SOLID PHASE PEPTIDE SYNTHESIS OF INSULIN USING SIDE CHAIN ANCHORED LYSINE

Abstract

The present application discloses the preparation of peptides, including insulin and insulin derivatives, using efficient methods for solid-phase and solution phase peptide synthesis.

Claims

1-12. (canceled)

13. A method for the solid phase synthesis of a protected, partially protected or unprotected insulin B-chain or an insulin B-chain derivative, the process comprising: preparing a lysine-resin conjugate comprising a resin and a lysine or a lysine derivative of the formula I, wherein: ##STR00012## W is a resin of formula IIIc: ##STR00013## wherein: each R.sup.1 and R.sup.3 is independently selected from H or is independently selected from the group consisting of 2-Cl, 2-C.sub.1-3 alkyl, 2-C.sub.1-3 alkoxy, 4-C.sub.1-3 alkyl, 4-C.sub.1-3 alkoxy, provided that only one of R.sup.1 and R.sup.3 is H; R.sup.2 is the solid phase of the resin; and Z is a bond or —C(═O)—; R is selected from the group consisting of —OH, a carboxyl protecting group, —NH.sub.2, —O—C.sub.1-6 alkyl, —O—C.sub.2-6 alkenyl, —O-tri-C.sub.1-3 alkyl silyl, a peptide residue selected from the group consisting of -Pro-OH, -Pro-NH.sub.2, -Pro-O—C.sub.1-6 alkyl, -Pro-O—C.sub.2-6 alkenyl, -Pro-O-tri-C.sub.1-3 alkylsilyl, Thr(Pr1), -Thr(Pr1)-Arg(Pr2)-Arg(Pr3)-OH, -Thr(Pr1)-Arg(Pr2)-Arg(Pr3)-NH.sub.2, -Thr(Pr1)-Arc(Pr2)-Arc(Pr3)-O—C.sub.1-6 alkyl, -Thr(Pr1)-Arg(Pr2)-Arg(Pr3)-O—C.sub.2-6 alkenyl, -Thr(Pr1)-Arg(Pr2)-Arg(Pr3)-O-tri-C.sub.1-3 alkylsilyl, -Thr(Pr1)-OH, -Thr(Pr1)-NH.sub.2, -Thr(Pr1)-O—C.sub.1-6 alkyl, -Thr(Pr1)-O—C.sub.2-6 alkenyl and -Thr(Pr1)-O-tri-C.sub.1-3 alkylsilyl, a peptide residue comprising 1 to 200 amino acids comprising optionally protected side chain and optionally protected terminal carboxyl group; wherein Pr1 is hydrogen or a -OH protecting group and each Pr2 and Pr3 is independently hydrogen or a guanidine protecting group; P is hydrogen, an amino protecting group, an N-terminus peptide residue comprising 1 to 200 amino acids comprising optionally protected side chain and optionally protected terminal amino group, wherein the N-terminus peptide residue comprises a C-terminus and an N-terminus; provided that when R is selected from the group consisting of —OH, a carboxyl protecting group, —NH.sub.2, —O—C.sub.1-6 alkyl, —O—C.sub.2-6 alkenyl, —O-tri-C.sub.1-3 alkyl silyl, a peptide residue selected from the group consisting of -Pro-OH, -Pro-NH.sub.2, -Pro-O—C.sub.1-6 alkyl, -Pro-O—C.sub.2-6 alkenyl, -Pro-O-tri-C.sub.1-3 alkylsilyl, Thr(Pr1), -Thr(Pr1)-Arg(Pr2)-Arg(Pr3)-OH, -Thr(Pr1)-Arg(Pr2)-Arg(Pr3)-NH.sub.2, -Thr(Pr1)-Arg(Pr2)-Arg(Pr3)-O—C.sub.1-3 alkyl, -Thr(Pr1)-Arg(Pr2)-Arg(Pr3)-O—C.sub.2-6 alkenyl, -Thr(Pr1)-Arg(Pr2)-Arg(Pr3)-O-tri-C.sub.1-3 alkylsilyl, -Thr(Pr1)-OH, -Thr(Pr1)-NH.sub.2, -Thr(Pr1)-O—C.sub.1-6 alkyl, -Thr(Pr1)-O—C.sub.2-6 alkenyl and -Thr(Pr1)-O-tri-C.sub.1-3 alkylsilyl, then P is not hydrogen or an amino protecting group; coupling the lysine-resin conjugate of the formula I, wherein R is selected from —OH, a carboxyl protecting group and a peptide residue comprising 1 to 200 amino acids comprising optionally protected side chain and optionally protected terminal carboxyl group, with a protected, partially protected or unprotected peptide residue Ib, wherein the peptide residue comprising 1 to 200 amino acids and the peptide residue Ib together comprise the insulin B-chain or the insulin B-chain derivative, to form the protected, partially protected or unprotected insulin B-chain or an insulin B-chain derivative; wherein the partially protected or unprotected insulin B-chain or an insulin B-chain derivative is SEQ ID NO 27 or 29, and analogs or derivatives thereof; further cleaving off the resin-bound protected, partially protected or unprotected insulin B-chain or an insulin B-chain derivative by contacting the resin-bound peptide under mild acidic condition using a mixture of an organic acid and an alcoholic solvent, or by heating the resin-bound peptide to an elevated temperature, or both using a mixture of an organic acid and an alcoholic solvent along with heating the resin-bound peptide at an elevated temperature for a sufficient period of time to cleave the insulin B-chain or an insulin B-chain derivative from the resin.

14. (canceled)

15. The method of claim 13, wherein P is selected from the group consisting of tert-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyloxy-carbonyl (carboxybenzyl or Z), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-ethyl (Dde), 2-nitrophenylsulfenyl (Nps) and allyloxycarbonyl (alloc).

16. The method of claim 13, and further cleaving of the resin-bound insulin B-chain or an insulin B-chain derivative by contacting the resin-bound peptide under mild acidic conditions using a mixture of an organic acid and an alcoholic solvent, or by heating the resin-bound peptide to an elevated temperature, or both using a mixture of an organic acid and an alcoholic solvent along with heating the resin-bound peptide at an elevated temperature for a sufficient period of time to cleave the insulin B-chain or an insulin B-chain derivative from the resin.

17. The method of claim 16, wherein the organic acid is selected from the group consisting of trifluoroacetic acid and acetic acid, and mixtures thereof, the alcoholic solvent is selected from the group consisting of trifluoroethanol, hexafluoro-isopropanol, methanol and mixtures thereof, and heating of the resin bound peptide is performed with microwaves.

18. The method of claim 16, wherein the partially protected or unprotected insulin B-chain or an insulin B-chain derivative is selected from the group consisting of an N-acylated derivative, a pegylated derivative, a biotinylated derivative, a derivative comprising a chromophore and a peptide residue comprising a natural amino acid residue, an unnatural amino acid residue, and mixtures thereof.

19-21. (canceled)

22. A method for preparing an insulin B-chain peptide selected from the group consisting of SEQ ID NOs: 27 and 29 comprising cleaving the peptide from a peptide-resin conjugate of the formula IV:
W-AA.sub.1-AA.sub.m  IV wherein: W is a resin of formula IIIc: ##STR00014## wherein: each R.sup.1 and R.sup.3 is independently selected from H or is independently selected from the group consisting of 2-Cl, 2-C.sub.1-3 alkyl, 2-C.sub.1-3 alkoxy, 4-C.sub.1-3 alkyl, 4-C.sub.1-3 alkoxy, with the proviso that only one of R.sup.1 and R.sup.3 is 2-Cl and only one of R.sup.1 and R.sup.3 is H; R.sup.2 is the solid phase of the resin; and Z is a bond or —C(═O)—; AA.sub.1 is a first peptide residue comprising a lysine residue or derivative thereof attached to W by the amino side chain of the lysine or lysine derivative; and AA.sub.m is the second to m number of residues where m is an integer from 1-200; by contacting the peptide-resin conjugate with a mixture of an organic acid and a solvent, or by heating the peptide-resin to an elevated temperature, or both using a mixture of an organic acid and a solvent, along with heating the resin-bound peptide at an elevated temperature for a sufficient period of time to cleave the peptide residue from the resin W.

23. The method of claim 22, wherein the peptide-resin conjugate is selected from the group consisting of SEQ ID NOs: 17, 22 and 24.

24. (canceled)

25. The method of claim 22, wherein the organic acid is selected from the group consisting of trifluoroacetic acid and acetic acid, and mixtures thereof, the alcoholic solvent is selected from the group consisting of trifluoroethanol, hexafluoro-isopropanol, methanol and mixtures thereof, and heating of the peptide-resin conjugate is performed at about 30 to 50° C.

26. The method of claim 13, wherein the resin is a 4-methoxytrityl resin.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0074] FIG. 1 shows the sequence of human insulin and various derivatives

[0075] FIG. 2 is a representative process scheme for a solid phase synthesis of bisoxidized human insulin A-chain isomers.

[0076] FIG. 3 is a representative scheme showing a step-by-step solid-phase synthesis of the insulin B-chain using a CTC-resin.

[0077] FIG. 4 is a representative scheme showing the synthesis of an insulin B-Chain using Fmoc-Lys-Thr(tBu)-OH attached through the side chain of Lys on an MMT-resin.

[0078] FIG. 5 is a representative scheme for the solid-phase synthesis of Fmoc-Lys-Thr(tBu)-Arg(Pbf)-Arg(Pbf)-OH (SEQ ID NO: 19) for Glargin B-Chain.

[0079] FIG. 6 is a representative scheme for the synthesis of insulin Glargin B-Chain using Fmoc-Lys-Thr(tBu)-Arg(Pbf)-Arg(Pbf)-OH attached through the side chain of Lys on a MMT-resin.

[0080] FIG. 7 is a representative scheme for the synthesis of insulin Detemir B-chain with N-terminal protection and using Fmoc-Lys attached through the side chain onto a MMT-resin.

[0081] FIG. 8 is a representative scheme for the synthesis of insulin Degludec B-Chain using Fmoc-Lys attached through the side chain on MMT-resin.

[0082] FIG. 9 is a representative scheme for the synthesis of insulin Degludec B-chain using N-terminal protection.

[0083] FIG. 10 is a representative scheme for the synthesis of biotinylated insulin B-chain.

[0084] FIG. 11 is a representative scheme for the synthesis of insulin and insulin analogs (Lispro and Aspart) by chain combination.

[0085] FIG. 12 is a representative scheme for the chain combination of an insulin analogs (Detemir and Degludec).

[0086] FIG. 13 is a representative scheme for the chain combination of an insulin analog Glargin.

SYNTHESIS OF A CHAINS

[0087] In one embodiment, there is provided a method for the solid-phase synthesis of the insulin bis-oxidized A-chain in which the cleavage of the protected peptide from the resin and its oxidation proceeds concurrently and in a short period of time. In this method, the synthetic problem due to the insolubility of the A-chain is overcome. In one aspect, this is achieved by applying solid-phase-synthesis using acid labile resins. Such resins include the trityl, diphenylmethyl and benzyl-type. The peptide chain is assembled using standard protocols for the solid phase synthesis of peptides using, for example, Fmoc-amino acids (FIG. 2). Oxidation of the resin-bound peptide maybe performed using iodine under mild acidic conditions, such as in halogenated hydrocarbons. Under these conditions, the cleavage of the protected peptide from the resin proceeds concurrently with the oxidation reaction, and the precipitation of the A-chain is avoided. In one aspect, solutions of trifluoroacetic acid or acetic acid in dichloromethane that are mixed with alcohols, such as trifluoroethanol or methanol, were used as the solvent for the concurrent oxidation and cleavage from the resin. In one embodiment, 2-chlorotrityl resin was used for the solid-phase chain assembly of the protected A-chain. The bis-oxidized protected linear A-chain that was obtained was deprotected by contacting the A-chain with various acidic solvent solutions, including DCM, TFA, TES and thioethers, and mixtures thereof. In one aspect, the present application discloses all three expected isomers of the bis-oxidized insulin A-chain (FIG. 2). The isomers can be separated by HPLC but can be also isolated and purified as a mixture of isomers.

SYNTHESIS OF B CHAINS

[0088] In one embodiment, the present application discloses a synthesis of the insulin B-chain in a step-by-step manner on acid sensitive resins of the trityl-type, such as the 2-chlorotrityl resin (FIG. 3). The application of Fmoc-amino acids suitably protected at their side chains with acid sensitive groups was also employed.

[0089] The present application also discloses the synthesis of insulin B-chain peptides and its derivatives such as the des-Thr(B30) insulin B-chain may be prepared where the side chain of Lys(B29) is attached to the resin instead of the chain's carboxyl group (FIG. 4). Similarly the B-chain for Glargin may be synthesized either by solid phase attachment of the protected arginine through the carboxyl group (FIG. 5) or using side group attachment through the lysine (FIG. 6). Furthermore, the side chain of the lysine that is attached to the resin may be used to prepare the Insulin Detemir B-chain (FIG. 7), and Insulin Degludec B Chain (FIG. 8, 9).

[0090] The present application further discloses that if the resin used for the side chain attachment of Lys is relatively labile, such as the 2-chlorotrityl resin or the 4-methoxytrityl resin, the partially protected insulin B-chain can be readily obtained by mild acidic or thermal treatment of the resin-bound peptide, where the peptide is cleaved from the resin with selectively deprotected Lys side-chain amino function. The partially deprotected insulin B-chains, their shorter or longer fragments and derivatives, can be selectively acylated in solution at the lysine side chain providing a variety of important B-chain derivatives.

[0091] The present application further discloses the synthesis of Lys(15-myristoyl)-des-Thr(30) human insulin B-chain, the synthesis of Lys(15-carboxypentadecanoyl-γ-glutamyl)B(29)-des-Thr(B30) human insulin B-chain and the selective pegylation and biotinylation (FIG. 11) of the side chain of Lys(B29) as well as the solid-phase-synthesis of selectively at the Lys(B29) side chain branched Insulin B-chain peptides.

[0092] The present application also discloses the selective acylation of the side chain of the Lys(B29)-human insulin B-chain and its shorter and longer peptide analogues and their derivatives. This can be performed by preparing and isolating the insulin B-chain protected at its amino terminal function by the Fmoc-group or a Z-type group. After the removal of the side chain protecting groups of the insulin B-chain derivative, the free amino function of the Lys(B30)-insulin may be acylated.

Combining Bisoxidized Chain A with B

[0093] The present application further discloses that the combination of the insulin chains using the bisoxidized A-chain of human or animal insulin and their derivatives, with the B-chain of human or animal insulin and their derivatives proceed smoothly in aqueous solutions buffered with the addition of various salts, such as sodium, calcium, zinc, iron salts etc. Furthermore, the solution may contain organic solvents such as alcohols, DMSO, acetonitrile etc. at various pH, including at pH>7, and at different temperatures, including from about 0-5° C., 2-6° C. and 5-10° C. The bis-oxidized A-chain and the B-chain can be reacted at different ratios, such as a ratio where the A-chain/B-chain ratio is >1, such as 1.05:1, 1.1:1, 1.2:1, 1.3:1, 1.5:1 and 2:1. In one aspect, the reaction provides a mixture of mono-oxidized A-chain, oxidized B-chain and B-chain dimers, A-chain dimmers and mixtures thereof. The product mixture may be separated by HPLC and recycled after the oxidation of the mixture of mono and di-oxidized A-chain, or converted to different insulin products by equilibrating with a redox system such as cysteine cysteine, or oxidized and reduced glutathione etc. Using the present method, combination yields of >60% may be obtained. Alternatively, a mild oxidant is added to the combination mixture, such as DMSO or the redox mixture oxidized and reduced glutathione, to re-oxidize the A-chain. In the resulting mixture, a mild reducing agent, such as thiols, including thiolamine, dithiotreitol or a redox system, may be added. Using the present method, the total yield of insulin and insulin analogs may be improved by 5-25% (FIGS. 11-13).

[0094] The preparation of insulin, insulin analogues and acylated insulin analogues are described in the examples that follows. The reaction and purification schemes described are generally applicable to the preparation of various different insulin derivatives, but the reactions conditions and sequences may not be applicable to all peptides, including certain insulin analogues and derivatives, as would be readily recognised by those skilled in the art. In these cases, the reactions can be successfully performed by usual modifications known to those skilled in the art in peptide synthesis, that is, by appropriate protection of interfering groups, changing to other conventional reagents or routine modification of reaction conditions and reaction sequences.

EXAMPLES

Example 1

[0095] Solid-phase synthesis of insulin A chain, B chain and of their protected segments General procedure:

[0096] A1. Preparation of Loaded 2-Chlorotrityl Resins

[0097] 2-Chlorotrityl chloride resin (CTC-Cl) (100 g; loading 1.6 mmol/g) of CBL-Patras, was placed in a 2 L peptide synthesis reactor and swelled with 700 mL dichloromethane (DCM) for 30 min at 25° C. The resin was filtered and a solution of 100 mmol Fmoc-amino acid and 300 mmol diisopropylethylamine (DIEA) in 500 mL DCM was added. The mixture was stirred under nitrogen for 2 hours at 25° C. The remaining active sites of 2-CTC resin were neutralised by adding 10 mL of methanol (MeOH) and reacting for 1 hour. The resin was filtered and washed twice with 400 mL DMF. The resin was filtered and treated twice with 500 mL 25% by volume of piperidine in DMF for 30 min. The resin was washed four times with 500 mL DMF. The resin was unswelled with 3 washes with 500 mL of isopropanol (IPA); and dried to constant weight. 70-95% of the mmol of the used amino acid was bound on the resin.

[0098] A2. Preparation of Loaded MBH-Resins, a General Method

[0099] MBH-Br resin (100 g; 190 mmol) was placed in a 2 L peptide synthesizer and swollen with 700 mL DCM for 30 min at 25° C. The resin was filtered and then a solution of Fmoc-amino acid and DIEA in 500 mL DCM was added. The mixture was stirred under nitrogen for 6 h at 25° C. Then the remaining active sites of the MBH resin were bound by adding 10 mL MeOH and stirring for 24 h. The resin was then filtered and washed twice with 400 mL DMF. The resin was filtered and reacted twice with 500 mL of a solution of 25% by volume of piperidine in DMF for 30 min. The resin was then washed four times with 500 mL DMF. The resin was diswelled with three washes with 500 mL IPA. The resin was then dried to constant weight under vacuum (15 torr, 25° C.). 60-90% of the mmol of the used amino acid were bound onto the resin.

[0100] B. Solid-Phase Synthesis, a General Protocol

[0101] The solid-phase synthesis was performed at 24° C., with 1.0 g amino acid esterified to the CTC or MBH resin as described in Part A of Example 1. During the whole synthesis the following protocol was used.

[0102] B1. Swelling of the Resin

[0103] The resin was placed in a 15 ml reactor and treated twice with 7 mL NMP, followed by filtration.

[0104] B2. Activation of the Amino Acid

[0105] The amino acid (3.0 equiv.) and 1-hydroxybenzotriazol (4.0 equiv.) was dissolved in a reactor with 2.5 times their volume in NMP and cooled to 0° C. DIC was then added (3.0 equiv.) and the mixture was stirred for 15 min.

[0106] B3. Coupling Reaction

[0107] The solution which was prepared in B2 was then added to the B1 reactor. The reactor was washed once with one volume of DCM and was added to the reactor which was stirred for 1-3 h at 25°-30° C. A Kaiser Test was performed to determine the completion of the reaction. If the coupling reaction was not completed after 3 h (positive Kaiser Test), the reaction mixture was filtered and recoupled with a fresh solution of activated amino acid. After completion of the coupling the reaction mixture was filtered and washed 4 times with NMP (5 volumes per wash).

[0108] B4. Removal of the Fmoc-Group

[0109] The resulting resin in B3 was filtered and then treated for 30 min with 5 mL of a solution which contained 25% by volume of piperidine. The resin is washed 3×5 mL NMP.

[0110] B5. Elongation of the Peptide Chain

[0111] After the incorporation of each amino acid the steps B1-B5 were repeated until the desired peptide chain was formed.

[0112] The following Fmoc-amino acids were used for coupling of the individual amino acid or amino acid fragments: Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Mmt)-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(Clt)-OH, Fmoc-Asn-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH and Fmoc-Cys(Acm)-OH; and the following Boc-amino acids: Boc-Phe-OH, and Boc-Gly-OH.

[0113] C. General Method for the Cleavage from the CTC-Resin of the Partially Protected Insulin Peptides and of their Protected Fragments which Contain Fmoc- or Boc-Groups on their N-Terminus and are Selectively Deprotected at a Lysine Side Chain.

[0114] The resin-bound peptide or peptide segment which was produced as described above in B1-B5 and was protected at a specific Lys side chain with Mmt or Mtt, was washed 4 times with 5 mL NMP, 3 times with 5 ml IPA and finally 5 times with 7 ml DCM to remove completely any residual NMP or other basic components. The resin was then cooled to 0° C., filtered from DCM and was treated six times with a solution of 10 mL 1.0-1.5% TFA in DCM/TES(95:5) at 5° C. The mixture was then stirred 20 min at 0° C. and filtered. The resin is then washed three times with 10 mL DCM. Pyridine is then added to the filtrates (1.3 equiv. relative to TFA) to neutralize the TFA. The cleavage solution in DCM was then mixed with an equal volume of water. The resulting mixture was distilled at reduced pressure to remove DCM (350 torr at 28° C.). The peptide or peptide fragment precipitated after the removal of DCM. The resulting peptide was washed with water and ether and dried at 30-35° C. under 15 Torr vacuum. Alternatively DCM was removed in vacuum and the partially protected peptide was precipitate by the addition of ether.

[0115] D. Cleavage from the CTC-Resin and Simultaneous Mono-Oxidation of Protected Peptides with Iodine. Preparation of Bis-Oxidized Insulin A-Chains. General Procedure.

[0116] The resin bound on the N— and on the side chains protected peptide, obtained as described above, was washed 4×5 mL NMP, 3×5 ml IPA and finally 5 times with 7 ml DCM to remove completely NMP and other basic components. The resin was then cooled to 0° C. After filtration of DCM the resin was processed twice at 5° C. with a solution of 10 mL 1%-TFA in DCM containing 20 equivalents (equiv.) of iodine in relation to the on the resin bound peptide. The resulting mixture was stirred for 5 min at 0° C. and filtered (instead of 1% TFA the same volume of a mixture of dichloromethane/acetic acid/trifluoroethanol can be used with similar results). The resin was then washed three times with 10 mL DCM. The combined filtrates were heated to 15° C. and stirred for further 30 min. Pyridine was then added to the filtrates (1.3 equiv. relative to TFA) to neutralize TFA. The cleavage solution in DCM was then mixed with an equal volume of 3%-sodium thiosulphate in water in order to remove the excess iodine. This was indicated by the discoloration of the mixture. The resulting mixture was distilled at low pressure to remove DCM (350 torr at 28° C.). The resulting peptide or peptide fragment precipitated out after the removal of DCM. The resulting peptide was washed with water and dried at 30-35° C. under vacuum of 15 Torr.

Example 2

[0117] Deprotection of the Bis-Oxidized Insulin A-Chains Described in FIG. 2. General Method:

[0118] The protected insulin chain A obtained as described above in Example 1 (0.01 mmol) were treated with 10 mL TFA/TES/thioanisol/water (85:5:5:5) for 3 h at 5° C. and for 1 h at 15° C. The resulting solution was concentrated in vacuum and then the deprotected peptide was precipitated by the addition of diisopropylether and washed three times with 10 mL diisopropylether. The resulting solid was dried in vacuum (25° C., 15 Torr) until constant weight.

Example 3

[0119] Deprotection of the Bisoxidized Insulin B-Chains. General Method:

[0120] The protected insulin chain B obtained as described above in Example 1 (0.01 mmol) was treated with 10 mL TFA/DTT/water (90:5:5) for 3 h at 5° C. and for 1 h at 15° C. The resulting solution is concentrated in vacuum and then the deprotected peptide was precipitated by the addition of diisopropylether and washed with 3×10 mL diisopropylether. The resulting solid was dried in vacuum (25° C., 15 Torr) until constant weight.

Example 4

[0121] Synthesis of Peptides Attached on Resins Through the Side Chain of Lysine.

[0122] 1 mmol of a Lys side chain deprotected amino acid or peptide, which can be obtained as described in the example 1C, was dissolved in 15 ml DCM. Then, 1.5 mmol DIPEA was added and 1 g 4-methoxytrityl chloride resin (1.2 mmol/g) and the mixture was stirred over night. 1 ml methanol was added and the mixture was stirred for additional 4 h at RT. The resin was then filtered, washed 3×DCM, 3×DMF, 3×iPrOH and 3× hexanes and dried in vacuum to constant weight.

Example 5

[0123] Synthesis of Selectively at the Lysine Side Chain Acylated Peptides. General Procedure.

[0124] 1 mmol of a Lys side chain deprotected amino acid or peptide, which can be obtained as described in the example 1C, was dissolved in 15 ml DMF. Then, 1.2 mmol DIPEA were added and 1 equivalent of the electrophilically activated agent and the mixture was stirred for 1-12 h at RT. The mixture was then poured into ice cold water and the resulting precipitate was washed with water and ether, deprotected as described under example 3 and purified as described under example 6 below.

Example 6

[0125] Purification of the Deprotected Peptides. General Procedure.

[0126] Crude deprotected trifluoroacetic acid salts of the insulin chains and of the bicyclic chain A derivatives were dissolved in 15% acetonitrile in water and loaded on a semi-preparative column 10×25 mm loaded with Chromasil; Phase A=1%-TFA in acetonitrile, phase B=1%-TFA in water; Linear gradient from 25%-A to 65%-A in 30 min. The purification yield varied from 30 to 90%.

Example 7

[0127] Synthesis of Insulin Like Peptides and of their Derivatives by the Linear Combination of the Bicyclic A-Chain of the Insulin and of its Derivatives and of the Linear B-Chain of Insulin and of its Derivatives: General Procedure.

[0128] Deprotected bicyclic A-chain of insulin like peptide or of its derivatives (0.006 mmol) and of linear chain-B of insulin like peptides or derivatives (0.005 mmol) was dissolved in 4 ml of a buffer of sodium glycinate/6-N guanidine hydrochloride (4:1) at pH=10.5. Then 1 ml DMSO was added gradually within 12 hours and then the mixture was stirred for additional 4 h at 15° C. From the resulting solution, the insulin-like peptides were isolated by purification performed as described in Example 4. The average yield of three experiments on insulin-like peptides was 5-80% calculated on the applied B-chain.

[0129] While a number of exemplary embodiments, aspects and variations have been provided herein, those of skill in the art will recognize certain modifications, permutations, additions and combinations and certain sub-combinations of the embodiments, aspects and variations. It is intended that the following claims are interpreted to include all such modifications, permutations, additions and combinations and certain sub-combinations of the embodiments, aspects and variations are within their scope. The entire disclosures of all documents cited throughout this application are incorporated herein by reference.