Dipeptide Comprising a Non-Proteogenic Amino Acid

Abstract

Described is a dipeptide comprising a non-proteogenic amino acid, methods of making such and methods of using said dipeptide in a process of making a polypeptide or protein comprising one or more non-proteogenic amino acids.

Claims

1. A method of preparing a final polypeptide or protein, comprising: (a) providing a polypeptide or protein containing one or more non-proteogenic amino acids; (b) providing a dipeptide having a free unprotected imidazolyl moiety, wherein the dipeptide structure consists of ##STR00008## and wherein R1 and R2 are selected from the group consisting of (i) R1 is H or an amino protecting group and R2 is an amino protecting group, (ii) R1 is a removable alkyl group and R2 is H or a removable alkyl group, and (iii) R1 and R2 jointly form a ring; R3 is selected from the group consisting of H, a secondary ammonium cation, a tertiary ammonium cation, and a metal cation forming a salt with the carboxylate group; and R4 is an anion that forms an acidic salt with the imidazole group via a salt bond; (c) activating the dipeptide by contacting the dipeptide with a phosphonium-based coupling reagent; and (d) reacting the activated dipeptide with the -N-terminal of the polypeptide or protein to obtain the final polypeptide or protein.

2. The method according to claim 1, wherein the amino protecting group is selected from the group consisting of: tert-Butoxycarbonyl (Boc), Triphenylmethyl (Trt), 2-(p-biphenylyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethyloxycarbonyl (Fmoc), 2-(4-Nitrophenyl)sulfonylethoxycarbonyl (Nsc), Benzyloxycarbonyl (Cbz), Allyloxycarbonyl (Alloc), o-Nitrobenzenesulfonyl (oNBS), p-Nitrobenzenesulfonyl (pNBS), 2,4-Dinitrobenzenesulfonyl (dNBS), 1-(4,4-Dimethyl-2,6-dioxocyclo-Hexylidene)-3-methylbutyl (ivDde) and o- or p-Nitrophenylsulfenyl (Nps).

3. The method according to claim 1, wherein the removable alkyl group is selected from the group consisting of Benzyl and tert-Butyl.

4. The method according to claim 1, wherein R1 and R2 jointly form a ring is selected from the group consisting of Phatalimide and 1,3,5-dioxazine.

5. The method according to claim 1, wherein (i) R1 is H or an amino protecting group selected from the group consisting of Boc, Trt, Bpoc, Fmoc, Nsc, Cbz, Alloc, oNBS, pNBS, dNBS, ivDde and Nps, and R2 is an amino protecting group selected from the group consisting of Boc, Trt, Bpoc, Fmoc, Nsc, Cbz, Alloc, oNBS, pNBS, dNBS, ivDde, and Nps; or R1 is a removable alkyl group selected from the group consisting of Benzyl and tert-Butyl, and R2 is H or a removable alkyl group selected from the group consisting of Benzyl and tert-Butyl; or R1 and R2 jointly form a ring selected from the group consisting of Phatalimide and 1,3,5-dioxazine; (ii) R3 is selected from the group consisting of H, a secondary ammonium cation, a tertiary ammonium cation, an alkali metal cation, and an alkaline earth metal cation forming a salt with the carboxylate group; and (iii) R4 is selected from the group consisting of the anion of a salt of TFA, the anion of a salt of HCl, the anion of a salt of HBr, and the anion of a salt of hydrogensulfate.

6. The method according to claim 1, wherein R4 is selected from the group consisting of the anion of a salt of TFA, the anion of a salt of HCl, the anion of a salt of HBr, and the anion of a salt of hydrogensulfate.

7. The method according to claim 1, wherein the dipeptide is ##STR00009## wherein * indicates the chiral center of the dipeptide and R4 is selected from the group consisting of the anion of a salt of TFA, the anion of a salt of HCl, the anion of a salt of HBr, an anion of a salt of hydrogensulfate and the anion of a salt of HOAc.

8. The method according to claim 1, wherein the dipeptide is Fmoc-His-Aib-OH, TFA, wherein His is histidine, Aib is the artificial amino acid 2-aminoisobutyric acid, Fmoc is the protection group 9-fluorenylmethyloxycarbonyl, and TFA is trifluoroacetic acid.

9. The method according to claim 1, wherein the dipeptide is attached to the polypeptide or protein of (a) at a histidine residue.

10. The method according to claim 9, wherein the histidine residue is a L-histidine.

11. The method according to claim 1, wherein the phosphonium-based coupling reagent is (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP).

12. The method according to claim 1, wherein the activating further comprises dissolving the dipeptide in an aprotic organic solvent before the reacting.

13. The method according to claim 12, wherein the aprotic organic solvent is selected from the group consisting of DMF, NMP, DMAC, DMSO, acetonitrile, dioxane, and mixtures thereof.

14. The method according to claim 1, wherein one or both of R1 and R2 are removed in a deprotection step under basic conditions.

15. The method according to claim 1, wherein the reacting is performed in aqueous media at a pH between 8.7 and 9.4.

Description

EXAMPLES

List of Abbreviations

[0240] ADO: 8-Amino-3,6-dioxaoctanoic acid [0241] Aib: 2-aminoisobutyric acid [0242] Alloc: Allyloxycarbonyl [0243] Boc: tert-Butoxycarbonyl [0244] Bpoc: 2-(p-biphenylyl)-2-propyloxycarbonyl [0245] Cbz: Benzyloxycarbonyl [0246] DCM: Dichloromethane [0247] DIC: N,N-Diisopropylcarbodiimide [0248] DIPEA: N,N-Diisopropylethylamine [0249] DME: Dimethyl ether [0250] dNBS: 2,4-Dinitrobenzenesulfonyl [0251] EtOH: Ethanol [0252] Fmoc: 9-fluorenylmethyloxycarbonyl [0253] HBr: Hydrobromic acid [0254] HCl: Hydrochloric acid [0255] His: Histidine [0256] HOAc: Acetic acid [0257] HOBt: Hydroxybenzotriazole [0258] ivDde: 1-(4,4-Dimethyl-2,6-dioxocyclo-Hexylidene)-3-methylbutyl [0259] Lys: Lysine [0260] MeCN: Acetonitrile [0261] Mtt: 4-Methyltrityl [0262] NMP: N-Methyl-2-pyrrolidone [0263] Nps: o- or p-Nitrophenylsulfenyl [0264] Nsc: 2-(4-Nitrophenyl)sulfonylethoxycarbonyl [0265] oNBS: o-Nitrobenzenesulfonyl [0266] OtBu: tert-Butoxy [0267] pNBS: p-Nitrobenzenesulfonyl [0268] PyBOP: (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate [0269] TBME: tert-Butyl methyl ether [0270] TBTU: O-(Benzotriazol-1-yl)-N,N,N,N-tetramethyluronium tetrafluoroborate [0271] TEA: Trimethylamine [0272] TFA: Trifluoroacetic acid [0273] TIPS: Triisopropylsilane [0274] Trt: Triphenylmethyl

Example 1

2-[(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(1H-imidazol-4-yl)-propanoylamino]-2-methyl-propanoic acid trifluoracatate (alternative name: Fmoc-His-Aib-OH, TFA)

[0275] ##STR00006##

[0276] Fmoc-His(Trt)-Aib-OH (1 moleq., starting material) was suspended in DCM (1.5 mL/g) and TIPS (1.7 moleq.) was added. Cooled (0-10 C.) TFA (3 mL/g) was added and the mixture was stirred at ambient temperature until the reaction was completed (High-performance liquid chromatography (HPLC) conversion). DME (1 mL/g starting material) and TBME (11 mL/g starting material) was added leading to an increase in temperature. The temperature was slowly allowed to return to room temperature (rt) resulting in precipitation of a white solid. The mixture was stirred for an additional 3 hr and filtered. The precipitate was washed twice with TBME (3 mL/g starting material) and dried overnight in vacuo affording the de-tritylated dipeptide as TFA-salt in 90% yield.

[0277] Stability studies at freeze (<15 C.5 C.), fridge (5 C.3 C.) , and room temperature (20 C. 3 C.) shows more than 24 month stability.

TABLE-US-00001 NMR data: [00007] Coupling Chemical Shift Coupling Constants .sup.1H (ppm) Inte-gral Pattern .sup.nJ.sub.HH (Hz) H18 1.34 3 H singlet ND H19 1.38 3 H singlet ND H14 2.93 1 H Double dublet .sup.1J.sub.HH = 14, .sup.3J.sub.HH = 9 H14 3.08 1 H Double dublet .sup.2J.sub.HH = 14, .sup.3J.sub.HH = 5 H9 4.20 1 H Multiplet ND H10, H10 4.25 2 H Multiplet ND H13 4.37 1 H multiplet ND H2, H7 7.33 2 H Triplet .sup.3J.sub.HH = 7 H15 7.33 1 H Singlet ND H3, H6 7.42 2 H Triplet .sup.3J.sub.HH = 7 H1, H8 7.68 2 H Dublet .sup.3J.sub.HH = 7 H12 7.71 1 H Dublet .sup.3J.sub.HH = 8.5 H4, H5 7.90 2 H dublet .sup.3J.sub.HH = 7 H17 8.17 1 H singlet ND H16 8.97 1 H singlet ND H20 12.5 1 H br.singlet ND

Example 2

[(S)-(22,40-dicarboxy-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatetracontan-1-oyl)][Aib.SUP.8.,Arg.SUP.34.]GLP-1-(7-37) peptide (alternative name: N.SUP.26 .[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]ethoxy}-ethoxy) -acetylamino]-ethoxy}-ethoxy)-acetyl][Aib.SUP.8.,Arg.SUP.34.]GLP-1-(7-37) peptide)

Step 1

[0278] In Situ Activation of dipeptide Fmoc-His-Aib-OH,TFA (Mixture I):

[0279] To a mixture of Fmoc-His-Aib-OH,TFA (4 moleq.) and HOBt*H.sub.2O (4 moleq.) was added NMP (4.7 mL/g dipeptide) at ambient temperature. To the stirred solution TEA was added until pH 8, while the temperature of the solution was kept at ambient temperature using an ice-bath. A solution of PyBOP (3.9 moleq.) in NMP (2.4 mL/g dipeptide) was added to the solution containing the dipeptide at ambient temperature. The pH of the reaction mixture was adjusted to pH 8 by use of TEA. The mixture was stirred at ambient temperature for 20 min. prior to the addition to mixture II.

Preparation of Peptide

[0280] (N.sup.26 [(S)-(22,40-dicarboxy-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatetracontan-1-oyl)] [Arg.sup.34]GLP-1-(9-37) peptide (alternative name: N.sup.26 [2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy -heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]ethoxy}-ethoxy)-acetyl][Arg.sup.34]GLP-1-(9-37) peptide for acylation (Mixture II): N.sup.26 [(S)-(22,40-dicarboxy-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatetracontan-1-oyl)] [Arg.sup.34]GLP-1-(9-37) was suspended in 40 w/w % H.sub.2O in NMP (37 g peptide/L solvent mixture). To the cooled suspension was added TEA until pH 9.3.

Step 2:

[0281] Acylation of peptide N.sup.26 [(S)-(22,40-dicarboxy-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatetracontan-1-oyl)] [Arg.sup.34]GLP-1-(9-37) peptide (Mixture III):

[0282] Mixture I was added dropwise to mixture II at ambient temperature. After addition the pH was readjusted to pH 9.3 (pH-meter) by TEA. The mixture was stirred until optimal conversion (measured by HPLC).

Step 3:

Removal of Protecting Group (Fmoc)

[0283] To the mixture III was added piperidine (20 moleq./dipeptide) and the mixture was stirred for 40 min at rt.

[0284] Orbitrap m/z 1028.7 (4+) 1371.4 (3+)

Example 3

Preparation of N.SUP..26,N.SUP..37-bis[(S)-(22-carboxy-33-(4-carboxyphenoxy)-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatritriacontan-1-oyl)][Aib8,Arg34,Lys37]GLP-1-(7-37) peptide (alternative name: N.SUP.26.-[2-[2-[2-[[2-[2-[2[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-, N.SUP.37.-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxyl-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib.SUP.8.,Arg.SUP.34.,Lys.SUP.37.]-GLP-1-(7-37)-peptide)

Step 1: Sidechain Acylation

[0285] [Arg.sup.34,Lys.sup.37]-GLP-1-(9-37)-peptide as an isoprecipitated pellet (15 g, peptide content of approximately 13% w/w, purity of 93%) was suspended in water (50 mL) and NaOH (aq) (1 M; 1150 L) was added to dissolve the peptide. The resulting solution (57 mL) was transferred to a 150 mL reaction chamber in a titrater setup. The pH of the solution was measured to 10.6. The pH was by the titrator adjusted to 11.3 with dilute NaOH (aq) (0.5 M, 0.67 mL) and the volume adjusted with water to 60 mL giving a final peptide concentration of approximately 33 mg/mL. Assaying the solution to a standard curve of [Arg.sup.34,Lys.sup.37]-GLP-1-(9-37)-peptide gave a corrected peptide content of 1.71 g. Using 1.71 g peptide gave a corrected concentration of the solution of 28.5 mg/mL. Activated sidechain 2,5-dioxopyrrolidin-1-yloxy (S)-(22-carboxy-33-(4-carboxyphenoxy)-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatritriacontanate (alternative name: (4-[9-((S)-1-Carboxy-3-{2-[2-({2-[2-(2-hydroxy-5-oxo-pyrrolidin-1-yloxycarbonylmethoxy)-ethoxy]-ethylcarbamoyl}-methoxy)-ethoxy]ethylcarbamoyl}-propylcarbamoyl)-nonyloxy]-benzoic acid) as 83% active ester, 1.73 g 3.3 eq) was dissolved in NMP (4.8 mL) giving 5.20 mL of solution (0.63 eq/mL). The NMP solution of the sidechain was added slowly from a syringe pump at a constant speed keeping pH constant at 11.3 by titrator controlled addition of aq. NaOH (aq) (0.5 M). 3.90 mL (2.4 eq) of sidechain was added over 1 h and 10 minutes 2 eq/h). Ultra Performance Liquid Chromatography (UPLC) analysis showed the acylation to be almost complete and addition was continued to a total of 2.8 eq of sidechain (4.43 mL). During addition a total of (16.71 mL; 8.4 mmol) 0.5 N NaOH (aq) was added by the titrator.

Step 2: Isoprecipitation

[0286] The reaction mixture was transferred with water to 450 mL centrifuge vials (22 mL in each) and pH in each was adjusted to 4.8 by addition of conc. acetic acid to give a white precipitate. EtOH (2.2 mL to a total of app. 10% v/v) was added. The precipitate was centrifuged and used without further purification in the next step.

Step 3: Ligation of Dipeptide

[0287] The isoprecipitate was suspended in NMP (48 mL; 50 mg peptide/mL) and DIPEA (656 L) was added. pH of the resulting solution was measured to pH=9.6 in a sample of 100 L of the mixture diluted with 900 L water.

[0288] The water content of the slurry was measured to 1.4% by Karl Fischer titration. Water (9.12 mL) was added to give a water content of app. 20%.

[0289] Fmoc-His-Aib-OH, TFA; 1058 mg, 3.5 eq) was activated with HOBt (248 mg; 3.5 eq), PyBOP (907 mg, 3.325 eq) and triethylamine (545 L) in NMP for 15 minutes. pH was 4-5 measured by a wet pH stick. The mixture was added to the peptide solution and pH was adjusted with triethylamine to pH 9.3 (by measuring a sample of the reaction mixture (100 L) added to water (900 L). After 1 h UPLC showed almost complete conversion to the desired product.

Step 4: Fmoc Deprotection

[0290] To the reaction mixture from the ligation step was added piperidine (3.35 mL, (5% v/v)) and the mixture was stirred for 25 minutes after which UPLC analysis showed complete conversion to the product. Water was added to give a 1:1 NMP-water solution and the pH was adjusted to 8.5 with acetic acid and the product purified by standard chromatography.

Analysis:

[0291] RP-UPLC: BEH C18, 150*1 mm@45 C. and 0.1 ml/min; gradient from 30 to 60% 0.04% TFA in MeCN (B eluent) in 30 min then up to 90% B, total run time 38 min. UV@215 nm, 5 Hz Synapt High Definition Mass Spectrometry (HDMS): positive ES-MS mode from m/z 200-2500 (1 Hz). V(cap): 3 kV; V(cone): 28V; Desolvation gas 250 C.@750 l/h; cone gas 50 l/h@110 C.

[0292] Rt=15.18 min

[0293] Exact mass: 4885, 4477 Da; Found: M/4: 1222.35; M/3: 1629.45

Example 4

Preparation of N.SUP.26.[(S)-(22-carboxy-33-(4-carboxyphenoxy)-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatritriacontan-1-oyl)][Aib.SUP.8.,Arg.SUP.34.,Lys.SUP.37.]GLP-1-(7-37) peptide (alternative name: N.SUP.26.-[2-[2-[2-[[2-[2-[2-[[(4S)-4carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib.SUP.8.,Arg.SUP.34.,Lys.SUP.37.]-GLP-1-(7-37)-peptide):

[0294] The peptide was synthesised using solid phase peptide synthesis:

[0295] To the 1.04 g resin Fmoc-Lys(Boc)-Wang-LL (eq. 0.24 mmol/g) was added in a stepwise manner 4 moleq. Standard Fmoc/OtBu protocol protected amino acids or Ser-Ser pseudoproline or Fmoc-L-Lys(Mtt)-OH. The activation amino acid (4 moleq.) in cartridge was reacted for 7 minutes with 4 moleq DIC and 4 moleq. HOBt in NMP. The resulting peptide was transferred to a reaction vessel with resin and reacted for 30 min. DIPEA (4 moleq.) was added and the reaction was continued for 30 min. The resin was flowwashed with NMP and subsequently deprotected using 20% Piperidine (10 ml, 20 min). The resin was again flowwashed with NMP.

MTT Deprotection:

[0296] The resin was washed in DCM. 1,1,1,3,3,3-hexafluoro-2-propanol (10 ml for 10 min) was added and the resin was drained. The deprotection procedure was repeated overall four times. The resin was washed with DCM, followed by NMP.

[0297] To a mixture of (S)-22-(tert-butoxycarbonyl)-33-(4-(tert-butoxycarbonyl)phenoxy)-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatritriacontan-1-oic acid, HOBt (4 moleq.) and DIPEA (4 moleq.) in NMP (10 ml) was added, and TBTU (3.8 moleq.) was added as a solid, and the mixture was shaken for 15 min. before added to the resin. After 2 hr. the resin was drained and flowwashed with NMP and DCM.

[0298] The peptidyl resin was swelled in NMP (10 mL) for 10 min at ambient temperature. The vessel was drained. 20 vol % piperidine in NMP (20 mL) was added to the resin. The mixture was swelled for 20 min at ambient temp. The dipeptide Fmoc-L-His-Aib-OH,TFA (580 mg) and HOBt.H.sub.2O (153 mg) were placed in a 20 mL vial. NMP (4 mL) was added. To the mixture was added TEA (650 L) until pH 8 (pH-stick). To the reaction mixture was added a solution of PyBOP (500 mg) in NMP (4 mL). To the reaction mixture was again added TEA (200 L) until pH 8 (pH-stick). The mixture was stirred for 35 min at ambient temp. The vessel was drained. 20 vol % piperidine in NMP (20 mL) was again added to the resin (double deprotection). The mixture was swelled for 20 min at ambient temp and the resin was drained. The resin was flow washed with NMP (50 mL), DCM (50 mL) and 3 times NMP (50 mL). Finally the resin was flowwashed with DCM and drained. The peptide was cleaved from the resin by a mixture of TFA, H.sub.2O and TIPS (95%, 2.5%, 2.5%) for 3 hr. The resulting cleaved peptide was precipitated in diethylether and isolated by filtration.

[0299] TOF MS ES+: m/z, found m/4 (1045.54), calculated m/4 (1045.5)

[0300] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.