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CAS No. : | 109425-55-0 | MDL No. : | MFCD00065668 |
Formula : | C25H30N2O6 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | JOOIZTMAHNLNHE-NRFANRHFSA-N |
M.W : | 454.52 | Pubchem ID : | 2756114 |
Synonyms : |
|
Num. heavy atoms : | 33 |
Num. arom. heavy atoms : | 12 |
Fraction Csp3 : | 0.4 |
Num. rotatable bonds : | 13 |
Num. H-bond acceptors : | 6.0 |
Num. H-bond donors : | 3.0 |
Molar Refractivity : | 123.33 |
TPSA : | 113.96 Ų |
GI absorption : | High |
BBB permeant : | No |
P-gp substrate : | Yes |
CYP1A2 inhibitor : | No |
CYP2C19 inhibitor : | No |
CYP2C9 inhibitor : | Yes |
CYP2D6 inhibitor : | Yes |
CYP3A4 inhibitor : | Yes |
Log Kp (skin permeation) : | -6.16 cm/s |
Log Po/w (iLOGP) : | 3.04 |
Log Po/w (XLOGP3) : | 4.1 |
Log Po/w (WLOGP) : | 4.28 |
Log Po/w (MLOGP) : | 2.6 |
Log Po/w (SILICOS-IT) : | 3.39 |
Consensus Log Po/w : | 3.48 |
Lipinski : | 0.0 |
Ghose : | None |
Veber : | 1.0 |
Egan : | 0.0 |
Muegge : | 0.0 |
Bioavailability Score : | 0.56 |
Log S (ESOL) : | -4.65 |
Solubility : | 0.0101 mg/ml ; 0.0000223 mol/l |
Class : | Moderately soluble |
Log S (Ali) : | -6.2 |
Solubility : | 0.000287 mg/ml ; 0.000000633 mol/l |
Class : | Poorly soluble |
Log S (SILICOS-IT) : | -6.56 |
Solubility : | 0.000125 mg/ml ; 0.000000275 mol/l |
Class : | Poorly soluble |
PAINS : | 0.0 alert |
Brenk : | 1.0 alert |
Leadlikeness : | 3.0 |
Synthetic accessibility : | 4.41 |
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 |
---|---|---|
100% | With benzotriazol-1-ol; diisopropyl-carbodiimide; In DMF (N,N-dimethyl-formamide); dichloromethane; at 40℃; | Fmoc-Orn (Boc) -OH (100.2 mg, 454.52 g/mol, 0.22 mmol, 1 eq), DIC (34. 4 mul, 126.20 G/MOL, 0.806 G/CM3, 0.22 MMOL, 1 eq) and HOBt (29.7 mg, 135.12 g/mol, 0.22 mmol, 1 eq) were dissolved in dry DMF/DCM (1/1,4 ml). After 10 minutes (R)-1-(2-NAPHTHYL) ETHYLAMINE (37.7 mg, 171.24 g/mol, 0.22 mmol, 1 eq, Acros) was added to the reaction mixture. After overnight stirring, temperature was raised to 40C and kept there for 2 hours. Solvent was then evaporated and residue purified with flash chromatography. 5- (N-BOC-AMINO)- (S)-2-(N-FMOC-AMINO)-N-((R)-1-(2-NAPHTHYL) ETHYL) pentanamide was obtained with quantitative yield. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: 3mmol of Fmoc-protected aminoacid was dissolved in DMF with 3mmol HBTU and 3mmol Cl-HOBt. After cooling themixture to about 0 C, 6mmol DIEPA was added in one portion and mixed thoroughly about 1min then transferred to microwave peptide reaction vessel containing 1mmol of Rink amide AM resin (Fmoc deprotected form). Reaction was carried out under micro wave radiation (30W, 60C, 5min). Resin was drained and washed with DMF. Deprotection was carried out by adding 5-8ml 20% piperidine in DMF and microwave assisted reacting for 3 min (20W, 50C, twice) followed by washing with DCM and DMF and continuing to next amino acid coupling. Orthogonal protected diamino acid was coupled to resin bound peptide at designated position, Fmoc was removed by microwave assisted deprotection as described. After thoroughly washing, 2equiv. of 1b was added to the resin and pyridine was added at minimum volume which could dissolve 1b and swell the resin. The mixture was transferred to standard peptide synthesis vessel and allowed to rotation overnight. Reaction resin was drained and washed twice with DCM, combined filtrate was concentrated in vaco and directly added to other peptide resin which ought to be guanidinylated or recrystalled by ethyl ether to recover 1b. Resin was cleaved by adding 10mlof TFA:TESi:H2O(95:2.5:2.5) and allowed reacting for 2h. Resin was drained and filtrate was collected and concentrated in vaco followed by diluting with acetonitrile to 10mmol/ml concentration and adding 1 equiv. TEA to catalyze reaction. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
19 mg | Example 3 Dat1-D-Ala2-Asp3-Ala4-Ile5-Phe6-Thr7-Asn8-Ser9-Tyr10-Arg11-Orn12-Val13-Leu14-Abu15-Gln16-Leu17-Ser18-Ala19-Arg20-Orn21-Leu22-Leu23-Gln24-Asp25-Ile26-Nle27-Asp28-Arg29-NH-CH3 (Peptide 27400). [Dat1, D-Ala2, Orn12, Abu15, Orn21, Nle27, Asp28]hGHRH(1-29)NH-CH3 The synthesis is conducted in a stepwise manner using manual solid phase peptide synthesis equipment. Briefly, [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin (Nova Biochem, La Jolla, Calif.) (750 mg, 0.50 mmol) is deprotected with 20% piperidine in DMF for 5 and 15 minutes and washed according to the protocol described in Table 3. The solution of Fmoc-Arg(Pbf)-OH (975 mg, 1.5 mmol) in DMF is shaken with the washed resin and DIC (235 muL, 1.5 mmol) in a manual solid phase peptide synthesis apparatus for 1 hour. After washing the resin three times with DMF, the coupling reaction was repeated as described above. After the repeated coupling and after the completion of the reaction is proved by negative ninhydrin test, the deprotection and neutralization protocols described in Table 3 are performed in order to remove the Fmoc protecting group and prepare the peptide-resin for coupling of the next amino acid. The synthesis is continued and the peptide chain is built stepwise by coupling the following protected amino acids in the indicated order on the resin to obtain the desired peptide sequence: Fmoc-Asp(OBut)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-AspfOBuVOH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-AspBu1)-OH, Fmoc-D-Ala-OH, Dat-OH. These protected amino acid residues (also commonly available from Novabiochem, Advanced Chemtech, Bachem, and Peptides International) are represented above according to a well accepted convention. The suitable protecting group for the side chain functional group of particular amino acids appears in parentheses. The OH groups in the above formulae indicate that the carboxyl terminus of each residue is free. The protected amino acids (1.5 mmol each) are coupled with DIC (235 muL, 1.5 mmol) with the exceptions of Fmoc-Asn(Trt)-OH and Fmoc-Gln(Trt)-OH which are coupled with HBTU reagent. In order to cleave the peptide from the resin and deprotect it, a portion of 250 mg of the dried peptide resin is stirred with 2.5 mL cleavage cocktail (94% TFA, 3% H2O, 1.5% m-cresol, and 1.5% phenol) at room temperature for 3 hours. To induce peptide precipitation, the cleavage mixture is added dropwise to cold (preferably -20 C.) ether. The precipitated material is collected by filtration or centrifugation and is washed three times with cold ether. The cleaved and deprotected peptide is dissolved in 50% acetic acid and separated from the resin by filtration. After dilution with water and lyophilization, 118 mg crude product is obtained. The crude peptide is checked by analytical HPLC using a Hewlett-Packard Model HP-1090 liquid chromatograph equipped with a Supelco Discovery HS C18 reversed-phase column (2.1 mm×5 cm, packed with C18 silica gel, 300 pore size, 3 mum particle size) (Supelco, Bellefonte, Pa.). Linear gradient elution (e.g., 40-70% B) is used with a solvent system consisting of (A) 0.1% aqueous TFA and (B) 0.1% TFA in 70% aqueous MeCN, and the flow rate is 0.2 mL/min. Purification is performed on a Beckman System Gold HPLC system (Beckman Coulter, Inc., Brea, Calif.) equipped with 127P solvent Module; UV-VIS Detector, model 166P; Computer workstation with CPU Monitor and printer, and 32-Karat software, version 3.0. 118 mg of crude peptide is dissolved in AcOH/H2O, stirred, filtered and applied on an XBridge Prep OBD reversed phase column (4.6×250 mm, packed with C18 silica gel, 300 A pore size, 5 mum particle size) (Waters Co., Milford, Mass.). The column is eluted with a solvent system described above in a linear gradient mode (e.g., 40-60% B in 120 min); flow rate 12 mL/min. The eluent is monitored at 220 nm, and fractions are examined by analytical HPLC. Fractions with purity higher than 95% are pooled and lyophilized to give 19 mg pure product. The analytical HPLC is carried out on a Supelco Discovery C18 reversed-phase column described above using isocratic elution with a solvent system described above with a flow rate of 0.2 mL/min. The peaks are monitored at 220 and 280 nm. The product is judged to be substantially (>95%) pure by analytical HPLC. Molecular mass is checked by electrospray mass spectrometry, and the expected amino acid composition is confirmed by amino acid analysis. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
16 mg | Example 4 N-Me-Tyr1-D-Ala2-Asp3-Ala4-Ile5-Phe6-Thr7-Gln8-Ser9-Tyr10-Arg11-Orn12-Val13-Leu14-Abu15-Gln16-Leu17-Ser18-Ala19-Arg20-Orn21-Leu22-Leu23-Gln24-Asp25-Ile26-Nle27-Asp28-Arg29-NH-CH2-CH3 (Peptide 28420) N-Me-Tyr1, D-Ala2, Gln8, Orn12, Abu15, Orn21, Nle27, Asp28]hGHRH(1-29)NH-CH2-CH3. The synthesis is conducted in a stepwise manner using manual solid phase peptide synthesis equipment. Briefly, 3-[(Ethyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin (Nova Biochem, La Jolla, Calif.) (610 mg, 0.50 mmol) is deprotected with 20% piperidine in DMF for 5 and 15 minutes and washed according to the protocol described in Table 3. The solution of Fmoc-Arg(Pbf)-OH (975 mg, 1.5 mmol) in DMF is shaken with the washed resin and DIC (235 muL, 1.5 mmol) in a manual solid phase peptide synthesis apparatus for 1 hour. After washing the resin three times with DMF, the coupling reaction was repeated as described above. After the repeated coupling and after the completion of the reaction is proved by negative ninhydrin test, the deprotection and neutralization protocols described in Table 3 are performed in order to remove the Fmoc protecting group and prepare the peptide-resin for coupling of the next amino acid. The synthesis is continued and the peptide chain is built stepwise by coupling the following protected amino acids in the indicated order on the resin to obtain the desired peptide sequence: Fmoc-Asp(OBut)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBut)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-AspfOBuVOH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH. These protected amino acid residues (also commonly available from Novabiochem, Advanced Chemtech, Bachem, and Peptides International) are represented above according to a well accepted convention. The suitable protecting group for the side chain functional group of particular amino acids appears in parentheses. The OH groups in the above formulae indicate that the carboxy-terminus of each residue is free. The protected amino acids (1.5 mmol each) are coupled with DIC (235 muL, 1.5 mmol) with the exceptions of Fmoc-Asn(Trt)-OH and Fmoc-Gln(Trt)-OH which are coupled with HBTU reagent. In order to cleave the peptide from the resin and deprotect it, a portion of 250 mg of the dried peptide resin is stirred with 2.5 mL of cleavage cocktail (94% TFA, 3% H2O, 1.5% m-cresol, and 1.5% phenol) at room temperature for 3 hours. To induce peptide precipitation, the cleavage mixture is added dropwise to cold (preferably -20 C.) ether. The precipitated material is collected by filtration or centrifugation and is washed three times with cold ether. The cleaved and deprotected peptide is dissolved in 50% acetic acid and separated from the resin by filtration. After dilution with water and lyophilization, 110 mg crude product is obtained. The crude peptide is checked by analytical HPLC using a Hewlett-Packard Model HP-1090 liquid chromatograph equipped with a Supelco Discovery HS C18 reversed-phase column (2.1 mm×5 cm, packed with C18 silica gel, 300 pore size, 3 mum particle size) (Supelco, Bellefonte, Pa.). Linear gradient elution (e.g., 40-70% B) is used with a solvent system consisting of (A) 0.1% aqueous TFA and (B) 0.1% TFA in 70% aqueous MeCN, and the flow rate is 0.2 mL/min. Purification is performed on a Beckman System Gold HPLC system (Beckman Coulter, Inc., Brea, Calif.) equipped with 127P solvent Module; UV-VIS Detector, model 166P; Computer workstation with CPU Monitor and printer, and 32-Karat software, version 3.0. 110 mg of crude peptide is dissolved in AcOH/H2O, stirred, filtered and applied on an XBridge Prep OBD reversed phase column (4.6×250 mm, packed with C18 silica gel, 300 A pore size, 5 mum particle size) (Waters Co., Milford, Mass.). The column is eluted with a solvent system described above in a linear gradient mode (e.g., 40-60% B in 120 min); flow rate 12 mL/min. The eluent is monitored at 220 nm, and fractions are examined by analytical HPLC. Fractions with purity higher than 95% are pooled and lyophilized to give 16 mg pure product. The analytical HPLC is carried out on a Supelco Discovery C18 reversed-phase column described above using isocratic elution with a solvent system described above with a flow rate of 0.2 mL/min. The peaks are monitored at 220 and 280 nm. The product is judged to be substantially (>95%) pure by analytical HPLC. Molecular mass is checked by electrospray mass spectrometry, and the expected amino acid composition is confirmed by amino acid analysis. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Whatman Chr 1 filter paper (8.5 cm 12.5 cm) was swollen inDMF (10 min) before Fmoc-b-Ala-OH activated in situ with DIC inthe presence of NMI (2 mmol/L each, 10 mL DMF) was coupled atroom temperature (RT, 2 h) [35]. Free hydroxyl groups were cappedwith acetic anhydride (10% v/v) in DMF (10 min). The Fmoc-groupwas cleaved here and for all following residues with piperidine(20% v/v) in DMF twice for 5 min each. The HMBA linker wascoupled using HBTU in the presence of DIPEA as base (0.2 mol/Leach, 10 mL DMF, RT, 1 h) [36]. Fmoc-Leu-OH (0.4 mol/L) wascoupled using DIC (0.2 mol/L) and DMAP (8 mmol/L) in DMF(10 mL) overnight. The remaining free anchors were capped withbenzoic anhydride (0.2 mol/L) dissolved in a mixture of pyridine(40 mmol/L) and DMF (10 mL, 2 h).The remaining residues were coupled on a Syro2000 equippedwith a SPOT synthesis block (MultiSyntech GmbH, Witten, Germany).Amino acids (0.16 mol/L) were pre-activated using HBTU (0.16 mol/L) in the presence of DIPEA (0.33 mol/L) and spotted(0.48 mL) three times allowing a reaction time of 5 min before thereagent was spotted again. Side chains of trifunctional amino acidswere protected with 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf) for Arg, tert-butyl (tBu) forAsp, Glu, Ser, Thr, and Tyr, tert-butyloxycarbonyl (Boc) for Lys, Trp,Orn, and trityl (Trt) for Cys, His, Asn, and Gln. Unreacted aminogroups were capped using acetic anhydride (10% v/v) in DMF andthe Fmoc-group cleaved (20% v/v) piperidine in DMF, two times5 min, 0.8 mL each, RT). After final Fmoc deprotection, the membranewas stained with bromophenol blue (0.002% w/v in methanol)to visualize the peptide spots (Supplement, Fig. S1), wasdried, and spots were labeled with a soft pencil. The membranewasswollen in DMF and incubated with a solution of HBTU and NMM(0.4 mol/L each) in DMF (10 mL, RT, 2 h) to obtain N,N,N0,N0-tetramethyl-guanidino-groups at the N-termini. The procedure wasrepeated once using fresh reagents for 5 h. Permanent protectinggroupswere cleaved in two steps using first a mixture of TFA,water,TIS, and phenol (90/6/3/1, v/v/v/w, 25 mL, RT) for 30 min and then amixture of TFA, DCM, TIS, water, and phenol (50/44/3/2/1, v/v/v/v/w, 25 mL, RT) for 3 h. The membrane was washed with DCM (10times) and dried before the marked spots were punched out andtransferred into a 96-well plate. Peptides were cleaved off themembrane with aqueous trimethylamine (45% w/v, 200 mL/well,RT) overnight and dried under vacuum with membrane spots beingpresent. Peptides were dissolved (two times 30 min, 150 mL/well) inaqueous acetonitrile (3% v/v) containing TFA (0.1% v/v) and transferredinto a new 96-well plate. Products were analyzed by matrixassistedlaser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS; 4700 Proteomics Analyzer, Applied Biosystems,Weiterstadt, Germany) using a-cyano-4-hydroxycinnamicacid as matrix (4 g/L in aqueous acetonitrile (60% v/v) containingTFA (0.1% v/v). Api137 was always synthesized on at least two spotsof each array. Api137 released from one spot was analyzed by RPHPLCon a Jupiter C18-column (ID: 2 mm) using a linear aqueousacetonitrile gradient containing TFA (0.1% v/v) as ion pair reagent.The second Api137 spot and a spot with only one amino acidcoupled were used as positive and negative controls in minimalinhibitory concentration (MIC) testing, respectively. Peptides weredried and stored at 20 C. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
[Dat1, D-Ala2, Orn12, Abu15, Orn21, Nle27, Asp28]hGH-RH(1-29)NH-CH3 The synthesis is conducted in a stepwise manner using manual solid phase peptide synthesis equipment. Briefly, [3-[(Methyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin (Nova Biochem, La Jolla, Calif.) (750 mg, 0.50 mmol) is deprotected with 20% piperidine in DMF for 5 and 15 minutes and washed according to the protocol described in Table 3. The solution of Fmoc-Arg(Pbf)-OH (975 mg, 1.5 mmol) in DMF is shaken with the washed resin and DIC (235 muL, 1.5 mmol) in a manual solid phase peptide synthesis apparatus for 1 hour. After washing the resin three times with DMF, the coupling reaction was repeated as described above. After the repeated coupling and after the completion of the reaction is proved by negative ninhydrin test, the deprotection and neutralization protocols described in Table 3 are performed in order to remove the Fmoc protecting group and prepare the peptide-resin for coupling of the next amino acid. The synthesis is continued and the peptide chain is built stepwise by coupling the following protected amino acids in the indicated order on the resin to obtain the desired peptide sequence: Fmoc-Asp(OBut)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBut)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBut)-OH, Fmoc-D-Ala-OH, Dat-OH. (0249) These protected amino acid residues (also commonly available from Novabiochem, Advanced Chemtech, Bachem, and Peptides International) are represented above according to a well accepted convention. The suitable protecting group for the side chain functional group of particular amino acids appears in parentheses. The OH groups in the above formulae indicate that the carboxyl terminus of each residue is free. (0250) The protected amino acids (1.5 mmol each) are coupled with DIC (235 muL, 1.5 mmol) with the exceptions of Fmoc-Asn(Trt)-OH and Fmoc-Gln(Trt)-OH which are coupled with HBTU reagent. (0251) In order to cleave the peptide from the resin and deprotect it, a portion of 250 mg of the dried peptide resin is stirred with 2.5 mL cleavage cocktail (94% TFA, 3% H2O, 1.5% m-cresol, and 1.5% phenol) at room temperature for 3 hours. To induce peptide precipitation, the cleavage mixture is added dropwise to cold (preferably -20 C.) ether. The precipitated material is collected by filtration or centrifugation and is washed three times with cold ether. The cleaved and deprotected peptide is dissolved in 50% acetic acid and separated from the resin by filtration. After dilution with water and lyophilization, 118 mg crude product is obtained. (0252) The crude peptide is checked by analytical HPLC using a Hewlett-Packard Model HP-1090 liquid chromatograph equipped with a Supelco Discovery HS C18 reversed-phase column (2.1 mm×5 cm, packed with C18 silica gel, 300 pore size, 3 mum particle size) (Supeico, Bellefonte, Pa.). Linear gradient elution (e.g., 40-70% B) is used with a solvent system consisting of (A) 0.1% aqueous TFA and (B) 0.1% TFA in 70% aqueous MeCN, and the flow rate is 0.2 mL/min. Purification is performed on a Beckman System Gold HPLC system (Beckman Coulter, Inc., Brea, Calif.) equipped with 127P solvent Module; UV-VIS Detector, model 166P; Computer workstation with CPU Monitor and printer, and 32-Karat software, version 3.0. 118 mg of crude peptide is dissolved in AcOH/H2O, stirred, filtered and applied on an XBridge Prep OBD reversed phase column (4.6×250 mm, packed with C18 silica gel, 300 pore size, 5 mum particle size) (Waters Co., Milford, Mass.). The column is eluted with a solvent system described above in a linear gradient mode (e.g., 40-60% B in 120 min); flow rate 12 mL/min. The eluent is monitored at 220 nm, and fractions are examined by analytical HPLC. Fractions with purity higher than 95% are pooled and lyophilized to give 19 mg pure product. The analytical HPLC is carried out on a Supelco Discovery C18 reversed-phase column described above using isocratic elution with a solvent system described above with a flow rate of 0.2 mL/min. The peaks are monitored at 220 and 280 nm. The product is judged to be substantially (>95%) pure by analytical HPLC. Molecular mass is checked by electrospray mass spectrometry, and the expected amino acid composition is confirmed by amino acid analysis. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
N-Me-Tyr1, D-Ala2, Gln8, Orn12, Abu15, Orn21, Nle27, Asp28]hGH-RH(1-29)NH-CH2-CH3 The synthesis is conducted in a stepwise manner using manual solid phase peptide synthesis equipment. Briefly, 3-[(Ethyl-Fmoc-amino)methyl]-indol-1-A-acetyl AM resin (Nova Biochem, La Jolla, Calif.) (610 mg, 0.50 mmmol) is deprotected with 20% piperidine in DMF for 5 and 15 minutes and washed according to the protocol described in Table 3. The solution of Fmoc-Arg(Pbf)-OH (975 mg, 1.5 mmol) in DMF is shaken with the washed resin and DIC (235 muL, 1.5 mmol) in a manual solid phase peptide synthesis apparatus for 1 hour. After washing the resin three times with DMF, the coupling reaction was repeated as described above. After the repeated coupling and after the completion of the reaction is proved by negative ninhydrin test, the deprotection and neutralization protocols described in Table 3 are performed in order to remove the Fmoc protecting group and prepare the peptide-resin for coupling of the next amino acid. The synthesis is continued and the peptide chain is built stepwise by coupling the following protected amino acids in the indicated order on the resin to obtain the desired peptide sequence: Fmoc-Asp(OBut)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBut)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asp(OBut)-OH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH. (0290) These protected amino acid residues (also commonly available from Novabiochem, Advanced Chemtech, Bachem, and Peptides International) are represented above according to a well accepted convention. The suitable protecting group for the side chain functional group of particular amino acids appears in parentheses. The OH groups in the above formulae indicate that the carboxy-terminus of each residue is free. (0291) The protected amino acids (1.5 mmol each) are coupled with DIC (235 mul, 1.5 mmol) with the exceptions of Fmoc-Asn(Trt)-OH and Fmoc-Gln(Trt)-OH which are coupled with HBTU reagent. (0292) In order to cleave the peptide from the resin and deprotect it, a portion of 250 mg of the dried peptide resin is stirred with 2.5 mL of cleavage cocktail (94% TFA, 3% H2O, 1.5% m-cresol, and 1.5% phenol) at room temperature for 3 hours. To induce peptide precipitation, the cleavage mixture is added dropwise to cold (preferably -20 C.) ether. The precipitated material is collected by filtration or centrifugation and is washed three times with cold ether. The cleaved and deprotected peptide is dissolved in 50% acetic acid and separated from the resin by filtration. After dilution with water and lyophilization, 110 mg crude product is obtained. (0293) The crude peptide is checked by analytical HPLC using a Hewlett-Packard Model HP-1090 liquid chromatograph equipped with a Supelco Discovery HS C18 reversed-phase column (2.1 mm×5 cm, packed with C18 silica gel, 300 pore size, 3 mum particle size) (Supelco, Bellefonte, Pa.). Linear gradient elution (e.g., 40-70% B) is used with a solvent system consisting of (A) 0.1% aqueous TFA and (B) 0.1% TFA in 70% aqueous MeCN, and the flow rate is 0.2 mL/min. Purification is performed on a Beckman System Gold HPLC system (Beckman Coulter, Inc., Brea, Calif.) equipped with 127P solvent Module; UV-VIS Detector, model 166P; Computer workstation with CPU Monitor and printer, and 32-Karat software, version 3.0. 110 mg of crude peptide is dissolved in AcOH/H2O, stirred, filtered and applied on an XBridge Prep OBD reversed phase column (4.6×250 mm, packed with Ci8 silica gel, 300 pore size, 5 mum particle size) (Waters Co., Milford, Mass.). The column is eluted with a solvent system described above in a linear gradient mode (e.g., 40-60% B in 120 min); flow rate 12 mL/min. The eluent is monitored at 220 nm, and fractions are examined by analytical HPLC. Fractions with purity higher than 95% are pooled and lyophilized to give 16 mg pure product. The analytical HPLC is carried out on a Supelco Discovery C18 reversed-phase column described above using isocratic elution with a solvent system described above with a flow rate of 0.2 mL/min. The peaks are monitored at 220 and 280 nm. The product is judged to be substantially (>95%) pure by analytical HPLC. Molecular mass is checked by electrospray mass spectrometry, and the expected amino acid composition is confirmed by amino acid analysis. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20% piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1% CF3COOH in the gradient system: 30 min gradient,30-100% CH3CN-H2O-0.1% CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: In a reaction vessel, Fmoc-protected Rink amide MBHA resinwas first swelled in DMF for fifteen min. A solution of 20percent piperidinein DMF was added and mixture shaken mechanically for15 min resulting in the removal of Fmoc group. The required Fmocprotected amino acids and coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) were placed in amino acid vessels sequentially. DMF was added to theamino acid vessel, which was subsequently added (by positivepressure of N2) to the reaction vessel containing the resin, followedby addition of N,N-diisppropylethylamine (DIEA). After 3 h of mechanicalshaking at ambient temperature, the solvent was drainedand the resin washed with DMF (3 x 5 min) followed by methanol(2 x 5 mL). The cycles of deprotection and coupling were repeatedtill the desired peptide chain length was obtained. The resin-boundpeptide was transferred to a round bottom flask, and simultaneousremoval of resin and protective groups was achieved by using acocktail combination of TFA:triisopropylsilane (TIPS):H2O[95:2.5:2.5] for 3 h. The crude peptide was filtered and purified onpreparative HPLC system, and analyzed using solvent system ofCH3CN-H2O-0.1percent CF3COOH in the gradient system: 30 min gradient,30-100percent CH3CN-H2O-0.1percent CF3COOH at 215 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: As a solid support 2-Chlor-tritylchloride resin (1.6meq /g, 100-200 mesh) was used. This resin was loaded with 0.6eq of amino acid and 8eq DIPEA in dry DCM overnight at RT. After extensive washing with DMF and DCM, the Fmoc-group was cleaved off with a mixture of 50% Piperidine in DCM/DMF (1:1) in DMF (freshly prepared) for 30mm at RT. After washing withDMF, DCM and MeOH the resin was dried under vacuum at RT overnight. The resin loading was determined via weight increase. The second amino acid was coupled with 4eq Mukaiyama-Reagent as coupling reagent, 6eq DIPEA in DMF/DCM (1:1) overnight at RT. The resin was extensively washed with DMF and DCM and the coupling rate was controlled by a test-cleavage. The Fmoc-group from the dipeptide was cleaved with a mixture of 50% Piperidine (25%)/DCM (25%) in DMF for maximally 5 mm followed by washings with DMF and DCM. The cleavage rates were again controlled by test-cleavage. The third amino acid was coupled using an excess of 4eq using 4eq HATU as coupling reagent and 6eq DIPEA. Complete couplings were accomplished at RT for 2-4 hours with thecoupling rate again controlled by a test-cleavage. The Fmoc-group from the tripeptide was cleaved with a mixture of 20% Piperidine in DMF for 2 x 15-20mm at RT followed by washings with DMF and DCM (test-cleavage). Resin was swollen in THF (ca. lOml/g resin). l2eq DIPEA were added and the reaction mixture was shaken at RT for 15mm. 3 eq 2-nitrobenzene-1- 1 sulfonylchloride were added and the resin was shaken at RT overnight. Resin was then drained, washed with DCM and DMF. The coupling rate was controlled via atest-cleavage. For the second step the Resin was suspended in DMF, l2eq MTBD (7-methyl-1,5,7- triazabicyclo [4.4.0]dec-5-ene) were added and the reaction mixture was shaken at RT for 10mm. Then 3eq Methyl-4-nitrobenzenesulfonate was added and the sluffy was shaked at RT. After 30mm. The resin was drained and washed with DMF and DCM. The coupling rate was controlled via a test-cleavage. For removal of the 2-nitrobenzene-1-lsulfonamide protecting group, the resin was suspended in DMF, l2eq DBU were added, the sluffy shaken for 5mm, then l2eqmercaptoethanol was added and the reaction mixture was shaken at RT for lh. The resin was drained and washed with DMF and DCM. The deprotection rate was controlled via a test-cleavage. Resin with tripeptide was washed with DCM, the coffesponding Intermediate dissolved in amixture of NMP/TMOF/AcOH (49.7/49.7/0.6) and the solution was added to the resin. Themixture was shaken at RT for 30mm up to 3h, then lOeq NaCNBH3 were added and the reaction mixture was shaken at RT overnight. Finally, the resin was washed with DMF, DCM, MeOH/DCM (1:1) and DMF. The Fmoc-group on the third amino acid was cleaved with a mixture of 20% Piperidine inDMF for 2x 15-20mm at RT followed by washings with DMF and DCM (test-cleavage). A cleavage-cocktail of 20% HFIP in DCM was added to the resin and the mixture was stuffed for2h at RT. The resin was filtered off and the solution was evaporated to dryness. The residue wasdissolved in water/acetonitrile and lyophilized. The obtained crude linear compound was cyclized by dissolving the powder in DMF. 1 .2eq HATU and Seq DIPEA were added and the reaction mixture stuffed at RT. Progress of the reaction was monitored by HPLC. After completion, the solvent was evaporated, the resulting residue taken up in water/acetonitrile (1:1) and lyophilized. Peptide macrocycles were purified using reversed phase high-performance liquid chromatography (RP-HPLC) using a Reprospher 100 C18-TDE column (250 x 20 mm, Sum particle size) as a stationary phase and water/acetonitrile as eluent (Gradient 40-100% MeCN over 60 mm). Fractions were collected and analyzed by LC/MS. Pure product samples werecombined and lyophilized. Product identification was obtained via mass spectrometry.Final BOC-deprotection was achieved by 50% TFA (DCM) treatment for 2h at RT. The reaction solution was concentrated down and the residue freeze-dried to yield the deprotected product as TFA salt. All peptides were obtained as white powders with a purity >90%; Example 15 was prepared according to the General Procedure for Peptide Macrocycle Synthesis using the following starting materials:Amino acids:1. Fmoc-L-6-Cl-Trp-OH, followed by on-bead N-methylation of the Trp alpha-N,2. Fmoc-L-Lys(BOC)-OH,3. Fmoc-L-Orn(BOC)-OH.Tether: Intermediate 4. MS (M+H): expected 703.3; observed 704.3 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Tetrapeptides 3a-d were synthesized by manual solid-phase peptide synthesis (SPPS) using 2-chlorotritylchloride polymer resin (2-CTC, 1.63mmol/g) in a 10-mL disposable syringe fitted with a porous polyethylene filter and N-terminal fluorenylmethyloxycarbonyl-protected amino acids. Coupling of the first amino acid: Fmoc-propargyl-Gly-OH (0.8 eq.) to the 2-CTC resin (1.0 eq.) was performed in anhydrous DCM (7.5mL) in the presence of DIEA (3.0 eq.) with shaking at room temperature for 2h. Then, MeOH (2mL) was added to cap the unreacted resin and the mixture was shaken for 30min. The resin was filtered, washed with several portions of DCM, DMF and again DCM and dried under high vacuum, overnight. Loading of the starting material was calculated via increase in dry weight. Next, the resin swelling was performed in DMF for 20min, Fmoc protecting group was removed by treatment with 20% piperidine in DMF (2×3mL, 5+20min) and the polymer was washed with DMF (5×3mL, 5×2min). Then, coupling of the appropriate amino acid: Fmoc-AA-OH (4.0 eq.) was performed in DMF (3mL) using HBTU (4.0 eq.), in the presence of DIEA (8.0 eq.) with shaking at room temperature. In the case of Fmoc-Ala-OH for 4h, with Fmoc-Orn(Boc)-OH, Fmoc-Thr (tBu)-OH and Fmoc-N-Me-Thr (tBu)-OH overnight and with Fmoc-Arg (Pbf)-OH and Fmoc-His (Trt)-OH the double coupling was performed (after 2h the resin was washed with DMF and treated with a freshly made solution of Fmoc-AA-OH, HBTU and DIEA in DMF and shaken for 2h). After each coupling, the resin was washed with DMF (4×4mL) and subsequently de-protected and washed as described above. At the end of the coupling cycle the resin was washed with several portions of DMF, MeOH and DCM and dried under high vacuum, overnight. Method (A) for coupling of the tail via HBTU activation: The coupling of the tails at the N-terminus of the synthesized tetrapeptides was performed using the acid (4 eq.) in DMF (2mL), in the presence of DIEA (8 eq.) and HBTU (4 eq.) at room temperature, shaking overnight. The resin was then washed extensively with several portions of DMF, MeOH and DCM and dried under high vacuum. Method (B) for coupling of the tail via mixed anhydride: Step 1. Heterocyclic acid derivative (0.52mmol, 1 eq.) was reacted with Boc anhydride (2.11mmol, 4 eq.) in dry tetrahydrofuran in presence of pyridine (2.64mmol, 5 eq.) at r. t. overnight. After full consumption of starting material volatiles were removed in vacuo. Aqueous 0.5M hydrochloric acid solution was added and the product was extracted with ethyl acetate. The organic layer was washed with water, separated and dried over sodium sulfate. Volatiles were removed in vacuo yielding a crude product as yellowish oil, which was used in the next step without further purification. Step 2. The 2-CTC resin containing 3a (0.13mmol, 1 eq.) was suspended in DMF (3mL) containing mixed anhydride prepared in Step 1 (presumed 0.52mmol, 4 eq.) and DIPEA (1.82mmol, 14 eq.). The reaction mixture was shaken at r. t. overnight. The resin was filtered, washed with DMF (5×2mL), DCM (5×2mL) and THF (5×4mL). General method for cleavage from the solid support. 2-CTC resin containing oligopeptides was added into a mixture of HFIP/DCM 1:4 (4mL) and the reaction mixture was shaken at room temperature for 1.5h. The resin was filtered off and washed with DCM and THF. The volatiles were evaporated in vacuo. Purification: The crude peptides were dissolved in THF (2.5mL) and H2O/MeCN (1:1, 2.5mL), filtered through a 0.45mum nylon membrane filter and purified in 1-3 runs by preparative RP-HPLC. The reported yields are based on calculated loading of the oligopeptide on 2-CTC resin. |
Tags: 109425-55-0 synthesis path| 109425-55-0 SDS| 109425-55-0 COA| 109425-55-0 purity| 109425-55-0 application| 109425-55-0 NMR| 109425-55-0 COA| 109425-55-0 structure
Precautionary Statements-General | |
Code | Phrase |
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Prevention | |
Code | Phrase |
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P265 | Wash skin thouroughly after handling. |
P270 | Do not eat, drink or smoke when using this product. |
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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. |
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Response | |
Code | Phrase |
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P321 | |
P322 | |
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P378 | |
P380 | Evacuate area. |
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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. |
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P305 + P351 + P338 | IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. |
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P335 + P334 | Brush off loose particles from skin. Immerse in cool water/wrap in wet bandages. |
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Code | Phrase |
H200 | Unstable explosive |
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H260 | In contact with water releases flammable gases which may ignite spontaneously |
H261 | In contact with water releases flammable gas |
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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 |
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H351 | Suspected of causing cancer |
H360 | May damage fertility or the unborn child |
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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 |
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