Tyr Trp Research Articles

Anticoagulant Oligonucleotide-Peptide Conjugates: Identification of Thrombin Aptamer Conjugates with Improved Characteristics.

Oligonucleotide–peptide conjugates (OPCs) are a promising class of biologically active compounds with proven potential for improving nucleic acid therapeutics. OPCs are commonly recognized as an efficient instrument to enhance the cellular delivery of therapeutic nucleic acids. In addition to this application field, OPCs have an as yet unexplored potential for the post-SELEX optimization of DNA aptamers. In this paper, we report the preparation of designer thrombin aptamer OPCs with peptide side chains anchored to a particular thymidine residue of the aptamer. The current conjugation strategy utilizes unmodified short peptides and support-bound protected oligonucleotides with activated carboxyl functionality at the T3 thymine nucleobase. The respective modification of the oligonucleotide strand was implemented using N3-derivatized thymidine phosphoramidite. Aptamer OPCs retained the G-quadruplex architecture of the parent DNA structure and showed minor to moderate stabilization. In a series of five OPCs, conjugates bearing T3–Ser–Phe–Asn (SFN) or T3–Tyr–Trp–Asn (YWN) side chains exhibited considerably improved anticoagulant characteristics. Molecular dynamics studies of the aptamer OPC complexes with thrombin revealed the roles of the amino acid nature and sequence in the peptide subunit in modulating the anticoagulant activity.

Formation and characterization of crosslinks, including Tyr-Trp species, on one electron oxidation of free Tyr and Trp residues by carbonate radical anion.

Dityrosine and ditryptophan bonds have been implied in protein crosslinking. This is associated with oxidative stress conditions including those involved in neurodegenerative pathologies and age-related processes. Formation of dityrosine and ditryptophan derives from radical–radical reactions involving Tyr˙ and Trp˙ radicals. However, cross reactions of Tyr˙ and Trp˙ leading to Tyr–Trp crosslinks and their biological consequences have been less explored. In the present work we hypothesized that exposure of free Tyr and Trp to a high concentration of carbonate anion radicals (CO3˙−), under anaerobic conditions, would result in the formation of Tyr–Trp species, as well as dityrosine and ditryptophan crosslinks. Here we report a simple experimental procedure, employing CO3˙− generated photochemically by illumination of a Co(iii) complex at 254 nm, that produces micromolar concentrations of Tyr–Trp crosslinks. Analysis by mass spectrometry of solutions containing only the individual amino acids, and the Co(iii) complex, provided evidence for the formation of o,o′-dityrosine and isodityrosine from Tyr, and three ditryptophan dimers from Trp. When mixtures of Tyr and Trp were illuminated in an identical manner, Tyr–Trp crosslinks were detected together with dityrosine and ditryptophan dimers. These results indicate that there is a balance between the formation of these three classes of crosslinks, which is dependent on the Tyr and Trp concentrations. The methods reported here allow the generation of significant yields of isolated Tyr–Trp adducts and their characterization. This technology should facilitate the detection, and examination of the biological consequences of Tyr–Trp crosslink formation in complex systems in future investigations.

Incorporation of Putative Helix-Breaking Amino Acids in the Design of Novel Stapled Peptides: Exploring Biophysical and Cellular Permeability Properties.

Stapled α-helical peptides represent an emerging superclass of macrocyclic molecules with drug-like properties, including high-affinity target binding, protease resistance, and membrane permeability. As a model system for probing the chemical space available for optimizing these properties, we focused on dual Mdm2/MdmX antagonist stapled peptides related to the p53 N-terminus. Specifically, we first generated a library of ATSP-7041 (Chang et al., 2013) analogs iteratively modified by L-Ala and D-amino acids. Single L-Ala substitutions beyond the Mdm2/(X) binding interfacial residues (i.e., Phe3, Trp7, and Cba10) had minimal effects on target binding, α-helical content, and cellular activity. Similar binding affinities and cellular activities were noted at non-interfacial positions when the template residues were substituted with their d-amino acid counterparts, despite the fact that d-amino acid residues typically ‘break’ right-handed α-helices. d-amino acid substitutions at the interfacial residues Phe3 and Cba10 resulted in the expected decreases in binding affinity and cellular activity. Surprisingly, substitution at the remaining interfacial position with its d-amino acid equivalent (i.e., Trp7 to d-Trp7) was fully tolerated, both in terms of its binding affinity and cellular activity. An X-ray structure of the d-Trp7-modified peptide was determined and revealed that the indole side chain was able to interact optimally with its Mdm2 binding site by a slight global re-orientation of the stapled peptide. To further investigate the comparative effects of d-amino acid substitutions we used linear analogs of ATSP-7041, where we replaced the stapling amino acids by Aib (i.e., R84 to Aib4 and S511 to Aib11) to retain the helix-inducing properties of α-methylation. The resultant analog sequence Ac–Leu–Thr–Phe–Aib–Glu–Tyr–Trp–Gln–Leu–Cba–Aib–Ser–Ala–Ala–NH2 exhibited high-affinity target binding (Mdm2 Kd = 43 nM) and significant α-helicity in circular dichroism studies. Relative to this linear ATSP-7041 analog, several d-amino acid substitutions at Mdm2(X) non-binding residues (e.g., d-Glu5, d-Gln8, and d-Leu9) demonstrated decreased binding and α-helicity. Importantly, circular dichroism (CD) spectroscopy showed that although helicity was indeed disrupted by d-amino acids in linear versions of our template sequence, stapled molecules tolerated these residues well. Further studies on stapled peptides incorporating N-methylated amino acids, l-Pro, or Gly substitutions showed that despite some positional dependence, these helix-breaking residues were also generally tolerated in terms of secondary structure, binding affinity, and cellular activity. Overall, macrocyclization by hydrocarbon stapling appears to overcome the destabilization of α-helicity by helix breaking residues and, in the specific case of d-Trp7-modification, a highly potent ATSP-7041 analog (Mdm2 Kd = 30 nM; cellular EC50 = 600 nM) was identified. Our findings provide incentive for future studies to expand the chemical diversity of macrocyclic α-helical peptides (e.g., d-amino acid modifications) to explore their biophysical properties and cellular permeability. Indeed, using the library of 50 peptides generated in this study, a good correlation between cellular permeability and lipophilicity was observed.

A NOVEL MUTATION IN YWTD DOMAIN OF LRP6 IMPAIRS ENDOTHELIAL CELL FUNCTION AND LEAD TO FAMILIAL CORONARY ARTERY DISEASE

ObjectivesPURPOSE: Our purpose is to identify the causal gene and it's mechanism for familial coronary artery disease in Chinese Han Population.MethodsWe recruited a large Chinese Han family with coronary artery...

Rapid in situ synthesis of [ 11C]methyl azide and its application in 11C click-chemistry

We synthesized [ 11C]methyl azide ([ 11C]MeA) by reacting [ 11C]methyl iodide ([ 11C]MeI) in situ with an azide-donor and used it in the synthesis of 11C-labeled 1,2,3-triazoles. A one-pot click approach comprised the infusion of gaseous [ 11C]MeI into a mixture of NaN 3, ethynylbenzene, and CuI in water at a temperature of 100 °C yielding the 11C-triazole in radiochemical yields (RCY) of 25%. In a two-step labeling protocol, we synthesized the [ 11C]MeA in acetonitrile in advance to the click step. Using the more soluble Na + / 18 -crown- 6 / N 3 - complex as source of N 3 - , a much higher trapping efficiency of [ 11C]MeI in this solvent ensured an almost quantitative conversion of [ 11C]MeI to [ 11C]MeA within 5–10 min at room temperature. The [ 11C]MeA was thereafter reacted with ethynylbenzene at 100 °C yielding 1-[ 11C]methyl-4-phenyl-1 H-1,2,3-triazole in preparative RCY of 60%. As a final proof of applicability, we used 11C-click-chemistry for the labeling of N-terminal 4-ethynylbenzene derivatized d-Glu– d-Tyr–[Cys–Tyr–Trp–Lys–Thr–Cys]–Thr, a cyclic water-soluble Tyr 3-octreotate derivative.

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His–Tyr–Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1 E)-2-(5-imidazolyl)ethenyl]-4-( p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His–Tyr–Gly, Trp–Tyr–Gly, Phe–Trp–Gly, Tyr–Trp–Gly, Asn–Tyr–Gly, Phe–Tyr–Gly, and Tyr–Tyr–Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore Trp–Tyr–Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn–Tyr–Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.

Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met–Tyr–Trp cross-linked adduct

Mycobacterium tuberculosis catalase-peroxidase ( Mtb KatG) is a bifunctional enzyme that possesses both catalase and peroxidase activities and is responsible for the activation of the antituberculosis drug isoniazid. Mtb KatG contains an unusual adduct in its distal heme-pocket that consists of the covalently linked Trp107, Tyr229, and Met255. The KatG(Y229F) mutant lacks this adduct and has decreased steady-state catalase activity and enhanced peroxidase activity. In order to test a potential structural role of the adduct that supports catalase activity, we have used resonance Raman spectroscopy to probe the local heme environment of KatG(Y229F). In comparison to wild-type KatG, resting KatG(Y229F) contains a significant amount of 6-coordinate, low-spin heme and a more planar heme. Resonance Raman spectroscopy of the ferrous–CO complex of KatG(Y229F) suggest a non-linear Fe–CO binding geometry that is less tilted than in wild-type KatG. These data provide evidence that the Met–Tyr–Trp adduct imparts structural stability to the active site of KatG that seems to be important for sustaining catalase activity.

IR-LD spectroscopic characterization of l-Tryptophan containing dipeptides

IR-spectroscopic and stereo-structural analysis of aromatic l-Tryptophan containing dipeptides l-Tryptophan– l-Tryptophan (Trp–Trp), l-Tyrosine– l-Tryptophan (Tyr–Trp) and cyclo(Trp–Trp) have been carried out by means of solid-state linear-dichroic infrared (IR-LD) spectroscopy of oriented as suspension in nematic liquid crystal solids. The experimental data have been compared with analogous ones of the simple amino acids l-Tyrosnine ( l-Tyr) and l-Tryptophan ( l-Trp). In cyclo(Trp–Trp), the IR-spectral changes towards the IR-ones of acyclic Trp–Trp have been determined. A theoretical analysis of Tyr–Trp at Hartree–Fock level of theory and 6-31G** basis set is also applied.

Functional mono- and dinuclear peptide complexes featuring chemospecific κ S- or η 6-coordination of organometallic half-sandwich fragments

Treatment of the dipeptides HTyrTrpOH, HTrpTyrOH, HTrpPheOH and HPheTyrOH with [(η 6-cymene)Ru(acetone) 3]·(CF 3SO 3) 2 ( 1) in CF 3COOH leads to chemospecific η 6-coordination in the order of electron-donating ability Trp>Tyr>Phe in respective complexes of the type [(η 6-cymene)Ru(η 6-dipeptide)](CF 3SO 3) 2 ( 3– 6) after 24–36 h at 20 °C. An analogous thermodynamic chemospecificity was also established for the complexes [(η 5-Cp*)Ir(η 6-dipeptide)](CF 3SO 3) 2 ( 7, HPheTrpOH; 8, HPheTyrOH). However, a time-dependent 1H NMR study of the reaction between [(η 5-Cp*)Ir(acetone) 3]·(CF 3SO 3) 2 ( 2) and HTyrPheOH confirms an initial kinetic preference for η 6-coordination of the C-terminal arene, as is also apparent from product mixtures obtained after treating 2 with HTrpPheOH or HTrpTyrOH for 1– 2 d at 50 °C. Dinuclear sandwich complexes of the type [(η 5-Cp*)Ir(μ 2-HMetXOHη 6-X:κ 2 N M, S)Pt(en)](CF 3SO 3) 4 ( 9– 11, X=Phe, Tyr, Trp) can be prepared in CF 3COOH following initial N-terminal κ 2 N M, S chelation of the (en)Pt II fragment by the methionine residue of the dipeptide. Chemospecific κ S coordination of the [(η 5-Cp*)Ir(dppz)] 2+ fragment in [(η 5-Cp*)Ir(dppz) (H 2HArgMetNH 2κ S)] 4+ ( 12), [{(η 5-Cp*)Ir(dppz)} 2(μ-H 2MetMetOHκ S:κ S′)] 5+ ( 13) and [(η 5-Cp*)Ir(dppz)(μ-HGlyMetOHκ S:κ N G)Pt(terpy)] 4+ ( 14) affords effective metallointercalators for DNA with binding constants K b in the range 1.3–3.3×10 6 M −1 and marked melting temperature shifts Δ T m of respectively 10.2, 15.2 and 11.2 °C.

UV protolysis of aromatic amino acids and related dipeptides and tripeptides

A comparison of the photochemical properties of three aromatic amino acids (tyrosine (Tyr), trytophan (Trp), phenylalanine (Phe)), five dipeptides (LeuTyr (Leu=leucine), TyrLeu, TyrTrp, TrpTyr, TyrTyr and one tripeptide (TyrTyrTyr) was carried out using high performance liquid chromatography; continuous wave low intensity and picosecond laser high intensity UV irradiation were employed. The photochemical sensitivity, photolysis quantum yield and quantum yield of peptide bond scission during laser UV irradiation were determined. A number of important relations for the quantum yield of the two-quantum photoreaction were derived.

NH 2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH 2-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH 2-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH 2-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA 76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala 2Val 3 residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH 2-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH 2-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH 2-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.

Amino-terminal sequence of baboon type C virus p30

The NH 2-terminal amino acid sequence of a baboon Type C virus p30 was determined to be: ProLeuArgThrValAsnAgrThrValGlnTyrTrp  XPhe. This is identical, assuming that X = Pro, to the terminal 14 residues of the endogenous feline virus, RD 114, p30,