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CAS No. : | 71989-38-3 | MDL No. : | MFCD00037129 |
Formula : | C28H29NO5 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | JAUKCFULLJFBFN-VWLOTQADSA-N |
M.W : | 459.53 | Pubchem ID : | 10895791 |
Synonyms : |
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Signal Word: | Warning | Class: | N/A |
Precautionary Statements: | P261-P305+P351+P338 | UN#: | N/A |
Hazard Statements: | 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 |
---|---|---|
450 g | With sodium carbonate In tetrahydrofuran; water | 5. Add 500 g of H2O and about 60 g of THF (tetrahydrofuran) to the reaction flask,Then, 299 g of L-Tyr (tBu) (O-tert-butyl-L-tyrosine), 150 g of Na2CO3 (sodium carbonate)After stirring, adding 300 g of 9-fluorenylmethoxycarbonyl chloride, the control system pH = 9,Reaction TLC (TLC) detection confirmed that the L-Tyr (tBu) reaction was complete,To obtain Fmoc-Tyr (tBu) (N- (9-fluorenylmethoxycarbonyl) -O-tert-butyl-L-tyrosine). After completion of the reaction, the product was extracted with 300 g of AcOEt (ethyl acetate). The ester layers were pooled and washed several times with saturated brine. The pH was adjusted to 3 with citric acid and driAfter completion of the reaction, the product was extracted with 300 g of AcOEt (ethyl acetate). The ester layers were pooled and washed several times with saturated brine. The pH was adjusted to 3 with citric acid and dried with 50 g Na2SO4. Filtered and desalted, and the filtrate was concentrated in hot water to a solution of 1/4. Cooling, adding 200g PET (petroleum ether) stirring crystallization filtration, drying the product 450g, the total yield: 80.1percent; product appearance: white powder;Product purity: 99percent (HPLC area normalized); Melting point mp: 150 ° C; Moisture (K, F): 1percent; Specific rotation: -18.9; Single impurity HPLC = 0.3 (areapercent); Optical purity: 99.98 percent. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 4.1.1. Peptide synthesis; 4.1.2; Solid-phase peptide synthesis (SPPS) was performed with standardFmoc chemistry on rink amide resin using an automated peptidesynthesizer (Syro I, Multisyntech). The resin was loaded into a5 mL reactor with a frit at the bottom. Swelling was performed bydispensing 1 mL DMF and incubating for 15 min (2) with 10 sshaking every minute. Fmoc deprotection was achieved by treatmentwith 40percent piperidine DMF for 3 min and 20percent piperidine inDMF for 12 min (10 s/min shaking). Peptide couplings were carriedout by double couplings with Fmoc-protected amino acids(5 equiv), HBTU (5 equiv), HOBt (5 equiv) and DIPEA (10 equiv) inDMF for 40 min (10 s/min shaking). At the respective position,Fmoc-F2Pmp-OH (3 equiv) was coupled in DMF (1 mL) by manualaddition using TBTU (3 equiv), HOBt (3 equiv) and DIPEA (6 equiv)for 3 h, after 3 min preactivation. In case of the sequences for which side-chain labeling with biotinor carboxyfluorescein was planned, an additional 4-methyltrityl-(Mtt-) protected lysine was coupled to the N-terminus. Toselectively remove the Mtt group the resin was washed for 1 minwith DCM (3), deprotection was then achieved by treatment with1.8percent TFA in DCM for 3 min (10). During the deprotection the DCMsolution turned yellow.For fluorescein-labeling of the amine side-chain 5(6)-carboxyfluorescein(3 equiv), HATU (3 equiv), HOAt (3 equiv) andDIPEA (6 equiv) were dissolved in DMF and pre-activated for3 min. The solution was aspirated and coupling was allowed toproceed for 1 h. This step was repeated 4 times.For biotin-labeling of the amine side-chain the resin waswashed for 1 min in NMP (3). D-(+)-Biotin (3 equiv), HATU(3 equiv), HOAt (3 equiv) and DIPEA (6 equiv) were dissolved inNMP and pre-activated for 3 min. The solution was aspirated andcoupling was allowed to proceed for 2 h. This step was repeated2 times. N-terminal acetylation (where applicable) was achieved by dispensing800 lL of a mixture of acetic anhydride/pyridine (1:9) andreaction twice for 5 min (10 s/min shaking). After each deprotection,coupling or acetylation step, 5 washings (1 min each) withDMF were performed (10 s/min shaking).After synthesis the resin was transferred in a 5 mL syringeequipped with a frit, washed with DCM for 1 min (3) and driedin high vacuum for at least 30 min. For cleavage 1 mL of a mixtureof TFA and TIS (20:1) was added. The syringe with the mixture waskept on a shaker for 3 h. Then the liquid phase was filtered into20 mL of ice-cold Et2O. Formed precipitate was centrifuged,washed with ice-cold Et2O (2 20 mL) and purified by HPLC. 4.1.2. Azide functionalization of the N-terminus; To the peptides with the longer carbon linker, 6-azidohexanoicacid was coupled (with standard coupling conditions) to the Nterminalamine.The N-terminal amine of the peptides with the shorter linkerwas converted to an azide functionality directly on solid support.Using the compound imidazole-1-sulfonyl-azide*HCl (synthesissee beneath) and modified conditions, which were reported forsolution phase chemistry from Goddard?Borger and Stick:8 Theresin was washed for 1 min each with DCM (2), DCM/MeOH(2) and MeOH (3). Then (for 40 mg resin, loading= 0.62 mmole/g) 1.4 equiv of imidazole-1-sulfonyl-azide*HClin 1 mL MeOH and 100 ll of a saturated and centrifuged solutionof CuSO4*5H2O was added. After 1 min, DIPEA (1.8 equiv) wasadded and the coupling was allowed to proceed for 1 h andrepeated once more with an intermediate washing with MeOH(3 1 min). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 4.1.1. Peptide synthesis; 4.1.2; Solid-phase peptide synthesis (SPPS) was performed with standardFmoc chemistry on rink amide resin using an automated peptidesynthesizer (Syro I, Multisyntech). The resin was loaded into a5 mL reactor with a frit at the bottom. Swelling was performed bydispensing 1 mL DMF and incubating for 15 min (2) with 10 sshaking every minute. Fmoc deprotection was achieved by treatmentwith 40percent piperidine DMF for 3 min and 20percent piperidine inDMF for 12 min (10 s/min shaking). Peptide couplings were carriedout by double couplings with Fmoc-protected amino acids(5 equiv), HBTU (5 equiv), HOBt (5 equiv) and DIPEA (10 equiv) inDMF for 40 min (10 s/min shaking). At the respective position,Fmoc-F2Pmp-OH (3 equiv) was coupled in DMF (1 mL) by manualaddition using TBTU (3 equiv), HOBt (3 equiv) and DIPEA (6 equiv)for 3 h, after 3 min preactivation. In case of the sequences for which side-chain labeling with biotinor carboxyfluorescein was planned, an additional 4-methyltrityl-(Mtt-) protected lysine was coupled to the N-terminus. Toselectively remove the Mtt group the resin was washed for 1 minwith DCM (3), deprotection was then achieved by treatment with1.8percent TFA in DCM for 3 min (10). During the deprotection the DCMsolution turned yellow.For fluorescein-labeling of the amine side-chain 5(6)-carboxyfluorescein(3 equiv), HATU (3 equiv), HOAt (3 equiv) andDIPEA (6 equiv) were dissolved in DMF and pre-activated for3 min. The solution was aspirated and coupling was allowed toproceed for 1 h. This step was repeated 4 times.For biotin-labeling of the amine side-chain the resin waswashed for 1 min in NMP (3). D-(+)-Biotin (3 equiv), HATU(3 equiv), HOAt (3 equiv) and DIPEA (6 equiv) were dissolved inNMP and pre-activated for 3 min. The solution was aspirated andcoupling was allowed to proceed for 2 h. This step was repeated2 times. N-terminal acetylation (where applicable) was achieved by dispensing800 lL of a mixture of acetic anhydride/pyridine (1:9) andreaction twice for 5 min (10 s/min shaking). After each deprotection,coupling or acetylation step, 5 washings (1 min each) withDMF were performed (10 s/min shaking).After synthesis the resin was transferred in a 5 mL syringeequipped with a frit, washed with DCM for 1 min (3) and driedin high vacuum for at least 30 min. For cleavage 1 mL of a mixtureof TFA and TIS (20:1) was added. The syringe with the mixture waskept on a shaker for 3 h. Then the liquid phase was filtered into20 mL of ice-cold Et2O. Formed precipitate was centrifuged,washed with ice-cold Et2O (2 20 mL) and purified by HPLC. 4.1.2. Azide functionalization of the N-terminus; To the peptides with the longer carbon linker, 6-azidohexanoicacid was coupled (with standard coupling conditions) to the Nterminalamine.The N-terminal amine of the peptides with the shorter linkerwas converted to an azide functionality directly on solid support.Using the compound imidazole-1-sulfonyl-azide*HCl (synthesissee beneath) and modified conditions, which were reported forsolution phase chemistry from Goddard?Borger and Stick:8 Theresin was washed for 1 min each with DCM (2), DCM/MeOH(2) and MeOH (3). Then (for 40 mg resin, loading= 0.62 mmole/g) 1.4 equiv of imidazole-1-sulfonyl-azide*HClin 1 mL MeOH and 100 ll of a saturated and centrifuged solutionof CuSO4*5H2O was added. After 1 min, DIPEA (1.8 equiv) wasadded and the coupling was allowed to proceed for 1 h andrepeated once more with an intermediate washing with MeOH(3 1 min). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Example 1The following amino acids were used: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc- Dap(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH and mono-tBu succinate. MS (M+H+): expected 971.1; observed 971.2Peptide Synthesis:The peptide was synthesized using CEM Microwave technology with coupling times of 5 minutes per amino acid at elevated temperature (78 °C) and a 0.25mmol scale. The synthesis is carried out using the TentalGel-S RAM resin as a solid support (0.24 meq /g). All amino acids used were dissolved in DMF to 0.2 mol concentration. A mixture of HOBT/HBTU 1: 1 (0.5 mol /L) 4 eq. and DIPEA 4eq. was used to activate the amino acids. Fmoc-Cleavage was achieved with Piperidine in DMF (20 percent) for 3 min. Fmoc-cleavage was repeated.General Synthesis Description:The cyclic peptides can be generated either via on-bead cyclisation (Allyl/Aloc strategy METHODE A) or as fully deprotected linear peptides via solution phase cyclisation (METHODE B).Linear peptides were either synthesized manually or using microwave technology via state-of- the-art solid phase synthesis protocols (Fmoc-chemistry) as referenced by e.g.: Kates and Albericio, Eds., "Solid Phase Synthesis: A practical guide", Marcel Decker, New York, Basel, 2000. As a solid support TentaGel-S-RAM resin (0.24 meq /g) was used. All Fmoc-amino acids were added in a 4-fold excess after activation with HOBT/HBTU 1 : 1 (0.5 mol/L in DMF) and 4 eq of DIPEA (2 mol/L in NMP). Fmoc-cleavage was achieved with 20percent Piperidine in DMF.Allyl/Aloc- Cleavage & Lactam- Cyclisation:(METHODE A: The resin-linked peptides were treated manually with a solution of 20 eq phenylsilane in DCM and 0.05 eq of tetrakis triphenylphosphine palladium for 30 min at RT. This procedure was repeated. The resin was washed with a solution of 0.5percent sodium dithiocarbamate in DMF. For the on-bead lactam formation, again activation reagent was added to the resin and shaken for additional 8h at RT. Completion of cyclisation was verified via Ninhydrin-test.METHODE B: Peptides were cyclized in solution after deprotection and cleavage from the resin and standard work-up. Crude peptides were treated with standard peptide activation regents in DMF. The cyclisation was monitored via HPLC.Cleavage & work-up:A cleavage-cocktail of trifluoroacetic acid, triisopropylsilane and water (95/2.5/2.5) was added to the resin and shaken for lh at RT. Cleaved peptides were precipitated in cold Ether (-18°C ). The peptides were centrifuged and the residue washed twice with cold ether. The residues were again dissolved in water/ acetonitrile and lyophilized.Purification :Peptides were purified using reversed phase high performance liquid chromatography (RP- HPLC) using a Reprospher 100 C18-T Columm (100 x 4.6 mm, 5u particle size) as a stationary phase and water/acetonitrile as eluent (0053) (Gradient 1-50 percent MeCN over 30 min). Fractions were collected and analyzed by LC/MS. Pure product samples were combined and lyophilized. All peptides were obtained as white powders with a purity >85 percent. Product identification was obtained via mass spectrometry. All standard amino acids were purchased from CEM. Fmoc-SAR-OH and Fmoc-N-Cyclopropyl- Glycine were purchased from Bachem and Enamine respectively. Mono-tBu-Succinate were purchased from Sigma- Aldrich |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Single-Coupling Procedure To the reaction vessel containing resin from the previous step was added piperidine:DMF (20:80 v/v, 2.0mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit.To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.0 mL). The mixture was periodically agitatedfor 3 minutes and then the solution was drained through the frit. The resin washed successively six times asfollows: for each wash, DMF (2.0 mL) was added to top of the vessel (not through the bottom frit) and theresulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. Tothe reaction vessel was added the amino acid (0.2M in DMF, 1.0 mL, 2 eq), then HATU (0.2M in DMF, 1.0mL, 2 eq), and finally DIPEA (0.4M in DMF, 1.0 mL, 4 eq). The mixture was periodically agitated for 15minutes, then the reaction solution was drained through the frit. The resin washed successively four times asfollows: for each wash, DMF (2.0 mL) was added to top of the vessel (not through the bottom frit) and theresulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. Tothe reaction vessel was added acetic anhydride (2.0 mL). The mixture was periodically agitated for 10minutes, then the solution was drained through the frit. The resin washed successively four times as follows:for each wash, DMF (2.0 mL) was added to top of the vessel (not through the bottom frit) and the resultingmixture was periodically agitated for 90 seconds before the solution was drained through the frit. Theresulting resin was used directly in the next step. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: General procedure for LYRAXaa-NHNH2: 250mg CTC-NHNH2 resin (loading capacity is 0.25 mmol) was added into the reaction flask of peptide synthesizer. The scale of amino acid is 1 mmol. In each coupling reaction, 2.5 mL of 0.4 M HATU, 2.5 mL of 0.8 DIPEA and 2.5 mL DMF were added and the mixture was kept shaking for 70 minutes. The resin was then treated with 20% piperidine in DMF and the removed Fmoc was monitored by a UV detector until completion. Then, the resin was washed with DMF and subject to next synthetic cycle with another amino acid and repeated above procedures. After completion of peptide synthesis, we finished deprotection and cleavage. When the first amino acid is Asp, Gln and Asp(Bn), the peptide resins were cleaved and deprotected with 95% TFA in water for 1 hour. When the first amino acid is Asn, complete cleavage with 1% TFA in DCM for1 hour then carry out deprotection with 95% TFA in water for 1 hour. Then, filtered and removed most of TFA by N2 gas blowing, then ethyl ether was added to precipitate the crude peptide. The crude peptide was subject to the HPLC analysis and purification by semi-preparativeHPLC. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The material was synthesized on a Protein Technologies Symphony X ® with amino acids dissolved in DMF at 0.5M. Fmoc-Cys(Trt)-OH was dissolved into a solution containing 0.5 M oxyma pure in DMF. HCTU was dissolved in NMP to 0.5 M, and a 1.0 M DIPEA in NMP was also used. DIC was dissolved in NMP to 0.5 M. Fmoc-deprotection was performed using a solution containing 20percent piperidine in DMF. [000159] To couple the peptide to the resin, Rink amide resin (0.2 mmol, 0.24 mmol/g) was treated with 20percent piperidine in DMF (3 x 6 mL, 3 min, 1 x 6 mL, 10 min). The resulting resin was washed with DMF (6 x 10 mL, 30 s). A solution containing Fmoc-Xaa-OH, HCTU, and DIPEA in NMP that has been pre-mixed for 30 seconds was added and the mixture was agitated for 30 minutes. The resin was filtered and washed once with NMP. A second solution containing Fmoc-Xaa-OH, HCTU, and DIPEA in NMP that has been pre-mixed for 30 seconds was added and the mixture was agitated again for 30 minutes. In the case of Fmoc-Cys(Trt)-OH, the amino acid/oxyma pure solution was mixed with DIC in NMP for 5 minutes, added to the resin, and agitated for 45 min. The resin was filtered and washed once with DMF. The resulting resin was filtered and washed with DMF (6 x 10 mL). The material was subjected to the aforementioned protocol and the peptide was elongated until macrocyclization. [000160] To couple the diamino acid, the resin was treated with 20percent piperidine in DMF (3 x 6 mL, 3 min, 1 x 6 mL, 10 min). The resulting resin was washed with DMF (6 x 10 mL, 30 s). A solution containing alloc-HCys((Fmoc-Ala-OH)-3-yl)-all (227 mg, 0.4 mmol), PyAOP (209 mg 0.4 mmol) and DIPEA (139mu1, 125 mg, 0.8mmol) in 5 mL of NMP was added to the resin. After 90 min, the resin was filtered and washed with DMF (6 x 10 mL). The peptide was then elongated using the aforementioned protocol. [000161] To perform Allyl-Alloc deprotection, the resin (0.2 mmol) was suspended in 10 mL of DMF and a solution containing Pd(PPh3)4 (300 mg, 0.26 mmol) in 10 mL of CH2C12 was added followed by 0.25 mL (2 mmol) of phenyl silane. The resulting mixture was shaken in the absence of light for 2 hours. A small sample was cleaved to ensure complete deprotection. The resulting resin was filtered and washed with CH2C12 (3 x 10 mL) and DMF (3x 10 mL). The resin was treated with a solution containing 0.5percent sodium diethyldithiocarbamate in DMF (10 mL, 4 x 15 min), and washed with DMF (3x 10 mL). [000162] To perform macrocyclization, the resin was treated with a 20percent piperidine in DMF solution (2 x 5 min, 1 x 10 min, 15 mL) and washed with DMF (6 x 15 mL). A solution containing 521 mg (1 mmol) of PyAOP in 15 mL of DMF was added and after 1 minute, 0.35 mL (2 mmol) of DIPEA was added and shaken for 60 minutes. A small sample was taken for analysis. The resulting resin was washed with DMF (3 x 15 mL) and placed back onto the SymphonyX to complete the synthesis. [000163] To cleave the peptide from the resin, the resin was treated with a solution containing 90:5:3:2 TFA-TIPS-DODT-H20 (20 mL). After 2 hours, the resin was filtered and washed with TFA (3 mL) and concentrated by 50percent. Cold (-78 °C) ether was added to the solution (50 mL) and the resulting mixture was centrifuged 3500 rpm for 10 minutes. The ether was decanted and the solid was subject to 2 additional washes and centrifuged with cold (-78 °C) ether. The resulting solid was dried under diminished pressure, dissolved into 1 : 1 H20-ACN, frozen and lyophilized. The peptides were then purified on a Waters autopure system using 0.1percent TFA in water and 0.1percent TFA in acetonitrile on a Waters PST CI 8 RP column (250 x 30 mm, 10 mu, 130 A) at a flow rate of 40 mL/min. A linear gradient was used 5-45percent acetonitrile over 40 or 60 minutes. Fractions containing the desired product were pooled and oxidized. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Peptide Synthesis: (0036) The peptide was synthesized using CEM Microwave technology with coupling times of 5 minutes per amino acid at elevated temperature (78° C.) and a 0.25 mmol scale. The synthesis is carried out using the TentalGel-S RAM resin as a solid support (0.24 meq/g). All amino acids used were dissolved in NMP to 0.2 mol concentration. A solution of 4 eq. COMU in DMF (0.5 mol/L) and DIPEA was used to activate the amino acids. Fmoc-Cleavage was achieved with Piperidine in DMF (20percent) for 3 min. Fmoc-cleavage was repeated. Cleavage from Resin: (0037) 10 ml of a cleavage-cocktail consisting of 95/2.5/2.5 Trifluoroacetic acid, Triisopropylsilane, and water was added to the resin and shaken for 3 h at RT. Cleaved peptide was precipitated in cold Et2O (?18° C.). The peptide was centrifuged 2×50 ml polypropylene tubes. The precipitates were washed two times with cold ether. Afterwards the precipitate was dissolved in H2O/Acetonitrile and lyophilizied to yield 88 mg white powder. Cyclization: (0038) Crude peptide was dissolved in DMF (15 ml). 1 eq of coupling reagents PyoAP (0.5 mol/L) in DMF and DIPEA in NMP (2 mol/1) were added. The reaction mixture was stirred at RT for 1 h. After the reaction was completed (LCMS control) the DMF content was concentrated down to approximately 2 ml. The residue was precipitated in cold (?18° C.) diethyl ether (40 ml). The peptide was centrifuged and the precipitate washed with cold ether. Purification: (0039) The crude peptide was purified by preparative HPLC on a Reprospher 100 C18-T Column (100×4.6 mm, 5 um particle size). As eluent system a mixture of 0.1percent TFA/water/acetonitrile was used with a gradient of 0-100percent acetonitrile within 0-75 min. The fractions were collected and checked by analytical HPLC. Fractions containing pure product were combined and lyophilized. 27 mg of white powder were obtained. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
20 mg | General procedure: The general procedure described in Example 2 was followed for the coupling of HOOC- PEG3-COOH and tripeptide (5) to resin bound-protected CCK8 peptide, 1, except 2 equivalents of <strong>[31127-85-2]HOOC-PEG3-COOH</strong> and tripeptide (5) were used instead of Fmoc-AEEP-OH, Fmoc- Cys(Trt)-OH, Fmoc-Asp(OiBu)-OH, Boc-Dap(Fmoc)-OH and Ac20. Resin cleavage and purification were performed as described in Example 2 to yield desired peptide EC1981 (6) (20 mg, 3%). [M+H]+ = Calculated 1641.6, found 1643. Fmoc-Sieber-resin (l.Og, 0.69mmol) was placed in a peptide synthesis vessel, and washed with DMF (3 x 10 mL). Initial Fmoc deprotection was performed using 20% piperidine in DMF (3 x 10 mL) solution for 10 mins per cycle. The resin was further washed with DMF (3 x 10 mL) and z'-PrOH (3 x 10 mL), and a Kaiser test was conducted to determine that the reaction was complete. The resin was washed again with DMF wash (3 x 10 mL), and a solution of Fmoc-Phe-OH (0.57 g, 1.38 mmol, 2.0 eq.) in DMF, PyBOP (0.72 g, 1.38 mmol, 2.0 eq.) and DIPEA (0.37 mL, 2.07 mmol, 3.0 eq.) were added to the vessel. The resulting solution was bubbled with Argon for 1 hour. The coupling solution was filtered, the resin was washed with DMF (3 x 10 mL) and z'-PrOH (3 x 10 mL), and a Kaiser test was conducted to determine that the reaction was complete. The process was repeated for each additional coupling according to the reagent amounts listed in Table 1. Resin bound-protected CCK8 peptide, 1, (0.8g, 0.28mmol) was placed in a peptide synthesis vessel, and was subjected to solid phase synthesis as described in Example 1 for the coupling of Fmoc-AEEP-OH (Fmoc-9-amino-4,7-dioxanonanoic acid), Fmoc-Cys(Trt)-OH, Fmoc-Asp(OiBu)-OH, Boc-Dap(Fmoc)-OH (Na-Boc-^-Fmoc-L-2,3-diaminopropionic acid) and Ac20 using to the reagent amounts shown in Table 2. Resin cleavage was performed with a cocktail of 94% CF3C02H, 2.5% EDT, 2.0% triisopropylsilane and 1.5% H20. The cleavage cocktail (10 mL) was poured onto the resin and bubbled with Argon for 30 mins, followed by filtration into a clean flask. Further cleavage was performed two times with fresh cleavage cocktail and 10 mins of Argon bubbling. The combined filtrate was poured onto cold diethyl ether, and the precipitate that formed was collected by centrifugation at 4000 rpm for 5 mins (3x). The precipitate was obtained following decanting and drying of the solid under vacuum; the product was then purified by preparative HPLC (mobile phase A = lOmM Ammonium acetate, pH = 5; Organic phase B = Acetonitrile; Method; 10% B to 100%B in 30 mins) to yield EC1825 (2) (30 mg, 7%). 1H NMR (500 MHz DMSO- 6) Pivotal signals: delta 7.49 (d, J = 7.9 Hz, 1H), 7.31 (d, / = 8.1 Hz, 1H), 7.23 - 7.14 (m, 5H), 7.13 (s, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.96 - 6.89 (m, 2H), 6.60 (d, J = 8.4 Hz, 2H), 4.50 (dt, J = 9.1, 6.6 Hz, 2H), 4.42 - 4.33 (m, 2H), 4.28 (td, J = 9.8, 8.8, 4.7 Hz, 2H), 4.23 (dd, = 8.7, 5.2 Hz, 2H), 4.17 (dd, = 9.0, 5.0 Hz, 1H), 1.95 (s, 3H), 1.95 (s, 3H), 1.82 (s, 3H). [M+H]+ = Calculated 1567.6, found 1569.3 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
21% | General procedure: Solid-phase peptide synthesis was carried out on Fmoc-cappedpolystyrene rink amide MBHA resin (100-200 mesh, 0.05-0.15 mmol scale). The following amino acidderivatives suitable for Fmoc SPPS were used: Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH,Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-aPhe-OH, Fmoc-aVal-OH,Fmoc-aTyr(tBu)-OH, Fmoc-(N-Me)-Phe-OH, Fmoc-D-Ser(TBS)-OH, Fmoc-D-hSer(TBS)-OH, Boc-Gly-OH. Dry resin was washed with DMF 3x and allowed to swell in DMF for 2 h prior to use. Allreactions were carried out using gentle agitation. Fmoc deprotection steps were carried out by treating theresin with a solution of 20percent piperidine/DMF (15 min x 2). Coupling of Fmoc-protected amino acids aswell as (N2-Boc)-hydrazino acids was effected using 5 equiv. HATU (0.5 M in DMF), 10 equiv. DIEA(1.0 M in DMF), and 5 equiv. of the carboxylic acid in DMF at 50 oC (1 h). Coupling of residues Nterminalto the hydrazino acids was carried out with 30 equiv. collidine and 10 equiv. of pre-formed Fmocamino acid chlorides (or 10 equiv. of Fmoc amino acids with 3.3 equiv. triphosgene) in THF at rt (1 h x2).3 After each reaction the resin was washed with DMF 2x, DCM 1x, then DMF 1x. Peptides undergoingMitsunobu reactions were capped with Boc-Gly-OH, washed with DCM 3x, and treated with 5 equiv.TBAF in THF for 3 h at rt. After the reaction the resin was washed with DCM 3x and then treated with 5equiv. triphenylphosphine in THF followed by 5 equiv. of DIAD, then strirred overnight at rt. Peptideswere cleaved from the resin by incubating with gentle stirring in 2 mL of 95:5 TFA:H2O at rt for 2 h. Thecleavage mixture was filtered and the resin was rinsed with an additional 1 mL of cleavage solution. Thefiltrate was treated with 8 mL of cold Et2O to induce precipitation. The mixture was centrifuged and thesupernatant was removed. The remaining solid was washed 2 more times with Et2O and dried undervacuum. Cysteine-containing peptides were purified, lyophilized, dissolved in 10mM phosphate buffer(pH 8.9, 5percent v/v DMSO), stirred until analytical HPLC and MS showed complete conversion to the cyclicdisulfide (1-2 d), and then repurified. Peptides were analyzed and purified on C12 RP-HPLC columns(preparative: 4mu, 90A, 250 x 21.2 mm; analytical: 4mu, 90A, 150 x 4.6 mm) using linear gradients ofMeCN/H2O (with 0.1percent formic acid), then lyophilized to afford white powders. All peptides werecharacterized by LCMS (ESI), HRMS (ESI-TOF), and 1H NMR. Analytical HPLC samples for all purifiedpeptides were prepared as 1 mM in H2O containing 20 mM phosphate buffer at pH 7.0. Linear gradientsof MeCN in H2O (0.1percent formic acid) were run over 20 minutes and spectra are provided for lambda = 220 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The Wang resin (0.3 -0.6 mmol/g, loading capacity) was loaded to peptide synthesis vessel, washed twice with 10 v of MDC, decanted the washings, added 10 v of MDC and kept for swelling for 1 h. Fmoc-Gly-OH (3.0 - 5.0 eq.) was dissolved in MDC, added minimum quantity of DMF to obtain clear solution and the mixture was transferred to reaction vessel. Added DIPC (3.0 - 6.0 eq.) followed by DMAP (0.01- 0.1 eq.) to the reaction vessel and stirred for 1.0? 3.0 h, at rt. Drained the reaction mass and washed the amino acid loaded resin twice with MDC followed by DMF. Capping of the unreacted functional sites were carried out using acetic anhydride and DIPEA. Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 15-25 percent piperidine in DMF two times for 5 and 10 min. followed by the resin was washed with 3-5*8 v 0.01? 0.1 M HOBt in DMF. The Fmoc-Arg(Pbf)-OH (2.0? 4.0 eq.), was coupled using coupling agents such as HBTU, COMU, DEPBT, and DIC, preferably DEPBT (2.0 - 4.0 eq.) and oxymapure, HOBt, preferably oxymapure (2.0? 4.0 eq.) and DIPEA, NMM, TMP, preferably DIPEA (5.0 -8.0 eq.) and MgCl2, ZnCl2, preferably MgCl2 (0.01? 0.1 eq) and DMF/NMP mixture as solvent. The reaction was performed in nitrogen atmosphere and r.t. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin with 3 x 10 v DMF |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: The Wang resin (0.3 -0.6 mmol/g, loading capacity) was loaded to peptide synthesis vessel, washed twice with 10 v of MDC, decanted the washings, added 10 v of MDC and kept for swelling for 1 h. Fmoc-Gly-OH (3.0 - 5.0 eq.) was dissolved in MDC, added minimum quantity of DMF to obtain clear solution and the mixture was transferred to reaction vessel. Added DIPC (3.0 - 6.0 eq.) followed by DMAP (0.01- 0.1 eq.) to the reaction vessel and stirred for 1.0? 3.0 h, at rt. Drained the reaction mass and washed the amino acid loaded resin twice with MDC followed by DMF. Capping of the unreacted functional sites were carried out using acetic anhydride and DIPEA. Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 15-25 percent piperidine in DMF two times for 5 and 10 min. followed by the resin was washed with 3-5*8 v 0.01? 0.1 M HOBt in DMF. The Fmoc-Arg(Pbf)-OH (2.0? 4.0 eq.), was coupled using coupling agents such as HBTU, COMU, DEPBT, and DIC, preferably DEPBT (2.0 - 4.0 eq.) and oxymapure, HOBt, preferably oxymapure (2.0? 4.0 eq.) and DIPEA, NMM, TMP, preferably DIPEA (5.0 -8.0 eq.) and MgCl2, ZnCl2, preferably MgCl2 (0.01? 0.1 eq) and DMF/NMP mixture as solvent. The reaction was performed in nitrogen atmosphere and r.t. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin with 3 x 10 v DMF |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Peptide synthesis was based on Fmoc chemistry, using a Symphony peptide synthesiser manufactured by Peptide Instruments and a Syro II synthesiser by MultiSynTech. Standard Fmoc- amino acids were employed (Sigma, Merck), with the following side chain protecting groups: Arg(Pbf); Asn(Trt); Asp(OtBu); Cys(Trt); GIu(OtBu); Gln(Trt); His(Trt); Lys(Boc); Ser(tBu); Thr(tBu); Trp(Boc); and Tyr(tBu) (Sigma). The coupling reagent was HCTU (Pepceuticals), diisopropylethylamine (DIPEA, Sigma) was employed as a base, and deprotection was achieved with 20percent piperidine in DMF (AGTC). Syntheses were performed using 0.37 mmol/gr Fmoc-Rink amide AM resin (AGTC), Fmoc-amino acids were utilised at a four-fold excess, and base was at a four-fold excess with respect to the amino acids. Amino acids were dissolved at 0.2M in DMSO, HCTU at 0.4M in DMF, and DIPEA at 1.6M in N-methylpyrrolidone (Alfa Aesar). Conditions were such that coupling reactions contained between 20 to 50percent DMSO in DMF, which reduced aggregation and deletions during the solid phase synthesis and enhanced yields. Coupling times were generally 30 minutes, and deprotection times 2 x 5 minutes. Fmoc-N-methylglycine (Fmoc- Sar-OH, Merck) was coupled for 1 hr, and deprotection and coupling times for the following residue were 20 min and 1 hr, respectively. After synthesis, the resin was washed with dichloromethane, and dried. Cleavage of side-chain protecting groups and from the support was effected using 10 mL of 95:2.5:2.5:2.5 v/v/v/w TFA/H20/iPr3SiH/dithiothreitol for 3 hours. Following cleavage, the spent resin was removed by filtration, and the filtrate was added to 35 mL of diethylether that had been cooled at -80°C. Peptide pellet was centrifuged, the etheric supernatant discarded, and the peptide pellet washed with cold ether two more times. Peptides were then resolubilised in 5-10 mL acetonitrile-water and lyophilised. A small sample was removed for analysis of purity of the crude product by mass spectrometry (MALDI-TOF, Voyager DE from Applied Biosystems). Following lyophilisation, peptide powders were taken up in 10 mL 6 M guanidinium hydrochloride in H20, supplemented with 0.5 mL of 1 M dithiothreitol, and loaded onto a C8 Luna preparative HPLC column (Phenomenex). Solvents (H20, acetonitrile) were acidified with 0.1 percent heptafluorobutyric acid. The gradient ranged from 30-70 percent acetonitrile in 15 minutes, at a flowrate of 15-20 mL /min, using a Gilson preparative HPLC system. Fractions containing pure linear peptide material (as identified by MALDI) were used for preparation of the bicycle derivatives by coupling to a scaffold molecule as described further below.A bicycle peptide designated 17-69-07-N434 was made corresponding to the bicycle peptide of Example lwith an N-terminal SarlO spacer similar to that of Reference Example 1, and conjugating group PYA (4-pentynoic acid, for "click" derivatisation with toxin). The structure of this derivative is shown schematically in Fig. 5. The linear peptide used to form this bicycle was as follows:(PYA)-(B-Ala)-SarlO-A(Dap)(D-Ala)NE(lNal)(D-Ala)CEDFYD(tBuGly)(Dap)The linear peptide and the bicycle peptide had the following LCMS Characteristics: |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Peptide synthesis was based on Fmoc chemistry, using a Symphony peptide synthesiser manufactured by Peptide Instruments and a Syro II synthesiser by MultiSynTech. Standard Fmoc- amino acids were employed (Sigma, Merck), with the following side chain protecting groups: Arg(Pbf); Asn(Trt); Asp(OtBu); Cys(Trt); GIu(OtBu); Gln(Trt); His(Trt); Lys(Boc); Ser(tBu); Thr(tBu); Trp(Boc); and Tyr(tBu) (Sigma). The coupling reagent was HCTU (Pepceuticals), diisopropylethylamine (DIPEA, Sigma) was employed as a base, and deprotection was achieved with 20percent piperidine in DMF (AGTC). Syntheses were performed using 0.37 mmol/gr Fmoc-Rink amide AM resin (AGTC), Fmoc-amino acids were utilised at a four-fold excess, and base was at a four-fold excess with respect to the amino acids. Amino acids were dissolved at 0.2M in DMSO, HCTU at 0.4M in DMF, and DIPEA at 1.6M in N-methylpyrrolidone (Alfa Aesar). Conditions were such that coupling reactions contained between 20 to 50percent DMSO in DMF, which reduced aggregation and deletions during the solid phase synthesis and enhanced yields. Coupling times were generally 30 minutes, and deprotection times 2 x 5 minutes. Fmoc-N-methylglycine (Fmoc- Sar-OH, Merck) was coupled for 1 hr, and deprotection and coupling times for the following residue were 20 min and 1 hr, respectively. After synthesis, the resin was washed with dichloromethane, and dried. Cleavage of side-chain protecting groups and from the support was effected using 10 mL of 95:2.5:2.5:2.5 v/v/v/w TFA/H20/iPr3SiH/dithiothreitol for 3 hours. Following cleavage, the spent resin was removed by filtration, and the filtrate was added to 35 mL of diethylether that had been cooled at -80°C. Peptide pellet was centrifuged, the etheric supernatant discarded, and the peptide pellet washed with cold ether two more times. Peptides were then resolubilised in 5-10 mL acetonitrile-water and lyophilised. A small sample was removed for analysis of purity of the crude product by mass spectrometry (MALDI-TOF, Voyager DE from Applied Biosystems). Following lyophilisation, peptide powders were taken up in 10 mL 6 M guanidinium hydrochloride in H20, supplemented with 0.5 mL of 1 M dithiothreitol, and loaded onto a C8 Luna preparative HPLC column (Phenomenex). Solvents (H20, acetonitrile) were acidified with 0.1 percent heptafluorobutyric acid. The gradient ranged from 30-70 percent acetonitrile in 15 minutes, at a flowrate of 15-20 mL /min, using a Gilson preparative HPLC system. Fractions containing pure linear peptide material (as identified by MALDI) were used for preparation of the bicycle derivatives by coupling to a scaffold molecule as described further below.A bicycle peptide designated 17-69-07-N385 was made corresponding to the bicycle region of the peptide ligand of Reference Example 1, minus the b-Ala -SarlO tail, and with replacement of the first and third cysteine residues by DAP residues forming alkylamino linkages to the TBMB scaffold. The structure of this derivative is shown schematically in Fig. 3.The linear peptide used to form this bicycle was as follows:Ac-A(Dap)(D-Ala)NE(lNal)(D-Ala)CEDFYD(tBuGly)(Dap)The linear peptide and the bicycle peptide had the following LCMS Characteristics: |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Peptide synthesis was based on Fmoc chemistry, using a Symphony peptide synthesiser manufactured by Peptide Instruments and a Syro II synthesiser by MultiSynTech. Standard Fmoc- amino acids were employed (Sigma, Merck), with the following side chain protecting groups: Arg(Pbf); Asn(Trt); Asp(OtBu); Cys(Trt); GIu(OtBu); Gln(Trt); His(Trt); Lys(Boc); Ser(tBu); Thr(tBu); Trp(Boc); and Tyr(tBu) (Sigma). The coupling reagent was HCTU (Pepceuticals), diisopropylethylamine (DIPEA, Sigma) was employed as a base, and deprotection was achieved with 20percent piperidine in DMF (AGTC). Syntheses were performed using 0.37 mmol/gr Fmoc-Rink amide AM resin (AGTC), Fmoc-amino acids were utilised at a four-fold excess, and base was at a four-fold excess with respect to the amino acids. Amino acids were dissolved at 0.2M in DMSO, HCTU at 0.4M in DMF, and DIPEA at 1.6M in N-methylpyrrolidone (Alfa Aesar). Conditions were such that coupling reactions contained between 20 to 50percent DMSO in DMF, which reduced aggregation and deletions during the solid phase synthesis and enhanced yields. Coupling times were generally 30 minutes, and deprotection times 2 x 5 minutes. Fmoc-N-methylglycine (Fmoc- Sar-OH, Merck) was coupled for 1 hr, and deprotection and coupling times for the following residue were 20 min and 1 hr, respectively. After synthesis, the resin was washed with dichloromethane, and dried. Cleavage of side-chain protecting groups and from the support was effected using 10 mL of 95:2.5:2.5:2.5 v/v/v/w TFA/H20/iPr3SiH/dithiothreitol for 3 hours. Following cleavage, the spent resin was removed by filtration, and the filtrate was added to 35 mL of diethylether that had been cooled at -80°C. Peptide pellet was centrifuged, the etheric supernatant discarded, and the peptide pellet washed with cold ether two more times. Peptides were then resolubilised in 5-10 mL acetonitrile-water and lyophilised. A small sample was removed for analysis of purity of the crude product by mass spectrometry (MALDI-TOF, Voyager DE from Applied Biosystems). Following lyophilisation, peptide powders were taken up in 10 mL 6 M guanidinium hydrochloride in H20, supplemented with 0.5 mL of 1 M dithiothreitol, and loaded onto a C8 Luna preparative HPLC column (Phenomenex). Solvents (H20, acetonitrile) were acidified with 0.1 percent heptafluorobutyric acid. The gradient ranged from 30-70 percent acetonitrile in 15 minutes, at a flowrate of 15-20 mL /min, using a Gilson preparative HPLC system. Fractions containing pure linear peptide material (as identified by MALDI) were used for preparation of the bicycle derivatives by coupling to a scaffold molecule as described further below.The Bicyclic Peptide chosen for comparison of thioether to alkylamino scaffold linkage was designated 17-69-07-N241. It is a bicycle conjugate of a thioether- forming peptide with a trimethylene benzene scaffold. The structure of this bicycle derivative is shown schematically in Fig. 2. The linear peptide before conjugation has sequence:H-( -Ala)-SarlO-Ala-Cys-(D-Ala)-Asn-Glu-(lNal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBuGly)- Cys-NH2 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Peptide synthesis was based on Fmoc chemistry, using a Symphony peptide synthesiser manufactured by Peptide Instruments and a Syro II synthesiser by MultiSynTech. Standard Fmoc- amino acids were employed (Sigma, Merck), with the following side chain protecting groups: Arg(Pbf); Asn(Trt); Asp(OtBu); Cys(Trt); GIu(OtBu); Gln(Trt); His(Trt); Lys(Boc); Ser(tBu); Thr(tBu); Trp(Boc); and Tyr(tBu) (Sigma). The coupling reagent was HCTU (Pepceuticals), diisopropylethylamine (DIPEA, Sigma) was employed as a base, and deprotection was achieved with 20percent piperidine in DMF (AGTC). Syntheses were performed using 0.37 mmol/gr Fmoc-Rink amide AM resin (AGTC), Fmoc-amino acids were utilised at a four-fold excess, and base was at a four-fold excess with respect to the amino acids. Amino acids were dissolved at 0.2M in DMSO, HCTU at 0.4M in DMF, and DIPEA at 1.6M in N-methylpyrrolidone (Alfa Aesar). Conditions were such that coupling reactions contained between 20 to 50percent DMSO in DMF, which reduced aggregation and deletions during the solid phase synthesis and enhanced yields. Coupling times were generally 30 minutes, and deprotection times 2 x 5 minutes. Fmoc-N-methylglycine (Fmoc- Sar-OH, Merck) was coupled for 1 hr, and deprotection and coupling times for the following residue were 20 min and 1 hr, respectively. After synthesis, the resin was washed with dichloromethane, and dried. Cleavage of side-chain protecting groups and from the support was effected using 10 mL of 95:2.5:2.5:2.5 v/v/v/w TFA/H20/iPr3SiH/dithiothreitol for 3 hours. Following cleavage, the spent resin was removed by filtration, and the filtrate was added to 35 mL of diethylether that had been cooled at -80°C. Peptide pellet was centrifuged, the etheric supernatant discarded, and the peptide pellet washed with cold ether two more times. Peptides were then resolubilised in 5-10 mL acetonitrile-water and lyophilised. A small sample was removed for analysis of purity of the crude product by mass spectrometry (MALDI-TOF, Voyager DE from Applied Biosystems). Following lyophilisation, peptide powders were taken up in 10 mL 6 M guanidinium hydrochloride in H20, supplemented with 0.5 mL of 1 M dithiothreitol, and loaded onto a C8 Luna preparative HPLC column (Phenomenex). Solvents (H20, acetonitrile) were acidified with 0.1 percent heptafluorobutyric acid. The gradient ranged from 30-70 percent acetonitrile in 15 minutes, at a flowrate of 15-20 mL /min, using a Gilson preparative HPLC system. Fractions containing pure linear peptide material (as identified by MALDI) were used for preparation of the bicycle derivatives by coupling to a scaffold molecule as described further below.A bicycle peptide designated 17-69-07-N426 was made corresponding to the bicycle peptide of Example lwith replacement of the DAP residues by N-MeDAP residues. The structure of this derivative is shown schematically in Fig. 4. The linear peptide used to form this bicycle was as follows:Ac-A(Dap(Me))(D-Ala)NE(lNal)(D-Ala)CEDFYD(tBuGly)(Dap(Me))The linear peptide and the bicycle peptide had the following LCMS Characteristics: |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Peptide synthesis was based on Fmoc chemistry, using a Symphony peptide synthesiser manufactured by Peptide Instruments and a Syro II synthesiser by MultiSynTech. Standard Fmoc- amino acids were employed (Sigma, Merck), with the following side chain protecting groups: Arg(Pbf); Asn(Trt); Asp(OtBu); Cys(Trt); GIu(OtBu); Gln(Trt); His(Trt); Lys(Boc); Ser(tBu); Thr(tBu); Trp(Boc); and Tyr(tBu) (Sigma). The coupling reagent was HCTU (Pepceuticals), diisopropylethylamine (DIPEA, Sigma) was employed as a base, and deprotection was achieved with 20percent piperidine in DMF (AGTC). Syntheses were performed using 0.37 mmol/gr Fmoc-Rink amide AM resin (AGTC), Fmoc-amino acids were utilised at a four-fold excess, and base was at a four-fold excess with respect to the amino acids. Amino acids were dissolved at 0.2M in DMSO, HCTU at 0.4M in DMF, and DIPEA at 1.6M in N-methylpyrrolidone (Alfa Aesar). Conditions were such that coupling reactions contained between 20 to 50percent DMSO in DMF, which reduced aggregation and deletions during the solid phase synthesis and enhanced yields. Coupling times were generally 30 minutes, and deprotection times 2 x 5 minutes. Fmoc-N-methylglycine (Fmoc- Sar-OH, Merck) was coupled for 1 hr, and deprotection and coupling times for the following residue were 20 min and 1 hr, respectively. After synthesis, the resin was washed with dichloromethane, and dried. Cleavage of side-chain protecting groups and from the support was effected using 10 mL of 95:2.5:2.5:2.5 v/v/v/w TFA/H20/iPr3SiH/dithiothreitol for 3 hours. Following cleavage, the spent resin was removed by filtration, and the filtrate was added to 35 mL of diethylether that had been cooled at -80°C. Peptide pellet was centrifuged, the etheric supernatant discarded, and the peptide pellet washed with cold ether two more times. Peptides were then resolubilised in 5-10 mL acetonitrile-water and lyophilised. A small sample was removed for analysis of purity of the crude product by mass spectrometry (MALDI-TOF, Voyager DE from Applied Biosystems). Following lyophilisation, peptide powders were taken up in 10 mL 6 M guanidinium hydrochloride in H20, supplemented with 0.5 mL of 1 M dithiothreitol, and loaded onto a C8 Luna preparative HPLC column (Phenomenex). Solvents (H20, acetonitrile) were acidified with 0.1 percent heptafluorobutyric acid. The gradient ranged from 30-70 percent acetonitrile in 15 minutes, at a flowrate of 15-20 mL /min, using a Gilson preparative HPLC system. Fractions containing pure linear peptide material (as identified by MALDI) were used for preparation of the bicycle derivatives by coupling to a scaffold molecule as described further below.A bicycle peptide designated 17-69-07-N474 was made corresponding to the bicycle peptide of Example 1 with replacement of the Cys6 by Dap(Me). The linear peptide used to form this bicycle was as follows: Ac-A(Dap(Me))(D-Ala)NE(lNal)(D-Ala)(Dap(Me))EDFYD(tBuGly)(Dap(Me))The structure of the TBMB derivative with the N385 peptide is shown schematically in Fig. 10. |
Tags: 71989-38-3 synthesis path| 71989-38-3 SDS| 71989-38-3 COA| 71989-38-3 purity| 71989-38-3 application| 71989-38-3 NMR| 71989-38-3 COA| 71989-38-3 structure
Precautionary Statements-General | |
Code | Phrase |
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Code | Phrase |
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Response | |
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P378 | |
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P391 | Collect spillage. Hazardous to the aquatic environment |
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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. |
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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.
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