Unusual amino acids: synthesis and introduction into naturally occurring peptides and biologically active analogues

This review covers our recent advances in the synthesis of unusual amino acids in optically pure form, and their introduction into naturally occurring peptides with specific biological properties, or into modified bioactive peptides, aiming to obtain analogues displaying enhanced performances in term of activity, bioavailability and resistance to enzymatic hydrolysis.     

Tumor-cell-targeted methionine-enkephalin analogues containing unnatural amino acids: design, synthesis, and in vitro antitumor activity

A series of new peptides (8-25) containing different unnatural amino acids of the adamantane type (1-6), was synthesized. Possible cytotoxic activity on human cervical adenocarcinoma (HeLa), larynx carcinoma (HEp-2), colon carcinomas (HT-29, Caco-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), mammary gland adenocarcinoma (MCF-7), and melanoma (HBL) cells were tested by the MTT assay. The results were compared with the effect of methionine-enkephalin (Tyr-Gly-Gly-Phe-Met, or opioid growth factor, OGF), and its shorter N-terminal fragments. Peptide analogues containing C(alpha alpha)-dialkylated glycine (Aaa1, 1) or C(alpha)-alkylated glycine (Aaa2, 2) amino acid residues showed antitumor activity against melanoma, larynx carcinoma, colon carcinomas, and colon metastasis cell lines in vitro. The pentapeptide Tyr-(R,S)-Aaa2-Gly-Phe-Met (18) was the most effective analogue especially against the most antitumor drug-resistant cell lines HEp-2 and SW-620. Apoptosis as a mode of cell death was confirmed in these tumor cells after exposure to pentapeptide 18.     

Structure-activity relationships of dermorphin analogues containing N-substituted amino acids in the 2-position of the peptide sequence

A series of dermorphin analogues containing an N-alkylated amino-acid residue Xaa in the 2-position of the peptide sequence was synthesized (Xaa =N-methylalanine, proline, pipecolic acid, N-methylphenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [Tic]). These peptides have the potential of assuming a cis Tyr^l-Xaa² peptide bond. Their in vitro opioid activity profiles were determined in μ and δ-receptor-representative binding assays and bioassays. Aside from [D-Pro²]dermorphin, all analogues showed high affinity for μ and/or δ-opioid receptors. Whereas most compounds were found to be full μ-agonists in the guinea pig ileum (GPI) assay, [Tic²]dermorphin (compound 7) was a partial μ-agonist. Replacement of Gly⁴in 7 with Phe resulted in an analogue (8) with weak μ-antagonist activity. Furthermore, analogues 7 and 8 both were potent § antagonists (K_c= 3–40 nM) against the §-agonists Leuenkephalin, DPDPE and deltorphin I in the mouse vas deferens (MVD) assay. Compound 3, containing l -Pro in the 2-position, turned out to be one of the most μ receptor-selective linear dermorphin analogues reported to date. Low-temperature HPLC experiments using micropellicular octadecyl silica as stationary phase revealed conformational heterogeneity of the dermorphin analogues which was ascribed to cis-trans isomerization around the Tyr^l-Xaa²-and Tyr⁵-Pro⁶ peptide bonds. In the case of analogue 7 four separate peaks corresponding to the four possible isomers were apparent at -5°C. Since opioid peptide analogues with a non-N-akylated l -amino acid residue in the 2-position are nearly inactive and cannot assume a cis peptide bond at the 1–2 position, these results support the hypothesis that the bioactive conformation of opioid peptides containing an N-alkylated l -amino acid residue in position 2 is characterized by a cis Tyr^l-Xaa² peptide bond.     

Biological activity of silylated amino acid containing substance P analogues

Abstract: The need to replace natural amino acids in peptides with nonproteinogenic counterparts to obtain new medicinal agents has stimulated a great deal of innovation on synthetic methods. Here, we report the incorporation of non‐natural silylated amino acids in substance P (SP), the binding affinity for the two hNK‐1 binding sites and, the potency to stimulate phospholipase C (PLC) and adenylate cyclase of the resulting peptide. We also assess the improvement of their stability towards enzyme degradation. Altogether, we found that replacing glycine with silaproline (Sip) in position 9 of SP leads to a potent analogue exhibiting an increased resistance to angiotensin‐converting enzyme hydrolysis.     

Design, synthesis, and evaluation of the antipsychotic potential of orally bioavailable neurotensin (8-13) analogues containing non-natural arginine and lysine residues

Neurotensin (NT) and its active fragment NT(8–13) elicit behavioral responses typical of clinically used antipsychotic drugs when administered directly to the brain. However, limited peptide stability and oral bioavailability have prevented these compounds from being developed as relevant pharmaceuticals. Recently, our laboratory designed and studied a first-generation NT(8–13) derivative, KK13, that elicited key pharmacokinetic and behavioral responses typical of clinically used antipsychotic drugs when administered to rats parenterally. This compound was the basis for the rational design of a series of second-generation NT(8–13) analogues (KH1–KH30) studied in this paper. Initial screening of these analogues for CNS activity by monitoring hypothermia induction after peripheral administration defined several compounds (KH11, KH24, KH26, and KH28–KH30) that warranted further investigation. Each compound maintained binding affinity for NTR₁, however, only KH24, KH26, and KH28 (as well as KK13) elicited significant hypothermic responses after oral administration. Of these, KH28 demonstrated an oral activity 3-fold greater than any other analogue; hence it was further characterized in a series of rat behavioral assays. KH28 attenuated d-amphetamine induced hyperlocomotion, a hallmark of current clinically effective antipsychotic drugs, after both IP and oral administration. In addition, tolerance to the compound did not develop after repeated daily dosing, as measured by hypothermic induction as well as attenuation of d-amphetamine induced hyperlocomotion. Finally, KH28 did not produce catalepsy, a deleterious side-effect elicited by classical antipsychotic drugs. KH28 is considered to be an ideal compound for further development as a potential novel antipsychotic.     

Acylamino boronic acids and difluoroborane analogues of amino acids: potent inhibitors of chymotrypsin and elastase

A series of 1-acylamino boronic acids (IA-VA), analogues of the amino acids phenylalanine, phenylglycine, alanine, valine, and isoleucine, were prepared as potential transition-state inhibitors of the serine proteases alpha-chymotrypsin and elastase, by a boronate homologation reaction. The corresponding difluoroboranes (IB-VB), produced from the boronic acids by treatment with HF, were more easily purified than the boronic acids. Since the difluoroboranes readily hydrolyze in water, they proved to be convenient precursors for the boronic acids. The phenylalanine and phenylglycine analogues I and II were good competitive inhibitors of alpha-chymotrypsin (Ki = 0.3-8 microM), and the alanine, valine, and isoleucine analogues (III-IV) proved to be good inhibitors of elastase (Ki = 0.1-35 microM). On the basis of their high affinity and the tendency of boronic acids to form borate complexes, these acylamino boronic acids may be behaving as transition-state inhibitors.     

NMR and molecular modeling characterization of RGD containing peptides

The tripeptide sequence arginine-glycine-aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO-d₆ of the cyclic pentapeptides, Ac-Cys-Arg-Gly-Asp-Cys-OH (CRGDC), a potent fibrinogen receptor antagonist, and Ac-Cys-Arg-Gly-D-Asp-Cys-OH (CRGdC), a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. ¹H-¹H distance constraints derived from two-dimensional NOE spectroscopy and torsional angle constraints obtained from ³JNH-h α coupling constants, combined with computer-assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well-defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.     

Synthesis of dipeptide 4-nitroanilides containing non-proteinogenic amino acids

A series of tert-butyloxycarbonyl amino acid 4-nitroanilides, including N-alkylated amino acids and (R)- thiazolidine-4-carboxylic acid, (S)-oxazolidine-4-carboxylic acid. (4S,5S)-5-methyloxazolidine-4-carboxylic acid, (4S,5R)-5-methyloxazolidine-4-carboxylic acid, (S)-azetidine-2-carboxylic acid, (S)-pipecolic acid and (S)-3,4-dehydroproline, were prepared conveniently by the isocyanate method or the mixed anhydride procedure. The resulting amino acid 4-nitroanilides were extended to corresponding dipeptide 4-nitroanilides with tert-butyloxycarbonyl-(S)-alanine. In the case of sterically hindered amino acid 4-nitroanilides the mixed anhydride procedure with diphenylphosphinyl chloride was successful.     

Synthesis and anticandidal properties of polyoxin L analogues containing alpha-amino fatty acids

Analogues of polyoxin L containing amino acids with saturated fatty acid like side chains were synthesized from the benzyloxycarbonyl-protected alpha-amino fatty acid p-nitrophenyl ester and uracil polyoxin C. Transfer hydrogenolysis using palladium black and formic acid gave diastereomeric, dipeptidyl polyoxin L analogues containing alpha-aminooctanoic acid (3), alpha-aminododecanoic acid (4), or alpha-aminohexadecanoic acid (5) as the amine terminal residue in 40-60% yield. Diastereomers of 3 and 5 were resolved by using high-performance liquid chromatography on a reversed-phase column and designated as 3a, 3b and 5a, 5b. Analogues 3-5 were excellent inhibitors of chitin synthetase from Candida albicans; 4, the best inhibitor, had an ID50 of 0.5 microM. The L,L diastereomers of 3 and 5 were 1-2 orders of magnitude more potent chitin synthetase inhibitors than their D,L homologues. None of the synthetic polyoxin L analogues inhibited transport of trimethionine, but 3a, 4, and 5b caused decreases of 71%, 87%, and 83%, respectively, in the initial rate of uptake of dileucine. Compounds 3-5 were significantly more stable to peptidase degradation than polyoxin L analogues containing naturally occurring alpha-amino acids. Compound 4 inhibited growth of C. albicans in culture at 40-80 micrograms/mL. All other analogues were less potent antifungals. The results suggest that synthetic polyoxins can be designed to have increased affinity for a peptide transport system and to have increased stability against intracellular degradation in C. albicans.     

Biologically active analogues of arginine vasopressin containing conformationally restricted dipeptide fragments

In this study we described the synthesis and pharmacological properties of five new analogues of arginine vasopressin (AVP). Four of these analogues contained ethylene-bridged dipeptide Phe-Phe in positions 2 and 3; one had two N-Me-Phe residues. All new peptides were tested for vasopressor and antidiuretic activities. We also estimated the uterotonic activities of these compounds in vitro. Three analogues were highly potent V₁-antagonists. One of them, namely [Cpa¹,(Phe-Phe)^2,3,Val⁴]AVP, which seemed to not interact with either V₂ and oxytocic receptors, was outstandingly selective. It is interesting that the high antipressor potency of our second peptide, [(N-Me-Phe)^2,3]AVP, was achieved without modification of position 1. Our results open new possibilities for the design of very potent and selective V₁-antagonists of AVP.     

Development of a new pharmacophore model that discriminates active compstatin analogs

Compstatin and its active peptide analogs can potentially be used for therapeutic purposes because their binding to the third component of complement prohibits its conversion into the proteolytically activated form of the third component of complement, thus inhibiting complement cascades in all three complement pathways. Mallik and Morikis built three quasi-dynamic pharmacophore models for compstatin peptide analogs before, but only nine compstatin peptide analogs were incorporated in their study and the most active compstatin analog had only medium inhibitory activity. Since then, many more compstatin analogs have been synthesized and their inhibitory activities tested. Furthermore, the X-ray structure of AcCompNH2-V4W-H9A bound to the third component of complement has become available (PDB ID: 2QKI). In this paper, we utilized all the new information and built a new pharmacophore model using a distinct approach. Our model demonstrated good performance in a separate test set of 82 compstatin analogs: it accurately identified 70% of the analogs of medium or high inhibitory activities and misclassified only 8.5% of the analogs of low or no inhibitory activities. The results proved our pharmacophore model to be a filter of great sensitivity and specificity.     

Colorimetric determination of some amino acids containing a sulfur group

A simple and rapid colorimetric method for the determination of some amino acids containing a sulfur group is described. The studied compounds are cysteine, cystine and methionine. The method is based on the formation of ferroin, from the reaction of the studied drugs with a mixture of iron(III) and 1,10-phenanthroline and measurement of the absorbance at 512 nm. The procedure has been successfully applied to the assay of the pharmaceutical preparations of the studied drugs after thinlayer Chromatographic separation and the results of the studied compounds compare favourably with the official methods.     

Determination of methionine and tryptophan level in veterinary infusions containing amino acids

Method has been developed for determination of methionine in veterinary infusions containing amino acid. Methionine was measured on the basis of color reaction with sodium pentacyano-nitrosyl-ferrate in alkaline solution. Reproducibility of the method was good (recovery: 95.9-101.9%, r.s.d. = 2.3%, p = 0.95, n = 6). Tryptophane was determined on the basis of its UV absorbency at 280 nm, methionine did not interfere on this wavelength. Reproducibility of tryptophane determination was with an order better than that of methionine determination (recovery: 97.7-98.3%, r.s.d. = 0.15%, p = 0.95, n = 9). Methionine could be regained from infusions that had been heated at 120 degrees C for sixty minutes in the same quantity as from infusions which had not been heated. On the other hand tryptophane could be regained with 10% positive error from the heated infusions. The error can mainly be attributed to absorbency increase of glucose which is present in the infusion.     

Functional assessment of angiotensin II and bradykinin analogues containing the paramagnetic amino acid TOAC

This study characterized pharmacologically the functional responses to agonists angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label at the N-terminal (TOAC1-AngII and TOAC0-BK) and internal (TOAC3-AngII and TOAC3-BK) positions of these vasoactive peptides. Affinity constants of the ligands for AT1 and B2 receptors were evaluated in vitro by binding assays and biological effects by extracellular acidification rates and in vivo by blood pressure responses. In contrast to internally labeled analogues (TOAC3-AngII or TOAC3-BK), the TOAC1-AngII and TOAC0-BK derivatives dose-dependently increased the extracellular acidification rate in adherent cultured Chinese hamster ovary (CHO) cells expressing AT1 or B2 receptors, respectively. In addition, TOAC(1)-AngII induced an increase in blood pressure when injected intravenously in awaken rats although with a potency four times smaller when compared to native AngII. Similarly to BK, TOAC0-BK dose-dependently decreased blood pressure when injected intra-arterially in rats with a lower potency when compared to the native peptide. On the contrary, TOAC3-AngII or TOAC3-BK did not provoke any alteration in blood pressure levels. In summary, our results confirmed that the insertion of TOAC-probe in the N-terminal region of peptides does not significantly modify the affinity or biological activity in vitro and in vivo conditions and could be an important tool to evaluate peptide-receptor interaction mechanism. Conversely, possibly due to the unique bend-inducing property of the cyclic TOAC probe, its insertion at position 3 in both AngII and BK structures seems to restrict the interaction and the activation of the AT1 and B2 receptors.     

Synthesis of functionalized non-natural amino acid derivatives via amidoalkylation transformations

Synthetic routes have been developed for the preparation of functionalized amino acid derivatives in which the α-substituent at carbon 2 is either an aromatic or a heteroaromatic group. The α-substituent was introduced using an amidoalkylation reaction using boron trifluoride etherate and proceeded in moderate yield with excellent regioselectivity. This protocol permitted the employment of the acid sensitive heterocycles: pyrrole, benzofuran, and indole. The scope and limitations of this procedure have been evaluated.     

NMR regulatory analysis: determination and characterization of Chinese-herb aristolochic acids

1H NMR methodology for the simultaneous determination and characterization of the nephrotoxic components of Aristolochia plants aristolochic acid I (AA-I) and aristolochic acid II (AA-II) was developed utilizing a 400 MHz spectrometer without the need of reference standards. The developed methodology is able to differentiate and assess chemical structures of these toxic injurious compounds. The quantity of each was calculated on the basis of the integrals for the signals of the H-7 and H-8 of the phenanthrene ring of AA-I and AA-II at delta7.38 and delta8.31, respectively, and the vinylic protons of the internal standard maleic acid at delta6.06. The accuracy of the method was established through the analysis of synthetic mixtures containing the internal standard maleic acid, with purified AA-I or combined AA-I and AA-II sodium salts. Excellent agreements were verified between the assay results and the quantities in the mixtures. The mean +/- SD recovery values for purified AA-I and combined AA-I and AA-II from two sets of 10 synthetic mixtures were 99.8 +/- 0.6% and 99.6 +/- 0.8%, respectively. The assay of 4 lots of commercial aristolochic acid by 1H NMR spectroscopy indicated AA-I and AA-II contents in the ranges 45.3-97.1% and 0-15.4%, respectively.     

Synthesis and structural studies of aza analogues of functionalized amino acids: new anticonvulsant agents

We have reported that functionalized amino acids 1 display potent anticonvulsant activities in mice and rats, and that the activity resides primarily in the D-isomer. In this study we investigated whether selectively replacing the C(2) tetrahedral atom with a trivalent nitrogen provides compounds with comparable activity. Six functionalized N(2)-substituted semicarbazides (3) were prepared. X-ray crystallographic analysis of 1-acetyl-4-benzyl-2-(thiazol-2-yl)semicarbazide (13) showed that it lost asymmetry and adopted a configuration midway between the corresponding D- and L-amino acid derivatives. Evaluation of 3 in both mice (ip) and rats (po) showed that the compounds exhibited significant anticonvulsant activities but in most cases at levels lower than their amino acid counterparts. One of the semicarbazides, 13, displayed excellent activity in mice and rats that compared favorably to that of phenytoin.