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The effect of the addition of amino acids and amino acid derivatives on the crystallization of hemoglobin and ribonuclease A has been evaluated. The results showed that certain types of additives expand the concentration conditions in which crystals are formed., Determination of the appropriate conditions for protein crystallization remains a highly empirical process. Preventing protein aggregation is necessary for the formation of single crystals under aggregation-prone solution conditions. Because many amino acids and amino acid derivatives offer a unique combination of solubility and stabilizing properties, they open new avenues into the field of protein aggregation research. The use of amino acids and amino acid derivatives can potentially influence processes such as heat treatment and refolding reactions. The effect of the addition of several amino acids, such as lysine, and several amino acid derivatives, such as glycine ethyl ester and glycine amide, on the crystallization of equine hemoglobin and bovine pancreatic ribonuclease A has been examined. The addition of these amino acids and amino acid derivatives expanded the range of precipitant concentration in which crystals formed without aggregation. The addition of such additives appears to promote the crystallization of proteins.

Six analogs (peptides 1–6) of the potent substance P (SP) derivative known as ‘Antagonist D’ were synthesized by substituting constrained amino acids Aib or Acp (cycloleucine, 1-amino cyclopentane carboxylic acid) at different positions in the Antagonist D sequence: D-Arg1-Pro2-Lys3-Pro4-D-Phe5-Gln6-D-Trp7-Phe8-D-Trp9-Leu10-Leu11-NH2. In the preliminary in vitro antiproliferative screening of the analogs on different human cancer cell lines by 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide (MTT) assay, peptide 1 was found to be the most active. Further, peptide 1 was butanoylated (analog 5) or octanoylated (analog 6) at the N-terminus. SP analogs 1, 5, and 6 were evaluated in vivo in a xenograft model of human primary colon tumor (PTC) cell line in athymic nude mice and were found to cause tumor regression. This study investigates if the use of the constrained amino acids Aib and Acp in the designed SP analogs can retain the in vitro and in vivo anticancer activities, which could be useful in cancer therapy and drug targeting. Further, the strategy of incorporation of Aib or Acp in biologically active peptides can be exploited in determining the receptor-bound conformation and in transforming these bioactive peptides into pharmacologically useful drugs. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

Three groups of chloroenyne-modified amino acids were synthesized. Chloroenyne moiety was attached at the N- or C-terminal amino acid (Tyr, Phe, Val, Gly, Lys) position carrying different protecting groups. Prepared derivatives will be used as building blocks in the synthesis of enediyne-peptide conjugates. Furthermore, reactivity of modified amino acids in the peptide bond formation reaction was tested.

Recently, it has become possible to reprogram the protein synthesis machinery such that numerous noncanonical amino acids can be translated into target sequences yielding tailor-made proteins. The canonical amino acid tryptophan (Trp) encoded by a single nucleotide triplet (UGG) is a particularly interesting target for protein engineering and design. Trp-residues can be substituted with a variety of analogs and surrogates generated biosynthetically or by organic chemistry. Among them, nitrogen-containing tryptophan analogs occupy a central position, as they have distinct chemical properties in comparison with aliphatic amines and imines. They resemble purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. These special properties of the analogs can be directly transmitted into related protein structures via in vivo ribosome-mediated translation. Proteins expressed in this way are further endowed with unique properties like new spectral, altered redox and titration features or might serve as useful biomaterials. We present and discuss current works and future developments in protein engineering with nitrogen-containing tryptophan analogs and related compounds as well as their relevance for academic and applicative research.

In vivo incorporation of monofluorinated noncanonical amino acids into recombinant proteins has been well-established for decades. Proteins fluorinated in this way proved to be useful tools for many practical applications. In contrast, trifluorinated amino acids have been incorporated in only a few peptides and relatively small proteins by using expression systems in living cells. A novel class of proteins with a fluorous core can be envisaged only if full replacement of the core-building hydrophobic and aliphatic amino acids such as leucine or methionine with the related analogues trifluoromethionine and trifluoroleucine would be feasible. However, our systematic efforts to introduce these amino acids in larger proteins (over 10 Da) that contain different structural motifs clearly show that only partial substitutions are possible. The reasons are high toxicity of these substances and difficulties to accommodate them into the compact cores of natural proteins without adverse effects on their structural integrity. Therefore, engineering of such three dimensional 'Teflon'-like structures would require, besides an expansion of the amino acid repertoire of the genetic code, a de novo protein design as well.

Recently, we have succeeded in incorporating various nonnatural amino acids into proteins by using four-base codon-anticodon pairs in Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system. Here, the reaction was conducted under various conditions in order to optimize the incorporation efficiency. The optimal concentration of aminoacyl-tRNA, reaction temperature, and reaction time were 2 nM, 25 degrees C, and 1.5 hr, respectively.

Amino acid derivatives of a modified indole-3-acetic acid have been synthesised. Fourteen new dipeptide-like compounds 3–4 were obtained and their structures were elucidated based on the IR, 1H NMR, MS spectra.

We have studied amino acid formation by UV (193 nm) irradiation to organic molecules (amines, alcohols and amides) in aqueous solution. Among several types of detected amino acids, small aliphatic amino acids (Gly and alpha-, beta-Ala and alpha-, beta-, gamma-ABA) were quantitatively identified. Among these small aliphatic amino acids, certain amino acids were formed in its free form, even before hydrolysis, contrary to the results of UV irradiation to a gas mixture of CO, NH3, and H2O, where amino acids were hardly detected before hydrolysis. The species distribution of identified amino acids showed a dependence on the starting organic molecules, and also on the presence of ammonia. The formation processes of the identified small aliphatic amino acids were investigated with the aid of electrospray ionization (ESI) MS and MS/MS measurements of photoproducts. Possible formation processes of these amino acid precursors from each starting molecules are proposed. By identifying the amino acid precursor, which has a chiral carbon atom, a new possibility is suggested for asymmetric photosynthesis of amino acid from achiral organic molecules.

Amino acids enter the body from dietary protein and are generated by the breakdown of tissue proteins. Nonessential amino acids can be synthesized from other metabolic intermediates. Amino acid disposal is dominated by protein synthesis, but amino acids are also oxidized by a series of complex pathways, some of which generate other useful small molecules. The nitrogen from amino acids is converted to urea in the liver and excreted in the urine. Amino acids circulate freely in the plasma and are distributed in all tissues. A series of specific proteins are responsible for transporting amino acids across cell membranes.

Purines bearing carbon substituents attached to carbon atoms of purine ring (positions 2, 6 and/or 8) are of great interest due to potential applications in medicinal chemistry and chemical biology. Transition metal-catalyzed cross-coupling reactions are a powerful tool [1] for the synthesis of these compounds but, in most cases, were only used for an introduction of simple unfunctionalized alkyl, alkenyl, alkynyl, aryl and hetaryl substituents. An extension of this methodology to introduction of functionalized C-substituents, is a very challenging target. One of the prominent functionalized substituents of high biological relevance is an amino acid residue. Our goal is to develop approaches towards the synthesis of a novel class of compounds: carbon-carbon linked conjugates of amino acids and purines. We have developed a facile and efficient synthesis of two classes of purines with amino acid attached in position 6 of purine scaffold, (purin-6-yl)alanines [2] and (purin-6yl)phenylalanines.[3] These derivatives were prepared by palladium catalyzed cross-coupling reactions of 6-halopurines with organometallic reagents based on protected amino acid residue. Conditions of cross-coupling reactions were optimized to avoid racemization in cases when enantiopure amino acid reagents were used. Final complete deprotection of the products was performed under mild conditions in order to prevent racemization or cleavage of acidolabile nucleosidic bonds. We obtained free purine bases, purine nucleosides and 9alkylated purines bearing free amino acid in position 6.

Protein-protein interactions are the key to organizing cellular processes in space and time. The only direct way to identify such interactions in their cellular environment is by photo-cross-linking. Here we present a new strategy for photo-cross-linking proteins in living cells. We designed two new photoactivatable amino acids that we termed photo-methionine and photo-leucine based on their structures and properties closely resembling the natural amino acids methionine and leucine, respectively. This similarity allows them to escape the stringent identity control mechanisms during protein synthesis and be incorporated into proteins by the unmodified mammalian translation machinery. Activation by ultraviolet light induces covalent cross-linking of the interacting proteins, which can be detected with high specificity by simple western blotting. Applying this technology to membrane protein complexes, we discovered a previously unknown direct interaction of the progesterone-binding membrane protein PGRMC1 with Insig-1, a key regulator of cholesterol homeostasis.

An efficient procedure has been developed for the preparation of previously unknown N-(3-trimethylsilyl-2-propynoyl) amino acids. The procedure is based on the reaction of 3-trimethylsilyl-2-propynoyl chloride with silylated amino acids which are generated in situ.

Treatment of N-(4-nitrophenoxycarbonyl)amino acid with an equimolar amount of amine in the absence of a coupling agent gave the corresponding α-amino acid amide in high yield.

Amino acids, peptides and proteins are important constituents of food. They supply the required building blocks for protein biosynthesis. In addition, they directly contribute to the flavor of food and are precursors for aroma compounds and colors formed during thermal or enzymatic reactions in production, processing and storage of food. Other food constituents, e.g. carbohydrates, also take part in such reactions. Proteins also contribute significantly to the physical properties of food through their ability to build or stabilize gels, foams, emulsions and fibrillar structures.

This chapter presents procedures for the synthesis of α-vinyl amino acids, in which the usual α-proton is replaced by an unsubstituted vinyl group (Fig. 1). The parent member of this family, α-vinylglycine (R≠H), is a natural product (1,2) and acts as a suicide substrate for a number of PLP-dependent enzymes (4-9). Higher members of this family (R≠H) have also been synthesized (10-12). Several including α-vinyl-m-tyrosine (13-15), α-vinyl-DOPA (13-15), α-vinylglutamate (16), α-vinylornithine (17), α-vinyllysine (18), and α-vinylarginine (18) are Trojan horse inhibitors of their cognate amino acid decarboxylases (AADCs). Such (appropriately labeled) AADC inhibitors may also have potential as reagents for positron emission tomography (19). Fig. 1. Generic structure for α-vinyl amino acids.

Synthesis of 4-substituted indolizidin-9-one amino acid by cyclization of pipecolic acid derivatives has been described.

New methods to unnatural amino acids continue to be developed. This review covers methods, published in 2001, that employ an asymmetric hydrogenation, a transition metal-catalyzed reaction, an enzymatic resolution or a chiral auxiliary.

Eight analogs 4a-7a and 4b-7b of philanthotoxin (PhTX) from wasp venom and nephilatoxin-8 (NPTX-8) from spider venom whose tyrosine or asparagine linker is replaced by squaryl (sq) amino acid or 4-amino squaryl (4-asq) amino acid have been synthesized in an efficient manner via coupling of N-acyl squaryl amino acid intermediate 19 or 26 with the corresponding polyamine part. Preliminary bioassay using crickets revealed that the analogs substituted by glutamate-type squaryl amino acid-containing NPTX 7a and 7b showed more potent paralytic activities than that of NPTX-8.

Mammalian cationic amino acid transporters (CAT) differ in their substrate affinity and sensitivity to trans-stimulation. The apparent Km values for cationic amino acids and the sensitivity to trans-stimulation of CAT-1, -2B, and -3 are characteristic of system y+. In contrast, CAT-2A exhibits a 10-fold lower substrate affinity and is largely independent of substrate at the trans-side of the membrane. CAT-2A and -2B demonstrate such divergent transport properties, even though their amino acid sequences differ only in a stretch of 42 amino acids. Here, we identify two amino acid residues within this 42-amino acid domain of the human CAT-2A protein that are responsible for the apparent low affinity of both the extracellular and intracellular substrate-binding sites. These residues are located in the fourth intracellular loop, suggesting that they are not part of the translocation pathway. Rather, they may be responsible for the low affinity conformation of the substrate-binding sites. The sensitivity to trans-stimulation is not determined by the same amino acid residues as the substrate affinity and must involve a more complex interaction between individual amino acid residues. In addition to the 42-amino acid domain, the adjacent transmembrane domain X seems to be involved in this function.

Thus,the incorporation of CAAs into peptides or peptidomimeticsinduce conformational restrictions and provide importantstructural effects. Furthermore, CAAs in peptide surrogateshave been used for structural and biomechanistic investi-gation as well as for probing the structural requirements ofreceptor-bound ligand conformations. This report providesan overview of the preparation of conformationallyconstrained CAAs and their derivatives.2. a-AA derivatives2.1. Cyclopropane AA derivativesThe cyclopropane ring system is particularly interestingbecause it affects the chemical and biological properties inpeptides through significant conformational restrictions inthe AA residues. They are found in a wide class of naturallyoccurring products, and synthetic 2,3-methanoamino acidsare structurally related to those cyclopropane AAs found inproteins. Racemic 2,3-methanoamino acids (4) have beensynthesized by several methods as reviewed by Stammer

Publisher Summary This chapter analyzes the probing of the organization of transcription complexes using photoreactive 4-Thio-substituted analogs of uracil and thymidine. The use of 4-thio-substituted UTP or dTTP analogs provides a powerful method to probe the organization of transcription complexes. These analogs may be placed at defined positions in the transcript or the DNA, and on UV activation form cross-links to nearby regions of the protein with high efficiency. A number of analogs have been employed in these studies. Subsequent mapping of the site of the cross-link by peptide mapping provides important information concerning the organization of the complex and the trajectory of the nucleic acid components over the surface of the protein. The preparation of transcription complexes with specifically positioned photoreactive analogs is experimentally designed. The design of the template is dictated by the purpose of the experiment and the position in the template or product that is being analyzed. The exact strategy for mapping the site of the cross-link depends on the suitability of various chemical or enzymatic cleavage reagents with the particular protein being examined.

This chapter focuses on amino acids, which are the monomeric units, or building blocks of proteins joined by a specific type of covalent linkage. The properties of proteins depend on the characteristic sequence of component amino acids, each of which has distinctive side chains. Amino acid polymerization requires elimination of a water molecule, as the carboxyl group of one amino acid reacts with the amino group of another amino acid to form a covalent amide bond. The repetition of this process with many amino acids yields a polymer, known as a polypeptide. The amide bonds linking amino acids to each other are known as peptide bonds. The sequence of amino acids in a protein is dictated by the sequence of nucleotides in a segment of the DNA in the chromosomes, and the uniqueness of each living organism is due to its endowment of specific proteins. Almost all of the naturally occurring amino acids in proteins are L-α-amino acids. The principal 20 amino acids in proteins have an amino group, a carboxyl group, a hydrogen atom, and an R-group attached to the α-carbon. A useful classification of the amino acids is based on the solubility (that is, ionization and polarity) characteristics of the side chains (R-groups). The R-groups fall into four classes, which include nonpolar (hydrophobic); polar, negatively charged (acidic); polar, positively charged (basic); and polar, neutral (unionized). Glycine is the smallest amino acid and has an H atom as its R-group. It is the only α-amino acid that is notoptically active. Glycine is used for the biosynthesis of many nonprotein compounds, such as porphyrins and purines.

Hydrophobicity is a useful concept to rationalize the role played by amino acid residues in terms of buried or exposed conformation with regard to the aqueous environment in proteins. The relationship of this concept with distinct approaches to represent the molecular surface is analyzed by computing reliable surface areas for three definitions namely the van der Waals, solvent-accessible, and solvent-excluded molecular surfaces. The surface areas are obtained for all of the naturally occurring amino acids by first setting a proper reference standard state and then calculating their values for a database of proteins containing a total of 4297 residues. Despite the great differences in these molecular surfaces, proper indexes are here defined for handling the information of interest to study the hydrophobic behavior of amino acids provided by such surfaces.

We report here on the cloning and functional characterization of the third subtype of amino acid transport system A, designated ATA3 (amino acid transporter A3), from a human liver cell line. This transporter consists of 547 amino acids and is structurally related to the members of the glutamine transporter family. The human ATA3 (hATA3) exhibits 88% identity in amino acid sequence with rat ATA3. The gene coding for hATA3 contains 16 exons and is located on human chromosome 12q13. It is expressed almost exclusively in the liver. hATA3 mediates the transport of neutral amino acids including alpha-(methylamino)isobutyric acid (MeAIB), the model substrate for system A, in a Na(+)-coupled manner and the transport of cationic amino acids in a Na(+)-independent manner. The affinity of hATA3 for cationic amino acids is higher than for neutral amino acids. The transport function of hATA3 is thus similar to that of system y(+)L. The ability of hATA3 to transport cationic amino acids with high affinity is unique among the members of the glutamine transporter family. hATA1 and hATA2, the other two known members of the system A subfamily, show little affinity toward cationic amino acids. hATA3 also differs from hATA1 and hATA2 in exhibiting low affinity for MeAIB. Since liver does not express any of the previously known high-affinity cationic amino acid transporters, ATA3 is likely to provide the major route for the uptake of arginine in this tissue.

For the past several years, our research laboratory has been developing chemical models of protein β-sheets in which molecular templates induce β-sheet structure in attached peptide strands [1]. We have termed these structures artificial β-sheets and call the templates a molecular scaffold and a β-strand mimic to reflect their structural roles. The molecular scaffold holds the peptide and peptidomimetic groups in proximity; the β-strand mimic duplicates the hydrogen-bonding functionality of a peptide β-strand and helps align the peptide strand by hydrogen bonding to it. A few years ago, we asked the deceptively simple question: “What happens when we move the β-strand mimic to the bottom edge of an artificial β-sheet?” [2] Its answer proved both exciting and significant. Not only did the molecules fold to adopt β-sheet-like structures, but these structures also formed well-defined hydrogen-bonded dimers in chloroform solution. This result is noteworthy, both because peptides generally do not form well-defined dimers (they aggregate) and because many proteins do.