NADH Model Reaction: Asymmetric Reduction of Non-activated Carbonyl Compounds with N-Anionized Hantzsch Ester

As model compounds for NADH coenzymes, three chiral Hantzsch ester-type dihydro-pyridines were prepared. By their use and 2,6-dimethyl-3,5-dicarbo-( — )-menthoxy-1,4-dihydropyridine that had already been prepared, asymmetric reductions of unactivated prochiral ketones were conducted under the action of alkali metal derivatives at room temperature in a non-polar solvent, and the alcohol product were obtained in a 36~80% chemical yield and 30~60% enantiomeric excess.     

Racemization of l-Proline during Trifluoroacetylation Detection of Unexpected Stereoisomers in Trifluoroacetylprolyl-valine t-Butyl Ester

The genome sequencing project on alkaliphilic Bacillus halodurans C-125 revealed a putative endo-β-N-acetylglucosaminidase (Endo-BH), which consists of a signal peptide of 24 amino acids, a catalytic region of 634 amino acids exhibiting 50.1% identity with the endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A), and a C-terminal tail of 220 amino acids. Transformed Escherichia coli cells carrying the Endo-BH gene exhibited endo-β-N-acetylglucosaminidase activity. Recombinant Endo-BH hydrolyzed high-mannose type oligosaccharides and hybrid type oligosaccharides, and showed transglycosylation activity. On deletion of 219 C-terminal amino acid residues of Endo-BH, the wild type level of activity was retained, whereas with deletions of the Endo-A homolog domain, the proteins were expressed as inclusion bodies and these activities were reduced. These results suggest that the enzymatic properties of Endo-BH are similar to those of Endo-A, and that the C-terminal tail does not affect the enzyme activity. Although the C-terminal tail region is not essential for enzyme activity, the sequence is also conserved among endo-β-N-acetylglucosaminidases of various origins.     

Reaction of pyridoxamine-5-P with pyrroloquinoline quinone (coenzyme PQQ)

PQQ catalyzes the oxidation of pyridoxamine (PM) and pyridoxamine-5-P (PMP) to pyridoxal and pyridoxal-5-P (PLP) at 37 degrees C in the absence of micelles and proteins. The time course of conversion of PMP into PLP was monitored by absorption spectroscopy; a rate of 10 nmol PLP/min was determined. The product of the reaction was identified by TLC, HPLC and its ability to restore the catalytic activity of apoaspartate aminotransferase. The conversion of PMP into PLP by free PQQ is more efficient than reactions catalyzed by the enzymes plasma amine oxidase and pyridoxamine-5-P oxidase at optimal pH values.     

Racemization in the Coupling Reaction with the Several Methods beside the Activated Ester Methods

The degree of racemization in the coupling reaction, Pro-Val + Pro, with the several other methods than the activated ester methods was measured and the results were compared with that in the coupling reaction, Leu-Phe + Val, as well as in the previous paper.¹⁾ In the azide and the mixed anhydride methods, no or almost no racemization was detected, whereas in the other tested methods of peptide synthesis (Pachornik-, DCCD- and phosphazo-methods) the significantly large racemization was observed. It can be attributed to the strong nucleo- philic N-atom in the penultimate amino acids (Pro) and the steric hindrance of C-terminal amino acid (Val), which are favourable to the formation of the oxazolone ring.

This assumption was further systematically confirmed in the synthesis of the other several tripeptides with the DCCD method. The separation of the diastereoisomers (LLL and LDL) of the resulting tripeptides by gas chromatography with a packed column was also here reported.     

Induction of the Acrosome Reaction in Starfish

In the starfish, Asterias amurensis , at least two distinct components of the egg jelly are required for inducing the acrosome reaction: a sulfated glycoprotein named acrosome reaction-inducing substance (ARIS) and a diffusible organic substance(s) named Co-ARIS. The following evidence suggested that ARIS and Co-ARIS cooperatively activate CA-channels of the sperm plasma membrane and eventually induce dramatic changes in sperm morphology, the acrosome reaction. 1) Pronase digest of ARIS (P-ARIS) and Co-ARIS, either as a pure or a crude preparation (Fraction M₈), were fully effective in combination for induction of the acrosome reaction in normal sea water, although they were not effective individually. P- ARIS alone induced the acrosome reaction fully in high Ca²⁺ sea water and markedly at high pHs, whereas Fraction M₈ alone did not induce the reaction even in these conditions. The reaction was not induced by increase in either the Ca²⁺ concentration or the pH of sea water, but was markedly induced in the absence of jelly components by raising both the pH and Ca²⁺ concentration together. 2) The ionophore A23187 induced the acrosome reaction appreciably when present alone and fully in the presence of monensin or Fraction M₈. Monesin alone was ineffective. 3) The jelly or a combination of ARIS and Fraction M₈ caused abrupt Ca²⁺ -uptake by the sperm. The Ca-channel blockers verapamil and diltiazem inhibited the jelly-induced acrosome reaction.     

l-Proline reduces IgG dimer content and enhances the stability of intravenous immunoglobulin (IVIG) solutions

Liquid intravenous immunoglobulin (IVIG) products offer improved convenience in preparation but often lack sufficient stability to allow room temperature storage. Furthermore, clinical tolerability may be affected due to formation of idiotype/anti-idiotype IgG dimers and/or aggregates. Here we report on the development of a 10% IVIG formulation with optimized stability achieved by the use of l-proline. The stability of concentrated liquid IVIG was strongly pH dependent. Aggregate formation, yellowish discoloration of the solution and loss of anti-hepatitis B surface antigen (HBs) antibody activity was minimal at intermediate pH (pH 4.8–5.3). Fragmentation of IgG was highest at low pH (pH 4.1). Idiotype/anti-idiotype IgG dimer formation was highest at neutral pH and was reduced with decreasing pH. The presence of l-proline further improved stability by inhibiting protein aggregation, reducing loss of anti-HBs antibody activity and decreasing coloring, particularly compared with glycine formulations. The IgG dimer content was up to 30% lower in solutions containing l-proline compared with those containing glycine or other stabilizers. In conclusion, a weakly acidic pH of approximately 5 and l-proline as stabilizer are optimal conditions for long-term stability of a liquid IVIG. l-proline, an amphiphilic, naturally occurring amino acid, is superior to glycine in restricting IgG dimer formation.     

Induction of Reaction Wood in Terminalia (Combretaceae): Roles of Gravity and Stress

Reaction wood (RW) as determined by the presence of gelatinousfibres (tension wood) was induced by experimental manipulationsof leaders and lateral branch axes of Terminalia catappa (Combretaceae).Axes were repositioned with respect to gravity with or withoutapplied bending. Other axes were bent without gravitationalreorientation. All gravitational reorientations caused developmentof RW on the side toward the normal position for the axis, evenwhen tensile stress was assumed to be on the opposite side.In axes bent without repositioning with respect to gravity,RW formed on the side with tensile stress. Bark girdling inducedRW proximal to the girdle and in distally directed tongues ofpartially isolated regions of intact bark. It is concluded thatboth reorientational changes and imposed stress can cause RW,but that the former stimulus may dominate over the latter whenthe two stimuli conflict. Auxin levels are presumably affectedby these two stimuli and by bark removal. Reaction wood, tension wood, gravity, mechanical stress, xylem development     

Racemization in the Coupling Reaction,Pro-Val+Pro,with the Activated Ester Methods

The degree of racemization in the several activated ester methods of the peptide synthesis was measured in using the critical racemization test, Pro-Val+Pro, with help of gas chromatography. The results were compared with that in the coupling reaction, Leu-Phe+Val, in which no racemization had been reported in the corresponding reaction conditions by F. Weygand et al., when the activated dipeptide esters had been prepared from Z-Leu+Phe-activated esters. The significantly higher racemization was observed in the methods of N-hydroxypiperidine ester and thiophenyl ester, even when the activated dipeptide esters were prepared from Z-Pro+Val-activated esters. On the other hand, almost no racemization was observed in the N-hydroxysuccinimide ester and p-nitrophenyl ester methods. A great extent of the racemization was detected when the activated dipeptide esters were prepared directly from Z-Pro-Val-OH.     

Generating Symmetry in the Asymmetric ATP-binding Cassette (ABC) Transporter Pdr5 from Saccharomyces cerevisiae

Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely “regenerated” Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters.     

Enzymatic Synthesis of N-formyl-L-aspartyl-L-phenylalanine Methyl Ester (Aspartame Precursor) Utilizing an Extractive Reaction in Aqueous/Organic Biphasic Medium

N-Formyl-L-aspartyl-L-phenylalanine methyl ester (N-formyl aspartame, F-AspPheOMe) was synthesized enzymatically utilizing an extractive reaction in an aqueous/organic biphasic system. The N-formyl aspartame yield in a pure aqueous monophasic system was, in general, ca. 3% , however, it was over 80 % in a water/1-butanol biphasic system using a simultaneously extractive operation of an enzymatic reaction in an aqueous phase and a product separation from an aqueous to an organic phases.     

Induction of human high K(M) 5'-nucleotidase in cultured 293 cells

Human 293 cells were stably transfected with a plasmid introducing a receptor for the ecdysone analog muristerone. The cells were further stably transfected with muristerone-inducible expression vectors carrying either the cDNA for the human high K(M) 5'-nucleotidase or the coding sequence of the nucleotidase linked to the 5'-end of the sequence for the green fluorescent protein. Upon induction, both types of transfectants overproduced nucleotidase activity in a time- and dose-dependent manner. Western blots gave values close to the expected subunit molecular masses of 65 and 92 kDa, respectively, excluding processing of the induced proteins. Cells induced to overexpress the nucleotidase showed a decreased growth rate and contained smaller pools of each of the four common ribonucleoside triphosphates. They showed no increased resistance to the toxicity of 2-chlorodeoxyadenosine.     

Induction of 2',5' oligo(A) synthetase in tumor-bearing mice with encephalomyocarditis (EMC) virus or poly(I)poly(C)

Infection of 13 month-old C3H mice with EMC virus or inoculation with the interferon inducer poly(I)poly(C) results in elevated levels of the enzyme 2',5' oligo(A) synthetase only in animals with spontaneous tumors (breast cancer or hepatomas). High enzymatic activities are detected in homogenates from liver, spleen, plasma and neoplastic cells of the animals with breast carcinomas and only in the neoplastic liver cells of the animals with hepatomas.     

Double Asymmetric Induction During the Addition of (RP)‐Menthyl Phenyl Phosphine Oxide to Chiral Aldimines

P,C‐Stereogenic α‐amino phosphine oxides were prepared from the addition of (R_P)‐menthyl phenyl phosphine oxide to chiral aldimines under neat condition at 80 °C in up to 91:9 dr_C and 99% yields. The diastereoselectivity was mainly induced by chiral phosphorus that showed matched or mismatched induction with (S)‐ or (R)‐aldimines, respectively. Chirality 28:132–135, 2016. © 2015 Wiley Periodicals, Inc.     

Reaction products from platinum(IV) amine compounds and 5'-GMP are mainly bis(5'-GMP)platinum(II) amine adducts

The reaction products obtained from mixtures of 5'-GMP and platinum(IV) compounds with formula Pt(IV)Cl4(LL) and Pt(IV)Cl2(OH)2(LL) (LL representing two monodentate or one bidentate amine ligand) have been characterized by proton NMR spectroscopy. The amines used are NH3, H2N-CH2-CH2-NH2 (ethylenediamine, en), H2N-CH2-C(CH3)2-CH2-NH2 (2,2-dimethyl-1,3-diaminopropane, dmdap), and HC(CH3)2-NH2 (isopropylamine, ipa). Conditions varied during the reaction are pH (values of 4, 7, and 10), effect of visible light, and addition of vitamin C as a reducing agent. In all cases, the major product appeared to be the bis(5'-GMP)(LL)Pt(II) compound. The pH effect is limited; i.e., at pH 4 the reactions proceed somewhat faster than at neutral pH, while at pH 10 slower reactions occur. The illumination with visible light also induces only slight differences in the yields of the products. On the other hand, when vitamin C is present, the reactions proceed quite rapidly, resulting in the same main product but in higher yields (up to 80%). The facts that apparently no Pt(IV) adducts with 5'-GMP can be observed under these conditions and that the major products are bis(5'-GMP)(LL)Pt(II) compounds clearly support the hypothesis that the antitumor activity of certain platinum(IV) compounds is based upon in vivo reduction to the corresponding platinum(II) compounds.