The synthesis of peptides and proteins containing non-natural amino acids

Methods for the incorporation of non-natural amino acids into proteins have advanced significantly over recent years and in this tutorial review we aim to give a general overview of the area. These techniques offer the possibility of modulating the structures and functions of proteins and thus permit the generation of novel designed systems for both biocatalytic and mechanistic studies. Four complementary approaches are discussed in detail along with examples of their application. The advantages and disadvantages of each technique are also discussed.     

Helix and H-bond formations of alanine-based peptides containing basic amino acids

We studied comprehensively the helicity and H-bonding evolutions during the folding processes of Lys- and Arg-containing alanine-based peptides. The evolution of α-helical conformation concerning the entire sequence and each amino acid residue was examined, as well as the helix-forming propensities were characterized. The formation of various types of the intramolecular H-bonds was also investigated, pointing out the helix-stabilizing role of local interactions and the destabilizing role of non-local interplays. Our study led to the observation that the non-local H-bonds affected the evolution of helical conformations, as well as the entire folding processes.     

Helix propensity of highly fluorinated amino acids

Highly fluorinated amino acids have been used to stabilize helical proteins for potential application in various protein-based biotechnologies. To gain further insight into the effect of these highly fluorinated amino acids on helix formation exclusively, we measured the helix propensity of three highly fluorinated amino acids: (S)-5,5,5,5',5',5'-hexafluoroleucine (Hfl), (S)-2-amino-4,4,4-trifluorobutyric acid (Atb), and (S)-pentafluorophenylalanine (Pff). We have developed a short chemoenzymatic synthesis of Hfl with extremely high enantioselectivity (>99%). To measure the helix propensity (w) of the amino acids, alanine-based peptides were synthesized, purified, and investigated by circular dichroism spectroscopy (CD). On the basis of the CD data, the helix propensity of hydrocarbon amino acids can decrease up to 24-fold (1.72 kcal.mol-1.residue-1) upon fluorination. This difference in helix propensity has previously been overlooked in estimating the magnitude of the fluoro-stabilization effect (which has been estimated to be 0.32-0.83 kcal.mol-1.residue-1 for Hfl), resulting in a gross underestimation. Therefore, the full potential of the fluoro-stabilization effect should provide even more stable proteins than the fluoro-stabilized proteins to date.     

Oxidative coupling of peptides to a virus capsid containing unnatural amino acids

This Communication describes the chemo- and site-selective coupling of cell type-specific targeting peptides to a virus capsid containing aminophenylalanine residues.     

Nucleophilicities of amino acids and peptides

The kinetics of the reactions of amino acids with stabilized diarylcarbenium ions (Ar(2)CH(+)) have been studied photometrically in aqueous solution at variable pH. In the range of 10.5 < pH < 12, the amino acids react much faster than the competing nucleophiles hydroxide and water. Though the pK(aH) values of the amino acids vary by almost four units, the nucleophilic reactivities of all primary amino groups differ by less than a factor of 4. The secondary amino group of proline is 10(2) times more reactive, and the thiolate site in cysteine exceeds the reactivities of the primary amino groups by a factor of 10(4). Nucleophilicity parameters N as defined by the correlation log k(20 degrees C) = s(N + E) have been determined in order to include amino acids into the most comprehensive nucleophilicity scales presently available, which provide a direct comparison of n-, pi-, and sigma-nucleophiles.     

Nanomolar binding of peptides containing noncanonical amino acids by a synthetic receptor

This paper describes the molecular recognition of phenylalanine derivatives and their peptides by the synthetic receptor cucurbit[7]uril (Q7). The 4-tert-butyl and 4-aminomethyl derivatives of phenylalanine (tBuPhe and AMPhe) were identified from a screen to have 20-30-fold higher affinity than phenylalanine for Q7. Placement of these residues at the N-terminus of model tripeptides (X-Gly-Gly), resulted in no change in affinity for tBuPhe-Gly-Gly, but a remarkable 500-fold increase in affinity for AMPhe-Gly-Gly, which bound to Q7 with an equilibrium dissociation constant (K(d)) value of 0.95 nM in neutral phosphate buffer. Structure-activity studies revealed that three functional groups work in a positively cooperative manner to achieve this extraordinary stability (1) the N-terminal ammonium group, (2) the side chain ammonium group, and (3) the peptide backbone. Addition of the aminomethyl group to Phe substantially improved the selectivity for peptide versus amino acid and for an N-terminal vs nonterminal position. Importantly, Q7 binds to N-terminal AMPhe several orders of magnitude more tightly than any of the canonical amino acid residues. The high affinity, single-site selectivity, and small modification in this system make it attractive for the development of minimal affinity tags.     

Studies on synthesis and molecular dynamics simulation of dendrimers containing amino acids and peptides

A series of poly(ether-amide) dendrimers with amino acids and peptides as the peripheral functional groups was synthesized, and their structures were confirmed by nuclear magnetic resonance (NMR) and electrospray ionization-mass spectrometry (ESI-MS) spectrometry. Molecular dynamics simulation of the peptide dendrimers in solution was performed, indicating that, the prior conformations of the dendrimers were atom number dependent, i.e., with the increases of the atom number, the prior conformations were more spherical. Also, the amino acid α-C atom radial distribution indicated that, with larger peripheral groups, more back-folding of the dendrimers occurred. __________ Translated from Acta Chimica Sinica, 2007, 65(1): 21–26 [译自: 化学 通报]     

Synthesis and antidiabetic activity of novel triazole derivatives containing amino acids

New series of triazole derivatives coupled with amino acids 1a-h were obtained via multicomponent reaction of 2-hydroxy benzaldehyde or 2-hydroxy acetophenone with thiosemicarbazide and different amino acids. The obtained compounds were reacted with p-toluinesulfonyl chloride 2 to give the corresponding sulfonamides 3a-h . Compound 1b was allowed to react with different aromatic aldehydes or cyclic ketone under alkaline conditions to afford the expected imino compounds 4a-d and 6a-c , respectively. These compounds were allowed to react with ethyl glycolate to yield the expected thiazolidinone derivatives 5a-d or 7a-c , respectively. Structures of the newly synthesized compounds were found to be in accordance with their elemental analyses and spectral data. The obtained compounds exhibited very prominent in vitro and in vivo antihyperglycemic effect at a dose of 40 mg/kg body weight compared to the standard drug gliclazide and control. The antidiabetic effect was investigated using oral glucose tolerance test in normal and non-insulin-dependent diabetes mellitus (NIDDM) in STZ-rat model. Compounds 3a - h , 5b , 5c , 5d , 7a , 7b , and 7c showed significant activity in lowering blood glucose (more than 80%) compared to the NIDDM control.     

Conformational analysis of some C2-symmetric cyclic peptides containing tetrahydrofuran amino acids

Cyclic oligomers of tetrahydrofuran amino acids, cyclo-(Taa1-Leu-Val)2 (left), cyclo-(Taa2-Leu-Val)2 (middle), and cyclo-(Taa2-Phe-Leu)2 (right), displayed well-defined intramolecularly hydrogen-bonded structures with distorted "beta-beta corner" motifs similar to the tennis ball seam.     

Infrared spectra of amino acids and peptides

Summary The ionization constants of glycine, glycylglycine, DL--alanine, and -alanine have been determined from their IR spectra.Khimiya Priodnykh Soedinenii, Vol. 3, No. 2, pp. 197–201, 1967     

Organophosphorus analogs of amino acids and peptides

A method for obtaining phosphorus analogs of homoproline was developed, and the complexing properties of these compounds were studied. An effective method for obtaining optically active 1-aminoalkylphosphonic acids that is based on the stereoselective enzymatic hydrolysis of their N-acyl derivatives is proposed. New approaches to the synthesis of phosphonopeptides using privalyl chloride as the condensing agent, silylated amino phosphonic acids as the amino components of peptide synthesis, and the proteinase papain as the stereoselective catalyst for the formation of the peptide bond were developed.Institute of Bioorganic Chemistry and Petrochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 27, No. 3, pp. 288–293, May–June, 1991. Original article submitted February 8, 1991.     

Prodrugs of HIV protease inhibitors-saquinavir, indinavir and nelfinavir-derived from diglycerides or amino acids: synthesis, stability and anti-HIV activity

With the aim of improving the pharmacological properties of current protease inhibitors (PIs), the synthesis of various acyl and carbamate amino acid- or diglyceride-containing prodrugs derived from saquinavir, indinavir and nelfinavir, their in vitro stability with respect to hydrolysis and their anti-HIV activity in CEM-SS and MT4 cells have been investigated. l-Leucine (Leu) and l-phenylalanine (Phe) were connected through their carboxyl to the PIs while l-tyrosine (Tyr) was conjugated through its aromatic hydroxyl via various spacer units. Hydrolysis of the prodrug with liberation of the active free drug was crucial for antiviral activity. The Leu- and Phe-PI prodrugs released the active free drug very rapidly (half-lives of hydrolysis in buffer at 37 degree C of 3-4 h). The Tyr-PI conjugates with a -C(O)(CH(2))(4)- linker exhibited half-lives in the 40-70 h range and antiviral activities in the 21-325 nM range (from 2 to 22 nM for the free PIs). The chemically very stable carbamate "peptidomimetic" Tyr-PI prodrugs (no hydrolysis detected after 7 days in buffer) displayed a very low anti-HIV activity or were even inactive (EC(50) from 2300 nM to >10 microM). A very low antiviral activity was measured for the diglyceride-substituted saquinavir and for all of the disubstituted indinavir and nelfinavir prodrugs. All these prodrugs probably released the active parent PI too slowly under the antiviral assay conditions. These results combined with those from transepithelial transport studies (Rouquayrol et al., Pharm. Res., 2002, 19, 1704-1712) indicate that conjugation of amino acids (through their carboxyl) to the PIs constitutes a most appealing alternative which could improve the intestinal absorption of the PIs and reduce their recognition by efflux carriers.     

Synthetic studies towards cyclic peptides. Concise synthesis of thiazoline and thiazole containing amino acids

Concise and efficient syntheses of optically pure thiazoline and thiazole containing amino acids of the constitution (26) and (27), based on simple condensation reactions between cysteine esters and N-protected imino ethers (22) and (25) derived from chiral amino acids, are described. The synthetic procedures are compatible with a range of amino acid side chains and protecting groups, and allow the preparation of a variety of small optically pure peptides i.e. (32) and (34) suitable for elaboration to naturally occurring cyclic peptides e.g. the lissoclinamides (3) and (4).     

Multiply charged ions in field desorption mass spectra of peptides containing basic amino acids

Multiply charged ions up to [M+4]⁴⁺ were observed in the field desorption mass spectra of five different peptides. The relationship between the charge-multiplicity and the number of basic sidechains is discussed.     

Thermal stability of amino acids

Thermal decomposition of L-α-amino acids RCH₂(NH₂)COOH where R = Me₂CH, Me₂CHCH₂, MeEtCH, and C₆H₅CH₂ was studied at temperatures below the melting points of their crystals. From the effective rate constants of the first order reactions energy parameters in the Arrhenius equation were calculated. Correlations between the reaction rate constants k _R and the inductive constants σ* of substituents R and also between the rate constants of the reactions and the dipole moments of amino acids was established. Value of ρ* parameter +8.8 in the Taft equation indicates the heterolytic mechanism of transformation of the amino acids. Chromato-mass spectrometric analysis of decomposition products shows that condensation, decarboxylation, and deamination of the amino acids take place.     

Preparation of hydrazides of amino acids and peptides

A new method of synthesizing hydrazides of amino acids and peptides is considered which involves the hydrazinolysis of the silyl esters of the corresponding compounds. A number of hydrazides of derivatives of amino acids and peptides has been obtained in high purity with yields close to quantitative. The physicochemical characteristics of the compounds synthesized are given.     

Preparation of hydrazides of amino acids and peptides

A new method of synthesizing hydrazides of amino acids and peptides is considered which involves the hydrazinolysis of the silyl esters of the corresponding compounds. A number of hydrazides of derivatives of amino acids and peptides has been obtained in high purity with yields close to quantitative. The physicochemical characteristics of the compounds synthesized are given.All-Union Scientific-Research Institute of the Technology of Blood Substitutes and Hormones Preparations, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 684–686, September–October, 1979.