- Coumarinic acid-based cyclic prodrugs of opioid peptides that exhibit metabolic stability to peptidases and excellent cellular permeability
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Purpose. To evaluate the cellular permeation characteristics and the chemical and enzymatic stability of coumarinic acid-based cyclic prodrugs 1 and 2 of the opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. Methods. The rates of conversion of the cyclic prodrugs 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH 7.4 (Caco-2 cell transport buffer) and in various biological media having measurable esterase activity were determined by HPLC. The cell permeation characteristics of [Leu5]-enkephalin, DADLE and cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto micropores membranes and monitored by HPLC. Results. In HBSS, pH 7.4, cyclic prodrugs 1 and 2 degraded chemically to intermediates that further degraded to [Leu5]-enkephalin and DADLE, respectively, in stoichiometric amounts. In 90% human plasma and rat liver homogenate, the disappearance of cyclic prodrugs 1 and 2 was significantly faster than in HBSS, pH 7.4. The half- lives in 90% human plasma and in rat liver homogenate were substantially longer after pretreatment with paraoxon, a known inhibitor of serine- dependent esterases. When applied to the AP side of a Caco-2 cell monolayer, cyclic prodrug 1 exhibited significantly greater stability against peptidase metabolism than did [Leu5]-enkephalin. Cyclic prodrug 2 and DADLE exhibited similar stability when applied to the AP side of the Caco-2 cell monolayer. Prodrug 1 was 665-fold more able to permeate the Caco-2 cell monolayers than was [Leu5]-enkephalin, in part because of its increased enzymatic stability. Prodrug 2 was shown to be approximately 31 fold more able to permeate a Caco- 2 cell monolayer than was DADLE. Conclusions. Cyclic prodrugs 1 and 2, prepared with the coumarinic acid promoiety, were substantially more able to permeate Caco-2 cell monolayers than were the corresponding opioid peptides. Prodrug 1 exhibited increased stability to peptidase metabolism compared to [Leu5]-enkephalin. In various biological media, the opioid peptides were released from the prodrugs by an esterase-catalyzed reaction, which is sensitive to paraoxon inhibition.
- Gudmundsson, Olafur S.,Pauletti, Giovanni M.,Wang, Wei,Shan, Daxian,Zhang, Huijuan,Wang, Binghe,Borchardt, Ronald T.
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Read Online
- The pivaloylglycol anchor group: A new platform for a photolabile linker in solid-phase synthesis
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We have designed a new photolabile linker (2) based on 2- pivaloylglycerol for the solid-phase synthesis of acids. The linker was prepared in six steps and anchored to the support via an amide bond. Photocleavage is a two-step process, in which the immobilized acids are released by photolyric generation of a radical center and subsequent spontaneous β-C,O bond scission. The pivaloyl linker (2) was found to cleave with high yields and purities the acids in various solvents (THF, CH2Cl2, dioxane, DMSO) by irradiation with light above 320 nm. Using this linker, we have demonstrated the solid-phase synthesis of test compounds by peptide synthesis, palladium-catalyzed cross coupling, and epoxidation. The linker proved to be stable toward the treatment with acids and bases. The photolysis rates of our pivaloyl linker (2) were compared with the rates of a o- nitrobenzyl photolinker (1) and proved to be superior.
- Peukert, Stefan,Giese, Bernd
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Read Online
- Solid-phase peptide synthesis in water using microwave-assisted heating
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An approach using water as a solvent (coupling and deprotection) was developed for the solid-phase synthesis of peptides using the most common Boc-amlno acid derivatives. Key aspects of this methodology are the use of a PEG-based resin, EDC-HONB as a coupling method, and microwave Irradiation as an energy source.
- Galanis, Athanassios S.,Albericio, Fernando,Grotli, Morten
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Read Online
- Peptide synthesis on fluorous support
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New fluorous supports were synthesized and used to prepare a peptide having a C-terminal COOH based on fluorous chemistry. The hexakisfluorous chain-type support was suitable for the synthesis of a pentapeptide or a peptide derivative on a fluorous support whose fluorine content is over 40 w/w%. A bioactive peptide, Leu-enkephalin, was easily synthesized using an Fmoc-strategy based on fluorous chemistry.
- Mizuno, Mamoru,Goto, Kohtaro,Miura, Tsuyoshi,Matsuura, Takeshi,Inazu, Toshiyuki
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Read Online
- Environmentally Conscious In-Water Peptide Synthesis Using Boc Strategy
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Seeking a more environmentally balanced method of peptide synthesis, we are focusing on developing organic solvent-free synthetic methods using water, an environmentally friendly solvent. In current peptide synthesis, the most common building blocks are Boc- and Fmoc-protected amino acids, which are highly soluble in organic solvents. We previously reported a technique for solid-phase peptide synthesis in water that utilizes Fmoc-amino acids converted to water-dispersible nanoparticles. The Boc strategy is well-known to be suitable for the industrial chemistry and green chemistry, because only gases are generated without any other by-products in the deprotection step of Boc group. Here we summarize in-water both liquid and solid-phase method using Boc-amino acids based on MW-assisted coupling reaction of nanosized reactants, nanoparticles and nanomicelles.
- Fujiwara, Suzuko,Hojo, Keiko,Inai, Hoshito,Manabe, Yuki,Tsuda, Yuko
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- Sustainable Peptide Synthesis Enabled by a Transient Protecting Group
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The growing interest in synthetic peptides has prompted the development of viable methods for their sustainable production. Currently, large amounts of toxic solvents are required for peptide assembly from protected building blocks, and switching to water as a reaction medium remains a major hurdle in peptide chemistry. We report an aqueous solid-phase peptide synthesis strategy that is based on a water-compatible 2,7-disulfo-9-fluorenylmethoxycarbonyl (Smoc) protecting group. This approach enables peptide assembly under aqueous conditions, real-time monitoring of building block coupling, and efficient postsynthetic purification. The procedure for the synthesis of all natural and several non-natural Smoc-protected amino acids is described, as well as the assembly of 22 peptide sequences and the fundamental issues of SPPS, including the protecting group strategy, coupling and cleavage efficiency, stability under aqueous conditions, and crucial side reactions.
- Avrutina, Olga,Knauer, Sascha,Koch, Niklas,Kolmar, Harald,Meusinger, Reinhard,Uth, Christina
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supporting information
p. 12984 - 12990
(2020/06/01)
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- TSBP auxiliary group, and liquid-phase total synthesis method of group-assisted enkephalin and derivatives thereof
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The invention relates to a TSBP auxiliary group, and a liquid-phase total synthesis method of group-assisted enkephalin and derivatives thereof. The synthesis method comprises the following steps: replacing solid-phase resin by TSBP to be connected with the C-end of N-end PG protected amino acid (PG-Xaa-OH, Xaa is any amino acid); carrying out separation and purification, removing N-terminal PG, carrying out polypeptide coupling, repeatedly coupling corresponding amino acids, and constructing an amino acid sequence H2N-Tyr (OtBu)-Gly-Gly-Phe-Xaa-TSBP of the enkephalin or the derivative precursor of the enkephalin; and carrying out side chain deprotection, and carrying out separation and purification on the enkephalin and derivatives thereof to obtain the enkephalin and derivatives thereofH2N-Tyr-Gly-Gly-Phe-Xaa-ZH (Z = O, S, NH and the like). Compared with the existing synthesis method, the method disclosed by the invention has the advantages of both liquid-phase and solid-phase synthesis methods, so that the glutathione can be synthesized and prepared more simply, conveniently, quickly, economically and efficiently; besides, the phosphate carrier can be recycled and directly reused, so that the waste of raw materials is reduced, the waste pollution is reduced, the cost is saved, and the method is beneficial to environmental protection.
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Paragraph 0057-0058; 0114; 0115-0120
(2020/07/02)
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- Exploration of the fifth position of leu-enkephalin and its role in binding and activating delta (DOP) and mu (MOP) opioid receptors
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Enkephalins are pentapeptidic endogenous ligands that regulate nociception by binding to mu (MOP) and delta (DOP) opioid receptors. To further explore the role of the leucine residue of Leu-enkephalin, 12 peptidomimetic analogs were synthesized by systematically replacing this residue with non-natural amino acids. The analogs were tested for their ability to bind DOP and MOP. We also investigated the potency of these analogs to inhibit cAMP production and to recruit β-arrestin 2 via both receptors. We found that replacement of the leucine residue by substituted non-natural amino acid derivatives of alanine, cycloleucine, or isoleucine was generally well tolerated. By contrast, substituting leucine with homoproline greatly reduced the affinity for DOP and, to a lesser extent, for MOP. Interestingly, when compared to Leu-enkephalin, analogs containing either aza-β-homoleucine or cycloleucine showed a bias toward inhibition of cAMP production through the activation of DOP but not MOP. By contrast, derivatives containing 4,5-dehydroleucine orD-allo-isoleucine conferred a bias toward β-arrestin 2 at MOP, but not DOP. Our results suggest that position 5 in Leu-enkephalin analogs can be further exploited to develop compounds with the potential to produce bias toward G protein or β-arrestin 2.
- Ndong, Dominique Bella,Blais, Véronique,Holleran, Brian J.,Proteau-Gagné, Arnaud,Cantin-Savoie, Isabelle,Robert, William,Nadon, Jean-Fran?ois,Beauchemin, Sophie,Leduc, Richard,Pi?eyro, Graciela,Guérin, Brigitte,Gendron, Louis,Dory, Yves L.
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- METHOD FOR PREPARING PEPTIDES
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The invention relates to a method for preparing peptides comprising the step of forming a peptide bond wherein the carboxyl group of a first amino acid or first peptide is activated and an amino group of the first activated amino acid or first peptide is protected by a protecting group having a water-solubility enhancing group and the activated carboxyl group of the first amino acid or first peptide is reacted with an amino group of a second amino acid or second peptide wherein said carboxyl group of the first amino acid or first peptide is activated in the absence of the second amino acid or second peptide. The invention further relates to peptides comprising a protecting group having a water-solubility enhancing group being bound to the amino group and an activated or free carboxyl group.
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Page/Page column 64-65
(2019/06/11)
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- N-Guanidyl and C-Tetrazole Leu-Enkephalin Derivatives: Efficient Mu and Delta Opioid Receptor Agonists with Improved Pharmacological Properties
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Leu-enkephalin and d-Ala2-Leu-enkephalin were modified at their N- and C-termini with guanidyl and tetrazole groups. The resulting molecules were prepared in solution or by solid phase peptide synthesis. The affinity of the different analogues at mu (MOP) and delta opioid receptors (DOP) was then assessed by competitive binding in stably transfected DOP and MOP HEK293 cells. Inhibition of cAMP production and recruitment of β-arrestin were also investigated. Finally, lipophilicity (logD7.4) and plasma stability of each compound were measured. Compared to the native ligands, we found that the replacement of the terminal carboxylate by a tetrazole slightly decreased both the affinity at mu and delta opioid receptors as well as the half-life. By contrast, replacing the ammonium at the N-terminus with a guanidyl significantly improved the affinity, the potency, as well as the lipophilicity and the stability of the resulting peptides. Replacing the glycine residue with a d-alanine in position 2 consistently improved the potency as well as the stability of the analogues. The best peptidomimetic of the whole series, guanidyl-Tyr-d-Ala-Gly-Phe-Leu-tetrazole, displayed sub-nanomolar affinity and an increased lipophilicity. Moreover, it proved to be stable in plasma for up to 24 h, suggesting that the modifications are protecting the compound against protease degradation.
- Beaudeau, Jean-Louis,Blais, Véronique,Holleran, Brian J.,Bergeron, Alexandre,Pineyro, Graciela,Guérin, Brigitte,Gendron, Louis,Dory, Yves L.
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p. 1615 - 1626
(2019/02/01)
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- Diethylphosphoryl-oxymab (DEPO-B) as a solid coupling reagent for amide bond formation
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A new phosphonium based coupling reagent DEPO-B has been synthesized from 5-(hydroxyimino)-1,3-dimethylpyrimidine-2,4,6 (1H,3H,5H)-trione (Oxyma B) and diethyl chlorophos-phate in presence of base. It is a solid material and the hydrolytic stability and solubility was evaluated for confirming its capability for usage in automated peptide synthesizer.
- Kumar, Ashish,Jad, Yahya E.,El-Faham, Ayman,de la Torre, Beatriz G.,Albericio, Fernando
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- A PROCESS FOR PREPARATION OF A PEPTIDE
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The present invention relates to a novel process for preparation of peptides having amino acid chain length in the range of 2-40 comprises the steps: i) attaching an end-blocked amino acid with an ionic liquid based solid support in presence of an ionic solvent to obtain an end-terminal blocked amino acid attached ionic liquid; ii) removing end-terminal blocking agent from the end-terminal blocked amino acid attached ionic liquid of step i) followed by work up to obtain an amino acid attached ionic liquid; iii) repeating steps i) through ii) one or more times to obtain a polypeptide attached ionic liquid; and iv) detaching the polypeptide from the polypeptide attached ionic liquid of step iii) to obtain the polypeptide. Said process does not use any auxiliary reagents like dehydrating agent or activating agent. The use of ionic liquids as supports as well as solvents result in the faster kinetics of the process, the separation issues are reduced, and the process has no racemization issues.
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Page/Page column 14; 15-16
(2018/07/29)
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- Recyclable hypervalent-iodine-mediated solid-phase peptide synthesis and cyclic peptide synthesis
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The system of the hypervalent iodine(III) reagent FPID and (4-MeOC6H4)3P was successfully applied to solid-phase peptide synthesis and cyclic peptide synthesis. Four peptides with biological activities were synthesized through SPPS and the bioactive cyclic heptapeptide pseudostellarin D was obtained via solution-phase peptide synthesis. It is worth noting that FPID can be readily regenerated after the peptide coupling reaction.
- Liu, Dan,Guo, Ya-Li,Qu, Jin,Zhang, Chi
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supporting information
p. 1112 - 1119
(2018/06/04)
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- Modulation of the Interaction between a Peptide Ligand and a G Protein-Coupled Receptor by Halogen Atoms
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Systematic halogenation of two native opioid peptides has shown that halogen atoms can modulate peptide-receptor interactions in different manners. First, halogens may produce a steric hindrance that reduces the binding of the peptide to the receptor. Second, chlorine, bromine, or iodine may improve peptide binding if their positive σ-hole forms a halogen bond interaction with negatively charged atoms of the protein. Lastly, the negative electrostatic potential of fluorine can interact with positively charged atoms of the protein to improve peptide binding.
- Rosa, Mònica,Caltabiano, Gianluigi,Barreto-Valer, Katy,Gonzalez-Nunez, Verónica,Gómez-Tamayo, José C.,Ardá, Ana,Jiménez-Barbero, Jesús,Pardo, Leonardo,Rodríguez, Raquel E.,Arsequell, Gemma,Valencia, Gregorio
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supporting information
p. 872 - 876
(2015/08/24)
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- A prodrug nanoparticle approach for the oral delivery of a hydrophilic peptide, leucine5-enkephalin, to the brain
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The oral use of neuropeptides to treat brain disease is currently not possible because of a combination of poor oral absorption, short plasma half-lives and the blood-brain barrier. Here we demonstrate a strategy for neuropeptide brain delivery via the (a) oral and (b) intravenous routes. The strategy is exemplified by a palmitic ester prodrug of the model drug leucine5-enkephalin, encapsulated within chitosan amphiphile nanoparticles. Via the oral route the nanoparticle-prodrug formulation increased the brain drug levels by 67% and significantly increased leucine 5-enkephalin's antinociceptive activity. The nanoparticles facilitate oral absorption and the prodrug prevents plasma degradation, enabling brain delivery. Via the intravenous route, the nanoparticle-prodrug increases the peptide brain levels by 50% and confers antinociceptive activity on leucine 5-enkephalin. The nanoparticle-prodrug enables brain delivery by stabilizing the peptide in the plasma although the chitosan amphiphile particles are not transported across the blood-brain barrier per se, and are excreted in the urine.
- Lalatsa, Aikaterini,Lee, Vivian,Malkinson, John P.,Zloh, Mire,Schaetzlein, Andreas G.,Uchegbu, Ijeoma F.
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experimental part
p. 1665 - 1680
(2012/09/08)
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- Nicotinoyl peptide derivatives and cosmetic composition comprising the same
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The present invention relates to nicotinoyl peptide derivatives, wherein neuropeptides are connected to nicotinic acid, and a cosmetic composition comprising the same. The peptide derivatives according to the present invention have good collagen generation and anti-inflammatory activities to show an excellent anti-wrinkle effect, little cytotoxicity, water-solubility, and good stability during long-term storage. Therefore, they can be effectively used for preparing an anti-aging cosmetic composition.
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- Neutrophil-mediated oxidation of enkephalins via myeloperoxidase-dependent addition of superoxide
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Neutrophils play a major role in acute inflammation in part by generating superoxide and an array of other reactive species. These white blood cells also contribute to protection against inflammatory pain by releasing opioid peptides. The biochemical interactions of enkephalins with neutrophil-derived oxidants are not well understood. In this investigation we reveal that neutrophils use myeloperoxidase to oxidize enkephalins to their corresponding tyrosyl free radicals, which react preferentially with the superoxide to form a hydroperoxide. In methionine enkephalin, rapid intramolecular oxygen transfer from the hydroperoxide to the Met sulfur results in the formation of a sulfoxide derivative. This reaction may occur at sites of inflammation where enkephalins are released and neutrophils generate large amounts of superoxide. Hydroperoxide formation destroys the aromatic character of the Tyr residue by forming a bicyclic structure via conjugate addition of the terminal amine to the phenol ring. As the N-terminal Tyr and its amino group are essential for their opiate activity, we hypothesize that oxidative modification of this residue should affect the analgesic activity of enkephalins.
- Nagy, Péter,Kettle, Anthony J.,Winterbourn, Christine C.
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scheme or table
p. 792 - 799
(2011/11/12)
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- THAL, a sterically unhindered linker for the solid-phase synthesis of acid-sensitive protected peptide acids
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(Chemical Equation Presented) The 5-(4-hydroxyphenyl)-3,4- ethylenedioxythienyl alcohol (THAL, Thiophene Acid Labile) is described as a new linker for the solid-phase synthesis of peptide carboxylic acids. It is based on the electron-rich 3,4-ethylenedioxythenyl (EDOTn) moiety and allows the obtention of free and tert-butyl-protected peptides by cleavage with 90% and 0.5% TFA, respectively. This very high acid lability makes it useful for the synthesis of sensitive peptides. Free and tert-butyl- protected Leu-enkephalins have been synthesized as models to demonstrate the utility of the linker.
- Isidro-Llobet, Albert,Boas, Ulrik,Jensen, Knud J.,Alvarez, Mercedes,Albericio, Fernando
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experimental part
p. 7342 - 7344
(2009/04/18)
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- A nonacid degradable linker for solid-phase synthesis
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Synthesis and applications of two new nonacid degradable linkers as an alternative to the Wang linker for solid-phase synthesis are described. Resin from linker 2 looks superior to linker 1 in terms of yields for both anchoring of the first building block and cleavage and in terms of higher purity of the final product. Use of linker 2 avoids side reactions associated with the use of Wang resin due to an undesired cleavage during final acid treatment.
- Colombo, Aina,De La Figuera, Natalia,Fernandez, Joan Carles,Fernandez-Forner, Dolors,Albericio, Fernando,Forns, Pilar
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p. 4319 - 4322
(2008/03/11)
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- MW-enhanced high-speed deprotection of Boc group using p-TsOH and concommitant formation of N-Me-amino acid benzyl ester p-TsOH salts
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A high-speed, complete deprotection of Boc group from Boc amino acids and protected peptide esters employing p-TsOH in toluene under microwave irradiation is found to be complete in 30s. The deprotection can be carried out in methanol and acetonitrile also. Under the present conditions, C-peptide benzyl esters and O-benzyl ethers have been found to be stable. This has permitted us to carry out the synthesis of [Leu]enkephalin employing the Boc/Bzl-group strategy. Further more, it has been found that both Nα-Fmoc and N α-Z groups are completely stable. The present conditions can be extended for the concomitant removal of the Boc group and the formation of C-benzyl amino acid esters as well. This has been utilized for the synthesis of N-Me amino acid benzyl esters starting from Boc-N-Me amino acids in a single step. Copyright Taylor & Francis, Inc.
- Suresh Babu, Vommina V.,Patil, Basanagoud S.,Vasanthakumar, Ganga-Ramu
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p. 1795 - 1802
(2007/10/03)
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- Photogenerated reagents
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This invention describes reagent precursors and methods for chemical and biochemical reactions. These reagent precursors that can be activated in solution upon irradiation to generate reagents required for the subsequent chemical reactions. Specifically, photogenerated reagents (PGR) are useful for controlling parallel combinatorial synthesis and various chemical and biochemical reactions.
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Page/Page column 22-23
(2008/06/13)
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- Studies on the synthesis and anti-Osteoporosis of estrogen-GHRPs linkers.
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The linkers of estrogen-GHRPs were prepared by the combination of estradiol, estrone, TyrGlyGlyPheLeuOH, and TyrGlyGlyPheLeuOH. Their anti-osteoporosis effect was evaluated by analyzing the data, for instance the weight of the body, femur, femur ash, the content of calcium and phosphor in the femur, the content of calcium and ALP activity in the serum, obtained from the corresponding bioassay in vivo. The results indicated that the anti-osteoporosis potency for estradiol, estrone, TyrGlyGlyPheLeuOH and TyrGlyGlyPheLeuNH(2) may be totally enhanced each other via the corresponding linkers.
- Wang, Chao,Cui, Weina,Zhao, Ming,Yang, Jian,Peng, Shiqi
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p. 143 - 146
(2007/10/03)
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- Studies on the synthesis of estrogen-GHRPS linkers
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A series of linkers consisted of estrone, estradiol, and GHRPS (growth hormone releasing peptides) were prepared with carboxyl-methyl or succinyl as the conjugated group. As a protective group in the side chain of Tyr, the Dcb (2,6-dichlorobenzyl) in the intermediates which is unstable to HF can be removed in high yield in the presence of Pd/C (10%), H2, and 4.4% formic acid in methanol.
- Wang, Chao,Zhao, Ming,Cui, Weina,Yang, Jian,Peng, Shiqi
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p. 1633 - 1641
(2007/10/03)
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- Sequentially photocleavable protecting groups in solid-phase synthesis
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(Matrix presented) A sequential solid-phase peptide synthesis was developed using both photolabile linker and protecting groups. The chromatic sequential lability between a tert-butyl ketone-derived linker (sensitive to irradiation at 305 nm) and a nitroveratryloxycarbonyl (NVOC) group (sensitive at 360 nm) was exploited to prepare Leu-Enkephalin in a 55% overall yield. This new strategy allows the preparation of peptides in essentially neutral medium, by avoiding the use of common deprotection reagents such as trifluoroacetic acid or piperidine.
- Kessler, Martin,Glatthar, Ralf,Giese, Bernd,Bochet, Christian G.
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p. 1179 - 1181
(2007/10/03)
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- Amino-protecting groups subject to deblocking under conditions of nucleophilic addition to a Michael acceptor. Structure-Reactivity studies and use of the 2-(tert-Butylsulfonyl)-2-propenyloxycarbonyl (Bspoc) group
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A new type of amino-protecting group is described in which a Michael acceptor is incorporated into the protectant so that treatment with a nucleophile will trigger deblocking. Comparison of various Michael acceptors showed that for several key electron-withdrawing groups, the order of reactivity was C6H5SO2 > Me3CSO2 > COOEt > C6H5SO > C6H4NO2-p. The reactivity of the nucleophile (e.g., primary and secondary aliphatic amines) followed an order related to both intrinsic basicity and steric effects. β- Substituents in the Michael acceptor caused significant retardation of the deblocking process. The Bspoc function was chosen for initial elaboration into a practical system for use in peptide synthesis. Bspoc amino acid chlorides were used as coupling agents and silica-tethered secondary amines as deblocking agents. With the latter, deblocking occurs cleanly and no byproducts remain in the organic solvent in which the deblocking is executed.
- Carpino, Louis A.,Philbin, Michael
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p. 4315 - 4323
(2007/10/03)
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- Phenylpropionic acid-based cyclic prodrugs of opioid peptides that exhibit metabolic stability to peptidases and excellent cellular permeation
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Purpose. To evaluate the cellular permeation characteristics and the chemical and enzymatic stability of phenylpropionic acid-based cyclic prodrugs 1 and 2 of opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly-Phe- Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. Methods. The rates of conversion of cyclic prodrugs 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH 7.4 (Caco-2 cell transport buffer) and in various biological media having measurable esterase activity were determined by HPLC. The cell permeation characteristics of [Leu5]-enkephalin, DADLE, and cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto microporus membranes and monitored by HPLC. Results. In HBSS, pH 7.4, cyclic prodrugs 1 and 2 degraded to [Leu5]-enkephalin and DADLE, respectively, in stoichiometric amounts. In 90% human plasma, the rates of disappearance of cyclic prodrugs 1 and 2 were slightly faster than in HBSS, pH 7.4. These accelerated rates of disappearance in 90% human plasma could be reduced to the rates observed in HBSS, pH 7.4, by pretreatment of the plasma with paraoxon, a known inhibitor of serine-dependent esterases. In homogenates of Caco-2 cells and rat liver, accelerated rates of disappearance of cyclic prodrugs 1 and 2 were not observed. When applied to the AP side of a Caco-2 cell monolayer, cyclic prodrug 1 exhibited significantly greater stability against peptidase metabolism than did [Leu5]-enkephalin. Cyclic prodrug 2 find DADLE exhibited stability similar to prodrug 1 when applied to the AP side of the Caco-2 cell monolayers. Prodrug 1 was 1680 fold more able to permeate the Caco-2 cell monolayers than was [Leu5]-enkephalin, in part because of its increased enzymatic stability. Prodrug 2 was shown to be approximately 77 fold more able to permeate a Caco-2 cell monolayer than was DADLE. Conclusions. Cyclic prodrugs 1 and 2, prepared with the phenylpropionic acid promoiety, were substantially more able to permeate Caco-2 cell monolayers than were the corresponding opioid peptides. Prodrug 1 exhibited increased stability to peptidase metabolism compared to [Leu5]- enkephalin. In 90% human plasma but not in Caco-2 cell and rat liver homogenates, the opioid peptides were released from the cyclic prodrugs by an esterase-catalyzed reaction that is sensitive to paraoxon inhibition. However, the rate of this bioconversion appears to be extremely slow.
- Gudmundsson, Olafur S.,Nimkar, Kalpana,Gangwar, Sanjeev,Siahaan, Teruna,Borchardt, Ronald T.
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- Acyloxyalkoxy-based cyclic prodrugs of opioid peptides: Evaluation of the chemical and enzymatic stability as well as their transport properties across Caco-2 cell monolayers
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Purpose. To evaluate the chemical and enzymatic stability, as well as the cellular permeation characteristics, of the acyloxyalkoxy-based cyclic prodrugs 1 and 2 of the opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly- Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. Methods. The rates of conversion of 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, were measured by HPLC in HBSS, pH = 7.4, and in various biological media (e.g., human plasma and Caco-2 cell and rat liver homogenates) having measurable esterase activity. The cellular permeation and metabolism characteristics of [Leu5]-enkephalin, DADLE and the cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto microporous membranes and monitored by HPLC. Results. Cyclic prodrugs 1 and 2 degraded slowly but stoichiometrically to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH = 7.4. In homogenates of Caco-2 cells and rat liver, as well as 90% human plasma, the rates of disappearance of the cyclic prodrugs were significantly faster than in HBSS. The stabilities of the cyclic prodrugs 1 and 2 were increased significantly in 90% human plasma and Caco-2 cell homogenates when paraoxon, a potent inhibitor of serine-dependent esterases, was included in the incubation mixtures. A similar stabilizing effect of paraoxon was not observed in 50% rat liver homogenates, but was observed in 10% homogenates of rat liver. When applied to the AP side of a Caco-2 cell monolayer, DADLE and cyclic prodrugs 1 and 2 exhibited significantly greater stability than [Leu5]-enkephalin. Based on their physicochemical properties (i.e., lipophilicity), cyclic prodrugs 1 and 2 should have exhibited high permeation across Caco-2 cell monolayers. Surprisingly, the AP-to-BL apparent permeability coefficients (P(App)) for cyclic prodrugs 1 and 2 across Caco-2 cell monolayers were significantly lower than the P(App) value determined for the metabolically stable opioid peptide DADLE. When the P(App) values for cyclic prodrugs 1 and 2 crossing Caco-2 cell monolayers in the BL-to-AP direction were determined, they were shown to be 36 and 52 times greater, respectively, than the AP-to-BL values. Conclusions. Cyclic prodrugs 1 and 2, prepared with an acyloxyalkoxy promoiety, were shown to degrade in biological media (e.g., 90% human plasma) via an esterase-catalyzed pathway. The degradation of cyclic prodrug 1, which contained an ester formed with an L-amino acid, degraded more rapidly in esterase-containing media than did prodrug 2, which contained an ester formed with a D-amino acid. Cyclic prodrugs 1 and 2 showed very low AP-to-BL Caco-2 cell permeability, which did not correlate with their lipophilicities. These low AP-to-BL permeabilities result because of their substrate activity for apically polarized efflux systems.
- Bak, Annette,Gudmundsson, Olafur S.,Friis, Gitte J.,Siahaan, Teruna J.,Borchardt, Ronald T.
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- Backbone amide linker (BAL) strategy for solid-phase synthesis of C-terminal-modified and cyclic peptides
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Peptide targets for synthesis are often desired with C-terminal end groups other than the more usual acid and amide functionalities. Relatively few routes exist for synthesis of C-terminal-modified peptides-including cyclic peptides - by either solution or solid-phase methods, and known routes are often limited in terms of ease and generality. We describe here a novel Backbone Amide Linker (BAL) approach, whereby the growing peptide is anchored through a backbone nitrogen; thus allowing considerable flexibility in management of the termini. Initial efforts on BAL have adapted the chemistry of the tris(alkoxy)benzylamide system exploited previously with PAL anchors. Aldehyde precursors to PAL, e.g. 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid, were reductively coupled to the α-amine of the prospective C-terminal amino acid, which was blocked as a tert-butyl, allyl, or methyl ester, or to the appropriately protected C-terminal-modified amino acid derivative. These reductive aminations were carried out either in solution or on the solid phase, and occurred without racemization. The secondary amine intermediates resulting from solution amination were converted to the 9-fluorenylmethoxycarbonyl (Fmoc)-protected preformed handle derivatives, which were then attached to poly(ethylene glycol)-polystyrene (PEG-PS) graft or copoly(styrene - 1% divinylbenzene) (PS) supports and used to assemble peptides by standard Fmoc solid-phase chemistry. Alternatively, BAL anchors formed by onresin reductive amination were applied directly. Conditions were optimized to achieve near-quantitative acylation at the difficult step to introduce the penultimate residue, and a side reaction involving diketopiperazine formation under some circumstances was prevented by a modified protocol for Nα-protection of the second residue/ introduction of the third residue. Examples are provided for the syntheses in high yields and purities of representative peptide acids, alcohols, N,N-dialkylamides, aldehydes, esters, and head-to-tail cyclic peptides. These methodologies avoid postsynthetic solution-phase transformations and are ripe for further extension.
- Jensen, Knud J.,Alsina, Jordi,Songster, Michael F.,Vágner, Josef,Albericio, Fernando,Barany, George
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p. 5441 - 5452
(2007/10/03)
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- Synthesis and use of amino acid fluorides as peptide coupling reagents
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The present invention is directed to the process of preparing a peptide comprising reacting a first amino acid or peptide with an amino acid fluoride of the formula: STR1 or the acid fluoride salts thereof wherein BLK is an N-amino protecting group AA is an amino acid residue and X is H or a protecting group useful, and the first amino and peptide have a free amino group and a blocked carboxy end.
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- Phosphorylation of enkephalins enhances their proteolytic stability
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Pharmacological action of enkephalins as opioid peptides is limited because of their rapid degradation by endoproteases. A novel approach is used in this study to prolong the life of those peptides. Phosphorylation of N-terminal tyrosine residue is found to have a profound influence in improving the stability of [Met]enkephalin and [Leu]enkephalin against the action of aminopeptidase M. Whereas, breakdown of [Met]enkephalin and [Leu]enkephalin is essentially complete in less than one min when incubated at 37°C with purified aminopeptidase M (EC 3.4.11.2; substrate:enzyme = 1:0.1) in Tris buffer (pH 7.02), the corresponding phospho analogs are still detected 60 min after start of incubation. The rate of disappearance of phospho-[Met]enkephalin and phospho-[Leu]enkephalin follows first-order kinetics with half-lives of 7.3 and 8.8 min, respectively.
- Dass, Chhabil,Mahalakshmi
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p. 1039 - 1045
(2007/10/03)
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- A Novel Amino Protection-Deprotection Procedure and Its Application in Solid Phase Peptide Synthesis
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N-4,4-Dimethyl-2,6-dioxocyclohexylidenemethyl (Dcm) amino acids are readily prepared and the protecting group can be removed with hydrazine at room temperature; their potential in solid phase peptide synthesis is illustrated.
- Bycroft, Barrie W.,Chan, Weng C.,Chhabra, Siri Ram,Teesdale-Spittle, Paul H.,Hardy, Paul M.
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p. 776 - 777
(2007/10/02)
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- Synthesis and application of N,N-bis-(1-adamantyloxycarbonyl) amino acids.
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The preparation of novel bis-(1-adamantyloxycarbonyl) amino acid derivatives has been undertaken and their properties studied. Among them, the p-nitrophenyl esters were subsequently applied to the stepwise synthesis of Leu-enkephalin. In the last coupling step, some hydantoin formation was encountered but it could be nearly completely overcome by working with more concentrated solution. The preparation of a tyrosine derivative presented special problems owing to the existence of the phenolic group in the precursor. The relative stability of 1-adamantyloxycarbonyl as N- and O-protecting groups was also studied.
- Nyasse,Ragnarsson
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p. 374 - 379
(2007/10/02)
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- Acetol: A useful new protecting group for peptide synthesis
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Acetol can be conveniently used as a reagent for the protection of carboxylic acids in peptide synthesis. It has been found to be inert and stable to hydrogenolytic and acidolytic conditions normally used in peptide synthesis. Deblocking has been selectively achieved under mild conditions using Bu4NF .3H2O in THF.
- Kundu, Bijoy
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p. 3193 - 3196
(2007/10/02)
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- Total Masking -Gly Bonds by Highly Lipophilic and Chromophoric Ferrocenylmethyl Residue in Peptide Syntheses of Hexaglycine and Leu-Enkephalin
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Highly lipophilic and chromophoric ferrocenylmethyl residue is applied to syntheses of peptides hexaglycine and Leu-enkephalin, masking therein all -Gly bonds.Thereby Fem groups influence properties of Fem-peptide derivatives advantageously both in chemosyntheses and cleaning operations, leading to constantly high yields et each step.Despite of its volumous dimension the Fem residue can be introduced into each peptide bond of oligoglycine by succeeding one another of building blocks of H-Fem-Gly-OMe.Strong alkaline conditions during the hydrolyses of methyl esters occurring several times in peptide derivatives do not influence the Gly-Gly-bond at all.Total protected derivatives BOC-(Fem-Gly-)6-OMe and BOC-Tyr(t-Bu)-(Fem-Gly-)2Phe-Leu-OtBu even are soluble in hexane/ethylacetate (1:1).
- Eckert, Heiner,Forster, Barbara,Seidel, Christoph
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p. 339 - 352
(2007/10/02)
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- The use of p-toluenesulfonic acid for removal of the N-T-butoxy-carbonyl protecting group in solid phase peptide synthesis
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The usefulness of p-toluenesulfonic acid in THF/CH2Cl2 for Boc removal in solid phase peptide synthesis has been demonstrated through the syntheses of Leu-5 and Met-5 enkephalin acids.
- Brinkman,Landi Jr.,Paterson Jr.,Stone
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p. 459 - 465
(2007/10/02)
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- PENTAFLUOROPHENYL DIPHENYLPHOSPHINATE A NEW EFFICIENT COUPLING REAGENT IN PEPTIDE CHEMISTRY
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Pentafluorophenyl diphenylphosphinate was found to be a new efficient coupling reagent for the racemization-free synthesis of peptides.It has been applied in both the solution and the solid phase peptide synthesis.
- Chen, Shaoqing,Xu, Jiecheng
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p. 6711 - 6714
(2007/10/02)
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- A new technique for rapid peptide coupling
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The present invention is directed to a process for forming a peptide bond between a first AMINO ACID containing a free N-terminal amino group and an acylating N-terminal amino protected second AMINO ACID which comprises reacting under amide forming conditions said first AMINO ACID with said second AMINO ACID in the presence of a basic and catalytic effective amount of a mixture comprising a tertiary amine of the formula: and a N-hydroxy nitrogen containing heterocycle or the quaternary ammonium salt of said mixture, wherein, R1, R2, and R3 are independently lower alkyl, or R1 and R2 together with the nitrogen to which they are attached form a heterocyclic ring.
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- SYNTHESIS OF ENKEPHALINS BY THE METHOD OF POLYMERIC ACTIVATED ESTERS BASED ON 4-HYDROXY-3-NITROBENZOPHENONE
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Leucine- and methionine-enkephalins have been synthesized by the successive growth of the peptide chain from the C-end by the method of polymeric activated esters based on 4-hydroxy-3-nitrobenzophenone with yields of 90 and 70percent, respectively, calculated on the initial C-terminal amino acid.Polystyrene with 2percent of divinylbenzene was used as the polymeric matrix.Using the synthesis of methionine-enkephalin as an example, the possibility has been shown of using polymeric activated esters for the synthesis of peptides with a free carboxy group.
- Grigor'ev, E. I.,Chernova, S. V.
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p. 468 - 474
(2007/10/02)
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- Investigation of the Reaction between Amino Acids or Amino Acid Esters and 9-Formylfluorene and Its Equivalents. Possible Utility of the Derived Enamines as Amino Group Protectants
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Treatment of 9-(hydroxymethylene)fluorene/9-formylfluorene (storable as the hemiacetal with methanol, 7) with amino acids and amino acid esters yields the corresponding enamines 8, which may be considered to be hydrocarbon analogues of N-formyl amino acid derivatives.Attempted coupling of the free acids 8 (R'=H) with amino acid esters failed, suggesting insufficient reduction in basicity of the amino group due to the enamine residue.The introduction of electron-withdrawing substituents into the fluorene ring decreases the basicity sufficiently to allow normal peptide coupling reactions, as for example with the 2,7-dichloro analogues derived from 17.Thus phenylalanine derivative 18 treated with leucine methyl ester and DCC gave dipeptide 19.The DC-FM-bar group could be removed by catalytic transfer hydrogenolysis.Mild acid hydrolysis represents a second general deblocking technique for the FM-bar function.It was demonstrated in a model study involving the highly sensitive amino acid α-phenylglycine that the FM-bar protecting group was less prone to cause racemization than the benzyloxycarbonyl function.It was demonstrated that the simple pentapeptide leucine enkephalin 29 could be synthesized using α-DC-FM-bar protection along with tert-butyl-based side chain protecting groups.
- Carpino, Louis A.,Chao, Hann Guang,Tien, Jien-Heh
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p. 4302 - 4313
(2007/10/02)
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- PROTECTION OF THE CARBOXY GROUP IN THE FORM OF THE 2-CYANOETHYL ESTERS IN SYNTHESIS OF PEPTIDES
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With the aim of studying the suitability of the 2-cyano group for the protection of carboxylic functions in peptide synthesis, we have obtained the 2-cyanoethyl esters of a number of amino acids and have studied their behavior under the conditions of peptide synthesis.The synthesis of the pentapeptide leucine-enkephalin has been performed with the use of 2-cyanoethyl protection for C-terminal carboxy groups throughout.The physicochemical characteristics of the compounds synthesized are given.
- Kalashnikov, V. V.,Samukov, V. V.
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p. 351 - 354
(2007/10/02)
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- Synthesis of peptide analogs
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A novel polymeric disc, wafer or similarly shaped resin for carrying out the synthsis of peptide analogs via solid phase peptide synthesis techniques is provided. The polymeric disc or wafer of the invention may be made out of those resin materials presently used in bead form in solid phase peptide synthesis, such as, benzhydrylamine resins, Boc-aminoacyl-4-(oxymethyl)-phenyacetamidomethyl (Pam) resin, polyamide resins and chloromethyl resins. The disc or wafer of the invention should, preferably, have a thickness of 200-400 μm and may be of any suitable length or width. A process for the synthsis of peptide analogs utilizing the polymeric disc or wafer of the present invention is also disclosed.
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- AMINO ACIDS AND PEPTIDES. VIII. A WATER - SOLUBLE ACTIVE ESTER, PHENOLSULFONIC ACID DERIVATIVE
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N-protected amino acid 4-phenolsulfonic acid derivative esters, such as 2,6-dibromo-4-sulfophenyl ester sodium salt, 2,6-dichloro-4-sulfophenyl ester potassium salt and 2-nitro-4-sulfophenyl ester sodium salt, were prepared.These esters are water - soluble active esters, and were applied for rhe synthesys of Leu-enkephalin.Keywords - water - soluble active ester; 2,6-dichloro-4-sulfophenyl ester; 2,6-dibromo-4-sulfophenyl ester; 2-nitro-4-sulfophenyl ester; peptide synthesis; Leu-enkephalin
- Kawasaki, Koichi,Tsuji, Toshiki,Maeda, Misuko,Matsumoto, Tatsuya,Hirase, Katsuhiko
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p. 1044 - 1048
(2007/10/02)
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- Anchoring of Amino Acids on Hydroxy Group-Containing Resins and Their Application to Peptide Synthesis Using N-Tritylamino Acid 1-Benzotriazolyl Esters
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Trt- and Fmoc-amino acids has been attached to p-alkoxybenzylalcohol resin (ABAR, 1) and diphenylmethanol resin (DPMR, 2) using triphenylphosphan (TPP) and diethyl azodicarboxylate (DEAD) at 0 deg C in tetrahydrofuran.The derived resins have been applied in "solid-phase" peptide synthesis using N-tritylamino acid 1-benzotriazolyl esters 9.
- Barlos, Kleomenis,Gatos, Dimitrios,Kallitsis, John,Papaioannou, Dionysios,Sotiriou, Petros,Schaefer, Wolfram
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p. 1031 - 1036
(2007/10/02)
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- A Novel Preparation of Amino Acid Diphenylmethyl Esters and Their Application in Peptide Synthesis
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Trityl amino acid diphenylmethyl esters 2 were prepared by Mitsunobu condensation of tritylamino acids 1 with diphenylmethanol using excess triphenylphosphane and diethyl azodicarboxylate.The esters 2 were converted into the corresponding p-toluenesulfonates 3 on treatment with p-toluenesulfonic acid.The removal of the N-trityl group in the presence of the diphenylmethyl ester group is studied by the preparation of leucine-enkephalin (12).Best selectivity is obtained when 1-hydroxybenzotriazole in trifluoroethanol is used as detritylation reagent.
- Barlos, Kleomenis,Kallitsis, John,Mamos, Petros,Patrianakou, Stella,Stavropoulos, Georg
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p. 633 - 636
(2007/10/02)
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- AMINO ACIDS AND PEPTIDES. VII. PREPARATION AND APPLICATION OF A WATER-SOLUBLE ACTIVE ESTER, p-TRIMETHYLAMMONIOPHENYL ESTER
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A water-soluble active ester, p-trimethylammoniophenyl ester iodide, and its nitro derivative were prepared from N-procteted amino acid and the corresponding phenol derivative by the dicyclohexylcarbodiimide method.The synthetic esters were applied to the synthesis of Leuenkephalin.Keywords--water-soluble active ester; p-trimethylammoniophenyl ester iodide; Leuenkephalin; peptide synthesis
- Tsuji, Toshiki,Okusada, Satoshi,Maeda, Mitsuko,Kawasaki, Koichi
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p. 2214 - 2217
(2007/10/02)
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- A Three-Dimensional Orthogonal Protection Scheme for Solid-Phase Peptide Synthesis under Mild Conditions
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Several Nα-dithiasuccinoyl (Dts) amino acids (1) were esterified, by use of N,N'-dicyclohexylcarbodiimide (DCC) and without racemization, to tert-butyl 4-(hydroxymethyl)-3-nitrobenzoate (8).The resultant handle derivatives 4 were treated with trifluoroacetic acid to yield crystalline 4-(Nα-Dts-aminoacyloxymethyl)-3-nitrobenzoic acids (3), which were quantitatively incorporated onto aminomethylcopoly(styrene-1percent divinylbenzene)-resins by DCC-mediated couplings to give the starting point for stepwise solid-phase synthesis of peptides anchored as o-nitrobenzyl (ONb) esters.Assembly of the protected leucine-enkephalin-resin derivative Dts-Tyr(t-Bu)-Gly-Gly-Phe-Leu-ONb-resin (2) was achieved from the appropiate Dts-amino acids by a highly efficient protocol.By carrying out, in each conceivable order, either in solution or on the solid phase, one two, or all three of the following orthogonal treatments , the common resin-bound intermediate 2 became the source of four partially or fully deblocked leucine-enkephalin derivatives.These four, namely Dts-Tyr(t-Bu)-Gly-Gly-Phe-Leu-OH, Dts-Tyr-Gly-Gly-Phe-Leu-OH, H-Tyr(t-Bu)-Gly-Gly-Phe-Leu-OH, and H-Tyr-Gly-Gly-Phe-Leu-OH, were each obtained pure in good yields and were characterized by amino acid composition, HPLC, 300-MHz 1H NMR, and fast atom bombardment mass spectrometry.The protected dipeptidyl sequence Prot-D-Val-L-Pro-ONb-resin was assembled with three different Nα-amino protecting groups and exposed to the recommended deblocking reagents.Loss of chains from the resin by diketopiperazine formation was very rapid with Prot=9-fluorenylmethoxycarbonyl (Fmoc) and also substantial with Prot=tert-butoxycarbonyl (Boc), but rather negligible wit h Prot=Dts.Thus, these experiments demonstrate the feasibility and benefits of a mild three-dimensional orthogonal protection scheme based on Dts for Nα-amino protection, tert-butyl derivatives for side chains, and o-nitrobenzyl esters for anchoring.
- Barany, George,Albericio, Fernando
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p. 4936 - 4942
(2007/10/02)
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