Synthesis and physicochemical characterization of the lanthionine analog of somatostatin[1–14]

Summary The synthesis of the lanthionine analog of somatostatin[1–14] on a Kaiser-oxime resin is described. The 12-residue peptide segment [3–14] was assembled and cyclized on the resin by using the method of peptide cyclization on an oxime resin (PCOR); the product was obtained with good yield (41%) and purity (94%). The Fmoc protecting group on the N-terminus was cleaved with DBU, followed by a 2+12 segment condensation in solution. The chromatographic (HPLC, CZE) and spectral (UV, NMR) properties of the lanthionine and the natural somatostatins have been studied and compared. Preliminary biological tests show that the lanthionine and the natural somatostatins exhibit similar binding affinities to somatostatin receptor SSTR2.Abbreviations Ala_L one end of a lanthionine unit - Boc tert-butyloxycarbonyl - BOP benzotriazol-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - Bzl benzyl - Cbz benzyloxycarbonyl - DQF-COSY double-quantum-filtered correlated NMR spectroscopy - CZE capillary zone electrophoresis - DBU 1,8-diazabicyclo[5.4.0]undec-7-ene - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - DMSO-d₆ hexadeuterated dimethylsulfoxide - EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - Fmoc 9-florenylmethoxycarbonyl - For formyl - HMPA hexamethylphosphoramide - HOBt N-hydroxybenzotriazole - HOHAHA homonuclear Hartmann-Hahn experiment - HPLC high-performance liquid chromatography - ROESY rotating frame nuclear Overhauser enhancement spectroscopy - TFA trifluoroacetic acid - PCOR peptide cyclization on an oxime resin - Tmac₂O trimethylacetic or pivalic anhydride - Tos p-toluenesulfonyl     

The photooxidation of glyoxylate by envelope-free spinach chloroplasts and its relation to photorespiration

Large quantities of CO₂ are released within many photosynthesizing tissues in the light by the process of photorespiration. This CO₂ arises largely from the carboxylcarbon atom of glycolate, which is synthesized in chloroplasts during photosynthesis. Glyoxylate is then produced by the glycolate oxidase reaction. The glyoxylate may be directly decarboxylated to CO₂, but some investigators believe the glyoxylate must first be converted to glycine before CO₂ is released during photorespiration. Spinach chloroplasts with their envelope membranes removed in dilute buffer solution have now been shown to carry out the oxidative decarboxylation of [1-¹⁴C]glyoxylate, in the presence of light and manganous ions in an atmosphere containing oxygen, to yield 1 mole each of ¹⁴CO₂ and formate. Rates of enzymatic decarboxylation exceeding 50 μmoles of ¹⁴CO₂ mg chlorophyll⁻¹ hr⁻¹ were obtained at pH 7.6; hydrogen peroxide is probably the oxidant in the reaction. Heated chloroplasts are inactive under the standard conditions and there is an almost absolute requirement for each of the components listed above. Conditions for some other nonenzymatic decarboxylations of glyoxylate have also been described. [1-¹⁴C]Glycine is decarboxylated by the enzymatic system at only 1% of the rate of [1-¹⁴C]glyoxylate. Maize chloroplast preparations are much less active than spinach chloroplasts. The high rates of CO₂ produced by the spinach system directly from glyoxylate, as well as the need for light and oxygen, suggest that this reaction functions in photorespiration, and that CO₂ arises during photorespiration without glycine as a mandatory intermediate.     

Stereoselective synthesis of lanthionine derivatives in aqueous solution and their incorporation into the peptidoglycan of Escherichia coli

The three diastereoisomers—(R,R), (S,S) and meso—of lanthionine were synthesized in aqueous solution with high diastereoselectivity (>99%). The (S) and (R) enantiomers of two differently protected sulfamidates were opened by nucleophilic attack of (R) or (S)-cysteine. Acidification and controlled heating liberated the free lanthionines. Using the same chemistry, an α-benzyl lanthionine was also prepared. The proposed method, which avoids the need of enrichment by recrystallization, opens the way to the labelling of these compounds with ³⁵S. Furthermore, in vivo bioincorporation into Escherichia coli W7 was studied. No incorporation of α-benzyl lanthionine was observed. In contrast, meso-lanthionine can effectively replace meso-diaminopimelic acid in vivo, while in the presence of (R,R)-lanthionine the initial increase of bacterial growth was followed by cell lysis. In the future, meso-[³⁵S]lanthionine could be used to study the biosynthesis of peptidoglycan and its turnover in relation to cell growth and division.     

A simple apparatus for the continuous monitoring of 14CO2 production from several small reaction mixtures

A simple apparatus is described which permits the continuous monitoring of ¹⁴CO₂ production from ten separate reaction mixtures simultaneously. The device is relatively simple and inexpensive to construct, makes use of small disposable incubation vials, and allows complete trapping of all ¹⁴CO₂ evolved in scintillation vials, where it can be easily counted. The use of this apparatus to determine the rates of metabolism by glomeruli of ¹⁴C-labeled substrates to ¹⁴CO₂ is described.     

Ureide Catabolism in Soybeans : III. Ureidoglycolate Amidohydrolase and Allantoate Amidohydrolase Are Activities of an Allantoate Degrading Enzyme Complex

We demonstrate that allantoate is catabolized in soybean seedcoat extracts by an enzyme complex that has allantoate amidohydrolase and ureidoglycolate amidohydrolase activities. Soybean seedcoat extracts released ¹⁴CO₂ from [ureido-¹⁴C]ureidoglycolate under conditions in which urease is not detectable. CO₂ and glyoxylate are enzymically released in a one to one ratio indicating that ureidoglycolate amidohydrolase is the responsible activity. Ureidoglycolate amidohydrolase has a K_m of 85 micromolar for ureidoglycolate. Glyoxylate and CO₂ are enzymically released from allantoate at linear rates in a one to 2.3 ratio from 5 to 30 min. This ratio is consistent with the degradation of allantoate to two CO₂ and one glyoxylate with approximately 23% of the allantoate degraded reacting with 2-mercaptoethanol to yield 2-hydroxyethylthio, 2′-ureido, acetate (RG Winkler, JC Polacco, DG Blevins, DD Randall 1985 Plant Physiol 79: 787-793). That [¹⁴C]urea production from [2,7-¹⁴C]allantoate is not detectable indicates that allantoate-dependent glyoxylate production is enzymic and not a result of nonenzymic hydrolysis of a ureido intermediate (nonenzymic hydrolysis releases urea). These results and those from intact tissue studies (RG Winkler DG Blevins, JC Polacco, DD Randall 1987 Plant Physiol 83: 585-591) suggest that soybeans have a second amidohydrolase reaction (ureidoglycolate amidohydrolase) that follows allantoate amidohydrolase in allantoate catabolism. The rate of ¹⁴CO₂ release from [2,7-¹⁴C]allantoate is not reduced when the volume of the reaction mixture is increased, suggesting that the release of ¹⁴CO₂ is not dependent on the accumulation of free intermediates. That [2,7-¹⁴C]allantoate dependent ¹⁴CO₂ release is not proportionally diluted by unlabeled ureidoglycolate indicates that the reaction is carried out by an enzyme complex. This is the first report of ureidoglycolate amidohydrolase activity in any organism and the first in vitro demonstration in plants that the ureido-carbons of allantoate can be completely degraded to CO₂ without a urea intermediate.     

A method for the determination of glucose-6-phosphatase activity in rat liver with [U-14C]glucose 6-phosphate as substrate.

A method is described for measuring the activity of glucose-6-phosphatase (EC 3.1.3.9) in rat liver. [U-¹⁴C]Glucose 6-phosphate, as substrate, is converted by the enzyme to [¹⁴C]glucose and inorganic phosphate. The addition of ZnSO₄ and Ba(OH)₂ at the end of the reaction precipitates phosphate and the unreacted [¹⁴C]glucose 6-phosphate, whereas [¹⁴C]glucose is not precipitated. After centrifugation, the amount of [¹⁴C]glucose formed is determined in a liquid scintillation counter.     

Use of 14C-carboxyl-luciferin in determining the mechanism of the firefly luciferase catalyzed reactions

The use of ¹⁴C-carboxyl-labelled luciferin as a substrate for the firefly luciferase catalyzed reaction produces ¹⁴CO₂ as a product. We have studied this reaction in the presence of ¹⁷O₂ and H¹⁸OH, using an excess of luciferin over luciferase. The initial collection of CO₂ contained close to one oxygen from ¹⁷O₂ for each molecule of ¹⁴CO₂ derived from luciferin, which is consistent with a cyclic peroxide mechanism. About half of the ¹⁴CO₂ remained bound to the enzyme and was collected after acidification of the medium. This CO₂ contained less than 0.1 of an atom of oxygen from ¹⁷O₂ for each molecule of ¹⁴CO₂ derived from luciferin. Exchange of medium CO₂-HCO₃ su? with water was not sufficiently great to account for the loss of any ¹⁷O previously incorporated. The most likely explanation appears to be a preferential exchange of oxygens of enzyme-bound CO₂ with water oxygens. Such exchange, and dilution of CO₂ from luciferin by medium CO₂, may explain previous results in which little incorporation of atmospheric oxygen was noted.     

Simple Determination of the CO(2)/O(2) Specificity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by the Specific Radioactivity of [C]Glycerate 3-Phosphate

A new method is presented for measurement of the CO₂/O₂ specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [¹⁴C]3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. ¹⁴CO₂ fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO₂ in O₂-saturated water and carboxylase only with 160 micromolar CO₂ under N₂. Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the [¹⁴C]PGA in the same peak. The specificity factor of Rubisco from spinach (Spinacia oleracea L.) (93 ± 4), from the green alga Chlamydomonas reinhardtii (66 ± 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods.     

A facile method for the direct synthesis of lanthionine containing cyclic peptides

A series of disulfide bridged peptides were designed as potential inhibitors of protein-protein interactions. Following solid phase synthesis, completely deprotected linear peptides were first oxidized to their disulfide analogs and then transformed into their lanthionine equivalents via a base-assisted reaction in water. Peptides consisting of cystine bridges of length i, i+3, with and without discrimination of the chiral centers, were studied for this transformation. Lanthionine peptides were also obtained directly from the reduced linear peptides under mild alkaline treatment, and the reaction proceeded via disulfide bond formation. The extent of conversion of a disulfide bridge into its lanthionine counterpart varied according to the primary sequence. Product characterization revealed diastereomeric lanthionine formation. The presence of D-amino acids, peptide conformation, and/or position of the cystine bridge are among the factors determining the facility of this reaction. Elimination of the backbone proton beta to the sulfur atom followed by intramolecular thiol Michael addition is the most likely mechanism for this transformation.     

Mechanism of glycolate transport in spinach leaf chloroplasts

The incorporation of ¹⁴CO₂ into glycolate by intact spinach leaf (Spinacia oleracea L. var. Kyoho) chloroplasts exposed to ¹⁴CO₂ (NaH¹⁴CO₃, 1 millimolar) in the light was determined as a function of O₂ concentrations in the reaction media. A hyperbolic saturation curve was obtained, apparent K_m (O₂) of 0.28 millimolar, indicating that glycolate is produced predominantly by ribulose-1,5-bisphosphate carboxylase/oxygenase. A concentration gradient of glycolate was invariably observed between chloroplast stroma and the outside media surrounding chloroplasts during photosynthetic ¹⁴CO₂ fixation under an O₂ atmosphere.     

Anaerobic formation of succinate from glucose and bicarbonate in resting cells of Leishmania donovani

The cell suspension of Leishmania donovani incorporates ¹⁴CO₂ resulting in the formation of [¹⁴C]-succinic acid under anaerobic conditions. The results showed that the [¹⁴C]-succinate formation from [1-¹⁴C]-glucose is much greater than that from [6-¹⁴C]-glucose. [¹⁴C-pyruvate] takes part in the production of succinic acid under anaerobic conditions without decarboxylation. The anaerobic formation of succinate appears to involve the production of malate, which is then converted to succinate via the reduction of fumarate by the reversal of the tricarboxylic acid cycle. Evidence indicated that the active species in this carboxylation reaction was CO₂ although HCO₃ was active to some extent.     

Extraction and partial characterization of the glycine decarboxylase multienzyme complex from pea leaf mitochondria

Glycine decarboxylase has been successfully solubilized from pea (Pisum sativum) leaf mitochondria as an acetone powder. The enzyme was dependent on added dithiothreitol and pyridoxal phosphate for maximal activity. The enzyme preparation could catalyze the exchange of CO₂ into the carboxyl carbon of glycine, the reverse of the glycine decarboxylase reaction by converting serine, NH₄⁺, and CO₂ into glycine, and ¹⁴CO₂ release from [1-¹⁴C]glycine. The half-maximal concentrations for the glycine-bicarbonate exchange reaction were 1.7 millimolar glycine, 16 millimolar NaH¹⁴CO₂, and 0.006 millimolar pyridoxal phosphate. The enzyme (glycine-bicarbonate exchange reaction) was active in the assay conditions for 1 hour and could be stored for over 1 month. The enzymic mechanism appeared similar to that reported for the enzyme from animals and bacteria but some quantitative differences were noted. These included the tenacity of binding to the mitochondrial membrane, the concentration of pyridoxal phosphate needed for maximum activity, the requirement for dithiothreitol for maximum activity, and the total amount of activity present. Now that this enzyme has been solubilized, a more detailed understanding of this important step in photorespiration should be possible.     

The reaction of 14C-labelled platinum ethylenediamine dichloride with adenine compounds and DNA

Various derivatives of adenine have been studied with regard to their rate of reaction with ¹⁴C-labelled platinum ethylenediamine dichloride, Pt(¹⁴C-en)Cl₂. The reactivities have been calculated from the “rate of disappearance” of Pt(¹⁴C-en)Cl₂ using chromatographic separation of reactants and products.Adenine and adenosine react very slowly at 37° whereas other adenine derivatives react much more readily in the order: poly A > AMP > ApA > poly d(AT). From the numerical values of the rate constants it is concluded that the presence of a phosphate group increases the reaction rate considerably. This is partly the explanation of the rapid reaction of poly A which possesses terminal phosphate groups. However adjacent adenine moieties such as those in polyadenylic acid (poly A) and adenosyl-3′5′-adenosine (ApA) may also react by another mechanism which involves the 6-NH₂ groups.The energies of activation of the second order reaction with platinum ethylenediamine dichloride (PtenCl₂) are 12.9, 18.8, 19.0 kcal/mole for poly A, AMP and ApA respectively.In DNA, no free phosphate groups are present, and the occurrence of adjacent adenines will be low. The reaction of PtenCl₂ with DNA seems to involve a rapid attack on deoxyguanosine (GdR) and a slow reaction with deoxyadenosine (AdR) and deoxycytidine (CdR).     

Silk—A new substrate for UDP-d-xylose: Proteoglycan core protein β-d-xylosyltransferase

The formation of most connective tissue polysaccharides is initiated by transfer of d-xylose from UDP-d-xylose to specific serine residues in the core proteins of the putative proteoglycans. The substrate specificity of the xylosyltransferase catalyzing this reaction has not yet been examined in detail, but it appears that a -Ser-Gly- pair is an essential part of the substrate structure. Since the preparation of the known acceptors (e.g., Smith-degraded or HF-treated cartilage proteoglycan) involves a substantial effort, we have searched for readily available proteins with the -Ser-Gly-sequence, which might serve as alternative substrates. In the present work, it was found that silk fibroin from Bombyx mori, which consists, in large part, of the repeating hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly, is an excellent substrate for the xylosyltransferase from embryonic chick cartilage. Pieces of silk were used directly in the reaction mixtures, and [¹⁴C]xylose transferred from UDP-d-[¹⁴C]xylose was measured by liquid scintillation spectrometry after rinsing the silk in 1 m NaCl and water. Substantially greater incorporation was observed with preparations of silk or fibroin which had been dissolved in 60% LiSCN and subsequently dialyzed exhaustively or diluted appropriately. Under standard reaction conditions, the V_max for fibroin was 531 pmol/h/mg enzyme protein, as compared to 223 pmol/h/mg for Smith-degraded proteoglycan. K_m values were 182 mg/liter (fibroin) and 143 mg/liter (Smith-degraded proteoglycan). The product of [¹⁴C]xylose transfer to silk was alkali labile, and [¹⁴C]xylitol was formed when [¹⁴C]xylosylsilk was treated with borohydride in alkali. Proteolytic digestion with papain, Pronase, leucine aminopeptidase, and carboxypeptidase A yielded a radioactive product which was identified as [¹⁴C]xylosylserine by electrophoresis and chromatography. The identity of the isolated [¹⁴C]xylosylserine was further supported by its resistance to treatment with alkali (0.5 m KOH: 100°C; 8h) and by acid hydrolysis which yielded [¹⁴C]xylose. Tryptic and chymotryptic fragments from fibroin were also good xylose acceptors and had V_max values 60–70% of that observed for the intact protein. Substantial acceptor activity was displayed also by the sericin fraction of silk and by the silk sequence hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly; the latter had a V_max value close to 20% of that of intact fibroin.     

Catabolism of 5-Aminolevulinic Acid to CO(2) by Etiolated Barley Leaves

The in vivo oxidation of the C₄ and C₅ of 5-aminolevulinic acid (ALA) to CO₂ has been studied in etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness. The rate of ¹⁴CO₂ evolution from leaves fed [4-¹⁴C]ALA is strongly inhibited by aminooxyacetate, anaerobiosis, and malonate. The rate of ¹⁴CO₂ evolution from leaves fed [5-¹⁴C]ALA is also inhibited by these treatments but to a lesser extent. These results suggest that (a) one step in ALA catabolism is a transamination reaction and (b) the C₄ is oxidized to CO₂ via the tricarboxylic acid cycle to a greater extent than is the C₅.     

Anaerobic degradation of phenol via carboxylation to 4-hydroxybenzoate: in vitro study of isotope exchange between 14CO2 and 4-hydroxybenzoate

Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-¹⁴C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn²⁺ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol ¹⁴CO₂ exchange · min^-1 · mg^-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min^-1 · mg^-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.     

The occurrence of a glycine cleavage system in mammalian brain

A major catabolic route for glycine in liver is its conversion to methylene tetra-hydrofolate (methylene THF), CO₂ and NH₃, catalysed by the glycine cleavage system described by Yoshida and Kikuchi (1970). In view of the role of glycine as a putative neuro-transmitter, the occurrence of this system in mammalian brain was investigated. Our studies demonstrated: (a) that the production of ¹⁴CO₂ from [1-¹⁴C]glycine required the presence of tetrahydrofolate, NAD, dithiothreitol and pyridoxal phosphate in the reaction mixture; (b) that besides CO₂l -serine was the other major product of the reaction; (c) that glyoxylate did not function as an intermediate in this reaction; and (d) that enzymatic activity appeared to be associated with membranes. All of these properties resembled those previously described for the hepatic system.     

The small-scale production of [U-C]acetylene from BaCO3: Application to labeling of ammonia monooxygenase in autotrophic nitrifying bacteria

A small-scale method has been adapted from an established procedure for the generation of [U-¹⁴C]acetylene from inexpensive and commonly available precursors. The method involves the fusing of Ba¹⁴CO₃ with excess barium metal to produce Ba¹⁴C₂. The BaC₂ is reacted with water to generate acetylene which is then selectively dissolved into dimethyl sulfoxide (DMSO). The results presented demonstrate the effect of Ba:BaCO₃ ratio on the concentrations of various gases released during the hydrolysis reaction and quantify the selectivity of the DMSO-trapping process for each gas. [U-¹⁴C]-Acetylene generated by this method has been used to inactivate ammonia monooxygenase in three species of autotrophic nitrifying bacteria: Nitrosomonas europaea, Nitrosococcus oceanus, and Nitrosolobus multiformis. Our results demonstrate that acetylene inactivation of this enzyme in all three species results in the covalent incorporation of radioactive label into a polypeptide of apparent M_r of 25,000–27,000, as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography.     

Inorganic Carbon Uptake during Photosynthesis : I. A Theoretical Analysis Using the Isotopic Disequilibrium Technique

Equations have been developed which quantitatively predict the theoretical time-course of photosynthetic ¹⁴C incorporation when CO₂ or HCO₃⁻ serves as the sole source of exogenous inorganic carbon taken up for fixation by cells during steady state photosynthesis. Comparison between the shape of theoretical (CO₂ or HCO₃⁻) and experimentally derived time-courses of ¹⁴C incorporation permits the identification of the major species of inorganic carbon which crosses the plasmalemma of photosynthetic cells and facilitates the detection of any combined contribution of CO₂ and HCO₃⁻ transport to the supply of intracellular inorganic carbon. The ability to discriminate between CO₂ or HCO₃⁻ uptake relies upon monitoring changes in the intracellular specific activity (by ¹⁴C fixation) which occur when the inorganic carbon, present in the suspending medium, is in a state of isotopic disequilibrium (JT Lehman 1978 J Phycol 14: 33-42). The presence of intracellular carbonic anhydrase or some other catalyst of the CO₂-HCO₃⁻ interconversion reaction is required for quantitatively accurate predictions. Analysis of equations describing the rate of ¹⁴C incorporation provides two methods by which any contribution of HCO₃⁻ ions to net photosynthetic carbon uptake can be estimated.     

<Superscript>153</Superscript>Sm and <Superscript>166</Superscript>Ho complexes with tetraaza macrocycles containing pyridine and methylcarboxylate or methylphosphonate pendant arms

A set of tetraaza macrocycles containing pyridine and methylcarboxylate (ac₃py14) or methylphosphonate (MeP₂py14 and P₃py14) pendant arms were prepared and their stability constants with La³⁺, Sm³⁺, Gd³⁺ and Ho³⁺ determined by potentiometry at 25 °C and 0.10 M ionic strength in NMe₄NO₃. The metal:ligand ratio for ¹⁵³Sm and ¹⁶⁶Ho and for ac₃py14, MeP₂py14 and P₃py14, as well as the pH of the reaction mixtures, were optimized to achieve a chelation efficiency higher than 98%. These radiocomplexes are hydrophilic and have a significant plasmatic protein binding. In vitro stability was studied in physiological solutions and in human serum. All complexes are stable in saline and PBS, but 20% of radiochemical impurities were detected after 24 h of incubation in serum. Biodistribution studies in mice indicated a slow rate of clearance from blood and muscle, a high and rapid liver uptake and a very slow rate of total radioactivity excretion. Some bone uptake was observed for complexes with MeP₂py14 and P₃py14, which was enhanced with time and the number of methylphosphonate groups. This biological profile supports the in vitro instability found in serum and is consistent with the thermodynamic stability constants found for these complexes.