Purity | Size | Price | VIP Price | USA Stock *0-1 Day | Global Stock *5-7 Days | Quantity | |||||
{[ item.p_purity ]} | {[ item.pr_size ]} |
{[ getRatePrice(item.pr_usd, 1,1) ]} {[ getRatePrice(item.pr_usd,item.pr_rate,item.mem_rate) ]} |
{[ getRatePrice(item.pr_usd, 1,1) ]} | Inquiry {[ getRatePrice(item.pr_usd,item.pr_rate,item.mem_rate) ]} {[ getRatePrice(item.pr_usd,1,item.mem_rate) ]} | {[ item.pr_usastock ]} | Inquiry - | {[ item.pr_chinastock ]} | Inquiry - |
* Storage: {[proInfo.prStorage]}
CAS No. : | 135248-89-4 | MDL No. : | MFCD03788178 |
Formula : | C18H17NO4S | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | RMTDKXQYAKLQKF-INIZCTEOSA-N |
M.W : | 343.40 | Pubchem ID : | 11024349 |
Synonyms : |
|
Num. heavy atoms : | 24 |
Num. arom. heavy atoms : | 12 |
Fraction Csp3 : | 0.22 |
Num. rotatable bonds : | 7 |
Num. H-bond acceptors : | 4.0 |
Num. H-bond donors : | 2.0 |
Molar Refractivity : | 93.1 |
TPSA : | 114.43 Ų |
GI absorption : | High |
BBB permeant : | No |
P-gp substrate : | No |
CYP1A2 inhibitor : | Yes |
CYP2C19 inhibitor : | No |
CYP2C9 inhibitor : | Yes |
CYP2D6 inhibitor : | No |
CYP3A4 inhibitor : | No |
Log Kp (skin permeation) : | -6.28 cm/s |
Log Po/w (iLOGP) : | 2.2 |
Log Po/w (XLOGP3) : | 2.98 |
Log Po/w (WLOGP) : | 2.91 |
Log Po/w (MLOGP) : | 2.32 |
Log Po/w (SILICOS-IT) : | 2.69 |
Consensus Log Po/w : | 2.62 |
Lipinski : | 0.0 |
Ghose : | None |
Veber : | 0.0 |
Egan : | 0.0 |
Muegge : | 0.0 |
Bioavailability Score : | 0.56 |
Log S (ESOL) : | -3.75 |
Solubility : | 0.0604 mg/ml ; 0.000176 mol/l |
Class : | Soluble |
Log S (Ali) : | -5.05 |
Solubility : | 0.00308 mg/ml ; 0.00000898 mol/l |
Class : | Moderately soluble |
Log S (SILICOS-IT) : | -5.0 |
Solubility : | 0.0034 mg/ml ; 0.00000991 mol/l |
Class : | Moderately soluble |
PAINS : | 0.0 alert |
Brenk : | 1.0 alert |
Leadlikeness : | 0.0 |
Synthetic accessibility : | 3.77 |
Signal Word: | Warning | Class: | N/A |
Precautionary Statements: | P261-P305+P351+P338 | UN#: | N/A |
Hazard Statements: | H302-H315-H319-H335 | Packing Group: | N/A |
GHS Pictogram: |
* 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 |
---|---|---|
73% | With trifluoroacetic acid; zinc; In methanol; at 20℃; for 14.0h; | N,N'-Bis(Fmoc)-L-cystine (12.0 g, 17.5 mmol) was dissolved in methanol (300 mL). Granular zinc (12.0 g) was added to this solution and the resulting mixture was vigorously stirred using a magnetic stirrer. Trifluoroacetic acid (75 mL, 1 mol) was added dropwise into the reaction mixture, over period of 2 h and stirred at room temperature for a period of 12 h. The reaction was monitored by C-18 reverse phase high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC, chloroform/methanol/acetic acid=30:1:0.1, v/v). Upon disappearance of <strong>[135273-01-7]N,N'-bis(Fmoc)-L-cystine</strong>, the reaction mixture was filtered and concentrated on a rotary evaporator to reduce the volume to approximately 100 mL. Dichloromethane (400 mL) was added and the mixture was washed with 2N aqueous hydrochloric acid. The aqueous layer was extracted with dichloromethane and the combined organic layers were dried over magnesium sulfate. Concentration of the solution gave N-(Fmoc)-L-cysteine, 8.8 g 73%) (FIGS. 3 and 4, structure B) as a white powder. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The peptides were prepared by microwave assisted solid phase peptide synthesis based on Fmoc chemistry. All peptides were assembled on a Peg rink amide resin (0.35mmol/g) and different microwave protocols were applied to the coupling steps depending on the amino acid type. In the standard protocol, couplings were performed using a five-fold excess of Fmoc-amino acids (0.2M in NMP), HOBT/HBTU (5eq., 0.45M in DMF) as activators, and DIPEA (10eq., 1M in NMP) as a base with 5min microwave irradiation at 75 C. Fmoc deprotection was achieved using piperidine (20% in DMF) with 3min microwave irradiation at 75 C. In order to avoid racemization of cysteine and histidine, their couplings were performed at 50 C. For arginine, which possesses a low reactivity toward peptide couplings, we adopted a double coupling approach (first coupling: 30 min at 75 C without microwave energy, second coupling: 5 min at 75C with microwave energy). Cleavage was performed using 10ml of Reagent K (TFA/phenol/water/thioanisole/EDT; 82.5/5/5/5/2.5) for 180 min. After cleavage, peptides were precipitated out and washed using ice-cold anhydrous ethyl ether. All peptides were purified by RP-HPLC using a gradient elution of 5-70% solvent B (solvent A: water/acetonitrile/TFA 94.9/5/0.1; solvent B: water/acetonitrile/TFA 5/94.9/0.1) over 20 min at a flow rate of 20ml/min. The purified peptides were freeze-dried and stored at 0 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 1 g (1.4 mMol) 2-chlorotritylchloride resin (1.4 mMol/g; 100 - 200 mesh, copoly(styrene-1% DVB) polymer matrix; Barlos et al. Tetrahedron Lett. 1989, 30, 3943-3946) was filled into a dried flask. The resin was suspended in CH2Cl2 (5 mL) and allowed to swell at room temperature under constant shaking for 30 min. A solution of 0.98 mMol (0.7 eq) of the first suitably protected amino acid residue (see below) in CH2Cl2 (5 mL) mixed with 960 mul (4 eq) of diisopropylethylamine (DIEA) was added. After shaking the reaction mixture for 4 h at 25 C, the resin was filtered off and washed successively with CH2Cl2 (1x), DMF (1x) and CH2Cl2 (1x). A solution of CH2Cl2/MeOH/DIEA (17/2/1, 10 mL) was added to the resin and the suspension was shaken for 30 min. After filtration the resin was washed in the following order with CH2Cl2 (1x), DMF (1x), CH2Cl2 (1x), MeOH (1x), CH2Cl2 (1x), MeOH (1x), CHO (2x), Et2O (2x) and dried under vacuum for 6 hours. Loading was typically 0.6-0.7 mMol/g. The following preloaded resins was prepared: Fmoc-Pro-2-chlorotrityl resin. The synthesis was carried out employing a Syro-peptide synthesizer (MultiSynTech) using 24-96 reaction vessels. In each vessel 0.04 mMol of the above resin was placed and the resin was swollen in CH2Cl2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: [tabl0001-en] Step Reagent Time 1 DMF, wash 2x1 min 2 20% piperidine/DMF 1x5 min, 1x15 min 3 DMF, wash 5x1 min 4 5 eq Fmoc amino acid/DMF +5 eq Py-BOP/DMF, 10 eq DIEA/DMF 1x60 min 5 DMF, wash 3x1 min Step 4 was repeated once. Unless indicated otherwise, the final coupling of an amino acid was followed by Fmoc deprotection by applying steps 1-3 of the above described reaction cycle. After completion of the synthesis, the resin (0.04 mMol) was suspended in 1 mL (0.13 mMol, 3.4 eq) of 1% TFA in CH2Cl2 (v/v) for 3 minutes, filtered, and the filtrate was neutralized with 1 mL (0.58 mMol, 14.6 eq) of 10% DIEA in CH2Cl2 (v/v). This procedure was repeated three times to ensure completion of the cleavage. The filtrate was evaporated to dryness and a sample of the product was fully deprotected by using a cleavage mixture containing 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane (TIS) to be analyzed by reverse phase-HPLC (C18 column) and ESI-MS to monitor the efficiency of the linear peptide synthesis. The HPLC-retention time (minutes) was determined using the analytical method as described above (UV-purity[after preparative HPLC]: 95%; RT: 1.56; [M+3H]/3 = 685.7). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
40% | SPPS was performed on Rink amide resin (0.1 mmol) as described in the general procedures. The first four amino acids, Ser-Ala-Asn-Met, were coupled on the peptide synthesizer using a standard protocol. After the completion of the synthesis, a manual coupling was carried out using Nalpha-FmocSer-(AcO3-alpha-D-O-GlcNAc)OH (0.2 mumol, 131 mg), with O-(7-azabenzotriazol-1-yl)-N,N,N?,N?-tetramethyl-uronium hexafluorophosphate (HATU; 0.2 mumol, 76 mg), 1-hydroxy-7-azabenzotriazole (HOAt; 0.2 mumol, 27 mg) and diisopropylethylamine (DIPEA; 0.4 mumol, 70 mul) in NMP (5 ml) for 12 h. The coupling reaction was monitored by standard Kaiser test. The resin was then washed with NMP (6 ml) and methylene chloride (DCM; 6 ml), and resubjected to the same coupling conditions to ensure completion of the coupling. The glycopeptide was then elongated on the peptide synthesizer after which the resin was thoroughly washed with NMP (6 ml), DCM (6 ml) and MeOH (6 ml) and dried in vacuo. The resin was swelled in DCM (5 ml) for 1 h and then treated with hydrazine (60%) in MeOH (10 ml) for 2 h and washed thoroughly with NMP (5 ml×2), DCM (5 ml×2) and MeOH (5 ml×2) and dried in vacuo. The resin was swelled in DCM (5 ml) for 1 h, after which it was treated with reagent K (TFA (81.5%), phenol (5%), thioanisole (5%), water (5%), EDT (2.5%), TIS (1%)) (30 ml) for 2 h at room temperature. The resin was filtered and washed with neat TFA (2 ml). The filtrate was then concentrated in vacuo to approximately of its original volume. The peptide was precipitated using diethyl ether (0 C.) (30 ml) and recovered by centrifugation at 3000 rpm for 15 min. The crude peptide was purified by RP-HPLC on a semi preparative C-8 column using a linear gradient of 0 to 100% solvent B in solvent A over a 40 min period and the appropriate fractions were lyophilized to afford 64 (118 mg, 40%). C129H204N32O40S2, MALDI-ToF MS: observed [M+], 2907.5916 Da; calculated [M+], 2905.4354 Da ( |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Representative Peptide Synthesis (0159) To deprotect the resin, a portion of resin (50 mg) was first swelled by suspension in CH2Cl2 (2 mL) for 30 min at rt and then drained. The resin was then treated with TFA (50% in CH2Cl2, 2 mL, 2×2 min, rt) and washed with dimethylformamide (DMF; 3×2 mL). To couple each amino acid, Fmoc-protected amino acids (2 equiv. relative to resin), 2-(1Hbenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU; 2 equiv.), and diisopropylethylamine (DIPEA; 2 equiv.) were dissolved in DMF (2 mL). The solution was allowed to pre-activate for 1 min prior to being added to the resin and agitated for 1 h at rt. After each coupling step, the resin was drained and washed with DMF (2×2 mL). To remove the Fmoc-protecting group after each coupling, the resin was treated with piperidine (2 mL of 20% in DMF, 2×10 min) and washed with DMF (3×2 mL). To acetylate the amino terminus, acetic anhydride (10 equiv.) and DIPEA (7 equiv.) were dissolved in DMF (2 mL), and the solution was added to the resin and agitated for 15 min. The resin was then drained and washed with DMF (2×2 mL). Upon synthesis of a complete linear peptide sequence, the resin was washed with diethyl ether (1×2 mL) and dried under vacuum for 48 h. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Peptide monomers of the present invention were synthesized using the Merrifield solid phase synthesis techniques on Protein Technology's Symphony multiple channel synthesizer. The peptides were assembled using HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), Diisopropylethylamine(DIEA) coupling conditions. For some amino acid couplings PyAOP(7-Azabenzotriazol- 1 -yloxy)tripyrrolidinophosponium hexafluorophosphate) and DIEA conditions were used. Rink Amide MB HA resin (100-200 mesh, 0.57 mmol/g) was used for peptide with C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid was used for peptide with C-terminal acids. The coupling reagents (HBTU and DIEA premixed) were prepared at lOOmmol concentration. Similarly amino acids solutions were prepared at 100 mmol concentration. Peptide inhibitors of the present invention were identified based on medical chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties.[00611] The peptides were assembled using standard Symphony protocols. The peptide sequences were assembled as follows: Resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4ml of DMF followed by treatment with 2.5ml of 20percent 4-methyl piped dine (Fmoc de- protection) for lOmin. The resin was then filtered and washed two times with DMF (4ml) and re -treated with N-methyl piperifine for additional 30 minute. The resin was again washed three times with DMF (4 ml) followed by addition 2.5ml of amino acid and 2.5ml of HBTU-DIEA mixture. After 45min of frequent agitations, the resin was filtered and washed three timed with DMF (4 ml each). For a typical peptide of the present invention, double couplings were performed. After completing the coupling reaction, the resin was washed three times with DMF (4 ml each) before proceeding to the next amino acid coupling. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Peptide monomers of the present invention were synthesized using the Merrifield solid phase synthesis techniques on Protein Technology's Symphony multiple channel synthesizer. The peptides were assembled using HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), Diisopropylethylamine(DIEA) coupling conditions. For some amino acid couplings PyAOP(7-Azabenzotriazol- 1 -yloxy)tripyrrolidinophosponium hexafluorophosphate) and DIEA conditions were used. Rink Amide MB HA resin (100-200 mesh, 0.57 mmol/g) was used for peptide with C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid was used for peptide with C-terminal acids. The coupling reagents (HBTU and DIEA premixed) were prepared at lOOmmol concentration. Similarly amino acids solutions were prepared at 100 mmol concentration. Peptide inhibitors of the present invention were identified based on medical chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties.[00611] The peptides were assembled using standard Symphony protocols. The peptide sequences were assembled as follows: Resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4ml of DMF followed by treatment with 2.5ml of 20percent 4-methyl piped dine (Fmoc de- protection) for lOmin. The resin was then filtered and washed two times with DMF (4ml) and re -treated with N-methyl piperifine for additional 30 minute. The resin was again washed three times with DMF (4 ml) followed by addition 2.5ml of amino acid and 2.5ml of HBTU-DIEA mixture. After 45min of frequent agitations, the resin was filtered and washed three timed with DMF (4 ml each). For a typical peptide of the present invention, double couplings were performed. After completing the coupling reaction, the resin was washed three times with DMF (4 ml each) before proceeding to the next amino acid coupling. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Peptide monomers of the present invention were synthesized using the Merrifield solid phase synthesis techniques on Protein Technology's Symphony multiple channel synthesizer. The peptides were assembled using HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), Diisopropylethylamine(DIEA) coupling conditions. For some amino acid couplings PyAOP(7-Azabenzotriazol- 1 -yloxy)tripyrrolidinophosponium hexafluorophosphate) and DIEA conditions were used. Rink Amide MB HA resin (100-200 mesh, 0.57 mmol/g) was used for peptide with C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid was used for peptide with C-terminal acids. The coupling reagents (HBTU and DIEA premixed) were prepared at lOOmmol concentration. Similarly amino acids solutions were prepared at 100 mmol concentration. Peptide inhibitors of the present invention were identified based on medical chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties.[00611] The peptides were assembled using standard Symphony protocols. The peptide sequences were assembled as follows: Resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4ml of DMF followed by treatment with 2.5ml of 20% 4-methyl piped dine (Fmoc de- protection) for lOmin. The resin was then filtered and washed two times with DMF (4ml) and re -treated with N-methyl piperifine for additional 30 minute. The resin was again washed three times with DMF (4 ml) followed by addition 2.5ml of amino acid and 2.5ml of HBTU-DIEA mixture. After 45min of frequent agitations, the resin was filtered and washed three timed with DMF (4 ml each). For a typical peptide of the present invention, double couplings were performed. After completing the coupling reaction, the resin was washed three times with DMF (4 ml each) before proceeding to the next amino acid coupling. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Peptide monomers of the present invention were synthesized using the Merrifield solid phase synthesis techniques on Protein Technology's Symphony multiple channel synthesizer. The peptides were assembled using HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), Diisopropylethylamine(DIEA) coupling conditions. For some amino acid couplings PyAOP(7-Azabenzotriazol- 1 -yloxy)tripyrrolidinophosponium hexafluorophosphate) and DIEA conditions were used. Rink Amide MB HA resin (100-200 mesh, 0.57 mmol/g) was used for peptide with C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid was used for peptide with C-terminal acids. The coupling reagents (HBTU and DIEA premixed) were prepared at lOOmmol concentration. Similarly amino acids solutions were prepared at 100 mmol concentration. Peptide inhibitors of the present invention were identified based on medical chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties.[00611] The peptides were assembled using standard Symphony protocols. The peptide sequences were assembled as follows: Resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4ml of DMF followed by treatment with 2.5ml of 20% 4-methyl piped dine (Fmoc de- protection) for lOmin. The resin was then filtered and washed two times with DMF (4ml) and re -treated with N-methyl piperifine for additional 30 minute. The resin was again washed three times with DMF (4 ml) followed by addition 2.5ml of amino acid and 2.5ml of HBTU-DIEA mixture. After 45min of frequent agitations, the resin was filtered and washed three timed with DMF (4 ml each). For a typical peptide of the present invention, double couplings were performed. After completing the coupling reaction, the resin was washed three times with DMF (4 ml each) before proceeding to the next amino acid coupling. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
17.4 mg | Phenethylamine-AMEBA resin (Sigma Aldrich, 0.1 mmol, 1.0 mmol/g) was subjected to solid phase peptidesynthesis on an automatic peptide synthesizer (CEM Liberty Blue Microwave) with standard double Arg for the Arg residues and DNIe coupled double time. Amino acids were prepared as 0.2 M solutions in DMF. A standard coupling cycle was defined as follows:Amino acid coupling: AA (5 eq.), HA11J (5 eq.), DIEA (25 eq.)Washing: DMF (3x7 mE)Fmoc Deprotection: 20% Piperidine/0. 1 M HOBt (2x7 mE)Washing: DMF (4x7 mE then 1 x5 mE) 55Intermediate 41a (276 mg, 0.1 mmol) was combined with 4 mE TFA solution (37 mE TFA, 1 mE H20, 1 mE TIPS, 3.06 g DTT) and shaken at r.t. for 3 hours. The solution was removed from the resin and precipitated into 40 mE cold Et20. The solution was vortexed and let stand over ice for 10 minutes before centriffiging at 4000 rpm for 5 minutes. Thesolvent was removed and the white solid was washed twice 60 more with cold Et20 (40 mE each time), centrifuged (5 minutes each time) and decanted. The solid was dried under vacuum overnight yielding Intermediate 41b-batch 1 (17.4 mg, 0.012 mmol). ECMS (5Q2 ProductAnalysis-AcidicPeptide-Polar, Acquity UPEC HER C18 column, 130 A, 1.7 65 pm, 2.1 mmx50 mm, 50 C.): R=1.83 minutes, MS [M+R]1513.5. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
80% | Phenethylamine-AMEBA resin (Sigma Aldrich, 0.25 mmol, 1.0 mmol/g) was subjected to solid phase peptide synthesis on an automatic peptide synthesizer (CEM Liberty Blue Microwave) with standard double Arg for the Arg residues and D-Nle andAzidolysine coupled double time. Amino acids were prepared as 0.2 M solution in DMF. A standard coupling cycle was defined as follows:Amino acid coupling: AA (5 eq.), HATU (5 eq.), DIEA (25 eq.)Washing: DMF (3x7 mL)Fmoc Deprotection: 20% Piperidine/0.1 M HOBt (2x7 mL)Washing: DMF (4x7 mL then 1x5 mL) After the assembly of the peptide, the resin was washed with DMF (2x50 mL) and DCM (2x50 mL) then dried under vacuum to give Intermediate 42a (770 mg, 0.250 mmol). Intermediate 42a (770mg, 0.250 mmol) was divided inhalf and each sample was combined with 6 mE TFA solution (37 mE TFA, 1 mE R20, 1 mE TIPS, 2.569 g (20 eq.) DTT) and shaken at ri. for 3 hours. The solution was removed from the resin and precipitated into 40 mE cold Et20. The solution wasvortexed and let stand over ice for 10 minutes before centrifuging at 4000 rpm for 5 minutes. The solvent was removed and the white solid was washed twice more with cold Et20 (40 mE each time), centrifuged (5 minutes each time) and decanted. The solid was dried under vacuum overnight andpurified via M-triggered RPEC yielding Intermediate 43b as a white powder (80 mg, 0.045 mmol, 80%). ECMS (5Q2 ProductAnalysis-Acidic-Peptide-Polar, Acquity UPEC HER C18 column, 130 A, 1.7 tm, 2.1 mmx50 mm, 50 C.):R=2.32 minutes, MS [M+R+2/2] 888.0. |
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. 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. 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. 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. 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... |
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 |
---|---|---|
6% | General procedure: The two azido derivative peptides 10 and 11 (Scheme 2) weresynthesised according to the general procedure by microwaveassistedFmoc-SPPS with 2 5 min couplings for each Fmoc-aminoacid (4.2 equiv) using HATU (4.0 equiv) and DIPEA (4.2 equiv). Thetemperature was set at 70 C (20 W, 10 min) for all amino acids,except for Cys and His, which were coupled at 50 C (20 W,10 min). For His, Cys, and Arg, initial coupling was performed atrt for 3 min before microwave-assisted couplings.31 Each aminoacid coupling cycle consisted of Fmoc-deprotection with 20%piperidine in DMF at 70 C (twice, 2.5 min and 5 min), 2 minDMF flow wash, followed by 10 min coupling with the pre-activatedamino acid. In the case of Fmoc-deprotection for Asp, 0.1 MHOBT in 20% piperidine/DMF was used. Upon completion of synthesis,the resin was washed with DMF, DCM, MeOH, and vacuumdried. The peptide was cleaved from the resin by stirring in a solutionof TFA (99%)/triisopropylsilane/water (95:2.5:2.5) for 4 h. Thecrude products (10 and 11) were purified by RP-HPLC.Peptide 10 yield: 6%. Purity: >95%. Molecular weight: 5446.2.ESI-MS: [M+4H]4+ m/z 1362.1 (calcd: 1362.6), [M+5H]5+ m/z1090.1 (calc: 1090.3), [M+6H]6+ m/z 908.8 (calcd: 908.7), [M+7H]7+ m/z 779.0 (calcd: 779.0), [M+8H]8+ m/z 691.9 (calcd:681.8). Rt = 23.8 min (25-45% solvent B, 20 min, C18-column). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
12% | General procedure: The two azido derivative peptides 10 and 11 (Scheme 2) weresynthesised according to the general procedure by microwaveassistedFmoc-SPPS with 2 5 min couplings for each Fmoc-aminoacid (4.2 equiv) using HATU (4.0 equiv) and DIPEA (4.2 equiv). Thetemperature was set at 70 C (20 W, 10 min) for all amino acids,except for Cys and His, which were coupled at 50 C (20 W,10 min). For His, Cys, and Arg, initial coupling was performed atrt for 3 min before microwave-assisted couplings.31 Each aminoacid coupling cycle consisted of Fmoc-deprotection with 20%piperidine in DMF at 70 C (twice, 2.5 min and 5 min), 2 minDMF flow wash, followed by 10 min coupling with the pre-activatedamino acid. In the case of Fmoc-deprotection for Asp, 0.1 MHOBT in 20% piperidine/DMF was used. Upon completion of synthesis,the resin was washed with DMF, DCM, MeOH, and vacuumdried. The peptide was cleaved from the resin by stirring in a solutionof TFA (99%)/triisopropylsilane/water (95:2.5:2.5) for 4 h. Thecrude products (10 and 11) were purified by RP-HPLC.Peptide 10 yield: 6%. Purity: >95%. Molecular weight: 5446.2.ESI-MS: [M+4H]4+ m/z 1362.1 (calcd: 1362.6), [M+5H]5+ m/z1090.1 (calc: 1090.3), [M+6H]6+ m/z 908.8 (calcd: 908.7), [M+7H]7+ m/z 779.0 (calcd: 779.0), [M+8H]8+ m/z 691.9 (calcd:681.8). Rt = 23.8 min (25-45% solvent B, 20 min, C18-column). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
15% | General procedure: iRGDK peptide (Fmoc-Ahx-c(CRGDRGPDC)-Ahx-K-NH2, iRGDK) was synthesized manually on Fmoc-Rink amide core-shell type resin (0.2 g, 0.30 mmol/g) [35] using Fmoc/tBu solid-phase procedure. For coupling reaction, the resin was treated with pre-activated amino acid solution, which was prepared with Fmoc-amino acid (0.18 mmol, 3 equiv.), HBTU (0.18 mmol, 3 equiv.), HOBt (0.18 mmol, 3 equiv.) and DIPEA (0.36 mmol, 6 equiv.) in NMP (4 mL). All amino acid couplings were performed for 1 h at 25 C. After the coupling reaction, the resin was washed with NMP (5 mL × 3), CH2Cl2 (5 mL × 3), and MeOH (5 mL × 3). The Fmoc group was removed using 20% piperidine/NMP (4 mL, 5 min + 10 min). Completion of each coupling step was monitored by the Kaiser test.After the last Fmoc deprotection, the peptide was cleaved from the resin by 4 mL of TFA:thioanisole:1,2-ethanedithiol:anisole (90:5:3:2) solution for 1 h and was recovered by ether precipitation. The crude peptide was treated with the methanolic 2,2-dipyridyl disulfide solution (10 mL, 1 mg/mL) for 1 h. Disulfide formation was monitored by HPLC. The final peptide was obtained by deprotection of t-butyl on the C-terminal cysteine with TFA:TFMSA:thioanisole:EDT (80:8:8:4, v/v). HPLC conditions for analysis and purification of the peptide were the same as used for iRGDK (1). After HPLC purification, the lyophilized peptide was obtained by freeze drying of HPLC fraction containing the product (12 mg). The yield was calculated based on the initial loading level of resins (yield: 15%). iRGDC (2) was analyzed by HRMS (calculated exact mass = 1304.5662 for C50H86N19O16S3 (iRGDC) [M + H]+ and [M + 2H]2 +, found 1304.5789 and 652.7883). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
25% | General procedure: iRGDK peptide (Fmoc-Ahx-c(CRGDRGPDC)-Ahx-K-NH2, iRGDK) was synthesized manually on Fmoc-Rink amide core-shell type resin (0.2 g, 0.30 mmol/g) [35] using Fmoc/tBu solid-phase procedure. For coupling reaction, the resin was treated with pre-activated amino acid solution, which was prepared with Fmoc-amino acid (0.18 mmol, 3 equiv.), HBTU (0.18 mmol, 3 equiv.), HOBt (0.18 mmol, 3 equiv.) and DIPEA (0.36 mmol, 6 equiv.) in NMP (4 mL). All amino acid couplings were performed for 1 h at 25 C. After the coupling reaction, the resin was washed with NMP (5 mL × 3), CH2Cl2 (5 mL × 3), and MeOH (5 mL × 3). The Fmoc group was removed using 20% piperidine/NMP (4 mL, 5 min + 10 min). Completion of each coupling step was monitored by the Kaiser test. After the last amino acid coupling, on-bead cyclization (S-S) was performed by treating the resin containing linear peptide with the S-Acm protecting groups with Tl(tfa)3 (0.12 mmol, 2 equiv.) in NMP (4 mL) for 2 h. The final peptide was cleaved with 4 mL of TFA:thioanisole:TIS:H2O (85:5:5:5, v/v) solution for 1 h and was recovered by ether precipitation as a crude white solid powder. For the peptide analysis, a flow rate of 1.0 mL/min and a 20 min-gradient of 10-80% of solvent B followed by a 5 min-constant flow of 100% solvent B (solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in acetonitrile) was used with XBridge BEH C18 Column (10 mum, 150 mm × 4.6 mm). For the peptide purification, a flow rate of 4.0 mL/min and a 20 min-gradient of 10-80% of solvent B followed by a 5 min-constant flow of 100% solvent B (solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in acetonitrile) was used with XBridge BEH130 Prep C18 (10 mum, 250 mm × 10 mm). Absorbance was measured at 230 nm. The lyophilized peptide was obtained by freeze drying of HPLC fraction containing the product (23 mg). The yield was calculated based on the initial loading level of resins (yield: 25%). iRGDK (1) was analyzed by MALDI-TOF (calculated exact mass = 1551.7201 for C68H103N20O18S2 (iRGDK) [M + H]+, found 1551.2295). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
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%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
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%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
14% | A glycopeptide solid phase was synthesized using a solid phase support in the form of TentaGel S RAM resin (0.24 mmol/g, 100 mg, 24 mumol). The amino acid extension reaction was conducted under conditions of microwave irradiation (40 W, 2,450 MHz, 50 C.) by reacting Fmoc amino acid derivative (96 mumol), HBTU (96 mumol), HOBt (96 mumol) and DIEA (144 mumol) in a DMF solution for six minutes. The sugar chain substitution amino acid extension reaction was conducted by reacting Fmoc-Thr(Ac3GalNacalpha)-OH:N-alpha-Fmoc-O-(2-acetamide-2-deoxy-3,4,6-tri-O-acetyl-alpha-D-galactopyranosyl)-L-threonine (29 mumol), HBTU (29 mumol), and HOBt (29 mumol) and DIEA (72 mumol) in a DMF solution for 10 minutes with microwave irradiation. HBTU (29 mumol) and HOBt (29 mumol) were added and the mixture was reacted for 10 minutes with microwave irradiation. The mixture was treated for 1 minute at room temperature with an acetic anhydride/DIEA/DMF (10:5:85, v/v/v) solution to acetylate the unreacted amino groups. Next, with microwave irradiation (40 W, 2,450 MHz, 50 C.), a 20% piperidine/DMF treatment was conducted for 3 minutes to remove the Fmoc group protection. In glycopeptide synthesis, the three steps of (1) extension with various Fmoc amino acids, (2) acetylation treatment, and (3) Fmoc removal were repeatedly sequentially conducted. The solid phase resin obtained was treated for 2 hours with trifluoroacetic acid:water (95:5, v/v). The reaction solution was filtered, ether was added to induce precipitation, and coarse crystals were obtained. The coarse product was dissolved in methanol, 1 N sodium hydroxide aqueous solution was added to adjust the solution to pH 12.0 to 12.5, and processing was conducted for 1 hour at room temperature. To this was added 10% acetic acid to adjust the solution to the vicinity of pH 7, after which the solvent was distilled off. The residue obtained was purified by reverse-phase high-performance liquid chromatography, yielding Compound 2 in the form of a freeze-dried powder (6.4 mg, yield 14%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
21% | A glycopeptide solid phase was synthesized using a solid phase support in the form of TentaGel S RAM resin (0.24 mmol/g, 100 mg, 24 mumol). The amino acid extension reaction was conducted under conditions of microwave irradiation (40 W, 2,450 MHz, 50 C.) by reacting Fmoc amino acid derivative (96 mumol), HBTU (96 mumol), HOBt (96 mumol) and DIEA (144 mumol) in a DMF solution for six minutes. The sugar chain substitution amino acid extension reaction was conducted by reacting Fmoc-Thr(Ac3GalNacalpha)-OH (29 mumol), HBTU (29 mumol), and HOBt (29 mumol) and DIEA (72 mumol) in a DMF solution for 10 minutes with microwave irradiation. HBTU (29 mumol) and HOBt (29 mumol) were added and the mixture was reacted for 10 minutes with microwave irradiation. The mixture was treated for 1 minute at room temperature with an acetic anhydride/DIEA/DMF (10:5:85, v/v/v) solution to acetylate the unreacted amino groups. Next, with microwave irradiation (40 W, 2,450 MHz, 50 C.), a 20% piperidine/DMF treatment was conducted for 3 minutes to remove the Fmoc group protection. In glycopeptide synthesis, the three steps of (1) extension with various Fmoc amino acids, (2) acetylation treatment, and (3) Fmoc removal were repeatedly sequentially conducted. The solid phase resin obtained was treated for 2 hours with trifluoroacetic acid:water (95:5, v/v). The reaction solution was filtered, ether was added to induce precipitation, and coarse crystals were obtained. The coarse product was dissolved in methanol, 1 N sodium hydroxide aqueous solution was added to adjust the solution to pH 12.0 to 12.5, and processing was conducted for 1 hour at room temperature. To this was added 10% acetic acid to adjust the solution to the vicinity of pH 7, after which the solvent was distilled off. The residue obtained was purified by reverse-phase high-performance liquid chromatography, yielding Compound 4 in the form of a freeze-dried powder (10.0 mg, yield 21%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
31% | A glycopeptide solid phase was synthesized using a solid phase support in the form of TentaGel S RAM resin (0.24 mmol/g, 100 mg, 24 mumol). The amino acid extension reaction was conducted under conditions of microwave irradiation (40 W, 2,450 MHz, 50 C.) by reacting Fmoc amino acid derivative (96 mumol), HBTU (96 mumol), HOBt (96 mumol) and DIEA (144 mumol) in a DMF solution for six minutes. The sugar chain substitution amino acid extension reaction was conducted by reacting Fmoc-Thr(Ac3GalNacalpha)-OH (29 mumol), HBTU (29 mumol), and HOBt (29 mumol) and DIEA (72 mumol) in a DMF solution for 10 minutes with microwave irradiation. HBTU (29 mumol) and HOBt (29 mumol) were added and the mixture was reacted for 10 minutes with microwave irradiation. The mixture was treated for 1 minute at room temperature with an acetic anhydride/DIEA/DMF (10:5:85, v/v/v) solution to acetylate the unreacted amino groups. Next, with microwave irradiation (40 W, 2,450 MHz, 50 C.), a 20% piperidine/DMF treatment was conducted for 3 minutes to remove the Fmoc group protection. In glycopeptide synthesis, the three steps of (1) extension with various Fmoc amino acids, (2) acetylation treatment, and (3) Fmoc removal were repeatedly sequentially conducted. The solid phase resin obtained was treated for 2 hours with trifluoroacetic acid:water (95:5, v/v). The reaction solution was filtered, ether was added to induce precipitation, and coarse crystals were obtained. The coarse product was dissolved in methanol, 1 N sodium hydroxide aqueous solution was added to adjust the solution to pH 12.0 to 12.5, and processing was conducted for 1 hour at room temperature. To this was added 10% acetic acid to adjust the solution to the vicinity of pH 7, after which the solvent was distilled off. The residue obtained was purified by reverse-phase high-performance liquid chromatography, yielding Compound 4 in the form of a freeze-dried powder (14.4 mg, yield 31%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
14% | A peptide solid phase was synthesized using a solid phase support in the form of TentaGel S RAM resin (0.24 mmol/g, 200 mg, 48 mumol, obtained from Rapp Polymere, GmbH). The amino acid extension reaction was conducted under conditions of microwave irradiation (40 W, 2,450 MHz, 50 C.) by reacting Fmoc amino acid derivative (192 mumol), HBTU (192 mumol), HOBt (192 mumol) and DIEA (288 mumol) in a DMF solution for six minutes. The sugar chain substitution amino acid extension reaction was conducted by reacting Fmoc-Thr(Ac3GalNacalpha)-OH (58 mumol), HBTU (58 mumol), and HOBt (58 mumol) and DIEA (144 mumol) in a DMF solution for 10 minutes with microwave irradiation. HBTU (58 mumol) and HOBt (58 mumol) were added and the mixture was reacted for 10 minutes with microwave irradiation. The mixture was treated for 1 minute at room temperature with an acetic anhydride/DIEA/DMF (10:5:85, v/v/v) solution to acetylate the unreacted amino groups. Next, with microwave irradiation (40 W, 2,450 MHz, 50 C.), a 20% piperidine/DMF treatment was conducted for 3 minutes to remove the Fmoc group protection. In glycopeptide synthesis, the three steps of (1) extension with various Fmoc amino acids, (2) acetylation treatment, and (3) Fmoc removal were repeatedly sequentially conducted. The solid phase resin obtained was treated for 2 hours with trifluoroacetic acid:water (95:5, v/v). The reaction solution was filtered, ether was added to induce precipitation, and coarse crystals were obtained. The coarse product was dissolved in methanol, 1 N sodium hydroxide aqueous solution was added to adjust the solution to pH 12.0 to 12.5, and processing was conducted for 1 hour at room temperature. To this was added 10% acetic acid to adjust the solution to the vicinity of pH 7, after which the solvent was distilled off. The residue obtained was purified by reverse-phase high-performance liquid chromatography, yielding Compound 3 in the form of a freeze-dried powder (13.0 mg, yield 14%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
11% | A peptide solid phase was synthesized using a solid phase support in the form of TentaGel S RAM resin (0.24 mmol/g, 200 mg, 48 mumol, obtained from Rapp Polymere, GmbH). The amino acid extension reaction was conducted under conditions of microwave irradiation (40 W, 2,450 MHz, 50 C.) by reacting Fmoc amino acid derivative (192 mumol), HBTU (192 mumol), HOBt (192 mumol) and DIEA (288 mumol) in a DMF solution for six minutes. The mixture was treated for 1 minute at room temperature with an acetic anhydride/DIEA/DMF (10:5:85, v/v/v) solution to acetylate the unreacted amino groups. Next, with microwave irradiation (40 W, 2,450 MHz, 50 C.), a 20% piperidine/DMF treatment was conducted for 3 minutes to remove the Fmoc group protection. In glycopeptide synthesis, the three steps of (1) extension with various Fmoc amino acids, (2) acetylation treatment, and (3) Fmoc removal were repeatedly sequentially conducted. The solid phase resin obtained was treated for 2 hours with trifluoroacetic acid:water (95:5, v/v). The reaction solution was filtered, ether was added to induce precipitation, and coarse crystals were obtained. The coarse product was purified by reverse-phase high-performance liquid chromatography, yielding Compound 1 in the form of a freeze-dried powder (9.0 mg, yield 11%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
15% | A glycopeptide solid phase was synthesized using a solid phase support in the form of TentaGel S RAM resin (0.24 mmol/g, 100 mg, 36 mumol). The amino acid extension reaction was conducted under conditions of microwave irradiation (40 W, 2,450 MHz, 50 C.) by reacting Fmoc amino acid derivative (144 mumol), HBTU (144 mumol), HOBt (144 mumol) and DIEA (216 mumol) in a DMF solution for six minutes. The sugar chain substitution amino acid extension reaction was conducted by reacting Fmoc-Thr(Ac3GalNacalpha)-OH (43 mumol), HBTU (43 mumol), and HOBt (43 mumol) and DIEA (108 mumol) in a DMF solution for 10 minutes with microwave irradiation. HBTU (43 mumol) and HOBt (43 mumol) were added and the mixture was reacted for 10 minutes with microwave irradiation. The mixture was treated for 1 minute at room temperature with an acetic anhydride/DIEA/DMF (10:5:85, v/v/v) solution to acetylate the unreacted amino groups. Next, with microwave irradiation (40 W, 2,450 MHz, 50 C.), a 20% piperidine/DMF treatment was conducted for 3 minutes to remove the Fmoc group protection. In glycopeptide synthesis, the three steps of (1) extension with various Fmoc amino acids, (2) acetylation treatment, and (3) Fmoc removal were repeatedly sequentially conducted. The solid phase resin obtained was treated for 2 hours with trifluoroacetic acid:water (95:5, v/v). The reaction solution was filtered, ether was added to induce precipitation, and coarse crystals were obtained. The coarse product was dissolved in methanol, 1 N sodium hydroxide aqueous solution was added to adjust the solution to pH 12.0 to 12.5, and processing was conducted for 1 hour at room temperature. To this was added 10% acetic acid to adjust the solution to the vicinity of pH 7, after which the solvent was distilled off. The residue obtained was purified by reverse-phase high-performance liquid chromatography, yielding Compound 2 in the form of a freeze-dried powder (13.3 mg, yield 15%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
17% | A glycopeptide solid phase was synthesized using a solid phase support in the form of TentaGel S RAM resin (0.24 mmol/g, 200 mg, 48 mumol). The amino acid extension reaction was conducted under conditions of microwave irradiation (40 W, 2,450 MHz, 50 C.) by reacting Fmoc amino acid derivative (192 mumol), HBTU (192 mumol), HOBt (192 mumol) and DIEA (288 mumol) in a DMF solution for six minutes. The sugar chain substitution amino acid extension reaction was conducted by reacting N-alpha-Fmoc-O-[2?,3?,4?,6?-tetra-O-acetyl-D-galactopyranosyl-beta(1?3)-2-acetamide-2-deoxy-4,6-di-O-acetyl-alpha-D-galactopyranosyl]-L-threonine (58 mumol), HBTU (58 mumol), and HOBt (58 mumol) and DIEA (144 mumol) in a DMF solution for 10 minutes with microwave irradiation. HBTU (58 mumol) and HOBt (58 mumol) were added and the mixture was reacted for 10 minutes with microwave irradiation. The mixture was treated for 1 minute at room temperature with an acetic anhydride/DIEA/DMF (10:5:85, v/v/v) solution to acetylate the unreacted amino groups. Next, with microwave irradiation (40 W, 2,450 MHz, 50 C.), a 20% piperidine/DMF treatment was conducted for 3 minutes to remove the Fmoc group protection. In glycopeptide synthesis, the three steps of (1) extension with various Fmoc amino acids, (2) acetylation treatment, and (3) Fmoc removal were repeatedly sequentially conducted. The solid phase resin obtained was treated for 2 hours with trifluoroacetic acid:water (95:5, v/v). The reaction solution was filtered, ether was added to induce precipitation, and coarse crystals were obtained. The coarse product was dissolved in methanol, 1 N sodium hydroxide aqueous solution was added to adjust the solution to pH 12.0 to 12.5, and processing was conducted for 1 hour at room temperature. To this was added 10% acetic acid to adjust the solution to the vicinity of pH 7, after which the solvent was distilled off. The residue obtained was purified by reverse-phase high-performance liquid chromatography, yielding Compound 2 in the form of a freeze-dried powder (22.0 mg, yield 17%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
22% | A peptide solid phase was synthesized using a solid phase support in the form of TentaGel S RAM resin (0.24 mmol/g, 50 mg, 12 mumol). The amino acid extension reaction was conducted under conditions of microwave irradiation (40 W, 2,450 MHz, 50 C.) by reacting Fmoc amino acid derivative (48 mumol), HBTU (48 mumol), HOBt (48 mumol) and DIEA (72 mumol) in a DMF solution for six minutes. The mixture was treated for 1 minute at room temperature with an acetic anhydride/DIEA/DMF (10:5:85, v/v/v) solution to acetylate the unreacted amino groups. Next, with microwave irradiation (40 W, 2,450 MHz, 50 C.), a 20% piperidine/DMF treatment was conducted for 3 minutes to remove the Fmoc group protection. In glycopeptide synthesis, the three steps of (1) extension with various Fmoc amino acids, (2) acetylation treatment, and (3) Fmoc removal were repeatedly sequentially conducted. The solid phase resin obtained was treated for 2 hours with trifluoroacetic acid:water (95:5, v/v). The reaction solution was filtered, ether was added to induce precipitation, and coarse crystals were obtained. The coarse product was purified by reverse-phase high-performance liquid chromatography, yielding Compound 1 in the form of a freeze-dried powder (6.3 mg, yield 22%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
15% | A glycopeptide solid phase was synthesized using a solid phase support in the form of Rink Amide-ChemMatrix resin (0.48 mmol/g, 50 mg, 24 mumol). The amino acid extension reaction was conducted under conditions of microwave irradiation (40 W, 2,450 MHz, 50 C.) by reacting Fmoc amino acid derivative (96 mumol), HBTU (96 mumol), in a DMF solution of HOBt (96 mumol) and DIEA (144 mumol) for 9 minutes. The sugar chain substitution amino acid extension reaction was conducted by reacting Fmoc-Thr(Ac3GalNacalpha)-OH] (28 mumol), PyBOP (28 mumol), and HOBt (28 mumol) and DIEA (72 mumol) in a DMF solution for 10 minutes with microwave irradiation. PyBOP (28 mumol) and HOBt (28 mumol) were added and the mixture was reacted for 10 minutes with microwave irradiation. The mixture was treated for 1 minute at room temperature with an acetic anhydride/DIEA/DMF (10:5:85, v/v/v) solution to acetylate the unreacted amino groups. Next, with microwave irradiation (40 W, 2,450 MHz, 50 C.), a 20% piperidine/DMF treatment was conducted for 3 minutes to remove the Fmoc group protection. In glycopeptide synthesis, the three steps of (1) extension with various Fmoc amino acids, (2) acetylation treatment, and (3) Fmoc removal were repeatedly sequentially conducted. The solid phase resin obtained was treated for 1 hours with trifluoroacetic acid:water (95:5, v/v). The reaction solution was filtered, ether was added to induce precipitation, and coarse crystals were obtained. The coarse product was dissolved in methanol, 1 N sodium hydroxide aqueous solution was added to adjust the solution to pH 12.0 to 12.5, and processing was conducted for 1 hour at room temperature. To this was added 10% acetic acid to adjust the solution to the vicinity of pH 7, after which the solvent was distilled off. The residue obtained was purified by reverse-phase high-performance liquid chromatography, yielding Compound 12 in the form of a freeze-dried powder (9.0 mg, yield 15%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Macrocycles were chemically synthesized using a Syro Wave automated peptide synthesizer (Biotage, Charlotte, NC) by Fmoc solid-phase peptide synthesis as previously described (Morimoto et al., Angew Chem. Int. Ed. Engl.51:3423-3427, 2012; Yamagata et al., Structure 22:345-352, 2012). Briefly, the chloroacetyl group or acetyl group was coupled onto the N-terminal amide group for the formation of cyclic or linear peptide analogs respectively after the automated synthesis. Peptides were cleaved by a solution of 92.5%trifluoroacetic acid (TFA), 2.5% water, 2.5% triisopropylsilane, and 2.5% ethanedithiol and precipitated by diethyl ether. To conduct the cyclization reaction, peptide pellet was dissolved in 10 mL DMSO/0.1%TFA in water (1:1), adjusted the pH>8 by addition of triethylamine and incubated for 1 h at 25C. This cyclization reaction was quenched by addition of TFA to acidify the peptide suspensions. Then peptides were purified by reverse-phase HPLC (RP-HPLC) and molecular masses were verified by MALDI-TOF mass spectrometry, using a microflex or ultraflex instrument (Bruker Daltonics, Billerica, MA) (FIG.5 and Table 2).All peptides were chemically synthesized on a 25 mumole scale using a Syro Wave automated peptide synthesizer (Biotage) by Fmoc solid phase peptide chemical synthesis (SPPS). Firstly, ^ ^ ^ ^ NovaPEG Rink Amide resins were incubated with N,N-dimethylformamide (DMF) with rotation at ambient temperature for 30 min and washed 5 times with DMF. Coupling of each Fmoc-protected amino acid was performed on the engorged resin with a solution of 300 muL 0.5 M Fmoc-protected amino acid, 300 muL 0.5 M 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt), and 150 muL 0.5 M N,N-diisopropylethylamine (DIPEA) in DMF and reacted for 1 hour at ambient temperature. After washing the resins with 1 mL DMF five times, Fmoc-deprotection was performed by incubating the resin with 600 muL 40% piperidine in DMF (vol/vol) and reacted for 30 min at ambient temperature. Each peptide was synthesized using the appropriately protected amino acid monomers corresponding to sequences in Tables 6 and 8 by repeating the Fmoc-protected amino acid coupling and Fmoc-deprotection steps accordingly. The N- terminal alpha-amino group of the synthesized peptides on the resin was chloroacetylated by incubating with a solution of 500 muL 0.5 M chloroacetyl N-hydroxysuccinimide (NHS) ester in N- methylpyrrolidone (NMP) with rotation for 60 min at ambient temperature. For the synthesis of Ce-L2 and Ce-L2d, the N-terminal alpha-amino group was acetylated by incubating with a solution of 500 muL 0.5 M acetic anhydride and 0.25 M DIPEA in NMP with rotation for 60 min at ambient temperature. After washing the resin with 5 x 1 mL DMF, peptides were fully deprotected and cleaved from resin by incubating with a solution of 2 mL trifluoroacetic acid (TFA), water, triisopropylsilane (TIS) and ethanedithiol (EDT) (92.5:2.5:2.5:2.5) with rotation for 3 hours at ambient temperature andprecipitated with diethyl ether. The peptide pellet was dissolved in 10 mL DMSO/0.1%TFA in water (1:1), and the pH adjusted to >8 by addition of triethylamine (TEA), and incubated at ambient temperature for 1 h to enhance the cyclization via a thioether bond formation between N-terminal chloroacetamide group and cysteine sulfhydryl group. Peptide mass and cyclization was confirmed by MALDI-TOF MS analysis. The cyclization reaction was quenched by addition of TFA to acidify the peptide suspensions. Peptides were then purified by reverse-phase HPLC (Table 4), molecular masses were verified by MALDI-TOF MS analysis (Table 4), using a microflex or autoflex instrument (Bruker Daltonics). Ring junction confirmed by MSMS spectrum and fragment analysis (FIG.5). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
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. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
The synthesis of A chain (InsA(G)) was conducted on 0.1 mmol Rink amide ChemMatrix resin (0.54 mmol/g) using a Biotage automated microwave peptide synthesizer. The C-terminal amino acid Asn was linked to the resin as Fmoc-Asp-OtBu through its side chain carboxyl group. Isoacyl-dipeptide Boc-Ser[Fmoc-Thr(tBu)]-OH was used as a single residue and coupled by a standard protocol as others. The peptide bound resin was treated with fresh 25% b-mercaptoethanol (15 mL) for 1.5 h twice and thoroughly washed with DMF (15 mL x 3) and DCM (15 mL x 3). Then DTNP (310 mg, 10 eq.) in 10 mL DCM was added to the resin and shaken for 1 h. The resin was washed again with DMF (15 mL x 3) and DCM (15 mL x 3) and treated with 1% TFA, 5% TIS in DCM (10 mL) for 2 min 5 times. Finally the resin was shaken in DCM (10 mL) for 1 h before it was cleaved by TFA/TIS/H20 (9 mL/500 pL/500 pL) for 2 h. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: SPPS was performed on a Prelude Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.) at 250-pmol or 400-pmol scale using six fold excess of Fmoc-amino acids (300 mM in NMP with 300 mM OXYMA PURE) relative to resin loading (typical loading of hydrazine-resin was 0.3 mmol/g). Fmoc-deprotection was performed using 20% piperidine in NMP. Coupling was performed using 3 : 3 : 3 : 4 amino acid/OXYMA PURE/DIC/collidine in NMP. NMP and DCM top washes (7 ml, 0.5 min, 2 x 2 each) were performed between deprotection and coupling steps. Coupling times were generally 60 minutes.Some amino acids including, but not limited to Fmoc-Arg(Pbf)-OH, Fmoc-Aib-OH, Fmoc-Cys(Trt)-OH or Boc-His(Trt)-OH were "double coupled", meaning that after the first coupling (e.g. 60 min), the resin was drained and more reagents were added (amino acid, OXYMA PURE, DIC, and collidine), and the mixture allowed to react again (e.g. 60 min). Cleavage of resin bound peptide hvdrazides and purificationAfter synthesis the resin was washed with DCM, and the peptide was cleaved from the resin by a 3 hour treatment with TFA/TIS/2-mercaptoethanol/water (87.5/5/5/2.5) followed by precipitation with diethylether. The peptide was dissolved in a suitable solvent (such as e.g., 10/90 acetic acid/water) and purified by standard RP-HPLC on a C18, 5 mM column, usingacetonitrile/water/TFA. The fractions were analysed by a combination of UPLC, MALDI-MS and LCMS methods, and the appropriate fractions were pooled.Cyclization of peptide hydrazides, disulfide oxidation and purificationMethod : NCL_M 1The pooled fractions from RP-FIPLC purification of the peptide hydrazides were diluted with water to 80: 20 water/acetonitrile. Disodium phosphate was added to a final concentration of 0.2 M and the pH was adjusted to 3.0 with concentrated hydrochloric acid (aq). The mixture was cooled to 0C and sodium nitrite (10 eq, 0.2 M in water) was added, and the mixture was stirred for 20 minutes at 0C. Sodium 2-mercaptoethanesulfonate (20 eq) was added and the pH was adjusted to 7.0 with 1 M NaOH (aq). The reaction mixture was stirred at 25C for 16 hours and then purified by standard RP-FIPLC on a C18, 5mM column, usingacetonitrile/water/TFA. The fractions were analysed by a combination of UPLC, MALDI-MS and LCMS methods, and the appropriate fractions were pooled and lyophilized. |
Tags: 135248-89-4 synthesis path| 135248-89-4 SDS| 135248-89-4 COA| 135248-89-4 purity| 135248-89-4 application| 135248-89-4 NMR| 135248-89-4 COA| 135248-89-4 structure
[ 135273-01-7 ]
(2R,2'R)-3,3'-Disulfanediylbis(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid)
Similarity: 0.91
[ 136050-67-4 ]
(2R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-[(4-methylphenyl)methylsulfanyl]propanoic acid
Similarity: 0.90
Precautionary Statements-General | |
Code | Phrase |
P101 | If medical advice is needed,have product container or label at hand. |
P102 | Keep out of reach of children. |
P103 | Read label before use |
Prevention | |
Code | Phrase |
P201 | Obtain special instructions before use. |
P202 | Do not handle until all safety precautions have been read and understood. |
P210 | Keep away from heat/sparks/open flames/hot surfaces. - No smoking. |
P211 | Do not spray on an open flame or other ignition source. |
P220 | Keep/Store away from clothing/combustible materials. |
P221 | Take any precaution to avoid mixing with combustibles |
P222 | Do not allow contact with air. |
P223 | Keep away from any possible contact with water, because of violent reaction and possible flash fire. |
P230 | Keep wetted |
P231 | Handle under inert gas. |
P232 | Protect from moisture. |
P233 | Keep container tightly closed. |
P234 | Keep only in original container. |
P235 | Keep cool |
P240 | Ground/bond container and receiving equipment. |
P241 | Use explosion-proof electrical/ventilating/lighting/equipment. |
P242 | Use only non-sparking tools. |
P243 | Take precautionary measures against static discharge. |
P244 | Keep reduction valves free from grease and oil. |
P250 | Do not subject to grinding/shock/friction. |
P251 | Pressurized container: Do not pierce or burn, even after use. |
P260 | Do not breathe dust/fume/gas/mist/vapours/spray. |
P261 | Avoid breathing dust/fume/gas/mist/vapours/spray. |
P262 | Do not get in eyes, on skin, or on clothing. |
P263 | Avoid contact during pregnancy/while nursing. |
P264 | Wash hands thoroughly after handling. |
P265 | Wash skin thouroughly after handling. |
P270 | Do not eat, drink or smoke when using this product. |
P271 | Use only outdoors or in a well-ventilated area. |
P272 | Contaminated work clothing should not be allowed out of the workplace. |
P273 | Avoid release to the environment. |
P280 | Wear protective gloves/protective clothing/eye protection/face protection. |
P281 | Use personal protective equipment as required. |
P282 | Wear cold insulating gloves/face shield/eye protection. |
P283 | Wear fire/flame resistant/retardant clothing. |
P284 | Wear respiratory protection. |
P285 | In case of inadequate ventilation wear respiratory protection. |
P231 + P232 | Handle under inert gas. Protect from moisture. |
P235 + P410 | Keep cool. Protect from sunlight. |
Response | |
Code | Phrase |
P301 | IF SWALLOWED: |
P304 | IF INHALED: |
P305 | IF IN EYES: |
P306 | IF ON CLOTHING: |
P307 | IF exposed: |
P308 | IF exposed or concerned: |
P309 | IF exposed or if you feel unwell: |
P310 | Immediately call a POISON CENTER or doctor/physician. |
P311 | Call a POISON CENTER or doctor/physician. |
P312 | Call a POISON CENTER or doctor/physician if you feel unwell. |
P313 | Get medical advice/attention. |
P314 | Get medical advice/attention if you feel unwell. |
P315 | Get immediate medical advice/attention. |
P320 | |
P302 + P352 | IF ON SKIN: wash with plenty of soap and water. |
P321 | |
P322 | |
P330 | Rinse mouth. |
P331 | Do NOT induce vomiting. |
P332 | IF SKIN irritation occurs: |
P333 | If skin irritation or rash occurs: |
P334 | Immerse in cool water/wrap n wet bandages. |
P335 | Brush off loose particles from skin. |
P336 | Thaw frosted parts with lukewarm water. Do not rub affected area. |
P337 | If eye irritation persists: |
P338 | Remove contact lenses, if present and easy to do. Continue rinsing. |
P340 | Remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P341 | If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P342 | If experiencing respiratory symptoms: |
P350 | Gently wash with plenty of soap and water. |
P351 | Rinse cautiously with water for several minutes. |
P352 | Wash with plenty of soap and water. |
P353 | Rinse skin with water/shower. |
P360 | Rinse immediately contaminated clothing and skin with plenty of water before removing clothes. |
P361 | Remove/Take off immediately all contaminated clothing. |
P362 | Take off contaminated clothing and wash before reuse. |
P363 | Wash contaminated clothing before reuse. |
P370 | In case of fire: |
P371 | In case of major fire and large quantities: |
P372 | Explosion risk in case of fire. |
P373 | DO NOT fight fire when fire reaches explosives. |
P374 | Fight fire with normal precautions from a reasonable distance. |
P376 | Stop leak if safe to do so. Oxidising gases (section 2.4) 1 |
P377 | Leaking gas fire: Do not extinguish, unless leak can be stopped safely. |
P378 | |
P380 | Evacuate area. |
P381 | Eliminate all ignition sources if safe to do so. |
P390 | Absorb spillage to prevent material damage. |
P391 | Collect spillage. Hazardous to the aquatic environment |
P301 + P310 | IF SWALLOWED: Immediately call a POISON CENTER or doctor/physician. |
P301 + P312 | IF SWALLOWED: call a POISON CENTER or doctor/physician IF you feel unwell. |
P301 + P330 + P331 | IF SWALLOWED: Rinse mouth. Do NOT induce vomiting. |
P302 + P334 | IF ON SKIN: Immerse in cool water/wrap in wet bandages. |
P302 + P350 | IF ON SKIN: Gently wash with plenty of soap and water. |
P303 + P361 + P353 | IF ON SKIN (or hair): Remove/Take off Immediately all contaminated clothing. Rinse SKIN with water/shower. |
P304 + P312 | IF INHALED: Call a POISON CENTER or doctor/physician if you feel unwell. |
P304 + P340 | IF INHALED: Remove victim to fresh air and Keep at rest in a position comfortable for breathing. |
P304 + P341 | IF INHALED: If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P305 + P351 + P338 | IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. |
P306 + P360 | IF ON CLOTHING: Rinse Immediately contaminated CLOTHING and SKIN with plenty of water before removing clothes. |
P307 + P311 | IF exposed: call a POISON CENTER or doctor/physician. |
P308 + P313 | IF exposed or concerned: Get medical advice/attention. |
P309 + P311 | IF exposed or if you feel unwell: call a POISON CENTER or doctor/physician. |
P332 + P313 | IF SKIN irritation occurs: Get medical advice/attention. |
P333 + P313 | IF SKIN irritation or rash occurs: Get medical advice/attention. |
P335 + P334 | Brush off loose particles from skin. Immerse in cool water/wrap in wet bandages. |
P337 + P313 | IF eye irritation persists: Get medical advice/attention. |
P342 + P311 | IF experiencing respiratory symptoms: call a POISON CENTER or doctor/physician. |
P370 + P376 | In case of fire: Stop leak if safe to Do so. |
P370 + P378 | In case of fire: |
P370 + P380 | In case of fire: Evacuate area. |
P370 + P380 + P375 | In case of fire: Evacuate area. Fight fire remotely due to the risk of explosion. |
P371 + P380 + P375 | In case of major fire and large quantities: Evacuate area. Fight fire remotely due to the risk of explosion. |
Storage | |
Code | Phrase |
P401 | |
P402 | Store in a dry place. |
P403 | Store in a well-ventilated place. |
P404 | Store in a closed container. |
P405 | Store locked up. |
P406 | Store in corrosive resistant/ container with a resistant inner liner. |
P407 | Maintain air gap between stacks/pallets. |
P410 | Protect from sunlight. |
P411 | |
P412 | Do not expose to temperatures exceeding 50 oC/ 122 oF. |
P413 | |
P420 | Store away from other materials. |
P422 | |
P402 + P404 | Store in a dry place. Store in a closed container. |
P403 + P233 | Store in a well-ventilated place. Keep container tightly closed. |
P403 + P235 | Store in a well-ventilated place. Keep cool. |
P410 + P403 | Protect from sunlight. Store in a well-ventilated place. |
P410 + P412 | Protect from sunlight. Do not expose to temperatures exceeding 50 oC/122oF. |
P411 + P235 | Keep cool. |
Disposal | |
Code | Phrase |
P501 | Dispose of contents/container to ... |
P502 | Refer to manufacturer/supplier for information on recovery/recycling |
Physical hazards | |
Code | Phrase |
H200 | Unstable explosive |
H201 | Explosive; mass explosion hazard |
H202 | Explosive; severe projection hazard |
H203 | Explosive; fire, blast or projection hazard |
H204 | Fire or projection hazard |
H205 | May mass explode in fire |
H220 | Extremely flammable gas |
H221 | Flammable gas |
H222 | Extremely flammable aerosol |
H223 | Flammable aerosol |
H224 | Extremely flammable liquid and vapour |
H225 | Highly flammable liquid and vapour |
H226 | Flammable liquid and vapour |
H227 | Combustible liquid |
H228 | Flammable solid |
H229 | Pressurized container: may burst if heated |
H230 | May react explosively even in the absence of air |
H231 | May react explosively even in the absence of air at elevated pressure and/or temperature |
H240 | Heating may cause an explosion |
H241 | Heating may cause a fire or explosion |
H242 | Heating may cause a fire |
H250 | Catches fire spontaneously if exposed to air |
H251 | Self-heating; may catch fire |
H252 | Self-heating in large quantities; may catch fire |
H260 | In contact with water releases flammable gases which may ignite spontaneously |
H261 | In contact with water releases flammable gas |
H270 | May cause or intensify fire; oxidizer |
H271 | May cause fire or explosion; strong oxidizer |
H272 | May intensify fire; oxidizer |
H280 | Contains gas under pressure; may explode if heated |
H281 | Contains refrigerated gas; may cause cryogenic burns or injury |
H290 | May be corrosive to metals |
Health hazards | |
Code | Phrase |
H300 | Fatal if swallowed |
H301 | Toxic if swallowed |
H302 | Harmful if swallowed |
H303 | May be harmful if swallowed |
H304 | May be fatal if swallowed and enters airways |
H305 | May be harmful if swallowed and enters airways |
H310 | Fatal in contact with skin |
H311 | Toxic in contact with skin |
H312 | Harmful in contact with skin |
H313 | May be harmful in contact with skin |
H314 | Causes severe skin burns and eye damage |
H315 | Causes skin irritation |
H316 | Causes mild skin irritation |
H317 | May cause an allergic skin reaction |
H318 | Causes serious eye damage |
H319 | Causes serious eye irritation |
H320 | Causes eye irritation |
H330 | Fatal if inhaled |
H331 | Toxic if inhaled |
H332 | Harmful if inhaled |
H333 | May be harmful if inhaled |
H334 | May cause allergy or asthma symptoms or breathing difficulties if inhaled |
H335 | May cause respiratory irritation |
H336 | May cause drowsiness or dizziness |
H340 | May cause genetic defects |
H341 | Suspected of causing genetic defects |
H350 | May cause cancer |
H351 | Suspected of causing cancer |
H360 | May damage fertility or the unborn child |
H361 | Suspected of damaging fertility or the unborn child |
H361d | Suspected of damaging the unborn child |
H362 | May cause harm to breast-fed children |
H370 | Causes damage to organs |
H371 | May cause damage to organs |
H372 | Causes damage to organs through prolonged or repeated exposure |
H373 | May cause damage to organs through prolonged or repeated exposure |
Environmental hazards | |
Code | Phrase |
H400 | Very toxic to aquatic life |
H401 | Toxic to aquatic life |
H402 | Harmful to aquatic life |
H410 | Very toxic to aquatic life with long-lasting effects |
H411 | Toxic to aquatic life with long-lasting effects |
H412 | Harmful to aquatic life with long-lasting effects |
H413 | May cause long-lasting harmful effects to aquatic life |
H420 | Harms public health and the environment by destroying ozone in the upper atmosphere |
Sorry,this product has been discontinued.
Home
* Country/Region
* Quantity Required :
* Cat. No.:
* CAS No :
* Product Name :
* Additional Information :