Prebiotic thermal polymerization of crystals of amino acids via the diketopiperazine reaction

In this work, we continue our studies on the thermal prebiotic oligomerization of amino acids. The next step is to consider all four types of electromagnetic interactions that our model may admit. In addition, only the polymerization of amino acids via the formation of diketopiperazine, which arises from the cyclodehydration of two amino acids, will be considered. By assuming that only one residue group of two will predominate in the diketopiperazine molecule, it is possible to reduce the three-body problem to a simpler situation with the two objects that we have already solved.     

15N-NMR spectroscopy. 34. Stereospecificity of the polymerization of D,L-leucine N-carboxyanhydride and γ-OMe-D,L-glutamic acid N-carboxyanhydride

D ,L -Leucine-N-carboxyanhydride (D ,L -Leu-NCA) and γ-methyl-D ,L -glutamic acid N-carboxyanhydride (γ-OMe-D ,L -Glu-NCA) were synthesized with ca. 2.5% ¹⁵N enrichment. Their polymerizations were conducted under a variety of conditions using benzylamine, triethylamine potassium tert-butanolate, and pyridine as initiators. The 40.55-MHz ¹⁵N-nmr spectra of the resulting stereocopolypeptides measured in trifluoroacetic acid display at least four signals, representing the isotatic, syndiotactic, and two heterotactic triads. From the signal intensities it was concluded that these NCAs behave nearly identically. With benzylamine initiation the formation of isotactic blocks is slightly favored, and they are still more predominant when strong bases are used as initiators. Initiation by pyridine favors the formation of syndiotactic sequences. However, in all cases the average lengths of the stereoblocks never exceeded 4 monomer units. The low stereospecificity of most polymerizations of D ,L -NCAs is confirmed by the high degree of solubility of the resulting poly(D ,L -amino acids) in aprotic solvents. Penultimate effects are weak or absent, so that most polymerizations follow Bernoullian type statistics. Deviations from these statistics were found for polymerizations in pyridine.     

Interindividual variability in the enantiomeric disposition of ibuprofen following the oral administration of the racemic drug to healthy volunteers

The plasma disposition of the enantiomers of ibuprofen has been investigated following the oral administration of the racemic drug (400 mg) to 24 healthy male volunteers. The plasma elimination of (R)-ibuprofen was found to be more rapid than that of the S-enantiomer [plasma half-life: (R) 2.03 h; (S) 3.05 h; 2P less than 0.001], resulting in a progressive enrichment in the plasma content of this isomer, some 64% of the total area under the plasma concentration time curves (AUC) being due to the pharmacologically active enantiomer. The influence of dose on the pharmacokinetic characteristics of the enantiomers of ibuprofen, over the range 200-800 mg, was investigated in three subjects. Examination of dose-normalized AUC values and oral clearance indicate the dose dependence of (R)-ibuprofen disposition.     

Selective enzymatic degradation of self-assembled particles from amphiphilic block copolymers obtained by the combination of N-carboxyanhydride and nitroxide-mediated polymerization

Combining controlled radical polymerizations and a controlled polypeptide synthetic technique, such as N-carboxyanhydride (NCA) ring-opening polymerization, enables the generation of well-defined block copolymers to be easily accessible. Here we combine NCA polymerization with the nitroxide-mediated radical polymerization of poly(n-butyl acrylate) (PBA) and polystyrene (PS), using a TIPNO and SG1-based bifunctional initiator to create a hybrid block copolymer. The polypeptide block consists of (block) copolymers of poly(L-glutamic acid) embedded with various quantities of L-alanine. The formed superstructures (vesicles and micelles) of the block copolymers possessed varying degrees of enzyme responsiveness when exposed to elastase and thermolysin, resulting in controlled enzymatic degradation dictated by the polypeptide composition. The PBA containing block copolymers possessing 50% L-alanine in the polypeptide block showed a high degradation response compared to polymers containing lower L-alanine quantities. The particles stabilized by copolypeptides with L-alanine near the hydrophobic block showed full degradation within 4 days. Particles containing polystyrene blocks revealed no appreciable degradation under the same conditions, highlighting the specificity of the system and the importance of synthetic polymer selection. However, when the degradation temperature was increased to 70 °C, degradation could be achieved due to the higher block copolymer exchange between the particle and the solution. A number of novel biohybrid structures are disclosed that show promise as enzyme-responsive materials with potential use as payload release vehicles, following their controlled degradation by specific, target, enzymes.     

Organization of the enantiomeric and racemic forms of an amphiphilic resorcinol derivative at the air-water and graphite-1-phenyloctane interfaces

This article describes a study of the outcome of racemate condensation in different types of monolayers. The study was performed on a resorcinol surfactant bearing an octadecyl chain and a lactate group which formed a monolayer at the interface of graphite and 1-phenyloctane as well as a Langmuir film at the air-water interface. Control experiments with the enantiopure materials provided the characteristics of the chiral organizations. The results obtained on the racemate show that on graphite the molecule forms chiral domains, indicating that spontaneous resolution takes place at the surface, a phenomenon that has been rationalized using molecular modeling. The X-ray crystal structure of the DMSO solvate of one of the enantiomers shows a similar type of packing to this monolayer. On the other hand, in the Langmuir layer it appears that the formation of a racemic compound is favoured, as it is in the solid state in three dimensions. The work shows how the symmetry restrictions in different environments can have a critical influence on the outcome of racemate organization, and underline the tendency of graphite to favour symmetry breaking in monolayers formed at its surface.     

Cysteine-independent polymerization of metallothioneins in solutions and in crystals

Polymerization of metallothioneins is one of the usually encountered puzzles during the research process of metallothioneins' structure and function. Our work focuses on the cysteine independently occurred polymerization from metallothioneins monomers in different milieus, while it leaves out the aggregation caused by the oxidation of cysteine, because the latter circumstance is the result of purification lapsus. After the purification of metallothioneins monomers, a dynamic light-scattering technique is used to detect the polymerized states of rabbit liver metallothionein I and II in different buffers, which is the first systematical detection of polymerized states of metallothioneins in solutions. The effects of different compositions of each buffer are discussed in details. Steric complementarity, hydrophobic, and electrostatic interaction characteristics are studied, following the modeling of monomers and relevant polymers of rat metallothionein II, rabbit liver metallothionein I and II. These theoretical calculations are the first complete computer simulations on different factors affecting metallothioneins' polymerization. A molecular recognition mechanism of metallothioneins' polymerization in solutions is proposed on the bases of experimental results and theoretical calculations. Preliminary X-ray studies of two crystal forms of rabbit liver metallothionein II are compared with the crystal structure of rat metallothionein II, and the polymerized states in crystal packing are discussed with the knowledge of polymerization of metallothioneins in solutions. The hypothesis, which is consistent with theoretical calculations and experimental results, is expected to construct a connection between the biochemical characteristics and physiological functions of metallothioneins, and this research may give some enlightenment to the topics of protein polymerizations.     

Enantiomer selection in the reaction of N-methyl-α-amino acid N-carboxyanhydride and 3-methyl-5-substituted hydantoin: A model reaction for the stereoselective polymerization of α-amino acid N-carboxyanhydride

The addition reaction to N-methyl-(S)-alanine or N-methyl-(S)-phenylalanine N-car-boxyanhydride (NCA) of 3-methyl-5-substituted hydantoin (HDT) catalyzed by a tertiary amine was investigated as a model reaction for the propagation reaction of NCA according to the activated-NCA mechanism. Several activated HDTs having the (S)-configuration of the asymmetric carbon atom were found to react more rapidly than their activated enantiomers. This experimental result indicates that the enantiomer selection by terminal-unit control takes place in the propagation reaction according to the activated-NCA mechanism in which an activated NCA is added to a terminal acylated NCA ring of the growing chain. The enantiomer excess of the HDT recovered from the reaction mixture of N-methyl-(S)-phenylalanine NCA and racemic HDTs activated by a tertiary amine was determined. The extent of the enantiomer selection in the polymerization was found to be 3–10 times as large as that in the model reaction. From these results, it was concluded that the chirality of the penultimate unit, as well as that of the terminal NCA ring, plays an important role in determining the enantiomer selection in the NCA polymerization.     

Specificity in the recognition of crystals by antibodies

We show that IgG molecules isolated from the serum of rabbits injected with crystals of monosodium urate monohydrate, magnesium urate octahydrate and allopurinol, can each catalyze the nucleation of the same type of crystal to which they were exposed. These results generalize previous findings related to monosodium urate monohydrate and assess the idea that the invasion of a foreign crystal into an organism may amplify a population of antibodies which bear in their binding sites an imprint of the crystal surface structure. Such antibodies further act as nucleating templates which accelerate crystal formation in vitro. Antibodies from rabbitscan injected with sodium urate crystals do not cross-react or cross-react only to a low extent with antibodies isolated from rabbits injected with crystals of either magnesium urate or allopurinol. These results indicate a high specificity of the elicited antibodies, as these can distinguish between nuclei of crystals having similar molecular and structural characteristics.     

The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin and non-heme iron-dependent enzyme that hydroxylates L-Phe to l-Tyr using molecular oxygen as additional substrate. A dysfunction of this enzyme leads to phenylketonuria (PKU). The conformation and distances to the catalytic iron of both L-Phe and the cofactor analogue L-erythro-7,8-dihydrobiopterin (BH2) simultaneously bound to recombinant human PAH have been estimated by (1)H NMR. The resulting bound conformers of both ligands have been fitted into the crystal structure of the catalytic domain by molecular docking. In the docked structure L-Phe binds to the enzyme through interactions with Arg270, Ser349 and Trp326. The mode of coordination of Glu330 to the iron moiety seems to determine the amino acid substrate specificity in PAH and in the homologous enzyme tyrosine hydroxylase. The pterin ring of BH2 pi-stacks with Phe254, and the N3 and the amine group at C2 hydrogen bond with the carboxylic group of Glu286. The ring also establishes specific contacts with His264 and Leu249. The distance between the O4 atom of BH2 and the iron (2.6(+/-0.3) A) is compatible with coordination, a finding that is important for the understanding of the mechanism of the enzyme. The hydroxyl groups in the side-chain at C6 hydrogen bond with the carbonyl group of Ala322 and the hydroxyl group of Ser251, an interaction that seems to have implications for the regulation of the enzyme by substrate and cofactor. Some frequent mutations causing PKU are located at residues involved in substrate and cofactor binding. The sites for hydroxylation, C4 in L-Phe and C4a in the pterin are located at a distance of 4.2 and 4.3 A from the iron moiety, respectively, and at 6.3 A from each other. These distances are adequate for the intercalation of iron-coordinated molecular oxygen, in agreement with a mechanistic role of the iron moiety both in the binding and activation of dioxygen and in the hydroxylation reaction.     

Solvent effect in the stereoselective polymerization of α-amino acid N-carboxyanhydride and some considerations on the mechanism of stereoregulation

In the Polymerization of phenylalanine N-carboxyanhydride (NCA) in No₂Oh initiated by MeNHBzl, L -,D -, and DL -NCA As were polymerized at the same rate, and no stereoselectivity was observed. When the same experiment was carried out in HCONEt₂, however, L - and D -NCA were both polymerized at a rate which was about twice as large as that of DL -NCA. In this case, the polymerization is stereoselective, ascribable to a preferable reaction between the optical enantiomorphs of the terminal residue of the growing chain and the NCA of the same chirality. On the other hand, the polymerization initiated by SarNMe₂ and MeNH(CH₂)₂CONMe₂ were stereoselective in NO₂Ph and HCONEt₂, but they were not stereoselective in m-(MeO)₂Ph. These findings indicate that the polymerizations initiated by a strong base in highly dipolar solvents are stereoselective. Apparently, the reaction between a chiral, cyclic terminal of growing chain and a chiral, cyclic activated NCA in the activated-NCA mechanism is highly stereoselective. In addition, from a kinetic investigation on on the copolymerization between L - and D -NCAs, the penultimate chiral centers were also suggested to contribute to the stereoselection. Stereoselection by the α-helical conformation of the growing chain and by a chiral, linear terminal amine have been considered so far, and the contribution from the present type of stereoselection must have been overlooked.     

Effect of degree of polymerization and steric configuration of poly(2-vinylpyridine) as catalyst in the polymerization of phenylalanine NCA

Despite its being weaker base poly(2-vinylpyridine) polymerized DL -β-phenylalanine NCA at a much faster rate than pyridine and α-picoline. Poly(2-vinylpyridine) adsorbs NCA by hydrogen bonding with the cooperation of a few pyridine groups. This results in a high local concentration of NCA. The syndiotactic configuration of pyridine group seemed to be least suitable for the cooperative hydrogen bonding. Adsorbed NCA is activated to form an “activated” NCA which in turn reacts with an NCA adsorbed on the same polymer chain. Since the polymer chain is flexible, this intramolecular reaction takes place frequently, resulting in the acceleration of polymerization. The intramolecular reaction along the polymer chain is dependent on the degree of polymerization of polymer catalyst. A suitable model was proposed for the intramolecular reaction to explain the effect of degree of polymerization.     

Contribution from the chirality of the growing chain to the enantiomer selectivity in activated-NCA polymerization of α-amino acid N-carboxyanhydride

As a model compound for the growing chain in the activated-NCA type of polymerization of α-amino acid N-carboxyanhydride (NCA), 3-[ω-acetylglycyl-poly(α-amino acid) acyl]-α-amino acid NCA (called the prepolymer) having various degrees of polymerization (DPs) was synthesized by the polymerization of Phe, Val, Glu(OEt), and Asp(OBzl) NCA in the presence of AcGly NCA by the tertiary amine. Activated (S)-Phe, Val, Glu(OEt), and Asp(OBzl) NCA were added to the terminal cyclic group of the corresponding (S)- or (R)- prepolymer, and the enantiomer selectivity in the reaction was investigated. With prepolymers having DPs ranging from 1 to 15, the addition reaction always took place preferentially between species having the same configuration, and the degree of the enantiomer selection increased with increasing DP of the prepolymer. With prepolymers having DP = 1 and 2, we found contributions from the chiral terminal unit and the chiral penultimate unit to the enantiomer selection, respectively. Prepolymer having DP = 5 was shown to take a β-type conformation, which led to higher enantiomer selection; and prepolymers having DP = 10 and 15 were shown to take an α-helix conformation, which led to much higher enantiomer selection than did the β-type conformation. In the present investigation the mechanisms of terminal-unit control, penultimate-unit control and conformational control of the enantiomer selection in the activated-NCA type of polymerization were clearly observed.     

Application of a quartz crystal nanobalance to the molecularly imprinted recognition of phenylalanine in solution

A quartz crystal nanobalance (QCN) biosensor was developed for the selective determination of phenylalanine (Phe) in aqueous solutions. A Phe imprinted copolymer was synthesized using polyacrylonitrile and acrylic acid [poly(AN-co-AA)]. The copolymer was then coated on quartz crystal electrode to form complementary structures for the template recognition of Phe. The composite electrode was then used to determine Phe levels in solution. Determinations were based on frequency shifts of molecularly imprinted polymer (MIP) modified quartz crystal electrode caused by Phe adsorption. The frequency shifts were linearly dependent on Phe concentration over the range 50∼500 mgL⁻¹. The results obtained show that the imprinted poly(AN-co-AA) modified biosensor had higher sensitivity (0.5839 Hz/mgL⁻¹) than a non-molecularly imprinted copolymer (0.2724 Hz/mgL⁻¹). Furthermore, good reproducibility, R.S.D. = 1.84% (n = 7) was observed, and the detection limit was 45 mgL⁻¹. The selectivity of the imprinted poly(AN-co-AA) modified biosensor was examined using a number of analytes similar to Phe, i.e., dopamine (DA), ascorbic acid (AscA), vanillylmandelic acid (VMA), uric acid (UA), tryptophan (Trp), and tyrosine (Tyr), and the results obtained showed a size dependent selective effect.     

Mutational analysis of phenylalanine beta 85 in the valine beta 6 acceptor pocket during hemoglobin S polymerization.

Hemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at beta 85 were made and expressed in yeast in an effort to evaluate the role of Phe-beta 85 in the acceptor pocket during polymerization of deoxy Hb S. The four Hb S variants have the same electrophoretic mobility as Hb S, and these beta 85 substitutions do not significantly affect heme-globin interactions and tetramer helix content. Hb S containing Trp-beta 85 had decreased oxygen affinity, whereas those with Leu-, Ala-, and Thr-beta 85 had increased oxygen affinity. All four supersaturated beta 85 variants polymerized with a delay time as does deoxy Hb S. This is in contrast to deoxy Hb S containing Phe-beta 88, Ala-beta 88, Glu-beta 88, or Glu-beta 85, which polymerized with no clear delay time (Adachi K, Konitzer P, Paulraj CG, Surrey S, 1994, J Biol Chem 269:17477-17480; Adachi K, Reddy LR, Surrey S, 1994, J Biol Chem 269:31563-31566). Leu substitution at beta 85 accelerated deoxy Hb S polymerization, whereas Ala, Thr, or Trp substitution inhibited polymerization. The length of the delay time and total polymer formed for these beta 85 Hb S variants depended on hemoglobin concentration in the same fashion as for deoxy Hb S: the higher the concentration, the shorter the delay time and the more polymer formed. Critical concentrations required for polymerization of deoxy Hb SF veta 85L, Hb SF beta 85A, Hb SF beta 85T, and Hb SF beta 85W are 0.65-, 2.2-, 2.5- and 3-fold higher, respectively, than Hb S.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in Taxol production in plant cell culture via manipulation of the phenylalanine ammonia lyase pathway

One approach to increasing secondary metabolite production in plant cell culture is to manipulate metabolic pathways to utilize more resources toward production of one desired compound or class of compounds, such as diverting carbon flux from competing secondary pathways. Since phenylalanine provides both the phenylisoserine side chain and the benzoyl moiety at C-2 of Taxol, we speculated that blockage of the phenylpropanoid pathway might divert phenylalanine into Taxol biosynthesis. We used specific enzyme inhibitors to target the first enzyme in the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), the critical control point for conversion of L-phenylalanine to trans-cinnamic acid. Cinnamic acid acted quickly in reducing PAL activity by 40-50%, without affecting total protein levels, but it generally inhibited the taxane pathway, reducing Taxol by 90% of control levels. Of the taxanes produced, 13-acetyl-9-dihydro-baccatin III and 9-dihydrobaccatin III doubled as a percentage of total taxanes in C93AD and CO93P cells treated with 0.20 and 0.25 mM cinnamic acid, when all other taxanes were lowered. The PAL inhibitor alpha-aminooxyacetic acid (AOA) almost entirely shut down Taxol production at both 0.5 and 1.5 mM, whereas L-alpha-aminooxy-beta-phenylpropionic acid (AOPP) had the opposite effect, slightly enhancing Taxol production at 1 microM but having no effect at 10 microM. The discrepancy in the effectiveness of AOA and AOPP and the lack of effect with addition of phenylalanine or benzoic acid derivatives further indicates that the impact of cinnamic acid on Taxol is related not to its effect on PAL but rather to a specific effect on the taxane pathway. On the basis of these results, a less direct route for inhibiting the phenylpropanoid pathway may be required to avoid unwanted side effects and potentially enhance Taxol production.