA (v/v) was used in all cases.

Example 1

[0663] 1d and 1e were synthesized according to the following scheme:

##STR00052##

[0664] A solution of tert-butyl-N-(benzyloxy)carbamate (4.00 g, 17.9 mmol) in DMF (20 mL) was added slowly to a suspension of sodium hydride (60% in mineral oil) (0.932 g; 23.3 mmol) in DMF (52 mL) at 0° C. The reaction was stirred at 0° C. for 1 h, then ethyl 6-bromohexanoate (15.9 mL, 89.6 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The reaction was quenched with sat. ammonium chloride solution (130 ml) and the mixture was extracted with DCM (3×200 ml). The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The obtained crude material (1a) was used in the next step without further purification.

[0665] 1a was dissolved in methanol (140 mL). The solution was split in two equal parts and two reactions were carried out in parallel. 10% Pd/C (190 mg, 0.18 mmol) was added to each solution. The reaction mixtures were purged with H.sub.2 and stirred under H.sub.2-atmosphere (balloon) for 15.5 h. The reaction mixtures were filtered through a plug of celite, which was washed with MeOH. The volatiles were removed in vacuo. The crude product was purified by flash chromatography to yield 1b as colorless oil.

[0666] Yield: 4.73 g, 96% over 2 steps.

[0667] To a solution of 1b (4.70 g, 17.1 mmol) in DCM (150 mL), DMAP (688 mg, 5.63 mmol), DIPEA (5.95 mL, 34.1 mmol) and N,N-diethylcarbamoyl chloride (6.49 mL, 51.2 mmol) were added. The reaction mixture was stirred at room temperature for 14 h. The reaction was quenched with sat. ammonium chloride solution (180 ml) and the layers were separated. The aqueous phase was extracted with DCM (3×180 ml). The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The crude product was purified by flash chromatography to yield 1c as colorless oil.

[0668] Yield: 6.27 g, 98%.

[0669] 1c (6.21 g, 16.6 mmol) was dissolved in a mixture of THF (186 mL), MeOH (62 mL) and water (62 mL). Lithium hydroxide (1.11 g, 46.4 mmol) was added and the reaction mixture was stirred for 4 h at room temperature. The reaction-mixture was cooled down in an ice bath and 1 M HCl was added until the pH reached pH 2. The mixture was extracted with 375 ml of ethyl acetate three times. The combined organic phase was dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The crude product was purified by flash chromatography to yield 1d as slightly yellow oil.

[0670] Yield: 5.27 g, 92%.

[0671] To a solution of 1d (1.97 g, 5.69 mmol) in DCM (15.8 mL), NHS (982 mg, 8.53 mmol) and EDC.HCl (1.64 g, 8.53 mmol) were added. The reaction mixture was stirred for 1.5 h at room temperature. The reaction mixture was diluted with 210 ml DCM and the solution was washed two times with 210 ml of 0.1 M aq. HCl and once with 210 ml of brine. The organic phase was dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The residue (1e) was used without further purification.

[0672] Yield: 2.72 g.

Example 2

[0673] Linker reagent 2f was synthesized according to the following scheme:

##STR00053##

[0674] To a solution of N-methyl-N-Boc-ethylenediamine (2.05 ml, 11.48 mmol) and NaCNBH.sub.3 (685 mg, 10.9 mmol) in MeOH (20 mL) was added 2,4,6-trimethoxybenzaldehyde (2.14 g, 10.9 mmol) as a solution in MeOH/DCM 1:1 v/v (40 ml) over 2 h. The mixture was stirred at room temperature for 1 h, acidified with 0.4 M HCl (60 mL) and stirred further 30 min. The reaction mixture was extracted 4 times with 150 mL ethyl acetate. The combined organic phase was washed with sat. NaHCO.sub.3 solution (200 mL) and brine (90 mL), dried over Na.sub.2SO.sub.4 and the solvents were evaporated under reduced pressure. The resulting N-methyl-N-Boc-N′-Tmob-ethylenediamine 2a was dried in vacuo and used in the next reaction step without further purification.

[0675] Yield: 4.02 g, 99%, colorless oil.

[0676] To a solution of Fmoc-N-Me-Asp(OBzl)-OH (4.77 g, 10.4 mmol) in DCM (147 mL), EDC.HCl (2.58 g, 13.48 mmol), OxymaPure (2.06 g, 14.5 mmol) and 2,4,6-collidine (2.6 mL, 19.7 mmol) were added. The mixture was stirred for 5 minutes. A solution of 2a (3.97 g, 11.2 mmol) in DCM (36.8 mL) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with 300 mL of 0.1 M HCl. The aqueous phase was extracted twice with 80 mL DCM. The organic phases were combined and washed with sat. NaHCO.sub.3 (1×140 mL, 2×70 mL) and brine (80 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtrated and the residue concentrated in vacuo. 2b was purified using flash chromatography.

[0677] Yield: 6.73 g, 82%.

[0678] 2b (6.71 g, 8.43 mmol) was dissolved in THF (67 mL). DBU (1.48 mL, 9.90 mmol) was added. The reaction mixture was stirred for 12 minutes. The reaction mixture was directly subjected to flash chromatography to yield 2c.

[0679] Yield: 4.63 g, 96%.

[0680] 1d (800 mg, 2.31 mmol) was dissolved in DCM (7 ml). PyBOP (1.20 g, 2.31 mmol) and DIPEA (366 μL, 2.10 mmol) were added. Last, a solution of 2c (1.20 g, 2.10 mmol) in DCM (8 ml) was added. The reaction was stirred for 72 h. The reaction mixture was diluted with DCM (180 ml) and the solution was washed three times with 120 ml of 0.1 M aqueous HCl and one time with 120 ml of brine. The organic phase was dried over Na.sub.2SO.sub.4 and filtered. The solvent was evaporated. 2d was purified using flash chromatography.

[0681] Yield: 1.69 g, 89%.

[0682] 2d (1.65 g, 1.83 mmol) was dissolved in a mixture of isopropanol (20 mL) and water (20 mL). LiOH (131 mg, 5.49 mmol) was added and the reaction stirred for 3 h. The reaction mixture was diluted with DCM (200 ml) and the solution was washed three times with 120 ml of 0.1 M aqueous HCl. The aqueous phase was extracted with 100 ml of dichloromethane and the organic phases were combined. The organic phase was washed with 120 mL of brine, dried over Na.sub.2SO.sub.4 and filtered. The solvent was evaporated. 2e was purified using flash chromatography.

[0683] Yield: 706 mg, 48%.

[0684] 2e (363 mg, 0.45 mmol) was dissolved in DCM (4.36 mL). NHS (77 mg, 0.67 mmol), and EDC.HCl (129 mg, 0.67 mmol) were added. The reaction mixture was stirred for 5.7 h at room temperature. The reaction mixture was diluted with 100 ml DCM and the solution was washed two times with 66 ml of 0.1 M aqueous HCl and once with 66 ml of brine. The organic phase was dried over Na.sub.2SO.sub.4 and was filtered. The solvent was evaporated. 2f was purified using flash chromatography.

[0685] Yield: 319 mg, 78 %.

Example 3

[0686] Linker reagent 3e was synthesized according to the following scheme:

##STR00054##

[0687] Four reactions were carried out in parallel. To a solution of compound 2b (60 g, 75 mmol) in CH.sub.2Cl.sub.2 (300 mL) was added piperidine (58 g, 0.68 mol, 67 mL). The reaction mixture was stirred at room temperature for 4 h. The four reactions which were performed in parallel were combined for work-up. The reaction mixture was diluted with H.sub.2O (500 mL) and adjusted with a 0.5 N HCl solution to pH=3-4. The organic phase was separated and the aqueous phase was extracted with CH.sub.2Cl.sub.2 (800 mL). The combined organic phases were washed with brine (400 mL) and 5% saturated NaHCO.sub.3 solution (400 mL). Then, the combined organic phases were dried over Na.sub.2SO.sub.4, filtered and the solvent was evaporated in vacuo. 3a was purified by chromatography on silica (100-200 mesh) with DCM/MeOH (20/1 to 4/1).

[0688] Yield: 150 g, 87%.

[0689] Two reactions were carried out in parallel. To a solution of Fmoc-Lys(Fmoc)-OH (79 g, 0.13 mol), 3a (70 g, 0.12 mol), 4-ethyl-morpholine (70 g, 0.61 mol, 77 mL) in MeCN (850 mL), T.sub.3P (50% in EtOAc; 140 g, 0.22 mol) was added dropwise over a period of 30 min. After addition, the reaction mixture was stirred at room temperature for 18 h. The two reactions which were performed in parallel were combined for work-up. The reaction mixture was diluted with H.sub.2O/CH.sub.2Cl.sub.2 (1:1, 2 L) and then adjusted with 0.5 N HCl solution to pH=3-4. The organic phase was separated and the aqueous phase was extracted with CH.sub.2Cl.sub.2 (1 L). The combined organic phases were washed with brine (800 mL) and 5% NaHCO.sub.3 solution (800 mL) in turn. Then, the combined organic phases were dried over Na.sub.2SO.sub.4, filtered and the solvent was evaporated in vacuo. 3b was purified by chromatography on silica (100-200 mesh) with petroleum ether/ethyl acetate (5/1 to 1/1).

[0690] Yield: 160 g, 57%.

[0691] Two reactions were carried out in parallel. To a solution of 3b (60 g, 52 mmol) in MeOH (1.2 L) was added 10% Pd/C (18 g) in a 2 L vessel. The reaction mixture was degassed and purged 3× with H.sub.2 and then stirred at 25° C. under H.sub.2-atmosphere (45 psi) for 2.5 h. The two reactions which were performed in parallel were combined for work-up. The reaction mixture was filtered by diatomite and the filtrate was concentrated in vacuo to give crude 3c. 3c was purified by chromatography on silica (100-200 mesh) with DCM/MeOH (200/1 to 100/3).

[0692] Yield: 70 g, 63%.

[0693] MS: m/z 1056.4=[M+H].sup.+, (calculated monoisotopic mass=1056.50).

[0694] 3c (1.10 g, 1.05 mmol) was dissolved in DCM (5.56 mL) and DBU (312 μL, 2.09 mmol) was added. The reaction mixture was stirred for 40 min. A solution of 1e (1.50 g, 3.14 mmol) in DCM (5.56 mL) was added. The reaction mixture was stirred for 30 min. The reaction mixture was diluted with ethyl acetate (240 mL) and washed three times with a mixture of 0.1 N HCl (150 mL) and brine (45 mL). The organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. 3d was purified using flash chromatography.

[0695] Yield: 1.07 g, 81%.

[0696] 3d (1.04 g, 0.82 mmol) was dissolved in DCM (12.5 mL). NHS (142 mg, 1.23 mmol) and EDC.HCl (236 mg, 1.23 mmol) were added. The reaction mixture was stirred for 3.5 h. The reaction mixture was diluted with 180 ml DCM and the solution was washed two times with 120 ml of 0.1 M aqueous HCl and once with 120 ml of brine. The organic phase was dried over Na.sub.2SO.sub.4 and was filtered. The solvent was evaporated. 3e was purified using flash chromatography.

[0697] Yield: 518 mg, 43%.

Example 4

[0698] Synthesis of CNP Linker Hydroxylamine:

[0699] Protected CNP was synthesized according to International application PCT/EP2017/050220 example 39c. The used CNP has the amino acid sequence of SEQ ID NO:3: LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC, wherein the cysteines at position 22 and 38 are connected through a disulfide-bridge. The linker was attached to lysine at position 26 according to the following scheme:

##STR00055##

[0700] General procedure: Protected CNP (1.0 eq) and the respective linker (2f or 3e, 1.1 eq) were dissolved in DMF (6.7 ml/g protected CNP). DIPEA (3 eq) was added and the reaction mixture was stirred for 20 h. The peptide was precipitated by addition of diethyl ether. The precipitate was washed twice with diethyl ether. The precipitate was dissolved in TFE/DCM and deprotected in TFA/TES/water/thioanisole 95:3:2.5:1 v/v/v/v for 1 h. The peptide was precipitated again with diethyl ether. The crude was purified by RP-HPLC.

[0701] 4a: Yield: 10.6 mg, 30%, 10× TFA salt.

[0702] MS: m/z 1119.10=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1119.10).

[0703] 4b: Yield: 5.7 mg, 20%, 10× TFA salt.

[0704] MS: m/z 1208.16=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1208.16).

Example 5

[0705] ##STR00056##

[0706] Potassium 5-hydroxypentanoyltrifluoroborate (50 mg, 0.24 mmol) and bis(pentafluorophenyl) carbonate (189 mg, 0.48 mmol) were dissolved in NMP (1 ml). The solution was cooled to 0° C. in an ice-bath and potassium carbonate (133 mg, 0.96 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The suspension was filtered and the precipitate was washed with NMP (0.5 ml). The filtrate was added to 45 ml heptane/diethyl ether 1:1. A white precipitate formed, and the suspension was kept at −20° C. for 30 minutes. The suspension was centrifuged, the supernatant was discarded and the residue washed once with 45 mL ice-cold heptane/diethyl ether 1:1. The residue was dried in vacuo. The crude material was purified by RP-HPLC with water/acetonitrile (no TFA) to yield 5.

[0707] Yield: 24 mg, 24%.

Example 6

[0708] Synthesis of KAT Functionalized 20 kDA-PEG

##STR00057##

[0709] Sunbright® GL2-200PA (20 kDa mPEG amine, 642 mg, 32 μmol) was dissolved in NMP (6.5 ml). Compound 5 (134 mg, 0.32 mmol) was dissolved in NMP (1.25 mL) and DIPEA (56 μL, 0.32 mmol) was added. Both solutions were mixed and stirred for 30 min. The product was precipitated in MTBE, dissolved in DCM and precipitated again from MTBE. The residue was dried in vacuo. 295 mg of this residue were further purified by dialysis (MWCO 3500) (3×0.1 M potassium phosphate buffer pH 7, 3× water). The product was lyophilized, dissolved in water, filtered and the filtrate was lyophilized.

[0710] Yield: 240 mg, 67%.

Example 7

[0711] Synthesis of H-GP(161)-OH

[0712] Fmoc-GP(80)-OH was synthesized by standard solid phase peptide synthesis. By coupling H-Gly-OtBu to the Fmoc-GP(80)-OH fragment in solution using PyBOP and DIPEA in DMF followed by Fmoc deprotection (20% piperidine in DMF), polypeptide H-GP(80)G-OtBu was obtained.

[0713] Fragment coupling of Fmoc-GP(80)-OH and H-GP(80)G-OtBu using PyBOP and DIPEA in DMSO and cleavage of protecting groups (Fmoc: 20% piperidine in DMF, tBu: 50% TFA in DCM) yielded polypeptide H-GP(161)-OH (7). Each intermediate and final polypeptide 7 were purified by RP-HPLC.

[0714] Yield: 87 mg of H-(GGPGGPGPGGPGGPGPGGPG).sub.8G-OH*1×TFA (7)

[0715] MS: m/z 818.52=[M+14H].sup.14+, (calculated monoisotopic mass for [M+14H].sup.14+=818.17).

Example 8

[0716] Synthesis of Ac-GP(80)-Lys-GP(81)-OH

[0717] Ac-GP(80)-OH and Fmoc-Lys(Cbz)-GP(80)-OH were synthesized on solid phase. H-Gly-OtBu was coupled to Fmoc-Lys(Cbz)-GP(80)-OH (PyBOP, DIPEA, NMP) in solution followed by Fmoc deprotection (20% piperidine in DMF) to yield H-Lys(Cbz)-GP(80)G-OtBu.

[0718] Fragment coupling of Ac-GP(80)-OH and H-Lys(Cbz)-GP(80)G-OtBu (PyBOP, DIPEA, DMSO) followed by global deprotection (Fmoc: 20% piperidine in DMF, tBu: 50% TFA in DCM, Cbz: H.sub.2, Pd/C, DMF) produced polypeptide Ac-GP(80)-Lys-GP(81)-OH (8). Each intermediate and final product 8 were purified by RP-HPLC.

[0719] Yield: 133 mg of Ac-(GGPGGPGPGGPGGPGPGGPG).sub.4K(GGPGGPGPGGPGGPGPGGPG).sub.4G-OH*1×TFA (8)

[0720] MS: m/z 1659.76=[M+7H].sup.7+, (calculated monoisotopic mass for [M+7H].sup.7+=1659.63).

Example 9

[0721] Synthesis of KAT Functionalized GP-Polypeptides 9a and 9b:

##STR00058##

[0722] Polypeptide 7 or 8 respectively (20 mg, 1.70 μmol) was dissolved in DMSO (250 μl). Potassium carbonate (2.4 mg, 17 μmol) was added. KAT reagent 5 (1.4 mg, 3.4 μmol) in 250 μl DMSO was added and the reaction stirred for 15 min. The resulting polypeptide (9a or 9b) was precipitated with diethyl ether (10 ml). The suspension was centrifuged, the supernatant decanted and the precipitate washed again with diethyl ether (10 ml). The residue was dried in vacuo and purified by RP-HPLC.

[0723] 9a (using 7): 26.7 mg.

[0724] MS: m/z 894.57=[M-KF+13H].sup.13+, (calculated monoisotopic mass for [M-KF+13H].sup.13+=894.57).

[0725] 9b (using 8): 9.5 mg.

[0726] MS: m/z 907.66=[M-KF+13H].sup.13+, (calculated monoisotopic mass for [M-KF+13H].sup.13+=907.65).

Example 10

[0727] The following conjugates were synthesized:

##STR00059## ##STR00060##

[0728] 10a: KAT functionalized polypeptide 9a (13.4 mg, 1.15 μmol) was used as stock solution in water (424 μl). CNP linker hydroxylamine 4a (4.5 mg, 0.8 μmol) was dissolved in ACN/water 1:1 containing 0.1% TFA v/v/v (5 ml). The KAT reagent solution was successively added until all CNP linker hydroxylamines reacted (total of 331 μl, 1 h reaction time) while the reaction was agitated. The product 10a was purified by RP-HPLC.

[0729] Yield: 4.1 mg, 30%.

[0730] MS: m/z 938.19=[M+17H].sup.17+, (calculated m/z for [M+17H].sup.17+=938.25).

[0731] 10b: KAT functionalized polypeptide 9b (5 mg, 0.42 μmol) was used as stock solution in water (169 μl). CNP linker hydroxylamine 4a (2.0 mg, 0.35 μmol) was dissolved in ACN/water 1:1 containing 0.1% TFA v/v/v (140 μl). 28 μl 1 M sodium citrate buffer (pH 4) was added, then the KAT reagent solution (140 μl) was added and the reaction agitated for 15 min. The product 10b was purified by RP-HPLC.

[0732] Yield: 4.2 mg, 69%.

[0733] MS: m/z 947.98=[M+17H].sup.17+, (calculated m/z for [M+17H].sup.17+=948.26).

[0734] 10c: KAT functionalized polypeptide 9a (7.7 mg, 0.5 μmol) was used as stock solution in water (100 μl). CNP linker hydroxylamine 4b (1.4 mg, 0.24 μmol) was dissolved in ACN/water 1:2 containing 0.1% TFA v/v/v (7.5 ml). The KAT reagent solution was successively added until all CNP linker hydroxylamines reacted (total of 82.5 μl, 5 h reaction time) while the reaction was agitated. The product 10c was purified by RP-HPLC.

[0735] Yield: 2 mg, 29%.

[0736] 10d: KAT functionalized polypeptide 9b (4.3 mg, 0.36 μmol) was used as stock solution in water (100 μl). CNP linker hydroxylamine 4b (1.1 mg, 0.19 μmol) was dissolved in ACN/water 1:2 containing 0.1% TFA v/v/v (5.7 ml). The KAT reagent solution was successively added until all CNP linker hydroxylamines reacted (total of 95.2 μl, 1.5 h reaction time) while the reaction was agitated. The product 10d was purified by RP-HPLC.

[0737] Yield: 1.8 mg, 34%.

[0738] MS: m/z 1221.65=[M+23H].sup.23+, (calculated m/z for [M+23H].sup.23+=1222.23).

[0739] 10e: PEG-KAT 6 was used as stock solution in water (50 mg/ml). CNP linker hydroxylamine 4b (4.2 mg, 0.7 μmol) was dissolved in 100 mM sodium citrate buffer (pH 4, 140 μl). 17.5 μl 1 M sodium citrate buffer (pH 3.9) containing 40 mM methionine was added to 160 μl of the PEG-KAT solution, then the solution of 4b (15 μl, 0.08 μmol) was added and the reaction agitated for 22 h. The product 10e was purified by SEC on an Aekta Purifier 100 system, using a Superdex 200 10/30 GL column and pH 7.4 buffer (10 mM phosphate, 140 mM NaCl, 3 mM KCl, 3 mM EDTA, 10 mM methionine, 0.03% Tween® 20) as eluent.

Example 11

[0740] Release Kinetics In Vitro

[0741] CNP conjugates 10a-d were dissolved in 60 mM sodium phosphate, 20 mM methionine, 3 mM EDTA, pH 7.4 at a concentration of 0.14 mg/mL. 10e was obtained from SEC in 10 mM phosphate, 140 mM NaCl, 3 mM KCl, 3 mM EDTA, 10 mM methionine, 0.03% Tween® 20, pH 7.4. The five solutions were incubated at 37° C. At various time points aliquots were withdrawn and analysed by RP-HPLC and ESI-MS. UV-signals were integrated and the ratio of the integral of liberated CNP to the total integrated area was plotted against incubation time.

[0742] Curve-fitting software was applied to estimate the corresponding half-life and plateau of release:

TABLE-US-00001 compound Half-life plateau 10a 3.5 d 98% 10b 3.7 d 97% 10c 4.9 d 98% 10d 4.9 d 98% 10e 8.7 d 98%

[0743] The plateaus show near quantitative release of CNP.

Example 12

[0744] Synthesis of Carboxylic Acid 12

##STR00061##

[0745] Carboxylic acid 12 was synthesized according to JACS 2014, 136, 5611 (supporting information pages S11-12).

Example 13

[0746] Synthesis of Hydroxylamine Functionalized TransCon Linker 13b

##STR00062##

[0747] Building block 12 (1.2 eq), 3a (1 eq), COMU (2 eq) and DIPEA (4 eq) are stirred in CH.sub.2Cl.sub.2 until full conversion is observed by LCMS. The reaction mixture is diluted with CH.sub.2Cl.sub.2 and washed 3 times with 0.1 M HCl and 3× with brine. The combined aqueous phases are extracted with CH.sub.2Cl.sub.2. The combined organic phases are dried over Na.sub.2SO.sub.4, filtrated and concentrated. The crude material is purified using flash chromatography to yield 13a.

[0748] To a solution of 13a (45 g, 52 mmol) in MeOH (1.2 L) is added 10% Pd/C (18 g) in a 2 L hydrogenated bottle. The reaction mixture is degassed and purged three times with H.sub.2 and then stirred at 25° C. under H.sub.2-atmosphere (45 psi) for 2.5 h. The reaction mixture is filtered through diatomite and the filtrate is concentrated in vacuo to give crude 13b. 13b is purified by flash chromatography with CH.sub.2Cl.sub.2/MeOH.

Example 14

[0749] Synthesis of Linker Conjugate 14

##STR00063##

[0750] Product 14 is synthesized by Fmoc solid phase peptide synthesis starting with Ramage Resin (e.g. IRIS Biotech GmbH). GRF(1-29) refers to the following peptide sequence: H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH.sub.2. Protected amino acids and hydroxyl amine building block 13b are coupled with TBTU (2.5 eq) and DIPEA (5 eq) in DMF. Fmoc deprotections are performed with 2:2:96 piperidine/DBU/DMF. Side chain protecting groups and the peptide-resin bond are cleaved by stirring the protected product-resin in a cleavage cocktail consisting of 90:5:5 TFA/TES/H.sub.2O. The crude material is purified by preparative HPLC. The product containing fractions are pooled and lyophilized to yield linker conjugate 14.

Example 15

[0751] Synthesis of KAT Reagent 15d

##STR00064##

[0752] Acetic acid anhydride (5 eq) is added to a solution of PA (1 eq) and DIPEA (10 eq) in DMF. The reaction mixture is stirred for 30 min at room temperature and the acetylated PA 15a is isolated by precipitation.

[0753] Ac-PA (1 eq), N-Boc-ethylene diamine (1.1 eq), PyBOP (1.5 eq) and DIPEA (3 eq) are dissolved in DMF and stirred until full conversion is observed. The product is purified by preparative HPLC. A solution of TFA/CH.sub.2Cl.sub.2 (1:1) is added to Ac-PA-NHCH.sub.2CH.sub.2NHBoc and stirred for 1 h at room temperature. The solution is concentrated and dried under high vacuum to yield 15b.

##STR00065##

[0754] Potassium 5-hydroxypentanoyltrifluoroborate (1 eq), 4-nitrophenyl chloroformate (1 eq) and triethylamine (2 eq) are stirred in NMP. After 2 h product 15c is precipitated with Et.sub.2O. Activated KAT reagent 15c is dissolved in NMP and 15b (1 eq) and NEt.sub.3 (8 eq) in NMP are added and the reaction is stirred at room temperature. The product 15d is isolated by preparative HPLC using ACN/water as eluent.

Example 16

[0755] Synthesis of TransCon PA GRF(1-29)

##STR00066##

[0756] Building block 14 (1 eq) is reacted with KAT polymer 15d (1 eq) in potassium citrate buffer (pH 4) at room temperature. The mixture is stirred until good conversion is observed by LCMS. The product is purified by HPLC.

[0757] Accordingly, the reaction can be performed with HA-KAT reagent 17.

Example 17

[0758] Synthesis of HA-KAT Reagent 17b

##STR00067##

[0759] Hyaluronic acid sodium salt (116 kDa, 1.00 g; 2.49 mmol; 1 eq.) was dissolved in a solution of 0.4 M 1.3-diaminopropane in 100 mM MES buffer (pH 5.5, 125 mL) under vigorous stirring. To the clear, colorless and viscous solution HOBt (1.15 g; 7.48 mmol; 3.00 eq.) was added. The mixture was treated in an ultrasonic bath until all lumps moldered and a grey suspension was formed. EDC.HCl (98.48 mg; 513.72 μmol; 0.21 eq.) was added. Upon dissolution of the carbodiimide, the suspension was stirred at room temperature overnight. Sodium acetate trihydrate (16.97 g; 124.69 mmol; 50.00 eq.) was added and a colorless, viscous and clear solution was formed immediately. After sodium acetate trihydrate dissolved completely, the solution was partitioned between fourteen 50 mL Falcon tubes (10 mL each). The HA was precipitated by addition of absolute EtOH (ad 50 mL). The tubes were closed, vigorously shaken and centrifuged at 8000 rpm for 3 minutes. After discarding the supernatant, the pellets were successively washed with EtOH, combined and dried under high vacuum. The obtained white pellets were dissolved in water (80.00 mL) to yield a clear, colorless and viscous solution. To this solution 4 M NaOH (26.62 mL) was added stirred at room temperature. Acetic acid (6.09 mL) was added, the pH was checked with pH-paper: pH 8.5 and the solution was filtered through a 150 mL bottletop filter into a 150 mL Corning bottle. The filtered solution was partitioned between eleven 50 mL Falcon tubes (10 mL each). The HA was precipitated by addition of absolute EtOH (ad 50 mL). The tubes were closed, vigorously shaken and centrifuged at 8000 rpm for 3 minutes. After discarding the supernatant, the obtained white pellets were washed with EtOH. After discarding the supernatant, the pellets were combined and dried under high vacuum to yield 17a.

[0760] Yield: 905 mg; 90%, white powder.

[0761] Amine content: 0.083 mmol/g.

[0762] Accordingly, hyaluronic acids with up to 60% derivatization can be synthesized.

[0763] Aminofunctionalized HA 17a (1 eq) is dissolved in buffer (pH 8). Compound 15c (5 eq) is added and the reaction mixture is stirred at room temperature until sufficient conversion is observed. KAT functionalized HA 17b is purified by precipitation.

Example 18

[0764] Synthesis of PA-Hydroxylamine Reagent 18

##STR00068##

[0765] Polymer 15b (1 eq), carboxylic acid 12 (2 eq) and COMU (2 eq) are dissolved in DMF. DIPEA (4 eq) is added and the reaction mixture is stirred at room temperature. After full conversion was detected by LCMS, polymer 18 was purified by preparative HPLC.

Example 19

[0766] Synthesis of HA-Hydroxylamine Reagent 19

##STR00069##

[0767] Hyaluronic acid 17a (1 eq), carboxylic acid 12 (0.3 eq), EDC.HCl (0.3 eq) and HOBt (3 eq) are stirred in MES buffer (pH 5.5) overnight. The functionalized HA intermediate is purified by precipitation.

[0768] Functionalized HA (228.00 mg; 1 eq.) is dissolved in water (11.40 ml) in a 50 ml-Falcon tube under vigorous shaking to yield a viscous solution (2% w/v HA) within 3.5 hours. TFA (3.80 ml; 0.05 mol; 88.96 eq.) is added to the HA solution and the reaction mixture is shaken vigorously. After 60 minutes the HA is precipitated from the viscous solution by addition of acetone. The precipitate is washed with EtOH and dried under high vacuum to yield 19.

[0769] Reagents 18 and 19 can be used to attach polymers to KAT functionalized linker-drug conjugates. If these linkers are designed to be cleavable (as in example 5), large prodrugs with extended plasma half-lives are formed.

[0770] Abbreviations: [0771] Ac acetyl [0772] Ac-PA N-terminal acetylated polypeptide containing proline and alanine residues [0773] ACN acetonitrile [0774] aq. aqueous [0775] Asp aspartate [0776] Bn benzyl [0777] Boc tert-butyloxycarbonyl [0778] Bzl benzyl [0779] Cbz benzyloxycarbonyl [0780] CNP C-type natriuretic peptide [0781] COMU (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate [0782] DBU 1,8-diazabicyclo (5.4.0)undec-7-ene [0783] DCM dichloromethane [0784] DIPEA diisopropylethylamine [0785] DMAP dimethylaminopyridine [0786] DMF dimethylformamide [0787] eq equivalent [0788] EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [0789] EDTA ethylene diamine tetraacetic acid [0790] Et ethyl [0791] Fmoc fluorenylmethyloxycarbonyl [0792] GRF growth hormone-releasing factor [0793] GP(80) glycine proline polypeptide (number of amino acids indicated in bracket) [0794] HA hyaluronic acid [0795] HOBt 1-hydroxybenzotriazole [0796] HPLC high performance liquid chromatography [0797] KAT potassium acyltrifluoroborate [0798] LC liquid chromatography [0799] LCMS liquid chromatography mass spectrometry [0800] Lys lysine [0801] Me methyl [0802] MeOH methanol [0803] MeCN acetonitrile [0804] MES 2-(N-morpholino)ethanesulfonic acid [0805] MTBE methyl-tert-butylether [0806] MWCO molecular weight cut-off [0807] MS mass spectrum/mass spectrometry [0808] NHS N-hydroxysuccinimide [0809] NMP N-methyl-2-pyrrolidone [0810] OxymaPure ethyl-(hydroxyimino)cyanoacetate [0811] PA proline/alanine polypeptide [0812] PEG polyethylene glycol [0813] pNP para-nitrophenyl [0814] PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate [0815] RP reversed phase [0816] sat. saturated [0817] SEC size exclusion chromatography [0818] TBTU N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate [0819] T.sub.3P 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide [0820] tBu and t-Bu tert-butyl [0821] TES triethylsilane [0822] TFA trifluoroacetic acid [0823] TFE trifluoroethanol [0824] THF tetrahydrofuran [0825] TLC thin layer chromatography [0826] Tmob 2,4,6-trimethoxybenzyl [0827] UPLC ultra performance liquid chromatography