Design, synthesis and antifungal activity of threoninamide carbamate derivatives via pharmacophore model.

Thirty-six novel threoninamide carbamate derivatives were designed and synthesised using active fragment-based pharmacophore model. Antifungal activities of these compounds were tested against Oomycete fungi Phytophthora capsici in vitro and in vivo. Interestingly, compound I-1, I-2, I-3, I-6 and I-7 exhibited moderate control effect (>50%) against Pseudoperonospora cubensis in greenhouse at 6.25 µg/mL, which is better than that of control. Meanwhile most of these compounds exhibited significant inhibitory against P. capsici. The other nine fungi were also tested. More importantly, some compounds exhibited remarkably high activities against Sclerotinia sclerotiorum, P. piricola and R. solan in vitro with EC50 values of 3.74-9.76 µg/mL. It is possible that the model is reliabile and this method can be used to discover lead compounds for the development of fungicides.

Chemical Synthesis of Oligoribonucleotide (ASL of tRNALys T.†brucei) Containing a Recently Discovered Cyclic Form of 2-Methylthio-N6 -threonylcarbamoyladenosine (ms2 ct6 A).

The synthesis of the protected form of 2-methylthio-N6 -threonylcarbamoyl adenosine (ms2 t6 A) was developed starting from adenosine or guanosine by using the optimized carbamate method and, for the first time, an isocyanate route. The hypermodified nucleoside was subsequently transformed into the protected ms2 t6 A-phosphoramidite monomer and used in a large-scale synthesis of the precursor 17nt ms2 t6 A-oligonucleotide (the anticodon stem and loop fragment of tRNALys from T. brucei). Finally, stereochemically secure ms2 t6 A→ms2 ct6 A cyclization at the oligonucleotide level efficiently afforded a tRNA fragment bearing the ms2 ct6 A unit. The applied post-synthetic approach provides two sequentially homologous ms2 t6 A- and ms2 ct6 A-oligonucleotides that are suitable for further comparative structure-activity relationship studies.

Total synthesis and stereochemical revision of xiamenmycin A.

The relative and absolute configurations of xiamenmycin A, a benzopyran compound isolated from Streptomyces xiamenensis 318 with a highly potent anti-fibrotic activity, have been characterized through the total synthesis. The key steps include the construction of the 3-chromanol moiety via Sharpless epoxidation followed by regio- and diastereo-selective cyclization and introduction of the threonine moiety at a later stage via Pd-catalysed aminocarbonylation in a one-pot procedure. The stereochemical assignment of natural xiamenmycin A has been accordingly revised to be 2R, 3S, 3'S, 4'R.

Total synthesis of xiamenmycin C and all of its stereoisomers: stereochemical revision.

Xiamenmycin C, a potent anti-fibrotic natural product, and all of its stereoisomers have been synthesized and their structures were fully characterized. Based on this study, the originally proposed structure of xiamenmycin C has been accordingly revised to be 2R,3S.

Asymmetric synthesis of chloroisothreonine derivatives via syn-stereoselective Mannich-type additions across N-sulfinyl-α-chloroimines.

Mannich-type reactions of O-Boc glycolic esters across chiral N-sulfinyl-α-chloroaldimines resulted in the efficient and syn-stereoselective synthesis of new γ-chloro-α-hydroxy-ß-amino esters (dr > 99 : 1). The α-coordinating ability of the chlorine atom was of great importance for the diastereoselectivity of the Mannich-type reaction and overruled the chelation of the sulfinyl oxygen with the lithium ion of the incoming E-enolate in the transition state model. These novel chloroisothreonine derivatives proved to be excellent building blocks in asymmetric synthesis of novel syn-ß,γ-aziridino-α-hydroxy esters and biologically relevant trans-oxazolidinone carboxylic esters.

Development and application of serine/threonine ligation for synthetic protein chemistry.

Chemical synthesis of proteins, especially those with post-translational modifications, has offered new opportunities to study the protein structure-function relationship. In the past four years, we have developed the serine/threonine ligation (STL), which involves the chemoselective reaction between peptide salicylaldehyde esters and peptides with N-terminal serine or threonine. The method has been successfully applied to the synthesis of both linear and cyclic peptides/proteins.

Preparation of optically active allothreonine by separating from a diastereoisomeric mixture with threonine.

A simple procedure is described to obtain D- and L-allothreonine (D- and L-aThr). A mixture of N-acetyl-D-allothreonine (Ac-D-aThr) and N-acetyl-L-threonine (Ac-L-Thr) was converted to a mixture of their ammonium salts and then treated with ethanol to precipitate ammonium N-acetyl-L-threoninate (Ac-L-Thr·NH(3)) as the less-soluble diastereoisomeric salt. After separating Ac-L-Thr·NH(3) by filtration, Ac-D-aThr obtained from the filtrate was hydrolyzed in hydrochloric acid to give D-aThr of 80% de, recrystallized from water to give D-aThr of >99% de. L-aThr was obtained from a mixture of the ammonium salts of Ac-L-aThr and Ac-D-Thr in a similar manner.

Synthesis of Lipoamino acids and their activity against cerebral ischemic injury.

A series of lipoamino acids were synthesized and their neuroprotective effect against brain ischemia induced by oxygen-glucose deprivation (OGD) on rat cerebral slices was evaluated. Among these compounds, N-stearoyl-L-tyrosine (4), N-stearoyl-L-serine (5) and N-stearoyl-L-threonine (6) exhibited good neuroprotective activity. We found that the neuroprotective activity of lipoamino acids depended on the acyl group, the presence of a free carboxylic function and a free hydroxyl group at the branched chain of the amino acids. The results also showed that 5 was the most active compound, protecting rat brain slices against OGD as well as hydrogen peroxide (H(2)O(2)) insult at the range of 1-10 M.

Efficient synthesis of suitably protected beta-difluoroalanine and gamma-difluorothreonine from L-ascorbic acid.

[reaction: see text] Fluorinated amino acids are useful building blocks for the preparation of biologically active peptides and peptidomimetics with increased metabolic stability. We report here the synthesis of two fluorinated amino acids, beta-difluoroalanine and gamma-difluorothreonine, as analogues of Ser and Thr, respectively. These compounds were suitably protected for Fmoc-based solid-phase peptide synthesis. Once incorporated into peptides, they may serve as alternative substrates or inhibitors of lantibiotic synthetases that posttranslationally dehydrate Ser and Thr residues to dehydroalanine and dehydrobutyrine, respectively.

Towards preparative asymmetric synthesis of beta-hydroxy-alpha-amino acids: L-allo-threonine formation from glycine and acetaldehyde using recombinant GlyA.

The diastereospecific formation of L-allo-threonine, catalyzed by the serine hydroxymethyltransferase GlyA form Escherichia coli, was studied with regard to the application in continuous processes. Process design will rely on a suitable description of enzyme stability and kinetics under relevant process conditions. Therefore, the effects of addition of organic co-solvents--methanol and acetonitrile--to the reaction mixtures on activity, stability, and diastereoselectivity were investigated. A series of progress curves from batch reactions at 35 degrees C in 50mM sodium phosphate buffer pH 6.6 and 50mM sodium phosphate buffer pH 6.6 in 20% methanol was used to estimate the respective kinetic parameters for an appropriate kinetic model. The experimental data agreed well with a kinetic model for an ordered reaction mechanism of the type bi-uni including the formation of a ternary complex and a pseudo-equilibrium assumption. The model was then applied in order to simulate the performance of the enzyme in an enzyme membrane reactor (EMR) and gave an excellent agreement with the corresponding experimental data. A space time yield of 227g L(-1)d(-1) was achieved in a continuous running EMR without significant loss of enzyme activity over 120 h of operation.

Fluoro amino acids: a rarity in nature, yet a prospect for protein engineering.

Fluoro amino acids are highly valuable compounds constantly gaining relevance in diverse fields of the biosciences as well as in the pharmaceutical industry. The value of these compounds can be attributed to the properties of the extremely electronegative fluorine atom. This atom forms a highly polarized bond of extraordinary strength with carbon. The formation of the fluorine-carbon bond is challenging: its chemical synthesis demands harsh reaction conditions and to date only one class of enzyme has been found capable of introducing the fluoride ion into an organic compound. Most of these fluorinating enzymes participate in the biosynthesis of 4-fluoro-L-threonine, the only fluoro amino acid of natural origin discovered so far. Despite their scarcity in nature, fluoro amino acids are valuable tools to fluorinate proteins. The fluoro protein variants often show improved stability and folding as well as altered activity and fluorescence characteristics. This review details the biosynthesis of 4-fluoro-L-threonine with a special focus on the fluorinating enzymes. Moreover, we elaborate on the application of fluoro amino acids as building blocks for fluorinated protein variants. Insight into different techniques to incorporate fluoro amino acids into proteins is also provided. We highlight prospects and the current relevance of fluoro amino acids as a tool to engineer proteins with novel traits.

Dipeptides of O-methyl-L-threonine as potential antimalarials.

L-Leucyl-O-methyl-L-threonine, O-methyl-L-threonyl-L-leucine, and O-methyl-L-threonyl-O-methyl-L-threonine were prepared and compared with O-methyl-L-threonine and L-leucine for antimalarial activity against Plasmodium berghei in mice. O-Methyl-L-threonine significantly prolonged survival time at doses of 160, 320, and 640 mg/kg. O-Methyl-L-threonyl-O-methyl-L-threonine was less active, significantly prolonging survival time only at 640 mg/kg. L-Leucine, as well as the other two dipeptides, exhibited no activity in this test.

Synthesis and some pharmacological properties of deamino(4-threonine,8-D-arginine)vasopressin and deamino(8-D-arginine)vasopressin, highly potent and specific antidiuretic peptides, and (8-D-arginine)vasopressin and deamino-arginine-vasopressin.

Deamino[4-threonine,8-D-arginine]vasopressin (dTDAVP), deamino[8-D-arginine]vasopressin (dDAVP), [8-D-arginine[vasopressin (DAVP), and deamino-arginine-vasopressin (dAVP) were synthesized by the solid-phase method and tested for their biological activities. dTDAVP has an antidiuretic potency of 793+/-95 units/mg and undetectable vasporessor activity, less than 0.02unit/mg. The antidiuretic-pressor (A/P) ratio of dTDAVP is greater than 39 000. dDAVP has an antidiuretic potency of 1200+/-126 units/mg and a vasopressor potency of 0.39+/-0.02; its A/P ratio is thus 3000. DAVP has an antidiuretic potency of 253+/-44 units/mg, a vasopressor potency of 1.1+/-0.04 units/mg, and an A/P ratio of 240. The A/P ratios of dDAVP and DAVP are much higher than those originally reported. dAVP has an antidiuretic potency of 1745+/-385 units/mg, a vasopressor potency of 346+/-13, and an A/P ratio of 5; values are in general agreement with those in the literature. Threonine subsitution has thus brought about a significant enhancement in antidiuretic specificity, a finding entirely consistent with earlier observations that enhancement of lipophilicity at position 4 alone or in combination in arginine-vasopressin can lead to enhanced antidiuretic specificity.

Some properties of synthetic fucopyranosylserines and -threonines.

alpha Fuc-Ser, alpha Fuc-Thr, beta Fuc-Ser, and beta Fuc-Thr were synthesized stereoselectively in good yields, and their properties were investigated. beta-Fucosylserine (and -threonine) was synthesized by Koenigs-Knorr reaction of per-O-acetylfucopyranosyl bromide with N-carbobenzoxy-L-serine (and -threonine) benzyl ester using mercuric cyanide, followed by hydrogenolysis and deacetylation. alpha-Fucosylserine (and -threonine) was synthesized by the halide-ion catalyzed reaction of per-O-benzylfucopyranosyl bromide with the serine (and threonine) derivative described above, followed by hydrogenolysis. These substances each gave a single peak on an amino acid analyzer. The anomeric configurations of these compounds were determined by 1H-NMR spectroscopy, optical rotation measurement and enzymatic degradations of these compounds. On acid hydrolysis, alpha Fuc-Ser and alpha Fuc-Thr were more rapidly hydrolyzed than the corresponding beta-series compounds. Under mild alkaline conditions in the presence of sodium borohydride, per-protected beta Fuc-Thr released about 50% of fucose but free beta Fuc-Thr did not However, free alpha Fuc-Thr and beta Fuc-Thr were cleaved to give acetylated fucose during acetylation with acetic anhydride-pyridine. alpha-L-Fucosidases, which were purified from abalone and rabbit livers by affinity chromatography, acted very well on alpha Fuc-Ser and alpha Fuc-Thr, but not on the beta-series compounds.

Synthesis of dinitrophenyl derivatives of 3-O-fatty acyl esters of serine and threonine.

The synthesis of 2,4-dinitrophenyl derivatives of the 3-O-oleoyl and 3-O-palmitoyl esters of serine and threonine are described. The derivatives were purified by preparative thin-layer chromatography (TLC) and characterized by 1H-nuclear magnetic resonance (NMR) spectroscopy. These derivatives may be useful for researchers interested in characterizing covalently bound fatty acids on serine and threonine hydroxyl groups of cellular proteins.

Synthesis and conformational and NMR studies of alpha-D-mannopyranosyl and alpha-D-mannopyranosyl-(1----2)-alpha-D-mannopyranosyl linked to L-serine and L-threonine.

alpha-D-Mannopyranosyl and alpha-D-mannopyranosyl-(1----2)-alpha-D- mannopyranosyl linked to L-serine and L-threonine have been synthesised as model substances for the linkage region in certain O-linked glycoproteins. Metropolis Monte Carlo simulations were performed with a modified version of the GESA program, to yield theoretical NOEs and interatomic distances as ensemble-average values, and these were compared with results from steady-state NOE experiments. The NOEs were determined as ensemble-average and as global minimum values. NMR chemical shift differences, obtained for signals of the glycopeptides relative to those of the respective monomers, were interpreted in terms of short inter-residue atomic distances as found within the global minima, and on the basis of averaged distances derived from Monte Carlo simulations.

An efficient access to protected disialylated glycohexaosyl threonine present on the leukosialin of activated T-lymphocytes.

The total synthesis of the threonine-linked core 2 class disialylated hexasaccharide in a completely protected form was accomplished for the first time. The L-threonine conjugate, N-(9-fluorenylmethoxycarbonyl)-O-[(5-acetamido-4,7,8,9-tetra-O-ben zyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2-->3)-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-2-acetam ido-2-deoxy-3,6-di-O-benzyl-beta-D-glucopyranosyl-(1-->6)-[(5-acetamido- 4,7,8,9-tetra-O-benzyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulo pyranosylonic acid)-(2-->3)-2,6-di-O-benzyl-beta-D-galactopyranosyl-(1-->3)]-2-acetami do-2-deoxy-alpha-D-galactopyranosyl-(1d-->4c:1f-->4e)-dilactone ]-L-threonine allyl ester was synthesized via stereocontrolled glycosylations employing readily accessible monosaccharidic blocks; t-butyl-diphenylsilyl-2-azido-2-deoxy-3,6-di-O-benzyl-beta-D-gluco pyranose, N-(9-fluorenylmethoxycarbonyl)-O-(2-azido-6-O-t-butyldimethylsilyl -2-deoxy-alpha-D-galactopyranosyl)-L-threonine allyl ester, 8, 9 and N-(9-fluorenylmethoxycarbonyl)-O-(2-azido-4,6-O-benzylidene-3-O-ch loroacetyl-2-deoxy-alpha-D-galactopyranosyl)-L-threonine allyl ester. For the introduction of the amino acid, the azide group was used to temporarily mask the amino group of GalNAc so as to obtain an alpha-glycosidic linkage without participation from the C-2 substituent. The threonine was attached to the sugar unit at the monosaccharide stage to avoid loss of oligosaccharide at a later stage. The Fmoc and allyl ester protected amino acid at the reducing end facilitates efficient glycopeptide synthesis on solid-phase support.

The use of synthetic lysine in the diet of lactating sows.

We conducted an experiment to determine the proportion of the lysine requirement of lactating sows that can be met using L-lysine x HCl. A total of 247 Pig Improvement Company (PIC) sows (parity one to four) were randomly allotted to one of five experimental diets containing .79% apparently digestible lysine. The first four diets contained 0, .075, .150, and .225% L-lysine x HCl replacing the intact lysine, primarily derived from soybean meal. Dietary crude protein was reduced from 17.9 to 16.9, 15.8, and 14.8% respectively. The fifth diet contained .174% L-lysine x HCl (15.5% CP) with added synthetic methionine, threonine, and tryptophan to restore the ratios of these amino acids to lysine to those in the control diet with no synthetic amino acids. The average lactation length was 15.7 +/- .3 d. Diet did not affect ADFI, sow backfat loss, sow loin eye area loss, or weaning-to-mating interval. Sows consumed an average of 4.6 kg/d and were provided 36 g/d of digestible lysine. Replacing soybean meal with increasing levels of L-lysine x HCl did not affect sow weight change. The number of pigs weaned decreased and preweaning mortality increased linearly (P = .08) with increasing levels of L-lysine x HCl. Litters from sows fed the .174% L-lysine x HCl with added methionine, threonine, and tryptophan grew slower and had a higher mortality rate than litters from sows fed no synthetic amino acids (P < .05). The addition of synthetic methionine, threonine, and tryptophan to the .174% L-lysine x HCl diet did not improve litter growth rate, but it did increase preweaning mortality (P = .05) and decrease the number of pigs weaned (P = .06) compared to the .15% L-lysine x HCl with no additional synthetic amino acids. These additions also resulted in an increased sow weight loss (P = .10). These results suggest that when more than .075% L-lysine x HCl is used to meet the lysine requirement preweaning mortality is increased and the number of pigs weaned is decreased. Supplementation with methionine, threonine, and tryptophan failed to ameliorate the negative response associated with L-lysine x HCl, which suggests that other amino acids may be limiting.

Serine/threonine ligation for the chemical synthesis of proteins.

Advances in the development of efficient peptide ligation methods have enabled the total synthesis of complex proteins to be successfully undertaken. Recently, a Ser/Thr ligation has emerged as a new tool in synthetic protein chemistry. The chemoselective reaction between an N-terminal serine or threonine of an unprotected peptide segment and a C-terminal salicylaldehyde ester of another unprotected peptide segment gives rise to an N,O-benzylidene acetal linked product, which upon acidolysis produces a native peptide bond at the site of ligation. Ser/Thr ligation has been used for the synthesis of the human erythrocyte acylphosphatase protein and MUC1 glycopeptide segments, semisynthesis of peptoid/PEG-RNase S protein hybrids, and cyclic peptide synthesis including cyclic tetrapeptides, cyclomontanin B, yunnanin C, mahafacyclin B, and daptomycin.

New chemistries for chemoselective peptide ligations and the total synthesis of proteins.

The identification of fast, chemoselective bond-forming reactions is one of the major contemporary challenges in chemistry. The requirements of the native chemical ligation - an N-terminal cysteine and C-terminal thioesters - have encouraged a search for alternative amide-forming ligation reactions. Among successful alternatives to native chemical ligation, are the α-ketoacid-hydroxylamine ligation with 5-oxaproline and, serine/threonine ligation, and potassium acyltrifluoroborate (KAT) ligation. In addition, the KAT ligation, along with the non-amide forming alkyne-azide ligation, is very useful for synthetic conjugations. All of these recent ligation methods were applied to synthesize different proteins, and have allowed chemists to incorporate unnatural amino acids, or to modify the peptide backbone.