METHOD FOR PEPTIDE SYNTHESIS AND APPARATUS FOR CARRYING OUT A METHOD FOR SOLID PHASE SYNTHESIS OF PEPTIDES

Abstract

The invention relates to a method for peptide synthesis, wherein said method comprises the steps of reacting a first amino acid or a first peptide with an α-amine protected second amino acid in a solvent selected from the group consisting of water, alcohol, and a mixture of water and alcohol, and removing the α-amine protecting group with a deprotecting solution. The invention further relates to protective agents, their use and an apparatus for carrying out a method for solid phase synthesis of peptides.

Claims

1. A method of forming protecting groups on functional groups during water based peptide synthesis, the method comprising using a protective agent, wherein the protective agent comprises a) a backbone structure, b) at least one water-solubility enhancing functional group and c) at least one reactive group, wherein the backbone structure comprises a moiety selected from the group consisting of 9-methylfluorene, t-butane and/or mono-, di or triphenylmethane, wherein the water-solubility enhancing functional group is selected from the group consisting of SO.sub.3.sup.−, PO.sub.3.sup.2−, N(CH.sub.3).sub.2, N(CH.sub.3).sub.3.sup.+, CN, OSO.sub.3.sup.− ester, OPO.sub.3.sup.2− ester and combinations thereof, and wherein the water-solubility enhancing functional group and the reactive group are attached to the backbone structure via at least one covalent bond, for protecting a functional group in a chemical reaction, wherein the agent is used for forming protecting groups on functional groups during water based peptide synthesis.

2. The method according to claim 1, wherein the agent is used for forming protecting groups on functional groups on a peptide and/or amino acid during water based peptide synthesis.

3. The method according to claim 2, wherein the functional group to be protected is selected from amine, alcohol, thiol and carboxyl groups.

4. The method according to claim 3, wherein the reactive group is selected from the group consisting of oxycarbonyl halogenide, oxycarbonyl Oxyma ester, oxycarbonyl O-succinimide, oxycarbonyl anhydride, halogenide, hydroxide and thiol groups.

5. The method according to claim 4, wherein the water solubility-enhancing functional group is SO.sub.3.sup.−.

6. The method according to claim 5, wherein the functional group to be protected is present on an amino acid, peptide or protein and the chemical reaction is peptide or protein synthesis in a solvent selected form water, alcohol or a mixture of water and alcohol.

7. Protective agent suitable for forming protecting groups on functional groups on a peptide and/or amino acid during water based peptide synthesis, wherein the protective agent comprises a) a backbone structure, b) at least one two water-solubility enhancing functional group and c) at least one reactive group, wherein the backbone structure comprises a moiety selected from the group consisting of 9-methylfluorene, t-butane and/or mono-, di or triphenylmethane, wherein the water-solubility enhancing functional group is selected from the group consisting of SO.sub.3.sup.−, PO.sub.3.sup.2−, N(CH.sub.3).sub.2, N(CH.sub.3).sub.3.sup.+, CN, OSO.sub.3.sup.− ester, OPO.sub.3.sup.2− ester and combinations thereof, and wherein the water-solubility enhancing functional group and the reactive group are attached to the backbone structure via at least one covalent bond.

8. (canceled)

9. A method for peptide synthesis, wherein said method comprises the steps of reacting a first amino acid or a first peptide with an α-amine protected second amino acid or second peptide in a solvent selected from the group consisting of water, alcohol, and a mixture of water and alcohol, and removing the α-amine protecting group with a deprotecting solution, characterized in that the α-amine protecting group is formed by reaction of a protective agent with a functional group on an amino acid or peptide, wherein the protective agent is suitable for forming protecting groups on functional groups during water based peptide synthesis, wherein the protective agent comprises a) a backbone structure, b) at least one water-solubility enhancing functional group and c) at least one reactive group, wherein the backbone structure comprises a moiety selected from the group consisting of 9-methylfluorene, t-butane and/or mono-, di or triphenylmethane, wherein the water-solubility enhancing functional group is selected from the group consisting of SO.sub.3.sup.−, PO.sub.3.sup.2−, N(CH.sub.3).sub.2, N(CH.sub.3).sub.3.sup.+, CN, OSO.sub.3.sup.− ester, OPO.sub.3.sup.2− ester and combinations thereof, and wherein the water-solubility enhancing functional group and the reactive group are attached to the backbone structure via at least one covalent bond.

10. The method according to claim 9, characterized in that the C-terminus of said first amino acid or first peptide is anchored to an insoluble support and that the method further comprises the step of cleaving the resulting peptide or protein from the polymeric support with a cleaving composition.

11. The method according to claim 10, characterized in that any reactive side chain functional group of said first amino acid or first peptide and said second amino acid or second peptide is protected with a side chain protecting group comprising at least one water solubility enhancing functional group and that the method further comprises the step of removing the side chain protecting groups.

12. The method according to claim 11, wherein the α-amine protecting group comprises a fluorescent structure and the method further comprises the step of monitoring the degree of formation of peptide bonds and/or monitoring the degree of removal of the α-amine protecting group by measuring fluorescence that is generated by the α-amine protecting groups coupled to the first amino acid or first peptide.

13. An apparatus for carrying out a method for solid phase synthesis of peptides or proteins according to claim 12, said apparatus comprising a reaction vessel for receiving an insoluble support material and a fluorimeter arranged for monitoring the changes in fluorescence intensity that is generated by a protecting group coupled to the insoluble support material.

14. Modified amino acid, peptide, protein or salt thereof comprising a protecting group formed by reaction of protective agent with a functional group of the amino acid, peptide or protein, wherein the functional group is selected from α-amino, side chain amino, thiol, carboxyl and hydroxy, wherein the protective agent comprises a) a backbone structure, b) at least one water-solubility enhancing functional group and c) at least one reactive group, wherein the backbone structure comprises a moiety selected from the group consisting of 9-methylfluorene, t-butane and/or mono-, di or triphenylmethane, wherein the water-solubility enhancing functional group is selected from the group consisting of SO.sub.3.sup.−, PO.sub.3.sup.2−, N(CH.sub.3).sub.2, N(CH.sub.3).sub.3.sup.+, CN, OSO.sub.3.sup.− ester, OPO.sub.3.sup.2− ester and combinations thereof, and wherein the water-solubility enhancing functional group and the reactive group are attached to the backbone structure via at least one covalent bond.

15. A method of peptide synthesis, wherein a capping agent is used for capping of free amines after a coupling step in order to prevent the formation of side products, the capping agent comprising a) a backbone structure, b) at least one water-solubility enhancing functional group and c) at least one reactive group, wherein the backbone structure comprises a moiety selected from the group selected from short chain alkyl, preferably C.sub.1 to C.sub.8 alkyl, cyclic alkyl chains or aromatic compounds, more preferably C.sub.1 to C.sub.4 alkyl or benzyl, wherein the water-solubility enhancing functional group is selected from the group consisting of SO.sub.3.sup.−, PO.sub.3.sup.2−, N(CH.sub.3).sub.2, N(CH.sub.3).sub.3.sup.+, CN, OSO.sub.3.sup.− ester, OPO.sub.3.sup.2− ester and combinations thereof, and wherein the water-solubility enhancing functional group and the reactive group are attached to the backbone structure via at least one covalent bond.

16. (canceled)

17. (canceled)

18. The method according to claim 6, wherein the protective agent has the following formula ##STR00069## wherein R2 and R7 are SO.sub.3.sup.− and R1, R3 to R6 and R8 are hydrogen, or R3 and R6 are SO.sub.3.sup.− and R1, R2, R4, R5, R7 and R8 are hydrogen, and wherein R99 is selected from the group consisting of oxycarbonyl halogenide, oxycarbonyl O-succinimide, oxycarbonyl Oxyma ester, oxycarbonyl anhydride, halogenide, oxymethyl halogenide, hydroxide and thiol groups, wherein R99 is preferably selected from oxycarbonyl halogenide, oxycarbonyl Oxyma ester and oxycarbonyl O-succinimide.

19. Protective agent according to claim 7, having the following formula ##STR00070## wherein R2 and R7 are SO.sub.3.sup.− and R1, R3 to R6 and R8 are hydrogen, or R3 and R6 are SO.sub.3.sup.− and R1, R2, R4, R5, R7 and R8 are hydrogen, and wherein R99 is selected from the group consisting of oxycarbonyl halogenide, oxycarbonyl O-succinimide, oxycarbonyl Oxyma ester, oxycarbonyl anhydride, halogenide, oxymethyl halogenide, hydroxide and thiol groups, wherein R99 is preferably selected from oxycarbonyl halogenide, oxycarbonyl Oxyma ester and oxycarbonyl O-succinimide.

20. The method according to claim 15, wherein after the completion of the reaction the reaction mixture is passed through an affinity column in order to purify the reaction mixture.

Description

[0112] The invention will be further described, by way of illustration, with reference to the accompanying drawing in which FIG. 1 is a schematic illustration of an apparatus according to the present invention.

[0113] FIG. 1 illustrates schematically the overall layout of automatic solid phase peptide synthesizer apparatus for use in carrying out a preferred method as described above. The apparatus comprises a reaction vessel 1 containing a first amino acid or a first peptide anchored to an insoluble polymeric support, a reagent container 2 including multiple reservoirs for providing coupling reagents, capping reagents, cleaving reagents etc., a solvent container 3, and an amino acids and/or peptide container 4 including multiple reservoirs for providing protected amino acids and/or peptides.

[0114] The reaction vessel 1 and the containers 2, 3, 4 are connected via tubes and valves arranged in such a way that specified quantities of specified reagents, amino acids and/or peptides can be delivered to the reaction vessel 1 in a specified sequence under control of control means (not shown) in a conventional manner.

[0115] The apparatus includes a fluorimeter 5 which is arranged for measuring monitoring the changes in fluorescence intensity of the contents of the reaction vessel 1. It is understood that alternatively, the reaction vessel 1 can comprise an integrated fluorimeter 5.

[0116] The reaction vessel is equipped with a waste outlet 6 for removing the waste stream after each washing step and a product outlet 7 for removing the product stream after the cleavage step. The waste outlet 6 is connected via a waste tube 8 to a chamber 9 where the pH of the stream is adjusted for subsequent separation and to a first ion exchange column 10. The product outlet 7 is connected via a product tube 11 to a second ion exchange column 12.

[0117] The ion exchange columns 10, 11 are arranged for retaining sulfonated waste compounds, such as side products, excess reagents and protection group residues that subsequently must be disposed. The purified solvent stream from the first ion exchange column 10 can be conducted to the sewer system or recycled. The purified product stream from the second ion exchange column 12 is used for obtaining the target protein.

[0118] The invention is illustrated but not limited by the following examples.

EXAMPLES

Example 1: Synthesis of 9-(3,6-disulfo)fluorenylmethyloxycarbonyl chloride (Smoc-Cl)

[0119] 2 g (7.73 mmol) of Fmoc-chloride was treated with 20 mL of concentrated sulfuric acid. After work up of the reaction mixture 2.96 g (7.07 mmol, 91.4%) of crude Smoc-chloride was obtained in form of a slightly yellow solid.

##STR00057##

[0120] Analytical data of Smoc-chloride:

[0121] .sup.1H NMR (500 MHz, D.sub.2O) δ=7.80 (s, 2H), 7.69 (d, J=7.9 Hz, 2H), 7.48 (d, J=7.8 Hz, 2H), 3.84 (d, J=4.8 Hz, 2H), 3.45 (t, J=4.7 Hz, 1H).

[0122] .sup.13C NMR (126 MHz, D.sub.2O) δ=145.57, 142.54, 141.65, 125.16, 121.61, 120.90, 62.54, 49.65.

Example 2: Synthesis of 9-(2,7-disulfo)fluorenylmethyloxycarbonyl chloride (Smoc-Cl)

[0123] 2 g (7.73 mmol) of Fmoc-chloride were treated with 20 mL of concentrated sulfuric acid and heated to 100° C. Sulfuric acid was neutralised with NaOH (pH9.5) and solvent removed under reduced pressure and NMR analytics confirmed formation of target intermediate. The intermediate was dissolved again in 20% sulfuric acid in water, stirred for 6 h to form 9-(2,7-disulfo)fluorenylmethanol. Sulfuric acid was neutralised with NaOH (pH 6.7) and the solvent removed under reduced pressure. A solution of 1.2 eq. phosgene in 25 ml of DCM was cooled to 0° C. and 9-(2,7-disulfo)fluorenylmethanol was added slowly under stirring (Carpino and Han, The Journal of Organic Chemistry 1972,37,(22), 3404-3409). The solution was stirred for 1 h in the ice bath and then let stand for 4 h at ice-bath temperature. Solvent and excess phosgene were removed under reduced pressure giving the corresponding product.

##STR00058##

[0124] NMR intermediate:

[0125] .sup.1H NMR (300 MHz, D.sub.2O) δ: 6.09 (s, 2H), 7.23-7.40 (m, 2H), 7.72 (s, 2H), 7.95 (d, J=6.2 Hz, 2H).

[0126] .sup.13C NMR (75 MHz, D.sub.2O) δ: 142.61, 132.99, 131.74, 130.23, 129.28, 127.22, 125.57, 124.69.

[0127] LC-APCI-MS for 9-(2,7-disulfo)-fluorenylmethyloxycarbonyl chloride:

[0128] LC-APCI-MS calculated for C.sub.15H.sub.9ClO.sub.22. m/z: 256.03. Measured m/z: 256.94 [M−H−2×SO.sub.3].sup.−.

Example 3: Synthesis of Smoc-Gly-OH (Smoc-glycine)

[0129] 2.5 g (8.41 mmol) of Fmoc-glycine were treated with 30 mL of concentrated sulfuric acid. After work up of the reaction mixture 3.7 g (8.09 mmol, 96.2%) of crude Smoc-glycine was obtained in form of a slightly yellow powder.

##STR00059##

[0130] Analytical data of Smoc-glycine:

[0131] LC-APCI-MS calculated for C.sub.17H.sub.14NO.sub.10S.sub.2.sup.− m/z: 456.01. Measured m/z: 455.85 [M−H].sup.−.

[0132] .sup.1H NMR (500 MHz, D.sub.2O) δ=8.01 (s, 2H), 7.87 (d, J=7.9 Hz, 2H), 7.79 (d, J=8.0 Hz, 2H), 5.95 (s, NH), 4.41 (d, J=6.2 Hz, 2H), 4.01 (t, J=6.2 Hz, 1H), 3.67 (s, 2H).

[0133] .sup.13C NMR (126 MHz, D.sub.2O) δ=158.21, 144.94, 142.41, 141.88, 125.51, 122.09, 121.09, 65.75, 46.95, 44.39.

Example 4: Synthesis of Smoc-L-Ala-OH (Smoc-alanine)

[0134] 2.5 g (8.03 mmol) of Fmoc-L-alanine were treated with 30 mL of concentrated sulfuric acid. After work up of the reaction mixture 3.8 g (7.64 mmol, 95.1%) of crude Smoc-L-alanine was obtained in form of a white powder.

##STR00060##

[0135] Analytical data of Smoc-alanine:

[0136] LC-APCI-MS calculated for C.sub.18H.sub.16NO.sub.7S. m/z: 390.06. Measured m/z: 389.96 [M− HSO.sub.3].sup.−.

[0137] .sup.1H NMR (500 MHz, MeOD) δ=7.78 (d, J=19.6 Hz, 2H), 7.57 (d, J=8.1 Hz, 2H), 7.55 (dd, 2H), 5.94 (s, NH), 3.95 (m, OH+2H), 3.85 (q, J=7.4 Hz, 1H), 2.93 (t, J=1.64 Hz, 1H), 1.01 (d, J=7.3 Hz, 3H).

[0138] .sup.13C NMR (126 MHz, MeOD) δ=175.11, 158.10, 146.21, 145.89, 145.44, 143.54, 127.09, 124.04, 123.90, 121.40, 67.23, 51.05, 48.51, 17.58.

Example 5: Synthesis of Smoc-L-Ile-OH (Smoc-isoleucine)

[0139] 2.5 g (7.07 mmol) of Fmoc-L-isoleucine were treated with 30 mL of concentrated sulfuric acid. After work up of the reaction mixture 3.8 g (6.82 mmol, 96.4%) of crude Smoc-L-isoleucine was obtained in form of a white powder.

##STR00061##

[0140] Analytical data of Smoc-isoleucine:

[0141] LC-APCI-MS calculated for C.sub.21H.sub.22NO.sub.7S. m/z: 432.11. Measured m/z: 432.06 [M− HSO.sub.3].sup.−.

[0142] .sup.1H NMR (500 MHz, MeOD) δ=7.79 (d, J=16.5 Hz, 2H), 7.57 (d, J=8.0 Hz, 2H), 7.55 (dd, J=8.0, 1.5 Hz, 2H), 4.15-3.89 (m, OH+2H), 3.77 (d, J=6.2 Hz, 1H), 2.93 (t, J=1.6 Hz, 1H), 1.50 (dtd, J=13.2, 10.1, 6.7 Hz, 1H), 1.20-1.05 (m, 1H), 0.97-0.81 (m, 1H), 0.55 (d, J=6.86 Hz, 3H), 0.54 (t, J=7.43 Hz, 3H).

[0143] .sup.13C NMR (126 MHz, MeOD) δ=173.98, 158.49, 146.15, 145.45, 143.48, 127.09, 124.02, 123.95, 121.38, 67.33, 60.30, 48.55, 38.31, 26.30, 15.98, 11.66.

Example 6: Synthesis of Smoc-L-Leu-OH (Smoc-leucine)

[0144] 2.5 g (7.07 mmol) of Fmoc-L-leucine were treated with 30 mL of concentrated sulfuric acid. After work up of the reaction mixture 3.6 g (7.01 mmol, 99.1%) of crude Smoc-L-leucine was be obtained in form of a white powder.

##STR00062##

[0145] Analytical data of Smoc-leucine:

[0146] LC-APCI-MS calculated for C.sub.21H.sub.22NO.sub.7S. m/z: 432.11. Measured m/z: 432.06 [M− HSO.sub.3].sup.−.

[0147] .sup.1H NMR (500 MHz, MeOD) δ=7.80 (d, J=18.0 Hz, 2H), 7.58 (d, J=8.1 Hz, 2H), 7.57 (dd, J=8.3, 4.1 Hz 2H), 4.11-3.93 (m, OH+2H), 3.86 (dd, J=9.9, 5.3 Hz, 1H), 2.94 (t, 1H), 1.41-1.31 (m, 1H), 1.30-1.17 (m, 2H), 0.57 (dd, J=14.7, 6.5 Hz, 6H).

[0148] .sup.13C NMR (126 MHz, MeOD) δ=175.08, 158.41, 146.18, 145.95, 145.38, 143.52, 127.10, 124.07, 123.97, 121.40, 67.27, 53.99, 48.54, 41.47, 25.86, 23.27, 21.78.

Example 7: Synthesis of Sulfo-Trt

[0149] In a heated out round bottom flask under nitrogen atmosphere, 237 mg (9.75 mmol, 3.0 eq.) magnesium were suspended in 10 mL THF. To this mixture 1/10 of a solution of 1.878 g (9.75 mmol, 3 eq.) 4-chlorobenzenesulfonic acid in 15 mL THF was added under vicious steering. To start the Grignard reaction, the mixture was heated to reflux and a drop of bromine was added. Afterwards the remaining part of the 4-chlorobenzenesulfonic acid solution was added dropwise and the mixture was refluxed for an additional 30 minutes before allowed to cool to room temperature.

[0150] In a second step, Kochi's coupling method was employed. For this purpose, a three neck flask was put under nitrogen atmosphere and cooled to −78° C. by using a suspension of dry ice in methanol. Afterwards 50 mL of THF were added, followed by 0.361 mL (3.25 mmol, 1 eq.) carbon tetrachloride and 0.325 ml (0.03 mmol, 0.01 eq., 0.1 mol/L solution in THF) dilithium tetrachlorocuprate(II). To this solution the previously prepared Grignard reagent was added dropwise. After stirring for 1 h at −78° C. and 6 h at 0° C., the mixture was allowed to warm to room temperature and stirred for an additional 18 h. The solution was quenched by adding 20 mL of water. Volatile components were removed under reduced pressure, resulting in 1.69 g of crude 4,4′,4″-(hydroxymethanetriyl)tribenzenesulfonate (Sulfo-Trt), in form of a brown solid. The product was isolated by semi-preparative RP-HPLC. Treating of 4,4′,4″-(hydroxymethanetriyl)tribenzenesulfonate with thionyl chloride (SOCl.sub.2) leaded to formation of tri(4-sulfophenyl)methyl chloride (Sulfo-Trt chloride). The solvent was removed under reduced pressure.

##STR00063##

[0151] Analytical data of Sulfo-Trt:

[0152] .sup.1H NMR (500 MHz, D.sub.2O) δ=7.68 (d, J=8.6 Hz, 6H), 7.44 (d, J=8.6 Hz, 6H).

[0153] .sup.13C NMR (126 MHz, D.sub.2O) δ=140.99, 136.88, 128.96, 127.02, 75.45.

Example 8: Synthesis of 2-hydroxy-2-methylpropane-1-sulfonate (tBuS-OH)

[0154] 6.00 g 2-methyl-2-propen-1-sulfonic acid sodium salt was diluted in 50 ml water. 10 ml sulfuric acid was added and the reaction stirred at room temperature overnight. Sulfuric acid was removed by CaCO.sub.3 precipitation. The solvent was removed by reduced pressure. The product was obtained as white powder. LC-MS and NMR analytics confirmed target compound.

##STR00064##

[0155] Analytical data of tBuS-OH:

[0156] ESI-MS calculated for C.sub.4H.sub.9O.sub.4S.sup.− m/z: 153.18, measured m/z: 152.9[M−H].sup.−.

[0157] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 1.18 (s, 6H), 2.67 (s, 2H), 5.20 (s, 1H).

[0158] .sup.13C NMR (126 MHz, DMSO) δ: 67.84, 61.81, 39.52, 29.65.

Example 9: Synthesis of 2-bromo-2-methylpropane-1-sulfonate (tBuS-Br)

[0159] 6.00 g 2-methyl-2-propen-1-sulfonic acid sodium salt was diluted in 20 ml HBr (48% w/w). The solvent was removed under reduced pressure. The product was obtained as a whitebrown powder. LC-MS and NMR analytics confirmed target compound.

##STR00065##

[0160] Analytical Data of tBuS-Br:

[0161] ESI-MS calculated for C.sub.4H.sub.8BrO.sub.3S.sup.− m/z: 216.07, measured m/z: 216.8[m-h]−.

Example 10: Synthesis of 2-mercapto-2-methylpropane-1-sulfonate (StBuS)

[0162] 3.00 g 2-methyl-2-propen-1-sulfonic acid sodium salt were diluted in 25 ml HCl conc. and the reaction solution stirred overnight. Afterwards the acid was neutralised with NaOH (pH=6.8) and the solvent was removed by reduced pressure. In a heated out round bottom flask under nitrogen atmosphere, 233.67 mg magnesium were suspended in 20 mL THF. A suspension of 1.5 g tBuS-CI in 15 mL THF was added under vicious steering. To start the Grignard reaction, the mixture was heated to reflux (70° C.) and a drop of bromine was added and the mixture was refluxed for additional 90 minutes before allowed to cool to room temperature. 4 eq. sulphur was added to the Grignard reagent and stirred for 8 h. Afterwards 200 ml water were added. The mixture was stirred for 2 h at room temperature. Subsequently, the solvent was removed under reduced pressure and the resulting white powder was analysed by NMR.

##STR00066##

[0163] Analytical data of StBuS:

[0164] .sup.1H NMR (500 MHz, Deuterium Oxide) δ: 1.56 (s, 6H), 2.18 (s, J=1.3 Hz, 1H), 3.67 (s, 2H).

[0165] .sup.13C NMR (126 MHz, D.sub.2O) δ: 61.66, 32.13, 28.48.

Example 11: Synthesis of (9-BBN)-Lys(SBoc)

[0166] 1.68 g Lysine was suspended in dry THF and 24.28 ml 9-Borabicyclo(3.3.1)nonan (9-BBN) in THF (0.5 mol/L) was added under inert gas. The resulting suspension was refluxed for 18 h at 100° C. The solvent was removed under reduced pressure. (9-BBN)-Lys was diluted in 20 ml THF and 1.97 g 1,1′-carbonyldiimidazole and 2.54 g tBuS-OH was added under inert gas. The resulting solution was stirred for 5 h. The solvent was removed under reduced pressure and the resulting crude product was washed tree times with DCM to remove imidazole residues.

##STR00067##

[0167] Analytical Data of (9-BBN)-Lys(SBoc):

[0168] ESI-MS calculated for C.sub.19H.sub.33BN.sub.2O.sub.7S.sup.2− m/z: 444.35, measured m/z: 443.2[M−H].sup.−.

Example 12: Synthesis of (9-BBN)-Tyr(tBus)

[0169] 2.00 g tyrosine was suspended in dry THF and 25 ml 9-BBN in THF (0.5 mol/L) was added under inert gas. The resulting suspension was refluxed for 18 h at 100° C. The solvent was removed under reduced pressure. (9-BBN)Tyr was suspended in 20 ml THF and 2.39 g tBuS-Br was added under inert gas. The resulting solution was stirred for 2 d. The solvent was removed under reduced pressure and the resulting crude product washed tree times with THF to remove bromide residues.

##STR00068##

[0170] Analytical Data of (9-BBN)-Tyr(tBuS):

[0171] ESI-MS calculated for C.sub.21H.sub.31BNO.sub.6S.sup.− m/z: 436.35, measured m/z: 436.2[M+H].sup.+, 453.2[M+H.sub.2O].sup.+.

Example 13: Stability Test of Smoc-Protected Amino Acids

[0172] To determine the stability of Smoc-protected amino acids in an aqueous environment, Smoc-glycine and Smoc-L-leucine were dissolved in water and incubated at ambient temperature for eight days. The condition of the amino acids was checked after 3, 6 and 8 days using analytical RP-HPLC. The compounds had to be stable in aqueous environment for a considerable amount of time. To prove this stability, Smoc-glycine and Smoc-leucine were dissolved in a minimal amount of water and kept at ambient temperature. The results of the experiments were monitored by RP-HPLC, performed after 3, 6 and 8 days. As a result, no significant change in the composition of the amino acids was observed.

Example 14: N-Terminal Deprotection of Smoc-Protected Amino Acids

[0173] For the usage in SPPS, protective groups must be easily and efficiently deprotectable under safe conditions. To determine the deprotection conditions of Smoc-protected amino acids, a series of experiments were performed by dissolving 1 mg of Smoc-glycine in 1 mL of water or ethanol. To this solution, the one of the following bases [0174] ammonia (10% aq.), [0175] ethanolamine (50% aq.), [0176] ethylendiamine (50% aq.) or [0177] piperidine (50% aq.)
was added and the mixture was incubated under shaking for 5 min at room temperature. The result was analysed by analytical RP-HPLC and ESI-MS.

[0178] The results showed that solutions of ammonia or amine in water are sufficient for Smoc deprotection at room temperature.

[0179] Additionally to piperidine which is the standard base used in Fmoc-SPPS deprotection appeared possible using water-soluble bases, such as ammonia, ethanolamine, and ethylenediamine. This allows access to significantly cheaper and easier procedures compared to the piperidine-based deprotection.

Example 15: Purification by Affinity Chromatography

[0180] Smoc-alanine was mixed with unprotected alanine as an artificial impurity, dissolved in 1M formic acid and loaded to a DEAE Sephadex A-25 ion exchange column. After washing the column with additional formic acid, the Smoc-alanine was eluted with a 4M solution of ammonium formate. The solution before loading, the solution after loading and the eluated fraction were analysed by RP-HPLC.

[0181] The results show that unprotected alanine passed the ion exchange column unhindered while the Smoc-protected alanine bound to the column. With the change of the mobile phase to a 4M solution of ammonium formate, the Smoc-alanine was eluted from the column without any impurity remaining. Thereby it has been shown that the Smoc-group can be successfully used in affinity chromatography for purification purposes.

Example 16: Synthesis of the Test Peptide H-V-G-G-V-G-OH Following the Smoc Approach

[0182] Couplings of Smoc-protected amino acids were performed as follows. Thus, 3 eq. of the respective Smoc-protected amino acid, 2.8 eq. of the activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 3.0 eq N-hydroxysuccinimide (NHS) and 6 eq. of sodium hydrogen carbonate as a general base were dissolved in a minimal amount of water and preactivated for 3 min. A typical coupling took 60 min at ambient temperature. N-terminal deprotection of a peptide resin was performed as double deprotection step with 10% aq. ammonia. After cleavage from the support, the test peptide was analysed by RP-HPLC and ESI-MS.

[0183] Analytical data of the peptide H-V-G-G-V-G-OH:

[0184] RP-HPLC, 10.fwdarw.60% MeCN, t.sub.R=19.1 min.

[0185] ESI-MS calc. for C.sub.16H.sub.29N.sub.5O.sub.6 m/z: 387.21 meas. 387.0 [M+H].sup.+

TABLE-US-00004 TABLE 1 Fluorescence-monitored SPPS of peptide H-L-V-A-I-G-OH Smoc-Gly- Smoc-Ile- Smoc-Ala- Smoc-Val- Smoc-Leu- PEGA PEGA Smoc PEGA Smoc PEGA Smoc PEGA Smoc PEGA Smoc Water resin 0.05 mM deprot. 0.05 mM deprot. 0.05 mM deprot. 0.05 mM deprot. 0.05 mM deprot. 1 3 108 1498 258 1964 362 1814 326 1422 345 1392 331 2 3 106 1670 237 1933 389 2117 478 1386 467 1350 345 3 2 99 1812 284 2186 384 1925 346 1549 346 1344 365 mean value 2.67 104.33 1660.00 259.67 2027.67 378.33 1952.00 383.33 1452.33 386.00 1362.00 347.00 standard 0.58 4.73 157.24 23.54 137.99 14.36 153.29 82.59 85.63 70.15 26.15 17.09 deviation

[0186] The fluorescence of water, solid support (PEGA resin) and PEGA resin-peptide was determined after each coupling step and the following deprotection steps. The results are shown in Table 1. Thus, after each coupling step, the fluorescence value rises due to the coupled Smoc group, and after each deprotection steps the fluorescence value decreases again revealing that real-time monitoring of reaction progress is possible.