- Optimization and anti-cancer properties of fluoromethylketones as covalent inhibitors for ubiquitin C-terminal hydrolase L1
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The deubiquitinating enzyme (DUB) UCHL1 is implicated in various disease states including neurodegenerative disease and cancer. However, there is a lack of quality probe molecules to gain a better understanding on UCHL1 biology. To this end a study was carried out to fully characterize and optimize the irreversible covalent UCHL1 inhibitor VAEFMK. Structure-activity relationship studies identified modifications to improve activity versus the target and a full cellular characterization was carried out for the first time with this scaffold. The studies produced a new inhibitor, 34, with an IC50 value of 7.7 μM against UCHL1 and no observable activity versus the closest related DUB UCHL3. The molecule was also capable of selectively inhibiting UCHL1 in cells and did not demonstrate any discernible off-target toxicity. Finally, the molecule was used for initial probe studies to assess the role of UCHL1 role in proliferation of myeloma cells and migration behavior in small cell lung cancer cells making 34 a new tool to be used in the biological evaluation of UCHL1.
- Chen, Hao,Das, Chittaranjan,Flaherty, Daniel P.,Galardy, Paul J.,Hewitt, Chad S.,Hussain, Sajjad,Imhoff, Ryan D.,Krabill, Aaron D.,Muli, Christine S.,Wendt, Michael K.
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- One-step C-terminal deprotection and activation of peptides with peptide amidase from stenotrophomonas maltophilia in neat organic solvent
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Chemoenzymatic peptide synthesis is a rapidly developing technology for cost effective peptide production on a large scale. As an alternative to the traditional C→N strategy, which employs expensive N-protected building blocks in each step, we have investigated an N→C extension route that is based on activation of a peptide C-terminal amide protecting group to the corresponding methyl ester. We found that this conversion is efficiently catalysed by Stenotrophomonas maltophilia peptide amidase in neat organic media. The system excludes the possibility of internal peptide cleavage as the enzyme lacks intrinsic protease activity. The produced peptide methyl ester was used for peptide chain extension in a kinetically controlled reaction by a thermostable protease.
- Arif, Muhammad I.,Toplak, Ana,Szymanski, Wiktor,Feringa, Ben L.,Nuijens, Timo,Quaedflieg, Peter J. L. M.,Wu, Bian,Janssen, Dick B.
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p. 2197 - 2202
(2014/07/21)
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- α-N-Protected dipeptide acids: A simple and efficient synthesis via the easily accessible mixed anhydride method using free amino acids in DMSO and tetrabutylammonium hydroxide
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The importance of dipeptides both in medicinal and pharmacological fields is well documented and many efforts have been made to find simple and efficient methods for their synthesis. For this reason, we have investigated the synthesis of α-N-protected dipeptide acids by reacting the easily accessible mixed anhydride of α-N-protected amino acids with free amino acids under different reaction conditions. The combination of TBA-OH and DMSO has been found to be the best to overcome the low solubility of amino acids in organic solvents. Under these experimental conditions, the homogeneous phase condensation reaction occurs rapidly and without detectable epimerization. The present method is also applicable to side-chain unprotected Tyr, Trp, Glu, and Asp but not Lys. This latter residue is able to engage two molecules of mixed anhydride giving the corresponding isotripeptide. Moreover, the applicability of this protocol for the synthesis of tri- and tetrapeptides has been tested. This approach reduces the need for protecting groups, is cost effective, scalable, and yields dipeptide acids that can be used as building blocks in the synthesis of larger peptides.
- Verardo,Gorassini
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p. 315 - 324
(2013/06/05)
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- Design, synthesis and biological evaluation of potent azadipeptide nitrile inhibitors and activity-based probes as promising anti-Trypanosoma brucei agents
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Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas disease and African sleeping sickness, respectively. There is an urgent need for the development of new drugs against both diseases due to the lack of adequate cures and emerging drug resistance. One promising strategy for the discovery of small-molecule therapeutics against parasitic diseases has been to target the major cysteine proteases such as cruzain for T. cruzi, and rhodesain/TbCatB for T. brucei. Azadipeptide nitriles belong to a novel class of extremely potent cysteine protease inhibitors against papain-like proteases. We herein report the design, synthesis, and evaluation of a series of azanitrile-containing compounds, most of which were shown to potently inhibit both recombinant cruzain and rhodesain at low nanomolar/picomolar ranges. A strong correlation between the potency of rhodesain inhibition (i.e., target-based screening) and trypanocidal activity (i.e., whole-organism-based screening) of the compounds was observed. To facilitate detailed studies of this important class of inhibitors, selected hit compounds from our screenings were chemically converted into activity-based probes (ABPs), which were subsequently used for in situ proteome profiling and cellular localization studies to further elucidate potential cellular targets (on and off) in both the disease-relevant bloodstream form (BSF) and the insect-residing procyclic form (PCF) of Trypanosoma brucei. Overall, the inhibitors presented herein show great promise as a new class of anti-trypanosome agents, which possess better activities than existing drugs. The activity-based probes generated from this study could also serve as valuable tools for parasite-based proteome profiling studies, as well as bioimaging agents for studies of cellular uptake and distribution of these drug candidates. Our studies therefore provide a good starting point for further development of these azanitrile-containing compounds as potential anti-parasitic agents. Copyright
- Yang, Peng-Yu,Wang, Min,Li, Lin,Wu, Hao,He, Cynthia Y.,Yao, Shao Q.
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supporting information; experimental part
p. 6528 - 6541
(2012/07/13)
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- Modulation of the inhibitor properties of dipeptidyl (acyloxy)methyl ketones toward the CaaX proteases
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Dipeptidyl (acyloxy)methyl ketones (AOMKs) have been identified as mechanism-based inhibitors of certain cysteine proteases. These compounds are also inhibitors of the integral membrane proteins Rce1p and Ste24p, which are proteases that independently mediate a cleavage step associated with the maturation of certain isoprenylated proteins. The enzymatic mechanism of Rce1p is ill-defined, whereas Ste24p is a zinc metalloprotease. Rce1p is required for the proper processing of the oncoprotein Ras and is viewed as a potential target for cancer therapy. In this study, we synthesized a small library of dipeptidyl AOMKs to investigate the structural elements that contribute to the inhibitor properties of this class of molecules toward Rce1p and Ste24p. The compounds were evaluated using a fluorescence-based in vitro proteolysis assay. The most potent dipeptidyl AOMKs contained an arginine residue and the identity of the benzoate group strongly influenced potency. A 'warhead' free AOMK inhibited Rce1p and Ste24p. The data suggest that the dipeptidyl AOMKs are not mechanism-based inhibitors of Rce1p and Ste24p and corroborate the hypothesis that Rce1p is not a cysteine protease.
- Dechert, Anne-Marie R.,MacNamara, James P.,Breevoort, Sarah R.,Hildebrandt, Emily R.,Hembree, Ned W.,Rea, Adam C.,McLain, Duncan E.,Porter, Stephen B.,Schmidt, Walter K.,Dore, Timothy M.
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scheme or table
p. 6230 - 6237
(2010/10/03)
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- Evaluation of α,β-unsaturated ketone-based probes for papain-family cysteine proteases
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The field of activity-based proteomics makes use of small molecule active site probes to monitor distinct subsets of enzymatic proteins. While a number of reactive functional groups have been applied to activity-based probes (ABPs) that target diverse families of proteases, there remains a continual need for further evaluation of new probe scaffolds and reactive functional groups for use in ABPs. In this study we evaluate the utility of the, α,β-unsaturated ketone reactive group for use in ABPs targeting the papain-family of cysteine proteases. We find that this reactive group shows highly selective labeling of cysteine cathepsins in both intact cells and total cell extracts. We observed a variable degree of background labeling that depended on the type of tag and linker used in the probe synthesis. The relative ease of synthesis of this class of compounds provides the potential for further derivatization to generate new families of cysteine protease ABPs with unique specificity and labeling properties.
- Yang, Zhimou,Fonovic, Marko,Verhelst, Steven H.L.,Blum, Galia,Bogyo, Matthew
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experimental part
p. 1071 - 1078
(2009/09/25)
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- Influence of solvent viscosity on the rate of hydrolysis of dipeptides by carboxypeptidase Y
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The influence of solvent viscosity on the rate of enzymatic hydrolysis of a series of dipeptides (Z-Phe-Gly, Z-Phe-Sar, Z-Phe-Ala, Z-Phe-NMeAla, Z-Phe-Aib and Z-Phe-Pro) by carboxypeptidase Y was investigated. The effect of solvent viscosity on the enzymatic hydrolysis revealed that whereas all Kcat values decreased with viscosity, those of the N-alkyl peptides decreased more than those of the N-H peptides. The kinetic behaviour implies the involvement of conformational changes of the enzyme in terms of the 'induced-fit' process. Copyright
- Kanosue, Yoshifumi,Kojima, Satoshi,Ohkata, Katsuo
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p. 448 - 457
(2007/10/03)
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- Relationship between the hydrophobicity of dipeptides and the Michaelis-Menten constant Km of their hydrolysis by carboxypeptidase-Y and carboxypeptidase-A
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The enzymatic hydrolysis of dipeptides by carboxypeptidase-Y and carboxypeptidase-A was investigated. In the enzymatic hydrolysis of the dipeptides, a good linear relationship (r = 0.997 and 0.999) was found between the Michaelis-Menten constant (Km) and the hydrophobicity of the substrates evaluated from relative elution volume in reversed-phase HPLC. The correlation suggests that the hydrophobicity of the C-terminal amino acid is a major factor in governing the stability of the enzyme-substrate complex. The difference in the slope of the linear-regression lines seems to reflect the degree of relative hydrophobicity of the binding pockets in carboxypeptidase-Y and carboxypeptidase-A.
- Kanosue, Yoshifumi,Kojima, Satoshi,Hiraga, Yoshikazu,Ohkata, Katsuo
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p. 1187 - 1193
(2007/10/03)
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- Highly diastereoselective peptide chain extensions of unprotected amino acids with N-(Z-α-aminoacyl)benzotriazoles
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Coupling an unprotected amino acid or dipeptide in partially aqueous solution with a readily available N-(Z-α-amino-acyl)benzotriazole or N-(Z-α-aminopetidoyl)benzotriazole affords N-terminal-protected di-, tri-, and tetrapeptides in yields of 85-98% (average 95% for 2a-i, 93% for 4a-f and 4a′, 86% for 5a-b) with minimal epimerization.
- Katritzky, Alan R.,Suzuki, Kazuyuki,Singh, Sandeep K.
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p. 2645 - 2652
(2007/10/03)
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- α-chymotrypsin-catalysed segment condensations via the kinetically controlled approach using carbamoylmethyl esters as acyl donors in organic media
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The superiority of the carbamoylmethyl ester as an acyl donor for the α-chymotrypsin-catalysed segment condensations via the kinetically controlled approach is demonstrated in several model systems carried out in organic media with low water content. Furthermore, this approach is successfully applied to the construction of the Leu-enkephalin sequence via a 4 + 1 segment coupling.
- Miyazawa, Toshifumi,Ensatsu, Eiichi,Hiramatsu, Makoto,Yanagihara, Ryoji,Yamada, Takashi
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p. 396 - 401
(2007/10/03)
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- Superiority of the carbamoylmethyl ester as an acyl donor for the protease-catalyzed kinetically controlled peptide synthesis in organic media: Application to segment condensations
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The superiority of the carbamoylmethyl ester as an acyl donor for the α-chymotrypsin-catalyzed kinetically controlled peptide synthesis was demonstrated in several segment condensations carried out in organic media with low water content. Then this approach was successfully applied to the construction of the Leuenkephalin sequence via the 4 + 1 segment condensation.
- Miyazawa, Toshifumi,Ensatsu, Eiichi,Tanaka, Kayoko,Yanagihara, Ryoji,Yamada, Takashi
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p. 1013 - 1014
(2007/10/03)
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- ARYLOXY AND ARYLACYLOXY METHYL KETONES AS THIOL PROTEASE INHIBITORS
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Thiol protease inhibitors are disclosed having the formula: STR1 or an optical isomer thereof, or a pharmaceutically acceptable salt thereof, wherein:n is 0 or 1;m is 0, 1 or 2;X is H or an N-protecting group; each Y is independently an optionally protected α-amino acid residue;R is an optionally protected α-amino acid side chain that is H or CH 3 or that is bonded to the α-carbon atom to which it is attached by a methylene, methine or phenyl radical; andR' is optionally substituted aryl.
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- NOUVELLE METHODE DE PROTECTION DU CARBOXYLE DES ACIDES a-AMINES: ESTERS 9-FLUORENYLIQUES
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The novel 9-fluorenyl esters of various L-a-aminoacids (N-protected or N-free) are easily obtained by the action of 9-diazofluorene on the aminoacid in the appropriate organic solvent.Such esters are cleanly cleaved by mild acidolysis or hydrogenolysis and can serve as amino-components in peptide synthesis.
- Proussios, Cleanthis,Kolovos, Miltiadis
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p. 3413 - 3414
(2007/10/02)
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- Efficient 1,4-Asymetric Induction Utilizing Electrostatic Interaction between Ligand and Substrate in the Asymmetric Hydrogenation of Didehydrodipeptides
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Electrostatic interaction between the amino group of the achiral 3-dimethylaminopropylidenebismethylenebis(diphenylphosphine) (1) and the carboxy group of the substrate enable an effective 1,4-asymmetric induction in the RhI-catalysed hydrogenation of didehydrodipeptides, to give (S,S)-or (R,R)-products selectively.The selectivity reached up to 94percent diastereoisomeric excess with acetyl didehydrodipeptides and 92percent with benzyloxycarbonyl substrates.
- Yamagishi, Takamichi,Ikeda, Satoru,Yatagai, Masanobu,Yamaguchi, Motowo,Hida, Mitsuhiko
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p. 1787 - 1790
(2007/10/02)
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- Allyl Esters as Selectively Removable Carboxy-protecting Functions in Peptide and N-Glycopeptide Syntheses
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Allyl esters are advantageous in protection of the carboxylic function in peptide and glycopeptide syntheses and can easily be synthesized from amino acids.They are stable under acidic conditions used for the removal of the Boc and Z groups.Under neutral or weakly basic conditions allyl esters are cleaved smoothly on treatment with catalytic amounts of tris(triphenylphosphane)rhodium(I) chloride in ethanol/water (9:1) leaving the N-protecting function and the N-glycosidic bond untouched.Selective deblocking of the α-carboxylic function of protected N-glycosylated asparagine derivatives 15 carried out in this way is exploited in C-terminal chain extension in the synthesis of N-glycotripeptides 17,e.g 17c, the latter representing a partial sequence of a human immunoglobulin G1.
- Waldmann, Herbert,Kunz, Horst
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p. 1712 - 1725
(2007/10/02)
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- Synthesis of Amides of Dipeptides and Kinetics of Papain-catalysed Hydrolysis of These Amides
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The N-benzyloxycarbonylated amides of the dipeptides L-alanyl-L-phenylalanine, L-leucylleucine, L-leucyl-L-phenylalanine, L-phenylalanyl-L-alanine and L-phenylalanyl-L-leucine have been synthesised and the kinetic parameters for the papain-catalysed hydrolysis of these substrates and their ester analogs have been determined at pH 8 and 9.All the amide substrates are hydrolysed at the amide linkage except N-benzyloxycarbonyl-L-alanyl-L-phenylalanine amide which hydrolyses to the extent of 45percent at the amide linkage and 55percent at the peptide linkage.For amide hydrolysis acetylation and deacetylation rate constants are approximately equal.
- Zaher, M. R.,El-Sharief, A. M. Sh.
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p. 740 - 743
(2007/10/02)
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- THE OXAZOLE-TRIAMIDE REARRANGEMENT. APPLICATION TO PEPTIDE SYNTHESIS
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The carboxyl group of a N-acylated amino acid may be protected by conversion to an oxazole derivative which, on photooxygenation, regenerates the carboxyl group in activated (triamide) form for peptide synthesis.
- Wasserman, H. H.,Lu, T.-J.
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p. 3831 - 3834
(2007/10/02)
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