A Charge-Deficient Analogue of Spermine with Chelating Properties

1,12-Diamino-3,6,9-triazadodecane, a new isosteric and charge-deficient analogue of spermine, is synthesized. Unlike spermine, the new analogue is an excellent chelator of Cu²⁺ ions. Possible applications of this compound for studying enzymes of polyamine metabolism and cellular functions of spermine are discussed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 303–311.Original Russian Text Copyright © 2005 by Khomutov, Grigorenko, Skuridin, Demin, Vepsalainen, Casero, Woster.     

A charge-deficient analogue of spermine with chelating properties

1,12-Diamino-3,6,9-triazadodecane, a new isosteric and charge-deficient analogue of spermine, is synthesized. Unlike spermine, the new analogue is an excellent chelator of Cu2+ ions. Possible applications of this compound for studying enzymes of polyamine metabolism and cellular functions of spermine are discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

Novel isosteric charge-deficient spermine analogue—1,12-diamino-3,6,9-triazadodecane: synthesis, pK a measurement and biological activity

Ionic interactions are essential for the biological functions of the polyamines spermidine and spermine in mammalian physiology. Here, we describe a simple gram scale method to prepare 1,12-diamino-3,6,9-triazadodecane (SpmTrien), an isosteric charge-deficient spermine analogue. The protonation sites of SpmTrien were determined at pH range of 2.2–11.0 using two-dimensional ¹H-¹⁵N NMR spectroscopy, which proved to be more feasible than conventional methods. The macroscopic pK _a values of SpmTrien (3.3, 6.3, 8.5, 9.5 and 10.3) are significantly lower than those of 1,12-diamino-4,9-diazadodecane (spermine). The effects of SpmTrien and its parent molecule, 1,8-diamino-3,6-diazaoctane (Trien), on cell growth and polyamine metabolism were investigated in DU145 prostate carcinoma cells. SpmTrien downregulated the biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosyl-l-methionine decarboxylase and decreased intracellular polyamine levels, whereas the effects of Trien alone were minor. Interestingly, both SpmTrien and Trien were able to partially overcome growth arrest induced by an ODC inhibitor, α-difluoromethylornithine, indicating that they are able to mimic some functions of the natural polyamines. Thus, SpmTrien is a novel tool to influence polyamine interaction sites at the molecular level and offers a new means to study the contribution of the protonation of spermine amino group(s) in the regulation of polyamine-dependent biological processes.     

Inhibition of acetylpolyamine and spermine oxidases by the polyamine analogue chlorhexidine

Acetylpolyamine and spermine oxidases are involved in the catabolism of polyamines. The discovery of selective inhibitors of these enzymes represents an important tool for the development of novel anti-neoplastic drugs. Here, a comparative study on acetylpolyamine and spermine oxidases inhibition by the polyamine analogue chlorhexidine is reported. Chlorhexidine is an antiseptic diamide, commonly used as a bactericidal and bacteriostatic agent. Docking simulations indicate that chlorhexidine binding to these enzymes is compatible with the stereochemical properties of both acetylpolyamine oxidase and spermine oxidase active sites. In fact, chlorhexidine is predicted to establish several polar and hydrophobic interactions with the active site residues of both enzymes, with binding energy values ranging from ?7.6 to ?10.6 kcal/mol. In agreement with this hypothesis, inhibition studies indicate that chlorhexidine behaves as a strong competitive inhibitor of both enzymes, values of K_i being 0.10 μM and 0.55 μM for acetylpolyamine oxidase and spermine oxidase, respectively.     

New oxaanalogues of spermine

A new isosteric charge-deficient spermine analogue, 1,12-diamino-4,9-diaza-5-oxadodecan, and O-(7-amino-4-azaheptyl)oxime of 3-aminopropanal, a stable analogue of the Schiff base intermediate in the enzymatic oxidation of spermine, were synthesized. The possible use of these compounds for the inhibition of spermine oxidase is discussed.     

A New Synthesis of α-Methylspermidine

A five-step synthesis of -methylspermidine (1,8-diamino-5-azanonane), the first polyamine analogue preventing pathological consequences of spermidine depletion in transgenic rats overproducing spermine/spermidine N ¹-acetyltransferase, from ethyl 3-aminobutyrate was achieved in a high overall yield.     

Characterization of spermine uptake by Ehrlich tumour cells in culture

Summary. Spermine is taken up by Ehrlich ascites tumour cells through a specific, saturable, temperature and energy-dependent transport system with a remarkably low affinity constant for spermine (around 1 μM). In the absence of a potassium ion gradient through the plasma membrane, spermine uptake remains saturable but the value of the K_m for spermine is much higher (153 μM). Difluormethylornithine treatment (3 mM for 48 h) induces significant increases in V_max values (up to 9-fold) and changes in the K_m values with scarce statistical significance. Among the biogenic amines tested, only spermidine and, partly, agmatine seem to share the same transport system with spermine. No difference is observed in the rate of spermine transport when assays are carried out in the presence of 50-fold excess of ornithine or calcium, or 100-fold excess of glutamine. Received April 25, 2000 Accepted November 1, 2000     

Polyamines and its analogue modulates amyloid fibrillation in lysozyme: A comparative investigation

BackgroundPolyamines can induce protein aggregation that can be related to the physiology of the cellular function. Polyamines have been implicated in protein aggregation which may lead to neuropathic and non neuropathic amyloidosis.Scope of reviewChange in the level of polyamine concentration has been associated with ageing and neurodegeneration such as Parkinson's disease, Alzheimer's disease. Lysozyme aggregation in the presence of polyamines leads to non neuropathic amyloidosis. Polyamine analogues can suppress or inhibit protein aggregation suggesting their efficacy against amyloidogenic protein aggregates.Major conclusionsIn this study we report the comparative interactions of lysozyme with the polyamine analogue, 1-naphthyl acetyl spermine in comparison with the biogenic polyamines through spectroscopy, calorimetry, imaging and docking techniques. The findings revealed that the affinity of binding varied as spermidine > 1-naphthyl acetyl spermine > spermine. The biogenic polyamines accelerated the rate of fibrillation significantly, whereas the analogue inhibited the rate of fibrillation to a considerable extent. The polyamines bind near the catalytic diad residues viz. Glu35 and Asp52, and in close proximity of Trp62 residue. However, the analogue showed dual nature of interaction where its alkyl amine region bind in same way as the biogenic polyamines bind to the catalytic site, while the naphthyl group makes hydrophobic contacts with Trp62 and Trp63, thereby suggesting its direct influence on fibrillation.General significanceThis study, thus, potentiates, the development of a polyamine analogue that can perform as an effective inhibitor targeted towards aggregation of amyloidogenic proteins.     

Rectification of the Olfactory Cyclic Nucleotide-Gated Channel by Intracellular Polyamines

Polyamine-induced inward rectification of cyclic nucleotide-gated channels was studied in inside-out patches from rat olfactory neurons. The polyamines, spermine, spermidine and putrescine, induced an `instantaneous' voltage-dependent inhibition with K <sub>d</sub> values at 0 mV of 39, 121 μm and 2.7 mm, respectively. Hill coefficients for inhibition were significantly < 1, suggesting an allosteric inhibitory mechanism. The Woodhull model for voltage-dependent block predicted that all 3 polyamines bound to a site 1/3 of the electrical distance through the membrane from the internal side. Instantaneous inhibition was relieved at positive potentials, implying significant polyamine permeation. Spermine also induced exponential current relaxations to a `steady-state' impermeant level. This inhibition was also mediated by a binding site 1/3 of the electrical distance through the pore, but with a K _d of 2.6 mm. Spermine inhibition was explained by postulating two spermine binding sites at a similar depth. Occupation of the first site occurs rapidly and with high affinity, but once a spermine molecule has bound, it inhibits spermine occupation of the second binding site via electrostatic repulsion. This repulsion is overcome at higher membrane potentials, but results in a lower apparent binding affinity for the second spermine molecule. The on-rate constant for the second spermine binding saturated at a low rate (∼200 sec⁻¹ at +120 mV), providing further evidence for an allosteric mechanism. Polyamine-induced inward rectification was significant at physiological concentrations. Received: 17 February 1999/Revised: 27 April 1999     

Partial Deletion of both the Spermine Synthase Gene and thePexGene in the X-linked Hypophosphatemic, Gyro (Gy) Mouse

Gy, along withHyp, is a dominant mutation of the normal genePexcausing X-linked hypophosphatemia in the mouse. HemizygousGymale mice, however, have greater defects in survival, bodily growth, skeletal mineralization, and neurological function than those found in heterozygousGyfemales or inHypmice. Since the gene for spermine synthase is immediately upstream of the homologous human genePEX, we compared the effects of theGyandHypmutations on both the spermine synthase gene and thePexgene. Barely detectable levels of spermine (<5 % of normal) with elevated levels of its precursor, spermidine, were found in organs ofGymale mice compared to normal male littermates. NeitherGyfemales norHypmale mice were significantly affected. Four missing introns of the spermine synthase gene were identified inGymale mice, suggesting extensive gene disruption. A pseudogene for spermine synthase was also identified in the mouse genome.PexmRNA was found in several but not all tissues studied in adult normal mice.PexmRNA was altered in bothGyandHypmice. All maleHypmice were lacking the 3′ end of thePexmessage, whereas all maleGymice were deficient at the 5′ end. In summary, theGymutation is associated with a recessively expressed mutation of the spermine synthase gene, leading to spermine deficiency, and a dominantly expressed mutation of thePexgene, leading to hypophosphatemia. Alterations in two contiguous genes inGymay explain the additional phenotypic abnormalities present in theGymale mouse.     

Novel metabolically stable and functionally active mimetic of spermidine

Earlier unknown 1,8-diamino-3-methyl-4-azanonane (γ-MeSpd) was synthesized. The analogue was a substrate of neither spermine/spermidine N ¹-acetyltransferase nor spermine synthase, but was capable to support the growth of DU145 cells having depleted polyamine pools. Such a combination of γ-MeSpd properties discloses novel opportunities to study cellular functions of catabolically unstable and easily interconvertible spermine and spermidine.     

Biological activity of cholecystokinin (30–33) tetrapeptide analogues

Analogues of the endogenous peptide corresponding to the 30–33 sequence of cholecystokinin (Trp-Met-Asp-Phe-NH₂) were synthesized, and their biological activity was studied. It was shown that, in rats, the N-succinylated Nle² analogue of this tetrapeptide exhibits increased anxiolytic properties in the dark-light chamber test and an enhanced alcohol intake by both the control animals and the alcohol-dependent animals under the conditions of free choice. Introduction of an isopropyl residue into the C-terminal amide of the Nle² analogue resulted in the appearance of anxiogenic and antialcohol activity and the ability to increase the morphine analgesic effect in the tail-flick test on rats. The two synthesized analogues retained an affinity for cholecystokinin receptors.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 130–139.Original Russian Text Copyright © 2005 by Proskuryakova, Bespalova, Palkeeva, Petrichenko, Pankratova, Shokhonova, Anokhina.     

Polyamines are essential for the synthesis of 2-ricinoleoyl phosphatidic acid in developing seeds of castor

The major fatty acid component of castor (Ricinus communis L.) oil is ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid), and unsaturated hydroxy acid accounts for >85% of the total fatty acids in triacylglycerol (TAG). TAG had a higher ricinoleate content at position 2 than at positions 1 and 3. Although lysophosphatidic acid (LPA) acyltransferase (EC 2.3.1.51), which catalyzes acylation of LPA at position 2, was expected to utilize ricinoleoyl-CoA preferentially over other fatty acyl-CoAs, no activity was found for ricinoleoyl-CoA in vitro at concentrations at which other unsaturated acyl-CoAs were incorporated rapidly. However, activity for ricinoleoyl-CoA appeared with addition of polyamines (putrescine, spermidine, and spermine), while polyamines decreased the rates of incorporation of other acyl-CoAs into position 2. The order of effect of polyamines on LPA acyltransferase activity was spermine > spermidine >> putrescine. At concentrations of spermine and spermidine of >0.1 mM, ricinoleoyl-CoA served as an effective substrate for LPA acyltransferase reaction. The concentrations of spermine and spermidine in the developing seeds were estimated at ∼0.09 and ∼0.63 mM, respectively. These stimulatory effects for incorporation of ricinoleate were specific to polyamines, but basic amino acids were ineffective as cations. In contrast, in microsomes from safflower seeds that do not contain ricinoleic acid, spermine and spermidine stimulated the LPA acyltransferase reaction for all acyl-CoAs tested, including ricinoleoyl-CoA. Although the fatty acid composition of TAG depends on both acyl-CoA composition in the cell and substrate specificity of acyltransferases, castor bean polyamines are crucial for incorporation of ricinoleate into position 2 of LPA. Polyamines are essential for synthesis of 2-ricinoleoyl phosphatidic acid in developing castor seeds.     

Synthesis of 2,11-bis(methylidene)spermine,a new inhibitor of spermine oxidase

Earlier unknown 1,12-diamino-2,11-bis(methylidene)-4,9-diazadodecane, which may be considered as an enzyme-activated inhibitor of spermine oxidase, was synthesized in 6 steps starting from 2-chloromethyl-3-chloropropene-1 in a high yield. Application of newly synthesized spermine analogue for the inhibition of spermine oxidase was discussed.     

Effect of polyamines on the inhibition of peptidyltransferase by antibiotics: revisiting the mechanism of chloramphenicol action

Chloramphenicol is thought to interfere competitively with the binding of the aminoacyl-tRNA 3′-terminus to ribosomal A-site. However, noncompetitive or mixed-noncompetitive inhibition, often observed to be dependent on chloramphenicol concentration and ionic conditions, leaves some doubt about the precise mode of action. Here, we examine further the inhibition effect of chloramphenicol, using a model system derived from Escherichia coli in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes, under ionic conditions (6 mM Mg²⁺, 100 mM NH₄⁺, 100 µM spermine) more closely resembling the physiological status. Kinetics reveal that chloramphenicol (I) reacts rapidly with AcPhe-tRNA·poly(U)·70S ribosomal complex (C) to form the encounter complex CI which is then isomerized slowly to a more tight complex, C*I. A similar inhibition pattern is observed, if complex C modified by a photoreactive analogue of spermine, reacts in buffer free of spermine. Spermine, either reversibly interacting with or covalently attached to ribosomes, enhances the peptidyltransferase activity and increases the chloramphenicol potency, without affecting the isomerization step. As indicated by photoaffinity labeling, the peptidyltransferase center at which chloramphenicol binds, is one of the preferred cross-linking sites for polyamines. This fact may explain the effect of spermine on chloramphenicol binding to ribosomes.     

An investigation of the dynamics of spermine bound to duplex and quadruplex DNA by <Superscript>13</Superscript>C NMR spectroscopy

A detailed analysis of the ¹³C relaxation of ¹³C-labelled spermine bound to duplex and quadruplex DNA is presented. T₁, T₂ and heteronuclear NOE data were collected at four ¹³C frequencies (75.4, 125.7, 150.9 and 201.2 MHz). The data were analyzed in terms of a frequency-dependent order parameter, S ²(ω), to estimate the generalized order parameter and the contributions to the relaxation from different motional frequencies in the picosecond–nanosecond timescale and from any exchange processes that may be occurring on the microsecond–millisecond timescale. The relaxation data was surprisingly similar for spermine bound to two different duplexes and a linear parallel quadruplex. Analysis of the relaxation data from these complexes confirmed the conclusions of previous studies that the dominant motion of spermine is independent of the macroscopic tumbling of the DNA and has an effective correlation time of ∼50 ps. In contrast, spermine bound to a folded antiparallel quadruplex had faster relaxation rates, especially R ₂. As with the other complexes, a fast internal motion of the order of 50 ps makes a substantial contribution to the relaxation. The generalized order parameter for spermine bound to duplex DNA and the linear quadruplex is small but is larger for spermine bound to the folded quadruplex. In the latter case, there is evidence for exchange between at least two populations of spermine occurring on the microsecond–millisecond timescale. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The polyamine spermine induces cystocarp development in the seaweed <Emphasis Type="Italic">Grateloupia</Emphasis> (Rhodophyta)

Polyamines such as putrescine, spermidine and spermine are ubiquitous aliphatic amines involved in reproductive events in plants and algae, and first become evident through changes in endogenous levels during reproductive development. To examine whether the differences observed in polyamines, during carposporogenesis, in the red alga Grateloupia, followed a specific pattern as is seen in other organisms, infertile axes (i.e. not showing cystocarps) were excised from the same holdfast of female fertilized individuals (i.e. showing cystocarps in other axes), and cultivated until the cystocarps became visible. Changes in the endogenous levels of free putrescine, spermidine and spermine were monitored over the 8 days of culture. The activity of enzymes related to polyamine metabolism, such as l-ornithine decarboxylase (ODC), diamine oxidase and polyamine oxidase, was measured at the beginning and end of the experimental period. Up to 50% of the infertile axes became fertile and produced cystocarps at a density of 1.91 ± 0.1 cystocarps mm⁻² after 8 days. The endogenous content of spermine increased markedly over the first 5 days of culture, then decreased to the initial level by day 8. Spermidine followed a similar pattern to spermine, whereas putrescine remained at high levels, until day 5 when it decreased abruptly. The activity of ODC was less on day 8 than on day 0, whereas the activities of diamine oxidase and polyamine oxidase increased. In parallel experiments with explants from infertile axes, exogenously added spermine (10⁻⁶ M) increased the number of cystocarps, and reversed the effect of cyclohexylamine (CHA), which is known to inhibit polyamine synthesis in Grateloupia. Serial sectioning and microscopic observation of specimens from explants cultivated in 10⁻⁶ M spermine indicated that cystocarp development was induced. The results suggest that, during transition from infertile to fertile spermine is accumulated, thus favouring the development of cystocarps, given the presumed role of spermine as an inducing agent.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation and alkaloids of <Emphasis Type="Italic">Hippeastrum vittatum</Emphasis>

Different concentrations of methyl jasmonate, spermine, casein hydrolysate, or progesterone combined with 16 mg/l 2 iP + 4 mg/l naphthalene acetic acid (NAA) were investigated in order to obtain higher multiplication rate and growth of Hippeastrum vittatum bulbs in vitro on MS basal medium. The highest multiplication rate (8.2 bulbs/explant) was attained with 80 mg/l spermine, while the highest bulb fresh weight (1.23 g/bulblet) was obtained with 4 mg/l methyl jasmonate. Progesterone at 20 mg/l or casein hydrolysate at 2.0 g/l gave the highest leaf length (14.1 and 13.2 cm, respectively). So, it can be advised to use 80 mg/l spermine combined with 16 mg/l 2 iP + 4 mg/l NAA to obtain the highest number of bulbs per explant with moderate leaf length and bulb fresh weight. Chemical analysis showed alternations in the alkaloid type ratio and number of compounds in the bulbs treated with methyl jasmonate (4 mg/l).     

Polycationic amphiphiles based on triethylenetetramine and their transfection efficacy

Novel polycationic amphiphiles derived from triethylenetetramine, a spermine analogue, containing cholesterol or dialkylglycerol residues as hydrophilic domains were synthesized. The amphiphiles and a helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) served for the preparation of cationic liposomes, physicochemical properties of which were evaluated. A comparative study of cytotoxicity and transfection efficiency demonstrated that the replacement of spermine by triethylenetetramine decreased transfection activity of cationic liposomes.