Trifunctional Amino Acid Research Articles

Acid-Modulated Peptide Synthesis for Application on Oxide Biosensor Interfaces.

In this paper we report an acid-modulated strategy for novel peptide microarray production on biosensor interfaces. We initially selected a controlled pore glass (CPG) as a support for solid-phase peptide synthesis (SPPS) to implement a chemistry that can be performed at the interface of multiple field effect transistor (FET) sensors, eventually to generate label-free peptide microarrays for protein screening. Our chemistry uses a temporary protection of the N-terminal amino function of each amino acid building block with a tert-butyloxycarbonyl (Boc) group that can be removed after each SPPS cycle, in combination with semi-permanent protection of the side chains of trifunctional amino acid residues. Such a protection scheme with a well-proven record of application in conventional, batchwise SPPS has been fine-tuned for optimal performance on CPG and, from there, translated to SPR chips that allow layer-by-layer monitoring of amino acid coupling. Our results validate this acid-modulated synthesis as a feasible approach for producing peptides in high yields and purity on flat glass surfaces, such as those in bio-FETs.

Chemical linguistics: Reading the modified proteome

In this issue of Molecular Cell, Lin etal. (2021) develop a tri-functional amino acid probe for the discovery and characterization of protein domains that sense or "read" protein post-translational modifications, a chemical tool that can facilitate our understanding of how signaling networks act at the molecular level.

A tri-functional amino acid enables mapping of binding sites for posttranslational-modification-mediated protein-protein interactions

Posttranslational modification (PTM), through the recruitment of effector proteins (i.e., "readers") that signal downstream events, plays key roles in regulating a variety of cellular processes. To understand how a PTM is recognized, it is necessary to find its readers and, importantly, the location of the binding pockets responsible for PTM recognition. Although various methods have been developed to identify PTM readers, it remains a challenge to directly map the PTM-binding regions, especially for intrinsically disordered domains. Here, we demonstrate a photo-crosslinkable, clickable, and cleavable tri-functional amino acid, ADdis-Cys, that when coupled with mass spectrometry (ADdis-Cys-MS) can not only identify PTM readers from complex proteomes but also simultaneously map their PTM-recognition modules. Using ADdis-Cys-MS, we successfully identify the binding sites of several reader-PTM interactions, among which we discover human C1QBP as a histone chaperone. This robust method should find wide applications in examining other histone or non-histone PTM-mediated protein-protein interactions.

Modifying perovskite solar cells with l(+)-cysteine at the interface between mesoporous TiO2 and perovskite

A typical trifunctional amino acid, l(+)-cysteine, is employed to modify the interface between mesoporous TiO2 and perovskite film, and promotes the power conversion efficiency of perovskite solar cells from 11.5% to 14.4%.

Beyond Heparinization: Design of Highly Potent Thrombin Inhibitors Suitable for Surface Coupling

During extracorporeal circulation, when blood comes in contact with artificial surfaces, patients receive a standard treatment with anticoagulants to avoid blood coagulation. Dialysis patients in particular are systemically treated with heparin up to four times a week, causing a high burden for the body. For potential anticoagulant modification of external materials, such as dialysis equipment, a series of highly potent thrombin inhibitors was developed. All inhibitors share the general formula arylsulfonyl-P3-Pro-4-amidinobenzylamide, where P3 is glycyl or a trifunctional amino acid residue in L-configuration. Among this series, several derivatives inhibit thrombin with Ki values of less than 1 nM. Specificity measurements revealed that this inhibitor type is highly specific for thrombin with negligible activity against related trypsin-like serine proteases. X-ray analysis of the most potent analogue in complex with thrombin demonstrated that the N-terminal arylsulfonyl group occupies the aryl binding site, whereas the P3 side chain is directed into the solvent and therefore is well suited for further coupling. Based on their in vitro profile, these inhibitors are suitable candidates for the development of hemocompatible materials with anticoagulant properties.

A series of metal–organic coordination polymers containing multiple chiral centers

Two new trifunctional amino acid derivatives, (S-2-(pyridine-4- ylmethylamino)succinic acid (L1) and S-2-(pyridin-4-ylmethylamino)pentanedioic acid (L2)), containing carboxylate, achiral amine and pyridine groups, were used as effective bridging ligands for the synthesis of six homochiral coordination networks of [ZnL1(H2O)2]·H2O (1), [CoL1(H2O)2]·H2O (2), [ZnL2(H2O)2] (3), [NiL1(H2O)]·H2O (4), [Cd2L122(H2O)]·3H2O (5) and [CdL2]·H2O (6). Compounds 1, 2 and 3 adopt 1D polymeric linear networks, and compound 4 presents a 2D lattice framework, while compounds 5 and 6 comprise 3D networks containing small voids occupied by water molecules. The achiral amine groups within all six compounds are induced by the neighbouring chiral carbon centres into homochiral units to coordinate transition metal cations. Hydrogen bonding contacts play very important roles for the formation and stabilization of these homochiral coordination networks. The magnetic properties of 2 and 4, and the photoluminescent properties of 1, 3, 5 and 6 in solid state were investigated.

Different approaches to synthesis of N-carboxyanhydrides of amides of trifunctional amino acids

Original method for synthesis of N-carboxyanhydrides of trifunctional α-amino acids containing substituents attached by amide bonds at the ω-functional group is suggested.

PEGylated Dendrimers with Core Functionality for Biological Applications

The synthesis of a variety of core functionalized PEGylated polyester dendrimers and their in vitro and in vivo properties are described in this report. These water-soluble dendrimers have been designed to carry eight functional groups on their dendritic core for a variety of biological applications such as drug delivery and in vivo imaging as well as eight solubilizing groups. Using a common symmetrical aliphatic ester dendritic core and trifunctional amino acid moieties, a library of dendrimers with phenols, alkyl alcohols, alkynes, ketones, and carboxylic acid functionalities has been synthesized without the need for column chromatography. The amines were PEGylated, leaving the other functionality of the amino acid available for further manipulation such as the attachment of drugs and/or labels. Radiolabeling experiments with the PEGylated dendrimers showed that they had a long circulation half-life in mice, confirming the potential of this class of dendrimers for therapeutic and/or diagnostic applications. A carboxylic acid functionalized dendrimer was elaborated to carry doxorubicin bound via a hydrazone bond. The drug-loaded carrier accumulated more in tumors and less in healthy organs than the clinically used PEGylated liposomal formulation Doxil. The efficient synthesis, high versatility, and favorable biological properties make these PEGylated polyester dendrimers promising structures for therapeutic and/or imaging applications.

Cholic acid-based fluorescent probes for enantioselective recognition of trifunctional amino acids

The ditopic fluorescent photoinduced electron transfer (PET) amino acid sensory probes and were designed and synthesized from cholic acid. To confer the probes with specific binding ability, an amidothiourea moiety and a cyclic diamino-chiral receptive site were introduced on the C17 side chain and the C7 and C12 hydroxyl pendants, respectively. In acetonitrile, the probes demonstrated differential binding toward trifunctional amino acids like serine, lysine, threonine and tyrosine against other simple amino acids. Enantioselectivities (KD/KL) of up to 8.9 and sensitivities in the micromolar range with the probes were observed for trifunctional amino acids.

Synthesis of Peptide‐Oligonucleotide Conjugates by Diels‐Alder Cycloaddition in Water

Peptide-oligonucleotide conjugates incorporating all the nucleobases and trifunctional amino acids are obtained by Diels-Alder reaction between diene-modified oligonucleotides (2'-deoxyribo- or ribo-) and malemide-derivatized peptides. Both reagents are easily synthesized by on-column derivatization of the corresponding peptides and oligonucleotides. The cycloaddition reaction is carried out under mild conditions, in aqueous solution at 37 degrees C, affording the desired peptide-oligonucleotide conjugate with high purity and yield. The speed of the reaction depends on the size and composition of both reagents, but it is accelerated by the presence of positively charged amino acids in the peptide fragment. However, a small excess of maleimide-derivatized peptide may be required in some cases to complete the reaction within 8 to 10 hr.

New developments in the synthesis of oligonucleotide-peptide conjugates

The stability of oligodeoxynucleotides to trifluoroacetic acid is studied. Pyrimidine oligonucleotides were stable in the conditions used for the removal of t-butyl groups. Oligonucleotide-3′-peptide conjugates carrying pyrimidine oligonucleotides are prepared stepwise using peptide-supports and Fmoc, t-butyl strategy. Using this strategy we have prepared an oligonucleotide-peptide conjugate containing as peptide the leucine-rich fragment of FOS, a transcription factor involved in many important cellular processes. This conjugate has a long peptide sequence with a large number of trifunctional amino acids.

Receptor Subtype-specific Docking of Asp6.59 with C-terminal Arginine Residues in Y Receptor Ligands

Y receptors (YRs) are G protein-coupled receptors whose Y(1)R, Y(2)R, and Y(5)R subtypes preferentially bind neuropeptide Y (NPY) and peptide YY, whereas mammalian Y(4)Rs show a higher affinity for pancreatic polypeptide (PP). Comparison of YR orthologs and paralogs revealed Asp(6.59) to be fully conserved throughout all of the YRs reported so far. By replacing this conserved aspartic acid residue with alanine, asparagine, glutamate, and arginine, we now show that this residue plays a crucial role in binding and signal transduction of NPY/PP at all YRs. Sensitivity to distinct replacements is, however, receptor subtype-specific. Next, we performed a complementary mutagenesis approach to identify the contact site of the ligand. Surprisingly, this conserved residue interacts with two different ligand arginine residues by ionic interactions; although in Y(2)R and Y(5)R, Arg(33) is the binding partner of Asp(6.59), in Y(1)R and Y(4)R, Arg(35) of human PP and NPY interacts with Asp(6.59). Furthermore, Arg(25) of PP and NPY is involved in ligand binding only at Y(2)R and Y(5)R. This suggests significant differences in the docking of YR ligands between Y(1/4)R and Y(2/5)R and provides new insights into the molecular binding mode of peptide agonists at GPCRs. Furthermore, the proposed model of a subtype-specific binding mode is in agreement with the evolution of YRs.

Fmoc-Protein Synthesis: Preparation of Peptide Thioesters Using a Side-Chain Anchoring Strategy

A side-chain anchoring approach for preparation of peptide thioesters by Fmoc SPPS is reported. This strategy involves the side-chain anchoring of trifunctional amino acids, such as Lys, Glu, Gln, Asp and Asn, for peptide elongation and the post-chain assembly introduction of thioester functionality. This approach allows for the use of standard nucleophilic Fmoc peptide synthesis cycles, which are generally incompatible with thioester-based resin-linkages. The strategy was successfully demonstrated by the straightforward Fmoc syntheses of a model RANTES(1--33) thioester peptide. The Fmoc prepared RANTES(1--33) thioester peptide was then ligated to RANTES(34--68), folded and purified to give the RANTES protein.

My Contributions to Science and Society

My Contributions to Science and Society

LGR8 Signal Activation by the Relaxin-like Factor

The relaxin-like factor (RLF) is thought to be responsible for the intra-abdominal migration of the testis during mammalian development. Our latest studies of RLF and LGR8 have revealed that the N-terminal region of the A chain is not required for receptor binding but is indispensable for cyclic AMP generation. RLF derivatives with six residues deleted from the N terminus of the A chain are active, whereas further truncation, up to the first A chain cysteine (A-10), yields tightly binding ligands devoid of signaling activity. These derivatives are specific competitive inhibitors (RLFi) of RLF. Although receptor binding is dependent upon B chain residues, the N-terminal region of the A chain is a generic trigger of the trans-membrane signaling activity.

Synthesis of structural analogs of the 13 – 19 sequence of human growth hormone releasing factor

Development of the principles of synthesis of bioregulators responsible for the secretion of natural hormones in the organism is among currently important tasks. One particular basic problem consists in finding previously unknown peptide agents stimulating the secretion of insulin, a hormone playing a key role in the regulation of metabolic processes in higher mammals and humans. One possible approach to solving this problem is via the synthesis of peptide structures capable of participating in hormone – receptor interactions and enhancing insulin secretion by cells in Largenhans’ islets in the pancreas. A real pathway to such peptide stimulators of insulin secretion can be based on the synthesis of previously unknown structural analogs of the amino end region of somatoliberin, the hypothalamic releasing factor of human growth hormone [1, 2]. Our previous investigations showed that this R-factor is capable of indirectly influencing insulin biosynthesis and secretion by pancreatic islet cells. However, from the standpoint of pharmacology, a significant disadvantage of using natural somatoliberin as a bioregulator of insulin secretion is its relatively low activity and insufficient selectivity. In addition to weakly stimulating the secretion of insulin, natural somatoliberin enhances (to a much greater extent) production of growth hormone and activates the secretion of glucagon. For this reason, much better prospects in solving the problem under consideration can be related to the amino end region of somatoliberin. This conclusion agrees with the results of computer simulation of hormone – receptor interactions involved in the biosynthesis and secretion of insulin. In this context, we have undertaken a special investigation aimed at the development of a method for the synthesis of previously unknown structural analogs of the amino end region of somatoliberin, which can be of interest as potential stimulators of insulin secretion. After a series of preliminary experiments and a comparative analysis of the results, it was concluded that the most promising approach is that based on the use of activated esters of N-protected amino acids. This approach leads to a high yield of the target compounds, excludes undesired side reactions, allows effective control over the racemization processes, and is less tedious than the other possible schemes. As a result, we have developed an effective method for the synthesis of previously unreported structural analogs of the 13 – 19 somatoliberin peptide sequence, which is a part of the amino end of somatoliberin. Distinguishing features of the proposed method are as follows: (i) use of benzyl esters of oligopeptides as amino components; (ii) use of p-nitrophenyl or pentafluorophenyl esters of N-protected amino acids as carboxy components; (iii) use of an acid-labile tert-butyloxycarbonyl protection for masking -amino groups in the carboxy components; (iv) stepwise peptide synthesis in the C N direction. An important advantage of the proposed method is the possibility of using protected derivatives of trifunctional amino acids for the synthesis of hydrophobic analogs of somatoliberin. It was also established that the isolation of target compounds from the reaction mixtures is considerably simplified by conducting the condensation stage in DMF. In this case, the target peptides can be isolated in a crystalline state and purified by recrystallization. The structure of peptides obtained using the proposed scheme is uniquely determined by the scheme of synthesis. The synthetic analogs of the terminal region of somatoliberin can be obtained in an analytically pure form.

Optical characteristics of responsive biopolymers; co-polycondensation of tri-functional amino acids and Cy-3 bis-amine with diacylchlorides

The synthesis and fluorescence behaviour of a range of amphiphilic poly( l-lysine co-bis-amine-Cy-3 iso-phthalamide) polymers that incorporate low levels of Cy-3 dye co-monomer (0.5–2.1% (w/w)) in their backbone have been investigated in aqueous solution over a range of pH values. The desired functionality of these biocompatible, hydrophobically modified polyelectrolytes was achieved by incorporating pendant hydrophilic carboxyl groups along the polymer backbone, via the l-lysine moiety, balanced by a degree of hydrophobicity introduced via the iso-phthaloyl moiety. Thus, the polymer changes from an extended conformation at high degrees of ionisation to a compact conformation stabilised by hydrophobic association at low degrees of ionisation and eventually precipitates from solution. At intermediate degrees of ionisation, such polymers exhibit amphiphilic properties. A bis-functional cyanine fluorophore derivative, co-polymerised within the polymer backbone, is demonstrated to act as a fluorescent reporter on the conformational state of the polymer. The materials have higher intrinsic fluorescence intensity per fluorophore monomer over a broad range of concentrations than bis-amine Cy-3 and their maximum fluorescence intensity is up to eight fold higher than the maximum intensity of bis-amine Cy-3, which is limited by quenching. It is also shown that the free fluorophore can be used to probe the conformation of unlabelled poly( l-lysine iso-phthalamide) through electrostatic interaction with the polymer. The technique allows rapid spectrophotometric determination of polymer conformation and offers the potential of an environmentally sensitive molecular pH probe for in vivo use.

Analysis of the transport properties of side chain modified dipeptides at the mammalian peptide transporter PEPT1.

This study was initiated to examine systematically the effect of side chain modifications at dipeptides on their transport via PEPT1. We synthesized a series of Xaa(R)-Ala and Ala-Xaa(R) dipeptides with the functional groups of the side chains modified by structurally different blocking groups R. Recognition and transport of these derivatives by PEPT1 was measured in Caco-2 cells, in transgenic Pichia pastoris cells and in Xenopus laevis oocytes expressing PEPT1. The dipeptide derivatives displayed K(i) values between 0.002 and 4 mM. Electrophysiological analyses showed that the Ala-Xaa(R) derivatives were transported by PEPT1. In contrast, most Xaa(R)-Ala derivatives--although recognized--did not show significant transport rates. Substitution of a terminal phenyl residue in the side chain blocking group by a p-nitrophenyl residue enhanced the affinity of several dipeptide derivatives for interaction with PEPT1. However, none of these compounds showed electrogenic transport in oocytes. With a K(i) value of 0.002 mM, Lys[Z(NO(2))]-Val displayed the highest affinity to PEPT1 ever reported. We conclude that the transport of side chain modified dipeptides into enterocytes depends (a) on the position of the modified trifunctional amino acid in the dipeptide, (b) the distance between its alpha-carbon and the side chain blocking group and (c) the hydrophobic character of the side chain modification.

Solid-phase approach to the synthesis of cyclen scaffolds from cyclotetrapeptides

Cyclen derivatives, as coordinating ligands, have recovered considerable interest for the development of diagnostic and therapeutic drugs, mimicking the binding site of metalloproteins. We demonstrate that the on-resin reduction of head-to-tail cyclotetrapeptides, anchored to a solid support by the side-chain of a trifunctional amino acid, is an efficient synthetic strategy. The proposed approach leads to the cyclen scaffold still bound to the resin, ready for further decorations.

Modified proteinases in peptide synthesis in organic media

We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp.