INSOLUBLE SUPPORT FOR SOLID PHASE SYNTHESIS

Abstract

The present invention relates to an insoluble support comprising distal binding sites, the support comprising a homogeneous polymeric matrix and constructs, the constructs covalently bound to the polymeric matrix, wherein the constructs comprise at least one branching agent selected from aminoalkanoic acids comprising at least 2 amino groups and from 3 up to 10 carbon atoms, cleavable linkers and at least one spacer coupled to at least one branching agent via an amide bond, the cleavable linkers providing the distal binding sites.

Claims

1. An insoluble support (resin) in particulate form comprising distal binding sites, the support comprising a homogeneous polymeric matrix and constructs, the constructs covalently bound to the polymeric matrix, wherein the constructs comprise at least one branching agent selected from aminoalkanoic acids comprising at least 2 amino groups and from 3 up to 10 carbon atoms, cleavable linkers and at least one spacer coupled to at least one branching agent via an amide bond, the cleavable linkers providing the distal binding sites.

2. The insoluble support according to claim 1, wherein the at least one branching agent is selected from aminoalkanoic acids comprising at least 2 but not more than 3 amino groups and from 3 up to 10 carbon atoms.

3. The insoluble support according to claim 1, wherein the at least one branching agent is selected from diaminoalkanoic acids comprising from 3 up to 10 carbon atoms.

4. The insoluble support according to claim 1, wherein the at least one branching agents are selected from diaminoalkanoic acids comprising from 3 up to 8 carbon atoms.

5. The insoluble support according to claim 1, wherein the at least one branching agent is selected from 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutyric acid and 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid, suitably the branching agent is selected from 2,4-diaminobutyric acid and 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid.

6. The insoluble support according to claim 1, wherein the at least one branching agent is lysine.

7. The insoluble support according to claim 1, wherein all branching agents are identical.

8. The insoluble support according to claim 3, wherein the number of branching agents d of a construct is given by the formula d(n)=2.sup.n1 and the number of linkers providing distal binding site 1 is given by the formula l(n)=2.sup.n, where n denotes the number of generations of branching agents and n being from 1 to 10.

9. The insoluble support according to claim 8, wherein n is from 2 to 10.

10. The insoluble support according to claim 9, wherein the at least one spacer is positioned between all branching agents.

11. The insoluble support according to claim 3, wherein the constructs are selected from: (A) [polymeric matrix]-BA(1)-LK.sub.2; (B) [polymeric matrix]-BA(1)-BA(2).sub.2-LK.sub.4; (C) [polymeric matrix]-BA(1)-BA(2).sub.2-BA(3).sub.4-LK.sub.8; (D) [polymeric matrix]-BA(1)-BA(2).sub.2-BA(3).sub.4-BA(4).sub.8-LK.sub.16; (E) [polymeric matrix]-BA(1)-BA(2).sub.2-BA(3).sub.4-BA(4).sub.8-BA(5).sub.16-LK.sub.32; where BA denotes a branching agent and the integer in brackets indicates the generation of branching agent, and LK denotes cleavable linkers.

12. The insoluble support according to claim 1; wherein the at least one spacer molecule is selected from organic molecules comprising two binding sites.

13. The insoluble support according to claim 1, wherein the at least one spacer molecule is selected from organic molecules comprising two binding sites selected form any one of carboxylic acids, amines, hydroxyls.

14. The insoluble support according to claim 1; wherein the at least one spacer molecule is selected from organic molecules comprising two binding sites selected form amino acids and polyethylene glycol.

15. The insoluble support according to claim 1, wherein at least one spacer is selected from amino acids comprising two binding sites, suitably glycine and alanine.

16. The insoluble support according to claim 1, wherein the linkers are selected form Rink amide, Wang, 2-chlorotrityl, PAM, PAL, HMPB, Sieber and Ramage.

17. The insoluble support according to claim 1; wherein the polymeric matrix is selected from homogeneous polymeric matrices comprising primary binding sites distributed throughout the polymeric matrix.

18. The insoluble support according to claim 1, wherein the polymeric matrix is selected from homogeneous polymeric matrices formed by emulsion polymerization comprising at least styrene and divinylbenzene (DVB).

19. The insoluble support according to claim 1, wherein the polymeric matrix is selected from homogeneous polymeric matrices formed from a polymerization composition comprising at least styrene and divinylbenzene (DVB) and DVB being present in an amount of below 4.0 wt %.

20. (canceled)

21. (canceled)

22. A method for forming an insoluble support as defined by claim 1, the method comprising providing a polymeric matrix comprising primary binding sites, and wherein the construct is formed by a divergent synthesis approach, a convergent synthesis approach or a combined divergent and convergent synthesis approach.

23. A method for forming an insoluble support as defined by claim 1, the method comprising providing a polymeric matrix comprising primary binding sites, and wherein the constructs are formed by a method comprising at least the steps: a) optionally coupling at least one spacer to the primary binding sites, b) coupling a branching agent to the primary binding sites of the base matrix, or optionally coupling the branching agent to at least one spacer, where the at least two amino groups are protected by protecting groups, c) removal of the protecting groups, where steps a), b) and c) may be repeated, and d) coupling of linkers to binding sites of distal branching agents.

24. A solid phase peptides synthesis protocol, solid phase morpholino oligomer synthesis and solid phase oligonucleotides synthesis for the synthesis of polypeptides, morpholino oligomers and oligonucleotides, the protocol comprising using an insoluble support as defined by claim 1.

25. A solid phase peptides synthesis protocol for the synthesis of polypeptides, the protocol comprising using an insoluble support as defined by claim 1.

26. The solid phase peptides synthesis protocol according to claim 25, wherein the polypeptides have at least 15 amino acids.

Description

DETAILED DISCLOSURE OF CERTAIN ASPECT OF THE INVENTION

[0215] Hereinafter is disclosed insoluble supports comprising constructs proving two 2, four 4, seven 7, eight 8 and sixteen-fold 16 increase of the initial (primary) binding sites of the polymeric matrix. Lysine (K or Lys) is used throughout as the branching agent. Where applicable glycine (G or Gly), polyethylene glycol (PEG.sub.6) or beta-alanine (Ala) are used as a spacer. PEG.sub.6 denotes that the PEG contains six individual PEG moieties covalently bound by peptide bonds.

[0216] Aminomethyl (AMS) polystyrene resins are used as the starting polymeric matrix for the synthesis of all insoluble supports presented below. All AMS resins used are amino methyl modified polystyrene resin cross-linked with 1% divinylbenzene. The AMS resins are provided in beads in the 35-150 m (100 to 200 mesh) range with a degree of substitution of 0.6, 0.93 and 1.95 mmol/gram, respectively

[0217] As Lysine is used as the branching agent, the constructs of the disclosed insoluble supports in this section can be denoted as branched polypeptides synthesized on a polymeric matrix.

[0218] For all insoluble supports lysine in the S-configuration (L amino acid) and R-configuration (D amino acid) has been used.

[0219] For all insoluble supports (2(Gly), 4(Gly), 8(Gly), 16(Gly)) except 7 linear the branching agent lysine is provided with two Fmoc groups for the protection of the two amines, i.e. the branching agent is provided as Fmoc-Lys(Fmoc)-OH.

[0220] The 7 linear is a linear construct. In the synthesis of the linear 7 insoluble support two classes of lysine have been used differing in terms of amine protection groups. One class of lysines has both amine groups protected by Fmoc: Fmoc-Lys(Fmoc)-OH. The other class of lysines have the alpha amine protected by Fmoc and the epsilon amine protected by Mtt: Fmoc-Lys(Mtt)-OH.

Presentation of the Structures of Some of the Reactants:

[0221] Structural formulas of Fmoc-Lys(Fmoc) (R and S configuration) where both amine groups are Fmoc protected:

##STR00004##

[0222] Structural formulas of Fmoc-Lys(Mtt)-OH (S configuration) where the N-terminal, alpha amine is Fmoc protected and the side chain, epsilon amine group is Mtt or Mmt protected.

##STR00005##

[0223] Fmoc-Rink Amide linker (Fmoc-Rink-OH) has the following chemical structure:

##STR00006##

Fmoc Ramage Linker (MW: 505.58):

##STR00007##

HMPB TBDMS and HMPB Linkers:

##STR00008##

[0224] The glycine spacer is provided as an Fmoc protected glycine. The PEG is provided as 2-[2-[2-(Fmoc amino) ethoxy]ethoxy] acetic acid (EAAE).

##STR00009##

[0225] Comments to the nomenclatures of the exemplified insoluble supports:

[0226] The number in parenthesis with respect to the branching agent lysine K (or Lys) denotes the layer or generation whereas the subscript denotes the total number of lysines of each layer/generation. E.g. K(2).sub.2 or Lys(2).sub.2 denotes that the construct of the insoluble support has two secondary lysines.

Disclosure of Solid Supports

[0227] Solids supports 2(Gly), 4(Gly) and 8(Gly) based on a 0.60 mmol/g AMS polymeric matrix (without linkers) were synthesized using the following protocol:

[0228] Starting AMS resin with an initial loading of 0.60 mmol/g (30.0 g, table 1) was introduced into 1 L glass reactor equipped with mechanical stirrer and swollen in DMF (8 mL/g of starting resin, 21 h). All amino acid couplings were carried out using a molar ratio of 1:1:1.25 of Fmoc amino acid (4.0 eq.) in DMF (0.50M), Oxyma (4.0 eq.) and NN-Diisopropylcarbodiimide (5.0 eq.). Fmoc amino acids (Fmoc-Gly-OH or Fmoc-Lys(Fmoc)-OH) were dissolved in DMF (0.50M) followed by the addition of Oxyma and NN-Diisopropylcarbodiimide and pre-activated for 30 min at room temperature before being transferred to the glass reactor. Couplings were carried out for 2 h at room temperature. Following coupling, the resin was washed with DMF (28 mL/g), capped using DMF/acetic anhydride (Ac.sub.2O)/Diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 min and re-washed using DMF (78 mL/g). Fmoc groups were deprotected prior to each coupling step using 20% piperidine in DMF (110 min+120 min) at room temperature. Following Fmoc-deprotection, the resin was washed using DMF (78 mL/g of starting resin).

[0229] Following the completion of the 2-Gly-AMS synthesis, the resin was washed with CH.sub.2Cl.sub.2 (3), iPrOH (3) and MTBE (3) prior to its drying under vacuum overnight. of the obtained resin was conserved while the other (28.2 g, table 1) were used for the synthesis of 4-Gly-AMS in the same way as 2-Gly-AMS.

[0230] Following the completion of the 4-Gly-AMS synthesis, the resin was washed with CH.sub.2Cl.sub.2 (3), iPrOH (3) and MTBE (3) prior to its drying under vacuum overnight. of the obtained resin was conserved while the other was used for the synthesis of 8-Gly-AMS in the same way as 2-Gly-AMS.

[0231] Following the completion of the 8-Gly-AMS synthesis, the resin was washed with CH.sub.2Cl.sub.2 (3), iPrOH (3) and MTBE (3) prior to its drying under vacuum overnight.

TABLE-US-00001 TABLE 1 Mass of starting Mass of polymeric Mass of modified matrix modified polymeric (AMS 0.60 polymeric matrix mmol/g) matrix (insoluble Type of of each (insoluble support) insoluble synthesis support) conserved support [g] [g] [g] 2X(Gly)-AMS 0.6 30.0 of AMS-0.6 42.30 14.10 4X(Gly)-AMS 0.6 28.2 of 2X(Gly)-AMS-0.6 42.60 28.40 8X(Gly)-AMS 0.6 14.2 of 4X(Gly)-AMS 0.6 20.38 20.38

[0232] Solids supports 2(Gly) and 4(Gly) based on a 0.93 mmol/g AMS polymeric matrix and 1.95 mmol/g AMS polymeric matrix respectively (without linkers) were synthesized using the following protocol:

[0233] Starting AMS resin with an initial loading of 0.93 or 1.95 mmol/g (1.0 or 2.0, tables 2 and 3) was introduced into 60 mL plastic syringes and swollen in DMF (8 mL/g of starting resin, 21 h). Syntheses were carried out manually where syringes were shaken with an Activo-PLS 44 synthesizer purchased from Activotec at 400 rpm in horizontal position. All amino acid couplings were carried out using a molar ratio of 1:1:1.25 of Fmoc amino acid (4.0 eq.) in DMF (0.50M), Oxyma (4.0 eq.) and NN-Diisopropylcarbodiimide (5.0 eq.). Fmoc amino acids (Fmoc-Gly-OH or Fmoc-Lys (Fmoc)-OH) were dissolved in DMF (0.50M) followed by the addition of Oxyma and NN-Diisopropylcarbodiimide and pre-activated for 30 min at room temperature before being transferred to the glass reactor. Couplings were carried out for 2 h at room temperature. Following coupling, the resin was washed with DMF (28 mL/g), capped using DMF/acetic anhydride (Ac.sub.2O)/Diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 min and re-washed using DMF (78 mL/g). Fmoc groups were deprotected prior to each coupling step using 20% piperidine in DMF (110 min+120 min) at room temperature. Following Fmoc-deprotection, the resin was washed using DMF (78 mL/g of starting resin).

[0234] 2-Gly-AMS and 4-Gly-AMS soluble supports synthesized using AMS resin of 0.93 mmol/g were isolated starting from the same reactor batch of starting AMS resin (2.0 g). Following the completion of the 2-Gly-AMS synthesis, the resin was washed with CH.sub.2Cl.sub.2 (3), iPrOH (3) and MTBE (3) prior to its drying under vacuum overnight. of the obtained resin was conserved while the other was used for the synthesis of 4-Gly-AMS in the same way as 2-Gly-AMS. Following the completion of the 4-Gly-AMS synthesis, the resin was washed with CH.sub.2Cl.sub.2 (3), iPrOH (3) and MTBE (3) prior to its drying under vacuum overnight.

[0235] 2-Gly-AMS and 4-Gly-AMS soluble supports synthesized using AMS resin of 1.95 mmol/g were isolated starting from starting AMS resin batches of 1.0 g and 2.0 g, respectively. Following the completion of their synthesis, resins were washed with CH.sub.2Cl.sub.2 (3), iPrOH (3) and MTBE (3) prior to their drying under vacuum overnight.

TABLE-US-00002 TABLE 2 Mass of Mass of starting Mass of modified polymeric modified polymeric matrix polymeric matrix (AMS 0.93 matrix (insoluble Type of mmol/g) of (insoluble support) insoluble each support) conserved support synthesis [g] [g] [g] 2X(Gly)-AMS 0.93 2.0 of AMS 0.93 3.52 1.76 4X(Gly)-AMS 0.93 1.76 of 2X(Gly)-AMS 0.93 2.67 2.67

TABLE-US-00003 TABLE 3 Mass of Mass of starting Mass of modified polymeric modified polymeric matrix polymeric matrix (AMS 1.95 matrix (insoluble Type of mmol/g) (insoluble support) insoluble of each support) conserved support synthesis [g] [g] [g] 2X(Gly)-AMS 1.95 1.0 of AMS 1.95 2.40 2.40 4X(Gly)-AMS 1.95 2.0 of AMS 1.95 7.80 7.80

Synthesis Protocols of Different Fmoc-Rink Amide Insoluble Supports

[0236] 2(Gly): (Fmoc-Rink).sub.2-Lys(1)-Gly-AMS [0237] 4(Gly): (Fmoc-Rink).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS [0238] 8(Gly): (Fmoc-Rink).sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS

[0239] Relevant soluble supports presented above were modified by the coupling of Rink amide using the following protocol:

[0240] Starting resins (1.0 g each) were introduced into 25 mL syringes and swollen in DMF (6.5 mL/g, 21 h). Syntheses were carried out manually where syringes were shaken with an Activo-PLS 44 synthesizer purchased from Activotec at 400 rpm in horizontal position. Fmoc groups were deprotected prior to rink amide linker coupling using 20% piperidine in DMF (110 min+120 min) at room temperature. Following Fmoc-deprotection, resins were washed using DMF (76.5 mL/g of starting resin). All rink amide couplings were carried out using 1:1:2 molar ratio of Rink amide linker in DMF (0.50M), PyAOP and Diisopropylethylamine (DIEA) at a 5-fold molar excess compared to synthesis scale. Rink amide was dissolved in DMF (0.50M) followed by the addition of PyAOP and stirred for 10 min at room temperature before adding DIEA. Next, the solution was introduced into the resin-containing syringes. Couplings were carried out overnight at room temperature. Following coupling, resins were washed with DMF (28 mL/g) and capped using DMF/acetic anhydride (Ac.sub.2O)/Diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 min. Next, resins were washed using DMF (78 mL/g), isopropanol (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0241] Table 4 presents the resin substitution before and after Rink amide coupling.

TABLE-US-00004 TABLE 4 Base resin/ Resin Resin polymeric substitution substitution matrix Resin type/ before Rink after Rink [substitution insoluble amide coupling amide coupling in mmol/g] support [mmol/g] [mmol/g] AMS (0.60) AMS 0.60 0.48 AMS (0.60) 2X(Gly)-AMS 0.90 0.68 AMS (0.60) 4X(Gly)-AMS 1.20 0.90 AMS (0.60) 8X(Gly)-AMS 1.76 0.98 AMS (0.93) AMS 0.93 0.62 AMS (0.93) 2X(Gly)-AMS 1.06 0.82 AMS (0.93) 4X(Gly)-AMS 1.48 1.10 AMS (1.95) AMS 1.95 1.08 AMS (1.95) 2X(Gly)-AMS 1.56 1.14 AMS (1.95) 4X(Gly)-AMS 1.80 1.20

Synthesis Protocols of Different Fmoc-Ramage Insoluble Supports

[0242] 2(Gly): (Fmoc-Ramage).sub.2-Lys(1)-Gly-AMS [0243] 4(Gly): (Fmoc-Ramage).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS [0244] 8(Gly): (Fmoc-Ramage).sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS

[0245] Relevant soluble supports presented above were modified by the coupling of Ramage using the following protocol:

[0246] Starting resins (1.0 g each) were introduced into 25 mL syringes and swollen in DMF (6.5 mL/g, 21 h). Syntheses were carried out manually where syringes were shaken with an Activo-PLS 44 synthesizer purchased from Activotec at 400 rpm in horizontal position. Fmoc groups were deprotected prior to Ramage linker coupling using 20% piperidine in DMF (110 min+120 min) at room temperature. Following Fmoc-deprotection, resins were washed using DMF (76.5 mL/g of starting resin). All ramage couplings were carried out using an equimolar ratio 1:1:1 of Ramage linker in DMF (0.15M-0.40M), Oxyma and NN-Diisopropylcarbodiimide (DIC) at a 2-fold molar excess compared to synthesis scale. Ramage was dissolved in DMF (0.15M-0.40M) followed by the addition of Oxyma and stirred for 10 min at room temperature before adding DIC. Next, the solution was introduced into the resin-containing syringes. Couplings were carried out overnight at room temperature. Following coupling, resins were washed with DMF (28 mL/g) and capped using DMF/acetic anhydride (Ac.sub.2O)/Diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 min. Next, resins were washed using DMF (78 mL/g), isopropanol (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0247] Table 5 presents the resin substitution before and after Ramage coupling.

TABLE-US-00005 TABLE 5 Base resin/ Resin Resin polymeric substitution substitution matrix Resin type/ before Rink after Rink [substitution insoluble amide coupling amide coupling in mmol/g] support [mmol/g] [mmol/g] AMS (0.60) AMS 0.60 0.48 AMS (0.60) 2X(Gly)-AMS 0.90 0.62 AMS (0.60) 4X(Gly)-AMS 1.20 1.12 AMS (0.60) 8X(Gly)-AMS 1.76 1.16 AMS (0.93) AMS 0.93 0.49 AMS (0.93) 2X(Gly)-AMS 1.06 1.00 AMS (0.93) 4X(Gly)-AMS 1.48 1.04 AMS (1.95) AMS 1.95 0.82 AMS (1.95) 2X(Gly)-AMS 1.56 0.94 AMS (1.95) 4X(Gly)-AMS 1.80 0.90

Synthesis Protocols of Different Fmoc-Leu-HMPB Insoluble Supports

[0248] 4(Gly): (Fmoc-Leu-HMPB-Leu).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS [0249] 8(Gly): (Fmoc-Leu-HMPB-Leu).sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS Relevant soluble supports presented above were modified by the coupling Fmoc-Leu-HMPB using the following protocol:

[0250] Starting resins (1.0 g each) were introduced into 25 mL syringes and swollen in DMF (6.5 mL/g, 21 h). Syntheses were carried out manually where syringes were shaken with an Activo-PLS 44 synthesizer purchased from Activotec at 400 rpm in horizontal position. Fmoc groups were deprotected prior to TBDMS-HMPB coupling using 20% piperidine in DMF (110 min+120 min) at room temperature. Following Fmoc-deprotection, resins were washed using DMF (76.5 mL/g of starting resin). AII TBDMS-HMPB couplings were carried out using 1:1:2 molar ratio of TBDMS-HMPB in DMF (0.50M), PyOxim and Diisopropylethylamine (DIEA) at a 2-fold molar excess compared to synthesis scale. TBDMS-HMPB was dissolved in DMF (0.50M) followed by the addition of PyOxim and stirred for 10 min at room temperature before adding DIEA. Next, the solution was introduced into the resin-containing syringes. Couplings were carried out overnight at room temperature. Following HMPB coupling, resins were washed with DMF (28 mL/g) and capped using DMF/acetic anhydride (Ac.sub.2O)/Diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 min. Next, resins were washed using DMF (78 mL/g), isopropanol (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0251] Starting HMPB-resins (300 mg each) were introduced into 12 mL syringes and swollen in THF (6.5 mL/g, 21 h). Syntheses were carried out manually where syringes were shaken with an Activo-PLS 44 synthesizer purchased from Activotec at 400 rpm in horizontal position. TBDMS group was deprotected prior to Fmoc-Leu-OH coupling using Et3N.3HF (12 equiv. compared to synthesis scale) in THF (0.22M) for 2 h at room temperature. Following TBDMS-deprotection, resins were washed using DIEA (1) then using DMF until neutral PH.

[0252] Prior to their coupling with Fmoc-Leu-OH, resins were swollen in DMF (6.5 mL/g, 21 h). All couplings were carried out using 1:1:1:0.25 molar ratio of Fmoc-Leu-OH in DMF (0.45M), Oxyma, NN-Diisopropylcarbodiimide (DIC) and 4-Dimethylaminopyridine (DMAP) at a 2.4-fold molar excess compared to synthesis scale. Fmoc-Leu-OH was dissolved in DMF (0.45M) followed by the addition of Oxyma, DIC and DMAP and stirred for 5 min at room temperature before being introduced into the resin-containing syringes. Couplings were carried out overnight at room temperature. Following coupling, resins were washed with DMF (28 mL/g) and capped using DMF/acetic anhydride (Ac.sub.2O)/Diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 min. Next, resins were washed using DMF (78 mL/g), isopropanol (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0253] Table 6 presents the resin substitution before and after Leu-HMPB coupling.

TABLE-US-00006 TABLE 6 Base resin/ Resin Resin polymeric substitution substitution matrix Resin type/ before Rink after Rink [substitution insoluble amide coupling amide coupling in mmol/g] support [mmol/g] [mmol/g] AMS (0.60) 4X(Gly)-AMS 1.20 0.88 AMS (0.60) 8X(Gly)-AMS 1.76 0.90 AMS (0.93) AMS 0.93 0.49

Synthesis Protocols of Different Fmoc-Gly-HMPB Insoluble Supports

[0254] 4(Gly): (Fmoc-Gly-HMPB).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS [0255] 8(Gly): (Fmoc-Gly-HMPB-).sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS

[0256] Relevant soluble supports presented above were modified by the coupling Fmoc-Gly-HMPB using the following protocol:

[0257] Starting resins (1.0 g each) were introduced into 25 mL syringes and swollen in DMF (6.5 mL/g, 21 h). Syntheses were carried out manually where syringes were shaken with an Activo-PLS 44 synthesizer purchased from Activotec at 400 rpm in horizontal position. Fmoc groups were deprotected prior to TBDMS-HMPB coupling using 20% piperidine in DMF (110 min+120 min) at room temperature. Following Fmoc-deprotection, resins were washed using DMF (76.5 mL/g of starting resin). All TBDMS-HMPB couplings were carried out using 1:1:2 molar ratio of TBDMS-HMPB in DMF (0.50M), PyOxim and Diisopropylethylamine (DIEA) at a 2-fold molar excess compared to synthesis scale. TBDMS-HMPB was dissolved in DMF (0.50M) followed by the addition of PyOxim and stirred for 10 min at room temperature before adding DIEA. Next, the solution was introduced into the resin-containing syringes. Couplings were carried out overnight at room temperature. Following HMPB coupling, resins were washed with DMF (28 mL/g) and capped using DMF/acetic anhydride (Ac.sub.2O)/Diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 min. Next, resins were washed using DMF (78 mL/g), isopropanol (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0258] Starting HMPB-resins (300 mg each) were introduced into 12 mL syringes and swollen in THF (6.5 mL/g, 21 h). Syntheses were carried out manually where syringes were shaken with an Activo-PLS 44 synthesizer purchased from Activotec at 400 rpm in horizontal position. TBDMS group was deprotected prior to Fmoc-Gly-OH coupling using Et3N.3HF (12 equiv. compared to synthesis scale) in THF (0.22M) for 2 h at room temperature. Following TBDMS-deprotection, resins were washed using DIEA (1) then using DMF until neutral PH.

[0259] Prior to their coupling with Fmoc-Gly-OH, resins were swollen in DMF (6.5 mL/g, 21 h). All couplings were carried out using 1:1:1:0.25 molar ratio of Fmoc-Gly-OH in DMF (0.45M), Oxyma, NN-Diisopropylcarbodiimide (DIC) and 4-Dimethylaminopyridine (DMAP) at a 2.4-fold molar excess compared to synthesis scale. Fmoc-Gly-OH was dissolved in DMF (0.45M) followed by the addition of Oxyma, DIC and DMAP and stirred for 5 min at room temperature before being introduced into the resin-containing syringes. Couplings were carried out overnight at room temperature. Following coupling, resins were washed with DMF (28 ml/g) and capped using DMF/acetic anhydride (Ac.sub.2O)/Diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 min. Next, resins were washed using DMF (78 ml/g), isopropanol (IPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0260] Table 7 presents the resin substitution before and after Gly-HMPB coupling.

TABLE-US-00007 TABLE 7 Base resin/ Resin Resin polymeric substitution substitution matrix Resin type/ before Rink after Rink [substitution insoluble amide coupling amide coupling in mmol/g] support [mmol/g] [mmol/g] AMS (0.60) AMS 0.60 0.40 AMS (0.60) 4X(Gly)-AMS 1.20 0.88 AMS (0.60) 8X(Gly)-AMS 1.76 1.00 AMS (0.93) AMS 0.93 0.53

(Fmoc-Linker).SUB.2.-Lys(1)-Gly-AMS Insoluble Support [2(Gly)]

[0261] Linkers: Fmoc-Rink and Fmoc-Ramage
Molecular Weight of the Respective Constructs without Support: [0262] Fmoc-Rink: Molecular weight (g/mol): 1245.40 [0263] Fmoc-Ramage: Molecular weight (g/mol): 1291.47

[0264] The constructs of the insoluble supports comprise one lysine, one glycine spacer and two Fmoc-Rink-linkers or two Fmoc-Ramage-linkers coupled to the amines of the first generation (first layer) of lysine, also referred to as distal lysine (distal branching agent). The glycine spacer is positioned between the polymeric matrix and the lysine branching agent.

[0265] The (Fmoc-Rink).sub.2-Lys(1)-Gly and (Fmoc-Ramage).sub.2-Lys(1)-Gly constructs contain one layer (first generation) of lysine, a primary layer, i.e. primary lysine. Each construct provides two distal binding sites available for peptide synthesis. Two Rink-linkers or two Ramage-linkers are bound to the amine groups (N-terminal alpha amine and side chain epsilon amine) of each lysine. The lysine is bound to the glycine and the glycine bound to the amines of the polymeric matrix.

[0266] Only lysine has been used where both amine groups of lysine are Fmoc protected.

[0267] The polymeric matrices used for modification are AMS 0.6, AMS 0.93 and AMS 1.95.

[0268] Reactants: Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-Rink-OH, Fmoc-Ramage-OH

Structural Formula of the (Fmoc-Rink).SUB.2.-Lys(1)-Gly Construct

##STR00010##

Structural Formula of the (Fmoc-Ramage).SUB.2.-Lys(1)-Gly Construct

##STR00011##

Synthesis Scheme for (Fmoc-Linker).SUB.2.-Lys(1)-Gly-AMS:

[0269] Linkers: Rink and Ramage

TABLE-US-00008 Resin-AMS + Fmoc-Gly-OH Coupling Resin-AMS-Gly-Fmoc deprotection Fmoc, coupling Fmoc-Lys(Fmoc)-OH Resin-AMS-Gly-Lys(Fmoc)-Fmoc deprotection Fmoc, coupling Fmoc-Linker-OH Resin-AMS-Gly-Lys(Fmoc-Linker)-Fmoc-Linker (Resin-AMS-Gly-Lys(1)-(Fmoc-Linker).sub.2
(Linker).sub.4-Lys(2).sub.2-Gly-Lys(1)-Gly-AMS Insoluble Support [4(Gly)] [0270] Linkers: Fmoc-Rink, Fmoc-Ramage and HMPB TBDMS
Molecular Weight of the Respective Constructs without Support: [0271] Fmoc-Rink: Molecular weight (g/mol): 2658.99 [0272] Fmoc-Ramage: Molecular weight (g/mol): 2522.93 [0273] HMPB TBDMS: Molecular weight (g/mol): 1461.66

[0274] The construct of these modified resins comprises Gly (G) in addition to three lysines and four linkers coupled to the second generation lysines. The Gly or beta-Ala spacers are positioned between first and second generation of lysines and between the first generation lysines and polymeric matrix.

[0275] The (Fmoc-Rink).sub.4-Gly(2).sub.2-Gly.sub.2-Lys(1)-Gly, (Fmoc-Ramage).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly and (HMPB TBDMS).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly constructs contain two layers (generations) of lysines, a primary and secondary layer, i.e. primary and secondary lysines, the secondary layer of lysines being the most distal lysines with respect to the polymeric matrix. The second layer of two secondary lysines provides in total four secondary binding sites available for peptide synthesis. Two Fmoc-Rink-linkers, two Fmoc-Ramage-linkers or two HMPB TBDMS)-linkers are bound to the two amine groups (N-terminal alpha amine and side chain epsilon amine) of each of the secondary lysines. Thus, the constructs have four linkers.

[0276] The polymeric matrices used for modification are AMS 0.6, AMS 0.93 and AMS 1.95.

[0277] Only lysine has been used where both amine groups of lysine are Fmoc protected. [0278] Reactants: Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-beta-Ala-OH, Fmoc-Rink-OH, Fmoc-Ramage-OH, HMPB TBDMS-OH
Structural Formula of the (Fmoc-Rink).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly Construct:

##STR00012##

Structural Formula of the (Fmoc-Ramage).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly Construct:

##STR00013##

[0279] Structural formula of the (TBDMS-HMPB).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly construct:

##STR00014##

[0280] For the characterization of 4 constructs a (NH.sub.2-Rink).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-NH.sub.2 and (NH.sub.2-Rink).sub.4-Lys(2).sub.2-Ala.sub.2-Lys(1)-Ala-NH.sub.2 were synthesized using Fmoc chemistry on a Symphony multi-channel peptide synthesizer. In order to cleave the construct from the polymeric matrix a sieber amide resin was used (Cas 915706-90-0). Branching agent Fmoc-Lys(Fmoc)-OH, Oxyma, Dic (Diisopropylcarbodiimide) and spacers (Gly, Ala=Beta alanine) were used at a 3-fold molar excess over the theoretical free amino groups. The amide couplings were allowed to proceed for 3 hours at RT. The obtained constructs were characterized by LC/MC.

(NH.sub.2-Rink).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-NH.sub.2 [0281] Calculated mass: 1770.99 [0282] Calculated: (M+2H)2+=886.49 [0283] Observed: (M+2H)2+=885.48

##STR00015##

(NH.sub.2-Rink).sub.4-Lys(2).sub.2-Ala.sub.2-Lys(1)-Ala-NH.sub.2 [0284] Calculated mass: 1810.89 [0285] Calculated (M+2H)2+=906.46 [0286] Observed: (M+2H)2+=907.36,

##STR00016##

Synthesis Scheme for (Linker).sub.4-K(2).sub.2-G.sub.2-K(1)-G-AMS): [0287] Linkers: Fmoc-Rink, Fmoc-Ramage and HMPB TBDMS

TABLE-US-00009 Resin-AMS + Fmoc-Gly-OH Coupling, deprotection Fmoc Resin-AMS-Gly-H coupling Fmoc-Lys(Fmoc)-OH Resin-AMS-Gly-Lys(Fmoc)-Fmoc deprotection Fmoc, coupling Fmoc-Gly-OH Resin-AMS-Gly-Lys(Gly-Fmoc)-Gly-Fmoc Deprotection Fmoc, coupling Fmoc-Lys(Fmoc)-OH Resin-AMS-Gly-Lys(Gly-Lys(Fmoc)-Fmoc)-Gly- Lys(Fmoc)-Fmoc deprotection Fmoc, coupling Linker-OH Resin-AMS-Gly-Lys(Gly-Lys(Linker)-Linker)-Gly- Lys(Linker)-Linker Resin-AMS-G-K(1)-G.sub.2-K(2).sub.2-(Linker).sub.4
(Linker).sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-K(1)-Gly-AMS Insoluble Support [8(Gly)] [0288] Linkers: Fmoc-Rink, Fmoc-Ramage and HMPB TBDMS [0289] Fmoc-Rink: Molecular weight (g/mol): 5486.17 [0290] Fmoc-Ramage: Molecular weight (g/mol): 4370.12 [0291] HMPB TBDMS: Molecular weight (g/mol): 4005.64

[0292] The construct of this insoluble support increases each primary binding site of the base AMS resin theoretically 8-fold. The construct comprises seven lysines, seven glycines and eight linkers. The construct has three layers/generations of lysines, one primary (proximal) lysine, two secondary (intermediate) lysines and four tertiary (distal) lysines. All secondary intermediate lysines between the primary (proximal lysine) and distal lysines (here tertiary lysines) are all bound to three different lysines signifying that the construct is symmetrical.

[0293] The polymeric matrix used for modification is AMS 0.6.

[0294] All lysines are exclusively of the type Fmoc-Lys(Fmoc)-OH. [0295] Reactants: Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-Rink-OH, Fmoc-Ramage-OH, TBDMS-HMPB-OH
Structure of the (Fmoc-Rink).sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS Insoluble Support

##STR00017##

Structure of the (Fmoc-Ramage).sub.8-K(3).sub.4-G.sub.4-K(2).sub.2-G.sub.2-K(1)-G-AMS Insoluble Support

##STR00018##

Structure of the (TBDMS-HMPB).sub.8-K(3).sub.4-G.sub.4-K(2).sub.2-G.sub.2-K(1)-G-AMS Insoluble Support

##STR00019##

Synthesis scheme for (Linker).sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS [0296] Linkers: Fmoc-Rink, Fmoc-Ramage, TBDMS-HMPB

TABLE-US-00010 Resin-AMS + Fmoc-Gly-OH Coupling, deprotection Fmoc Resin-AMS-Gly-H coupling Fmoc-Lys(Fmoc)-OH Resin AMS-Gly-Lys(Fmoc)-Fmoc deprotection Fmoc, coupling Fmoc-Gly-OH Resin AMS-Gly-Lys(Gly-Fmoc)-Gly-Fmoc deprotection Fmoc, coupling Fmoc-Lys(Fmoc) Resin AMS-Gly-Lys(Gly-Lys(Fmoc)-Fmoc)-Gly- Lys(Fmoc)-Fmoc deprotection Fmoc, coupling Fmoc-Gly-OH Resin AMS-Gly-Lys(Gly-Lys(Gly-Fmoc)-Gly-Fmoc)-Gly- Lys (Gly-Fmoc)-Gly-Fmoc deprotection, coupling Fmoc-Lys(Fmoc)-OH Resin AMS-Gly-Lys(Gly-Lys(Gly-Lys(Fmoc)-Fmoc)-Gly- Lys(Fmoc)-Fmoc)-Gly-Lys(Gly-Lys(Fmoc)-Fmoc)-Gly- Lys(Fmoc)-Fmoc deprotection Fmoc coupling Linker-OH Resin AMS-Gly-Lys(Gly-Lys(Gly-Lys(Linker)-Linker)- Gly-Lys(Linker)-Linker)-Gly-Lys(Gly-Lys(Linker)- Linker)-Gly-Lys(Linker)-Linker (Resin-AMS-Gly-Lys(1)-Gly.sub.2-Lys(2).sub.2-Gly.sub.4-Fmoc-Lys(3).sub.4- Linker.sub.8
(Fmoc-Rink).sub.4-(PEG.sub.6).sub.4-Lys(2).sub.2-Lys(1)-AMS Insoluble Support [4(PEG)]

[0297] The construct of this modified insoluble support comprises PEG.sub.6 spacers in addition to three lysines and four Fmoc-Rink-linkers. The PEG.sub.6 spacers are positioned between the distal lysines and the Fmoc-Rink-linkers.

[0298] The PEG spacer is provided as 2-[2-[2-(Fmoc amino) ethoxy]ethoxy] acetic acid (EAAE). Thus, the PEG.sub.6 spacer is provided by coupling 6 AEEA (PEG): s to each amine group of the secondary (distal) lysines.

##STR00020##

[0299] The polymer matrix used for modification is AMS 0.6.

[0300] Only lysine has been used where both amine groups are Fmoc protected. [0301] Reactants: Fmoc-Lys(Fmoc)-OH, AEEA, Fmoc-Rink-OH
Synthesis of Insoluble Support (Fmoc-Rink).sub.4-(PEG.sub.6).sub.4-Lys(2).sub.2-Lys(1)-AMS

[0302] Fmoc protected amino acids (K) and AEEA (PEG) are coupled by recurrent reaction steps comprising deprotection of the Fmoc groups followed by the coupling of relevant Fmoc protected amino acids and AEEA. After the consecutive coupling of the last AEEA the linkers are coupled. The amide bonds are formed using equimolar ratio of Fmoc-Lys(Fmoc)-OH, AEEA and Fmoc-Rink-OH, respectively and Oxyma pure (Ethyl cyano (hydroxyimino)acetate: Novabiochem) and diisopropylcarbodiimide (DIC) at 3-fold excess over the theoretical free amino groups (calculated from the first AMS binding site) in DMF. Fmoc groups are removed prior to each coupling step using two treatments of 25% (v/v) piperidine in DMF (5 and 20 minutes respectively). The amide coupling procedure is allowed to proceed for 2 hours at room temperature RT.

Synthesis scheme for (Fmoc-Rink).sub.4-(PEG.sub.6).sub.4-LysK(2).sub.2-LysK(1)-AMS:

TABLE-US-00011 Resin-AMS + Fmoc-Lys(Fmoc)-OH Coupling, deprotection Fmoc Resin-AMS-Lys-H coupling Fmoc-Lys(Fmoc)-OH Resin-AMS-Lys-(Lys(Fmoc)-Fmoc).sub.2 Deprotection Fmoc, coupling AEEA Resin-AMS-Resin-AMS-Lys-Lys.sub.2-(PEG6).sub.4 deprotection Fmoc, coupling Fmoc-Rink-OH Resin-AMS-Resin-AMS-Lys-Lys.sub.2-(PEG6).sub.4-(Rink- Fmoc).sub.4
Structure of the (Fmoc-Rink).sub.4-(PEG.sub.6).sub.4-Lys(2).sub.2-Lys(1)-NH.sub.2Construct:

##STR00021## [0303] Molecular weight 5971.62

(Linker).SUB.7.-LysGly-Linear-Gly-AMS Insoluble Support [7 Linear]

[0304] Linker: Fmoc-Rink, Fmoc-Ramage and HMPB TBDMS
Molecular Weight of the Respective Constructs without Support: [0305] Fmoc-Rink: Molecular weight (g/mol): 4608.22 [0306] Fmoc-Ramage: Molecular weight (g/mol): 4370.12 [0307] TBDMS-HMPB: Molecular weight (g/mol): 3312.76

[0308] The construct of this insoluble support contains six lysines, three glycines as spacers, and seven linkers selected from Fmoc-Rink-OH, Fmoc-Ramage, HMPB TBDMS, said linkers coupled to four of the six lysines.

[0309] In the synthesis of the linear 7 insoluble support two classes of lysine have been used differing in terms of protection groups. One class of lysines have both amine groups protected by Fmoc: Fmoc-Lys(Fmoc) epsilon amine protected by Mtt: Fmoc-Lys(Mtt)-OH.

[0310] The construct of the 7 insoluble support is synthesized by first synthesizing a main chain by only coupling Fmoc-Lys(Mtt)-OH. The main chain is the chain where all amide bonds involve an alfa amine. After the synthesis of the main chain comprising three lysines and three glycines, the Mtt is cleaved of with a solution of 30% hexafluoroisopropanol HFIP in DCM for 1 hour at 25 C. In a subsequent step Fmoc-Lys (Fmoc) is coupled to the epsilon amines of each lysine of the main chain. Thereafter, all Fmoc groups are cleaved of using two treatments of 25% (v/v) piperidine in DMF (5 and 20 minutes respectively). Finally, linkers are coupled to the lysines. The seven form 7 secondary (distal) binding sites.

[0311] The side chain amines of each lysine of the main chain are only coupled to one lysine, respectively, providing a construct with a configuration which herein is referred to as linear.

[0312] The amide bonds are formed using equimolar ratio of Fmoc-Lys(Fmoc)-OH, Fmoc-Lys(Mtt)-OH, and linker, respectively and Oxyma pure (Ethyl cyano (hydroxyimino)acetate: Novabiochem) and diisopropylcarbodiimide (DIC) at 3-fold excess over the theoretical free amino groups (calculated from the first AMS binding site) in DMF. Where applicable, Fmoc groups are removed prior to each coupling step using two treatments of 25% (v/v) piperidine in DMF (5 and 20 minutes respectively). The amide coupling procedure is allowed to proceed for 2 hours.

[0313] The polymeric matrix used for modification is AMS 0.6.

Synthesis Scheme for 7 Linear:

TABLE-US-00012 Resin-AMS + Fmoc-Gly-OH Coupling, deprotection Fmoc Resin-AMS-Gly-H coupling Fmoc-Lys(Mtt)-OH Resin-AMS-Gly-Lys(Mtt)-Fmoc deprotection Fmoc, coupling Fmoc-Gly-OH Resin-AMS-Gly-Lys(Mtt)-Gly-Fmoc deprotection Fmoc, coupling Fmoc-Lys(Mtt)-OH Resin-AMS-Gly-Lys(Mtt)-Gly-Lys(Mtt)-Fmoc deprotection Fmoc, coupling Fmoc-Gly-OH Resin-AMS-Gly-Lys(Mtt)-Gly-Lys(Mtt)-Gly-Fmoc deprotection Fmoc, coupling Fmoc-Lys(Mtt)-OH Resin-AMS-Gly-Lys(Mtt)-Gly-Lys(Mtt)-Gly-Lys(Mtt)- Fmoc deprotection Mtt (30% HFIP in DCM) Resin-AMS-Gly-Lys-Gly-Lys-Gly-Lys-Fmoc coupling Fmoc-Lys(Fmoc)-OH Resin-AMS-Gly-Lys(Lys(Fmoc)-Fmoc)-Gly- Lys(Lys(Fmoc)-Fmoc)-Gly-Lys(Lys(Fmoc)-Fmoc)-Fmoc deprotection Fmoc and coupling Fmoc-Linker-OH Resin-AMS-Gly-Lys(Lys(Fmoc-Linker)-Fmoc-Linker)- Gly-Lys(Lys(Fmoc-Linker)-Fmoc-Linker)-Gly- Lys(Lys(Fmoc-Linker)-Fmoc-Linker)-Fmoc-Linker Resin-AMS-Gly-Lys(1)(Fmoc-Linker-Lys(2)(Fmoc- Linker)-Gly-Lys(2)(Fmoc-Linker-Lys(3)(Fmoc-Linker))- Gly-Lys(3)(Fmoc-Linker)-Lys(4)(Fmoc-Linker)-Fmoc- Linker = (Fmoc-Rink)7-LysGly-linear-Gly-AMS)

[0314] This modified solid resin has a construct comprising lysines which are only bound to one other lysine, where applicable through a Glycine spacer. The presence of lysines only bound to one other lysine (lysine with other identity) provides an odd number of secondary binding sites: here seven secondary binding sites.

Structural Formula of the (Fmoc-Rink).SUB.7.-LysGly-Linear-Gly-AMS Insoluble Support:

##STR00022##

Structural Formula of the (Fmoc-Ramage).SUB.7.-LysGly-Linear-Gly-AMS Insoluble Support:

##STR00023##

Structural Formula of the (TBDMS-HMPB).SUB.7.-LysGly-Linear-Gly-AMS Insoluble Support:

##STR00024##

(Fmoc-Rink).sub.16-Lys(4).sub.8-Gly.sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS [16(Gly)] [0315] Molecular weight (g/mol): 1112.49

[0316] The 16(Gly) insoluble support comprises a construct providing a 16-fold increase of the primary binding sites of the base AMS resin. The construct comprises 15 lysines, 15 glycine spacers and 16 Fmoc-Rink-linkers. This construct is symmetrical containing four layers (generations) of lysines, i.e. eight quaternary lysines Lys (4), four tertiary lysines Lys (3), two secondary lysines Lys (2) and a primary lysine Lys(1). Furthermore, all lysines (secondary Lys (2) and tertiary Lys (3)) between the lysine bound to the primary binding site of the base resin and the quaternary lysines Lys (4) are bound to three different lysines. E.g. each of the two secondary lysines Lys (2) are bound to the primary lysine Lys(1) and to two tertiary lysines Lys (3). If all lysines except the distal (distal lysines are in this case the quaternary lysines Lys (4)) and primary are all bound to three lysines then the construct is symmetrical. Also, the number of secondary binding sites are a function of the following equation: Y=2, where [0317] n denotes the number of layers of lysines in the construct. Here we have 4 layers and, hence, 16 secondary binding sites.

[0318] The polymeric matrix used for modification is AMS 0.6.

[0319] All lysines are exclusively Fmoc-Lys(Fmoc). [0320] Reactants: Fmoc-Lys(Fmoc), Fmoc-Gly, Fmoc-Rink
Structural Formula of the (Fmoc-Rink).sub.16-Lys(4).sub.8-Gly.sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS Insoluble Support:

##STR00025##

Synthesis of the (Fmoc-Rink).sub.16-Lys(4).sub.8-Gly.sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS

[0321] The peptide construct is synthesized on a manual peptide synthesis reactor.

[0322] Fmoc protected Lysine is coupled to the amines of the AMS. The amide bonds are formed using equimolar ratio of Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-Rink-OH respectively and Oxyma pure (Ethyl cyano (hydroxyimino)acetate: Novabiochem) and diisopropylcarbodiimide (DIC) at 3-fold excess over the theoretical free amino groups (calculated from the first AMS binding site) in DMF. Fmoc groups are removed prior to each coupling step using two treatments of 25% (v/v) piperidine in DMF (5 and 20 minutes respectively). The amide coupling procedure is allowed to proceed for 2 hours.

Synthesis Scheme for 16(Gly)

TABLE-US-00013 Resin-AMS + Fmoc-Gly-OH Coupling, deprotection Fmoc Resin-AMS-Gly-H coupling Fmoc-Lys(Fmoc)-OH Resin AMS-Gly-Lys(Fmoc)-Fmoc deprotection Fmoc, coupling Fmoc-Gly Resin AMS-Gly-Lys(Gly-Fmoc)-Gly-Fmoc deprotection Fmoc, coupling Fmoc-Lys(Fmoc) Resin AMS-Gly-Lys(Gly-Lys(Fmoc)-Fmoc)-Gly- Lys(Fmoc)-Fmoc deprotection Fmoc, coupling Fmoc-Gly Resin AMS-Gly-Lys(Gly-Lys(Gly-Fmoc)-Gly-Fmoc)-Gly- Lys(Gly-Fmoc)-Gly-Fmoc deprotection, coupling Fmoc-Lys(Fmoc)-OH Resin AMS-Gly-Lys(Gly-Lys(Gly-Lys(Fmoc)-Fmoc)-Gly- Lys(Fmoc)-Fmoc)-Gly-Lys(Gly-Lys(Fmoc)-Fmoc)-Gly- Lys(Fmoc)-Fmoc deprotection Fmoc, coupling Fmoc-Gly Resin AMS-Gly-Lys(Gly-Lys(Gly-Lys(Gly-Fmoc)-Gly- Fmoc)-Gly-Lys(Gly-Fmoc)-Gly-Fmoc)-Gly-Lys(Gly- Lys(Gly-Fmoc)-Gly-Fmoc)-Gly-Lys(Gly-Fmoc)-Gly- Fmoc deprotection Fmoc, coupling Fmoc-Lys(Fmoc)-OH Resin AMS-Gly-Lys(Gly-Lys(Gly-Lys(Gly-Lys(Fmoc)- Fmoc)-Gly-Lys(Fmoc)-Fmoc)-Gly-Lys(Gly-Lys(Fmoc)- Fmoc)-Gly-Lys(Fmoc)-Fmoc)-Gly-Lys(Gly-Lys(Gly- Lys(Fmoc)-Fmoc)-Gly-Lys(Fmoc)-Fmoc)-Gly-Lys(Gly- Lys(Fmoc)-Fmoc)-Gly-Lys(Fmoc)-Fmoc deprotection Fmoc, coupling Fmoc-Rink Resin AMS-Gly-Lys(Gly-Lys(Gly-Lys(Gly-Lys(Fmoc- Rink)-Fmoc-Rink)-Gly-Lys(Fmoc-Rink)-Fmoc-Rink)-Gly- Lys(Gly-Lys(Fmoc-Rink)-Fmoc-Rink)-Gly-Lys(Fmoc- Rink)-Fmoc-Rink)-Gly-Lys(Gly-Lys(Gly-Lys(Fmoc- Rink)-Fmoc-Rink)-Gly-Lys(Fmoc-Rink)-Fmoc-Rink)-Gly- Lys(Gly-Lys(Fmoc-Rink)-Fmoc-Rink)-Gly-Lys(Fmoc- Rink)-Fmoc-Rink (Resin-AMS-Gly-Lys(1)-Gly2-Lys(2).sub.2-Gly.sub.4-Fmoc- Lys(3).sub.4-Fmoc-Gly.sub.8-Fmoc-Lys(4).sub.8-Fmoc-Rink.sub.16)

Synthesis of Polypeptides Using the Insoluble Support of the Invention

[0323] Template polypeptide sequences PP1 to PP 6 were synthesized using the following insoluble supports (in detail presented above):

Supports with Rink Linker: [0324] 1: (Fmoc-Rink)-AMS 0.6 (resin without construct) [0325] 1: (Fmoc-Rink)-AMS 0.93 (resin without construct) [0326] 1: (Fmoc-Rink)-AMS 1.95 (resin without construct) [0327] 2(Gly): (Fmoc-Rink).sub.2-Lys(1)-gly-AMS 0.6 [0328] 2(Gly): (Fmoc-Rink).sub.2-Lys(1)-Gly-AMS 0.93 [0329] 2(Gly): (Fmoc-Rink).sub.2-Lys(1)-Gly-AMS 1.95 [0330] 4(Gly): (Fmoc-Rink).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS 0.6 [0331] 4(Gly): (Fmoc-Rink).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS 0.93 [0332] 4(Gly): (Fmoc-Rink).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS 1.95 [0333] 8(Gly): (Fmoc-Rink).sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS 0.6
Supports with Ramage Linker: [0334] 1: (Fmoc-Ramage)-AMS 0.6 (resin without construct) [0335] 1: (Fmoc-Ramage)-AMS 0.93 (resin without construct) [0336] 1: (Fmoc-Ramage)-AMS 1.95 (resin without construct) [0337] 2(Gly): (Fmoc-Ramage).sub.2-Lys(1)-Gly-AMS 0.6 [0338] 2(Gly): (Fmoc-Ramage).sub.2-Lys(1)-Gly-AMS 0.93 [0339] 2(Gly): (Fmoc-Ramage).sub.2-Lys(1)-Gly-AMS 1.95 [0340] 4(Gly): (Fmoc-Ramage).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS 0.6 [0341] 4(Gly): (Fmoc-Ramage).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS 0.93 [0342] 4(Gly): (Fmoc-Ramage).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS 1.95
Supports with HMPB TBDMS Linker: [0343] 1: HMPB TBDMS AMS 0.6 (resin without construct) [0344] 1: HMPB TBDMS AMS 0.93 (resin without construct) [0345] 4(Gly): (HMPB TBDMS).sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS 0.6 [0346] 8(Gly): (HMPB TBDMS).sub.8-Lys(3).sub.4-Gly.sub.4-Lys(2).sub.2-Gly.sub.2-Lys(1)-Gly-AMS 0.6

[0347] The reference resin is an amino methyl (AM/AMS) polystyrene resin cross-linked with 1% divinylbenzene in the 100-200 mesh size range (75-150 m) with a degree of substitution of 0.6, 0.93 and 1.95 mmol/gram respectively.

[0348] The following polypeptides were synthesized:

TABLE-US-00014 PP1:WLFAGGPSSGAPPPS(15mer) PP2:YAEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPPPS (39mer) PP3:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (39mer) PP4:fFPRPGGGGNGDFEEIPEEYL(20mer) PP5:FVQYLIQG(8mer) PP6:HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG(31mer)

Constructs:

[0349] 1: No construct only unmodified resin with linker as indicated below. [0350] 2(Gly): -Gly-Lys(1)-Linker.sub.2 [0351] 4(Gly): -Gly-Lys(1)-Gly.sub.2-Lys(2).sub.2-Linker.sub.4 [0352] 8(Gly): -Gly-Lys(1)-Gly.sub.2-Lys(2).sub.2-Gly.sub.4-Lys(3).sub.4-Linkers

[0353] Tables 8 to 14 present various data related to the polypeptide synthesis.

[0354] In some columns the assembly yield [%] and cleavage yield [%] are indicated. These parameters are calculated as follows:

[00001]$\begin{array}{cc}\mathrm{Assembly}\mathrm{yield}=\frac{m\left(\mathrm{peptide}-\mathrm{resin}\right)-m\left(\mathrm{resin}\right)}{\mathrm{MW}(\mathrm{protected}\mathrm{peptide}\mathrm{scale}}100& \left(1\right)\end{array}$$\begin{array}{cc}\mathrm{Cleavage}\mathrm{yield}=\frac{m\left(\mathrm{crude}\mathrm{peptide}\right)}{\mathrm{MW}\left(\mathrm{peptide}\mathrm{salt}\right)\mathrm{scale}}100& \left(2\right)\end{array}$

Synthesis Protocol for PP 1 with DMF as Solvent

[0355] Starting resins (400 mg each) were introduced into 25 mL syringes and swollen in DMF (6.5 mL/g, 21 h) for initial swelling measurements prior to their transfer to Symphony X reactors. Fmoc groups were deprotected prior to each coupling step using 20% piperidine in DMF (110 min+120 min) at room temperature. All amino acid couplings were carried out using an equimolar ratio (1:1:1) of Fmoc amino acid (0.30M in DMF), Oxyma (0.90M in DMF) and NN-Diisopropylcarbodiimide (0.90M in DMF) at 5-fold molar excess compared to synthesis scale. Couplings were carried out without pre-activation for 1 h 30 min at room temperature.

[0356] After syntheses completion, resins were transferred again to the 25 mL syringes to measure their final swellings then washed with isopropanol (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0357] Dried Fmoc-protected peptidyl resins were swollen again in DMF (6.5 mL/g, 21 h). Fmoc groups were deprotected using 20% piperidine in DMF (110 min+120 min) at room temperature. Next, resins were washed with DMF (7), iPrOH (3) and MTBE (3) prior to their re-drying under vacuum overnight.

[0358] Dried Fmoc-deprotected peptides were cleaved from resins using a cleavage cocktail containing TFA (Trifluoroacetic acid)/H.sub.2O/DTT (Dithiothreitol) (13 mL/g of peptidyl resin, 85/5/5 v/v/w) for 2 h at room temperature. TIS (5% v) was then added and stirring was continued for another 1 h. The resin was filtered out and washed with TFA (3 mL/g of peptidyl resin). Peptides were precipitated with MTBE (10-folds the volume of TFA) at 0 C. The resulting suspensions were transferred to 50 ml conical centrifuge tubes and centrifuged at 2500 rcf for 10 min prior to supernatants' decantation. The crude solid products were washed again with cold MTBE (5) and dried under vacuum overnight. [0359] Molecular weight: 1426.60 [0360] Calculated mass: +2/2=714.30 [0361] Observed mass (AMS 0.93 2(Gly)) +2/2=714.05 [0362] Observed mass (AMS 1.95 2(Gly)): +2/2=714.43 [0363] Observed mass (AMS 1.95 4(Gly)): +2/2=714.64

[0364] All 15 amino acids used were Fmoc-protected.

[0365] Side chains protecting groups involved: tBu, Boc: S=Ser(tBu) and W=Trp(Boc) [0366] Molecular weight of protected PP 1:1917.28 g/mol [0367] Molecular weight of unprotected PP 1:1426.60 g/mol

TABLE-US-00015 TABLE 8 Data synthesis of 15mer polypeptide: WLFAGGPSSGAPPPS: PP 1 Solvent for the synthesis: DMF Mass of matrix at the beginning of synthesis after coupling of Rink linker: 400 mg Theoretical Final mass Final volume Substitution mass of Fmoc of Fmoc of Fmoc Unmodified at beginning Synthesis protected protected protected matrix/resin Type of of synthesis scale peptidyl resin peptidyl resin peptidyl resin [mmol/g] construct [mmol/g] [mmol] [mg] [mg] [mL] AMS 0.6 1X 0.48 0.192 725 660 4.6 AMS 0.6 2X(Gly) 0.68 0.272 861 800 4.3 AMS 0.6 4X(Gly) 0.90 0.360 1010 930 4.0 AMS 0.6 8X(Gly) 0.98 0.392 1065 987 3.7 AMS 0.93 1X 0.62 0.248 820 760 4.3 AMS 0.93 2X(Gly) 0.82 0.328 956 788 3.6 AMS 0.93 4X(Gly) 1.10 0.440 1146 680 3.3 AMS 1.95 1X 1.08 0.432 1132 990 3.8 AMS 1.95 2x(Gly) 1.14 0.456 1173 995 3.3 AMS 1.95 4X(Gly) 1.20 0.480 1214 684 2.5 SPPS reactor Theoretical throughput HPLC purity crude peptide (mass after of crude Weight Assembly Cleavage mass after Crude peptide cleavage/final peptide gain yield yield cleavage after cleavage volume) [%] [mg] [%] (1) [%] (2) [mg] [mg] [g/L] 82.4 303 82 71 296 211 45.9 89.3 460 88 88 419 370 86.1 81.3 610 89 88 555 486 114.0 78.2 674 90 87 604 523 141.4 74.4 415 87 66 382 250 58.1 85.5 461 73 66 505 330 91.7 84.5 378 45 64 678 430 130.3 81.8 686 83 69 666 460 121.1 83.7 696 80 70 703 490 148.5 83.2 391 43 58 740 429 171.6

[0368] Implementation of support of invention significantly increases throughput while essentially maintaining purity or even increasing purity.

Synthesis Protocol for PP 1 with DMSO/EtOAc (3:7 v/v) and DMF as Solvent

[0369] Starting resins (400 mg each) were introduced into 25 mL syringes and swollen in DMF (6.5 mL/g, 21 h). Syntheses were carried out manually where syringes were shaken with an Activo-PLS 44 synthesizer purchased from Activotec at 400 rpm in horizontal position. Fmoc groups were deprotected prior to each coupling step using 20% piperidine in DMSO/EtOAc (3:7, v/v) or DMF (110 min+120 min) at room temperature. Following Fmoc-deprotection, resins were washed using DMSO/EtOAc (3:7, v/v) or DMF (76.5 mL/g of starting resin). All amino acid couplings were carried out using an equimolar ratio (1:1:1) of Fmoc amino acid in DMSO/EtOAc (3:7, v/v) or DMF (0.30M), Oxyma and NN-Diisopropylcarbodiimide at 2-fold molar excess compared to synthesis scale. Fmoc amino acids were dissolved in the corresponding solvent/solvent mixture (0.30M) followed by the addition of Oxyma and NN-Diisopropylcarbodiimide and stirred for 5 min at room temperature before being introduced into the resin-containing syringes. Couplings were carried out for 1 h 30 min at room temperature. Following coupling, resins were washed using DMSO/EtOAc (3:7, v/v) or DMF (26.5 mL/g of starting resin).

[0370] After syntheses completion, final resin swellings were measured then resins were washed with (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0371] Dried Fmoc-protected peptidyl resins were swollen again in DMF (6.5 mL/g, 21 h). Fmoc groups were deprotected using 20% piperidine in DMF (110 min+120 min) at room temperature. Next, resins were washed with DMF (7), iPrOH (3) and MTBE (3) prior to their re-drying under vacuum overnight.

[0372] Dried Fmoc-deprotected peptides were cleaved from resins using a cleavage cocktail containing TFA/H.sub.2O/DTT (13 mL/g of peptidyl resin, 85/5/5 v/v/w) for 2 h at room temperature. TIS (5% v) was then added and stirring was continued for another 1 h. The resin was filtered out and washed with TFA (3 mL/g of peptidyl resin). Peptides were precipitated with MTBE (10-folds the volume of TFA) at 0 C. The resulting suspensions were transferred to 50 ml conical centrifuge tubes and centrifuged at 2500 rcf for 10 min prior to supernatants' decantation. The crude solid products were washed again with cold MTBE (5) and dried under vacuum overnight.

[0373] Mass of resin at beginning of synthesis after coupling of Rink Amide linker: 400 mg

[0374] Side chains protecting groups involved: tBu, Boc: S=Ser(tBu) and W=Trp(Boc) [0375] Molecular weight of protected PP 1:1917.28 g/mol [0376] Molecular weight of unprotected PP 1:1426.60 g/mol

TABLE-US-00016 TABLE 9 Data Synthesis of 15mer polypeptide: WLFAGGPSSGAPPPS: PP 1 Solvent for the synthesis: DMSO/EtOAc (3:7) except first row AMS 0.6 with DMF Mass of matrix at the beginning of synthesis after coupling of Rink linker: 400 mg Theoretical Final mass Final volume Theoretical Substitution mass of Fmoc of Fmoc of Fmoc crude peptide Unmodified at beginning Synthesis protected protected protected mass after Assembly matrix/resin Type of of synthesis scale peptidyl resin peptidyl resin peptidyl resin cleavage yield [mmol/g] construct [mmol/g] [mmol] [mg] [mg] [mL] [mg] [%] (1) AMS 0.6 1X 0.48 0.192 725 650 4.3 296 77 DMF AMS 0.6 1X 0.48 0.192 725 640 2.7 296 80 AMS 0.6 2X(Gly) 0.68 0.272 861 808 2.8 419 90 AMS 0.6 4X(Gly) 0.90 0.360 1010 950 2.8 555 91 AMS 0.6 8X(Gly) 0.98 0.392 1065 1006 2.7 604 92 SPPS reactor throughput HPLC purity (mass after of crude Cleavage Crude peptide cleavage/final peptide Weight gain yield after cleavage volume) [%] [mg] Solvent [%] (2) [mg] [g/L] 83.8 283 DMF 87 264 61.4 85.1 293 DMSO/EtOAc 264 258 95.6 81.5 468 DMSO/EtOAc 93 388 138.6 81.9 630 DMSO/EtOAc 96 530 189.3 80.0 693 DMSO/EtOAc 99 595 220.4
Synthesis Protocol for PP 2 (YAEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPPPS) with DMF as Solvent

[0377] Starting resins (200 mg each) were introduced into 12 mL syringes and swollen in DMF (6.5 mL/g, 21 h) for initial swelling measurements prior to their transfer to Symphony X reactors. Fmoc groups were deprotected prior to each coupling step using 20% piperidine in DMF (110 min+120 min) at room temperature. All amino acid couplings were carried out using an equimolar ratio (1:1:1) of Fmoc amino acid (0.30M in DMF), Oxyma (0.90M in DMF) and NN-Diisopropylcarbodiimide (0.90M in DMF) at 5-fold molar excess compared to synthesis scale. Couplings were carried out without pre-activation for 2 h at room temperature except for Thr5 which was coupled during 8 h and Ile.sup.12 and 17 which were coupled during 6 h.

[0378] After syntheses completion, resins were transferred again to the 12 mL syringes to measure their final swellings then washed with (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0379] Dried Boc-protected peptides were cleaved from resins using a cleavage cocktail containing TFA/H.sub.2O/DTT/TIS (13 mL/g of peptidyl resin, 85/5/5/5 v/v/w/v) for 3 h at room temperature. The resin was filtered out and washed with TFA (3 mL/g of peptidyl resin). Peptides were precipitated with MTBE (10-folds the volume of TFA) at 0 C. The resulting suspensions were transferred to 50 ml conical centrifuge tubes and centrifuged at 2500 rcf for 10 min prior to supernatants' decantation. The crude solid products were washed again with cold MTBE (5) and dried under vacuum overnight. [0380] Molecular weight: 4041.54 [0381] Calculated mass: +3/3=1348.26; +4/4=1011.38 [0382] Observed mass (AMS 0.6 2(Gly)): +3/3=1347.98; +4/4=1011.70 [0383] Observed mass (AMS 0.93 4(Gly)): +3/3=1348.6; +4/4=1011.67

TABLE-US-00017 TABLE 10 Data Synthesis of 39mer polypeptide: YAEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPPPS: PP 2 Solvent for the synthesis: DMF Mass of matrix at the beginning of synthesis after coupling of Ramage linker: 200 mg Theoretical Final mass Final volume Substitution mass of Boc of Boc of Boc Unmodified at beginning Synthesis protected protected protected matrix/resin Type of of synthesis scale peptidyl resin peptidyl resin peptidyl resin [mmol/g] construct [mmol/g] [mmol] [mg] [mg] [mL] AMS 0.6 1X 0.39 0.078 619 560 1.9 AMS 0.6 2X(Gly) 0.62 0.124 866 840 2.3 AMS 0.6 4X(Gly) 1.28 0.224 1404 1060 2.4 AMS 0.6 8X(Gly) 1.16 0.232 1447 870 1.9 AMS 0.93 1X 0.49 0.098 727 692 2.5 AMS 0.93 2X(Gly) 1.00 0.200 1275 880 2.2 AMS 0.93 4X(Gly) 1.04 0.208 1318 1002 2.1 AMS 1.95 1X 0.82 0.164 1081 1030 2.7 AMS 1.95 2x(Gly) 0.94 0.188 1210 823 2.2 AMS 1.95 4X(Gly) 0.90 0.180 1167 421 1.0 SPPS reactor Theoretical throughput crude peptide (mass after mass after Crude peptide HPLC purity cleavage/final cleavage after cleavage after cleavage volume) [mg] [mg] [%] [g/L] 333 280 54.3 147.4 533 500 57.7 217.4 962 680 56.3 283.3 991 550 23.4 289.5 418 380 54.5 152.0 854 661 54.8 300.5 888 640 39.1 304.8 700 650 49.6 240.8 803 674 24.1 306.4 769 295 10.6 295.0 Weight gain Assembly yield Cleavage yield [mg] [%] (1) [%] (2) 377 86 84 668 96 94 910 73 71 722 56 56 514 94 91 725 65 75 848 73 72 867 95 93 665 63 84 261 26 38
Synthesis Protocol for PP 3 (HGEGTFTSDLSKQMEEEAVRLFXEWLKNGGPSSGAPPPS) with DMF as Solvent

[0384] Starting resins (200 mg each) were introduced into 12 mL syringes and swollen in DMF (6.5 mL/g, 21 h) for initial swelling measurements prior to their transfer to Symphony X reactors. Fmoc groups were deprotected prior to each coupling step using 20% piperidine in DMF (110 min+120 min) at room temperature. All amino acid couplings were carried out using an equimolar ratio (1:1:1) of Fmoc amino acid (0.30M in DMF), Oxyma (0.90M in DMF) and NN-Diisopropylcarbodiimide (0.90M in DMF) at 5-fold molar excess compared to synthesis scale. Couplings were carried out without pre-activation for 2 h at room temperature except for Thr5 which was coupled during 8 h and Ile.sup.12 and 17 which were coupled during 6 h.

[0385] After syntheses completion, resins were transferred again to the 12 mL syringes to measure their final swellings then washed with (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0386] Dried Boc-protected peptides were cleaved from resins using a cleavage cocktail containing TFA/H.sub.2O/DTT/TIS (13 mL/g of peptidyl resin, 85/5/5/5 v/v/w/v) for 3 h at room temperature. The resin was filtered out and washed with TFA (3 mL/g of peptidyl resin). Peptides were precipitated with MTBE (10-folds the volume of TFA) at 0 C. The resulting suspensions were transferred to 50 ml conical centrifuge tubes and centrifuged at 2500 rcf for 10 min prior to supernatants' decantation. The crude solid products were washed again with cold MTBE (5) and dried under vacuum overnight. [0387] Molecular weight: 4186.63 [0388] Calculated mass: +3/3=1396.54; +4/4=1047.65 [0389] Observed mass (AMS 0.6 2(Gly)): +3/3=1397.12; +4/4=1048.11 [0390] Observed mass (AMS 0.6 4(Gly)): +3/3=1396.89; +4/4=1047.65

[0391] All amino acids used were Fmoc-protected except for the last amino acid (Tyr) which was Boc-protected. [0392] Side chains protecting groups involved: tBu, Boc, Trt: S=Ser(tBu), W=Trp(Boc), Q=Gln(Trt), K=Lys(Boc), D=Asp(OtBu), Y=Tyr(tBu), T=Thr(tBu), E=Glu(OtBu) [0393] Molecular weight of protected 39-mer: 5596.22 g/mol [0394] Molecular weight of unprotected 39-mer: 4041.54 g/mol

TABLE-US-00018 TABLE 11 Data Synthesis of 39mer polypeptide: HGEGTFTSDLSKQMEEEAVRLFXEWLKNGGPSSGAPPPS: PP 3 Solvent for the synthesis: DMF Mass of matrix at the beginning of synthesis after coupling of Ramage linker: 200 mg Theoretical Final mass Final volume Substitution mass of Boc of Boc of Boc Unmodified at beginning Synthesis protected protected protected matrix/resin Type of of synthesis scale peptidyl resin peptidyl resin peptidyl resin [mmol/g] construct [mmol/g] [mmol] [mg] [mg] [mL] AMS 0.6 1X 0.39 0.078 674 360 2.4 AMS 0.6 2X(Gly) 0.62 0.124 953 690 2.9 AMS 0.6 4X(Gly) 1.28 0.224 1560 993 3.1 SPPS reactor Theoretical throughput crude peptide (mass after mass after Crude peptide HPLC purity cleavage/final cleavage after cleavage after cleavage volume) [mg] [mg] [%] [g/L] 362 339 53.3 141.3 576 603 52.4 207.9 1040 885 43.5 275.8 Weight gain Assembly yield Cleavage yield [mg] [%] (1) [%] (2) 167 34 94 518 66 99 843 60 82

[0395] The implementation of a support of the invention significantly increases throughput at good/commercially relevant/useful purity.

Synthesis Protocol for PP 4 (fPRPGGGGNGDFEEIPEEYL) with DMF as Solvent

[0396] Starting resins (200 mg each) were introduced into 12 mL syringes and swollen in DMF (6.5 mL/g, 21 h) for initial swelling measurements prior to their transfer to Symphony X reactors. Fmoc groups were deprotected prior to each coupling step using 20% piperidine in DMF (110 min+120 min) at room temperature. All amino acid couplings were carried out using an equimolar ratio (1:1:1) of Fmoc amino acid (0.30M in DMF), Oxyma (0.90M in DMF) and NN-Diisopropylcarbodiimide (0.90M in DMF) at 5-fold molar excess compared to synthesis scale. Couplings were carried out without pre-activation for 1 h 30 min at room temperature.

[0397] After syntheses completion, resins were transferred again to the 12 mL syringes to measure their final swellings then washed with (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0398] Dried Boc-protected peptides were cleaved from resins using a cleavage cocktail containing TFA/H.sub.2O/DTT/TIS (13 mL/g of peptidyl resin, 85/5/5/5 v/v/w/v) for 3 h at room temperature. The resin was filtered out and washed with TFA (3 mL/g of peptidyl resin). Peptides were precipitated with MTBE (10-folds the volume of TFA) at 0 C. The resulting suspensions were transferred to 50 ml conical centrifuge tubes and centrifuged at 2500 rcf for 10 min prior to supernatants' decantation. The crude solid products were washed again with cold MTBE (5) and dried under vacuum overnight. [0399] Molecular weight: 2180.32 [0400] Calculated mass: +2/2=1091.16 [0401] Observed mass (AMS 0.6 4(Gly)): +2/2=1091.02 [0402] Observed mass (AMS 0.6 8(Gly)): +2/2=1091.06

[0403] All amino acids used were Fmoc-protected except for the last amino acid (D-Phe) which was Boc-protected. [0404] Side chains protecting groups involved: tBu, Trt, Pbf: Y=Tyr(tBu), E=Glu(OtBu), D=Asp(OtBu), N=Asn(Trt), Arg(Pbf) [0405] Molecular weight of protected 20-mer: 3233, 86 g/mol [0406] Molecular weight of unprotected 20-mer: 2180.32 g/mol

TABLE-US-00019 TABLE 12 Data Synthesis of 20mer polypeptide: fPRPGGGGNGDFEEIPEEYL: PP 4 Solvent for the synthesis: DMF Mass of matrix at the beginning of synthesis after coupling of HMPB-leucine linker: 200 mg Theoretical Final mass Final volume Substitution mass of Boc of Boc of Boc Unmodified at beginning Synthesis protected protected protected matrix/resin Type of of synthesis scale peptidyl resin peptidyl resin peptidyl resin [mmol/g] construct [mmol/g] [mmol] [mg] [mg] [mL] AMS 0.93 1X 0.49 0.098 484 166 2.4 AMS 0.6 4X(Gly) 0.88 0.176 710 451 2.5 AMS 0.6 8X(Gly) 0.90 0.180 722 490 1.7 SPPS reactor Theoretical throughput crude peptide (mass after mass after Crude peptide HPLC purity cleavage/final cleavage after cleavage after cleavage volume) [mg] [mg] [%] [g/L] 236 135 78.8 56.3 424 320 76.5 128.0 433 290 46.1 170.6 Weight gain Assembly yield Cleavage yield [mg] [%] (1) [%] (2) 62 20 57 290 53 76 330 59 67

[0407] Application of support of invention significantly increases throughput while essentially preserving purity.

Synthesis Protocol for PP 5 (FVQYLIQG) with DMF as Solvent

[0408] Starting resins (100 mg each Fmoc-HMPB-Gly-AMS 0.60) were introduced into 12 mL syringes and swollen in DMF (6.5 mL/g, 21 h) for initial swelling measurements prior to their transfer to Symphony X reactors. Fmoc groups were deprotected before prior to each coupling step using 20% piperidine in DMF (110 min+120 min) at room temperature. All amino acid couplings were carried out using an equiamolar ratio (1:1:1) of Fmoc amino acid (0.30M in DMF), Oxyma (0.90M in DMF) and NN-Diisopropylcarbodiimide (0.90M in DMF) at 5-fold molar excess compared to synthesis scale. Couplings were carried out without pre-activation for 1 h 30 min at room temperature.

[0409] After syntheses completion, resins were transferred again to the 12 mL syringes to measure their final swellings then washed with (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0410] Dried Fmoc-protected peptide fragments were cleaved from resins using a soft cleavage cocktail containing 3% TFA in dichloromethane (DCM) for 15 min. This treatment was done 3 times and each time the resin was filtered out and resulting solution was collected. Combined collected solutions were evaporated under vacuum and the obtained concentrated oil was precipitated in MTBE/Heptane (1:1, v/v) prior to filtration and drying under vacuum overnight.

[0411] All amino acids used were Fmoc-protected. [0412] Side chains protecting groups involved: tBu, Trt: Q=Gln(Trt), Y=Tyr(tBu) [0413] Molecular weight of protected 8-mer: 1730, 1 g/mol

TABLE-US-00020 TABLE 13 Synthesis of 8mer polypeptide: FVQYLIQG: PP 5 Solvent for the synthesis: DMF Mass of matrix at the beginning of synthesis after coupling of HMPB-Glycine linker: 100 mg Theoretical Final mass Final volume Substitution mass of Fmoc- of Fmoc of Fmoc Unmodified at beginning Synthesis protected protected protected matrix/resin Type of of synthesis scale peptidyl resin peptidyl resin peptidyl resin [mmol/g] construct [mmol/g] [mmol] [mg] [mg] [mL] AMS 0.6 1X 0.40 0.04 160 132 0.7 AMS 0.6 8X(Gly) 1.00 0.10 251 187 0.5 SPPS reactor Theoretical throughput crude peptide (mass after mass after Crude peptide HPLC purity cleavage/final cleavage after cleavage after cleavage volume) [mg] [mg] [%] [g/L] 69 66 93 94.3 173 116 87 232.0 Weight gain Assembly yield Cleavage yield [mg] [%] (1) [%] (2) 41 59 96 109 63 67

[0414] Application of support of invention significantly increases throughput while essentially preserving purity.

Synthesis Protocol for PP 6 (HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG) with DMF as Solvent

[0415] Starting resins (200 mg each) were introduced into 12 mL syringes and swollen in DMF (6.5 mL/g, 21 h) for initial swelling measurements prior to their transfer to Symphony X reactors. Fmoc groups were deprotected prior to each coupling step using 20% piperidine in DMF (110 min+120 min) at room temperature. All amino acid couplings were carried out using an equimolar ratio (1:1:1) of Fmoc amino acid (0.30M in DMF), Oxyma (0.90M in DMF) and NN-Diisopropylcarbodiimide (0.90M in DMF) at 5-fold molar excess compared to synthesis scale. Couplings were carried out without pre-activation for 1 h 30 min at room temperature.

[0416] After syntheses completion, resins were transferred again to the 12 mL syringes to measure their final swellings then washed with (iPrOH, 3) and methyl tert-butyl ether (MTBE, 3) prior to their drying under vacuum overnight.

[0417] Dried Boc-protected peptides were cleaved from resins using a cleavage cocktail containing TFA/H.sub.2O/DTT/TIS (13 mL/g of peptidyl resin, 85/5/5/5 v/v/w/v) for 3 h at room temperature. The resin was filtered out and washed with TFA (3 mL/g of peptidyl resin). Peptides were precipitated with MTBE (10-folds the volume of TFA) at 0 C. The resulting suspensions were transferred to 50 ml conical centrifuge tubes and centrifuged at 2500 rcf for 10 min prior to supernatants' decantation. The crude solid products were washed again with cold MTBE (5) and dried under vacuum overnight. [0418] Molecular weight: 3383.73 [0419] Calculated mass: +3/3=1128.91; +4/4=846.93 [0420] Observed mass (AMS 0.6 4(Gly)): +3/3=1129.01; +4/4=847.06 [0421] Solvent used for the synthesis of PP 6 on Symphony X: DMF [0422] Mass of resin at beginning of synthesis after coupling of HMPB-Glycine linker: 200 mg.

[0423] All amino acids used were Fmoc-protected except for the last amino acid (His) which was Boc-protected. [0424] Side chains protecting groups involved: tBu, Boc, Trt, Pbf: R=Arg(Pbf), W=Trp(Boc), E=Glu(OtBu), K=Lys(Boc), Q=Gln(Trt), Y=Tyr(tBu), S=Ser(tBu), D=Asp(OtBu), T=Thr(tBu), H=His(Trt) [0425] Molecular weight of protected PP 6:5234.46 g/mol [0426] Molecular weight of unprotected PP 6:3383.73 g/mol

TABLE-US-00021 TABLE 14 Data synthesis of PP 6 polypeptide: HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG Theoretical Final mass Final volume Substitution mass of Boc of Boc of Boc Unmodified at beginning Synthesis protected protected protected matrix/resin Type of of synthesis scale peptidyl resin peptidyl resin peptidyl resin [mmol/g] construct [mmol/g] [mmol] [mg] [mg] [mL] AMS 0.93 1X 0.53 0.106 731 479 1.7 AMS 0.60 4X(Gly) 0.88 1.176 1082 728 2.1 SPPS reactor Theoretical throughput crude peptide (mass after mass after Crude peptide HPLC purity cleavage/final cleavage after cleavage after cleavage volume) [mg] [mg] [%] [g/L] 407 288 30 169.4 676 500 28 280.1 Weight gain Assembly yield Cleavage yield [mg] [%] (1) [%] (2) 303 55 71 567 62 74