Synthesis of phosphopeptides via global phosphorylation on the solid phase: Resolution of H-phosphonate formation

The formation of the H-phosphonate by-products from the global phosphorylation of a Thr-containing peptide resin using both di-t-butyl and dibenzyl N,N-diethylphosphoramidite was identified to result from 1H-tetrazole-mediated cleavage of the t-butyl or benzyl from the intermediate dialkyl phosphite triester and re-arrangement of the resultant hydroxy phosphite diester to the H-phosphonate form. This side reaction was rectified by the use of aqueous iodine for the oxidation step in which the H-phosphonate is oxidised to the benzyl phosphorodiester which, on acidolytic treatment, gives the desired dihydrogen phosphate.     

Preparation of O-phosphotyrosine-containing peptides by Fmoc solid-phase synthesis: Evaluation of several Fmoc-Tyr(PO3R2)-OH derivatives

Summary The synthesis of two model Tyr(P)-containing peptides using Fmoc-Tyr(PO₃ tBu₂)-OH, Fmoc-Tyr(PO₃Bzl₂)-OH and Fmoc-Tyr(PO₃H₂)-OH established that the t-butylphosphate-protected derivative was the preferred derivative for use in Fmoc solid-phase peptide synthesis, since it afforded phosphopeptides in high purity and with the lowest amount of Tyr-peptide contamination. In addition, this study confirmed that commercially available Fmoc-Tyr(PO₃H₂)-OH is also suitable for use in Fmoc solid-phase synthesis but gives less pure phosphopeptides, along with the generation of 1–4% of the tyrosine-containing peptide for the model sequences studied. In view of the good performance of Fmoc-Tyr(PO₃ tBu₂)-OH, a large-scale three-step synthetic procedure was developed which involved phenacyl protection of the carboxyl group, phosphite-triester phosphorylation of the tyrosyl hydroxyl using di-t-butyl N,N-diethylphosphoramidite, and final removal of the phenacyl group by zinc reduction in acetic acid.Abbreviations BOP benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate - tBu t-butyl - Bzl benzyl - DBU 1,8-diazabicyclo[5,4,0]undec-7-ene - DMF N,N-dimethylformamide - EDT ethanedithiol - Fmoc 9-fluorenylmethoxycarbonyl - HOBt N-hydroxybenzotriazole - HPLC high performance liquid chromatography - NMM N-methylmorpholine - Pac phenacyl - TFA trifluoroacetic acid - THF tetrahydrofuran - Tyr(P) O-phosphotyrosine     

Production of N,N′-diacetylchitobiose from chitin using temperature-sensitive chitinolytic enzyme preparations of Aeromonas sp. GJ-18

Summary N,N′-diacetylchitobiose was produced from chitin as a major hydrolytic product by controlling the ratio of β-N-acetylglucosaminidase to N,N′-diacetylchitobiohydrolase activities in the crude enzyme preparation of Aeromonas sp. GJ-18. When the enzyme preparation was preincubated at 50 °C, β-N-acetylglucosaminidase was nearly inactivated, while the N,N′-diacetylchitobiohydrolase was still active. Thus, the composition of chitin oligosaccharides depended on the preincubation temperature of the crude enzyme preparations. Typically, after 7 days of incubation with the substrate chitin, 78.9 and 56.6% of N,N′-diacetylchitobiose yields were obtained from swollen α-chitin and powdered β-chitin, respectively, with enzyme preparations that had been pretreated at 50 °C for 60 min.     

Synthesis and use of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-di-Boc-glutamate γ-semialdehydes and related aldehydes

Summary.  This review article focuses on the synthesis and reactions of N,N-di-Boc glutamate and aspartate semialdehydes as well as related aldehydes. These building blocks are prepared according to various strategies from glutamic and aspartic acids and find interesting synthetic applications. In the first part, the methods for the synthesis of N,N-di-Boc-amino aldehydes are summarized. The applications of these chiral synthons for the synthesis of unnatural amino acids and other bioactive compounds are discussed in the second section. Received April 24, 2002 Accepted August 13, 2002 Published online January 30, 2003 Authors' address: Prof. Violetta Constantinou-Kokotou, Chemical Laboratories, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece, E-mail: vikon@aua.gr Abbreviations: AcNH-TEMPO, 4-acetamido-2,2,6,6-tetramethyl-1-piperidinyloxy free radical; AIBN, 2,2′-azobis(2-methylpropionitrile); Aliquat, methyltrioctylammonium chloride; Bn, benzyl; Boc, tert-butoxycarbonyl; Bu^t, tert-butyl; m-CPBA, 3-chloroperoxybenzoic acid; DAST, diethylaminosulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; (R,R)-(+)-DET, (R,R)-(+)-diethyltartrate; DIBALH, diisobutyl aluminium hydride; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; Et₃N, triethylamine; KHMDS, potassium bis(trimethylsilyl)amide; (S)-LLB, lanthanium-lithium-bis-metallic binaphthol catalyst; MsCl, methanesulfonyl chloride; NEM, N-ethylmorpholine; NMO, 4-methylmorpholine N-oxide; PPA, propylphosphonic acid anhydride; TBHP, tert-butyl hydroperoxide; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSI, 1-(trimethylsilyl)imidazole; Trt, trityl.     

Kinetics of the action of three selenides on Staphylococcus aureus growth as studied by microcalorimetry

The effect of three kinds of selenide on Staphylococcus aureus growth was studied by means of microcalorimetry. Differences in their capacities to inhibit the metabolism of this bacterium were observed. The rate constant k (in the log phase) in the presence of the compounds decreased with increasing concentrations of the compounds. The relationship of k and c is nearly linear for the selenium compounds. Judged from the rate constant, k, and the half-inhibitory concentration IC₅₀, the experimental results reveal that the sequence of antibiotic activity of the three tested selenides compounds is (2-hydroxy benzyl imino)ethyl n-hexyl selenide> n-butyl(2-hydroxy benzyl imino)ethyl selenide>bis[(2,4-dihydroxy benzyl imino)ethyl] selenide.     

Synthesis of symmetric and asymmetric diamides of citric acid and amino acids

Summary A convenient method for the synthesis of symmetric and asymmetric diamides of amino acids including DOPA and citric acid from 2-tert-butyl-1,3-di(N-hydroxysuccinimidyl)citrate and 1-tert-butyl-2,3-di(N-hydroxysuccinimidyl)citrate is described.Abbreviations AcOtBu tert-butyl acetate - i-Bu iso-butyl - tBu tert-butyl - Bzl benzyl - p-OH-Bzl p-hydroxybenzyl - m,p-(OH)₂-Bzl m,p-dihydroxybenzyl - DCCI dicyclohexylcarbodiimide - Et ethyl - Me methyl - Su succinimidyl - SuOH N-hydroxysuccinimide - Ph phenyl     

Studies on peptides CLVIII. Model experiments for the synthesis of open-chain unsymmetrical cystine-peptides

Treatment of a mixture of Cys(R)(O) and Cys(R) with an acid was found to generate cystine in fairly good yields, when suitable R, R, and an acid were selected. An unsymmetrical cystine peptide was prepared by treatment of a mixture of Z(OMe)-Cys(R) (0)-Ala-NH₂ (R=Acm or MBzl) and Z(OMe)-Cys(MBzl)-Gly-OBzl with TFA or 1 M TFMSA/TFA.³ Oxytocin was obtained in an excellent yield by TFA treatment of the protected peptide containing Cys(Acm)(0) and Cys(MBzl). Thus, formation of the disulfide bond was found feasible at the position of Cys(R) (0).The following abbreviations are used Boc t-butyloxycarbonyl - Z(OMe) p-methoxybenzyloxycarbonyl - MBzl p-methoxybenzyl - Acm acetamidomethyl - Bzl benzyl - Ad l-adamantyl - tBu t-butyl - TFA trifluoroacetic acid - TFMSA trifluoromethanesulfonic acid - TMSOTf trimethylsilyl trifluoromethane sulfonate     

Characterization of a putative thioredoxin peroxidase prx1 of <Emphasis Type="Italic">Candida albicans</Emphasis>

In this study, we characterized a putative peroxidase Prx1 of Candida albicans by: 1) demonstrating the thioredoxin-linked peroxidase activity with purified proteins, 2) examining the sensitivity to several oxidants and the accumulation of intracellular reactive oxygen species with a null mutant (prx1Δ), a mutant (C69S) with a point mutation at Cys69, and a revertant, and 3) subcelluar localization. Enzymatic assays showed that Prx1 is a thioredoxin-linked peroxidase which reduces both hydrogen peroxide (H₂O₂) and tert-butyl hydroperoxide (t-BOOH). Compared with two other strong H₂O₂ scavenger mutants for TSA1 and CAT1, prx1Δ and C69S were less sensitive to H₂O₂, menadione and diamide at all concentrations tested, but were more sensitive to low concentration of t-BOOH. Intracellular reactive oxygen species accumulated in prx1Δ and C69S cells treated with t-BOOH but not H₂O₂. These results suggest that peroxidase activity of Prx1 is specified to t-BOOH in cells. In both biochemical and physiological cases, the evolutionarily conserved Cys69 was found to be essential for the function. Immunocytochemical staining revealed Prx1 is localized in the cytosol of yeast cells, but is translocated to the nucleus during the hyphal transition, though the significances of this observation are unclear. Our data suggest that PRX1 has a thioredoxin peroxidase activity reducing both t-BOOH and H₂O₂, but its cellular function is specified to t-BOOH.     

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Summary The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg⁻¹) as compared to PCSIR-102 (77 IU mg⁻¹) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K _m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K _m value) which may show the superiority of the latter in terms of better enzyme properties.     

The peptides of α-aminosuberic acid I New intermediates for synthetic studies

Summary. The paper describes the synthesis of α-aminosuberic acid derivatives suitable for the synthesis of peptides. These include Z-, Boc- and Fmoc-protection on the α-amino group, benzyl ester, Boc-hydrazide and Z-hydrazide as well as the free carboxylic function in the side chain, and methyl ester, benzyl ester or free α-carboxylic group. Their use is demonstrated on the synthesis of the respective derivatives of Asu-Val-Leu. The enzyme catalyzed reaction was succesfully used both as a route to L-Asu from the D,L-compound as well as for the direct synthesis of the optically active tripeptide derivative from the Z-D,L-Asu-OH. Received February 5, 1999     

Age and growth of lanternfishes, Symbolophorus californiensis and Ceratoscopelus warmingii (Myctophidae), in the Kuroshio–Oyashio Transition Zone

To estimate the age and growth of dominant lanternfishes in the Kuroshio–Oyashio Transition Zone, we examined the sagittal otolith microstructure of Symbolophorus californiensis (n = 30) and Ceratoscopelus warmingii (n = 93) collected from the western North Pacific during 1997–2003. Age of S. californiensis ranged from 81 to 541 days corresponding to postmetamorphosis stage (juveniles and adults), and the von Bertalanffy model was fitted: L _t  = 128[1 − exp{−0.003(t − 1.52)}], where L is the standard length (mm) and t is age in days. Age of C. warmingii ranged from 6 to 416 days, and growth before metamorphosis was linear (L _t  = 0.346t + 1.51), and the von Bertalanffy model was fitted to the postmetamorphosis stage: L _t  = 80.8 [1 − exp{−0.00769(t − 34.4)}]. Growth of these two lanternfishes was faster than that of other lanternfishes in previous studies but considerably slower than that of Japanese sardine (Sardinops melanostictus) and anchovy (Engraulis japonicus) distributed in the Kuroshio–Oyashio Transition Zone. Temperature and prey availability are discussed in relation to this difference in growth rate.     

Stereochemistry of the Reductive Amination of 4-Oxoproline Derivatives with Glycine Esters

An amination of 4-oxoproline derivatives with glycine methyl or benzyl ester and sodium cyanoborohydride led to the mixtures of corresponding diastereomeric 4-cis- and 4-trans-glycinoproline derivatives. We found that the ratio of diastereomers mainly depends on the structure of 4-oxoproline ester groups and, to a lesser extent, on the structure of N-acyl substituents. The best results were achieved with tert-butyl ester group; it ensured good yields of the amination products and the greatest prevalence of 4-cis-isomers. The structure of ester group in glycine molecule only scarcely affected the resulting ratio of N-(N-benzyloxycarbonylglycyl)-4-glycinoprolines.     

The synthesis of isomeric 4-prolinylamines and 4,4′-diprolinylamines

Starting from the previously describedtert-butyl esters of 4-epimericN-benzyloxycarbonyl-4-hydroxyprolines andN-benzyloxycarbonyl-4-trans- and 4-cis-trifluoroacetaminoprolinetert-butyl esters, the corresponding uprotected 4-aminoprolines and a number of their partially protected derivatives were synthesized via the intermediate 4-O-mesyl and 4-azide derivatives. The reductive amination ofN-benzyloxycarbonyl-4-oxoprolinetert-butyl ester with ammonium acetate led toN-benzyloxycarbonyl-4-cis-4′-cis- and 4-cis-4′-trans-diprolinylamines. The¹H NMR and CD spectra of the synthesized compounds are described.     

Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124

Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Galβ1→3GlcNAc, LNB) and galacto-N-biose (Galβ1→3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBP_Bl). In the presence of α-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a k _cat/K _m value for GNB that was approximately 50 times higher than that for LNB, whereas LNBP_Bl showed similar k _cat/K _m values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-α-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.     

Killing bacterial spores by organic hydroperoxides

Killing of wild-type spores of Bacillus subtilis by t-butyl hydroperoxide, cumene hydroperoxide and peracetic acid was not through DNA damage, as shown by the absence of mutations in the survivors and the identical sensitivity of spores of strains with or without a recA mutation. In contrast, B. subtilis spores (termed α⁻β⁻) lacking the DNA protective α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to t-butyl hydroperoxide and cumene hydroperoxide, and their killing was in large part through DNA damage, as shown by the high frequency of mutations in the survivors and the greater sensitivity of α⁻β⁻ recA spores. Analysis of t-butyl hydroperoxide-treated spores showed that generation of DNA damage in α⁻β⁻ spores was more rapid than in wild-type spores; α/β-type SASP also protected against DNA strand breakage in vitro caused by t-butyl hydroperoxide. α/β-Type SASP appeared to play no role in protection of spores from killing by peracetic acid; wild-type and α⁻β⁻ spores exhibited identical peracetic acid sensitivity and their killing by this agent appeared to be not through DNA damage. Received 17 December 1996/ Accepted in revised form 13 March 1997     

Global phosphorylation at Ser16 of the 32-residue cytoplasmic domain of phospholamban: Comparison of di-t-butyl- and dibenzyl-N,N-diisopropylphosphoramidites

Summary Phospholamban (PLB), a 52-residue integral membrane protein of the cardiac sarcoplasmic reticulum, is known as the regulator of the Ca²⁺-ATPase pump via its phosphorylation-dephosphorylation at Ser¹⁶. In order to investigate the structural effects of the phosphorylation on the cytoplasmic domain of PLB, we synthesized PLB 2–33 in its nonphosphorylated and phosphorylated forms using Fmoc chemistry. PLB 2–33 was phosphorylated using the global phosphorylation method. Phosphorylation with di-t-butyl-N,N-diisopropylphosphoramidite led to an incomplete reaction (nonphosphorylated peptide was recovered) and to the formation of an H-phosphonate. In contrast, the use of dibenzyl-N,N-diisopropylphosphoramidite yielded the desired phosphorylated peptide quantitatively and did not give rise to the H-phosphonate. Our results show the effectiveness of the dibenzyl-protected phosphoramidite when the site to be phosphorylated is particularly hindered.     

Preparation of 3-ketovalidoxylamine A C–N lyase substrate: <Emphasis Type="Italic">N</Emphasis>-<Emphasis Type="Italic">p</Emphasis>-nitrophenyl-3-ketovalidamine by <Emphasis Type="Italic">Stenotrophomonas maltrophilia</Emphasis> CCTCC M 204024

3-Ketovalidoxylamine A C–N lyase is one of three key enzymes in the production of valienamine, which is a potent glucosidase inhibitor from validamycin A. N-p-Nitrophenyl-3-ketovalidamine, used as the substrate of 3-ketovalidoxylamine A C–N lyase, was prepared from N-p-nitrophenylvalidamine with free cells of Stenotrophomonas maltrophilia CCTCC M 204024. The yield and selectivity of N-p-nitrophenyl-3-ketovalidamine from cells were improved by treatment with 10 mM ethylenediaminetetraacetic acid. The optimal pH and temperature for N-p-nitrophenyl-3-ketovalidamine formation was pH 6.0 and 30°C, respectively. N-p-Nitrophenyl-3-ketovalidamine was formed with a yield of 0.68 in the first batch.     

Isolation and Structure Elucidation of Epipolasterol and 22,23-Dihydroepipolasterol from the Marine Sponge Epipolasis sp.

Two new sterols, epipolasterol and 22(23)-dihydroepipolasterol, have been isolated from the marine sponge Epipolasis sp. These are unusual metabolites as they both contain a t-butyl group in the sterol side chain. In addition, the presence of two degrees of unsaturation in the side chain of epipolasterol is rare. The known sterol, 22-dehydro-24-isopropylcholesterol was also found in this sponge.     

Effects of environmental settings on MTBE removal for a mixed culture and its monoculture isolation

A mixed culture was utilized to evaluate methyl tert-butyl ether (MTBE) removal under various conditions and to isolate a MTBE-degrading pure culture. The results showed that high MTBE removal efficiencies can be reached even in the presence of other substrates. The biodegradation sequence of the target compounds by the mixed culture, in order of removal rate, was toluene, ethyl benzene, p-xylene, benzene, MTBE, ethyl ether, tert-amyl methyl ether, and ethyl tert-butyl ether. In addition, preincubation of the mixed cultures with benzene and toluene showed no negative effect on MTBE removal; on the contrary, it could even increase the degradation rate of MTBE. The kinetic behavior showed that the maximum specific growth rate and the saturation constant of the mixed culture degrading MTBE are 0.000778 h⁻¹ and 0.029 mg l⁻¹, respectively. However, a high MTBE concentration (60 mg l⁻¹) was slightly inhibiting to the growth of the mixed culture. The pure culture isolated from the enrichments in the bubble-air bioreactor showed better efficiency in MTBE removal than the mixed culture; whereas, tert-butyl alcohol was formed as a metabolic intermediate during the breakdown of MTBE.