All amino acid derivatives, and Rink-amide MBHA resins were purchased from IRIS Biotech GmbH (Marktredwitz, Germany), whereas N,N–diisopropylethylamine (DIEA), 1–hydroxybenzotriazole (HOBt), N,N'–diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), 1.8–diazabicyclo[5.4.0]undec–7–ene (DBU), thioanisole, 5(6)–carboxyfluorescein (Cf), 5(6)–carboxytetramethylrhodamine (Rh) and 1,2–ethanedithiol were FLUKA (Buchs, Switzerland) and Sigma Aldrich (Budapest, Hungary) products. Dabcyl acid [4–((4-(dimethylamino)phenyl)azo)benzoic acid] was ordered from (AAT Bioquest, Inc., Sunnyvale, CA). Solvents for syntheses and purification were obtained from Molar Chemicals Kft (Budapest, Hungary). The buffers were prepared with ion exchanged distilled water.
Rink amide mbha resin
Manufactured by Iris Biotech
Sourced in Germany
Rink-amide MBHA resin is a solid-phase resin used in the synthesis of peptides. It provides a linker for the attachment of the first amino acid, allowing the peptide chain to be built up on the resin support. The resin is based on a polystyrene matrix and contains a Rink-amide linker, which facilitates the cleavage of the synthesized peptide from the resin.
Automatically generated - may contain errors
Lab products found in correlation
Dabcyl acid 4 4 dimethylamino phenyl azo benzoic acid, by AAT Bioquest (2 mentions)
N n dimethylformamide dmf dichloromethane dcm, by Molar Chemicals (2 mentions)
Diethyl ether et2o, by Molar Chemicals (2 mentions)
Centrifuge 5702, by Eppendorf (1 mentions)
Hplc grade acetonitrile mecn, by Avantor (1 mentions)
Fmoc amino acids, by Iris Biotech (1 mentions)
Kinetex xb c18, by Phenomenex (1 mentions)
Trifluoroacetic acid tfa, by Carl Roth (1 mentions)
Diethyl ether, by Molar Chemicals (1 mentions)
N n dimethylformamide dmf dichloromethane dcm, by Merck Group (1 mentions)
O 7 azabenzotriazol 1 yl n n n n tetramethyluronium hexafluorophosphonate hatu, by Iris Biotech (1 mentions)
N ethyldiisopropylamine diea, by Merck Group (1 mentions)
Fmoc gly wang resin, by Iris Biotech (1 mentions)
Trifluoroacetic acid tfa, by Iris Biotech (1 mentions)
Delta 600 pump, by Waters Corporation (1 mentions)
Lcms 2010ev, by Shimadzu (1 mentions)
Acetic anhydride, by Merck Group (1 mentions)
α cyano 4 hydroxycinnamic acid, by Merck Group (1 mentions)
Triisopropylsilane tis, by Merck Group (1 mentions)
1 2 ethanedithiol edt, by Merck Group (1 mentions)
14 protocols using rink amide mbha resin
1
Solid-Phase Peptide Synthesis Reagents
2
Solid-Phase Peptide Synthesis Protocols
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All amino acid derivatives, and Rink-amide MBHA resins were purchased from IRIS Biotech GmbH (Marktredwitz, Germany), whereas N,N-diisopropylethylamine (DIEA), 1-hydroxybenzotriazole (HOBt), N,N′-diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), 1.8-diazabicyclo[5.4.0]undec-7-ene (DBU), thioanisole, 5(6)-carboxyfluorescein (Cf), triisopropylsilane (TIS) and 1,2-ethanedithiol were FLUKA (Buchs, Switzerland) and Sigma Aldrich (Budapest, Hungary) products. Dabcyl acid [4-((4-(dimethylamino)phenyl)azo)benzoic acid] was ordered from (AAT Bioquest, Inc., Sunnyvale, CA). Solvents for syntheses and purification were obtained from Molar Chemicals Kft (Budapest, Hungary). The buffers were prepared with ion exchanged distilled water.
3
Oxime-Based Peptide Synthesis
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Amino acid derivatives, K-Oxyma Pure® and Rink-Amide MBHA resin were obtained from Iris Biotech GmBH (Marktredwitz, Germany), Novabiochem®/Merck-Millipore (Darmstadt, Germany) and Bachem (Bubendorf, Switzerland). Boc-aminooxyacetic acid (Boc-Aoa–OH), aminooxyacetic acid, scavengers, coupling agents (1-hydroxybenzotriazole hydrate (HOBt), N,N′-diisopropylcarbodiimide (DIC)), and cleavage reagents (triisopropylsilane (TIS), piperidine, 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU), trifluoroacetic acid (TFA), diisopropylethylamine (DIPEA), acetic anhydride (Ac2O), methanol (MeOH), n-butyric anhydride and solvent for HPLC acetonitrile (ACN) were purchased from Sigma-Aldrich Kft (Budapest, Hungary). Daunorubicin hydrochloride was provided from IVAX (Budapest, Hungary). N,N-Dimethylformamide (DMF), dichloromethane (DCM) and diethyl ether (Et2O) were purchased from Molar Chemicals Kft (Budapest, Hungary). All reagents and solvents were of analytical grade or highest available purity.
4
Peptide Synthesis on Rink-Amide Resin
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All the peptides were synthesised manually on a Rink-amide MBHA resin (loading capacity: 0.69 mmol/g—Iris Biotech) with a Fmoc/tBu strategy. Each coupling was performed with 3 eq of amino acid, 3 eq of HOBt and 3 eq of DIC, for 1 h. The final coupling of the isopropylidene-protected aminooxyacetic acid (≥Aoa-OH) was performed with 3 eq of Aoa derivative, 3 eq of HOBt and 9 eq of DIC, for 1 h. The peptides containing the GFLG spacer were cleaved from the resin with a cocktail composed of TFA, TIS and dH2O (95:2.5:2.5 v/v%), for 1.5 h, RT. The ones containing the LRRY spacer were cleaved by adding the cleavage reagents TFA, crystalline phenol, thioanisole, dH2O, EDT (85:6:4:4:1 v/m/v/v/v%), for 2 h, RT. Peptides were precipitated in a 10× excess of ice-cold Et2O. The precipitates were then centrifuged and washed three times (3 min, 4400rpm Eppendorf Centrifuge 5702) with fresh Et2O, dissolved in dH2O:MeCN (0.1% TFA) 1:1 (v/v%) and freeze-dried.
5
Peptide Synthesis Using MBHA Resin
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Rink-amide MBHA resin and all amino acid derivatives were purchased from Iris Biotech GmBH (Marktredwitz, Germany), except for N-Fmoc-L-Statine, which was obtained from Fluorochem Ltd. (Hadifield, UK). Aminooxyacetic acid, scavengers, coupling agents (1-hydroxybenzotriazole hydrate (HOBt), N,N′-diisopropylcarbodiimide (DIC)), and cleavage reagents (triisopropylsilane (TIS), piperidine, 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU), 1,2-ethandithiol (EDT), thioanisole), diisopropylethylamine (DIPEA) and acetic anhydride (Ac2O) were purchased from Sigma Aldrich Kft. (Budapest, Hungary). Daunorubicin hydrochloride was provided by IVAX (Budapest, Hungary). N,N-Dimethylformamide (DMF), dichloromethane (DCM), diethyl ether (Et2O), trifluoroacetic acid (TFA) and HPLC grade acetonitrile (MeCN) were purchased from VWR Chemicals (Debrecen, Hungary). All reagents and solvents were of analytical grade or highest available purity.
6
Synthesis and Purification of INAD and dCRY Peptides
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The peptide corresponding to INAD230–243 was prepared by stepwise solid-phase synthesis using Fmoc strategy on a MultiSynTech semi-automated peptide synthesizer. Fmoc-amino acids and rink amide MBHA resin were purchased from Iris Biotech GmbH (Marktredwitz, DE). Cleavage reactions were performed in a TFA/TIS/H2O mixture (95:2.5:2.5) for 1 h at room temperature. The crude peptide was purified by accelerated chromatographic isolation (IsoleraTM Spektra, Biotage, Uppsala, S). Eluted fractions were verified by analytical HPLC and ESI mass spectrometry and lyophilized. The dCRY490–516 peptide (purity >99%) was purchased from ThermoFisher Scientific (Waltham, MA, USA). Both peptides were acetylated at the N-terminus and amidated at the C-terminus, to mimic the protein environment and remove extra charges.
7
Peptide Synthesis and Purification
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All chemicals were purchased from Sigma (Saint Louis, USA) if not denoted
otherwise. Rink amide MBHA resin was obtained from Iris Biotech (Marktredwitz,
Germany). 9-fluorenylmethoxy-carbonyl- (Fmoc) protected amino acid derivatives
and
O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU) were purchased from Merck (Darmstadt, Germany).
Trifluoroacetic acid (TFA) was obtained from Roth (Karlsruhe, Germany).
For HPLC separations solvents consisting of water (solvent A) and ACN (solvent
B), both containing 0.1% TFA, were used. Analytical runs were performed on an
Agilent 1100 HPLC (Boeblingen, Germany) with a quaternary pump, a well-plate
autosampler and a variable wavelength detector. Separations were performed on a
3.0 × 50 mm reversed phase
column (Phenomenex Kinetex XB C-18, 2.6 μm) with a
flow-rate of 0.6 mL/min. A Merck-Hitachi High Speed LC system (Darmstadt,
Germany) with a Merck Hibar Li Chrospher® RP-8 column
(250–25 mm, 5 μm) was used for
preparative separations (flow-rate: 8 mL/min). Eluted compounds were
analyzed by MALDI mass spectrometry. NMR spectroscopy was carried out using
Varian Gemini 2000 spectrometer in deuterated chloroform.
otherwise. Rink amide MBHA resin was obtained from Iris Biotech (Marktredwitz,
Germany). 9-fluorenylmethoxy-carbonyl- (Fmoc) protected amino acid derivatives
and
O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU) were purchased from Merck (Darmstadt, Germany).
Trifluoroacetic acid (TFA) was obtained from Roth (Karlsruhe, Germany).
For HPLC separations solvents consisting of water (solvent A) and ACN (solvent
B), both containing 0.1% TFA, were used. Analytical runs were performed on an
Agilent 1100 HPLC (Boeblingen, Germany) with a quaternary pump, a well-plate
autosampler and a variable wavelength detector. Separations were performed on a
3.0 × 50 mm reversed phase
column (Phenomenex Kinetex XB C-18, 2.6 μm) with a
flow-rate of 0.6 mL/min. A Merck-Hitachi High Speed LC system (Darmstadt,
Germany) with a Merck Hibar Li Chrospher® RP-8 column
(250–25 mm, 5 μm) was used for
preparative separations (flow-rate: 8 mL/min). Eluted compounds were
analyzed by MALDI mass spectrometry. NMR spectroscopy was carried out using
Varian Gemini 2000 spectrometer in deuterated chloroform.
8
Synthesis of Daunorubicin Conjugates
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All amino acid derivatives and Rink-Amide MBHA resin were purchased from Iris Biotech GmBH (Marktredwitz, Germany). Boc-aminooxyacetic acid (Boc-Aoa-OH), coupling agents and cleavage reagents (1-hydroxybenzotriazole hydrate (HOBt), N,N′-diisopropylcarbodiimide (DIC), triisopropylsilane (TIS), piperidine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), trifluoroacetic acid (TFA)), diisopropylethylamine (DIPEA), n-butyric anhydride, and acetonitrile (MeCN) for HPLC were obtained from Sigma-Aldrich Kft. (Budapest, Hungary). Daunorubicin hydrochloride was a gift from IVAX (Budapest, Hungary). Dimethylformamide (DMF), dichloromethane (DCM) and diethyl ether were purchased from Molar Chemicals Kft (Budapest, Hungary). All reagents and solvents were of analytical grade or highest available purity.
9
Peptide Synthesis Using Fmoc Methodology
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All solvents and reagents were used as supplied. Fmoc amino acid derivatives were purchased from Novabiochem (Billerica, MA, USA). O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphonate (HATU), the Rink Amide MBHA resin (0.69 mmol/g), Fmoc-Gly-Wang resin (0.73 mmol/g), and trifluoroacetic acid (TFA) were obtained from IrisBiotech (Marktredwitz, Germany). Solvents for peptide synthesis (N,N-dimethylformamide (DMF), dichloromethane (DCM), and (N-ethyldiisopropylamine (DIEA)) were obtained from Sigma Aldrich (St. Louis, MO, USA).
10
Solid-Phase Synthesis of Peptides
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The studied peptides (N-Ac-X1X2X3-NH2) were assembled in a solid-phase synthesizer Liberty Blue (CEM, USA) by stepwise coupling of the corresponding Fmoc-amino acids to the growing chain on Rink Amide MBHA resin (100–200 mesh, 0.67 mmol g−1) purchased from IRIS, Biotech GmbH, Marktredwitz, Germany. Fully protected peptide resins were synthesized according to a standard procedure involving cleavage of the Nα-Fmoc protecting group with 20% piperidine in DMF and coupling, mediated by mixtures of coupling reagents DIC/Oxyma in DMF. On completion of synthesis, the deprotection and detachment of linear peptides from the resins were carried out simultaneously using a TFA/H2O/TIS (95 : 2.5 : 2.5) cleaving mixture. Each of the resins was washed with DCM, and the combined TFA filtrates were evaporated at room temperature. The precipitated residues were triturated with tert-butyl-methylether, collected by suction, and dried by lyophilization. The linear peptides were purified by HPLC using a Waters instrument with a Delta 600 pump, and a 2489 UV/VIS detector. The purity and identity of all peptides were determined by analytical HPLC and by the ESI MS technique.
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