LINKER MOLECULE AND USE THEREOF IN METHODS FOR PURIFYING PEPTIDES

Abstract

The present invention relates to a method for the purification of peptides which are produced by solid phase peptide synthesis (SPPS) and corresponding linker molecules for use in said method.

Claims

1. Compound of the formula
X.sub.1-L-X.sub.2(1), wherein X.sub.1 is selected from ##STR00027## wherein each R.sup.1 and R.sup.2 is independently from each other selected from H or B, wherein at least R.sup.1 or R.sup.2 is B, wherein R.sup.3 is selected from H or B, wherein B is an acid labile amine protecting group, wherein R.sup.4 is selected from H, C.sub.1-C.sub.12-alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid labile protecting group, L is selected from functional linkers, that are cleavable nucleophilically from X.sub.2 under basic conditions, in particular L is of the form -T-U, wherein T is a spacer between X.sub.1 and U, wherein in particular T is selected from substituted or unsubstituted C.sub.1-C.sub.12-alkyl-, in particular C.sub.1-C.sub.6-alkyl, in particular C.sub.1-C.sub.3-alkyl, R.sup.5C(O)NHR.sup.6, R.sup.5C(O)OR.sup.6, R.sup.5C(O)O, C(O)OR.sup.6, C(O)NHR.sup.6, C(O), C(O)O, R.sup.5-phenyl-R.sup.6, R.sup.5-phenyl-, -phenyl-R.sup.6, -phenyl-, wherein R.sup.5 and R.sup.6 are independently from each other selected substituted or unsubstituted C.sub.1-C.sub.12-alkyls, in particular C.sub.1-C.sub.6 alkyls, particularly C.sub.1-C.sub.3 alkyls, and wherein U is the cleavage activating part of the functional linker, wherein the activating part is formed to stabilize an anion formed during an basic cleavage from X.sub.2, X.sub.2 is of the form YZ, wherein Y is selected from OC(O) or S(O).sub.2, and Z is an electron-withdrawing leaving group.

2. Compound according to claim 1, wherein B is selected from Boc (COOtBu), trityl (C(Ph).sub.3), Mmt (C(Ph).sub.2C.sub.6H.sub.4OMe), DMT (C(Ph)(C.sub.6H.sub.4OMe).sub.2), Cbz (COOCH.sub.2Ph), benzylideneamine (CPh), phtalimides ((CO).sub.2C.sub.6H.sub.4), p-toluenesulfonamides (SO.sub.2C.sub.6H.sub.4Me), benzylamine (CH.sub.2Ph), acetamides (COMe), trifluoroacetamide (COCF.sub.3), Dde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), wherein particularly B is Boc, and/or the acetal- or ketal protecting groups are selected from ##STR00028## wherein r is 0 to 12, in particular 0, 1 or 2.

3. Compound according to claim 1, wherein T is selected from C.sub.1-C.sub.12-alkyl-, R.sup.5C(O)NHR.sup.6, R.sup.5C(O)OR.sup.6, R.sup.5C(O)O, C(O)OR.sup.6, C(O)NHR.sup.6, C(O), C(O)O, wherein R.sup.5 and R.sup.6 are independently from each other selected C.sub.1-C.sub.6-alkyls, in particular C.sub.1-C.sub.2-alkyls, in particular T is selected from CH.sub.2, CH.sub.2C(O)NH(CH.sub.2).sub.2, (CH.sub.2)C(O)O(CH.sub.2).sub.2, CH.sub.2C(O)O, C(O)O, C(O)O(CH.sub.2).sub.2, C(O), in particular CH.sub.2, CH.sub.2C(O)NH(CH.sub.2).sub.2, C(O)O(CH.sub.2).sub.2, CH.sub.2C(O)O, C(O).

4. Compound according to claim 1, wherein U of the moiety T-UY is selected from the moieties according to the formulas (5), (6), (7), (8), (9), (10) and (11), in particular from (5), (6), (8), (9) and (10), ##STR00029## wherein R.sup.8 is selected from C.sub.1-C.sub.6-alkyl, CF.sub.3, CH.sub.2CF.sub.3, ##STR00030## in particular from Boc-Lys(Boc)-, wherein R.sup.7.sub.n, R.sup.9.sub.m, R.sup.10.sub.p, R.sup.11.sub.q, R.sup.13.sub.r and R.sup.14.sub.s is selected from C.sub.1-C.sub.6-alkyl or I and/or -M-effects generating substituents, in particular C.sub.1-C.sub.3-alkyls, F, Cl, Br, I, CNNO.sub.2, N.sub.3, CF.sub.3, SO.sub.3H, CO.sub.2H, wherein n equals 0, 1, 2, 3 or 4, in particular n is 0 oder 1, in particular 0, wherein m equals 0, 1, 2 or 3, in particular m is 0 oder 1, in particular 0, wherein p equals 0, 1, 2, 3 or 4, in particular p is 0 oder 1, in particular 0, wherein q equals 0, 1, 2 or 3, in particular q is 0 oder 1, in particular 0, wherein r equals 0, 1, 2 or 3, in particular r is 0 oder 1, in particular 0, wherein s equals 0, 1, 2 or 3, in particular s is 0 oder 1, in particular 0.

5. Compound according to claim 1, wherein Z is selected from the group F, Cl, Br, I, N.sub.3, SR.sup.12, OCF.sub.3, OCH.sub.2CF.sub.3, OSO.sub.2CF.sub.3, SO.sub.2C.sub.6H.sub.4CH.sub.3, SO.sub.2CF.sub.3, SO.sub.2CH.sub.3 ##STR00031## in particular Cl, ##STR00032## in particular ##STR00033## wherein R.sup.12 is a C.sub.1-C.sub.6-alkyl-, an aryl- or a benzyl residue.

6. Compound according to claim 1, wherein X.sub.1 is a moiety of formula (2) or (3), in particular of formula (3), wherein R.sup.3 is H, R.sup.1 and R.sup.2 comprise a Boc protecting group or R.sup.1 is H and R.sup.2 is a Boc protecting group, wherein in particular R.sup.1 is H and R.sup.2 is a Boc protecting group.

7. Compound according to claim 1, wherein Y is of the form OC(O).

8. Compound according to claim 1, wherein T is of the form (CH.sub.2)C(O)NH(CH.sub.2).sub.2, (CH.sub.2)C(O)O(CH.sub.2).sub.2, C(O)O(CH.sub.2).sub.2, in particular C(O)O(CH.sub.2).sub.2 and (CH.sub.2)C(O)NH(CH.sub.2).sub.2, in particular (CH.sub.2)C(O)NH(CH.sub.2).sub.2, wherein U is a moiety of formula (5) or (6), in particular of formula (6), ##STR00034##

9. Compound according to claim 1, wherein T is of the form CH.sub.2C(O)O, C(O)O, in particular CH.sub.2C(O)O, wherein U is a moiety of formula (7), ##STR00035## wherein R.sup.7 is selected from C.sub.1-C.sub.6-alkyl or I and/or -M-effect generating substituents, in particular C.sub.1-C.sub.3-alkyl, F, Cl, Br, I, CNNO.sub.2, N.sub.3, CF.sub.3, SO.sub.3H, CO.sub.2H wherein n equals 0, 1, 2, 3 or 4, in particular 0 or 1, in particular 0.

10. Compound according to claim 1, wherein T is of the form CH.sub.2, wherein U is a moiety of formula (8), ##STR00036## wherein R.sup.8 is Boc-Lys(Boc) and r equals 0.

11. Compound according to claim 1, wherein T is of the form CH.sub.2, (CO), in particular CH.sub.2, wherein U is a moiety of formula (9), wherein s equals 0, ##STR00037##

12. Compound according to claim 1, wherein T is of the form C(O), wherein U is a moiety of formula (10), wherein m equals 0, ##STR00038## wherein Y is of the form SO.sub.2, and wherein Z is Cl.

13. (canceled)

14. An intermediate compound of formula X.sub.1-L-Y-PEP (12), wherein X.sub.1, L and Y is defined according to claim 1, and wherein PEP comprises a full-length peptide bound to X.sub.2 via its N-terminus.

15. (canceled)

16. Method for the purification of peptides, in particular of peptides prepared by solid phase peptide synthesis (SPPS), comprising the following steps: i. contacting a composition of a full-length peptide to be purified and at least one impurity, in particular at least one acetylated truncated sequence, with a capture compound according to claim 1, and subsequent reaction to a compound of formula (12), ii. cleavage of the acid labile protecting groups by addition of an acid, iii. contacting the composition of ii. with a surface-modified solid support, wherein a covalent hydrazone or oxime bond is formed between the capture compound and the solid support, and a compound of formula (13) is provided, iv. cleavage of the full-length peptide from the solid support.

17. Method according to claim 16, wherein the solid support comprises on its surface the functional groups aldehyde, in particular OCH.sub.2CHO, ketone, hydroxylamine, in particular ONH.sub.2, and hydrazine, in particular N.sub.2H.sub.3.

18. Method according to claim 16, wherein after or during cleavage of the full-length peptide from the solid support, the solid support D is cleaved from the residue X.sub.1-L of the capture compound and the solid support is regenerated.

19. An intermediate compound of formula D-X.sub.1-L-Y-PEP (13), wherein D is a surface-modified solid support, which is characterized in that the surface is modified by synthetic or natural polymers, in particular modified polysaccharides, in particular aldehyde- or hydrazine-modified sepharose/agarose or cellulose, wherein X.sub.1 is of the form NHO, NHNH or C(O), wherein L, Y and PEP are defined according to claim 12.

Description

DESCRIPTION OF THE FIGURES

[0149] FIG. 1 shows a schematic representation of the method according to the invention to illustrate the same. i) SPPS with acetylation after each coupling, n-fold repetition; ii) addition of molecule X1-L-X2, iii) immobilization; iv) washing; v) basic cleavage; vi) filtration; vii) regeneration. 1: purification resin, 2: capture molecule, 3: native peptide (desired product), 4: truncated sequences, x=1 to n, n: full-length peptide, n-x: truncated sequences.

[0150] FIG. 2 shows the absorption at 278 nm when measuring the supernatant after cleaving off the peptide (compound 3 in FIG. 6) with 0.5 vol. % N.sub.2H.sub.4 to demonstrate the reversibility of the hydrazone bond from example 1. Abs.: Absorption of the supernatant of peptide-loaded aldehyde-modified agarose support at 278 nm. t: Time elapsed after the addition of a 0.5% hydrazine solution in minutes (min). n: Amount of peptide in the supernatant in nmol calculated on the basis of absorption. Dashed line: Non-linear regression according to Hill with the formula: y=0.142*x/(3.16+x), R.sup.2=92, t.sub.1/2=3.16 min.

[0151] FIG. 3 shows chromatograms of the individual phases upon purification according to the invention of a peptide after Native Chemical Ligation (NCL) from Example 2. Chromatogram 1 shows the desired product (P) in the reaction mixture of the NCL after 24 hours. Chromatogram 2 shows the supernatant after 30 minutes of immobilization. Chromatogram 3 shows the desired product (P) after 30 minutes of washing and cleavage. P: Peak of the desired product.

[0152] FIG. 4 shows chromatograms upon purification according to the invention of peptides 7-13 after solid phase peptide synthesis from example 4. The chromatograms, which are labeled with supernatant, show the impurities which could be separated by the method. FM=capture molecule according to invention; (a) peptide 7 (Taul); (b) peptide 8 (Tau2); (c) peptide 9 (GNRH), (d) peptide 10 (Magainin); (e) peptide 11 (Terts72Y); (f) peptide 12 (bivalrudine); (g) peptide 13 (TAT), (h) peptide 14 (research peptide). Each upper diagram shows the peptide after synthesis and each lower diagram shows the peptide after purification.

[0153] FIG. 5 shows a schematic representation of the method according to the invention to illustrate the same. FIG. 5 shows an alternative representation to FIG. 1.

[0154] FIG. 6 shows Scheme 3: Reversibility of the hydrazone bond between a peptide and a purification resin. i) 0.1 M NH.sub.4OAc, pH=4, 0.1 M PhNH.sub.2, 30 min; ii) 0.5% N.sub.2H.sub.4, 5 mM TCEP; the peptide sequence (white characters in black area) is assigned to SEQ ID NO: 1.

[0155] FIG. 7 shows Scheme 4: Purification of a peptide after native chemical ligation i) 0.1 M Na.sub.2HPO.sub.4, 3 M Gdn*HCl, 20 mM TCEP, 50 mM MesNa, 1% (v/v) PhSH, pH=7, 15 h; ii) Addition sepharose resin and 2-fold vol. of 0.1 M NH.sub.4OAc pH=2.5, pH=4, 30 min; iii) Washing H.sub.2O, EtOH; iv) N.sub.2H.sub.4, 4 mM TCEP, 30 min. The peptide sequences are assigned to SEQ ID NO: 1 (white characters in black area) and SEQ ID NO: 2 (YENDRIK in white area).

[0156] FIG. 8 shows Scheme 5: Purification according to the invention of a peptide mixture after solid phase peptide synthesis (SPPS) i) SPPS with acetylation after each coupling, n-fold repetition; ii) Addition of molecule 2; iii) Cleavage with TFA; iv) Immobilization on purification resin by addition of Ph-NH.sub.2 at pH 3-4; v) Washing with water and buffer; vi) Base 5% NH.sub.4OH; vii) Regeneration of the purification resin 1 by addition of H.sub.2O/acetone/TFA (49,5/49,5/1). 1: purification resin, 2: capture molecule, 3: native peptide (desired product), 4: truncated sequences, x=1 to n, n: full-length peptide, n-x: truncated sequences.

[0157] FIG. 9 shows Scheme 6: Functionalization of agarose beads i) NaOH, NaBH.sub.4, H.sub.2O, r.t, o/n, 18 h; ii) 20 mM NaIO.sub.4, H.sub.2O, r.t, 1 h.

[0158] FIG. 10 shows Scheme 7: Synthesis of the base-labile linker i) N.sub.2H.sub.4*H.sub.2O; ii) 2.2 eq. pNO.sub.2PhCO.sub.2Cl; iii) mCPBA; n: peptide.

[0159] SEQ ID Nos: 1 to 10 show peptides from examples 1, 2 and 4.

EXAMPLES

Example 1. Demonstration of the Reversibility of the Hydrazone/Oxime Bond

[0160] The reversible binding of the peptide to aldehyde-modified agarose beads is demonstrated in the following using the example of hydrazone binding; due to the electronic similarity (see also A. Dirksen, P. Dawson, Bioconjugate Chem. 2008, 19, 2543-2548.), the results are applicable to the oxime bond. It is shown that the equilibrium can be controlled by the addition of hydrazine (N.sub.2H.sub.4).

[0161] Peptide 3 was bound to the support 1 in the conjugation buffer (0.1 M NH.sub.4OAc, 0.1 M PhNH.sub.2, pH=3) in 30 min (FIG. 6: Scheme 3). Subsequently, it was washed with water and the supernatant removed. Then a solution of 0.5 volume percent hydrazine hydrate was added to the beads and the absorption was measured at 278 nm of the supernatant (200 L) at a time interval (with the ND-I000 spectrophotometer from NanoDrop Technologie). It was found that after 10 min, 80% of the peptide can be measured in the supernatant. After another 50 minutes, 86% of the peptide was recovered. A half-life of three minutes was determined by non-linear regression, FIG. 2 shows the adsorption at 278 nm when measuring the supernatant after addition of N.sub.2H.sub.4.

Example 2: Purification of a Peptide after Native Chemical Ligation (NCL)

[0162] The purification of a peptide from a complex system containing a mixture of peptide material as well as organic and inorganic impurities was performed.

[0163] The mixture to be purified was obtained after an NCL (FIG. 7: scheme 4). This reaction is performed in an aqueous buffer system and is used to synthesize larger peptides and protein domains. After 24 h reaction time of the NCL the raw mixture is obtained, which essentially contained the desired ligation product and the thioester (H-YENRIK-MESA), which was used in excess. This can be seen in the chromatogram (UPLC-MS from Waters, Acquity H-Class PDA/QDa, on Polaris C18 A 5 m 250/4 column) in FIG. 3.

[0164] Twice the volume of conjugation buffer was added to the ligation buffer (0.1 M Na.sub.2HPO.sub.4, 20 mM TCEP, 50 mM MesNa, pH=7). Modified sepharose beads were subsequently added and the two-phase system was shaken for 30 minutes. The supernatant of the sepharose gel was analysed using UPLC-MS (FIG. 3, chromatogram 2); no mass that could be assigned to the ligation product could be found in the connected mass spectrometer. However, since an absorption signal can be observed at the retention time of the product, it also becomes apparent that impurities can be removed that could not be separated by means of HPLC. After washing with water and some ethanol and acetonitrile, 0.5 vol. % hydrazine hydrate in water was added to the peptide-loaded sepharose beads and the supernatant was analysed after 30 minutes, wherein the chromatogram 3 (FIG. 3) was obtained. This shows a high peptide purity (desired product) of over 90%.

Example 3: Exemplary Purification of a Peptide Mixture after Solid Phase Peptide Synthesis

[0165] The reaction scheme (scheme 5) of purification according to the invention is shown in FIG. 8.

[0166] After solid phase synthesis (SPPS), a capture molecule 2 (FIG. 8) is added to the last coupled amino acid. An important prerequisite for this is that acetylation was complete in the previous steps. Afterwards, all truncated sequences and the capture-molecule-modified full-length peptide are cleaved from the resin. Subsequently, non-peptide impurities are removed by ether precipitation and the crude peptide mixture is dissolved in an acetate buffer which has a pH value of 3-4 and to which 0.1 M aniline is added as catalyst. This solution is now added to functionalized sepharose beads 1 (FIGS. 8 and 9). This material is applied in protein purification as a material for affinity chromatography. Sepharose has the advantage of being easily penetratable by the peptides. The sepharose is previously aldehyde modified (see FIG. 9), the aldehyde modification of the sepharose is known from: J. Guisan, Enzyme Microb. Technol. 1988, 10, p. 375.

[0167] Hereby, only peptides that carry the capture molecule with the hydrazide function are anchored to the solid support. Amines, which theoretically can also react with aldehydes, are protonated at the pH value to be used and therefore not nucleophilic enough for an attack on aldehyde. The truncated sequences that are still in the sepharose can be washed out with water. Treatment of sepharose with a basic solution, e.g. ammonia in water, causes the capture molecule to decompose and the full-length peptide dissolves. The solution can then be lyophilized, wherein ammonia is removed. Subsequently, the peptide is obtained in pure form as a solid. One advantage of the method is its rapid immobilization and broad applicability to different peptides.

Example 4: Exemplary Regeneration of the Purification Resin

[0168] After peptide purification with capture molecule 2 (FIG. 8), the original aldehyde function of 1 remains blocked with hydrazine, in order to make the purification resin available again for a new purification cycle, the resin needs to be regenerated and thus the aldehyde function needs to be restored. This is achieved by shifting the equilibrium by adding aldehydes or ketones. The exemplary feasibility was shown as follows. The purification resin 1 was divided into three equal aliquots (I, II, III) and two aliquots (II, III) were treated with hydrazine in conjugation buffer for 30 min. Subsequently, it was washed with water and Aliquot III was washed seven times with a mixture of water, acetone and TFA (49.5:49.5:1). Then all aliquots were treated with FmocN.sub.2H.sub.3 in conjugation buffer for 14 h and then each washed five times with water, DMF and water. This was followed by two treatments with DMF/Piperidine (20%) for 4 min each and then the UV absorption of the resulting fluoren piperidine adduct was measured at 301 nm in the supernatant (SmartSpecPlus Spectrophotometer from BioRad in 1 mL semi-micro quartz cuvettes from Hellma). The untreated aliquot I showed a load of 27 mol/g; the aliquot II without regeneration a load of 18 mol/g (67% of I), and the regenerated resin showed a load density of 25 mol/g (93% of I). This experiment shows the successful regeneration of the purification resin.

TABLE-US-00001 TABLE 1 UV-absorption and loading density of purification resins after different regeneration treatments Aliquot A (301 nm) S (mol/g) Proportion [%] I 0.286 27 100 II 0.199 18 67 III 0.267 25 93

Example 5: Purification of a Peptide Mixture

[0169] The method according to the invention for the purification of peptides was applied to seven peptides of different polarity, which were H-TLADEVSASLAK-OH (SEQ ID NO: 3) (7) fragment 427-438 of the tau protein relevant to Alzheimer's disease, H-ATLADEVSASLAK-NH.sub.2 (SEQ ID NO: 4) (8) fragment 427-439 of tau, the cysteinyl peptide H-CQWSLHRKRHLARTLLTAAREPRPAPPSSNKV-NH.sub.2 (SEQ ID NO: 5) (8) from protein progona-doliberin-2 (9), H-GIGKFLHSAKKFGKAFVGEIMNS-NH.sub.2 (SEQ ID NO: 6) Magainin (10), H-YLFFYRKSV-NH.sub.2 Terts72Y (SEQ ID NO: 7) (11), H-FPRPGGGGNGDFEEIPEEYL-NH.sub.2 (SEQ ID NO: 8) Bivalirudin (12) and H-GRKKRRQRRRPQ-NH.sub.2 TAT (SEQ ID NO: 9) (13).

[0170] The crude peptide mixture was dissolved in the conjugation buffer and added to the sepharose beads 1 within 30-60 min, subsequent washing with water and neutral aqueous solutions (4M urea, 1M table salt) all acetylated truncated sequences and other impurities could be removed. The cleavage of linker 2 (FIG. 10: scheme 7) was performed with 5% ammonia solution in water for 20 minutes, it was then neutralized in-situ with acetic acid and the solubility was increased.

[0171] The purity of the individual phases was assessed by means of the UPLC-MS. The chromatograms of the non-purified (without capture molecule FM) and purified peptides as well as of the supernatant that contained the impurities are shown in FIG. 4. A peptide purity of 85% (originally 39%) was achieved for 7, of 93% (originally 39%) was achieved for 8, of 80% (originally 24%) was achieved for 9, of 90% (originally 23%) was achieved for 10, of 87% (originally 60%) was achieved for 11, of 90% (originally 40%) was achieved for 12, of 95% (originally 37%) was achieved for 13 (see FIG. 4).

TABLE-US-00002 TABLE 2 Purity and yield of different peptides after application of the purification meethod according to the invention Purity Purity Length Hydrophobic crude after Name No. Application Status in aa aa product treatment Yield Tau1 7 Alzheimer's preclinical 13 62% 40% 85% 70% disease Tau2 8 Alzheimer's preclinical 12 58% 39% 93% 69% disease GNRH 9 fertilisation approved 32 59% 35% 80% 42% Magainine 10 antibiotic approved 23 65% 23% 90% 62% Terts72Y 11 lung cancer phase II 9 77% 60% 87% 92% Bivalrudin 12 anti- approved 20 65% 40% 90% 89% coagulation TAT 13 HIV phase II 12 17% 37% 95% 83% Testpeptid 14 research 13 46% 45% 60%

[0172] The synthesis of the base-labile linker 2 was performed according to scheme 7 in FIG. 10.

Materials and Methods

Solid Phase Peptide Synthesis and Purification

[0173] The automated solid phase peptide synthesis was performed in 25 mol batches with a MultiPep RS peptide synthesis machine from Intavis AG. The syntheses of peptide amides were performed on Tentagel R RAM resin (0.2 mmol-g-1) from Rapp Polymer. Before the beginning of the synthesis, the resin was transferred to 3 mL syringe reactors (PE reactor from Multisyntech) and soaked in DMF. Unless otherwise specified, the specification of equivalents refers to the initial loading of the resin used.

Fmoc-Removal:

[0174] To remove the temporary Fmoc protecting groups, the resin was treated once for 4 min and once for 6 min with 400 L piperidine/DMF (4:1) and subsequently washed five times with 800 L DMF and it was continued with the coupling of the Fmoc amino acid derivatives.

Coupling of Fmoc Amino Acid Derivatives:

[0175] A solution of 5 eq. amino acid in DMF (0.3 M) was pre-activated for 1 min at room temperature with solutions of 4.5 eq. HCTU in DMF (0.3 M) and 10 eq. NMM in DMF (0.6M) and then added to the resin. After 30 min reaction time, the resin was washed three times with 800 L DMF and it was continued with blocking the termination sequences.

Blocking the Truncated Sequences:

[0176] The resin was treated once for 5 min with 400 l AC.sub.2O/2,6-Lutidin/DMF solution (5:6:89) and subsequently washed three times with 800 L DMF each.

Last Step Coupling of the Capture Molecule:

[0177] As the last step of the solid phase peptide synthesis, the capture molecule 2 was coupled to the desired target peptide. The synthesis resin was mixed with a solution of 5 eq. capture molecule 2 in DMF (0.3 M) and 5 eq. Oxyma in DMF (0.3 M) and 12 eq. DIPEA in DMF (0.7 M) mixed. After 60 min reaction time, it was washed twice with 800 L DMF each.

Release from Polymeric Support:

[0178] The resin was with 2 mL of a solution of 96% TFA, 2% water, 2% triisopropylsilane, in the case of thiol-containing amino acids (cysteine or methionine) in the target sequence 0.5% ethandithiol and 0.5% thioanisole were added to the solution, wherein the amount of TFA was reduced by 1%. The synthesis resin was treated with this cleaving mixture and shaken for 2 h at room temperature. Afterwards, the cleaving solution was collected and the resin was washed twice with 1 mL TFA each. The cleaving solution was combined with the washing solutions and precipitated in 50 ml cold diethyl ether. This suspension was then centrifuged and the organic supernatant was discarded.

Immobilization on Purification Resin:

[0179] After centrifugation, the crude precipitate was dissolved in 3 ml of the conjugation buffer (0.1 M NH.sub.4OAc, 0.1 M aniline, pH=3), if the mixture did not dissolve completely, acetonitrile was added. This solution was transferred into a 6 ml syringe from bBraun with a filter insert PE 25 m pore size from Multisyntech, in this syringe was one gram functionalized sepharose. Immobilization was performed for 30 to 60 minutes. It was then washed 5 times with deionized water of MilliQ purity, 5 times with a 4M urea solution and 5 times with water. Then the desired peptide was cleaved basically with 5 v % NH.sub.4OH and 1 v % mercaptoethanol in water from the resin. Lyophilization provided the desired peptides as a white flaky solid.

Synthesis of Capture Molecule 2 (Compound 2 in Scheme 7Corresponds to Compound (25))

Tert-butylhydrazine Carboxylate 5 (Compound 5 in Scheme 7)

[0180] Hydrazine monohydrate (80%, 32.5 g, 520 mmol) was mixed with isopropanol (100 ml) at 0 C., with a solution of Boc.sub.2O (50.0 g, 230 mmol) in isopropanol (50 ml) drop by drop. The reaction mixture became turbid after addition and stirring was continued at room temperature for 2 h. The solvent was removed, the residue dissolved in dichloromethane and dried over magnesium sulphate. Then the solvent was evaporated and the residue was recrystallized from hexane, resulting in the title compound 5 (22.8 g, 75%) as colorless crystals. Smp 36-37 C. Rf (EtOAc/hexane 1:1) 0.20. .sup.1H-NMR (300 MHz, CDCl.sub.3): 6.16 (s, 1H, NH), 3.67 (s, 2H, NH.sub.2), 1.42 (s, 9H, C(CH.sub.3).sub.3). .sup.13C-NMR (75 MHz, CDCl.sub.3, TMS): 158.3, 77.2.28.5. The analytical data are consistent with the literature data (A. Bredihhin, U. Maeorg, Tetrahedron 2008, 64, 6788-6793).

Bis(4-nitrophenyl)(thiobis(ethane-2,1-diyl))bis(carbonate) 6 (Compound 6 in Scheme 7)

[0181] 6.72 (33 mmol) 4-nitrophenyl chloroformate was added to a solution of 1.87 ml (2.12 g, 15 mmol) 2,2-thiodiethanol in 40 ml anhydrous dichloromethane. Then 2.68 ml (33 mmol) of anhydrous pyridine was slowly added drop by drop with ice cooling and vigorous stirring. The reaction mixture was stirred for 1 h at room temperature. The reaction solution was mixed with 100 ml saturated ammonium chloride solution, extracted three times with 100 ml chloroform and dried over anhydrous magnesium sulphate.

[0182] The organic phases were combined and constricted in a vacuum. The residue was absorbed into ethyl acetate and the product was precipitated with a small amount of cyclohexane. After filtration, 5.43 g (12 mmol, 80%) was obtained as white solid. Melting point: 136.5 C., Rf (EtOAc/cyclohexane 1:1) 0.78. .sup.1H NMR (300 MHz, DMSO) 8.30 (d, J=9.2 Hz, 2H, ArH), 7.55 (d, J=9.3 Hz, 2H, ArH), 4.42 (t, J=6.4 Hz, 2H, CH.sub.2), 2.96 (t, J=6.5 Hz, 2H, CH.sub.2). .sup.13C-NMR (75 MHz, CDCl.sub.3, TMS): 155.22, 151.93, 145.17, 125.41, 122.56, 67.85, 29.63.

2-[2-(1-((tert-butyl)oxy-carbonyl)oxy-carbonyl)-hydrazyl-ethylsulfanyl]-ethyl 4-nitrophenyl carbonate 7 (Compound 7 in Scheme 7)

[0183] 1.97 g (4.31 mmol) bis(4-nitrophenyl) (thiobis(ethane-2,1-diyl))bis(carbonate) 6 were added to 20 ml dry dichloromethane and at 0 C. 1 eq. (0.58 g, 4.31 mmol) tert-butyl hydrazine carboxylate 5 with 3 eq. (1.13 ml, 6.66 mmol) DIPEA was slowly added dropwise for one hour. The reaction solution was stirred for another 12 hours and then mixed with water. The product was extracted three times with 100 ml dichloromethane and dried over anhydrous magnesium sulphate. The organic phases were combined and constricted in a vacuum. The residue was purified by column chromatography (EtOAc/cyclohexane 2:1), after which 1.03 g (2.31 mmol, 53%) of a transparent oil was obtained. Rf (EtOAc/Cyc10hexane 1:1) 0.20. .sup.1H NMR (300 MHz, CDCh) 8.28 (d, J=9.1 Hz, 2H, ArH), 7.39 (d, J=9.1 Hz, 1H, ArH), 6.64 (s, 1H, NH), 6.33 (s, 1H, NH), 4.44 (t, J=6.8 Hz, 2H, CH.sub.2), 4.33 (t, J=6.6 Hz, 2H, CH.sub.2), 2.92 (t, J=6.8 Hz, 2H, CH.sub.2), 2.84 (t, J=6.6 Hz, 2H, CH.sub.2), 1.46 (s, 9H). .sup.13C NMR (75 MHz, CDCl.sub.3) 155.52, 152.57, 125.47, 121.96, 82.09, 68.06, 65.34, 64.18, 31.10, 30.59, 28.27, 27.03.

2-[2-(1-((tert-butyl)oxy-carbonyl)oxy-carbonyl)-hydrazyl-ethylsulfonyl]-ethyl4-nitrophenyl carbonate 2 (Compound 2 in Scheme 7)

[0184] To a solution of thioether 7 (0.52 g, 1.1 mmol) in 50 ml dichloromethane, 77% (489 g, 2.2 mmol) of m-CPBA was slowly added at room temperature. After stirring for 12 h the reaction mixture was mixed with 1M NaHCO.sub.3 solution and the organic phase was extracted three times with 50 ml dichloromethane. The combined organic phases were dried with magnesium sulphate and the solvent was removed from the rotary evaporator, after which the product was precipitated as white amorphous solid 0.52 mg (1.1 mmol, quantitative). .sup.1H NMR (300 MHz, CDCl.sub.3) 8.29 (d, J=9.2 Hz, 2H, ArH), 7.41 (d, J=9.2 Hz, 2H, ArH), 6.89 (s, 1H, NH), 6.36 (s, 1H, NH), 4.74 (t, J=5.9 Hz, 2H, CH.sub.2), 4.63 (t, J=5.3 Hz, 2H, CH.sub.2), 3.54 (t, J=5.9 Hz, 2H, CH.sub.2), 3.45 (t, J=5.5 Hz, 2H, CH.sub.2), 1.45 (s, 9H). .sup.13C NMR (75 MHz, CDCl3) 155.85, 155.18, 152.29, 145.79, 125.56, 122.00, 82.32, 62.25, 59.45, 54.01, 53.24, 28.23.

[0185] The synthesis of the compounds according to formula (14), (15), (17), (18), (19): was based on a modular principle, which is shown in the general synthesis scheme (Scheme 1).

Syntheses for the Preparation of the Compound of Formula (14)

N,N-Bis-(tert-butoxycarbonyl)-aminooxyacetyl-N-hydroxylsuccinimide Ester (BS2: XO, R.SUP.1.=Boc)

[0186] Add N-hydroxylsuccinimide (0.41 g, 3.20 mmol) and dicyclohexylcarbodiimide (0.67 g, 0.32 mmol) at 0 C. to a solution of commercially available N,N-bis-boc-amino-oxyacetic acid (1.00 g, 3.20 mmol) in 11 ml ethyl acetate/dioxane (1:1). At room temperature the solution was allowed to stir for 3 hours and the suspension was filtered over Celite and washed with ethyl acetate. The filtrate was concentrated under vacuum to dry and dissolved again in 100 ml ethyl acetate. It was washed with 5% NaHCO.sub.3 solution, saturated NaCl solution and water (100 ml each). The organic phase was dried with MgSO.sub.4 and evaporated under vacuum wherein 1.24 g (3.20 mmol) product was obtained as a white solid. Yield: 1.24 g (quant.); Rf (cyclohexane/ethyl acetate, 1:1) 0.50; .sup.1H NMR (300 MHz, CDCl.sub.3) 4.86 (s, 2H), 2.85 (s, 4H), 1.53 (s, 18H).

N,N-Bis-(tert-butoxycarbonyl)-aminooxyacetyl-1-((2-aminoethyl)thio)propan-2-ol amide (BS3: XO, R.SUP.1.=Boc)

[0187] BS2 (XO, R.sub.1=Boc, 1.00 g, 2.55 mmol) was mixed in 25 ml dichloromethane with 1-((2-aminoethyl)thio)propan-2-ol (0.38 g, 2.55 mmol) and diisopropyl-ethylamine (DIPEA, 0.53 ml, 3.06 mmol) and stirred overnight. It was washed with 5% NaHCO.sub.3 solution, saturated NaCl solution and water (100 ml each). The organic phase was dried with MgSO.sub.4 and vacuum-constricted wherein 1.04 g (2.55 mmol) product was obtained as a white solid. Yield: 1.04 g (quant.); Rf (CH.sub.2Cl.sub.2/MeOH, 98:2) 0.35; .sup.1H NMR (300 MHz, CDCl.sub.3) 7.95 (s, 1H), 4.44 (s, 2H), 3.92-3.82 (m, 1H), 3.53 (qd, J=6.7, 1.5 Hz, 2H), 2.83-2.65 (m, 4H), 2.49 (dd, J=13.7, 8.7 Hz, 1H), 1.55 (s, 18H), 1.25 (d, J=6.2 Hz, 3H); .sup.13C NMR (75 MHz, CDCl.sub.3) 167.94, 150.57, 85.41, 65.98, 41.86, 38.81, 32.21, 28.19, 22.23.

2,2-Dimethylpropanoyloxy-[2-[2-[2-[2-(4-nitrophenoxy)carbonyloxypropylthionyl]ethylamino]-2-oxo-ethoxy]amino] 2,2-dimethylpropanoate P (XO, R.SUB.1.=Boc, R.SUB.2.=OC.SUB.6.H.SUB.4.pNO.SUB.2.)

[0188] Bis(4-nitrophenyl)carbonate (1.01 g, 4.95 mmol) were added to a solution of BS3 (XO, R.sub.1=Boc) (1.52 g, 3.30 mmol) in 5 ml dry CH.sub.2Cl.sub.2. Dry pyridine (0.40 ml, 4.95 mmol) was then added under ice cooling. The reaction solution was stirred for 18 hours. The precipitate was filtered off and was washed with 50 ml DCM. The filtrate was washed with saturated NH.sub.4Cl solution (50 ml) and the aqueous phase was extracted with 50 ml CHCl.sub.3. After drying with MgSO.sub.4, the combined organic phases were constricted on the rotary evaporator and the residue was purified by column chromatography (cyclohexane/etOAc, 2:1). Yield: 1.27 g (67%); Rf (cyclohexane/etOAc, 1:1) 0.44; .sup.1H NMR (300 MHz, CDCl.sub.3) 8.27 (d, J=9.2 Hz, 2H), 7.89 (s, 1H), 7.39 (d, J=9.2 Hz, 2H), 5.00 (dd, J=12.5, 6.3 Hz, 1H), 4.43 (s, 2H), 3.53 (dd, J=13.3, 6.5 Hz, 1H), 2.86-2.69 (m, 2H), 1.53 (s, 18H), 1.47 (d, J=6.3 Hz, 3H).

2,2-Dimethylpropanoyloxy-[2-[2-[2-(4-nitrophenoxy)carbonyloxypropylsulfonyl]ethylamino]-2-oxo-ethoxy]amino]2,2-dimethylpropanoates Formula (14)

[0189] Slowly add m-CPBA (0.96 g, 4.30 mmol) at room temperature to a solution of P (XO, R.sub.1=Boc, R.sub.2=OC.sub.6H.sub.4pNO.sub.2) (1.27 g, 2.15 mmol) in 21 ml dichloromethane. After 12 hours of stirring, the reaction mixture was washed twice with a saturated NaHCO.sub.3 solution (15 ml) and the organic phase was concentrated in a vacuum after drying with MgSO.sub.4. The sulfone was preserved as a white solid. Yield: 1.26 g (97%); Rf (cyclohexane/etOAc, 1:1) 0.29; .sup.1H NMR (300 MHz, CDCl.sub.3) 8.28 (d, J=9.3 Hz, 1H), 8.15 (t, J=5.8 Hz, 1H), 7.41 (d, J=9.3 Hz, 1H), 5.48-5.39 (m, 1H), 4.44 (s, 1H), 3.83 (dd, J=6.2, 4.2 Hz, 1H), 3.56 (dd, J=14.9, 8.3 Hz, 1H), 3.37 (t, J=6.5 Hz, 1H), 3.27 (dd, J=14.9, 3.9 Hz, 1H), 1.57 (d, J=6.4 Hz, 1H), 1.54 (s, 7H). .sup.13C NMR (75 MHz, CDCl.sub.3) 168.58, 155.42, 151.58, 150.52, 145.67, 125.46, 122.04, 85.60, 77.16, 76.62, 70.63, 58.19, 53.67, 33.07, 28.16, 20.21; ESI-MS: (calculated MNa.sup.+: 628.16 g/mol, found: 628.17 m/z).

Syntheses for the Preparation of the Compound of Formula (18)

((tert-butoxycarbonyl)amino)glycine (BS1 XNH, R.SUB.1.H)

[0190] Bromoacetic acid (1.48 g, 10.4 mmol) was added to a methanolic solution (10 ml) of NaOH (0.70 g, 17.4 mmol) and Boc-hydrazine (1.17 g, 8.7 mmol) at 0 C. The solution was heated for 5 hours under reflux. Then MeOH was removed and 50 ml water was added. The aqueous phase was extracted three times with ethyl acetate (50 ml). The aqueous phase was then brought to pH 2 with citric acid and extracted three times with 50 ml ethyl acetate. The combined organic phases were dried with MgSO.sub.4 and the solvent was removed under reduced pressure. Yield: 0.85 g (51%) white solid; Rf (CH.sub.2Cl.sub.2/MeOH, 8:2) 0.15. .sup.1H NMR (300 MHz, DMSO) 8.55 (s, 2H), 8.17 (s, 2H), 3.40 (s, 2H), 1.37 (s, 9H); ESI-MS: (calculated MH+: 191.10 g/mol, found: 191.33 m/z).

N-(tert-butoxycarbonyl)-N-((tert-butoxycarbonyl)amino)glycine (BS1 X=NBoc, R.SUB.1.H)

[0191] Boc.sub.2O (5.74 g, 26.03 mmol) was added as a solid to a solution of ((tert-butoxycarbonyl)amino)glycine (5.00 g, 26.0 mmol) and NaOH (1.57 g, 39.04 mmol) in 104 ml dioxane/H.sub.2O (1:1). The solution was stirred overnight at room temperature for 18 hours and the dioxane was then removed under reduced pressure. Add 100 ml saturated NaHCO.sub.3 solution to the aqueous residue and wash twice with 100 ml Et.sub.2O. The aqueous phase was brought to pH 2 with citric acid. The white suspension was extracted three times with 150 ml ethyl acetate. After drying with MgSO4 the solvent was removed wherein a white solid formed. Yield: 7.56 g (quant.); Rf (CH.sub.2Cl.sub.2/MeOH, 9:1) 0.75; .sup.1H NMR (300 MHz, DMSO) 12.34 (s, 1H), 9.24 (s, 1H), 3.56 (s, 2H), 1.46-1.32 (m, 18H).

N-(tert-butoxycarbonyl)-N-((tert-butoxycarbonyl)amino)glycinyl-N-hydroxylsuccinimide (BS2 X=NBoc, R.SUB.1.H)

[0192] N-(tert-butoxycarbonyl)-N-((tert-butoxycarbonyl)amino)glycine (1.36 g, 4.45 mmol) in 15 ml ethyl acetate/dioxane (1:1) was added at 0 C. to N-hydroxylsuccinimide (0.52 g, 4.45 mmol) and dicyclohexylcarbodiimide (DCC, 0.93 g, 4.45 mmol). At room temperature, the solution was allowed to stir for 15 hours. Afterwards the suspension was filtered over Celite and washed with ethyl acetate. The filtrate was concentrated under vacuum to dry and dissolved again in 100 ml ethyl acetate. It was washed with 5% NaHCO.sub.3 solution, saturated NaCl solution and water (100 ml each). The organic phase was dried with MgSO.sub.4 and evaporated under vacuum wherein 1.24 g (3.20 mmol) product was obtained as white foam. Yield: 1.51 g (88%); Rf (cyclohexane/ethyl acetate, 1:1) 0.45; .sup.1H NMR (300 MHz, CDCl.sub.3) 4.67 (s, 1H), 4.19 (s, 2H), 2.87 (s, 4H), 1.49 (m, 18H).

N-(tert-butoxycarbonyl)-N-((tert-butoxycarbonyl)amino)glycinyl-1-((2-aminoethyl)thio)propan-2-ol Amide (BS3: X=NBoc, R.SUB.1.H)

[0193] BS2 (X=NBoc, R.sub.1H, 0.36 g, 0.92 mmol) was mixed in 10 ml dichloromethane with 1-((2-aminoethyl)thio)propan-2-ol (0.13 g, 0.92 mmol) and DIPEA (0.18 ml, 1.01 mmol) and stirred overnight. It was washed with 5% NaHCO.sub.3 solution, saturated NaCl solution and water (100 ml each).

[0194] The organic phase was dried with MgSO.sub.4 and vacuum-constricted wherein 0.27 g (0.65 mmol) product was obtained as white foam. Yield: 0.27 g (quant.); Rf (CH.sub.2Cl.sub.2/MeOH, 98:2) 0.36; .sup.1H NMR (300 MHz, CDCl3) 8.34 (s, 1H), 6.66 (s, 1H), 4.05 (s, 2H), 3.90-3.81 (m, 1H), 3.49 (dd, J=10.4, 4.0 Hz, 2H), 2.78-2.63 (m, 3H), 2.47 (dd, J=13.7, 8.7 Hz, 1H), 1.49 (s, 9H), 1.46 (s, 9H), 1.23 (d, J=6.2 Hz, 3H).

[2-(2,2-dimethylpropanoyloxy)-2-[2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxycarbonyloxypropylthionyl]ethylamino]-2-oxo-ethyl]hydrazino] 2,2-dimethylpropanoate P (XO, R.SUB.1.=Boc, R.SUB.2.ONO.SUB.2.C.SUB.2.H.SUB.4.)

[0195] N,N-disuccinimidyl carbonate (0.19 g, 0.72 mmol) was added to a solution of BS3 (X=NBoc, R.sub.1H) (0.25 g, 0.72 mmol) in 5 ml dry CH.sub.2Cl.sub.2. Dry pyridine (0.06 ml, 0.73 mmol) was then added under ice cooling. The reaction solution was stirred for 17 hours. 50 ml DCM was added to the solution. The organic phase was washed with 10% citric acid solution and dried with MgSO.sub.4. The combined organic phases were constricted at the rotary evaporator and the residue was purified by column chromatography (CH.sub.2Cl.sub.2/MeOH, 19:1). Yield: 1.27 g (67%); Rf (CH.sub.2Cl.sub.2/MeOH, 9:1) 0.60; .sup.1H NMR (300 MHz, CDCl.sub.3) 8.53 (d, J=95.5 Hz, 1H), 7.07 (s, J=9.0 Hz, 1H), 4.01 (s, 2H), 3.82 (ddd, J=8.2, 6.1, 3.9 Hz, 1H), 3.41 (d, J=6.1 Hz, 4H), 2.67 (dt, J=13.2, 9.3 Hz, 4H), 2.46 (dd, J=13.7, 8.2 Hz, 2H), 1.44 (s, 9H), 1.41 (s, 9H), 1.18 (t, J=6.8 Hz, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3) 169.71, 162.47, 154.43, 77.16, 66.04, 50.53, 41.57, 39.13, 32.22, 28.18, 28.12, 25.57, 22.06, 18.88.

[2-(2,2-dimethylpropanoyloxy)-2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxycarbonyloxypropylsulfonyl]ethylamino]-2-oxo-ethyl]hydrazino] 2,2-dimethylpropanoate (formula (18))

[0196] m-CPBA (0.81 g, 0.36 mmol) was slowly added at room temperature to a solution of P (X=NBoc, R.sub.1 H, R.sub.2ONO.sub.2C2H.sub.4) (0.10 g, 0.18 mmol) in 5 ml dichloromethane. After stirring for 14 hours, the reaction mixture was washed three times with 5% NaHCO.sub.3 in a saturated NaCl solution (33 ml each) and the organic phase was then dried with MgSO.sub.4. The solvent was removed in a vacuum and the sulfone was preserved as a white amorphous solid. Yield: 0.08 g (76%); Rf (CH.sub.2Cl.sub.2/MeOH, 9:1) 0.45 1H NMR (300 MHz, CDCl.sub.3) 8.48 (s, 1H), 6.95 (s, 1H), 4.39 (dt, J=15.7, 7.8 Hz, 1H), 4.06 (s, 2H), 3.73 (d, J=4.3 Hz, 2H), 3.48-3.17 (m, 4H), 3.41 (d, J=6.1 Hz, 2H), 3.02 (d, J=13.2 Hz, 1H), 2.85-2.68 (m, 3H), 1.46 (s, 9H), 1.43 (s, 9H), 1.30 (d, J=6.4 Hz, 3H); .sup.13C NMR (75 MHz, CDCl.sub.3) 170.18, 167.99, 154.71, 133.30, 131.86, 130.13, 129.83, 128.20, 77.16, 62.88, 53.37, 33.53, 28.23, 28.16, 25.57, 23.25. ESI-MS: (calculated MNa.sup.+: 603.19 g/mol, found: 603.06 m/z).

Syntheses for the Preparation of the Compound of Formula (15)

N-(tert-butoxycarbonyl)-aminooxyacetyl-1-((2-aminoethyl)thio)propan-2-ol amide (BS3: XO, R.SUB.1.H)

[0197] Commercially available 2-((((tert-butoxycarbonyl)amino)oxy)acetic acid (1.00 g, 4.73 mmol) was dissolved in dry CH.sub.3CN (47 ml). Add N-hydroxysuccinimide (0.66 g, 5.68 mmol) and DCC (1.18 g, 5.68 mmol) successively to the solution and stir the resulting reaction mixture at room temperature for 1 hour. Then add a solution of 1-((2-aminoethyl)thio)propan-2-ol (0.85 g, 5.68 mmol) in 3 ml dry CH.sub.3CN and stir the resulting reaction mixture at room temperature for 18 hours. The CH.sub.3CN was removed and the concentrate absorbed in 50 ml ethyl acetate. It was washed with 10% citric acid solution (50 ml) and saturated NaCl solution. The residue obtained was purified by chromatography on a silica gel column with a step gradient of MeOH (1-8%) in CH.sub.2Cl.sub.2 as a mobile phase. The desired building block was obtained as white foam. Yield: 0.26 g (16%); Rf (CH.sub.2Cl.sub.2/MeOH, 9:1) 0.50; .sup.1H NMR (300 MHz, acetones) 8.14 (s, 1H), 4.22 (s, 2H), 3.85 (t, J=1.6 Hz, 1H), 3.53-3.38 (m, 2H), 2.72-2.66 (m, 2H), 2.62-2.58 (m, 2H), 1.46 (s, 9H), 1.19 (d, J=6.1 Hz, 3H).

[2-[2-[2-(4-nitrophenoxy)carbonyloxypropylthionyl]ethylamino]-2-oxo-ethoxy]amino] 2,2-dimethylpropanoate P (XO, R.SUB.1.H, R.SUB.2.=OC.SUB.6.H.SUB.4.pNO.SUB.2.)

[0198] Bis(4-nitrophenyl)carbonate (0.276 g, 0.90 mmol) were added to a solution of BS3 (XO, R.sub.1H) (0.24 g, 0.75 mmol) in 5 ml dry CH.sub.2Cl.sub.2. Dry pyridine (0.07 ml, 0.75 mmol) was then added under ice cooling. The reaction solution was stirred for 18 hours. The precipitate was filtered off and was washed with 50 ml DCM. The filtrate was washed with saturated NH.sub.4Cl solution (50 ml) and the aqueous phase was extracted with 50 ml CHCl.sub.3. After drying with MgSO.sub.4, the combined organic phases were constricted on the rotary evaporator and the residue was purified by column chromatography (cyclohexane/etOAc, 2:1). Yield: 0.27 g (96%); Rf (cyclohexane/etOAc, 1:1) 0.34; .sup.1H NMR (300 MHz, CDCl.sub.3) 8.35 (s, 1H), 8.27 (d, J=9.1 Hz, 2H), 7.58 (s, 1H), 7.40 (d, J=9.1 Hz, 2H), 5.06-4.94 (m, 1H), 4.32 (s, 2H), 3.54 (d, J=6.3 Hz, 2H), 2.87-2.73 (m, 4H), 1.48 (d, J=4.1 Hz, 3H), 1.47 (s, Hz, 9H).

[2-[2-[2-(4-nitrophenoxy)carbonyloxypropylsulfonyl]ethylamino]-2-oxo-ethoxy]amino] 2,2-dimethylpropanoates Formula (15)

[0199] m-CPBA (0.263 g, 1.18 mmol) was slowly added at room temperature to a solution of P (XO, R.sub.1=Boc, R.sub.2=OC.sub.6H.sub.4pNO.sub.2) (0.30 g, 0.59 mmol) in 5 ml dichloromethane. After 12 hours of stirring, the reaction mixture was washed twice with a saturated NaHCO.sub.3 solution (15 ml) and the organic phase was concentrated in a vacuum after drying with MgSO.sub.4. The sulphone was preserved as white foam. Yield: 0.250 g (84%); Rf (cyclohexane/etOAc, 1:1) 0.05; 1H NMR (300 MHz, CDCl3) 8.51 (s, 1H), 8.28 (d, J=9.2 Hz, 2H), 7.64 (s, 1H), 7.41 (d, J=9.2 Hz, 3H), 5.47-5.35 (m, 1H), 4.33 (s, 2H), 3.88-3.79 (m, 2H), 3.57 (dd, J=14.9, 8.3 Hz, 1H), 3.41-3.34 (m, 2H), 3.28 (dd, J=14.8, 3.8 Hz, 1H), 1.57 (d, J=6.4 Hz, 3H), 1.48 (s, 9H); .sup.13C NMR (75 MHz, CDCl.sub.3) 168.58, 155.42, 151.58, 150.52, 145.67, 125.46, 122.04, 85.60, 77.16, 76.62, 70.63, 58.19, 53.67, 33.07, 28.16, 20.21; ESI-MS: (calculated MH+: 528.16 g/mol, found: 528.15 m/z).

Syntheses for the Preparation of the Compound of Formula (16)

Sodium 4-carboxy-2-nitrobenzenesulfonate

[0200] 4-sulfamylbenzoic acid was dissolved in a mixture of 5 ml fuming HNO.sub.3 and 10 ml H.sub.2SO.sub.4 (95%). The reaction solution was stirred overnight at 90 C. and then diluted with 100 ml water. At 0 C. the acid was neutralized by adding Na.sub.2CO.sub.3. Subsequently, acidification was carried out by adding HCl until the pH value was 2. The water was removed and the residue was extracted with EtOH/iPrOH (1:1). Subsequently, the organic solvent was removed wherein the product was obtained as a brown solid. Yield: 6.86 g (61%); Rf (CH.sub.2C12/MeOH/AcOH, 7:2:1) 0.05; 1H NMR (300 MHz, MeOD) 8.23 (d, J=1.6 Hz, 1H), 8.17 (d, J=1.6 Hz, 1H), 8.16 (s, 1H); ESI-MS (neg.): (calculated (M-Na): 245.97 g/mol, found: 296.00 m/z).

Sodium 4-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-2-nitrobenzenesulfonate

[0201] A solution of t-butyl carbazate (1.91 g, 14.27 mmol) and sodium 4-carboxy-2-nitrobenzenesulfonate (3.84 g, 14.27 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.76 g, 14.27 mmol) was mixed overnight at room temperature in 60 ml methanol/H.sub.2O (1:1). The solvents were removed under reduced pressure and the residue was purified by column chromatography (CH.sub.2Cl.sub.2/MeOH, 9:1). A solution of t-butyl carbazate (1.91 g, 14.27 mmol) and sodium 4-carboxy-2-nitrobenzenesulfonate (3.84 g, 14.27 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.76 g, 14.27 mmol) was mixed overnight at room temperature in 60 ml methanol/H.sub.2O (1:1). The solvents were removed under reduced pressure and the residue was purified by column chromatography (CH.sub.2C12/MeOH, 9:1). Yield: 6.86 g (61%); Rf (CH.sub.2C12/MeOH/AcOH, 7:2:1) 0.30; .sup.1H NMR (300 MHz, DMSO) 10.46 (s, 1H), 9.04 (s, 1H), 8.01 (d, J=1.5 Hz, 1H), 7.99 (d, J=1.5 Hz, 1H), 7.96 (s, 1H), 1.43 (s, 9H); ESI-MS (neg.): (calculated (M-Na): 360.05 g/mol, found: 360.00 m/z).

tert-butyl 2-(4-(chlorosulfonyl)-3-nitrobenzoyl)hydrazine-1-carboxylate

[0202] Cyanuric chloride (0.26 g, 1.42 mmol) was added to a solution of sodium 4-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-2-nitrobenzenesulfonate (0.55 g, 1.42 mmol) and 18-crown-6 ether (0.02 g, 0.07 mmol) in dry acetone. The solution was heated for 18 h under reflux. After cooling, the reaction mixture was filtered over Celite and purified by column chromatography (CH.sub.2Cl.sub.2/MeOH, 9:1). Yield: 0.23 g (43%); .sup.1H NMR (300 MHz, CDCl.sub.3) 10.18 (s, 2H), 8.22 (d, J=0.5 Hz, 1H), 8.22 (dd, J=1.7, 0.5 Hz, 1H), 8.18 (d, J=1.6 Hz, 1H), 8.15 (d, J=0.6 Hz, 1H), 1.23 (s, 9H); ESI-MS (neg.): (calculated (M): 379.02 g/mol, found: 378.92 m/z).

Syntheses for the Preparation of the Compound of Formula (20)

6-Azidoisobenzofuran-1(3H)-on

[0203] A clear solution of 6-amino-phtalid (2.50 g, 15.92 mmol) in 1M HCl (28 ml) was cooled to 0 C. and mixed with 3 ml of an aqueous solution of NaNO.sub.2 (1.66 g, 13.89 mmol) drop by drop. The resulting suspension was stirred for 10 min at 0 C. and mixed with 10 ml of a solution of NaN.sub.3 (2.09 g, 31.85 mmol) drop by drop at 0 C. (strong HN.sub.3 gas development!, foaming). The foamy suspension was stirred at 0 C. for one hour. The precipitate was sucked off and washed several times with a total of 300 ml of water. The brown solid was crushed and dried overnight in a drying cabinet. It was then dissolved in 300 ml CH.sub.2Cl.sub.2 and filtered off. The filtrate was freed under vacuum from the solvent wherein a light brown solid was obtained. Yield: 2.53 g (91%); Rf (CH.sub.2C12/MeOH/AcOH, 7:2:1) 0.30; .sup.1H NMR (300 MHz, CDCl.sub.3) 7.58 (d, J=1.9 Hz, 1H), 7.47 (dd, J=8.2, 0.7 Hz, 1H), 7.31 (dd, J=8.2, 2.1 Hz, 1H), 5.31 (s, 2H); ESI-MS: (calculated (MH).sup.+: 176.05 g/mol, found: 176.23 m/z).

5-azido-2-(hydroxymethyl)benzohydrazide

[0204] 6-Azidoisobenzofuran-1(3H)-on (0.50 g, 2.83 mmol) were dissolved in 6 ml dimethylformamide (DMF) and hydrazine hydrate (0.71 ml, 14.13 mmol) was added and the solution was stirred at 70 C. for 3 hours. The DMF and hydrazine hydrate were removed under vacuum. The residue was purified by column chromatography (CH.sub.2C12/MeOH, 9:1), wherein a light yellow powder was obtained. Yield: 0.10 g (17%). Rf (CH.sub.2Cl.sub.2/MeOH, 9:1) 0.53; .sup.1H NMR (300 MHz, DMSO) 9.62 (s, 1H), 7.57 (d, J=8.3 Hz, 1H), 7.20 (dd, J=8.3, 2.4 Hz, 1H), 7.09 (d, J=2.4 Hz, 1H), 5.31-5.21 (m, 1H), 4.57 (d, J=5.7 Hz, 2H), 4.50 (s, 2H), 3.33 (s, 2H), ESI-MS: (calculated (MH)+: 208.08 g/mol, found: 207.90 m/z), (calculated (MNa).sup.+: 230.07 g/mol, found: 230.01 m/z).

tert-butyl 2-(5-azido-2-(hydroxymethyl)benzoyl)hydrazine-1-carboxylate

[0205] 5-Azido-2-(hydroxymethyl)benzohydrazide (0.10 g, 0.48 mmol) were dissolved in Dioxan/EtOAc/iPrOH (1:1:1) 10 ml and mixed with Boc anhydride (0.105 g, 0.48 mmol) and DIPEA (0.10 ml, 0.57 mmol). The solution was stirred at room temperature for 12 hours and then the solvent was removed under reduced pressure. The residue was absorbed in 50 ml CH.sub.2Cl.sub.2 and washed twice with a 10% citric acid solution (50 ml each). After drying with MgSO.sub.4 and removing the organic solvent, a yellowish oil was obtained. Yield: 0.10 g (67%). Rf (CH.sub.2Cl.sub.2/MeOH, 9:1) 0.75; .sup.1H NMR (300 MHz, DMSO) 10.06 (s, 1H), 9.04 (d, J=30.3 Hz, 1H), 7.69-7.58 (m, 1H), 7.25 (dd, J=8.3, 2.4 Hz, 1H), 7.10 (d, J=1.3 Hz, 1H), 5.28 (t, J=5.7 Hz, 1H), 4.62 (d, J=5.6 Hz, 2H), 1.43 (s, 9H); ESI-MS: (calculated (MNa)+: 330.12 g/mol, found: 330.29 m/z).

tert-Butyl 2-(5-azido-2-(((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)methyl)benzoyl)hydrazin-1-carboxylate

[0206] N,N-disuccinimidyl carbonate (0.105 g, 0.41 mmol) was added to a solution of tert-butyl 2-(5-azido-2-(hydroxymethyl)benzoyl)hydrazine-1-carboxylate (0.105 g, 0.34 mmol) in 5 ml dry dimethylformamide. Then dry pyridine (0.03 ml, 0.41 mmol) was added. The reaction solution was stirred for 17 hours at room temperature. The solvent was removed under vacuum. 50 ml DCM was added to the solution. The organic phase was washed with 10% citric acid solution (250 ml) and dried with MgSO.sub.4. The combined organic phases were constricted at the rotary evaporator and the residue was purified by column chromatography (CH.sub.2C12/MeOH, 19:1). Yield: 0.06 g (40%). Rf (CH.sub.2Cl.sub.2/MeOH, 19:1) 0.45; .sup.1H NMR (300 MHz, DMSO) 10.06 (s, J=10.9 Hz, 1H), 8.99 (s, J=8.4 Hz, 1H), 7.62 (d, J=8.3 Hz, 1H), 7.53 (s, 1H), 7.25 (dd, J=8.3, 2.4 Hz, 1H), 5.76 (s, 2H), 3.34 (s, 4H), 1.43 (s, 9H), ESI-MS: (calculated (MNa)+: 471.12 g/mol, found: 471.25 m/z).

Syntheses for the Preparation of the Compound of Formula (21)

6-Methoxyisobenzofuran-1 (3H)-on

[0207] A mixture of 3-methoxybenzoic acid (10.00 g, 65.07 mmol), 37% formalin solution (7.5 ml, 80 mmol), 37% HCl (8.00 ml) and 75 ml 100% acetic acid was heated for 18 hours with reflux. After cooling, the clear solution is switched off and left at this temperature for 14 hours. The acetic acid was removed in the air stream at 80 C. The residue was absorbed in 150 ml toluene and concentrated to 40 ml. The 80 C. hot solution was washed with 40 ml portions of 20% Na.sub.2CO.sub.3 solution (3 times) and 40 ml water. After adding 3 ml morpholine, the organic phase was stirred for 2 h at 80 C. and then washed with 50 ml portions of 10% H.sub.2SO.sub.4 (3 times) and water. To crystallize the product, the mixture was concentrated to 25 ml and the mixture stirred. The product was obtained by filtering in the form of white crystals. Yield: 3.52 g (33%). .sup.1H NMR (300 MHz, DMSO) 7.58 (dd, J=8.3, 0.7 Hz, 1H), 7.35 (dd, J=8.3, 2.4 Hz, 1H), 7.31 (d, J=2.2 Hz, 1H), 5.34 (s, 2H), 3.84 (s, 3H).

6-Hydroxyisobenzofuran-1(3H)-on

[0208] Nitrogen atmosphere, 6-methoxyisobenzofuran-1(3H)-on (3.00 g, 18.09 mmol) was dissolved in anhydrous dichloromethane (100 ml). The resulting mixture was magnetically stirred and cooled in an ice bath for 10 minutes. Then BBr3 (3.46 ml, 36.18 mmol) was added. The reaction mixture was then heated to room temperature and stirred for 12 hours. Then 5 ml water was added and the mixture was transferred to a separating funnel and extracted with ethyl acetate (3100 ml). The combined organic extracts were dried over magnesium sulphate, filtered and concentrated under reduced pressure, wherein a white solid was obtained. Yield: 1.52 g (56%). .sup.1H NMR (300 MHz, DMSO) 10.08 (s, 1H), 7.47 (dd, J=8.3, 0.6 Hz, 1H), 7.19 (d, J=2.3 Hz, 1H), 7.16 (d, J=2.3 Hz, 1H), 7.10 (d, J=2.0 Hz, 1H), 5.28 (s, 2H); ESI-MS: (calculated (MH)+: 151.04 g/mol, found: 151.05 m/z).

5-azido-2-(hydroxymethyl)benzohydrazide

[0209] Dissolve 6-hydroxyisobenzofuran-1(3H)-on (0.43 g, 2.83 mmol) in 6 ml dimethylformamide (DMF) and add hydrazine hydrate (1.42 ml, 28.26 mmol) and stir the solution at 100 C. for 3 hours. The DMF and hydrazine hydrate were removed under vacuum. The residue was purified by column chromatography (CH.sub.2Cl.sub.2/MeOH, 9:1), wherein a light yellow powder was obtained. Yield: 0.10 g (17%). Rf (CH.sub.2C12/MeOH, 9:1) 0.40; .sup.1H NMR (300 MHz, DMSO) 10.08 (s, 1H), 9.73 (s, 1H), 7.47 (d, J=8.3 Hz, 1H), 7.18 (dd, J=8.3, 2.3 Hz, 1H), 7.10 (d, J=2.3 Hz, 1H), 4.57 (d, J=5.7 Hz, 2H), 4.50 (s, 2H), 3.33 (s, 2H), ESI-MS: (calculated (MH)+: 183.08 g/mol, found: 183.15 m/z).

Exemplary Purification with Capture Molecule (14)

[0210] The purification was performed with another peptide example 14 (AKADEVSLHKWYG; SEQ ID NO: 10) and a linker of formula (14) (Fngermolekl 14) on commercially available, aldehyde-modified agarose (High Density Glyoxal, 6BCT from ABT).

Solid Phase Peptide Synthesis and Purification

[0211] The automated solid phase peptide synthesis was performed in 100 mol batches with a MultiPep RS peptide synthesis machine from Intavis AG. The synthesis was performed on a Wang-resin (1.0-1.4 mmol/g) from Carl Roth. Before the beginning of the synthesis, the corresponding amount of peptide synthesis resin was weighed in 5 mL syringe reactors (PE reactor from Intavis) and soaked in DMF. The weight of equivalents of amino acid was refers to the initial loading of the resin used, unless otherwise stated.

Fmoc-Removal:

[0212] To remove the temporary Fmoc protecting groups, the resin was treated once for 5 min and once more for 8 min with 1500 L piperidine/DMF (4:1) and subsequently washed seven times with 10.2 mL DMF and it was continued with the coupling of the Fmoc amino acid derivatives.

Coupling of Fmoc Amino Acid Derivatives:

[0213] A solution of 5 eq. amino acid in DMF (0.3 M) was pre-activated for 1 min at room temperature with solutions of 4.5 eq. HCTU in DMF (0.3 M) and 10 eq. NMM in DMF (0.6 M) and then added to the resin. After 30 min reaction time, the resin was washed three times with 10.2 mL DMF and it was continued with blocking the truncated sequences.

Blocking the Truncated Sequences:

[0214] The resin was treated twice for 5 min with 1.5 mL AC.sub.2O/2,6-lutidine/DMF solution (5:6:89) and subsequently washed seven times with 10.2 mL DMF each.

Last Step Coupling of the Capture Molecule:

[0215] As the last step of the solid phase peptide synthesis, the capture molecule (14) was coupled to the desired target peptide (50 mol). The synthesis resin was mixed with a solution of 4 eq. capture molecule (0.3 M), 6 eq. HOBt in DMF (0.4 M) and 4 eq. DIPEA in DMF (0.3 M) mixed. After a reaction time of 60 min, it was washed twice with 2 mL DMF each, twice with 2 mL CH.sub.2Cl.sub.2 and then washed again twice with 2 mL DMF.

Alternative Last Step of Acetylation of the Full-Length Peptide:

[0216] Analogously to the protocol for blocking the truncated sequences, the full-length peptide as a control sample was also acetylated (peptide acetylated).

Release from Polymeric Support:

[0217] The resin was treated with 3 mL of a solution of 95% TFA, 2.5% water and 2.5% triisopropylsilane. The synthesis resin was mixed with this cleaving mixture and shaken for 3 h at room temperature. Afterwards, the cleaving solution was collected and the resin was washed twice with 1 mL TFA each. The cleaving solution was combined with the washing solutions and concentrated by argon flow to approx. 1 mL volume. Afterwards, it was precipitated with 10 mL cold diethyl ether and the precipitate was centrifuged. The supernatant was discarded. In FIG. 4h (above) the chromatogram of the peptide without linker before purification is shown with a purity of 45%.

Purification

[0218] The crude precipitate (about 5 mol theoretical yield) was dissolved in conjugation buffer (0.1 M NH.sub.4OAc, 0.1 M aniline, pH=3.8). If the mixture did not dissolve completely, acetonitrile was added. In a 3 mL syringe reactor with a 25 m PE prefilter 400 L (160 mg) agarose were added. The purification resin was then conditioned by washing 3 times with conjugation buffer (0.1 M NH.sub.4OAc, 0.05 M aniline, pH=3.8). The peptide solution was then added to the purification resin. Immobilization was then performed for 60 minutes. Afterwards, it was washed three times with conjugation buffer, three times with a 5 M urea solution, three times with 70% ethanol and finally five times with water. The mixture was then treated basically with 5 v % NH.sub.4OH in water to cleave conjugated peptide from the resin. Lyophilization provides the peptide as a white flaky solid.

Proof of Immobilization

[0219] To provide clear evidence of immobilization, acetylated peptide and peptide with bound linker were purified on modified as well as on pure agarose (6% B Agarose Bead STANDARD, ABT) with PEC in this experiment. The eluate after the left linker cleavage was assessed with UPLC-UV. The results showed that after cleavage, a significant signal of the product mass is only detected in peptide with bound linker on modified purification resin. In addition, it can be seen that also with pure agarose about 2.3% of the product is obtained compared to modified agarose.

TABLE-US-00003 TABLE 3 Integrals of the product peak for the peptides after purification with modified and unmodified agarose Peptide linker Peptide acetylated Agarose Modified agarose Agarose Modified agarose Integral 17759 763044 197 131 (V * s) Proportion 2.31% 100% 0.03% 0.02%

Regeneration of the Purification Resin

[0220] After peptide purification with the capture molecule (14), the original aldehyde function of 1 remains blocked with the hydroxyl-modified capture molecule. To make the purification resin available for a new purification cycle, the resin must be regenerated and thus the aldehyde function restored. This is achieved by shifting the equilibrium by adding aldehydes or ketones. Regeneration for repeated purification cycles was demonstrated as follows:

[0221] Two purifications of peptide 14 were performed simultaneously (purification I). The purification resin was then washed four times each with a mixture of water, acetone and TFA (ketone, 49.95:49.95:0.1), or water, acetaldehyde and TFA (aldehyde, 89.95:9.95:0.1) and five times with water for regeneration. Afterwards the purification including the conditioning was performed in the same way as described above. The regeneration and purification was performed three times (purification II-IV) and the lyophilized product was taken up in equal volumes of water, acetonitrile and TFA (69.9:29.9:1) and measured with UPLC-MS.

TABLE-US-00004 TABLE 4 Integrals of the product peak and percentage proportion for the peptides after purification with modified agarose (purification I) and after three regeneration cycles (purification II-IV). Aldehyde Ketone Integral Purification Integral (V * s) Proportion Purity (V * s) Proportion Purity I 4100144 100% 58.9% 2138955 100% 58.2% II 2044538 50% 58.5% 911365 43% 58.7% III 1719147 42% 57.8% 567140 27% 58.6% IV 381717 9% 61.5% 576371 27% 59.8%

[0222] The results showed that the resin can be regenerated in both cases. Regeneration with a ketone only decreases significantly in the third cycle (purification IV). In contrast, in aldehyde regeneration, the purification capacity remains at about a quarter of the initial capacity, but decreases to this value already after the second cycle (purification III). In both experiments a purity of about 60% was achieved, which remains constant during the regeneration cycles (example chromatogram for purification I, aldehyde, FIG. 4h (below)).