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A digestibility trial was carried to assess the apparent digestibility coefficient (ADC) of crude protein (CP) and amino acids (AA) in two feather meals (FeM) of different origin (FeM1 and FeM2) and in their enzymatically pre‐treated ingredient stream (PTFeM1 and PTFeM2) in rainbow trout. The ADC of CP was 11%–17% higher (p 0.05) of ADC of CP among FeMs and among PTFeMs. The FT‐Raman analysis showed a reduction in concentration and conformational stability of disulphide bonds in the PTFeMs when compared to their FeM counterparts. Moreover, important discrepancies in disulphide concentration were observed between FeMs and between PTFeMs. These observations suggest that the ADC of CP in FeM is not only governed by disulphide bond content and other variables should be considered. Higher concentrations of the cross‐linked amino acids (CLAA) lanthionine, lysinoalanine and β‐aminoalanine were measured in FeM1 and PTFeM1 as compared to FeM2 and PTFeM2 respectively. Previous studies have suggested inverse relationships between CLAAs and the digestibility as well as bioavailability of AAs in feed ingredients. A previous study conducted in our laboratory showed a lower (p

Alloviroidin is a cyclic heptapeptide, produced by several species of Amanita mushrooms, that demonstrates high affinity for F-actin as is characteristic of virotoxins and phallotoxins. Alloviroidin was synthesized via a [3 + 4] fragment condensation of Fmoc-d-Thr(OTBS)-d-Ser(OTBS)-(2 S,3 R,4 R)-DHPro(OTBS)2-OH and H-Ala-Trp(2-SO2Me)-(2 S,4 S)-DHLeu(5-OTBS)-Val-OMe to form bond A. The linear heptapeptide favored a turn conformation, facilitating cyclization between Val1 and d-Thr2 (position B). Global deprotection and HPLC purification afforded alloviroidin with NMR spectra in excellent agreement with the natural product.

Hydrogen bonds (H bonds) play a major role in defining the structure and properties of many substances, as well as phenomena and processes. Traditional H bonds are ubiquitous in nature, yet the demonstration of weak H bonds that occur between a highly polarized C-H group and an electron-rich oxygen atom, has proven elusive. Detailed here are linear and nonlinear IR spectroscopy experiments that reveal the presence of H bonds between the chloroform C-H group and an amide carbonyl oxygen atom in solution at room temperature. Evidence is provided for an amide solvation shell featuring two clearly distinguishable chloroform arrangements that undergo chemical exchange with a time scale of about 2 ps. Furthermore, the enthalpy of breaking the hydrogen bond is found to be 6-20 kJ mol-1 . Ab-initio computations support the findings of two distinct solvation shells formed by three chloroform molecules, where one thermally undergoes hydrogen-bond making and breaking.

The microbisporicins are the most potent lantibiotics isolated to-date. Cyclic tetra-, hexa- and octapeptides, inspired by this family of antimicrobial agents, have been synthesized from linear peptides. Generalized reaction conditions are reported for the two-step conversion of linear peptides to thioether macrocycles: formation of a disulfide followed by reductive desulfurization. 1H NMR analysis of the reduction reaction mixture indicated the intermediacy of a dehydroalanine when excess hexamethylphosphorus triamide (HMPT) was employed for the reduction. Maintaining a stoichiometric amount of HMPT, in dilute methanolic solution, gave the corresponding thioethers, retaining stereochemical integrity.

The dramatic increase in the prevalence of antibiotic-resistant bacteria has necessitated a search for new antibacterial agents against novel targets. Moiramide B is a natural product, broad-spectrum antibiotic that inhibits the carboxyltransferase component of acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid synthesis. Herein, we report the 2.6 Å resolution crystal structure of moiramide B bound to carboxyltransferase. An unanticipated but significant finding was that moiramide B bound as the enol/enolate. Crystallographic studies demonstrate that the (4S)-methyl succinimide moiety interacts with the oxyanion holes of the enzyme, supporting the notion that an anionic enolate is the active form of the antibacterial agent. Structure–activity studies demonstrate that the unsaturated fatty acid tail of moiramide B is needed only for entry into the bacterial cell. These results will allow the design of new antibacterial agents against the bacterial form of carboxyltransferase.

An orthogonally protected τ-histidinoalanine residue was synthesized via regioselective alkylation of optically pure Boc- l -His-OTCE (TCE = trichloroethyl) with a sulfamidate electrophile derived from Fmoc- d -Ser-OBn. The goal was the synthesis of a non-natural theonellamide, invoking readily accessible variants of the other 10 amino acids. Peptide fragments corresponding to the east and west rings of “theonellamide X″ were synthesized in solution. Each ring was formed independently, providing insights into protecting group limitations, side reactions, and the optimal order of events to approach the formation of the bicyclic system. Ultimately, an undecapeptide was prepared, with the eastern ring formed. The twelfth amino acid, an l -α-aminoadipic acid building block, was prepared and preliminary investigations into its attachment to the undecapeptide are reported.

Peptides that exhibit enzymatic or hormonal activities are regulatory factors and desirable therapeutic drugs because of their high target specificity and minimal side effects. Unfortunately, these drugs are susceptible to enzymatic degradation, leading to their rapid elimination and thereby demanding frequent dosage. Structurally modified forms of some peptide drugs have shown enhanced pharmacokinetics, improving their oral bioavailability. Here, we discuss a novel glycomimetic approach to modify lysine residues in peptides. In a model system, the ε-amine of Ts-Lys-OMe was reductively alkylated with a glucose derivative to afford a dihydroxylated piperidine in place of the amine. A similar modification was applied to H-KPV-NH2, a tripeptide derived from the α-melanocyte stimulating hormone (α-MSH) reported to have antimicrobial and anti-inflammatory properties. Antimicrobial assays, under a variety of conditions, showed no activity for Ac-KPV-NH2 or the α- or ε-glycoalkylated analogs. Glycoalkylated peptides did, however, show stability toward proteolytic enzymes.

Kalata B1 (4) is a prototypical, 29-residue, Mobius cyclotide with a cis prolyl peptide bond in loop 5. Two analogs were synthesized in which Pro24 was substituted by trans-4-hydroxy-l-proline (peptide 5) and cis-4-hydroxy-l-proline (peptide 6). Linear peptides were assembled by solid phase peptide synthesis using Fmoc/tBu chemistry. Head-to-tail cyclization was performed using HATU, side-chain protecting groups removed and the cyclic peptides 2 and 3 isolated by RP-HPLC. Oxidation led to the formation of peptides 5 and 6, each incorporating three disulfide bonds. Analysis of TOCSY and NOESY spectra of the purified peptides enabled assignment of the backbone amide and Hα resonances. These showed a striking correlation with those of native kalata B1, indicating that folding had produced the same disulfide bridge topology. While somewhat surprising that stereoelectronic effects introduced by the hydroxyl substituents in this key region of the peptide had little impact, this reflects the strong thermodynamic driving force toward formation of the cyclic cystine knot scaffold.

Streptomyces ipomoeae is the causal agent of Streptomyces soil rot of sweet potato, a disease marked by highly necrotic destruction of adventitious roots, including the development of necrotic lesions on the fleshy storage roots. Streptomyces potato scab pathogens produce a phytotoxin (thaxtomin A) that appears to facilitate their entrance into host plants. S. ipomoeae produces a less-modified thaxtomin derivative (thaxtomin C) whose role in pathogenicity has not been examined. Here, we cloned and sequenced the thaxtomin gene cluster (txt) of S. ipomoeae, and we then constructed targeted txt mutants that no longer produced thaxtomin C. The mutants were unable to penetrate intact adventitious roots but still caused necrosis on storage-root tissue. These results, taken in context with previous histopathological study of S. ipomoeae infection, suggest that thaxtomin C plays an essential role in inter- and intracellular penetration of adventitious sweet potato roots by S. ipomoeae. Once inside the plant host, the pathogen uses one or more yet-to-be-determined factors to necrotize root tissue, including that of any storage roots it encounters.

Previous syntheses of histidinoalanine (HAL) have led to mixtures of regioisomers and/or stereoisomers. For example, opening of N-Cbz-D-serine-β-lactone (6) with Boc-L-His-OMe (5) gave a 2:1 mixture of τ- and π-regioisomers. The sulfamidate 10, derived from N-benzyl-D-serine methyl ester (11), was reacted with Boc-L-His-OMe (5) to give the τ-HAL derivative 17 as a single isomer in 57% yield. A similarly prepared τ-HAL 19, bearing protecting groups that were all hydrogenolytically labile, led to the free bis-amino acid, τ-L-histidinyl-D-alanine (τ-4), as a salt-free standard for amino acid analysis.

Skp1 is a subunit of E3SCFUbiquitin-ligases and other protein complexes (1). In the social soil amoeba Dictyostelium discoideum, Skp1 contains a novel hydroxyproline (Hyp)1-linked pentasaccharide. The Hyp-glycosylation pathway consists of P4H1, a non-heme Fe(II)-dependent dioxygenase that modifies Skp1 at Pro143, and five sequentially acting cytoplasmic glycosyltransferase activities encoded by three genes (2). P4H1 may serve as an O2 sensor, as the enzyme has a high Km for O2 in vitro (3), elevated O2 levels are required for P4H1-null cells to culminate into fruiting bodies, and reduced O2 is sufficient for P4H1-overexpression cells (4). Reverse genetic analyses suggest that the sequential post-translational glycosylation events modulate the effect of hydroxylation in hierarchical fashion (5). Based on biochemical and genetic studies, Skp1 is the only substrate for P4H1 and the glycosyltransferases in cells, and mediates the role of the pathway enzymes on culmination and other developmental transitions (6). As recently summarized (2), the Hyp-glycosylation pathway appears to be widely expressed in unicellular protists including some significant human and plant pathogens. Among known prolyl 4-hydroxylases, P4H1 is most related to the sequences of Egl-9 (Eggless-9), an enzyme involved in O2-regulation in C. elegans, and the 3 human homologs that sense O2 in humans. Egl-9 and the human PHDs (prolyl hydroxylase domain proteins; also known as EGLNs or HPHs) modify hypoxia-inducible factor-α (HIFα), a subunit of the HIFα-HIF® transcriptional factor heterodimer whose accumulation directly induces hypoxia response genes that support glycolysis, angiogenesis, and erythropoiesis (7). Hydroxylation of Pro402 or Pro564 of HIFα results in recognition by the von Hippel-Lindau subunit of the E3VBCUb-ligase followed by degradation within the 26S-proteasome. Egl-9 and human PHD1-3 are thought to serve as direct O2-sensors, in part due to their relatively high Km values for O2 as a substrate. Their activity in cells is promoted by the availability of the co-substrate αKG (α-ketoglutarate, a Krebs cycle intermediate), ascorbate, and Fe(II), and inhibited by the product succinate, other Krebs cycle metabolites, and reactive oxygen species (e.g., 8,9). Much remains to be learned about the regulation of PHDs, and the significance of additional PHD targets in animal cells (10). Whereas HIFα and possibly other proteins mediate PHD/Egl-9-dependent hypoxic responses in animal cells, Dictyostelium apparently lacks HIFα and the VBC-complex that recognizes it, and P4H1-dependent hypoxic responses appear to be mediated by Skp1 (6). Conversely, though Skp1 is highly conserved in eukaryotes, the equivalent of Pro143 is notably absent in chordates. Interestingly, Skp1 is evolutionarily related, not to HIFα, but to elongin C, a subunit of the E3VBCUb-ligase that targets HIFα for degradation. The homologous E3SCFUb ligases, conserved from yeast to humans, contain the SCF subcomplex whose Skp1 adaptor links the scaffold protein cullin-1 to an F-box protein, which presents targets such as cell cycle and other regulatory proteins for polyubiquitination. Analysis of the human and Dictyostelium genomes predicts more than 50 F-box proteins (unpublished data), potentially diversifying the pool of SCF complexes, and thereby suggesting a global role in regulation of the proteome (11). Interestingly, there is no evidence that P4H1 regulates the stability of its Skp1 target (6). A biochemical analysis of substrate selection by P4H1 was initiated to provide insight into mechanism of this evolutionary shift between protists and animals, and to reveal clues about regulation of its hydroxylase activity. As shown here, adoption of a direct P4H1 assay revealed similar sensitivity to metabolic inhibitors, and 1H-NMR studies demonstrated formation of (2S,4R)-4-hydroxy-L-proline (a.k.a. trans-4-hydroxyproline, Hyp), as for human PHD2 (12–14). However, whereas PHD2 recognizes truncated oxygen-dependent degradation domains (ODDs) of HIFα, and peptides as short as 15-mers have successfully been employed as substrates, P4H1-processing of Skp1 depended on global structural attributes. Remarkably, substrate recognition is conserved in animal Skp1s. The implication that P4H1 recognition depends on conserved features important for SCF-complex formation was supported by loss of substrate activity when Skp1 was complexed with an F-box protein. Such a recognition mechanism could explain P4H1’s apparent high degree of specificity for Skp1, and suggests how Skp1 modification may be regulated in cells. A similar dependence on Skp1 features was observed for the Hyp-capping activity of Gnt1, whose gene is phylogenetically co-distributed with protist P4H1 and in some genomes appears to be encoded as a separate domain of the same protein (2). Whereas animal PHD’s render global effects by regulating the half-life of HIFα with transcriptional consequences on many genes, global effects of Dictyostelium P4H1 and its partner Gnt1 may be rendered by the targeting of Skp1 alone that controls the half-lives of many proteins via the large family of E3SCFUb-ligases.

Nα-tert-Butoxycarbonyl-L-trans-4-hydroxyproline allyl ester (Boc-Hyp-OAll) was glycosylated with 2,3,5-tri-O-benzyl-L-arabinose p-cresylthioglycoside in 60% yield with 4:1 β:α stereoselectivity. Deprotection of N- and C-terminii independently gave a prolyl amine and prolyl carboxylate respectively that were coupled under standard conditions with 1-[bis-(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5,b]-pyridininium hexafluorophosphate 3-oxide (N-HATU) to give the dimer 1 in 46% yield. These results represent the first steps toward the production of homogeneous oligomers to determine the minimal epitope of the Art v 1 allergen.

Reaction of 1,2-O-dibutylstannylene-3,4-6-tri-O-benzyl-beta-D-mannopyranose with Nalpha-fluorenylmethoxycarbonyl-cis-4-trifluoromethanesulfonyloxyproline allyl ester led to formation of a beta-mannoside of trans-4-hydroxyproline. Subsequent manipulation of the C2 hydroxy group gave rise to beta-D-Glc and beta-D-GlcNAc derivatives.

The synthesis of two naturally-occurring isomers of 3,4-dihydroxyproline is reported. l -2,3-cis-3,4-trans-3,4-Dihydroxyproline was synthesized from l -arabinose in 10 steps and 31% overall yield. The same series of reactions was employed to convert l -xylose to l -2,3-trans-3,4-trans-3,4-dihydroxyproline. Orthogonally protected versions of these amino acids were produced on gram scale, en route to the free amino acids, and these will serve as versatile intermediates in peptide synthesis. This synthetic strategy involved Nα-Fmoc protection and protection of the C3 and C4 secondary alcohols as methoxyethoxymethyl (MEM) ethers.

[reaction: see text] NMR was used to study the thermodynamics of the cis --trans isomerization for prolyl amide bonds in the compounds shown. The magnitude of K(t/c) for C-terminal esters is greater than for the corresponding amides, signifying stronger backbone stereoelectronic effects in esters. Increasing the steric bulk of the N-terminal residue from Ac- to Ac-Gly- favors the trans conformation. Incorporation of a Phe residue N-terminal to Pro, however, shifts the equilibrium in favor of the cis conformation, via a stabilizing aromatic-proline interaction.

Readily available, crystalline penta- O -propanoyl-β- d -glucofuranose is shown to be a suitable glycosylating agent for the acid-catalysed, direct synthesis of O -, S - and N -glucofuranosyl compounds. β-Linked products are formed with good selectivity. Reaction with cyanotrimethylsilane gave the 1,2- O -(1-cyanopropylidene)acetal rather than the C -glycosyl cyanide. By selective acid-catalysed hydrolysis, the title compound was converted to the 1-hydroxy analogue from which the trichloroacetimidates were made as further potential glycosylating agents.

Hydrated zirconium molybdate (ZMh) is known to precipitate from solutions of dissolved spent nuclear fuel, particularly from the waste fission product solution after the uranium and plutonium have been extracted during reprocessing. Its precipitation can cause major problems during waste treatment, and therefore a complete understanding of its chemical behaviour, especially with regard to its role in the nuclear fuel cycle, is desirable. We have used a number of complementary analytical techniques to elucidate the hitherto incompletely understood chemistry of formation of ZMh in synthetic fuel reprocessing solutions. We have demonstrated that ZMh formation was governed by multi-step surface reactions and does not involve the formation of colloids or particulates in solution. The first step in the deposition of ZMh onto surfaces is the formation of an amorphous film with a Zr ∶ Mo ratio close to unity. It is followed by the formation, growth and nucleation of ZMh particles of varying degrees of crystallinity with a Zr ∶ Mo ratio close to 0.5. The X-ray diffraction pattern of deposited ZMh particles is in agreement with the reported crystallographic data. The structural features of the film and ZMh were also examined at the nanometer scale.

There is an urgent demand for the development of new antibiotics due to the increase in drug-resistant pathogenic bacteria. A novel target is the multifunctional enzyme acetyl-CoA carboxylase (ACC), which catalyzes the first committed step in fatty acid synthesis and consists of two enzymes: biotin carboxylase and carboxyltransferase. Covalently attaching known inhibitors against these enzymes with saturated hydrocarbon linkers of different lengths generated dual-ligand inhibitors. Kinetic results revealed that the dual-ligands inhibited the ACC complex in the nanomolar range. Microbiology assays showed that the dual-ligand with a 15-carbon linker did not exhibit any antibacterial activity, while the dual-ligand with a 7-carbon linker displayed broad-spectrum antibacterial activity as well as a decreased susceptibility in the development of bacterial resistance. These results suggest that the properties of the linker are vital for antibacterial activity and show how inhibiting two different enzymes with the same compound increases the overall potency while also impeding the development of resistance.

Prolyl hydroxylation and subsequent glycosylation of the E3(SCF) ubiquitin ligase subunit Skp1 affects its conformation and its interaction with F-box proteins and, ultimately, O2-sensing in the organism. Taking a reductionist approach to understand the molecular basis for these effects, a series of end-capped Thr-Pro dipeptides was synthesized, tracking the sequential post-translational modifications that occur in the protein. The conformation of the pyrrolidine ring in each compound was gauged via coupling constants ((3)JHα,Hβ) and the electronegativity of the Cγ-substituents by chemical shifts ((13)C). The equilibrium between the cis-trans conformations about the central prolyl peptide bond was investigated by integration of signals corresponding to the two species in the (1)H NMR spectra over a range of temperatures. These studies revealed an increasing preference for the trans-conformation in the order Pro < Hyp < [α-(1,4)GlcNAc]Hyp. Rates for the forward and reverse reactions, determined by magnetization transfer experiments, demonstrated a reduced rate for the trans-to-cis conversion and a significant increase in the cis-to-trans conversion upon hydroxylation of the proline residue in the dipeptide. NOE experiments suggest that the Thr side chain pushes the sugar away from the pyrrolidine ring. These effects, which depended on the presence of the N-terminal Thr residue, offer a mechanism to explain altered properties of the corresponding full-length proteins.

An efficient, convergent solution phase synthesis of monomer, dimer, trimer and tetramer of the β-l-arabinofuranosylated hydroxyproline (β-l-Araf-Hyp) glycocluster is described. This motif constitutes the carbohydrate-specific epitope of Art v 1, the major allergen of mugwort pollen. While a single monomeric unit was proposed at the outset, poor yields for the seemingly trivial steps of end-capping to replace protecting groups with N-terminal acetamides and C-terminal methyl amides led to the introduction of N-terminal, central and C-terminal β-l-Araf-Hyp building blocks. Dimer 2 was obtained in 60% yield by coupling of two monomers, followed by hydrogenolysis of benzyl ether protecting groups. Trimer 3 was obtained in 35% yield via a [2 + 1] coupling and tetramer 4 in 15% yield via a [2 + 2] fragment condensation. Circular dichroism spectra show that monomer 1 displays no organized structure, whereas compounds 2-4 show a strong negative band at 200 nm and a weak positive band at ∼220 mn, as is characteristic of the polyproline II helix.

A selection of natural lipids, milk fat, cocoa butter and soybean oil, and three synthetic triglycerides 1,3-dipalmitoyl-2-oleoyl-rac-glycerol (POP), 1(3),2-palmitoyl-3(1)-oleoyl-rac-glycerol (PPO), and 1,3-dipalmitoyl-2-butyryl-rac-glycerol (PBP) were used as substrates for calf pregastric lipase (CPGL) catalysed hydrolysis at pH 6.5, 37.5 °C. CPGL preferentially releases short-chain fatty acids at the sn-3 position and unsaturated fatty acids during the hydrolysis of lipids containing long-chain fatty acids, and its activity increases with increasing unsaturation in the carbon chain of the long-chain fatty acids. The relative rate constants of hydrolysis were 1.56, 0.99, 0.73 h−1 for C4:0, C6:0, C8:0 esters, respectively, at sn-3; 0.0097, 0.0047 h−1 for the C16:0, C18:0 esters at sn-1,3, respectively; 0.0094 h−1 for the C18:1 ester at sn-2; and 0.0073, 0.0085, 0.012 h−1 for C18:1, C18:2, C18:3 esters, respectively, at sn-1,2,3. These last results confirmed that CPGL enzyme does not exhibit positional selectivity towards lipid substrates. The rate constant of hydrolysis was 1.14 h−1 for the C4:0 ester at sn-2. PBP was synthesised in four steps from commercially available glycerol as starting material. Glycerol was first selectively protected as a bis-silylether at the primary alcohol (sn-1,3) positions. The free alcohol at the sn-2 position was then esterified with butyric anhydride. The protecting groups at C1 and C3 were then removed, and the resultant hydroxyl groups esterified with palmitic acid.

A mixture of the two diastereoisomers of (2R)-1,2-di-O-butyl-sn-glycerol-3-O-p-nitrophenyl-n-propylphosphonate, varying only in their configuration at phosphorus, was synthesised from (R)-3-O-benzyl-sn-glycerol. The diastereoisomers were separated by normal phase HPLC and fully characterised. Both diastereoisomers were irreversible inhibitors of the activity of lamb pregastric lipase (LPGL), although the isomer which eluted first in the HPLC separation was significantly more effective.

Whereas hemoglobin (Hb) E-beta thalassemia is recognized as probably the most common serious hemoglobinopathy worldwide, its natural history remains poorly defined. The interaction of hemoglobin E and beta-thalassemia result in a wide spectrum of clinical disorders, some indistinguishable from thalassemia major and some milder and not transfusion-dependent. Partially as a result of this wide range of phenotypes, clear guidelines for approaches to transfusion and to iron-chelating therapy for patients with Hb E-beta thalassemia have not been developed. By contrast, data that have accumulated during the past 10 years in patients with beta-thalassemia permit a quantitative approach to the management of iron overload and provide guidelines for the control of body iron burden in individual patients treated with iron-chelating therapy. These guidelines may be applicable to patients with Hb E-beta thalassemia. Preliminary evidence from our studies of iron loading in affected patients with Hb E-beta thalassemia in Sri Lanka suggest that this disorder may be associated with variable, but accelerated, gastrointestinal iron absorption, and that the iron loading associated with chronic transfusions in patients with Hb E-beta thalassemia is similar to that observed in patients with beta-thalassemia. These data, in the only cohort of patients with Hb E-beta thalassemia to have undergone quantitative assessment of body iron burden, suggest that the principles that guide assessment of iron loading and initiation of chelating therapy in patients with beta-thalassemia may be generally applicable to those with Hb E-beta thalassemia. Further quantitative studies in both nontransfused and transfused patients will be necessary to permit firm conclusions.

erythro -Hydroxyasparagine ( e HyAsn, 1 ) occurs in a number of naturally occurring peptides and proteins. Previous syntheses have relied on enzyme-catalyzed reactions to produce relevant, optically active intermediates. We report herein a completely chemical synthesis that intercepts Boger’s synthesis of the diastereomer ( t HyAsn, 4 ), utilizing a Sharpless asymmetric aminohydroxylation reaction to introduce the two stereocenters. Boc-HyAsn(OTBS)-OH ( 10 ) was coupled effectively with phenylalanine methyl ester using EDC/HOBt.

A series of phosphonochloridates was prepared from the corresponding phosphonate monoesters, and their reactions with alcohols, amines, and the bisnucleophile 4-aminobutan-1-ol have been investigated using 31P NMR spectroscopy. In the conversion of phosphonate monoesters to phosphonochloridates via the addition of thionyl chloride or oxalyl chloride, pyrophosphonate anhydrides were found to be formed readily as byproducts. The anhydrides reacted readily with alcohols, but more slowly than the corresponding phosphonochloridates, and only sluggishly, if at all, with amines. Therefore, when phosphonamides are prepared, anhydride formation must be suppressed. This is accomplished when the monoester is added to the chloridating agent. Unhindered phosphonochloridates reacted predominantly with the amino function of 4-aminobutan-1-ol to furnish the phosphonamidates, whereas a sterically hindered phosphonochloridate demonstrated a preference for O-coupling. This result indictes that the energy gained during P−O b...

The anionic phospho-Fries rearrangement of phosphoric acid (3,5-di-isopropoxy)phenyl ester diethyl ester ( 11 ) gave rise to (2-hydroxy-4,6-di-isopropoxy-phenyl)phosphonic acid diethyl ester ( 12 ) in excellent yield. The phenol functionality of 12 was converted to the corresponding triflate which was coupled with vinyltributylstannane, under Stille conditions, to give a styrene. This molecule is intended to serve as the aromatic fragment in the synthesis of a phosphorus-based transition-state analogue for the hydrolysis of the S -(−)-zearalenone lactone.

Phosphonate diester 2, a hapten recently employed to induce the first monoclonal catalytic antibodies capable of catalyzing peptide bond formation, was synthesized via elaboration of scalemic α-amino phosphonate 4.

Monoclonal antibodies, induced with a phosphonate diester hapten, catalyzed the coupling of p-nitrophenyl esters of N-acetyl valine, leucine, and phenylalanine with tryptophan amide to form the corresponding dipeptides. All possible stereoisomeric combinations of the ester and amide substrates were coupled at comparable rates. The antibodies did not catalyze the hydrolysis of the dipeptide product nor hydrolysis or racemization of the activated esters. The yields of the dipeptides ranged from 44 to 94 percent. The antibodies were capable of multiple turnovers at rates that exceeded the rate of spontaneous ester hydrolysis. This achievement suggests routes toward creating a small number of antibody catalysts for polypeptide syntheses.

Glycosides of hydroxyproline (Hyp) in the plant cell wall matrix were discovered by Lamport and co-workers in the 1960s. Since then, much has been learned about these Hyp-rich glycoproteins. The intent of this review was to compare and contrast some less common structural motifs, in nontraditional roles, to uncover themes. Arabinosylation of short-peptide plant hormones is essential for growth, cell differentiation and defense. In a very recent development, prolyl hydroxylase and arabinosyltransferase activity has been shown to have a direct impact on the growth of root hairs in Arabidopsis thaliana. Pollen allergens of mugwort and ragweed contain proline-rich domains that are hydroxylated and glycosylated and play a structural role. In the case of mugwort, this domain also presents a significant immunogenic epitope. Major crops, including tobacco and maize, have been used to express and produce recombinant proteins of mammalian origin. The risks of plant-imposed glycosylation are discussed. In unicellular eukaryotes, Skp1 (a subunit of the E3(SCF) ubiquitin ligase complex) harbors a key Hyp residue that is modified by a linear pentasaccharide. These modifications may be involved in sensing oxygen levels. A few studies have probed the impact of glycosylation on the structure of Hyp-containing peptides. These have necessarily looked at small, synthetic molecules, since natural peptides and proteins are often isolable in only minuscule amounts and/or are heterogeneous in nature. The characterization of native structural motifs, together with the determination of glycopeptide conformation and properties, holds the key to rationalizing nature's architectural design.

The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O(2) regulation of development, suggesting the existence of an ancient O(2)-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O(2) signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. (1)H NMR demonstrated formation of 4(trans)-hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFα by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3(SCF)ubiquitin ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily related E3(VBC)ubiquitin ligases.

Protected glycopyranosylcyanides were transformed into the corresponding N - tert -butoxycarbonyl-glycopyranosyl methylamines in a one-pot transformation in high yields. This general method is applicable to glycopyranosylcyanides bearing common O - and N -protecting groups frequently used in carbohydrate chemistry.

Phosphonate diester 2, a hapten recently employed to induce the first monoclonal catalytic antibodies capable of catalyzing peptide bond formation, was synthesized via elaboration of scalemic α-amino phosphonate 4.

Mefp1 is a protein produced by the marine mussel, Mytilus edulis, which helps the organism to adhere to surfaces in turbulent waters. To better understand the nature of the adhesion process, we sought to synthesize homogeneous oligopeptides based on the repeating decapeptide unit of the protein. The fully protected decapeptide 10 has been synthesized from appropriately protected amino acid building blocks using a fragment condensation strategy. A key feature of the strategy is the late incorporation of the synthetically valuable dihydroxyproline residue. This synthesis of the orthogonally protected repeating decapeptide unit of Mefp1 represents an important first step toward producing useful quantities of homogeneous oligopeptides related to the protein.

Protected glycopyranosylcyanides were transformed into the corresponding N - tert -butoxycarbonyl-glycopyranosyl methylamines in a one-pot transformation in high yields. This general method is applicable to glycopyranosylcyanides bearing common O - and N -protecting groups frequently used in carbohydrate chemistry.

A general strategy is proposed, wherein a pentose sugar γ-lactone can be converted, via a series of nine reactions, to a 3,4-dihydroxyproline, suitably protected for use in peptide synthesis. Thus, d-ribonolactone (6) has been converted to N-fluorenylmethoxycarbonyl-3,4-di-O-tert-butyldimethylsilyloxy-d-2,3-cis-3,4-cis-proline (7) in 18.9% overall yield. Likewise, l-arabinonolactone (11) has been converted to N-fluorenylmethoxycarbonyl-3,4-di-O-tert-butyldimethylsilyloxy-l-2,3-cis-3,4-trans-proline (36) in 13.7% overall yield and l-lyxonolactone (12) to N-fluorenylmethoxycarbonyl-3,4-di-O-tert-butyldimethylsilyloxy-l-2,3-trans-3,4-cis-proline (37) in 11.2% overall yield. These building blocks have also been fully deprotected to give the free amino acids. We believe that this series of reactions ought to be applicable to the synthesis of any of the eight stereoisomers of 3,4-dihydroxyproline, by judicious selection of the pentose starting material.