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The BI-3802 was designed by Boehringer Ingelheim and could be obtained free of charge through the Boehringer Ingelheim open innovation portal opnMe.com, associated with its negative control.
Synonyms: Fmoc-L-Arginine
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Batch number can be found on the product's label following the word 'Batch'.
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Batch number can be found on the product's label following the word 'Batch'.
Search for reports by entering the product batch number.
Batch number can be found on the product's label following the word 'Batch'.
Search for reports by entering the product batch number.
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CAS No. : | 91000-69-0 |
Formula : | C21H24N4O4 |
M.W : | 396.44 |
SMILES Code : | [H][C@@](CCCNC(N)=N)(NC(=O)OCC1C2=CC=CC=C2C2=C1C=CC=C2)C(O)=O |
Synonyms : |
Fmoc-L-Arginine
|
MDL No. : | MFCD00051770 |
InChI Key : | DVBUCBXGDWWXNY-SFHVURJKSA-N |
Pubchem ID : | 2724631 |
GHS Pictogram: |
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Signal Word: | Warning |
Hazard Statements: | H302-H315-H319-H332-H335 |
Precautionary Statements: | P261-P280-P305+P351+P338 |
* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Solid phase peptide synthesis was performed on a CEM Liberty Peptide Synthesizer using standard Fmoc chemistry. TentaGel S Ram resin (1 g; 0.25 mmol/g) was swelled in NMP (10 ml) prior to use and transferred between tube and reaction vessel using DCM and NMP. Coupling (0148) An Fmoc-amino acid in NMP/DMF/DCM (1:1:1; 0.2 M; 5 ml) was added to the resin in a CEM Discover microwave unit together with HATU/DMF or COMU/DMF (0.5 M; 2 ml) and DIPEA/NMP (2.0 M; 1 ml). The coupling mixture was heated to 75° C. for 5 min while nitrogen was bubbled through the mixture. The resin was then washed with NMP (4×10 ml). Deprotection (0149) Piperidine/DMF (20percent; 10 ml) was added to the resin for initial deprotection and the mixture was heated by microwaves (30 sec; 40° C.). The reaction vessel was drained and a second portion of piperidine/NMP (20percent; 10 ml) was added and heated (75° C.; 3 min.) again. The resin was then washed with DMF (6×10 ml). Side Chain Acylation (0150) Fmoc-Lys(ivDde)-OH or alternatively another amino acid with an orthogonal side chain protective group was introduced at the position of the acylation. The N-terminal of the peptide backbone was then Boc-protected using Boc2O or alternatively by using a Boc-protected amino acid in the last coupling. While the peptide was still attached to the resin, the orthogonal side chain protective group was selectively cleaved using freshly prepared hydrazine hydrate (2-4percent) in NMP for 2×15 min. The unprotected lysine side chain was first coupled with Fmoc-Glu-OtBu or another spacer amino acid, which was deprotected with piperidine and acylated with a lipophilic moiety using the peptide coupling methodology as described above. Alternatively, the acylation moiety was introduced as a premade building block e.g. Fmoc-Lys(hexadecanoyl-gamma-Glu)-OH where gamm-Glu is the coupling of Glutamic acid through the side-chain. Abbreviations employed are as follows: COMU: 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]methanaminium hexaflourophosphate ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methyl-butyl Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl DCM: dichloromethane DMF: N,N-dimethylformamide (0151) DIPEA: diisopropylethylamine EtOH: ethanol Et2O: diethyl ether HATU: N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide MeCN: acetonitrile NMP: N-methylpyrrolidone (0152) TFA: trifluoroacetic acid TIS: triisopropylsilane Cleavage (0153) The resin was washed with EtOH (3×10 ml) and Et2O (3×10 ml) and dried to constant weight at room temperature (r.t.). The crude peptide was cleaved from the resin by treatment with TFA/TIS/water (95/2.5/2.5; 40 ml, 2 h; r.t.). Most of the TFA was removed at reduced pressure and the crude peptide was precipitated and washed three times with diethylether and dried to constant weight at room temperature. HPLC Purification of the Crude Peptide (0154) The crude peptide was purified to greater than 90percent by preparative reverse phase HPLC using a PerSeptive Biosystems VISION Workstation equipped with a C-18 column (5 cm; 10 mum) and a fraction collector and run at 35 ml/min with a gradient of buffer A (0.1percent TFA, aq.) and buffer B (0.1percent TFA, 90percent MeCN, aq.). Fractions were analyzed by analytical HPLC and MS and relevant fractions were pooled and lyophilized. The final product was characterized by HPLC and MS. (0155) The synthesized compounds are shown in Table 1 and Table 2 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: tGLP-1 and its analogues 2?13 were all synthesized using general solid-phase peptide synthesis of N-Fmoc/tBu chemistry. 63Fmoc Rink Amide-MBHA resin (0.1 mmol) was added to a 25 ml peptide synthetic vessel and swollen with DMF for 40 min. After deprotected by 25percent piperidine in DMF, a solution of Fmoc-AA-OH (0.4 mmol), HATU (0.4 mmol), HoAt (0.4 mmol) and DIPEA (0.8 mmol) in DMF was added to the vessel. After reacted for 1 h, the resin was washed three times with DMF and three times with CH2Cl2, then qualitative ninhydrin testing was performed to monitor whether some free amino groups still existed on the resin ornot. If not, the resin was washed three times with DMF again and repeated the procedures of deprotection and coupling. Forthe coupling of some unnatural amino acids, NMM instead of DIPEA and NMP instead of DMF were used. Besides, the reaction time was prolonged to 4 h. Following the final deprotection of N-terminus, the target peptide was cleaved from resin with Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5) for 2 h atroom temperature. After filtration, the residue solution was concentrated, precipitated with cold diethyl ether and centrifuged for three times. The residue was dissolved in water and purified by Waters 2545 preparative RP-HPLC system. Sephadex G-25 was used for the further purification to remove some short peptide impurities. The molecular mass of the target peptide was confirmed by MALDI-TOF. The purity of peptide was tested with analytical RP-HPLC, and the conditions were as follows: a linear gradient of 20percent mobile phase A and 80percent mobile phase B to 80percent mobile phase A and 20percent mobile phase B (A: acetonitrile containing 0.1percent TFA; B: H2O containing 0.1percent TFA) in 30 min, at a flow rate of 1 mL/minute with UV detection at 214 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: tGLP-1 and its analogues 2-13 were all synthesized using general solid-phase peptide synthesis of N-Fmoc/tBu chemistry. 63Fmoc Rink Amide-MBHA resin (0.1 mmol) was added to a 25 ml peptide synthetic vessel and swollen with DMF for 40 min. After deprotected by 25% piperidine in DMF, a solution of Fmoc-AA-OH (0.4 mmol), HATU (0.4 mmol), HoAt (0.4 mmol) and DIPEA (0.8 mmol) in DMF was added to the vessel. After reacted for 1 h, the resin was washed three times with DMF and three times with CH2Cl2, then qualitative ninhydrin testing was performed to monitor whether some free amino groups still existed on the resin ornot. If not, the resin was washed three times with DMF again and repeated the procedures of deprotection and coupling. Forthe coupling of some unnatural amino acids, NMM instead of DIPEA and NMP instead of DMF were used. Besides, the reaction time was prolonged to 4 h. Following the final deprotection of N-terminus, the target peptide was cleaved from resin with Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5) for 2 h atroom temperature. After filtration, the residue solution was concentrated, precipitated with cold diethyl ether and centrifuged for three times. The residue was dissolved in water and purified by Waters 2545 preparative RP-HPLC system. Sephadex G-25 was used for the further purification to remove some short peptide impurities. The molecular mass of the target peptide was confirmed by MALDI-TOF. The purity of peptide was tested with analytical RP-HPLC, and the conditions were as follows: a linear gradient of 20% mobile phase A and 80% mobile phase B to 80% mobile phase A and 20% mobile phase B (A: acetonitrile containing 0.1% TFA; B: H2O containing 0.1% TFA) in 30 min, at a flow rate of 1 mL/minute with UV detection at 214 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th... |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th... |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. (0997) The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedur... |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. (0997) The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedur... |
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Coupling of the First Protected Amino Acid Residue to the Resin 0.5 g of 2-chlorotritylchloride resin (100-200 mesh, copoly(styrene-1% DVB) polymer matrix, Cat. No. 01-64-0114, Novabiochem, Merck Biosciences Ltd.) (Barlos et al. Tetrahedron Lett. 1989, 30, 3943-3946) (1.4 mMol/g, 0.7 mmol) was filled into a dried flask. The resin was suspended in CH2Cl2 (2.5 ml) and, allowed to swell at room temperature under constant stirring for 30 min. The resin was treated with 0.49 mMol (0.7 eq) of the first suitably protected amino acid residue and 488 mul (4 eq) of diisopropylethylamine (DIEA) in CH2Cl2 (2.5 ml), the mixture was shaken at 25 C. for 4 hours. The resin was shaken (CH2Cl2/MeOH/DIEA: 17/2/1), 30 ml for 30 min; then washed in the following order with CH2Cl2 (1×), DMF (1×), CH2Cl2 (1×), MeOH (1×), CH2Cl2 (1×), MeOH (1×), CH2Cl2 (2×), Et2O (2×) and dried under vacuum for 6 hours. Loading was typically 0.6-0.9 mMol/g. The following preloaded resin was prepared: Fmoc-Pro-2-chlorotritylresin. Synthesis of the Fully Protected Peptide Fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel were placed approximately 60 mg (weight of the resin before loading) of the above resin. The following reaction cycles were programmed and carried out: Steps 3 to 6 are repeated to add each amino-acid. Analytical Method: Analytical HPLC retention times (RT, in minutes) were determined using a Jupiter Proteo 90 A column, 150×2.0 mm, (cod. 00E-4396-B0-Phenomenex) with the following solvents A (H2O+0.1% TFA) and B (CH3CN+0.1% TFA) and the gradient: 0 min: 95% A, 5% B; 0.5 min: 95% A, 5% B; 20 min: 40% A, 60% B; 21 min: 0% A, 100% B; 23 min: 0% A, 100% B; 23.1 min: 95% A, 5% B; 31 min: 95% A, 5% B. Formation of Disulfide beta-Strand Linkage After formation of the disulfide beta-strand linkage, the resin was suspended in 1 ml (0.14 mMol) of 1% TFA in CH2Cl2 (v/v) for 3 minutes and filtered, and the filtrate was neutralized with 1 ml (1.15 mMol) of 20% DIEA in CH2Cl2 (v/v). This procedure was repeated twice to ensure completion of the cleavage. The resin was washed three times with 1 ml of CH2Cl2. The CH2Cl2 layer was evaporated to dryness. The volatiles were removed and 8 ml dry DMF were added to the tube. Then 2 eq. of HATU in dry DMF (1 ml) and 4 eq. of DIPEA in dry DMF (1 ml) were added to the peptide, followed by stirring for 16 h. The volatiles were evaporated to dryness. The crude cyclised peptide was dissolved in 7 ml of CH2Cl2 and extracted with 10% acetonitrile in H2O (4.5 ml) three times. The CH2Cl2 layer was evaporated to dryness. To deprotect the peptide fully, 3 ml of cleavage cocktail TFA:TIS:H2O (95:2.5:2.5) were added, and the mixture was kept for 2.5 h. The volatiles were evaporated to dryness and the crude peptide was dissolved in 20% AcOH in water (7 ml) and extracted with isopropyl ether (4 ml) for three times. The aqueous layer was collected and evaporated to dryness, and the residue was purified by preparative reverse phase HPLC. After lyophilisation the products were obtained as white powders and analysed by the HPLC-ESI-MS analytical method described above. The analytical data comprising purity after preparative HPLC and ESI-MS are given. The peptide was synthesized starting with the amino acid L-Pro which was grafted to the resin. Starting resin was Fmoc-Pro-2-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Pro-DPro-Lys-Gln-Tyr-Cys-Tyr-Arg-Dab-DPro-Ala-Ser-Cys-Ala-His-Tyr. A disulfide beta-strand linkage was introduced as described above. The product was cleaved from the resin, cyclized, deprotected and purified as indicated by preparative reverse phase LC-MS. After lyophilisation the product was obtained as white powder and analysed by the HPLC-ESI-MS analytical method described above ([M+2H]2+: 933.1; RT: 10.47; UV-purity: 72%). |
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Coupling of the First Protected Amino Acid Residue to the Resin 0.5 g of 2-chlorotritylchloride resin (100-200 mesh, copoly(styrene-1% DVB) polymer matrix, Cat. No. 01-64-0114, Novabiochem, Merck Biosciences Ltd.) (Barlos et al. Tetrahedron Lett. 1989, 30, 3943-3946) (1.4 mMol/g, 0.7 mmol) was filled into a dried flask. The resin was suspended in CH2Cl2 (2.5 ml) and, allowed to swell at room temperature under constant stirring for 30 min. The resin was treated with 0.49 mMol (0.7 eq) of the first suitably protected amino acid residue and 488 mul (4 eq) of diisopropylethylamine (DIEA) in CH2Cl2 (2.5 ml), the mixture was shaken at 25 C. for 4 hours. The resin was shaken (CH2Cl2/MeOH/DIEA: 17/2/1), 30 ml for 30 min; then washed in the following order with CH2Cl2 (1×), DMF (1×), CH2Cl2 (1×), MeOH (1×), CH2Cl2 (1×), MeOH (1×), CH2Cl2 (2×), Et2O (2×) and dried under vacuum for 6 hours. Loading was typically 0.6-0.9 mMol/g. The following preloaded resin was prepared: Fmoc-Pro-2-chlorotritylresin. Synthesis of the Fully Protected Peptide Fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel were placed approximately 60 mg (weight of the resin before loading) of the above resin. The following reaction cycles were programmed and carried out: Steps 3 to 6 are repeated to add each amino-acid. Analytical Method: Analytical HPLC retention times (RT, in minutes) were determined using a Jupiter Proteo 90 A column, 150×2.0 mm, (cod. 00E-4396-B0-Phenomenex) with the following solvents A (H2O+0.1% TFA) and B (CH3CN+0.1% TFA) and the gradient: 0 min: 95% A, 5% B; 0.5 min: 95% A, 5% B; 20 min: 40% A, 60% B; 21 min: 0% A, 100% B; 23 min: 0% A, 100% B; 23.1 min: 95% A, 5% B; 31 min: 95% A, 5% B. Formation of Disulfide beta-Strand Linkage After formation of the disulfide beta-strand linkage, the resin was suspended in 1 ml (0.14 mMol) of 1% TFA in CH2Cl2 (v/v) for 3 minutes and filtered, and the filtrate was neutralized with 1 ml (1.15 mMol) of 20% DIEA in CH2Cl2 (v/v). This procedure was repeated twice to ensure completion of the cleavage. The resin was washed three times with 1 ml of CH2Cl2. The CH2Cl2 layer was evaporated to dryness. The volatiles were removed and 8 ml dry DMF were added to the tube. Then 2 eq. of HATU in dry DMF (1 ml) and 4 eq. of DIPEA in dry DMF (1 ml) were added to the peptide, followed by stirring for 16 h. The volatiles were evaporated to dryness. The crude cyclised peptide was dissolved in 7 ml of CH2Cl2 and extracted with 10% acetonitrile in H2O (4.5 ml) three times. The CH2Cl2 layer was evaporated to dryness. To deprotect the peptide fully, 3 ml of cleavage cocktail TFA:TIS:H2O (95:2.5:2.5) were added, and the mixture was kept for 2.5 h. The volatiles were evaporated to dryness and the crude peptide was dissolved in 20% AcOH in water (7 ml) and extracted with isopropyl ether (4 ml) for three times. The aqueous layer was collected and evaporated to dryness, and the residue was purified by preparative reverse phase HPLC. After lyophilisation the products were obtained as white powders and analysed by the HPLC-ESI-MS analytical method described above. The analytical data comprising purity after preparative HPLC and ESI-MS are given. The peptide was synthesized starting with the amino acid L-Pro which was grafted to the resin. Starting resin was Fmoc-Pro-2-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Pro-DPro-Lys-Gln-Tyr-Cys-Tyr-Arg-Dab-DPro-Ala-Ser-Cys-Tyr-His-Tyr. A disulfide beta-strand linkage was introduced as described above. The product was cleaved from the resin, cyclized, deprotected and purified as indicated by preparative reverse phase LC-MS. After lyophilisation the product was obtained as white powder and analysed by the HPLC-ESI-MS analytical method described above ([M+2H]2+: 978.6; RT: 10.95; UV-purity: 82%). |
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General procedure: Sufficient quantity (50-100 mg) of Fmoc-PAL-PEG-PS resin or Fmoc-Rink amide MB HA resin, loading: 0.5-0.6 mmol/g was swelled in DMF (1-10 ml /100 mg of resin) for 2-10 minutes. The Fmoc-group on resin was removed by incubation of resin with 10-30 % piperidine in DMF (10- 30 ml / 100 mg of resin), for 10-30 minutes. Deprotected resin was filtered and washed excess of DMF, DCM and ether (50 ml X 4). Washed resin was incubated in freshly distilled DMF (1 ml / 100 mg of resin), under nitrogen atmosphere for 5 minutes. A 0.5 M solution of first Fmoc- protected amino acid (1-3 eq.), pre-activated with HOBt (1-3 eq.) and DIPCDI (1-2 eq.) in DMF was added to the resin, and the resin was then shaken for 1-3 hrs, under nitrogen atmosphere. Coupling completion was monitored using a qualitative ninhydrin test. After the coupling of first amino acid, the resin was washed with DMF, DCM and Diethyl ether (50 ml X 4). For the coupling of next amino acid, firstly, the Fmoc-protection on first amino acid, coupled with resin was deprotected, using a 10-20% piperidine solution, followed by the coupling the Fmoc- protected second amino acid, using a suitable coupling agents, and as described above. The repeated cycles of deprotection, washing, coupling and washing were performed until the desired peptide chain was assembled on resin, as per general (Scheme 1) above. Finally, the Fmoc- protected peptidyl-resin prepared above was deprotected by 20% piperidine treatment as described above and the peptidyl-resins were washed with DMF, DCM and Diethyl ether. Resin containing desired peptide was dried under nitrogen pressure for 10-15 minutes and subjected for cleavage/ deprotection. Using above protocol and suitable variations thereof which are within the scope of a person skilled in the art, the short chain peptides designed in the present invention were prepared, using Fmoc-SPPS approach. Furthermore, resin bound short chain peptides were cleaved and deprotected, purified and characterized using following protocol. CLEAVAGE AND DEPROTECTION: The desired short chain peptides were cleaved and deprotected from their respective peptidyl- resins by treatment with TFA cleavage mixture as follows. A solution of TFA / Water / Triisopropylsilane (95: 2.5: 2.5) (10 ml / 100 mg of peptidyl-resin) was added to peptidyl-resins and the mixture was kept at room temperature with occasional starring. The resin was filtered, washed with a cleavage mixture and the combined filtrate was evaporated to dryness. Residue obtained was dissolved in 10 ml of water and the aqueous layer was extracted 3 times with ether and finally the aqueous layer was freeze-dried. Crude peptide obtained after freeze-drying was purified by preparative HPLC as follows: PREPARATIVE HPLC PURIFICATION OF THE CRUDE SHORT CHAIN PEPTIDES: Preparative HPLC was carried out on a Shimadzu LC-8A liquid chromatography. A solution of crude peptide dissolved in DMF or water was injected into a semi-Prep column (Luna 10mu; C18; 100 A0), dimension 250 X 50 mm and eluted with a linear gradient of ACN in water, both buffered with 0.1 % TFA, using a flow rate of 15 -50 ml / min, with effluent monitoring by PDA detector at 220 nm. A typical gradient of 20 % to 70 % of water- ACN mixture, buffered with 0.1 % TFA was used, over a period of 50 minutes, with 1% gradient change per minute. The desired product eluted were collected in a single 10-20 ml fraction and pure short chain peptides were obtained as amorphous white powders by lyophilisation of respective HPLC fractions. HPLC ANALYSIS OF THE PURIFIED SHORT-CHAIN PEPTIDES After purification by preparative HPLC as described above, each peptide was analyzed by analytical RP-HPLC on a Shimadzu LC-10AD analytical HPLC system. For analytical HPLC analysis of short chain peptides, Luna 5mu; C18; 100 A , dimension 250 X 4.6 mm column was used, with a linear gradient of 0.1 % TFA and ACN buffer and the acquisition of chromatogram was carried out at 220 nm, using a PDA detector. CHARACTERIZATION BY MASS SPECTROMETRY Each peptide was characterized by electrospray ionisation mass spectrometry (ESI-MS), either in flow injection or LC/MS mode. Triple quadrupole mass spectrometers (API-3000 (MDS-SCIES, Canada) was used in all analyses in positive and negative ion electrospray mode. Full scan data was acquired over the mass range of quadrupole, operated at unit resolution. In all cases, the experimentally measured molecular weight was within 0.5 Daltons of the calculated monoisotopic molecular weight. Quantification of the mass chromatogram was done using Analyst 1.4.1 software. Following table l(i) is the list of short chain peptides synthesized using the SPPS approach as described above. Mentioned Seq. ID. No 1 in the list was taken as a reference from WO 2011027257. |