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Yeast Ssb proteins (Ssbp) are ribosome-associated Hsp70 chaperones that function in translation. Elevated levels of Ssbp enhance the ability of over-expressed Hsp104 chaperone to eliminate the yeast [PSI+] prion, while depletion of Ssbp reduces this effect. Millimolar concentrations of guanidine in the growth medium cure yeast cells of prions by inactivating Hsp104. Guanidine is also toxic to yeast, irrespective of the status of Hsp104 and [PSI+]. Strains that lack Ssbp are hypersensitive to guanidine toxicity. Here we show that ssb- cells have normal numbers of [PSI+] "seeds", but can be cured of [PSI+] using one-sixth of the guanidine concentration required to eliminate [PSI+] from SSB cells. Correspondingly, the level of intracellular guanidine was eight-fold higher in ssb- cells than in wild-type cells, which explains all effects of Ssbp depletion on susceptibility to guanidine. The sensitivity of wild-type cells to the effects of guanidine also correlated with guanidine uptake, which was enhanced at low temperature. Guanidine sensitivity of strains mutated in any of 16 ABC membrane transporters, which are implicated in multidrug resistance, was normal. We found that an erg6 mutant that has an altered membrane lipid composition was hypersensitive to guanidine toxicity, but the lipid composition of ssb- cells was identical to that of wild-type cells. Our results suggest that Ssbp depletion does not affect prion seed regeneration, and that elevated guanidine uptake by ssb- cells may be due to increased retention rather than to an alteration in active or passive transport of the compound.
An efficient method for the solid-phase synthesis of cyclic guanidines from N-acylated amino acid amides, bis cyclic guanidines from N-acylated dipeptides derived from orthogonally protected diamino acids, and N-acylated guanidines from disubstituted cyclic guanidines is described. The exhaustive reduction of N-acylated amino acid amides yields diamines that on treatment with cyanogen bromide lead to the formation of cyclic guanidines. Resin-bound orthogonally protected diamino acids (i.e., N(alpha)-Fmoc-N(x)-(Boc)-diamino acid, x = beta, gamma, delta, epsilon) were N-acylated following removal of the Fmoc group. Removal of the Boc functionality from the side chain then generated a primary amine. Subsequent coupling of Boc amino acids, followed by removal of the Boc group, generated dipeptides that were N-acylated. Exhaustive reduction of amide bonds of the N-acylated dipeptides generated tetraamines having four secondary amines, which upon cyclization with cyanogen bromide afforded the resin-bound trisubstituted bis cyclic guanidines. Treatment of the resin-bound disubstituted cyclic guanidines with carboxylic acids gave N-acylated guanidines. On the basis of their high yield and purity, bis cyclic guanidines derived from N(alpha)-Fmoc-N(epsilon)-Boc-lysine and N-acylated guanidines were chosen for preparation of mixture-based combinatorial libraries. Details of the preparation of these positional scanning libraries using the "libraries from libraries" concept are presented.
The presence of low concentrations of guanidine . HCl has a pronounced effect on the overall rate of reactivation of lactic dehydrogenase from pig muscles after preceding dissociation and deactivation in various denaturants. The obseverd attenuation is a function of the amount of guanidine . HCl present during reconstitution. At a given guanidine concentration in the reactivation buffer the yield, but not the rate of reactivation, is influenced by the extent of denaturation caused initially in the process of deactivation and dissociation. As a possible explanation for the influence of guanidine . HCl on the kinetics of reconstitution, binding of the ligand to intermediates of folding and association is considered. This hypothesis is corroborated by the observation that guanidine . HCl in the relevant concentration range does bind to native lactic dehydrogenase without inactivating the enzyme or disrupting its quaternary structure. A kinetic model comprising guanidine binding to both the native enzyme and structured intermediates is proposed to describe the observed effects of guanidine . HCl on the rate of reactivation. In addition, the dissociation constants for guanidine binding to intermediates of reconstitution and to native lactic dehydrogenase are estimated.
An operationally straightforward and efficient method for the alkylation of carbamate-protected guanidines with various alkyl halides and mesylates is described. This protocol proceeds via deprotonation of the acidic N-carbamate hydrogen of the guanidine under biphasic conditions using a catalytic amount of a tetrabutylammonium salt as a phase-transfer catalyst. In this manner, highly functionalized guanidines can be obtained. The reaction is tolerant of a wide range of functional groups on both the alkyl halide and guanidine component. In addition, the reaction is sufficiently mild such that simple aqueous workup and filtration through a short silica gel column yields the substituted guanidines in high purity. In conjunction with the EDCI-mediated guanylation of disubstituted thioureas with amines, phase-transfer catalyzed alkylation of guanidines via a one-pot, three-component synthesis of substituted guanidines was achieved.
Spectrophotometric titration curves were obtained at 242 nm for native and fully guanidinated horse-heart ferricytochrome c. The cytochrome c data were fit over the pH range 9-12 (I = 0.35) by a theoretical curve with pK' values of 10.35 and 11.70. The slope of the experimental data increases sharply above pH 12.5 suggesting that two tyrosine residues with pK' values greater than 12.5 are exposed by conformation change. The guanidinated cytochrome c data after correction for the alkaline spin-state transition were fit over the entire pH range 9-13.6 (I = 0.35) by a theoretical curve with pK' values 10.37, 10.78, 11.50, and 13.60. These results along with viscosity measurements indicate that the unfolding transition occurs at higher pH in the guanidinated derivative. N-Acetylimidazole was used to acetylate specific tyrosyl groups of guanidinated cytochrome c. Assignments of acetylated tyrosine residues were confirmed by peptide mapping of 14C-labelled derivatives. Spectrophotometric titrations with rapid data acquisition of two monoacetylated derivatives allowed assignments of pK'1 (10.37) to Tyr-67 and pK'4 (13.60) to Tyr-97. The basis for the large differences in acidity and chemical reactivity of these two residues is not obvious from the crystallographic structure and may arise from differences in solvent access due to motions of the polypeptide chain.
With 3-bromo-4-fluorotoluene as starting material, [4-fluoro-3-(trimethylsilyl)benzyl]guanidine was prepared in five steps in 1.5% overall yield. Radioiodination of this silicon precursor using N-chlorosuccinimide in trifluoroacetic acid at room temperature for 5 min gave (4-fluoro-3-[131I]-iodobenzyl)guanidine ([131I]FIBG) in 50-60% radiochemical yield. A byproduct which had a retention time in two HPLC systems similar to that of (m-iodobenzyl)guanidine (MIBG) was formed in about 30% yield. [131I]FIBG was stable up to 3 h under these conditions of iodination, indicating that the byproduct is not generated as a result of [131I]FIBG degradation. Using hydrogen peroxide as the oxidant in aqueous medium and a reaction time of 30 min at 50 degrees C, yields of [131I]FIBG could be increased to 75-80%, with less than 7% of the byproduct formed under these conditions. Astatination of the silicon precursor using N-chlorosuccinimide in trifluoroacetic acid at 70 degrees C gave 65-70% radiochemical yield of (3-[211At]astato-4-fluorobenzyl)guanidine ([211At]AFBG) in 10-15 min; about 17% of the byproduct formation was seen. Astatination of the silicon precursor under aqueous conditions using hydrogen peroxide was not successful.
Crambescidins and batzelladines, novel marine guanidine alkaloids, have unique pentacyclic and tricyclic guanidine core structures, respectively. They display a considerable array of biological activity and not surprisingly have attracted considerable synthetic interest. The first total synthesis of crambescidin 359 (7) and stereoselective total synthesis of batzelladine D (11) were accomplished based on a successive 1,3-dipolar cycloaddition reaction strategy. During synthetic studies of 7, the absolute stereochemistry was revealed. Based on the structure of 7, the novel C2-symmetric pentacyclic guanidine compounds 69a-d were designed and synthesized as guanidine organocatalysts. The catalyst 69b works efficiently as an asymmetric catalyst of the alkylation reaction of the glycynate-benzophenone Schiff base 73, which gives 74 with 80-90% ee.
Simple preparation methods of modified guanidines have been explored as potential chiral superbases. Thus, 3,7,8-trisubstituted and 3,6,7, 8-tetrasubstituted 1,4,6-triazabicyclooctene systems were prepared from (1S,2S)-1,2-diphenylethylenediamine through stepwise 2-chloro-1, 3-dimethylimidazolinium chloride (DMC)-induced cyclizations of protected thioureas to the corresponding 2-iminoimidazolidines and then of 2-(2-hydroxyethylimino)imidazolidines to the bicyclic systems. Linear guanidines with a 2-hydroxyethyl functional group were prepared by the reaction of carbodiimides with 2-amino alcohols. Reaction of linear-type guanidines with DMC followed by base treatment afforded 1,4-disubstitued 2-iminoimidazolidines. Furthermore, another type of 1,4,6-triazabicyclooctene was also prepared through double DMC-induced cyclization of guanidines with two 2-hydroxyethyl substituents.
meta-Iodobenzyl guanidine (MIBG) combines the structural properties of the neuron-blocking agents bretylium and guanethidine and is being used increasingly for various clinical applications. Different samples of MIBG were assayed for possible contamination with benzyl guanidine (BG). Fast-atom-bombardment mass spectrometry (FAB-MS) analysis showed a prominent but variable m/z 150 signal, corresponding to a protonated BG. The MS/MS fragmentation pattern of these [M + H]+ ions was similar to that obtained from FAB-MS-generated, protonated BG, confirming the proposed molecule and associated structures. RP-HPLC analysis of both guanidines, however, excluded the possibility of contamination of MIBG with BG. It was therefore concluded that the BG signal was an artifact of the FAB-MS procedure. In addition, the importance of the meta-substituted iodine for the biological activity of MIBG was investigated. Three different biochemical and cell-biological properties of MIBG were compared with those of its precursor MIBA and BG. The assays used were: inhibition of the catecholamine "Uptake I" system in SK-N-SH neuroblastoma and PC-12 pheochromocytoma cells, inhibition of mitochondrial respiration, and general cytotoxicity in L1210 leukemia cells. Of the drugs tested, MIBG was the most efficient in Uptake I inhibition and was more toxic in survival assays, but as compared with BG it was almost equipotent in inhibiting mitochondrial respiration. These findings contribute to a further elucidation of the mechanism by which MIBG exerts its various actions.
The microsomal N-hydroxylation of the strongly basic guanidinium group (debrisoquine) to N-hydroxyguanidine (N-hydroxydebrisoquine) and the retroreduction of the N-hydroxyguanidine are demonstrated for the first time. The reduction of the N-hydroxyguanidine by liver homogenates and hepatocytes is catalysed by a microsomal NADH-dependent system that is strongly inhibited by hydroxylamine or N-methylhydroxylamine. In the presence of these alternate substrates for the reductase the microsomal catalysed N-hydroxylation of debrisoquine is readily characterized. The oxidation was inhibited by antibodies against NADPH cytochrome P450 reductase and the role of the P450 monooxygenase was further verified by studies with partially purified and purified P450 2C3 reconstituted systems. The transformation of N-hydroxydebrisoquine to the corresponding urea derivative was also detected in in vitro experiments with microsomal fractions and enriched P450 fractions as well as with flavin-containing monooxygenase (FMO). Experiments with catalase, superoxide dismutase and H2O2 have shown that the H2O2 or O2-, respectively, formed from the respective enzyme and the substrate, apparently participated in the reaction. Whereas the N-hydroxylation of the guanidine involves the usual monooxygenase activity of cytochrome P450 the resultant N-hydroxyguanidine decouples monooxygenases (cytochrome P450, FMO) and the H2O2 and, above all, O2- thus formed transform the N-hydroxyguanidine further to the corresponding urea derivative. The possibility for the N-hydroxylation of non-physiological guanidines to N-hydroxyguanidines and subsequent oxidative conversion to the respective urea is comparable to the physiological transformation of arginine to citrulline via N-hydroxyarginine with the liberation of nitric oxide (endothelial derived relaxing factor) and could, therefore, contribute to the efficacy of drugs containing guanidine and similar functional groups.
The titanium dinitrogen complex, [[(Me(2)N)C(N(i)Pr)(2)]( 2)Ti](2)(N(2)) (2), was synthesized by reduction of the dichloride precursor, [(Me(2)N)C(N(i)Pr)(2)](2)TiCl(2) (1). The dinitrogen complex reacts with phenyl azide to yield the titanium imido complex, [(Me(2)N)C(N(i)Pr)(2)](2)TiNPh (3). The fluxional behavior of the guanidinate ligands in compounds 1-3 was investigated using variable temperature and two-dimensional NMR techniques; guanidinate ligand rotation and racemization reactions were observed. Rearrangement of the guanidinate ligand to an asymmetrical bonding mode utilizing the dimethylamino and amide-nitrogen atoms is observed in the bridging oxo and sulfido derivatives (4 and 5). These compounds are formed by the reactions of 2 with pyridine N-oxide and propylene sulfide, respectively. The ligand rearrangement was observed to be reversible for the bridging sulfido complex 5; the structure of this compound is sensitive to temperature and solvent. The solid-state and solution structures of compounds 1-5 are discussed.
cDNA fragments representing the region in polypeptide 2C containing mutations in a guanidine-resistant or -dependent mutant were cloned into the wild-type background of an infectious clone. Transfection of COS-1 cells with these plasmids yielded viruses that were either completely resistant to 2.0 mM guanidine hydrochloride or dependent on this concentration of drug for growth.
We have carefully examined the frequency of guanidine-resistant revertants in six different clonal pools of guanidine-dependent mutants of type 1 poliovirus. The mutation frequency was (6.5 +/- 6.3) x 10(-4) (with all amino acid substitutions occurring at position 227). The minimal corrected base substitution frequency per single nucleotide site in the codon for amino acid 227 was (2.1 +/- 1.9) x 10(-4).
Effect of two classical and potent denaturants, guanidine hydrochloride (GuHCl) and guanidine thiocyanate (GuHSCN) on purified wheat germ lipase has been studied. Lipase was found to be active only up to 5 M GuHCl and 1.5 M GuHSCN. The extent of interaction was determined by the measurement of apparent partial specific volume of the enzyme in presence of these two denaturants. While the preferential interaction parameter (zeta 3) has values of 0.08 +/- 0.02 and 0.14 +/- 0.03 g/g, the interaction parameter (delta m3/delta m2)T,mu 1, mu3 has values of 35 +/- 9 and 50 +/- 10 mole/mole for GuHCl and GuHSCN, respectively. The number of denaturant molecules bound to the enzyme, A3, obtained experimentally were 0.486 +/- 0.020 and 0.348 +/- 0.020 g/g and the calculated values were 0.459 +/- 0.023 and 0.567 +/- 0.030 g/g for 6 M GuHCl and 3 M GuHSCN, respectively. The volume change occurring upon denaturation results in -420 +/- 42 and -462 +/- 84 ml/mole in 6 M GuHCl and 3 M GuHSCN, respectively. The denaturation is accompanied by exposure of hydrophobic groups to the bulk solvent as confirmed by fluorescence emission measurements of the enzyme. The Tm measurements indicated a control value of 56 +/- 1 degree C. In presence of 6 M GuHCl/3 M GuHSCN, the value was 42 +/- 1 degree C. These results explain the retention of lipase activity even at 5 M GuHCl from a mechanistic point of view.
MNNG is a strong topically acting carcinogen. Various single oral doses (150, 120, 60 mg/kg) were investigated regarding their effect on the mitotic activity of epithelial tissues in male Sprague-Dawley rats. MNNG inhibits cell proliferation in the forestomach, jejunum and esophagus. Necroses were observed in the forestomach and in the liver (150 mg/kg). Independent of any tissue lesions MNNG produces an elevated mitotic activity of the hepatocytes and in the adrenal cortex. These proliferative effects are not observed when the structurally related noncarcinogenic compounds guanidine carbonate (GC) and guanidine nitrate (GN) are used.
Long chain free fatty acids interfere with the inhibitory action of phenethylbiguanide and related compounds on mitochondrial respiration in vitro. This interference depends on binding of fatty acids to mitochondria and diminishes with decreasing chain length. Reversal of guanidine-derivative inhibition by fatty acids differs from that caused by dinitrophenol in that the effect of fatty acid is achieved without alteration in coupling or respiratory control. The binding of phenethylbiguanide to mitochondria is inhibited by both fatty acid and dinitrophenol. Serum albumin potentiates the inhibitory potency of guanidine derivatives, probably by removing endogenous mitochondrial free fatty acids.
Modified guanidines were explored as potential chiral superbases. Thus, chiral 1,3-dimethyl-2-iminoimidazolidines with or without 4, 5-diphenyl groups, their guanidinium salts, and the 2-iminoimidazolidines with (S)-1-phenylethyl groups on the ring nitrogens were prepared by treatment of 2-chloroimidazolinium chlorides with appropriate amines. Bicyclic guanidines were also prepared from a prolinamide using a similar procedure.